Design, Synthesis and In-silico study of Isatin Derivatives as Potential Anti-Asthmatic Activity

Heterocyclic chemistry, essential in drug design, studies cyclic compounds containing heteroatoms like nitrogen, oxygen, or sulfur¹. These compounds, including Isatin derivatives, contribute significantly to medicinal chemistry. Isatin (1H-indole-2,3-dione) and its derivatives have been extensively studied due to their diverse biological activities. Discovered in 1841 by Erdmann and Laurent, Isatin has a versatile chemical structure that allows for multiple substitutions, leading to various therapeutic applications. Among these, the anthelmintic and antifungal properties of its derivatives are particularly significant. Mannich and Schiff bases, derived from Isatin, have also gained attention. A Mannich base is a β-amino ketone produced through the Mannich reaction, where an active hydrogen compound reacts with formaldehyde and an amine derivative. Schiff bases, on the other hand, are nitrogen analogs of aldehydes or ketones, formed by condensing an aldehyde or ketone with a primary amine^2,3 . Historically, Isatin research has evolved significantly. After its isolation from indigo in 1841, Adolf von Baeyer in the 1870s studied its synthesis and properties. Subsequent developments include the Stolle and Sandmeyer syntheses in the early 20th century, followed by discoveries of its antimicrobial and anticancer properties from the 1950s to the 1970s. Isatin derivatives exhibit antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, and antioxidant activities. Additionally, they show potential in treating central nervous system disorders like anxiety and depression^4,5. Asthma, a chronic inflammatory airway disease, results from genetic and environmental factors. It involves airway obstruction, eosinophilic inflammation, and hyperresponsiveness, triggered by allergens like dust and smoke. It progresses in two phases: an immediate histamine release and a later IgE-mediated inflammatory response^6,7,8. Molecular docking, a key computational technique in drug discovery, predicts the optimal binding orientation of molecules, helping assess drug efficacy. It involves protein-ligand interactions through search algorithms and scoring functions. Despite challenges in receptor flexibility, docking plays a crucial role in hit identification, lead optimization, and bioremediation^9,10.

2. DRUG PROFILE¹¹

Isatin (1H-Indole-2,3-Dione) is a yellow-orange crystalline compound first derived from indigo in the 19th century. With a molecular formula of C?H?NO? and a weight of 147.13 g/mol, it melts at 201°C. It is sparingly soluble in water but dissolves in ethanol, acetone, and DMSO. Stable between pH 3-9, it requires dry, airtight storage.

3. MATERIALS AND METHODS

3.1 MATERIALS

Chemicals and Solvents Used: Isatin, Benzylamine, Glacial acetic acid, Dimethlyamine, Piperazine, Pthalimide, Diphenylamine, 1-Methly piperazine, Morpholine, Formaldehyde, Ethanol, Methanol, Chloroform, DMSO, Ethyl acetate, Hexane, Benzene,Pet. Ether, Silica gel G.

Instruments: Single Pan Digital Balance

3.2 METHODS:

3.2.1 SYNTHESIS

3.2.1.1 Synthesis of benzylimino-isatin Sciff Base

3.2.1.2. Synthesis of benzylimino- isatin mannich bases

The previously synthesized compound, 3-(benzylimino)-1,3-dihydro-2H-indol-2- one (0.01 mol), was dissolved in 10 mL of methanol. To this solution, the appropriate secondary amine (0.01 mol) was added separately, followed by the addition of formaldehyde (0.01 mol, 37%) with continuous stirring. The mixture was stirred using a magnetic stirrer for 3 hours, after which it was left to stand at room temperature for 24 hours. The resulting precipitate was collected, recrystallized from methanol, and dried in a hot air oven. The purity of the final product was confirmed using thin-layer chromatography (TLC), with an (C2) Rf value of 0.60, (C3) Rf value of 0.30.

Compound 2

Compound 3

3.2.2 THIN LAYER CHROMATOGRAPHY (TLC) ANALYSIS OF ISATIN DERIVATIVES¹²

Qualitative Analysis via TLC

Thin Layer Chromatography (TLC) was employed for the preliminary analysis of synthesized isatin derivatives using silica gel-coated plates. The method allowed the separation and identification of compounds based on polarity. Different solvent systems were tested to achieve optimal resolution.

Method:

Compounds were applied to TLC plates using capillary tubes and placed in a chamber containing a suitable mobile phase. After development and air drying, plates were examined under UV light at 254 nm and 366 nm. Selected plates were treated with spraying reagents and gently heated to enhance color visibility. The retention factor (Rf) was calculated as:

Rf = Distance traveled by compound / Distance traveled by solvent front

Detection:

Spots were visualized using iodine vapors and further detected with spraying reagents. UV light observation and Rf values helped identify and characterize the separated compounds.

3.2.3 IN SILICO STUDY¹³

PASS Online (Prediction of Activity Spectra for Substances) is a computational tool used to predict the biological activities of chemical compounds. It evaluates pharmacological effects, mechanisms of action, toxicity, enzyme interactions, metabolism, and gene expression based on a compound’s 2D structural formula. The software aims to identify new biological targets and assess the bioactivity potential of novel or existing substances. PASS employs a structure-based approach and can screen large compound libraries quickly, often providing results in under a minute. It accepts input in MOL format and delivers output as plain text. The Way2Drug PASS Online platform (http://www.pharmaexpert.ru/passonline) is commonly used to evaluate phytoconstituents. The prediction is based on Structure-Activity Relationship (SAR) analysis, using a training set of over 205,000 compounds with more than 3,750 known biological activities. It generates two probability scores: Probable Activity (Pa) and Probable Inactivity (Pi), each ranging from 0.000 to 1.000. These values are independent and aid in interpreting a compound’s likely bioactivity. A higher Pa value indicates a greater chance of experimental confirmation.

3.2.4 Molecular Docking Experimental Methods

Molecular docking is a computational technique widely used in drug discovery to predict the interaction between small molecules (ligands) and target proteins (receptors). In this study, AutoDock Tools version 1.5.6 was employed to create input files and analyze docking results using protein 3G45 as the target. The docking evaluation was based on binding affinity (kcal/mol), inhibition constant (µM), intermolecular energy, and hydrogen bonding interactions^14,15,16.

Step 1: Ligand Preparation: Synthesized compounds were drawn using ChemSketch. The 2D structures were converted to 3D using Avogadro and energy-minimized using force fields in AutoDock Tools. The structures were saved in PDBQT format for docking.

Step 2: Protein Preparation: The protein structure (3G45) was obtained from the Protein Data Bank (www.rcsb.org). Using Molegro Molecular Viewer, water molecules and non-protein ligands were removed, and hydrogens were added. The cleaned file was converted to PDBQT format using AutoDock Tools.

Step 3: Grid Preparation: A grid box was set around the active site residues of 3G45 using AutoGrid4. The ligand and protein were selected, and grid parameters saved as a .gpf file.

Step 4: Docking Execution: AutoDock4 was used with the Lamarckian Genetic Algorithm (LGA) to perform docking. Ligands and receptors in PDBQT format were selected, and docking results saved as .dlg files.

Step 5: Analysis: Docking results were analyzed by ranking conformations based on binding energy. The final ligand-protein complex was visualized using Molegro Molecular Viewer and Discovery Studio, highlighting hydrogen bonds, hydrophobic interactions, and π-π stacking^17,18.

4. RESULTS AND DISCUSSION

4.1 RESULTS

4.1.1 THIN LAYER CHROMATOGRAPHY OF ISATIN DERVATIVES

The TLC of sythesis of isatin dervatives was carried out to detect the presence of C1,C2,C3 are the Rf values are tabulated in Table-3.

Table 1 : Detection of synthesis of Isatin derivatives

4.1.2 PASS REPORT FOR SYNTHESIS OF ISATIN DERVATIVES

4.1.2.1 Pass Report of Synthesis of {(3Z) 3 -(Benzylimino)-1,3-dihydro-2H-Indol-2-one} (C1);

The Pa (Probability of activity) and Pi (Probability of inactivity) values indicate the likelihood of a compounds biological activity bases on its molecular structure.

 4.1.2.2 Pass Report of Synthesis of [(3Z)-3-(benzylimino)-1-[(dimethylamino) methyl]-1, 3- dihydro- 2H-indol-2-one](C2)

4.1.2.3. Pass Report of Synthesis of[(3Z)-3-(benzylimino)-1-[(4- methylpiperazin-1-yl) methyl]- 1,3- dihydro- 2H-indol-2-one](C3):

4.1.3 MOLECULAR DOCKING

RESULTS FOR FINAL DOCKING SCORE