All chemical reagents and solvents used were of reagent grade or purified using standard methods. TLC analyses were conducted on silica gel plates (Sorbent Silica G UV254). Column chromatography was carried out on flash silica gel (Sorbent 230–400 mesh). NMR spectra were recorded at 1H (400 MHz) and 13C (100 MHz) on a Bruker instrument. Coupling constants (J) and chemical shifts (δ) are given in hertz and ppm respectively, using TMS (1H NMR) and solvents (13C NMR) as internal standards.
General procedure for the synthesis of (2a-f)
Our previously published[15] procedure was followed. Briefly, to a solution of ethanol (25 mL) and appropriate aldehyde (RCHO, 5 mmol) was added ethyl cyanoacetate (0.5 mL, 5 mmol), thiourea (0.38 g, 5 mmol) and piperidine (1.0 mL, 10 mmol). The mixture was heated at reflux overnight and then cooled to room temperature. The precipitate was dissolved in 0.5 M NaOH (20 mL) and washed with ethyl acetate (15 ml ⋅3). Then the aqueous solution was acidified to pH ~ 2 by slow addition of 1 M HCl. This caused the product to precipitate, which was then filtered using vacuum filtration.
General procedure for the synthesis of (3a-o)
To a solution of 2-mercapto-6-oxo-4-(4-(phenylthio)phenyl)-1,6-dihydropyrimidine-5-carbonitrile derivatives (1.36 mmol) in CH3CN (10 ml), K2CO3 (6.79 mmol) was added and the resulting mixture was stirred for 10–15 min. To this was added the appropriate (bromomethyl)phenyl derivatives (1.22 mmol) and the reaction was stirred at room temperature for 16-18h. Upon completion, the reaction mixture was cooled to ambient temperature and the solvent removed in vacuo. The dried residue was washed by H2O (pH = 9–10, 20 mL ⋅ 2) and brine (15 ml ⋅ 2) followed by product extraction in ethyl acetate (20 ml). The solvent was evaporated in vacuo to obtain crude product, which was purified using silica gel column chromatography.
2-Mercapto-6-oxo-4-(4-(phenylthio)phenyl)-1,6-dihydropyrimidine-5-carbonitrile (2a). Yield: 58%.
1H NMR (DMSO-d6): δ 13.26 (bs, 1H), 12.91 (s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.50 (m, 5H), 7.31 (d, J = 8.4 Hz, 2H); 13C NMR (DMSO-d6): δ 177.6, 161.5, 159.5, 142.3, 133.8, 132.0, 131.9, 131.2, 130.8, 130.5, 130.1, 130.1, 129.5, 128.5, 127.9, 115.8, 90.1 ppm. HRMS (ESI-TOF) (m/z): Calcd. for C17H10N3OS2, [M-H+]: 336.0260; found: 336.0263.
4-(4-(Benzylthio)phenyl)-2-mercapto-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (2b). Yield: 45%.
1H NMR (DMSO-d6): δ 13.81 (s, 1H), 13.17 (bs, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 7.2 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.25 (m, 1H), 4.37 (s, 2H); 13C NMR (DMSO-d6): δ 178.1, 162.1, 159.9, 142.4, 137.3, 129.7, 129.3, 129.0, 128.1, 127.7, 126.6, 116.2, 89.5, 35.8. HRMS (ESI-TOF) (m/z): Calcd. for C18H12N3OS2, [M-H+]: 350.0416; found: 350.0420.
2-Mercapto-6-oxo-4-(4-(phenoxymethyl)phenyl)-1,6-dihydropyrimidine-5-carbonitrile (2c). Yield: 36%.
1H NMR (DMSO-d6): δ 13.31 (bs, 1H), 13.14 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 6.96 (t, J = 7.2 Hz, 1H), 4.37 (s, 2H); 13C NMR (DMSO-d6): δ 176.8, 161.3, 159.2, 158.6, 142.1, 130.0, 129.5, 127.8, 121.5, 115.4, 91.1, 68.9. HRMS (ESI-TOF) (m/z): Calcd. for C18H12N3O2S, [M-H+]: 334.0645; found: 334.0645.
(E)-2-Mercapto-6-oxo-4-(4-styrylphenyl)-1,6-dihydropyrimidine-5-carbonitrile (2d). Yield: 56%.
1H NMR (DMSO-d6): δ 13.32 (bs, 1H), 12.93 (s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 7.2 Hz, 2H), 7.37–7.48 (m, 4H), 7.33 (d, J = 6.0 Hz, 1H); 13C NMR (DMSO-d6): δ 177.5, 161.7, 159.5, 141.1, 137.0, 131.5, 129.7, 129.4, 129.2, 128.9, 128.7, 127.7, 127.3, 126.6, 115.8, 90.0. HRMS (ESI-TOF) (m/z): Calcd. for C19H12N3OS, [M-H+]: 330.0696; found: 330.0698.
2-Mercapto-6-oxo-4-(4-(phenylethynyl)phenyl)-1,6-dihydropyrimidine-5-carbonitrile (2e).[15] Yield: 58%.
1H NMR (DMSO-d6): δ 10.03 (s, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.57 (t, J = 8.0 Hz, 2H), 7.39 (t, J = 8.0 Hz, 2H); 13C NMR (DMSO-d6): δ 171.4, 135.4, 132.1, 131.8, 129.6, 129.0, 128.5, 122.5, 93.4, 88.5. HRMS (ESI-TOF) (m/z): Calcd. for C19H10N3OS, [MH-]: 328.0545; found: 328.0541.
N-Benzyl-4-(5-cyano-2-mercapto-6-oxo-1,6-dihydropyrimidin-4-yl)benzamide (2f). Yield: 58%.
1H NMR (DMSO-d6): δ 13.41 (s, 1H), 13.20 (s, 1H), 9.26 (t, J = 6.0 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 4.4 Hz, 4H), 7.28 (m, 1H), 4.51 (d, J = 6.0 Hz, 2H); 13C NMR (DMSO-d6): 176.6, 165.8, 160.7, 158.8, 139.8, 137.8, 132.1, 129.5, 129.4, 128.7, 127.8, 127.6, 127.2, 115.0, 91.6, 43.2. HRMS (ESI-TOF) (m/z): Calcd. for C19H13N4O2S, [M-H+]: 361.0754; found: 361.0756.
2-((4-Azidobenzyl)thio)-6-oxo-4-(4-(phenylthio)phenyl)-1,6-dihydropyrimidine-5-carbonitrile (3a). Yield: 90%.
1H NMR (DMSO-d6): δ 7.83 (d, J = 8.0 Hz, 2H), 7.49 (m, 3H), 7.43 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 4.51 (s, 2H); 13C NMR (DMSO-d6): δ 169.2, 166.3, 166.6, 166.4, 140.4, 138.6, 135.2, 134.8, 133.2, 132.8, 131.0, 130.3, 129.8, 129.0, 128.6, 119.5, 118.4, 91.1, 33.9. HRMS (ESI-TOF) (m/z): Calcd. for C24H15N6OS2, [M-H+]: 467.0749; found: 467.0751.
Methyl4-(((5-cyano-6-oxo-4-(4-(phenylthio)phenyl)-1,6-dihydropyrimidin-2-yl)thio)methyl)benzoate (3b). Yield: 97%.
1H NMR (DMSO-d6): δ 7.87 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.41–7.49 (m, 5H), 7.31 (d, J = 8.4 Hz, 2H), 4.42 (s, 2H), 3.83 (s, 3H); 13C NMR (DMSO-d6): δ 170.0, 166.6, 166.4, 144.6, 139.7, 135.2, 133.0, 130.3, 129.7, 129.6, 129.6, 128.9, 128.8, 128.6, 119.0, 90.5, 52.5, 34.0. HRMS (ESI-TOF) (m/z): Calcd. for C26H18N3O3S2, [M-H+]: 484.0795; found: 484.0774.
6-Oxo-4-(4-(phenylthio)phenyl)-2-((4-(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidine-5-carbonitrile (3c). Yield: 90%.
1H NMR (DMSO-d6): δ 7.79 (d, J = 8.4 Hz, 2H), 7.64 (qd, J = 8.4 Hz, 4H), 7.43–7.51 (m, 5H), 7.31(d, J = 8.0 Hz, 2H), 4.49 (s, 2H); 13C NMR (DMSO-d6): δ 168.6, 166.7, 166.0, 143.4, 140.7, 134.5, 133.3, 130.3, 130.0, 129.8, 129.1, 128.5, 128.2, 127.9, 126.0, 125.6, 123.3, 118.0, 91.4, 55.3, 33.8. HRMS (ESI-TOF) (m/z): Calcd. for C25H15F3N3OS2, [M-H+]: 494.0614; found: 494.0598.
2-((4-Azidobenzyl)thio)-4-(4-(benzylthio)phenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (3d). Yield: 40%.
1H NMR (DMSO-d6): δ 7.90 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 7.6 Hz, 4H), 7.33 (t, J = 8.0 Hz, 2H), 7.26 (m, 1H), 7.06 (d, J = 8.4 Hz, 2H), 4.52 (s, 2H), 4.39 (s, 2H); 13C NMR (DMSO-d6): δ 166.6, 165.7, 161.5, 144.8, 142.8, 139.0, 137.2, 133.9, 132.1, 131.1, 129.3, 128.9, 127.7, 126.8, 119.6, 116.4, 92.8, 35.7, 34.1. HRMS (ESI-TOF) (m/z): Calcd. for C25H17N6OS2, [M-H+]: 481.0905; found: 481.0911.
Methyl 4-(((4-(4-(benzylthio)phenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio)methyl) benzoperoxoate (3e). Yield: 68%.
1H NMR (DMSO-d6): δ 7.88 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 6.4 Hz, 4H), 7.31 (t, J = 7.2 Hz, 2H), 7.24 (m, 1H), 4.34 (s, 2H), 4.32 (s, 2H); 13C NMR (DMSO-d6): δ 171.6, 170.8, 166.6, 166.5, 145.6, 139.3, 137.6, 135.0, 129.6, 129.3, 129.2, 129.0, 128.9, 128.4, 127.6, 127.2, 127.0, 120.5, 89.1, 52.5, 36.3, 33.8. HRMS (ESI-TOF) (m/z): Calcd. for C27H20N3O3S2, [M-H+]: 498.0946; found: 498.0953.
4-(4-(Benzylthio)phenyl)-6-oxo-2-((4-(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidine-5-carbonitrile (3f). Yield: 59%.
1H NMR (DMSO-d6): δ 7.83 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 4H), 7.44 (d, J = 7.2 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.25 (m, 1H), 4.59 (s, 2H), 4.38 (s, 2H); 13C NMR (DMSO-d6): δ 166.7, 165.8, 142.5, 137.2, 132.2, 130.1, 129.6, 129.3, 128.9, 127.7, 126.8, 125.8, 125.7, 117.02, 92.9, 35.8, 33.9. HRMS (ESI-TOF) (m/z): Calcd. for C26H17F3N3OS2, [M-H+]: 508.076; found: 508.0773.
2-((4-Azidobenzyl)thio)-6-oxo-4-(4-(phenoxymethyl)phenyl)-1,6-dihydropyrimidine-5-carbonitrile (3g). Yield: 66%.
1H NMR (DMSO-d6): δ 7.96 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 7.6 Hz, 2H), 7.45 (d, J = 8.0 Hz, 4H), 7.31 (t, J = 7.6 Hz, 2H), 7.05 (t, J = 8.8 Hz, 4H), 6.96 (t, J = 6.8 Hz, 1H), 5.22 (s, 2H), 4.53 (s, 2H); 13C NMR (DMSO-d6): δ 167.4, 166.01, 161.6, 158.5, 141.6, 139.1, 135.0, 133.8, 131.1, 130.0, 129.4, 127.9, 127.8, 121.3, 119.6, 116.3, 115.2, 93.6, 68.8, 34.1. HRMS (ESI-TOF) (m/z): Calcd. for C25H17N6O2S, [M-H+]: 465.1134; found: 465.1149.
Methyl-4-(((5-cyano-6-oxo-4-(4-(phenoxymethyl)phenyl)-1,6-dihydropyrimidin-2-yl)thio)methyl)benzoate (3h). Yield: 68%.
1H NMR (DMSO-d6): δ 7.90 (d, J = 8.0 Hz, 4H), 7.62 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.04 (d, J = 7.6 Hz, 2H), 6.96 (t, J = 7.2 Hz, 1H), 5.02 (s, 2H), 4.57 (s, 2H), 3.84 (s, 3H); 13C NMR (DMSO-d6): δ 171.4, 167.5, 166.4, 158.6, 143.1, 141.5, 135.2, 130.0, 129.7, 129.3, 127.9, 121.3, 115.2, 108.6, 93.4, 68.9, 52.6, 34.2. HRMS (ESI-TOF) (m/z): Calcd. for C27H20N3O4S, [M-H+]: 482.1175; found: 482.1175.
6-Oxo-4-(4-(phenoxymethyl)phenyl)-2-((4-(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidine-5-carbonitrile (3i). Yield: 65%.
1H NMR (DMSO-d6): δ 7.90 (d, J = 8.0 Hz, 2H), 7.62–7.69 (m, 6H), 7.31 (t, J = 7.6 Hz, 2H), 7.04 (d, J = 8.0 Hz, 1H), 6.95 (t, J = 7.2 Hz, 1H), 5.22 (s, 2H), 4.61 (s, 2H); 13C NMR (DMSO-d6): δ 168.6, 167.4, 165.9, 158.5, 142.4, 141.6, 135.0, 130.2, 130.0, 129.3, 127.9, 125.8, 125.7, 121.3, 116.4, 115.2, 93.7, 68.8, 33.9. HRMS (ESI-TOF) (m/z): Calcd. for C26H17F3N3O2S, [M-H+]: 492.0994; found: 492.0989.
(E)-2-((4-Azidobenzyl)thio)-6-oxo-4-(4-styrylphenyl)-1,6-dihydropyrimidine-5-carbonitrile (3j). Yield: 18%.
1H NMR (DMSO-d6): δ 8.00 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 7.6 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.40 (m, 3H), 7.33 (d, J = 6.4 Hz, 2H), 7.08 (d, J = 5.2 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.55 (s, 2H); 13C NMR (DMSO-d6): 167.6, 166.9, 165.9, 141.1, 139.1, 137.1, 134.0, 131.5, 131.1, 129.7, 129.2, 128.9, 128.6, 128.3, 127.8, 127.3, 126.9, 119.7, 93.0, 34.1. HRMS (ESI-TOF) (m/z): Calcd. for C26H17N6OS, [M-H+]: 461.1185; found: 461.1179.
(E)-Methyl 4-(((5-cyano-6-oxo-4-(4-styrylphenyl)-1,6-dihydropyrimidin-2-yl)thio)methyl) benzoate (3k). Yield: 34%.
1H NMR (DMSO-d6): δ 7.94 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 7.6 Hz, 2H), 7.79 (d, J = 7.6 Hz, 2H), 7.66 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 7.6 Hz, 2H), 7.42 (d, J = 6.4 Hz, 2H), 7.39 (d, J = 5.2 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.61 (s, 2H), 3.84 (s, 3H); 13C NMR (DMSO-d6): δ 167.0, 166.5, 165.9, 162.1, 143.2, 141.1, 137.1, 134.4, 131.4, 129.7, 129.2, 129.0, 128.7, 127.7, 127.2, 126.9, 116.7, 93.1, 52.6, 34.1. HRMS (ESI-TOF) (m/z): Calcd. for C28H20N3O3S, [M-H+]: 478.1225; found: 478.1240.
6-Oxo-4-(4-(phenylethynyl)phenyl)-2-((4-(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidine-5-carbonitrile (3l). Yield: 51%.
1H NMR (DMSO-d6): δ 7.93 (d, J = 8.0 Hz, 2H), 7.64–7.75 (m, 6H), 7.65 (m, 2H), 7.46(m, 3H), 4.60 (s, 2H); 13C NMR (DMSO-d6): δ 166.6, 162.5, 142.6, 135.6, 132.0, 131.9, 130.2, 129.6, 129.4, 129.3, 125.9, 125.8, ,122.3, 116.5, 93.7, 92.3, 89.1, 33.9. HRMS (ESI-TOF) (m/z): Calcd. for C27H16F3N3OS, [M-H+]: 486.0888; found: 486.0884.
4-(2-((4-Azidobenzyl)thio)-5-cyano-6-oxo-1,6-dihydropyrimidin-4-yl)-N-benzylbenzamide (3m). Yield: 55%.
1H NMR (DMSO-d6): δ 9.24 (t, J = 6.0 Hz 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 4.0 Hz, 4H), 7.26 (m, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 4.51 (m, 4H); 13C NMR (DMSO-d6): δ 167.1, 166.7, 166.0, 162.0, 139.9, 139.0, 138.2, 137.3, 134.1, 131.1, 129.2, 128.7, 127.9, 127.2, 119.6, 116.3, 93.9, 43.2, 34.1. HRMS (ESI-TOF) (m/z): Calcd. for C26H18N7O2S, [M-H+]: 492.1248; found: 492.1232.
Methyl 4-(((4-(4-(benzylcarbamoyl)phenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio) methyl)benzoate (3n). Yield: 63%.
1H NMR (DMSO-d6): δ 9.24 (t, J = 6.0 Hz 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.88 (dd, J = 8.0 Hz, 4H), 7.55 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 4.4 Hz, 4H), 7.25 (m, 1H), 4.51 (d, J = 6.0 Hz, 2H), 4.44 (s, 2H); 13C NMR (DMSO-d6): δ 170.3, 168.0, 166.9, 166.5, 166.1, 144.7, 139.9, 139.7, 136.2, 129.6, 128,7, 128.6, 127.6, 127.2, 118.9, 91.0, 52.5, 43.1, 34.0. HRMS (ESI-TOF) (m/z): Calcd. for C28H21N4O4S, [M-H+]: 509.1289; found: 509.1271.
N-Benzyl-4-(5-cyano-6-oxo-2-((4-(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidin-4-yl)benzamide (3o). Yield: 68%.
1H NMR (DMSO-d6): δ 9.21 (t, J = 6.0Hz 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.66 (qd, J = 8.0 Hz, 4H), 7.34 (d, J = 4.4 Hz, 4H), 7.26 (m, 1H), 4.58 (s, 2H), 4.52 (d, J = 6.0 Hz, 2H); 13C NMR (DMSO-d6): δ 167.2, 167.1, 166.0, 163.1, 142.8, 139.9, 138.5, 137.1, 130.2, 129.1, 128.7, 128.4, 128,1, 127.8, 127.2, 126.0, 125.7, 123.3, 116.8, 93.5, 43.2, 33.9. HRMS (ESI-TOF) (m/z): Calcd. for C27H18F3N4O2S, [M-H+]: 519.1097; found: 519.1091.
ATPase assays
Inhibition on ATPase activity of EcSecAN68 was determined by malachite green colorimetric assay as previously described.[15] IC50 is defined as the concentration of the compound that inhibits 50% of ATPase activity.
Bacteriostatic effects
Bacteriostatic effects were evaluated at 37oC in 96-well microtitier plates as previously described.[15] Minimum inhibitory concentration (MIC) is the lowest concentration of compounds at which bacterial cells were not able to grow at tested condition.