In vitro microbiological evaluation of 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5,1-(4H,5H)-diyl))diethanones, novel bis acetylated pyrazoles

2.1 Chemistry

Performing TLC assessed the reactions and the purity of the products. All the reported melting points are taken in open capillaries and were uncorrected. Sonication is performed on a Life Care-Fast Ultrasonic system (Life Care Equipments Pvt. Ltd., Mumbai, India) operating at a frequency of 45 kHz. The reaction flask is located in the maximum energy area in the bath and the addition or removal of water controlled the temperature of the water bath. IR spectra are recorded in KBr (pellet forms) on a Thermo Nicolet-Avatar-330 FT-IR spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, US) and note worthy absorption values (cm^-1) alone are listed. ¹H and ¹³C NMR spectra are recorded at 400 and 100 MHz, respectively, on Bruker AMX 400 NMR spectrometer (Bruker Biospin International, Ag, Aegeristrasse, Switzerland) using CD Cl₃ as solvent. The ESI +ve MS spectra are recorded on a Varian Saturn 2200 MS spectrometer (Varian Inc., Palo Alto, USA). Satisfactory microanalyses are obtained on Carlo Erba 1106 CHN analyzer (Thermo Fisher Scientific Inc., Waltham, MA, US). By adopting the literature precedent, bis chalcones 1-6 [23] and 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5,1-(4H, 5H)-diyl))diethanones 7-12 [21] are prepared.

2.2. Microbiology

All the clinically isolated bacterial strains namely Staphylococcus aureus, β-Heamolytic streptococcus, Micrococcus luteus, Bacillus subtilis, Salmonella typhii, Shigella flexneri, Vibreo cholerae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, MTB H₃₇Rv, INH-resistant MTB and fungal strains namely Aspergillus flavus, Aspergillus niger, Mucor indicus, Rhizopus arrhizus, and Microsporum gypsuem are obtained from the Faculty of Medicine, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India.

2.3. In vitro antibacterial and antifungal activity by disc diffusion method

The in vitro activities of the compounds were tested in Sabourauds dextrose broth (SDB) (Hi-media, Mumbai) for fungi and nutrient broth (NB) (Hi-media, Mumbai) for bacteria by the disc diffusion method following the reported method [24]. The respective hydrochlorides of the test compounds 7-12 were dissolved in water to obtain 1 mg/mL stock solution and the different concentrations (100, 200, 500 ppm) were prepared from the stock solution. Seeded broth (broth-containing microbial spores) was prepared in NB from 24-h-old bacterial cultures on nutrient agar (Hi-media, Mumbai) at 37 ± 1°C while fungal spores from 1 to 7-day-old Sabourauds agar (Hi-media, Mumbai) slant cultures were suspended in SDB. Sterile paper disc of 5-mm diameter was saturated with the three different concentrations and such discs were placed in each seeded agar plates. The petri plates were incubated in BOD incubator (Sigma Instruments, Chennai, India) at 37°C for bacteria and at 28°C for fungi. The zone of inhibition was recorded by visual observations after 24 h of inhibition for bacteria and after 72-96 h of inhibition for fungi. Moreover, the zone of inhibition was measured by excluding the diameter of the paper disc. Ciprofloxacin was used as standard for bacteria and fluconazole as standard for fungi under analogous conditions.

2.4. In vitro antibacterial and antifungal activity by twofold serial dilution method

MIC in μg/mL values was carried out by twofold serial dilution method [25]. The respective test compounds 7-12 were dissolved in dimethyl sulphoxide (DMSO) to obtain 1 mg/mL stock solution. Seeded broth (broth-containing microbial spores) was prepared in NB from 24-h-old bacterial cultures on nutrient agar (Hi-media, Mumbai) at 37 ± 1°C while fungal spores from 1 to 7-day-old Sabourauds agar (Hi-media, Mumbai) slant cultures were suspended in SDB. The colony forming units (cfu) of the seeded broth were determined by plating technique and adjusted in the range of 10⁴-10⁵ cfu/mL. The final inoculums size was 10⁵ cfu/mL for antibacterial assay and 1.1-1.5 × 10² cfu/mL for antifungal assay. Testing was performed at pH 7.4 ± 0.2 for bacteria (NB) and at a pH 5.6 for fungi (SDB). Exactly 0.4 mL of the solution of test compound was added to 1.6 mL of seeded broth to form the first dilution. One milliliter of this was diluted with a further 1 mL of seeded broth to give the second dilution and so on till six such dilutions were obtained. A set of assay tubes containing only seeded broth was kept as control. The tubes were incubated in BOD incubators at 37 ± 1°C for bacteria and 28 ± 1°C for fungi. MICs were recorded by visual observations after 24 h (for bacteria) and 72-96 h (for fungi) of incubation. Ciprofloxacin was used as standard for bacteria studies and fluconazole was used as standard for fungal studies.

2.5. In vitro antitubercular activity by luciferase reporter phage assay method

The preliminary antitubercular activity screening was conducted against M. tuberculosis H₃₇Rv, INH-resistant M. tuberculosis by luciferase reporter phage assay method [26] at two different concentrations (1.00 and 2.00 mg/mL). Fifty microliter bacterial suspension equivalent to MacFarlands No. 2 standard was added to 400 mL of G7H9 with and without the test compound. For each sample, two drug-free controls and two drug concentrations were prepared and this setup was incubated for 72 h at 37°C. After incubation, 50 mL of the high titer Luciferase reporter phage (PhAE129) and 40 mL of 0.1 M CaCl₂ were added to all the vials and this setup was incubated at 37°C for 4 h. After incubation, 100 mL of the mixture was taken from each tube into a luminometer cuvette and equal amount of working D-Luciferin (0.3 mM in 0.05 M sodium citrate buffer, pH 4.5) solution was added. The RLU was measured after 10 s of integration in the Luminometer (Monolight 2010, Pegasus Scientific Inc., Rockvillae, USA). Duplicate readings were recorded for each sample and the mean was calculated. The percentage reduction in the RLU was calculated for each test sample and compared with control. The experiment was repeated when the mean RLU of the control was less than 1,000.

3.1. Chemistry

Synthesis of 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5,1-(4H,5H)-diyl))diethanones 7-12 is carried out in excellent yields (Scheme 2 and Table 1) by the reaction of bis chalcones 1-6 with hydrazine hydrate catalyzed by anhydrous sodium acetate/acetic anhydride under ultrasonic irradiation method at 45°C within 10-20 min. It has been observed in the traditional classical method, the reaction mixture of bis chalcones 1-6 with hydrazine hydrate catalyzed by anhydrous sodium acetate in refluxing acetic anhydride for 5-8 h yield compounds 7-12 in moderate yields. However, when this reaction is performed under sonication method [27], the reaction takes place rapidly within 10-20 min with excellent yields (Table 1). In this study, acetic anhydride is the best solvent for the facile synthesis of bis pyrazoles, 7-12 in excellent yields without any solubility problem. In addition, in situ acetylation occurs in the course of the reaction because of solvent, acetic anhydride under the reaction conditions. The structures of the synthesized 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5,1-(4H,5H)-diyl))diethanones 7-12 are confirmed by FT-IR, MS, ¹H NMR, and ¹³C NMR spectral studies and elemental analysis [21].

3.2. Antimicrobial activity of 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5,1-(4H, 5H)-diyl))diethanones by disc diffusion method 7-12

3.3. Antimicrobial activity of 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5,1-(4H, 5H)-diyl))diethanones by twofold serial dilution method 7-12

3.4. Antitubercular activity of 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5, 1-(4H, 5H)-diyl))diethanones by luciferase reporter phage assay method 7-12