Synthesis of Novel 2-(Substituted amino)alkylthiopyrimidin-4(3H)-ones as Potential Antimicrobial Agents

5-Alkyl-6-(substituted benzyl)-2-thiouracils 3a,c were reacted with (2-chloroethyl) diethylamine hydrochloride to afford the corresponding 2-(2-diethylamino)ethylthiopyrimidin-4(3H)-ones 4a,b. Reaction of 3a–c with N-(2-chloroethyl)pyrrolidine hydrochloride and/or N-(2-chloroethyl)piperidine hydrochloride gave the corresponding 2-[2-(pyrrolidin-1-yl)ethyl]-thiopyrimidin-4(3H)-ones 5a–c and 2-[2-(piperidin-1-yl)ethyl]thiopyrimidin-4(3H)-ones 6a,b, respectively. Treatment of 3a–d with N-(2-chloroethyl)morpholine hydrochloride under the same reaction conditions formed the corresponding 2-[2-(morpholin-4-yl)ethyl]thiopyrimidines 6c–f. On the other hand, 3a,b were reacted with N-(2-bromoethyl)phthalimide and/or N-(3-bromopropyl)phthalimide to furnish the corresponding 2-[2-(N-phthalimido)ethyl]-pyrimidines 7a,b and 2-[3-(N-phthalimido)-propyl]pyrimidines 7c,d, respectively. Compounds 3a–d, 4a,b, 5a–c, 6a–f and 7a–d were screened against Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Bacillus subtilis NRRL 4219 and Bacillus cereus), yeast-like pathogenic fungus (Candida albicans ATCC 10231) and a fungus (Aspergillusniger NRRL 599). The best antibacterial activity was displayed by compounds 3a, 3b, 4a, 5a, 5b, 6d, 6f, 7b and 7d, whereas compounds 4b, 5b, 5c, 6a, 6b and 6f exhibited the best antifungal activity.

In a previous study [34], we synthesized a series of 2-(substituted amino)ethylthiopyrimidines analogues of S-DABOs to be screened as reverse transcriptase inhibitors against human immunodeficiency virus (HIV-1). We found it of interest to evaluate the antimicrobial activity for such pyrimidine derivatives. In the present work, and as a part of our continuing interest in the chemistry of pyrimidines [30,[34][35][36][37][38][39][40][41][42], the synthesis and antimicrobial evaluation of some novel 2-(substituted amino)alkylthiopyrimidin-4(3H)-one derivatives have been investigated.
Although several compounds showed strong antibacterial and antifungal activities, none of them were found to be superior to the reference drugs. Compounds 3a, 3b, 4a, 5a, 5b, 6b, 6d, 6f, 7b and 7d displayed a relatively broad spectrum activity, accordingly, their MIC values were determined. The MIC values for compounds 3a, 3b, 4a, 5a, 5b, 6b, 6d, 6f, 7b and 7d against the most sensitive tested microorganisms, Staphylococcus aureus and Candida albicans are represented in Table 2. According to the above results, the antimicrobial activity seemed to be dependent on the nature of substituents. Compounds containing a 4-chlorobenzyl substituent at C-6 of the pyrimidine ring showed the best antibacterial activity, whereas, the best antifungal results were given by compounds containing 2-(pyrrolidin-1-yl)ethylthio and 2-(piperidin-1-yl)ethylthio substituents at C-2 of the pyrimidine ring. Concerning compounds 7a-d, the ethyl group at C-5 of the ring was found to improve the antimicrobial activity.

General
Melting points (°C) were measured in open glass capillaries using a Branstead 9100 Electrothermal melting point apparatus and are uncorrected. NMR spectra were obtained on a Bruker AC 500 Ultra Shield NMR spectrometer (Fällanden, Switzerland) operating at 500.13 MHz for 1 H and 125.76 MHz for 13 Compounds 3a-d, 4a, 5a,b and 6a were reported in our previous studies [34,42].

General Procedure for Preparation of 2-(Substituted amino)ethylthiopyrimidines 4b, 5c and 6b-f
To a solution of the appropriate compound 3a-d (1 mmol) in anhydrous DMF (5 mL), was added anhydrous potassium carbonate (0.304 g, 2.2 mmol) followed by the appropriate 2-chloroethyl substituted amine hydrochloride (1.1 mmol). The mixture was stirred at room temperature for 24 h, then was diluted with H 2 O (100 mL) and extracted with diethyl ether (3 × 50 mL). The combined organic extract was washed with H 2 O (3 × 50 mL), dried (MgSO 4 ) and evaporated under reduced pressure. The residue was chromatographed on silica gel column with CHCl 3 to afford the target compounds.

General Procedure for Preparation of 2-[2-(N-Phthalimido)ethyl]thiopyrimidin-4(3H)-ones 7a,b and 2-[3-(N-Phthalimido)propyl]thiopyrimidin-4(3H)-ones 7c,d
Anhydrous potassium carbonate (0.152 g, 1.1 mmol) was added to a solution of the appropriate compound 3a,b (1 mmol) in DMF (5 mL), followed by addition of N-(2-bromoethyl)phthalimide and/or N- (3-bromopropyl) phthalimide (1.1 mmol). The reaction mixture was stirred at room temperature for 24 h and worked up as described above for the preparation of compounds 4-6.    [43] Sterile nutrient, Czapek's dox and malt extract agar media were inoculated, separately, with 100 µL cell suspension of the chosen microorganism, bacteria, fungi and yeast-like fungi, respectively, and poured into Petri-dishes (20 cm diameter). The test compounds (200 µg/10 mm diameter disc) were placed onto the surface of the agar Petri-dishes. The antimicrobial activities were expressed as the diameter of the growth inhibition zone in mm. [44] The minimal inhibitory concentrations (MICs) of the test compounds were determined using serial dilutions technique. Different concentrations ranging 50.0-200.0 µg/mL for each compound in dimethyl sulphoxide (DMSO) were placed on filter paper disc (1 cm diameter). The discs were deposited on the surface of inoculated agar plates and kept at low temperature before incubation which favours diffusion over microbial growth to detect the inhibition zone clearly. The plates were incubated at 30 °C for 24 h for bacteria and yeast and for 48 h for fungi.