Heterocycles [h]-fused to 4-oxoquinoline-3-carboxylic acid. Part XI: Synthesis and antibacterial activity of 4-fluoro-6-oxoimidazo[4,5-h]quinoline-7-carboxylic acids

2.1 Chemistry

The synthesis of 2-arylimidazo[4,5-h]quinolones 3a–f is achieved in good yields via microwave-assisted cyclocondensation of 2 with the particular hetarene carboxaldehyde as outlined in Scheme 1. While adsorbed on silica gel in the presence of nitrobenzene as oxidizing agent, the two reactants were irradiated with a power of 350 W for 8–10 min at 140 °C. This versatile method, as applied in this study, has recently been reported for the synthesis of several 2-arylbenzimidazoles [12, 13]. Acid-catalyzed hydrolysis of the ethyl esters 3a–f furnished the corresponding 9-cyclopropyl-4-fluoro-6-oxo-6,9-dihydro-1H-imidazo[4,5-h]quinoline-7-carboxylic acids 4a–f in high yield and in pure form.

The structures of the new heterocycles 3a–f and 4a–f are supported by spectral (IR, MS, and NMR) data (details are given in Experimental Section). The IR spectra showed absorption bands arising from stretching vibrations at 1612–1638 cm^–1 (conjugated keto group), 1697–1722 cm^–1 (carbonyl of conjugated CO₂Et/CO₂H), and 3088–3233 cm^–1 (N–H). In addition, compounds 4a–f displayed strong absorption bands at 3395–3445 cm^–1 (O–H). The mass spectra of 3a–f and 4a–f displayed the correct molecular ion peaks as suggested by their molecular formulas and for which the high-resolution mass spectra (HRMS; measured by electrospray ionization [ESI]) data are in good agreement with the calculated values. ¹H and ¹³C signal assignments to the different protons and carbons are based on DEPT and 2D (COSY, HMQC, and HMBC) experiments, which showed correlations consistent with these assignments. Thus, carbon–hydrogen connectivities, as revealed by HMQC experiments, established precise signal assignments to the various carbons and their attached hydrogens. The skeletal carbons of the benzofused entity (ring B) in 3a–f and 4a–f are recognizable by their doublet signals (with varying J values) originating from scalar (through-bond) coupling with the neighboring fluorine atom at carbon-4. In HMBC experiments for 3a–f and 4a–f, distinct “three-bond” (¹H–¹³C) correlations are observed between 5-H and each of C-3a, C-9a, and C-6, and between 8-H and each of C-9a, C-1′, C-6, and CO₂H/CO₂Et.

2.2 Antimicrobial activity

The antimicrobial properties of 2-hetaryl-4-fluoro-9-cyclopropyl-6-oxo-1H-imidazo[4,5-h]quinoline-7-carboxylic acids 4a–f were evaluated in vitro against four bacterial strains and three fungi, together with those of 6-oxo-1H-imidazo[4,5-h]quinoline-7-carboxylic acid 1a, unsubstituted at position 2 of the imidazole ring and of its 2-aryl derivatives 1d–g. The results, reported in Table 1, show that 1a was found to possess the best antibacterial activity against both, Gram-positive and Gram-negative bacteria: minimum inhibitory concentration (MIC) values range from 0.15 to 3.0 μg mL^–1 for Bacillus subtilis and Staphylococcus aureus and from 0.7 to 3.0 μg mL^–1 for Haemophilus influenzae and Escherichia coli. Among arylimidazofluoroquinolones 1d–g, a remarkable inhibition of employed Gram-positive bacteria and H. influenzae was exhibited by the p-chlorophenyl derivative 1g (MICs 0.7–3.0 μg mL^–1) and by the p-fluorophenyl compound 1f (MICs 3–12 μg mL^–1), whereas unsubstituted 1d and methyl-substituted compound 1e were selective only against B. subtilis at concentrations of 6–12 μg mL^–1.

Of the hetarylfluoroimidazoquinolones 4a–f, furan derivatives 4e and 4f were found to have the highest antibacterial effectiveness against all tested bacteria. Their broad-spectrum activity follows the order B. subtilis (MIC 1.5 μg mL^–1) > H. influenzae (MICs 3–6 μg mL^–1) > S. aureus (MICs 12–25 μg mL^–1) > E. coli (MIC 25 μg mL^–1). The highest activity of thiophene derivatives 4c and 4d was recorded against B. subtilis at concentrations of 1.5–3.0 μg mL^–1 and against H. influenzae at 12 μg mL^–1. Compound 4c showed also a mild inhibition of S. aureus at 50 μg mL^–1. Meanwhile, pyridine derivatives were totally unable to show any inhibition (MICs > 100 μg mL^–1). B. subtilis and H. influenzae were found to be the most sensitive microorganisms. However, in all cases, the antibacterial activity of the tested compounds is lower than that of the standard drug ciprofloxacin.

Concerning antifungal properties, none of the tested compounds exhibited any activity against Candida tropicalis, Saccharomyces cerevisiae, and Aspergillus niger, the MICs being always higher than 100 μg mL^–1 (data not shown; MICs of miconazole reference drug 6, 12, and 3 μg mL^–1, respectively).

From the obtained results, it was revealed that the presence of aromatic (compounds 1d–g) and heteroaromatic (compounds 4a–f) nuclei decreases the antibacterial effectiveness of compound 1a. However, among arylfluoroimidazoquinolones, fluorine and especially chlorine substitutions (1f, 1g) play an important role in activity as well as furan (compounds 4e–f) among hetarylfluoroimidazoquinolones. It is noteworthy that derivatives 4e and 4f, together with 1a, were the only compounds inhibiting the growth of Gram-negative E. coli. In general, no significant differences in the efficacy were detected between the 2- and 4-pyridine isomers or between the 2- and 3- furan isomers, whereas the activity of 2-thiophene derivative 4c against S. aureus was higher than that of the 3-thiophene derivative 4d. However, the antibacterial properties expressed by the tricyclic imidazoquinolone 1a are lower as compared to the previously investigated thiadiazole analogues [11], tetracyclic quinolones [8], and thienopyrrolo-quinolones [7].

4.1 General procedure for the synthesis of ethyl 2-(hetaryl)-9-cyclopropyl-4-fluoro-6-oxo-6,9-dihydro-1H-imidazo[4,5-h]quinoline-7-carboxylate (3a–f)

To a homogeneous solution of 2 (0.61 g, 2 mmol) in 10 mL CHCl₃-MeOH (1:4, v/v), the particular aromatic aldehyde (2 mmol), nitrobenzene (0.32 g, 2.6 mmol), and silica (3 g) were added successively. The resulting slurry was evaporated to dryness under reduced pressure and placed in a 10-mL glass vial. The vial was sealed tightly with an aluminum-Teflon crimp top, and the mixture was irradiated with an irradiation power of 250 W for 20–25 min at a pre-selected temperature of 200 °C. Thereafter, the vial was cooled to 20 °C by gas jet cooling, and the reaction product was then purified by flash column chromatography, eluting at first with CHCl₃, followed by CHCl₃-MeOH (9:1, v/v).

4.2 General procedure for the preparation of 9-cyclopropyl-4-fluoro-2-hetaryl-6-oxo-6,9-dihydro-1H-imidazo[4,5-h]quinoline-7-carboxylic acids (4a–f)

A stirred solution of the particular ester 3a–f (2 mmol) in 30 mL 12 % aq. HCl and 15 mL ethanol was heated at 80–85 °C under reflux conditions. Progress of the ester hydrolysis was monitored by silica gel TLC and was completed within 24 h. Thereafter, the reaction mixture was cooled, poured onto ice water and neutralized with 12 % aq. sodium hydroxide solution. The resulting precipitate was collected, washed with water, and dried.

4.3 Antimicrobial activity

The antibacterial and antifungal properties of acids 1a, 1d–g, and 4a–f were determined by broth two-fold dilution procedure [14–16]. The Gram-positive bacteria B. subtilis ATCC 6633 and S. aureus ATCC 25923, Gram-negative E. coli ATCC 8739 and H. influenzae ATCC 19418, yeasts C. tropicalis ATCC 1369 and S. cerevisiae ATCC 9763, and mold A. niger ATCC 6275 were used throughout the study. Stock solutions of the tested compounds were prepared in dimethyl sulfoxide. Serial dilutions were then made in the media (Haemophilus test medium for H. influenzae, Mueller-Hinton broth for other bacteria, Sabouraud liquid medium for fungi, Oxoid, Basingstoke, Hampshire, UK) to obtain test concentrations ranging from 100 to 0.0015 μg mL^–1. In all cases, the amount of dimethyl sulfoxide did not exceed 1 % v/v. The test tubes were inoculated with 5 × 10⁵ bacteria per mL and incubated at 37 °C for 24 h or with 1 × 10³ fungi per mL and incubated at 25 °C for 48 h. Ciprofloxacin and miconazole were used as standard antibacterial and antifungal drugs, respectively. The MICs (μg mL^–1) were taken as the lowest concentration of compound that inhibits visible growth of the tested microorganisms.