Design,Synthesis And Bological Activity Of C-7 Derivatives Of Four Cephalosporin Parent Cores

Cephalosporins with a broad antibacterial spectrum and low toxicity have been widely used in the treatment of infections.The currently marketed cephalosporin drugs are roughly divided into five generations,which are mainly based on the antibacterial spectrum and the time to market.The cephalosporin drug molecule generally consists of three parts:the cephalosporin parent core,the C-7 amino modification group,and the C-3 substituent（or no substituent）;the cephalosporin parent core,there are 7-ACA,7-ACCA,7-ANCA,7-MAC,7-MCA（derived from 7-MAC by removal of dibenz-hydryl group）commonly;the C-7 amino modification group of cephalosporin can be simple thiopheneacetyl,L-phenylglycyl group,cysteinyl group,cyanomethyl mercapto-acetyl,aminothiazolylacetyl,etc.,or it can have a very complex structural fragment,such as 2-furyl-（Z）-2-methoxyiminoacetyl,2-（4-amino）thiazole-（Z）-2-methoxyimino-acetyl group.The C-3 modified group,the more common ones are acetoxymethyl,chlorine atom,and（1-methyl-1H-tetrazol-5-yl）thio)methyl.The main differences between the first to fifth generation cephalosporin drugs are those between the C-7 amino side chains and the C-3substituents.In view of this,this thesis tries to select four kinds of cephalosporin parent core make different modifications to the C-7 position of the parent core,and convert the ester group to carboxyl group,hoping to find new molecule（s）with better activity than the cephalosporin parent core,or than similar cephalosporin drugs,or else with unreported activity,laying a key foundation for the future study of the biological activity of the cephalosporin parent core.1.Design and synthesis of C-7 derivatives of four kinds of cephalosporin parent coresFour kinds of cephalosporin parent cores,7-MAC,7-ACA,7-ACCA,and 7-ANCA were selected for molecular design and synthesis.At the beginning of this study,we learned from the experience of fluoroquinolone research and adopted a simple modification strategy to modify the C-7 amino group of four kinds of cephalosporin parent cores with different haloacyl chlorides to obtain TM1 series molecules（25）,and perform biological activity assays to investigate the feasibility of simple modification;further,selected the highly active molecules in TM1,derivatized with azoles,and obtained 7-MAC mercaptoazole derivatives TM2-TM4（45）with A-Linker-B mode.Afterwards,the C-7 amino group of 7-MAC was reacted with aromatic aldehydes,and the resulting Schiff bases were reduced by Na BH₄/BF₃ in acetic anhydride to produce the new cephalosporin derivatives TM5（23）and TM6（23）by a tandem reaction of imime reducing,the C-7 methoxy group falling off and then the ethyl group and different aromatic methyl group introducing at C-7 position,which laid a substance foundation for exploring the biological activity of non-acyl modified products of the C-7 amino group.Finally,the four kinds of cephalosporin parent cores were directly coupled with the marketed drug oxaprazine to obtain the A-B model cephalosporin parent core derivative TM7a-TM7e（5）by the amide synthesis method;Furthermore,Linker was designed as amino acids of different lengths,and A-Linker-B model cephalosporin parent core derivatives TM7f-TM7n（9）were designed and synthesized,thereby obtaining novel cephalosporin parent core derivatives modified by oxaprazine.Through the exploration of experimental conditions,combined with column chromatography and thin layer chromatography,7 series,33 intermediates and 130 target compounds were synthesized through different synthetic routes;The chemical structures of the synthesized target compounds were confirmed by ¹H NMR,¹³C NMR and HR MS.During the research process,the problem of the 7-MAC double bond shift was solved,the simple reduction of the imine was realized,and the simple preparation method of the C-7 dialkylamino cephalosporin derivatives was discovered,which has remarkable innovation.2.Study on the biological activity of C-7 derivatives of four kinds of cephalosporin parent coresSeven strains of human pathogenic bacteria,one strain of Pichia pastoris and three strains of Citrus pathogenic bacteria were selected to perform the antibacterial activity assays of some intermediates and all target molecules.Some molecules were tested for the inhibitory activity of one strain of Mycobacterium smegmatis and one strain of HCT116 tumor cells.At present,the main focus is on antibacterial activity testing.Activity against human pathogenic bacteria:In general,the target compounds showed different strengths of inhibitory activity against the different strains tested.（1）For E.coli,TM1f and TM1g are equivalent to the marketed drugs cefoxitin sodium and ceftizoxime sodium（MIC,2μg/m L,the same below）,8 times that of cephalothin;inhibitory activity of TM4 is significantly stronger than those of TM2/TM3,of which TM4b/TM4o（2μg/m L）,TM4c/TM4n（8μg/m L）are equivalent to the bacteriostatic activity of listed cephalosporin drugs;in TM5,TM5t/TM5u with benzyl aromatic ring as dichloro substituted phenyl had the best inhibitory activity（2μg/m L）;the C-4 carboxyl type molecules TM6 had stronger antibacterial activity than the corresponding ester type molecules TM5,among which TM6b/TM6c/TM6d/TM6p/TM6v have the strongest inhibitory activities（2μg/m L）,which is equivalent to the inhibitory activity of the first-generation cephalosporin drug cefalotin,the second-generation cephalosporin drug cefoxitin sodium,the third-generation cephalosporin drug ceftizoxime sodium and aztreonam;the inhibitory activities of TM7e/TM7j/TM7k against E.coli（2μg/m L）were equivalent to cephalothin,cefoxitin sodium,ceftizoxime sodium and aztreonam.（2）For S.enteritidis,the inhibitory activities of TM1k/TM1n（32/64μg/m L）were twice or equivalent to that of the marketed drug cephalothin（64μg/m L）;the inhibitory activities of TM7c/TM7d（4/32μg/m L）were 8 times or equivalent to that of the marketed drug cefalotin（32μg/m L）.（3）For A.baumannii,TM5p（16μg/m L）/TM5q（8μg/m L）/TM5r（8μg/m L）had excellent inhibitory activity,which were 8 times,8 times and 16 times of cephalothin/ceftoxime sodium/ceftizoxime sodium respectively,which were also stronger or equivalent to that of aztreonam against this bacteria strain.（4）For P.aeruginosa,the inhibitory activity ofΔ³normal structure and carboxyl type TM4 was far greater than that ofΔ²isomer and ester type TM2/TM3;structure-activity relationship:benzoxazole derivatives>non-benzoxazole derivatives,benzothiazole derivatives（1-4μg/m L）>benzimidazole derivatives（1-8μg/m L）≈benzoxazole derivatives（4-8μg/m L）;TM4d/TM4l/TM4m had excellent inhibitory activity（1μg/m L）,which was better than the all tested cephalosporin drugs;the inhibitory activity（16μg/m L）of TM7b against the bacteria strain was equivalent to cephalothin and 4 times that of ceftizoxime sodium.（5）For M.luteus,the 25 haloacyl products TM1 all showed excellent inhibitory activity,of which 16 compounds had an MIC value of 2μg/m L;TM4g/TM4h/TM4i/TM4k/TM4l had super strong inhibitory activity（0.03125μg/m L）against this bacteria strain,which was much stronger than those of the tested cephalosporins;TM7b has a significant inhibitory effect on the bacteria（8μg/m L）,which was 2 times that of cephalothin and 4times that of ceftizoxime sodium.（6）For S.aureus ATCC 25129,ATCC 14125,TM1a and TM1k in TM1（16～64μg/m L）had good antibacterial activity;For S aureus ATCC14125,TM4i/TM4k/TM4m showed super inhibition activity（0.03125μg/m L）,which is8 times that of cefalotin,64 times that of cefoxitin sodium,64 times that of ceftizoxime sodium,and 16 times that of meropenem;TM7c/TM7d/TM7e showed strong inhibitory activity（0.25μg/m L）,which was equivalent to that of cefalotin and 8 times that of cefalotin/ceftizoxime sodium.（7）The inhibitory activities（16,8μg/m L）of TM1a and TM1k against Bacillus subtilis were 4 times and 8 times that of cefalotin（64μg/m L）;the activity of TM1a is equivalent to that of cefalotin（16μg/m L）,and the inhibitory activity of TM1k was twice that of cefoxitin sodium.Activity against Pichia pastoris:The tested compounds were not sensitive to Pichia pastoris（mostly MIC is 0.256μg/m L）,but TM1a（32μg/m L）and TM1f（64μg/m L）showed significant inhibitory activity,which were 8 times and 4 times stronger than that of Cefalotin,respectively.Activity against citrus pathogens:The test results showed that the results showed that the inhibitory activity of most compounds against citrus canker decreased with the decrease of the concentration of the compounds,and had a good activity-dose relationship;At the concentrations of 2μg/m L and 1μg/m L,IM1g/IM1h/IM1j/IM1n had the best antibacterial activity（inhibition rates were 60%～69%,48～59%,respectively）,which were stronger than that of 7-MAC and positive control cefalotin（inhibition rates were 10-12%,-1-22%,respectively）.Although most of the synthesized compounds have no inhibitory activity against Colletotrichum gloeo-sporioides Co.3 and Alternaria alternate Al.6,the inhibition rate of TM1s at 4μg/m L on the Alternaria alternate Al.6 was as high as 90%,which was equivalent to the positive control prochloraz and stronger than the same kind of drug cephalothin.At the concentration of 4μg/m L,the intermediate IM1k/IM1v also had excellent inhibitory activities（inhibition rate of 100%,90%,respectively）against Alternaria alternate Al.6,which is stronger than or equivalent to the positive control prochloraz;At the concentration of 1μg/m L,the inhibitory activities of IM1k/IM1v（inhibition rate of 70%,60%,respectively）were still stronger or equivalent to prochloraz（inhibition rate of 50%）,and stronger than the same kind of drug cephalothin（inhibition rate was 50%）.In this project,it was discovered for the first time that some derivatives of cephalosporin parent cores have the inhibitory activity against citrus pathogens,which deserves further study.Activity against Mycobacterium smegmatis activity:So far,the inhibitory activities of some cephalosporin derivatives against Mycobacterium smegmatis have been evaluated.The results showed that TM4i/TM4k/TM4m all showed significant inhibitory activity（15.625μg/m L）,which was comparable to the positive control isoniazid（8μg/m L）.This subject is a preliminary attempt by our laboratory to derive the cephalosporin parent cores.We have explored a feasible method to prevent the shift of the 7-MAC double bond,established an effective method for C-4 and C-7 derivation,and discovered a simple method for the preparation of C-7 site dialkylamine cephalosporin derivatives;The biological activities of all target molecules against human pathogens and citrus pathogens were evaluated,and the anti-tuberculosis activities of some of the target molecules were also tested,the effects of the changes of C-7,C-4 and C-3 groups and double bond shift on these activities were investigated.This project has discovered that some molecules have super antibacterial activity,which achieved the goal of discovering new molecules with better antibacterial activity than the cephalosporin parent cores,or better antibacterial activity than cephalosporins with similar structure,or with unreported activity.This study verified the design ideas derived from the cephalosporin parent cores,established the research methods,and discovered the lead molecules,which laid a solid theoretical and experimental foundation for the follow-up research.Other activities of the designed molecules in this subject are still under study.