Design,Synthesis And Structure-activity Study Of Small Molecule Drugs Based On PI3K/AKT/mTOR Signaling Pathway And Telomerase Reverse Transcriptase Target

PI3K/Akt/mTOR signaling pathway is the main signal pathway regulating autophagy.Telomerase is a significant target for tumor therapy.When telomerase's activity declined,abonormal growth of cell's proliferation will be inhibited,which also causes those cell's apoptosis.In our study,based on the existing chemical structure information of active compouinds,through computer aided drug design software,we synthesized series compounds targeting PI3K/Akt/mTOR signaling pathway and telomerase reverse transcriptase respectively,based on the theory of drug design,we synthesized several series of compounds and tested their biological activities.Shikonin is the main component of Chinese herbal medicine Arnebia euchroma,which exhibit high activity against tumor cell lines.However,because of its poor selectivity and high cytotoxicity,not only cancer cells but also normal cells are also strongly inhibited,there is still a long way from clinical application.So to maintain its high activity against tumor cells and reduce its toxicity have been the main problem to be solved.We hope to explore a more efficient and less toxic antitumor compound.Through the analysis of the pharmacore of shikonin,we found that the side chain hydroxyl was not necessary for the activity,but the main modification site.Acylation and etherification can maintain strong anti-tumor activity at low concentration,and the introduction of double bond can significantly improve the activity.Based on the molecular docking(the Docking Energy of selected compound is-15.7597 k Cal/M and shikonin is 6.13917 k Cal/M)and pharmacophore analysis of shikonin,23 derivatives were designed and synthesized.In addition,the structure activity relationship was discussed,most of the derivatives are effective against selected tumor cells,especially compound 12 whose IC₅₀on SGC-7901 was 4.1�2.6?M.Meanwhile the anticancer mechanism of compound 12 on SGC-7901 cells had been investigated by Annexin V/PI staining,immunofluorescence,western blot assay and molecular docking.These results indict compound 12 may induced cell apoptosis and cell autophagy through regulating the PI3K signal pathway,which plays a significant role on apoptosis of the compound.Telomerase have significance to abonormal cells.Disorder of telomerase activity can cause cell cycle arrest and this will cause death of tumor cells gradually.Telomerase has become one important target of anticancer drug design.Because of the telomeres'unceasing repairegerm cells and tumor cells,the expected critical telomere length associated with functional replicative senescence cannot be achieved after long-term treatment in vitro.Therefore,there are still some problems in the inhibition of small molecule telomerase,such as the mechanism of telomerase activation and inhibition is not clear,and the side effects of telomerase inhibition on germ cells,stem cells and embryonic stem cells still exist.Many pyrazole derivatives have important pharmacological activities whose skeleton become key point during drug research.Traditional drug design methods play an important role in the history of drug synthesis.On the other hand,new technologies such as computer-aided drug design can not be ignored.With a large number structural analysis of the cocrystall structure of proteins and small molecule complexes,it provides specific method for drug molecules desig oriented targets.In our research,based on traditional drugs design principles,such as skeleton transition and splicing principle,together with new technology,using telomerase reverse transcriptase as the target,according to its structural characteristics,the core skeleton of dihydropyrazole and pyrimidine was firstly determined.At the same time,according to the results of molecular docking,the left and right hydrophobic regions of dihydropyrazole and pyrimidine were modified with multi-functional groups,and 67 novel dihydropyrazole pyrimidine compounds were designed and synthesized,In order to solve the problem of high toxicity of existing telomerase inhibitors,and to develop new lead compounds of telomerase reverse transcriptase inhibitors with high biological activity and low toxicity.CCK-8 test kits were used to test the cell proliferation and telomerase activity of the synthesized compound.The mechanism of action of target compound 91 was detected by cell cycle and apoptosis.Firstly,4-methylpyrazole and pyrimidine compounds were designed based on the literature and the results of our previous work.Through screening,it was found that many compounds had good antitumor activity and telomerase inhibitory activity.The IC₅₀ of 5-(1-benzyl-4-methyl-4,5-dihydro-1h-pyrazol-3-yl)-N-(3-methoxybenzyl)pyrimidin-2-amine compound 33 on MCF7,SKOV3 and a-375 cells were 6.91�0.43,16.37�0.83 and 10.06�0.34 mm,respectively,with little cytotoxicity.The inhibitory activity of compound 33 on telomerase was 1.25�0.48 nm.At the same time,compared with the docking results of compound 33 with existing drugs,it also shows that the skeleton has a good interaction with telomerase.On the basis of compound 33,the effect of methyl group on the activity of these compounds was discussed.It is found that pyrazole methyl is not a necessary group in biological activity.When the pyrimidine substituent is piperazine,it can further increase the related biological activity,but the cytotoxicity also increases.Following the direction of anti proliferation and telomerase inhibitory activity,we constantly modified and modified each group to obtain the expected compound 91(2-(4-ethylpiperazine-1-yl)-5-(1-phenyl-4,5-dihydro-1h-pyrazole-3-yl)pyrimidine),which is our expected target compound.The cell proliferation activity was 2.06�0.88 mm,3.26�0.43 mm and 0.97�0.29 mm,which reached the level of doxorubicin in the positive control group.The telomerase inhibitory activity was 0.075�0.55 nm.The mechanism was verified by cell cycle and apoptosis experiments.It is proved that the compound can inhibit the activity of tumor cells by inducing apoptosis.The highest docking energy of compound 91 with telomerase protein was 22.852 k J/m,the lowest was 15.7847 k J/m,and the average was 19.06663k J/M.The results indicate which the number of interaction energy are in accordance with the experimental results.We concluded our overall opinion that the methyl group on the dihydropyrazole ring is not necessary,but the directional group with?interaction is beneficial to the interaction with protein molecules.When the substituents of pyrimidine ring are different substituted benzyl,it has better biological activity,but its cytotoxicity is also higher.When the ring is replaced by piperazine,it not only improves the activity of these molecules,but also greatly reduces the cytotoxicity,probably because piperazine ring can increase the interaction between these molecules and DNA.The final target compound 91 has good biological activity and low toxicity.It is a potential telomerase reverse transcriptase inhibitor and has great significance for the development of new telomerase inhibitors.