Synthesis of Certain Pyrimidine Derivatives as Antimicrobial Agents and Anti-Inflammatory Agents

A variety of novel bicyclic and tricyclic pyrimidine derivatives was obtained via reaction of 6-amino-2-thioxo-1H-pyrimidine-4-one (1) with a different reagents. The antimicrobial and anti-inflammatory activities of some of the synthesized compounds were tested.

Reaction of 1 with aromatic aldehydes in methanol containing few drops of hydrochloric acid (pH = 5-6) led to the formation of 9-substituted-1,3,6,8,9,10-hexahydro-2,7-dithioxopyrido[2,3-d:6,5 d`] dipyrimidine-4,6-diones 3a-e. In this reaction, the polar and acidic condition of solvent make the reaction occur through the formation of a 6-imino group that leads to higher nucleophilicity of C-5 whereby attack occurs at the aldehyde carbonyl group (Scheme 1).   On the other hand, when we used a non-polar and slightly acidic solvent (DMF and few drops of acetic acid , pH = 6.5-7), the imino group wasn't formed so the reaction proceeds via nucleophilic attack of the amino group on the aldehyde carbonyl group leading to the 6-{[1-aryl-methylidene]-amino}-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one condensation products 4a,b and not 4a′,b′, as reported earlier [12]. The formation of the isomeric imino compounds 4a′,b′ is not plausible because the amino group is much more nucleophilic than the CH at position 5 of 6-amino-2-thioxo-1Hpyrimidine-4-one (1); in addition the 1 H-NMR showed one proton signal at δ = 5.26 ppm for the pyrimidine H-5 and one proton signal at δ = 7.84 ppm for the azamethine proton (N=CH), confirming fortmation of the structures 4a,b.

Antimicrobial activity
The in vitro antimicrobial activity of the newly synthesized compounds against Escherichia coli (ATCC 25922) as an example of Gram negative bacteria, Bacillus subtilis (ATCC-6633) as a Gram positive bacteria and Candida albicans (ATCC-10231) as a fungus was investigated using the diskdiffusion method [14] at the Department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University, Helwan, Egypt. Ampicillin was used as an antibacterial standard and nystatin as a standard antifungal agent. The results are given in Table 1. Table 1. Antimicrobial activity of the synthesized compounds expressed as size of the inhibition zone (mm/mg sample).

E. coli (ATCC-25922)
-18 11 Ampicillin 12 --Nystatin -: No activity, DMSO: dimethylsulfoxide (used as solvent) [15] The anti-inflammatory activity of compounds 2c, 3c, 4b, 5b was investigated in comparison with ibuprofen as standard anti-inflammatory agent (Table 2, Figure 1).  The carrageenan-induced rat paw oedema assay was carried out using a modified Winter's method as a preliminary screening test [16]. The rats (in groups of five animals weighing 120-170 g, young adult male Sprague-Dawley) were housed in a controlled environment and provided with standard rodent chow and water for 24 h before a dose of the test compounds (50 and 70 mg/kg sc) was administered. One hour later, the volume of the right hind paw was measured, and 0.05 mL of a 1% suspension of carrageenan in sterile pyrogen-free 0.9% NaCl solution was injected subcutaneously into planter aponeurosis of the hind paw. One hour after the injection of carrageenan, the paw volume was again measured by a water pletysmometer. The mean increase of paw volume at each time interval was compared with that of control group (five rats treated with carrageenan, but without test compounds) at the same time intervals. The percentage inhibition values were calculated according to the formula: % Anti-inflammatory activity = (1-Rt/Rc)*100 (Rt = result of tested group; Rc = result of control group).

Anti-inflammatory activity
All experiments involving animals were carried out using protocols approved by the Animal Committee, University of Barcelona (Spain). Animal care was in compliance with Generalitat of Catalunya regulations on protection of animals used for experimental and other scientific purposes.

General
All melting points are uncorrected. Elemental analyses were carried out in the Microanalytical Center, Cairo University, Giza, Egypt. IR spectra (KBr) were recorded on Pye Unicam SP 1200 Spectrophotometer. 1 H-NMR spectra were recorded in DMSO-d 6 on a 90 MHz Varian NMR spectrometer using TMS as an internal standard and chemical shifts are expressed as δ ppm units. The mass spectra were determined with HP model MS-5988 at electron energy 70 eV. The homogeneity of all compounds synthesized was checked by TLC on 2.0 cm × 6.0 cm aluminum sheets coated to a thickness of 0.25 µm with silica gel 60 containing a fluorescent indicator. Characterization data of the various compounds prepared are given in Tables 3 and 4.   (7) -2-thioxo-2,3,5,6,7,8-hexahydro-1H-pyrimido[4,5-d]
Concerning the anti-inflammatory activity, analysis of the results obtained using one-way analysis of variance (ANOVA) shows that there is a significant difference of all compounds from control at 3 h and 4 h At 3 h, compounds 3c, 4b and 5b show higher activity than ibuprofen, while compound 2c shows less activity. At 4 h, compounds 3c, 4b show higher activity than ibuprofen while compound 5b joined 2c in showing less activity. This lesser activity of compounds 2c, 5b may be due to cyclisation of the 5,6-positions of 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one (1) and the higher activity of compounds 3c, 4b may be due to the presence of an indolyl group as substituent.