Design, Synthesis And Antibacterial Evaluation Of Novel3-MBA Derivatives Targeting FtsZ

The extensive use and misuse of antibiotics have resulted in the emergence and prevalence of bacterial resistance, which has seriously threatened human health. Nowadays, the two most representative widespread-resistant bacteria MRSA and MDRSA have posed a significant threat, both in hospitals and, more recently, in the community. Many originally powerful antibiotics in clinic have showed either weak or no activity against multidrug-resistant pathogenic bacteria, which contributes to the enormous difficulty in fighting against bacterial infections. As a result, new approaches and targets for discovering the next generation of antibacterials are urgently needed to combat the increasing number of life-threatening bacterial infections.The major obstacle to the development of new antibacterials is the search for novel molecular targets. Essential bacterial cell division proteins, in particular FtsZ (Filamentous temperature-sensitive protein Z), represent the most attractive ones, because almost all proteins in the bacterial cell-division pathway are necessary for bacterial viability. There are about sixteen essential bacterial cell divisions proteins remaining largely unexploited, which are widely conserved. During bacterial cell division, FtsZ undergoes GTP-dependent polymerization at the mid-cell to form a Z-ring polymerizes. Meanwhile, FtsZ recruits other downstream cell division proteins. Finally, FtsZ and other cell division proteins form the septum that enables the daughter cells to separate.The compounds which affect both the polymerization and the GTPase activity of FtsZ or only interfere with one aspect could be developed as potential antibacterials. This class of compounds, especially natural products, have been reported, which block bacterial cell division by inhibiting the biochemical activity of FtsZ. However, most FtsZ inhibitors have no antibacterial activity in vivo and none of them has entered clinical evaluation. So far, only PC190723, a potent3-methoxybenzamide (3-MBA) derivative, has demonstrated efficacy in models of bacterial infection and is moving toward the clinic.3-MBA is one of the most promising and enlightening FtsZ inhibitors for the development of antibacterial drugs. In contrast to other starting points such as A-189and534f6,3-MBA has two key advantages. First, though possessing relatively weak antibacterial activity against Staphylococci,3-MBA is able to bind to FtsZ at high efficiency and specificity. Second,3-MBA could easily penetrate bacterial cells, which are often barriers for other FtsZ inhibitors.The findings from the docking model by computer has indicated that a cleft existed between the C-terminal domain and helix-7on FtsZ is considered as the binding site of3-MBA derivatives, different from the GTP binding site located in the GTPase domain. Actually, benzamide group of3-MBA derivatives binds in the cleft adjacent to R191, Q192, N263, V307, and T309. The phenoxy ether group of3-MBA interacts with R191and Q192in the cleft through hydrogen bonds. And the3-ether substituents combines in the hydrophobic channel consisted of the amino acid residues1172, E185, N188,1228, and1230. Nowadays, none of the3-MBA derivatives acts with the core helix H7. Scientists predicted that interaction of3-MBA derivatives with H7could significantly contribute to the inhibition of the polymerization and the GTPase activity of FtsZ.In this thesis, the discovery and characterization of novel3-MBA derivatives with potent activity against staphylococci are presented. Our objective was to increase the antibacterial activity and the pharmaceutical profile of3-MBA derivatives. Three series of3-MBA derivatives (series A, B and C) targeting FtsZ were designed, synthesized and evaluated in vitro antibacterial activity. Afterwards, according to structure-activity relationship, three compounds with more favorable antibacterial activity were selected as starting points and further modified with drug-like substituents to create series D and E. The structures of target compounds have all been confirmed by MS,1H NMR, and IR.In vitro antibacterial activity of all the target compounds were presented as the minimum inhibitory concentrations determined by the broth microdilution method and summarized as belows.1. Antibacterial activity against three species of Staphylococcus aureus.1) Series A:Compared with3-MBA, all the derivatives of series A demonstrated remarkablely improved activity against MRSA, MSSA and penicillin-resistant S. aureus. The MICs of derivatives of series A were no more than128μg/ml. Among them, the most predominant A2and A7showed8μg/ml against both susceptible and resistant strains, exhibiting more than256-fold higher activity than the parent3-MBA.2) Series B:All the compounds of series B except B1and B9showed excellent activity against all three species of S. aureus, with the MICs in the range of4to128μg/ml. The two most preferable compound B5and B12exhibited more than256-fold enhanced activity than3-MBA.3) Series C:The activity against S. aureus of series C was weaker than series A and B. Only C6and C12displayed favorable activity against all the three phenotypes of S. aureus, with MICs between8and32μg/ml.4) Series D and E:Compared with3-MBA, most of the compounds of series D and E showed little improvement in activity against both susceptible and resistant S. aureus, with the MICs more than128μg/ml.2. Antibacterial activity against six species of Streptococcus spp. Compared with3-MBA, all the derivatives of series A to E demonstrated little improved activity against all six species of Streptococcus spp., including four strains of Streptococcus pneumoniae and two strains of Streptococcus pyogenes., with all MICs more than128μg/ml.Structural modification and structure-activity relationships of3-MBA derivatives were summarized as follows.1) A majority of the3-MB A derivatives showed potent activity against S. aureus. However, nearly all of the3-MBA derivatives exhibited little activity against Streptococcus spp. These phenomenons are consistent with previous studies by Stokes et al., but the specific reasons are not known.2) Resistant strains such as MRS A and penicillin-resistant S. aureus are still sensitive to3-MBA derivatives.3) The methyl group of3-MBA can be changed with other substituents to generate new derivatives with remarkably improved activity. The introduction of alkyl and halogenated hydrocarbons substituents to the phenoxy group of3-MBA demonstrates remarkably increase in activity against both susceptible and resistant S. aureus. The appropriate distance from the phenoxy group to the terminal halogens is4carbon atoms.4) Derivatives composed of benzamide and aromatic moieties joined together by an appropriate ether linkage, exhibit remarkablely improved activity. The length of ether linkage and the kind of aromatic ring could significantly affect the antibacterial activity. Compared with polar groups, the introduction of non-polar groups to the phenoxy group of3-MBA is more beneficial for efficacy against pathogens. The reason may be that3-ether substituents of3-MBA bind to the hydrophobic channel on FtsZ.In summary, FtsZ is an attractive but as yet underexploited target for the discovery of novel antibacterials. Synthetic FtsZ inhibitors have been explored and3-MBA is found to be the most attractive for development into an antibacterial agent. It’s very hopeful that introducing side chains to the phenoxy group of3-MBA, with appropriate length and terminal aromatic moiety, could result in potent activity against both susceptible and resistant staphylococci. Further development of3-MBA derivatives described in this thesis should lead to the generation of compounds with significant therapeutic value.