SYNTHESIS, DOCKING STUDIES AND EVALUATION OF ANTIMICROBIAL AND IN VITRO ANTIPROLIFERATIVE ACTIVITY OF 5H-CHROMENO 4,3-D PYRIMIDIN-2-AMINE DERIVATIVES Original Article RAJESH B. PATIL*, SANJAY D. SAWANT Sinhgad Technical Education society’s, Smt. Kashibai Navale College of Pharmacy, Pune Saswad Road, Kondhwa (Bk), Pune-411048, Maharashtra, India. Email: rajshama1@yahoo.com Received: 26 Nov 2014 Revised and Accepted: 20 Dec 2014 ABSTRACT Objective: Docking studies and synthesis of 4-aryl-5H-chromeno [4,3 -d]pyrimidine-2-amine derivatives to evaluate antimicrobial and in vitro cytotoxicity activity. Methods: Docking studies were performed on Autodock Vina. Computational work was carried out with UCSF Chimera, Argus lab, Marvin beans. Antimicrobial activity was carried out with the agar cup plate method on two gram-positive organisms viz. Bacillus Subtilis and Staphylococcus Aureus and two gram-negative organisms viz. Escherichia Coli and Pseudomonas Aeruginosa. In vitro cytotoxicity was performed on HeLa cell lines with Sulfo Rhodamine B (SRB) assay method. Results: In docking studies compounds CHR 7, CHR 8 and CHR 9 gave highest docking score (binding free energy) and moderate antimicrobial activity against gram positive organisms. All the synthesized compounds showed poor antimicrobial activity against gram negative organisms. In vitro cytotoxicity activity, in terms of growth inhibitory concentration 50 % (GI50) was in the range 37.9 - 57.1 µM. Though synthesised compounds possess moderate GI50, in comparison to standard Adrinamycin the compounds are inactive. Conclusion: A series of 4-aryl-5H-chromeno[4,3 -d]pyrimidine-2-amine derivatives were synthesized and evaluated for antimicrobial and in vitro cytotoxicity studies. The compound CHR 9 was found most active among all the synthesised compounds. Keywords: Chromenes, Chromeno[4,3-d]pyrimidine-2-amine, Antimicrobial, HeLa cell lines, Autodock Vina. INTRODUCTION Chromans, chromenes are important class of bioactive molecules consisting of benzene and pyran fused ring called benzopyrans. The flavones, isoflavones, flavanoids and coumarins have been extensively studied phyto constituents containing benzopyran ring. Chromene derivatives possess diverse pharmacological activities including antitumor [1, 2], antivascular [3], antimicrobial [4, 5], antioxidant [6], TNF-α inhibitor [7], antifungal [8], anticoagulant [9], antispasmolytic [10, 11], estrogenic [12, 13], antiviral [14], anthelminthic [15], anti-HIV [16], antitubercular [17, 18], anti- inflammatory [19, 20], herbicidal [21], analgesic [22] and anticonvulsant [23,24] activity. The emergence of resistant strains of microorganisms has made the development of antimicrobial agents challenging. Amongst many antimicrobials, pyrimidines and their annelated derivatives are of particular importance being widely spread in nature in the form of nucleobases viz. cytosine, thymine, uracil, adenine and guanine. Aminocoumarin derivatives like novobiocin, coumermycin Al, clorobiocin and their analogues exert antibiotic effect by binding tightly to B subunit of bacterial DNA gyrase but show poor activity towards gram-negative bacterial pathogens [25, 26]. Many ring annelated chromene derivatives have been reported to possess cytotoxic effects [1, 27]. Two families of such ring annelated chromenes viz. 2,4-diaryl- 4H,5H-pyrano[3,2-c]benzopyran-5-one derivatives and 1- benzopyrano[3,4-b][1,4]benzothiazine-6-one derivatives reported by Collota et al [28] possess potent cytotoxic effects. This prompted us to synthesize 4-phenyl-5H-chromeno[4,3-d]pyrimidin-2-amine derivatives in which chromene ring is annelated with pyrimidine-2- amine ring. Computer docking technique helps in finding the important binding modes of ligand with its target protein. The analysis of important interactions like hydrogen bonds formed with important residues, hydrophobic interactions facilitate drug design process. In the present paper, we report the synthesis, characterization and docking studies of 4-phenyl-5H-chromeno[4,3- d]pyrimidin-2-amine derivatives. The cytotoxicity of synthesized compounds was investigated on HeLa cell lines (human cervical adenocarsinoma cell lines) by Sulfo Rhodamine B (SRB) assay. The antimicrobial activity was investigated on two gram-positive organisms viz. Bacillus Subtilis and Staphylococcus Aureus and two gram-negative organisms viz. Escherichia Coli and Pseudomonas Aeruginosa. MATERIALS AND METHODS Docking studies In the present study, the X-ray crystal structure of the antimicrobial agent Clorobiocin bound to topoisomerase II DNA gyrase was obtained from the RCSB Protein Data Bank (PDB ID: 1KZN). Resolution of protein structure with 205 amino acid residues was 2.30 A 0 . The protein was further processed by removing water and clorobiocin. The resulted clean protein was further refined by energy minimization in UCSF Chimera [29] with Amber ff12SB force field. Combination of 10,000 steepest descent and conjugate gradient steps with 0.02 A 0 step size were used during energy minimization. The energy minimized protein structure was used for docking procedure. 2D structures of all the synthesized compounds (see scheme of synthesis) were drawn and converted to 3D structures using Marvin Sketch (a structure drawing program). Geometry optimization was carried out in ArgusLab 4.0.1 (from Thomson and Planaria Software LLC) on semi empirical quantum mechanical basis with parameterized model number 3 (PM3) hamiltonian, until restricted closed shell hartree-fock self consistent field formalism converses to 10 - [10] kcal/mol and steepest descent geometry search criteria until gradient converses to 10 -6 kcal/mol. Gasteiger partial atomic charges of optimized molecules were computed in UCSF chimera and were updated in 3D structures. Docking simulation was carried out in Autodock Vina [30]. Polar and aromatic hydrogens and gasteiger charges were added in the protein using MGLtools1.5.4 [31] and the pdb file was subsequently International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 7, Issue 2, 2015 Innovare Academic Sciences