Pyrimidine-5-carbonitriles – part III: synthesis and antimicrobial activity of novel 6-(2-substituted propyl)-2,4-disubstituted pyrimidine-5-carbonitriles

Chemical synthesis

The reaction of the aliphatic aldehydes 3-methylbutanal (1a) or (±)-3-phenylbutanal (1b) with ethyl cyanoacetate (2) and thiourea (3) in ethanol, in the presence of potassium carbonate, yielded the target 6-substituted-2-thiouracil-5-carbonitriles 4a,b in 28% and 42% yields, respectively. Compounds 4a and 4b were allowed to react with benzyl bromide, 4-fluorobenzyl chloride, 4-chlorobenzyl chloride, and 4-nitrobenzyl bromide in the presence of potassium carbonate, in N,N-dimethylformamide (DMF) at room temperature for 12 h to yield the target derivatives 5a–h in 72–90% yields. Similarly, compounds 4a and 4b were reacted with 2-bromoethyl methyl ether, benzyl chloromethyl ether, 2-bromomethyl-5-nitrofuran, and anhydrous potassium carbonate in DMF or acetone to yield the corresponding 2-methoxyethylthio, benzyloxymethylthio, or 5-nitrofuran-2-ylmethylthiol analogues 6a–h, 7, and 8a,b, respectively (Scheme 1).

Scheme 1

Synthesis of compounds 4a, 4b, 5a–h, 6a, 6b, 7, 8a and 8b.

The pronounced reactivity of 4-halopyrimidines toward nucleophilic reagents was utilized for the synthesis of 4-arylthiopyrimidine-5-carbonitriles. In our initial studies, an attempted reaction of the 4-chloropyrimidine 9 with thiophenol or 2-, 3-, or 4-thiocresols in ethanol, in the presence of potassium carbonate through prolonged heating for up to 24 h, did not yield the expected 4-arylthio derivatives 10a–d, and the starting materials were recovered unchanged. In contrast, carrying out the reaction in pyridine with heating for 3 h yielded the target compounds 10a–d in 75–85% yields. Aromatic amines and 1-phenylpiperazine or 1-benzylpiperazine reacted smoothly with compound 9 in boiling ethanol in the presence of potassium carbonate to yield the corresponding 4-arylamino and piperazino derivatives 11a–d and 12a–d (Scheme 2).

Scheme 2

Synthesis of compounds 9, 10a–d, 11a–d, 12a, and 12b.

The structures of all the newly synthesized compounds were confirmed by elemental analyses, ¹H NMR, ¹³C NMR, and electrospray ionization mass spectral (ESI-MS) data, in addition to the X-ray spectra of compounds 4a [35], 6a [36], 10a [37], and 11c [38].

In vivo antimicrobial activity

Compounds were tested for in vitro growth inhibitory activity against the standard strains of the Institute of Fermentation of Osaka (IFO), namely Staphylococcus aureus IFO 3060, Bacillus subtilis IFO 3007 (Gram-positive bacteria), Escherichia coli IFO 3301, Pseudomonas aeruginosa IFO 3448 (Gram-negative bacteria), and the yeast-like pathogenic fungus Candida albicans IFO 0583. The primary screening was carried out using the agar disc-diffusion method using Müller-Hinton agar medium [39]. The results are shown in Table 1 and reveal varying degrees of inhibition against the tested microorganisms. Potent antibacterial activity was noted for the compounds 5e, 5f, 5g, 5h, 7, 8a, 8b, and 12a, which produced growth inhibition zones >18 mm against one or more of the tested microorganisms. In addition, compounds 5b, 5c, and 6a showed moderate activity (growth inhibition zones 14–18 mm). Compounds 5a, 11a, 11d, and 12b exhibited weak activity (growth inhibition zones 10–13 mm), and the remaining compounds were practically inactive against the tested microorganisms. In general, it would be concluded that the Gram-positive bacteria S. aureus and B. subtilis are considered the most sensitive among the tested microorganisms. In addition, all the tested compounds were devoid of activity against the yeast-like pathogenic fungus C. albicans. The potent and broad-spectrum activity of the compounds 8a and 8b may be attributed to the synergistic effect of the pyrimidine nucleus and the 5-nitro-2-furyl moieties [40, 41]. The introduction of an arylthiol or arylamino substituents (compounds 10a–d and 11a–d) at the C-4 pyrimidine carbon dramatically diminished the antibacterial activity compared with their 4-oxopyrimidine analogues.

Conclusion

New pyrimidine derivatives that are structurally related to known pyrimidine antimicrobial drugs were synthesized and tested for inhibitory activity against a panel of Gram-positive and Gram-negative bacteria and the yeast-like pathogenic fungus C. albicans. The results of the antimicrobial testing revealed that eight compounds show potent antibacterial activity against one or more of the tested bacterial strains. None of the tested compounds is active against C. albicans.

General

Melting points were measured in open glass capillaries using a Barnstead 9100 Electrothermal melting point apparatus (Denmark) and are uncorrected. NMR spectra were obtained on a Bruker AC 500 Ultra Shield NMR spectrometer (Fällanden, Switzerland) operating at 500 MHz for ¹H and 125 MHz for ¹³C. ESI-MS data were recorded on an Agilent 6410 (Santa Clara, CA, USA) Triple Quad tandem mass spectrometer at 4.0 and 3.5 kV for the positive and negative ions, respectively. Monitoring the reactions and checking the purity of the final products were carried out by thin layer chromatography using silica gel precoated aluminum sheets (60 F₂₅₄, Merck) and visualization with ultraviolet light. The bacterial strains and C. albicans fungus were obtained from the IFO (Osaka, Japan). The reference drugs ampicillin trihydrate (CAS 7177-48-2), gentamicin sulfate (CAS 1405-41-0), and clotrimazole (CAS 23593-75-1) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).

6-(2-Substituted propyl)-2-thiouracil- 5-carbonitriles (4a,b)

A mixture of the appropriate aldehyde 1a,b (0.01 mol), ethyl cyanoacetate (2, 1.13 g, 0.01 mol), thiourea (3, 0.76 g, 0.01 mol), and potassium carbonate (1.38 g, 0.01 mol), in ethanol (30 mL), was heated under reflux for 6 h. On cooling, the separated precipitate was filtered, washed with diethyl ether, and dried. The obtained solid was added to water (20 mL) and the mixture was heated at 80–90°C until a clear solution was obtained. After cooling, the solution was acidified with acetic acid and stirred for 30 min. The deposited precipitate was filtered, washed with cold water, dried, and crystallized from acetic acid.

2-Arylmethylthio-6-(2-substituted propyl)-3,4-dihydro-4-oxopyrimidine-5-carbonitriles (5a–h), 6-(2-substituted propyl)-2-(2-methoxyethylthio)-3,4-dihydro-4-oxopyrimidine-5-carbonitriles (6a,b), and 2-benzyloxymethylthio-6-(2-methylpropyl)-3,4-dihydro-4-oxopyrimidine-5-carbonitrile (7)

To a solution of the appropriate 6-substituted-2-thiouracil-5-carbonitrile 4a,b (0.01 mol) in DMF (10 mL), benzyl bromide, 4-fluorobenzyl chloride, 4-chlorobenzyl chloride, 4-nitrobenzyl bromide, 2-bromoethyl methyl ether or benzyl chloromethyl ether (0.01 mol), and anhydrous potassium carbonate (1.38 g, 0.01 mol) were added and the mixture was stirred at room temperature for 12 h. Water (15 mL) was added, and the mixture was stirred for an additional 30 min. The separated crude product was filtered, washed with cold water, dried, and crystallized from ethanol or aqueous ethanol.

2-(5-Nitrofuran-2-ylmethylthio)-6-(2-substituted propyl)-3,4-dihydro-4-oxopyrimidine-5-carbonitriles (8a,b)

To a solution of the appropriate 6-substituted-2-thiouracil-5-carbonitrile 4a,b (0.01 mol), in acetone (15 mL), 2-bromomethyl-5-nitrofuran (2.06 g, 0.01 mol) and anhydrous potassium carbonate (1.38 g, 0.01 mol) were added and the mixture was stirred at room temperature for 12 h. The solvent was then distilled in vacuo at room temperature. Water (15 mL) was added to the residue and the mixture was stirred for an additional 30 min. The obtained solid was filtered, washed with cold water, dried, and crystallized.

2-(4-Chlorobenzylthio)-4-chloro-6- (2-methylpropyl)pyrimidine-5-carbonitrile (9)

2-(4-Chlorobenzylthio)-6-(2-methylpropyl)-3,4-dihydro-4-oxopyrimidine-5-carbonitrile (5c, 16.69 g, 0.05 mol) was added portionwise to a mixture of phosphorus oxychloride (19.2 mL) and N,N-dimethylaniline (10.3 mL) over a period of 10 min with stirring. The mixture was then heated under reflux for 1 h. On cooling, the mixture was poured onto crushed ice (200 g), stirred for 30 min, and extracted with diethyl ether (2 × 200 mL). The ethereal extract was dried over anhydrous sodium sulfate and concentrated under a reduced at room temperature to yield the crude products as an oil. The crude product was purified by flash silica gel column chromatography using chloroform as an eluent; yield 67%; mp 49–51°C; ¹H NMR (DMSO-d₆): δ 0.91 (d, 6H, CH₃, J = 7.0 Hz), 2.13 (m, 1H, CH), 2.73 (d, 2H, CH₂, J = 7.0 Hz), 4.45 (s, 2H, CH₂S), 7.34 (d, 2H, Ar-H, J = 8.0), 7.45 (d, 2H, Ar-H, J = 8.0); ¹³C NMR: 22.0 (CH₃), 27.9 (CH), 34.1 (CH₂CH), 44.9 (CH₂S), 104.0 (C-5), 113.7 (CN), 128.3, 130.8, 132.0, 135.8 (Ar-C), 161.2 (C-4), 173.9 (C-6), 174.5 (C-2); ESI-MS, m/z (rel. int.): 356.2 (M+4+H, 11)⁺, 354.2 (M+2+H, 71)⁺, 352.2 (M+H, 100)⁺. Anal. Calcd for C₁₆H₁₅Cl₂N₃S: C, 54.55; H, 4.29; N, 11.93; S, 9.10. Found: C, 54.21; H, 4.26; N, 11.85; S, 8.92.

4-Arylthio-2-(4-chlorobenzylthio)-6-(2-methylpropyl)pyrimidine-5-carbonitriles (10a–d)

To a solution of compound 9 (3.52 g, 0.01 mol) in dry pyridine (5 mL), the appropriate arylthiol (0.01 mol) was added and the mixture was heated under reflux for 3 h. On cooling, the solvent was distilled in vacuo, and water (10 mL) was added to the residue. The precipitate was filtered, washed with water, dried, and crystallized from ethanol or aqueous ethanol.

4-Arylamino-2-(4-chlorobenzylthio)-6- (2-methylpropyl)pyrimidine-5-carbonitriles (11a–d)

To a solution of compound 9 (705 mg, 0.002 mol) in ethanol (8 mL), the appropriate aromatic amine (0.002 mol) and anhydrous potassium carbonate (276 mg, 0.002 mol) were added and the mixture was heated under reflux for 6 h. On cooling, the solvent was distilled in vacuo, and water (10 mL) was added to the residue. The separated precipitate was filtered, washed with cold water, dried, and crystallized from ethanol.

2-(4-Chlorobenzythio-6)-(2-methylpropyl)-4-(4-substituted piperazino)pyrimidine-5-carbonitriles (12a,b)

To a solution of compound 9 (705 mg, 0.002 mol) in ethanol (8 mL), the appropriate 1-substituted piperazine (0.002 mol) and anhydrous potassium carbonate (276 mg, 0.002 mol) were added and the mixture was heated under reflux for 3 h. On cooling, the solvent was distilled in vacuo, and water (10 mL) was added to the residue. The separated precipitate was filtered, washed with cold water, dried, and crystallized from ethanol.

Determination of antimicrobial activity (agar disc-diffusion method)

The sterile filter paper disc (8 mm diameter) was moistened with the compound solution in dimethyl sulfoxide of specific concentration (200 μg/disc), the antibacterial antibiotic gentamicin or ampicillin trihydrate (100 μg/disc), or the antifungal drug clotrimazole (100 μg/disc) and was carefully placed on the agar culture plate that had been previously inoculated separately with the microorganism. The plates were incubated at 37°C, and the diameter of the growth inhibition zones were measured after 24 h in case of bacteria and 48 h in case of C. albicans.

Determination of the minimal inhibitory concentration (MIC)

Compounds 5e,f,g,h, 7, 8a,b, and 12a, gentamicin, and ampicillin trihydrate were dissolved in dimethyl sulfoxide at concentration of 128 μg/mL. Twofold dilutions of the solution were prepared (128, 64, 32, …, 0.5 μg/mL). The microorganism suspensions at 10⁶ colony-forming units/mL concentrations were inoculated to the corresponding wells. The plates were incubated at 36°C for 24 h. The MIC values were determined as the lowest concentration that completely inhibited visible growth of the microorganism as detected by the naked eye.