Chapter 7 Bioactive flavaglines: Synthesis and pharmacology Christine Basmadjian,1,2 Qian Zhao,1,2 Armand de Gramont,2,3 Maria Serova,2,3 Sandrine Faivre, 3 Eric Raymond,3 Stephan Vagner,4 Caroline Robert,4 Canan G. Nebigil,5 Laurent Désaubry1* 1 Therapeutic Innovation Laboratory (UMR 7200), CNRS/University of Strasbourg, Faculty of Pharmacy, 67401 Illkirch cedex, France 2 AAREC Filia Research, 1 place Paul Verlaine, 92100 Boulogne-Billancourt, France 3 Departments of Medical Oncology, Beaujon University Hospital, INSERM U728/AP- HP, 92110 Clichy, France 4 Gustave Roussy Institute, INSERM U981 and Department of Dermato-Oncology, 94805 Villejuif, France 5 Biotechnology and Cell Signaling Laboratory (UMR7242), CNRS/University of Strasbourg, 67412 Illkirch cedex, France * To whom correspondence should be addressed. Phone: 33-368-854-141. Fax: 33-368-854-310. E-mail: [email protected] Running title: Bioactive flavaglines Abstract 1 The flavaglines represent a family of more than 100 cyclopenta[b]benzofurans that are found in medicinal plants of the genus Aglaia in South-East Asia. These compounds display potent anti-inflammatory, neuroprotective, cardioprotective and above all, anticancer activities. The most amazing feature of the flavaglines is their ability to kill cancer cells without affecting normal cells. Additionally, flavaglines protect neurons and cardiac cells from many types of stresses. Such a selective cytotoxicity to cancer cells and cytoprotection to normal cells, which occur both at nanomolar concentrations, is unprecedented. This unique pharmacological profile of activity begins to be rationalized with the recent discovery of their molecular targets, the scaffold proteins prohibitins and the translation initiation factor eIF4A. This chapter aims to describe the synthetic routes to flavaglines, their mechanism of action, the evaluation of their biological potency and ongoing effort to provide novel therapeutic agents. Key words: cancer; cytoprotection; prohibitins, eIF4A, protein synthesis, rohinitib, HSF1, TXNIP. Contents 1. Introduction 2. Biosynthetic aspects 3. Synthesis of flavaglines 3.1 Chemical syntheses 3.2 Biomimetic synthesis 3.3 Synthesis of silvestrol 4. Pharmacological properties of flavaglines 4.1 Anticancer activity 4.2. Anti-inflammatory and immunosupressant activities 4.3 Cytoprotective activity 4.4 Antimalarial activities 5. Structure-activity relationships 6. Concluding remarks Abbreviations References 1. Introduction 2 A recent survey of the first-in class medicines approved over the last decades indicates that a quarter of them are natural products derivatives [1]. This report clearly demonstrates the importance of these compounds to develop new drugs. Indeed, natural compounds display certain notable advantages compared to fully synthetic drugs [2]. Firstly, natural products are secondary metabolites that were selected by evolution to act as chemical weapons or signaling molecules, with the ability to reach their receptor in the targeted organism. As such, they often have the ability to cross biological membranes. Many of them are suspected to be substrate of membrane transporters. This is an important issue, because natural compounds that are identified based on in vitro pharmacological assays, are often also active in vivo. Secondly, natural products have in general more chiral centers, more varied ring systems, a higher ratio of Csp3/Csp2, less nitrogen and more oxygen atoms than fully synthetic drugs. This structural complexity provides excellent opportunities to explore new areas of the chemical space and to generate original, therefore patentable, compounds. Although natural products had traditionally been invaluable as a source of medicines, the development of new natural compounds in oncology was interrupted for over ten years with the advent of targeted therapies [2]. After the approval of Topotecan in 1996, the development of other natural products was essentially stopped because of the nearly exclusive focus on targeted therapies by the pharmaceutical industries. However, because targeted therapies did not fulfill all their expectations, natural product derivatives return to the front stage, which has been manifested by the approval of fourteen of these compounds between 2007 and 2013 in oncology [2]. Since the initial report of rocaglamide (1) by King and coll. in 1992 [3], there has been growing interest from chemists and biologists on this unique class of cyclopenta[b]benzofurans called flavaglines (or sometimes rocaglamides or rocaglates). Figure 1 offers a glimpse of certain significant natural and synthetic flavaglines. 3 Figure 1: Representative natural (1-6) and synthetic (7-13) flavaglines The flavaglines are a family of more than 100 cyclopenta[b]benzofurans found in Asian plants of the genus Aglaia (Meliaceae). These compounds display potent insecticidal, antifungal, anti-inflammatory, neuroprotective, cardioprotective and above all anticancer activities. Their most intriguing feature is the selectivity of their cytotoxicity toward cancer cells. Indeed, as far as we know, all cancer cell lines and transformed cell lines are sensitive to this cytotoxicity, while primary cell cultures of non-cancerous cells are not affected. This selective cytotoxicity was first described by Marian and coll. [4]. It has also been observed 4 that flavaglines promote the survival of neurons and cardiac cells toward many types of stresses. This unique feature is not rationalized with our current state of knowledge. It seems that these compounds target a feature that is consubstantial to the nature of the cancer itself. The chemistry and pharmacology of flavaglines has been the object of several reviews [5- 9]. Over the last year, we observed acceleration in the pharmacological investigation of the flavaglines, marked in particular by the identification of their molecular targets. The purpose of this article is to highlight the most recent advances on these exciting anticancer agents, with a special emphasis on their mode of action. 2. Biosynthetic aspects Along with flavaglines, aglaforbesins 18 and aglains 19 are also characteristic metabolites of the genus Aglaia (Scheme 1). The term “flavagline” proposed by Harald Greger from the University of Vienna, originally covered these three groups of compounds [10], but over time it has tended to solely refer to cyclopenta[b]benzofurans due to their distinctive pharmacological activities. These three families of secondary metabolites display the same patterns of substitution and stereochemical relationships. Proksch was the first to propose a biosynthetic pathway that begins with the condensation of hydroxyflavone (14) with a cinnamic amide (15) to afford an aglain (16) that may undergo an α-ketol rearrangement to yield a flavagline 17 [11]. Reduction of intermediary ketones, possibly by NADPH, would generate the diols 18, 19 and 20. Aglaforbesins are also probably generated by the same process, but with an addition of the cinnamic amide on the hydroxyflavone that occurs with the opposite orientation. 5 Scheme 1. Hypothetical biosynthesis of flavaglines and related aglaforbesins and aglains 3. Synthesis of flavaglines 3.1 Chemical syntheses The first total synthesis of a flavagline, rocaglamide (1), was described in 1990 by Trost et al. [12]. Since then, multiple laboratories were attracted by this challenge due to the complexity of this structure characterized by two contiguous quaternary chiral centers and two adjacent aryl groups in cis-orientation on the cyclopentane ring (Scheme 2). 6 Scheme 2: Trost’s enantioselective synthesis of rocaglamide [12] Trost’s approach relies on the enantioselective [3+2]-cycloaddition of a trimethylenemethane derivatives generated from 22 with the oxazepinedione 21 (Scheme 2). Subsequent transformations, including the condensation of 3,5-dimethoxyphenol to cyclopentanone 23, gave access to the flavaglines skeleton but with an incorrect configuration compared to the natural product. Additional six steps afforded rocaglamide (1) with the desired stereochemistry via dehydro-intermediate 25. Despite these long and multiple steps, this synthesis remains the only enantiospecific one to date. Since Trost’s synthesis, more than ten syntheses of flavaglines have been described. In 1992, Taylor et al. developed a racemic synthesis of rocaglamide which was later improved by Dobler’s group in 2001 [13,14]. Taylor’s strategy begins with a Hoesch reaction between cyanohydrin 26 and phloroglucinol to afford benzofuranone 27 (Scheme 3) [13]. Aldehyde 28, obtained by a Michael addition, was converted to the cyclopentanone 29 after cyclisation and Swern oxidation. Silylation followed by enolate formation, sequential addition of carbon 7 disulfide and iodomethane, and treatment with sodium methoxide gave β-keto ester 30 which was converted to rocaglamide (1) in the nest two steps. Scheme 3: Taylor’s synthesis of rocaglamide (1) [13] Dobler and colleagues modified the previous strategy using cyanohydrin 31 in an umpolung reaction to generate after deprotection the cyclopentanone 29 (Scheme 4) [14]. This ketone was treated with Stiles’ reagent to give the ester 30 which was then transformed into rocaglamide (1) in three steps. Scheme 4: Dobler’s racemic synthesis of rocaglamide [14] In 2008, Qin and his group further modified Taylor’s synthesis by introducing a methoxycarbonyl to the Michael acceptor, therefore circumventing Stiles carboxylation [15]. 8 Condensation of benzofuranone 27 with the dimethyl 2-benzylidenemalonate