Design,Synthesis And Activity Evaluation Of Epigenetic Enzyme G9a Indole Derivatives Inhibitors

Objective:In the face of a deteriorating environment and increasing pressure on life,the incidence of cancer is rising year by year,and it is imperative to find a definitive anti-tumor drug.Studies have shown that abnormal expression of protein methyltransferase leads to imbalance of histone methylation patterns and promotes the development of human cancer.Among them,anti-tumor drugs targeting histone methyltransferases(HMTs),especially G9a(autochromatin histone lysine N-methyltransferase,EHMT2),are the current research hotspots.However,the current research on G9a inhibitors is still in the basic research stage,and there have been no reports of G9a inhibitors entering clinical research.The purpose of this research is to find a new G9a active lead compound based on the work of virtual screening,and further design and synthesize the corresponding series of structural derivatives by computer-assisted drug design method,through in vitro enzyme inhibition activity screening and anti-tumor activity evaluation.And structure-activity relationship analysis,design and synthesis of G9a inhibitors with higher activity and selectivity,and then expand the structure type of G9a inhibitors,which provides reference for the development of next-generation inhibitors.At present,the synthesis of the target compound and the preliminary screening of anti-tumor activity have been completed,and the target compound has been studied on the targeting and mechanism of G9a.Method:1.Computer Virtual Screening and Determination of Leading CompoundsAccording to the crystal structure of G9a(PDB:4NVQ)and its ligand A-366,a virtual screening model based on molecular docking was established,and 50 compounds with higher scores were preliminarily screened(see appendix 4).The low-energy conformations of the 50 compounds were overlapped,and the pharmacophore model of G9a was preliminarily established for screening and matching.The anti-proliferation activity of H1299 cell line was evaluated and validated.Compounds with high evaluation index and prominent evaluation of initial screening activity were used as lead compounds.2.Structural optimization and synthesis based on lead compoundsThe docking model was comprehensively analyzed,the structure of the lead compound was optimized and the synthetic route was designed.In this paper,the key intermediates were synthesized from indole or 3-chloromethoxyanisaldehyde via Wilsmeier-Hack reaction and Henry reaction.Finally,two series of target compounds,30 indole alkaloids,were synthesized via Kett-Schopengler reaction.The structures of the target compounds were confirmed by high resolution mass spectrometry(LC-MS),1H-NMR and 13C-NMR.3.Evaluation of the Activity of Target CompoundsThe antitumor activity of 30 target compounds was detected by SRB method.This method used human non-small cell lung cancer cell line H1299 as the test cell line,G9a inhibitors BIX01294 and UNC0642 as positive drugs,and evaluated the anti-tumor activity of the compounds with the inhibition rate of cell proliferation.Result:1.Fifty compounds with G9a inhibitory potential were obtained by virtual screening.At the initial screening concentration of 10 μM,the cell inhibition of the positive drugs BIX01294 and UNC0642 were 92.8%、76.7%respectively,while the cell inhibition of the candidate compounds 11687,27368 and 464990 were 95.5%、72.9%、68.8%respectively,which were comparable to that of the positive drugs.The IC50 of compounds 11687、27368 and 46490 were 2.99±0.26μM,7.81 ±1μM、less than 0.78μM respectively.Compound 27368 was used as the lead compound according to the preliminary screening results of pharmacophore evaluation index and antiproliferative activity.2.Thirty indole small molecule compounds were synthesized in this paper.42 intermediates and 30 target compounds were designed and synthesized.Among them,42 compounds were not reported by scifinder.Compounds 1,2,3,4,13,14,15,16,17,19,22,23,24,25,26 have significant inhibitory effects on non-small cell lung cancer H1299.Conclusion:Thirty structural derivatives of G9a inhibitors were optimized and synthesized by means of virtual screening,and their structures were characterized by computer-aided drug design.Compounds 1,2,3,4,13,14,15,16,17,19,22,23,24,25 and 26 were found to have antineoplastic activities in vitro.It has obvious inhibitory effect.The above results provide reference value for further study of G9a inhibitors.