Development and characterization of a CNS-penetrant benzhydryl hydroxamic acid class IIa histone deacetylase inhibitor

We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and ～150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC₅₀ levels for ～8?h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.     

Design,synthesis and biological evaluation of novel hydroxamic acid based histone deacetylase 6 selective inhibitors bearing phenylpyrazol scaffold as surface recognition motif

In recent years, inhibition of HDAC6 became a promising therapeutic strategy for the treatment of cancer and HDAC6 inhibitors were considered to be potent anti-cancer agents. In this work, celecoxib showed moderate degree of HDAC6 inhibition activity and selectivity in preliminary enzyme inhibition activity assay. A series of hydroxamic acid derivatives bearing phenylpyrazol moiety were designed and synthesized as HDAC6 inhibitors. Most compounds showed potent HDAC6 inhibition activity. 11i was the most selective compound against HDAC6 with IC₅₀ values of 0.020 µM and selective factor of 101.1. Structure-activity relationship analysis indicated that locating the linker group at 1′ of pyrazol gave the most selectivity. The most compounds 11i (GI₅₀ = 3.63 μM) exhibited 6-fold more potent than vorinostat in HepG2 cells. Considering of the high selectivity against HDAC6 and anti-proliferation activity, such compounds have potential to be developed as anti-cancer agents.     

Design and synthesis of a new generation of substituted purine hydroxamate analogs as histone deacetylase inhibitors

Histone deacetylase inhibitors have been proved to be great potential for the treatment of cancer. Recently, we designed and modified a series of substituted purine hydroxamate analogs as potent HDAC inhibitors based on our previous studies. The target compounds were investigated for their in vitro HDAC inhibitory activities and anti-proliferative activities. Results indicated that these compounds could effectively inhibit HDAC and possess obvious anti-proliferative activity against tumor cells. Promisingly, target compounds 4m and 4n outperformed SAHA in both enzymatic inhibitory activity and cellular anti-proliferative activity assay.     

Discovery of novel N-hydroxy-2-arylisoindoline-4-carboxamides as potent and selective inhibitors of HDAC11

N-Hydroxy-2-arylisoindoline-4-carboxamides are potent and selective inhibitors of HDAC11. The discovery, synthesis, and structure activity relationships of this novel series of inhibitors are reported. An advanced analog (FT895) displays promising cellular activity and pharmacokinetic properties that make it a useful tool to study the biology of HDAC11 and its potential use as a therapeutic target for oncology and inflammation indications.     

Property based optimization of δ-lactam HDAC inhibitors for metabolic stability

The novel δ-lactam based HDAC inhibitor, KBH-A118 (3) shows a good HDAC enzyme and cancer cell growth inhibitory activities but has undesirable pharmacokinetics profiles because of instability in mouse liver microsome. To improve metabolic stability, various analogues were prepared with substituents on aromatic ring of cap group and various chain lengths between the cap group and δ-lactam core. The newly prepared analogues showed moderate to potent in vitro activities. Among them six compounds (8a, 8e, 8j, 8n, 8t, and 8v) were evaluated on mouse liver microsome assay and it turned out that the microsomal stabilities were dependent on lipophilicity and the number of the rotatable bonds. Finally, the animal pharmacokinetic profiles of 8e displayed improving oral exposure and oral bioavailability.     

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.     

Exploring hydroxamic acid inhibitors of HDAC1 and HDAC2 using small molecule tools and molecular or homology modelling

Hydroxamic acid compounds 1–10 containing a N-hydroxycinnamamide scaffold and a 4-(benzylamino)methyl cap group that was either unsubstituted (1) or substituted with one (2–4) or two (5–10) methoxy groups in variable positions were prepared as inhibitors of Zn(II)-containing histone deacetylases (HDACs). The 3,4- (9) and 3,5- (10) bis-methoxy-substituted compounds were the least potent against HeLa nuclear extract, HDAC1 and HDAC2. Molecular modelling showed methoxy groups in the 3-, 4- and 5-position, but not the 2-position, had unfavourable steric interactions with the G32-H33-P34 triad on a loop at the surface of the HDAC2 active site cavity. An HDAC1 homology model showed potential ionic (E243..K288) and cation-pi (K247..F292) interactions between helix 10 and helix 11 that were absent in HDAC2 ((G243..K288) and (K247..V292)). This surface-located interhelical constraint could inform the design of bitopic HDAC1 and HDAC2 selective ligands using an allosteric approach, and/or protein-protein interaction (PPI) inhibitors.     

Design,synthesis and evaluation of novel indirubin-based N-hydroxybenzamides,N-hydroxypropenamides and N-hydroxyheptanamides as histone deacetylase inhibitors and antitumor agents

Several novel indirubin-based N-hydroxybenzamides, N-hydropropenamides and N-hydroxyheptanamides (4a-h, 7a-h, 10a-h) were designed using a fragment-based approach with structural features extracted from several previously reported HDAC inhibitors, such as SAHA (vorinostat), MGCD0103 (mocetinostat), nexturastat A and PXD-101 (belinostat). The biological results reveal that our compounds showed excellent cytotoxicity toward three common human cancer cell lines (SW620, PC-3 and NCI-H23) with IC₅₀ values ranging from 0.09 to 0.007 µM. The cytotoxicity of the compounds was equipotent or even up to 10-times more potent than adriamycin and up to 205-times more potent than SAHA. Among the series of N-hydroxypropenamides, compounds 10a-d were the most potent HDAC inhibitors as well as cytotoxicity toward the cell lines tested. In addition, the strong inhibitory activites toward HDAC of our compounds were observed with IC₅₀ values of below-micromolar range. Especially, compound 4a inhibited HDAC6 with an IC₅₀ value of 29-fold lower than that against HDAC2 isoform. Representative compounds 4a and 7a were found to significantly arrest SW620 cells at G0/G1 phase. Compounds 7a and 10a were found to strongly induce apoptosis in SW620 cells. Docking studies revealed some important features affecting the selectivity against HDAC6 isoform. The results clearly demonstrate the potential of the indirubin-hydroxamic acid hybrids and these compounds should be very promising for further development.     

Utilization of tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinone as a cap moiety in design of novel histone deacetylase inhibitors

A series of novel 5,6,7,8-Tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one derivatives bearing a hydroxamic acid, 2-aminoanilide and hydrazide moieties as zinc-binding group (ZBG) were designed, synthesized and evaluated for the HDAC inhibition activity and antiproliferative activity. Most of the tested compounds displayed strong to moderate HDAC inhibitory activity. Some of these compounds showed potent anti-proliferative activity against human HepG2, MCF-7 and HCT-116 cell lines. In particular, compounds IVa, IVb, IXa and IXb exhibited significant anti-proliferative activity against the three cell lines tested compared to SAHA as a reference. Compound IVb is equipotent inhibitor for HDAC1 and HDAC2 as SAHA. It is evident that the presence of free hydroxamic acid group is essential for Zn binding affinity with maximal activity with a linker of aliphatic 6 carbons. Docking study results revealed that compound IVb could occupy the HDAC2 binding site and had the potential to exhibit antitumor activity through HDAC inhibition, which merits further investigation.     

Design and synthesis of aryl ether and sulfone hydroxamic acids as potent histone deacetylase (HDAC) inhibitors

A series of novel hydroxamic acid based histone deacetylases (HDAC) inhibitors with aryl ether and aryl sulfone residues at the terminus of a substituted, unsaturated 5-carbon spacer moiety have been synthesized for the first time and evaluated. Compounds with meta- and para-substitution on the aryl ring of ether hydroxamic acids 19c, 20c, 19e, 19f and 19g are potent HDAC inhibitors with activities at low nanomolar levels.     

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.     

Novel N-hydroxybenzamides incorporating 2-oxoindoline with unexpected potent histone deacetylase inhibitory effects and antitumor cytotoxicity

In our search for novel small molecules targeting histone deacetylases, we have designed and synthesized two series of novel N-hydroxybenzamides incorporating 2-oxoindolines (4a–g, 6a–g). Biological evaluation showed that these benzamides potently inhibited HDAC2 with IC₅₀ values in sub-micromolar range. In three human cancer cell lines the synthesized compounds were up to 4-fold more cytotoxic than SAHA. Docking experiments indicated that the compounds tightly bound to HDAC2 at the active binding site with binding affinities much higher than that of SAHA. Our present results demonstrate that these novel and simple N-hydroxybenzamides are potential for further development as anticancer agents and further investigation of similarly simple N-hydroxybenzamides should be warranted to obtain more potent HDAC inhibitors.     

Discovery of novel 4-aryl-thieno[1,4]diazepin-2-one derivatives targeting multiple protein kinases as anticancer agents

A series of 4-aryl-thieno[1,4]diazepin-2-one were synthesized and evaluated for their antiproliferative activities against the A375P melanoma and U937 hematopoietic cell lines. Several compounds showed very potent antiproliferative activities toward both cell lines and the activities were better than that of sorafenib, the reference standard. Derivatives were made as amide (8a–8i, 9a–9m) and urea (10a–10d, 11a–11d) with diverse hydrophobic moieties. One of the most potent inhibitor 10d, 1-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-(4-(2-oxo-2,3-dihydro-1H-thieno [3,4-b][1,4]diazepin-4-yl)phenyl)urea was found to be very potent inhibitor of multi-protein kinases including FMS kinase (IC₅₀?=?3.73?nM) and is a promising candidate for further development in therapeutics for cancer.     

Design,synthesis and evaluation of novel N-hydroxybenzamides/N-hydroxypropenamides incorporating quinazolin-4(3H)-ones as histone deacetylase inhibitors and antitumor agents

In our search for novel small molecules targeting histone deacetylases, we have designed and synthesized several series of novel N-hydroxybenzamides/N-hydroxypropenamides incorporating quinazolin-4(3H)-ones (4a-h, 8a-d, 10a-d). Biological evaluation showed that these hydroxamic acids were generally cytotoxic against three human cancer cell lines (SW620, colon; PC-3, prostate; NCI-H23, lung cancer). It was found that the N-hydroxypropenamides (10a-d) were the most potent, both in term of HDAC inhibition and cytotoxicity. Several compounds, e.g. 4e, 8b-c, and 10a-c, displayed up to 4-fold more potent than SAHA (suberoylanilide hydroxamic acid, vorinostat) in term of cytotoxicity. These compounds also comparably inhibited HDACs with IC₅₀ values in sub-micromolar range. Docking experiments on HDAC2 isozyme revealed some important features contributing to the inhibitory activity of synthesized compounds, especially for propenamide analogues. Importantly, the free binding energy computed was found to have high quantitative correlation (R² ∼ 95%) with experimental results.     

Design,synthesis and activity evaluation of indole-based double – Branched HDAC1 inhibitors

Histone deacetylases inhibitors (HDACIs) represents effective treatments for cancer. In continuing our efforts to develop novel and potent HDACIs, a series of N-hydroxycinnamamide-based HDACIs with aromatic ring and various aliphatic linker have been successfully designed and synthesized. Biological evaluations established that compounds 4h, 4i, 4j, 4l, 4r showed superior inhibition on histone deacetylase and antiproliferative activity in some solid tumor cell lines [HeLa, SK-N-BE(2), PC-3] compared to the known inhibitor SAHA. Among these analogs, 4l exhibited selectivity to HDAC1.     

Design,synthesis and evaluation of antiproliferative activity of melanoma-targeted histone deacetylase inhibitors

The clinical validation of histone deacetylase inhibition as a cancer therapeutic modality has stimulated interest in the development of new generation of potent and tumor selective histone deacetylase inhibitors (HDACi). With the goal of selective delivery of the HDACi to melanoma cells, we incorporated the benzamide, a high affinity melanin-binding template, into the design of HDACi to generate a new series of compounds 10a-b and 11a-b which display high potency towards HDAC1 and HDAC6. However, these compounds have attenuated antiproliferative activities relative to the untargeted HDACi. An alternative strategy furnished compound 14, a prodrug bearing the benzamide template linked via a labile bond to a hydroxamate-based HDACi. This pro-drug compound showed promising antiproliferative activity and warrant further study.     

Design,synthesis and anticancer evaluation of acridine hydroxamic acid derivatives as dual Topo and HDAC inhibitors

Multitarget inhibitors design has generated great interest in cancer treatment. Based on the synergistic effects of topoisomerase and histone deacetylase inhibitors, we designed and synthesized a new series of acridine hydroxamic acid derivatives as potential novel dual Topo and HDAC inhibitors. MTT assays indicated that all the hybrid compounds displayed good antiproliferative activities with IC₅₀ values in low micromolar range, among which compound 8c displayed potent activity against U937 (IC₅₀?=?0.90?μM). In addition, compound 8c also displayed the best HDAC inhibitory activity, which was several times more potent than HDAC inhibitor SAHA. Subsequent studies indicated that all the compounds displayed Topo II inhibition activity at 50?μM. Moreover, compound 8c could interact with DNA and induce U937 apoptosis. This study provides a suite of compounds for further exploration of dual Topo and HDAC inhibitors, and compound 8c can be a new dual Topo and HDAC inhibitory anticancer agent.     

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.     

Cyclic tetrapeptides with –SS– bridging between amino acid side chains for potent histone deacetylases’ inhibition

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose –SS– bond is reduced within the cell. Here, cyclic peptides with –SS– bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-l-amino acid-l-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-l-amino acid-d-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was also a potent HDAC inhibitor.     

Discovery of N-(2,3,5-triazoyl)mycophenolic amide and mycophenolic epoxyketone as novel inhibitors of human IMPDH

Syntheses of ten derivatives of mycophenolic acid (MPA) at C-6′ position, and structure–activity relationship study among these derivatives, MPA and mycophenolic hydroxamic acid (MPHA) led to discovery of N-(2,3,5-triazolyl)mycophenolic amide 4, (7′S) mycophenolic epoxyketone 9 and (7′R) mycophenolic epoxyketone 10 having potent inhibitory activity against human inosine-5′-monophosphate dehydrogenase (IMPDH) type I and II as well as antiproliferative activity on human leukemia K562 cells. Compounds 4, 9, and 10 showed induction activity of erythroid differentiation in K562 cells. Inhibitory effects of 4 and 10 against IMPDH were attenuated by supplemental guanosine in K562 cells. In contrast, attenuation effect by supplemental guanosine was not significant in the case of 9. Compound 9 weakly inhibited the enzyme activity of HDAC in the nuclear lysate of K562 cells at 10 μM. These observations suggest that the primary target of 4, 9, and 10 is IMPDH, whereas compound 9 partially inhibits a certain type of HDAC.