Search

Chemical manipulations performed on aroyl-pyrrolyl-hydroxyamides (APHAs) led to (aryloxopropenyl)pyrrolyl hydroxamates 2a−w, and their inhibition against maize HDACs and their class I or class II HDAC selectivity were determined. In particular, from these studies some benzene meta-substituted compounds emerged as highly class II (IIa)-selective HDAC inhibitors, the most selective being the 3-chloro- and 3-fluoro-substituted compounds 2c (SI = 71.4) and 2f (SI = 176.4). The replacement of benzene with a 1-naphthyl ring afforded 2s, highly active against the class II homologue HD1-A (IC50 = 10 nM) but less class II-selective than 2c,f. When tested against human HDAC1 and HDAC4, 2f showed no inhibitory activity against HDAC1 but was able to inhibit HDAC4. Moreover, in human U937 acute myeloid leukaemia cells 2f did not produce any effect on apoptosis, granulocytic differentiation, and the cell cycle, whereas 2s (that retain class I HDAC inhibitory activity) was 2-fold less potent than SAHA used as reference.

A new series of 2-, 3-, and 4-acylaminocinnamyl-N-hydroxyamides 1-3 have been prepared, and their anti- HDAC (against maize HD2, HD1-B, and HD1-A enzymes) activities have been assessed. Cinnamyl-hydroxyamides bearing acylamino substituents at the C2 position of the benzene ring (compounds 1a-g) showed very low HDAC inhibiting activities, with IC50 values in the high micromolar range. By shifting the same acylamino groups from C2 to C3 (compounds 2a-g) as well as C4 (compounds 3a-f) position of the benzene ring, a number of highly potent HDAC inhibitors have been obtained. In the anti-HD2 assay 3c (IC50 11 nM) was the most potent compound, being >11600-, 4.5-, and 10-fold more potent than sodium valproate, SAHA, and HC-toxin, respectively, and showing the same activity as trapoxin. HD1-B and HD1-A assays have been performed to screen the inhibitory action of 1-3 against mammalian class I (HD1-B) and class II (HD1- A) HDAC homologous enzymes. From the corresponding IC50 data, a selectivity ratio has been calculated. In general, compounds 1-3 showed no or little selectivity towards the class II homologue HD1-A, the most selective being 2a with class II selectivity ratio 4.3. About the inhibitory potency, the 4-(2-naphthoylamino)cinnamyl-N-hydroxyamide 3f showed the highest inhibiting effect against the two enzymes (IC50-HD1-B 36 nM IC50-HD1-A 42 nM). Selected 2 and 3 compounds will be evaluated to determine their antiproliferative and cyto differentiating activities on HL-60 cells.

Aroyl-pyrrole-hydroxy-amides (APHAs) are a new class of synthetic HDAC inhibitors recently described by us. Through three different docking procedures we designed, synthesized, and tested two new isomers of APHA lead compound 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1), compounds 3 and 4, characterized by different insertions of benzoyl and propenoylhydroxamate groups onto the pyrrole ring. Biological activities of 3 and 4 were predicted by computational tools up to 617-fold more potent than that of 1 against HDAC1; thus, 3 and 4 were synthesized and tested against both mouse HDAC1 and maize HD2 enzymes. Predictions of biological affinities (Ki values) of 3 and 4, performed by a VALIDATE model (applied on either SAD or automatic DOCK or Autodock results) and by the Autodock internal scoring function, were in good agreement with experimental activities. Ligand/receptor positive interactions made by 3 and 4 into the catalytic pocket, in addition to those showed by 1, could at least in part account for their higher HDAC1 inhibitory activities. In particular, in mouse HDAC1 inhibitory assay 3 and 4 were 19- and 6-times more potent than 1, respectively, and 3 and 4 antimaize HD2 activities were 16- and 76-times higher than that of 1, 4 being as potent as SAHA in this assay. Compound 4, tested as antiproliferative and cytodifferentiating agent on MEL cells, showed dose-dependent growth inhibition and hemoglobin accumulation effects.

Previous SAR studies (Part 1:  Mai, A.; et al. J. Med. Chem. 2003, 46, 512−524) performed on some portions (pyrrole-C4, pyrrole-N1, and hydroxamate group) of 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1a) highlighted its 4-phenylacetyl (1b) and 4-cynnamoyl (1c) analogues as more potent compounds in inhibiting maize HD2 activity in vitro. In the present paper, we investigated the effect on anti-HD2 activity of chemical substitutions performed on the pyrrole-C2 ethene chains of 1a−c, which were replaced with methylene, ethylene, substituted ethene, and 1,3-butadiene chains (compounds 2). Biological results clearly indicated the unsubstituted ethene chain as the best structural motif to get the highest HDAC inhibitory activity, the sole exception to this rule being the introduction of the 1,3-butadienyl moiety into the 1a chemical structure (IC50(2f) = 0.77 μM; IC50(1a) = 3.8 μM). IC50 values of compounds 3, prepared as 1b homologues, revealed that between benzene and carbonyl groups at the pyrrole-C4 position a hydrocarbon spacer length ranging from two to five methylenes is well accepted by the APHA template, being that 3a (two methylenes) and 3d (five methylenes) are more potent (2.3- and 1.4-fold, respectively) than 1b, while the introduction of a higher number of methylene units (see 3e,f) decreased the inhibitory activities of the derivatives. Particularly, 3a (IC50 = 0.043 μM) showed the same potency as SAHA in inhibiting HD2 in vitro, and it was 3000- and 2.6-fold more potent than sodium valproate and HC-toxin and was 4.3- and 6-fold less potent than trapoxin and TSA, respectively. Finally, conformationally constrained forms of 1b,c (compounds 4), prepared with the aim to obtain some information potentially useful for a future 3D-QSAR study, showed the same (4a,b) or higher (4c,d) HD2 inhibiting activities in comparison with those of the reference drugs. Molecular modeling and docking calculations on the designed compounds performed in parallel with the chemistry work fully supported the synthetic effort and gave insights into the binding mode of the more flexible APHA derivatives (i.e., 3a). Despite the difference of potency between 1b and 3a in the enzyme assay, the two APHA derivatives showed similar antiproliferative and cytodifferentiating activities in vivo on Friends MEL cells, being that 3a is more potent than 1b in the differentiation assay only at the highest tested dose (48 μM).

Chemical manipulations performed on aroyl pyrrolyl hydroxyamides, a new class of HDAC inhibitors previously reported by us, led to (aryloxopropenyl)pyrrolyl hydroxyamides 3a−g. Such compounds, showing better inhibitory activity against maize HD1-A than HD1-B (two homologues of mammalian class IIa and I HDACs, respectively), are the first class of IIa-selective inhibitors (fold selectivity:  7−78). They could be useful as tools for probing the biology of these enzymes and eventually as new anticancer agents with low toxicity.

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 N1-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 N1-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 μM) and 9 (IC50 = 1.0 μM) 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 μM (IC50 of 1 = 4.9 μM), 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 deacetylases (HDACs) are a new class of anticancer agents that affect gene regulation. We had previously reported the first simple synthetic HDAC inhibitors with in vitro activity at submicromolar concentrations. Here, we present structure−activity data on modifications of a phenylalanine-containing lead compound including amino acid amides as well as variations of the amino acid part. The compounds were tested for inhibition of maize HD-2, rat liver HDAC, and for the induction of terminal cell differentiation and inhibition of proliferation in Friend leukemic cells. In the amide series, in vitro inhibition was potentiated up to 15-fold, but the potential to induce cell differentiation decreased. Interestingly, an HDAC class selectivity was indicated among some of these amides. In the amino acid methyl ester series, a biphenylalanine derivative was identified as a good enzyme inhibitor, which blocks proliferation in the submicromolar range and is also a potent inducer of terminal cell differentiation.

The binding mode of 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides 1a−c, belonging to a recently reported class of synthetic histone deacetylase (HDAC) inhibitors (Massa, S.; et al. J. Med. Chem. 2001, 44, 2069−2072), into the new modeled HDAC1 catalytic core is presented, and enzyme/inhibitor interactions are discussed. HDAC1 X-ray coordinates were obtained by virtual “mutation” of those of histone deacetylase-like protein, a bacterial HDAC homologue. In in vitro antimaize HD2 as well as antimouse HDAC1 assay, compounds 1a−c showed inhibitory activities in the low micromolar range. Similarly, 1a−c are endowed with anti-HDAC activity in vivo:  on mouse A20 cells, 1a−c induced histone hyperacetylation leading to a highly increased acetylation level of H4 as compared to control histones. Results obtained with acid−urea−triton polyacrylamide gel electrophoresis have been confirmed by Western Blot experiments. Finally, compound 1a, chosen as a representative member of this class of HDAC inhibitors, resulted endowed with antiproliferative (45 and 85% cell growth inhibition at 40 and 80 μM, respectively) and cellular differentiation (18 and 21% of benzidine positive cells at the same concentrations) activities in murine erythroleukemic cells.

To analyze mechanisms of senescence‐associated gene expression, we have investigated histone deacetylases (HDACs) in human fibroblasts undergoing replicative senescence. We found that the overall acetylation pattern of histones does not vary detectably with replicative senescence. By Northern blot and Western blot, we found a significant decrease in the abundance of HDAC‐1 in senescent cells. Biochemical analysis of deacetylase activities in extracts from old and young cells revealed a striking difference. While by anion exchange chromatography we found a single peak of activity in extracts from young cells, which coincided with the elution of both HDAC‐1 and HDAC‐2, in senescent cells a second peak of activity was found. This second peak of activity is associated with HDAC‐2 but does not contain HDAC‐1. These results suggest that HDAC‐2 is present in at least two distinct forms, one of which is specific for senescent cells. Further biochemical characterization of the enzyme activity revealed that addition of nicotinamide adenine dinucleotide (NAD) did not detectably influence the activity of any fraction, suggesting that NAD is not an essential co‐factor for the analyzed HDACs from diploid human fibroblasts.

Novel 3-(4-aroyl-2-pyrrolyl)-N-hydroxy-2-propenamides are disclosed as a new class of histone deacetylase (HDAC) inhibitors. Three-dimensional structure-based drug design and conformational analyses into the histone deacetylase-like protein (HDLP) catalytic core suggested the synthesis and biological evaluation of compounds 7a−h. Experimental pKi values are in good agreement with VALIDATE predicted pKi values of new derivatives. All compounds 7a−h show HDAC inhibitory activity in the micromolar range, with 7e as the most potent derivative (IC50 = 1.9 μM). The influence of the 4‘-substituent in the aroyl moiety is not significant for the inhibitory activity, as all compounds 7a−g show IC50 values between 1.9 and 3.9 μM. Otherwise, the unsaturated chain linking the pyrrole ring to the hydroxamic acid group is clearly important for the anti-HDAC activity, the saturated analogue 7h being 10-fold less active than the unsaturated counterpart 7a.

To analyze mechanisms of senescence-associated gene expression, we have investigated histone deacetylases (HDACs) in human fibroblasts undergoing replicative senescence. We found that the overall acetylation pattern of histones does not vary detectably with replicative senescence. By Northern blot and Western blot, we found a significant decrease in the abundance of HDAC-1 in senescent cells. Biochemical analysis of deacetylase activities in extracts from old and young cells revealed a striking difference. While by anion exchange chromatography we found a single peak of activity in extracts from young cells, which coincided with the elution of both HDAC-1 and HDAC-2, in senescent cells a second peak of activity was found. This second peak of activity is associated with HDAC-2 but does not contain HDAC-1. These results suggest that HDAC-2 is present in at least two distinct forms, one of which is specific for senescent cells. Further biochemical characterization of the enzyme activity revealed that addition of nicotinamide adenine dinucleotide (NAD) did not detectably influence the activity of any fraction, suggesting that NAD is not an essential co-factor for the analyzed HDACs from diploid human fibroblasts.

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.

The cell-cycle progression of germinating embryos of maize (Zea mays L.) was studied from 0 to 72 h after the start of imbibition using DNA flow cytometry on isolated nuclei, and analyses of thymidine kinase activity, histone biosynthesis and levels of proliferating cell nulcear antigen (PCNA). At the start of germination, 75% of the cells were in G1, but this population had decreased to 25% by 72 h. The concomitant increase of cells in S-phase did not occur continuously, but stepwise, indicating that during germination most of the cells enter S-phase as a partially synchronized population. Within the initial 60 h of embryo germination the cells passed through one S-phase; the start and duration of this period of replicative DNA synthesis was further substantiated by the analysis of S-phase-associated events, the biosynthesis of core histones and the enzyme activity of thymidine kinase, which both began to increase at about 12 h after the start of differentiation. Thymidine kinase fluctuated periodically during germination with a transient maximum at 30 h and a second peak at 72 h; histone biosynthesis was not detectable until 12 h after the start of germination. The levels of PCNA protein closely resembled the pattern of thymidine kinase during germination. Together with the cytometric data this allows a clear assignment of cell cycle events to different times of embryo differentiation.

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. To study the activity of HD and the effect of potential inhibitors in vitro so far only radio-active assays have existed. For the search of new inhibitors and for the use in HD identification and purification we established a simple, non-radioactive assay that allows screening of large numbers of compounds. The assay is based on an aminocoumarin derivative of an Omega-acetylated lysine as enzyme substrate.