Design,Synthesis And Mechanism Study Of Novel Quinoxaline-1,4-di-N-oxides With Antibacterial Activity

In the early stage,quinoxaline-1,4-di-N-oxides(Qd NOs)were used as antibacterial drug in veterinary clinical,recent studies have demonstrated a spectrum of pharmacological properties,including anticancer,antiviral,antimycobacterial,antiparasitic and antifungal.Particularly,the discovery and structural modification of novel Qd NOs against Mycobacterium tuberculosis(M.tb)and protozoan have become one of the hotspots in the field of pharmaceutical chemistry.Previous research has demonstrated that the Reactive Oxygen Species(ROS)generation following their biotransformation,ROS react with DNA,leading to breaks in the DNA strands,which eventually result in death of the bacteria.Previous researches have demonstrated that the pathway in which Qd NOs exert its biological activity was the generation of hydroxyl radical(OH·),Qd NOs possess strong hypoxia-selective,it could react with reductase and obtain one-electron,releasing OH·,one of the highly ROS,OH·directly reacts with DNA,which is often irreversible,leading to degradation of DNA.However,there are few studies on the antituberculosis mechanism of Qd NOs.Studies of the antimycobacterial mechanism based on M.tb mainly focus on the regulation of various pathways in macrophages,including ROS and autophagy.ROS can act on the electron transport chain(ETC)of M.tb,leading to a disturbance in energy homoeostasis.High levels of ROS may also induce autophagy,thereby restricting the growth and replication of intracellular M.tb.In this study,we used the strategy of pharmacophore fusion,and the pharmacophores of Qd NOs were retained.Then we focused on the structural modification of C2 and C6 positions of quinoxaline ring,and a series of novel quinoxaline-1,4-di-N-oxides derivatives were obtained.Antibacterial activities,as well as the structure-activity relationship(SAR),of the compounds were assayed.Finally,the mechanism of antibacterial action of Qd NOs was elucidated from ROS,DNA synthesis and repair,and autophagy.1.Design,synthesis and activity study of novel thiazolidinone-containing Quinoxaline-1,4-di-N-oxidesIn this study,a series of novel thiazolidinone-containing quinoxaline-1,4-di-N-oxides were designed and synthesized,SAR was perfect,and potential lead compounds were screened out to lay a foundation for the research and development of Qd NOs.Previous research has demonstrated that the substituents on C2 and C7positions of quinoxaline ring have a great influence on the activity of Qd NOs,and the SAR of Qd NOs was analyzed,results also found that the C2 substituents was important to the activity.In recent years,the structural modification of compounds mainly relies on the methods of pharmacophore fusion,compound skeleton transition and electron isosteric,to obtain lead compounds with potential activity.Thiazolidinone derivatives possessing wide biological activities and pharmacological properties,it also provides a new idea for the design and modification of Qd NOs,In this study,the strategy of pharmacophore fusion was used,and a series of novel Qd NOs containing various thiazolidinone moieties at the C2 position were designed,after oxidation,Beirut reaction,hydrolysis,aldehydes amine condensation and cyclization condensation,26 novel thiazolidinone-containing quinoxaline-1,4-di-N-oxides(TZN1～26)were obtained.The antibacterial,antifungal and antituberculosis activities of compounds TZN1～26 were determined by broth microdilution and MABA methods,the results showed that:compounds TZN4,TZN5,TZN10,TZN11,TZN15,TZN16,TZN20,TZN21,TZN25 and TZN26 exhibited better antibacterial activity against G~+,the activity increased 2-8 times compared with olaquindox,such as compounds TZN20and TZN21,exhibited potential activities against S.aureus(ATCC29213,MIC=16μg/m L).Compounds TZN19-26 exhibited good antifungal activities(MIC≤8μg/m L)against C.albicans,C.tropicalis,A.fumigatus and C.neoformans.Compounds TZN20,TZN21,TZN25 and TZN26 showed the best antimycobacterial activity(MIC=1.56μg/m L).Further analysis of the activity showed that,the presence of an electron-withdrawing moiety at position C7 or C4 can significantly improve the antibacterial activity of compounds.However,an electron-releasing group reduces the activity of the compounds.Based on the 3D-QSAR results,we can draw some conclusions.For the C7position of the quinoxaline ring and the C4 position of the phenyl group,sterically bulky substituents,hydrophilic substituent or electronegative groups are preferred to increase the activities of compounds.Electropositive substituents in the C7 position would decrease the affinity of compounds,the activities increased dramatically.For the C2 side chain of the quinoxaline ring,more hydrogen bond donor substituents should be considered.2.Design,synthesis and activity study of novel nitrogenous heterocyclic-containing quinoxaline-1,4-di-N-oxidesThere are few structural modifications on the C6 position of quinoxaline ring,only a few studies have shown that the introduction of a halogen atom or methyl group increases the activity of compounds against M.tb,leading to the vacancy of the SAR at this position.In this study,the strategy of pharmacophore fusion was used,and a series of novel Qd NOs containing various nitrogenous heterocyclic moieties at the C6 position were designed,meanwhile,ester or acyl groups were introduced in the C2 position,methyl or trifluoromethyl were introduced in the C3 position and a fluorine atom was attached on C7 position,after oxidation,Beirut reaction and nucleophilic substitution reaction,33 novel nitrogenous heterocyclic-containing quinoxaline-1,4-di-N-oxides(NCH1～33)were obtained.The antibacterial and antituberculosis activities of compounds NCH1～33 were determined by broth microdilution and MABA methods,the results showed that:compounds NCH1～33 showed lower activities against E.coli(ATCC25922),only compounds NCH5,NCH6 and NCH25 have good activities,with an MIC between 4and 8μg/m L.Compared with ATCC25922,compounds NCH16,NCH20,NCH24,NCH28 and NCH29 remain the activities against drug-resistant E.coli,which indicated that the mode of antibacterial action of Qd NOs may be different from that of fluoroquinolones.The MIC of compounds NCH1～33 against Actinobacillus pleuropneumoniae(ATCC27090)down to 0.25μg/m L,against HPS0165 the MIC down to 1μg/m L.Compounds NCH1～33 exhibited better antibacterial activity against S.aureus(ATCC29213),the activity increased 256 times compared with mequindox,MIC down to 0.5μg/m L.The MIC of compounds NCH1～33 against clinical isolation of drug-resistant S.aureus down to 1μg/m L,against MRSA the MIC down to 4μg/m L.Compounds NCH16,NCH20,NCH28 and NCH29 exhibited the most potent antimycobacterial activity against M.tb strain H37Rv(MIC≤0.25μg/m L),the activity increased 16～32 times compared with mequindox.Compound NCH29 also exhibited excellent antimycobacterial activity in an M.tb-infected macrophage model,compound NCH29 could indeed reduce the bacterial load inside the macrophages and after 4 d of exposure at 40 times the MIC concentration,showing 2.73-log-reduction in growth of M.tb.When the infected cells were exposed to compound NCH29 for 1 d,the reduction was ranged from 0.1 to 1.69-log in growth of M.tb.Further analysis of the activity showed that,the presence of ethyl ester and benzyl ester group at position C2 can significantly improve the antibacterial activity of compounds.The introduction of a trifluoromethyl group at the C3 position also increased antimycobacterial activity.When an imidazole or 1,2,4-triazole group was introduced into C6 position,the compounds exhibited excellent activities.And a fluorine atom at the R7 position of quinoxaline ring increases activityBased on the 3D-QSAR results,we can draw some conclusions.For the C6position of the quinoxaline ring,hydrophilic substituent or electronegative groups are preferred to increase the activities of compounds.For the C3 position,hydrophobic substituents could increase the activities of compounds.For the C2/C6 side chain,more hydrogen bond donor substituents should be considered.3.Antibacterial mechanism of quinoxaline-1,4-di-N-oxidesStudy on the mechanism of action against E.coliBased on the previous studies,we hypothesized the mechanism of action of Qd NOs:The ROS generation following Qd NOs biotransformation,react with DNA,leading to breaks in the DNA strands;meanwhile,Qd NOs can also interfere with the process of bacterial DNA repair.In this study,DNA synthesis and repair related enzymes were selected to clarify the relationship between Qd NOs and DNA damage repair system,including DNA polymerase I,DNA ligase and DNA topoisomerase(DNA gyrase and DNA topoisomerase IV),the results showed that:(1)Qd NOs had no obvious inhibitory effects on DNA ligase and DNA topoisomerase;(2)Qd NOs had obvious inhibitory effects on DNA polymerase I,at 128μg/m L,the inhibition rates of quindoxine,tirapazamine,olaquindox and mequindox on DNA polymerase I were 80.2%,78.7%,50.8%and 54.5%,respectively;(3)Qd NOs can compete with d NTPs for the active site of DNA polymerase I,and the N-oxide groups in Qd NOs play a vital role in the activity of Qd NOs.We can conclude that Qd NOs play a role by inhibiting the activity of DNA polymerase I.Study on the mechanism of action against Mycobacterium tuberculosisIn this study,the influence of Qd NOs on energy homoeostasis of M.tb were investigated by measure the membrane integrity,ATP depletion and m RNA level,the results showed that:compound NCH29 was able to disrupt the membrane integrity of M.tb,trigger a significant reduction in ATP levels and rapid down-regulation of m RNA(ndh and ndh A)levels.Based on the above results,after compounds NCH29incubate with M.tb,it can disrupt the membrane integrity,causing homeostasis disbalance;NCH29 could further act on ETC and disturb the function of NDH-2,disrupt the redox homeostasis,block the transfer of electrons in ETC and interfere with M.tb energy metabolism,resulting in M.tb death.To further study on the mechanism of action after Qd NOs incubate with M.tb-infected macrophages,we evaluated the intracellular ROS and relative mitochondrial ROS levels after treatment with compound NCH29 using the DCFH-DA and Mito SOX assays by multifunctional microplate reader,and the LC3 II level were determined by western blot and immunofluorescence staining,the results showed that:compound NCH29 stimulates the production of cellular ROS in M.tb-infected macrophages;compound NCH29 treatment is capable of inducing autophagy and LC3 accumulation in M.tb-infected macrophages.Based on the above results,when compound NCH29 incubate with M.tb-infected macrophages,on the one hand,compound NCH29 noticeably increased cellular ROS levels,ROS attack M.tb,resulting in M.tb death;on the other hand,high level ROS subsequently induced autophagy in M.tb infected macrophages,inhibit the growth of M.tb,further exert antimycobacterial effect.In summary,59 novel quinoxaline-1,4-di-N-oxides were designed and synthesized,antibacterial activities,as well as the SAR,of the compounds were assayed.The results of this study indicated that Qd NOs could inhibit the activity of DNA polymerase I,interfere with energy metabolism and induce autophagy to exert antibacterial effect.This study further clarified the mechanism of action of Qd NOs and established the SAR,which lay a foundation for the design and modification of Qd NOs.