Design, Synthesis, And Molecular Docking Study of Novel Diclofenac Hybridized Heterocyclic -1,2,4-Triazol Derivative as Potent Anti-Inflammatory Anti-Cancer Agent

Inflammation and cancer are among the most significant health challenges worldwide. Chronic inflammation has been recognized as a major factor in the initiation, progression, and metastasis of various cancers. Persistent inflammatory responses lead to the production of cytokines, reactive oxygen species (ROS), and other mediators that contribute to DNA damage, uncontrolled cell proliferation, angiogenesis, and inhibition of apoptosis. Therefore, the development of novel therapeutic agents possessing both anti-inflammatory and anticancer activities has become an important area of pharmaceutical research [2,6].

Diclofenac is a widely used non-steroidal anti-inflammatory drug (NSAID) that exerts its pharmacological action primarily through inhibition of cyclooxygenase (COX) enzymes, thereby reducing the synthesis of prostaglandins responsible for pain and inflammation. Recent studies have demonstrated that diclofenac and its derivatives possess additional biological activities, including anticancer properties, making it an attractive scaffold for the development of multifunctional therapeutic agents [1,2]. Structural modification of diclofenac has been extensively investigated to improve its efficacy, selectivity, and safety profile while exploring new therapeutic applications [2].

Heterocyclic compounds occupy a prominent position in medicinal chemistry due to their diverse biological activities and favorable pharmacokinetic properties. Among them, 1,2,4-triazole derivatives have attracted considerable attention because of their broad spectrum of pharmacological activities, including anti-inflammatory, antimicrobial, antioxidant, anticonvulsant, and anticancer effects [6,8,9]. The 1,2,4-triazole ring system is considered a privileged scaffold in drug design owing to its ability to form hydrogen bonds and other molecular interactions with biological targets, thereby enhancing biological activity [10,11].

Several studies have reported the successful synthesis and biological evaluation of novel 1,2,4-triazole derivatives exhibiting significant anticancer activity against various cancer cell lines [7,10]. Furthermore, triazole-containing compounds have demonstrated promising anti-inflammatory potential through modulation of inflammatory mediators and enzyme inhibition pathways [6]. These findings suggest that incorporation of the triazole pharmacophore into existing drug molecules may lead to the development of more potent therapeutic agents.

The concept of molecular hybridization has emerged as an effective strategy in modern drug discovery. Molecular hybridization involves the combination of two or more pharmacologically active moieties within a single molecular framework to produce compounds with enhanced biological activity, improved selectivity, and reduced adverse effects. Hybrid molecules often exhibit synergistic effects arising from the combined pharmacological properties of the parent structures [2,4]. Therefore, the hybridization of diclofenac with a biologically active 1,2,4-triazole nucleus represents a promising approach for the development of novel anti-inflammatory and anticancer agents.

Advances in synthetic organic chemistry have facilitated the efficient synthesis of heterocyclic compounds through environmentally friendly and sustainable methodologies. Green chemistry approaches, including one-pot synthesis, multicomponent reactions, catalytic processes, and solid-supported synthesis, have gained significant importance in medicinal chemistry research due to their reduced environmental impact and improved synthetic efficiency [14–20]. These strategies contribute to the development of economically viable and environmentally sustainable pharmaceutical compounds.

In recent years, molecular docking has become an indispensable computational tool in drug discovery and development. Molecular docking enables the prediction of binding modes, interaction patterns, and binding affinities of synthesized compounds with specific biological targets, thereby facilitating the identification of potential lead molecules before extensive biological evaluation [12]. The integration of molecular docking studies with synthetic and biological investigations provides valuable insights into structure-activity relationships and mechanisms of action.

Based on these considerations, the present research work focuses on the design, synthesis, characterization, and molecular docking evaluation of novel diclofenac hybridized heterocyclic-1,2,4-triazole derivatives. The synthesized compounds are expected to exhibit enhanced anti-inflammatory and anticancer activities through the synergistic contribution of both diclofenac and triazole pharmacophores. The study aims to explore the therapeutic potential of these hybrid molecules and establish their structure-activity relationships through computational and experimental investigations.

Structure of Diclofenac Sodium

Figure.  Structure of Sodium [2-(2,6-dichloroanilino) phenyl] acetate

General Information Diclofenac Sodium

Table No 1. General Information of drug

2. METERIAL & METHODS:

2.1 Experimental part:

All chemical reagents and solvents are purchased from commercial suppliers (HIMIDIA) and were without further purification. Melting points were determination on a capillary melting point apparatus and are uncorrected.

The identification and characterization of the synthesized compounds were carried out by the following procedure to ascertain that all the compounds were of parent compound.

• Melting Point
• Thin Layer Chromatography (TLC)
• FT-Infrared Spectroscopy (FTIR)
• Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR)
• UV Spectroscopy 
• Mass Spectroscopy

2.1.1Synthetic Scheme

Procedure:

Step 1: Preparation of Diclofenac Free Acid

• Diclofenac sodium (3.18 g, 0.01 mol) was dissolved in 50 mL of distilled water with continuous stirring. Dilute hydrochloric acid was added dropwise until the pH of the solution reached approximately 2.
• A white precipitate formed immediately indicating the formation of diclofenac free acid.
• The precipitate was filtered under vacuum and washed with cold distilled water to remove traces of hydrochloric acid.
• The product was dried in a desiccator.
• Yield 88 %   Melting Point 165-167°C

Step 2: Synthesis of Ethyl-2-(2,6-dichloroanilino) phenylacetate

• Diclofenac acid (2.96 g, 0.01 mol) was refluxed with thionyl chloride (5 mL) for about two hours in a round bottom flask fitted with a reflux condenser.
• The reaction mixture was then cooled and excess thionyl chloride was removed by evaporation under reduced pressure.
• The resulting acid chloride intermediate was dissolved in absolute ethanol (30 mL) and refluxed for three hours.
• After completion of the reaction, the mixture was poured into ice-cold water which resulted in the formation of a solid ester product.
• The crude product was filtered and recrystallized using ethanol.
• Yield: 82 %    Melting Point: 118-120°C

Step 3: Synthesis of Diclofenac Acetohydrazide

The ester derivative (3.24 g, 0.01 mol) was dissolved in ethanol (25 mL).

• Hydrazine hydrate (5 mL) was added slowly with constant stirring.
• The mixture was refluxed for 4 hours.
• Reaction progress was monitored using TLC.
• After completion, the reaction mixture was cooled and poured into ice water.
• A solid precipitate formed which was filtered and recrystallized from ethanol.
• Yield 78 % Melting Point 178-180°C

Step 4: Synthesis of Diclofenac-1,2,4-Triazole Derivatives

• Acetohydrazide (2.82 g, 0.01 mol) was dissolved in ethanol (30 mL).
• Substituted benzaldehyde (0.01 mol) was added to the solution followed by ammonium acetate (0.77 g) and zirconyl oxychloride octahydrate catalyst.
• The reaction mixture was refluxed for 5-6 hours.
• Reaction progress was monitored using TLC.
• After completion, the mixture was poured into ice-cold water.
• The solid product obtained was filtered and recrystallized from ethanol.
• Yield 65-75 %

2.2 THE IUPAC NAMES OF SYNTHESIZED COMPOUNDS ARE AS FOLLOWS:

1. 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline 
2. 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline
3. 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

2.3 METHOD:

2.3.1 SCHEME:

DERIVATIVES:

1. 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline 

Physical Properties

• Molecular formula:  C21H15Cl2N5O2
• Molecular weight:   440.284
• Appearance: Pale yellow crystalline solid
• Melting Point: 205–210 °C
• Yield: 72–88%
• Solubility: Soluble in DMSO, DMF; sparingly soluble in ethanol

2. 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Physical Properties

• Appearance:          Pale yellow crystalline solid
• Yield:    72–80 %
• Melting Poit          : 198–204 °C
• Solubility:  Soluble in DMSO, DMF; slightly soluble in ethanol
• Molecular Formula:          C??H??Cl?N?O
• Molecular Weight:           ~425.31 g/mol

3. 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Physical Properties

• Appearance: Pale yellow crystalline solid
• Melting Point: 205–210 °C
• Yield: 72–88%
• Solubility: Soluble in DMSO, DMF; sparingly soluble in ethanol
• Molecular Formula::
• Molecular Weight::

2.4 DOCKING STUDIES:

Structure Based Drug Design:

When the structure of the target is known (available), usually from X-ray crystallography, the most commonly used virtual screening method is molecular docking. Molecular docking can also be used to test possible hypotheses before conducting costly laboratory experiments. Molecular docking programs try to predict how a drug candidate binds to a protein target without performing a laboratory experiment. X-ray crystallographic techniques have been applied in medicinal chemistry to perform structural elucidation of large complex molecules. NMR techniques have also been used in the determination of the structure of proteins and enzymes. The basic assumption in SBDD is that good inhibitors must possess significant structural and chemical complementarity to their target receptor.

2.5 ADME PREDICTION:

The Qikprop Tool of Schrodinger_2015 (Maestro 10.2 version) is a quick, accurate, and easyto-use Absorption, Distribution, Metabolism, and Excretion (ADME) prediction program designed by Professor William L. Jorgensen. It predicts physically significant descriptors and pharmaceutically relevant properties of organic molecules, either individually or in batches. In addition to predicting molecular properties, QikProp provides ranges for comparing particular molecules' properties with those of 95% of known drugs.

3. RESULTS AND DISCUSSION

1. 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline 

Fig.No.1 IR Spectrum of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Observation Table for FTIR Spectrum

Table no.2. Observation table of IR Spectrum of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Fig No. 2  ¹H NMR Spectrum of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Table no. 3 Observation table of ¹H NMR Spectrum of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Fig No. 4. Mass Spectrum of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Table no.5. Observation table of Mass Spectrum of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Fig No.5 Calibration of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Table no.6  Observation table of Calibration of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Fig No.6 UV–Visible Spectrum of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

Table no.7 Observation table of  UV–Visible Spectrum of 2,6-dichloro-N-(2-{[5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl)aniline

2. 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No.7  IR Spectrum of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no.8 Observation table of IR Spectrum of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No.8 ¹H NMR Spectrum of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no.9 Observation table of ¹H NMR Spectrum of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No.9 Mass Spectrum of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no. 10 Observation table of Mass Spectrum of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No.10  Calibration of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no. 11 Observation table of Calibration of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No.11 UV Visible Spectrum of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no. 12 Observation table of UV Visible Spectrum of 2,6-dichloro-N-(2-{[5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

3. 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No. 12 IR Spectrum of  2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no. 13 Observation table of IR Spectrum of  2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No.13 ¹H NMR Spectrum of  2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no. 14 Observation table of ¹H NMR Spectrum of  2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No. 14 Mass  Spectrum of 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no. 15 Observation table of Mass  Spectrum of 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No. 15 Calibration of 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no. 16 Observation table of Calibration of 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Fig No. 16 UV Visible spectrum of 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Table no. 17 Observation table of UV Visible spectrum of 2,6-dichloro-N-(2-{[5-(4-fluorophenyl)-4H-1,2,4-triazol-3-yl]methyl}phenyl) aniline

Molecular Docking:

Table No. 18. Structures, scores and key interactions of 12 selected compounds along with standard Diclofenac & Dacomitinib

Figure No.17 1a & b Docked images of 3 compounds (PDB: 5F19).

Figure No. 18 1a & b Docked images of 3 compounds over EGFR (PDB: 4I23).

ADME PREDICTION:

Table No. 19. Structures, scores and key interactions of 12 selected compounds along with standard Diclofenac & Dacomitinib