Design,Synthesis And Preliminary Biological Evaluation Of Tyrosine Derivatives As Mcl-1 Inhibitors

Apoptosis is remarkably regulated at gene level,which can effectively remove damaged cells caused by DNA damage or during normal development,thus preventing tumor growth.Cancer occurs when apoptosis and cell proliferation are not controlled.For this reason,disorder of apoptosis is considered to be a hallmark of cancer.The mechanism of apoptosis is very complex,involving numerous signaling pathways.There are two main pathways of apoptosis through the death receptor-mediated extrinsic pathway and the intrinsic mitochondrial pathway.Both pathways eventually activated the caspase cascade,causing changes in cell morphology and biochemical characteristics of apoptosis.The Bcl-2 family of proteins exist a series of protein-protein interactions which regulate the intrinsic pathway.The balance between pro-apoptotic and anti-apoptotic proteins is the key to apoptosis.The anti-apoptotic Bcl-2 proteins,such as Bcl-2 and Mcl-1,are generally overexpressed in many human cancers.Without doubt,targeting anti-apoptotic Bcl-2 proteins to restore the tumor cells sensitivity is an effective anti-cancer strategy.Venetoclax is currently the only Bcl-2 selective inhibitor approved by the FDA in 2016,which of drug resistance is related to the overexpression of Mcl-1.Moreover,overexpression of Mcl-1 is the main reason of resistance to radiation and chemotherapy.Therefore,the development of Mcl-1 selective inhibitors is not only beneficial for tumor treatment,but also can overcome the resistance of Bcl-2 inhibitors.Based on our group's work,a series of compounds A1-A31 targeting Mcl-1 were designed and synthesized by using tyrosine derivative L28 as lead compound.Subsequently,the structures of target compounds were confirmed by 1H-NMR,13C-NMR and HRMS.We determined the affinity of Mcl-1 protein using fluorescence polarization.We obtained compound A7 with Ki value of 0.18±0.04 ?M.Representative compounds were selected to determine the affinities of Bcl-2 and Bcl-xL to explore the selectivity.The affinities of A7,A9 and A17 to Mcl-1 was 5-7fold more potent than Bcl-2,all of them existed no activity to Bcl-xL.Surprisedly,both A26 and A27 had no activity to Bcl-2 and Bcl-xL.Molecular docking to study the binding mode of compound A7 to Mcl-1 confirmed that A7 can mimic hot residues of Bim-BH3 domain.Interestingly,compound A7 occupied the Mcl-1 hydrophobic pocket through 3,5-dimethyl-4 chlorobenzene,formed hydrogen bonding interactions with Arg263 through the nitro and sulfonamide and ?-? interactions with Phe270 by the phenylalkylamine group.In addition,we analyzed the differences of binding pockets in Mcl-1,Bcl-2 and Bcl-xL to explain that 1-naphthyl group in R2 position may result in high selectivity against Mcl-1 over Bcl-2 and Bcl-xL.MTT assay revealed that compounds A7,A9,A17,A26 and A27 showed anti cell proliferation activity.Gratifyingly,compound A7 showed better anti-proliferation activity than UMI-77 in HepG2 and KM3 cells.Further studies indicated that compound A7 could dose-induce HepG2 cell apoptosis through the mitochondrial pathway using Annexin V-FITC/PI apoptosis assay,TUNEL assay and Mitochondrial membrane potential assay.After transfection by siMcl-1,the expression of Mcl-1 and the apoptosis of HepG2 cells was detected.It was found that the apoptosis rate of HepG2 cells after silencing the expression of Mcl-1 is 12.53%,which was similar to the apoptosis rates of HepG2 cells treated by A7 and A26 with 10 ?M.It was confirmed that our compound may lead to apoptosis by inhibiting Mcl-1.