Novel quinazolinone derivatives: Synthesis and Antimicrobial Activity

This work involves to synthesis novel organic compounds and studies their pharmacological. Approach: The title compound quinazolinone derivatives were prepared by reacting 3 amino of 3-amino-2-methylquinazolin-4(3H)-one (first) with various aldehydes and ketones, and second coupling through diazonium salts with resorcinol or with ethylacetoacetate then cyclized with hydrazine. The starting material 3-amino-2-methylquinazolin-4(3H)-one was synthesized by reacting hydrazine with 2-methyl-4H-benzo[d][1,3]oxazin-4-one, which in turn was prepared from anthranilic acid. The chemical structures of the synthesized compounds were confirmed on the basis of their spectral data (FT-IR, UV/visible, HNMR, spectra and CHN analyss). All synthesized compounds were tested in vitro against a number of microorganisms (Staphylococcus aurous, E.coli, Proteus vulgaris, Pseudomonas, and Klebsiella) and two fungal Aspergillus niger and Candida albicans in order to assess their antimicrobial properties. Results: The study indicates that these compunds have high activity against tested bacteria. Aim: We aim to synthesize quinazolinone derivatives having active moieties to evaluate their antimicrobial activities. Conclusion/Recommendations: Based on the reported results, it may be concluded that 3-amino-2-methylquinazolin-3(4H)-one act as synthones for Schiff bases and for diazonium coupling. [Researcher 2010;2(4):82-88]. (ISSN: 1553-9865).


Experimental and Methods
All chemical used were of reagent grade (supplied by either Merck or Fluka) and used as supplied. The FTIR spectra in the range (4000-400) cm-1 were recorded as KBr disc on FTIR 8300 Shimadzu Spectrophotometer. Proton NMR spectrum was recorded on Bruker-DPX 300 MHz spectrometer with TMS as internal standard in Jordan University. The UV-Visible spectra were measured in ethanol using Shimadzu UV-Vis. 160 A spectrophotometer in the range (200-1000) nm. Elemental microanalysis was carried out using CHNOS elemental analyzer model 5500 Carlo-Erba instruments(Italy made). Gallen Kamp M.F.B.600.010 F melting point apparatus was used to measure the melting point of all the prepared compounds.

Method B:
A mixture of orth-acetamido(ethyl )benzoate (0.01 mole) and (0.01 mole) of hydrazine hydrate in 150mL. of ethanol were refluxed for 12h. then left to cool at room temp. The reaction mixture was washed with water, the recrystalized from ethanol.

Synthesis of diazocompounds (II and III):
A solution of the 3-amino-2-methylquinazolin-4(3H)-one (0.05 mole) in a mixture of (25ml) of hydrochloric acid and (50ml) of glacial acetic acid was diazotized at 0 o C with sodium nitrite solution (0,055 mole) dissolved in 10 ml of water. After Keeping at this temperature for 3 hours the resulting solution of diazonium salt was filtered and added to a mixture of 75ml. of glacial acetic acid, (0.05mole) of ethyl acetoacetate EAA (or resorcinol) and (0.37mole) of sodium acetate at 0 o C to 5 o C and pH=5.5. The mixture was left for 10hr , then an equal volume of water was added and the crude product was collected, washed with ethanol, then water, and recrystallized from ethanol.

Synthesis of Schiff bases (V-IX):
A mixture of 0.0lmole of 3-amino-2-methylquinazolin-4(3H)-one and 0.0l mole of aldehyde or ketone in (10 ml) absolute ethanol was refluxed in water bath for (30min ) then left to cool in ice -water. The solid was filtered, washed with 2%HCl then water and recrystallized twice from ethanol.

Polymerization:
The resin was synthesized by methods reported earlier (Gabilondo, 2002;Young and others, 2003;Waage, and Elder, 1991). The IX-formaldehyde resins were synthesized in molar ratio by adding IX and formaldehyde (37% aqueous solution). The pH (9.0) was adjusted by using of sufuric acid. The reaction mixture was refluxed for 30 minutes at 90 o C.
2.6 Biological activities 2.6.1Antibacterial activity: The Test Organisms used were: Staphylococcus aureus as gram positive bacteria, and Escherichia coli, Proteus vulgaris, Klebsiella and Pseudomonas aeruginosa as gram negative bacteria. Hole diffusion method was used to measure the inhibitory activity as indicated by the diameter of the inhibition zone. Concentration of 1mg/mL of test compounds were prepared by dissolving the compounds in dimethyl sulfoxide (DMSO), for each concentration, 0.2 ml of synthesized compounds II-X (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°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.

Antifungal activity:
The test organisms used were Aspergillus niger and Candida albicans. Samples II-X were dissolved in DMSO 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°C. The inoculated medium was poured into sterile Petri-dishes, allowed to solidify, and incubated at 25°C for seven days. The plates were examined for evidence of inhibition of growth.

Results and Discussion 3-amino-2-methylquinazolin-4(3H)-one (I):
Compound (I) is the key intermediates for the compounds synthesized later in this work. It has been prepared by the two methods, first by the reaction of 2-methyl-4H-benzo[d] [1,3]oxazin-4-one with hydrazine by using of ethanol as asolvent. Yield 50% and m.p. These data show clearly that these tow atoms are the most reactive toward the electrophilic substitution reaction on benzene ring, in compound (I). The determined bond angle and twist angle and 3D-geometrical structure, indicate that this molecule is planar.

Synthesis of diazocompounds ((E)-ethyl 2-((2-methyl-4-oxoquinazolin-3(4H)-yl)diazenyl)-3-o xobutanoate(II) and (E)-3-((2,4-dihydroxyphenyl)diazenyl)-2-methylquin azolin-4(3H)-one (III)):
Diazotisation is carried out in concentrated acid is used for the diazo component, since hydrolysis of the diazonium salt occurs in dilute acid. Here, the acid of choice was concentrated hydrochloric acid. used in amixture with glacial acetic acid. A particularly important reagent combination is the nitrosyl hydrochloric acid which is used extensively as a nitrosating agent for compound (I). Coupling is usually accompanied by some evidence of decomposition however, by careful addition of diazonium salt solution at 0-5 o C to a solution of coupling component in acetic acid, 30, 45% yield of azoquinazolin was usually obtained. To complete the coupling, particularly for reactions using nitrosyl hydrochloric acid in the diazotisation, the pH of the reaction mixture was adjusted to approximately 4-5. Thus, an appropriate amount of 10 % sodium acetate solution was slowly added below 5 o C. The yield of compound (II) was 30% (and m.p.=135 o C), but the yield of compound (III) was 70% (and m.p. =125 o C). The IR spectra of (II and III) showed the disappearance of the NH group absorption bands and the presence of bands at 1495 cm -1 and 1470 cm -1 for (N=N), and the absorption band for C=O groups (II and III) were 1710 and 1700 cm -1 respectively. In addition of 3175 cm -1 for O-H group for the compound (III).

Synthesis of Schiff Bases (V-IX ):
In general, Schiff 's bases are prepared by refluxing equi molar amounts of the primary amines (aromatic or aliphatic or related derivatives) with carbonyl compounds (aldehyde or ketones, aromatic or aliphatic derivatives) in appropriate solvent (cyclohexane, benzene and preferentially ethanol). Sometimes, the reaction is catalyzed by few drops of glacial acetic or piperidine and refluxing for 2 hours. The physical and spectral data are shown in table 1. Proton NMR for the compound (VII): ( S. 1.6(3H) for CH 3 , S. 1.9(3H) for CH 3  The yield of compound (X) was 55% (and m.p.=166 o C). The IR spectra of (IV) showed absorption band for C=O group at 1723 cm -1 Biological Activity: The antimicrobial screening data show that the compounds exhibit antimicrobial properties and it is important to note that the new derivatives exhibit more inhibitory effects than the orginal molecule (I). From table (2) it is clear that the zone of inhibition against the gram-negative bacteria and gram-positive bacteria. The increased activity of the new derivatives can be explained that act as more powerful and potent bactericidal agents, thus killing more of the bacteria than the orginal molecule (I). The π-electron delocalization over the new derivatives increases the lipophilic character and favours its permeation through the lipoid layer of the bacterial memberanes. It was reported that 3H-quinazolin-4-one derivatives have interesting antimicrobial activity against different species of Gram positive bacteria, Gram negative bacteria and pathogenic Fungi. Schiff's bases have been widely reported to be biologically versatile compounds having antifungal, fungicidal, herbicidal and plant growth regulating properties. The presence of imino linkage ( -N=C-) in these compounds has been regarded as being assential for the enhancement of antibacterial and antimicrobaial activities (Rajesh, and Greech, 1988