Discovery And Activity Evaluation Of Novel Arylazole Benzamide Antibacterial Lead Compounds Based On FtsZ

Infections caused by bacteria have long been a common and multiple disease that threatens human health,and even severely deprives human life.The discovery of penicillin in 1928 and the subsequent application of various antibacterial drugs saved millions of lives and made an indelible feat for human health.However,the use of antibacterial drugs will inevitably lead to the emergence of bacterial resistance.Especially in recent years,the widespread use or even abuse of antibacterial drugs greatly shortened the time for bacterial resistance,however,the development of new antibacterial drugs is far from catching up with this pace.Nowadays,the increasingly serious bacterial resistance has undoubtedly aggravated the threat of clinical infection.We are worried that no drug will be effective against those multi-drug resistant bacteria in the near future,and humans will return to the no-penicillin age before the 20th century.Natural selection,survival of the fittest.Bacteria have evolved resistance due to the emergence of antibacterial drugs.Therefore,the rational use of antibacterial drugs can reduce the evolutionary pressure of bacteria and slow down the development of bacterial resistance.In addition,the discovery of novel effective drugs by acting on new antibacterial targets will also be a more direct and effective means of attacking resistant bacteria.The filamentous temperature-sensitive protein Z(FtsZ)is a key protein involved in bacterialcell division.Intervention of its normal biological function will result in the inhibition of bacterial division and eventually the death of bacteria.Therefore,FtsZ can be a promising new target for the development of antibacterial drugs,and various FtsZ inhibitors have been reported to exhibit excellent antibacterial activity.Among them,the 2,6-difluorobenzamide derivatives modified from 3-methoxybenzamide(3-MBA)is a very representative class of FtsZ inhibitors,and their targeting effect,molecular mechanism,biological activity in vivo and in vitro,crystal structure of protein-ligand,etc.have been reported to varying degrees.Therefore this kind of 3-MBA derivatives have attracted extensive attention from antibacterial research groups and institutions worldwide.However,at present,such compounds are only in preclinical studies,further screening and optimization of their structures are still of great significance.In view of this,starting from 3-MBA analogues,we have designed and synthesized series of compounds with a wide range of structure variety through structure-based rational drug design and computer-aided drug design strategy.A total of 182 compounds in 21 series were evaluated for their in vitro and in vivo antibacterial activity,bacterial morphology,targeting effect,and biostability,etc.Previous studies have found that the formamide bond in the structure of benzamide FtsZ inhibitors contributes greatly to protein binding and antibacterial activity,so structural modification of its key pharmacophore,formamide,for the study of the interaction mode between this type of compound and FtsZ protein,exploration of new types of antibacterial drugs are of great significance.We replaced the formamide with triazole,oxadiazole,hydrazide,urea and other structures by bioisosteric strategy,designed and synthesized a total of 52 compounds from A to K series.After in vitro activity screening,we found that the derivative B6 substituted with 1H-1,2,3-triazole had good antibacterial activity against the four positive bacteria tested(B.subtilis,sensitive and resistant S.aureus).The MIC of S.aureus is between 2-4 μg/mL.However,the activity of the formamide parent compound 6 was only 16 μg/mL.It is indicated that 1,2,3-triazole exhibits stronger antibacterial activity than formamide in this series of structures,and provides a high quality lead for the development of novel antibacterial drugs.The drugs targeting FtsZ can cause the bacterial morphology to become larger or longer.However,on the targeting test we found that B6 did not cause significant changes in bacterial morphology;and the structure-activity relationship of this phenyltriazoles B-series was also inconsistent with the conclusions of the benzamides,so we conclude that the target of B series compounds may not be FtsZ.To this end,we further explored its potential target protein.PharmMapper search system predicted that peptide deformylase(PDF)may be its potential target.Subsequent prediction of the potential binding pocket of the protein by LigsitCSC system,as well as computer docking analysis,found that B6 can produce strong interaction with the binding site of PDF.The triazole and ether oxygen atoms form hydrogen bonds with surrounding amino acid residues,the long alkyl chain interacts with the hydrophobic region.The binding mode can well explain the structure-activity relationship of B series.It is speculated that the derivative of phenyltriazole structure exerts antibacterial activity by acting on PDF,which provides a preliminary direction for the further research on the action mechanism of lead B6.After replacing the formamide led to off-target effects,we retained the formamide structure and explored its 3-lipophilic side chain.Firstly,a five-membered hybridization of isoxazole was introduced as an intermediate linking ring to form 3-arylisoxazole-benzamide derivatives,namely series L total 35 compounds.The activity screening highlighted a compound L25.Molecular docking analysis showed that the amino acid residue of Asp199 near the isoxazole ring in the structure might be a potential binding site.By calculating the distance between the N,O atoms on the ring and the carboxyl residue of Asp 199,it is speculated that if the positions of the N and O atoms are exchanged,the N atom is 3.4 A from the carboxyl group of Asp 199,and this may form an ion dipole interaction.Thus,M series total 21 compounds containing 5-arylisoxazole-benzamide was designed,in which the positions of N,O atoms on isoxazole ring in L series were interchanged.The activity test of this series compounds found that compound M14 has better antibacterial activity than L25,and the MICs against B.subtilis and B.pumilus is decreased from 0.03,0.25 μg/mL to 0.015,0.03 μg/mL,activity was increased by 2-8 times;MICs against three S.aureus strains(sensitive,penicillin-resistant,methicillin-resistant)is decreased from 8,4,8 μg/mL to 2,2,4 μg/mL,activity was increased by 2-4 times.By computer docking analysis,the N atom on the isoxazole ring in M14 did interact with the amino acid residue of Asp 199.In the FtsZ protein polymerization experiment,the same concentration of M14 can promote FtsZ polymerization effect stronger compared to L25,indicating that the binding of M14 with FtsZ is stronger.Subsequently,in the mouse blood infection model,M14 showed good antibacterial activity in vivo,and intraperitoneal injection of 150 mg/kg M14 divided into five dose can significantly reduce the bacteria amount of the blood in mice.The IC50 of M14 against mammalian Hela cell line is greater than 128 μg/mL,which is much higher than its antibacterial concentration,and this also indicates that M14 has no inhibitory effect on mammalian tubulin indirectly.Subsequently,structural optimization of the intermediate connecting ring were further carried out,and the N to S series total 44 compounds containing 1,3,4-oxadiazol-2-one were designed.The evaluation test found that the antibacterial activity of N14 against four kinds of S.aureus,including three resistant bacteria,was between 0.5-1 μg/mL,which was the best active compound in the series.Exchanging the substitution on both sides of 1,3,4-oxadiazol-2-one ring getting Q series will cause the activity to disappear,indicating that the intermediate heterocyclic ring has a specific adaptation to the target site of the protein.The introduction of a methylene group in the molecule to extend the length of the molecule getting O and P series will also result in a decrease in antibacterial activity,which may be related to the binding pocket limitation of the target.However,the degree of activity reduction is smaller after being introduced into the-O-C-bridge to become an-O-C-C-bridge,which is presumed to be related to the flexibility and scalability of the three atomic chains.Considering the potential biostability of the lactone bond in the 1,3,4-oxadiazol-2-one ring,we designed R,S series derivatives that the O atom in the ring was replaced with N and C atoms.In vitro plasma stability experiments showed that the design strategy greatly improved the molecular biological stability.The content of compound R1 was almost unchanged after 24 hours,while the plasma half-life of N14 was only 2.5 hours.However,the antibacterial activity of this two series compounds disappeared.It was found that the computational Log P value ofN14 was 3.41,after the O was replaced by N and C,the log P was 2.74 and 2.69 respectively,indicating the polarity was increased significantly.Since the binding site is a hydrophobic cavity,it is speculated that the disappearance of the antibacterial activity of R and S series is mainly caused by the decrease in lipophilicity and the spatial volume effect.The T and U series total 30 compounds have been greatly modified in molecular conformation,in which the isoxazole ring of the L and M series was changed to the isoxazoline ring.The substitution of C-C double bond to C-C single bond produces a chiral carbon on the ring and creates a non-coplanar structure.The conformation of the entire molecule is more flexible and varied,in order to study the induced binding mode of configuration-curved molecular and FtsZ protein.The activity screening found that T16 has the strongest antibacterial activity,and the MICs against four kinds of S.aureus including three resistant bacteria was between 0.25-0.5 μg/mL,which was superior to the control PC 190723 and clinical drugs ciprofloxacin,linezolid,erythromycin.Since T16 is a racemate,it is speculated that the antibacterial activity of the R or S configuration will be better.The molecular docking results of T16-R,T16-S with FtsZ showed that the binding conformations almost verlap with each other;the docking score showed that the binding force of R configuration was slightly better than S configuration.Unlike the X-ray bonding mode of PC 190723 which has a plane conformation,the T16 molecule binds to the target site in a similar"M" type conformation,which is consistent with our original design.Under induced-binding mode,this slightly curved molecular structure may be more compatible with the rugged surface of the amino acid residue in the binding pocket.Preliminary cytotoxicity experiments showed that the racemate T16 had rarely toxic effects on mammalian Hela cells,IC50>64 μg/mL,which was much higher than its MIC.Subsequent chirality and activity,toxicity characteristics should be further explored to determine its clinical prospects.From the 21 series a total 182 compounds,and their bioactivity evolution,we found three benzamide derivatives M14,N14,T16 containing 4-tert-butylphenylisoxazole,4-bromophenyloxadiazolone,4-n-butylphenyl isoxazoline structure have the best antibacterial activity.The SAR analysis showed that the benzamide group which formed the main hydrogen bonds is attached to the intermediate heterocycle on the left,and the substituted benzene ring which formed the main hydrophobic interaction is attached to the intermediate heterocycle on the right.It seemed like that this five-membered heterocyclic ring only serves to link the left and right pharmacophores,but in fact the spatial volume and structural characteristics of the intermediate five-membered heterocyclic ring have an important effect on the activity.For example,the size of the substituent and the composition of the hetero atom on the ring not only play an important role in the binding of the target site,but also affect the biological stability and pharmacokinetic properties of the entire molecule.In particular,the structure and properties of the pharmacophores on the left and right sides have been basically determined,and the transformation space is limited.But there is still a lot of room for the structural transformation of the intermediate connecting ring,which will also be important for further improving the antibacterial activity and the druggability properties of the entire molecule.In this study,based on the structure of benzamide FtsZ inhibitor,we designed and synthesized a series of compounds based on bioelectronic isosteric principles,structure-based skeletal optimization,and computer-assisted drug design,and found a number of lead agents with outstanding antibacterial activity in vivo,in vitro and along with FtsZ-targetting activity.The active molecules with many skeletal diversity in this study not only provide reliable structural instruction for further studying the binding mode between benzamide inhibitors and FtsZ target proteins,designing antibacterial compounds targeting FtsZ,but also provide high-quality lead compounds for further optimizing bioactivity and druggability to develop new generations of antibacterial drugs with novel structures and unique mechanisms of action,in order to counter the increasingly serious problem of drug-resistant bacteria around the world.