Design, Synthesis And Biological Evaluation Of Cinnamamides As FtsZ-targeted Antibacterial Agents

Currently, bacterial infections remain to be a major public health concern worldwide, threating the human health and even lives seriously. One of the main reasons is the widespread use and misuse of antibacterial agents, resulting in the prevalence of resistant bacteria; the other is the development of new antibacterial agents far behind the speed of bacterial resistance evolution in recent years. Bacterial resistance has led to the reduced or completely lost efficacy of many effective antibacterial agents in clinic. With the increasing bacterial resistance, we almost face a fact of no effective alternative agents. It is an urgent need to develop novel antibacterial agents to meet the currently growing medical needs.As the effective modification of the existing antibacterial agents is becoming more and more difficult, targeting the new and important proteins of bacteria to develop novel antibacterial agents has aroused the growing interests of many scientific researchers. We have researched and analyzed nearly 10 new antibacterial targets, and have found that bacterial cell division protein FtsZ has the good potential to be an antibacterial target and it has been confirmed as a proming target for the development of novel antibacterial agents.We have selected the bacterial cell division protein FtsZ as the new target for the development of novel antibacterial agents in this project. Through molecular modeling and literature analysis, we found that FtsZ proteins from different sources (nearly 10 different strains) had a high similarity, and then we selected the FtsZ protein form Staphylococcus aureus as a representative and systematically investigated its binding pockets. In addition, we also carried out the study in the known more than 50 natural FtsZ inhibitors and more than 200 synthetic FtsZ inhibitors. Through these studies, we found that cinnamaldehyde not only had the good properties, but also had the clear binding pocket. What’s more, we found that the binding pocket of cinnamaldehyde was near that of the known FtsZ inhibitor PC 190723, and that there was no drug development currently reported targeting both regions at the same time. Therefore, we intented to design the novel compounds acting on the two adjacent binding pockets of FtsZ at the same time to improve the activity of cinnamaldehyde with remaining its broad antibacterial spectrum. Furthermore, it was also reported that cinnamic acid as the derivative of cinnamaldehyde had the preliminary FtsZ-targeted antibacterial activity, which would be an important reference to our drug design.In this project, the benzaldehyde derivatives were selected as the starting materials, converted to four series of 102 compounds via Knoevenagel condensation reaction, acylation reaction and amidation reaction. And the synthetic routes of all the key intermediates were also constructed in the meantime. The structures of all the target compounds were characterized by multiple spectrums. The antibacterial evaluation of all the target compounds and the controls, such as cinnamic acid (CA), curcumin (Cur), ciprofloxacin hydrochloride (Cipro) and oxacillin sodium (OS), was conducted using the broth dilution method against seven Gram-positive strains and two Gram-negative strains. The bacterial cell division inhibitory assay was also performed against two representative Gram-positive strains and two Gram-negative strains. Additionally, GTPase assay as well as the light scattering assay was carried out for the further confirmation of their target in the molecular level.The biological activities of cinnamamides were summarized as follows: Compound C12 showed potent biological activities. In the antibacterial assay, it exhibited good activity against Staphylococcus epidermidis with an MIC value of 4 μg/mL, which was 32-fold or over 32-fold better than those of CA, Cur and Cipro; it also displayed strong activity against Staphylococcus aureus ATCC25923 with an MIC value of 4 μg/mL, which was 32-fold or over 32-fold more potent than those of CA and Cur; in addition, this compound exerted good activity against penicillin-resistant S. aureus with an MIC value of 16 ug/mL, which was more than 8-fold better than those of CA, Cur, Cipro and OS; In the cell division inhibitory assay, compound C12 possessed a strong inhibition against the cell division of S. aureus ATCC25923 with a minimum inhibitory concentration of 1 μg/mL, which was 128-fold or more than 128-fold better than those of the FtsZ-targeted controls CA and Cur. In the biological evaluation, compound D3 showed a good potential. In the antibacterial assay, it inhibited the proliferation of penicillin-resistant S. aureus with an MIC value of 2 μg/mL, which was over 64-fold more stronger than those of CA, Cur, Cipro and OS; it displayed a good activity against S. aureus ATCC25923 with an MIC value of 2 μg/mL, which was 4-fold to over 64-fold better than those of CA, Cur, Cipro and OS; this compound also showed a good potential in the activity against Bacillus subtilis with an MIC value of 4 μg/mL, which was 8-fold to more than 32-fold better than those of CA and Cur. In the cell division inhibitory assay, compound D3 had a strong cell division inhibitory activity against B. subtilis with a minimum inhibitory concentration of 4 μg/mL, which was 4-fold to over 32-fold better than those of the FtsZ-targeted controls CA and Cur; it also exerted a good inhibition against the cell division of S. aureus ATCC25923 with a minimum inhibitory concentration of 16 μg/mL, which was 8-fold or over 8-fold more potent than those of CA and Cur. In the biological evaluation, compound D15 also showed good activities. In the antibacterial assay, it inhibited the proliferation of penicillin-resistant S. aureus with an MIC value of 4 μg/mL, which was over 32-fold better than those of CA, Cur, Cipro and OS. In the bacterial cell division inhibition assay, this compound inhibited the cell division of B. subtilis with a minimum inhibitory concentration of 1 μg/mL, which was 16-fold to over 128-fold more potent than those of the FtsZ-targeted controls CA and Cur; it also showed a strong inhibitory activity against S. aureus ATCC25923 with an MIC value of 2 μg/mL, which was 64-fold or over 64-fold better than those of CA and Cur. In the GTPase assay and light scattering assay, all the tested representatives inhibited the activity of FtsZ significantly, basically consistent with their antibacterial activities as well as bacterial cell division inhibitory activities, which indicated that cinnamamides exerted their antibacterial avtivities by targeting FtsZ. In this study, we have got some excellent lead compounds (A46, A66, A610, C1, C7, C8, C12, D3, D4 and D15). These leads have great value in the design and discovery of novel cinnamamides as well as the other novel chemical types of FtsZ-targeted antibacterial agents.Through the biological activity analysis of the target compounds, we summarized the following structure-activity relationships (SAR):In the series A, the activities of compounds would change significantly against most of the tested strains if either of the substituents changed on the phenyl ring and on the amide N in the scaffold. We speculated that the substituents on the phenyl ring of the scaffold might contribute to the anchor of the compounds in T7-loop region of the hydrophobic pocket, and the substituents on the amide N needed to meet the request of the space in the binding site, and then the compounds could exert a good activity. In the series B, phenyl ring in the scaffold of the compounds was substituted by the substituents with different volumes, electrical properties, lipophilicities, etc, while amide N was substituted by the (1H-benzimidazol-2-yl)methyl fragment. The compounds in this series exhibited a poor biological activity on the whole, which might be due to the benzimidazole fragment on the amide N probably not being a good fit with the receptors on the spatial orientation, affecting the binding of the compounds to the receptor, and resulting in the weak activities of most compounds in this series. In the series C, amide N in the scaffold of the compounds was the same N at the 1 position of the 2-methyl-1H-benzimidazole fragment. The biological activities of the compounds in this series had improved significantly on the whole compared with those of the compounds in the series A and B, which indicated that the spatial orientation of the benzimidazole fragment might play an important role in the receptor affinities and the biological activities of the compounds. However, the introduction of several methoxyl groups to the phenyl ring of the scaffold would lead to the reduced activities, which might be due to the increased steric hindrance resulted from the introduction of methoxyl groups, impacting the effective binding of the compounds to the receptor. In the series D, the amide N in the scaffold of the compounds was the same N on the 1 position of 2-substituents-1H-benzimidazole fragment. The compounds in this series showed significantly different biological activities. The C-2 position of benzimidazole fragment could only hold substituents with appropriate volume and property, indicating there might be a small space in the binding sites. In addition, C-6 substituted compound showed a good activity, which might be due to that the substituents at this position probably could extend up to the PC sites or binding sites of guanidyl methyl diaryl compounds.In summary, we have successfully designed and discovered a library of novel FtsZ-targeted cinnamamides with good biological activities in the cellular level and molecular level for the first time. The virtual docking studies suggested that these compounds could bind to the T7-loop region of FtsZ and also partly to the binding pocket of PC 190723, having achieved the purpose of multi-targeted drug design in theory, while the confirmation of the more specific binding sites was on the way. The study not only has led to the discovery of several novel lead compounds with good activities, but also has deepened our understanding in the spatial structure of the FtsZ. It plays an important role in the discovery of more excellent cinnamamides as well as the other chemical types of FtsZ-targeted antibacterial agents, and lays a solid foundation for the further study in the structure and function of FtsZ in the future.