Preparation, Analysis and Antibacterial Evaluation of Some New 1,3,5-Trisubstituted Pyrazole Derivatives

The goal of this study was to develop, synthesise, and characterise a novel 1,3,5-trisubstituted-2pyrazoline derivative, as well as test its antibacterial activity. The reaction of chalcone derivatives with succinic hydrazide in the presence of pyridine yielded the 1,3,5-tri-substituted-2-pyrazolines derivatives. The IR, 1HNMR, and mass spectral analyses were used to characterise a total of 20 substances. Antibacterial activity of the compounds was tested on five Gm+Ve bacterial strains (Staphylococcus aureus, S. Faecalis, Bacillus Substilis, P. Vulgaris, and B. Pumilus) and two GmVe bacterial strains (Staphylococcus aureus, S. Faecalis, Bacillus Substilis, P. Vulgaris, and B. Pumilus) and two Gm-Ve i.e. Escherichia Coli, Klebsiella Penumoniae in two different concentration i.e. 50 and 100 μg/ml by Agar-diffusion method using Cup-plate method. Standard antimicrobial drugs were Norfloxacin and Ciprofloxacin. The antibacterial activity of substances against Gm+Ve bacterial strains (Staphylococcus Aureus, Staphylococcus Faecalis, Bacillus Substilis, Pseudomonas Vulgaris, and B. Pumilus) suggested the following order of action: BR-3 >BR-2>BR1>CL-4>BR-4>CL-3> CL-2>CL-5>CL-6>ME-3>ME-2>ME-4>ME-5>ME-6>ME-7>CL-7>CL-8>CL1>ME-8>ME-1>ME-8>ME-1>ME-8>ME-1>ME-8>ME-1>ME-8>ME-1>ME-8>ME-1>ME-8>ME1>ME-8>ME-1>ME-8>ME-1>ME-8>ME The chemicals of the BR-1 to BR-4 series have the most Original Research Article Sharma et al.; JPRI, 33(53A): 100-116, 2021; Article no.JPRI.78008 101 action. ME-8, CL-8, CL-7, CL-1, ME-5, ME-6, and ME-1 have light activity, whereas CL-2, CL-5, ME-4, CL-6, ME-3, ME-2, and ME-7 have moderate activity. The compounds BR-3, BR-2, BR-1, CL-4, BR-4, and CL-3 have been shown to have excellent action. The result data of antibacterial activity suggested that Cl, Br, F, and Nitro substitution at third and Fifth position may enhance the antbacterial activity of the compounds but the methyl and methoxy substitution may resulted in reduction of the activity.


INTRODUCTION
The study of infectious illnesses and transmission has reduced the death rate of numerous diseases as mankind has progressed over time [1][2][3][4]. Today, we have a far better understanding of how to treat and survive infectious disorders. Bacterial pathogenesis and virulence factors, on the other hand, have allowed bacterial infections to persist and become troublesome [5][6][7]. Bacterial infections are of particular importance because, in the absence of the complexity that multicellular creatures possess, bacteria have devised a novel means of survival [8]. Bacteria have the intrinsic capacity to spontaneously modify their DNA while reproducing in response to harmful conditions, and hence transmit this survival drive on to their progeny [9].
Antimicrobial medications have played a significant role in human health and have substantially helped human living since their discovery. Antimicrobial agents, on the other hand, are among the most commonly mishandled medications by doctors. The widespread and indiscriminate use of antibiotics has led in the establishment of drug resistance and multidrug resistance (MDR) among microorganisms, which has reached alarming levels in many areas of the world, particularly in poor nations [10][11][12]. The short lifespan of currently employed antimicrobial medications; genetic and metabolic changes; a quicker pace of evolution as global temperatures changes; Scientists and physicians are concerned about well-documented negative effects from long-term antimicrobial usage, as well as the expensive expense of clinical research and medication development. Antibiotic drug resistance is a critical challenge that necessitates a fresh effort to create new and effective antibiotic classes with unique or changed modes of action. Drug resistance for their indicated antibiotic therapy has been found in parasitic protozoa such as trypanosoma, plasmodium, toxoplasma gondii, leishmania, and entamoeba in recent investigations [13][14][15][16]. Early on in the development of medicinal chemistry, The isolation of therapeutic compounds found in plants was a major issue for scientists. Today's researchers are equally concerned with the development of novel synthetic medicine molecules [17]. The medications that are used to treat bacterial infections are becoming increasingly ineffective, due to bacterial strain resistance. As a result, there is an increased need for novel medications or chemically modified moieties that are efficient against bacterial infection. We discovered the diazoles, which are claimed to be utilised as antibacterial agents, while searching for moieties that must be efficient against bacterial illness [18][19][20][21][22]. Pyrazole has a chemical behaviour that is unusual not just among heterocyclic compounds, but also among related diazoles. Pyrazole derivatives have been known for over 80 years, but research into their chemistry has been sluggish. Earlier research focused mostly on the creation of synthetic approaches [23]. The study of chemical properties, particularly the peculiarities of the behaviour of pyrazole derivatives and the clarification of their physicochemical features, has recently gotten a lot of interest. Pyrazole derivatives have a long history of usage as herbicides and insecticides in agriculture, as well as in the pharmaceutical business as antipyretics and anti-inflammatory agents [24]. A wide range of biological activity has been described for compounds containing pyrazole nucleus, including antibacterial, antifungal, antioxidant, anti-amoebic, analgesic, antitubercular, neuroprotective, anticancer, antiproliferative, antiviral, anticonvulsant, and muscarinic action [25]. The pyrazoles ring is an important synthesis pathway in the pharmaceutical sector because of its wide variety of biological activities. Chalcone is a 1,3diphenyl-2-propene-1-one compound with two aromatic rings connected by a three-carbon,unsaturated carbonyl system [26]. The reaction of 1,3,5-pyrazoline with chalcone derivatives was used to create novel pyrazoline derivatives, which were then tested for antibacterial efficacy against both gram-positive and gram-negative microorganisms. The goal of the study was to develop, synthesise, and characterise novel 1,3,5-trisubstituted-2-pyrazolines derivatives, as well as to assess their antibacterial properties.

MATERIALS AND METHODS
The chemicals p-chloroacetophenone, pbromoacetophenone, and p-methylacetophenone were donated by Hi-media in New Delhi. Benzaldehyde, 4-fluorobenzaldehyde, 4chlorobenzaldehyde, 4-bromobenzaldehyde, 4nitrobenzaldehyde, 4-methyl benzaldehyde, and 4-methoxy benzaldehyde were all given by CDH (Chemical Drug House) in New Delhi, India. The succinic acid was given by Sigma Aldrich in New Delhi. In the trials, synthetic-grade chemicals were used. The melting temperatures of the generated compounds were evaluated in open glass capillaries. The IR spectra were recorded using a Bruker-alpha IR Spectrometer. The findings of the element analysis were found to be within 0.4 percent of the theoretical value. 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.
Equimolar quantity (0.05 M) of p-chloro acetophenone and p-methyl acetophenone was taken and mixed with equimolar quantity of benzaldehyde and substituted benzaldehyde. The mixture was dissolved in ethanol. The mixture was stirrer for 5 minutes and added 50% aqueous solution of potassium hydroxide was added slowly with continues stirring at room temperature for 24 hrs. The completion of the reaction was monitored by the TLC. Then the synthesis is completed, the mixture was poured into the crushed ice, solid product was obtained but if the solid product was not obtained so acidified with dilute hydrochloric acid [28]. 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 II: Synthesis of succinic hydrazide
Succinic acid (0.05M) can be easily converted to succinichydrazide by reaction with hydrazine hydrate (0.05M) in alcohol, then the reaction mixture was cooled to room temperature, succinichydrazide separates as solid which was recrystallized using ethanol. The IR spectra denotes the peak at 3500.66 (primary-N-Hstr.); 3313.58 (Secondary N-Hstr.); 1658.32(C=O); 1430-3046.55 (CH-CH). The reaction was monitored by the TLC using Hexane: ethyl acetate as mobile phase. Obtained compounds were characterized by IR, 1HNMR and were found consistent with an expected structure ( Fig. 1).

Scheme III: Synthesis of 1,3,5-tri substituted pyrazole
The synthesized chalcone derivatives with equimolar quantity (0.005 M) was mixed with succinic hydrazide (0.005M) in absolute alcohol and addition of small amount of pyridine (0.01M). The reaction mixture was refluxed at 65°C up to 2-6 hrs. The reaction was monitored by the TLC using ethyl acetate: hexane as mobile phase. The solvent was completely evaporated and then was poured into the ice cold water with constant stirring, that covert liquid form into solid product, that resulted into the corresponding synthesized product [29]. The synthesis was shown in scheme-II (Fig. 2). This solid was filtered under vacuum and dried. The synthesized compound purified by the column chromatography and were obtained as pale-yellow solid colour powder. Norfloxacin and Ciprofloxacin were used as standard drugs for the compare the efficacy of synthesized compounds against gram positive and gram negative bacteria respectively [30].
Nutrient agar broth medium was used for the preparation of inoculum of the bacteria and nutrient agar was used for the screening method.
The test organisms were subcultured using nutrient agar medium. The tubes containing sterilized medium were inoculated with the respective bacterial strain. After incubation at 37ºC ±1ºC for 18 hours, they were stored in a refrigerator. The nutrient agar medium was sterilized by autoclaving at 121ºC (15 lb/sq.inch) for 15 min. The petriplates, tubes and flasks plugged with cotton were sterilized in hot-air oven at 160ºC, for an hour. Into each sterilized petri plate (20 cm diameter), was poured about 125 ml of molten nutrient agar medium which was already inoculated with the respective strain of bacteria (5 ml of inoculum to 250 ml of nutrient agar medium) aseptically. The plates were left at room temperature aseptically to allow the solidification [31]. After solidification, the cups of each of 7 mm diameter were made by scooping out medium with a sterilized cork borer from a petri dish and labelled accordingly.
Each test compound (5 mg) was dissolved in dimethyl sulfoxide (5 ml AR grade) to give a concentration of 1000 g/ml. Norfloxacin solution were also prepared to give a concentration of 1000 µg/ml in sterilized distilled water. The pH of all the test solutions and control was maintained in between 2 to 3 by using conc. HCl. All the compounds were tested at dose levels of 50 g (0.05 ml) and 100 g (0.1 ml) and DMSO used as a control. The solutions of each test compound, control and reference standard (0.05 ml and 0.1 ml) were added separately in the cups and the plates were kept undisturbed for at least 2 hours in a refrigerator to allow diffusion of the solution properly into nutrient agar medium. Petri dishes were subsequently incubated at 37±1°C for 24 hrs. After incubation, the diameter of zone of inhibition surrounding each of the cups was measured with the help of an antibiotic zone reader [32,33]. The same procedure adopted for the gram-negative bacteria screening and Ciprofloxacin was used as a standard drug.

Scheme-I
Infrared spectroscopy and proton NMR spectroscopy were used to analyse the produced compounds, and they were confirmed to be trustworthy with a likely structure. The obtained compounds were analysed using IR and 1HNMR and found to have a structure that was compatible with what was predicted. The IR spectra show the peak at 1650-1658 (C=O); 1500-1580 (Ar C=C Quadrant), 761 (mono substituted benzene); 1105 (C-F str), 825 (C-Cl str), 1015 (C-Br str), and 1160 (C-Br str) (OCH3 str). These compounds were further validated by proton NMR, which revealed the chalcone system's typical ethylene protons in the range of 7.60 (C=O-CH str), 6.68-7.90 (Ar-H str), and 8.05 (=CH-Ar str). TLC was used to monitor the reaction, which used Hexane: ethyl acetate as the mobile phase.

Scheme-III
The synthesized compounds were characterized by the Infra-red spectroscopy and proton NMR spectroscopy and were found reliable with probable structure. Obtained compounds were characterized by IR, 1HNMR and were found consistent with an expected structure. The IR

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 str., aromatic) 1510. 25

CONCLUSION
All the 2-pyrazolines have been evaluated for their antibacterial activity against Staphylococcus Aureus, Staphylococcus Faecalis, Bacillus Substilis, P. Vulgaris and B. Pumilus (Grampositive) and Escherichia coli, Proteus vulgaris (Gram-negative), using Agar diffusion method. The results of this evaluation have been compared by taking benzyl penicillin, Ciprofloxacin was used as standard. The antibacterial activity data of 2-pyrazolines (BR-3>BR-2>BR-1>CL-4>BR-4>CL-3>CL-2) indicated that the compounds have significant inhibitory activity on all the bacteria at both 50 µg (0.05 ml) and 100 µg (0.1 ml) dose levels when compared with standard. Among all the compounds tested, compounds BR-3, BR-2, BR-1, CL-4, BR-4 and CL-3 possessed maximum activity. These compounds possessed the halogens on the aromatic ring and thus reveal the positive contribution of electron withdrawing groups to the antibacterial activity.
Presence of electronegative group (Br, Cl, F and NO 2 ) either at 3 rd and 5 th position of 1,3,5pyrazoline ring is required for the potent antimicrobial activity. Presence of electronegative group (Br, Cl) at 3 rd and 5 th position may necessary for the best activity against bacterial strains but the addition of F, NO 2 has shown the moderate activity but in case of -CH 3 and -OCH 3 substitution may diminish the activity.
The series BR-1 to BR-4 is most active compound of the synthesized compounds. This evident that the presence of bromine in the third and Fifth position of pyrazole is essential for the antimicrobial activity and -Cl, -Br, -F and -NO 2 group attached at phenyl ring enhance the antimicrobial activity. The result data of antimicrobial activity suggested that Cl, Br, F, and NO 2 substitution at 3 rd and 5 th position may enhance the antimicrobial activity of the compounds but the CH 3 and OCH 3 substitution may resulted in reduction of the activity.

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
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

ACKNOWLEDGEMENT
I would like to thank SAIF, Punjab University, Chandigarh for carried out the IR, 13C NMR, mass spectroscopy for characterization of synthesized compounds.