Design And Synthesis Of Novel Azole Compounds And Evaluation For Antimicrobial Activities

Azole compounds are an important class of nitrogen heterocycles with electron-rich property. This special structure endows azole-based derivatives easily bind with the enzymes and receptors in organisms through noncovalent interactions such as hydrogen bonds, coordination bonds, ion-dipole, cation-?, ?-? stacking and hydrophobic effect as well as van der Waals force etc., thereby possessing various applications in medicinal chemistry. The design, synthesis and antimicrobial activity of azole derivatives have been extensively investigated and have become one of the highly active highlights in recent years, and the progress is quite rapid. In particular, a large number of thiazole and imidazole antimicrobial agents have been penetratingly studied as candidates and even some of them have been used in clinic, which have shown the great potential and development value. In this thesis, based on the current investigation and development in thiazole and imidazole derivatives, a series of novel thiazole and imidazole compounds were designed and synthesized. The structures of all the newly synthesized compounds were characterized by modern spectra. The preliminary structure-activity relationships, preparative methods and conditions were discussed. Further interaction of the synthesized active compound with DNA and human serum albumin(HSA) was investigated by fluorescence and UV-vis absorption spectroscopy to evaluate their transportation and pharmacokinetic properties. The main contents were summarized as follows:(1) Preparation of novel carbazole aminothiazoles: The commercially available carbazole II-1 was reacted with various alkyl bromides to give intermediate N-alkylcarbazole compounds II-2a-j, and then the later were further reacted with chloroacetyl chloride in dichloromethane at r.t. to produce compounds II-3a-j. The further treatment of N-alkylcarbazole II-3a-j with thiourea in refluxing absolute ethanol afforded the N-alkylcarbazole aminothiazole II-4a-j.(2) Preparation of novel 12-imidazole substituted berberrubines: The commercially available berberine hydrochloride III–1 was placed in the round bottom flask and in a vacuum(20 mm Hg), stirred at 190 oC for 30 min to give the crude product of berberrubine III-2. The berberrubine III-2 was further reacted with the appropriate imidazoles and formaldehyde in dry n-butanol at 110 °C to produce the 12-substituted azolylberberrubine derivatives III-3a-j.(3) All the newly synthesized compounds were characterized by 1H NMR, 13 C NMR, IR, MS and HRMS spectra.(4) The prepared intermediates and target compounds were evaluated for their in vitro antimicrobial activities. The biological assays indicated that some synthesized compounds could significantly inhibit the growth of tested microorganisms and some of them displayed equipotent or superior efficacies to the current clinical drugs. Notably, 1-amylcarbzaole aminothiazole compound II–4d showed excellent activity against P. aeruginosa with MIC value of 2 μg/mL, which was far better than Norfloxacin(MIC = 6 μg/mL) and Chloromycin(MIC = 32 μg/m L). Heptyl substituted carbzaole aminothiazole II–4f displayed the best anti-MRSA activities(MIC = 4 μg/mL), which was potent than Norfloxacin(MIC = 8 μg/m L) and Chloromycin(MIC = 16 μg/m L). Furthermore, some highly active carbazole aminothiazoles showed appropriate ranges of Clog P, log P and molar refractivity to pharmacokinetic behaviors and no obvious toxicity to HepG2 cells. In addition, the imidazole-based berberrubine derivative III–3a gave potent activities and broad antibacterial spectrum against the tested bacterial strains with MIC values ranging from 1 to 64 μg/mL, especially against B. typhi and B. subtilis with inhibitory concentrations of 1 and 8 μg/m L, which were far potent than Norfloxacin and Chloromycin.(5) The preliminary structure-activity relationship showed that alkyl chain lengths had great effects on biological activities of N-alkylcarbazole aminothiazole derivatives. In addition, azolyl moiety and its bulk substituent exerted an important effect on the antimicrobial activity of azole-based berberrubine.(6) Combination of carbazole aminothiazole II–4d and II–4f respectively with antibacterial Chloromycin, Norfloxacin or antifungal Fluconazole exhibited better antimicrobial efficiency than the separated use of them alone. These results demonstrated that the majority of combinations could highly enhance antimicrobial activity, overcome drug resistance and broaden spectra, which might be attributed to the different binding sites of these compounds towards the tested microorganism.(7) The preliminary interactive investigations of compound II–4f with calf thymus DNA by fluorescence and UV-vis spectroscopic methods revealed that compound II–4f could effectively intercalate into DNA to form compound II–4f–DNA complex which might block DNA replication and thus exert its antimicrobial activities.(8) The highly bioactive compound III–3a was further examined for cytotoxic properties on HepG2 lines using the colorimetric cell proliferation MTT assay. Berberine was selected as a positive control. Cytotoxic results showed that the cell viability of compound III–3a against both HepG2 cells was at least more than 87% within concentration of 100 μg/m L, which was better than clinic drug Berberine.(9) By applying fluorescence microscopy, it was demonstrated that the target compound III–3a with potent antimicrobial activity showed lower ROS generation to Berberine. Therefore, the introduction of imidazole ring into 12-position of berberrubine increases the biological activity and decreases the toxicity.(10) The siginificant interaction of target compound III–3a with calf thymus DNA displayed that compound III–3a could intercalate into DNA to form III–3a–DNA complex which might further block DNA replication to exert its powerful antibacterial and antifungal activities. The binding investigations with HSA showed that HSA could generate fluorescent quenching by III–3a as a result of the formation of ground-state III–3a–HSA complex, and the calculated parameters revealed that the binding process should be spontaneous hydrophobic contacts, specific electrostatic interactions and hydrogen bonds played a significant role in the formation of compound III–3a–HSA. Molecule docking results demonstrated that compound III–3a partly occupies subdomain IIA, resulting in fluorescence quenching of Trp-214. Hydrophobic interaction exists between the aromatic rings of compound III–3a and HSA. Though the interactions are dominated by hydrophobic contacts, specific electrostatic interactions and hydrogen bonds between imidazole ring of compound III–3a and Lys-195 in HSA are also involved in the binding process.