Synthesis , Analgesic and Anti-inflammatory Activities of 3-Ethyl-2-substituted Amino-3 H-quinazolin-4-ones

Purpose: To synthesize a series of novel 3-ethyl-2-substituted amino-quinazolin-4(3H)-ones and evaluate them for their analgesic and anti-inflammatory activities. Methods: The compounds, 3-ethyl-2-substituted amino-quinazolin-4(3H)-ones, were synthesized by reacting the amino group of 3-ethyl-2-hydrazino quinazolin-4(3H)-one with a variety of aldehydes and ketones. The synthesized compounds were characterized by Fourier transform infrared (FTIR), protonnuclear magnetic resonance spectroscopy (H-NMR) and mass spectrometry. The purity of the compounds was determined by elemental analysis. Test for analgesic activity was performed by tail-flick technique using Wistar albino mice while anti-inflammatory activity was evaluated by carrageenaninduced paw oedema test in rats. Diclofenac sodium was used as positive control for both analgesic and anti-inflammatory activities. Results: The compound, 3-ethyl-2-(cyclohexylidene-hydrazino)-3H-quinazolin-4-one (AS1), emerged as the active analgesic (activity, 63.89 %) and anti-inflammatory (activity, 60.00 %) compound of the series, and compared well with the reference standard, diclofenac sodium, which exbited analgesic and anti-inflammatory activities of 62.04 and 65.11 %, respectively Conclusion: The compound (AS1) can serve as a lead molecule for further development to a clinically useful novel class of analgesic and anti-inflammatory agents.


INTRODUCTION
Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for the treatment of acute and chronic inflammation, pain and fever.Most NSAIDs act via inhibition of cyclooxygenase, thus preventing prostaglandin biosynthesis.However, this mechanism of action is also responsible for their main undesirable effects, gastrointestinal (GI) ulceration and, less frequently, nephrotoxicity.Increase in hospitalization and deaths due to GI-related disorders parallels increased use of NSAIDs.
Therefore, the discovery of new safer antiinflammatory drugs represents a challenging goal for such a research area [1][2][3][4].
The present work is an extension of our ongoing efforts towards the development and identification of new molecules for analgesic and anti-inflammatory activities.The objective of the present study was to synthesize a series of 3ethyl-2-substitutedamino-quinazolin-4(3H)-ones and evaluate them for their analgesic and antiinflammatory activities.).The NMR spectra of the synthesized compounds were recorded in deuterated chloroform (CDCl 3 ) (unless specified) with tetramethyl silane (TMS) as internal reference (chemical shift in , ppm) using Varian 300 MHz and Bruker 500 MHz (Washington, USA) spectrometers.The mass spectra of the compounds were obtained on Jeol GC mate instrument (Masspec, Japan).Elemental analyses were performed in Perkin-Elmer 2400 CHN elemental analyzer (Waltham, USA).The progress of the reaction was monitored on readymade silica gel plates (Merck) using chloroform : methanol (9:1) as a solvent system.Iodine was used as a developing agent.

3-Ethyl-2-thioxo-2,3-dihydro-1H-quinazolin-4one (4)
A solution of ethylamine (1.31 g, 0.02 mol) in dimethyl sulfoxide (10 mL) was stirred vigorously.To this was added carbon disulphide (1.6 mL) and aqueous sodium hydroxide (1.2 mL of 20 M solution) dropwise during 30 min with stirring.Dimethyl sulphate (2.5 g, 0.02 mol) was then added gradually, keeping the reaction mixture in freezing mixture with stirring which was continued for further 2 h.The reaction mixture was then poured into ice-water and the solid obtained was filtered, washed with water, dried and recrystallized from 95 % ethanol.
Methyl anthranilate (3) (1.5 g; 0.01 mol) and the above prepared N-(ethyl) methyl dithiocarbamic acid (0.01 mol) were dissolved in ethanol (20 mL).To this anhydrous potassium carbonate (100 mg) was added and refluxed for 23 h.The reaction mixture was cooled in ice and the solid separated was filtered and purified by dissolving in 10 % alcoholic sodium hydroxide solution (2N) and re-precipitating by treatment with 20 % dilute hydrocholoric acid.The solid obtained was filtered, washed with water, and dried.It was recrystallized from 95 % ethanol to afford (4).

Animal studies
The synthesized compounds were evaluated for analgesic and anti-inflammatory activities.

Analgesic activity
Test for analgesic activity was performed by tailflick technique [15,16] using Wistar albino mice (25 -35 g) of either sex selected by random sampling.Diclofenac sodium, at dose levels of 5, 10 and 20 mg/kg, was administered orally by gastric lavage as reference drug for comparison.
The test compounds at three dose levels (5, 10, 20 mg/kg) were administered orally.Reaction time was recorded at 30 min, 1, 2 and 3 h after treatment, and cut-off time was 10 s.Analgesic activity (AA, %) was calculated as in Eq 1.
where T 1 is the reaction time (s) before treatment, and T 2 the reaction time (s) after treatment.

Anti-inflammatory activity
Anti-inflammatory activity was evaluated by carrageenan-induced paw oedema test in Wistar Albino rats [17].Diclofenac sodium (5, 10 and 20 mg/kg) was administered as a standard drug for comparison.The test compounds were administered orally at three dose levels of 5, 10 and 20 mg/kg.Paw volume was measured using mercury displacement technique with the aid of a plethysmograph immediately before and 30 min, 1, 2 and 3 h after carrageenan injection.Inhibition (I, %) of paw oedema was calculated as in Eq 2.
where x is mean paw volume of the rats before administration of carrageenan and test compounds or reference compound (test group), a is the mean paw volume of rats after the administration of carrageenan in the test group (drug-treated), b is the mean paw volume of rats after the administration of carrageenan in the control group, y is the mean paw volume of rats before administration of carrageenan in the control group.

Statistical analysis
Statistical analysis of the biological activities of the synthesized compounds in the animals was carried out using one-way analysis of variance (ANOVA).In all cases, post-hoc comparisons of the means of individual groups were performed using Tukey's test.A significance level of p < 0.05 denote significant difference in all cases.All values are expressed as mean ± standard deviation (SD).GraphPad Prism 3.0 version.(GraphPad GraphPad Software, Inc.11452 El Camino Real, #215, San Diego, CA 92130 USA) was used for the statistical analysis.

RESULTS
The key intermediate H NMR spectra of 4 showed multiplet around δ 1.0-2.0 and 7.10-7.50for ethyl (5H) protons and aromatic (4H) protons respectively; and a singlet at δ 10.55 indicating the presence of NH.Data from elemental analysis were in conformity with the assigned structure.Further, the molecular ion recorded in the mass spectra is also in agreement with the molecular weight of the compound.
The IR spectra of compound 5 showed disappearance of NH and C=S stretching signals of cyclic thiourea.It showed a peak for carbonyl (C=O) stretching at 1680 cm -1 .The 1 H NMR spectra of compound 5 showed singlets due to SCH 3 , at δ 2.70, multiplet around 7.20-7.60for aromatic (4H) protons, respectively.Data from the elemental analyses and molecular ion recorded in the mass spectra further confirmed the assigned structure.
Nucleophilic displacement of methylthio group of 5 with hydrazine hydrate was carried out using ethanol as solvent to afford 3-ethyl-2-hydrazino-3H-quinazolin-4-one 6.The formation of 6 was confirmed by 1 H NMR spectra which showed singlets at  4.56 and 10.31 due to NH 2 and NH, respectively, a multiplet at δ 1-1.5 and 7. 15-7.30for ethyl (5H) protons and aromatic (4H) protons respectively.The NH and NH 2 signals at 3330, 3205 cm -1 are appeared in the IR spectra.It also showed a peak for carbonyl (C=O) at 1670 cm -1 .Data from the elemental analyses are in conformity with the assigned structure.Further, the molecular ion recorded in the mass spectra is also in agreement with the molecular weight of the compound.
The compounds, 3-ethyl-2-substituted amino-3Hquinazolin-4-ones AS1 -AS5 were obtained by the condensation of amino group of 3-ethyl-2hydrazino-3H-quinazolin-4-one (6) with a variety of aldehydes and ketones.Formation of product is indicated by the disappearance of peak due to NH 2 of the starting material in IR and 1 H NMR spectrum of all the compounds AS1 -AS5.The IR 1 H NMR spectra of these compounds showed the presence of peaks due to (N=CR 1 R 2 ) carbonyl (C=O), NH and aryl groups.The mass spectra of the the compounds show molecular ion peaks corresponding to their molecular formula.In the mass spectra of compounds AS1 -AS5, a common peak at m/z 144 corresponding to quinazolin-4-one moiety appeared.Elemental (C, H, N) analysis satisfactorily confirmed rhe elemental composition and purity of the synthesized compounds.
Anti-inflammatory activity data (Table 2) indicate that all the test compounds protected rats from carrageenan-induced inflammation moderately at 30 min of reaction time with increased activity at 1 h that reached a peak level at 2 h.Decline in activity was observed at 3 h.

Figure 1 :
Figure 1: Some lead molecules of quinazolin-4-one EXPERIMENTAL Chemistry Melting points (mp) were taken in open capillaries on a Thomas Hoover melting point apparatus and are uncorrected.IR spectra of the synthesized compounds were recorded by FT-IR (Shimadzu, Japan) using KBr pellet ( max in cm -1

Table 2 :
Anti-inflammatory activity of the synthesized compounds (AS1 -AS5) cData expressed as mean  SD from six different experiments, each carried out in duplicate; a p < 0.05, b p < 0.01and c p < 0.001 a compared with their respective positive control