Synthesis, Activity Evaluation Of Novel TERT Inhibitors And Preliminary Research On Anticancer Activity Mechanisms

Telomerase is specifically expressed in 85%-90%of tumor cells to maintain telomere extension.And the intracellular quantity of telomerase reverse transcriptase is increased.However telomerase is almost impossible to be detected in normal cells.At present telomerase has become one of new popular targets for the development of highly effective and specific anti-tumor drugs.Based on the structural characteristics of compound BIBR1532 and its possible binding mode with telomerase,and referenced with structural features of small molecule telomerase inhibitors that have been reported,a total of seventy-six target compounds with three different classes and other structural related characteristics were designed and synthesized in the paper.The structures of all target compounds were confirmed by ¹H NMR and HRMS.No telomerase activity was reported for all target compounds in this paper,sixty of which have not been reported in literatures.The molecular weights of seventy-two target compounds were all below 400.As far as their molecular weights were concerned,most target compounds still had a large space for structural optimization as lead compounds.In this paper,the inhibitory activities of all target compounds on human melanoma cells A375 and other three cancer cells were determined.Among them,forty-two compounds had moderate or strong inhibitory activities on cancer cells such as human melanoma cells A375 in vitro with IC₅₀0 values ranged from 1.87?M to 88.60?M.Their activities were weaker than that of adriamycin,but much stronger than that of BIBR1532.Among all target compounds,compound 2t showed the strongest inhibitory activity against human melanoma A375 cells with the IC₅₀0 value of 1.87?M,which was about 2.4 times the IC₅₀0 value of adriamycin.Based on the inhibitory activities against cancer cells,the preliminary structure-activity relationships of various types target compounds were clarified.The elementary structure-activity relationships of acrylamide derivatives were as follows:on 3-position,substituted phenyl groups were more advantageous than the2'-naphthalene group,electron-withdrawing groups on phenyl group may be detrimental to the enhancement of activity,and the cyano group was more superior than the cyclopropyl substitution;when the hydrogen atom in the amide group was replaced by the methyl or ethyl group,the molecules'activities can also be maintained or even improved.The fundamental structure-activity relationships of compounds 5a-5e and7a-7l were as following:the hydroxyloxime group was more helpful than the ketone group;the naphthalene ring was more beneficial for the activity improvement than substituted benzene or thiazole rings on the 3-position.Compared with other title compounds,acrylamide derivatives 2a-2x,3a-3g and but-2-en-1-one-oxime derivatives5a-5e were more active against tested cancer cells.To determine cell selection of title compounds and provide directions for the next studies,we selected several title compounds with strong anti-cancer activities to study their toxicities against human normal gastric epithelial cells GES-1 and human immortalized normal liver cells L-02.It was found that some compounds belonging to2a-2x and 3a-3g were less toxic to GES-1 and L-02 cells within the tested concentration range.Compounds 5a and 6a were electron-rich unsaturated diaryl compounds which were more toxic to the normal cells.In our study,thirty-nine representative compounds were selected for telomerase inhibitory activity test in vitro.Twenty-seven compounds showed potent telomerase inhibitory activities in vitro with IC₅₀0 values between 0.62?M and 25.50?M.The activities of twenty-one compounds were better than that of the positive control astrosporin,but weaker than that of BIBR1532.The IC₅₀0 value of compound 2e on telomerase activity in vitro reached 0.62?M,which was about twice the IC₅₀0 value of BIBR1532.Based on the telomerase inhibitory activities of the target compounds,thepreliminary structure-activity relationships of title compounds with different types were obtained in the paper.The preliminary structure-activity relationships of acrylamide derivatives 2a-2x,3a-3g and 4a were as follows:on 3-position,the cyano group was more superior than the cyclopropyl group,benzene ring bearing electron donor groups with suitable steric hindrance was beneficial to improve the activities of the compounds,and the binding sites of telomerase may be tolerant for 3-position substitutions with certain steric hindrance groups such as the cyclopropyl group;the hydrogen atom in the amino group was not necessary for the telomerase inhibitory activity.The preliminary structure-activity relationships of compounds 5a-5e and 7a-7l were as following:the hydroxyloxime group was more advantageous than the ketone group;3-substituents with electron rich groups such as the naphthyl group,were favorable for the activity enhancement.Compared with other target compounds,acrylamide derivatives 2a-2x,3a-3g and but-2-en-1-one-oxime derivatives 5a-5e showed stronger telomerase inhibitory activities in general.Based on the co-crystal structure of telomerase and BIBR1532,the interactions between some active compounds and telomerase were studied by molecular docking in silico.The docking results showed that the binding modes of the above compounds and BIBR1532 in telomerase were basically the same.In the paper,we investigated the inhibitory mechanisms of compounds 2e and 2h against gastric cancer MGC-803 and SMMC-7721 cells,respectively.It was found that both compounds could block the cell cycles of cancer cells in G2/M phase and reduce the protein expression level of TERT in a dose-dependent manner along with inhibiting the activity of telomerase and inducing apoptosis of cancer cells.Based on the above results,it was believed that acrylamide derivatives were the TERT inhibitors,which were consistent with the expected target.In summary,based on rational design,we have obtained a class of acrylamide inhibitors with novel structures and potent inhibitory activities against both certain cancer cells and telomerase.These achievements were consistent with the prior goal that BIBR1532analogs have stronger anti-cancer activities against solid tumors.Considering the activities,toxicities and molecular weights of the target compounds,the more active compounds of acrylamide derivatives 2a-2x and 3a-3g,such as 2t and 3c,could be identified as leads for the next round of chemical modification and activity evaluation.