Synthesis, Characterization of Some New 1,3,5- Trisubstituted Pyrazole Derivatives for Their Antifungal Potential

The aim of the study was to develop, synthesis, and characterise a novel 1,3,5-trisubstituted-2pyrazolines derivative, as well as to evaluate its antifungal activity. The reaction of chalcone derivatives with succinic hydrazide in the presence of pyridine yielded the 1,3,5-tri-substituted-2pyrazolines derivatives. Total 20 compounds has been synthesized and characterized by the IR, 1HNMR and mass spectral analysis. Antifungal activity of the compounds carried out onfour fungal strains i.e. Saccharomyces cerevisiae, A. Niger, C. Albicans and R. Oryzae in two different concentration i.e. 50 and 100 μg/ml by Agar-diffusion method using Cup-plate method. The usual antifungal medicine was ketoconazole. All of the synthesized 1,3,5-trisubstituted pyrazole compounds (ME1-ME8, CL1-CL8, BR1-BR4) showed medium to best action against examined organisms, according to antifungal activity data. The antifungal activity of compounds against fungal strains (Saccharomyces cerevisiae, A. Niger, Candida albicans, and R. Oryzae) revealed the following order of action: CL-4 > BR-4 > CL-3 > CL-2 > ME-3> ME-2> CL-5 > CL-6 > ME-4 > ME-5 > ME-6 > ME-7 > CL-7 > CL-8>ME-8 >CL-1 >ME-1 > CL-5 > CL-6 > ME-4 > ME-5 > ME-6 > ME-7 > CL-7 > CL-8>ME-8 >CL-1 >ME-1. Electronegative groups (Br, Cl, F, and NO2) must be present at the third and fifth positions of the 1,3,5-pyrazoline ring for significant antifungal action. The presence of an electronegative group at the third and fifth positions may be required for the best Original Research Article Sharma et al.; JPRI, 33(57B): 211-223, 2021; Article no.JPRI.78006 212 action against bacterial and fungal strains, however the addition of F, NO2 has demonstrated moderate activity, while the substitution of methyl and methoxy groups may reduce the activity. The synthesized compounds in the BR-1 through BR-4 class are the most active.


INTRODUCTION
In the contemporary period, sickness from fungal and bacterial origins has increased dramatically. There is an increased need for novel medications or chemically modified moieties that are effective against bacterial and fungal illness. We discovered the diazoles, which are claimed to be employed as antifungal medicines, while searching for moieties that must be efficient against fungal infection [1]. Anti-fungals are used to treat fungal diseases including athlete's foot, ringworm, and candidiasis (thrush), as well as dangerous systemic infections such cryptococcal meningitis. Antifungal kills the fungal organism without harming the host by taking advantage of distinctions between mammalian and fungal cells. As a result, novel moieties are being sought to aid in the treatment of fungal infections [2]. Pyrazole derivatives have the interest of chemists due to their vast variety of biological characteristics and many practical uses. Pyrazole has been shown to have a wide range of biological effects, including antifungal, anticancer, anti-inflammatory, antifungal, antiproliferative, anticonvulsant, antioxidant, and antitubercular properties. The pyrazole ring is a useful synthetic target in the pharmaceutical industry because of its wide variety of biological activities [3]. Pyrazole-containing products are commercially available. Celecoxib is an NSAID used to treat rheumatoid arthritis, osteoarthritis, acute pain, and menstruation; Phenzone (phenazon or antipyrine) is an analgesic and antipyretic; Lonazolac is an NSAID; Betazole is an H2 receptor agonist. It is used to assess stomach secretory function in clinical settings. Fipronil is a broad-spectrum pesticide that affects the central nervous system of insects by inhibiting chloride ions from passing through the gamma-Aminobutyric acid (GABA) receptor and glutamate-gated chloride channels, which are both central nervous system components [4]. Chalcone is made through claisen-schmidt condensation of an aromatic aldehyde and a ketone, which is catalyzed by a base or an acid, and then dehydration. Antifungal action is mediated by the presence of a,-unsaturated functional group, which can be changed depending on the kind of substituent on the aromatic rings [5]. As they undergo a range of chemical processes, they also serve as a backbone for the production of numerous heterocyclic compounds. As a result, chalcone plays a crucial role in the production of therapeutic substances [6]. Antifungal agents, antioxidants, anti-inflammatory activity, cytotoxic activity, hypoglycaemic activity, anti-hepatotoxic, antimalarial, antileishmanial, tyrosine inhibitors, and antitumor activities are among the pharmacological activities of many chalcone derivatives of natural or synthetic origin, according to a review of the literature [7].
The combined synthesis reaction of chalcone with pyrazole moieties may be useful in synthesizing a novel pyrazole derivative, which will aid in the hunt for new antifungal compounds. The aim of the study was to see how effective novel 1,3,5-trisubstituted-2-pyrazolines derivatives in fighting fungus.

MATERIALS AND METHODS
Hi-media, New Delhi, provided the chemicals pchloroacetophenone, p-bromoacetophenone, and p-methylacetophenone. CDH (Chemical Drug House) in New Delhi, India, provided benzaldehyde, 4-fluorobenzaldehyde, 4chlorobenzaldehyde, 4-bromobenzaldehyde, 4nitrobenzaldehyde, 4-methylbenzaldehyde, and 4-methoxybenzaldehyde. Sigma Aldrich, New Delhi, provided the succinic acid. Chemicals of synthetic grade were utilized in the experiments. In open glass capillaries, the melting points of the produced compounds were determined. The Bruker-alpha IR Spectrometer was used to record IR spectra. Elemental analysis was carried out, and the results were determined to be within 0.4 percent of the theoretical values. 1HNMR spectra were recorded on Bruker Avance 400 spectrophotometer at 400 MHz, 5mm multi-nuclear inverse probe head, low and high-temperature facility. Mass Spectra were recorded using Mass Spectrometers Jeol SX-102 (FAB) by ESI.

Scheme I: Synthesis of chalcones by claisenschmidt condensation
An equimolar amount of substituted acetophenone (0.05 M) was combined with an equimolar amount of benzaldehyde and substituted benzaldehyde. In ethanol, the mixture was dissolved. The mixture was stirred for 5 minutes before adding a 50 percent aqueous potassium hydroxide solution slowly and stirring for 24 hours at room temperature. The thin layer chromatography (TLC) was used to monitor the reaction's completion. The reaction was then finished and poured over crushed ice, yielding a solid product; however, if the solid product was not produced, it was acidified with dilute hydrochloric acid [8]. The obtained solid was separated by filtration, dried and purified by column chromatography using solvent system (hexane: ethyl acetate). The reaction was shown in synthesis Scheme I.

Scheme III: Synthesis of 1,3,5-tri substituted pyrazole
The synthesized chalcone derivatives were combined in absolute alcohol with equimolar amounts of succinic hydrazide (0.005 M) and a little amount of pyridine (0.01M). The reaction mixture was refluxed for 2-6 hours at 65°C. TLC was used to monitor the reaction, which used ethyl acetate:hexane as the mobile phase. The solvent was entirely evaporated before being placed into ice cold water and constantly stirred to turn the liquid into a solid product, which yielded the synthesized product [9]. This substance was filtered and dried under vacuum. The synthesized compound purified by the column chromatography and were obtained as pale yellow solid colour powder. The synthesis was shown in Scheme-III.

Evaluation of Antifungal Activity
In order to study and evaluate the antifungal activity, reliable and reproducible methods must be used to evaluate them. Agar Diffusion method was used for evaluation of the antifungal activity using Cup-plate method [10-13].

Antifungal Screening of the Synthesized Compounds
The antifungal activity of all 20 produced compounds was tested. Saceharomyces cerevisiae, Aspergillus NIGER, Rhizopus oryzae, and Candida albicans were obtained from IMTECH Chandigarh for the screening. As a standard, ketoconazole was used. Potato-DextroseAgar (PDA) medium was used to subculture the test organisms [14][15][16][17]. To acquire growth, the tubes containing sterilized media were injected with test fungus and stored at room temperature. They were then refrigerated at 4 degrees Celsius. Cup plate technique was used to test the activity of the derivatives at various concentration levels. The typical medicine was ketoconazole. Each test chemical (5 mg) was diluted in 5 mL Analar grade dimethyl sulfoxide to provide a 1000 g/mL concentration. Each test compound's solution, control, and reference standards (0.05 ml and 0.1 ml) were put individually in the cups, and the plates were left undisturbed in the refrigerator for at least 2 hours to allow appropriate diffusion of the solution into the PDA medium. After that, the petri dishes were stored at room temperature for 48 hours. After that, using an antibiotic zone reader, the diameter of the zone of inhibition in mm surrounding each of the cups was measured.

RESULTS AND DISCUSSION
Scheme I: The synthesized compounds was characterized by the Infra-red spectroscopy and proton NMR spectroscopy and was found reliable with probable structure. Obtained compounds were characterized by IR, 1HNMR and were found consistent with an expected structure. The IR spectra denotes the peak at 1650-1658 (C=O); 1500-1580 (C=C Quadrant of Ar), 761 (mono substituted benzene); 1105 (C-F), 825 (C-Cl), 1015 (C-Br), and 1160 (OCH 3 ). These compound further confirmed by proton NMR revealed the characteristic ethylene protons of the chalcone system in between δ 7.60 (C=O-CH), 6.68-7.90 (Ar-H) and 8.05 (=CH-Ar) confirm the compound. The reaction was monitored by the TLC using Hexane: ethyl acetate as mobile phase.

Scheme III:
The synthesized compounds was characterized by the Infra-red spectroscopy and proton NMR spectroscopy and was found reliable with probable structure. Obtained compounds were characterized by IR, 1HNMR and were found consistent with an expected structure. The IR spectra demotes the peak at 3205.66 (C-H    372.14 (Quassi-molecular ion peak (M+H)+)   376.11 (Quassi-molecular ion peak (M+H)+).    388.13 (Quassimolecular ion peak (M+H)+)

CONCLUSION
Using the Agar diffusion technique, all of the 2pyrazolines were tested for antifungal activity against Saccharomyces cerevisiae, A. niger, C. albicans, and R. oryzae. The outcomes of this study were compared using ketoconazole as a benchmark. When pyrazoline was compared to standard, the antifungal activity results showed that the compounds exhibit considerable inhibitory effect on all fungal strains at both 50 g (0.05 ml) and 100 g (0.1 ml) dosing levels. Chemicals BR-3, BR-2, BR-1, CL-4, BR-4, and CL-3 had the highest activity of all the compounds examined. These compounds have halogens on the aromatic ring, indicating that electron withdrawing groups to the antifungal activity.
Electronegative groups (Br, Cl, F, and NO 2 ) must be present at the third and fifth positions of the 1,3,5-pyrazoline ring for significant antifungal action. The presence of an electronegative group (Br, Cl) at the third and fifth positions may be required for the best action against bacterial and fungal strains, however the addition of F, NO 2 has demonstrated moderate activity, while the replacement of -CH3 -OCH3 may reduce the activity. The synthesized compounds in the BR-1 through BR-4 class are the most active. This shows that bromine in the third and fifth positions of pyrazole is required for antifungal action, and that the presence of chloro-, bromo-, fluoro-, and nitrogen groups linked to the phenyl ring enhances antifungal activity. The antifungal activity data revealed that Cl, Br, F, and Nitro substitutions at the third and fifth positions may increase the antifungal activity of the compounds, whilst methyl and methoxy substitutions may decrease it.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT AND ETHICAL APPROVAL
It is not applicable.