Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors

The structure–activity and structure–kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11 Å channel leading to the Zn²⁺ catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.     

Design and synthesis of novel benzimidazole derivatives as phosphodiesterase 10A inhibitors with reduced CYP1A2 inhibition

A novel class of phosphodiesterase 10A (PDE10A) inhibitors with reduced CYP1A2 inhibition were designed and synthesized starting from 2-{[(1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (1). Introduction of an isopropyl group at the 2-position and a methoxy group at the 5-position of the benzimidazole ring of lead compound 1 resulted in the identification of 2-{[(2-isopropyl-5-methoxy-1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (25b), which exhibited potent PDE10A inhibitory activity with reduced CYP1A2 inhibitory activity compared to compound 1.     

Novel N-hydroxyfurylacrylamide-based histone deacetylase (HDAC) inhibitors with branched CAP group (Part 2)

Histone deacetylases (HDACs) are significant enzymes involved in tumor genesis and development. Herein, we report a series of novel N-hydroxyfurylacryl-amide-based HDAC inhibitors, which are marked by introducing branched hydrophobic groups as the capping group. The inhibitory activity of the synthesized compounds against HDACs and several tumor cell lines are firstly determined. Fifteen compounds with promising activities are selected for further evaluation of target selectivity profile against recombinant human HDAC1, HDAC4 and HDAC6. Compounds 10a, 10b, 10d and 16a exhibit outstanding selectivity against HDAC6. Analysis of HDAC4 X-ray structure and HDAC1, HDAC6 homology model indicates that these enzyme differ significantly in the rim near the surface of the active site. Although TSA has been known as a pan-HDAC inhibitor, it exhibits outstanding selectivity for HDAC6 over HDAC4. For further physicochemical properties study, six compounds are chosen for determination of their physicochemical properties including log D_7.4 and aqueous solubility. The results suggest that compounds with a smaller framework and with hydrophilicgroups are likely to have better aqueous solubility.     

Design and synthesis of novel HDAC8 inhibitory 2,5-disubstituted-1,3,4-oxadiazoles containing glycine and alanine hybrids with anti cancer activity

Oxadiazole is a heterocyclic compound containing an oxygen atom and two nitrogen atoms in a five-membered ring. Of the four oxadiazoles known, 1,3,4-oxadiazole has become an important structural motif for the development of new drugs and the compounds containing 1,3,4-oxadiazole cores have a broad spectrum of biological activity. Herein, we describe the design, synthesis and biological evaluation of a series of novel 2,5-disubstituted 1,3,4-oxadiazoles (10a–10j) as class I histone deacetylase (HDAC) inhibitors. The compounds were designed and evaluated for HDAC8 selectivity using in silico docking software (Glide) and the top 10 compounds with high dock score and obeying Lipinski’s rule were synthesized organically. Further the biological HDAC inhibitory and selectivity assays and anti-proliferative assays were carried out. In in silico and in vitro studies, all compounds (10a–10j) showed significant HDAC inhibition and exhibited HDAC8 selectivity. Among all tested compounds, 10b showed substantial HDAC8 inhibitory activity and better anticancer activity which is comparable to the positive control, a FDA approved drug, vorinostat (SAHA). Structural activity relation is discussed with various substitutions in the benzene ring connected on 1,3,4-oxadizole and glycine/alanine. The study warranted further investigations to develop HDAC8-selective inhibitory molecule as a drug for neoplastic diseases. Novel 1,3,4-oxadizole substituted with glycine/alanine showed HDAC8 inhibition.     

Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis,biological activity and cellular uptake kinetics

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.     

Discovery of adamantane based highly potent HDAC inhibitors

Herein, we report the development of highly potent HDAC inhibitors for the treatment of cancer. A series of adamantane and nor-adamantane based HDAC inhibitors were designed, synthesized and screened for the inhibitory activity of HDAC. A number of compounds exhibited GI₅₀ of 10-100 nM in human HCT116, NCI-H460 and U251 cancer cells, in vitro. Compound 32 displays efficacy in human tumour animal xenograft model.     

Discovery of novel phenoxybenzamide analogues as Raf/HDAC dual inhibitors

Histone deacetylase (HDAC) inhibitors as an important epigenetic therapeutic strategy affect signaling networks and act synergistically with kinase inhibitors for the treatment of cancer. Herein we presented a series of novel phenoxybenzamide analogues with inhibition of Raf and HDAC. Among them, compound 10e showed potent antiproliferative activities against Hepg2 and MDA-MB-468 in cellular assays. This work may lay the foundation for developing novel dual Raf/HDAC inhibitors as potential anticancer therapeutics.     

Novel HDAC6 isoform selective chiral small molecule histone deacetylase inhibitors

In an effort to identify HDAC isoform selective inhibitors, we designed and synthesized novel, chiral 3,4-dihydroquinoxalin-2(1H)-one and piperazine-2,5-dione aryl hydroxamates showing selectivity (up to 40-fold) for human HDAC6 over other class I/IIa HDACs. The observed selectivity and potency (IC₅₀ values 10–200 nM against HDAC6) is markedly dependent on the absolute configuration of the chiral moiety, and suggests new possibilities for use of chiral compounds in selective HDAC isoform inhibition.     

Design,synthesis, and biological evaluation of novel histone deacetylase 1 inhibitors through click chemistry

We report the design, synthesis, and biological evaluation of a new series of HDAC1 inhibitors using click chemistry. Compound 17 bearing a phenyl ring at meta-position was identified to show much better selectivity for HDAC1 over HDAC7 than SAHA. The compond 17 also showed better in vitro anticancer activities against several cancer cell lines than that of SAHA. This work could serve as a foundation for further exploration of selective HDAC inhibitors using the compound 17 molecular scaffold.     

Novel N-hydroxybenzamide-based HDAC inhibitors with branched CAP group

Ongoing effort to gather further knowledge about the structural requirements on histone deacetylase inhibitors led to the synthesis of novel N-hydroxybenzamide-based HDAC inhibitors 1a–o, introducing branched hydrophobic groups at the capping group, and their inhibition activity against HDACs and anti-proliferation activity in four tumor cell lines were determined. Compounds 1j–o were further tested against recombinant human HDAC1 and HDAC4 to evaluate their selectivity profile. This work further suggests that the chemical nature of the capping group is critical for subtle discrimination between the class I and the class II HDAC isoforms.     

Design and synthesis of novel hybrid benzamide–peptide histone deacetylase inhibitors

We designed and synthesized a series of novel hybrid histone deacetylase inhibitors based on conjugation of benzamide-type inhibitors with either linear or cyclic peptides. Linear tetrapeptides (compounds 13 and 14), cyclic tetrapeptides (compounds 1 and 11), and heptanediamide–peptide conjugates (compounds 10, 12, 15 and 16) were synthesized through on-resin solid-phase peptide synthesis (SPPS). All compounds were found to be moderate HDAC1 and HDAC3 inhibitors, with IC₅₀ values ranging from 1.3 μM to 532 μM. Interestingly, compound 15 showed 19-fold selectivity for HDAC3 versus HDAC1.     

The design of a novel near-infrared fluorescent HDAC inhibitor and image of tumor cells

Histone deacetylases (HDACs) have been found to be biomarkers of cancers and the corresponding inhibitors have attracted much attention these years. Herein we reported a near-infrared fluorescent HDAC inhibitor based on vorinostat (SAHA) and a NIR fluorophore. This newly designed inhibitor showed similar inhibitory activity to SAHA against three HDAC isoforms (HDAC1, 3, 6). The western blot assay showed significant difference in compared with the negative group. When used as probe for further kinematic imaging, Probe 1 showed enhanced retention in tumor cells and the potential of HDAC inhibitors in drug delivery was firstly brought out. The cytotoxicity assay showed Probe 1 had some anti-proliferation activities with corresponding IC₅₀ values of 9.20 ± 0.96 μM on Hela cells and 5.91 ± 0.57 μM on MDA-MB-231 cells. These results indicated that Probe 1 could be used as a potential NIR fluorescent in the study of HDAC inhibitors and lead compound for the development of visible drugs.     

Design,synthesis, and bioactivity evaluation of novel Bcl-2/HDAC dual-target inhibitors for the treatment of multiple myeloma

Multiple myeloma (MM) is the second most common haematological malignancy. Almost all patients with MM eventually relapse, and most recommended treatment protocols for the patients with relapsed refractory MM comprise a combination of drugs with different mechanisms of action. Therefore novel drugs are in urgent need in clinic. Bcl-2 inhibitors and HDAC inhibitors were proved their anti-MM effect in clinic or under clinical trials, and they were further discovered to have synergistic interactions. In this study, a series of Bcl-2/HDAC dual-target inhibitors were designed and synthesized. Among them, compounds 7e–7g showed good inhibitory activities against HDAC6 and high binding affinities to Bcl-2 protein simultaneously. They also displayed good growth inhibitory activities against human MM cell line RPMI-8226, which proved their potential value for the treatment of multiple myeloma.     

Design,synthesis and evaluation of novel HDAC inhibitors as potential antitumor agents

Phenyl imidazolidin-2-one was introduced as the linker for novel HDAC inhibitors. A focused library of 20 compounds was designed and synthesized, among which eight compounds showed equivalent or higher potencies against HDAC1 as compared to vorinostat. In vitro antitumor activity assays in HCT-116, PC-3 and HL-60 cancer cells revealed six compounds with potent antitumor activities, and compound 1o showed 6- to 9-fold higher potencies compared to vorinostat. In an HCT-116 nude mice xenograft model, compound 1o displayed significant antitumor activity in both continuous and intermittent dosing schedules.     

Hydroxamates of para-aminobenzoic acid as selective inhibitors of HDAC8

A series of hydroxamates (4a–4l) were prepared from p-aminobenzoic acid to inhibit HDAC8. The idea is to substitute rigid aromatic ring in place of less rigid piperazine ring of hydroxamates reported earlier by our group. It is expected to increase potency retaining the selectivity. Result obtained suggested that the modifications carried out retained the selectivity towards HDAC8 isoform and increasing the potency in very few cases. Increase in potency is also associated with variation in cap aryl region. Two compounds (4f & 4l) were found to inhibit HDAC8 at concentrations (IC₅₀) less than 20 μM.     

Isoxazole moiety in the linker region of HDAC inhibitors adjacent to the Zn-chelating group: Effects on HDAC biology and antiproliferative activity

A series of hydroxamic acid based histone deacetylase inhibitors 6–15, containing an isoxazole moiety adjacent to the Zn-chelating hydroxamic acid, is reported herein. Some of these compounds showed nanomolar activity in the HDAC isoform inhibitory assay and exhibited micro molar inhibitory activity against five pancreatic cancer cell lines.     

Anti-tumor activity evaluation of novel tubulin and HDAC dual-targeting inhibitors

Histone deacetylases (HDACs) have proven to be promising targets for the development of anti-cancer drugs. In this study, we reported a series of novel chalcone based tubulin and HDAC dual-targeting inhibitors. Three compounds inhibited the activities of HDAC and tubulin polymerization simultaneously and displayed anti-proliferative activities toward eleven human tumor cell lines. Compound 8a remarkably induced growth inhibition, apoptosis and G2/M phase arrest of A549 tumor cells. Finally, the inhibitory activities of 8a against HDAC6 and tubulin were rationalized by molecular docking studies.     

Merging of ruxolitinib and vorinostat leads to highly potent inhibitors of JAK2 and histone deacetylase 6 (HDAC6)

Inhibition of more than one pathway in a cancer cell with a single molecule could result in better therapies with less complex dosing regimens. In this work multi-component ligands have been prepared by joining together key pharmacophores of two different enzyme inhibitors in a way which increases potency against the individual pathways. Selective JAK1/2 inhibitor, ruxolitinib (3), and pan-HDAC inhibitor vorinostat (4) were linked together by a single nitrogen atom to create a new series of compounds with very potent JAK2 and HDAC6 inhibition with selectivity against HDAC1. A preferred compound, 13b, had unprecedented sub-nanomolar JAK2 potency with an IC₅₀ of 41?pM and a sub-nanomolar IC₅₀ against HDAC6 of 200?pM. Binding models show a good fit into both JAK2 and HDAC6.     

Discovery of novel 9H-purin derivatives as dual inhibitors of HDAC1 and CDK2

HDAC and CDK inhibitors have been demonstrated to be synergistically in suppressing cancer cell proliferation and inducing apoptosis. In this work, we incorporated the pharmacophore groups of HDACs and CDKs inhibitors into one molecule to design and synthesize a series of purin derivatives as HDAC/CDK dual inhibitors. The lead compound 6d, showing good HDAC1 and CDK2 inhibitory activity with IC₅₀ values of 5.8 and 56 nM, respectively, exhibited attractive potency against several cancer cell lines in vitro. This work may lead to the discovery of a novel scaffold and potential dual HDAC/CDK inhibitors.     

Lactam based 7-amino suberoylamide hydroxamic acids as potent HDAC inhibitors

A series of SAHA-like molecules were prepared introducing different lactam-carboxyamides in position 7 of the suberoylanilide skeleton. The activity against different HDAC isoforms was tested and the data compared with the corresponding linear products, without substituent in position 7. In general, this modification provided an effective reinforcement of in vitro activity. While the lactam size or the CO/NH group orientation did not strongly influence the inhibition, the contemporary modification of the suberoylamide fragment gave vary active variants in the lactam series, with compound 28 (ST8078AA1) that showed IC₅₀ values between 2 and 10 nM against all Class I HDAC isoforms, demonstrating it to be a large spectrum pan-inhibitor. This strong affinity with HDAC was also confirmed by the value of IC₅₀ = 0.5 μM against H460 cells, ranking 28 as one of the most potent HDAC inhibitors described so far.