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INTRODUCTION
Inflammation is a critical physiological response triggered by harmful agents such as pathogens, toxins, and damaged tissues. It is part of the innate immune defense system designed to eliminate the injurious stimuli and initiate the healing process. While acute inflammation is protective and short-lived, aiding in tissue repair and pathogen clearance, chronic inflammation is persistent and pathological, contributing to the development of several serious diseases such as rheumatoid arthritis, atherosclerosis, type 2 diabetes, Alzheimer’s disease, and various types of cancer (Medzhitov, 2008; Libby, 2007). Commonly used anti-inflammatory therapies, including Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) and corticosteroids, are effective but often associated with adverse outcomes. Prolonged use of NSAIDs can result in gastrointestinal ulcers, renal impairment, and increased cardiovascular risk, whereas corticosteroids may cause immune suppression and hormonal imbalance (Vane, 1998; Rainsford, 2007). These drawbacks underscore the necessity to develop safer and more selective anti-inflammatory drugs with minimal side effects. With recent progress in computational biology and molecular modeling, drug discovery processes have become more efficient and targeted. In particular, molecular docking has emerged as a valuable in silico approach that allows researchers to study the interaction of bioactive compounds with specific molecular targets. Docking simulations help predict how small molecules bind to the active sites of key enzymes or receptors involved in inflammation, estimating binding affinity, interaction types, and binding conformations. These insights support the virtual screening and rational design of potential therapeutic agents before experimental validation (Kitchen et al., 2004; Lionta, 2014). Several biomolecular targets are widely recognized in the regulation of inflammatory responses:
- Cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis involved in pain and swelling.
- 5-Lipoxygenase (5-LOX), important in leukotriene formation contributing to allergic and chronic inflammatory responses.
- Nuclear factor kappa B (NF-κB), a transcription factor regulating pro-inflammatory gene expression.
- Inducible Nitric Oxide Synthase (iNOS), which produces nitric oxide in inflammatory tissues.
- Tumor Necrosis Factor-alpha (TNF-α), a central cytokine mediating systemic inflammation (Lawrence, 2009; Singh and Agrawal, 2008).
The integration of computational drug discovery tools such as docking, pharmacophore modeling, and virtual screening accelerates the identification of novel lead molecules with potential anti-inflammatory effects. This study aims to explore the anti-inflammatory activity of selected natural and/or synthetic compounds by conducting molecular docking against well-characterized pro-inflammatory targets. The results from this computational evaluation will provide valuable insights into binding mechanisms, interaction patterns, and their possible therapeutic relevance in treating inflammatory diseases.
AIM
To synthesize and characterize a series of 4, 6-diphenylpyrimidin-2-amine derivatives, evaluate their potential anti-inflammatory activity through in silico molecular docking studies against key pro-inflammatory targets such as Taurine dehydrogenase, and validate their anti-inflammatory efficacy using the carrageenan-induced rat paw edema model.
MATERIALS AND METHODS
Materials
All reagents, solvents, and synthetic starting materials used in the study were of analytical grade and obtained from commercial sources without further purification. The synthesis and characterization of the target compounds were performed using the following standard analytical techniques:
- Determination of physical constants (melting point),
- Thin-Layer Chromatography (TLC),
- Elemental analysis,
- Fourier-Transform Infrared (FT-IR) spectroscopy,
- Proton Nuclear Magnetic Resonance (¹H NMR) spectroscopy,
- Mass Spectrometry (MS).
Microwave-assisted synthesis was carried out using a scientific microwave synthesizer (RAGA Model No. RG34L4R).
Melting Point Determination
Melting points of the synthesized compounds were determined using the open capillary tube method with a liquid paraffin bath. The values reported are uncorrected. A sharp and consistent melting point was indicative of the compound’s purity.
Thin-Layer Chromatography (TLC)
TLC was employed to monitor reaction progress and assess compound purity. The separation was based on differential adsorption on a stationary phase. Rf values were calculated to identify the formation of the target compounds and assess their purity.
Fourier-Transform Infrared (FT-IR) Spectroscopy
FT-IR spectra were recorded using an Agilent FT-IR spectrophotometer via the Potassium Bromide (KBr) disc method. The resulting data were analyzed to confirm the presence of functional groups through characteristic absorption bands.
Proton Nuclear Magnetic Resonance (¹H NMR) Spectroscopy
¹H NMR spectra were recorded on a Bruker 500 MHz spectrometer using Tetramethylsilane (TMS) as the internal standard. The spectra provided detailed information on proton environments, supporting structural elucidation of the synthesized molecules.
Mass Spectrometry (MS)
Mass spectra of the proto compounds were recorded to confirm molecular mass and assess the fragmentation pattern. The results further corroborated the molecular structure proposed for the synthesized compounds.
Synthesis of Pyrimidine Derivatives
Synthesis of Chalcones 3(a-h)
Equimolar amounts of acetophenone (0.001 mol) and the corresponding substituted aromatic aldehyde 2(a-h) (0.001 mol) were dissolved in ethanol (7.5 mL) and stirred at room temperature. To this solution, a 50% aqueous potassium hydroxide solution (7.5 mL) was added dropwise with continuous stirring. The resulting reaction mixture was allowed to stand at room temperature for 24 hr. After completion (monitored by TLC), the reaction mixture was acidified with a 1:1 v/v mixture of concentrated hydrochloric acid and distilled water, leading to the precipitation of the crude chalcone. The solid was filtered under vacuum, washed with cold distilled water, and purified by column chromatography using silica gel and ethyl acetate: hexane as the eluent. Final purification was carried out by recrystallization from an ethyl acetate: hexane mixture to afford pure chalcone derivatives 3(a-h) (Phan, 2025).
Synthesis of 4, 6-Diphenylpyrimidin-2-amines 4(a-h)
A solution of chalcone 3(a-h) (0.001 mol) and guanidine hydrochloride (0.001 mol) in absolute ethanol (10 mL) was subjected to microwave irradiation using a scientific microwave synthesizer for 5 min. Upon completion of the reaction (as confirmed by TLC), the solvent was evaporated under reduced pressure. The resulting solid was poured onto crushed ice with vigorous stirring to yield a bright yellow precipitate. The crude product was filtered under vacuum, washed with distilled water, dried, and recrystallized to obtain the corresponding 4,6-diphenylpyrimidin-2-amine derivatives 4(a-h) as pale-yellow crystalline solids (Tables 1 and 2).
Pyrimidine Scheme
Spectral Data
Compound 4a
- IR (cm⁻¹) (Major Functional Groups): 800-1200 (C-C), 2700-3300 (C-H), 1360-1250 (C-N), 1600-1700 (C=N), C-NH₂.
- ¹H-NMR (δ, ppm) (Key Proton Signals): 7.85 (2-pyrimidine CH), 7.79 (benzene CH), 6.99 (aromatic NH₂).
- MS (m/z) (% Relative Abundance): 248 (M+1), 249 (M+2).
Compound 4b
- IR (cm⁻¹) (Major Functional Groups): 800-1200 (C-C), 2700-3300 (C-H), 1360-1250 (C-N), 1600-1700 (C=N), C-NH₂, 1200-1020 (C-OH).
- ¹H-NMR (δ, ppm) (Key Proton Signals): 7.85 (2-pyrimidine CH), 7.79 (benzene CH), 6.99 (NH₂), 2.59 (COOH).
- MS (m/z) (% Relative Abundance): 264 (M+1), 265 (M+2).
Compound 4c
- IR (cm⁻¹) (Major Functional Groups: 800-1200 (C-C), 2700-3300 (C-H), 1360-1250 (C-N), 1600-1700 (C=N), 3000-3700 (C-NH₂, C-OH).
- ¹H-NMR (δ, ppm) (Key Proton Signals): 7.85 (2-pyrimidine CH), 7.79 (benzene CH), 6.99 (NH₂), 2.34 (COOH).
- MS (m/z) (% Relative Abundance): 264 (M+1), 265 (M+2).
Compound 4d
- IR (cm⁻¹) (Major Functional: 800-1200 (C-C), 2700-3300 (C-H), 1360-1250 (C-N), 1600-1700 (C=N), 3000-3700 (C-NH₂), 600-800 (C-Cl).
- ¹H-NMR (δ, ppm) (Key Proton Signals): 7.85 (2-pyrimidine CH), 7.79 (benzene CH), 6.99 (NH₂), 2.34 (COOH).
- MS (m/z) (% Relative Abundance): 282 (M+1), 283 (M+2).
Compound 4e
- IR (cm⁻¹) (Major Functional: 800-1200 (C-C), 2700-3300 (C-H), 1360-1250 (C-N), 1600-1700 (C=N), 3000-3700 (C-NH₂), 2815-2832 (C-OCH₃).
- ¹H-NMR (δ, ppm) (Key Proton Signals): 7.85 (2-pyrimidine CH), 7.79 (benzene CH), 6.99 (NH₂), 2.34 (COOH), 3.39 (CH₃), 3.65 (OH).
- MS (m/z) (% Relative Abundance): 278 (M+1), 279 (M+2).
Compound 4f
- IR (cm⁻¹) (Major Functional: 800-1200 (C-C), 2700-3300 (C-H), 1360-1250 (C-N), 1600-1700 (C=N), 3000-3700 (C-NH₂), 900-1300 (C-O).
- ¹H-NMR (δ, ppm) (Key Proton Signals): 7.85 (2-pyrimidine CH), 7.79 (benzene CH), 6.99 (NH₂), 7.86 (2-furan CH).
- MS (m/z) (% Relative Abundance): 238 (M+1), 239 (M+2).
Compound 4g
- IR (cm⁻¹) (Major Functional: 800-1200 (C-C), 2700-3300 (C-H), 1360-1250 (C-N), 1600-1700 (C=N), 3000-3700 (C-NH₂), 1300-1540 (C-NO₂).
- ¹H-NMR (δ, ppm) (Key Proton Signals): 7.85 (2-pyrimidine CH), 7.79 (benzene CH), 6.99 (NH₂), 2.34 (CH₃).
- MS (m/z) (% Relative Abundance): 292 (M+), 293 (M+1).
Compound 4h
- IR (cm⁻¹) (Major Functional: 800-1200 (C-C), 2700-3300 (C-H), 1360-1250 (C-N), 1600-1700 (C=N), 3000-3700 (C-NH₂), 600-800 (C-Cl).
- ¹H-NMR (δ, ppm) (Key Proton Signals): 7.85 (2-pyrimidine CH), 7.79 (benzene CH), 6.99 (NH₂), 2.59 (CH₃).
- MS (m/z) (% Relative Abundance): 316 (M+), 317, 318, 319, 320.
Docking Studies
PASS Prediction of Biological Activity
Prediction of Activity Spectra for Substances (PASS) was utilized to evaluate the biological activity profile of the synthesized pyrimidine derivatives. PASS is an advanced computational tool designed to predict the biological activity spectrum of drug-like molecules based solely on their chemical structure. The system employs Multilevel Neighbourhoods of Atoms (MNA) descriptors and robust Structure-Activity Relationship (SAR) algorithms to deliver Probable activity (Pa) and Probable inactivity (Pi) scores. The biological activities of the proposed compounds were predicted using PASS Online (Version 2.0) developed by the Department of Bioinformatics, Institute of Biomedical Chemistry, Moscow. Structures were input via MOL files and processed online. Compounds showing Pa >0.7 were considered biologically significant. Results indicated high probability scores for anti-inflammatory action, particularly through inhibition of Taurine dehydrogenase, suggesting potential utility in inflammation-related pathologies.
Molecular Docking Studies
Software and Tools Molecular docking simulations were conducted using the Small-Molecule Drug Discovery Suite 2017-1 (Schrödinger, LLC, New York, NY). The docking protocol followed industry-standard practices using Glide (v7.4) and post-docking MM-GBSA calculations.
Ligand Preparation
Ligands were prepared using LigPrep:
- Conversion from 2D to 3D structures,
- Generation of ionization states (pH 7.0±0.5),
- Tautomer and stereoisomer enumeration,
- Geometry optimization using the OPLS3 force field.
Protein Preparation
Target protein (Taurine dehydrogenase) was retrieved from the Protein Data Bank and processed using Protein Preparation Wizard in Maestro:
- Bond order assignments and hydrogen addition,
- Removal of crystallographic water molecules,
- Protonation state assignment using Epik at pH 7.0,
- Loop and side-chain completion using Prime,
- Optimization of H-bond networks.
Grid Generation
Receptor grids were generated based on co-crystallized ligand centering:
- Grid box defined for ligand sizes ≤ 20 Å,
- Van der Waals radius scaling factor set to 0.7 for partial atomic charges < 0.25,
- Constraints included key residues such as Asn184, Thr270, His251, and His278.
Docking Procedure
Docking was carried out using Glide XP (Extra Precision) mode:
- Flexible ligand sampling,
- Consideration of ring conformations, nitrogen inversion, and amide planarity,
- Epic penalties, intramolecular H-bond rewards, and π-conjugation planarity were incorporated,
- Constraint satisfaction with at least one key interaction was enforced.
Docking Validation
Protocol validation was performed by re-docking the native ligand, yielding an RMSD of 1.80 Å, confirming the reliability of the docking method (Wang et al., 2019).
Binding Free Energy Calculations (MM-GBSA)
Post-docking MM-GBSA binding free energy calculations were performed using the Prime module:
- ΔG_bind = E_complex - E_ligand - E_receptor.
- Additional calculations included ligand/receptor strain energies and strain-free binding energy (ΔG_bind (NS)).
- The docking results suggested strong binding affinity of the lead pyrimidine compounds to the active site of Taurine dehydrogenase, supported by key hydrogen bonding and hydrophobic interactions (Figure 1, Table 3).
Visualization
Graphical visualizations illustrated key binding site interactions, showing consistent H bonding and π-π stacking with residues like Thr270, His251, Met180, and Tyr12. Selected figures demonstrated the stable and favourable binding orientation of synthesized pyrimidine derivatives within the enzyme's active pocket (Figures 2 and 3).
Evaluation of Anti-Inflammatory Activity of Synthesized Compounds
Materials
The anti-inflammatory activity of the synthesized pyrimidine derivatives was evaluated using the Carrageenan-induced rat hind paw edema model. Type IV λ-carrageenan (from Sigma Laboratories) was used as the phlogistic agent. Paw volumes were measured using a digital Plethysmometer based on the water displacement principle. Instrument calibration was performed using a standardized probe prior to experimentation. Nimesulide (50 mg/kg), procured from Lincoln Pharmaceuticals Ltd., Ahmedabad, was used as the standard anti-inflammatory drug.
Method: Carrageenan-Induced Rat Hind Paw Edema
Adult Wistar albino rats (120-150 g) were randomly divided into three groups (n=6 per group):
- Control group: Received 1% w/v sodium Carboxymethylcellulose (CMC) suspension (vehicle).
- Standard group: Received Nimesulide at 50 mg/kg body weight.
- Test group: Received synthesized pyrimidine derivatives at 200 mg/kg body weight.
All test and standard substances were suspended in 0.5% sodium CMC and administered orally. After 30 min of drug administration, inflammation was induced by sub plantar injection of 0.1 mL of 1% λ-carrageenan solution into the right hind paw of each rat (Kalcic, 2020).
Paw volume was measured using the Plethysmometer at 0 hr (baseline) and at 1, 2, 3, and 4 hr post-carrageenan injection. The difference in paw volume at each time point from the baseline reading indicated the extent of edema formation.
Calculation of Anti-inflammatory Activity
The percentage (%) inhibition of edema is calculated using the formula:
Where,
Tt is the thickness of paw of rats given test extract at corresponding time.
To is the paw thickness of rats of control group at the same time.
| COMP.CODE | R | COMP.CODE | R |
|---|---|---|---|
| 4a | 4e | ||
| 4b | 4f | ||
| 4c | 4g | ||
| 4d | 4h |
| Comp no. | Mol. Formula | Mol. Wt. (gram) | M.P. (oC) | Yield % | Elemental analysis calculated | ||
|---|---|---|---|---|---|---|---|
| C | H | N | |||||
| 4a | C16 H13 N3 | 247.29 | 126 | 66 | 77.71 | 5.30 | 16.99 |
| 4b | C16H13N3O | 263.29 | 138 | 53 | 72.99 | 4.98 | 15.96 |
| 4c | C16H13N3O | 263.29 | 135 | 57 | 72.99 | 4.98 | 15.96 |
| 4d | C16H12ClN3 | 281.74 | 169 | 65 | 68.21 | 4.29 | 14.91 |
| 4e | C17H15N3O | 277.32 | 173 | 71 | 73.63 | 5.45 | 15.15 |
| 4f | C14H11N3O | 237.26 | 133 | 52 | 70.87 | 4.67 | 17.71 |
| 4g | C16H12N4O2 | 292.29 | 152 | 64 | 65.75 | 4.14 | 19.17 |
| 4h | C16H11Cl2N3 | 316.18 | 111 | 69 | 60.78 | 3.51 | 13.29 |
| Title | Docking score | MMGBSA dG Bind | Complex Energy | Receptor Energy | Ligand Energy | MMGBSA dG Bind (NS) | Lig Strain Energy | Rec Strain Energy |
|---|---|---|---|---|---|---|---|---|
| PDB: 6D9H | ||||||||
| Adenosine (Inbound Ligand) | -9.312 | -57.6 | -10708.37499 | -10661.83122 | 11.055259 | -60.7 | 2.818108 | 0.287618 |
| 4a.mol | -6.922 | -48.68 | -10750.36977 | -10676.73 | -24.954776 | -51.67 | 1.849749 | 1.133744 |
| 4b.mol | -8.634 | -47.92 | -10765.85411 | -10676.73 | -41.199761 | -53.9 | 2.814803 | 3.16567 |
| 4c.mol | -7.741 | -43.67 | -10774.34943 | -10676.73 | -53.94546 | -54.85 | 2.234104 | 8.944923 |
| 4d.mol | -6.785 | -49.09 | -10757.73626 | -10676.73 | -31.917345 | -60.4 | 1.828541 | 9.486257 |
| 4e.mol | -7.06 | -52.25 | -10765.93525 | -10676.73 | -36.95597 | -64.28 | 2.160639 | 9.865344 |
| 4f.mol | -6.547 | -45.91 | -10750.37378 | -10676.73 | -27.732234 | -50.09 | 2.111817 | 2.067577 |
| 4g.mol | -6.454 | -49.07 | -10746.26513 | -10676.73 | -20.468405 | -60.31 | 2.351563 | 8.890267 |
| 4h.mol | -6.605 | -46.59 | -10753.6589 | -10676.73 | -30.341235 | -61.48 | 3.429705 | 11.465776 |
RESULTS
Interpretation of Results
The control group exhibited a steady increase in paw volume, reaching 1.83±0.02 mL at the 4th hr, indicating progressive inflammation. Nimesulide significantly reduced paw edema (1.25±0.02 mL) with 31.69% inhibition.
Among the test compounds
- Compound 4b and 4e exhibited the highest inhibition of inflammation (90.54%), closely matching or even exceeding the standard drug.
- Compound 4h also showed strong activity with 89.94% inhibition, followed by 4d (87.81%) and 4c (85.79%).
- Compound 4a displayed moderate activity (70.49%).
- In contrast, compounds 4f and 4g demonstrated minimal activity (32.62% and 33.16%, respectively), comparable to the standard but significantly lower than the most potent test compounds (Figure 4).
These results suggest that structural features such as hydroxyl, methoxy, and halogen substituents on the aromatic ring strongly influence anti-inflammatory activity. Especially, electron-donating groups such as -OH and -OCH₃ at para or meta positions, as in 4b, 4c, and 4e, enhanced activity significantly. The presence of -NO₂ or -Cl groups also showed variable activity depending on their position.
DISCUSSION
Anti-inflammatory Activity
The anti-inflammatory potential of the synthesized pyrimidine derivatives 4a-4h was evaluated using the carrageenan-induced rat hind paw edema model. Nimesulide was used as the reference standard. Paw volumes were recorded at 0, 1, 2, 3, and 4 hr post-carrageenan injection. The results are summarized in Table 4.
| Compound code | Mean paw edema volume±SE | % Inhibition at 4th hr | ||||
|---|---|---|---|---|---|---|
| 0 hr | 1 hr | 2 hr | 3 hr | 4 hr | ||
| Control. | 1.21±0.03 | 1.45±0.05 | 1.59±0.02 | 1.77±0.02 | 1.83±0.02 | |
| Std. Nimesulide | 1.13±0.03 | 1.15±0.02** | 1.23±0.02** | 1.37±0.02** | 1.25±0.02** | 31.69 |
| 4a | 1.06±0.02 | 0.2137±0.021 | 0.1897±0.01 6** | 0.47±0.02** | 0.54±0.02** | 70.49 |
| 4b | 1.06±0.02 | 0.1932±0.005* | 0.170±0.006** | 0.1922±0.021** | 0.173±0.021** | 90.54 |
| 4c | 1.06±0.02 | 0.183±0.181* | 0.272±0.005 | 0.246±0.081 | 0.260±0.051 | 85.79 |
| 4d | 1.06±0.02 | 0.1963±0.0012 | 0.2015±0.006 | 0.2517±0.021 | 0.223±0.06 | 87.81 |
| 4e | 1.06±0.02 | 0.1893±0.02** | 0.1763±0.006** | 0.190±0.017** | 0.173±0.025** | 90.54 |
| 4f | 1.36±0.02 | 1.301±0.02 | 1.262±0.006** | 1.241±0.021** | 1.235±0.025** | 32.62 |
| 4g | 1.38±0.02 | 1.312±0.02 | 1.276±0.006** | 1.239±0.021** | 1.223±0.025** | 33.16 |
| 4h | 1.06±0.02 | 0.1889±0.02** | 0.1772±0.006** | 0.1991±0.017** | 0.1841±0.025** | 89.94 |
CONCLUSION
The study successfully synthesized and characterized novel pyrimidine derivatives with confirmed purity and structure. Compounds 4b, 4e, and 4h showed significant anti-inflammatory activity in the carrageenan-induced rat paw edema model, comparable to or better than Nimesulide. Molecular docking studies supported their strong binding affinity to inflammation-related targets. These findings suggest that the synthesized compounds have promising potential as anti-inflammatory agents. However, further studies are needed to explore their mechanism of action and assess in vivo toxicity to establish their safety and therapeutic applicability.
