Borinostats: solid-phase synthesis of carborane-capped histone deacetylase inhibitors with a tailor-made selectivity profile

Abstract

The elevated expression of histone deacetylases (HDACs) in various tumor types renders their inhibition an attractive strategy for epigenetic therapeutics. One key issue in the development of improved HDAC inhibitors (HDACis) is the selectivity for single HDAC isoforms over unspecific pan inhibition to minimize off-target toxicity. Utilizing the carborane moiety as a fine-tuning pharmacophore, we herein present a robust solid phase synthetic approach towards tailor-made HDACis meeting both ends of the selectivity spectrum, namely pan inhibition and highly selective HDAC6 inhibition.

Fig. 1. Pharmacophore model of selective HDACis: zinc-binding group (ZBG), hydrophobic alkyl or aryl linker and a sterically demanding, hydrophobic cap group.

Scheme 1. Synthesis of the carborane building blocks. Reagents: (a) (i) n-BuLi, hexane, 0 °C; (ii) C₂H₄O, 0 °C; (iii) AcOH, MeOH, rt, 45%; (b) CrO₃, acetone, AcOH, H₂O, rt, 69%; c) (i) n-BuLi, Et₂O, −78 °C; (ii) CO_2(g), −78 °C; (iii) H₂O, rt, quant.

Scheme 2. Synthesis of the modified resins 7a–d with four different linkers. Reagents: (a) PhthN-OH, NEt₃, DMF, 24 h, rt; (b) N₂H₄·H₂O, MeOH, 30 min, rt; (c) Fmoc-linker-COOH, HATU, HOBt·H₂O, DIPEA, DMF, 18 h, rt.

Scheme 3. Fmoc deprotection of the modified resins 7a–d, amide coupling with the carboranyl cap groups and cleavage off the resin. Reagents: (a) 20% piperidine in DMF, rt; (b) 3, HATU, DIPEA, DMF, 18 h, rt, (c) 4, COMU, DIPEA, DMF, 18 h, rt; (d) 5% TFA, DCM, 1 h, rt, 74–99%. For definition of X see Scheme 2.

Fig. 2. Target inhibition indicated by IC₅₀ values (μM) of carborane-based hydroxamates 8a–d, 9a–d and control compounds SAHA, 10 and 11 grouped by linker against HDAC1 and HDAC6, determined by fluorogenic HDAC inhibition assays.

Fig. 3. 8c (A) and 9d (B) docked into the human HDAC6 catalytic domain 2 (PDB ID: 5EDU). HDAC6 is shown in gray. Residue numbering according to PDB ID: 5EDU. 8c (C) docked into human HDAC1 (PDB ID: 5ICN). HDAC1 is shown in grey. Residue numbering according to PDB ID: 5ICN. Superimposed HDAC6 backbone trace is shown in light blue for comparison (PDB ID: 5EDU). B: orange, C: gray/green, N: blue, O: red, Zn: purple. Hydrogen atoms are omitted. Ligand–receptor interaction and Zn²⁺ coordination is shown as dashed lines.

Fig. 4. (A) Immunoblotting with anti-acetyl SMC3 (HDAC8 inhibition), anti-acetyl-α-tubulin (HDAC6 inhibition) and anti-acetyl-histone H3 (HDAC1 inhibition) antibodies on HL60 cell lysates obtained after 24 h treatment at 1 μM with compounds 8c, 9d, 10, 11 or vorinostat (SAHA) as a control. (B) Comparative cellular viability (log IC₅₀ μM) of three leukemic cell lines originated from different lineages (K562, HPBALL and HL60) after 72 h treatment with compounds 8c, 9d, 10 or 11. The IC₅₀ data (n = 3) plotted as a heat map with each box of the heat map representing the mean of three independent experiments (n = 3), whereas the color of the individual cell is related to its position along with a log IC₅₀ (μM) gradient. The table below is depicting actual IC₅₀ values used for plotting these heat maps.

Fig. 5. (A)–(C) Illustrative synergy map of 8c, 9d and vorinostat (SAHA) after 72 h co-treatment of the acute myeloid leukemia (AML) cell line HL60 with bortezomib (proteasome inhibitor) at depicted concentrations. The mean synergy score calculations were based on the ZIP model and visualizations were performed using Synergy Finder webtool.