Novel Antimicrobial Agents: Fluorinated 2-(3-(Benzofuran-2-yl) pyrazol-1-yl)thiazoles

A new series of 2-pyrazolin-1-ylthiazoles 8a–d and 13–16 was synthesized by cyclization of N-thiocarboxamide-2-pyrazoline with different haloketones and 2,3-dichloroquinoxaline. The structures of the new compounds were confirmed by elemental analyses as well as NMR, IR, and mass spectral data. The newly synthesized compounds were evaluated for their antimicrobial activities, and also their minimum inhibitory concentration (MIC) against most of test organisms was performed. Amongst the tested ones, compound 8c displayed excellent antimicrobial activity.


Introduction
Pyrazolines are nitrogen-containing heterocyclic compounds, well known for their pronounced biological activity. These biological activities include antibacterial [1], antifungal [2], herbicidal [3], and insecticidal activities [4]. It was demonstrated that the combination of pyrazole with azole ring, linked to each by one sigma bond, led to more biologically active targets; for example, pyrazolylthiazoles showed excellent antimicrobial activities [5]. Continuing our work in this research field [6][7][8][9] and in an attempt to identify new and potent antimicrobial agents, we tried here to generate new benzofuryl 2-pyrazolin-1-ylthiazoles as antimicrobial agents using simple methods.

Results and Discussion
2.1. Chemistry. The starting pyrazoline-1-carbothioamide 5 was prepared by treatment of 2-acetylbenzofuran 1 with equivalent of 4-flurobenzaldehyde 2 in the presence of 10% alcoholic NaOH in 90% ethanol with stirring at room temperature to give chaconne 3. Reaction of chalcone 3 with equivalent amount of thiosemicarbazide was performed in ethanol in the presence of 2.5 equivalent of sodium hydroxide to the target precursor 5.
The reaction product structures were elucidated by means of NMR, MS spectroscopy, and elemental analyses (Table 1).
For example, 1 H NMR spectra of 8a-d contained two doublet-doublet and one triplet signals due to the presence of CH 2 adjacent asymmetric carbon. The mass spectra of 8a-d showed the molecular ion peaks at m/z 481, 499, 515 and 561, respectively in agreement with the calculated masses.

Antimicrobial Activity.
Chemical compounds were individually tested against a panel of Gram-positive and Gramnegative bacterial pathogens and yeast. Antimicrobial tests were carried out by the agar well diffusion method [15] using 100 L of suspension containing 1 × 10 8 CFU/mL of pathological tested bacteria and 1 × 10 6 CFU/mL of yeast spread on nutrient agar (NA) and Sabourund dextrose agar (SDA), respectively. After the media had cooled and solidified, wells (10 mm in diameter) were made in the solidified agar and loaded with 100 L of tested compound solution prepared by dissolving 100 mg of the chemical compound in one mL of dimethyl sulfoxide (DMSO). The inculcated plates were then incubated for 24 h at 37 ∘ C for bacteria and at 28 ∘ C for yeast. Negative controls were prepared using DMSO employed for dissolving the tested compound. Ciprofloxacin (50 g/mL) and ketoconazole (50 g/mL) were used as standard for antibacterial and antifungal activities respectively. After incubation time, antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms and compared with that of the standard. The observed zone of inhibition is presented in Table 2. Antimicrobial activities were expressed as inhibition diameter zones in millimeters (mm). The experiment was carried out in triplicate, and the average zone of inhibition was calculated.

Minimal Inhibitory Concentration (MIC) Measurement.
The bacteriostatic activity of the active compounds (having inhibition zones (IZ) ≥ 18 mm) was then evaluated using the twofold serial dilution technique [16]. Twofold serial dilutions of the tested compounds solutions were prepared using the proper nutrient broth. The final concentration of the solutions was 200, 100, 50, and 25 g/mL. The tubes were then inoculated with the test organisms, grown in their suitable broth at 37 ∘ C for 24 hours for bacteria (about 1 × 10 8 CFU/mL and 1 × 10 6 CFU/mL of yeast), and each 5 mL received 0.1 mL of the above inoculum and incubated at 37 ∘ C for 24 hours. The lowest concentration showing no growth was taken as the minimum inhibitory concentration (MIC).

Conclusion
Novel pyrazolylthiazoles, with potential antimicrobial activity, were prepared from available 2-acetylbenzofuran. Firstly, chalcone 3 was obtained by condensation with 4-fluorobenzaldehyde in alcoholic NaOH. This precursor reacted with thiosemicarbazide in strong basic medium to afford the Npyrazoline thioamide 5. Reaction of the latter with different haloketones and 2,3-dichloroquinoxaline gave the target pyrzolinylthiazoles 8-16. The new compounds were tested for their antimicrobial activities and significant activities due to presence of three nucleuses: benzofuran, pyrazole, and thiazole. Also, some substituent increases the antimicrobial activities such as chloro substituent in compounds 8c and 13a.