Design, Synthesis, Molecular Docking & Evaluation of Antioxidant Activity of Novel Quinoline-Azetidinone Hybrid Compounds

Heterocyclic compounds occupy a central and irreplaceable position in medicinal chemistry, owing to their prevalence in natural products and synthetic pharmaceuticals, as well as their broad spectrum of biological activities¹. A heterocyclic compound is defined by the presence of a ring structure that contains at least one atom other than carbon—commonly nitrogen, oxygen, or sulfur integrated into the cyclic framework. These heteroatoms contribute unique electronic, structural, and steric properties that make heterocycles highly versatile and functionally diverse. As a result, heterocyclic scaffolds have been widely explored for the design of bioactive molecules with applications across a multitude of therapeutic areas².

A remarkable proportion of drugs in clinical use today are built upon heterocyclic frameworks. These compounds are not only integral to the pharmacophore—the part of a molecule responsible for biological activity—but also influence a drug’s solubility, metabolic stability, binding affinity, and ability to cross biological membranes. Indeed, a survey of FDA-approved small-molecule drugs reveals that more than 75% contain at least one heterocyclic moiety³. The therapeutic importance of heterocycles is further highlighted by their occurrence in many biologically significant natural products, such as alkaloids (e.g., quinine, morphine), vitamins (e.g., thiamine, riboflavin), antibiotics (e.g., penicillin, cephalosporin), and nucleic acids (e.g., purines, pyrimidines)⁴.The incorporation of heteroatoms into the cyclic structure imparts several beneficial properties. For example, nitrogen-containing heterocycles often possess basicity, which can enhance interactions with biological targets such as enzymes, receptors, or nucleic acids through hydrogen bonding or ionic interactions. Similarly, oxygen and sulfur heteroatoms contribute to the polarity and electron density distribution of the molecule, impacting both reactivity and binding characteristics. Furthermore, heterocyclic rings can serve as bioisosteres—functional groups that mimic the physical or chemical properties of another group—to modulate activity, reduce toxicity, or overcome resistance⁵.

Role of Hybrid Molecules in Modern Drug Discovery

The growing complexity of human diseases and the limitations of traditional single-target drug design have prompted a paradigm shift towards the development of hybrid molecules in modern medicinal chemistry. A hybrid molecule is a single chemical entity that incorporates two or more distinct pharmacophores—each with its own defined biological activity—into a unified structure⁶. The rationale behind this strategy is to combine the beneficial properties of individual pharmacophores to produce compounds capable of exerting multiple biological effects simultaneously. This approach offers a promising route for the development of next-generation therapeutics, particularly for multifactorial diseases such as cancer, Alzheimer’s disease, diabetes, and infections caused by multidrug-resistant pathogens⁷.

Hybrid molecules represent an evolution from combination therapy, in which two or more separate drugs are administered concurrently to achieve synergistic or additive therapeutic effects. While combination therapy has been successful in several settings—such as antiretroviral treatment for HIV and multidrug regimens for tuberculosis—it also presents challenges including complex pharmacokinetics, potential drug-drug interactions, and patient non-compliance⁸. Hybrid molecules address these issues by integrating multiple pharmacophores into a single structure, ensuring coordinated delivery, uniform distribution, and synchronized action at the target site.

Quinoline is a nitrogen-containing heteroaromatic compound consisting of a benzene ring fused to a pyridine ring at the 2,3-positions, resulting in a bicyclic aromatic system with the molecular formula C?H?N. This fusion imparts distinctive electronic and physicochemical properties that are crucial for its interaction with various biological targets⁹. The structure can be described by its systematic IUPAC name, 1-aza-naphthalene, and is represented by a planar, rigid framework where the nitrogen atom occupies the position equivalent to the 1-position of naphthalene¹⁰.

Fig. 1.2 Structures of Quinoline

The azetidinone ring system, commonly referred to as the β-lactam ring, is a four-membered cyclic amide characterized by significant ring strain due to its small size and deviation from ideal bond angles. The core structure consists of three carbon atoms and one nitrogen atom forming a square planar arrangement, with the carbonyl group (C=O) located at the 2-position of the ring¹¹. This strained configuration results in enhanced reactivity, particularly towards nucleophiles, as the amide bond is much more susceptible to cleavage compared to that in typical six-membered lactams.

Fig. 1.7 Structures of β-lactam Ring

MATERIALS AND METHODS:

All reagents and chemicals used in this study were of analytical grade and utilized without further purification. Glacial acetic acid, Chloro-acetylchloride, Dragendorff’s reagent, Ethyl acetate, Acetic acid, Toluene, Benzene, Dimethyl formamide, DMSO, Calcium Carbonate, Chloroform, n-Hexane, Ethanol, Methanol,  Acetone etc.

MOLECULAR DOCKING

To gain insights into the binding interactions and potential antioxidant mechanisms of the synthesized quinoline–azetidinone hybrid compounds, a molecular docking approach was adopted. Docking simulations were carried out using AutoDock Vina 4.2, a widely accepted and robust tool for virtual screening, integrated with MGL Tools 1.5.7 for protein and ligand preparation¹². The enzyme Cytochrome c peroxidase (PDB ID: 2X08), a heme-containing protein implicated in reactive oxygen species metabolism, was selected as the target receptor owing to its relevance in oxidative stress-mediated pathways. Ascorbic acid, a well-known natural antioxidant, was used as the reference standard to benchmark the docking performance of the designed molecules¹³.

Selection and Preparation of Target Protein

The three-dimensional crystal structure of Cytochrome c peroxidase (PDB ID: 2X08) was retrieved from the Protein Data Bank (PDB) (https://www.rcsb.org/). The structure was examined for resolution, missing residues, and co-crystallized ligands or water molecules. The protein, originally complexed with native ligands and crystallographic waters, was subjected to preparatory steps using MGL Tools (AutoDock Tools 1.5.7). All heteroatoms, water molecules, and any cofactors not essential for binding were removed to prevent interference in the docking process¹⁴. Hydrogen atoms, especially polar hydrogens, were added to the protein structure to maintain the correct ionization and tautomeric states at physiological pH.

Ligand Preparation

All designed quinoline–azetidinone hybrid compounds were constructed using ChemDraw Ultra 12.0 and saved in .mol format. The two-dimensional structures were then converted into three-dimensional conformations using Open Babel. Energy minimization of ligands was performed using the MMFF94 force field to obtain stable, low-energy conformers suitable for docking studies. Subsequently, these optimized structures were imported into AutoDock Tools, where torsion angles were assigned, non-polar hydrogens were merged, and Gasteiger charges were applied. Each ligand was then saved in PDBQT format, ready for docking.Ascorbic acid, the standard antioxidant, was prepared following the same protocol for comparative docking analysis¹⁵.

Grid Box Configuration

To ensure accurate and consistent docking, a grid box was defined around the active site of the target protein. The center of the grid box was set based on the coordinates of the heme group and surrounding catalytic residues. The grid dimensions were carefully adjusted to encompass the entire active site pocket, allowing flexibility for ligand binding. The typical grid box dimensions were set to 40 × 40 × 40 points with a spacing of 0.375 Å, ensuring adequate coverage of the receptor surface.

Docking Protocol Using AutoDock Vina

Docking simulations were conducted using AutoDock Vina 4.2, which employs a sophisticated scoring function to predict binding affinities and ligand conformations within the active site. The docking parameters were kept at default, with the exhaustiveness set to 8, providing a balance between computational time and accuracy. Each ligand was allowed full rotational flexibility during docking, and multiple binding poses (up to nine conformers per ligand) were generated for evaluation¹⁵.

Post-Docking Analysis

Docked complexes were visualized and analyzed using Discovery Studio Visualizer and PyMOL. Key interactions between ligands and active site residues, such as hydrogen bonding, π–π stacking, hydrophobic contacts, and metal coordination with the heme group, were mapped. The strength and type of interactions were evaluated to understand the binding behavior and antioxidant potential of each compound.

Binding energy values, hydrogen bond counts, and interaction distances were recorded and compared with the standard drug ascorbic acid. Compounds exhibiting binding affinities equal to or greater than ascorbic acid and showing favorable interactions within the active site were considered promising leads¹⁶.

Validation of Docking Protocol

To ensure the reliability of the docking methodology, a re-docking validation was performed. The co-crystallized ligand (if available) from the original 2X08 PDB file was re-docked into the binding site using the same protocol. The root-mean-square deviation (RMSD) between the experimental and re-docked poses was calculated. An RMSD value below 2.0 Å was considered acceptable, indicating that the docking protocol could reliably reproduce native ligand conformations.

Scoring and Ranking of Compounds

The docked compounds were ranked based on their binding affinities and interaction profiles. Emphasis was placed on those exhibiting multiple hydrogen bonds with catalytically important residues and those occupying the hydrophobic groove near the hemecenter. Special attention was given to ligands forming stable π–π interactions with aromatic residues such as tryptophan and phenylalanine or coordination bonds with the iron center of the heme group¹⁷.

Structure-activity relationship (SAR) analysis was performed to correlate the structural features of the quinoline–azetidinone hybrids with their binding efficiency. Substituent effects, ring orientation, and flexibility were evaluated for their impact on docking performance.

The docking scores ranged from –6.7 to –7.2 kcal/mol, with ascorbic acid, the reference antioxidant, showing a lower binding affinity of –6.0 kcal/mol. This indicates that the synthesized compounds potentially bind more strongly to the CcP active site than the standard, suggesting better stability and inhibitory potential.Among all, compound 5e exhibited the best docking score of –7.2 kcal/mol, highlighting its strong binding affinity. It formed multiple interactions, including hydrogen bonds with LYS:93, VAL:94, and LEU:96, π-sigma interaction with GLU:91, halogen interaction with GLY:82, and π-alkyl interaction with ARG:95. These diverse binding interactions contribute significantly to the high stability of the ligand–protein complex¹⁸.

Compounds 5b and 5c both showed docking scores of –7.1 kcal/mol, forming hydrogen bonds and π-anion/π-H interactions, notably involving GLU:91, GLU:16, and ARG:86. These residues lie within the active pocket of CcP, contributing to favorable electrostatic and van der Waals interactions.Compound 5a showed a docking score of –6.7 kcal/mol, with hydrogen bonding involving ARG:86, and π-alkyl and π-cation interactions with LEU:96 and GLU:16, respectively. Although its score is slightly lower, the presence of halogen atoms (Cl and Br) may contribute to specific non-covalent interactions, enhancing binding stability.Compound 5d, which also scored –6.7 kcal/mol, formed hydrogen bonds with LEU:96, π-sigma interactions with GLU:91, and halogen bonding with GLA:17, along with additional C–H interactions with GLY:82 and ARG:86. These cumulative interactions suggest a moderately stable complex¹⁹.

In comparison, ascorbic acid, used as the reference molecule, exhibited a lower docking score of –6.0 kcal/mol, and formed hydrogen bonds with GLU:16, GLU:91, ARG:86, and GLY:82. Despite its multiple H-bonds, its overall weaker binding affinity may be attributed to the absence of extended aromatic or halogen functionalities that enhance π and hydrophobic interactions.

Table 1 :Molecular docking score and interaction

The docking results were cross-referenced with experimental antioxidant data (e.g., DPPH assay results) to assess the predictive value of molecular docking. Compounds with high binding affinities and favorable interactions were expected to show potent free radical scavenging activity²⁰. This correlation helped validate the proposed mode of action, whereby ligand binding to Cytochrome c peroxidase may mimic or enhance the antioxidant effect observed in vitro.

Target Protein: Cytochrome c Peroxidase (PDB ID: 2X08)

Cytochrome c peroxidase (CcP) is a heme-dependent oxidoreductase enzyme predominantly found in the mitochondria of Saccharomyces cerevisiae (yeast). It plays a pivotal role in cellular redox regulation by catalyzing the reduction of hydrogen peroxide (H?O?) into water, utilizing electrons supplied by cytochrome c. This enzymatic action constitutes a key component of the cell’s defense mechanism against oxidative stress, contributing to the detoxification of reactive oxygen species (ROS) and preservation of cellular integrity²¹.

Table 2: Target Protein

Target Protein	PdbID	Structure
Cytochromec peroxidase (CcP)	2X08

Structural Information

The three-dimensional structure of Cytochrome c peroxidase is available in the Protein Data Bank under the accession code 2X08. This crystal structure was determined using X-ray diffraction at a high resolution of 1.98 Å, offering an accurate model suitable for computational and docking studies. The structure comprises a single polypeptide chain of 294 amino acids and includes a heme prosthetic group (HEM), which serves as the redox-active center necessary for enzymatic function.

• Protein Data Bank ID: 2X08
• Organism: Saccharomyces cerevisiae
• Method: X-ray Crystallography
• Resolution: 1.98 Å
• Chain: A
• Functional Group: Heme (Fe-protoporphyrin IX)

Functional Role in Redox Biology

CcP mediates the transfer of electrons from reduced cytochrome c to hydrogen peroxide, effectively neutralizing the peroxide by converting it into water. The catalytic reaction proceeds through transient intermediate states known as Compound I and Compound II, which feature high-valent iron-oxo species within the heme cofactor. These redox intermediates facilitate electron flow and ensure rapid turnover of substrate.

The enzyme's function is crucial for preventing oxidative damage to proteins, lipids, and nucleic acids, thereby maintaining mitochondrial and cellular homeostasis²².

Catalytic and Binding Site Characteristics

The active site of CcP is organized around its hemecenter, with iron coordination and nearby residues contributing to catalytic activity and substrate binding. Key amino acids include:

• His175: Acts as the axial ligand to the heme iron (proximal side)
• His52: Positioned on the distal side, involved in proton transfer and stabilization of peroxide
• Trp191: Plays a role in intramolecular electron transfer during catalysis
• Arg48: Participates in hydrogen bonding and stabilization of reactive intermediates

These residues are highly conserved and essential for the peroxidase mechanism. The accessibility and chemical environment of the heme cavity make it an ideal docking site for antioxidant candidates.

Relevance to Antioxidant Drug Discovery

Given its central function in oxidative defense, Cytochrome c peroxidase is frequently studied as a target protein for evaluating antioxidant activity of small molecules. Modulating its function through small-molecule interactions can offer therapeutic potential in diseases where oxidative imbalance is a contributing factor, including neurodegeneration, inflammation, and cancer.

 The selection of PDB ID: 2X08 for molecular docking was based on several factors

• High-resolution crystal structure, ideal for precise modeling of ligand-protein interactions
• Well-defined heme binding cavity, critical for assessing antioxidant-like interactions
• Biological significance in redox metabolism and ROS detoxification
• Structural compatibility with virtual screening tools such as AutoDock Vina

This structure provides a biologically relevant platform for in silico docking simulations aimed at identifying compounds that can mimic or enhance the enzyme’s antioxidant function.

Docking Interaction Profiles of Synthesized Compounds

General procedure for synthesis of 2-chloroquinoline-3-carbaldehyde(2)

To a well-stirred solution of anhydrous dimethylformamide (DMF, 0.03 mol) maintained at a temperature of 0–5°C, phosphorus oxychloride (POCl?, 0.09 mol) was added dropwise under cooling to control the exothermic nature of the reaction. The mixture was stirred for an additional 5 minutes to ensure complete formation of the Vilsmeier–Haack reagent. Subsequently, acetanilide (0.01 mol) was added slowly to the reaction flask, and the entire reaction mass was heated under reflux at 75–80°C for 8 hours to promote cyclization and formylation.

Upon completion of the reflux period, the reaction mixture was allowed to cool to room temperature and then cautiously poured onto crushed ice with vigorous stirring. This quenching step led to the immediate precipitation of a pale yellow solid, identified as 2-chloro-3-formylquinoline (Compound 2). The solid product was collected by vacuum filtration, washed thoroughly with chilled water to remove residual acidic by-products, and recrystallized from ethyl acetate to afford the purified compound in good yield²³.

General procedure for synthesis of (Z)-1-(2-chloroquinolin-3-yl)-N-(substituted)methanimine (4a-e)

A mixture of 2-chloroquinoline-3-carbaldehyde (0.01 mol) and an equimolar quantity of substituted aromatic amine (3a–e, 0.01 mol) was dissolved in absolute ethanol (50 mL) in a round-bottom flask. To this reaction mixture, 2 mL of concentrated hydrochloric acid was added as a catalyst to facilitate the condensation process. The reaction mixture was then heated under reflux for 8 hours, ensuring uniform mixing and effective interaction of the reactants.

The progress of the reaction was monitored at regular intervals using thin-layer chromatography (TLC) on silica gel plates with a suitable solvent system (e.g., chloroform:ethyl acetate, 7:3), and the development of a new spot indicated the formation of the desired Schiff base²⁴.

Upon completion, the reaction mixture was allowed to cool to room temperature and then poured onto crushed ice with continuous stirring. The resulting solid precipitate was separated by vacuum filtration, washed thoroughly with chilled water to remove unreacted acid and by-products, and recrystallized from hot ethanol to obtain the final product in a purified crystalline form.

Table 3: FTIR spectra Interpretation of Compound 4-(5d) 2

Fig. 1: ¹H-NMR Spectra of Compound 4-(5d)

Table 4:¹H-NMR spectra Interpretation of Compound 4-(5d)

Fig.2: FTIR Spectra of Compound 4-(5d)

Table 5: FTIR spectra Interpretation of Compound 4-(5d)

Fig. 3: ¹H-NMR Spectra of Compound 4-(5d)

Table 6:¹H-NMR spectra Interpretation of Compound 4-(5d)

Fig. 4: ¹H-NMR Spectra of Compound 4-(5d)

Table 7:¹H-NMR spectra Interpretation of Compound 4-(5d)

EVALUATION OF ANTIOXIDANT ACTIVITY BY DPPH RADICAL SCAVENGING METHOD

The antioxidant potential of the synthesized quinoline–azetidinone derivatives (5a–5e) was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, a widely accepted method for measuring the hydrogen-donating capacity of compounds. The method is based on the ability of antioxidants to reduce the deep violet-colored DPPH radical to a yellow-colored diphenyl picryl hydrazine upon electron or hydrogen atom donation.

Preparation of DPPH Solution

To prepare the DPPH working solution, 4 mg of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was accurately weighed and dissolved in 100 mL of methanol or ethanol, yielding a 0.004% (w/v) or approximately 0.1 mM solution. The mixture was stirred until complete dissolution was achieved. Since DPPH is light-sensitive and prone to degradation upon exposure to sunlight, the solution was either freshly prepared prior to use or stored in a dark amber bottle at low temperature to maintain its stability throughout the experiment²⁵.

Preparation of Test Samples

Each synthesized compound was weighed accurately and dissolved in methanol to prepare a stock solution of 1 mg/mL. From this, serial dilutions were made to obtain a range of concentrations (typically 20, 40, 60, 80, and 100 µg/mL) for activity evaluation. A known antioxidant, ascorbic acid, was used as a standard under identical conditions for comparative purposes.

Assay Procedure

In a clean, labeled test tube, 2.0 mL of DPPH solution was mixed with 2.0 mL of each test compound at different concentrations. The mixture was vortexed gently to ensure uniform mixing and then incubated in the dark at room temperature (25?±?2°C) for 30 minutes to allow the reaction to proceed. A control sample containing 2.0 mL of DPPH solution and 2.0 mL of methanol (without test compound) was also prepared to represent maximum absorbance. A blank consisting of methanol alone was used to zero the spectrophotometer²⁶.

After incubation, the absorbance of each sample was measured at 517 nm using a UV–Visible spectrophotometer. The decrease in absorbance indicates the scavenging ability of the compound toward the DPPH radical.Brand-Williams et al. [61]

Calculation of Antioxidant Activity

The percentage of DPPH radical scavenging activity was calculated using the following formula:

Scavenging Activity (%) =AT - AsA0× 100

• A_T? is the absorbance of the control
• As? is the absorbance of the test sample

Each sample was tested in triplicate to ensure reproducibility, and the results were expressed as mean ± standard deviation. A plot of % inhibition versus concentration was constructed to determine the IC?? value (concentration required to inhibit 50% of DPPH radicals), which reflects the compound's antioxidant potency. A lower IC?? value indicates stronger antioxidant activity

The percent inhibition was measured at increasing concentrations (20–100 µg/mL), and the results were compared with ascorbic acid, which served as the standard reference.As expected, ascorbic acid demonstrated the highest radical scavenging activity across all concentrations, reaching 98.13% inhibition at 100 µg/mL. Among the synthesized compounds, compound 5a exhibited the most potent antioxidant activity, showing a concentration-dependent increase and achieving 91.04% inhibition at the highest dose tested. This high activity may be attributed to the presence of both chloro and bromo substituents, which may enhance the radical stabilization through inductive effects and halogen-based interactions.

Compound 5e was the second most active, displaying 88.90% inhibition at 100 µg/mL. The presence of a para-nitro substituent in 5e likely contributes to its activity by acting as a strong electron-withdrawing group, thereby facilitating effective hydrogen or electron donation to neutralize free radicals.Compound 5d, containing 3,4-dimethoxy groups on the phenyl ring, showed moderate antioxidant activity, with 69.02% inhibition at 100 µg/mL. The methoxy substituents, known to be electron-donating, may enhance resonance stability of the resulting phenoxy radicals, although their impact appears less significant compared to halogen or nitro substituents.In contrast, compounds 5b and 5c exhibited lower antioxidant potential, with maximum inhibitions of 63.89% and 59.67%, respectively. Compound 5b contains a meta-nitro group, which may be less effective in delocalizing the unpaired electron through resonance, while 5c, with a para-methoxy substituent, shows limited hydrogen-donating capacity under the assay conditions.²⁷

Table 8: Percentage Inhibition of synthesized derivative

Fig. 5.1 Graph representing percentage inhibition