The Optimize Synthesis And Bioassay Of Novel Lead Compound Based On The Structure Of GlmU

The intracellular catalytic enzyme GlmU which act as a very important bifunctional enzyme in bacterial peptidoglycan and lipopolysaccharide biosynthesis, has acetyltransferase and uridyltransferase activity. Though GlmU is a good target enzyme, the research on inhibitors against GlmU is very little, until now the screened inhibitors usually have good effects aginst GlmU, but have no bactericidal activity. The hydrazide compounds own extensive biological and medicinal activity include anti-viral activity, anti-tumor activity, anti-malarial activity, insecticide activity, sterilization activity and so on.In this study, based on lead compound N,N’-di-2-naphthyloxy-acetyl-hydrazine (against GlmU IC50=14.23±2.12 μmol/L) found by Min, we used theoretical computer simulation analysis technology to optimize the structure of the lead compound on the basis of characteristic of the GlmU active cavity and the structure of the substrate and lead compound. On the basis of keeping hydrazide activity structure, hydrophobic halogen was introduced to naphthalene ringon and shorted the chain length, which lead to the compound had better binding conformation in the active sites. Also we synthesized the novel unreported halogen-containing dinaphthyloxy amino aliphatic diamide compounds and dinaphthyloxy amino aryloxycarboxylic diamide compounds, with the aim to seek the noval GlmU inhibitors with bactericidal activity. And the basic structure of contemplated synthesis three types lead compounds are as follow:This paper mainly contains three parts as follow:1. Synthesis and Characterization:14 novel unreported halogen-containing dinaphthyloxy amino aliphatic diamide compounds and dinaphthyloxy amino aryloxycarboxylic diamide compounds were synthesized used halogen-containing alpha-naphthol (beta-naphthol) as raw material by etherification, acylation and diacylation. The structures of the compounds were confirmed by IR, 1H NMR.2. The enyme and bacteria level test of target compounds:The HPLC method was adopted to test GlmU enzyme activities of target compounds. The preliminary bioassay indicated that:target compounds possessed good activities against GlmU enzyme, the comparisons of the inhibitory activity between the compounds 2-16 and lead compound 1 showed that IC50 values of the compounds 2-16 were less than 30 μmol/L, while compounds 4,5,7-16 owed-IC50 values less than 20 μmol/L, compared with the lead compound, activity against GlmU of the target compounds improved 29.77%-67.40%, wherein compounds 4,8,10-13,15,16, which activity against GlmU were increased by more than 50% compared with the lead compound; Compared with adipoyl dihydrazine compounds, succinyl dihydrazine compounds and isophtholoyl dihydrazine compounds had higher activity against GlmU enzyme; the sixth substituted naphthlyl ring could enhance GlmU enzyme inhibitive activity for adipoyl dihydrazine compounds and succinyl dihydrazine compounds. The diluted broth method was adopted to test inhibitive activities of target compounds against Xoo(Xanthomonas oryzae pv. Oryzae). The preliminary bioassay indicated that:target compounds possessed a certain extent activities against Xoo, the comparisons of the inhibitory activity between the compounds 2-16 and lead compound 1 analysed that MIC values of the compounds 2-5,7-16 were less than 350μg/mL; the sixth substituted naphthlyl ring could enhance inhibitive activity against Xoo for adipoyl dihydrazine compounds and succinyl dihydrazine compounds.3. Computer theoretical simulation analysis:the Surflex-dock was adopted to find the binding sites of the target compounds in the active cavity, as well as the comparisons of the conformation of target compounds with the substrate, expole the mechanism of target compounds acting on GlmU enzyme. The results showed that most of the compounds had a good combinations with the active cavity, and formed hydrogen bonds with key amino acids (Asp 103, Lys23). Two different mechanisms, verified the design idea of the lead compound. And we initially concluded that compoundsl,3,4,5,11,12,13 might be competitive inhibitors for the double substrates; compounds 2,6,15 might be competitive inhibitors for GlcNAc-1-P and anti-competitivethe inhibitors for UTP; compounds 7,8,9,10,14 might be competitive inhibitors for UTP and anti-competitive inhibitors for GlcNAc-1-P.In this study, we expored the biological activity of the target compounds, docked them with GlmU uridyltransferase active cavity, explored mechanism in the active cavity, as well as compared the conformations of them with the substrate. The experiment changed the traditional method which depended on the characteristics of small molecular structure to design and synthesis the target enzyme inhibitor lead compounds. We not only avoided the blindness and reduced unnecessary waste, also shorted cycle of find potential lead compounds to improve the research of new, highly efficient inhibitor compounds.