Bioactive Natural Products [11, 1 ed.] 0444636021, 9780444636027, 9780444636102, 0444636102

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Contributors Numbers in Parentheses indicate the pages on which the author’s contributions begin.

A.F. Barrero (1), Institute of Biotechnology, University of Granada, Avda. Fuentenueva, Granada, Spain R. Braz-Filho (231), Laborato´rio de Ci^encias Quı´micas, Centro de Ci^encias e Tecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes; ICE, Universidade Federal Rural do Rio de Janeiro, Seropedica, Brazil S.M. Cardoso (65), CERNAS, School of Agriculture, Polytechnic Institute of Coimbra Bencanta, Coimbra; University of Aveiro, Aveiro, Portugal M.D. Catarino (65), CERNAS, School of Agriculture, Polytechnic Institute of Coimbra Bencanta, Coimbra, Portugal G. Chen (209), School of Life Science and National Glycoengineering Research Center, Shandong University, Jinan; Center for Gene and Cell Engineering, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China K. Chen (209), School of Life Science and National Glycoengineering Research Center, Shandong University, Jinan; Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Wannan Medical College, Wuhu, PR China A.R. de Carvalho Jr. (231), Laborato´rio de Ci^encias Quı´micas, Centro de Ci^encias e Tecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Brazil M.G. de Carvalho (231), ICE, Universidade Federal Rural do Rio de Janeiro, Seropedica, Brazil D. Dı´ez (137), Facultad de Ciencias Quı´micas, Universidad de Salamanca, Salamanca, Spain P.W. Dhore (287), Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, India V. Domingo (1), Institute of Biotechnology, University of Granada, Avda. Fuentenueva, Granada, Spain I. Duttagupta (29), Indian Association for the Cultivation of Science, Kolkata, West Bengal, India B. Fenert (399), Apteka “Dbam o Zdrowie”, Szczecinek, Poland H. Gao (347), Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, PR China

xi

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Contributors

K.C. Ghosh (29), Indian Association for the Cultivation of Science, Kolkata, West Bengal, India A. Gil-Meso´n (137), Facultad de Ciencias Quı´micas, Universidad de Salamanca, Salamanca, Spain J.N. Jacob (101), Organomed Corporation, Coventry, RI, United States V. Karuppiah (417), Marine Biotechnology Laboratory, State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China D.M. Kokare (287), Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, India U.R. Lal (263), Birla Institute of Technology, Ranchi, Jharkhand, India C. Li (209), Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Wannan Medical College, Wuhu, PR China J. Li (347), State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, PR China Z. Li (417), Marine Biotechnology Laboratory, State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China H. Makabe (323), Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Kami-ina, Nagano, Japan I.S. Marcos (137), Facultad de Ciencias Quı´micas, Universidad de Salamanca, Salamanca, Spain R.F. Moro (137), Facultad de Ciencias Quı´micas, Universidad de Salamanca, Salamanca, Spain J.F. Quilez del Moral (1), Institute of Biotechnology, University of Granada, Avda. Fuentenueva, Granada, Spain A. Rabahi (65), Centre de Recherche Scientifique et Technique en Analyses PhysicoChimiques CRAPC, Bou-Ismail, Tipaza, Algeria N.A. Raut (287), Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, India S.D. Saoji (287), Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, India A.M.S. Silva (65), University of Aveiro, Aveiro, Portugal A. Singh (263), Herbal Consultant, Phase-VII, Mohali, Punjab, India S. Sinha (29), Indian Association for the Cultivation of Science, Kolkata, West Bengal, India W. Sun (417), Marine Biotechnology Laboratory, State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China O. Talhi (65), University of Aveiro, Aveiro, Portugal I.J.C. Vieira (231), Laborato´rio de Ci^encias Quı´micas, Centro de Ci^encias e Tecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Brazil

Contributors

xiii

M.B. Zarzycka (399), Apteka “Bursztynowa”, Koszalin, Poland P.K. Zarzycki (399), Section of Toxicology and Bioanalytics, Koszalin University of Technology, Koszalin, Poland H. Zhao (347), Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, PR China J. Zou (347), Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, PR China

Preface The exploration of bioactive molecules from natural resources continues to play a major role in developing novel natural products of therapeutic significance. Volume 48 of Studies in Natural Product Chemistry presents many interesting classes of natural products with exciting biological activities. In the past century, remarkable creativity has been achieved in the synthesis of structurally complex natural products. In the first chapter, Domingo et al. have presented some recent accomplishments in the total synthesis of different families of natural products through C–H functionalization strategies. Nonribosomal peptides (NRPs) comprise a large group of pharmacologically important natural products, mostly isolated from microorganisms. Nowadays, special importance has been given to their isolation and chemical synthesis for their use as antibiotics, immunosuppressants, etc. Sinha et al. have discussed the chemical synthesis of these NRPs in Chapter 2. Flavonoids are polyphenolic compounds with very diverse roles. They occur ubiquitously in food plants and vegetables. In Chapter 3, Cardoso et al. have discussed the basic chemistry of flavonoids, structure–affinity relationship between flavonoids and key inflammatory markers, as well as an approach to some synthetic strategies targeting the enhancement antiinflammatory properties of flavonoids. The two major rhizomes, turmeric (Curcuma longa) and ginger (Zingiber officinale), originated from the same family, Zingiberaceae, are known to have a variety of medicinal and biological properties. In Chapter 4, Jacob has provided a comparative account of the structural and biochemical properties of volatile and nonvolatile fractions of turmeric and ginger. Marcos et al. have discussed a complete biogenetic classification of diterpenes that have a 7-6-5 tricarbocyclic system in Chapter 5. A biosynthetic approach to each group and synthetic approaches that have not been reported previously are presented. Fructooligosaccharides (FOSs) are oligosaccharides that are composed of linear chains of fructose units, linked by beta (2-1) bonds. They exhibit a number of interesting properties, including a low sweetness intensity; they are also calorie free, noncariogenic and are considered as soluble dietary fibers. Chen et al. have reviewed the mechanism of action and effects of FOSs with emphasis on the relationship between the prebiotic effects and the benefits to health in Chapter 6.

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Preface

In Chapter 7, Vieira et al. have provided a review on the world’s largest genus, Psychotria, with nearly 2000 species, mainly found in tropical and subtropical regions of the globe. They have presented the chemical, biological, and synthetic aspects of compounds found in this genus. The diseases that are mostly prevalent in tropical and subtropical regions can be cured efficiently by the natural product-based drugs. Lal and Singh have discussed, in Chapter 8, the trends during the last 5 years (2008–13) in the discovery and development of natural product-based drugs against tropical diseases. The epidemic rise in the number of patients suffering from Diabetes mellitus requires prompt action for discovering new remedies especially from bioactive natural resources. In Chapter 9, Raut et al. have presented a review focusing the importance of bioactive natural products for the treatment of this disease. Prodelphinidins have many significant biological activities such as antitumor, antiviral, and antiinflammatory. A comprehensive discourse on isolation, synthesis, and biological activities of prodelphinidins is presented by Makabe in Chapter 10. Gao et al. have presented a detailed review on the source, chemistry, bioactivities, and biosynthetic pathway of natural products from endolichenic fungi in Chapter 11. Cyanobacteria (blue-green algae) are photosynthetic prokaryotes that are excellent source of vitamins and proteins. Many cyanobacteria produce compounds with potent biological activities. In Chapter 12, Zarzycki et al. have provided general information about active metabolites from cyanobacteria and particularly about the use of such biological materials as the ingredients in food and pharmaceutical formulations. Actinomycetes are one of the most efficient groups of secondary metabolite producers which play a significant role in pharmaceutical applications. Around 150 natural products have been isolated so far from these ubiquitous actinobacteria. Li et al. have presented a review on the natural products isolated from marine actinomycetes. I am confident that this volume will prove to be of great interest to scientists working in the field of medicinal chemistry and natural product chemistry. I would like to thank Ms. Taqdees Malik and Ms. Humaira Hashmi for their assistance in the preparation of this volume. I am also grateful to Mr. Mahmood Alam for the editorial assistance. Atta-ur-Rahman, FRS International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry University of Karachi Karachi 75270 Pakistan

Chapter 1

Recent Accomplishments in the Total Synthesis of Natural Products Through C–H Functionalization Strategies V. Domingo*, J.F. Quilez del Moral and A.F. Barrero* Institute of Biotechnology, University of Granada, Avda. Fuentenueva, Granada, Spain

Chapter Outline Introduction Radical C–H Functionalization (
)-Leuconoxine Ouabagenin Gracilioether F Metal-Catalyzed C–H Functionalization Podophyllotoxin Dictyodendrin A and Dictyodendrin B

1 3 3 5 7 9 9

Miscellaneous Azidation of Tetrahydrogibberellic Acid (+)-Myrrhanol C Complanadines A and B (+)-Hongoquercin A Concluding Remarks References

15 15 17 20 23 25 26

10

INTRODUCTION C–H functionalization is the term used to describe the ability to cleave a C–H bond in a molecule regio- and stereoselectively and thereby transform it into new C–C, C–X (X ¼ nitrogen, oxygen, or halogen), or C–metal bonds. These transformations can be also classified according to the chemical species generated as radical, radical-cationic, radical-anionic, carbenic, cationic, or metal-catalyzed transformations (the term C–H activation is used in the last case). A considerable number of reactions and their applications in synthesis have already been described and reviewed covering different types of processes [1–5]. The field of C–H functionalization appears to offer the synthetic chemist the opportunity to find new disconnections almost at *

Co-authors of this chapter.

Studies in Natural Products Chemistry, Vol. 48. http://dx.doi.org/10.1016/B978-0-444-63602-7.00001-1 © 2016 Elsevier B.V. All rights reserved.

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Studies in Natural Products Chemistry

will within a multitude of covalent C–H bonds. Most C–H transformations have been uncovered relatively recently. As a result, these modes of activation are being applied at present in material science, agrochemistry, drug development, perfumery, and the realm of natural products discussed in this chapter (Fig. 1.1). The steady growth and refinement of C–H functionalization is based mainly on the high level of originality and creativity in the disconnection approach to complex organic architectures. In another sense, the transformation of a C–H bond into a bond with a different function is nothing new. It has been present in natural processes for millennia and in natural product synthesis we are simply trying to emulate the level of synthetic efficiency found in the natural world. Nature can generate hydroxyls (P450 oxidases) [6–9], halides (halogenases) [10–12], amines (transaminase and aminoacid dehydrogenase enzymes) [13–16] or construct new C–C bonds (cyclases) [17–21] with surgical precision. Technologically, the most powerful synthetic strategy would be to take an enzyme and use it in every reaction in a synthetic sequence as a simple chemical. However, enzyme engineering has not yet achieved the levels of efficiency that are needed to transform diverse molecular cores and experimentation in enzyme engineering and direct evolution are still mandatory [22,23]. Historically, the application of C–H functionalization in natural products dates from the early studies of steroids. In the early 1960, the Nobel prize winner Sir Derek Barton (1969 for his contribution to “conformational analysis”) found a wide range of new reactions applicable to hydrocarbons. In particular, the discovery of a remote nitrite radical functionalization enabled him to obtain aldosterone acetate in scalable amounts for the first time [24]. The birth of C–H functionalization was also assisted at that stage by other chemists including Breslow, Corey, Arigoni, and Woodward (For a review see Ref [25] and references cited therein). From those times to the present era, C–H functionalization has been increasingly exploited in the synthesis of natural products and reviewed recently [3,26,27], so the aim of this chapter is to highlight the most recent achievements in this field. A number of molecules have been selected to illustrate the

Pharmaceutical targets

Perfumery

Natural product synthesis

C–H activation

C–C, C–X FIG. 1.1 C–H activation field.

Material science

Agrochemistry

Recent Accomplishments in the Total Synthesis Chapter

1

3

diversification that has taken place in terms of the range of natural products (their biosynthetic origin) and the C–H functionalization strategies executed in their construction. Although highly desirable, it is impossible to cover all the contributions that have appeared, and the authors apologize for any omissions.

RADICAL C–H FUNCTIONALIZATION (2)-Leuconoxine (
)-Leuconoxine (1) is grouped as a monoterpene indole alkaloid (over 2000 described) [28] that is characterized by a singular congested diaza[5.5.6.6] fenestrane skeleton (Fig. 1.2). Leuconoxine (1) belongs to the subfamily of Aspidosperma alkaloids, and this natural product was first found in the leaves and stems of plants of the genus Leuconotis (eugenifolius and griffithii) in Malaysia and Indonesia [29,30]. Due to their important range of bioactivities (anticancer, antimalarial, and antiarrhythmic) as well as the challenging scaffolds, indole alkaloids have stimulated the synthetic community and the interest in (
)-leuconoxine (1) has resulted in five syntheses [31]. In 2015, Gaich’s group synthesized this molecule using as its key strategy a novel photoinduced domino macrocyclization/transannular cyclization [31] (Scheme 1.1). The congested ABC-ring system of 1 was assembled in a late stage using a Witkop cyclization, a photochemical C–H functionalization process. The synthetic sequence commenced with the synthesis of the quiral methyl ketone 1a using previously described methods. 1a was afterward transformed into the b-ketosulfide 1b via conversion to the silyl enol ether, electrophilic addition of bromine, and treatment of the bromoketone with dimethylsulfide (90% yield, three steps) (Scheme 1.1). Then, 1b was submitted to Gassman’s indole synthesis conditions obtaining the indole core 1c in excellent yield (77%). After experimenting with several conditions to remove the thiol in 1c and applying a routine set of reactions, they were ready to orchestrate the Witkop photochemical reaction in the a-chloroacetamide 1f. Irradiation of 1f at 254 nm enabled the Witkop photocyclization, affording a regioisomeric mixture of compounds, including indolophanes (2,4) 1g and

Witkop cyclization O H

N N

BN C

A N D

O (–)-Leuconoxine (1)

FIG. 1.2 (
)-Leuconoxine.

Diaza [5.5.6.6] fenestrane skeleton

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Studies in Natural Products Chemistry

O

(a) NEt3, TMSOTf, CH2Cl2

O

O

(b) NBS, THF (c) SMe2, toluene, 80°C

OEt

(d) aniline, –45°C, CH2Cl2, MeCN, tBuOCl then NEt3, then H3PO4

O

MeS

OEt

90%

SMe O OEt N H

77%

1a

1b

1c O

CN

(e) TFA, thiosalicylic acid, 88% (f) DIBAL-H; (g) DMSO, SO3.Py (h) Ph3P K CHCN, toluene, 80°C

NH

(l) BH3.SMe2, THF,

Cl

then NaBO3, 51%

(i) Mg, MeOH, 85% N H

73% (3 steps)

1d

N H

(j) LiAlH4, Et2O O (k) Cl OH DIC, DMAP

1e

73% (2 steps) O NH

Cl

Cl

N H

49% 5:2.5:1:1

HO

O

O

(m) hu (254 nm), Na2CO3, MeOH, rt

Witkop cyclization

HN N H

–Cl

N H

–

HO

HO

1f

Transannular cyclization

HN

O NH O O Transannular cyclization

H

OH

HN HN

N H

1h

1g

HN

HO

N H HO

+

O

H

O

H N

Transient iminium ion

N

N H

N H OH

1i

HO 1j

SCHEME 1.1 Synthetic sequence. O

H

N

O

H N

N H

O

H N

(n) TPAP, NMO, MeCN

N N

50% HO

HO 1j

O (–)-Leuconoxine (1)

SCHEME 1.2 (
)-Leuconoxine end game.

(2,7) 1h together with the desired cyclized product at position C-3 in the heterocycle 1j. The key C–H functionalization step features a photoinduced electron transfer from the excited state of the indolic system to the chloroacetamide moiety. Thus, the diradical cation cyclises forming a transient iminium ion that is trapped intramolecularly affording compounds 1i and 1j (Scheme 1.2).

Recent Accomplishments in the Total Synthesis Chapter

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5

It should be noted that the global yield for this transformation was modest; however, in this case, the power of the C–H functionalization lies in the rapid assembly of the terpene-indolic system in one chemical operation in a cascade fashion. At the end of the synthesis to obtain (
)-leuconoxine (1), 1j was oxidized with catalytic TPAP (tetrapropylammonium perruthenate) which establishes the final fenestrane skeleton of (
)-leuconoxine in 50% yield (Scheme 1.2).

Ouabagenin Ouabagenin (2) is the aglycon of ouabain. The glycoside steroid was isolated in 1888 from the bark of the African ouabio tree (Acokanthera ouabio), and the aglycon was subsequently isolated in 1942 by Mannich and Siewert [32–34]. Ouabagenin (2) is encompassed in the family of the cardenolides (from Greek kardia ¼ heart), a subtype of the family of steroids. Steroids are described as modified (nor) polycyclic triterpenoids lacking from three methyl groups (two in position C-4 in the A ring and one at the junction of the B–C rings), and they are biosynthetically formed from the mevalonic acid pathway (Fig. 1.3). From the medicinal point of view, ouabagenin (2) displays cardiotonic activity through inhibition of the Na+/K+-ATPase, an enzyme found in the plasma membrane of all eukaryotic cells. For this reason, it has proved to be of great value in developing potent inotropic agents for the treatment of congestive heart failure. Topologically speaking, cardenolides possess a 6-6-6-5 carbocyclic skeleton where both A/B and C/D ring fusions are cis in contrast to the typical steroid structure. In the D ring, a characteristic butenolide framework is found at C-17, and this motif is in part responsible for the high bioactivity of this molecules. However, despite its promising bioactive profile, the first total synthesis of this molecule was not achieved until 2008 due to its complexity [35]. The impressive synthetic composition carried out by Deslongchamps et al. employed a polyanionic cyclization as key step that afforded a few milligrams of product at the end of the sequence. O

C–H oxidations O

O 19

O

11

HO HO HO

C

1

HO

OH

B

H A

14

H D

5

HO Ouabagenin (2) FIG. 1.3 Ouabagenin.

Steroid skeleton (cardenolide)

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Studies in Natural Products Chemistry

As an example of the advantages of employing “C–H functionalization logic,” Baran et al. recently accomplished a scalable synthesis of ouabagenin (>100 mg obtained, >500 mg obtained using the more direct precursor) by using a classical semisynthetic approach starting from a cheap precursor, cortisone acetate. Shortly afterward, they published a full account describing the synthesis of 2 and the approach toward C-19-hydroxylated corticosteroid analogs [36,37]. The authors tackle the synthesis through two relay elements: redox relay (oxidations set at C-19, C-14) and oxidative stereochemical relay (oxidations set at C-1, C-5) (Scheme 1.3). By means of this strategy, the degree of oxidation and complexity is progressively increased in the carbon skeleton of the steroid during the synthesis. O

O

O

H

(a)

H+,

H

ethylenglycol 81%

H

H

O

O

(b) hυ 3–5 days solid-state (68%) solution (43%)

Adrenosterone (2a)

O (d) TiCl4; AgOAc HO O 19 71%

H

(e) H2O2

HO

(h) Al-Hg, H2O 56%

H

5

O

H H O

(i) PPTS, Me2CO (j) LiBEt3H

H

63% (2 steps)

HO

O

H H

O

H 2f

O HO O

H

O

H

H 69% (2 steps)

O B Et

2g

(k) Li, NH3 (l) PPTS, Me2CO

O

O

O

O

H

O

2e

O

O

O

H

H O

O

O

H

(g) H2O2 O HO 50% (3 steps) O

O

O

H 2d

H

H 2c

(f) SeO2

O

H

O

O

O

O

O

O

2b

O

HO HO

I

(c) NIS, Li2CO3 85%

O

H

H

O

O

HO H

O B Et

2h

H

O 2i

O O

O

O (m) TMSOTf, PdII (n) SiO2, DIPEA

HO O

F3C

F

F

F

H

O

O

(o) O2, Co(acac)2

14

H

F

HO O

86%

O B Et

H O

OH

H H

42% (2 steps)

O

O

B Et

2j

17

H

O

(p) N2H4; I2; Et3N HO (q) CuTC, O Pd(PPh3)4, A O

O B Et

2k

2l

(Protected ouabageninone)

F 55% (2 steps)

O

O

O

O

(r) [Co2B]

O

NtBu

(s) Me2N

NMe2

O A

HO O

H

O H

70% (2 steps)

HO HO HO OH

(t) HCl 90%

O

O B Et

Bu3Sn

H H

OH

HO HO Ouabagenin (2)

2m

SCHEME 1.3 Synthetic sequence.

OH

Recent Accomplishments in the Total Synthesis Chapter

1

7

Adrenosterone (2a) obtained from cortisone acetate was the starting point of the synthetic sequence (Scheme 1.3). Ketalization of the less-hindered ketones (rings A and D) and abstraction of the g hydrogen in a Norrish type II photochemical transformation functionalized the C-19 position obtaining 2b in 68%. The radical transformation was carried out in solid state yielding better yields than in solution. The strained cyclobutanol ring was then selectively fragmented after exposure of 2b to oxidative reaction conditions using N-iodosuccinimide (NIS), and 2c was generated in 85% yield. Deprotection of the A-ring ketal and hydrolysis of the C-19 iodide was achieved in a two-step protocol TiCl4/AgOAc to afford 2d in 71% creating the new hydroxyl function. The stereochemistry of the primary hydroxyl achieved in this intermediate 2d would serve afterward to direct the hydroxyl functions with facial selectivity at C-1 and C-5 in 2g. As result of epoxidation of enones 2d and later 2e, the diepoxide 2f was obtained (50% three steps). Exposure of 2f to the amalgam Al-Hg promoted the epoxide opening, and 2g was obtained in 56% yield. After several transformations, enone 2j was obtained using Saegusa’s protocol; remarkably, after extensive survey of conditions, employment of perfluorotoluene gave a productive transformation from 2i to 2j in 55% yield, while suppressing epimerization at C-14. The last hydroxyl at C-14 (C–D rings junction) was inserted using Mukaiyama hydration onto enone 2j in 86% yield, and the protected key intermediate 2k was used to construct a library of ouabagenin analogs by exchanging the characteristic butenolide framework of the natural product for other subunits. The last steps comprised the transformation of the ketone at C-17 into the vinyl iodide and Stille cross-coupling (Fu¨rstner modification) after which the cross-coupling product 2l was obtained in 42%. Reduction of the dienoate 2l and isomerization of the resulting olefin (albeit dr ¼ 3:1 was observed in this step) provide 2m (70% yield two steps). Finally, global deprotection of 2m procured stereoselectively ouabagenin (2). Despite the fact that the classical chemistry of steroids has been extensively exploited, this example shows us that the beautiful frameworks of these compounds designed by nature allow different types of C–H functionalization reactions. These reactions in final term are crucial in supplying scalable amounts of material no previously achieved by conventional methods in some fully elaborated members of the family of steroids.

Gracilioether F Gracilioether F (3) is an oxygenated polyketide natural product isolated from the apolar extracts of the marine sponge Plakinastrella mamillaris. The marine invertebrate was first collected in the Fiji Islands, and this organism has been a rich source of polyketides showing antimicrobial, antitumor, antifungal, and antiparasitic properties [38,39]. Gracilioether F (3) possesses an unusual tricylcic core and five contiguous stererocenters which distinguish it

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Studies in Natural Products Chemistry

from other gracilioethers with fewer rings that have been found in the sponge Agelas gracilis. Thus a plausible biosynthetic origin of the gracilioether family from a common furanylidene motif was postulated by Perkins et al. [40,41] (Fig. 1.4). In Brown’s synthetic endeavor, two key steps were employed: (a) a new [2 +2] cycloaddition between an alkene and monosubstituted ketene catalyzed by Lewis acid and (b) a late-stage oxidation (C–H functionalization step) to build the lactone ring [42]. The advanced intermediate cyclobutanone 3c was assembled through a ketene–alkene [2 + 2] cycloaddition. The methodology developed by the same authors generated the alkenyl ketene in situ from an unsaturated acid chloride 3a to give 3c in 65% yield and good diastereomeric ratio (20:1) after condensation (Scheme 1.4). Then alkylation was carried out to obtain 3d in high C–H oxidation Et

H

Et

O R1

O Et

O

O

R

O

Et

Et

H

R

H

O

H

Et R

O Gracilioether F (3)

Gracilioether skeleta

FIG. 1.4 Gracilioether F.

O Ph

Cl Me

(a) AlMe3 (2.5 equiv) i-Pr2NEt (1.1 equiv)

Me

Et

[2 + 2] Cycloaddition 65% yield, 20:1 d.r.

CH2Cl2, –78 to –45°C

3a

H

O

3b

H

Ph

Generation of ketenes in situ

+

Et

AlMe3 O C

Et

H Me

Ph

3c

2 equiv 3b

Et (d) O3, CH2Cl2, –78°C then Et

H

O

THF, –78 °C, EtI, –78 to 22°C 91% yield, 20:1 d.r. Gram scale

(c) H2O2, NaOH MeOH, 22°C

Et

(b) KHMDS Et

H

H

Et

H

Ph

3d

3e H

Et

O

H 4

O Et

H HO

O

3f

SCHEME 1.4 Synthetic sequence.

O O

61% yield

Me

Et

Et

(e) NaClO2, NaH2PO4, t-BuOH, Et isobutylene 22°C

Et

83% yield Me

Et

H

O O

Et

H O

Et O

Gracilioether F (3)

O O

H HO 3f

Ph

(f) Cu(OAc)2 (1 equiv), H2O2, MeCN, 0°C 10%, 88% brsm

Et

H

Et O

Recent Accomplishments in the Total Synthesis Chapter

1

9

diastereoselectivity, and the alkylated product was submitted to Baeyer– Villiger conditions, providing the lactone 3e in 61% yield over two steps. Compound 3f, crucial for the success of the synthesis through application of the late stage C–H oxidation, was prepared after performing a one-pot ozonolysis and quenching under Pinnick oxidation conditions in 83% yield. Finally, attempts were made to oxidize the C–H bond at C-4 exploring a range of conditions (RuO4, TFDO, Fe(OAc)2, etc.) and bearing in mind the privileged stereoelectronic position of this bond. However, gracilioether (3) was only achieved when the oxidation was carried out with the combination of Cu(OAc)2 and H2O at 0°C in a 10% (88% recovered starting material) using as starting material the carboxylic acid 3f.

METAL-CATALYZED C–H FUNCTIONALIZATION Podophyllotoxin Podophyllotoxin (4) is a lignan natural product originated from the shikimate pathway. In this biosynthetic route, coniferyl alcohol is generated and used as the basic unit of construction to elaborate more complex lignans through further cyclizations and enzymatic modifications [43]. Podophyllotoxin (4) can be found in the rhizome and roots of Podophyllum hexandrum (P. emodi) or P. peltatum (Berberidaceae). This family of compounds displays important bioactivities, for example, etoposide and teniposide, derived from 4 by anchoring glucosides to the oxygen placed in C-4, which have found clinical application in cancer theraphy [44] (Fig. 1.5). The brevity of the synthesis of 4 in five steps reported by Maimone and coworkers stems from the successful application of a diastereoselective Pd catalyzed C (sp3)–H arylation reaction enabled by a conformational biasing element. The method used highlights the importance of C–H activation in preparing a family of different analogs by modifying the arene coupling counterpart [45].

OH H 4

O

R O

Ar1

O H

O

O

O H

C–H arylation MeO

OMe

H

O

O

Ar2

OMe Podophyllotoxin (4) FIG. 1.5 Podophyllotoxin.

Podophyllum lignans

10

Studies in Natural Products Chemistry

Structurally, podophyllotoxin (4) has four contiguous stereocenters in a 1,2-cis- 2,3-trans-configuration which are crucial to reveal biological activity and hitherto the ease with which isomerization forms the cis lactone and leads to a loss of bioactivity that has limited synthetic approaches to some extent. From the synthetic point of view, one of the key achievements in the new route was the preparation of the intermediate 4d bearing the pendant directing group to drive the C–H activation event. To make 4d in 41% yield (three steps), cycloaddition of the o-quinodimethane generated after deprotonation of 4b with the amide 4c was carried out, then the resulting condensation product 4c was reduced and the diol treated with 2,2 dimethoxypropane under acidic conditions (Scheme 1.5). The intermediate 4d was protected as an acetal form since previous investigations with other protecting groups over the same carbocyclic substrate afforded mainly the b-lactam (competitive reductive elimination product in the C–H activation event). The authors proposed the higher reactivitiy of 4d in terms of conformational locking of the PdIV intermediate; thus, the cyclohexyl ring is fixed into a half-chair-like conformation releasing the steric crowd around the palladium center and facilitating the productive aryl transfer through the achievement of the correct organometallic geometry. Among the parameters under study in the cross-coupling event, the directing group (2-thiomethylaniline), bases, solvents, etc. were evaluated, proving the importance of the addition of dibenzyl phosphate that was turned into a 58% for the diastereoselective installation of the aryl moiety. Finally, removal of the directing group under acidic conditions resulted in 43% of podophyllotoxin (4) together with C-4-epi-4 in 33% yield. The latter has the same configuration as etoposide.

Dictyodendrin A and Dictyodendrin B The search for natural products in marine and terrestrial environments has led to the discovery of a number of biologically active indole alkaloids which includes the family of dictyodendrins, isolated from a Japanese marine sponge, Dictyodendrilla verongiformis in 2003 [46] (Fig. 1.6). Among the biologically active properties that have been found are the first telomerase inhibitory natural products (showing 100% inhibition at 50 mg/mL) of marine origin as well as promising anticancer compounds. It is also important to underline that in March 2012, chemical analysis of a southern Australian marine sponge, an Ianthella sp., yielded dictyodendrins F–J as new examples of these rare types of marine alkaloids [47]. Dictyodendrins F and H–J exhibited significant protease b-secretase (BACE) inhibitory activity (IC50 1–2 mM), with the differential cytotoxicity displayed by F–I against two human colon cancer cell lines (IC50 2–16 mM) marking them as both cytotoxins and probable substrates for the multidrug resistance efflux pump P-glycoprotein.

S O

CHO

O

Br 4a

I

(a)

O

DCM/ NaOH BnNEt Cl

OH

O

3

(c) KHMDS −78 to 1°C

MeO2C H N

2 steps 1 recrystallization 4b

(b) n-BuLi, MgBr2

OK O

CO2Me

O

O HN

SMe

MeS

O 4c

(d) LiEt3BH –78°C MeO

OMe

(e) TsOH, THF (41% yield, overall 3 steps)

O H H HN MeS

4d

O H

(f) 15% mol Pd(OAc)2, K2CO3 (1.5 equiv), 5-Iodo-1,2,3-trimethoxybenzene (2 equiv), 40% (BnO)2PO2H t-AmOH (0.1 M), 50 h, 110°C 58% yield

O

(g) TFA/ H2O THF 25 °C

H

4

O

O H

O

Pd catalyzed C–H activation SCHEME 1.5 Synthetic sequence.

OH

MeO

O OMe

OMe 4 43% + 33% 4-epi

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Studies in Natural Products Chemistry

HO OSO3Na

C–H arylation

NH

Suzuki–Miyaura coupling

HO

NH

MeO2C C–H insertion

OSO3Na

C–H arylation

Suzuki–Miyaura coupling

O N

OH

OH C–H amination

N C–H insertion

HO

C–H arylation

HO OH OH Dictyodendrin A (5a)

OH OH Dictyodendrin B (5b)

FIG. 1.6 Dictyodendrins.

These tyramine-derived alkaloids feature a characteristic and unique pyrrolo[2,3-c]carbazole moiety that is decorated with electron-rich arene rings and carries at least one sulfate group on the periphery that has been shown to be essential for telomerase inhibitory activity. Overall, the scarcity of the dictyodendrins, the physiological properties of which are still not fully understood, the promise of efficient telomerase inhibition, the significant BACE inhibitory activity and their compact, and demanding structural characteristics render these marine natural products formidable targets for a synthesis-driven investigation at the chemistry/biology interface. Between 2005 and 2006, Fuerstner and coworkers reported the first total syntheses of dictyodendrins B, C, and E [48,49] followed by Tokuyama and coworkers in 2010 with the first total synthesis of dictyodendrin A and the syntheses of the B–E analogs [50,51]. For all the above reasons, dictyodendrins have been synthetic targets for relevant groups using C–H functionalization planning as key element in their construction very recently [52,53]. Yamaguchi, Itami, and Davies based their approach toward dictyodendrin A (5a) on sequential functionalization over the pyrrole nucleus [52], in contrast to Gaunt’s synthesis of dictyodendrin B (5b) which decorates the readily available 4-bromoindole nucleus [53]. The synthesis of dictyodendrin A (5a) commenced with the precursor N-alkyl pyrrole 5c synthesized under Paal–Knorr conditions (Scheme 1.6). In several studies, the double carbenoid insertion failed over 5c and these failed attempts drove the authors to conduct the key simultaneous C–H functionalization process on 5d. It should be noted that 5d was obtained in gram scale after the first Rh catalyzed C–H functionalization event at C-3 in 52% yield. To obtain high reactivity and site selectivity between C4- and C5-positions in 5d, several parameters were screened. The choice of the catalyst proved crucial and a sterically demanding and rigid catalyst (Rh2(STCPTAD)4) gave the best results. The double alkylation reaction afforded 5e in a notable 70% yield when two portions (8 h difference) of 1 mol% of Rh catalyst and 1.25 equiv of diazoester were added in one pot. Electrophilic

Recent Accomplishments in the Total Synthesis Chapter

MeO

I

2

(b)

N

4

1 mol% RhCl(CO)L2 Ag2CO3

N

5

MeO 1 mol% Rh2(S-TCPTAD)4; NBS

L = [P(OCH(CF3)2]3

Br MeO2C

CO2Me N

70% One pot, gram scale

52% Gram scale

5c

CO Me 2

OMe

OMe

MeO

N

(a)

MeO 5d

OMe 5e

OMe OBn

OMe OBn

OBn MeO

MeO (c)

13

1

N Boc

N Boc

N Boc

Bpin 10% Pd[P(t-Bu)3]2

MeO2C

CO2Me N

K3PO4, dioxane/water 47% MeO

OMe

(d) LDA, THF, –78°C 6 π electrocyclization 47% (e) MeI, K2CO3 93%

OX N

OMe

MeO 5g X = H 5h X = Me OMe

OMe

5f

MeO2C

OSO3Na

OH

HO

MeO

N H

N H (f) CF3CO2H:Pd(OH)2/C H2

Refs. [50–51]

MeO2C

OMe N

89%

OMe

MeO OMe 5i

MeO2C

OH N

3 steps

HO

OH

OH Dictyodendrin A (5a)

SCHEME 1.6 Synthetic sequence.

bromination with NBS of the pyrrole nucleus gave 5e. Addition of indole-3boronic acid pinacol ester under Suzuki–Miyaura coupling catalysis gave 5f in 47% isolated yield. The synthetically important pyrrolo[2,3-c]carbazole scaffold 5g was obtained upon LDA treatment, and ring closure was achieved in a pericyclic fashion in 47% (6 p electrocyclization). Then, the phenol ring was methylated to generate 5h. Finally, removal of Boc protecting group and debenzylation gave 5i in 89% in two steps, an intermediate previously obtained in Tokuyama’s synthesis, thus the completion of dictyodendrin A (5a) followed the previously described three last steps [50,51]. Taking advantage of the intermediate 5g, dictyodendrin F was also synthetized after oxidation of the indolic nucleus, hydrolysis, and deprotection [52]. In reference to dictyodendrin B (5b), Gaunt’s group have synthesized the marine natural product using six direct C–H functionalizations starting from the commercially available indole nucleus (Scheme 1.7). The synthesis begins with a method previously discovered in the same laboratory, copper catalyzed C–H arylation using iodonium salts [54]. The first

14

Studies in Natural Products Chemistry

I BF4

MeO

(a)

2

1. 1.2 equiv CuCl (5 mol%) 1.2 equiv 2,6-dtbp CH2Cl2 (0.2 M) 35°C, 48 h 68% [42 g scale]

Br 3

4 5

2

N H

O

(b) MeO

5k

MeO

O 2. 1.1 equiv Bi(OTf)3 (5 mol%) MeNO2 (0.5 M) rt, 24 h 57% [33 g scale] (Crystallized)

MeO

I

MeO

MeO NO2 Br

N

Bpin 1.5 equiv

O N

PdCl2(dppf) (5 mol%) 5 equiv 2 M K2CO3 (aq) dioxane (0.2 M) MeO 90°C

MeO OMe

20 h 93% [1.5 g scale]

5m

5 equiv 7 equiv K2CO3 DMF (0.1 M) 100°C, 16 h 83% [2.0 g scale]

5l OMe

(g) 1. 1.1 equiv NBS DMF (0.1 M), rt then 30 equiv NaOMe OtBu 3 equiv CuI 80°C 81% NO2 [1.5 g scale]

(f)

O

Br MeO

O

3 equiv PdCl2(dppf) (5 mol%) 5 equiv 2 M KOH(aq) 80°C, 30 min 63% [4.7 g scale]

OtBu

MeO

(e) Br

N H (d) MeO

Cl

5j (3.6 $/g)

MeO

N H

6 7

(c) [IrCl(COD)]2 (1.5 mol%) dtbpy (3 mol %) 1.5 equiv (Bpin)2 THF (0.2 M) 90°C Br μW, 1h

MeO

MeO

OtBu N3 H

O

6

N

OMe

(h) Pd(OH)2/C (10 mol%) 10 bar H2, MeCN (0.2 M) 24 h MeO then OMe MeO 5n

30 equiv AcOH 1.5 equiv tBuONO 1.2 equiv TMSN3 95% [1.3 g scale]

OMe MeO 5o

SCHEME 1.7 Synthetic sequence.

p-methoxy aryl moiety was placed at the C-3 position of the cheap indole 5j in 68% yield on 42 g scale using a 5 mol% load of catalyst. Later, the doubly substituted indole 5k was obtained in 57% under Friedel–Crafts conditions using Bi(III) triflate and the corresponding acyl chloride. Next transformation used the directing group ability of the unprotected N–H to perform an iridium catalyzed C–H borylation at position C-7. Over the same crude reaction, a Suzuki–Miyaura cocktail was subsequently added in a one-pot process obtaining the third iterative functionalization 5l in 63% after 30 min. 5l was transformed into 5m after nitrogen alkylation in presence of K2CO3 in 83% yield. The last aryl moiety in the structure of dictyodendrin B (5b) was introduced in an excellent 93% yield in 5n by means of Suzuki–Miyaura coupling. In this step, dropwise addition of nitrophenol-derived boronic ester (prepared from 1-fluoro-3-iodo-2-nitrobenzene) was needed in order to avoid secondary processes. The aryl moiety bearing the nitro group in 5n proved fundamental for the installation of the oxygen at the C-6 position since electronic deactivation favor the electrophilic bromination in 5n at the desired position. Again, a one-pot protocol was employed and NaOMe in methanol and copper(I) iodide were added to the bromination crude mixture to yield in 81% the methoxy ether placed in 5o.

Recent Accomplishments in the Total Synthesis Chapter

MeO

OtBu N3 H

O N

OMe

MeO

OSO3NH4

OtBu

(i) PhMe (0.1 M) 180°C 30 min

NH

O

NH

O N

OMe End game Refs. 49, 51

N

OH

HO

MeO

OMe

OH

OMe

MeO 5o

15

HO

MeO

(62%) [Continuous flow] [1.5 g scale]

1

MeO

5p > 1 g prepared

HO Dictyodendrin B (5b)

SCHEME 1.8 End game dictyodendrin B.

The final steps comprised the challenging carbazole ring closure, and after extensive experimentation, the chosen route was three steps: reduction of the nitro group, diazotization of the amine, and azidation providing the precursor 5o in 95% yield (Scheme 1.7). Thermal decomposition of the azide to obtain the carbazole 5p (62%) was carried out under controlled conditions by means of flow chemistry (1 g/30 min) (Scheme 1.8). Thus, the dangers associated with the exothermicity of the reaction were avoided. Moreover, 5p was achieved without the necessity of performing tedious work-ups to remove high-boiling point solvents often used under standard conditions [55]. The end game to obtain over 1 g of dictyodendrin B (5b) followed the previously described four steps of deprotection and sulfonylation [49,51]. To sum up, a modular synthesis of dictyodendrin B (5b) was completed and tailored using a wide range of C–H functionalization reactions. This approach makes possible the diversification of the natural product in other analogs in multigram scale, and the synthetic sequence is also complemented by the use of safe flow chemistry techniques.

MISCELLANEOUS Azidation of Tetrahydrogibberellic Acid Although it is not being applied in total synthesis in order to go more deeply into the importance of developing C–H functionalization, we wish to describe briefly the recent advance made in the field by Hartwig [56] (Fig. 1.7). The rapid construction of libraries of bioactive analogs based on natural products to access to potential therapeutics constitutes one of the paradigms in chemical sciences. It is of paramount importance to design novel catalysts and reactions in this area and a recent example is a striking metal-catalyzed azidation of tertiary C–H bonds. The methodology was applied in the azidation of tetrahydroacetogibberellic acid.

16

Studies in Natural Products Chemistry

O

H O

OH

O H O

OH

N3

C–H azidation

8-Azido tetrahydrocetogibberellic acid (6) FIG. 1.7 Tetrahydrogibberellic acid.

H H CO2H 6a

H OH H CO2H H Enzyme 6b

> 100 GAs H CO2H CHO 6c

[O]

SCHEME 1.9 Biosynthetic processes.

Gibberellins (GAs) are functionalized diterpenoids found in microorganisms and higher plants acting as phytohormones in the growth and development of plants [57]. The name of these compounds is derived from Gibberella fujikuroi, a fungus that colonized rice plants and in which the secondary metabolite was found at the end of the 19th century. Biosynthetically, GAs derive from kaurenoic acid 6a, a diterpene originated from the mevalonic acid and methylerythritol phosphate biochemical routes. Ring contraction of 6a and enzymatic modifications afford a common precursor 6c. The 6-5-6-5 carbocyclic intermediate 6c is further transformed in higher functionalized GAs. Importantly, the degree and selectivity of the functionalization in the final diterpenoids depends on the type organism (Scheme 1.9). Recently, a late stage modification of gibberellins was reported as a direct application of reaction discovery. The optimized conditions employed 3 equiv of thermally stable azidoiodinane 6e in combination with iron diacetate and a tridentate bisoxazolidine ligand at 50°C to obtain a 75% yield of 6a (Scheme 1.10). The reported conditions were found after experimenting with metal complexes (porphyrins), various nitrogen ligands, and combinations with oxidants (eg, tert-butylhydroperoxide). The reaction transforms a C–H bond into a C–N bond, and the mechanism is postulated by the authors to go through a tertiary radical catalyzed by iron acetate. This mechanism was supported by the different reactivity shown in relation to various substrates and radical clocks (TEMPO). The mild intermolecular reaction was also applied to a series of hydrocarbons in good yields and selectivity for tertiary C–H bonds and the C–H cleavage proved to be the rate determining step (KIE 5.0  0.3). Finally, to test the possibilities of the new chemical tool, the resulting azides were transformed into heterocycles, amides, amines, and heteroarenes.

Recent Accomplishments in the Total Synthesis Chapter

O

H O

N3

OH

O

I O

Fe(OAc)2 10 mol%, CH3CN, 50°C

O

O

1

H O

OH

O

H

17

H

O

OH

H O

2 equiv

N

N 6d

6e

11% mol

iPr

O

O

O

N

iPr

OH

N3

75% yield 6a

H O

OH

O H O

OH

O

N

N N

O Fluorescent tag

3

6f

SCHEME 1.10 Fluorescent tag applications.

Focusing on compound 6a, a derivatization was performed using a Huisgen cycloaddition to anchor a fluorescent tag to give 6f, a compound with potential application in cell imaging and bioconjugation [56].

(+)-Myrrhanol C Highly selective C–H functionalizations are also achieved by enzymes in nature (vide supra). The environmentally friendly conditions of these transformations together with their efficiency mean that these natural processes can compete favorably with the analogous synthetic transformations [9]. Among the most common enzymes applied in chemical synthesis, the hemecontaining cytochromes P450 occupy a prominent position, with aliphatic C–H hydroxylations being by far the most common functionalization reported for these enzymes [58]. A representative example of the potentiality of this catalytic C–H functionalization was the conversion of 11-deoxycortisol to hydrocortisone, a remote and highly selective hydroxylation performed by P450lun in Curvularia lunata [59] (Scheme 1.11) (Fig. 1.8). (+)-Myrrhanol C (7) is a natural triterpene that was isolated from mastic gum (the resin of Pistacia lentiscus) [60], a substance which possesses recognized medicinal properties [61]. Furthermore, this gum was used to embalm corpses in ancient Egypt. Although this gum was already known to be active against different cancers [62], in 2011 Simmet et al. reported that myrrhanol C (7) triggers apoptosis in chemoresistant, PC-3 androgen-independent human prostate cancer cells in vitro and in vivo [63]. This molecule is therefore a

18

Studies in Natural Products Chemistry

O

O

OH OH

HO

OH OH

P450 O

O

Hydrocortisone

11-Deoxycortisol SCHEME 1.11 Selective hydroxylation by P450lun.

B-Alkyl Suzuki–Miyaura Microbial C–H oxidation OH HO

H (+)-Myrrhanol C (7)

FIG. 1.8 Myrrhanol C.

promising antiprostate cancer lead, which should be underlined since prostate cancer is the second most common and lethal type of cancer [64]. Barrero et al. conceived a convergent approach to (+)-myrrhanol (7) where a bicyclic moiety is coupled to an acyclic counterpart using a B-alkyl Suzuki– Miyaura reaction. Key in this synthesis is the employment of a microbial stereo- and regioselective late stage C–H oxidation of 7a, easily available from sclareol, the starting substrate that can be isolated on a multigram scale from extracts of Salvia sclarea [65]. Best conditions for incubation of 7a with Mucor plumbeus were found after 24 h, where up to a 52% of 7b was obtained (Scheme 1.12). It should be noted that longer periods of incubation led to ketone 7c, resulting from the oxidation of the hydroxyl group. However, the stereospecific reduction of 7c to desired triol 7b turns this keto-derivative into a valuable synthetic intermediate. After selective protection of the primary alcohol in 7b, the required onecarbon homologation was achieved after Swern oxidation and Wittig olefination of the advanced intermediate 7f (Scheme 1.13). On the other hand, the acyclic counterpart 7i was prepared from commercial geranyl bromide, which was converted to alkyne 7h following a described procedure [66]. A Negishi carbometalation of 7h led to the required E-vinyl iodide intermediate 7i in 82% yield. Then precursor 7i was reacted via palladium catalyzed B-alkyl Suzuki–Miyaura with bicyclic 7g to give the expected coupled product efficiently in 90% yield, where the geometry of the alkene was maintained.

Recent Accomplishments in the Total Synthesis Chapter

OH

OH

OH

19

1

OH

Mucor plumbeus OH

OH H

H

C–H oxidation

OH HO

7b

7a

(–)-Sclareol (ca. $51/g)

OH O

H

H 7c

time

52% 24 h incubation

SCHEME 1.12 C–H oxidation by Mucor plumbeus. OH

OH

(a) PivCl, DMAP Py: CH2Cl2(1:1) 0°C

OH

76%

HO

OPiv

HO

7b

OMEM TBSO

(c) MEMCl, Hunig´s base, DMF rt, 92%

7d OH

(d) LiAlH4, THF, rt, 90%

OPiv

(b) TBSCl, imidazole, DMAP, DMF, rt, 98%

7e

(e) Swern OMEM

OMEM (f) PPh3CH3Br, t-BuOK, toluene, rt, 85% over two steps

TBSO 7f

TBSO 7g

(g) AlMe3, Cp2ZrCl2 DCM, –30°C

Geranyl bromide

(h) I2, THF

7h

82%

7i

(k) HCl, AcOH, THF, rt 80%

(i) 9-BBN, THF, reflux 7g (j) 7i, Pd(dppf)Cl2, AsPh3, Cs2CO3, H2O, DMF, 25°C, 30 min, 90%

I

OMEM TBSO 7j

OH HO (+)-Myrrhanol C (7)

SCHEME 1.13 Synthetic sequence.

It should be noted that harsh conditions were necessary in the hydroboration reaction due to the sterically congested environment of the vinyl group in 7g. Finally, deprotection of 7j with AcOH and HCl in THF led to target (+)-myrhanol C (7).

20

Studies in Natural Products Chemistry

Complanadines A and B Complanadines A (8a) and B (8b) are Lycopodium pyridine-type alkaloids isolated from the methanolic extracts of Lycopodium complanatum by Kobayashi in 2000 and 2005, respectively [67,68] (Fig. 1.9). The biosynthetic pathway that produces the unusual lycodine units (8c) that then dimerize to form 8a and 8b is still not fully understood [69], although some conclusions have been obtained from isotope labeling experiments. Moreover, 8a has exhibited bioactive profiles for Alzehimer’s treatment (inducement of secretion of neurotrophic factors). Thus, the obscure biosynthetic origin and promising medicinal activity make them important targets for chemical synthesis. Tsukano’s plan to construct the rare alkaloids 8a and 8b was to couple both monomeric scaffolds using the privileged position of the C–H at C-2 in the pyridine 8c (Scheme 1.14) [70]. However, this strategy is even more challenging when both monomers are different and give an unsymmetrical dimer as in the case of complanadine B (8b) that possesses a carbonyl in one of its subunits. The synthesis began with the chiral isolation of (+)–8d performed on an amylase chiral column in 48% from the racemic mixture. Then 8d was transformed into 8c after reduction of the thioketal of 8d with Raney Ni. 8f and 8e were prepared afterward, once the absolute stereochemistry (X-ray characterizatrion) and optical rotation of licodine obtained from 8c were secured. The pyridine N-oxide 8e was obtained by MCPBA oxidation in 92% yield and the 3-bromolycodine 8f after borylation and bromination of 8c in 62% yield. The next key step in the synthesis was the coupling of both monomers, and therefore a survey of conditions was undertaken to optimize this step. The most important factor was found to be the addition of pivalic acid, Cs2CO3, and the choice of the ligand; the more electron-rich and bulkier tBuDavePhos increased the yield by 10% compared with tBu3P.

R N H

N

Direct arylation HN

N R = H2, Complanadine A (8a) R = O, Complanadine B (8b) FIG. 1.9 Complanadines A and B.

R

(a) MCPBA, CH2Cl2, 0°C to RT 92%

H N Cbz

N

N Cbz

R = H2 (8c) R = O (8d)

H

H

H N

O

N H

N

N H

HN

H (8a)

H N

(d) Pd(OAc)2, (8e) tBuDavePhos, Cs2CO3, pivalic acid mesitylene, 130°C, 62%

(8f)

SCHEME 1.14 Synthetic sequence.

H (8b)

N

H N

HN

N (g) K2CO3, MeOH DMP, CH2Cl2 (76% 3 steps) (h) 6 M aq. HCl, 70°C, 78%

(e) Pd(OH)2/C, HCO2NH4, MeOH, 67%

(c) CuBr2, MeOH/H2O (1:1), reflux, 60% (2 steps)

Br

N

H

(8e)

(b) [{Ir(cod)(OMe)}2], tBudpy, [B(pin)]2, THF, reflux

Cbz

O

N Cbz

H O Cbz N N

(f) Ac2O, 125°C, d.r. = 3:1

N Cbz

OAc N

Cbz

H N (8g)

N H

N (8h)

22

Studies in Natural Products Chemistry

The optimized conditions gave rise to the key dimer 8g in 62% that was transformed in (
)-complanadine A (8a) after deprotection of the piperidine ring with Pd(OH)2 and ammonium formate. Complanadine B (8b) was prepared upon treatment of the precursor N-oxide 8g with acetic anhydride at reflux, promoting a Claisen-type rearrangement. Then, hydrolysis of the acetate in 8h, Dess–Martin oxidation, and acidic deprotection afforded 8b in 60% yield for the four last steps. At the end of the synthesis, the authors reexamined the sign of the optical rotation reported for complanadine A and they determined it to be levorotatory, in contrast to the one described previously in the isolation of the natural product and other total syntheses [67–72]. It must to be highlighted that a similar disconnection approach using C–H functionalization was previously performed in Sarpong’s synthesis [71]. Both pyridine precursors 8j and 8l were prefunctionalized to carry out a Suzuki cross-coupling to obtain 8a. Indeed, 8j was prepared by a Hartig–Miyaura borylation protocol (Scheme 1.15).

N

cat. [Ir(COD)(OMe)]2/dtBu-dipy [B(pin)]2, THF, 80°C

N

75%

N O

N

B

Boc

Boc (8i)

H N

(8j)

Tf2O, pyridine –78°C to rt

O

N

O

N

72%

OTf

N

Boc

Boc (8l)

(8k)

PdCl2(dppf), K3PO4 HCl, 70°C (8j) + (8l) 42% two steps

H N Cbz

N

HN H N

Complanadine A (8a) SCHEME 1.15 End game.

Recent Accomplishments in the Total Synthesis Chapter

1

23

(+)-Hongoquercin A Professors Baran and Yu envisioned the synthesis of (+)-hongoquercin (9) by taking advantage of the invention of a number of C–H functionalizations of benzoic acids derivatives developed in the laboratories of Professor Yu [73–77]. Apart from hongoquercin, the authors showed how C–H functionalizations proved to be a valid strategy to “the divergent functionalization of natural product cores” [78]. Thus, a number of hongoquercin derivatives were prepared using this approach (Fig. 1.10). (+)-Hongoquercin A (9) was isolated from the extracts of an unidentified fungus in 1998. This substance exhibits activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium [79]. The synthetic route toward hongoquercin commenced with (+)-chromazonarol (9a), a compound available from commercial (+)-sclareolide [80]. Hydroxycarbonylation of the corresponding triflate of 9a afforded the required benzoic acid derivative 9b. Pd-mediated C–H methylation required extensive research to optimize of reaction conditions, a study carried out with m-toluic acid as model. It was found that the presence of both Boc-Phe-OH as ligand and 1,4-benzoquinone was essential for the success of the transformation. Under the optimized conditions shown in Scheme 1.16, monoalkylated derivative 9c was produced in 60% yield. At this point, only a C–H hydroxylation was needed to reach the target (+)-hongoquercin (9). However, when 9c was subjected to the previously reported protocol for the Pd(II)-catalyzed hydroxylation of arenes [77], no (+)-hongoquercin (9) was detected, and only a 10–15% yield of this compound was detected when the pressure of O2 was raised to 10 atm. The authors then considered previous studies by Professor Yu showing that electron-deficient aryl amides can enable C–H activation where other directing groups failed [81]. In the event, once the corresponding amide 9d formed, C–H methylation took place in reasonable yield. At this point, and after extensive

C–H oxidation HO

CO2H Me

O

O

C–H methylation

H

(+)-Hongoquercin (9) FIG. 1.10 Hongoquercin A.

OH

H

meroterpene skeleton

OH

CO2H

(a) Tf2O O

(b) Pd(OAc)2, dppf, KOAc 1 atm CO, DMSO

H

O H

80% 9a

9b CO2H Me

(a) MeBF3K, Pd(OAc)2 BoC-Phe-OH, 1,4-benzoquinone t-BuOH, 90°C 9b

O

HO

(b) O2, (10 atm), Pd(OAc)2 KOAc, DMA

H

45%

(c) (COCl)2 (d) C6F5NH2

CO2H Me

O H

15%

(i) BF3·OEt2, MeOH, 105°C (j) 6 M NaOH, THF, 80°C 71%

9c

(+)-Hongoquercin (9)

76% CONHC6F5

O

CONHC6F5 Me

(g) MeBF3K, Pd(OAc)2 Boc-Phe-OH, 1,4-benzoquinone THF, 120°C

H

9d

SCHEME 1.16 Synthetic sequence.

60%

O H

9e

AcO

(h) PhI(OAc)2, Pd(OAc)2 NaOAc, Ac2O, DCE

CONHC6F5 Me

O H

54% 9f

Recent Accomplishments in the Total Synthesis Chapter

25

C6F5

X

O

1

NH

O

O

N CF3 O

O

H

O

H Alkylation

O

X

O

C6F5

Amination

NH

O

CF3

O H

O

Lactonization

H

O

Arylation

H

X = OH or NHC6F5 CO2H OH

Carbonylation O

Hydroxylation

Vinylation

C6F5 N O

O

CO2H

H

Ph

O H

O H

SCHEME 1.17 Diversification in C–H functionalization.

experimentation, the final C–H oxidation was able to proceed using Pd(OAc)2 as a catalyst and stoichiometric quantities of PhI(OAc)2, Ac2O, and NaOAc to give 9f in 54% yield. Regeneration of the acidic function together with the reduction of the acetate group rendered (+)-hongoquercin (9). Finally, the feasibility of functionalizing these benzoic acids and amides made it possible to produce a number of hongoquercin derivatives, thus broadening the possible applicability of these types of interesting molecules (Scheme 1.17).

CONCLUDING REMARKS The aim of this chapter is to show that C–H functionalization has been applied extensively and successfully in the synthesis of natural products. Terpenes, polyketides, alkaloids, lignans, and natural products which involve combining several biosynthetic routes can be built in a straightforward manner by disconnection of pervasive C–H bonds. The increasing widespread use of C–H functionalization tactic is not simply due to the many advantages it provides shortening the length of synthetic processes, reducing waste, supplying material in larger quantities (scalability), and facilitating analog diversification. Its real potential lies in its ability to refresh and complement conventional

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Studies in Natural Products Chemistry

rethrosynthetic analysis. If there is no other option, we will still have to use classical synthetic sequences which are sometimes long and tedious. However, it is becoming increasingly possible to speed these synthetic processes up by taking advantage of new strategies based on C–H functionalization.

REFERENCES [1] G. Dyker, Handbook of C–H Transformations: Applications in Organic Synthesis, vols. 1–2, Wiley-VCH, Weinheim, Germany, 2005. [2] A.E. Shilov, G.B. Shul’pin, Chem. Rev. 97 (1997) 2879–2932. [3] Chem. Soc. Rev. 40 (2011) 1845–2040. [4] T.W. Lyons, M.S. Sanford, Chem. Rev. 110 (2010) 1147–1169. [5] J. Wencel-Delord, F. Glorius, Nat. Chem. 5 (2013) 369–375. [6] J. Bicas, D. Lemos, P. Ana Paula, M. Glaucia, Chem. Rev. 109 (2009) 4518–4531. [7] S. Kille, F.E. Zilly, J.P. Acevedo, M.T. Reetz, Nat. Chem. 3 (2011) 738–743. [8] R. Fasan, ACS Catal. 4 (2012) 647–666. [9] J.C. Lewis, P.S. Coelho, F.H. Arnold, Chem. Soc. Rev. 40 (2011) 2003–2021. [10] E. Yeh, L.C. Blasiak, A. Koglin, C.L. Drennan, C.T. Walsh, Biochemistry 46 (2007) 1284–1292. [11] S. Flecks, E.P. Patallo, X. Zhu, A.J. Ernyei, G. Seifert, A. Schneider, C. Dong, J.H. Naismith, K.H. van Pe´e, Angew. Chem. Int. Ed. 47 (2008) 9533–9536. [12] D.R.M. Smith, S. Gru¨schow, R.J.M. Goss, Curr. Opin. Chem. Biol. 17 (2013) 276–283. [13] T.K. Hyster, C.C. Farwell, A.R. Buller, J.A. McIntosh, F.H. Arnold, J. Am. Chem. Soc. 136 (2014) 15505–15508. [14] S. Matthew, H. Yun, ACS Catal. 2 (2012) 993–1001. [15] M. Heberling, B. Wu, S. Bartsch, D.B. Janssen, Curr. Opin. Chem. Biol. 17 (2013) 250. [16] N.J. Turner, Curr. Opin. Chem. Biol. 15 (2011) 234–240. [17] J. Degenhardt, T.G. Kollner, J. Gershenzon, Phytochemistry 70 (2009) (1621–1637). [18] I. Abe, M. Rohmer, G.D. Prestwich, Chem. Rev. 93 (1993) 2189–2206. [19] I. Abe, G.D. Prestwich, in: D.H.R. Barton, K. Nakanishi (Eds.), Comprehensive Natural Products, Elsevier, Oxford, UK, 1999, p. 267. [20] K. Poralla, in: D.H.R. Barton, K. Nakanishi (Eds.), Comprehensive Natural Products, Elsevier, Oxford, UK, 1999, p. 299. [21] V. Domingo, J.F. Arteaga, J.F. Quilez del Moral, A.F. Barrero, Nat. Prod. Rep. 26 (2009) 115–134. [22] N.J. Turner, E. O’Reilly, Nat. Chem. Biol. 9 (2013) 285–288. [23] J.D. Keasling, A. Mendoza, P.S. Baran, Nature 492 (2012) 188–189. [24] D.H.R. Barton, J.M. Beaton, J. Am. Chem. Soc. 82 (1960) 2641. [25] W. Gutekunst, P.S. Baran, Chem. Soc. Rev. 40 (2011) 1976–1991. [26] D.Y.-K. Chen, S.W. Youn, Chem. Eur. J. 31 (2012) 9452–9474. [27] J. Yamaguchi, A.D. Yamaguchi, K. Itami, Angew. Chem. Int. Ed. 36 (2012) 8960–9009. [28] S.E. O’Connor, J.J. Maresh, Nat. Prod. Rep. 23 (2006) 532–547. [29] F. Abe, T. Yamauchi, Phytochemistry 35 (1994) 169–171. [30] C.-Y. Gan, Y.-Y. Low, N.F. Thomas, T.-S. Kam, J. Nat. Prod. 5 (2013) 957–964. [31] M. Pfaffenbach, T. Gaich, Chem. Eur. J. 21 (2015) 1–4. [32] L.F. Fieser, M. Fieser, Steroids, Reinhold, New York, NY, 1959 (Chapter 20). [33] A. Arnaud, C. R. Hebd. Seances Acad. Sci. 106 (1888) 1011.

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1

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[34] C. Mannich, G. Siewert, Ber. Dtsch. Chem. Ges. A 75 (1942) 737–750. [35] H. Zhang, M. Sridhar Reddy, S. Phoenix, P. Deslongchamps, Angew. Chem. Int. Ed. 47 (2008) 1272–1275. [36] H. Renata, Q. Zhou, P.S. Baran, Science 339 (2013) 59–63. [37] H. Renata, Q. Zhou, G. Dunstl, J. Felding, R. Merchant, C.-H. Yeh, P.S. Baran, J. Am. Chem. Soc. 137 (2015) 1330–1340. [38] C. Festa, S. De Marino, M.V.D. Auria, E. Deharo, G. Gonzalez, C. Deyssard, S. Petek, G. Bifulco, A. Zampella, Tetrahedron 68 (2012) 10157–10163. [39] C. Festa, C.D. Amore, B. Renga, G. Lauro, S. Marino, M.D. Auria, G. Bifulco, A. Zampella, S. Fiorucci, Mar. Drugs 11 (2013) 2314–2327. [40] M.D. Norris, M.V. Perkins, Tetrahedron 69 (2013) 9813–9818. [41] M.D. Norris, M.V. Perkins, E.J. Sorensen, J. Erik, Org. Lett. 17 (2015) 668–671. [42] C.M. Rasik, M.K. Brown, Angew. Chem. Int. Ed. 53 (2014) 14522–14526. [43] P.M. Dewick, Medicinal Natural Products: A Biosynthetic Approach, second ed., WileyVCH, Chichester, UK, 2002. [44] C. Canel, R.M. Moraes, F.E. Dayan, D. Ferreira, Phytochemistry 54 (2000) 115–120. [45] C.P. Ting, T.J. Maimone, Angew. Chem. Int. Ed. 53 (2014) 3115–3119. [46] K. Warabi, S. Matsunaga, R.W.M. van Soest, N. Fusetani, J. Org. Chem. 68 (2003) 2765–2770. [47] H. Zhang, M.M. Conte, Z. Khalil, X.-C. Huang, R.J. Capon, RSC Adv. 2 (2012) 4209–4214. [48] A. Fuerstner, M.M. Domostoj, B. Scheiper, J. Am. Chem. Soc. 128 (2006) 8087–8094. [49] A. Fuerstner, M.M. Domostoj, B. Scheiper, J. Am. Chem. Soc. 127 (2005) 11620–11621. [50] H. Tokuyama, K. Okano, H. Fujiwara, T. Noji, T. Fukuyama, Chem. Asian J. 6 (2011) 560–572. [51] K. Okano, H. Fujiwara, T. Noji, T. Fukuyama, H. Tokuyama, Angew. Chem. Int. Ed. 49 (2010) 5925–5929. [52] A.D. Yamaguchi, K.M. Chepiga, J. Yamaguchi, K. Itami, H.M.L. Davies, J. Am. Chem. Soc. 137 (2015) 644–647. [53] A.K. Pitts, F. O’Hara, R.H. Snell, M.J. Gaunt, Angew. Chem. Int. Ed. 127 (2015) 1–6. [54] R.J. Phipps, N.P. Grimster, M.J. Gaunt, J. Am. Chem. Soc. 130 (2008) 8172–8174. [55] J.C. Pastre, D.L. Browne, S.V. Ley, Chem. Soc. Rev. 42 (2013) 8849–8869. [56] A. Sharma, J.F. Hartwig, Nature 517 (2015) 600–604. [57] L.N. Mander, Nat. Prod. Rep. 20 (2003) 49–69. [58] M.K. Julsing, S. Cornelissen, B. Buhler, A. Schmid, Curr. Opin. Chem. Biol. 12 (2008) 177–186. [59] M. Bureik, R. Bernhardt, in: R.D. Schmid, V.B. Urlacher (Eds.), Modern Biooxidation— Enzymes, Reactions, and Applications, Wiley-VCH, Weinheim, Germany, 2007, pp. 155–176. [60] F.J. Marner, A. Freyer, J. Lex, Phytochemistry 30 (1991) 3709–3712. [61] F.U. Huwez, D. Thirlwell, A. Cockayne, D.A. Ala’Aldeen, N. Engl. J. Med. 339 (1998) 1946. [62] H. Sakagami, K. Kishino, M. Kobayashi, K. Hashimoto, S. Iida, A. Shimetani, Y. Nakamura, K. Takahashi, T. Ikarashi, H. Fukamachi, K. Satoh, H. Nakashima, T. Shimizu, K. Takeda, S. Watanabe, W. Nakamura, In Vivo 23 (2009) 215–223. [63] S.A.F. Morad, C. Schmidt, B. Buechele, B. Schneider, M. Wenzler, T. Syrovets, T. Simmet, J. Nat. Prod. 74 (2011) 1731–1736. [64] American Cancer Society, Cancer Facts & Figures 2012, American Cancer Society, Atlanta, GA, 2012.

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[65] A.F. Barrero, E.A. Manzaneda, J. Altarejos, S. Salido, J.M. Ramos, M.S. Simmonds, W.M. Blaney, Tetrahedron 51 (1995) 7435–7450. [66] V. Domingo, L. Silva, H.R. Dieguez, J.F. Arteaga, J.F. Quı´lez del Moral, A.F. Barrero, J. Org. Chem. 74 (2009) 6151–6156. [67] J. Kobayashi, Y. Hirasawa, N. Yoshida, H. Morita, Tetrahedron Lett. 41 (2000) 9069–9073. [68] H. Morita, K. Ishiuchi, A. Haganuma, T. Hoshino, Y. Obara, N. Nakahata, J. Kobayashi, Tetrahedron 61 (2005) 1955–1960. [69] X. Ma, D.R. Gang, Nat. Prod. Rep. 21 (2004) 752–772. [70] L. Zhao, C. Tsukano, E. Kwon, Y. Takemoto, M. Hirama, Angew. Chem. Int. Ed. 52 (2013) 1722–1725. [71] D.F. Fischer, R. Sarpong, J. Am. Chem. Soc. 132 (2010) 5926–5927. [72] C. Yuan, C.-T. Chang, A. Axelrod, D. Siegel, J. Am. Chem. Soc. 132 (2010) 5924–5925. [73] R. Giri, N. Maugel, J.-J. Li, D.-H. Wang, S.P. Breazzano, L.B. Saunders, J.-Q. Yu, J. Am. Chem. Soc. 129 (2007) 3510–3511. [74] D.-H. Wang, T.-S. Mei, J.-Q. Yu, J. Am. Chem. Soc. 130 (2008) 17676–17677. [75] K.M. Engle, P.S. Thuy-Boun, M. Dang, J.-Q. Yu, J. Am. Chem. Soc. 133 (2011) 18183–18193. [76] D.-H. Wang, K.M. Engle, B.-F. Shi, J.-Q. Yu, Science 327 (2010) 315–319. [77] Y.-H. Zhang, J.-Q. Yu, J. Am. Chem. Soc. 131 (2009) 14654–14655. [78] B.R. Rosen, L.R. Simke, P.S. Thuy-Boun, D.D. Dixon, J.-Q. Yu, P.S. Baran, Angew. Chem. 125 (2013) 7458–7461. [79] D.M. Roll, J.K. Manning, G.T. Carter, J. Antibiot. 51 (1998) 635–639. [80] D.D. Dixon, J.W. Lockner, Q. Zhou, P.S. Baran, J. Am. Chem. Soc. 134 (2012) 8432–8435. [81] M. Wasa, K.M. Engle, J.-Q. Yu, J. Am. Chem. Soc. 131 (2009) 9886–9887.

Chapter 2

Synthetic Studies Toward Nonribosomal Peptides I. Duttagupta, K.C. Ghosh and S. Sinha Indian Association for the Cultivation of Science, Kolkata, West Bengal, India

Chapter Outline Introduction 29 Biological Activities of Nonribosomal Peptides 33 Chemical Synthesis of NRPs 34 Synthesis of Gramicidine S, 1 34 Synthesis of Actinomycin D, 2 35 Synthesis of Bacitracin A, 3 37 Synthesis of Polymyxin B1, 34 39 Synthesis of Daptomycin, 44 40 Synthesis of Tyrocidine A, 46 41 Synthesis of Lysobactin (Katanosin B), 52 42

Synthesis of Thiocillin 1, 74 Synthesis of Polytheonamide B, 5 Synthesis of Cyclosporin A, 6 Synthesis of Enterobactin, 7 Synthesis of Vibriobactin, 117 Synthesis of Microcystin LA, 9 Synthesis of Antillatoxin, 138 Concluding Remarks Acknowledgments References

46 48 51 54 55 56 58 59 60 62

INTRODUCTION Nonribosomal peptides (NRP) comprise a large group of pharmacologically important natural products, mostly isolated from microorganisms (bacteria or fungi). Members of this family can contain nonproteinogenic amino acids, which are not naturally encoded by genetic code during ribosomal protein synthesis. There are some eight significant categories of NRP, classified according to their utility. 1. Antibiotics: NRPs like actinomycin, bacitracin, vancomycin, etc., have antibiotic properties and, hence, are classified under antibiotics; other examples include Gramicidin S 1, Actinomycin D 2, Bacitracin A 3, and Vancomycin 4 (Fig. 2.1). A review on vancomycin has already been published [1]. 2. Cytostatics: Compounds that have the ability to inhibit cell growth are known as cytostatics and due to this unique ability these classes of Studies in Natural Products Chemistry, Vol. 48. http://dx.doi.org/10.1016/B978-0-444-63602-7.00002-3 © 2016 Elsevier B.V. All rights reserved.

29

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Studies in Natural Products Chemistry

H2N O N H

N

Ph

N

N

HN

NH

O

O O

N

O O

N

H N

N H

O

Ph

O

O

H N

N H

H N

N H

O

O

O

O

H N

O

N

O O

O O

HN

O NH2

1. Gramicidin S

O O

N

O O N

O NH

N

NH2

O

O

2. Actinomycin D

OH O2C H N

O HN

O H O N O H3N

N H

O

O

N H

H N NH

H N

HO

N

O Ph

HO O

H N

O

S HO

O NH

N H

H N

O

Cl O OH O

N H O H2N

O

H N O

N H

HN

O

H

3. Bacitracin A

O

Cl

H

N

O O O

O

H3N

NH2

O OH

HO

CO2

H N

O

N H

O H

H2N

HN

O

HO

OH

OH

4. Vancomycin

FIG. 2.1 NRP antibiotics.

3.

4.

5. 6.

compounds find their use in chemotherapeutics, eg, bleomycin A2 [2], Polytheonamide B 5, etc. (Fig. 2.2). Immunosuppressants: Compounds that either inhibit or reduce the activity of the immune system are known as immunosuppressants, generally administered for the treatment of autoimmune disorders or during organ transplants, eg, Cyclosporin 6 (Fig. 2.3). Siderophores are small iron chelating compounds secreted by microorganisms to scavenge iron from mineral phases by formation of usable Fe3+ complexes to be taken up by active transport, eg, Enterobactin 7a and Bacillibactin 7b (Fig. 2.4). Pigments: Indigoidine is a typical example of pigment with a royal blue color in acidic solutions, eg, Indigoidine 8 (Fig. 2.5). Toxins: Compounds synthesized by living cells capable of causing disease are termed as toxins. Microcystin 9 and Nodularin 10 are hepatotoxic NRP (Fig. 2.6).

OH O

O

H N

H N

N H

O 1

O

H N

N H

O

O N H

O

O

H N

H N

N H

6 O

O

H N

N H

O

O N H O 12

O

H2N O

H N

MeHN O

O

N H 32

O NH

O OH

O HN O

O

H N

N H O

O

H N

N H O

O OH NHMe

O

H N

N H

O

O

H N 25

O O

H N

O N H O OH 38 O NHMe

H N

O N H NHMe

H N O HO

O N H

H N

N H 44

O O NH2

NH 19 O

N H

O

NHMe O

H N

O O

H N

N H O

O

O

N H NHMe HN O

OH

S

O

H N

N H

O

O

NHMe

NHMe O NH

H N

O

H N

N H

O

O

O N H

O O

NH2 H N O HO

HO

O N H 48

OH O

NH2 Polytheonamide B, 5

FIG. 2.2 NRP cytostatic.

O NH O

32

Studies in Natural Products Chemistry

HO H N

N N

N

N

O

O

H N

O

O

O

O

N

O N O

O

O

O

NH

N

N H 6. Cyclosporin

FIG. 2.3 NRP immunosuppressant.

OH OH

O

O

HN O

H N

HO

NH OH

O

O

OH

OH O HN O

OH OH

O

O

O

O HN

O

O

O

HN

N H

HO HO 7a. Enterobactin

O OH O

O HO

O O

O NH O

NH

7b. Bacillibactin

FIG. 2.4 NRP siderophores.

NH2

O HN

O

O NH H2N

O 8. Indigoidine

FIG. 2.5 NRP pigment.

7. Nitrogen storage polymers: Cyanophycin (Fig. 2.7) is a amino acid polymer consisting of aspartic acid backbone and arginine side groups, it is used as a nitrogen–carbon-storage compound. 8. Phytotoxins: Compounds that kill or retard the growth of a plant is known as phytotoxin, eg, HC-toxin 11 and Victorin 12 (Fig. 2.8).

Synthetic Studies Toward Nonribosomal Peptides Chapter

CO2H

O N

HN OMe

CO2H NH

Ph

H N

O

H N O

N

O

OMe

O NH

O

HN

O

O

33

2

O

HN

NH Ph

O

O CO2H HN

CO2H

O

NH2

9. Microcystin LA

NH

H N

N H

10. Nodularin

FIG. 2.6 NRP toxins.

NH

O

H2N

O

HN

H N NH

O

OH HN O

O NH

HO

H N

O Cyanophycin

NH2 NH

FIG. 2.7 NRP nitrogen storage polymers.

O

OH

Cl

O N

O

NH

Cl HO HN

O

O NH HN

O

O HN O 11. HC toxin

HO

O O

NH

CO2H NH

O O

NH OH H2N

O Cl

12. Victorin

FIG. 2.8 NRP phytotoxins.

BIOLOGICAL ACTIVITIES OF NONRIBOSOMAL PEPTIDES Following the discovery of penicillin and its antibiotic properties, these classes of compounds have attracted a lot of interest. NRPs are naturally synthesized by enzymes called “nonribosomal peptide synthetases” (NRPSs). These are large enzymes that activate amino acids in order to synthesize peptides by a thiotemplate mechanism. These are secondary metabolites, though these are not essential for growth or reproduction of the organism but perform other important tasks like defense, communication, or exportation. The presence

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Studies in Natural Products Chemistry

of nonproteinogenic amino acids and their unique structure grant them an edge over other peptides in terms of pharmacokinetics and bioavailability.

CHEMICAL SYNTHESIS OF NRPs Solid-phase peptide synthesis is the most commonly used strategy for the synthesis of the linear and/or cyclic polypeptide structure, a few solution phase synthesis has also been reported. The key step lies in the design and synthesis of the structurally diverse nonproteinogenic amino acids. Amidst many NRPs, synthetic studies of few important NRPs are discussed below.

Synthesis of Gramicidine S, 1 Gramicidin S or Gramicidin Soviet was discovered by Russian microbiologist Georgyi Frantsevitch Gause in 1942. It is a cyclodecapeptide containing two identical pentapeptides connected head to tail, written as cyclo(-L-Val-LOrn-L-Leu-D-Phe-L-Pro-)2, has antibiotic and antifungal properties. Two units of D-phenylalanine are the only nonproteinogenic amino acids present in the sequence. Wadhwani et al. [3] in 2006 reported a solid-phase approach using Fmoc chemistry for the synthesis of Gramicidin S. In this report, 2-chlorotrityl resin loaded with Fmoc protected D-phenylalanine (0.63–0.87 mmol/g) was chosen as the starting material. Peptide elongation was carried out using standard piperidine (22% in DMF, 30 min) mediated Fmoc deprotection followed by sequential addition of Fmoc-amino acids preactivated with 6-Cl-HOBt and HCTU in the presence of the base DIPEA in N-methyl pyrrolidone (NMP). The on resin linear decapeptide 13a was cleaved from resin using TFA/iPr3SiH/H2O. The bis-N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde at ornithine residue) protected free linear decapeptide 13b was then subjected to PyBOP/HOBT-mediated cyclisation in DCM. Cyclisation followed by hydrazine (2%)-mediated Dde deprotection yielded Gramicidin S in 69% overall yield (Scheme 2.1). Solution phase synthesis of two of the natural homologues of Gramicidin S (S 2 and S 3) was previously reported by Nozaki et al. [4] in 1985. Contrary to Gramicidin S, the Gramicidin S 2 had an L-amino butyric acid instead of valine and Gramicidin S 3 has both the valines replaced by L-a-amino butyric acid. The Boc (N-terminal) and benzyl (O-terminal) protected tetrapeptide 14 was common to both the gramicidins and were synthesized by “Hold in solution” method [5]. The tetrapeptide on Boc deprotection followed by EDC/HOBt-mediated peptide coupling with requisite Boc-protected amino acid yielded the pentapeptides 15. The pentapeptides on saponification yielded the C-terminal free pentapeptides 16. A portion of 16 was treated with HCl in dioxane to generate N-terminal and C-terminal free pentapeptides 17. Cross coupling between peptides 16 and 17 yielded the linear decapeptides 18.

Synthetic Studies Toward Nonribosomal Peptides Chapter

2

35

O HOOC

DIPEA, CH2Cl2

NHFmoc

NHFmoc

Cl

Standard Fmoc solid-phase peptide synthesis, 9 cycles

Resin loading Ph

Ph NH(Dde) O O

H N

HN

N H

O

O

H N

N H

O

Ph

(92.5 / 5 / 2.5 v/v)

N

H N

N H

TFA / iPr3SiH / H2O

O

O

O

O

Ph

H N

N H

O

13a

NH(Dde) NH(Dde)

O H N

HN

N H

O N H

O O

HO C 2 N H Ph

O

H N O

N H

Ph

H N

O

O N

H N

1. PyBOP, HOBt, DIPEA, CH2Cl2 2. N2H4 (2% in THF)

Gramicidin S, 1

O NH(Dde)

13b

SCHEME 2.1 Wadhwani’s synthesis of Gramicidin S, 1.

These decapeptides on Boc deprotection and EDC/HOBt-mediated peptide coupling in Pyridine-DMF (1:1) mixture at a concentration of 3 mM yielded the Cbz-protected (at ornithine residue) cyclic decapeptides 19. Subsequent Cbz deprotection yielded Gramicidin S 2 1a and Gramicidin S 3 1b (Scheme 2.2).

Synthesis of Actinomycin D, 2 Actinomycin D or Dactinomycin was isolated by Selman Waksman and his coworker Woodruff [6] in 1940 from soil bacteria of the genus Streptomyces. It was the first antibiotic also have anticancer activity. Sarcosine, D-valine, and N-methylvaline are the nonproteinogenic amino acids present in Actinomycin D. It is a dimer of two cyclopentadepsipeptide joined at 2-nitro-3-benzyloxy-4-methylbenzoyl group. Johannes Meienhofer in 1970 reported a synthesis of Actinomycin D [7] starting from the esterificatiom of the carboxyl group of N-Cbz-protected N-methylvaline and the hydroxyl group of N-Boc protected threonine to yield 20. In the following step, hydrogenolysis and mixed anhydride-mediated coupling with Cbz-protected sarcosine yielded 21. Subsequent chain elongation was carried out at the C-terminal using the same mixed anhydride strategy with O-t-butyl D-valyl-L-prolinate to yield O-(benzyloxycarbonylsarcosylL-N-methylvalyl)-N-t-butyloxycarbonyl-L-threonyl-D-valyl-L-proline-t-butyl

36

Studies in Natural Products Chemistry

O BocHN

H N

N H

O

R

O

COOBn HCl/Dioxane

N

NHCbz

R

R

O

H N

NaOH, EtOH BocHN

O

H N

N H

O

O

H N

17

O

Ph

15a, R = CH3 15b, R = H

HCl/Dioxane

Ph

CO2H

Ph 17a, R = CH3 17b, R = H

R2

BocHN

O

O

H N

H N

O

H N

N H

O

Et3N

N H

N

O

O

CbzHN R1 H N

HN

N 1. HCl/Dioxane

O N H

O

O O N H Ph

O

H N O CbzHN

N H

H N

R2

O

CO2H N

O 18a, R1 = H. R2 = CH3 18b, R1 = R2 = H

Ph

O H2/Pd,

O N

H N

H N

Ph NHCbz

NHCbz

O

O N H

Ph

2. EDC, HOBt, Py

O

16a, R = CH3 16b, R = H

R1

16

COOBn N

N

NHCbz

EDC, HOBt,

CO2H

O

H N

N

O

NHCbz

R

O

H N

N H

O

ClH.H2N

N H

O

HO2C

14

NHCbz

BocHN

BocHN, EDC, HOBt, Et3N

Ph

O

H N

HCl

Gramicidin S2, 1a R1 = H. R2 = CH3 Gramicidin S3, 1b R1 = R2 = H,

O 19a, R1 = H. R2 = CH3 19b, R1 = R2 = H

SCHEME 2.2 Nozaki’s synthesis of Gramicidins S2 and S3.

ester 22. Boron trifluoride-mediated t-butyl ester and N-Boc deprotection followed by 2-nitro-3-benzyloxy-4-methylbenzoyl chloride treatment yielded O-(benzyloxycarbonylsarcosyl-L-N-methylvalyl)-N-(2-nitro-3-benzyloxy-4methylbenzoyl)-L-threonyl-D-valyl-L-proline 23. The compound 23 was treated with di-p-nitro phenyl sulfite in pyridine to yield 24. HBr-mediated Cbz deprotection yielded the precursor which was cyclized in pyridine using the high dilution technique to yield the crystalline cyclic pentapeptide lactone derivative, (2-nitro-3-hydroxy-4-methylbenzoyl)-L-threonyl-D-valyl-L-prolylsarcosyl-L-N-methylvaline (threonine hydroxyl) lactone 25. Catalytic reduction of the aromatic nitro group followed by potassium ferricyanide-mediated oxidation (dimerization) yielded Actinomycin D 2 (Scheme 2.3).

Synthetic Studies Toward Nonribosomal Peptides Chapter

O

OH O

O

OH NHBoc

HN

CbzN

O BocHN

O

H2/Pd

O

BocHN

O

NCbz HO

20

O

Cbz O N

HO

O

O

N 1. Isobutyl chloroformate

O

BocHN

O O

HN

2. D-Val-L-Pro-OtBu O

O

O

OtBu O

Cbz N

N

O

HO

O O

37

2

N

Cbz N

O

BocHN

21

22

O O

HO2C N

O O

HN

1. BF3-acetic acid

Cbz N Bis-p-nitrophenyl sulphite

O

HN

2. 2-nitro-3-benzyloxy-4methylbenzoyl chloride

N

O NO2

O 23

OBn O

O

O

O2N

O

O

N

O O

HN

N

Cbz N

1. HBr-Dioxane

O NO2 OBn 1. H2/Pd 2. K3Fe(CN)6

N HN

2. Et3N

O

HN

N

O

O O

N

O

O

N H

NO2 OBn

O 24

25 Actinomycin D, 2

SCHEME 2.3 Meienhofer’s synthesis of Actinomycin D, 2.

Takumi Tanaka et al. in 1980 [8] reported an aziridine ring open up mediated synthesis of Actinomycin D. The dipeptide D-valyl-L-proline t-butyl ester on DCC coupling with the aziridine 26 yielded the tripeptide 27. Trityl deprotection and coupling with 2-nitro-3-benzyloxy-4-methylbenzoic acid NHS ester yielded 28. Aziridine ring open up using benzyloxycarbonylsarcosinyl-N-methyl-L-valine yielded the ester linkage containing peptide 29 that was then converted to Actinomycin D, 2 using the following protocol (Scheme 2.4).

Synthesis of Bacitracin A, 3 Bacitracin (a mixture of closely related peptides) was first isolated by Goorley [9] from a girl named Tracy in 1945. It is produced by soil bacteria of the group Bacillus subtilis. Bacitracin A is the most effective and widely studied antibiotic that interferes with the cell wall and peptidoglycan synthesis.

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Studies in Natural Products Chemistry

CO2tBu O N

t

O

BuO2C

HO2C

+

DCC

1. 85% HCOOH

N

N Trt 26

H 2N

27

O t

O

BuO2C

t

O

BuO2C

Cbz N

N HN

N O

N

O

O O

HN N Me

CO2H

NO2

O O

O

N

O

NO2

O

Cbz N

O

HN

OBn O

28

2. 2-nitro-3-benzyloxy-4methylbenzoic acid-NHS ester

NTrt

HN

29

OBn N O2N

O O

HN

1. TFA

NO2 O

O

1. HBr-Dioxane O

O

OBn

Cbz N

O

HN

2. Bis-p-nitrophenyl sulphite

N

2. Et3N

24

N

N HN O

O O

O

1. H2/Pd

O

N N H

2. K3Fe(CN)6

NO2

Actinomycin D, 2

OBn

25

SCHEME 2.4 Tanaka’s synthesis of Actinomycin D, 2.

D-aspartic acid, D-phenylalanine, D-ornithine, D-glutamic acid, and a thiazoline containing amino acid are the nonproteinogenic amino acids present in Bacitracin A. Lee et al. in 1996 reported [10] a solid-phase method for the total synthesis of Bacitracin A. PAL resin linked to the side chain of the O-allyl protected L-asparagine was used as the starting point for the solid-phase synthesis. Chain elongation to yield the linear decapeptide 30 was done using Fmoc strategy. The sixth amino acid residue in the backbone was side chain N-allyloxycarbamate-protected Fmoc-L-lysine-OH. Allyl ester and allyl carbamate were then deprotected followed by cyclization on resin yielded the core structure 31. The Boc-protected thiazoline amino acid 32 was synthesized by the condensation of L-cysteine methyl ester and an iminoether derived from Boc L-isoleucine following a procedure previously reported by Pattenden [11]. Fmoc deprotection of the resin bound decapeptide and peptide coupling with 32 resulted in the formation of resin bound Boc-protected Bacitracin A, which was then treated with TFA and triethylsilane to remove the solid support as well as the Boc protection (Scheme 2.5).

Synthetic Studies Toward Nonribosomal Peptides Chapter

2

39

Sequential deprotection/coupling of 1. Fmoc D-Asp(OtBu)-OH 2. Fmoc L-His(Trt)-OH 3. Fmoc D-Phe-OH 4. Fmoc L-Ile-OH 5. Fmoc D-Orn(Boc)-OH 6. Fmoc L-Lys(Alloc)-OH

O NHFmoc

O HO NH2

O

PAL

PAL

HBTU / DIEA

t

O NHAlloc

O

BuO2C H N O H

H N PAL

O H O N

HN O

7. Fmoc L-Ile-OH 8. Fmoc D-Glu(OtBu)-OH 9. Fmoc L-Leu-OH

O

t

O N H

O

Trt N NH

HN

N

H N

1. Pd(Ph3P)4

O

O Ph

Boc H N O

O N H

O

Boc H N

O

N H

O N H

Trt N NH

H N

N

O

O Ph

CO2tBu

HN 30

O

H

O H

O H O N

HN

2. PyBOP/HOBt

NHFmoc

BuO2C H N

H N

PAL

CO2tBu

HN

O

N H

NHFmoc

O HN

O

O N H

31 NHFmoc

H 1. Piperidine NHBoc

−

2. LiO

N

O

S HATU/PyBOP

H 32

Bacitracin A, 3

3. TFA

SCHEME 2.5 Jinho Lee’s synthesis of Bacitracin, 3.

Synthesis of Polymyxin B1, 34 Polymyxin B is an antibiotic derived from the bacterium Bacillus polymyxa, primarily used for the treatment of resistant Gram-negative infections. It is a mixture of a number of identical compounds varying in their fatty acid group. Six units of L-diaminobutyric acid (Dab), D-phenylalanine, and S-(+)-4methyloctanoic acid are the nonproteinogenic amino acids present in Polymyxin B. Sharma et al. reported an Fmoc solid-phase approach for the total synthesis of Polymyxin B1 [12]. The N-terminal side chain S-(+)-6-methyloctane1-carboxylic acid was synthesized by malonic ester synthesis followed by decarboxylation and saponification of the mesylate of commercially available S-(+)-4-methyl-1-hexanol. Starting from Fmoc-Thr(tBu)-SASRIN resin, sequential chain elongation with respective protected amino acid and finally coupling with S-(+)-6-methyloctane-1-carboxylic acid yielded the resinlinked-protected linear peptide 33. Selective deprotection of the Dde-protecting

40

Studies in Natural Products Chemistry

group at the desired position by 2% hydrazine in DMF followed by resin unloading by 1% TFA in DCM yielded the resin-free linear peptide. Diphenoxyphosphoryl azide (DPPA)-mediated cyclization between the carboxyl group of threonine and the side chain amino group of the selectively deprotected L-diaminobutyric acid yielded the protected peptide, which on TFA-mediated deprotection yielded Polymyxin B1 34 in 64% yield (Scheme 2.6).

Synthesis of Daptomycin, 44 Daptomycin is a lipopeptide antibiotic isolated from Streptomyces roseosporus administered in cases of serious Gram-positive infections. Kynurenine (Kyn) and 3-methylglutamic acid (3-mGlu) are the two nonproteinogenic amino acids present and are critical for the bioactivity of Daptomycin. Total synthesis of daptomycin has only recently been reported by Lam et al. that makes use of both solution phase as well as solid-phase peptide synthesis [13]. The nonproteinogenic amino acid kynurenine was synthesized by ozonolysis of protected L-tryptophan and 3-methylglutamic acid was synthesized from L-pyroglutamic acid [14]. NHBoc Ph

OtBu O

NHFmoc

Sequential deprotection/coupling 1. Fmoc L-Dab(N-Boc)-OH 2. Fmoc L-Dab(N-Boc)-OH 3. Fmoc L-Leu-OH 4. Fmoc D-Phe-OH 5. Fmoc L-Dab(N-Boc)-OH 6. Fmoc L-Dab(Dde)-OH 7. Fmoc L-Dab(N-Boc)-OH

O

NH

O

O N H

N H

NHBoc

O HN

NH-Dde

NH

HN BocHN

O O

HN

8. Fmoc L-Thr(tBu)-OH 9. Fmoc L-Dab(N-Boc)-OH 10. S-(+)-6-methyloctanoic acid

O

O OtBu

HN

NHBoc OtBu

O O

33

NH O

BocHN

N H

H3N O OH

O

H3 N

NH HN 1. 2% Hydrazinne/DMF 2. 1% TFA/CH2Cl2 3. DPPA/DIEA/CH3CN

O O

NH

NH3

HN

OH O

4. 95% TFA/H2O

NH

O NH

NH

NH HN

O

O O

NH HN Ph

O NH3

O NH3

SCHEME 2.6 Sharma’s total synthesis of Polymyxin B1, 34.

Polymyxin B1, 34

Synthetic Studies Toward Nonribosomal Peptides Chapter

2

41

Allyl ester of glycine was taken as the starting material for the synthesis of the peptide fragment 35 containing an ester linkage with kynurenine. Glycine allyl ester on peptide coupling with Fmoc-L-Thr(tBu)-OH yielded a dipeptide, which on tBu group removal and silylation yielded 36. Fmoc deprotection and another peptide coupling followed by another Fmoc removal, diazo transfer reaction and TBS deprotection yielded the azido tripeptide 37. Esterification of 37 with Fmoc-L-Trp(Boc)-OH yielded the depsipeptide 38 which after palladiumcatalyzed deallylation, followed by ozonolysis gave 35 as product (Scheme 2.7). The synthesis of the other fragment, ie, trityl-resin-linked pentapeptide 39 (Fmoc-L-Orn(Boc)-L-Asp(tBu)-D-Ala-L-Asp(tBu)-Gly) was assembled via standard Fmoc strategy, which was then coupled with N3-L-Asp(tBu)-L-Thr[O-Kyn(Boc,CHO)-Fmoc]-Gly-OH 35. The resin-linked peptide 40 was then coupled with Fmoc-3-mGlu(tBu)-OH and Boc-D-Ser(tBu)-OH. After reduction of the azide group, further chain elongation and resin detachment afforded the whole linear sequence 41 (Scheme 2.8). a,a-Dimethoxysalicylaldehyde was introduced at the C-termini of 41 as a part of the cyclization strategy and the protecting groups were removed to yield 42. The deprotected linear peptide underwent serine ligation to yield 43, an N,O-benzylidene acetal-linked cyclic product which on TFA treatment afforded daptomycin 44 in 67% yield (Scheme 2.9).

Synthesis of Tyrocidine A, 46 Tyrocidine is an active ingredient of tyrothricin which was isolated by American microbiologist Rene Dubos in 1939 from the soil microbe Bacillus brevis. It is a

1. TFA 2. Fmoc-Thr(tBu)-OH, BocHN

CO2All

O N3

O

1. DEA, DCM 2. Fmoc-Asp(tBu)-OH, 3. Diethylamine

CO2All

4. Imidazole-1-sulfonyl azide 5. TBAF

36

O

OH H N

N H O CO2tBu

FmocHN

3. TFA 4. TBSCl, imidazole

OTBS H N

CO2All

Fmoc-Trp(N-Boc)-OH

N3

NBoc O

HN CO2tBu

37

NHFmoc

O

NH O

CO2All

NHFmoc O 1. Pd(PPh3)4, THF 2. O3, −78°C

O N3

BocN O

N H HN CO2tBu

CHO

O O CO2H

35

SCHEME 2.7 Hiu Yung Lam’s synthesis of Daptomycin, fragment 35.

38

42

Studies in Natural Products Chemistry

O

O NHFmoc

O

O

NHBoc

Fmoc-SPPS

O

NH HN HN O

H2N

CO2tBu

HN CO2tBu

35

O O 39

BocHN O O N3

NH

N H

O N H O CO2tBu

O

H N

O 1. Fmoc-SPPS 2. DTT, DIEA, DMF

O N H O HN BuO2C O t BuO2C O HN

t

3. Fmoc-SPPS 4. AcOH, TFE

O NHFmoc O

40

BocHN

NBoc

O

CHO NH O C9H19

NH O

N H

H N

O

O N N H H O CONHTrt O NH HN t BuO2C O H O t BuO2C N N O HN t H O BuO2C O O O CO2tBu OH H N N NHBoc H O O 41 CO2tBu NBoc CHO

SCHEME 2.8 Hiu Yung Lam’s synthesis of Daptomycin linear fragment 41.

cyclic decapeptide and has two units of nonproteinogenic D-phenylalanine. Tyrocidine disrupts the cell membrane function of bacteria but has also been found to be toxic toward some human cells. Bu et al. in 2002 reported an Fmoc strategy based solid-phase approach for the total synthesis of Tyrocidine A [15]. Their previously reported on resin cyclization of thioesters with ammonium hydroxide [16] was effectively used as the key step for this synthesis. L-Leucine Fmoc loaded on Tenta Gel-OH resin with a succinic ester and amino thiol linker was used as the starting material and chain elongation the resin linked linear thioesters decapeptide 45. Protecting groups of side chains of constituting amino acids were removed followed by the treatment with 7 M aq. ammonia on resin decapeptide yielded Tyrocidine A 46 (Scheme 2.10).

Synthesis of Lysobactin (Katanosin B), 52 Lysobactin is a cyclic depsipeptide antibiotic isolated in 1988 and produced by a species of Lysobacter [17] and Cytophaga [18]. It targets cell wall biosynthesis and is active against Gram-positive bacteria. 3-Hydroxy-leucine,

Synthetic Studies Toward Nonribosomal Peptides Chapter

2

43

H2N O

41

NH

1. α,α-Dimethoxy salicylaldehyde

CONH2 O NH O H N N H O O CONH2

O

2. TFA C9H19

NH

N H

O

N H

O

O

H N O HO2C

N H

N H

O Pyridine Acetate

C9H19

N H

NH

NH

O

O

O

N H

43

H2N O NH O C9H19

NH

N H

CONH2 O O H N N H O CO2H

NH

O

N H

O

O

O HO2C

N H O NH2

N H

O HN O

HO2C

TFA

NH O

H N

N O

CO2H

O

O HO

O

H N

O HO2C HO2C O

42

O

H N

O NH2

OHC

NH2

O

O CONH2 O H N N H O CO2H

O NH2

CO2H

NH2

N H

O HN

O

O

NH

O

HO2C

HO H N

O

O HN

HO H N O

N H HN

O O NH Daptomycin, 44

N H

O

CO2H

SCHEME 2.9 Hiu Yung Lam’s total synthesis of Daptomycin, 44

3-hydroxy-asparagine, allo-threonine, and 3-hydroxy-phenylalanine are the nonproteinogenic amino acids present in Lysobactin. Von Nussbaum and coworkers published the first total synthesis of Lysobactin [19] in 2007. DCC-mediated solution phase peptide coupling of N-Cbz and O-2-(trimethylsilyl)ethyl (TMSE) protected tripeptide 47 with suitably protected L-serine followed by Boc deprotection yielded the ester linkage containing tetrapeptide 48. The compound 48 was coupled with Boc-Glyhydroxyasparagine-OH, Boc deprotection yielded the fragment 49 (Scheme 2.11). In a similar fashion, the other fragment was synthesized from the benzyl ester of L-serine. Sequential solution phase peptide coupling with Boc-LIle-OH, Boc-L-Arg-OH, Boc-L-Leu-OH, Boc-3-hydroxy-L-leucine-OH, and

44

Studies in Natural Products Chemistry

O O O

Standard Fmoc solid-phase

S

N H

NHF moc

Peptide synthesis, 9 cycles

O O

O

O N H

45

H2N S

O

O

O

H N

N H

O

H2N

O

H N

N H

H2N

O

N H O

O

O H N

O

H N O

N H

N

O

NH2

HO

O N

O NH 7M NH3.H2O

O HN O

NH

O

Resin cleavage and cyclization

NH HN O

O

O NH

H N

HN O

O

H2N

NH

H2N

O

O

H2N Tyrocidine A, 46

OH

SCHEME 2.10 Xianzhang Bu’s total synthesis of Tyrocidine A, 46

O CbzHN

N H

H N O

47

Ph

CbzHN

2. TFA

N H

O O TMSE

48 O

1. Boc-Gly-HyAsn-OH 2. TFA

O

1. Boc-Ser(tBu)-OH OH

CbzHN

N H

H N O

O

Ph

O O

t

O BuO

TMSE

H2NOC H N O

H N O

O

Ph

O O

NH2

O tBuO

TMSE

OH O NH2

N H 49

SCHEME 2.11 Franz von Nussbaum’s total synthesis of Lysobactin, fragment 49.

hydrogenolysis afforded 50. Peptide coupling between the fragments followed by TMSE and Boc deprotection yielded the linear Lysobactin precursor 51. This precursor on cyclization yielded Lysobactin 52 (Scheme 2.12). Another solution phase synthesis of Lysobactin 52 was reported by Guzman-Martinez et al. in the same year [20]. b-Hydroxyphenylalanine methyl ester 53 was synthesized using Sharpless asymmetric aminohydroxylation [21] which was then coupled with Boc-D-Leu-L-Leu-OH using 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) to provide

Synthetic Studies Toward Nonribosomal Peptides Chapter

HO BnO2C

NH2

HO

H N

N H

HOOC

3. Boc-Leu-OH 4. Boc-3-hydroxy-Lue-OH 5. H2, Pd/C

NH

O

45

H N

H2N

Boc strategy Solution phase 1. Boc-Ile-OH 2. Boc-D-Arg-OH

2

HO H N

O N H

O

NHBoc O

50

O

CbzHN

HN Ph

NH O HOOC

O H2NOC NH

O

OH O

HO O

1. 49, HATU

HO

HN

H2N

NH

NH

O

2. TBAF

O

i

3. Pr3SiH, TFA

HN

HN NH

51

O O

HO

HN NH

HO

CONH2

O OH

O

NH2

NH NH O

O

1. HATU, NMM 2. H2, Pd/C

H 2N

N H

H N

O

HN

O

OH

O HO

NH

O O

HN

Lysobactin, 52

HN

Ph O

O

O

O N H

NH

H N

NH2 NH

SCHEME 2.12 Franz von Nussbaum’s total synthesis of Lysobactin, 52.

the tripeptide 54. The tripeptide 54 on methyl ester hydrolysis and coupling with H-3-OTBS-Leu-Leu-OAll yielded the pentapeptide 55. Compound 55 on esterification with Fmoc-L-Ser(tBu)-OH followed by Fmoc removal, coupling with Fmoc-hydroxyasparagine-(Trt)-OH and allyl ester cleavage provided the depsipeptide 56 (Scheme 2.13). The other fragment was synthesized from D-allo-threonine methyl ester. D-allo-Thr-OMe was coupled with Boc-L-Ile-OH and H-Gly-OAllyl to yield a tripeptide 58 that was saponified and again coupled with Fmoc-D-Arg (Boc)2-OH to provide a tetrapeptide 59. The tetrapeptide 59 on Fmoc removal and coupling with fragment 56 afforded the linear depsipeptide 60. Removal of the allyl ester and Fmoc followed by coupling and Boc/tBu removal produced Lysobactin 52 in 33% yield (Scheme 2.14).

46

Studies in Natural Products Chemistry

Boc-D-LeuLeu-OH

NH2 Ph

MeO

O

HN

OH 53

O

Ph

NH

54

OMe

1. Fmoc-L-Ser(OtBu) 2. Piperidine

OH

O O O TBSO

1. LiOH 2. H-3-OTBS-LeuLeu-OAll,

OH

O

H N

HN BocHN

Ph

HN O

BocHN

H N

O

HN

BocHN 3. Fmoc-HyAsn(Trt)-OH OAll 4. Pd(PPh3)4, PhSiH3

O

TrtHNOC O H N

t NH BuO H N

O O O TBSO

O

55

Ph

H N

O

OH NHFmoc

O OH

O

56

SCHEME 2.13 Martinez synthesis of depsipeptide, 56.

HO O

HO

OH 1. SOCl2, MeOH

HO

O

2. Boc-L-Ile-OH,

NH2

O

O

OMe

1. LiOH

N H

HN

2. H-Gly-OAllyl

OAll OAll

1. 4N HCl

NBoc

O

HN FmocHN

H N

O

58

O 1. Piperidine

OH

HN

H N

2. Fmoc-D-Arg(Boc)2-OH 59

NHBoc

NHBoc

57

BocHN

O N H

2. 56 O

O

O

HN BocHN

H N O O O TBSO

Ph O

TrtHNOC O H N

t NH BuO H N

O

O N H

O

OH OAll O

HN NHFmoc R

H N

OH

HN

1. Pd(PPh3)4, PhSiH3 2. Piperidine 3. DEPBT, DIEA 4. TFA

O

O

60

Lysobactin, 52

NBoc R=

N H

NHBoc

SCHEME 2.14 Martinez Total synthesis of Lysobactin, 52.

Interestingly in both the approaches D-Leu-L-Leu fragments were introduced at an early stage, ie, before the formation of adjacent ester linkage in order to minimize the possibility of O to N acyl migration.

Synthesis of Thiocillin 1, 74 Thiocillin 1 is a thiopeptide antibiotic isolated from Bacillus cereus. Six thiazole containing units and a pyridine unit are present in this compound. The first total synthesis of Thiocillin was reported by Ciufolini and coworkers [22] utilizing Bohlmann-Rahtz pyridine synthesis [23] as one of the key steps.

Synthetic Studies Toward Nonribosomal Peptides Chapter

47

2

Selenium dioxide-mediated oxidation of a methyl thiazole 61 yielded the corresponding aldehyde 62 that was converted to an ynone 63 using Grignard reaction followed by Dess–Martin Periodinane oxidation. Bohlmann-Rahtz reaction between ynone 63 and a previously reported thiazole compound 64 derived from L-threonine [24] yielded the pyridine containing core 65 of Thiocillin 1. To introduce orthogonality among the two protecting groups, the acetyl protecting group was replaced by TBS followed by ester hydrolysis and N-Boc protection to yield 66. The protected acid on coupling with the known segment 67 [25] followed by elimination, TBS deprotection and a two step oxidation afforded the fragment 68 (Scheme 2.15). Parekh–Doering oxidation of the known R-alcohol 69 [26] and condensation with methyl cysteinate hydrochloride, in Shioiri thiazole synthesis condition [27] followed by aromatization yielded the N-Boc protected thiazole 70. The compound 70 on repetitive solution phase Boc–peptide coupling strategy with previously reported acid [25b] and 71 [28] followed by selective elimination of the secondary hydroxyl group and Boc deprotection yielded the other fragment 72 of Thiocillin 1 (Scheme 2.16). For the total synthesis, the fragments 68 and 72 were coupled followed by saponification and Boc deprotection to afford the linear peptide 73.

O N

S

S

OHC

SeO2

N

S

COOEt

61

N

S

1.

N

2. Dess–Martin

63 +

H

N H H

O

NH AcOH

S

COOEt

1. K2CO3 2. TBSCl

N

S

N

O

O

N

S N

O

N N

O

S 63

AcO NH4OAc

S

N

COOEt

62

S

TBSO

MgBr

3. LiOH 4. Boc2O

N

S 65

64

N

S

EtOOC CO2H

S

TBSO

NH2

N N

N

3. TBAF 4. Two step oxidation

67

S

N

1. BOPCl 2. (a) MsCl, (b) DBU

OAc

H N

OH O

N

S

+ S

66 O

O

NBoc O

NH

S

HO2C

HN

S N

N N

S

N NBoc

O

O

SCHEME 2.15 Ciufolini’s synthesis of fragment, 68.

S

N

O

68

OAc

48

Studies in Natural Products Chemistry

1. Solution phase Bocpeptide coupling COOH COOMe 1. SO3.pyr, DMSO 2. methyl cysteinate.HCl

HO OH NHBoc

69

HO

TFA.H2N

N H OH

NHBoc

N H

S

70

OH

O

N

NBoc

71

S

3. MnO2 O

2. O

N

3. (a) MsCl (b) DBU 4. TFA

72

N

COOMe

S

SCHEME 2.16 Ciufolini’s synthesis of fragment, 72.

S

O H H N N H H

S

OH

N

68 + 72

1. BOPCl 2. LiOH 3. TFA

O

O OH

N

H

S

S

S HN

O

O

N

HO

N

N

N

TFA.H2N

NH

S

N

NH

CO2H 73

H OH S

DPPA

O H H N N H O H OH N O

S N OH H N

NH N

HO

H

N H

Thiocillin 1, 74

N

HS

S

O H

OH

N H

S

N

O

N H N

S

O

SCHEME 2.17 Ciufolini’s total synthesis of Thiocillin 1, 74.

This linear depsipeptide on cyclization with DPPA yielded Thiocillin 1, 74 (Scheme 2.17).

Synthesis of Polytheonamide B, 5 Polytheonamides (A and B) 5 are cytotoxic linear peptides, isolated from the marine sponge Theonella swinhoei, and are believed to be produced by symbiotic microorganisms. Among the 48 amino acids sequence 13 of them are nonproteinogenic amino acid residues (Fig. 2.9). The only total synthesis was reported by Inoue and coworkers in 2010 [29]. In their retrosynthetic plan (Scheme 2.18), they identified four fragments 81, 82, 83, and 84 mostly having a glycine at their C-termini in order to eliminate any chance of racemization during peptide coupling, which they planned to assemble in order to complete their total synthesis. Fmoc

Synthetic Studies Toward Nonribosomal Peptides Chapter

H2N

H2N

OH

H2N

OH

H2N

O

O

D-Ala

H2N

H2N

OH

O

O MeHN OH H2N

OH

OH OH

O

O

D-Tle

D-Ser

O D-Asn

L-Tle

OH

OH

MeHN

OH

H2N

OH

OH

H2N

O

O

OH

H2N

O

77a

76

O

S

OH

H2N

OH

H2N

O

77b

O 75 O

OH

H2N

O

O

79

78

OH

OH H N 2

O N-Me-D-Asn

H2N

O

49

2

80

FIG. 2.9 Nonproteinogenic amino acid residues in polytheonamide.

Polytheonamide B, 5

O

O

H N O

O

H N

N H

O

O

H N

N H

O

O

H N

N H

O

O

H N

N H

H N

N H

O

CO2H

O Fragment 81

O HO2C

O

H N

N H

O OH

N H O

O

H N

O OH NHMe

H N

N H O

NHFmoc

OH

O FmocHN

NHMe

Fragment 83

NHMe

O

H N

HO2C

N H

O O

O NH

H N

N H O

O

N H

O

OH

O

O Ot Bu

NHMe

O

CO2H

HN

HN O

NH

NHMe HN O NH

O

H N

O

N H

O O

Fragment 82

O

O

H N

N H

O

H2N

H2N

O

H N

NHMe O NH

O O

H N

S

O N H OH NHMe

H N

O N H

O O NHMe

H N O

O N H Ot Bu

Fragment 84

H N

O

t BuO

O N H

O O NHTr

H N

O

O N H

O O NHTr

O NH

O NHTr

SCHEME 2.18 Retrosynthetic analysis of Polytheonamide B, 5.

strategy was their approach for the solid-phase synthesis as acidic conditions required in case of Boc strategy which may cause side reactions (elimination) in the b-hydroxy amino acids. Initial task of the nonproteinogenic amino acids synthesis (76–80) and installing suitable protection was achieved following general synthetic

50

Studies in Natural Products Chemistry

MeO2C 1. Sharpless AA

1. RuCl3, NaIO4

MeHNOC

2. FmocCl

FmocHN

OMe O

OMe OH

BocHN

S N

BnON

2. TEPMO NaClO2

88

90

S N BnOHN

1. Katsuki oxidation 2. TFA

2. FmocCl

FmocHN

1. H2, Pd(OH)2/C 2. NaOH FmocHN

1. ZnBr2 CO2t Bu

2. FmocOSu

H2NOC FmocHN

Boc-79-O-t Bu

O S

HO BocHN

CO2t Bu

CO2H

CO2H

Fmoc-79 1. Tf2O, 2-6-lutidine 2. NaSMe 3. HCl 4. FmocCl

MeS CO2t Bu

FmocHN

89

NHFmoc

CO2H

Fmoc-78

BocHN

3. H2, Pd(OH)2/C 4. Boc2O

CO2H Fmoc-77a

3. FmocOSu

O

H2NOC

1. KN(SiMe3)2, MeI 2. DIBAL CO2t Bu

O

O

86

MeO2C PhFIHN

BocHN CO2H Boc-77a

Prenyl Bromide

NH3

OH

1. TFA

Zn

CO2t Bu N Boc 87

O

OH

O

85

CO2H

Fmoc-76

1. MeMgBr

O

O

OH

90

Fmoc-80

SCHEME 2.19 Syntheses of nonproteinogenic amino acids for Polytheonamide B.

protocol summarized below (Scheme 2.19). Sharpless asymmetric aminohydroxylation reaction of methyl 4-methoxycinnamate followed by a series of protection/deprotection and RuCl3 oxidation yielded the Fmoc-protected amino acid 76 [30]. Boc-L-Ser-OMe on treatment with methyl magnesium bromide and a two step oxidation yielded the Boc-protected 77a which was then converted to Fmoc-77a, similarly Boc-D-Ser-OMe yielded Fmoc-77b under similar conditions. Zinc-mediated 1–4 addition of prenyl bromide to the camphorsultam containing compound 85 yielded the alkene 86. The alkene 86 was hydrogenated, hydrolyzed, and Fmoc protected to produce Fmoc-78. Aqueous ammonia mediated open up of N-Boc protected, tBu ester of lactam 87, synthesized from pyroglutamic acid [13,31a] yielded N-Boc and O-tBu protected 79. Deprotection followed by N-Fmoc protection resulted in Fmoc-79. 9-Phenylfluorenyl (PhFl) protected aspartate 88 on dimethylation at b-carbon, reduction of methyl ester and replacing PhFl with Boc produced the alcohol 89. The alcohol was converted to the corresponding triflate and in situ substitution with sodium methanethiolate to yield a methyl sulfide 90. The compound 90 on N-protecting group manipulation, stereoselective Katsuki oxidation [32] and TFA treatment yielded Fmoc-80. The primary amides at residues 43, 45, and 46 were trityl protected and the alcohols at 41, 47, and 48 residues were tBu protected. Four of the fragments

Synthetic Studies Toward Nonribosomal Peptides Chapter

O FmocHN

O O

1. Solid-phase peptide synthesis (a) Nα-Fmoc-amino acid, HBTU, HOBt, i-Pr2NEt, NMP (b) 22% piperidine/NMP 2. TFA/H2O

2

51

Ncap-[81]-S(CH2)2CO2Et, (A)

Fmoc-[82]-S-(CH2)2CO2Et, (B)

3. HS(CH2)2CO2Et, HOBT,DCC Fmoc-[83]-S-(CH2)2CO2Et, (C) 1. Solid-phase peptide synthesis (a) Nα-Fmoc-amino acid, HBTU, HOBt, i-Pr2NEt, NMP (b) 22% piperidine/NMP

O H2N t

O

H-[84-(t Bu)3(Tr)3]-OH, (D)

2. (CF3)2CHOH/DCM BuO

D

1. C, AgNO3, HOOBt

H-[26-48(t Bu)3(Tr)3]-OH

H-[12-48(t Bu)3(Tr)3]-OH

2. piperidine

2. piperidine 1. A, AgNO3, HOOBt

1. B, AgNO3, HOOBt

Ncap-[1-48]-OH

2. TFA/H2O

TFA H2O

Polytheonamide B, 5

SCHEME 2.20 Masayuki Inoue’s total synthesis of Polytheonamide B, 5.

ca 81, 82, 83, and 84 were assembled employing SPPS, resin cleavage and finally conversion to their corresponding thioesters of ethyl 3-mercaptopropionate. In case of the fourth fragment 84 due to the presence of tBu and Trityl groups resin cleavage was affected using trifluoroethanol instead of TFA. Final assembly of the peptides were done in solution phase using Aimoto condition (silver nitrate and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine) [33] following which careful deprotection afforded Polytheonamide B 5 (Scheme 2.20).

Synthesis of Cyclosporin A, 6 Cyclosporin or Cyclosporin A 6 was isolated from the fungus Tolypocladium inflatum in January 1969 by Hans Peter Frey and its immunosuppressive effect was discovered on 31st January 1972 by Hartmann F. Sta¨helin. Cyclosporin is an undecacyclopeptide in which 7 out of the 11 amino acids are N-methylated and has one interesting (2S,3R,4R,6E)-3-hydroxy-4-methyl2-methylamino-6-octenoic acid (MeBmt) amino acid which is important for its biological activity. Apart from the previously reported synthetic studies toward Cyclosporin A analogues [34] Danishefsky and coworkers recently reported the total synthesis of Cyclosporin A [35] using isonitrile coupling reactions as a key step for the generation of N-methyl amino acid containing peptides. The MeBmt segment was synthesized following a procedure reported by Evans [36] from N-hexenoyloxazolidinone, 91. N-hexenoyloxazolidinone on stereoselective methylation reduction and Swern oxidation yielded the aldehyde 92. The aldehyde on treatment with Cbz-Sar-OMe enolate and ester hydrolysis produced the oxazolidine 93. Hydrolysis of the oxazolidine

52

Studies in Natural Products Chemistry

followed by an acetonide protection furnished the required MeBmtoxazolidine 94 (Scheme 2.21). The tetrapeptide 95 was synthesized by condensation of two dipeptides 99 and 100. N-methyl containing dipeptide 98 was synthesized by using isonitrile coupling reactions between 96 and 97. The terminal N-methylation of the dipeptide to yield 99 was achieved by carbamate cleavage followed by retro-aza-Diels-Alder methylation [37]. The other dipeptide 100 was similarly synthesized, ie, by coupling thioacid and an isonitrile followed by radical reduction. Condensation of the two fragments yielded the desired tetrapeptide 95 (Scheme 2.22). The other tetrapeptide was synthesized in a very similar fashion where Microwave irradiation of azido acid 101 [38] and L-leucine isonitrile 102 [39] afforded N-formyl amide 103. The compound 103 on O-tBu deprotection, coupling with L-alanine benzyl ester, followed by sequential reduction of the formyl and the azido group and another peptide coupling with methyl-leucine methyl ester yielded the tetrapeptide 104 (Scheme 2.23). The mixture of the thioacid 105, N-Me-Gly-OBn, and cyclohexyl isocyanide [40] yielded the dipeptide 106 which was debenzylated and converted to its corresponding thioacid 107 using Lawesson’s reagent [41]. The thioacid

1. NaHMDS, MeI

O N 91

Bn

OHC

2. LAH 3. Swern Oxdn

HO2C

CO2Me 1. N , LDA Cbz

O O

92

H

N

2. Hydrolysis 3. Crystallization

O O

93

CO2H 1. KOH

94

N

2. Acetone

O

SCHEME 2.21 Synthesis of Me-Bmt Acetonide 94.

O BocHN

SH +

1. CHCl3 CN

CO2Bn

96

97

SH

BocHN

+

CN

CO2Bn

1. CHCl3 2. Bu3SnH, AIBN

O

O BocHN

2. TBTH N AIBN BnO2C

O N

N

N

CO2Bn

O 95

SCHEME 2.22 Synthesis of Tertapeptide 95.

1. TFA 2. Cyclopentadiene, HCHO

NHBoc

H.HCl N N

3. TFA

O 98

N

BocHN 100

O

BnO2C

O

CO2Bn

1. H2, 10%Pd/C 2. 99, DEPBT, DIPEA

99

Synthetic Studies Toward Nonribosomal Peptides Chapter

TFA.HN 1. HCOOH 2. H2N-Ala-OBn, O 3. LiBH4

OtBu

O N3

OH + NC

DCE, μwave

OtBu O

101

O N

O

102

N3

103

O N

4. Tf2O, Et3SiH 5. PPh3 6. Boc-MeLeu-OH, 7. TFA

CHO

NH

53

2

NH O

CO2Bn

104

SCHEME 2.23 Synthesis of Tertapeptide 104.

O SH

BocHN

+

O

c-(C6H11)-NC

HN

N

BocHN

OBn

O

O

150

106

HN

HO

O

N N

BocHN

1. 104, c-(C6H11)-NC 2. TFA

O

SH

O N NH

3. 94 4. HCl

107

2. Lawesson's reagent

O

MeHN

O

1. H2, 10% Pd/C OBn

O

O N

108

NH

O

O OBn

HO H N

N N 1. H2, 10%Pd/C

O

N O

1. NaOH 2. TFA

N

O

BnO NHBoc

O

O

O

O

O

2. 95, PyBOP

N

N

O N H

3. c-(C6H11)-CN, μwave

OO N

NH 109

HO H N

N N

N

N

O

O

H N

O

O

O

O

N

O N O

O

O N H

N

O NH Cyclosporin A, 6

SCHEME 2.24 Danishefsky’s total synthesis of Cyclosporin A, 6.

107 on similar sequence of reactions with tetrapeptide 104 as amine and coupling with N-methyl oxazolidine of MeBmt 94 as acid afforded the heptapeptide fragment 108. Debenzylation followed by coupling with the fragment 95 yielded the linear undecapeptide 109. The linear undecapeptide 109 on saponification, Boc deprotection followed by cyclohexyl isonitrile mediated concerted coupling and N-methylation yielded Cyclosporine A, 91 (Scheme 2.24).

54

Studies in Natural Products Chemistry

Synthesis of Enterobactin, 7 Enterobactin 7, a siderophore was discovered by Gibson and Neilands in 1970 from Escherichia coli and Salmonella typhimurium [42]. It is the strongest known siderophore and constitutes of three units each of 2,3-dihydroxybenzoic acid and L-serine. Bacillibactin, another siderophore, is structurally analogous to enterobactin. Corey and coworkers reported the synthesis of enterobactin in 1977 from N-benzyloxycarbonyl-L-serine [43]. Cbz-L-Ser-OH was converted to its p-bromophenacyl ester and the side chain hydroxyl group was protected with DHP to yield 111. Phenacyl deprotection and treatment with 2,2ʹ (4-tertbutyl-1-isopropylimidazolyl) disulfide [44] followed by simultaneous treatment with Cbz-L-ser-phenacylester 110 yielded the diserine derivative 112. Repeating the same steps as above the compound 112 was converted to the triserine derivative 113. Phenacyl and THP deprotection of the triserine derivative followed by 2,2ʹ (4-tert-butyl-1-isopropylimidazolyl) disulfide assisted cyclization yielded the cyclic depsipeptide 114. Cbz deprotection followed by excess 2,3-dihydroxybenzoyl chloride treatment led to the formation of Enterobactin 7 (Scheme 2.25). A very similar strategy for the synthesis of Enterobactin was reported by Rastetter and coworkers in 1981 [45]. Shanzer et al. reported an efficient organotin template assisted synthesis of enterobactin [46]. N-trityl protected b-lactone 115 synthesized from L-serine following modified Sheehan’s method [47] on treatment with distannoxane yielded the Enterobactin framework 116. Trityl deprotection followed by

NHCbz HO

1. p-Bromophenacyl bromide,KHCO3

NHCbz O

HO

O

2. DHP, p-TsOH

COOH O

NHCbz THPO

110

Br

1. Zn/AcOH

O

2. tBu

O

i

N S

O 111

N

Br

O

NHCbz

NHCbz

O

O

O

Br

O

O

O

O

112

O O

O

Bu

NHCbz THPO

NHCbz

O

t

N

Pr

3. 110

Repeat previous step

N S

i

NHCbz THPO

Pr

Br

113

O CbzHN

1. AcOH 2. Zn/AcOH t 3. Bu

NHCbz

O

1. H2, Pd/C O

i

O

Pr

N S N Pr

NHCbz N

2. 2,3-Dihydroxybenzoyl chloride

O

N S

i

O 114

t

Bu

SCHEME 2.25 Corey’s total synthesis of Enterobactin, 7.

Enterobactin, 7

Synthetic Studies Toward Nonribosomal Peptides Chapter

55

O

O 1. TrtCl HO

2

OH NH2

TrtHN

2. DIC, DMAP

O

[Bu2Sn(OCH2CH2O)]2

O 115

TrtHN

NHTrt

O O

O

NHTrt 1. HCl-EtOH 2.

PhH2CO

116

Enterobactin, 7

O O OCH2Ph

O

O

NO2

3. H2, Pd/C

SCHEME 2.26 Shanzer’s total synthesis of Enterobactin, 7.

acylation with nitrophenyl ester of 2,3-bis(benzyloxy)benzoic and hydrogenolysis provided Enterobactin 7 as the product (Scheme 2.26). Another organotin based approach for the synthesis of Enterobactin was reported later by Gutierrez and coworkers in 1981 also from N-protected b-lactone 115 synthesized from serine [48].

Synthesis of Vibriobactin, 117 Vibriobactin 117 an iron chelator was isolated from Vibrio cholerae in 1983 by Griffiths and coworkers [49]. It accommodates three 2,3-dihydroxybenzoyl residues and two L-threonine derived oxazolidines. Parabactin, Agrobactin, Fluviabactin, and Vulnibactin are a few siderophore that are structurally very similar to that of Vibriobactin. Bergeron and coworkers reported the total synthesis of Vibriobactin in 1985 [50]. N-4 benzyl and N-1-Boc protected norspermidine 118 synthesized from norspermidine [51] was N-acylated with 2,3-dimethoxybenzoyl chloride to yield 119. The compound was deprotected, treated with Boc-L-Thr-OH, and again treated with TFA to provide N1,N4-bis[(L)-threonyl]-N7-(2,3dimethoxybenzoy1)norspermidine 120. Compound 120 was completely deprotected by BBr3 treatment, and it was then treated with 2,3-dihydroxybenimidate to yield Vibriobactin 117 (Scheme 2.27). The previous synthesis identified the 2-(o,m-dihydroxyphenyl)oxazoline synthesis as the most critical step, hence to facilitate the formation of the same Ishihara and coworkers [52] devised a molybdenum (VI) oxide catalyzed synthesis of Vibriobactin 117. O-xylylene protected 2,3-dihydroxy benzoic acid 121 was coupled with L-Thr-OMe to yield the compound 122 which was converted to the corresponding oxazoline methyl ester 123 using Mo (VI) catalyst (Scheme 2.28). The other fragment 124 was synthesized by Sb (lll) catalyzed coupling [53] of O-xylylene protected 2,3-dihydroxy benzoic acid and norspermidine. This fragment on coupling with the acid of 123 and xylylene deprotection yielded

56

Studies in Natural Products Chemistry

OMe BocHN

N Bn

2,3-Dimethoxybenzoyl chloride

NH2

OMe BocHN

118

OH

O N H

NH2

O HO N O

O

119

N

1. BBr3

OMe N H

O

2. Ethyl 2,3-dihydroxybenimidate 120

OH NH

HO

O

H2N

3. Boc-L-Thr-OH 4. TFA

N H

OMe

OH

1. TFA 2. H2, Pd/C

N Bn

O

OH

N

H N

N

OH

OH

O

Vibriobactin, 117

O

SCHEME 2.27 Bergeron’s total synthesis of Vibriobactin, 117

COOMe 1. NaOH 2. SOCl2

O

COOMe NH Mo(VI) = O cat

O

3. H-L-Thr-OMe, DMAP

O

121

O O

HO O

O

O

122

COOMe N 123

SCHEME 2.28 Synthesis of oxazoline, 123.

Vibriobactin 117 (Scheme 2.29). This methodology was also used for the synthesis of fluviabactin.

Synthesis of Microcystin LA, 9 Microcystin LA 9 represent a class of hepatotoxin that include over 80 closely related structures and are produced by Microcystis or Planktothrix, genus of cyanobacteria [54]. Microcystin LA consist of nonproteinogenic amino acids like D-alanine, N-methyl-dehydroalanine, iso-D-glutamic, and iso-D-b-methyl aspartic acid and (all-S, all-E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) 126. Chamberlin and co workers reported a convergent total synthesis of Microcystin LA in 1996 [55]. In their retrosynthetic analysis, they identified three fragments ca (all-S, all-E)-3-amino-9-methoxy-2,6,8-trimethyl-10phenyldeca-4,6-dienoic acid 126, 127, and 128 which were to be synthesized

Synthetic Studies Toward Nonribosomal Peptides Chapter

2

57

COOMe O Sb(OEt)3

NH2

H2N

+

O

2

O

O

H N O

O

O

O

H N

NH2

124

O O

123, Sb(OEt)3

1. Acid of 123

O

N HN

Vibriobactin, 117

2. H2, Pd/C

O HN

NH

125

SCHEME 2.29 Ishihara’s total synthesis of Vibriobactin, 117.

CO2H HN OMe

N

HN

NH

O

O

O

COOMe

O N

OMe

NH

O

O

O NH Ph

O

H N

H N O

9

O

NHBoc

HN Ph

H N

OTce H N

O CO2H

O

CO2H NHBoc + O

OTce

H N

NH2 O

Ph Boc-ADDA, 126

127

BocHN +

O COOMe

O

COOMe OMe

HN

O N

NH

O

COOMe 128

O HN HO2C

SCHEME 2.30 Chamberlin’s retrosynthetic analysis of Microcystin LA, 9

and assembled in the final stages for the completion of the synthesis (Scheme 2.30). The Boc protected nonproteinogenic amino acid Boc-ADDA [56] 126 [(allS, all-E)-3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid] was synthesized from the corresponding Weinreb amide of a known Evans aldol product 129 [57]. The Weinreb amide 129 on methyl ether formation and DIBAL reduction furnished the aldehyde 130 which was then converted to the alkene 131 employing Corey–Fuchs protocol [58]. The compound 131 was converted to its corresponding boronic acid 132 which on Suzuki coupling and hydrolysis yielded the Boc-ADDA 126 (Scheme 2.31). The synthesis of N-methyl dehydroalanine containing fragment 128 initiated with the acid catalyzed condensation of methyl glyoxylate hemiacetal 133 [59] and N-methyl benzyl carbamate followed by its conversion to phosphonylsarcosine derivative which on saponification yielded the acid 134. The acid on peptide coupling (2 cycles) with D-Ala-L-Leu-O-tBu and

58

Studies in Natural Products Chemistry

OMe O

OH O Ph

1. NaH/MeI NMe

Ph

2. DIBAL

OMe

129

1. PPh3/CBr4 H 2. BuLi, MeI

OMe 1. HBBr2

Ph

2. Citrate buffer

130

131

CO2Me 1. OMe Ph

B(OH)2

I

NHBoc

Pd(Ph3)4

CO2H

OMe Ph

NHBoc

2. Hydrolysis 126

132

SCHEME 2.31 Chamberlin’s synthesis of Boc-ADDA, 126

HO

H+

HO COOMe MeO

133

O

P(OMe)2

N CO2H Cbz 134

+

COOMe NCbz

CbzNHMe Ether

1. D-Ala-L-Leu-OtBu 2. H2, Pd/C 3. Boc-D-Glu-OMe 4. CH2O 5. TFA 6. Boc2O

1. MsCl, NEt3 2. P(OMe)3/ NaI 3. NaOH

128

SCHEME 2.32 Synthesis of fragment, 128.

Boc-D-Glu-OMe and a Wittig type olefination at the phosphonate center followed by TFA treatment yielded the fragment 128 (Scheme 2.32). N-benzyl-phenylfluorenyl-dimethyl-D-aspartate [60] was alkylated and selectively saponified to yield 135. Replacing the benzyl and phenylfluorenyl groups with Boc, coupling with L-alanine trichloroethyl ester and Boc deprotection yielded the fragment 127 (Scheme 2.33). Coupling the two fragments 127 and 128 yields a hexapeptide 136 which on Boc deprotection and another coupling with Boc-ADDA-OH 126 yields the linear precursor 137. Compound 137 on deprotection, pentafluorophenol ester (Pfp ester) mediated cyclization and saponification yielded Microcystin LR 9 (Scheme 2.34).

Synthesis of Antillatoxin, 138 Antillatoxin 138, an ichthyotoxic lipopeptide isolated from the marine cyanobacterium Lyngbya majuscula [61], it has a conjugated diene with a tBu group and the isolated terminal olefin which is an important aspect for its bioactivity. The actual structure was established after total synthesis proved pointed out the ambiguity in the proposed structure. Fumiaki Yokokawa and coworkers in 1999 reported the first total synthesis of Antillatoxin [62] and were the first to point out the ambiguity in proposed structure [63]. Antiselective boron-mediated asymmetric aldol reaction [64]

Synthetic Studies Toward Nonribosomal Peptides Chapter

CO2Me NBnPhFl

1. LHMDS, MeI

NBnPhFl HO2C

2. LiOH

CO2Me

1. H2, Pd/C 2. Ala-OTce

59

2

127

3. TFA CO2Me

135 SCHEME 2.33 Synthesis of fragment, 127.

O

COOMe N

NH

H 2N O 127 + 128

1. 126 2. Zn/AcOH

1. HATU/collidine O

2. TFA OTce

H N

H N

O O

136

O COOMe

COOMe

O N

HN OH

3. DCC/Pfp-OH

HN

NH

O

O

O

NHBoc Ph

OTce

H N

H N

O 137

O

HN

1. TFA 2. NaHCO3 3. LiOH

Microcystin LA, 9

O COOMe

SCHEME 2.34 Chamberlin’s total synthesis of Microcystin LA, 9.

between the aldehyde 139 and ester 140 followed by TES protection of the newly formed alcohol, chiral auxiliary deprotection, and reduction of the ester group yielded the alcohol 141. The alcohol on oxidation, Still–Horner olefination [65] afforded a (Z)-ester which on TFA treatment underwent spontaneous lactonization and the lactone on stereoselective phenylselenylethyl insertion yielded the substituted lactone 142. Cleavage of the lactone, allylation of the carboxylic acid followed by esterification with Alloc-S-ala-S-Me-Val-Gly-OH yielded the precursor 143. Oxidative elimination of the phenylselenyl group followed by alloc/allylester deprotection and DPPA-mediated cyclization afforded Antillatoxin 138 (Scheme 2.35). Following this description, other groups also reported the synthesis of Antillatoxin 138 [66], keeping the general protocol same and with the focus being on the development of the diene fragment.

CONCLUDING REMARKS There are over 1600 NRPs reported in the literature [67], the number of which continues to grow by the day. Armed with unique structural features, higher bioavailability, and specificity these molecules are currently contributing a

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tBu

Ph

O

+ Bn

N

2. TESOTf 3. DIBAL

SO2Mes

CHO 139

OH

OTES

O

1. LiOH 2. allyl bromide

tBu

O

N

3 .Alloc-S-ala-S-Me -val-gly-OH

O

142

3. TFA 4. PhSeCH2Li

141

140 SePh

O

N H O NHAlloc AllylO2C

O

tBu

O

SePh 143

O N H

1. NaIO4 2. Pd(PPh3)4 3. DPPA

1. TPAP 2. (EtO)2P(O)CH2CO2Me

tBu

1. (c-Hex)2BOTf

O

O

tBu

N O

H N Antillatoxin, 138 O

SCHEME 2.35 Yokokawa’s total synthesis of Antillatoxin, 138.

lot in the pharmaceutical industry, a trend that will only grow in the near future. Though most of the marketed NRPs are semisynthetically prepared using recombinant technology but chemical synthesis on the other hand provides a powerful tool to artificially prepare them in a much larger scale, also the structure and the structure activity relationship studies can only be possible with something as efficient as chemical synthesis.

ACKNOWLEDGMENTS S.S. thanks DST, India, for financial support by a grant [SR/S1/OC/0087/2012]. I.D.G. is thankful to IACS and K.C.G. is thankful to CSIR for their fellowships.

ABBREVIATIONS AIBN Ala All Alloc Arg Asp Bn Boc2O BOPCl Cbz Dab DBU DCC

2,20 -azobis(2-methylpropionitrile) alanine allyl allyloxycarbonyl arginine aspartic acid benzyl di-tert-butyl dicarbonate bis(2-oxo-3-oxazolidinyl)phosphinic chloride carbobenzyloxy 2,4-diaminobutyric acid 1,8-diazabicyclo[5.4.0]undec-7-ene N,Nʹ-dicyclohexylcarbodiimide

Synthetic Studies Toward Nonribosomal Peptides Chapter

DCE DCM Dde DEA DEPBT DHP DIBAL DIC DIPEA DMAP DMF DMSO DTT EDC FmocCl Glu Gly HATU HBTU HCTU His HOBT Ile LAH LDA Leu LHMDS Lys NaHMDS NHS Orn PAL Phe Pro PyBOP Ser TBAF TBSCl TBTH t Bu

2

1,2-dichloroethane dichloromethane bis-N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl N,N-diethylaniline 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one 3,4-dihydro-2H-pyran diisobutylaluminum hydride N,N0 -diisopropylcarbodiimide N,N-diisopropylethylamine 4-dimethylaminopyridine dimethylformamide dimethylsulfoxide dithiothreitol 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides 9-fluorenylmethyl chloroformate glutamic acid glycine 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxid hexafluorophosphate N,N,N0 ,N0 -tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate O-(6-chlorobenzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium hexafluorophosphate histidine hydroxybenzotriazole isoleucine lithium aluminum hydride lithium diisopropylamide leucine lithium bis(trimethylsilyl)amide lysine sodium bis(trimethylsilyl)amide N-hydroxysuccinimide ornithine peptide amide linker phenylalanine proline (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate serine tetrabutylammoniumfluoride tert-butyldimethylsilyl chloride trybutyltin hydride tertiary butyl

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Tce TEMPO TES Tf2O TFA TFE THF THP Thr TPAP Trp Trt Val

trichloroethyl 2,2,6,6-tetramethyl-1-piperidinyloxy triethylsilane trifluoromethanesulfonic anhydride Trifluoroacetic acid 2,2,2-trifluoroethanol tetrahydrofuran tetrahydropyranyl threonine tetrapropylammonium perruthenate tryptophan trityl valine

REFERENCES [1] H. Herzner, K.R. Braun, in: H.G. Schmalz (Ed.), Organic Synthesis Highlights IV, Wiley, Wienheim, 2000, pp. 281–288. [2] D.L. Boger, H. Cai, Angew. Chem. Int. Ed. 38 (1999) 448. [3] P. Wadhwani, S. Afonin, M. Ieronimo, J. Buerck, A.S. Ulrich, J. Org. Chem. 71 (2006) 55. [4] S. Nozaki, I. Muramatsu, Bull. Chem. Soc. Jpn. 58 (1985) 331. [5] S. Nozaki, I. Muramatsu, Bull. Chem. Soc. Jpn. 52 (1982) 2165. [6] S.A. Waksman, H.B. Woodruff, Proc. Soc. Exper. Biol. 45 (1940) 609. [7] J. Meienhofer, J. Am. Chem. Soc. 92 (1970) 3771. [8] T. Tanaka, K. Nakajima, K. Okawa, Bull. Chem. Soc. Jpn. 53 (1980) 1352. [9] B. Johnson, H. Anker, F. Meleney, Science 102 (1945) 376. [10] J. Lee, J.H. Griffin, J. Org. Chem. 61 (1996) 3983. [11] M. North, G. Pattenden, Tetrahedron 46 (1990) 8267. [12] S.K. Sharma, A.D. Wu, N. Chandramouli, C. Fotsch, G. Kardash, K.W. Bair, J. Peptide Res. 53 (1999) 501. [13] H.Y. Lam, Y. Zhang, H. Liu, J. Xu, C.T.T. Wong, C. Xu, X. Li, J. Am. Chem. Soc. 135 (2013) 6272. [14] (a) C. Milne, A. Powell, J. Jim, M. Al Nakeeb, C.P. Smith, J. Micklefield, J. Am. Chem. Soc. 128 (2006) 11250. (b) K.C. Woo, K. Jones, Tetrahedron Lett. 32 (1991) 6949. (c) C. Herdeis, H.P. Hubmann, Tetrahedron Asymmetry 3 (1992) 1213. (d) C. Herdeis, H. P. Hubmann, H. Lotter, Tetrahedron Asymmetry 5 (1994) 351. [15] X. Bu, X. Wu, G. Xie, Z. Guo, Org. Lett. 4 (2002) 2893. [16] X. Bu, G. Xie, C.W. Law, Z. Guo, Tetrahedron Lett. 43 (2002) 2419. [17] (a) J. O’Sullivan, J.E. McCullough, A.A. Tymiak, D.R. Kirsch, W.H. Trejo, P.A. Principe, J. Antibiot. 41 (1988) 1740. (b) D.P. Bonner, J. O’Sullivan, S.K. Tanaka, J.M. Clark, R. R. Whitney, J. Antibiot. 41 (1988) 1745. [18] J. Shoji, H. Hinoo, K. Matsumoto, T. Hattori, T. Yoshida, S. Matsuura, E. Kondo, J.Antibiot. 41 (1988) 713. [19] F. Von Nussbaum, S. Anlauf, J. Benet-Buchholz, D. Haebich, J. Koebberling, L. Musza, J. Telser, H. Ruebsamen-Waigmann, N.A. Brunner, Angew. Chem. Int. Ed. 46 (2007) 2039. [20] A. Guzman-Martinez, R. Lamer, M.S. VanNieuwenhze, J. Am. Chem. Soc. 129 (2007) 6017.

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Chapter 3

The Antiinflammatory Potential of Flavonoids: Mechanistic Aspects M.D. Catarino*, O. Talhi†, A. Rabahi{, A.M.S. Silva† and S.M. Cardoso*,† *

CERNAS, School of Agriculture, Polytechnic Institute of Coimbra Bencanta, Coimbra, Portugal University of Aveiro, Aveiro, Portugal { Centre de Recherche Scientifique et Technique en Analyses Physico-Chimiques CRAPC, Bou-Ismail, Tipaza, Algeria †

Chapter Outline Chemistry of Flavonoids and Occurrence in Nature The Inflammatory Process The Antiinflammatory Potential of Flavonoids Inflammatory Biochemical Mediators Inflammatory Enzymes

65 70 73 75 79

Transcription Factors and Signaling Pathways Synthetic Strategies Toward Enhanced Antiinflammatory Flavonoids Concluding Remarks Acknowledgments References

87

90 95 95 96

CHEMISTRY OF FLAVONOIDS AND OCCURRENCE IN NATURE Flavonoids are a large group of phenolic compounds having a benzopyranone or a benzopyran structure, which are widely distributed in the plant kingdom [1]. These compounds are important secondary metabolites of plants synthesized by the phenylpropanoid pathway and can be found in the vacuoles of flowers, leaves, stems, and roots [2]. They serve vital roles in plants physiology, including regulation of cell functions and signaling, ultraviolet light protection, and reproduction and survival as well. Moreover, they can act either as attractants or repellants and/or cytotoxic agents, drawing pollinators and symbionts, while deterring herbivores and pathogenic microorganisms [3]. Chemically, flavonoids are low molecular weight polyphenolic substances based on a 15-carbon skeleton, consisting of two aryl rings (A- and B-rings) connected by a heterocyclic pyran ring (C-ring) and forming the C6–C3–C6 flavan nucleus depicted in Fig. 3.1 [4,5]. Studies in Natural Products Chemistry, Vol. 48. http://dx.doi.org/10.1016/B978-0-444-63602-7.00003-5 © 2016 Elsevier B.V. All rights reserved.

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5′ 4′

6′ 8

B

O 2

3′

7 A

C

5

4

6

2′ 3

FIG. 3.1 Schematic structure of the flavan nucleus, the basic flavonoid skeleton.

O O

O

Flavonoids

Isoflavonoids

O

O Chalcones

O Aurones

Neoflavonoids

O OH

O Auronols

FIG. 3.2 Major classes of flavonoids.

The position of the aryl substituent divides the flavonoid family into different classes, namely flavonoids (2-arylflavan), isoflavonoids (3-arylflavan), and neoflavonoids (4-arylflavan) (Fig. 3.2). In addition, flavonoids may frequently occur as flavan-opened chain compounds to give chalcones or as the 5-membered C-ring derivatives aurones and auronols (Fig. 3.2). Usually, variations in the level of oxidation and substitution patterns of the C-ring involve a ketone function (pyranone or furanone) and/or a 3-hydroxyl group on the flavan nucleus, giving rise to diverse classes including flavones, flavon-3-ols, flavan-3-ols, flavanones, dihydroflavon-3-ols, and anthocyanins (Fig. 3.3). The majority of the naturally occurring flavonoids share various substituents on their A- and B-rings, such as hydroxyl and methoxyl groups [6]. Flavones and flavon-3-ols, which differ on the absence/presence of a 3-hydroxyl group in the pyranone ring, are the most abundant categories of flavonoids in nature. Flavanones and dihydroflavon-3-ols are saturated derivatives (lack of unsaturated C2]C3 function on the pyranone ring) of flavones and flavon-3-ols, respectively. Flavanols have a skeleton similar to that of flavanones, with exception on the 4-keto group that is absent in the former ones. Instead, anthocyanins are distinguished from the others due to their flavylium (2-arylchromenylium) ion skeleton [6,7].

The Antiinflammatory Potential of Flavonoids Chapter

O

O

67

O

O

OH

OH O Flavones

3

O Flavon-3-ols

Flavan-3-ol O+

O OH

O Flavanones

O Flavanon-3-ols

Anthocyanins

FIG. 3.3 Major flavonoid categories.

Naturally, flavonoids may occur as free aglycones (basic flavonoid structure), or modified by O-glycosylation or C-glycosylation [8]. Sugar residues appearing in flavonoids are usually glucose, rhamnose, glucorhamnose, galactose, and arabinose [6] that are frequently acylated with aliphatic or aromatic acid groups. Most common C- and/or O-glycosylation sites occur at C-3, C-5, C-7, C-30 , and C-40 [9]. Flavonoids are deeply integrated in our daily diet since they can be abundantly found in vegetables, fruits, nuts, seeds, and foods of plant origin, such as tea, wine, olive oil, etc. [10]. In fact, they play an important role in some foods sensorial aspects, such as the red color and the astringency sensation, respectively, assigned to anthocyanins and tannins that are present in red wines [11,12]. It is estimated that the dietary intake of flavonoids is around 1–2 g/day. Table 3.1 shows the average content of flavonoids of selected foods (Fig. 3.4). Epidemiological and medical data indicate that dietary flavonoids play key roles in prevention and controlling of chronic diseases such as cancer, diabetes, neurodegenerative, cardiovascular, and inflammatory conditions [9,13,14]. However, correlations between the intake of flavonoids and their in vivo effects are still under debate, as their bioavailability and accumulation in body tissues are very complex topics and not fully understood. The absorption of food phenolics is primarily dictated by their chemical structure, as globally determined by several features (eg, the degree of glycosylation, acylation, conjugation with other phenolics, molecular size, degree of polymerization, and hydrophobicity), but is also dependent on other intrinsic and extrinsic factors [15]. Reinforced attention is recently being paid to the role of gut microbial flora, which is thought to have the ability to transform some flavonoids into a series of absorbable low-molecular-weight phenolic metabolites [16], thus improving their bioavailability and consequently, their bioactivity.

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TABLE 3.1 Average Flavonoid Content of Selected Foods

Food Source

Category

Flavonoid

Average Content (mg/100 g wet edible portion)

Flavon-3-ols

Kaempferol

0.13

Myricetin

0.15

Quercetin

0.45

Flavones

Luteolin

0.19

Flavanones

Hesperetin

27.3

Naringenin

15.3

Myricetin

0.5

Quercetin

1.14

Flavones

Luteolin

1.90

Flavanones

Eriodictyol

21.4

Hesperetin

27.9

Naringenin

0.55

Kaempferol

0.14

Quercetin

4.01

Flavones

Luteolin

0.12

Flavanols

()-Epicatechin

7.53

()-Epigallocatechin

0.26

()-Epigallocatechin 3-gallate

0.19

(+)-Catechin

1.30

Anthocyanidins

Cyanidin

1.57

Flavon-3-ols

Kaempferol

0.50

Quercetin

1.11

Flavanones

Naringenin

0.26

Flavanols

()-Epicatechin

0.42

()-Epicatechin 3-gallate

0.15

()-Epigallocatechin

0.78

(+)-Catechin

3.11

Fruits Orange (Citrus sinensis)

Lemon (Citrus limon)

Apple (with skin) (Malus domestica)

Strawberries (Fragaria X ananassa)

Flavon-3-ols

Flavon-3-ols

Continued

The Antiinflammatory Potential of Flavonoids Chapter

3

69

TABLE 3.1 Average Flavonoid Content of Selected Foods—Cont’d

Food Source

Category

Flavonoid

Average Content (mg/100 g wet edible portion)

Anthocyanidins

Cyanidin

1.68

Delphinidin

0.31

Pelargonidin

24.9

Isorhamnetin

5.01

Kaempferol

0.65

Quercetin

20.3

Isorhamnetin

23.6

Kaempferol

46.8

Quercetin

22.6

Kaempferol

7.84

Quercetin

3.26

Flavones

Luteolin

0.80

Flavon-3-ols

Kaempferol

1.49

Myricetin

14.8

Quercetin

0.28

Apigenin

215.5

Luteolin

1.09

Myricetin

2.10

Quercetin

7.30

Apigenin

2.57

Luteolin

1.00

Apigenin

0.55

Luteolin

2.00

Naringenin

24.9

Vegetables Onions (Allium cepa)

Kale (Brassica oleracea)

Broccoli (Brassica oleracea var. italica)

Flavon-3-ols

Flavon-3-ols

Flavon-3-ols

Spices Parsley (Petroselinum crispum)

Flavones

Oregano (unspecified)

Flavon-3-ols

Flavones

Rosemary (Rosmarinus officinalis)

Flavones

Flavanones

Adapted from S. Bhagwat, D.B. Haytowitz, J.M. Holden, U.S. Dep. Argiculture (2011) 1.

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R3

R4

R4

5

R R2

R2

O

O

R6 R3

1

Flavanones R1

O

Flavones

R

Chrysin Apigenin Luteolin Diosmetin

Hesperetin R1 = R2 = R3= OH, R4 = OCH3 R1 = R2 = OH, R3 = R4 = R5 = R6 = H Eriodictyol R1 = R2 = R3 = R4 = OH R1 = R2 = R5 = OH, R3 = R4 = R6 = H 1 2 4 5 3 6 Naringenin R1 = R2 = R4 = OH, R3 = H R = R = R = R = OH, R = R = H R1 = R2 = R4 = OH, R5 = OCH3, R3 = R6 = H

O

HO

O

Flavon-3-ols Galangin Kaempferol Myricetin Quercetin Isorhamnetin Fisetin

R1 = R2 = R3 = OH, R4 = R5 = R6 = H R1 = R2 = R3 = R5 = OH, R4 = R6 = H R1 = R2 = R3 = R4 = R5 = R6 = OH R1 = R2 = R3 = R4 = R5 = OH, R6 = H R1 = R2 = R3 = R5= OH, R6= H, R4= OCH3 R2 = R3 = R4 = R5 = OH, R1 = R6 = H

OH

O

Isoflavones

OH Genistein

R1 R2

OH OH HO

O

OH HO HO

O

OH

OH OH (+)-Catechin

OH (−)-Epicatechin

HO

OH

O

OH

Anthocyanins Cyanidin

OH

R3

OH

OH

Flavan-3-ol

O+

R1 = R2 = OH, R3 = H

Delphinidin R1 = R2 = R3 = OH OH Pelargonidin R2 = OH, R1 = R3 = H OH

OH O HO

O

OH

OH

OH

O

OH

OH

OH (−)-Epigallocatechin

(−)-Epigallocatechin

OH 3-gallate

FIG. 3.4 Structures of the main natural flavonoids existing in our daily food.

THE INFLAMMATORY PROCESS The inflammatory process is a complex and coordinated immunological response of the organism to harmful stimuli, including pathogen agents, internal injuries, or irritants [17]. From this process, two distinct yet interconnected defense mechanisms can be considered, ie, an unspecific response (innate immunity) and a highly specific one (adaptive immunity) [18]. The first

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mechanism is present in all individuals and is activated by a large number of stimuli. It is also quickly triggered and it involves physical, chemical, and biological barriers, specialized cells and soluble molecules. In turn, the activation of adaptive immune system causes the increment of microbial-specific leukocytes, a process that is highly effective and specific, but that takes days to fully develop [19]. In general, immune recognition relies on three different mechanisms, ie, recognition of “microbial non-self,” recognition of “missing self” and recognition of “induced or altered self” [20,21]. The basis of “microbial non-self” recognition lies in the ability of the host to recognize unique microbial antigens and/or epitopes that are exogenous proteins. This strategy allows the innate immune system to discriminate between “infectious non-self” and “non-infectious self” [22]. The second strategy, recognition of “missing self”, relies on the detection of diverse markers produced by normal cells [23]. The third strategy (recognition of induced self ) is based on the detection of markers of abnormal self. When stressed (eg, infected or transformed), cells upregulate the expression of endogenous ligands that tag them for recognition and elimination by the immune system [21]. With exception of the “missing self” recognition, which is mediated through the major histocompatibility complex (MHC), ie, transmembrane molecules designated as self-recognizing molecules [24], the other two mechanisms of recognition are carried out by pattern recognition receptors (PRRs) that are expressed in the surface of both circulating and residing cells (found in the blood circulation and extracellular matrix, respectively) allowing them to detect either exogenous or endogenous antigens. When activated, these PRRs will trigger a series of signaling cascades that culminate in changes on the vasculature of the damaged tissue, ending up in an incremented blood flow, elevated cellular metabolism, vasodilatation, release of soluble mediators, extravasation of fluids and cellular influx, which overall cause the visible hallmarks of acute inflammation, ie, calor (warmth), rubor (redness), tumor (swelling), and dolor (pain) [25,26]. While a controlled inflammatory process is self-limited and usually beneficial for the host, an inflammation out of control often results in chronic diseases. Examples of chronic inflammatory diseases include arthritis, autoimmune disorders, degenerative joint diseases, rheumatism, atherosclerosis, diabetes, and even cancer [27]. Therefore two stages of inflammatory responses can be differentiated, ie, acute and chronic inflammation. The acute phase begins immediately upon injury and rapidly turns severe, mainly involving neutrophils and some macrophages, which represent the first line of defense. Notwithstanding, this event lasts for a short period of time, ending up in the resolution of the inflammatory event. When this process lasts longer and/or gets deregulated, neutrophils are replaced by macrophages and other cells, including the T cells (a subclass of leukocytes that plays a central role in the regulation of the immune system, in particular in the adaptive immunity),

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establishing a chronic inflammation that leads to the progressive degeneration of the tissue, causing inflammatory pathologic conditions [28]. All the events of acute and/or chronic inflammation are mediated and controlled by chemical mediators, including histamine, serotonin, cytokines, chemokines, eicosanoids, and adhesion molecules among many others (Fig. 3.5) [29]. From these mediators, tumor necrosis factor-a (TNF-a) plays one of the most important roles in the inflammatory signaling cascades. This is an extremely pleiotropic cytokine due to the ubiquity of its receptors and its ability to activate a number of signal transduction pathways that ultimately affect the expression of a broad range of genes [30,31]. Nuclear factor-kB (NF-kB) is such one of the pivotal transcription factor that is activated by TNF-a. Its activation enhances the induction of

Stimulus Propagation of inflammation

Cell membrane

Receptor

MKK IKK

ERK

JNK

Cytokines and NO• Prostanoids other mediators

p38

IkBα

IkBα NF-kB

AP-1

AA

L-Arg

iNOS

NF-kB

NF-kB

AP-1

LOX

COX-2

Nucleus

FIG. 3.5 Schematic representation of the inflammatory signaling cascade. The proinflammatory stimulus (cytokines or microbial antigens) bind to the cell receptor triggering the nuclear factorkB (NF-kB) or mitogen-activated protein kinase (MAPK) signaling cascades. The former begins with the activation of the inhibitor of kB kinase (IKK) that ultimately phosphorylates inhibitor of kB-a (IkBa) and marks it to be degraded. Upon degradation of IkBa, the NF-kB is released and able to translocate to the nucleus where it binds to specific DNA regions, enhancing the transcription of several proinflammatory genes encoding for other biochemical mediators including cytokines and the enzymes inducible nitric oxide synthase (iNOS), lipoxygenase (LOX), and cyclooxygenase-2 (COX-2). The function of iNOS is to produce nitric oxide (NO) from L-arginine (L-Arg), while the other two enzymes convert the arachidonic acid (AA) into several prostanoids including the prostaglandins, thromboxanes, and leukotrienes. All these mediators will then carry their functions contributing for the propagation of inflammation.

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proinflammatory genes encoding for the synthesis of newly cytokines (including TNF-a itself ), chemokines, cell adhesion molecules, and proinflammatory enzymes including cyclooxygenase (COX)-1 and COX-2, 5-lipoxygenase 5-(LOX), and inducible nitric oxide synthase (iNOS) [32–34]. Notably, with exception for COX-1 (that despite its involvement in inflammation, is mainly constitutive), COX-2, 5-LOX, and iNOS are three pivotal enzymes that control the biosynthesis of crucial proinflammatory mediators [prostanoids, leukotrienes, and nitric oxide (NO ), respectively], which in turn are responsible for different events such as vasoconstriction or dilation, vasopermeability, coagulation, pain, fever, cytotoxicity for foreign agents, etc. [35–37]. In fact, well-known drugs in the market (eg, aspirin, ibuprofen or naproxen) exert their antiinflammatory, antipyretic, and analgesic functions through inhibition of COX-1 and COX-2, with major affinity for the latter [38]. Besides activation of NF-kB signaling pathway, which is central in inflammatory process, TNF-a also interacts with other signaling cascades such as activator protein-1 (AP-1), the mitogen-activated protein kinases (MAPKs) and the apoptotic pathways, contributing for the regulation of proliferation, gene expression, differentiation, cell survival, and cell death [39]. Notwithstanding, it should be highlighted that besides TNF-a, many other important cytokines carry important functions during the inflammatory immune response. Indeed, this process encloses a cocktail of more than 100 different types of cytokines [40]. From these, interleukin (IL)-1b, IL-6, interferon (IFN)-g are examples of other well-known proinflammatory cytokines involved in the inflammatory process, while IL-10, IL-2 and transforming growth factor (TGF)-b are important antiinflammatory cytokines responsible for the immune tolerance [41,42]. 

THE ANTIINFLAMMATORY POTENTIAL OF FLAVONOIDS Several harmful side effects are associated to the long-term usage of antiinflammatory and immunomodulatory drugs used in the conventional (allopathic) system of medicine. Therefore, there is a need to search for alternative drugs with low or no toxicity [43]. In this field, flavonoids are emerging as potential candidates, since over the last years they have been reported to target proinflammatory mediators such as the biochemical messengers (eg, cytokines, chemokines, adhesion molecules, prostanoids, and NO ), enzymes (eg, COX-2, LOX, and iNOS), and signaling pathways and transcription factors [29,44,45]. An example that demonstrates the potential of these compounds is the so-called French Paradox, which relies on the epidemiological observation that French people have a relatively low incidence of coronary heart disease (CHD), despite their diet rich in saturated fats. Presumably, this fact is associated to their higher rates of wine intake [46] and in turn, to the wine’s high content in polyphenols, particularly in flavonoids [47]. Among them, 

74

Studies in Natural Products Chemistry

malvidin, mostly in form of 3-O-b-glucoside, is the main flavonoid present in red wine, and also responsible for its color [48]. This compound has been reported to dramatically decrease IL-6, TNF-a, macrophage inflammatory protein, IL-1 and IL-8 secretion in CD23-stimulated human monocyte-derived macrophages, through downregulation of their gene expression, as well as the iNOS-mediated NO production through blockage of iNOS mRNA transcription [25]. Moreover the treatment of LPS-stimulated RAW 264.7 with malvidin has been demonstrated to block the activation and binding of NF-kB to the correspondent DNA binding site, as well as to hamper ERK1/2, p38, and JNK activities, which are the three main protagonists in the MAPKs signaling pathway [49]. Atherosclerosis, one of the most relevant CHD, is characterized by an inflammatory condition resultant from the accumulation of foam cells (ie, fat-laden macrophages) on artery walls, causing their thickness [50,51]. Cellular adhesion molecules including E-selectin, intra, and vascular cell adhesion molecules (ICAM-1 and VCAM-1, respectively) are central players in this events since they mediate the adhesion of leucocytes to the endothelial cells and are upregulated upon cytokine stimulation, in an inflammatory environment [51]. It is important to remark that both cyanidin and quercetin, which are also abundant in red wine, have been demonstrated to inhibit monocyte adhesion to TNF-a-induced human aortic endothelial cells (HAEC), thus effectively attenuating VACM-1 expression. In this model, quercetin was also able to significantly attenuate ICAM-1 and E-selectin expressions [52]. The three central groups of proinflammatory enzymes, ie, iNOS, COX-2, and LOX, have also been demonstrated to be potently inhibited by quercetin (both at mRNA and protein expression level), and consequently decrease their respective down-products NO , prostaglandins, and leukotrienes [53,54]. Other chemical mediators including TNF-a, IL-1b, IL-6, and MCP-1 have also been reported to be downregulated in several quercetin-treated stimulated macrophages such as RAW 264.7 or PMBC macrophages [25]. Additionally, both quercetin and cyanidin notably reduced NF-kB expression and binding to DNA, as well as the ERK1/2 and p38-MAPK expression, consequently impairing NF-kB and MAPK signaling pathways on stimulated HAEC [52,55]. Several studies have also shown that flavonoids have the ability to enhance endogenous antiinflammatory chemical mediators (eg, IL-10) and diverse antioxidant and detoxification defensive enzymes [25,29,56,57]. It is important, however, to retain that different flavonoids have distinct structural features, and consequently act differently toward the inflammatory mediators. Up to present, reported studies regarding the structure–antiinflammatory relationships of flavonoids have been mostly focus on the key enzymes/pathways of the process, ie, COX-2, LOX, iNOS, and signaling pathways NF-kB and MAPK. According to that, this manuscript revises the relevant available information on this theme. 



The Antiinflammatory Potential of Flavonoids Chapter

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75

Inflammatory Biochemical Mediators As pointed before, cytokines are one of the most important groups of chemical mediators in inflammation and thus are considered central therapeutic targets in inflammatory-associated diseases. Despite the limited number of studies focusing the structure-modulatory activities of flavonoids on these mediators (Table 3.2), some clues could already been taken. In particular, as demonstrated by Comalada et al. [58] in LPS-stimulated bone marrowderived mice macrophages (BMDM), 30 ,40 -dihydroxylated flavonoids (quercetin and luteolin) were the most potent ones regarding the inhibitory ability toward TNF-a. At a lower degree, this ability was also observed for flavonoids with a 40 -hydroxylation (genistein, kaempferol, apigenin, and hesperetin), independently of the presence/absence of the unsaturated C2]C3 function, of the 3-hydroxylation or of an iso position of the B-ring. In turn, the absence of hydroxyl groups in the B-ring virtually abolished this inhibitory capacity. Nonetheless, the effects associated with some of these features remain unclear. Indeed, in opposition to the work of Comalada et al. [58], Herath and coworkers [59] suggested that the lack of unsaturated C2]C3 function in eriodictyol might contribute to the suppression of its inhibitory ability toward TNF-a when compared with other flavonoids, as evaluated in LPS-stimulated mouse macrophage J774.1 cells. A similar conclusion was taken by Lo´pez-Posadas [60] when observing the weak inhibitory effects of the flavanones hesperetin and naringenin in parallel to the potent effects of apigenin and diosmetin. In the same study, the authors also observed that the inhibitory capacity of isoflavonoids toward TNF-a was lower than that of the majority of flavonoids. The 30 ,40 -dihydroxylation in the B-ring of flavonoids has also been suggested as the most favorable requirement for inhibition of other proinflammatory cytokines, namely the macrophage colony-stimulating factor (M-CSF), IL-6, and monocyte chemotactic protein-1 (MCP-1). In this regard, Comalada et al. [58] demonstrated that amongst nine flavonoids, quercetin and luteolin were the most effective inhibitors of M-CSF, as evaluated in BMDM cells isolated from 6-week-old Balb/c mice. The comparative results of flavones (apigenin, chrysin, luteolin and diosmetin) and flavonols (quercetin and kaempferol) with isoflavones (genistein and daidzein) and flavanones (hesperetin) also allowed the authors to conclude that inhibition toward M-CSF was negatively affected either by the iso position of the B-ring or by the absence of the unsaturated C2]C3 function. In addition, During et al. [61] described that luteolin and 30 ,40 dihydroxyflavone (DHF) exhibited improved inhibitory effects against IL-6 and MCP-1 in IL-1b-stimulated Caco-2 cells when compared to chrysin, apigenin, and quercetin. Interestingly, apigenin also demonstrated strong inhibitory effects, which suggests that, even though the optimal conditions were observed for the flavonoids with the catechol group (30 ,40 -dihydroxyphenyl

TABLE 3.2 Overview of the Importance of Certain Structural Features for Interactions Between Flavonoids and Inflammatory Biochemical Mediators Target

Influence of the Flavonoid Structural Features

Best 0

Molecule

Model

Test Conditions

Compounds

C2]C3

4-keto

3 -OH

40 -OH

5-OH

7-OH

3-OH

3-B-ring

Other

Reference

TNF-a

LPS-stimulated

Treatment with

Quercetin,



n.d.

+

+

n.d.

n.d.





IA dependent on

[58]

BMDM cells

nine flavonoids

luteolin

the number of OH

(25–100 mM) LPS-stimulated

Treatment with

Flavonols and

J774.1 cells

12 flavonoids

flavones

+

n.d.





n.d.

n.d.





Flavones may

[59]

depend of the Nr of

(0.3–30 mM)

OH, but not flavonols

Concanavalin

Treatment with

Quercetin,

A-stimulated rat

14 flavonoids

fisetin,

splenocytes

(50 mM)

luteolin,

+

n.d.

+

+

+

n.d.





3-Gly # IA

[60]

+

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.



Independent on the

[58]

apigenin, diosmetin, genistein M-CSF

M-CSF-stimulated

Treatment with

Quercetin,

BMDM cells

nine flavonoids

luteolin

Nr of OH

(25–100 mM) n.d.

n.d.

+

+







n.d.

30 -OMe "

MCP-1 and

IL-1b-stimulated

Treatment with

Luteolin,

IL-6

Caco-2 cells

10 flavonoids

dimethoxy-

inflammation;

(50 mM)

chrysin

5-, 7-OMe " IA;

[61]

3-OMe abolish IA IL-4 and

Purified basophils

Treatment with

Fisetin,

IL-13

stimulated with

18 flavonoids

luteolin,

anti-IgE-antibody

(1–30 mM)

apigenin

n.d.

n.d.

+

+

+

+

n.d.

n.d.

[62]

E-selectin,

TNF-a-stimulated

Treatment with

Apigenin,

ICAM-1,

HAEC

eight flavonoids

chrysin,

(5–50 mM)

galangin,

VCAM-1

+

+





+

+



n.d.

+

+

+

+

+

+



n.d.

[63]

quercetin kaempferol ICAM-1

TNF-a-stimulated

Treatment with

Kaempferol,

A549

nine flavonoids

chrysin,

(1–30 mM)

apigenin,

50 -OH # inhibitory

[64]

activity

luteolin Histamine

n.d.

+

+

+

+

n.d.

3-, 7-Gly # IA;

Treatment with

Diosmetin,

stimulated RBL-

52 flavonoids

fisetin,

6-OH " activity;

luteolin

more than two OH

2H3 cells

+



DNP-BSA-

[65]

in B-ring abolish IA; 3-OMe # IA 



50 -OH does not

DNP-BSA-

Treatment with

Luteolin,

stimulated RBL-

22 flavonoids

apigenin,

influence; 6-OH "

2H3 cells

(20 mM)

diosmetin,

IA; 7-Gly # IA

n.d.

n.d.

+

+

+

n.d.

[66]

fisetin, quercetin

A549, adenocarcinomic human alveolar basal epitelial cells; BMDM, bone marrow-derived mice macrophages; Caco-2, colorectal adenocarcinoma cells; DNP-BSA, 2,4-dinitrophenylatedbovine serum albumin; Gly, glycoside; HAEC, human aortic endothelial cells; IA, inhibitory activity; ICAM-1, intercellular adhesion molecule-1; IgE, immunoglobulin E; IL, interleukin; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; OH, hydroxyl group; OMe, methoxyl group; RBL-2H3, basophilic leukemia cells; TNF-a, tumor necrosis factor-a; VCAM-1, vascular adhesion molecule-1; (+), positive influence; (), negative influence; (), irrelevant influence; n.d., not determined.

78

Studies in Natural Products Chemistry

moiety), 40 -hydroxylation is sufficient for good activity. In contrast, quercetin (structurally related to luteolin with an extra 3-OH group) exhibited less effective inhibition toward IL-6 and MCP-1, thus suggesting that the 3-OH group produces unfavorable conditions for this activity. In turn, 5,7-dimethoxylation of chrysin (ie, 5,7-dimethylchrysin) potentiated the inhibition of IL-6 and MCP-1 in IL-1b-stimulated Caco-2 cells [61]. Remarkably, the authors also observed that methoxylation of the hydroxyl groups of quercetin and of luteolin (but not those of apigenin), caused a significant increase of the MCP-1 secretion, thus suggesting that the presence of methoxyl groups on C-30 (but not on C-40 ), promotes proinflammatory properties on the flavones. In addition, the 3-methoxyl substituent on the C-ring of quercetin obliterated either anti- or proinflammatory effects. The cytokines IL-4 and IL-13 are produced by mastocytes, basophiles, and eosinophils during Th2 response (ie, a series of events that lead to the humoral immunity), which is known for counteracting the proinflammatory events occurring during inflammation. However, the dysregulation of this response leads to overproduction of these two cytokines, a fact that is known to be the basis of allergic reactions and autoimmune diseases including allergic rhinitis, atopic dermatitis, and asthma [67,68]. Hirano et al. [62] stimulated basophils with anti-IgE antibodies in order to create an in vitro model of allergic reaction to test a set of 18 flavonoids against the overproduction of IL-4 and IL-13. Among these, fisetin, luteolin, and apigenin revealed the most potent inhibitory activities, allowing the authors to conclude that the essential structural requirements for optimal inhibitory activity toward IL-4 and IL-13 were hydroxylation at C-7 and C-40 , in the presence of an additional OH group in either position C-3 (fisetin) or C-5 (luteolin and apigenin). The authors also concluded that the presence/ absence of a 3-hydroxyl group did not interfere with the inhibitory effects. Cell adhesion molecules, ie, cell surface proteins responsible for the binding of cells to the extracellular matrix or to other cells, are overexpressed during inflammation so that more leukocytes can be recruited for the injured region. This event is particularly relevant during atherosclerosis development [69], and hence, the influence of dietary flavonoids on cell adhesion molecules have been often evaluated in cellular models of this disease [14,70,71]. Lotito and Frei [63], when evaluating the effectiveness of various flavonoids toward three cell adhesion molecules on TNF-a-stimulated primary human aortic endothelial (HAECn), observed that the expression of E-selectin, ICAM-1 and VCAM-1 were only inhibited by hydroxyflavones such as apigenin and chrysin, and flavonols such as galangin, kaempferol, and quercetin, whereas the flavanone naringenin and the flavanol epicatechin were ineffectual. Based on their results, the authors could conclude that the unsaturated C2]C3 function was a striking feature for the inhibitory activity toward cell adhesion molecules and that this activity was favored in the

The Antiinflammatory Potential of Flavonoids Chapter

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79

presence of the 4-keto group and the 5- and 7-hydroxyl groups. These requisites were also supported by the work of Chen and coworkers [64], when comparing the inhibitory effects of three flavonols (kaempferol, quercetin, and myricetin) and six flavones [flavone, chrysin, apigenin, luteolin, baicalein (6-hydroxychrysin), and baicalin (7-O-glucuronide baicalein)] on the TNFa-induced ICAM-1 expression on adenocarcinomic human alveolar basal epithelial cells (A549). The authors also suggested that other positive factors include the OH group at C-40 or at C-30 and C-40 while the opposite was proposed for hydroxylation at C-3 or at C-50 . Histamine is another important inflammatory mediator that is released by mast cells upon a stimulus of an antigen, in an event known as degranulation, triggering several systemic effects, such as vasodilation, mucous secretion, nerve stimulation, and smooth muscle contraction, causing the classical allergy symptoms [72]. Mastuda and colleagues [65] showed that diosmetin, fisetin, and luteolin were the most effective flavonoids (amongst 52) in inhibiting the degranulation process of antigen-IgE-stimulated rat basophilic leukemia (RBL-2H3) cells, as assessed by the b-hexosaminidase release. Based on the comparison of the results, the authors concluded that unsaturated C2]C3 function of flavones and flavonols was a striking structural requirement for this activity. Also, this ability was potentiated by the increment of OH groups in one of the positions C-30 , -40 , -5, -6, and -7. Moreover, better results were observed for flavones than for flavonols, suggesting that 3-hydroxylation hampers this inhibitory activity. Other features have been shown to have deleterious effects on flavonoids performance including the 3-methylation of flavonols, 5-hydroxylation, and glycosylation in the positions C-7 and C-3 [65]. This data corroborates previous studies reported in Cheong et al. [66] and Kawasaki et al. [73].

Inflammatory Enzymes A variety of enzymes, including COXs, LOXs, and iNOS, work together in order to initiate and perpetuate the inflammatory process. These enzymes are involved in the production of arachidonic acid, prostaglandins, leukotrienes, and NO , which are crucial mediators of inflammation, in signal transduction and cell activation processes [59]. According to literature, the inhibition of these enzymes is probably one of the most important targets of flavonoids determining their antiinflammatory properties [74]. Relevant works focusing the structural requirements of flavonoids for inhibitory activity of these enzymes are described below and data is summarized in Table 3.3 (biological studies) and Table 3.4 (docking studies). COX-2 catalyzes the conversion of arachidonic acid into prostaglandins and thromboxanes. This is one of the most important proinflammatory enzymes and is also an excellent target for antiinflammatory drugs, as it is only expressed on cells undergoing an inflammatory process. 

TABLE 3.3 Overview of the Importance of Certain Structural Features for Interactions Between Flavonoids and Inflammatory Enzymes and Transcription Factors Target Molecule COX-2

Influence of the Flavonoid Structural Features

Test Model

Conditions

Best Compounds

C2]C3

4-keto

3 -OH

40 -OH

5-OH

7-OH

3-OH

3-B-ring

Other

Reference

Quercetin



+

+

+

?

+

n.d.

n.d.

More than two OH

[75]

TGF-a-

Treatment

stimulated

with

DLD-1 cells

12 flavonoids

0

in B-ring abolish IA

(0–500 mM) 



LPS-stimulated

Treatment

rat peritoneal

with

in B-ring abolish IA;

macrophages

39 flavonoids

glycosidic forms # IA

Flavones

+

+

+

+

n.d.

n.d.

More than two OH

[76]

(0.4–40 mM)

LOX

dimethoxychrysin

Caco-2 cells

10 flavonoids

7-OMe " IA; 3-OMe

(50 mM)

abolish IA

Treatment with

with A23187

24 flavonoids

and AA

(0.15–40 mM)

Luteolin, quercetin

+

+

n.d.

n.d.

30 -OMe

Luteolin,

with

neutrophils

n.d.



Treatment

Human isolated

n.d.



IL-1bstimulated

[61]

" inflammation; 5-,

+

n.d.

+

+







n.d.

Planarity influence

[77]

the IA

treatment Monitoring of

Screening of

Luteolin, fisetin,

RR and SB

18 flavonoids

quercetin

+

+











n.d.

Individual OHs do not influence IA,

15-LOXs

but the presence of

activity in vitro

a catechol either on A- or B-ring " IA

[78]

iNOS

LPS-stimulated

Treatment

Quercetin,

BMDM cells

with nine

luteolin

+

n.d.





n.d.

n.d.





IA dependent on the

[58]

Nr of OH

flavonoids (25–100 mM) 



Glycosylation # IA;

LPS-stimulated

Treatment

Diosmetin,

mouse

with

apigenin, tetra-O-

more than two OH

peritoneal

73 flavonoids

methylluteolin,

in B-ring # IA; 3-,

hexa-O-

5-, or 40 - OMe " IA

macrophages

+

n.d.

+

+

+

n.d.

[79]

methylmyricetin LPS-stimulated

Treatment

RAW 264.7

with

Luteolin

n.d.

n.d.

+

+

+

+

n.d.

n.d.

OMe at 30 - or

[80]

40 -position " IA

25 flavonoids (2.5–50 mM) Concanavalin

Treatment

Quercetin, fisetin,

A-stimulated rat

with

luteolin, apigenin,

splenocytes

14 flavonoids

diosmetin,

(50 mM)

genistein

NF-kB

LPS-stimulated

Treatment

Quercetin,

(through

IEC18

with nine

hesperetin,

flavonoids

genistein, apigenin

IkBa)

+

n.d.



+

+

n.d.





3-Gly # IA;

[60]

40 -OMe # IA;



n.d.

n.d.

+

+

n.d.

?



+

n.d.





n.d.

n.d.

n.d.



[81]

(50 mM) LPS-stimulated

Treatment

Quercetin,

BMDM

with nine

luteolin

IA dependent on the

[58]

Nr of OH

flavonoids (25–100 mM)

Continued

TABLE 3.3 Overview of the Importance of Certain Structural Features for Interactions Between Flavonoids and Inflammatory Enzymes and Transcription Factors—Cont’d Target

Influence of the Flavonoid Structural Features

Test 0

Molecule

Model

Conditions

Best Compounds

C2]C3

4-keto

3 -OH

40 -OH

5-OH

7-OH

3-OH

3-B-ring

Other

Reference

p38-MAPK

Monitoring of

Screening of

Flavonols

+

+

+

+



+

+

n.d.

7-OMe " IA; 3-OMe

[82]

enzyme activity

42 flavonoids

or -Gly not

in vitro

(0.1–100 mM)

tolerated; 20 - and 50 -OH " IA

LPS-stimulated

Treatment

Quercetin,

IEC18

with nine

hesperetin,

flavonoids

genistein, apigenin



n.d.

n.d.

+

+

n.d.

+

n.d.

[81]

(50 mM)

A23187, calcium ionophore; AA, arachidonic acid; BMDM, bone marrow-derived mice macrophages; Caco-2, colorectal adenocarcinoma cells; COX-2, cyclooxygenase-2; Gly, glycoside; IA, inhibitory activity; IkBa, inhibitor of kB-a; iNOS, inducible nitric oxide synthase; IEC18, intestinal epitelial cells; LOX, lipoxygenase; LPS, lipopolysaccharide; NF-kB, nuclear factor-kB; OH, hydroxyl group; OMe, methoxyl group; p38-MAPK, class of mitogen activated proteins; RAW264.7, mouse leukaemic monocyte macrophage cells; RR, rabit reticulocytes; SB, soybean; TGF-a, transforming growth factor-a; (+), positive influence; (), negative influence; (), irrelevant influence; (?), inconclusive; n.d., not determined.

TABLE 3.4 Overview of the Interactions Between Flavonoids and the Specific Sites at the Target Molecules Based on Docking Studies Target Molecule COX-2

Model

Conditions

Best Compounds

Observations

Reference

Docking studies

Analysis of 11 flavonoids interactions with COX-2

Baicalein, quercetin

Higher G-scores for the 2 selected compounds;

[83]

Quercetin: HB of 30 -, 40 -OH with Tyr385 and Ser-530 Baicalein: HB of catechol-like group with Tyr-385 and Ser-530 LOX

Analysis of baicalein and quercetin interactions with 12- and 15-LOX

Quercetin

Baicalin: Biding of 5-, 6-OH with iron atom; HB of 7-OH with Glu-356

iNOS

Analysis of 15 quercetin derivatives interactions

Quercetin-3-O-acetate, 6,8-dichloroquercetin-3-O-acetate, 6-bromo-3-O-acetate

Formation of HB of 4-keto with Gln-365 and Trp-366, common to all the compounds; better energy binding values obtained for the selected compounds

[85]

JNK1

Analysis of quercetin interactions

–

HB formation of 5-OH, 30 -OH, and 4-keto groups with Met-111, Ile-32, and Glu-109, respectively; HI between the B-ring and Ile-32, Asn-114 and Val-158

[86]

[84]

Quercetin: Biding of 3-OH with iron atom; HB of C-ring with Arg-402 and Gln-406; more stable linkage

Continued

TABLE 3.4 Overview of the Interactions Between Flavonoids and the Specific Sites at the Target Molecules Based on Docking Studies—Cont’d Target Molecule

JNK3

Model

Conditions

Best Compounds

Observations

Reference

Analysis of rhamnetin interactions

–

HB formation of 4-keto and 5-OH group with Met-111; HB-nw formation of 30 -, 40 -OH groups with Lys-55 and Asp-169; HI between A-ring and Val-158 and Leu-168, and B-rings and Ile-32 and Val-40.

[87]

Analysis of 3,6-dihydroxyflavone interactions

–

HB formation of 6-OH and 4-keto with Met-111; HI between B-ring and Val-40 and Leu-168; HI between A-ring and Ile-32; Leu-110 and Val-158

[88]

Analysis of 42 flavonoids interactions

Flavones and flavonols

HB formation of 4-keto group with Met149;

[82]

Flavones: HB formation of 7-OH and 40 -OH with and Gln-75 and Asn-152, respectively; Flavonols: HB formation of 30 -, 40 -OH groups with Gln-75; HB formation of 7-OH with Asn-152

p38MAPK

Analysis of rhamnetin interactions

–

HB-nw formation of 30 -, 40 -OH with Lys-53 and Asp-168; HB formation of 5-OH with Met-109; HI between B-ring and Val-38 and Phe-169; HI between A-ring and Ala-157 and Leu-167

[87]

Analysis of 42 flavonoids interactions

Flavonols

Quercetin: HB formation of 30 -, 40 -OH with Lys-53 and Asp-168; HB formation of 5-, 3-OH, and 4-keto with Gly-110, His-107, and Met-109, respectively;

[82]

Myricetin: HB-nw formation of 40 -, 50 -OH with Thr-106, Met-109, and His-107; HB formation of 3-OH and 4-keto with Asp168 and Lys-53, respectively; HB-nw of 7-OH with Gly-36 and Ser-32 Ala, alamine; Arg, arginine; Asn, asparagine; COX-2, cyclooxygenase-2; Gln, glutamine; Glu, isoleucine; HB, hydrogen bond; HB-nw, hydrogen bond network; HI, hydrophobic interactions; His, histidine; JNK, c-Jun N-terminal kinases; iNOS, inducible nitric oxide synthase; Leu, leucine; LOX, lipoxygenase; Lys, lysine; Met, methionine; OH, hydroxyl group; p38-MAPK, class of mitogen activated proteins; Phe, phenylalanine; Ser, serine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.

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Mutoh et al. [75] investigated 12 flavonoids in TGFa-induced DLD-1 human colon adenocarcinoma cells for their suppressive action on COX-2. The results demonstrated that in general, flavonoids with a 4-keto group could efficiently inhibit COX-2. Instead, catechin and epicatechin, which are devoid of this group, only exhibited weak activities. Moreover, the most potent inhibitors of COX-2 (quercetin and rhamnetin) had in common the presence of 30 ,40 -dihydroxyl (catechol) moiety in the B-ring and a low electron density in the 7-oxygen group in the A-ring, allowing to conclude that these features may enhance the inhibition toward this enzyme. In turn, as observed for myricetin and epigallocatechin, the presence of three OH groups in B-ring completely abolished the inhibitory effects over COX-2, regardless the presence of the 4-keto group. Concordant results were reported by Takano-Ishikawa et al. [76] when testing the effects of 39 distinct flavonoids on the prostaglandin production in LPS-stimulated peritoneal macrophages. These authors reported that catechins and anthocyanidins, which lack the 4-keto functionality were inefficient, indicating once more that this is a pivotal structural requirement for strong COX-2 inhibitory activity. In addition, this ability was potentiated by the unsaturated C2]C3 function, such as in chrysin, apigenin, and baicalin with respect to naringenin, eriodictyol, and hesperetin. Moreover, it was observed that genistein (with 5,7-dihydroxyl substituents) exhibited higher inhibitory effects than daidzein and genistin (with monohydroxylations in C-5 and C-7 positions, respectively), thus supporting the importance of these features, as described before. In contrast, the comparison of experimental data from quercetin with those of its glycoside derivatives, rutin and quercitrin, allowed to conclude that 3-glycosylation abolished COX-2 inhibitory ability. In agreement with the results of Takano-Ishikawa et al. [76], During and Larondelle [61] also observed that the catechol in B-ring of flavones favored their inhibitory activity toward COX-related PGE2 in IL-1b-stimulated Caco-2 cells. Additionally the authors reported that O-methylation of both 5- and 7-hydroxyl groups on the A-ring (5,7-dimethylchrysin vs. chrysin) could potentiate this activity while the opposite tendency was observed by 3-glycosylation of flavonols (rutin or quercitin vs. quercetin). Further elucidation of flavonoids-COX-2 interactions were achieved by docking studies. As reported, the COX-2 active site is divided into three regions: (i) a hydrophobic pocket defined by Tyr-385, Trp-387, Phe-518, Ala-201, Tyr-248, and Leu-352; (ii) a hydrophilic residues arranged to form a hydrogen bond network, namely Arg-120, Glu-524, Tyr-355; and (iii) a side pocket defined by several conserved residues including His-90 and the nonconserved residues Arg-513 and Val-523 [89]. In their study, Mello et al. [83] concluded that amongst 11 flavonoids, including flavones, flavonols, flavanones and isoflavonoids, baicalein, and quercetin were the ones with highest G-scores (a value that indicates the binding strength). The authors also concluded that the slightly-decreased G-score value of quercetin

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regarding to that of bacailein was probably due to their differences on the binding pattern. While the B-ring of quercetin was oriented to the hydrophobic pocket forming hydrogen bonds between catechol groups and the Tyr-385 and Ser-530 residues, baicalein instead bound to these same amino acids through its catechol-like function in the A-ring. iNOS, ie, the enzyme that catalyzes the oxidative deamination of L-arginine to produce the potent proinflammatory mediator NO , and 5-LOX, ie, the enzyme that catalyzes the production of leukotrienes from arachidonic acid, are also pivotal in the inflammation process [36,90]. In general, structure–activity relationship in vitro studies performed in biological models indicated that the striking structural features to inhibit these enzymes are similar to those of COX-2, ie, the catechol group in ring-B, the unsaturated C2]C3 function, the 4-keto group, and the hydroxylations at C-5 and -7 [58,77,79,91]. The effects of the 3-OH group remain, however, inconclusive. While the presence of this group has been clearly shown to hamper the inhibitory activity of quercetin toward 5-LOX, as compared to luteolin [77], this has not been supported by the studies of Va´zquez-Agell et al. [92] whom have described that this group is important for a stable binding of quercetin to this enzyme. Still, some of the consensus requirements were also supported in silico through docking studies. In particular, in distinct isoforms of LOX (12- and 15-), Mascayano et al. [84] confirmed the importance of the catechol group in the B-ring or a catechol-like group in the A-ring, as well as the hydroxylation at the positions C-5 and -7, since these features are involved in important interactions and hydrogen bond formations with LOX active site. It was further confirmed in silico that the 4-keto group of different flavonoids always connect with iNOS with the same hydrogen bonds [85]. 

Transcription Factors and Signaling Pathways As previously mentioned, NF-kB is the central transcription factor triggered during inflammatory events. In normal conditions, an inhibitor of kB (ie, IkBa) protein keeps the subunits (p50 and p65) of this transcription factor sequestered in an inactive state in the cytoplasm. When stimulated by TNF-a, a series of complex signaling cascades triggers the activation of IkB kinase (IKK) that in turn phosphorylates IkBa resulting in the dissociation of this inhibitor from NF-kB [32]. When released, NF-kB translocates to the nucleus where it binds to DNA specific binding sites and enhancer regions, inducing transcription of several proinflammatory genes that encode for several biochemical mediators and enzymes [93]. Despite the numerous studies reporting the positive effects of flavonoids against the activation of NF-kB, very few have focused on the structural aspects underlying its activity. In their study, Lo´pez-Posadas [81] tested nine flavonoids in LPSstimulated intestinal epithelial cells, in order to evaluate their ability to inhibit the IkBa phosphorylation and the consequent NF-kB activation.

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The comparison of the obtained results obtained for apigenin vs. chrysin and genistein vs. daidzein allowed the authors to conclude that the presence of 40 OH and 5-OH were clearly the most important structural features for inhibition of IkBa phosphorylation. Moreover, by comparing genistein vs. apigenin and diosmetin vs. luteolin performances, it was suggested that both the B-ring position (C-2 vs. C-3) and the 40 -methoxylation have irrelevant interferences on this biological activity. Finally, the comparison of the interactions between IkBa phosphorylation with diosmetin and that of hesperetin allowed the authors to conclude that unsaturated C2]C3 function might impair this activity. Yet, the contribution regarding the 3-OH group was inconclusive, since it appeared to favor the inhibitory ability when comparing the effects of quercetin vs. luteolin, while the opposite was observed for the results from kaempferol vs. apigenin [81]. The importance of the 5-OH group for NF-kB inhibition was further corroborated by Shin et al. [94], when measuring the NF-kB-dependent transcriptional activity on TNF-a-induced HCT116 cells and further analyzing the three-dimensional quantitative relationships (3D-QSAR) of 30 flavonoid derivatives. Overall, the authors have concluded that the most ideal structure of flavonoids for optimal NF-kB inhibition should contain an electronegative substituent at the C-5 position of the A-ring, along with a bulky substituent at the meta-position of the B-ring, as well as a hydrophobic substituent at the meta-position of the B-ring. MAP kinases are central components of signal transduction pathways that lead to the enhanced expression of important mediators of inflammation. Nuclear translocation of activated MAPKs assist the modulation of gene transcription via the induction and/or transactivation of transcription factors [39]. From these, c-Jun N-terminal kinases (JNK) are responsible for the phosphorylation and activation processes of transcription factors and of other cellular factors that upregulate the expression of many genes encoding cytokines (TNF-a, IL-2), growth factors, cell surface receptors, cell adhesion molecules (E-selectin) among others [43]. Several reports have previously demonstrated good inhibitory capacity of quercetin toward c-Jun N-terminal kinase 1 (JNK1, ie, one of the JNKs isoforms). In this regard, based on docking studies (Table 3.4), Lee et al. [86] established that the 5-OH and the 4-keto groups of quercetin could form hydrogen bonds with the backbone of Met-111 and Glu-109, respectively, while a third hydrogen bond was formed by the 30 -OH group with the Ile32 residue. Hydrophobic interactions between the B-ring and Ile-32, Asn114, and Val-158 were also observed [86]. Based on the binding model of quercetin with JNK1, the authors also proposed that rhamnetin could be a potent inhibitor of this enzyme [86]. This theory was recently supported by Jnawali et al. [87], where the authors have observed the same interactions formed by the 4-keto group and 5-OH of rhamnetin with the Met-111, as well as the same hydrophobic interactions

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of A- and B-rings to the protein. Additionally, a hydrogen bond network was formed between the 30 - and 40 -OH groups and the side chains of Lys-55 and Asp-169. This binding model of rhamnetin was remarkably similar to that obtained for 3,6-DHF and JNK1, which only differ in the hydrogen bond formations with Lys-55 and Asp-169, since the OH groups of the B-ring are absent in 3,6-DHF [88]. Previous results demonstrated a similar binding behavior of flavonols with JNK3 [82]. In fact, the authors reported high-inhibitory activity against JNK3 for both flavonols and flavones, hence highlighting the importance of the 4-keto group for this biological activity. Indeed, the authors remarked a hydrogen bond formed in between this group and the Met-149 of the JNK3 backbone, regardless opposite orientations of flavonols and flavones when interacting with the enzyme (ie, interaction with flavones are established in between 7-OH with Gln-75 and 30 ,40 -OH with Asn-152, while interaction of 7-OH and 30 ,40 -OH of flavonols are established with interact with Asn-152 and Gln-75, respectively). p38 MAPK, another member of the MAPKs pathway, mediates cellular functions, including cell migration, cell survival, and cell death and is also believed to be a critical regulator of the inflammatory response, since encoding genes for TNF-a, IL-1b, IL-6, IL-8, and COX-2 are regulated by this pathway [43]. Based on a screening with a set of 42 flavonoids, Goettert et al. [82] have concluded that the necessary requirements for a flavonoid to interact with p38-MAPK are the presence of the unsaturated C2]C3 function, the 4-keto, and the 3-OH groups. Substituents at the C-5 and C-6 positions do not seem to have any influence in the flavonoids activity, while the substitution of the C-3 position for other groups besides OH (eg, O-glycosides or OMe) are not tolerated. Moreover, the hydroxyl groups at the positions C-20 , -30 , -40 , and -50 , and a 7-methoxy group are features that favor the inhibitory effectiveness [81,82]. For more detailed clarifications, the interaction of the flavonol rhamnetin with p38-MAPK has been simulated through docking studies, revealing a binding network between the 30 - and 40 -hydroxylation groups and the Lys53 and Asp-168 of the p38-MAPK, while the 5-OH formed a hydrogen bond with Met-109. Additionally, the B-ring formed hydrophobic interactions with Val-38 and Phe-169, while those of the A-ring were established with Ala-157 and Leu-167 [87]. These interactions are similar to that observed between quercetin and p38a-MAPK, since the catechol group of this flavonol also established hydrogen bonds with Lys-53 and Asp-168 side chains. However, in this case, the amino backbone of Met-109 was linked to the 4-keto group, while the 5-OH group connected to Gly-110. The 3-OH was shown to be important for this interaction, since it linked to the carbonyl function of His-107. The fact that the 7-methoxyl of rhamnetin was oriented to a hydrophobic region of the p38a-MAPK suggested that this substituent could display a more efficient interaction [82].

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Interestingly, the flavonol myricetin binds to the p38a-MAPK ATP binding site with an opposite orientation to that of rhamnetin or quercetin. While the 40 -, 50 -OH groups formed a hydrogen bond network with Thr-106, Met109, and His-107, the 3-OH and the 4-keto groups formed hydrogen bonds with Asp-168 and amino group of Lys-53 side chains, respectively. Further hydrogen binding interactions can be establish between the 7-OH and the carbonyl functions of Gly-36 and Ser-32 as well as to the amino function of Ser-32 [82].

SYNTHETIC STRATEGIES TOWARD ENHANCED ANTIINFLAMMATORY FLAVONOIDS Nature has always supplied us with the beneficial flavonoids suitable to promote our health maintenance, but due to their relatively low bioavailability, tracking different and efficient methods for their synthesis and structural configuration is still requested. Hence, naturally extracted flavonoids exhibiting powerful antiinflammatory activity can be selected as lead structures and starting point for the chemists in order to elaborate new flavonoid derivatives fitting the required biological activity. Looking to their simple structure, the high throughout biological screening of natural low-molecular-weight flavonoids becomes more attractive in biology and medicinal chemistry areas [95,96]. Updates on the chemistry of flavonoid was recently a subject of interest; Verma et al. [96] have covered most of the synthetic methods applied for the biologically active flavones and their structural modifications with diverse functional groups, like the thoroughly utilized hydroxyl, methoxyl, glycosyl, and prenyl groups with variable number and at different positions on the basic skeleton. The authors have notably outlined the commonly used procedures for the preparation of the flavone basic skeleton, known as the Baker–Venkataraman rearrangement. This reports the conversion of an ortho-hydroxyacetophenone phenolic ester 1 into a b-diketone 2, via an intramolecular base-catalyzed Claisen condensation. The b-diketones 2 are indeed good precursors for the synthesis of flavones 3 through their facile intramolecular cyclization; the most outstanding methodology was very well used in our laboratory involving the application of molecular iodine in dimethylsulfoxide (DMSO) (Scheme 3.1) [95,96]. Within the context of organic synthetic strategies, a priceless literature data have been published showing convenient procedures using various synthons. Chalcones 6, for instance, are easily obtained by Claisen–Schmidt condensation of ortho-hydroxyacetophenones 4 and benzaldehydes 5 under basic media. Very recently, Hatnapure et al. [97] applied the Algar–Flynn– Oyamada cyclisation of chalcones 6 to generate a new series of novel methoxylated flavonols 7 with biological interests. Accordingly, quercetin or kaempferol can systematically be synthesized through a similar organic pathway as depicted in Scheme 3.2. The same research group aimed the

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SCHEME 3.1 The Baker–Venkataraman route toward flavone synthesis.

SCHEME 3.2 Claisen–Schmidt condensation to form chalcones 6 and Algar–Flynn–Oyamada cyclisation in to the antiinflammatory flavonols 7.

construction of novel piperazine–chromone analogues 8 and 9 for further biological purposes. The undertaken synthetic route is composed of several modification steps applied to the acetophenone precursors, as shown in Scheme 3.3. Most of the piperazine-modified chromones 8 and 9 have displayed remarkable antiinflammatory activity [98]. It is noteworthy that chemistry allows a flexible modification of the flavone basic structure with variable degree of substituents such as hydroxyl (or methoxyl) groups at different positions and such important tool permits a rapid targeting of more effective molecular models for medicine. Following the previously mentioned synthetic strategies, Silva et al. [99] have elaborated a series of flavonoids capable to inhibit COX-1 and COX-2 enzymes, as well as the production of the cytokines and a chemokine, in a complex matrix implicated in the systemic inflammatory process in the blood. Other structure–activity relationship data reveal that among a series of synthesized chlorinated flavones, 8-chloro-30 ,40 ,5,7-tetrahydroxyflavone has evidenced the highest antiinflammatory ability [100].

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SCHEME 3.3 Synthesis of piperazine–chromone analogues as TNF-a and IL-6 inhibitors.

A quite large number of chemical modifications can be applied on the flavonoid scaffolds and/or their synthetic precursors, such as the O- and C-glycosylation and C-prenylation [95], in order to modulate the whole structure for specific biological activation. Apart from the well-established reactivity of flavonoids (reduction, oxidation, aromatic substitution, and rearrangement reactions) [96], the combination of other nitrogen (or oxygen) containing heterocycles such as pyrazoles, imidazoles, pyridines, etc. with flavonoids is rather a promising strategy in medicinal chemistry which brought fruitful results in terms of enhanced biological activity. In this way, Chavan et al. [101] have designed a novel pyrazole–flavone dyad 12 which is synthesized from an ortho-hydroxyacetophenone-pyrazole 10. This key starting material 10 is subjected to the Claisen–Schmidt condensation, to produce chalcones 11 which were readily converted to the expected flavones 12 using the I2/DMSO reagent system (Scheme 3.4). The structures 12 were finely characterized by 2D NMR (including COSY, HSQC and HMBC experiments) and their biological assessment have demonstrated that they possess potent inhibitory effects against both COX-1 and COX-2, being some of them highly selective against COX-2.

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etc.

SCHEME 3.4 Synthesis of pyrazole–flavone dyads as potent COX-1 and COX-2 inhibitors.

The structural modulation of flavone with potential pharmacophores has deserved more attention in the medicinal chemistry field. Based on the well-known multiplicity of biological actions associated with aminoflavones, Moorkoth et al. [102] proposed the attachment of the imidazolidinone moiety to the flavone basic structure at position 6. The synthesis of 19 compromises five steps as represented in Scheme 3.5, starting by the Friedel–Craft acylation of paracetamol 13 to afford the acetophenone precursor 14, which is brought to the reaction sequence of Claisen–Schmidt condensation to chalcones 15 and subsequent cyclodehydrogenation to flavone 16 using refluxing the I2/DMSO reagent system. In fact, paracetamol 13 was strategically used because of its protected amino group; the acetyl protecting group was easily removed by acid hydrolysis to generate the 6-aminoflavone 17 and this was directly attached to the highly substituted phenolic aza-lactone branches 18, thus yielding the imidazolidinone-flavone compounds 19. The in vivo anticancer screening studies carried out on EAC-induced mice have shown prominent activity while the antiinflammatory activity evaluation showed that most of the synthesized compounds 19 possess significant levels of activity. The well-documented antiinflammatory agent “chrysin” has been chemically modified by adding a pyridin-4-yl moiety to position 8, in order to afford 5,7-dihydroxy-8-(pyridin-4-yl)flavone 22. Compound 22 has been prepared by the Suzuki–Miyaura C–C cross-coupling reaction of 8-iodo-5,7dimethoxyflavone 20 with an appropriate pyridin-4-yl-boronic acid 21 followed by demethoxylation by treatment with BBr3 (Scheme 3.6) [103,104]. Among various other combinations of 8-substituted heteroaryl-chrysin derivatives (such as furan-3-yl, thiophen-3-yl, among other) synthesized by similar procedure, 8-(pyridin-4-yl)chrysin 22 was the candidate possessing the highest antiinflammatory ability. With help of western blot and reverse transcriptase-polymerase chain reaction analysis, it was convincely explained that the chrysin-pyridine combination is the most compatible to the antiinflammatory activity by adopting simultaneous and different mechanisms, namely, the inhibition of PGE2 production (via COX-2 downregulation and

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SCHEME 3.5 Synthesis of pyrazole–flavone dyads 19 with antiinflammatory effects.

SCHEME 3.6 Enhanced antiinflammatory effects by chemical modification of chrysin bearing a pyridine moiety.

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COX-2 inhibition), NO production (via downregulation of iNOS expression), TNF-a, and IL-6 production. Interestingly, it was discovered how a very simple alteration of the pyridine radical position from 8-(pyridin-4-yl)chrysin 22 to 8-(pyridin-3-yl)chrysin can eventually cause less than 50% inhibition of PGE2 and NO production [104]. 

CONCLUDING REMARKS Notably, the antiinflammatory capacities of flavonoids are exerted through the modulation of distinct biochemical mediators, enzymes, and signaling pathways involved in the inflammatory process. Despite the structural requirements for their effectiveness slightly vary with the target molecule, several common features can be pointed. These include the hydroxylations at the 30 -, 40 -, 5-, 7-positions, the unsaturated C2]C3 function, and 4-keto group. At present, the influence of the hydroxylation at the position C-3 is rather doubtful since many studies report contradictory effects. We have seen from the present literature survey that the flavone basic skeleton is synthetically accessed via simple procedures which relay on the Claisen–Schmidt condensation to chalcones or the Baker–Venkataraman route to b-diketones and their consecutive cyclisation pathways to the pyran-4-one heterocycle. From a biological point of view, the structural combination of flavone with various nitrogen heterocycles constitutes an interesting strategy toward enhanced antiinflammatory activity of the parent compound. The variation in the substituent on the ring A and/or B of the flavone can also have additive or synergetic effects affecting the required biological activity. The quest for a best structural configuration is not a difficult task for a chemist, thereby; determining new antiinflammatory candidates is foreseen.

ACKNOWLEDGMENTS The authors gratefully acknowledge the Portuguese Foundation for Science and Technology (FCT), European Union, QREN, FEDER, COMPETE, for funding CERNAS (Project PEst-OE/AGR/UI0681/2011) and the Organic Chemistry Research Unit (QOPNA) (project FCT UID/QUI/00062/2013; FCOMP-01-0124-FEDER-037296). We would also like to thank the General Directorate for Scientific Research and Technological Development-DGRSDT of Algeria for the financial support. O.T. also thanks the project New Strategies Applied to Neuropathological Disorders (CENTRO-07-ST24-FEDER-002034), co-funded by QREN, “Mais Centro-Programa Operacional Regional do Centro” and EU, FEDER for his post-doctoral position. Conflict of Interest: The authors confirm that this article content has no conflicts of interest.

ABBREVIATIONS 3D-QSAR A549

three-dimensional quantitative relationships Alveolar basal epithelial cells

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BMDM CHD COX DHF DMSO HAEC IFN-g IKK IL iNOS JNK LOX MAPKs MCP-1 M-CSF MHC NF-kB NO PRRs RBL-2H3 TGF-b TNF-a 

bone marrow-derived mice macrophages coronary heart disease cyclooxygenase dihydroxyflavone dimethylsulfoxide human aortic endothelial cells interferon-g inhibitor of kB kinase interleukin inducible nitric oxide synthase c-Jun N-terminal kinases lipoxygenase mitogen-activated protein kinases monocyte chemotactic protein-1 macrophage colony-stimulating factor major histocompatibility complex nuclear factor-kB nitric oxide pattern recognition receptors rat basophilic leukemia transforming growth factor-b tumor necrosis factor-a

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Chapter 4

Comparative Studies in Relation to the Structure and Biochemical Properties of the Active Compounds in the Volatile and Nonvolatile Fractions of Turmeric (C. longa) and Ginger (Z. officinale) J.N. Jacob Organomed Corporation, Coventry, RI, United States

Chapter Outline Introduction Chemistry of Turmeric and Ginger Chemistry of Turmeric Diphenylheptanoid Structures Related to Curcumin Phenyl Propene (Cinnamic Acid-Type) Derivatives Diphenylpentanoid Structures Turmeric Oil (TO) Chemistry Chemistry of Ginger Ginger oleoresin compounds Volatile Compounds from Ginger Structural Similarities of Turmeric and Ginger Compounds Gene Expression in the Rhizomes of Ginger and Turmeric

102 103 104 105 105 105 106 108 110 110 118 118

Biological Properties of Turmeric (Curcumin) and Ginger in Relation to Structure Biological properties of turmeric Antiplatelet activity of curcumin Antimicrobial activity of curcumin Anticancer property of curcumin Biological Properties of Ginger Anticancer Property: Ginger Antiinflammation, curcumin, and ginger Anticancer and Antiinflammatory properties of turmeric oil

Studies in Natural Products Chemistry, Vol. 48. http://dx.doi.org/10.1016/B978-0-444-63602-7.00004-7 © 2016 Elsevier B.V. All rights reserved.

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102 Studies in Natural Products Chemistry Antiemetic effects of ginger, receptor interactions 126 Anti-Alzheimer’s properties for turmeric and ginger 127 Diabetes-turmeric and ginger 127

Side-Chain Structure and Anticancer Activity Conclusion References

128 130 131

INTRODUCTION A wide variety of compounds present in spices that are extensively used as food seasoning agents possess potent antioxidant, antiinflammatory, antimutagenic, and cancer preventive properties. Turmeric and ginger have been used as spices in Indian and south Asian cooking and were attributed a number of medicinal properties in Ayurvedic medicines for treating digestive disorders and several ailments. Turmeric (Curcuma longa) and ginger (Zingiber officinale) are rhizomes and they originate from the same family, Zingiberaceae. These two major natural products are known to have a variety of medicinal and biological properties. The rhizomes of turmeric and ginger contain pungent vanillyl ketones and have been reported to possess strong antiinflammatory activity [1]. This review is a closer look at the structural parameters of the active compounds present in these two natural products that lead to common biological properties. The volatile oils and the nonvolatile oleoresins are the chief sources for these active ingredients. This review examines the structural features and the molecular mechanisms underlying biological effects of the active compounds in these natural products in terms of their effects on intracellular signaling cascades. The common structural aspects also shed light into the common biological properties and the structural similarities and differences of these compounds are discussed in relation to the biological and medicinal properties. It is not an exhaustive literature review for the two natural products, but I have tried to include the most relevant literature to compare the chemical and biological properties of these two related but different products. Turmeric belongs to the genus Curcuma which consists of several species with underground stem, rhizome. The name derives from terra merita or meritorious earth and the plant reaches a height of 1 m. The rhizomes are yellow or orange in color and the primary rhizome is an extension of the stem and long cylindrical multibranched secondary rhizomes grow from the primary rhizome [1]. Turmeric is widely used as a food colorant. Turmeric powder is a major ingredient in curry powder and paste. It has been used in Ayurveda and Chinese medicine for skin disorders and wound healing [2]. It has been used for centuries in Ayurvedic medicine and Unani systems of medicine for treating inflammatory disorders, including hepatitis and arthritis. Other medicinal properties include blood purifier, a remedy for liver, stomach and dental problems, contraception, bactericide, germicide, beauty, deodorizing agent, disinfectant, a cure for leach and insect bites, cough, cold, sneeze, and various types of skin diseases. Based on these traditional applications,

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4 103

dietary supplements containing turmeric rhizome and extracts are also being used in western countries to prevent and treat arthritis [3,4]. Turmeric and its constituent curcumin have several healing properties. Recently it was reported that curcumin acts as an antidepressant by interaction with dopamine receptors with D1 and D2 receptors as well as elevates the brain dopamine level [5]. Curcumin reduces blood glucose levels through antihyperglycemic and insulin sensitizer effects. Multidrug bacterial pathogens are a major threat in the treatment of bacterial infections. C. longa extracts along with a few other natural products extracts exhibit high antibacterial activity [6]. Ginger, Z. officinale Roscoe (Zingiberaceae), is a native of Asia and is grown in many tropical countries. It is a slender perennial 60–100 cm in height. Like turmeric, ginger has a thick underground stem called rhizome [7]. The active components of ginger are the volatile oils and the pungent phenol compounds, gingerols and shogaols. Ground dried ginger is used in a variety of foods, especially in bakery products. Ginger is used in various traditional systems of medicine against different ailments such as arthritis, rheumatism, infectious diseases, and vomiting [8]. Ginger is used in prevention of chemotherapy induce nausea and vomiting [9]. Ginger has been mentioned in traditional medicine in China and India and has been used for more than 25 centuries [10]. Several studies showed that ginger is effective against different types of cancer cells including lung, ovarian, colon, breast, skin, prostate, and pancreatic cancer cells [11].

CHEMISTRY OF TURMERIC AND GINGER Curcuma is an important genus in the family Zingiberaceae. About 20 Curcuma species have been studied of which the most investigated species are C. longa, C. xanthorrhiza, and C. zdvaria [4]. Mainly three classes of compounds are found in these species. 1. Diphenyl alkanoids (nonvolatile). 2. Phenylpropene (cinnamic acid type) derivatives (nonvolatile). 3. Turmeric oil (TO) containing terpenoids (volatile). Ginger (Z. officinale) also belongs to the family Zingiberaceae. In addition to Z. officinale, the genus Z. boehmer contain other minor species such as Z. cassumunar (grown in Thailand), Z. mioga (grown in Japan), and Z. zerumbet (grown in Asian tropics) [12]. Ginger also contains three classes of compounds. 1. Diphenyl heptanoids (nonvolatile). 2. Gingerols and shogaols (nonvolatile phenyl alkane derivatives). 3. Ginger oil (GO) containing terpenoids (volatile). The odor and much of the flavor of turmeric and ginger are determined by the volatile oil.

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CHEMISTRY OF TURMERIC The diphenylalkanoids can be further classified as diphenylheptanoids (1) (curcuminoids) and diphenylpentanoids depending on the chain length connecting the phenyl groups. The presence of about 235 compounds have been identified in turmeric of which 109 sesquiterpenes [13]. Over hundred diarylheptanoids have been discovered [2]. Among the diarylheptanoids three are curcuminoids, the major pharmacologically active ingredients of turmeric. The curcuminoid-type compounds are common in the family, Zingiberaceae. The diphenylheptane compounds in Curcuma species differ in the substitution pattern on the phenyl groups with phenolic and methoxy groups and in the heptane chain by functional groups such as carbonyl and hydroxyl groups, unsaturation of the chain. Curcumin (Curcumin I), demethoxycurcumin (Curcumin II), and bis-demethoxycurcumin (Curcumin III) (Fig. 4.1) are the most widely distributed hepta-diterpenoids in the genus Curcuma and they are responsible for the yellow color of the rhizome [14]. Oxygenation is normally on C-30 and C-40 ring carbons except in one case 0 5 -methoxycurcumin (5) [14a]. Cyclized diphenylheptanoid to form a pyrone unit on the chain, cyclocurcumin (6) (Fig. 4.2), has been found in C. longa [15,16]. This compound has the same molecular formula as curcumin and is an intramolecular Michael addition product of the enol oxygen to the enone group.

OH

O

R1

R2

R

R 1

OH

HO Curcumin I R1 = R2 = OCH3 (2) Curcumin II R1 = OCH3, R2 = H (3) Curcumin III R1 = R2 = H (4)

FIG. 4.1 Diphenylheptanoid structure and natural curcumin analogs.

O OH

O

R1

R2 CH3O

HO

O

OH

R1 = R2 = OCH3 OCH3 5

FIG. 4.2 50 -Methoxy curcumin and cyclocurcumin.

OCH3

OH

HO 6

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Diphenylheptanoid Structures Related to Curcumin Several diarylheptanoids had been purified from Curcuma comosa extract, and their pharmacological effects were reported having antiinflammatory and estrogenic like function [17,18]. Two specific molecules, (4E,6E)1,7-diphenylhepta-4,6-dien-3-ol (7) and (6E)-1,7-diphenylhept-6-en-3-ol (8) (Fig. 4.3), were reported as the major compounds, which comprised 16.02% and 30.66% of the crude extract, respectively [19].

Phenyl Propene (Cinnamic Acid-Type) Derivatives Both mono and dimeric phenyl propene derivatives are reported from Curcuma. Caffeic acid, cinnamic acid, p-methoxy cinnamic acid, ethyl p-methoxy cinnamic acid, cinnamaldehyde, and Calebin A are present in this genus [20–22]. Calebin A (9) and its analog (10) (Fig. 4.4) are reported as novel curcuminoids isolated from turmeric (C. longa) [19,20]. Calebin A is an ester with some similarity to curcuminoid structure.

Diphenylpentanoid Structures There are two diphenylpentanoid compounds (Fig. 4.5) from C. longa, (1E,4E)1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one and (1E,4E)-1-(4hydroxy-3-methoxyphenyl)-5-(4-hydroxyphenyl)-1,4-pentadien-3-one [14]. OH

OH

7

8 (6E)-1,7-Diphenyl-6-en-3-ol

(4E,6E)-1,7-Diphenylhepta-4,6-dien-3-ol

FIG. 4.3 Diphenylheptanoid structures. OH

O

OH

O

O O HO

OCH3

(10)

HO

Calebin A (9) O

O

OCH3

FIG. 4.4 Cinnamic acid-type structures. O R

HO

R′

11

OH

(1E,4E)-1,5-bis(4-Hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one (R=R′ = OMe) (1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(4-hydroxyphenyl)-1,4-pentadien-3-one R = H, R′ = OMe)

FIG. 4.5 Diphenylpentanoid structures.

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Turmeric Oil (TO) Chemistry Turmeric Oil (TO) contains terpenoids such as mono- and sesquiterpenes. Monoterpenes (Fig. 4.6) contain two isoprene units with molecular formula, C10H16, and they may be acyclic or cyclic compounds. Curcuma essential oils contain about 30 different monoterpenes. The following are a few examples [14]. Sesquiterpenes are compounds with molecular formula C15H24 and their derivatives containing three isoprenoid units, and as in monoterpenes, they can be acyclic or cyclic compounds. Over 140 different sesquiterpenes have been isolated from the genus Curcuma. These compounds fall into one of the three major categories, bisabolanes (Fig. 4.7), germacranes (Fig. 4.8), and guaianes (Fig. 4.9). C. aromatica, C. longa, C. xanthorrhiza, and C zedoaria are the major sources of these compounds. ar-Turmerone is the most widely distributed among these, while a-curcumene, b-curcumene, and zingiberene are found in four or more species [14]. About 30 different guaiane-type sesquiterpenes are reported, among them isocurcuminol is the most widely distributed. They have fused cyclopentane and cycloheptane ring systems and some of the typical examples are in Fig. 4.13. Isocurcumenol is the most widely distributed compound. Curcumenol (44), isocurcumenol (45), 4-epicurcumenol (46), etc., contain a 5,8-cyclic ether. Sapthulenol and isospathulenol contain fused cyclopropane ring also. Curcumenone (47) is a caraborane-type sesquiterpenes [23]. Several novel compounds are reported from genus Curcuma. 2-Methoxy5-hydroxybisabola-3,10-diene-9-one and 2,8-epoxy-5-hydroxybisabola-3,10diene-9-one, one new monoterpene, 2-(2,5-dihydroxy-4-methylcyclohex-3-enyl) Monoterpenes from turmeric oil CH3

CH2

CH2

CH3

CH3

CH2

H3C

CH3

Myrcene (12)

H3C

CH3

-Phellandrene (13)

CH3

H3C

CH3

-Phellandrene (14)

CH3

H3C

CH3

p-Cymene (15)

H3C

CH2

Limonene (16)

CH2 CH3

CH2 CH3

H3C H3C

CH3

-Terpinene (16)

H3C

CH3

-Terpinene (17)

FIG. 4.6 Monoterpenes.

H3C

CH3

D-Sabinene (18)

H3C CH3

-Pinene (19)

-Pinene (20)

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Sesquiterpenes from turmeric oil CH3 H

CH3 H

O

CH3

CH3 H

O

α-Turmerone (21)

O

CH2

β-Turmerone (22)

O

CH3

ar-Turmerone (23)

CH3

Curlone (24)

CH2

CH3

CH3

CH3

CH3 CH3

CH3

CH3

CH3

CH3

CH3

α-Zingiberene (25)

ar-Curcumene (26)

CH3

CH3

CH2

CH3

CH3

β-Bisabolene (27)

CH3

cis-γ-Bisbolene (28)

OH CH3

CH3

OH

O

O

CH3 CH3

CH3

CH3 CH3

CH3

CH3

Turmeronol A (29)

CH3

CH2

CH3

CH3

α-Turmerol (31)

Turmeronol B (30)

CH3

CH2

OH CH3 H

OH

CH3

β-Sesquiphellandrene (32) CH3 OH

HO

O

CH3

O

CH3

H3C

H3C

5-Hydroxyl-ar-turmerone (33)

CH3

CH3 CH3

H3C

Turmeronol B (34)

O

CH3

CH3

H3C

Bisabolone (35)

CH3

Bisabolone-4-one (36)

H CH3

CH3

CH3

O

OH

O

H

OH

O O

H3C

CH3

CH3

H3C

H3C

CH3

(38) (39) (New sesquiterpenes compounds) [22]

Turmeronol A (37)

FIG. 4.7 Bisabolane-type compounds.

O O

O

Curdione (40)

O

O

Dehydrocurdione (41)

FIG. 4.8 Germacane-type compounds.

O O

O

1,10:4,5-Diepoxy-7 (11)germacren-8-one (42)

O O

3,4-Epoxy-6,9-germacrane dione (43)

108 Studies in Natural Products Chemistry

H

H

H O

O O

OH

Curcumenol (44)

O

Isocurcumenol (45)

O

OH

OH

4-Epicurcuminol (46)

Curcumenone (47)

FIG. 4.9 Guaiane-type compounds.

H

OH

NH

O

O

O

OH

O O

O

OH H

(48)

H

(49)

H

H

(50)

(51)

H O

OH

(52)

O

(53)

OH

O

OH

(54)

FIG. 4.10 Other novel compounds.

propanoic acid, bisacurone A, and 4-methylene-5-hydroxybisabola-2,10diene-9-one were isolated [24]. Novel compounds were also isolated from the rhizomes of Curcuma wenyujin, namely elema-1,3,7(11),8-tetraen-8,12lactam (48), 7b,8a-dihydroxy-1a,4aH-guai-9,11-dien-5b,8b-endoxide (49), hydroxy isogermafurenolide (50), isogermafurenolide (51), curcumenol (52), 4-epicurcumenol (53), and neocurcumenol (54) (Fig. 4.10) [25].

CHEMISTRY OF GINGER Ginger oleoresin components: The pungent property of ginger is due to gingerols, shogoals, paradols, and gingerone (Fig. 4.11). Gingerols have the relatively high pungent value followed by shogoals and then gingerone [26]. Liao et al. reported the isolation of a new compound, O-methyldehydrogingerol, from the ether extract of the Z. officinale along with 28 known compounds [27–29]. Ginger contains up to 3% essential oil, accounting for 20–25% of the oleoresin. The major compounds are camphene, b-phellandrene, and 1,8cineol [30]. Other constituents include ()-a-zingiberene, ()-b-bisabolene, (+)-ar-curcumene, ()-b-sesquiphellandrene, and acyclic a-farnesene [31]. The ratio of (+)-ar-curcumene (increasing with storage time) and ()-azingiberene plus ()-b-sesquiphellandren (decreasing with storage time) and the viscosity of the essential oil (increasing with storage time) help to obtain information on the quality of the herbal preparation. The oil also contains sesquiterpenes and sesquiterpene alcohols, the latter having an impact on the smell

Structure and Biochemical Properties of the Active Compounds Chapter

O

OH

CH3O

O CH3 (CH2)n

CH3O

HO

CH3 (CH2)n

HO

Gingerols n = 4: n = 6: n = 8:

4 109

55

Shogaols n = 4: n = 6: n = 8:

[6]-Gingerol [8]-Gingerol [10]-Gingerol

56

[6]-Shogaol [8]-Shogaol [10]-Shogaol

O CH3O

O CH3 (CH2)n

CH3O CH3

HO

HO

Paradols 57

Zingerone

58

n = 6: [6]-Paradol, n = 8: [8]-Paradol, n = 10:[10]-Paradol O

OH

H3CO

O

HO

HO

O-methyldehydrogingerol 59 O

60

6-Dehydrogingerdione

O

O

H3CO

H3CO

HO

HO

11,11-Isodehydrogingerdione 61

OH

[6]-Gingerol

O

62 OH

H3CO

OH

H3CO

HO

[8]-Shogaol

O

H3CO

HO

63

[18],[6]-Gingerdiol 64 OH

OH

O

H3CO

OCH3

OH

HO

Hexahydrocurcumin

65

OH

2,5-Dihydroxybisabola-3,10-diene 66

FIG. 4.11 Ginger oleoresin components.

of ginger. The taste of ginger is mainly affected by monoterpenes (camphene, limonene, myrcene, b-phellandrene, and a-pinene, borneol, 1,8-cineol, citronellol, geranial, geraniol, geranylacetate, linalool, neral, and others). The high content of neral and geranial corresponds to the lemon smell of Australian ginger [32].

110 Studies in Natural Products Chemistry

Major constituents of ginger oil (GO) are monoterpenes (a-thujene, a-pinene, camphene, b-pinene, sabinene, myrcene, a-phellandrene, limonene, b-phellandrene, a-terpinene), sesquiterpenes (b-bisabolene, cis-g-bisabolene, bcaryophyllene, ar-curcumene, a-zingiberene, b-sesquiphellandrene), aldehydes (neral, geranial, citronellal), esters (a-terpenylacetate, bornyl acetate, geranyl acetate, citronellyl acetate), and alcohols (linalool, a-terpineol, geraniol, citronellol, elemol, zingiberenol (I), cis-sesquisabinene hydrate, trans-a-sesquiphellandrol, nerolidol, 2-heptanoldodecatrienol) [27]. Miscellaneous: 1,8-cineole, a-naginatene, zingerone, [6]-shogaol, sulfonated compounds: (4)-gingesulfonic acid and shogasulfonic acids A, B, C [33].

Ginger oleoresin compounds Three new compounds containing an adenine group attached to the gingeroltype carbon skeleton, an unusual structural feature in higher plants, were identified from ginger and characterized through catch-and-release methodology using ion-exchange resins, allowing for fast and selective resolution of the basic, acidic, and neutral components (Fig. 4.12) [34].

Volatile Compounds from Ginger GO contains a number of volatile compounds comprising of mono and sesquiterpenes. Essential oils from ginger rhizome differ markedly in their composition from region to region. The major sesquiterpene hydrocarbon is a-zingiberene (Fig. 4.13). Several of the volatile compounds are similar in both TO and GO. However, the smell of these oils is different because of the variation in the proportion of the constituents. Table 4.1 gives a list of volatile compounds in both turmeric and ginger for comparison [14,29]. There are common structural features in the curcuminoids in turmeric and ginger as shown in Table 4.2 [1,2,7,29].

NH2 N N O

N

N

CH3O (CH2)nCH3 HO

(67) n = 4, [6]-Zingerine; n = 6, [8]-Zingerine; n = 8, [10]-Zingerine 5-(6-amino-9H-purin-9-yl) analogs of [6]-, [8]-, and [10]-gingerols, respectively. FIG. 4.12 Gingerol-adenine compounds.

4 111

Structure and Biochemical Properties of the Active Compounds Chapter

CH3

H

CH3

CH3

H

CH3

CH3

α-Zingiberene (68) H

CH3

CH3

CH3

CH3

CH3

CH2

CH3

ar-Curcumene (69)

CH3

CH3 CH3

β-Bisbolene (70)

CH3

CH3

β-Sesquiphellandrene (71) CH3

CH3

CH2OH

CH2 CH3 H2C

OH CH3

CH3

β-Zingiberene (72)

CH3

CH3

Nerolidol (73)

CH3

CH3

Geraniol (74)

FIG. 4.13 Volatile compounds from ginger.

TABLE 4.1 Volatile Components of Turmeric Oil and Ginger Oil: Terpenoids Turmeric

Ginger

Monoterpenes

Monoterpenes

α-Pinene

α-Pinene

β-Pinene

β-Pinene

Limonene

Limonene

α-Phellandrene

Camphene

β-Phellandrene

Sabinene

D-Sabinene

Myrcene

α-Terpinene

α-Phellandrene

γ-Terpinene

β-Phellandrene

Borneol

α-Thujene

Isoborneol

α-Terpinene

Camphor

γ-Terpinene

Camphene

α-Cymene

β-3-Carene

Myrtenal

1,8-Cineol

Phellandral

p-Cymene

Neral Continued

112 Studies in Natural Products Chemistry

TABLE 4.1 Volatile Components of Turmeric Oil and Ginger Oil: Terpenoids—Cont’d Turmeric

Ginger

p-Cymene-8-ol

Geranial

Carvacrol

Citronellal

Terpinolene

Cryptone

Terpinen-4-ol

Camphor

α-Terpineol

Carvotanacetone

Perillene

α-Fenchyl acetate

Citronellol

α-Terpenyl acetate

Linalool

Isobornyl acetate

Myrcene

Bornyl acetate

(E)-β-Ocimene

Geranyl acetate

Linalool

Citronellyl acetate

Nerol(Z)-β-Ocimene

Linalool Borneol Menth-2-en-1-ols Terpin-4-ol α-Terpineol Isopulegol Geraniol Citranellol

Sesquiterpenes

Sesquiterpenes

α-Atlantone

δ-Elemene

β-Atlantone

γ-Elemene

β-Bisabolene

β-Elemene

Bisacurone

(Z or E)-b-Farnesene

Bisacurone A

β-Bisabolene

Bisacurone B

α-Copaene

Bisacurone C

α-Bisabolene

Bisacurol

cis-g-Bisabolene Continued

Structure and Biochemical Properties of the Active Compounds Chapter

4 113

TABLE 4.1 Volatile Components of Turmeric Oil and Ginger Oil: Terpenoids—Cont’d Turmeric

Ginger

Bisacumol

β-Gurjunene

Bisacurone epoxide

β-Caryophyllene

1,10-Bisaboladiene-3,4-diol

γ-Muurolene

2,10-Bisaboladiene-1,4-diol

δ-Cadienene

1,3,5,10-Bisabolapentaen-9-ol

δ-Selinene

1,3,5,10-Bisabolatetraene

ar-Curcumene

1,3,5,11-Bisabolatetraene

α-Zingiberene

Bisabola-3,10-diene-2-one

(E)-b-Bergamotene

2,5-Dihydroxybisabola-3,10-diene

Germacrene B

4,5-Dihydroxybisabola-2,10-diene

Germacrene D

4-Hydroxy-3-methoxy-2,10-bisaboladien-9-one

(Z and E)-Calamenenes

3-Hydroxy1,10-bisaboladien-9-one

Pentylcurcumene

4-Hydroxybisabola-2,10-dien-9-one

Elemol

4-Methoxy-5-hydroxybisabola-2,10-diene-4-one

α-Bisabolol

α-Curcumene

β-Bisabolol

β-Curcumene

Zingiberenol (I)

γ-Curcumene

Zingiberenol (II)

Curlone

Aromadendrenol

Turmerone

Cubebol

ar-Turmerone

10-a-Cadinol

α-Turmerone

cis-Sesquisabinene hydrate

β-Turmerone

α-Eudesmol

Turmeronol A

β-Eudesbol

Turmeronol B

trans-a-Sesquiphellandrol

Xanthorrhizol

Nerolidol

Zingiberene

10-epi-g-Eudesmol

Cadalenequinone α-Calacorene Continued

114 Studies in Natural Products Chemistry

TABLE 4.1 Volatile Components of Turmeric Oil and Ginger Oil: Terpenoids—Cont’d Turmeric

Ginger

8-Hydroxycadalene α-Cadinol Curazeone Pyrocurzerenone Curcurabranol A Curcurabranol B Curcumenone 4S-Dihydrocurcumenone Curmadione Curcumadione Curmenone Isocurcumadione Curzerene Curzerenone 5-epi-Curzerenone β-Elemene γ-Elemene Elemol β-Elemenone Curcolonol Curcolone β-Dietyopetrol β-Eudesmol α-Silenene (a-Eudesmene) β-Silenene 1,4-Dihydroxy furanoeremophilan-6-one Acetoxyneocurdione Curdione Continued

Structure and Biochemical Properties of the Active Compounds Chapter

4 115

TABLE 4.1 Volatile Components of Turmeric Oil and Ginger Oil: Terpenoids—Cont’d Turmeric

Ginger

Dehydrocurdione 13-Hydroxydehydrocurdione 13-Acetoxydehydrocurdione 3,4-Epoxy-6,9-germacranedione (1R,10R)-Epoxy-(-)-1,10-dihydrocurdione Furanodiene Furanodienone Furanogermenone Germacrone Germacrone-13-al Germacrene Germacrone-4,5-epoxie Glechomanolide (1S,10S),(4S,5S)-Germacrone-1(10),4-diepoxide 4,5-Epoxy-12-acetoxy-7a,11a-dihydrogermacradien8-one 13-Hydroxygermacrone Isofuranodienone Neocurdione Wenjine Zederone Aerugidiol Alismoxide Curcumol Curcumenol epi-Curcumenol 4-epi-Curcumenol Isocurcumenol Continued

116 Studies in Natural Products Chemistry

TABLE 4.1 Volatile Components of Turmeric Oil and Ginger Oil: Terpenoids—Cont’d Turmeric Oxycurcumenol Neocurcumenol Procurcumenol Curcumadiol Procurcumenol 1-epi-Procurcumenol Isoprocurcumenol 9-oxo-Neoprocurcumenol Neoprocurcumenol Oweicurculactone Spathulenol Isospathulenol 4-Hydroxy-7(11),10(14)-guaiadien-8-one 7α,11α-Epoxy-5b-hydroxy-9-guaiaen-8-one Zedoarondiol Sozedoarondiol Zedoalacetone A Zedoalactenone B (1β,4β,5β,10β)-Zedoaronediol Methylzedoarondiol Zedoarol Zedoarolide A Zedoarolide B Parviflorene A Parviflorene B Previflorene E Parviflorene F Difurocumenone

Ginger

4 117

Structure and Biochemical Properties of the Active Compounds Chapter

TABLE 4.2 Comparison of Curcuminoids in Turmeric and Ginger Curcuminoids (Turmeric) O

Curcuminoids (Ginger)

O

O

R

R′

HO

OH

OH R2

R1O HO

OH

Curcumin (R = R´ = OMe) (2)

R3

Demethoxycurcumin (R = Ome; R´ = H) (3) Bisdemethoxycurcumin (R = R´ = H) (4) O

O

R′

OR

OH

(E)-1,7-Bis-(4-hydroxy-3-methoxyphenyl)1-heptene-3,5-dione (R = H; R´ = OMe) (75) O

HO

(R1 = CH3; R2 = OH; R3 = H) (81) Demethylated hexahydrocurcumin (R1 = R3 = H; R2 = OCH3) (82) Demethoxylated hexahydrocurcumin (R1 = CH3; R2 = R3 = H) (83)

OH

MeO

(R1 = CH3; R2 = OCH3; R3 = H) (80) Demethylated hexahydrocurcumin

R′

HO

Hexahydrocurcumin

OMe

Methoxylated hexahydrocurcumin

OH

(R1 = CH3; R2 = R3 = OCH3) (84)

Dihydrocurcumin (76) O

OH

OH

R

R′

HO

OH

OH

MeO

OMe

HO

OH

(85) OAc OAc

Tetrahydrodemethoxycurcumin (R = H; R´ = OMe) (77)

R1

R3 OH

HO

O

R2 OH

HO

(1E,4E,6E)-1,7-Bis-(4-hydroxyphenyl)1,4,6-heptatriene-3-one (78) OH

R1 = R3 = OCH3; R2 = H (86) R1 = R2 = R3 = OCH3 (87) R1 = OCH3; R2 = H; R3 = OH (88) R1 = R3 = OH; R2 = H (89) O R1

MeO

(1E,3E)-1,7-Diphenyl-1,3-heptadiene5-ol (79)

OH

HO R2

Gingerone A R1 = OCH3; R2 = H (90) Methoxylated gingerone A R1 = R2 = OCH3 (91) Demethoxylated gingerone A R1 = R2 = H (92)

118 Studies in Natural Products Chemistry

STRUCTURAL SIMILARITIES OF TURMERIC AND GINGER COMPOUNDS The oleoresins of ginger show similar structures as that of curcumin. The common structural features are evident in the following compounds (Figs. 4.14 and 4.15). Dehydrozingerone, a structural analog of curcumin, is a phenolic compound, isolated from ginger rhizomes [35,36].

GENE EXPRESSION IN THE RHIZOMES OF GINGER AND TURMERIC It was reported that ginger and turmeric accumulate important pharmacologically active metabolites at high levels in their rhizomes and a significant set of genes were found to be exclusively or preferentially expressed in the rhizome of ginger and turmeric. Large classes of enzymes involved in specialized metabolism were also found to have apparent tissue-specific expression, suggesting that gene expression itself may play an important role in regulating metabolite production in these plants [37,38]. The gingerols in ginger and the curcuminoids in turmeric appear to be derived from the phenylpropanoid O

O

OCH3

CH3O

HO

OH

Curcumin

O O CH3O CH3O (Half curcumin structure)

HO

HO Dehydrozingerone

Zingerone

O

OH

OH OCH3

CH3O

OH

HO

O

OCH3

HO

OH

OH

OAc OCH3

CH3O

OH

HO

OH

CH3O

OAc

CH3O

OCH3

OH

HO

OH O CH3O

HO

O

O

OCH3 CH3O

OH

HO

FIG. 4.14 Structural features of curcumin and curcuminoid compounds from ginger.

OCH3

OH

O

O OCH3

CH3O

HO

Curcumin

O

O

CH3O

HO

CH3O

Paradol

Shogoals

O

HO

O

OH

O

CH3O

CH3O

HO

OH

Gingerol

HO

FIG. 4.15 Structural features of curcumin and non-volatile pungent compounds from ginger.

1-Dehydro-10-gingerdione

120 Studies in Natural Products Chemistry

pathway. Assays for enzymes in the phenylpropanoid pathway identified the corresponding enzyme activities in protein crude extracts and these enzymes included phenylalanine ammonia lyase, polyketide synthases, p-coumaroyl shikimate transferase, p-coumaroyl quinate transferase, caffeic acid O-methyltransferase, and caffeoyl-CoA O-methyltransferase and they have potential roles in controlling production of certain classes of gingerols and curcuminoids [39].

BIOLOGICAL PROPERTIES OF TURMERIC (CURCUMIN) AND GINGER IN RELATION TO STRUCTURE Biological properties of turmeric Biological properties of turmeric and ginger and their effect on various diseases are given in Table 4.3 for comparison [1,39,40]. Turmeric contains mainly curcuminoids and TO as the active components. Most of the biological studies are reported on curcumin. Curcumin possess a diverse profile of biological actions that result in changes in oxidative stress, inflammation, and cell-death pathways. Curcumin has been studied for its potential therapeutic applications in cancer, aging, endocrine, immunological, gastrointestinal, and cardiac diseases. In addition, data in animal models and in humans have also begun to be collected in stroke, Alzheimer’s disease, and Parkinson’s disease and its effect on stress and mood disorders [41–43]. Ginger contains nonvolatile pungent components gingerols, shogaols, etc., and the GO components which have terpenes similar to TO.

Antiplatelet activity of curcumin The traditional medicinal practice of Z. officinale is to promote blood circulation for removing blood stasis through antiplatelet aggregation activity [44]. Liao et al. studied the pharmacological activity including antiplatelet aggregation and vasorelaxing effects of several compounds from the ether extract of ginger and found that [6]-gingerol and [6]-shogaol exhibited more potent antiplatelet aggregation bioactivity. The authors concluded that the compounds which possessed a carbonyl group at the 6-position of the alkyl chain in phenylalkanoids and phenylalkenoids would be good lead drug candidates and the function of ginger to promote the blood circulation for removing blood stasis could be related to antiplatelet aggregation and vasorelaxing mechanism [45].

Antimicrobial activity of curcumin The polyphenolic compound curcumin has been subjected to a variety of antimicrobial investigations due to extensive traditional uses and low side effects. Antimicrobial activities for curcumin and rhizome extract of C. longa against different bacteria, viruses, fungi, and parasites have been reported [45]. The

TABLE 4.3 Biological and Medicinal Properties of Turmeric and Ginger Biological and Medicinal Properties of Turmeric (Curcumin) and Effect on Diseases

Biological and Medicinal Properties of Ginger and Effect on Diseases

Carminative stimulant

Carminative stimulant

Digestive disorders

Digestive properties

Skin diseases, ring worm, scabies

Fever

Biliary disorders

Anorexia

Anorexia

Cough

Sinusitis

Dyspnea

Wound healing

Vomiting

Antibacterial

Cardiac complaints

Antiprotozoal

Constipation

Antifungal

Flatulence

Antiviral

Colic

Gastroprotective

Swelling

& Prokinetic

Elephantiasis

Immunomodulatory

Disuria

Pro-apoptotic

Diarrhea

Antiapoptotic

Cholera

Neuroprotective

Dyspepsia

Antiinflammatory

Diabetes

Antiangiogenic

Tympanitis

Antioxidant

Neurological disorders

Anticarcinogenic

Antiinflammatory properties

Cardioprotective

Rheumatism

Antidiabetic

Arthritis

Cystic fibrosis

Inflammation of liver

Multiple sclerosis

Phlegmatic conditions

Alzheimer disease

Respiratory problems such as asthma and cough

Liver injury Nephrotoxicity Inflammatory bowel diseases Arthritis Gall stone formation

Antiemetic and antinauseant properties Chemoprotective properties Hypolipidemic effect

Cataract formation

Antimicrobial and insecticidal properties

Chemopreventive

Anxiolytic-like effect

Chemotherapeutic

Androgenic activity

Stimulates muscle regeneration

Antivertigo activity

Cardiovascular diseases

122 Studies in Natural Products Chemistry

promising results for antimicrobial activity of curcumin make it a good candidate to enhance the inhibitory effect of existing antimicrobial agents through synergism [46].

Anticancer property of curcumin Curcumin has been shown to induce apoptosis in a variety of cancer cells, inhibit tumors in animal models of carcinogenesis, and act on a variety of signal transduction pathways and molecular targets involved in the development of cancer [47]. Curcumin is a multiedge sword in fighting cancer by asserting its antitumor activity by altering the deregulated cell cycle via cyclin-dependent, p53-dependent, and p53-independent pathways. “The cell cycle is divided into four distinct phases (G1, S, G2, and M). The progression of a cell through the cell cycle is promoted by CDKs, which are positively and negatively regulated by cyclins and CKis, respectively” (Fig. 4.16) [48]. These anticancer effects of curcumin are predominantly mediated through its negative regulation of various transcription factors, growth factors, inflammatory cytokines, and protein kinases, and it abrogates proliferation of cancer cells by arresting them at different phases of the cell cycle and/or by inducing their apoptosis [49,50]. Deregulated inflammatory pathways lead to cancer and the mechanism by which chronic inflammation drives cancer initiation and progression is via increased production of proinflammatory mediators, such as cytokines, chemokines, reactive oxygen species (ROS), overexpression of oncogenes, cyclo-oxygenase-2 (COX-2), matrix metalloproteinase (MMP), intracellular signaling pathway mediators, and transcription factors [50].

FIG. 4.16 Cell cycle. Adapted from and thanks to G. Sa, T. Das, Cell Div. 3 (2008) 14. doi:10.1186/1747-1028-3-14. http://creativecommons.org/licenses/by/2.0.

Structure and Biochemical Properties of the Active Compounds Chapter

4 123

Curcumin fulfills the characteristics for an ideal chemopreventive agent with its low toxicity, affordability, and easy accessibility even though its use is limited by the low bioavailability. Curcumin modulates multiple molecular pathways involved in the lengthy carcinogenesis process to exert its chemopreventive effects through several mechanisms: promoting apoptosis, inhibiting survival signals, scavenging ROS, and reducing the inflammatory cancer microenvironment [51]. Curcumin exhibits in vitro and in vivo chemopreventive and chemotherapeutic effects on various cancer cell types and animal models [52–55]. It is a lipophilic diphenolic molecule with multiple mechanisms by which it may mediate chemotherapy and chemopreventive effects on cancer. Its safety and tolerance are high with little or no side effects. The activation of the nuclear factor kB (NFkB) is crucial in inflammation and cancer [56]. This dietary compound has been shown to inhibit several cell signaling pathways [57], including NFkB, activating protein-1, tumor necrosis factor (TNF), and metastatic and angiogenic pathways. The compound also inhibits certain enzymes, including COX-2 and MMPs [58–61]. Several enzymes are involved that are essential in carcinogenesis and are also targeted by curcumin, aldo-keto reductase family 1 member B10, serine/threonine-protein kinase, protein kinase C, MMP-9, COX-1, and epidermal growth factor receptor, to gain further insight into the mechanism of action [62–64]. A lipophilic derivative of curcumin, diacetyl curcumin (DAC) (93), and a hydrophilic derivative, diglutaryl curcumin (DGC) (94), (Fig. 4.17) were synthesized, and their in vivo analgesic and antiinflammatory activities were compared with those of curcumin and aspirin. The in vitro anticancer activities of curcumin and the two derivatives against three cell cancer lines were compared with those against a noncancerous cell line. The inhibitory effects were comparable to each other and nearing that of curcumin, while they showed low inhibitory effect toward the noncancerous cell line. The mouse tail flick assay showed that curcumin, DAC, and DGC increased latency time. DGC was most effective as an analgesic, even more so than aspirin. The maximum percentage effect was highest with DGC at 3 h. The carrageenan induced paw edema model indicated antiinflammatory activity of all three curcumin formulations. The percentage inhibition of paw edema was maximum for DAC, followed by aspirin and curcumin [65].

O

CH3O

OH OCH3

R1 O

O

R1

Diacetyl curcumin (R1 = OAc) (93); Diglutaryl curcumin (R1 = HOOC(CH2)3CO) (94) FIG. 4.17 Curcumin derivatives.

124 Studies in Natural Products Chemistry

Bisdemethoxycurcumin (BDMC) is a natural curcumin derivative and it has shown potent anticancer effects and the anticancer effects are sometimes greater than that of curcumin. BDMC could inhibit proliferation and survival of several types of cancer cells including breast, colon, glioma, and leukemia cells and suppress cancer invasion and metastasis by reducing the expression of metalloproteinase (MMPs) [66–70]. BDMC inhibited MCF-7 cell proliferation and induced G2/M cell cycle arrest [71].

Biological Properties of Ginger Ginger contains several pungent phenolic compounds like [6]-gingerol, [6]shagol, [6]-paradol etc. Many of these compounds contribute toward the biological and medicinal properties of ginger. They have been studied for their anti-bacterial, anti-oxidant, anti-inflammatory and anti-tumor properties.

Anticancer Property: Ginger Several studies have been conducted on the anticancer properties of ginger against various cancers. The growth-inhibiting and apoptosis-inducing properties of ginger extract (GE) in in vitro and in vivo prostate cancer models have been reported. The antiproliferative efficacy of the most-active GE biophenolics as single-agents and in binary combinations was studied and the data demonstrated that binary combinations of ginger phytochemicals synergistically inhibit proliferation of PC-3 cells. Combining GE with its constituents (in particular, [6]-gingerol) resulted in significant augmentation of GE’s antiproliferative activity [72]. The anticancer properties of ginger are attributed to the presence of certain pungent vallinoids, viz., [6]-Gingerol and [6]-paradol, as well as some other constituents like shogaols and zingerone. A number of mechanisms that may be involved in the chemopreventive effects of ginger and its components have been reported from the laboratory studies in a wide range of experimental models [73]. [6]-Gingerol has been studied for its cytotoxic effects in various cancer cell lines and was shown to induce cell death in cervical cancer cell line, HeLa, by caspase 3-dependent apoptosis and autophagy [74]. Cancer chemopreventive effects of [6]-gingerol, the major pungent component of ginger, and its impact on different steps of the metastatic process were reported [75]. Ginger extracts containing gingerols have weaker

HO

O

OH O Pinocembrin (95) FIG. 4.18 Flavonoid compound.

Structure and Biochemical Properties of the Active Compounds Chapter

OH

4 125

O

O

1,7-Diphenyl-5-hydroxy-4-4,6-heptadien-3-one (96)

Piperitenone (97)

FIG. 4.19 Diaryl heptanoid and monoterpenoid compounds.

activity against colorectal cancer compared to turmeric extracts containing curcumin [76]. Pinocembrin (95) (Fig. 4.18), a natural flavonoid compound in ginger roots, has shown antiinflammatory and neuroprotective effects as well as the ability to reduce ROS, protect the blood–brain barrier, modulate mitochondrial function, and regulate apoptosis [77]. Phytochemical analysis of the rhizome extract of Curcuma ecalcarata, a hitherto uninvestigated south Western Ghats endemic species, contains the diarylheptanoid trans, trans-1,7-diphenyl-5-hydroxy-4,6-heptadiene-3-one, steroid b-sitosterol, flavanone pinocembrin, and monoterpenoids piperitenone and 8hydroxy piperitone (Fig. 4.19). The study highlights the plant as a rich source of the flavanone pinocembrin and the volatile aroma compound piperitenone [78].

Antiinflammation, curcumin, and ginger Several research reports have established that curcumin is a potent antiinflammatory agent [90–98]. Curcumin has the ability to inhibit inflammatory cell proliferation, invasion, and angiogenesis through multiple molecular targets and mechanisms of action. The antiinflammatory effects of curcumin were evaluated relative to various chronic inflammatory diseases [92]. The curcuminoid fraction of turmeric has been used on rheumatoid arthritis (RA) model animals in preventing arthritis [93]. The antidepressant effects of curcumin in animal models of depression and its influence on neurotransmitters such as serotonin and dopamine are reported [94] and are also associated with its antiinflammatory property. COX-1 and COX-2 catalyze the conversion of arachidonic acid to the endoperoxide prostaglandin (PG)H2 which can form a variety of prostaglandins, thromboxanes, and prostacyclin through catalysis by non-rate-limiting enzymes [95] or by nonenzymatic rearrangement. They are essential for the maintenance of the renal and gastrointestinal tract functions. They are the targets of widely used nonsteroidal antiinflammatory drugs (NSAIDs). Curcumin inhibited the expression of COX-2 in different tumor cells and in animal models [43]. Purified 10-gingerol, 8-shogaol, and 10-shogaol inhibited COX-2 and no inhibition of COX-1 was detected. Therefore, these compounds might be responsible, in part, for the antiinflammatory activity of ginger [28]. Comparison of structural units of shogaol (99) and gingerol (100) shows a,bunsaturation vs b-hydroxy ketone structures (Fig. 4.20).

126 Studies in Natural Products Chemistry

O CH3O

HO

O

OH

CH3O α,β-Unsaturation

HO

( )n β-Hydroxy ketone side chain

Shogaol (99)

Gingerol (100)

FIG. 4.20 Nonvolatile compounds—side-chain variation and anticancer activity.

Metabolism of gingerol and curcumin: It was shown that 10-gingerol is extensively metabolized in zebra fish embryos through a reductive path way into two isomeric metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol. 10-Gingerol gives a similar metabolic profile in humans as it did in zebrafish embryos, since these two metabolites were also found in human urine [99a]. Several in vivo studies have identified number of intestinal metabolites in human and rats including curcumin glucuronide, curcumin sulfate, tetrahydrocurcumin, and hexahydrocurcumin [99b].

Anticancer and Antiinflammatory properties of turmeric oil TO has a number of terpenes, among them the bisabola sesquiterpenes such as the turmerones are biologically active. Among them the antitumor properties of ar-turmerone have been known for very long time and its absolute configuration and SAR have been studied [97]. TO contains sesquiterpenes and fish oil has eicosapentaenoic acid and docosahexaenoic acid, possessing antiinflammatory activity. The antiinflammatory and analgesic properties of these two natural products were compared with aspirin as a standard. The percent inhibition as a measure of paw edema for TO and fish oil at 100 mg/kg was 76% and 31%, respectively, while the percent inhibition by the combination of the two at 100 mg/kg was 62%, which was the same as that of aspirin at the same dose. The inhibitory activity of fish oil at 50 mg/kg was 86% and with an increase in dose the activity decreased [98]. Fractional distillation and chromatographic separation of TO gave column fractions having biological activity against the PANC-1 pancreatic cancer cell line, with an EC50 in the range of 23–33 mg/mL. These fractions were analyzed by NMR and GCMS and found to contain the sesquiterpenes, ar-curcumene, 7-epi-zingiberene, b sesquiphellandrene, curlone, a-turmerone, b-turmerone, and ar-turmerone. The ability of TO components to induce cell death was independent of caspase activity. Potency was higher at lower cell density and was reduced by increasing serum concentration, the latter indicating serum binding of active components [99,100].

Antiemetic effects of ginger, receptor interactions Ginger is effective to alleviate nausea and emesis due to chemotherapy with emetogenic drugs [101–105]. The cytotoxic chemotherapy drugs increase

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O

HO OCH3

101

FIG. 4.21 Cysteine-conjugated metabolite of shogaol.

5-HT release in the intestine and augment activity of visceral afferent vagus nerves [106,107]. Administration of 5-HT type 3 (5-HT3) receptor antagonists suppresses cytotoxic drug-induced vomiting in an animal model [108]. Pungent constituents of ginger inhibited 5-HT3 receptor function in neuroblastoma cells and suggest that the antiemetic efficacy of ginger may contribute to its 5-HT3 receptor blocking activity [102,109–111]. The chemotherapeutic drugs increase 5-HT release in the intestine [106,107,112]. Ginger could be used as a safe antiemetic drug at postoperation [103,113–116]. Structural comparison for antiemetic activity shows the common structural unit 101 (Fig. 4.21). The exact mechanism of action of ginger in relation to its antiemetic properties is unclear, although it appears to inhibit serotonin receptors and to exert antiemetic effects at the level of the gastrointestinal system and in the central nervous system. Ginger components [6]-, [8]-, and [10]-gingerol as well as [6]-shogaol were shown in different in vivo studies to be at least partly responsible for the drug’s antiemetic properties [117].

Anti-Alzheimer’s properties for turmeric and ginger Both curcumin (turmeric) and ginger have been reported to have strong antiAlzheimer’s properties [29,118–120]. Curcumin has neuroprotective and antioxidant effects, and it protects neuron-like PC-12 rat cells and umbilical endothelial cells against Ab toxicity and reduces tau hyper phosphorylation [121]. [6]-Gingerol pretreatment protected against Ab25–35-induced cytotoxicity [122]. Ginger root extract exhibits antiinflammatory properties by suppressing the transcription of inflammatory mediator genes through the MAPK and NFkB signaling pathways [123]. The similar structural features for curcumin and gingerol help them to elicit anti-Alzheimer’s effect. Ginger compounds interact with various targets and the structural features eliciting antiAlzheimer’s effect indicate different domains having hydrogen bonding sites and hydrophobic domain connected by a cyclic or linear carbon spacer [29].

Diabetes-turmeric and ginger Curcumin is shown to be effective in animal models for diabetes, including insulin resistance, hyperglycemia, hyperlipidemia, and islet apoptosis and

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necrosis, and prevent the complications due to diabetes. However, clinical trials of curcumin are reported only in using curcumin to treat diabetic nephropathy, microangiopathy, and retinopathy [124]. Turmeric extract (0.5% in diet, ethanol, and/or hexane extraction) for 4 weeks significantly reduced the blood glucose levels in type 2 diabetic KK-A(y) mice [10]. Ginger and its main components, gingerols and shogaols, can inhibit synthesis of several proinflammatory cytokines including IL-1, TNF-a, and IL-8 along with inhibiting prostaglandin (PG) and leukotriene synthesis enzymes [125]. Chronic hyperglycemia increases circulating levels of inflammatory biomarkers such as IL-6 (IL6), TNF-a, and C-reactive protein (CRP). TNF-a and IL-6, as the major cytokines, initiate inflammatory responses and cause the production of CRP as an acute-phase reactant [126]. Oral ginger supplementation ameliorated inflammation through reduction in levels of TNF-a and hs-CRP concentrations in blood samples of the patients with type 2 diabetes mellitus [127–130].

SIDE-CHAIN STRUCTURE AND ANTICANCER ACTIVITY [6]-Gingerol and [6]-shogaol (6-SHO) differ in the side chain (Fig. 4.22). Gingerol contains b-hydroxyketone, while shogaol is a dehydration product with an a,b-unsaturated ketone. This difference in side-chain structure marks a difference in their activities depending on the cancer cell line. [6]-Gingerol inhibits the metastasis of MDA-MB-231 breast cancer cells and induced apoptosis in LNCaP prostate cancer cells inhibits the growth of several types of murine tumors such as melanomas, renal cell carcinomas, and colon carcinomas by enhancing the infiltrations of tumor-infiltrating lymphocytes CD4 and CD8 T-cells and B220+ B-cells [76–79]. [6]-Gingerol was also shown to inhibit the progression of phorbol ester-induced skin tumor in ICR mice [79]. A few studies have been published on the anticancer properties of [6]-gingerol and its mechanism of action against colon cancer [80–82]. Radhakrishnan et al. reported the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer [83,86]. In the case of human breast cancer, the combination chemotherapeutic protocol, cyclophosphamide, doxorubicin, and 5-fluorouracil [6]-gingerol showed the highest anticancer potency that is superior to that of the combination protocol in the breast cancer

O

OH

O

H3CO

H3CO

HO

HO

[6]-Gingerol (55) FIG. 4.22 Structural comparison for shogoal and gingerol.

[6]-Shogaol (56)

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cell model, MCF-7. Structure–activity relationship (SAR) studies have shown that the acquisition of free hydroxyl group in the aliphatic side chain of [6]-gingerol is essential for the antibreast cancer activity and that the length of aliphatic side chain in [6]-gingerol is optimum for its anticancer activity because any decrease in the side-chain length resulted in a dramatic loss of anticancer activity [84]. Additionally, it was shown that allylation of phenolic group resulted in antibreast cancer activity superior to that of [6]-gingerol, while methylation or isoprenylation of phenolic group has led to a potential decrease in the anticancer activity and loss of aromaticity resulted in a complete loss of [6]-gingerol’s cytotoxic activity [84]. 6-SHO effectively reduced survival and induced apoptosis of cultured human (LNCaP, DU145, and PC-3) and mouse (HMVP2) prostate cancer cells [85]. 6-SHO inhibited tumor growth and induced apoptosis in U937 xenograft mouse model [86]. Cysteine-conjugated metabolite of [6]-shogaol, 5-cysteinyl-[6]-shogaol (Fig. 4.23), is bioactive by acting as a carrier of [6]shogaol in both cancer cells and mice [87]. The [8]- and [10]-shogaols and their respective cysteine-conjugated metabolites also showed apoptotic activity toward colon cancer cells [88]. 6-SHO was more effective than [6]-gingerol, and [6]-paradol at reducing survival of prostate cancer cells and reducing STAT3 and NFkB signaling, and also showed significant tumor growth inhibitory activity in an allograft model using HMVP2 cells; thus it may have potential as a chemopreventive and/or therapeutic agent for prostate cancer [85]. Among the ginger components, including [6]-shogaol, [6]-paradol, and [6]-gingerol, [6]-shogaol showed the greatest inhibitory effects on the non-small cell lung cancer (NSCLC) cell proliferation and anchorage-independent growth. [6]-Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis [89].

O H2N OH O

S

HO OCH3

5-Cysteinyl-[6]-shogaol (98) FIG. 4.23 Common structural unit.

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CONCLUSION Natural products are a rich source of organic compounds and among them turmeric and ginger belong to the same family, Zingiberaceae. The rhizomes or underground stems of these plants have proven to be very valuable in providing a large number of small-molecule organic compounds with a variety of medicinal properties. The structural similarities of these compounds may have contributed to their similarity in biological properties. Terpenoids and diarylheptanoids, including the curcuminoids, and the gingerol-related compounds are believed to be responsible for most of the medicinal properties of these two natural products and a significant set of genes were found to be exclusively or preferentially expressed in the rhizome of ginger and turmeric [37]. Several terpenes in the volatile fractions of both turmeric and ginger are common and the structures of oleoresin components of these two natural products have striking similarities. These multitargeted agents might be less likely to encounter problems of drug resistance. These phenolic and terpenes compounds are endowed with a remarkable number of different biochemical mechanisms to fight against several diseases, yet with limited toxicity for normal cells. The structural correlation and biochemical properties of these compounds will help the design and development of drug targets and lead compounds for new drug discovery. The studies on these natural compounds are expected to lead to novel drug development for the treatment and prevention of several diseases and for healthy living.

ABBREVIATIONS AND ACRONYMS TO GE GO CDK ROS MMP TNF NFkB DAC DGC BDMC 6-SHO SAR NSCLC NSAIDs CRP

Turmeric Oil Ginger Extract Ginger Oil Cyclin-Dependent Kinase Reactive Oxygen Species Matrix Metalloproteinase Tumor Necrosis Factor Nuclear Factor kB Diacetyl curcumin Diglutaryl curcumin Bisdemethoxycurcumin [6]-shogaol Structure–activity relationship Non-Small Cell Lung Cancer NonSteroidal Antiinflammatory Drugs C-reactive protein

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Chapter 5

7-6-5 Tricarbocyclic Diterpenes: Valparanes, Mulinanes, Cyathanes, Homoverrucosanes, and Related Ones I.S. Marcos, R.F. Moro, A. Gil-Meso´n and D. Dı´ez Facultad de Ciencias Quı´micas, Universidad de Salamanca, Salamanca, Spain

Chapter Outline Introduction Classification: Structural Types of 7-6-5 Tricarbocyclic Diterpenes and Related Ones Valparanes and Valparolanes [5,6] Mulinanes and Azorellanes Cyathane Family Verrucosanes Family [120–144] Other 7-6-5 Tricarbocyclic Diterpenes Biosynthesis Valparanes and Valparolanes Mulinanes and Azorellanes Cyathanes, Cyanthiwigins, and Verrucosanes Family Syntheses and Synthetic Approximations Oltra Synthesis of Valparadiene

138

140 140 143 149 163 171 172 175 177 178 181 181

Synthetic Approach to Cyathanes and Cyanthiwigins 181 Cha Syntheses of (
)-Cyathin A3 and (
)-Cyathin B2 184 Gademann Synthesis of Cyrneine A 186 Kanoh Synthesis of ()-Scabronine G 187 Nakada Syntheses of ()-Scabronines G and A, and ()-Episcabronine A 189 Nakada Synthesis of ()-Scabronine D 190 Nakada Synthesis of ()-Cyathin 191 B2 Stoltz Syntheses of ()-Cyanthiwigins B, F, and G 192 Gao Syntheses of (
)-Cyanthiwigins A, C, G, and H 194

This review is dedicated to Prof. P. Basabe for her encourage, inspiration, and friendship all over the years. Studies in Natural Products Chemistry, Vol. 48. http://dx.doi.org/10.1016/B978-0-444-63602-7.00005-9 © 2016 Elsevier B.V. All rights reserved.

137

138 Studies in Natural Products Chemistry Piers Synthesis of (
)-Verrucosan-2S-ol, (
)-Neoverrucosan-5R-ol, and (
)-Homoverrucosa-2-en-5S-ol 196 Synthetic Approach to Gagunin Diterpenoids 197

Guerrero Synthesis of Mulinanes 36, 43, 46, and 54 198 Conclusions 199 Acknowledgment 200 References 201

INTRODUCTION Valparanes, mulinanes, cyathanes, and homoverrucosanes are diterpenes that are broadly distributed in nature and have a 7-6-5 tricarbocyclic system (Fig. 5.1). In contrast with other families or groups of tricyclic diterpenes (pimaranes, abietanes, totaranes, or cassanes) with the podocarpane tricyclic system 6-6-6 [1], which appear in excellent diterpenoid reviews [2] and are grouped and classified according to their biogenetic origin, 7-6-5 tricyclic diterpenes have never been studied together. This review presents a classification of these types of diterpenes, organized according to their biogenetic origin, that allows us to observe the structural relationship among them. In this review, we will examine the 7-6-5 tricyclic diterpenes valparanes, mulinanes, cyathanes, and homoverrucosanes and their epi-derivatives, such as epihomoverrucosanes and gagunins, which have been included with other biogenetic-related groups, including valparolanes, azorellanes, verrucosanes, neoverrucosanes, and epi-neoverrucosanes (Fig. 5.1). Among compounds with a cyathane skeleton are, can be found the cyanthiwigins, which differentiate from the rest in the stereochemical relationship syn between the angular methyls at C6 and C9, while the rest of cyathanes present these methyls in anti relationship. The same difference is in the verrucosane family, being gagunins group the only one that present a syn relationship between the angular methyls at C7 and C10, the rest of family components show an anti relationship. Until now, the reviews refer mainly to the synthesis of cyathanes, see Wright and Whitehead [3] and Enquist and Stoltz [4]. To the best of our knowledge, there is no review of the synthesis of these compounds from other groups. 7-6-5 Tricyclic diterpenes and biogenetically related ones proceed from very different sources, such as plants, fungi, and sponges. The biological activities that these compounds show (antibiotics, antitumorals, and the nerve growth factor (NGF)—a capacity that implicates their potential as therapeutic agents to treat neurodegenerative disease such as Alzheimer’s or Parkinson’s—and the structural diversity) account for the great synthetic and biological interest roused by these compounds. In this review, the diterpenoids of each known type are classified according to their biogenesis. A biosynthetical approach for each group is proposed

5 139

7-6-5 Tricarbocyclic Diterpenes Chapter

2 19

1

20

A

11 18 6

10

3 4

13

B H

16

8

H

5

9

7

C

14 13

12

12

11

20

18 4

A

19

6

C

15 12 11

3

17

10

8

B

5

H

17

9

2

3 18

19

1

A

4

2

1

7

13

6

5

H 10

16

14

7

B H

8 9

C

16

15

17

15

14

20

Mulinane

Valparane

20

17

1

16

15

2

11

4

6

9

18

6 3

Cyathane

16

8

H

H

7

19

15 14

8 9

13 12

16

4

5

10

17 9

H

2

1

Azorellane

Valparolane

H

19

3

11

17

12

6

18

20

H

Cyanthiwigin

19 6

5

8

7

4 3

H

H

20

10

H

H

14 11 12

H

2

18

7

9

H 10

1

13

13

13

16

15 17

Homoverrucosane

epi-Homoverrucosane

19

19

6

8

5 4 18

A

B

7 1

3 2

H

6

C H 10 14

D

8

5

9

B

20 18

4

1

A

11

7

2

3 16

Gagunin (Bis-epi-homoverrucosane)

13 15 17

Verrucosane

12 16

H

C 9 H 10 14

D

13 15

20

H

11

H

13

12

17

Neoverrucosane

epi-Neoverrucosane

FIG. 5.1 7-6-5 Tricyclic diterpenes skeletons and biogenetically related ones.

and a revision of the synthesis and synthetical approaches that have not been reported previously in other reviews is included. In this work, identical representation for all 7,6,5 diterpenes has been used (Fig. 5.1) in order to better compare their structures, but the numbering and naming of the cyclic systems is kept as it appears in the references.

140 Studies in Natural Products Chemistry

CLASSIFICATION: STRUCTURAL TYPES OF 7-6-5 TRICARBOCYCLIC DITERPENES AND RELATED ONES The structures of the different compounds classified in each group and table show the isolation sources, biological activity, and references.

Valparanes and Valparolanes [5,6] Valparanes 1–26 and valparolanes 27–30, Figs. 5.2 and 5.3, respectively, constitute a natural products group that is isolated from the Cistaceae family of plants; in particular, from Halimium viscosum and Halimium verticillatum as shown in Tables 5.1 and 5.2, respectively. As curiosity H. viscosum presents several chemotypes [7,9,11–13].

2

20

1

4

7

H

12

3

16

8

H

O

9

18

6

3

O

1 2

11

19

15

H

14

Valparene, 1

H

H

17

15

H

H

H

H

H

13

Valpara-2,13-diene, 2

2S,3R-Epoxyvalpar-15-ene, 4

Valpara-1,3,15-triene, 3

Valpar-15-en-2-one, 5

OH HO HO HO

H

H

H

Valpara-1,15-dien-3R-ol, 6

H

Valpara-1,15-dien-3S-ol, 7

H

OH

H

H

Valpara-2,15-dien-1R-ol, 8

H

H

H

Valpara-3(19),15-dien -2S-ol, 10

Valpar-2-en-15-ol, 9 OMe

O MeO H

MeO

H

Valpara-1,3,13-triene, 11

H

H

H

3R-Methoxy-valpara-1,15diene, 12 O

H

3S-Methoxyvalpara-1,15-diene, 13 O

H

H

H

1R-Methoxy-valpara -2,15-diene, 14

H

Valpara-3,15-dien2-one, 15

HO

O

HO

HO H

H

H

O

Valpara-1,13-dien-3S-ol, 16

H

Valpara-2,15-dien1,4-dione, 17

O

H

H

3S,4S-Epoxy-valpar15-en-2-one, 18

AcO

H

H

H

3S-Hydroxy-valpara4,15-dien-2-one, 20

Valpara-3,15-dien-2S-ol, 19

O

HO H

H

2S-Acetoxy-valpara4,15-dien-3S-ol, 21

(MeO)2HC

H

Valpara-1,3,5,15-tetraene, 22

MeOOC

MeO

H

H

(3R)-4S-Methoxy-valpar -15-en-2-one, 23

MeOOC

O

MeOOC H

H

H

H

O 2,2-Dimethoxy-2,3-seco -valpar-15-en-3-one, 24

FIG. 5.2 Valparane diterpenoids.

H

H

O Dimethyl 3,19-dinor-2,3-secovalpar-15-ene-2,4-dioate, 25

H

Methyl 3,15-dioxo-16-nor -2,3-seco-valpar-2-oate, 26

7-6-5 Tricarbocyclic Diterpenes Chapter

5 141

20 1 2 19

4

H

16

8 7

H

AcO

AcO

9

18

6

3

O

11

15 14

17

12

Valparolone, 27

H

H

O

H

H

H O

O

Valparola-2(4),13-dien -3-one, 28

(4S)-2S-Acetoxy-valparol -15-en-3-one, 29

(4S)-2S-Acetoxy-valparol -8(14)-en-3-one, 30

FIG. 5.3 Valparolane diterpenoids.

TABLE 5.1 Natural Sources and Chemotypes of Valparanes Compounds

Natural Sources

Chemotypes

References

Valparene, 1

Halimium viscosum

Valparaiso, Spain

[5,7,8]

(Valpara-2,15-diene)

H. verticillatum Valpara-2,13-diene, 2

H. viscosum

Valparaiso

[7,9,10]

Valpara-1,3,15-triene, 3

H. viscosum

Valparaiso

[10]

2S,3R-Epoxy-valpar-15-ene, 4

H. viscosum

Valparaiso

[7–10]

H. verticillatum Valpar-15-en-2-one, 5

H. viscosum

Valparaiso

[10]

Valpara-1,15-dien-3R-ol, 6

H. viscosum

Valparaiso

[10]

Valpara-1,15-dien-3S-ol, 7

H. viscosum

Valparaiso

[10,11]

Valpara-2,15-dien-1R-ol, 8

H. viscosum

Valparaiso

[10]

Valpar-2-en-15-ol, 9

H. viscosum

Valparaiso

[10]

Valpara-3(19)-15dien-2S-ol, 10

H. viscosum

Valparaiso

[9–11]

Valpara-1,3,13-triene, 11

H. viscosum

Villarino de los Aires, Spain

[9]

3R-Methoxy-valpara-1,15diene, 12

H. viscosum

Villarino de los A

[9]

3S-Methoxy-valpara-1,15diene, 13

H. verticillatum

Villarino de los A

[9]

1R-Methoxy-valpara-2,15diene, 14

H. viscosum

Villarino de los A

[7,9]

Valpara-3,15-dien-2-one, 15

H. viscosum

Villarino de los A

[7,9]

Valpara-1,13-dien-3S-ol, 16

H. viscosum

H. verticillatum

[9] Continued

142 Studies in Natural Products Chemistry

TABLE 5.1 Natural Sources and Chemotypes of Valparanes—Cont’d Compounds

Natural Sources

Chemotypes

References

Villarino de los A Valpara-2,15-dien-1,4-dione, 17

H. viscosum

Celorico da Beira, Portugal

[7]

3R,4R-Epoxy-valpar-15en-2-one, 18

H. viscosum

Celorico da Beira

[7]

Valpara-3,15-dien-2S-ol, 19

H. viscosum

Celorico da Beira

[11]

3S-Hydroxy-valpara-4,15dien-2-one, 20

H. viscosum

Celorico da Beira

[11]

2S-Acetoxy-valpara-4,15dien-3S-ol, 21

H. viscosum

Celorico da Beira

[11]

Valpara-1,3,5,15-tetraene, 22

H. viscosum

Sa˜o Joao da Pesqueira, Portugal

[12]

(3R),4S-Methoxy-valpar15-en-2-one, 23

H. viscosum

Sa˜o Joao da Pesqueira

[12]

2,2-Dimethoxy-2,3-secovalpar-15-en-3-one, 24

H. viscosum

Valparaiso

[13]

Dimethyl 3,19-dinor-2,3-secovalpar-15-ene-2,4-dioate, 25

H. viscosum

Sa˜o Joao da Pesqueira

[12]

Methyl 3,15-dioxo-16-nor2,3-seco-valp-2-oate, 26

H. viscosum

Sa˜o Joao da Pesqueira

[12]

Valparanes The valparane structure was determined spectroscopically, including bidimensional experiments such as 13C/13C NMR INADEQUATE of valparene 1 [5], X-ray diffraction of cycloheptenone 5, and chemical correlations between them (Fig. 5.2; Table 5.1). As can be observed (Fig. 5.1), these compounds shown a 7-6-5 tricyclic skeleton with trans–anti–trans ring junctions. The absolute configuration of these compounds was established by circular dichroism (CD) experiments [6–13]. In the valparane group can be included seco-derivatives 24–26, possible biogenetic precursors of valparolanes, as will be shown later on.

7-6-5 Tricarbocyclic Diterpenes Chapter

5 143

TABLE 5.2 Natural Sources and Chemotypes of Valparolanes Natural Sources

Chemotypes

References

Valparolone, 27

Halimium viscosum

Valparaiso, Spain

[11,13]

Valparola-2(4),13-dien-3one, 28

H. viscosum

Villarino de los Aires, Spain

[9]

(4S)-2S-Acetoxy-valparol15-en-3-one, 29

H. viscosum

Celorico da Beira, Portugal

[11]

(4S)-2S-Acetoxy-valparol-8 (14)-en-3-one, 30

H. viscosum

Celorico da Beira

[11]

Compounds

Valparolanes These compounds are 6-6-5 tricyclic diterpenes, but are included in this review as valparanes derivatives as they proceed biogenetically from them. Their structures were established spectroscopically and by chemical correlations with valparanes [6,9,11,13]. The absolute configuration of these compounds was established by CD of a cyclohexanone derivative [13] (Fig. 5.3; Table 5.2).

Mulinanes and Azorellanes The diterpenes with mulinane and azorellane skeletons constitute a diterpene group isolated from plants that grow in the Andes, mainly from species of the genus Azorella, Laretia, and Mulinum (Apiaceae, Umbelliferae). Next are shown, compounds with mulinane (Fig. 5.4) and azorellane (Fig. 5.5) skeletons isolated until know and in Tables 5.3 and 5.4 the natural sources from which have been isolated, the biological activities, and references. These compounds are synthetic precursors, of a series of mulinanes and azorellanes derivatives in which biological activities have been carried out, but they are not reported in this review [14].

Mulinanes [15–19,21–37] Structurally, these compounds (or nearly of all them) are characterized by having an acid group in C-20, and a seven-member ring C functionalized. They show a wide variety of biological activities, such as antituberculosis, antimalaric, antibacterial, spermatostatic, and antihyperglycemic effects (Fig. 5.4; Table 5.3). Structure of mulinic acid was established by a combination of spectroscopic and X-ray diffraction [15]. As can be observed in

144 Studies in Natural Products Chemistry

14

15

17

16 12

11

CH2OAc O

7

O O

13

8 9

COOH

6

H

H

10

20

H

18

5

4

O

COOH

H

COOH

H

H

H

3

COOH

H

HO

H

H

O

19 1

O O

COOH

2

Isomulinic acid, 32

Mulinic acid, 31

Mulinenic acid, 34

17-Acetoxymulinic acid, 33

Mulinolic acid, 35

CH2OH COOH

H

COOH

H

CH2OH

H

HO

H

H

COOH

H

H

HO

COOH

H

H

H

O Mulina-11,13-dien -20-oic acid, 36

Mulinol, 38

17-Hydroxy -mulina-11,13-dien20-oic acid, 37

OH

HO

O COOH

H

HO

COOH

H

H

CH2OAc HOH C 2

H

14-Oxo-mulin12-en-20-oic acid, 42

20-Hydroxy-mulina11,13-dienyl acetate, 43

H

OHC

H

O

CH2OAc

AcO

COOH

H

HO

H

H

COOH

H

COOH

H

H O

O

11-Oxo-mulina-12,14-dien-20-oic acid, 46

COOH

7α-16-Dihydroxy-mulina11,13-dien-20-oic acid, 45

16-Hydroxy-mulina11,13-dien-20-oic acid, 44 O

COOH

H

H H

H

O COOH

H

COOH HOH2C

H

H

H

13α,14α-Dihydroxy-mulin -11-en-20-oic acid, 41

O

13-epi-Mulinolic acid, 40

11,12α-Epoxy-mulin-13-en-20-oic acid, 39

16-Oxo-mulin-12-en -20-oic acid, 48

11,14-Dioxo-mulin-12,-en-20-oic acid, 47

Mulinone A, 50

13α-Hydroxy-14-oxo -mulin-11-en-20 oic acid, 49

OAc CH2OAc H

HO

COOH

CH2OH HOOC

H

H

H

H

H

COOH CH2OAc

H H

OAc 7β-Hydroxy-mulina -9,12-dienyl acetate, 52

Mulinone B, 51 OAc

Mulina-11,13-dien -20-ol, 54

Mulin-13β-ol, 53 OAc

HO

HO H

H

COOH

H

COOH

H

COOH

H

H

H

H

18-Acetoxy-mulina-11,13dien-16,20-dioic acid, 55

O H H

OH 7β-Acetoxy-9-epimulin-11-en-13β-ol, 56

14α-Hydroxy-mulina-11,13(16) -dien-20-oic acid, 57

11α-Hydroxy-14-oxomulin-12-en-20-oic acid, 59

15α-Acetoxy-mulina-11,13 -dien-20-oic acid, 58

H

HO

Mulina-11,13-diene, 60

H

H

H OAc

2S-Acetoxy-mulin -11-ene-13R-ol, 61

OAc 2S-Hydroxy-mulina -11,13-dienyl acetate, 62

FIG. 5.4 Mulinane diterpenoids.

Fig. 5.4, these compounds show a interannular junction, cis–anti–trans. Recently, compound 56 has been isolated and characterized as the the first 9-epi-mulinane—the only mulinane with interanular junction trans–syn– trans [24]. Mulinanes 36, 43, 46, and 54 structures have been confirmed by synthesis, as will be discussed later.

Azorellanes [14,20,38–50] These compounds do not present a functionalized C-20 but in C-13 is always present an oxygenated functionality. Mulinanes show a great variety of

5 145

7-6-5 Tricarbocyclic Diterpenes Chapter

OAc

17 15 8 13 9

16

HO

7

OH

6

20

H 10 1

12 11

H

18

5 3

4

19

H HO

Azorellan-13α-ol, 64

Azorellanol, 63

H

HO

HO Azorellan-13β-ol, 65

7-Deacetyl-azorellanol, 66

O O

OAc

HO O

H

O

H

H

H

HO

HO Azorellolide, 67

Dihydro-azorellolide, 68

Azorellanone, 69

13-epi-Azorellanol, 70

OAc CHO

CH2OAc H

H

HO

O

H

H3CO

HO

17-Acetoxyazorellan-13α-ol, 71

H

OH CH2OH

Azorellanol methyl ether, 72

Yaretol, 74

Azorelaldehyde, 73

FIG. 5.5 Azorellane diterpenoids.

TABLE 5.3 Mulinanes, Natural Sources, and Biological Activity Compounds Mulinic acid, 31

Natural Sources Mulinum crassifolium

Activity

References

Antituberculosis

[14–17]

Azorella compacta Isomulinic acid, 32

M. crassifolium

17-Acetoxymulinic acid, 33

M. crassifolium

Antituberculosis

[14,18]

Mulinenic acid, 34

M. crassifolium

Antituberculosis

[14,19,20]

[15]

Spermatostatic Mulinolic acid, 35

Mulina-11,13-dien20-oic acid, 36

M. crassifolium

Antihyperglycemic

Laretia acaulis

Antituberculosis

A. compacta

Gastroprotective

M. spinosum

Trypanocidal

A. compacta

Antihyperglycemic

L. acaulis

Antituberculosis

[14,17,20–25]

Spermatostatic

[14,17,22–28]

Gastroprotective Continued

146 Studies in Natural Products Chemistry

TABLE 5.3 Mulinanes, Natural Sources, and Biological Activity—Cont’d Compounds

Natural Sources

Activity

References

17-Hydroxymulina-11,13-dien20-oic acid, 37

A. compacta

[26]

Mulinol, 38

A. compacta

11,12a-Epoxymulin-13-en-20-oic acid, 39

A. compacta

[29]

13-epi-Mulinolic acid, 40

L. acaulis

[22,24]

13a,14aDihydroxy-mulin11-en-20-oic acid, 41

M. spinosum

Antimalaric

A. compacta

Antituberculosis

14-Oxo-mulin-12en-20-oic acid, 42

M. spinosum

20-Hydroxymulina-11,13dienyl acetate, 43

A. compacta

16-Hydroxy-mulin11,13-dien-20-oic acid, 44

A. compacta/ Mucor plumbeus

[32]

7a,16-Dihydroxymulina-11,13-dien20-oic acid, 45

A. compacta/ M. plumbeus

[32]

11-Oxo-mulina12,14-dien-20-oic acid, 46

A. compacta

Antibacterial

[33]

11,14Dioxo-mulin12en-20-oic acid, 47

A. compacta

Antibacterial

[33]

16-Oxo-mulin-12en-20-oic acid, 48

A. madreporica

Antibacterial

[34]

13a-Hydroxy-14oxo-mulin-11en-20-oic acid, 49

A. madreporica

Antibacterial

[34]

Mulinone A, 50

M. crassifolium

[35]

Mulinone B, 51

M. crassifolium

[35]

Antituberculosis

[14,16]

[14,30,31]

[30] Antimalaric

[31]

7-6-5 Tricarbocyclic Diterpenes Chapter

5 147

TABLE 5.3 Mulinanes, Natural Sources, and Biological Activity—Cont’d Compounds

Natural Sources

Activity

References

7b-Hydroxymulina-9,12-dienyl acetate, 52

M. compacta

[35]

Mulin-13b-ol, 53

Azorella compacta

[17]

Mulina-11,13dien-20-ol, 54

A. compacta

[17]

18-Acetoxy-mulina11,13-dien-16,20dioic acid, 55

A. compacta

7b-Acetoxy-9-epimulin-11-en-13bol, 56

A. trifurcata

[24]

14a-Hydroxymulina-11,13(16)dien-20-oic acid, 57

A. trifurcata

[24]

15a-Acetoxymulina-11,13-dien20-oic acid, 58

A. trifurcata

[24]

11-a-Hydroxy-14oxo-mulin-12en-20-oic acid, 59

A. trifurcata

[24]

Mulina-11,13diene, 60

A. compacta

2S-Acetoxy-mulin11-en-13R-ol, 61

A. spinosa

[37]

2S-Hydroxymulina-11,13dienyl acetate, 62

A. spinosa

[37]

Gastroprotective

Gastroprotective

[17,24]

[36]

biological activities, including trypanocidal, analgesic, antiinflammatory, antiNF-kB (antinuclear factor kappa-light-chain-enhancer of activated B cells), and antituberculosis (Fig. 5.5; Table 5.4). The absolute stereochemistry of azorellolide, 67, has been proposed as shown in Fig. 5.5 based in X-ray diffraction [46]. Between these compounds can be considered the nor-diterpene

148 Studies in Natural Products Chemistry

TABLE 5.4 Azorellanes, Natural Sources, and Biological Activity Compounds

Natural Sources

Activity

References

Azorellanol, 63

A. compacta

Trypanocidal

[14,17,28,38–41]

Analgesic Antiinflammatory Anti-NF-kB Antituberculosis Azorellan13a-ol, 64

A. madreporica

Azorellan13b-ol, 65

A. yareta

7-Deacetylazorellanol, 66

Laretia acaulis

Antituberculosis

[14,17,24,42,43]

Trichomonacidal

[17,24,44]

Toxoplasmacidal

[14,39,40,45]

A. compacta

A. compacta

Analgesic Antiinflammatory Anti-NF-kB Antituberculosis

Azorellolide, 67

A. cryptantha

Dihydroazorellolide, 68

A. cryptantha

Azorellanone, 69

A. yareta

Spermatostatic

[20,46] [46]

Spermatostatic

[14,39,47]

Analgesic Antiinflammatory Antituberculosis 13-epiAzorellanol, 70

Laretia acaulis

Antituberculosis

[14,24,40]

17-Acetoxyazorellan13a-ol, 71

A. madreporica

Antituberculosis

[14,41,48]

Azorellanol methyl ether, 72

A. compacta

[17]

Azorelaldehyde, 73

A. cryptantha

[49]

Yaretol, 74

A. madreporica

Antituberculosis

[14,50]

7-6-5 Tricarbocyclic Diterpenes Chapter

5 149

yaretol 74, a rearranged mulinane whose structure was confirmed by X-ray diffraction but not its absolute configuration [50]. This structure can be understood through oxidation, followed by the cyclopropane ring opening of an azorellane.

Cyathane Family Tricyclic diterpenes of the cyathane family are divided into six groups; how they are divided mainly depends on their natural origins, which names these compound groups. They have been isolated from both terrestrial and marine sources. Many of these compounds show antibacterial/antimicrobial activity and many are antitumorals against human cancer cell lines, but perhaps their more important therapeutic potential is derived from their ability to induce NGF release from glial cells. NGF is a specific neurotrophin for joining the basal forebrain cholinergic neurons, associated with Alzheimer’s disease (AD). It has been comprobed by preclinic and clinic studies that by therapies carried out with NGF is achieved a notable reduction of the neuronal loss and disminution of the cognitive loss [51]. It will be further described when examining the different groups of cyathane derivatives, with indication of the more interesting characteristic of each case. Figs. 5.6–5.11 show their structures, and Tables 5.5–5.10 show their natural sources, biological activities, and references.

Cyathins, Cyafrins, and Derivatives [51–65] In 1971 [118], Ayer and coworkers communicated the isolation and preliminary characterization of a mixture of the first compounds of this class with antibiotic activity found in the bird’s nest fungus Cyathus helenae Brodie extract, but they could not be totally characterized (Fig. 5.6; Table 5.5). In 1972, Ayer and Taube published the first fully-characterized structure of these kinds of compounds called cyathin A3 75 and allocyathin B3 76 [52]. The absolute configuration of cyathin A3 75 was assigned as shown in Fig. 5.6 by the exciton chirality method [54]. In order to describe the first series of these compounds, Ayer and coworkers proposed a nomenclature [55] based on the different unsaturation degrees of the compounds. The natural products with 20 carbons and 30 hydrogens are classified as cyathins A series; the ones with 28 hydrogens are series B, and ones with 26 hydrogens are classified as series C. The subscript after the letter refers to the number of oxygens in the molecules. Isomers of the first cyathins isolated were designed as allocyathins. For example, cyathin B3 has the same empirical formula as allocyathin B3. Since the 1970s, a large number of compounds with a cyathane skeleton have been isolated, so this nomenclature became obsolete; and, as the number of diterpenes increased they were incorporated into new groups with different names, such as cyafrins, sarcodonins, scabronines, erinacines,

150 Studies in Natural Products Chemistry O

O

O

O

O

16 13

HOH2C

14 6 5

12 11 10

15

7 4

8 17

9

HOH2C

OHC

1

H

HO

H

HO

H

HO

H

HO

2

3 18

19

HOH2C

OHC H

HO

20

CH2OH Allocyathin B3, 76

Cyathin A3, 75

Cyathin B3, 77

OH

O

HOH2C

OHC

O

O

O

O

Cyathin A4, 79

Cyathin C3, 78

HOH2C

HOH2C

HOH2C

H

OH O

H

HO

H

H

HO

H

HO

O

H

HO O

COOH Neoallocyathin A4, 80

Cyathin C5, 81

OH

Allocyafrin B4, 84

Cyafrin B4, 83

Cyafrin A4, 82

OH

OH

OH

OH

O HOH2C

O

15

H

HO

Cyafrin A5, 85

AcOH2C

AcOH2C

HOH2C OH H

HOH2C

11

H

AcO

11,15-Diacetylcyathatriol, 87

Cyathatriol, 86

H

HO

15-Acetylcyathatriol, 88

O

OH

H

AcO

11-Acetylcyathatriol, 89 OH

O 14

OHC

OHC

12

12

H

H

H

H 3

Allocyathin B2, 90

Cyathin B2, 91

HO

HOH2C

HOH2C

HOH2C

O

H

OH

HOH2C

OH

HO

H

H

Cyathin I, 103

OH

HO

Cyathin J, 104 OH

OH

HO HOH2C

HOH2C

HOH2C

AcO

O

Cyathin H, 102

Cyathin G, 101

H

H

H

OH

OH

Cyathin L, 106

Cyathin K, 105

Cyathin M, 107

OH

OH

HO

OH

H

MeO

H O

Cyathin N, 108

Cyathin O, 109 MeO

OH

OHC

OHC

O

H

H

O Cyathin P, 110

Cyafrin D, 111

FIG. 5.6 Cyathin, cyafrin, and derivative diterpenoids.

O

O

O

HOH2C

OH

HOH2C

O

H

MeO

O

HOOC

HO

HO

HO

OH

HOH2C

Cyathin E, 99

Cyathin D, 98 OH

H

Cyathin F, 100

H

OHC

O

H

OHC MeO

97

OHC

OHC

HOOC

OH

OH

MeO

OH

H

96

11-Acetylcyathin A3, 95

OH

O

H

Erinacol, 94

Cyatha-3,12-dien14-one, 93

OH

HO

AcO

MeO

Cyatha-3,12-diene, 92

OH

O

3

Cyafrin E, 112

7-6-5 Tricarbocyclic Diterpenes Chapter

5 151

TABLE 5.5 Cyathanes, Natural Sources, and Biological Activity Compounds

Natural Sources

Activity

References

NGF synthesis

[51–53]

Cyathin A3, 75

Cyathus helenae

Allocyathin B3, 76

C. helenae

[52–54]

Cyathin B3, 77

C. helenae

[55]

Cyathin C3, 78

C. helenae

[55]

Cyathin A4, 79

C. helenae

[56]

Neoallocyathin A4, 80

[56]

Cyathin C5, 81

C. helenae

[56]

Cyafrin A4, 82

Cyathus africanus

[54]

Cyafrin B4, 83

C. africanus

[54]

Allocyafrin B4, 84

C. africanus

[54]

Cyafrin A5, 85

C. africanus

[54]

Cyathatriol, 86

Cyathus earlei

[57]

11,15Diacetyl-cyathatriol, 87

C. earlei

[57]

15-Acetylcyathatriol, 88

C. earlei

Inhibition NO

[57,58]

Antiinflammatory Antitumor 11-Acetylcyathatriol, 89

C. earlei

Allocyathin B2, 90

C. earlei

Cyathin B2, 91

C. earlei

[57]

Cyatha-3,12-diene, 92

Hericium erinaceum

[59]

Cyatha-3,12dien-14-one, 93

H. erinaceum

Erinacol, 94

H. erinaceum

11-Acetylcyathin A3, 95

H. erinaceum

96

Strobilurus tenacellus

[57,58] Antibacterial

NGF synthesis

[57]

[60] [61] [61]

Antimicrobial

[62]

Continued

152 Studies in Natural Products Chemistry

TABLE 5.5 Cyathanes, Natural Sources, and Biological Activity—Cont’d Compounds

Natural Sources

Activity

References

97

S. tenacellus

Antitumor

[62]

Cyathin D, 98

Cyathus africanus

[58]

Cyathin E, 99

C. africanus

[58]

Cyathin F, 100

C. africanus

Inhibition NO

[58]

Antiinflammatory Cyathin G, 101

C. africanus

Cyathin H, 102

C. africanus

[58] Inhibition NO

[58]

Antiinflammatory Cyathin I, 103

Cyathin J, 104

Cyathin K, 105

Cyathus hookeri

Inhibition NO

Cyathus gansuensis

Inhibition NO

C. gansuensis

Inhibition NO

[63]

Antiinflammatory [64]

Antiinflammatory [64]

Antiinflammatory Cyathin L, 106

C. gansuensis

Inhibition NO

[64]

Antiinflammatory Cyathin M, 107

C. gansuensis

Inhibition NO

[64]

Antiinflammatory Cyathin N, 108

C. gansuensis

Inhibition NO

[64]

Antiinflammatory Cyathin O, 109

C. gansuensis

Inhibition NO

[64]

Antiinflammatory Cyathin P, 110

C. gansuensis

Inhibition NO

[64]

Antiinflammatory Cyafrin D, 111

Cyathus africanus

Antitumor

[65]

Cyafrin E, 112

C. africanus

Antitumor

[65]

7-6-5 Tricarbocyclic Diterpenes Chapter

5 153

striatals, striatins, glaucopines, cyrneines, nigernins, onychiols, and cyanthiwigins. As will be shown later on, compounds 75, 76, 90, and 91 have been synthesized.

Sarcodonins, Neosarcodonins [66–72,75], and Scabronines [73,74,76–82] Sarcodonins, neosarcodonins, and scabronines are compounds with cyathane skeletons, isolated from the fungus Sarcodon scabrosus (Fig. 5.7; Table 5.6). Unfortunately, in the literature for these compounds, mistakes appear regarding C18 configuration (for some derivatives), as well as mistakes referencing the names or denominations of some group components. It has been found that the same compound will be given different names; or that the same name will be used to refer to different compounds. Here, we will try to resolve this misunderstanding by indicating the correct structure and denomination. The main characteristic of sarcodonins and neosarcodonins is that all of them present functionalized C-19, with a hydroxy group or esterified (Fig. 5.7; Table 5.6). The first members of this group (sarcodonin A 113 and sarcodonin G 114) were isolated from the fungus S. scabrosus in 1989 by Shibata and coworkers [66]. In this publication, there is a misrepresentative drawing of the sarcodonin G C18 configuration, which lead to errors for several groups. However, in the same publication, it includes a perspective drawing obtained by X-ray analysis where it clearly states the configuration at C18 of sarcodonin G as S. The configuration at C18 as S, of these compounds, was confirmed by Piers and coworkers through X-ray diffraction of a sarcodonin G derivative [67]. In 2000, the same authors described the synthesis of (
) sarcodonin G 114 [68]. The configuration of scabronines K–M 135–137 [80,81] and secoscabronine M, 138 [82] derivatives at C18 was proposed according to biogenetic reasons, and through comparison with sarcodonin G 114. As this compound, 114, has S configuration at C18, in Fig. 5.7 appears the correct structures for scabronines K–M 135–137 and secoscabronine M, 138, for reference. However, C18 configuration of some compounds in these groups has not been established or proposed. In 1998, the isolation of sarcodonin L and M [75] was communicated without establishing the configuration at C11 and C14, respectively. In the same year, the structures of scabronines B 125 and C 126 appeared in literature, stating that scabronine B 125 was identical to sarcodonin M and scabronine C 126 was identical to sarcodonin L [76,80] (Fig. 5.7). In the same year, scabronines A 123, episcabronine A 124, scabronine G 130, and scabronine J 133 [74] were reported by Ohta and coworkers; and, in this year, this group also communicated the structure determination of scabronine A 123 [73] and scabronines B–F 125–129 [76].

154 Studies in Natural Products Chemistry OH

O

OHC

OH

OHC

OHC

CH2OH

H 18

H

H

19 CH2OH

CH2OH

Sarcodonin A, 113

OH

OHC

OHC

OHC

H

H

H CH2O-oleoyl

CH2O-linoleoyl Linoleoyl-sarcodonin A, 116

CH2O-stearoyl Estearoyl-sarcodonin A, 118

Oleolyl-sarcodonin A, 117 OH

OH

MeO O

OHC H

OHC H

MeO

H

MeO CH2OH

CH2OH Neosarcodonin A, 119

MeO

O COOH

H

COOH

PhCOOH2C H

PhCOO

COOH

COOH

Scabronine C, 126 (Sarcodonin L)

OH

OHC

COOH

H

PhCOO

Scabronine B, 125 (Sarcodonin M)

Episcabronine A, 124 OAc

O

PhCOOH2C

COOH 11

H

OH

PhCOOH2C

O

14

MeO

Scabronine A, 123

Neosarcodonin O, 122

OH

O

H

H

CH2OH

Neosarcodonin C, 121

HO

MeO

H

CH2OH

Neosarcodonin B, 120

HO

OH

MeO

HO

OHC

MeO

Sarcodonin I, 115

OH

OH

MeO

CH2OH

Sarcodonin G, 114

O

OHC

COOH

OHC

COOH

H

H

Scabronine E, 128

Scabronine D, 127 OH

Scabronine G, 130

Scabronine F, 129

OH

OMe

OH

14

PhCOOH2C

PhCOOH2C

OHC

11

H

HO

H

HO

OHC

COOH

MeO

CH2OH

CH2OH

19 CH2OH

Scabronine J, 133

Scabronine H, 132

15-Benzoyl-cyatha-3,12 -diene-11R,14S,19-triol, 131 (Ma's scabronine G, 131 ) O

OH

17,19-Dihydroxy-14S-methoxy -cyatha-3,5(10),11-trien-15-al, 134 (Ma's scabronine J, 134)

O MeO

CH2OH

OHC H H

CH2OH

PhCOOH2C

H CH2OH

Scabronine K, 135

OHC

CH2OH Scabronine L, 136

O

HO

H

HO

CH2OH

H

H

O

H MeO

H CH2OH Scabronine M, 137

FIG. 5.7 Sarcodonin, neosarcodonin, and scabronine diterpenoids.

CH2OH

H O

Secoscabronine M, 138

O

7-6-5 Tricarbocyclic Diterpenes Chapter

5 155

TABLE 5.6 Sarcodonins, Neosarcodonins and Scabronines, Natural Sources, and Biological Activity Compounds Sarcodonin A, 113

Sarcodonin G, 114

Natural Sources

Activity

References

Sarcodon scabrosus

Antibacterial

[66–68]

S. scabrosus

Antibacterial

Antiinflammatory [66–69]

Antiinflammatory Antiproliferative Sarcodonin I, 115

S. scabrosus

Linoleoyl-sarcodonin A-116

S. scabrosus

Antiinflammatory

[71]

Oleoyl-sarcodonin A-117

S. scabrosus

Antiinflammatory

[71]

Estearoyl-sarcodonin A-118

S. scabrosus

Antiinflammatory

[71]

Neosarcodonin A, 119

S. scabrosus

Antiinflammatory

[72]

Neosarcodonin B, 120

S. scabrosus

Antiinflammatory

[72]

Neosarcodonin C, 121

S. scabrosus

Antiinflammatory

[72]

Neosarcodonin O, 122

S. scabrosus

Inhibition NO

[58,71]

[70]

Antiinflammatory Antitumor Scabronine A, 123

Sarcodon scabrosus

NGF synthesis

[73,74]

Episcabronine A, 124

S. scabrosus

NGF synthesis

[74]

Scabronine B, 125

S. scabrosus

NGF synthesis

[75,76]

Antibacterial Scabronine C, 126

S. scabrosus

NGF synthesis

[75,76]

Antibacterial Scabronine D, 127

S. scabrosus

NGF synthesis

[76]

Scabronine E, 128

S. scabrosus

NGF synthesis

[76]

Scabronine F, 129

S. scabrosus

NGF synthesis

[76]

Scabronine G, 130

S. scabrosus

NGF synthesis

[74]

15-Benzoyl-cyatha3,12-diene-11R,14S,19triol, 131

S. scabrosus

Antibacterial

[77,78]

Antifungal

Continued

156 Studies in Natural Products Chemistry

TABLE 5.6 Sarcodonins, Neosarcodonins and Scabronines, Natural Sources, and Biological Activity—Cont’d Compounds

Natural Sources

Activity

References

S. scabrosus

Antibacterial

[77,78]

(Ma’s scabronine G, 131) Scabronine H, 132

Antifungal Scabronine J, 133

S. scabrosus

[74]

17,19-Dihydroxy-14Smethoxy-cyatha-3,5 (10),11-trien-15-al, 134

S. scabrosus

[79]

Scabronine K, 135

S. scabrosus

[80]

Scabronine L, 136

S. scabrosus

[80]

Scabronine M, 137

S. scabrosus

Secoscabronine M, 138

S. scabrosus

(Ma’s scabronine J, 134)

NGF synthesis

[81] [82]

In 2004, Ma and coworkers reported the isolation and structure determination of two epimers (131 and 132) called scabronine G and scabronine H, respectively [77]. Unfortunately, the name of scabronine G had been already assigned by Ohta and coworkers [74] (see Fig. 5.7; Table 5.6). So, in order to avoid confusion on this subject, compound 131 appears as its systematic name as a cyathane derivative: 15-benzoyl-cyatha-3,12-diene-11,14,19-triol, 131 (Ma’s scabronine G, 131), and as a suggestion to call it episcabronine H. A similar problem happened with scabronine J 133, described in 1998 by Ohta and coworkers [74]. In 2008, Ma and coworker [79] gave the same name to compound 134 (see Fig. 5.7; Table 5.6). In order to avoid confusion, we will refer to compound 134 as its systematic name: 17,19-dihydroxy-14Smethoxy-cyatha-3,5(10),11-trien-15-al, 134 (Ma’s scabronine J, 134). The absolute configuration of scabronine A 123 [73] was determined by the Mosher method, and the one of scabronines B–F 125–129 [76] and secoscabronine M 138 by CD (Fig. 5.7; Table 5.6). The synthesis of 114, 123, 124, 127, and 130 will be shown later on.

7-6-5 Tricarbocyclic Diterpenes Chapter

5 157

Erinacines, Striatals, and Striatins [83–96,119] Erinacines, striatals, and striatins are cyathanes characterized for containing a pentose unit joint to the cyathane aglycone (Fig. 5.8; Table 5.7). Erinacines are cyathane-xylosides that show biological activity as stimulators of NGF synthesis and could be useful as medicines for degeneratives neuronal disorders, such as AD and peripheral nerve regeneration. They are isolated mainly from fungi as Hericium erinaceum, Hericium ramosum, Cyathus striatus, and Gerronema fibula. The erinacine A 139 structure was determined by hydrolysis with b-glucosidase that renders D-xylose and the aglycon that was identified as allocyathin B2 90 [83]. The erinacine E 143 structure was corroborated by X-ray diffraction [87]. Although the stereochemistry sugar part of erinacine F 144 could not be determined, what has been established is that erinacine G 145 is its 3,4-secoderivative [87]. The erinacine P 150 [91], also known as herical [92,93], absolute configuration was established by chemical correlation with erinacine A 139 and erinacine B 140, and the biogenesis of erinacine and the striatal family is proposed in these works. Erinacine Q 151 [94] was chemically correlated with erinacine P 150 and biosynthetically with erinacine C 141. Erinacine R 152 is the only compound of the family that has functionalized the six-member ring B. New analogs of erinacine E 142, such as CJ-15,544 153 and CJ-14,258 162, have been isolated from H. ramosum CL24240 when cultivated in different media. The natural product compound 153 is the oxidation product of erinacine E 143 in C30 . Erinacine I 147 is a cyathane derivative isolated from the cultured mycelia of H. erinaceum together with erinacine H 146. Erinacine J 148 can be envisioned as a secoderivative of compound 162, CJ-14,258. Striatals and striatins form a group of structurally related cyathanexyloxides. Striatins are artifacts formed in the natural products extraction with methanol, and, on mild treatment with acids, are reconverted to the corresponding striatals [92]. Nearly all of them are biologically actives as antifungal, antibacterial, and inhibitors of NGF synthesis. Later on, the synthesis of compounds 139, 140, and 143 are shown. Glaucopines and Cyrneines [97–103] Glaucopines are cyathanes’ derivatives isolated from Sarcodon glaucopus that show antiinflammatory biological activity. Cyrneines are cyathanes isolated from the mushroom Sarcodon cyrneus. Cyrneine A 166 was identified as 1-hydroxyallocyathin B2 and cyrneine B 167 as 4-hydroxyglaucopine C (Fig. 5.9; Table 5.8). The structures of the novel diterpenoids were determined by analysis of spectroscopic data and confirmed by synthesis of cyrneine A 166 and will be covered later on.

158 Studies in Natural Products Chemistry HO

HO

HO O

HO O

O

O

O

HO

HOH2C

OHC

4

HO

O

O

OHC

O

HO

HO HO

HO O

O

OHC H

EtO 3

Erinacine A, 139

Erinacine C, 141

Erinacine B, 140

Erinacine D, 142 HO

O HO

O

H

HO

O

HO

O

H

HO

HO

O

O

H

HO

HO

O

O

HO

HO

HO

H

HO H

HO

H

HO

H

HO

H

NaOOC H O O

Erinacine E, 143

Erinacine F, 144 HO

O HO

OH

HO

H

HO

O

O

HO

HO H

H

O

HO

H

O

O

HO

O H AcOH2C

Erinacine H, 146

Erinacine G, 145

AcOH2C

H

O

HO OHC

O

H

HO

H

AcO

O Erinacine I, 147

Erinacine J, 148

Erinacine K, 149

Erinacine P, 150 (Herical)

HO

HO

HO

O

O

O

HO

HO HO

O HO

O

O O

HOH2C

H

AcO

O

H

AcO

H

O

AcO

O H

MeO

Striatin A, 159

O

H

MeO

Striatin B, 160

Striatin C, 161

O

H

HO

O

H O

H MeO H OH

FIG. 5.8 Erinacines, striatals, and striatins.

Striatal D, 158 HO

O

HO

H H

H

Striatal C, 157

O HO

O HO

H

OHC H OH

HO

O

HO

O HO

O

H

OHC

H

H

O HO

H OH

Striatal B, 156

H

HO

HO

O

H

OHC

Striatal A, 155

O

H

HO O HO

H

AcO

O

O HO H

H

154

CJ-15,544, 153

O

O HO

O

H EtO

Erinacine R, 152

O

OHC

OHC

HO

Erinacine Q, 151

O

HO

3'

HO

H

AcO

O

HO

O

OHC H

AcO

O

H

HO

H H OH

HO

H

CJ-14,258, 162

H

O

7-6-5 Tricarbocyclic Diterpenes Chapter

5 159

TABLE 5.7 Erinacines, Striatals and Striatins, Natural Sources, and Biological Activity Compounds

Natural Sources

Activity

References

Erinacine A, 139

Hericium erinaceum

NGF synthesis

[83–85]

Erinacine B, 140

H. erinaceum

NGF synthesis

[83,85]

Erinacine C, 141

H. erinaceum

NGF synthesis

[83,85]

Erinacine D, 142

H. erinaceum

NGF synthesis

[85,86]

Erinacine E, 143

H. erinaceum

NGF synthesis

[85,87,88]

k-Opioid receptor agonist Erinacine F, 144

H. erinaceum

NGF synthesis

[85,87]

Erinacine G, 145

H. erinaceum

NGF synthesis

[87]

Erinacine H, 146

H. erinaceum

NGF synthesis

[89]

Erinacine I, 147

H. erinaceum

NGF synthesis

[85,89]

Erinacine J, 148

H. erinaceum

Erinacine K, 149

H. erinaceum

Antibiotic

[85,90]

Erinacine P, 150

H. erinaceum

Antifungal

[85,91–93]

(Herical)

Hericium ramosum

Antibacterial

[85,90]

Cytotoxic Hemolytic

Erinacine Q, 151

H. erinaceum

[85,94]

Erinacine R, 152

H. erinaceum

[85,95]

CJ-15,544, 153

H. ramosum

154

H. erinaceum

Striatal A, 155

Cyathus striatus

k-Opioid receptor agonist

[88] [60]

Antifungal

[91,92]

Antibacterial Leishmanicidal Cytotoxic Striatal B, 156

C. striatus

Antifungal

[92]

Antibacterial Leishmanicidal Cytotoxic Continued

160 Studies in Natural Products Chemistry

TABLE 5.7 Erinacines, Striatals and Striatins, Natural Sources, and Biological Activity—Cont’d Compounds

Natural Sources

Activity

References

Striatal C, 157

C. striatus

Antifungal

[92]

Antibacterial Cytotoxic Striatal D, 158

Gerronema fibula

Striatin A, 159

Cyathus striatus

[92] Antibacterial

[92,96]

Antifungal Striatin B, 160

C. striatus

Antibacterial

[92,96]

Antifungal Striatin C, 161

C. striatus

Antibacterial

[92]

Antifungal CJ-14,258, 162

k-Opioid receptor agonist

Hericium ramosum

MeO

OH

[88]

OH

OH O

O

OHC

OHC

OHC H

H O

4

H MeO

Glaucopine A, 163

OH

HOH2C OH

Cyrneine A, 166

O

OH

OHC

Glaucopine C, 165

Glaucopine B, 164

OH 4

OHC O

Cyrneine B, 167

O

O

Cyrneine C, 168

O

OHC

OH

OHC

H H

H

Cyrneine D, 169

O

H

O

Cyrneine E, 170

FIG. 5.9 Glaucopines and cyrneines.

Nigernins, Onychiols, and Other Derivatives [104–111] Nigernins A–F 171–176 are cyathanes isolated from the fruiting bodies of basideomycete Phellodon niger. Nigernin F 176 is a 3,4-seco cyathane derivative that appears through oxidation of nigernin E 171. The structures of these compounds were established by spectroscopic methods.

7-6-5 Tricarbocyclic Diterpenes Chapter

5 161

TABLE 5.8 Glaucopines and Cyrneines, Natural Sources, and Biological Activity Compounds

Natural Sources

Activity

References

Glaucopine A, 163

Sarcodon glaucopus

Antiinflammatory

[97]

Glaucopine B, 164

S. glaucopus

Antiinflammatory

[97]

Glaucopine C, 165

S. glaucopus

Antiinflammatory

[98,99]

Cyrneine A, 166

Sarcodon cyrneus

NGF

[100–102]

Cyrneine B, 167

S. cyrneus

NGF

[100,102]

Cyrneine C, 168

S. cyrneus

NGF

[102]

Cyrneine D, 169

S. cyrneus

NGF

[102]

Cyrneine E, 170

S. cyrneus

[103]

From the fern Onychium japonicum, have been reported a series of cyathane derivatives including onychiol B, 177 and glycosyl derivatives of unknown absolute configuration 178–181 [106,107]. Cyathin derivatives 182–185 have been isolated from Higginsia sp. sponges, showing the same tricyclic system trans–anti–trans as nigernin B 172 (Fig. 5.10; Table 5.9).

Cyanthiwigins [112–117] The structure and absolute configuration of the first cyanthiwigins isolated, cyanthiwigins A–D 186–189, determined spectroscopically, confirmed by X-ray diffraction and Mosher ester analysis, were published in 1992 by Kashman and coworkers. Currently, 37 cyanthiwigin compounds are known, isolated mainly from Epipolasis reiswigi and Myrmekioderma styx. Structurally, these compounds have the tricyclic skeleton of cyathanes, present stereochemistry cis between rings A and B, respectively, and the angular methyls disposition on carbons C6 and C9 is syn in contrast with the rest of cyathanes with disposition anti of these methyls. Cyanthiwigins can be divided into six groups: (a) cyanthiwigins A–G 186–192 and cyanthiwigin AF 217 that possess a double bond in C12; (b) compounds with an epoxide in C11–C12 or C12–C13 of cyanthiwigins H, J, K, M–Q (193, 195, 196, 198–202), cyanthiwigin AB 213, cyanthiwigin AE 216, cyanthiwigin AG 218, cyanthiwigins 219, 220, epipolone 221, and epipolol 222; (c) compounds with double bond in C13–C14 as the cyanthiwigin R–X (203–209); (d) cyanthiwigins that possess an a,b-unsaturated ketone

MeO

O

MeO

O

O HOOC

HOOC H

H

HOOC

HOOC H

H

Nigernin A, 171

O

H

Nigernin B, 172

H

Nigernin D, 174

Nigernin C, 173

O

O

MeO

MeO O

O

HOOC

HOOC

H

H

O

O

Nigernin F, 176

Nigernin E, 175 OH OH

OH OH

OH OH O

H

HO

H

OH

H

H

HO HO

O

H

O

H

OH O

H

OH

H

HO HO

178

Onychiol B, 177

179

OH OH

HO

H

OH O

OH HO

H

OH HO HO

HO HO H

OH O

H

OH

H O

H O

180 O

181 O

HOH2C

H

H

OH O

O

AcOH2C

H

H OH

182

H

H

H OH

183

H OH

H

184

185

FIG. 5.10 Nigernins, onychiols, and other derivatives.

TABLE 5.9 Nigernins, Onychiols, and Other Derivatives Compounds

Natural Sources

Nigernin A, 171

Phellodon niger

[104]

Nigernin B, 172

P. niger

[104]

Nigernin C, 173

P. niger

[105]

Nigernin D, 174

P. niger

[105]

Nigernin E, 175

P. niger

[105]

Nigernin F, 176

P. niger

[105]

Onychiol B, 177

Onychium japonicum

Activity

Inhibitory action on smooth muscle

References

[106,107]

7-6-5 Tricarbocyclic Diterpenes Chapter

5 163

TABLE 5.9 Nigernins, Onychiols, and Other Derivatives—Cont’d Compounds

Natural Sources

178

O. japonicum

[108]

179

O. japonicum

[108]

180

O. japonicum

[108]

181

O. japonicum

[108]

182

Higginsia sp.

[109–111]

183

Higginsia sp.

[109]

184

Higginsia sp.

[109]

185

Higginsia sp.

[111]

Activity

References

in the seven-member ring such as cyanthiwigin Y–AA (210–212); (e) cyanthiwigin I 194 and cyanthiwigin L 197 which show an exocyclic double bond between C12 and C15; and (f ) rearranged analogs of cyanthiwigin such as the spirocyclic cyanthiwigin AC 214 and cyanthiwigin AD 215. Epipolone 221 and epipolol 222 are isolated from the sponge E. reiswigi, but their absolute configuration has not been established [117] (Fig. 5.11; Table 5.10). This cyathane group is one of the most studied due to their member’s interesting properties, as will be seen later on in the synthesis of several members, such as 186–188, 191–193, 206, 207, 211, and 214.

Verrucosanes Family [120–144] The verrucosane diterpenes family includes verrucosane, neoverrucosane, epi-neoverrucosane, homoverrucosane, epi-homoverrucosane, and gagunin, or 10,13-bis-epi-homoverrucosane (Fig. 5.12). They are isolated mainly from the liverworts (Hepaticae) of genus Mylia, Scapania, Gyrothyra, Schistochila, Plagiochila, Jamesoniella, and Heteroscyphus (Jungermanniales) and Fossombronia (Metzgeriales), and they cannot be found in plants nor in fungus although recently they have been found in sponges as Epipolasis kushimotoensis [132] or in Phorbas sp. [140], and in the phototrophic bacterium Chloroflexus aurantiacus [136]. Generally, they have been obtained from wild specimens, and some from Plagiochila adianthoides grown in an axenic culture [135].

164 Studies in Natural Products Chemistry 16 14 15

11

O

7

6 5

12

17

H 9

10

H

4

18

3

OH

H O

H O

H

1

OH

H OH

H

H OH

H

O

H

2

20 19

Cyanthiwigin B, 187

Cyanthiwigin A, 186

Cyanthiwigin C, 188

Cyanthiwigin D, 189

Cyanthiwigin E, 190

HO O

O

H

H

H

H O

H

Cyanthiwigin F, 191

Cyanthiwigin G, 192

H

H O

H

O

H

Cyanthiwigin H, 193

HO

OH

O

Cyanthiwigin I, 194

Cyanthiwigin J, 195

HO

HO

OH

H

O O

O H

H OH

H

H OH

H

Cyanthiwigin K, 196

O

Cyanthiwigin L, 197

O

H O

OH

H

Cyanthiwigin M, 198

H O

O

H

O

H

Cyanthiwigin O, 200

Cyanthiwigin N, 199

O

HO

O H

H O

O

H

O

O

H

OH

H

HO

O

H

Cyanthiwigin Q, 202

Cyanthiwigin P, 201

O

H

HOO

H O

H

HOO

Cyanthiwigin S, 204

Cyanthiwigin R, 203

O

H

Cyanthiwigin T, 205 O

O H HO

O

H

OH

H

Cyanthiwigin U, 206

OH

H

HO

H

H HO

OH

H

OH

H

Cyanthiwigin W, 208

Cyanthiwigin V, 207

O

H

HO

OH

H

Cyanthiwigin Y, 210

Cyanthiwigin X, 209

O OH

O H

H OH

H

O

H

Cyanthiwigin Z, 211

O

O

H

Cyanthiwigin AA, 212

OHC

H

H

HO

HO

Cyanthiwigin AB, 213

Cyanthiwigin AC, 214

H O

H

Cyanthiwigin AD, 215

OH O

O

O

HOH2C

H H

Cyanthiwigin AE, 216

O

O

Cyanthiwigin AG, 218

O

O

Epipolone, 221

H

H

219

220

OH

O H

H H

H OAc

O

H

Cyanthiwigin AF, 217

O

H

H

H

FIG. 5.11 Cyanthiwigins.

OAc

O

O

O

H

H

Epipolol, 222

7-6-5 Tricarbocyclic Diterpenes Chapter

5 165

TABLE 5.10 Cyanthiwigins, Natural Sources, and Biological Activity Compounds

Natural Sources

Activity

References

Cyanthiwigin A, 186

Epipolasis reiswigi

Anticancer

[112,113]

Myrmekioderma styx

Antituberculosis

E. reiswigi

Anti-HIV anticancer

Cyanthiwigin B, 187

M. styx

[112,113]

Antituberculosis Cyanthiwigin C, 188

E. reiswigi M. styx

HBV hepatitis B virus

[112–114]

Antibiotic Anticancer Antituberculosis Cyanthiwigin D, 189

E. reiswigi

Anticancer

M. styx

Antituberculosis

Cyanthiwigin E, 190

M. styx

Anticancer

[112]

Cyanthiwigin F, 191

M. styx

Anticancer

[112]

Cyanthiwigin G, 192

M. styx

[112]

Cyanthiwigin H, 193

M. styx

[112]

Cyanthiwigin I, 194

M. styx

Cyanthiwigin J, 195

M. styx

[112]

Cyanthiwigin K, 196

M. styx

[112]

Cyanthiwigin L, 197

M. styx

[112]

Cyanthiwigin M, 198

M. styx

[112]

Cyanthiwigin N, 199

M. styx

[112]

Cyanthiwigin O, 200

M. styx

[112]

Cyanthiwigin P, 201

M. styx

[112]

Cyanthiwigin Q, 202

M. styx

[112]

Cyanthiwigin R, 203

M. styx

[112]

Cyanthiwigin S, 204

M. styx

[112]

Cyanthiwigin T, 205

M. styx

[112]

Cyanthiwigin U, 206

M. styx

[112]

Cyanthiwigin V, 207

M. styx

[112]

Anticancer

[112,113]

[112]

Continued

166 Studies in Natural Products Chemistry

TABLE 5.10 Cyanthiwigins, Natural Sources, and Biological Activity—Cont’d Compounds

Natural Sources

Activity

Cyanthiwigin W, 208

M. styx

[112]

Cyanthiwigin X, 209

M. styx

[112]

Cyanthiwigin Y, 210

M. styx

[112]

Cyanthiwigin Z, 211

M. styx

Cyanthiwigin AA, 212

M. styx

[112]

Cyanthiwigin AB, 213

M. styx

[115]

Cyanthiwigin AC, 214

M. styx

[115]

Cyanthiwigin AD, 215

M. styx

[115]

Cyanthiwigin AE, 216

Streptomyces spheroides

Antimicrobial

[116]

Cyanthiwigin AF, 217

S. spheroides

Antimicrobial

[116]

Cyanthiwigin AG, 218

S. spheroides

Antimicrobial

[116]

219

Myrmekioderma styx

Anticancer

[114]

220

M. styx

Anticancer

[114]

Epipolone, 221

Epipolasis reiswigi

[117]

Epipolol, 222

E. reiswigi

[117]

Anticancer

References

[112]

Verrucosanes, Neoverrucosanes, and epi-Neoverrucosanes The structure of these compounds was determined spectroscopically and by chemical correlation, and, in the case of verrucosa-2S,8R-diol 232, by X-ray diffraction and its absolute configuration by CD of its di-p-bromobenzoate derivative [125]. In a similar manner, the structure and stereochemistry of neoverrucosan-5R-ol, 235 [127] and 13-epi-neoverrucosan-5R-ol, 242 by X-ray diffraction of their p-bromobenzoyl derivatives [128] (Fig. 5.13; Table 5.11) were confirmed. As can be seen in Fig. 5.13, all of them show a 3,6,6,5 tetracyclic ring system in a cis–trans–anti–trans configuration. Verrucosanes 223, 235, and 254 have been synthesized as will be seen later on.

5 167

7-6-5 Tricarbocyclic Diterpenes Chapter

19 6

5

8

7

4

H

2

18

16

9

H 10

1

3

H

H

20

H

14 11 12

H

13

10 13

13 15 17

Homoverrucosane

epi-Homoverrucosane

19

19

6

8

6

5

4 18

C 9 7 A B H 10 1 3 2 H 14 D 16

Gagunin (Bis-epi-homoverrucosane)

13 15

B

20 18

4

A

11 3

2

C 9 H 10 1 H 14 D

7

12 16

17

Verrucosane

8

5

13 15

20 11

H H

13

12

17

Neoverrucosane

epi-Neoverrucosane

FIG. 5.12 Verrucosane-type skeletons.

Umabanol 253 could be considered as a rearranged verrucosane or neoverrucosane as will be seen in the biosynthesis part, with its structure determined by spectroscopic analysis.

Homoverrucosanes and epi-Homoverrucosanes Verrucosanes, neoverrucosanes, and 13-epi-neoverrucosanes are related with the homoverrucosanes and 13-epi-homoverrucosanes series 254–259 because, in smooth conditions, the opening of the cyclopropane ring permits the change from tetracyclic to tricyclic systems. As can be seen in Fig. 5.14, these compounds present a 7,6,5 tricarbocyclic ring system in the trans–anti–trans configuration. From liverwort Jamesoniella tasmanica together with the epi-neoverrucosane 243 are isolated butenolides 260–262 that show the cyathane skeleton, although the authors consider them as epi-neohomoverrucosenolides [129] (Fig. 5.14; Table 5.12). Gagunins Gagunins, 263–279, are compounds that can be classified as 10,13-bis-epihomoverrucosanes. They are isolated from Phorbas sp., sponges and present the two angular methyls at C7 and C10 in syn disposition, by contrary to the rest of the verrucosane family. These compounds show activity against the human leukemia cell line (K-562) (Fig. 5.15; Table 5.13). The cytotoxicity of the gagunins is modulated by the differential hydroxylation, or esterification, level present in a specific gagunin. Gagunin E is the most potent

168 Studies in Natural Products Chemistry 19 6

5

3

9

20

H

2

14 11 12

H OH

OAc

O

9

H 10

4 2 1 18

OH

8

7

H

H

H

H

OH

H

2

H OAc

H

OH

OH

OH

11

13 15

16 17

Verrucosa2S,9R-diol, 224

Verrucosan-2S-ol, 223

2S- Acetoxyverrucosan-11S-ol, 227

2S-Hydroxyverrucosan-9-one, 226

9R- Acetoxyverrucosan-2S-ol, 225

OH OAc

OH 9

9

H

2

H

11

H

2

OAc

H

Verrucosa2S,9R,13R-triol, 229

H

2

H

H 13

OH

OH

OH 11S- Acetoxyverrucosan-2S-ol, 228

H

2 13

OH

13

OH

8

9

H

2

H

OH

O

OH

OH 2S,13R-Dihydroxy -verrucosan-9-one, 231

9R-Acetoxy-verrucosa -2S,13R-diol, 230

Verrucosa2S,8R-diol, 232

19

OH

HO

9

5

2

H

2

H

4 2

H

OH

H H

13 15 17

Neoverrucosan -5R-ol, 235

(15S)-2S,16S-Epoxyverrucosan-16-ol, 234

HO

9

5 20

14 11 12

H 3

HO

HO

OH

9

H 10

1

16

Verrucos-13-ene -2S,9R-diol, 233

HO

8

7

18

O

13

6

HO

OH

OH

Neoverrucosa -5R,9S-diol, 236

O

O

OH

OH

5

H HOH2C

HOH2C

H

18

OHC

H

5

H

H

H

H H

H

H

9

12

OH

Neoverrucosa -5R,18-diol, 237

Neoverrucosa -5R,9S,18-triol, 238

5R,9S-Dihydroxy -neoverrucosan -18-al, 239

9S-Hydroxy -neoverrucosan -5-one, 240

O

O

9S-12S-Dihydroxy -neoverrucosan -5-one, 241 OAc

HO

HO 5

H

H

H H

13

8

5

H

H

O

5

CH2OAc

H H

13

H

13-epiNeoverrucosan -5-one, 244

O 8

13S-Hydroxy-13-epineoverrucosan -5-one, 245

8S-Acetoxy-13S-hydroxy13-epi-neoverrucosan -5-one, 246

O

HO

8

5

H

H H

OH

OAc

OH O 5

13

OH

20-Acetoxy-13-epineoverrucosa5R,12S-diol, 243

(+)-13-epiNeoverrucosan -5R-ol, 242

H

13

OH

H

13

OH

16

H

H H

H

13

13

13-epiNeoverrucosa5R,20-diol, 249

12-Acetoxy-13-epineoverrucosan-5-one, 250 19

HO

AcO

6

H H H

H OSO3Na

H

18 5 4

OSO3Na HO

H

7 1

2

3 16

H

H 14

20 10 13

15 17

5R-Hydroxy-13-epi11R-neoverrucosanylsulfate, 251

12

OAc

OH CH2OAc

8S,16-Diacetoxy13R-hydroxy-13-epineoverrucosan-5-one, 248

8S,13S-Dihydroxy -13-epi-neoverrucosan -5-one, 247

5

CH2OH

5R-Acetoxy-13-epi11R-neoverrucosanylsulfate, 252

Umabanol, 253

FIG. 5.13 Verrucosane, neoverrucosane, and epi-neoverrucosane diterpenoids.

11

7-6-5 Tricarbocyclic Diterpenes Chapter

5 169

TABLE 5.11 Verrucosane, Neoverrucosane and epi-Neoverrucosane, Natural Sources, and Biological Activity Compounds

Natural Sources

Verrucosan-2S-ol, 223

Mylia verrucosa

Activity

References [120,133,136,138]

Scapania bolanderi Chloroflexus aurantiacus Verrucosa-2S,9R-diol, 224

M. verrucosa

[120,121,123]

9R-Acetoxy-verrucosa-2Sdiol, 225

M. verrucosa

[120,122]

2S-Hydroxy-verrucosa-9one, 226

M. verrucosa

[120]

2S-Acetoxy-verrucosan-11Sol, 227

M. verrucosa

[120,122]

11S-Acetoxy-verrucosan-2Sol, 228

M. verrucosa

[120,122]

Verrucosa-2S,9R,13R-triol, 229

M. verrucosa

[124]

9R-Acetoxy-verrucosa2R,13R-diol, 230

M. verrucosa

[124]

2S,13R-Dihydroxyverrucosan-9-one, 231

M. verrucosa

[124]

Verrucosa-2S,8R-diol, 232

Gyrothyra underwoodiana

[125]

Verrucos-13-ene-2S,9R-diol, 233

Scapania bolanderi

[133]

2S,16S-epoxy-verrucosan16-ol, 234

M. taylorii

[126]

Neoverrucosan-5R-ol, 235

M. verrucosa

Antitumor

[127,132–134]

Scapania bolanderi Schistochila rigidula Neoverrucosa-5R,9S-diol, 236

Saprospira grandis

[142]

Neoverrucosa-5R,18-diol, 237

S. grandis

[142]

Continued

170 Studies in Natural Products Chemistry

TABLE 5.11 Verrucosane, Neoverrucosane and epi-Neoverrucosane, Natural Sources, and Biological Activity—Cont’d Compounds

Natural Sources

Activity

References

Neoverrucosa-5R,9S,18triol, 238

S. grandis

[142]

5R,9S-Dihydroxyneoverrucosan-18-al, 239

S. grandis

[142]

9S-Hydroxy-neoverrucosan5-one, 240

Saprospira sp.

[143]

9S,12S-Dihydroxyneoverrucosan-5-one, 241

Saprospira sp.

[143]

13-epi-Neoverrucosan-5Rol, 242

Plagiochila stephensoniana

[128,134]

Schistochila nobilis 20-Acetoxy-13-epineoverrucosa-5R,12S-diol, 243

Jamesoniella tasmanica

[129]

13-epi-Neoverrucosan5-one, 244

Fossombronia alaskana

[130]

13S-Hydroxy-13-epineoverrucosan-5-one, 245

F. alaskana

[130]

8S-Acetoxy-13S-hydroxy13-epi-neoverrucosan-5-one, 246

F. alaskana

[130,131]

8S,13S-Dihydroy-13-epineoverrucosan-5-one, 247

F. alaskana

[130]

8S,16-Diacetoxy-13-epineoverrucosan-5-one, 248

F. alaskana

[130]

13-epi-Neoverrucosa-5R,20diol, 249

Heteroscyphus planus

[137]

12-Acetoxy-13-epineoverrucosan-5-one, 250

Hamigera tarangaensis

[139]

5R-Hydroxy-13-epi-11Rneoverrucosanyl-sulfate, 251

Axinyssa tethyoides

[144]

5R-Acetoxy-13-epi-11Rneoverrucosanyl-sulfate, 252

A. tethyoides

[144]

Umabanol, 253

Epipolasis kushimotoensis

[132]

5 171

7-6-5 Tricarbocyclic Diterpenes Chapter

HO

HO

19 5

H

6

4

20

14 11 12

H

2

18

9

H 10

1

3

H

8

7

13

16

15 17

Homoverrucosa 2-en-5S-ol, 254

OH

HO

OH O

HO

6

5

13-epiHomoverrucosa 2-en-5S-ol, 255

5

6

H

2

18

H

2

CH2OAc

H 10

1 2

H

14

2

12

O 16

O 20-Acetoxy-4S,5R-epoxy-13-epineohomoverrucos15(17)-en-16,12S-olide, 260

H

13-epi-Homoverrucosa -2-en-5S,6R,8R-triol, 259

H

H 13

14 13

12

12

HO 15

CH2OAc

OH

CH2OAc

H

13 15

8

O

O

3

H

20

7

6

H

6R-Hydroxy-13-epihomoverrucosa -2-en-3-one, 258

19 5

5

H

5S,6R-Dihyhroxy-13-epihomoverrucosa -2-en-8-one, 257

13-epi-Homoverrucosa -2en-5S,6R-diol, 256

O

5

8

OH OH

HO

6

H

H 2

OH

O

15

O

16

16

O

O 20-Acetoxy-4S,5R-epoxy-13Rhydroxy-13-epi-neohomoverrucos15(17)-en-16,12S-olide, 261

O

20-Acetoxy-4S,5R-epoxy-14Rhydroxy-13-epi-neohomoverrucos15(17)-en-16,12S-olide, 262

FIG. 5.14 Homoverrucosane and epi-homoverrucosane diterpenoids.

cytotoxic of this group [140,141]; its structure has been determined on the basis of combined chemical and spectroscopic methods.

Other 7-6-5 Tricarbocyclic Diterpenes Gukulenins 280–285 [145,146] are a tetraterpenoid group isolated from the marine sponge Phorbas gukhulensis. Gukulenins are pseudodimers that show a skeleton with a bis-tropolone moiety, rings A–C are a rearranged variant of the polyoxygenated tricyclic system of gagunins isolated from the same organisms (Fig. 5.16; Table 5.14). Gukulenins exhibited cytotoxicity against different tumor cell lines in the range of 0.05–0.80 mM. Among other compounds with a 7-6-5 tricyclic system that have been isolated from different sources are the brown alga Dictyota dichotoma (Dictyoxetane, 286) [147] and the soft coral Sinularia dissecta (Mandapamate, 287) [148], or Sinularia maxima (Isomandapamate, 288) [149] (Fig. 5.17). However, their structures are not related biogenetically with the ones of this review and, for this reason, have not been considered.

172 Studies in Natural Products Chemistry

TABLE 5.12 Homoverrucosane and epi-Homoneoverrucosanes, Natural Sources, and Biological Activity Compounds

Natural Sources

Activity

References

Homoverrucosa-2-en-5S-ol, 254

Schistochila rigidula

Antitumor

[132,134]

13-epi-Homoverrucosa-2en-5S-ol, 255

S. nobilis

[134,135]

Plagiochila cristata Plagiochila adianthoides

13-epi-Homoverrucosa-2en-5S,6R-diol, 256

P. cristata

[135]

5S,6R-Dihydroxy-13-epihomoverrucosa-2-en-8-one, 257

P. cristata

6R-Hydroxy-13-epihomoverrucosa-2-en-3one, 258

P. cristata

13-epi-Homoverrucosa2-en-5S,6R,8R-triol, 259

P. cristata

20-Acetoxy-4S,5R-epoxy13-epi-neohomoverrucos-15 (17)-en-16,12S-olide, 260

Jamesoniella tasmanica

[129]

20-Acetoxy-4S,5R-epoxy13R-hydroxy-13-epineohomoverrucos-15(17)en-16,12S-olide, 261

J. tasmanica

[129]

20-Acetoxy-4S,5Repoxy-14R-hydroxy-13-epineohomoverrucos-15(17)en-16,12S-olide, 262

J. tasmanica

[129]

P. adianthoides [135]

P. adianthoides [135]

P. adianthoides [135]

P. adianthoides

BIOSYNTHESIS All 7-6-5 tricyclic diterpenes and their tetracyclic analogs 3-6-6-5 proceed from geranylgeranyl pyrophosphate (GGPP) (Scheme 5.1). The t,t,t-GGPP isomerize to R/S-geranyllinaloyl pyrophosphate (GLPP) and the late, through anti-Markovnikov cyclization, can generate the enantiomers ions 289 and 290 [131,150]. Ion 289 is the precursor of valparanes and valparolanes, and the rest proceed from the ion 290 via the bicyclic system 291.

5 173

7-6-5 Tricarbocyclic Diterpenes Chapter

O

O

nPr O

AcO HO

nPr

H

HO

nPr

H

O

H

2

O O

5

HO

O

O nPr

Gagunin B, 264

O O AcO

O

6

5

AcO

H O

OAc

HO

Gagunin G, 269

O

nPr

H

HO

H

2

H

H

2

O

2

H

AcO

H

OH

2

5

O

6

2

nPr O

H

O

O

O

O

O

O nPr

nPr

nPr

Gagunin N, 276

Gagunin M, 275

O

H

OH

H

nPr

O

O

HO O

6

5

AcO

H

nPr Gagunin L, 274

nPr

Et

O

O

HO O

6

O O

nBu

O nPr O

O

OH

H

O Gagunin K, 273

O

AcO

2

nPr Gagunin J, 272

nPr

H

O

O nPr

Gagunin I, 271

HO

O

6

5

O

O

O

5

nPr

H

O

O

HO

HO

Et

H

O

O

O

AcO

O O

6

5

nPr

nPr

O

O

AcO

O O

6

5

O

nPr

nPr

O

O

AcO O O

Gagunin H, 270

O

nPr

O

6

2

nPr

Gagunin F, 268

O

5

O

nPr

Gagunin E, 267

AcO

O

O nPr

O

O

H

2

O

O

nPr

nPr

H

H

2

O O

6

5

HO

H

H

2

O

Et

O

O

AcO

6

5

AcO

H

H

2

O nPr

nPr

O

O

HO

O

6

5

Gagunin D, 266

O nPr

nPr

O

O

HO

nPr

Gagunin C, 265

O nPr

nPr

O

OH

O

O nPr

nPr

H

2

O

O

Gagunin A, 263

H

OAc

O

O

O

6

5

HO

H H

2

nPr

O

O

AcO

O

6

O

H

2

nPr

nPr

O

O

AcO

6

5

O

nPr

nPr

O

O

AcO

O O

6

5

O

nPr

nPr

O

Gagunin O, 277 O

AcO HO

5

nPr

H

HO

5

nPr

O

O

EtO O O

6

2

nPr

nPr

O

OAc

O O

6

nPr

H

O

H

2

O

H

O

O

O

O nPr

Gagunin P, 278

nPr Gagunin Q, 279

FIG. 5.15 Gagunin diterpenoids.

Markonikov cyclization of 289 (Scheme 5.1) lead to the bicyclic system 292 (which already possess the skeleton of the bicyclic diterpenes sphenelobane [151–155] and tormesane [156–158]) that, by ring expansion and ulterior cyclization, lead to the diterpene 7-6-5 tricyclic system of valparanes and valparolanes, as can be seen in subsequent schemes.

174 Studies in Natural Products Chemistry

TABLE 5.13 Gagunins, Natural Sources, and Biological Activity Compounds

Natural Sources

Activity

References

Gagunin A, 263

Sponge Phorbas sp.

Antitumor

[140]

Gagunin B, 264

Sponge Phorbas sp.

Antitumor

[140]

Gagunin C, 265

Sponge Phorbas sp.

Antitumor

[140]

Gagunin D, 266

Sponge Phorbas sp.

Antitumor

[140]

Gagunin E, 267

Sponge Phorbas sp.

Antitumor

[140]

Gagunin F, 268

Sponge Phorbas sp.

Antitumor

[140]

Gagunin G, 269

Sponge Phorbas sp.

Antitumor

[140]

Gagunin H, 270

Sponge Phorbas sp.

Antitumor

[141]

Gagunin I, 271

Sponge Phorbas sp.

Antitumor

[141]

Gagunin J, 272

Sponge Phorbas sp.

Antitumor

[141]

Gagunin K, 273

Sponge Phorbas sp.

Antitumor

[141]

Gagunin L, 274

Sponge Phorbas sp.

Antitumor

[141]

Gagunin M, 275

Sponge Phorbas sp.

Antitumor

[141]

Gagunin N, 276

Sponge Phorbas sp.

Antitumor

[141]

Gagunin O, 277

Sponge Phorbas sp.

Antitumor

[141]

Gagunin P, 278

Sponge Phorbas sp.

Antitumor

[141]

Gagunin Q, 279

Sponge Phorbas sp.

Antitumor

[141]

Ion 290 has led by cyclization to the bicyclic system 291 and is precursor of all the rest of 7-6-5 tricyclic systems. Depending on the sigmatropic rearrangement that takes place in 291, according to the bond that participates in the ring expansion intermediates 293 and 294/295 are produced. Via a rearrangement of ion 291, lead to only one stereoisomer ion 293 with angular methyls syn precursor of mulinanes and azorellanes. By contrast ring B expansion of 291, via b, gives the epimer bicyclic systems 294 and 295 with angular methyls anti and syn, respectively. Being ion 294 precursor of the cyathanes numerous group, except for cyanthiwigins, and verrucosanes family, except for gagunins. The other ion, epimer 295, is the precursor of cyanthiwigins and gagunins. In ulterior schemes will be detailed the routes that lead to each one of these groups.

5 175

7-6-5 Tricarbocyclic Diterpenes Chapter

O

O

O

H

H

H OH

OH

OHC

OAc

OH O

OH

OAc

HOH2C

O

H

AcO

C

OH

OH

H

O

OMe

H

H

OH

OH HO

OH H N

B H

OH

H

HO

H

HO

A O

O

O

Gukulenin B, 281

Gukulenin A, 280

O

Gukulenin C, 282

O

H

O

H

H

OH H N AcO

OH

H

OH O

OH

OH

O

H

OH O

NH

HO

OHC

OAc OH

HO

H

OH H

OH

OH HO3S

H

HO

O

HO

O

Gukulenin D, 283

OH H

O

Gukulenin E, 284

Gukulenin F, 285

FIG. 5.16 Gukulenin diterpenoids.

TABLE 5.14 Gukulenins, Natural Sources, and Biological Activity Compounds

Natural Sources

Activity

References

Gukulenin A, 280

Phorbas gukhulensis

Antitumor

[145]

Gukulenin B, 281

P. gukhulensis

Antitumor

[145]

Gukulenin C, 282

P. gukhulensis

Antitumor

[146]

Gukulenin D, 283

P. gukhulensis

Antitumor

[146]

Gukulenin E, 284

P. gukhulensis

Antitumor

[146]

Gukulenin F, 285

P. gukhulensis

Antitumor

[146]

Valparanes and Valparolanes Once established, a general diagram for the biosynthesis of all the review compounds will proceed—in detail—with different group biosynthesis.

176 Studies in Natural Products Chemistry

OH

H O

H MeOOC

O

OMe

H MeOOC

H

Mandapamate, 287

Dictioxetane, 286

COOMe

H

HO

O

H OH

OH

COOMe

H

HO

O

OMe

H

Isomandapamate, 288

FIG. 5.17 7-6-5 Diterpenoids with different biogenetic origin.

PPO

PPO

PPO

t,t,t-GGPP

R-GLPP

+

S-GLPP

b

a

H a

291

+

289

+

+

290

H

b

b

293

H 292

Hαoβ + (Tormesane Hβ) (Sphenolobane Hα)

Mulinane Azorellane

Valparane Valparolane

e

Gagunin

Cyathane

H

294

295 g

c

+

+

H

f

Cyanthiwigin

epi-Neoverrucosane epi-Homoverrucosane

d

Verrucosane Neoverrucosane Homoverrucosane

SCHEME 5.1 7-6-5 Tricyclic diterpenes and their tetracyclic analogs biogenesis.

Ion 292 (Scheme 5.2) that comes from the cyclization of R-GLPP, by 1,2rearrangement with ring expansion, gives intermediate 296. Markovnikov cyclization of 296 lead to the tricyclic ion 297 with trans–anti–trans rings stereochemistry and syn disposition of hydrogens H8 and H14. This ion is stabilized by hydrogen loss, giving valparane skeleton diterpenes (1–26), as valparene 1. This ion 297 could also be understood by a cascade cyclization of the R-GLPP, which is two consecutives anti-Markovnikov cyclizations, to form rings A and B followed by the cyclization of the side chain to obtain the 7-6-5 tricyclic system. No experimental evidence for another route is known, but the coexistence of tormesanes, derived from 292, in the extracts of H. viscosum where valparanes are isolated [13] lead us to propose the bicyclic system as a precursor of the 7-6-5 tricyclic system of valparanes. As will be see later on, in the biosynthesis of other tricyclic systems, has been experimentally confirmed that they proceed from bicyclic systems, indicating that effectively the bicyclic system should participate in the biogenetic route.

7-6-5 Tricarbocyclic Diterpenes Chapter

5 177

H

+

Hαo β

PPO

H

R-GLPP

+

296

292

PPO

H

11 H

6 7

H

8

H

H

14

+

Valparene, 1 (Valparanes, 1–26)

297

R-GLPP

O

O

OHC H H O

H

O

9 Valparolone, 27 (Valparolanes, 27–30)

H H

H

H

299

H

298

SCHEME 5.2 Valparanes and valparolanes biogenesis.

Ring A oxidation of valparene 1 with singlet oxygen, 1O2, followed by cleavage of C–C bond of 298 lead to secovalparanes 299 that, by condensation, gives valparolanes (27–30), as valparolone 27.

Mulinanes and Azorellanes Bicyclic system 291 (Scheme 5.3) that comes from the cyclization of S-GLPP, by B ring expansion via a analogously to the one that takes place for valparanes lead to intermediate 293. This one, by cyclization with the side chain, gives the tricyclic system 300 with trans–anti–trans rings stereochemistry, and disposition anti between H3 and H5 hydrogens. This ion has all the adequately disposed substituents to experiment a 1,2 hydride and methyl rearrangement, which leads to ion 301. This intermediate 301 is the precursor of mulinanes (31–62) and azorellanes (63–74). This biogenetic proposal is more reasonable than the one indicating labdanes as the biogenetical precursors of mulinanes [15]. However, as in valparanes, there is no experimental evidence that precludes the possibility of the tricyclic system 300 being formed by a cyclization cascade—as the one indicated in Scheme 5.3. In any case, independently the procedence of 301, could happen two different routes. In via h, takes place the carbon vecinal hydride migration being stabilized finally by proton loss giving mulinanes (31–62), as mulinic acid 31, and in via i it is an allylic proton the one that enables the cyclopropane ring formation that will lead to azorellanes (63–74), as azorellanol 63. Norditerpenoid

178 Studies in Natural Products Chemistry

a

+

PPO H

+

a

H

291

S-GLPP

293

5

PPO

3

H

O

H

H

+

300

S-GLPP

H

OH CH2OH

Yaretol, 74 Ox. -Me

[1,2]

OAc O O

H

COOH

H

H

h H

h H

+

H

i

17

H

H

i

HO H

H

Mulinic acid, 31

Mulinanes

301

(31–62)

Azorellanes

Azorellanol, 63

(63–74)

SCHEME 5.3 Mulinanes and azorellanes biogenesis.

yaretol 74 formation is explained by the oxidation of an azorellane intermediate with loss of C17 methyl and the opening of the cyclopropane ring.

Cyathanes, Cyanthiwigins, and Verrucosanes Family The cyathane family is constituted by the groups of cyathins and cyafrins, sarcodonins, neosarcodonins, scabronines, erinacines, striatals, striatins, glaucopines, cyrneines, nigernins, onychiols, and cyanthiwigins; while the verrucosane family is formed by verrucosanes, neoverrucosanes, epi-neoverrucosanes, homoverrucosanes, epi-homoverrucosanes, and gagunins. The last group of each family, cyanthiwigins and gagunins, show (as structural characteristics) a trans–anti–cis rings stereochemistry in the 7-6-5 tricyclic systems, and a disposition syn of the angular methyls and can be considered as 9-epi-cyathanes and 10,13-epi-homoverrucosanes derivatives, respectively. Their own biogenetic route will be shown later on. As it has been said before, ring B expansion of 291, via b produces the epimeric bicyclic systems 294 and 295 with the angular methyls in anti and syn relationships, respectively (Scheme 5.4). From 295 cyanthiwigins and gagunins are formed, and from 294 the rest of cyathane and verrucosane families are formed. By cyclization, ion 294 evolves into the intermediates 302 and 303 (Scheme 5.4), both with stereochemistry trans–anti–trans of the 7-6-5 tricyclic ring system, but differ in the orientation of the isopropylic chain (in syn or anti) with respect to the angular vicinal hydrogen, respectively. That cyclization requires a boat, or chair, conformation of the side chain fragment that participates in the process [131,138]. The evolution of 302 produces by 1,2-hydride rearrangement, via c, to the most numerous group of this tricyclic systems, the cyathanes, or by 1,5-hydride rearrangement via d, the verrucosanes family as it will be see later on. In fact, the 1,2 hydride rearrangement, via c, of

5 179

7-6-5 Tricarbocyclic Diterpenes Chapter

H b

b

+

H

H

+

H

+

+

H

H

H

d

H

H

+

g

305 H

H

H

e

H

H

H

+

H

307

235 Neoverrucosan-5R-ol (235–241)

H

H H

H

H

306

[1,5 H– ]

+ H

H H

H

+

303

H

312

HO

+ + H

H

H

[1,2 H– ] f

H

H H

H

+

g

H

Cyathanes (75–185)

[1,5 H– ]

H

[1,5 H– ]

H

f H

H +

304

302

294

H

[1,2 H– ]

c

d

291

295

H c

b

H

+

H

+

H

H OH

H H

313

Cyanthiwigins (186–222)

223

308

310

309

H

2

H

Verrucosanes (223–234)

O nPr

HO

5

nPr

O

O

AcO

O O

6

H 2

H

H H

H

314

H

13

H

+ H

H

311

13-epi-Homoverrucosanes (255–259)

242 epi-Neoverrucosanes (242–252)

H

H

H

O

Gagunins (263–279)

HO

HO

HO H

+

O

H

O

263

H

nPr

255 Homoverrucosan -5S-ol, 254

SCHEME 5.4 Cyathanes, cyanthiwigins, and verrucosanes family biogenesis.

302 proportionates the intermediate 304 that it is stabilized by loss of a proton forming the diterpene cyathane family (75–185), except for cyanthiwigins. Biosynthetic studies utilizing [1-13C] acetate, [2-13C] acetate, and [1,2-13C] acetate indicate that cyathane is formed from acetate via isoprenoid pathways. The labeling experiments are consistent with a route involving cyclization rearrangement of geranylgeranyl pyrophosphate, such as the one described [159]. This is confirmed with experiments carried out with alltrans-[1-2H2, 2-2H1] geranylgeranyl diphosphate by a cell-free system prepared from basidiomycete Hericium erinaceum [59]. In the same way, the experiments done with 13C-labeled glucose show that the diterpenoid part of the striatals/striatins is formed via the mevalonate pathway [92]. The cyanthiwigins group can be understood by the cyclization of 295 (Scheme 5.4) that lead to the 7-6-5 tricyclic system 305, with a trans–anti– cis rings stereochemistry typical of this group. The 1,2 hydride rearrangement, via f, of ion 305 followed by loss of a proton of the cyclopentane ring lead to these compounds (186–222). Once the cyathanes biosynthesis is explained, we proceed with the verrucosanes family. While the 1,2 hydride rearrangement via c of 302 lead to cyathanes, the 1,5 rearrangement via d of 302 gave verrucosanes, neoverrucosanes, and homoverrucosanes. The 1,5 rearrangement via e of 303 lead to the 13-epi-neoverrucosanes and 13-epi-homoverrucosanes (Scheme 5.4). Effectively, the 1,5 rearrangement via d of the intermediate 302 generates ion 306, which leads to the tetracyclic system cyclopropylcarbinyl ion 307, and from it to the neoverrucosane group (235–241) as neoverrucosan-5R-ol 235. A sigmatropic rearrangement of 307 to intermediate ion 308 facilitates

180 Studies in Natural Products Chemistry

verrucosanes formation (223–234) [131,132]. The tricyclic system of homoverrucosan-5S-ol, 254, is from the last compound (or ion 308) by ring expansion. Intermediate 303 by 1,5 hydride rearrangement via e lead to ion 309, which, by cyclization, gave the tetracyclic system 310 of epi-neoverrucosanes (242–252) as 13-epi-neoverrucosan-5R-ol 242. By rearrangement of epi-neoverrucosane, ion 310 can generate the new cyclopropylcarbinyl ion 311; and this one, by ring expansion, lead to the 7-6-5 tricyclic system of the 13-epihomoverrucosanes (255–259) as 13-epi-homoverrucosan-5S-ol 255. The explaination the biosynthesis of gagunins or 10,13-bis-epihomoverrucosanes remains. The 1,2 hydride rearrangement of 305 lead to the cyanthiwigin group, while the 1,5 rearrangement via g lead to ion 312, and from it, the tetracyclic system cyclopropylcarbinyl ion 313 is generated. A sigmatropic rearrangement of 313 produces the intermediate ion 314, which, by ring expansion and ulterior oxidations and esterifications, lead to gagunins 263–279. Biosynthetic studies on verrucosan-2S-ol 223, carried out in the phototropic eubacterium Chloroflexus auranticus [138] using in vivo incorporation of [1-13C] acetate, [2-13C] acetate, and [1,2-13C] acetate, have confirmed the biosynthetic route, and that this compound is synthesized via the mevalonate pathway. In this manner, the biosynthetic studies carried out on 8S-acetoxy-13S-hydroxy-13-epi-neoverrucosan-5-one 246, in the liverwort Fossombronia alaskana [131] using [1-13C] glucose and [U-13C6] glucose as precursors confirm the biosynthetic route of Scheme 5.4. These studies conclude that the neoverrucosane diterpene is generated via 1-deoxyxylulose 5-phosphate in the above liverwort and confirm the postulated 1,5 hydride shift by an incorporation experiment with [6,6-2H2] glucose. The tetracyclic system of umabanol 253 could be formed from verrucosanes or from neoverrucosanes by ring expansion of the cyclopropane ring [132] (Scheme 5.5).

HO

H H

H H

Verrucosan-2S-ol, 223

H

Cyanthiwigin U, 206

H

Umabanol, 253

H HO

H

H

HO

OH

H

OHC

H O

O HO

Cyanthiwigin AC, 214

Neoverrucosan -5R-ol, 235

HO

H

H H

Cyanthiwigin AD, 215

O

H

Cyanthiwigin A, 186

SCHEME 5.5 Biogenetical proposal for umabanol and cyanthiwigins AC and AD.

O

7-6-5 Tricarbocyclic Diterpenes Chapter

5 181

Finally, the espiranic system of cyanthiwigin AC 214 and the tricyclic system of cyanthiwigin AD 215 have been proposed to be formed from cyanthiwigin U 206 and cyanthiwigin A 186, respectively [115] (Scheme 5.5).

SYNTHESES AND SYNTHETIC APPROXIMATIONS Fig. 5.18 appears the tricyclic diterpenes with a 7-6-5 system not synthesized until now. Among them can be seen a valparane derivative, a considerable number of cyathane derivatives, and three verrucosane derivatives. Until 2009, cyathane syntheses have been collected in the excellent reviews of Wright and Whitehead [3] and Enquist and Stoltz [4]. Synthesized cyathanes are (
)-allocyathin B2 and (+)-erinacine A [160,161], (
)-allocyathin B2 [162,163], (+)-allocyathin B2 [164,165], (
)-sarcodonin G [68], (
)-allocyathin B3 [166–168], ()-cyathin A3 [168,169], ()-erinacine B [170], ()-erinacine E [171], ()-scabronine G [172], (+)-cyanthiwigin U [173], (+)-cyanthiwigin W [173], ()-cyanthiwigin Z [173], (+)-cyanthiwigin AC [174], and ()cyanthiwigin F [175]. This work reported the synthesis and synthetic approach concerning cyathane derivatives that have appeared later to 2009. The synthesis of cyanthiwigin B, F, and G [176], cyathin A3 and cyathin B2 [177], cyrneine A [178], scabronines G [179,180], and A [180], episcabronine A [180], cyanthiwigin A, C, G, and H [181] and scabronine D [182], cyathin B2 [183], and the synthesis of a valparane derivative [184] and verrucosane derivatives [185] such as (
)-verrucosan-2S-ol, (
)-neoverrucosan-5R-ol, and (
)-homoverrucosa-2-en-5S-ol, will be shown. Very recently, the first synthesis of 36, 43, 46, and 54 [186] mulinanes has been reported.

Oltra Synthesis of Valparadiene In 2005 Cuerva, Oltra, and coworkers [184] describe the synthesis (Scheme 5.6) of valparane diterpenoid valpara-2,14-diene 315, from geranyllinaloyl acetate in only three steps, which include a selective epoxidation [187] to 316; and, as key step, a radical cyclization mediated by titanocene(III) in a closing 7-endo-trigonal that lead to 317 with a 21% overall yield. Treatment with PCl5 lead to 315 whose spectroscopical properties coincide with the compound obtained from natural valparene 1, isolated by Urones and coworkers [13].

Synthetic Approach to Cyathanes and Cyanthiwigins Sarpong and coworkers carried out a synthetic approach to cyathanes type diterpenes and cyanthiwigins (Scheme 5.7) using a tandem reaction that involves a stereoselective cyclopropanation/Cope rearrangement of a racemic

182 Studies in Natural Products Chemistry O

OH

O

HOH2C

OHC

O

HOH2C H

HO

OHC H

HO

H H CH2OH

Allocyathin B2, 90

Allocyathin B3, 76

Cyathin A3, 75

HO

Sarcodonin G, 114

HO O

O

HO

O

HO O

HO

HO

O

O

H

HO

O

O

HO OHC

H

OHC

OHC

HO

Erinacine A, 139

H

Scabronine G, 130

Erinacine E, 143

Erinacine B, 140

COOH

H

O

H HO

OH

H

O

OH

H

Cyanthiwigin W, 207

Cyanthiwigin U, 206

H

H

H

HO

O

H

HO

Cyanthiwigin AC, 214

Cyanthiwigin Z, 211

O O

O

H

MeO

O

H

Cyanthiwigin F, 191 MeO

HO

O

H

H

Cyathin B2, 91

Cyanthiwigin G, 192

Cyanthiwigin B, 187

O

OHC

H

H

H

OH

O

OH

HO COOH

COOH

H

MeO

MeO

Scabronine A, 123

PhCOOH2C

OHC

COOH

O

OH

H

H

Scabronine D, 127

Episcabronine A, 124

Cyrneine A, 166

O H

H O

H

Cyanthiwigin A, 186

H OH

H

Cyanthiwigin C, 188

Cyanthiwigin H, 193 19

HO 2

3

5

6

8

7

H

H

H

5

9

H 10

4 2 1

OH

14 11 12

6

4 2

9

H 10

1

H

20

14 11 12

H 3 16

HO

8

7

18

13

13

O

H

H

13 15 17

Valpara-2,13-diene, 2

H

Verrucosan-2S-ol, 223

COOH

H

H

Neoverrucosan -5R-ol, 235

CH2OH

H

H

Homoverrucosa 2-en-5S-ol, 254

CH2OAc

H

H

COOH

H

O 11-Oxo-mulina-12,14-dien-20-oic acid, 46

Mulina-11,13-dien -20-ol, 54

20-Hydroxy-mulina11,13-dienyl acetate, 43

Mulina-11,13-dien -20-oic acid, 36

FIG. 5.18 Cyathane and mulinane derivatives, which synthesis or synthetic approaches have appeared later to 2009.

7-6-5 Tricarbocyclic Diterpenes Chapter

AcO

1. NBS, H2O 2. K2CO3

AcO

5 183

Cp2TiCl2 Mn dust

O

H H

2,4,6-Collidine CH3SiCl, THF

OH

Geranyllinaloyl acetate

316

317 PCl5

HI/ H

H

H

H

1

315

SCHEME 5.6 Oltra synthesis of valparadiene.

OTBS O

H

O

H

OMOM

(±)-318

OMOM (±)-320

(±)-319

H

O

N2

Rh

SO2

Catalyst, [Rh2((R)-dosp)4] (1 mol %)

COOMe

N C12H25

OTBS

1. LiHMDS, 2-PyNTf2 2 Bu3SnCH=CH2 [Pd(PPh3)4] LiCl, CuCl

O 6 step

O

321

Rh

pentane

4 [Rh2((R)-dosp)4]

OTBS

OTBS

(Catalyst) MeOOC

+ 5

H

MeOOC

OMOM

H

H

(-)-322

(+)-323

7 1

1 4

[Rh2((R)-dosp)4] [Rh2((S)-dosp)4]

OTBS MeOOC

OTBS +

5

H OMOM

H

(+)-322

[Rh2((R)-dosp)4] [Rh2((S)-dosp)4]

OMOM

H

1 5

MeOOC

H OMOM

H

(-)-323 5 1

SCHEME 5.7 Sarpong and coworkers synthetic approach to cyathanes type diterpenes and cyanthiwigins.

184 Studies in Natural Products Chemistry

TABLE 5.15 Rh(II)-Catalyzed PKR of Diene (
)-258 Ratio

e.r.

e.r.

(+)-322/ (2)-322

(2)-323/ (+)-323

Entry

Catalyst

322/323

1

Rh2(OOct)4

1:1

2

[Rh2((R)-dosp)4]

1:1

12:88

88:12

3

[Rh2((S)-dosp)4]

1:1

89:11

15:85

diene in the presence of a catalyst of Rh(II) that allows for a parallel kinetic resolution (PKR), and to establish the stereocenter at C5 on the BC ring junction [188]. The main difference between cyathanes and cyanthiwigin diterpenes is the stereochemical relationship between the angular methyls at C6 and C9 that are in disposition anti in cyathanes and syn in cyanthiwigins. This difference implies that the hydrogen at C5 has a syn relationship with the methyl group at C9 in cyathanes, and is anti in the tricyclic system of cyanthiwigins. In this synthetic approach, starting from the Hajos–Parrish racemic ketone 318, is obtained in six steps intermediate (
)-319. From this compound is prepared (
)-320 by vinyl triflate formation and subsequent Stille coupling with vinyltributyltin. After several trials, the authors by reaction of (
)-320 with vinyldiazoacetate 321 in the presence of Davies’s dirhodium tetraprolinate catalysts [Rh2((R)-dosp)4] and [Rh2((S)-dosp)4] achieved 322 and 323 each in 85:15 e.r. as outlined in Scheme 5.7 (Table 5.15 entries 2 and 3). At the same time, by taking advantage of PKR, the tricyclic system of cyathin 75 and cyanthiwigin 186 diterpenes is obtained using a common racemic diene precursor. The key step involves a stereoselective cyclopropanation and a subsequent stereospecific divinylcyclopropane rearrangement, which allows control of the stereochemistry at C5.

Cha Syntheses of (
)-Cyathin A3 and (
)-Cyathin B2 The stereoselective synthesis of cyathin A3 75 and cyathin B2 91 (Scheme 5.8) published by Cha and coworker [177] uses a Prins-type reaction of a cycloalkenyl cyclopropanol and subsequent manipulation of the spirocyclobutane ring used to achieve the required diastereocontrol in the quaternary carbon of the trans 6/7 ring system, with respect to the methyl group at C-9. For the elaboration of the seven-member ring, a RCM catalyzed by a secondgeneration Grubbs catalyst is used.

7-6-5 Tricarbocyclic Diterpenes Chapter

1. EtMgBr, Ti(OiPr)4 2. TMSCl

Br

MeO TiCl4

iPr 324

Br OMe iPr 326 (X-ray)

O

H

O

RCM Grubbs II

9

H

Cyathin B2, 91

H

HO

332

OH H

OH

iPr 328 R = p-MeOC6H4 O

O PhCO2H TBDPSO PPh3 DIAD

4 steps

R

iPr 329 R = p-MeOC6H4

O

TBDPSO

Ti(OiPr)4 iPrMgBr

H

iPr 330

1. Davis' reagent 2. TFAA 3. Ac2O, Hg(OCOCF3)2

OHC

HO

H

iPr 331

iPr 327 R = p-MeOC6H4

R

HO 1. PhSH, AIBN 2. PPh3·Br2

1. K2OsO4·2H2O PhS 2. Pb(OAc)4 3. [RhCl(PPh3)3]

O

Br

Ar O

OH H

1. Zn 2. LHMDS, ArCHO

iPr 325

PhS

R

O

OMe

OTMS MeOOC

5 185

1. TBAF 2. KOH

HO 9

H

BzO

333

HO

H

Cyathin A3, 75

SCHEME 5.8 Cha syntheses of (
)-cyathin A3 and (
)-cyathin B2.

For this synthesis of cyathin A3 and cyathin B2, Cha and coworkers used—as a starting material—the known dienoate 324 (Scheme 5.8). Kulinkovich cyclopropanation [189], with EtMgBr of the late compound and subsequent silylation, delivered 325. Treatment of 325 with acetal 2-bromo-1,1-dimethoxyethane in the presence of TiCl4 lead mainly to 326, which stereochemistry was corroborated by X-ray diffraction. Reductive elimination with Zn dust followed by condensation with ArCHO produces 327. The addition to this substrate of an adequate organometallic diene by the more hindered face of the cyclobutanone ring lead to 328, with the aim to carry out the ring-closing metathesis (RCM). Effectively, the RCM of 328 by the second-generation Grubbs catalyst furnishes 329. Addition of thiophenol and subsequent Grob fragmentation of cyclobutanone ring provided 330. Under sharpless asymmetric dihydroxylation conditions, regioselective dihydroxylation of 330 gives the corresponding diol, which by oxidative cleavage with lead(IV) acetate, and subsequent decarbonylation of the resulting aldehyde with the Wilkinson catalyst, delivered 331. Oxidation of 331 with Davis’s oxaziridine gives the corresponding sulfoxide, which, in the presence of trifluoroacetic anhydride, produces a reaction mixture that found cyathin B2 91—although in low yield. When that mixture is treated without separation and it is made to react with acetic anhydride (Ac2O) followed by hydrolysis with Hg(OCOCF3)2 provided cyathin B2 91 in 90% yield.

186 Studies in Natural Products Chemistry

Cyathin B2 91 is transformed into cyathin A3 75 by the reaction sequence shown in Scheme 5.8. Reduction of the aldehyde with L-selectride, subsequent epoxidation of the resulting allylic alcohol to give the corresponding a-epoxide (10:1) as major isomer, primary alcohol protection with tertbutylchlorodiphenylsilane (TBDPSCl) and treatment with 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) lead to 332. Mitsunobu inversion of 332 with benzoic acid gives 333 that by successive deprotections lead to cyathin A3 75.

Gademann Synthesis of Cyrneine A To carry out the first synthesis of cyrneine A 166, Gademann and coworkers [178] used a reductive Knoevenagel/Heck cyclization strategy, which established the 35two quaternary stereogenic centers of the molecule and a regioselective reductive desymmetrization (Scheme 5.9). Other interesting steps are a ring expansion by a carbene rearrangement, and a reductive palladiummediated carbonylation. For cyrneine A 166 synthesis, start with the preparation of intermediate 336, which already has the required functionalization and stereochemistry. Synthesis of 336 from ()-(R)-carvone was done in eight steps. The stereoselective reduction of ()-(R)-carvone and tert-butyldimethylsilyl (TBS) protection, followed by exocyclic double bond reduction, ozonolysis, and reductive O CHO O

OTBS

O

4 steps

OTBS

OTBS

O O

Ph-Me 175°C

334

(−)-(R)-carvone

O

OTBS

3 steps

OHC

O

O

3 steps

335

336

337 (X-ray) NaBH4 CeCl3

O

OH

OTBS

Pd(OAc)2

4 steps

O

O

CH2Br2 LiTMP

OTBS

TfO

OTBS

O

O OTBS

OTBS 4

338

OH

BuLi

HO

O

339

OTBS

OTBS

OTBS

O

340

341 (X-ray)

5 6

1. (MeSO2)2, Py 2. LiBr, Li2CO3

OTBS

OTBS

1. PhNTf2, HHMDS 2. [Pd(PPh3)4], LiCl OHC CO, n-Bu3SnH 3. TBAF

Br2HC 342

343

SCHEME 5.9 Gademann synthesis of cyrneine A.

Cyrneine A, 166

OH

7-6-5 Tricarbocyclic Diterpenes Chapter

5 187

work-up give 334. The ring cyclization to a five-member by piperidinium acetate, followed by reduction and etherification, lead to 335. This compound is adequate for Claisen rearrangement that allows elaboration of one of the quaternary stereocenters. In this manner, 336 was obtained in a 55% global yield via the eight steps. Knoevenagel condensation of aldehyde 336 with cyclohexa-1,3-dione in the presence of L-proline, and in situ reduction with Hantzsch ester, of the unsaturated intermediate and ulterior methylation in the presence of methyl iodide and ozonolysis lead to 337, whose structure was confirmed by X-ray diffraction. Diastereoselective reduction of 337 under Luche conditions (CeCl3, NaBH4) were produced with high regioselectivity and the 5R,6R diastereoisomer 338 as the major one (4.2:1) was obtained. Mesylation of 338 and elimination produced 339; cyclohexanone reduction, protection, cyclopentanone triflate formation followed by allylic oxidation gave the a,b-unsaturated ketone 340. Tricyclic system cyclization was carried out by palladiummediated Heck reaction to give 341, with the required configuration being confirmed by X-ray diffraction. Ring expansion was done in two steps. First, 341 was made to react with dibromomethane and lithium 2,2,6,6-tetramethylpiperidine-1-ide (LiTMP). Second, the obtained dibromide 342 was made to react with n-BuLi at a low temperature, giving the subsequent rearrangement with formation after expansion of cycloheptenone 343. In order to introduce the C12-aldehyde group was done first to the corresponding enol triflate and this intermediate by reductive, palladium-catalyzed carbonylation followed by the corresponding deprotection reaction gave cyrneine 166, whose structure was corroborated by X-ray diffraction.

Kanoh Synthesis of (2)-Scabronine G In 2011, Kanoh and coworkers described the synthesis of ()-scabronine G 130 [179]. The scabronine G 130 tricyclic system with two quaternary carbon centers was carried out via diastereoselective intramolecular double Michael (IDM) reaction and Prins cyclization (Scheme 5.10). After an adequate study of the IDM reaction with a racemic substrate, the cyclization was optimized using the methodology developed by Ihara and coworkers [190], and the synthesis of scabronine G was started with the known chiral alcohol (+)-344 (Scheme 5.10). Protection of 344 as TBS ether, followed by selective bromination and halogen exchange, gave iodide 345, which was coupled with the lithium enolate of 3-methyl-2-cyclohexenone at 78°C to obtain 346 in 54% yield. The IDM reaction of 346 with trimethylsilyliodide-hexamethyldisilazane (TMSI-HMDS) gives 347 in 75% yield in an enantiomerically pure form (99% ee).

188 Studies in Natural Products Chemistry

O

O

I

TBSO

Li

TMSI HMDS

O

3 steps

OTBS

OH EtOOC

EtOOC

OTBS

5

2 steps

OH

H

O OTf H

OTBS

O

O

O

4 steps

H

349

I

OTBS H

H

346

O

350

H

EtO

EtOOC

O

O

4 steps

LDA HMPA

345

344

O

O

H H OH

OTBS 5

5 steps

H

H

COOEt 347

348

OTBDPS 351

O

3 steps

OH

OHC COOMe

TBDPSO

O TBDPSO OTBDPS

H 352

H

COOMe

Me2AlCl

H

353

354 1. DMP 2. PhSeCl, H2O2

1. (CH2OH)2, TsOH 2. NaOH (aq.), MeOH then HCl

O OHC

5

COOH

H

Scabronine G, 130

O

O

OHC

5

H

Scabronine G methyl ester, 356

OHC

COOMe DBU

COOMe H

355

SCHEME 5.10 Kanoh synthesis of ()-scabronine G.

The C5 configuration obtained was not the adequate one, so it was necessary to invert it. First of all, ketone at C17 was protected as its cyclic acetal and ester at C10 was transformed by reduction with lithium aluminum hydride (LAH), followed by a 9-azabicyclo[3.3.1] nonane N-oxyl (ABNO)-catalyzed oxidation with phenyliodine(III) diacetate (PIDA) to give the aldehyde derivative. The epimerization was achieved with tetra-N-butylammonium fluoride (TBAF) at 40°C, and then lactol formation—at the same time of the TBS group deprotection—gave 348. Reduction of 348 followed by selective protection of the primary hydroxyl group and 2-azaadamantane N-oxyl (AZADO)-catalyzed oxidation of the secondary alcohol with PIDA and ulterior formation of the triflate derivative was obtained 349. A Kumada coupling reaction followed by removal of the silyl ether renders 350. In order to introduce C11–C13 alkenyl side chain 351, compound 350 was oxidized with o-iodoxybenzoic acid (IBX) and the obtained aldehyde was coupled with allyl iodide 351 in the presence of CrCl2 by a Nozoki–Hiyama [191] coupling reaction. The hydroxy derivative obtained was reduced into ketal 352 using the standard Barton–McCombie [192] procedure. Deprotection of the acetal followed by Davis’s oxaziridine oxidation gave the

5 189

7-6-5 Tricarbocyclic Diterpenes Chapter

corresponding acyloin that, by Pb(OAc)4 oxidation, led to 353—an adequate substrate for Prins cyclization. Reaction of 353 with Me2 AlCl gave the desired 7-6-5 tricyclic systems 354 as a 1:1 mixture of isomers E/Z. The following steps required deprotection, oxidation, and hydrolysis. Oxidation of 354 followed by selenylation and oxidation gave 355 and ()-scabronine G methyl ester 356. Finally, using the sequence previously developed by Danishefsky and coworkers [172] (isomerization, aldehyde protection, methyl ester hydrolysis, and deprotection) afford ()-scabronine G 130 that was identical to the natural product.

Nakada Syntheses of (2)-Scabronines G and A, and (2)-Episcabronine A ()-Scabronine A 123 displays six contiguous stereogenic centers in the seven-member ring and can be considered the most complex structure of the scabronine family (Scheme 5.11). Scabronines A 123 and G 130 show a

OH CHO

(EtO)2(O)PO OTIPS BnO

OH

BnO

6 steps

357

OMe

1. iPrMgBr CuCN·2LiCl 2. TBAF

OMe

OMe

HO 1. PIDA 2. H2, Pd/C

OMe

· O 360 (X-ray)

359

358

O

1. Swern Ox. 2. Ph3PCH3Br, tBuOK 3. NaBH4, then 3N HCl 4. H2C=C(CH2Br)CH2OTBDPS Zn

O

OHC

COOH H

OMe

TBDPSO CHO

1. TBAF 2. NaClO2 3. DMP

H

1. PIDA 2. Grubbs II

OH

OTBDPS

OH 361

362

(−)-Scabronine G, 130 4 steps

O

TBDPSO

COOMe

O H

O

O

OHC 3 steps

11

HO

363

OH

O

MeO

TBDPSO

DBU

MeO

OH

O

H

HO

H

1. NaOMe 2. HCl

13

H

H

HO

365

364

COOMe

11

366

4 steps

OHC BzO

MeO

O

COOH

H MeO

368

H

1. NaOMe, MeOH 2. HCl, MeOH 3. 2N NaOH(aq.) MeOH then 3N HCl (aq.)

HO H

(−)-Episcabronine A, 124

1. MeI, NaH 2. 3N HCl (aq.) THF

11

MeO

O

MeO

HO

COOMe

H MeO

1. MeI, NaH 2. 2N NaOH(aq.) MeOH then 3N HCl (aq.)

H

(−)-Scabronine A methyl ester, 367

O

HO

COOH

H MeO

H

(−)-Scabronine A, 123

SCHEME 5.11 Nakada syntheses of ()-scabronines G and A, and ()-episcabronine A.

190 Studies in Natural Products Chemistry

strong effect on the production and excretion of neurotrophic factors including NGF in 1321N1 human astroglial cells [81]. The syntheses of scabronines G 130 and A 123 [180,193] (Scheme 5.11) start with phosphate 358 preparation, in six steps from aldehyde 357. Reaction of 358 with iPrMgCl and CuCN2LiCl and ulterior deprotection gave (quantitatively) the chiral allene 359. Reaction of 359 with PIDA in methanol gave rise to an o-benzoquinone mono-dimethylacetal that slowly (by an intramolecular inverse-electron-demand Diels–Alder (IEDDA)) gave a single product with 95% ee that, by treatment with H2 Pd/C, produced partial hydrogenation and hydrogenolysis of the benzyl group giving 360—whose structure was corroborated by X-ray diffraction. Swern oxidation of 360 followed by Wittig reaction, and later ketone reduction and acid hydrolysis generate a hydroxyketone that, by treatment with the adequate organozinc reagent (in situ prepared), supply intermediate 361 as the only isomer. Reaction of 361 with PIDA provides the suitable ketoaldehyde to achieve the RCM with the Grubbs II catalyst and obtains compound 362 with the required 7-6-5 tricyclic system. Deprotection and successive Pinnick and Dess-Martin oxidations of 362 give ()-scabronine G 130 (19 steps from 357 and 21% overall yield). For ()-scabronine A 123 synthesis was used intermediate 362. Oxidation of 362 and methylation provided a methyl ester that, by reaction with osmium tetraoxide and triphosgene, lead to carbonate 363. Treatment of 363 with DBU gives 364, Corey–Bakshi–Shibata (CBS) catalyst reduction, deprotection, and selective oxidation of the primary hydroxyl group supply 365. Transformation of 365 into ()-scabronine A 123 (or its methyl ester 367) requires an oxa-Michael/acetalization cascade. For that reaction, 365 was made to react with sodium methoxide in methanol and the intermediate was treated directly with acidic methanol. In this manner, the intermediate methylacetyl 366 was obtained, which, by treatment with MeI/NaH, permits the methylation of the hydroxyl group at C11. Alkaline hydrolysis of the methyl ester followed by treatment in acidic media gives ()-scabronine A 123. The same treatment of 366, without doing the hydrolysis of the ester, allows us to obtain ()-scabronine A methyl ester 367. Finally ()-episcabronine A 124 was obtained from compound 364, which, by reaction with BzCl, reduction with CBS, deprotection, and PIDA oxidation, gives 368. Treatment of 368 with NaMeO/MeOH and ulterior treatment with HCl in methanol and alkaline hydrolysis leads to ()-episcabronine A 124, by a b-elimination/oxa-Michael cascade reaction.

Nakada Synthesis of (2)-Scabronine D ()-Scabronine D 127 (Scheme 5.12) has (besides the five stereogenic centers in the 7-6-5 tricyclic system typical of cyathanes) the presence of an oxygenbridged hemiacetal between the C11 and C14 positions.

7-6-5 Tricarbocyclic Diterpenes Chapter

O

OMe

TBDPSO

OMe

HO COOMe

OH CHO 357

O

COOMe

1. TBAF 2. HCl, MeOH

H

HO

5 191

364

H 369 1. NaOH, MeOH

OtBu 2.

OMe

OH 14

BzO

BzO O 11

COOH H

(−)-Scabronine D, 127

1. TFA 2. HCl 3N

N H

N

OMe HO

O

COOtBu H

371

O BzCl DMAP

COOtBu H

370

SCHEME 5.12 Nakada synthesis of ()-scabronine D.

For ()-scabronine D 127 synthesis [182] (Scheme 5.12), the tricyclic intermediate 364 was used that appeared in ()-scabronine A 123 synthesis (Scheme 5.11). TBAF deprotection of 364 followed by methyl acetal formation provides 369. Basic conditions hydrolysis of 369 followed by esterification with N,N0 -diisopropyl-O-tert-butyl isourea provides 370. Treatment of 370 with BzCl gives 371 that by reaction with TFA hydrolyze the tert-butyl ester, maintaining the methyl acetal, so the intermediate was made to react with 3N HCl in THF at 60°C giving ()-scabronine D 127 (28 steps from 357, 13% overall yield).

Nakada Synthesis of (2)-Cyathin B2 Nakada and coworkers achieved the first enantioselective total synthesis of ()-cyathin B2 91 [183] (Scheme 5.13). To carry out the synthesis of ()-cyathin B2 91, they use a common intermediate 362 that appeared in ()-scabronines G 130 and A 123 syntheses (Scheme 5.11). The main problem to transform 362 into ()-cyathin B2 91 consists in the transformation of the formyl group at C17 into a methyl, preserving the seven-member ring ketone. Reduction of 362 with zinc borohydride in THF at 5°C for 1 h gives 372 in a 99% yield. In order to remove the resultant primary hydroxy group Barton–McCombie deoxygenation was used, transforming the alcohol into its thiocarbonate derivative first and then reducing the tributyltin hydride in the presence of azobisisobutyronitrile (AIBN), obtaining 373. Then, TBAF deprotection followed by Dess–Martin periodinane (DMP) reagent oxidation afforded ()-cyathin B2 91, whose spectroscopical data matched the data reported by Ayer and coworker [57], being [a]D and the melting point reported for the first time.

192 Studies in Natural Products Chemistry

O

O

TBDPSO

TBDPSO

17

CHO H

CH2OH

Zn(BH4)2

362

H

372 1. PhOCSCl, DMAP 2. nBu3SnH, AIBN

O

O TBDPSO

OHC H

1. TBAF 2. DMP

(−)-Cyathin B2, 91

H

373

SCHEME 5.13 Nakada synthesis of ()-cyathin B2.

Stoltz Syntheses of (2)-Cyanthiwigins B, F, and G The main strategy that developed Enquist and Stoltz [175] in their elegant synthesis of ()-cyanthiwigin F 191, was used for the syntheses of ()cyanthiwigin B 187 and G 192 [176]. In their synthesis, an enantioselective Pd-catalyzed double alkylation reaction was used as the pivotal step by which stereochemistry is established about the central B ring of the cyanthiwigin framework (Scheme 5.14). On the monocyclic substrate, a tandem ring-closing cross-metathesis reaction was carried out, followed by an aldehyde–olefin radical cyclization process that lead to the tricyclic ring system of the cyanthiwigin. Autocondensation of diallyl-succinate 374, with NaH by a Claisen– Dieckmann process, followed by methylation with methyl iodide in the presence of potassium carbonate, lead to bis(b-ketoester) 375 as a 1:1 racemic mixture (R,R)-375 plus (S,S)-375 and meso-diastereomer (that can be carried out on a multigram scale). Treatment of a 1:1 diastereoisomeric mixture of bis(b-ketoester) 375 with Pd(dmdba)2 (dmdba ¼ 3,5dimethoxydibenzylideneacetone) and enantiopure tert-butyl phosphinooxazoline (PHOX) ligand 376 in Et2O affords the bisalkylated products enantioenriched 377 and meso-377 as a 4.4:1 mixture. All carbon quaternary stereocenters were simultaneously constructed in a single-step procedure with excellent enantioselectivity. Selective monoenolization of diketone 377 with potassium bis(trimethylsilyl) amide (KHMDS) and trapping of the resulting enolate gave the trifluoromethanesulfonate 378, which was transformed into

5 193

7-6-5 Tricarbocyclic Diterpenes Chapter

O O O

O

O

1. Allyl alcohol NaH, PhMe reflux

O

O

+

Pd(dmdba)2 Et2O, 25°C

O

(R,R)-377 99% e.e.

1:1 mixture of racemic:meso diastereomers

N

O

O 382 t-BuSH AIBN PhH 80°C

O

H

379

PhH, 60°C NaBO3, THF, H2O

O H

KHMDS, PhN(Tf)2 THF, –78°C

4 3

H

I Zn, TMSCl 1,2-dibromoethane THF, 65°C then Pd(PPh3)4

B O 381

O 5

H

383 (X-ray) 1. KHMDS, THF, –78°C allyl chloroformate 2. Pd2(pmdba)3, MeCN, 80°C 3. CeCl3, i-PrLi, THF, –78°C

O

i-PrMgCl CuCN,THF Pd(dppf)Cl2

CH2Cl2

H

H H

TfO

O H H R

1. NaBH4, 25°C MeOH, CH2Cl2 2. MnO2, CH2Cl2 3. Martin´s sulfurane CDCl3

O

PCC

O

378

384 TfO

O

H

4.4:1 d.r.

Me

380

OHC

meso-377

KHMDS, PhN(Tf)2 THF, –78°

N

Me Cl2Ru O iPr

O

O

375

374

O

O

t-Bu

376 O

O

2. K2CO3, MeI acetone, reflux

O

PPh2 N

O

R = i-Pr, (–)-Cyanthiwigin F, 191 R = H, 385

H H

O

OH 386

(–)-Cyanthiwigin B, 187

(–)-Cyanthiwigin G, 192

SCHEME 5.14 Stoltz syntheses of ()-cyanthiwigins B, F, and G.

tetraene 379 via a Negishi cross-coupling procedure. Zinc dust was treated with 1,2-dibromoethane and trimethylsilyl chloride and alkyl iodide (4-iodo-2-methylbut-1-ene) was added to this mixture, followed by a THF solution containing palladium (0) catalyst and triflate 378. In this manner, the tetraolefin 379 was obtained. At this point, it is necessary to mention the construction of the seven-member ring as well as the elaboration of the allylic isopropyl group on the A ring. The two transformation could be achieved with a single catalytic operation by treating 379 with modified Grubbs–Hoveyda catalyst 380 and vinylboronate species 381, in this manner it is achieved the RCM to form the seven member ring and cross-metathesis with boronate 381 that by oxidative work-up lead to aldehyde 382 in only one step. The tricyclic ring system of cyanthiwigin was achieved by using a

194 Studies in Natural Products Chemistry

radical intramolecular cyclization of the aldehyde 382. Treatment with tertBuSH and azobis (isobutylnitrile) (AIBN) lead to the tricyclic diketone 383 as a single diastereomer, which stereochemistry was corroborated by X-ray diffraction. The selective enol triflate formation of 383 gave 384. This compound, by coupling the reaction with the corresponding lithium organocuprate—catalyzed by Pd—afforded ()-cyanthiwigin F191, together with a reduction product 385 (1.8:1). The natural product ()-cyanthiwigin F 191 was obtained in this manner, in nine steps (from diallyl-succinate and 1.9% overall yield, without the use of protecting groups). The cyathane core 383, accessed in seven steps and 6.2% overall yield, is used in the synthesis of ()-cyanthiwigin B 187 and ()-cyanthiwigin G 192. From 383 the enolcarbonate as intermediate is formed and made to react with catalytic palladium (0) to provide an enone that, by reaction with isopropyl lithium in the presence of cerium trichloride (Luche-type activation conditions), leads to 383 as a mixture of diastereomers. Oxidation of 386 with pyridinium chlorochromate (PCC) produces ()-cyanthiwigin B 187 (37% yield from 383). Reduction of 187 followed by allylic oxidation and dehydration with Martin’s sulfurane gives ()-cyanthiwigin G 192 (7% yield from 187).

Gao Syntheses of (
)-Cyanthiwigins A, C, G, and H In 2013, Gao and coworkers published the synthesis of cyanthiwigins A 186, C 188, G 192, and H 193 [181] using a series of reactions that are highly stereospecific to building the tricyclic system typical of these compounds (Scheme 5.15). These reactions include a formal [4 + 2] cycloaddition modification, 1,4 addition, alkylation, and RCM. Cyanthiwigins A 186, C 188, G 192, and H 193 were synthesized using cis-hydrindanone 392 as intermediate corresponding. In order to prepare the desired cis-hydrindadone, a sequential Michael addition followed by oxonium ion-promoted cyclization was done in the following manner. Reaction of 387 enolate with 388 gave the Michael addition product 389. p-toluenesulfonic acid (PTSA) added to 389, without purification, lead to 391, through oxonium intermediate 390, which cyclized and eliminated methanol to give 391. Decarboxylation of 391 given to 392 was carried out with Raney Nickel in an acceptable yield, with its structure confirmed by the X-ray diffraction method. For cycloheptene ring formation, it was necessary to protect the C3 carbonyl group of 392 followed by 1,4 addition of 3-methylbut-3-en-1-yl magnesium bromide in the presence of CuI and TMSCl to give the corresponding silyl enol ether, which, by reaction with methyl lithium and allylic iodide, lead to the mixture of O and C-alkylation products 393 and 394. The heating in

5 195

7-6-5 Tricarbocyclic Diterpenes Chapter

OMe OMe LDA

OMe O 387

PTSA

O BnOOH2C

BnOOC

–MeOH

BnOOC O 391

O 389

388

PTSA

O BnOOH2C

O

O

O

Me+O

MeO

390

Raney Ni

O

O

+

H

Toluene ref.

H

O 392

O 393

RCM

1. KHMDS PhNTf2 2. PdCl2(dppf) Et3SiH

H

ICu, TMSCl 3. MeLi, THF allylic iodide

O

394

H

O

H

O O

1. Ethylene glycol p-TsOH 2. MgBr

1. NaBH4 2. KHMDS, CS2, IMe 3. nBu3SnH, AIBN 4. PPTS 5. LDA, TMSCl 6. Pd(OAc)2

O H

H

H H

O

O

O

O

O 397

1. iPrLi 2. PCC

395

1. PPTS 2. LDA, TMSCl 3. Pd(OAc)2 3. iPrLi 5. PCC

H

Cyanthiwigin G, 192

NaBH4

m-CPBA

O H

H O

H

Cyanthiwigin H, 193

396

H

H O

H

O

Cyanthiwigin A, 186

H

OH

Cyanthiwigin C, 188

SCHEME 5.15 Gao syntheses of (
)-cyanthiwigins A, C, G, and H.

toluene of the mixture gave 394 by 3,3-sigmatropic rearrangement. RCM of 394—using Grubbs II catalyst—gave the tricarbocyclic intermediate 395. Reduction of 395, followed by Barton’s protocol deoxygenation, deprotection, and sequential Saegusa–Ito oxidation [194] gave enone 396. Treatment of 396 with isopropyllithium lead to the tertiary alcohol that, by PCC oxidation, gives cyanthiwigin A 186. Luche reduction (NaBH4CeCl3) of cyanthiwigin A 186 lead to cyanthiwigin C 188 in 90% yield and selective oxidation of 186 with m-CPBA gave cyanthiwigin H 193 and its diastereomer in a 6:1 ratio in 98% combined yield. Transformation of 395 in its vinyl triflate derivative and ulterior Pd-catalyzed reduction with Et3SiH gives 397, which, in a fivestep procedure (deprotection, silyl enol ether formation, Pd(OAc)2 oxidation, iPrLi addition, and PCC oxidation), achieves cyanthiwigin G 192.

196 Studies in Natural Products Chemistry

Piers Synthesis of (
)-Verrucosan-2S-ol, (
)-Neoverrucosan5R-ol, and (
)-Homoverrucosa-2-en-5S-ol In 1997, Piers and coworker [185] reported the first synthesis of verrucosane, neoverrucosane, and homoverrucosane diterpenoids (Scheme 5.16). For the syntheses of (
)-verrucosan-2S-ol 223, (
)-neoverrucosan-5R-ol 235, and (
)-homoverrucosa-2-en-5S-ol 254, the known enone 398 was used as the starting material. Conjugate addition of organocuprate 399 (in the presence of CH3SiCl) to enone 398 gave the epimers mixture 400 and 401 in approximately 11:1 ratio, that, by equilibration with NaOMe in methanol, lead to a new mixture in a 1:14 ratio (95%). Alkylation of 401 enolate with 2-(t-butyldimethylsilyloxy)-4-iodobutane followed by methylation gave ketone 402. Deprotection of 402, followed by oxidation and condensation with bases promoted ring closure to the tricyclic enone 403, that, by metal–ammonia reduction, followed by hydrogenation of the isopropenyl group and oxidation with dimethyldioxirane and protection gave 404. Methylenation of 404 with Tebbe’s reagent and silyl ether deprotection led to allylic alcohol 405, in a very high overall yield. Oxidation of 405, followed by treatment of the resultant enone with RhCl3 in EtOH, provided the enone 406, which is intermediate in the syntheses of (
)-verrucosan-2S-ol 223 and (
)-neoverrucosan-5R-ol 235. Reduction of 406 followed by stereoselective cyclopropanation gives OTBS 2

Cu(CN)Li2 399

O

1. TBAF 2. PCC 3. NaOEt EtOH

+

H

NaOMe MeOH

O

CH3SiCl

H 2 steps

400

398

H

O

O

H O

402

401

403 1. Li, NH3 2. H2, Pt 3. KHMDA dimethyldioxirane 4. TBDMSCl, imidazole

HO 5 4 2 3

1

H

H

1. NaBH4, CeCl3 2. Et2Zn, ICH2Cl ClCH2CH2Cl

O

TBSO

HO H

H

1. TPAP, NMO 2. RhCl3·3H2O

H 406

H 405

1. Cp2TiCH2AlClMe2 2. TBAF

H O

H 404

(±)-Neoverrucosan -5R-ol, 235 1. Dimethyldioxirane 2. H2NNH2, MeOH, AcOH 3. Et2Zn, ICH2Cl ClCH2CH2Cl

H2SO4 H2O, Me2CO

HO H H

(±)-Homoverrucosa 2-en-5S-ol, 254

H2SO4 H2O, Me2CO

3

5 4 2 1

H

H OH

(±)-Verrucosan-2S-ol, 223

SCHEME 5.16 Piers synthesis of (
)-verrucosan-2S-ol, (
)-neoverrucosan-5R-ol, and (
)homoverrucosa-2-en-5S-ol.

5 197

7-6-5 Tricarbocyclic Diterpenes Chapter

(
)-neoverrucosan-5R-ol 235. On the other side, direct epoxidation of 406 with dimethyldioxirane followed by treatment with NH2NH2 in Wharton’s [195] conditions and ulterior face-selective cyclopropanation gave (
)-verrucosan-2S-ol 223. Treatment of (
)-verrucosan-2S-ol 223 and (
)-neoverrucoin acetone led to san-5R-ol 235 with aqueous H2SO4 (
)-homoverrucosa-2-en-5S-ol 254 in both cases.

Synthetic Approach to Gagunin Diterpenoids In 2013, Stoltz and coworkers [196] published an enantioselective synthesis of the carbocyclic core of the gagunin diterpenoids. For the synthesis of gagunin E 267 was planned a synthetic strategy similar to the one used for their cyanthiwigins synthesis (Scheme 5.17). The enol triflate intermediate 378 obtained in Scheme 5.14 is made to react by Heck reaction with silyl ketene acetal 407 in the presence of Pd(PPh3)4 and LiOAc afforded methyl ester 408. Protection of 408 followed by Weinreb amide formation and the addition of vinyl magnesium chloride furnished 409. RCM of 409 in the presence of Hoveyda–Grubbs II generation catalyst 380 produced adduct 410. After enol carbonate formation, Wacker’s oxidation gives ketone 411, which was diazotized using Danheiser’s conditions to produce 412. In order to form the carbocyclic system of gagunin E 267, 412 was treated with Rh2(OAc)4 to obtain the 1. Ethylene glycol p-TsOH 2. N,O-dimethyl hydroxylamine hydrochloride 3. CH2CHMgCl

OTMS O 407

O

Pd(PPh3)4 LiOAc

TfO

O

O

O O O

O 408

378

409 N

N

Me Cl2Ru

1. LHMDS CF3COOCH2CF3

iPr

2. p-ABSA, Et3N H2O, CH3CN

O

O

O

412

O

O O

411

N2

410 nPr

O O

K2CO3 MeOH

O H O

O O 413 (X-ray)

O O

H

O

O AcO

H

O

6

H 2

H O

O

O

O 414

5

O

+

nPr

O

OH

O

Rh2(OAc)4

O

O

O O

O

O

2. O2, PdCl2, Cu(OAc2)

O

O

380

1. LHMDS methyl chloroformate

O

O

Me

O

nPr

O 415

SCHEME 5.17 Stoltz synthetic approach to gagunin diterpenoids.

Gagunin E, 267

198 Studies in Natural Products Chemistry

cyclopropane 413 in 71% yield. Enol carbonate cleavage and cyclopropane opening was achieved with K2CO3 in methanol, giving the expected product 414 and an unexpected (and unstable to chromatographic conditions) rearranged product 415. As can be observed, compound 414 already has a full gagunin tricyclic skeleton and from it can be prepared gagunins and analogs.

Guerrero Synthesis of Mulinanes 36, 43, 46, and 54 During the referee process, Guerrero and coworkers published the first synthesis of mulinane diterpenoids, specifically dienone 46 and dienes 54, 43, and 36 [186] (Scheme 5.18). In order to establish the relative configuration at C8, the authors used a diastereoselective anionic oxy-Cope rearrangement and an unprecedented vinylogous Saegusa dehydrogenation reaction to address C-ring functionality. The synthesis starts with the b-ketoester 416 that by Michael addition to methyl vinyl ketone, and ulterior Robinson annulation by treatment in acidic media gives enone 417, allowing the control of one of the two quaternary stereocenters present in the mulinane objectives. Treatment of 417 with Stiles’ reagent (methyl magnesium carbonate) in dimethylformamide at 130°C gives an unsaturated b-ketoacid intermediate that, by esterification with diazomethane and ulterior Pd-catalyzed heterogeneous hydrogenation, gave b-ketoester 418 in good yield and complete diastereoselectivity and established the mulinanes trans-hydrindane core. COOEt O

1. MVK, K2CO3 O 2. pTsOH, Dean–Strark

COOEt

1. Stiles' reagent 2. CH2N2 3. H2, Pd/C

O COOEt

H

1. NaH, ClP(O)(OEt)2 2. LiCu(CH3)2

MeO

O

O 417

416

COOEt

H MeO

418

419 1. i-Bu2AlH 2. DMP 3. Allyl-MgBr

H

COOEt

H

Grubbs′ 2nd generation catalyst

8 11

H

9

COOEt

8

MgCl 9

H

COOEt

OH

422 11S 423 11R

424 11S 425 11R

HK 18-crown-6

11

OH

OH

COOEt

H

OHC

H

421

420

DMP

H

COOEt

H

1. KH(TMS) PhNTf2 2. Pd(PPh3)4 LiCl, Et3SiH

H

COOEt

H

LAH, THF reflux

CH2OH

H H

Ac2O DMAP Et3N

H

CH2OH

H

O 426

429

43

54 1. DMP 2. NaClO2, NaH2PO4 2-methyl-2-butene

TMSOTf 2,6-lut.

H

COOEt

Pd(OAc)2 DBU

H

H OTMS 427

H

COOEt

LiI 2,4,6-collidine µW

H

COOH

H

H H

O

O 428

46

SCHEME 5.18 Guerrero synthesis of mulinanes 36, 43, 46, and 54.

36

COOH

7-6-5 Tricarbocyclic Diterpenes Chapter

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Treatment of 418 with diethyl chlorophosphate in the presence of sodium hydride and ulterior nucleophilic methylation with lithium dimethylcuprate lead to 419 (with the C8 methyl group of the mulinane skeleton already in place). From 419, diene 420 was achieved, where an anionic oxi-Cope rearrangement was tested in order to obtain the C8 quaternary center. Transformation of 419 to 420 was achieved following the sequence i-Bu2AlH reduction, DMP oxidation, and allylmagnesium bromide addition. In this manner, the 3:2 allylic alcohols diastereomeric mixture 420 was achieved. Potassium hydride added to a solution of 420 and 18-crown-6 in toluene gave aldehyde 421 in 73% yield. The seven-membered ring of the mulinane skeleton synthesis was achieved via RCM, using 421 as intermediate. Treatment of 421 with methallylmagnesium chloride led to a 3:1 alcohols diastereomeric mixture 422 and 423. Reaction of these compounds (separately) with Grubbs’ second-generation Ru-alkylidene catalyst led to the tricyclic alcohols 424 and 425, respectively. Oxidation of the epimers 424 and 425 with DMP gave the same ketone 426. Treatment of 426 with trimethylsilyl trifluoromethanesulfonate and 2,6-lutidine gave the enol silane intermediate 427, which enabled the examination of a vinylogous Saegusa oxidation used for the first time in the synthesis of natural products. In this manner, reaction of 427 with a stoichiometric amount of Pd(OAc)2 in CH3CN and an ulterior addition of 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU) gave dienone 428, which, by treatment with a large excess of lithium iodide, in 2,4,6-collidine at 200°C under microwave irradiation gave—after acidulation—the mulinane 46. For the mulinanes 54, 43, and 36 synthesis, 426 was used as an intermediate. Enolization of 426 with potassium bis (trimethylsilyl) amide (KN(TMS)2) and enolate trapping with N-phenyl-bis (trifluoromethanesulfonimide) (PhNTf2) gave the triflate intermediate that, by treatment with Pd(PPh3)4 and Et3SiH in LiCl, gave diene 429, which, by AlLiH4 (LAH) reduction in refluxing THF, led to mulinane 54. Acetylation of 54 gave the natural product 43 and the DMP oxidation of 54, followed by Lindgre–Kraus–Pinnick oxidation led to mulinane 36.

CONCLUSIONS In this review, the 7-6-5 tricarbocyclic diterpenes: valparanes, mulinanes, cyathanes, homoverrucosanes, epi-homoverrucosanes, gagunins, and related ones (valparolanes, azorellanes, verrucosanes, neoverrucosanes, and epineoverrucosanes) have been gathered together and classified. The precedence of natural sources, biological activities, and the biosynthetic routes are described. The natural diterpenes in this review proceed biogenetically from GLPP and present a common biogenetic route. The formation of a [5,3,0] decane bicyclic system and ulterior rearrangement and cyclization gives the 7-6-5 tricyclic system that evolution by a proton loss (valparane family), by hydride or/and alkyl rearrangements 1,2 (cyathane and mulinane families) or 1,5

200 Studies in Natural Products Chemistry

(verrucosane family). The synthesis and synthetic approaches to the valparane, mulinanes, and verrucosanes families that have appeared up until now, as well as cyathanes from 2009 to present, have all been included.

ACKNOWLEDGMENT The authors would like to thank, MINECO (SAF2014-59716-R, CTQ2015-68175-R) Junta de Castilla and Leo´n-ESF (UC21, BIO/SA59/15), for the financial support, and Universidad de Salamanca.

ABBREVIATIONS ABNO Ac2O AD AIBN anti-NF-kB AZADO BzCl CBS CD DBU dmdba DMP GGPP GLPP IBX IDM IEDDA KHMDS LAH LiTMP m-CPBA NGF PCC PhNTf2 PHOX PIDA PKR PTSA RCM TBAF

9-azabicyclo[3.3.1] nonane N-oxyl acetic anhydride Alzheimer’s disease azobisisobutyronitrile antinuclear factor kappa-light-chain-enhancer of activated B cells 2-azaadamantane N-oxyl benzoyl chloride Corey–Bakshi–Shibata circular dichroism 1,8-diazabicyclo[5.4.0] undec-7-ene 3,5-dimethoxydibenzylideneacetone Dess–Martin periodinane geranylgeranyl pyrophosphate geranyllinaloyl pyrophosphate o-iodoxybenzoic acid intramolecular double Michael inverse-electron-demand Diels–Alder potassium bis(trimethylsilyl) amide lithium aluminum hydride lithium 2,2,6,6-tetramethylpiperidine-1-ide meta-chloroperbenzoic acid nerve growth factor pyridinium chlorochromate N-phenyl-bis (trifluoromethanesulfonimide) tert-butyl phosphinooxazoline phenyliodine(III) diacetate parallel kinetic resolution p-toluenesulfonic acid ring-closing metathesis tetra-N-butylammonium fluoride

7-6-5 Tricarbocyclic Diterpenes Chapter

TBDPSCl TBS TFA THF TMSCl TMSI–HMDS

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tert-butylchlorodiphenylsilane tert-butyldimethylsilyl trifluoroacetic acid tetrahydrofurane trimethylsilylchloride trimethylsilyliodide–hexamethyldisilazane

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Chapter 6

Fructooligosaccharides: A Review on Their Mechanisms of Action and Effects G. Chen*,†, C. Li‡ and K. Chen*,‡ *

School of Life Science and National Glycoengineering Research Center, Shandong University, Jinan, PR China † Center for Gene and Cell Engineering, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China ‡ Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Wannan Medical College, Wuhu, PR China

Chapter Outline Introduction 209 Fermentation of FOS by Gut Microbiota 210 Beneficial Effects of FOS and Mechanisms 212 Immunomodulation 212 Improvement of Gastrointestinal Condition 213 Protection Against Colorectal Cancer 218

Weight Management and Obesity-Related Disorders Improvement of Bioavailability and Uptake of Mineral Conclusions Acknowledgments References

219

220 220 222 223

INTRODUCTION Fructooligosaccharides (FOS), also sometimes called oligofructose or oligofructan, is a common name of oligosaccharides with beta (2!1) fructosylfructose glycosidic bonds. They have a degree of polymerization (DP) of 2–10 and are generally denoted as Fn or GFn (G referring to the terminal glucose unit, F referring to fructose units, and n designating the number of fructose units in the fructan chain) [1]. In plants, they occur in many organs (eg, leaves, fruit, and rhizome) served as carbohydrate reserve. FOS can be prepared by hot water extraction and ultrasound-assisted extraction from

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plants, fruits, and vegetables (eg, Cichorium intybus, Arctium lappa, and Allium cepa), possibly followed by chemical degradation and/or enzymatic hydrolysis, or enzymatically synthesized (transglycosylation of fungal enzyme beta-fructosidase) from sucrose [2–4]. The presence of b (2 ! 1) fructosyl-fructose glycosidic bonds endows FOS with unique physicochemical and physiological properties, rendering them resistance to hydrolysis by digestive enzyme of the human upper gastrointestinal tract. Thus, FOS was classified as “nondigestible oligosaccharides” that are distinguished from other carbohydrates [5]. In recent years, various nondigestible oligosaccharides (eg, FOS, galactooligosaccharide, isomaltooligosaccharides, soybean oligosaccharides, xylooligosaccharides, and maltitol) have been tested for their prebiotic effects on the microbial community. They present important physiological properties beneficial to the consumer’s health, and these substances were widely used as food ingredients [6]. FOS fulfills the criteria for prebiotic classification: (1) resistance to gastric acidity, to hydrolysis by mammalian enzymes, and to gastrointestinal absorption; (2) fermentation by intestinal microflora; and (3) selective stimulation of the growth and/or activity of those intestinal bacteria that contribute to health and well-being [7].

FERMENTATION OF FOS BY GUT MICROBIOTA The human gut microbiota inhabits a complex ecosystem and plays fundamental role in the well-being of host. It is sometimes considered as our “forgotten organ” [8]. The human large intestine is one of the most diversely colonized and metabolically active organs in the human body [9]. Up to 1000 different species of bacteria reside in the colon with microbial populations comprising approximately 1011–1012 cfu/g of contents. Because of the high residence time of colonic contents and favorable colonic condition, it is appropriate for microbial colonization [10]. As the strictly anaerobic environment, obligate anaerobes are preponderant in the intestinal flora. The two main types of fermentation in the gut are saccharolytic and proteolytic. Because the formation of beneficial bioactive substances [eg, short-chain fatty acids (SCFAs), defensins, and vitamin], most of the saccharolytic bacteria can be considered potentially beneficial (especially lactobacilli and bifidobacteria). The main materials for gastrointestinal bacterial growth are dietary nondigestible carbohydrates. The symbiotic bacteria disassemble dietary nondigestible carbohydrates by a series of carbohydrate hydrolyzing enzymes. However, almost all carbohydrates (eg, dietary fiber, resistant starch, and sugar alcohols) that reach the large intestine will be fermented by the colonic bacteria and affect their growth and metabolic activities in a nonspecific and generalized pattern. In batch cultures inoculated with fecal slurries, the addition of inulin and FOS selectively increased the number of viable bifidobacteria with a parallel decrease in fecal pathogens (eg, Bacteroides, Clostridia, and Coliforms) [11,12]. Due to their chemical structure, FOS escape upper

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intestinal hydrolysis and absorption and enter the large intestine, where can be selectively utilized by beneficial bacteria (eg, Bifidobacterium and Lactobacillus) and decreased growth of harmful microbes (eg, Escherichia coli and Clostridium), thereby improving gastrointestinal condition [13,14]. Indigestibility in the small intestine and fermentability in the colon are two key properties of FOS, which provide a basis for their physiological functions. The fermentation products of FOS by intestinal probiotics, principally SCFAs, are absorbed and utilized by colonocytes and other bacteria to exert variety of physiological activities. SCFAs are important energy sources of host and account for about 10% of daily energy intake. SCFAs play a multifaceted role at intestinal functions and overall health, including inflammation, nutrition, energy metabolism, and colon cancer [15]. Prebiotic effects are defined as “the selective stimulation of growth and/or activity(ies) of one or a limited number of microbial genus(era)/species in the gut microbiota that confer(s) health benefits to the host” [16]. The vital roles of gut microbiota in human physiology and pathophysiological conditions have been well illuminated [17]. The supplementation of FOS can establish a beneficial condition for host health by altering composition and/or activity of the gut microflora, in which potentially health-promoting dominant microorganisms are raised and/or activated with a parallel decrease of potentially harmful ones [13,18]. As one of the most widely commercially available prebiotic compounds, the prebiotic effects of FOS have attracted the interest of academic and industrial scientists. The potential benefits of FOS on health are summarized in Table 6.1.

TABLE 6.1 Potential Beneficial Effects of FOS on Health Potential Beneficial Effects

Reference

Selectively stimulate growth and/or activity(ies) of beneficial microbial genus(era)/species

[13,14,19]

Activate immune system

[20–23]

Reduce morbidity and duration of infectious and antibiotic-associated diarrhea

[24–27]

Alleviate symptoms of irritable bowel syndrome and inflammatory bowel disease

[28–30]

Reduce risk of colon cancer

[31–33]

Decrease appetite

[34–36]

Reduce the risk of obesity-related disorders

[37,38]

Enhance bioavailability and uptake of mineral

[39–41]

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BENEFICIAL EFFECTS OF FOS AND MECHANISMS Immunomodulation The host immune system is comprised of multifunctional cell types enabling to eliminate a large variety of pathogenic organisms [42]. It has been well known that the host–bacterial association is important for the development human immune system [43]. The gut-associated lymphoid tissue (GALT) is a major component of mucosal immune system and resists the invasion of pathogens, while tolerates self-antigens and the commensal nonpathogenic microorganisms. GALT is compartmentalized into inductive sites [eg, Peyer’s patches and noduli lymphoidei aggregati intestini tenuis] and effector sites (eg, lamina propria) forming a multilayered immune network [44]. A large number of documents have reported the beneficial effects of FOS on the innate immunity in animal models. The diet supplemented with 5% (w/w) FOS increased concentrations of total immunoglobulin A (IgA) in the jejunum, ileum, and colon, and raised expression of polymeric immunoglobulin receptor (pIgR) in intestines of infant mice [22]. DNA microarray data revealed that genes related to intestinal immune responses were significantly influenced by FOS administrationseem [21]. These results from animal studies suggested that the GALT seems to be the primary target of FOS to activate mucosal immunity. In aquatic animals, the intake of FOS with a nutrient supplement increased immunoglobulin M (IgM) levels, phagocytic activity and survival rate after challenged with Aeromonas hydrophila [45]. Moreover, dietary supplementation with FOS could also attenuate allergic disorders by reducing the serum allergen-specific IgG1 level and expression of interleukin (IL)-5 and eotaxin in mice [46,47]. FOS could alleviate 2,4dinitrofluorobenzene-induced contact hypersensitivity response (CHS) with lower expression of IL-10, IL-12 p40, and IL-17 in the lesional site [48]. And Bifidobacterium pseudolongum in the intestinal tract might play a role in the reduction of 2,4-dinitrofluorobenzene-induced CHS by dietary supplementation with FOS [49]. In free-living Chilean elderly, supplement of FOS (6 g/day mixed in a special nutritional supplement) increased activity of natural kill cell and decreased infections compared with non-supplemented individuals, while the production of IL-2 by peripheral blood mononuclear cells and the proportion of natural kill cell was not changed [20]. In a randomized, double-blind, placebo-controlled trial, FOS-supplemented formula (4 g/L FOS) increased the concentration of fecal bifidobacteria while being well tolerated, and specific IgA tended to be higher in the FOS group compared with the placebo group (4 g/L maltodextrins) [23]. A mixture of GOS and FOS had protective effects against both allergic manifestations and infections [50]. A synbiotic combination of FOS plus Lactobacillus salivarius significantly decreased

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childhood atopic dermatitis intensity, medication use frequency and eosinophil cationic protein compared with FOS intake alone [51]. FOS can modulate the colonic and systemic immune system to reduce the risk of miscellaneous pathologies directly or indirectly. FOS primarily activated immunocytes in Peyer’s patches and caused other immunologic changes including increase of secretory IgA in ileum and caecum. Dietary FOS improves the human gut ecosystem most likely through changes in metabolic profile and composition of microbiota [19]. Bacterial cell surface macromolecules are key factors in the microorganism–host cross talk. The FOS-induced composition change of the intestinal microflora may shift the presence of pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPSs), lipoteichoic acids, bacterial flagellin, and unmethylated CpG DNA. The gut-associated lymphatic tissue and enterocytes recognize PAMPs through pattern-recognition receptors (PRRs), mainly including the toll-like receptors (TLRs) and C-type lectin receptors (CLRs), AIM2-like receptors (ALRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), and NOD-like receptors (NLRs) [52,53]. As one of the most extensively studied and best-characterized PRR families, TLRs are the main receptors respond to sensing the PAMPs of probiotics and invading pathogens [54]. These interactions manipulate intricate downstream signaling pathways, including NF-kB and mitogen-activated protein kinases (MAPKs) signaling pathways, which orchestrate the expression of multiple immune-related genes [55,56]. Consistent with such a role, NF-kB and MAPKs pathways are employed to manipulate the signal output of heterologous receptors respond to probiotics [57,58]. In addition, FOS may directly interact with TLR receptors, resulting in activation of downstream signaling pathways of immune responses [59,60]. SCFAs can activate extracellular signal-regulated kinase 1/2 and p38 MAPK signaling pathways in intestinal epithelial cells via G-protein-coupled receptors (GPRs) leading to the production of chemokines and cytokines [61]. Plausible hypotheses have shown that FOS may potentially affect the immune system as a direct or indirect result of the change in the composition and/or fermentation profile of the microbiota. More studies addressing the correlation between the microbial composition and immune markers are needed to understand the impact of FOS on our immune system.

Improvement of Gastrointestinal Condition Human gut is colonized with myriad bacteria, eukaryotes, and viruses. The microbiota plays vital roles in the development and optimal functioning of immune system [62]. The intestinal dysbacteriosis is involved in pathological process of various diseases. Metagenomic and other analyses confirmed that the gastrointestinal flora imbalance is associated with the development of some gastrointestinal disorders [63–66].

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Prevention of Gastrointestinal Infections The supplement with FOS-riched yacon flour had a protective effect against intestinal infection of Salmonella enteritidis serovar Typhimurium and decreased translocation to spleen and liver from 15 to 30 days of treatment [25]. This effect would be mediated by increased total S-IgA, IL-6, and macrophage inflammatory proteins-1a + cells and expression of the receptors CD206 and TLR4, which indicated enhanced nonspecific immunity [25]. In vitro, pretreated with FOS-enriched inulin (100 mg/ml) significantly stimulated bactericidal capacity and decreased IL-1b expression in the chicken macrophage HD11 cell line challenged with Salmonella enteritidis, whereas showed nonsignificant influence on phagocytosis, expression of inducible nitric oxide synthase, C-C motif chemokine ligand 4 and LPS-induced tumor necrosis factor-a [67]. However, diets containing FOS improved the colonization resistance of rats to Salmonella enteritidis, but concomitantly impaired the colonic epithelium and increased intestinal permeability which enhanced translocation of pathogens [68,69]. In a randomized, controlled study, after the cessation of Clostridium difficile-associated diarrhea, patients receiving FOS (12 g/day) for 30 days presented lower relapse rate than those taking the placebo [26]. In a multicentric double-blind vs placebo study, prophylactic treatment with FOS plus Lactobacillus sporogenes significantly reduced morbidity and duration of diarrhea with active infections requiring antibiotics therapy [24]. However, results of a multicenter trial suggested that the administration of inulin and FOS was not effective for preventing diarrhea and antibiotic-associated diarrhea in children [70]. In elderly patients receiving broad-spectrum antibiotics, consumption of FOS cannot prevent antibiotic-associated diarrhea, even though FOS was well tolerated and increased fecal bifidobacterial concentrations [27]. Besides immunomodulation properties, other potential abilities are employed by prebiotics to defense pathogens. The presence of an inducible b-fructofuranosidase enzyme and specific transport systems for fructooligosaccharide (DP < 8) enable probiotics, especially Bifidobacteria, to efficiently ferment inulin-like fructans [71,72]. This endows probiotics with advantage in competition for nutrients and ecological niches, which in turn suppress growth and adhesion to intestinal epithelial cells of pathogens. Moreover, some probiotics, mainly lactic acid bacteria, are able to secrete bacteriocin which is effective to kill or incapacitate pathogenic microorganism [73–76]. In this regard, some fermentation products (eg, lactic acid and SCFAs) reduce luminal pH to suppress the growth of less acid-resistant species, which are often pathogenic microorganisms. More recently, in a coculture system of Pseudomonas aeruginosa with IEC18 eukaryotic cells, FOS appeared to reduce pathogenicity through decreasing intracellular exotoxin A levels [77]. The attachment of microbes to carbohydrate moieties on the enterocyte is

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the crucial step of successful colonization and infection. It has been reported that dietary oligosaccharides mimic the sugar chains on the glycoproteins and glycolipids present on gut epithelial cells, thereby blocking the adhesion of pathogenic microorganisms [78]. From the above-mentioned experiments, daily diet supplemented with FOS is able to reduce intestinal pathogen colonization by improving the mucosal immunity, facilitating the growth of probiotics, and reducing virulence and adhesion of pathogens. However, it is noted that FOS fermentation by colonic bacteria may trigger the intestinal damage in some situations.

Treatment for Function Bowel Disorders Functional bowel disorder (FBD) is a functional gastrointestinal disorder with symptoms attributable to the mid or lower gastrointestinal tract, including the irritable bowel syndrome (IBS), functional abdominal bloating, functional constipation, functional diarrhea, and unspecified FBD. The exact pathophysiology of FBD is multifactorial and still not completely understood. IBS is one of the major types of FBD and characterized by abdominal pain, disturbed defecation, and bloating in the absence of any obvious organic cause [79]. IBS affects up to 15% of western population and significantly impacts quality of patients’ lives. The potential role of gastrointestinal microbiota in pathogenesis of IBS has lead to several studies investigating the effectiveness of prebiotics and probiotics for its management. In a randomized comparative double-blind trial, supplemented with FOS (5 g/day for 6 weeks) significantly decreased the intensity of digestive disorder and daily activities as compared to the placebo product among patients diagnosed with minor FBDs [30]. According to the data of functional digestive disorders quality of life questionnaire, the discomfort item scores increased in the FOS group [30]. Nevertheless, some studies have demonstrated adverse effects of dietary FOS in patients with IBS. Olesen et al. reported that IBS symptoms worsened in the FOS group (20 g FOS) compared with the placebo group [80]. At the endpoint of the trial, there were no significant differences between intervention and placebo group, and the authors concluded that such high dose of FOS should not be recommended for IBS patients [80]. In a double-blind crossover trial, intake of FOS at a dose of 2 g (three times daily) for 4 weeks produced no significant change in fecal weight and pH, whole-gut transit time and fasting breath hydrogen concentrations, and failed to show therapeutic value in 21 patients with IBS [81]. The exact pathophysiology of IBS is multifactorial. The traditional theories consider that abnormal gastrointestinal motility and visceral hypersensitivity are the major mechanisms associated with pathophysiology of IBS. Recently, inflammation/immune activation and alterations in fecal flora (decrease of bifidobacteria) were observed in IBS patients compared to

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healthy subjects [82,83]. The alteration of gut microbiota and immune dysfunction may be additional potential mechanisms of IBS. Due to the potential of modulating intestinal microbiota and mucosal immunity, intake of FOS seems to be beneficial for the symptomatic control of IBS patients. However, the fermentable carbohydrate can stimulate colonic gas production and luminal distention, which might increase flatulence and bloating resulting in abdominal discomfort and pain in the context of visceral hypersensitivity. High-fermentable oligo-, di-, and monosaccharides, and polyols diet induces gastrointestinal symptoms and lethargy in IBS patients but only flatulence in the healthy subjects [84]. Recently, some studies have focused on managing functional gastrointestinal symptoms in patients with IBS by restricting fermentable carbohydrate. There is a high-quality evidence to support its use as effective strategy to reduce functional gastrointestinal symptoms [85–87]. Although the prebiotics showed a beneficial role in symptomatic control of IBS in many individuals, the use of FOS to treat IBS is still uncertain and awaits more trials.

The Control of Inflammatory Bowel Disease Inflammatory bowel disease (IBD) is a relapsing and remitting disorder characterized by chronic inflammation of the gastrointestinal tract. Crohn’s disease (CD) and ulcerative colitis (UC) are the principal forms of IBD. As many as 1.4 million persons in the United States and 2.2 million persons in Europe are affected by these diseases [88]. A complex interaction of environmental and host factors are involved in the development of IBD. Although the exact etiology of IBD remains unclear, it appears that IBD results from abnormal mucosal immune response to the commensal gastrointestinal microbiota in genetically susceptible individuals [89]. CD causes discontinuous transmural inflammation throughout the gastrointestinal tract primarily affects the ileocolic region of the intestines [90]. Symptoms of CD include diarrhea, abdominal cramps, fever, weight loss, and perianal manifestations, which have profound adverse effects on patients’ nutritional status and quality of life [91]. Type 1 T-helper lymphocyte (Th1) and Th17-related cytokines (eg, IL-12, IL-23, and IL-27) are selectively activated in Crohn’s disease [92]. A number of treatment options for CD are available, such as blockers of proinflammatory cytokines (eg, TNF-a, IL-6, and IL-12) and integrin a-4, steroids, immunosuppressants, antibiotics, and anti-T-cell therapy [93]. The intestinal microbiota play a vital role in the inflammation associated with CD disease [94]. Prebiotics, such as FOS, with capacity to increase fecal and mucosal Bifidobacteria, can interact with the mucosal immune system and modulate mucosal dendritic cell (DC) function in patients with CD. In an uncontrolled study, the daily intake of FOS (15 g/day for 3 weeks) significantly decreased Harvey Bradshaw index and remarkably increased the percentage of mucosal DCs expressing IL-10 with a paralleled

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increase of Bifidobacteria concentration in feces of patients with Crohn’s disease [29]. Similarly, the consumption of 15 g/day FOS for 4 weeks restored immunity homeostasis through increasing IL-10-positive DC and reducing percentage of lamina propria DC staining of IL-6 but no change in IL-12 p40 production in patients with active Crohn’s disease [28]. Even though FOS impacted on DC function, no significant differences were found in clinical response or inflammatory markers or selected microbiota (Bifidobacteria and Faecalibacterium prausnitzii) between the groups after the 4-week intervention [28]. UC causes continuous mucosal inflammation which is restricted to the colon, and it is characterized by ulceration of the colon and rectum with bleeding, mucosal crypt abscesses, and inflammatory pseudopolyps. In a prospective, randomized, placebo-controlled pilot trial showed that dietary supplementation with FOS (12 g/day) for 2 weeks is well tolerated and significantly reduced dyspeptic symptoms compared with placebo group in patients with active ulcerative colitis [95]. Intragastric FOS (1 g/day for 14 days) reduced intestinal inflammatory activity and injury with increased concentrations of lactate and butyrate in trinitrobenzene sulfonic acid-induced colitis rats [96]. There is convincing evidence that intestinal dysbiosis is implicated in the initiation and perpetuation of inflammatory processes observed in IBD patients [97]. In patients with IBD, alteration of intestinal microflora is characterized by lower proportions of Bifidobacteria, F. prausnitzii, Firmicutes, and Clostridium leptum, and a larger proportion of Enterobacteriaceae [98–100]. A positive correlation between decrease of disease activity and increase of number of Bifidobacterium longum was observed in Crohn’s disease patients receiving FOS-enriched inulin [101]. FOS has the ability to modulate the composition and activity of the intestinal microbiota in a beneficial way. The prebiotic effects of FOS may contribute to the decreased symptom of IBD and encourage for follow-up studies in treatment for IBD patients. The reduced carbohydrate fermentation and high protein fermentation in the large intestine also correlated to intestinal inflammation [102]. Commensal bacteria are capable of modulating the host innate immune system and programming the adaptive immune system. The probiotics (especially Bifidobacteria and Lactobacilli) regulate the activation of NF-kB pathway and the release of immunoregulatory cytokine (eg, IL-10) which suppresses inflammation and predisposition to allergies served as an antiinflammatory cytokine [103,104]. The depletion of butyrate was considered as a prominent feature of CD and UC patients when compared with healthy subjects [105,106]. The increased butyrate can modulate the transcription of inflammatory genes through suppression of NF-kB activation, which is one of the major mechanisms of anti-inflammatory effect of butyrate [107]. Furthermore, the association of oxidative stress with IBD is evidenced by increased permanent oxidative damage in IBD [108]. The antioxidant capacity

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of FOS has been confirmed in vitro and in vivo models [109,110]. FOS can prevent human colonic muscle cell impairment induced by LPS and this effect appears to be related to the protection of FOS against LPS-induced damage [111]. Thus, FOS seems to alleviate symptoms of IBD by reducing oxidative stress injury.

Protection Against Colorectal Cancer Colorectal cancer (CRC) is the fourth cause of cancer death throughout the world with a 5-year survival rate of 64.9% [112]. In 2015, about 49,700 Americans will die of CRC [113]. Surgical resection, chemotherapy, and radiation therapy are the major treatments for CRC patients. These treatments often present some undesirable effects, including an increased risk for infections, nausea, vomiting, hair loss, and fatigue, which decline the quality of patients’ lives. Several gene mutations have been linked to a high susceptibility of CRC [114]. Emerging evidence suggested that the chronic inflammation and gastrointestinal microflora linked with colon cancer risk [115–117]. Reactive oxygen species produced by the colonic bacteria Enterococcus faecalis and inflammatory cells will directly cause DNA damages in enterocytes, which may provide a mechanistic basis for CRC [108,118]. FOS has long been appreciated for their antigenotoxicity and anticarcinogenicity. The favorably alteration of the microbiota could elevate the levels of xenobiotic metabolizing enzymes and decreases bacterial enzymes involved in the synthesis of carcinogen. In healthy persons, intervention with FOS tended to increase the total amount of SCFA and significantly suppressed the production of carcinogens [32,33]. Several studies have shown some benefits of FOS in the management of CRC and toxicant-induced carcinogenesis. In a multicenter study, the feeding of FOS (10 g/day) significantly increased the fecal butyrate concentration in the patients with large colorectal adenomas, where decreased fecal lithocholic acid and increased concentrations of cholic acid, total primary bile acids, chenodeoxycholic acid, and ursodeoxycholic acid in feces in subjects without adenoma [31]. The intervention synbiotic (containing FOS, Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus GG) showed prebiotic effects (Bifidobacterium and Lactobacillus increased and Clostridium perfringens decreased) and significantly reduced colorectal proliferation and the cytotoxicity of fecal water on colonic cells [119]. They also reported improvement of epithelial barrier function and a decreased exposure to genotoxins in the polyp patients [119]. Clostridia and Eubacteria are more inclined to synthesis carcinogens and tumor promoters derived from the food components by comparison to Bifidobacterium and Lactobacillus [120]. Butyrate is able to induce cell differentiation, apoptosis, and proliferation inhibition [121]. Furthermore, butyrate is contributed to reduction of toxicity of chemotherapy drug against CRC [122]. So far the

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studies about the direct effect of butyrate on CRC in vivo animal models have yielded conflicting results [123]. The paradoxical effects of butyrate may be determined by its intracellular concentration, cells under different microenvironmental conditions, different energy sources and the state of activation of the cells [124,125]. Aberrant crypt foci (ACF) are the putative preneoplastic lesions in the colonic mucosa and acted as a marker for the risk of developing CRC [126]. FOS was also effective in preventing the ACF incidence in the middle and distal colon of 1,2-dimethylhydrazine (DMH)-challenged BALB/c mice, which may be partly attributed to the upregulated expression of p16 in the distal colon [127]. But it should be noted that 6-month intervention of FOS-enriched inulin did not yield a significant influence on the rectal ACF number, mucosal proliferation, and apoptosis compared with control group among subjects at increased risk for sporadic CRC [128]. The mechanisms behind these effects are not fully understood and require further investigations.

Weight Management and Obesity-Related Disorders Prebiotics (primarily FOS) intake is associated with the control of obesity and related metabolic disorders in a number of animal models [38,129–131]. In human studies, FOS treatment (30 g/day) increased serum short-chain fatty acids and plasma peptide YY (PYY) levels and decreased hunger and appetite in overweight and obese persons [34]. In women with type 2 diabetes, consuming 10 g/day of FOS-enriched inulin modulated some inflammatory markers [37]. However, inconsistent or negative results have been reported. Verhoef et al. reported that 16 g/day FOS can effectively reduce energy intake in health subjects, perhaps due to higher glucagon-like peptide-1 (GLP-1) and PYY levels [36]. In contrast, Pedersen et al. reported a dose-dependent increase in PYY but no significantly influences on appetite profile, energy intake, glucose, insulin, or GLP-1 concentrations after FOS supplementation (15, 25, 35, 45, and 55 g daily) in nonobese humans [35]. Due to low calorie and potential to modulate gut hormone release, dietary FOS supplementation is beneficial to reducing energy intake and improving insulin sensitivity. The SCFAs can mediate release of the gastrointestinal peptides (eg, PYY, ghrelin and oxyntomodulin) which may contribute to the weight management and energy metabolism [132–134]. Besides regulation of gut hormone release, the acetate can directly affect hypothalamic arcuate nucleus and induce anorectic signal [135]. Recently, some research illustrated the important role of symbiotic gut microbiota in the control of host energy metabolism [136–138]. In genetic obese and diet-induced leptin-resistant mice, feeding with FOS (0.3 g/mouse/day) improved glucose homeostasis and leptin sensitivity, which are associated with decrease of Firmicutes and increase of Bifidobacterium spp. and Bacteroidetes phyla [139]. The overweight children are further linked with the microbiota

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aberrancy (a greater number of Staphylococcus aureus and a lower number of bifidobacteria) [140]. The Bacteroidetes and the Firmicutes are the two dominant populations of beneficial bacteria in human gut. In obese people, the relative proportion of Bacteroidetes is decreased compared with lean people [141]. The reestablishment of harmonious gut microbial ecology with a higher proportion of bifidobacteria and a lower proportion of S. aureus might be beneficial to obesity-associated disorders.

Improvement of Bioavailability and Uptake of Mineral Many studies have shown that FOS intake enhanced absorption of minerals, increased bone mineral density, and prevented osteopenia in both animal models and clinical research [39,40,142,143]. However, no effect was observed on bone mineral density in postmenopausal women as well as calcium absorption and retention in adolescent girls [144,145]. These inconsistent results may depend from their physiological status. The dietary FOS can increase mineral bioavailability and absorption by influencing cecal environment (increasing mineral absorbability, crypt fission, and cell vitality and modulating the expression of transport protein in the cecal mucosa) [41,146]. The stimuli of growth and development of the gut’s absorptive area are also involved in the enhanced mineral absorption [147]. SCFAs and other organic acids derived from the colonic fermentation of FOS lower luminal pH in the large intestine and modify ion speciation, which enhance the solubility in the luminal fluid, thereby facilitating the transfer and bioavailability of ion. Butyrate may also stimulate the active calcium transport via upregulation of transient receptor potential vanilloid type 6 and calbindin-D9k [148,149]. Two transcription factors, vitamin D receptor and cdx-2, appeared to be involved in the regulation of calbindin-D9k gene expression in the colorectal segment but not in the proximal small intestine during the process through which the FOS diet enhanced calcium absorption [150]. Further, a Ca–H exchanger is also involved in facilitation of calcium absorption by SCFAs in proximal colon [151].

CONCLUSIONS FOS fulfill all the criteria for prebiotic classification and possesses a variety of beneficial effects to the health of consumers through multiple mechanisms (Fig. 6.1). Although some promising results have been observed, the specific therapeutic role for FOS remains to be further defined. Regarding the broad applications of FOS, there is a clear need for more efforts to explore the optimal dosages and treatment duration of FOS as a prophylactic or therapeutic treatment, and improve understanding of the causal relationship between the alterations of gut microbial ecology and the beneficial effects of FOS.

Prebiotic 0000

0000

6 1

Bacteriocins

4

5

0000

Defensins

Pathogen slgA

0000

0000

0000

0000

0000

2 IEL

7 TGFβ RA

DC B

Mφ

Mφ

10

Mφ

NK cell

9 Mφ

Mast cell

Mφ DC

B

TGFβ IL-10

12

Immune regulation Tolerance!

12

12

Tc 12 Th17 Th2

Lymphoid tissue B

B

B

FIG. 6.1 See legend on next page.

B B

B

TNFα IL-1β IL-6

IL-12 IL-23 IL-4

B 11

IgE

Immune stimulation

Th1 Treg

IEL

GoC

M 8

3

GoC

CMI : Intracellular pathogens Inflammation CMI : Fungal protection Inflammation Humoral : Extracellular pathogens Ab-mediated pathology/allergy

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ACKNOWLEDGMENTS This study was financially supported by the Major State Basic Research Development Program of China (973 Program) (No. 2012CB822102) and the High Technology Research and Development Program of China (863 Program) (No. 2012AA021501).

ABBREVIATIONS ACF CHS CRC DC DMH DP FBD FOS

aberrant crypt foci contact hypersensitivity response colorectal cancer dendritic cell 1,2-dimethylhydrazine degree of polymerization functional bowel disorder fructooligosaccharides

FIG. 6.1 Prebiotics and probiotics exert a range of effects on mucosal barrier function and the responses of the underlying immune tissue of the GALT. Probiotic and commensal microbes (light blue (light gray in the print version) rectangles), assisted by prebiotics (light blue (light gray in the print version) strings), out-compete pathogenic organisms (red (dark gray in the print version) rectangles) for nutrients and binding sites on the epithelial cell surface (1). This barrier function is enforced by the ability of probiotics to influence mucin expression and mucus secretion from the goblet cells (GoC, 2) and by pathogen neutralization by IgA in the mucus layer, facilitated by pIgR-mediated transcytosis of sIgA through the epithelial cell (3). Probiotic bacteria can induce antimicrobial peptides against pathogens either directly, as bacteriocins (4) or activation of epithelial cells to secrete defensins (5). Luminal gut contents are tasted by three mechanisms: directly via DCs extending dendrites through the tight junction and into the lumen (6), epithelial cell pinocytosis of microbiota (7), or by selective transfer of luminal contents via specialized epithelial cells, microfold (M) cells (8). Interspersed between epithelial cells, intraepithelial lymphocytes (IELs—predominantly CD8+) contribute to the cytotoxic, killing response of the epithelial barrier (9). The innate killing response can be activated in NK cells via APC production of IL-12 and the production of IL-15 by epithelial cells (10). Immunity to extracellular parasites is elicited through B cell class switching to IgE production and the sensitization of mast cells/granulocytes, which upon secondary exposure, release primary amines such as histamine (type I hypersensitivity) (11). Finally, the adaptive response elicited is dependent of the presentation responses of 6, 7, and 8. If these APCs present safe commensal/probiotic (blue (gray in the print version)) peptides, then tolerogenic mechanisms driven by TGFb, IL-10, and retinoic acid are initiated-resulting in suppression of T effector responses (Th1, Th2, Th17, and Tc) and IgA production. If APCs present pathogenic peptides (red (dark gray in the print version)), then the default setting of tolerance is bypassed and as a result of the immune stimulatory cytokine environment, effector responses are initiated: Th1-CMI to intracellular pathogens, Th17-CMI to fungal infection, and Th2-humoral responses to extracellular pathogens (12). Probiotics modulate this on/off switch of the mucosal immune system in a strain-dependant manner. Inappropriate modulation by probiotics or pathogenic subversion of mucosal immunity can result in immunopathology: allergy, inflammatory bowel disease, and cancer. This figure is cited from H. Hardy, J. Harris, E. Lyon, J. Beal, A.D. Foey, Nutrients 5(2013): 1869–1912.

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gut-associated lymphoid tissue glucagon-like peptide-1 inflammatory bowel disease irritable bowel syndrome immunoglobulin interleukin lipopolysaccharide mitogen-activated protein kinase nuclear factor kappa-light-chain-enhancer of activated B cells pathogen-associated molecular patterns polymeric immunoglobulin receptor pattern-recognition receptors peptide YY short-chain fatty acids T-helper toll-like receptors tumor necrosis factor-a ulcerative colitis

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Chapter 7

Psychotria Genus: Chemical Constituents, Biological Activities, and Synthetic Studies A.R. de Carvalho Jr.*, M.G. de Carvalho†, R. Braz-Filho*,† and I.J.C. Vieira* *

Laborato´rio de Ci^ encias Quı´micas, Centro de Ci^ encias e Tecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Brazil † ICE, Universidade Federal Rural do Rio de Janeiro, Serope´dica, Brazil

Chapter Outline Introduction Chemical Constituents Dimeric and Polyindoline Alkaloids Isolated from Psychotria Species Monoterpene Indole Alkaloids Isolated from Psychotria Species Other Classes of Alkaloids Isolated from Psychotria Species Triterpenoids from Psychotria Species Other Classes of Metabolites from Psychotria Species Biological Activities

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Cytotoxic Activity Analgesic Activity Antioxidant Activity Antimicrobial Activity Synthesis of Some Compounds from Psychotria Species Quadrigemine C (2) and Psycholeine (3) Psychotrimine (9) 1-Hydroxybenzoisochro manquinone Concluding Remarks References

242 248 250 251 252 252 252 252 252 258

INTRODUCTION Natural products have been employed in the treatment of several diseases for thousands of years [1]. Even recently, despite availability of synthetical drugs, plants remain widely used for medicinal purposes [2]. The diversity of biologically active compounds in plants has motivated chemical studies of various species. Recently, many plant-derived drugs, including semisynthetics compounds, have either been introduced to the market or are involved in clinical trials [3,4], highlighting the importance of medicinal plants in drug discovery. Studies in Natural Products Chemistry, Vol. 48. http://dx.doi.org/10.1016/B978-0-444-63602-7.00007-2 © 2016 Elsevier B.V. All rights reserved.

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The family Rubiaceae Juss. comprises 620 genera totaling about 13526 species, distributed worldwide [5]. Psychotria L. is the largest of the Rubiaceae, possessing more than 2000 species, mainly found in tropical and subtropical regions [6]. Based on its morphological features and geographical distribution, the genus was divided into three subgenera: Psychotria (pantropical), Heteropsychotria (neotropical species), and Tetramerae (some African and Madagascan species) [7]. However, Nepokroeff et al. proposed the reorganization of the genus based on a molecular phylogenetic study [8]. Several Psychotria species are widely used in folk medicine around the world for the treatment of various illnesses. Flowers and fruits of P. colorata, for example, are used by “caboclos” from the Amazon to treat earache and abdominal pain, respectively [9]. In Malaysia, leaves of P. rostrata are employed for the treatment of constipation [10]. P. viridis is used as an ingredient in the hallucinogenic beverage called ayahuasca [11], owing to the presence of N,N-dimethyltryptamine, an indole alkaloid structurally related to neurotransmitter serotonin [12]. Intestinal infections, coughs, respiratory, and stomach disorders are other examples of illnesses which have been treated using other Psychotria species [13].

CHEMICAL CONSTITUENTS Many studies have examined the chemical composition of the species of the genus Psychotria (Rubiaceae). Since 1974 several works have shown that Psychotria is a potential source of alkaloids. Approximately 52% of the metabolites reported were characterized as alkaloids (about 87% belong to the subgroup of indole alkaloids), followed by triterpenes (12%), flavonoids (6%), along with constituents of other classes. Since Psychotria is taxonomically complex, alkaloids can be an important tool to distinguish its species from others which belong to genera with similar features such as Cephaelis Sw. and Palicourea Aubl. [14]. Moreover, these metabolites have shown a range of biological activities, increasing interest in the study of this genus, with the aim of discovering new natural medicines.

Dimeric and Polyindoline Alkaloids Isolated from Psychotria Species The main alkaloids found in pantropical Psychotria (subgenus Psychotria) are polyindole alkaloids, which are characterized by the presence of several N-methyl tryptamine moieties in their structures [15], such as psychotridine (1). This alkaloid, derived from five N-methyltryptamine units, was isolated from P. beccarioides, P. forsteriana, P. oleoides, and P. colorata [16–19]. Quadrigemine C (2) is another example of a polyindole alkaloid identified in this genus having been isolated from P. colorata and P. oleoides [9,16,18,20–22]. Four polyindoline alkaloids, named quadrigemines A (4) and B (5), psychotridine (1), and isopsychotridine C (6), were isolated from leaves of

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7 233

P. forsteriana [19,23]. Besides these compounds, meso-chimonanthine (7), a dimeric indole alkaloid, was also isolated from the same species. It was the first isolation of a dimeric isomer of calycanthine, which are commonly present in the genus Calycanthus, from Psychotria [24]. The chemical study of P. rostrata leaves led to the isolation of two new alkaloids: psychopentamine (8) and psychotrimine (9). Compound 8 was the first example of a polymeric pyrrolidinoindoline alkaloid which contains a C-3a/C-50 bond. This group of compounds generally display two types of common linkages: C-3a/C-3a0 bond and C-3a/C-70 bond [10]. Other examples of polyindoline alkaloids isolated from Psychotria, along with the compounds mentioned above, are summarized in Table 7.1 and their structures are shown in Fig. 7.1.

Monoterpene Indole Alkaloids Isolated from Psychotria Species Monoterpene indole alkaloids (MIAs) are biosynthesised by the coupling of tryptophan and the iridoid terpene secologanin. These kinds of metabolites have exhibited several biological activities, being used, for example as anticancer, antimalaria, and antiarrhythmia agents [30]. MIAs are the main type of alkaloids found in the subgenus Heteropsychotria (neotropical species) [31], being considered chemotaxonomic markers for this subgenus [32]. The chemical investigation of P. correae led to the isolation of seven MIAs, along with other kinds of metabolites. Among them, six alkaloids were reported for the first time: correantoside (22), 10-hydroxycorreantoside (23), correantine A (24), correantine B (25), 20-epi-correantine B (26), and correantine C (27) [33]. Henriques et al. [34] reported the isolation of N-b-D-glucopyranosyl vincosamide (28) from leaves of P. leiocarpa. It was the first report of an N-glycosylated monoterpenoid indole alkaloid. The authors also concluded that the accumulation of this alkaloid depends on the age of the plant and light exposure, being restricted to the aerial parts of P. leiocarpa [34]. The chemical study of P. brachyceras led to the isolation of a new MIA named brachycerine (29). According to Nascimento et al. [35], this alkaloid belongs to a new subclass of MIAs since its terpenoid moiety is probably derived from epiloganin rather than secologanin, as is the usual case. Subsequent studies also showed that the concentration of brachycerine (29) increases on UV-B radiation exposure and osmotic/oxidative stress, suggesting that this compound may play a role in plant defense mechanisms [35,36]. From leaves of P. umbellata, an unusual alkaloid named psychollatine (30) was isolated. This compound is mainly accumulated in aerial parts of the plant but low amounts were also found in its roots [37,38]. Other studies regarding P. umbellata have been made leading to the isolation of three new MIAs: 3,4-Dehydro-18,19-b-epoxy-psychollatine (31), N4-[1-((R)-2-hydroxypropyl)]psychollatine (32), and N4-[1-((S)-2-hydroxypropyl)]-psychollatine (33) [39]. Table 7.2 provides information concerning these compounds, as well as other MIAs isolated from Psychotria species. Their structures are displayed in Fig. 7.2.

234 Studies in Natural Products Chemistry

TABLE 7.1 Dimeric and Polyindoline Alkaloids Isolated from Psychotria Species Compound

Species

Part

References

Psichotridine (1)

P. forsteriana

Leaves

[16–19]

P. oleoides

Leaves

P. colorata

Leaves

P. beccarioides

Leaves

P. colorata

Flowers and leaves

P. oleoides

Leaves

Psycholeine (3)

P. oleoides

Leaves

[21]

Quadrigemine A (4)

P. forsteriana

Leaves

[19]

Quadrigemine B (5)

P. forsteriana

Leaves

[16,19,25]

P. colorata

Leaves

P. rostrata

Leaves and twigs

Isopsychotridine C (6)

P. forsteriana

Leaves

[19]

Meso-chimonanthine (7)

P. forsteriana

Not specified

[16,24]

P. muscosa

Leaves

Psichopentamine (8)

P. rostrata

Leaves

[10]

Psychotrimine (9)

P. rostrata

Leaves

[10]

Psichotriasine (10)

P. calocarpa

Leaves

[26]

Hodgkinsine (11)

P. colorata

Flowers and leaves

[9,16,17,22,25]

P. oleoides

Leaves

P. lyciiflora

Leaves

P. muscosa;

Leaves

P. beccarioides

Branches and twigs

P. rostrata

Flowers and leaves

P. colorata

Flowers

P. muscosa

Leaves

Quadrigemine C (2)

(+)-Chimonanthine (12)

[9,15,18,20–22]

[9,16,25]

Continued

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

7 235

TABLE 7.1 Dimeric and Polyindoline Alkaloids Isolated from Psychotria Species—Cont’d Compound

Species

Part

References

P. rostrata

Branches and twigs

(13)

P. henryi

Leaves and twigs

[27]

(14)

P. henryi

Leaves and twigs

[27]

Nb-demetyl-mesoChimonantina (15)

P. lyciiflora

Leaves

[22]

Psychohenin (16)

P. henryi

Leaves and twigs

[28]

Quadrigemine I (17)

P. oleoides

Leaves

[22]

Isopsychotridine B (18)

P. oleoides

Leaves

[18,22]

Oleoidine (19)

P. oleoides

Leaves

[22]

Caledonine (20)

P. oleoides

Leaves

[22]

Psychotripine (21)

P. pilifera

Leaves

[29]

Other Classes of Alkaloids Isolated from Psychotria Species Besides the above-mentioned compounds, other kinds of alkaloids have also been reported for the genus Psychotria. From aerial parts of P. glumerullata, for example, three new quinoline alkaloids, named glomerulatine A to C (63– 65, Table 7.3, Fig. 7.3) were isolated [53]. The chemical study of P. klugii led to the isolation of two new benzoquinolizidine alkaloids: klugine (66) and 70 -O-demethylisocepaheline (67). In addition, cephaeline (68), isocephaeline (69), and 7-O-methylipecoside (70) were also isolated from stem bark of P. klugii [54].

Triterpenoids from Psychotria Species Some studies have also reported the isolation of tritepenoids from Psychotria species, such as psychotrianosides A to G (72–78) and other common compounds as lupeol (83), betulin (84), friedelin (88), and so on. In Table 7.4 we can find information regarding these and other triterpenoids and Fig. 7.4 shows their structures.

236 Studies in Natural Products Chemistry Me N

H

H

Me

Me

N

H Me

Me

N

N N

Me

N

N Me

H

H

N

N H Me (2)

Me

H

H

N Me

(3)

H

N

N

N Me

H

N

H

H Me

Me

H

N

N

N

N

N

H

Me

H N

Me

N

(1)

H N

N

H

N

N

Me

H

N

H

H N

H N

N

N

Me

N N

N

N

Me Me

H N N

Me

Me

Me

N

H

N

N

N

H

N

H

N

Me

N

N H

N

N

N Me

H

H

N N Me

N H

N N

N

Me

Me

N

N

(4)

N

H

(6)

Me

H H

(5)

H

H

N

H

Me

N

N Me

N NMe

N H

H (7)

H

H N

N

N

(9) Me N

Me

N

H

Me

H Me (8)

NHMe

N

N

N Me

N

N

Me

H

H

NH

N

N

N Me

N

N Me

H N

H N

H

Me

H N

H N

N

N Me (10)

NH H

N N H Me H (11)

N N H Me H (12)

FIG. 7.1 Dimeric and polyindoline alkaloids isolated from Psychotria species.

H

H

Me

7 237

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

Me

N

H H Me N N

H N

N Me

N

N N

N

N

N

N N H H Me (14)

N Me

H (13)

H

H

Me Me

Me N H

Me

N

H

N

N

n

N

N

N

Me N H

N

H

NH

N Me

H (17)

N

N Me

H

N

Me N

N N

N

NH Me

(16)

(15)

n = 3 (18) n = 4 (19) n = 5 (20)

N

N

H

Me

(21)

FIG. 7.1—CONT’D

TABLE 7.2 Monoterpene Indole Alkaloids Isolated from Psychotria Species Compound

Species

Part

References

Correantosideo (22)

P. correae

Leaves

[33]

10-hidroxicorreantosideo (23)

P. correae

Leaves

[33]

Correantine A (24)

P. correae

Leaves

[33]

Correantine B (25)

P. correae

Leaves

[33]

20-epi-correantine B (26)

P. correae

Leaves

[33]

Correantine C (27)

P. correae

Root

[33]

N-b-D-glucopiranosil vincosamide (28)

P. leiocarpa

Aerial parts

[34]

Brachycerine (29)

P. brachyceras

Leaves

[40]

Psichollatine (30)

P. umbellata

Leaves

[37,39]

3,4-Dehydro-18,19-b-epoxyPsychollatine (31)

P. umbellata

Leaves

[39]

N4-[1-((R)-2-hydroxypropyl)]psychollatine (32)

P. umbellata

Leaves

[39]

N4-[1-((S)-2-hydroxypropyl)]Psychollatine (33)

P. umbellata

Leaves

[39]

Continued

238 Studies in Natural Products Chemistry

TABLE 7.2 Monoterpene Indole Alkaloids Isolated from Psychotria Species—Cont’d Compound

Species

Part

References

5a-carboxystrictosidine (34)

P. acuminata

Leaves

[41,42]

P. bahiensis

Aerial parts

P. acuminata

Leaves

P. bahiensis

Aerial parts

Desoxicordifoline (36)

P. acuminata

Leaves

[41]

Lagamboside (37)

P. acuminata

Leaves

[41]

(E/Z)-vallesiachotamine (38 + 39)

P. acuminata

Leaves

[41–44]

P. bahiensis

Aerial parts

P. laciniata

Leaves

P. suterella

Leaves

P. acuminata

Leaves

P. barbiflora

Leaves

P. myriantha

Leaves

P. myriantha

Aerial pars

Strictosidine (41)

P. acuminata

Leaves

[41]

Palicoside (42)

P. acuminata;

Leaves

[41]

Bahienoside A (43)

P. bahiensis

Aerial parts

[42]

Angustine (44)

P. bahiensis

Aerial parts

[42,43]

P. laciniata

Leaves

P. bahiensis;

Aerial parts

P. nuda;

Leaves

P. prunifolia;

Leaves

P. suterella

Leaves

P. laciniata

Leaves

Bahienoside B (35)

Strictosidinic acid (40)

Strictosamide (45)

[41,42]

[6,41,45,46]

[14,42,44,47,48]

Continued

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

7 239

TABLE 7.2 Monoterpene Indole Alkaloids Isolated from Psychotria Species—Cont’d Compound

Species

Part

References

Isodolichantoside (46)

P. correae

Leaves

[33]

10-hydroxy-iso-deppeaninol (47)

P. prunifolia

Branches

[49]

10-hydroxy-antirhine (48)

P. prunifolia

Branches

[49]

N-oxide-10-hydroxyantirhine (49)

P. prunifolia

Branches

[49]

14-oxoprunifoleine (50)

P. prunifolia

Branches

[49]

17-Vinil-19-oxa-2-azonia12-azapentaci-clo [14.3.1.02,14.05,13.06,11] icosa-2(14),3,5(13),6 (11),7,9-hex-aeno (51)

P. prunifolia

Leaves

[47]

17-vinil-19-oxa-2-azonia-12azapentaci-clo [14.3.1.02,14.05,13.06,11] icosa-2(14),3,5(13),6(11),7,9hex-aeno (52)

P. prunifolia

Leaves

[47]

N-demethyl-correantoside (53)

P. stachyoides

Leaves

[50]

Naucletine (54)

P. suterella

Leaves

[48]

Croceaine A (55)

P. umbellata

Leaves

[37]

Umbellatine (56)

P. umbellata

Leaves

[51]

Correantosine E (57)

P. stachyoides

Leaves

[32]

Correantosine F (58)

P. stachyoides

Stem bark

[32]

Stachyoside (59)

P. stachyoides

Aerial parts

[52]

Nor-methyl-23-oxocorreantoside (60)

P. stachyoides

Aerial parts

[52]

Lyaloside (61)

P. laciniata

Leaves

[44]

Aerial parts

[46]

P. suterella Myrianthosine (62)

P. myriantha

R

H N

NMe

H

NMe

H N

H

O

N

H

CO2Me

HO H O

H

NMe

N

OGlc H R = H (22) R = OH (23)

N

H

CHO

O H HO

R2

O

R1

Me H R1 = H; R2 = CHO (25) R1 = CHO; R2 = H (26) NH

H O

N H Glc

H (27)

H

O

O Me (24) O

H

NMe

H

H

N H

OGlu

H

HO

OGlc O

H

CO2Me

(29)

(28)

HO N

H NH

H (30)

H

O

H

N

OGlc

H

O

N CO2Me H

O

H

OGlc H

NH CO2Me H N

O

MeO2C

CO2H

N H H GlcO

CO2Me

(32)

(31)

N

H

O

CO2Me

CO2CH3

HO H

N H H GlcO

NH H GlcO

NH H

GlcO

N

H

O

O

H

H

OGlc

CO2Me (33)

(35)

(34)

CO2H N CO2Me H

N O

N H

N CO2Me

N H Glc

OGlc

H

(37)

(36)

H

H

H

O

N

CHO

Me N CO2H H

NH CO2Me H

OGlc (40)

H

(38 + 39)

NH CO2H H N

CH2OH

CO2Me N H

H

H

O OGlc

N

H

H

(41)

FIG. 7.2 Monoterpene indole alkaloids isolated from Psychotria species.

O OGlc

(42)

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

7 241

H OGlc O

H

N

N MeO2C H

O

H CO2Me

N N

N

H

N

H OGlc (43)

N

OGlc

(45)

(44)

NMe HO

H OH HO

N

H N H

N

H

N

H O

H OH

H

OGlc (47)

(46)

(48)

− O

+ N

N+

HO

H

H

HO MeO2C

O

H H

H

O

H

O

N

H O H N

H N

H

H

OH

N H O

H

H

(49)

O

N H

(50)

O (51) O

N N

H O

N H

N O

H H

O

H (53)

N

N

H

H (52)

N

OGlc

O (54)

FIG. 7.2—CONT’D

Other Classes of Metabolites from Psychotria Species Other types of metabolites isolated from this genus are summarized in Table 7.5 and their structures are displayed in Fig. 7.5.

BIOLOGICAL ACTIVITIES Many studies have evaluated the biological properties of extracts, fractions, and isolated compounds from Psychotria species. These plants have been shown to have mostly cytotoxic, analgesic, antioxidant, and antimicrobial activities, as described in the next sections.

242 Studies in Natural Products Chemistry

H NH

H N H N

NH GlcO H

N

OGlc

H

N O

H

H

O H

H

CO2CH3

CO2CH3 (55)

(56) 3,4

Δ

O

O

N H H

H

H OGlu O

H3CO2C

H O O

H

N

H

N

N

(57) (58)

O

H N H N

OGlc

O

(61)

OGlc H

OGlc

(59) H

(60)

NH N H O

OGlu

HO O (62)

FIG. 7.2—CONT’D

Cytotoxic Activity Roth et al. [19] evaluated cytotoxic activities of four polyindoline alkaloids, isolated from P. forsteriana on rat hepatoma cells (HTC line). Quadrigemines A (4) and B (5), psychotridine (1), and isopsychotridine C (6) exhibited higher cytotoxity than vincristine, used in antitumor chemotherapy. OH

N

N

N H MeO2C MeO

N O

Vincristine

OAc H HO CO2Me H

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

7 243

TABLE 7.3 Other Classes of Alkaloids Isolated from Psychotria Species Compound

Species

Part

References

Glomerulatine A (63)

P. glumerulata

Aerial parts

[53]

Glomerulatine B (64)

P. glumerulata

Aerial parts

[53]

Glomerulatine C (65)

P. glumerulata

Aerial parts

[53]

Klugine (66)

P. klugii

Stem bark

[54]

7ʹ-O-demethylisocefaeline (67)

P. klugii

Stem bark

[54]

Cephaelina (68)

P. klugii

Stem bark

[54]

Isocephaelina (69)

P. klugii

Stem bark

[54]

7-O-methylipecoside (70)

P. klugii

Stem bark

[54]

Harmane (71)

P. barbiflora

Leaves

[6,55]

P. suerrensis

Me N

H N

N

N

N

N Me

(63)

N

N Me

(64)

OH

N COCH3

MeO

H

OMe

MeO

OH

N

OGlc H

O

MeO (66)

HH H N

MeO

O

MeO

H N H

Me

(71)

(70)

H

R2O R1 = R2 = H (68) R1 = Me, R2 = H (69)

(67)

H

OMe

MeO

H

OR1

MeO

N H H H N

Me

N

H

H H H N

N

(66)

HO

N

HO

Me H N N

N

OH

FIG. 7.3 Other classes of alkaloids isolated from Psychotria species.

244 Studies in Natural Products Chemistry

TABLE 7.4 Triterpenoids Isolated from Psychotria Species Compound

Species

Part

References

Psychotrianoside A (72)

P. sp

Whole plant

[56]

Psycotrianoside B (73)

P. sp

Whole plant

[56]

Psycotrianoside C (74)

P. sp

Whole plant

[56]

Psycotrianoside D (75)

P. sp

Whole plant

[56]

Psycotrianoside E (76)

P. sp

Whole plant

[56]

Psycotrianoside F (77)

P. sp

Whole plant

[56]

Psycotrianoside G (78)

P. sp

Whole plant

[56]

Ardisianoside D (79)

P. sp

Whole plant

[56]

Barbinervic acid (80)

P. stachyoides

Leaves

[50]

a-amirin (81)

P. stachyoides

Leaves

[50,57]

P. adenophylla

Leaves

P. adenophylla

Leaves

Ursolic acid (82)

[57,58]

P. mariniana Lupeol (83)

Betulin (84)

P. mariniana;

[58,59]

P. vellosiana

Aerial parts

P. adenophylla

Leaves

[57,58]

P. mariniana Betulinic acid (85)

P. adenophylla

Leaves

[57]

Bauerenol (86)

P. adenophylla

Leaves

[57]

Bauerenol acetate (87)

P. adenophylla

Leaves

[57]

Friedelin (88)

P. adenophylla

Leaves

[57]

In this essay, the authors concluded that the concentrations necessary to promote 100% cellular mortality (after 24 h of incubation) were 2.5, 5, 5, and 10 mM for compounds 1, 4, 6, and 5, respectively [14]. In a subsequent study, quadrigemine B (5) also showed time- and dose-dependent cytotoxic activity against HEp-2 cells [69]. Hayashi et al. [64] reported that psychorubrin (123), a new naphthoquinone isolated from P. rubra, showed cytotoxic activity in the KB cell assay (ED50 ¼ 3.0 mg/mL). In addition, another four naphthoquinone derivatives (137–140) were prepared as a way to establish its structure–activity

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

R1

R2

OH

R1

7 245

R2

O H

H R3

H R4O

(72) (73) (74) (75) (76) (77) (78) (79)

H HO

HO

R1 CH2OH CH3 CH3 CH3 CH3 CH2OH CHO CH3

R2 CH3 CHO CH3 CH3 CH3 CH3 CH3 CH3

R3 α-ΟH β-OH =O α-OH α-OH α-OH α-OH α-OH

R3

COOH

R4 Ara β-D-Xyl(1 β-D-Xyl(1 α-L-Rha(1 β-D-Xyl(1 β-D-Xyl(1 α-L-Ara β-D-Xyl(1

(78)

R1 (81) (82)

OH

R2 H CH3

R3 CH3 CH3 H CO2H

2)-b-D-Glc(1 4)-α-L-Ara 2)-b-D-Glc(1 4)-α-L-Ara 2)-β-D-Glc-α- L-(1 4)-β-D-Glc-β- D-Xyl(1 4)-Ara 4)-β-D-Glc(1 2)-β-D-Glc(1 2)-α-L-Ara 4)-α-L-Rha(1 2)-β-6-Acethyl-Glc(1 4)-α-L-Ara 2)-β-D-Glc(1

4)-α-L-Ara

R H RO

HO

H

O (88)

(86) R = H (87) R = Ac

(83) R = H (84) R = OH (85) R = CO2H

FIG. 7.4 Triterpenoids isolated from Psychotria species.

relationships. All derivatives exhibited higher cytotoxity than psychorubrin (ED50 ranging from 0.3 to 0.6 mg/mL). The authors concluded that extension of conjugation (observed for compounds 137 and 140) (Fig. 7.6) is not sufficient to increase cytotoxic activity, since compound 141 (another naphthoquinone tested) was not active. Thus, other factors must be considered [64]. The in vitro cytotoxic activity of klugine (66), cephaelin (68), and isocephaelin (69), isolated from P. klugii, was evaluated against four human cancer cells lines, SK-MEL, KB, BT-549, and SK-OV-3. In this assay, doxorubicin and 5-fluorouracil were used as positive controls, whereas DMSO was used as a negative control. O

OH

O

OH

F

H

N

OH O OH H O

OMe O

Doxorubicin

Me

HO NH2

O

N

O

H

5-Fluorouracil

246 Studies in Natural Products Chemistry

TABLE 7.5 Other Classes of Metabolites Isolated from Psychotria Species Compound

Species

Part

References

Blumenol A (89)

P. yunnanensis

Aerial parts

[60]

Drummondol (90)

P. yunnanensis

Aerial parts

[60]

3b-Hydroxy-5a, 6a-epoxi-7megastimen-9-one (91)

P. yunnanensis

Aerial parts

[60]

Salicylic acid (92)

P. yunnanensis

Aerial parts

[60]

Resorcinol (93)

P. yunnanensis

Aerial parts

[60]

(-)-Loliolide (94)

P. yunnanensis

Aerial parts

[60]

(6S)-Menthiafolic acid (95)

P. yunnanensis

Aerial parts

[60]

4-hydroxybenzoic acid (96)

P. yunnanensis

Aerial parts

[60]

Vanillic acid (97)

P. yunnanensis

Aerial parts

[60]

Siringic acid (98)

P. yunnanensis

Aerial parts

[60]

Ethyl protocatechuate (99)

P. yunnanensis

Aerial parts

[60]

3-Hydroxy-1-(3,5-dimethoxy-4hydroxyphenyl)propan-1-one (100)

P. yunnanensis

Aerial parts

[60]

b-Hydroxypropiovanillone (101)

P. yunnanensis

Aerial parts

[60]

()-Butin (102)

P. yunnanensis

Aerial parts

[60]

2-(4-hydroxy-3-metoxyphenil)-3(2-hydroxy-5-metoxyphenyl)-3oxo-1-propanol (103)

P. yunnanensis

Aerial parts

[60]

(+)-siringaresinol (104)

P. yunnanensis

Aerial parts

[60]

Feoforbı´deo A (105)

P. acuminata

Leaves

[61]

Pirofeoforbı´deo A (106)

P. acuminata

Leaves

[61]

b-Sitosterol (107)

P. adenophylla

Leaves

[57–60,62]

P. hainanensis

Leaves

P. mariniana; P. vellosiana

Aerial parts

b-Sitosterol glycosylated (108)

P. stachyoides

Leaves

[50]

Stigmasterol glycosylated (109)

P. stachyoides

Leaves

[50]

Stigmasterol (110)

P. vellosiana

Aerial parts

[59]

Psicotramida A (111)

P. sp.

Stem

[63]

Psicotramida B (112)

P. sp.

Stem

[63] Continued

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

7 247

TABLE 7.5 Other Classes of Metabolites Isolated from Psychotria Species—Cont’d Compound

Species

Part

References

Psicotramida C (113)

P. sp.

Stem

[63]

Psicotramida D (114)

P. sp.

Stem

[63]

Psicorubrina (115)

P. rubra

Stem

[64]

Stearic acid (116)

P. hainanensis

Leaves

[62]

6-hydroxy-luteolin-7-O-rutinoside (117)

P. rubra

Aerial parts

[65]

Luteolin-7-O-rutinoside (118)

P. rubra

Aerial parts

[65]

Quercetin (119)

P. hainanensis;

Leaves

[13,62]

P. spectabilis

Leaves

Kaempferol-7-O-glucopyranoside (120)

P. hainanensis

Leaves

[62]

Kaempferol-3-O-glucopyranoside (121)

P. hainanensis

Leaves

[62]

Rutin (122)

P. hainanensis

Leaves

[62]

Psychorubrin (123)

P. rubra

Aerial parts

[65]

6a-hydroxygeniposide (124)

P. rubra

Aerial parts

[65]

Daucosterol (125)

P. hainanensis

Leaves

[62]

Psycacoraone (126)

P. yunnanensis

Aerial parts

[66]

Scopoletin (127)

P. vellosiana

Aerial parts

[59]

Squalene (128)

P. vellosiana

Aerial parts

[59]

Cyclopsychotride A (129)

P. longipes

Whole plant

[67]

Deoxysolidagenone (130)

P. spectabilis

Leaves

[13]

Solidagenone (131)

P. spectabilis

Leaves

[13]

Coumarin (132)

P. spectabilis

Leaves

[13]

Umbelliferone (133)

P. spectabilis

Leaves

[13]

Psoralene (134)

P. spectabilis

Leaves

[13]

Benz[g]isoquinoline-5,10-dione (135)

P. camponutans

Wood

[68]

1-hydroxybenzoisochromanquinone I (136)

P. camponutans

Wood

[68]

248 Studies in Natural Products Chemistry

Compound 66 was more potent against these human cancer cell lines (IC50 values of 0.25, 0.3, 0.86, and 0.18 mg/mL, respectively) than doxorubicin (IC50 values of 1.57, 1.7, 1.0, and 1.3 mg/mL, respectively). On the other hand, compounds 66 and 69 did not show cytotoxic activity against these cell lines [54].

Analgesic Activity Aiming at discovering new painkillers, some researchers have investigated the analgesic properties of extracts and isolated compounds (mostly alkaloids) from Psychotria species, such as P. colorata, used by Amazonian Cablocos to treat earache and abdominal pain. The analgesic activity of an alkaloid extract from P. colorata was assessed by the formalin, writhing, and tail-flick methods, confirming the opioid-like analgesic activity of this species [70]. In a subsequent study, it was reported that the alkaloids from this plant exhibited inhibitory activity on [3H]naloxone binding in rat striata membranes [71]. Other work related to P. colorata was carried out by Both et al. [51] in order to evaluate the analgesic activity of hodgkinsine (11), a major alkaloid isolated from this plant. The authors concluded that this compound presented dose-dependent analgesic activity in mice, probably mediated by opioid and glutamate receptors, suggesting that it participates in the analgesia previously reported for P. colorata [72]. Umbellatine (56), an alkaloid from OH

OH

O

O

OH

OH O (90)

(89)

OH HO O

CO2H

(91)

HO

(96)

H3CO

(99)

OCH3 (98) O

OH

H3CO

OH

HO

HO

OH

HO OCH3 (97)

O O

HO

H3CO

HO

HO

O HO

O OH

OH

(95)

(93)

O

OH (94)

CO2H

(92)

O

O O

OH

O

OCH3 (1 100)

FIG. 7.5 Other classes of metabolites isolated from Psychotria species.

(1 101)

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

7 249

OH H3CO

OH

OCH3

OH OH

O

HO

OH O

OCH3

H3CO O (103)

O (102)

O

OH OCH3

H3CO OH (104)

N

NH

OH O

N

HN

NH

RO

R

HO O

(105) (106)

(107) (108) (109) (110)

O R CO2Me H

R R R R

= = = =

H Glu Glu, Δ22 H

HO

( )n

OH (

)14

OH (111) n = 16 (113) n = 15 (112) n = 14 (114) n = 13

OH O O

H 3C

(CH2)

R2

16

OH O (115)

O

R1O

O OH

R3

R OH

(116)

(117) (118) (119) (120) (121) (122)

O R OH H H H H H

R1 R2 Rut; OH; Rut; OH; H OH; Glc H H H H OH

R3 H H OH OH O-Rut O-Rut

FIG. 7.5—CONT’D

P. umbellata, is other example of a compound which showed analgesic properties [51]. Both et al. [73] evaluated the analgesic activity of alkaloid extracts from three Psychotria species classified as P. myriantha, P. nuda, and P. pubigera. In this work, it was reported that only P. myriantha showed this property (hot plate method) [73].

250 Studies in Natural Products Chemistry O

OH

OH

O

O HO

O

O

O

O

OH

OH

OH HO

O

O HO

OH

(123)

OH

O

OH

O

O

RO

OH

(124)

(125)

OH O

OH (126)

(127)

(128)

(129)

R

O

O

O

O

R O

R (130) H (131) OH

OH

O R (132) H (133) OH

O O

O (134)

N

O O (135)

O (136)

FIG. 7.5—CONT’D

Antioxidant Activity Fragosos et al. [74] evaluated antioxidant and antimutagenic potentials of psychollatine (30) and the crude foliar extract of P. umbellata. Antioxidant properties were assessed in strains of Saccharomyces cerevisiae deficient in superoxide dismutase and/or catalase (exposed to H2O2 and paraquat) and by the hypoxanthine/xanthine oxidase assay. Psychollatine (30) was more efficient in protection of strains treated with paraquat, whereas the crude foliar extract

Chemical Constituents, Biological Activities, and Synthetic Studies Chapter

O

O O

O (137)

O O

O (138)

O

7 251

O

O (139) O

O

O

O

O

(140)

(141)

FIG. 7.6 Structures of compounds 137–141.

showed better results for strains treated with H2O2. In the hypoxanthine/ xanthine oxidase assay both psychollatine (30) and the crude extract showed a marked dose-dependent antioxidant activity, but the crude extract was more active (possibly owing to the presence of flavonoids) than the isolated compound. Both the crude extract and psychollatine (30) showed antimutagenic effects on strains of S. cerevisiae (mutagenesis was induced by H2O2) [74]. Brachycerine (29), isolated from P. brachyceras, is another example of an MIA which possessed antioxidant and antimutagenic activities [75]. The in vitro antioxidant activity of fruits, stems, and leaf extracts of P. nilgiriensis was investigated by DPPH, ABTS+, and FRAP assays. An acetone extract of fruits, had the highest total phenolics (505.74 mg GAE/g extract), tannin (460.78 mg GAE/g extract), and flavonoids (67.78 mg RE/g extract). In addition, this extract presented higher values for DPPH (IC50 ¼ 20.0 mg/mL), ABTS (41,343.51 mmol TE/g extract), and FRAP (4,713.33 mmol Fe (II)/mg extract) assays [76].

Antimicrobial Activity The antimicrobial activity of methanolic extracts of leaves, roots, and stem barks of P. microlabastra against bacteria, protozoa, and fungi, was evaluated by the disk diffusion method. In addition, fractions obtained by partition of these extracts with petrol, dichloromethane, and ethyl acetate were also tested. In this work, the authors reported that all extracts displayed activity against all bacteria and protozoa tested, especially ethyl acetate fractions [77]. Three Psychotria species, along with other Rubiaceae and Meliaceae species, were studied in order to investigate their antimicrobial properties by the disk diffusion method. Extracts of leaves and bark of P. gardineri, P. nigra,

252 Studies in Natural Products Chemistry

and P stenophylla were prepared using n-hexane, dichloromethane, and methanol as solvents and tested against Saccharomyces cerevisiae, Ustilago maydis, Escherichia coli, Micrococcus luteus, Bacillus subtilis, Bacillus cereus, and Aspergillus niger. P. gardineri and P. stenophylla showed a broad antimicrobial activity against six of the seven microorganisms tested while P. nigra was active against four species [78]. The biological activities reported for Psychotria species are summarized in Table 7.6.

SYNTHESIS OF SOME COMPOUNDS FROM PSYCHOTRIA SPECIES Quadrigemine C (2) and Psycholeine (3) The total synthesis of quadrigemine C (2) and psycholeine (3), isolated from P. oleoides [15], was performed by Lebsack et al. [87]. The route reported starts with meso-chimonanthine (7) which was obtained by reaction of oxindole with isatin in 13 steps, as had been reported in previous work [88]. After some steps, quadrigemine C (2) was obtained and acid-catalysed isomeration led to the formation of psycholeine (3), as described in Fig. 7.7.

Psychotrimine (9) The first total synthesis of psychotrimine (9) was proposed by Matsuda et al., involving 16 steps [89]. On the other hand, Newhouse and Baran [90] carried out the total synthesis of ()-psycotrimine in four steps, using 7-bromotryptamine as starting material. According to them, there had been no methodology, up to that point, to construct that kind of C–N bond [90], as can be seen in Fig. 7.8.

1-Hydroxybenzoisochromanquinone This benzoquinone was isolated from the wood of P. camponutans. Its synthesis, performed by Jacobs et al. [91], was achieved with a phthalide annulation reaction using 3-cyano-1(3H)-isobenzofuranone (142) and 5,6-dihydropyran2-one (143), followed by reduction of the lactone moiety. This synthetic route is described in Fig. 7.9 [91].

CONCLUDING REMARKS The genus Psychotria presents a wide chemical diversity, comprising mainly alkaloids. The more abundant alkaloids of the subgenus Psychotria are polyindole alkaloids whereas MIAs are predominant in the subgenus Heteropsychotria. Terpenoids, flavonoids, and other compounds are well known for their biological properties and although a suite of compounds belonging to

TABLE 7.6 Biological Activities Reported for Psychotria Species Species

Plant Part

Extract or Compound

Activity

References

P. rubra

Stem

Psychorubrin

Cytotoxic activity

[64]

P. rostrata

Bark and twigs

Quadrigemine B

Cytotoxic activity

[69]

P. forsteriana

Leaves

Psychotridine, auadrigemines A and B, isopsychotridine, and chimonanthine

Cytotoxic activity

[19,79]

P. camponutans

Wood

Benz[g]isoquinoline-5,10-dione

Cytotoxic activity

[68]

1-hydroxybenzoisochromanquinone P. spectabilis

Leaves

Solidagenone and psoralene

Cytotoxic activity

[13]

P. colorata

Leaves and flowers

Aqueous and alkaloid extracts

Analgesic activity

[70,71]

P. colorata

Flowers

Hodgkinsine

Analgesic activity

[72]

P. brachypoda

Leaves

Ethanol extract

Analgesic activity

[80]

P. umbellata

Leaves

Umbellatine

Analgesic activity

[51]

P. myriantha

Leaves

Alkaloid extract

Analgesic activity

[73]

P. nilgiriensis

Stem and fruit

Acetone extract

Analgesic and antioxidant activities

[76]

P. sarmentosa

Leaves and stems

Aqueous extract

Analgesic activity

[81]

P. umbellata

Leaves

Methanol extract and umbellatine

Antioxidant and antimutagenic activities

[74]

Continued

TABLE 7.6 Biological Activities Reported for Psychotria Species—Cont’d Species

Plant Part

Extract or Compound

Activity

References

P. brachyceras

Leaves

Methanol extract and brachycerine

Antioxidant and antimutagenic activities

[75]

P. leiocarpa

Leaves

N,b-D-glucopyranosyl vincosamide

Antioxidant activity

[82]

P. microlabastra

Leaves, stem, and roots bark

Methanol extract, petrol, and ethyl acetate fractions

Antimicrobial activity

[77]

P. gardineri

Branches and leaves

Dichloromethane and methanol extracts

Antimicrobial activity

[78]

P. nigra

Branches and leaves

Dichloromethane, hexane and methanol extracts

Antimicrobial activity

[78]

P. reevesii

Aerial parts

Methanol extract

Antimicrobial activity

[83]

P. spectabilis

Leaves

Coumarin, deoxysolidagenone, psoralene, and solidagenone

Antifungal activity

[13]

P. prunifolia

Branches

Ethanol extract, strictosamide, and 14-oxoprunifoleine

Antiprotozoal activity

[49]

P. serpens

Not specified

Ethanol extract

Inhibition of herpes simplex virus (HSV-1) replication

[84]

P. klugii

Stem bark

Klugine, 7ʹ-O-demethylisocephaeline, cephaeline, isocephaeline, and 7-Omethylipecoside

Antiparasitic activity

[54]

P. laciniata

Leaves

Alkaloid fraction, lyaloside, and strictosamide

Monoamine oxidase inhibition

[44]

P. suterella

Leaves

Alkaloid extract and (E/Z)-vallesiachotamine

Monoamine oxidase inhibition

[44]

P. myriantha

Leaves

Strictosidinic acid

Monoamine oxidase inhibition

[45]

P. myriantha

Aerial parts

Alkaloid extract, myrianthosine, and strictosidinic acid

Antichemotactic activity

[46]

P. leiocarpa

Leaves

Aqueous extract

Allelopathic activity

[85]

P. capitata

Leaves

Ethanol extract

P. leiocarpa

Leaves

Ethanol extract

Antimycobacterial activity

[86]

P. glaziovii

Leaves

Ethanol extract

Antimycobacterial activity

[86]

P. nuda

Leaves

Ethanol extract

Antimycobacterial activity

[86]

P. pubigera

Leaves

Ethanol extract

Antimycobacterial activity

[86]

P. racemosa

Leaves

Ethanol extract

Antimycobacterial activity

[86]

P. ruelliifolia

Leaves

Ethanol extract

Antimycobacterial activity

[86]

P. suterella

Leaves

Ethanol extract

Antimycobacterial activity

[86]

P. vellosiana

Leaves

Ethanol extract

Antimycobacterial activity

[86]

256 Studies in Natural Products Chemistry H H N NMe

O N H Oxindole

H H N NMe

I

1. BuLi, TMEDA, Et2O78°C

13 Steps

2. ICH2CH2I, Et2O N −78°C to 0°C N H H Me I Meso-chimonanthine SnBu

+

O O

X=

N N H H Me

3

Bn N

N H Isatin

O

X Pd2(dba)3.CH3, P(2-furyl)3, CuI, NMP, rt

NMeTs

NMeTs Bn N

O

O H H N NMe

H H N NMe

NBn

TsMeN

Pd(OAc)2, R-ToI-BINAP, TfO

TsMeN

PMP, MeCN, 80°C N N H H Me

O

O

N Bn

N N H H Me NBn TfO

TsMeN

Pd(OH)2, EtOH, MeOH H2 (100 psi), 80°C Me Bn N

H N

O

MeN

H H N NMe TsMeN

Na, NH3, THF, −78°C

TsMeN

H N

N

N H

100°C

N N H H Me

N N R H Me

N H

O

N

0.1 NAcOH

NH4Cl

N Bn

Me

R H N NMe

H

H N

N Me

N H

(2)

N Me

N Me

H

(3)

FIG. 7.7 Synthesis of quadrigemine C (2) and psycholeine (3) proposed by Lebsack et al. [87].

I I

NHCO2Me

HN H

HN

N H

NCO2Me

1. NIS, Et3N I

Br

Br

N H

NHCO2Me

N

Br

NH2

2. Pd(OAc) Na2CO3 LiCl, X

TMS

= X NHCO2Me

3. CuI N

N H

MeNH

NMe

4. Red-Al

NCO2Me

K2CO3

R

N

N

N H

R = CH2CH2NHCO2Me

NHMe

H

MeO2CNH

NCO2Me

N H

N H N

NHMe

H

MeO2CNH

NHMe

N H Br

NHCO2Me

FIG. 7.8 Synthesis of psychotrimine (9) proposed by Newhouse and Baran [90].

O

O O

OH O

+

O

1.1 equiv. t-BuOLi

O

(142)

OMe O O

Acetone, Δ, 2h

THF −60°C, 3h; rt, 14h

CN

5 equiv. K2CO3 2.2 equiv. Me2SO4

OH

OMe

OMe OH

O

(143)

OMe O O

1.2 equiv. DIBAL-H

O

Toluene, −60°C, 2.5 h OMe

3 equiv. CAN

O

CH3CN/H2O1/2 rt, 30 min OMe

O (136)

FIG. 7.9 Synthesis of 1-hydroxybenzoisochromanquinone proposed by Jacobs et al. [91].

OH

258 Studies in Natural Products Chemistry

these phytochemical classes has been isolated from the Psychotria genus, few have been subjected to pharmacological assays. From two thousand species, only forty-seven have been examined so far. There is a perception that extensive research work has been done with some species of this genus; however, a large number of species are still chemically and/or pharmacologically unknown. While this review has attempted to unite the relevant information about Psychotria species, the bioactivity profiles from the genus, and its alkaloids as the main bioactive compounds, clearly suggest future research priorities. The presence of alkaloids makes the species of Psychotria extremely promising, considering that this class of metabolites has shown a range of biological activities. Moreover, these compounds can be used as models to obtain more potent and effective synthetic derivatives.

ABBREVIATIONS Ac CAN Et MIAs NIS Pd2(dba)3 PMP Rt THF TMEDA TMSOTf

acetyl cerium (IV) ammonium nitrate ethyl monoterpene indole alkaloids N-iodosuccinimide tris(dibenzylideneacetone)dipalladium (0) 1,2,2,6,6-pentamethylpiperidine room temperature tetrahydrofuran tetramethylethylenediamine trimethylsilyl trifluoromethanesulfonate

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Chapter 8

Recent Developments in Natural Product-Based Drug Discovery in Tropical Diseases U.R. Lal* and A. Singh† * †

Birla Institute of Technology, Ranchi, Jharkhand, India Herbal Consultant, Phase-VII, Mohali, Punjab, India

Chapter Outline Introduction Alkaloids Phenolics Quinones

263 264 268 272

Terpenes Miscellaneous Concluding Remarks References

274 278 282 282

INTRODUCTION Protozoan parasites that are infectious to humans represent a significant threat to health and cause more than a million deaths annually [1]. They also threaten the lives of billions worldwide and are associated with significant morbidity and large economic impacts [2–7]. The protozoan disease burden is focused in tropical and subtropical regions of the world; more temperate regions of our globe, including North America and the Asia Pacific region, are impacted by protozoan diseases. The significant burden of human protozoan infections has been exacerbated by the lack of a licensed vaccine for any of the diseases these parasites cause. Treatment and prophylaxis has therefore been dependent on drugs, many of which have become less effective necessitating the search for replacements [8]. Tropical diseases caused by bacteria, fungi, and viruses are still a major threat to public health, despite the tremendous progress in human medicine. Their impact is particularly large in developing countries due to the relative unavailability of medicines and the emergence of widespread drug resistance. As a result of the continuous evolution of microbial pathogens toward antibiotic resistance, there have been demands for the development of new and effective antimicrobial compounds.

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Further, there is also a risk of coinfection with diseases like HIV/AIDS, and diseases like tuberculosis and malaria have high risk of relapsing due to increased resistance to the drugs [9,10]. There is an urgent need to discover new treatments against these ailments or to kill their vectors because of the development of resistance and/or the side effects or costs of available treatments. Most people still rely on traditional medicine for their prevention or treatment. As the plants are a recognized source of new medicines with great potential, there is a need to search new drugs based on traditional system of medicine that can help in eliminating the burden of tropical diseases [11]. Recent reviews have been published in this area but are localized to a particular area or to a particular class of compounds [12,13]. In this review we discuss the bioactive natural products discovered from January 2008 to the beginning of 2013 from natural sources including plants, marine, and microorganisms.

Alkaloids Among the various hapalindole-related alkaloids, isolated from cultured cyanobacterium Fischerella ambigua (UTEX 1903), Fischambiguine B (1) displayed a strong inhibitory activity against Mycobacterium tuberculosis with an MIC (minimum inhibitory concentration) value of 2 mM, with no detectable cytotoxicity in a Vero cell line [14]. Bis-indole alkaloids flinderole A–C (2, 2a, 3) from Australian plant Flindersia acuminata and from the Papua New Guinean plant Flindersia amboinensis (flinderoles B 2a and C 3) were found to have selective antimalarial activities with IC50 values between 0.15 and 1.42 mM [15]. Tryptophan–polyketide hybrid, codinaeopsin (4), isolated from an endophytic fungus was found to be active against Plasmodium falciparum (IC50 ¼ 2.3 mg/mL or 4.7 mM) [16]. Novel antiplasmodial isoquinoline alkaloids cassiarins A (5) and B (6) were isolated from the leaves of Cassia siamea, out of which 5 showed a potent antiplasmodial activity. Other cassiarins C–E (7, 8, 9) from flowers of same plant showed moderate antiplasmodial activity against P. falciparum 3D7. Cassiarin D (8) is a dimeric compound consisting of 5-acetonyl-7-hydroxy-2-methylchromone and cassiarin C (7), and cassiarin E (9) is a dimer of cassiarins A and C [17,18]. Alkaloids from Australian tree, Mitrephora diversifolia, azafluorenone (5, 8-dihyroxy-6-methoxyonychine) (10) and 5-hydroxy-6-methoxyonychine showed antispasmodial activity against two different strains of the parasite P. falciparum (3D7 and Dd2). 5-Hydroxy-6-methoxyonychine displayed IC50 values of 9.9 mM (for 3D7) and 11.4 mM (for Dd2), respectively, while compound 10 showed minimal activity, citing the importance of hydroxylation in selective positions for the activity [19]. Fig. 8.1A constitutes all the structures from 1 to 10. Alstiphyllanines A–D (11–14), isolated from Alstonia macrophylla showed moderate antiplasmodial activity against P. falciparum [20]. Antimalarial benzylisoquinoline alkaloid (15) and (s) isocrydine were isolated

8 265

Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

from the rainforest tree Doryphora sassafras [21]. Isomeric tropane alkaloids 3-a-(1-methylitaconyl)-6-b-senecioyloxytropane (16), (1-methylmesaconyl)-6b-angeloyloxytropane (17), and 3-a-(1-methylmesaconyl)-6b-tigloyloxytropane (18) isolated from the aerial parts of Schizanthus tricolor showed marginal inhibition of P. falciparum strain K1 with IC50 values of 22.8, 24.8, and 36.0 mM and displayed no cytotoxicity on MRC-65 cells (IC50 > 64 mM). Interestingly, the alkaloid mixture exhibited slightly higher activity (IC50 17.0 mM) than the pure compounds, indicating some synergy between the different isomers [22]. Various indolizidine derivatives have been isolated from Prosopis glandulosa var. glandulosa as antiinfectives and antiparasitic compounds. Prosopilosidine (2,3-dihydro-1H-indolizinium chloride) (19) showed potent in vitro antifungal activity against Cryptococcus neoformans and Aspergillus fumigatus (IC50 values ¼ 0.4 and 3.0 mg/mL, respectively) and antibacterial activity against methicillin-resistant Staphylococcus aureus and Mycobacterium intracellulare (IC50 values of 0.35 and 0.9 mg/mL, respectively). 19–22 (along with their dihydrochloride salts 24–26) were also found to be fungicidal against C. neoformans (MFCs ¼ 0.63–1.25 mg/mL) and 20, 21, 22, and 27 against A. fumigatus

A

Cl

R

OH

N

N

CN

N

N

R

N

O

N

NH

NH

NH (2) R= H (2a) R= CH3

(1)

(3) HO

O HO

O N H

O

(4)

O

N

O

O

N H

HO

N

HO N

O

O

(6)

(7)

(5) O

O

O

N

OH

OH

O

O

N

N

O

OH

OH N HO

O (8)

(9)

O

(10)

FIG. 8.1 (A) Alkaloids with activity against tropical diseases (structures 1–10). (Continued)

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B

O O

H3CO

COOCH3

N

N

H3CO

O

H3CO

O

O

O

H3CO COOCH3

H3CO

COOCH3

H3CO

O N

N

(11)

O

H3CO

N

(12)

H3CO

O

H3CO H3CO

(13)

O

O

N

O

O O

(15)

(14)

(16) N

O O

O O

O (17) R1

O

O

O O

O (18)

O

O

R1 N

N

R

OH NH

X=

(CH2)9

OH Y=

O

O

N

R2

O

O

HO

O N

N

N

CF3OO

COOCH3

H3CO

O N

NH

(19) R=R1=Y; (22) R=X/Y; R1=Y/X (24) R=R1=Y.HCl; (27) R=X; R1=X (20) R=R1=Y; (21) R=X/Y; R1=Y/X (23) R=X; R1=X (25) R=R1=Y.HCl; (26) R=X.HCl/Y.HCl; R1=Y.HCl/X.HCl

(CH2)9

FIG. 8.1—Cont’d (B) Alkaloids with activity against tropical diseases (structures 11–26).

(MFCs ¼ 0.63–2.5 mg/mL) were similar to amphotericin B. 19 and 23 also exhibited potent activity and high selectivity index (SI) values against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of P. falciparum, with IC50 values of 39 and 95 ng/mL and 42 and 120 ng/mL, respectively (chloroquine, IC50 ¼ 17 and 140 ng/mL). 19 also showed in vivo antimalarial activity, with an ED50 value of 2 mg/kg/day/ip against Plasmodium berghei-infected mice after 3 days of treatment [23]. Structures of the constituents 11–26 are given in Fig. 8.1B. Guanidine alkaloids (28–31) together with the known batzelladines A, F, H, and L, ptilomycalin A, and fromiamycalin isolated from the Caribbean

Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

C

NH N H N

H2N

O

O

NH O

N

O

(CH2)9

N

O

H N

H2N

XN

O

NH

N

(CH2)6

N H

N H

(CH2)8

(29)

(28)

NH

8 267

OH

O N

HO N

O

N (CH2)6

N H

N H

O N

7 O

N

N

((30)

(CH2)7

N H

(31)

Br N O

OH O

N

H N

Br

H N

N H

NH2

O

(34)

(35) O

OCH3

N H

O

N

OCH3 OCH3 N H O

N H

O (38)

(37)

(36) N

HO

R2

(32) R1 = H, R2 = CH3 (33) R1 = R2 = CH3

O

HN

N O OR1

N H O

O (39)

O O

N H

N

O

(40)

FIG 8.1—Cont’d (C) Alkaloids with activity against tropical diseases (structures 27–40).

marine sponges Monanchora arbuscula and Clathria calla exhibited potent antimalarial activity. Aromatization in the tricyclic core of dehomodehydrobatzelladine C (29) (compared to norbetzelladine A (28), and L (31)) and 29 (compared to batzelladine L) did not alter the antimalarial activity. Batzelladine A was as active as norbatzelladine A (28), batzelladine L, and norbatzelladine L (IC50 0.2–0.4 mM, FcB1). Ptilomycalin A, with one pentacyclic guanidine core, was the most active (IC50 0.1 mM), while compound 29 and clathriadic acid (30), with one aromatic tricyclic guanidine core, exhibited weak antimalarial activities, IC50 with 2.3 and 4.5 mM, respectively. Batzelladine L and ptilomycalin A exhibited similar antimalarial activities against the FcB1 strain than against the D6 and W2 strains. Batzelladine L (0.3 mM) showed a comparable IC50 with ptilomycalin A (0.1 mM) but with a higher SI value [24]. 1,4-Dihydroxy-5-phenyl-2-pyridinone alkaloids from Septoria pistaciarum 32 exhibited moderate in vitro antiplasmodial (antimalarial)

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activity against chloroquine-sensitive (D6) and -resistant (W2) strains of P. falciparum and cytotoxic activity to Vero cells. Compound 33 was moderately active against both methicillin-sensitive and methicillin-resistant strains of S. aureus [25]. Aurachin E (34), contrary to other aurachins, combines a high in vitro antiplasmodial activity with low cytotoxicity and absence of mitochondrial respiratory inhibition [26]. Oroidin (35) was isolated from the marine sponge Agelassventres and inhibited the activity and function of Pdr5P, an enzyme responsible for multidrug resistance phenotype in Saccharomyces cerevisiae. This compound may help in the development of new drugs that reverse this dangerous phenotype of pathogenic yeast and fungi [27]. Among known alkaloids 8-hydroxy-noracronycine (36) and 1,5-dihydroxy 2,3-dimethoxy 10-methyl-9-acridone (37) was found to be potent antimalarial constituents from constituents of Citropsis articulata root bark in an in vitro study. Catharanthine (38), 11-(OH)-coronaridine (39), vobasine (40), and hispolone also displayed antiplasmodial activity against PfK1 (IC50 ¼ 0.996–3.63 mg/mL) [28]. Structures of compounds 28–40 are given in Fig. 8.1C.

Phenolics Prenylated isoflavanones sophoronol C 41 and sophoronol E 42 isolated from the roots of Sophora mollis exhibited moderate antiplasmodial activity against the CQS D10 strain of P. falciparum, with IC50values of 12.9 and 12.8 mM, respectively [29]. Prenylated chalcones, bipinnatones A (43) and B (44), from aerial parts of the Queensland shrub Boronia bipinnata inhibited hemoglobinase II with IC50 values of 64 and 52 mM, respectively [30]. Three phenolic glycosides, robustasides E (45), F (46), G (47) and robustaside D (47a) isolated from Greveilla species “Porinda Queen” exhibited potent antimalarial (IC50 5.4–55.4 mM) activity [31]. The structures of compounds 41–47a are given in Fig. 8.2A. Naphthalene glycosides rumexneposide A (48) rumexneposide B (49) and phenolics such as aloesin and epicatechin 3-gallate from Rumex species exhibited antitubercular activity (MIC: 1.0–20.0 mM) when isoniazid was used as the positive control (MIC: 2.04 mM) [32]. Phloroglucinol derivative tomentosone A 50 and tomentosone B 50a isolated from Rhodomyrtus tomentosa leaves inhibited the growth of chloroquine-resistant and -sensitive strains of the malaria parasite P. falciparum, with IC50 values of 1.49 and 1.0 mM, respectively [33]. Azaphilone derivatives Biscogniazaphilone A 51 and Biscogniazaphilone B 52 from endophytic fungus Biscogniauxia formosana exhibited antimycobacterial acticity with MIC values of 5.12 and 2.52 mg/mL, respectively [34]. Phenylphenalenones haemodorone (53), haemodorol (54), and haemodorose (55) were isolated from the Australian plant Haemodorum simplex by bioactive fractionation; the crude extract of both aerial and bulbaceous

Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

8 269

components of the plant exhibited varying degrees of antibacterial, antifungal, and antiviral activity [35]. The structures (48–55) are given in Fig. 8.2B. Benzoic acid derivatives (from Piper glabratum and P. acutifolium) have shown leishmanicidal activity and trypanocidal activity in vitro. Among the evaluated compounds methyl 3,4-dihydroxy-5-(30 -methyl-20 -butenyl) benzoate (56) exhibited leishmanicidal effect (IC50 13.818.5 mg/mL) against the three Leishmania strains used, and methyl 3,4-dihydroxy-5-(2-hydroxy-3methylbutenyl)benzoate (57), methyl 4-hydroxy-3-(2-hydroxy-3-methyl-3butenyl)benzoate (58), showed significant trypanocidal activity, with IC50 values of 16.4, 15.6, and 18.5 mg/mL, respectively [36]. Methoxylated flavonoid derivatives from Beilschmiedia zenkeri, (2S,4R)-5,6,7-trimethoxyflavan-4-ol (59), (2S,4R)-4,5,6,7-tetramethoxyflavan (60), beilschmieflavonoid A (61), and A O

O

OH

O

OCH3

HO

O

OH O

OH

(41)

O

OCH3 OH O

(42) HO

HO

OH

HO

OH O

OH

OH

O

O

O O

HO HO OH

OH

OH

O

OH

O

(45) O

HO HO

O OH

OH

O O

HO HO

O O

O

O

O O

O

O

HO

OH OH

OH

OH

O

O

HO

O

O

OH

(44)

O (43)

HO

OH

O

O (46)

O

HO HO (47a)

OH

(47)

O

FIG. 8.2 (A) Phenolics with activity against tropical diseases (structures 41–47). (Continued)

270 Studies in Natural Products Chemistry

B

O

O

O

O

O

HO HO

O

OH

O

OH

O O

HO HO

O

OH

O

OH O

(48)

O

O

O

(50) O

O

O

O

O

O

O

HO

O

O

O

O

O O

O

OH

(49)

O

O

O

O

O

O

OH

HO HO

(50a) HO

OH

(52)

OH

(51)

O

O

O OH

O

O

O O

O

O O (53)

O

O (54)

O (55)

FIG. 8.2—Cont’d (B) Phenolics with activity against tropical diseases (structures 48–55).

beilschmieflavonoid B (62) showed antibacterial activity against three strains of bacteria, Pseudomonas agarici, Bacillus subtilis, and Streptococcus minor, and antiplasmodial activity against P. falciparum, chloroquine-resistant strain W2 [37]. Cyclolignene derivatives from Pycnanthus angolensis roots, namely pycnanthulignene A (63), pycnanthulignene B (64), pycnanthulignene C (65), and pycnanthulignene D (66) showed antimicrobial activity against a panel of drug resistant pathogens [38]. Isoflavanone, Platyisoflavanone A (67), isolated from the stem bark of Platycelphium voe¨nse showed anti-TB activity [39]. Ericifolin (eugenol 5-O-b-(60 -O-galloylglucopyranoside) 68, 3-methoxyellagic acid 4-O-rhamnopyranoside, and 2-O-p-hydroxybenzoyl-6-O-galloyl-glucoose have been isolated from antibacterial leaves extract of Melaleuca ericifolia [40]. One of the rare natural products substituted with a dioxane/tetrahydropyran moiety rarely found in nature was isolated from marine-derived fungus Chaetomium species known as chaetoxanthones A, B, and C (69, 70, 71). Compound 71 is a chlorinated xanthone substituted with a tetrahydropyran ring. Compound 70 showed selective activity against P. falciparum with an IC50 value of 0.5 mg/mL without being cytotoxic toward cultured eukaryotic cells, while 69 and 70 displayed a moderate activity against Trypanosoma cruzi with an IC50

Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

C

COOCH3

COOCH3

COOCH3

OH HO

O

OH

HO OH

OH

(56) O

O

O

O

(59) R = H (60) R = CH3 R1

O

O

R

(63) (64) (65) (66)

O R2

O R2 O

HO

O

OH

O

O

O

OH

R

R O OH

O

O O

HO HO

O

O

OH

O

O

OH

HO H3CO

O

HO

O

CH2

OH

OH

OR

(72) R = H (73) R = SO4H

(71)

HO H3CO

O

HO4SO

O

OH

OH

OH HO

O

OH

HO

OH

O

OH

(75)

HO

O

OH

HO

O (78)

HO

O

O OH

O

OH OH

OH (79)

OH

(77)

OH

OH OH

O

OH

O

(76)

OH

OH

O (74)

OH O

CH2

(69) R = H (70) R = OH

OH

(68)

OCH3

OH

O

H3CO

O

OCH3

(67) H3CO

R3

HO HO

HO

R1 = OH, R2 = H R1 =R2 =OCH2O R1 R2 = H, R3 =OCH3 R1 =R2 =OCH3; R3 =OH

R2

R1

O

(61) R = CH3 (62) R = H

OH

O

(58)

O

O

O

O

OH

(57)

O

8 271

OH OH

O (80)

FIG. 8.2—Cont’d (C) Phenolics with activity against tropical diseases (structures 56–80).

272 Studies in Natural Products Chemistry

value of 1.5 mg/mL [41]. a-Mangostin (72), a prenylated xanthone isolated from the fruit hull of Garcinia mangostana L., and its metabolites as mangostin 3-sulfate 73 and mangostanin 6-sulfate 74 (metabolized by two fungi, Colletotrichum gloeosporioides (EYL131) and Neosartorya spathulata (EYR042)) were active as antimycobacterial agent. 73 exhibited higher antimycobacterial activity than the substrate 72 [42]. Coumarin derivative 8-hydroxy-6-methoxy-pentylisocoumarin (75) from Xylosma longifolia (Flacourtiaceae) exhibited an MIC value of 40.5 mg/mL against M. tuberculosis [43]. The flavonoids isorhamnetin, myricetin (76), luteolin (77), quercetin (78), epicatechin (79), kaempferol (80), rutin, and taxifolin were found to modulate isoniazid susceptibility in fast-growing mycobacteria. Myricetin 76 was the most potent compound and could reduce the minimum inhibitory concentration of isoniazid 64-fold at a concentration of 16 mg/mL [44]. The structures of compounds are given in Fig. 8.2C.

Quinones Alisiaquinones A–C (81–83) and alisiaquinol (84), isolated from a New Caledonian deep-water sponge, displayed micromolar range activity on two enzymatic targets of importance for the control of malaria, the plasmodial kinase Pfnek-1, and a protein farnesyl transferase, as well as on different chloroquine-sensitive and -resistant strains of P. falciparum. Alisiaquinone C 83 displayed a submicromolar activity on P. falciparum and a competitive selectivity index on the different plasmodial strains [45]. Antiplasmodial anthrone-anthraquinone Albopunctatone (85) was isolated from Australian Ascidian Didemnum albopunctatum and was found to be moderately active (IC50 5.3 value of mM) [46]. Naphthoquinone derivatives goniothalaminone A (86) and goniothalaminone B (87) isolated from Goniothalamus scortechinii had moderate antiplasmodial, antimycobacterial, and cytotoxic activities [47]. Ehretiquinone (88) and prenylhydroquinone isolated from Ehretia longiflora showed antitubercular activity. Ehretiquinone 88 also exhibited antiinflammatory activity [48]. Engelharquinone (89), isolated as naturally occurring products from a plant source, Engelhardia roxburghiana exhibited moderate antitubercular activity against M. tuberculosis 90-221387 [49]. A naphthopyrone derivative, lasionectrin (90) isolated from fermentations of an Acremonium-like fungus provisionally identified as a Lasionectria sp. (Ascomycota, Hypocreales), was found to inhibit the growth of P. falciparum with an IC50 value of 11 mM [50]. Pseudodimeric meroterpenoids scabellone D (91) containing benzo[c]chromene-7,10-dione scaffold (rare among natural products) was identified as a moderately potent, nontoxic inhibitor of P. falciparum [51]. The structures of compounds 81–91are given in Fig. 8.3A. Naphthohydroquinones, named busseihydroquinones A–D (92–95) and prenylated dihydronaphthoquinone, isolated from the CH2Cl2/MeOH (1:1)

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Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

A

HN

R

O

O

O

O

O OH

O

OH

O HO

O

OCH3 (88)

(86) R= H (87) R= OCH3

(85) O

O

O

OH

O

O

O

O OH

OH

O

OH

(89)

OH

O

O

O OH

(90)

B

O O

O

OH

O

(84)

NH2

OH

O OH

O

R

O

OH

O

O OH

(83) OH

O

OH

O

(81) R = H; (82) R=OCH3

OH

HO

SO2

(91)

OH O OH OH O O

OH O OH

O

O

OH O

O

O

O

O (92)

(93)

OH O

(94)

O

OH N

O O

(95)

H N S

(96)

H N

N NH

NH Br

O

O

OH

Br

O

(97)

FIG. 8.3 (A) Quinones with activity against tropical diseases (structures 81–91). (B) Quinones with activity against tropical diseases (structures 92–97).

274 Studies in Natural Products Chemistry

extract of the roots of Pentas bussei showed only marginal activities against the chloroquine-sensitive (D6) and the chloroquine-resistant (W2) strains of P. falciparum despite of the fact that genus Pentas is frequently used by traditional healers for the treatment of malaria [52]. Brominated pyrroloiminoquinones, dihydrodiscorhabdin B (96) and discorhabdin Y (97) displayed potent and selective in vitro antiprotozoal activity, however P. berghei-infected mice did not respond to these metabolites due to their toxicity in vivo [53]. For structures 91–97 see Fig 8.3B.

Terpenes A abeitane diterpenoid (13S,15S)-6b,7a,12a,19-tetrahydroxy-13b,16-cyclo8-abietene-11,14-dione (98), from the plant Plectranthus porcatus, showed moderate activity (MIC value was 62.50 mg/mL) against S. aureus ATCC 6538 [54]. Bioassay-guided fractionation of tropical marine sponge Cymbastela hooperi afforded diterpene formamides (99–102). Compound 99 is one of the very few examples of a natural product that contains both formamide and isonitrile functionalities within the same molecule. In in vitro antiplasmodial bioassays, 99 was found to have moderate activity (IC50 0.5 mg/mL), 100 had weak activity (IC50 14.8 mg/mL), and 101–102 were inactive indicating the importance of –CN– or NH–CO– group for the activity [55]. Naturally occurring sesquiterpenes elemol (103), transnerolidol (104) and fokienol (104a) contained in essential oils from two plants, amyris (Amyris balsamifera) and Siam-wood (Fokienia hodginsii), showed contact repellency against the yellow fever mosquito, Aedes aegypti [56]. Dihydro-b-agarofuran polyesters, 1a,9b-difuranoyloxy-2-oxo-dihydro-b-agarofuran (105), 1a,9bdifuranoyloxy-2-oxo-3-enedihydro-b-agarofuran (106), and 1a,9b-difuranoyloxy dihydro-b-agarofuran (107), isolated from the CHCl3 extract of the root bark of Osyris lanceolata, displayed antifungal activity against Candida albicans. Compounds 105 and 107 showed antibacterial activity against the Grampositive B. subtilis and S. aureus and Gram-negative Escherichia coli and Pseudomonas aeruginosa. Compound 106 displayed weak antibacterial activity only against E. coli [57]. Bromophycolides, R–U (107–111), isolated from the Fijian red alga Callophycus serratus. These compounds expand the known structural variety of diterpene-benzoate macrolides and exhibited modest cytotoxicity toward selected human cancer cell lines. Bromophycolide S (108) also showed submicromolar activity against the human malaria parasite P. falciparum in comparison to others [58]. For structures 98–111 see Fig 8.4A. Corallolide A (112) and corallolide B (113) isolated from Carrabean gorgonian octoral Pseudopterogorgia bipinnata were shown to have antiparasitic and antitubercular activity [59]. Okundoperoxide (114), a bicyclic cyclofarnesylsesquiterpene endoperoxide was isolated by bioassayguided fractionation of extracts from Scleria striatinux with moderate antiplasmodial activity [60]. Bioassay-guided fractionation of extracts from a

8 275

Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

Fijian red algae afforded diterpenoid benzoate class of compound bromophycoic acid A (115) and E (116), and these compounds display a range of activities against malarial parasites and bacterial pathogens including low micromolar suppression of MRSA and VREF [61]. Ursolic acid (117), a common secondary metabolite in plants, was found to stimulate suicidal death of erythrocytes (cells devoid of mitochondria and nuclei) by cell scrambling. It may be helpful in combating malarial parasite as they thrive on erythrocytes [62]. Bryonolic acid (118), another triterpenoid found in cucurbitaceae family, induces marked increase in expression of Heme oxygenase-1 which also has translational implications in malarial disease expression [63]. A

OH O O

NC

HO

OH

N H

H

O O

H

HO

(103)

H

(105)

(104a)

O O

O

O

O

(102)

O O

O

O

OH

O

O

O O

(104)

O

H

(101) OH

N H

N H

(100)

(99)

(98)

O

O

O HN CHO N H H

OH

O

O

O O

O

O

O

O

HO

O Br

O

(106)

(107)

O

O

Br

O

(108) Br

O

Br (109)

O

O

O

HO

O

HO

O

Br

HO

O

Br (110)

Br (111)

FIG. 8.4 (A) Several terpenes with activity against tropical diseases (structures 98–111). (Continued)

276 Studies in Natural Products Chemistry

B

O

OH

O

HO

HO OO

O

O (112)

O

O OH

O

(114)

O

(113) OH

H O

O HO

O

H HO

Br

(117)

Br

HOOC (115) (116)

O

HO

O O O (119)

O

(118)

OH

HO

H3CCOO

OH

OCOCH3

(120)

OAc

OH

O

O

O

OAc

O

OAc (121)

O

OH (122)

FIG. 8.4—Cont’d (B) Several terpenes with activity against tropical diseases (structures 111–122).

Among the triterpenes derived from the stem bark of Ekebergia capensis, namely 7-deacetoxy-7-oxogedunin (119), 2-hydroxymethyl-2,3,22,23tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene 120 showed IC50 values of 6 and 7 mM, respectively. Compound 120 at a dose of 500 mg/kg showed moderate parasitemia suppression of 52.9% against P. berghei NK 65 in a mouse model. Other triterpenoids from E. capensis (ekeberin A and D1) had moderate activity against the same strain [64].

Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

8 277

C

O

HO HO

O OH

H3COOC HO O

O

O

O

(123) R=H (124) R=CH3

OH

OH

O HO O HO

O HO

HO HO HO HO OH

O

O

O

O OH O OH HO

O

O

OH

(125) FIG. 8.4—Cont’d (C) Several terpenes with activity against tropical diseases (structures 123–125).

Antiplasmodial triterpenoids from the fruits of neem (Azadirachta indica) were isolated and characterized as neemfruitins A (121) and neemfruitins B (122), azadirone and gedunin (known triterpenoids). Their activity (in vitro) provides useful information to the structure activity relationship to limnoid class of compounds [65]. For structures 111–122 see Fig. 8.4B. Antiprotozoal steroidal saponins pandaroside G (123) and its methyl ester (124) isolated from the marine sponge Pandaros acanthifolium potently inhibited the growth of Trypanosoma brucei rhodesiense (IC50 values 0.78 and 0.038 mM, respectively) and Leishmania donovani (IC50 1.3 and 0.051 mM, respectively) [66]. Racemoside A (125), a water-soluble steroidal saponin purified from the fruits of Asparagus racemosus, was found to be effective against antimonialsensitive (strain AG83) and -unresponsive (strain GE1F8R) L. donovani promastigotes, with IC50 values of 1.15 and 1.31 mg/mL, respectively. Incubation of promastigotes with racemoside A 125 caused morphological alterations including cell shrinkage, an aflagellated ovoid shape and chromatin condensation. Racemoside A 125 also showed significant activity against intracellular amastigotes of AG83 and GE1F8R at a seven- to eightfold lower dose, with IC50 values of 0.17 and 0.16 mg/mL, respectively, and was nontoxic to murine peritoneal macrophages up to a concentration of 10 mg/mL. Hence, racemoside A is a potent antileishmanial agent that merits further pharmacological investigation [67], see Fig. 8.4C.

278 Studies in Natural Products Chemistry

Miscellaneous Diterpene alkaloids agelasine J (126), agelasine K (127), and agelasine L (128) isolated from the marine sponge Agelas cf. mauritiana collected in the Solomon Islands displayed in vitro antimalarial activity against P. falciparum [68]. Monamphilectine A (129), a diterpenoid b-lactam alkaloid showed potent antimalarial activity, was isolated in very less (in milligrams) quantity by bioassay-directed extraction of a Puerto Rican marine sponge Hymeniacidon sp. [69]. Flavaglines of the cyclopenta-[bc]-benzopyran type isolated from the leaves of Aglaia forbesii named desacetylpyramidaglains A, C, D (130– 132), only 132 was tested for the antitubercular activity and was found to be comparable with the control (isoniazid) [70]. Bioassay-directed fractionation of extracts from the fermentation broth and mycelium of the fungus Edenia sp. led to the isolation of antileishmanial compounds, preussomerin EG1 (133), palmarumycin CP2 (134), palmarumycin CP17 (135), palmarumycin CP18 (136), and CJ-12,371 (137). All metabolites caused significant inhibition of the growth of L. donovani in the amastigote form, with IC50 values of 0.12, 3.93, 1.34, 0.62, and 8.40 mM, respectively. Compounds 133–137 were inactive when tested against P. falciparum or Trypanasoma cruzi at a concentration of 10 mg/mL indicating that they have selective activity against Leishmania parasites. They also showed weak cytotoxicity to Vero cells (IC50 of 9, 162, 174, 152, and 150 mM, respectively); however, the therapeutic window of these compounds is quite significant with 75, 41, 130, 245, and 18 times (respectively) more antileishmanial activity than cytotoxicity [71]. Fagraldehyde (138), a seco-iridoid isolated from Fagraea fragrans, was weakly active against P. falciparum [72]. Compound 139 was isolated from the ethanolic leaf extract of Momordica charantia showed a moderately strong antitubercular activity against M. tuberculosis H37Rv (MIC ¼ 14 mg/mL) according to the MABA susceptibility assay [73]. The compounds physalins D (140) and physalin F (141) isolated from Physalis angulate were investigated using the similarity ensemble approach database which predicted the antimalarial activity of each of these compounds, which were shown using an in vitro assay against P. falciparum. However, treatment of P. berghei-infected mice with 141 increased parasitemia levels and mortality, whereas treatment with 140 was protective, causing a parasitemia reduction and a delay in mortality in P. berghei-infected mice. The exacerbation of in vivo infection by treatment with 141 was probably due to its potent immunosuppressive activity, which is not evident for 140 [74]. The structures of the bioactive compounds 126–141 are given in Fig 8.5A. From the petroleum ether extract of the root bark of Cussonia zimmermannii four polyacetylenes, 142 and 143, were isolated and were active against T. brucei rhodesiense, T. cruzi, P. falciparum, and L. donovani [75]. Five-membered polyketide endoperoxides, epiplakinic acid F methyl ester

8 279

Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

(144) and epiplakinidioic acid, and a peroxide-lactone, plakortolide J (145) from the Puerto Rican sponge Plakortis halichondrioides, when assayed for antimalarial and antitubercular activity, showed strong activity against the pathogenic microbes P. falciparum and M. tuberculosis [76]. Viridamides A (146) and viridamide B (147) lipodepsipeptides with antiprotozoal activity were isolated from the marine cyanobacterium Oscillatoria nigro-viridis Viridamide A showed antitrypanosomal activity with an IC50 of 1.1 mM and antileishmanial activity with an IC50 of 1.5 mM [77]. Alkynoic lipopeptides have been isolated from a Panamanian strain of the marine cyanobacterium Lyngbya majuscule namely Carmabin A (148), dragomabin (149), and A Cl−

N

N

Cl−

N

N

N

N N

N

NH2

Cl−

N

N

O

N

H N

N

N

O

NH2

NH2

N O

HN (126)

(127)

(128) O

O

O

OH

OH

OH

O

O

O

O

O

O

O

R

O

(130) R1 =OH, R2 = H (131) R1 =H, R2 =OH (132) R1 =OH(b), R2 = H

NH

HO R1

O

O

O O

C (129)

R2

O

OH

O

O O

O O

O (134) R=H (135) R=OH

O (133)

O (138)

(136)

H O

OH

O O

O

HO

(137)

H O

O O O

O

O

HO

(139)

O (140)

O

O O

O OH OH

H

OH

O

O (141)

FIG. 8.5 (A) Miscellaneous compounds having activity against tropical disease (structures 126–141). (Continued)

280 Studies in Natural Products Chemistry

B

OH

OH

O O

O O

(143)

O

(142)

O

O

O O O

(144)

O O

(145)

O

O

O

H N

N

O N

N H

O

O O

N

O

O

O

O

O

(146) O

O

O

H N

N

O N

N H

O

O

N

O

O

O

O

(147) O

O

H N

N

O N

N

O

H3CO NH2

O

(148) O N

H3CO OCH3

O N

N

O

O NH2

O

H N

N O

O

O

O

O N

N O

(150)

O

OO

(149)

O

O

O

O N

OCH3

O OCH3

NH2

H3CO OCH3

OCH3 (151)

FIG. 8.5—Cont’d (B) Miscellaneous compounds having activity against tropical disease (structures 142–151).

Natural Product-Based Drug Discovery Against Tropical Diseases Chapter

C

O

OH OH

S OH

O

N

(152)

O

HO

8 281

O

O

O

(153)

(154)

O

O

O O

O (155)

O

O OH

O

O

(156)

FIG. 8.5—Cont’d (C) Miscellaneous compounds having activity against tropical disease (structures 152–156).

dragonamide A (150) showed good antimalarial activity (IC50 4.3, 6.0, and 7.7 mM, respectively) whereas the nonaromatic analog, dragonamide B was inactive [78]. Cyanolide A (151), isolated from extracts of a Papua New Guinea collection of Lyngbya bouillonii, is a new and highly potent molluscicidal agent against the snail vector Biomphalaria glabrata (LC50 ¼ 1.2 mM) [79]. Molluscicides can play an important role in the control of schistosomiasis because snails of the genus Biomphalaria act as intermediate hosts for the parasite. Thiopalmyrone (152) and palmyrrolinone (153), metabolites isolated from extracts of a Palmyra Atoll environmental assemblage of two cyanobacteria, cf. Oscillatoria and Hormoscilla spp., represent new and potent molluscicidal chemotypes against B. glabrata (LC50 ¼ 8.3 and 6.0 mM, respectively). A slight enhancement in molluscicidal effect (LC50 ¼ 5.0 mM) was observed when these two natural products were utilized as an equimolar binary mixture [80]. On the same line to control the vectors which may be another route to decrease the growth of parasitic disease, essential oil components from ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils (0.1 mg/mL) were found to be active against the mosquito, A. aegypti and exhibited 100% and 97.5% larval mortality, respectively. Major constituents (+)-camphene, benzoic acid, thymol, carvacrol, benzyl benzoate, and benzyl trans-cinnamate caused 100% mortality. Study also indicated that thymol and benzyl benzoate were major contributors to the larvicidal activity of the artificial blend. The results show that the essential oils of ajowan and Peru balsam and some of their constituents have potential as botanical insecticides against mosquito larvae [81]. The structures of compounds 142–151 are given in Fig. 8.5B. Antiprotozoan compounds, gracilioethers A–C (154–156), isolated from the marine sponge Agelas gracilis showed antimalarial activity against

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P. falciparum with IC50 values of 0.5–10 mg/mL, whereas gracilioether B (155) also showed antileishmanial activity [82]. The structures of active gracilioethers are given in Fig. 8.5C.

CONCLUDING REMARKS At present, there is an urgent requirement of discovering new antiprotozoal agents especially in view of emerging resistance to existing drugs as well as increase in cases of HIV-leishmania coinfection and coinfection with other tropical diseases. Exploration of natural sources taking clues from traditional knowledge holds great promise for finding new leads, as has been clear from successes of such studies in the past. This is particularly meaningful today as huge strides have been made in understanding the biology of tropical disease parasites and more biochemical targets are known today. This coupled with advances in technology for isolation, structure elucidation, and biological evaluation makes the search for novel agents from natural sources even more exciting.

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Chapter 9

Selected Bioactive Natural Products for Diabetes Mellitus N.A. Raut, P.W. Dhore, S.D. Saoji and D.M. Kokare Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, India

Chapter Outline Introduction Bioactive Natural Remedies Flavonoids Polyphenols Curcumin Xanthones Thiosugar Derivatives Tannins

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Terpenoids Chalcones Phenolic Acids Miscellaneous Amino Acids Alkaloids Conclusion References

304 306 308 311 311 312 314 315

INTRODUCTION Diabetes mellitus is a life-threatening disorder affecting large number of population. This is a group of metabolic disorder associated with impaired carbohydrate, protein, and fat metabolism. Hyperglycemia is the main characteristic cause due to deficient release of insulin from the pancreas either inherited or acquired defect or due to development of resistance to insulin. The persistent hyperglycemia in diabetes may result in impaired and damage functioning of certain vital organs such as eyes, blood vessels, heart, kidneys, and nerves [1]. At the verge of converting into diabetic, normal individual progresses through impaired glucose tolerance (IGT) and impaired fasting glycemia (IFG). During this transition, individuals are at high risk to convert into diabetic and therefore individuals with IGT or IFG should take proper precaution [2]. In 600 BC, the Hindu physician, Ayurved Susruta, described diabetes as a state in which urine is sweet. He described diabetes as a condition, “madhumeha” in which the person passes urine, which resembles honey. Susruta advised starvation and avoidance of sweet food substances to the patient suffering from madhumeha [3]. The Egyptian Papyrus Ebers (1500 BC) illustrated diabetes as a disease related with the excessive thirst and urination. Greek physician, Aretaeus (AD 130), mentioned it as the “melting down Studies in Natural Products Chemistry, Vol. 48. http://dx.doi.org/10.1016/B978-0-444-63602-7.00009-6 © 2016 Elsevier B.V. All rights reserved.

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of the flesh into urine,” involving muscle wasting and polyuria with sweet and sticky urine. Although sweet urine was recognized as a part of diabetic syndrome, it was Wills (1674) who observed it as infused with honey and sugar. The word “Diabetes” is derived from the Greek word “Diabainein” which means to pass through (Diabetes ¼ flow through + Mellitus ¼ honey). Mathew Dabson (1784) demonstrated that sweetness of urine in diabetes was due to sugar and the sugary matter which was not formed in the kidney but existed in the blood stream [3]. Ancient Arabic, Avicenna, described diabetes in detail with the symptoms such as increased appetite, polydipsia, polyuria, tendency for development of gangrene and inheritance. Earlier, in 1889, German scientists, Joseph von Mering and Oskar Minkowski, showed that surgical removal of pancreas produces diabetes in dog. During the early part of this century, several investigations discovered the pancreatic substance that reduced blood glucose. In 1906, Adolf Kussmaul coined the term “acidosis” for formation of acid in diabetic coma [3]. In an ancient Chinese culture, diabetes is named as ta´ng niǎo bı`ng, which means “sugar-urine disease.” Korean and Japanese have also used the same Chinese name for diabetes. Indeed, sweet taste of urine is because of the presence of sugar which has been confirmed by Matthew Dobson in 1776 [4]. Insulin, oral hypoglycemic agents, and several natural remedies are used to treat the metabolic aberrations caused by diabetes mellitus. In the year 1921, Banting et al. extracted insulin [5], first time obtained as pure crystals in 1926 and its amino acid sequence was determined by Sanger in 1955 [6]. Insulin is the first hormone for which radioimmunoassay was developed [7]. It is secreted by b-cells of islets of langerhans in pancreas and acts primarily on glucose sensor [8]. Insulin consists of two amino acid chains namely A and B, linked by disulfide bridges, which are essential for its biological activity. Sulfonylureas cause hypoglycemia by stimulating insulin release from pancreatic b-cells. However, their effects in the treatment of diabetes are more complex. The acute administration of sulfonylurea to noninsulin-dependent diabetes mellitus patients increases insulin release from the pancreas. Furthermore, it may increase insulin levels by reducing hepatic clearance of the hormone, which also causes stimulation of somatostatin release and slight suppression of glucagon secretion. Sulfonylureas block the ATP-sensitive K+ channels on the cell membrane of pancreatic b-cells and initiate its action [9]. Biguanides have extra pancreatic spectrum of action and do not depend upon exogenous or endogenous insulin in the body. They stimulate the peripheral utilization of glucose, increase the sensitivity of the muscles to insulin action, and reduce the intestinal absorption of glucose. Thiazolidinediones were introduced in 1997, which bind to peroxisome proliferator-activated gamma receptor and resulting in increased glucose uptake in muscle and reduced endogenous glucose production. In the year 2000, first agent of this

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class, troglitazone was withdrawn from US market because of its hepatotoxicity. The two other agents rosiglitazone and pioglitazone are used worldwide [10]. As insulin is ineffective orally, the search was continued for an orally effective agent, Synthalin-A, as biguanide was the earliest oral hypoglycemic agent to be used in therapy but was found to be toxic. Interestingly Janbon (1942) led to the discovery of the hypoglycemic action of sulfonamides. Frank and Fuchs had confirmed this in 1955 and observed the blood glucose-lowering effect of carbutamide, a sulfonamide, during its trial in infectious diseases. Since then, many sulfonylureas were used successfully as oral antidiabetic agents. In 1957, biguanides staged a comeback in diabetes therapy when Unger introduced phenformin as an effective and less toxic oral antidiabetic agent [11]. Presently, diabetes is usually controlled by regular physical exercise, proper diet, and use of synthetic oral hypoglycemic agents. Though the oral hypoglycemic agents proved excellent in controlling blood sugar level, they cannot take care of every complication associated with diabetes. However, herbal remedies with multiple phytoconstituents not only control the blood sugar but also ameliorate the diabetic complications. Therefore, this review aims to gather systematic information about the bioactive natural remedies, mostly from herbal sources.

BIOACTIVE NATURAL REMEDIES From time immemorial, man is using natural resources for the treatment or cure of ailments. Several plants have been used in the treatment of diabetes based on their traditional knowledge and scientific reports. Usually, diabetes can be controlled by regular exercise, balanced diet, and chemotherapy. In chemotherapy, oral hypoglycemic drugs are used, but they are associated with several adverse effects. In the recent years consumption of natural remedies has immensely increased due to their less or no adverse effects and similar degree of efficacy as compared to oral hypoglycemic agents. The WHO also endorsed the use of medicinal plants in the treatment of diabetes [12] and encouraged the documentation of ethnomedical data of medicinal plants on the basis of their traditional knowledge [13,14]. Hitherto, hundreds of plants have been screened for the treatment of diabetes and several compounds are isolated from them. Effectiveness of natural products is particularly attributed to large number of bioactive compounds they hold. The bioactive compounds such as polyphenols, flavonoids, tannins, chalcones, and carotenoids are mostly together responsible for efficacy of natural products [13,15,16]. Plants utilize phenolic phytochemicals, having high antioxidant activity [17] to protect themselves from abiotic and biotic stresses. These phenolic compounds are equally beneficial for preventing and combating human chronic diseases linked to oxidative stress [18]. The phenolic

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compounds in plant foods are mainly correlated with significant antioxidant activities along with high amounts of a-amylase and a-glucosidase inhibitors. This plays a very important role in managing hyperglycemia and related complications of hypertension [17,19,20]. Individually, these bioactive molecules may have specific therapeutic activity and might produce certain adverse effects. This can be explained on the basis of structure–activity relationship (SAR), and therefore, it is imperative to discuss their chemistry and bioactivity along with specific structure– function benefits [19,20]. Consumption of these bioactive molecules together in the form of natural product is beneficial and safe. This is because of the fact that the cofactors or other biomolecules negate the adverse effects of each other and therefore considered to be safe. This review aims to gather the information of such bioactive natural products related to the chemistry and biological activities with an attempt to correlate the pharmacological action and the chemical structure. This structure–activity correlation of bioactive natural products effective in diabetes will be helpful in planning various approaches for the control of diabetes. And the same may be useful for the identification and development of lead compounds for diabetes. Some major bioactive molecules and their chemistry, biological activity, and uses have been discussed.

Flavonoids Introduction and Phytochemistry Flavonoids are a class of bioactive compounds having varied chemical structures and properties. They found in fruits, vegetables, herbs, cocoa, chocolate, tea, soy, red wine, and other plant food and beverage products [21]. They represent a large class of no less than 6000 phenolic compounds comprising 15 carbons, with 2 aromatic rings A and B. Ring A condensed with sixmembered heterocyclic ring C, a phenylbenzopyran functionality. This group can be divided broadly into the flavonoids (2-phenylbenzopyrans), isoflavonoids (3-benzopyrans), and neoflavonoids (4-benzopyrans) on the basis of position of attachment of aromatic ring to the benzopyrano moiety (Fig. 9.1). In general, they are divided into six subclasses, such as flavonols, flavones, flavanones, flavan-3-ols (or catechins), isoflavones, and 3⬘ 2⬘ 8 7

1 O

A

C

5

4

6

1⬘ 2

4⬘ B 5⬘ 6⬘

3

FIG. 9.1 Basic structure and numbering system of flavonoids.

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anthocyanidins (Fig. 9.2; Table 9.1). Within each subclass, individual flavonoids and isoflavones are identified and characterized by hydroxylation and conjugation pattern of rings A, B, and C [22]. The basic flavonoid nucleus possesses diverse variety of substituents with the presence of hydroxyl groups at 40 -, 5-, and 7-positions. Majority of flavonoids exist as glycosides with sugar moiety as substituent. Sugars and hydroxyl substituent on basic skeleton of flavonoids increase the water solubility, whereas substituents, like methyl groups and isopentyl units, are responsible for the lipophilicity of the flavonoids [23]. Flavones lack oxygenation at C-3 but otherwise may have a wide range of substituents such as hydroxyl, methyl, O- and C-alkyl group, and glycosylation at different positions. Flavones are present in celery, parsley, and some herbs [24] with apigenin and luteolin, as major dietary flavones. Polymethoxylated flavones, such as tangeretin and nobiletin, have been found in citrus species with 7-O-glycosides [25]. Although it is possible to replace all 10 hydrogen atoms on the flavones, so far only 7 substituents have been reported. Compounds with four, five, and six hydroxyl groups are common, and those with five are abundant [26]. Most commonly occurring flavonoids are flavonols found in every plant except algae and fungi. The main dietary flavonols such as kaempferol, quercetin, fisetin, isorhamnetin, and myricetin are present in the form of O-glycosides [24]. Conjugation occurs at position 3 of the ring C and other substitutions can also occur at position 5-, 7-, 30 -, 40 -, and 50 -carbons. There are numerous flavonols conjugate with different sugar moieties; aglycones are limited. Kaempferol alone has more than 200 different sugar conjugates [25].

A

B

C

D

E

F

FIG. 9.2 Subclasses of flavonoids, (A) flavonol, (B) flavone, (C) flavanones, (D) flavanol, (E) isoflavone, and (F) anthocyanidins.

TABLE 9.1 Substituents on Various Flavonoids

Flavonoid Subclass

B-Ring Connection to C Ring (Position on C Ring)

Flavonols

2

Flavones

Flavanones

Flavanol

2

2

2

Substituents

C-Ring Unsaturation

C-Ring Functional Groups

2–3 Double bond

3-Hydroxy, 4-oxo

Prominent Antidiabetic Flavonoids

3

5

7

30

40

50

Kaempferol

OH

OH

OH

H

OH

H

Myricetin

OH

OH

OH

OH

OH

OH

Quercetin

OH

OH

OH

OH

OH

H

Isorhamnetin

OH

OH

OH

OH

OCH3

H

Apigenin

H

OH

OH

H

OH

H

Luteolin

H

OH

OH

OH

OH

H

Eriodictyol

H

OH

OH

OH

OH

H

Hesperetin

H

OH

OH

OH

OCH3

H

Naringenin

H

OH

OH

H

OH

H

3-Hydroxy

(+)-Catechin (C)

OH

OH

OH

OH

OH

H

3-Ogallate

(+)-Gallocatechin (GC)

OH

OH

OH

OH

OH

OH

(+)-Epicatechin (EC)

OH

OH

OH

OH

OH

H

(-)-Epigallocatechin (EGC)

OH

OH

OH

OH

OH

OH

2–3 Double bond

4-Oxo

None

4-Oxo

None

Isoflavones

Anthocyanidins

3

2

2–3 Double bond

1–2, 3–4 Double bonds

4-Oxo

3-Hydroxy

(-)Epicatechin-3-gallate (ECG)

G

OH

OH

OH

OH

H

(-)-Epigallocatechin-3-gallate (EGCG)

G

OH

OH

OH

OH

OH

Daidzein

H*

H

OH

H

OH

H

Genistein

H*

OH

OH

H

OH

H

Glycitein

H*

H

OH

H

OH

H

Biochanin A

H*

OH

OH

H

OCH3

H

Formononetin

H*

OH

OH

H

OCH3

H

Cyanidin (Cy)

OH

OH

OH

OH

OH

H

Delphinidin (Dp)

OH

OH

OH

OH

OH

OH

Petunidin (Pt)

OH

OH

OH

OCH3

OH

OH

Peonidin (Pn)

OH

OH

OH

OCH3

OH

OH

Pelargonidin (Pg)

OH

OH

OH

H

OH

H

Malvidin (Mv)

OH

OH

OH

OCH3

OH

OCH3

G ¼ gallate. H* indicates the attachment of H at position 2 instead of position 3, as rings B and C linked at position 3 in isoflavones.

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Flavones and flavonols are classified on the nature of the benzene ring (A) in the benzopyrone part of the molecule as it contains four replaceable hydrogen atoms at positions 5, 6, 7, and 8. The number of hydroxyl groups may vary from zero to four and hence there are five main divisions. In each division, number of compounds can be derived depending upon the condition of the side phenyl nucleus (C). The naturally occurring flavonols of monohydroxy-benzopyrone group are fisetin and robinetin. Fisetinis or fisetins reported as activator of sirtuins [27] and inhibitor of DNA methyltransferase enzyme [28]. Naturally occurring flavones of dihydroxy-benzopyrone group show the most favored orientation for two hydroxyl group substitutions, ie, 5:7 and therefore very widely distributed. Among flavones the first three members are chrysin, apigenin, and luteolin. Anthocyanins (of the Greek anthos ¼ flower and kianos ¼ blue), a subgroup of flavonoids, are the most important pigment of the vascular plants. This pigment can easily be incorporated in aqueous media and therefore makes them interesting to use as natural water-soluble colorants [29]. Primarily they occur as glycosides of aglycon anthocyanidin chromophores [30] with the sugar moiety attached at position 3 on ring C or 5-,7-positions on the ring A. The most common sugars that are bonded to anthocyanidins in the form of mono-, di-, or tri-saccharides are glucose, galactose, arabinose, rhamnose, and xylose except for the 3-deoxyanthocyanidins such as luteolinidin and apigeninidin in sorghum [31]. Until now, about 17 aglycon forms of anthocyanins and anthocyanidins have been identified from the nature. The most commonly occurring aglycons are only 6, such as cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin [32]. They can be differentiated from each other on the basis of number of hydroxyl groups, the number and nature of bonded sugars at various positions, the aliphatic or aromatic carboxylates bonded to the sugar moieties, and the position of these bonds [33]. More than 600 anthocyanins occur in nature, which have sugar moieties acylated by a variety of aliphatic or aromatic acids [34,35]. They differ in extent of sugar acylation and the identity of the acylating agent, number, and positions at which the sugars are attached and the number and position of hydroxyl and methoxyl groups on the basic anthocyanidin skeleton. Usually, chemical structures of commonly occurring anthocyanidins differ with the positions of substituent at 30 and 50 of ring B. Flavan-3-ols represent the major class of naturally occurring C6–C3–C6 monomeric flavonoids which are present in many fruits, teas, cocoa, and chocolate. They exist as monomers (catechin and epicatechin) or oligomers (proanthocyanidins). The other flavan-3-ols like epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epigallocatechin (EGC), and gallocatechin occur mostly in grapes, tea, and certain leguminous seeds [35]. Structurally most complex subclass of flavonoids are flavan-3-ols with saturated C-3 element and therefore nonplanar. The simple monomers like

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(+)-catechin and its isomer ()-epicatechin can be hydroxylated to form gallocatechins. It also undergoes esterification with gallic acid. Flavan-3-ols form oligomeric and polymeric proanthocyanidins by C–C linkage, which cannot be broken by hydrolysis, are known as condensed tannins. The two chiral centers at C-2 and C-3 of the flavan-3-ols produce four isomers for each level of B-ring hydroxylation, two of which, (+)-catechin and ()-epicatechin, are widespread in nature, whereas ()-catechin and (+)-epicatechin are rare [36]. Rutin is a flavonoid glycoside of quercetin, found in many plant species, and its name comes from the plant Ruta graveolens which also contains rutin (Fig. 9.3). It is also known as quercetin-3-O-rutinoside, rutoside, and sophorin with chemical name 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[a-Lrhamnopyranosyl-(1 ! 6)-b-D-glucopyranosyloxy]-4H-chromen-4-one [37]. The proanthocyanidins have been classified, by Hemingway and Karchesy [38] based on their ring A, into two categories such as phloroglucinol and resorcinol. Proapigeninidins, propelargonidins, proluteolinidins, procyanidins, prodelphinidins, and protricetinidin are the examples of ring A-type phloroglucinols. The resorcinol-type ring A are proguibourtinidin, proteracacidin, profisetinidin, promelacacidin, and prorobinetinidin. Proapigeninidins lack a hydroxyl group at C-3, whereas proluteolinidins occur rarely in the nature. The most common phloroglucinol-type proanthocyanidins found in nature are procyanidins and prodelphinidins having characteristic substitution patterns of the ring B with 30 ,40 -dihydroxy or 30 ,40 ,50 -trihydroxy groups [39]. Isoflavones are the flavones found exclusively in leguminous plants with attachment of ring B at position C-3 instead of C-2, which occurs in soy bean (Glycine max) with the highest concentrations [40]. Daidzein and genistein are the major dietary isoflavones, which occur primarily in soy foods [25]. A number of human, animal, and cell-culture studies reported the antidiabetic effects of genistein [41–50]. The flavanones are having nonplanar geometry with a chiral center at C-2 and ring C is attached to the ring B at C-2 in the a-configuration of all the OH O

HO

OH OH HO O

OH

O

O H3C HO

O HO

FIG. 9.3 Rutin.

OH

O

OH

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flavanones occurring naturally. The basic difference between flavones, flavonols, and flavanones is that dihydroflavones (flavanones) lack the double bond between carbons 2 and 3 in the ring C of the flavonoid skeleton. Thus, in flavanones, C-2 bears one hydrogen atom in addition to the phenolic ring B and C-3 two hydrogen atoms. Since C-2 is a center of asymmetry (epimeric center), two stereoisomeric forms of each flavanone structure are possible. Thus, ring B can be either in the (2S)- or (2R)-configuration. Enzymatically catalyzing reaction that converts chalcones to flavanones is stereospecific and thus isolated flavanones from plants are levorotatory. Citrus fruits contain high concentration of flavanones and most abundantly found two major flavanones are naringenin and hesperitin. The bitter flavor of grapefruit is because of naringenin which is reported to possess antidiabetic activity [51].

Pharmacological Actions The chronic hyperglycemia due to diabetes causes long-term damage, dysfunction, and failure of various vital organs, and flavonoids can correct these abnormalities [52]. Intermediate conditions in the transition between normal and diabetes such as IGT and IFG, flavonoids can improve the situation in some cases. Most of the actions of the flavonoids are due to their antioxidant property. In order to reduce the incidence, progression, and severity of complications caused by continuous diabetic hyperglycemia, it is essential to control blood sugar level. The routine hyperglycemic period is difficult to avoid, which is mainly responsible for deleterious effects due to glucose toxicity. Hence, supplementary remedy, which will take care of certain pathological processes such as glycating agents, inhibition of enzymes and oxidants in the body, will be required for attenuation of harmful effects of glucose. Flavonoids affect various causative factors responsible for the development of diabetes and thereby prevent further complications such as cataract [53–56]. It has also been reported that level of blood glucose and methylglyoxal reduced in streptozotocin-induced diabetes in rats fed on high-isoflavone soy protein [57]. The regulation of hyperglycemia by apigenin has been investigated by Panda and Kar [58], in which administration of apigenin for 10 days increased level of serum insulin with decrease in the level of glucose and hepatic glucose 6-phosphatase activity. It suggests that apigenin regulates hyperglycemia by increasing serum insulin level [58]. It has been reported that antioxidant property of anthocyanins is responsible for the prevention of diabetes [59]. Rutin is reported to enhance the Akt phosphorylation which plays a key role in glucose metabolism. It is known to improve insulin resistance and increase glucose uptake by enhancing the activities of many enzymes such as catalase, glutathione reductase, glutathione peroxidase, and superoxide dismutase [60–62]. Some studies correlated antioxidant effect of rutin, effect of rutin on insulin receptor kinase (IRK) activity in the presence of insulin, and

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alteration in Akt pathways with the increased uptake of glucose [62,63]. Many studies have highlighted the effect of rutin on the molecules responsible for downstreaming of insulin-signaling pathways including Akt and AMPK. On the other hand, Hsu et al. [64] have shown that rutin directly causes upstreaming of insulin-signaling pathway via phosphorylation of IRK. So, it is one of the potential bioactive agents available from natural sources for glycemic control which enhances IRK activity and insulin-signaling pathway for improvement of glucose uptake [64].

POLYPHENOLS Curcumin Introduction and Phytochemistry Curcumin is a natural yellow pigment and a key bioactive constituent isolated from the rhizomes of Curcuma longa (turmeric). It is a hydrophobic type of polyphenolic bis-a,b-unsaturated b-diketone which exhibits keto–enol tautomerism and having IUPAC nomenclature as 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadien-3,5-dione [65–67]. In curcumin two aryl moieties having oxy-substitutions and unsaturation are attached to each other by a seven-carbon chain and it is known as diarylheptanoid. Usually, the 7C linkage is unsubstituted but having unsaturation, oxo and hydroxyl substitution as an exception in which it exists in an E-configuration (trans C]C bonds) (Fig. 9.4). The aryl ring substitutions mostly oxy type (hydroxy or methoxy) may be symmetrical or asymmetrical [68]. Curcuminoids are the collective terms used to encompass three structural analogs obtained from turmeric such as curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) (Table 9.2). The three structural analogs differ in methoxy substitution on the aromatic ring. Whereas A

OH

HO R1

R2 O

OH

O

O

β

B Aromatic

OCH3

H3CO HO

Olefinic linker

OH

FIG. 9.4 Structure of (A) curcuminoid and (B) curcumin indicating functional regions.

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TABLE 9.2 Various Curcuminoids Substituents Name of Compounds

R1

R2

Curcumin

OCH3

OCH3

Demethoxycurcumin (DMC)

OCH3

H

Bisdemethoxycurcumin (BDMC)

H

H

curcumin possesses two symmetric o-methoxyphenols linked through a,bunsaturated b-diketone moiety, BDMC is lacking two o-methoxy substitutions. DMC has an asymmetric structure with one of the phenyl rings having o-methoxy substitution. Among these three curcuminoids in turmeric, most abundant is the curcumin than DMC and BDMC [68].

Pharmacological Actions There are many studies conducted to ascertain blood glucose-lowering effects of turmeric and curcuminoids in varying experimental models. Both are effective to control hyperglycemia and the activities of enzymes involved in glucose metabolism. Furthermore, these findings also revealed that, as compared to turmeric, curcuminoids possess better blood glucose-lowering activity in diabetic rats [67]. Curcumin by reducing hyperglycemia also reported to subside the oxidative stress caused by reactive oxygen species and lipid peroxidation. It also enhances endogenous antioxidant machinery such as catalase, glutathione peroxidase, superoxide dismutase, and reduced glutathione [69–71]. Aziz et al. [69] conducted a series of work on antidiabetic and antioxidant effects of curcumin and novel curcumin derivatives (NCD) on isolated pancreatic islets and in type 1 diabetes. According to his studies, HO-1 gene expression and HO activity in the pancreas are increased by NCD which also reported to decrease lipid peroxidation in pancreas and liver, causing superior oxidative status and improved endogenous defenses. The studies suggest that the NCD can appreciably improve experimental type 1 diabetes. This study claims and provides evidence for pancreatic islets regeneration in diabetic rats after treatment with NCD for 40 days [69].

Xanthones Introduction and Phytochemistry Xanthones or 9H-xanthen-9-ones (dibenzo-g-pirone) are tricyclic polyphenols, which consist of two benzene rings attached through a carbonyl group

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O 8

9

1 2

7 6 5

O 4a

3 4

FIG. 9.5 Structure of 9H-xanthen-9-one.

and oxygen which restricts free rotation about the carbon–carbon bonds. They have structural similarities with flavonoids and studies have shown the biosynthetic relationship between them. The “Xanthone” is a combination of “xanth,” which means yellow, and “one” which refers to keto group in the structure. The bioactivities of xanthones are specifically dependent on the unique tricyclic backbone along with various substituents at different positions (Fig. 9.5) [72,73]. Usually, the numbering of the carbons is done according to a biosynthetic convention as the symmetrical nature of the xanthone nucleus coupled with its mixed biogenetic origin in higher plants. The numbering system is based on the basic skeleton as xanthene-9-one and the lowest numbers are used, except for the cases where only ring B is oxygenated [66,67]. Mangiferin, 1,3,6,7tetrahydroxyxanthone-C2-b-D-glycoside, is widely distributed in higher plants. Mangiferin is a first xanthone to be investigated for pharmacological potential and it exhibits antidiabetic property [74].

Pharmacological Actions The biological or therapeutic activities of xanthones are dependent on the basic tricyclic nucleus, but the activities may differ with varying nature and position of the various substituents [72,73]. Some of the examples are discussed in the later text. The mangiferin itself [75–80] and its glycoside (mangiferin-7-O-b-glycoside) [75,78] have been reported to possess antidiabetic activity. This activity of mangiferin has been attributed to two mechanisms such as increasing sensitivity [75] and/or decreasing resistance [78] for the insulin. Further investigations endorsed the inhibitory activity of mangiferin and its glycoside against several carbohydrate-metabolizing enzymes [80–83] for its antidiabetic activity.

Thiosugar Derivatives Introduction and Phytochemistry Thiosugars are the compounds in which the oxygen atom in the five-membered carbohydrate ring is replaced with sulfur. The derivatives of thiosugars exhibit strong inhibitory activity against alpha glucosidases. An important member of thiosugar group is salacinol, which occurs naturally in the plant Salacia reticulata. It consists of thiocyclopentane, a five-membered ring with trivalent sulfur

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(sulfonium ion) and O-sulfate as part of erythritol side chain with ability to act as counterion. The X-ray crystallographic analysis revealed that it has spiro-like configuration with1-deoxy-4-thioarabinofuranosyl cation attached through sulfur to a 10-deoxyerythrosyl-30-sulfate anion [84]. Salacinol, potent a-glucosidases inhibitor, is a thiosugar sulfonium sulfate comprising 1-deoxy-4-thio-D-arabinofuranosyl cation and 30-sulfate anion. The permanent positive charge borne by this is responsible for a-glucosidase inhibitory activity. In 1997, salacinol was isolated from S. reticulata, traditionally used plant in Sri Lanka and south region of India for the treatment of diabetes [85]. The potency of salacinol is the same as that of voglibose and acarbose, the widely used clinical antidiabetic drugs [86,87]. In the subsequent years, other potent glucosidase inhibitors such as kotalanol [88], ponkoranol [89,90], salaprinol [90], and de-O-sulfonated derivatives of all four parent compounds [91–95] were isolated from the same plant. The structure of this class of sulfonium-ion compounds is unique and differs only in the length of the polyhydroxylated side chain (Fig. 9.6). For example, salaprinol has a three-carbon side chain, whereas kotalanol has a seven-carbon side chain. The configurations around the heterocyclitol moiety and also the configurations up to the C-30 position in the side chain are conserved in all of these compounds. Mohan and coworkers [96] have shown that changing the configurations at any of these positions, including that at the stereogenic sulfur atom, leads to complete or partial loss of inhibitory activities against the human intestinal R-glucosidases [96,97]. A

C

B

D

FIG. 9.6 Thiosugar derivatives (A) salacinol, (B) kotalanol, (C) salaprinol, and (D) ponkoranol.

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Pharmacological Actions Glucosidases are largely involved in the human metabolic system and its inhibition is considered to be an efficient approach in the treatment of diabetes [98]. The inhibitory action on various glucosidases can be achieved by blocking active sites present on enzymes by using positively charged moieties. Many naturally occurring and synthesized azasugars are believed to carry a positive charge at physiological pH and may bind to active sites of glucosidase enzymes [99–102]. The permanent positive charge at a suitable position in a potent glucosidase inhibitor imitates the oxacarbenium ion-like transition state in the enzyme-catalyzed reaction [85,103–105] and thereby exerts its action.

Tannins Introduction and Phytochemistry Tannins are the polyphenolic secondary metabolites of higher plants also known as plant polyphenols (vegetable tannins), imparting astringent and tanning properties and therefore termed as “tannin.” All parts of the plants including bark, leaves, roots, seeds, and fruits contain tannins in abundance. As the tannins play a crucial role in the defense mechanism of plants for the protection against microbial infection, insects, or animal herbivory, the production of tannin moves up. Tannins are shiny, amorphous, white and light yellow, or nearly colorless powder possessing characteristic strange smell and astringent taste [106]. The common characteristic features of tannins are binding to proteins, pigments, tanning leather, and ascertain the quantitative determination. These are basic in nature and exhibit antioxidant properties with high molecular weight ranging from 500 to 30,000 Da. These characteristics of tannins set them apart from other polyphenolic compounds. Enormous variety of compounds with vast structural divergence make their classification, a difficult task. Therefore, classification based on special structural features and chemical properties provides valuable input. “Hydrolyzable tannins” are those, which can be hydrolyzed and usually grouped into gallotannins and ellagitannins (ETs), whereas some ETs cannot be hydrolyzed because of the presence of CdC coupling of its polyphenolic residue with polyol unit. In gallotannins, galloyl units or their metadepsidic derivatives are bound to polyols, triterpenoid, or catechin units. ETs are complex polyphenolic compounds composed of one hexahydroxydiphenoyl (HHDP) moiety esterified to sugar (mainly glucose). They do not have glycosidically linked to catechin units but their two galloyl units are CdC coupled to each other. HHDP-sugar linkage is responsible for the structural variability of ETs [107]. Ellagitannins occurrence is abundant in some fruits, nuts, and seeds such as almonds, black raspberries, pomegranates, strawberries, and walnuts [108]. Dietary ETs hydrolyze to ellagic acid (EA) in the human gastrointestinal tract. Both EA and ETs produce urolithin A (3,8-dihydroxy-6Hdibenzopyran-6-one) and its monohydroxylated analog known as urolithin

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B [109]. This might probably be due to cleavage of lactone ring, decarboxylation, and dehydroxylation reactions [110]. ETs are hydrolyzed at ester bond producing HHDP, which are spontaneously rearranged to EA, a dimeric derivative of gallic acid, with poor aqueous solubility [111]. The derivatives of EA existing in plants are formed by methylation, glycosylation, and methoxylation of hydroxyl groups [112]. “Condensed tannins” include nonhydrolyzable oligomeric and/or polymeric proanthocyanidins, produced due to coupling of C-4 and C-8 or C-6 linkage between monomeric catechin. Polymeric flavano-tannins which are coupled flavan-3-ol (catechin) units also belong to condensed tannins. Tannins containing C-glycosidic catechin units in addition to the characteristic structural element HHDP of the monomeric ETs are termed as “complex tannins” [113]. Derivatives of hexahydroxydiphenic acid are supposed to be formed by oxidative coupling of vicinal galloyl ester groups in galloyl-D-glucose ester (Fig. 9.7).

Ellagitannin metabolites are: l l

Monomeric in nature: Formed by intramolecular CdC oxidative coupling Oligomeric in nature: Formed by intermolecular CdO coupling

High molecular weight tannins form less digestible complexes with starch and digestive enzymes by covalent bonding, electrostatic attraction, hydrogen bonding, and hydrophobic interactions [114]. Kaki-tannin with chief functional constituents such as epicatechin, EGC, epicatechin-3-O-gallate, and epigallocatechin-3-O-gallate is useful for prevention of hyperinsulinemia in type 2 diabetes [115].

Pharmacological Actions Earlier tannins have been considered as antinutrients because of their ability to precipitate proteins, enzyme inhibition, lowering absorption of minerals and vitamins from gastrointestinal tract [116]. Nevertheless, tannins have HO

OH HO

OH

O HO

OH

O

OH

HO HO

HO

OH

−2H

O

OH

HO

O

HO

OH OH

Galloyl ester G FIG. 9.7 Oxidative coupling of galloyl ester groups.

Hexahydroxydiphenoyl ester G-G

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become one of the most important classes of natural constituent from plant origin because of the fact that several health benefits have been explored from it [117]. Gallotannins such as 1,3,6-tri-O-galloyl-2-chebuloyl-b-D-glucopyranoside and 1,3,6-tri-O-galloyl-2-chebuloyl ester-b-D-glucopyranoside act on the insulin pathway for exerting antidiabetic action [118]. 1,2,3,4,6-Penta-Ogalloyl-b-D-glucose inhibits 11b-hydroxysteroid dehydrogenases type 1 (11bHSD-1) activity and improves high-fat diet (HFD)-induced diabetes [119]. An exhaustive SAR study has been conducted by Ren et al. [120] on galloyl esters of glucose such as 1,2,3,4,6-penta-O-galloyl-D-glucopyranose (PGG) and its analogs relevant to antidiabetic potential. Further, they revealed that both D-glucose and galloyl substitution are essential for antidiabetic activity. The interesting fact is that per se D-glucose or gallic acid and its analogs do not possess glucose clearance activity. Galloyl groups with unprotected phenyl hydroxyl groups must be substituted on core of D-glucose, as it provides an optimal scaffold for the spatial orientation of galloyl groups. This may be interacting with the receptor to exert antidiabetic activity by inducing glucose uptake. Furthermore, out of two anomeric forms of PGG (a-PGG and b-PGG), an a-anomeric form is reported to possess potent antidiabetic activity. The galloyl groups at positions 1, 2, 3, 4 of glucose and all the five galloyl groups at positions 1, 2, 3, 4, 6 of b-PGG are essential for antidiabetic activity. If any of galloyl group from these positions is removed, the resulting galloyl esters lack the bioactivity in both a- and b-anomeric forms of glucose. On the contrary, galloyl group at C-6 position of a-PGG is not essential and its replacement with chloride (Cl) results in enhanced antidiabetic activity as compared to a-PGG. Furthermore, PGG also enhances glucose uptake and mimics insulin activity in vitro, which suggests its possible mechanism behind the antidiabetic activity [120]. Tannins or polyphenols produce denaturation of proteins and thereby promote the nonspecific inhibition of a-glucosidase. Corilagin and EA are dose-dependent competitive inhibitors of a-glucosidase [121], whereas ellagitannins and gallotannins are noncompetitive a-amylase inhibitors. Ellagitannins and gallotannins have potential to retard starch digestion and thereby reduce the postprandial hyperglycemia in diabetic patients. The hydrolyzed products of these tannins, such as ellagic acid and gallic acid, do not have affinity for a-amylase causing complete loss of a-amylase inhibitory activity [114]. The a-amylase inhibitory effect of ETs is because of galloyl group (a-galloyl, b-galloyl, or free hydroxyl group) which is essential for activity. The a-amylase is inhibited by procyanidins (condensed tannins), and if the degree of polymerization increases, higher inhibition can be achieved. Procyanidins form insoluble aggregate and stable complex with a-amylase and thereby might reduce the postprandial blood glucose levels [122].

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Terpenoids Introduction and Phytochemistry Terpenoids, also called as isoprenoids, are class of diversified natural products similar to terpenes, made up of isopentenyl diphosphate (five-carbon isoprene) produced from assembled mevalonic acid (Fig. 9.8). The terpenes and terpenoids differ from each other by functional groups. Terpenoids are the chemically modified terpenes by oxidation (usually addition of oxygen), removal or shifting of methyl groups, or rearrangement of isoprene carbon skeleton. Most of the times the term terpene is used in general by the authors to include terpenoids [123,124]. The isoprene units (IU) essentially consist of five carbons having two unsaturated bonds and a branched chain. Terpenoids are classified depending on the number of isoprene units linked with each other from head to tail, as hemiterpenoids (1 IU, 5C), monoterpenoids (2 IU, 10C), sesquiterpenoids (3 IU, 15C), diterpenoids (4 IU, 20C), sesterterpenoids (5 IU, 25C), triterpenoids (6 IU, 30C), tetraterpenoids (8 IU, 40C), and polyterpenoid (with a larger number of isoprene units) [124]. Pentacyclic triterpenes (Figs. 9.9 and 9.10) are widely distributed in the natural resources especially plants and are responsible for variety of biological activities. Based on structural dissimilarities, these compounds are classified into three different classes [125]: (i) Oleanane-type triterpenes (ii) Ursane-type triterpenes (iii) Lupane-type triterpenes

Pharmacological Actions The terpenoids have a broad range of pharmacological activities against cancer, diabetes, inflammation, malaria, and a variety of infectious diseases (viral and bacterial). Ursolic and oleanolic acids inhibit the enzyme tyrosine phosphatase 1B (PTP1B), which subsequently shows the negative regulation and inhibition of insulin signaling. It is also known that, there is an enhancement of insulin sensitivity in muscle and liver of PTP1B knockout mice [126]. This suggested the use of specific PTP1B inhibitors in the treatment of diabetes. The triterpenoids ursolic acid and oleanolic acid are found in most of the A

B

C

FIG. 9.8 Structure of (A) isoprene, (B) mevalonic acid, and (C) isopentenyl diphosphate.

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FIG. 9.9 Structure of (A) oleanolic acid, (B) corosolic acid, (C) maslinic acid (D) hyptadienic acid, (E) rosamultic acid, and (F) tormentic acid.

FIG. 9.10 2a,19a-Dihydroxy-3-oxo-12-ursen-28-oic acid b-D-glucopyranoside ester.

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plants used therapeutically in Asia against the ailments such as hepatitis, inflammation, tumor, and diabetes [127–129]. Ursolic acid and its derivatives stimulate glucose uptake by increasing phosphorylation of insulin receptors [130]. Oleanolic acid and its derivatives such as 3-b-(2-carboxybenzoyloxy)-oleanolic acid (NPLC441) also competitively inhibit PTP1B and stimulate glucose uptake through multitarget pathways [131]. They also inhibit a-glucosidase [129] and possess specific and potent agonistic activity for TGR5, a G-protein-coupled receptor. This receptor intercedes endocrine functions of bile acids and assumed to be a good target for antidiabetic drugs. It prevents the development of obesity and insulin resistance in HFD-fed mice through activated mitochondrial function. Therefore, potent antihyperglycemic activity of oleanolic acid can be attributed to TGR5 agonistic activity [129]. Hyptadienic acid, tormentic acid, rosamultic acid, and 2a,19a-dihydroxy3-oxo-12-ursane-28-oic acid have been derived from Potentilla fulgans and possess inhibitory a-glucosidase activity. Hyptadienic and rosamultic acids are pentacyclic triterpenes with contracted ring A for antidiabetic activity [132–135]. Maslinic and corosolic acids are pentacyclic triterpenes and structural analog of each other. Both these compounds can be synthesized from oleanolic acid and ursolic acid, respectively. They inhibit adrenaline, increased plasma glucose in mice, and used for hypoglycemic activity. Corosolic acid also reported to inhibit glycogen phosphorylases, which is responsible to diminish postchallenge serum glucose levels in humans [136]. Irrespective of the structural differences, triterpenoids possess ability to inhibit glycogen phosphorylase. Hydroxyl groups attached at different positions in structural skeleton of terpenoids and the change in their positions or removal can affect the antidiabetic potency. Presence of hydroxyl group at (C-2) 2a and 2b positions causes complete loss of antidiabetic activity of oleanolic acid and ursolic acid than the parent compound. In corosolic acid, if hydroxyl group is introduced at C-23 position, it results in slight enhancement of the potency. The position of hydroxyl group change from 2a and 3b of maslinic acid and corosolic acid resulted in the reduced antidiabetic potency. Similarly, if configuration of 3b-hydroxyl group changes to 3a-position, it causes complete loss of potency of oleanolic acid and ursolic acid. Besides, replacement of 3b-hydroxyl group with carbonyl moiety retains the potency of oleanolic acid and ursolic acid [125].

Chalcones Introduction and Phytochemistry Chalcones are a,b-unsaturated ketones (trans-1,3-diaryl-2-propen-1-ones), consisting of two aromatic rings (A and B) attached by a,b-unsaturated carbonyl system with variety of substituents (Figs. 9.11 and 9.12). It is a

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FIG. 9.11 Structure of (A) 20 ,40 -dihydroxy-4-methoxydihydro chalcone and (B) 4-hydroxyderricin.

FIG. 9.12 Structure of xanthoangelol. 3⬘ 2⬘ 1⬘

3 R1

CH3 O

+

R2

H O

KOH (50% w/v) C2H5OH, 25°C

4 R1

2

A

5

4⬘

B

R2 5⬘

6⬘

1

6 O R1= −H, −OH, −F, Cl, Br, l R2= −H, −OMe, −OBn, −OCH2O−

FIG. 9.13 Synthesis of chalcones.

biosynthetic product of the plants synthesized by shikimate pathway. Being the precursors of flavonoids, they are considered as open-chain flavonoids found abundantly in edible plants. Many heterocycles of biological importance, such as 1,4-diketones, benzothiazepine, flavones, and pyrazolines, can be synthesized using chalcones as key precursors. The two rings A and B which are linked by a,b-unsaturated carbonyl system are the unique features of chalcones, as no other antidiabetic drugs have this type of linkage. The bright yellow-colored chalcones found in many plants and in some families contribute significantly in the pigmentation of corolla. Chalcones can be synthesized in the laboratory by aldol condensation between a benzaldehyde and an acetophenone in the presence of base (Fig. 9.13) [137].

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Pharmacological Actions Chalcones and its derivatives possess several pharmacological activities such as antibacterial [138,139], anticancer [140–144], antimitotic [141], anticonvulsant [145], antifilarial [146], antifungal [147,148], antiinflammatory [149,150], antimalarial [151–154], antimicrobial [155], antioxidant [156–158], antiprotozoal (antileishmanial and antitrypanosomal) [159], and larvicidal [160]. They have also been reported to show inhibitory activity against many enzymes including cyclooxygenase [161], mammalian alphaamylase [162], and monoamine oxidase [163]. The antidiabetic potential of chalcone has been predicted from the antiatherosclerotic activity of 2-hydroxy-40 -methoxychalcone, which is reported to stimulate PPAR-g mRNA and protein expression in human aortic smooth muscle cells [164]. Through PPAR activation, expression and secretion of various adipocytokines in adipose tissues have been modulated by naringenin. It is a member of chalcone group and known to enhance the sensitivity to insulin. So, the insulin resistance is decreased with the chalcones by enhancing insulin sensitivity [165]. Investigations carried out using 3T3-L1 adipocyte culture medium showed that for promoting cellular glucose consumption substitution on ring A is essential, particularly at position 2. Hydroxy, bromo, chloro, fluoro, and iodo substituents at this position result in good activity. The highest activity is exhibited by iodo substitution compared with other substitutions. Presence of iodo group at position 3 on ring A and methoxy or benzyloxy substitution on ring B of chalcone also exhibit good activity [164]. Insulin-resembling activity has been reported for chalcones. 4-Hydroxyderricin and xanthoangelol act on different targets wherein 4-hydroxyderricin enhances glucose uptake as compared to xanthoangelol [166]. Chalcone also inhibits aldose reductase, enzyme converting glucose into sorbitol, when 40 position in ring A substituted with 20 ,40 -dihydroxy group or hydroxyl group [167].

Phenolic Acids Introduction and Phytochemistry Phenolic acids are naturally occurring nonflavonoid polyphenolic secondary metabolites and extensively distributed in all vegetables and fruits. Phenolic acids are derivatives of benzoic acid and cinnamic acid consists of an aromatic ring carrying one (phenol) or more hydroxyl (polyphenol) groups (Fig. 9.14) [168]. Hydroxycinnamic acid compounds produced simple esters with glucose or hydroxy carboxylic acids. These compounds form a diverse group that includes the widely distributed hydroxybenzoic and hydroxycinnamic acids (Table 9.3) [169]. Biological activity of these compounds is by virtue of its lipophilicity which depends on ring substitution [170].

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FIG. 9.14 Structure of (A) hydroxybenzoic acid, (B) cinnamic acid, (C) caffeic acid, (D) p-coumaric acid, (E) ferulic acid, and (F) gallic acid.

TABLE 9.3 Cinnamic Acid and Its Derivatives Group Positions Chemical Name

X1

X2

X3

X4

Cinnamic acid

H

H

H

H

O-Hydroxycinnamic acid

OH

H

H

H

m-Hydroxycinnamic acid

H

OH

H

H

p-Hydroxycinnamic acid

H

H

OH

H

O-Methoxycinnamic acid

OCH3

H

H

H

m-Methoxycinnamic acid

H

OCH3

H

H

p-Methoxycinnamic acid

H

H

OCH3

H

Ferulic acid

H

OCH3

OH

H

4-Methoxy-trans-cinnamic acid

H

H

OCH3

H

4-Methoxy-trans-cinnamic acid ethyl ester

H

H

OCH3

C2H5

Caffeic and cinnamic acids are two other phenolic acids that occur in many beverages, vegetables, and fruits including apple, artichoke, blueberry, cherry, chicory, cider, coffee, kiwi, pear, and plum [21,171]. Cinnamic acid (3-phenylprop-2-enoic acid) has C6–C3 skeleton and commonly exists in ester forms of quinic acid, shikimic acid, and tartaric acid. Caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid are commonly available cinnamic acid derivatives [172,173].

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Gallic acid is present in many herbs and fruits in the form of free acids, esters, catechin derivatives, and hydrolyzable tannins and consumed in free state or as a part of tannin molecule. Gallic acid (3,4,5-trihydroxybenzoic acid) is one of the polyphenolic compounds with many beneficial biological effects [174]. Gallic acid is also produced from hydrolysis of tannic acid [175].

Pharmacological Actions The most important activity possessed by phenolic acids is an antioxidant which is responsible for many of the pharmacological potentials. The hypoglycemic effect of ferulic acid and phenolic acid fractioned from rice bran is considered due to antioxidant activity. Furthermore, they have been reported to elevate glucokinase activity and production of glycogen in the liver, thereby regulating blood glucose levels in type-2 diabetes [176]. Hydrangeic acid increased significantly adiponectin release into the medium, the translocation of glucose transporter 4 (GLUT4), and 2-deoxyglucose uptake into the cells. It possesses antidiabetic activity but not directly activates PPARg [177]. Cinnamic acid is reported to have a beneficial role in diabetes by means of having different mechanisms. Cinnamic acid and its derivatives possess insulin-secreting activity [178] and also activate insulin-mediated glucose transport [179]. Hydroxyl groups at meta and para positions of cinnamic acid are essential for effective in-vivo and in-vitro action of insulin release. The most potent action of insulin secretion among all the cinnamic acid derivatives obtained with p-hydroxy and m-methoxy substitution on cinnamic acid/ferulic acid structure [180]. Ferulic acid possesses ability to promote the proliferation of pancreatic b-cells, restore insulin secretion, and suppress blood glucose level [181,182]. Isoferulic acid suppresses the hepatic gluconeogenesis and thereby reduces the serum glucose concentration [172]. Cinnamic acid and its derivatives reported to promote and modulate glycogenesis pathways thereby improve glucose utilization and inhibit gluconeogenesis [183]. Caffeic acid and cinnamic acid are reported to have various effects for their antidiabetic activity. Some of them include activation of glycogen synthesis, increased glucose uptake and utilization, improvement of insulin sensitivity, and expression of glycogen synthase in TNFa-induced insulin-resistant cells [184]. para-Methoxycinnamic acid (p-MCA) enhances glucose-stimulated insulin secretion without causing hypoglycemia, thereby lower fasting serum glucose [185]. It is most potent among cinnamic acid derivatives and noncompetitively inhibits a-glucosidase. Daily administration of p-MCA increases the activities of glycolytic enzymes such as glucokinase, hexokinase, and phosphofructokinase in the liver of diabetic rats and suppresses the activity of glucose 6-phosphatase [186,187].

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para-Coumaric acid is an activator for 50 -adenosine monophosphateactivated protein kinase (AMPK) and enhances glucose uptake. This suggests that p-coumaric acid can significantly prevent or improve insulin resistance and modulate glucose metabolism in type-2 diabetes [188]. Antidiabetic potential of phenolic acids such as coumaric acid, ferulic acid, and gallic acid is less compared to caffeic acid and cinnamic acid. Glucose uptake by gallic acid and cinnamic acid is less compared to hydroxycinnamic acid, including caffeic acid, coumaric acid, and ferulic acid. Caffeic acid, a derivative of cinnamic acid, is more potent than those containing single hydroxy group (coumaric acid and ferulic acid). The above findings suggest that the hydroxy groups on the cinnamic moiety might be responsible for enhanced glucose uptake (Fig. 9.15). It has been observed that gallic acid induces glucose uptake by stimulating the GLUT4 translocation by phosphorylation of insulin receptor-mediated phosphoinositide 3-kinase [189].

MISCELLANEOUS Amino Acids Introduction and Phytochemistry S-allylcysteine (SAC) is a sulfur-containing amino acid, derived from garlic (Fig. 9.16). Oral administration of SAC decreases the blood glucose level in diabetic rats. The proposed mechanism of action of SAC includes promotion of glucose uptake and metabolism, and it inhibits the process of hepatic gluconeogenesis to show insulin-like effect on peripheral tissues. Further, SAC

FIG. 9.15 Basic structure of cinnamic acid derivatives.

NH2

S

O

CH2 HO FIG. 9.16 Structure of S-allylcysteine.

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may produce insulin, probably by inhibition of some substances competing with insulin for their –SH group [190].

Pharmacological Actions The digestion of poly- and oligosaccharides depends on a-glucosidase which converts them to absorbable monosaccharides. Inhibition of a-glucosidase delay digestion of poly- and oligosaccharides, causing reduction in glucose absorption thereby decrease the blood glucose levels and subsequently attenuate the rise of postprandial hyperglycemia [186].

Alkaloids Introduction and Phytochemistry Alkaloids are bioactive natural nitrogen-containing compounds, usually basic in nature having diverse pharmacological activities. They have complex structure with high molecular mass with nitrogen in a heterocyclic ring and may present in the form of primary, secondary, tertiary, or even as quaternary ammonium form. The name “alkaloids” has been derived from two words “alkali” and “loid” which means “alkali like,” ie, it refers alkaline or basic nature of the compound. This name was introduced by German chemist Carl Friedrich Wilhelm Meibner in 1819 [191]. Berberine (C20H19NO5), naturally occurring isoquinoline alkaloid (Fig. 9.17), is the major constituent of Chinese medicinal herb Rhizoma coptidis. For the clinical uses, it has been reported to be isolated from many plants such as Berberis aquifolium (Oregon grape), Berberis aristata (tree turmeric), Berberis vulgaris (barberry), Coptis chinensis (Coptis or goldenthread), and Hydrastis canadensi (goldenseal). Medicinal uses of berberine are well documented in Ayurvedic and Chinese system of medicine. It exists in many forms, but most widely used form is hydrochloride salt [192]. Pharmacological Actions Though alkaloids possess diversified pharmacological activities, there are very few reports on antidiabetic activity of alkaloids. Some of them are O O

N

CH3O CH3O

FIG. 9.17 Berberine.

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discussed here with their probable mechanism by which they show antidiabetic activity. Hypoglycemic activity of alkaloid preparation of leaves of Aegle marmelos has been reported by Ponnachan et al. [193]. They reported that oral treatment with alkaloid extract to alloxan diabetic albino rats decreases the blood glucose levels by promoting glycogenesis [193]. The hypoglycemic activity of total alkaloid of Lepidium sativum Linn on alloxan diabetic rats has been reported. This study suggested that the various alkaloidal constituents in plants may act by various mechanisms such as repair or proliferation of pancreatic b-cells, stimulation of secretion of insulin, increased sensitivity to insulin and decreased resistance, increased glycogenesis, and inhibition of gluconeogenesis [194]. Antioxidant and antidiabetic activity of alkaloids isolated from Catharanthus roseus (L.) has been reported by Tiong et al. [195]. They revealed that four alkaloids have been isolated and all of them possess hypoglycemic activity. The study conducted by them showed that this activity may be due to increase in glucose uptake, antioxidant activity, and PTP-1B inhibitory activity [195]. Berberine has been reported to possess antidiabetic activity in STZnicotinamide induced type 2 diabetes in rats, may be due to decrease in level of glucogenic enzymes [196]. The antihyperglycemic effect of berberine is the serendipitous discovery in 1980 during the treatment of Chinese diabetic patient suffering from diarrhea. This miraculous discovery of berberine effect has made the physicians to use this molecule as an antihyperglycemic agent. There are several clinical reports in the literature mentioning antidiabetic activity of berberine [197]. It prevents glucose absorption through intestine and Pan et al. [198] demonstrated that it acts by inhibiting alpha-glucosidase as that of acarbose and miglitol. The absorption of complex carbohydrates in small intestine is delayed by this class of drugs; thereby cause reduction of postprandial glucose level and long-term complications of diabetes. Sucrase and maltase activities were inhibited by berberine similar to acarbose but at higher concentration. Berberine also effectively inhibited the activity of disaccharidases in Caco-2 cells. One group reported that berberine was hardly absorbed through rat intestine [198,199]. It is reported that berberine increases insulin sensitivity and thereby lowers its blood level in type 2 diabetes with prominent insulin resistance. Conversely, it is reported to increase insulin secretion by repair of damaged or exhausted islets in type 1 diabetes or the late stage of type-2 diabetes which is characterized by poor b-cell function. This activity of berberine has been attributed to its antioxidant and antilipid peroxidation properties [200]. There are several studies conducted in-vitro in cell models (cell lines) such as hepatocytes (HepG2 cell line), muscle cells (L6 and C2C12 cell lines), and adipocytes (3T3-L1 cell line) which describes the insulin-independent glucose consumption by berberine, similar to metformin [197,201–205]. Berberine is known as an AMPK activator. Its insulin-independent hypoglycemic effect is related to inhibition of mitochondrial function, stimulation of glycolysis,

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and activation of AMPK pathway. Cheng et al. [202] studied the effect of berberine on glucose uptake by L6 myotubes and demonstrated the involvement of AMPK and p38 MAPK pathway for its antihyperglycemic action. AMPK is an enzyme which is considered as the metabolic switch for fatty acid oxidation, glucose uptake, lipolysis, and lipid metabolism through phosphorylation of involved target proteins [206,207].

CONCLUSION From the foregoing, it is understood that plants have great potential to produce numerous bioactive molecules. These bioactive molecules have diversified biological/pharmacological activities, particularly antidiabetic, with varying mode of action. Antidiabetic efficacy of these natural products is usually attributed to number of bioactive compounds of the same or different chemical classes including polyphenols, flavonoids, tannins, chalcones, and carotenoids. Together, these bioactive molecules show excellent antidiabetic activity with lesser or no adverse effects. Among, some bioactive molecules may potentiate the effect of particular class/group of compounds through enhancement of bioavailability and counteraction of adverse effects. The genesis of these vast varieties of bioactive compounds in plants ascribed to their constant exposure toward biotic and abiotic stresses. Therefore, these bioactive compounds have been proven to be effective in chronic human diseases/disorders linked to stresses. Diabetes is one such disorder and several bioactive natural products are used for its treatment. The efficacy and potency of the bioactive molecules used in the treatment of diabetes are mainly related to its chemical structure. The future potential of the bioactive natural products used in diabetes will be based on the modification of structures of biologically active compounds (leads), which is a primary requirement for drug development. The SAR of bioactive compounds effective in diabetes is of immense importance for gaining idea on enhancement of bioavailability and intrinsic potency, providing oral applicability and elimination of adverse effects. Attempt made in this review about lead optimization is not only essential for selection of best candidate for further drug development but also beneficial for choosing the line of action for control of diabetes.

ABBREVIATIONS AMPK BDMC Cy DMC Dp

adenosine monophosphate-activated protein kinase bisdemethoxycurcumin cyanidin demethoxycurcumin delphinidin

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EA EC ECG EGC EGCG ETs GC GLUT HFD HHDP IFG IGT IRK IU Mv NCD Pg PGG p-MCA Pn PPAR Pt PTP PTP1B SAC SAR STZ WHO

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ellagic acid epicatechin epicatechin gallate epigallocatechin epigallocatechin-3-gallate ellagitannins gallocatechin translocation of glucose transporter high-fat diet hexahydroxydiphenoyl impaired fasting glycemia impaired glucose tolerance insulin receptor kinase isoprene units malvidin novel curcumin derivatives pelargonidin 1,2,3,4,6-penta-O-galloyl-D-glucopyranose para-methoxycinnamic acid peonidin peroxisome proliferator-activated receptor petunidin tyrosine phosphatase protein-tyrosine phosphatase 1B S-allylcysteine structure–activity relationship streptozocin World Health Organization

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Chapter 10

Studies on Prodelphinidins: Isolation, Synthesis, and Their Biological Activities H. Makabe Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Kami-ina, Nagano, Japan

Chapter Outline Introduction Isolation and Determination of the Structures of Prodelphinidins Synthesis of Prodelphinidins Mass Spectrometry

323

324 324 332

Biological Activities of Prodelphinidins Antitumor Activity Antiinflammatory Activity Conclusion References

336 336 342 343 344

INTRODUCTION Proanthocyanidins are known as condensed or nonhydrolyzable tannins whose structures are consisted of flavan-3-ol units [1,2]. These compounds are widely distributed in the nature such as vegetables, fruits, crops, and plants. Proanthocyanidins have been reported to exhibit strong free-radical scavenging and antioxidative activities. Many significant biological activities such as antitumor, antiviral, and antiinflammatory were reported. Thus proanthocyanidins are increasingly recognized as possessing beneficial health effects for humans. Among the proanthocyanidins, the reports of the studies on prodelphinidins which possess (+)-gallocatechin and/or (
)-epigallocatechin units are limited to dimers and trimers probably due to the difficulty to obtain high degree of oligomers from the nature. The structural diversity of these compounds arises through configurational differences such as C3- and C4 chiral centers and the regioisomers and stereochemistries of the interflavan bonds (C4 to C8). In addition, various degrees of polymers exist. Because their identification as well as purification is extremely difficult even using modern methods of isolation techniques, further investigation of the biological Studies in Natural Products Chemistry, Vol. 48. http://dx.doi.org/10.1016/B978-0-444-63602-7.00010-2 © 2016 Elsevier B.V. All rights reserved.

323

324 Studies in Natural Products Chemistry

activities, ie, mechanism of action of the bioactivities, remains unknown. Quite recently, in order to obtain pure prodelphinidins, Makabe and coworkers reported the total synthesis of prodelphinidin B1–B4 (1–4), and C2 (5) and their antitumor activity [3,4]. Here the author wishes to review the isolation, the recent synthetic approaches, and biological activities (Fig. 10.1).

ISOLATION AND DETERMINATION OF THE STRUCTURES OF PRODELPHINIDINS From the 1970s to the 1990s, isolation of prodelphinidins was reported. As to the dimeric prodelphinidins, a number of isolations from plants have been reported: prodelphinidin B1 (1) from Cistus incanus [5], Lotus pedunculatus [6] and Stryphnodendron adstringens [7], prodelphinidin B2 (2) from L. pedunculatus [6] and S. adstringens [7], prodelphinidin B3 from Sorghum vulgare seeds [8] and Quercus dentata [9], and prodelphinidin B4 (4) from Ribes nigrum [10], Vicia faba [11], and S. adstringens [7]. Here the author wishes to describe the isolation of prodelphinidin B1 (1) and B2 (2) from the stem bark of S. adstringens [7]. The procedure of isolation is as follows. Air-dried stem bark of S. adstringens (480 g) was extracted with acetone– water (7:3, 4.8 L). The combined extracts were filtered and the filtrate was concentrated to 0.5 L and lyophilized (183 g). This fraction was dissolved into H2O (5 L) and extracted with EtOAc (27 L). After concentration in vacuo, the EtOAc extract and remained H2O phase afforded dark brown solids of 31 and 152 g, respectively. A portion (15 g) of the EtOAc extract was subjected to column chromatography on Sephadex LH-20 [71 cm  5 cm, eluents; 50% EtOH (5 L), EtOH (5 L), 50% MeOH (3.5 L), MeOH (9.7 L), and 70% acetone (2.6 L); 15 mL fractions] to furnish 18 main fractions. Each main fraction was further separated by multilayer coil countercurrent chromatography (MLCCC; EtOAc–n-PrOH–H2O ¼ 140:8:80). Subfractions were purified using HPLC (reverse-phase C18 column). Fractions 6 and 7 gave prodelphinidin B1 (1, 16 mg) and B2 (2, 68 mg), respectively (Fig. 10.2). As to the trimeric prodelphinidins, Outtrup and coworkers characterized the structure of prodelphinidin C2 (5) in 1986 [12]. Qa´dan and coworkers also reported the isolation of epigallocatechin–gallocatechin–catechin (6) and epigallocatechin–gallocatechin–gallocatechin (7) from Cistus albidus (Fig. 10.3) [13]. They also isolated an oligomer of prodelphinidin. The molecular size of the oligomer was estimated to be six to seven flavan-3-ol units by analysis of 13C NMR (nuclear magnetic resonance) spectra.

SYNTHESIS OF PRODELPHINIDINS Because purification and identification of prodelphinidins from plants are very difficult, the mechanism of their biological activities remains unknown. Therefore syntheses of prodelphinidins are quite important to obtain pure materials for evaluating their biological activities. Many examples of the

P

P

P

P

P

FIG. 10.1 The structures of prodelphinidins.

326 Studies in Natural Products Chemistry

FIG. 10.2 Isolation of prodelphinidin B1 (1) and B2 (2) from the stem bark of Stryphnodendron adstringens.

FIG. 10.3 The structures of prodelphinidin trimers isolated from Cistus albidus.

Studies on Prodelphinidins Chapter

10 327

syntheses of procyanidins were reported in this decade including our synthesis [14–17]; however, synthetic studies on prodelphinidins were quite limited due to difficulty in obtaining (+)-gallocatechin or (
)-epigallocatechin as synthetic starting materials. Although (+)-gallocatechin or (
)-epigallocatechin is commercially available, both of these compounds are very expensive. Thus it was necessary to prepare sufficient quantities of (+)-gallocatechin or (
)-epigallocatechin derivatives according to the reported procedure. Here the author wishes to describe the synthesis of (+)-gallocatechin and (
)-epigallocatechin derivatives by Chan and coworkers [18]. Friedel–Crafts alkylation of 3,5-dibenzyloxyphenol (8) with cinnamyl alcohol 9 followed by protection as tert-butyldimethylsilyl (TBDMS) ether afforded 10. Compound 10 was dihydroxylated with the Sharpless asymmetric dihydroxylation using AD-mixa to give diol 11. Cyclization of diol 11 under the orthoformate in the presence of pyridinium p-toluenesulfonate (PPTS) followed by basic hydrolysis gave protected (+)-gallocatechin derivative 12. Compound 12 was oxidized with Dess–Martin periodinane to give ketone 13. Stereoselective reduction of the carbonyl group of 13 using L-selectride afforded (
)-epigallocatechin derivative 14 (Scheme 10.1). Barron [19], Suzuki [20], Zhang [21], and Krohn et al. [22] also synthesized (+)-gallocatechin and/or (
)-epigallocatechin derivatives. In 1986, Delcour and coworker reported synthesis of prodelphinidin B3 (3) and C2 (5) starting from (+)-catechin and (+)-dihydromyrcetin. When excess (+)-catechin (5.5 eq.) was used the condensed product was prodelphinidin B3 (3). Equimolar amounts of (+)-catechin and (+)-dihydromyrcetin afforded prodelphinidin C2 (5) as a main product (Scheme 10.2) [23]. The recent example of the synthetic study on prodelphinidin B3 (3) using Lewis acid was reported by Krohn and coworkers [24]. They prepared gallocatechin nucleophile 12 and catechin electrophile 15. The condensation between 15 and 16 smoothly proceeded to give 17 in 98% yield using TMSOTf as a Lewis acid at
78°C. The ratio of trans:cis was more than 50:1 by careful analysis of 1H NMR (Scheme 10.3). In 2013, Makabe and coworkers reported the first total synthesis of prodelphinidin B3 (3) and C2 (5) [3]. The gallocatechin-derived electrophile 19 was constructed as Chan and coworkers reported with slight modification followed by 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) oxidation in the presence of ethoxyethanol. The equimolar coupling between catechin nucleophile 18 and gallocatechin electrophile 19 using Yb(OTf )3 as Lewis acid afforded condensed product 20 in good yield. The acetyl group of the condensed product 20 was removed to give diol 17 using n-Bu4NOH [25]. Finally deprotection of the benzyl ethers of 17 using Pearlman’s catalyst and subsequent lyophilization afforded prodelphinidin B3 (3) in high yield. The 1H NMR spectral data of peracetate of 3 (21) were in good agreement with the reported values (Scheme 10.4) [23]. The synthesis of prodelphinidin C2 (5) was reported by the same group [3]. The key reaction was equimolar condensation between nucleophile 17

OBn

BnO

OH (1) H2SO4 (SiO2)

+ BnO

OBn

8

BnO

41% (from 9)

BnO

OBn

OBn

OBn

OBn

OH OH

10

OBn

9

OBn TBS O

OBn

CH(OEt)3, PPTS

BnO

O

OBn

75%

OH OBn

11

12 OBn

OBn

OBn OBn

BnO

O

OBn

L-Selectride

BnO

O

OBn

80% (from 12) OH

O OBn 13

OBn AD-mixα, MeSO2NH2

(2) TBSCl, Imidazole

BnO

OBn

OBn

TBS O

OH

OBn 14

SCHEME 10.1 Synthesis of (+)-gallocatechin and (
)-epigallocatechin derivatives by Chan and coworkers.

Dess–Martin periodinane

OH OH HO

OH OH HO

O

OH

O

OH

OH +

HO

(1) NaBH4 (2) AcOH

O

OH

OH

OH OH HO

OH

O

OH

OH

OH

OH O

(+)-Catechin (5.5 eq.)

OH OH

(+)-Dihydromyrcetin (1.0 eq.)

Prodelphinidin B3 (3) OH OH HO

O

OH OH

OH OH HO

O

OH

OH +

HO

O

OH OH (+)-Catechin (1.0 eq.)

OH

(1) NaBH4 (2) AcOH

OH OH HO

O

OH OH O (+)-Dihydromyrcetin (1.0 eq.)

OH OH OH

OH HO

O

OH OH Prodelphinidin C2 (5)

SCHEME 10.2 Synthesis of prodelphinidin B3 (3) and C2 (5) from (+)-catechin and (+)-dihydromyrcetin.

OH OH

OBn

OBn OBn

BnO

OBn DDQ, BnOH

O

OBn

BnO

O

OH OBn

OH OBn OBn 15

12 OBn OBn

BnO

OBn +

O

OBn

OBn

85%

BnO

OBn 16 (3 eq.)

OBn BnO

O

OBn

TMSOTf

O

OH

OBn

OBn OH

OBn OBn

–78°C 98%

OH OBn BnO

O

15 (1 eq.)

OBn OH

OBn 17 SCHEME 10.3 Synthesis of prodelphinidin B3 derivative.

OBn

OBn OBn OBn OBn BnO

O

OBn +

BnO

OBn BnO

Yb(OTf)3

O

OBn

OH

86%

OAc

OBn

O OAc OBn BnO

OBn

20 OAc

OH OBn

OH OBn BnO

O

OBn

OBn

HO OBn

O

H2, Pd(OH)2/C 96%

O

OAc

OH

OBn

OBn

OH

O

OH

17

O

OAc

Ac2O, Py. DMAP OAc AcO 28%

OAc

Prodelphinidin B3 (3)

OAc

O

OAc OAc

OH OH

SCHEME 10.4 Synthesis of prodelphinidin B3 (3).

AcO

OH OH

OH HO

OH OBn

82%

OH

19

O

n-Bu4NOH OBn

OBn OEE

18

BnO

OBn

OAc 21

332 Studies in Natural Products Chemistry

and electrophile 22. In this condensation, they found that AgOTf as Lewis acid was effective for the construction of catechin trimer derivatives. The acetyl group of the condensed product 23 was removed to afford triol 24 using n-Bu4NOH [24]. Finally deprotection of the benzyl ethers of 24 and subsequent lyophilization afforded prodelphinidin C2 (5) in high yield. The 1 H NMR spectral data of peracetate 25 were in good agreement with the reported values (Scheme 10.5) [23]. In 2013, Makabe and coworkers also synthesized prodelphinidin B1, B2, and B4 [4]. The gallocatechin-derived nucleophile 12 was constructed as Chan and coworkers reported [18]. Gallocatechin-derived electrophiles 19 and 22 were prepared as they previously reported [3]. The epigallocatechinderived nucleophile 14 was prepared according to the Chan and coworkers’ method [18]. DDQ oxidation of 14 in the presence of methanol or ethoxyethanol followed by acetylation gave epigallocatechin-derived electrophiles 26 and 27, respectively (Scheme 10.6). They examined the condition of equimolar condensation between gallocatechin nucleophile 12 and epigallocatechin electrophile 26 or 27 to construct prodelphinidin B1 (1) skeleton. Using 27 as an electrophile and Yb(OTf )3 as a Lewis acid afforded condensed product 28 in 66% yield. Next, they investigated the condition of equimolar condensation between epigallocatechin nucleophile 14 and epigallocatechin electrophiles 26 or 27 to construct the prodelphinidin B2 (2) skeleton. The electrophile containing methoxy group 26 afforded condensed product 29 in good yield when AgBF4 was used as Lewis acid. Yb(OTf )3 also gave 29 in good yield. In this case, the methoxy group at the C4 position of electrophile was quite important for Lewis acid-mediated condensation. They further investigated the condition of equimolar condensation between epigallocatechin nucleophile 14 and gallocatechin electrophile 19 or 22 to construct prodelphinidin B4 (4) skeleton. 4-(200 -Ethoxyethoxy) derivative 19 afforded condensed product 30 in 78% yield when Yb(OTf )3 was used as Lewis acid. On the other hand, the reaction using methoxy derivative 22 gave 30 in low yield (Scheme 10.7). The condensed products 28–30 were transformed into diols 31, 33, and 35 using n-Bu4NOH [24]. Finally deprotection of the benzyl ethers of 31, 33, and 35 and subsequent lyophilization afforded prodelphinidin B1 (1), B2 (2), and B4 (4) in good yield. The 1H and 13C NMR spectral data of peracetate of 1 (32) [6], 2 (34) [6], and 4 (36) [11] were in good agreement with the reported values (Scheme 10.8).

MASS SPECTROMETRY Krohn and coworkers reported electrospray ionization (ESI) mass spectral data of O-benzyl protected prodelphinidin B3 [24]. They found retro-Diels– Alder fragmentation occurred with regioselectivity at the benzoannelated pyran ring (Scheme 10.9).

OBn OBn BnO OBn OBn BnO

O

OBn OH

OBn BnO

O

OBn OBn

OBn OBn BnO

O

OAc

OBn AgOTf

+

OBn

n-Bu4NOH

O

OBn OH

OBn OMe O

OBn

OBn BnO

22

OBn

17

23

HO

OBn OBn OH

O

H2, Pd(OH)2/C OBn OH

OH HO

Ac2O, Py.

O

OH OH

OBn

OH HO

24

SCHEME 10.5 Synthesis of prodelphinidin C2 (5).

OAc OAc

OH

OAc AcO

OH Prodelphinidin C2 (5)

OAc

O

OAc

DMAP 27%

OAc OAc AcO

OAc

O

OAc OAc

OH

OH OBn

OH

O

O OAc

OH

96% OBn

O

AcO

OH OH OH

OBn

O

OAc

OH

OBn O

OAc

OH

OBn

OBn BnO

OBn OH

OBn

OBn BnO

OBn

O

OH

BnO

77%

73%

OAc

OBn

OBn BnO

OAc 25

334 Studies in Natural Products Chemistry OBn

BnO

OBn Ref. [18]

O

OBn

OBn OBn OBn

BnO

O

OH

OBn

OBn (1) DDQ, ROH (2) Ac2O, DMAP, py. BnO

O

OBn OAc

OH OBn OR

OBn

OBn 12

14

R = Me: 26 (39%, 2 steps) EE: 27 (70%, 2 steps)

Ref. [3] OBn OBn BnO

O

OBn OAc

OBn OR R = EE: 19 Me: 22

SCHEME 10.6 Synthesis electrophiles.

of

gallocatechin

and/or

epigallocatechin

nucleophiles

and

OBn OBn OBn

OBn

O

BnO

OBn

OBn

OBn

OAc BnO

O

OBn

BnO

O

OBn

+

CH2Cl2 3h 66%

OAc

OH OBn OEE 27

OBn 12

Yb(OTf)3

OBn

OBn

OBn

BnO

O

OBn OH

OBn 28 OBn OBn OBn

OBn BnO

O OH

OBn

OBn

OBn BnO

O

BnO

OAc

O

OBn +

OBn OAc

CH2Cl2 3h 76%

OBn OMe

OBn 14

AgBF4

26

OBn

OBn

OBn O

BnO

OBn OH

OBn 29 OBn OBn OBn

OBn

O

OBn

BnO

14

OAc

O

OBn Yb(OTf) 3

+ OAc

OH OBn

OBn

OBn

OBn BnO

O

BnO

OBn OEE 19

CH2Cl2 2h 78%

OBn

OBn

OBn O

BnO

OBn OH

OBn

30

SCHEME 10.7 Condensation of gallocatechin and/or epigallocatechin nucleophiles and electrophiles to construct prodelphinidin B1 (1), B2 (2), and B4 (4) skeleton.

OBn

OBn

OBn BnO

O OAc OBn BnO

BnO

OBn OBn OBn

OBn BnO

OBn

OBn

OBn

OBn

OH OBn BnO

90%

O

OBn OBn

n-Bu4NOH

OBn

O

OH

OAc 32

OBn

29

33

OAc OAc

OH

(1) H2, Pd(OH)2 OBn (2) lyophilization 80%

O

AcO

OH OH

OH OH HO

OH

O

BnO

O

OAc

Prodelphinidin B2 (2)

34

OAc OBn BnO

OBn BnO OBn

n-Bu4NOH 79%

O

OBn

O OH OBn BnO

30

HO

O

(1) H2, Pd(OH)2 (2) lyophilization

OBn OBn OBn

O

OH OBn

OAc OH

OBn

OBn OBn

OAc OAc

OH OH

OBn

quant.

OH OH HO

OH

OAc AcO

OH OH OH OH

O

Ac2O, DMAP, Py. 27%

O

OBn

OAc OAc AcO

OH OH

35

Prodelphinidin B4 (4)

OAc OAc OAc

O

OAc OAc

OAc

OBn

SCHEME 10.8 Synthesis of prodelphinidin B1 (1), B2 (2), and B4 (4) and their peracetates.

OAc

O

OH OBn

OAc OAc

OAc OAc AcO

OH

OBn

O

Ac2O, DMAP, Py. 16%

OH

OBn

OAc OAc

OH

HO

O

OAc

O

OH

OBn BnO

OAc AcO

22%

OAc OAc

OAc

Prodelphinidin B1 (1) OBn

OBn OBn

Ac2O, DMAP, Py.

OH

31

OAc

OH

O

OH

OBn

OBn BnO

OH

OBn

28

OH OH

O

OH

OBn

O

AcO

O

OH HO

OBn

OH

BnO

HO (1) H2, Pd(OH)2 (2) lyophilization quant.

O

OAc

OH

OBn OBn

OH

90% O

O

n-Bu4NOH

OAc

OH OBn

36

336 Studies in Natural Products Chemistry

Na+

Na+

OBn

OBn

OBn BnO

O

CID

OBn

OBn OH

OBn

O

OBn

O

BnO

OBn OH

OBn BnO

OBn

OBn BnO

O

+

OBn HO

OH OBn

OBn

SCHEME 10.9 Specific fragmentation through retro-Diels–Alder fragmentation.

Makabe and coworkers also found that ESI-TOF (time of flight) MS/MS spectrum of synthetic prodelphinidin C2 (5) [3] gave interesting fragmentations through retro-Diels–Alder reaction (Fig. 10.4). The fragmentation observed suggested not only the cleavage of C4–C8 flavan bond but also a retro-Diels–Alder reaction. The detail is shown in Fig. 10.5 [26]. The product ions m/z 747, 731 suggest that a retro-Diels–Alder reaction took place. The product ion 443 revealed that the precursor ion, [M + H]+, was cleaved of the C4–C8 flavan bond followed by a retro-Diels– Alder reaction. The product ion m/z 611 suggests a dimer of gallocatechin, and 595, gallocatechin–catechin or catechin–gallocatechin. The molecular formula and mass of this molecule were C45H38O20 and 898 Da. Compared to the ESI-TOF mass spectrum of prodelphinidin C2 (5), procyanidins afforded the fragmentations only by the cleavage of C4–C8 flavan bonds. The ESI-TOF MS of synthetic catechin pentamer is as follows (Fig. 10.6) [26]. Fragmentation was occurs only by cleavage of internal C4 to C8 flavan bonds. The molecular formula of the [M + H]+ ion is C75 H63 O30 + . Five product ions, m/z 1443, 1155, 867, 579, and 291, showed the cleavage of internal C4 to C8 flavan bonds (Fig. 10.7).

BIOLOGICAL ACTIVITIES OF PRODELPHINIDINS Antitumor Activity Cell Proliferation Assay In 2004, Lin and coworkers reported antiproliferative activity of prodelphinidin B2 3,3ʹ-di-O-gallate (PDB2-3,3ʹOG) isolated from the bark of Myrica rubra [27]. Their results showed that PDB2-3,3ʹOG inhibited the proliferation of A549 cell by cell cycle arrest in the G0/G1 phase and inducing apoptosis. enzyme-linked immunosorbent assay (ELISA) showed that G0/G1 phase arrest was due to increase in the expression of p21/WAF1. They also found that an enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand and soluble Fas ligand, might be responsible for the apoptotic effect induced by PDB2-3,3ʹOG.

19-Oct-2012 11:35:28 1: TOF MSMS 899.20ES+ 6.38e4

PDC2-20V 20121019_03 480 (13.177) Cm (453:529) 595.1481 142004

100

899.2141 65240

609.1281 112775

305.0687 51636

610.1321 42199

%

900.2179 38953

443.0999 25999 731.1682 22779

441.0839 21453

747.1633 15713 291.0895 9999

306.0724 9432 317.0673 4643

151.0401 1607

423.0741 4799

444.1039 6355

567.1172 8883

748.1679 7599 713.1568 2591

483.0963 2806

179.0347 1456

901.2203 13577

611.1348 10453

318.0723;813

749.1685 2225

881.2031 1638

902.2224 3057 903.2096;294

639.1703;154

m/z

0 150

200

250

300

350

400

450

500

FIG. 10.4 ESI-TOF MS/MS spectrum of prodelphinidin C2 (5).

550

600

650

700

750

800

850

900

950 1000 1050

338 Studies in Natural Products Chemistry OH

OH OH

HO

O

OH HO

OH OH OH

OH HO

OH HO

OH OH

O OH

OH

O

OH OH HO

OH

O

OH

OH

O

HO

OH OH OH

OH

O

OH HO

O

OH HO

OH

OH OH

OH HO

O

OH

O

OH

OH

OH

OH

OH

m/z 899

HO

O

OH

OH OH

OH OH HO

OH HO

OH

O

HO

O

or

O

OH

OH HO

OH

OH

OH HO

O

OH

OH

OH

OH OH

OH OH

OH

O

OH

m/z 291

OH

OH

OH

m/z 611

m/z 443

m/z 595 OH

O

O OH

OH O OH

OH HO

OH HO

OH

OH OH

OH

OH

O

OH OH

OH OH HO

O

OH OH

OH

OH

OH

OH

O

HO

OH

OH

OH OH OH

OH HO

OH OH OH

OH HO

O

OH

O

OH

O

HO

OH OH OH

OH HO

OH OH

OH HO

O

OH

O

OH

O

HO

OH OH

OH

HO

OH

OH

OH HO

OH

OH HO

OH

OH

OH OH

HO

O OH

OH

O

HO

OH

OH OH

OH HO

OH

OH

OH O

m/z 731

OH

OH

HO

OH

m/z 747

Retro-Diels–Alder reaction

FIG. 10.5 Assignment of ESI-TOF MS/MS spectrum of prodelphinidin C2 (5).

20130321_02 138 (22.777) Cm (131:149) 867.2144 5951

100

%

577.1335 2866

868.2195 2480 1443.3450 1995

579.1493 1526

229.1576 291.0875 256 2

0 100

200

300

580.1536 474 425.0851 16

400

500

600

865.1995 1318

715.1697 31

700

800

1155.2795 1105 869.2222 1153.2690 600 628 1003.2166;10

900

1000

1100

1156.2881 1291.2859 649 43

1200

1300

1400

1445.3545 1027 1446.3544 357

1500

1600

FIG. 10.6 ESI-TOF MS/MS spectrum of catechin pentamer.

In 2013, Makabe and coworkers accomplished the total synthesis of prodelphinidin B3 (3) (PDB3) and C2 (5) (PDC2) [3]. They also reported antitumor activity of PDB3 and PDC2 through cell cycle arrest and caspase-3 activation. To examine the antitumor activity against PC-3 prostate cancer cell lines together with procyanidin B3 (PCB3), C1 (PCB1), and C2

10 339

Studies on Prodelphinidins Chapter

OH 1

HO

O

OH

2 4

OH HO

8⬘

3

OH O

OH

6⬘ 4⬘

OH HO

HO

OH

2⬘

7⬘ 5⬘

OH

1⬘

3⬘OH

O

O

OH OH

OH OH HO

OH OH

HO

O

OH HO

OH

O

OH OH

OH

O

OH

OH HO

OH

O

OH

OH HO

OH

O

OH

OH

OH

O

OH OH

OH HO

OH HO

OH OH

OH

OH

OH

O

OH OH

OH HO

OH

OH

OH

m/z 1443

OH

O

OH OH

OH

m/z 867

m/z 1155

OH HO

O

OH OH

OH HO

OH

O

OH

OH HO

O

OH OH

m/z 579

OH OH

OH

m/z 291

FIG. 10.7 Assignment of ESI-TOF MS/MS spectrum of catechin pentamer.

(PCB2), which were prepared by us previously [28,29], cell count measurement and 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, yellow tetrazole (MTT) assay were performed. Epigallocatechin gallate (EGCG), which is well known as an antitumor agent isolated from green tea, was used as a positive control. As shown in Fig. 10.7, EGCG, PDB3, and PDC2 exhibited significant cytotoxic activity with IC50 values below 50 mmol/L. Comparison of the activity between PDB3 and procyanidin B3 suggested that the cytotoxic effects were obviously associated with the presence of the additional hydroxy group of the B ring (pyrogallol moiety) of PDB3. They found that pyrogallol moiety of PDB3 greatly affected the level of the cytotoxic effect. As to PDC2 and procyanidin C1 or C2, the pyrogallol moiety was essential for the activity as in the case of PDB3 and procyanidin B3. Comparisons between PDB3 and either PDB2 or EGCG suggested that the number of pyrogallol moieties did not seem to affect the activity. This phenomenon was also observed in the MTT assay (Fig. 10.8). In 2013 Makabe and coworkers also reported the antitumor effects of prodelphinidin B1 (1), B2 (2), and B4 (4) [4]. As shown in Fig. 10.9, EGCG, prodelphinidin B1 (1) (PDB1), B2 (2) (PDB2), and B4 (4) (PDB4) showed significant cytotoxic activities. These results also suggested that pyrogallol moieties are essential factors of cytotoxicity against tumor cell. In addition

A

B

1.2 0 μM

10 μM

25 μM

50 μM

100 μM

0 μM

*

*

*

*

*

Relative cell viability (sample/vehicle)

Relative cell number (sample/vehicle)

* * 0.6

* * 0.4

25 μM

50 μM

*

1.2 1 0.8

*

*

*

*

* 0.6 0.4

0.2

100 μM

*

1.4

1 0.8

1.6

* * *

0.2 0

0 EGCG

PCB3

PDB3

PCC1

PCC2

PDC2

EGCG

PCB3

PDB3

PCC1

PCC2

PDC2

FIG. 10.8 Effects of various concentrations of test compounds on cell proliferation. Asterisks indicated a significant difference between the control- and test-compound-treated cells, as analyzed by Student’s t-test (p < 0.001) A: cell count measurement. B: MTT assay.

Studies on Prodelphinidins Chapter

10 341

1.2

1

Relative cell number (sample/control)

* 0.8

(μM)

*

*

0 10 25

* 0.6

* 0.4

*

50 100

* *

* *

0.2

*

*

*

*

0 EGCG

PDB1

PDB2

PDB3

PDB4

FIG. 10.9 Effects of various concentrations of test compounds on cell proliferation as analyzed Student’s t-test (P