Recent Advances in the Synthesis of organic Compounds to combat Neglected Tropical Diseases [1 ed.] 9781608059027, 9781608059034

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Recent Advances in the Synthesis of Organic Compounds to Combat Neglected Tropical Diseases Edited By

Adilson Beatriz & Dênis Pires de Lima Institute of Chemistry Federal University of Mato Grosso do Sul Campo Grande, MS Brazil

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DEDICATION This book is dedicated to those who suffered and are still suffering from neglected diseases. Equally, it is devoted to researchers that with the commitment to their work routinely contribute by seeking, in many and different ways, for new drugs to treat those diseases.

CONTENTS Foreword

i

Preface

ii

List of Contributors

iii

CHAPTERS 1.

CHAGAS DISEASE

3

An Overview of Recent Advances in the Search for Synthetic Organic Compounds with Trypanocidal Activity Ana Camila Micheletti, Edson dos Anjos dos Santos and Felicia Megumi Ito 2.

DENGUE FEVER

32

Recent Advances in the Discovery of Small Organic Molecules for the Prevention and Treatment of Dengue Fever Lucas Cunha Dias de Rezende, Victor Hugo Aquino and Flavio da Silva Emery 3.

LEISHMANIASIS

63

An Overview of New Synthetic Antileishmanial Candidates Nubia Boechat and Luiz Carlos da Silva Pinheiro 4.

LEPROSY Chasing Potential Antileprotic Compounds Dênis Pires de Lima and Davidson Pires de Lima

122

5.

LYMPHATIC FILARIASIS

149

Therapeutic Arsenal and Drug Discovery for Lymphatic Filariasis from a Synthetic Point of View Gabriela Bianchi dos Santos, Ana Patrícia Yatsuda Natsui and Flavio da Silva Emery 6.

MALARIA

172

Synthesis of Hybrid Molecules and Inhibitors of Falcipain for Use as Therapeutic Agents in Treatment of Malaria Renata Barbosa de Oliveira, Marcela Silva Lopes and Saulo Fehelberg Pinto Braga 7.

SCHISTOSOMIASIS

196

Recent Progresses in Synthesis and Evaluation of Bioactive Compounds with Molluscicidal Activity - Molluscicidal Activity of Synthetic and Natural Products Ronaldo Nascimento de Oliveira and Ricardo A. W. Neves Filho 8.

TUBERCULOSIS

231

Synthetic Organic Compounds as Potential Antitubercular Drugs: A Review of the Progress Made in the Last Five Years Jean Leandro Santos, Guilherme F. dos Santos Fernandes, Priscila Longhin Bosquesi, Leonardo B. Marino, Clarice Q. F. Leite, Chung M. Chin and Fernando Rogério Pavan Subject Index

267

i

FOREWORD Half the disease burden of the developing world – 80% of the world’s population – is due to infectious and parasitic diseases, most of which are preventable and/or treatable. More than 1 billion people is affected by at least one neglected tropical diseases (NTDs), primarily poor populations living in tropical and subtropical climates, with an approximated 534 000 deaths per year. These diseases have a huge impact on labor productivity and costs low-income countries billion of dollars a year, keeping up the vicious circle of poverty. Yet despite these alarming numbers, between 1975 and 1999 only thirteen new drugs were approved that targeted NTDs. Fortunately, over the past decade the tide has began to turn, in both recognizing the lack of new medicines dedicated to the diseases of the most neglected populations and in initiating efforts to redress the problem. In recent years, there has been a remarkable commitment on the part of ministries of health in endemic countries, global health initiatives, funding agencies and philanthropists escalated, as did donations of medicines from pharmaceutical companies and the engagement of the scientific community, having efforts towards the infectious diseases of the developing world. In this scenario, medicinal and synthetic chemists are of upmost importance, for designing, synthetizing and evaluating new drug candidates for parasitic and infectious diseases. This eBook is dedicated to the theme of “Recent Advances in the Synthesis of Organic Compounds to Combat Neglected Tropical Diseases” and features contributions from a Brazilian team of synthetic chemists and pharmaceutical researchers that work in the field of synthesis of compounds that are potentially bioactive against the causative agents of NTDs.

Carolina Horta Andrade LabMol - Laboratory for Molecular Modeling and Drug Design Faculdade de Farmácia Universidade Federal de Goiás Rua 240, Qd. 87 Setor Leste Universitário, 74605-170 Goiânia - GO Brazil

ii

PREFACE In the website of WHO, 14 diseases are reported to occur exclusively or specially in tropics. Most of them are infectious diseases affecting mainly poor populations and include leishmaniosis, dengue fever, malaria and Chagas disease. These diseases affect around 1 billion people and, according to WHO, 100% of developing countries are affected by at least five of these diseases. Accordingly, there is an urgent necessity for increasing the arsenal of drugs to combat neglected diseases in order to guarantee relief and well-being to many patients. The situation requires powerful research and improvement of politics to support investigation in this area of knowledge. There is a great volume of scientific work dealing with biology, immunology and genetics of parasites which cause these diseases. Taking in account the present situation, the role of Organic Chemistry in the pharmaceutical industry continues to be one of the main propellers for the discovery of new drugs. Medicinal organic chemists have contributed by seeking new bioactive compounds. Nowadays, there is a considerable global effort to evoke awareness to this problem and overcome the persisting inequality in the focus of drug discovery. Therefore, this eBook gathers important scientific research performed by scientists worldwide showing the state of the art of Organic Medicinal Chemistry dedicated to the synthesis of compounds that are potentially bioactive against the causative agents of neglected diseases. The eBook intends to be a valuable source of information to researchers in the area of Medicinal Chemistry, Organic Synthesis, as well as to undergraduate and graduate students interested in this subject. The eBook is composed of eight chapters, each of which is dedicated to one type of neglected disease. Essentially, the authors compiled important work concerning the synthesis and biological evaluation, according to their targets, of lead compounds in the process of developing new pharmaceuticals.

Adilson Beatriz & Dênis Pires de Lima Institute of Chemistry Federal University of Mato Grosso do Sul Campo Grande – MS Brazil

iii

List of Contributors Aquino, Vitor Hugo Department of Clinical, Toxicological and Bromatological Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirao Preto – SP, Brazil Boechat, Nubia Department of Organic Synthesis, Institute of Drug Technology - Farmanguinhos, Oswaldo Cruz Foundation - Fiocruz, Rio de Janeiro-RJ, Brazil Bosquesi, Priscila Longhin Faculty of Pharmaceutical Sciences, Paulista State University- UNESP, Araraquara-SP, Brazil Braga, Saulo Fehelberg Pinto Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte-MG, Brazil Chin, Chung M. Departamento de Fármacos e Medicamentos, Faculty of Pharmaceutical Sciences, Paulista State University- UNESP, Araraquaara-SP, Brazil de Lima, Davidson Pires Department of Clinical Medicine, Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte-MG, Brazil de Lima, Dênis Pires Institute of Chemistry, Federal University of Mato Grosso do Sul, Campo Grande-MS, Brazil Emery, Flávio da Silva Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto – FCFRP, University of São Paulo - USP, Ribeirao Preto – SP, Brazil

iv

Fernandes, Guilherme F. dos Santos Faculty of Pharmaceutical Sciences, Paulista State University- UNESP, Araraquara-SP, Brazil Ito, Felicia Megumi Federal Institute of Mato Grosso do Sul. Rua Tanure Salime, s/n, 79400-000, Coxim-MS, Brazil Leite, Clarice Q. F. Laboratory of Mycobacteriology “Prof. Dr. Hugo David”, Department of Biological Science, Faculty of Pharmaceutical Sicences, Paulista State University - UNESP, Jaú-SP, Brazil Lopes, Marcela Silva Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte-MG, Brazil Marino, Leonardo B. Laboratory of Mycobacteriology “Prof. Dr. Hugo David”, Department of Biological Science, Faculty of Pharmaceutical Sicences, Paulista State University - UNESP, Jaú-SP, Brazil Micheletti, Ana Camila Institute of Chemistry, Federal University of Mato Grosso do Sul, Campo Grande-MS, Brazil Natsui, Ana Patricia Yatsuda Department of Clinical, Toxicological and Bromatological Analyses, Faculty of Pharmaceutical Sciences of Ribeirao Preto, São Paulo University - USP, Ribeirão Preto-SP, Brazil Neves Filho, Ricardo A.W. Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg, Germany

v

Oliveira, Renata Barbosa de Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte-MG, Brazil Oliveira, Ronaldo Nascimento de Laboratory of Synthesis of Bioactive Compounds, Department of Molecular Sciences, Federal Rural University of Pernambuco, Recife-PE, Brazil Pavan, Fernando Rogério Laboratory of Mycobacteriology “Prof. Dr. Hugo David”, Department of Biological Science, Faculty of Pharmaceutical Sicences, Paulista State University - UNESP, Jaú-SP, Brazil Pinheiro, Luiz Carlos da Silva Department of Organic Synthesis, Institute of Drug Technology - Farmanguinhos, Oswaldo Cruz Foundation - Fiocruz, Rio de Janeiro-RJ, Brazil Rezende, Lucas Cunha Dias Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Riberão Preto – FCFRP, University of São Paulo - USP, Ribeirao Preto – SP, Brazil Santos, Edson dos Anjos dos Federal Technological University of Paraná, Apucarana-PR, Brazil Santos, Gabriela Bianchi dos Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto - FCFRP, São Paulo University - USP, Ribeirão Preto-SP, Brazil Santos, Jean Leandro Faculty of Pharmaceutical Sciences, Paulista State University- UNESP, Araraquara-SP, Brazil

Send Orders for Reprints to [email protected] Organic Compounds to Combat Neglected Tropical Diseases, 2014, 3-31

3

CHAPTER 1 CHAGAS DISEASE An Overview of Recent Advances in the Search for Synthetic Organic Compounds with Trypanocidal Activity Ana Camila Micheletti1,*, Edson dos Anjos dos Santos2 and Felicia Megumi Ito3 1

Federal University of Mato Grosso do Sul, Institute of Chemistry, Av. Senador Filinto Muller, 1555, 79074-460, Campo Grande-MS, Brazil; 2Federal Technological University of Paraná, Campus Apucarana, Rua Marcílio Dias, 635, CEP 86812-460, Apucarana-PR, Brazil and 3Federal Institute of Mato Grosso do Sul. Rua Tanure Salime, s/n, 79400-000, Coxim-MS, Brazil Abstract: Chagas disease was discovered over 100 years ago, and continues to be a major public health problem, with 16 to 18 million people infected with Trypanososma cruzi, its causative agent, and 21,000 deaths associated with this disease every year. Nifurtimox (Nfx) and benznidazole (Bnz) are even today the only clinically available drugs for the chemotherapy of Chagas disease, but they show significant side effects, involve long-term treatment and there are other problems, like emergence of resistant strains. This chapter aims to highlight recent contributions from the organic synthesis field on developing new compounds with trypanocidal activity, including discussion on important structural features and molecular targets.

Keywords: Chagas disease, antichagasic activity, organic synthesis, trypanocidal activity. 1. INTRODUCTION The year 2009 was 100 years since the discovery of Chagas disease, but it continues to be a serious public health problem almost completely ignored by the pharmaceutical market, and is therefore, classified as a neglected disease. Neglected diseases, such as Chagas disease, leishmaniasis, schistosomiasis, malaria, filariosis and sleeping sickness affect a quarter of the world and are prevalent throughout the tropics and subtropics, representing a major public health problem in regions least able to deal with the economic burden. Millions of people *Address correspondence to Ana Camila Micheletti: Federal University of Mato Grosso do Sul, Institute of Chemistry, Av. Senador Filinto Muller, 1555, 79074-460, Campo Grande-MS, Brazil; Tel/Fax: +55 67 3345 3558; E-mail: [email protected] Adilson Beatriz and Dênis Pires de Lima (Eds) All rights reserved-© 2014 Bentham Science Publishers

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worldwide still die of diseases that can be prevented and treated. Daily, over 35,000 people die from infectious and neglected diseases. Very little investment is devoted to research and development of drugs to treat diseases affecting poor populations. The lack of interest from pharmaceutical companies searching for new drugs for certain diseases is directly connected to the low purchasing power of these populations [1, 2]. Moreover, parasitic protozoa are eukaryotic; so they share many common features with their mammalian host, making the development of effective and selective drugs a hard task [3]. Most of the drugs used in therapies were discovered at least five decades ago. They are difficult to administer, have high toxicity, and the treatments are timeconsuming and costly. The urgency in finding new medicines for neglected diseases has motivated research and development in various countries around the world [1, 2]. Chagas disease, also known as American trypanosomiasis, is caused by the flagellate protozoan parasite Trypanosoma cruzi, and is responsible for a considerable human mortality and morbidity, especially in Latin America, where it is endemic. An estimated 16 to 18 million people are infected with T. cruzi worldwide and more than 100 million people are at risk of acquiring the disease. Recent surveys indicate that about 200,000 new cases and 21,000 deaths are associated with this disease every year [2, 4]. The easy migration of infected individuals due to globalization is of great concern since the disease is being spread to non-endemic regions [5]. T. cruzi presents three main morphological forms in a complex life cycle, which necessarily involves crossing a vertebrate host (mammals, including man) and invertebrate host (hematophagous insects of the subfamily Triatominae): the epimastigote and metacyclic trypomastigote forms found in insects, besides the amastigote and trypomastigote blood forms responsible for the multiplication and spread of infection in man, respectively [2]. The vectors are bugs (family Reduviidae), hematophagous insects, popularly known as kissing bugs or weevils. Any mammal can harbor the parasite, while birds and reptiles are refractory to infection. The main reservoirs in the sylvatic cycle are opossums, armadillos, dogs, cats, rats etc. In the domestic cycle, due to the proximity of the dwellings of

Chagas Disease

Organic Compounds to Combat Neglected Tropical Diseases 5

man with the wild environment, the reservoirs are humans and domestic or synanthropic mammals such as dogs, cats, rats and pigs [6]. Chagas disease is transmitted by triatomine feces (90%), through contact with broken skin or mucous membranes of the eye, blood transfusion, organ transplantation, laboratory accidents, and oral or congenital contamination. Vectorial and transfusional transmission have steadily declined through multinational initiatives focused on vector control (Triatoma infestans) and blood donor screening [5], but the sanitary problems associated with those already infected still remains a challenge [7]. The disease is characterized by three clinical phases named acute, indeterminate and chronic, that differ in symptoms and morbidity. The acute phase can be identified or not (acute Chagas disease - ACD) shows a tendency to progression to chronic forms if not treated early with specific drug. In humans, in the absence of treatment, the symptoms persist for about two months, with a mortality of 2% to 8%, especially among children. After the acute phase, approximately 60% - 70% of the infected patients will progress to an indeterminate form, without any clinical manifestation. The remaining 30% to 40% develop chronic clinical forms, divided into three types according to the presented complications: cardiac, digestive or mixed (with cardiac and digestive) [2, 6]. The old and quite unspecific drugs nifurtimox (Nfx, 1), a nitrofuran derivative, and benznidazole (Bnz, 2) (Fig. 1), a nitroimidazole derivative, are even today the only clinically available drugs for the chemotherapy of Chagas disease. Both drugs act via the reduction of the nitro group. Nfx and Bnz show trypanocidal effects on all forms of the parasite, curing around 80% of acute cases. However, patients should undergo long-term treatment, about 60 days, with 2-3 doses per day; the drugs are not accessible for patients, in some cases being restricted to specialized clinics when medical monitoring during the course of treatment is required. There are also other problems, for example, some strains of T. cruzi are resistant to treatment, low antiparasitic activity of these drugs in chronic disease, besides significant side effects that both drugs induce, like include anorexia, nausea, vomiting, headache, central nervous system depression, maniacal symptoms, seizures, vertigo, peripheral polyneuropathies and dermatitis, probably

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because of non-selective bioreduction, affecting the parasite and the mammalian host [2, 7-10]. N CH3 N

O S

N

N

O

O

NO 2

NH

NO2

O

nifurtimox (1)

benznidazole (2)

Figure 1: Current drugs for the chemotherapy of Chagas disease.

Because of the urgent need for new therapies for Chagas disease, many research groups around the world are focusing on developing molecules with antiparasitic potential with more appropriate treatment regimens. In this chapter we gather information published since 2009 on the advances of organic synthesis in creating new molecules with antichagasic activity. 2. NEW SYNTHETIC MOLECULES 2.1. Naphthoquinones Quinones are widely distributed in nature and naphthoquinone derivatives are considered privileged structures in medicinal chemistry on the basis of their biological activities and structural properties. In folk medicine, plants containing naphthoquinones are usually employed for the treatment of different diseases [11, 12]. Their fundamental feature is their easy reduction and, therefore, the ability to act as an oxidizing or dehydrogenating agent, and their biological activity is related to the acceptance of 1 and/or 2 electrons to form the corresponding radical anion or dianion species [11, 13]. The redox-cycling of quinones may be initiated by either one- or two-electron reduction. The one-electron reduction of quinones is catalyzed by NADPH–cytochrome P450 reductase, generating unstable semiquinones. These transfer electrons to molecular oxygen and return to their original quinoidal form, thus generating superoxide anion radical (•O2−) that can form other highly reactive oxygen species (ROS) (H2O2, •HO). These ROS may react directly with DNA or other cellular macromolecules, such as lipids and proteins, leading to cell damage [11].

Chagas Disease

Organic Compounds to Combat Neglected Tropical Diseases 7

Many groups have been synthesizing molecules with a quinone scaffold with the aim to produce new trypanocidal agents. Ramos et al. [13] synthesized 2,3diphenyl-1,4-naphthoquinone (3, Fig. 2) that proved to be active against trypomastigotes of T. cruzi in an in vitro assay, with an LD50 of 2.5 μM. The compound was studied in vivo as well, in the murine model, showing activity with low toxicity. The study revealed that 3 acts by producing oxidative radicals, but caused no genomic DNA fragmentation, also being a competitive inhibitor of lipoamide dehydrogenase of the parasite (TcLipDH). During evolution, these parasites developed a specific thiol redox metabolism based on the trypanothione system. In addition, they possess LipDH, an enzyme that is structurally and mechanistically closely related to trypanothione reductase (TR). Da Silva Jr. et al. [14] synthesized and evaluated the trypanocidal activity of βlapachone -based 1,2,3-triazoles, 3-arylamino-nor-β-lapachones, 3-alkoxy-nor-βlapachones and imidazole anthraquinones. 2,2-Dimethyl-3-(2,4-dibromophenylamino)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (IC50/24 h 24.9 ± 7.4) and 4azido-3-bromo-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (4, Fig. 2) with 23.4 ± 3.8 μM showed a trypanocidal activity higher than benznidazole, against bloodstream trypomastigote forms of the parasite. The group continued the work and prepared other quinoidal compounds [5]. Based on a nor--lapachone 1,2,3-triazole phenyl-substituted derivative as a prototype, novel triazolic naphthofuranoquinones with different electron donating or withdrawing groups in the phenyl ring were synthesized and evaluated for anti-T. cruzi activity. Also, lapachol was used as starting material for the synthesis, by structural modifications. The insertion of a 1,2,3-triazole group into the 1,4-naphthoquinoidal structure efficiently enhanced its pharmacological activity, and five substances were identified as potent trypanocidal compounds, with derivatives more active than benznidazol (IC50/24 h, from 10 to 80 μM) (5, Fig. 2). Ferreira et al. [12] also have prepared 16 derivatives based on β-lapachone. The compounds presented a broad spectrum of lytic effects upon bloodstream trypomastigotes of T. cruzi, with five derivatives having IC50/24 h in the range of 20 (6, Fig. 2) to higher than 4000 M. Ubiquinone (coenzyme Q - CoQ) is a quinoidal compound that occurs in the lipid core of mitochondrial membranes and functions in the electron transport chain as

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a carrier. A structural feature common to CoQ mimics is a lipophilic side chain, which increases permeability in the inner phospholipid bilayers of mitochondria, where electron transport complexes are embedded. Phosphonium cations are used extensively as molecular probes for studying mitochondrial function and have demonstrated utility as mediators of transport into mitochondria. The attachment of a triphenylphosphonium group to an alkyl or alkenyl side chain can guide the lipocation into energized mitochondria by electrostatic attraction and leads to increased drug concentrations in the mitochondrial matrix. Long et al. [15] hypothesized that if a phosphonium moiety were bound to the lipid substituent in antagonists of the Plasmodium and Trypanosoma electron transport chains, higher drug concentrations would be achieved inside the mitochondrion and would lead to increased antiparasitic effects. With this aim, they examined the effects on antiparasitic activity of phosphonium group attachment to structural analogs of CoQ containing phthalimide and 1,4-naphthoquinone platforms. The compounds were assessed for efficacy against Vero cell-infected T. cruzi amastigotes. The IC50 values for inhibition of parasite development ranged from 1.6 (7, Fig. 2) to 5.4 μM and variable degrees of Vero cell toxicity was observed.

O O O

O N

N

-

+

N

N O O

Br H3C

(3)

O

CH3

(5)

(4) O O

O +

P

O Br

9 CH3

(6)

O

(7) Cl

Figure 2: Active quinoidal compounds.

N N

Chagas Disease

Organic Compounds to Combat Neglected Tropical Diseases 9

2.2. Nitrofuranes and Nitrothiophenes 5-Nitrofuryl derivatives possess high and selective anti-T. cruzi activity in vitro and in vivo, and one of the mechanisms of action of this family of compounds is the induction of oxidative stress in the parasite [16]. Gerpe et al. [16] have synthesized 5-nitrofuran and 5-nitrothiophene -derivatives with allylamide and (3phenyl)allylamine moieties, using connection via thiosemicarbazone, semicarbazone, or amide linkers. Allylamine derivatives potently inhibit squalene epoxidase (SE), a microsomal mono-oxygenase that catalyzes the conversion of squalene to 2,3-oxidosqualene using molecular oxygen. SE is essential for the synthesis of cholesterol in mammals and ergosterol in fungi, and some allylamines have also shown to be potent in vitro and in vivo T. cruzi growth inhibitors, acting by selective reduction of the parasite’s endogenous membrane sterol levels. Among synthesized derivatives, many have activity against T. cruzi (like 8, with IC50 of 1.9 μM), and also the ability to accumulate squalene, a property favored by the presence of the 5-nitro moiety. Herrera et al. [10] prepared, based on the cytotoxic characteristics and potential pharmacophoric effect of the class, thienyl-2-nitropropene compounds that proved to be trypanocidal. The IC50 of the most active compound (9, Fig. 3) on epimastigotes and amastigotas in vitro ranged from 0.6 to 1.3 μM. NO2 CH2 N O2N

N H

CH2 CH3

N H

O

S

(8)

(9)

Figure 3: Active nitrofurans and nitrothiophenes.

2.3. Metal Complexes The many activities of metal ions in biology have stimulated the development of metal-based chemotherapeutics in different fields of medicine. Although emphasis has been placed mainly on cancer treatment as a result of the great success of cisplatin, recent studies have also included parasitic diseases. A significant number of studies have provided evidence of the usefulness of applying the

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complexation of transition metals with antitrypanosomal ligands as a strategy for enhancing pharmacological properties (efficacy, bioavailability) or the desired chemical properties (such as lipophilicity and reactivity). This approach takes advantage of the emerging medicinal chemistry paradigm on drug discovery to develop agents that could modulate multiple targets simultaneously with the aim of enhancing efficacy relative to drugs that address only a single target. It is useful to modify the ligand’s pharmacological profile and/or obtain compounds that could act through a dual mechanism of action combining the pharmacological properties of the metal and ligand in a cooperative effect. Biological properties of the metal-ligand species will depend not only on the nature of the ligand and the metal but also on the co-ligands and especially on the structural and physicochemical characteristics of the complex [7, 17, 18]. Ruthenium and vanadium seem to be privileged among metals to construct complexes as anti-protozoal agents. Ruthenium has proved to be an excellent choice for the development of anti-T. cruzi metal complexes, because of their redox stability, excited state lifetime and rate of ligand exchange [18]. Pagano et al. [7] have synthesized ruthenium complexes containing thiosemicarbazones with a 5-nitrofuryl moiety, which is considered to be a pharmacophore for trypanocidal activity. They have synthesized three series of mixed-ligand ruthenium complexes of the formula [RuCl2(HL)2], [RuCl3(DMSO)(HL)] and [RuCl(PPh3)(L)2] (where L means ligand) with bioactive 5-nitrofuryl-containing thiosemicarbazones (10, Fig. 4), and although coordination to ruthenium seems to decrease the anti-T. cruzi activity of the ligand, the effect of the presence of different co-ligands on the activity and related physicochemical properties was evident. In the light of this, while a high lipophilicity seems to favor the trypanocidal effect, the free radical scavenger capacity of the complexes could be correlated to a lower biological activity. The most active compound showed 80% of growth inhibition of T. cruzi epimastigote cells at 25 μM (growth inhibition of the parasite under treatment with nifurtimox was taken as 100%). It had been stated that the mechanism of action of the complexes is the same as that of the ligands, i.e. bioreduction and redox cycling. Donnici et al. [18] described the preparation and biological characterization of eight new ruthenium complexes [Ru-Cl2(ATZ)(COD)], where COD represents 1,5-cyclooctadiene, with aryl-4-

Chagas Disease

Organic Compounds to Combat Neglected Tropical Diseases 11

oxothiazolylhydrazons (ATZ) ligands, deemed to be non-classical bioisosters of thiosemicarbazones. This second ligand possesses great ability to interact with biological targets by hydrophobic interaction and provides chemical stability to the metal complex. In almost all cases, the organometallic compound was more active than the ligand alone on trypomastigotes and epimastigotes of T. cruzi, and many of them were more effective than benznidazole and nifurtimox. The most active compound was 11 (Fig. 4), with an IC50 of 1.8 μM over the epimastigote form and it can be considered as a prototype for an antitrypanosomal drug. Ru

S

N O 2N

R N H

O N

Cl S

NH

S

NH Ru

N

HL

Cl

HN

NHR

S

Br

Ru

SN

R= H, methyl, ethyl or phenyl

N

R N

NH

O

L (10)

(11)

Figure 4: Active ruthenium complexes.

The control of T. cruzi infection in vertebrate hosts is dependent on the activation of macrophages and NO production, which is involved in intracellular parasite destruction. Thus, the pharmacological modulation of the host’s immune response against T. cruzi through control of the levels of NO has been gaining substantial interest as a potentially valuable chemotherapeutic approach. Silva et al. [19] have prepared a new class of ruthenium NO donors, cis-[Ru(NO)(bpy)2L]n+, cis[Ru(H2O)(bpy)2L]n+ and cis-[Ru(NO2)(bpy)2L]n+ where L = imidazole (imN), 1methylimidazole (1-miN) or sulphite ion (SO32-), and evaluated their effects in cell cultures and animal models. Ruthenium NO donors are able to lyse trypomastigotes of T. cruzi present in the bloodstream (BT), by an NO-dependent mechanism, and can also inhibit the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the glycolytic cascade that has been implicated in intracellular events mediated by NO. The enzyme possesses important structural differences when compared to the homologous protein from the mammalian host

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(about 45% sequence identity) and, because of that, is an attractive target for the development of novel antitrypanosomatid agents. In vitro and in vivo trypanocidal compounds were those which effectively donate NO, and one of the possible mechanisms responsible for this effect is the inhibition of GAHPD (IC50 values from 89 (cis-[Ru(NO)(bpy)2imN](PF6)3) to 153 M - in vitro assay). But, because these complexes react easily with glutathione and potentially with trypanothione, it is reasonable to suggest that they can also act through the trypanothione/ trypanothione reductase system. Benítez and coworkers have reported, in the last few years, three series of vanadyl complexes (VIV), using diverse polypyridyl [17, 20, 21] and salicylaldehyde semicarbazones [17, 21] as ligands. Metabolic pathways of kinetoplastid parasites (Trypanosomaparasites) are similar to those present in tumor cells leading to a correlation between anti-trypanosomal and anti-tumor activities. In addition, it has been proposed that compounds that efficiently interact with DNA in an intercalative mode, like polypyridyl chelators (Npy,Npy donors) capable of intercalating DNA, could also show anti-trypanosomal activity [17]. First, the authors studied a polypyridyl system as ligand, the dipyrido[3,2-a:2’, 3’ -c]phenazine (dppz), together with salicylaldehyde semicarbazones (Salsem -2H), as coligands. The latter are versatile ligands which present a wide range of bioactivity [17]. 2,2’-Bipyridine (bipy) was also used as a polypyridyl system, but in this case, the complexes had just moderate activity. Compounds containing both dppz and salicylaldehyde semicarbazone or 5-bromosalicylaldehyde semicarbazone ligands showed IC50 values of 13 and 19 μM on epimastigotes of the Dm28c strain of T. cruzi, respectively, by interacting with DNA, and introducing conformational changes of the biomolecule. Then, dppz was used again, in a complex with formula [VO(SO4)(H2O)2(dppz)].2H2O (12, Fig. 5). The compound was significantly trypanocidal, with an IC50 of 3 μM and, in addition, showed the ability to stimulate apoptosis in tumor human cell lines by an interaction with DNA [20]. Changing the pyridine moiety to 1,10-phenantroline and using another pool of semicarbazones, with methoxy, ethoxy and bromine groups substituting the ortho- and para-positions of the phenolic ring, the group has synthesized another five vanadyl complexes [VO(salsen-2H)(phen)], much more active than the others, with IC50 values between 1.6 and 3.8 μM (13, Fig. 5) [21].

Chagas Disease

Organic Compounds to Combat Neglected Tropical Diseases 13 N N N

OH2

N

N

N

N

O

OH2

O

V O

O

V

NH2

O

O4S

N CH3

(13)

(12)

Figure 5: Active vanadyl complexes.

Other metals and ligands have been used in recent years to prepare trypanocidal compounds, and examples are summarized in Table 1. Table 1: Other trypanocidal complexes Metal (Reference)

Ligands

Pt (II) [22]

3-(5-nitrofuryl)acrolein thiosemicarbazones

Pt (II) [22]

3-(5-nitrofuryl)acrolein thiosemicarbazones (14, Fig. 6)

Molecular Formula

IC50 (μM)

Mode of Action

[PtCl2(HL)]

T. cruzi Talahuen 2: 8.6 - > 25 T. cruzi Dm28c: 24.40 – 63.23

Production of free radicals and interaction with DNA

[Pt(L)2]

T. cruzi Talahuen 2: 9.1 - >25 T. cruzi Dm28c: 5.89 – 48.22

Production of free radicals and interaction with DNA

Ni (II) [23]

Anionic form of 4,6dimethyl-1,2,3triazolo[4,5d]pyrimidin-5,7-dione (dmax – 15, Fig. 6)

Ni(dmax)2(A)n(H2O)m A= aliphatic or aromatic amine

Pd [24]

N,N-dimethyl-1phenethyl-amine (DMPA), 1,2-ethane-bis (diphenylphosphine) (DPPE) (16, Fig. 6)

[Pd2 (S(−)C2,NDMPA)2 (mDPPE)]Cl2

15x10

Mn (II) [25]

Nitrobenzaldehyde, nitroacetophenone and nitrobenzophenone thiosemicarbazones (17, Fig. 6)

[Mn(L)2Cl2]

19,21 - > 200 (trypomastigotes)

L= ligand.

229 μM to 11 μM (44, Fig. 15), and a selectivity index < 2 in almost all cases. O O

CH3 N

CH3 N

O

CH3 CH3

N

CH3 N N CH3

(42)

(41)

O CH3

O

CH3 CH3

N

CH3 N N

(43)

Figure 14: Cyclic amines 41 to 43.

Megazol (45) is a nitro 1,3,4-thiadiazole that has been shown to be highly active against T. cruzi, including strains resistant to benznidazole, in vitro and in vivo and has become a core structure for the design of new leads for the treatment of Chagas disease. It has been described as a scavenger of trypanothione, the cofactor for trypanothione reductase, but despite its noteworthy trypanocidal activity, megazol (45) development was discontinued due to reports of in vitro mutagenic and genotoxic effects. Salomão et al. [43] synthesized 1,3,4thiadiazole-2-arylhydrazone derivatives of megazol (45) in order to find potential trypanocidal agents, exploring the hypothesis that the introduction of a radical

Chagas Disease

Organic Compounds to Combat Neglected Tropical Diseases 23

scavenger arylhydrazone moiety in the heterocyclic framework of megazol (45) could modulate the production of toxic nitro anion radical species, thus avoiding potentially mutagenic properties. The study identified new possible prototypes; IC50/2 day values for the active compounds were 2.9 ± 0.6, and 1.6 ± 1.0 μM (46, Fig. 15) on intracellular amastigotes, with good selectivity indexes.

N

N

N

CH3

N N

NH2

S

O

N Cl

CH3

O2N

CH3

(44)

(45) NO2

NH N

N

N

N S

N

N H

CH3

CH3

N

O

N

F O

CH3

N O2N

N

CH3

F

n-Bu CH3 F

(46)

(47)

OH Cl

F

O

O N O

OAc

AcO

AcO OAc

(48)

Figure 15: Active nitrogen heterocycles 44 to 48.

1,2,3-Triazole-based tetrafluorophenoxymethyl ketones were prepared by Brak et al. [32] with the aim to obtain cruzain inhibitors. Because of the ability of 1,2,3-triazoles to function as rigid linking units that mimic the atom placement and electronic properties of a peptide bond without the susceptibility to

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hydrolysis, many known 1,2,3-triazoles possess biological activity. The compounds differ in the number of carbon atoms (1 to 6) and type (straight, branched and/or cyclic) of side chains linked to the same scaffold, and many of them proved to be cruzain inhibitors, as well as being active against intracellular parasites. Derivatives with an n-butyl chain attached to an α-carbonyl position near the triazolic moiety (47, Fig. 15) have the best interaction with the enzyme, and the more potent inhibitors all had improved activity in cell culture (IC50 = 3.1 - 4.2 μM). Bouchikhi et al. [44] have synthesized isoindigo derivatives, simpler nitrogen heterocycles, bearing an acetyl-sugar residue attached to one of the indolin-2-one nitrogens and diversely substituted on the aromatic rings. The best activities against T. cruzi (TulahuenC2C4) were found for compounds with IC50 values in the range of 10–30 M, with 48 (Fig. 15) being the most active. The study suggests that these scaffolds could be of value for the discovery of new molecules useful as lead compounds for tropical diseases. 2.5. Nitrogenated Open Chain Compounds Classes of nitrogenated open chain compounds like hydrazides, hydrazones, amidines, diamines and others, some of them containing sulfur, also play important roles in the search for novel trypanocidal agents. Borchhardt et al. [45] reported the synthesis of 3,4,5-trimethoxybenzohydrazides containing different aromatic substituents such as nitro-, bromo-, methoxyphenyl, naphthyl etc., as cruzain inhibitors. These derivatives inhibited the activity of the enzyme at a range of concentration from 40 μM (49, Fig. 16) to 60 μM. Arylimidamides, diamidines and a guanylhydrazones with trypanocidal activity are described by Pacheco et al. [46], all containing aromatic moieties and/or O and S heterocycles. The arylamidine 50 (Fig. 16) showed striking anti-parasitic activity (IC50 = 4.05 μM against bloodstream trypomastigotes and IC50 = 0.76 μM against intracellular forms) and low toxicity (selectivity index near 100). Putrescine and 1,3-propanediamine are reported to show activities against P. falciparum, L. donovani and T. cruzi, probably interfering with polyamine metabolism, validating diamines derivatives as an attractive scaffold for the

Chagas Disease

Organic Compounds to Combat Neglected Tropical Diseases 25

development of new anti-parasitic drugs. Caminos et al. [1] synthesized a series of N,N-substituted diamines, covering different chain lengths (between the two nitrogen atoms) and degrees of substitution in the aromatic moiety. Compounds were assayed on epimastigotes of T. cruzi, and many derivatives showed activity below 10 μM (some of them showed submicromolar activities). The best trypanocidal agents, such as derivative 51 (Fig. 16) with IC50 = 0.73 μM and a good selectivity index, regarding cytotoxicity on Vero cells, have a bulky group para to the nitrogenated chain (O-benzyl). When the substituent is smaller (OMe), the compounds are inactive. O

CH3 O

O

N

HN

N H CH3

NH

HN

O

NH

O CH3

(49)

(50)

CH3 O N

O

H N

H N NH2 S

N H

CH3 O O O

(51)

O CH3

CH3

(52)

Figure 16: Nitrogenated open chain trypanocidal compounds.

Thiosemicarbazones have been described as having parasitocidal action against T. cruzi. This class of small molecules has been evaluated over the last 50 years as antiviral, antibacterial and anticancer compounds. Thiosemicarbazones derived from 1-indanones were synthesized by Caputto et al. [47], based on data from the literature, that exhibited, in some cases, relevant anti-T. cruzi activities (epimastigote form), the most active being the 4,5-dimethoxy-substituted derivative 52 (Fig. 16) (IC50 = 1.8 μM, SI= 989.0). It is interesting to note that, for this group, chloro and nitro derivatives were not active against T. cruzi in culture. Compound 52 inhibited cruzipain, the major cysteine protease expressed in all the life cycle stages of the parasite, pointing to this as the target of action. This endoproteinase is the most

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abundant member among the cysteine-, serine-, threonine-, and metallo-proteinases, and it is essential for infection of host cells, replication and metabolism of the parasite and it plays multiple roles in disease pathogenesis. 2.6. Sugars, Phosphorus Compounds, Phenolic Derivatives T. cruzi trans-sialidase (TcTS) is involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. Scavenging sialic acid from the host glycoconjugates aids the recognition and attachment of the parasite to host cells, and allows its evasion from the host’s immune response, resulting in host cells invasion where the parasite can complete its life cycle [48, 49]. Campo et al. [49] have synthesized 1,2,3-triazole sialic acid-based neoglycoconjugates to evaluate their activity in inhibition of TcTS, by mimicking of the terminal sugars of T. cruzi mucins. The target sialylmimetic neoglycoconjugates are represented by 1,4disubstituted 1,2,3-triazole-sialic acid derivatives containing a galactose modified at either the C-1 or C-6 positions, glucose or gulose at the C-3 positions, and by the amino acid derivative 1,2,3-triazole fused threonine-3-O-galactose. The study concludes that sialic acid and galactosyl units are relevant for TcTS inhibition, and the best trypanocidal agent showed had IC50 = 260 M (53, Fig. 17). OH

HO

COOH O

HO

O

CH3

OH

O

N

P

HO

N N

HO

OH H

(53)

H

HO H

N H

HO

H

OH

P

(54)

H

OH O

Cl

CN O l-Oytubosi l-Oytubosi

O

P

N

N H

O

(55)

Figure 17: Antichagasic compounds 53 to 56.

CH3

O

O

OH

8

Cl

(56)

Chagas Disease

Organic Compounds to Combat Neglected Tropical Diseases 27

Rosso et al. [50] have prepared 1-[2-(alkylamino)ethyl] bisphosphonates with alkyl groups having 9, 10, 11, 12, 14, or 18 carbon atoms and one with a benzyl moiety, which were designed on the basis of literature data about antichagasic activity of aminobisphosphonates. The compounds were tested for inhibition of farnesyl diphosphate synthase of T. cruzi (Tc FPPS), which has been demonstrated to be the target of bisphosphonates that have activity in vitro and in vivo against T. cruzi, and solanesyl diphosphate synthase, another important prenyltransferase in T. cruzi (Tc SPPS), which is involved in the synthesis of ubiquinone. Compound 54 (Fig. 17) proved to be an extremely potent growth inhibitor against the clinically more relevant form of T. cruzi exhibiting an IC50 value of 0.67 M, significantly more potent than benznidazole. This cellular activity was associated with the inhibition of enzymatic activity towards the target enzyme Tc FPPS with an IC50 value of 0.81 M. Nogueira et al. [51] synthesized a new series of dialkylphosphorylhydrazones, designed according to the concepts of molecular hybridization of the functional hydrazonic groups of nifurtimox (1) and miltefosine phosphate, an anticancer drug, active against Leishmania. Butyl and isobutyl substituents were linked to the phosphoryl group, whereas nitro, cyano, carboxy, chloro groups substituted the aromatic moieties. The activity of these compounds was just moderate against epimastigotes of T. cruzi, with IC50 values ranging from 8.3 (55, Fig. 17) to 83.5 M. Besides the synthesis of 3,4,5-trimethoxybenzohydrazides, Borchhardt et al. [45] have prepared chalcones to evaluate their activity as cruzain inhibitors, as the class is described in the literature as inhibitors of malarial cysteine proteases. The most potent compounds had IC50 values between 20 (56, Fig. 17) and 25 μM and represent a new class of lead candidates for further development. Furthermore, almost all active chalcone derivatives possess the methylenedioxyphenyl (1,3benzodioxole) moiety, which suggests a significant contribution of this group to the observed activity. ACKNOWLEDGEMENTS The authors are indebted to Professor Derrick L. J. Clive (University of Alberta, Canada) for the great assistance with English review of this book chapter.

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nitrobenzaldehyde, N4-methyl-4-nitroacetofenone, and N4-methyl-4-nitrobenzophenone thiosemicarbazone : Investigation of in vitro activity against Trypanosoma cruzi. Polyhedron 2010, 29, 2232 – 2238. Muscia, G. C.; Cazorla, S. I.; Frank, F. M.; Borosky, G. L.; Buldain, G. Y.; Asís, S. E.; Malchiodi, E. L. Synthesis, trypanocidal activity and molecular modeling studies of 2alkylaminomethylquinoline derivatives. Eur. J. Med. Chem. 2011, 46, 3696 – 3703. Romeiro, N. C.; Aguirre, G.; Hernández, P.; González, M.; Cerecetto, H.; Aldana, I.; PérezSilanes, S.; Monge, A.; Barreiro, E. J.; Lima, L. M. Synthesis, trypanocidal activity and docking studies of novel quinoxaline -N-acylhydrazones, designed as cruzain inhibitors candidates. Bioorg. Med. Chem. 2009, 17, 641 – 652. Chennamaneni, N. K.; Arif, J.; Buckner, F. S.; Gelb, M. H. Isoquinoline-based analogs of the cancer drug clinical candidate tipifarnib as anti-Trypanosoma cruzi agents. Bioorg. Med. Chem. Lett. 2009, 19, 6582 – 6584. Kraus, J. M.; Tatipaka, H. B.; McGuffin, S. A.; Chennamaneni, N. K.; Karimi, M. Second generation analogues of the cancer drug clinical candidate tipifarnib for anti-Chagas disease drug discovery. J. Med. Chem. 2010, 53, 3887 – 3898. Kraus, J. M.; Verlinde, C. L. M. J.; Karimi, M.; Lepesheva, G. I.; Gelb, M. H.; Buckner, F. S. Rational modification of a candidate cancer drug for use against Chagas disease. J. Med. Chem. 2009, 52, 1639 – 1647. Ancizu, S.; Moreno, E.; Torres, E.; Burguete, A.; Pérez-Silanes, S.; Benítez, D.; Villar, R.; Solano, B.; Marín, A.; Aldana, I.; Cerecetto, H.; González, M.; Monge, A. Heterocyclic-2carboxylic acid (3-cyano-1,4-di-N-oxidequinoxalin-2-yl)amide derivatives as hits for the development of neglected disease drugs. Molecules 2009, 14, 2256 – 2272. Brak, K.; Kerr, I. D.; Barrett, K. T.; Fuchi, N.; Debnath, M.; Ang.; K.; Engel, J. C.; McKerrow, J. H.; Doyle, P. S.; Brinen, L. S.; Ellman, J. A. Nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors as promising new leads for Chagas disease chemotherapy. J. Med. Chem. 2010, 53, 1763 – 1773. Sirawaraporn, W.; Sertsrivanich, R.; Booth, R. G.; Hansch, C.; Neal, R. A.; Santi, D. V. Selective inhibition of Leishmania dihydrofolate reductase and Leishmania growth by 5benzyl-2,4-diaminopyrimidines. Mol. Biochem. Parasitol. 1988, 31, 79 – 86. Cavalli, A.; Lizzi, F.; Bongarzone, S.; Brun, R.; Krauth-Siegel, R. L.; Bolognesi, M. L. Privileged structure-guided synthesis of quinazoline derivatives as inhibitors of trypanothione reductase. Bioorg. Med. Chem. Lett. 2009, 19, 3031 – 3035. Schormann, N.; Velu, S. E.; Murugesan, S.; Senkovich, O.; Walker, K.; Chenna, B. C.; Shinkre, B.; Desai, A.; Chattopadhyay, D. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase. Bioorg. Med. Chem. 2010, 18, 4056 – 4066. Suryadevara, P. K.; Olepu, S.; Lockman, J. W.; Ohkanda, J.; Karimi, M.; Verlinde, C. L. M. J.; Kraus, J. M.; Schoepe, J.; Voorhis, W. C. V.; Hamilton, A. D.; Buckner, F. S.; Gelb, M. H. Structurally simple inhibitors of lanosterol 14-alpha-demethylase are efficacious in a rodent model of acute Chagas disease. J. Med. Chem. 2009, 52, 3703 – 3715. Castro, D.; Boiani, L.; Benitez, D.; Hernández, P.; Merlino, A.; Gil, C.; Olea-Azar, C.; González, M.; Cerecetto, H.; Porcal, W. Anti-trypanosomatid benzofuroxans and deoxygenated analogues: synthesis using polymer-supported triphenylphosphine, biological evaluation and mechanism of action studies. Eur. J. Med. Chem. 2009, 44, 5055 – 5065. Filho, J. M. S.; Leite, A. C. L.; Oliveira, B. G.; Moreira, d. R. M.; Lima, M. S.; Soares, M. B. P.; Leite, L. F. C. C. Design, synthesis and cruzain docking of 3-(4-substituted-aryl)-1,2,4-

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[51]

Organic Compounds to Combat Neglected Tropical Diseases 31

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Organic Compounds to Combat Neglected Tropical Diseases, 2014, 32-62

CHAPTER 2 DENGUE FEVER Recent Advances in the Discovery of Small Organic Molecules for the Prevention and Treatment of Dengue Fever Lucas Cunha Dias de Rezende1, Victor Hugo Aquino2 and Flavio da Silva Emery1,* 1

Department of Pharmaceutical Sciences, FCFRP/USP, Ribeirao Preto - SP, Brazil, Av. do Café s/n. CEP: 14040-903, Ribeirão Preto, SP, Brazil; 2 Department of Clinical, Toxicological and Bromatological Analysis, FCFRP/USP, Ribeirao Preto - SP, Brazil, Av. do Café s/n. CEP: 14040-903, Ribeirão Preto, SP, Brazil Abstract: Dengue fever (DF) is the most important mosquito-borne viral disease, affecting around 100 million people mainly in tropical and subtropical countries. The dengue virus (DENV) is a member of the Flaviviridae family and possesses a single positive-stranded RNA genetic material encoding a single polyprotein, which is cleaved into structural and nonstructural proteins. The consequences of DENV infections range from fever to haemorrhagic manifestations, and the treatment of the disease is restricted to symptomatic relief. Medicinal chemistry approaches allied with synthetic methods can be of great importance for developing effective treatment for this disease, and the scientific literature provides interesting results of studies that mainly targeted viral processes to give rise to novel drugs. Viral entry, RNA replication, and polyprotein cleavage have been the main, although not the only, focus in the development of novel anti-viral compounds for the prevention and treatment of DF. In this chapter, we focus on the new trends in the development of bioactive small molecules.

Keywords: Dengue fever, dengue hemorrhagic fever, antiviral, drug discovery. 1. TREATMENT A specific treatment for DF is not yet available and the clinical management of the disease is restricted to rehydration of the patient and the administration of

*Address correspondence to Flavio da Silva Emery: Department of Pharmaceutical Sciences, FCFRP/USP, Ribeirao Preto - SP, Brazil. Av. do Café s/n. CEP: 14040-903, Ribeirão Preto, SP, Brazil; Tel/Fax: +55 16 3602-0658; E-mail: [email protected] Adilson Beatriz and Dênis Pires de Lima (Eds) All rights reserved-© 2014 Bentham Science Publishers

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Organic Compounds to Combat Neglected Tropical Diseases 33

paracetamol to relieve fever and headache, with the main objectives being to avoid the development of severe disease and to relieve the patient's symptoms [1]. Reinforcing this scenario, there is an urgent need for specific therapeutic treatments directed against DENV, for example treatments that inhibit flavivirus infection or involve vaccination against it. Various synthetic and natural compounds, that have inhibitory effects on different flaviviruses, have been studied in vitro and in vivo with respect to their effects on DENV. However, these studies have only resulted in experimental therapeutics; the compounds failed to different extents in their clinical trials. Although they have been unsuccessful to date, structure- and ligand-based studies have provided a lot of helpful information for the ongoing search on small organic molecules for the treatment of DF [2]. 2. A MEDICINAL CHEMISTRY OVERVIEW OF DENGUE Regarding the target of treatments, there are 2 common approaches for discovering novel small organic molecules with therapeutic potential against the DF pathologic agent: the viral target-based approach, in which compounds are designed to interact directly with viral biomacromolecules, hampering their activity; and the host target-based approach, in which human proteins, essential for the DENV life cycle and that can be given as drugs, are targeted [3]. Among the most important viral processes and biomacromolecules targeted in the development of anti-flavivirus agents are viral entry, viral RNA polymerase, and viral protease [4, 5]. As for host proteins, cellular receptors, through which the virus gains entry into the host, and proteins reported to bind to the viral RNA and enable viral replication, are robust targets for anti-viral drug development. 2.1. Viral Serine Protease Inhibitors Serine endoprotease NS3 is a viral enzyme that is activated when complexed in the protease domain (NS3pro) to the integral membrane protein NS2B (essentially in the hydrophilic central portion NS2BH), forming the complex NS2B-NS3 and, consequently, the protease active site. The complex NS2B-NS3 is involved in the processing of polyproteins, playing a pivotal role in viral replication, and has therefore emerged as a suitable target for the development of therapeutics for DF [4,

34 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al.

6]. Besides the screening of small molecule libraries, the most prominent approach for the development of an NS2B-NS3 inhibitor has been to explore the medicinal chemistry of peptidomimetics similar to the protease catalytic substrate [6]. Among the natural products, 2 compounds isolated from Boesenbergia rotunda have been reported to have strong inhibitory activity against NS3 protease. Even at low concentrations, the cyclohexenyl chalcone derivatives panduratin A and 4hydroxypanduratin A were reported to have inhibitory activities towards the DENV NS3 protein. In general, these natural compounds and similar derivatives can be efficiently synthesised through the Diels–Alder cycloaddition of ringsubstituted chalcones and (E)-ocimene (Scheme 1) [7, 8]. The activity of these compounds has been the subject of theoretical studies aimed at the design of novel lead compounds [7]. OH

OH O

O

O

HO OH

OH

Panduratin A

4-Hydroxypanduratin A

OH O

OH O

24 h, 150ºC

R

+

R

85 - 95%

OH

OH

Scheme 1: Top: The structures of panduratin A and 4-hydroxypanduratin A. Bottom: Application of the Diels-Alder reaction to synthesise these compounds.

Compounds with α-ketoamides have also been evaluated in terms of their inhibitory effects against DENV serine proteases. Some of these evaluations have arisen on the basis of the rationale that the oxygen nucleophile in serine proteases can be covalently bound to various electrophiles, resulting in enzymatic inhibition. Several examples of the successful development of serine protease

Dengue Fever

Organic Compounds to Combat Neglected Tropical Diseases 35

inhibitors by using the strategy of covalent protease inhibition are described in the scientific literature, providing support for this approach. In this context, aldehyde is a common electrophilic functional group known to act as an electrophilic trap interacting with the nucleophilic serine residues in the enzyme’s active site. The more electrophilic α-ketoamide is present in several protease inhibitors, pharmaceuticals, and natural products, including inhibitors of the serine protease from HCV. Consequently, it is also a prominent approach for developing small molecules to covalently inhibit DENV NS2B-NS3 [9]. a) Aldolic Condensation O

O Ar

KOH/MeOH

OH

+ H

HNR1R2, HATU, HOAt, NEt3, DMF

O OK

Ar

O

O

R1 N

Ar

O

R2

O

b) Direct Coupling O

O Ar

R1 N

+ H

DBU or Cu(OTf)2, CH(OMe)3, S: CH2Cl2

R2

R3

H

+

R2

R1 N

Ar

R2

O

O c) Passerini Reaction O

O

OH

SiCl4, CH2Cl2

N

R3

H N

O

Dess–Martin CH2Cl2

R2

O O

R1 N O

Most active core

R2

O O

H N

R2 HN

H N

R3

O

O

H N

O O

O HO

Promising Compounds

Scheme 2: Synthetic approaches to obtain a diversity-oriented library of α-ketoamides. The most active core and the most promising compounds are boxed.

In the search for DENV NS2B-NS3 inhibitors, a very elegant study looked for ideal structural cores bearing α-ketoamide derivatives. Applying 3 different synthetic routes (Scheme 2): aldolic condensation (a), direct aldehyde coupling (b), and the Passerini reaction (c), a library of 53 compounds bearing the β,γunsaturated α-ketoamide and similar derivatives were synthesised and assayed for their anti-dengue activity. The application of structure–activity relationship (SAR) analysis to 11 prototype compounds, with punctual modifications such as reduction of the linker length between the electrophilic trap and the cyclic

36 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al.

structure, reduction/epoxidation of the ketones, and methylation of the linker, allowed the definition of a core structure that had optimal inhibitory characteristics, which was the initially designed structure (Scheme 2) [9]. In a second SAR approach keeping the most active core intact and modifying the aromatic substituent, 28 compounds were assayed for their anti-dengue activity and the electron-donating residues (e.g. methoxy and pyridyl) were observed to promote inhibitory activity while the electron-withdrawing residues (e.g. nitro, cyano, and fluoro) diminished inhibitory activity in most cases. Additionally, indole substituents greatly enhanced the anti-dengue activity. The effects of various substituents on the ketoamide nitrogen were studied and it was observed that the substituent size could be extended to longer alkyl chains, but cyclohexyl and phenyl groups were less tolerated. Based on the assumption that the substituent on the alpha-ketoamide nitrogen occupied the S10-pocket, moieties resembling serine were explored and remarkable activity was observed (Scheme 2) [9]. Based on the enzyme structure, a 3-aryl-2-cyanoacrylamide scaffold was designed as anchor pharmacophore for inhibitors of the DENV protease. This core shares similarities to β,γ-unsaturated α-ketoamides but contains a nitrile group, which can act as an electrophilic trap for the enzymatic serine. Using a synthetic approach where 2-cyanoacetamide derivatives were coupled with carbonyl compounds (Scheme 3), 86 analogues were obtained and applied in a SAR study. This new class of 3-aryl-2-cyanoacrylamides was found to inhibit DENV NS2BNS3 protease. Higher activity was observed with a para-substituted aromatic system with high electron density, an amide or acid residue, and planar molecule geometry. Pronounced activity and selectivity towards the viral enzyme was observed in a primary para-hydroxy cyanoacrylamide derivative (Scheme 3) [10]. An aminobenzamide -based scaffold was explored as an inhibitor of DENV protease. A library was synthesised from para and meta aminobenzoates, which were converted to isocyanates followed by functionalization with propargylamine and click chemistry -based coupling with several azides (Scheme 4). The compound that had the highest activity showed an IC50 for protease inhibition of 6.82 ± 0.09 µM [11].

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Organic Compounds to Combat Neglected Tropical Diseases 37

O O

O

O

HR3 (amine)

R1

R3 N

O

R2

R3

base - rt or reflux 2 - 20 h

rt, 20 h N

R1 R2

N

O NH2 HO

N Most promising compound

Scheme 3: The synthetic approach used to obtain a library of compounds containing the 3-aryl-2cyanoacrylamide scaffold and the most promising compound in terms of its activity.

O

O OMe

O

O

Trichloromethyl chloroformate

propargyl amine

NH2

C O

H N

N

substituted azide and ascorbate

C O

O

NH

N N N

C O

H N

N N N

C O

NH

R1

O NH R2

H N

OM e

OMe

OMe

OH CDI/ Amine

NH

H N N N N

R1

LiOH

C O

NH

R1

O O

NH

O C NH NH

N

N N

F IC50 = 6.82µM

Scheme 4: The synthetic approach used to obtain anti-dengue aminobenzamide derivatives and the most active compound from the series (boxed).

The application of the EUDOC docking program in an in silico screen for 2 enzymatic sites using the Mayo Clinic small molecule database resulted in 3 hits [12]. One of the promising compounds, the anthraquinone derivative ARDP0006,

38 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al.

which competitively inhibits DENV protease, was further studied. Using similar anthraquinoidal compounds that were commercially available, 4 compounds were found to have better activity than the lead compound (Scheme 5) [13]. Acridone, a tricyclic molecular scaffold that resembles an anthraquinone, was also explored. A series of N-substituted acridones synthesised using anilino-benzoic acids as the starting material were assayed. The compound that was most active against DENV in this series showed an EC50 of 2.5 µM in a viral inhibition assay (Scheme 6).

NH2 O OH O

NO2

OH

NO2

NH2 OH O

OH O HO

ARDP0006

OH

N N

O2 N NH2 O

NO2 O

NH2

OH

OH

O

O

Structurally related compounds showing higher enzymatic inhibition

Scheme 5: Left: ARDP0006, an anthraquinone derivative obtained from computational screening. Right Anthraquinoidal derivatives that had higher activity. R1

R4

CO2H +

R2

Cl

H2N

K2CO3/Cu

R1

CO2H

R2

N H

H2SO4

R3

O

Cl

N

O

R4

R2

R3

O R4

R1 R2

R1

R4 N H

R3

Allyil bromide Ethanone

N R3

EC50=2.5µM

Scheme 6: The synthetic methodology employed to obtain N-substituted acridones and the most active from in the series (boxed).

Another strategy for developing inhibitors of the NS2B-NS3 enzyme complex is the peptidomimetic approach, an example of substrate-based drug design in which

Dengue Fever

Organic Compounds to Combat Neglected Tropical Diseases 39

compounds are rationally designed to mimic the substrate peptide. From a series of 17 tripeptide aldehydes synthesised using solution-phase chemistry, good inhibition of DENV protease was observed for Lys-Arg-Arg with a phenylacetyl substitution (Table 1) [14]. Other results obtained from the peptidomimetic approach that were also assayed included peptides synthesised through N-terminal acetylation and C-terminal amidation that shared similarities with viral polyprotein [15], conformationally restricted cyclic peptides [16], and retropeptide hybrids [17] (Table 1). A fused-bicyclic structure is commonly applied to the design of peptidomimetic compounds, including commercially available anti-viral compounds such as Danuravir, which bears the bis-tetrahydrofuranyl moiety. Isomannide has a fused bicyclic structure and has already been used as a starting point for the construction of peptidomimetic serine protease inhibitors. Isomannide hydroxyls was tosylated with tosyl chloride, following which the tosyl was substituted for an azide with NaN3, and then palladium-catalyzed reduction of the azide moiety to an amine was performed with molecular hydrogen. The bis-amino derivative of isomannide was then coupled with several oxazolone derivatives commonly used as amino acid precursors. Even though the library was not tested against DENV NS2BNS3, its inhibitory activity against serine protease was assayed in a hepatitis C virus (HCV) serine protease and the novel scaffold and synthetic approach could be modified to achieve good anti-dengue lead compounds (Scheme 7) [18]. Table 1. Peptidemimetic inhibitors of DENV NS3-NS2B protease Entry

Peptide

IC50

References

1

GLPVCGSEESRRGCNTPGCRRSWPVCTRR Cyclic peptide - isomer A

4.3 µM

[14]

2

GLPVCGSEESRRGCNTPGCRRSWPVCTRR Cyclic peptide - isomer B

9.3 µM

[14]

3

Benzoyl-KRR

9.5 µM

[16]

4

Phenylacetyl-KRR

6.7 µM

[16]

5

Ac-RTSKKR-CONH2

12.1 µM

[15]

6

Ac-KKR-CONH2

22.3 µM

[15]

7

Ac-FAAGRK-CONH2

25.9 µM

[15]

40 Organic Compounds to Combat Neglected Tropical Diseases HO O

1. TsCl 2.NaN3 3. H2,Pd/C

O O

O OH

O

H

H 2N

NH 2

R1

Rezende et al.

N H

R1 O

R2 R2

N H

H N O

O

O O

N H

HN

R1 O R2

Scheme 7: The synthetic approach used for the development of a fused-bicyclic peptidomimetic compound from isomannide.

Five-membered multi-heterocyclic rings are somewhat controversial in the medicinal chemistry field due to their ability to interact with a variety of biological targets. DENV NS2B-NS3, together with other enzymatic targets, was explored in a screen of 163 derivatives of 5-membered multi-heterocyclics (Scheme 8). Thiohydantoin and rhodanine derivatives, obtained from the cyclization of diverse amino acids, were shown to be most potent against NS2BNS3. The aromaticity of substituents bonded to the multiheterocyclic rings seemed to be fundamental to their activity; actually, the vast majority of thiohydantoin and rhodanine cycles substituted with aromatic cycles showed at least intermediary inhibition of NS2B-NS3 while all non-aromatic derivatives were virtually inactive [19].

Scheme 8: The synthetic procedure used to obtain thiohydantoin and rhodanine.

The inhibition of DENV NS2B-NS3 protease by benzo-[d]-isothiazol-3(2H)-one derivatives bearing a triazole cycle synthesised via click chemistry coupling of propargyl-functionalized isothiazolone heterocycles, and structurally diverse azides to form derivatives was the objective of a study with interesting observations (Scheme 9). Through this synthetic strategy, a library comprising 33 compounds was obtained; for 6 of these somewhat promising enzymatic and viral inhibition was observed (Scheme 9) [20].

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Organic Compounds to Combat Neglected Tropical Diseases 41

2.2. Viral Entry Inhibitors Any virus needs to gain access to the cellular machinery of its host cell in order to replicate. The inhibition of viral access to the host cell cytoplasm is a well explored strategy for treating viral infections [21] and some efforts have been made to develop novel drugs against DENV that could use this mechanism of action. Among the structural proteins encoded by the DENV genome, 3 are considered structural proteins: capsid (C), premembrane (prM), and the envelope (E) proteins. The DENV E-protein plays an important role in viral entry, mediating the fusion between the viral membrane and the host membrane; consequently, it has emerged as a potential therapeutic target. Molecular docking of the E-protein has been utilized as a strategy to design small organic compounds with the ability to preclude viral access to the host cell's cytoplasm. The β-OG binding pocket of this protein is a hydrophobic pocket described by Modis et al. to be involved in the pH-dependency of the fusion of DENV to the host cell [22] and has been employed in theoretical studies aimed at the rational design of anti-dengue agents. A high-throughput docking virtual screening experiment using the β-OG binding pocket enabled the selection of 111 compounds as possible therapeutic agents from a library of almost 600,000 compounds. Two compounds that had positive results in the biological assays were selected for detailed SAR analysis to increase the potency of this class of compounds against DENV (Scheme 10) [23]. The findings suggested that the amine group R1 and aryl group R2 were crucial for the activity; additionally it was observed that several substitutions were tolerated on the aryl ring R2 and that a change of the pyrimidine ring to a quinazoline ring improved the potency. Based on this information from the SAR analysis, a compound bearing high efficacy against all serotypes of laboratory strains and clinical isolates of DENV was synthesised. Further characterization showed that this compound interfered early in the DENV life cycle, blocking its entry to the host cell cytoplasm after its internalization into endosomes, probably by targeting E-protein [23].

42 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al. O

H2N

O

sodium ascorbate/ CuSO4/BuOH/H2O

N S

CDI/THF

N S

NH

R1 N3

NH

O

O

N

R1

N

N

O N S

OH O

S

S

N

N NH O

CDI/THF

NH

sodium ascorbate/ CuSO4/BuOH/H2O

O

O

O

R2 N3

H2N

O

O R2

O

O

O R2

NH

O

O

N

N

N

NH

NH R1/R2

NH O

O O

Scheme 9: Top: Synthetic scheme of benz[d]isothiazol-3(2H)-one derivatives. Bottom: The substituents present in the most active compounds.

Another virtual screening of the same pocket of E-protein, applying a different search protocol to a different compound library, resulted in 3 hits and these contained the thiazole moiety (Scheme 11) [24]. Several analogues bearing this central thiazole moiety were synthesised, tested for their activity against another kind of flavivirus—yellow fever virus — [25], and a highly promising derivative containing 2 bromine atoms was synthesised (Scheme 11). This brominated compound was later applied as a lead compound to develop novel agents against DENV. Non-brominated derivatives were also synthesised and tested for their activity against flavivirus. Novel non-brominated phenylthiazole compounds with a similar capacity to inhibit DENV were obtained and a novel SAR model of phenylthiazoles with anti-flaviviral activity was proposed (Scheme 11) [26, 27].

Dengue Fever

Organic Compounds to Combat Neglected Tropical Diseases 43 B

A

H N

N

N

N

R1

NH

H N

N

N

S

N

C H N R1

NH

N S

S N

O

NH2

R2

R2

Cl EC50: 0.07

EC50: 0.9

EC50: 1.69

Scheme 10: A and B: The most active compounds discovered from virtual screening of the Eprotein pocket and the testing of 111 hits. C: The most active compound discovered from SAR analysis of the compounds derived from A and B. A

H N

O H N

S

N HN

H N

S

B

Cl

Br

R1

R3

C N

O

O

N

O

Br atom not necessary

Polar Group

S

N

S Br

Linear lipophilic Lenght: 4units

99.6%

R2 Cl

D

N S

Cl

N

N

N

S

SMe

OH

OMe

O

O

O

O N

S

N

S

N

S

N HN

NH2 NH

NH2 S

N Cl S nBu 91,9%

nBu 94.1%

nBu 99.8%

nBu 99.2%

Scheme 11: A: Hits containing a central thiazole ring obtained from the virtual screening of the βOG pocket of the DENV E-protein [24]. B: The highly anti-flaviviral phenylthiazole derivative and its ability to inhibit DENV (shown as % of DENV inhibition) [25, 27]. C: The SAR model of phenylthiazoles as anti-flaviviral agents [27]. D: Highly anti-flaviviral non-brominated phenylthiazole derivatives and their ability to inhibit DENV (shown as % of DENV inhibition) [27].

During the process of cellular entry, DENV and other enveloped viruses bind to heparan sulphate in the cellular membrane. Sulphated polysaccharides are known inhibit enveloped viruses, hampering their access to the cytoplasm [28]. Among

44 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al.

the examples of sulphated polysaccharides are the over-sulphated algal mannans and xylomannans [29], obtained using SO3-pyridine, and glucans and galactomannans from Gastrodia elata [30], Leucaena leucocephala, and Mimosa scabrella [31] sulphated using chlorosulphonic acid. A similar concept was observed in the in vitro inhibition of DENV by various carbohydrate-binding agents [32] and zosteric [33] acid, which hampered virus-cell adhesion. 2.3. Inhibitors of RNA Replication Among the non-structural proteins, NS5 protein has 2 enzymatic functions essential for viral propagation: methyltransferase (MTase) and the RNAdependent RNA polymerase (RdRp). RdRp is responsible for the synthesis of viral RNAs, a particular function that is not provided by the host cell’s machinery; consequently, the viral RdRp presents as an attractive anti-viral target [34]. Viral polymerase activity can be targeted using either nucleoside analogs, which are phosphorylated to a nucleotide before they compete with natural nucleotides for the enzymatic active site and terminate replication, or non-nucleoside compounds, which target allosteric sites in the protein. Nucleoside inhibitors of viral polymerases are the prevailing commercially available class of drugs for treating viral infections.

Scheme 12: Top: Acetylene-substituted deaza-adenosine compounds with potent and specific antiflaviviral activity. Bottom: The strategy used for the functionalisation of the ribose with the acetylene group using Grignard reagent.

Dengue Fever

Organic Compounds to Combat Neglected Tropical Diseases 45

To develop inhibitors of DENV RdRp, over 90 adenosine analogs were analysed, resulting in the identification of an acetylene-substituted deaza-adenosine compound with potent and specific anti-flaviviral activity (NITD008), synthesised through the oxidation of the hydroxyl group of ribose to a carbonyl followed by acetylene functionalization using Grignard reagent (Scheme 12, above) [35]. The anti-viral actions of this compound were thoroughly characterised and several lines of evidence support the proposition that its anti-viral activity resulted from RNA chain termination. Later, a similar compound (NITD449) was shown to have important anti-flaviviral activity but poor oral availability. To overcome this problem, its diisobutyryl analogue (NITD203) was synthesised resulting in an enhancement of its bioavailability (Scheme 12) [36].

O

O

OH

HO

O OH OH

O

O

HO

OH O

OH OH

O

O

O

Flacourtoside B

Flacourtoside A

OH

HO

O O

O

O

HO O

HO

OH OH

O

HO

O OH

O

O

OH

HO O

O

OH

OH OH

O

OH O

O

O

O

Flacourtoside D

Flacourtoside C O HO O

O

O

HO

OH O

O OH O Bz

Flacourtoside E

O

OH

HO O OH

O

HO

OH O

O

O

O

O OH OH

Bz

OH O

Flacourtoside F

Scheme 13: The structure of glycosides extracted from Flacourtia ramontchi that inhibited DENV polymerase.

46 Organic Compounds to Combat Neglected Tropical Diseases

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Molecular hybridization, the synthesis of novel compounds through the fusion of 2 active chemical groups, has also been applied as an approach to developing novel compounds for fighting DENV infection. The search for novel adenosine analogs of DENV reflects the fact that purine is widely known to be a potentially anti-viral chemical group. It was proposed the chemical linkage of purine scaffold with a β-lactam ring that, besides its primary uses as an antibacterial moiety; possess other pharmacological activities, including antiviral. Although promising lead compounds were obtained for other viral infections, none of the hybrids showed activity against DENV [37]. In the field of non-nucleoside polymerase inhibitors, natural products have shown some promise. From extracts obtained from different parts of 400 plants randomly collected in Madagascar, the tree Flacourtia ramontchi was selected for a bioassayguided purification project. Six phenolic glycosides, named flacourtosides A–F (Scheme 13), were isolated from F. ramontchi and their inhibitory activity against DENV was described to be via polymerase inhibition [38]. O B O

R1

O

CsCO 3 , CH 3 CN ;

O

Br

N NH

S

HOOC HN

O

1. 2.

R2

R2

MeOOC

Br MeOO C

R2 R1

+

O Cl

N N

B

HN

pyridine

O S

O

P d(P h 3 ) 4 , K 2 CO 3 , D MF; LiOH , MeO H

N N R1

O S

O

NH 2 Br

S

N N

O

R1

R2

H

Benzyl

0.7

H

Methyl-2-naphtalene

0.37

F

Methyl-2-naphtalene

0.26

IC50(uM )

Cl Triaryl pyrazoline

Scheme 14: The synthetic procedure used to obtain sulfonylanthranilic acid derivatives and the IC50 values for the most active compounds. The triaryl pyrazoline scaffold is boxed.

Another effort to develop DENV inhibitors was published by the company Novartis. Applying a scintillation assay developed by the company, high-

Dengue Fever

Organic Compounds to Combat Neglected Tropical Diseases 47

throughput screening of the corporate compound archive resulted in the identification of N-sulfonylanthranilic acid as a potentially active compound. Employing a synthetic approach based on the Suzuki coupling of modified pyrazole boronic esters with 3-bromobenzenesulfonamides, several derivatives were synthesised [39]. The assaying of the obtained compounds for viral and human polymerases identified 3 derivatives that were highly specific to, and active against, DENV (Scheme 14) [39]. In a similar approach, a small library of 200 small molecular compounds with diverse structures was screened for activity against flavivirus. Triaryl pyrazoline was identified as an inhibitor of DENV through its actions on viral replication. 2.4. Other Relevant Compounds Several studies that aimed to treat DENV infection by designing and screening drugs with alternative or unknown mechanisms of action have been reported in the literature. Inosine monophosphated (IMPDH) is an enzyme that acts during a rate-limiting step in the synthesis of GTP and has been used as a target for several diseases, including viral diseases. Some long-known compounds, such as mycophenolic acid, a non-nucleoside inhibitor of IMPDH [40] isolated from the fermentation of Penicilium stoloniferum [41] and clinically useful in organ transplantation [42], and Vx-497, a phenyloxazole derivative that is a potent reversible inhibitor of IMPDH, have been valued as anti-dengue compounds with promising results in vitro [43]. Among the novel synthetic compounds with the capacity to inhibit IMPDH, a uracil-based compound synthesised primarily via the Gould–Jacobs reaction has been obtained and shown to be a potent in vitro inhibitor of DENV (Scheme 15) [44]. Another nucleotide-derived core, 6-methylmercaptopurine, seemed to inhibit DENV in a cell culture by interacting with IMPDH, but, due to pharmacokinetic issues, in vivo experiments do not support the use of this drug, even though it may be suitable for modification [45]. CO2C2H5

O O

N O

N

NH

O

CO2C2H5

O NaOEt, EtOH

O

N O

N

N H

CO2C2H5

1.Dowtherm A 2. HCl1N

OH O

N O

OH N

N

CO2C2H5

Scheme 15: The synthesis of compounds active against IMPDH via the Gould-Jacobs reaction.

48 Organic Compounds to Combat Neglected Tropical Diseases

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High-throughput screening of the Novartis corporate chemical library gave rise to 2 potential lead compounds for treating dengue: NITD-982 and NITD2636, which inhibited in vitro DENV production with EC90s of 5.2 nM [46] and 1.7 µM [47], respectively. Both compounds seemed to act by inhibiting viral translation. NITD982 was shown to inhibit dihydroorotate dehydrogenase, an enzyme of the pyrimidine synthesis pathway, but no in vivo activity was reported, probably due to pyrimidine intake. Even though NITD-2636 inhibited both host and viral translation, its mechanism of action is not yet fully understood. Interestingly, however, it was observed to have significant in vivo activity (Scheme 16). An NITD-2636 derivative bearing a cyano group, NITD-451, was shown to have enhanced activity against DENV. R1 N HO

Tf2O,

N

Zn(CN)2 Pd(PPh3)4

OTf

N N

F

F

NITD-2636

NITD-451 Cl N O Cl O

CF3

Cl

N O

N

NITD-982

Scheme 16: Top: The synthetic approach used to convert NITD-2636 to NITD-451. Bottom: The molecular structure of NITD-982.

The compound 5-methoxyquinazoline-2,4-diamine was identified by the screening of a compound library, using a DENV -replicon cell line, as a moderate inhibitor of DENV with a low cytotoxicity [48]. The synthesis of a library of similar compounds enabled the development of derivatives with better activity. Initially, compounds with different substituted positions were obtained and better results were obtained when position 5 was substituted for an electron-donating group (Scheme 17). The activity was optimized by varying the substituents at position 5 and the compound substituted with a tert-butoxy group showed a higher activity in the DENV-replicon cell line with a selectivity index (SI) of over

Dengue Fever

Organic Compounds to Combat Neglected Tropical Diseases 49

1000 (Scheme 17). Even though the mechanism of action of this compound has not been elucidated it seems to be a promising compound for the future development of anti-dengue drugs [48]. D-glucose was used as a starting material for the synthesis of azole derivatives. Among several compounds obtained by the synthetic procedures used, only 2 showed a moderate inhibition of DENV and are potential lead compounds obtained from renewable sources (Scheme 18) [49]. No mechanistic hypothesis for the observed results was made by the authors. Br R

N

Guanidine carbonate,

R

CN

or

NH2

N

N

F

N

NH2

R

NH2

O

NH2

CN

EC50 = 2.8 nM SI > 1000

Position 5: Best results - electron-donating

Scheme 17: Left: The method used for the synthesis of 5-methoxyquinazoline-2,4-diamine derivatives. Right: The structure of the most active compound (boxed). Br O Br

S N

N HOHO

O MeO

NBS, PPh3 O

O

Me Me

Br HO

O MeO

IBX/DMSO O

O

Me Me

Br O

O MeO

N O

O S N

O

N

Me Me

O

O MeO

O HO

Halo benzoic hydrazide O

O

N H

O

Me Me

N

O HO

Br

PIDA/MeOH O

O

Me Me

N N

O

Me Me

EC50: 29.9 uM

Br O

O

O O HO

O

O

Me Me

EC50: 64.6 uM

Scheme 18: The synthetic approach used to obtain 2 active derivatives from glucose.

The DENV proteins prM/M, E, and NS1 are known to possess sites for N-linked glycosylation and, during translation, the oligosaccharide (Glc)3(Man)9(GlcNAc)2 is transferred to specific asparagine (Asn) residues in a cotranslational process

50 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al.

catalysed by oligosaccharyltransferase. In the endoplasmic reticulum (ER), this glycoprotein is processed by α-glucosidases I and II before it is associated with calnexin and calreticulin, which are crucial proteins for glycoprotein folding. This makes α-glucosidase a suitable target for anti-viral treatment [50]. Castanospermine (CP) is a polyhydroxylated indolizidine alkaloid isolated from Castanospermum australae [51] and also obtained by total synthesis using different approaches (Scheme 19) [52]. It is structurally an azasugar related to Dglucose, known as a ER glucosidase I inhibitor, consequently reducing viral secretion and infection. In vivo studies have shown its ability to improve the survival of mice [53]. b GP OH H OH transannular GP O O cyclization H O N N b a GP

HO HO

GP OGP O

O

RCM

H N

O GP

O GP

a Bn GP

O O

Bn

O O

GP

O

O O

H

N O

O

GP GP O O

zinc-mediated fragmentation PG

PG

H N

I GP

O

O

O

O PG

O PG

OH O PG concerted ring opening

OBn BnO N

OGP O

O

R H

nitrenium ion cyclization

OBn BnO

H N

OGP

R

O

O

Scheme 19: Synthetic approaches to the synthesis of castanospermine.

Some iminosugars, such as deoxynorijimycin, are glucose mimetics that act as competitive inhibitors for α-glucosidase. Deoxynorijimycin, an N-containing sugar isolated from Morus sp. [54], has shown limited in vivo DV inhibition [55, 56]. However, continuous N-alkylation towards higher and more lipophilic compounds led to the discovery of CM-10-18 [55, 57], an active compound in the more lethal dengue haemorrhagic fever (DHF), but with a high toxicity. The evolution towards an aromatic ether showed an in vivo active compound with a good pharmacokinetic profile and low cytotoxicity (Scheme 20) [58].

Dengue Fever

Organic Compounds to Combat Neglected Tropical Diseases 51 OH

OH HO

OH N H

OH

HO

OH

OH

OH HO

OH

N (CH2)2CH3

HO

OH

HO

deoxynorijimycin

N (CH2)5

OH HO

OH N (CH2)5

OH

CM-10-18

OH HO

OH N (CH2)6 O

OH

F F

Scheme 20: The structural evolution of iminosugar analogues from deoxynorijimycin.

The N-terminal domain of the flavivirus NS5 protein contains an Sadenosylmethionine (SAM)-dependent RNA methyltransferase that is involved in the final steps of the capping process; in particular, it is responsible for catalysing the nucleophilic mechanism of methylation of the N-7 of the guanosine cap and the 2-O’ of the ribose subunit, yielding S-adenosylhomocysteine (SAH) as a byproduct (Scheme 21) [59]. Because this process is essential for viral replication, it represents a valid target for anti-viral drug discovery and some active inhibitors of DV methyltransferase (DVMTase) have been described. Despite the need for a complete mechanistic elucidation, a potentially promising inhibitor is a good starting point for the further design of lead compounds for this target. Ribavarin -5-triphospate, a GTP analogue [60], sinefungin (SIN), a SAM analogue [61, 62], and aurintricarboxylic acid (ATA), are able to inhibit DV 2-O’-MTase. SIN is also able to inhibit N7MTase [63], as can some SAH analogues that can bind both N7 and 2-O’-MTases. Ribavirin is a synthetic broad spectrum anti-viral used to treat RNA infections [64]. The classical synthesis process involves acid catalyzed coupling of cyano1,2,4-triazole, or trimethylsylil derivatives of the triazole, with the respective ribofuranose (Scheme 22) [65]. A common metabolite of this anti-viral is 5′triphosphate (RBT) [66]. RBT can be obtained synthetically [60] by an ‘one-pottwo-step’ procedure using POCl3 and trimethylphosphate, in the presence of the

52 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al.

strong base proton sponge [67], forming an dichloridate intermediate, which is converted to RBT by the addition of pirophosphate solution (Scheme 22). H2N

N

N

N N

NH2

SAM O

O

HO2C

OH O N7

O- 5' P P O H O O- O

-O

O

O

O

P

O

5'

N7

OH

S Me

N

H OH

Methyltransferase

NH NH2

N

H

catalysis

OO O P P O H O O- O P

-O

O

H OH

H OH

NH H

H OH

NH2

N +

H2N

N OH N

HO

Base

N

O

S

HO2C

NH2 N

SAH O O2' O P OH

CO2H

O Base

NH2

O O2'H O P OH

O

O

N

Me S HO

O

N N

HO

NH2 N

N

SAM

Scheme 21: The capping process mediated by nucleophilic attack at SAM.

N H

N

CN

N N

O

AcO

OAc

+ AcO

O

1.

H+, acid catalyzed fusion 2. NH4OH HO HO

OAc

N

N

O NH2

OH Ribavirin

CN

N N Si

Ribavirin

1.

N

POCl3,(CH3O)3PO Proton Sponge

O P HO Cl O

O

AcO

+

AcO

N O

N

Cl

N

OH

Br OAc

O NH2

pyrophosphate

H2N OO O P N -O O O O N N O P -O O P OH HO O OH RBT

Scheme 22: Ribavarin and the synthesis of RBT.

SIN is a nucleoside obtained from the fermentation broth of Streptomyces griseolus [62, 68] and also by total synthesis [69, 70]. One of the stereocontrolled

Dengue Fever

Organic Compounds to Combat Neglected Tropical Diseases 53

synthetic approaches involves a Curtius rearrangement towards amino functionality incorporation and an asymmetric hydrogenation (Approach A in Scheme 23) [70]. Another strategy (Approach B in Scheme 23) involves 2 nitroaldol condensation for chain elongation and further amino functionality introduction [69]. Approach A O

HO

OMe HO

HO2C

Swern oxidation/Horner-Emmons olefination

O O

OH H

O

H2N HO H EtO2C

Curtius rearrangement

OMe

O OMe O

O

NH2 NH2

OH N

R H N

O

N

N

Cbz

HO2C

N

NH2

S Me HO

O

N OH N

N NH2 N

SAM

(+)-Sinefungin reduction

Approach B

NH2 N N O

N N

NH2 OHC H

N

CO2Bn

Nitro-Aldol condensation

O O

NH2

NO2 H RO BnO2C NH2

N

N

N

NH2 N

Oxidation and Nitro-Aldol condensation

O O O

O2N

N

N

N

O O OH

O

Scheme 23: Sinefungin (SIN) synthesis and structural analogy to SAM.

SAH and its analogues are selective competitive inhibitors of MTases based on the structure of SAM with substitution on the adenine moiety to occupy a cavity located above the SAM-binding pocket (Scheme 24) [71]. Interestingly, the modified SAH showed a high selectivity for virus MTase. This is a serious limitation of this therapeutic approach, because this class of enzyme is involved in different kinds of biological activities in humans; these compounds, however, feature a promising structure to be explored in the search for anti-virals. There are several other examples of DENV inhibitors with different mechanisms of action, which were discovered in the biological screening of older drugs (Fig. 1). Teicoplanin, an glycopeptide antibiotic, and the adamantine derivative LCTA-

54 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al. HO

HO

O

N H

O Cl

HO

O

Cl

O

O

O HO

HN

N H

O N O H H

H N

H H N

O

HN

HO

O

O

HO HO

HN

NH 2

O

O

NH

N H

O

O

O

OH

OH

H

H NH H O

OH

OH O O

NH

LCTA-949

O

O

NH

O

HO

OH

O

O

Cl

HO O H H N

O HO

O O O

NH O

O

O

O

HO

Boc NH

HN

Cl

N H

O

O

HO

HO

OH

OH HO

HO O OH

HO OH

OH

teicoplanin OH

HO OH

O

OH

OH H

O H

OH

OH H O

O

O

O

O narasin

O HO HO

O

O H

O

O

O

O H O

O

O H

O O

H O OH

ivermectin

H

O

lovastatin O

H

OH

Figure 1: Known drugs with alternative anti-viral activity.

949 inhibited DENV entry in vitro with a low cytotoxicity [72]. Narasin [73] is a natural polyether antibiotic used in prevention of coccidiosis in chicken [74], which has shown a tendency to disrupt DENV protein synthesis [75]. Lovastatin, a marketed hypocholesteremic agent, showed an ability to reduce DENV infection by interfering with the transport of secretory proteins from the ER to the Golgi apparatus, affecting the virus assembly [76]. The movement of essential proteins is also affected by Ivermectin [77], a semisynthetic anti-helmintic which inhibits importins , proteins involved in the nuclear import of viral proteins [78]. Ivermectin also inhibits viral replication as a consequence of the inhibition of NS3 helicase [79].

Dengue Fever O

HO

N

N

HO

Organic Compounds to Combat Neglected Tropical Diseases 55

OH N

N

N

HO

NIS

R1

O

I

PPh3, DMF

H N

NH2

N

OH N

H N

N

HO

l-homocysteine R1

H2O, dioxane

O

S O

HO

N

OH N

N

H N N

R1

SAH, R1=H NH2

NHFmoc MeO

O

S O

AcO

N

Cl

OAc N

HO

1) p-chloro-aniline

N

LiOH

O

S O

HO

N

N

OH N

N

H N N Cl

Scheme 24: SAH and the synthesis of its analogues.

As we have seen, several natural products are available for anti-viral studies, especially for studies on DV. Within the secondary metabolites, flavonoids are widely described in the literature as DENV inhibitors. Although mechanistic evaluation is required for some of these products, they represent good potential starting material for the development of innovative anti-virals. Quercetin [80], Glabranine, and 7-O-Glabranine [81] showed potential DENV inhibition with low cytotoxicity in the in vitro models used. Other flavonoids were identified as DENV NS5 polymerase inhibitors, such as Chartaceone D [82], isolated from the bark of Cryptocarya chartacea, and podocarpusflavone A, extracted from Dacrydium balansae, both of which had low in vitro cytotoxicity (Fig. 2). OH OH O

O

O

HO

O

O OH

O

OH O

OH O

O

quercetin

7-O'-Methyl-Glabranine

Glabranine

OH O

O OH HO

HO HO

O

O O OH

O OH O

Chartaceone D O

O OH

Figure 2: Known anti-viral flavonoids.

OH

podocarpusflavone A

56 Organic Compounds to Combat Neglected Tropical Diseases

Rezende et al.

CONCLUSIONS Although the ongoing efforts in the search for DENV inhibitors have resulted in a panel of diverse potential therapeutics, none of these have been effective in the clinical trials. This situation, reinforced by the fact that there is no vaccine against DENV, demonstrates that the only way to decrease the disease incidence at the present time is to control the vector. Despite the short duration of the symptoms of this disease, which could lead to questions in terms of our investment in drug development projects, the lethality of the hemorrhagic form of the disease and the serotype evolution of the virus could enhance the severity of DENV as a health issue, especially in developing and tropical countries. Therefore, the development of more rational approaches towards drug design, together with a deeper knowledge of the biochemistry of DENV, could increase the chances of success in developing effective DENV inhibitors. In this context, all the findings described in the literature will allow the development of a library of hits and lead compounds useful for classical or modern medicinal chemistry -driven drug discovery. ACKNOWLEDGEMENTS We acknowledge Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support to the authors. CONFLICT OF INTEREST The authors confirm that this chapter contents have no conflict of interest. REFERENCES [1] [2]

[3]

Organization, W. W. H., Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control. 2009; p 157. Ray, D.; Shi, P.-Y., Recent Advances in Flavivirus Antiviral Drug Discovery and Vaccine Development. Recent Patents on Anti-Infective Drug Discovery 2006, 1 (1), 45-55; Ghosh, D.; Basu, A., Present perspectives on flaviviral chemotherapy. Drug Discovery Today 2008, 13 (13-14), 619-624. Lescar, J.; Luo, D.; Xu, T.; Sampath, A.; Lim, S. P.; Canard, B.; Vasudevan, S. G., Towards the design of antiviral inhibitors against flaviviruses: The case for the

Dengue Fever

[4]

[5] [6]

[7] [8]

[9]

[10] [11]

[12] [13] [14] [15]

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CHAPTER 3 LEISHMANIASIS An Overview of New Synthetic Antileishmanial Candidates Nubia Boechat* and Luiz Carlos da Silva Pinheiro Oswaldo Cruz Foundation – Fiocruz, Institute of Drug Technology Farmanguinhos, Department of Organic Synthesis. 21041-250, Rio de Janeiro, RJ, Brazil Abstract: Leishmaniasis is considered by the World Health Organization to be one of the world’s most neglected diseases. The current drugs used to treat leishmaniasis were developed more than 40 years ago and are toxic. In addition, they generate resistance, and coinfection with the AIDS virus has worsened the situation of this disease. The purpose of this chapter is to review the various synthetic compounds described between 2002 and 2012 that have been tested against Leishmania spp. and that could be considered possible prototypes for treating this disease. Compounds were organized into 20 chemical classes.

Keywords: Leishmaniasis, prototypes, synthesis. 1. INTRODUCTION Leishmaniasis is considered by the World Health Organization (WHO) to be one of the world’s most neglected diseases. This disease threatens approximately 350 million people, of which 12 million are currently infected. Approximately 2 million new infections occur each year in 88 countries of tropical and temperate regions [1]. This disease can be caused by 20 different species of Leishmania and can be transmitted by the female phlebotomine sandfly of the genus Phlebotomus or Lutzomyia [2, 3]. Leishmaniasis can manifest in the following three ways: cutaneous (CL), mucocutaneous (MCL) and visceral (VL, also called “kala-azar”) *Address correspondence to Nubia Boechat: Oswaldo Cruz Foundation – Fiocruz, Institute of Drug Technology - Farmanguinhos, Department of Organic Synthesis. 21041-250, Rio de Janeiro, RJ, Brazil; Tel/Fax: +55 21 39772458; E-mail: [email protected] Adilson Beatriz and Dênis Pires de Lima (Eds) All rights reserved-© 2014 Bentham Science Publishers

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Boechat and Pinheiro

[4]. Leishmania parasites that cause CL and MCL are L. Mexicana, L. braziliensis, L. tropica and L. major. While L. donovani, L. infantum, L. chagasi are those that are responsible for VL, the most lethal form of the disease, which, if untreated, can be fatal. 1.1. Current Drugs in Use Treatment of leishmaniasis is complicated because there are different causative species and various clinical manifestations. A serious problem is related to coinfection with HIV/AIDS. Over the past few years, various drugs that were previously developed for other diseases have also been tested and showed variable efficacy [5-11]. Since the early 20th century, the pentavalent antimonial compounds, Glucantime® (meglumine antimoniate) (1) and Pentostam® (sodium stibogluconate) (2) were the first choice for leishmaniasis treatment despite their high cost and serious side effects [12]. The misuse of those drugs, incomplete treatment and inappropriate prescription guidelines have been described as the major reasons of the emergence of resistance of Leishmania parasites against pentavalent antimonials [13-17]. H3C

HO

HO H H

OH O Sb OH OH O

O

HO HO

Na (1) OH O

HO

Na OH O O O O O Sb Sb O O O

OH

NH

O

OH OH

OH

O

O (2)

OH

OH H H

O Na

.9H2O

OH

OH O

O

OH

CO2H O

(3) HO

OH NH2

Amphotericin B (3) is widely used for visceral leishmaniasis. More effective and less toxic are the following three new pharmaceutical formulations of amphotericin B: liposomal ampho B (L-AmB: Ambiosome®), amphotericin B

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 65

colloidal dispersion (ABCD: Amphocil®) and amphotericin B lipid complex (ABL: Abelcit®) [18-22]. In the diamidine class of compounds, pentamidine (4) was first synthesized in the late 1930s and is currently produced by Sanofi-Aventis. Its efficacy has gradually declined over the years, and serious adverse effects are associated with this drug [23, 24]. Miltefosine (5) is orally administered and was initially developed as an antitumor drug, but it showed activity against leishmaniasis and has been registered in India for the treatment of visceral leishmaniasis. Early clinical studies demonstrated a 94% cure rate, but the efficacy is variable for Leishmania spp. Miltefosine presents certain limitations such as high-level teratogenicity, clinical resistance and high cost [25-27]. Paromomycin (6) is indicated for the treatment of acute and chronic intestinal amebiasis and acts synergistically with antimonials in vitro against leishmaniasis. It is well tolerated, showing results comparable to amphotericin B [28, 29]. O H2 N

O

O P O O O-

NH2 (4)

NH

NH

NH2

HO

O

HO

H2N HO

O

OH

O

OH

O

NH2

H3C

O N

O OH

O

+

(5)

OH H2 N

N

H

N

N (7)

(6) OH

NH2

Sitamaquine (7), an orally active drug in development by the Walter Reed Army Institute in collaboration with GlaxoSmithKline, is in preclinical and clinical tests. Sitamaquine has demonstrated oral efficacy against L. donovani, and studies are

66 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

being conducted to characterize its pharmacokinetic profile, safety and tolerability when compared with amphotericin B [30-33]. 2. NEW SYNTHETIC CANDIDATES

COMPOUNDS

AS

ANTILEISHMANIAL

Several functional groups and heterocyclic rings have been associated as pharmacophoric groups with leishmanicidal activity. Groups such as amidine, diamidine, guanidine and polyamine as well as small heterocyclic moieties, including the following imidazole, pyrazole, thiadiazole, triazole, dioxazole and piperazine, are frequently found in leishmanicidal compounds. Activity has also been associated with quinolines, pyrazolopyrimidines, triazolopyrimidines, imidazopyrimidines, chalcones and naphthoquinones. Here, we present various synthetic compounds described between 2002 and 2012 that have been tested against Leishmania and that could be considered possible prototypes. This review is organized by the chemical class of the compounds. 2.1. Azole Derivatives Azoles represent one of the most important classes of antiparasitic drugs. The imidazolic antifungals ketoconazole (8) and triazolic itraconazole (9) have been used to treat cutaneous leishmaniasis with variable success rates [34]. They inhibit the C14 demethylation of lanosterol, which interferes with the production of leishmanial ergosterol, an essential component of their membrane structure. Thus, azoles such as imidazole, pyrazole, triazole, thiadiazole, oxazole and oxadiazole have become important synthetic targets in the development of drugs for parasitic diseases. N

N

O

N

O

O

N

H

N

O Cl

(8)

Cl

N N

N O

N

O

O

N

H (9)

N

N

O Cl

Cl

2.1.1. Triazole Derivatives 1,4-Dicyanophenyl-1H-1,2,3-triazoles (12) were prepared by the copper(I)catalyzed 1,3-dipolar cycloaddition reaction of 3- or 4-azidobenzonitriles (10) and suitable ethynylbenzonitriles (11). Using the modified Pinner method, the

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 67

resulting dinitriles 12 were transformed to imidate esters, which reacted with ethanolic solutions of ammonia, isopropylamine or ethylenediamine followed by treatment with aqueous HCl to afford congeners 13 as dihydrochloride salts. In this paper, the authors reported that the type and placement of cationic moieties as well as the nature of aromatic substituents can influence the antiprotozoal activity. The compounds were tested in vitro against L. donovani and showed IC50 values (concentration for 50% growth inhibition) in a range of 4.3-58 µM [35]. N3 N N N

R1 +

(i)

(10)

NC

CN

(ii)

N N N

R

R

R1 R2

R1 R2 (12)

(13)

R2 R1= R2= H, OH, OMe

(11)

NH , NH2

R=

NH

N

,

N H

HN

Reagents and conditions: (i) sodium ascorbate, CuSO4.5H2O, DMF/H2O or t-BuOH/H2O, 24-48 h; (ii) 1,4-dioxane-EtOH, HCl(g), rt, and then: amines, EtOH, rt, 4 days.

Tekwani and coworkers reported the reaction of azido acid 14 with O-tritylhydroxylamine to give O-tritylated hydroxamates (15). A subsequent cycloaddition reaction between these derivatives and terminal alkynes resulted in O-trityl protected aryltriazolylhydroxamates (16), which was followed by the deprotection of the O-trityl group, giving new aryltriazolylhydroxamates (17). The effects of derivatives of 17 on the viability of the promastigote stage of L. donovani showed in vitro antileishmanial activities with IC50 values in the range of 4.55-40 µg/mL [36]. O N

3

(i) OH

O N3

(14)

N H

OTrityl

R

(ii)

N N N

(15)

O N H

(16)

(iii)

n = 6-9 R= Ph, p-N,N-dimethylanilyl, 2-pyridyl, 2-thienyl, 3-biphenyl, 2-quinolinyl

R

N N N

OTrityl

O N H

OH

(17) o

Reagents and conditions: (i) NH2–O–Trityl, IBCF, THF, -15 C, 2 h; (ii) CuI, Hunig’s base, THF, rt; (iii) BF3.OEt2, THF, rt, 20 min, TFA/thioanisole (1:1), 0 oC, 3 h.

68 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

Boechat and coworkers reported the synthesis of 1,2,3-triazolic compounds from the reaction of diazomalonaldehyde (18) with appropriate aromatic amine hydrochlorides (19) to yield N-substituted-phenyl-1,2,3-triazole-4-carbaldehyde (20). The reaction of derivatives of 20 with DAST (N,N-diethylaminosulfur trifluoride) yielded 4-difluoromethyl-1,2,3-triazoles derivatives (21). The carbaldehyde 20 (3-Cl) and the difluoromethylated 21 (3-Cl) showed IC50 values of 2.8 and 2.6 µM, respectively, when tested against L. amazonensis [37]. CHO + NH3Cl

N2 O

O H H (18)

(i)

+

N N N

(19)

R1

R1

(ii) (20)

N N N

CHF2

(21)

R1

R1= 4-Cl; 4-Br; 4-CH3; 4-OCH3; 2,5-OCH3; 3-Cl; 3,5-Cl; 3-CN; 4-CN; 4-NO2; 2-OCH3; 3,4-OCH3

Reagents and conditions: (i) H2O, rt, 24h, (ii) DAST, CH2Cl2, rt, 24h

2.1.2. Imidazole Derivatives Boechat and coworkers also reported the condensation between N-aryl-amidines (22) and 2-bromomalonaldehyde (23) leading exclusively to 5-carbaldehyde imidazoles (24). Using DAST, the carbaldehyde group was readily converted into a difluoromethyl group generating the derivatives 25. When tested against L. amazonensis, the difluoromethylated derivative 25 (4-Cl) showed an IC50 of 1.7 µM [37]. NH 2

N

N

Br HO +

R

(22)

O H

H (23)

N N

(i)

R

CHO

N

(ii) (24)

R

CHF 2

(25)

R= H; 4 -Cl; 4 -Br; 4 -F; 4 -NO 2 ; 4 -CF 3 ; 4 -CN; 4 -CH 3 ; 4 -OCH 3 ; 2 ,6 -F; 2 -CH 3 ; 3 -OCF 3

Reagents and conditions: (i) AcOH, TEA, i-PrOH, rt, 17h; (ii) DAST, CH2Cl2, rt, 24h.

Siddiqui and coworkers used N-arylation of imidazole (26) with different aryl halides (27) using hexadecyltrimethylammonium bromide as a catalyst to synthesize the compound 4-(1H-imidazol-1-yl) benzaldehyde (28), which reacted with a number of substituted aromatic acetophenones to yield 3-[4-(1H-imidazol1-yl) phenyl]prop-2-en-1-ones (29). All the compounds were tested in vitro

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 69

against L. major promastigotes and showed significant activities with IC50 values between 0.63-0.99 µg/mL [38]. N F N N H (26)

+

O

N R

(ii)

(i)

N CHO (27)

N (29)

CHO (28)

R= H, F, Cl, Br, I, OCH3

Reagents and conditions: (i) K2CO3, C16H33(CH3)3N+Br, DMF, 100 oC; (ii) MeOH, NaOH, 25 oC.

Bhandari and coworkers [39] synthesized a series of imidazoles (30 and 31), which were assayed against L. donovani. Ketones 32 reacted with pyrrolidine and formaldehyde under asymmetric Mannich conditions in the presence of L-proline to give the corresponding products 33. Subsequent replacement of the pyrrolidine with imidazole followed by reduction gave the hydroxyl intermediates 34. Condensation of the 34 isomer with substituted aryl halides furnished the required ethers 30. To form imidazoles 31, a regioselective ring opening of styrene epoxide (35) or phenoxy methyl oxirane (36) with imidazole gave the corresponding alcohols 37. A nucleophilic aromatic substitution with aryl fluoride derivatives generated the targeted aryloxy ethers (31). The imidazole derivatives 30 and 31 inhibited the promastigote form at 94-100% in a concentration of 10 μg/mL, whereas against the amastigote form, they showed an IC50 value in the range of 0.47-4.85 μg/mL [39]. In another paper, Bhandari and coworkers [39] described the synthesis of a new series of bis and mono imidazoles. The Mannich reaction on cyclohexanone (38) with pyrrolidine gave 2,6-bis-pyrrolidin-1-ylmethyl-cyclohexanone (39) which, on reaction with imidazole followed by reduction, gave the corresponding hydroxy derivative 40. The reaction of 40 with aryl halides furnished the desired 2,6-bisimidazolylmethyl-cyclohexyl aryl ethers (41). For the synthesis of the imidazoles 42, cyclohexanone (38) was reacted with pyrrolidine under Mannich conditions to give 2-pyrrolidin-1-ylmethyl-

70 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

cyclohexanone (43). The subsequent replacement of the pyrrolidine with imidazole followed by reduction led to the cis/trans mixture of 2-imidazol-1ylmethyl-cyclohexanol (44). The trans-isomer 44 was condensed with substituted aryl halides to obtain the corresponding ethers 42.

R O

(ii), (iii) R

N

R

(i) (32)

N

(33)

(iv)

(34)

N

R O

OH

O

N

R3

N

R3

R3

R2

R1

(30)

R= CH 3, Ph; R 1 = H, F, NO 2 , CF 3, R 2 = H, NO 2, CF 3; R 3 = H, CH 3 O N

(v)

(35)

N R

O

(36)

N

R O

OH (37) O

R2 (iv) R1

N

(31)

(v) R= Ph, PhOCH 2 ; R 1 = H, F, NO 2 ; R 2 = H, NO 2 , CF3

Reagents and conditions: (i) pyrrolidine, (HCHO)n, L-proline/DMSO, 6-8 h; (ii) corresponding Mannich salt, imidazole, EtOH:H2O, 5 h; (iii) NaBH4/MeOH, 2 h; (iv) K(t-OBu), DMSO, aryl halides, 2-3 h; (v) imidazole, EtOH, reflux, 5 h.

Bromination of cyclohexanone (38) gave the 2,6-dibromo derivative (45), which, upon reaction with imidazole followed by reduction, gave the 2,6-di-imidazol-1-yl cyclohexanol (46). Condensation of this hydroxy compound with substituted aryl halides gave the required aryloxy bisimidazolyl (47). All bisimidazolyl 41 showed high activities against L. donovani amastigotes with IC50 values in a range of 0.588 to 2.437 μg/mL. The derivatives 47 exhibited interesting activity (IC50 of 3.00 and 3.02 μg/mL), whereas monoimidazolyl analogs 42 showed inhibitory activity with IC50 values ranging from 0.71 to 4.57 μg/mL [40]. A simple and efficient approach to synthesize 2H-benzimidazole 1,3-dioxide derivatives 48 is the reaction of benzofuroxan analogs 49 with different nitroalkanes. Compounds 48 were tested against L. braziliensis with ID50 (50% inhibitory dose) values in a range of 3.5-50 mM and showed better or similar activities than the reference drugs nifurtimox and miltefosine [41, 42].

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 71 N

(i)

N HCl

O (38) (v)

N O (39)

N

(ii), (iii)

N

N OH

OH

O

(44)

(vii), (iii)

O (38)

Br O

N

N

R1

N

N O

(46)

N

R

N

OH (ix)

(41)

R= H, F, NO2, CF3, R1= H, NO2, CF3

R1

(viii)

N

(45)

R

(42)

N Br

O

(40)

N

R

(vi)

N

N

(iv)

N

O (43)

N

(iv)

N

HCl

N

N

N

(ii), (iii)

R1

(47)

Reagents and conditions: (i) (HCHO)3, pyrrolidine, i-PrOH; (ii) imidazole, EtOH:H2O; (iii) NaBH4; (iv) K(t-OBu), DMSO, aryl halides; (v) (HCHO)3, pyrrolidine, L-proline, DMSO; (vi) Br2, CCl4; (vii) imidazole, DMF; (viii) NaH, DMF, aryl halides. R1 (i) O N O N

R1 R 2

(49)

R2

(i)

R1

(i)

R2

O N N O O N

O N

R1= Cl, CH=NOH, (E/Z)CH=CH-Ph R2= H, Cl

R1= H, Cl, Br, CH3, OCH3, CH=NOH R2= H, Cl

N O R

N O

R= (CH2)3CH3, Ph, 4-OCH3Ph

(48)

Reagents and conditions: (i), 2-nitroalkanes, piperidine, THF, rt.

A series of benzyloxy furanyl and benzyloxy thiophenyl azoles was synthesized by Bhandari and coworkers and screened in vitro for antileishmanial activity. Compound 50 emerged as the most active compound against L. donovani amastigotes with an IC50 of 3.04 μM and was more potent than the reference drugs miltefosine and miconazole [43]. A novel series of trans-2-aryloxy-1,2,3,4,-tetrahydronaphthyl azoles and related cyclohexyl azoles was synthesized and evaluated in vitro against L. donovani. Compound 51 was identified as the most active analog with an IC50 of 0.64

72 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

μg/mL and was more potent than the reference drugs sodium stibogluconate and paromomycin [44]. N

Cl

N

O

O

O

N Cl

N (50)

(51)

Cl

2.1.3. Pyrazole Derivatives Bernardino and coworkers synthesized a series of pyrazoles from the key intermediates 5-amino-1-aryl-1H-pyrazole-4-carbonitriles (52) that were subjected to aprotic deamination with t-butyl nitrite to generate 1-aryl-1Hpyrazole-4-carbonitriles (53). The reaction of 53 with HCl followed by treatment with NH3 produced 1-aryl-1H-pyrazole-4-carboximidamides (54). The compounds 53 were treated with CS2 and ethylenediamine to produce 5-amino-1aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (55). The derivatives 54 showed poor activity against L. amazonensis promastigotes with an IC50 value of 105 μM, whereas the compounds 55 presented good activities against promastigotes of L. amazonensis with an IC50 value of 15.5 μM and lower activities against L. braziliensis and L. infant [45,46]. HN CN N

(i) N

NH2

R (52) R= Br, NO2, 3,5-diCl

N

(ii) N

R

N NH2

CN

(53)

N

NH N

N

R (54) (iii)

N

R

(55)

Reagents and conditions: (i) t-butyl nitrile, THF, reflux, 6h; (ii) a)HCl, EtOH, 0 oC-rt, 5 days; b) NH3, EtOH, rt, 7 days; (iii) ethylenediamine, CS2, 14-15 h, 115 oC.

The antiprotozoal activity of a series of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3yl]morpholine derivatives was assayed in vitro. The compound 56 was active against L. donovani with an IC50 value of 2.3 μM [47].

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 73

A series of 1H-pyrazole-4-carbohydrazides was synthesized and tested against the Leishmania parasite. The most active were derivatives 57 and 58, and they were shown to be more effective against the promastigote form of L. amazonensis than against L. chagasi and L. braziliensis species. Both compounds were less toxic than pentamidine and ketoconazole [48]. O

N N

O N N (56)

Boc

N

N

H

N H N Y

H X

(57) X = Br, Y = NO2 (58) X = NO2, Y = Cl

2.1.4. Thiadiazole Derivatives Foroumadi and coworkers described the synthesis of a series of compounds derived from 1,3,4-thiadiazole [49]. The treatment of 5-nitroimidazole (59), 5nitrofurane (60) and 5-nitrothiophene (61) with thiosemicarbazide followed by cyclization gave 2-amino-1,3,4-thiadiazole (62). The reaction of amine 62 with NaNO2 followed by HCl gave 2-chloro-1,3,4-thiadiazole (63). The reaction of compound 63 with piperazine gave N-piperazinyl compound followed by Naroylation with appropriate benzoyl chlorides or thiophen-2-carbonyl chlorides afforded target compounds 64. The most potent compounds against the promastigote form of L. major were found to be an N-(5-chloro-thiophen-2-yl) carbonyl derivative and N-benzoyl analog with IC50 values of 9.35 and 10 μM, respectively [50]. Continuing the study of the synthesis and in vitro evaluation against L. major, Foroumadi and coworkers maintained the same synthetic strategy to obtain a series of compounds derived from 5-nitrofuran -2-yl-1,3,4-thiadiazol (65) and 5nitrothiophen-2-yl-1,3,4-thiadiazol (66) by exchange of the imidazole ring by furan and thiophene, respectively. The compound nitrofuran, containing 2chlorobenzoyl on the piperazine ring of this series, was potent against the promastigote form of L. major [51] and the 2,5-disubstituted-1,3,4-thiadiazoles are active against drug-resistant wild-type species related to cutaneous and visceral leishmaniasis caused by L. major, L. infantum and L. tropica, even

74 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

though they exhibited different growth inhibition profiles at different concentrations [52]. Canto-Cavalheiro and coworkers reported a series of salts of mesoionic compounds 1,3,4-thiadiazolium-2-phenylamine (67) derivatives that were evaluated against promastigotes of L. amazonensis, L. braziliensis and L. chagasi. The halogen derivatives exhibited very potent antileishmanial activity with ED50 approximately 10 µM for the three species [53]. N O2N (59)

O2N

CHO N Y (i) CH3 Z (ii) or O2N Y Z

N N S

N N

Y

NH2 (iii)

S

Z O2N

(62)

(63)

Cl (iv) (v) O2N

S

Z

N

N

R O

(64) Z= N-CH3; Y= N (65) Z=O; Y=CH (66) Z= S, Y=CH

CH(OAc)2

(60) Z=O; Y=CH (61) Z= S, Y=CH

N N

Y

Cl

Cl

R=

Cl

S

S

Cl

S

Br

Reagents and conditions: (i) thiosemicarbazide, EtOH, HCl, reflux; (ii) ammonium ferric sulfate, H2O, reflux; (iii) NaNO2, HCl, Cu; (iv) piperazine, EtOH, reflux; (v) appropriate thiophen-2carbonyl chlorides or benzoyl chlorides, benzene, pyridine, rt. Cl

-

N N R

S

H

(67) R= H, F, Cl, Br, OH, OCH 3 , NO 2, CN, OCH 2CH 3

2.1.5. Oxadiazole Derivatives Cerecetto and coworkers described the in vitro activity of furoxans and furazans against the promastigote form of L. braziliensis and L. pifanoi strains. Derivatives 68-71 showed ID50 values between 1.0 and 50.0 µM. Miltefosine was used as the reference drug [54]. 2.2. Pyridinone and Dihydropyridine Derivatives Tempone and coworkers described the in vitro activity of eight clinically used 1,4-dihydropyridines. They were tested against Leishmania and T. cruzi parasites

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 75

and showed that these derivatives represent an important class of compounds. Further investigations into their mode of action could be useful in the development of new agents against leishmaniasis and Chagas’ disease [55]. O

O Ph

S R

N

O

N

Ph O

Ph

N

S R

N O

(69)

O

O

O

S R

N

O

(68) R=

O

O

O

Ph

N

N

O

N

(71)

(70)

-Ph, -CH=CH 2, -CH 2CH 2OEt, -CH 2CH 2 SEt,

NH 2

S

(O) n

n= 0,1

-CH 2CONH 2, -CH 2 CH 2NH(p-OCH 3Ph)

The synthesis of the pyridinones from γ-pyrone 72 was described by Suryawanshi and coworkers. Compound 73 was prepared from the reaction between the γpyrone 72 and dibromopentane. The reaction of 73 with aniline furnished dimeric pyridinone 74 and monomeric pyridinone 75. The pyridinones 74 and 75 were subjected to in vitro antileishmanial screening against L. donovani promastigotes and amastigotes. The dimeric pyrone 73 showed good leishmanicidal activity, and the potency of this compound was greatly enhanced by the N-substitution of an oxygenated function of pyrone. The pyridinones tested against promastigotes showed significant activity, with an IC50 of 4.04-9.98 µg/mL, whereas against the amastigote stage in macrophages, they showed an IC50 from 5.32 to 9.64 µg/mL. The activities of those compounds were higher when compared with miltefosine but lower in relation to amphotericin B [56]. O

O OH

O

O

O

O

O

O

(73)

(72)

O

O

O

(i)

(ii)

O

+ N R

(74)

N R

O

O

O N R

O O (75)

R= Ph, Bz, 2,6-diCH 3Ph, 2,3-diCH 3, 4-NO 2Ph, 2 -OCH 3 Ph, 2-OCH 3Ph, 4-FPh, 4 -ClPh, c-hex

Reagents and conditions: (i) K2CO3, DMF, rt, 12h; (ii) amines, EtOH, 120-125 ºC, 24h.

76 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro R2

R2 N

N R3O

OR3 O

O O

O

O OR1

R1= H, CH3, CH2Ph

OR3

R3O

R3= CH3, CH2CH3

O

R2= H, 4-CH3, 2-OCH3, 4-OCH3, 4-F, 4-Cl, 4-Br, 3,4-diCl (76)

O O

O

O

O O

(77)

Tripathi and coworkers described 1-phenyl-4-glycosyl-dihydropyridines (76 and 77) as potent antileishmanial agents. The synthesis of these compounds started with the reaction of xylofuranosyl dialdoses or pyranosyl with methylacetoacetate and the appropriate aniline in the presence of tetrabutylammonium hydrogen sulfate. The compounds were screened in vitro and in vivo against L. donovani and showed IC50 values ≤15 mg/mL and a better selectivity index (SI) than the standard drugs pentamidine and miltefosine [57]. 2.3. Pyrimidine and Pyrimidone Derivatives Commercially available chloro-pyrimidines (78) and desired amines were used by Whitlock and coworkers to synthesize 2-pyridyl pyrimidine derivatives 79. The antileishmanial activity of these compounds was then determined on L. donovani. Sixteen compounds were equally active or more active than the standard agent miltefosine, with IC50 values from 0.26-0.70 µM [58]. R1

R1 R

Cl

R N

N

(i)

R2 N

N N

N (78)

(79)

R= CH3, CF3 R1= H, Cl R2= amines

Reagent and condition: (i) amines, Et3N, EtOH, reflux.

Suryawanshi and coworkers also described the synthesis and in vivo activity of 4N-substituted terpenyl pyrimidines derivatives against L. donovani. The reaction of ketene dithioacetal 80 with guanidine furnished terpenyl pyrimidine 81. Compound 81 displayed 66% inhibition at 50 mg/kg in a hamster model [59-61].

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 77 (i) SMe O

SMe

SMe

N (81)

(80)

N NH2

Reagents and conditions: (i) guanidine, i-PrOH.

The synthesis of a series of 2,4,6-trisubstituted pyrimidines and 1,3,5-triazines were described by Chauhan and coworkers. The reaction of compounds 82 with cyanuric chloride gave the corresponding triazinyl derivatives 83, which, when treated with different amines, afforded the final target compounds 84. To synthesize pyrimidinyl derivatives, the raw material 82 was oxidized to the corresponding sulfone 85, which was treated with various amines to yield target compounds 86. These compounds were screened for in vitro and in vivo antileishmanial activity against L. donovani. The pyrimidines showed in vitro inhibition of 80-100% against promastigotes, whereas against amastigotes, they showed IC50 values in a range of 0.89-9.68 mg/mL. The in vivo assays showed moderate inhibition of 48-56% [62]. OCH 3

OCH 3 (i)

H 3CO

(ii) SCH 3

R

OCH 3

HN

SCH 3 N

(82) R= H, OCH 3 R 1= amines

NH 2

OCH 3 (iii)

N

N

N

OCH 3 SO 2CH 3

R N

(84)

N

Cl

H 3CO

(85)

N

HN

Cl

N N

(83)

N

SCH 3

R

N

N

H 3CO R

H 3 CO

N NH 2

(iv)

R1 N

R1

H 3 CO R1

R (86)

N

N NH 2

Reagents and conditions: (i) cyanuric chloride, K2CO3, THF, reflux; (ii) amines, K2CO3, THF, reflux; (iii) m-CPBA, DCM, 0 oC-rt; (iv) amines, THF, 100 oC, closed steel vessel.

Fan and coworkers reported a green and efficient preparation of pyrano[3,2c]pyridone and pyrano[4,3-b]pyran derivatives in a one-pot, three-component reaction of aldehyde 87, 4-hydroxy-pyridin-2(1H)-one (88) and malononitrile (89). The hybrid compound 90 inhibited L. donovani growth and showed an IC50 of 10.6 μM [63].

78 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro OH

HO O CHO

HN HO

O

N

O

O H N

+

HN O

+ NC

O

CN

(i)

O O

(89) OH

OH

N

CN

HN

(88)

O

(87)

NH 2

(90)

Reagents and conditions: (i) [bmim][BF4], 80 oC.

2.4. Quinoline and Isoquinoline Derivatives Tempone and coworkers described the synthesis of quinoline derivatives from enaminone 91. The cyclization of 91 yielded 4-hydroxyquinolines (92), which, after treatment with phosphorus oxychloride, gave the 4-chloroquinoline derivatives 93. The compounds were tested against L. chagasi and showed IC50 values in a range of 0.091-18.78 μM and 3.55->15 μM for promastigotes and amastigotes, respectively [64]. OH N

H

R

(i)

O

(92)

(91) R=

R

(ii)

N

OEt R

Cl

N (93)

,

Reagents and conditions: (i) PhO2. 250 oC; (ii) POCl3

The synthesis of amodiaquine analogs was reported by Bertinara and coworkers. The target molecules were prepared by the reaction of 4-amino-2(hydroxymethyl)phenol (94) with 4,7-dichloroquinolone (95). The intermediate 96 was treated with tert-butylcarbamate followed by hydrolysis to give the amine intermediate 97. The target compound 98 was obtained by reaction of 97 with phenylisocyanate. The synthesized compounds proved to be active against L. donovani intracellular amastigotes, with IC50 values in the 1.83-17.7 μM range. Compound 96 showed the best IC50 value of 1.83 μM [65].

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 79 OH OH OH

NH 2

H

Cl + N

Cl

.HCl

Cl

(ii) N (96)

(95)

(94)

H Et

Cl

N (97)

.3 CF 3 CO 2 H

.HCl

OH

N N

(iii) (iv)

Cl

OH H

N

(i)

N

(v) NH 2

Et Cl

N (98)

N

O N H

N H

Reagents and conditions: (i) EtOH, reflux, 2.5 h; (ii) conc. HCl, reflux, 18 h; (iii) EtNHCH2CH2NHBoc, Et3N, CH3CN, rt, 18 h; (iv) CF3CO2H, CH2Cl2, rt, 6 h; (v) Et3N, PhNCO, CH3CN, rt.

Jain and coworkers reported the synthesis of 8-quinolinamines from commercially available 6-methoxy-8-nitroquinoline (99), which was converted to 8-quinolinamine (100) in four steps. Starting from 4-methoxy-2-nitro-5pentyloxyaniline (101), the 8-quinolinamine (102) was synthesized in three steps. Compounds 100 and 102, upon reaction with suitably side chain-protected Cbz/Boc-L/D-amino acids followed by deprotection, provided the 8-quinolinamine derivatives 103 and 104, respectively. Compounds 103 and 104 were tested in vitro against L. donovani promastigotes and exhibited antileishmanial activity with IC50 values of 2.7-19 μg/mL. The activity was comparable to pentamidine but less potent than amphotericin B [66]. Borne and coworkers reported the synthesis of chloroquine analogs from the reaction of 4,7-dichloroquinoline (95) with isoquinuclidine-6-carbonitrile (105) to give 106, which, following reduction of the nitrile group, gave 107. The secondary amine 108 was obtained by dimethylation. The analogue 109 was prepared from 105, where the quinoline ring is bonded to the isoquinuclidine through a methyleneamine bridge. Methylation of 105 with formaldehyde and formic acid gave 110, which was reduced to yield 111. The amine 111 reacted with 95 to yield the target product 109. Compounds 108 and 109 displayed potent in vitro activity against L. donovani with IC50 values of 1.9 and 3.0 μg/mL, respectively, comparable to pentamidine, but are 10- to 40-fold less potent than amphotericin B [67].

80 Organic Compounds to Combat Neglected Tropical Diseases H 3CO

H 3CO

H 3CO N NO 2

Boechat and Pinheiro

N

NH

(100) OC 5 H 11 H 3 CO NH 2

H 11 C 5O H 3CO

(103) H 11C 5 O

C 2H 5

C 2H 5

N

N NH NH 2

(102)

NH 2 O

H 3CO

NH

(101)

R

H N

NH 2

(99)

NO 2

C(CH 3 )3

N

C(CH 3 )3

NH

(104)

H N

R NH 2 O

R = L/D- amino acid

Silva and coworkers reported the synthesis of quinolin-8-ylarylsulfonamides (113) from the reaction of 8-aminoquinolines (112) with various arylsulfonylchlorides. The quinolinylarylsulfonamides 113 were active against promastigote forms of L. amazonensis and L. chagasi with IC50 values ranging from 2.12 to 2.85 µM and 0.45 to 0.56 µM, respectively, 2-fold than the control amphotericin B in the L. chagasi bioassay [68]. The synthesis of benzenesulfonyl-2-methyl-1,2,3,4-tetrahydroquinoline derivatives was described Mazzieri and coworkers. The preparation of tetrahydroquinoline derivatives was carried out by reduction of quinoline with NaBH4/NiCl2.6H2O followed by condensation with the appropriate benzenesulfonyl chloride. The derivatives 114 and 115 were tested against L. donovani and showed moderate growth inhibition with IC50 values of 18.87 and 21.35 μM, respectively [69]. Mondal and coworkers described the synthesis of binuclear derivatives of quiniline 116 by reaction of 8-hydroxyquinoline with dihaloalkanes. The in vivo investigation of all synthesized compounds against L. donovani promastigotes and amastigotes suggested the importance of the presence of a halogen substituent, which was found to be more important than the hydrophobic chain length. The absence of Cl afforded a less active bis-quinoline derivative. The activity of a compound with a CH2 unit was higher than that of hydrophobic chains with 3-5 carbon atoms in the ether bridge between quinoline moieties [70].

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 81 H 2N

NC Cl

N

HN

N (95)

CN

N

(i)

+ Cl

N

Cl

(105)

N (106)

Cl

N (107)

(iii)

(iv) Cl

N (108)

N

N

CN

N (iii) or

(ii)

N

(ii)

H

(i)

N

CH 2NH 2

(110)

(111)

Cl

N (109)

Reagents and conditions: (i) K2CO3, DMF, 140 oC, 24 / 48 h; (ii) LAH, rt, 18 h; (iii) HCO2H, HCHO, reflux, 70 oC, 20h; (iv) CH3CHO, reflux, 70 oC, 20h. (i) N NH2

H

(112)

N O

N O S

R=

Br

,

N Br

,

Cl F

F

S

Br

Ar (113)

Reagents and conditions: (i) arylsulfonylchlorides, py, 0 oC, 90 min and then, rt, overnight. Cl N O S O R (114) R= Cl, (115) R= Br

Cl O

O

N

N (116)

Ablordeppey and coworkers described the synthesis of benzothienoquinolines and benzofuroquinolines. Substituted anthranilic acids 117 were acylated with suitable acyl chlorides (118). In a double intramolecular cyclization reaction, the acylated intermediates were converted to substituted-11-quinolones 119, which were subsequently converted to benzofuroquinolines and benzothienoquinolines 120 by chlorination with POCl3 followed by hydrogenation. The targeted final products 121 were then obtained by methylation reaction. All the derivatives 121 showed significant activities against L. donovani promastigotes with IC50 values between 0.23 to >40 μg/mL, and pentamidine was used as the standard drug [71]. Moll and coworkers reported the cyclocondensation of the benzopyrylium salt 122 and different substituted aromatic amines to yield arylisoquinolinium derivatives 123. The derivatives had IC50 values from 0.54-19.7 μM. Compound 123 showed

82 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

synergistic action with amphotericin B in vitro, making it a potential candidate for further in vivo experiments [72]. H N

O

CO2H NH2

X

Cl

+

(i)

X

(ii)

R (117)

N (iii) R

O

(118)

(119)

X

(iv)

R

(120) (v) I

X= O, S

R= H, Cl, CN, OCH3, CO2H, CONH2

N (121)

X

R

o

o

Reagents and conditions: (i) NaOH, H2O, rt; (ii) PPA, 130 C, 3 h; (iii) POCl3, 110 C, 24 h; (iv) H2, Pd/C, MeOH; (v) CH3I, TMS, 110 oC, reflux, 12 h. H 3 CO O+ OCH 3 (122)

X

(i)

H 3CO N

-

+

X

-

OCH 3

X= TFA

(123)

Reagents and conditions: (i) amines, AcOH, rt.

2.5. Quinazoline and Quinoxaline Derivatives The synthesis of 2,4-disubstituted quinazolines derivatives was described by Sahu and coworkers. 2,4-Dichloroquinazoline (124) reacted with suitable arenes to produce 2-chloro-4-aryl quinazolines (125). The derivative 125 was reacted with different cyclic amines to afford the desired compounds 126, which exhibited IC50 values of 025-6.2 and 4.17-25.14 μg/mL against promastigotes and intracellular amastigotes of L. donovani, respectively [73]. (i)

N N (124)

R

R

Cl

Cl

N N (125)

N

(ii) Cl

N

N

(126)

X

R= 3-indolyl, 1-methyl-3-indolyl, 2,4,6-triOCH3-C6H2, 2,4-diOCH3-C6H3 X= NH, NCH3, CH2, O, N4-FPh, 2-Py, Ph

Reagents and conditions: (i) Ar-H, AlCl3, DCE, N2, 75-80 oC, 2-3 h; (ii) TEA, dioxane, 90-95 oC.

Agarwal and coworkers described a synthetic strategy for obtaining quinazoline derivatives. Tetralone 127 was used as starting material to react with various

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 83

aldehydes, which yielded benzylidenes 128. The cyclization of 128 with guanidine sulfates gave the quinazoline derivatives 129 and 130. The dihydro- and tetrahydroquinazoline derivatives were tested against L. donovani promastigote and amastigote forms. The compounds with better promastigote inhibition were screened against amastigotes and exhibited IC50 values in a range of 2.65-8.95 μg/mL. The reference drugs used were sodium stibogluconate and pentamidine [74]. R2

O

O

R

R1

(129) (ii)

(i)

+ R2

R

(127)

R1

N

N

(128)

N

N

R= H, F, Cl; R 1= H, Cl; R 2 = cyclic amines (130)

H

R1

R

Reagents and conditions: (i) aldehydes, 5% alcoholic KOH, ethanol, 0 oC, rt, 2 h; (ii) substituted guanidine sulfates, t-BuOK, methanol, reflux, 24 h.

A Clauson-Kaas reaction starting from the 2-nitroanilines was performed to prepare the intermediates 1-(2-nitrophenyl)pyrroles 131 that were subsequently reduced and treated with various acid chlorides to generate the acetamides 132. The 4-substituted pyrrolo[1,2-a]quinoxalines 133 were prepared by cyclization of these amides according to the Bischler-Napieralski reaction. The quinoxalines were tested for their in vitro antileishmanial activities on the L. amazonensis and L. infantum strains and showed IC50 values from 0.5-36 and 0.5-25 µM, respectively [75]. R1

R1 R2 NO2

N

R2

(i) (ii) N

O

N H

(iii) R

R2

N

R1

N

R

(133) (131)

(132)

R1= R2= H, OCH3; R= alkyl, alkenyl, alkynyl, aryl, cis-trans oxiranyl

Reagents and conditions: (i) BiCl3, NaBH4, EtOH; (ii) RCOCl, Py, dioxane; (iii) a) POCl3, toluene, reflux, 4h; b) NaHCO3, H2O, rt.

84 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

Monge and coworkers reported a series of quinoxaline N,N’-dioxides that were tested in vitro against the promastigote form of L. braziliensis. The compounds 134 showed ID50 values between 1.0 and 50.0 μM, and miltefosine was used as the reference drug [76]. The sulfonamide derivatives 135 showed IC50 values between 2.1-3.1 μM and were almost 10-15-fold less active than the reference drug amphotericin B [77]. O N

R R

N O

O N

Cl

R1 R2

N O

CN O2 S N H

R

(135)

(134) R= H, F, Cl,

R= 2-NO2, 4-NO2

R1= SPh, COPh, CO2CH2CH3 R2= CH3, CF3, Ph

2.6. Indole Derivatives Indoles comprise a wide variety of biologically active compounds, and some of them have been described as antileishmanial agents [78]. An N-alkylation reaction of tetracyclic indoles 136 with ω-dihaloalkanes provided different indolyl N-alkyl halide derivatives that were treated with tetraethylmethylene bisphosphonate to yield the indol bisphosphonates 137. The bisphosphonates were tested in vitro and showed more than 80% inhibition against both the promastigote and amastigote stages of L. donovani. The IC50 values ranged from 1.69-24 μg/mL and were significant when compared with sodium stibogluconate as reference drug [79]. X

X

(i) N H

(ii)

(136) X= S, CH 2 ; n= 4-6;

N EtO OEt P ( ) n O O P OEt OEt

(137)

Reagents and conditions: (i) ω-dihaloalkane, NaH, DMF; N2, 0 oC, 2h (ii) CH2[P(O)(OCH2CH3)2]2, NaH, THF, N2, 0 oC, 1.5h.

Chauhan and coworkers described the synthesis of indole derivatives. From the reaction of indole-3-carbaldehyde (138) with (p-cyanophenoxy)pentylbromide compound 139 was obtained. The condensation of 139 with 2-thiohydantoin

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 85

furnished compound 140. Compound 140 showed growth inhibition against the amastigotes of L. donovani with an IC50 value of 18.2 μM [80]. Br O O

O O

5

H

H N

H N

NC N H

HN S

N H

O O 5

(i)

S

N H N

(ii)

(140)

(139)

(138)

O 5 CN

CN

Reagents and conditions: (i) toluene, NaOH, TBAB, rt; (ii) ethanolamine, EtOH, 60 oC.

Homodimers of bis-β-carbolinium were obtained from the reaction of symmetrical dihalogeno-linkers with 6-chloro-norharmane. The most active compound was the dimer 141 with an IC50 value of 0.5 μM. The meta substituted aryl linker was 100fold more active than the corresponding dimer with the para substituted linker. This suggests that relative orientation of the two nostocarboline units has an impact on the activity against L. Donovani [81, 82]. Cl

Cl

N H

N X

N (141)

X

N H

Indolylglyoxylamide derivatives have been synthesized and evaluated in vitro against the amastigote form of L. donovani. Compound 142 (D/cis) has been identified as the most active analog with an IC50 value of 5.17 μM and was several fold more potent than the standard drugs sodium stibogluconate and pentamidine [83]. A series of 2-(pyrimidin-2-yl)-1-phenyl-2,3,4,9-tetrahydro-1H-β-carboline derivatives was synthesized and evaluated for antileishmanial activity against L. donovani. Compound 143 exhibited the best antileishmanial activity with an IC50 value of 1.93 mg/mL against amastigotes and was more active than the reference drugs sodium stibogluconate and pentamidine [84].

86 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro Cl

NH

O O O

N N

N

N

Cl

N H

O

OCH3

H3CO

N H

(143) (142)

In another paper, Chauhan and coworkers described the synthesis and in vitro antileishmanial activity against L. donovani for a series of [1,2,4]triazinoindoletriazine derivatives. Compound 144 showed an IC50 value of 4.01 μM and exhibited 20- and 10-fold more selectivity when compared with the standard drugs pentamidine and sodium stibogluconate, respectively [85]. The synthesis and antiparasitic activities of the acetylated glycosylisoindigo derivatives were describe by Moreau and coworkers. The compounds were tested in vitro against the following causative agents of tropical diseases : malaria, Chagas' disease, human African trypanosomiasis and leishmaniasis. The most active compound against L. donovani was the azaisoindigo 145 with an IC50 value of 0.84 μΜ [86]. H N

N O N N N N

N

N S

(144)

Br

N N

O

N

N O AcO

OAc OAc OAc

(145)

2.7. Purine, Pyrazolopyrimidine, Pyrazolopyridine and Thienopyridine Derivatives Silva and coworkers described the reaction of the key intermediate 6-thiopurine 146 to obtain new leishmanicidals. Thiopurine was reacted with 1-bromo-3chloropropane and subsequently treated with thioethylamine to produce derivative 147. Compound 146 was also treated with 2-bromoacetate followed by hydrolysis

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 87

with KOH, yielding the derivative 148. The corresponding derivative 149 was produced by several steps from 146. All compounds were evaluated against L. amazonensis and L. chagasi and showed activity only against promastigotes of L. amazonensis (IC50 29-50 μM). Derivative 149 was the most active compound, with an IC50 value of 29 μM [87]. N N

H N

SH

O HO

N

N

N S

S

N OCH 3 (iii)

(i)

(ii) N H (146) (iv)

S

NH 2

N

N N

N H (147)

OH SCH 2CO 2H

(149)

N

N

N H

N

(148)

Reagents and conditions: (i) 1-bromo-3-chloropropane, EtOH, 0 oC for 30 min-1h (rt); (ii) thioethylamine 48 h, 25 oC; (iii) 2-bromoacetate, DMF, 0 oC, 30 min (iv) KOH, MeOH / H2O, 24 h, 25 oC.

Jorda and coworkers reported the ability of pyrazolo[4,3-d]pyrimidine and purines derivatives to inhibit leishmanial CRK3 protein kinase activity and to kill axenic amastigotes of L. donovani. Pyrazolopyrimidines 150 were markedly more potent inhibitors of CRK3/CYC6 than the corresponding purines and showed IC50 values of 57.9 and >100 μM, respectively [88]. H N

HN

R N

N N

(150) R= 4-FPh, 3-ClPh, 2-OHBn, Adamantan-1-yl

A series of 1H-pyrazolo[3,4-b]pyridine derivatives was synthesized by Echevarria and coworkers using the condensation reaction between the appropriate aniline with 4-chloro-1H-pyrazolo[3,4-b]pyridine (151), which yielded the target

88 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

compounds 152 and 153. The Mannich reaction is also an alternative to obtain the molecule 153. These compounds were tested against promastigote forms of L. amazonensis and showed IC50 values of 0.39 and 0.12 μM, respectively [89]. N HO

HO R

Cl

O

R

(i) O

N N

N

NH O

R

(ii) O

N

N (151)

NH O

O

N N

N

N

(152)

R= CH3, Ph

(153)

(iii)

Reagents and conditions: (i) 4-hydroxyaniline, Δ; (ii) (CH3CH2)2NH, HCOH, 2-propanol; (iii) diethylaminomethyl-4-acetylaminophenol.

Castro and coworkers described the synthesis of thieno[2,3-b]pyridine derivatives through the reaction of appropriate anilines with 4-chlorothieno[2,3-b]pyridine-5carbonitrile (154) without solvents, producing the derivatives 4(phenylamino)thieno[2,3-b]pyridine-5-carbonitriles (155), which were subsequently treated with ethylenediamine and CS2 to yield the target compounds 156. Inhibition of 95.4% of the L. amazonensis was achieved with 50µM of 156, which has better efficacy than glucantime [90]. H3CO

H3CO Cl

S

N (154)

NH N

NH CN

CN (ii)

(i) S

N (155)

N H S

N (156)

Reagents and conditions: (i) anilines, 140 oC, 4h; (ii) NH2(CH2)2NH2, CS2, HCl 6N, reflux, 24h.

The synthesis of tetrahydro[1]benzothieno[2,3-d]pyrimidine derivatives (BTP) and their in vitro evaluation against T. cruzi, M. tuberculosis, L. amazonensis, and six human cancer cell lines was described by Hammond and coworkers. The synthesis of the BTP derivative began with the preparation of 2-amino-4,5,6,7tetrahydrobenzo[b]thienophene-3-carboxylic acid ethyl ester (157) using a Gewald reaction. Compound 157 reacted with an excess of formamide yielded the cyclic

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 89

pyrimidinone 158, which underwent chlorination with POCl3 to give 159. Nucleophilic displacement with aqueous hydrazine formed the corresponding aromatic hydrazine derivative 160. The synthesis of BTP 161 was concluded with the formation of a Schiff base between 160 and an appropriate aldehyde or ketone. Compound 161 was highly selective in vitro against L. amazonensis with an IC50 value of 1.7 μM [91]. CO2Et NH2

S

Cl

O (i)

(157)

(ii)

NH S

Cl

N

N

(158)

N (iii)

NH

HN

(iv)

N S

N S

(159)

NH2 N

S

N (161)

N

(160)

Reagents and conditions: (i) diethyl amine, EtOH, rt, sonication, 30 min; (ii) formamide, reflux, 6 h; (iii) POCl3, reflux, 3 h; (iv) hydrazine, MeOH, reflux, 3 h; (v) aldehyde or ketone, EtOH, reflux, 2-72 h.

2.8. Benzimidazole, Derivatives

Benzothiazole,

Benzoxadiazole

and

Benzoxazole

Navarrete-Vázquez described the synthesis of benzimidazole-pentamidine hybrids. The alkylation of 4-hydroxybenzaldehydes with 1,5-dibromopentane yields the bis-aldehydes 162. The conversion of the resulting bis-aldehydes to the respective benzimidazole 163 was performed by the following two different methods: 1) by treatment with adequately substituted 1,2-phenylenediamine; 2) one-pot reduction-cyclization reaction of 2-nitroanilines in the presence of sodium dithionite. The derivatives 163 were up to 26-fold more active than pentamidine and exhibited high bioactivity against L. mexicana, with IC50 values between 0.36-1.06 μM [92]. R2

R2 O

OHC

O n n=5

(i) or (ii)

R1

N N H

CHO (162)

O

CH2 2

R2 (163)

R1 = H, CH3, NO2, OCH3, CF3 o

R2= H, OCH3

Reagents and conditions: (i) Na2S2O5, DMF, 90 C, 20 h; (ii) Na2S2O4, H2O:EtOH, 80 oC, 10-14 h.

90 Organic Compounds to Combat Neglected Tropical Diseases

Boechat and Pinheiro

Ihara and coworkers reported the synthesis of fluorinated rhodacyanine derivatives by the reaction of rhodanine 164 with N,N-diphenylformamidine followed by treatment with acetic anhydride to yield 165, which was treated with N-methyl-2-methylbenzothiazolium salt (166) to provide merocyanine (167). The reaction of 167 with methyl p-toluenesulfonate followed by condensation with 168 afforded the target compound 169. Compound 169 exhibited an IC50 value of 0.011 µM for in vitro activity against L. donovani and an IC50 value of 0.353 µM in an in vitro assay in macrophages. In vivo ∼95% inhibition was observed at dosages ranging from 1.3 to 12.5 mg/kg [93]. Ph N

S

S

Ac S

(i), (ii)

O

S O

S

F

N

+

S (168)

N

TsO

(iii)

S (165)

N

(167)

+

O (164)

S

S

TsO

F

(166)

(iv), (v), (vi)

S

N S

S O

N

Cl

S (169)

Reagents and conditions: (i) N,N-diphenylformamidine (ii) Ac2O / TEA; (iii) Ac2O / TEA; (iv) TsOMe, DMF, toluene, 150 oC; (v) MeCN, TEA, 75 oC; (vi) HCl, MeOH, 80 oC.

The synthesis of 2-(arylthio)vinylbenzo[1,2-c]1,2,5-oxadiazole N-oxide was accomplished by Porcal and coworkers, who described an efficient Wittig reaction performed in mild conditions with polymer-supported triphenylphosphine (PSTPP). Treatment of tiophenol 170 with paraformaldehyde and HBr followed by PS-TPP yielded the corresponding polymer-bound phosphonium salt 171. The reaction of the salt 171 with NaH followed by the condensation with formylbenzofuroxan and the concomitant cleavage of the resin resulted in the compounds 172. The oxidation of these benzofuroxanyl derivatives with m-CPBA gives 2-(arylsulfinyl)vinylbenzo[1,2-c]1,2,5-oxadiazole N-oxide (173). Benzofuroxans 172 and 173 were assayed in vitro against L. pifanoi (IC50 4.3-4.6 µM). The compounds 172 and 173 showed better results against L. braziliensis, with IC50 values in the range of 0.9-4.6 µM [94]. The synthesis and antiprotozoal activity of benzoxazole derivatives were reported by Kozikowski and coworkers. The screening for inhibition of L. donovani

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 91

revealed that compound 174 had an IC50 value of 0.08 µM and was 3-fold more active than miltefosine [95]. Navarrete-Vazquez described the synthesis and in vitro antileishmanial activity of compound 175 (IC50 value of 1.3 μM), which was 4.5-fold more active than nitazoxanide and tizoxanide and almost 10-fold more potent than pentamidine against promastigotes of L. Mexicana [96]. Br Ph + Ph P S

SH (i) R

-

(iii)

(ii) (170)

S

N

O N

R (iv) R

(171)

O

(172) (v)

R = H, F (Z and E isomers), R = Cl (E isomer)

O S

N

O O

N (173)

Reagents and conditions: (i) CH2O, HBr, toluene, 50-60 oC, 3h; (ii) PS-TPP, toluene, 50 oC, 48h; (iii) NaH, THF, RT, 1h; (iv) formylbenzofuroxan, THF, 50 oC, 8-12 h; (v) m-CPBA, CH2Cl2, -78 at 0 ºC, 2 h. O

OH N CO2H

HN

O

O OH

O N

N

NH O2N

O (174)

S (175)

2.9. Acridine Derivatives Di Giorgio and coworkers described the synthesis of symmetric and nonsymmetric diaminoacridines by mono- or di-acylation. The reaction of proflavine (176) with different benzoyl chlorides or different anhydrides yielded the corresponding amides 177. The derivatives acridinyl acetamide and acridinyl benzamide showed antileishmanial activities against L. infantum with IC50 values between 0.11-341.5 µM and 0.03-12.8 µM for the promastigote and amastigote forms, respectively. Symmetric conformation with acetylamino or benzoylamino substituents was essential for antileishmanial activity [97].

92 Organic Compounds to Combat Neglected Tropical Diseases (i) H 2N

N

H

NH 2

N

N

N O

R

(176)

Boechat and Pinheiro

R

(177)

H O

R= CH 3 , Ph

Reagents and conditions: (i) appropriate anhydrides or benzoylchlorides, py, TEA, 1h, 80 oC.

2.10. Coumarine and Pyrane Derivatives Azas and coworkers synthesized a series of 4-arylcoumarin derivatives via a ligand-coupling reaction of the coumarin ring with substituted phenyl derivatives. The treatment of the appropriate 4-hydroxycoumarins (178) with triflic anhydride gave 4-trifluoromethylsulfonyloxycoumarin (179). In modified Suzuki conditions, the C-4 arylation was performed with arylboronic acids in the presence of a palladium catalyst and CuI as a cocatalyst. The 4-(3,4-dimethoxyphenyl)-6,7dimethoxycoumarin (180) exhibited potent activity against L. donovani amastigotes with an IC50 value of 1.1 µM and a selectivity index twice than the reference amphotericin B [98]. H 3CO

O

O

H 3 CO

O

(ii)

(i) H 3 CO

H 3 CO OH (178)

H 3 CO

O

OSO 2 CF3 (179)

O

O

H 3 CO (180)

OCH 3 OCH 3

Reagents and conditions: (i) (CF3SO2)2O, Et3N, CH2Cl2; (ii) ArB(OH)2, Pd(PPh3)4, CuI, Na2CO3, toluene, 110 oC.

Copp and coworkers described the synthesis of pseudopyronines A and B. The target acylpyrone skeletons 181 and 182 were obtained by the cyclization of βoxo acids using carbonyldiimidazole. Reduction of the α-acyl group of the 2Hpyran-2-one (181) utilizing NaCNBH3 afforded the natural product 183. Deacylation of 182 with H2SO4 yielded 6-heptyl-4-hydroxy-2-pyrone (184) with subsequent acylation by hexanoyl chloride affording the 2H-pyran-2-one (185), which, after reduction, provided pseudopyronine B (186). 3-Acylpyrones 181 and 185 were the most active compounds against L. donovani, with IC50 values of 0.46 and 0.55 mg/mL, respectively [99].

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 93 OH

O

OH C 5H 11

C5H1 1

O O (181) OH

C 5H 11

O

O O (183) OH

OH

O C 7H 15

C 7 H 15

C 6H 13

(i)

C 7H 15

O

O

(iii)

(ii) O

(182)

C 7H 15

O

OH

O

O

C 6H 13

(i)

C 5 H 11

C 7H 15

O

(185)

(184)

O (186)

Reagents and conditions: (i) NaCNBH3, THF, 2 M HCl, rt, 2.5 h; (ii) 90% H2SO4, 130 oC, 1 h; (iii) hexanoyl chloride, TFA, reflux, 3 h.

2.11. Azepine Derivatives Compounds containing the azepine unit possess a wide range of biological activities, and some derivatives are active against promastigotes and intracellular amastigotes of L. Mexicana [100] and L. chagasi [101]. Palma and coworkers [100] reported the synthesis of azepine analogs starting from the available N-benzyl-α-naphthylamines, which, on reaction with allyl bromide, were converted into N-allyl-N-benzyl-substituted-α-naphthylamines (187). The introduction of an allyl moiety at the o-position of the amino group was carried out via an aromatic amino-Claisen rearrangement of the N-allyl derivatives yielding the products 188. Oxidation and subsequent intramolecular 1,3-dipolar cycloaddition of 188 was performed to obtain 1,4-epoxytetrahydronaphtho[1,2-b]azepines (189). Reductive cleavage of the N-O bond of 189 gave exclusively cis-2-aryl-4hydroxytetrahydronaphtho[1,2-b]azepines (190). The compounds showed activity against the intracellular form of L. chagasi promastigotes with IC50 values between 6.0-183.8 μM. R2 R1

N

(i)

N H

R (187)

(188)

(iii) N H (190)

H

R2

R2

R1

(ii)

O N

R2

R

R1 (189)

R

(R, R1, R2) = H, F, Cl, Br, OCH3, CH3, NO2 R1

R

Reagents and conditions: (i) BF3.OEt2, 115-125 oC, 1-4 h; (ii) a) H2O2, Na2WO4.2H2O, methanol, 0-25 oC, 45-50 h; b) toluene, reflux, 6-10 h; (iii) Zn, 80% AcOH, 80-82 oC, 2-5 h.

94 Organic Compounds to Combat Neglected Tropical Diseases

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Kunick and coworkers reported a novel type of antileishmanial agents, namely 2(3-aryl-3-oxopropenyl)-9-tert-butyl-paullones (191). The authors assume that the substructure of the chalcones linked in the paullone is an important moiety for antileishmanial activity. Several compounds of this class showed activity against L. donovani axenic amastigotes with a growth inhibition (GI50) of 50% < 1 μM and against parasites in host macrophages without exhibiting toxicity for human host cells [102]. H N

O

O

HN (191)

2.12. Porfirine Derivatives A rational approach in the search for new drugs is to exploit biochemical differences between the parasite and its mammalian host. One specific example in the case of Leishmania relates to the biosynthesis of heme, a critical prosthetic group for proteins involved in metabolism and electron transport. Like other Trypanosomatids, Leishmania parasites require heme or porphyrins to be preformed for them. Thus, the heme biosynthetic pathway is essential to the survival of Leishmania, and drugs that can interfere with the heme acquisition processes of the parasite may prove useful in treatment of leishmaniases [103]. Boechat and coworkers described the use of porphyrins as photosensitizers and suggested that they may be essential for good results in treating leishimanioses [104, 105].

Cl

-

OR N OR H N+ H N

(192) R= CH 3 ; CH 2 CH 3

Jones and coworkers described the growth curves of L. tarentolae exposed to several different concentrations (1.1-4.2 µM) of carbaporphyrin ketals (192) and

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 95

indicated that these compounds have great promise as potent inhibitors of Leishania [106]. 2.13. Biguanidines, Amidines and Diamidines Aromatic diamidine -containing compounds have a long history of use in the treatment of human African trypanosomiasis and leishmaniasis [107-112]. The main representative of such classes is pentamidine, which is one of the current therapies for leishmaniasis. Due to their cationic character, diamidines frequently exhibit high toxicity, such as cardiotoxicity, nephrotoxicity, and pancreatic complications, and display poor oral bioavailability. To overcome these limitations, new aromatic dications and their prodrugs 193 have been synthesized and screened against a number of pathogens including the protozoan parasite species Trypanosoma and Leishmania [113-115]. Amidine 194 and diamidine groups have been recognized as pharmacophoric group in antiparasitic drugs. The “reversed” amidines 195 are compounds that have the imino group of the amidine attached to an aromatic, forming an “anilino”- nitrogen in contrast to the original amidino aromatic or heteroaromatic, in which the imino group is directly attached to the aryl ring. This type of chemical modification has brought the best results for antiparasitic activity. R

2

R

X

X O n O

1

n = 3-6 X = CH, N

NH

R2

NH

,

NH 2

N

NH ,

HN

HN

,

N H

R 2=

R1

NH N H

(193)

Ph

NH

,

,

N H

,

N N

N NH 2

N OH

NH 2

NH 2

,

N N N N H

,

,

N OCH 3 NH 2

R 1 = H, F, Cl, OCH 3, NO 2, OH, CO 2Et, CO 2H, CONH 2, CN

NH

NH Ar

NH2 (194)

Ar

N H (195)

Ph

Tidwell and coworkers reported the synthesis and antiprotozoal activities of cationic bisbenzofurans and phenylbenzofurans. In this series of compounds, some derivatives displayed activity against L. donovani superior to that of pentamidine [116, 117].

96 Organic Compounds to Combat Neglected Tropical Diseases

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The synthesis and antiprotozoal activity of dicationic bis(phenoxymethyl) benzenes, bis(phenoxymethyl)naphthalenes, and bis(benzyloxy)naphthalenes were reported. The reaction between cyanophenol 196 and α,α’-dibromoxylene (197) gave the dinitrile 198. The nitriles underwent the Pinner reaction to give the amidine derivative 199, which was active against L. donovani (IC50 value of 1.3 μM) [118]. NH NC

CN HO

(196) (i)

+

H 2N

O

O

(ii)

Br O

O (198)

(197)

(199)

Br CN

NH H2N

Reagents and conditions: (i) K2CO3 or Cs2CO3, DMF; (ii) HCl, EtOH, 1,4-dioxane, then appropriate amine.

Boykin and coworkers described the syntheses of the azaterphenyl derivatives by Suzuki coupling of the appropriate aryl halides 200 with the corresponding aryl boronic acids or esters to form teraryl bis-nitriles (201). The bis-nitriles were converted to the diamidines by reaction with LiN(TMS)2 followed by hydrolysis. Compounds 202 and 203, which have pyridines instead of a phenyl group, showed a 3- to 4-fold increase in activity over the corresponding terphenyl. This system can be represented by the 2 most active compounds, 202 and 203, with IC50 values of 0.063 and 0.084 μM, respectively. The authors found that the diamidine system, which is highly active, showed a consistent and distinctive structure activity relationship against L. donovani axenic amastigotes [119]. A series of compounds with amidoxime group was synthesized using the Buchwald-Hartwig and Heck reactions. Two amidoximes, 204 and 205, showed interesting in vitro activities toward L. donovani promastigotes with IC50 values of 8.3 and 8.8 μM, respectively [120].

Leishmaniasis

Organic Compounds to Combat Neglected Tropical Diseases 97 (i) (ii) R

X

NH

HN H2N

N

N (202)

+ NC

NH2

(HO)2B

N

Y= Cl, Br

R= B(OH)2, Cl, Br

(i), (ii)

(201)

(200)

(iii)

HN

NH

H2 N

N

NH2

(203)

Reagents and conditions: (i) Pd(PPh3)4, Na2CO3, toluene, 80 oC; (ii) a) LiN(TMS)2, THF, rt, overnight; b) HCl (gas), ethanol, rt, overnight; (iii) 4-cyanophenylboronic acid, Pd(PPh3)4, Na2CO3, toluene, 80 oC.

HO O O

N NH2HN O

S

S

O

O N (204)

O

OH

H2N

N (205)

H2N

OH

2.14. Sulfonamide Derivatives González-Rosende reported a series of sulfonamides (206-215) obtained by reaction of the amines with the corresponding sulfonyl chloride. The leishmanicidal activity of the compounds was evaluated in vitro against the cultured promastigotes of L. infantum. The pyrimidines, pyrazine, pyridine, thiazoles, isoxazole, pyrazole, indazole, indanes and aniline derivatives were found to be active compounds with IC50 values between 10 [9]. SI was defined as the median inhibitory concentration (IC50)/MIC. 2.1. ANTI-Mycobacterium Tuberculosis Drugs The antitubercular drugs retrieved by this search are summarized in Table 1. Several molecular modification strategies have been used to discover novel antitubercular drugs. Ahsan et al. described the synthesis and evaluation of new pyrazoline derivatives using thiacetazone as a backbone [16]. Specifically, the authors proposed that the rigidity of N-acyl hydrazone be increased by molecular annealing with other compounds (Fig. 1). This strategy reduces the number of conformers and it has been used to discover several new compounds. S N O H3C

N NH

NH

NH2 S

anelation

H3C H3C

O

N

bioisosteric replacement to oxygen NH2

O CH3

Figure 1: Production of the pyrazoline derivative by molecular modification of N-acyl hydrazone.

Ahsan et al. also proposed isosteric substitution of sulfur (thiocarbonyl) with oxygen (carbonyl), which increased the antimycobacterial activity by about four times and the SI values were > 161 [16, 17]. The same group also reported that oxadiazole derivatives were active against MTB. In particular, they reported that the compound 4-[(5-[(4-fluor-ophenylamino]-1,3,4-oxadiazol-2-yl)methylamino]1,2-dihydro-1,5-dimethyl-2-phenylpyrazol-3-one inhibited mycobacterial growth

234 Organic Compounds to Combat Neglected Tropical Diseases

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at a concentration of 0.78 µg/mL and isoniazid-resistant MTB at a concentration of 3.12 µg/mL. This oxadiazole derivative did not violate Lipinski’s parameters. Lipinski’s parameters, also known as the “rule of five”, is widely used as a filter for drug-like properties based on the following essential drug-like properties: 1) > 5 H-bond donors; 2) molecular weight > 500 kDa; 3) LogP > 5; 4) and > 10 H-bond acceptors [18]. Ajay et al. reported the synthesis and evaluation of two 4-alkylaminoaryl phenyl cyclopropyl methanones derivatives against MTB. The compounds synthesized from 4-fluorochalcones were active against MTB strain H37Rv with MIC values of 3.12–12.5 µg/mL. Interestingly, these compounds were also able to inhibit the growth of Plasmodium falciparum in vitro with IC50 values as low as 0.080 and 0.035 µg/mL and SI values of 4975 and 6948, respectively [19]. Some compounds with MICs < 0.10 µM against MTB have been reported. For example, Ali et al. described a series of substituted phenyl-5,6-dimethoxy-1-oxo2,5-dihydro-1H-2-indenyl-methanone analogs with MICs ranging from 0.1–6.62 µM and an IC50 of 62.5 µg/mL in Vero cells [20]. Of these compounds, 5,6-dimethoxy1-oxo-2,5-dihydro-1H-2-indenyl-4-fluorophenylmethanone was the most promising compound against MTB strain H37Rv and isoniazid-resistant MTB with an MIC of 0.10 µM in both strains (Fig. 2). Structure–activity relationship studies have shown that substitution of phenyl groups with an electron-accepting group, such as 4-fluoro, increases antimycobacterial activity whereas substitution with an electron-donating group decreases antimycobacterial activity. Quinoxaline and quinoxaline, 1,4-di-N-oxide derivatives, display antiviral, anticancer, antibacterial, and antiparasitic effects. Ancizu et al. synthesized and evaluated 36 new 1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid aryl amide derivatives against MTB [23]. After confirming their antimycobacterial properties, the cytotoxicity of each molecule was evaluated using Vero cells. If the SI was ≥ 10, the compound was considered to be active against MTB. Based on the structures of these molecules, the authors concluded that electron-accepting groups (i.e. chloro) in the quinoxaline ring generally increase the antimycobacterial activity of the derivatives, while electro-donating groups reduce antimycobacterial activity (Fig. 3) [23].

Tuberculosis

Organic Compounds to Combat Neglected Tropical Diseases 235 CH3

O

O

O

O CH3 F electron withdrawing groups increase antimycobacterial activity

Figure 2: 5,6-dimethoxy-1-oxo-2,5-dihydro-1H-2-indenyl-4-fluorophenylmethanone is active at a concentration of 0.10 µM in isoniazide-resistant and non-resistant MTB.

Furo[3,2-f]chromene derivatives were reported to inhibit the growth of M. tuberculosis [21], and displayed cytotoxicity against Vero cells despite the absence of mutagenic effects of the test compounds. Cytotoxicity is a common undesirable effect observed in drug discovery that needs to be decreased to obtain safe compounds for clinical use. Some molecular modification strategies, such as the development of prodrugs, have been proposed to decrease the cytotoxicity of these compounds [22]. O Cl

Cl

-

O

O

+

N

NH

+

N

O

-

CH3

Cl

-

O

+

N

NH

+

N

O

CH3

-

Figure 3: Structures of quinoxaline 1,4-di-N-oxide derivatives with MICs of 500 against MTB.

De Logu et al. described the synthesis and antimycobacterial activities of N-aryl phenazine-1-carboxamide derivatives against MTB strain H37Rv and drugresistant ATCC MTB strains [24]. Interestingly, the authors also evaluated the intracellular activity of the test compounds against MTB phagocytosed by macrophages. They reported that the compound [N-(4-bromophenyl)-5,10dihydrophenazine-1-carboxamide] was able to inhibit the growth of MTB strain H37Rv at a concentration of 0.19 µg/mL (Fig. 4). The compound also demonstrated activity against strains resistant to pyrazinamide, streptomycin, and rifampicin. The compound was not cytotoxic. When administered at 5 mg/L, it reduced the growth of mycobacterium phagocytosed in J774A.1 cells by 91.6%.

236 Organic Compounds to Combat Neglected Tropical Diseases

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The authors reported that the N-aryl phenazine-1-carboxamide subunit was the effective pharmacophore, and its MICs ranged from 0.19 to 0.79 mg/L. The authors also suggested that the antibacterial activity was mediated by the inhibition of cellular superoxide dismutase. Variable Subunit pharmacophore subunit

O

NH

Br

N

N

N-aryl or heteroaryl substituents

Figure 4: Structure of N-(3-bromophenyl)-5,10-dihydrophenazine-1-carboxamide, which had an MIC of 0.19 µg/mL against MTB strain H37Rv and had low cytotoxicity.

Our review revealed that quinolone derivatives are the most active antitubercular compounds with MICs < 0.3 µM. Dinakaran et al. synthesized and evaluated the activities of 34 derivatives of 2-(sub)-3-fluoro/nitro-5,12-dihydro-5oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid against MTB strain H37Rv and multidrug-resistant MTB (MDR-MTB) [25]. The MICs of these compounds ranged from 0.18 to 12.82 µM against MTB. The MICs of 2-(3(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo [3,2-a]quinoline-6-carboxylic acid against MTB and MDR-MTB were 0.18 and 0.08 µM, respectively (Fig. 5). In vivo, oral administration of 50 mg/kg body weight of this compound reduced the bacterial load in the lung and spleen to 2.78 and 3.12 log10 protection, respectively [25]. In a similar study, the same research group synthesized 30 derivatives of 9-fluoro-2,3-dihydro-8,10-(mono/di-sub)-3methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (ofloxacin derivatives). These compounds had MICs of 0.18–12.82 against MTB. Of note, the compound 10-[2-carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl]-9-fluoro-2,3dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij] quinolone -6-carboxylic acid had MICs of 0.19 µM and 0.09 µM against MTB and MDR-MTB, respectively. The SI for this compound was 695. In vivo experiments revealed that this compound inhibited mycobacterial growth by decreasing the mycobacterial

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Organic Compounds to Combat Neglected Tropical Diseases 237

load in the lung and spleen by 1.91 and 2.91 log10 protections. The authors suggested that the antitubercular effects of this compound involved inhibition of the supercoiling activity of DNA gyrase. Meanwhile, structure–activity relationship studies revealed that the antimycobacterial activity of this compound was increased by 1.1–8.8 times by introducing a nitro group at position C8 [26] (Fig. 5). electron withdrawing substituent

electron withdrawing substituent

O

O

F

NO2

N

N

anelation

N O

N

O

OH

N

S N

H3C

O

F

OH

N

O

CH3 anelation

HO O

H3C

MIC = 0,19 uM

MIC = 0,39 uM (Dinakaran, Senthilkumar et al., 2008a)

(Dinakaran, Senthilkumar et al., 2008b)

electron withdrawing substituent

O F H3C O

OH

N N

H3C

O

N O

CH3 NO2

MIC = 0,16 uM (Sriram, Yogeeswari et al., 2011)

Figure 5: Structures of fluoroquinolone derivatives with MICs of 0.16–0.39 µM against MTB.

Sriram et al. described the development of several fluoroquinolone derivatives [27]. One of these compounds, namely 1-cyclopropyl-7-(3,5-dimethyl-4-(3-nitropropanoyl)piperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic

238 Organic Compounds to Combat Neglected Tropical Diseases

Santos et al.

acid (Fig. 5), was active in vitro with MICs of 0.16 and 0.04 µM against MTB in the logarithmic growth phase and in starved culture conditions, respectively. Based on the results of docking studies, the authors suggested that this compound inhibits MTB isocitrate lyase, an enzyme involved in the glyoxylate pathway, which incorporates carbon during the growth of microorganisms cultured with acetate or fatty acids as the primary carbon sources [27]. The two most active compounds identified in this review were reported by Senthilkumar et al. in two papers published in 2009 [26, 28]. In the first report, the authors synthesized and evaluated 51 derivatives of 1-(cyclopropyl/2,4difluorophenyl/t-butyl)-1,4-dihydro-6-fluoro-7-(sub secondary amino)-4oxoquinoline-3-carboxylic acid, and evaluated their effects against MTB strain H37Rv and MDR-MTB. The authors found that 7-(3-(diethylcarbamoyl) piperidin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (Fig. 6) was the most active compound in vitro with an MIC of 0.09 µM against both MTB and MDR-MTB. In vivo, oral administration of 50 mg/kg body weight of this compound decreased the mycobacterial load in the lung and spleen tissue to 2.53 and 4.88 log10 protections, respectively. Structure–activity relationship studies demonstrated that the antimycobacterial activity imparted by the N1 substituent was in the order of cyclopropyl > tert-butyl > 4-fluorophenyl. Substitutions at position C7 yielded the following activity order: substituted piperidines > substituted piperazines > fused piperazines and piperidines > (thio)-morpholines. Furthermore, previous studies conducted by Dinakaran et al. [26] showed that substitution with a nitro group increased the antimycobacterial activity. Considering these structure–activity relationships, the authors synthesized and tested the antimycobacterial activity of the compound 7-(4((ben-zo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-1-cyclopropyl-1,4-dihydro6-nitro-4-oxoquinoline-3-carboxylic acid. Of the compounds originally evaluated, this compound showed the greatest activity in vitro with MICs of 0.08 and 0.16 µM against MTB and MDR-MTB, respectively (Fig. 5). The SI of this compound was > 1580. In vivo studies also showed that this compound decrease the bacterial load in the lung and spleen to 2.78 and 4.15 log10 protections, respectively, after administration at a dose of 50 mg/kg body weight [28].

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Organic Compounds to Combat Neglected Tropical Diseases 239

Electron-withdrawing substituent

O

O

O2N

OH N

N

Cl

MIC = 0,09 uM

N N H

cyclopropyl > tert-butyl > 4-flurophenyl

O

piperidines > substituted piperazines > fused piperazines and piperidines > (thio) morpholines (Senthilkumar, Dinakaran, Yogeeswari, China et al., 2009)

O O2N O O

N

O OH

N

MIC = 0,08 uM

N

(Senthilkumar, Dinakaran, Yogeeswari, Sriram et al., 2009)

Figure 6: Structure–activity relationship of piperidine and piperazine quinolone derivatives active against MTB with MICs < 1 µM.

Pyrazinamid is a prodrug that undergoes biotransformation in vivo by the enzyme pyrazinamidase to generate an active metabolite, pyrazinoic acid. Pyrazinoic acid accumulates inside the bacillus at acidic pH, disrupting the membrane potential and interfering with energy production. Pyrazinoic acid also inhibits the enzyme fatty acid synthase-I, which is required for the synthesis of fatty acids in bacteria. Therefore, synthesis and evaluation of novel pyrazinamid analogs is an important strategy to identify candidate drugs that show greater activity than the parent compound. Dolezal et al. synthesized 20 amide derivatives and found that lipophilic substitution of the phenyl group with 3,4-methyl or trifluoromethylphenyl groups, for example, increases antimycobacterial activity (Fig. 7).

240 Organic Compounds to Combat Neglected Tropical Diseases

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However the authors did not test whether the compounds are converted to pyrazinoic acid in vivo [29]. lipophilic substituents O N

O

molecular modification

N

NH2

N

NH

CF 3

N MIC = 6.25 ug/mL

pyrazinamide

Figure 7: Synthesis of pyrazinamide derivatives that are more active against MTB than the parent drug.

Gosh et al. reported several glycosyl thioacetamide derivatives with MICs of 1.56 µg/mL and SI > 10. The authors found that changes in the stereochemistry of the sugar moiety hardly affected the MIC and proposed that sulfonyl acetamide derivatives could mimic the tetrahedral transition state of fatty acid biosynthesis. However, it was also reported that lipophilicity is an important physicochemical property for antitubercular drugs and the authors did not conduct studies to test the important of this property [30]. The compound 2-[4-(1H-[1,2,4]-triazol-1yl)phenyl]-1-(3-fluorobenzyl)-4,6-difluoro-1H-benzo[d]imidazole synthesized by Jadhav et al., and had an MIC of 0.36 µg/mL and SI of 156, which suggested it was an interesting derivative for further studies [31]. Beyond its antimycobacterial activity, the compound was also active in vitro against several bacterial species, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Salmonella typhosa. Structure–activity relationship studies revealed that the presence of a halogen at position 3 in the benzyl subunit increases antibacterial activity (Fig. 8) [31]. The molecular hybridization approach was used by Kakwani et al. to synthesize several cinnamide derivatives. The authors used 1,2-ethylenediamine as the parent compound to design hybrid compounds containing α,β-unsaturated carbonyl moiety (trans-cinnamic). α,β-unsaturated compounds such as trans-cinnamic acid possess antimycobacterial activities and had synergistic effects when administered in

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Organic Compounds to Combat Neglected Tropical Diseases 241

combination with rifampicin. The authors also tested isosteric replacement of the 1,2-ethylenediamine subunit to obtain piperazine, homopiperazine and 2-(piperazin1-yl)ethanamide derivatives. Using this approach, the authors synthesized a hybrid compound with an MIC of 5.1 µM and an SI of 121 (Fig. 9) [32]. F N

N N

N

N

F

MIC = 0,36 ug/mL

F

Electron withdrawn substituents at phenyl subunit

Figure 8: Structure of an imidazole derivative with antitubercular and antibacterial activities.

H3C

O NH

HO

OH NH

NH

NH

R

O

H3C ethambutol

R Isosteric replacement

H N

NH

NH

N

NH N H

O NH

NH O

H2N

MIC = 5,1 uM

Hybrid compound

SI = 121

Figure 9: Use of molecular hybridization to synthesize cinnamide derivatives.

Molecular modification of isoniazid yielded several active compounds with MICs of < 0.77 µM active against MDR-MTB strains (Fig. 10). Interestingly, some modifications in the isoniazid structure yielded compounds that were active against resistant strains but their antimycobacterial activities were weaker than the parent compound [33]. Manjashetty et al. used microwave-assisted one-pot reactions to synthesize several derivatives of isoniazide, including benzaldehydes

242 Organic Compounds to Combat Neglected Tropical Diseases

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and a series of dimedone compounds. One of these compounds,N-[9-[2(benzyloxy)phenyl]-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8,9-octahydro-10 (1H) -acridinyl]isonicotinamide, was a promising candidate with MICs < 0.17 µM against MTB and 0.69 µM against MDR-MTB [34]. The isonicotinoyl hydrazinocarbothioamide derivative 2-isonicotinoyl-N-[2(trifluoromethyl)phenyl]hydrazinecarbothioamide, which had an MIC of 0.58 μM against MTB strain H37Rv and isonicotinoyl hydrazide (INH)-resistant MTB, was synthesized by reacting INHwith substituted phenyl thiocarbamate. This derivative (Fig. 10) was 1.24 and 157 times more active against MTB strain H37Rv and INH-resistant MTB, respectively, than isoniazid. The SI for this compound was > 359 [35]. O NH N

NH2

isoniazide

isoniazid analogs obtained by molecular modification

NH

N

N

N

NH

HO

O

N

H3C

NH

CH3 OH

CF3

MIC: 0,77 uM

MIC: 0,0004 uM

N

N

N

N

N

O

O

O

MIC: 0,56 uM

N

N H

O

N

NH

CF3

O

CH3

N

N

N MIC: 0,25 ug/mL

O

MIC: 0,45 uM

S

NH NH

O

H3C H3C

N

CH3 CH3

HN O

F

MIC < 0,17 uM

N

Figure 10: Structures of isoniazide derivatives that were obtained by molecular modification (adapted from: Manjashetty, Yogeeswari et al., 2011).

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Organic Compounds to Combat Neglected Tropical Diseases 243

The combinatorial chemical approach using libraries containing large numbers of individual compounds and mixtures is an important drug discovery strategy that allows researchers to screen a large number of diverse compounds and has led to the identification of several potential candidate compounds. Using this approach, Nefzi et al. evaluated a series of chiral pentaamines and bis-heterocyclic compounds that achieved inhibitory rates of 90%–100% against MTB strain H37Rv, with MICs ranging from 2 to 7 µg/mL [36]. Singh et al. synthesized α,α’-(EE)-bis(benzylidene)-cycloalkanones with MICs ranging from 6.25 to 1.56 µg/mL and evaluated their antitubercular activities [37]. The authors synthesized the compounds by reacting cycloalkanones with different aromatic aldehydes using ethanolic KOH, with good yields. The EE configuration of diastereoisomers was characterized by nuclear Overhauser effect nuclear magnetic resonance experiments. The authors also have found that more active compounds could be synthesized by substituting the phenyl ring with electronaccepting groups, such as bromo and nitro groups (Fig. 11) [37]. O O NH

NH

NH

S

N

CF3

R

MIC = 0.58 uM

R

R = Br (MIC = 1.56 ug/mL); NO2 (MIC = 3.12 ug/mL)

(Sriram, Yogeeswari et al., 2009)

(Sign, Pandey et al., 2011)

-

O

O O

+

S N

O

N CH3

NH O

N NH

N

O

O

O O CH3

N

N

O sulfonylurea derivative

CH3

MIC = 1.56 ug/mL (MDR-MTB) (Sohn, Lee et al., 2008)

Figure 11: Structures of compound with different MICs values.

MIC = 0.19 uM (Tawari, Bhuva et al., 2010)

244 Organic Compounds to Combat Neglected Tropical Diseases

Santos et al.

Searches for new targets are essential to design new candidate drugs for the treatment of TB. Acetohydroxy acid synthase is an enzyme that catalyzes the conversion of two molecules of pyruvate to 2-acetolactate and CO2, a reaction that is involved in the biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine, which are essential for mycobacterial survival. Sohn et al. evaluated a series of sulfonylurea derivatives capable of inhibiting this enzyme [38]. One sulfonylurea derivative displayed antimycobacterial activity comparable with that of streptomycin, and was also active against intracellular MTB, including drug-resistant MTB strains, with an MIC of 3.1 µg/mL. Several 4-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives that were synthesized by condensation of 5-nitro-2-furaldehyde with various ketones under acidic conditions were found to be potent antitubercular compounds. In particular, (E)-3(5-nitrofuran -2-yl)-1-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one (Fig. 11) had an MIC of 0.19 µM, which was much lower than that of isoniazide (1.8 µM) used as a control. The SI was > 1800. Structure–activity data revealed that the nitrofuran subunit was essential for the antimycobacterial activity of this compound [39]. Our literature search also identified several compounds with MICs of < 1 μM or < 1 μg/mL. Trivedi et al. [40] described the synthesis and pharmacological properties of dihydropyrimidines as potential inhibitors of dihydrofolate reductase (DHFR), a promising drug target for the treatment of mycobacterial infections. DHFR plays an important role in the folate cycle and inhibition of DHFR interrupts the supply of thymidine, which is essential for DNA biosynthesis. Of 30 dihydropyrimidines evaluated in their study, [ethyl 4-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate] and [ethyl 4-[3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl]-6-methyl-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylate] (Fig. 12) were the most active compounds with MICs of 0.02 μg/mL [40]. In 2011, the same group published a similar study in which they substituted the N-pyrimidine ring at position N-1 position. In this study, the authors synthesized the compound diethyl 1-(2-chlorophenyl)-1,4-dihydro-2,6-dimethyl-4-(1,3-diphenyl-1H-pyrazol-4-yl)pyridine-3,5dicarboxylate (Fig. 12), which had an MIC of 0.02 μg/mL [41].

Tuberculosis

Organic Compounds to Combat Neglected Tropical Diseases 245

Quinoxalines and their mono- and di-N-oxide derivatives display a broad range of biological activities, including antiprotozoal and anticancer properties. This is partly due to its chemical structure, which undergoes bioreduction under hypoxic conductions, causing DNA damage. Based on this mechanism of action, Vicente et al. synthesized a series of 3-phenylquinoxaline 1,4-di-N-oxide derivatives and tested their activities against MTB. Interestingly, 34 of 70 compounds tested in that study had an MIC of < 0.2 μg/mL. The most active compounds contained a fluorine substitution at position 7 of quinoxaline or in the para position of the phenyl group (Fig. 12) [42]. The compounds 11-alkoxylated and 11-aminated benzofuro[2,3-b]quinoline showed excellent antitubercular activities with MICs of < 0.2 μM. These compounds were prepared in a single reaction step from anthranilic acid and 2coumaranone in phosphorus oxychloride. The compounds obtained in this step were then reacted with alcohols and amines to yield 11-alkoxylated and 11aminated benzofuro[2,3-b]quinoline derivatives. The compound 11dimethylaminobenzofuro[2,3-b]quinoline (Fig. 11) had an MIC of 0.2 μg/mL and showed low cytotoxicity against Vero cells with an IC50 of > 30 μg/mL [43]. Paul et al. also reported the synthesis and evaluation of bisquinoline derivatives, such as 2-(3-bromophenyl)-6-chloro-3-[4-(6-chloro-4-phenyl-2-quinolyl)phenoxy]-4phenylquinoline, which was very potent with an MIC of 1.1 μM and displayed low cytotoxicity [44]. Vavrikova et al. described the preparation of isoniazid hydrazones, although the compounds were less active than the parent drug [45]. Therefore, the authors used molecular modification methods to increase the antimycobacterial activities of these compounds. They designed several hybrid drugs containing fluoroquinolone or pyrazinamide subunits. These compounds had MICs of < 0.5 μg/mL and high SI values [45]. Dobrikov et al. used molecular modification methods to synthesize 47 ethambutol derivatives with MICs ranging from 0.65 μM to 14.03 µM. Some of these derivatives were more potent than the parent drug. The “duplicated” compound (Fig. 11) had an MIC of 0.65 μM and an SI > 375 [46].

246 Organic Compounds to Combat Neglected Tropical Diseases

Santos et al. R

R N

N N

N

H N

CH3 Cl

O

O

EtOOC

NH O

COOEt

N H3C

H3C

H3C R = F or NO2 (MIC = 0.02 uM)

MIC = 0.02 uM

(Trivedi, Bhuva et al., 2010)

(Trivedi, Dodiya et al., 2011)

-

H3C

O

+

F

N

N

CH3

CN

+

N

N

-

O

F

O

MIC < 0.2 ug/mL (SI > 150) (Yang, Tseng et al., 2010)

MIC = 0.02 ug/mL (SI > 124) (Vicente, Perez-Silanes et al., 2009)

ethambutol derivative subunit

F

O

O

CH3

O

F

OH

OH

NH N O

NH

N

isoniazid derivative subunit

N

NH

CH3

N O quinolone subunit

OH

MIC = 0.5 ug/mL MDR-TB (SI > 1200) (Vavrikova, Polanc et al., 2011)

(Dobrikov, Valcheva et al., 2012)

MIC = 0.65 uM (SI > 375)

Figure 12: Structures of potent antimycobacterial compounds with MICs ≤ 0.0.2 μM.

The tables presented in this review provide an extensive summary of the most promising antitubercular compounds that were obtained in the last 5 years using organic synthesis methods. It is possible to compare the antitubercular activities of each compound, including the MIC against MTB strain H37Rv, IC50 against Vero cells (Table 1) or other cell types (Table 2), and SI.

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Organic Compounds to Combat Neglected Tropical Diseases 247

Table 1: Class compounds of different synthetic organic compounds and the activity against MTB H37Rv, VERO cell and the calculation of selectivity index of their best compound MIC

Class Compounds

Refs.

MW

Best Compound

SI

IC50

μg/m L

μM

μg/mL

μM

IC50/ MIC

1.16

3.12d

62.50e

168.30

54

0.29

0.83d

>47.62

0.78d

1.98

62.50e

S N

[17] H3C H3C

NH2

N

O

371,4 3-(4-fluorophenyl)-6,7-dimethoxy-3a,4-dihydro2 3H-indeno[1,2-c]pyrazole-2-carbothioamide

O F O N

[16] H3C H3C

NH2

N

O

355.3 6

O

3-(4-fluorophenyl)N-(4-chlorophenyl)-6,7-dimethoxy-3a,4dihydro-3H-indeno [1,2-c] pyrazole-2carboxamide

>134.00 e

>161

158.47

80

F CH3 N H3C

[18]

N

NH

N

O

N

NH

4-[(5-[(4-fluorophenylamino]394.4 1,3,4-oxadiazol-2-yl)methylamino]-1,2-dihydro0 1,5-dimethyl-2-phenylpyrazol-3-one

O

F

248 Organic Compounds to Combat Neglected Tropical Diseases MIC

Class Compounds

Refs.

Santos et al.

MW

Best Compound

337.8 0

SI

IC50

μg/m L

μM

μg/mL

μM

IC50/ MIC

(2-(4-Chlorophenyl)cyclopropyl)(4-(furan-2ylmethylamino) phenyl)methanone

3.12d

9.23

256.00e

757.84

82

5,6-dimethoxy-1-oxo-2,5-dihydro1H-2-indenyl-4-fluorophenylmethanone

0.03

0.10d

62.5e

198.85

1988

Dispiropyrrolothiazole derivative

0.21d

0.36

Cl

[19] O O H2N

CH3

O

O [20]

O 314.3 0

O CH3 F N O

O

[47]

S

O N

O NH

F 3C

O

CH3 CH3

583.5 8

>62.50e >107.10 >297

Tuberculosis

Organic Compounds to Combat Neglected Tropical Diseases 249 MIC

Class Compounds

Refs.

MW

Best Compound

364.4 3

(E)-3-(2-(4,5,7,7-Tetramethyl-8,9-dihydro-7Hfuro[3,2-f]chromen-2-yl)vinyl)pyridine

μg/m L

μM

5.00a

13.72

SI

IC50 μg/mL

μM

IC50/ MIC

>274.40

>20

CH3 CH3 O

HN

[21] H3C

O

N

O

CH3

>100.00 e

CH3

7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2>100.00 carboxylic 496 e acid 2-phenylethylamide

[23]

359.8 0

[24]

319.3 3

N-(4-fluorophenyl)-5,10-dihydrophenazine-1carboxamide

0.19d

0.59

[25]

495.5 6

2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2a]quinoline-6-carboxylic acid

0.19

0.39d

e

>626.31

>105 2

64.42

130.00e

333

>200.00

250 Organic Compounds to Combat Neglected Tropical Diseases

Refs.

Class Compounds

Santos et al. MIC

MW

Best Compound

[26]

475.3 8

[29]

SI

IC50

μg/m L

μM

μg/mL

μM

IC50/ MIC

10-[2-carboxy-5,6-dihydroimidazo[1,2a]pyrazin-7(8H)-yl]-9-fluoro-2,3-dihydro-3methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4ij]quinoline-6-carboxylic acid

0.09

0.19d

62.75

132.00e

695

295.2 5

N-(3-trifluoromethylphenyl)pyrazine-2carboxamide

6.25b

21.17 >62.50e >211.68

>10

[30]

479.4 5

glycosyl thioacetamide derivative

1.56d

3.25

>15.60g

>32.54

>10

[31]

405.3 7

2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-(3fluorobenzyl)-4,6-difluoro-1-H-benzo-[d]imidazole

0.36b

0.89

50.00f

123.34

139

Tuberculosis

Refs.

[32]

[34]

[33]

Organic Compounds to Combat Neglected Tropical Diseases 251 Class Compounds

MIC MW

Best Compound

320.3 8

(2E,2'E)-N,N'-ethane-1,2-diylbis(3-phenylprop2-enamide)

μg/m L

μM

μg/mL

μM

IC50/ MIC

1.63

5.10a

198.00

618.00e

121

N-[9-[2-(benzyloxy)phenyl]-3,3,6,6-tetramethyl1,8-dioxo-2,3,4,5,6,7,8,9-octahydro-10(1H)77.82

>108.00 e

>158.00 e

>635

>64

252 Organic Compounds to Combat Neglected Tropical Diseases MIC

Class Compounds

Refs.

Santos et al.

MW

Best Compound

SI

IC50

μg/m L

μM

μg/mL

μM

IC50/ MIC

(4S)-4-benzyl-1-((25)-1-(2-(((S)-5-benzyl-2645.9 iminoimidazolidin-1-yl)methyl)pyrrolidin-1-yl)2 3-phenylpropan-2-yl)-3(cicloheptylmethyl)imidazolidin-2-imino

3.90

6.04

39.48

61.12

10

[48]

1-cyclopropyl-7-(8-(4-methoxybenzyl)577.6 3,4,5,6,7,8-hexahydroisoquinolim-2-(1H)-yl-1,40 dihydro-6-fluoro-8-methoxy-5-nitro-4oxoquinoline-3-carboxylic acid

0.09

0.16d

62.38

108.00e

675

[49]

532.0 0

0.05

0.09d

>77.67

>146.00 >155 e 3

HN

[36]

N

N

N

NH N

NH

Quinolone derivative

Tuberculosis

Organic Compounds to Combat Neglected Tropical Diseases 253 Class Compounds

Refs.

MIC MW

Best Compound

7-(4-((benzo[ 492.4 d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-18 cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinoline3-carboxylic acid

[28]

H2N

SI

IC50

μg/m L

μM

μg/mL

μM

IC50/ MIC

0.04

0.08d

62.54

127.00e

1563

3.12d

7.23

31.12g

72.12

10

3.136.31g

7.63- ≥300.00 ≥731.00 g 15.37

N N

Naphthalen-2-yl-9-naphthalen-2-ylmethylene6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2ylamine

[37]

431.5 1

[38]

4-(cyclopropylcarbonyl)-N-[(4,6410.4 dimethoxypyrimidin-2-yl)carbamoyl]-1-methyl1H-pyrazole-5-sulfonamide

≥96 or ≥48

254 Organic Compounds to Combat Neglected Tropical Diseases

Refs.

[35]

Class Compounds

Santos et al. MIC

MW

Best Compound

2-(pyridin-4-ylcarbonyl)-N-[2356.2 (trifluoromethoxy)phenyl]hydrazinecarbothioam 2 ide

1-cyclopropyl-7-(3,5-dimethyl-4-(3nitropropanoyl) piperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4dihydroquinoline-3-carboxylic acid

SI

IC50

IC50/ MIC

μg/m L

μM

μg/mL

0.21

0.58d

>208.00

0.08

0.16d

62.29

127.00e

779

e

μM

>583.90 >990

[27]

490.4 8

[39]

326.3 4

(2E)-3-(5-nitrofuran -2-yl)-1-[4-(piperidin-1yl)phenyl]prop-2-en-1-one

0.06

0.19a

113.89

349.00e

1837

[40]

419.4 5

ethyl 4-[3-(4-fluorophenyl)-1-phenyl1H-pyrazol-4-yl]-6-methyl-2-oxo-1,2,3,4tetrahydropyrimidine-5 carboxylate

0.02b

0.05

>10.00e

>23.84

>500

256 Organic Compounds to Combat Neglected Tropical Diseases

Refs.

Class Compounds

Santos et al. MIC

MW

Best Compound

[51]

4’-[5-(4-fluorophermyl)pyridin-3-yl]-1-methyl502.5 dispiro-[indan-2,2- pyrrolidine-3,2-indan]-1,3,13 ´trione

[43]

262.3 0

1,1-dimethylaminobenzofuro-[2,3-b]-quinoline

(a) REMA; (b) MABA; (c) BACTEC; (d) Agar dilution method (NCCLS); (e) MTT; (f) Neutral Red; (g) Other.

SI

IC50

μg/m L

μM

μg/mL

0.06

0.12d

>62.50e >124.37

μM

IC50/ MIC

>103 6

114.37 >150

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Organic Compounds to Combat Neglected Tropical Diseases 257

Table 2: Class compounds of different synthetic organic compounds and the activity against MTB H37Rv, cytotoxicity to cells others than VERO and the calculation of selectivity index of their best compound Refs.

Class Compounds

MW

MIC Best Compound

IC50

SI

μg/mL

μM

μg/mL

μM

IC50/ MIC

3.25

>15.60g

>32.54

>10

J774 Macrophage cell line from mouse 479.45

glycosyl thioacetamide derivative

1.56d

222.31

2-acetylpyridine N4(ethyl) thiosemicarbazone

3.13ª

14.80ª 625.00ª

2811.00ª

190

[53]

380.37

4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N(pyrimidin-2-yl)benzenesulfonamide

3.90a

10.25 312.50a

821.57

80

[37]

431.51

Naphthalen-2-yl-9-naphthalen-2-ylmethylene6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2ylamine

3.12d

7.23

31.12g

72.12

10

[30]

[52]

N

S N

NH

NH

CH3

CH3

H2N

N N

258 Organic Compounds to Combat Neglected Tropical Diseases Refs.

Class Compounds

MW

Santos et al. MIC Best Compound

IC50

SI

μg/mL

μM

μg/mL

μM

IC50/ MIC

56.13

162.00g

20

HepG2 hepato cell line from human liver carcinoma [54]

OH

N HO

346.51

1-({1-[3-(4-hydroxyphenyl)-1methylpropyl]piperidin-4-yl}methyl)piperidin-4-ol

2.70

7.80d

482.45

1-Cyclopropyl-6-fluoro-4-oxo-7-{4-[4(fluorobenzoyl)hydrazonomethyl] piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic acid

1.00g

2.07

>617.54 >1280.00e >618

25.00

N CH3

[45]

PBMC peripheral blood mononuclear cells from human [55]

320.42

6-Isopentyl-1,2,3,4-tetrahydrobenzo[a]phenazin5(7H)-one

>78.20

>8

Tuberculosis

Refs.

Organic Compounds to Combat Neglected Tropical Diseases 255 Class Compounds

MIC MW

Best Compound

[41]

506.0 0

[50]

[42]

SI

IC50

μg/m L

μM

μg/mL

μM

IC50/ MIC

Diethyl 1-(2-chloro-phenyl)-1,4-dihydro-2,6dimethyl-4-(1,3-diphenyl-1h-pyrazol-4-yl) pyridine-3,5-dicarboxylote

0.02b

0.04

>10.00e

>19.76

>500

566.6 2

3-Benzyl-2-[4-fluoro-2-(1-imidazol-1-yl-ethyl)phenoxy]-6-(4-phenyl-[1,2,3]triazol-1-yl)quinoline

3.13c

5.52

>176.48

32

295.2 6

7-methyl-3-(4’-fluoro)phenylquinoxaline-2carbonitrile 1,4-di-N-oxide

338.68

>500

>100.00 g