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. 2012 May 24;55(10):4664-82.
doi: 10.1021/jm300178u. Epub 2012 May 10.

Design of Bcl-2 and Bcl-xL inhibitors with subnanomolar binding affinities based upon a new scaffold

Haibin Zhou  1 , Jianfang Chen, Jennifer L Meagher, Chao-Yie Yang, Angelo Aguilar, Liu Liu, Longchuan Bai, Xin Cong, Qian Cai, Xueliang Fang, Jeanne A Stuckey, Shaomeng Wang
Affiliations

Design of Bcl-2 and Bcl-xL inhibitors with subnanomolar binding affinities based upon a new scaffold

Haibin Zhou et al. J Med Chem. .

Erratum in

  • J Med Chem. 2012 Jun 28;55(12):5987

Abstract

Employing a structure-based strategy, we have designed a new class of potent small-molecule inhibitors of the anti-apoptotic proteins Bcl-2 and Bcl-xL. An initial lead compound with a new scaffold was designed based upon the crystal structure of Bcl-xL and U.S. Food and Drug Administration (FDA) approved drugs and was found to have an affinity of 100 μM for both Bcl-2 and Bcl-xL. Linking this weak lead to another weak-affinity fragment derived from Abbott's ABT-737 led to an improvement of the binding affinity by a factor of >10 000. Further optimization ultimately yielded compounds with subnanomolar binding affinities for both Bcl-2 and Bcl-xL and potent cellular activity. The best compound (21) binds to Bcl-xL and Bcl-2 with K(i) < 1 nM, inhibits cell growth in the H146 and H1417 small-cell lung cancer cell lines with IC(50) values of 60-90 nM, and induces robust cell death in the H146 cancer cell line at 30-100 nM.

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Figures

Figure 1
Figure 1
Chemical structures of previously reported potent and specific Bcl-2/Bcl-xL inhibitors.
Figure 2
Figure 2
Crystal structure of Bcl-xL with five key residues of BAD BH3 peptide at the binding site. Centroids of hydrophobic pharmacophores are shown in spheres. The pharmacophore model based on three residues at Site 1 binding pocket (purple spheres in red circle) was used in pharmocophore search.
Figure 3
Figure 3
Three classes of scaffolds identified based on the pharmacophores in the Bad BH3 peptide. The three dimensional pharmocophore model is based on two aromatic rings (blue color) and one hydrophobic group (red color). The distance between the aromatic ring was set to 5 ± 1 Å and the distances between the aromatic ring to the hydrophobic group were set to 6 ± 1 Å.
Figure 4
Figure 4
Structure-based design of a new scaffold as the initial lead compound 4 and its crystal structure in complex with Bcl-xL. (A), (B) Rank 1 pose of Lipitor and Celecoxib with Bcl-xL using the Bad BH3 peptide bound Bcl-xL structure (PDB ID: 2BZW). (C) Identification of the core scaffold from the FDA approved drugs database for a new class of Bcl-2/Bcl-XL inhibitors. (D) Co-crystal structure of 4 in complex with Bcl-xL (1.7 Å).
Figure 5
Figure 5
Computational structure-based design of a new class of Bcl-2/Bcl-xL inhibitors by linking two fragments with weak binding affinities. (A) Superposition of the crystal structure of 4 (green) onto the crystal structure of 1 (yellow) in complex with Bcl-xL. (B) Measurement of the distance (8.2 Å) between 4 and 5. The 1 bound Bcl-xL structure was used in the surface representation. (C) Chemical structures of 5 and the new designed Bcl-2/Bcl-xL inhibitors with different linkers.
Figure 6
Figure 6
Chemical structures of designed Bcl-2 and Bcl-xL inhibitors.
Figure 7
Figure 7
Chemical structures of 7 and its analogues.
Figure 8
Figure 8
Binding models between (A) 20, (B) 21 and Bcl-XL. The Bcl-XL protein from the crystal structure between 1 and Bcl-XL was used in the docking simulations. The highest ranked poses of both compounds were selected as the binding models. The added ethyl group to 20 was denoted by the red circle. Residues of Bcl-XL at the binding site were labeled.
Figure 9
Figure 9
(A). Cell death induction by 20 and 21 in the H146 cancer cell line. Cells were treated for 24 h and cell death was analyzed by trypan blue assay. (B). Induction of cleavage of PARP and caspase-3 by 20 and 21 in the H146 cell line. Cells were treated for 24 h and caspase-3 and PARP were probed by western blotting.
Scheme 1
Scheme 1
Synthesis of compound compound 4 Reagents and conditions: a) i. K2CO3, MeOH, reflux; ii. CNCH2COOEt, t-BuOK; b) (S)-4-(2-iodoethyl)-2,2-dimethyl-1,3-dioxolane, K2CO3; c) i. KOH, H2O/THF/MeOH; ii. 3-(4-methylpiperazin-1-yl)propan-1-amine, EDCI, HOBt, DIEA, DCM; iii. 4 M HCI in dioxane, MeOH.
Scheme 2
Scheme 2
Synthesis of compound 6 Reagents and conditions: a) i. K2CO3, MeOH, reflux; ii. CNCH2COOEt, t-BuOK; b) (S)-4-(2-iodoethyl)-2,2-dimethyl-1,3-dioxolane, K2CO3; c) i. KOH, H2O/THF/MeOH; ii. 3-(4-methylpiperazin-1-yl)propan-1-amine, EDCI, HOBt, DIEA, DCM; d) i. 27, Pd(dba)2, tri-tert-butylphosphine, sodium tert-butoxide, DMF, toluen; ii. 4 M HCI in dioxane, MeOH, 10 min
Scheme 3
Scheme 3
Synthesis of compounds 7,13,14 and 15 Reagents and conditions: a) ferf-butyl 4-(4-aminophenyl)piperazine-1-carboxylate, pyridine; b) i. (R)-N1,N1-dimethyl-4-(phenylthio)butane-i, 3-diamine, DIEA, DMF; ii. TFA, CH2CI2; c) i. 26, Pd(dba)2, tri-terf-butylphosphine, sodium terf-butoxide, DMF, toluen; ii. 4 M HCI in dioxane, MeOH, 10 min; d)4'-chloro-[1,1'-biphenyl]-2-carbaldehyde, Na(OAc)3BH, CICH2CH2CI; e) Aniline, pyridine; f) (R)-N1, N1-dimethyl-4-(phenylthio)butane-1,3-diamine, DIEA, DMF.
Scheme 4
Scheme 4
Synthesis of compounds 9 and 10 a) 4-ethynylbenzoic acid, EDCI, DMAP, CH2CI2, b) i. 26, Pd(PPh3)4, Cul, Et3N, DMF; ii. 4 M HCI in dioxane, MeOH, 10 min; c) i. 4-ethynylaniline, Pyridine, ii. (R)-N1,N1-dimethyl-4-(phenylthio)butane-1,3-diamine, DIPEA, DMF;
Scheme 5
Scheme 5
Synthesis of compound 8 a) NaN3, Cul, L-proline, NaOH, DMSO, 70 °C; b) i. 31, CuSO4-5H2O, Sodium L-ascorbate, H2O/t-BuOH; ii. 4 M HCI in dioxane, MeOH, 10 min
Scheme 6
Scheme 6
Synthesis of compounds 12 and 11. Reagents and conditions: a) i. K2CO3, MeOH, reflux; ii. CNCH2COOEt, t-BuOK; b) (S)-4-(2-iodoethyl)-2,2-dimethyl-1,3-dioxolane, K2CO3; c) 1-(4-nitrophenyl)piperazine, Cul, L-Proline, K2CO3, 80 °C, 2h; d) i. KOH, H2O/THF/MeOH; ii. 3-(4-methylpiperazin-1-yl)propan-1-amine, EDCI, HOBt, DIEA, DCM; e) i. H2, Pd/C; ii. 4-fluoro-3-nitrobenzene-1-sulfonyl chloride, pyridine; iii. (R)-N1,N1-dimethyl-4-(phenylthio)butane-1,3-diamine, DIPEA, DMF; iv. 4 M HCI in dioxane, MeOH; f) N1-(4-nitrophenyl)ethane-1,2-diamine, Cul, L-Proline, K2CO3, 80 °C, overnight;
Scheme 7
Scheme 7
Synthesis of compounds 20,16,17,18 and 19 Reagents and conditions: a) Mel, K2CO3; b) 1-(4-nitrophenyl)piperazine, Cul, L-Proline, K2CO3, 80 °C, 2h; c) i. KOH, H2O/THF/MeOH, reflux; ii. H2, Pd/C; iii. 4-fluoro-3-nitrobenzene-1-sulfonyl chloride, pyridine; iv. (R)-N1,N1-dimethyl-4-(phenyltriio)butane-1,3-diamine, DIPEA, DMF; d) 3-(4-methylpiperazin-1-yl)propan-1-amine, EDCI, HOBt, DIEA, DCM; e) methyl amine, EDCI, HOBt, DIEA, DCM, f) TFA, CH2CI2; g) EtOH, N,N'-Diisopropylcarbodiimide, 4-(dimethylamino)pyridine, THF.
Scheme 8
Scheme 8
Synthesis of compound 21 Reagents and conditions: a) i. NBS, DMF; ii. Mel, K2CO3; b) 1-(4-nitrophenyl)piperazine, Cul, L-Proline, K2CO3, 80 °C, 2h; c) Ethynyltrimethylsilane, Pd(PPh3)4, Cul, Et3N, DMF, 85 °C; d) KOH, Dioxane, EtOH, H2O, reflux, 2h; e) i. H2, Pd/C; ii. 4-fluoro-3-nitrobenzene-1-sulfonyl chloride, pyridine; iii. (R)-N1,N1-dimethyl-4-(phenylthio)butane-1,3-diamine, DIPEA, DMF.
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