Progress in the Chemistry of Organic Natural Products 115 3030648524, 9783030648527

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Progress in the Chemistry of Organic Natural Products

A. Douglas Kinghorn · Heinz Falk · Simon Gibbons · Yoshinori Asakawa · Ji-Kai Liu · Verena M. Dirsch Editors

115 Progress in the Chemistry of Organic Natural Products

Progress in the Chemistry of Organic Natural Products

Series Editors , College of Pharmacy, The Ohio State University, Columbus, OH,

A. Douglas Kinghorn USA Heinz Falk

, Institute of Organic Chemistry, University Linz, Linz, Austria , School of Pharmacy, University of East Anglia, Norwich, UK

Simon Gibbons

, Faculty of Pharmaceutical Sciences, Tokushima Bunri University,

Yoshinori Asakawa Tokushima, Japan

Ji-Kai Liu , School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, China Verena M. Dirsch Austria

, Department of Pharmacognosy, University of Vienna, Vienna, Wien,

Advisory Editors Giovanni Appendino , Department of Pharmaceutical Sciences, University of Eastern Piedmont, Novara, Italy Roberto G. S. Berlinck São Carlos, Brazil

, Instituto de Química de São Carlos, Universidade de São Paulo,

Jun’ichi Kobayashi, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Agnieszka Ludwiczuk Lublin, Poland C. Benjamin Naman China

, Department of Pharmacognosy, Medical University of Lublin, , Department of Marine Pharmacy, Ningbo University, Zhejiang,

Rachel Mata , Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Distrito Federal, Mexico Nicholas H. Oberlies , Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, NC, USA Deniz Tasdemir , Marine Natural Products Chemistry, GEOMAR Helmholtz Centre for Ocean Research, Kiel, Schleswig-Holstein, Germany Dirk Trauner

, Department of Chemistry, New York University, New York, NY, USA

Alvaro Viljoen , Department of Pharmaceutical Sciences, Tshwane University of Technology, Pretoria, South Africa Yang Ye , State Key Laboratory of Drug Research and Natural Products Chemistry Department, Shanghai Institute of Materia Medical, Shanghai, China

The volumes of this classic series, now referred to simply as “Zechmeister” after its founder, Laszlo Zechmeister, have appeared under the Springer Imprint ever since the series’ inauguration in 1938. It is therefore not really surprising to find out that the list of contributing authors, who were awarded a Nobel Prize, is quite long: Kurt Alder, Derek H.R. Barton, George Wells Beadle, Dorothy Crowfoot-Hodgkin, Otto Diels, Hans von Euler-Chelpin, Paul Karrer, Luis Federico Leloir, Linus Pauling, Vladimir Prelog, with Walter Norman Haworth and Adolf F.J. Butenandt serving as members of the editorial board. The volumes contain contributions on various topics related to the origin, distribution, chemistry, synthesis, biochemistry, function or use of various classes of naturally occurring substances ranging from small molecules to biopolymers. Each contribution is written by a recognized authority in the field and provides a comprehensive and up-to-date review of the topic in question. Addressed to biologists, technologists, and chemists alike, the series can be used by the expert as a source of information and literature citations and by the non-expert as a means of orientation in a rapidly developing discipline. All contributions are listed in PubMed.

More information about this series at http://www.springer.com/series/10169

A. Douglas Kinghorn Heinz Falk Simon Gibbons Yoshinori Asakawa Ji-Kai Liu Verena M. Dirsch •

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•

•

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Editors

Progress in the Chemistry of Organic Natural Products Volume 115

With contributions by Bernd Schmidt Søren Brøgger Christensen
Henrik Toft Simonsen
Nikolai Engedal
Poul Nissen
Jesper Vuust Møller
Samuel R. Denmeade
John T. Isaacs Jiří Pospíšil
Daniela Konrádová
Miroslav Strnad Steven D. Shnyder
Colin W. Wright

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Editors A. Douglas Kinghorn College of Pharmacy Ohio State University Columbus, OH, USA

Heinz Falk Institute of Organic Chemistry Johannes Kepler University Linz, Oberösterreich, Austria

Simon Gibbons School of Pharmacy University of East Anglia Norwich, UK

Yoshinori Asakawa Faculty of Pharmaceutical Sciences Tokushima Bunri University Tokushima, Japan

Ji-Kai Liu School of Pharmaceutical Sciences South Central University for Nationaliti Wuhan, China

Verena M. Dirsch Department of Pharmacognosy University of Vienna Wien, Austria

ISSN 2191-7043 ISSN 2192-4309 (electronic) Progress in the Chemistry of Organic Natural Products ISBN 978-3-030-64852-7 ISBN 978-3-030-64853-4 (eBook) https://doi.org/10.1007/978-3-030-64853-4 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021, corrected publication 2021 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

It is a great pleasure to announce that with effect from the present Volume 115, Univ. Prof. Dr. Verena M. Dirsch, University Professor and Head, Department of Pharmacognosy, University of Vienna, Vienna, Austria has become a new Series Editor of “Progress in the Chemistry of Organic Natural Products”. Prof. Dirsch has published widely, and has worked in particular on bioassay method development and mechanism-of-action studies on naturally occurring molecules in medicinal plants. She has been a member of the Editorial Advisory Board of this book series since 2008 (Volume 92). Prof. Dirsch was responsible for organizing the seven chapters from internationally renowned authors of Volume 110 (“Cheminformatics in Natural Product Research”; 2019), with the purpose of this volume being to show how “big data” from natural product collections and databases can be mined by computerized methods for molecular target identification. In addition to being an active scientist, Prof. Dirsch has had a number of additional senior administrative roles at the University of Vienna, including Vice-Dean of the Faculty of Life Sciences (2008–2014) and Deputy Speaker of the Center of Pharmaceutical Sciences (2014–present). Prof. Dirsch is warmly welcomed into her new role for our book series. Professor Jun’ichi Kobayashi, of the Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan has stepped down from his past position as Series Editor for this book series, and will become a member of the Editorial Advisory Board. The other Series Editors and Springer are very grateful for his many contributions to the book series for Volumes 92–114 (2008–2021). Prof. Kobayashi has had an illustrious academic career in the natural products area, and has published prodigiously on the structure elucidation of bioactive molecules from all of marine organisms, terrestrial microbes, and higher plants. He has also been a research supervisor to over 100 graduate students in the past. In Volume 115, there are four chapters altogether that feature a varied group of natural products. In the first of these, Prof. Dr. Bernd Schmidt, of the University of Potsdam, Germany, has written on the role that total synthesis may play in revising the structures proposed for decanolides, which are ten-membered lactones found primarily in fungi, frogs, and termites. Prof. Søren Brøgger Christensen, of the v

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Preface

University of Copenhagen, Denmark, and colleagues describe the development of the intriguing plant-derived sesquiterpene lactone, thapsigargin, a potent inhibitor of the enzyme, SERCA (sarco-endoplasmic Ca2+ ATPase), with potential as a lead compound to treat cancer. The third chapter was written by a team headed by Prof. Miroslav Strnad, of the Czech Academy of Sciences, Institute of Experimental Botany, and Palacký University, Olomouc, Czech Republic. This covers the potential of various plant phenolic compounds for treating the tropical and sub-tropical infectious disease, leishmaniasis. In the final chapter, Dr. Steven Shnyder and Prof. Colin Wright, of the University of Bradford, U.K. describe recent advances on the plant alkaloid, cryptolepine, which is of particular interest as a lead for the treatment of malaria, trypanosomiasis, and cancer. Columbus, OH, USA Linz, Austria Norwich, UK Tokushima, Japan Wuhan, China

Douglas Kinghorn Heinz Falk Simon Gibbons Yoshinori Asakawa Ji-Kai Liu

Contents

The Role of Total Synthesis in Structure Revision and Elucidation of Decanolides (Nonanolides) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bernd Schmidt From Plant to Patient: Thapsigargin, a Tool for Understanding Natural Product Chemistry, Total Syntheses, Biosynthesis, Taxonomy, ATPases, Cell Death, and Drug Development . . . . . . . . . . . . . . . . . . . . . Søren Brøgger Christensen, Henrik Toft Simonsen, Nikolai Engedal, Poul Nissen, Jesper Vuust Møller, Samuel R. Denmeade, and John T. Isaacs

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Antileishmanial Activity of Lignans, Neolignans, and Other Plant Phenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Jiří Pospíšil, Daniela Konrádová, and Miroslav Strnad Recent Advances in the Chemistry and Pharmacology of Cryptolepine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Steven D. Shnyder and Colin W. Wright Correction to: Antileishmanial Activity of Lignans, Neolignans, and Other Plant Phenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jiří Pospíšil, Daniela Konrádová, and Miroslav Strnad

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The Role of Total Synthesis in Structure Revision and Elucidation of Decanolides (Nonanolides) Bernd Schmidt

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Recurring Methods in Decanolide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Esterification and Macrolactonization Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Ring-Closing Olefin Metathesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Olefin Cross-Metathesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Examples of the Synthesis-Assisted Structure Elucidation of Ten-Membered Lactone Natural Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Fusanolides, Modiolides or Curvulalic Acid? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Stagonolides and Curvulide A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Seimatopolides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Pheromones from Madagascan Mantellid Frogs . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 A Potential Pheromone from the Queen of the Termite Silvestritermes minutus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 7 9 11 16 18 18 23 34 41 46 49 50

Abbreviations Ac BINAP CD CM COSY

Acetyl 2,2 -Bis(diphenylphosphino)-1,1 -binaphthyl Circular dichroism Cross-metathesis Correlation spectroscopy

B. Schmidt (B) Institut für Chemie, Universität Potsdam, Karl-Liebknecht-Strasse 24–25, 14476 Potsdam-Golm, Germany e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. D. Kinghorn, H. Falk, S. Gibbons, Y. Asakawa, J.-K. Liu, V. M. Dirsch (eds.), Progress in the Chemistry of Organic Natural Products, Vol. 115, https://doi.org/10.1007/978-3-030-64853-4_1

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2

CSA DCC DDQ DET DMAP ECM EDC ee EI EOM ESI GC-FTIR

B. Schmidt

Camphor sulfonic acid Dicyclohexyl carbodiimide 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone Diethyl tartrate 4-N,N-Dimethylamino pyridine Exciton chirality method 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Enantiomeric excess Electron ionization Ethoxymethyl Electrospray ionization Gas chromatography—Fourier transform infrared spectroscopy (-coupling) GC-MS Gas chromatography—mass spectrometry (-coupling) GCxGC-TOFMS Two-dimensional gas chromatography/time-of-flight mass spectrometry (-coupling) HMBC Heteronuclear multiple bond correlation HRMS High-resolution mass spectrometry HSQC Heteronuclear single quantum coherence Ipc Isopinocampheyl IR Infrared (-spectroscopy) LAH Lithium aluminum hydride m-CPBA meta-Chloroperbenzoic acid MNBA 2-Methyl-6-nitrobenzoic anhydride MOM Methoxymethyl NHC N-Heterocyclic carbene NHK Nozaki–Hiyama–Kishi (-coupling) NMR Nuclear magnetic resonance (-spectroscopy) 1D-NMR One-dimensional nuclear magnetic resonance (-spectroscopy) 2D-NMR Two-dimensional nuclear magnetic resonance (-spectroscopy) NOE Nuclear Overhauser effect NOESY Nuclear Overhauser enhancement and exchange spectroscopy PMB p-Methoxybenzyl PPAR Peroxisome proliferator-activated receptors RCM Ring-closing metathesis ROESY Rotating frame Overhauser effect spectroscopy salen 2,2 -Ethylenebis(nitrilomethylidene)diphenol (-ligand) TBAF Tetrabutylammonium fluoride TBDPS tert-Butyldiphenylsilyl TBS tert-Butyldimethylsilyl TEMPO 2,2,6,6-Tetramethylpiperidine-1-oxyl TES Triethylsilyl OTf Trifluoromethanesulfonate Tos p-Tolylsulfonate

The Role of Total Synthesis in Structure …

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1 Introduction Decanolides, sometimes referred to as nonanolides, are naturally occurring tenmembered lactones. The first compound with this general structure was isolated from jasmine oil in 1942 [1], and later named jasmine ketolactone (1). The tenmembered lactone structure could not be assigned until 1964; this assignment was based on IR and NMR spectroscopic investigations, but elucidation of the absolute and relative configuration required a four-step conversion into the known (–)-methyl jasmonate (2), and comparison with the previously published specific rotation of 2 [2] (Scheme 1). In their 1996 landmark review on ten-membered lactone natural products [3], Dräger et al. named the diplodialides as the first examples of decanolides, probably because these metabolites are, like the majority of decanolides known to date (and in contrast to jasmine ketolactone (1)), pentaketides and originate from a microbial source, the plant pathogenic fungus Diplodia pinea [4]. Diplodia pinea is the causal agent of the diplodia tip blight, a fungal disease that affects pines and other conifers when exposed to stressed conditions (Plates 1 and 2). The first three decanolides from this source were discovered in 1975 by Ishida and Wada and named diplodialides A–C [4], and a fourth metabolite was isolated in the following year in very small quantities and named diplodialide D [5]. The strategy for structure elucidation of the diplodialides in the 1970s was very similar to that employed for jasmine ketolactone (1) a decade earlier: the ten-membered lactone structure and the (E)-configuration of the endocyclic C=C double-bond were established through a combination of IR and NMR spectroscopy. However, in contrast to the work on jasmine ketolactone (1) ten years before, high-resolution mass spectrometry (HRMS) had entered the scene and provided valuable information for elucidating the constitution. Assignment of absolute configurations to the stereocenters at

Scheme 1 Historical elucidation of absolute and relative configuration of (–)-jasmine ketolactone (1)

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Plate 1 Browning of needles of a pine tree caused by diplodia tip blight. Photograph courtesy of Pflanzenschutzamt Berlin. Copyright Pflanzenschutzamt Berlin. Reproduced with permission

Plate 2 Black fruiting structures of Diplodia pinea. Photograph courtesy of Pflanzenschutzamt Berlin. Copyright Pflanzenschutzamt Berlin. Reproduced with permission

The Role of Total Synthesis in Structure …

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C-3 and C-9 still required synthetic degradation to known compounds and comparison with their published specific rotations, occasionally after derivatization [6]. By oxidation of the secondary alcohol of diplodialide B (4) and hydrogenation of the C-4=C-5 double bond, Ishida and Wada were able to prove that the absolute configuration at C-9 is identical for diplodialides A–C. The (R)-configuration assigned to 3–5 was proven by reductive ozonolysis, which gave (R)-hexane-1,5-diol (7), a compound that could be compared to its known enantiomer. The configuration of diplodialides B (4) and C (5) at C-3 was elucidated by conversion of 4 to the known p-nitrobenzoate 9 of dimethyl malate in five steps: acetylation of 4 furnished the acetate 8, which underwent oxidative ozonolysis, basic cleavage of the acetate and the lactone, methylation with diazomethane and eventually derivatization as a para-nitrobenzoate 9 (Scheme 2). In their original publications, Wada and Ishida presented the structures of diplodialides A–D (3–6) as shown in Scheme 2, without assigning any absolute or relative configurations for diplodialide D (6) [5, 6]. Insufficient amounts of material prevented a further investigation at that time. Although it appeared highly likely that the configurations at C-3 and C-9 should be the same as for diplodialides B (4) and C (5), this was not established unambiguously until 2018, when the first enantioselective total synthesis of diplodialide D (6) was published by Ramanujan and Kumar [7]. These researchers started from (S)-glycidol tosylate (10), an enantiomerically pure building block with a reliably assigned absolute configuration. Epoxide 10 is available via Sharpless epoxidation of allyl alcohol [8] and was initially converted to compounds 11 and 12 in two and five steps, respectively. Both electrophilic coupling partners

Scheme 2 Historical structure elucidation of diplodialides A–C

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were successively coupled with lithiated 1,3-dithiane (13) in a Corey–Seebach reaction to furnish 14 [9]. In three steps the ω-hydroxycarboxylic acid 15 was obtained, which was cyclized to the ten-membered lactone 16 through a Yamaguchi macrolactonization [10, 11] and oxidative cleavage of the protecting group. From lactone 16, diplodialide C (5) was accessible via reductive cleavage of the dithiane, and diplodialide D (6) via hydrolytic cleavage of the dithiane (Scheme 3). Analytical data obtained for synthetic diplodialides C and D, including specific rotations, matched those previously reported by Wada and Ishida for the compounds isolated from the natural source [5, 6]. This eventually corroborated the assumed, but not proven, absolute configurations of the stereocenters and completed the structure elucidation of diplodialide D (6) after four decades. Over the twenty years following the isolation and structure elucidation of the diplodialides, ca. 50 decanolides were isolated from natural sources. The status of decanolide research in the mid-1990s, including a survey of compounds isolated between 1975 and 1995, results from biosynthesis studies, and investigations into their biological properties and an overview of strategies for their total synthesis have been summarized in the first review dedicated exclusively to the topic of decanolides

Scheme 3 Enantioselective total syntheses of diplodialides C (5) and D (6) and confirmation of assigned absolute configurations

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[3]. In this review, Dräger et al. noted that more than 80% of the decanolides known at that time originate from microbial sources (mostly fungi), where 15% were isolated from animals and just one decanolide was isolated from a plant, that is, jasmine ketolactone (1). In 2012, Sun et al. published a second review dedicated to the same topics that specifically covered the period after publication of the first review by Dräger et al. until mid-2011 [12]. Sun et al. noted the isolation and description of 63 new decanolides in the chemical literature during this time. Fungi remained the main source of decanolides. Other reviews were not intended to be comprehensive but covered specific sub-topics, for example, synthesis studies of specific decanolides [13]. Since the 1970s, instrumental analytical techniques have seen tremendous progress, which has changed the strategies used for structure elucidation of natural products fundamentally. Synthetic interconversion and degradation of novel metabolites to known compounds are used rarely nowadays to assign structures; in particular, because very sensitive high-field NMR spectrometers are routinely available, and elaborate 1D- and 2D-NMR experiments can be performed in a short time. In addition, the substantial expansion of ionization techniques available for mass spectrometry facilitates the measurement of high-resolution mass spectra and the reliable determination of a molecular formula, double-bond equivalents and ring closures. In light of this technological progress, it appears somewhat surprising that until the present time the structural assignment of newly isolated decanolides has remained incomplete occasionally and has turned out to be in part erroneous. This can—as in Wada’s and Ishida’s case of diplodialide D forty years ago—often be explained by the very small amounts of new metabolites obtained by isolation from the natural source, which makes purification and hence interpretation of the NMR spectra often very difficult. In such cases, the total synthesis of natural products from enantiopure starting materials with reliably assigned absolute configurations (either obtained exchiral pool or corroborated otherwise) can assist in structure elucidation. This may be done by proving an assumed structure, by completing structural information (which is mostly the case when configurations of one or more stereocenters remain elusive by NMR spectroscopy), or by revising erroneously assigned structures. Herein, the contribution of total synthesis to the structure elucidation of naturally occurring decanolides is discussed by representative case studies. Results from isolation studies and investigations into the biosynthesis and bioactivities of certain compounds will be mentioned briefly, if appropriate, but they are not the primary focus of this chapter.

2 Recurring Methods in Decanolide Synthesis In their major review from 1996, Dräger et al. classified synthesis approaches to decanolides as follows: “(i) oxidative fragmentation of annulated decanolides, (ii) ring closure by C–C-bond formation, (iii) ring closure by macrolactonization” [3].

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The methods for ring closure by C–C-bond formation used at that time were Pdcatalyzed allylic substitutions, SmI2 -mediated Reformatsky reactions or intramolecular radical additions to C=C double-bonds. These methods, as well as the oxidative fragmentation of annulated decanolides, no longer play a significant role in the total synthesis of ten-membered lactones due to the development of efficient olefin metathesis catalysts starting in the mid-1990s. Comparison of the review on decanolides in general by Sun et al. from 2012 [12] and with Ishigami’s review on the synthesis of selected ten-membered lactone natural products from 2009 [13] reveals how olefin metathesis has revolutionized the total synthesis of decanolides. However, macrolactonization remains a method of constant importance over the decades; in particular, Yamaguchi’s method has been, and is, used routinely in decanolide synthesis [14]. As a notable example for the efficient use of an oxidative ring expansion strategy [15] in contemporary decanolide total synthesis, the cephalosporolides should be mentioned [16–18]. Many contemporary total syntheses of decanolide natural products proceed via either of these two general strategies: (i) an ω-enoic acid 17 and an ω-alkenol 18 are esterified (most commonly using Steglich’s esterification or a variant [19]) and then cyclized by ring-closing olefin metathesis (RCM) or (ii) an ω-enoic acid 17 and an ω-alkenol 18 are connected through cross-metathesis (CM) and the resulting secoacid 20 is then cyclized using Yamaguchi macrolactonization [14] or an alternative mixed-anhydride method (Scheme 4). An alternative way to connect two advanced fragments to an unsaturated secoacid 20, which is subsequently cyclized via macrolactonization, involves carbonyl olefination methods. The Julia–Kocienski olefination, for example, often shows very high levels of (E)-stereoselectivity [20, 21]. This method has been used as a key step in the synthesis of the plant metabolites cytospolides C and D [22]. In this section a brief overview of methods often used in the synthesis of naturally occurring decanolides is given. Scheme 4 General strategies for contemporary decanolide total synthesis

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2.1 Esterification and Macrolactonization Reactions Steglich’s reaction [19] is the most commonly used esterification method in decanolide total synthesis. The method relies on the use of dicyclohexylcarbodiimide (DCC) in combination with a catalytic amount of N,N-dimethylaminopyridine (DMAP) in a moderately polar aprotic solvent, such as dichloromethane. A typical example is the esterification step in Prisinzano’s synthesis of aspinolide A ((5R)-25) and 5-epi-aspinolide A ((5S)-25) [23]. Sometimes 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDCI), which is normally employed as its hydrochloride (EDCI•HCl), is used advantageously [24]. In contrast to DCC, the use of EDCI allows aqueous workup at pH 4–6 to remove the urea co-product formed during the esterification. An example is the coupling of carboxylic acid 26 and alcohol 27 to ester 28, a precursor of the ten-membered lactone pinolide [25]. In both examples, a carboxylic acid and an alcohol can be used in equimolar or near equimolar amounts (Scheme 5). The carbodiimides DCC and EDCI•HCl were originally introduced as peptide coupling reagents. An overview of these and related reagents is given in a study on the assessment of their thermal stability [26]. Although Yamaguchi’s method has occasionally been used for difficult intermolecular esterifications instead of Steglich’s conditions, this method shows its strength best in its intramolecular version, the lactonization. Yamaguchi’s reaction relies on the formation of an activated mixed anhydride of the carboxylic acid to be coupled with a nucleophile and 2,4,6-trichlorobenzoyl chloride [14]. In contrast

Scheme 5 Examples of Steglich esterifications

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to intermolecular esterifications, high- or pseudo-high dilution conditions have to be applied for medium-sized macrolactones [27]. For example, in a synthesis of stagonolide B (31), the hydroxycarboxylic acid 29 underwent lactonization to 30 under Yamaguchi conditions when a solution of the mixed anhydride (formed in situ from 29 and 2,4,6-trichlorobenzoylchloride) was added to a highly diluted solution of DMAP in toluene via syringe pump. The effective concentration was 0.5 mM in this case [28]. Occasionally, Yamaguchi’s macrolactonization conditions have been reported to produce only dimers or polymers, or the reagent reacts with other nucleophilic groups in the lactonization precursor. All of these problems occurred in a total synthesis of cytospolide D (35) when Yamaguchi’s conditions were tested for the lactonization of 32 [29, 30], but could be overcome by using Shiina’s reagent, 2methyl-6-nitrobenzoic anhydride (MNBA), instead [31]. Like Yamaguchi’s reagent MNBA also reacts with the carboxylic acid to a mixed anhydride. In this particular case the elimination product 34 was observed as a side product, which presumably is formed through intermediate formation of a β-lactone [29] (Scheme 6). In a follow-

Scheme 6 Examples of the application of Yamaguchi and Shiina macrolactonization

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up study on the synthesis of cytospolide D analogs, it was discovered that small differences in the structures of the precursors can be crucial for the success of a macrolactonization. If, for instance, the C-3 hydroxy group of 32 is protected as a TBS ether and/or if the C-2 epimer of 32 is used, the lactonization fails completely, even under optimized Shiina conditions [30]. Contemporary methods for ester coupling reactions, their mechanisms and representative applications in the synthesis of complex natural products have been reviewed [27].

2.2 Ring-Closing Olefin Metathesis Ring-closing olefin metathesis (RCM) has emerged as one of the most versatile C–C-bond forming reactions used in the synthesis of carbo- and heterocyclic target molecules [32, 33], and was triggered by the discovery of stable and defined homogeneous precatalysts based on Ru- [34, 35] and Mo-carbenes [36]. The general mechanism of an RCM reaction leading to an unsubstituted ten-membered lactone is shown in Scheme 7. It proceeds through a series of (2+2)-cycloaddition/cycloreversion reac-

Scheme 7 General mechanism of an RCM reaction

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B. Schmidt

tions. The catalytically active species, a metal alkylidene complex, reacts with one of the two double-bonds to a metallacyclobutane, which undergoes a cycloreversion with liberation of ethene. The new metal alkylidene reacts intramolecularly with the second C=C double-bond of the starting material to a bicyclic metallacyclobutane, which eventually undergoes a cycloreversion to regenerate the catalyst and furnish the product. Ring-closing metathesis reactions are entropy-driven and rely on the formation of a volatile co-product, which in most cases is ethene. In principle, all steps (and the overall reaction) are reversible, but removal of ethene shifts the equilibrium to the formation of the product [37]. Due to their robustness, low sensitivity toward air and moisture and high functional group tolerance in particular, the Ru-based precatalysts introduced by Grubbs and co-workers have found many applications in the total synthesis of natural products [38–42]. First-generation catalysts (A) are less active (which can be an advantage in some cases, as will be discussed below) but are cheaper and more conveniently accessible. Second-generation catalysts (B) have one N-heterocyclic carbene (NHC) ligand. They are more active and therefore lower catalyst loadings are required. These catalysts are also suitable for the generation of triple- and even tetra-substituted double bonds, but they are more expensive and their synthesis requires additional steps. The two most commonly used metathesis catalysts are A1 [34] and B1 [35]. While the formation of five- and six-membered rings through RCM proceeds without difficulties in most cases at substrate concentrations >0.1 M and catalyst loadings 99.9% ee and converted in six routine steps to acid 72. The C-7–C-9 fragment (R)-74 was synthesized from 73 through a similar sequence of yeast-mediated reduction and lipase-catalyzed kinetic resolution. Both fragments were connected via Yamaguchi esterification conditions followed by cleavage of the acetal. The resulting aldehyde

Scheme 16 Enantioselective total synthesis of modiolide A (61)

22

B. Schmidt

75 was used in an intramolecular Nozaki–Hiyama–Kishi (NHK) coupling [85] which proceeded with fair diastereoselectivity to give 76. Removal of the minor diastereomer, the C-7-epimer, was accomplished by chromatography. Introduction of the C-2=C-3 double-bond (via phenylselenylation and elimination) and removal of the protecting groups completed the first enantioselective total synthesis of modiolide A (61). The absolute configurations of the two enantiomerically pure key fragments, (S)-71 and (R)-74, were secured by comparison of their specific rotations with previously reported literature values. All analytical data of synthetic modiolide A (61) matched those reported for the natural product, including the sign of specific rotation, which confirmed the absolute configuration originally assigned via the exciton chirality method. In a formal total synthesis of modiolide A (61) the absolute configuration at C-9 was derived ex-chiral pool [86] (Scheme 17). A C-6–C-9 fragment 80 was synthesized from l-malic acid (79) in ten steps and then coupled with acid 78 using Steglich conditions. The C-1–C-5 fragment 78 was obtained in six steps from the known epoxide 77, which had previously been synthesized via Sharpless epoxidation and therefore has a reliably assigned absolute configuration. After cleavage of both PMBprotecting groups in ester 81, the resulting diol 82 underwent RCM in the presence of second-generation catalyst B1 cleanly to 83 (the natural product stagonolide C, cf. Section 3.2) with the required (E)-configuration of the newly formed double-bond. Protection of 83 as a bis-TBS ether furnished 84, an exact intermediate of the previous

Scheme 17 Total synthesis of stagonolide C (83) and formal total synthesis of modiolide A (61)

The Role of Total Synthesis in Structure …

23

modiolide A synthesis [84]. Compound 84 was identical to that intermediate in every regard, including the sign and value of the specific rotation. A close inspection of the analytical data reported for natural [68] and synthetic [84] modiolide A (61) with those reported for fusanolide B (60) by Shimada et al. in 2002 [64] revealed only very small differences that cannot be plausibly explained by the different configuration at C-4. The only notable differences were observed for the value of the specific rotations, but all three were dextrorotatory (see Chart 1 and Scheme 16). As first pointed out by Quang et al. in 2018 [82], who isolated modiolides A (61) and B (62) together with the four new modiolides D–G from the marine fungus Paraconiothyrium sp. VK-13, it has been proved that the originally assigned structure of fusanolide B (60) is incorrect and that actually modiolide A (61) was isolated from Fusarium sp. by Shimada et al. [64]. Interestingly, two of the four new modiolides isolated and characterized by Quang et al., modiolide F (65) and modiolide G (66), have an all-cis relative configuration (deduced from H,Hcoupling constants and NOESY spectra) as originally proposed for fusanolide B (60). A comparison of the analytical data obtained for the new metabolite modiolide G (66) [82] with those reported for “fusanolide B” [64] and modiolide A (61) [68, 84] shows substantial differences that underline the necessity to revise the structure of “fusanolide B”. Remarkably, the new modiolides F (65) and G (66) have been assigned the (S)-configuration at C-9, in contrast to the other modiolides (or most ten-membered lactone natural products in general). The absolute configuration at C4 was determined as (R) for modiolide G (66), based on Mosher’s method, and from there the absolute configurations at C-7 and C-9 were deduced to be the opposite of those in modiolide A (61) [82].

3.2 Stagonolides and Curvulide A Stagonolide A (85), originally named stagonolide, was first isolated and characterized in 2007 by Yuzikhin et al. from culture filtrates of Stagonospora cirsii [87]. This fungus is a pathogen that affects Cirsium arvense, the field or Canada thistle, a perennial weed that is difficult to control in agriculture by mechanical means or with common herbicides [88]. Stagonolide A (85) was shown to have strong phytotoxic activity against C. arvense by inhibiting root growth and photosynthesis, and was therefore proposed as a lead structure for novel herbicides derived from fungal plant pathogens [87, 88] (Chart 2). Shortly after this seminal investigation, several other ten-membered lactones were isolated from Stagonospora cirsii, structurally characterized and tested for their phytotoxic activity against C. arvense: stagonolides B–F (83, 86–89) [89] and stagonolides G–I (90–92) [78] were discovered in 2008 by Evidente et al.; stagonolides J (93) and K (94), the most recent members of the stagonolide family, were isolated in 2019 by Dalinova et al. [90]. The ten-membered lactone curvulide A (95) has not been isolated from cultures of Stagonospora cirsii, but from the marine

24

B. Schmidt

Chart 2 Originally published structures of stagonolides and curvulide A

fungus Curvularia sp. [79]. It is mentioned in this context because it is the C-4–C5-epoxide of stagonolide E (88) or its C-6-epimer and therefore structurally related (Chart 2). The original structural assignment of stagonolide A (85) was based on a combination of HRMS, IR spectroscopy, 1D- and 2D-NMR methods and chemical modification [87]: reduction of stagonolide A (85) with NaBH4 proceeds highly diastereoselectively and furnished a compound that was in all regards identical with herbarumin

The Role of Total Synthesis in Structure …

25

I (41) [91] (Scheme 18). While this proves the constitution and relative configuration assigned to stagonolide A (85), it is not conclusive evidence for the assigned absolute configuration, although the commonly assumed (9R)-configuration appears to be highly likely as it is found in most other stagonolides and the herbarumins. As no specific rotation or chiroptical data have been reported for stagonolide A (85) or its reduction product, semisynthetic herbarumin I (41) [87], a comparison with the published specific rotation of natural herbarumin I [91] is not possible. To date, four enantioselective total syntheses [92–95] and one formal synthesis [96] of stagonolide A (85) have been published. In all the total syntheses negative specific rotations [with values in the range from −40°cm2 (10 g)−1 to −60°cm2 (10 g)−1 ] were reported for synthetic 85. These data can be used to corroborate the assigned absolute configuration once the value for stagonolide A (85) isolated from the natural source is available. As a representative example for an enantioselective approach to stagonolide A, the first synthesis reported in the literature by Srihari et al. is highlighted below [92]. The starting point of this route is the enantiomerically pure epoxide 96, which had previously been synthesized from d-ribose by the same group in three steps in the course of another total synthesis project [97]. Copper-mediated Scheme 18 Chemical modification of stagonolide A (85) and its relation to herbarumin I (41)

Scheme 19 First enantioselective total synthesis of stagonolide A (85): ex-chiral pool approach from d-ribose

26

B. Schmidt

opening of epoxide 96 with ethyl magnesium bromide was used to introduce the C-9propyl side chain and Steglich esterification of the secondary alcohol gave the RCM precursor 97. Ring-closing metathesis of 97 proceeded with moderate diastereoselectivity ((E):(Z) = 4:1). Chromatographic removal of the undesired (Z)-isomer was only possible after cleavage of the acetal. The resulting product, herbarumin I (41), was selectively oxidized at C-7, the allylic position, to furnish stagonolide A (85). In summary, two out of three configurationally stable stereocenters in the ex-chiral pool starting material d-ribose were retained in the product stagonolide A (Table 1, entry 1 and Scheme 19). The other total syntheses of stagonolide A rely on very similar synthetic strategies. Two of these also use carbohydrates (d-ribose: Table 1, entry 2 [93] or d-glucose: Table 1, entry 3 [94]) as ex-chiral pool starting materials, while a third synthesis makes use of the well-established aldol methodology based on thiaoxazolidinone auxiliaries to control the absolute and relative configurations at C-8 and C-9 (Table 1, entry 4) [95]. As in the first synthesis, ring closure is accomplished in the other three syntheses by RCM. By using one or two benzyl ethers as hydroxy protecting groups at C-7 and C-8 and second-generation catalysts, the RCM step can be notably improved with regard to diastereoselectivity, yield and catalyst loading [94, 95]. The structures of stagonolides B–K (83, 86–94) were elucidated via HRMS, IR spectroscopy, and 1D- and 2D-NMR spectroscopic methods. While (in contrast to stagonolide A (85)) specific rotations were reported for all these natural products, the absolute configurations were not assigned based on chiroptical methods, suitable NMR methods or X-ray-crystallography, but mostly by analogy to structurally related decanolides, such as the herbarumins [78, 89, 90]. As outlined in the case of one of the most recently discovered stagonolides, stagonolide J (93), application of Mosher’s method gives ambiguous results for vicinal diols, which is a serious limitation if absolute configurations need to be determined [90]. So far, no total syntheses of stagonolides H–J (91–93) have been reported (Table 1, entries 36–38). The originally assigned absolute and relative configurations of stagonolides B (86) (Table 1, entries 5–8), C (83) (Table 1, entries 9–15) and E (88) (Table 1, entries 20–26) could be confirmed via enantioselective total syntheses. In the case of stagonolide D (original structure: 87) total synthesis led to a revision of the absolute and relative configuration (revised structure: 98) (Table 1, entries 16–19) [98], and stagonolide G (original structure: 90) turned out to be a γ-butyrolactone 99 rather than the ten-membered lactone 90 (Table 1, entries 31–35) [99]. In the case of stagonolide F (89) the results from a synthetic study suggest that the constitution was erroneously assigned, but no revised structure has so far been proposed. It has, however, been suggested to use the name (–)-5-epi-aspinolide A rather than stagonolide F for structure 89 in the future (Table 1, entries 27–30) [23]. The total syntheses of stagonolides isolated from Stagonospora cirsii and their implications for structure elucidation are summarized in Table 1. The most notable erroneous structural assignment in the stagonolide family is probably that of stagonolide G as a ten-membered lactone. This misassignment was discovered as a result of an enantioselective total synthesis published by AnguloPachón et al. [99] (Table 1, entry 32 and Scheme 20): the two key fragments, alcohol

d-Mannitol l-Diethyl tartrate d-Mannitol; Sharpless kinetic resolution

11

12

13

l-Malic acid; Sharpless epoxidation Lipase- and Ru-cat. dynamic kinetic resolution

9

Fully confirmed

Enzymatic hydrocyanation; asymmetric dihydroxylation

8

Stagonolide C (83)

d-Ribose; lipase-cat. kinetic resolution

7

10

d-Ribose; Co-salen cat. kinetic resolution

d-Ribose

6

Fully confirmed

d-Glucose

d-Ribose

d-Ribose

(continued)

[105]

[104]

[103]

[102]

[86]

[28]

[93]

[101]

[100]

[95]

[94]

[93]

[92]

Source of chirality in total syntheses Ref.

5

Confirmed or revised structure based on synthesis

Chiral auxiliary mediated aldol addition

Constitution and relative configuration trans confirmed; confirmation of absolute configuration pending

Status of structure elucidation after total synthesis

4

3

Stagonolide B (86)

Stagonolide A (85)

1

2

Stagonolide

Entry

Table 1 Summary of total synthesis-based structure elucidation of stagonolides A–K

The Role of Total Synthesis in Structure … 27

Lipase-cat. kinetic resolution; asymmetric LAH-binol carbonyl reduction d-Mannitol; lipase-Ru-catalyzed dynamic kinetic resolution

21

22

Co-salen cat. kinetic resolution; Sharpless epoxidation

20

Fully confirmed

(R)-Glycidol; alpine-borane carbonyl reduction (synthesis of stagonolide D; revised absolute and relative configuration confirmed

19

Stagonolide E (88)

d-Xylose (synthesis of ent-stagonolide D; revision of configuration to (4S,7R,8S,9R) proposed)

18

(continued)

[66]

[112]

[111]

[110]

[98]

d-Mannitol (synthesis of originally [109] assigned structure and confirmation)

[108]

[107]

17

Sharpless asymmetric epoxidation (synthesis of originally assigned structure and confirmation)

Originally assigned structure 87 was revised to 98 (stereocenters are (4S,7R,8S,9R)-configured)

d-Glyceraldehyde

Stagonolide D (98)

[106]

Source of chirality in total syntheses Ref.

16

Confirmed or revised structure based on synthesis

15

Status of structure elucidation after total synthesis Proline cat. aminooxylation; Jorgensen epoxidation

Stagonolide

14

Entry

Table 1 (continued)

28 B. Schmidt

Co-salen cat. epoxide hydrolytic [117] resolution and Sharpless epoxidation (synthesis of published structure and confirmation proposed)

Asymmetric organocat. [119] α-hydroxylation and epoxidation (synthesis of published structure and confirmation proposed) (R)-Propylene oxide; separation of [23] diastereomers after Nozaki-Hiyama-Kishi-cyclization (synthesis of structure 89, but significant discrepancies with data published for natural and synthetic stagonolide F reported; re-naming of 89 to (–)-5-epi-aspinolide A proposed)

29

30

(continued)

Asymmetric dihydroxylation; [118] R-propylene oxide (synthesis of published structure and confirmation proposed)

28

Structure assigned for stagonolide F No revised structure proposed so has been questioned; renaming to far (–)-5-epi-aspinolide A was proposed

27

Stagonolide F (89)

(S)-5-Hexene-2-ol; asymmetric dihydroxylation (formal synthesis)

26

[116]

d- and l-Proline cat. α-hydroxylation [115]

25

[113]

Source of chirality in total syntheses Ref.

d- and l-Proline cat. α-hydroxylation [114]

Confirmed or revised structure based on synthesis Lipase-cat. kinetic resolution

Status of structure elucidation after total synthesis

24

Stagonolide

23

Entry

Table 1 (continued)

The Role of Total Synthesis in Structure … 29

Stagonolide J (93)

Stagonolide K (94)

39

Absolute configuration assigned based on previous synthesis

No synthesis reported

–

d-Ribose

–

–

38

–

–

–

Stagonolide I (92)

37

No synthesis reported

Stagonolide H (91)

36

No synthesis reported

Co-salen cat. epoxide hydrolytic [123] resolution; asymmetric dihydroxylation (revised structure 99 confirmed)

35

[124]

–

–

–

[122]

d-Glyceraldehyde (revised structure 99 confirmed)

34

[99]

[120]

d-Glyceraldehyde; asymmetric [121] dihydroxylation (published structure confirmed)

d-Glucose (published structure confirmed)

Source of chirality in total syntheses Ref.

33

Originally assigned structure 90 was revised to 99

Confirmed or revised structure based on synthesis

Asymmetric allylboration (revision to 99 proposed)

Stagonolide G (99)

31

Status of structure elucidation after total synthesis

32

Stagonolide

Entry

Table 1 (continued)

30 B. Schmidt

The Role of Total Synthesis in Structure …

31

Scheme 20 Total synthesis leading to the revised structure of stagonolide G (99)

102 and carboxylic acid 106, were synthesized using well-established and highly enantioselective allylborations of acetaldehyde (100) and aldehyde 103 with (–)isopinocampheyl-((–)-Ipc)-boranes 101 and 104, respectively. The TBDPS-protected allylboration product 105 was converted in four routine steps into carboxylic acid 106, which was coupled with 102 under Yamaguchi conditions to give the RCM precursor 107. RCM of 107 proceeded with high (Z)-selectivity, as required, and gave the protected decanolide 108. Cleavage of both acetal protecting groups (EOM = ethoxymethyl) was accomplished under particularly mild Lewis-acidic conditions and furnished compound 90. The structure of 90 was corroborated by 1D- and 2DNMR methods. In particular, 2D-heteronuclear long-range correlation spectroscopy (HMBC) was found to be a valuable tool for proving the ten-membered lactone structure assigned to 90. However, a comparison with the spectral data published for stagonolide G [78] revealed differences that were so substantial that the originally assigned structure 90 had to be revised. For these reasons, Angulo-Pachón et al. synthesized a diastereomer of 90, which also had notably different analytical data

32

B. Schmidt

than stagonolide G. By chance, the authors discovered that re-measurement of an NMR sample of 90 in CDCl3 gave NMR spectra with an additional set of signals. The additional set of signals turned out to be a good match for the originally reported NMR data of stagonolide G. This led to the hypothesis that the ten-membered lactone 90 might undergo an acid-catalyzed rearrangement, which was eventually verified on a preparative scale by treating the ten-membered lactone 90 with a catalytic amount of CSA (camphor sulfonic acid). By application of 1D- and 2D-NMR methods, again in particular by HMBC spectroscopy, the rearrangement product was identified as γbutyrolactone 99. Apart from the value of specific rotation value, all other analytical data (including the sign of specific rotation) matched those reported for stagonolide G very well. Intriguingly, prior to the total synthesis of Angulo-Pachón et al. three other total syntheses had been reported, which all confirmed the originally assigned tenmembered lactone structure 90 (Table 1, entries 31, 33, 34) [120–122]. In these cases, the final step of the total synthesis involved a Lewis-acid-mediated or reductive cleavage of benzyl-protecting groups. Most likely, these deprotection steps proceeded with a rearrangement to the five-membered lactone, which was not noticed because its spectral data agreed with those reported for the natural product. Based on the available data, it cannot be excluded conclusively that the actual structure of stagonolide G is that of a ten-membered lactone 90, which underwent ring contraction to 99 during isolation from the natural source, e.g. promoted by chromatography on acidic silica gel. The results from the total syntheses only confirmed that the compound described as stagonolide G in the isolation paper [78] is indeed a γ-butyrolactone rather than a ten-membered lactone. A curiosity is, in some regard, how the structure assignment of the recently discovered stagonolide K was accomplished [90]. The constitution and relative configuration were elucidated by HR-ESI-MS, IR-spectroscopy, and 1D- and 2D-NMR methods. Crystals suitable for single-crystal X-ray analysis were obtained, but in the absence of any heavy atoms determination of the absolute configuration through anomalous dispersion is not possible and therefore X-ray analysis could only serve as a confirmation of the results obtained from NMR analysis. Gratifyingly, Maram et al. had isolated and characterized a—at that time—non-natural C-9 epimer in the course of their total synthesis of herbarumin III a few years before [124]. Their synthesis started from d-ribose, which was converted in six steps into a 3:2 mixture of epimers of 108. While (R)-108 served as an enantiopure starting material for herbarumin III, its epimer (S)-108 was converted to 9-epi-herbarumin III in five steps. The product obtained after ring-closing metathesis turned out to be identical with stagonolide K (94), including the sign of specific rotation, which led to the assignment of a (7R,9S)-configuration to stagonolide K (Table 1, entry 39 and Scheme 21). Curvulide A (95) is the C-4–C-5-epoxide of stagonolide E (88) or of a stagonolide E epimer. It is covered in this section due to its structural resemblance and possible structural connection with stagonolide E, although the producing organisms are quite different: curvulide A (95) was isolated from the marine fungus Curvularia sp., which is associated with a red alga [79]. Upon its isolation, the structure of curvulide A (95) could only be partly resolved: a combination of HR-ESI-MS, IR spectroscopy, and

The Role of Total Synthesis in Structure …

33

Scheme 21 Synthesis of 9-epi-herbarumin III (stagonolide K (94))

1D- and 2D-NMR methods (in particular COSY, HSQC and HMBC) secured the constitution shown in Chart 2 and the relative (trans)-configuration of the epoxide at C-4 and C-5. A characteristic Cotton-effect in the CD spectrum of curvulide A, which resembled the spectrum of modiolide A (cf. Scheme 16), suggested that the configuration at C-9 is identical for both natural products, that is (9R). However, the NOE-spectra remained inconclusive due to the high flexibility of the ten-membered lactone structure, and therefore, an assignment of the configuration at C-6 (and hence at C-4 and C-5) was not possible [79]. Structure elucidation was eventually completed through total synthesis from the chiral-pool-derived starting material d-mannitol (Scheme 22) [66]. d-Mannitol was converted in three steps to the enantiomerically pure C 2 -symmetric diol 111, a versatile chiral building block. From 111, butenoate 112 was synthesized in six steps and submitted to the conditions of a highly diastereoselective RCM-elimination sequence to furnish seco-acid 113. Yamaguchi macrolactonization and cleavage of the MOM-protecting group furnished stagonolide E (88). Stagonolide E (88) was subjected to Sharpless epoxidation conditions: based on the mnemonic device developed for predicting the outcome of such epoxidation reactions [8], it was expected that the combination of stagonolide E (88) and (–)d-diethyltartrate (DET) should be a matched pair and yield the (4R,5R)-configured epoxide, while the use of (+)-l-diethyltartrate (DET) should result in a slowly reacting mismatched pair and give the (4S,5S)-epoxide. Indeed, no conversion was observed with (+)-l-DET after several days, whereas with (–)-d-DET the starting material was fully consumed after 48 hours. The product isolated from the reaction mixture had identical analytical data, including the sign of specific rotation, and a value of specific rotation in the same order of magnitude as reported for naturally occurring curvulide A. A detailed NMR spectroscopic analysis

34

B. Schmidt

Scheme 22 Enantioselective total syntheses of stagonolide E (88) and curvulide A (95); completion of structure assignment for curvulide A

of synthetic curvulide A (95) confirmed the relative configuration of stereocenters C-4 and C-6 shown in Scheme 22. Thus, 95 is (4R,5R,6R,9R)-configured.

3.3 Seimatopolides Seimatopolides A (114) and B (115) are ten-membered lactones with a nonyl substituent at C-9. They were isolated in 2012 by Hiep et al. via bioactivity-guided fractionation from the culture broth of the fungus Seimatosporium discosioides [125]. This culture broth displayed notable activity in a reporter gene assay for the activation of the γ-subtype of peroxisome proliferator-activated receptors (PPAR-γ). The PPARs contribute to the regulation of lipid and lipoprotein metabolism, glucose and fatty acid homeostasis, and the inflammatory response [125]. Activators of these receptors might therefore be lead structures for potential anti-inflammatory or antidiabetic drugs. The isolated seimatopolides A and B were evaluated for their activating ability toward PPAR-γ, and half-maximal effective concentrations (EC 50 ) of 1.15 μM for seimatopolide A (114) and 11.05 μM for seimatopolide B (115) were measured. Troglitazone, an antidiabetic drug that acts as a PPAR-γ agonist but was discontinued due to severe side effects, was used as positive control. Its EC 50 value was determined as 0.44 μM and is therefore in the same potency range as that of seimatopolide A [125].

The Role of Total Synthesis in Structure …

35

Chart 3 Originally published structures of seimatopolides A and B

The constitutions and relative configurations of both metabolites were elucidated through the combined use of HR-ESI-MS, IR spectroscopy and 1D- and 2DNMR methods. As in several other examples discussed before, 2D long range C–Hcorrelation spectroscopy (HMBC) was found to be particularly useful for elucidating the connectivity of the carbon atoms. The relative configurations were established through a detailed analysis of vicinal coupling constants and NOE spectroscopy. A (9S)-configuration was assigned to both seimatopolides (Chart 3) based on a modified Mosher method that analyzes 1 H-NMR chemical shift differences of the corresponding (R)- and (S)-Mosher esters. Less than five months after the original report on the isolation and structure elucidation of seimatopolides A (114) and B (115), three total syntheses of seimatopolide A were published within one week (Table 2, entries 1–3) and five further total syntheses within the following year (Table 2, entries 4 to 8). A synthesis that claims to be the “first stereoselective total synthesis of seimatopolide A” in the title uses the RCM of a triple-MOM-ether as the cyclization step. The RCM precursor was obtained through Steglich-esterification of a MOM-protected 3-hydroxy-hex-5enoic acid and an appropriately substituted and protected secondary alcohol. In this approach, all stereocenters are established via stereoselective synthesis: the stereocenter C-9 results from an enantioselective Keck-allylation [133, 134], the stereocenters at C-6 and C-7 were established by Os-catalyzed enantioselective dihydroxylation [135] and the stereocenter at C-3 results from an asymmetric Sharpless epoxidation [8]. These methods are well-established, reliable and furnish enantiomerically pure building blocks in predictable configurations. As a result of their synthesis, the authors confirm the assigned structure and report a negative specific rotation for synthetic seimatopolide A that is in the same range as that of the natural product [126] (Table 2, entry 1). A simultaneously published synthesis used (S)-4-pentene1,2-diol (obtained from the well-known (S)-glycidol) as one enantiomerically pure starting material. A Prins reaction [136] with decanal proceeded with high diastereoselectivity and led to a product that contained the stereocenters at C-9 and C-7 in the required configurations. The stereocenter at C-6 was established via enantioselective organocatalytic amino-oxylation using d-proline [137]. The second building block is the same (R)-configured MOM-protected 3-hydroxy-hex-5-enoic acid used in the synthesis discussed above (Table 2, entry 1). The final steps (Steglich-esterification, RCM, deprotection) are also identical with that approach. As a conclusion from this

Target structure

114

114

114

114

ent-114

ent-114

114

ent-114

Entry

1

2

3

4

5

6

7

8

Revised (3S,6S,7S,9R) configuration confirmed

l-Tartrate (C-6, C-7); oxazoborolidine catalyzed ketone reduction (C-9); lipase-Ru-cat. dynamic resolution (C-3)

−27.4, CH3 OH

Revised (3S,6S,7S,9R) configuration confirmed Revised (3S,6S,7S,9R) configuration confirmed

−28.9, CH3 OH

−27.1, CH3 OH

Sharpless kinetic resolution (C-7); +21.7, CH3 OH diastereoselective ketone reduction (C-9, C-6); aldol addition with thiaoxazolidinone auxiliary (C-3)

Two asymmetric dihydroxylations (C-9; C-7, C-6); Ru-BINAP-diamine cat. carbonyl reduction (C-3)

l-(+)-Tartrate (C-6, C-7); (−)-d-DET-mediated Sharpless epoxidation (C-9); lipase-mediated resolution (C-3)

Absolute configuration revised to (3S,6S,7S,9R)

Absolute configuration revised to (3S,6S,7S,9R)

Fully confirmed

−59.3, CH3 OH

+30.0, CH3 OH

Fully confirmed

−143.5, CH3 OH

[132]

[131]

[130]

[129]

[128]

[127]

[126]

Conclusion for structure assignment Ref.

Specific rotation/°cm2 (10 g)−1

d-Mannitol (C-6, C-7); +30.9, CH3 OH (+)-l-DET-mediated Sharpless epoxidation (C-9); lipase-mediated resolution (C-3)

l-Aspartic acid (C-9, C-3); asymmetric dihydroxylation (C-6, C-7)

(S)-4-Pentene-1,2-diol; diastereoselective Prins reaction (C-9, C-7); d-proline cat. aminooxylation (C-6); Sharpless epoxidation (C-3)

Ti-BINOL-mediated allylation (C-9); asymmetric dihydroxylation (C-6, C-7); Sharpless epoxidation (C-3)

Source of chirality (stereocenter)

Table 2 Summary of total syntheses of seimatopolide A

36 B. Schmidt

The Role of Total Synthesis in Structure …

37

study, the originally assigned structure 114 for seimatopolide A was also confirmed and a negative specific rotation, albeit with a significantly lower value, was recorded [127] (Table 2, entry 2). A third synthesis (that was actually the first based on the “received” and “published online” dates of the original publication) led to a different conclusion with regard to the absolute configuration of seimatopolide A [128] (Table 2, entry 3). The authors reported the synthesis of seimatopolide A with its assigned structure 114, but they found a positive specific rotation for this compound, which suggested that the (3R,6R,7R,9S)-configuration of the natural product was erroneously assigned and should be revised to (3S,6S,7S,9R) (ent-114). In contrast to the syntheses summarized above (Table 2, entries 1–2), this approach utilized a starting material from the chiral pool with a reliably assigned absolute configuration, that is l-aspartic acid (Scheme 23). l-Aspartic acid was converted to epoxide 116, a well-established chiral building block, following a previously published procedure [138]. The enantiomerically pure allyl alcohol 117 was obtained via epoxide opening with a sulfur ylide, and the PMBprotected alcohol was converted to the C-1–C-4-building block, carboxylic acid 118,

Scheme 23 Enantioselective total synthesis of ent-seimatopolide A (114)

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in three routine steps. The same enantiomerically pure starting material 116 was also used for the synthesis of the C-5–C-9 fragment: ring opening of 116 with the Liacetylide of 1-octyne, protection of the resulting secondary alcohol as a TBS-ether, hydrogenation of the triple bond and concomitant cleavage of the PMB-ether, oxidation of the primary alcohol to an aldehyde and finally a Wittig-olefination furnished the unsaturated ester 119. The stereocenters C-6 and C-7 were installed in the required configuration using Sharpless asymmetric dihydroxylation to yield 120, which was protected as an acetonide, followed by a one-pot reduction of the ester to an aldehyde and Wittig olefination, cleavage of the TBS-ether, and Yamaguchi esterification with 118. Ring-closing metathesis of the product, diene 121, was investigated but required a very high catalyst loading of 20 mol% of second-generation Grubbs’ catalyst B1. Ring-closing metathesis of a fully deprotected precursor (obtained after cleavage of the TBS ether and the acetonide) gave a complex mixture of products. The best result was obtained if the TBS-ether was cleaved prior to the RCM step, and the acetonide was removed after RCM. Via this route the catalyst loading could be lowered to 10 mol% and the product 114 was obtained with high (E)-selectivity. In contrast to the original report by Hiep et al. and the two simultaneously published total syntheses (Table 2, entries 1 and 2) compound 114 was found to have not only a significantly different value of specific rotation but also a positive sign of specific rotation, whereas all other analytical data (in particular NMR data) matched those previously reported for seimatopolide A very well. From these observations the authors conclude that the absolute configuration of seimatopolide A was erroneously assigned and that the actual structure is ent-114, with a (3S,6S,7S,9R)-configuration of the stereocenters. The proposed revision of absolute configuration was shortly afterwards confirmed by total syntheses of both enantiomers of seimatopolide A using chiral-pool-derived starting materials (Table 2, entries 4 and 5) [129]. Starting from l-(+)-tartrate the C 2 -symmetric diol (S,S)-111 was synthesized in four steps following a previously published route [139]. The C 2 -symmetry of this enantiomerically pure building block was exploited for differentiation of the C=C-double bonds: selective monoprotection was accomplished with the sterically demanding OH-protecting group triphenylmethyl (step 1). The nonyl substituent at C-9 was installed by cross-metathesis with 1-undecene (step 2), catalyzed by the phosphine-free Umicore-M51 catalyst B3, which is particularly well suited for CM reactions. Sharpless epoxidation of the newly generated C=C double-bond (step 3), followed by regioselective reductive epoxide cleavage with sodium bis(2-methoxyethoxy)aluminum hydride (Red-Al®) and acid-catalyzed cleavage of the triphenylmethyl-protecting group (step 4) established stereocenter C-9. Finally, the C-6–C-7-diol was regioselectively protected as an acetonide. In the next step, the C-5–C-9-fragment 122 was coupled with carboxylic acid (S)-118, the C-1–C-4 fragment, using Shiina’s conditions (cf. Scheme 6). RCM of diene 123 proceeded with very high (E)-selectivity. Simultaneous deprotection of the C-6–C-7-acetonide and the C-3-TBS-ether was accomplished with trifluoroacetic acid. All analytical data obtained for the product ent-114 matched those reported for natural seimatopolide A, including the sign of specific rotation. The non-natural enantiomer of seimatopolide A, ent-seimatopolide A (114), was synthesized through the

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Table 3 Summary of total syntheses of seimatopolide B Entry

Target structure

Source of chirality (stereocenter)

Specific rotation/°cm2 (10 g)−1

Conclusion for structure assignment

1

115

Three Co-salen catalyzed hydrolytic epoxide cleavage reactions (C-3, C-6, C-9)

−212.6, CH3 OH

Fully confirmed [141]

2

115

Two Co-salen +16.6, CH3 OH catalyzed hydrolytic epoxide cleavage reactions (C-3, C-6); l-proline-catalyzed amino-oxylation (C-9)

3

ent-115

Two Co-salen −13.4, CH3 OH catalyzed hydrolytic epoxide cleavage reactions (C-3, C-6); d-proline-catalyzed amino-oxylation (C-9)

4

ent-115

Asymmetric allylboration (C-9); oxazoborolidine cat. ketone reduction (C-6); lipase-Ru-catalyzed dynamic resolution (C-3)

−13.4, CH3 OH

Ref.

Absolute configuration revised to (3S,6R,9R)

[142]

Revised (3S,6R,9R) configuration confirmed

[132]

same sequence of steps from (R,R)-111 (accessible from d-mannitol, cf. Scheme 22) and (R)-118 (Scheme 24) [129]. Two further syntheses of levorotatory seimatopolide A (ent-114) (Table 2, entries 6 and 8) [130, 132] and one synthesis of dextrorotatory ent-seimatopolide A (114) (Table 2, entry 7) [131] corroborated the revision of the absolute configuration of seimatopolide A from (3R,6R,7R,9S) as originally proposed by Hiep et al. [125] to (3S,6S,7S,9R). Parallel to the disclosure of the first total syntheses of seimatopolide A, a correction of the original isolation paper was published that corrected the absolute configuration of seimatopolide A to (3S,6S,7S,9R) and its specific rotation to –20.8°cm2 (10 g)−1 . The absolute configuration of seimatopolide B was revised to (3S,6R,9R) and the specific rotation to –12.6°cm2 (10 g)−1 [140]. Seimatopolide B (115) has attracted less attention from the synthetic community but was still the target of three total syntheses (Table 3). The first synthesis confirmed the originally assigned (3R,6S,9S)-configuration of naturally occurring (–)-seimatopolide (Table 3, entry 1) [141]. However, the second total synthesis led to the conclusion that (3R,6S,9S)-configured 115 is actually the non-natural enantiomer, (+)-seimatopolide B. Therefore, the structure of seimatopolide B was corrected to

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Scheme 24 Enantioselective total synthesis of seimatopolide A (ent-114)

ent-115 with a (3S,6R,9R)-configuration (Table 3, entries 2 and 3) [142], which was confirmed by a third synthesis (Table 3, entry 4) [132].

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3.4 Pheromones from Madagascan Mantellid Frogs Although frogs primarily communicate through acoustic signals, non-volatile (e.g. water-soluble proteins) and more recently volatile pheromones have been shown to play an important role for distinguishing conspecies from closely related species. Over the past few years macrolactones have been identified as a particularly important group of semiochemicals in frogs. They are derived biosynthetically from fatty acids that are oxidized at the ω-terminus. Cyclization increases the lipophilicity and hence the volatility [143]. Male frogs of the family Mantillidae secrete a species-specific cocktail of volatile organic compounds from glands at the inner side of their shanks (Plate 3). Separating and identifying the individual components of these mixtures of semiochemicals is hampered by the extremely small amounts in which they are present [144]. In practice, isolation of the individual compounds on a scale that would allow NMR spectroscopic analysis is normally not possible. Gas chromatography-MS has therefore evolved as the method of choice to study the composition of cocktails of volatile semiochemicals, not only from frogs but also from insects. A plethora of mass spectra has been stored in databases that can be searched for matching or

Plate 3 Male frogs of the family Mantillidae: Mantidactylus multiplicatus (left) and Gephyromantis decaryi (right), and a view at the gland of G. decaryi (bottom right). Reproduced with permission from Poth D, Wollenberg KC, Vences M, Schulz S [144] Copyright Wiley-VCH GmbH, 2012

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similar spectra. The detailed analysis of mass spectra of diverse macrolactones from natural sources by Schulz et al. has helped to elucidate characteristic fragmentation mechanisms and deduce rules that allow a tentative assignment of the ring size and to some extent the position of endocyclic double bonds and the location of substituents [145]. There are, however, some limitations: stereoisomers normally do not have distinctive fragmentation patterns and the elucidation of substitution patterns can also remain ambiguous after analysis of the mass spectra. In these cases stereoselective synthesis can provide valuable assistance by supplying reference compounds with varying absolute and relative configurations that are used for comparison in GC-MS experiments and GC on chiral stationary phase for assigning absolute configurations. As an example, the synthesis-assisted identification of the volatile constituents secreted by the femoral glands of the Madagascan frog Mantidactylus femoralis (Plate 4) is discussed in the following paragraphs [146]. Analysis of the gland extracts by GC-MS revealed the presence of two major components that were identified as ten-membered lactones, based on their mass spectra. One of these is phoracantholide I (124), a known decanolide that was identified by comparison with published mass spectral data [144, 147]. (R)Phoracantholide I ((R)-124) is a defensive semiochemical produced by the beetle Phoracantha synonyma [147]. Both enantiomers had been synthesized previously from (R)- and (S)-propylene oxide (67) [144], respectively, and were available for comparison using GC on chiral stationary phase. It turned out that Mantidactylus femoralis produces exclusively (S)-phoracantholide I ((S)-124). For the second, hitherto unknown major constituent the structure of a dimethylated ten-membered lactone

Plate 4 Mantidactylus femoralis. Reproduced with permission from Poth D, Peram PS, Vences M, and Schulz S [146] Copyright American Chemical Society, 2013

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Chart 4 (S)-Phoracantholide I ((S)-124) and possible structures of the second constituent of the gland extracts from Mantidactylus femoralis

was proposed based on its mass spectrum. Out of all constitutional isomers plausible, biosynthesis pathways exist for the four isomers 125–128, which were then synthesized as mixtures of stereoisomers using RCM and hydrogenation (Chart 4). Comparison with the GC-MS trace of the gland extract excluded structures 125, 127 and 128, and pointed to 4-methyl-9-decanolide (126) as the correct constitution of the second major gland extract component. In the next step all four stereoisomers of 126 were synthesized selectively and submitted to GC-analysis on a chiral stationary phase. This revealed that the structure of the novel natural product, for which the name mantidactolide A was proposed, is (4R,9S)-126 (Scheme 25).

Scheme 25 Stereoselective synthesis of mantidactolide A ((4R,9S)-126) as a reference sample

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For the synthesis of mantidactolide A ((4R,9S)-126) (+)-β-citronellene ((S)-129) and (S)-propylene oxide ((S)-67) were used as enantiomerically pure starting materials. (+)-β-Citronellene ((S)-129) was converted into the carboxylic acid (S)-130 in three steps and the alcohol 131 was obtained by Cu-mediated epoxide opening of (S)-67. Steglich esterification gave diene 132, which underwent RCM in the presence of second-generation Grubbs’ catalyst to furnish selectively the (Z)-configured product. Hexafluorobenzene was added in this step to enhance the activity of the metathesis catalyst [148]. Hydrogenation of the double-bond gave mantidactolide A ((4R,9S)-126). Interestingly, specimens of M. femoralis collected at other locations in Madagascar did not contain (S)-124 and (4R,9S)-126 as volatile pheromones, but a cocktail of branched alkanols and 8-methyl-9-decanolide (128), for which the name mantidactolide B was proposed and a relative (8R*,9R*)-configuration was assigned tentatively. The identification of mantidactolide B was accomplished through a strategy similar to that outlined above for mantidactolide A [146]. The femoral gland extract of two specimens of the Madagascan frog Gephyromantis luteus was investigated recently [149]. GC-MS showed that just two compounds were present, and due to the relatively large amount of material (ca. 500 μg per frog) NMR spectroscopic analysis of the mixture was possible. One compound was identified as the known sesquiterpene frogolide (133) [150]. For the other compound a ten-membered lactone structure 134 with a 9-ethyl substituent and two methyl groups at C-4 and C-8 were tentatively assigned, based on the mass spectrometric fragmentation pattern and H,H-COSY and HMBC correlations observed in the NMR-spectra obtained from the mixture (Chart 5). To verify the constitutional structure 134 proposed for the novel natural product and to elucidate the relative and absolute configuration, a stereoselective synthesis was undertaken [149]. Considering the structure of the semiochemicals previously

Chart 5 Components of the femoral gland extract from Gephyromantis luteus

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isolated from Madagascan frogs an all-cis-configuration of the three substituents appeared to be the most likely and therefore (4R,8S,9S)-134 and its enantiomer (4S,8R,9R)-134, were synthesized first. For the synthesis of (4R,8S,9S)-134 the carboxylic acid 135 (obtained in four steps from (S)-citronellal) and the alcohol 137 (obtained via asymmetric crotylboronation of propanal (136) [151]) were coupled under Steglich conditions to furnish the RCM precursor 138. RCM of 138 was accomplished using the second-generation Grubbs’ catalyst B1 and tetrafluoro-1,4benzoquinone as an isomerization inhibitor. Hydrogenation of 139 required Rh/C as a catalyst instead of the more common catalyst Pd/C to avoid epimerization and obtain (4R,8S,9S)-134 as a single stereoisomer. The enantiomer (4S,8R,9R)-134 was synthesized along the same sequence of steps, using (R)-(+)-citronellal as a starting material and (–)-(Ipc)2 BOCH3 as a chiral reagent for the crotylboration step (Scheme 26). For both enantiomers of 134, gas chromatographic analyses on a chiral stationary phase were performed. Comparison with the extract from Gephyromantis luteus revealed that the natural product is levorotatory (4R,8S,9S)-134, for which the name luteolide has been proposed by the authors [149]. It turned out that a previously discovered, but up to that point, unidentified volatile component from the femoral gland extracts of the related species Gephyromantis moseri [152] is identical to luteolide ((4R,8S,9S)-134) and that this compound also occurs in the frog Mantidactylus betsileanus.

Scheme 26 Stereoselective synthesis of luteolide ((4R,8S,9S)-134)

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3.5 A Potential Pheromone from the Queen of the Termite Silvestritermes minutus Approximately 3000 species of termites are known to date and new species are discovered each year. Termite colonies can consist of up to seven million individuals, but they have normally only one pair of fertile members, named the queen and king. Communication within a colony is of utmost importance and is accomplished through semiochemicals. The cuticular hydrocarbons (CHCs) are a well-investigated group of pheromones, but low-molecular weight alcohols also do play a significant role. In a comprehensive review article on the chemistry of secondary termite metabolites recently published in this series [153], macrolactones (macrolides) are primarily discussed as defensive chemicals. A recent investigation into the queenspecific volatiles [154] of the termite Silvestritermes minutus (Plates 5 and 6) revealed that a ten-membered lactone appears to play a significant role in this species [155]. Queen pheromones suppress, for example, the fertility of other colony members and cause tending behavior toward the queen [153]. Investigation of the headspace of a sample of living termites and hexane extracts of tissues from dissected queens of S. minutus by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GCxGC-TOFMS) revealed the presence of a low-molecular weight constituent for which no matching mass spectra could be found in the databases consulted [155]. High-resolution mass spectral (HRMS) data, fragmentation patterns that were characteristic for macrolides [145], and microhydrogenation experiments and GC-Fourier-transform IR-spectroscopy (GC-FTIR) pointed to the presence of an unsaturated ten-membered lactone with a pentyl-substituent at C-9 and a (Z)-configured C=C double-bond. The location of the double-bond could be further narrowed down by methylthiolation and MS analysis of the addition product; the observation of some characteristic fragments suggested Plate 5 Queens with workers and soldiers of S. minutus. Photograph courtesy of Robert Hanus. Copyright Robert Hanus. Reproduced with permission

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Plate 6 King (center) and queens of S. minutus. Photograph courtesy of Robert Hanus. Copyright Robert Hanus. Reproduced with permission

that the double-bond is located between C-5 and C-6 [155]. To verify the hypothesized constitutional structure of 140 and to elucidate the absolute configuration, all four stereoisomers (5E,9R)-140, (5Z,9R)-140, (5E,9S)-140 and (5Z,9S)-140 were synthesized from a common enantiomerically pure starting material, (S)-glycidyl tosylate ((S)-10), in a stereodivergent approach (Scheme 27). For the synthesis of (9R)-configured stereoisomers, (S)-glycidyl tosylate ((S)-10) was first reacted with allylmagnesium bromide in a Cu-catalyzed epoxide opening reaction. The product, alcohol (S)-141a, was coupled with butylmagnesium bromide in the presence of a catalytic amount of CuCl to give (R)-142. The enantiomer, (S)-142, was obtained from (S)-glycidyl tosylate ((S)-10) by inverting the order of the first two steps. Both enantiomers of alcohol 142 were coupled with hex-5-enoic acid (143) under Steglich conditions to yield dienes (R)-144 and (S)-144, respectively. Ring-closing metathesis was accomplished for both precursors using the phosphine-free metathesis catalyst B4 and led to 4:1 mixtures of (E)- and (Z)-isomers of 140. The isomers were separated by repeated chromatography [155].

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Scheme 27 Synthesis of the queen-specific volatile from Silvestritermes minutus ((5Z,9S)-140)

Gas chromatographic analysis on a chiral stationary phase and comparison of the mass spectrometric fragmentation patterns under different ionization conditions with those observed for the natural product showed that its structure is (5Z,9S)-140. Exposure of this compound to the termites did not trigger any behavioral changes in first experiments, but the synthetic accessibility of (5Z,9S)-140 did not only help in confirming and completing the structural assignment, but will also pave the way for

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future investigations into the biological role of this queen-specific volatile from S. minutus.

4 Conclusions Elucidating the structure of natural products is often a laborious and time-consuming endeavor due to the minute amounts isolated from the natural source, limited stability of the metabolites or difficult purification processes that can make it almost impossible to obtain reasonably pure samples. But even in those cases where a natural product has been isolated in pure form, NMR spectroscopic analysis might remain ambiguous, for example, due to high flexibility of the molecule which makes it impossible to draw reliable conclusions from NOE experiments or from an analysis of the coupling constants. In such cases, as the examples highlighted in this chapter hopefully illustrate, modern organic synthesis can serve as a valuable tool in confirming or revising a structural proposal. If the amounts of material available from the biological source are so small that NMR analysis is impossible and the proposed structure is solely based on mass-spectrometric investigations, stereoselective synthesis even becomes an indispensable tool for providing single isomers as reference compounds. This chapter is not intended to be a comprehensive survey, but provides the reader with an illustrative overview. We have highlighted the role of organic synthesis for the structure elucidation of natural products for the example of ten-membered lactones, but it can be safely assumed that organic synthesis plays an important role for the structure elucidation of many other classes of natural products. A surprising result from this compilation of examples is that in a notable number of cases the synthesisbased re-examination of a structural assignment is itself erroneous and confirms a wrong structure proposal. For this reason, it is justified to embark on the total synthesis of a natural product even if one or more previous syntheses exist. Acknowledgments The contributions of our group to the field have been generously supported by the Deutsche Forschungsgemeinschaft (DFG grant Schm1095/6). The author thanks Dr. Robert Hanus, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, for helpful discussions and for providing photographs of S. minutus, Prof. Stefan Schulz and Prof. Miguel Vences, TU Braunschweig, for providing high-resolution photographs of mantellid frogs and the Pflanzenschutzamt Berlin for providing high-resolution photographs of Diplodia tip blight infected pine trees.

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115. Dey S, Sudalai A (2015) A concise enantioselective synthesis of marine macrolide stagonolide E via organocatalysis. Tetrahedron: Asymmetry 26:344 116. Liu Z, Xu C, del Pozo J, Torker S, Hoveyda AH (2019) Ru-based catechothiolate complexes bearing an unsaturated NHC ligand: effective cross-metathesis catalysts for synthesis of (Z)α,β-unsaturated esters, carboxylic acids, and primary, secondary, and Weinreb amides. J Am Chem Soc 141:7137 117. Perepogu AK, Raman D, Murty USN, Rao VJ (2009) Concise synthesis of stagonolide F by ring closing metathesis approach and its biological evaluation. Bioorg Chem 37:46 118. Chinnababu B, Reddy SP, Babu KS, Venkateswarlu Y (2014) Asymmetric total synthesis of stagonolide F. Synth Commun 44:2886 119. Shelke AM, Suryavanshi G (2015) An efficient organocatalytic route for asymmetric total synthesis of stagonolide F. Tetrahedron Lett 56:6207 120. Srihari P, Kumaraswamy B, Bhunia DC, Yadav JS (2010) First stereoselective total synthesis of stagonolide G. Tetrahedron Lett 51:2903 121. Ramesh D, Rajaram S, Prabhakar P, Ramulu U, Kumar Reddy D, Venkateswarlu Y (2011) Asymmetric total synthesis of stagonolide G. Helv Chim Acta 94:1226 122. Pavan Kumar CNSS, Ravinder M, Naveen Kumar S, Jayathirtha Rao V (2011) Stereoselective synthesis of stagonolide G from d-mannitol. Synthesis:451 123. Rajendra Prasad K, Venkanna A, Babu KS, Prasad AR, Rao JM (2014) Stereoselective synthesis of revised structure of stagonolide G. Tetrahedron Lett 55:616 124. Maram L, Parigi RR, Das B (2016) Chiral approach to total synthesis of phytotoxic and related nonenolides: (Z)-isomer of (6S,7R,9R)-6,7-dihydroxy-9-propylnon-4-eno-9-lactone, herbarumin-III and their C-9 epimers. Tetrahedron 72:7135 125. Hiep NT, Choi Y-h, Kim N, Hong SS, Hong S-B, Hwang BY, Lee H-J, Lee S-J, Jang DS, Lee D (2012) Polyhydroxylated macrolides from Seimatosporium discosioides and their effects on the activation of peroxisome proliferator-activated receptor gamma. J Nat Prod 75:784 126. Sabitha G, Yagundar Reddy A, Yadav JS (2012) First stereoselective total synthesis of seimatopolide A. Tetrahedron Lett 53:5624 127. Reddy BP, Pandurangam T, Yadav JS, Reddy BVS (2012) A novel strategy for the chiral 2,4,5-triol moiety and its application to the synthesis of seimatopolide A and (2S,3R,5S)-(–)2,3-dihydroxytetradecan-5-olide. Tetrahedron Lett 53:5749 128. Reddy CR, Rao NN, Reddy MD (2012) Total synthesis of (+)-seimatopolide A. Eur J Org Chem 4910 129. Schmidt B, Kunz O, Petersen MH (2012) Total syntheses of naturally occurring seimatopolide A and its enantiomer from chiral pool starting materials using a bidirectional strategy. J Org Chem 77:10897 130. Kavitha N, Kumar VP, Reddy CS, Chandrasekhar S (2013) Total synthesis of (–)seimatopolide A. Tetrahedron: Asymmetry 24:1576 131. Prasad KR, Revu O (2014) Total synthesis of (+)-seimatopolide A. J Org Chem 79:1461 132. Rej RK, Pal P, Nanda S (2014) Asymmetric synthesis of naturally occurring (−)seimatopolides A and B. Tetrahedron 70:4457 133. Keck GA, Tarbet KH, Geraci LS (1993) Catalytic asymmetric allylation of aldehydes. J Am Chem Soc 115:8467 134. Abdul Fattah T, Saeed A (2017) Applications of Keck allylation in the synthesis of natural products. New J Chem 41:14804 135. Kolb HC, VanNieuwenhze MS, Sharpless KB (1994) Catalytic asymmetric dihydroxylation. Chem Rev 94:2483 136. Han X, Peh G, Floreancig PE (2013) Prins-type cyclization reactions in natural product synthesis. Eur J Org Chem 1193 137. Brown SP, Brochu MP, Sinz CJ, MacMillan DWC (2003) The direct and enantioselective organocatalytic α-oxidation of aldehydes. J Am Chem Soc 125:10808 138. Frick JA, Klassen JB, Bathe A, Abramson JM, Rapoport H (1992) An efficient synthesis of enantiomerically pure (R)-(2-benzyloxyethyl)oxirane from (S)-aspartic acid. Synthesis 1992:621

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Bernd Schmidt studied chemistry at the RWTH Aachen and received his diploma degree in 1991 and his Dr. rer. nat. degree from the same institution in 1994 with a dissertation on the field of organoboron chemistry. From 1994–1995 he joined the group of Prof. Philip Kocienski, at that time at the University of Southampton, England, as a postdoctoral fellow, where he came into contact with the vast field of natural product synthesis. After returning to Germany, he started his independent academic career at the Technical University of Dortmund, where he received his habilitation in 2001 and was appointed as Privatdozent. His early career stages were supported by fellowships from the Deutsche Forschungsgemeinschaft (DFG) and the Fonds der Chemischen Industrie (FCI). In 2006, Prof. Schmidt accepted a call to a professorship in organic chemistry at the University of Potsdam, Germany, where he has been ever since. His research interests are centered around organic synthesis and range from the development of synthesis methodology (with a focus on transition metal-catalyzed reactions, in particular Pdcatalyzed couplings and olefin metathesis) to target molecule synthesis. Apart from research, Bernd Schmidt is dedicated to teaching organic chemistry on all academic levels for both chemistry minors and majors. He has been a member of the Board of the Faculty of Science for more than ten years, a member of the Academic Senate of the University of Potsdam for four years, and Dean of Studies of the Faculty of Science for more than six years.

From Plant to Patient: Thapsigargin, a Tool for Understanding Natural Product Chemistry, Total Syntheses, Biosynthesis, Taxonomy, ATPases, Cell Death, and Drug Development Søren Brøgger Christensen, Henrik Toft Simonsen, Nikolai Engedal, Poul Nissen, Jesper Vuust Møller, Samuel R. Denmeade, and John T. Isaacs

S. B. Christensen (B) Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark e-mail: [email protected] H. T. Simonsen Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Bld 223, 2800 Kgs. Lyngby, Denmark e-mail: [email protected] N. Engedal Department of Tumor Biology, Institute for Cancer Research, University Hospital, Montebello, 0379 Oslo, Norway e-mail: [email protected] P. Nissen Department of Molecular Biology and Genetics, Danish Research Institute of Translational Neuroscience – DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark e-mail: [email protected] J. V. Møller Department of Biomedicine, Aarhus University, Ole Worms Allé 3, Bld 1182, Room 114, 8000 Aarhus C, Denmark e-mail: [email protected] S. R. Denmeade · J. T. Isaacs Department of Oncology, Prostate Cancer Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Maryland, The Johns Hopkins University School of Medicine, Baltimore, The Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, MD 21231, USA e-mail: [email protected] J. T. Isaacs e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. D. Kinghorn, H. Falk, S. Gibbons, Y. Asakawa, J.-K. Liu, V. M. Dirsch (eds.), Progress in the Chemistry of Organic Natural Products, Vol. 115, https://doi.org/10.1007/978-3-030-64853-4_2

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Contents 1 2 3 4 5

Introduction: Silphium and Thapsia garganica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Naturally Occurring Thapsigargins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Taxonomy of the Genus Thapsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochemistry of Thapsia garganica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Transtaganolides and Basiliolides from Species Belonging to Thapsia . . . . . . . . 5.2 The Essential Oil of Thapsia garganica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Tethered Lipids from Thapsia garganica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Chemistry and Chemical Synthesis of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Chemistry at O-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Chemistry at Lactone C-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Chemistry at O-2 and O-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Chemistry at O-7 and O-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Sustainable Supply of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Thapsigargin from Plants and Plant Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Synthesis of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Partial Synthesis of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Pharmacology of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Effects on Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Effects on Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Effect on Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Molecular Pharmacology of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Calcium Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Thapsigargin Induces Cell death, ER Stress and Growth Arrest, and Inhibits Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 The Pharmacophore of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Substituent at O-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Substituent at O-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Substitution at C-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Substitution at O-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 The Carbonyl Group at C-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 The Two Hydroxy Groups HO-7 and HO-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Thapsigargin as a Probe for Purification of P2A Ca2+ ATPases . . . . . . . . . . . . . . . . . . . . . 12 Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1 Targeting of Chemotherapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Targeting of Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Sarco-Endoplasmic Reticulum ATPase (SERCA) . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Thapsigargin and Other P-type Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Thapsigargin Prodrug Design, Preparation, and Preclinical Evaluation . . . . . . . . 12.6 Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61 64 65 66 67 67 68 70 70 70 72 74 75 77 77 78 79 80 80 80 81 81 81 83 84 86 86 86 87 87 87 88 89 90 92 94 95 95 98 100 101

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1 Introduction: Silphium and Thapsia garganica Silphium (Latin) or σ´ιλϕιoν (silphion, Greek) was a resin exported from the ancient Greek settlement Cyrene located on the northern coast of Libya [1, 2]. The product was sold for its weight in silver, with one source even referring to its weight in gold, making Cyrene a prosperous society from about 600 BC to about 100 BC. Several coins depict the plant from which silphium was prepared. These pictures clearly reveal that the parent plant must have belonged to the Apiaceae family. Overharvesting of the population extinguished the plant and in the third century AD the export ceased [1, 2]. Several theories on the identity of the producing plant have been presented, and, in particular, species belonging to the genus Ferula have gained acceptance in this regard. The ruins of Cyrene were visited by the Swedish botanist Söderling-Brydolf in the 1960’s and a large population of Thapsia garganica L. (Apiaceae) (Fig. 1) made her suggest that this was the parent plant [3]. From an ancient tradition, the meat of sheep fed with silphion should experience a particular attractive taste [4]. However, the toxicity of T. garganica in particular against mammals undermines the theory that sheep could have been fed with the plant [5]. The generic name of Thapsia comes from the Ancient Greek θαψ´ια (thapsia), as the Greeks believed the genus was discovered on the island of Thapsos (Sicily, Italy) [6]. The English common name for Thapsia species became “deadly carrots”, from

Fig. 1 Thapsia garganica with ripened fruits (Ibiza, Spain)

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14

O R3

2

O

1 5

3

9

10

8 6 7

4 11

O

15

12

O

R2 O OH OH 13

1

1 R = CH3(CH2)6CO, R2 = CH3(CH2)2CO, R3 = CH3CO (thapsigargin) [15, 17, 23, 24] 2 R1 = CH3(CH2)4CO, R2 = CH3(CH2)2CO, R3 = CH3CO (thapsigargicin) [15, 17, 23, 24] R2 = 2-MeBut, R3 = CH3CO (thapsitranstagin) [27–30] 3 R1 = iVal, 4 R1 = Ang, R2 = Sen, R3 = CH3CO (thapsivillosin A) [28, 29] R2 = 2-MeBut, R3 = CH3CO (thapsivillosin B) [27–29] 5 R1 = Ang, R3 = CH3CO (thapsivillosin C) [28, 30] 6 R1 = CH3(CH2)6CO, R2 = 2-MeBut, R2 = Sen, R3 = CH3CO (thapsivillosin D) [28, 30] 7 R1 = 6-MeOct, 8 R1 = 6-MeOct, R2 = 2-MeBut, R3 = CH3CO (thapsivillosin E) [28] R2 = 2-MeBut, R3 = CH3CO (thapsivillosin G) [28] 9 R1 = 6-MeHep, R2 = Sen or Ang, R3 = C H3CO (thapsivillosin H) [28] 10 R1 = Ang or Sen, R2 = C H3(CH2)2CO, R3 = CH3CO (thapsivillosin I) [27, 28] 11 R1 = Ang, 12 R1 = iVal, R2 = C H3(CH2)2CO, R3 = CH3CO (thapsivillosin J) [27, 28] R2 = CH3(CH2)2CO, R3 = CH3CO (thapsivillosin K) [27, 28] 13 R1 = Sen, R2 = 2-MeBut, R3 = CH3CO (acetyltrilobolide) [25] 14 R1 = CH3CO, R2 = 2-MeBut, R3 = H (2-hydroxy-10- O-deacetyl trilobilide) [25] 15 R1 = H, O O

O

O

O

O

O O

O O 16 O iVal =

O O

O 2-MeBut =

2

Sen =

O Ang =

O 6-MeOct =

6-MeHep =

6

6

O

Fig. 2 Hexaoxygenated guaianolides containing the thapsigargin skeleton isolated from plants belonging to the genera Thapsia and Laser. The epoxide 16 is a synthon prepared for structure elucidation (Section 6.4) [15, 17, 23–25, 27–30]

reports of the plants being poisonous [7]. The root bark and the resin of Thapsia have been used in Arabic and European medicine, with Hippocrates first describing the skin-irritant effects of Thapsia around 400 BC. Since then, the roots, in particular, and the resin have been used in traditional medicine. Theophrastus (circa 327–287 BCE) described the properties of the roots of Thapsia as being able to “purge both upwards and downwards” if ingested, and that, while domestic cattle in Greece did

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not touch it, imported cattle would feed on it and perish of diarrhea [8, 9]. The resin was prepared by extracting the roots with ethanol followed by concentration of the solution. The bark was removed from roots collected from December to March and used in traditional medicine for the treatment of leanness, chronic diseases of the lungs, and sterility. By carefully avoiding contact with the other parts of the roots than the bark the Arabs were able to avoid skin eruptions whereas the Kabyles were covered in suppurating sores [10, 11]. The resin was used extensively as a counterirritant [10, 12] and was described in several pharmacopoeias including the French Pharmacopoeia from 1937. Examination of the descriptions of the effects of plasters containing the resin, however, indicates that the plasters must have been very unpleasant remedies [10, 12, 13]. The biological activities of botanicals from T. garganica provoked scientific interest in phytochemicals from this species. Intensive research inspired by the late Professor Finn Sandberg (University of Uppsala, Sweden), but initiated at the Royal Danish School of Pharmacy (Copenhagen) in 1973 revealed that the species was a source of unique compounds with outstanding chemical and pharmacological properties. The major active principle was isolated in 1978 [14], and elucidated structurally as the hexaoxygenated guaianolide 1 [15–17], and named thapsigargin (Fig. 2). In addition to 1, a number of other polyoxygenated guaianolides were isolated from extracts of the roots. Since harvesting of the roots meant killing of the plant it was preferred in our later studies, which involved extraction of gram amounts of 1, to isolate these compound from the fruits of the plant (Fig. 1) [18]. Collection of the fruits is a non-invasive procedure and much easier than collecting the roots. Stress induced by damaging plants has also highlighted the apparent ecological function of thapsigargin (1) and its derivatives. Thapsigargins are thought to be a group of defense compounds against herbivory and are generally toxic to all animals as they inhibit SERCA in both vertebrates and invertebrates [19]. This activity is responsible for the potent toxicity of these plants. The biosynthesis and production of 1 and related compounds is induced upon damaging the plant. Indeed, there is a significant change in a plant’s chemical profile after plant damage, and models suggest that thapsigargins represent both a constitutive and induced intraspecific defense. Thapsigargins are clearly the dominant defense compounds in these plants, and they seem to be produced through a common biosynthetic pathway with little diversity [20, 21]. The potent skin irritation provoked by purified thapsigargin (1) led to investigations of its effects on isolated cells (Section 8.3). Thapsigargin (1) was found to induce release of histamine from mast cells and mediators from other cells belonging to the immune system if the cells were incubated in Ca2+ -containing media. Persuasion of Dr. Ole Thastrup to assay if these effects could be related to an influence of the Ca2+ homeostasis led to the discovery that 1 inhibits SERCA pumps with a subnanomolar affinity (Section 12.3). Today, thapsigargin (1) has become an important tool for investigating Ca2+ homeostasis, ER stress responses, and cell death.

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2 Naturally Occurring Thapsigargins Phytochemical studies on Thapsia garganica were initiated just after HPLC and highfield NMR spectroscopy (270 MHz) had become available in Copenhagen in the early 1970s. This enabled the separation of the very closely related thapsigargins from this species and recording of their high-field NMR spectra. The structure elucidation of thapsigargin was facilitated by comparison of its spectroscopic data with those of trilobolide (compound 17) [22], enabling establishment of the molecular constitution (Figs. 2 and 3) [23]. Even though thapsigargin (1) since its first isolation in 1978 [14] has been isolated several times and in gram amounts, the compound has never been crystallized, preventing X-ray analysis to establish the relative and absolute configurations. Instead, the crystalline epoxide 16 (Fig. 2) was prepared and an Xray structure revealed the relative configuration, albeit not at the two tertiary alcohol carbons, C-7 and C-11 [15]. The absolute configuration was established by analyzing exciton coupling between the allylic ester and the α,β-unsaturated angelic acid [17]. The absolute configuration at C-7 was the same as that of C-7 in the closely related trilobolide (17) (Fig. 3) which is crystalline [24]. The diversity of the genus Thapsia inspired a phytochemical investigation of the different species leading to isolation of 14 hexaoxygenated guaianolides containing the skeleton of thapsigargin and two additional pentaoxygenated guaianolides possessing the same skeleton as trilobolide (for a review see [19]). Later studies have revealed that hexaoxygenated guaianolides (Fig. 2, compounds 14 and 15) also are present in the genus Laser (Figs. 2 and 3) [25]. A patent claims that Laserpitium archangelica Wulf. in Jacq. and Laserpitium siler L. in addition to Laser trilobum Borkh., contain trilobolide (17) in substantial amounts [26]. In conclusion, penta- and hexaoxygenated guaianolides are only found in a very few and closely-related plants belonging to the genera Thapsia, Laser, and possibly Laserpitium. O 14

O

2

O O

9 1 5

3

10 8 6 7

4 15

O

11

12

O

R O OH OH 13

17 R = 2-MeBut (trilobolide) [27, 29, 31] 18 R = CH3(CH2)2CO (nortrilobolide) [27, 32] 19 R = Sen (thapsivillosin F) [29, 33]

Fig. 3 Pentaoxygenated guaianolides containing the skeleton of trilobolide isolated from plants belonging to the genera Thapsia and Laser [27, 29, 31–33]

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3 Biosynthesis of Thapsigargin If thapsigargin (1) should be produced by biotechnological techniques, an absolute requirement is a knowledge of the enzymes involved in the biosynthesis. All guaianolides, a subgroup of γ-sesquiterpene lactones, are formed from farnesyl pyrophosphate (Scheme 1) [34, 35]. Farnesyl pyrophosphate is produced from isopentenyl pyrophosphate, which reacts with two molecules of dimethylallyl pyrophosphate [34]. Facilitated by sesquiterpenoid synthase, TgPS2 farnesyl pyrophosphate is converted into epikunzeaol [36]. This enzyme has also been described by other Thapsia species, and the formation of epikunzeaol is believed to be the first step for the complex sesquiterpene lactones in the genus Thapsia [37]. After oxidation of one of the methyl groups into a carboxylic acid, the γ-lactone is formed spontaneously. The subsequent cyclization yields the guaianolide skeleton, but the sequence of oxidations to give the hexaoxygenated guaianolide skeleton and the esterification steps currently are not fully elucidated (Scheme 1) [38]. An interesting observation is that in all guaianolides, H-7 is always in the αposition. In contrast, H-1 is in general in the α-position if the compound is isolated from a plant belonging to the Asteraceae, whereas H-1 in guaianolides isolated from plants belonging to the Apiaceae, in general, is β-disposed [35, 39]. TgFPPS OPP

OPP

OPP

+ 2x

DMAP

IPP

FPP

spontanous

TgTPS2

CYP76AE2

HO HOOC

O O

HO epikunzeaol

O

O O

O

O O

O 1

O O OH OH

O

Scheme 1 Biosynthesis of thapsigargin (1). IPP: isopentenyl pyrophosphate, DMAP: dimethylallyl pyrophosphate, FPP: farnesyl pyrophosphate, TgFPPS: T. garganica farnesyl pyrophosphate synthase, thapsigargin TPS2: T. garganica terpene synthase 2, CYP76AE2: T. garganica epikunzeaol 11 hydroxylase

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4 Taxonomy of the Genus Thapsia The genus Thapsia belongs to the family Apiaceae. According to the “Flora Europaea 1968” [40], the genus encompasses three species: T. garganica L., T. maxima Mill. and T. villosa L. In August 2019, these three species were the only accepted names according to “The Plants List” (http://www.theplantlist.org/), but the taxonomy of the genus Thapsia is still unresolved. Phytochemical studies and the morphological appearance of the different varieties of the Thapsia species severely question the present state of knowledge. In particular, the two different species of T. maxima and T. villosa have very different morphologies, with some being about 0.5 m and others being 1.5 m tall herbs [41]. Chemotaxonomic investigations revealed a surprising regularity in that plants producing polyoxygenated guaianolides like thapsigargin (1) (Fig. 2) and trilobolide (17) (Fig. 3) do not produce compounds possessing the unique thapsane skeleton (Fig. 4, 20–33), and vice versa [41, 42]. Apparently, this production 18 10

1 2 3 11

O

7

9 45

6 13

12

O R2

17

15 14

R1

O

16

8

19

O 21 R1 = H R2 = H 22 R1 = H R2 = Fer 1 23 R = SenO R2 = H

20

R1

SenO

R3

O R R2

O

O HO

24 R = H 25 R = Ac

OSen R2

O

26 R1 = H 27 R1 = H 28 R1 = H 29 R1 = H 30 R1 = SenO 31 R1 = AngO 32 R1 = TigO 33 R1 = H

R2 = H R2 = H R2 = H R2 = H R2 = H R2 = H R2 = H R2 = AngO

R3 = AngO R3 = SenO R3 = pCoumO R3 = Fer R3 = H R3 = H R3 = H R3 = H

O

O

O O

pCoum = HO O

OAng 34

Fer =

HO O

Fig. 4 Thapsanes isolated from species of T. maxima and T. villosa [43–46]

Tig =

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of either thapsanes or polyoxygenated guaianolides is dependent on the number of chromosomes [41]. A new taxonomic investigation based on sequences of the nuclear ribosomal internal transcribed spacer (nrITS) region led to a suggestion that the genus Thapsia should be divided into 14 species: T. asclepium, T. garganica, T. gummifera, T. gymnesica, T. laciniata, T. leucotricha, T. maxima, T. minor, T. scabra, T. smittii, T. tenuifolia, T. thapsioides, T. transtagana, and T. villosa [42]. This taxonomic revision of Thapsia is generally well accepted and supported. However, the full resolution of this genus and its sister genera is still ongoing.

5 Phytochemistry of Thapsia garganica 5.1 Transtaganolides and Basiliolides from Species Belonging to Thapsia Besides thapsigargin (1), several SERCA-inhibitory transtaganolides have been isolated from plants belonging to the genus Thapsia (Fig. 5, compounds 35a–35d, 36a, and 36b) [47, 48]. In the first report [47] compounds 35a and 35b isolated from T. transtagana were named transtaganolides C and D, and compounds 36a and 36b transtaganolides A and B. In a second report [48], the compounds 35a–35d as isolated from T. garganica were named basiliolides A1, A2, C, and D. The basiliolides were claimed to be SERCA inhibitors because of their ability to drain endoplasmic reticulum Ca2+ from sea urchin eggs [48] and from Jurkat cells [49]. These compounds, however, are, unlike thapsigargin (1) [50], unable to provoke apoptosis after 18 h incubation with Jurkat cells [48], and they have never been tested Fig. 5 Basiliolides 35a–35d and transtaganolides 36a and 36b

O O O

O O

8

R

35a R = CH3, C-8: α-CH3, β-vinyl (basiliolide A1) 35b R = CH3, C-8: α-vinyl, β-CH3 (basiliolide A2) 35c R = COOCH3, C-8: α-CH3, β-vinyl (basiliolide B) 35d R = CH2OOAc, C-8: α-CH3, β-vinyl (basiliolide C) O O O O

O O

36a C-8: α-CH3, β-vinyl (transtaganolide A) 36b C-8: β-CH3, α-vinyl (transtaganolide B)

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Scheme 2 Suggested biosynthesis of basilolides and transtaganolides

O O

OH

O

O

O

O O OH

O

O O O

37

O

O O OH

O

O

35

O

O

O

O

O

O O

O

O

O

38

O

OH

37

Scheme 3 Claisen rearrangement of geranyloxycoumarin 38 to give 37

on isolated SERCA. The unusual carbon skeleton of these compounds is suggested to originate from the deoxygenated geranyl derivative of coumarin 36 (Scheme 2) [48]. Even though 37 has not been found as a natural product to date, this compound might arise by a Claisen rearrangement of 38 (Scheme 3), which is the first compound that was isolated from T. garganica [51]. In the biomimetic synthesis performed on basiliolides (35), 38 was not used as a starting material, but related rearranged compounds were utilized for the construction of the carbon skeleton [52, 53].

5.2 The Essential Oil of Thapsia garganica The essential oils of the fruits of T. transtagana and T. garganica are blue [54, 55]. The appearance of this color is explained by the isolation of proazulenic slovanolides (Fig. 6) from the fruits of these plants. Two different azulenes may be formed depending on the structure of the starting material. The two 11-hydroxyslovanolides (39 and 40) afforded 1,4-dimethylazulene upon heating [56]. A possible mechanism of action for the formation of an azulene from the two 11-hydroxyslovanolides 39 and 40 is a retrograde Prins reaction [58] followed by three cis-eliminations of carboxylic acids [59] (Scheme 4). The 11-O-acylslovanolides 41–44 are converted into 8-acetyl-1,5dimethylazulene, as suggested in Scheme 5. This reaction path involves a

From Plant to Patient: Thapsigargin, a Tool for Understanding … Fig. 6 Slovanolides isolated from fruits of T. garganica [54, 57]

69 O 14

1

R O

9 2

3

O

1 5

O

8 6 7

4 15

R2

10

11

O 12

O

O

R3

13

39 R1 = 2-MeBut, R2 = CH3(CH2)2CO, R3 = H [54, 57] 40 R1 = iVal, R2 = 2-MeBut, R3 = H [54] R3 = CH3CO [54] 41 R1 = CH3CO, R2 = 2-MeBut, 1 2 3 R = CH3CO [54] 42 R = CH3CO, R = 2-Sen, R2 = 2-MeBut, R3 = C H3CO [54] 43 R1 = H, 44 R1 = CH3CO, R2 = 2-Sen, R3 = CH3CO [54] O

R1OH

O

1

R O

R1

O

H

R

R

O

O O

O

O H

O

OH

O

O

O

OH

O

O

45 O

O

O

OH

Scheme 4 Rearrangements of 11-hydroxyslovanolides to give 1,5-dimethylazulene (45) O O

R1 O

O

O

O

O

R1 O

R O

O

O

O

O

R

– CO

O

O

O

O

O

R1 O

R

O

HO

O

CO

O

O

O H O

O O

HO R1COOH

R1

R HO

O 46

O

O

O

R O

O

O

O HO

Scheme 5 Rearrangement of 11-O-acyl-slovanolide to give 8-acetyl-1,5-dimethylazulene (46)

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S. B. Christensen et al. O O

O HO O O

O OH O

O

O

O O HO

47

O 48

49

Scheme 6 Racemization of tethered lipids

decarbonylation of a 2-acyloxyester, a reaction that is very poorly described [60, 61].

5.3 Tethered Lipids from Thapsia garganica A tethered lipid was discovered when thapsigargin (1) was isolated in large amounts [62]. Purified thapsigargin has very often been found to be contaminated in this manner. The contaminating compound was determined as a cyclic ester of glycerol and hexadecanedioic acid. The poor optical rotation of the isolated lipid 47 indicated that the compound had partly had racemized by acyl migrations, as indicated in Scheme 6. The total stereospecific synthesis of 49 confirmed this hypothesis [62]. Except for the genus Thapsia, tethered lipids have only been found in archaebacteria, where they occur as ethers of glycerol [63].

6 Chemistry and Chemical Synthesis of Thapsigargin A major requirement for the success of thapsigargin (1) as a tool for understanding Ca2+ homeostasis and for drug development was knowledge of the chemistry of the molecule to enable structural modifications and the preparation of drug candidates. An early overview of this work has been given by Christensen et al. [19]. An updated review was published in 2015 [64].

6.1 Chemistry at O-8 A major breakthrough in the chemistry of thapsigargin was the discovery that the ester group at O-8 is much more sensitive to solvolysis than are the other ester groups. Thus, methanolysis takes place within minutes if the compound is dissolved in methanolic trimethylamine (Scheme 7). No solvolysis of the other ester groups is detectable [65]. If the 11-hydroxy group is masked, the solvolysis of the ester at O-8

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O

O O

O O

O

a b

O OH OH

O

O

O

O

O

O

O

O

OH OH OH

O

O

O 50

Scheme 7 Solvolysis of thapsigargin (1). a (CH3 CH2 )3 N, CH3 OH; b NaOCH2 CH3 , CH3 CH2 OH

proceeds much slower, indicating the anchimeric assistance of this hydroxy group for the cleavage of the ester [66]. For the large-scale production of debutanoyl thapsigargin (50), the solvolysis has been performed with ethanolic sodium ethoxide at −15°C [67]. The secondary alcohol at C-8 can be oxidized to the corresponding ketone 51 (Scheme 8) [65]. Use of appropriate reductants can afford selective reduction to the α-alcohol 50 with sodium borohydride [65] or to the epimeric β-alcohol 52 with sodium triacetoxyborohydride [68]. Tritiation of thapsigargin (1) has been performed using tritiated sodium borohydride as a reductant to give 1 tritiated at C-8 [65]. O

O O

O O

OH OH OH

O O O

50 b

a

O

O O

O O

O OH OH

O O O

51 c

O

O O

O O

OH OH OH

O O 52

Scheme 8 a CrO3 ; b NaBH4 ; c Na(CH3 COO)3 BH

O

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6.2 Chemistry at Lactone C-12 If stronger bases than trimethylamine are used at room temperature, the lactone ring opens, and a mixture of the O-6 (50) and the O-8 (53) lactones are obtained (Scheme 9) [15]. In order to obtain a derivative of thapsigargin that cannot open the lactone ring, the carbonyl at C-12 was reduced to a methylene group via a mixture of the two epimeric lactols (54) [69], which was converted to a thioketal (55). Treatment with triphenyltin hydride afforded the target compound 56 (Scheme 10). Lactol formation also has been achieved using bis(2-methoxyethoxy)ethoxyaluminum hydride instead of sodium borohydride [66]. Unfortunately, the radical conditions for the last reaction step also induced isomerization of the angelate residue to a tiglate residue (Scheme 10) [70]. The 7,11,12-trihydroxy system of the lactols 54 showed some unexpected chemical properties. Attempts to acylate the 12-hydroxy group using triethyl orthoformate as a general procedure for acetalization [71], however, also formed are the two orthoformates 57 besides the acetal 58 (Scheme 11) [66]. If triethyl orthoacetate is used as a reagent, only the 12-O-acetate 59 is formed, probably via the orthoester of acetic acid (Scheme 11) [66]. If the 11-O,12-O-diacetate 59 is dissolved in a nitrile and a Lewis acid, the oxazoline 60 is formed probably via the intermediates I and II (Scheme 12) [69]. Similar oxazoline formations have been seen when acetals of sugars have been reacted with nitriles in the presence of Lewis acids. However, in these cases, the hydroxy group serving as a donor for the oxygen atom in the ring has not been masked as an ester [72, 73]. O

O O

O

O

O O

O OH OH

O 1

O

a

O

O

O

O

O

O

O

O

O

O O

OH OH OH

O

O

O HO O H O H

O 50

53

Scheme 9 Relactonization of thapsigargin (1). a NaHCO3 , H2 O, and CH3 OH

O

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O

O

O

O OH OH

O O

O

O a

O OH OH

O

OH

O

1

O

O

O

O

O

O

O

O

54

b

O

O

O

O

O

O

O

O O

O

O

c

O OH OH

O

O

O O

O OH OH

O S

56

55

Scheme 10 a lithium borohydride in diethyl ether; b CH3 CH2 SH added HCl; c (C6 H5 )3 SnH 2,2´–azo-isobutyronitrile O

O O

O

O

O O OH

O O

O

O O

O O O

O OH OH

O

O O

O

O

57 a

+

OH

54

O

O O

O

O

O

O OH OH

O

b

O O O

O O

O

O

O O

O OH OH

O O

58

O

59

Scheme 11 Chemistry of the lactols 54. a triethyl orthoformate p-toluenesulfonic acid; b triethyl orthoacetate

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O O

O

O a

O OH OH

O

O

O

O

O O

O

O

O

O OH O

O O

O

O

OH 54

59

O

CH3CN b O

O

O

O

O

O

O

O

O

O O OH O

O

O N C

O

O O

O OH

O

O O

O

SnCl4

O

II

CH3C N

O

O SnCl4

O I

O

O O

O

O

O O OH

O O

O N

60

Scheme 12 Oxazoline formation of 60. a (CH3 CO)2 O; b SnCl4

6.3 Chemistry at O-2 and O-10 The re-lactonization depicted in Scheme 9 prevents preparative hydrolysis of the esters at the other oxygen atoms, O-2 and O-10, since stronger bases are needed for saponification of these ester groups. However, masking of O-8 and O-11 as an acetonide enables the chemistry at these oxygens to be determined (Scheme 13) [18, 68, 74]. The acetonide prevents relactonization and, consequently, the use of stronger bases enables selective deacylation at O-2 and O-10. By taking advantage of the faster acylation of the secondary alcohol at C-2, reacylation only occurs at O-2. In the example below this, the latter step is only performed after reacylation at O-8. The reaction pathway was used for the preparation of a probe for affinity chromatography of P2A Ca2+ ATPases (Section 11) [75]. The same strategy has been used for synthesizing other compounds in which the O-2 acyl group has been replaced [76]. Selective acylation at the tertiary alcohol O-10 can be afforded using isopropenyl acetate [74]. However, attempts to introduce other acyl groups in this position failed

From Plant to Patient: Thapsigargin, a Tool for Understanding … O

O O

O

O

O

O

O

O

O OH OH OH

O O

O

b

O

O OH

O

O

61

O N (CH2)11

O OH O

O

a

O

50

O

75

N (CH2)11COOH

O

OH

HO O

O

O OH O

O O

O

O

63

O OH O

O c

O

62

d O

O

O N (CH2)11 O O

e

OH

O N (CH2)11

OH

O

O O

OH OH OH

O

O

O O

O OH OH

O O

64

O

O

65 f O

O

O N (CH2)11

O

O O

O

O O

O OH OH

O O 66

Scheme 13 Selective acylation at O-2 and O-10. a dimethoxypropane and p-toluenesulfonic acid in acetone; b KOH CH3 OH; c DCC and DMAP in DCM; d HCl in CH3 OH; e (CH3 CH2 CH2 CO)2 O and pyridine in DCM; f isopropenyl acetate added p-toluenesulfonic acid

if isopropenyl esters were used. In these cases, better results were obtained if acid anhydrides were used in the presence of p-toluenesulfonic acid (Scheme 13) [18].

6.4 Chemistry at O-7 and O-11 Thapsigargin (1) has never been isolated in a crystalline form, thus preventing X-ray analysis of this compound. However, crystal structures at 2.1–2.2 Å resolution have been determined for SERCA1a with bound thapsigargin [77], and conformational

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analyses of 1 have revealed that the torsion angles between C-1—C-5, C-5—C-6, and C-6—C-7 show very little variability whereas the remaining torsion angels in the seven-membered ring may vary [78]. In the NMR spectra of 1, the coupling constants between H-8 and the two protons at C-9 reveal a staggered conformation, in which H-8 has a torsion angle to both protons of about 60° (Fig. 7). In this conformation, the torsion angle between O-7 and O-11 is approximately 180°. This antiperiplanarity of the two hydroxy groups is also found in 1 when coordinated to SERCA [79]. This might explain the unexpected epoxide formation if 1 is treated with thionyl chloride (Scheme 14, 16). Probably the reaction proceeds through the intermediate I. An initial reaction at O-11 is likely since this hydroxy group appears to be the most reactive [18, 69]. Analogous dehydration was observed earlier for trilobolide (17) [31]. In contrast, most other vicinal diols react with thionyl chloride either by forming sulfite esters or by elimination [80]. Thapsigargin analogs in which O-7 selectively has been acylated have been prepared using the acetonide 61 as starting material (Scheme 13).

Fig. 7 Conformation of thapsigargin (1). The two oxygen atoms O-7 and O-11 are marked as green spheres and the two hydrogens as yellow spheres. The grey spheres are carbon, the red spheres oxygen and the white spheres hydrogen atoms

From Plant to Patient: Thapsigargin, a Tool for Understanding … O

O O

O O

O OH OH

O 1

O

O O

O

O

O

77

a

O

O O

O O H Cl O O S O O I

O

O

O O

O

O

O O

O O O O 16

Scheme 14 Epoxide formation of thapsigargin. a SOCl2

7 Sustainable Supply of Thapsigargin 7.1 Thapsigargin from Plants and Plant Cell Cultures If attempts to use a derivative of thapsigargin (1) as a chemotherapeutics agent are to be successful, an annual demand of approximately one ton of 1 is to be expected. During the performed preclinical and clinical studies that have been conducted to date, 1 was isolated from fruits of a wild population of the producing plant. However, attempts to use fruits of T. garganica cultivated in Ibiza (Spain) have afforded yields of about 1% of the weight of the dry fruits [18]. A small company was initiated in Ibiza (ThapsIbiza S. L.) to supply GenSpera (the company that tested the derivative mipsagargin) with fruits to isolate 1 for drug production. During 2014–2017, ThapsIbiza managed to germinate well over 1000 seeds of T. garganica and also supplied samples for biosynthesis studies performed at the University of Copenhagen [38]. As GenSpera has terminated their activities, so has ThapsIbiza. Thus, the current supply of 1 for Sigma and other vendors now again relies solely on collecting wild plants. A procedure for isolating 1 in yields of 0.3% from fruits of Sardinian T. garganica has been published [81]. Proper extraction procedures of the roots from Algerian T. garganica have afforded almost 5% w/w of 1 and 2% w/w of thapsigargicin (2) [82]. Even though these figures are impressive, it is still unrealistic for one ton of 1 to be obtained each year from wild populations of T. garganica. The use of in vitro plant cell cultures leading to shooting multiplication and rooting of T. garganica was successful, and the company Alkion Bioscience and the University of Copenhagen managed to establish a production of thapsigargins in temporary immersion bioreactors (TIBs). Through the use of inducers like methyl jasmonate (MeJA), total production of 1 and nor-trilobolide (18) reached 2.5% of the

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plant dry weight [83]. Thus, this method with shoot regeneration from leaf explants of T. garganica in temporary immersion bioreactors (TIBs) could represent a future successful production of 1 until the missing knowledge of the enzymes involved can provide a heterologous production of this sesquiterpenoid.

7.2 Synthesis of Thapsigargin Total synthesis of thapsigargin (1) from easily available starting materials would, in principle, enable access to several kg amounts provided that it is possible to scale up the protocol. A number of successful syntheses have been published [84–86]. The Ley approach involved 42 steps, making this route of limited value for producing 1 in ton amounts. A major problem is the construction of the polyoxygenated sesquiterpene lactone nucleus with the correct stereochemistry. In the Baran approach, this problem was solved by taking advantage of the santonin-photosantonin rearrangement. Irradiation of santonin dissolved in glacial acetic acid with UV light affords a guaianolide with the same stereochemistry as 1 (Scheme 15, 68) [87]. All the oxygen atoms except O-6, O-7, and O-2 have been introduced in the Baran starting material 67, enabling an elegant formation of the synthon 68 by irradiation with UV light. Compound 68 may be converted into 1 in a few steps. This procedure overcame the problem of the introduction of the three oxygens at C-7, C-8, and C11, which previously had been a limiting factor for the scalable preparations of 1 [88–90]. The introduction of O-2 was performed using the procedure developed for converting nortrilobolide into 1 (Section 7.3). A 12-step total synthesis of 1 was published by Evans et al. Here, the carbon skeleton was established by a pinacol coupling of a C15 molecule containing both a carbonyl and a formyl group (Scheme 16). This C15 molecule was constructed in a biomimetic alkylation of a C10 molecule generated from carvone and a C5 molecule similar to the way geranyl diphosphate and isoprenyl diphosphate react in vivo in the plant [85]. The last steps involved the addition of acetic acid to the 10,14 olefin, inversion of the stereochemistry at C-8, and finally, the introduction of an octanoyloxy group at C-2. The last step was performed, as described by Crestey et al. [91].

Scheme 15 The santonin-photosantonin rearrangement

O O O

O O

CH3COOH hν OH

O O

O

OH

O O

67

68

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Cl O Bn

TBS

O

O Bn

TBSO

O

O

O O

O TBS

TBS

O

O

O O

O O

O O TBS

O O

TBS

O

O O

O

O

O

TBSO O

O OH OH

O

O Bn OH O

O O

TBS

O 1

Scheme 16 Chen and Evans total synthesis of thapsigargin (1) [85]

7.3 Partial Synthesis of Thapsigargin Trilobolide (17) is available readily from Laser trilobum Borkh. (Apiaceae), which can be grown in large amounts in the Czech Republic [26]. This compound can be crystallized from a plant extract without using chromatography [26]. A four-step procedure for converting trilobolide into a thapsigargin analog has been developed using nortrilobolide as starting material. This method has later been performed using trilobolide as starting material. A crucial step in this conversion is the selective solvolysis of the angelic acid substituent at O-3. Previous attempts to remove this acid involved permanganate oxidation of the angelic acid moiety to introduce a vicinal diol followed by periodate oxidation to afford a pyruvate ester, which is cleaved easily by solvolysis [68]. This procedure, however, only afforded a modest yield of the target compound. Much higher yields were obtained by solvolysis in an acidic aqueous medium to give the labile 3-hydroxy derivative, which in situ was oxidized into the 3-carbonyl derivative (Scheme 17). It has been reported that L. trilobum can be grown in the Czech Republic in fields and a patent describes that 0.95 g trilobolide might be isolated from 60 g of dried fruits, even though a detailed description is missing [26]. If trilobolide (17) is used as a starting material the 2-methylbutanoic acid unit at O-8 has to be replaced with butanoic acid when thapsigargin (1) is the target compound. However, as described later, thapsigargin analogs with an aminoacyl group at O-8 are preferred for the preparation of prodrugs, so 1 would have to be converted into a relevant prodrug. Consequently, this four-step synthesis might be a sustainable way of producing starting materials for the preparation of prodrugs in ton amounts.

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O

O O

O

O

O

O O

a

O OH OH

O

O

O

6

b

O OH OH

O

O

O

O

O O

O OH OH

O O

O

17 O

O

O 6

c

O O

O

O

HO

O OH OH

O

6

d

O

O

O

O

O OH OH

i O

O

O

O 1

Scheme 17 Introduction of O-2. a solvolysis with aqueous hydrofluoric acid in the presence of chromic acid; b introduction of octanoyloxy group by treatment with manganese(III)acetate in the presence of octanoic acid; c reduction with zinc borohydride; d angeloylation with angelic acid, benzoyl chloride, and triethylamine [91]

8 Pharmacology of Thapsigargin 8.1 Effects on Animals Thapsia garganica is reported to cause several forms of intoxications to sheep and dromedaries [5, 92]. Only one other plant species, Hyoscyamus muticus, causes more plant poisoning in Algeria [5]. No doubt the thapsigargins are a major cause of the toxicity since the lethal dose of thapsigargin (1) in mice is only 0.2–0.8 mg/kg [93, 94]. The skin-irritant properties of 1 provoked the hypothesis that thapsigargins like the 4β-phorbol esters [95, 96] were co-carcinogenic agents. This idea was confirmed experimentally [97]. However, whereas the phorbol esters are activators of the protein kinase C enzyme family [98–100], 1 is an inhibitor of SERCA.

8.2 Effects on Muscles Incubation of the rat aorta with thapsigargin (1) causes a dual effect. In the presence of endothelium, a relaxant effect on aorta precontraction with potassium ions is noted. In the absence of an endothelium, an increased contraction is observed [101]. The increased contraction is explained by an increase in cytosolic Ca2+ concentrations and the relaxant effect by the release of endothelium-dependent relaxant factors from the endothelial cells. Thapsigargin increased the contractile force of spontaneous mechanical activity but had no effect on the amplitude. This effect was blocked by nitrendipine, which blocks voltage-dependent Ca2+ channels in vascular smooth

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muscles. In contrast, 1 had no significant effect on the right atrium [102]. This might be explained by a higher concentration of SERCA in the cardiac muscle, requiring a higher dose of 1 [103, 104].

8.3 Effect on Cells Based on the idea that the thapsigargin-triggered skin irritation was caused by the secretion of histamine from mast cells, thapsigargin (1) was investigated for its ability to provoke the release of mediators from rat mast cells. An efficient release of histamine was observed if the mast cells were incubated with submicromolar concentrations of 1 in a Ca2+ -containing medium. No release was observed in the absence of Ca2+ in the extracellular medium [105]. Subsequent experiments confirmed that 1 not only releases histamine from mast cells but also provokes secretions of mediators from a broad spectrum of cells belonging to the immune system [106]. Thapsigargin (1) has been shown to induce histidine decarboxylase expression in murine macrophage RAW 264.7 cells affording a higher content of histamine in the cells [107]. The discovery that 1 was a cytotoxin initiated all the work for developing it into a potential drug [108].

9 Molecular Pharmacology of Thapsigargin 9.1 Calcium Homeostasis In a resting cell, the cytosolic Ca2+ concentration is about 60–100 nM, the concentration in the endoplasmic/sarcoplasmic reticulum (ER/SR) 100–600 μM, and the concentration in the extracellular medium approximately 1 mM [109–112]. Calcium ions are present either as free ions or complexed with calcium-binding proteins [113, 114]. The steady-state with several orders of magnitude difference between the Ca2+ concentrations in the ER, the extracellular environment and the cytosol are essential for the survival of the cells and for enabling the cellular response through calcium signaling coupled to external stimuli (Fig. 8) [109–111]. The concentration gradients are mainly maintained by Ca2+ ATPases, ion exchangers and release of Ca2+ mediated by calcium channels. Channels allow the Ca2+ facilitated down gradient diffusion through membranes [110]. The ATPases enable Ca2+ to be pumped against the concentration gradient into the ER/SR, the Golgi apparatus, or to the external medium by the use of energy obtained by hydrolysis of ATP to ADP [115–117]. After stimulation of the cell by the interaction of an agonist with a G-protein coupled receptor (GPCR) a cascade, the reaction occurs [112, 114, 118]. The activation of the receptor causes protein lipase C to cleave phosphatidyl-inositol-4,5-bisphosphate (PIP2 ) to inositol trisphosphate (IP3 ) and diacylglycerol (DAG) [118, 119]. IP3 opens

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Fig. 8 Cell activation after interaction of an agonist with a G-protein coupled receptor

IP3 receptor-operated channels in the ER/SR membrane enabling Ca2+ to diffuse into the cytosol [112, 114, 118]. Depletion of Ca2+ from the ER has been suggested to release STIM1 from the ER membrane. Subsequently, STIM1 opens the Ca2+ channel Orai1 located in the plasma membrane to induce the store-operated Ca2+ entrance (SOCE) [120, 121]. In cardiac tissue and in muscles, contraction involves increased Ca2+ in the cytosol, but the mobilization is different from the activation through G-coupled protein receptors [122]. Activation of the cell occurs when the Ca2+ concentration in the cytosol reaches 1 μM. DAG provokes enzymes belonging to the protein kinase C family to phosphorylate various proteins [118, 119]. The major enzymes responsible for reestablishing the Ca2+ compartmentation after cell activation are the SERCA-ATPase family pumping Ca2+ into the ER or SR [117, 123], plasma membrane calcium ATPases (PMCA-family), and Na+ -Ca2+ (NCX) exchanger transporting Ca2+ out to the extracellular medium [109]. Low Ca2+ concentrations in the ER increase the velocity of the SERCA pump [114]. The increased signaling responses induced by simultaneous Ca2+ mobilization by thapsigargin (1) and PKC activation by 12-O-tetradodecanoylphorbol-13-acetate (TPA) indicate that 1 and TPA may synergistically amplify the effects of each other. This has indeed been confirmed in macrophages [107, 124] and rat mast cells [125, 126]. Patents for using the principle in chemotherapy have been issued [127, 128].

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9.2 Thapsigargin Induces Cell death, ER Stress and Growth Arrest, and Inhibits Autophagy Inhibition of SERCA causes efflux of Ca2+ from the ER into the cytosol [129]. The ER Ca2+ depletion provokes SOCE and an inflow of extracellular Ca2+ to give a high nanomolar cytosolic concentration. Studies at the single-cell level have indicated that after the initial Ca2+ rise, the cytosolic Ca2+ levels decrease to baseline, but after 12 to 36 h a second rise to micromolar cytosolic concentration occurs [130–132]. This second rise in Ca2+ concentration occurs asynchronously within the cell population and is associated with rapidly ensuing morphological and biochemical changes related to apoptosis [130–133]. These studies, together with general indications of the cytotoxic actions of increased cytosolic Ca2+ levels [134], suggested a role for cytosolic Ca2+ in thapsigargin-induced cell death, which may involve calmodulin activation [132]. However, this mechanism may be restricted to certain cell types since studies in S49 T-lymphoma cells, LNCaP and PC3 prostate cancer cells, and MCF7 breast cancer cells indicate that a rise in cytosolic Ca2+ levels is not required for thapsigargin to induce cell death [135–138]. Instead, more general critical initiating factors of thapsigargin-induced cell death appear to be ER Ca2+ depletion and the resulting unfolded protein response (UPR) [129, 138, 139]. The absence of Ca2+ in the ER prevents the proteins from folding correctly. Initially, the cell stops protein expression, degrades misfolded proteins and mobilizes chaperones involved in the protein-folding process [140]. Prolonged ER stress, however, initiates the apoptotic switch. Thapsigargininduced apoptosis requires prolonged ER Ca2+ depletion and a sustained UPR [129, 138] and involves distinct contributions from UPR components (Fig. 9) [139]. The factors ATF4 and CHOP upregulate the expression of death receptor 5 [139], which is strictly required for thapsigargin-mediated activation of caspase-8, caspase-3, and cell death in LNCaP cells and HCT116 colorectal cancer cells [139, 141, 142]. The UPR transcription factors ATF4 and CHOP also upregulate MAP1LC3B (LC3B), which through a non-autophagic mechanism, contributes to caspase-8 activation [139]. For thapsigargin-induced cell death, PERK is required in both LNCaP and HCT116 cells but acts independently of ATF4, CHOP, DR5, and LC3B [139]. Also, IRE1 appears to play a cell-type-dependent role since it is required for thapsigargininduced cell death in LNCaP cells [139] and mouse embryonic fibroblasts [143], but not in HCT116 cells [139] or MCF10A breast epithelial cells [144]. Part of the explanation for this is suggested by the recent discovery of an IRE1-XBP1-dependent pathway that leads to sustained activation of JNK, which acts in a pro-apoptotic manner in LNCaP cells but not in HCT116 cells [139, 145]. Treatment of cells with thapsigargin blocks the intracellular lysosomal degradation pathway autophagy via perturbation of intracellular Ca2+ , and independently of apoptosis and the UPR [146]. Thapsigargin also inhibits cell proliferation, and it can do this even at sub-cytotoxic concentrations [128, 138]. Detailed analyses of thapsigargin-mediated effects in LNCaP and PC3 cells indicate that partial ER Ca2+ depletion (obtained with low concentrations of thapsigargin) is sufficient to

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1 SERCA Ca2+ SERCA ER/SR

SERCA

Ca2+ Ca2+ ATF4 CHOP

DR5

LC38

IRE1

PERK Casp8

XPB1

Casp3

JNK

APOPTOSIS

Fig. 9 Apoptosis after UPR provoked by thapsigargin (1)

induce cytostatic growth arrest, whereas a near-complete depletion is required for thapsigargin to inhibit autophagy, induce the UPR, and trigger apoptosis [129]. Interestingly, the ability of thapsigargin to kill cancer cells is – unlike classical chemotherapeutic agents – independent of cellular proliferation status [133, 147]. This suggests that thapsigargin would be very efficient in killing all the cancer cells in a tumor, and this prompted the idea of generating thapsigargin-based pro-drugs for targeted cancer therapy.

10 The Pharmacophore of Thapsigargin As realized in 1990, thapsigargin (1) binds in a 1:1 ratio in a cavity in SERCA found near the cytosolic surface of the pump [104, 148–150]. The ligand interacts with the M3, M5, and M7 transmembrane segments. Pro-827, which has an affinity for the

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butanoate group, is situated in the L6-L7 loop (Fig. 10). Superimposed structures of free SERCA and SERCA bound to 1 reveal that only small changes in the conformation of any of the amino acid residues occur upon binding, implying that 1 fits almost like a key to a lock [79]. In particular, Phe-256 is of importance for the binding since mutation of this to other amino acids leads to functional pumps that have a reduced affinity for 1. Also, mutations of the amino acids Ile-765 and Tyr-837 cause reductions in affinity [148]. The pharmacophore model of 1 involving the butanoate, the acetate, the angelate and to some extent the guaianolide group, as apparent from this investigation, has been confirmed by making derivatives and investigating their biological activity (i.e., structure-activity relationship (SAR) studies). An unspoken assumption for all structure-activity relationships is that all the ligands bind to the same site in the target molecule. In the case of 1 as an inhibitor of SERCA, this assumption was verified by X-ray analysis of the pump interacting with a number of inhibitors derived from 1 [79, 151].

Fig. 10 The binding site of thapsigargin (1). Phe-256 and Gln-259 (blue) interact with the guaianolide skeleton, the acetate group interacts with Phe-834 (green). Ile-765, Asn-768, Val-769 and Val263 (red) interact with the angelate moiety, and Pro-827, Leu-828 and Ile-829 (yellow) interact with the butanoate group. The octanoate extends into the lipid layer with weak interactions with Met-838 (brown) and Val-769 (red). Leu-828 and Ile-829 (yellow) are acceptors for water-mediated hydrogen bonds from the carbonyl of the butanoate, and Glu-255 (dark green) is an acceptor for a water-mediated hydrogen bridge to HO-11. The gray and red spheres depict the carbon and oxygen atoms, respectively, of 1 [79]. Transmembrane regions are marked with a M followed by a number

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10.1 Substituent at O-8 The pharmacophore model emphasizes that a flexible lipophilic side chain at O-8 is needed for a low K D value. Unexpectedly, even very long and bulky side chains did not significantly decrease the affinity although the binding cavity was too small for these side chains [138]. X-ray analysis revealed that the side chains penetrate the openings between the M3 and M5 transmembrane cavities, allowing the voluminous molecule to create a space to enable binding [151]. The advantage of this discovery was taken to construct prodrugs of thapsigargin (1) [94, 152]. As expected, removal of the O-8 side chain substantially reduces the affinity from the sub-nanomolar K D value for 1 to 10 nM for 8-O-debutanoyl-1 [79]. Replacement of the butanoate groups with non-flexible acyl groups also decreases the affinity as compared to 1, and as would be expected since these long side chains cannot penetrate the cavities between the M3 and M5 transmembrane segments [152]. Nevertheless, thapsigargin analogs with such replacements and which maintain a K D value below 10 nM, have been generated [138], thus demonstrating the feasibility of generating prodrugs that can unmask highly cytotoxic thapsigargin analogs with high affinity for SERCA. Inversion of the stereochemistry at O-8 afforded an analog with a negligible affinity for SERCA [153].

10.2 Substituent at O-10 Systematic replacement of the substituent at O-10 of thapsigargin revealed that this side chain could only be varied to a very limited extent without severely impairing SERCA-binding. Replacement with a butanoate group reduced the affinity about 100 times, and the introduction of a p-phenyl benzoate substituent reduced the affinity to be outside the windows that were measured [18].

10.3 Substitution at C-2 A lipophilic substituent in this position is not important for SERCA binding, as evidenced by the affinity of trilobolide (17), in which C-2 is a methylene group. The affinity constant K D of trilobolide to SERCA is significantly higher than that of thapsigargin, but is still in the nanomolar range [79]. Even a sidechain anchored to a bead shows so large an affinity that affinity chromatography for isolating 1 from a cell lysate failed because it was not possible to remove the ligand from the stationary phase (Section 11) [75].

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10.4 Substitution at O-3 Removal of the lipophilic angelate group strongly decreased the affinity for SERCA [79]. Inversion of the stereochemistry at C-3 also afforded an analog with severely decreased affinity [153]. However, lipophilic acyl groups with chain lengths of the same size as thapsigargin afforded potent analogs. The importance of the sizes of the acyl groups at this position is illustrated by the observation that the affinities for 3O-benzoate, 3-methylbenzoate and 4-methylbenzoate severely drops with the length of the side chain. The 3-O-p-phenylbenzoate ester shows no affinity in the range measured. In contrast, the 3-O-p-phenylphenylacetate still possesses a significant affinity [18].

10.5 The Carbonyl Group at C-12 The carbonyl group at C-12 of thapsigargin (1) is not essential for activity since the reduction of this group to a methylene group affords a compound equipotent to 1 [70].

10.6 The Two Hydroxy Groups HO-7 and HO-11 Initial studies revealed that the epoxide of thapsigargin (16), as well as the 7,11-di-Oacetate, were unable to release histamine from mast cells [33]. This result indicated that the 7,11-hydroxy groups are essential for provoking cellular responses. Later results, however, have revealed that the 11-O-butanoate still has a significant affinity for the SERCA pump [18]. Other experiments additionally have confirmed that the epoxide 16 also has an affinity for the pump, although considerably poorer [69]. A similar observation has been made for the epoxide of trilobolide [154]. Interestingly, trilobolide (17) can inhibit SERCA activity up to 90% in vitro [138], and although 17 is unable to provoke histamine release from mast cells [29], it is able to induce growth arrest, ER Ca2+ depletion, UPR, and cell death at high concentrations in prostate and breast cancer cells [129, 138]. Docking studies indicate that the ester carbonyl at O-8 forms a hydrogen bond to Leu828 and Ile829 via a water molecule and HO-11 hydrogen bonding to Glu255 via two water molecules. These bindings might be of importance for the orientation of the ligand in SERCA [69]. In conclusion, theoretical studies, as well as empirical studies using available thapsigargin analogs and derivatives, have revealed the pharmacophore depicted in Fig. 11.

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O

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Fig. 11 Pharmacophore model of thapsigargin (1). The red groups a, b, and c are important for a high affinity for SERCA; the blue lipophilic group increases the activity. The distances between carbonyls a, b, and c in 1 are carbonyl a to carbonyl b 0.9 nm, carbonyl b to carbonyl c 0.5 nm, and carbonyl a to carbonyl c 0.7 nm. The three acyl groups must be linked by a lipophilic skeleton. The two light blue oxygen groups and the carbonyl group in alkyl b might be important for hydrogen bonding via water bridges to the SERCA pump

11 Thapsigargin as a Probe for Purification of P2A Ca2+ ATPases The affinity of thapsigargin (1) for Ca2+ pumps of the P2A type inspired the development of affinity chromatographic columns for the isolation of such pumps by irreversibly binding a thapsigargin analog to Sepharose beads [75]. Since the long side chain at O-8 has been shown to penetrate the space between the transmembrane residues, this side chain was excluded as a linker between the pharmacophore and the beads of the column. Instead, a linker anchored at O-2 was constructed (72, Scheme 18). The procedure for purifying P2A Ca2+ ATPases was successful for purifying wildtype human SPCA. The pump efficiently bound to the thapsigargin-labeled Sepharose beads and could be eluted with a 10 mM Ca2+ solution. Thapsigargin (1) has an affinity for SPCA of 7.7 μM, approximately 104 times higher than that of the affinity for SERCA [76]. Human SERCA with a sub-nM affinity constant also very efficiently bound to the beads labeled with 1, but could not be eluted. However, a SERCA mutant I765A with at much lower affinity for 1 could be purified with this technique [75].

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Scheme 18 Immobilization of a thapsigargin derivative. a CF3 COOH

12 Prostate Cancer Prostate cancer is the form of this disease that causes the most deaths among men in high-income countries. In low-income countries, lung cancer mortalities exceed those from prostate cancer [155, 156]. Many cancer chemotherapeutic agents cause mitotic arrest, eventually causing cell death. If cancer develops fast, a major part of the cells is in a proliferating state, and consequently, they are sensitive towards agents that target mitosis such as vincristine, vinblastine, paclitaxel, and epothilone [157, 158]. Unfortunately, many malignant cells proliferate fast in vitro but very slowly in humans [157]. Since some of our normal cells also divide fast, these also are sensitive towards the chemotherapeutic agents applied, and, consequently, the dose cannot be increased uncritically [159]. Unfortunately, prostate cancer is an example of cancer disease in which malignant cells often proliferate very slowly, thus complicating treatment [147, 160]. Prostate cancer in the early stage consists mainly of androgen-dependent cells. Androgen ablation therapy will give a positive response in 80–90% of patients [161]. All of these patients, eventually, will relapse to a castration-resistant state (mCRPC) with a slow proliferation rate [161]. At present, the slow growth of the tumors means that no cure for the treatment of mCRPC is available [159].

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12.1 Targeting of Chemotherapeutics Classical prodrugs are made by masking functional groups in the drug, like carboxylic acids or alcohols, to obtain a more efficient form of drug delivery [162]. In the case of thapsigargin, however, advanced use of prodrugs was attempted by targeting the toxin against the tumors [163]. Targeting toxins can involve antibody-directed enzyme prodrug therapy (ADEPT) [164], gene-directed enzyme prodrug therapy (GDEPT), antibody-drug conjugated therapy (ADC), or taking advantage of enzymes with unique substrate specificity overexpressed in the tumors [163]. The prodrug thus consists of a carrier, a linker and a payload. In ADEPT, an enzyme (e.g., a carboxypeptidase) is conjugated to an antibody to proteins present on the surface of the cancer cells. After administration of the conjugate to the patient, the antibodyenzyme conjugate is bound to the cancer cells. Subsequently, a prodrug consisting of a payload conjugated to a substrate for the enzyme is administered. The enzymebound via the antibody to the surface of the cancer cell will cleave the payload from the prodrug only in the vicinity of cancer cells. Even though some promising clinical results have been obtained, no drug based on this principle is in the clinic at present (Fig. 12) [163, 164]. In GEPDT, a gene encoding for a unique enzyme is introduced to the tumor by a vector. After expression of the enzyme on the surface of the tumor, the cells will be sensitive for prodrugs consisting of a toxin conjugated to a substrate for the enzyme (Fig. 13) [163].

Fig. 12 Antibody-directed enzyme prodrug therapy

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Fig. 13 Gene-directed enzyme prodrug therapy

toxin

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In antibody-drug conjugates, the payload is conjugated with an antibody via a linker. The prodrug is administered to a patient and after binding to the cancer cell surface, the prodrug is internalized by the cell and the cytotoxin released. Three ADC’s, gemtuzumab ozogamicin, brentuximab vedotin and inotuzumab ozogamicin have been approved to treat hematologic cancers and trastuzumab emtansine has been approved for the treatment of breast cancer [165, 166]. Even though these drugs show beneficial effects, the toxicity profiles of these are comparable to those of other cancer chemotherapeutics, meaning that the expected selectivity is not obtained. In spite of these problems, a number of other ADCs are under development (Fig. 14) [167].

12.2 Targeting of Thapsigargin In the case of thapsigargin (1), selective activation of prodrug forms was attempted by taking advantage of enzymes with particular specificity present in tumors: human kallikrein-related peptidase 2 (KLK2, former human kallikrein 2 or hK2) and prostate-specific antigen (PSA), also known as human kallikrein-related peptidase 3 [168]. Later, prostate-specific membrane antigen (PSMA) was added as a tool for targeting 1 [94]. Prostate cancer cells, like normal prostate epithelial cells, express and secrete PSA. The serine protease PSA belongs to the human kallikrein protease family [169, 170]. In the fluids of the gland, PSA cleaves semenogelins that mediate gel formation in the semen. After diffusion into the circulation, the majority of PSA is inactivated either by cleavage or by complexation with proteins [169]. The increased level of PSA, because of production in prostate cancer tumors as well as in the prostate gland, is used as a marker for prostate cancer [169, 170]. The observation that PSA is only active in the vicinity of prostate cancer tumors, besides the prostate gland, has led to the idea that conjugation of a cytotoxin like doxorubicin to a peptide cleaved by PSA via a linker might afford a prodrug that is only cleaved in the vicinity of prostate cancer cells and consequently is targeted towards prostate cancer tumors (Fig. 15) [171, 172]. A drawback with doxorubicin is that the compound is a topoisomerase II inhibitor and consequently most active when the cells are in a highly proliferative state [173]. Kallikrein-related peptidase 2 is also secreted from both the prostate and prostate cancer cells [174, 175]. Like PSA, the blood level of this enzyme might be used as a marker for prostate cancer, and like PSA, KLK2 is inactivated after entering the blood [176]. Prostate-specific membrane antigen (PSMA) is a putative class II transmembranous glycoprotein. It efficiently cleaves poly-γ-glutamyl sequences [177]. The active site of the enzyme is located on the outside of the cell enabling it to cleave prodrugs in the vascular tissue (Fig. 16). The enzyme is found in the prostate gland and is highly expressed in the neovascular tissue of a number of tumors, including

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Fig. 15 In the prostate, PSA is active and cleaves the prodrug. In blood vessels, PSA in inactivated by binding to proteins

Fig. 16 Prodrug cleaved at the surface of PSMA-producing cells to give promoiety (extracellular) and toxin intracellular after penetration of the cell membrane

prostate, brain, and lung cancer, but is not found in significant amounts outside the prostate gland in healthy humans [94, 178–181]. The unusual substrate specificity of this enzyme for cleaving a very polar substrate and the overexpression of PMSA in many tumors makes it an interesting target for prodrugs.

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12.3 Sarco-Endoplasmic Reticulum ATPase (SERCA) The SERCA protein family consists of at least ten isoforms, SERCA 1a and 1b, SERCA 2a–2c, and SERCA 3a–3f [182]. The affinity between the different SERCA isoforms and thapsigargin (1) varies with K i values of 0.21 nM towards SERCA1b, 1.3 nM towards SERCA2b, and 12 nM towards SERCA3a [108, 130, 133, 183–185]. By complexing to the binding site (Fig. 10), 1 makes movements of the transmembrane segment impossible. The immobility of the enzyme prevents Ca2+ transportation. The pump is locked in an E2 conformation without Ca2+ ions [186]. Accordingly, cells incubated with 1 in a sub- or low μM concentrations quickly undergo apoptosis. Being a P-type ATPase, the pump transports two ions of calcium against the concentration gradient into the ER by the energy released by phosphorylation of an aspartate residue [187]. Two to three hydrogen ions are counter-transported, meaning that the pump creates an electrochemical voltage over the membrane [187, 188]. The pumping cycle is well understood, even though some details still are missing [189]. The cycle consists of six key states, two E1 stages with high affinity for Ca2+ and four E2 stages with low affinity for Ca2+ (Fig. 17) [115, 189]. In the first state, nH+ E2 ATP

Fig. 17 The pumping cycle of SERCA

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(1), two or three H+ and an ATP are bound to the pump. The pump exchanges the H+ with two cytosolic Ca2+ ions and is transformed to the 2Ca2+ E1 ATP state (2). After phosphorylation of Asp-351 the high energy [2Ca2+ ]E1P ADP (3) occluded state is formed. ADP is exchanged with ATP to give a low energy phosphorylated occluded intermediate [2Ca2+ ]E2P ATP state (4). The two calcium ions are exchanged with luminal protons after the opening of a channel to the lumen to give nH+ E2P-ATP, in which n equals 2 or 3 (5). After closing the luminal channel, the occluded state [nH+ ]E2P ATP (6) is formed, in which no channel to either the lumen or the cytosol is open. The cycle is concluded by dephosphorylation to give nH+ E2ATP. The ratelimiting steps are the E1P to E2P [190] and the E2 to E1 transformations (Fig. 17) [191]. A slightly deviating model has been suggested [192]. Among the known SERCA inhibitors thapsigargin is by far the most potent [184, 193–195]. Thapsigargin (1) locks SERCA in an E2 stage. The X-ray structures of the thapsigargin-SERCA complex with and without nucleotide have been solved, enabling analysis of the topography of the binding site [196].

12.4 Thapsigargin and Other P-type Pumps The P-type pumps are a major family consisting of a large number of different pumps [117]. All of these are characterized by the presence of an aspartate moiety, which is phosphorylated by ATP to enable the pump to transport typically an ion against a concentration gradient. Some pumps in addition, are flipases, establishing different concentrations of lipids in the inner and outer plasma membrane. Thapsigargin (1) also shows an affinity for the SPCA pump [76]. The SPCA pump transports Ca2+ /Mn2+ into the Golgi compartment. The affinity of 1 for the SPCA is about 1,000 times lower (7.7 μM) than for the SERCAs (0.2–13 nM). Structure-activity relationship studies have revealed that 1 binds to SPCA differently from the way it binds to SERCA [76]. Even though there are structural similarities between all the P-type ATPases, 1 has a poor to almost vanishingly small activity against mammalian Ptype ATPases like PMCA and the Na+ /K+ ATPases and some Ca2+ -ATPases from nematodes [104, 150, 197, 198].

12.5 Thapsigargin Prodrug Design, Preparation, and Preclinical Evaluation The potency of thapsigargin (1) as a cytotoxin made this compound an interesting choice as a potential cancer chemotherapeutic agent. Thapsigargin (1) is extremely cytotoxic and kills cells in all stages, in contrast to many chemotherapeutics, which are targeted against cells in the proliferative states [108, 199–204]. The preparation of thapsigargin prodrugs, however, is not straightforward. First, the absence of amino

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groups prevents conjugation to any peptide. In addition, the following problems must be addressed: (1) The prodrug should be hydrophilic, preventing diffusion into cells and thereby making interaction with the intracellular SERCA pump impossible. (2) The prodrug must be a substrate for the protease, in this case, either PSA, KLK2 or PSMA. (3) The drug cleaved from the prodrug after interaction with the enzyme must still be a toxin and be able to reach and neutralize the target (SERCA). According to structure-activity relationships, a flexible acyl group attached to O-8 should enable potent interaction of the analog with SERCA. A long chain affords a distance between the bulky guaianolide skeleton and the peptide binding, enabling the amide group to come into the active site cleft, which in all three proteases are located in a cavity inside the enzyme [205–208]. Consequently, the butanoyl group at O-8 was substituted with ω-aminoacyl groups to give thapsigargin analogs possessing an amino group for conjugating with an appropriate peptide. Among the many analogs prepared, the 8-(12-aminododecanoate) (Fig. 18, compound 73) showed the same cytotoxicity as 1 [152]. The high cytotoxicity is explained mainly by the structural similarities with 1 and by the ability of the O-8 acyl group to penetrate the transmembrane sections enabling the molecule to fit into the binding site [79, 94]. The high hydrophilicity of the peptide moieties of the molecules 74–76 was hypothesized to prevent penetration of the cell membrane and thereby make it impossible for the molecules to reach the SERCA enzyme. Since advantage was taken of the expression of PSA [160], KLK2 [176, 209], and PSMA [210] in prostate cancer tumors, substrates for these enzymes were conjugated with the amino group. Based on kinetic studies, compound 75 was found to be an excellent substrate for KLK2 [176], 74 for PSA [93], and 76 for PSMA [210–213]. Cleavage of the relevant peptide bond in 75 with KLK2 and in 74 with PSA gives the lipophilic toxin 77, which penetrates the cell membrane (Fig. 19). In both cases, the C-terminal amino acid (Leu) was not removed by the enzyme. However, a C-terminal Leu was essential for the peptide to become a substrate [176]. In the case of PSMA, 76 was found to be an optimal substrate. The prodrug is rapidly cleaved by PSMA to give 78. PSMA is expressed in prostate cancer cell lines and in neovascular tissues in a number of tumors. Toxin 78 is somewhat slowly converted to 79 by PSMA. A C-terminal Asp in the peptide is required for cleavage by PSMA [94]. Preclinical studies were performed particularly on prodrugs 74 and 76. In vitro studies of 74 revealed that the prodrug was cleaved efficiently by PSA-producing LNCaP prostate cancer cells, to give the toxin 77, meaning that the C-terminal Leu was not removed. Encouragingly, 77 efficiently killed PSA-producing cells [176, 214]. Non-PSA-producing cell lines like HCT116 were not affected by the prodrug 74. The compound was stable in human plasma. In vivo studies revealed that injection of 7 mg/kg in mice afforded a plasma concentration of 15 μM and a half-life of 2.8 hours. Only trace amounts of the toxin 74 was observed in the plasma. Continuous injection of the prodrug caused complete growth inhibition of PSA-producing xenograft tumors and had no effect on non-PSA producing xenografts. No general toxicity was seen [93].

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Fig. 18 8-O-(12-aminododecanoyl)-8-O-debutanoyl thapsigargin (73) and the three prodrugs cleaved by KLK2 (74), PSA (75), and PSMA (76), respectively. The cleavage sites are marked with an arrow

Prodrug 76 was much more toxic toward PSMA-producing prostate cancer cell lines than toward non-PSMA producing cells. However, high concentrations of prodrug 76 did show some toxicity towards non-PSMA producing cells, indicating that the prodrug, to a minor extent, is able to penetrate the cell membrane. If the activity of PSMA was blocked with an inhibitor, high concentrations were required for cytotoxicity [94]. Kinetic studies revealed no cleavage of 76 in benign tissue outside the prostate. The cleavage product 79 was twice as potent in cell killing as 78. A 3-day course with injections of 56 mg/kg of prodrug 76 caused a 50% regression of LNCaP xenografts over 30 days in mice. A weight reduction of 15% was observed after a week, but the mice regained their weight after three weeks. Wholebody studies in mice revealed a much higher concentration of 76 in tumor tissue than

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Fig. 19 Toxins released from the prodrugs 74 and 75 after cleavage with KLK2 and PSA (77) and toxins released from prodrug 76 after cleavage with PSMA (78 and 79)

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in other tissues. No abnormal changes were observed in rats and monkeys after three injections with 10 mg/kg of prodrug for three consecutive days [94].

12.6 Clinical Trials Clinical trials, and bringing a drug to the market, are so encompassing and funddemanding projects that no typical department at a university could undertake such a challenge. Thus, the biotech company GenSpera Inc. was formed. The company took over all the intellectual rights of the researchers and universities involved. Transfer agreements were signed by all involved parties. Before clinical trials were started, prodrug 76 was given the name “mipsagargin” and the code G202. The GLP synthesis of mipsagargin, required for initiating clinical trials, was developed by GenSpera [67]. Good laboratory practice toxicity tests were performed in rats and cynomolgus monkeys by Ricerca Biosciences, LLC. Based on the results, an institutional review board recommended clinical phase 1 clinical studies. Initially, 44 patients were given mipsagargin in doses up to 88 mg/m2 . Two patients revealed an infusion-related syndrome and two patients had increased creatinine levels. Adverse effects were nausea, fatigue, rash, and pyrexia. The clinical response was not measured, but some patients suffering from advanced hepatocellular carcinoma (HCC) had prolonged disease stabilization [215, 216]. The presence of PSMA in neovascular HCC tumors explains such a clinical effect, since mipsagargin is expected to destroy the vascular tissue and thereby is effectively functioning similar to angiogenesis inhibitors [94]. In a clinical phase 2 trial, 25 patients suffering from progressive advanced hepatocellular carcinoma previously treated with sorafenib were treated with mipsagargin 40 mg/m2 on day 1 and 2 and 66.8 mg/m2 in a 28-day

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cycle. Of 25 treated patients, 19 were evaluable for efficacy, 12 had the best response for stable disease and 12 showed radiologic progression, and seven were censored. A phase 2 clinical trial revealed that mipsagargin was relatively well tolerated and increased the period of disease stabilization in patients with HCC that had advanced during treatment with sorafenib [217]. Unfortunately, these results did not show sufficient efficacy for continued support to enable GenSpera to perform a phase 3 clinical trial needed for the potential registration of mipsagargin. Later, the company changed its name to Inspyr Therapeutics, Inc., but no further development of the drug has been performed. No attempts have been made to treat patients with higher doses. Probably this would cause general toxic effects in the body of the patient since, at high concentrations, mipsagargin in spite of the hydrophilic peptide moiety, appears to be able to penetrate cell membranes and consequently block the SERCA pump not only in cancer cells but also in benign tissue [94, 159]. The possibility of using a 4β-phorbol conjugated with the peptide moiety β-Asp(γ-Glu)4 as a prodrug (Fig. 20, compounds 80–82), inspired by the mipsagargin approach, was investigated. Fig. 20 Prodrugs 80–82 of 4β-12-Odetetradodecanoate-12-O-Nacyl-12-aminododecanoate phorbol acetate

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These phorbol prodrugs were designed for coadministration with 74–76, since reports of synergistic enhancement of cell death induced by phorbol and thapsigargin derivatives have been reported [124–126]. No selectivity for protease-producing and non-protease producing prostate cancer cells were observed, suggesting that the cell membrane could not prevent the compounds from interacting with SERCA [218].

13 Perspective Thapsigargin (1) was isolated in order to explain skin-irritant properties of the ancient traditional drug Resina Thapsiae, consisting of the resin from the umbelliferous plant T. garganica. A number of the active principles, all polyoxygenated guaianolides, were discovered. The major compound obtained was named thapsigargin (1). An explanation of its skin-irritant properties is that 1 very efficiently releases histamine from mast cells and mediators from other cells of the immune system. Molecular pharmacology studies demonstrated that 1 is an extremely potent inhibitor of the Ca2+ ATPases situated in the membrane of the endoplasmic or in muscle cells of the sarcoplasmic reticulum (the SERCA pumps). Thapsigargin (1) has later become a standard compound for biological assays on Ca2+ homeostasis. A search in SciFinder® (Chemical Abstracts Service, Columbus, Ohio, USA; June 2020) with the keyword thapsigargin gave almost 19,000 hits and it also gave over 50.000 hits in Google Scholar. This indicates the importance of this plant-derived compound. Complexation of 1 with SERCA also enabled crystallization of the SERCA pump. This study, when combined with several later studies, has enabled an understanding of the kinetics and mechanism of action of the SERCA pump and other P-type ATPases. Studies of thapsigargin-induced apoptosis have offered biologists new knowledge of the cellular mechanisms behind Ca2+ imbalance- and ER stress-induced cell death. Mipsagargin, a derivative of 1, has been tested in phase 2 clinical trials as a drug for the treatment of advanced hepatocellular carcinoma. Unfortunately, the targeting of the drug by conjugation with peptides selectively cleaved by proteases present primarily in cancer tissue did not sufficiently prevent systemic toxic effects, thus preventing the potential use of the compound as a drug. However, attempts to conjugate 1 with albumin might resolve the problem of selectivity [159]. Alternatively, other thapsigargin prodrugs, or other SERCA inhibitors, might be used. Such inhibitors may be designed by computational chemistry or might be found in Nature. Other SERCA inhibitors than those related to 1 have been found as natural products [195, 219]. Unfortunately, neither of these is sufficiently potent for the prodrug approach. In addition to the biological studies, intensive studies of 1 have disclosed new fascinating insight into the guaianolides and their organic chemistry. The targeting studies of 1 have revealed new and surprising facets of prodrug development, and the compound has offered invaluable new knowledge on cell function and death. It has often been mentioned that it is easy to perform important research when one has been lucky enough to find a unique compound. The answer to this statement is that

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luck is not a characteristic feature of a good scientist. The characteristic feature of a good scientist is recognition of the importance of the discovery and relevant actions. A compound with similar inhibitory effects on the SERCA pump, trilobolide (17), was isolated a few years before 1. In this case, the discoverers did not initiate intensive chemical and biological studies but just shelved the compound. In the case of 1, such studies were initiated, leading to fruitful and important new scientific knowledge. The story of 1 raises the question as to whether other natural products with valuable biological properties are being left forgotten in the storerooms of natural product chemists?

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165. Coats S, Williams M, Kebble B, Dixit R, Tseng L, Yao N-S, Tice DA, Soria J-C (2019) Antibody-drug conjugates: future directions in clinical and translational strategies to improve the therapeutic index. Clin Cancer Res 25:5441 166. Nicolaou KC, Rigol S (2019) Total synthesis in search of potent antibody-drug conjugate payloads. From the fundamentals to the translational. Acc Chem Res 52:127 167. Halford B (2020) Antibody-drug conjugates make a comeback. Chem Eng News 98:16 168. Denmeade SR, Isaacs JT (2005) The SERCA pump as a therapeutic target: making a “smart bomb” for prostate cancer. Cancer Biol Ther 4:14 169. Balk SP, Ko YJ, Bubley GJ (2003) Biology of prostate-specific antigen. J Clin Oncol 21:383 170. Lilja H (2003) Biology of prostate-specific antigen. Urology 62(Suppl 1):27 171. Denmeade SR, Nagy A, Gao J, Lilja H, Schally AV, Isaacs JT (1998) Enzymatic activation of a doxorubicin-peptide prodrug by prostate-specific antigen. Cancer Res 58:2537 172. Brady SF, Pawluczyk JM, Lumma PK, Feng DM, Wai JM, Jones R, Feo-Jones D, Wong BK, Miller-Stein C, Lin JH, Oliff A, Freidinger RM, Garsky VM (2002) Design and synthesis of a pro-drug of vinblastine targeted at treatment of prostate cancer with enhanced efficacy and reduced systemic toxicity. J Med Chem 45:4706 173. Denny WA (2006) Deoxyribonucleic acid topoisomerase inhibitors. Compr Med Chem II 7:111 174. Darson MF, Pacelli A, Roche P, Rittenhouse HG, Wolfert RL, Young CY, Klee GG, Tindall DJ, Bostwick DG (1997) Human glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma: a novel prostate cancer marker. Urology 49:857 175. Darson MF, Pacelli A, Roche P, Rittenhouse HG, Wolfert RL, Saeid MS, Young CY, Klee GG, Tindall J, Bostwick DG (1999) Human glandular kallikrein 2 expression in prostate adenocarcinoma and lymph node metastases. Urology 53:939 176. Janssen S, Rosen DM, Ricklis RM, Dionne CA, Lilja H, Christensen SB, Isaacs JT, Denmeade SR (2006) Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (hK2)-activated thapsigargin prodrug. Prostate 66:358 177. Pinto JT, Suffoletto BP, Berzin TM, Qiao CH, Lin S, Tong WP, May F, Mukherjee B, Heston WDW (1996) Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic carcinoma cells. Clin Cancer Res 2:1445 178. Wang H-L, Wang S-S, Song W-H, Pan Y, Yu H-P, Si T-G, Liu Y, Cui X-N, Guo Z (2015) Expression of prostate-specific membrane antigen in lung cancer cells and tumor neovasculature endothelial cells and its clinical significance. PLoS One 10:e0125924 179. Nomura N, Pastorino S, Jiang P, Lambert G, Crawford JR, Gymnopoulos M, Piccioni D, Juarez T, Pingle SC, Makale M, Kesari S (2014) Prostate specific membrane antigen (PSMA) expression in primary gliomas and breast cancer brain metastases. Cancer Cell Int 14:26 180. Kasoha M, Unger C, Solomayer E-F, Bohle RM, Zaharia C, Khreich F, Wagenpfeil S, JuhaszBoess I (2017) Prostate-specific membrane antigen (PSMA) expression in breast cancer and its metastases. Clin Exp Metast 34:479 181. Nimmagadda S, Pullambhatla M, Chen Y, Parsana P, Lisok A, Chatterjee S, Mease R, Rowe SP, Lupold S, Pienta KJ, Pomper MG (2018) Low-level endogenous PSMA expression in nonprostatic tumor xenografts is sufficient for in vivo tumor targeting and imaging. J Nucl Med 486 182. Periasamy M, Kalyanasundaram A (2007) Serca pump isoforms: their role in calcium transport and disease. Muscle Nerve 35:430 183. Casemore D, Xing X (2015) SERCA as target for cancer therapies. Int Cancer Sci Ther 2:100 184. Michelangeli F, East JM (2011) A diversity of SERCA Ca2+ pump inhibitors, recent advances in membrane biochemistry. Biochem Soc Symp 78:131 185. Chin TY, Lin HC, Kuo JP, Chueh SH (2007) Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells. Am J Physiol Cell Physiol 292:C383 186. Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the calcium pump of the of sarcoplasmic reticulum at 2.6 Å resolution. Nature 405:647 187. Bublitz M, Morth JP, Nissen P (2011) P-type ATPases at a glance. J Cell Sci 124:2515

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208. Mesters JR, Barinka C, Li W, Tsukamoto T, Majer P, Slusher BS, Konvalinka J, Hilgenfeld R (2006) Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer. EMBO J 25:1375 209. Janssen S, Jakobsen CM, Rosen DM, Ricklis RM, Reineke U, Christensen SB, Lilja H, Denmeade SR (2004) Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer. Mol Cancer Ther 3:1439 210. Denmeade SR, Isaacs JT (2012) Engineering enzymatically activated “molecular grenades” for cancer. Oncotarget 3:666 211. Denmeade SR, Isaacs JT, Christensen SB (2010) Methods and compositions for the detection and treatment of cancer using targeted activation of peptide prodrugs and imaging agents. WO2010107909A2 212. Denmeade SR, Isaacs JT, Christensen SB (2014) Methods and compositions for the detection of cancer. US 8,772,226 B2 213. Isaacs JT, Denmeade SR, Christensen SB, Lilja H (2003) Prostate-specific prodrugs. US 6,504,014 B1 214. Vander Griend DJ, Antony L, Dalrymple SL, Xu Y, Christensen SB, Denmeade SR, Isaacs JT (2009) Amino acid containing thapsigargin analogues deplete androgen receptor protein via synthesis inhibition and induce the death of prostate cancer cells. Mol Cancer Ther 8:1340 215. Mahalingam D, Tubb B, Neumunaiitis JJ, Cen P, Rowe JH, Sarantopoulos J, Kurman MR, Allgood V, Campos LT (2015) Clinical activity and correlative DCE-MRI imaging of G-202, a thapsigargin-based prostate-specific membrane antigen-activated prodrug, in progressive hepatocellular cancer. J Clin Oncol 33(Suppl 3):301 216. Mahalingam D, Wilding G, Denmeade S, Sarantopoulas J, Cosgrove D, Cetnar J, Azad N, Bruce J, Kurman M, Allgood VE, Carducci M (2016) Mipsagargin, a novel thapsigarginbased PSMA-activated prodrug: results of a first-in-man phase I clinical trial in patients with refractory, advanced or metastatic solid tumours. Br J Cancer 114:986 217. Mahalingam D, Mahalingam D, Arora SP, Sarantopoulos J, Peguero J, Campos L, Cen P, Rowe J, Allgood V, Tubb B (2019) A phase II, multicenter, single-arm study of mipsagargin (G202) as a second-line therapy following sorafenib for adult patients with progressive advanced hepatocellular carcinoma. Cancers 11:6 218. Zimmermann T, Christensen SB, Franzyk H (2018) Preparation of enzyme-activated thapsigargin prodrugs by solid-phase synthesis. Molecules 23:1463 219. De Ford C, Heidersdorf B, Haun F, Murillo R, Friedrich T, Borner C, Merfort I (2016) The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling. Cell Death Dis 7:e2070

Søren Brøgger Christensen obtained his Ph.D. degree in pharmaceutical sciences at the Royal Danish School of Pharmacy in 1975, supervised by Professor P. Krogsgaard-Larsen. He undertook Visiting Scientist positions at UCLA (Professor R. V. Stevens, 1977) and University College London (Professor John Foreman 1984). He was appointed as Assistant Professor (1976), Reader (1991), and Professor 2008 at the Royal Danish School of Pharmacy, which in 2000 was merged with the University of Copenhagen. He has focused his research on natural products with biological activities. Licochalcone A was isolated from Glycyrrhiza inflata and shown to be a potent lead compound against malaria parasites. Analogues were prepared in order to develop an optimized drug, with the intellectual property rights of the biotech company Lica Pharmaceuticals Ltd. being based on this work. Thapsigargin was isolated from

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S. B. Christensen et al. Thapsia garganica and the chemical and pharmacological properties were investigated leading to the intellectual properties of right behind the biotechnical company GenSpera Ltd. In addition, a number of other natural products have been investigated. He is co-author of over 185 publications in international scientific journals and has been named as co-inventor of more than 10 patents. From 2010 to 2015, he managed the project, “A New Sustainable Platform for Production of Natural Products as Illustrated by Production of Thapsigargin in Moss”, supported by the Danish Research Council for Strategic Research. Henrik Toft Simonsen obtained his Ph.D. degree in 2002 from the Royal Danish School of Pharmacy within the subject of Pharmacognosy. He then did two years of postdoctoral research at the Department of Chemistry, University of Cambridge, Cambridge, UK, before he returned to Denmark and spent another postdoctoral period at the Agricultural University, later the University of Copenhagen. In 2008, he was employed as first Assistant, then (after six months) Associate Professor at the Department of Plant and Environmental Sciences, University of Copenhagen. In 2016 he moved to his current position as Associate Professor at the Department of Biotechnology and Biomedicine, Technical University of Denmark. Also in 2016, he co-founded Mosspiration Biotech to commercialize previous research, and today Mosspiration markets the brand Mossebelle. He has published more than 70 peer-reviewed papers and submitted five patent applications. His research group routinely characterizes enzymes involved in the biosynthesis of terpenoids including thapsigargin, utilizing moss and Nicotiana for in planta characterization. The aim is to advance the understanding of terpenoid biochemistry to provide new possibilities in bioengineering. Both mosses and algae provide a robust platform for “in one go” combinatorial biochemistry studies that can be utilized directly for the discovery of novel biochemistry and industrial production. Nikolai Engedal obtained a B.Sc. in chemistry in 1996, followed by a M.Sc. in biochemistry in 1999, and a Ph.D. degree in cell biology in 2005 at the University of Oslo (Norway) under the guidance of Professor Heidi Kiil Blomhoff. After postdoctoral work in the group of Professor Kirsten Sandvig at the Institute for Cancer Research, the Norwegian Radium Hospital (2006–2010), he moved to a second postdoctoral position in the group of Professor Ian G. Mills at the Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership (2010–2014). In 2014, he received a Young Research Talent award from the Research Council of Norway, and he led the independent research group “the Autophagy Team” at NCMM in the period 2014–2020. Currently, Dr. Engedal holds a researcher position at the Institute for Cancer Research, Oslo University Hospital. His main research focus is on the cellular process of autophagy, intracellular signaling, epigenetics, and cell death in relation to cancer. This has

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included a series of studies on the biological effects of the plant-derived compound thapsigargin, and various chemical analogs thereof, in human cancer cells. Poul Nissen obtained his Ph.D. degree in structural biology in 1997 from Aarhus University (AU), supervised by Prof. Jens Nyborg and working on translation GTPases. He then held a postdoctoral position from 1997 to 2000 with Professor Thomas A. Steitz at Yale University (Nobel laureate 2009) on the ribosome structure. After that, he initiated his own research at AU on membrane transporters, in particular P-type ATPases, first through a Ole Rømer stipend of the Danish Research Council, and since 2006 as a Professor of Protein Biochemistry at AU. He is currently the Director of the Danish Research Institute of Translational Neuroscience – DANDRITE of the Nordic EMBL Partnership for Molecular Medicine and Director of the Danish research infrastructure for cryo-EM research. The main topics of his research program are on membrane transporters in brain cells and higher-order membrane structures in neurons. Jesper Vuust Møller graduated as Master of Medicine in 1966 and as Dr. Med. in 1968 from Aarhus University. He was Associate Professor from 1966 until 1990 and from 1990 to 2008 he was Professor at the Department of Biophysics at Aarhus University. His employment at this university was interrupted temporarily when he was Visiting Professor at Manchester University, UK 1968–1969 (with W.D. Stein), and from 1973 to 1974 when he was Visiting Professor at Duke University Medical Center, USA (with C. Tanford). He has been Chairman of the Danish Biomembrane Center (1993–1996). His studies have been focused on transport over membranes, in particular, of SERCA, and the role of this intracellular Ca2+ -ATPase as a target to combat prostate cancer. After his retirement from Aarhus University, he has continued his research on transport over cell membranes as Professor Emeritus. He has authored or coauthored more than 175 articles and reviews in international scientific journals. Samuel R. Denmeade obtained his medical M.D. degree from Columbia University in New York City. He completed a residency in Internal Medicine at the University of Chicago. He then completed a clinical Medical Oncology Fellowship at Johns Hopkins University, Baltimore, MD in 1995 and then pursued a postdoctoral fellowship in prostate cancer research in the laboratory of Dr. John Isaacs. He joined the faculty of Johns Hopkins in 1998 and is currently Professor of Oncology, Urology, Pharmacology, and Molecular Sciences. He is also the Director of the Genitourinary Oncology Program at Johns Hopkins. As a laboratory scientist, Dr. Denmeade leads a team focused on developing targeted therapies for prostate cancer. He is the author of 170 papers and co-inventor on 22 US patents. A PSMA-activated prodrug, a PSA-activated protein toxin, and a

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S. B. Christensen et al. PSMA-targeted nanoparticle developed in his laboratory have been out-licensed and tested in Phase I-III clinical trials. As a clinician scientist, Dr. Denmeade has been the Principal Investigator on a number of clinical trials with a particular focus on hormonal therapy-based trials. Most recently he has completed an innovative trial demonstrating that administration of a highdose testosterone could be safely given to men with castrationresistant prostate cancer while producing a significant therapeutic effect. John T. Isaacs obtained a Ph.D. degree in Biochemistry in 1978 from Emory University, Atlanta, GA, under the direction of Professor Francis Binkley. He did his postdoctoral fellowship training in Pharmacology and Experimental Therapeutics with Professor Donald S. Coffey at the Johns Hopkins University School of Medicine (JHSOM) where he became a faculty member in 1980. Since 1993, he has been a Full Professor in three departments in the JHSOM (i.e. Urology. Oncology, Pharmacology, and Molecular Sciences) and in the Chemical and Molecular Engineering Department in the Johns Hopkins University, Kreiger School of Arts and Science, Baltimore, Maryland. He also is the past Director of the Cellular and Molecular Medicine Graduate Program at JHSOM, Baltimore, Maryland. He has served as a member of editorial boards on seven journals in oncology, including being the Editor-in-Chief of “The Prostate”. His long-term goal is to develop effective therapies to decrease the death rates due to cancer, particularly prostate cancer. This goal requires not only pre-clinical development and validation of specific approaches, but the translation of these basic science findings into clinical development and human trials. In this regard, his group has an established record of translating pre-clinical discoveries into clinical trials (i.e., four drugs taken into clinical trials). Presently, a series of agents that his group invented/developed are currently in various phases of clinical testing. He has contributed to 325 peer-reviewed manuscripts, 17 patents, and 70 book chapters. Throughout his career, he has received various awards and honors, including from the Sigma Xi (Emory University Chapter) Most Outstanding Graduate Student Research, CaPCure Foundation Competitive Awards, Past President of the Society for Basic Urologic Research, the Life Achievement Award from the Society for Basic Urologic Research, and the Prostate Cancer Foundation Movember Challenge Award. He is a member of the National Academy of Inventors (USA).

Antileishmanial Activity of Lignans, Neolignans, and Other Plant Phenols Jiˇrí Pospíšil, Daniela Konrádová, and Miroslav Strnad

Contents 1 2 3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secondary Metabolites of the Shikimate Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Phenylpropanoids and Their Impact on Human Health . . . . . . . . . . . . . . . . . . . . . 3.3 Bioactive Lignans and Neolignans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 Introduction Small specialized molecules not essential for life, yet “the game changers in the survival fight” is a simplified definition for plant secondary metabolites (SMs) [1]. Compared with the generally uniform pathways leading toward primary metabolites, some SM biosynthesis pathways differ across the plant kingdom. Indeed, the production and occurrence of SMs within plants is a compromise of the plant’s needs The original version of this chapter was revised (Fig. 3 and Fig. 14 were updated). The correction to this chapter is available at https://doi.org/10.1007/978-3-030-64853-4_5 J. Pospíšil · D. Konrádová (B) · M. Strnad Laboratory of Growth Regulators, The Czech Academy of Sciences, Institute of Experimental Botany & Palacký University, Šlechtitel˚u 27, CZ-78371 Olomouc, Czech Republic e-mail: [email protected] J. Pospíšil e-mail: [email protected] M. Strnad e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021, corrected publication 2021 A. D. Kinghorn, H. Falk, S. Gibbons, Y. Asakawa, J.-K. Liu, V. M. Dirsch (eds.), Progress in the Chemistry of Organic Natural Products, Vol. 115, https://doi.org/10.1007/978-3-030-64853-4_3

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and the surrounding environment. Consequently, the secondary plant metabolome is always time and place specific [2–4]. Although SMs are not directly involved in the plant growth–development–reproduction (GDR) cycle, the diversity of speciesspecific SMs provides the plant with a unique opportunity not only to “survive” but also gives them an advantage both in their survival and in the need for adaptation [5]. The structural diversity of SMs is generated due to the wide variety of abiotic and biotic stresses that plants face in each day in the fight for their survival [2, 6]. Once produced, SMs take an important role in various plant responses (e.g., as antiviral, antibacterial or antifungal protective agents), or they participate in plant/environment interactions (e.g., serving as signal messengers in communication/competition within plants and/or symbiotic microorganisms, or as pollinator attractants) [7]. Certain regulatory roles of SMs have been reported in the case of pathogen-induced cell death, oxidative cell burst, auxin transport, and in cell division [8–11]. Based on their purpose, SMs can be present in a specific plant tissue/organ in the form of an active compound (direct use), or in the form of an inactivated precursor (to be used when needed, e.g., in the cases of infection, wounding, or stress). In some cases, the activation can be elicited by external sources (e.g., herbivore digestioninitiated activation, or in methyl salicylate-to-salicylic acid transformation in the livers of herbivores) [7]. Herbivore digestion-related activation of SMs [7], based on the close plant/herbivore coexistence, is of great interest. Indeed, some SMs of plants have developed over time the ability to mimic the function of endogenous molecules (hormones, substrates, etc.) of herbivores, and therefore to interact exclusively with herbivore-based molecular targets located within membranes and enzymes. However, the effects of plant SMs on herbivores are not only negative, since numerous positive effects of SMs on human/animal health have been reported [12–14]. In addition, many SMs have served as starting points in drug development campaigns [13–16]. In the present contribution, the authors would like to draw the reader’s attention toward the role that phenylpropanoid-based SMs play in the treatment of a humanrelated parasite caused disease — leishmaniasis. After a brief introduction to both leishmaniasis and phenylpropanoid biosynthesis, the biological activity of phenylpropanoids in general will be covered. As apparent from Section 2, leishmaniasis is a disease that is difficult to treat. This is a illness where many apparently unrelated features such as poor nutrition and coinfection by other diseases can interfere with the healing process. For this reason, the present contribution covers several not necessarily related aspects of lignan and neolignan biological activity. Phenylpropanoids, as phenolic SMs, are present to a substantial degree in the daily diet, and therefore should be considered not only as potential sources of future drugs (or as interesting structural motifs to develop new drugs) but also for their influence on the treatment or prevention of various diseases from the nutritional point of view. Phenylpropanoids that are ingested by humans each day, can have, and presumably do have, a big influence on the efficacy of medical treatments and on healing processes.

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2 Leishmaniasis Leishmaniasis, a disease listed in 2010 by the World Health Organization (WHO) as one of the neglected diseases, is caused by parasites of the genus Leishmania [17]. The WHO has attempted to control and eliminate leishmaniasis not only with the development of effective ways of treatment, but, perhaps more importantly, by prevention and control tools and programs. The list of actions taken is summarized in the Global Vector Control Response 2017–2030 of WHO [18]. Leishmaniasis belongs to the parasitic, vector-borne group of diseases. The phlebotomine sandfly serves as the vector and more than 800 individual species of such sandfly vectors have been identified to date. Only a small portion of them (approximately 100 species) can transmit parasites pathogenic to humans [19, 20]. Leishmaniasis-related sandfly species are Lutzomyia, called “new world” species (widespread in areas of Central and South America), and Phlebotomus, referred to as “old world” species (widespread in areas of Europe, Asia, and Africa) [21]. In terms of the development of leishmaniasis, the role of the sandfly vector is particularly important since the inner environment of the sandfly modulates the parasite’s development and influences the final manifestation of the disease. Overall, the extent of the disease manifestation results not only from the vector but also by variations in the leishmanial parasite. To give a brief overview, linked below are different leishmanial species with the clinical manifestations of the illness and the area of occurrence (Table 1) [22–24]. For this purpose, a simplified taxonomy of the Leishmania genus has been employed that divides it into two main subgroups, Paraleishmania and Euleishmania, which are further divided into the main subgenera, L. (Leishmania) and L. (Viannia) [25]. The subgenus L. (Leishmania) relies on humans as the main host reservoir, whereas in the case of L. (Viannia), humans are only an accidental host [22–24]. From the life cycle viewpoint, leishmanial parasites are dimorphic organisms inhabiting two different environments during their development. Within these two periods, the parasite alternates between the promastigote and amastigote forms, with the actual form determined by the environment. In the sandfly vector Phlebotomus or Lutzomyia, the parasite lives in the form of promastigotes as a flagellated, extracellular organism, which needs to survive in conditions full of proteolytic enzymes necessary for blood digestion [26–28]. After several specific interactions between the sandfly gut and the parasite, the procyclic promastigotes are transformed into its metacyclic form. The metacyclic form is covered with specific phosphoglycans that gives the promastigote the necessary protection and also the means of interaction (sandfly/parasite) [29–32]. In the metacyclic form, the parasite is motile, flagellated and fully virulent due to the specific surface proteins (HASPs, SHERP or LPGlipophosphoglycans) [33–35]. During the time of procyclic/metacyclic transformation, the parasite migrates in the sandfly gut to the stomodeal valve. After reaching this, the parasite destroys the stomodeal valve and causes a reflux of the parasite [36]. Consequently, when the sandfly is fed, the highly infective promastigote forms are

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Table 1 Simplified taxonomy of the Leishmania genus and the clinical manifestations caused. VL-visceral, CL-cutaneous, DCL-diffuse cutaneous, RL-recidivans, PKDL-post-kala-azar dermal syndrome, DsCL-disseminated cutaneous L., MCL-mucocutaneous [22–24] Subgenus

Species

Selected associated species

Spectrum of clinical manifestation in humans

Occurrence area

L. (Leishmania)

L. donovani

L. archibaldi, L. chagasi, L. infantum

VL, PKDL, CL

India, Bangladesh, Ethiopia, Sudan, East Africa

L. major

L. arabica, L. gerbilli, L. turanica

CL

Iran, Saudi Arabia, north Africa, Middle East, central and west Africa

L. tropica

L. aethiopica, L. killicki

CL, DCL, LR, (VL-rare)

Middle East, northern and southern Africa, eastern Mediterranean

L. mexicana

L. amazonensis, L. aristidesi, L. forattinii, L. garnhamii, L. pifanoi, L. venezuelensis, L. waltoni

DCL, CL, DsCL South America

L. braziliensis

L. peruviana, L. panamensis, L. shawi

CL, MCL, DCL, South America LR, DsCl,

L. (Viannia)

L. guyanensis L. lindenbergi L. utingensis L. lainsoni L. naiffi

inoculated into the host skin. In the host, the metacyclic promastigotes are immediately taken by phagocytic cells (neutrophils, macrophages) [37] and their transformation from the promastigote to the nonflagellated amastigote begins. Along with the parasite, the inoculated content that is injected into the host also contains sandfly saliva, the exosomes, the microbiome of the sand fly and PSG (promastigote secretory gel). It was found that PSG can modulate the virulence of the leishmanial parasites and thus influence the clinical form of the leishmaniasis [38, 39]. The immune response of the human host differs according to the parasite species, the clinical form of leishmaniasis, and the original state of health of the patient. The literature in general divides the clinical forms of leishmaniasis to two major clinical forms, visceral (VL) and cutaneous (CL) leishmaniasis, and recognizes several

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postdisease syndromes or complications connected with VL and CL as post-kalaazar dermal syndrome (PKDL), the mucocutaneous form (MCL), the disseminated form (DsCL), the diffuse form (DCL), coinfection (AIDS/leishmaniasis), and others [17]. In general, the widely spread L. donovani (India, Africa regions), L. infantum (Mediterranean areas) and L. chagasi (Latin America) cause visceral leishmaniasis (VL). While visceral leishmaniasis is endemic in more than 60 countries, 90% of all cases are reported from just seven countries: Brazil, Ethiopia, Kenya, Somalia, South Sudan, Sudan, and India [21]. In the case of L. donovani, the main host reservoir is the human, and for L. infantum, the domestic dog, but other mammalian reservoirs also exist [40]. The incubation period of the parasite ranges from 2 weeks up to 8 months, and the beginning of incubation can be sudden, asymptomatic, or variable, while the disease itself (leishmaniasis) can develop even a year after incubation when the patient immune system is suppressed. If not treated, VL is fatal and the host is killed often due to a secondary bacterial infection within 2 years after the parasite incubation [41]. The characteristic clinical features of VL are irregular and persistent fever and splenomegaly (Fig. 1). Additional symptoms, such as pancytopenia, hepatomegaly, hypergammaglobulinemia, and weight loss occur later on. In the case of children, fever, weakness, night sweats, weight loss, pallor, diarrhea, and growth dysfunction can also be observed [42, 43]. In the Indian subcontinent, VL disease can cause hyperpigmentation due to an increased production of adrenocorticotropic hormone. Such an observation has served as an inspiration for the Hindi name of VL disease, namely, kala-azar (black fever) [44, 45]. Cutaneous leishmaniasis (CL) is a zoonotic disease with numerous mammalian reservoirs, although in highly agglomerated areas of India, Afghanistan, and Sudan

Fig. 1 Child patient with visceral leishmaniasis with visible hepatosplenomegaly, and a male patient with post-kala-azar dermal leishmaniasis, which was previously treated, and cured from visceral leishmaniasis. Source: World Health Organization, https://www.who.int/campaigns/world-healthday/2014/photos/leishmaniasis/en/

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Fig. 2 Male patient with mucocutaneous leishmaniasis with noticeable destruction of nose (left). Child patient (Kabul, Afghanistan) with cutaneous leishmaniasis, characterized by raised borders and ulcerated center (right). Source: World Health Organization, https://www.who.int/campaigns/ world-health-day/2014/photos/leishmaniasis/en/

it can easily become anthroponotic [46–48]. Both L. (Leishmania) and L. (Viannia) can cause CL. Clinical features of CL are diverse and are parasite dependent. In comparison with VL, CL is not a lethal disease, but the scarification of large skin areas can lead to unpleasant social and physical burdens for CL patients. In immunocompromised patients, CL can develop into the diffuse cutaneous form (DCL) of the illness [49]. The clinical manifestation of CL can range from single, chronic ulcerative lesions (often with the appearance of various ulcers) to nonulcerative, disseminated nodular lesions (Fig. 2). From the treatment viewpoint, CL, in most cases, heals spontaneously after persisting for months or years and leaves atrophic scars as a “memento” of its presence. In contrast, the DCL form, a rare variant of CL caused by L. aethiopica, L amazonensis, and L. mexicana that is characterized by satellite lesions, may persist for even decades before being healed [46, 50]. Mucosal leishmaniasis associated with L. braziliensis is characterized by mucosal lesions of the nose, mouth, or larynx areas. It develops months after the primary skin lesions are healed. The mucosal tissue inflammation is followed by ulceration and perforation of the septum (Fig. 2). It is also nonfatal disease, but the tissue destruction may have a very heavy social impact [46]. Transmission of leishmanial parasites is vector dependent, and therefore its propagation should, in principle, be “controllable.” Unfortunately, the main outbreaks of this disease are in the poorest areas of the world. Thus, malnutrition and the lack of adequate healthcare and of other most basic commodities (clean water, drugs, protective nets, etc.) are the main reasons why to date all preventive actions and vector-spread control programs fail. Since these failures, and as no current efficient

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Table 2 Recommended drugs for the first-line treatment and drugs under the clinical trials. (Adapted from [52]). CL (cutaneous leishmaniasis), VL (visceral leishmaniasis) Leishmaniasis form

Drug options

Treatment schedule

VL

Pentostam (1), glucantime (2) amphotericin B (3) liposomal amphotericin B (3) pentamidine (4)

First line

VL

Miltefosine (5) paromomycin (6) sitamaqine (7) amphotericin B (3) (different types)

Clinical trials

CL

Pentostam (1), glucantime (2) amphotericin B (3) pentamidine (4) paromomycin (6) (with urea, methylbenzethonium chloride)

First line

CL

Miltefosine (5) paromomycin (6) sitamaqine (7) imiquimod (8) ketoconazole (9) fluconazole (10) itraconazole (11)

Clinical trials

vaccine is available, the main weapon against leishmaniasis is chemotherapy [51, 52] (Table 2). Current chemotherapy relies on a few key drugs — antimonial-based compounds (1 and 2), amphotericin B (3), pentamidine (4), miltefosine (5), paromomycin (6), and sitamaquine (7) (Table 3). Amphotericin B (3) is a macrolide-based antifungal antibiotic, and the antileishmanial activity of this compound was first recognized in the early 1960s. From the biological mode of action viewpoint, it is believed that amphotericin B (3) increases the permeability of the parasitic membrane by targeting ergosterol receptors located on the surface of promastigotes and amastigotes. The influx of ions to the parasite is then the cause of parasite death [53]. Antimonybased compounds, known under the names of pentosam ((1), sodium stibogluconate) and glucantime ((2), meglumine antimonate), are the oldest treatments used against leishmaniasis. Their mode of action is believed to be based on the interference of an antimony complex with enzymes involved in fatty acid oxidation and glycolysis [54]. Pentamidine (4), on the other hand, is thought to interfere with replication and transcription mechanisms in parasite mitochondria [55]. Miltefosine (5) was originally an antitumor agent that was latterly approved for leishmaniasis treatment. It is considered that 5 interrupts the proliferation of the parasite via interference with sterol and phospholipid-dependent cell signaling pathways. Some other studies suggest that the proliferation interruption is caused by the interaction with enzymes of the lipid metabolic pathway [56]. Paromomycin (6), originally an antibiotic, was shown to be an efficient antileishmanial drug if used in combination with antimonial

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Table 3 Structure, administration, and the mode of action (presumed) of the main antileishmanial drugs. (IV, intravenous; IM, intramuscular; per os, oral administration) Structure

Drug

Administration mode of action

Pentostam (1) IV/IM

interference with energy metabolism of parasite, interference with parasitic DNA topoisomerase I

Liposomal Amphotericin B (3)

IV

targets ergosterol in the surface membrane of promastigotes and amastigotes, increases the membrane permeability and ion influx

Pentamidine (4)

IV (IM)

interference with parasite DNA

Miltefosine (5)

per os (oral)

Interference with lipid dependent cell signaling pathways (continued)

drugs (via a synergistic effect). Under such conditions, it proved to be active against intestinal protozoa, and a wide range of bacteria. It is believed that in protozoal, metabolism, paromomycin (6) inhibits protein synthesis by binding to the 30S ribosomal subunit [57]. Sitamaquine (7) was developed to treat visceral leishmaniasis by oral administration. It is known that it accumulates inside the parasite, although the precise mode of action (or even a reliable hypothesis) is still unknown. The drug has no significant activity against CL [58]. Complications connected with the administration of the above-mentioned drugs such as toxicity and increasing drug resistance are forcing medical personnel in the “front line” to use various combinations of the above-mentioned “first line drugs.” Necessity has even driven the testing of drugs that have been shown previously to be inefficient against leishmaniasis. The driving force behind such an approach is the hope of finding synergistic effects between administered drugs. Some successes

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Table 3 (continued) Ketoconazole (9)

per os (oral)

Fluconazole (10)

per os (oral)

Paromomycin Local (6)

Inhibition of the cytochrome P450-mediated 14α-demethylation of lanosterol and blocking ergosterol synthesis

Antibiotic – broad spectrum of action

in treating CL and VL were found for the antimycotic drugs ketoconazole (9), fluconazole (10), and itraconazole (11) [59, 60]. The proper choice of drug(s) for the treatment of leishmaniasis is influenced by many factors, and one has to take in account the species of parasite, the clinical form of the disease, the general health state of the patient, possible coinfections, and the resistance to those drugs administered in a given region. Even though the mode of action of various drugs differs substantially, in general, efficacious compounds for antileishmanial treatment all in some way enhance the immune system of the patient, e.g., by activating/generating nitrogen and oxygen metabolites toxic to amastigote parasites [61]. The urgency of developing new antileishmanial agents is now considerable, as the incidence of leishmaniasis in Europe, China, and the southern USA has gradually increased in recent years. This situation is related to global climate changes (e.g., the presence of natural vectors such as P. papatasi, P. sergenti s.l., and P. tobbi has been observed in Mediterranean forests [22, 62, 63]) and the socioeconomic migration of people from areas having an endemic occurrence of leishmaniasis. In addition, resistance to first-choice drugs is slowly increasing in most of the regions affected and is increasing dramatically the need to search for new treatments. Several chemical entities have been reported recently with promising antileishmanial activities that have

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originated from laboratory research [64, 65] were inspired by traditional medicines [66]. Unfortunately, many of these compound leads are only at the beginning of their journey from the laboratory to patients [64–66]. As will be suggested in the next sections of this chapter, it is believed that structural design based on the skeletons of phenylpropanoid dimers can bring an answer to the search for new antileishmanial drugs.

3 Secondary Metabolites of the Shikimate Pathway Behind the colorful and diverse world of phenylpropanoid (PP) metabolites is the shikimate pathway (SP) of plant biosynthesis, the producer of aromatic compounds. The shikimate pathway can be seen as a seven-step enzymatic process that interconnects the primary metabolism products, phosphoenolpyruvate ((13) from glycolysis) and d-erythrose-4-phosphate ((12) from the pentose phosphate pathway) [67] with phenylpropanoid metabolites (Fig. 3). In short, the aldol condensation of 12 and 13 that is catalyzed with 3-deoxy-d-arabino-heptulosanate 7-phosphate synthase (DAHPS) produces 3-deoxy-d-arabino-heptulosanate 7-phosphate (14) (the metabolic regulation of this process in plants seem to be preferentially controlled at genetic level) [67]. Generation of a phosphate-intermediate is followed by enzymatic transformation with the participation of five enzymes [68, 69]. In the second step of transformation, enolization/intramolecular aldolization catalyzed with monomeric 3-dehydroquinate synthase and requiring NAD+ , yields the tertiary alcohol-containing 3-dehydroquinic acid (15) [68]. Compound 15 is then dehydrated to 3-dehydroshikimate (16). The reaction is catalyzed by 3-dehydroquinate dehydratase that exists in two forms, type I and II. These differ in their secondary structure (50% vs. 75% of α-helix) and in the mechanism whereby H2 O is eliminated (synvs. anti-elimination) [70]. Type I (characterized in Escherichia coli), uses Schiff base (lysine) formation in the active site to eliminate the H2 O molecule. This site is not present in type II 3-dehydroquinate dehydratase (characterized in Aspergillus nidulans) and is known to eliminate the water molecule via an anti-elimination mechanism [71, 72]. Next, the reduction of 16 to shikimate (17) is carried out. The reduction is catalyzed by the bi-functional enzyme complex DHQ/shikimate dehydrogenase and NADP-oxidoreductase, which uses NADP (in plants) as a cofactor [69, 73, 74]. Shikimate (17) is phosphorylated by shikimate kinase and generates the shikimate-3-phosphate (18). Irreversible phosphorylation of 17 is driven by ATP substrate formation and is linked with the chloroplast environment [75, 76]. At this stage, phosphoenolpyruvate (12) becomes involved and reversible condensation with 18 results in the formation of 5-enolpyruvylshikimate-3-phosphate (19) and HPO4 2– . The reaction is mediated by EPSP-synthase (the only target for the glyphosate herbicide that is supposed to make this minimally toxic for human and environment) [77]. Finally, a “deoxygenation” step generates the key intermediate of AAA metabolism in plants (chorismate (20)). The chorismate (20) generation is characterized by an anti-1,4-elimination of the phosphate group from 19 [78]. Chorismate synthase is

OH

OH

O O OPO3H2

O

13 (phosphoenol pyruvate)

H2 C

OH

OH

OH

O CO2H

O

CO2H

CO2H

CO2H

CO2H

20 ((3R,4R)-chorismate)

HO

O

19 ((3R,4S,5R)-5-enolpyruvyl shikimate-3-phosphate)

H2O3PO

HO

14 ((4R,5S,6R)-3-deoxy-arabinoheptulosonate-7-phosphate)

H2O3PO O

OH

CO2H

HO2C

OH

OPO3H2

OH

21 (L-tryptophan)

CO2H

NH2

OH

OH

HO2C

OH

OH

CO2H

25 (L-phenylalanine)

H2N

23 ((1S,4R)-1-(S)-arogenate)

OH

NH2 CO2H

CO2H

HO

arogenate dehydratase

17 ((3R,4S,5R)-shikimic acid)

OH

shikimate-5-dehydrogenase

prephenate amniotransferase

shikimate kinase

22 ((1S,4S)-prephenate)

OH

O CO2H

CO2H

HO2C

O

16 ((4S,5R)-3-dehydroshikimic acid)

3-dehydroquinate dehydratase

18 ((3R,4S,5R)-shikimate-3-phosphate)

chorismate mutase

N H

HO

HO

15 ((1R,3R,4S)-3-dehydroquinic acid)

3-dehydroquinate synthase

3-deoxy-D-arabino-heptulosonate-7-phosphate synthase

12 ((2S,3S)-erythro-4-phosphate)

plastid

Fig. 3 Shikimate pathway – from chorismic acid to aromatic amino acids and phenylpropanoids [88–90]

shikimate pathway

H2O3PO

CO2H

24 (L-tyrosine)

H 2N

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Fig. 3 (continued)

phenylpropanoid metabolites pathway

cytoplasma

OH

O

33

HO

B

cell wall

isoflavonoids

O

O

A

32 ((2R,3R)-epi anthocyanidins

HO

OH

OH

34

lignans

C

HO

OH

OH

flavonoids

O

O

31 ((E stilbenes

OH

OH HO

OH

OH

COSCoA

CO2H

CO2H

O

CO2H

37 ((E)-sinapic acid)

HO

O

36 ((E)-caffeic acid)

HO

HO

35 ((E)-ferulic acid)

HO

O

30 ((E)-4-coumaroylCoA)

HO

3-malonyl-CoA

OH

29 coumarins

HO

HO 28 ((E)-4-coumaric acid)

CO2H

O

O

26 ((E)-cinnamic acid)

CO2H

phenylalanine ammonia lyase

OH

27 (salicylic acid)

CO2H

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responsible for the last step of the shikimate pathway, using flavin that serves as an electron donor for 19 [79, 80]. The shikimic pathway is an important active carbon transport machinery, and, since it is exclusively located in fungi, bacteria and plants, it could be an appropriate target for novel antibiotic or herbicide development (an ideal and “safe” target for novel drug candidates) [69, 79, 81–83]. By chorismate (20) generation, the shikimate pathway enters a crossroad from which three aromatic acids, tryptophan (21), and especially tyrosine (24) and phenylalanine (25) (via prephenate (22) and arogenate (23), catalyzed by chorismate mutase and prephenate aminotransferase), are derived. Phenylalanine (25), as one of the key final products of the shikimate pathway, is then transformed to the key phenylpropanoid group member, (E)-cinnamic acid (26). This transformation is catalyzed by one of the most well-studied plant enzymes, phenylalanine ammonia-lyase (PAL). The key function of this enzyme is the deamination of phenylalanine to yield (E)-cinnamic acid (26). Further transformation of 26 mediated by cinnamate-4-hydroxylase (C4H) and 4-coumarate/coenzyme A ligase (4CL) yields (E)-4-coumaric acid (28) and (E)-4-coumaroyl-CoA (30), respectively. All additional phenylpropanoid-based structures, regardless of their complexity, originate from these compounds. Such complexity can be either introduced via “oxidative” modification of the original structure (phenylpropanoid acids, coumarins), dimerization and subsequent modification of dimers (lignans and neolignans), or via condensation of the phenylpropanoid unit with malonyl coenzyme A (e.g. flavonoids). Overall, these transformations rely on the vast variety of enzymes such as chalcone synthase (CHS), chalcone flavanone isomerase (CFI), flavone 3-hydroxylase (F3H), or dihydroflavonol reductase (DFR), to name just a few, and yield important groups of secondary metabolites such as lignans, sinapate esters, coumarins, isoflavonoids, stilbenes, aurones, flavones, flavonols, and anthocyanins. The regulation of these processes is maintained by transcription factors that are closely linked to the biotic and abiotic factors that surround the plant [84, 85]. The overall process is then carried out in the outer membrane of the endoplasmic reticulum [84].

3.1 Phenolics During their long evolution, plants have had to adapt to various and dramatically changing biotic and abiotic conditions. As previously mentioned, their answer to such challenges was the development of a massive arsenal of secondary metabolites (SM). The “move” of plants from aquatic conditions to the point where they were fully adapted to life on land, was accompanied with a huge burst in the biosynthesis of phenolic compounds. A main driving force for this is that phenolics can protect plants against deadly UV irradiation [86, 87]. This might also be the reason why phenolics became one of the most widely distributed groups of secondary metabolites in plants (these are uncommon in bacteria, fungi, and algae) [88–90]. To be classified as “a phenolic compound,” it must contain at least one phenolic unit, and these vary from simple compounds to complex

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OH

C6-C2-C6 stilbenes anthraquinones (C6)n catechol melanins

C6 simple phenols benzoquinones

C6-C1 phenolic acids

C6-C1-C6 xanthones

C6-C2 acetophenones phenylacetic acids

C6-C4 naphthoquinones C6-C3 hydroxycinnamic acids coumarins, phenylpropenes chromones

C6-C3-C6 flavonoids isoflavonoids

(C6-C3)2 lignans neolignans

(C6-C3-C6)2 biflavonoids

(C6-C1)2 hydrolyzable tannins (C6-C3)n lignins

(C6-C3-C6)n condensed tannins

Fig. 4 Known phenolic secondary metabolites classified according to their carbon content (Cx)n [88, 90]

polyphenolic molecules such as highly polymerized derivatives. The classification of phenolics that is based on the C6 (benzene ring) carbon content divides this complex group to 14 subgroups (Fig. 4) [88–90]. Each structural feature (group) can then have quite different roles in plants. Indeed, phenolics are involved in pigmentation (mostly in red, blue and purple pigments), in growth and reproduction processes, and in the mechanisms of resistance to pathogens. They also actively protect plants against bacterial (antibiotic purposes) and herbivore (toxic compounds) attacks, and play a key role in plant/plant and plant/animal communications/relationships (Fig. 5) [88, 90]. The specific role of phenolics then determines the place of their accumulation/localization within the plant tissues and organelles. In general, they can be found in central vacuoles, epidermal and subepidermal cells (leaves and shoots), attached to cell walls (covalently) or as part of waxes. They can also be found in nuclei as complexes with DNA (e.g., flavonoids that protect against oxidative stress) [91–96].

3.2 Phenylpropanoids and Their Impact on Human Health The biochemical shikimate pathway (SP) is present only in microorganisms and plants, although metabolites related to those from the SP, such as aromatic amino acids [69], are also essential to animals. Thus, plants and SP-containing microorganisms are the only source of such compounds. It is not surprising then that phenolics as such abundant plant secondary metabolites are consumed by animals daily in

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Fig. 5 Schematic overview of plant/environment interactions that are mediated by phenolic compounds

large quantities. Again, not surprisingly, some plant secondary metabolites interfere with animal (human) metabolism and are able to mimic, for example, human hormones (e.g., lignans as phytoestrogens). Due to the wide diversity of SM in plants, their biological activities are diverse and sometimes unexpected. Already, to date, several hundred SM-related compounds are used in the agrochemical, cosmetic, and pharmaceutical industries daily [97]. In the present contribution, it is intended to highlight primarily the pharmacological benefits of lignans and neolignans for their important roles in the drug discovery and development process. Another important aspect to be discussed is the beneficial activities of natural compounds (lignans and neolignans in particular) on organisms (animals/humans). The present authors believe that such information is a key to new drug development and may help eradicate or reduce the increasing incidence of drug resistance and other negative side effects apparent in current chemotherapy. Indeed, the composition of SMs, and of phenylpropanoid metabolites in particular, may change with time depending on several factors [1, 4, 97]. Thus, the composition of the nutrients consumed by humans may change as well and can positively or negatively influence the above-mentioned aspects of drugs on patients. If the basic biological activities of various classes of SM are known, such information in combination with the amounts of SMs in various nutrients may influence the effects of

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administered drugs on human health. In the case of leishmaniasis, it has been established that the impact of nutrition and coinfection on drug treatment is high. Thus, lignan and neolignan structures that may have a positive value in the search for new biologically active antileishmanial compounds are indicated.

3.3 Bioactive Lignans and Neolignans Lignans and neolignans, as phenylpropanoid dimers, may have a beneficial effect on human health, in terms of both potential drug therapy and disease prevention [98–100]. The term “beneficial to human health” includes not only direct biological activities related to specific compounds or drugs but also to the ability of such molecules in helping prolong human life by direct or indirect interaction with additional compounds or receptors in the body [101]. The great potential of phenylpropanoid dimers results from their capacity, alone or in a synergistic way with several other phenolics, to interact with enzymes, receptors, or even directly with DNA (or other so-far-unknown targets) [90, 102, 103]. Up to the present, more than 8,000 phenylpropanoids have been detected and characterized. However, only a few have been fully characterized with respect to their biological activity profiles. Most importantly, their effects (short- or longer-term) on human health are mostly unknown [104]. In this part of the chapter will be mentioned numerous lignans and neolignans with various biological activities that potentially are beneficial to human health. These will be dealt with according to the system or organ that may benefit from the presence of such molecules. The effects of various structurally rather similar compounds are wide. From the literature, this range of biological activities is referred to in terms of the “scavenger theory” [105, 106]. This theory assumes that phenolic compounds in general are capable of “disabling” free radicals via the formation of stabilized chemical complexes [107]. Some other studies have suggested that plant phenols may interfere with the way that peroxo/nitro radicals are formed and thus diminish the impact of oxidative stress on the organism [107]. Another theory indicates that the previously observed interference of phenolics (lignans and neolignans) with oxidative stress or radical generation/scavenging has an impact on the immune response of the organism and serves as an endogenous modulator [108]. Unfortunately, a clear mode of action is still unknown for many phenolic compounds of plant origin [101]. What is interesting is that all currently used models suggest that the mode of action of these phenols, inclusive of lignans and neolignans, starts with immune response modulation. Depending on the model, this modulation is either direct (lignan receptor) or indirect (interaction with radicals or peroxides, for example) [105–108]. This is thought to constitute the mechanism of most of the currently used antileishmanial compounds, so that in some way they all enhance or regulate the immune system of a patient [61].

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Neuroprotective activities

Neurodegenerative diseases affect many aspects of human life. Memory, cognition, the ability to speak and to understand, and/or the unique personality of patients may be affected. The neurodegenerative impact on the central nervous system (CNS) is a result of a gradual and progressive loss of neuronal cells, or is a consequence of intra- or extracellular protein aggregate accumulation. To date, more than 600 neurological disorders have been described and recognized, but the CNS-related “prominent” disorders are Alzheimer’s disease (AD), Parkinson’s disease (PD), cerebrovascular disease, multiple sclerosis (MS), and epilepsy [109, 110]. Concerning their pathophysiological mechanisms, oxidative stress and mitochondrial dysfunction or neuroinflammation have been suggested as the causes of these neurodegenerative diseases [109, 110]. Phenylpropanoids and phenylpropanoid dimers have neuroprotective properties, and therefore might play a crucial role in neurological disease prevention. Moreover, it has been observed that some of these compounds may have a positive impact in the treatment of such diseases by removing or alleviating certain of their symptoms [111, 112]. Previously, it was demonstrated experimentally that the flavonoids myricetin (40) and quercetin (41) (Fig. 6), when present a Ginkgo biloba extract, have protective properties against dementia development [113]. It was observed that these compounds can modify neural gene expression in mice. However, it is unclear how these compounds might enter the human brain [114–116]. Similarly, long-term administration of the phenylpropanoid ferulic acid (36) enhances the resistance of the organism to toxic effects of the β-amyloid peptide [117]. This β-amyloid peptide is believed to induce oxidative stress and inflammation in the brain, processes that play important roles in the pathogenesis of Alzheimer’s disease [117]. Curcumin (38), resveratrol (31), and some catechins (e.g., epigallocatechin gallate (39)) might also have protective properties against Alzheimer’s disease and other types of dementia, since they are known for their antioxidant and immunomodulatory properties. Thus, OH OH O

O

O

OH

OH O

HO

OH O

HO

O

OH

O OH OH 39((2R,3R )-epigallocatechin gallate)

38 (curcumin) OH

O

O

OH HO

OH

O

OH OH

40 (myricetin)

OH OH

HO HO

O

OH

OH

HO

41 (quercetin)

Fig. 6 Selected phenylpropanoids with neuroprotective activity

42 (honokiol)

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possibly they also can protect neurons against the neurotoxic effects of β-amyloid protein accumulation. However, in these cases, such effects have not been proven [118–122]. Honokiol (42) (a neolignan extracted from species of the genus Magnolia) also possesses CNS-protective activity. Honokiol is found in a number of traditional Chinese medicine preparations that are utilized for their beneficial effects. In the case of neuroprotective activity, it is considered that the protective properties of honokiol (42) result from the inhibition of prostaglandin E2 production in the brain [123–125]. In addition, compound 42 is also a suppressor of proinflammatory cytokines, which influence the oxidative damage of tissues, and exhibits antiviral properties (against dengue virus type 2), and also has antitumor potential and improves learning abilities and memory damage in mice [126, 127].

3.3.2

Antioxidative Activity

It has been known for some time that reactive oxygen species (ROS) have a harmful impact on cells, since they cause structural or functional damage to lipids, proteins, or nucleic acids [128, 129]. Phenylpropanoids [130, 131] and other plant phenols [132] behave as strong antioxidants with the ability to transform a wide range of reactive chlorine, nitrogen, or oxygen radical species to harmless atoms and functional groups. In addition, they are also strong chelating agents with the propensity of chelating various metal ions. Thus, it seems logical that they could be used in a protective capacity (e.g., as nutritional supplements), with the aim of retarding or suppressing age-related degenerative diseases. It should be pointed out, however, that the real impact of such compounds on age-related degenerative illnesses is so far speculative, since none of the studies focused on this topic is conclusive [133, 134]. It was also suggested that ROS play a key role in various mechanisms that lead to nephropathy [135]. Some scientific studies have produced indirect evidence that phenylpropanoids and especially more complex phenolic compounds could play an important role in diminishing the quantities of ROS in the human body [136, 137]. Resveratrol (31) (Fig. 7), a stilbene-type phenolic compound, possesses antiinflammatory, antioxidative, antiaging, and antidiabetic effects, among other biological activities. In terms of its mechanism of action, it has been observed that 31 acts as NAD-dependent deacetylase sirtuin-1 activator and also is an activator of various antioxidant enzymes via interacting with the nuclear factor erythroid 2-(Nrf2)- Kelch-like ECH-associated protein 1 (Keap1) [138, 139]. In addition, 31 can modulate nuclear factor kappa-B (NF-κB), nitric oxide synthase (iNOS), and inhibit cyclooxygenase [138, 139]. Moreover, it was observed that 31 may decrease the risk of proteinuria, hypoalbuminemia, and hyperlipidemia [138, 139]. However, for these activities, the mechanism of action in each case is not clear. Accordingly, it is apparent that 31 “interacts” in the human body in many ways. To summarize its type of activity, a comprehensive list of the effects of 31 is provided according to the tissue where it acts, or in the case of cardiovascular prevention, according to the manner it is believed to act (Fig. 8) [140–144].

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Fig. 7 The biological activity of resveratrol related to muscle, liver, adipose and pancreatic tissue, and its cardiovascular preventive actions

Fig. 8 Selected phenylpropanoids and phenols with antioxidant activity

OH

O OH

HO

HO

OH

O HO OH

O OH O HO

O

O

O OH

OH

44 (gallic acid)

OH

OH

OH O

O

HO

HO OH

HO

OH OH

43 (α-D-glucosyl-rutin)

O

OH

HO

45 ((2R,3S)-catechin)

The flavonoids quercetin (41) and α-glucosyl-rutin (43) were also demonstrated as potent antioxidative compounds, since they are able to scavenge small free-radicals • such as HOO• , O•– 2 , and NO . Additionally, these molecules display other biological activities including renoprotective [145], antidiabetic, and antitumor activities [146–148]. Similarly, phenolic compounds found in wine, such as anthocyanins, resveratrol (31), gallic acid (44), catechin (45), myricetin (40), quercetin (41) and many other related compounds, may be expected to be potent antioxidants and have a role in producing diminished levels of ROS in human plasma [140–144, 149, 150].

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Indeed, it is probable that all previously mentioned plant phenolic compounds do not interact with cellular targets as such. It is likely that after their absorption into the bloodstream they are glycosylated, methylated, or glucuronidated. Accordingly, the newly generated conjugates would then be responsible for the biological activities observed [151, 152].

3.3.3

Biological Activities Related to Cancer, Inclusive of Inhibition of Cellular Proliferation and Induction of Apoptosis

Angiogenesis is a fundamental process that is coordinated by various growth factors and the cell adhesion of molecules (in endothelial and mural cells) that leads to premature degradation of extracellular matrixes, and to the migration and proliferation of endothelial cells [153, 154]. Degradation of the extracellular matrix is mediated by metalloproteinases that allows the generation of new and abundant blood vessels [155, 156]. From the cancer viewpoint, angiogenesis plays an important part in tumor mass generation, and therefore it is essential in tumor expansion. Not surprisingly, the United States Food and Drug Administration (FDA) has approved certain angiogenesis inhibitors as a novel treatment option for cancer [157]. Recently, it was discovered that mixtures of phenols in red wine and green tea prevent thrombin-induced activation of MMP2 in vascular smooth muscle cells. Epigallocatechin-3-gallate (39) and epicatechin-3-gallate (46) (both green tea phenolic esters, Fig. 9) can mimic a MMP-2-activation inhibitory effect [158, 159]. It was also discovered that these phenolic substances prevent Vascular Endothelial Growth Factor (VEGF) expression. This major proangiogenic factor can stimulate endothelial cell migration, proliferation, and the formation of new blood vessels. Red wine phenols as well as anthocyanins such as delphinidin and cyanidin can prevent VEGF expression and its release [160, 161]. The effects of dietary phenols on vascular endothelial and smooth muscle cells (increase in vasodilatation, antiproliferative effects, antithrombotic factors, etc.) suggest their antiatherogenic and vascular-protective role [154]. Such compounds also exhibit a role in mediating the migration and proliferation of vascular cells. In this context, resveratrol (31) (increasing the expression of the tumor suppressor gene, protein p53), delphinidin (47) (through the cyclin D1 and A dependent pathways), and epigallocatechin-3-gallate (39) (induction of apoptosis), have shown some effectiveness in this regard [161–163]. Interestingly, it was also demonstrated that the antioxidant activity of the abovementioned phenols is maintained even if they are administered orally. In such cases, the naturally occurring forms of these compounds are possibly activated by bacterial (intestines) and human (oral) enzymes [164]. Catechin (32) (present in tea) when activated via this mechanism was shown to inhibit production of the metalloprotease enzymes, inducing cell arrest and apoptosis [165–167]. Other mechanisms of action concerning such activation have been shown by methoxylated flavonoids (present in citrus fruits, pepper, and betel). The newly generated metabolites inhibit the formation of DNA adducts. These types of adducts are generally formed upon the influence

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OH HO

O

O OH

O

HO

OH HO

OH

O

OH

OH

OH O

OH

R

O

OH Cl

OH

OH

OH

OH

46 ((2R,3R)-epicatechin-3-gallate)

47 (delphinidin, 48 (cyanidin, R = H)

49 (kaempferol)

HO

HO

OH

O O

O

O

O

O

O

O

O

O

O O

O

O

O

O

O O

OCH3

O O O

54 (tuberculatin)

50 (tangeretin)

OH HO O

OH

S O

O

OH O

55 (justicidin A)

O

O

O

O

O

O

O

O

O

O

OH

O

and O

O

O O

O O

O O

O

O OH

51 (podophyllotoxin)

52 (teniposide)

O

O OH

53 (etoposide)

Fig. 9 Selected examples of phenolic compounds with important antioxidant activity

of tobacco nitrosamines [168]. Such activity was observed to a differing extent for each of anthocyanins, catechins, flavanols, flavones, and isoflavones [169], and their effects were shown in colon, prostate, epithelial, and endometrial cells, and, in one case, breast cancer cells [101, 170, 171]. Quercetin (41), a common flavonoid and one of the most well-studied phenolic compounds, may be used as a dietary supplement during treatments for cancer, diabetes, or digestion-related diseases. In addition, 41 also shows anti-inflammatory, antiviral (against influenza A virus), antiulcer, antiallergenic, and antiproliferative activity, and can modulate cardiovascular diseases [172–175]. It was also demonstrated that quercetin can inhibit the growth of human ovarian (OVCA 433) and promyelocytic leukemia (HL60) cells [176–178]. In addition, the metastatic potential of breast adenocarcinoma cells was diminished upon

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administration of 41 (rat adenocarcinoma cells). Quercetin (41) also inhibited proliferation in case of non-small cell lung carcinoma cell lines and hepatoma cell growth (ML-3 murine hepatoma cells) [179–181]. Tangeretin (50) (a methoxylated flavone) and genistein (33) (an isoflavone) were shown to suppress the growth of HL-60 cells. Genistein (33) and kaempferol (49, a widely studied flavonol) were found to inhibit the proliferation of human colon cancer cells (Caco-2 and HT29) [182–185]. The potential protective effects of kaempferol (49) have been described further for cardiovascular diseases, obesity, and inflammation. Based on mechanistic studies, it was concluded that 49 may reduce adipose tissue accumulation and improve the symptoms of diabetes and hyperlipidemia (in mice) [186–191]. It was demonstrated that 49 can also mediate apoptosis in pancreatic cancer cells via inhibition of the EGFR/p38 signaling pathway, and also upregulate p21 expression and downregulate cyclin B1 expression in cancer cells, leading to apoptotic cell death [192–194]. The most prominent phenylpropanoid dimer with antineoplastic activity is podophyllotoxin (51). This aryltetralin lignan may be extracted from Podophyllum peltatum and was originally used medicinally in an ointment to treat genital warts and molluscum contagiosum [195, 196]. Lignan 51 has the ability to bind to tubulin and disrupt microtubule synthesis. Since this compound itself has severe secondary toxic effects on the human body, structural modification was required to decrease its toxicity while maintaining unchanged the original antiproliferative activity (see the structures of etoposide (53) and teniposide (52), Fig. 9). These two epipodophyllotoxin glycoside derivatives (52 and 53) were approved by the U.S. FDA as novel anticancer drugs for the treatment of various types of cancer (including Kaposi’s sarcoma, certain lymphomas, lung cancer, and testicular cancer) [197]. The mode of action of both etoposide (53) and teniposide (52) is based on their interaction with the topoisomerase II enzyme, which prevents the religation of DNA strands [198–201]. Justicidin A (55) is structurally similar to podophyllotoxin and was isolated from the plant Justicia procumbens L. (Acanthaceae). This plant is used in Chinese traditional medicine to treat fever, pain (via pharyngolaryngeal swelling), and also tumor formation [202]. The mode of action of justicidin A (55) seems to be the same as that of tuberculatin (54) and presumably activates tumor necrosis factor R [203].

3.3.4

Anti-inflammatory Activity

Inflammation is the basic response of the immune system to an irritant stimulus (e.g., from bacteria, viral or fungal pathogens, radiation, chemical damage, or wounding of tissues). The external characteristics of inflammation include redness, heat, swelling, pain, and loss of function, but not all of these symptoms are always present. During this process, the immune system can identify both the damage made and the pathogen responsible and to isolate these and promote healing. However, if the immune system is not working properly, the wrong tissue can be attacked (repetitively) and undesired (chronic) damage can take place [204]. Various phenolic compounds act to prevent such toxic effects (Fig. 10). In general, they are able to restore the redox balance via interactions with ROS. Therefore, the

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OH

O

O

O

O

O

O

O O

O O

O

O

O O

O

O

O

O

O

57 ((3S,4S)-5''-methoxy-yatein)

56 ((3R,4R)-arctigenin)

58 (taiwanin C)

OH

O

O

O

O

O

OH

OH

O HO

O

O

O

O

O

O

O

OH HO

59 ((R,Z)-savinin)

60 ((6R,8R)-alashinol A)

61 ((1R,3aS,4S,6aS)-asarin B)

OH

O

O

OH O

O

O

HO

O

HO

OH

HO

O

O

O

OH

OH O

O

O

62 ((1S,2R)-neoasarinin A)

64 ((1S,2S)-neoasarinin C)

63 ((1R,2S)-neoasarinin B)

HO HO

OH O

O O

O HO O

HO

OH OH

HO

65 ((1S,2R)-neoasarininoside A)

O O

OH

O HO

O

O

OH

HO

O

O

HO

O

O

OH

OH

O

HO

OH

O O

HO O

OH OH

HO

66 ((1S,2R)-neoasarininoside B)

O OH

HO

67 (secoisolariciresinol diglucoside)

Fig. 10 Examples of phenolic compounds with reported cytotoxic activity

level of oxidative stress in tissues can be regulated and the response of the organism to the irritation may be moderated [170]. A well-known plant compound with such properties is the diarylheptanoid curcumin (38), which has the ability to suppress the expression of proinflammatory cytokines (TNF, IL-1, ICAM (intracellular adhesion molecule-1)) and VCAM (vascular cell adhesion molecule-1) in human umbilical vein endothelial cells (Fig. 10). Curcumin (38) is also able to suppress enzymes that are important in the inflammation process (COX, LOX, MAPK and KIK) [205].

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From the lignan compound class, (–)-arctigenin (56) (extracted from Arctium lappa) shows interesting activity due its capability to decrease NO• levels and those of a pro-inflammatory cytokine. Other biological activities that have been determined for (–)-arctigenin (56) include antiviral (against influenza A), antioxidant, and antiproliferative activities (inhibition of protein kinase B in PANC-1 pancreatic cancer cells). It was also observed that compound 56 is, upon metabolism by intestinal bacteria, transformed to a group of structurally related bioactive metabolites [206–210]. The antiproliferative activity of lignan 56 is believed to be caused by inhibition of the transcription factor STAT3. In addition, several other cytotoxic activities were reported for arctigenin (56), against breast cancer (MDA-MB-231, -435S, -453, and -468, with IC 50 values ranging from 0.285 μM to 3.756 μM), gall bladder cancer (by modulation of epidermal growth factor pathway), and human glioma (by induction of G0/G1 cell cycle arrest) cells [197, 211–213]. The biosynthetically related lignans, 5 -methoxy-yatein (57) and taiwanin C (58), were shown to interact via COX-2 (the cyclooxygenase-2 enzyme) to inhibit the transformation of arachidonic acid to prostaglandin E2 (PGE2). Prostaglandins are required to trigger the inflammation process [214]. Savinin (59) modulates the inflammatory process via the inhibition of tumor necrosis factor-alpha (TNF-α) and as a result of the proliferation of T cells [215]. Alashinol A (60) inhibits the production of TNF-α and interleukin-6 [216]. The lignans asarinin B (61), neoasarinin A–C (62–64), and neoasarininoside A (65) and B (66), isolated from Asarum heterotropoides, also possess anti-inflammatory activities, which are associated with the inhibition of PAF (platelet activation factor), based on the cell-based assay used [217]. Secoisolariciresinol diglucoside (67) (a lignan glycoside extracted from flax seeds) may be able to reduce inflammation in the brain [218].

3.3.5

Antiallergenic Activity

“Allergy,” as a definition of a collection of diseases, was first introduced in 1906. Over time, this definition has undergone minor modifications, to refer presently to an exaggerated immune sensitivity to certain environmental compounds or allergens. It is a hypersensitive response mechanism of the immune system to these allergens, acting as antigens, occurring in susceptible individuals. Most allergic reactions are mediated by a type I (anaphylaxis) immune mechanism. The term “atopy” is used if a patient has a hereditary predisposition toward the development of certain hypersensitivity reactions. From a mechanistic viewpoint, atopy refers to the hereditary predisposition to produce immunoglobulin type E antibodies against common environmental allergens causing atopic diseases (allergic rhinitis, asthma, and atopic eczema) [219]. Since this condition results from the overreaction of the immune system, it is perhaps not surprising that many plant phenols (Fig. 11) possess activities potentially useful for allergy treatment. Table 4 contains a simplified overview of several lignans and neolignans that have shown allergy-related activities [220–226].

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O

O

O

O

O

O

HO

O

OH

O

O

O

HO

68 ((2R,3R,E)-maceneolignan A)

70 (myristicin)

71 ((2R,3R,4S,5S)-verrucosin)

O HO

O

O O

HO

72 ((2R,3R,4S,5S)-nectandrin B)

OAc

HO

O

O

O

OH

O

O

O

O

73 ((2R,3R)-maceneolignan A)

74 ((2S,3S)-maceneolignan D)

OH

OH

O

O

O

O

OH

OH

O O

O

75 ((1R,2S)-maceneolignan H)

76 ((+)-(2R,3R)-licarin A)

O

O O

O

O

O

O

O

O

O

OH

O HO

O

O

O

78 ((+)-(1S,3aR,4S,6aR)-kobusin)

79 ((+)-(1S,3aR,4S,6aR)-aschantin)

77 (malabaricone C)

O O O O

O O

O

O O

HO

O

O

O O

O

80 ((+)-(2R,3S,4S,5S)-veraguensisn)

81 ((±)-(2R,3R,4S,5S)-galgravin)

O O

82 ((2S,3S,4R,5R)-nectandrin A)

OH O

O O

83 ((2R,3R,4S,5S)-futokadsurin C)

HO

84 (magnolol)

Fig. 11 Examples of lignans and neolignans with reported antiallergy activity

3.3.6

Lignans and Neolignans with Antimicrobial and Antiviral Activities

Bacteria and viruses are omnipresent in each and every single stage of human life. Risks related to these microbes therefore are not connected only with an

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Table 4 Selected lignan and neolignan compounds with reported antiallergy activity Compound

Activity

Ref.

Several lignans from Myristica fragrans

Inhibitors of CC chemokine receptor 3 (important role in allergic manifestation)

[220]

Maceneolignan A (68)

Inhibition of CCR3-mediated chemotaxis

[221] [220]

Inhibition of CCR3-mediated chemotaxis and inhibition of degranulation

[221]

(+)-Licarin (69) Myristicin (70) Verrucosin (71) Nectandrin B (72)

Maceneolignan A, D, and H (73, 74, 75) Inhibition of degranulation

[221]

(+)-Licarin A (76) Malabaricone C (77) Lignans from Magnoliae Flos

Inhibition of interleukin-2 cytokine

(+)-Kobusin (78)

Interaction with Jurkat T-cells [222] (immortalized line of human T-lymphocytes. Important cells for studying acute T-cell leukemia expression and various chemokine receptors susceptible to viral entry)

(+)-Aschantin (79) (+)-Veraguensin (80) (±)-Galgravin (81) Nectandrin A (82)

[222]

Futokadsurin C (83) Neolignans from Palhinhaea cernua

Inhibition of xanthine oxidase associated with gout

[223]

Secoisolariciresinol diglucoside (67)

Tryptophan metabolites interaction, anticolitis activity

[224] [225]

Antiulcerative colitis activity

[226]

Magnolol (84) Aryl tetralin lignan gylcosides isolated from Lespedeza cuneata

unclean or an unsafe environment, but also from invasive pathogens. Along with numerous pathogenic bacteria and viruses, there are also many bacterial species that are nonpathogenic or even beneficial. However, even the latter types of organisms may become a threat if a host becomes too sensitive to their presence or if immunity is suppressed. Modern humanity is equipped with many means of withstanding viral or bacterial infections, including the use of chemotherapeutic drug regimens. However, the growing resistance by pathogenic microorganisms to commonly used drug therapy is threatening World Health Organization programs focused on the prevention and suppression of bacterial, viral, parasitic, and fungal infections. Perhaps in an extreme case in the near future, none of today’s drugs will be effective against such infections. Thus, novel structurally unrelated drug candidates are being actively sought. For example, catechins from green tea (e.g., (–)-epigallocatechin gallate (39), (–)-epigallocatechin (85), (–)-epicatechin-3-gallate (46)) possess not only cancer-related properties and protective properties against cardiovascular or neurodegenerative diseases but also have antibacterial potential [148, 227, 228]. From the available biological data, it seems that such compounds are highly active against

Antileishmanial Activity of Lignans, Neolignans …

141 OH OH

OH OH HO

O

HO

O

OH

OH

O OH

OH

OH

O

OH

OH OH

85 ((–)- (2R,3R)-epigallocatechin)

86 ((–)-(2S,3R)-gallocatechin-3-gallate)

OH

OH

OH HO

HO

O

OH O

O OH

O

OH OH

OH

O

OH

O

OH

OH

OH

OH

87 ((–)-(2R,3R)-epigallocatechin-3-gallate)

O

88 ((–)-(2S,3R)-catechin-3-gallate)

OH

HO OH O

OH

OH

OH

HO

OH HO

O O

HO

O O HO

O OH

HO

O HO

OH

O

O

HO

89 ((2R,3R,2'R,3'R)-theaflavin-3'-gallate)

HO

OH HO

OH

90 ((2R,3R,2'R,3'R)-theaflavin-3'-gallate)

Fig. 12 Selected phenols with antimicrobial activity

Gram-positive bacteria, and recent studies also suggest that they can be used to control common oral infections (e.g. dental caries, periodontal diseases) [229–231]. From a mechanistic viewpoint, these compounds have the capability of inhibiting various enzymes. For example, (–)-epigallocatechin gallate (39) is able to inhibit phosphorylation of c-Jun N-terminal kinases, mitogen-activated protein kinases MEK1/2, extracellular signal-regulated kinases ERK1/2, ELK1 protein, dihydrofolate reductase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and carbonyl reductase [232–235]. In addition, 39 can modulate the lipid organization on the bacterial plasma membrane and therefore affect the distribution of proteins within the cell. It also interacts with the epidermal growth factor receptor, the hepatocyte growth factor receptor (HGFR), and the 67 kDa laminin receptor (67LR) [236–238]. Additional studies have also demonstrated that compound 39 increases anti-inflammatory activity in liver cells via decreasing iNOS and COX-2 expression [239, 240]. (–)-Gallocatechin-3-gallate (86), (–)-epigallocatechin-3-gallate (87), (–)catechin-3-gallate (88), epicatechin-3-gallate (46), theaflavin-3 -gallate (89),

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O O

O

O

O

O OH

O

OH

O

HO

O HO

O O

O

92 ((3R,4R)-hinokinin)

91 ((–)-(3R,4R) -nortrachelogenin)

O

93 (dehydroguaiaretic acid)

O O

O

O

O O

O

O

O

O

O

OH

94 ((2S,3S)-melaleucin A)

O

95 ((2S,3S)-melaleucin B)

96 ((2S,3S)-melaleucin C)

O O

OH

O

O

O

OH O HO

OH

O O

O O

OH

O

O

97 ((1S,2S)-myrislignan)

98 ((1R,2R)-myrislignanometin E)

99 ((2S,3S)-licarin B) O

O OH

O

OH

O O

HO

O

O

O

AcO O

O

HO

O

OH

O

100 ((2S,3S)-5'-methoxylicarin B)

101 ((2R,3S,4S,5S)-verrucosin) O

O

102 ((2S,3S)-difenneolignan A)

NH2 O

OH

OH

HO O

HO O

OH O

HO OH

103 ((2S,3S)-difenneolignan B)

OH

O

104 ((2S,3S)-pahangine A)

Fig. 13 Selected examples of lignans and neolignans with antimicrobial activity

and theaflavin-3-gallate (90) (Fig. 12) were shown to possess nanomolar activities against Bacillus cereus (a food-borne pathogen causing vomiting and diarrhea), but their mechanism of action was not established. (–)-Epigallocatechin-3-gallate (88) was also found to be active against Legionella pneumophila, probably due to a selective immunomodulatory action during cytokine formation. Compound 87 was determined as being protective against tobacco smoke-induced damage of alveolar macrophages, and it also showed activity against Mycobacterium tuberculosis by down-regulation of the host tryptophan-aspartate-containing coat (TACO) gene that restricts the entry and survival of tuberculosis infection [229]. Additional biological activities of (–)-epigallocatechin-3-gallate (87) were found against bacteria such as

Antileishmanial Activity of Lignans, Neolignans …

143

Mycoplasma pneumonia, Mycoplasma orale, and Mycoplasma salivarum, as well as other pathogenic bacteria [241–243]. Kaempferol (49) and quercetin (41) glycosides may inhibit the toxic manifestation of botulinum neurotoxins (Clostridium botulinum) [229, 244]. Selected examples of antimicrobial activity of the theaflavins [102, 245–271] are summarized in Table 5 (Fig. 12). Potent lignans and neolignans with noteworthy antimicrobial activity [272–276] are presented in Table 6 [197] (Fig. 13). Over the past two decades, several interesting lignans and neolignans with good activity against numerous viruses were isolated and identified from various sources (Fig. 13). A few representative examples include difenneolignans A (102) and B Table 5 Selected examples of plant phenolic compounds with antimicrobial and antitoxic activity (modified from [229]) Organism

Adverse effect

Phenolic inhibitor(s)

Ref.

B. anthracis

Anthrax

(+)-Epigallocatechin-3-gallate (39)

[245]

B. cereus

Food poisoning; emesis

Catechins, theaflavins, tea

[246, 247]

C. difficile

Severe diarrhea

Tea phenolics

[248]

C. tetani

Tetanus

Thearubigin

[249]

E. coli strains

Food poisoning; diarrhea

(+)-Epigallocatechin-3-gallate (39)

[102, 250–252]

H. pylori

Ulcers; chronic gastritis

Epicatechin-3-gallate (46), (–)-epigallocatechin-3-gallate (87), tea

[253–257]

L. pneumophila

Legionellosis; pneumonia

(–)-Epigallocatechin-3-gallate (87)

[257–259]

Mycobacterium tuberculosis

Tuberculosis

Catechins

[260, 261]

Toxins

Adverse effect

Phenolic inhibitors

Ref.

Vero (Shiga)

Diarrhea

(–)-Epigallocatechin-3-gallate (87), (–)-gallocatechin-3-gallate (86)

[262]

Tetanus neurotoxin Neurotoxicity

Catechins

[249]

Cholera

Cholera

Catechins; theaflavins

[263, 264]

Viruses

Adverse effect

Phenolic inhibitors

Ref.

Adenovirus

Respiratory infections

Catechins; tea

[265]

Bovine coronavirus Gastroenteritis in (–)-Epigallocatechin-3-gallate cattle (87)

[266]

Epstein-Barr

Infectious mononucleosis

(–)-Epigallocatechin-3-gallate (87)

[267]

HIV

AIDS

Catechins, theaflavins

[268–271]

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Table 6 Selected examples of lignans and neolignans with antimicrobial activity (modified from [197]) Compound

Sensitive bacterial strain

(–)-Nortrachelogenin (91)

Various bacterial strains, antibiotic resistant strains

Hinokinin (92)

Staphylococcus aureus (SA), Methicillin-resistant SA

Skin and respiratory infections

[273]

Dehydroguaiaretic acid (93)

Micrococcus luteus, Staphylococcus albus

Hospital related infections

[274]

Melaleucins A–C (94–96)

Staphylococcus aureus (SA), Methicillin-resistant SA

Skin and respiratory infections

[275]

Myrislignan (97)

Streptococcus pneumoniae

Meningitis, pneumonia

[276]

Myrislignanometin E (98)

Disease

Ref. [272]

Maceneolignan H (75) Licarin A (76) Licarin B (99) 5 -Methoxylicarin B (100) Verrucosin (101)

(103) (isolated from Illicium difengpi), having EC 50 values of 2.26 and 2.16 mg/cm3 , respectively, and patentiflorin A with IC 50 values of 14–32 nM (as tested on several different isolates) against HIV [277, 278]. Pahangine A (104), another neolignan with antiviral activity, showed activity against dengue virus type 2. Neolignan 104 was found to interact with viral proteases (NS2B/NS3) and result in their inhibition [279]. Coxsackieviruses (types A and B) are small nonenveloped viruses from the Picornaviridae family, and are causative agents for aseptic meningitis, spastic paralysis, and HFM diseases (hand–foot–mouth diseases) [280]. Isatindolignanoside A was found to be active against Coxsackievirus B3, with an IC 50 value of 25.9 μmol/dm3 [281]. The dibenzylic lignan trachelogenin may offer a possible treatment against hepatitis C, a disease caused by the hepatitis C virus (HCV), and for which no vaccine is yet available. This compound was shown to interact with the host CD81 protein, and thus prevented the entry of HCV to hepatocytes (IC 50 = 0.325 mg/cm3 (for the HCV cc model) and 0.259 mg/cm3 (for the HCV pp model) [282]. Another important target studied in the context of plant phenolic compounds is their interaction with the gut microbiome. Current hypotheses suggest strongly that these compounds can actively inhibit invasive bacterial species and prevent them from their involvement with the gut of the patient. Unfortunately, the precise mechanism as to how this occurs is not established at this stage [283, 284]. For example, it has been demonstrated that a blueberry (Vaccinium angustifolium) extract containing phenolic constituents promotes the growth of bifidobacteria (beneficial), while green tea extracts can modify the growth of pathogenic bacteria, inclusive of Clostridium difficile, Escherichia coli, and Salmonella typhimurium [285–287]. Some other studies further suggest that the above-mentioned interactions with the gut microbiome are due to modulating insulin levels, leading to hepatoprotective and

Antileishmanial Activity of Lignans, Neolignans …

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atheroprotective properties, and hence may help to reduce the risks associated with chronic diseases [288–291].

3.3.7

Antidiabetic and Antiobesity Effects

Diabetes is a group of metabolic diseases caused by defects in insulin production, insulin regulation, or a combination of both, which results in hyperglycemia or chronic hyperglycemia. Both these diseases have unpleasant consequences, sometimes inclusive of the failure of the eyes, kidneys, nervous system, blood vessels, or heart. Pathological processes that lead to such consequences vary from autoimmune destruction of β-cells to abnormalities linked with insulin resistance. Hyperglycemia is a causative agent of polyuria, polydipsia, weight loss, polyphagia, or blurred vision. The acute symptoms are life threatening and include ketoacidosis or nonketotic hyperosmolar syndrome. Chronic hyperglycemia relates to retinopathy and loss of vision, nephropathy and renal failure, peripheral neuropathy, and dysfunction of the intestinal and urinary tracts and the cardiovascular system [292]. In general, if a patient suffers from insulin resistance and type 2 diabetes, obesity is also evident. Obesity may trigger coronary heart diseases, certain forms of cancer, sleep-breathing disorders, and complicate the treatment of other diseases due to the inflammation of metabolic tissues [293, 294]. In the case of plant phenolic compounds, some have proven to be valuable in ameliorating the symptoms of diabetes type 2. Flavonoid derivatives such as various anthocyanins, and catechin (45), epicatechin (32), and kaempferol (49) and/or their glycosides, as well as procyanidins, may have a preventive role and can be used in nutrition-based cotreatments during the stabilization of diabetes symptoms [101, 295]. Their general mechanisms of action are based on the protection of β cells against glucose toxicity and on the regulation of glucose transport (glycemic management) [122, 295–297]. Another well-studied compound is the flavonol diglycoside, rutin (105), which possesses antioxidant, anticarcinogenic, antihypertensive, and antihypercholesterolemic activity, which makes it a compound of interest in influencing the progression of obesity and diabetes [298, 299]. Interestingly, its biological activities are not dependent on one single mechanism. Clinical studies suggest that 105 increases HDL (high-density lipoprotein), VLDL (very-low density lipoprotein), TGL (triglycerides), and TC (total cholesterol) levels, while decreasing LDL (lowdensity lipoproteins) levels. Rutin (105) also increases the secretion of insulin, and therefore decreases glucose levels (in hyperglycemic rats) and moderates the Ca+2 uptake (in rat pancreatic islets) [299, 300]. The stilbenoid, resveratrol (31), in laboratory animals and humans, may improve glycemic control. It affects the energy metabolism and mitochondrial function, and in animal models acts as a calorie restriction mimetic. Compound 31 can activate 5 -adenosine monophosphate-activated protein kinase (AMPK), increase SIRT1 expression, and modulate peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1 alpha protein levels, and also it increases citrate synthase activity

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HO OH

OH

HO

HO OH

O O

HO

O

O

adipogenesis suppresion osteogenesis stimulation mitochondrial activity modulation DNA damage modulation xenobiotic metabolism modulation glutamate metabolism modulation

O

OH

O

OH

HO

45 (catechin)

49 (kaempferol)

anti-cancerous

anticancerous

OH

OH

105 (rutin)

metastasis inhibition redox status modulation

OH

HO

HO

HO

angiogenesis modulation

O

HO HO

O OH

O

31 (resveratrol)

proliferation/apoptosis modulation

OH OH

antioxidant activity

anti-hyperglycemic

anticancerous

cardiovascular protection

antihypertension

pancreas protective

antikidney productive hypercholesterolemia antiobesity

skeletal muscle protective

antidiabetic

sciatic nerve protective

cardiovascular protection

antidiabetic

antiaging

neuro-protective

antioxidant activity

antiobesity

degenerative disease protection

antiinflamation

antibactrial activity

antihyperlipidemia

oral infection protection

antibacterial

anti-inflammtory

autophagy modulation

Fig. 14 Structurally important phenolic compounds — resveratrol, rutin, catechin, and kaempferol — and their biological activities

and decreases glucose levels in circulating blood [301]. Resveratrol (31) regulates signaling molecules and key adipogenic genes (PPARy2, C/EBPα, leptin) in epididymal adipose tissues in mice [302]. In addition to such diverse antiobesityrelated effects, resveratrol, like other plant phenolic compounds, demonstrates multitarget activity and interferes with signaling pathways related to other diseases. For example, compound 31 has been investigated for its effects on various cancer lines (pancreatic, colorectal, lymphoma, breast, prostate, and leukemia), in terms of its ability to inhibit cell proliferation, cell cycle progression, and to increase apoptosis. An overview of the biological activities of 31 and other selected plant phenols is summarized in Fig. 14 [148, 303–309]. Several lignans and neolignans, such as secoisolariciresinol diglucoside (106) might also contribute to the treatment of obesity or diabetes in the future (Fig. 15). It was demonstrated that diglucoside 106 improves lipid and glucose metabolism by enhancing the insulin signaling pathway via AMP-activated protein kinase activation in the liver. In addition, it also possesses the ability to protect rats with metabolic syndrome against the consequences of oxidative stress [310, 311]. Gomisin N (107), a lignan isolated from Schisandra chinensis, was shown in the concentration range of 10–100 μM to inhibit adipogenesis and the differentiation of 3T3-L1 preadipocytes, processes that can cause high-fat induced obesity [312]. The previously mentioned arctigenin (56) is also able to suppress adipogenesis and fat accumulation (in differentiated 3T3-L1 cells) via the reduction of adipogenic transcription factor expression [212]. The lignans manassantins A (108) and B (109) activate AMPK and inhibit mitochondrial complex I. In addition, these compounds could be used to increase the sensitivity to insulin by activating its metabolism [313]. A brief summary of the activity types in which lignans and neolignans may serve as antiobesity agents is shown in Table 7 [313–319].

Antileishmanial Activity of Lignans, Neolignans … HO

OH HO

O O

HO

O

HO

O

147 O

O

O O

OH

O O

OH

O

O

OH

O

O

OH

HO

O

OH

OH

106 ((2R,3R)-secoisolariciresinol di- D-glucoside)

107 ((6S,7R)-gomisin N)

110 ((2R,3R,4S,5S)-nectandrin B)

O O HO

O

O

O

HO

OH

O

O OH

O

O

OH

O

O OH

O

O O

O

O O

108 (manassantin A)

OH

111 (syringaresinol-4-O-ß-D-glucoside)

O

O O HO

O

O

O O

O

O

OH O

O

O O

O

112 ((2S,3S)-phyllanthin)

O

109 (manassantin B)

O

O

O

O

O

O

O O

OH

HO

O

O

O O

O

O

O

O

113 ((6R,7S)-schisandrin B)

114 (5R,8R,11R)-isocubebinic ether

115 (selamoellenin A)

Fig. 15 Selected lignans and neolignans with antidiabetic and antiobesity activity

3.3.8

Antiprotozoal Activity

As a result of recent climatic changes, parasitic diseases such as trypanosomiasis, malaria, and leishmaniasis are becoming increasingly threatening to human populations in many countries [320, 321]. Despite efforts being made to eliminate parasitic diseases by prevention, control and eradication programs, the overall situation is not improving, and, for example, malaria has made a great comeback over the past 25 years and still remains as one of the greatest threats to human health and well-being [320]. Unfortunately, the situation regarding the chemotherapeutic treatment of parasitic diseases is no better, since the use of the majority of available drugs may be accompanied by one or more of the following limitations: toxic side effects, an inappropriate mode of administration, a high price, and the requirement for long-term treatment (a problem in poor and developing countries). The whole situation is made

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Table 7 Selected lignans and neolignans possessing the antidiabetic and antiobesity activities Phenol

Mode of action

Ref.

Licarin B (99)

Improvement of insulin sensitivity (in 3T3-L1 adipocytes), by activation of GLUT4 in the IRS-1/PI3K/AKT pathway

[313]

Syringaresinol-4-O-β-d-glucoside (111)

Modulation of glucose and lipid metabolism

[314]

Phyllanthin (112)

Protection against diet-induced metabolic disorders (in mice), decreased adipogenic gene expression and increased lypolytic gene expression, reduction of serum and liver tryglycerides, and counteracted inflammation and insulin resistance

[315]

Schisandrin B (113)

In long term use shows beneficial activities [316] against non-alcoholic fatty liver disease (NAFLD) in obese mice

Nectandrin B (72)

Activation of Nrf2/ARE pathways and stimulation of antioxidant enzymes (HepG2 cells)

[317]

Isocubebinic ether (114)

Antidiabetic role due to activity in the uptake of glucose by 3T3-L1 adipocytes

[318]

Selamoellenin A (115)

Preventive against high-glucose induced injury for human umbilical cells, repairing vascular endothelial dysfunction

[319]

even more complex by an increasing number of cases where coinfections and drug resistance play crucial roles in antiparasitic drug administration [322–324]. One of the oldest parasitic diseases is malaria, which causes nearly 500 million new clinical cases and up to 2.7 million deaths each year worldwide. The enormous cost in human life has led to the incorporation of various prevention and medical programs focused on the eradication of malaria and its causes, Plasmodium falciparum and its natural vectors (mosquitoes) [321, 325]. Since the eradication of malaria or its vectors seems to be extremely difficult due to the formation of novel Plasmodium falciparum-resistant strains and the inefficiency of the insecticides employed, the development of novel chemotherapeutic regimens appears to be the only available possibility [326]. Fortunately, it was found that along with the well-known molecule quinine (from the bark of Cinchona spp., Rubiaceae) [327], the natural product artemisinin ((116), an endoperoxide sesquiterpene lactone) isolated from Artemisia annua (Asteraceae), can also be used to treat malaria (Fig. 16). For artemisinin (116), a decade-long campaign of several pharmaceutical companies has allowed its production and those of its currently clinically used semisynthetic derivatives to occur at a reasonable price. Thus, a reliable treatment for malaria is now available even for developing countries. From the mechanistic viewpoint, 116 has

Antileishmanial Activity of Lignans, Neolignans … Fig. 16 Artemisinin, a potent antimalarial compound, and its more bioavailable derivatives

149

O

H 3C

O O

O

O

O O

O O

O

O

O

O

O O O

O O

CO2

O O

O

Na CO2H

116 (artemisinin)

117 (artesunate)

118 (artemether)

119 (artelinate)

the ability to react with hemin, which is present in red blood cell membranes (Plasmodium parasites are rich in hemin) and to create an adduct that further undergoes oxidation of thiol-containing proteins within the parasite [328–330]. Unfortunately, the situation concerning other parasite-triggered human diseases is far from being solved. Human African trypanosomiasis (HAT), known as sleeping sickness, caused by Trypanosoma brucei gambiense and Trypanosoma brucei rhodiense, respectively, which produce either chronic (T.b. gambiense) or acute (T.b. rhodiense) infections (if untreated both are fatal), can be medicated only with the use of arsenic-based drugs at this time [322]. Chagas’ disease, triggered by Trypanosoma cruzi that affects nearly 90 million people each year (7 million get infected) can be treated effectively only with nitroimidazoles (benznidazole) [331, 332]. Similarly, leishmaniasis, is a further neglected parasitic disease, and together with Chagas’ disease and HAT is endangering more than 350 million people in 88 countries of the tropics and subtropical areas of the world per year [333]. As mentioned earlier, leishmaniasis symptoms range from localized self-healing lesions to severe lethal visceral forms that attack internal organs [334, 335]. Similarly, as in the case of the two previously mentioned parasitic diseases, the arsenal of antileishmanial drugs is far from optimal and most treatments are accompanied by severe side effects. In addition, the recent rise of resistance to administered treatments (drugs) has been observed. Thus, in all three major types of protozoal infection, novel drug candidates are needed desperately. In this context, plant phenols are potentially useful compounds for future antiprotozoal disease treatment. Many members of this compound group have demonstrated noteworthy biological properties with respect to Trypanosoma, Leishmania, and Plasmodium parasites [321]. In the case of trypanocidal activity, several phenolic compounds have been shown to be quite promising (Fig. 17). The lignan (–)methylpluviatolide (120) was active against T. cruzi in animal models and had the same effect on two T. cruzi strains [336]. The flavonoids sakuranetin (121) and 7-methoxyaromadendrin (122) both displayed trypanocidal activity at concentration of 500 μg/cm3 , where they caused 100% lysis of the parasites in an in vitro assay. It is believed that the antioxidant activity of these flavonoids is responsible for this activity [337]. A mixture of two flavones, 3 ,4 -methylenedioxy-5,6,7,5 tetramethoxyflavone (123) and 3 ,4 -methylenedioxy-5,7-dimethoxyflavone (124), inhibited completely (at the high concentration of 100 μg/cm3 ) the enzymatic activity

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O

O

OH

O

OH

HO

O

O

O

O

O

O HO

HO

O O

120 ((–)- (3R,4R)-methylpluviatolide O

122 ((2R,3R)-7-methoxy-aromadendrin)

121 ((S)-sakuranetin)

O O

O

O O

O

O

O

O O

O

O

O

HO

OH

O

O

123 (3',4'-methylenedioxy5',5,6,7-tetramethoxyflavone) O

124 (3',4'-methylenedioxy5,7-dimethoxyflavone)

125 ((2S,6R)-O-methylcentrolobine)

O

OH

O

O HO

O

OH O

O

126 (demethocycurcumin)

O

OH

128 (2',6'-dihydroxy-4'methoxychalcone)

O

O O HO

O HO

O

OH

O

OH

127 (klaivanolide)

129 (licochalcone A)

OH

O

130 ((+)-(R,Z)-nyasol) OH

OH

O OH

O HO

O

HO

OH

O

O

O

HO

131 (luteolin)

132 (3-methoxy-carpachromene)

O

O

133 (diospyrin)

Fig. 17 Selected lignans and neolignans with antiprotozoal activity (Part 1)

of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme of T. cruzi [338, 339]. Many naturally occurring plant phenols display antileishmanial activity (Table 8) [65, 66, 340–343]. In vivo experiments have demonstrated the potential activity of Omethylcentrolobine (125) and demethoxycurcumin (126) (34.5% and 65.5% decrease of lesion size in treated mice after 45 days, respectively). It was proposed that the activity observed is related to the β-dicarbonyl system or the α,β-unsaturated system present in the compounds tested. Klaivanolide (127) showed in vitro antileishmanial activity against L. donovani for both amphotericin B-sensitive (IC 50 = 1.75 μM) and amphotericin B-resistant strains (IC 50 = 3.12 μM) [344]. The neolignan (3,4dimethoxy)-2-(4-methylthiophenoxy) propiophenone (128) demonstrated a significant antileishmanial activity against L. donovani (in vivo) with the ability to reduce liver amastigotes by 42% after 5 days (at the dosage of 100 mg/kg/day) [345].

Antileishmanial Activity of Lignans, Neolignans …

151

Table 8 Plant phenolic compounds with determined antileishmanial activity [65, 66, 340–343] Active compounds

Activity

Diospyrin (133), plumbagin (134)

Generate parasite-deadly oxygen free radicals

Jacaranone (135)

Toxic to peritoneal mice macrophages at concentration ED50 = 0.02 mM

Hydropiperone (136)

Causes total lysis of parasites at concentration 100 μg cm−3

Amarogentin (137)

At concentration higher than 60 μM can inhibit catalytic activity of topoisomerase I

2 ,6 -Dihydroxy-4 -methoxychalcone (138) Able to affect the parasite macrophages (proposed mode of action is based on the interaction with parasite mitochondria and effects on the respiratory chain) Sulfuretin (139)

EC 50 = 0.09–0.011 μg cm−3 against promastigotes

Medioresinol (140), (–)-lirioresinol B (141) Active against amastigotes with survival index of parasites 40% at 60 μg cm−3 , (+)-Nyasol (130)

In vitro inhibition of L. major IC 50 = 12 μM

(–)-Sanguinolignan A (142)

In vitro inhibition of L. amazonensis with IC 50 = 36.7± 3.08 μg cm−3

5-Methyl-coumarin (143)

In vitro inhibition of L. major with IC 50 = 13.4 μg cm−3

Licochalcone A (129) possesses a selective ability to inhibit fumarate reductase and other enzymes of the parasite respiratory chain and therefore inhibit parasitical mitochondrial respiration [346]. (+)-Nyasol (130) showed antimalarial activity against Plasmodium falciparum (IC 50 = 49 μM) and activity against Leishmania major (IC 50 = 12 μM) [347]. Luteolin (131), on the other hand, demonstrated only antiplasmodial activity with an IC 50 value of 4.9 μM. It was also toxic for Vero cells at a concentration greater than 10.6 μM [348]. 3-Methoxycarpachromene (132), a flavone isolated from Pistacia atlantica, demonstrated activity with an IC 50 value of 3.4 μM against the P. falciparum strain K1 [347]. The lignan hinokinin (92) was already mentioned to exhibit cytotoxic activity against various cancer cell lines (murine gastric adenocarcinoma (MK-1), human cervical (HeLa), murine metastatic melanoma (B16F10), human colon adenocarcinoma (HT-29) and murine lymphocytic leukemia (P-388)), and to show anti-inflammatory activity. As a constituent of Chamaecyparis obtusa, hinokinin (92) was also active toward Trypanosoma cruzi parasites with an IC 50 value 0.7 μM (against free amastigotes). This activity was equivalent to that of the currently used standard treatment of Chagas disease, benznidazole (IC 50 0.8 μM against free amastigotes) [349] (Fig. 18).

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O O O OH

O OH

O

134 (plumbagin)

OH

136 (hydropiperone)

135 (jacaranone)

OH

OH

O O

O O

O

OH

HO O

HO

OH

O

OH

OH

HO

O

OH

O

O

OH

138 (2',6'-dihydroxy4'-methoxychalcone)

137 (amarogentin)

OH

O

139 (sulfuretin)

O

O

O

O

O

O

O O O

O

O

O

O OH

O

O

O O

HO

140 ((1S,3aR,4S,6aR)-medioresinol)

O

O

142 ((–)- (3S,4R,4'S)sanguinolignan A)

141 ((–)-(1S,3aR,4S,6aR)-lirioresinol B)

O O O

O

143 (5-methyl-coumarin)

S

O

144 ((3,4-dimethoxy)-2-(4methylthiophenoxy)propiophenone)

Fig. 18 Selected lignans and neolignans with antiprotozoal activity (Part 2)

3.3.9

Miscellaneous Plant Phenols with Interesting Biological Effects

From previous sections, it was argued that plant phenols, in general, and especially lignans and neolignans, are truly fascinating molecules with interesting biological effects. Even though their biological activities differ, from the mode-of-action viewpoint these are often related to the modulation of the immune system or to antioxidant activity (interactions with ROS). Nevertheless, all relevant interesting aspects of their biological activities have not been covered earlier in this chapter. Thus, some additional relevant biological effects of plant phenols will be mentioned below. Osteoporosis is a disease of the bones where they become more porous and therefore weakened, leading to an increased risk of fractures. The lignans baicalenosides A–C (145–147), extracted from Scutellaria baicalensis, showed in vitro antiosteoporotic activity by activating osteoprotegerin transcription required for the bone

Antileishmanial Activity of Lignans, Neolignans …

153

remodeling process (Fig. 19) [350]. In turn, dehydrodiconiferyl alcohol (148) showed a capacity to inhibit osteoclast cell differentiation and therefore to enhance bone cell formation [351, 352]. Sesamin (149) displayed promising activity on the chondrogenic differentiation of human mesenchymal stem cells that are useful in cartilage regeneration [353]. HO

HO

HO O

O

O

O

O

O

OH

O O

HO HO

O

O O

O

O

HO

O

HO

OH

145 (baicalensinoside A) O

O

HO

OH

OH

OH

OH

O

O

HO

OH

O

OH

OH

146 (baicalensinoside B)

147 (baicalensinoside C)

O

O

OH

O

O OH

HO

O O

O

O

OH

148 ((2S,3R)-dehydrodiconiferyl alcohol)

O

O

OH OH

O

149 ((1S,3aR,4S,6aR)-sesamin)

150 ((2S,3S)-americanin A) OAc

O O

O

O

O

O O

O

O O

O AcO

(+)-151 ((+)-(2S,3R,4S,5S)-zuonin A)

O

(–)-151 (–)-(2R,3R,4S,5R)-zuonin A)

O

O

O

O O

152 ((S)-galanganol D diacetate)

O

O

O

O

OH

O

O

O O

O O O

O

O

O OH

153 ((5S,6S,7S)-micratherin A) O O O O

O

O

O H3CO

O O

O O OH

O

155 ((6S,7S)-schisandrin)

154 ((6S,7R)-gomisin M2)

O

HO

O

OCH3

O OH

O O

156 ((5S,6S,7S)-angeloylgomisin R)

157 ((6S,7R)-schisantherin A)

158 ((2R,3R,4S,5S)-nectandrin B)

Fig. 19 Selected lignans and neolignans with biological activity associated with protective ability against osteoporosis, melanogenesis, and liver damage

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Melanogenesis is a physiological process connected with melanin production that is carried out in melanosomes by melanocyte cells. Thus, specialized dendritic cells (melanocytes) synthesize and transfer pigment to recipient cells. However, if malignant melanocytes upregulate melanogenesis, the overall process can become dangerous and, in some cases, even life threatening [354]. Several phenolic compounds have demonstrated biological effects that may allow for the regulation of this process. For example, when americanin A (150) was applied at a concentration of 20 μM, it downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase (TYR) expression in melanocytes [355]. Gomisin N (107) at concentrations of 1–10 μM regulated the phosphatidylinositol 3kinase/serine/threonine protein kinase B (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK) signaling pathway [356]. (+)-Zuonin A ((+)-151), when applied at concentrations ranging from 1 to 20 μM, inhibited melanogenesis by decreasing levels of tyrosinase and tyrosinaserelated proteins (TRP-1 and 2). Interestingly, its enantiomer, (–)-zuonin A ((–)-151), inhibited c-Jun N-terminal kinases that are associated with several diseases such as cancer and neurological disorders, but did not interact with tyrosinase and tyrosinaserelated proteins [357, 358]. Galanganol D diacetate (152) inhibited melanogenesis (IC 50 = 2.5 μM), presumably via a TYR, TRP-1, and 2 mRNA expression inhibition mechanism [359]. The liver, the largest solid organ in the human body, is a vital metabolic and immune “factory.” Its primary functions are to transform and “dismantle” substances, mine energy, and detoxify the organism. The human liver has an enormous capacity to regenerate, but even with such a regenerative capacity, can be harmed by many diseases, toxins, and pathogens. It was shown that several plant phenols have potentially important hepatoprotective activity [360]. The lignan arctigenin (56) was found to reduce via an immunosuppressive mechanism acute hepatitis [361]. Micrantherin A (153), gomisin M2 (154), and schizandrin (155), obtained from Schisandra chinensis, showed an ability to protect liver tissue against paracetamol damage (tested on HepG2 human liver carcinoma cells) [362]. Angeloylgomisin R (156) and schisantherin A (157), from Schisandra pubescens, on the other hand, increased the survival of QSG7701 hepatocyte cells in the presence of d-galactosamine (d-GalN-induced cell damage) [363]. As a final example, nectandrin B (158), a lignan isolated from nutmeg, demonstrated an ability to prevent oxidative damage via Nrf2 activation [317].

4 Concluding Remarks It is the hope of the authors that the reader has been brought to the conclusion that plant phenols inclusive of lignans and neolignans have great potential value in the treatment of neglected tropical diseases such as leishmaniasis (Fig. 20) [364]. Indeed, their range of biological activities, which are extremely broad for this family of natural compounds, is supportive of their possible future use. Only subtle changes

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Fig. 20 Overview of the most important biological activities attributed to natural phenolic products (modified from [364])

in a given chemical structure may dramatically change the resultant biological activities observed. One may conclude that most of the reported biological interactions are triggered by the oxido-redox properties of these phenols. The remainder of the chemical structure involved is then responsible for the “delivery” of the phenolic part to appropriate active biological site. Identified biologically active phenolic compounds along with their reported activities are included in many readily available databases that can be searched, including Phenol-Explorer, eBASIS-EuroFIR, the USDA Database for the Flavonoid Content of Selected Foods, and ADReCS-Target. In most cases, while their mechanisms of action are still unknown, the plant phenols that have been evaluated in vitro may interact with cell signaling pathways, gene transcription, and proliferative mechanisms, and overall may modulate diverse biological targets, as summarized in Fig. 21 [365].

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Fig. 21 Overview of selected molecular targets of phenol compounds related to cardiovascular diseases, diabetes, cancer, and neurologic diseases. Color code defines the interaction with the molecular target: inhibition (red) or stimulation (green)

Nevertheless, it should also be pointed out that biological test results obtained directly from natural product screening programs should be looked at with some skepticism, particularly those conducted in in vitro bioassays. One should not forget that plant phenols undergo important structural modifications once absorbed by the organism (GIT, liver), both during the conjugation/deconjugation processes or in being transported into cells [365]. The potential preventive and therapeutic benefits that phenols offer as nutrients are commonly discussed and presented in many scientific publications and are also the subject of media reports. However, the negative effects of one or two groups of plant phenols may appear to outweigh the overall potential benefits of these compounds as a whole as additives to the human diet [366]. Questionable concentration-dependent effects of plant phenols have been published. For example, at high concentration levels, green tea catechins demonstrate hepatic and gastrointestinal toxicity, as shown in a study on dogs [367]. Another example is the possibility that a flavonoid-based diet for pregnant women may induce the risk of leukemia in their offspring, a suggestion based on the interactions of these flavonoids with topoisomerase-II [368]. Regardless if these two cases are actually clinically relevant or not, the biological activities of plant phenols in humans, and especially lignans and neolignans, must continue to be studied carefully in the future. Indeed, widely used nutrient supplements of plant origin tend to include many phenolic compounds (Fig. 22). Hence, a main research direction on which to focus, should involve the development of short and efficient ways on how to prepare phenylpropanoids, lignans, and neolignans that are needed for further research (e.g., as analytical standards or as compounds for biological testing) [369, 370].

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Fig. 22 Overview depicting the presence of various phenols in natural or processed sources

Once such pure compounds are available more widely, nutrient preparations can be screened with the aim of detecting the presence of phenylpropanoid derivatives [371], and, independently, to assess their biological activity against relevant biological targets, including their antiprotozoal activity. The authors of this chapter are working currently on the design of lignan-inspired antileishmanial compounds [372, 373], that are inspired by the naturally occurring (–)-sanguinolignan A (142) [342], a lactone-containing lignan found in a plant used by Peruvian shamans to treat leishmaniasis. Potentially, its mechanism of its action determination can pave the way toward the design of new potent antileishmanial drugs. Acknowledgments This work was financed by the European Regional Development Fund-Project “Centre for Experimental Plant Biology” (grant number CZ.02.1.01/0.0/0.0/16_019/0000738). For their continual support, J.P. is grateful to Prof. M. Strnad, Dr K. Doležal, and Prof. J. Hlaváˇc (Palacky University).

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Jiˇrí Pospíšil is a Group Leader of the Natural Product Synthesis Group (pospisilab.com) in the Laboratory of Growth Regulators, Institute of Experimental Botany ASCR & Palacký University in Olomouc, Czech Republic (rustreg.upol.cz). He is also an Associate Professor of Organic Chemistry at the Department of Organic Chemistry, Faculty of Science, Palacky University (orgchem.upol.cz). Pospíšil’s current research program focuses on new organic synthetic strategy development, including the development of new tunable Brønsted acids for organocatalysis and the design of various polyfunctional synthetic building blocks for Diversity Oriented Synthesis, with the aim of using these in the context of plant secondary metabolite and phytohormone synthesis. The main target compounds in the field of natural product synthesis are lignans, neolignans, and gibberellins. He received his undergraduate education at Masaryk University in Brno, Czech Republic, where he also obtained his Master’s degree in organic chemistry under the guidance of Prof. Milan Potáˇcek. After finishing his initial

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studies, he undertook his Ph.D. degree as a Rhodia-Organics Fellow at Université catholique de Louvain, Belgium, with Prof. István E. Markó. He then moved to the laboratory of Prof. Alois Fürstner at the Max-Planck Institute für Kohlforschung, Germany. In October 2008, he started his independent research career as a FSR-FNRS research fellow at the Université catholique de Louvain. From October 2012, he worked as a CNRS research fellow in the group of Professor Samir Z. Zard at the Ecole Polytechnique in Paris, France, before he accepted an offer from Palacky University in January 2014 to take up the Group Leader position referred to above. In 2016, he was appointed as Assistant Professor at the Department of Organic Chemistry at Palacky University, and in April 2020, he was promoted to Associate Professor of Organic Chemistry. He has received two main awards thus far (Incentive Award 2012 (C.G.B-C.B.B) and Alfred Bader Prize for Organic Chemistry 2014 (Czech Chemical Society)), and has published three chapters in books and 41 papers in peer-reviewed journals. Daniela Konrádová received her Ph.D. degree in Experimental Biology from Palacky University, Olomouc, Czech Republic, under the guidance of Assoc. Prof. Jiˇrí Pospíšil, Ph.D. Her dissertation was on the border of two disciplines (organic chemistry and antiparasitic activity). For her doctoral work, she extended the topic of her Master’s Thesis (phenylpropanoid compounds) to the field of phenolic compounds inspired by plant secondary metabolites with potential antileishmanial activities. During the early stages of her doctorate she focused on organic synthesis with the aim of preparing a medium-sized chemical library of phenolic compounds suitable for bioactivity screening. The results of this work have been published in the “European Journal of Organic Chemistry” (2017) and the “Journal of Organic Chemistry” (2018). In 2018, Dr Konrádová received support from the Palacký University Endowment Found and underwent a short research stay in Belgium at Université catholique de Louvain, Groupe de Recherche en Physiologie Végétale (GRPV). There, she extended her knowledge on plant metabolites. In 2019, with the support of the Mobility Program from Palacký University she went for six months to The Hebrew University in Jerusalem, Israel, where she worked in the Kuvin Centre for the Study of Tropical and Infectious Diseases, National Centre for Leishmaniasis — Hadassah Medical School. Currently, Dr. Konrádová is employed at the Czech Academy of Science, Institute of Experimental Botany and her main interest is focused on bioactive phenylpropanoid synthesis and their applications in the context of antileishmanial research.

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J. Pospíšil et al. Miroslav Strnad is Professor in the Laboratory of Growth Regulators, Institute of Experimental Botany ASCR and Palacký University in Olomouc, Czech Republic (rustreg.upo l.cz). Strnad’s current focus is on the research and development of a new generation of compounds with antiviral, antiproliferative, antiangiogenic and antisenescence properties, the molecular mechanisms of their action, and potential combination therapies based on these compounds, as well as new phytohormone-derived cosmetics and plant growth regulators for plant biotechnology and agriculture. He graduated in Phytotechnologies from the Faculty of Agronomy, Mendel University, Brno, in 1982 (Ing.). In 2001, was promoted to Professor of Palacký University. In 1998, he was awarded the Rhone-Poulenc Rorer Award by the Phytochemical Society of Europe (PSE), in recognition of his work on the identification, analysis, and biochemistry of the topolin phytohormones. Prof. Strnad has published widely (11 chapters in books; more than 410 papers in peer-reviewed journals; three books; eight Czech and 49 international patents; citations >13,500; Hirsch index: 58). He was PSE President during the period 2014–2016, and became Vice-President in 2018.

Recent Advances in the Chemistry and Pharmacology of Cryptolepine Steven D. Shnyder and Colin W. Wright

Contents 1 2 3 4 5 6 7 8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis of Indoloquinoline Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis of Cryptolepine and Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibacterial and Antifungal Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anticholinesterase and β-Amyloid Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antidiabetic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anti-inflammatory Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryptolepine as a Lead to New Antiprotozoal Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Amebiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1 Ethnopharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2 Development of Antimalarial Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Cytotoxic and Antineoplastic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Cytotoxic Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Antineoplastic Agents: In Vitro Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Antineoplastic Agents: In Vivo Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Pharmacokinetics and Toxic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Cryptolepine Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Toxic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Miscellaneous Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

178 179 180 184 185 186 186 187 187 187 187 187 189 191 192 192 193 195 196 196 197 198 198 198

S. D. Shnyder Institute of Cancer Therapeutics, School of Pharmacy and Medical Sciences, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK e-mail: [email protected] C. W. Wright (B) School of Pharmacy and Medical Sciences, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 A. D. Kinghorn, H. Falk, S. Gibbons, Y. Asakawa, J.-K. Liu, V. M. Dirsch (eds.), Progress in the Chemistry of Organic Natural Products, Vol. 115, https://doi.org/10.1007/978-3-030-64853-4_4

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1 Introduction Cryptolepine (5-methyl-10H-indolo[3,2-b]quinoline (1)) (Fig. 1) is the principal indoloquinoline alkaloid found in the dried roots of the West African climbing shrub Cryptolepis sanguinolenta (Lindl.) Schltr. (Apocynaceae) (Plate 1) [1], synonymous with C. triangularis N.E.Br and Pergularia sanguinolenta Lindl. [2]. Although more than 40 species of Cryptolepis are recognized [2], C. sanguinolenta is the only one so far reported to contain 1. While this alkaloid has also been found in the aerial parts of Indian Sida acuta Burm.f. (Malvaceae), the quantity was small (0.017% of a basic extract) [3], and is much lower than the amount present in C. sanguinolenta roots (ca. 1%) [1]. Fig. 1 Structures of cryptolepine and indoloquinoline isomers

4 6

N

5N 1

9

10

N

11

N 1a (cryptolepine, alternative form)

1 (cryptolepine)

N

N

N N

2 (neocryptolepine)

3 (isocryptolepine)

N N 4 (isoneocryptolepine, synthetic)

Plate 1 Cryptolepis sanguinolenta; photograph courtesy Dr. Naalamle Amissah, Department of Crop Science, College of Basic and Applied Science, University of Ghana

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In the literature, the structure of cryptolepine is depicted in two resonance forms, 1 and 1a. Structure 1, in which N-5 is quaternary, is preferred by the authors because cryptolepine has only one protonatable nitrogen (N-10) and one pKa ; the quaternary nature of N-5 is also supported by the large downfield shift of the N-methyl group signal in the 1 H-NMR spectrum (δ 4.7 ppm in CDCl3 ) [1]. The single protonatable nitrogen in 1 has implications for the antiplasmodial potency of this compound and its analogs, as discussed in Sect. 8.3. In addition to 1, about 14 minor alkaloids have been isolated from C. sanguinolenta, including the indoloquinoline isomers, neocryptolepine (2), and isocryptolepine (3) (Fig. 1) [1]. A fourth member of this series, isoneocryptolepine (4) (Fig. 1), has not been found in Nature but has been synthesized [4]. The dried roots of C. sanguinolenta are widely used in West African traditional medicine as a decoction for the treatment of many non-infectious and infectious diseases including malaria [1]. The chemistry and biology of 1 were reviewed previously in 2008 [1], and the aim of this chapter is to discuss progress made over the last decade especially with respect to the therapeutic potential of 1 and its semisynthetic and synthetic analogs. To date, the main interest has been the development of novel antimalarials (Section 8.3), reflecting the widespread traditional use of C. sanguinolenta for the treatment of malaria.

2 Biosynthesis of Indoloquinoline Alkaloids Plausible biosynthesis pathways for the indoloquinoline alkaloids 1, 2, and 3 have been proposed by Parvatkar and Parameswaran in 2016 [5], and the pathway for 1 is illustrated in Figs. 2 and 3. Biosynthesis from chorismate, (5) via indole-3-glycerol Fig. 2 Biosynthesis of indoloquinoline precursors; adapted from [5]

O O

O

HO

P O O O

OH O OH

N H

O

5 (chorismate)

6 (indole-3-glycerol phosphate)

N H 7 (indole) O

O

O O

N H 8 (indoxyl)

N H 9 (isatin)

OH NH2 10 (anthranilic acid)

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Fig. 3 Biosynthesis of 1; adapted from [5]

O

NH

H 2N imine formation

+

O

O

OH

N H 8 (indoxyl)

OH

N H 11

10 (anthranilic acid)

NH

N

HSCoA O

SCoA

N H

intramolecular cyclization N H

O

13

12

HN

N SAM

N H

O

O

N H

14 (quindolinone)

15 (cryptolepine-11-one)

NADPH reduction and dehydration

N

N SAM N-methylation

N H

N

16 (quindoline)

1 (cryptolepine)

phosphate, (6), leads to indole (7), the precursor of indoxyl (8) and isatin (9), from which anthranilic acid (10) is derived (Fig. 2). Condensation of 8 and 10 gives rise to 3-anthraniloyl indole (11) that may be converted to its thioester 12 by co-enzyme A followed by cyclization to form 13, which isomerizes to quinolinone 14 (Fig. 3). Reduction of 14 by NADPH followed by dehydration yields quindoline (16), and selective SAM-type N-methylation of the quinoline nitrogen then results in the formation of 1. N-Methylation of 14 may also take place, leading to cryptolepine-11-one, (15), of interest as a constituent of C. sanguinolenta and as a probable human metabolite of 1, as discussed in Section 9. The biosynthesis of neocryptolepine (2) and isocryptolepine (3) may arise from the reaction of anthranilic acid 10 with oxindole or 3-formylindole, respectively [5]. A number of indoloquinoline alkaloid syntheses are to some extent biomimetic.

3 Synthesis of Cryptolepine and Analogs For reviews of earlier synthesis routes to 1, see [1] and [6]. In most schemes, the precursor, quindoline (16) is synthesized and then methylated to give 1. Some more recent methods have employed “domino” or “tandem” processes involving two or

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more bond formations under identical reaction conditions [5, 7, 8], although it should be noted that in the earlier method of Holt and Petrow published in 1947 [9] (Fig. 4), the condensation of O,N-diacetylindoxyl (17) and isatin, (9) to form quindoline11-carboxylic acid (18), involves the formation of two bonds in the same step. This reaction gives good yields but the original method required ten days for the condensation reaction at room temperature, whereas a modified method in which the reaction mixture is refluxed requires only four hours [10]. “Domino” approaches to 1, 2, and 3 have been described [5, 7]. A number of cryptolepine (1) analogs substituted at the 2, 7, and/or 11 positions, have been synthesized using a four-step method incorporating tandem reductive cyclization [7], as illustrated in Figs. 5 and 6. In this method, 2-substituted indole derivatives such as 21 are first formed by reacting lithiated N-sulfylindole derivatives (19) with electrophiles (20) (Fig. 5). Reductive cyclization of 21 with triphenyl phosphine and MoO2 Cl2 (dmf)2 as a catalyst in refluxing toluene yields the corresponding 10(phenylsulfonyl)-indolo[3,2-b]quinolines (22). Desulfonation of 22 followed by N-5 methylation of the resultant 10H-indolo[3,2-b]quindolines (23), using methyl iodide in sulfolane gives cryptolepine analogs as hydroiodide salts (24). The possible mechanisms involved in the formation of 22 from 21 are illustrated in Fig. 6. These may involve either electrophilic substitution of the in situ-formed nitrene (25) to produce an ionic intermediate 26, (route a, Fig. 6) which is converted spontaneously to 27, or else nitrene (25) may directly undergo C–H insertion to furnish intermediate 27 (route b, Fig. 6). Dehydration of 27 yields the aromatized products (22). Limitations of this process are that the reduction–cyclization–aromatization step gives relatively low yields of 20–38% and that only a few analogs have been prepared [7]. The yields of linear indoloquinolines may be increased by employing microwave-assisted reductive cyclization, although the major compound formed was spiro[2H-indole-2,3 -oxindole, which is present as the “core” of various alkaloids [8]. Several other novel routes to 1 have also been reported in recent years. Two consecutive microwave-assisted palladium-catalyzed reactions have formed the basis

O

OAc

+

O N H

N Ac

N KOH, N2 reflux, 4 h

9 (isatin)

17 (O,N-diacetylindoxyl)

COOH

N H

18 (quindoline-11-carboxylic acid) diphenylether reflux, 3 h —CO2

N

N

N 1 (cryptolepine)

Fig. 4 Synthesis of 1 adapted from [9]

sulfolane, CH3I 60°C, 12 h

N H 16 (quindoline)

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R1

O2N

R1

R3 OH SO2Ph

2. O N 2

N SO2Ph

N

R3

19

R2 O

21

20 R3

N PPh3M, MoO2Cl2(dmf)2,

R2

R1

aq. NaOH, MeOH,

toluene, reflux, 16 h

80°C, 3 h

R2

N SO2Ph 22

I R3

N R

CH3I, sulfolane,

1

R3

N R1

55°C, 16 h N H

R2

R2

N H

22

23

Fig. 5 Synthesis of 1; involving tandem reductive cyclization; adapted from [7] Fig. 6 Suggested mechanisms of reductive cyclization; adapted from [7]

O2N

N

OH

SO2Ph 24 (R groups omitted) PR3 PR3=O H

H

N

:N

or N

N

OH

SO2Ph

OH

SO2Ph

25

route a

route b

H N

N

H

OH

N SO2Ph 26

OH

N SO2Ph 27

– H 2O 22

Recent Advances in the Chemistry and Pharmacology of Cryptolepine Fig. 7 Palladium-catalyzed synthesis of 16

I

N

183 H N

aniline, Pd(OAc2)2, Xantphos,

Br

CsCO3, toluene 120°C, Mw

Br

N

28

29 H N

PdCl2(PPh3)2, NaOAc, DMA, 150°C, Mw

N 16 (quindoline)

of novel syntheses of the four indoloquinoline skeletons 1–4 [11]. The synthesis of quindoline 16 from bromo-iodoquinoline (28) via 29 is illustrated in Fig. 7. In 2016, Wippich et al. reported an efficient synthesis of 1 from 3-fluoro-2phenylquinoline (30), involving rhodium-catalyzed N-boc amination to give 31, followed by annulation with pyridine hydrochloride to form 16, which is easily methylated to 1 [12] (Fig. 8). Yonekura et al. in 2018 described the synthesis of 2-, 3-, and 11-substituted cryptolepine analogs (35), by indium-catalyzed annulation of o-acylanilines (33) with 3-acetyloxyindole (32), yielding quindoline analogs (34) that were then methylated to give 35 (Fig. 9) [13]. A route to 8-substitued quindolines (40) has been described by Shuvalov et al. in 2019, as shown in Fig. 10 [14]. The 2-aryl-3-nitro-tetrahydroquinolines (38) were prepared by fusing 2-hydroxymethylene-cyclohexanone (36), with nitroacetophenone enamines (37). Reductive Cadogan cyclization of 37 was then carried out using 1,2-bis(diphenylphosphino)ethane (DPPE) to yield δ-carbolines (39), which were then aromatized to the corresponding quindoline derivatives (40). A number of analogs of 1 may potentially be prepared by methylation of 39 and 40. NHBoc BocN3, K3PO4, 5mol% (Cp*RhCl2)2, Ar,

N

N

20mol% AgSbF6, 80°C, 14 h, (DCE) F

F 30

31

N

N

pyridine.HCl 220°C, 4 h

1. MeI, r.t., 24 h (sulfolane), N H

16 (quindoline)

Fig. 8 Rhodium-catalyzed synthesis of 16

2. Na2CO3, r.t., 1 h, CHCl3/H2O

N 1 (cryptolepine)

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OAc

R3

H2N

+ O N H

R2

N InBr3, (5mol%),

R2

PhCl or o-C6H4Cl2

34

33

32

R1

N H

R1

R3 R2

N

OTf MeOTf, solvent, 30–50°C, 24 h

R1

N H 35

Fig. 9 Indium-catalyzed synthesis of 35

OH NO2

O

NO2

80°C, 72 h

+ H 2N

N R

36

DPPE, 150°C

R 38

37

H N

H N Ph2O, Pd/C, Δ N

N

R

R

40

39 R = CH3, OCH3, Cl, or Br

Fig. 10 Synthesis of 8-substituted quindoline 40

4 Antibacterial and Antifungal Activity In a number of earlier studies, cryptolepine (1) was shown to have in vitro activities against several bacteria and fungi with MIC values of 5.0 and 3.4 μM against the sensitive and resistant parasites, respectively), while the IC 50 value of 43 against mouse macrophages was 9.7 μM, indicating some selective toxicity against the amastigote form.

8.3 Malaria 8.3.1

Ethnopharmacology

In 2018, malaria was responsible for an estimated 405,000 deaths of which 94% occurred in Africa and 67% of global deaths were in children under the age of five years [33]. Although the global incidence of malaria declined significantly from

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2010 to 2018, the rate of improvement has slowed since 2014, and there were an estimated 228 million cases worldwide in 2018. Meanwhile, the most important malaria parasite, Plasmodium falciparum, responsible for 99.7% cases globally, has developed resistance to artemisinin derivatives in some areas, especially along the Thai– Cambodia border [34, 35], so that there is an urgent need for new antimalarial drugs. In addition, attention is again being given to the eradication of malaria, which will require antimalarial drugs with activity against the liver and gametocyte stages of the parasite as well as the blood stages targeted by most currently available antimalarial agents. The aqueous root extract of C. sanguinolenta is used widely in West Africa for the treatment of malaria. In Ghana, the herbal product, Phyto-laria, is marketed for the treatment of malaria in the form of tea bags and a number of other Cryptolepis-based aqueous formulations are also available. The efficacy of Phyto-laria was assessed in a prospective, open clinical study in patients over the age of 11, with clinical symptoms of malaria and a diagnosis of malaria by examination of blood films [36]. Parasitemia in all of the patients evaluated cleared within seven days, with two patients showing recrudescence within 28 days, although this could have been due to re-infection. Hematological and biochemical abnormalities present before being treated generally improved during treatment but blood alkaline phosphatase levels were elevated persistently during the study compared with pre-treatment values, and serum uric acid levels showed a significant rise during treatment. The study suggests that Phyto-laria may be effective for the treatment of malaria in patients aged between 11 and 50 years. However, an important limitation of the study was the exclusion of young children, who are the most susceptible to serious complications and death due to malaria. Hence, it cannot be assumed that Phyto-laria would be effective in this group, as those under five years of age especially are likely to have little immunity to malaria. Pregnant women, who are also at increased risk of severe malaria, were also excluded from this study [36]. In a pharmacovigilance study in which 14 Cryptolepis-based aqueous formulations were examined, it was found that the content of cryptolepine (1) was variable and could not be detected in two brands [37]. Only three brands passed the microbial limit test and perhaps most worrying, 11 of the 14 products contained artesunate. None of the brands complied with packaging and labeling requirements. Another concern with respect to herbal products containing C. sanguinolenta is that the roots are sourced entirely from the wild leading to declining populations [38]. Some attempt is being made to domesticate C. sanguinolenta and to determine the optimum growing conditions and time of harvest for maximum yields of cryptolepine. Thus, it has been found that at 289 days after planting, the content of 1 in the dried roots reached a maximum of 1.84% w/w [38]. Cryptolepine (1) has moderately potent antiplasmodial activity against both chloroquine-sensitive (HB3) and resistant (K1) strains of Plasmodium falciparum cultured in red blood cells (IC 50 = 0.27 and 0.44 μM against 3D7 and K1 strains, respectively). However, it was not able to cure mice infected with P. berghei berghei when given orally (80.5% suppression of parasitemia compared to untreated infected controls at 50 mg/kg/day) whereas 100% suppression is required for a cure [39].

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Cryptolepine (1) was found to be toxic when injected by intraperitoneal (i.p.) injection following two daily doses of 20 mg/kg [40]. It is likely that toxicity is related to DNA-intercalation, inhibition of DNA synthesis, and inhibition of topoisomerase II [41]. X-ray crystallography of cryptolepine crystallized with specific DNA oligomers showed that 1 selectively intercalates between non-alternating C-G base pairs [42]. The failure of oral cryptolepine (1) to cure malaria in mice is probably due to metabolism and/or pharmacokinetic factors as discussed in Section 10.1. Recently, compound 1 has been shown to have potent activity against P. falciparum (N54) late stage (IV/V) gametocytes (IC 50 = 1.97 μM) [43], which could be significant for the elimination of malaria as antimalarials with activity against the sexual stages as well as the asexual stages (liver and asexual red blood cell stages) that will be required [44]. In vitro combination studies against asexual blood stages of P. falciparum indicated that 1 exhibits synergism with amodiaquine, additive effects with chloroquine and lumefantrine but antagonism with mefloquine [43]. In vitro synergies between 1 and artemisinins (artesunate, artemether, and dihydroartemisinin) as well as in vivo synergy between 1 and artesunate in P. berghei (NK-65) infected mice have been reported with no significant toxicity observed [45]. In another study, 1 was shown to act synergistically with the diterpene xylopic acid when administered orally to mice infected with P. berghei [46]. No significant toxicity to the kidney, liver, and spleen was observed but the testes were affected adversely at higher doses.

8.3.2

Development of Antimalarial Agents

The antiplasmodial mode of action of 1 has been shown to be due, at least in part, to the inhibition of hemozoin formation, raising the possibility that it may be possible to prepare cryptolepine analogs that retain potent activity against Plasmodium spp. but without the DNA-intercalating ability that could be responsible for toxicity [40]. A number of halogenated analogs of 1 were prepared by employing methodology based on that of Holt and Petrow from 1947 [9] (Fig. 4), using substituted derivatives of 9 and/or 17 as starting materials [10, 40]. Several di-halogenated cryptolepine analogs were found to be up to 10-fold more potent than the parent compound against P. falciparum in vitro, and also suppressed parasitemia in P. berghei-infected mice by 90% when given by intraperitoneal injection at 25 mg/kg/day without apparent toxicity to the mice [10]. The best studied compound is 2,7-dibromocryptolepine (43) (Fig. 11) which, in common with 1, inhibits the formation of hemozoin but, in contrast, does not appear to intercalate into DNA, as shown by thermodenaturation studies (T m values 4 and 9°C for 43 and 1, respectively, with values below 5°C considered to be due to non-specific binding to DNA) [10]. Experiments have indicated that the increased potency of 43 is neither due to more potent inhibition of hemozoin formation nor due to increased accumulation of this basic compound into the acidic parasite food vacuole compared to 1, suggesting that a second, currently unknown mechanism of action may be operating [10]. When treating malaria, two drugs with differing modes of action are usually given together in order to reduce the risk of malaria parasite resistance developing. Dual-acting compounds such as

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43 may have a significant advantage over drugs with a single mode of action with respect to resilience to parasite resistance and as a simpler, more economical therapy. A number of other compounds based on 1 have been synthesized as potential antimalarials. Since the antimalarial mode of action of 1 involves the inhibition of hemozoin formation, it was suggested that structural modifications such as the addition of another protonable nitrogen resulting in higher accumulation into the parasite’s acidic food vacuole may be expected to result in more potent antiplasmodial activity [10]. This hypothesis was tested with the synthesis of a series of analogs incorporating alkyl diamine side chains at C-11, for example, 44, which showed significantly increased activities against chloroquine-resistant P. falciparum in vitro, when compared to the parent compound, and with improved cytotoxic/antiplasmodial selectivity indices [47]. However, fluorescence microscopy of parasite-infected red blood cells showed that these compounds accumulated into the parasite nucleus and that most of the compounds were able to bind to double-stranded DNA as well as to hematin, suggesting that both of these targets are involved in their antiplasmodial action. Knowledge of protonatable sites in cryptolepine analogs is clearly important for understanding their antiplasmodial action, and 1 H-NMR spectroscopy has been employed to identify them and determine their dissociation constants [48]. In another study, Behzadi et al. in 2011 explored the relationship between the antiplasmodial activities of 2-bromo- and 2,7-dibromocryptolepine (43) and shielding tensors, which may be relevant to the design of new analogs [49]. The possibility remains that cryptolepine analogs incorporating basic groups but which are unable to intercalate into DNA could be potentially interesting lead to antimalarial compounds. In a series of indolo[3,2-b]-C11-carboxamides, 2-bromo-N-[2-(piperidin-1yl)ethyl]-10H-indolo[3,2b]quinoline-11-carboxamide (Fig. 11, 45) was the most promising compound (IC 50 = 1.3 μM against P. falciparum in vitro) [50]. This compound is interesting in that the nitrogen at N-5 is not quaternary as in cryptolepine, a feature considered important for antiplasmodial activity [40], and, in addition to inhibiting hemozoin formation, it has been shown to inhibit malaria parasite hemoglobin uptake. However, although orally active with predicted favorable pharmacokinetics, the suppression of parasitemia in mice infected with P. berghei was no more than 35% when compared to untreated infected mice [50]. Further syntheses of novel cryptolepine analogs aimed at increasing antiplasmodial potency, optimizing pharmacokinetic properties and with activities against liver and sexual stage malaria parasites would be worthwhile. However, from an environmental perspective, organic synthesis is not generally eco-friendly and as the majority of malaria patients are poor, the affordability of antimalarial drugs is an important consideration. An alternative approach that has the potential to address these issues could be to cultivate C. sanguinolenta and then extract 1 from the roots as a precursor for the production of semi-synthetic analog leads for novel antimalarial drugs. Recent work has resulted in the development of a simple and efficient alkaloid extraction process using non-toxic environmentally friendly solvents for the isolation of 1, and preliminary experiments have shown that 1 may be halogenated directly to yield semi-synthetic analogs such as 46 with potent antiplasmodial activities [51].

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8.4 Trypanosomiasis Species of Trypanosoma are responsible for Human African Trypanosomiasis (HAT) as well as Chagas’ disease in South America. The currently available antitrypanosomal drugs are of limited effectiveness, are often toxic to patients, and parasite resistance is widespread [52]. Cryptolepine (1) and eight synthetic analogs have been assessed for in vitro activity against T. brucei (strain 427) trypanosomes [52]. All these compounds displayed marked activity, with IC 50 values