Class I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and Therapeutic Efficacy in Acute Myeloid Leukemia

Abstract

The HDAC inhibitor 4-tert-butyl-N-(4-(hydroxycarbamoyl)phenyl)benzamide (AES-350, 51) was identified as a promising preclinical candidate for the treatment of acute myeloid leukemia (AML), an aggressive malignancy with a meagre 24% 5-year survival rate. Through screening of low-molecular-weight analogues derived from the previously discovered novel HDAC inhibitor, AES-135, compound 51 demonstrated greater HDAC isoform selectivity, higher cytotoxicity in MV4-11 cells, an improved therapeutic window, and more efficient absorption through cellular and lipid membranes. Compound 51 also demonstrated improved oral bioavailability compared to SAHA in mouse models. A broad spectrum of experiments, including FACS, ELISA, and Western blotting, were performed to support our hypothesis that 51 dose-dependently triggers apoptosis in AML cells through HDAC inhibition.

Figure 1

Vorinostat (1), belinostat (2), panobinostat (3), romidepsin (4), and AES-135 (5).

Scheme 1. Synthesis of Compounds 35–40

Key: (a) R²SO₂Cl, ⁱPr₂NEt, CH₂Cl₂, 16 h, rt, N₂; (b) pentafluorobenzyl bromide, Cs₂CO₃, MeCN, 16 h, rt; (c) CF₃CO₂H/CHCl₃ (1:3), 24 h, rt; (d) appropriate aniline, PPh₃Cl₂, CHCl₃, 90 min, 100 °C, MW, N₂; (e) MeI, Cs₂CO₃, MeCN, 20 h, rt; (f) H₂, 10% Pd/C, THF/MeOH (2:1), 16 h, rt; (g) (i) (COCl)₂, THF, DMF, 2 h, 0 °C, N₂; (ii) O-benzylhydroxylamine, ⁱPr₂NEt, THF, 16 h, rt, N₂. R¹, R², and R³ are variable depending on the molecule and shown below in Table 1.

Scheme 2. Synthesis of Compounds 46, 47, 51, and 54

Key: (a) (i) 4-tert-butylbenzaldehyde, THF/TFE (4:1), 16 h, rt, (ii) NaBH₄, MeOH, 6 h, rt; (b) (Boc)₂O, DMAP, MeCN, 2 h, 80 °C, MW; (c) H₂, 10% Pd/C, THF/MeOH (2:1), 16 h, rt; (d) (i) (COCl)₂, THF, DMF, 2 h, 0 °C, N₂; (ii) O-benzylhydroxylamine, ⁱPr₂NEt, THF, 16 h, rt, N₂; (e) CF₃CO₂H/CHCl₃ (1:3), 18 h, rt; (f) 4-tert-butylbenzoic acid, PPh₃Cl₂, CHCl₃, 90 min, 100 °C, MW, N₂; (g) AcCl, CH₂Cl₂, 24 h, 0 °C to rt.

Figure 2

(A) Dot plots and (B) stacked bar graphs representing the distribution of MV4-11 cells classed as healthy, early apoptosis, and late apoptosis 18 h postdosing with varying concentrations of SAHA and 51 (AES-350) using FACS.

Figure 3

(A) Docking of 51 with zebra fish HDAC6 CD2 (light blue) (PDB: 6CSR). (B) Ligand interaction diagram depicting the key interactions of 51 in HDAC6 CD2. (C) Docking of 51 in the human HDAC8 catalytic domain (green) (PDB: 6HSK). (D) Ligand interaction diagram depicting the key interactions of 51 in the HDAC8 catalytic domain. (E) Overlap of 51 (gray) with quisinostat (red) in the human HDAC8 catalytic domain (PDB: 6HSK). In A, C, and E, 51 is illustrated by gray (C), white (H), blue (N), red (O), and yellow (Zn²⁺). Other interactions are shown as follows: π-stacking (green dashed lines), H-bonds and salt bridges (yellow dashed lines), and Zn²⁺ chelation (yellow/red dots).

Figure 4

(A) HDAC6 inhibition profile with varying concentrations of 51 in HeLa cell lysates (IC₅₀ = 0.58 ± 0.13 μM, n = 2). (B) Western blots probing for Ac-α-tubulin, Ac-Histone H3, and HSC70 from MV4–11 after 6 h with varying concentrations of 51 (AES-350) and SAHA.

Figure 5

Mean plasma concentrations of 51 in CD-1 mice following PO (20 mg/kg, n = 3) and IV administration (5 mg/kg, n = 3).