Design,Synthesis And Antibacterial Evaluation Of Benzothiazolyl And Indolyl Phenanthridium Derivatives As FtsZ Inhibitors

Infectious diseases caused by drug-resistant bacteria have been a huge threat to human health.With the increasing number and types of drug-resistant bacteria in recent years,vancomycin,the last line of defense of antibiotics,has also been broken through.Therefore,the research and development of new antibacterial drugs with novel mechanisms of action has become an unavoidable task.Filamentous temperature-sensitive protein Z(FtsZ)is an important functional protein in the process of bacterial division.Since it is widespread and highly conserved in bacterial cells,FtsZ has becomea new antibacterial target with promising prospect.In this thesis,the PC hydrophobic slit of the FtsZ protein was used as the target binding site.By simplifying the reported FtsZ inhibitor sanguinarine,a natural product,benzothiazolyl and indolyl phenanthridium derivatives(A-C series)were designed and synthesized.Besides,their antibacterial activity and FtsZ targeting were systematically evaluated.The relevant research results are summarized as follows:Among the benzothiazolyl phenanthridium compounds(A series),only A4 and A5 had moderate antibacterial activity(MIC=1-64 μg/mL)against one or some of four Gram-positive susceptible bacteria strains(B.subtilis ATCC9372,B.pumilus CMCC63202,S.aureus ATCC25923 and S.pyogenes EMA-S)and five Gram-positive drug-resistant bacteria strains(methicillin-resistant S.aureus ATCC43300,penicillin-resistant S.aureus ATCC31007,S.aureus CI,penicillin-resistant S.epidermidis,erythromycin-resistant S.pyogenes),which were typical bacteriostatic agents.The other compounds did not have obvious antibacterial activity.However,all compounds had no antibacterial activity against two Gram-negative strains(E.coli ATCC25922 and P.aeruginosa ATCC27853).Based on above studies,we further designed and synthesized indolyl phenanthridium derivatives(B and C series).These two series of compounds showed excellent antibacterial activity against the above nine Gram-positive drug-susceptible and-resistant bacteria strains,some of them had significant antibacterial activity against vancomycin-susceptible and-resistant E.faecium and E.faecalis.Among B series,for example,B9 and B10 possessed significant breakthroughs in the activity against penicillin-resistant S.epidermidis(MIC=2 μg/mL),which was 4 and 2-fold better than sanguinarine and linezolid,respectively.The MICs of B9 and B10 against vancomycin-resistant E.faecium(Van-A resistant)and E.faecalis(Van-B resistant)were both 4μg/mL,which was 8-fold more powerful than sanguinarine.Among C series,C5 had the best activity(MIC=1-8μg/mL)against S.aureus ATCC25923,methicillin-resistant S.aureus,two strains of penicillin-resistant S.aureus,penicillin-resistant S.epidermidis and erythromycin-resistant S.pyogenes,which were superior to ciprofloxacin and berberine.However,these two series of compounds had no efficacy on two Gram-negative bacteria strains(E.coli ATCC25922 and P.aeruginosa ATCC27853).Thus,C3 and C5 with significant antibacterial activity were selected as representative compounds for further evaluation.The time-killing curve revealed that C3 and C5 had the characteristics of bactericidal kinetics,whose bactericidal effect was concentration-dependent manner.Biofilm formation inhibition assay indicated that the two compounds did not have any inhibitory effect on biofilm formation of P.aeruginosa.Multipassage resistance selection studies indicated that C3 and C5 were not easy to develop resistance to sensitive S.aureus during the induction of 15 passages.In addition,the hemolytic test showed that C3 and C5 had no hemolytic effect on mice red blood cells.Preliminary pharmacodynamic assay in vivo showed that C5 had good antibacterial activity in mice,but is weaker than linezolid.According to the above results,C5 was used to further study the mechanism of action.The optical microscope assay proved that C5 could inhibit the cell division of bacteria,which indicated that the compound had a preliminary targeting,while the light scattering experiment revealed that C5 promoted the polymerization of FtsZ protein in a concentration dependent manner.The transmission electron microscope assay indicated that C5 could promote protein polymerization,but could not orderly polymerize into bundles.This further confirmed that C5 specifically acted on FtsZ protein.The structure-activity relationships of benzothiazolyl and indolyl phenanthridium derivatives are summarized as follows:①2-(Benzothiazol-2’-yl)-5-methylphenanthridin-5-ium compounds(A series):Different substituents were introduced at the 7,8,9 and 10 positions,and their order of activity was methyl>methoxy or hydrogen.Their order of activity against susceptible bacteria strains is 9-methyl>10-methyl>8-methyl>7-methyl;their order of activity against resistant bacteria strains is 10-methyl>9-methyl>8-methyl>7-methyl.②5,9-Dimethyl-2-(indol-6’-yl)phenanthridin-5-ium compounds(B series):Substituent was introduced at the 1’-N position of indole,and their order of antibacterial activity is benzyl>isobutyl≈phenylpropyl>butyl>methyl>propyl.That is,as the length of alkyl chain increased,the activity of the target compound first decreased and then increased,and the more sterically hindered substituents had better antibacterial activity.When the alkyl chain was further extended or branched alkanes were introduced,the activity against sensitive bacteria strains did not change significantly,but the activity against resistant bacteria strains was further enhanced.The activity against penicillin-resistant S.epidermidis and vancomycin-resistant E.faecalis ATCC51299 was significantly enhanced by introducing substituents at the para position of benzyl group,while the activity against other bacteria strains had no significant change.③5,10-Dimethyl-2-(indol-6’-yl)phenanthridin-5-ium compounds(C series):Substituent was introduced at the 1’-N position of indole,and their order of antibacterial activity is benzyl>butyl≈isobutyl>phenylpropyl>methyl>ethyl,whose structure-activity relationships were similar to B series.The activity against sensitive bacteria and resistant bacteria decreased slightly by introducing substituents into the para position of benzyl group.④When the length of alkyl chain at the 1’-position was short or the steric hindrance was small,the activity of C series was better than that of B series in the activity against drug-resistant or-sensitive bacteria strains.However,with the increase of alkyl chain or the substituent steric hindrance,the antibacterial activity of B series was better than C series in some strains.These bacteria strains include B.subtilis ATCC9372,erythromycin-susceptible S.pyogenes,Penicillin-resistant S.aureus CI,erythromycin-resistant S.pyogenes,E.faecium ATCC19434,and Van-A resistant E.faecium ATCC51559.In conclusion,we designed and synthesized 27 benzothiazolyl and indolyl phenanthridium derivatives(A-C series)based on the structural simplification of the natural product sanguinarine with FtsZ protein as the target.On this basis,we found that indolylphenanthridium derivatives had significant antibacterial activity through MIC,MBC,time-killing curve,biofilm formation inhibition assay,multipassage resistance assay,hemolysis test and preliminary pharmacodynamics assay in vivo.In particular,C5 had the strongest activity against sensitive S.aureus(MIC=1μg/mL),which was 4 fold as much as sanguinarine and ciprofloxacin,and 2 fold as much as linezolid;its activity against five drug-resistant strains(methicillin-resistant S.aureus,two penicillin-resistant S.aureus,penicillin-resistant S.epidermidis and erythromycin-resistant S.pyogenes)(MIC=2-8μg/mL)were better than ciprofloxacin and berberine.Preliminary pharmacodynamic assay in vivo indicated that C5 showed good antibacterial effect,and it was not easy to induce drug resistance of S.aureus.Further mechanism studies showed that C5 could inhibit bacterial cell division and promote the polymerization of FtsZ protein,leading to disorderly polymerization of FtsZ protein and presenting disordered knots,which indicated that C5 specifically acted on FtsZ protein.Molecular docking study predicted the binding mode of C5:C5 could enter the PC hydrophobic slit of FtsZ protein and form hydrophobic interactions with amino acid residues such as Gln 192、Gly 196、Asp 199、Leu200、Val203、Gly227、Ile228、Asn263、Thr265、Val297、Thr309、Val310 and Ile311.In a ward,this study not only provides a theoretical basis for the design of novel FtsZ inhibitors,but also lays a good foundation for the further research and development of 2-indolyl-5-methylphenanthridin-5-ium compounds as antibacterial agents.