Design, Synthesis And Biological Evaluation Of Hydroxamic Acids As Histone Deacetylase Inhibitors

Histone deacetylase (HDAC) is one of the most important anti-cancer target. It is present in eukaryotic cells widely. At present, there are 18 subtypes has been found. They can be divided into two categories on the basis of homology with the yeast’s histone deacetylase. One kind of categories is the Zn2+-dependent histone deacetylase (including Ⅰ, Ⅱ,Ⅳ class HDAC), another kind is the NAD+-dependent histone deacetylase (class Ⅲ HDAC).HDAC inhibitors can inhibit the side chain of histone to deacetylate, make the histone acetylate, thereby change the structure of DNA, has played the role of regulation of gene expression, and can enhance the stability of the DAN genome, the normal gene mutation probability reduced. Increasing the acetylation of HDAC can also make the cell division cycle arrest, induction of cell differentiation and apoptosis, inhibition of tumor angiogenesis. Structure of HDAC inhibitors include the aniline, hydroxamic acids, electrophilic ketones and so on. Vorinostat and Romidepsin have approved for the treatment of cutaneous T cell lymphoma (CTCL) respectively in 2006 and 2009 by American FDA as HDAC inhibitors. More compounds get into clinical research. It indicates that the HDAC inhibitor has broad development prospects as a kind of new antitumor drugs.HDAC inhibitors of hydroxamic acid may cause blood disorders, taste disorders, fatigue, diarrhea. Due to the poor stability of hydroxamic acid groups, the application of these drugs is restricted. HDAC inhibitors of aniline are usually showing a low activity than hydroxamic acids. We chose HDAC-2 (MAX) enzyme and computer-aided drug design,18 compounds of N-hydroxy-N-phenyl-4-benzamido-benzamides were designed. The benzene ring or a substituted benzenering connect to the benzene ring on the nitrogen atom of the hydroxamic acid group. The substituted benzene ring enter into 14A hydrophobic cavity at the bottom of the zincion, so that the structure of molecules combine with catalytic active center more better. This structure reduces the acidity of the hydroxamic acid, the purpose of reducing side effects is achieved. Substituted nitrobenzene,4-nitrobenzoic acid,4- chlorobenzoyl chloride, benzoyl chloride and phenylacetyl chloride were used as the raw material to synthesize target compounds.4-benzoylamido benzoylchloride was synthesized via esterification, reduction, benzoyl, hydrolysis and acylation of 4-nitrobenzoic acid. Then connected with N-hydroxy-substituted aniline which obtained by reduction of substituted nitrobenzene under Raney nickel catalyzed. The HRMS,13CNMR and ’HNMR were used to confirm the structure.In the enzyme inhibition assay, the target compounds’median inhibitory concentrations of the enzyme extract from HeLa cells is in the micromolar level. Ortho-substituted compounds a2, b2, c2 have a low activity than ortho-chloro-substituted compounds. The activity will increase when using chlorine or methyl to replace para position of benzene ring, for example the compounds a4, a6, c4, c6 have a better inhibitory activity (IC50<10μM) than the positive control drug SAHA (IC50= 10.8μM). We also evaluated the activity of histone deacetylase inhibiting in vitro. Activity of inhibition Cell proliferation was detected on cervical Hela cells, A-549 cells, EC-109 cells, Hep G2 cells and HFF cells by MTT method. According to the measurement result, biological activity and structure-activity relationship was studied. Compound al, b1, b4, c4, C6 had a stronger activity than SAHA, but had a less activity than SAHA on normal fibroblasts. Then the acute toxicity tests in mice and anti-tumor experiments in vivo was did. Compounds a1, b1,b4, c4, C6, IC50>3.3mmol/kg, is higher than SAHA IC50= 2.92mmol/kg, so the experimental compounds’ toxicity is much smaller than SAHA. In vivo antitumor experiments, half of the inhibition quantity of compounds b4, C6 is 197.4μmol/kg and 136.4μmol/kg. It is less than the value of the control drug SAHA 294.6μmol/kg, the inhibitory activity of compound c6 is superior to b4 and is twice of SAHA.This article reveals the structure of N-hydroxy-N-phenyl-4-benzoylamido-benzamides compound has anti-tumor effect and security advantages. The antitumor mechanism is studied also, but further mechanism, sub-acute toxicity, pharmacokinetics and other pharmaceutically further study need to continue study.