Design,Synthesis And Anti-tumor Activity Evaluation Of Novel 1,3-diaryl-1,2,4-triazole Derivatives As HDAC6 Inhibitors

Histone deacetylase 6(HDAC6),as a unique member of HDACs family,contains two tandem catalytic domains,CD1 and CD2.Meanwhile,CD2 has a wider range of substrates and is able to catalyze several non-histone substrates,including α-tubulin and HSP90,while CD1 is highly specific for peptide substrates containing C-terminal acetyllysine residues.Owing to this unique protein structure and diverse substrate types,HDAC6 performs a wide range of biological functions,and its abnormally high expression in cells is positively correlated to the occurrence and development of various diseases,especially tumors.Over the past few decades,the role played by HDAC6 inhibitors as anticancer agents has sparked great interest of biochemists worldwide.While,the currently reported HDAC6 inhibitors are still limited with several problems,such as weak subtype selectivity,low response rate towards solid tumors,and poor developability as drugs.It is important to develop novel HDAC6 inhibitors that are highly efficient,selective,and effective in enhancing sensitivity to solid tumors when used alone or in combination with chemotherapeutic agents,and to further enrich the structural types of HDAC6 inhibitors.Through an initial in-house library compounds screening,we fortunately identified a class of compounds with 1,3-diaryl-1,2,4-triazole backbone that exhibited potential HDAC6 inhibitory activity.Meanwhile,literatures have revealed that most of the reported HDAC6 inhibitors contained amide units in their Cap groups.Inspired by this,we attempted to utilize the 1,2,4-triazole nucleus,which is a recognized bioisostere of amides,as the Cap group,benzylamine as the Linker region,and amide,carboxylic acid and hydroxamic acid as the ZBG region to design and synthesize a series of 1,3diaryl-1,2,4-triazole derivatives(total 40 compounds).The structure-activity relationship(SAR)studies revealed that:1)Hydroxamic acid-based ZBG displayed the best potency;2)The modification of N1 phenyl of the 1,2,4-triazole-based Cap group was unfavorable for activity;3)The modification of C3 phenyl should take into account the steric hindrance of the substituents,as well as the substituted site.Current studies proved that Cl substitution at the para position was best for activity.Amongst,compound Ⅰ-10k displayed the best inhibition effect on HDAC6 with IC50 value of 3.12 nM,with 352-、24-、45-and 1542-fold selectivity over HD AC1、HDAC2、HDAC3 and HDAC8,respectively,that was much more potent than the control SAHA.Docking studies revealed the binding models of compound Ⅰ-10k showed similarity with the ligand HPB in the active site of HDAC6-CD2,suggesting the series Ⅰ derivatives represented by Ⅰ-10k might inhibit HDAC6 activity by interacting with CD2.Analysis of the TCGA(The Cancer Genome Atlas)database revealed that HDAC6 mRNA was significantly more expressed in cancerous tissues of esophageal cancer patients than in normal tissues,and positively correlated with poorer prognosis of patients.The biological evaluations demonstrated Ⅰ-10k showed antiproliferative activity(IC50=2.77-12.58μM)towards several esophageal cancer cells with different degrees of differentiation(TE-1,Kyse-30,EC-109,Kyse-510,Kyse-140,and Kyse150),and displayed the best inhibitory activity against EC-109 cells.Further mechanism studies showed Ⅰ-10k could concentration-dependently inhibit the invasion of EC-109 cells,and induce apoptosis,without significant effect on cell cycle arrest.Subsequent pharmacodynamic evaluations demonstrated Ⅰ-10k could effectively inhibit the growth of tumor cells in human EC-109 xenograft mouse model within a safe dose range,and exhibited good pharmacokinetic properties,with potential for further development.The above findings have encouraged us to carry out further structural modifications of such 1,3-diaryl-1,2,4-triazole derivatives to explore the underlying effects on the inhibitory activity towards HDAC6.So we conducted systematic conformational studies on the Cap and Linker regions with the help of CADD(Computer-aided drug design),scaffold hopping,and bioisosteric replacement strategies,and synthesized a total of 90 compounds involving seven new series of compounds:1)Series Ⅱ compounds focused on the alkylation substitution of N1 phenyl of the Cap region;2)Series Ⅲ compounds focused on heteroaromatic substitution of C3 phenyl;3)Series Ⅳ/Ⅴ/Ⅵ were designed to discuss the change of the activity after the scaffold hopping of 1,2,4-triazole backbone;4)Series Ⅶ-alkylation of NH in the Linker region;5)Series Ⅷ-scaffold hopping of the Linker region.The systematic structural modifications and SAR studies further dissected the structural features of 1,3-diaryl-1,2,4-triazole derivatives targeting HDAC6 and provided ideas for designing single-targeted HDAC6 or dual-targeted HDAC6/1 inhibitors on the basis of this backbone.In conclusion,this paper provided a SBDD(Structure-based drug design)approach to design and synthesize eight series of 130 structurally novel derivatives,which enriched the structural types of HD AC inhibitors;And the effects of changes in the derivatives Cap,Linker,and ZBG regions on the inhibitory efficacy and selectivity of HDAC6 were systematically investigated via SAR studies,providing a reference for the construction of targeted HDAC inhibitors.Meanwhile,the evaluation of antiproliferation activity of representative compound Ⅰ-10k against esophageal cancer cells EC-109 in vitro and in vivo also provided a new candidate molecule for the treatment of esophageal cancer,as well as an important theoretical and experimental basis for the discovery of anti-digestive tract tumor drugs targeting HDACs.