Design,Synthesis And Preliminary Bioactivity Studies Of Novel Indazole-3-acylsulfonamide Derivatives As Mcl-1 Inhibitors

Cancer,that is,malignant tumor,is a type of diseases caused by abnormal growth of cells and human body dysfunction,which seriously affects human health.Evading apoptosis is one of the important characteristics of cancer,which is also one of the main reasons for the resistance of tumor cells to traditional therapeutic drugs.Apoptosis pathways include endogenous and exogenous apoptosis pathways,wherein,B-cell lymphoma leukemia-2（Bcl-2）family proteins play an important role in the endogenous apoptosis pathway.Myeloid leukemia factor-1（Mcl-1）is overexpressed in a variety of tumor cells.which is not only closely related to the development of tumors,but also assists tumor cells to be resistant to a variety of traditional anti-tumor drugs such as Paclitaxel,Vincristine and so on.For the Mcl-1protein,some active molecules with novel structures have been developed based on various drug design methods and strategies,and have good Mcl-1 inhibitory activities.However,the development of Mcl-1 inhibitors is still relatively slow,and no marketed drugs have been reported.In addition,the existing Mcl-1 inhibitors still have obvious defects,such as low drug-like properties,relatively low cell activity,lack of in vivo activity data and other problems in terms of physical and chemical properties.Therefore,it is urgent for researchers to find more structurally novel and more active Mcl-1 inhibitors,which could provide more chemical entities for clinical research.Through literature review,acylsulfonamide is a common structural part in organic chemistry,which has excellent hydrogen bond properties（as hydrogen bond donors or acceptor）.In the past few decades,many compounds containing acylsulfonamide moiety have been developed,which have a wide range of biological properties,such as anti-viral,anti-cancer and anti-bacterial activitives.Additionally,indazole is an important aromatic system in heterocyclic chemistry.Due to its unique physical,chemical and biological properties,it has been used as an important skeleton for the design of anti-cancer drug.Therefore,we hybridized the indazole ring with the acylsulfonamide moiety to design a series of new indazole-3-acylsulfonamide derivatives based on the principle of molecular hybridization.In this work,we synthesized a total of 27 new compounds,including 20 target compounds.And the new compounds were structurally confirmed by nuclear magnetic resonance spectra(¹H NMR and ¹³C NMR)and high-resolution mass spectra（HRMS）,and their melting points and other physical parameters were also tested.Subsequently,we conducted preliminary activity studies of target compounds.The binding affinities of the target compound to the anti-apoptotic Bcl-2 proteins（Mcl-1,Bcl-2 and Bcl-x L）were mainly detected by fluorescence polarization（FPA）.The MTT assay wasused to test the anti-proliferative activitives of target compounds on tumor cells;Annexin V-FITC/PI dual staining assay was used to detect the ability of target compounds to induce apoptosis in tumor cells.The abilities of target compounds to activate Caspase-3 activity in K562 cells were evaluated by the Caspase-3 fluorescence assay kit.In addition,we used SYBYL-X 2.1software to conduct molecular docking studies of representative compounds.The test results demonstrated that most compounds showed good binding affinities with Mcl-1.Among them,compound 15（K_i=0.43μM）showed the best binding affinity with Mcl-1,which was slightly better than the positive control AT-101（K_i=0.45μM）.Selective experiments have shown that representative compounds 15,17 and 21 showed good selectivity for Mcl-1.In particular,compound 15 has a more than 40 times binding affinity with Mcl-1 over both with Bcl-2 and Bcl-x L,which has the potential to avoid thrombocytopenia caused by the inhibition of Bcl-x L.The molecular docking results showed that compound 15 occupied the key pocket P2 of Mcl-1 through 3,4-dichlorobenzyl moiety.In addition,five hydrogen bonds were formed between the acylsulfonamide fragment and3-nitro-4-chlorophenyl group and the amino acid residues Arg263,Thr266 and His224,respectively,which enhanced the interactions between Mcl-1 and the acylsulfonamide fragment.The MTT assay results showed that three representative compounds 15,17 and 21had good inhibitory activities against MDA-MB-231 and K562 cells,of which compound 15exhibited better inhibitory activity than the other two compounds(IC₅₀=12.3,10.6 and 6.62μM,respectively).Happily,they had no effect on the normal cells LO2,outperforming the positive control drug AT-101.The apoptosis results showed that compound 15 could effectively induce apoptosis in Mcl-1-dependent tumor cells K562 cells,and the apoptosis rate（36.8%）of K562 cells was slightly higher than that of AT-101（34.3%）at 12μM.In addition,it activated Caspase-3 activity in K562 cells in a dose-dependent manner,further validating its mechanism of action by inducing apoptosis to exert antitumor activity in K562cells.The above results might provide a certain experimental basis and theoretical for the future discovery new Mcl-1 inhibitors with higher potential and selectivity.