Toward an HDAC6 inhibitor: synthesis and conformational analysis of cyclic hexapeptide hydroxamic acid designed from alpha-tubulin sequence

A cyclic hexapeptide hydroxamic acid inhibitor for HDAC6 has been designed and synthesized on the basis of the facts that alpha-tubulin is the substrate of HDAC6 and of the excellent inhibitory activity of cyclic tetrapeptide hydroxamic acids (CHAPs) for HDACs. Unexpectedly, cyclic hexapeptide hydroxamic acid showed very low HDAC inhibitory activity. To explain the low activity, we have carried out conformation analysis and compared it to the crystal structure of alpha-tubulin. The conformation around the acetylated lysine of the cyclic hexapeptide substrate or the aminosuberate hydroxamic acid [Asu(NHOH)] of cyclic hexapeptide inhibitor is different from that around alpha-tubulin's lysine-40. The difference in the conformation seems to cause some steric hindrance at the capping site resulting in poor binding capacity.     

Mutagenicity of pyridine- and quinoline-carbohydroxamic acid derivatives

11 pyridine- and 6 quinoline-carbohydroxamic acids were tested for mutagenicity on Salmonella typhimurium TA100 and TA98. The results are compared with those obtained for benzohydroxamic acid and 4 naphthohydroxamic acids. Most of them were mutagenic on both these tester strains. Of the pyridine derivatives, pyridine-2-carbohydroxamic acid was the most potent mutagen. Quaternarization of the pyridine-ring nitrogen prevented the induction of mutation to a marked extent. Among the quinoline derivatives, quinoline-6-carbohydroxamic acid showed potent mutagenicity similar to that of 2-naphthohydroxamic acid. The present study supports the proposal made previously that the mechanism for mutagenicity of hydroxamic acids involves Lossen rearrangement of the acid conjugates produced by enzymic acylation (or perhaps phosphorylation or sulfation) of the hydroxamic acids, followed by carbamoylation of the target molecule in the cell by the resultant isocyanate. The multiplicity of factors determining the mutagenic potency of hydroxamic acids is discussed.     

Inhibition of urease activity by hydroxamic acid derivatives of amino acids.

Hydroxamic acids have been reported to be potent and specific inhibitors of urease (EC 3.5.1.5) activity of plant and bacterial origin. The present investigation was performed on the inhibitory effect of hydroxamic acid derivatives of naturally occurring amino acids on the urease activity of the Jack Bean and the alimentary tracts of rats. Methionine-hydroxamic acid was the most powerful inhibitor (I50=3.9 X 10(-6) M) among nineteen alpha-aminoacyl hydroxamic acids. Phenylalanine-, serine-, alanine-, glycine-, histidine-, threonine-, leucine-, and arginine-hydroxamic acids followed, in order of decreasing inhibitory power. The inhibition proceeded with time at a comparable rate to fatty acyl hydroxamic acid inhibition. The I50 values of alpha-aminoacyl hydroxamic acids were found to be almost equal to those of the corresponding fatty acyl hydroxamic acids. This fact shows that the alpha-amino group did not affect inhibitory power. However, aspartic-beta-, lysine-, and glutamic-gamma-hydroxamic acids, in descending order, were much less inhibitory, probably due to the presence of a carboxyl or omega-amino group. Furthermore, the pH optimum of the inhibition shifted to lower pH in the presence of a carboxyl group, and to a higher pH in e presence of an amino group. The results suggest that the dissociation of an acidic or a basic group reduces the inhibitory power of hydroxamic acid. Hydroxamic acid inhibits urease activity with strict specificity, excpet for aspartic-beta-hydroxamic acid, which inhibited asparaginase competitively. Hydroxamic acid derivatives of amino acids inhibited not only the urease activity of the Jack Bean, but also that of the caecum and ileum parts of the rat intestine.     

Photoinduced DNA cleavage by anthracene based hydroxamic acids

Two different series of naphthalene and anthracene based hydroxamic acids having amino acid derivatives were synthesized. Single strand DNA cleavage was achieved on irradiation of newly synthesized hydroxamic acids by UV light (≥350nm). Both reactive oxygen species (ROS) and generated radicals from hydroxamic acids were shown to be responsible for the DNA cleavage. Further, DNA cleaving ability of hydroxamic acids was found to be dependent on its concentration and on its structure.     

Antiproliferative activities of a library of hybrids between indanones and HDAC inhibitor SAHA and MS-275 analogues

New compounds derived from inhibitors of histone deacetylases (HDACs) have been synthesized and their antiproliferative activities towards non small lung cancer cell line H661 evaluated. Their design is based on hybrids between indanones to limit conformational mobility and other known HDAC inhibitors (SAHA, MS-275). The synthesis of these new derivatives was achieved by alkylation of appropriate indanones to introduce the side chain bearing a terminal ester group, the latter being a precursor of hydroxamic acid and aminobenzamide derivatives. These new analogues were found to be moderately active to inhibit H661 cell proliferation.     

Synthesis and biological evaluation of N-aryl salicylamides with a hydroxamic acid moiety at 5-position as novel HDAC-EGFR dual inhibitors

A novel series of N-aryl salicylamides with a hydroxamic acid moiety at 5-position were synthesized efficiently. Their activities against EGFR kinase and HDACs were evaluated. All compounds displayed inhibitory activity against EGFR and HDACs. The antiproliferative activities of synthesized compounds were evaluated by MTT method against human cancer cell lines A431, A549 and HL-60. Compound 1o showed the most potent inhibitory activity against A431 and A549. Compounds 1k and 1n exhibited higher potency against HL-60 than gefitinib and SAHA. N-Aryl salicylamides with a hydroxamic acid moiety at 5-position is another new HDAC-EGFR dual inhibitors.     

Study of reactions induced by hydroxylamine treatment of esters of organic acids and of 3-ketoacids: application to the study of urines from patients under valproate therapy

Hydroxylamine used at alkaline pH as oximating agent in the search for organic aciduria by gas chromatography/mass spectrometry (GC/MS) induces other chemical reactions. Esters are partially transformed in their corresponding hydroxamic acids. GC/MS characteristics of the trimethylsilylated derivatives of the hydroxamic acids arising from alpha-unsaturated esters are here reported. Their mass spectral fragmentation helps in the recognition of peaks arising from the glucuronides of 2-ene- and probably 2,3'-diene-valproic acid. By heating in the injection port of the gas chromatograph, part of some trimethylsilylated hydroxamic acids are transformed to the corresponding isocyanates by a Lossen-like rearrangement. In addition to the corresponding hydroxamic acids, hydroxylamine treatment of alpha-unsaturated esters forms 2-isoxazolidin-3-ones by intramolecular Michael addition. GC/MS characteristics of the trimethylsilylated derivatives of these compounds are reported. Submitted to hydroxylamine, 3-ketoacids forms 2-isoxazolin-5-ones by cyclization of the oximes after acidification. This explains the existence of two GC peaks observed from urine extracts of patients under valproate therapy, which correspond to two tautomers of 2-isoxazolin-5-one originating from the oximes of the 3-keto-valproic acid.     

Resistance of cereals to aphids: Interaction between hydroxamic acids and the aphid Sitobion avenae (Homoptera: Aphididae)

The hydroxamic acids in the seedlings of four varieties of winter wheat were extracted initially in boiling methanol in order to avoid enzymatic hydrolysis. The increase in numbers of aphids on these varieties of wheat were recorded. HPLC analysis of hydroxamic acids in the extracts showed the presence of DIMBOA-glucoside, DIMBOA-aglucone and its benzoxazolinone (MBOA). There was a highly significant negative correlation between the concentration of DIMBOA-aglucone in the seedlings and aphid performance. However, the association between aphid performance and DIMBOA-aglucone in the tips of the seedlings was weaker. In general aphid infestation of wheat seedlings slightly increased the concentration of all the hydroxamic acid derivatives. Moreover, aphids ingested hydroxamic acids and were able to detoxify some of the DIMBOA-aglucone they ingested.     

Synthesis and dual biological effects of hydroxycinnamoyl phenylalanyl/prolyl hydroxamic acid derivatives as tyrosinase inhibitor and antioxidant

We previously reported that caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) acted as both a good antioxidant and tyrosinase inhibitor, in particular when caffeic acid was conjugated with proline or amino acids having aromatic ring like phenylalanine. Here, various hydroxycinnamic acid (HCA) derivatives were further conjugated with phenylalanyl hydroxamic acid and prolyl hydroxamic acid (HCA-Phe-NHOH and HCA-Pro-NHOH) to study the structure and activity relationship as both antioxidants and tyrosinase inhibitors. When their biological activities were evaluated, all HCA-Phe-NHOH and HCA-Pro-NHOH exhibited enhanced antioxidant activity compared to HCA alone. Moreover, derivatives of caffeic acid, ferulic acid, and sinapic acid inhibited lipid peroxidation more efficiently than vitamin E analogue (Trolox). In addition, derivatives of caffeic acid and sinapic acid efficiently inhibited tyrosinase activity and reduced melanin content in melanocytes Mel-Ab cell.     

Thiol-based SAHA analogues as potent histone deacetylase inhibitors

In order to find novel nonhydroxamate histone deacetylase (HDAC) inhibitors, a series of thiol-based compounds modeled after suberoylanilide hydroxamic acid (SAHA) was synthesized, and their inhibitory effect on HDACs was evaluated. Compound 6, in which the hydroxamic acid of SAHA was replaced by a thiol, was found to be as potent as SAHA, and optimization of this series led to the identification of HDAC inhibitors more potent than SAHA.     

Design and synthesis of a novel class of histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACs) have emerged as a novel class of antiproliferative agents. Utilizing structure-based design, the synthesis of a series of sulfonamide hydroxamic acids is described. Further optimization of this series by substitution of the terminal aromatic ring yielded HDAC inhibitors with good in vitro and in vivo activities.     

HRP-catalyzed bioactivation of carcinogenic hydroxamic acids. The greater reactivity of glycolyl- versus acetyl-derived hydroxamic acids

An analysis of the hydroxamic acid oxidation reaction by H2O2 and horseradish peroxidase (HRP) was made with three pairs of hydroxamic acids. Each pair consisted of the aceto- and glycolhydroxamic acid derivatives from one of three different arylhydroxylamines. The parent arylhydroxylamines were the known carcinogens, N-hydroxy-2-aminofluorene and N-hydroxy-4-aminobiphenyl and the noncarcinogen 4-chlorophenyl-hydroxylamine. All the hydroxamic acids appeared to be converted to products that were expected on the basis of the previously-proposed mechanism of this peroxidative reaction. Each acetohydroxamic acid gave the corresponding nitroso compound and O-acetyl ester of the starting material in approximately equal amounts. The glycolhydroxamic acids gave the corresponding nitroso compound and a relatively unstable product that was proposed, by analogy, to be the O-glycolyl ester of the starting material. A comparison of the initial rates of reaction of each hydroxamic acid pair showed that the glycolhydroxamic acid was much more susceptible to the peroxidation reaction than was the corresponding acetohydroxamic acid. The initial rate of the reaction was also highly dependent upon the nature of the aromatic ring in the order fluorene greater than biphenyl greater than 4-chlorophenyl. The relative degree of HRP-catalyzed covalent binding to DNA of the aceto- and glycolhydroxamic acids in the fluorene series was studied and found to parallel the relative rates of reaction of these substrates in the H2O2/HRP system. It was proposed that glycolhydroxamic acids are likely to be more genotoxic than are acetohydroxamic acids when subjected to peroxidative bioactivation conditions.     

Identification of a potent non-hydroxamate histone deacetylase inhibitor by mechanism-based drug design

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, we synthesized several suberoylanilide hydroxamic acid (SAHA)-based compounds designed on the basis of the catalytic mechanism of HDACs. Among these compounds, 5b was found to be as potent as SAHA. Kinetic enzyme assays and molecular modeling suggested that the mercaptoacetamide moiety of 5b interacts with the zinc in the active site of HDACs and removes a water molecule from the reactive site of the deacetylation.     

Self-assembly and cytotoxicity study of waterwheel-like dinuclear metal complexes: the first metal complexes appended with multiple free hydroxamic acid groups

Two waterwheel-like dinuclear complexes [M(2)(PHA)(4)(H(2)O)(2)] (M = Cu(II) (1), Zn(II) (2); HPHA = phthal-hydroxamic acid) appended with four free hydroxamic acid groups, namely, free hydroxamic acid metal complexes (FHAMCs) have been synthesized and characterized. The crystal structure of complex 1 was determined by single crystal X-ray diffraction, which adopts the paddlewheel motif with four bidentate carboxylate ligands joining two Cu(II) ions. The relative cytotoxicities of compounds 1 and 2 against SMMC-7721 and HO-8910 cell lines are similar and more predominant than HPHA (IC(50): Cu(II)>Zn(II)>HPHA). The synergic effect of the bound water molecules, multiple free hydroxamic acid groups and dimetal active sites with bridging carboxylate may have significant impacts on their pharmacological activity. As the prototype for a new class of hydroxamic acid derivatives, the self-assembly of FHAMCs presents a promising new strategy in designing multiple hydroxamic acids with remarkable bioactivities.     

Zinc binding groups for histone deacetylase inhibitors

Zinc binding groups (ZBGs) play a crucial role in targeting histone deacetylase inhibitors (HDACIs) to the active site of histone deacetylases (HDACs), thus determining the potency of HDACIs. Due to the high affinity to the zinc ion, hydroxamic acid is the most commonly used ZBG in the structure of HDACs. An alternative ZBG is benzamide group, which features excellent inhibitory selectivity for class I HDACs. Various ZBGs have been designed and tested to improve the activity and selectivity of HDACIs, and to overcome the pharmacokinetic limitations of current HDACIs. Herein, different kinds of ZBGs are reviewed and their features have been discussed for further design of HDACIs.     

Design and synthesis of novel histone deacetylase inhibitor derived from nuclear localization signal peptide

We describe herein the synthesis and characterization of a new class of histone deacetylase (HDAC) inhibitors derived from conjugation of a suberoylanilide hydroxamic acid-like aliphatic-hydroxamate pharmacophore to a nuclear localization signal peptide. We found that these conjugates inhibited the histone deacetylase activities of HDACs 1, 2, 6, and 8 in a manner similar to suberoylanilide hydroxamic acid (SAHA). Notably, compound 7b showed a threefold improvement in HDAC 1/2 inhibition, a threefold increase in HDAC 6 selectivity and a twofold increase in HDAC 8 selectivity when compared to SAHA.     

Design and synthesis of non-hydroxamate histone deacetylase inhibitors: identification of a selective histone acetylating agent

A series of suberoylanilide hydroxamic acid (SAHA)-based non-hydroxamates was designed, synthesized, and evaluated for their histone deacetylase (HDAC) inhibitory activity. Among these, methyl sulfoxide 15 inhibited HDACs in enzyme assays and caused hyperacetylation of histone H4 while not inducing the accumulation of acetylated alpha-tubulin in HCT116 cells.     

Pyrimidine-2,4,6-Triones: a new effective and selective class of matrix metalloproteinase inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that have been implicated in various disease processes. Different classes of MMP inhibitors, including hydroxamic acids, phosphinic acids and thiols, have been previously described. Most of these mimic peptides and most likely bind in a similar way to the corresponding peptide substrates. Here we describe pyrimidine-triones as a completely new class of metalloprotease inhibitors. While the pyrimidine-trione template is used as the zinc-chelating moiety, the substituents have been optimized to yield inhibitors comparable in their inhibition efficiency of matrix metalloproteinases to hydroxamic acid derivatives such as batimastat. However, they are much more specific for a small subgroup of MMPs, namely the gelatinases (MMP-2 and MMP-9).