Design, Synthesis, Computer Modeling and Analgesic Activity of Some New Disubstituted Quinazolin-4(3H)-ones

Quinazolines derivatives exhibited a vital role in many pharmacological activities [1-8] including anti-inflammatory, [9] antibacterial, [10] and anticonvulsant, [11] activities. Schiff 's bases have generated a great deal of attention due to their interesting pharmaceutical activities include possess potent analgesic and antiinflammatory activities [12]. In the view of these facts and to develop earlier reporting [6] quinazoline-4(3H)-ones series that drawn great attention in the field of synthetic medicinal chemistry because it shown good analgesic and anti-inflammatory activities therefore, our aim was oriented to design derivatives of existing clinically used NSAIDs that has ability to inhibit the cyclooxygenase (COX) and as a result in safety when taking paracetamol [13] because it used in medication to treat pain and fever [14] through acting by inhibition of cyclooxygenase (COX), and recent findings suggest that it is highly selective for COX2 [15]. As part of our ongoing medicinal chemistry research program we found that quinazolines [6] especially quinazolin-4(3H)-ones with 2, 3-disubstitution that reported [16] to possess significant analgesic, anti-inflammatory activities. Based on these findings it is rationalized to synthesis and design new substituted quinazolin-4(3H)-ones and screen their anti-inflammatory and analgesic activities (Scheme 1).


Introduction
Quinazolines derivatives exhibited a vital role in many pharmacological activities [1][2][3][4][5][6][7][8] including anti-inflammatory, [9] antibacterial, [10] and anticonvulsant, [11] activities. Schiff 's bases have generated a great deal of attention due to their interesting pharmaceutical activities include possess potent analgesic and antiinflammatory activities [12]. In the view of these facts and to develop earlier reporting [6] quinazoline-4(3H)-ones series that drawn great attention in the field of synthetic medicinal chemistry because it shown good analgesic and anti-inflammatory activities therefore, our aim was oriented to design derivatives of existing clinically used NSAIDs that has ability to inhibit the cyclooxygenase (COX) and as a result in safety when taking paracetamol [13] because it used in medication to treat pain and fever [14] through acting by inhibition of cyclooxygenase (COX), and recent findings suggest that it is highly selective for COX-2 [15]. As part of our ongoing medicinal chemistry research program we found that quinazolines [6] especially quinazolin-4(3H)-ones with 2, 3-disubstitution that reported [16] to possess significant analgesic, anti-inflammatory activities. Based on these findings it is rationalized to synthesis and design new substituted quinazolin-4(3H)-ones and screen their anti-inflammatory and analgesic activities (Scheme 1).
for C 8

Synthesis of substituted 3-Amino-2-methylthio Quinazolin-4(3H)-one (VI)
A solution of 3-amino-2-mercaptoquinazolin-4(3H)-one 1.93 g (0.01 mol) in sodium hydroxide 10 ml (20% w/v) was obtained by warming on a water bath. It was clarified by filtration while in warm condition, cooled and treated with dimethyl sulphate 1.26 g (0.01 mol) under constant stirring. The solution was stirred at room temperature for 12 h. The solid obtained was filtered off, washed with cold water, dried and recrystallized from chloroform/ethanol.

Pharmacology
All the newly synthesized compounds VIII 1-12 were preliminarily evaluated for their analgesic and Anti-inflammatory activities (using writhing test) using paracetamol as writhing protective stander. The analgesic activity of the newly synthesized compounds VIII 1-12 compared to paracetamol (Sigma Chemical Co., St. Louis, MO, USA) as a reference was measured after and 30, 60, 90, 120, 150, and 180 after p-benzoquinone (Aldrich) subcutaneous injection. All the tested compounds significantly showed highly percentage of protection against writhing compared with the control untreated group.

Analgesic screening
Adult albino mice of either sex weighing 20-25 gm which was obtained from animal house of Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. Mice's were divided into twelve groups; each group consists of six mice's per cage. The mice's were kept under constant temperature 30°C and 12 hours light/dark cycle. All animals were acclimatized in the animal facility for at least two weeks prior the experiments. The animals were kept fastened for 24 hours prior to the experiment, but they were allowed free access to water [20,21]. The animal experiments described below comply with the ethical principles and guidelines for the care and use of laboratory animals adopted by the National Egyptian Community. The equipment used was Dial micrometer model (120 -1206 Baty, Sussex, England). The test compounds as well Paracetamol were suspended in water by the aid of few drops of Tween-80 (Sigma) to produce 2% suspension. And p-benzoquinone (Aldrich) was dissolved in water for injection containing a few drops of Tween-80 to produce 0.02% solution and was used as writhing inducer.

Analgesic activity
The analgesic action of some newly synthesized compound was determined using the writhing method on mice [22]. The mice were randomly arranged in groups each of 10 animal's one group was kept as control. The animals of another group were given paracetamol subcutaneously in a dose of 30 mg/kg body weight. Mice of the other groups were blindly injected subcutaneously with test compounds in a dose of 150 mg/kg body weight. After 30 minutes, each animal of each group was injected with 0.25 ml of 0.02% aqueous solution of p-benzoquinone and was observed for writhing after 30, 60, 90, 120, 150 and 180 minutes. Animals protected from writhing were recorded in each group and the analgesic potency of the test compounds was determined as percentage of protection against writhing. The results are presented in Table 1.

Molecular Modeling
Docking studies were carried out to examine the analgesic effect of compounds VIII 1-12 which subjected to docking using Molecular Operating Environment (MOE) program [23] on the 3D structure of the cyclooxygenase-2 enzyme (COX-2) in a trial to predict their analgesic action drugs and the aim of the flexible docking calculations is prediction of correct binding geometry for each binder.

Preparation of the target protein
The protein target needs to be prepared and modeled according to the format requirements of the docking algorithms used. Thus the required protein was downloaded from Protein Data Bank (PDB) (code 4COX) using discovery Studio 2.5 software. Water molecules were removed from downloaded protein. Crystallographic disorders and unfilled valence atoms were corrected using alternate conformations and valence monitor options. Protein was subjected to energy minimization by applying CHARMM force fields for charge, and MMFF94 force field for partial charge. Inflexibility of structure is obtained by creating fixed atom constraint. The binding site of the protein was defined and prepared for docking.

Tested compounds preparation
The designed compounds 2D structures were sketched using ChemBio Draw Ultra 14.0 and saved in MDL-SDfile format. SD file opened, 3D structures were protonated and energy minimized by applying CHARMM force fields for charge, and MMFF94 force field for partial charge, then prepared for docking by optimization of the parameters.

Results and Discussion
The present work, involves the synthesis of new derivatives of substituted-2-Benzylamino-3-(substituted benzylidene amino) quinazolin-4(3H)-one VIII 1-12 via starting with key intermediates through bromination and chlorination of methylanthranilate using reported method [17][18][19] to get compound I 1-4 . Compound I 1-4 underwent reaction with carbon disulfide and sodium hydroxide to afford II 1-4 that suspected to dimethylsulfate resulting the compound III 1-4 that when reacted with hydrazine hydrate it yielded compound V 1-4 . The structures of such new compounds were confirmed by both elemental and spectral analyses. The IR spectra of V 1-4 in KBr showed carbonyl stretching around 1700 cm -1 in addition, to NH 2 stretching around 3300 cm -1 . The 1 H NMR spectra of V 1-4 in DMSO-d6, showed singlet of one proton, at 3.20-3.29 ppm due to SH group and NH 2 group appeared as abroad singlet at 5.21-5.69 ppm which is D 2 O exchangeable moreover, In the 13 C-NMR spectra of these compounds showed C=O peak at about 160 ppm corresponded to carbonyl groups of quinazoline ring that confirm the cyclization of intermediate IV into V. Consequently compound VI was obtained from V upon treatment with dimethyl sulfate in sodium hydroxide and the structure of the resulting compounds clearly confirmed from 1 HNMR spectra that showed disappearance of SH signal and appearance of singlet signal around 2.5 ppm belong SCH 3 . Frequently, replacement of alkylthio group at 2-position with benzyl amine in simple electrophilic substitution reaction it produce new quinazoline compounds VII [1][2][3][4] where the elemental analysis and spectral data confirm the existence of this substitution reaction where the 1 HNMR of new compound contain two signals one at 4.40-4.80 ppm and another one at 5.40-5.80 ppm that exchangeable with D 2 O which belong to NH and NH 2 respectively, also the structure of some the compounds were established from the spectral data of the resulting compounds. The title compounds substituted-2-Benzylamino-3-(substituted benzylidene amino) quinazolin-4(3H)-one VIII 1-12 were obtained by the condensation of amino group of 3-amino-2-substituted-benzylamino Quinazolin-4(3H)-one VII 1-4 with a different aromatic aldehydes that afford new Schiff 's bases. The assignment of the produced Schiff 's bases were based on spectral and elemental analysis where 1 H-NMR spectrum of all the compounds VIII 1-12 showed disappearance of signal due to NH 2 group in addition, the 1 H-NMR spectrum of all the compounds VIII 1-12 exhibit singlet of one proton around 8.50-8.97 ppm which belong the (N=CH) proton. The IR spectrum of titled compounds VIII 1-12 showed the presence of peak carbonyl (C=O), NH and Aryl groups. Because titled compounds contain halogen atom(s) its mass spectrum showed molecular ion peaks corresponding to their molecular formula in addition, to its isotopic peak moreover, in some compounds containing two halogen the mass spectrum showed peaks of M+, M+2, and M+4 that clearly observed and consequently, proven the resulting product. Finally, the structure of the newly synthesized product compounds VIII 1-12 was proven on the basis of their elemental and spectral data. From the previous mentioned discussion it was observed that our synthetic strategies adopted to obtain the newly synthesized quinazolin-4(3H)-one depending using whether simple synthetic procedure or simple reagent(s). The results of analgesic testing indicate that the test compounds exhibited excellent significant analgesic activity and docking study revealed that the synthesized compounds have potential analgesic activity and can be further optimized and developed as a lead compound. The rationalized steps depend on ligand based drug design particularly a molecular hybridization approach that involves the coupling of two or more groups with relevant biological properties.

Docking Discussion
The obtained results indicated that all studied ligand have similar position and orientation inside the putative binding site of the COX -2 enzyme. The selected compounds VIII 12 , VIII 9 , VIII 3 , and VIII 11 showed good binding energies ranging from -37.18 to -39.12 kcal/mol ( Table 2).

Conclusions
We have synthesized newly derivatives of disubstituted quinazolin-4(3H)-ones that showed analgesic activity. From the data obtained in Table 1 it was found that all derivative VIII 1-12 have excellent significant analgesic activity. In addition to, the molecular docking for all compounds was performed on the active site of COX-2 enzyme in a trial to predict their mode of action as analgesic drugs, in which the compounds showed several interactions leading to the conclusion that they might exert their action through inhibition of COX-2 enzyme. The biological analgesic screening was performed in Pharmacology Department, Faculty of Pharmacy Al-Azher University, Cairo, Egypt.