Design,Synthesis And Biological Evaluation Of Novel Cinnamic Anhydride Derivatives As FtsZ Protein Targeted Antibacterial Agents

The emergence of methicillin-resistant Staphylococcus aureus,vancomycin-resistant Enterococcus,and other multidrug-resistant strains is a serious threat to global public health and safety.There are currently two strategies to overcome this problem:Firstly,extensively modifying antibacterial drugs and changing the interaction with drug targets to maintain their antibacterial activity;secondly,developing new antibacterial agents that can interact with new targets.Recent studies have indicated that filamentous temperature-sensitive protein Z(FtsZ)is an essential cell division protein for bacteria and plays a key role in bacterial cell division.Thus it has attracted increasing attention and is expected to become a target for the development of new antibacterial agents.Eleven compounds of A series were designed and synthesized by scaffold hopping and preferred skeleton fragment construction in the thesis,and based on the binding sites of the long gap between the H7 helix and the C-terminal subdomain in the FtsZ protein.However,preliminary antibacterial activity results indicated that the antibacterial activity of A series was not excellent against whether six kinds of Gram-positive bacteria or two kinds of Gram-negative bacteria.This demonstrated that the planar rigid structure of 1,2,4-triazolo[3,4-a]phthalazine skeleton didn’t interact wih the binding sites of the long gap between the H7 helix and the C-terminal subdomain,which is not conducive to the production of antibacterial activity.Based on the above research,we found that natural product inhibitors targeting FtsZ protein chrysophaentins A,hemi-chrysophaentins and curcumin owned similar structural units with phenylpropane structure.Compound with such structural characteristics,such as cinnamaldehyde,can interact with the binding site of the long gap between the H7 helix and the C-terminal subdomain in the FtsZ protein.In view of this,twenty-three cinnamic anhydride derivatives of B series were designed and synthesized.Generally,anhydrides are considered to be unstable and easy to hydrolyze,so there are few reports about anhydrides as drugs.However,this kinds of cinnamic anhydride derivatives are stable in aqueous solution,so it is of great significance to further study this kind of derivatives.Various substituted benzaldehydes reacted with malonic acid under the catalytic amount of piperidine through Knoevenagel-Doebner-condensation generate various substituted cinnamic acids.Subsequently,these cinnamic acids were acylated by oxalyl chloride to give the corresponding cinnamyl chlorides,which reacted with another cinnamic acid derivative to product the target compounds.This synthetic route was simple and had a yield of 31-83%.Then we determined the related antibacterial activities of the target compounds.The results are summarized as follows:In the cinnamic anhydride derivatives of B series,compounds B6、B10、B11 and B17 showed better activity than other compounds and control curcumin against whether Gram-positive or Gram-negative bacteria.This indicated that the introduction of methoxy,methoxycarbonyl and methylenedioxy groups in the symmetrical structure can increase antibacterial activity.In particular,compound B11 displayed the best antibacterial activity against the above bacterial stains.Compared with that of curcumin,for example,the activity of compound B11 against S.aureus ATCC25923,S.epidermidis,methicillin-resistan S.aureus ATCC43300 and penicillin-resistant S.aureus PR increased 2 times respectively;its activity against B.subtilis ATCC9372 and B.pumilus ATCC63202 increased 4 times respectively;its activity against against E coli ATCC25922 and P.aeruginosa ATCC27853 was equivalent to that of curcumin.In addition,the antibacterial activity of B series compounds is better than that of intermediate 6a(cinnamic acid),which indicated that the chemical properties of B series compounds are stable in the determination of antibacterial activity.The results of minimum bactericidal concentration(MBC)showed that this series of compounds played an antibacterial role by inhibiting the growth of bacteria.Next,we investigated the targeting activity of B series compounds.The effect of compound B11 possessing the best antibacterial activity on the morphology of B.subtilis and S.aureus cells was observed by the phase contrast microscope.The results exhibited that the cell length of B.subtilis cells and the cell volume of S.aureus increased after compound B11 treatment.Based on these observations,we preliminarily confirmed that compound B11 targeted FtsZ protein.In order to observe the binding mode of the cinnamic anhydride derivatives with FtsZ protein more intuitively,we used the computer aided drug design software(sybyl-x 2.0)to study the molecular docking of compound B11 with SaFtsZ protein.It was found that compound B11 could occupy completely the nucleotide-binding pocket in the N-terminal subdomain of the FtsZ protein,thereby forming hydrogen bonds with Gly20,Gly21,Ala73,Gly 108,Arg 143 and Thr109,and producing hydrophobic interactions with Gly20,Leu69,Gly70,Ala73,Gly104,Thr109,Pro135,Glul39,Arg143,Ala186 and Asp187.This is almost consistent with curcumin occupying the binding site of FtsZ protein.Due to the additional hydrogen bond between the oxygen atom on the para-methoxy of the benzene ring of compound B11 and the amino acid residue Thr109,the afinity between the compound and FtsZ protein was further enhanced,thereby resulting in its better antibacterial activity than that of curcumin.In summary,we designed and synthesized thirty-four compounds and determined that the target compound had excellent antibacterial activity and could target FtsZ protein by minimum inhibitory concentration(MIC)experiment,(MBC)experiment,cell morphology experiment and molecular docking.Among them,compound B11 had the best antibacterial activity against B.pumilus ATCC63202 and B.subtilis ATCC9372,the minimum inhibitory concentration(MIC)of which was as low as 8 μg/mL.Therefore,we believe that the cinnamic anhydride derivatives can be considered as lead compounds of small molecule inhibitors targeting FtsZ protein for further study.These findings also provide new ideas and methods for the design and synthesis of novel FtsZ inhibitors as antibacterial agents.