Synthesis and Antibacterial Activity of 6-Amino-2-[ ( E )-2-( 4-hydroxy-3-methoxyphenyl ) ethenyl ]-3 , 4-dihydroquinazolin-4-one and its Intermediate Compounds

Quinazolinone ring system is known to have a broad spectrum of antibacterial activity against positive Gram and negative Gram bacteria [1]. The antibacterial activity is due to its similarity to the structure of bacterial dihydrofolate reductase (bDHFR) inhibitor [2-4]. Several compounds are known to inhibit the bacterial dihydrofolate reductase enzyme in vitro. One is 6-amino-3-benzyl-4H-quinazolinone. This compound has shown good activity against Staphylococcus aureus and inhibited bacterial dihydrofolate reductase at IC50 40 μM. The amino group at position 6 plays an important role of the activity. Compound analogues without the amino substituent at position 6 lacks the antibacterial activity [5]. Another compound is 3-(5-bromothiazol-2-yl)-2-(E)styrylquinazolin-4-one. This compound’s activity can be compared with the positive control which was used. The styryl (phenylethenyl) group at position 2 and substituents at position 3 play an important role in the activity [6]. This study aimed to combine the chracteristic of the two compounds to make a new quinazolinone derivate which has antibacterial activity. Several methods are possible for synthesizing quinazolinones, but microwave and ultrasonic irradiation has attracted considerable attention for rapid synthesis of organic compounds [7]. Synthesis and Antibacterial Activity of 6-Amino-2-[(E)-2-(4-hydroxy-3methoxyphenyl)ethenyl]-3,4-dihydroquinazolin-4-one and its Intermediate Compounds


INTRODUCTION
Quinazolinone ring system is known to have a broad spectrum of antibacterial activity against positive Gram and negative Gram bacteria [1]. The antibacterial activity is due to its similarity to the structure of bacterial dihydrofolate reductase (bDHFR) inhibitor [2][3][4]. Several compounds are known to inhibit the bacterial dihydrofolate reductase enzyme in vitro. One is 6-amino-3-benzyl-4H-quinazolinone. This compound has shown good activity against Staphylococcus aureus and inhibited bacterial dihydrofolate reductase at IC50 40 µM. The amino group at position 6 plays an important role of the activity. Compound analogues without the amino substituent at position 6 lacks the antibacterial activity [5]. Another compound is 3-(5-bromothiazol-2-yl)-2-(E)styrylquinazolin-4-one. This compound's activity can be compared with the positive control which was used. The styryl (phenylethenyl) group at position 2 and substituents at position 3 play an important role in the activity [6]. This study aimed to combine the chracteristic of the two compounds to make a new quinazolinone derivate which has antibacterial activity. Several methods are possible for synthesizing quinazolinones, but microwave and ultrasonic irradiation has attracted considerable attention for rapid synthesis of organic compounds [7].
All the solvents, reagents and chemicals were analytical or synthesis grade and used without further purification. Meting points were determined in capillary tube using melting point apparatus (Stuart Scientific) and are uncorrected. 1 H NMR, 13 C NMR and other 2-D spectra were recorded on a JEOL JNM 500 MHz FTNMR spectrometer; chemical shifts are expressed in δ (ppm) with reference to TMS. Infrared (IR) spectra were recorded on a FTIR spectrophotometer (8400S, Shimadzu). Mass spectral (MS) data were obtained on a LC-MS (Mariner Biospectrometry) using Q-tof mass spectrometer fitted with an ESI source. Full-scan mode from m/z 100 to 1200 was performed with a source temperature of 140 °C. HPLC column (Supelco 5µ C18, 250 × 2 mm i.d.) was used for the analysis. Solvent A was water with 0.3 % acetic acid; solvent B was methanol with 0.3 % acetic acid. Solvents were delivered at a total flow rate of 1 mL/min. The solvent running by isocratic elution. Thin layer chromatography was performed on precoated (0.25 mm) silica gel GF254 plates (E. Merck, Germany), compounds were detected with 254 nm UV lamp. Microwave irradiation was performed using modified home microwave oven (Sharp) equipped with reflux condensor.
Synthesis of 2-methyl-6-nitro-3,4-dihydroquinazolin-4-one (2): Compound 2 was synthesized by nitration of compound 1 according to reported method for nitro derivatives of quinazolin-4-one [9]. To the solution of compound 1 (8 g, 50 mmol) in 133.26 mL concentrated sulphuric acid (98 %) was added dropwise of fuming nitric acid (11.18 mL, 250 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 4 h and then poured into crushed ice (50 g). The precipitate was filtered and washed with cold purified water and dried in vacuum drying oven at 70 °C for 1 h. The solid was recrystallized from ethanol.

Synthesis of 6-amino-2-[(E)-2-(4-hydroxy-3-methoxyphenyl)ethenyl)-6-nitro-3,4-dihydroquinazolin-4-one (4):
Compound 4 was synthesized by reduction of compound 3 using modified method of aryl nitro reduction as reported earlier [11,12]. To a suspension of compound 3 (101.72 mg, 0.3 mmol) in 30 mL the mixture of hydrochloric acid-ethanol (1:1) was added reduced iron powder (251.28 mg, 4.5 mmol). The suspension was exposed to ultrasonic irradiation for 2 h at 55 °C with TLC analysis monitoring for the completion of the reaction. The reaction mixture was filtered to remove the iron residue and the filtrate obtained diluted with ethanol and added concentrated ammonia solution to precipitate salts. The mixture was filtered through cellite and solvent of the filtrate was evaporated under reduced pressure. The solid was washed with ethanol and purified water dried in vacuum drying oven at 70 °C for 1 h.
Antibacterial activity: Antibacterial activity test was performed by disc diffusion method with nutrient agar medium [13]. Antibacterial activity was performed against Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 14028 and Escherichia coli ATCC 25922. Inoculum was prepared by suspending bacteria (in the form of freeze-dried) into nutrient broth medium and diluted to obtain a concentration of 1 × 10 6 bacteria/mL. 5 mg of synthesized compound and positive control (trimethoprim) were carefully weighed and dissolved in 5 mL of DMSO and then diluted with 45 mL sterile purified water in order to obtain a concentration of 100 mg/mL. The solution was sterilized by filtration and created a serial dilution with mixture of DMSO-H2O (1:1) to obtain the following concentrations 50 mg/mL; 25 mg/mL; 12.5 mg/mL; 6.25 mg/mL. Compound 3 was made in the same way but with the solvent DMSO. Negative control was made in form of DMSO. 20 µL of each dilution test solution and control solution were dripped on the 6 mm paper disc. The paper discs were placed in a petri dish containing nutrient agar and inoculated bacteria and then incubated for 18-24 h at 37 °C.

RESULTS AND DISCUSSION
The titled compound was synthesized stepwise as shown by Scheme-I. In first step, anthranilic acid was reacted with acetic anhydride (Ac 2 O) under microwave irradiation for 20 min at power level 50 % (400 Watt) to obtain an intermediate compound 2-methylbenzoxazin-4-one. Ammonium acetate was added to the mixture (30 %) and the microwave irradiation was continued for 20 min at power level (240 Watt) to provide compound 1 [8]. This compound was treated with fuming nitric acid in concentrated sulphuric acid at room temperature for 4 h in order to obtain compound 2 [9].
Then compound 2 was treated with vanillin in glacial acetic acid in presence of sodium acetate as catalyst and irradiated with microwave to obtain compound 3. The irradiation was performed for 10 times for 10 min at intervals of 5 min each. Physical data and analytical data of the synthesized compounds is given in Table-1. The UV-visible and IR spectra data of the synthesized compounds are given in Table-2. The NMR spectra data of the synthesized compounds are given in Table-3
None of the synthesized compounds showed any antibacterial activity. The result of the susceptibility assay of the synthesized compounds is given in Table-4. The lack of antibacterial activity by the tested compound might be caused by several factors. One is the solubility of the tested compound: solubility in water was important, because the compounds should be spread in the medium. The process of distribution of compounds in the medium was important for the success of the disc diffusion method [13]. The disc diffusion method was chosen instead of the turbidimetry method, because the tested compounds, especially compounds 2 and 3 will undergo salting out in an aqueous medium.
The other factor is the ability of tested compounds to penetrate the bacterial cell walls. The tested compounds was supposed to target the bacterial dihydrofolate reductase enzyme found in the cytoplasm of bacteria. Therefore, the tested compound had to be able to penetrate the bacterial cell wall. The penetration ability of the tested compound was influenced by the physical and chemical properties of these compounds [2].
The results indicated that the tested compounds were not be able to penetrate the bacterial cell wall.