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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2013 October 11.
Published in final edited form as:
PMCID: PMC3495162
NIHMSID: NIHMS412127

Structure-Based Discovery of BM-957 as a Potent Small-Molecule Inhibitor of Bcl-2 and Bcl-xL Capable of Achieving Complete Tumor Regression

Abstract

Bcl-2 and Bcl-xL anti-apoptotic proteins are attractive cancer therapeutic targets. We have previously reported the design of 4,5-diphenyl-1H-pyrrole-3-carboxylic acids as a class of potent Bcl-2/Bcl-xL inhibitors. In the present study, we report our structure-based optimization for this class of compounds based upon the crystal structure of Bcl-xL complexed with a potent lead compound. Our efforts accumulated into the design of compound 30 (BM-957), which binds to Bcl-2 and Bcl-xL with Ki <1 nM and has low nanomolar IC50 values in cell growth inhibition in cancer cell lines. Significantly, compound 30 achieves rapid, complete and durable tumor regression in the H146 small-cell lung cancer xenograft model at a well-tolerated dose-schedule.

Introduction

Apoptosis is a tightly regulated cellular process to eliminate damaged and unwanted cells and plays a critical role in normal development and homeostasis of multicellular organisms. Evasion of apoptosis is a hallmark of human cancer 13 and targeting key apoptotic regulators with the goal of restoring apoptosis in tumor cells is a promising cancer therapeutic strategy. 2, 4

The B-cell lymphoma 2 (Bcl-2) family proteins are key regulators of apoptosis and consist of both pro-apoptotic and anti-apoptotic members.57 Pro-apoptotic Bcl-2 proteins are structurally subdivided into two groups: those like Bax, Bak and Bok, which contain three Bcl-2 homology (BH) domains (BH1-BH3) and Bad, Bid, Bim, Bik, Puma, Noxa, and others that contain only the BH3 domain.8 Although the precise mechanism of the Bcl-2 and Bcl-xL pro-survival function is not completely understood, it is clear that these proteins inhibit apoptosis by directly binding to and sequestering their pro-death counterparts such as Bim, Bid and Bad.9 Bcl-2 and Bcl-xL are frequently overexpressed in cancer cell lines and human cancer tissues. Such overexpression helps cancer cells suppress a variety of apoptotic stimuli including those associated with cancer chemotherapeutic agents and confers on tumor cells resistance to current therapeutic agents.10, 11 Thus, inhibition of these anti-apoptotic Bcl-2 family members offers an attractive approach for the development of new cancer therapeutics.

Experimentally determined three-dimensional structures of Bcl-2 and its closely related homologous protein Bcl-xL showed that the BH1, BH2 and BH3 domains in these proteins form a well-defined hydrophobic surface binding groove (the BH3 binding groove), which interacts with BH3-only pro-apoptotic proteins such as Bad, Bid, and Bim.1214 It has been proposed that small-molecule inhibitors designed to bind to the BH3 surface binding groove in Bcl-2 and Bcl-xL will inhibit their anti-apoptotic function and promote apoptosis in tumor cells with high expression of these proteins. In the past ten years, a number of classes of non-peptide, small-molecule inhibitors have been designed to target the BH3 binding grooves in Bcl-2 and Bcl-xL proteins.1536 ABT-737 (1) and its analogue ABT-263 (2) (Figure 1), from Abbott Laboratories, represent two of the most potent small molecule inhibitors of Bcl-2 and Bcl-xL proteins reported to date. Both compounds bind to Bcl-2 and Bcl-xL with very high affinities (Ki < 1 nM) and efficiently induce tumor regression in multiple xenograft tumor models.2432 In Phase I/II clinical trials, ABT-263 shows evidence of promising antitumor activity in patients in chronic lymphocytic leukemia37 but has a very limited single-agent activity in patients with small cell lung cancer38.

Figure 1
Chemical structures of previously reported representative potent Bcl-2/Bcl-xL inhibitors.

Our laboratory has recently reported the design of compound 3 as a potent Bcl-2 and Bcl-xL inhibitor.39 Compound 3 binds to Bcl-2 and Bcl-xL with Ki values of <1 nM and potently inhibits cancer cell growth with IC50 values of approximately 10 nM in multiple cancer cell lines. Compound 3 is also capable of achieving a strong in vivo antitumor activity in the H146 xenograft model in mice at a well-tolerated dose-schedule. Although compound 3 shows strong in vivo antitumor activity and in fact induces tumor regression during the treatment in the H146 xenograft tumor model, it fails to achieve durable tumor regression. After the treatment was stopped, tumors quickly regrew, suggesting that further optimization is needed toward our goal of obtaining a highly optimized Bcl-2/Bcl-xL inhibitor for clinical development. In the present study, we have performed further structure-based optimization for this class of compounds based upon a co-crystal structure of Bcl-xL complexed with compound 4 (BM-903), an analogue of compound 3 (Figure 2). Our efforts accumulated into the design of a new, highly potent Bcl-2/Bcl-xL inhibitor, which is capable of achieving complete and durable tumor regression in the H146 xenograft tumor model.

Figure 2
(A)–(D). Co-crystal structure of compound 4 (BM-903) complexed with Bcl-xL. Compound 4 is colored in yellow and the surface representation of Bcl-xL is shown. Residues interact with the nitro-phenyl and the thio-phenyl groups on the compounds ...

Results and Discussion

Analysis of the co-crystal structure of compound 4 complexed with Bcl-xL showed that the nitro group in 4 binds to a hydrophobic pocket, which can accommodate a larger group than the nitro group (Figure 2B). We have therefore modified the nitro group to determine structure-activity relationship at this site. Since 4 binds to Bcl-2 and Bcl-xL with very high affinities (Ki value <1 nM to both Bcl-2 and Bcl-xL), exceeding the lower limits of our binding assays for these two proteins, we designed and synthesized compound 5 (BM-916, Table 1) as a less potent but more soluble compound and used it as the template for further modifications of the nitro group. Compound 5 has a Ki value of 31.3 nM to Bcl-2 and 37.7 nM to Bcl-xL, respectively (Table 1).

Table 1
Structure-activity relationships of the nitro group replacements.

To determine the contribution of the nitro group, we first synthesized compound 6, in which the nitro group is replaced with a hydrogen atom. Compound 6 is 18- and 30-times less potent than 5 in its binding affinities to Bcl-2 and Bcl-xL, respectively, confirming the importance of the nitro group.

Since the nitro group inserts into a small hydrophobic pocket in Bcl-xL in the crystal structure of 4 complexed with Bcl-xL (Figure 2), we designed and synthesized a series of analogues of 5 by replacing the nitro group with a small hydrophobic group. These include 7-9, in which the nitro group is replaced with a halogen atom (F, Cl and Br), 10-13, in which the nitro group is replaced with a small alkyl group, 14-17 with a small alkyloxyl group, and 18-21 with an alkylsulfonyl group. The binding data (Table 1) show that with the exception of compounds 9 and 18, in which the nitro group is replaced by Br and trifluoromethylsulfonyl, respectively, these analogues bind to Bcl-2 and Bcl-xL 5-20 times less potently than 5. While 9 is still slightly less potent than 5, 18 is as potent as 5 in binding to both Bcl-2 and Bcl-xL.

Having identified trifluoromethylsulfonyl as a suitable replacement group for the nitro group for effective interaction with both Bcl-2 and Bcl-xL, we next synthesized 22, in which the nitro group in 4 is replaced with trifluoromethylsulfonyl. Compound 22 binds to Bcl-2 and Bcl-xL with high affinities (Ki = 2.1 nM for Bcl-2 and < 1 nM for Bcl-xL). Similar to 3 and 4, compound 22 has no appreciable binding to Mcl-1 at concentrations as high as 5 μM, indicating that it is a potent and specific Bcl-2/Bcl-xL inhibitor.

We next evaluated 22 for its activity in inhibition of cell growth in the H1417 and H146 small-cell lung cancer cell lines, which are sensitive to potent and specific Bcl-2/Bcl-xL inhibitors such as compounds 1-4.24, 27, 40 Compound 22 potently inhibits cell growth in the H1417 and H146 cancer cell lines, with IC50 values of 151 nM and 98 nM, respectively. The binding and cellular data thus indicated that 22 is a promising lead compound for further modifications.

The co-crystal structure (Figure 2C) also showed that the dimethylamino group of compound 4 forms hydrogen bonds with side chains of E96 and Y195 residues of Bcl-xL. We next modified the dimethylamino group in 22 using different sized nitrogen containing rings with or without a hydroxyl group. Compound 23 with morpholinyl shows weaker binding affinity than 22 to Bcl-2, but all other compounds have similar potencies to both Bcl-2 and Bcl-xL, as compared to 22. Consistent with its weaker binding affinity to Bcl-2, compound 23 is 3–5 times less potent than 22 in inhibition of cell growth in both H146 and H1417 cancer cell lines. All other compounds show similar potencies with IC50 values of 100–200 nM in inhibition of cell growth in the H1417 and H146 cell lines.

In our subsequent in vivo evaluation, we found that compound 28 is effective in inhibition of tumor growth in the H146 tumor xenograft model at a well-tolerated dose-schedule (see below). Therefore, we focused subsequent modifications on 28.

The co-crystal structure (Figure 2D) shows that the N-methyl group in the core structure of compound 4 inserts into the well-defined hydrophobic pocket formed by L112, V126 and F146 in Bcl-xL and there is more space available in this pocket to accommodate a larger hydrophobic group than methyl. We therefore probed this hydrophobic binding pocket by replacing the methyl in compound 28 with ethyl, propyl, isopropyl and butyl groups (Table 3). Compound 30 with ethyl and compound 31 with isopropyl bind to both Bcl-2 and Bcl-xL with very high affinities. While 30 and 31 bind to Bcl-2 with IC50 values of 5.4 and 4.0 nM, respectively (Ki values = 1.2 and 0.8 nM, respectively), they bind to Bcl-xL with IC50 values of 6.0 and 3.9 nM, respectively (Ki values < 1 nM). However, compound 32 with propyl and compound 33 with butyl have lower affinities to Bcl-2 than 30 and 31. Interestingly, these four compounds have similar high binding affinities to Bcl-xL. Similar to 22, compounds 2833 have no appreciable binding to Mcl-1 at concentrations as high as 5 μM.

Table 3
Modifications of the N-methyl site in compound 28.

Testing of compounds 3033 in the H1417 and H146 cell lines showed that these compounds are very potent in inhibition of cell growth and are 5–10 times more potent than 28. While 30 has IC50 values of 21 nM and 22 nM, respectively, in these two cancer cell lines, compound 31 has IC50 values of 9 and 13 nM, respectively. However, compounds 32 and 33 are >10-times less potent than 30 and 31.

Further in vitro evaluations of potent Bcl-2 and Bcl-xL inhibitors

We next tested compounds 28, 30 and 31 for their ability to induce cell death in the H146 cancer cell line, in direct comparison to compounds 1 and 2. The results are shown in Figure 3.

Figure 3
Induction of cell death by compounds 1, 2, 28, 30 and 31 in the H146 cell line. Cells were treated with different concentrations of the compounds for 24 hr. Cell viability was determined using a trypan blue exclusion assay.

All these compounds induce cell death in a dose-dependent manner but have different potencies. While 28 is somewhat less potent than 1 and 2, 30 and 31 are several times more potent than 1 and 2. For example, 30 and 31 at 10 nM with 24 hr–treatment induces >50% of the H146 cells to undergo cell death, whereas 1 and 2 at 30–100 nM have a similar effect.

We further tested compounds 2, 28 and 30 in the H146 cell line for their ability to induce cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3, two key biochemical markers of apoptosis. The results are shown in Figure 4. Compound 30 at 10 nM, 28 at 100 nM and 2 at 30 nM all induce clear cleavage of PARP and activation of caspase-3 and have similar effects. Hence, the potencies for these three compounds in induction of cleavage of PARP and activation of caspase-3 in the H146 cells are consistent with their potencies in induction of cell death.

Figure 4
Western blot analysis of biochemical markers of apoptosis in H146 cancer cells treated with compounds 2, 28 and 30. H146 cancer cells were treated with indicated concentrations of each compound for 24 hr. PARP, cleaved PARP (Cl PARP), caspase-3 and cleaved ...

In vivo evaluation of compounds 28, 30 and 31

We determined the maximum tolerated dose (MTD) for compounds 28, 30 and 31 in severe combined immunodeficiency (SCID) mice. It was found that 28 at 50 mg/kg, 30 at 25 mg/kg and 31 at 10 mg/kg, daily, intravenous dosing, 5 days a week for 2 weeks were well tolerated in SCID mice and the animals had less than 10% of weight loss. Higher doses of these compounds (75 mg/kg for 28, 50 mg/kg for 30 and 25 mg/kg for 31) caused more than 10% of weight loss. These experiments established the MTDs for these three compounds in SCID mice for our subsequent pharmacodynamics (PD) and efficacy experiments. Based upon the toxicity data, we decided to further evaluate compounds 28 and 30 for their in vivo antitumor activity.

We tested compounds 28 and 30 for their ability to induce cleavage of PARP and activation of caspase-3 in the H146 xenograft tumor tissue in mice at their respective MTD in a pharmacodynamics experiment. Mice bearing H146 tumors were given a single i.v. dose of 28 at 50 mg/kg or 30 at 25 mg/kg. The mice were then sacrificed at 3-, 6- and, 24-h time points and tumors were harvested for analysis. Western blotting analysis showed that both 28 and 30 effectively induce robust cleavage of PARP and caspase-3 in H146 tumor tissues at 3- and 6-h time-points, indicative of strong apoptosis induction by both compounds in tumor tissues (Figure 5).

Figure 5
Induction of cleavage of PARP and caspase-3 in H146 xenograft tumors by compounds 28 and 30. SCID mice bearing H146 xenograft tumors (100–200 mm3) were treated with vehicle control, single dose of 28 (50 mg/kg, i.v.) or 30 (25 mg/kg, i.v.). Mice ...

Based upon the encouraging in vivo pharmacodynamic data, we next evaluated 28 and 30 for their antitumor efficacy in the H146 xenograft tumor model (Figure 6). Our data showed that although compound 28 at 50 mg/kg effectively inhibits tumor growth, it fails to induce tumor regression. In contrast, compound 30 at 25 mg/kg is capable of achieving complete tumor regression. Of 7 mice treated with 30, all mice were tumor-free at day 47 (15 days post-treatment) and five (71%) remained tumor-free on day 58 (28 days post-treatment). Similar to the data obtained from our MTD experiment, both compounds 28 and 30 are well tolerated in tumor-bearing animals. All treated animals experienced less than 10% weight loss compared to the vehicle control and all regained their weight quickly after the treatments were finished. This in vivo experiment thus established that 30 achieves complete and durable tumor regression in the H146 xenograft tumor model and is more efficacious than 28.

Figure 6
Antitumor activity of compounds 28 and 30 in H146 xenograft tumor model. SCID mice bearing xenograft tumors (100 mm3) were treated with vehicle control, compound 28 at 50 mg/kg, i.v. or compound 30 at 25 mg/kg, i.v., daily, 5 days a week for two weeks. ...

Synthesis of Designed Bcl-2 family protein inhibitors

The general synthetic route to compounds in Table 1 and Table 2 is shown in Scheme 1. Compound 34, which was prepared according to a previously reported method 39, was coupled with 3-(4-Methyl-piperazin-1-yl)-propylamine to give phenylnitro 35. Reduction of the phenylnitro under hydrogen atmosphere in the presence of Pd/C yielded the corresponding aniline, which was subjected to different m-substituted benzenesulfonyl chloride in pyridine to generate compounds in Table 1.

Scheme 1
Synthesis of target compounds in Table 1 and Table 2.
Table 2
Structure-activity relationships of the tail soluble group.

Using a converged synthetic strategy, compounds in Table 2 were prepared by coupling amine 37a-f with highly activated fluoro containing intermediate 38. Amine 37a-f were obtained by treatment of 36 with different amines, followed by removal of the Fmoc group with Et2NH and reduction with borane. Phenylnitro 34 was reduced by hydrogenation and then treated with commercially available 4-fluoro-3-(trifluoromethylsulfonyl)benzenesulfonyl chloride to yield 38.

The general synthetic route for compounds in Table 3 is shown in Scheme 2. Briefly, condensation of 39 with primary amines resulted in pyrrole 40a-b. Phenylnitro 41a-b were prepared by Ullman coupling of 1-(p-nitrophenyl)piperazine with 40a-b, followed by hydrolysis of these ethyl esters, which yielded free carboxylic acid 42a-b. Reduction of the nitro group of 42a-b and 42c-d39, followed by coupling of 4-fluoro-3-(trifluoromethylsulfonyl)benzene-1-sulfonyl chloride, yielded sulfonanilide 43a-d, treatment of which with 37e in the presence of DIPEA in DMF, gave compounds in Table 3.

Scheme 2
Synthesis of target compounds in Table 3.

Summary

We have performed further structure-based optimization for a new class of Bcl-2/Bcl-xL inhibitors containing 4,5-diphenyl-1H-pyrrole-3-carboxylic acid as the core structure. Our efforts accumulated into the design of compound 30 (BM-957), which binds to Bcl-2 and Bcl-xL with the Ki values <1 nM and shows potent activity in cell growth inhibition in cancer cell lines, with IC50 values of ~20 nM against the H1147 and H146 small-cell lung cancer cell lines. Compound 30 induces robust cleavage of PARP and caspase-3 in 24 h at concentrations as low 10 nM in the H146 cell line. In vivo, compound 30 achieves complete and durable tumor regression in H146 xenograft tumors and is thus more efficacious than compound 3 in the same tumor model. The efficacy data thus suggest that compound 30 has a superior pharmacokinetic property to compound 3. Taken together, compound 30 represents a promising Bcl-2/Bcl-xL inhibitor for extensive evaluations as a new anticancer agent.

Experimental Section

Chemistry

General Methods

Unless otherwise noted, all reactions were performed under nitrogen atmosphere in dry solvents under anhydrous conditions and reagents were used as supplied without further purification. NMR spectra were acquired at a proton frequency of 300 MHz and chemical shifts are reported in parts per million (ppm) relative to an internal standard. The final products were purified by a C18 reverse phase semi-preparative HPLC column with solvent A (0.1% of TFA in H2O) and solvent B (0.1% of TFA in CH3CN) as eluents. All the target compounds have purities of >95% based upon UPLC analysis. Compounds 1 (ABT-737) and 2 (ABT-263) were purchased from SellechBio.com and their purity was confirmed to be >95% based upon UPLC analysis.

5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(3-(4-(4-nitro phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (35)

A solution of 34 (1.06 g, 2.0 mmol), 3-(4-methylpiperazin-1-yl)propan-1-amine (472 mg, 3.0 mmol), EDCI (768 mg, 4.0 mmol), and DMAP (244 mg, 2.0 mmol) in CH2Cl2 (10 mL) was stirred for 3 h until no 34 was observed by TLC. The mixture was diluted with EtOAc (200 mL), washed sequentially with 1 M HCl (50 mL), H2O (50 mL), and brine (20 mL), dried (Na2SO4), filtered, and concentrated. The concentrate was flash chromatographed on silica gel with 5% MeOH/CH2Cl2 to provide 1.15 g (86%) of 35. 1H NMR (300 MHz, CDCl3): δ 8.14 (d, J = 10.8 Hz, 2H), 7.27-7.05 (m, 5H), 6.89-6.68 (m, 5H), 3.53-3.47 (m, 4H), 3.42 (s, 3H), 3.26-3.19 (m, 6H), 2.61 (s, 3H), 2.45-2.09 (m, 8H), 2.24 (s, 3H), 2.11 (t, J = 6.4, 2H), 1.50-1.41 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 165.99, 154.57, 150.26, 138.54, 135.84, 133.68, 133.31, 132.28, 130.58, 129.41, 129.17, 128.42, 125.88, 122.37, 121.04, 118.58, 114.83, 114.33, 112.67, 55.90, 55.07, 53.48, 53.03, 48.39, 46.72, 46.00, 37.63, 31.58, 26.47, 11.40. MS (ESI): m/z 670.92 (M + H)+.

General Procedure I

5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl) propyl)-4-(3-(4-(4-(3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (5)

To a solution of 35 (100 mg, 0.15 mmol) in 15 mL of methanol was added 10% wt. Pd/C (10 mg, 0.1 eq. m/m). The solution was stirred under hydrogen atmosphere for about 20 min at room temperature until no 35 was observed by TLC. The reaction mixture was filtered and the filtrate was concentrated in vacuum. The residue was used for next step directly without purification. To the solution of this aniline in pyridine, 3-nitrobenzene-1-sulfonyl chloride (40 mg, 0.18 mmol) was added at 0 °C. The mixture was stirred at 0°C to room temperature for 1 hour until no aniline was observed by TLC. H2O (10 mL) was added and the mixture was extracted with EtOAc (2×30 mL). The EtOAc solution was washed with brine (50 mL), dried over Na2SO4 and concentrated in vacuo. The concentrate was purified by HPLC to give the pure product 5 (as the trifluoroacetate salt, 89 mg), yield 72% over two steps. The gradient ran from 80% of solvent A and 20% of solvent B to 40% of solvent A and 60% of solvent B in 40 min. 1H NMR (300 MHz, CD3OD): δ 8.44-8.39 (m, 2H), 8.09-8.06 (m, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.32-7.22 (m, 3H), 7.14-7.04 (m, 7H), 6.86 (t, J = 2.2 Hz, 2H), 3.51 (br, 4H), 3.42-3.34 (m, 17H), 2.97 (t, J = 7.3 Hz, 2H), 2.92 (s, 3H), 2.45 (s, 3H), 1.87-1.83 (m, 2H). MS (ESI): m/z 826.00 (M + H)+.

5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(3-(4-(4-(phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (6)

6 was prepared from 35 and benzenesulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.76-7.74 (m, 2H), 7.60-7.45 (m, 3H), 7.34-7.23 (m, 3H), 7.16-7.04 (m, 7H), 6.90-6.86 (m, 2H), 3.52-3.9 (m, 21H), 3.00 (t, J = 7.4 Hz, 2H), 2.93 (s, 3H), 2.47 (s, 3H), 1.89-1.84 (m, 2H). MS (ESI): m/z 780.75 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(3-fluorophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (7)

7 was prepared from 35 and 3-fluorobenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.50-7.36 (m, 3H), 7.26-7.18 (m, 4H), 7.13-6.95 (m, 7H), 6.87-6.81 (m, 2H), 3.52-3.22 (m, 21H), 2.99 (t, J = 7.0 Hz, 2H), 2.88 (s, 3H), 2.37 (s, 3H), 1.82-1.75 (m, 2H). MS (ESI): m/z 799.00 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(3-chlorophenylsulfonamido)phenyl)piperazin-1-yl)phenyl) -1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (8)

8 was prepared from 35 and 3-chlorobenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.67-7.40 (m, 4H), 7.30-7.20 (m, 3H), 7.11-7.00 (m, 7H), 6.87-6.84 (m, 2H), 3.52-3.26 (m, 21H), 2.98 (t, J = 7.4 Hz, 2H), 2.90 (s, 3H), 2.42 (s, 3H), 1.87-1.82 (m, 2H). MS (ESI): m/z 815.42 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(3-bromophenylsulfonamido)phenyl)piperazin-1-yl)phenyl) -1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (9)

9 was prepared from 35 and 3-bromobenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.81-7.64 (m, 3H), 7.38-7.21 (m, 4H), 7.11-7.00 (m, 7H), 6.88-6.82 (m, 2H), 3.53-3.26 (m, 21H), 2.99 (t, J = 6.9 Hz, 2H), 2.91 (s, 3H), 2.42 (s, 3H), 1.87-1.82 (m, 2H). MS (ESI): m/z 859.92 (M + H)+.

5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(3-(4-(4-(3-(tri fluoromethyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (10)

10 was prepared from 35 and 3-trifluoromethylbenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.94-7.86 (m, 3H), 7.68 (t, J = 8.0 Hz, 1H), 7.32-7.01 (m, 10H), 6.88-6.86 (m, 2H), 3.52-3.28 (m, 21H), 2.98 (t, J = 7.5 Hz, 2H), 2.91 (s, 3H), 2.44 (s, 3H), 1.91-1.81 (m, 2H). MS (ESI): m/z 849.08 (M + H)+.

5-(4-Chlorophenyl)-1,2-dimethyl-4-(3-(4-(4-(3-methylphenylsulfonamido)phenyl)piper azin-1-yl)phenyl)-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (11)

11 was prepared from 35 and 3-methylbenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.56-7.51 (m, 2H), 7.38-7.21 (m, 5H), 7.13-7.00 (m, 7H), 6.87-6.84 (m, 2H), 3.52-3.28 (m, 21H), 2.98 (t, J = 7.1 Hz, 2H), 2.91 (s, 3H), 2.44 (s, 3H), 2.33 (s, 3H), 1.90-1.81 (m, 2H). MS (ESI): m/z 795.00 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(3-ethylphenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (12)

12 was prepared from 35 and 3-ethylbenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.58-7.34 (m, 6H), 7.24-7.16 (m, 3H), 7.06-6.94 (m, 5H), 6.80-6.75 (m, 2H), 3.46 (s, 3H), 3.30-3.05 (m, 18H), 2.83 (s, 3H), 2.71-2.64 (m, 4H), 2.50 (s, 3H), 1.77-1.73 (m, 2H), 1.20 (t, J = 7.6, 3H). MS (ESI): m/z 809.00 (M + H)+.

4-(3-(4-(4-(3-tert-Butylphenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-5-(4-chloro phenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (13)

13 was prepared from 35 and 3-tert-butylbenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.58-7.50 (m, 3H), 7.36-7.18 (m, 4H), 7.07-7.01 (m, 7H), 6.87-6.84 (m, 2H), 3.53-3.22 (m, 21H), 2.99 (t, J = 7.2 Hz, 2H), 2.88 (s, 3H), 2.38 (s, 3H), 1.85-1.81 (m, 2H), 1.17 (s, 9H). MS (ESI): m/z 837.00 (M + H)+.

5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(3-(4-(4-(3-(trifluoromethoxy)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (14)

14 was prepared from 35 and 3-trifluoromethoxylbenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.70-7.43 (m, 4H), 7.29-7.20 (m, 3H), 7.10-7.01 (m, 7H), 6.87-6.85 (m, 2H), 3.52-3.25 (m, 21H), 2.98 (t, J = 7.3 Hz, 2H), 2.90 (s, 3H), 2.41 (s, 3H), 1.89-1.79 (m, 2H). MS (ESI): m/z 864.92 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(3-methoxyphenylsulfonamido)phenyl)piperazin-1-yl) phenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (15)

15 was prepared from 35 and 3-methoxybenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.35-7.00 (m, 14H), 6.88-6.84 (m, 2H), 3.71 (s, 3H), 3.54-3.24 (m, 21H), 3.01 (t, J = 6.7 Hz, 2H), 2.91 (s, 3H), 2.40 (s, 3H), 1.89-1.82 (m, 2H). MS (ESI): m/z 811.00 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(3-ethoxyphenylsulfonamido)phenyl)piperazin-1-yl) phenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (16)

16 was prepared from 35 and 3-ethoxybenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.37-7.02 (m, 14H), 6.88-6.86 (m, 2H), 4.01-3.94 (m, 2H), 3.54-3.28 (m, 21H), 3.00 (t, J = 7.1 Hz, 2H), 2.93 (s, 3H), 2.44 (s, 3H), 1.92-1.82 (m, 2H), 1.34 (t, J = 7.0 Hz, 3H). MS (ESI): m/z 825.00 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(3-isopropoxyphenylsulfonamido)phenyl)piperazin-1-yl) phenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (17)

17 was prepared from 35 and 3-isopropoxylbenzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.42-6.95 (m, 14H), 6.81-6.78 (m, 2H), 4.59-4.51 (m, 1H), 3.47 (s, 3H), 3.38-3.10 (m, 18H), 2.85 (s, 3H), 2.75 (t, J = 7.3 Hz, 2H), 2.50 (s, 3H), 1.77-1.75 (m, 2H), 1.28 (d, J = 6.0 Hz, 6H). MS (ESI): m/z 839.08 (M + H)+.

5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(3-(4-(4-(3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (18)

18 was prepared from 35 and 3-(trifluoromethylsulfonyl)benzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 8.29 (t, J = 6.4 Hz, 2H), 8.22 (s, 1H), 7.96 (t, J = 7.9 Hz, 1H), 7.37-7.15 (m, 5H), 7.04-7.00 (m, 5H), 6.83-6.79 (m, 2H), 3.46 (s, 3H), 3.40-3.22 (m, 18H), 2.88 (s, 3H), 2.82 (t, J = 7.3 Hz, 2H), 2.50 (s, 3H), 1.83-1.79 (m, 2H). MS (ESI): m/z 913.25 (M + H)+.

5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(3-(4-(4-(3-(methylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (19)

19 was prepared from 35 and 3-(methylsulfonyl)benzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 8.12-8.02 (m, 3H), 7.74 (t, J = 7.8 Hz, 1H), 7.31-7.23 (m, 3H), 7.12-7.02 (m, 7H), 6.93-6.91 (m, 2H), 3.56-3.36 (m, 21H), 3.06-3.01 (m, 5H), 2.93 (s, 3H), 2.42 (s, 3H), 1.92-1.83 (m, 2H). MS (ESI): m/z 859.00 (M + H)+.

5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-ethylpiperazin-1-yl)propyl)-4-(3-(4-(4-(3-(methylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (20)

20 was prepared from 35 and 3-(ethylsulfonyl)benzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 8.10-8.03 (m, 3H), 7.78 (t, J = 7.9 Hz, 1H), 7.34-7.24 (m, 3H), 7.16-7.05 (m, 7H), 6.92-6.89 (m, 2H), 3.54-3.29 (m, 21H), 3.14 (dd, J = 14.8, 7.41 Hz, 2H), 3.01 (t, J = 7.2 Hz, 2H), 2.94 (s, 3H), 2.46 (s, 3H), 1.90-1.85 (m, 2H), 1.09 (t, J = 7.4 Hz, 3H). MS (ESI): m/z 873.00 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(3-(cyclopropylsulfonyl)phenylsulfonamido)phenyl) piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (21)

21 was prepared from 35 and 3-(cyclopropyl)benzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CD3OD): δ 8.09-8.02 (m, 3H), 7.80 (t, J = 7.8 Hz, 1H), 7.65-6.71 (m, 11H), 6.25 (s, 1H), 5.89 (d, J =1.7 Hz, 1H), 3.46 (s, 3H), 3.37-3.23 (m, 17H), 2.92 (br, 1H), 2.79 (s, 3H), 2.60 (t, J =3.9 Hz, 2H), 2.50 (s, 3H), 1.83 (s, 2H), 1.70-1.68 (m, 2H), 1.32 (s, 2H). MS (ESI): m/z 885.17 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(4-fluoro-3-(trifluoromethylsulfonyl)phenylsulfonamido) phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic Acid (38)

38 was prepared from 34 and 4-fluoro-3-(trifluoromethylsulfonyl)benzene-1-sulfonyl chloride according to general procedure I. 1H NMR (300 MHz, CDCl3): δ 8.29 (dd, J = 6.0, 2.0 Hz, 1H), 8.08-8.04 (m, 1H), 7.35-6.62 (m, 13H), 3.40 (s, 3H), 3.13-3.07 (m, 8H), 2.60 (s, 3H). MS (ESI): m/z 791.64 (M + H)+.

General Procedure II

(R)-4-(azetidin-1-yl)-1-(phenylthio)butan-2-amine (37a)

A solution of 36 (1.0 g 2.3 mmol), azetidine (394 mg, 6.9 mmol), EDCI (662 mg, 3.5mmol), and HOBt (466mg, 3.5 mmol) in DCM (12 mL) was stirred for 4 h at room temperature until no 36 was observed by TLC, then diluted with DCM (100 mL), washed sequentially with 1M HCl (50 mL), H2O (50 mL), and brine (20 mL), dried (MgSO4), filtered, and concentrated. The concentrate was flash chromatographed on silica gel with 50% EtOAc/hexanes to provide (R)-(9H-fluoren-9-yl)methyl 4-(azetidin-1-yl)-4-oxo-1-(phenylthio)butan-2-ylcarbamate, 957mg (88%). The amide was dissolved in MeCN (20 mL) and treated with Et2NH (2.1 mL, 20 mmol). The solution was stirred for 2 h at room temperature until no starting material was observed by TLC and concentrated. The concentrate was flash chromatographed on silica gel with 5% MeOH/CH2Cl2 to provide (R)-3-amino-1-(azetidin-1-yl)-4-(phenylthio)butan-1-one, 497 mg (98%). A mixture of this free amine and 1M BH3 in THF (5 mL) was stirred for 16 h at room temperature, treated with MeOH (1.5 mL) and concentrated HCl (0.5 mL), stirred at 80°C for 3 h, cooled to room temperature, adjusted to pH 10 with 4M Na2CO3, diluted with CH2Cl2 (150 mL), washed with H2O (50 mL) and brine (10 mL), dried (MgSO4), filtered, and concentrated. The concentrate was flash chromatographed on silica gel with 15% MeOH/CH2Cl2 to provide 37a, 445 mg (95%). 1H NMR (300 MHz, D2O), δ 7.53 (d, J = 7.2 Hz, 2H), 7.49-7.38 (m, 3H), 4.32-4.18 (m, 2H), 4.03-3.82 (m, 2H), 3.54-3.15 (m, 5H), 2.61-2.51 (m, 1H), 2.44-2.39 (m, 1H), 2.07-1.90 (m, 2H). 13C NMR (75 MHz, D2O): δ 132.99, 131.13, 129.78, 128.10, 54.91, 54.77, 50.64, 48.53, 35.92, 26.45, 15.82. MS (ESI): m/z 237.66 (M + H)+.

(R)-1-(Phenylthio)-4-(pyrrolidin-1-yl)butan-2-amine (37b)

37b was prepared from 36 and pyrrolidine according to general procedure II. 1H NMR (300 MHz, D2O): δ 7.55 (d, J = 7.2 Hz, 2H), 7.46-7.37 (m, 3H), 3.54-3.48 (m, 3H), 3.39-3.05 (m, 4H), 3.02-2.89 (m, 1H), 2.87-2.68 (m, 1H), 2.25-1.94 (m, 6H). 13C NMR (75 MHz, D2O): δ 133.02, 131.13, 129.78, 128.09, 54.38, 54.08, 50.74, 48.77, 35.85, 27.70, 22.58. MS (ESI): m/z 251.26 (M + H)+.

(R)-1-(Phenylthio)-4-(piperidin-1-yl)butan-2-amine (37c)

37c was prepared from 36 and piperidine according to general procedure II. 1H NMR (300 MHz, D2O): δ 7.59 (d, J = 7.3 Hz, 2H), 7.50-7.39 (m, 3H), 3.50-3.00 (m, 7H), 2.90-2.72 (m, 2H), 2.30-2.07 (m, 2H), 2.01-1.58 (m, 5H), 1.54-1.35 (m, 1H). 13C NMR (75 MHz, D2O): δ 133.02, 131.20, 129.78, 128.11, 53.52, 53.19, 52.68, 48.88, 35.88, 25.89, 22.74, 20.93. MS (ESI): m/z 265.38 (M + H)+.

(R)-1-(3-Amino-4-(phenylthio)butyl)azetidin-3-ol (37d)

37d was prepared from 36 and azetidin-3-ol according to general procedure II. 1H NMR (300 MHz, D2O): δ 7.55 (d, J = 7.1 Hz, 2H), 7.50-7.39 (m, 3H), 4.72-4.63 (m, 1H), 4.51-3.69 (m, 4H), 3.51-3.43 (m, 1H), 3.38-3.20 (m, 4H), 2.08-190 (m, 2H). 13C NMR (75 MHz, D2O): δ 132.92, 131.13, 129.78, 128.15, 63.27, 62.52, 58.73, 50.71, 48.44, 35.93, 26.66. MS (ESI): m/z 253.60 (M + H)+.

(R)-1-(3-Amino-4-(phenylthio)butyl)piperidin-4-ol (37e)

37e was prepared from 36 and piperidin-4-ol according to general procedure II. 1H NMR (300 MHz, D2O): δ 7.51 (d, J = 3.5 Hz, 2H), 7.41-7.33 (m, 3H), 4.09-3.77 (m, 1H), 3.46-2.69 (m, 9H), 2.12-2.08 (m, 3H), 1.87 (br, 2H), 1.69-1.57 (m, 1H). 13C NMR (75 MHz, D2O): δ 132.99, 131.18, 129.76, 128.04, 64.59, 52.58, 51.38, 48.87, 47.89, 35.88, 30.88, 28.85, 26.16. MS (ESI): m/z 281.46 (M + H)+.

(R)-1-(3-Amino-4-(phenylthio)butyl)-4-methylpiperidin-4-ol (37f)

37f was prepared from 36 and 4-methylpiperidin-4-ol according to general procedure II. 1H NMR (300 MHz, D2O): δ 7.44 (d, J = 6.8 Hz, 2H), 7.40-7.34 (m, 3H), 3.55-3.47 (m, 1H), 3.39-2.81 (m, 8H), 2.67-2.04 (m, 2H), 1.81-1.76 (m, 4H), 1.27 (s, 3H). 13C NMR (75 MHz, D2O): δ 133.09, 131.26, 129.81, 128.15, 65.02, 52.64, 49.10, 48.96, 48.56, 35.84, 34.62, 28.45, 25.88. MS (ESI): m/z 295.36 (M + H)+.

General Procedure III

(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl) piperazin-1-yl)phenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic Acid (22)

To a solution of 38 (79 mg, 0.1 mmol) and (R)-N1,N1-dimethyl-4-(phenylthio)butane-1,3-diamine (44 mg, 0.2 mmol) in DMF (1 mL) was added DIPEA (0.5 mL). The solution was stirred for 4 hours at room temperature until no 38 was observed by TLC. The reaction mixture was concentrated in vacuo and the residue was purified by HPLC to give the pure product 22 (trifluoroacetic acid salt, 81 mg, yield 81%). The gradient ran from 60% of solvent A and 40% of solvent B to 20% of solvent A and 80% of solvent B in 40 min. 1H NMR (300 MHz, CD3OD): δ 7.84 (d, J = 2.0 Hz, 1H), 7.70 (dd, J = 9.1, 2.2 Hz, 1H), 7.26-6.96 (m, 16H), 6.86-6.78 (m, 2H), 3.98-3.91 (m, 1H), 3.42-3.31 (m, 11H), 3.20-3.02 (m, 4H), 2.80 (s, 6H), 2.57 (s, 3H), 2.25-1.98 (m, 2H). MS (ESI): m/z 996.30 (M + H)+.

(R)-5-(4-Chlorophenyl)-1,2-dimethyl-4-(3-(4-(4-(4-(4-morpholino-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxylic Acid (23)

23 was prepared from 38 and (R)-4-morpholino-1-(phenylthio)butan-2-amine according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.93 (d, J = 2.1 Hz, 1H), 7.74 (dd, J = 9.2, 2.22 Hz, 1H), 7.44-6.89 (m, 17H), 6.83 (d, J = 9.4 Hz, 1H), 4.03-3.98 (m, 3H), 3.78-3.71 (m, 2H), 3.56-3.36 (m, 11H), 3.25-3.09 (m, 8H), 2.64 (s, 3H), 2.32-2.09 (m, 2H). MS (ESI): m/z 1037.80 (M + H)+.

(R)-4-(3-(4-(4-(4-(4-(Azetidin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethyl sulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-5-(4-chlorophenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic Acid (24)

24 was prepared from 38 and 37a according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.90 (d, J = 2.0 Hz, 1H), 7.60 (dd, J = 9.1, 2.0 Hz, 1H), 7.31-7.26 (m, 4H), 7.24-6.96 (m, 13H), 6.88-6.82 (m, 1H), 4.25-4.15 (m, 2H), 4.03-3.97 (m, 3H), 3.43 (s, 3H), 3.40-3.11 (m, 12H), 2.67 (s, 3H), 2.54-2.35 (m, 2H), 2.07-1.85 (m, 2H). MS (ESI): m/z 1007.08 (M + H)+.

(R)-5-(4-Chlorophenyl)-1,2-dimethyl-4-(3-(4-(4-(4-(1-(phenylthio)-4-(pyrrolidin-1-yl)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxylic Acid (25)

25 was prepared from 38 and 37b according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.95 (d, J = 1.9 Hz, 1H), 7.74 (dd, J = 9.1, 2.1 Hz, 1H), 7.30-7.01 (m, 14H), 6.84 (d, J = 9.2 Hz, 1H), 6.58-6.42 (m, 3H), 3.99 (br, 1H), 3.80-3.53 (m, 2H), 3.46 (s, 3H), 3.44-2.95 (m, 14H), 2.61 (s, 3H), 2.29-1.68 (m, 6H). MS (ESI): m/z 1021.75 (M + H)+.

(R)-5-(4-Chlorophenyl)-1,2-dimethyl-4-(3-(4-(4-(4-(1-(phenylthio)-4-(piperidin-1-yl)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxylic Acid (26)

26 was prepared from 38 and 37c according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 8.36 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 9.2, 2.2 Hz, 1H), 7.20-6.92 (m, 15H), 6.53-6.38 (m, 3H), 4.12 (br, 1H), 3.42 (s, 3H), 3.36-3.17 (m, 11H), 2.88 (s, 5H), 2.61 (s, 3H), 2.29-2.19 (m, 8H). MS (ESI): m/z 1035.92 (M + H)+.

(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(3-hydroxyazetidin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic Acid (27)

27 was prepared from 38 and 37d according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.89 (d, J = 1.9 Hz, 1H), 7.75 (dd, J = 9.0, 2.0 Hz, 1H), 7.36-7.09 (m, 13H), 7.01-6.66 (m, 5H), 4.03 (br, 1H), 3.81-3.50 (m, 2H), 3.44 (s, 3H), 3.40-2.89 (m, 15H), 2.62 (s, 3H), 2.28-1.73 (m, 2H). MS (ESI): m/z 1024.00 (M + H)+.

(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxypiperidin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic Acid (28)

28 was prepared from 38 and 37e according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.86 (s, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.27-7.25 (m, 4H), 7.20-6.79 (m, 14H), 4.03-3.75 (m, 2H), 3.49-3.31 (m, 13H), 3.14-2.89 (m, 6H), 2.58 (s, 3H), 2.26-1.88 (m, 5H), 1.67-1.63 (m, 1H). 13C NMR (75 MHz, CD3OD): δ 167.60, 151.31, 146.91, 144.09, 137.99, 137.13, 136.10, 134.72, 133.49, 133.40, 132.64, 131.05, 130.84, 130.39, 128.98, 128.37, 128.14, 127.35, 126.82, 123.13, 122.17, 121.99, 117.62, 116.34, 113.93, 110.08, 108.62, 64.34, 59.71, 53.60, 52.62, 51.06, 50.69, 48.51, 37.97, 31.40, 30.84, 29.54, 28.24, 27.94, 10.71. MS (ESI): m/z 1051.30 (M + H)+.

(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxy-4-methylpiperidin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl) piperazin-1-yl)phenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic acid (29)

29 was prepared from 38 and 37f according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.79 (d, J = 1.6 Hz, 1H), 7.66 (dd, J = 9.1, 1.7 Hz, 1H), 7.20-6.75 (m, 18H), 4.03-3.88 (m, 1H), 3.45-3.33 (m, 10H), 3.25-2.95 (m, 9H), 2.51 (s, 3H), 2.22-1.93 (m, 2H), 1.80-1.66 (m, 4H), 0.97 (s, 3H). MS (ESI) m/z 1066.16 (M + H)+.

General Procedure IV

Ethyl 5-(4-chlorophenyl)-4-(3-iodophenyl)-2-methyl-1-propyl-1H-pyrrole-3-carboxylate (40a)

To the solution of 39 (2.0 g, 4.1 mmol) in MeOH (20 mL) was added propylamine (1.0 mL ,12.3 mmol) and the solution was stirred for 6 h at room temperature until no starting material was observed by TLC. The solution was acidified with 1M HCl (50 mL) and extracted with EtOAc (2 × 50 mL). The combined EtOAc solutions were washed with brine (50 mL), dried (Na2SO4) and concentrated in vacuo. The concentrate was flash chromatographed on silica gel with 10% EtOAc/hexane to provide 1.89 g of 40a (91%). 1H NMR (300 MHz, CDCl3): δ 7.52 (t, J = 1.6 Hz, 1H), 7.46-7.42 (m, 1H), 7.28-7.24 (m, 2H), 7.08-6.94 (m, 3H), 6.84 (t, J = 7.8 Hz, 1H), 4.08 (dd, J = 14.3, 7.1 Hz, 2H), 3.73 (t, J = 7.7 Hz, 2H), 2.61 (s, 3H), 1.58-1.46 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H), 0.75 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 165.60, 139.89, 138.20, 136.00, 134.64, 133.88, 132.60, 130.30, 130.17, 129.77, 128.79, 128.56, 122.57, 111.21, 92.91, 59.30, 45.83, 24.01, 14.03, 11.69, 11.07.

Ethyl 1-butyl-5-(4-chlorophenyl)-4-(3-iodophenyl)-2-methyl-1H-pyrrole-3-carboxylate (40b)

40b was prepared from 39 and butylamine according to general procedure IV. 1H NMR (300 MHz, CDCl3): δ 7.52 (s, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.1 Hz, 1H), 6.84 (t, J = 7.7 Hz, 1H), 4.07 (dd, J = 14.4, 7.3 Hz, 2H), 3.76 (t, J = 7.6 Hz, 2H), 2.62 (s, 3H), 1.53-1.43 (m, 2H), 1.22-1.09 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H), 0.78 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 165.60, 139.90, 138.21, 135.96, 134.63, 133.88, 132.62, 130.29, 130.14, 129.78, 128.79, 128.55, 122.56, 111.21, 92.92, 59.30, 44.05, 32.77, 19.79, 14.04, 13.55, 11.70.

General Procedure V

Ethyl 5-(4-chlorophenyl)-2-methyl-4-(3-(4-(4-nitrophenyl) piperazin-1-yl)phenyl)-1-propyl-1H-pyrrole-3-carboxylate (41a)

Ester 40a (1.52 g, 3.0 mmol), 1-(4-nitrophenyl)piperazine (1.87 g, 9.0 mmol), CuI (57 mg, 0.3 mmol), L-proline (173 mg, 1.5 mmol) and K2CO3 (1.24 g, 9.0 mmol) were dissolved in 30 mL of DMSO. This solution was stirred for 8 h under a nitrogen atmosphere at 80°C until no 40a was observed by TLC. The reaction mixture was cooled, saturated NH4Cl solution (50 mL) was added and the solution extracted with EtOAc (2 × 50 mL). The combined EtOAc solutions were washed with brine (50 mL), dried (Na2SO4) and concentrated in vacuo. The concentrate was flash chromatographed on silica gel with 40% EtOAc/hexane to provide 1.44 g of 41a (82%). 1H NMR (300 MHz, CDCl3): δ 8.14-8.09 (m, 2H), 7.26-7.06 (m, 5H), 6.86-6.62 (m, 5H), 4.09 (dd, J = 14.2, 7.08 Hz, 2H), 3.75 (t, J = 7.7 Hz, 2H), 3.48 (t, J = 4.9 Hz, 4H), 3.15 (t, J = 5.3 Hz, 4H), 2.62 (s, 3H), 1.61-1.48 (m, 2H), 1.05 (t, J = 7.1 Hz, 3H), 0.76 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 165.88, 154.75, 149.55, 138.55, 136.55, 135.45, 133.55, 132.68, 131.00, 129.94, 128.43, 127.88, 125.93, 124.32, 123.39, 119.32, 114.08, 112.69, 111.39, 59.26, 49.01, 46.90, 45.86, 24.06, 14.06, 11.7, 11.08. MS (ESI): m/z 587.50 (M + H)+.

Ethyl 1-butyl-5-(4-chlorophenyl)-2-methyl-4-(3-(4-(4-nitrophenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxylate (41b)

41b was prepared from 40b according to general procedure V. 1H NMR (300 MHz, CDCl3): δ 8.11 (d, J = 9.3 Hz, 2H), 7.26-7.06 (m, 5H), 6.84-6.63 (m, 5H), 4.09 (dd, J = 14.3, 7.1 Hz, 2H), 3.79 (t, J = 7.6 Hz, 2H), 3.48 (t, J = 4.7 Hz, 4H), 3.14 (t, J = 5.3 Hz, 4H), 2.62 (s, 3H), 1.55-1.45 (m, 2H), 1.22-1.10 (m, 2H), 1.05 (t, J = 7.1 Hz, 3H), 0.79 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 165.88, 154.75, 149.54, 138.52, 136.55, 135.41, 133.54, 132.71, 130.98, 129.91, 128.42, 127.88, 125.93, 124.31, 123.38, 119.33, 114.07, 112.69, 111.38, 59.26, 49.00, 46.89, 44.06, 32.82, 19.79, 14.07, 13.55, 11.74. MS (ESI): m/z 601.82 (M + H)+.

General Procedure VI

5-(4-Chlorophenyl)-2-methyl-4-(3-(4-(4-nitrophenyl)piperazin-1-yl)phenyl)-1-propyl-1H-pyrrole-3-carboxylic acid (42a)

To a solution of 41a (1.00 g, 1.7 mmol) in 30 mL of 1:1:1 dioxane, EtOH, and H2O was added NaOH (680 mg, 17.0 mmol) and the solution was refluxed for 20 hours until no 41a was observed by TLC. After cooling the reaction was neutralized with 1M HCl and the compound was extracted with EtOAc. The EtOAc solution was washed with brine, dried (Na2SO4) and concentrated in vacuo to produce 900 mg of compound 42a as a white solid (95% yield, used for next steps directly without purification). 1H NMR (300 MHz, CDCl3): δ 8.13 (d, J = 9.3 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 7.09-7.03 (m, 3H), 6.84-6.64 (m, 5H), 3.74 (t, J = 7.5 Hz, 2H), 3.48 (t, J = 4.5 Hz, 4H), 3.15 (t, J = 5.2 Hz, 4H), 2.62 (s, 3H), 1.57-1.50 (m, 2H), 0.76 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 169.73, 154.75, 149.50, 138.56, 137.41, 135.73, 133.77, 132.67, 130.70, 130.50, 128.51, 128.14, 125.93, 124.55, 123.26, 119.87, 114.30, 112.67, 109.84, 48.92, 46.79, 45.94, 23.98, 12.09, 11.04. MS (ESI): m/z 559.17 (M + H)+.

1-Butyl-5-(4-chlorophenyl)-2-methyl-4-(3-(4-(4-nitrophenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxylic Acid (42b)

42b was prepared from 41b according to general procedure VI. 1H NMR (300 MHz, CDCl3): δ 8.11 (d, J = 9.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 7.03 (t, J = 7.7 Hz, 1H), 6.82-6.63 (m, 5H), 3.78 (t, J = 8.0 Hz, 2H), 3.44 (t, J = 4.8 Hz, 4H), 3.13 (t, J = 5.4 Hz, 4H), 2.61 (s, 3H), 1.54-1.44 (m, 2H), 1.20-1.10 (m, 2H), 0.79 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 170.66, 154.77, 149.44, 138.47, 137.36, 135.79, 133.74, 132.74, 130.75, 130.52, 128.49, 128.03, 125.92, 124.68, 123.29, 119.99, 114.22, 112.65, 109.95, 48.94, 46.75, 44.15, 32.73, 19.78, 13.51, 12.13. MS (ESI): m/z 573.80 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(4-fluoro-3-(trifluoromethylsulfonyl)phenylsulfonamido) phenyl)piperazin-1-yl)phenyl)-2-methyl-1-propyl-1H-pyrrole-3-carboxylic Acid (43a)

43a was prepared from 42a according to general procedure I. 1H NMR (300 MHz, CDCl3): δ 8.31 (dd, J = 5.9, 1.8 Hz, 1H), 8.09-8.04 (m, 1H), 7.37 (t, J = 8.9 Hz, 1H), 7.25-6.64 (m, 12H), 3.75 (t, J = 7.4 Hz, 2H), 3.18-3.11 (m, 8H), 2.64 (s, 3H), 1.58-1.51 (m, 2H), 0.77 (t, J = 7.3 Hz, 3H). MS (ESI): m/z 819.00 (M + H)+.

1-Butyl-5-(4-chlorophenyl)-4-(3-(4-(4-(4-fluoro-3-(trifluoromethylsulfonyl)phenylsulfon amido)phenyl)piperazin-1-yl)phenyl)-2-methyl-1H-pyrrole-3-carboxylic Acid (43b)

43b was prepared from 42b according to general procedure I. 1H NMR (300 MHz, CDCl3): δ 8.31 (dd, J = 5.9, 2.2 Hz, 1H), 8.09-8.04 (m, 1H), 7.37 (t, J = 8.8 Hz, 1H), 7.26-6.64 (m, 12H), 3.78 (t, J = 8.0 Hz, 2H), 3.21-3.08 (m, 8H), 2.64 (s, 3H), 1.55-1.45 (m, 2H), 1.23-1.10 (m, 2H), 0.79 (t, J =7.2 Hz, 3H). MS (ESI): m/z 833.52 (M + H)+.

5-(4-Chlorophenyl)-1-ethyl-4-(3-(4-(4-(4-fluoro-3-(trifluoromethylsulfonyl)phenylsulfon amido)phenyl)piperazin-1-yl)phenyl)-2-methyl-1H-pyrrole-3-carboxylic Acid (43c)

43c was prepared from 42c39 according to general procedure I. 1H NMR (300 MHz, CDCl3): δ 8.32 (dd, J = 6.0, 2.0 Hz, 1H), 8.08-8.03 (m, 1H), 7.35 (t, J = 8.9 Hz, 1H), 7.26-6.64 (m, 12H), 3.83 (dd, J = 14.0, 6.9 Hz, 2H), 3.17-3.08 (m, 8H), 2.64 (s, 3H), 1.63 (t, J = 7.1 Hz, 3H). MS (ESI): m/z 805.66 (M + H)+.

5-(4-Chlorophenyl)-4-(3-(4-(4-(4-fluoro-3-(trifluoromethylsulfonyl)phenylsulfonamido) phenyl)piperazin-1-yl)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic Acid (43d)

43d was prepared from 42d39 according to general procedure I. 1H NMR (300 MHz, CDCl3): δ 8.30 (dd, J = 5.9, 1.6 Hz, 1H), 8.10-8.07 (m, 1H), 7.35 (t, J = 8.9 Hz, 1H), 7.26-6.68 (m, 12H), 4.42-4.33 (m, 1H), 3.18-3.13 (m, 8H), 2.73 (s, 3H), 1.41 (d, J = 10.0 Hz, 6H). MS (ESI): m/z 819.52 (M + H)+.

(R)-5-(4-Chlorophenyl)-1-ethyl-4-(3-(4-(4-(4-(4-(4-hydroxypiperidin-1-yl)-1-(phenylthio) butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl) phenyl)-2-methyl-1H-pyrrole-3-carboxylic Acid (30)

30 was prepared from 43c and 37e according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.86 (s, 1H), 7.71 (d, J = 9.1 Hz, 1H), 7.29-6.96 (m, 16H), 6.87-6.79 (m, 2H), 4.04-3.77 (m, 4H), 3.49-3.28 (m, 8H), 3.17-2.94 (m, 8H), 2.60 (s, 3H), 2.05-1.69 (m, 6H), 1.10 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CD3OD): δ 169.03, 152.67, 148.36, 145.43, 139.23, 137.46, 137.39, 136.08, 135.09, 134.77, 134.28, 132.37, 132.02, 131.76, 131.61, 130.35, 129.66, 129.54, 128.73, 128.19, 124.95, 124.51, 123.26, 118.97, 117.66, 115.29, 111.72, 109.98, 61.10, 53.98, 52.42, 52.05, 51.22, 50.93, 40.10, 39.34, 32.77, 30.90, 28.19, 27.32, 25.25, 25.06, 24.62, 24.57, 16.14, 13.88, 11.87. MS (ESI) m/z 1066.26 (M + H)+.

(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxypiperidin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic Acid (31)

31 was prepared from 43d and 37e according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.84 (d, J = 1.8 Hz, 1H), 7.70 (dd, J = 9.2, 2.1 Hz, 1H), 7.26-6.95 (m, 17H), 6.80 (d, J = 9.2 Hz, 1H), 4,42-4.33 (m, 1H), 4.02-3.73 (m, 2H), 3.48-3.31 (m, 10H), 3.25-2.88 (m, 6H), 2.67 (s, 3H), 2.20-1.87 (m, 5H), 1.66-1.62 (m, 1H), 1.38 (d, J = 7.1 Hz, 6H). MS (ESI) m/z 1080.30 (M + H)+.

(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxypiperidin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-2-methyl-1-propyl-1H-pyrrole-3-carboxylic Acid (32)

32 was prepared from 43a and 37e according to general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.91 (d, J = 1.8 Hz, 1H), 7.78 (dd, J = 9.2, 2.2 Hz, 1H), 7.46-7.03 (m, 17H), 6.87 (d, J = 9.1 Hz, 1H), 4.02-4.00 (m, 2H), 3.86 (t, J = 7.6 Hz, 2H), 3.51-2.95 (m, 16H), 2.65 (s, 3H), 2.27-2.11 (m, 3H), 1.93 (br, 2H), 1.72-1.63 (m, 1H), 1.61-1.51 (m, 2H), 0.75 (t, J = 5.1 Hz, 3H). MS (ESI): m/z 1080.28 (M + H)+.

(R)-1-Butyl-5-(4-chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxypiperidin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl) piperazin-1-yl)phenyl)-2-methyl-1H-pyrrole-3-carboxylic acid (33)

33 was prepared from 43b and 37e according to general procedure III. 1H NMR (300 M Hz, CD3OD): δ 7.91 (s, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.33-6.81 (m, 18H), 4.08-3.79 (m, 4H), 3.53-2.94 (m, 16), 2.63 (s, 3H), 2.33-2.19 (m, 1H), 2.17-2.04 (m, 2H), 1.98-1.87 (m, 2H), 1.75-1.58 (m, 1H), 1.54-1.44 (m, 2H), 1.23-1.11 (m, 2H), 0.78 (t, J = 7.3 Hz, 3H). MS (ESI): m/z 1094.36 (M + H)+.

Fluorescence polarization based binding assays

Details of the expression and purification of Bcl-2, Bcl-xL and Mcl-1 proteins and determination of Kd values of fluorescent probes to proteins are provided in the SI. IC50 and Ki values for the interaction with Bcl-2/Bcl-xL of our designed compounds and reference compounds were determined in competitive binding experiments, in which an inhibitor in serial dilutions was allowed to compete with a fixed concentration of a fluorescent probe for a fixed concentration of a protein. Mixtures of 5 μl of the tested compound in DMSO and 120 μl of pre-incubated protein/probe complex in the assay buffer were added to assay plates and incubated at room temperature for 2 h with gentle shaking. The final concentrations of the protein and probe were 1.5 nM and 1 nM for the Bcl-2 assay, 10 nM and 2 nM for the Bcl-xL assay, and 20 nM and 2 nM for the Mcl-1 assay, respectively. Controls containing protein/probe complex only (equivalent to 0% inhibition) or free probe only (equivalent to 100% inhibition), were included in each assay plate. FP values were measured as described above. IC50 values were determined by nonlinear regression fitting of the competition curves. The Ki value of a compound to a protein was calculated using the equation described previously 41 based upon the measured IC50 value, the Kd value of the probe to the protein, and the concentration of the protein and probe in the competitive assays. Ki values were also calculated using a published equation. 42 The values obtained from both equations were found to be in excellent agreement.

Cell death assay

Cell death assays were performed using trypan blue staining. Cells were treated with the indicated compounds. At the end of treatment, cells were collected and stained with trypan blue. Cells that stained blue or the morphologically unhealthy cells were scored as dead cells. At least 100 cells were counted for each sample.

Western blotting

Cells were lysed using radioimmunoprecipitation assay lysis buffer (PBS containing 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with 1 μmol/L phenylmethylsulfonyl fluoride and 1 protease inhibitor cocktail tablet per 10 mL on ice for 20 min, and lysates were then cleared by centrifugation before protein concentration determination using the Bio-Rad protein assay kit according to the manufacturer’s instructions. Proteins were electrophoresed onto 4–20% SDS-PAGE gels (Invitrogen) and transferred onto polyvinylidenedifluoride membranes. Following blocking in 5% milk, membranes were incubated with a specific primary antibody, washed, and incubated with horseradish peroxidase–linked secondary antibody (Amersham). The signals were visualized with the chemiluminescent horseradish peroxidase antibody detection reagent (Denville Scientific). Rabbit antibodies against PARP and caspase-3 are from Cell Signaling Technology. Rabbit anti-GAPDH is from Santa Cruz Biotechnology.

Efficacy studies

When tumors reached tumor volumes between 40–110 mm3, mice were randomized into different groups, 8 mice per group, with a mean tumor volume of 70 mm3. Mice were treated with compound 28 at 50 mg/kg, or compound 30 at 25 mg/kg, intravenously, daily, 5 days a week for 2 weeks, or vehicle control. Tumor sizes and animal weights were measured 3 times a week during the treatment and twice a week after the treatment. Data are presented as mean tumor volumes ± SEM. Statistical analyses were performed by two-way ANOVA and unpaired two-tailed t test, using Prism (version 4.0, GraphPad, La Jolla, CA). P < 0.05 was considered statistically significant. The efficacy experiment was performed under the guidelines of the University of Michigan Committee for Use and Care of Animals.

Acknowledgments

This research was supported in part by a grant from the National Cancer Institute, National Institutes of Health (U19CA113317). Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (Grant 085P1000817). Coordinates for Bcl-xL complexed with 4 were deposited into the Protein Data Bank with accession numbers 3SPF.

Abbreviations

Bcl-2
B-cell lymphoma 2
Bax
Bcl-2 related protein X
Bak
Bcl-2 antagonist/killer
Bok
Bcl-2-related ovarian killer protein
Bad
Bcl-2 antagonist of cell death
Bid
BH3 interacting death domain
Bim
Bcl-2 interacting mediator
Bik
bcl-2 interacting killer
Puma
p53 upregulated modulator of apoptosis
Bcl-xL
B-cell lymphoma x long
BH
Bcl homology
PARP
poly(ADP-ribose) polymerase
SCID
severe combined immunodeficiency
EDCI
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
HOBT
1-Hydroxybenzotriazole hydrate
DIPEA
N,N-Diisopropylethylamine
FPA
fluorescence polarization assay

References

1. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000;21:485–495. [PubMed]
2. Reed JC. Apoptosis-based therapies. Nat Rev Drug Discov. 2002;1:111–121. [PubMed]
3. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. [PubMed]
4. Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents. Nature. 2000;407:810–816. [PubMed]
5. Adams JM, Cory S. Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol. 2007;19:488–496. [PMC free article] [PubMed]
6. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008;9:47–59. [PubMed]
7. van Delft MF, Huang DC. How the Bcl-2 family of proteins interact to regulate apoptosis. Cell Res. 2006;16:203–213. [PubMed]
8. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2002;2:647–656. [PubMed]
9. Willis SN, Fletcher JI, Kaufmann T, van Delft MF, Chen L, Czabotar PE, Ierino H, Lee EF, Fairlie WD, Bouillet P, Strasser A, Kluck RM, Adams JM, Huang DC. Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science. 2007;315:856–859. [PubMed]
10. Green DR, Evan GI. A matter of life and death. Cancer Cell. 2002;1:19–30. [PubMed]
11. Amundson SA, Myers TG, Scudiero D, Kitada S, Reed JC, Fornace AJ., Jr An informatics approach identifying markers of chemosensitivity in human cancer cell lines. Cancer Res. 2000;60:6101–6110. [PubMed]
12. Petros AM, Medek A, Nettesheim DG, Kim DH, Yoon HS, Swift K, Matayoshi ED, Oltersdorf T, Fesik SW. Solution structure of the antiapoptotic protein bcl-2. Proc Natl Acad Sci U S A. 2001;98:3012–3017. [PubMed]
13. Sattler M, Liang H, Nettesheim D, Meadows RP, Harlan JE, Eberstadt M, Yoon HS, Shuker SB, Chang BS, Minn AJ, Thompson CB, Fesik SW. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science. 1997;275:983–986. [PubMed]
14. Petros AM, Nettesheim DG, Wang Y, Olejniczak ET, Meadows RP, Mack J, Swift K, Matayoshi ED, Zhang H, Thompson CB, Fesik SW. Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies. Protein Science. 2000;9:2528–2534. [PubMed]
15. Wang JL, Liu D, Zhang ZJ, Shan S, Han X, Srinivasula SM, Croce CM, Alnemri ES, Huang Z. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci U S A. 2000;97:7124–7129. [PubMed]
16. Degterev A, Lugovskoy A, Cardone M, Mulley B, Wagner G, Mitchison T, Yuan J. Identification of small-molecule inhibitors of interaction between the BH3 domain and Bcl-xL. Nat Cell Biol. 2001;3:173–182. [PubMed]
17. Tzung SP, Kim KM, Basanez G, Giedt CD, Simon J, Zimmerberg J, Zhang KY, Hockenbery DM. Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3. Nat Cell Biol. 2001;3:183–191. [PubMed]
18. Enyedy IJ, Ling Y, Nacro K, Tomita Y, Wu X, Cao Y, Guo R, Li B, Zhu X, Huang Y, Long YQ, Roller PP, Yang D, Wang S. Discovery of small-molecule inhibitors of Bcl-2 through structure-based computer screening. J Med Chem. 2001;44:4313–4324. [PubMed]
19. Kutzki O, Park HS, Ernst JT, Orner BP, Yin H, Hamilton AD. Development of a potent Bcl-x(L) antagonist based on alpha-helix mimicry. J Am Chem Soc. 2002;124:11838–11839. [PubMed]
20. Wang G, Nikolovska-Coleska Z, Yang CY, Wang R, Tang G, Guo J, Shangary S, Qiu S, Gao W, Yang D, Meagher J, Stuckey J, Krajewski K, Jiang S, Roller PP, Abaan HO, Tomita Y, Wang S. Structure-based design of potent small-molecule inhibitors of anti-apoptotic Bcl-2 proteins. J Med Chem. 2006;49:6139–6142. [PubMed]
21. Tang G, Ding K, Nikolovska-Coleska Z, Yang CY, Qiu S, Shangary S, Wang R, Guo J, Gao W, Meagher J, Stuckey J, Krajewski K, Jiang S, Roller PP, Wang S. Structure-based design of flavonoid compounds as a new class of small-molecule inhibitors of the anti-apoptotic Bcl-2 proteins. J Med Chem. 2007;50:3163–3166. [PMC free article] [PubMed]
22. Tang G, Yang CY, Nikolovska-Coleska Z, Guo J, Qiu S, Wang R, Gao W, Wang G, Stuckey J, Krajewski K, Jiang S, Roller PP, Wang S. Pyrogallol-based molecules as potent inhibitors of the antiapoptotic Bcl-2 proteins. J Med Chem. 2007;50:1723–1726. [PMC free article] [PubMed]
23. Tang G, Nikolovska-Coleska Z, Qiu S, Yang CY, Guo J, Wang S. Acylpyrogallols as inhibitors of antiapoptotic Bcl-2 proteins. J Med Chem. 2008;51:717–720. [PubMed]
24. Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ, Joseph MK, Kitada S, Korsmeyer SJ, Kunzer AR, Letai A, Li C, Mitten MJ, Nettesheim DG, Ng S, Nimmer PM, O’Connor JM, Oleksijew A, Petros AM, Reed JC, Shen W, Tahir SK, Thompson CB, Tomaselli KJ, Wang B, Wendt MD, Zhang H, Fesik SW, Rosenberg SH. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature. 2005;435:677–681. [PubMed]
25. Petros AM, Dinges J, Augeri DJ, Baumeister SA, Betebenner DA, Bures MG, Elmore SW, Hajduk PJ, Joseph MK, Landis SK, Nettesheim DG, Rosenberg SH, Shen W, Thomas S, Wang X, Zanze I, Zhang H, Fesik SW. Discovery of a potent inhibitor of the antiapoptotic protein Bcl-xL from NMR and parallel synthesis. J Med Chem. 2006;49:656–663. [PubMed]
26. Bruncko M, Oost TK, Belli BA, Ding H, Joseph MK, Kunzer A, Martineau D, McClellan WJ, Mitten M, Ng SC, Nimmer PM, Oltersdorf T, Park CM, Petros AM, Shoemaker AR, Song X, Wang X, Wendt MD, Zhang H, Fesik SW, Rosenberg SH, Elmore SW. Studies leading to potent, dual inhibitors of Bcl-2 and Bcl-xL. J Med Chem. 2007;50:641–662. [PubMed]
27. Park CM, Bruncko M, Adickes J, Bauch J, Ding H, Kunzer A, Marsh KC, Nimmer P, Shoemaker AR, Song X, Tahir SK, Tse C, Wang X, Wendt MD, Yang X, Zhang H, Fesik SW, Rosenberg SH, Elmore SW. Discovery of an orally bioavailable small molecule inhibitor of prosurvival B-cell lymphoma 2 proteins. J Med Chem. 2008;51:6902–6915. [PubMed]
28. Wendt MD, Shen W, Kunzer A, McClellan WJ, Bruncko M, Oost TK, Ding H, Joseph MK, Zhang H, Nimmer PM, Ng SC, Shoemaker AR, Petros AM, Oleksijew A, Marsh K, Bauch J, Oltersdorf T, Belli BA, Martineau D, Fesik SW, Rosenberg SH, Elmore SW. Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo. J Med Chem. 2006;49:1165–1181. [PubMed]
29. Park CM, Oie T, Petros AM, Zhang H, Nimmer PM, Henry RF, Elmore SW. Design, synthesis, and computational studies of inhibitors of Bcl-XL. J Am Chem Soc. 2006;128:16206–16212. [PubMed]
30. Shoemaker AR, Oleksijew A, Bauch J, Belli BA, Borre T, Bruncko M, Deckwirth T, Frost DJ, Jarvis K, Joseph MK, Marsh K, McClellan W, Nellans H, Ng S, Nimmer P, O’Connor JM, Oltersdorf T, Qing W, Shen W, Stavropoulos J, Tahir SK, Wang B, Warner R, Zhang H, Fesik SW, Rosenberg SH, Elmore SW. A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo. Cancer Res. 2006;66:8731–8739. [PubMed]
31. Shoemaker AR, Mitten MJ, Adickes J, Ackler S, Refici M, Ferguson D, Oleksijew A, O’Connor JM, Wang B, Frost DJ, Bauch J, Marsh K, Tahir SK, Yang X, Tse C, Fesik SW, Rosenberg SH, Elmore SW. Activity of the Bcl-2 family inhibitor ABT-263 in a panel of small cell lung cancer xenograft models. Clin Cancer Res. 2008;14:3268–3277. [PubMed]
32. Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, Johnson EF, Marsh KC, Mitten MJ, Nimmer P, Roberts L, Tahir SK, Xiao Y, Yang X, Zhang H, Fesik S, Rosenberg SH, Elmore SW. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 2008;68:3421–3428. [PubMed]
33. Vogler M, Dinsdale D, Dyer MJ, Cohen GM. Bcl-2 inhibitors: small molecules with a big impact on cancer therapy. Cell Death Differ. 2009;16:360–367. [PubMed]
34. Chonghaile TN, Letai A. Mimicking the BH3 domain to kill cancer cells. Oncogene. 2008;27 (Suppl 1):S149–S157. [PMC free article] [PubMed]
35. Kang MH, Reynolds CP. Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res. 2009;15:1126–1132. [PMC free article] [PubMed]
36. Petros AM, Huth JR, Oost T, Park CM, Ding H, Wang X, Zhang H, Nimmer P, Mendoza R, Sun C, Mack J, Walter K, Dorwin S, Gramling E, Ladror U, Rosenberg SH, Elmore SW, Fesik SW, Hajduk PJ. Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR. Bioorg Med Chem Lett. 2010;20:6587–6591. [PubMed]
37. Roberts AW, Seymour JF, Brown JR, Wierda WG, Kipps TJ, Khaw SL, Carney DA, He SZ, Huang DC, Xiong H, Cui Y, Busman TA, McKeegan EM, Krivoshik AP, Enschede SH, Humerickhouse R. Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease. J Clin Oncol. 2012;5:488–496. [PubMed]
38. Rudin CM, Hann CL, Garon EB, Ribeiro de Oliveira M, Bonomi PD, Camidge DR, Chu Q, Giaccone G, Khaira D, Ramalingam SS, Ranson MR, Dive C, McKeegan EM, Chyla BJ, Dowell BL, Chakravartty A, Nolan CE, Rudersdorf N, Busman TA, Mabry MH, Krivoshik AP, Humerickhouse RA, Shapiro GI, Gandhi L. Phase II study of single-agent navitoclax (ABT-263) and biomarker correlates in patients with relapsed small cell lung cancer. Clin Cancer Res. 2012;18:3163–3169. [PMC free article] [PubMed]
39. Zhou H, Aguilar A, Chen J, Bai L, Liu L, Meagher JL, Yang CY, McEachern D, Cong X, Stuckey JA, Wang S. Structure-Based Design of Potent Bcl-2/Bcl-xL Inhibitors with Strong in Vivo Antitumor Activity. J Med Chem. 2012 [PMC free article] [PubMed]
40. Zhou H, Aguilar A, Chen J, Bai L, Liu L, Meagher JL, Yang CY, McEachern D, Cong X, Stuckey JA, Wang S. Structure-Based Design of Potent Bcl-2/Bcl-xL Inhibitors with Strong in Vivo Antitumor Activity. Journal of Medicinal Chemistry. 2012;55:6149–6161. [PMC free article] [PubMed]
41. Nikolovska-Coleska Z, Wang R, Fang X, Pan H, Tomita Y, Li P, Roller PP, Krajewski K, Saito NG, Stuckey JA, Wang S. Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal Biochem. 2004;332:261–273. [PubMed]
42. Huang X. Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand. J Biomol Screen. 2003;8:34–38. [PubMed]
43. Lee EF, Czabotar PE, Smith BJ, Deshayes K, Zobel K, Colman PM, Fairlie WD. Crystal structure of ABT-737 complexed with Bcl-xL: implications for selectivity of antagonists of the Bcl-2 family. Cell Death Differ. 2007;14:1711–1713. [PubMed]
44. Sybyl; a molecular modeling system; is supplied by Tripos, I., St. Louis, MO 63144.
45. Case DA, Darden TA, Cheatham TE, I, Simmerling CL, Wang J, Duke RE, Luo R, Crowley M, Walker RC, Zhang W, Merz KM, Wang B, Hayik S, Roitberg A, Seabra G, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Mathews DH, Seetin MG, Sagui C, Babin V, Kollman PA. AMBER 10. University of California; San Francisco: 2008.
46. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Zakrzewski VG, Jr, Montgomery JA, Stratmann RE, Burant JC, Dapprich S, Millam JM, Daniels AD, Kudin KN, Strain MC, Farkas O, Tomasi J, Barone V, Cossi M, Cammi R, Mennucci B, Pomelli C, Adamo C, Clifford S, Ochterski J, Petersson GA, Ayala PY, Cui Q, Morokuma K, Malick DK, Rabuck AD, Raghavachari K, Foresman JB, Cioslowski J, Ortiz JV, Baboul AG, Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Gomperts R, Martin RL, Fox DJ, Keith T, Al-Laham MA, Peng CY, Nanayakkara A, Challacombe M, Gill PM, Johnson B, Chen W, Wong MM, Andres JL, Gonzalez C, Head-Gordon M, Replogle ES, Pople JA. Gaussian 98, Revision A-11. Gaussian, Inc; Pittsburgh PA: 2001.
47. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics. 1983;79:926–935.
48. Ryckaert JP, Ciccotti G, Berendsen HJC. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. Journal of Computational Physics. 1977;23:327–341.
49. Darden T, York D, Pedersen L. Particle Mesh Ewald - an N. Log(N) Method for Ewald Sums in Large Systems. Journal of Chemical Physics. 1993;98:10089–10092.