Design And Synthesis Of Novel Benzimidazoles And Evaluation For Their Antimicrobial Activities

Benzimidazole is an important aromatic fused heterocycle containing benzene ring and imidazole, and it has larger conjugated system and stronger electron transport abilities than those of benzene or imidazole, which leads its derivatives possessing extensive medicinal applications. Particularly, benzimidazoles could readily bind with a variety of biological active sites in biological system through various weak interactions to show a diverse range of biological activities including antiparasitic, antibacterial, antifungal, anticancer, analgesic, anti-inflammatory, antioxidant antidiabetic, antihypertensive ones etc. Recently, the increasing incidence of multidrug-resistant strains, intractable pathogenic microorganisms and newly emerging pathogens is a global human threat. Therefore, better understanding the mechanisms of currently available antibiotics and exploring alternative antibacterial drugs have emerged as an important strategy in the development of new antimicrobial therapies. Benzimidazole is structurally similar to purine, and its derivatives could compete with purines, distinctly inhibiting the synthesis of nucleic acids and proteins, thereby killing bacterial strains or inhibiting their growth. Therefore, the development of benzimidazoles as antibacterial agents has become one of the highly active highlights. Based on the current investigation in benzimidazole compounds, a series of novel benzimidazole derivatives as potential antimicrobial agents were designed and synthesized. All the newly synthesized compounds were evaluated for their antibacterial and antifungal activities. The preparative methods, preliminary structure-activity relationships and conditions were discussed. Further binding behaviors between the prepared active compound and HSA were investigated by UV-vis absorption and fluorescence spectroscopy to evaluate their transportation and pharmacokinetic properties. The preliminary antimicrobial mechanism was also studied. The main work was summarized as follows:(1) Preparation of novel 5-fluorouracil(5-FU) benzimidazoles: Commercially available 5-FU was reacted with chloroacetic acid in presence of aqueous potassium hydroxide to afford analogue II–2. Conjugates II–3a–c were efficiently prepared by cyclization of compound II–2 and o-phenylenediamine or its derivatives. The N-alkylation of intermediate II–3a with a series of alkyl bromides in acetonitrile at 70 °C with potassium carbonate as base respectively afforded target alkyl benzimidazole compounds II–4a–h with yields ranging from 61% to 77%, suggesting that the length of aliphatic chain in alkyl bromides played a slight role in forming target compounds. Halobenzyl halides and intermediate II–3a underwent N-alkylation to produce compounds II–5a–l with a large difference in yields of 47-78%.Mono-substituted 5-FU derivatives II–8a–d and II–9a–g were directly prepared from compound II–2. The reaction of o-phenylenediamine or its derivatives with alkyl or halobenzyl halides efficiently provided compounds II–6a–d and II–7a–g in the presence of potassium carbonate and DMF, and the resulting products were subsequently reacted with acid II–2 in polyphosphate(PPA) at 180 °C to produce target hybrids II–8 and II–9.(2) Preparation of novel sulfonyl benzimidazoles: Commercial acetaniline was reacted with chlorosulfonic acid to produce the intermediate N-protected sulfonyl chloride III–2, and then further treated with sodium sulfite and sodium bicarbonate in water at 80 oC to give sodium 4-acetamidobenzenesulfinate III–3 in an excellent yield. Stirring a mixture of sodium compound III–3 and tetrabutylammonium iodide in 2-(chloromethyl)oxirane at 80 oC afforded compound III–4 and III–5. o-Phenylene diamines were reacted with the carboxylic acids to produce the intermediate benzimidazoles, which were able to open the compound III–4 to afford target compounds III–6a–g. Intriguingly, 1H-benzimidazole-2-thiol also could break C–O bond to give III–7.(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 manifested that some prepared compounds exhibited good or even superior antibacterial and antifungal activities against the tested strains to the reference drugs chloromycin, norfloxacin and fluconazole. Hybrids II–4a, II–8a and II–8b showed significant inhibition against all tested strains with low inhibitory concentrations in range of 2–64 μg/m L. Especially, benzimidazole derivative II–5c was efficacious in inhibiting MRSA(MIC = 2 μg/m L) and B. proteus(MIC = 4 μg/m L). Towards fungi S. cerevisiae, it gave the highest antifungal activity(MIC = 1 μg/mL) among these prepared conjugates, which was 16-fold higher than fluconazole. Moreover, compound III-6e with ethyl group gave the best anti-S. aureus activity with MIC value of 1 μg/m L, which was 16-fold and 2-fold less than control drug chloromycin and norfloxacin, respectively. Compound III-6c with methyl gave the best antifungal efficiencies with MIC values of 1–16 μg/mL towards the corresponding fungi strains, especially for C. albicans. and S. cerevisia.(5) The preliminary structure-activity relationships showed that the length of alkyl chain had significant influence on biological activities. In addition, not only the type and position of substitutions at benzene ring but also the number of halobenzyl group exerted important effect. The introduction of benzimidazole fragment was conductive to the biological activities.(6) Experimental research revealed that compound II–5c could effectively intercalate into calf thymus DNA to form compound II–5c–DNA complex which might block DNA replication and thus exert antimicrobial activities. The specific interactions of compound III-6c with DNA were studied by UV-vis absorption spectroscopy. Experimental results displayed that compound III-6c could intercalate DNA to form compound III-6c–DNA complex which might further block DNA replication to exert their powerful antibacterial and antifungal activities. The binding behavior of compound III-6c with human serum albumin(HSA) revealed that noncovalent interaction played important roles in the association of compound III-6c-HSA.(7) Molecular docking indicated that the fluorine atom of 5-FU in compound II–5c was in close vicinity to the residue Lys 423 of the Topo IA through hydrogen bonds with distance of 2.1 ?, which manifested the necessity of the fluorine for the increased bioactivity. Furthermore, molecule II–5c could also form two hydrogen bonds with Lys 423 through the oxygen atoms at carbonyl groups with distance of 2.5 ?, which might be the crucial reason that compound II–5c displayed strong inhibitory efficacy against test strains.Fifty nine compounds were successfully synthesized in this thesis. Forty two compounds were new, including twenty two intermediates, twelve alkyl compounds, eighteen halogen benzyl compounds, six benzimidaolyl ethanols and one benzimidazole-2-thiol.