INTRODUCTION
Over the past two decades, microbial infections have become a major cause of morbidity and a frequent immune system suppressor. Microbes cause numerous poisonous syndromes and common epidemics in human civilizations. In recent years, microbial diseases including tuberculosis (TB), pneumonia, cholera, typhoid, diphtheria, and plague have claimed a heavy toll on humanity. Because they are bacteriostatic and not bactericides, many of the readily available antimicrobial medications today are toxic and cause disease recurrence. Due to the prolonged administration periods, they may also cause resistance to increase (Bhatt and Sharma, 2013).
Of all the infectious diseases, TB causes the highest number of deaths each year. The number of people who have an Mycobacterium tuberculosis (MTB) infection that is latent is also estimated to be around 1.7 billion. These people have no symptoms and are not contagious, but they are at risk of developing an infection at some point in their life. A 2-month intense phase of pyrazinamide, ethambutol, rifampicin, and isoniazid, the administration is followed by a 4-month continuation phase of rifampicin, and isoniazid administration is the current TB therapy regimen. The formation of drug-resistant (DR-) strains is influenced by several variables, including the administration of suboptimal drug concentration, inadequate tolerability, and poor patient compliance. It is believed that 3.5% of cases of TB that have just been identified and 18% of TB that have already been treated have MDR-TB, characterized by resistance to both RIF and INH. 8.5% of MDR-TB cases are classified as extensively DR TB (XDR-TB), which is defined as having resistance to RIF and INH in addition to at least one fluoroquinolone and a second-line injectable medication. Drug-susceptible TB, XDR-TB, and MDR-TB patients have relative cure rates of 82%, 34%, and 55%. Therefore, it is essential to create shorter, better-tolerated medication regimes to eradicate DS- and DR-TB completely (Arora et al., 2020).
Structure-based drug design is a technique that makes use of computational chemistry techniques to find or create new chemical compounds that potentially bind to a target and inhibit that target protein. Molecular docking is the term used to describe the computational process of making molecules “fit” the binding site. This determines how a compound will conform and orient at the desired binding site (SBDD, 2022).
Recently, heterocycles containing nitrogen atoms in their structural motif, particularly pyrazoles and their derivatives, have attracted the attention of researchers. These agents are effective against numerous microbial infections and resistance developed by microorganisms. Pyrazoles have numerous biological activities such as antioxidant (Ambethkar et al., 2015; Gressler et al., 2010; Mardiana et al., 2017), anticancer (Inceler et al., 2013; Nitulescu et al., 2010; Prasad et al., 2013; Rai et al., 2015), antimicrobial (Chandna et al., 2014; Thumar and Patel, 2012; Vijesh et al., 2011), cyclin-dependent kinase inhibitor (Sun et al., 2013), tissue non-specific alkaline phosphatase inhibitor (Sidique et al., 2009), antiproliferative (Huang et al., 2012), antihepatotoxicity (Khalilullah et al., 2011; Khan et al., 2006), antileishmanial (Dardari et al., 2006; Dos Santos et al., 2011a, 2011b), antiinflammatory (Kendre et al., 2013; Malladi et al., 2012; Tewari et al., 2014), monoamine oxidase inhibitor (Chimenti et al., 2010; Peyssonnaux and Eychène, 2001), antitubercular (Khunt et al., 2012; Pathak et al., 2012), anticonvulsant (Abdel-Aziz et al., 2009; Kaushik et al., 2010), and analgesic (Vijesh et al., 2013).
Given the above-mentioned pharmacological importance, we have designed target compounds. Several species of bacteria, including two Gram-positive, two Gram-negative, and one mycobacterium as well as one fungus, were screened against the newly synthesized hetero cyclic derivatives.
MATERIALS AND METHODS
General
All laboratory-grade chemicals and reagents were procured from commercial suppliers and used without additional purification. Remi Electrothermal capillary melting point apparatus employed for the melting point determination. Mass spectra were recorded on a Shimadzu MS-QP2010 Ultra apparatus. 1H and 13CNMR spectra were acquired from the Bruker spectrophotometer model ultra-shield.
Chemistry
The scheme for the synthesis of the designed pyrazole-4-carboxamide derivative was depicted in Figure 1. The detailed synthetic procedure was enumerated below:
Step-1: Synthesis of ethyl 3-hydroxy-1-phenyl-1H-pyrazole-4-carboxylate (3)
Phenylhydrazine (0.242 g, 2.24 mmol) was added drop by drop to an ethanolic solution of sodium ethoxide (0.61 g, 4.5 mmol), diethyl ethoxy methylene malonate (0.486 g, 2.25 mmol), and they were all cooled in an ice-cold water bath for 10 minutes. The mixture was agitated for 1 hour at 0°C and 2 hours at room temperature. The resultant mixture was added to a solution of 1 N hydrochloric acid. The precipitated solid was filtered out, separated by washing with water and hexane, then thoroughly dried to produce ethyl 3-hydroxy-1-phenyl-1H-pyrazole-4-carboxylate (Huang et al., 2017).
Step-2: 3-hydroxy-1-phenyl-1H-pyrazole-4-carbonyl chloride (4)
A mixture of 3 (0.3 g, 1.3 mmol), PCl3 (0.35 g, 1.3 mmol), I2 (0.03 g, 0.13 mmol), and DMF were combined in 2.0 ml of DCE and agitated at 100°C for 12 hours while in the open air. To obtain the pure chemical 4, the contents of the RBF were evaporated, worked up with water, extracted with hexane, and purified via column (Li et al., 2021).
Step-3: 1H-pyrazole-4-carboxamide (5a-5m) derivatives
In the presence of an acidic environment, 1 mmol of intermediate 4 was dissolved in ethanol (10 ml), and refluxed with a variety of substituted aromatic amines (a–m) (1 mmol, 1 equivalent). Reaction progress was monitored by TLC and after complete reaction, the crude mass was evaporated under vacuum and worked up with sodium bicarbonate solution followed by brine solution. Further, the pure compound was isolated by column chromatography with a 10% ethyl acetate–hexane solvent system to produce the 1H-pyrazole-4-carboxamide (5a–5m) derivatives.
Biological activity
Antibacterial activity
All of the bacterial strains utilized in this experiment were obtained from Osmania University’s Department of Microbiology and kept at 4°C. The designed hybrids (5a–5m) were tested for antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) using the disc-diffusion method. The reference antibiotic was Neomycin sulfate (10 μg/ml) in DMSO. The microorganisms were produced by inoculating 0.5 ml of spore suspension (108 spores/ml) culture broth into nutrient agar medium in pre-sterilized Petri dishes. DMSO was used to prepare a stock solution for each of the synthesized compounds (5a–5m). The Petri dishes were seeded with nutrient agar medium, the disc (6 mm in diameter) was filled with 60 μg/ml of each test solution, and the Petri dishes were incubated at 37°C for 24 hours. At the equal preceding concentration, DMF alone was utilized as the control. Each compound’s zone of inhibition was measured in millimeters. There were three duplicates of the experiment (Ericsson and Sherris, 1971; Jorgensen et al., 1999).
Antifungal activity
The Osmania University’s Department of Microbiology provided the fungal strains, which were stored at 4°C. The disc-diffusion method was used to assess the synthesized derivatives’ antifungal efficacy against fungus strains (Candid albicans and Aspergillus niger). The reference antibiotic used was Nystatin (10 μg/ml in DMSO). In pre-sterilized Petri plates, potato dextrose agar medium was placed, and microorganisms were cultured by inoculating the standard suspension of culture broth. DMSO was used to prepare a stock solution for each of the produced compounds (5a–5m). The Petri dishes were incubated at 28°C for 48 hours with the discs (6 mm in diameter) packed with 60 μg/ml of each test solution. The discs were then placed on the seeded potato dextrose agar medium. At the equal preceding concentration, DMF alone was utilized as the control. Each compound’s zone of inhibition was measured in millimeters. There were three duplicates of the experiment (NCCLS, 1992).
Antitubercular assay
Test organisms
MTB H 37Rv (ATCC 27294) strains, which are susceptible to isoniazid, were used to assess the antitubercular activity of the synthesized compounds.
 | Figure 1. Synthesis of pyrazole-4-carboxamide derivatives. [Click here to view] |
Preparation of inoculum
To have a fresh batch for the study, the bacterial strains were subcultured and given Muller Hinton broth for 2 weeks at 37°C. By dilution with normal saline solution, bacterial suspensions with 0.5 McFarland standard turbidity, equivalent to 108 CFU, were prepared. In a glass vessel, the liquid was vortexed for 30 seconds, and the particles were allowed to settle [46]. For the inoculation, 100 μl of the microbial suspension was employed.
Preparation of test samples
Stock solutions of the synthesized compounds at a concentration of 100 μg/ml were prepared in DMSO. Title compounds were serially diluted from their corresponding stock solutions to determine the minimal inhibitory concentration for each compound (50, 25, 12.5, 6.25, 3.12, 1.6, and 0.8 μg/ml).
Preparation of growth medium and screening of antitubercular activity screening
After being sterilized using an autoclave at 121°C for 15 minutes, the mycobacterium was grown on Middlebrook 7H11 agar medium with Oleic Albumin Dextrose Catalase. The medium was then diluted with various concentrations of synthesized (5a–o) compounds at different strengths (50, 25, 12.5, 6.25, 3.12, 1.6, and 0.8 μg /ml). Allowed solidify under laminar airflow with the lids slightly open, 5 ml of middle brook 7H11 agar medium was poured into each of the designated quadrants of sterile quad-plates using an aseptic technique.
After solidification, bacterial suspension from the culture broth was inoculated aseptically through a loop (3 mm internal diameter) and cultured for 21 days at 37°C. By counting the colonies that formed on the medium and comparing them with the controls, the minimum inhibitory concentration (MIC) was determined. As negative and positive controls, respectively, DMSO, isoniazid, and pyrazinamide were used (Alqahtani and Asaad, 2014).
RESULTS AND DISCUSSION
Chemistry
All the pyrazole-4-carboxamide derivatives were synthesized in moderate yields from the designed scheme of synthesis. The chemical shift values of each compound determined the structure of the compounds in 1H NMR and 13C NMR. The characteristic pyrazole ring hydrogen chemical shift values appeared around 8.3–8.4 ppm in all compounds. The carboxamide bond linkage C13 chemical shifts were observed in the region of 160–170 ppm in all the synthesized compounds. Further, the mass spectra of each compound agree with the respective m/z values confirming the formation of the pyrazole-4-carboxamide derivatives. The structural details, percentage yield, and melting points of the synthesized pyrazole-4-carboxamide derivatives were enumerated in Table 1.
The spectral data of the synthesized pyrazole-4-carboxamide derivatives were given below:
Compound 5a: 3-hydroxy-N,1-diphenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid,1H NMR(500 MHz, Chloroform-d): δ 7.07 (1H, tt, J = 7.8, 1.2 Hz), 7.18-7.34 (3H, 7.24 (tt, J = 7.4, 1.2 Hz), 7.27 (dddd, J = 8.2, 7.8, 1.4, 0.5 Hz)), 7.40-7.55 (4H, 7.47 (dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.48 (dddd, J = 8.2, 1.5, 1.2, 0.5 Hz)), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.41 (1H, s).13C NMR: δ 119.9 (2C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8-127.8 (2C, 127.8 (s), 127.8 (s)), 128.1-128.3 (4C, 128.2 (s), 128.2 (s)), 137.4 (1C, s), 139.7 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C16H13N3O2 ([M + H]+): 279.30, found 280.25.
 | Table 1. Sructural and physical data of pyrazole-4-carboxamide derivatives. [Click here to view] |
Compound 5b: N-(4-chlorophenyl)-3-hydroxy-1-phenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 7.24 (1H, tt, J = 7.4, 1.2 Hz), 7.35-7.54 (4H, 7.42 (ddd, J = 8.1, 1.6, 0.5 Hz), 7.47 (dddd, J = 8.1, 7.4, 1.5, 0.5 Hz)), 7.75 (2H, ddd, J = 8.1, 1.5, 0.5 Hz), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.41 (1H, s).13C NMR: δ 120.5 (2C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 128.9 (2C, s), 133.7 (1C, s), 137.4 (1C, s), 139.7 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C16H12ClN3O2 ([M + H]+): 313.74, found 314.65.
Compound 5c: N-(3-chlorophenyl)-3-hydroxy-1-phenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 7.09-7.65 (6H, 7.15 (dt, J = 8.1, 1.7 Hz), 7.24 (tt, J = 7.4, 1.2 Hz), 7.35 (td, J = 8.1, 0.5 Hz), 7.47 (dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.59 (dt, J = 8.2, 1.7 Hz)), 7.73-7.95 (3H, 7.78 (td, J = 1.7, 0.5 Hz), 7.88 (dtd, J = 8.1, 1.1, 0.5 Hz)), 8.41 (1H, s).13C NMR: δ 119.9 (1C, s), 120.2 (1C, s), 122.8 (2C, s), 127.0 (1C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 130.0 (1C, s), 132.3 (1C, s), 138.2 (1C, s), 139.7 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C16H12ClN3O2 ([M + H]+): 313.74, found 314.65.
Compound 5d: N-(2-chlorophenyl)-3-hydroxy-1-phenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 7.14-7.36 (3H, 7.22 (ddd, J = 8.0, 7.7, 1.4 Hz), 7.24 (tt, J = 7.4, 1.2 Hz), 7.29 (ddd, J = 8.0, 7.7, 1.6 Hz)), 7.39-7.54 (3H, 7.46 (ddd, J = 8.0, 1.6, 0.5 Hz), 7.47 (dddd, J = 8.1, 7.4, 1.5, 0.5 Hz)), 7.72-7.95 (3H, 7.78 (ddd, J = 8.0, 1.4, 0.5 Hz), 7.88 (dtd, J = 8.1, 1.1, 0.5 Hz)), 8.42 (1H, s).13C NMR: δ 121.8 (1C, s), 122.7-122.8 (3C, 122.8 (s), 122.8 (s)), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.1-128.3 (4C, 128.2 (s), 128.2 (s), 128.3 (s)), 129.2 (1C, s), 134.6 (1C, s), 139.7 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C16H12ClN3O2 ([M + H]+): 313.74, found 314.60.
Compound 5e: 3-hydroxy-N-(4-nitrophenyl)-1-phenyl-1H-pyrazole-4-carboxamide
Light brown color solid, 1H NMR(500 MHz, Chloroform-d): δ 7.18-7.54 (5H, 7.24 (tt, J = 7.4, 1.2 Hz), 7.35 (ddd, J = 8.7, 2.3, 0.4 Hz), 7.47 (dddd, J = 8.1, 7.4, 1.5, 0.5 Hz)), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.13 (2H, ddd, J = 8.7, 1.8, 0.4 Hz), 8.43 (1H, s).13C NMR: δ 116.6 (2C, s), 122.8 (2C, s), 125.0 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 137.4 (1C, s), 139.7 (1C, s), 147.3 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C16H12N4O4 ([M + H]+): 324.30, found 325.20.
Compound 5f: 3-hydroxy-N-(3-nitrophenyl)-1-phenyl-1H-pyrazole-4-carboxamide
Light browncolor solid, 1H NMR(500 MHz, Chloroform-d): δ 7.24 (1H, tt, J = 7.4, 1.2 Hz), 7.35-7.54 (4H, 7.42 (dt, J = 8.2, 1.5 Hz), 7.45 (ddd, J = 8.4, 8.2, 0.5 Hz), 7.47 (dddd, J = 8.1, 7.4, 1.5, 0.5 Hz)), 7.61 (1H, ddd, J = 8.4, 1.7, 1.6 Hz), 7.81-7.95 (3H, 7.87 (ddd, J = 1.7, 1.5, 0.5 Hz), 7.88 (dtd, J = 8.1, 1.1, 0.5 Hz)), 8.41 (1H, s).13C NMR: δ 112.0 (1C, s), 119.9 (1C, s), 122.8 (2C, s), 123.3 (1C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 129.6 (1C, s), 137.5 (1C, s), 139.7 (1C, s), 143.9 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C16H12N4O4 ([M + H]+): 324.30, found 325.25.
Compound 5g: 3-hydroxy-1-phenyl-N-(p-tolyl)-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 2.21 (3H, s), 7.02-7.16 (4H, 7.08 (ddd, J = 8.1, 1.6, 0.5 Hz), 7.09 (ddd, J = 8.1, 1.4, 0.5 Hz)), 7.24 (1H, tt, J = 7.4, 1.2 Hz), 7.47 (2H, dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.41 (1H, s).13C NMR: δ 21.3 (1C, s), 117.9 (2C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 129.6 (2C, s), 137.4 (1C, s), 139.7 (1C, s), 141.5 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C17H15N3O2 ([M + H]+): 293.33, found 294.25.
Compound 5h: 3-hydroxy-1-phenyl-N-(m-tolyl)-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 2.30 (3H, s), 6.90 (1H, ddd, J = 8.0, 1.8, 1.6 Hz), 7.15-7.30 (3H, 7.22 (ddd, J = 8.2, 8.0, 0.5 Hz), 7.22 (ddd, J = 1.6, 1.4, 0.5 Hz), 7.24 (tt, J = 7.4, 1.2 Hz)), 7.40-7.59 (3H, 7.47 (dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.53 (ddd, J = 8.2, 1.8, 1.4 Hz)), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.42 (1H, s).13C NMR: δ 21.3 (1C, s), 118.6 (1C, s), 119.9 (1C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.1-128.3 (3C, 128.1 (s), 128.2 (s)), 129.0 (1C, s), 133.7 (1C, s), 138.4 (1C, s), 139.7 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C17H15N3O2 ([M + H]+): 293.33, found 294.25.
Compound 5i: N-(3,5-dimethylphenyl)-3-hydroxy-1-phenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 2.31 (6H, s), 6.74-6.89 (3H, 6.80 (dd, J = 2.6, 1.3 Hz), 6.84 (t, J = 2.6 Hz)), 7.24 (1H, tt, J = 7.4, 1.2 Hz), 7.47 (2H, dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.42 (1H, s).13C NMR: δ 21.3 (2C, s), 118.6 (2C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.7-127.8 (2C, 127.7 (s), 127.8 (s)), 128.2 (2C, s), 138.3-138.5 (3C, 138.4 (s), 138.5 (s)), 139.7 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C18H17N3O2 ([M + H]+): 307.35, found 308.25.
Compound 5j: 3-hydroxy-N-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 3.76 (3H, s), 6.64 (2H, ddd, J = 8.8, 2.7, 0.5 Hz), 7.18-7.39 (3H, 7.24 (tt, J = 7.4, 1.2 Hz), 7.33 (ddd, J = 8.8, 1.7, 0.5 Hz)), 7.47 (2H, dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.41 (1H, s).13C NMR: δ 56.0 (1C, s), 114.5 (2C, s), 120.5 (2C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 137.4 (1C, s), 139.7 (1C, s), 158.3 (1C, s), 159.8 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C17H15N3O3 ([M + H]+): 309.33, found 310.15.
Compound 5k: 3-hydroxy-N-(3-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 3.72 (3H, s), 6.72 (1H, ddd, J = 8.3, 1.5, 1.3 Hz), 7.16-7.30 (2H, 7.22 (td, J = 8.2, 0.5 Hz), 7.24 (tt, J = 7.4, 1.2 Hz)), 7.33-7.54 (4H, 7.38 (td, J = 1.4, 0.5 Hz), 7.47 (dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.46 (dt, J = 8.2, 1.4 Hz)), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.41 (1H, s).13C NMR: δ 56.0 (1C, s), 106.4 (1C, s), 116.7 (1C, s), 119.9 (1C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 129.7 (1C, s), 139.3 (1C, s), 139.7 (1C, s), 158.1 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C17H15N3O3 ([M + H]+): 309.33, found 310.20.
Compound 5l: N-(3,5-dimethoxyphenyl)-3-hydroxy-1-phenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 3.73 (6H, s), 6.16 (1H, t, J = 1.8 Hz), 6.41 (2H, dd, J = 2.1, 1.8 Hz), 7.24 (1H, tt, J = 7.4, 1.2 Hz), 7.47 (2H, dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.41 (1H, s).13C NMR: δ 56.0 (2C, s), 101.7 (1C, s), 106.4 (2C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 138.7 (1C, s), 139.7 (1C, s), 158.3 (1C, s), 161.6 (2C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C18H17N3O4([M + H]+): 339.35, found 340.30.
Compound 5m: N-(4-ethylphenyl)-3-hydroxy-1-phenyl-1H-pyrazole-4-carboxamide
Pale yellow color solid, 1H NMR(500 MHz, Chloroform-d): δ 1.07 (3H, t, J = 7.5 Hz), 2.56 (2H, q, J = 7.5 Hz), 7.03-7.30 (5H, 7.09 (ddd, J = 8.1, 1.3, 0.6 Hz), 7.17 (ddd, J = 8.1, 1.4, 0.6 Hz), 7.24 (tt, J = 7.4, 1.2 Hz)), 7.47 (2H, dddd, J = 8.1, 7.4, 1.5, 0.5 Hz), 7.88 (2H, dtd, J = 8.1, 1.1, 0.5 Hz), 8.42 (1H, s).13C NMR: δ 14.6 (1C, s), 28.7 (1C, s), 117.9 (2C, s), 122.8 (2C, s), 127.3 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.2 (2C, s), 129.9 (2C, s), 137.4 (1C, s), 139.7 (1C, s), 144.2 (1C, s), 158.3 (1C, s), 163.2 (1C, s). ESI-MS: m/z Anal. Calcd. For C18H17N3O2 ([M + H]+): 307.35, found 308.25.
Antimicrobial activity
Antibacterial activity
The antibacterial potential of 13 synthesized novel pyrazole carboxamide derivatives (5a–5m) against 2 Gram-positive (S. aureus and B. subtilis) and 2 Gram-negative organisms (P. aeruginosa and E. coli) was examined in this study. The results of the antibacterial activity were listed in Table 2 and were expressed as the zone of inhibition (mm) of all compounds tested against Gram-negative and Gram-positive microorganisms. The antibacterial potential of all the title compounds was discovered using an in vitro antibacterial assay. The standard drug (Neomycin at 10 μg/ml) zone of inhibition was used to compare all compounds’ potential to inhibit microbial growth.
At a concentration of 60 μg/ml, the compounds 5e, 5i, 5j, and 5k showed considerably higher inhibition of Gram-positive bacteria growth than the other compounds. All the compounds under study demonstrated greater inhibitory power against B. subtilis than S. aureus among the two Gram-positive bacterial strains. The most potent compound is 5i, which showed nearly similar antibacterial activity at a dose of 60 μg/ml when compared to the standard neomycin at a dose of 10 μg/ml.
At a concentration of 60 μg/ml the compounds 5d, 5e, 5i, 5j, and 5k showed relatively higher inhibition of Gram-negative bacteria growth than the other compounds. All the compounds under study established greater inhibitory potential against E. coli than P. aeruginosa among the two Gram-negative bacterial strains. The most potent compound is compound 5k, and when compared with standard neomycin activity at a dose of 10 μg/ml, compound 5k showed equivalent antibacterial potency at a level of 60 μg/ml.
It is clear from the findings of the in vitro antibacterial assay that the substituent groups on the phenyl ring system have an impact on the basic pyrazole carboxamide nucleus’s ability to distinguish between Gram-positive and Gram-negative bacteria. The compounds that have electron-donating groups, like 5i (3,5-dimethyl), 5j (4-OCH3), 5k (4-OCH3), and compounds with strong electron-withdrawing groups, such as 5e (4-NO2), 5d, and (2-Cl) are active against both Gram-negative and Gram-positive bacterial strains. Relatively the pyrazole-4-carboxamide derivative displayed a good tendency toward the inhibition of Gram-positive bacteria rather than Gram-negative bacteria. This relative specificity of the pyrazole-4-carboxamides might be attributed to the better interactions of the compounds with the Gram-positive macromolecular network than the more stringent Gram-negative bacteria.
 | Table 2. Zone of inhibition (mm) of the compounds against Gram positive and Gram-negative bacterial strains. [Click here to view] |
Antifungal activity
The synthesized compounds’ antifungal profile was also developed against specific fungus strains (Table 3). Interestingly, compounds 5a, 5i, 5j, 5l, and 5m significantly inhibited the growth of the two tested fungi. Additionally, A. niger is found to be more susceptible to synthesized compounds than other strains. The most effective antifungal of all the derivatives, 5a, 5i demonstrated maximal growth inhibition with zones of inhibition of 21 and 19 mm against A. niger and C. albicans followed by 5j, 5l, and 5m. Compared to A. niger, all substances showed decreased sensitivity to C. albicans except 5j (zone of inhibition of 22 mm3 as shown in Table 3). The outcomes show that the pyrazole carboxamide derivatives may have antifungal properties. In comparison to compounds containing electron-withdrawing groups, those containing electron-donating groups are more effective against the tested fungal strains. Furthermore, the nonpolar substituents had greater effectiveness against the fungi than the remaining substituent-containing compounds.
Antitubercular activity
Using the middle brook 7H11 medium, 13 synthesized compounds (5a–5m) were tested for antitubercular activity against the MTB H 37Rv (ATCC 27294) strain at a range of doses (100, 50, 25, 12.5, 6.25, 3.12, 1.6, and 0.8 μg/ml). The antitubercular activity was enumerated as MIC and these values were compared to those of the Isoniazid and pyrazinamide reference drugs. The findings showed that just a few of the test substances had reasonably effective antitubercular activity against the MTB H 37Rv (ATCC 27294) strain, which is isoniazid sensitive. The outcomes are shown in Table 4.
 | Table 3. Zone of inhibition (mm) of the compounds against fungal strains. [Click here to view] |
Based on the observations, compounds 5e (MIC: 3.12 μg/ml) 5g and 5m (MIC: 6.25 μg/ml), and 5h (MIC: 12.5 μg/ml) exhibit significant antitubercular activity against MTB H 37Rv (ATCC 27294) strain, followed by compounds 5m, 5k, 5b, 5c, and 5f (MIC: 25 μg/ml), which exhibit moderate antitubercular activity. According to the findings, the new pyrazole carboxamide derivatives are not nearly as effective as the widely used drugs pyrazinamide and isoniazid. Except for 5j, and 5d, all the compounds have some antitubercular activity against the MTB H 37Rv (ATCC 27294) strain.
 | Table 4. MIC of synthesized derivatives against MTB strain. [Click here to view] |
CONCLUSION
In conclusion, the designed pyrazole-4-carboxamide derivatives were synthesized through the proposed synthetic scheme in moderate to good yields. The spectral analysis of the synthesized compounds by NMR and MASS techniques determined the structure of all compounds. All the synthesized compounds were tested for their antibacterial, antifungal, and antitubercular activities. Among the tested compounds the compounds with electron-donating groups displayed noticeable inhibition of bacterial growth against both Gram-negative and Gram-positive strains as well as tested fungal strains. In the case of antitubercular assay compounds 5e, 5g, and 5m displayed a promising bacterial inhibition. Further studies, regarding the molecular mechanism of antimicrobial activity of this pyrazole-4-carboxamide, are needed, to establish the complete mechanism of action.
AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.
FINANCIAL SUPPORT
There is no funding to report.
CONFLICT OF INTEREST
The authors disclose no conflict of interest.
ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
DATA AVAILABILITY
All data generated and analyzed are included in this research article.
PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation
REFERENCES
Abdel-Aziz M, Abuo-Rahma GEA, Hassan AA. Synthesis of novel pyrazole derivatives and evaluation of their antidepressant and anticonvulsant activities. Eur J Med Chem, 2009; 44:3480–7.
Alqahtani JM, Asaad AM. Anti-tuberculous drugs and susceptibility testing methods: current knowledge and future challenges. J Mycobac Dis, 2014; 4:140–51.
Ambethkar S, Padmini V, Bhuvanesh N. A green and efficient protocol for the synthesis of dihydropyrano[2,3-c]pyrazole derivatives via a one-pot, four component reaction by grinding method. J Adv Res, 2015; 6:975–85.
Arora G, Gagandeep Behura A, Gosain TP, Shaliwal RP, Kidwai S, Singh R. NSC 18725, a pyrazole derivative inhibits growth of intracellular Mycobacterium tuberculosis by induction of autophagy. Front Microbiol, 2020; 10:51–69; doi:10.3389/fmicb.2019.03051.
Bhatt H, Sharma S. Synthesis and antimicrobial activity of pyrazole nucleus containing 2-thioxothiazolidin-4-one derivatives. Arab J Chem, 2013; 10:S1590–6; doi:10.1016/j.arabjc.2013.05.029.
Chandna N, Kapoor JK, Grover J, Bairwa K, Goyal V, Jachak SM. Pyrazolylbenzyltriazoles as cyclooxygenase inhibitors: synthesis and biological evaluation as dual anti-inflammatory and antimicrobial agents. New J Chem, 2014; 38:3662.
Chimenti F, Carradori S, Secci D, Bolasco A, Bizzarri B, Chimenti P, Granese A, Yáñez M, Orallo F. Synthesis and inhibitory activity against human monoamine oxidase of N1-thiocarbamoyl-3,5-di(hetero)aryl-4,5-dihydro-(1H)- pyrazole derivatives. Eur J Med Chem, 2010; 45:800–14.
Dardari Z, Lemrani M, Sebban A, Bahloul A, Hassar M, Kitane S, Berrada M, Boudouma M. Antileishmanial and antibacterial activity of a new pyrazole derivative designated 4-[2-(1-(ethylamino)-2-methyl- propyl)phenyl]- 3-(4-methyphenyl)-1-phenylpyrazole. Arch Pharm (Weinheim), 2006; 339:291–8.
Dos Santos MS, Gomes AO, Bernardino AM, de Souza MC, Khan MA, de Brito MA, Castro HC, Abreu PA, Rodrigues CR, de Léo RM, Leon LL Synthesis and antileishmanial activity of new 1-aryl-1H-pyrazole-4-carboximidamides derivatives. J Braz Chem Soc, 2011a; 22:352–402.
Dos Santos MS, Oliveira ML, Bernardino AM, de Léo RM, Amaral VF, de Carvalho FT, Leon LL, Canto-Cavalheiro MM. Synthesis and antileishmanial evaluation of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazole derivatives. Bioorg Med Chem Lett, 2011b; 21:7451–64.
Ericsson HM, Sherris JC. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol Microbiol Scand, 1971; 217:90.
Gressler V, Moura S, Flores FC, Flores DC, Colepicolo P, Pinto E. Antioxidant and antimicrobial properties of 2-(4,5-Dihydro-1H-pyrazol1-yl)-pyrazole and 1-carboxamidino-1H-pyrazole derivatives. J Braz Chem Soc, 2010; 21:1477–83.
Huang D, Liu A, Liu W, Liu X, Ren Y, Zheng X, Pei H, Xiang J, Huang M, Wang X. Synthesis and insecticidal activities of novel 1H-pyrazole-5-carboxylic acid derivatives. Heterocycl Commun, 2017; 23:455–60; doi:10.1515/hc-2017-0110.
Huang YY, Wang LY, Chang CH, Kuo YK, Kaneko K, Takayama H, Kimura M, Juang SH, Wong FF. One-pot synthesis and antiproliferative evaluation of pyrazolo[3,4-d] pyrazole derivatives. Tetrahedron, 2012; 68:9658.
Inceler N, Yilmaz A, Baytas SN. Synthesis of ester and amide derivatives of 1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-4-carboxylic acid and study of their anticancer activity. Med Chem Res, 2013; 22:3109.
Jorgensen JH, Turnidge JD, Washington JA. Antibacterial susceptibility tests: dilution and disk diffusion methods. In: Versalovic J (Ed.). Manual of clinical microbiology, American Society for Microbiology, Washington, DC, vol. 7, pp 1526–43, 1999.
Kaushik D, Khan AS, Chawla G, Kumar S. N’-[(5-chloro-3-methyl-1-phenyl-1Hpyrazol-4-yl)methylene]2/4-substituted hydrazides: Synthesis and anticonvulsant activity. Eur J Med Chem, 2010; 45:3943–9.
Kendre BV, Landge MG, Jadhav WN, Bhusare SR. Synthesis and bioactivities of some new 1H-pyrazole derivatives containing an aryl sulfonate moiety. Chin Chem Lett, 2013; 24:325–34.
Khalilullah H, Khan S, Ahsan MJ, Ahmed B. Synthesis and antihepatotoxic activity of 5-(2,3-dihydro-1,4-benzodioxane-6-yl)-3- substituted-phenyl-4,5-dihydro-1H-pyrazole derivatives. Bioorg Med Chem Lett, 2011; 21:7251–4.
Khan SA, Ahmad B, Alam T. Synthesis and antihepatotoxic activity of some new chalcones containing 1, 4-dioxane ring system. Pak J Pharm Sci, 2006; 19:290–304.
Khunt RC, Khedkar VS, Chawda RS, Chauhan NA, Parikh AR, Coutinho EC. Synthesis, antitubercular evaluation and 3D-QSAR study of N-phenyl-3-(4- fluorophenyl)-4-substituted pyrazole derivatives. Bioorg Med Chem Lett, 2012; 22:666–78.
Li F, Wu X, Guo F, Tang Z, Xiao J. One-step conversion of amides and esters to acid chlorides with PCl 3. Eur J Org Chem, 2021; 30:4314–7.
Malladi S, Isloor AM, Shetty P, Fun HK, Telkar S, Mahmoud R, Isloor N. Synthesis and anti-inflammatory evaluation of some new 3, 6-disubstituted-1, 2, 4-triazolo-[3, 4-b]-1, 3, 4-thiadiazoles bearing pyrazole moiety. Med Chem Res, 2012; 21:3272–81.
Mardiana L, Bakri R, Septiarti A, Ardiansah B. The synthesis of 2-(5-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenol using sodium impregnated on activated chicken eggshells catalyst. IOP Conf Ser Mater Sci Eng, 2017; 188:12022.
NCCLS. Reference method for broth dilution antifungal susceptibility testing of yeasts, approved standard. National Committee for Clinical Laboratory Standards (NCCLS), M27-S3, Wayne, PA, vol. 28, no. 15, 1992.
Nitulescu GM, Draghici C, Missir AV. Synthesis of new pyrazole derivatives and their anticancer evaluation. Eur J Med Chem, 2010; 45:4914–9.
Pathak RB, Chovatia PT, Parekh HH. Synthesis, antitubercular and antimicrobial evaluation of 3-(4-chlrophenyl)-4-substituted pyrazole derivatives. Bioorg Med Chem Lett, 2012; 22:5129–33.
Peyssonnaux C, Eychène A. The raf/MEK/ERK pathway: new concepts of activation. Biol Cell, 2001; 93:53–62.
Prasad YR, Kumar GV, Chandrashekar SM. Synthesis and biological evaluation of novel 4,5-dihydropyrazole derivatives as potent anticancer and antimicrobial agents. Med Chem Res, 2013; 22:2061.
Rai U, Isloor AM, Shetty P, Pai KS, Fun HK. Synthesis and in vitro biological evaluation of new pyrazole chalcones and heterocyclic diamides as potential anticancer agents. Arab J Chem, 2015; 8:317.
Sidique S, Ardecky R, Su Y, Narisawa S, Brown B, Millán JL, Sergienko E, Cosford ND . Design and synthesis of pyrazole derivatives as potent and selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP). Bioorg Med Chem Lett, 2009; 19:222–35.
SBDD. Structure-based drug design (SBDD). 2022. https://revive.gardp.org/resource/structure-based-drug-design-sbdd/?cf=encyclopaedia (Accessed 7 September 2022).
Sun J, Lv XH, Qiu HY, Wang YT, Du QR, Li DD, Yang YH, Zhu HL. Synthesis, biological evaluation and molecular docking studies of pyrazole derivatives coupling with a thiourea moiety as novel CDKs inhibitors. Eur J Med Chem, 2013; 68:1–9.
Tewari AK, Singh VP, Yadav P, Gupta G, Singh A, Goel RK, Shinde P, Mohan CG. Synthesis, biological evaluation and molecular modeling study of pyrazole derivatives as selective COX-2 inhibitors and anti-inflammatory agents. Bioorg Chem, 2014; 56:8–15.
Thumar NJ, Patel MP. Synthesis, characterization and biological activity of some new carbostyril bearing 1H-pyrazole moiety. Med Chem Res, 2012; 21:1751–61.
Vijesh AM, Isloor AM, Shetty P, Sundershan S, Fun HK. New pyrazole derivatives containing 1,2,4-triazoles and benzoxazoles as potent antimicrobial and analgesic agents. Eur J Med Chem, 2013; 62:410–5.
Vijesh AM, Isloor AM, Telkar S, Peethambar SK, Rai S, Isloor NSynthesis, characterization and antimicrobial studies of some new pyrazole incorporated imidazole derivatives. Eur J Med Chem, 2011; 46:3531–6.