Design,Synthesis And Anti-resistant Bacterial Activity Study Of Novel Glycopeptide And Lipopeptide Antibiotics

Antibiotics are one of the greatest discoveries in medical history.However,bacteria gradually evolved resistance to antibiotics in less than 100 years,and the situation of resistance is getting worse.In recent years,antibiotic resistance kills an estimated 700000people each year worldwide,and some experts predict that number could rise to 10million by 2050 if efforts are not made to curtail resistance or develop new antibiotics.Therefore,antibiotic resistance is bound to be one of the greatest health threats facing human no matter now and in the coming decades.In the arsenal of antibiotics,glycopeptide antibiotics and cyclic lipopeptide polymycin antibiotics,as the last line of defense against the intractable resistant bacteria,are used as the last treatment in the emergency situation of severe drug-resistant infections.While with the appearance of glycopeptide-resistant and even polymyxin-resistant bacterial strains,this last line of defense is also at stake,and a"post-antibiotic era"of no drugs is looming.Under such a severe situation,it is particularly important to design and modify antibiotics from natural products sources reasonably and effectively to obtain corresponding safe and effective semi-synthetic derivatives,such as the approval of new generation of glycopeptide antibiotics telavancin,dalbavancin and oritavancin.At the same time,the current enthusiasm for the research on the semi-synthetic modification of glycopeptide antibiotics such as vancomycin and polymyxin-type antibiotics is conducive to accelerating the development of a new generation of antimicrobial drugs,enriching the currently scarce pipeline of antibiotic research and development,and temporarily dealing with the current serious problem of drug-resistant bacteria infection.The research work of this dissertation mainly started from three sources of natural products of antibiotics（vancomycin,norvancomycin,polymyxin B）,including five projects respectively in these three chapters of novel vancomycin derivatives,novel norvancomycin derivatives as well as novel polymyxin B analogues for the systematic investigation and research,a total of 219 new compounds were synthesized and prepared.In the first part of these research work,"Design,synthesis and pharmacological activities evaluation of novel vancomycin derivatives"is the main part of the full text with more in-depth research,which includes three different projects of glycosylated modification of vancomycin derivatives,pyrophosphate-targeting vancomycin derivatives as well as membrane-targeting vancomycin derivatives,vancomycin derivatives respectively based on three different strategies.Lipid Ⅱ as the precursor of peptidoglycan,the chief component of gram-positive bacterial cell wall,is called“Achilles’heel”of cell wall biosynthesis which is crucial for bacterial survival.The target for glycopeptide such as vancomycin is exactly Lipid Ⅱ,while the main reason for the drug resistance of such bacteria is that the mutation of the binding site of Lipid Ⅱ leading to the decrease of its affinity with the glycopeptide antibiotics such as vancomycin.Hence,how to increase the binding affinity with Lipid Ⅱ in drug-resistant bacteria is of great significance for combating vancomycin resistant bacteria.Lipophilic substitution on vancomycin is an effective strategy for the development of novel vancomycin analogues against drug-resistant bacteria by enhancing bacterial cell wall interactions.However,hydrophobic structures usually lead to long elimination half-life and accumulative toxicity;therefore,hydrophilic fragments were also introduced to the lipo-vancomycin to regulate their pharmacokinetic/pharmacodynamic properties.Here,in the project of extra glycosylated vancomycin derivatives,we synthesized 51 new vancomycin analogues carrying various sugar moieties on the seventh-amino acid resorcinol and lipophilic substitutions on vancosamine with extensive Structure-activity relationship analysis.The optimal analogues indicated128-1024-fold higher activity against many drug-resistant strains such as methicillin-resistant S.aureus（MRSA）,vancomycin-intermediate resistant S.aureus（VISA）,and vancomycin-resistant Enterococci（VRE）compared with that of vancomycin.In vivo pharmacokinetics studies demonstrated the effective regulation of extra sugar motifs,which shortened the half-life and addressed concerns of accumulative toxicity of lipo-vancomycin.The mechanism study revealed the possible interaction between extra sugar moiety and terminal dipeptide in Lipid Ⅱ,which also explained the antibacterial activity enhancement from another aspect.This project presents an effective strategy for lipo-vancomycin derivative design by introducing extra sugars,which leads to better antibiotic-like properties of enhanced efficacy,optimal pharmacokinetics,and lower toxicity.In the project of pyrophosphate-targeting vancomycin derivatives,we rationally designed and synthesized 39 novel vancomycin derivatives by respective or combined modifications using metal-chelating,lipophilic,and galactose-attachment strategies for extensive structure–activity relationship（SAR）analysis.In a proposed mechanism,the conjugation of building blocks carrying dipicolylamine on the seventh amino acid resorcinol position or C-terminus endowed the vancomycin backbone with binding capacity for the pyrophosphate moiety in lipid II while maintaining the intrinsic binding affinity for the dipeptide terminus of the bacterial cell wall peptidoglycan precursor.The in vitro antibacterial activities were evaluated,and the optimal compounds indicated 16-to 1024-fold higher activity against VRE than that of vancomycin.Meanwhile the synergistic effects brought about by combining two peripheral modifications was proved to produce particularly potent activity against VRE.The development of vancomycin derivatives targeting the cell membrane of bacteria is an effective strategy for bacteria.In addition,the complex components of the cell membrane of bacteria are crucial for the survival of bacteria,and it is generally difficult to mutate,which reduces the occurrence of drug resistance under this mechanism.In this“membrane-targeting vancomycin derivatives”project,we reported 31 novel vancomycin derivatives carrying a sulfonium moiety and 4 control compounds,which was an underestimated modification in previous studies and rarely reported in literature.The sulfonium-vancomycin analogues exhibited enhanced antibacterial activity against VRB both in vitro and in vivo potently.More interestingly,sulfonium could alter the innate feature of vancomycin and tune to widen its antibacterial activity spectrum against Gram-negative bacteria.The mechanism studies illustrated that sulfonium modification enhances the interaction of vancomycin with bacteria cell membrane and disrupts membrane integrity.In addition,we evaluated in vivo pharmacokinetic profile,stability,and toxicity of these derivatives,which demonstrated the good druggability of sulfonium modification.This project provides a promising strategy for combating drug-resistant bacterial infection,and also advances the knowledge on sulfonium derivatives for structural optimization and drug development.In the second part of these research work“Design,synthesis and pharmacological activity evaluation of novel norvancomycin derivatives”,which is the fourth project,we mainly designed and selectively introduced a series of hydrophobic fragments or sulfonium motif carrying lipophilic fragments at N-terminus to obtain corresponding32 single modified norvancomycin derivatives.In the in vitro antibacterial activity test against gram-positive bacteria,we found part of these above compounds could exert good antibacterial activity,especially these compounds with long alkyl groups,biphenyl groups as well as sulfonium motif carrying the long alkyl or biphenyl groups could exhibit 32-2048-fold higher antibacterial activity than vancomycin.Meanwhile the results also proved the N-terminus modification on norvancomycin is promising,the follow-up work will continue from here.The third part of these research work“Design,synthesis and pharmacological activity evaluation of novel polymyxin B analogues”is also the fifth project.The present serious multi-resistant gram-negative bacterial infection has come to the point where no antibiotics to use.Polymyxins as the corresponding“last line of defence”treatment had to be revived and reintroduced into clinic,but its toxic side effects has seriously hindered its extensive use,so we hope to use semisynthetic modification for polymyxin B to obtain the analogues with better efficacy and/or reduced toxicity.Herein,we introduced 20 kinds of natural amino acids through the semi-synthetic modification at the position of 2^nd and/or 10^th threonine hydroxyl group,and finally obtained 52polymyxin B analogues with single or double sites’modification.Although there was no improvement detected in the in vitro anti-gram-negative bacterial activity tests,it proved some certain chemical modification tolerance of these two sites.In vitro cytotoxicity test results showed that these polymyxin B analogues with hydrophilic and polar amino acid modification had higher safety.This will also provide certain guidance and reference for us to carry on the other semi-synthetic and total synthetic work on polymyxin B in the future.To sum up,this study aims to explore and study the semi-synthetic modification of vancomycin,norvancomycin and polymyxin B through different strategies,and to obtain a series of promising antibacterial drug candidate targeted at drug-resistant bacteria,which provides certain ideas for the development of glycopeptide antibiotics and lipopeptide antibiotics.