Design,Synthesis And Biological Mechanism Of Bis-Substituted Aromatic Sulfonamide Derivatives 3328

Based on previous work and extensive literature review,we designed and synthesized a series of sulfonamide derivatives by molecular hybridization approaches.The structure-activity relationship of the synthesized compounds was explored.The biological mechanism of compound 3328(compound 13g)with the most potent activity was discussed.The detailed research content is as follows:1.We designed and synthesized 31 sulfonamide derivatives by the molecular hybridization approaches:10a-10e,13a-13g,14a-14f,17a-17f,18a-18g;And the effects of anti-tumor activity of substituted groups,sulfonyl groups and benzimidazole groups were discussed.This observation suggested that the electron deficiency or richness of the A ring system may affect the antiproliferative activity to a certain extent and benzimidazole or benzothiophene motifs enhanced anticancer activity and selectivity.Then we explored the significance of the 3,4,5-trimethoxy group of phenyl ring(B ring).These inhibitory activity results indicated that the 3,4,5-trimethoxy group was very crucial for the antitumor.When the 3,4,5-trimethoxy group was replaced by other groups,compounds exhibited weak activity.The antitumor activity of the compounds in vitro was affected by the change of the C ring substituent.To our delight,the compounds with 4-methylbenzene sulfonyl group of C ring showed the most potent activity.2.The MTT method was used to detect the inhibitory activities of sulfonamide derivatives against human gastric cancer cells(MGC-803),human prostate cancer cells(PC-3)and human breast cancer cells(MCF-7).The results showed that some compounds exerted excellent inhibitory activity on three tumor cells,among which compound 3328 exhibited the best activity,and the IC50 values were 1.02μmol/L(MGC-803)and 3.34 μmol/L(PC-3),5.40 μmol/L(MCF-7).Based on the antitumor activity screening results in vitro,compound 3328 and human gastric cancer cell MGC-803 were selected for intensive antitumor mechanism research.3.The cell viability of compound 3328 was tested by clone formation experiment and growth curve experiment.The experimental results showed that compound 3328 significantly inhibited the growth of human gastric cancer cell MGC-803 in a time-dependent manner and colony formation.Therefore,we detected changes in the cell cycle by flow cytometry.The results showed that compound 3328 blocked the cell cycle in the G2/M phase.Western Blotting experiments detected the expression changes of G2 phase-related proteins CyclinB1,CDK1 and M-phase related protein p-Histone3(M-phase blocker molecular marker),which further confirmed this conclusion.4.Exploring the mechanism of compound 3328 inhibiting cell proliferation.Since MAPK/ERK signaling pathway was related to cell proliferation,we detected the proteins related to MAPK/ERK signaling pathway Ras,c-Raf,p-c-Raf,p-MEK,ERK,and p-ERK after treatment with compound 3328 by Western Blotting.The results showed that compound 3328 inhibited the protein expression of the Ras-Raf-MEK-ERK signaling pathway,and the downstream transcription factors Fox03a and c-Myc were also inhibited.The literature reported that the AKT/mTOR signaling pathway and the MAPK/ERK signaling pathway have a synergistic effect in the progression of gastric cancer.Therefore,we also detected the AKT/mTOR signaling pathway related proteins p-AKT,p-mTOR.The expression of these two proteins also showed a concentration-dependent down-regulation.These two pathways were related to tumor cell proliferation,which led to tumor cell proliferation inhibition when it was inhibited.Therefore,we believed that compound 3328 might inhibit the proliferation of human gastric cancer cell MGC-803 by MAPK/ERK and AKT/mTOR pathways.5.Apoptosis can induce tumor cells death.Therefore,we used FITC-AnnexinV/PI double staining method to detect whether compound 3328 could induce apoptosis of human gastric cancer cell line MGC-803.The morphological changes of cells were observed by DAPI fluorescence staining experiment.The results showed that after compound 3328 was applied to human gastric cancer cell MGC-803 for 48 hours,the apoptosis rate increased significantly in a concentration-dependent manner,the nuclear staining deepened,the cytoplasm contracted and ruptured,and the anti-apoptotic proteins Bcl-2 and MCL-1 were both down-regulated,the pro-apoptotic protein Noxa was up-regulated,Cleaved-Caspase-7 was activated,and its substrate PARP was also significantly cleaved.In addition,the expression of important endogenous apoptosis inhibitors c-IAP1 and XIAP were both down-regulated.These results indicated that compound 3328 could induce apoptosis of human gastric cancer cell line MGC-803.6.In order to further verify the reliability of the mechanism of compound 3328 in human gastric cancer cells,we selected two other human gastric cancer cells SGC-7901 and HGC-27 for the above studies.The results showed that compound 3328 also exhibited a potent inhibitory effect on human gastric cancer cells SGC-7901 and HGC-27,with IC50 values of 2.3μmolL and 1.61μmol/L,respectively,and could significantly inhibit the viability of two gastric cancer cells and the formation of clones.After treatment with compound 3328,the cell cycle was blocked in the G2/M phase,and the proteins in the two pathways of MAPK/ERK and AKT/mTOR showed a concentration-dependent down-regulation.The apoptosis rate of these two gastric cancer cells increased with increasing concentration,and the apoptosis-related proteins were consistent with the changes in human gastric cancer cell MGC-803.In addition,compound 3328 was less toxic to normal human gastric epithelial cells GES-1(IC50 value was 15.22μmol/L).The reliability of our experimental results were further verified.The above results showed that the sulfonamide derivative 3328 exhibited potent inhibitory activity against human gastric cancer cells.It inhibited the proliferation of human gastric cancer cells by inhibiting the MAPK/ERK pathway and AKT/mTOR pathway,arrested the cell cycle in the G2/M phase,and induced apoptosis of human gastric cancer cells MGC-803,SGC-7901 and HGC-27.The toxicity was no obvious against normal human gastric epithelial cells GES-1.These findings provided a basis for the research and development of this type of anti-gastric cancer drugs.