Design, Synthesis And Reversal Of Multdrug Resistance Evaluation Of 4-Substituted-2-Naphthoamide Derivatives Targeting AcrB

In recent years, due to the widespread use and abuse of antibiotics, bacterial resistance is a growing problem. Wherein multi-drug resistant Gram-negative bacterial infections has become an important factor in threating to human health seriously. Therefore, research and development of antibacterial drugs with novel mechanisms of action to combat drug-resistant infections is imminent. Overexpression of AcrB efflux pumps in multidrug-resistant Escherichia coli is the main reasons for Gram-negative bacteria resistances and pathogenicities. So the research on the target of AcrB has aroused people’s wide attention.Bacterial AcrB efflux pump inhibitor combined with antimicrobial agents could be an effective way to improve or restore the effectiveness of antimicrobial agents. According to the pre-screening and activity determination of the compounds from the chemical library, compounds 4-isopentyloxy-2-naphthalene carboxamide (WYA3) and 4-benzyl-2-naphthalene carboxamide (WYA9) have been proved to be the hit compounds that could inhibit the overexpression of the multidrug efflux pump AcrB of K coli to increase the bacteria sensitivity to antibiotics. But its antibacterial sensitizing potency may need further improvement before clinical application. Therefore,25 target compounds were designed and synthesized with the 4-substituted-2-naphthalene amide as the structure modification core, retaining the isopentyloxy and benzyloxy side chain, by introducing different substituents in its 2-linkers. The structure identification of all the target compounds have been conducted by MS and 1HNMR, and the interaction between the active compounds G3E and the AcrB target proteins have been analyzed through molecular docking.The hit compound 4-isopentyloxy-2-naphthalene carboxamide (WYA3) was as the positive control of compound in this thesis. Inherent antibacterial activity, synergistic antibacterial activity, Nile Red efflux inhibitiory activity and stability of inside and outside cell membrane of all the target compounds have been conducted, the related activity were summarized as follows:All the target compounds exhibited weak inherent antimicrobial activity with MIC values of 512 μg/mL against E. coli BW25113 (wild-type, AcrB overexpression) and E. coli BW25113 (ΔAcrB, AcrB deletion), thereby excluding the self influence on synergistic antibacterial activity when combined with the antibiotics. For known antibiotics, most of the target compounds exhibited stronger synergistic antimicrobial activity than the positive compound. Thereinto, compound G2C was best one with synergistic activity in the concentration of 256 μg/mL, which showed twice synergistic antibacterial efficacy to three antibiotics than the positive compound, G3B showed the best synergistic activity in the concentration of 128μg/mL to Erythromycin,4-fold better than the positive compound. Further Nile Red efflux inhibitory activity presented that, some of the target compounds showed a certain degree of Nile Red efflux inhibitory activity in different concentrations. Thereinto, compounds G2B, G2E and G3C presented effective Nile Red efflux inhibitory activity equal to the positive compound at a concentration of 200μM, compounds G3D, G3G and G3K showed weaker inhibitory activity than the positive control at a higher concentration of 500μM. The results of related activity of inner and outer membranes of bacterial cells against the compounds with synergistic antimicrobial activity and Nile efflux inhibitory activity showed that, most of the target compounds did not cause outer membrane permeability, further test screened that the compounds G2I, G3C, G3E and G3G did not cause inner membrane permeability, which meeting all the conditions of effective efflux pump inhibitors.Through the analysis of related activity results for G2 and G3 series, the structure-activity relationships were concluded as followed:In general, the overall activities of G3 series with 4-isopentyl-2-naphthalene carboxamide skeleton were better than that of G2 series with 4-benzyl-2-naphthalene carboxamide skeleton, which initially confirmed the 4-isopentyloxy group is the pharmacophore with reversal of multidrug resistant activity. By introducing aromatic ring, heterocyclic ring and various substituted benzyl side chain with large steric bulk in the 2-naphthyl carboxamide group, the compounds could enhance their synergistic antibacterial activity and Nile Red efflux inhibitory activity to some extent, which further corroborated the introduction of the side chain with large steric bulk in the 2-amide linkers could contribution to close binding for AcrB protein phenylalanine hydrophobic pocket; Subsequent effect on outer membrane and inner membrane screened compounds G2I, G3C, G3E and G3G with 4-methoxy-benzyl, cyclohexane and O-trifluoromethylphenyl side chains, which possessed desired efflux pump inhibitors conditions, indicating that the introduction of different substituted saturated or unsaturated six-membered ring particularly those with hydrophilic substituents binding for AcrB amino acid residues to form hydrogen bonds, could play a positive impact on the stability of the inner and outer membranes of the bacteria.