| Cancer has always been one of important problems affecting the healthy development of modern human society.In the course of the long struggle between human and cancer,a variety of methods have been used for clinics in which drug therapy is one of the main methods for the clinical intervention and treatment.According to the action mechanisms of recent new anticancer drugs,we can know that many drugs exert their therapeutic effects by inducing apopotosis of cancer cells.Scientists find that apopotosis is a normal physiological process of our body which plays an integral role in many biological events such as embryogenesis,tissue homeostasis and removing unwanted or infected cells.In the pathlogical condition,dysregulated apoptosis is closely related to the development of many human disorders,such as cancer,autoimmune,cardiovascular diseases and so on.Cells undergo apoptosis by two major apoptotic pathways,namely mitochondrial and death receptor-mediated pathways.In the mitochondrial-mediated pathway,the B-cell lymphoma-2(Bcl-2)protein family is a key regulator which consists of two major subfamilies,that is,pro-apoptotic Bcl-2 proteins(Bax,Bak,Bid,etc)and anti-apoptotic Bcl-2 proteins(Bcl-2,Bcl-xL,Mcl-1,etc).It was reported that the overexpression of anti-apoptotic Bcl-2 proteins could be observed in many cancer types.For example,Bcl-2 protein was overexpressed in 80%of B-cell lymphomas and 30-60%of prostate cancers,while overexpression of Bcl-xL could be seen in breast and lung cancers.Therefore,studying the inhibitors targeting against anti-apoptotic proteins could effectively inhibit their activities and induce tumor cells to undergo apoptosis to treat cancers.Many anti-apoptotic Bcl-2 proteins inhibitors are reported in recent decades,which consist of antisense oligonucleotides,BH3 peptides and small-molecule inhibitors.Because the two formers have some problems in the process of drug delivery,small molecule inhibitors with high efficiency and low toxicity has become the focus of this kind of drug development.With the development of modern structural biology.nuclear magnetic resonance,computer virtual screening and other modern advanced technologies,scientists developed many potent small-molecule anti-apoptotic Bcl-2 proteins inhibitors,such as ABT-199?ABT-263?(R)-Gossypol(AT-101)?GX015-070?HA14-1 and so on.In 2008,Professor Chengguo Xing discovered a rhodanine derivative as Bcl-2 protein inhibitor,WL-276,with a new structure,good activity,effectively inducing the apoptosis of PC-3 tumor cells and other advantages.In vivo,it exhibited excellent inhibitory activity against the growth of PC-3 tumor.On the basis of the previous cooperation with professor Xing,our group designed and synthesized a series of new 2-thioxo-4-thiazolidinone derivatives.The bioactivity results showed that the activities of compounds containing aromatic amino acid side chains were more potent than that of the aliphatic amino acid side chains;the aromatic ring replacing the biphenyl part reduced the activities;the methyl on the benzenesulfonamide was not essential for binding affinity for Bcl-2 protein.In this study,based on the scaffold of WL-276,we designed and synthesized a series of 2-thioxo-4-thiazolidinone derivatives(series A)to explore their SARs by using the aromatic amino acid sides such as 4-bromophenylalanine and tryptophan side chain to replace the phenylalanine and adding different substituents on the biphenyl part and benzenesu1 fonam i des..In our previous study,the new pyrrolidine derivative lead compound BPA2 exhibited a moderate Mcl-1 protein inhibition(Ki=8.4?M)which had poor inhibitory activities for other anti-apoptotic Bcl-2 proteins.As it displayed good water solubility,had a stable scaffold and did not like the previous WL-276 derivatives with poor water solubility and not stability in base.Therefore,based on the preliminary SARs and BPA2,we introduced benzenesulfonamides parts and made further modifications on the biphenyl,phenylalanine side chain and Boc parts to design and synthesize two series of pyrrolidine derivatives(series B and series C)in this study.As to 2-thioxo-4-thiazolidinone derivatives,L-amino acids were used as the starting materials to synthesis the scaffold of 2-thioxo-4-thiazolidinone by one-pot method,then obtained the target compounds by Knoevenagel and amide condensation reactions.During the synthesis of pyrrolidine derivatives,we used L-hydroxyproline as the starting material to generate the target compounds successively through Mitsunobu,Williamson and amide condensation reactions.Two classes of derivatives were designed and synthesized with a total of 140 new compounds which consisted of 78 target comounds and 62 new intermediates.All new compounds were identified with 1H-NMR,13C-NMR and MS.In addition,we also tested their melt points and other physical constants.In the bioactivity studies,the binding affinities for anti-apoptotic Bcl-2 proteins of these target compounds were tested by fluorescence polarization assays(FPAs).Then further evaluations were carried out for the active compounds so as to determine their antiproliferative activities against tumor cell lines by MTT assay.Finally,we tested the activity to induce PC-3 tumor cells to undergo apoptosis by Annexin V-FITC/PI assay.The results showed that most compounds of series A had good inhibitory activities for Bcl-2 protein and some compounds were more potent than WL-276.Among these selected active compounds,four compounds(A4,A15,A21 and A24)exhibited potency in inhibiting three anti-apoptotic Bcl-2 proteins(Bcl-2,Bcl-xL and Mcl-1)except A7 and A16.To our delight,A15(Ki= 63 nM)and A16(Ki = 74 nM)displayed nanomolar Ki values for Mcl-1 protein which were more potent than the lead compound WL-276(Ki = 250 nM).Especially,A16 had no inhibitory activity for Bcl-xL protein and exhibited good selectivity for Mcl-1 protein,which could avoid thrombocytopenia owing to the inhibition of Bcl-xL.In addition,it had similar antiproliferative activities against K562 tumor cells with AT-101.The bioactivity results of two series of pyrrolidine derivatives suggested most target compounds showed better inhibitory activities for Mcl-1 protein than the lead compound BPA2,and some compounds were almost the same as AT-101(Ki= 0.21?M),such as B14(Ki= 0.26.M)and B20(Ki=0.22 ?M).On this basis,eight active compounds(B14,B20,B23,B27,C10,C16,C17 and C18)were selected to test their binding affinities for Bcl-2 and Bcl-xL protein.The result showed that most compounds exhibited good inhibitory acitivities for Bcl-2 protein and displayed poor or no binding affinities for Bcl-xL protein.In the aspect of antiproliferative activities for tumor cells,these compounds had good inhibitory activities against PC-3 tumor cells.Among these compounds,the most active compound B27 displayed similar activities to induce PC-3 tumor cells to perform apoptosis with AT-101 at 8?M concentration during the apoptotic assay.In summary,based on the previous studies for rhodanine and pyrrolidine lead compounds,we further explored the SARs to design and synthesize three series of target compounds.Related bioactivity studies showed that some compounds not only exhibited good inhibitory activities for Mcl-1 protein,but also had poor binding affinities for Bcl-xL.These results will establish foundation for our group to search new anti-apoptotic Bcl-2 proteins inhibitors with high binding affinity and selectivity as well as have a certain reference value for developing new anti-cancer drugs through the mechanism of inducing apoptosis. |
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