Design,synthesis And Biological Evaluation Of BTK/HDAC Dual Target Inhibitors In Mantle Cell Lymphoma

Mantle Cell Lymphoma(MCL)is an invasive and incurable type of B-cell Lymphoma,accounting for approximately 3%to 10%of non-hodgkin’s Lymphoma(NHL).Most patients were in clinical stages Ⅲ or Ⅳ at the time of diagnosis with a higher incidence in men(male:female=2:1).Chromosome t(11;14)(q13;q32)translocation leads to the overexpression of cyclin D1,which is the core of the pathogenesis of MCL.In addition,disorders of the cell cycle,abnormal DNA damage repair,apoptosis,epigenetic modification and disorders of various signaling pathways caused by secondary genetic changes also play vital roles in the development of MCL.At present,with the in-depth study of MCL,targeted drug therapy has made certain progress.Abnormal and excessive activation of the B cell receptor(BCR)signaling pathway is a key factor for the growth and survival of various B-cell lymphomas.BTK(Bruton’s tyrosine kinase),overexpressed in MCL cells,is a pivotal kinase in the BCR signaling pathway.Thus,targeting to BTK is crucial treatment means for MCL.Ibrutinib(IBN),the first-generation irreversible BTK inhibitor,was approved by the FDA in 2013 for the treatment of relapsed/refractory MCL.Although,IBN is effective in the clinical application of MCL,there are also a variety of adverse reactions and IBN resistance.Therefore,it is of great significance to explore new treatment strategies for the treatment of MCL.The combination of mutant proteins which cause resistance to Ibrutinib and HSP90(Heat shock protein 90)increases the stability of the former,while binding of HSP90 to client proteins is regulated by histone deacetylase(HD AC).HD AC takes a crucial role in cancer by deacetylating histones and non-histones,making HD AC an important antineoplastic target.HDAC inhibitors(HDACIs)exert antitumor effects by regulating gene transcription,DNA damage repair,cell cycle,and apoptosis.At present,abundant HDACIs have been used in the treatment of hematologic tumors and shown good therapeutic effects.Besides,the combined application of HDACIs and other anticancer drugs has revealed synergistic effects in improving the anticancer effect.The combination of HDACIs and BTK inhibitors was of momentous impacts in varieties of B-cell lymphomas including IBN-resistant cells,producing a more potent anti-tumor effect than any single drug.Targeting BTK and HD AC can not only inhibit key signaling molecules of BCR signaling pathway,but also degrade key proteins of BCR and non-BCR signaling pathways,thus playing a dual inhibitory role and enhancing the inhibitory effect on B cell lymphoma.Our laboratory had designed and synthesized several series of BTK/HDAC dual-target inhibitors.Based on previous research,this paper further optimized the design to synthesize Yp series compounds.The work maintained 1,3-disubstituted-4-aminopyrazolopyrimidine as the basic skeleton of the BTK inhibitor,and used substituted pyridine aminophenyl or 4-phenoxyphenyl as hydrophobic groups of BTK inhibitor.Simultaneously,the basic skeleton was connected to the hydrophobic groups as the Surface Recognition Group(SRC)of the HD AC inhibitor,also hydroxamic acid was used as Zinc Binding Group(ZBG).Besides,the structure and length of linker were constantly changed to investigate the effects of different linkers and hydrophobic groups on biological activities.All compounds were confirmed by1H NMR,13C NMR and HRMS.All new compounds were evaluated for biological activities.At a concentration of 1 μM,most of the compounds had a BTK inhibition rate of greater than 80%,which were weaker than that of positive drug IBN.With the increase of the length of the Linker,the inhibitory activity on HDAC gradually increased.Among them,compound Ypll revealed the best HDAC inhibitory activity,which was equivalent to SAHA.Later,we performed assay on antiproliferative activity of the above-mentioned compounds in MCL cell lines.Many of them displayed higher inhibitory potency to MCL cell lines,of which dual target compounds Yp17(BTK IC50=99.6 nM,HDACs IC50=261.9 nM)and Yp21(BTK IC50=244.9 nM,HDACs IC50=157.5 nM)existed superior anti-proliferative activity against IBN.In particular,the antiproliferative activities were 5-14 folds compared with IBN in IBN-resistant cell lines(Maver-1,Z138,Rec-R,Mino-R).Moreover,compounds Ypll,Yp17,and Yp21 also had moderate activity against HepG2 cells.The results of this study provided insights into the discovery of novel BTK/HDAC dual-target inhibitors to treat MCL,laying the foundation for the development of candidates against MCL.In the future,we will conduct an in-depth study on their mechanisms of action.