Synthesis and Antimicrobial Activity of Some New Pyrimidinone and Oxazinone Derivatives Fused with Thiophene Rings Using 2-Chloro-6-ethoxy-4-acetylpyridine as Starting Material

A series of pyridines, pyrimidinones, oxazinones and their derivatives were synthesized as antimicrobial agents using citrazinic acid (2,6-dihydroxyisonicotinic acid) as a starting material. α,β-Unsaturated ketones 3a–c were condensed with cyanothio-acetamide in the presence of ammonium acetate to give 2-cyanopyridinethiones 4a–c, which were reacted with ethyl chloroacetate to yield the corresponding cyano esters 5a–c. The esters 5a–c were cyclized by action of sodium methoxide to aminoesters 6a–c, which were aminolyzed with ammonia to corresponding aminoamide derivatives 7a-c. Also, the esters 6a–c were hydrolyzed with NaOH to the corresponding sodium salt 8a–c, which were treated with acetic anhydride to afford 2-methyloxazinones 9a–c. The latter compounds were treated with ammonium acetate to afford 2-methylpyrimidinones 10a–c, followed by methylation with methyl iodide to yield 2,3-dimethyl-pyrimidinones 11a–c. The antimicrobial screening showed that many of these compounds have good antibacterial and antifungal activities comparable to streptomycin and fusidic acid used as reference drugs.


Synthesis
The starting materials 3a-c (Table 1) were prepared from 2,6-dihydroxyisonicotinic acid (1) via the corresponding 2-chloro-6-ethoxy-4-acetylpyridine 2 according to literature methods [1,19]. Acryloyl derivatives 3a-c were condensed with 2-cyanothioacetamide in the presence of ammonium acetate to give the corresponding cyanopyridine thione derivatives 4a-c ( Table 1). Treatment of 4a-c with ethyl chloroacetate in the presence of anhydrous K 2 CO 3 gave the corresponding ethyl ester derivative 5a-c (Table 1), which were cyclized by sodium methoxide in methanol to give the amino ester derivatives 6a-c ( Table 1). Aminolysis of compounds 6a-c by action of ammonia gas afforded the corresponding aminoamide derivatives 7a-c (Scheme 1, Table 1). The IR spectra of 6a-c showed the absence of ν (CN) for 5a-c and the presence of broad band corresponding to ν (NH 2 ). Also, the IR spectra of 7a-c showed the absence of ν(C=O, ester) for 6a-c and the presence of a broad band corresponding to ν(NH 2 ). Compounds 6a-c were hydrolyzed by refluxing with ethanolic sodium hydroxide (NaOH) to the corresponding sodium salts 8a-c, which was treated in situ with refluxing acetic anhydride to give the corresponding oxazinone derivatives 9a-c ( Table 2). Reaction of 9a-c with ammonium acetate in refluxing acetic acid afforded the corresponding pyrimidinone derivatives 10a-c (Table 2), which were treated with methyl iodide in N,N-dimethylformamide in the presence of anhydrous K 2 CO 3 to yield the corresponding 3-methyl-pyrimidinone derivatives 11a-c (Scheme 2, Table 2).

Antimicrobial Activity
The antimicrobial activities of some of the synthesized compounds were determined by the agar diffusion method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [20]. The compounds were evaluated for antimicrobial activity against bacteria, viz. Streptomyces sp., Bacillus subtilis, Streptococcus lactis, Escherichia coli, and Pseudomonas sp. and antifungal activity against various fungi, viz. Aspergillus niger, Penicillium sp and yeast Candida albican and Rhodotorula ingeniosa.
The concentrations of the tested compounds (10 µg/mL) were used according to a modified Kirby-Bauer's disk diffusion method. The sterile discs were impregnated with 10 µg/disc of the tested compound. Each tested compound was performed in triplicate. The solvent DMSO was used as a negative control and streptomycin/fusidic acid were used as standard calculated average diameters (for triplicates) of the zone of inhibition (in mm) for tested samples with that produced by the standard drugs. Four of the synthesized compounds 5a, 7b, 9b and 10b exhibited potent antibacterial and antifungal bioactivity compared with the standard drug used. The other tested compounds were found to exhibit a moderate to low antibacterial activity (Table 3). On the other hand, when different concentrations of compound 9a were used, it was exhibited a moderate antibacterial activity, but it exhibited very good antibacterial activity at higher concentrations (3× and 4×) ( Table 4), while different concentrations of compounds 5a and 10a exhibited very good antifungal activities (2× and 3×) ( Table 5). Table 4. Antibacterial activity of compound 9a at different concentrations.

Chemistry
Melting points were measured using Electrothermal 9100 digital melting point apparatus (Electrothermal, Essex, UK) and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1600 FTIR (Perkin-Elmer, Downers Grove, IL, USA) in KBr discs. 1 H-and 13 C-NMR spectra were measured on a Jeol 5000 MHz spectrometer (Jeol, Tokyo, Japan) in DMSO-d 6 , and chemical shifts were recorded in δ ppm relative to the internal standard TMS. The Mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer (Madison, WI, USA) using EI and the values of m/z are indicated in Dalton. Elemental analyses were performed on a Perkin-Elmer 2400 analyzer (Perkin-Elmer) and were found within ±0.4% of the theoretical values. All reactions were followed by TLC (Silica gel, Aluminum Sheets 60 F 254 , Merck, Darmstadt, Germany). Starting material 2 was prepared from citrazinic acid (1) according to published procedures [1,19]. Antimicrobial screening was carried out in Department of Microbial Chemistry, National Research Center, Cairo, Egypt.

Conclusions
A series of newly compounds 3-11 were prepared using citrazinic acid (2,6-dihydroxyisonicotinic acid) as a starting material. The obtained derivatives were screening as antimicrobial and antifungal agents. Four of the synthesized compounds 5a, 7b, 9b and 10b exhibited potent antibacterial and antifungal bioactivity compared with streptomycin and fusidic acid used as reference drugs. The other tested compounds were found to exhibit moderate to low antibacterial activity. On the other hand when higher concentrations (3× and 4×) of compound 9a, which exhibited a moderate antibacterial activity, were used, this compound exhibited very good antibacterial activity. While different concentrations of compounds 5a and 10a exhibited a very good antifungal activity (2× and 3×).