| Macrolide antibiotics are a class of weakly basic lipophilic macrolactone ring compounds,usually with a 14-16 membered lactone ring as the mother nucleus,to the macrolactone ring of which one or more deoxy sugars,typically L-clatinose or Ddesosamine,are attached.Macrolide antibiotics are commonly used in clinical treatment of infectious diseases,such as respiratory tract infection and soft tissue infection.However,due to their widespread use,bacteria are gradually induced to develop drug resistance,which severely weakens their efficacy and limits their clinical application.In order to combat the increasingly serious problem of bacterial resistance,the research and development of novel macrolide antibiotics against drug resistant bacteria has become the most urgent issue to be addressed.The studies of the mechanism have indicated that macrolides can bind to the peptide release channel in the peptidyl transferase center of the 50S subunit of bacterial ribosome,which can interfere with the elongation of nascent peptide chain,and restrain the synthesis of protein,thereby exerting antibactericl activity.Previously,our research team designed and synthesized a series of azithromycin derivatives,and found that they had excellent antibacterial activity.However,in the above study,only preliminary screening of antibacterial activity were conducted,and no systematic studies weree conducted on compounds with significant antibacterial activity.On this basis,we have conducted in-depth and systematic research on the azithromycin derivatives S2-S8 with excellent antibacterial activity.This thesis mainly includes two parts:optimization of the synthesis process of azithromycin derivatives S2-S8 and evaluation of their biological activities.Part I:The synthesis process of azithromycin derivatives S2-S8 was optimized.The effects of reaction conditions such as molar ratio,catalyst,solvent,temperature,and concentration,as well as post processing method on the reaction yield and product purity were investigated.The optimal synthesis process for compounds S2-S8 was obtained.Using azithromycin as raw material,the final product S2-S8 was obtained through six steps of reaction.Their overall yield was increased from 40%～60%to 80%～85%,with the purity of greater than 98.5%.In the synthetic optimization of intermediate I,under the premise of ensuring the yield,the reactant molar ratio and acid binding agent were adjusted,and the reactant concentration was improved to 2.5 times,thereby shortening the reaction time and improving the reaction efficiency.During the synthesis of intermediate Ⅱ,by trimming the molar ratio of reactants from 1:3 to 1:5.5 and reducing the amount of solvent by 1/2,the reaction time was shortened from 40 hours to 7.5 hours,and the yield was increased from 41%to 96%.In the synthetic optimization of intermediate Ⅲ,by adjusting the ratio of reactants and catalyst,while increasing the concentration of reactant to 2 times,the reaction time was shortened from 20 hours to 8 hours at room temperature,and the yield was enhanced to 96%.In the synthetic optimization of intermediate Ⅳ,when the concentration was heightened from 0.17 mol/L to 0.52 mol/L,the reaction time was shortened by 4 hours.At the same time,the aftertreatment process was optimized from silica gel column chromatography to recrystallization of ethanol and water for purification,significantly reducing the amount of solvent used,decreasing costs and saving time.In the synthetic optimization of intermediateV,under the premise of ensuring the yield,the molar ratio of intermediateⅣ to side chain was regulated from 1:1.51 to 1:1,the solvent ratio was reseted from 1.3:1 to 1:1,and the concentration was raised from 0.056 mol/L to 0.167 mol/L,thereby cutting the reaction time by 6 hours and increasing the yield from 86%to 92%.In the synthetic optimization of final product S,the molar ratio of reactants was modified from 1:30 to 1:25,and the molar ratio of catalyst was adjusted from 1:2 to 1:3.As a result,the reaction time was diminished by one day at room temperature and the yield reached 83%.Through the studies of the above synthesis process,we have obtained a synthetic process route that is simple in operation,low in cost,high in product purity,and high in yield.Part II:Systematicallyevaluation for the biological activity of S2-S8 to obtain more potential antibacterial agents.In vitro antibacterial activity study showed that compounds S2-S8 exhibited excellent antibacterial activity for susceptible Staphylococcus aureus,with an MIC value of 0.06-0.25 μg/mL,among which,the most active compound was S7 with an MIC value of 0.06 μg/mL,32-fold better than azithromycin(MIC=0.25 μg/mL),2133 times better than norfloxacin(MIC>128μg/mL),and even 8 times as much as vancomycin(MIC=0.5 μg/mL);For Bacillus pumilus,S3 and S8 had the best antibacterial activity,with an MIC value of 0.5 μg/mL,equivalent to azithromycin,but much stronger than ciprofloxacin and norfloxacin;The antibacterial activity of S2-S8 against Enterococcus faecalis and Enterococcus faecalis was also prominent,with the MIC values of 0.125-0.25 μg/mL,which were better than the positive controls.The best compounds were S2 and S8 with the anti-Enterococcus faecalis showing 8-fold more potent activity than azithromycin(MIC=1 μg/mL)and the anti-Enterococcus faecium reaching 64 times,which was far better than ciprofloxacin and penicillin sodium;Moreover,S2-S8 had excellent antibacterial activity against susceptible Streptococcus pyogenes,with the MIC values of 0.001μg/mL,displaying a 30-fold increase in antibacterial activity over the azithromycin(MIC=0.03 μg/mL).For drug-resistant bacteria,S4,S5 and S7 had a good antibacterial effect on penicillin-resistant Staphylococcus aureus,with a MIC value of 2 μg/mL,exerting more than 64-fold increased activity over the clarithromycin(MIC>128μg/mL),equivalent to linezolid and norfloxacin,while S2 and S8 also increased by 32 or 16 times than the control drugs.For erythromycin-resistant Streptococcus pyogenes,S4 and S6 showed the best antibacterial activity,with an MIC value of 1 μg/mL,128 times better than azithromycin and clarithromycin(MIC=128 μg/mL),equivalent to linezolid and penicillin sodium.In contrast,S2-S8 showed weak antibacterial activity against methicillin-resistant Staphylococcus aureus(MIC=64-256 μg/mL),comparable to azithromycin and clarithromycin.For the ermB+mefA resistant Streptococcus pneumoniae,S2-S8 produced strong antibacterial activity,with the MIC values of 0.250.5 μg/mL,displaying a 256 to 1024-fold increase in antibacterial activity over the azithromycin(MIC=256 μg/mL)and clarithromycin(MIC=128 μg/mL),which is equivalent to vancomycin and oxacillin sodium.For vancomycin-resistant Enterococcus faecium,the antibacterial activity of S2-S8 was better than those of the positive controls,among which,the best compound was S2,exerting more than 8-fold increased activity over the azithromycin(MIC>256 μg/mL),but still did not show antibacterial effect.Similarly,S2-S8 had no antibacterial effect on vancomycinresistant Enterococcus faecalis,which is equivalent to the positive controls.For gramnegative bacteria,S2-S8 showed no antibacterial activity against Escherichia coli and Pseudomonas aeruginosa(MIC value is 16-64 μg/mL),which is consistent with most positive controls.Overall,the target compound has good activity against Staphylococcus aureus,Enterococcus faecalis,Enterococcus faecium,Streptococcus pyogenes,penicillinresistant Staphylococcus aureus,erythromycin-resistant Streptococcus pyogenes,and the ermB+mefA resistant Streptococcus pneumoniae,with the MIC values of 0.001μg/mL and 0.25 μg/mL against the tested susceptible and resistant bacteria,respectively.Of all the target compounds,compound S4 exhibited the best in vitro antibacterial activity against the seven tested strains simultaneously,with the MIC values of 0.25,0.25,0.125,0.001,2,1 and 0.25 μg/mL respectively.The result of In vitro antibacterial activity indicated that compounds S2-S8 exhibited bacteriostatic effects and were typical antibacterial agents,with the most prominent compound being S4.Time-kill curve showed that at the concentrations of 1 × MIC、2 × MIC、4 × MIC,compound S4 exhibited a certain bactericidal effect within 0-12 hours,and is concentration and time dependent,while within 12-24 hours,it showed good bacteriostatic effect.Biofilm formation inhibition result indicated that compounds S2-S5 and S7-S8 showed extremely strong inhibitory effects on the formation of biofilms of Pseudomonas aeruginosa PAO1 at sub-inhibitory concentration,with inhibition rates ranging from 90.83%±0.42%to 74.63%±0.66%,indicating that the compounds killed bacteria by inhibiting the formation of bacterial biofilms.the result of multipassage resistance assay showed that compounds S2,S4 and S7 were not easy to induce drug resistance to the ermB+mefA resistant Streptococcus pneumoniae AB11 within 15 generations.Experimental studies of hemolysis showed that compounds S2-S8 did not cause hemolysis in mouse erythrocytes.Cytotoxicity study displayed that compounds S2,S4S6,and S8 had little cytotoxicity on HEK293 cells at normal concentrations.In vivo toxicity assay showed that within one week of administration,compound S4 with high safety did not cause serious toxicity to mice,preliminarily indicating that the compound solution was safe in vivo.In vivo pharmacodynamic sutdies showed that compound S4 at the concentration of 5 or 25 μg/mL,significantly reduced the number of viable bacteria in mice,demonstrating excellent antibacterial activity in vivo,with a significant difference compared to the control drugs azithromycin and linezolid.In summary,we have completed the optimization of the synthesis process of the azithromycin derivatives S2-S8,and systematically conducted experimental studies on in vitro antibacterial activity,bactericidal activity,time-killing kinetics,biofilm formation inhibition,multipassage resistance,hemolysis,cytotoxicity,in vivo toxicity and pharmacodynamics in vivo.The research results show that the compound S4 has excellent antibacterial activity in vitro and in vivo,with low cytotoxicity and high safety in vivo.The further demonstrates the therapeutic potential of compound S4 as a new antibacterial candidate drug,which will be further evaluated in the future. |
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