Synthesis, Computational studies and Biological Evaluation of Pyrimidine linked Coumarin derivatives for In-vitro Anti-Breast Cancer Activity

The present work was focused on the Synthesis, Computational studies and Biological evaluation of Pyrimidine linked Coumarin derivatives for in vitro anti-breast cancer activity. 7.1. PHASE-I (IN-SILICO STUDIES): 7.1.1. TARGET SELECTION: The present study was focussed on anti-breast cancer activity. Estrogen Receptor (PDB ID:3ERT) was chosen based upon various literature reviews. The corresponding enzyme was obtained from the RCSB Protein Data Bank 7.1.2. LEAD IDENTIFICATION: From various literatures reviewed, pyrimidine linked coumarin moiety has been selected for evaluating anti-breast cancer activity. Various substitutions were given to the lead molecules to enhance the study. 7.1.3. LEAD OPTIMISATION: Lead molecules which possess good ADME properties was selected for docking against ER (PDB ID:3ERT). 70 compounds with various substitutions were optimized using QIKPROP. Toxicity was also determined to predict the effectiveness of the drug 7.1.3. DOCKING: Compounds with good ADME properties was docked with Estrogen Receptor (PDB ID:3ERT). Docking studies were performed using Schrodinger Maestro v12.6 Based upon the docking score and binding interactions top ranked compounds was selected for synthesis using conventional methods. 7.2. PHASE II (SYNTHETIC STUDIES): 7.2.1. Synthesis of Ethyl 2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate: Urea, ethylacetoacetate and benzaldehyde were dissolved in 25 ml of ethanol along with 3 drops of conc. H2SO4 and refluxed for 90mins to yield the product Ethyl 2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate which is the Biginelli compound. 7.2.2. Synthesis of 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide: The Biginelli Compound produced was reacted with hydrazine hydrate to produce 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide. 7.2.3. Synthesis of 7-hydroxy 3-formyl Chromone: DMF and POCl3 was added with vigorous stirring at 500C and heated for 2hrs and stirred at 45-550C. To this solution Resacetophenone was added and to produce 7-hydroxy 3-formyl Chromone. 7.2.4. Synthesis of 2-hydroxy-6-methyl-4-phenyl substituted-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide-7-hydroxy-4H-1-benzopyran-4-one: The formed 7-hydroxy 3-formyl Chromone and 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide were refluxed to formed 2-hydroxy-6-methyl-4-phenyl substituted-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide-7-hydroxy-4H-1-benzopyran-4-one. 7.3.PHASE III (ANALYTICAL STUDIES): • Physicochemical Parameters such as Solubility, Melting point analysis, Thin Layer Chromatography were performed for the synthesized compounds. • Characterization of the synthesized compounds was done using UV spectroscopy, Infrared spectroscopy, Mass spectroscopy and 1H NMR spectroscopy. 7.4. PHASE IV (BIOLOGICAL SCREENING): The synthesized pyrimidine linked coumarin derivatives PC-1- PC7 has been screened for their in vitro cytotoxicity for antibreast cancer activity in MCF7 cell line using MTT assay and the results were compared to 5-fluorouracil. The IC50 values of the compounds PC1-PC7 was found to be 125, 94.66, 129, 141.3, 132.8, 163.3, and 97.86. Hence, the overall results showed that the percentage of viable cells, decreased with increasing the dose. The findings indicated that the test compounds showed least to good cytoxicity against the MCF 7 cell lines. CONCLUSION: The present study was focussed on developing the computational tools which helps in minimizing the process of drug discovery. • A set of 70 compounds were docked against the target Estrogen Receptor (PDB ID: 3ERT). • Among the 70 compounds, the top ranked 7 compounds were synthesized using the conventional Methods. • All the synthesized compounds were evaluated for their physicochemical properties such as solubility, melting point analysis and Thin layer Chromatography. • Characterization of the synthesized compounds was done using UV spectroscopy, Infrared spectroscopy, Mass spectroscopy,1H NMR spectroscopy. • The synthesized pyrimidine linked coumarin derivatives have been screened for their anti-breast activity in MCF-7 cell lines using MTT assay. • The findings indicated that the test compounds showed least to good activity. Among which PC-2 with nitro group at para position of phenyl group was found to be the most potent compound among other derivatives