Discovery And Development Of New Anti-cancer Drugs Based On Promoting Apoptosis

Apoptosis is a highly conserved physiological process which is vital for development and tissue homeostasis.Every day of life,about 50 to 70 million cells in adult bodies undergo apoptosis.Strict control mechanisms for apoptosis are very important because unlimited cell death can lead to severe consequences.A hallmark of cancer is escape of apoptosis.And in the last three decades,studies have elucidated how cancer cell escape apoptosis,which lays a foundation for apoptosis-targeted therapies.Apoptosis is regulated through two distinct pathways:the extrinsic apoptosis pathway and the intrinsic apoptosis pathway.Bcl-2 family proteins are key regulators of the intrinsic mitochondrial apoptosis pathway.Studies have revealed that cancer cells could upregulate anti-apoptotic Bcl-2 proteins and/or downregulate pro-apoptotic ones in various different ways to escape apoptosis.Accordingly,direct activation apoptotic pathway using anti-apoptotic Bcl-2 protein inhibitors or upregulation pro-apoptotic Bcl-2 family proteins by HDAC inhibitors could restore apoptosis and kill cancer cells.Studies on novel anti-apoptotic Bcl-2 protein inhibitorsMost of anti-apoptotic Bcl-2 protein inhibitors reported are BH3 minetics which mimic the BH3 domain of the pro-apoptotic BH3-only proteins to bind anti-apoptotic Bcl-2 ones.Compound 41 which is a tyrosine derivatives was found moderate ability to inhibit the binding of BH3 peptide to Bcl-2 protein with a Ki value of 5.2μM.Molecular docking studies showed that 41 occupied the p3 and p4 pocket of the bingding groove,and phenyl ring of the benzsulfamide moiety pointed toward the p2 pocket.Based on results of molecular docking studies and the structure-activity relationship of Bcl-2 protein inhibitors in literature,new compounds were designed by keeping tyrosine skeleton and the benzsulfamide moiety of compound 41,and then introducing substituents(nitro and R2)on the benzsulfamide moiety to occupy the p2 pocket so as to enhance the binding affinities.Besides,according to the principle of bioisosterism and homology,we replaced the biphenyl moiety of compound 41 by substituted phenyl groups(Ri)and the Boc moiety by acyl groups(R3)to investigate the effects of these two moieties on binding affinities.A series of tyrosine derivatives(series A)were designed and synthesized through the structural modification mentioned above.Furthermore,a series of tetrahydroisoquinoline derivatives(series B)were designed and synthesized by using the aromatic amino acid side cyclization strategy.For the same reasons,we also introduced substituents(nitro and R3)on the benzsulfamide moiety and replaced the biphenyl moiety of compound 41 by substituted phenyl groups(Ri)and the Boc moiety by acyl groups(R2).Results showed that fourteen compounds of series A and fourteen compounds of series B exhibited improved binding affinities to Bcl-2 protein compared to their predecessor 41,and some of them were even better than Gossypol(e.g.A3,A24,A28,B25 and B26).For series A compounds,The Boc group was vital for compound’s binding affinity to Bcl-2 protein and the removal of this Boc group led to the loss of compound’s activity(e.g,A14-A19).However,the binding affinities could be restored when acyl groups were introduced to this position(e.g.A20-A28).Substitutions on sulfonamide moiety were beneficial to compound’s potency.Compounds with substitutions on sulfonamide moiety behaved much more better(e.g.A3,A24 and A28).But this was a different situation when it came to compounds with cycloalkyl amino groups on sulfonamide moiety(e.g.A8,A9 and A27).In addition,changes on biphenyl part had a little influence on compound’s binding affinities compared with those on the Boc group and sulfonamide moiety(e.g,vs A3 vs A5,A6 vs A7 and A24 vs A28).For series B compounds,compounds with hydrogen on X position(B1-B5)or substituted amino group on R3 position(B10-B12,B18,B27-B29)showed no binding affinities,and compounds with substituted benzyl group on Ri position(e.g.B9,B19 and B25)showed better binding affinities than those with propyl group on this position(B6 and B7).In addition,the change of substitutions on R2 position had a less influence on the binding affinities of compounds except 4-phenyl benzyl group on this position(e.g.B13 and B20).Compounds A1,A24,A28,B19,B21,B25 and B26 were tested their binding affinities to Mcl-1 and Bcl-XL protein.Results showed that these compounds exhibited potent binding affinities to Mcl-1 protein,but had minor or none binding affinities to BcI-XL protein.MTT assays showed that compounds Al,A24,A28,B25 and B26 had certain anti-proliferative activities against tested cancer cell lines,but had less influence on the growth of normal human cell.Among them,compound A28 displayed similar anti-proliferative activities against cancer cells compared to Gossypol.Furthermore,it was found that compounds A28 and B25 induced apoptosis in a dose-dependent manner in the Jurkat cell line.Treatment of the Jurkat cells by 10 μM and 20 μM of A28 for 48 h results in 6.2%and 22.2%of apoptotic cells(early+late)respectively,as compared to 2.6%of apoptotic cells in the DMSO control.Treatment of the Jurkat cells by 15 μM and 30 pM of B25 for 48 h results in 7.3%and 21.3%of apoptotic cells(early+late)respectively,as compared to 3.2%of apoptotic cells in the DMSO control.Caspase-3 activation is a crucial process involved in the intrinsic apoptosis pathway,so we determined the ability of compounds A28 and B25 to induce caspase-3 activity in the Jurkat cell line.The obtained results showed that compounds A28 and B25 induced activation of caspase-3 activity in a dose-dependent manner.Studies on novel HDAC inhibitorsA series of 2,6-disubstituted purine HDAC inhibitors were designed and synthesized by our group.Studies revealed that several compounds had comparable or even better in vitro HDAC inhibitory activity than SAHA,but unfortunately;the anti-proliferative activity of these compounds was modest.Among them,compound H5r was the most active one with the IC50 value of 0.075 μM agaisnt HeLa extract(mainly HDAC1 and HDAC2).Using compound H5r as lead compounds,we replaced 2,6-disubstituted purine ring by 2,9-disubstituted purine ring and-CH2CONH-by-NH-to abtain a series of new HDAC inhibitors(series C).We also investigated the length of the linker on the effects of HDAC inhibitory activity.In vitro HDAC inhibitory activities showed that nine compounds exhibited similar or even better HDAC inhibitory activities than SAHA.Notably,three compounds C9,C13 and C14 exhibited very potent inhibitory activities with the IC50 values of 26nM,27nM and 30nM,respectively.The length of the linker and substituents on purine ring had an effect on the inhibitory activities against HDACs.As expected,compounds with shorter linkers(C1-C5 and C21)showed poor inhibition on HDAC,while compounds containing a five-or six-methylene linker(C6-C20,C22-C25)possessed better inhibitory activities.The substitution with amino or chloro on purine C2-position was superior to other substitutions or no substitution,such as C13 vs C19 and C12 vs C24.It seemed that bulky substitutions on purine C2-position resulted in a decrease in inhibitory activities.On the other hand,compounds with substitutions on purine Ng-position retained more potency than those with no substitution(e.g.C7 vs C6 and C9 vs C8).Further studies indicated that the most active compounds C9、C13 and C14 showed better inhibition to HDAC2 than to HDAC8 and possessed obvious anti-proliferative activities.Among them,target compound C13 exhibited better results compared with SAHA in all three tested cell lines.Besides,compound C13 increased the level of acetylated histone H3 in a dose-dependent manner in KG 1 cells.Compounds C13 was also found to induce apoptosis in a dose-dependent manner in the KG 1 cell line.Treatment of the KG1 cells by 1μM and 2μM of C13 for 24 h results in 20.7%and 50.2%of apoptotic cells(early+late)respectively,as compared to 14.2%of apoptotic cells in the DMSO control.Besids,Compound C13 could induce activation of caspase-3 activity in a dose-dependent manner.In conclusion,developing new agents to promote tumor cell apotosis is a new strategy in anti-cancer agents studies.In our studies,two series of anti-apoptotic Bcl-2 proteins inhibitors(series A and B)and a series of HD AC inhibitors(series C)have been developed based on the mechanism of tumor cell escaping apoptosis,A total of 147 new compounds were synthesized,and 82 of them were target compounds.Target compounds were identified by 1HNMR,13CNMR and HRMS,and also tested of their moilting points and purities.The preliminary biological evaluation found some active compounds which could be used as lead compounds for further study.