Design, Synthesis, and Evaluation of Novel 3-, 4-substituted, and 3,4-di Substituted Quinazoline Derivatives as Antimicrobial Agents

FARAG A. EL-ESSAWY*, ABDULRAHMAN I. ALHARTHI, MSHARI A. ALOTAIBI, NANCY E. WAHBA, NADER M. BOSHTA Preparatory Year Deanship, Basic Science Department, Prince Sattam Bin Abdulaziz University, 151 Alkharj 11942, KSA Chemistry Department, Faculty of Science, Menoufia University, Shebin El-Koam, Egypt Department of Chemistry, Collage of Science and Humanities, Prince Sattam Bin Abdulaziz University, 83Alkharj 11942, KSA

In the recent years, more and more interest has been focused on nitrogen heterocyclic systems and their applications. For example, the pharmaceutical reports reveal that nitrogen heterocycles are widely described as drug fragments. Among various nitrogen heterocyclic systems, quinazoline is one of the most prevalent heterocyclic rings found in the top 25 most frequent nitrogen heterocycles in U.S. FDA approved drug [1]. Moreover, it has been intensively studied due to their employment as building blocks in drug discovery showed good pharmacokinetics properties. Moreover, the ability to support a variable number of derivatives having six positions that can be substituted subsequently support the SAR study. In addition, the most of quinazoline derivatives having various biological activities including antimicrobial [2], analgesic and anti-inflammatory [3], anti-convulslant [4], anti-cancer [5], and anti-tubercular [6] activities. In addition, quinazoline is a core structure subunit in a variety of bioactive natural products [7,8]. Quinazoline derivatives have been reported to possess significant activity as antihypertensive [9], antifibrillatory, choleretic, antiphlogistic [10], antimitotic [11], antifungal [12] and anticonvulsant agents [13].
Method B: A solution of hydrazide (1 g, 4.6 mmol) in 50 % Acetic acid (37 ml) was cooled to 0-5 ˚C and a solution of sodium nitrite (0.4 g, 5.3 mmol) in water (1 mL) was added dropwise with vigorous stirring at such a rate that the reaction temperature did not exceed 5 ˚C. The reaction mixture was stirred at the same temperature for 1.5 h, the precipitate was filtered off and dried.
Method B: A mixture of 7 (1 g, 6.9 mmol) and hydrazine hydrate (0.5 g, 10.4 mmol) in (10 mL) absolute ethanol was heated under reflux for 12 h. The odour of ammonia must be finished. The reaction mixture was cooled and then poured onto ice water, filtered and dried to give 11 as a white solid, yield 0.5 g and 1 g (72%) and (91%)
Method B: A mixture of compound 11 (0.5 g, 3.2 mmol) and acetyl bromide (0.4 g, 3.2 mmol) in dry dioxane (32 ml) was heated under reflux for 10 h, the excess solvent was removed under reduced pressure. The product that separated out filtered off then dried.

General procedure for the preparation of compounds 17a-c:
To a solution of 16 (0.2 g, 0.7 mmol) in (2 mL) acetonitrile, piperidine and/ or diphenyl amine and/ or 2,5-dimethyl aniline (0.7 mmol) and potassium carbonate anhydrous (0.7 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. Then the formed solid collected by filtration.

Methyl [(4-oxoquinazolin-3(4H)-yl)methyl]carbamate (18h)
To a solution of azide 10 (1 g, 4.4 mmol) in absolute methanol (10 ml) was heated at reflux for 7h and the reaction mixture was evaporated under reduced pressure to dryness to give 18h as an off white solid, yield 0. General procedure for the preparation of compounds 18i,j: To a solution of azide 10 (1 g, 4.4 mmol) in dry dioxane (13 mL) the corresponding phenol (4.4 mmol) was added. The reaction mixture was heated at reflux for 10 h, then the solvent was removed under reduced pressure to give a solid.

General procedure for the preparation of compounds 29a-c:
To a suspension of 4-hydrazinylquinazoline 19 (0.16 g, 1 mmol) in dioxane (50 mL) was added acetic acid (0.1 ml) and the appropriate 5(4H)-oxazolone (28a-c) (1 mmol). The mixture was refluxed for an appropriate time (36-72 h) and then the mixture was poured into cold water (100 ml). The precipitate was filtered and recrystallized from ethyl acetate to give the corresponding imidazoloquinazolinone derivatives 29a-c as yellow crystals.   The synthetic strategies and the biological evaluations on the pyrido [1,2-c] quinazolinone derivatives are limited, only two synthetic approach were reported. The first one included the synthesis from ethyl 2-(quinazolone)-5-oxo-2,5dihydroisoxazole-4-carboxylates as starting materials, in which reaction first with NaN3 followed by base-catalyzed rearrangement [28]. The second method based on the ring closure of the 2-o-amino-phenyl-6-phenylpyrimidine-4(3H)one with formic acid [29]. Both producers are long synthetic way and limited to synthesize various derivatives. Since many analogues of this ring system showed marked biological activity [30], we have investigated the synthesis of this scaffold, and herein we present our results.
Construction of the final products 17a-c can be done by reaction between 2-chloro-N-(4-oxoquinazolin-3(4H)yl)acetamide (16) and the commercially available secondary amine or the aniline derivatives in the presence of potassium carbonate as a base in acetonitrile and stirring at room temperature for 24 h to afford the final products in 81% to 83% yield as showed in (Scheme 3).
The synthetic route used to prepare carbamides 18a-g and carbamates derivatives 18h-j is outlined in (Scheme 4). In which a Curtius rearrangement of the acyl azide 10 was carried out by refluxing in dioxane. In details, refluxing the acyl azide 10 with secondary amine or aniline derivatives gave the corresponding carbamides 18a to 18g, while the refluxing with methanol or phenol derivatives gave the corresponding carbamates 18h to 18j (Scheme 4 The construction of 5-substituted or not substituted 1,2,4-triazole moiety fused from the 3,4 side with quinazoline heterocyclic ring from the c side leads to 5-substituted or not substituted 1,2,4-triazolo [4,3-c]quinazoline having various biological activities. Different derivatives from 1,2,4-triazolo [4,3-c] quinazoline have been synthesized as follow, the starting material quinazoline-4-thiol produced 4-hydrazinylquinazoline (19) in moderate yield when subjected to condensation with hydrazine hydrate in alcoholic medium [36]. The key intermediate 4-hydrazinyl derivative 19 was readily converted to the fused triazolo derivatives 20-22 (Scheme 5) by reaction with different reagents, triethylorthoformate, acetic acid, and/or benzoyl chloride to give the triazoloquinazoline derivatives 20-22 in moderate yield 67-72%. The structures of all the newly synthesized compounds 20-22 were confirmed by 1 H NMR, 13 C NMR and element analysis data, explained in the experimental part.
In order to prepared the spyropyrazolo quinazoline derivatives, 4-hydrazinylquinazoline (19) was dissolved in ethanol and refluxed with ethyl-(ethoxymethylene)-cyanoacetate, ethoxymethylene-malonate, ethoxymethylenemalononitrile, acetyl acetone and ethylacetoacetate (in AcOH) afforded the corresponding substituted pyrazole derivatives 23-27, respectively (Scheme 5). The structures of the latter compounds were confirmed on the basis of their elemental analysis and spectral data (cf. Experimental). The IR spectra of compounds 23 and 25 showed absorption bands characteristic for NH2 and C≡N groups, while those of compound 24 and 27 revealed absorption bands characteristic for C=O (ester) and C=O (keton). Also, the 1 H NMR spectra showed signals at δ = 6.41-7.33 ppm due to NH2 for compounds 23 and 25 respectively. Scheme 5. Fomation of triazoloquinazoline and spyropyrazolo quinazoline derivatives 2-Phenyl-5(4H)-oxazolone derivatives (28a-c) were prepared separately from hippuric acid, acetic anhydride, sodium acetate and an appropriate aldehyde or ketone [37]. In the last step 4-hydrazinylquinazoline (19) was reacted with the 2-phenyl-5(4H)-oxazolone derivatives (28a-c) in the presence of acetic acid (Scheme 6). In this reaction, 4hydrazinylquinazoline acts as a nucleophile. Compound (19) attacks the carbonyl group of the oxazolone ring and the ring is cleaved, then the 4-imidazolin-5-one ring is formed. It was expected to obtain compound (30) but instead compound (29a-c) were formed (reaction mechanism in Scheme 6). This reaction cannot be carried out with aldehydes or ketones containing electron-withdrawing substituents such as 4-fluorobenzaldehyde and 2,2,2-trifluoro-1phenylethanone or basic substituents such as N,N-dimethylbenzaldehyde. Basic groups react with acetic acid and convert it to an electron-withdrawing group. Electron-withdrawing substituents prevent the formation of the 2hydroxy-imidazolidinyl ring (29) (Scheme 6). https://doi.org/10.37358/RC.20.2.7951 Scheme 6. Formation of Imidazoline derivatives

Antimicrobial activity
The antimicrobial activities of the 3-, 4-substituted, and 3,4-di substituted quinazoline derivatives were evaluated by the agar diffusion method under the regulations made by Clinical and Laboratory Standards Institute (CLSI) [38]. The inhibition zone for each derivative measured by ruler to determine its size (in mm) and compared with the inhibition zone produced by the standard drugs. The quinazoline derivatives were evaluated for antimicrobial activity against bacteria (Gram-positive bacteria: Bacillus subtilis and Staphylococcus aureus; Gram-negative bacteria: Escherichia coli and Proteus vulgaris), and fungi (Candida albicans and Aspergillus flavus). Gentamycin used as standard drug for the bacterial strains, while Ketoconazole was used as standard drug for the fungi strains (table 1).

Conclusions
In conclusion, a novel series of 3-, 4-substituted, and 3,4-di substituted quinazoline derivatives were designed and synthesized. The structure of the novel derivatives have been elucidated using different spectroscopic techniques (IR, NMR spectra and EI-MS). The antimicrobial activities of the most novel quinazoline derivatives have been evaluated against Gram-positive, Gram-negative bacteria, and fungi. The most active compound against all the tested strains was recorded for compound 6c. whereas compounds 13d, 15a, 17b, 18b, 18d, 25 and 29a-c of the newly synthesized compounds exhibited moderate antimicrobial activities. So that, the pyrimidoqunazoline derivative 6c could be useful as a hit scaffold for further modifications to synthesized more active derivatives.