Synthesis and Anti-inflammatory Activity of Newly Synthesized 1,7-Dimethyl-4H-Benzo [D] [1,3]-Oxazin-4-One and 3-Amino-1,7- Dimethyl-Quinazolin-4(3H)-One

General experimental procedure

All reagents and solvents were purchased from sigma-Aldrich, in Germany. Melting points were determined on a kofler hot stage apparatus and were uncorrected. IR spectra were recorded on a Buck scientific IR M500 instrument. The 1H and 13C NMR spectra were recorded in DMSO-d6 at 400MHz with HAZ VOLATILE V2. M Chemical shifts are reported in ppm relative to tetramethylsilane. Gas chromatography mass spectra were obtained on a Finingan MAT 44S mass spectrophotometer operating at 70eV. Elemental analysis was carried out with analytical. Thin layer chromatography (TLC) was used to monitor the reactions.

Synthesis of 7-methyl-1-methyl-4H-benzo [D] [1,3]-oxazin- 4-one (1)

This involved the condensation of Methyl-2-amino-7-methylbenzoate 2.11g(0.01mol) and 1.02g (0.01mol) acetic anhydride in 30ml ethanol medium were reacted. The reaction was heated under reflux with stirring using a magnetic stirrer until the reaction mixture showed no trace of starting material when the TLC was developed (2 hours). At the end of the reaction, work up was done. Ethanol was removed in vacuum and the crude mixture was poured into 50ml of ice water on a cold-water bath. The mixture was stirred for 30 minutes filtered and extracted into ethyl acetate and allowed to evaporate at room temperature to give solid products which were recrystallized from hexane or dichloromethane-hexane mixture. Yield was 2.01g (95%), mp: 148-150 oC (Figure 1).

Figure 1:

Synthesis of 3-amino-7-methyl-1-methyl quinazolin-4 (3H)-one. (2)

The condensation of equimolar amounts of 2-methyl-7-methyl- 4H-benzo [D] [1,3]-oxazine-4-one (1.06g, 0.005mol) and hydrazine hydrate (0.93g, 0.01mol) were added to 30ml boiling ethanol with stirring using a magnetic stirrer until the reaction mixture showed no trace of starting material when the TLC was developed (3 hours). At the end of the reaction, the reaction mixture was concentrated in vacuum under reduced pressure using rotary evaporator. The white precipitate formed was then filtered, washed three times with 20ml of distilled water [20mlx3]. The white crystals were dried and recrystallized from dimethylformamide (DMF) to give pure 3-amino-2-methylquinazolin-4 (3H)-one. Yield was 1.00g (94%) mp: 97-99 oC (Figure 2-4).

Figure 2:

Where: R₁ = H, R₂ = H and R₃ = CH₃

Figure 3:

Figure 4:

Where: R₁ = H, R₂ = H and R₃ = CH₃

Chemistry

The introduction of 2-amino substituent is a successful strategy to improve the chemical stability of benzoxazinone. Due to the pharmacological activities of 4(3H)-quinazolinone derivatives, 2,3-disubstituted derivative of quinazoline-4-one were synthesized via the interaction of the benzoxazinone derivative with nitrogen nucleophile with the aim of obtaining more pricise information about the course of the reaction and some interesting pharmaceutical compounds. The reaction of 4, 5-disubstituted derivatives of 2-amino-methyl-5-methylbenzoate and acetic anhydride yielded the cyclic compound 7-methyl-1-methyl-4H-benzo[d]-[1,3]-oxazin- 4-one. The reaction of this compound with hydrazine hydrate yielded 3-Amino-7-methyl-1-Methyl Quinazolin-4 (3H)-one.

Pharmacological evaluation

Anti-inflammatory activity: Anti-inflammatory activity will be measured using carrageenan-induced rat paw oedema assay [22,23]. Groups of 5 rats of both sexes (pregnant female excluded) were given a dose of an anti-inflammatory activity of synthesized compounds. After one hour 0.1ml, 1% Carrageenan suspension in 0.9% NaCl solution were injected into the sub-planter tissue of the right-hand paw. The linear paw circumference was measured at hourly interval for four hours [24]. Two groups of drugs treated rats and one control group were used each test day and the mean paw oedema value for the test group being compared with the mean value for the control group for that day. Anti-inflammatary activity [25] will be measured as the percentage reduction in oedema level where drug was present, relative to control. Indomethacin (10mg/ kg) was administered orally as reference drug, whereas 10% Tween 80 was uses as negative control.

Statistical analysis: All data were expressed as the mean + SEM, the student’s t-test was applied to determine the significance of the difference between the control group and the test compounds.

Elemental analysis: The compositions of the compounds are summarized in Table 1. The C and H contents (both theoretically calculated values and actual values) are indicated

Table 1:Characterization and physical data of synthesized compounds.

Table 2:¹³C-NMR of synthesized compounds.

Table 3:¹³C-NMR of synthesized compounds.

Table 4:Effect of the test compounds on acetic acid induced writhing in mice.

The in vivo analgesic activity of 7-chloro-2-methyl4[H]- benzo[d][1,3]-oxazin-4-one (1) and 3-amino-7- chloro-2-methylquinazolin- 4(3H)-one (2) was determined using mouse writhing assay and the results obtained are summarized in Table 1-4.

Characterization of 7-methyl-1-methyl-4H–benzo[d] [1,3]–oxazine–4–one (1)

¹H NMR (400MHz, DMSO) δ 7.16 (s, 1H), 6.40 (s, 1H), 3.78 (s, 6H), 3.68 (s, 3H), ¹³(NMR (400MHz, DMSO) δ 168.28, 155.80, 149.23, 140.28, 113.37, 100.56, 100.05, 56.94, 56.94, 56.13, 51.93, 16.95: IR (KBr, cm^-1) 3381, 3203, 3135, (NHz), 3018 (CH aromatic), 2951, 2871, 2718 (CH aliphatic), 1662 (C=0). Anal.Cal 1159 (c-0) for C¹¹H₁₁N0₄; C 62.20; H 5.18. Found: C 62.10, H 4.98.

Characterization of 3-amino-7-methyl 2-methyl quinazoline-4-(3H)-One (2)

 ¹H NMR (400 MHz, DMSO) δ 7.41 (s, 1H), 7.10 (s, 1H), 5.80 (s, 2H), 3.90 (s, 6H), 2.58 (s, 3H), ¹³C NMR (400MHz, DMSO) δ 160.28, 155.29, 154.57, 149.07, 143.77, 113.65, 108.24, 105.64, 56.80, 56.63, 22.58, IR (KBr, cm^-1) 3301 (NH₂), 1622 (C=0), Anal. Cal. for C11H13N303; C 56.11, H 5.53; Found, C 56.40, H 5.41.

The test investigated compounds exhibited significant antiinflammatory activity when compared with the control test sample. For the IR spectra, compound 1 were characterized by absence of υ NH2and presence of υ C-O stretch in 1159cm-1 region of the compound. Compound 2 was characterized by absence of υ C-O and presence of υNH2 in 3284cm-1and 3194cm-1 region of the compound. Among the prepared products, 3-Amino-7-methyl- 2-methyl-3H-quinazolin-4-one (2) was found to exhibits the most potent in vitro anti-inflammatory activity. The compounds synthesized exhibited promising anti-inflammatory drug. Compound 1 has Anti-inflammatory activities of 42.55% and 55.54% at 20mg/kg and 40mg/kg respectively while Compound 2 has Anti-inflammatory activities of 58.38% and 73.56% respectively. Compound 2 showed the highest activity at 40mg/ kg compared to the other compound 1, acetylsalicylic acid and indomethacin. It may be that the substitution of amino group at position three increases the activity. These compounds synthesized have a higher activity than acetylsalicyclic acid, which is a standard anti-inflammatory drug.
The acetic acid induced abdominal constriction method is widely used for the evaluation of peripheral antinociceptive activity [26]. It is very sensitive and able to detect antinociceptive effects of compounds at dose levels that may appear inactive in other methods like the tail-flick test [26,27]. Local peritoneal receptors are postulated to be partly involved in the abdominal constriction response [28]. The method has been associated with proteinoids in general, e.g., increased levels of PGE2 and PGE2a inter peritoneal fluids [29], as well as lipoxygenase products by some researchers [30,31]. Indomethacin (10mg/kg) was administered orally as reference drug while 10% olive oil was used as negative.

Compound 1 has Anti-inflammatory activities of 74.67% and 42.55% at 20 mg/kg and 54.45% at 40 mg/kg dose levels, while compound 2 has Anti-inflammatory activities of 58.38% and 73.56% at 20 mg/kg and 40mg/kg dose levels respectively. The compounds have high Anti-inflammatory activities. Compound 2 has a higher Anti-inflammatory activities compared to compound 1 and also has a higher Anti-inflammatory activity compared to Indomethacin, a standard Anti-inflammatory drug. It is therefore concluded that compound 2 could be a potential anti-inflammatory agent and a tool in pharmaceutical drug delivery.

The author hereby declares that the work presented in this article is original and that any liability for claims relating to the content of this article will be borne by them.

The author appreciates the assistance of the Department of Pharmaceutical Pharmacology at the University of Benin who supplied the Swiss mice and Ethic approval of animal use from Ethics committee of the Faculty of pharmacy, University of Benin, Benin City Nigeria who gave the Ethic approval of animal use.

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