Chemistry
All used solvents and reagents were of the greatest analytical reagent grade and were not further purified. Stuart Scientific SMP1was used to determine the melting points and are uncorrected. TLC was carried out on UV fluorescent Silica gel Merck 60 F254 plates, and the spots were identified with a UV lamp (254 nm). The SHIMADZU FTIR-Affinity-1S spectrometer was used to identify the main functional groups ranging from 400–4000 cm-1. While, Bruker spectrometer (400 MHz) was used to collect the NMR spectra using tetramethyl silane (TMS) as an internal reference. Elemental analyses were carried out with the help of a GmbH-Vario EL III Elementar Analyzer. The high-resolution mass spectroscopy (HRMS) was performed using the LCMS/MS impact II. GmbH-Vario EL III Elementar Analyzer was used to perform the elemental analyses.
Synthesis and characterization of sulfonamide-based 1, 2, 3-triazoles 3(a,b,c)
To a solution of propargyl amine (1 mmol) in DMSO (10 ml) was added a solution of copper sulphate (0.10 g) and sodium ascorbate (0.15 g) in water (10 ml) dropwise under stirring. The appropriate sulfa drug azide 2a-c (1 mmol) was then added to the reaction mixture, the stirring was continued for 6-10 hours at room temperature. The reaction was monitored by TLC (hexane-ethyl acetate), and once it was completed, crushed ice water was added to the mixture. Filtration was used to collect the precipitate formed, which was then washed with saturated ammonium chloride solution before being recrystallized from ethanol/DMF to yield the required 1,2,3-triazoles 3(a,b,c).
4-(4-(Aminomethyl)-1H-1,2,3-triazol-1-yl)-N-(pyrimidin-2-yl)benzenesulfonamide (3a). It was obtained in 86 % yield as yellow powder; Mp: 220-221 °C. IR (KBr) ύmax/cm-1: 3380-3450 (NH, NH2), 3045 (CHa5), 2940 (CHal), 1620 (C=N), 1560 (C=C). 1H-NMR (DMSO-d6, 400 MHz): δH = 4.25 (s, 2H, CH2), 6.84 (s, 2H, NH2), 7.04 (bs, 1H, Ar-H), 8.16-8.43 (m, 6H, Ar-H), 8.50 (s, 1H, H-5-triazolyl), 12.02 (s, 1H, NHSO2). 13C-NMR (DMSO-d6, 100 MHz): δC = 56.56 (CH2); 119.97, 120.12, 121.44, 122.75, 127.85, 128.33, 139.25, 146.36, 150.23, 158.64, 159.25 (Ar-C, C=N). Calculated for C13H13N7O2S: C: 47.12; H; 3.95; N; 29.59. Found: C: 47.29; H, 3.78; N, 29.34. HRMS (ESI): 331.0654 [M+].
4-(4-(Aminomethyl)-1H-1,2,3-triazol-1-yl)-N-(pyridin-2-yl)benzenesulfonamide (3b). It was obtained in 84 % yield as yellow solid; Mp: 248-250 °C. IR (KBr) ύmax/cm-1: 3350-3440 (NH, NH2), 3060 (CHar), 2920 (CHal), 1610 (C=N), 1550 (C=C). 1H-NMR (DMSO-d6, 400 MHz): δH = 4.42 (s, 2H, CH2), 6.85 (s, 1H, NH2), 7.23-7.51 (m, 2H, Ar-H), 7.76-8.05 (m, 4H, Ph-H), 8.39 (bs, 2H, Ar-H), 8.88 (s, 1H, H-5-triazolyl), 12.43 (s, 1H, NHSO2). 13C-NMR (DMSO-d6, 100 MHz): δC = 56.49 (CH2); 119.73, 121.22, 122.11, 123.87, 128.22, 129.45, 140.45, 148.76, 154.11, 158.07, 159.75 (Ar-C, C=N). Calculated for C14H14N6O2S: C: 50.90; H: 4.27; N: 25.44. Found: C: 50.78; H: 4.42; N: 25.67. HRMS (ESI): 330.0689 [M+].
N-(Diaminomethylene)-4-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)benzene-sulfonamide (3c). It was obtained in 88 % yield as yellow pale powder; Mp: 278-279 °C. IR (KBr) ύmax/cm-1: 3310-3460 (NH2), 3080 (CHar), 2960 (CHal), 1615 (C=N), 1580 (C=C). 1H-NMR (DMSO-d6, 400 MHz): δH = 4.43 (s, 2H, CH2), 6.78 (bs, 6H, 3 x NH2), 7.94-8.07 (m, 4H, Ar-H), 8.72 (s, 1H, H-5-triazolyl). 13C-NMR (DMSO-d6, 100 MHz): δC = 57.09 (CH2); 118.87, 121.09, 121.22, 124.87, 126.66, 128.23, 138.39, 147.45, 156.26, 155.45, 157.29, 158.45 (Ar-C, C=N). Calculated for C10H13N7O2S: C: 40.67; H: 4.44; N: 33.20. Found: C: 40.49; H: 4.67; N: 33.35. HRMS (ESI): 295.0599 [M+].
Maintenance of Toxoplasma strain:
Virulent T. gondii RH HXGPRT(-) strain was maintained in the Medical Parasitology Department, Faculty of Medicine, Alexandria University by serial intraperitoneal passages into mice. Peritoneal exudates were harvested on the fifth day post inoculation. Parasites were passed twice through a 27-gauge needle, washed twice by 1000 x g centrifugation for 10 minutes in RPMI 1640 without fetal bovine serum (FBS) (Gibco BRL). Then the parasites were suspended in the same medium to a density of 106 parasites/ml. The viability was evaluated using a dye-exclusion test with 0.2% Trypan blue [54, 55] .
Vero cell line
Vero cells were obtained from African green monkey kidney fibroblast cell (obtained from National Cancer Institute, Cairo, Egypt, and maintained in Medical Research Institute). A growth medium consisting of RPMI-1640 supplemented with 10 % FBS (Gibco BRL), 100 units/ml penicillin (Gibco BRL) and 100 μg/ml streptomycin (Gibco BRL). The cells were cultured and maintained at 37 °C in a humidified 5 % CO2 incubator [56]
Cytotoxicity tests
Cytotoxicity of the prepared sulfa drugs 3(a,b,c) on Vero cells was determined using 3-(4, 5-dimethylthiazol-2-yl) -2,5- diphenyl tetrazolium bromide MTT assay. Vero cells were seeded at a density of 2x104 cells/ml/well of a 96-well plate containing 200 µl of the growth medium. The plate was incubated at 37 ºC in a humidified 5% CO2 incubator, After 24 hours, the cells were treated with the prepared sulfa drugs 3(a,b,c), which were dissolved in (RPMI-1640) to obtain the final concentrations of 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 µg/ml.. Wells with sulfadiazine and culture media only served as the positive and negative control, respectively. After 24 hours of treatment, 200 µl of the medium was aspirated out and 100 µl of MTT - PBS (5 mg/ml) solution in RPMI medium in the ratio of 1: 9 was added to each well. The plate was then covered with aluminum foil and incubated for 4 hours in a 37 ºC incubator. The medium was then discarded and 100 µl of DMSO was added into each well to solubilize the dark-blue MTT formazan salt. The optical density was measured at 570 nm absorbance using a Benchmark Microplate Reader (Bio Rad). Cytotoxicity, expressed as CC50, was defined as the highest dilution of test samples that causes 50% destruction of cells [57]
Vero cell growth suppression (%) was estimated using the following equation:
using CompuSyn software (version, 1) [58, 59].
Effects of sulfadrugs on intracellular Toxoplasma gondii
For this purpose, Vero cells were cultured in 96well plates (2 × 104 cells/well/200 µl) for 24 hours in RPMI 1640 medium supplemented with 10% inactivated FBS at 37°C and 5% CO2. Next, the cells were infected with T. gondii tachyzoites (parasite: cell ratio = 10:1). After 24 hours, the medium was changed and the infected Vero cells were incubated with different concentrations (3.125–400 µg/ml) of the prepared sulfa drugs 3(a,b,c). Sulfadiazine (3.125–400 µg/ml) and RPMI 1640 were also used as positive and negative controls, respectively. MTT solution (5 mg/ml) was added to the cultures and incubated for 4 hours. Then, 200 µg/well of DMSO was added to all plates. After 15 minutes, the optical absorbance was measured at 570 nm wavelength. Growth inhibition (GI) was calculated as in the following equation,
GI (%) = [(At – Ac)/Ac] * 100
Where At and Ac are the absorbance of treated cells and control, respectively. In addition, IC50 is the 50% growth inhibition concentration .Selectivity Index (SI) of the samples was calculated using the IC50 and the hostcell cytotoxicity profiles (SI = CC50/IC50 [57] using CompuSyn software (version, 1) [58, 59].
Scanning Electron Microscopy (SEM)
Electron microscopic analysis was performed to further explore the anti-Toxoplasma mechanism of sulfa drugs 3(a,b,c), the ultrastructure of T. gondii tachyzoites treated in vitro with sulfa drugs 3(a,b,c) was observed using (SEM) (Joel JSM-53001A, Tokyo, Japan).
Tachyzoites were collected from peritoneal exudates of infected mice on the fifth day post inoculation as previously described [55]. Then tachyzoites were divided into four tubes, each contains 105 tachyzoites. The first tube was used as control (normal, nontreated group), while sulfa drugs 3(a,b,c) were added to the remaining three tubes respectively. Then, tachyzoites were incubated for 2 hours at room temperature. After that, tachyzoites were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) washed in cacodylate buffer and attached on a slide. Then the slide was post-fixed for 2–4 hours using 1–2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) at room temperature and dehydrated in graded ethanol dilutions (70, 80, 90, and 100%). They were dried using critical point method, mounted on stubs, coated with gold (20–30 nm), and then observed using SEM [60].