Uses of 2-Ethoxy-4(3H) quinazolinone in Synthesis of Quinazoline and Quinazolinone Derivatives of Antimicrobial Activity: The Solvent Effect

2- Ethoxy-4(3H) quinazolinone 1 was synthesized and allowed to react with various halides, namely: alkyl, benzyl, allyl, acyl, haloacetyl, crotonyl, benzoyl, 2-furoyl and 1-naphthalenesulphonyl halides affording quinazoline and quinazolinone derivatives. The reactions of compound 1 with phosphorus oxychloride, phosphorus pentasulfide, ethyl chloroformate, ethyl chloroacetate, α-bromoglucose tetraacetate, p-acylaminobenzenesulfonyl chloride, acrylonitrile, chalcone and chalcone oxide were also investigated. Depending on the reaction condition and reactant halide, the type of substituent (alkyl, acyl, aroyl, etc.) that will reside on either of the expected positions (3 or 4) on the quinazoline moiety can control the reaction pathway for synthesis of the promising products. The significant role of solvent responsible for determining both the reaction pathway and type of products synthesized was also discussed. Some derivatives were chosen for biological screening test against Gram (-ive) and Gram (+ive) bacteria and two strains of fungi.


Results and Disscussion
By the solvent free reaction of 2-ethoxy (4H)-3, 1-benzoxazin-4-one with ammonium acetate 2-ethoxy (4H)-3,1-quinazolin-4-one 1 was synthesized to be used as the starting material. In this paper we report its behavior towards various types of organic halides, with the aim of obtaining more precise information about the course of reaction and bearing into consideration the effects of changing the reaction conditions (dry solvent, solvent free, base, etc.) on the reaction pathway to obtain the promising products. Therefore the interaction of compound 1 with alkyl halides such as methyl iodide, ethyl iodide, benzyl chloride and ethyl chloroacetate, carried out in dry acetone and anhydrous potassium carbonate gave the corresponding N-substituted quinazolinones 2a-d (Scheme 1), respectively. The IR spectra of products 2a-d showed absorption bands in the range 1672-1791 attributable to υ max of C=O groups. But when the reaction was carried out using allyl bromide the O-substituted quinazoline 3 was obtained, confirming the allylic effect caused by the unsaturated moiety adjacent to the carbon atom bearing the halide. In addition, the IR spectrum of product 3 showed an absorption band at 1264 attributable to υ max of (C-O-C) and no any band for the C=O group. Compound 1 was reacted with acid chlorides, namely: acetyl, n-decanoyl, phthalimidoacetyl and 1-naphthalene sulfonyl chlorides in dry acetone / anhydrous K 2 CO 3 affording the 3-substituted 4a-d products, respectively, and with α,β-unsaturated or aromatic halides namely: crotonyl, furoyl and benzoyl chlorides affording the O-substituted derivatives 5e-g respectively. But when the reactions were done using oil bath, both the O-substituted 5a-g and N-substituted 4a-g, with lower yields of the latter. The IR spectra for 4a-d showed two groups of absorption bands in the ranges 1671-1691 and 1714-1738, which are attributable to the endocyclic and exocyclic carbonyl groups of quinazolinone derivatives, respectively, whereas the IR spectra of 5e-g showed absorption bands in the range 1763-1769 attributable to C=O groups of the ester. This can be a good proof in explaining why compound 1 when reacted, in dry acetone, with acid chlorides having the carbonyl groups directly linked to an aliphatic moiety (e. g. acetyl, decanoyl, phthalimidoacetyl) the amide form of quinazolinone enhanced the formation of the N-substituted quinazolinones, whereas its reactions with the α, β-unsaturated or aromatic acid chlorides (e. g. crotonyl, furoyl and benzoyl chlorides) allowed the imidol form to predominate enhancing the formation of Osubstituted quinazolines. Moreover, this behavior was opposed under solvent free condition. However, in dry pyridine only products 4a-g were obtained with the formation of the more favorable pyridinium chloride. This confirmed that the more basic pyridine, compared with the only polar acetone, preferred to interact with the slightly more acidic amide form affording the pyridinium salt according to the expected reaction pathway, and therefore enhancing the formation of the 3-substituted quinazolinones, independent of the type of acid chloride used (Scheme 2). The structures of 4a-g and 5a-g were based on the microanalytical and spectral data.
Compound 1 was reacted with p-acetylaminobenzenesulfonyl chloride, ethyl chloroformate and chloro-acetyl chloride in dry pyridine affording derivatives 6, 7 and 8 respectively (Scheme 3). The IR spectra of products 6-8 showed two types absorption bands in the ranges 1666-1672 and 1712-1762 attributable to υ max of the endocyclic C=O group of quinazolinone and exocyclic C=O groups of acetyl-aminophenyl, ethoxycarbonyl and chloroacetyl moieties, respectively. It is well known that the N-glycosylated heterocycles are of important significance because of the cytotoxic activitiy of their acetylated derivatives, which increases dramatically after hydrolysis [14]. Herein we discuss the interaction of derivative 1 with α-bromoglucose tetraacetate in 1,4-dioxane to afford α and β anomers of the N-substituted acetylated quinazolinone derivative 9 followed by the deacetylation using potassium carbonate /methanol to give α and β anomers of the deacetylated product 10 (Scheme 4). The IR spectra for 9 showed two absorption bands at 1662 and 1738 attributable to υ max of the endocyclic C=O group of quinazolinone and the acetyl groups of per-O-acetylated glucose moiety respectively. The H NMR showed no indication for the N-H of quinazolinone and elemental analysis showed no indication for the bromide atom which was originally attached to the glucose moiety. This confirmed its removal during the reaction of glucosamine with quinazolinone.
On the other hand, compound 1 was reacted with an alkyl halide having an N-glucosidated thiadiazole moiety, the 2-chloromethyl-5-glucopyranosylaminothiadiazole, in DMF for 8 h affording compounds 14. The H NMR spectrum of 14 showed a singlet at δ 5.29 for the N-CH 2 bonding. Acetylation of 14 afforded derivative 15. The IR spectrum was devoid any NH group band. Derivatives like 14 and 15 are believed to have biological activities similar to those of cyclic and acyclic nucleosides [18].

General disc diffusion (agar-based) method
Standard discs of tetracycline (antibacterial agent) and amphotericin B (antifungal agent) served as positive controls and references for antimicrobial activities respectively, but filter discs impregnated with 10µL of solvent (chloroform, ethanol, DMF) were used as a negative control. The agar used is Mueller -Hinton agar that is rigorously tested for composition and pH. The depth of the agar in the plate is a factor to be considered in this method. Blank paper discs (Schleicher and Schuell, Spain) with a diameter of 8.0 mm were impregnated with 10 µL of the tested concentration of the stock solutions. When a filter paper disc impregnated with a tested chemical is placed on agar, the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area susceptible to the chemical around the disc. This area of no growth around the disc is the "zone of inhibition" or "clear zone". For disc diffusion, the zone diameters were measured with slipping calipers of the National Committee for Clinical Laboratory Standards (NCCLS) [20]. Agar-based method is a good alternative method being simpler and faster than broth-based methods [21,22].

Antibacterial Activity
Concentration of 1 mg/mL of test compounds were prepared by dissolving the compounds in its proper solvent. For each concentration, 0.2 mL of synthesized compounds (1 mg/mL) was added to each hole. The plates were allowed to stand at room temperature for two hours and then incubated. The organisms were grown in nutrient agar at 37 o C for 24 hours. After incubation period, the growth inhibition zones diameters were carefully measured in mm. The clear zone around the wells was measured as inhibition zones. The absence of a clear zone around the well was taken as inactivity.
Results of antibacterial activity tested against E. Coli (G-) and S. Aureus (G+) showed that all of the selected compounds were antibacterially active and comparatively efficient.

Antifungal Activity
The samples were dissolved, each in its proper solvent, then 0.5 mL sample of each compound (1 mg/mL) plus 0.1 mL of the tested fungal suspension were mixed thoroughly with 20 mL of agar medium, which was maintained at 45 o C. The inoculated medium was poured into sterile Petri-dishes, allowed to solidify, and incubated at 25 o C for seven days. Results of antifungal activity tested showed that compounds 4c, 4d, 6, 9 and 10 were active against both fungi, none was active with A. flavus, 4f, 4g, 8 and 15 were active only with C. albicans, whereas the rest of compounds were totally inactive.
All the results for the antimicrobial evaluation are given in (Table 1) showing the inhibition zone diameter in mm/mg sample. Both compounds 9 and 10 showed the highest inhibition with S. aureus whereas compounds 6 and 9 showed the highest inhibition towards C. albicans.
In conclusion all the products 4b-d,f,g, 6-10 and 15 were antibacterially active and comparatively efficient. In addition, compounds 4c, 4d, 6, 9 and 10 were active against both fungi, 4f, 4g, 8 and 15 were active only with C. albicans, and the rest were inactive.
The antimicrobial activity of the products compared to those of tetracycline and amphotericin B are given in Fig 1.

Experimental
All melting points recorded are uncorrected. The IR spectra were recorded on a Pye Unicam SP1200 spectrophotometer using KBr wafer technique. The 1 H-NMR spectra were determined on a Varian FT-200, Brucker AC-200 MHz instrument using TMS as an internal standard. Chemical shifts (δ) are expressed in ppm. The mass spectra were determined by EI technique using MP model NS-5988 and Shimadzu single focusing mass spectrometer (70 eV). (1): 2-ethoxy(4H)-3,1-benzoxazin-4-one (0.01 mol) and ammonium acetate (0.01 mol) were fused using an oil bath for 2 h. The mixture was poured into an ice/water mixture and stirred. The off-white precipitate that separated out was filtered, washed, air-dried and crystallized from ethanol to give off-white crystals of compound General procedure for the synthesis of compounds 2a-d and 3.

2-Ethoxy-4(3H)quinazolinone
A mixture of quinazolinone 1 (0.01 mol) and any of the alkyl halides methyl iodide, ethyl iodide, benzyl chloride, ethyl chloroacetate or allyl bromide (0.01 mol) in dry acetone and anhydrous K 2 CO 3 (50 mL/2 g) was heated under reflux for about 24 h. The excess acetone was distilled off and the residue was poured into cold water with stirring. The solid that separated out was filtered by suction, washed with water, dried and crystallized from suitable solvent affording derivatives 2-3, respectively.  General procedure for the synthesis of compounds 4a-d and 5a-c.
To a solution of compound 1 (0.01 mol) in 50 mL of dry acetone was added the acid chloride, acetyl, n-decanoyl, phthalimidoacetyl, naphthalene sulphonyl, crotonyl, furoyl or benzoyl chlorides (0.04 mol) and anhydrous K 2 CO 3 (0.04 mol). The reaction mixture was refluxed for 24 h. The excess acetone was evaporated and the residue was poured into cold water with stirring. The separated solid was filtered off, washed with cold water, dried and crystallized from the proper solvent to give 4 and 5, respectively.  General procedure for the synthesis of compounds 6, 7, and 8.

Synthesis of Compounds 9 (acetylated α and β anomers) and 10 (deacetylated α and β anomers)
A crude mixture of compound 1 (0.05 mol) and α-bromoglucose tetraacetate (0.01 mol) in 1,4-dioxane (100 mL) was heated with stirring under reflux for 4 h. The mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was then dissolved in ethyl acetate, washed sequentially with saturated NaHCO3, dried over MgSO4. Purification and separation was achieved using column chromatography (3:1 EtOAc: Hexane) affording the acetylated derivative 9 as a white solid which was later on crystallized using dichloromethane-diethyl ether -hexane solvents. A solution of product 9 in MeOH (0.004 mol/ 65mL) was treated with sodium carbonate solution (0.002 mol). A white syrupy solid began to precipitate. The residue was purified using column chromatography (3:1, EtOAc:Hexane) to give the deacetylated product as a white solid, which was later on crystallized using dichloromethane-diethyl ether-hexane solvents to give the crystalline product 10 (α and β anomers).

2-(2-Ethoxyquinazolin
Preparation of the reagent for compound (14): crystallized from the proper solvent to afford 2-chloromethyl-5-aminothiadiazole. A mixture of this product and α-bromoglucose tetraacetate (0.01mol each) in anhydrous acetonitrile (300 mL) was left for 24 h at room temperature with occasional stirring. The suspension was filtered through Celite and the filtrate was taken to dryness. The resulting residue was purified by flash chromatography using a gradient of hexane/ethyl acetate (2:1 to 1:1) affording the acetylated α-anomer of the reagent, the 2-chloromethyl-5-N-(2,3,4,6-tetra-Oacetyl-β-glucopyranosyl) aminothiazole. A mixture of this reagent (0.01 mol) and quinazolinone 1 (5 eqv.) was heated under reflux in 1,4-dioxane (50 mL) for 8 h. The excess solvent was then evaporated and the residue was poured into cold water with stirring. The solid that separated out was filtered, washed with cold water, dried and crystallized from DMF to give light brown crystals of the acetylated product of derivative 14; m.p. above 300ºC. Deacetylation was achieved by adding CH3ONa solution (0.01mol) to this derivative (0.02mol) in methanol (65  mL