Structural Modification Based On Ibrutinib And Biologica Evaluation In Mantle Cell Lymphoma

Mantle Cell Lymphoma(MCL),with poor prognosis,aggressive and diagnosed,is an incurable non-Hodgkin’s Lymphoma(NHL),which accounts for about 3-6%of NHL.The median age of onset was 68 years old,most of which were in stage Ⅲ-Ⅳ at diagnosis,with a median survival of only 3-5 years.The chromosome t(11;14)(q13;q32)translocation is a genetic feature of MCL.The chromosome translocation leads to the overexpression of Cyclin D1,which is the center of MCL pathogenesis.Cell cycle disorder,genomic instability,abnormality of DNA damage repair mechanism and epigenetics also play important roles in the development of MCL.At present,the treatment of MCL is still based on traditional systemic chemotherapy,such as R-CHOP(rituximab+cyclophosphamide+doxorubicin+vincristine+prednisone),which can significantly improve the complete response(CR)and progression-free survival of patients.However,some patients developed drug resistance.The treatment of relapsed/refractory MCL has encountered new challenges.New drugs and treatment options are urgently needed to prolong the overall survival of patients.Based on the pathogenesis of MCL,the research and development of new target drugs play important roles in the treatment of MCL.The B cell receptor(BCR)signaling pathway is implicated in the pathogenesis of MCL.BTK plays a critical role in BCR signaling pathway,which regulates cell survival,proliferation,activation,differentiation and maturation of B cells.BTK is commonly overexpressed in MCL.In 2013,the first-in-class BTK-inhibitor ibrutinib(IBN)was approved by FDA for the treatment of MCL.However,another phase II clinical trial found that approximately 43%of MCL patients did not respond to IBN or were initially effective but relapsed within 12 months of treatment.Once relapsed after IBN treatment,the 1-year survival rate was only 22%.In addition to drug resistance,it has some undesirable side effects due to its unexpected off-target toxicities resulting from potential binding to non-target locations.In addition to targeting BTK,IBN interacts with several kinases involved in other important signaling pathways,such as EGFR,FGR,FRK,HER2,HER4,ITK,JAK3,LCK,BLK,and TEC.BTK inhibitors with high selectivity may be more tolerant to patients and reduce undesirable side effects.Therefore,the development of BTK inhibitors with higher selectivity and lower toxicity has important clinical significance.In this paper,four series BTK inhibitors(RA,RB,RC and RD)were designed and synthesized base on the principle of medicinal chemistry and means of computer-aided drug design.All compounds were determined the inhibitory activity against BTK kinase and the antiproliferative activity against MCL.Compared with IBN,a number of hit compounds with higher BTK selectivity,showed more potent anti-proliferative activity against MCL cell lines and primary tumor cells of MCL patients.Firstly,we designed and synthesized RA series compounds,using the fragment splicing design strategy.This series focused on the effects of hydrophobic groups and terminal substituents on biological activity.Many compounds exhibited strong BTK inhibitory activities.The IC50 values of compounds RA-6a,RA-6b,RA-12i,RA-13a,RA-13b,RA-13c and RA-13e were 27,50,47,21,64,52 and 45 nM,respectively.Compounds RA-6a,RA-13a,RA-13c and RA-13e demonstrated potent antiproliferative effects in MCL cell lines which surpassed those in IBN.Further experiments found that compounds RA-6a and RA-13e induced dose-dependent cell apoptosis in Z138 and Jeko-1 cells.Compound RA-13e treating Z138 cells for 24h could induce cell cycle arrest in the G1 phase.Western blotting of the Z138 cells with RA-6a displayed inhibition the phosphorylation of BTK and PLCy2.RA-6a also demonstrated more potent antiproliferative activity in primary MCL patient cells than IBN.In addition,RA-6a and RA-13e behaved higher selectivity than IBN.By computer-aided drug design we designed and synthesized the RB and RC series.In vitro enzyme assays,some compounds RB showed strong BTK inhibitory activity,while RC series completely lost the inhibitory activity against BTK kinase.Compound RB2 showed potent effect against MCL cells with IC50 values lower than 1μM,which is 3-39 folds higher than IBN.Then we performed the further experiments on compound RB2.Compound RB2 induced apoptosis in Z138 and Jeko-1 cells with a dose-dependent manner at low concentrations.In Z138 cells,RB2 can effectively reduce the phosphorylation level of BTK and downstream PLCy2 at low micromolar concentrations.In addition,RB2 could significantly reduce the survival of primary tumor cells in MCL patients,and its inhibitory effect was better than IBN.The results of kinase selectivity experim ents showed that RB2 had higher selectivity for BTK than IBN.The major off-target kinases of RB2 were those with cysteine residue in the ATP binding pocket,which could form irreversible bound by the covalent reactive group.RB2 showed high potency against Blk,Bmx,Tec,Txk,ErbB4 and EGFR with IC50 values of 10,6,36,104,147 and 292 nM,respectively.Compared to IBN,RB2 showed much lower inhibitory effect against other kinases,indicating improved BTK selectivity and potential safety improvement for the treatment of MCL.Although the RC series lost their inhibitory activity against BTK,most compounds of RC series showed potent anti-proliferative activity in MCL.Then we selected potent compound RC6 testing a panel of 24 kinases to research its BTK-independent mechanisms,while it had no inhibitory activity against them.Based on the open-loop strategy,we designed and synthesized the substituted pyrazole derivatives(RD series).These compounds lost the inhibitory activity against BTK,but they obtained anti-proliferative activity against MCL cell lines.RD12a,RD12b,RD15a and RD15b exhibited the best antiproliferative activity,with an IC50 value of lower than 1μM,which was 3-28 folds higher than IBN.These potent compounds can be studied as anti-cancer agents,and their exact mechanisms should further study.It has been reported that HDACs inhibitors could down-regulate the expression of c-Myc and they exhibited significantly anti-proliferative activity in MCL cells and IBNresistant MCL cells.HDACs inhibitors can also restore the expression of microRNAs targeting BTK,down-regulating BTK protein,blocking downstream signaling,and inducing apoptosis.In addition,combination of BTK and HD AC inhibitors exhibited synergistic anti-tumor effects in MCL.BTK/HDAC dual-target inhibitors have not been reported.In this paper,based on th e previous BTK inhibitor research,we designed and synthesized a series of BTK/HDAC dual-target inhibitors,testing the inhibition activities against BTK/HDAC and antiproliferative activity against MCL.The RI series focuses on the effects of linkers,hydrophobic groups and ZBG groups on biological activity.In RI-a series,the hydroxamic acid was ZBG group,investigating the SAR of hydrophobic groups and linkers.Unfortunately,these compounds with potent BTK inhibitory activity had no inhibitory effect on HDACs.The compounds RI-a5,RI-a8,RI-a9,RI-all,RI-a12 and RI-a13 exhibited higher BTK inhibitory activity IC50 values of 26,16,19,21,10 and 7 nM,respectively.RI-a9,RI-a12 and RI-a13 using piperidine and pyrrolidine as linkers had effective activity against BTK and MCL cells.While compound RI-a 14 with the methylene group linker completely loses the BTK inhibitory activity,which we speculated may be due to the short linker.The RI-b series used o-phenylenediamine as ZBG functional group to investigate the effect of linkers and hydrophobic groups on biological activity.Most compounds did not have desired inhibitory activity against HDACs.Although compound RI-b12 had no inhibitory effect on HDACs,it obtained better inhibitory activity against BTK than IBN(RI-b12 IC50=4nM,IBN IC50=8 nM).RI-b12 could be further developed as a BTK inhibitor.Compound RI-b3 showed strong inhibitory activity against both BTK and HDACs(BTK IC50=54nM,HDACs IC50=1.5μM),and its anti-proliferative activity against MCL was significantly stronger than IBN.Based on RD-a9 and RD-a14,we designed RD-c and RD-d series to study the SAR of the linkers’ length.Compounds obtained effective HD AC inhibitory activities,extending the length of linkers.Compounds RI-c3,RI-c4,RI-dl,RI-d2 and RI-d3 showed strong inhibitory activity against BTK and HDAC.RI-c4 and RI-d3 were the best agents(RI-c4 BTK IC50=31 nM,HDACs IC50=30 nM;RI-d3 BTK IC50=88 nM,HDACs IC50=37 nM).RI-c4 and RI-d3 also exhibited predominant antiproliferative activity against MCL.We selected the most active RI-b3,RI-c4 and RId3 for preliminary mechanism studies.RI-b3,RI-c4 and RI-d3 could effectively induce apoptosis in Jeko-1 and Z138 cells in a dose-dependent manner.The antiproliferative activities of the compounds RI-b3,RI-c4 and RI-d3 against Jeko-ko(BTK knock out Jeko-1)cells were significantly weaker than that against Jeko-1 cells,consistent with the results of IBN.It indicated that inhibition BTK was an important mechanism for anti-tumor effects of dual-target compounds.In addition,these BTK/HDAC dual-target compounds behaved strong anti-tumor activity on primary IBN-resistant MCL cell lines,which is beneficial for the treatment of MCL.In this paper,we synthesized 72 BTK inhibitors and 35 BTK/HDAC dual-target inhibitors.All compounds had not been reported and were confirmed by 1H NMR,13C NMR and MS.They were tested the inhibition activities against BTK/HDAC and antiproliferative activity against MCL.A number of compounds showed stronger antiproliferative activity against MCL cells than IBN.This study could serve as basis for the further optimization of targeted drugs,the study of anti-tumor mechanism,and the screening of anti-cancer drugs.