4(1H)-Quinolone derivatives as new antibacterial drug leads

This proposal is a follow-up to the on going FWF funded project (P21152-B18) "Evaluation of 4(1H)-quinolone derivatives as a new antimycobacterial drug leads`. Several new 4(1H)-quinolones having C7 -C20 alkyls, alkenyls, alkynyls at position 2 with different positions of unsaturation have been synthesized and tested against various strains of mycobacteria. Against the rapidly growing strains, some of the compounds showed four fold more potency compared to our hit compound evocarpin, isolated from the fruit of the Chinese medicinal plant Euodia rutaecarpa and two fold more potency than isoniazid. The compounds also displayed pronounced growth inhibition of M. tuberculosis H37Rv and M. bovis BCG. Very potent antibacterial potential of the synthetic derivatives was also recorded against EMRSA-15 and 16 compared to norfloxacin, but the quinolones failed to inhibit the growth of Gram-negative bacteria such as E. coli and P. aeruginosa. In the human diploid embryonic lung cell line, the alkynyls bearing quinolone derivatives displayed significantly less toxicity compared to their alkenyls containing analogous. In our effort to elucidate the mechanism of antitubercular activity, the most active compounds were tested against ATP dependent M. tuberculosis MurE ligase and preliminary results revealed that N-methyl-2- alkenyl-4(1H)-quinolones as potential MurE inhibitors in a micromolar range. The ease of synthesis of the quinolone derivatives together with improved selectivity to the mycobacteria makes N-methyl-2-alkynyl-4(1H)- quinolones attractive leads for further developments. In addition, these compounds will serve as important anti- MRSA drug leads because of their pronounced activities against EMRSA-15 and 16. In line with the outcomes of our on going project the second phase of the study is proposed with the objective of further optimization of the lead compounds targeting M. tuberculosis MurE ligase and EMRSA-15 and 16. In this study we wish to introduce groups such as piperazinyl, pyrollidinyl and amine groups and their derivatives in the unsaturated aliphatic side chain at position 2 and the aromatic ring system in positions 5 to 8, which could possibly interact with the amino acid residues of MurE substrate binding site. In addition, uracil and its derivatives will be introduced to the 4(1H)- quinolone nucleus as the result of our preliminary studies revealed the uracil recognition site of MurE as the probable interaction site of the enzyme. The compounds to be synthesized will be tested for their antibacterial, cytotoxic and M. tuberculosis MurE ligase inhibitory activities. This project will be carried out by a consortium of pharmacognosits, pharmaceutical chemists and microbiologists.

Although 4(1H)-quinolones have been used since the mid-1990s for the treatment of a broad range of clinical infection, we carried out further investigation on this group of commercially available antibiotics for the following two reasons. Firstly, our 4(1H)-quinolone derivatives, which displayed promising in vitro antibacterial potential, possess aliphatic groups at position 1 and 2 and no substituent at position 3, whereas all the antibiotic 4(1H)-quinolones, including fluoroquinolones possess a carboxyl group at position 3 and no substituents at position 2. Second, fluoroquinolones have enjoyed great success against both Gram-positive and Gram-negative bacteria and certain anaerobes, but their widespread overuse and misuse in agronomy and veterinary medicine led to the emergency of extensively drug resistant bacteria. Thus, new drugs with new modes of action are required to combat this crisis. HalO 54 Hal 6 4a 3 R = alkyl, alkenyl, bromoalkyl, cycloalkyl 72R` = alkenyl, alkynyl, phenylalkyl, phenylalkoxyl, bromoalkyl, 3`-aminealkyl Hal 8 8a N1R` Hal = Cl, Br, I HalR Figure 1. 4(1H)-quinolone In this regard, we have synthesized a series of compounds (Fig. 1) and evaluated their antibacterial, efflux inhibiting and cytotoxic properties in vitro. Results of our studies revealed that against the rapidly growing mycobacteria strains Mycobacterium fortuitum, M. smegmatis and M. aurum compounds having alkynyl groups at position 2 showed potent inhibitory effects compared to the antibiotics ethambutol and isoniazid. Efflux of antibiotics by transport proteins (efflux pumps) is part of the resistance strategy of bacterial cells. Selected compounds synthetized in course of this project were able at micromolar concentrations to inhibit efflux of ethidium bromide (a model substrate of efflux pumps) in M. aurum. These results pave the way to explore the combination of 4(1H)- quinolone derivatives with antibiotics in order to circumvent resistance development. Another important finding of this research project is that our 4(1H)-quinolones prevented the conjugal transfer of selected plasmids from the donor to acceptor serving as a plasmid-curing agent. Since conjugated plasmid transfer is responsible for most of the resistance found in human pathogens, curing of plasmid-mediated drug resistance in pathogenic strains of bacteria is of great practical significance to control the spread of antibiotic resistance. The potent conjugal plasmid transfer inhibitory effect of the 4(1H)-quinolone derivatives highlights a new avenue of our research in the fight against antibiotic resistance. It is also worth mentioning that our compounds displayed antimalarial activity against Plasmodium falciparum and antitrypanosomal activity against Trypanosoma brucei rhodesiense in vitro at low micromolar concentrations. T. b. rhodesiense is known to cause sleeping sickness.