INTRODUCTION:

The most significant objective of medicinal chemistry be in the direction of synthesize the compound that shows potential activity as therapeutic agents with minor level of toxicity. The Quinazolin-4-(3H)-one derivatives encompass be report which shows a broad spectrum of biological activity^1,2. The substituted quinazolin-4- one derivatives include be shown their having antitumor, anti-inflammatory, antihistaminic, antimicrobial, antitubercular, anticonvulsant, antiallergic and anti helminthitic, hypoglycemic, HIV-1 reverse transcriptase inhibitor³ Epidermal growth factor receptor which is a cellular transmembrane glycoprotein comprise one from four member of the erbB Family of tyrosine kinase receptor.

This receptor is playing a governing role in opening signals which give direction towards activities of epithelial cells, tumor of epithelial cell source. Further, the growth factor receptor Which one among four transmembrane growth factor receptor proteins which shows similar structure and function, also it is known as c-erbB-1 and consisting of extracellular receptor domain, A Transmembrane province intracellular domain with tyrosine kinase utility. The left behind member of this group are (c-erbB2), (c-cerbB-3), (c-erbB-4)⁴. EGFR near in ranges 40,000 to 1,00,000 receptors per normal cell⁵. The growth factor receptor is over articulated in a number of solid tumors, as well as renal cancer, ovarian cancer, breast cancer, non small cell lung cancer (NSCLC), Head and colon cancer, neck cancer⁶. The Ligands Which are bind to EGFR in close TGF-α (Transforming growth factor-alpha), epidermal growth factor, Amphiregulin, betaceluin, and heparin binding EGF, wherever related ligand binds EGFR which leads to autophosphorylation of tyrosine kinase receptor later activation of signal transduction pathway it acting significant part within adaptable cellular proliferation and differentiation, survival⁷. Present in two pharmacological avenue used for this growth factor receptor inhibition is the most part first monoclonal antibodies, small molecule antibodies. Monoclonal antibody plays an important task in blocking ligand bind in the extracellular field, while small molecule inhibitor exhibits their effects in intracellular portion of the receptor in the direction of prevent phosphorylation of tyrosine kinase with finally activation signal transduction pathway and epidermal growth factor receptor inhibitors have ability to produce anticancer activity⁸.

Tyrosine kinase (EGFR-TKs) acting crucial part in cancer cell migration, proliferation and survival, adhesion, differentiation. Over expression and mutation of EGFR have been related with a variety of cancers. Development of EGFR inhibitors has become a most important focus in antitumor drug campaigns. The more than a few drugs such as Lapatinib, afatinib, erlotinib and gefitinib have been permitted via (FDA) the food and drug administration for clinical use of EGFR inhibitors.⁹In silico model of diverse molecules determination done by using ChemDraw and molinspiration. The quinazolinone derivatives having drug likeness and molecular properties compared to reference molecules, which obey to Lipinski Rule of five will be preferred for lab synthesis^10-13. docking methods include be used as modern drug designing to appreciate for the drug receptor binding interaction and it encompass be revealed in literature that computational measures may strong evidence allow out the design of novel, added effective drug by means of informative of drug-receptor interaction mechanism¹⁴. In this study is a extension of our future efforts aiming to generate novel synthetic a lead compound and its further in vivo and invitro testing for future development as EGFR inhibitor.

MATERIAL AND METHODS:

General procedure designed for synthesis of Quinazolin 4(3H)-one^15,16

Anthranilic acid (0.01mol ml), 10ml of pyridine, add acetic anhydride (0.01mol) were refluxed in 100ml RBF under microwave at 300W for 5-15 minutes to produce 2-methyl benzoxacine-4-one which is intermediate product. The benzoxacine 4 one intermediate is irradiated with the solution of desired amines under microwave at 350 W for 25-60 minutes with continuously monitoring a reaction using thin layer chromatography and the cooled reaction mixture and poured into crushed ice and concentrated HCl with constant stirring for 30 minutes. the resulting solid react with substituted aromatic aldehydes using glacial acetic acid under reflux for 4-5hrs. The precipitate is then filter, washed by using cold water and the product is obtained, recrystallized by using ethanol. Melting point of synthesized compounds was determined by open capillaries method. The homogeneity, Purification of synthesized derivative was routinely monitored by TLC on siliga G plates, benzene: chloroform as mobile phase and visualization were done by iodine chamber.

Molecular Docking Study^17,18:

Auto Dock Tools is a program of automated docking tools, designed to determine how the small molecules bind within target protein of well known 3D-structure. Auto Dock vina used to identify the binding mode of newly synthesized derivatives responsible for activity to find out the binding energies of those compounds in the active sites. In addition the site of ligand within the enzyme binding position which can be view by discovery studio visualizer which can be useful for developing effective and prospective drug molecules for significant binding nature. Designed libraries were afforded for prediction of cytotoxic activity on Transferase EGFR TKs (1M17) by molecular docking study.

Software required:

Molecular graphics laboratory tools, Auto Dock vina PyRx virtual screening tool was downloaded in www.scrpps.edu, ChemDraw ultra 8.0 were used and biovia Discovery studio visualizer was downloaded from https://www.3dsbiovia.com/biovia-discovery. The Mol file of Ligand to PDB format translation were done by using Chem 3D Pro 8.0, protein to PDB format translation was carried out by using Molecular operating environment (MOE).

Methodology:

Computer Aided drug design which is one of the tool acting a crucial role in understanding the structure activity relationship, binding energy, interaction between the ligand and protein, binding affinity etc. On this program, Auto dock was widely used in evaluating the binding studies of our synthesized compound on targeted enzyme. The binding energy of the synthesised compounds (QOC1-QOC6) on the crystal structure of EGFR TKs [PDB ID: 1M17] was obtain in protein data bank (http://www.rcsb.org/pdp) place at Brookhaven National Laboratory in 1971.

Preparation of protein:

The crystal structure 3D of EGFR-TKs of Transferase (PDB code: 1M17) was retrieved in the RSCB protein data bank. Docking preparation tool of molecular operating environment (MOE) used to prepare enzyme for docking and python prescription (PyRx) 0.8 were used to keep protein molecule in pdbqt format contains hydrogen atoms at all polar residue.

Ligand preparation:

The 2D structures of Ligands were prepared by Chembiodraw (Cambridge, MA, USA). and converted into relevant 3D structures in the open Babel of Pyrx0.8.

Validation of docking:

In Ligand active site of crystal structure of EGFR TKs removed from the (MOE) molecular operating environment for the ligand were redocked, alignment between docked ligand in the crystal structure using biovia studio viewer.

Receptor Grid Generation:

The Receptor Lattice generations require a “equipped” structure:, all atom structure with suitable bond order and formal charge. Auto Dock searches carry the binding interactions with one or more ligand and receptor molecule, usually a protein shape and properties of the receptor is represented on a grid through numerous different sets of field which provide increasingly more accurate scoring of the ligand pose and option in every tab of the Receptor Grid making panel permit essential receptor structure by exclusive of any co-crystallized ligand which may be present, confirm the position, size of the active site will be represented using receptor grids, arrangement Auto Dock constraint , A grid area were made in the region of binding position of the receptor molecule.

Docking Analysis:

Docking study be performed by PyRx auto dock vina. The results are measured in expressions free of binding energy mode and value of higher binding energy resultant the RMSD value zero to be consider as binding affinity interaction of Ligand, pose dock analysis done by biovia discovery studio visualizer.

The prepared crystal structures of ligand, active site of various enzymes such as crystal structure of [PDB ID: 1M17] were subjected to Auto dock Vina for measuring binding energies. The grid box was set at approx. above 90 90 90, genetic algorithm (GA) in default setting was engaged for the studies. In investigate parameter; quantity of runs and the other setting were left as default. The result of docked study measurements seen in output were at word format.

Location, orientation of Ligands at protein receptor, interaction in amino acids which bounce to ligand was identified, visualize in Auto Dock tool. Throughout the docking process the top ten conformations were replicated for each of the compound after the minimization of the energy.

The binding energy of all the Ligands against EGFR-TKs macromolecule prediction done by using auto dock vina, one of the most generally used docking software in docking procedure, eight binding pose be produced, the binding pose with highest binding energy resultant in the RMSD value of zero measured as binding affinity of ligand.

Among, The newly synthesized compounds QOD1 and QOD2 (-8.9 kcal/mol) exist the highest binding energy and remaining compounds of quinazolinones however, produced better binding energy than the 5 fluoro Uracil which is used as reference drug I binding score (-4.5kcal/mol) and almost related affinity when compared to Gefitinib which is used as a reference drug 2 binding score (-9.1kcal/mol.) Almost all the compounds showed the excellent binding score (-7.5 to -8.9 kcal/mol) synthesized quinazolin-4-(3H)-ones. The amino acid residue interacting with synthesized quinazolin4 (3H)-one derivatives and all molecules showed hydrogen bond interactions, many having Vander walls attraction in different amino acid residue at the binding site. In universal, the synthesized compounds were found to include almost equal binding affinity to Gefitinib this is because of an increased number of hydrogen bond, vanderwalls attraction at the amino acids of binding site. The most active molecule has hydrogen bond interactions over the enzyme although pialkyl interactions, also pi-sigma interactions were analyzed.

ADME properties prediction:

A computational method in determination of ADME properties of synthesized molecules were functionalized .the absorption percentage calculated by Topological polar surface area (TPSA). Among, all the parameters which could exist experimentally all the synthesized molecules having better range of % ABS. all these parameters calculated by molinspiration toolkit¹⁹ the outcome located at table: 3 the absorption (%ABS) calculated by % ABS = 109-(0.345×TPSA)²⁰.

Bioactivity prediction:

During this analysis , for determination of bioactivity properties of synthesized analogues were carried out by the each and every one calculated parameters ,which be able to observed that all synthesized derivatives compared with reference drug Showed less affinity with GPCR ligand (G- protein coupled receptor) kinase inhibitor, ion channel modulator, nuclear receptor ligand, protease inhibitor and enzyme inhibitor , the toxological comparative studies of all synthesized derivatives with reference drug.

Fig. 1: Scheme of the synthesized compounds²¹

Table 1: Physiochemical Properties of Synthesized Compounds

Code	Molecular formula	Binding affinity (Kcal/mol)	RF Value	Elemental analysis (%)					% Yield
Code	Molecular formula	Binding affinity (Kcal/mol)	RF Value	C	H	O	N	Halogen	% Yield
QOD1	C₂₂H₁₆ClN₃O	-8.9	0.80	70.68	4.31	4.28	11.24	9.48	73
QOD2	C₂₂H₁₆N₄O₃	-8.9	0.78	68.74	4.20	12.49	14.58	---	70
QOD3	C₂₄H₂₂N₄O	-7.5	0.78	75.37	5.80	4.18	14.65	---	71
QOD4	C₂₂H₁₆FN₃O	-8.6	0.82	73.94	4.51	4.48	11.76	5.32	73
QOD5	C₂₃H₁₉N₃O₂	-7.8	0.72	74.78	5.18	8.66	11.37	---	69
QOD6	C₂₂H₁₇N₃O₂	-8.4	0.76	74.35	4.82	9.00	11.82	---	70
5FU	---	-4.5
Gefitinib	---	-9.1

Table 2: Docking result and various interactions of tested compounds on Transferase 1M17

Code	VDW	H-Bond	Pi-alkyl	Pi-sigma
QOCI	GLYA:697, GLYA:695, SERA:696, ASPA:831, GLYA:772	----	LEU A:820 VAL A:702	--
QOC2	GLYA:695 GLYA:697 SERA:813 ASPA:813 ARG A:817 ASNA:818 ASP A:831 GLY A:772	LEUA:694	VALA:702 LEUA:820	---
QOC3	ASPA:984, META:983	ASPA:988, VALA:987	ALA A:989	VAL A:986
QOC4	SERA:696 GLYA:697 GLYA:695 GLYA:772	LEUA:697	VALA:702 LEUA:820	---
QOC5	ALAA:989 ASPA:988 META:983 GLUA:981	VAL A:987	LEUA:977	---
QOC6	ALAA:989, ASPA:988, GLUA:981, META:983	VALA:989	LEUA:977	VAL A:986
REF1	ASPA:746 ALAA:743	TYRA:803, ARCA:807, ALAA:678, GLNA:677, VALA:745	---	---
REF2	GLNA:767 LEUA:753 THRA:766 ALA A:719 GLUA:738 CYSA:751 THRA:830 GLYA:697 SERA:696 LEUA:694 GLYA:695 META:769 GLYA:772 PHEA:699	ASPA:831	LYSA:721 META:742 LEUA:764	VAL A:702

Table 3: ADME Properties of synthesized compounds

Molecular properties	Ref1	Ref2	QOC1	QOC2	QOD3	QOD4	QOD5	QOD6
M Log P	-0.59	4.19	4.51	2.96	3.94	4.00	3.89	3.35
T-PSA	65.72	68.75	46.52	46.92	50.16	46.92	56.16	67.15
N atoms	9	31	27	27	29	27	28	27
n-ON acceptor	4	7	4	4	5	4	5	5
n-OHNH donor	2	1	1	1	1	1	1	2
n violations	0	0	0	0	0	0	0	0
n-rotb	0	8	4	4	5	4	5	4
MV	96.91	385.07	330.35	326.50	362.72	321.74	342.36	324.83
MW	130.08	446.91	375.86	340.41	384.48	359.40	371.44	357.41
%ABS (%of absorption)	86.33	85.28	92.95	92.81	91.695	92.81	89.62	85.833

Table 4: Bioactivity Score of synthesized compounds

Receptors	Ref1	Ref2	QOD1	QOD2	QOD3	QOD4	QOD5	QOD6
GPCR Ligand	-2.60	0.12	0.07	0.07	0.07	0.07	0.03	0.11
Ion channel modulator	-1.95	-0.04	-0.08	-0.07	-0.08	-0.08	-0.13	-0.03
Kinase inhibitor	-.2.62	0.66	-0.14	-0.12	-0.09	-0.09	-0.15	-0.08
Nuclear receptor ligand	-3.04	-0.21	-0.39	-0.36	-0.33	-0.35	-0.36	-0.24
Protease inhibitor	-3.15	-0.30	-0.28	-0.24	-0.24	-0.27	-0.29	-0.23
Enzyme inhibitor	-1.56	0.03	-0.21	-0.17	-0.17	-0.19	-0.21	-0.13

Fig. 2:

RESULTS AND DISCUSSION:

In computational analysis, for the determination of absorption, distribution, metabolism, elimination (ADME) properties of newly synthesized derivatives produced, percentage of absorption (%ABS) calculated by TPSA. Among the entire parameters can be noted, all synthesized molecules having the excellent %ABS from 85.8 to 92.9 and the substituted electron withdrawing group having more value of 92.81 and 92.95 %ABS than the both reference drug 1 and2 having range of 86.33 and 85.28 present in table 3.the newly synthesized quinazolin 4 (3H) one violated Lipinski’s parameters and bioactivity prediction Shown less affinity with GPCR than the reference drug 1 and 2 present in Table:4. Hence could make them potential promising agents for cytotoxic of EGFR inhibitors.

The docking pose was obtained based on their docking parameters, their resultant binding pockets. These evaluations should be supportive for understanding the binding interactions over the targeted enzyme. Molecular docking studies of synthesized quinazoline- 4- one were carried out, the docked scores of the synthesized derivatives drop within range of -7.5, -8.9kcal/mol which showed at table -1 All the synthesized Compounds were established to powerfully inhibit the EGFR TKs Transferase enzyme was completely proficient locate the target protein and outcome of docked result is showed all the docking study of Ligands include (high binding energy value) lower energy value when compared to the 5-Fluoro Uracil as a reference drug 1 as a result of binding energy value of -4.5kcal/mol. In addition the various interaction value of QOD1 to QOD6 and Ref drugs I and 2 which showed at table-2. and some docked Ligands including ref1, ref2 present in Fig-2. Represent the optimum low binding energy (high binding energy values) for the docked score of Ligands. Amongst six Ligands that were docked by EGFR-TKs Transferase enzyme, The substituted electron withdrawing group ligand chlorine and nitro ligand QOD1, QOD2 showed the majority effective with high binding score of -8.9 kcal/mol. The substituted electron withdrawing group ligand QOD4 the best docked value of -8.6kcal/mol, further, substituted electron donating methyl group ligand QOD3 the best docked score of -7.5 while substituted electron donating methoxy group ligand QOD5 -7.8kcal/mol and ligand QOD6 by the way of docking score of -8.4 kcal/mol likewise, docked ligand arrangement demonstrate Hydrogen bond and electrostatic interaction, Pi alkyl and Pi sigma interactions present in table-2.

CONCLUSION:

In preliminary Insilico study of Insilico showing of different analogues was performed to evaluate the drug like properties by molinspiration software. Bioactivity of the proposed analogues and Drug likeness properties were analyzed and all the synthesized derivatives obeyed towards Lipinski rule were elected for additional studies. Docking score of synthesized derivatives discovered based on scores of the docking study compound was preferred further analysis will be structural and pharmacological evaluation study.

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Received on 14.08.2020 Modified on 28.09.2020

Research J. Pharm. and Tech. 2021; 14(9):4849-4855.

DOI: 10.52711/0974-360X.2021.00842