Design,Synthesis And Activity Study Of UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine Deacetylase(LpxC)Inhibitors As Novel Antibiotic

BackgroundThe rapid increase of infections by Gram-negative pathogens along with the emergence of drug-resistant bacterial strains, posing the serious threat to the public health, is the great challenge in21century and demands the development of novel antibiotics directed against the previously unexploited targets.One of the promising targets in Gram-negative bacteria is the zinc-dependent metalloamidase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC catalyzes the first committed, second overall step in the biosynthetic pathway of lipid A, which is essential for the bacterial viability and toxicity. Additionally, LpxC is highly conserved among Gram-negative bacteria and shares no sequence homology with any other known zinc-metalloenzymes. Thus LpxC has become an attractive target for the structure-based drug design, and research on LpxC inhibitors is a very promising strategy in the development of current antibiotic therapy for Gram-negative bacteria.Design、synthesis and activity evaluation of target compoundsCurrently identified LpxC shared the same structural characters and almost all of the potent inhibitors contain a hydroxamate group for chelating the catalytic Zn2+and a side chain for effectively interacting with the hydrophobic tunnel. Based on the LpxC crystal structures and the binding mode of the known LpxC inhibitors in complex with LpxC, we designed4series of novel compounds as LpxC inhibitors with the strategies of CADD, conformational restriction and biosiostere. Before synthesizing the designed compounds, we docked our compounds with EcLpxC via FlexX of sybyl8.0. The result showed that almost all of the compounds had similar or higher score than the positive control, which, to some extent, ensured the rationality of our design strategy and supplied basement for our study.During the synthesis of series A, all the compounds designed were synthesized using L-hydroxyproline as starting material through a reaction sequence including esterification, Boc-protection or sulfonylation, SN2nucleophilic substitution, Suzuki couplingcon, condensation, Sonogashira coupling, mitsunobu reaction to obtain the key intermediate. During the synthesis of series B, all the compounds designed were synthesized using various L amino acids as starting material through a reaction sequence including Sonogashira coupling, hydrolysis, condensation, Galaser coupling to obtain the key intermediate. During the synthesis of series C, all the compounds designed were synthesized using (S)-methyl2-(4-ethynylbenzamido)-4-(methylthio)-butanoate as starting material through a reaction sequence including oxidation, Galaser coupling to obtain the key intermediate. The methyl ester groups of all the former compounds were converted to hydroxymate group to obtain the target compounds.Additionally, aiming for high activity compounds, preliminary activity assay was also carried out in vitro including MIC assay, antibacterial annulus and anti-LpxC assay.ResultsIn this research, all target compounds were obtained and identified by1H-NMR and HRMS spectra. Literature retrieval proved that all the compounds were new and not reported.In MIC assay, the results showed that four series of these compounds exhibited highly selective antibacterial activity against Gram-negative bacteria (E.coil ATCC 25922and P. aeruginosa ATCC27853) as compared with Gram-positive bacteria (S.A. ATCC25923�'�MRS.A. ATCC29213). Some potent compounds were also assayed for their antibacterial activities against E.coil (MDR), P. aeruginosa (MDR) and E.cloacae (MDR). Among them, a few target compounds showed slightly lower activity compared with positive control. While, the most potent compounds D1and D2were chosen to be assayed for their antibacterial activities against E. coil W3110(wild-type strain) and E. coil CMR300(membrane-compromised strain), which was finished in Duke University.Compounds D1and D2were also assayed for their antibacterial annulus against Ecoil ATCC25922and P. aeruginosa ATCC27853. The results showed that these two compounds was comparable to the positive control LPC009, Levofloxacin, Claforan towards these two strains.Additionally, the most potent compounds Dl and D2were also chosen to be carried out anti-LpxC assay. The results showed that these two compounds was comparable to the positive control LPC028towards EcLpxC and they can be researched deeply in the future work.ConclusionsIn conclusion, based on the crystal structure of LpxC and the binding mode of the known LpxC inhibitors with the enzyme, we designed and synthesized four series of the target compounds. Preliminary antibacterial activities assay and enzyme activity assay showed most compounds possess potential activity. These compounds are promising lead compounds for developing new LpxC inhibitors as novel antibiotic.