1. INTRODUCTION:

Microwave synthesis is recognized as a crucial technique in green chemistry due to its eco-friendly nature, providing a swift and an effective substitute to traditional methods of heating.¹ It offers benefits such as reduced reaction times, lower costs, simplicity in processing, less pollution, high yields, and no direct contact between the energy source and reaction vessels. This is how chemical synthesis will be a component of the sustainable development approach.² Heterocyclic compounds have primarily been synthesized due to their diverse biological activities.³ Considerable attention has been directed towards synthesizing aza-heterocycles.⁴ Pyrazolines are notable nitrogen(aza)-containing heterocyclic compounds that play a significant role in medicinal chemistry.⁵ Significant focus has been placed on pyrazoline derivatives because of their intriguing pharmacological properties.⁶ They have found to possess antifungal⁷, antibacterial^8-9, antidepressant¹⁰, anticonvulsant¹¹, anti-inflammatory^12-14, antitumor^15-17, antidiabetic¹⁸, antitubercular^19-20, and analgesic²¹ properties. The traditional synthesis of the title compounds involves Claisen-Schmidt condensation of aromatic aldehydes with ketones to form α, β-unsaturated ketones (chalcones). Chalcone is an aromatic ketone and an enone that serves as the building block for numerous significant biological molecules.²² The chalcone configuration consists of three fundamental components: a pair of phenyl rings and the α, β-unsaturated carbonyl framework that connects them.²³

These chalcones when cyclized with hydroxylamine hydrochloride in an alkaline medium produces isoxazole derivatives. Additionally, chalcones cyclized with phenyl hydrazine hydrochloride under alkaline conditions yield pyrazoline derivatives.^24-25 Pyrazolines can be synthesized through several methods, such as condensing chalcones with hydrazine, hydrazine derivatives, or thiosemicarbazide in either acidic or basic conditions.²⁶ Addressing infectious diseases remains a critical and challenging issue due to factors such as emerging pathogens and the increasing prevalence of multi-drug-resistant microorganisms. This problem is notably severe in hospitalized patients, as well as those with immunocompromised conditions such as AIDS, cancer treatments, or organ transplants. Despite the availability of a wide range of antibiotics and chemotherapeutics, the rise of both established and novel antibiotic resistances in recent decades underscores the urgent need for the development of new classes of antibacterial agents.^27-28 A promising approach to tackle antibiotic resistance is to create new compounds with unique mechanisms of action, thereby avoiding cross-resistance with current treatments. Infectious diseases continue to be a major cause of death globally. Over recent decades, new infectious diseases have emerged, and previously controlled ones have reappeared. Despite significant advancements in antimicrobial therapy, numerous challenges persist with the currently available drugs. Consequently, there is an ongoing need for the discovery of new antimicrobial agents with improved pharmacological profiles. The rise of multi-drug-resistant microorganisms has reached a critical level, presenting a serious challenge to the medical community. This has intensified the search for effective novel antimicrobial agents.^29-31

2. EXPERIMENTAL WORK:

2.1 Materials and methods:

Chemicals and solvents (ethanol and acetone) used were of laboratory grade. IR spectra (KBr, cm-1) were recorded on a FT-IR - 8400 S, Shimadzu Corporation, Kyoto, Japan. ¹H NMR spectral analysis was carried out at IICT, Hyderabad using an AVANCE-300 (300 MHz FT NMR) spectrometer. Mass spectra was recorded on Micromass Quatto II triple quadrapole Mass Spectrometer, IICT, Hyderabad, India. Reactions were carried out in a CATA’s Scientific Microwave Synthesis System Model No. RG 31L 2R, Power output 700 W, 2450 MHz, Catalyst Total Scientific Solutions.

2.2 Preparation of chalcone (I):

A solution containing substituted acetophenone (0.01 mol) and substituted benzaldehydes (0.02 mol) in dry ethanol (20 ml) was prepared in a beaker. Sodium hydroxide was added in catalytic amounts (1-2 pellets), and the reaction mixture was subjected to microwave irradiation for 2 minutes at 210 watts (30% microwave power). The mixture was then cooled in an ice bath. The resulting product was filtered, washed with ethanol (5 ml), followed by water, and recrystallized from acetone.

2.3 Procedure for the synthesis of 3-substituted phenyl-5-substituted phenyl-4,5-dihydro-pyrazol-1-carbothiamide (II):

A mixture of chalcone (0.022 mol) and thiosemicarbazide (0.02 mol) was dissolved in a combination of acetone (5 ml) and ethanol (5 ml). Potassium carbonate (K₂CO₃) was added, and the mixture was stirred vigorously before being irradiated in a microwave oven for 1 minute at 700 watts. The reaction mixture was then poured over crushed ice. The resulting product was filtered and recrystallized from a 1:1 (v/v) mixture of acetone and ethanol.

2.3.1: 3-(4-nitrophenyl)-5-phenyl-4, 5 dihydropyrazole-1-carbothiamide (IIa):

Brown solid, Yield: 75.62 %. mp 122-124°C Rf: 0.64 (Chloroform/methanol 1:9). IR (KBr, cm^-1): 3490.92 cm¹ (NH₂), 3363.62 (NH₂), 1577.66 (C=N), 3056.96 (Ar C-H).¹H-NMR: 7.02-7.71, (m, Ar-H), 2.03 (s, NH₂). EI-MS: 326.00.

2.3.2: 3-(4-hydroxyphenyl)-5-phenyl-4, 5 dihydropyrazole-1-carbothiamide (IIb):

Yellow solid, Yield: 72.22 %. mp 184-186°C Rf: 0.58 (Chloroform/methanol 1:9). IR (KBr, cm^-1): 3438.84 (NH₂), 3224.74 (NH₂), 1579.59 (C=N), 3064.68 (Ar C-H).¹H-NMR: 7.02-7.47 (m, Ar-H), 2.45 (s, NH₂). EI-MS: 297.14.

2.3.3: 3-(4-dimethylaminophenyl) 5-phenyl -4,5 dihydropyrazole-1-carbothiamide (IIc):

Dark brown solid, Yield: 70.87 %. mp 220-222°C Rf: 0.59 (Chloroform/methanol 1:9). IR (KBr, cm^-1): 3377.12 (NH₂), 3234.40 (NH₂), 1587.31 (C=N), 3078.18 (Ar C-H).¹H-NMR: 7.09-7.70 (m, Ar-H), 2.92 (s, NH₂). EI-MS: 324.00.

2.3.4: 3-phenyl-5-(4-bromophenyl) 4, 5 dihydropyrazole-1-carbothiamide (IId):

Off white solid, Yield: 72.56 %. mp 140-142°C Rf: 0.60 (Acetone/methanol 1:9). IR (KBr, cm^-1): 3427.27 (NH₂), 3236.33 (NH₂), 1585.38 (C=N), 3050.55 (Ar C-H).¹H-NMR: 7.09-7.44, m (Ar-H), 2.62 (s, NH₂). EI-MS: 360.60.

2.3.5: 3-(2-bromo phenyl)-5-phenyl- 4, 5 dihydropyrazole-1-carbothiamide (IIe):

Brown solid, Yield: 68.22 %. mp 168-170°C Rf: 0.55 (Acetone/methanol 1:9). IR (KBr, cm^-1): 3321.27 (NH₂), 3287.45 (NH₂), 1595.46 (C=N), 3056.65 (Ar C-H).¹H-NMR: 7.29-7.54, m (Ar-H), 3.12 (s, NH₂). EI-MS: 360.60.

2.3.6: 3-(3-nitrophenyl)-5-phenyl-4, 5 dihydropyrazole-1-carbothiamide (IIf):

Orange solid, Yield: 65.34 %. mp 180-182°C Rf: 0.61 (Acetone/methanol 1:9). IR (KBr, cm^-1): 3314.25 (NH₂), 3255.74 (NH₂), 1591.66 (C=N), 3055.46 (Ar C-H).¹H-NMR: 7.32-7.58, m (Ar-H), 2.34 (s, NH₂). EI-MS: 326.20.

2.3.7: 3-(3-bromo phenyl-5-phenyl-4, 5 dihydropyrazole-1-carbothiamide (IIg):

White solid, Yield: 71.15 %. mp 214-216°C Rf: 0.65 (chloroform/methanol 1:9). IR (KBr, cm^-1): 3338.56 (NH₂), 3267.41 (NH₂), 1592.23 (C=N), 3087.51 (Ar C-H).¹H-NMR: 7.23-7.47, m (Ar-H), 3.07 (s, NH₂). EI-MS: 360.40.

2.3.8: 3-(3-hydroxyphenyl)-5-phenyl-4, 5 dihydropyrazole-1-carbothiamide (IIh):

White solid, Yield: 73.37 %. mp 198-200°C Rf: 0.70 (chloroform/methanol 1:9). IR (KBr, cm^-1): 3436.44 (NH₂), 3269.48 (NH₂), 1595.23 (C=N), 3096.52 (Ar C-H).¹H-NMR: 7.21-7.33 m (Ar-H), 2.87 (s, NH₂). EI-MS: 297.20.

2.3.9: 3-phenyl-5-(4-nitrophenyl)-4, 5 dihydropyrazole-1-carbothiamide (IIi):

Off white solid, Yield: 67.45 %. mp 240-242°C Rf: 0.62 (chloroform/methanol 1:9). IR (KBr, cm^-1): 3338.56 (NH₂), 3267.41 (NH₂), 1592.23 (C=N), 3087.51 (Ar C-H).¹H-NMR: 7.23-7.47, m (Ar-H), 3.07 (d, NH₂). EI-MS: 326.80.

2.3.10: 3-(4-fluorophenyl)-5-phenyl-4, 5 dihydropyrazole-1-carbothiamide (IIj):

Off white solid, Yield: 69.87 %. mp 190-192°C Rf: 0.59 (Acetone/methanol 1:9). IR (KBr, cm^-1): 3421.16 (NH₂), 3168.56 (NH₂), 1587.28 (C=N), 3108.11 (Ar C-H).¹H-NMR: 7.23-7.47, m (Ar-H), 3.07 (d, NH₂). EI-MS: 299.20.

2.4 Antibacterial and Antifungal Screening:

The agar dilution method was conducted using Muller-Hinton agar (Hi-Media) for antibacterial activity and Sabouraud’s dextrose agar (Hi-Media) for antifungal activity. The media were sterilized by autoclaving at 15 psi for 30 minutes. A loopful of the stock culture was transferred into 10 mL of agar slant in sterilized test tubes, which were then incubated at 37°C for 24 hours for bacterial cultures and 72 hours for fungal cultures. After incubation, 3 mL of distilled water was added to each test tube, and the cultures were agitated to ensure a uniform suspension. Post-incubation, 3 mL of distilled water was introduced into each test tube, and the cultures were agitated to achieve uniform suspension. Test compounds (50 mg) were dissolved in 10 mL DMSO to create a 5 mg/mL solution. Serial dilutions were made with DMSO to obtain concentrations of 50, 100, 300, and 500 µg/mL. Similarly, standard drugs (ciprofloxacin and fluconazole) were diluted to the same concentrations. All procedures were performed under aseptic conditions. The sterile medium was melted in a water bath and maintained at 45°C. It was then poured into sterile petri dishes to a thickness of approximately 4-5 mm, and the inoculated microorganisms were added. The dishes were allowed to set at room temperature for 30 minutes. Wells of 6 mm diameter were created using a sterile stainless-steel bore, and 1 mL of each solution was added to the wells. The Petri dishes were then refrigerated for 30 minutes to facilitate the diffusion of the solutions. Following this, they were incubated at 37°C for 24 hours to evaluate antibacterial activity and for 72 hours to assess antifungal activity. The zones of inhibition were measured in millimeters, and the compounds were evaluated based on their effective concentrations.

2.5 Molecular Docking Study:

Molecular docking studies were conducted using AutoDock Vina 1.5.7 software. The crystal structure of S. aureus biotin protein ligase (PDB ID: 3V7R) with 2.30 Å resolution was downloaded from the protein data bank. After selection of protein, any water molecules, ions, or other non-protein entities were removed from the PDB file in PyMol, leaving only the protein structure. Polar Hydrogen atoms and Kollman charges were added to the protein structure using AutoDock Vina 1.5.7. The protein structure was inspected for missing atoms, incorrect bonds, or other structural issues, and any problems were repaired to ensure a valid and functional protein structure. The modified protein structure was saved in PDBQT format. This PDBQT file was used as the input for the docking simulation. Ligand molecules were designed in ChemDraw Ultra 12.0.2 and saved in a .sdf format then by the help of PyMol Software the extension was converted into .pdb format. Hydrogen atoms were added to the ligand using AutoDock Vina 1.5.7. The modified ligand structure was saved in PDBQT format. This PDBQT file was used as the input for the docking simulation. A text file named `config.txt` was created to specify the docking parameters and input files. This file included the size of the active site and its x, y, and z center, defining the 3D search space within the protein's active site or region of interest for the docking simulation. The exhaustiveness parameter was set to control the thoroughness of the docking search. The configuration file was saved in .txt format. A command prompt was opened. The directory containing the AutoDock Vina and the prepared protein, ligand, and configuration files was navigated. AutoDock Vina was executed with the input files. The docking simulation ran, and output files containing information about the predicted binding poses and their corresponding binding energies were generated by AutoDock Vina.

2.6 Drug- and lead-likeness, Pharmacokinetic, Pharmacogenomic Profile and ADMET Prediction

SMILES notations for the studied compounds were entered into the SwissADME web server (http://www.swissadme.ch/) to predict their pharmacokinetics and drug-likeness characteristics. The pkCSM database was used to assess the ADMET properties of the compounds, including human intestinal absorption (%), blood-brain barrier permeability, AMES toxicity, inhibition of hERG I and II, and hepatotoxicity.

3. RESULTS AND DISCUSSION:

3.1 Chemistry:

The protocol for the preparation of 3-substituted phenyl-5-substituted phenyl-4,5-dihydro-pyrazol-1-carbothiamide (IIa-j) is depicted in Scheme 1.

Scheme 1: Synthesis of 4, 5-dihydropyrazole derivatives

A solution of substituted acetophenone (0.01mol) and substitute benzaldehydes (0.02 mol) in presence of base catalyst were used in first step to produce chalcone. Chalcone were then reacted with thiosemicarbazide (0.02 mol) and K₂CO₃to produce compounds IIa-j. The compounds were prepared in good yield (65.34 to 73.37 %) and synthesis of compound was supported by spectral analysis viz., FTIR, ¹H NMR, and MS. The IR spectrum of synthesized derivatives IIa-j represented stretching band in between 3400-3200 cm^-1 confirmed the presence of primary amino group, stretching band at 1595.46-1577.66 cm^-1 indicates the presence of C=N group whereas stretching band at near 950 cm^-1 indicates the presence of C-S group. The presence of aromatic proton was evaluated by the ¹H NMR spectra which revealed multiplet at 7.02-7.58 ppm along with presence of singlet of NH₂in the range of 3.12-2.07 ppm. Each compound represented mass spectra in positive mode [M+H]⁺, against molecular weight and thus confirms synthesis of compound.

3.2 Biological Activity:

The antibacterial activity was performed against E. coli and S. aureus while the antifungal activity was tested against A. niger. Ciprofloxacin and Fluconazole served as standards for antibacterial and antifungal activity, respectively.

Table 1: Antibacterial and Antifungal activity data of synthesized derivatives IIa-j

Compounds	R₁	R₂	Zone of inhibition (mm)
			Antibacterial Activity								Antifungal Activity
			*E.coli*				*S . aureus*				*A. niger*
			50 μg/ ml	100 μg/ ml	300 μg/ ml	500 μg/ ml	50 μg/ ml	100 μg/ ml	300 μg/ ml	500 μg/ ml	50 μg/ ml	100 μg/ ml	300 μg/ ml	500 μg/ ml
IIa	p-NO₂	H	-	12	14	18	-	11	15	17	-	14	15	18
IIb	p-OH	H	11	15	18	25	12	15	18	26	-	12	18	26
IIc	p-N-(CH₃)₂	H	-	10	11	13	-	12	14	18	-	11	14	18
IId	H	p-Br	-	10	13	19	-	14	16	20	12	14	19	28
IIe	o-Br	H	-	10	12	18	-	12	14	18	-	11	13	20
IIf	p-NO₂	H	-	12	13	15	-	11	13	17	-	12	14	15
IIg	m-NO₂	H	-	13	14	16	-	12	15	18	-	13	16	18
IIh	m-Br	H	-	11	12	17	-	11	13	16	-	12	15	18
IIi	m-OH	H	-	12	15	17	10	15	19	25	-	13	18	22
IIj	H	p-NO₂	11	13	14	23	11	15	26	28	16	20	25	30
Cipro.	-	-	13	16	24	31	13	16	24	31	-	-	-	-
Fluco.	-	-	-	-	-	-	-	-	-	-	15	19	28	35

Cipro =Ciprofloxcin ; Fluco =Fluconazole

Table 2. List of intermolecular interactions between the ligand molecules docked with 3V7R (biotin protein ligase from S. aureus)

Compounds	Docking score	Interacting amino acids	Compounds	Docking score	Interacting amino acids
lla	-7.9	Arg122A, Trp127A, Lys187A, Ile224A	llg	-7.2	His126A, Ile224A
llb	-8.8	Arg122A, Ile224A, Trp127A	llh	-8.6	Asp180A, Ser128A, Ile224A, Trp127A
llc	-7.0	Asn179A, Arg122A, Met195A, Ile150A	lli	-8.6	Asp180A, Trp127A, Ile224A, Arg227A
lld	-8.3	Trp127A, Ile224A, Trp127A	llj	-9.1	Trp127A, Ile224A
lle	-7.9	Trp127A, Ser128A, Trp127A, Ile224A	Ciprofloxacin	-7.2	Asn154A, Ser318A, Ile317A
llf	-7.4	Trp127A, Asn212A, Al228A, Lys187A, Ile224A	Fluconazole	-7.3	Trp127A, Ile224A, Lys187A, Trp127A

Table 3: The drug-likeness features according to Lipinski’s rule of five and Veber, Ghose, Egan and Muegge rules applied for compounds IIa-j

Compound	Molecular Mass (<500 Da)	Rotatable bonds	No. of H-bond acceptors (<10)	No. of H-bond donars (<5)	XLog p (<5)	Surface area TPSA	No. of violations of Rule of Five	Lipinski	Ghose	Veber	Egan	Muegge
IIa	326.37	4	3	1	2.56	119.53	0	Yes	Yes	Yes	Yes	Yes
IIb	295.36	3	2	2	3.11	96.16	0	Yes	No	Yes	No	No
IIc	322.43	4	1	1	3.59	79.17	0	Yes	No	Yes	No	No
IId	358.26	3	1	1	4.16	75.93	0	Yes	Yes	Yes	Yes	Yes
IIe	358.26	3	1	1	4.16	75.93	0	Yes	No	Yes	No	No
IIf	326.37	4	3	1	2.56	119.53	0	Yes	Yes	Yes	Yes	Yes
IIg	358.26	3	1	1	4.16	75.93	0	Yes	No	Yes	No	No
IIh	295.36	3	2	2	3.11	96.16	0	Yes	No	Yes	No	No
IIi	326.37	4	3	1	2.56	119.53	0	Yes	Yes	Yes	Yes	Yes
IIj	297.35	3	2	1	3.57	75.93	0	Yes	No	Yes	No	No

Table 4. Computational pharmacokinetic and toxicity profiles for compounds

Compound	Intestinal absorption	BBB permeability	CYP1A2 Inhibitor	CYP2C19 Inhibitor	CYP2C9 Inhibitor	CYP2D6 Inhibitor	CYP3A4 Inhibitor	hERG I/II inhibitor	AMES toxicity	Hepatotoxicity
IIa	91.493	-0.22	No	Yes	Yes	No	Yes	No/No	No	No
IIb	90.766	-0.017	Yes	Yes	Yes	No	No	No/No	No	No
IIc	94.631	0.178	No	Yes	Yes	No	Yes	No/No	No	No
IId	91.767	0.217	Yes	Yes	Yes	No	No	No/No	No	No
IIe	91.535	0.259	Yes	Yes	Yes	No	No	No/No	No	No
IIf	91.865	-0.172	No	Yes	Yes	No	Yes	No/No	No	No
IIg	92.406	0.227	Yes	Yes	Yes	No	No	No/No	No	No
IIh	92.913	0.004	No	Yes	Yes	No	No	No/No	No	No
IIi	91.489	-0.189	No	Yes	Yes	No	Yes	No/No	No	No
IIj	92.818	0.22	Yes	Yes	Yes	No	No	No/No	No	No

3.4 Drug- and lead-likeness, Pharmacokinetic, Pharmacogenomic Profile and ADMET Prediction:

Based on Lipinski's Rule of Five, evaluated through the SWISSADME web server, all tested compounds adhere to the rule's criteria for good permeability and bioavailability, with rotatable bonds ranging from 3 to 4. Further analysis of compounds IIa-j was conducted using several medicinal chemistry rules—Lipinski, Ghose, Veber, Muegge and Egan—via SwissADME (Table 3). Additionally, pharmacogenomic profiles were assessed to determine whether the compounds might act as substrates or inhibitors of CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4. SMILES files for molecules IIa-j were uploaded to the pkCSM database to evaluate their ADMET properties, including human intestinal absorption (in %), blood-brain barrier (BBB) permeability and toxicity. (Table 4). All compounds are free from toxicities like AMES, cardiotoxicity and hepatotoxicity.

4. CONCLUSION:

In this study, substituted 4,5-dihydro-pyrazol-1-carbothiamide derivatives (IIa-j) were synthesized using microwave-assisted methods and assessed for their antibacterial activity against E. coli and S. aureus, as well as their antifungal activity against A. niger. The microwave-assisted synthesis provided several benefits, including a simple reaction setup, the use of commercially available and inexpensive reagents, straightforward workup, high product yield, product purity, and reduced reaction time. All synthesized derivatives showed mild to good activity against the tested strains. Specifically, compounds IIb and IIj demonstrated activity against E. coli, while compounds IIb, IIi, and IIj were active against S. aureus. Additionally, compounds IId and IIj exhibited strong antifungal activity against A. niger. Among the derivatives, compound IIj was found to be the most effective across all tested strains, and docking studies indicated that IIj achieved a good docking score (-9.1) against the 3V7R target. Therefore, compound IIj, with its broad-spectrum activity, has potential as a template for the development of future antimicrobial agents.

5. ABBREVIATIONS:

TLC: Thin layer chromatography; NMR: Nuclear magnetic resonance; FT-IR: Fourier Transform infrared spectroscopy; E. coli: Escherichia coli; S. aureus: Staphylococcus aureus; A. niger: Aspergillus niger.

6. CONFLICT OF INTEREST:

There was no conflict of interest among the authors.

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