3-Substituted-N-(4-hydroxynaphthalen-1-yl)arylsulfonamides as a novel class of selective Mcl-1 inhibitors: structure-based design, synthesis, SAR, and biological evaluation

Abstract

Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins, is a validated and attractive target for cancer therapy. Overexpression of Mcl-1 in many cancers results in disease progression and resistance to current chemotherapeutics. Utilizing high-throughput screening, compound 1 was identified as a selective Mcl-1 inhibitor and its binding to the BH3 binding groove of Mcl-1 was confirmed by several different, but complementary, biochemical and biophysical assays. Guided by structure-based drug design and supported by NMR experiments, comprehensive SAR studies were undertaken and a potent and selective inhibitor, compound 21, was designed which binds to Mcl-1 with a Ki of 180 nM. Biological characterization of 21 showed that it disrupts the interaction of endogenous Mcl-1 and biotinylated Noxa-BH3 peptide, causes cell death through a Bak/Bax-dependent mechanism, and selectively sensitizes Eμ-myc lymphomas overexpressing Mcl-1, but not Eμ-myc lymphoma cells overexpressing Bcl-2. Treatment of human leukemic cell lines with compound 21 resulted in cell death through activation of caspase-3 and induction of apoptosis.

Figure 1

(A) Structure of the HTS lead compound 1. (B) Putative binding mode of 1 to Mcl-1(PDB ID: 2NLA). Side chains residues of mNoxa peptide are shown in blue sticks. The surface of Mcl-1 protein is colored according to the chemical shift intensity. Significant shift (>0.09 ppm) is represented with purple, moderate shift (≥0.03 and ≤0.09 ppm) represented with pink. (C) Overlaid ¹⁵N–¹H HSQC spectra of Mcl-1 (red) and in the presence of 1 (Mcl-1:1 ratio of 1:2) (black), (Mcl-1:1 ratio of 1:1) (purple). Arrows show the direction of chemical shift changes upon binding of 1. (D) Plot of chemical shift changes of Mcl-1 amide upon addition of 1 (Mcl-1:1 ratio of 1:2) as a function of Mcl-1 residue numbers.

Scheme 1. Synthetic Route for 1 and Analogues

Reagents and conditions: (a) NIS, TFA, reflux, 24 h; (b) HS(CH₂)_nCOOCH₃ (n = 1, 2), Pd(OAc)₂, Xantphos, Cs₂CO₃, LiI, ZnCl₂, THF, 60 °C, overnight, or HS(CH₂)₃CH₃, Pd₂(dba)₃, Dppf, Et₃N, NMP, 80 °C, 2 h, or HCC(CH₂)_nOH (n = 1, 2), Pd(PPh₃)₂Cl₂, CuI, Et₃N/THF, 60 °C, 2 h (4:1), 60 °C, 2 h; (c) Fe, AcOH, 70 °C, 1 h, or Pd/C, H₂ 30 psi, EtOH/EtOAc (6:1), rt, overnight; (d) RSO₂Cl, pyridine, CH₂Cl₂, rt, overnight, or RCOCl, Et₃N, CH₂Cl₂, rt, overnight, or , CH₃CN, 80 °C, 15 h; (e) BBr₃, CH₂Cl₂, 0 °C to rt, 1 h, or BBr₃, CH₂Cl₂, 0 °C to rt, 1 h, quench with MeOH at 0 °C; (g) phenyl boronic acid, Pd(PPh₃)₄, Na₂CO₃, THF/H₂O, 60 °C, 2 h; (h) NH₄OH, rt, 1 h; (i) NaN₃, SiCl₄, CH₃CN, 80 °C, 15 h; (j) LiOH, THF, rt, 1 h; (k) H₃CCOCl, Et₃N, 0 °C, rt, 30 min.

Figure 2

Putative binding modes of (A) 16, (B) 17, (C) 18 to Mcl-1 (PDB ID: 2NLA). Surface of Mcl-1 protein is colored according to the chemical shift intensity. Significant shift (>0.09 ppm) is represented with purple, moderate shift (≥0.03 and ≤0.09 ppm) represented with pink. Plots of chemical shift changes of Mcl-1 amide upon addition of (D) 16 (Mcl-1:16 ratio of 1:2), (E) 17 (Mcl-1:17 ratio of 1:2), (F), and 18 (Mcl-1:18 ratio of 1:2) as a function of Mcl-1 residue numbers.

Figure 3

Overlaid ¹⁵N–¹H HSQC spectra of Mcl-1 (red) and in the presence of 16 (Mcl-1:16 ratio of 1:2) (purple), 17 (Mcl-1:17 ratio of 1:2) (blue), 18 (Mcl-1:18 ratio of 1:2) (green) for (A) Phe 228, (B) Met 250, and (C) Val 249 and Val 253. Arrows show the direction of chemical shift changes upon binding of compounds. (D) Overlay of putative binding modes of 16 (purple), 17 (blue), and 18 (green) to Mcl-1 (PDB ID: 2NLA) highlighting in red Val 249, Met 250, and Val 253 on helix 4, Phe 228 on helix 3 of Mcl-1.

Figure 4

Putative binding mode of 21. (A) Surface of the Mcl-1 protein (PDB ID: 2NLA) is colored according to the chemical shift intensity. Significant shift (>0.09 ppm) is represented with purple, moderate shift (≥0.03 and ≤0.09 ppm) represented with pink. (B) Plot of chemical shift changes of Mcl-1 amide upon addition of 21 (Mcl-1:21 ratio of 1:2) as a function of Mcl-1 residue numbers. (C) Overlaid ¹⁵N–¹H HSQC spectra of Mcl-1 (red) and in the presence of 21 (Mcl-1:21 ratio of 1:2) (black), (Mcl-1:21 ratio of 1:1) (purple). Arrows show the direction of chemical shift changes upon binding of 21. (D) Mcl-1 residues shown to be perturbed in HSQC NMR in the presence of 21 (Mcl-1:21 ratio of 1:2) (green).

Figure 5

Interaction of Mcl-1 inhibitors with endogenous Mcl-1 protein and Noxa. Biotin-labeled Noxa (BL-Noxa, 0.1 μM) was incubated with whole cell lysates of 2LMP cells with or without tested Mcl-1 inhibitors and Bim BH3 peptide as a positive control, followed by incubation with precleared streptavidin agarose beads. Eluted beads were subjected to Western blot analysis with anti-Mcl-1 antibody.

Figure 6

Cell death induced by Mcl-1 inhibitor 21 is Bax/Bak-dependent. MEFs deficient in Bax and Bak (gray bars) along with their wild-type counterpart (black bars) were exposed for 15 h to different concentrations of 21, and the cell viability was assessed with PI staining. Error bars represent the mean ± SEM. The significance was calculated using unpaired t test, and the number of data is shown for each tested concentration with corresponding significance: (**) p < 0.01 and (***) p < 0.001.

Figure 7

Sensitivity of Eμ-myc lymphoma cells overexpressing Mcl-1 and Bcl-2 antiapoptotic proteins to inhibitor-induced cell death. Eμ-myc/Mcl-1 and Eμ-myc/Bcl-2 lymphomas were treated for 15–18 h with increasing concentrations of 19, 21, 41, and ABT-263. Dead cells were assessed by LIVE/DEAD fixable dead cell stain kit (ViVID). The data shown represents means ± SEM from 3–7 independent experiments. The significance was calculated using unpaired t test, and the number of data is shown for each tested concentration with corresponding significance: (*) is p < 0.05, (**) p < 0.01, and (***) p < 0.001.

Figure 8

Cell death and apoptosis induction by Mcl-1 inhibitors in human leukemic cell lines. (A) Inhibition of cell growth by designed Mcl-1 inhibitors in the HL-60, MV4,11, and K-562 leukemia cell lines. Cells were treated for 3 days, and cell growth was determined using CellTiter Glo luminescent cell viability assay. (B) Analysis of apoptosis induced by 21 and 37 in the HL-60 leukemia cell line. Cells were treated with 21, 37, and 41 for 20 h using indicated concentrations, and apoptosis was analyzed with annexin-V and propidium iodide (PI) double staining by flow cytometry. Early apoptotic cells were defined as annexin-V positive/PI-negative, and late apoptotic cells as annexin-V/PI-double positive. Induction of the apoptosis by 37 was tested also in the presence of Z-VAD-FMK. (C) Induction of caspase-3 by 19 and 21 in the HL-60 cell line. Cells were treated for 20 h, and caspase-3 was detected with fluorometric-based assay. Results shown are the mean and SEM from at least three separate experiments.