Design,Synthesis And Biological Evaluation Of Benzothiazole,Benzoxazole And Furanone Derivatives As Quorum Sensing Inhibitors

Quorum sensing(QS)is a widely existing intercellular communication system in various microorganisms,which relies on the production and sensing of diffusible quorum sensing signaling molecules(QSSM),sometimes referred to as autoinducers(AI).Once the QSSM concentration in the surrounding environment of a bacterial population reaches a specific threshold,the transcription of multiple genes will synchronize,allowing the population to act collectively.This diffusible signal regulation controls a wide range of activities such as swarming movement,biofilm maturation,virulence factors,secondary metabolite production,and antibiotic resistance.In recent years,attempts to develop new antibacterial agents have included targeting specific virulence factors or virulence regulatory mechanisms,to minimize the selective pressure that leads to drug resistance.One strategy is to interfere with QS mediated signaling,disrupt bacterial communication to reduce virulence,and enable infected bacteria to be cleared by the host’s defense system.Compared with traditional antibiotics,the use of QS inhibitors(QSIs)that do not directly damage bacterial survival should exert less selective pressure on drug resistance.In view of this,this paper takes QS as the target,based on the structural characteristics of AIs and QSIs,20 series of 197 compounds were designed and synthesized using computer-aided drug design,molecular splicing,bioelectronic rearrangement principles,and other drug design strategies.The structure of the target compound was characterized and its biological activity was measured.We determined the effect of compounds on bacterial growth by measuring the MIC values of seven different Gram positive and Gram negative bacteria and the growth curve of Pseudomonas aeruginosa in bacterial culture medium containing certain concentrations of compounds.And the inhibition activity of various virulence factors(such as pyocyanin,rhamnolipids,protease,and swarming movement)and biofilms were measured.However,due to the fact that individual QSIs may not be sufficient to completely eliminate bacteria,we further measured the combination effect of the compound and antibiotics ciprofloxacin and clarithromycin,hoping to achieve the goal of not only eliminating bacteria but also addressing bacterial resistance.Finally,four fluorescent strains PAO1-lasB-gfp、PAO1-rhlA-gfp、PAO1-pqsA-gfp and PAO1-gfp were used to further determine the targeting of the compound to the three QS systems of Pseudomonas aeruginosa.Research on benzothiazole derivatives as QSIs.Based on the investigation of the structure of QSIs,we found that it is essential to have methylene between N-acyl and 1,2,3-triazole for QS inhibition activity.However,replacing the head of the compound with different functional groups can still maintain QS inhibition activity.Moreover,we found that benzothiazole compounds not only significantly reduced the levels of toxic metabolites produced by bacteria involved in QS and redox balance mechanisms,but also reduced their ability to cluster and adhere to the surface.Based on the principle of molecular hybridization,we fused these key pharmacophores with QS inhibition potential together,using 2-aminobenzothiazole as the mother nucleus and introducing N-acyl and 1,2,3-triazole nucleotides connected by methylene in the side chain,thus obtaining a novel mother nucleus structure as a QSI.Among them compound 51,6i and 7j has moderate pyocyanin inhibition activity.In the subsequent growth inhibition experiment,the growth trend of bacteria in the culture medium containing compounds remained basically consistent with the negative control group.These results further proved that these compounds did not have an impact on bacterial growth and did not generate selective pressure on bacteria.Moreover,in the later stage of measuring the inhibition activity of compounds on more virulence factors and biofilm formation,we found that these compounds exhibited good inhibition activity in the production of rhamnolipid,protease activity,and biofilm formation.Most compounds had a certain degree of inhibition effect,for example,the most active compound 7j was able to inhibit 51.55±4.79%of biofilm formation at 16μg/mL,and the scanning electron microscopy results further confirmed the inhibition effect of compound 7j on biofilm formation.In combination with traditional antibiotics CIP and CLA,they can enhance the antibacterial effect of antibiotics in a concentration dependent manner.In the safety evaluation,the compound hardly produces hemolysis at 256 μg/mL.What’s more,compound 7j can inhibit the fluorescence expression of PAO1-lasB-gfp and PAO1-pqsA-gfp in a concentration dependent manner in the fluorescence reporter strain inhibition experiment,.Research on benzoxazole derivatives as QSIs.We designed benzoxazole derivatives as QSIs using chlorzoxazone and CL as lead compounds.We first obtained 2-hydrazine benzoxazole as the aromatic head based on the principle of bioisosterism and obtained a new structure by fusing the hydrophobic tail pharmacophore of CL and investigated their activity.The results showed that the F series compounds did not inhibit bacterial growth and had moderate inhibition activity on the production of pyocyanin.The inhibitory rates of 19a-19f on the production of pyocyanin were all over 30%.Based on this structure of F series compounds,we further changed the number of carbon atoms in the side chain to explore the effect of chain length on QS activity.The results showed that reducing or increasing the number of carbon atoms would further improve the activity of the compound to some extent,but excessive chain length would actually cause the activity of the compound to disappear.The inhibition rate of compound 21b with the best activity can reach 53.52±4.14%.However,the inhibition activity of pyocyanin of the I series compounds obtained by adding two carbon atoms actually decreased,with only very weak inhibitory or agonistic activity.In order to further investigate the influence of the head and side chain groups of the compound on QS inhibition activity,we replaced the head or tail of the compound and obtained two series of compounds,J and K.Unfortunately,the activity did not increase further.The growth inhibition experiment proved that these compounds did not affect bacterial growth from another perspective,and they also exhibit strong or moderate inhibition activity on the production of rhamnolipids and the activity of protease.For example,compounds 21a and 21d can inhibit the production of rhamnolipids by more than 50%,with inhibition rates of 52.01±6.01%and 51.69±2.29%respectively.Crystal violet staining and scanning electron microscopy experiments have demonstrated the excellent inhibition effect of compounds on biofilm formation from different perspectives.Especially the inhibition rate of compound 20c can reach 76.60±2.39%at 32 μg/mL,with only 23.40±2.39%of the biofilm remaining.And the compound can also inhibit the swarming movement of Pseudomonas aeruginosa PAO1 in a concentration dependent manner and enhance the antibacterial activity of antibiotics CIP and CLA.In further targeting experiments,compound 20c was able to inhibit the fluorescence expression of PAO1-lasB-gfp and PAO1-pqsA-gfp in a concentration dependent manner,indicating that the compound may target the las and pqs QS systems.In the safety experiment,the measured compounds almost did not produce hemolytic effects on mouse red blood cells,fully demonstrating that the compounds have safety within the effective concentration.What’s more,compound 20c can effectively enhance the antibacterial effect of antibiotic CIP in a mouse model of bacteremia.Overall,these new structured compounds exhibit good QS inhibition activity,effectively inhibiting the production of virulence factors and biofilm formation.They can also be used in combination with antibiotics to treat bacterial infections,providing new ideas and methods for addressing bacterial resistance.Research on furanone derivatives as QSIs.We screened the small molecule database using the structure of the LasR ligand binding domain of P.aeraginosa that binds to autoinducers,and obtained hit compounds with moderate docking scores(PubChem CID:10567938).After further modification and optimization of the precursor compounds,we designed and synthesized novel 4-(substituted phenyl)-5methylene-2(5H)-furanone derivatives as QSIs,and evaluated their QS inhibition activities.Compounds 34a,34f,34h,37g,37h,37i,39b,39d,39e,39j,391,41g and 41h with strong inhibition activity against pyocyanin were selected for systematic research.Compound 39e exhibited the most prominent QS inhibition activity.For example,in the minimum inhibitory concentration and growth inhibition experiments,39e had almost no effect on bacterial growth,which would not exert significant selective pressure on bacteria like traditional antibiotics,thereby weakening the development of acquired drug resistance.On the other hand,compound 39e not only exhibited excellent inhibition activity against various virulence factors and effectively inhibited the formation of biofilms,but also significantly enhanced the inhibition activity of two antibiotics CIP and CLA against two strains of P.aeruginosa in vitro.For example,the protease inhibition activity of compound 39e was 30.54±7.2%at 102.4 μg/mL,and compound 39e resulted in only 50.79±5.25%of biofilm residue at 32μg/mL.Even more exciting is that in the bacteremia model infected with Paeruginosa PAO1,it significantly improved the antibacterial effect of CIP in vivo.In addition,compound 39e has almost no hemolytic activity on mouse red blood cells.The results of the GFP reporter fluorescence strain inhibition experiment showed that compound 39e simultaneously targets the las,rhl and pqs QS systems in P.aeruginosa.Therefore,compound 39e can be used as an effective QSI for further development of antibacterial infections.Next,we innovatively fused the structure of the furan ring with OdDHL and maintained the characteristics of the five point pharmacophore model.Based on the previous research,we used 3-methyl-4-(4-fluorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one as the parent nucleus and introduced amide or ester groups as polar domains at the 3-and 5-positions of the furan ring.Finally,we designed and synthesized 4-(4fluorophenyl)-5-hydroxy-3,5-dimethylfuran-2(5H)-one analogues(Q series)and 3(aminomethyl)-4-(4-fluorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one analogues(R series).After preliminary determination of the inhibition activity of these two series of compounds against pyocyanin,we synthesized the most active substituents in these two series,and further designed and synthesized the 3-(aminomethyl)-4-(4fluorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one analogues as S series.The results indicated that all three series of compounds can effectively inhibited the production of virulence factors and the formation of biofilms.Especially the S series compounds are the most prominent.Compound 46h can inhibit the production of 65.34±6.52%pyocyanin at 102.4 μg/mL and 60.33±2.11%biofilm formation at 64 μg/mL without affecting bacterial growth.In addition,in subsequent experiments,compound 46h was further demonstrated to inhibit the formation of biofilms in scanning electron microscopy experiments and inhibit bacterial swarming movement in a concentration dependent manner.In the studies combined with antibiotics,these compounds significantly enhanced the antibacterial activity of ciprofloxacin and clarithromycin in vitro in a concentration dependent manner.In addition,compound 46h can significantly improve the bacterial clearance rate of ciprofloxacin in mouse infection models.Further research on its mechanism suggests that compound 46h can inhibit the fluorescence expression of PAO1-lasB-gfp,PAO1-rhlA-gfp,and PAO1-pqsA-gfp in a concentration dependent manner,but does not interfere with PAO1-gfp,indicating that the compound directly targets the QS system.In order to further enhance the QS inhibition activity of furanone compounds,we innovatively designed T series compounds by introducing a hydroxamic acid structure onto the furanone ring.We hoped to enhance the QS inhibition activity by chelating with iron through hydroxamic acid.Among them,compound 48b can inhibit the production of 79.69±5.62%pyocyanin and 46.43±2.74%rhamnolipid at 102.4 μg/mL.What’s more,it has been proven to effectively inhibit the formation of biofilms.Specifically,48b can promote the fluorescence expression of iron carriers pyoverdine and pyochelin in a concentration dependent manner,demonstrating the iron chelation effect of this compound.Overall,we designed 4-fluorophenyl-5-methyl-2(5H)-furanone,3(aminomethyl)-4-(4-fluorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one derivatives and N-(4-(4-fluorophenyl)-1-hydroxy-5-methyl-2-oxo-2,5-dihydro-1H-pyrrole-3-yl)methyl)benzamide derivatives from different perspectives and measured their QS inhibition activity.The results showed that these new structured compounds have good QS inhibition activity and can effectively inhibit the production of virulence factors and the formation of biofilms,And it can also be used in combination with antibiotics to treat bacterial infections,providing new ideas and methods for solving bacterial resistance.In this study,we used QS system as the target and conducted extensive literature research,then we designed and synthesized 20 series of 197 compounds using computer-aided drug design,molecular splicing,and bioisosterism principles,and selected compounds with outstanding QS inhibition activity such as 7j,20c,39e,46h and 48b.We determined the effect of compounds on bacterial growth by measuring the MIC values of seven different Gram positive and Gram negative bacteria and the growth curve of P.aeruginosa in bacterial culture medium containing certain concentrations of compounds.And the inhibition activity of various virulence factors(such as pyocyanin,rhamnolipids,protease,and swarming movement)and biofilms formation were measured to determine the QS inhibition activity of the compound from multiple aspects.However,due to the fact that individual QSIs may not be sufficient to completely eliminate bacteria,we further measured the combination efficacy of the compound and antibiotics CIP and CLA,hoping to achieve the goal of not only eliminating bacteria but also addressing bacterial resistance.Finally,four fluorescent strains PAO1-lasB-gfp,PAO1-rhlA-gfp,PAO1-pqsA-gfp and PAO1-gfp were used to further determine the targeting of the compound to the three QS systems of P.aeruginosa.The numerous structurally diverse QSIs in this study injected new vitality into the fight against P.aeruginosa infection.