Synthesis , Characterization and Biological Activity of 2-Methyl-3-aminoquinazolin-4 ( 3 H )-ones Schiff Bases

O tratamento de 3-amino-2-metilquinazolina/6-bromo-2-metilquinazolina-4(3H)-onas, 2a,b, com 2,3-indolinediona na presença de traços de ácido acético glacial forneceu 3-{(2 -oxo-1 ,2 -dihidroindol-3 -ilideno)amino}-2-metilquinazolina/6-bromo-2-metilquinazolina-4-(3H)-onas, 3a,b, as quais foram condensadas com várias aminas secundárias e formaldeído em etanol para originar compostos do tipo 3-{(1 -alquil/arilaminometil-2 -oxo-1 ,2 -di-hidroindol-3 -ilideno)amino}-2metil-6-quinazolin-4-(3H)-onas, 4a1-6 e 4b1-6.Os compostos sintetizados foram caracterizados por análise elementar, espectroscopia no infravermelho, RMN H e espectrometria de massas. Adicionalmente, foram investigadas suas atividades antimicrobiana, analgésica, anti-inflamatória e anti-helmíntica. Os resultados das atividades biológicas revelaram que os compostos 4a3, 4a4 e 4b6 exibiram atividades analgésica e anti-inflamatória significativas. Os compostos 4b5 e 4b6 apresentaram atividade anti-helmíntica, quando testados frente a Pheretima posthuma.


Chemical synthesis
The starting material 3-amino-2-methylquinazolin-6bromo-2-methylquinazolin-4(3H)-ones 2a,b were prepared according to literature methods, 10,11 from the corresponding anthranilic acid.3-Aminoquinazolinone derivatives 2a,b were used as precursors for further reaction with the purpose of synthesizing several heterocyclic compounds attached to the 3-methylquinazolin-4(3H)-one moiety.Thus, condensation of 2a,b with 2,3-indolinedione in the presence of trace amount of glacial acetic acid, furnished the corresponding Schiff bases 3a,b.The Schiff bases thus obtained were treated in ethanol with formalin and various secondary amines to afford the corresponding Mannich bases 4a 1-6 and 4b 1-6 .Analytical data, FT-IR, 1 H NMR and mass spectra confirmed the structures of the new compounds 4a 1-6 and 4b 1-6 .Compounds 3a,b showed absorption bands at 3472 cm -1 for NH group and at 1727 and 1670 cm -1 due to C=O stretching vibrations.Also, the 1 H NMR spectra of compounds 3a and 3b showed a singlet at 10.63 and 10.53 ppm, respectively for the NH hydrogens, confirming the formation of Schiff bases 3a,b.
The mass spectra of 3a showed molecular ion peak M + at m/z 304 corresponding to the molecular formula C 17 H 12 N 4 O 2 .The infrared spectra of compounds 4a 1-6 and 4b 1-6 were characterized by the disappearance of the NH absorption band.Also, the 1 H NMR spectra of title compounds 4a 1-6 and 4b 1-6 showed the presence of methylene group and the aromatic hydrogens all appeared at the expected chemical shifts (see Experimental).The mass spectra of 4a 1 showed the molecular ion peak M + at m/z 361 corresponding to the molecular formula C 20 H 19 N 5 O 2 .

Antimicrobial activity
Compounds 4a 1-6 and 4b 1-6 were assayed for their antibacterial activity against Staphylococcus aureus, Staphylococcus faecalis, Escherichia coli, and Salmonella typhi and for antifungal activities against Candida albicans and Aspergillus niger by cup plate method. 12Further, their minimum inhibitory concentration values against these microorganisms were determined by serial dilution method. 13The test microorganisms were obtained from Department of Microbiology, OUAT, Orissa, India.Muller Hinton agar plates (37 o C, 24 h) and Sabouraud's dextrose agar plates (26 o C, 48-72 h) were used for the cultivation of bacteria and fungi, respectively.The results of antibacterial and antifungal activity tests are summarized in Tables 1 and  2 with standard drugs ciprofloxacin and clotrimazole for comparison.Most of the synthesized compounds were found to possess varied degrees of antibacterial activities as evident from their minimal inhibitory concentration (MIC).Compounds 4b 5 and 4b 6 were found to possess moderate activity against all the tested bacteria while all the tested compounds except 4b 5 and 4b 6 showed lower activity against Escherichia coli.Most of the synthesized compounds were found to possess moderate activity against tested fungi.However, no correlation was observed between different substituents at 1 position of 2,3-indolinedione ring and antifungal activity.It is noted that bromination at 6 position of 2-methylquinazolin-4(3H)-one ring does not have any significant effect on antimicrobial activity.

Analgesic and anti-inflammatory activity
The synthesized compounds 4a 1-6 and 4b 1-6 were evaluated for analgesic and anti-inflammatory activities.Students t-test were performed to ascertain the significance of the exhibited activities.The test compounds (100 mg kg -1 ) and the standard drug indomethacin (10 mg kg -1 ) were administered in the form of a suspension (1% carboxymethyl cellulose as vehicle) by oral route.The study of analgesic activity was done by tail immersion method, 14 using wistar albino mice.The percent analgesic activity was calculated and listed in Table 3 with a standard drug indomethacin for comparison.Compounds 4a 2 ,a 4 , and 4b 2 ,b 4 having diethylaminomethyl and dihexylaminomethyl groups, respectively at 1 position of 2,3-indolinedione nucleus showed significant percent analgesic activity ranging from 50 to 54.96 after 1 h of administration.Moreover, diethylaminomethyl group was found to be optimum for analgesic activity in comparison to other groups.It was noted that bromination at 6 position of 3-methylquinoxaline-4(3H)-one ring decreases the analgesic activity.Remaining compounds showed less analgesic activity in comparison to standard drug indomethacin.
In the present investigation the anti-inflammatory activity of test compounds 4a 1-6 and 4b 1-6 in acute conditions was evaluated using the carrageenan induced rat paw edema method of Winter et al. 15 The carrageenan induced inflammation model is a cyclooxygenases(COX) dependent reaction and is used to determine COX inhibition.For anti-inflammatory evaluation, adult albino rats of either sex were divided in groups of 6.The percent anti-inflammatory activity with indomethacin as a standard drug is presented in Table 4. Compounds 4a 2-a 4 and 4a 6 exhibited significant percent antiinflammatory activity ranging from 43.14 to 46.85 after 1h of administration while remaining compounds showed lower activity in comparison to indomethacin.It was observed that compounds 4a 3 and 4a 4 having bulky diphenylaminomethyl and dihexylaminomethyl groups, respectively at 1 position of 2,3-indolinedione nucleus showed maximum activity.It is evident from Table 4 that bromination at 6 position of 3-methylquinoxalin-4(3H)one nucleus decreases the anti-inflammatory activity.
Some of the compounds tested produced significant analgesic and anti-inflammatory effects.These compounds may exhibit both central and peripheral actions because analgesia and inflammation are central and peripheral processes, respectively.The compounds show suppressant activity on acute inflammation model of carrageenan induced inflammation in rats paw and produced significant analgesic activity.From these observations it can be indeed inferred that the endogenous chemical substances liberated during pain and inflammation like histamine, serotonin and arachidonic cascade metabolites are inhibited to produce analgesic and anti-inflammatory activities.

Antihelmintic activity
7][18] The method of Dash et al. 19 was followed and the results are presented in Table 5. Piperazine citrate, which is used in the treatment of roundworm and threadworm (cestoda) infections was selected as a reference standard.The antihelmintic activity data revealed significant activity of all the test compounds against the tested Pheretima posthuma.In the present study compounds 4a 3-6 and 4b 5,6 were found to be more potent than piperazine citrate.It is evident from results that compounds containing dihexylaminomethyl, piperazinylmethyl and morpholinomethyl groups at 1 position of 2,3-indolinedione ring are more active.It is also noted that bromination at 6 position of 2-methylquinazolin-4(3H)-one derivatives slightly increases the antihelmintic activity.The principal aim of the present study was to modify and optimize the structural features of 2, 3, 6-trisubstituted quinazolin-4(3H)-ones.

Experimental
Melting points were determined on a Tempstar apparatus and are uncorrected.Infrared spectra were recorded on a Jasco (410) FT-infrared spectrophotometer, measured as KBr disks. 1 H NMR were recorded on a Bruker DPX-300 MHz spectrometer in deuteriochloroform with trimethylsilane as internal standard (chemical shift in ppm).The mass spectral data were obtained with a Perkin-Elmer Hitachi RMU-6L MS-30 spectrometer at 70 eV and a 90 o C inlet temperature.Purity of all the compounds was checked on silica gel plates and spots were located in iodine vapours.Elemental analysis was performed on EURO EA (Italy) analyzer and the results were within 0.3% of calculated values.

General method for the preparation of Mannich bases 4a 1-6 and 4b 1-6
To a mixture of 3a,b (0.005 mol) and 37% formalin (1 mL) in ethanol (20 mL) was added drop wise appropriate secondary amines (0.005 mol) with stirring over 15 min.The stirring was continued for 1h at room temperature and the reaction mixture then warmed for 15 min on a water bath.The mixture was poured into ice-cold water and stored in a refrigerator.The solid thus separated was filtered, washed with water, dried and recrystallized from appropriate solvent.The following title compounds were prepared as just described:

Table 3 .
Analgesic activities of orally administered compounds 4a 1-6 and 4b 1-6 by tail immersion method Results are expressed in mean ± SEM (n = 6).Significance levels a P < 0.05, b P < 0.01, and c P < 0.001 as compared with respective control.

Table 4 .
Anti-inflammatory activities of orally administered compounds