Design,Synthesis And Antibacterial Evaluation Of 11-O-Alkaryltriazolcarbamoyl-3-O-descladinosyl-3-keto Clarithromycin Derivatives

Macrolide antibiotics are alkalophilic lipophilic compounds with lactone ring structure produced by Streptomyces,and have the advantages of strong antibacterial activity,wide antibacterial spectrum,and medication safety,etc.They can effectively treat respiratory tract and soft tissue infection,and are widely used in clinic.However,the emergence of bacterial resistance seriously limits the effectiveness of clinical application of macrolides.Therefore,it is an urgent problem to develop new macrolide antibiotics against various drug-resistant pathogens while eliminating the abuse of antibiotics.Macrolide antibiotics bind to the peptide exit channel entrance of the peptidyl transferase center in the bacterial ribosome 50S subunit 23 S rRNA,which can promote dissociation of peptidyl tRNA on ribosomes,inhibit the extension of peptide chain,block the biosynthesis of bacterial protein and produce antibacterial effect.The main reasons of bacterial resistance include the modification of drug target by methylation enzyme encoded by erm gene and the efflux pump protein encoded by mef gene to pump drugs out of the cell.Macrolide antibiotics have complex chemical structure and great space for chemical modification.According to their mechanism of action and bacterial resistance,structural modification of existing macrolide antibiotics is an important way to develop new antibacterial drugs.It is reported that desosamine at the C-5 pisition of the second generation of macrolide antibiotic clarithromycin is an essential group for antibacterial activity,and cladinose at the C-3 position cannot interact with the bacterial ribosome,and may induce the expression of resistant gene mef to make the bacteria develop resistance.There are very few studies on the modification of the C-11 position,which may be related to the poor reaction activity at this position.In our previous study,the C-11 side chain was found to bind to the nucleotide residue A752 in the domain II of 23S rRNA of 50S subunit in bacterial ribosome,which is beneficial to enhance the antibacterial activity,especially anti-resistant activity.Based on the above considerations,we designed 11-O-alkaryltriazolcarbamoyl-3-O-descladinosyl-3-keto clarithromycin derivatives by taking clarithromycin as the mother nucleus,retaining desosamine at the C-5 position necessary for antibacterial activity,removing the cladinose at the C-3 position and oxidizing resultant hydroxyl group at the C-3 position to ketone,and finally introducing various alkaryltriazolcarbamoyl side chains at the C-11 position.Then,using clarithromycin as the starting material,we established reasonable synthetic routes,and synthesized three series of 35 target compounds through nine reaction steps.The structures of the target compounds were characterized by MS,1H NMR and 13C NMR.The in vitro antibacterial activity of the target compounds against four sensitive bacteria and five resistant bacteria was determined by double broth dilution method.The results are as follows.For the four sensitive strains tested,the target compounds displayed moderate antibacterial activity against Bacillus subtilis,in which the best active compounds were 13f and 13i with the MIC values of 1 μg/mL,and showed slight antibacterial activity against Staphylococcus aureus,in which the most active compounds 13f and 13i had the MIC values of 4 μg/mL.In contrast,the activity of target compounds against Escherichia coli and Pseudomonas aeruginosa was poor.For the 5 drug-resistant strains tested,on the other hand,most of the target compounds exhibited significantly increased antibacterial activity against ermB resistant phenotype Streptococcus pneumoniae,mefA+ermB resistant phenotype Streptococcus pneumoniae and erythromycin-resistant Streptococcus pyogenes,among which compound 13f possessed the strongest activity against the above three drug-resistant strains with the MIC values of 16 μg/mL,which was>16-,8-and 16-fold better activity than clarithromycin(>256,128 and 256 μg/mL)and>16,16 and 16-fold better activity than azithromycin(>256,256 and 256 μg/mL),respectively.Moreover,the target compounds also showed effective antibacterial activity against methicillin-resistant Staphylococcus aureus and penicillin-resistant Staphylococcus aureus,in which compound 13e showed the best activity with the MIC values of 16 μg/mL,which were 16-and 16-fold stronger activity than clarithromycin(>256 and>256 μg/mL)and azithromycin(>256 and>256 μg/mL),respectivelyIn general,the target compounds exhibited better activity against Staphylococcus aureus,Bacillus subtilis,erythromycin-resistant Streptococcus pyogenes,ermB resistant phenotype Streptococcus pneumoniae and mefA+ermB resistant phenotype Streptococcus pneumoniae.The MIC values against the tested sensitive and resistant bacteria can reach 1 μg/mL and 16 μg/mL,respectively.Among all the target compounds,compound 13f showed the best in vitro antibacterial activity against the above five bacteria at the same time,with MIC values of 4,1,16,16,16 μg/mL,respectively.Compared with the control drugs,the target compounds showed weakened activity against sensitive bacteria and distinctly enhanced activity against resistant bacteria,among which,B series compounds were much better than A and C series compounds.It was worth noting that the antibacterial activity of the target compounds against ermB resistant Streptococcus pneumoniae and mefA+ermB resistant Streptococcus pneumoniae was obviously improved,which indicated that the combined C-3 and C-11 modification of clarithromycin overcame the active efflux of resistant bacteria,and improved the affinity for bacterial ribosome,thereby effectively inhibiting erm and mef resistant bacteria,which is consistent with our design ideasAccording to the in vitro activity data of the target compounds,the structure-activity relationships can be summarized as follows:1)The combined modification of C-3 and C-11 positions of clarithromycin changed the spatial structure of the 14-membered macrolide mother nucleus,and significantly improved the activity of against drug-resistant bacteria while partially retaining the activity clarithromycin against sensitive bacteria;2)when the distance between the C-11 atom and the 1,2,3-triazole in the side chain was 6 atoms,the compounds displayed the best combination with the bacterial ribosome,showing the best activity of anti-sensitive bacteria and anti-resistant bacteria;3)The substituent on the terminal benzene ring on the C-11 alkaryltriazolcarbamoyl side chain had great influence on the antibacterial activity:The electron-withdrawing groups could improve the antibacterial activity,while most of the electron-donating groups were unfavorable for the antibacterial activity;The aromatic substitution and long-chain alkyl substitution on the terminal benzene ring had beneficial effects on the antibacterial activity of the compounds,among which straight alkanes were better than branched alkanes;The substitution of halogen atoms had a favorable effect on the antibacterial activity,among them,chlorine atom was better than fluorine and bromine atoms;The position of substituents on the terminal benzene ring had little effect on the antibacterial activity of the compounds.In vitro bactericidal evaluation revealed that the target compounds exerted antibacterial effects against sensitive and resistant bacterial strains through bacteriostasis.The study of bactericidal kinetics further confirmed that compound 13i was a concentration-dependent bacteriostatic agent from a microscopic perspective.The molecular docking results showed that compound 13i had a similar binding mode to clarithromycin.In addition to retaining the hydrogen bonding between the C-2’hydroxyl group of clarithromycin and A2058 nucleotide residue A2058 in domain V of 23S rRNA of 50S subunit in the bacterial ribosome,the nucleotide residue A752 in the domain Ⅱ of 23S rRNA of 50S subunit in bacterial ribosome,the C-11 alkaryltriazolcarbamoyl side chain of clarithromycin not only formed hydrogen bonds with A752 and U790 nucleotide residues:in the domain Ⅱ.but also interacted with the pocket composed of by G2505,C2610 and C2611 nucleotide residues in the domain V,which improved the activity against resistant bacteria.