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Starting from the N-hydroxy-3-(4-(2-phenylbutanoyl)amino)phenyl)acrylamide (5 b) previously described by us as a HDAC inhibitor, we prepared four aza-analogues, 6-8, 9 b, as regioisomers containing the pyridine nucleus. Preliminary screening against mHDAC1 highlighted the N-hydroxy-5-(2-(2-phenylbutanoyl)amino)pyridyl)acrylamide (9 b) as the most potent inhibitor. Thus, we further developed both pyridylacrylic- and nicotinic-based hydroxamates (9 a, 9 c-f, and 11 a-f) and 2'-aminoanilides (10 a-f and 12 a-f), related to 9 b, to be tested against HDACs. Among them, the nicotinic hydroxamate 11 d displayed sub-nanomolar potency (IC 50 : 0.5 nM) and selectivity up to 34 000 times that of HDAC4 and from 100 to 1300 times that of all the other tested HDAC isoforms. The 2'-aminoanilides were class I-selective HDAC inhibitors, generally more potent against HDAC3, with the nicotinic anilide 12 d being the most effective (IC 50 HDAC3 =0.113 μM). When tested in U937 leukemia cells, the hydroxamates 9 e, 11 c, and 11 d blocked over 80 % of cells in G2/M phase, whereas the anilides did not alter cell-cycle progress. In the same cell line, the hydroxamate 11 c and the anilide 10 b induced about 30 % apoptosis, and the anilide 12 c displayed about 40 % cytodifferentiation. Finally, the most potent compounds in leukemia cells 9 b, 11 c, 10 b, 10 e, and 12 c were also tested in K562, HCT116, and A549 cancer cells, displaying antiproliferative IC 50 values at single-digit to sub-micromolar level., (© 2020 Wiley-VCH GmbH.)

We have screened our compound collection in an established cell based assay that measures the derepression of an epigenetically silenced transgene, the locus derepression assay. The screen led to the identification of 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzenecarbohydroxamic acid (9b) as an active which was found to inhibit HDAC1. In initial structure activity relationships study, the 1-methylbenzimidazole ring was replaced by the isosteric heterocycles benzimidazole, benzoxazole, and benzothiazole and the position of the hydroxamic acid substituent on the phenyl ring was varied. Whereas compounds bearing a para substituted hydroxamic acid (9a-d) were active HDAC inhibitors, the meta substituted analogues (8a-d) were appreciably inactive. Compounds 9a-d selectively inhibited HDAC6 (IC50 = 0.1-1.0 μM) over HDAC1 (IC50 = 0.9-6 μM) and moreover, also selectively inhibited the growth of lung cancer cells vs. patient matched normal cells. The compounds induce a cell cycle arrest in the S-phase while induction of apoptosis is neglible as compared to controls. Molecular modeling studies uncovered that the MM-GBSA energy for interaction of 9a-d with HDAC6 was higher than for HDAC1 providing structural rationale for the HDAC6 selectivity.

Four novel series of cinnamyl-containing histone deacetylase (HDAC) inhibitors 1-4 are described, containing hydroxamate (1 and 3) or 2-aminoanilide (2 and 4) derivatives. When screened against class I (maize HD1-B and human HDAC1) and class II (maize HD1-A and human HDAC4) HDACs, most hydroxamates and 2-aminoanilides displayed potent and selective inhibition toward class I enzymes. Immunoblotting analyses performed in U937 leukemia cells generally revealed high acetyl-H3 and low acetyl-α-tubulin levels. Exceptions are compounds 3 f-i, 3 m-o, and 4 k, which showed higher tubulin acetylation than SAHA. In U937 cells, cell-cycle blockade in either the G₂/M or G₁/S phase was observed with 1-4. Five hydroxamates (compounds 1 h-l) effected a two- to greater than threefold greater percent apoptosis than SAHA, and in the CD11c cytodifferentiation test some 2-aminoanilides belonging to both series 2 and 4 were more active than MS-275. The highest-scoring derivatives in terms of apoptosis (1 k, 1 l) or cytodifferentiation (2 c, 4 n) also showed antiproliferative activity in U937 cells, thus representing valuable tools for study in other cancer contexts., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aroyl-pyrrolyl-hydroxy-amides (APHAs) are a class of synthetic HDAC inhibitors described by us since 2001. Through structure-based drug design, two isomers of the APHA lead compound 1, the 3-(2-benzoyl-1-methyl-1H-pyrrol-4-yl)-N-hydroxy-2-propenamide 2 and the 3-(2-benzoyl-1-methyl-1H-pyrrol-5-yl)-N-hydroxy-2-propenamide 3 (iso-APHAs) were designed, synthesized and tested in murine leukemia cells as antiproliferative and cytodifferentiating agents. To improve their HDAC activity and selectivity, chemical modifications at the benzoyl moieties were investigated and evaluated using three maize histone deacetylases: HD2, HD1-B (class I human HDAC homologue), and HD1-A (class II human HDAC homologue). Docking experiments on HD1-A and HD1-B homology models revealed that the different compounds selectivity profiles could be addressed to different binding modes as observed for the reference compound SAHA. Smaller hydrophobic cap groups improved class II HDAC selectivity through the interaction with HD1-A Asn89-Ser90-Ile91, while bulkier aromatic substituents increased class I HDAC selectivity. Taking into account the whole enzyme data and the functional test results, the described iso-APHAs showed a behaviour of class I/IIb HDACi, with 4b and 4i preferentially inhibiting class IIb and class I HDACs, respectively. When tested in the human leukaemia U937 cell line, 4i showed altered cell cycle (S phase arrest), joined to high (51%) apoptosis induction and significant (21%) differentiation activity.

(Aryloxopropenyl)pyrrolyl hydroxamates were recently reported by us as first examples of class II-selective HDAC inhibitors and can be useful tools to probe the biology of such enzymes. Molecular modelling and 3-D QSAR studies have been performed on a series of 25 (aryloxopropenyl)pyrrolyl hydroxamates to gain insights about their activity and selectivity against both maize HD1-B and HD1-A, two enzymes homologous of mammalian class I and class II HDACs, respectively. The studies have been accomplished by calculating alignment-independent descriptors (GRIND descriptors) using the ALMOND software. Highly descriptive and predictive 3-D QSAR models were obtained using either class I or class II inhibitory activity displaying r(2)/q(2) values of 0.96/0.81 and 0.98/0.85 for HD1-B and HD1-A, respectively. A deeper inspection revealed that in general a bent molecular shape structure is a prerequisite for HD1-A-selective inhibitory activity, while straight shape molecular skeleton leads to selective HD1-B compounds. The same conclusions could be achieved by molecular docking studies of the most selective inhibitors.

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of gamma-globin and/or beta-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the gamma/(gamma+beta) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of gamma-globin mRNA and the gamma/(gamma+beta) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of gamma-globin and decreased by 2-fold that of beta-globin mRNA, increasing the gamma/(gamma+beta) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as gamma/(gamma+beta) mRNA inducers in erythroblasts obtained from patients with beta(0) thalassemia. Progenitor cells from patients with beta(0) thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low beta-globin levels but 10 times higher-than-normal levels of the alpha hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in beta(0) thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the gamma/(gamma+beta) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with beta(0) thalassemia.

A novel series of compounds containing a uracil moiety as the connection unit between a phenyl/phenylalkyl portion and a N-hydroxy-polymethylenealkanamide or -methylenecinnamylamide group (uracil-based hydroxamic acids, UBHAs) was tested against maize histone deacetylases (HDACs) and mouse HDAC1. Compounds with a phenyl/benzyl ring at the uracil-C6 position and bearing 4-5 carbon units as well as a m- or p-methylenecinnamyl moiety as a spacer were the most potent inhibitors. In cell-based human HDAC1 and HDAC4 assays, the two UBHAs tested inhibited the HDAC1 but not HDAC4 immunoprecipitate activity. When tested in human leukemia U937 cells, some UBHAs produced G1 phase arrest of the cell cycle. Moreover, 1j showed high antiproliferative and dose-dependent granulocytic differentiation properties. The tested UBHAs displayed weak p21WAF1/CIP1 induction in U937 cells, and 1d and 1j showed high histone H3 and alpha-tubulin acetylation effects.

Starting from the pharmacophore model for HDAC inhibitor design, a novel series of hydroxamates bearing a uracil moiety as connecting unit (CU) has been prepared and tested. Almost all compounds exhibited HDAC inhibiting activity at low nanomolar concentrations, the N-hydroxy-6-(3,4-dihydro-4-oxo-6-benzyl- and -6-phenyl-2-pyrimidinylthio)hexanamides 1d and 1l being more potent than SAHA in enzymatic assays. Such compounds also caused hyperacetylation in NIH3T3 cell core histones and were endowed with interesting antiproliferative and cytodifferentiating effects in human leukemia (HL-60) cells.

Recently we reported a novel series of hydroxamates, called 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides (APHAs), acting as HDAC inhibitors (Massa, S.; et al. J. Med. Chem. 2001, 44, 2069-2072). Among them, 3-(4-benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide 1 was chosen as lead compound, and its binding mode into the modeled HDAC1 catalytic core together with its histone hyperacetylation, antiproliferative, and cytodifferentiating properties in cell-based assays were investigated (Mai, A.; et al. J. Med. Chem. 2002, 45, 1778-1784). Here we report the results of some chemical manipulations performed on (i) the aroyl portion at the C4-pyrrole position, (ii) the N(1)-pyrrole substituent, and (iii) the hydroxamate moiety of 1 to determine structure-activity relationships and to improve enzyme inhibitory activity of APHAs. In the 1 structure, pyrrole N(1)-substitution with groups larger than methyl gave a reduction in HDAC inhibiting activity, and replacement of hydroxamate function with various non-hydroxamate, metal ion-complexing groups yielded poorly active or totally inactive compounds. On the contrary, proper substitution at the C4-position favorably affected enzyme inhibiting potency, leading to 8 (IC50 = 0.1 micro M) and 9 (IC50 = 1.0 micro M) which were 38- and 3.8-fold more potent than 1 in in vitro anti-HD2 assay. Against mouse HDAC1, 8 showed an IC50 = 0.5 micro M (IC50 of 1 = 4.9 micro M), and also in cell-based assay, 8 was endowed with higher histone hyperacetylating activity than 1, although it was less potent than TSA and SAHA. Such enhancement of inhibitory activity can be explained by the higher flexibility of the pyrrole C4-substituent of 8 which accounts for a considerably better fitting into the HDAC1 pocket and a more favorable enthalpy ligand receptor energy compared to 1. The enhanced fit allows a closer positioning of 8 hydroxamate moiety to the zinc ion. These findings were supported by extensive docking studies (SAD, DOCK, and Autodock) performed on both APHAs and reference drugs (TSA and SAHA).

Inhibitors of histone deacetylase (HD) bear great potential as new drugs due to their ability to modulate transcription and to induce apoptosis or differentiation in cancer cells. We have described previously analogues of the complex natural HD inhibitors trapoxin B and trichostatin A with activities in the submicromolar range. Here we report structure-activity relationship analyses of further analogues of trichostatin A with respect to in vitro inhibition of maize HD-2 and their ability to induce terminal cell differentiation in Friend leukemic cells. This is the first report that shows the correlation between HD inhibitory activity and action on cancer cells on a larger series of similar compounds. Only the compounds that inhibit HD induce differentiation and/or exert antiproliferative activities in cell culture. Our studies support the use of in vitro systems as screening tools and provide structure-activity relationships that merit further investigation of this interesting target.