Identification of Lead Compounds As Antagonists of Protein Bcl-x

Identification of Lead Compounds As Antagonists of Protein Bcl-x...
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7856 J. Med. Chem. 2009, 52, 7856–7867 DOI: 10.1021/jm9010687

Identification of Lead Compounds As Antagonists of Protein Bcl-xL with a Diversity-Oriented Multidisciplinary Approach Simone Di Micco,† Romina Vitale,§ Maurizio Pellecchia,‡ Michele F. Rega,‡ Renata Riva,§ Andrea Basso,*,§ and Giuseppe Bifulco*,† †

Dipartimento di Scienze Farmaceutiche, Universit a degli studi di Salerno, Via Ponte don Melillo, 84084 Fisciano (SA), Italy, ‡Burnham Institute for Medical Research, La Jolla, 92037 California, and §Dipartimento di Chimica e Chimica Industriale, Universit a degli Studi di Genova, Via Dodecaneso 31, 16146 Genova, Italy Received July 20, 2009

We report on the use of a diversity oriented synthesis (DOS) approach that resulted in the generation of a set of libraries of compounds presenting novel structural cores. These chemical cores have been employed to design potential antagonists of the antiapoptotic protein Bcl-xL through reiterated steps of molecular docking calculations followed by experimental verification of binding. Our data suggest that the DOS approach is suitable to generate novel scaffolds, which can be employed to target protein-protein interactions. Introduction a 1

Diversity oriented synthesis (DOS ) represents a new methodological approach that in recent years is gaining more and more importance in the field of organic synthesis and medicinal chemistry. While traditional target oriented synthesis is directed toward obtaining specific compounds, the goal of diversity oriented synthesis is the efficient preparation of collection of substances characterized by high diversity content, in terms of skeletons, appendages, and stereochemistry. One way to achieve this is to employ pluripotent substrates (PSs), which are molecules that can be synthetically elaborated, according to their functional groups, in many different ways, independent one to the other, each way leading to a different molecular skeleton, in analogy with pluripotent stem cells. Ideally, the PS is not a single entity but a collection of molecules with a common skeleton, generated in a combinatorial fashion, already displaying all or most of the final decorative elements: this library of compounds is then further elaborated to generate “n” different combinatorial libraries, each with a different core structure. The ground-breaking nature of this “star-burst” approach is that the final compounds, although deriving from a common substrate, are *To whom correspondence should be addressed. Phone: þ39-089969741 (G.B.); þ39-010-3536117 (A.B.). Fax: þ39-089-969602 (G.B.); þ39-010-3536118 (A.B.). E-mail: [email protected] (G.B.); andreab@ chimica.unige.it (A.B.). a Abbreviations: DOS, diversity oriented synthesis; Bcl-2, B-cell lymphoma 2; Bak, Bcl-2 antagonist/killer; Bcl-xL, B-cell lymphoma x long; FPA, fluorescence polarization assay; PSs, pluripotent substrates; ROM/RCM, ring-opening/ring-closing metathesis; BH, Bcl-2 homology; NOEs, Nuclear Overhauser effects; rmsd, root-mean-square deviation; MSMS, maximal speed molecular surface; OD, optical density; HPLC, high-performance liquid chromatography; 1D 1H NMR, onedimensional proton nuclear magnetic resonance spectroscopy; IPTG, Isopropyl β-D-1-tiogalattopiranoside; LB, lysogeny broth; TPPI, time proportional phase incrementation; PFG, pulse field gradient; HEPES, N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid; LAH, lithium aluminum hydride; MMFFs, Merck molecular force field; MCMM, Monte Carlo multiple minimum; GB/SA, generalized Born/surface area; PRCG, Polak-Ribier conjugate gradient; U-5C-4CR, Ugi five centre four component reaction.

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structurally different (therefore able to cover a higher area of the chemical space) and characterized by high novelty and complexity content (therefore potentially able to explore new regions of the chemical space). To differentiate DOS from total synthesis, the availability of chemical processes able to generate high complexity in a limited number of synthetic steps is very important. For these reasons, reactions able to create new C-C bonds, rings, stereocenters, or functional groups, possibly with fast kinetics, are highly favored;2 reactions possessing these requirements are, for example, cycloaddition, multicomponent, tandem, and domino reactions, molecular rearrangements. We have recently started to apply this approach to a library of oxabicyclic derivatives. As shown in Scheme 1, amino acid 1 has been reacted with various aldehydes, isocyanides, and alcohols according to a completely stereoselective intramolecular Ugi multicomponent reaction3 (U-5C-4CR) and a combinatorial library of oxabicyclic peptidomimetics 2 has been obtained, where decorative elements R1, R2, and R3 and R4 can be varied at will. Compounds 2 are highly functionalized substrates and can undergo a great number of transformations: for example, the oxabicyclic system can undergo a retro-Diels-Alder reaction with loss of furan, leading to sublibrary 3;4 if R1 and/or R4 possess an unsaturated double or triple bond ring-opening/ring-closing metathesis (ROM/ RCM), processes can generate sublibraries 4, 5, and 6;5 if R2 has an additional amino group, the library of bicyclic lactams 7 can be obtained instead. All these processes usually just require a single synthetic step, being therefore highly straightforward; in addition, they are independent and orthogonal one to the other and can be performed in sequence, as demonstrated by the generation of sublibrary 8 coupling a lactam formation followed by a ROM/RCM step. In addition, the sublibraries obtained in this way can be further elaborated, as demonstrated by the formation of library 9 from library 34 and library 10 from library 6.5 We have very recently introduced an additional transformation of 2, leading to two classes of regioisomeric cyclohexenols 11 and 12, r 2009 American Chemical Society

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Scheme 1. Diversity Oriented Synthesis (DOS) Applied to a Library of Oxabicyclic Pluripotent Substrates

involving a ring-opening process mediated by metal catalysts (Scheme 1). Ring-opening of heterobicyclic systems mediated by metal catalysts has been extensively explored by the group of Lautens6 mainly on symmetrical compounds; the substrates employed in this study, on the other hand, are not symmetrical and therefore can give two distinct regioisomeric products depending on which of the two C-O bonds is broken during the Sn20 process. In this way, our synthetic approach is convergent, because it allows the assemblage of complex entities in a few synthetic steps from simple substrates, and at the same time divergent, because it allows the formation of two distinct molecules in one operation. Although for the sake of clarity only one enantiomer is shown in Scheme 1, both enantiomerically pure forms of each scaffold are independently accessible by employing the correct bicyclic amino acid 1. It is worth noting that most of the libraries obtained possess an original core structure, and therefore their potential biological activity cannot be assigned by analogies with compounds reported in the relevant literature but has to be determined by the aid of virtual screening or by high throughput screening experiments. Our approach consisted of designing a number of virtual libraries originated from compounds of general formula 2, 4, 6, 8, and 10-12 (Scheme 1) and analyzing by virtual screening their ability to interact with a given biological target. The novel nature of the resulting scaffolds suggest that such libraries may be employed in the search of compounds capable of antagonizing protein-protein interactions, for which “traditional” drug-like libraries usually fail to provide suitable leads or even hit compounds.

To probe this hypothesis, we have chosen the antiapoptotic protein Bcl-xL, a member of the Bcl-2 family of proteins that contributes to the equilibrium between cell proliferation and cell death (apoptosis), as possible target. Apoptosis, or programmed cell death, is a highly controlled biological mechanism regulating the removal of aged, damaged, and unnecessary cells.7-11 The antiapoptotic proteins bind the proapoptotic counterparts and sequester them from the cellular environment inhibiting the apoptosis process. The analysis of experimental three-dimensional structures of some antiapoptotic proteins showed the presence of a hydrophobic surface groove, formed by BH1 (Bcl-2 homology domain 1), BH2, and BH3 domains and called BH3 binding groove. This hydrophobic crevice constitutes the binding cavity for the pro-apoptotic macromolecules.12-14 The up regulation of antiapoptotic members of this family (Bcl-2, Bcl-xL) is observed in many cancers. This overexpression protects the cancer cells from the activation of apoptosis, favoring their proliferation and their survival to the anticancer compounds.12-14 The design of small molecules, binding the BH3 domain of antiapoptotic proteins and able to inhibit the protein-protein interactions, can be a new strategy for the cancer therapy.15 Hence, we sought to use the newly derived DOS core structures to design new potential antagonists of Bcl-xL. Our strategy consists of iterative molecular docking studies followed by synthetic chemistry and experimental verification of binding by using in vitro assays. Results and Discussion Synthesis of 14a,c and 15a-c. As already anticipated in the introduction, ring-opening of oxabicyclic systems can be

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Scheme 2. The Synthetic Pathway to Polyfunctionalized Cyclohexenols 14 and 15

Scheme 3. Synthesis of Second Generation Cyclohexenols 19 and 20

performed with opportune nucleophiles in the presence of metal catalysts. Our major concern was related to the fact that this kind of reaction is often reported in the literature on simple substrates with a limited number of additional functional groups that could interfere with the reaction outcome. On the contrary, our Ugi-derived substrates possessed various functional groups, like amino, alcohol, or amide, which could interact with the catalyst or the nuclephile. After an optimization process, substrates 2a-c were prepared by condensation of enantiomerically pure 1 with opportune aldehydes and isocyanides in methanol according to the conditions previously described by us.3 Compounds 2a-c where then treated with LAH, furnishing pure 13a-c in nearly quantitative yield and leaving the other functionalities of 2 untouched. Alcohols 13a-c were then reacted, employing conditions similar to those described by Lautens,16 with phenylboronic acid in the presence of Pd(C6H5CN)2Cl2, a bis-phosphine ligand and K2CO3aq in methanol (Scheme 2). Interestingly the regioisomeric ratio for compounds 14 and 15 varied depending on the decorating elements introduced

during the U-5C-4CR, and in the case of compound 13b, only regioisomer 15b was found in the reaction mixture.17 All the described reactions were separately conducted on both enantiomers of 1, obtained according to a previously reported procedure,4 and each couple of regioisomers 14-15 was separated by the aid of flash chromatography. Structures were univocally assigned with the aid of NMR 2D experiments, NOEs, and J coupling constants. Synthesis of Compounds 19a,b, 20a,b, ent-19a,b and ent20a,b. In a first attempt, these compounds were prepared by reaction of the bicyclic amino acid 1 (R1 = H) with formaldehyde and an opportune isocyanide, however, this reaction failed to give the desired U-5C-4C adducts. As an alternative route, less efficient but more reliable, we reacted bicyclic amino ester 16 with bromoacetamides 17 and 18, previously prepared according to known procedures,18,19 and the resulting compounds were subjected to reduction and ring-opening according to the previously described methodologies (Scheme 3); it is worth noting that, although the final regioisomers 19a,b and 20a,b were isolated with

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Table 1. List of Substituents and Functionalities for All Members of the Small Virtual Libraries (A-G) Obtained from DOS Scaffolds of Scheme 1

acceptable yields, the bridgehead opening process was less smooth in the presence of a secondary amine, as already reported in the literature. The same synthetic pathway was followed to prepare the corresponding enantiomers. Molecular Docking and NMR Studies. The DOS derived scaffolds 2, 4, 6, 8, and 10-12 have been used to design seven small libraries A-G, inserting different substituent: aliphatic/aromatic groups and functionalities containing nitrogen, oxygen, or sulfur (reported in Table 1). The initial libraries were tested in silico for their ability to bind to the BH3 binding groove of Bcl-xL. The analysis of docking calculations and the comparison of all tested compounds (21-58, Table 1) revealed that the scaffolds 11 and 12 could be used to design promising molecules. In particular, based on visual inspection and on scoring function value, the structure 56 (KD = 3.14  10-11 M-1), containing an additional benzylamino substituent,

gave the best docking result and it was taken into account for the substituent choice. Unfortunately, while ring-opening reaction of substrates 2 with C nucleophiles proceeded smoothly, all synthetic efforts to perform the same reaction mediated by nitrogen or oxygen nucleophiles failed to give the desired amino-substituted products in acceptable yields. Thus, taking into consideration this synthetic limitation, compounds 59-61 and 62-64 (Scheme 4) were designed using scaffolds 11 and 12, respectively. Moreover, a pyridine was introduced in the molecules with the aim to have a bulky hydrophobic group containing a hydrogen bond acceptor able to interact with a macromolecular counterpart. To fully explore the possibility to establish hydrogen bonds by pyridine nitrogen, the substituent position on this heteroaromatic ring was changed, obtaining the isomers 59-61. The same considerations applied for 62-64. Compounds ent-59-64 (Scheme 4), enantiomers of 59-64, were

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Scheme 4. Molecular Structures of 59-64 and ent-59-64

also considered in the docking studies to verify the influence of the absolute configuration on the binding to the protein. The docking studies indicated that 59-64 and ent-59-64 presented a comparable predicted binding affinity for Bcl-xL (see Supporting Information Table S1). They showed a similar binding mode (overlaid docked structures are reported in Supporting Information Figures S35-S38), occupying equivalent spaces of the active site (Figures 1 and 2). In particular, the small molecules spanned their structural elements along the hydrophobic groove of the protein, establishing van der Waals interactions with the macromolecular counterparts, giving a significant contribution to the complex stability. Moreover, for 59-64 and ent-59-64, the analysis of the docked structures reveals, for all the pyridine regioisomers, that the pyridine nitrogen is not involved in any H-bond and consequently in the small collection of derivatives (Scheme 2) the pyridine was substituted by a phenyl ring. The 3D spatial arrangements of 59-61 docked conformations were equivalent, giving similar interactions with the protein (Figure 1a-c). In detail, the benzyl groups interacted with the positively charged side chain of Arg104, and small differences among the compounds regard the ability of forming hydrogen bonds by means of the CH2OH substituent of cyclohexene. Their enantiomers (ent-59-61) presented overlapped docking poses between them (Figure 1d-f). They differed from 59-61 for the inverted position of

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pyridine and p-methoxyphenyl ring along the protein groove. In particular, in our model, the pyridine ring seems to be able to establish cation-π interactions with the Arg143 side chain, and the CH2OH group seems to form hydrogen bonds with NH(CO) of Gly142 and with Arg143 side chain. The docked conformations of 62-64 resulted in being superimposable (Figure 2a-c). In our poses, the p-methoxyphenyl group and the benzyl ring faced the positively charged side chain of Arg104. The 62 established a hydrogen bond with NH of Gly142 by its CH2OH functionality, whereas the 63 by the same group interacted with carboxylic oxygen of Trp141. Their enantiomers (ent-62-64) showed docking poses similar to 62-64. The ent-62-64 rotated about 180° the positions of the amide bond, benzyl group, and the OH functionalities with respect to their enantiomers (Figure 2d-f). Moreover, ent-62 and ent-63 established a hydrogen bond with CO in the side chain of Asn140 by the CH2OH group. As found for 62-64, the remaining aromatic ring pointed toward the solvent exposed surface. The above-described theoretical results traced the structural features responsible for potential binding affinity and brought to the design and synthesis of compounds 14a,c and 15a-c (Scheme 2) and their enantiomers (ent-14a,c and ent15a-c). The ability of 14a,c, 15a-c, ent-14a,c, and ent-15a-c to bind Bcl-xL was tested by NMR-based binding assays. In detail, 1D 1H NMR spectra of unlabeled protein were recorded in the absence and presence of the putative ligands and the potential binding processes were detected by observing the shifts of Bcl-xL resonances in the aliphatic region of spectra: active site methyl groups of Ile, Leu, Thr, Val, or Ala (region between -0.8 and 0.3 ppm). It was possible to work with the unlabeled protein because the tested compounds did not display chemical shift signals in this upfield region of the spectrum and the protein signals were well resolved and critical residues previously assigned.20 Upon titration of test ligands, unfortunately, the analysis of 1D spectra did not reveal any significant shift of protein resonances indicating absence or at least a very low binding affinity. However, these compounds showed low solubility in the solvent medium used for NMR experiments, and so affecting the quality of spectra and the interpretation of the experimental data. Taking into account the NMR spectroscopy results and the solubility problems that could affect their eventual employment in pharmacological studies and eventually in therapy, 14a, 14c, 15a-c, ent-14a, ent-14c, and ent-15a-c were modified by obtaining a new cluster of the more hydrophilic analogues 19a,b and 20a,b (Scheme 3). The design of the new compounds resulted from the analysis of the docking poses of parent compounds 59-64 and ent-59-64 (Figure 1 and 2). However, it also appeared that the benzyl group and/or phenyl ring in the compound series were not directly involved in interactions with the protein, suggesting that those molecular groups could be removed without affecting activity but presumably resulting in compounds with increased solubility. The modified analogues underwent docking calculations in order to predict and rationalize their binding mode to the protein. The theoretical studies (Figure 3) highlighted that all compounds occupied equivalent active site portions, establishing hydrophobic contacts along the groove of Bcl-xL,

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Figure 1. Three-dimensional models of the interactions formed by 59-61 (respectively, (a), (b), and (c)) and ent-59-61 (respectively, (d), (e), and (f)) with Bcl-xL. The protein is represented by molecular surface and colored according to the hydrophobicity (dodger blue = hydrophilic, orange red = hydrophobic, see Experimental Section). All ligands are depicted by sticks (black for 59 and ent-59, dodger blue for 60 and ent-60, magenta for 61 and ent-61) and balls (colored by atom type: C, gray; polar H, white; N, dark-blue; O, red). The green lines indicate the hydrogen bonds between ligand and protein. The figure highlights essential interactions: the compounds establish hydrophobic contacts along the groove.

especially by their aromatic rings, which may possibly result in compounds with higher affinity for the protein. In particular, the analysis of docking studies revealed the potential ability of compounds 19a, ent-19b, 20a,b, and ent20a,b to interact with side chains of Asn140 and Arg143 by their CH2OH and CO groups, respectively. Using the same 1D 1H NMR strategy, the binding properties of 19a,b-20a,b, and ent-19a,b-20a,b for Bcl-xL were tested (Figure 4). The analysis of the experimental NMR data showed a weak binding with Bcl-xL, monitoring the chemical shift variation of aliphatic resonances of the biological target. In particular, there was the appearance of a signal at -0.06 ppm in the presence of 20b (Figure 4A), 19a (Figure 4A), and ent-19a (Figure 4C), together with a slight shift of the resonance at -0.12 ppm. This appeared signal

came from the overlapped methyl groups frequencies of Leu94 and Leu134 (-0.12 ppm). The same effect, but lower, was observed with the compounds ent-20a (Figure 4B). The ent-19b (Figure 4C), followed by 20a (Figure 4A) and 19a (Figure 4A), induced an appreciable shift of resonance of Val139 (-0.79 ppm). This residue is very close to Arg143, so then the shift of Val135 frequency is an indirect proof of the interaction with Arg143, as also assessed by previous studies.21 These results could be in agreement with docking studies that showed the possibility to form a hydrogen bond with Arg143 and the proximity to the above aliphatic protein residues (Figure 3). To confirm the data from 1D 1H NMR spectra, 2D-NOESY experiments of the compounds were performed in the absence and presence of protein. In the latter

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Figure 2. Three-dimensional models of the interactions formed by 62-64 (respectively, (a), (b), and (c)) and ent-62-64 (respectively, (d), (e), and (f)) with Bcl-xL. The protein is represented by molecular surface and colored according to the hydrophobicity (dodger blue = hydrophilic, orange red = hydrophobic, see Experimental Section). All ligands are depicted by sticks (yellow for 62 and ent-60, green for 63 and ent-63, turquoise for 64 and ent-64) and balls (colored by atom type: C, gray; polar H, white; N, dark blue; O, red). The green lines indicate the hydrogen bonds between ligand and protein. The figure highlights essential interactions: the compounds establish hydrophobic contacts along the groove.

case, intense cross-peaks of ligand were observable, indicating that transiently the small molecules bound the protein; due to the acquired macromolecular τc, positive cross peaks appeared (see Supporting Information, Figures S19-S34). In the absence of the macromolecule, the positive crosspeaks disappeared due to the fast tumbling of the small molecules. With these results in hand, we also tested compounds 19a, b and 20a,b in a fluorescence polarization assay in order to check if the ability of these molecules to bind the nude protein was accompanied also by their ability to displace the natural ligand (a fluoresceinated Bak peptide). However, these experiments did not show appreciable displacement at 200 μM concentration. This result was not completely unexpected due to the strong affinity of the competitive

BH3-peptide ligand (120 nM) and the relative small size of our molecules. On the other hand, the results obtained by means of combined NMR techniques and molecular modeling trace a route in the elaboration of new compounds endowed with increased activity. Conclusions The strategy of diversity oriented synthesis (DOS) has been used to obtain small libraries of compounds presenting original structural cores. A combined molecular docking-NMR spectroscopy iterative approach has led to the identification of novel potential inhibitory scaffolds against Bcl-xL. The synthetic schemes and the molecular docking studies, supported by experimental verification by

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Figure 3. Three-dimensional models of the interactions formed by 19a,b-20a,b (respectively, (a), (b), (c), and (d)) and ent-19a,b-20a,b (respectively, (e), (f), (g), and (h)) with Bcl-xL. The protein is represented by molecular surface and colored according to the hydrophobicity (dodger blue = hydrophilic, orange red = hydrophobic, see Experimental Section). All ligands are depicted by sticks (dark-slate-gray for 19a and ent-19a, turquoise for 19b and ent-19b, yellow for 20a and ent-20a, green for 20b and ent-20b) and balls (colored by atom type: C, gray; polar H, white; N, dark blue; O, red). The green lines indicate the hydrogen bonds between ligand and protein. The figure highlights essential interactions: the compounds establish hydrophobic contacts along the groove.

NMR spectroscopy, provide a robust platform onto which further optimized compounds can be obtained. The data also provide some validation of our central hypothesis that

the DOS generated libraries could provide novel and suitable starting scaffolds in targeting protein-protein interactions.

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Figure 4. Aliphatic region of the 1H 1D NMR spectrum of Bcl-xL reported in absence (black spectrum in (A), (B), and (C)) and presence of 19a (blue, A), 19b (red, B), ent-19a (red, C), ent-19b (green, C), 20a (red, A), 20b (green, A), ent-20a (green, B), and ent20b (blue, B). The gray row indicates the appeared signal coming from the overlapped methyl groups frequencies of L90 and L130 (-0.12 ppm, in the black spectrum). The gray dashed lines show the shifts of labeled diagnostic residues of the alone protein (black spectrum) in the presence of the ligands (red, green, and blue spectra). The residues number refers to the structure 2O2M.

Experimental Section Synthetic Procedures. Compounds 14a,c, 15a-c, ent-14a,c, ent-15a-c, 19a,b, 20a,b, ent-19a,b, and ent-20a,b were prepared from the corresponding bicyclic derivatives according to the

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following procedure: the substrate (103 μmol), [Pd(C6H5CN)2]Cl2 (10.3 μmol) 1,3 bis-diphenylphosphinopropane (11.3 μmol) were dissolved in MeOH (1.5 mL) together with water (25 μL) and K2CO3 (103 μmol) under argon. The reaction is heated at 50 °C for 2-5 h, and then the solvents were evaporated under vacuum and the crude taken up in DCM and washed with brine. Chromatographic purification is performed using EtOAc/PE mixtures. (2R)-N-Benzyl-2-{N-benzyl-N-[(1R,4R,5S,6R)-5-hydroxy-6(hydroxymethyl)-4-phenyl-2-cyclohexene-1-yl]amino}-3-phenylpropanamide 14a. 1H NMR (300 MHz): δ 1.62 (1H, tt, J = 10.6, 3.2), 1.97 (1H, d, J = 7.4), 3.12 (1H, d, J = 3.1), 3.19-3.38 (3H, m), 3.65-3.74 (3H,m), 3.94 (1H, d, J = 14.0), 4.12 (1H, d, J = 14.0), 4.07-4.15 (2H, m), 4.17 (1H, dd, J = 14.8, 5.6), 4.27 (1H, dd, J = 14.8, 6.0), 5.42 (1H, broad s), 5.88 (1H, ddd, J = 10.1, 5.0, 2.4), 6.18 (1H, d, J = 10.2), 6.88-7.27 (20H, m). 13C NMR (75 MHz): δ 33.27 (CH2), 42.20 (CH), 43.19 (CH2), 46.71 (CH), 52.23 (CH2), 55.43 (CH), 60.01 (CH2), 65.29 (CH), 69.12 (CH), 126-130 (12  CH, ArH), 129.57 (CH, olefine), 130.85 (CH, olefine), 137.71 (C, Ar), 138.43 (2  C, Ar), 140.36 (C, Ar), 172.76 (C). HPLC: elution time 14.6 min (100% at 220 nm). For all compounds, HPLC analyses were carried out on a HP 1100 instrument, using a gradient from H20/AcCN 9:1 to pure AcCN in 15 min (flow 0.4 mL/min) with a Gemini C6Phenyl column (150 mm  3 mm, 3um), confirming a purity g95%. HR-MS: expected for C36H38N2O3 547.2961; found 547.2958, -0.5 ppm. (2R)-N-Benzyl-2-{N-benzyl-N-[(1S,2R,5S,6R)-6-hydroxy-2(hydroxymethyl)-5-phenyl-3-cyclohexene-1-yl]amino}-3-phenylpropanamide 15a. 1H NMR: δ 2.77 (1H, broad s), 3.18 and 3.24 (2H, part AB of an ABX system Jax=3.4, Jbx=11.8, Jab = 11.8), 3.24-3.51 (3H, m), 3.66-3.75 (3H, m), 3.90 (1H, d, J=13.5), 4.18 (1H, dd, J = 14.8, 5.4), 4.31 (1H, dd, J=14.8, 4.3), 4.38 (1H, s), 4.57 (1H, d, J=13.5), 5.41 (1H, t, J=4.8), 5.67 (1H, d, J = 10.5), 5.80 (1H, dt, J=10.5, 1.9), 6.90-7.42 (20H, m). 13C NMR: δ 34.82 (CH2), 37.45 (CH), 43.22 (CH2), 49.69 (CH), 52.25 (CH2), 59.10 (CH), 63.84 (CH), 64.62 (CH2), 71.35 (CH), 126-129. (12  CH, ArH), 127.28 (CH, olefine), 131.45 (CH, olefine), 137.88 (C, Ar), 138.71 (C, Ar), 140.45 (C, Ar), 140.75 (C, Ar), 172.90 (C). HPLC: elution time 14.4 min (98% at 220 nm). HR-MS: expected for C36H38N2O3 547.2961; found 547.3001; 7.3 ppm. (2R)-N-Benzyl-2-{N-benzyl-N-[(1S,2R,5S,6R)-6-hydroxy-2-(hydroxymethyl)-5-phenyl-3-cyclohexene-1-yl]amino}-2-phenylacetamide 15b. 1H NMR: δ 2.81 (2H, broad s), 3.33 (1H, broad s), 3.70-3.90 (3H, m), 4.29-4.48 (5H, m), 4.83 (1H, t, J=6.3), 5.63 (1H, d, J=9.9), 5.81 (1H, t, J=5.4), 5.94 (1H, dt, J=9.9, 2.1), 6.84-7.47 (20H, m). 13C NMR: δ 37.35 (CH), 43.40 (CH2), 48.99 (CH), 52.82 (CH2), 58.50 (CH), 63.02 (CH2), 66.02 (CH), 71.74 (CH), 126-129 (12  CH, ArH), 127.70 (CH, olefine), 133.03 (CH, olefine), 137.63 (C, Ar), 138.07 (C, Ar), 140.58 (C, Ar), 140.93 (C, Ar), 172.94 (C). HPLC: elution time 14.3 min (100% at 220 nm). HR-MS: expected for C35H36N2O3 533.2804; found 533.2800; -0.8 ppm. (2R)-N-(4-Methoxyphenyl)-2-{N-benzyl-N-[(1R,4R,5S,6R)5-hydroxy-6-(hydroxymethyl)-4-phenyl-2-cyclohexene-1-yl] amino}-3-phenylpropanamide 14c. 1H NMR: δ 1.74 (2H, m), 2.88 (1H, broad s), 3.13 (1H, dd, J=11.9, 1.8), 3.43-3.56 (2H, m), 3.66-3.85 (3H, m), 3.74 (3H, s), 3.90 (1H, d, J=8.5), 3.98 (1H, d, J=13.8), 4.02-4.12 (1H, m), 4.14 (1H, d, J=13.8), 5.89 (1H, ddd, J=10.3, 4.9, 2.4), 6.22 (1H, d, J=10.3), 6.77 (1H, d, J= 9.0), 7.18-7.38 (19H, m, ArH). 13C NMR: δ 33.37 (CH2), 42.34 (CH), 46.59 (CH), 52.38 (CH2), 55.41 (CH3), 56.22 (CH), 61.13 (CH2), 65.44 (CH), 69.72 (CH), 114.10 (CH, ArH), 121.58 (CH, ArH), 126.37 (CH, ArH), 127.32 (CH, ArH), 127.39 (CH, ArH), 128-130 (6  CH, ArH), 129.63 (CH, olefine), 130.30 (CH, olefine), 130.50 (C, Ar), 137.94 (C, Ar), 138.77 (C, Ar), 139.96 (C, Ar), 156.41 (C, Ar), 171.08 (C). HPLC: 13.8 min (95% at 220 nm). HR-MS: expected for C36H38N2O4 563.2910; found 563.2899, -1.9 ppm.

Article

(2R)-N-(4-Methoxyphenyl)-2-{N-benzyl-N-[(1S,2R,5S,6R)-6hydroxy-2-(hydroxymethyl)-5-phenyl-3-cyclohexene-1-yl]amino}-3-phenylpropanamide 15c. 1H NMR: δ 1.51 (1H, d, J = 2.3), 2.87 (1H, broad s), 3.21-3.50 (5H, m), 3.70 (1H, broad s), 3.74 (3H, s), 3.77-3.80 (1H, m), 3.94 (1H, d, J = 13.7), 3.92-3.98 (1H, m), 4.42 (1H, broad s), 4.50 (1H, d, J=13.7), 5.66 (1H, d, J = 10.2), 5.78 (1H, dt, J = 10.2, 1.9), 6.77 (2H, d, J = 9.0), 7.12-7.43 (17H, m, ArH). 13C NMR: δ 34.78 (CH2), 37.39 (CH), 49.68 (CH), 52.30 (CH2), 55.42 (CH3), 60.00 (CH), 64.05 (CH), 65.29 (CH2), 70.51 (CH), 114.02 (CH, ArH), 121.79 (CH, ArH), 126.37 (CH, ArH), 126.93 (CH, olefine), 127.36 (CH, ArH), 127.50 (CH, ArH), 128-129 (6  CH, ArH), 130.54 (CH, olefine), 131.14 (C, Ar), 139.00 (C, Ar), 139.98 (C, Ar), 140.41 (C, Ar), 156.37 (C, Ar), 171.27 (C). HPLC: 13.6 min (96% at 220 nm). HR-MS: expected for C36H38N2O4 563.2910; found 563.2892; -3.2 ppm. N-Benzyl-2-{N-[(1R,4R,5S,6R)-5-hydroxy-6-(hydroxymethyl)4-phenyl-2-cyclohexene-1-yl]amino}acetamide 19a. 1H NMR: δ 1.83 (1H, m), 2.16 (3H, broad s), 3.16 (1H, d, J = 5.2), 3.39 (1H, d, J=17.0), 3.46 (1H, d, J=17.0), 3.59 (1H, broad s), 3.72 and 3.76 (2H, part AB of an ABX system Jax=4.0, Jbx=4.0, Jab = 9.7), 3.97 (1H, dd, J = 8.8, 5.4), 4.45 (2H, d, J=5.1), 5.78 (1H, ddd, J=10.0, 4.0, 1.4), 5.93 (1H, d, J=10.0), 7.27-7.37 (10H, m), 7.57 (1H, t). 13C NMR: δ 43.09 (CH), 43.16 (CH2), 45.96 (CH), 49.43 (CH2), 55.68 (CH), 62.55 (CH2), 70.10 (CH), 127-130 (6  CH, ArH), 128.39 (CH, olefine), 128.99 (CH, olefine), 138.14 (C, Ar), 138.21 (C, Ar), 171.59 (C). HPLC: elution time 10.3 min (100% at 220 nm). HR-MS: expected for C22H26N2O3 366.1943; found 366.1943; 0.0 ppm. N-Benzyl-2-{N-[(1S,2R,5S,6R)-6-hydroxy-2-(hydroxymethyl)5-phenyl-3-cyclohexene-1-yl]amino}acetamide 20a. 1H NMR: δ 2.43 (4H, broad s), 2.80 (1H, dd, J = 9.9, 1.7), 3.40 (2H, s), 3.56 (1H, broad s), 3.63 (1H, dd, J=10.6, 7.4), 3.87 (1H, dd, J = 10.6, 3.8), 3.99 (1H, broad s), 4.42 and 4.47 (2H, part AB of an ABX system Jax = 5.0, Jbx = 5.0, Jab = 13.7), 5.60 (1H,dt, J = 10.2, 2.3), 5.70 (1H, d, J=10.2), 7.16-7.32 (11H, m). 13C NMR: δ 39.24 (CH), 43.37 (CH2), 47.24 (CH), 49.42 (CH2), 61.54 (CH), 66.30 (CH2), 67.71 (CH), 127-129 (6  CH, ArH), 128.37 (CH, olefine), 128.57 (CH, olefine), 138.10 (C, Ar), 140.68 (C, Ar), 171.63 (C). HPLC: elution time 9.6 min (100% at 220 nm). HR-MS: expected for C22H26N2O3 366.1943; found 366.1911, -8.7 ppm. N-(4-Methoxyphenyl)-2-{N-[(1R,4R,5S,6R)-5-hydroxy-6-(hydroxymethyl)-4-phenyl-2-cyclohexene-1-yl]amino}acetamide 19b. 1 H NMR: δ 1.96 (1H, m), 2.05 (3H, broad s), 3.23 (1H, d, J=5.0), 3.48 (1H, d, J = 16.9), 3.55 (1H, d, J = 16.9), 3.61 (1H, t, J = 3.6), 3.78 (3H, s), 3.87 (2H, d, J = 5.7), 4.04 (1H, dd, J = 8.5, 5.4), 5.83 (1H, dd, J = 10.1, 3.9), 6.00 (1H, dt, J = 10.1, 1.8), 6.84 (2H, d, J= 9.0), 7.24-7.38 (5H, m), 7.47 (2H, d, J=9.0), 9.21(1H, s). 13C NMR: δ 43.44 (CH), 45.85 (CH), 49.91 (CH2), 55.45 (CH and CH3), 62.62 (CH2), 70.23 (CH), 114.13 (CH, ArH), 121.26 (CH, ArH), 127.49 (CH, olefine), 128.57 (CH, ArH), 128.62 (CH, olefine), 128.72 (CH, ArH), 129.92 (CH, ArH), 130.78 (C, Ar), 138.14 (C, Ar), 156.30 (C, Ar), 169.45 (C). HPLC: elution time 10.2 min (100% at 220 nm). HR-MS: expected for C22H26N2O4 382.1893; found 382.1890; -0.7 ppm. N-(4-Methoxyphenyl)-2-{N-[(1S,2R,5S,6R)-6-hydroxy-2-(hydroxymethyl)-5-phenyl-3-cyclohexene-1-yl]amino}acetamide 20b. 1 H NMR (CD3OD): δ 2.30 (1H, broad s), 2.90 (1H, dd, J = 9.8, 1.0), 3.45 (1H, d, J = 17.3), 3.51 (1H, d, J = 17.3), 3.60 (1H, broad s), 3.77 (3H, s), 3.82 (2H, dd, J = 10.8, 7.1), 3.95 (2H, dd, J=10.8, 3.8), 4.10 (1H, broad s), 5.69 (1H, d, J=11.5), 5.74 (1H, dt, J=11.5, 2.4), 6.84 (2H, d, J=9.0), 7.22-7.38 (5H, m), 7.52 (2H, d, J = 9.0). 13C NMR: δ 39.71 (CH), 47.57 (CH), 49.91 (CH2), 55.52 (CH3), 61.03 (CH), 66.15 (CH2), 67.80 (CH), 114.25 (CH, ArH), 121.31 (CH, ArH), 127.31 (CH, olefine), 128.39 (CH, olefine), 128.55 (CH, ArH), 128.60 (CH, ArH), 128.90 (2  CH, ArH), 131.11 (C, Ar), 140.47 (C, Ar), 156.45 (C, Ar), 169.55 (C). HPLC: elution time 9.5 min (100% at 220 nm). HR-MS: expected for C22H26N2O4 382.1893; found 382.1887; -1.5 ppm.

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Molecular Docking. All ligands structures were built and their geometries optimized through MacroModel 8.5 software22 package and using the MMFFs force field.23 MonteCarlo Multiple Minimum (MCMM) method (10000 steps) of the MacroModel package22 was used in order to allow a full exploration of the conformational space. The so obtained geometries were optimized using the Polak-Ribier conjugate gradient algorithm (PRCG, 9  107 steps, maximum derivative less than 0.001 kcal/mol). A GB/SA (generalized Born/surface area)24 solvent treatment was used, mimicking the presence of H2O, in the geometry optimization and in the conformational search steps. The proteins for the docking calculations were prepared using MacroModel software: all hydrogen were added, bond order and missing atoms were checked by visual inspection, and the charges of side chains were assigned considering their pKa. Autodock 3.0.5 software25 was used for all docking calculations belonging. Before performing the virtual screening of the libraries (Table 1), the validation of docking methodology was done by using as reference structure the NMR (2O2M.pdb)26 A 3D model of the complex formed by Bcl-xL and N-acylsulfonamide ligand was deposited in the Protein Data Bank (PDB).27,28 In detail, a blind docking was performed to verify the ability of search method to detect the well-known binding cavity. In a subsequent step, the conformational search parameters were validated docking the N-acylsulfonamide ligand in the binding site and evaluating the skill of the search method to reproduce the experimental bioactive conformation of the ligand from the NMR solution structure. For the blind docking calculations, a grid box size of 256  256  256 with spacing of 0.375 A˚ between the grid points, centered on the macromolecule, covering all protein surface, was used. Ten calculations consisting of 256 runs were performed, obtaining 2560 structures (256  10). The protein, derived from the NMR solution structure 2O2M (pdb archive code), was used as Bcl-xL model for the docking calculations. For all the docked structures, all bonds were treated as active torsional bonds except the amide bonds. A grid box size of 64  64  64 with spacing of 0.375 A˚ between the grid points was used, presenting a grid center with the following x, y, and z coordinates respectively: 3.675, 5.538, and 2.0. In the theoretical studies, we considered the amine functionality of the ligands as protonated at physiological pH and, consequently, positively charged in the calculations. To achieve a representative conformational space during the docking studies and taking into account the variable number of active torsions, from 8 to 10 calculations consisting of 256 runs were performed, obtaining 2048/2560 structures. The Lamarkian genetic algorithm was employed for dockings. An initial population of 600 randomly placed individuals. The maximum number of energy evaluations and of generations was set up on the number of active torsions presented by the different compounds. The first parameter ranged from 4  106 to 6  106 and the maximum number of generations from 5  106 to 7  107. A mutation rate of 0.02 and a crossover rate of 0.8 were used, and the local search frequency was set up at 0.26. Results differing by less than 2 A˚ in positional root-mean-square deviation (rmsd) were clustered together and represented by the result with the most favorable free energy of binding. All the 3D models were depicted using the UCSF Chimera package:29 molecular surfaces were rendered using Maximal Speed Molecular Surface (MSMS).30 The 3D models of the protein are represented by hydrophobicity surface showing the amino acid property in the Kyte-Doolittle scale.31 The hydrophobicity surface colors range from dodger blue for the most hydrophilic amino acid to orange-red for the most hydrophobic one, with the white representing the 0.0 value of the Kyte-Doolittle scale.31 NMR Experiments. NMR experiments were performed on a Bruker Avance 700 spectrometer fitted with a cryoprobe at T = 300 K. 1D 1H experiments were run with 500 μL of solution

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(deuterated buffer at pH = 7.5) of Bcl-xL at 20 μM, in the absence and presence of added compounds, everyone at 250 μM concentration. In the final concentrations of all NMR solutions, a 2% of DMSO-d6 99.95% was used. The same conditions were employed for 2D-NOESY experiments but with concentrations of Bcl-xL and ligands of 10 μM and 1 mM, respectively. The NOESY32 spectra in D2O were acquired with a mixing time of 500 ms, a number of 16 scans/t1, and a t1max value of 15.24 ms. All 2D spectra were acquired in the phase-sensitive mode, and the TPPI method33 was used for quadrature detection in the ω1 dimension. In all experiments, a watergate PFG sequence was used for water suppression. The unlabeled Bcl-xL samples were prepared and purified as reported in literature.12 Briefly, Escherichia coli strain BL21 was transformed with the pET-21b plasmid (Novagen) carrying the gene coding for Bcl-xL ΔTM (Bcl-xL deletion mutant lacking the transmembrane domain). Bacteria were grown on LB supported media. Induction of protein expression was carried out at OD600 = 0.8 with 1 mM IPTG for 4 h at 37 °C. Following cell harvest and lysis by sonication, the protein was purified using a Niaffinity column (Amersham). The eluate was extensively dialyzed against 40 mM phosphate buffer (pH = 7.5). Fluorescence Anisotropy. The formation of heterodimers was measured in terms of increased anisotropy (Polarion, Tecan Co.) using fluoresceinated Bak peptide as natural ligand. All the experiments were performed in polar buffer (HEPES pH 7, 100 mM; KCl, 250 mM; MgCl2, 15 mM; DTT, 5 mM; EDTA, 5 MM); the experiment volume was always 200 μL per well, and the plates used were 96-well black nonbonding plates (Corning). Protein and ligands were mixed at room temperature and maintained at target temperature (30 °C) in the dark for the whole duration of the experiments. Anisotropy measurements were done at different times of incubation to assess the stability of the reactions. Two sets of experiments were carried out with the following concentrations: experiment-1 Bcl-xL 200 nM, Fluo-Bak 20 nM, inhibitor 2 μM; experiment-2 Bcl-xL 100 nM, Fluo-Bak 10 nM, inhibitor 20 μM.

Acknowledgment. We thank MIUR (PRIN contract number 2006031151_005), and the EMBO for the financial support of SDM. Supporting Information Available: 1H- and 13C NMR spectra for compounds 14a,c, 15a-c, 19a,b, and 20a,b. 2D-NOESY spectra of 19a,b, 20a,b, ent-19a,b, and ent-20a,b in presence and absence of Bcl-xL acquired at 300 K (pH 7.5) in D2O solution; calculated inhibition constant of the complex formed by 19a, b-20a,b, ent-19a,b-20a,b, 59-64, and ent-59-64 with Bcl-xL; calculated fitness scores of the complex formed by 19a,b-20a,b, and ent-19a,b-20a,b with Bcl-xL; superimposition of docked poses of 59-64 and ent-59-64. This material is available free of charge via the Internet at http://pubs.acs.org.

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