Synthesis and characterization of fully substituted pyrimidines by using ketene dithioacetal as potent antimicrobial agent

Various ketene dithioacetals of acetoacetanilides were reacted with guanidine nitrate in the presence of base to produce the 2-amino-4-isopropyl-6-alkoxy- N -arylpyrimidine-5-carboxamide derivatives with good yields. All the synthesized compounds were characterized by mass, NMR and IR and also evaluated for antimicrobial activity against five different bacterial and fungal strains. The compounds 4i, 4k and 4l has found comparatively good active against all the bacterial strains


INTRODUCTION
From the beginning of pharmaceutical research nitrogen containing heterocyclic compounds have been considered as the privileged structure because of the different biological property and its tremendous applications e.g., anticancer, diuretic, anticonvulsant, anti-inflammatory and antihypertensive activities [1]. Particularly pyridine nucleus which is the important constructive part of many natural products and used as antimalarial [2], antitoxoplasma [3], fungicidal [4], antibacterial [5], anti-inflammatory and antitumor agent [6]. The Biginelli reaction [7] has been reported for the synthesis of many pyrimidine darivatives by the one-pot condensation of active methylene, urea and aldehydes. There are some other methods are also reported with some little modification in Biginelli reaction by the condensation of aldehydes, urea and arylketones in acetic acid using a catalytic amount of KHSO4 [8], in basic condition [9] and by microwave irradiation [10]. Now a days of literature have been reported for the synthesis of thiophene, furan, isooxazole, pyrazole, pyridine and pyrimidine etc. heterocycles by using dithioacetal in place of aldehyde [11,12]. Thus, above literature promote us to synthesize novel pyrimidine analogues using ketene dithioacetal and their biological screening.

RESULT AND DISCUSSION
In our current research variety of acetoacetanilides 2a-t were prepared from methyl 4methyl-3-oxopentanoate (Scheme 1). These acetoacetanilides on reaction with CS 2 and methyl iodide in presence of potassium carbonate produce ketene dithioacetal 3a-t (Scheme 2) on active methylene. These dithioacetals are behave as a precursor of aldehyde, which on reaction with guanidine nitrate in presence of sodium methoxide or sodium ethoxide lead to the formation of fully substituted pyrimidine 4a-t (Scheme 2). Here base replaces one thiomethoxy group and after cyclization methoxy or ethoxy group is introdused at the position of thiomethoxy group. Varoius ketenedithioacetal was used for the synthesis of pyrimidine, is listed in Table 1.

1. Experimental
Thin-layer chromatography was accomplished on 0.2-mm precoated plates of silica gel G60 F 254 (Merck). Visualization was made with UV light (254 and 365 nm). IR spectra were recorded on a FT-IR-8400 spectrophotometer using DRS prob. 1 H (400 MHz) and 13 C (100 MHz) NMR spectra were recorded on a Bruker AVANCE II spectrometer in CDCl 3 . Chemical shifts are expressed in δ ppm downfield from TMS as an internal standard. Mass spectra were determined using direct inlet probe on a GCMS-QP 2010 mass spectrometer (Shimadzu). Melting points were measured in open capillaries and are uncorrected.

1. 1. General procedure for the synthesis of substituted isopropylacetoacetanilide(2a-t)
A mixture containing the primary amine (10 mmol), methyl isobutyrylacetate (10 mmol) and catalytic amount of sodium or potassium hydroxide lie (10 %) was reflux at 110 °C for the approximately 15-20 h. The reaction was monitored by TLC. After completion of reaction, the solvent was removed under vacuum. The solid or oil was crystallized from methanol to give pure product 2a-t.

1. General procedure for the synthesis of ketene dithioacetals (3a-t)
A mixture of 4-methyl-3-oxo-N-arylpentanamide 2a-t, (10 mmol) and dried K 2 CO 3 (10 mmol) in DMF was stirred for 2 h at room temperature. Then CS 2 (30 mmol) was added and the mixture was stirred for an additional 2 h at room temperature. Methyl iodide (20 mmol) was added at 0-5 °C and reaction mixture was stirred again for 4 h at RT. After completion of reaction, reaction mixture was poured in to cold water (40 mL). The precipitated crude product was filtered and crystallized in EtOH.

1. 3. General procedure for the synthesis of 2-amino-4,6-disubstituted pyrimidine-5carboxamide derivatives (4a-t)
Ketene dithioacetals 3a-t (10 mmol) was added in the mixture of guanidine nitrate (10 mmol) and sodium methoxide or sodium ethoxide (20 mmol) in methanol or ethanol within 10-15 min. The resulting reaction mixtures was further stirred at room temperature for 15 min then refluxed for 6h. After completion of the reaction, the mixture was poured in to cold water. Precipitated solid was filtered, wash with water, dried and crystallization from EtOH to afford analytically pure products 4a-t.

ANTIMICROBIAL SCREENING OF SYNTHESIZED PYRIMIDINES
All the synthesized compounds were tested against different bacterial and fungal strains i.e. Pseudomonas aeruginosa, Proteus vulgaris, Escherichia Coli, Staphylococcus aureus, Candida albican for their in vitro antibacterial activity. Well Diffusion/Agar Cup Method was used and results are listed in Table 2. The investigation of antibacterial and antifungal screening data revealed that all the tested compounds 4a-t showed moderate to potent activity. The compounds 4i, 4k and 4l showed comparatively good activity against all the bacterial strains.