Novel Coumarins:Design, Synthesis And Their Relative Researches As Antimicrobial Agents And Artificial Probes For DNA

Coumarin compounds are an important type of aromatous oxygen-containing heterocycles with a fused structure of benzene ring and α-pyrone, and virtually contain π-π conjugated system with rich electron, strong charge-transport properties as well as good thermodynamic and photochemical stability. This kind of rigid and fused ring makes their derivatives easy be modified by a diversity of substituents, thus coumarins display wide potential applications in the fields of bromatology, perfume, material, pesticides, supramolecular chemistry and medicinal chemistry. Specially, coumarin compounds as medicinal agents have been attracting special interest due to their outstanding contributions in the prevention and treatment of diseases, and the related researches and developments have become an extremely attractive highlight. Some coumarin-based drugs such as warfarin, dicoumarol, acenocoumarol, ethyl biscoumacetate, armillarisin and carbocromen have been extensively used in clinic. In antiinfective field, coumarin-type of antibiotics like coumermycin A1, novobiocin and chlorobiocin have been in active investigations. They can not only inhibit the activity of ATPase, but also target topoismerase II or DNA gyrase to interfere the superhelix, reproduction and transcription of DNA as well as the isolation of chromosome, which further inhibit the growth of bacteria. In recent years, a lot of synthetic coumarin compounds have been reported to be with good and broad spectrum of antimicrobial efficiency, especially against MRSA. These exciting results attract a number of researchers to be engaged in the development of coumarin-based antimicrobials. Besides, coumarins are actively studied to be as probes. Much literature covered coumarin-derived biomolecule probes, which conduce to monitor timely enzyme activity, complex biological events as well as accurate pharmacological and pharmacokinetic properties in the biologically detective techniques. In view of these observations, based on the current situation in the researches of coumarin compounds as antimicrobials and probes for biologically important species and combining with the previous researches of antibacterial and antifungal coumarins in our group, this thesis designed and synthesized a series of novel coumarin compounds and explored the preparative methods and reaction conditions. These new compounds were evaluated for their in vitro antimicrobial activity, and the structure-activity relationships and antimicrobial action mechanisms were also discussed and presented. Partition coefficients and aqueous solubility of some target compounds were evaluated to predict their pharmacokinetic behaviors. Drug combination study, bactericidal kinetic assay, bacterial resistance study, molecular modeling experiments and quantum chemical studies as well as bacterial membrane permeabilization and the interaction between some target molecules and DNA were also investigated. The in vitro preliminary evaluation of coumarin-derived triazolyl ethanol as DNA probe was also studied. The main work was summarized as follows:(1) Preparation of novel coumarin-quinolone hybrids: The construction of coumarin rings II-3a-b, II-7 and II-8 was based on commercially available phenols. Aminocoumarins II-3a-b were treated with chloroacetyl chloride to give key intermediates II-4a-b, while hydroxyl coumarins II-7 and II-8 were reacted with alkyl dibromides to afford II-9a-c and II-10a-c in moderate to good yields. The intermediates were further reacted with quinolones in ethanol at reflux using NaHCO3 as base to yield the target coumarin-quinolone hybrids II-5a-f, II-11a-i and II-12a-i;(2) Preparation of novel coumarin-derived azolyl ethanols: Commercially available phenols were used as starting materials to construct coumarin rings III-2 and III-11, and then compounds III-2 and III-11 were further treated respectively with 2-(chloromethyl)oxirane to conveniently and efficiently afford epoxides III-3 and III-12. The desired target compounds III-4a-e, III-5, III-6a-b, III-7a-b, III-8, III-9, III-13a-e, III-14, III-15a-b, III-16a-b and III-17 were synthesized via the ring-opening of intermediates III-3 and III-12 by various azoles in ethanol using potassium carbonate as base;(3) Preparation of novel coumarin-derived chalcones: Compound IV-2 was constructed by using commercially available resorcinol as starting material. Compound IV-3 was obtained through multi-steps, and then further alkylation by bromoalkanes to give compounds IV-4a-c. The desired target coumarin-derived chalcones IV-8a-d and IV-9a-l were thus afforded by intermediates IV-3 and IV-4a-c treating with compounds IV-7a-d at 130 oC in toluene with glacial acetic acid and piperidine as catalyst;(4) Preparation of novel coumarin-derived benzimidazoles: Commercially available phenols were used as starting materials to construct coumarin rings V-2a-b and V-15. Compounds V-2a-b and V-15 were further acted through multi-steps to give V-3a-b and V-16. Target compounds V-4a-b, V-11a-b and V-17a-b were obtained by treating compounds V-3a-b and V-16 with o-phenylene diamines in DMF at 80 oC. Compounds V-4a, V-11 a and V-17 a were further alkylation in DMF with potassium carbonate as base to afford bis-substituted coumarin-benzimidazoles V-5a-b, V-12a-d and V-18a-b in DMF at 80 oC;Intermediate V-6 was synthesized via alkylation of V-3a by ethyl bromide. The desired mono-substituted coumarin-benzimidazoles V-7a"b, V-10a-j, V-13a-j and V-19a-d were conveniently and efficiently afforded by treating compounds V-3a-b, V-6 and V-16 with o-phenylene diamines in DMF at 80 oC;(5) All the newly synthesized compounds were characterized by IR, 1H NMR, 13 C NMR, MS/HRMS spectra.(6) The preparative condition(solvent, base and temperature) of coumarin-derived azole ethanol III was explored. The study results found that when ethanol was selected as solvent, potassium carbonate was used as base and the reaction temperature was 70 oC, the yield of target compound III was the highest.(7) The in vitro antibacterial and antifungal assays indicated that most target compounds in the series II could significantly inhibit the growth of all the tested bacteria and fungi. Especially, hybrids with quinolones linked 4-coumarins II-11a"i, their activities were generally good. The most highly active compound was II-12 g, which displayed superior antimicrobial activity to the current clinical drugs chlormycetin, norfloxacin and fluconazole, and equipotent efficiency to ciprofloxacin and clinafloxacin;(8) The drug combination study of compounds II-5b, II-12 d and II-12 g with standard drugs chlormycetin, norfloxacin, ciprofloxacin, clinafloxacin and fluconazole respectively was carried out. The results indicated that the combination effect of compound II-5b against Gram-positive bacteria was better than that of Gram-negative bacteria, and the best effect was obtained by combining with ciprofloxacin. When compound II-12 d was combind with norfloxacin, S. aureus was the most sensitive bacterium with fractional inhibitory concentration index(FICI) value of 0.062. The combination of compound II-12 g with chlormycetin exhibited synergism against all the tested bacterial strains except for S. aureus and M. luteus. More importantly, its combination effect towards MRSA was superior to II-5b and II-12 d, which showed the potentiality of compound II-12 g to overcome drug-resistance in drug combination therapy. Besides, compounds II-5b, II-12 d and II-12 g possessed relatively rapid killing effects against S. aureus and E. coli, and almost triggered no resistance to S. aureus and MRSA even after 12 passages. Molecular docking studies revealed that these three compounds could target GyrB, which might be a reason for their good antibacterial activity and low resistance;(9) The preliminary interactive investigations of compound II-12 g with calf thymus DNA by fluorescence and UV-vis spectroscopic methods revealed that compound II-12 g could effectively intercalate into DNA to form II-12g-DNA complex, which might block DNA replication and thus exerted its antimicrobial activity;(10) The in vitro antibacterial and antifungal assays for series III indicated that some prepared compounds with appropriate logPow values showed effective antibacterial and antifungal activities in comparison with the reference drugs chlormycetin and norfloxacin. Noticeably, compound III-14 with bis-triazolyl ethanol group exhibited low MIC value of 8 μg/mL against MRSA, which was comparable or even superior to reference drugs norfloxacin(MIC = 8 #g/mL) and chlormycetin(MIC = 16 #g/mL). It could also effectively inhibit the growth of the tested fungal strains compared to fluconazole. Further binding studies of coumarin III-14 with calf thymus DNA were investigated by UV-vis absorption and fluorescence spectroscopy. It was found that compound III-14 could interact with calf thymus DNA by groove binding to form III-14"DNA complex via both hydrogen bonds and van der Waals force, which might be the factor to exert the powerful antimicrobial activity;(11) The in vitro antibacterial and antifungal assays for series IV indicated that most coumarin-derived chalcones exhibited effective antibacterial and antifungal activities in comparison with the reference drugs chlormycetin and norfloxacin. The most active compound was IV-9h, which displayed strong antibacterial activities with MICs between 1 and 8 μg/mL against all the tested strains compared with chlormycetin(MIC = 8-32 μg/mL) and norfloxacin(MIC = 1-16 μg/mL). Moreover, except for C. albicans, compound IV-9h was more active towards the tested fungi contrast with fluconazole, especially for A. flavus, the inhibitory effect was 512-fold stronger than fluconazole. Compound IV-9h not only possessed relatively rapid killing effects against M. luteus and S. dysenteriae, but also triggered almost no resistance to S. aureus and MRSA even after 15 passages;(12) The in vitro antimicrobial data exhibited that the effect of alkyl groups on activity was more obvious than that of the substituents on benzene ring, which was based on the observation that non-alkyl and ethyl group modified compounds IV-8a-d and IV-9a-d were less active than butyl and hexyl moieties substituted coumarin-chalcones IV-9e-l. Therefore, compounds IV-8d, IV-9d, IV-9h and IV-9l were selected to further investigate the structure-activity relationship. Their different ability to bind with ct DNA demonstrated the influence order of alkyl group on antimicrobial activity was butyl > hexyl > ethyl. This result further clarified the reason why compound IV-9h was the most active one;(11) The in vitro antibacterial and antifungal assays for series V indicated that some coumarin-derived benzimidazoles exhibited effective antibacterial and antifungal activities in comparison with the reference drugs chlormycetin and norfloxacin. The most active and broad-spectrum compound was V-19 d. Especially against M. luteus and MRSA, the MIC value was 0.5 μg/mL, which was superior to chlormycetin(MIC = 8/32 μg/mL) and norfloxacin(MIC = 2/16 μg/mL). Furthermore, compound V-19 d showed aqueous solubility value of 26.65 mg/L, and triggered almost no resistance to S. aureus and MRSA even after 15 passages, demonstrating its potentiality to be further studied. Structure-activity relationship showed that the antimicrobial activity of 3-coumarin-benzimidazoles was stronger than that of 8-coumarin-benzimidazoles. This observation was further confirmed by the data analysis of quantum chemical studies of compounds V-10 g, V-13 g and V-19d;(14) The preliminary antimicrobial action mechanism of compound V-19 d was explored. The results indicated that V-19 d could effectively permeate the cell membrane of MRSA and E. coli. The IC50(the concentration of the compounds that inhibits 50% biofilm development) values of compound V-19 d against the biofilm formation of MRSA and E. coli were found to be 0.5 and 1 μg/mL, respectively. The EC50(the concentration of compound that reduces 50% bacterial titer of a preformed biofilm) values of coumarin-benzimidazole V-19 d were 3.2 μg/mL to MRSA and 6.25 μg/mL to E. coli. These results indicated the great potentiality of compound V-19 d as novel membrane active antimicrobial agent against drug resistant bacterial strains;(15) Coumarin-derived triazolyl ethanol VI-7 was successful synthesized and confirmed by IR, 1H NMR, 13 C NMR and HRMS spectra. Fluorescence and UV-vis spectroscopic methods were employed to study its potentiality as DNA probe. The results displayed that compound VI-7 preliminarily possessed the in vitro ability to be developed as DNA probe.This thesis successfully synthesized one hundred and forty one compounds totally, among them one hundred and two compounds are new, including twenty four coumarin-quinolone hybrids, twenty three coumarin-derived azolyl ethanols, sixteen coumarin-derived chalcones and thirty nine coumarin-derived benzimidazoles.