Design, Synthesis And Biological Evaluation Of [1,2,4] Triazolo [1,5-a]pyrimidines As Potent Lysine Specific Demethylase 1 (LSD1/KDM1A)Inhibitors

Epigenetic modification of histone modifications is a very important direction for research.Histone Lysine Specific Demethylase 1(LSD1,also known as KDM1A),the first identified histone lysine specific demethylase in 2004,can specifically remove H3K4 and H3K9 single or double methylation,which is involved in a highly conserved flavin adenine dinucleotide(FAD).Besides,LSD1 is able to remove the methylation of nonprotein substrates(such as transcription factor E2F1,tumor suppressor gene p53,DNA methyl transferase 1)to regulate the biological function of cells.Studies have shown that LSD1 activity or expression in many tumor cells(such as breast cancer,lung cancer,prostate cancer,gastric cancer,etc.)is higher than the corresponding normal cells,by inhibiting the activity or expression of LSD1 can effectively inhibit the growth of tumor cells.Therefore,it is of great significance to develop an efficient and highly specific LSD1 inhibitor for tumor therapy.A new series of [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1 inhibitors were designed,synthesized,and further evaluated for their cytotoxicity against MGC-803,EC109,A549 and PC-9 cells as well as the ability of inhibiting LSD1.Some of these compounds showed potent inhibition toward LSD1 and selectively inhibited growth of A549 and PC-9 cells.Compound 6l potently inhibited growth of PC-9 cells(IC50 = 0.59 μM),about 4-fold more potent than 5-FU.Further SARs studies led to the identification of compounds 6l-m,which had good growth inhibition against all the tested cancer cell lines and were much more potent than 5-FU and GSK2879552.Besides,compounds 5p,5q and 6i inhibited LSD1 potently(IC50 = 0.154,1.19 and 0.557 μM,respectively).Docking studies revealed that compound 5p formed arene-H interactions with Val333 and hydrogen bonds with surrounding Ala331,Met332,and Ala539 residues.Compound 5p significantly inhibited migration of A549 and PC-9 cells in a concentration-dependent manner,but had different effect on the expression of E-cadherin and N-cadherin.In addition,we used Annexin Ⅴ-FITC double staining to detect the effect of compound 61 on apoptosis of MGC-803 cells.We found that compound 6l could induce the apoptosis rate of MGC-803 in a concentration-dependent manner.We used compound 5p to regulate LSD1 and its substrate in PC-9 and MGC-803 tumor cells.The results showed that compound 5p could inhibit the expression of LSD1 at the cellular level,and the expression of LSD1 in the cells was significantly decreased.Compound 5p could specifically upregulate the expression of protein substrates H3K4me1 and H3K4me2 in cells,but not on H3K4me3 expression.The [1,2,4]triazolo[1,5-a]pyrimidine scaffold may serve as a starting point for developing potent LSD1 inhibitors for cancer therapy.In conclusion,the novel LSD1 inhibitor with [1,2,4]triazolo[1,5-a]pyrimidine as the basic skeleton was designed and synthesized and the basic skeleton of triazolopyrimidine was retained.By further enriching the diversity of different amino substituents,we could explore the structure-activity relationship,and then build a novel structure and diversity of the target small molecule compounds in order to obtain the LSD1 inhibitory activity of small molecules.Based on this design concept,we are surprised to find the three compounds 5p,5q and 6i with inhibitory activity against LSD1,which inhibited the IC50 values of LSD1 by 0.154,1.19 and 0.557 μM,respectively,and then evaluated them for a series of activities.This is sufficient to demonstrate the reliability of the design concept and provide a new way for the discovery of followers of LSD1 inhibitors and other target inhibitors.