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

Novel 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5,1-(4H,5H)-diyl))diethanones 7-12 were tested for their antimicrobial activity by disc diffusion and twofold serial dilution method against the tested bacterial and fungal strains. Compounds 7 against Micrococcus luteus, 8 against β-Heamolytic streptococcus, M. luteus, Klebsiella pneumonia, Microsporum gypseum, 9 against Staphylococcus aureus, Shigella flexneri, Vibreo cholerae, Pseudomonas aeruginosa, Aspergillus flavus, Mucor indicus, 10 against Salmonella typhii, S. flexneri, M. gypseum, 11 against K. pneumonia, M. gypseum, 12 against K. pneumonia, and M. gypseum show superior zone of inhibitions and exhibited excellent antibacterial and antifungal activities at a MIC value of 6.25 μg/mL. Moreover, all the tested compounds 7-12 revealed promising antitubercular activity against Mycobacterium tuberculosis H37Rv and INH-resistant M. tuberculosis. Compounds 8 against M. tuberculosis and 11 against INH-resistant M. tuberculosis exhibited the percentage of reduction in RLU at 89 and 85%, respectively.


Introduction
Mycobacterium tuberculosis (MTB) is a pathogenic bacterial species in the genus Mycobacterium and is the causative agent of most cases of tuberculosis. Tuberculosis is a common and often deadly infectious disease in humans [1,2]. Tuberculosis is the most common opportunistic disease in persons infected with human immunodeficiency virus [3]. The genome of MTB is rich in lipid-metabolizing and P450 enzymes. The cell envelope of MTB is unique and is associated with its pathogenicity [4]. Mycolic acids are the major constituents of the protective barrier of cell envelope of MTB and are essential for survival, virulence, and antibiotic resistance [5]. Inhibitors of mycolic acid biosynthesis, such as isoniazid (INH), ethambutol (EMB), and pyrazinamide (PZA), are still in the frontline of antitubercular drugs [6].
The discovery of the norfloxacin plays an important role in structure-activity relationships analysis of the fluoroquinolonic nucleus A-D, (Scheme 1) which led to the development of new derivatives with better solubility, higher antimicrobial activity, prolonged serum half-life, fewer adverse side effects, and both oral and parenteral routes of administration [7][8][9]. Naturally occurring bacterial DNA gyrase inhibitor such as novobiocin, a coumarin derivative E (Scheme 1), is known as antibacterial agents [10]. The coumarin drug inhibits ATPase activity of DNA gyrase by competing with ATP for binding to the subunit B of the enzyme. Owing to side effects, no pharmaceutically useful drug has been derived from the coumarins [11]. Although huge efforts have been dedicated to find a potent antibacterial agents that can overcome bacterial resistance, promising lead structures of DNA gyrase and topoisomerase IV enzyme inhibitors with novel mechanisms of action have not been found [12]. This reflects the inherent difficulties associated with the discovery and clinical testing of new candidates and the lack of significant pharmaceutical industry research in this area. Hence, the discovery and development of new drugs that effectively combat TB are accorded a great importance. In recent years, interest in pyrazoles has increased significantly because of their proven usefulness as intermediates in the preparation of new pharmaceuticals and agrochemicals [13][14][15]. Also, pyrazole derivatives F and G (Scheme 1) were identified as a new class of DNA gyrase and topoisomerase IV enzyme inhibitors [16]. Besides these, amides are well known for their therapeutic values since the amide group is an important pharmacophore. Antibiotics such as penicillins and cephalosporins have an amide group. The resistance toward available drugs is rapidly becoming a major worldwide problem. The necessity to design new compounds to overcome this resistance has become one of the most important areas of research today. Owing to our interest in synthesizing fascinating biologically active structurally diverse heterocycles [17][18][19][20], we recently reported the clean production of 1,1'-(5,5'-(1,4-phenylene)bis(3-aryl-1H-pyrazole-5,1-(4H,5H)-diyl)) diethanones, a novel series of bis pyrazole derivatives using sodium acetate/acetic anhydride triggered by ultrasound irradiation [21], which accelerated the chemical reaction and mass transferred via the process of acoustic cavitation [22]. Extending the research in this area, we decided to investigate the antibacterial, antifungal, and antitubercular activities of the target compounds with the hope to develop some promising antimicrobial and antimycobacterial agents.

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. 1 H and 13 C NMR spectra are recorded at 400 and 100 MHz, respectively, on Bruker AMX 400 NMR spectrometer (Bruker Biospin International, Ag, Aegeristrasse, Switzerland) using CDCl 3 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.

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 Scheme 1 Structure of novel antitubercular agents.
fungal spores from 1 to 7-day-old Sabourauds agar (Himedia, 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.

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 10 4 -10 5 cfu/mL. The final inoculums size was 10 5 cfu/mL for antibacterial assay and 1.1-1.5 × 10 2 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.

In vitro antitubercular activity by luciferase reporter phage assay method
The preliminary antitubercular activity screening was conducted against M. tuberculosis H 37 Rv, 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 CaCl 2 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.