Design,Synthesis And Anticancer Activity Study Of HDAC And VEGFR Dual-target Inhibitors

Histone acetylation is important for DNA chromatin structure and gene transcription regulation.The reversible acetylation of histone at the lysine residue is controlled by histone deacetylases(HDACs)and histone acetyltransferases(HATs).HDACs catalyze the deacetylation of nucleosomal histones,which favors condensation of chromatin structure,thus repressing the gene transcription.In addition,HDAC.s also catalyze the deacetylation of non-histone proteins(HSP90,tubulin,p53,Bcl-2,etc),thereby affecting protein stability,activity and protein-protein interaction.Altered expression and dyregulation of HDACs are always closely linked to tumor development HDAC inhibitors(HDACIs)can impact various cell functions by inhibiting the deacetylation of histone or non-histone proteins such as HSP90 and tubulin at the molecular level.thus causing cell cycle arrest,differentiation and apoptosis.Up to now,four HDACIs have been approved by the US Food and Drug Administration(FDA)for the treatment of cancer,which undoubtedly confirms the therapeutic value of HDACs as anticancer target.The growth of solid tumors depends on nutrients and oxygen supplied by angiogenesis,the process wherein new capillaries are formed from existing blood vessels.This is facilitated by a number of endogenous proteins among which VEGF is thought to be the key.VEGF is secreted by tumors and binds to VEGR receptors(VEGFRs)on the surface of nearby endothelial cells,which thereby induces dimerization and auto-phosphorylation of receptor.Subsequent activation of downstream signaling pathways then facilitates the formation of new blood.Thus inhibition of VEGF/VEGFR signaling pathway which blocks tumor angiogenesis could lead to tumor growth inhibition.At present,three antibodies and eight small molecular VEGFR inhibitors have been approved for the treatment of malignant tumors,which greatly confirms the therapeutic value of VEGF/VEGFR signaling pathway in the development of anticancer drugsThough,drugs targeting HDAC or VEGFR alone have achieved significant sucess in anticancer field,both HD AC inhibitors and VEGFR inhibitors have met problems in clinical trials or treatments,such as low response rate and acquired drug resistance.Combination therapies and development of multi-target inhibitors are the strategies to address such problems.Through literature research,we found that HDAC inhibitors and VEGFR inhibitors showed synergistic antitumor effects.And Pazopanib,an approved VEGFR inhibitor,has been proved to show synergistic antitumor effect with a variety of HDAC inhibitors in preclinical and clinical research.Based on the pharmacophore model of HDAC and VEGFR inhibitors and guided by the combination principles,a series of Pazopanib-based dual-target inhibitors of HDAC and VEGFR was designed.In the design of these compounds,we use the indazole pyrimidine amine structure of Pazopanib as Cap group,which is connected with hydroxamic acid or o-phenylenediamine as ZBG group via a suitable linker selected from HDACIs in clinical trials.In this paper,we designed and synthesized 23 novel compounds,whose structures were identified by 1H NMR,13C NMR and HRMS.We firstly used HeLa cell nuclear extract as HDAC enzyme resource to assess HDAC inhibitory activity of our compounds.Most of our compounds showed potent HDACs inhibition.Among the hydroxamate derivatives,compounds 13e,13f and 13g displayed superior HDAC inhibitory activity than the positive control SAHA.6d and 14e,the most potent compounds among o-phenylenediamine derivative,displayed equivalent HDAC inhibition as the positive control MS275.The results of HDAC isofom selectivity assay showed that compounds 13f exhibited more potent HDAC6 inhibition than the positive control SAHA,while compounds 6d and 14g tended to be Class I HDACs selective as MS275.In Western Blot assay,at the same concentration of 100 nM,compound 13f could result in more acetylation of tubulin than SAHA,which was in line with the results of HDAC isoform selectivity assay.While compound 6d could significantly enhance the acetylation of histone H4,which was equivalent to MS275.Meanwhile,representative compounds 6d,10a,13f and 14g showed comparable antiproliferative activities against multiple solid and hematologic tumor cell lines.As for the evaluation of antiangiogenic activity,we first screened our compounds against VEGFR-2 at 5μM.The results showed that compounds 6d,10a,13f and 14g exhibited comparable VEGFR-2 inhibitory activity to the positive control Pazopanib.Then subsequent HUVECs tuber formation and rat thoracic aorta rings assay demonstrated that representative compounds 6d and 14g displayed comparable tumor angiogenesis inhibition to Pazopanib.