Design, Synthesis, Bioactivity Evaluation, Crystal Structures, and In Silico Studies of New α-Amino Amide Derivatives as Potential Histone Deacetylase 6 Inhibitors

Abstract

Hydroxamate, as a zinc-binding group (ZBG), prevails in the design of histone deacetylase 6(HDAC6) inhibitors due to its remarkable zinc-chelating capability. However, hydroxamate-associated genotoxicity and mutagenicity have limited the widespread application of corresponding HDAC6 inhibitors in the treatment of human diseases. To avoid such side effects, researchers are searching for novel ZBGs that may be used for the synthesis of HDAC6 inhibitors. In this study, a series of stereoisomeric compounds were designed and synthesized to discover non-hydroxamate HDAC6 inhibitors using α-amino amide as zinc-ion-chelating groups, along with a pair of enantiomeric isomers with inverted L-shaped vertical structure as cap structures. The anti-proliferative activities were determined against HL-60, Hela, and RPMI 8226 cells, and 7a and its stereoisomer 13a exhibited excellent activities against Hela cells with IC₅₀ = 0.31 µM and IC₅₀ = 5.19 µM, respectively. Interestingly, there is a significant difference between the two stereoisomers. Moreover, an evaluation of cytotoxicity toward human normal liver cells HL-7702 indicated its safety for normal cells. X-ray single crystal diffraction was employed to increase insights into molecule structure and activities. It was found that the carbonyl of the amide bond is on the different side from the amino and pyridine nitrogen atoms. To identify possible protein targets to clarify the mechanism of action and biological activity of 7a, a small-scale virtual screen using reverse docking for HDAC isoforms (1-10) was performed and the results showed that HDAC6 was the best receptor for 7a, suggesting that HDAC6 may be a potential target for 7a. The interaction pattern analysis showed that the α-amino amide moiety of 7a coordinated with the zinc ion of HDAC6 in a bidentate chelate manner, which is similar to the chelation pattern of hydroxamic acid. Finally, the molecular dynamics simulation approaches were used to assess the docked complex's conformational stability. In this work, we identified 7a as a potential HDAC6 inhibitor and provide some references for the discovery of non-hydroxamic acid HDAC6 inhibitors.

Keywords: HDAC6 inhibitors; bioactivity evaluation; crystal structure; molecular dynamics simulation; non-hydroxamate; reverse docking; synthesis; α-amino amide.

Figure 1

HDAC inhibitors approved by the FDA, representative HDAC6 inhibitors, and potent non-hydroxamate-based HDAC6 inhibitors. HDAC inhibitors approved by FDA (1a); Representative HDAC6 inhibitors (1b); Potent non-hydroxamate based HDAC6 inhibitors (1c).

Figure 2

Active site analysis of HDAC6 (PDB ID: 5edu): (a) cross-sectional view of the active site (surface represented) with (R)-TSA (sticks represented); (b) surface representation of the cavity with (R)-TSA (sticks represented); (c) important amino acids around (R)-TSA (sticks represented). Zn²⁺ ion is shown as a cyan sphere and metal coordination interactions are indicated as red dashed lines. The color codes of carbon, oxygen, and nitrogen are green, red, and blue, respectively. The carbon atoms of (R)-TSA are colored in yellow.

Figure 3

The design of cap, linker, and zinc-binding group(ZBG).

Scheme 1

Reagents and conditions: (a) H₂O, Na₂CO₃, rt; (b) SOCl₂, MeOH, 80 °C; (c) Pd(Dppf)Cl₂, KOAC, bis(pinacolato)diborane, DMF, 90 °C; (d) Pd(PPh₃)_4, Na₂CO₃, DMF/H₂O, 80 °C; (e) zinc powder, AcOH, MeOH, 60 °C; (f) Fmoc-AAs, EDCI, HOBt, DMF, rt; (g) piperidine, DMF, rt.

Figure 4

A perspective view of 2 and 8, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level.

Figure 5

A perspective view of 7a, 7d, and 13a, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level.

Figure 6

Superimposition of the co-crystallized poses (yellow) and the docking pose (green) of the same ligands. (a) (HDAC1), (b) (HDAC2), (c) (HDAC4), (d) (HDAC6), (e) (HDAC7), (f) (HDAC8), (g) (HDAC10).

Figure 7

Two-dimensional diagram depicting HDAC6–ligand interaction of 7a (a), 7d (b), 13a (c), and TSN (d). Key amino acids and their binding interaction are identified.

Figure 8

Three-dimensional interaction pattern diagram of 7a (a), 7d (b), 13a (c), and TSN (d) with HDAC6. Hydrogen bonds are represented by yellow dashed lines; π–π stacking interactions are represented by green dashed lines; the zinc ion is shown as a cyan ball; coordination interactions are represented by red dashed lines and distances are given in Å. Co-crystallised ligand TSN colored in yellow.

Figure 9

(A): Plot presenting the stability of protein–ligand interaction (RMSD). (B): The protein conformation changes along its side chain is represented in the RMSF throughout the trajectory. (C,D) represent the bar graph and the 2D interaction between the ligand and the protein throughout trajectory.