4.2 Synthesis of 1,3-bis(benzotriazol-1-yl)-propan-2-ol (1)
In a Schlenk tube equipped with a reflux condenser, 1H-benzotriazole (2.500 g; 20.99 mmol), potassium hydroxide (1.211 g; 21.58 mmol), tetrabutylammonium bromide (0.997 g; 3.09 mmol), and water (20 mL) were stirred at 55 ºC for 45 min. Then, 1,3-dichloro-propan-2-ol (1.0 mL; 1.390 g; 10.78 mmol) and toluene (40 mL) were added, and the mixture was heated for 48 h at 85 ºC. A white solid was generated at the interface; therefore, the reaction mixture was allowed to cool at room temperature (rt) and filtered under vacuum. The filtrate was extracted with 4 portions of water (20 mL), separated and dried with sodium sulfate. The solution was concentrated to dryness under vacuum to give a yellow solid. Both solids were mixed and purified by recrystallization with tetrahydrofuran:pentane and the ligand was obtained as a white solid (structure of the ligand 1 is show in Fig. 4 with the respective atom numbering).
Yield 2.225 g (72.03%). M.p.: 185–187°C. FTIR (KBr, cm− 1): 3368, 3067, 2924, 1616, 1589, 1497, 1454, 1420, 1358, 1304, 1281, 1265, 1227, 1169, 1134, 1092, 1038, 1011, 922, 872, 849, 799, 779, 768, 737, 667, 625, 606, 575, 548, 517, 471, 428. Raman (cm− 1): 3369, 3314, 3168, 3071, 2991, 2950, 2918, 1762, 1590, 1498, 1379, 1270, 1233, 1165, 1122, 1003, 879, 768, 620, 542, 331, 181.
1H NMR (400.1 MHz, DMSO-d6) δ 8.04 (d, 2H, 4, 4’), 7.91 (d, 2H, 7, 7’), 7.56 (t, 2H, 6, 6’), 7.40 (t, 2H, 5, 5’), 5.62 (d, 1H, OH), 4.99 (dd, 2H, 2, 2’), 4.81 (dd, 2H, 2, 2’), 4.56 (s, 1H, 1). 13C NMR (DMSO-d6, 101 MHz) δ 145.15 (C3, C3’), 133.78 (C8, C8’), 127.17 (C6, C6’), 123.89 (C5, C5’), 119.04 (C4, C4’), 111.26 (C7, C7’), 68.90 (C1), 51.41(C2, C2’). MS-ESI (m/z, ES+) calcd. For [M + H]+: 295, found: 295. UV/Vis (bands λmax, nm (ε, L mol-1 cm-1): 204 (76272), 262 (23597), 280 (16771). Anal. Calcd. for C15H14N6O: C 61.21; H 4.79; N 28.55. Found: C 61.25; H 4.84; N 28.49%.
4.3 Synthesis of the complexes
4.3.1 Synthesis of [Co{1,3-bis(benzotriazol-1-yl)-propan-2-ol-N,N}Cl2] (2)
(1) (0.35 mmol; 103.4 mg) was dissolved in acetone (17 mL), and CoCl2·6H2O (0.34 mmol; 80.9 mg) in acetone (5 mL) was added to this mixture. The resulting solution was stirred for 3 h at rt. This mixture was centrifuged at 400 rpm for 9 min, washing with acetone and ethyl ether and removing the liquid phase between each wash. Then, the solvent was evaporated to dryness to give a blue solid.
Yield: 132.2 mg (91.7%). M.p.: 341–346°C (decomposition). FTIR (KBr, cm− 1): 3491, 3098, 2928, 1709, 1597, 1497, 1458, 1435, 1393, 1373, 1350, 1312, 1288, 1231, 1184, 1169, 1146, 1026, 949, 876, 853, 806, 779, 756, 741, 671, 621, 579, 540, 521, 428. Raman (cm− 1): 3074, 2968, 2933, 2920, 1599, 1494, 1388, 1364, 1293, 1226, 1126, 1008, 951, 875, 775, 664, 620, 571, 539, 497, 466, 343, 304, 193. UV/Vis bands λmax, nm (ε, L mol− 1 cm− 1): 204 (39461), 263 (12404), 280 (9304), 530 (25). Anal. Calcd. for C15H14Cl2CoN6O: C 42.48; H 3.33; N 19.81. Found: C 42.51; H 3.39; N 19.78%.
4.3.2 Synthesis of [Cu{1,3-bis(benzotriazol-1-yl)-propan-2-ol-N,N}Cl2] (3)
(1) (0.34 mmol; 99.6 mg) was dissolved in acetone (14 mL), and CuCl2·2H2O (0.33 mmol; 55.9 mg) in acetone (8 mL) was added to this mixture. The resulting solution was stirred for 3 h at rt. This mixture was centrifuged at 400 rpm for 9 min, washing with acetone and ethyl ether and removing the liquid phase between each wash. Then, the solvent was evaporated to dryness to give a pea-green solid.
Yield: 95.8 mg (67.7%). M.p.: 195–199°C (decomposition). FTIR (KBr, cm− 1): 3379, 1593, 1493, 1458, 1323, 1288, 1234, 1165, 1092, 1003, 945, 872, 779, 745, 667, 652, 575, 513, 432. Raman (cm− 1): 3448, 3329, 3293, 3172, 3070, 2946, 1751, 1589, 1490, 1456, 1373, 1281, 1233, 1170, 1126, 997, 937, 876, 777, 619, 544, 375, 265. UV/Vis bands λmax, nm (ε, L mol− 1 cm− 1): 203 (60623), 262 (17372), 276 (11714), 860 (106). Anal. Calcd. for C15H14Cl2CuN6O: C 42.02; H 3.29; N 19.60. Found: C 42.05; H 3.31; N 19.55%.
4.3.3 Synthesis of [Co{1,3-bis(benzotriazol-1-yl)-propan-2-ol-N,N}(H2O)2(CH3COO)2] (4)
(1) (0.34 mmol; 100.3 mg) was dissolved in tetrahydrofuran:methanol (4:1, 15 mL), and Co(CH3COO)2·4H2O (0.34 mmol; 83.7 mg) in tetrahydrofuran:methanol (3:2, 5 mL) was added to this mixture. A color change of the solution to orange was immediately observed. The resulting solution was stirred for 2 h at rt. The solution was concentrated to dryness under a vacuum to give a purple solid, which was washed with acetone and ethyl ether, and removed the liquid phase between each wash. Then, the solvent was evaporated to dryness to give a purple solid.
Yield: 131.1 mg (76.0%). M.p.: 162–166°C (decomposition). FTIR (KBr, cm− 1): 3367, 3067, 2924, 1558, 1497, 1416, 1342, 1304, 1227, 1165, 1134, 1096, 1015, 872, 779, 741, 667, 613, 548, 513, 471, 432. Raman (cm− 1): 3367, 3062, 2989, 2947, 2917, 1586, 1488, 1451, 1385, 1308, 1267, 1227, 1166, 1109, 998, 930, 878, 772, 615, 179, 74. UV/Vis bands λmax, nm (ε, L mol− 1 cm− 1): 203 (32949), 261 (10264), 280 (8426), 514 (28). Anal. Calcd. for C19H24CoN6O7: C 44.98; H 4.77; N 16.56. Found: C 44.99; H 4.80; N 16.53%.
4.3.4 Synthesis of [Cu{1,3-bis(benzotriazol-1-yl)-propan-2-ol-N,N}(CH3COO)2]⋅2H2O (5)
(1) (0.34 mmol; 100.5 mg) was dissolved in tetrahydrofuran:methanol (4:1, 15 mL), and Cu(CH3COO)2·H2O (0.33 mmol; 66.8 mg) in tetrahydrofuran:methanol (3:2, 5 mL) was added to this mixture. The resulting solution was stirred for 5 h at reflux. The reaction mixture was allowed to cool at rt and concentrated to dryness under vacuum to give a green solid, which was washed with acetone and ethyl ether, and removed the liquid phase between each wash. Then, the solvent was evaporated to dryness to give a dark green solid.
Yield: 156.1 mg (93.1%). M.p.: 199–203°C (decomposition). FTIR (KBr, cm− 1): 3368, 3067, 2920, 1609, 1497, 1423, 1300, 1281, 1227, 1165, 1134, 1084, 1003, 934, 899, 872, 779, 745, 683, 625, 548, 513, 432. Raman (cm− 1): 3335, 3173, 3066, 2928, 1590, 1532, 1495, 1447, 1379, 1287, 1267, 1229, 1165, 1122, 1003, 938, 884, 838, 775, 698, 625, 542, 511, 302, 220, 186, 116. UV/Vis bands λmax, nm (ε, L mol− 1 cm− 1): 204 (49112), 262 (13205), 278 (9498), 424 (192), 450 (262), 714 (38). Anal. Calcd. for C19H24CuN6O7: C 44.57; H 4.72; N 16.41. Found: C 44.62; H 4.81; N 16.31%.
4.4 Biological studies
4.4.1 Microorganisms and mammalian cells
The study was carried out on eight strains of Candida sp. Four reference strains obtained from the American Type Culture Collection-ATCC (C. albicans 90028; C. tropicalis 66029; C. glabrata MYA2950; C. parapsilosis 22019) and clinical isolates resistant to fluconazole donated and characterized genotypically and phenotypically by the Corporation para Investigaciones Biológicas – CIB, Medellín, Colombia (C. albicans CAPF-13; C. tropicalis CAPF-01; C. glabrata CAPF-07; C. parapsilosis 24754). All yeasts were cultured on saboraud agar (OXOID Ltd., Basingstoke, Hampshire, UK) at 35°C. Fresh cultures were used for each experiment.
Macrophage J774.A1 (ATCC® TIB-67™) was donated by Cellular and Functional Biology and Biomolecular Engineering Group from the Universidad Antonio Nariño, Colombia. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA), supplemented with 10% inactivated fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA) and kept under conditions of 95% humidity, 5% CO2 and 37°C.
4.4.2 Susceptibility on planktonic cells of Candida sp
The minimum inhibitory concentrations (MICs) for strains of Candida spp were determined by the microdilution method according to Clinical & Laboratory Standards Institute (CLSI) guidelines, protocol M27-A4. Fluconazole (FCZ) and itraconazole (ITZ) obtained from Sigma-aldrich were used as reference drugs. The minimum fungicidal concentration (CFM) was determined from subcultures on saboraud agar of the MIC and concentrations above the MIC. The CFM was the concentration of the compound in which the growth of ≤ 3 CFU was observed after 24 hours of incubation at 35ºC.
4.4.3 In vitro drug interaction assay
The modified fixed-ratio isobologram method described by Quinton L. Fivelman et al. was used [54]. Pharmacological interactions between drug A and drug B were prepared from MIC. Concentrations equal to 8X MIC, 4X MIC, 2X MIC, MIC, 1/2 MIC, and 1/4 MIC were prepared and combined inversely (Table S3, Supplementary Material).
U-bottom plates were inoculated with 0.5 x102 cells/mL − 2.5 x103 cells/mL of Candida sp, incubated for 24 h, 37°C. Fractional MICs were obtained visually as the concentration that inhibits 50% of the initial inoculum. Fractional inhibitory concentrations (FIC) are calculated from the MIC obtained using the formula:
FIC (drug) = MIC (drug in combination) / MIC (drug alone)
The ∑FIC index is obtained from the sum of the FIC of the drugs in each combination. Its value defines whether the interaction is synergistic (< 0.5), additive (0.51–0.99), indifferent (1–3.9), or antagonistic (> 4).
4.4.4 Antibiofilm activity
To evaluate the effect of cobalt (II) complexes on the resulting biofilm, 106 cells/ml of Candida spp were grown in RPMI 1640. 200 µl of each culture was added to 96-well flat-bottom microtiter plates and incubated for 24h at 37°C with shaking (50 rpm) to allow biofilm formation as was previously described [55]. Afterward, Candida biofilms were rinsed three times with PBS to remove planktonic cells. Different concentrations of cobalt (II) complexes were added to yield final concentrations among 1/2MIC-4XMIC. Plates were incubated without shaking for 24h, at 37°C. Untreated cells and RPMI 1640 without yeast were included as positive and negative controls, respectively. The metabolic activity of biofilms was determined using a semi-quantitative 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT, Cayman Chemical) reduction assay. In brief, stock solutions of XTT in PBS (0.5 g/L) and Menadione in acetone (10mM) were prepared and stored at -80°C. Prior to use XTT/menadione solutions were freshly prepared in a ratio 10:1. 100 µl of XTT/Menadione mixture was then added to each well. Plates were incubated for 3h, at 37°C, in the dark, and absorbance was measured at 490 nm. The sessile minimum inhibitory concentrations (SMIC50) were calculated.
4.4.5 Filamentation assay
The inhibitory effect of Co(II) complexes on the switch from yeast to hyphae of C. Albicans was tested, according to described by Sun et al., 2015 [56]. Briefly, cells were grown at 37°C on YPD broth (1% yeast extract, 2% peptone, and 2% glucose), with rotary shaking at 200 rpm, overnight. Then, cells were harvested by centrifugation and washed twice with ultrapure water. 2.5 x106 cells/mL were transferred to RPMI 1640 supplemented with 0.5% GlcNAc; 0.5% peptone, and 0.3% KH2PO4, with or without metallic complexes (control). The compounds were added in a concentration range of 7.8–62.5 µg/mL. The plates were incubated at 37°C during 4 h. Lastly; cell morphology was recorded by counting at least 200 cells, discriminating between yeast cells and hyphae. The results were expressed as the percentage of the mycelium, and the inhibition percent were calculated. Ten repetitions were established with each concentration. The assays were repeated in two independent moments.
4.4.6 In vitro cytotoxicity assay
The in vitro effect of the complexes, ligands, and salts on the viability of J774.A1 macrophages was determined by the colorimetric method using the tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich). In summary, a cell density of 1x105 cells/mL in monolayer was exposed for 72 hours with the metal complexes in concentration ranges 300 − 11.1 µg/mL. Then, MTT was added to each well (10%) for 4 hours, and the optical density determined at 595nm using an iMark™ Microplate Absorbance Reader (BioRad, Madrid, Spain). The cytotoxicity percentage was calculated with the equation: [(OD450nm control–OD450nm treatment)/OD450nm treatment)] x100. The results were expressed as Cytotoxic Concentration 50 (CC50) determined by sigmoidal regression using Msxlfit software (GO Business Solution, Guildford, UK).
4.4.7 Statistical analysis
Data analysis of filamentation assays was performed with the statistical package IBM SPSS Statistics version 25.0. The effect of different concentrations of Co(II) complexes on dimorphic transition of C. albicans were determined using the Kruskal-Wallis test. A p value < 0.05 was defined as statistically significant. The cytotoxic concentration 50 (CC50) and 90 (CC90) were calculated by sigmoidal regression from the percentages of inhibition using Msxlfit software (GO Business Solution, Guildford, UK). Graphs were generated using Microsoft Excel.