Synthesis and evaluation of antimicrobial properties of some azole derivatives

Purpose: To synthesize new azole derivatives and determine their antimicrobial properties. Methods: The reaction of the intermediates (2a-2c) with 3a-3c in acetone/potassium carbonate solution yielded 4a-4i, which were characterized using Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (H-NMR), carbon-13 nuclear magnetic resonance (C-NMR)) and mass spectrometry (MS). Compounds 4a-4i were assessed for their antibacterial and antifungal effects using the sequential dilution technique, relative to ofloxacin and ketoconazole. Results: The spectral data for 4a-4i were consistent with the assigned structures. The MIC of compound 4h (10 μg/ml) was similar to that of ketoconazole against Aspergillus flavus, Penicillium citrinum, and Aspergillus niger. The MIC value of compound 4b (10 μg/ml) for Penicillium citrinum was comparable to that of ketoconazole while the MIC value of compound 4d against Staphylococcus aureus and Escherichia coli (20 μg/ml) was equivalent to the corresponding MIC value for ofloxacin. Conclusion: The synthesized compounds bearing boronic acid moiety are good antimicrobial agents. Accordingly, further investigation into the thiazole-imidazole or thiazole-triazole derivatives bearing boronic acid moiety is suggested.


INTRODUCTION
Recent reports on microbial resistance and emergence of new microbial diseases pose serious challenges to the affected patients, as well as the health care community [1]. This challenge is further exacerbated by inappropriate antimicrobial therapy trends [2]. Therefore, scientists are making efforts to provide novel antimicrobial agents with diverse mechanisms of action from the known chemical classes of the existing antimicrobial drugs [3,4].
Azole is one of the important chemical classes of antimicrobial agents [5]. The development of azole antimicrobial agents is the focus of current research [6]. Many azole-based antimicrobial agents are already in clinical use, for example, ketoconazole, econazole, miconazole, posaconazole, fluconazole, voriconazole, and isavuconazole. Based on these facts [5][6][7][8][9][10], the present study was carried out to synthesize new azole derivatives, and to determine their antibacterial and antifungal properties.

EXPERIMENTAL Materials and reagents
Gallenkamp apparatus was used to determine the melting points of the synthesized compounds. The IR spectra determination (KBr; wave number in cm -1 ), NMR analysis (DMSO-d 6; δ in ppm), mass analysis (M + ; m/z), and elemental investigation (C, H and N analysis) were performed using Shimadzu spectrophotometer, Bruker DRX-300 spectrophotometer, Jeol-JMS-D-300 spectrometer, and VARIO El Elementer apparatus, respectively. The monitoring of reactions and assessment of purity were carried out using TLC. Compounds 3a-3c were purchased from Sigma Aldrich.

Synthesis of substituted phenacyl intermediates (2a-2c)
Compounds 2a-2c were prepared using the prior art process [11]. In general, a mixture of acetophenone (0.1 mole) in acetic acid (20 ml) was stirred at 80 o C with a solution of bromine (0.1 mole) in acetic acid (25 ml). The precipitate was filtered and recrystallized from ethanol.

Determination of antimicrobial activity
The sequential dilution technique [12,13] was used for determination of antimicrobial effects of the synthesized compounds. A similar procedure was described in previous publications [7][8][9][10]. Different concentrations of 4a-4i, ketoconazole, and ofloxacin were prepared and their MICs were determined using agar medium and sterile dimethyl sulfoxide (DMSO). The sterile DMSO also functioned as control or blank. The microorganisms tested are indicated in Table 2.

Statistical analysis
The data are expressed as mean ± standard error mean (S.E.M., n=3). Statistical analysis was done using SPSS-software (version 20). Statistical significance was assumed at p < 0.05. Figure 1 and Figure 2 depict the synthesis of compounds 4a-4f and 4g-4i, respectively. The reaction of 2a-2c with 3a-3c in acetone/K 2 CO 3 yielde 4a-4i, which were characterized through spectral analysis. Table 1 and Table 2 display the spectral data of compounds 4a-4i. The IR spectra of the different compounds in 4a-4i series exhibited characteristic IR bands. These bands included characteristics peaks for -OH groups of 4b, 4e, and 4h, starting from 3350 to 3360 cm -1 ; -NHgroups of 4a-4c and 4g-4i from 3120 to 3125 cm -1 ; and C=O groups of the acetoxy groups of 4a, 4d, and 4g, from 1730 to 1735 cm -1 . Compounds 4a-4i also exhibited peaks for C=O group from 1690 to 1695; C=N group from 1640 to 1645; and C=C assemblage from 1595 to 1600 cm -1 .

DISCUSSION
The structure-activity analysis of compounds 4a-4i revealed that compounds of the triazole series (4g-4i) were more potent antifungal agents than those of the imidazole series (4a-4c) and the thiazole series (4d-4f). This is in line with a previous report [14] which indicated that the azole ring must contain at least two nitrogen atoms for enhanced effects of the azole antifungal agents because the nitrogen atom in position 3 of imidazole ring and triazole ring is essential for the binding of fungal enzymes. This also accounts for the fact that compounds 4d-4f which lack nitrogen at position 3, were the least potent of the thiazole compounds against the tested fungal strains. It was also obvious that the thiazole derivatives (4d-4f) were more potent antibacterial agents than the imidazole (4a-4c) and triazole (4g-4i) compounds.
It has been established that benzothiazole-based compounds have promising antibacterial effects [15]. There is a possibility that the 4-phenylthiazole moiety of compounds 4d-4f might be working like benzothiazole-based compounds [15]. In addition, the most potent antifungal compound (4h), and another promising antifungal agent (4b) also contain boronic acid moieties, which are also supposed to increase their antifungal potencies [16]. No effect on antimicrobial effect was seen with compounds containing bromine atom. However, the fluorinated derivatives of 4a-4i must be assessed for their antimicrobial potential, since many fluorinated azole antifungal agents [16,17], and fluorinated antibacterial agents [18] are in clinical use. Finally, the presence of the acetoxy group in compounds 4a, 4d and 4g was not supposed to provide any additional antibacterial benefits.

CONCLUSION
Compounds 4h, 4b and 4d have been identified as promising antimicrobial agents. However, they need to be evaluated against other bacteria and fungi. It has been established that the presence of a fused thiazole-imidazole or thiazole-triazole ring system in the 4a-4i types of compounds, along with a boronic acid moiety, may provide better broad-spectrum antimicrobial agents. The incorporation of fluorine in the structure may also provide beneficial outcomes.