Design And Activity Evaluation Ofnovel 5-cyano-6-phenyl Pyrimidine LSD1 Inhibitors Based On Coenzyme FAD

Histone lysine specific demethylase 1(LSD1)is a member of the flavin adenine dinucleotide(FAD)-dependent amine oxidase family,which remove one and two methyl groups from mono-and dimethylated H3K4 and H3K9 by enzymatic oxidation.At present,LSD1 is found to be more highly expressed than normal cells in a variety of cancer cells,including gastric cancer,breast cancer,and leukemia.Inhibiting or inactivating LSD1 can significantly increase the accumulation of substrates and effectively inhibit the differentiation,proliferation,migration and invasion of cancer cells.Therefore,LSD1 has become a promising therapeutic target for a variety of cancers.The development of efficient,low-toxic,and highly selective LSD 1 inhibitors not only enriches the backbone types of LSD 1 inhibitors,but also provides data support for antitumor drug development targeting histones demethylase.As a heterocyclic compound with important pharmacological activity,pyrimidine derivatives such as cytosine and uracil play important biological functions in the body.Pyrimidine derivatives are also widely used in the treatment of various diseases,such as antiviral,antibacterial and antiturnor.Based on the previous work,we screened a lead compound 6 with potential inhibitory activity on LSD1.By modifying its structure,we designed and synthesized a series of new pyrimidine derivatives and evaluated their enzyme activities and preliminary biological mechanism studies.Specific work studies are as follows:1.Through reviewing relevant literatures,we grasped the protein structure,biological function,and correlation of LSD1 with various tumors.Then,the development status and main problems of LSD1 inhibitors were described in detail,which provided a theoretical basis for the work of this topic.2.Our group had constructed a small molecule compound library based on the pyrimidine skeleton in the early stage,and reported that several types of compounds based on pyrimidine skeleton had good inhibitory activity on LSD 1.We further conducted extensive LSD1 inhibitory activity tests on the internal pyrimidine compound library,and found that a new class of pyrimidine compounds 6 linked to methylthiotetrazole has acceptable inhibitory activity on LSD1.We used compound 6 as a leader,and analyzed the binding mode with LSD 1 protein to expand its structure appropriately,and designed and synthesized 63 new pyrimidine derivatives.3.All the new synthesized compounds were evaluated for LSD 1 inhibitory activity in vitro.Most compounds showed good inhibitory activity,of which compound 66 had the best activity with IC50=56.65 nM.Docking analysis showed that Compound 66 had a similar mode of action with FAD.The cyano group on the pyrimidine ring not only formed a key hydrogen bond with Lys661 through the water molecule 1032,but also formed a hydrogen bond with Met332.One of the nitrogen atoms on the tetrazolium ring and the left carboxyl group formed important hydrogen bonds with Val881 and Ser289,respectively.These amino acid residues were all important residues of FAD binding with LSD1,and it was initially confirmed that compound 66 might exert its activity by competing LSD1 with FAD.Further biological mechanism studies had shown that compound 66 binded to LSD1 in a reversible manner and effectively inhibited LSD1 activity at the cellular level.In addition,it significantly inhibited the migration and invasion of LSD 1 overexpressed MGC-803 cells.In summary,63 new pyrimidine compounds were synthesized and evaluated for inhibitory activity.Among them,21 compounds had IC50<200 nM,and the inhibitory activity of compound 66 was nearly 20 times higher than that of the lead compound.These findings not only enriched the structural types of pyrimidine-type LSD1 inhibitors,but also obtained a new class of LSD1 inhibitors that competitively binded to FAD,confirming the feasibility of ligand-based design schemes,which had important reference significance for the development of new modes of action of LSD1 inhibitors.