First Evidence That Oligopyridines, Î±-Helix ... - ACS Publications

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First Evidence That Oligopyridines, α‑Helix Foldamers, Inhibit Mcl‑1 and Sensitize Ovarian Carcinoma Cells to Bcl‑xL‑Targeting Strategies Céline Gloaguen,†,‡,§,∞ Anne Sophie Voisin-Chiret,*,†,∥ Jana Sopkova-de Oliveira Santos,†,∥ Jade Fogha,†,∥ Fabien Gautier,⊥,# Marcella De Giorgi,†,∥ Grégory Burzicki,†,∥ Serge Perato,†,∥ Cécile Pétigny-Lechartier,†,‡,§,∞ Karin Simonin-Le Jeune,†,‡,§,∞ Emilie Brotin,†,‡,§,∞ Didier Goux,†,∇ Monique N’Diaye,†,‡,§,∞ Bernard Lambert,‡,¶,∞ Marie-Hélène Louis,†,‡,§,∞ Laetitia Ligat,○,◆ Frédéric Lopez,○,◆ Philippe Juin,⊥,# Ronan Bureau,†,∥ Sylvain Rault,†,∥ and Laurent Poulain*,†,‡,§,∞ †

Normandie Université, 14032 Caen, France UNICAEN, BioTICLA Unit (Biologie et Thérapies Innovantes des Cancers Localement Agressifs), EA4656, 14032 Caen, France § Comprehensive Cancer Center François Baclesse, 14076 Caen, France ∞ Inserm, U1199 “Biologie et Thérapies Innovantes des Cancers localement Agressifs”, 14076 Caen, France ∥ UNICAEN, CERMN (Centre d’Etudes et de Recherche sur le Médicament de Normandie) - FR CNRS INC3M, 14032 Caen, France ⊥ Team 8, Nantes-Angers Centre for Cancer Research, UMR 892 Inserm 6299 CNRS, 44007 Nantes, France # Institut de Cancérologie de l’Ouest, Comprehensive Cancer Center René Gauducheau, 44805 Nantes, France ∇ UNICAEN, Centre de Microscopie Appliquée à la Biologie, IBFA, SF4206 ICORE, 14032 Caen, France ¶ CNRS (placed at the disposition of EA4656 by CNRS, Délégation régionale Ile-de-France Est), 94532 Thiais cedex, France ○ INSERM UMR1037-Plateforme Protéomique-Pôle Technologique du Centre de Recherches en Cancérologie de Toulouse, 2 avenue Hubert Curien, 31100 Toulouse, France ◆ Université Toulouse III-Paul Sabatier, UMR1037 CRCT, 31000 Toulouse, France ‡

S Supporting Information *

ABSTRACT: Apoptosis control defects such as the deregulation of Bcl-2 family member expression are frequently involved in chemoresistance. In ovarian carcinoma, we previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect cancer cells against apoptosis and their concomitant inhibition leads to massive apoptosis even in the absence of chemotherapy. Whereas Bcl-xL inhibitors are now available, Mcl-1 inhibition, required to sensitize cells to Bcl-xL-targeting strategies, remains problematic. In this context, we designed and synthesized oligopyridines potentially targeting the Mcl-1 hydrophobic pocket, evaluated their capacity to inhibit Mcl-1 in live cells, and implemented a functional screening assay to evaluate their ability to sensitize ovarian carcinoma cells to Bcl-xL-targeting strategies. We established structure−activity relationships and focused our attention on MR29072, named Pyridoclax. Surface plasmon resonance assay demonstrated that pyridoclax directly binds to Mcl-1. Without cytotoxic activity when administered as a single agent, pyridoclax induced apoptosis in combination with Bcl-xL-targeting siRNA or with ABT-737 in ovarian, lung, and mesothelioma cancer cells.

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INTRODUCTION

initiate apoptosis by either blocking the activity of antiapoptotic members or directly activating multidomain pro-apoptotic members, which is mediated via interaction of the BH3 domain of one protein with the hydrophobic pocket of another.2 Constant efforts are made to impede the activity of antiapoptotic members such as Bcl-2 or Bcl-xL, among which the development of potent BH3-mimetic molecules represent a promising way.3,4 These molecules bind to the BH3-binding

Apoptosis control defects are frequently involved in chemoresistance in both hematological malignancies and solid tumors, and the deregulation of Bcl-2 family member expression constitutes one of the most frequent and important events. These proteins share Bcl-2 homology domains (named BH domains). Antiapoptotic proteins (Bcl-2, Bcl-xL, etc.) contain the BH1 to BH4 domains, whereas pro-apoptotic proteins contain either the BH1 to BH3 domains (multidomain members such as Bax and Bak) or only the BH3 domain (BH3-only group such as Bad, Bid, Noxa, Puma, and Hrk).1 Under cellular stress, BH3-only proteins © XXXX American Chemical Society

Received: April 30, 2014

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DOI: 10.1021/jm500672y J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry

4g34 compounds or more specific and more active such as marinopyrrole A (maritoclax)35 or MIM1 compound.36 NMRbased screening of a large fragment library also identified a chemically distinct hit series that binds to different sites on Mcl-1.37,38 Very recently, Nikolovska-Coleska et al.39 disclosed a novel series of small-molecule Mcl-1 inhibitors as sulfonamides, discovered through high-throughput screening followed by the utilization of structure-based design. Few data are yet available concerning these molecules, and none of these Mcl-1 inhibitors have entered clinical trials, but their discovery provides a new basis for the development of future Mcl-1 inhibiting molecules. The BH3 domains correspond to an α helix. Mimicking of the BH3 domain could thus be realized by foldamers bearing substituents as recognition motifs able to interact with the hydrophobic pocket of the targeted antiapoptotic protein. By computational modeling, we thus designed small compounds based on a pyridyl scaffold, abiotic foldamers40,41 that potentially target the Mcl-1 hydrophobic binding pocket. These molecules were synthesized, and we evaluated their capacity to physically interact with Mcl-1 in cellulo (BRET assay) and designed a screening assay in order to evaluate their ability to sensitize ovarian carcinoma cells to Bcl-xL targeting strategies. Here, we present the results of these investigations and establish the first structure−activity relationships among 18 of these molecules. We focused our interest in the most promising of them, MR29072, which we named pyridoclax.

groove in antiapoptotic proteins of the Bcl-2 family and promote cell death through the release of pro-apoptotic Bcl-2 family members.5 ABT-737 (and its orally available derivative ABT-263, navitoclax, currently under phase II clinical investigations) allows efficient inhibition of the antiapoptotic activity of Bcl-2 and Bcl-xL.6,7 It has been shown to be able to induce apoptotic cell death as a single agent in hematologic malignancies8−10 and, to a lesser extent, in solid tumor cells.11−13 ABT-737 can also sensitize cancer cells to chemotherapy.6,8,14 However, its activity has been conditioned to the absence or inactivation of Mcl-1,15,16 whereas the strong expression and activity of Mcl-1 is associated with the absence of response to ABT-737.15,17,18 The expression and activity of the antiapoptotic protein Mcl-1 thus constitutes a major hurdle for the activity of ABT-737. Mcl-1 is overexpressed in ovarian carcinoma, and its expression has also been associated with chemoresistance.19 We previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect tumor cells against apoptosis, and that their concomitant inhibition leads to massive apoptosis even in the absence of chemotherapy, whereas the down-regulation of either Bcl-xL or Mcl-1 remains ineffective.20 In this context, we also showed that Mcl-1 down regulation or inactivation was required to sensitize ovarian cancer cells to Bcl-xL-targeting BH3-mimetic molecules such as HA14-121 or ABT-737.22 Importantly, the MCL1 locus is one of the most frequently amplified in human cancers, further pointing to its centrality in carcinogenesis and increasingly its importance as a high priority therapeutic target.23 Many strategies have been developed to inhibit Mcl-1 expression or to impede its activity through the activation of its endogenous inhibitors (i.e., BH3-only proteins such as Bim, Noxa, or Puma), in order to restore ABT-737 sensitivity. These strategies include the use of Mcl-1-targeting siRNA,24 Noxa gene transfer,13,25 or signaling pathway inhibition.26,27 As we previously demonstrated, platinum compound-based chemotherapy is also able to decrease Mcl-1 protein levels as well as to induce BH3only proteins in ovarian carcinoma, leading to a sensitization to ABT-737.21,22 However, the difficult application of such strategies in clinical practice, in part due to cumulative toxicities (conventional chemotherapies) or to in vivo inefficiency (siRNA, gene therapy), incites researchers to identify specific and potent Mcl-1 inhibitors. Like other antiapoptotic Bcl-2 proteins, Mcl-1 is able to sequester an α-helical fold but differs in the exposed surface of its binding groove. A number of residues in the binding groove differentiate Mcl-1 from its homologues,28 and its groove appears more electropositive than other antiapoptotic proteins (Bcl-xL’s groove is almost completely uncharged).29 Moreover, crystallographic comparative analysis of the hydrophobic grooves of Mcl-1 and Bcl-xL suggested that whereas the groove of Bcl-xL is extremely flexible allowing the interaction with various BH3 ligands, the groove of Mcl-1 is conformationally rigid.30 Such structural differences have strong impact on the development of specific Mcl-1 inhibitors and are probably responsible for the difficulties for the scientific community to design such inhibitors. The recent discovery of two BH3 peptides, one (BimS2A) constituted by a Bim variant31 and the other constituted by the BH3 domain of Mcl-1,32 both able to specifically inhibit Mcl-1 function through a Noxa-like mechanism, established a proof of concept that such specific inhibitors could be designed. Other approaches such as fragment nuclear magnetic resonance-based methods, screening structure−activity relationships, and library screening or stapled peptide-base competitive screening led to the discovery of some synthetic compounds.7 These molecules are either poorly specific such as the S133 or

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RESULTS Computational Modeling. BH3-only proteins, Mcl-1 natural inhibitors, anchor in the Mcl-1 cavity principally by hydrophobic residues situated on the contact face of the BH3only helix, that is, by the residues at positions i, i + 4, i + 7, and i + 11, and by an aspartic acid residue (i + 9) on the opposite side. This aspartic acid interacts with Arg263 of Mcl-1, which is fully conserved among the antiapoptotic proteins. Previously, our laboratory was interested in the synthesis42−46 and characterization of oligopyridyl abiotic foldamers, potential α-helical mimetics.40,41 Results of our studies have confirmed that these compounds are able to mimic selected side chain positions of an α helix. Among those oligopyridyl foldamers, MR29004 consisted of four pyridine rings substituted by methyl group on the ortho position of the two middle rings (Figure 1A). The preferential twist angles between the two substituted rings were predicted between 30° and 60° with a small energy barrier (3 kcal/mol), which can easily be comparable to the angle between side chains at positions i + 4 and i + 7 of a polyalanyl α helix (60°). Moreover, we observed that its two methyl substituents mimic well side chains at the desired positions, i + 4 and i + 7.40 In this study, starting from the modulation of MR29004, a potential Mcl-1 inhibitor (pyridoclax) was designed by conserving the MR29004 methyl substituent on the second ring and replacing the methyl group on the third ring by a bulkier hydrophobic substituent (a styryl substituent; Figure 1A), drawing inspiration from Noxa, a selective Mcl-1 ligand, which has a phenylalanine in this position, not conserved among all BH3-only proteins. Ab initio simulations and solved X-ray structure confirmed that despite introduction of the styryl group, pyridoclax has conserved the preferential twist angle values between successive rings, compared with its inferior analogue (Table 1). Recently, Fesik and collaborators have identified a small molecule (6-chloro-3-[3-(4-chloro-3,5-dimethylphenoxy)propyl]-1H-indole-2-carboxylic acid) with a significant binding affinity to Mcl-1 (Ki = 55 nM). The co-crystal structure of this B


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Figure 1. Computational modeling of pyridoclax. (A) ORTEP representation of MR29004 and pyridoclax MR29072 X-ray structures (left) and superposition of a polyalanyl α helix (in blue) and X-ray structure (in purple) of MR29004 and pyridoclax MR29072 (right). (B) Representative docking poses of pyridoclax in hMcl-1 (PDB ID 2KBW47). (C) The minimized average complex from MR29072/hMcl-1 dynamic study. The protein is presented in gray as surface (left) and as ribbon (right) and pyridoclax as stick.

compound bound to Mcl-1 (PDB ID 4HW248) showed that it occupies two hydrophobic pockets in Mcl-1 (corresponding to the hydrophobic pocket for i + 4 and i + 7), thus forming interactions with Met231, Thr266, Val253, Leu267, Phe270, Phe228, and Met250. The ligand carboxylic acid moiety of the compound interacts with Arg263. To predict potential pyridoclax/Mcl-1 interactions, docking studies were performed on human Mcl-1 protein (PDB ID 2KBW47). Different docking protocols were tested: pyridoclax docked with relatively good fitness scores, and it positioned well into the hydrophobic groove of Mcl-1. Two principals orientations emerged from the docking studies (Figure 1B). We observed that the styryl group of pyridoclax is always anchored in

the same Mcl-1 pocket in all poses even if the orientations of pyridoclax in the binding cavity changed. It establishes π−π interactions with Phe228 and His224 and a hydrophobic interaction with Met231 of Mcl-1. The methyl group points to different pockets or it is exposed on the surface in different poses. Moreover, one of the pyridines at the pyridoclax extremity was always close to Arg263 of Mcl-1. At the other extremity, the pyridine ring is located on a Mcl-1 pocket near Phe270. Interestingly, Mcl-1 residues involved in pyridoclax fixation are close to one observed in the Fesik’s inhibitor/Mcl-1 complex. The molecular dynamics simulation of 1 ns carried out on the two selected poses (Figure 1B) showed that pyridoclax fluctuated strongly in the binding cavity for the first pose in contrast to the C


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Table 1. Predicted Minimum Energy Conformers Obtained from PES at HF/2-31G Level between the Neighboring Rings

In order to realize a coupling reaction selectively at the α position of the nitrogen atom, compound 5 could be engaged in a halogen−halogen exchange reaction52 with sodium iodide and acetyl chloride in acetonitrile under microwave irradiation at 100 °C for 1 h with the aim to introduce an iodine atom in position α to nitrogen atom (compound 6) (the choice of one or another pathway depends on the product desired and on the selectivity of the het(aryl)boronic acid used in successive SMC reactions). The iodine compound 6 was engaged in a SMC reaction with the 6-bromo-5-methylpyridin-3-ylboronic acid (7) to afford selectively compound 8 in 86% yield. Finally, compound 8 is engaged in a SMC reaction by increasing the number of equivalents of boronic acid, which allows the expected derivative to be obtained in one step: MR29072 was therefore obtained in six steps with an overall yield of 39%. Surface Plasmon Resonance. We used surface plasmon resonance (SPR) technology to verify the direct interaction between Mcl-1 and pyridoclax and determine affinity constant (Kd). SPR experiments were performed in three independent conditions. With a first batch of pyridoclax, specificity of the interaction was evaluated in comparison with binding of pyridoclax to immobilized Bcl-xL. Binding studies were realized in duplicate with either 1000 RU (not shown) or 5000 RU of immobilized proteins. We thus verify that no interaction occurs between the antiapoptotic protein Bcl-xL and pyridoclax (Figure 2A). The sensorgram in Figure 2A shows a dose-dependent interaction between His-Mcl-1 coated on the BIAcore sensor chip (5000 RU) and pyridoclax used as an analyte. Specific binding kinetics parameters of pyridoclax to the immobilized His-Mcl-1 determine affinities between pyridoclax and the His-tagged Mcl-1 protein of 1.55 and 3.6 nM over 5000 and 1000 RU of

second one, which stays stable along whole dynamic. For this second pose, interactions observed in the docking study were maintained along the whole dynamic (Figure 1C). Chemistry. Recently, we have developed the Garlanding concept,46 which allows building a long linear chain from one ring by the implementation of iterative cross-coupling reactions between boronic species and dihalogenated compounds. This regioselective, flexible, highly reproducible, and efficient methodology permitted us to obtain oligopyridyl,43,44 oligophenylpyridyl,49,50 and oligothienylpyridyl foldamers.51 This Garlanding approach was applied to synthesize an original series of molecules belonging to the oligopyridine family that are of interest as Mcl-1 inhibitors. Pyridoclax (MR29072) was the first in the series to be synthesized according to the sequence shown in Scheme 1. In a second step, a family of new derivatives was prepared to define the first structure−activity relationships. In order to introduce the styryl group on the pyridine ring, a Suzuki−Miyaura cross-coupling (SMC) reaction between a trihalopyridine and the commercially available trans-2-phenylvinyl boronic acid was used. The 5-bromo-3-iodo-2-hydroxypyridine, 2, was prepared from the 5-bromo-2-hydroxypyridine, 1, by an iodination reaction with N-iodosuccinimide (NIS) in acetonitrile under reflux during 4 h. Then, the 5-bromo-3-iodo-2hydroxypyridine (2) underwent a chlorodehydroxylation reaction using dichlorophenylphosphonic acid at 160 °C for 3 h. The obtained compound 3 was then engaged in a SMC reaction with the trans-2-phenylvinyl boronic acid (4), commercially available, to obtain compound 5 in 93% yield. Compound 5 could be engaged directly in a first SMC reaction that will occurs on the 5 position rather than on the 2 position due to the reactivity of halogens toward SMC reaction (I > Br > Cl). D


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Journal of Medicinal Chemistry Scheme 1. Chemical Procedure To Synthesize MR29072 (Pyridoclax)a

a Reagents and conditions: (a) NIS (1.1 equiv), CH3CN, rfx, 4 h; (b) PhPOCl2, 160 °C, 4 h; (c) Pd(PPh3)4 (0.05 equiv), Na2CO3 (2.5 equiv), 1,4-dioxane, rfx, 24 h; (d) NaI (10 equiv), CH3COCl (1.5 equiv), CH3CN, 100 °C, 1 h, C = 0.25 M, microwave; (e) Pd(PPh3)4 (0.05 equiv), K3PO4 (2.5 equiv), DME, rfx, 20 h; (f) Pd(PPh3)4 (0.1 equiv), Na2CO3 (5 equiv), 1,4-dioxane, rfx, 24 h.

Figure 2. Real-time binding of pyridoclax and MR29004 on immobilized His fusion proteins. (A) Low mass weight-single cycle kinetics analysis was simultaneously performed on immobilized His fusion Mcl-1 (5000 RU) and His fusion Bcl-xL (5000 RU) with five injections of analyte at 1, 2, 4, 8, and 16 nM. Analyte injections lasted for 120 s each and were separated by 10 s dissociation phases. An extended dissociation period of 10 min followed the last injection. The pyridoclax sensorgram recorded for Mcl-1 was fitted globally to a 1:1 interaction (black curves). (B) Low mass weight-single cycle kinetics analysis was simultaneously performed on immobilized His fusion Mcl-1 (3500 RU) and His fusion Bcl-xL (3500 RU) with five injections of analytes (pyridoclax and MR29004) at 70, 140, 280, 560, and 1120 nM. Analyte injections lasted for 120 s each and were separated by 10 s dissociation phases. An extended dissociation period of 10 min followed the last injection. The MR29004 sensorgram was not fitted. Each sensorgram represents a differential response where response on an empty reference channel (A) or Bcl-xL-coated reference channel (B) has been subtracted and is expressed in RU as a function of time in seconds.

Pyridoclax Disrupts Mcl-1/Bim Interaction. Bioluminescence resonance energy transfer (BRET) technology was used to monitor the effect of pyridoclax on the binding properties of Mcl-1 within live cells. This technique allows us to detect and quantify specific protein−protein interactions by measuring energy transfer between a donor luciferase fusion protein and an acceptor YFP fusion protein. After activation by coelenterazine H, luciferase can promote emission of light from YFP provided that donor and acceptor are less than 10 nm apart. We measured interaction between luciferase fused BimL and YFP-fused Mcl-1.

immobilized Mcl-1, respectively, while affinity for Bcl-xL could not be determined. A second set of experiments was performed with a second batch of pyridoclax. With immobilized Bcl-xL used as reference surface for nonspecific binding measurements, a differential interaction about 10 RU was observed with pyridoclax, whereas no interaction occurs with MR29004 (Figure 2B). In these conditions, a calculated Kd of 25 nM (see Table 2) confirmed an affinity of binding in the nanomolar range. Binding parameters from three kinetic experiments are summarized in Table 2. E


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condensed or fragmented nuclei were detected (Figure 4A,B,C) in these conditions. In contrast, their combination led to massive cell death, as demonstrated by strong cell detachment, by the appearance of a strong sub-G1 peak on the DNA content histogram (over 50%), and by the appearance of a 60% fraction of annexin V positive cells (Figure 4A). It should be noted that this fraction is mainly double positive; that could be due to the time of analysis, since we studied annexin V/PI staining after a 24 h exposure, whereas apoptosis induction is observable as soon as 2 h after the beginning of the exposure, as demonstrated in Figure 4E. It should therefore be considered that these double-positive cells have undergone postapoptotic necrosis. Moreover, the viability evaluation showed that this combination led to a drastic decrease of the number of viable cells and to a concomitant increase of dead cell number (Figure 4B), and western blot showed a complete cleavage of PARP and caspase 3 (Figure 4C). DAPI staining and electron microscopy showed that this combination led to nuclear condensations and fragmentations highly evocative of apoptotic cell death (Figure 4D). We showed that these effects were optimal after exposure to 25 μM pyridoclax but were also observed to a lower extent in response to 10 μM (data not shown). The kinetic study of the effect of this combination showed that apoptosis was observed as soon as 2 to 4 h after the beginning of the exposure (37% of events in sub-G1 fraction after 4 h), and this observation is compatible with a pharmacologic Mcl-1 inhibition through BH3-mimetic activity (Figure 4E). Altogether, these elements show that pyridoclax strongly sensitizes ovarian cancer chemoresistant IGROV1-R10 cells to Bcl-xL targeting siRNA, their combination leading to massive apoptosis. Pyridoclax Sensitizes Various Cancer Cell Types to Bcl-xL Targeting siRNA. We therefore studied the effect of the combination of MR29072 with siXL1 in other ovarian carcinoma cell lines (Figure 5A), as well as in other cancer cell types (Figure 5B). We observed a similar response to this combination in all ovarian carcinoma cell lines, as well as in lung carcinoma (A549) and mesothelioma (MSTO-211H) cell lines. Otherwise, we used another ovarian cancer cell line, A2780, in which Mcl-1 expression is higher than in other ones, whereas Bcl-xL expression is clearly lower than in other cell lines (Figure 5C). As demonstrated by siRNA-mediated Mcl-1 silencing, this cell line is dependent on Mcl-1 for its survival since we observed apoptosis induction after transfection of Mcl-1-targeting siRNA

Table 2. Rate Constants and Affinities for Binding of Pyridoclax on Immobilized His-Mcl-1 Fusion Proteins immobilized ligand (RU) MCl-1 (1000 RU)

analyte

pyridoclax (batch 1) MCl-1 (5000 RU) pyridoclax (batch 1) MCl-1 (3500 RU) with pyridoclax (batch 2) subtracted Bcl-xL

ka, M−1 s−1

kd, s−1

Kd, nM

7.6 × 10

2.7 × 10−2

3.6

4.89 × 106

7.5 × 10−2

1.55

5

4.79 × 10

2

1.23 × 10

−5

25

We first ensured the specificity of the interaction evaluated by BRET by carrying out a donor saturating assay (Figure 3A) where the BRET signal is measured using a constant amount of donor and an increasing amount of acceptor. A saturation BRET level is clearly achieved for the untreated condition, thus attesting the specificity of the BRET signal, whereas pyridoclax treatment leads to a much flattened curve, underscoring the inhibitory effect of the compound. Histograms in Figure 3B show the influence of pyridoclax treatment on BimL/Mcl-1 interaction when measured under conditions that roughly yield 50% of the maximal BRET signal corresponding to the relative affinity between the two proteins. It clearly indicates that pyridoclax is able to disrupt BimL/Mcl-1 interaction in whole living cells. This experiment has been performed with 10, 25, and 50 μM in a second cell line, MCF7. It showed a concentration-dependent effect, beginning at 10 μM and reaching a maximum intensity at 25 μM since 50 μM did not increase the observed effect (Supplementary Data 1, Supporting Information). Effect of Pyridoclax as Single Agent or Combined with siRNA-Mediated Bcl-xL Inhibition. To demonstrate the interest of pyridoclax as a Mcl-1 inhibitor, we used a model of selective dependence on both Bcl-xL and Mcl-1 in which Bcl-xL expression was silenced by RNA interference 48 h before exposure. We used the ovarian carcinoma cell line IGROV1-R10, since we previously demonstrated that this cell line was highly sensitive to the concomitant inhibition of Bcl-xL and Mcl-120 but remained viable when only one of these targets was inhibited. As expected, neither 24 h of pyridoclax treatment alone nor the Bcl-xL targeting siRNA (siXL1) alone induced massive cell death on their own. We observed a slowed proliferation but neither cell detachment, strong sub-G1 peak, caspase 3 activation, nor

Figure 3. Effect of pyridoclax in whole living cells using BRET assay. (A) Donor saturation assay. Saturation curves were measured in Hela cells transiently cotransfected with increasing acceptor/donor (peYFP-Mcl-1/pRluc-BimL) ratio as described in Experimental Procedures and untreated or treated with pyridoclax. (B) Effect of pyridoclax treatment on a single ratio acceptor/donor (yielding 50% of the maximal BRET signal) after a 16 h exposure at 50 μM. F


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Figure 4. Effect of pyridoclax on IGROV1-R10 ovarian cancer cells, alone or associated with a Bcl-xL targeting siRNA (siXL1). Cells were transfected with 10 nM siRNA for 48 h before a 24 h (A, B, C, D) or 2, 4, or 6 h (E) exposure to 25 μM pyridoclax. (A) Cellular morphology, DNA content histograms, and annexin V/propidium iodide biparametric histograms. (B) Trypan blue exclusion assay. (C) Bcl-xL expression and PARP and caspase 3 cleavage assessed by western blot. (D) Nuclear morphology studied after DAPI staining (top) and cellular morphology studied by electron microscopy. (E) Short time effects of the combination pyridoclax/siXL1 (assessed 2, 4, and 6 h after the beginning of the exposure to pyridoclax). Cellular morphology and DNA content histograms (left panel) and PARP and caspase 3 cleavage assessed by western blot (right panel).

it should be noticed that the more active concentration of pyridoclax was then 50 μM, probably also due to the high level of Mcl-1 expression. Pyridoclax Sensitizes Chemoresistant Ovarian Cancer Cells to ABT-737. Because ABT-737 is one of the most potent Bcl-xL inhibiting BH3-mimetic molecules and because the response of ovarian cancer cells to ABT-737 is conditioned by the inhibition of Mcl-1, we evaluated the effect of the

in a concentration dependent manner. It should be noticed that we had to use a more elevated concentration of siRNA (30 nM) to efficiently silence Mcl-1 in this cell line compared with that for SKOV3 or IGROV1 cells, for instance (10−20 nM), probably due to the very high level of Mcl-1 expression in A2780 compared with these two other cell lines. As expected, in this Mcl-1-dependent cell line, the exposure to pyridoclax as single agent induced massive cell death, although G


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Figure 5. Effect of pyridoclax alone or combined with a Bcl-xL targeting siRNA (siXL1) on ovarian, lung, and mesothelial cancer cell lines. Cells were transfected with 10 nM siRNA for 48 h before a 24 h exposure to 25 μM pyridoclax. Cell detachment and sub-G1 proportion on DNA histograms were assessed by the observation of cellular morphology and by flow cytometry, respectively, in ovarian carcinoma cells (A) and lung or mesothelioma cancer cells (B). (C) expression of Bcl-xL and Mcl-1 in a panel of ovarian cancer cell lines. Note the strong ratio of Mcl-1/Bcl-xL expression in A2780 cells. (D and E) Effects of Mcl-1-targeting siRNA (siMCL1) and pyridoclax, respectively, on apoptotic cell death in Mcl-1-dependent ovarian cancer cell line A2780. H


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Figure 6. Effect of the combination of pyridoclax with ABT-737 on chemoresistant ovarian cancer cells IGROV1-R10 (top) and SKOV3 (bottom). Cells were concomitantly (A, E) or sequentially (B) exposed to 25 μM pyridoclax and 5 μM ABT-737, and cellular effects were assessed either in real-time (A, B, E) or after 24 h (C, D, F). (A, B, E) Real-time cellular activity assessed by impedancemetry (xCELLigence technology), after concomitant exposure (A, E) or sequential exposure (B; 24 h pyridoclax, then ABT-737). (C) Cellular morphology and DNA content histograms after a 24 h concomitant exposure. (D, F) PARP and caspase 3 cleavages after a 24 h concomitant exposure.

IGROV1-R10 and SKOV3 chemoresistant ovarian cancer cell lines. Indeed, when the two molecules were combined, cellular activity was drastically decreased in both cell lines as assessed by impedancemetry (xCELLigence technology) (Figure 6A,F).

combination of ABT-737 with pyridoclax (Figure 6). As described previously with siXL1, we observed that neither ABT-737 nor pyridoclax induced cell death as single agents, whereas their combination led to massive apoptotic cell death in both I


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The number of viable cells (right panel) was not strongly affected, and caspase 3 remained globally uncleaved in this condition. Therefore, it could be concluded that, although pyridoclax is able to induce ER stress, this event is not responsible for the observed apoptotic cell death when it is associated with Bcl-xL inhibiting strategies, thus clearly differentiating pyridoclax from numerous other presumed Mcl-1 inhibitors.

We also observed a strong cell detachment and an important sub-G1 fraction on DNA content histograms (Figure 6C), as well as complete PARP and caspase 3 cleavages after 24 h treatment (Figure 6D,F, right panel). It should be noticed that these effects were similar in concomitant exposure experiments and in sequential exposure (pyridoclax 24 h, then ABT-737; Figure 6A,B). Moreover, apoptosis was observed as soon as the cells were exposed to pyridoclax and ABT-737, arguing in favor of a BH3-mimetic activity. Pyridoclax Induces ER Stress and Modifies Bcl-2 Family Expression, but Its Cytotoxic Effect Does Not Require These Events. Since numerous presumed Mcl-1 inhibitors have been demonstrated to exert their effect through an ER stressmediated Noxa induction, we investigated whether pyridoclax was able to induce ER stress and subsequent BH3-only transcriptional activation. Our results showed that pyridoclax induced an increase of Mcl-1, Bim, and Noxa expression (Figure 7A), through a mechanism at least in part transcriptional that mimics the effect induced by the ER stress inducer thapsigargin (Figure 7B). Indeed, Mcl-1 and Bim mRNA increased about 1.2-fold and Noxa mRNA about 3.5-fold after a 24 h exposure to pyridoclax as well as to thapsigargin. We also observed a time-dependent increase of Noxa protein expression from 2 to 24 h (Figure 7C). Since it has been previously shown that ER stress could be responsible for such transcriptional activation of Bcl-2 family members, we assessed CHOP and HSP70 expression by western blot and showed that ER stress was strongly induced after a 24 h exposure to pyridoclax (Figure 7D). However, it should be noticed that when pyridoclax is combined with siXL1, neither CHOP nor HSP70 appeared induced, suggesting that apoptosis occurred before ER stress. Moreover, as shown on Figure 4, we observed apoptotic cell death as soon as 2 h after the beginning of the exposure, whereas Noxa induction is not strong yet (Figure 7C). The immunoprecipitation experiments performed after 6 h showed that ABT-737 released Bax, Bak, and Bim from Bcl-xL to be resequestered by Mcl-1. Pyridoclax did not modify Bax and Bak sequestration by Bcl-xL but increased Bim binding to Bcl-xL. In contrast, it drastically decreased the amount of Bim bound to Mcl-1. Whole lysate observation showed that Noxa expression was induced, as previously observed, and immunoprecipitation showed that it was associated with Mcl-1 (Figure 7E). As expected, when ABT-737 and pyridoclax were associated, Bim was then released from both Bcl-xL and Mcl-1, in agreement with Bim/Mcl-1 BRET experiments evoked previously. In order to study whether Noxa induction is required for pyridoclax-induced Mcl-1 inactivation and subsequent cell death when Bcl-xL is inhibited (siXL1 or ABT-737), we silenced Noxa expression through interference RNA strategy (siNOXA). We showed that siNOXA was able to abrogate the pyridoclaxinduced Noxa expression but was not able to protect IGROV1R10 cells from apoptosis induced by the combination of pyridoclax/siXL1 (Figure 7F). Similar results were obtained with the combination pyridoclax/ABT-737 (data not shown). Finally, we used thapsigargin to induce ER stress and observed its effect on the response to a Bcl-xL-inhibiting strategy (siXL1, ABT-737). A 24 h exposure to thapsigargin induced ER stress as shown by CHOP induction, strongly induced Noxa expression, but only poorly sensitized cells to ABT-737 (Figure 7G) compared with pyridoclax. Indeed, whereas in response to the combination pyridoclax/ABT-737 cells were completely detached, presented nearly only condensed and fragmented nuclei (left panel), and showed a completely cleaved caspase3 (middle panel), they remained mainly attached and mainly presented normal nuclei in response to the combination thapsigargin/ABT-737.

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QSAR We have demonstrated above that pyridoclax induces cell death through Mcl-1 inhibition (pro-apoptotic activity). In order to determine pyridoclax essential chemical functions for Mcl-1 inhibition property, we synthesized various derivatives presented in Schemes 2 and 3. Concerning the preparation of pyridoclax analogues, either they were synthesized using 2.5 equiv of corresponding (het)aryl boronic species in order to produce analogues with identical ends (pathway A, MR31321, MR31336, MR31322, MR31350, MR31361, MR30814, MR30846, MR30847, and MR30854) or they were prepared with two different (het)aryl boronic species in order to produce analogues with different ends (pathway B, MR31348 and MR31349; pathway C MR30849 and MR30850). The choice of the synthesis pathway was determined on the basis of the reactivity of different boronic species. In both cases, an iterative procedure as described for MR29072 was used. MR29071 was synthesized using the same procedure as described previously from the 5-bromo-2-iodo-3-(E)-styrylpyridine (6) and the 6-bromo-pyridin-3-yl boronic acid (12) to afford the intermediate dibromostyrylbipyridine (13) (pathway D). Another procedure was used to produce MR30820. First, we prepare in an excellent yield 5-bromo-2-(pyridin-3-yl)benzaldehyde (19) according to a SMC reaction from 2,5dibromobenzaldehyde (17) and 3-pyridyl boronic acid (18). The selectivity of the reaction is due to the presence of the aldehyde function, which allows orientation of the coupling reaction only toward the formation of the C−C bond in the ortho position. Then, the SMC reaction between compound 19 and boronic acid 16 gave the desired aldehyde 20. Finally, a Horner− Wadsworth−Emmons reaction was realized using aldehyde 20 in the presence of 1 equiv of diethyl benzylphosphonate and 2 equiv of potassium hydroxide in refluxing tetrahydrofuran during 19 h. We successfully obtained 2-(pyridin-3-yl)-5-(3-methyl-4-pyridin3-ylphenyl)-(E)-styrylphenyl as MR30820 in 77% yield. We evaluated the ability of these analogues to sensitize IGROV1-R10 cells to siRNA-mediated Bcl-xL silencing (Figure 8). The inhibition activities of derivative compounds were then compared with pyridoclax to evaluate the importance of individual pyridoclax chemical groups. We observed that some modifications did not affect the activity, whereas others decreased or abolished it completely. In the middle of the molecule, pyridoclax has a bipyridine core substituted by a methyl and a styryl group in the 5 position. The styryl presence in the 5 position seems to be crucial for the pro-apoptotic activity: when styryl was replaced by hydrogen (MR2900642) or methyl (MR29004), we did not observe proapototic activity. When the styryl group was moved to the 4 position (MR30820), apoptotic activity was reduced, even if a similar sub-G1 fraction has been observed. Indeed, despite comparable proportions of sub-G1 fraction in DNA content histograms after exposure to pyridoclax and MR30820, both FS/FL3 biparametric histograms (size/DNA content) and cellular and nuclear morphologies showed that the extent of cell death was quite different. It was nearly complete after exposure to pyridoclax, J


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Figure 7. Effect of pyridoclax on Bcl-2 family expression, ER stress, and association of these events with apoptosis in response to Bcl-xL targeting strategies. Cells were exposed to 25 μM pyridoclax, and Mcl-1, Bcl-xL, Bim, and Noxa expression were assessed at protein level after 24 h in response to pyridoclax (A) and at the mRNA level in response to pyridoclax and to the ER stress inducer thapsigargin (500 nM) (B). (C) Induction of Noxa expression by pyridoclax after 2, 4, 6, and 24 h exposure. (D) ER stress induction was assessed by the study of CHOP and HSP70 induction by western blot. (E) Immunoprecipitation of Bcl-xL and Mcl-1. Cells were exposed 24 h to ABT-737 and then 6 h to pyridoclax, and the binding of Bim, Noxa, Bax, and Bak to Bcl-xL and Mcl-1 was studied after immunoprecipitation (left panels). The expression of these proteins in whole lysate is presented on the right panel. (F) Bcl-xL and Noxa expression were silenced for 48 h using specific siRNA (siXL1 and siNOXA) and cells were then exposed to 25 μM pyridoclax. Cellular morphology and DNA content histogram (left panel), as well as PARP and caspase 3 cleavage (right panel), were studied after 24 h. G The effect of thapsigargin-induced ER stress on ABT-737 cytotoxicity was assessed by the study of cellular and nuclear morphology, DNA content histograms, trypan blue exclusion test, and caspase 3 cleavage after 24 h. CHOP and Noxa protein expression was studied by western blot. K


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Journal of Medicinal Chemistry Scheme 2. Chemical Procedure To Synthesize MR29072 and Its Analoguesa

a

With identical ends when blue color (het)aryl boronic species were used and with different ends when purple, red, magenta, or green (het)aryl boronic species were used. Reagents and conditions: (a) NIS (1.1 equiv), CH3CN, rfx, 4 h; (b) PhPOCl2, 160 °C, 4 h; (c) Pd(PPh3)4 (0.05 equiv), Na2CO3 (2.5 equiv), 1,4-dioxane, rfx, 24 h; (d) NaI (10 equiv), CH3COCl (1.5 equiv), CH3CN, 100 °C, 1 h, C = 0.25 M, microwave; (e) Pd(PPh3)4 (0.05 equiv), K3PO4 (2.5 equiv), DME, rfx, 20 h; (f) Pd(PPh3)4 (0.1 equiv), Na2CO3 (5 equiv), 1,4-dioxane, rfx, 24 h.

MR30814, lacking the two N-terminal nitrogens, does not present good activity. From these data, we hypothesized that the pyridine nitrogens play a particular role in the pyridoclax activity. Because the docking studies proposed that one terminal nitrogen is situated in the proximity of Arg263, we synthesized derivatives with only one terminal pyridine and we also varied the nitrogen

whereas it was moderate after exposure to MR30820 (Supplementary Data 2, Supporting Information). The methyl presence on the other middle ring seems necessary to the pro-apoptotic activity: the methyl absence (MR29071) abolished completely the activity. Terminal groups of pyridoclax are pyridines, each with nitrogen at the meta position of the junction link. The derivative L


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Journal of Medicinal Chemistry Scheme 3. Chemical Procedure To Synthesize MR30820a

a Reagents and conditions: (a) Pd(PPh3)4 (0.05 equiv), Na2CO3 (2.5 equiv), 1,4-dioxane, rfx, 4 h; (b) Pd(PPh3)4 (0.05 equiv), K3PO4 (2.5 equiv), DME/H2O, rfx, 20 h; (c) PhCH2P(O)(OEt)2 (1 equiv), KOH (2 equiv), THF, rfx, 19 h.

This approach to design compounds distributing their substituents at the key interacting side chain positions of the BH3 helix mimetic, was already applied for example to search for a Bak mimetic.53 Since these works, numerous abiotic foldamers with side chains mimicking peripheral functionalities of proteic α-helices have been published in the literature. For example, Hamilton et al. described a series of terphenyl scaffolds presenting 3,2′,2″ alkyl or aryl substituents on the three ortho positions of the phenyl rings in a spatial orientation that mimics the i, i + 3, or i + 4 and i + 7 residues on a native α helix.54 Fluorescence polarization assays showed that less hydrophobic terphenyls present lower affinity, emphasizing the importance of hydrophobic interactions for the binding to the recognition cleft.54−58 From a predictive computational study, Che et al. have proposed that a pyridyl-based scaffold should be more flexible in the α-helix mimicking regions with respect to a phenyl scaffold and that it should be superior as a helical surface mimetic.59 Subsequently, Hamilton and co-workers have synthesized terpyridyl scaffolds using sequential Bohlmann−Rahtz hetero annulation reactions with low yields. Recently, we have developed the Garlanding concept,46 which allows us to overcome the long and difficult synthesis of terpyridyl used by Hamilton and co-workers. This approach allows building of a long linear chain from one ring by the implementation of iterative SMC reactions between boronic species and dihalogenated compounds. It was applied to synthesize the oligopyridine family that is of interest as Mcl-1 inhibitors. Among these compounds, we were interested in MR29004, the pyridoclax inferior analogue, with a scaffold consisting of four pyridine rings substituted by methyl on the two central rings. We observed that methyl substituents can overlap helix side chains at the position i + 4 and i + 7 of an α helix. The comparative sequence analysis of the BH3 domain showed that the leucine residue in position i + 4 is fully conserved in all pro-apoptotic proteins. Next, pyridoclax was designed by replacing one methyl group of MR29004, by a more bulky hydrophobic substituent (a styryl), drawing inspiration from Noxa. Noxa, a selective Mcl-1 ligand, alone has a phenylalanine in position i + 7. Despite the introduction of a styryl in the inferior analogue, we predicted that pyridoclax has a conformation (twist angles) close to its inferior analogue and thus the substituents of pyridoclax can still mimic i + 4 and i + 7 α helix residues.

position in pyridines. Indeed, the absence of the nitrogen in just one terminal ring (MR31348 and MR31349) does not greatly affect the activity. In conclusion, pyridyl nitrogen on one terminal ring at least is necessary for good pro-apoptotic activity. We also observed that addition of a second nitrogen on the terminal ring (pyrimidine ring) in MR31361 or a switch of the nitrogen position (meta to para) in MR31350, decreased activity. Finally terminal pyridyl or phenyl rings were replaced by a smaller one, thienyl ring. For those derivatives, we observed a lack of activity; only MR31336 presents a reduced activity due to introduction of the methyl substituents on the thienyl rings. Similarly to the data presented for MR30820, sub-G1 fraction was not, on its own, representative of the extent of cell death, and other parameters such as the intensity of PARP cleavage, cell detachment, or nuclear condensations and fragmentation should be taken into account, as shown in the table presented in Supplementary Data 3, Supporting Information.

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DISCUSSION Mcl-1 is frequently amplified in human cancers and has been designed as a high priority therapeutic target in oncology.23 The cooperation between Bcl-2-like proteins and Mcl-1 to protect cancer cells has been well documented. In ovarian carcinoma, we clearly showed that Bcl-xL inhibition is highly cytotoxic provided that Mcl-1 is also inhibited.20 However, whereas great hopes are placed in the Bcl-xL or Bcl-2 inhibitors (ABT-263, ABT-199) that present some activity in hematological and solid cancers,8,13 Mcl-1 inhibitors are dramatically lacking. The development of such Mcl-1 inhibiting molecules thus constitutes a major challenge for the success of Bcl-xL targeting strategies in numerous malignancies. The solved structures of pro-apoptotic/antiapoptotic complexes showed that the complex formation is ensured by a BH3 amphiphatic helix, which lies in the hydrophobic groove formed by BH1, BH2, and BH3 domains of the second partner. The mimicking of the BH3 amphiphatic helix could thus be realized by foldamers bearing substituents (key recognition motifs) able to interact with hydrophobic pocket of the targeted antiapoptotic protein. Systematically, the BH3 helix face in contact with the antiapoptotic groove consists of hydrophobic residues corresponding to the face of the α helix, that is, the positions i, i + 4, i + 7, and i + 11. M


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Figure 8. Structure−activity relationships. Various oligopyridines derivatives from pyridoclax were synthesized, and their activity was evaluated as previously described for pyridoclax, that is, after silencing of Bcl-xL in IGROV1-R10. The cells were transfected with siRNA for 48 h and then exposed for 24 h to pyridoclax derivatives. Cellular morphology and DNA content histograms are presented for each molecule. Molecules are classified considering the chemical function that has been modified compared with MR29072 (pyridoclax).

Our docking studies have suggested that pro-apoptotic activity of pyridoclax can be due to direct pyridoclax/Mcl-1 interaction. The observed poses have been divided into two groups, the first

group with pyridoclax positions relative 180° to positions of the second one. According to the docking poses (Figure 1B), the styryl aromatic ring was situated in both groups in the same N


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Mcl-1 does not lead to cytotoxicity in the presence of pyridoclax 50 μM (data not shown), whereas it would have been the case if pyridoclax could act as a Bcl-xL inhibitor. Moreover, it could be argued that if pyridoclax would be a pan-inhibitor, it should induce cell death as single agent, which is not the case. These elements suggest that pyridoclax presents a relative selectivity for Mcl-1. We used surface plasmon resonance (SPR) assays to demonstrate that pyridoclax was able to directly and selectively interact with Mcl-1 (versus Bcl-xL). The results show a direct interaction between His-Mcl-1 coated on the BIAcore sensor chip with pyridoclax used as an analyte with a dose-dependent binding and an affinity about KD = 1.55 nM with 5000 RU of immobilized proteins. The same technology was used to verify that no interaction occurs with the antiapoptotic protein Bcl-xL. Moreover, using MR29004 as analyte in the same conditions, no interaction with Mcl-1 was recorded. Some BH3 mimetic molecules (ABT-737, HA14-1, Gossypol, 2-methoxy-Antimycin A3, S1) previously demonstrated either activity as a single agent or a sensitizing effect to ABT-737. However, it has been shown that only ABT-737 was able to inhibit Bcl-230 (for review) and that the other molecules acted through an ER-stress mediated induction of Noxa (via ATF3 and ATF4 induction) and subsequent Mcl-1 inhibition.63 One of them (Gossypol) also appears able to inhibit proteasome activity that consequently induces ER stress.63 Moreover, ABT-737 itself has been described to induce ER stress,64 even if the opposite has also been described.63 Relationships between ER stress and Bcl-2 family are complex because ER stress can lead to BH3-only transcriptional activation,65 and Bcl-2 family is known to modulate positively or negatively the unfolded protein response.66 Noxa and Mcl-1 induction have been also observed in response to other ER stress inducers, thapsigargine67 and celestratol,68 the latter study also showing that this mechanism sensitizes cells to ABT-737. Here we also observed a strong induction of Noxa after a 24 h exposure to pyridoclax, as well as a moderate induction of Mcl-1 and Bim, both at mRNA and protein level. Similar events were observed in our cell lines in response to the ER stress inducer thapsigargin, suggesting that they are at least in part due to ER stress induction. Moreover, since surface plasmon resonance experiments (recently introduced in the manuscript in response to the reviewers) suggest that Mcl-1/pyridoclax is difficult to disrupt, it could also be hypothesized that pyridoclax binding to Mcl-1 leads to the stabilization of the protein itself, thus leading to its accumulation. However, we studied the possibility that the observed effect would be nonspecific, due to the activation of ER stress previously described in response to numerous other BH3mimetic molecules.63 We observed that pyridoclax induced ER stress after 24 h. However, some elements argued in favor of an ER stress independent mechanism. Indeed, ER stress did not seem to occur when pyridoclax was combined with siXL1 or ABT-737, suggesting that rapid and massive apoptosis induced by these combinations did not allow the induction of ER stress. It could thus be hypothesized that ER stress that occurs in response to pyridoclax as a single agent cannot occur when it is combined with the inhibition of Bcl-xL, due to apoptosis induction. We also showed that the induction of Noxa was weak during the six first hours in response to pyridoclax alone and was quite absent when it was combined with siXL1 or ABT-737, arguing in favor of a Noxa independent mechanisms. This was verified by the use of a Noxa targeting siRNA. Noxa silencing did not affect the response to pyridoclax, thus

Mcl-1 cavity independently of scaffold orientation, and it stacked with Phe228. Docking studies have also shown that another type of pyridoclax/Mcl-1 interaction is present in the complex. One of the ending nitrogen rings was close to the Mcl-1 Arg263. This result is interesting according to previous structural studies. Indeed a solvent-exposed aspartic acid, conserved in all BH3only proteins, is crucial for the BH3-only/antiapoptotic complex formation. This aspartic acid forms a salt bridge with an arginine, fully conserved among all the antiapoptotic proteins (Arg263 in human Mcl-1). Moreover, in the recently published X-ray structure of an Mcl-1 inhibitor (6-chloro-3-[3-(4-chloro-3,5dimethylphenoxy)propyl]-1H-indole-2-carboxylic acid) complexed with hMcl-1, a hydrogen bond was established between the inhibitor carboxyl group and the nitrogen of Arg263.60,60 So this predicted that electrostatic interaction could be important for the complex formation pyridoclax/Mcl-1. Thus, modeling studies suggested that the pyridoclax could have good chemical properties to interact with Mcl-1 protein. To study the capacity of pyridoclax to disrupt Bim/Mcl-1 in live cells, we used a method that allows us to monitor in-cell interactions between one transiently transfected luciferase tagged protein (Bim in this work) and a second, yellow fluorescent tagged protein (Mcl-1 in this work) by bioluminescence resonance energy transfer (BRET). This method provides robust and reliable signals. It allows direct study of the effects of oligopyridines added in the culture medium on intracellular interactions. Since it has been shown that Bcl-2 proteins change their conformation after insertion into the cell membranes, as reviewed in ref 61, BRET assay constitutes a pertinent assay to study protein−protein interaction between these proteins under native conditions compared with other screening assays performed using soluble fragments of the target protein. BRET assay, previously used to study the disruption of the interaction Bax/Bcl-xL by polyphenol derivatives,62 demonstrated the capacity of pyridoclax to dissociate Mcl-1 from Bim in whole living cells. We further investigated the ability of pyridoclax to sensitize cancers cells to Bcl-xL inhibition strategies. We previously demonstrated the dependence of ovarian cancer cells to both Bcl-xL and Mcl-120 and the ability of Mcl-1 inhibition to sensitize chemoresistant ovarian cancer cells to Bcl-xL inhibiting strategies.21,22 These cell lines therefore constitute pertinent models for the screening of libraries of molecules with BH3mimetic potential. In particular, the IGROV1-R10 cell line demonstrated strong sensitivity to the concomitant inhibition of Bcl-xL and Mcl-1, whereas the inhibition of these proteins separately does not induce cytotoxic effects. Here we showed that pyridoclax did not induce apoptosis as a single agent in IGROV1-R10 cells but presented a great ability to sensitize these cells to Bcl-xL-targeting siRNA, as well as to ABT-737. This was true in ovarian cancer cells, as well as in lung cancer and mesothelioma cells. Other Mcl-1 inhibitors have been shown to be able, as a single agent, to kill leukemia cells dependent on Mcl-1, particularly MIM1.36 However, in our study, the cell lines used were not sensitive to the siRNAmediated inhibition of Mcl-1 alone, as presented previously.20,22 This observation is in agreement with the absence of effect of pyridoclax alone. In contrast, the exposure of the A2780 cell line (which is dependent on Mcl-1 for its survival) to pyridoclax as single agent led to massive cell death, as was also the case in response to Mcl-1 siRNA-mediated silencing. Interestingly, we also evaluated the association of pyridoclax with a Mcl-1-targeting siRNA (siMCL1), in order to evaluate the possible inhibition of Bcl-xL by pyridoclax. siRNA silencing of O


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inhibitors. Although further in vivo preclinical studies are now required, our study demonstrated the potential interest of these innovative molecules to sensitize ovarian cancer cells to Bcl-xL targeting strategies. Considering the dramatic situation of patients with tumors resistant to conventional chemotherapy, this opens interesting perspectives concerning the use of pyridoclax and its derivatives in combination with Bcl-xL targeting molecules such as the ABT-737-derived orally available molecule navitoclax.

demonstrating that ER stress mediated Noxa induction was not responsible for apoptotic cell death in response to pyridoclax/ siXL1 or ABT-737 combination. In contrast, the activity of obatoclax (described as pan-inhibitor of Bcl-2 like proteins, including Mcl-169) is in part inhibited when Noxa expression is silenced by siRNA strategy.70 Moreover, the use of the ER stress inducer thapsigargin and its combination with ABT-737 was drastically less efficient to induce cell death compared with the combination pyridoclax/ABT-737, despite the strong induction of Noxa, arguing in the same way. We also observed a decrease in Mcl-1 expression when pyridoclax was associated with a Bcl-xL targeting strategy, when a massive apoptotic cell death is observed. It is known that Mcl-1 could be degraded by caspases, thus explaining why, after a 24 h exposure, Mcl-1 appeared to be decreased. However, in both cell lines, pyridoclax strongly induced Mcl-1 expression, at least in part through transcriptional activation at least in part due to ER stress. When pyridoclax is associated with Bcl-xL inhibition (siRNA or ABT-737), this increase does not occur (neither ER stress) since the cells are rapidly orientated toward apoptosis. The level of Mcl-1 expression is thus comparable to the level observed in control cells when pyridoclax is combined with siXL1 and more clearly reduced when pyridoclax is combined with ABT-737 (apoptosis is in this case induced more quickly, and activated caspase 3 level appears to be more intense). The observed absence of Mcl-1 expression increase is thus probably the results of combined mechanisms, such as the absence of ER stress induction, the rapid orientation toward apoptosis, and Mcl-1 degradation by proteasome and caspases. The QSAR studies allowed us to highlight the importance of individual pyridoclax chemical functions. The methyl and styryl hydrophobic motifs situated both at 5 position are necessary for the pro-apoptotic activity, and the docking results showed that these two groups anchor in the Mcl-1 hydrophobic pockets. The nitrogen present on terminal pyridines also plays an important role. The absence of the nitrogen on both terminal rings abolished the pyridoclax pro-apoptotic activity. Our simulations showed that the absence of nitrogen on the terminal ring does not influence the global conformation of the compound with respect to the pyridoclax. From this data, we hypothesized that nitrogen itself could be involved in the binding site recognition. Indeed, the docking studies showed that one of the ending pyridine nitrogens, in both orientations observed in docking, is situated in the proximity of Mcl-1 Arg263 and an electrostatic interaction could occur. Therefore, a series of derivatives with only one terminal pyridine and various nitrogen positions in the pyridine were synthesized and evaluated. In conclusion, only one N-terminal pyridine is necessary for the pro-apoptotic activity (with little preference for the pyridine next to the styryl substituted pyridine). Indeed, the docking studies proposed the two opposite orientations of pyridoclax, which match well with these experimental results.

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EXPERIMENTAL PROCEDURES

Chemical Synthesis. Commercial reagents were used as received without additional purification. Only new compounds are described. Melting points were determined on a Kofler heating bench. IR spectra were recorded on a PerkinElmer BX FT-IR spectrophotometer. The band positions are given in reciprocal centimeters (cm−1). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a JEOL Lambda 400 spectrometer. Chemical shifts are expressed in parts per million downfield from tetramethylsilane as an internal standard and coupling constants in hertz. Chemical shifts are reported in parts per million (ppm) relative to the solvent resonance. Chromatography was carried out on a column using flash silica gel 60 Merck (0.063− 0.200 mm) as the stationary phase. The eluting solvent indicated for each purification was determined by thin layer chromatography (TLC) performed on 0.2 mm precoated plates of silica gel 60F-264 (Merck) and spots were visualized using an ultraviolet-light lamp. High resolution mass spectra were performed at 70 eV by electronic impact (HRMS/EI) or by positive or negative electrospray (HRMS/ESI) on a JEOL JMS GCMate spectrometer. Elemental analyses for new compounds were performed, and the data for C, H, and N were within ±0.4 of the theoretical values for all final compounds. Purities of samples subjected to biological testing were assessed using these two methods and shown to be ≥95%. General Procedure To Synthesize MR29072 and Its Analogues with Identical Ends. To 5-bromo-2-hydroxypyridine (1; 5 g, 29 mmol) was added solid N-iodosuccinimide (7.1 g, 32 mmol) in acetonitrile (120 mL). The solution was stirred at reflux for 4 h and followed by TLC. The solution was cooled to room temperature, filtered, and washed with methanol. 5-Bromo-2-hydroxy-3-iodopyridine (2) was obtained as a pink solid (yield 83%). Compound 2 (9 g, 30 mmol) was dissolved in phenylphosphonic dichloride 90% (100 mL, 0.3 mol). The mixture was stirred and heated (160 °C) for 4 h and followed by TLC. At room temperature, it was introduced dropwise into a 1 L vial filled with water and cooled at 0 °C. The solution was neutralized by addition of NH4OH solution. The mixture was extracted in ethyl acetate. The product, 5-bromo-2-chloro3-iodopyridine (3), was obtained as a white solid (yield 82%). To a reaction vessel (100 mL) in a nitrogen environment containing 3 (5 g, 15.7 mmol) were added trans-phenylvinylboronic acid (4; 2.7 g, 18 mmol), tetrakis(triphenylphosphine)palladium(0) (907 mg, 0.8 mmol), and sodium carbonate (4.2 g, 39 mmol) in 1,4-dioxane (100 mL). The mixture was stirred at reflux for 24 h followed by TLC. The product was cooled to room temperature and filtered on Celite. The solution was dried on MgSO4, filtered, and evaporated. The residue was purified by chromatography (c-hexane/ethyl acetate = 99:1, then 98:2) to afford 5-bromo-2-chloro-3-(E)-styrylpyridine (5) as a white solid (yield 93%). Mp: 104 °C. IR (KBr disc): 3036, 1627, 1530, 1494, 1447, 1389, 1152, 1116, 1063, 956, 897, 755, 719, 687, 491 cm−1. 1H NMR (CDCl3) δ 8.30 (d, 1H, J = 1.9 Hz), 8.03 (d, 1H, J = 1.9 Hz), 7.52 (d, 2H ortho Ph, J = 7.8 Hz), 7.38 (dd, 2H meta Ph, J = 7.8 Hz), 7.32 (d, 1H para Ph, J = 7.8 Hz), 7.26 (d, 1H, CHCH, J = 16.6 Hz), 7,04 (d, 1H, CH CH, J = 16,6 Hz). 13C NMR (CDCl3) δ 148.5, 148.4, 136.6, 135.8, 134.5, 133.6, 129.0, 128.8 (2C Ph), 127.1 (2C Ph), 121.8, 119.3. LCMS(EI): 293[M + H+], 295[M+ + 2], 297[M+ + 4]. To 5 (200 mg, 0.7 mmol) were added sodium iodide (1.05 g, 7 mmol), acetyl chloride (0.07 mL, 1 mmol), and acetonitrile (10 mL). The solution was stirred under microwave application for 1 h at 100 °C. At room temperature, the mixture was neutralized by a NaHCO3 solution. After an extraction and a wash with sodium bisulfite solid/water, the

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CONCLUSION In summary, we designed, synthesized, and identified new molecules among the oligopyridine family that could be efficiently used as Mcl-1 inhibitors, pyridoclax (MR29072) appearing as the lead compound of this family. We used SPR assays to demonstrate that pyridoclax was able to directly and selectively interact with Mcl-1. We also showed that pyridoclax induces ER-stress and subsequent Noxa expression, but that these events were not required for the observed sensitizing effect, thus differentiating this original molecule from most other Mcl-1 P


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IR (KBr disc): 2943, 1648, 1389, 866, 759, 696, 615 cm−1. 1H NMR (CDCl3) δ 8.89 (d, 1H, J = 1.92 Hz), 8.77 (d, 1H, J = 1.92 Hz), 8.21 (d, 1H, J = 1.92 Hz), 7.96 (1H, J = 1.92 Hz), 7.68−7.66 (dd, 2H, J = 2.92 Hz, J = 7.4 Hz), 7.57 (d, 1H, J = 7 Hz), 7.51 (d, 2H, J = 2.92 Hz), 7.47 (d, 2H, J = 7 Hz), 7.43−7.41 (dd, 2H, J = 2.92 Hz), 7.38−7.34 (dd, 2H, J = 7 Hz), 7.26−7.21 (m, 2H, CHCH), 2.68 (s, 3H, CH3). 13C NMR (CDCl3) δ 153.25, 152.67, 147.64, 146.56, 141.38, 139.75, 138.32, 136.60, 133.34, 132.37, 131.67, 131.63, 130.73, 130.53, 128.79 (3C), 128.30, 127.16, 126.81 (2C), 125.99, 125.30, 125.27, 125.12, 121.84, 26.86. LCMS(EI): 437 [M + H+]. 3-Methyl-2-(3-methylthiophen-2-yl)-5-(5-(3-methylthiophen-2-yl)3-(E)-styrylpyridin-2-yl)pyridine, MR31336. The general procedure was followed using 3-methylthiophen-2-yl boronic acid (401 mg, 2.82 mmol) and 8 (486 mg, 1.13 mmol) to yield the title compound (105 mg) as an orange oil (yield 20%). IR (KBr disc): 2943, 1648, 1389, 866, 759, 696, 615 cm−1. 1H NMR (CDCl3) δ 8.89 (d, 1H, J = 1.92 Hz), 8.77 (d, 1H, J = 1.92 Hz), 8.21 (d, 1H, J = 1.92 Hz), 7.96 (1H, J = 1.92 Hz), 7.68−7.66 (dd, 2H, J = 2.92 Hz, J = 7.4 Hz), 7.57 (d, 1H, J = 7 Hz), 7.51 (d, 2H, J = 2.92 Hz), 7.47 (d, 2H, J = 7 Hz), 7.43−7.41 (dd, 2H, J = 2.92 Hz), 7.38− 7.34 (dd, 2H, J = 7 Hz), 7.26−7.21 (m, 2H, CHCH), 2.68 (s, 3H, CH3). 13C NMR (CDCl3) δ 153.25, 152.67, 147.64, 146.56, 141.38, 139.75, 138.32, 136.60, 133.34, 132.37, 131.67, 131.63, 130.73, 130.53, 128.79 (3C), 128.30, 127.16, 126.81 (2C), 125.99, 125.30, 125.27, 125.12, 121.84, 26.86. LCMS(EI): 437 [M + H+]. 2-(5-Methyl-6-(pyridin-4-yl)pyridin-3-yl)-5-(pyridin-4-yl)-3-(E)styrylpyridine, MR31350. The general procedure was followed using 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (500 mg, 2.44 mmol) and 8 (421 mg, 0.98 mmol) to yield the title compound (233 mg) as a pale yellow solid (yield 56%). Mp: 168−170 °C. IR (KBr disc): 2928, 2675, 2487, 1630, 1600, 1446, 1419, 1384, 1110, 1031, 963, 819, 766, 748, 680, 623 cm−1. 1H NMR (CDCl3) δ 8.85 (d, 1H, J = 2.2 Hz), 8,80 (d, 1H, J = 1.9 Hz), 8.73−8.71 (dd, 2H, J = 1.7 Hz, J = 4.5 Hz), 8.70−8.68 (dd, 2H, J = 1.7 Hz, J = 4.5 Hz), 8.21 (d, 1H, J = 2.2 Hz), 7.97 (d, 1H, J = 1.9 Hz), 7.58−7.57 (dd, 2H, J = 1.7 Hz, J = 4.5 Hz), 7.48− 7.47 (dd, 2H, J = 1.7 Hz, J = 4.5 Hz),7.41 (d, 2H, J = 6.8 Hz), 7.32−7.29 (m, 2H), 7.26−7.25 (m, 1H), 7.23−7.16 (m, 2H, CHCH), 2.43 (s, 3H, CH3).13C NMR (CDCl3) δ 155.78, 154.09, 150.69 (2C), 149.95, 148.06, 147.59, 146.84, 144.59, 140.04, 136.41, 134.42, 133.37, 132.67, 132.06, 131.10, 128.89 (2C), 128.65, 126.88 (2C), 124.67, 124.52, 123.76 (2C), 121.61 (2C), 121.40, 19.91. LCMS (EI): 427.37 [M + H+]. 5-(6-(5-Methyl-6-(pyrimidin-5-yl)pyridin-3-yl)-5-(E)-styrylpyridin3-yl)pyrimidine, MR31361. The general procedure was followed using pyrimidine-5-boronic acid (345 mg, 2.79 mmol) and 8 (400 mg, 0.93 mmol) to yield the title compound (200 mg) as a pale yellow solid (yield 50%). Mp: >250 °C. IR (KBr disc): 3036, 1557, 1454, 1408, 1128, 1015, 967, 893, 744, 725, 690,627 cm−1. 1H NMR (CDCl3) δ 9.33 (s, 1H), 9.30 (s, 1H), 9.10 (s, 2H), 9.07 (s, 2H), 8.90 (d, 1H, J = 1.9 Hz), 8.88 (d, 1H J = 2.2 Hz), 8.25 (d, 1H, J = 2.2 Hz), 8.07 (d, 1H, J = 1.9 Hz), 7.48 (d, 2H, J = 7 Hz), 7.40−7.32 (m, 3H), 7.27 (d, 1H, J = 16 Hz, CHCH), 7.22 (d, 1H, J = 16 Hz, CHCH), 2.53 (s, 3H, CH3). 13C NMR (CDCl3) δ 158.50, 158.05, 156.92 (2C), 155.03 (2C), 154.00, 152.28, 148.47, 146.55, 140.10, 136.17, 134.54, 133.83, 133.72, 132.65, 132.48, 131.66, 131.11, 129.75, 128.95 (2C), 128.83, 126.92 (2C), 124.13, 22.13. LCMS(EI): 429.58 [M + H+]. 5-Methyl-5′,6-biphenyl-3′-(E)-styryl-3,2′-bipyridine, MR30814. The general procedure was followed using benzeneboronic acid (708 mg, 5.81 mmol) and 8 (1 g, 2.32 mmol) to yield the title compound (600 mg) as a white solid (yield 61%). Mp: 141 °C. IR (KBr): 3036, 3022, 2930, 2857, 2245, 1436, 1401, 1391, 1115, 852, 734, 672 cm−1. 1H NMR (CDCl3) δ 8.88 (s, 1H), 8.83 (s, 1H), 8.23 (s, 1H), 7.99 (s, 1H, H4), 7.71 (d, J = 7.8 Hz, 2H), 7.63 (d, J = 7.8 Hz, 2H), 7.57− 7.43 (m, 8H), 7.37−7.24 (m, 5H), 2.48 (s, 3H, CH3). 13C NMR (CDCl3) δ 158.4, 153.1, 146.9, 146.2, 140.3, 139.7, 137.5, 136.8, 135.9, 133.3, 132.5 (2C), 131.7, 130.6, 129.3 (2C), 129.2 (2C), 128.8 (2C), 128.3 (2C), 128.2 (2C), 128.1, 127.2 (2C), 126.8 (2C), 125.4, 20.1. LCMS (ESI): 424.55; [M + H+] 425.27. 5-(3,4-Dimethoxyphenyl)-2-(6-(3,4-dimethoxyphenyl)-5-methylpyridin-3-yl)-3 (E)-styrylpyridine, MR30846. The general procedure was followed using 3,4-dimethoxyphenylboronic acid (293 mg, 1.85 mmol) and 8 (320 mg, (0.67 mmol) to yield the title compound

organic layer was dried with MgSO4, filtered, and evaporated. The product was purified by chromatography (c-hexane/ethyl acetate = 98:2, then 95:5) to achieve 5-bromo-2-iodo-3-(E)-styrylpyridine 6 as white solid (yield 84%). Mp: 78 °C. IR (KBr disc): 3054, 3023, 1629, 1492, 1446, 1377, 1269, 1109, 1026, 952, 889, 751 cm−1. 1H NMR (CDCl3) δ 8.29 (d, 1H, J = 1.96 Hz), 7.83 (d, 1H, J = 1.96 Hz), 7.52 (d, 2H ortho Ph, J = 7.8 Hz), 7.38 (dd, 2H meta Ph, J = 7.8 Hz), 7.32 (d, 1H para Ph, J = 7.8 Hz), 7.09 (d, 1H, CHCH, J = 15.6 Hz), 6.94 (d, 1H, CHCH, J = 15.6 Hz). 13C NMR (CDCl3) δ 149.64, 139.64, 135.87, 135.11 (CH CH), 134.70 (CHCH), 128.99, 128.89 (2C), 128.09, 127.09 (2C), 121.06, 120.78. LCMS(EI): 258[M + H+], 260[M+ + 2]. To a reaction vessel (100 mL) in a nitrogen atmosphere containing 6 (1.4 g, 3.6 mmol) were added 6-bromo-5-methylpyridin-3-ylboronic acid (7; 978 mg, 0.45 mmol), tetrakis(triphenylphosphine)palladium(0) (210 mg, 0.18 mmol), and potassium phosphate (2.1 g, 9 mmol) in 1,2-dimethoxyethane (30 mL). The mixture was stirred at reflux for 20 h, followed by TLC. At room temperature, the solution was extracted with ethyl acetate. The organic layer was dried on MgSO4, filtered, and evaporated. The residue was purified by chromatography (c-hexane/ethyl acetate = 95:5, then 9:1 and 8:2) and 5,6′-dibromo-5′methyl-3-(E)-styryl-2,3′-bipyridine (8; yield 86%) was obtained as white solid. Mp: 143 °C. IR (KBr disc): 3031, 1393, 1111, 1050, 968, 898, 740, 685, 499 cm−1. 1H NMR (CDCl3): 8.65 (d, 1H, J = 1.9 Hz), 8,41 (d, 1H, J = 1.9 Hz), 8.17 (d, 1H, J = 1.9 Hz), 7.83 (d, 1H, J = 1.9 Hz), 7.42 (d, 2H, J = 7.8 Hz), 7.36 (dd, 2H, J = 7.8 Hz), 7.31 (d, 1H, J = 7.8 Hz), 7.12 (d, 1H, J = 16.6 Hz), 6,99 (d, 1H, J = 16.6 Hz), 2.46 (s, 3H, CH3). 13C NMR (CDCl3): 151.4, 149.4, 147.8, 144.9, 139.6, 136.5, 136.0, 135.2, 134.0, 133.9, 133.3, 128.9 (2C), 128.8, 126.9 (2C), 123.4, 120.4, 22.0. LCMS(EI): 428 [M + H+], 430[M+ + 2], 432[M+ + 4]. To a reaction vessel (100 mL) in a nitrogen atmosphere containing 8 (320 mg, 0.7 mmol) were added pyridin-3-yl boronic acid (156 mg, 1.7 mmol), tetrakis(triphenylphosphine)palladium(0) (78 mg, 0.07 mmol), and sodium carbonate (355 mg, 3.35 mmol) in 1,4-dioxane (20 mL). The mixture was stirred at reflux for 24 h, followed by TLC. At room temperature, the suspension was filtered on Celite, and the solution was extracted with ethyl acetate. The organic layer was dried on MgSO4, filtered, and evaporated. The product was purified by chromatography (c-hexane/ethyl acetate = 8:2, then 7:3 and 50:50) to achieve 5,6′di(pyridin-3-yl)-5′-methyl-3-(E)-styryl-2,3′-bipyridine as MR29072 or pyridoclax (yield 86%) as a white solid. Mp: 158 °C. IR (KBr): 2957, 1727, 1575, 1274, 1125, 1014, 967, 801, 770, 688 cm−1. 1H NMR (400 MHz, CDCl3): δ 8.98 (d, J = 1.9 Hz, 1H), 8.91 (d, J = 1.9 Hz, 1H), 8.87 (m, 2H), 8.72 (dd, J = 4.9 Hz, J = 1.9 Hz, 2H), 8.68 (dd, J = 4.9 Hz, J = 1.9 Hz, 1H), 8.24 (d, J = 1.9 Hz, 1H), 8.00 (m, 3H), 7.48 (d, J = 7.8 Hz, 3H), 7.44 (dd, J = 7.8 Hz, J = 1.9 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 16.6 Hz, 1H), 7.23 (d, J = 16.6 Hz, 1H), 2.51 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 155.3, 153.4, 149.9, 149.6, 149.2, 148.2, 148.0, 146.9, 139.8, 136.5, 136.3, 135.8, 134.4, 134.1, 133.1, 133.0, 132.9, 132.6, 132.0, 131.1, 128.8 (2C), 128.5, 126.8 (2C), 124.6, 123.8, 123.2, 20.0. LCMS (EI): m/z (%) [M + H]+ theoretical 427.53; experimental 427.32. Anal.Calcd for C29H22N4: C, 81.67; H, 5.20; N, 13.14. Found: C, 81.65; H, 5.25; N, 13.23. 3-Methyl-5-(3-(E)-styryl-5-(thiophen-2-yl)pyridin-2-yl)-2-(thiophen2-yl)pyridine, MR31321. The general procedure was followed using thiophen-2-yl boronic acid (445 mg, 3.48 mmol) and 8 (500 mg, 1.16 mmol) to yield the title compound (320 mg) as a yellow oil that crystallized (yield 63%). IR (KBr disc): 2922, 1638, 1618, 1451, 1437, 1222, 1150, 828, 701, 692 cm−1. 1H NMR (CDCl3) δ 8.90 (d, 1H, J = 1.96 Hz), 8.73 (d, 1H, J = 1.96 Hz), 8.18 (d, 1H, J = 1.96 Hz), 7.95 (d, 1H, J = 1.96 Hz), 7.58 (d, 1H, J = 3.87 Hz), 7.49−7.46 (m, 4H), 7.43 (d, 1H, J = 3.87 Hz), 7.38−7.34 (dd, 2H, J = 7 Hz), 7.30 (m, 1H, J = 7 Hz), 7.20 (s, 2H, CHCH), 7.19−7.17 (m, 2H), 2.68 (3H, CH3). 13C NMR (CDCl3) δ 152.85, 151.24, 147.70, 145.96, 145.06, 140.42, 140.12, 139.45, 136.71, 133.17, 132.82, 131.91, 131.29, 129.75, 129.59, 128.95, 128.57, 128.53, 128.03, 127.96, 127.54, 127.03, 126.47, 125.22, 124.74, 121.81, 21.77. LCMS(EI): 437 [M + H+]. 3-Methyl-5-(3-(E)-styryl-5-(thiophen-3-yl)pyridin-2-yl)-2-(thiophen3-yl)pyridine, MR31322. The general procedure was followed using thiophen-3-yl boronic acid (357 mg, 2.79 mmol) and 8 (400 g, 0.93 mmol) to yield the title compound (160 mg) as a brown oil (yield 49%). Q


Article

Journal of Medicinal Chemistry (300 mg) as a yellow solid (yield 82%). Mp: 80 °C. IR (KBr): 3054, 3026, 2931, 2835, 1722, 1602, 1587, 1516, 1422, 1253, 1139, 1025, 693, 541 cm−1. 1H NMR (CDCl3) δ 8.84 (s, 1H, H6), 8.80 (s, 1H), 8.17 (s, 1H), 7.98 (s,1H), 7.48 (d, J = 6.8 Hz, 2H), 7.36 (dd, J = 7.8, 7.1 Hz, 2H), 7.30 (d, J = 8.3 Hz, 1H), 7.28 (d, J = 16.4 Hz, 1H, CHCH), 7.27−7.21 (m, 4H), 7.18 (d, J = 15.9 Hz, 1H, CHCH), 7.04 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 4.01 (s, 3H, CH3O-meta), 3,97 (s, 9H), 2,50 (s, 3H, CH3). 13C NMR (CDCl3) δ 158, 152.7, 149.7, 149.6, 149.2, 148.8, 147.7, 147.1, 139.6, 136.8, 135.8, 133.5, 132.9, 132.4, 132.1, 131.6, 130.7, 130.3, 128.8, 128.4, 126.8, 125.5, 121.8, 119.7, 112.5, 111.7, 110.5 (2C), 110.3 (2C), 56.1, 56.0, 55.9, 55.8, 20.5. LCMS(ESI):545.64 [M + H+]. 5-(3,4,5-Trimethoxyphenyl)-6′-(3,4,5-trimethoxyphenyl)-5′-methyl3-(E)-styryl-2,3′-bipyridine, MR30847. The general procedure was followed using 3,4,5-trimethoxyphenylboronic acid (392 mg, 1.85 mmol) and 8 (400 mg, 0.84 mmol) to yield the title compound (355 mg) as a yellow solid (yield 70%). Mp 100 °C. IR (KBr): 2995, 2931, 2833, 1723, 1584, 1508, 1451, 1418, 1343, 1238, 1125, 747 cm−1. 1H NMR (CDCl3): δ 8.83 (s, 1H), 8.80 (s, 1H), 8.16 (s, 1H), 7.99 (s, 1H), 7.49 (d, J = 7.1 Hz, 2H), 7.35 (dd, J = 7.6, 6.8 Hz, 2H), 7.31 (d, J = 7.3 Hz, 1H), 7.28 (d, J = 15.6 Hz, 1H, CHCH), 7,21 (d, J = 15.6 Hz, 1H, CH CH), 6.86 (s, 2H), 6,84 (s, 2H), 3.99 (s, 6H, CH3O-meta), 3,93 (s, 3H, CH3O-para), 3,91 (s, 6H, CH3O-meta′), 3.82 (s, 3H, CH3O-para′), 2,50 (s, 3H, CH3). 13C NMR (CDCl3): δ 158.4, 153.1, 146.9, 146.2, 140.3, 139.7, 137.5, 136.8, 135.9, 133.3, 132.5 (2C), 131.7, 130.6, 129.3 (2C), 129.2 (2C), 128.8 (2C), 128.3 (2C), 128.2 (2C), 128.1, 127.2 (2C), 126.8 (2C), 125.4, 56.1 (2C), 56.0 (2C), 55.8 (2C), 20.1. LCMS(EI): 605.69 [M + H+]. 6′,5-(Dicyanophenyl)-5′-methyl-3-(E)-styryl-2,3′-bipyridine, MR30854. The general procedure was followed using 4-cyanophenylboronic acid (63 mg, 0.43 mmol) and 8 (130 mg, 0.29 mmol) to yield the title compound (130 mg) as a yellow solid (yield 95%). Mp: 226 °C. IR (KBr): 3029, 2957, 2925, 2854, 2225, 1731, 1606, 1450, 1419, 1383, 1277, 838,747, 694 cm−1. 1H NMR (CDCl3): δ 8.88 (d, J = 2.2 Hz), 8.85 (d, J = 2.2 Hz, 1H), 8.24 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 2.2 Hz, 1H), 7.79 (dd, J = 8.3, 1.9 Hz, 4H), 7.74 (dd, J = 8.5, 2.0 Hz, 4H), 7.45 (d, J = 8.0 Hz, 2H), 7.35 (d, J=8.1 Hz, 2H), 7.31 (d, J = 7.3 Hz, 1H), 7.17 (d, J = 16.3 Hz, 2H, CHCH), 2.49 (s, 3H, CH3). 13C NMR (CDCl3): δ 156.5, 153.8, 148.0, 147.0, 144.6, 141.9, 140.0, 136.2(2C), 134.5, 134.4, 133.5, 133.2, 132.8, 132.4 (2C), 132.2, 131.1, 130.0 (2C), 129.0, 128.9, 128.0 (2C), 127.0 (2C), 124.7(2C), 118.8, 118.6, 112.5, 112.0, 20.5. LCMS(ESI): 475.55 [M + H+]. General Procedure To Synthesize Compounds of Formula 9. 3-(6Chloro-5-(E)-styrylpyridin-3-yl)pyridine. To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-chloro-3-(E)-styrylpyridine (5; 1.5 g, 5.08 mmol) were added pyridin-3-yl boronic acid (781 mg, 6.35 mmol), tetrakis(triphenylphosphine)palladium(0) (5% mol), and Na2CO3 (2.5 equiv) in 1,4-dioxane. The mixture was stirred at reflux for 24 h, followed by TLC. The product was cooled to room temperature and filtered on Celite. The solution was dried on MgSO4, filtered, and evaporated. The residue was purified by chromatography (c-hexane/ ethyl acetate = 7:3) to afford the title compound (1.41 g) as a pale yellow solid (95% yield). Mp: 159 °C. IR (KBr disc): 3045, 3019, 2924, 2853, 1638, 1578, 1446, 1427, 1385, 1166, 1087, 1057, 1023, 951, 921, 804, 729, 714, 687 cm−1. 1H NMR (CDCl3): δ 8.88 (d, 1H, J = 2.2 Hz), 8,71− 8.69 (dd, 1H, J = 1.7 Hz, J = 4.76 Hz), 8.49 (d, 1H, J = 2.3 Hz), 8.14 (d, 1H, J = 2.3 Hz), 7.94−7.91 (m, 1H), 7.58 (d, 2H, J = 7.3 Hz), 7.40−7.26 (m, 5H), 7.13 (d, 1H, J = 16.4 Hz, CHCH). 13C NMR (CDCl3): δ 149.92, 149.70, 148.10, 146.09, 136.15, 136.10, 134.49, 133.99, 133.04, 132.91, 132.53, 132.30, 128.89 (2C), 127.06 (2C), 123.86, 122.72. LCMS(EI): 293.35 [M + H+], 295.34 [M+ + 2]. 2-Chloro-5-phenyl-3-(E)-styrylpyridine. The general procedure was followed using phenyl boronic acid (929 mg, 7.62 mmol) and 5-bromo2-chloro-3-(E)-styrylpyridine (5; 1.5 g, 5.08 mmol). The residue was purified by chromatography (c-hexane/ethyl acetate = 9:1) to yield the title compound (1.10 g) as a colorless oil (yield 74%). IR (KBr disc): 3058, 3033, 2924, 2850, 1630, 1601, 1580, 1546, 1494, 1448, 1417, 1407, 1390, 1322, 1222, 1210, 1162, 1116, 1085, 1054, 1008, 961, 903, 845, 762, 741, 696, 563 cm−1. 1H NMR (CDCl3): δ 8.54 (d, 1H, J = 2.4 Hz), 8,07 (d, 1H, J = 2.4 Hz), 7.59−7.24 (m, 11H), 7.11 (d, 1H, J = 16 Hz, CHCH). 13C NMR (CDCl3): δ 146.3, 143.8, 136.4, 135.3, 135.2,

134.6, 133.4, 132.5, 129.3 (2C), 128.8, 128.7 (2C), 128, 127.5 (2C), 127.4, 126.4 (2C). LCMS(EI): 292.35 [M + H+], 294.34 [M+ + 2]. General Procedure To Synthesize Compounds of Formula 10. 3(6-Iodo-5-(E)-styrylpyridin-3-yl)pyridine. To a solution of 3-(6-chloro5-(E)-styrylpyridin-3-yl)pyridine (1 g, 3.41 mmol) in acetonitrile at a concentration of 0.25 M, sodium iodide (10 equiv) and acetyl chloride (1.5 equiv) were added. The reaction mixture was heated under microwave irradiation at 100 °C for 60 min. It was carefully quenched with saturated NaHCO3 aqueous solution until pH = 8. Product was extracted with ethyl acetate and washed with saturated NaHSO3 aqueous solution. The organic layer was dried over MgSO4 and concentrated. The residue, a yellow solid (1.28 g, 98% yield), was subjected to 1H NMR, and integrations of some characteristic signals permitted us to obtain the percentage of conversion. It was used in the successive reaction without further purification. Mp: 160 °C. IR (KBr disc): 3079, 3045, 3023, 2924, 1634, 1574, 1544, 1495, 1453, 1420, 1381, 1163, 1087, 1060, 1023, 959, 914, 805, 729, 712, 680 cm−1. 1H NMR (CDCl3): δ 8.88 (d, 1H, J = 2.2 Hz), 8,71−8.69 (dd, 1H, J = 1.7 Hz, J = 4.76 Hz), 8.45 (d, 1H, J = 2.3 Hz), 7.94 (d, 1H, J = 2.3 Hz), 7.93− 7.90 (m, 1H), 7.59 (d, 2H, J = 7.3 Hz), 7.46−7.35 (m, 4H), 7.28 (d, 1H, J = 16 Hz, CHCH), 7.09 (d, 1H, J = 16 Hz, CHCH). 13C NMR (CDCl3): δ 149.1, 148, 144.8, 140.7, 135.4, 135.2, 134.1, 133.4, 133.2, 132.9, 128.7 (2C), 128, 127.4, 126.4 (2C), 124, 115.9. LCMS(EI): 385.11 [M + H+]. 2-Iodo-5-phenyl-3-(E)-styrylpyridine. The general procedure was followed using 2-chloro-5-phenyl-3-(E)-styrylpyridine (1 g, 3.42 mmol). The residue, a yellow oil (1.01 g, 77% yield), was used in the successive reaction without further purification. IR (KBr disc): 3060, 3030, 2932, 1623, 1570, 1491, 1449, 1406, 1110, 1080, 1034, 1008, 959, 906, 761, 696 cm−1. 1H NMR (CDCl3): δ 8.45 (d, 1H, J = 2.4 Hz), 7.95 (d, 1H, J = 2.4 Hz), 7.59−7.24 (m, 11H), 7.08 (d, 1H, J = 16 Hz, CH CH). 13C NMR (CDCl3): δ 144.8, 140.7, 136.4, 135.4, 135.2, 133.4, 132.9, 129.3 (2C), 128.8, 128.7 (2C), 128, 127.5 (2C), 127.4, 126.4 (2C), 114.9. LCMS(EI): 384.18 [M + H+]. General Procedure To Synthesize Compounds of Formula 11. 2Bromo-3-methyl-5-(5-(pyridine-3-yl)-3-(E)-styrylpyridin-2-yl)pyridine. To a reaction vessel in a nitrogen atmosphere containing 3-(6iodo-5-(E)-styrylpyridin-3-yl)pyridine (120 mg, 0.31 mmol) was added 6-bromo-5-methylpyridin-3-yl boronic acid (100 mg, 0.46 mmmol), followed by tetrakis(triphenylphosphine)palladium(0) (5% mol) and aqueous K3PO4 (2.5 equiv). The mixture was heated at 80 °C for 1 h and then under reflux until the complete consumption of aryl halide (TLC) (20 h). The reaction mixture was concentrated and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated. The residue was purified by chromatography on silica gel (cyclohexane/ethyl acetate, 5:5 to 3:7) to afford 130 mg of pure compound as a pale yellow solid (98% yield). Mp: 149 °C. IR (KBr disc): 3039, 1588, 1557, 1490, 1438, 1379, 1190, 1120, 1053, 1023, 970, 907, 804, 746, 708, 721, 686, 540 cm−1. 1H NMR (CDCl3): δ 8.94 (d, 1H, J = 2 Hz), 8.81 (d, 1H, J = 2.2 Hz), 8.71−8.69 (dd, 1H, J = 1.7 Hz, J = 4.8 Hz), 8.48 (d, 1H, J = 2 Hz), 8.19 (d, 1H, J = 2.2 Hz), 7.99−7.96 (m, 1H), 7.90 (d, 1H, J = 2.4 Hz), 7.67−7.62 (m, 1H), 7.45−7.43 (m, 2H), 7.37−7.33 (m, 2H), 7.31−7.29 (m, 1H), 7.20 (d, 1H, J = 16 Hz, CHCH), 7.12 (d, 1H, J  16 Hz, CHCH), 2.47 (s, 3H, CH3). 13C NMR (CDCl3): δ 152.61, 149.67, 148.22, 147.97, 146.97, 139.80, 136.26, 134.47, 134.40, 133.34, 132.71, 132.53, 132.13, 132.03, 131.95, 128.90 (2C), 128.66, 128.56, 128.43, 126.86, 124.37, 123.87, 22.12. LCMS(EI): 428.24 [M + H+], 430.29 [M+ + 2]. 2-Bromo-3-methyl-5-(5-phenyl-3-(E)-styrylpyridin-2-yl)pyridine. The general procedure was followed using 2-iodo-5-phenyl-3-(E)styrylpyridine (170 mg, 0.44 mmol) and 6-bromo-5-methylpyridin-3-yl boronic acid (1.5 equiv, 142 mg, 0.66 mmol). The residue was purified by chromatography (cyclohexane/ethyl acetate, 8:2) to afford 182 mg of pure compound as a pale yellow solid (97% yield). Mp: 175 °C. IR (KBr disc): 3030, 2921, 2853, 1578, 1555, 1442, 1385, 1061, 970, 898, 760, 694 cm−1. 1H NMR (CDCl3): δ 8.85 (d, 1H, J = 2.2 Hz), 8.50 (d, 1H, J = 2.2 Hz), 8.21 (d, 1H, J = 2.2 Hz), 7.93 (d, 1H, J = 2.2 Hz), 7.73−7.70 (m, 2H), 7.55−7,44 (m, 5H), 7.37−7.30 (m, 3H), 7.24 (d, 1H, J = 16 Hz, CHCH), 7.15 (d, 1H, J = 16 Hz, CHCH), 2.49 (s, 3H, CH3). 13C NMR (CDCl3): δ 144.7, 143.8, 143.6, 142.5, 137.7, 136.4, 136, 135.3, R


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1,2-dimethoxyethane. The mixture was heated at 80 °C for 1 h and then under reflux until the complete consumption of aryl halide (TLC) (24 h). The reaction mixture was concentrated and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated. The residue was purified by chromatography on silica gel (c-hexane/ethyl acetate, 8:2) to afford 80 mg of pure compound as a yellow solid (94% yield). Mp 170 °C. IR (KBr): 3040, 2987, 2937, 2825, 1586, 1415, 1343, 1237, 1130, 1009, 770, 750, 696 cm−1. 1H NMR (CDCl3): δ 8.73 (s, 1H), 8.69 (d, J = 2.2 Hz, 1H), 8.21 (s, 1H), 7.92 (s, 1H), 7.45 (d, J = 7.1 Hz, 2H), 7.35 (dd, J = 7.3, 6.8 Hz, 2H), 7.31 (d, J = 7.32 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.14 (s, 2H), 6.83 (d, J = 6.4 Hz, 1H), 3.98 (s, 9H, OCH3), 2.49 (s, 3H, CH3). 13C NMR (CDCl3): δ 158.4, 152.9 (2C), 152.5, 149.3, 147.3, 139.5, 138.1, 136.4, 136.2, 135.5, 133.4, 133.3, 133.0, 130.7, 128.8 (2C), 128.6, 126.9 (2C), 123.8, 120.0, 106.3 (2C), 60.9, 56.1, 29.6, 20.2. LCMS(EI): 518.41 [M + H+], 520.42 [M+ + 2]. 5-Bromo-6′-(3,4-dimethoxyphenyl)-5′-methyl-3-(E)-styryl-2,3′-bipyridine. The general procedure was followed using 2-bromo-3-methyl5-(5-(pyridine-3-yl)-3-(E)-styrylpyridin-2-yl) pyridine (250 mg, 0.52 mmol) and of 3,4-dimethoxyphenylboronic acid (105 mg, 0.58 mmol). The residue was purified by chromatography on silica gel (c-hexane/ethyl acetate, 8:2 to 5:5) to afford 180 mg of pure compound as a yellow solid (yield 93%). Mp: 158 °C. IR (KBr): 3083, 2994, 2949, 2914, 2819, 1606, 1594, 1519, 1419, 1406, 1276, 1135, 1026, 968, 926, 759 cm−1. 1H NMR (CDCl3): δ 8.73 (d, J = 1.9 Hz, 1H), 8.69 (d, J = 2.2 Hz, 1H), 8.20 (d, J = 2.2 Hz, 1H), 7.90 (d, J = 1.9 Hz, 1H), 7.45 (d, J = 7.3 Hz, 2H), 7.35 (dd, J = 7.6, 7.1 Hz, 2H), 7.31 (d, J = 7.4 Hz, 1H), 7.21−7.14 (m, 3H), 7.17 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 3.95 (s, 6H, OCH3), 2.49 (s, 3H, CH3).13C NMR (CDCl3): δ 158.2, 152.5 (2C), 149.3, 149.0, 148.7, 147.5, 139.5, 136.6, 136.4, 133.3, 133.2, 132.6, 132.4, 130.7, 129.0, 128.7 (2C), 127.1, 123.9 (2C), 121.8, 112.3, 110.6, 74.2, 55.7, 20.3 (CH3). LCMS(EI): 488.39 [M + H+], 491.41 [M+ + 2]. 5-(3,4-Dimethoxyphenyl)-6′-(3,4,5-trimethoxyphenyl)-5′-methyl3-(E)-styryl-2,3′-bipyridine, MR30849. The general procedure was followed using 5-bromo-6′-(3,4,5-trimethoxyphenyl)-5′-methyl-3-(E)styryl-2,3′-bipyridine (280 mg, 0.54 mmol) and 3,4-dimethoxyphenylboronic acid (108 mg, 0.60 mmol). The residue was purified by chromatography on silica gel (c-hexane/ethyl acetate, 8:2 to 5:5) to afford 200 mg of pure compound as a yellow solid (yield 64%). Mp: 190 °C. IR (KBr): 3033, 2994, 2959, 2937, 2839, 1587, 1520, 1451, 1409, 1342, 1266, 1239, 1172, 1141, 1127, 1026, 750 cm−1. 1H NMR (CDCl3): δ 8.84 (d, J = 2.2 Hz, 1H), 8.79 (d, J = 2.2 Hz, 1H), 8.17 (d, J = 2.2 Hz, 1H), 7.99 (d, J = 2.0 Hz, 1H), 7.48 (d, J = 7.3 Hz, 2H), 7.35 (dd, J = 7.8, 6.8 Hz, 2H), 7.30 (d, J = 7.1 Hz, 1H), 7.27 (d, J = 2.2 Hz, 1H), 7.27 (d, J = 16 Hz, 1H, CHCH), 7.23 (d, J = 16.2 Hz, 1H, CHCH), 7.19 (d, J = 2.2 Hz, 1H), 6.84 (s, 3H), 4.01 (s, 3H), 3.97 (s, 3H), 3.93 (s, 6H), 3.91 (s, 3H), 2.50 (s, 3H, CH3). 13C NMR (CDCl3): δ 158.2, 157.0, 152.5, 149.6, 149.5, 147.6, 147.0, 139.7, 138.0, 136.8 (2C), 135.9, 135.8 (2C), 133.8, 132.5, 132.3, 131.7, 130.6, 130.3, 128.8 (2C), 128.3, 126.8 (2C), 125.5, 119.8, 111.7, 110.2, 106.3, 61.0, 56.3 (2C), 56.2, 56.0, 20.3. LCMS(EI): 575.67 [M + H+]. 5-(3,4,5-Trimethoxyphenyl)-6′-(3,4-dimethoxyphenyl)-5′-methyl3-(E)-styryl-2,3′-bipyridine, MR30850. The general procedure was followed using 5-bromo-6′-(3,4-dimethoxyphenyl)-5′-methyl-3-(E)styryl-2,3′-bipyridine (180 mg, 0.37 mmol) and 3,4,5-trimethoxyphenylboronic acid (86 mg, 0.41 mmol). The residue was purified by chromatography on silica gel (c-hexane/ethyl acetate, 8:2 to 5:5) to afford 140 mg of pure compound as a yellow solid (yield 66%). Mp: 174 °C. IR (KBr): 2999, 2967, 2932, 2838, 1582, 1514, 1450, 1419, 1242, 1146, 1124, 1024, 749 cm−1. 1H NMR (CDCl3): δ 8.83 (d, J = 2.2 Hz, 1H), 8.80 (d, J = 2.2 Hz, 1H), 8.16 (d, J = 2.4 Hz, 1H), 7.98 (d, J = 1.7 Hz, 1H), 7.49 (d, J = 7.1 Hz, 2H), 7.36 (dd, J = 7,6, 7.1 Hz, 2H), 7.30 (d, J = 6.8 Hz, 1H), 7.21 (d, J = 17.8 Hz, 1H, CHCH), 7.20 (d, J = 17.8 Hz, 1H, CHCH), 7.20 (d, J = 8.3 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.86 (s, 2H), 3.99 (s, 6H), 3.97 (s, 3H), 3.96 (s, 3H), 3.94 (s, 3H), 2.50 (s, 3H, CH3). 13C NMR (CDCl3): δ 158.0, 153.8, 153.0, 149.0, 148.7, 147.5, 147.2, 139.9, 138.1, 136.6, 136.1, 133.6, 133.4, 132.8, 132.6, 132.5, 131.7, 131.0, 128.9 (2C), 128.5, 127.0 (2C), 125.2, 121.8, 112.1 (2C), 110.3 (2C), 104.2, 61.1, 56.4 (2C), 56.1, 55.9, 20.7. LCMS(EI): 575.67 [M + H+].

134.7, 133.7, 133.4, 132.9, 129.3 (2C), 129, 128.8, 128.7 (2C), 128, 127.5 (2C), 127.4, 126.4 (2C), 16.8. LCMS(EI): 427.20 [M + H+], 429.27 [M+ + 2]. 2-(5-Methyl-6-(pyridin-3-yl)-pyridin-3-yl)-5-phenyl-3-(E)-styrylpyridine, MR31348. To a reaction vessel in a nitrogen atmosphere containing 2-bromo-3-methyl-5-(5-phenyl-3-(E)-styrylpyridin-2-yl) pyridine (650 mg, 1.52 mmol) were added pyridin-3-yl boronic acid (28àmg, 2.28 mmol), tetrakis(triphenylphosphine)palladium(0) (5% mol), and aqueous K3PO4 (2.5 equiv) in 1,4-dioxane. The mixture was heated at 80 °C for 1 h and then under reflux until the complete consumption of aryl halide (TLC) (24 h). The reaction mixture was concentrated and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated. The residue was purified by chromatography on silica gel (cyclohexane/ethyl acetate, 5:5) to afford 310 mg of pure compound as a white solid (41% yield). Mp: 191 °C. IR (KBr disc): 2920, 2848, 1628, 1582, 1571, 1446, 1408, 1385, 1113, 1010, 760, 753, 692, 540 cm−1. 1H NMR (CDCl3): δ 8.90 (d, 1H, J = 1.9 Hz), 8,88 (d, 1H, J = 2.2 Hz), 8.86 (d, 1H, J = 1.9 Hz), 8.68−8.67 (dd, 1H, J = 1.7 Hz, J = 4.8 Hz), 8.24 (d, 1H, J = 1.9 Hz), 8.03 (d, 1H, J = 1.7 Hz), 7.98−7.97 (m, 1H), 7.72 (d, 2H, J = 8.3 Hz), 7.57−7.53 (m, 2H),7.49− 7.46 (m, 4H), 7.38−7.34 (m, 2H), 7.31−7.29 (m, 1H), 7.27 (d, 1H, J = 16.6 Hz), 7.22 (d, 1H, J = 16.6 Hz), 2.50 (s, 3H, CH3). 13C NMR (CDCl3): δ 155.14, 152.55, 149.97, 149.22, 148.14, 147.23, 139.45, 137.29, 136.64, 136.60, 136.12, 135.66, 134.45, 132.69, 132.59, 131.71, 131.12, 129.22 (2C), 128.83 (2C), 128.39, 128.30, 127.23 (2C), 126.81 (2C), 125.06, 123.25, 20.03. LCMS(EI): 426.58 [M + H+]. 2-(5-Methyl-6-phenylpyridin-3-yl)-5-(pyridin-3-yl)-3-(E)-styrylpyridine, MR31349. The general procedure was followed using 2-bromo-3methyl-5-(5-(pyridine-3-yl)-3-styrylpyridin-2-yl) pyridine (250 mg, 0.58 mmol) and phenyl boronic acid (64 mg, 0.53 mmol). The residue was purified by chromatography on silica gel (cyclohexane/ethyl acetate, 5:5) to afford 200 mg of pure compound as a white−pale yellow solid (yield 81%). Mp: 159 °C. IR (KBr disc): 3052, 2924, 2855, 1598, 1552, 1495, 1454, 1423, 1378, 1010, 972, 912, 806, 745, 696, 686 cm−1. 1H NMR (CDCl3): δ 8.90 (d, 1H, J = 1.9 Hz), 8,79 (d, 1H, J = 2.2 Hz), 8.75 (d, 1H, J = 2.2 Hz), 8.64−8.62 (dd, 1H, J = 1.2 Hz, J = 4.7 Hz), 8.15 (d, 1H, J = 2.2 Hz), 7.94−7.91 (m, 2H), 7.55 (d, 2H, J = 7 Hz), 7.44−7.33 (m, 6H),7.29−7.26 (m, 2H), 7.23−7.19 (m, 2H), 7.14 (d, 1H, J = 16 Hz, CHCH), 2.42 (s, 3H, CH3). 13C NMR (CDCl3): δ 158.44, 153.85, 149.46, 148.17, 147.51, 146.86, 139.90, 139.76, 136.43, 134.60, 133.41, 133.17, 132.97, 132.73, 132.58, 132.21, 131.97, 130.94, 129.08 (2C), 128.83 (2C), 128.49, 128.25 (2C), 126.88 (2C), 124.82, 123.91, 21.04. LCMS(EI): 426.42 [M + H+]. General Procedure To Synthesize MR29072 Analogues with Different Ends. 5-Bromo-6′-iodo-5′-methyl-3-(E)-styryl-2,3′-bipyridine, 14. To a solution of 5,6′-dibromo-5′-methyl-3-(E)-styryl-2,3′bipyridine, (8; 600 mg, 1.39 mmol) in acetonitrile at a concentration of 0.25 M, sodium iodide (10 equiv) and acetyl chloride (1.5 equiv) were added. The reaction mixture was heated under microwave irradiation at 100 °C for 60 min. It was carefully quenched with saturated NaHCO3 aqueous solution until pH = 8. Product was extracted with ethyl acetate and washed with saturated NaHSO3 aqueous solution. The organic layer was dried over MgSO4 and concentrated. The residue, a yellow solid (441 mg, 66% yield) was subjected to 1H NMR and integrations of some characteristic signals permitted us to obtain the percentage of conversion. It was used in the successive reaction without further purification. Mp: 147 °C. IR (KBr): 3029, 1576, 1449, 1428, 1389, 1112, 1089, 968, 900, 686, 498 cm−1. 1H NMR (CDCl3): δ 8.64 (d, J = 1.9 Hz, 1H), 8.38 (d, J = 1,9 Hz, 1H), 8.16 (d, J = 1.9 Hz, 1H), 7.73 (d, J = 1.9 Hz, 1H), 7.42 (d, J = 6.8 Hz, 2H), 7.35 (t, J = 7.8 Hz, 2H), 7.31 (d, J = 6.8 Hz, 1H), 7.11 (d, J = 16.6 Hz, 1H), 6.98 (d, J = 16.6 Hz, 1H), 2.46 (s, 3H, CH3). 13C NMR (CDCl3): δ 151.4, 149.5, 148.1, 139.2, 137.5, 136.5, 135.9, 134, 133.9, 133.3, 128.9 (2C), 128.8, 126.9 (2C), 125.6, 123.4, 120.4, 26.2. LCMS (ESI): [M + H+] calcd 478.15; found 479.06. General Procedure for the Synthesis of Compounds 15. 5-Bromo6′-(3,4,5-trimethoxyphenyl)-5′-methyl-3-(E)-styryl-2,3′-bipyridine. To a reaction vessel in a nitrogen atmosphere containing 5-bromo-6′iodo-5′-methyl-3-(E)-styryl-2,3′-bipyridine (14; 180 mg, 0.38 mmol) were added 3,4,5-trimethoxyphenylboronic acid (88 mg, 0.42 mmol), tetrakis(triphenylphosphine)palladium(0), and aqueous K3PO4 in S


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Journal of Medicinal Chemistry Procedure To Synthesize MR29071. 5,6′-Dibromo-3-(E)-styryl-2,3′bipyridine, 13. To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-iodo-3-(E)-styrylpyridine, (6; 780 mg, 2 mmol) were added 6-bromopyridin-3-yl boronic acid (12; 469 mg, 2.3 mmol), tetrakis(triphenylphosphine)palladium(0) (5% mol), and aqueous Na2CO3 in 1,4-dioxane. Then the mixture was heated to 80 °C until the starting material was consumed (TLC). After cooling to room temperature, the mixture was filtered on Celite and washed with CH2Cl2. The aqueous layer was extracted with EtOAc. Combined organic layers were dried over MgSO4. Solvents were removed in vacuo, and crude product was purified by chromatography (c-hexane/ethyl acetate 97:3 to 95:5) affording product as a white solid (540 mg, 64% yield). Mp: 135 °C. IR (KBr): 3034, 1573, 1451, 1426, 1393, 1089, 971, 898, 831, 740, 684, 489 cm−1. 1H NMR (CDCl3): δ 8,66 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H), 8.17 (d, J = 1.9 Hz, 1H), 7.83 (dd, J = 7.8 Hz, J = 1.9 Hz, 1H), 7.6 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 2H), 7.36 (t, J = 7.8 Hz, 2H), 7.32 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 16.6 Hz, 1H), 6.97 (d, J = 16.6 Hz, 1H). 13 C NMR (CDCl3): δ 150.3, 149.7, 148.5, 141.3, 138.4, 135.6, 134.9, 133.1, 132.8, 132.4, 127.9 (2C), 127.8, 126.8, 125.9 (2C), 122.3, 119.5. LCMS (ESI) 415 [M + H+], 417 [M+ + 2], 419 [M+ + 4]. 5,6′-Di(pyridin-3-yl)-3-(E)-styryl-2,3′-bipyridine, MR29071. To a reaction vessel in a nitrogen atmosphere containing 5,6′-dibromo-3-(E)styryl-2,3′-bipyridine (13; 0.250 mg, 0.6 mmol) were added 3-pyridyl boronic acid (185 mg, 1.5 mmol), tetrakis(triphenylphosphine)palladium(0) (10% mol), and aqueous Na2CO3 in 1,4-dioxane. Then the mixture was heated to 80 °C until the starting material was consumed (TLC). After cooling to room temperature, the mixture was filtered on Celite and washed with CH2Cl2. The aqueous layer was extracted with EtOAc. Combined organic layers were dried over MgSO4. Solvents were removed in vacuo, and crude product was purified by chomatography (ethyl acetate to CH2Cl2/MeOH 95:5) affording product as a white solid (180 mg, 73% yield). Mp: 224 °C. IR (KBr): 3027, 1585, 1451, 1415, 1011, 967, 809, 745, 704 cm−1. 1H NMR (CDCl3): δ 9.29 (d, J = 1.9 Hz, 1H), 9.07 (d, J = 1.9 Hz, 1H), 8.98 (d, J = 1.9 Hz, 1H), 8.88 (d, J = 1.9 Hz, 1H), 8.72 (dd, J = 4.9 Hz, J = 1.9 Hz, 1H), 8.69 (dd, J = 4,8 Hz, J = 1.9 Hz, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8,24 (d, J = 1.9 Hz, 1H), 8.18 (dd, J = 7.8 Hz, J = 1.9 Hz, 1H), 8,02 (d, J = 7.8 Hz, 1H), 7,92 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8 Hz, 2H), 7.46 (m, 2H), 7.36 (t, J = 7.8 Hz, 2H), 7,32 (d, J = 7.8 Hz, 1H), 7.25 (d, J = 16.6 Hz, 1H), 7.21 (d, J = 16.6 Hz, 1H). 13C NMR (CDCl3): δ 154.5, 153.4, 150.8, 150.1, 149.6, 148.3, 148.2, 146.9, 138.1, 136.3, 134.4 (2C), 134.3, 134.1, 133.1, 133.0, 132.9, 132.7, 132, 128.8 (2C), 128.5, 126.8 (2C), 124.7, 123.8, 123.6 120. LCMS(EI): 413.49 [M + H+]. Procedure To Synthesize MR30820. 5-Bromo-2-(pyridin-3-yl)benzaldehyde, 19. To a reaction vessel in a nitrogen atmosphere containing 2,5-dibromobenzaldehyde (17; 2 g, 7.58 mmol) were added 3-pyridyl boronic acid 18 (1.16 g, 9.47 mmol), tetrakis(triphenylphosphine)palladium(0) (5% mol), and aqueous Na2CO3 in 1,4-dioxane. Then the mixture was heated to 80 °C until the starting material was consumed (TLC). After cooling to room temperature, the mixture was filtered on Celite and washed with CH2Cl2. The aqueous layer was extracted with EtOAc. Combined organic layers were dried over MgSO4. Solvents were removed in vacuo, and crude product was purified by chomatography (c-hexane/ethyl acetate 9:1 to 5:5) affording product as a white solid (1.60 g, 80% yield). Mp 140 °C. IR (KBr): 3055, 2920, 2884, 1927, 1682, 1581, 1465, 1420, 1380, 1188, 1177, 1025, 888, 800, 714, 642, 559 cm−1. 1H NMR (CDCl3): δ 9.90 (s, 1H, CHO), 8.72 (dd, J = 4.9, 2.0 Hz, 1H), 8.65 (s, 1H), 8.19 (d, J = 2.0 Hz, 1H), 7.81 (dd, J = 7.8, 2.0 Hz, 1H), 7.70 (dd, J = 7.8, 2.0 Hz, 1H), 7.44 (dd, J = 7.8, 4.9 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H). 13C NMR (CDCl3): δ 189.7, 149.9 (2C), 149.7, 140.4, 137.0, 136.7, 134.9, 132.5, 131.2, 123.2, 123.1. MS (EI): m/z calcd 261.9868; found 261.9870. 2-(Pyridin-3-yl)-5-(3-methyl-4-pyridin-3-ylphenyl)benzaldehyde, 20. To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2(pyridin-3-yl)benzaldehyde (19; 100 mg, 0.38 mmol) were added 3-methyl-4-(pyridin-3-yl)phenyl boronic acid (16; 102 mg, 0.47 mmol), tetrakis(triphenylphosphine)palladium(0) (5% mol), and aqueous K3PO4 in 1,2-dimethoxyethane. The mixture was then refluxed until the starting material was consumed (TLC). After cooling to room temperature, the mixture was filtered on Celite and washed with CH2Cl2.

The aqueous layer was extracted with EtOAc. Combined organic layers were dried over MgSO4. The crude product was purified by chromatography (c-hexane/ethyl acetate 5:5 to EtOAc) affording the product as a white solid (130 mg, 97% yield). Mp 155 °C. IR (KBr): 3022, 2980, 2958, 2923, 2858, 1690, 1598, 1574, 1553, 1451, 1417, 1382, 1188, 1137, 1115, 1065, 1021, 999, 907, 874, 802, 763, 706, 678, 619, 565 cm−1. 1H NMR (CDCl3): δ 10.07 (s, 1H, CHO), 8.75 (s, 2H), 8.68 (d, J = 7.3 Hz, 2H), 8.35 (d, J = 2.0 Hz, 1H), 7.98 (dd, J = 7.8, 2.0 Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.65 (s, 1H), 7.61 (dd, J = 8.2, 1.7 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.49−7.44 (m, 2H), 7.37 (d, J = 7.4 Hz, 1H), 2.40 (s, 3H, CH3). 13C NMR (CDCl3): δ 191.2, 150.0, 149.4, 149.3, 147.9, 141.1, 140.7, 139.0, 137.9, 137.3, 136.8, 136.5, 134.2, 132.2, 131.6, 130.6, 129.3, 126.8 (2C), 124.8 (2C), 123.3 (2C), 20.5 (CH3). LCMS(EI): 351.41 [M + H+]. 2-(Pyridin-3-yl)-5-(3-methyl-4-pyridin-3-ylphenyl)-(E)-styrylphenyl, MR30820. To a stirred solution of diethyl benzylphosphonate (65 mg, 0.28 mmol) in tetrahydrofuran under nitrogen was added potassium hydroxide (32 mg, 0.57 mmol). After 30 min of stirring, 2-(pyridin-3-yl)5-(3-methyl-4-pyridin-3-ylphenyl)benzaldehyde (100 mg, 0.28 mmol) was added. The mixture was stirred at room temperature for 1 h and then refluxed until the starting material was consumed (TLC). Water was added, and the mixture was extracted with EtOAc. Organic layer was washed with saturated aqueous solution of NaCl and dried over MgSO4. Solvents were removed in vacuo and crude product was purified by chromatography (c-hexane/ethyl acetate 5:5) affording product as a yellow solid (50 mg, 42% yield). Mp: 139 °C. IR (KBr): 3036, 3022, 2930, 2857, 2245, 1436, 1401, 1391, 1115, 852, 734, 672 cm−1. 1H NMR (CDCl3): δ 8.64 (s, 1H), 8.57 (bs, 3H), 7.92 (s, 1H), 7.64−7.62 (m, 2H), 7.56−7.51 (m, 3H), 7.34−7.27 (m, 4H), 7.26−7.17 (m, 3H), 7.16−7.13 (m, 2H), 7.00 (d, J = 16.5 Hz, 2H, CHCH), 2.30 (s, 3H, CH3). 13C NMR (CDCl3): δ 150.1, 149.8, 148.4, 148.1, 140.7, 140.3, 137.4, 137.1, 137.0, 136.5, 136.2, 136.1, 132.0, 131.9, 131.0, 130.7, 130.4, 129.3, 128.6 (2C), 128.5, 128.4, 128.2 (2C), 127.8, 126.6 (2C), 126.5, 124.8, 123.0, 20.5 (CH3). LCMS(EI): 425.54 [M + H+]. X-ray Crystallography. Single crystals of pyridoclax suitable for X-ray crystallographic analysis were obtained by slow evaporation from DMSO. Data for crystal structure analysis were collected at 100 K with a Bruker-Nonius Kappa CCD area detector diffractometer with graphitemonochromatized Kα radiation (λ = 0.71073 Å). The structure was solved using direct methods and refined by full-matrix least-squares analysis on F2. Program SHELXS-9771 was used to solve structure and SHELXL-9772 to refine structure. The refinement was based on F2 for all reflections, and all non-hydrogen atoms were refined anisotropically. Hydrogen atom positions were determined via difference Fourier maps and refined with isotropic atomic displacement parameters. Crystallographic data for pyridoclax have been deposited at the Cambridge Crystallographic Data Centre, CCDC No. 996607. Copies of this information may be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (+44-1223-336408; E-mail: [email protected] or http://www.ccdc.cam.ac.uk). Molecular Modeling. The docking of pyridoclax and its derivatives into Mcl-1 target was carried out with the GOLD program (v5.0) using the default parameters.73,74 This program applies a genetic algorithm to explore conformational spaces and ligand binding modes. To evaluate the proposed ligand poses the ChemScore and Goldscore fitness functions were applied in the docking studies. For this docking study a tridimensional (3D) NMR structure of human Mcl-1 cocrystallized with BH3-only protein BID was used (PDB ID 2KBW;47 http://www.rcsb.org/). The binding site in the Mcl-1 was defined as an 8 Å sphere on Bid helix present in the 2KBW crystal structure. The docking was carried out with a geometrically optimized pyridoclax 3D structure built using Discovery Studio,75 as well as with two MR29072 conformers extracted from X-ray data. Molecular dynamics simulations were carried out on docking complexes between pyridoclax poses and MCL-1. All simulations were carried out using CHARMM version c29b276 with potential function parameter set 22 from Discovery Studio,75 and each system was surrounded by a rectangular box of TIP3P water molecules77 using CHARMmGUI solvator.78,79 Force field parameters for the pyridoclax were generated using Discovery Studio program applying MMFF partial T


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