Design And Synthesis Of Novel Quinolone Azoles And Their Antibacterial Study

Quinolones are an important and classical kind of synthetic antibacterial agents. The cellular targets for this type of antibacterial drugs are the bacterial gyrase and topoisomerase Ⅳ. The related structural modifications based on 3-carboxyl benzopyridone skeleton have become an extremely attractive area as they are generally well tolerated with good treatment effectiveness, broad antibacterial spectrum and favorable pharmacokinetic characteristics. Since the first quinolone drug was discovered in 1960s, four generations of quinolones have been successfully developed in succession and a large number of quinolone drugs have been successfully and widely used in clinic to treat genitourinary infections and common respiratory tract pathogens. Meanwhile, azole heterocyclic compounds, such as triazole, imidazole, thiazole and so on, have been paid special attention due to their potential applications as medicinal agents. These types of unique structures endow them to readily bind with a variety of enzymes and receptors in biological system via weak interactions, and thus display a broad spectrum of biological activities. A large number of researches have been focusing on azole compounds, the progress have been rapidly developing, and become one of highly active topics in recent years. Combining with authors’researches and referring to other works from literatures on quinolone in recent years, a series of novel quinolone azoles were designed and synthesized. These novel compounds were evaluated for their antimicrobial activity, and structure-activity relationships were also discussed and summarized. Cytotoxicity, ionization constants (pKa values), aqueous solubility and partition coefficients of some highly active target compounds were evaluated to predict their pharmacokinetics behaviors. Their interaction between highly active target molecules and human serum albumin and the preliminary antibacterial mechanism were also discussed. The main work was summarized as follows:(1) Preparation of triazolyl ethanol quinolones:Commercially available diethyl malonate was reacted with triethoxymethane in the presence of anhydrous zinc chloride to produce diethyl Ⅱ-1 in an almost quantitative yield of 97%, and then compound Ⅱ-1 was further treated with a series of substituted phenylamines in ethanol to afford diethyl 2-((phenylamino)methylene)malonate derivatives Ⅱ-2a-h, which were cyclized in oxydibenzene under reflux to give the desired quinolones Ⅱ-a-h. The latter was further N-alkylated by commercial 2-(chloromethyl)oxirane to yield quinolones Ⅱ-4a-h, subsequently the epoxy ring was opened by 1,2,4-triazole in ethanol using sodium bicarbonate as base to produce racemates Ⅱ-5a-h, and then were further hydrolyzed in water by 3% sodium hydroxide at 100℃ to afford the corresponding triazolyl ethanol quinolones II-6a-h.(2) Preparation of novel hybrids of metronidazole and quinolones:Commercial ethoxymethylene malonic ester was reacted with a series of substituted phenylamines in ethanol to afford intermediates Ⅲ-1a-h. The obtained compounds were then cyclized in phenoxybenzene under reflux to produce the desired quinolones 2a-h in good yields, which were further N-alkylated by racemic 2-(chloromethyl)oxirane to yield quinolone derivatives Ⅲ-3a-h. The epoxy rings of compounds Ⅲ-3a-h were opened by 4-nitroimidazole and 2-methyl-5-nitroimidazole respectively in ethanol using sodium bicarbonate as base to produce the corresponding racemates Ⅲ-4a-h and Ⅲ-5a-h. and then the later were further hydrolyzed by 3% sodium hydroxide to afford the target quinolone-metronidazole hybrids Ⅲ-6a-h and Ⅲ-7a-h.(3) Preparation of novel antibacterial 3-aminothiazolquinolones:Compound Ⅳ-1 was easily prepared by the reaction of commercial triethoxymethane, ethyl 3-oxobutanoate and propionic anhydride. The synthesized intermediate 1 was reacted with a series of substituted phenylamines in the absence of solvent to afford Ⅳ-2a-i in almost quantitative yields, and then were further cyclized in phenoxybenzene under reflux to produce the quinolones Ⅳ-3a-i. Compounds Ⅳ-3a-i were further N-alkylated by commercial bromoethane to yield quinolone derivatives Ⅳ-4a-i and further brominated by bromine in acetic acid to produce corresponding 3-(2-bromoacetyl) quinolones Ⅳ-5a-i. The cyclization of bromoacetyl groups at 3-positions of compounds Ⅳ-5a-i with thiourea in ethanol readily gave the target quinolone thiazoles Ⅳ-6a-i.Substitution of compound Ⅳ-4e with alicyclic amines in DMSO using triethylamine as base respectively afforded quinolones Ⅳ-7a-d and Ⅳ-10a-d, and then compounds Ⅳ-7a-d and Ⅳ-10a-d were reacted respectively with bromine in acetic acid to produce corresponding 3-(2-bromoacetyl) quinolones Ⅳ-8a-d and Ⅳ-11a-d, and then further cyclizations with thiourea in ethanol produced target aminothiazolquinolones Ⅳ-9a-d and Ⅳ-12a-d. The reaction of compounds Ⅳ-9d and Ⅳ-12d in the solution of trifluoroacetic acid-CH2Cl2 reduced Boc groups to produce target compounds Ⅳ-9e and Ⅳ-12e respectively.(4) The newly synthesized compounds were characterized by 1H NMR,13C NMR, IR, MS and HRMS spectra.(5) The in vitro antibacterial and antifungal assays indicated that most intermediates and target compounds in the series Ⅱ could significantly inhibit growth of all the tested microorganisms. Especially, 1-(oxiran-2-ylmethyl) quinolones Ⅱ-4a-i displayed equipotent or superior antifungal activities in comparison with the current clinical drugs. Notably, target compound Ⅱ-6b gave better inhibitory behaviors against the tested bacterial and fungal strains than reference drug norfloxacin and fluconazole.(6) The preliminary interactive investigations of compound Ⅱ-6b with calf thymus DNA by fluorescence and UV-vis spectroscopic methods revealed that compound Ⅱ-6b could effectively intercalate DNA to form compound Ⅱ-6b-DNA complex which might block DNA replication and thus exert its antimicrobial activities.(7) The in vitro antibacterial assays indicated that most of the prepared compounds in the series Ⅲ exhibited good or even stronger antimicrobial activities in comparison with reference drug norfloxacin. Notably, the hybrid of metronidazole and quinolone Ⅲ-7d showed significant inhibition against all the tested bacterial strains with low inhibitory concentrations (MIC=0.5-8 μg/mL). Furthermore, these highly active metronidazole-quinolone hybrids showed appropriate ranges of pKa, log P and aqueous solubility to pharmacokinetic behaviors and no obvious toxicity to A549 and human hepatocyte LO2 cells.(8) Their competitive interactions with metal ions to HSA revealed that the participation of Mg2+ ion in compound Ⅲ-7d-HSA association could result in a concentration increase of free compound Ⅲ-7d. Molecular modeling and experimental investigation of compound Ⅲ-7d with DNA suggested that possible antibacterial mechanism might be in relation with multiple binding sites between bioactive molecules and topo Ⅳ-DNA complex.(9) The in vitro antibacterial assays showed that most of target compounds and some intermediates in the series IV exhibited good antimicrobial activities in comparison with reference drug norfloxacin. Especially, the target compounds exhibited good anti-gram-positive bacterial activity including multi-drug resistant MRSA. Structure-activity relationship disclosed that the 2-aminothiazole fragment at 3-position of quinolone played an important role in exerting antibacterial activity, and that the substituents in benzene ring of quinolones could influence antibacterial spectrum.(10) Cell toxicity study suggested that the 3-aminothiazolquinolone Ⅳ-12b did not show cytotoxicity to A459 and human cell LO2 lines, which was better than reference drug norfloxacin. Notably, the MRSA strain was very sensitive to 3-aminothiazolquinolone Ⅳ-12b (MIC= 2.2 nM). Further research suggested that compound Ⅳ-12b could induce MRSA resistance more slowly in comparison with Norfloxacin. After 12 passages, the MIC values against MRSA just changed in the range between 1.3 nM and 3.1 nM.3-Aminothiazolylquinolone Ⅳ-12b also exhibited strong inhibitory activity to DNA gyrase in comparison with Norfloxacin.(11) Molecular modeling showed that the hydrogen bonds at resistance mutation region and the interaction in negatively supercoiled region might be the important reasons that compound Ⅳ-12b gave strong inhibitory efficacy against strains of quinolone-resistant bacteria. The preliminary interactive investigations of compound Ⅳ-12b with MRSA DNA revealed that the binding mode of compound Ⅳ-12b to MRSA DNA was the formation of ternary complex mediated by Cu2+ ions, in which copper ion acted as a bridge between the phosphate groups of nucleic acid and carbonyl and aminothiazole moieties of 3-aminothiazolquinolone Ⅳ-12b. The optimal concentration of Cu2+ ion for ternary complex formation was in the range of intracellular Cu2+ ion concentrations in bacteria, so that this binding mode might be biologically relevant. In addition, the preference exhibited by 3-aminothiazolquinolone Ⅳ-12b for single-stranded DNA indicated that additional stabilization might arise from stacking interactions between the backbone of 3-aminothiazolquinolone Ⅳ-12b and DNA bases.One hundred and fifty three compounds are successfully synthesized in this thesis, among them one hundred and eighteen compounds are new, including thirty six triazolyl ethanol quinolone, forty hybrids of metronidazole and quinolones, forty eight 3-(2-aminothiazol-4-yl) quinolones...