Antioxidants are chemicals that stop the oxidation of other molecules from harming cells. Numerous human diseases, including cellular necrosis, CVS disease, cancer, neurological disorder, Parkinson’s dementia, Alzheimer's disease, inflammatory disease, etc., are greatly influenced by oxidative stress. Compounds containing hydroxyl groups at the para position of the aromatic ring confer a better radical scavenging activity compared to those with hydroxyl groups in other positions [1]. 2-Aminopyrimidine has great importance as pyrimidine is widely spread in living organisms. Gabriel and Colman first isolated pyrimidine in 1899. 2-Aminopyrimidine and its derivatives have a broad spectrum of biological activities. A large number of heterocyclic compounds derived from chalcone group have been reported as active biological entities, where 2-aminopyrimidine play a vital role owing to their wide range of therapeutic activities [2]. 2-Aminobenzothiazole moiety is present in various bioactive molecules such as imaging agents for antitumor, antimicrobial, antifungal, orexin receptor antagonist and the Gram positive selective antibacterial. Benzothiazoles, which have benzene and thiazole rings, are utilized in numerous medicines worldwide. Chemical compounds like benzothiazoles and their heterocyclic derivatives have several biological impacts [3]. Indole-3-carboxaldehyde is an essential scaffold and intermediates enabling the generation of numerous synthetic and natural substances with therapeutical impacts, especially those retaining antitumor, antidepressant, antimicrobial, antiviral, anthelmintic and inhibitory effects on transcription and DNA replication as well as their muscle relaxant properties [4]. Aniline is an organic compound consisting of a phenyl group attached to an amino group, it is the simplest aromatic amine. Industrially significant commodity chemical, as well as a versatile starting material for fine chemical synthesis. In recent years, macrocyclization is a promising strategy in modern drug design, as it can minimize the entropic loss associated with the ligand adopting a favorable conformation, which may lead to a gain of potency and selectivity. "Schiff bases" emerged with the 1864 report of German scientist Hugo Schiff [5]. Drug discovery, drug design, testing, and development constitute the extensive and intricate steps involved in sketching a pharmacological molecule. A specific type of software called docking programs is often used in drug design. These programs facilitate in the binding of ligands, or small molecules, to proteins, or larger molecules with biological significance [6]. Thus, by keeping this above idea in our mind we had synthesized certain Novel Schiff bases containing the following Heteroaryl scaffolds viz., 2-aminopyrimidine, 2-aminobenzothiazole, Indole-3-carboxadehyde & aniline.

In this study we aim for the docking studies of the synthesized compounds using PyRx tools.

EXPERIMENTAL

MATERIALS AND METHODS

2-aminopyrimidine, 2-aminobenzothiazole, Indole-3-Carboxaldehyde and aniline were acquired from Sigma -Aldrich USA. Melting points of all the synthesized were determined by open capillary tube methods and values were uncorrected. The 2-aminopyrimidine derivatives [AB1-AB3] (Fig.2.1), 2-aminobenzothiazole derivatives [BT1-BT3] (Fig.2.2), Indole-3-carboxaldehyde derivatives [IC1-IC3] (Fig.2.3) and Aniline derivatives [AN1-AN3] (Fig.2.4), are synthesized.

ANTIOXIDANT ACTIVITY

PRINCIPLE

Antioxidants are the compounds capable to either delay or inhibit the oxidation processes which occur under the influence of atmospheric oxygen or reactive oxygen species. DPPH free scavenging method is based on reduction of DPPH in ethanol in presence of hydrogen donation due to the formation of non-radical form DPPH.

PROCEDURE

The antioxidant activity of sample is determined by DPPH scavenging assay method. A solution of DPPH was prepared by dissolving 12.5mg of DPPH in 50ml ethanol. The absorbance of stock solution was diluted with ethanol to an absorbance of 0.98 different concentrations (20, 40, 60,80,100µg/ml) of sample was prepared in ethanol, then added to 1ml of DPPH solution.

This reaction mixture was incubated at 37°C darkness for 20-30min. The absorbance was determined at 517nm. Ascorbic acid was used as standard.

 MOLECULAR DOCKING METHODOLOGY

In the current molecular simulation study, PyRx Software was used to constitute a ligand-based computer modeling program for forecasting binding energy of the selected compound [7]. The structures of all synthesized compounds are produced using Chemsketch software (http://www.acdlabs.com/resources/freeware). Chem3D pro 8.0 was utilized to optimize the structures and to minimize energy. Molecular docking was performed out using the optimized compounds.

1. PREPARATION OF GRID AND DOCKING PARAMETERS

The 3D structure of the molecular target was taken from Protein Data Bank (PDB) (www.rcsb.org). Loading the molecules in to PyRx workspace. Converting the pdb file to pdbqt files. Select the protein and ligand by simply clicking and run vina. Select vina search space. Enclose the labels within grid box. The active sites were selected using grid boxes around the bound cocrystal ligands, which was like this: number of grid points (60×60×60), center (xyz coordinates) and the grid point spacing was 0.375 Å. In order to correlate the test compounds for their respective activity and to examine their in-silico interaction, they were docked into the active site. Click forward button to start vina calculations. Once the calculations are done, results will be populated by giving binding affinity (Kcal/mol) values. Docking study was performed using PyRx Software and Discovery Studio is utilized for visualization.

The newly synthesized nitrogen containing aryl-heterocyclic derivatives were subjected to docking using PDB:1KXM (Antioxidant) with the aid of PyRx software. The docking results were shown in Table 5.14. In which Compound BT2 and AB2 shows maximum binding affinity of -9.7 and -8.7, Compound AN1 and AN2 shows minimum binding affinity of -7.5 and -7.6 against antioxidant protein (PDB ID: 1KXM) when compared with the standard drug Ascorbic acid.

Figure 4:  Histogram of Docking results of test compounds on Antioxidant activity

The newly synthesized compounds were screened for their invitro antioxidant activity by DPPH radical scavenging activity. The compounds were tested at various concentrations (0.2, 0.4, 0.6, 0.8, 1.0 µg/mL) and the Q(%) values had been determined for each compound and compared with control as well as standard antioxidants. l-ascorbic acid (AA) was used as the standard antioxidants. In DPPH radical scavenging activity assay, the purple chromogen radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) is reduced by antioxidant/reducing compounds to the corresponding pale-yellow hydrazine. The scavenging capacity is generally evaluated in organic media by monitoring the absorbance decrease at 515– 528nm until the absorbance remains constant or by electron spin resonance. DPPH radical is reduced by antioxidants and causing absorbance decrease at 515nm is the principle of measurement of this assay.

ANTIOXIDANT ACTIVITY OF TEST COMPOUND IC2