| Quinoxaline-1,4-di-N-oxides（QdNOs）have unique N→O groups and show a wide range of biological activities,such as antibacterial,antiprotozoal,antitubercular,antitumor,which have been the focused issues and trends of medicinal chemistry research.The QdNOs,including olaquindox,carbadox,mequindox and quinocetone,have good antibacterial effects on Escherichia coli,Salmonella,and Brachyspira hyodysenteriae,have good antibacterial activity,and were used as growth-promoting agents in veterinary clinics.In our laboratory,a series of QdNOs modified at the C6 positions were obtained,and the antibacterial results showed that the target QdNOs had stronger activity against Staphylococcus aureus（S.aureus）and reduced activity against Escherichia coli（E.coli）.This finding is quite different from the former researchs that QdNOs are mainly effective against Gram-negative bacteria.In this work,we propose to continue the design of QdNOs by introducing various different groups at the C6 positions,such as heterocycles,benzene rings and pyridine rings containing different substituents and different linkage arms,to obtain a series of novel QdNOs derivatives,determine their anti-S.aureus activity and establish antibacterial structure-activity relationship（SAR）.On this basis,the antibacterial mechanism of action was elucidated by morphological changes of bacteria,changes in ROS level,DNA damage,ATP level and membrane potential.By comparing the intracellular drug concentrations of the compounds in S.aureus and E.coli,we revealed that the chemical structure changes affected the intracellular accumulation capacity and thus led to the difference in antibacterial activity.This study provides the basis for the discovery of quinoxaline drug candidates with anti-S.aureus activity,offering new choise for the treatment of infections caused by S.aureus and drug-resistant S.aureus.1.Design,synthesis and antibacterial activity of novel quinoxaline-1,4-di-N-oxidesThe 6,7-substituted-2-quinoxaline-1,4-di-N-oxides was obtained through oxidation reaction and Beirut reaction.A total of 32 novel QdNOs derivatives,30 of which are unreported,were obtained by introducing various nitrogenous heterocyclic,benzene,pyridine,and amine substituents at the C6 positions of QdNOs via aromatic nucleophilic substitution reactions,click reactions,and suzuki coupling reactions.The antibacterial activity of the compounds was determined by micro-broth dilution method,and the results showed that the novel QdNOs were anaerobically selective.For S.aureus ATCC 29213,some of the compounds showed superior antibacterial activity（8-2μg/mL）under aerobic conditions,such as 4c,4d,4e,4f,4g,4i,4j,9a,comparable to cefoxitin and superior to MAQO（128μg/mL）;under anaerobic conditions,the activity of the above compounds reached 2-0.5μg/mL.For E.coli ATCC 25922,most of the compounds showed poor activity under aerobic conditions（128-64μg/mL）;some compounds such as 4b,4d,4e,4f,4g,4i,4j,9a,16 had moderate antibacterial activity under anaerobic conditions（16-8μg/mL）,which was worse than MAQO（1μg/mL）.The antimicrobial SAR showed that the compounds had poor antimicrobial activity when non-aromatic groups were substituted at the C6 position,such as 4n,4o,4q,4t,4v,4w,4x.Some compounds had superior anti-S.aureus ATCC 29213 activity when aromatic groups without linker arms were substituted at the C6 position,such as 4c,4d,4e,4f,4g,4i,4j,9a,but when the aromatic group is a six-membered ring such as phenyl or pyridyl,as in 9d,9c,or when the aromatic group is connected to the QdNOs by a linker arm,as in4l,4m,4p,4r,4s,4u,the activity of the compounds is poor or disappears.By comparing4c,4d,4e,4f,4g,4i,4j,9a with 10f,16,20,25,it was found that the presence of hydrogen atoms attached to basic nitrogen atoms in nitrogen-containing aromatic five-membered heterocyclic substituent groups was found to significantly reduce the antibacterial activity of the target compounds,while the presence of substituent had no significant effect,as in4e,4f,4g.In addition,the double substitution of the heterocyclic group at the C6 and C7positions rendered the compounds inactive,as in 5i.Quantitative structure-activity relationship model showed:R2=0.913,and adjusted R2=0.892,indicating that the model has better explanatory ability.Leave-one-out cross validation showed:RMSEP=-68.585,r=0.771,indicating that the model has good generalization ability.Descriptors that have a more important impact on the model are NaasN and piPC8,with normalized coefficients of 0.472 and 0.461,respectively,which are positively correlated with antibacterial activity;Mor17v,Mor09m,and GATS2v have slightly weaker effects on activity,with normalized coefficients of 0.231,0.259,-0.242,respectively,The former two are positively correlated with activity,and the latter are negatively correlated with activity.2.Mechanism research of novel quinoxaline-1,4-di-N-oxides against Staphylococcus aureusThe compound 9a with superior activity was selected for the study of anti-S.aureus mechanism.The bactericidal kinetic curves revealed that the novel QdNOs were bacteriostatic at 1×MIC and 2×MIC and bactericidal at 4×MIC,indicating that this class of compounds is concentration-dependent.Scanning electron microscopy（SEM）revealed that the division of bacteria was blocked with the treatment of 9a,and the bacterial surface showed a circular depression profile along the diameter,and some of the bacterial surface surfaces were wrinkled and deformed;the lysis diffusion of the cell wall was found by transmission electron microscopy（TEM）,and the boundaries of the cell wall,cell membrane and cytoplasmic components were blurred and the cytoplasm leaked.In this study,by detecting changes of membrane potential and ATP of S.aureus,it was found that the ATP decreased significantly under the treatment of 4×MIC compound 9a,which was comparable to the CCCP positive control,and the membrane potential also had significant changes,verifying the damage to the cell membrane of S.aureus by 9a.ROS and superoxide anion（O2·-）in the bacteria were detected by fluorescent probe method,and the results showed that ROS and O2·-in the bacteria increased significantly at 60 min and 30min,respectively,and the levels of both were positively correlated with the incubation time.The comet experiment results showed that the comet tail length of the experimental group（9.12±2.54μm）was significantly longer than that of the blank group（5.49±2.42μm）.The above findings suggest that the novel QdNOs exert their antibacterial activity by damaging the cell wall、cell membrane and DNA of S.aureus through the production of reactive oxygen species.3.Accumulation research of quinoxaline-1,4-di-N-oxides in the bacteriaAn LC-MS/MS method for the accumulation of compounds 4j and MAQO on S.aureus and E.coli was established,and the accumulation of both compounds in bacteria was measured and time-accumulation curves were established.The results showed that the accumulation of 4j and MAQO in both bacteria reached equilibrium around 20 minutes,but 4j accumulated rapidly in the first 10 minutes,almost close to 90%of the equilibrium point,while MAQO accumulated around 50%of the equilibrium point in the first 10minutes.4j had a higher accumulation than MAQO in both strains.Therefore,the modification of C6 position increased the accumulation ability of QdNOs on S.aureus and E.coli;however,the anti-E.coli activity of 4j was not superior to that of MAQO,so we believe that the difference in antibacterial spectrum and antibacterial activity of QdNOs may be mainly due to the difference in target affinity caused by the structural difference of the compounds.In summary,34 novel QdNOs derivatives were synthesized by structural modification of the C6 position of the quinoxaline,and 14 QdNOs with good anti-S.aureus activity（MIC≤4μg/mL）were identified;Based on the above compounds and activities,a quantitative structure-activity relationship model with better interpretation and generalization capabilities was established.The novel QdNOs derivatives have stronger intracellular accumulation and can exert antibacterial activity through producing ROS and disrupting the cell wall,cell membrane and damaging DNA of bacteria.These provide the scientific basis for the research and development of quinoxalines. |
