Design,Synthesis And Bioactivity Evaluation Of Novel N-11,C-12,C-13 And C-9 Aralkyl Clarithromycin Semisynthetic Derivatives

Macrolide antibiotics are relatively stable,weakly alkaline substances and usually contain 14 to 16-membered lactone rings,which are substituted by methyl,ethyl,hydroxy,carbonyl,glycosyl and others in different positions of the lactone rings.Macrolide antibiotics exhibit excellent antibacterial activity against various sensitive and resistant bacteria in vivo and in vitro,and also have good pharmacokinetic properties,such as high bioavailability,high blood concentration,short peak time and long half-life,etc.Thus,macrolide antibiotics are one of the most commonly used antibiotics in clinic,which are mainly used to treat various infectious diseases caused by Gram-positive bacteria such as S.aureus,S.pneumoniae and S.pyogenes.The binding sites of macrolide antibiotics are located in 23 S rRNA of bacterial ribosome.Macrolide antibiotics can bind to G2057,A2058,A2059,G2505,A2062 and U790 of 23S rRNA and block the nascent peptide release channel.Meanwhile,they can promote the dissociation of short acyl peptide tRNA from the bacteria ribosome,stop the prolongation of peptide chain and inhibit the synthesis of bacterial protein,thereby exhibiting antibacterial activity.The resistance of macrolide antibiotics is usually caused by modification or mutation of binding sites,active efflux and enzyme inactivation.The 23S rRNA methylation,ribosomal RNA mutation,ribosomal protein variation,active efflux of bacterial efflux pump and enzyme-based drug resistance are the most common resistance mechanisms of macrolide antibioticsSince the discovery of the first macrolide antibiotic picromycin in 1950,the development of macrolide antibiotics has gone through four generations.The development process of the first to the fourth generation of macrolide antibiotics indicates that the following significant improvement:(1)From the initial acid instability in vivo,side effects(such as erythromycin)to excellent pharmacokinetic properties,such as good stability in vivo,high bioavailability,high tissue concentration and long half-life(such as claritlrromycin and azithromycin);(2)From the initial narrow antibacterial spectrum and weak antibacterial activity against resistant bacteria(such as erythromycin,clarithromycin and azithromycin)to potent antibacterial activity against various respiratory pathogens,especially for the erm gene-mediated MLSB-and mef gene-mediated M-resistant bacteria(such as telithromycin,cethromycin and solithromycin).The above great progress and success have brought hope to effectively alleviate the problem of bacterial resistance.The research and development process of macrolide antibiotics shows that obtaining new macrolide nucleus and introducing aralkyl side chains are the main ways to develop new antibacterial drugs.Based on the design strategy of "fragment cutting and binding",we first opened the 14-membered lactone ring of clarithromycin by oxidation reaction,then introduced the appropriate aralkyl side chains,and finally established new 15-membered lactone ring by esterification.The stereoconformation of the new 15-membered macrolide nucleus changed to some extent to facilitate binding with the bacterial ribosome.Various aralkyl side chains could also be flexibly introduced into the lactone ring,and they can bind to 23 S rRNA domain II of bacterial ribosome.Therefore,five series of 103 N-11,C-12,C-13 and C-9 aralkyl clarithromycin semisynthetic derivatives were designed and synthesized,then their antibacterial activity,bactericidal activity,bactericidal kinetics and cytotoxicity were measured and evaluated.The minimum inhibitory concentration(MIC)of A,B,C,D and E series compounds was determined by broth microdilution method.Clarithromycin and azithromycin were used as controls in the evaluation of their antibacterial activity.The results are as follows.1.Antibacterial activity against sensitive bacteria.The C and D series compounds showed excellent antibacterial activity against sensitive S.aureus ATCC25923.Among them,compounds 49b,491 and 49v in D series displayed the strongest antibacterial activity(MIC≤0.25μg/mL),which was equivalent to clarithromycin(MIC<0.25μg/mL).The A,C and D series of compounds possessed potent antibacterial activity against B.subtilis ATCC9372.Among them,compounds 36g,36h and 36j(MIC≤0.25μg/mL)in C series and compounds 49b,49d,49f,491 and 49v(MIC≤0.25μg/mL)in D series exhibited the most potent antibacterial activity,which was equivalent to clarithromycin(MIC≤0.25μg/mL).In contrast,all the target compounds and clarithromycin exerted weak antibacterial activity against E.coli ATCC25922 and P.aeruginosa ATCC27853.In summary,compounds 49b,491 and 49v exhibited the strongest antibacterial activity among all the target compounds,and displayed excellent antibacterial activity(MIC≤0.25 μg/mL)against the sensitive S.aureus ATCC25923 and B.subtilis ATCC9372.In addition,compared with clarithromycin,these three compounds also exhibited a certain enhanced antibacterial activity against E.coli ATCC25922 and P.aeruginosa ATCC27853.2.Antibacterial activity against resistant bacteria.Many compounds in A,B,C and D series had strong antibacterial activity against resistant bacteria.In particular,compound llg(MIC=4 μg/mL)in A series exhibited the strongest antibacterial activity against resistant S.aureus ATCC31007,displaying a substantial improvement(64 fold increase)over clarithromycin(MIC>256 μg/mL).Compound llg(MIC=4μg/mL),and compound 20m(MIC=4 μg/mL)in B series showed the most potent antibacterial activity against resistant S.aureus ATCC43300,which was 64-fold stronger than that of clarithromycin(MIC>256 μg/mL).Compounds 11d,11f and 11g in A series,compounds 20m and 20n in B series,and compounds 36m and 36n in C series exhibited potential antibacterial activity against ermB resistant S.pneumoniae B1,whose MIC values were all 8 μg/mL,32-fold more active than that of clarithromycin(MIC>256 μg/mL).Compounds llg(MIC=4 μg/mL)and 36m(MIC=4 μg/mL)exhibited the strongest antibacterial activity against mefA+ermB resistant S.pneumoniae AB11,which was 32-fold better than that of clarithromycin(MIC=128μg/mL).Compounds lle-g in A series,compounds 20m and 20n in B series,and compounds 36k,36m and 36n in C series possessed the most potent antibacterial activity against resistant S.pyogenes 1,whose MIC values were all 8 μg/mL,32-fold stronger than that of clarithromycin(MIC=256 μg/mL).Overall,compounds 11f,11g,20m,20n,36m and 36n displayed potent and balanced antibacterial activity(MIC=4-8 μg/mL)against resistant S.aureus ATCC31007,resistant S.aureus ATCC43300,ermB resistant S.pneumoniae B1,mefA+ermB resistant S.pneumoniae AB11 and resistant S.pyogenes 1,which had the advantages of broad antibacterial spectrum.According to the above results of antibacterial activity,the structure-activity relationships of A-E series are summarized as follows:(1)Introduction of the aralkyl side chain at the C-13 position of the 15-membered macrolide nucleus has the best effect on improving the antibacterial activity of the compound against sensitive bacteria,followed by that at the N-11 position and the worst at the C-12 position;(2)Increasing the distance between aralkyl side chain and the 15-membered macrolide nucleus can enhance the antibacterial activity of the compound against sensitive bacteria;(3)Compared with the target compounds,clarithromycin is almost inactive against resistant bacteria,which indicates that the aralkyl side chain is an essential group to improve the antibacterial activity of resistant bacteria;(4)The introduction position of the aralkyl side chain on the lactone ring will show different antibacterial activity of A-E series.When the aralkyl side chain is connected to the N-11,C-12 and C-13 position,it is beneficial to improve the antibacterial activity against resistant bacteria,but it is connected to the C-9 position,the activity against resistant bacteria is basically lost;(5)When the group attached to the end of the aralkyl side chain is electron-donating group,electron-withdrawing group or unsubstituted group,the electron-donating group especially the alkyl group has the greatest contribution to the improvement of antibacterial activity against resistant bacteria.And the longer the alkyl chain is,the stronger antibacterial activity of resistant bacteria is.When the length reaches five carbon atoms,the antidrug resistant activity is the strongest.Compounds 11f and llg in A series,compounds 20m and 20n in B series,compounds 36k,36m and 36n in C series and compound 49g in D series were selected to determine their minimum bactericidal concentration(MBC)against mefA+ermB resistant S.pneumoniae AB11.The results showed that they exerted antibacterial activity by inhibiting the growth of bacteria.In addition,the MBC values of compounds 36g,36h and 36j in C series and compounds 49b,491 and 49v in D series against sensitive S.aureus ATCC25923 were also determined.The results indicated that they also played an antibacterial role through bacteriostatic mechanism.In order to study their bactericidal kinetics,the time bactericidal curves of compounds llg and 36k against mefA+ermB resistant S.pneumoniae AB11 were drawn.The results indicated that when its concentration was higher than or equal to 1 MIC,compound llg exhibited a certain degree of bactericidal effect,possessing both a concentration and time dependence in 0-12 h,and excellent bacteriostatic effect in 12-24 h.In the same way,compound 36k also exhibited similar bactericidal kinetic properties to 11g.In summary,the tested compounds are all excellent bacteriostatic agents.Compounds 36g,36h and 36j in C series and compounds 49b,491 and 49v in D series were selected to determine their toxicity to human breast cancer cells MCF-7 by MTT method,and clarithromycin was used as a control.The results showed that the tested compounds were effective against sensitive S.aureus ATCC25923 and B.subtilis ATCC9372,and the influence of toxic factors could be excluded.The full understanding of the development process of the four generations of macrolides antibiotics,the profound understanding of their mechanism of action and drug resistance,the precise understanding of the relationship between their structural modification and antibacterial activity,all lay a solid foundation for us to adopt the"fragment cutting and combining" design strategy.Therefore,we designed and synthesized five series of novel N-11,C-12,C-13 and C-9 aralkyl clarithromycin semisynthetic derivatives by changing the macrolide nucleus structure and introducing aralkyl side chains from clarithromycin as the raw material.A lot of compounds with excellent antibacterial activity were found in the evaluation of antibacterial activity.For example,compound llg(MIC=4μg/mL)exhibited significant antibacterial activity against mefA+ermB resistant S.pneumoniae AB11,much better than clarithromycin(MIC=128 μg/mL),which can be used as a lead compound for further study.On this basis,we analyzed and summarized the structure-activity relationships of the target compounds,then found the correlations between the antibacterial activity and macrolide lactone ring,the type of aralkyl side chain,the connection position of aralkyl side chain on the macrolide lactone ring and the configuration of chiral carbon at the connection position,which provided the theoretical basis for further structure optimization.Thus,in order to deal with the growing problem of bacterial resistance,the above findings are helpful to find more macrolide derivatives with new structural types.