Synthesis, Characterization, In-silico and Biological Evaluation of Indazole Derivatives as Potential Anti-Inflammatory and Antimicrobial Agents

Introduction

Various heterocyclic compounds with antibacterial properties have been used as anti-inflammatory drugs, urinary antiseptics, and herbicides. Among these, heterocyclic derivatives of triadimedes have found applications in the treatment of common ailments.¹ A carbocyclic compound is an organic compound that is cyclic and contains all carbon atoms arranged in a ring structure. Heterocyclic compounds are widely used in various biological fields and are considered crucial organic molecules due to their ability to combat a wide range of ailments.²

Pyrazole

Pyrazoles are a class of heterocyclic compounds containing a five-membered ring and are highly effective in organic synthesis. The presence of pyrazole nuclei in various structures enables a wide range of applications in agriculture, technology, and medicine. These substances have a varied range of pharmacological activities, including antiviral, antimicrobial, anticancer, antidepressant, anti-inflammatory, and anti-tuberculosis effects. They are also recognized as potent inhibitors of protein glycation, indicating their potential as therapeutic agents for the treatment of a variety of illnesses.^3,4

Indazole

Indazole (1H-benzopyrazole) is a fused bicyclic heterocycle consisting of a pyrazole ring fused with a benzene ring.^7-9 This scaffold has garnered significant interest in medicinal chemistry due to its broad spectrum of biological activities, including anti-inflammatory, antimicrobial, anticancer, and analgesic properties. The therapeutic relevance of indazole derivatives has been demonstrated through their action on diverse biological targets, making them promising candidates in drug discovery. The varied biological features of the nitrogen containing heterocycle family, particularly the distinct indazole nuclei, have piqued attention in the past and in the present.^8,10-12

Chemistry of Indazole

When indazole was first discovered, scientist Emil Fisher described it as a "pyrazole ring fused with the benzene ring". Due to its fascinating chemical and biological characteristics, it has been investigated in enormous detail.^10,12,13 The azole family, which includes nitrogen, hydrogen, and carbon atoms, includes indazole.¹⁴ Benzopyrazoles or isoindazolones are heterocyclic chemical compounds containing two nitrogen atoms, and are commonly referred to as indazoles.^15-18

The aromatic heterocyclic indazole system comprises ten electrons. Compared to pyrazole, indazole exhibits dual reactivity, demonstrating the characteristics of both pyridine and pyrrole. Three tautomeric versions of indazole are: 1H-, 2H-, and 3H-.^18,19

Structurally, indazole exhibits tautomerism and dual reactivity, displaying both pyridine-like (basic) and pyrrole like (aromatic) behaviour. This duality contributes to its ability to interact with various enzymes and receptors, thereby influencing a range of pharmacodynamic profiles.

Given the urgent need for novel agents against inflammatory disorders and drug-resistant bacterial infections, the present study targeted two clinically validated enzymes: cyclooxygenase-2 (COX-2) and bacterial DNA gyrase. COX-2 is a key enzyme involved in the biosynthesis of prostaglandins that mediate inflammation, making it a prominent target for anti-inflammatory therapy. DNA gyrase, a type II topoisomerase essential for bacterial DNA replication, is a validated antimicrobial target, particularly against Gram-negative pathogens.

In this context, we designed and synthesized novel indazole derivatives bearing a 1,3,4-oxadiazole moiety with sulfanyl substitution to enhance their biological activity. These hybrids were evaluated for anti-inflammatory and antibacterial potential using a combination of molecular docking and in vitro assays.

Materials and Methods

A series of 4-(4-methoxyphenyl)-6-methyl-5-(5-sulfanyl-1,3,4-oxadiazol-2-yl)-4,5,6,7-tetrahydro-1H-indazole-3,6-diol derivatives (K-1 to K-6) were synthesized following a multi-step synthetic strategy as described below.

Synthesis

The synthesis of 4-(4-methoxyphenyl)-6-methyl-5-(5- sulfanyl-1,3,4-oxadiazol-2-yl)-4,5,6,7-tetrahydro-1H indazole-3,6-diol derivatives (K-1 to K-6) was carried out via a multi-step reaction pathway. Initially, 3-oxobutanoic acid (0.1 mol) was reacted with hydrazine hydrate (0.12 mol) in ethanol (50 mL) under reflux conditions at 80 °C for 4-5 hours to obtain the corresponding hydrazide intermediate. The reaction mixture was then cooled, and the precipitated solid was filtered and dried. (Figure 1)

In the next step, the hydrazide intermediate (0.05 mol) was treated with carbon disulfide (0.06 mol) and potassium hydroxide (0.06 mol) in methanol (40 mL). The mixture was refluxed at 70 °C for six hours to form the 1,3,4-oxadiazole ring. After completion, the reaction mixture was acidified with dilute hydrochloric acid to pH 5-6, and the resulting precipitate was collected by filtration and washed with cold water.

The oxadiazole intermediate (0.02 mol) was then subjected to cyclization by reacting with 4-methoxybenzaldehyde (0.02 mol) and methanamide (0.05 mol) in ethanol (30 mL) under reflux at 85°C for four hours. The reaction was cooled, poured into ice-cold water, and the solid product was filtered and dried.

In the final step, the resulting intermediate (0.01 mol) was condensed with various substituted aromatic aldehydes (0.01 mol) in ethanol (20 mL) using a few drops of glacial acetic acid as a catalyst. The reaction mixture was refluxed for three hours at 75 °C. Upon cooling, the final products precipitated out, were filtered, washed with cold ethanol, and recrystallized using an ethanol-water mixture (3:1) to yield pure derivatives labelled K-I to K-VI.

The final compounds were characterised using thin-layer chromatography (TLC) (ethyl acetate:hexane (7:3 v/v)), FT-IR, ¹H NMR, ¹³C NMR, and mass spectroscopy.

Characterization

The synthesized indazole derivatives (K-1 to K-6) were characterized using standard analytical techniques. Melting points were determined using an open capillary method and are uncorrected. Thin-layer chromatography (TLC) was carried out on silica gel-G plates, and Rf values were determined using a solvent system of ethyl acetate:hexane (7:3 v/v). Spots were visualized under UV light (254 nm) (Table 1).

Infrared (IR) spectra were recorded using a Shimadzu FTIR spectrophotometer in the range of 4000-400 cm⁻¹ using KBr pellets. The key absorption bands included –NH (3378.88 cm⁻¹), –OH (3562.81 cm⁻¹), Ar–CH (2938.09 cm⁻¹), C–H (2722.58 cm⁻¹), C=O (1741.47 cm⁻¹), C–S (1269.64 cm⁻¹), and -CH₃ (959.14 cm⁻¹).

¹H NMR spectra were obtained using a Bruker Avance III 400 MHz spectrometer with CDCl₃ as solvent and Tetramethylsilane (TMS) as internal standard. Characteristic signals included δ 4.75 (–NH), δ 4.6 (CH₂), δ 6.5-8.1 (aromatic protons), and δ 9.94 (–OH). 13C NMR spectra showed peaks at δ 33.83 (CH₃), δ 75.01 (CH₂), δ 70-150 (aromatic carbons), and δ 163.39 (C=O).

Mass spectra were recorded using an Agilent liquid chromatography-mass spectroscopy (LC-MS) system. The molecular ion peak for the final compound was observed at m/z 541.4 (M⁺+H), confirming the expected molecular mass.

Melting points and Rf values for all synthesized compounds are provided in Table 2 and Table 3, respectively.

Docking Studies

Molecular docking studies were conducted to evaluate the binding interactions of the synthesized indazole derivatives with two key biological targets: cyclooxygenase-2 (COX-2, PDB ID: 1CX2) and bacterial DNA gyrase (PDB ID: 4URO). Docking simulations were carried out using AutoDock Vina integrated within the PyRx 0.8 virtual screening tool.

Protein and Ligand Preparation

Crystal structures of COX-2 and DNA gyrase were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). Prior to docking, proteins were prepared by removing all water molecules, co-crystallized ligands, and ions using Swiss PDB viewer. Polar hydrogens were added, and Kollman charges were assigned using AutoDock tools. The synthesized ligands were sketched in ChemSketch, converted to 3D conformations, and energy-minimized using the MMFF94 force field in BIOVIA Discovery Studio 2020. Ligands were then saved in Protein Data Bank, Quaternion, and Torsional degrees of freedom (PDBQT) format.

Docking Parameters

Docking was performed using the Lamarckian Genetic Algorithm in AutoDock Vina. A grid box was defined to include the active sites of both targets:

. COX-2: Grid center at (x = 22.5, y = 26.3, z = 28.9), grid size: 25 × 25 × 25 Å

. DNA Gyrase: Grid center at (x = –12.4, y = 8.2, z = 9.7), grid size: 24 × 24 × 24 Å

The default scoring function of AutoDock Vina, which estimates binding affinity in kcal/mol, was used (Table 2).

Docking Validation

To validate the docking protocol, a redocking study was performed by extracting and re-docking the native ligand from each crystal structure. The Root Mean Square Deviation (RMSD) value between the native and redocked pose was found to be <2.0 Å, confirming the reliability of the docking procedure.

Biological Evaluation

In Vitro Inflammatory Activity

A laboratory technique for testing the anti-inflammatory effects of the produced chemicals is to denature bovine serum albumin (BSA). The test chemicals were dissolved in small amounts of dimethyl sulfoxide and then diluted with phosphate buffer (0.2 M, pH 7.4) to obtain the required quantity. All solutions were assured to have a final dimethyl sulfoxide (DMSO) concentration of less than 2.5% using this method. After mixing 1 mL of the test mixture containing varying drug dosages with 1 mL of phosphate buffer solution containing 1mM albumin, the mixture was left to stand at 270±10⁰C for 15 minutes. To induce denaturation, the reaction mixture was incubated in a water bath at 60±10◦C for 10 minutes. Turbidity was evaluated at 660 nm using Shimadzu spectrometry after the mixture had cooled. To calculate the percentage inhibition of denaturation, the drug-free control was removed from the total. Following the completion of each experiment, out of a total of three, the average was determined. To determine the percentage of inhibition, the following formula was applied. A practicable standard solution was developed similar to the test solution, and ibuprofen served as the reference control.

% Inhibition =( Vt ) x 100

where Vt is the a v e r a g e a b s orbance of triplicate drugs.

Vc = Control absorbance of average in triplicate

Antibacterial Activity

Initially, the test compounds were dissolved in sterile dimethylformamide (DMF) for preliminary screening using the established method of E. coli inhibition. The substances were subsequently submerged in sterile 1,4-dioxane, which was filter-sterilized through a 0.2 μm membrane filter due to its thermal instability and boiling point below 121°C. The antibacterial activity of the chemicals was assessed at a concentration of 200 μg/mL. Ciprofloxacin, dissolved in sterile water, served as the standard reference. Additionally, 1,4-dioxane and water were used as controls in the experiment.

Results

Characterization of Synthesized Indazole Derivatives

The structural elucidation of the synthesized indazole derivatives was confirmed using melting point determination, thin-layer chromatography (TLC), FT-IR, 1H NMR, 13C NMR, and mass spectrometry.

The final compound, 3-acetoxy-5-((5-(3,6-dihydroxy- 4-(4-methoxyphenyl)-6-methyl-4,5,6,7-tetrahydro-1H-indazol-5-yl)-1,3,4-oxadiazol-2-ylthio)methyl)benzoic acid, exhibited a yield of 76.2% and a melting point in the range of 140-142 °C.

Melting Point and TLC

Melting points for all synthesized compounds were sharp and consistent, and the Rf values ranged from 0.62 to 0.72, confirming their purity. (Table 3)

The solvent system used was ethyl acetate:hexane (7:3), and visualization was performed under UV light at 254 nm. The reported values represent the mean of triplicate determinations.

Spectral Data

3-acetoxy-5-((5-(3,6-dihydroxy-4-(4-methoxyphenyl) 6-methyl-4,5,6,7-tetrahydro 1Hindazol-5-yl)-1,3,4-oxadiazol-2-ylthio)methyl)benzoic acid

Yield: 76.20%. melting point 140-142°C. IR: 3378.88: N-H stretching, 3562.81: O–H stretching, 2938.09: Aromatic C-H stretching, 2722.58: Aliphatic C-H, 1741.47: C=O (ester/carboxylic), 1269.64: C-S stretching, 959.14: CH₃ bending.

¹H- NMR (CDCL3): δ (ppm) δ 9.94 (s, 1H, OH), δ 8.10- 6.50 (m, 11H, aromatic protons), δ 4.75 (s, 1H, NH), δ 4.60 (s, 2H, CH₂), δ 3.85 (s, 3H, OCH₃), δ 2.12 (s, 3H, CH₃).

¹³C NMR (CDCL3): δ (ppm) δ 163.39 (C=O), δ 150- 70 (aromatic/heterocyclic carbons, 21 signals), δ 75.01 (CH₂), δ 33.83 (CH₃)

Mass Spectroscopy

The LC-MS spectrum of the compound exhibited a molecular ion peak at m/z 541.4 [M+H]+, consistent with the calculated molecular weight.

Biological Evaluation

In Vitro Inflammatory Activity

The anti-inflammatory activity of the synthesized indazole derivatives was evaluated in vitro using the bovine serum albumin (BSA) denaturation assay. Test compounds were prepared at concentrations of 50, 100, 250, and 500 μg/mL. Each compound was initially dissolved in dimethyl sulfoxide (DMSO), then diluted with phosphate buffer (0.2 M, pH 7.4), to ensure a final DMSO concentration of less than 2.5%.

For each test concentration, 1 mL of the test solution was mixed with 1 mL of BSA solution (1 mM) and incubated at 27 ± 1 °C for 15 minutes. The reaction mixture was then heated in a water bath at 60 ± 1 °C for 10 minutes to induce protein denaturation, followed by cooling at room temperature. Turbidity was measured at 660 nm using a Shimadzu UV-Vis spectrophotometer.

Ibuprofen (100 μg/mL) was used as the reference standard. All experiments were carried out in triplicate, and results were expressed as mean ± standard deviation (SD).

The percentage inhibition of protein denaturation was calculated using the following corrected formula:

% Inhibition=[Vc-Vt/Vc]x 100

Where, Vc is the absorbance of the control, and Vt is the absorbance of the test sample.

Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test to determine significant differences among the groups. A P value of <0.05 was considered statistically significant.

Among the tested compounds, K-3 and K-5 exhibited the highest anti-inflammatory activity, with K-3 showing 74.56% inhibition at 500 μg/mL and an average of 59.39 ± 2.12%, while ibuprofen showed 88.64 ± 1.04% inhibition. These results suggest that the tested derivatives demonstrate dose-dependent anti-inflammatory activity, with select compounds showing promising potential for further pharmacological development.

Table 4 dissipates the in vitro anti-inflammatory activity of derivatives of 4-(4-methoxyphenyl)-6-methyl-5-(5- sulfanyl-1, 3, 4-oxadiazol-2-yl)-4,5,6,7-tetrahydro-1H-indazole-3,6-diol.

Antibacterial Activity

The antibacterial activity of the synthesized indazole derivatives was evaluated using the agar disc diffusion method against two bacterial strains: Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923).

Sterile discs (6 mm diameter) were impregnated with 200 μg of each test compound dissolved in dimethylformamide (DMF), and allowed to dry. Mueller-Hinton Agar (MHA) plates were inoculated with standardized bacterial suspensions adjusted to 0.5 McFarland standard (approximately 1.5 × 10⁸ CFU/mL). The discs were placed onto the surface of the agar, and the plates were incubated at 37 °C for 24 hours.

Ciprofloxacin (10 μg/disc) was used as the positive control, while DMF alone served as the vehicle control. It was confirmed that DMF did not produce any zone of inhibition, indicating no inherent antibacterial effect.

The diameter of the zone of inhibition was measured in millimeters (mm) and recorded as mean ± standard deviation (SD) from three independent replicates.

Among the derivatives, K-3 exhibited the most potent antibacterial activity, with inhibition zones of 23.0±0.5 mm (E. coli) and 24.0±0.4 mm (S. aureus), comparable to ciprofloxacin (25.0±0.3 mm and 24.0±0.2 mm, respectively). Compounds K-5 and K49 also demonstrated significant inhibition against both strains, whereas K7 and K18 showed minimal activity.

These results suggest that specific substitutions on the indazole scaffold enhance antibacterial efficacy, particularly against Gram-positive and Gram-negative organisms. The promising activity of K-3 and K-5 highlights their potential as lead molecules for further antibacterial drug development.(Table 5)

Discussion

In the present study, a series of novel 4-(4-methoxy-phenyl)-6-methyl-5-(5-sulfanyl-1,3,4-oxadiazol-2-yl)- 4,5,6,7-tetrahydro-1H-indazole-3,6-diol derivatives were synthesized and evaluated for their antiinflammatory and antimicrobial activities. Structural modifications were aimed at enhancing biological efficacy through strategic substitution on the indazole-oxadiazole scaffold.

Anti-Inflammatory Activity

The in vitro anti-inflammatory evaluation was carried out using the BSA denaturation method, a well-established assay for detecting the ability of compounds to prevent protein denaturation-an essential step in inflammation. Among the synthesized compounds, K-3 and K-5 demonstrated the most significant inhibition of protein denaturation, showing 59.39% and 54.46% inhibition, respectively, at 500 μg/mL.

The structure-activity relationship (SAR) suggests that the presence of a para-methoxy group on the phenyl ring (as in K-3 and K-5) contributes to enhanced activity. This may be attributed to increased hydrogen bonding potential and improved membrane permeability, which facilitate better interaction with active site residues of inflammatory mediators such as COX-2.²⁰ Additionally, the sulfanyl-1,3,4-oxadiazole moiety may act as a pharmacophoric unit that enhances binding through electrostatic and van der Waals interactions.

When compared with the standard ibuprofen (88.64% inhibition at 100 μg/mL), the test compounds exhibited moderate anti-inflammatory activity, suggesting room for optimization through further derivatization or prodrug strategies.

Antibacterial Activity

The synthesized compounds also exhibited variable degrees of antibacterial activity against E. coli and S. aureus using the agar disc diffusion method. Compounds K-3 and K-5 again showed superior performance, with zones of inhibition comparable to ciprofloxacin (10 μg/ mL), indicating their broad-spectrum potential.

SAR analysis in this context reveals that electron-donating substituents such as methoxy groups improve antibacterial potency, likely by enhancing lipophilicity and promoting membrane penetration. The combination of the indazole core and the oxadiazole ring appears to favour interactions with bacterial enzymes such as DNA gyrase, as supported by docking results.

Literature reports highlight that drugs such as diclofenac and ibuprofen show potent anti-inflammatory activity through COX-2 inhibition, often exceeding 80% inhibition at lower doses. Likewise, ciprofloxacin, a fluoroquinolone antibiotic, is highly effective against both Gram-positive and Gram-negative bacteria, with inhibition zones generally ranging from 24-26 mm.^20,21 Although the synthesized compounds fall slightly short of these standards, they demonstrate promising activity, particularly in the case of K-3 and K-5, which may serve as lead scaffolds for further optimization.²²

While the results are encouraging, this study has certain limitations. The biological assays were limited to in vitro screening, and no cytotoxicity or in vivo anti-inflammatory or antibacterial studies were conducted. Further investigations, including mechanistic enzymatic assays, in vivo pharmacological validation, and Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiling, are essential to establish the therapeutic potential and safety profile of these compounds.

Conclusion

In this study, a series of novel indazole derivatives incorporating a 1,3,4-oxadiazole moiety were successfully synthesized and evaluated for their anti-inflammatory and antibacterial potential. Among them, compounds K-3 and K-5 emerged as promising dual inhibitors, demonstrating significant activity against both COX- 2 and DNA gyrase in molecular docking studies, with strong binding affinities and favorable interactions with key active site residues.

These findings were supported by in vitro assays, where K-3 and K-5 showed substantial protein denaturation inhibition and zone of bacterial inhibition, respectively. When compared with reference drugs such as ibuprofen (anti-inflammatory) and ciprofloxacin (antibacterial), the test compounds displayed comparable activity, suggesting that further structural optimization may yield therapeutically viable leads.

Overall, the study highlights the potential of methoxy-substituted indazole-oxadiazole hybrids as a valuable scaffold for the development of anti-inflammatory and antimicrobial agents. Future work will focus on in vivo validation, cytotoxicity profiling, and pharmacokinetic studies to further establish their drug-like potential.

Conflict of Interest

No

Acknowledgement

We would like to thank the principal and professors of the SSM College of Pharmacy and Study Center at BLDEA’s, Vijayapura and also The Oxford College of Pharmacy, Bengaluru for their steadfast encouragement and moral support during our research work.