Spiroindoline-Capped Selective HDAC6 Inhibitors: Design, Synthesis, Structural Analysis, and Biological Evaluation

Abstract

Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit 6a. We identified compound 6j as the most potent and selective hHDAC6 inhibitor of the series. Biological investigation of compounds 6b, 6h, and 6j demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. 6j induced HDAC6-dependent pSTAT3 inhibition.

Figure 1

Structures of FDA approved HDAC inhibitors: vorinostat (1), romidepsin (2), panobinostat (3), and belinostat (4), spiroindoline inhibitor 5, and novel HDAC6i 6a–j.

Scheme 1. Synthesis of the Final Compounds 6a–j

Reagents and conditions: (A) (a) Phenylhydrazine or (1,1-biphenyl)-2-ylhydrazine·HCl, AcOH, 80 °C, 2 h; (b) H₂, Pd/C, MeOH, 25 °C, 3 h; (c) paraformaldehyde or benzaldehyde, NaBH₃CN, MeOH, 25 °C, 8 h; (d) benzoyl chloride, TEA, DCM, 25 °C, 1 h; (e) 1 N HCl in MeOH, 25 °C, 15 min; (f) methyl 4-formyl benzoate, NaBH₃CN, MeOH, 25 °C, 8 h; (g) NH₂OH (50 wt % in H₂O), 4 M KOH in MeOH, DCM/MeOH, 25 °C, 2 h; (B) (h) NMO, MeCN, 25 °C, 12 h; (i) 10a, NaBH₃CN, MeOH, 25 °C, 8 h; (C) (j) 2-(4-(methoxycarbonyl)phenyl)acetic acid, EDCI, HOBt, DIPEA, DCM, 0 to 25 °C, 24 h; (k) methyl 4-(isocyanatomethyl)benzoate, TEA, dry THF, 45 °C, 2 h; (l) methyl 4-(hydroxymethyl)benzoate, CDI, DCM, 0 to 25 °C, 6 h.

Figure 2

Stereoview of a Polder omit map of the HDAC6-6a complex for which the atomic coordinates of 6a were omitted from the structure factor calculation (PDB 6V7A; contoured at 5.0 σ). Atoms are color-coded as follows: C = light blue (HDAC6 catalytic domain 2), light gray (symmetry mate), or wheat (inhibitor), N = blue, O = red, Zn²⁺ = gray sphere, and solvent = small red spheres. Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively.

Figure 3

Docked poses of 6a into HDAC1 (A) and HDAC6 (B). Compound 6a is represented by purple sticks, the residues in the active sites are represented by lines, and the protein is represented as a cartoon. Zn²⁺ is represented by a gray sphere. H-bonds are shown as black dotted lines, and the red solid lines represent the metal coordination bonds.

Figure 4

Docked poses of 6j into HDAC1 (A) and HDAC6 (B). Compound 6j is represented by orange sticks, the residues in the active sites are represented by lines and the protein is represented as cartoon. Zn²⁺ is represented by a gray sphere. H-bonds are shown as black dotted lines, and the red solid lines represent the metal coordination bonds.

Figure 5

(A) Immunoblot analysis of pSTAT3 in MEC-1 cells treated with 6a or 6j (5 or 10 μM) for 30 h. Actin was used as a loading control. The histogram shows the quantification by densiometric analysis of the levels of pSTAT3 relative to actin (n = 2). Data are presented as mean value ± SD. One way ANOVA; *p < 0.05. (B) Immunoblot analysis of pSTAT3 in U266 cells treated with 6j (25 μM) for 4, 8, 16, 24, and 48 h. Actin was used as a loading control. The blot is representative of three independent experiments with similar results.