All solvents were of analytical grade and reagents were purchased from Sigma-Aldrich. 1H and 13C NMR spectra were recorded on a Varian and Bruker WH-200 (400 MHz) spectrometer in CDCl3 or DMSO-d6 as solvent, using TMS as an internal standard and chemical shifts are expressed as ppm. Mass spectra were determined on a Shimadzu LC-MS. High resolution mass spectra were determined on a Bruker Daltonics instrument. The elemental analyses were carried out using an Elemental Vario Cube CHNS rapid Analyzer. The progress of the reaction was monitored by TLC pre-coated silica gel G plates.
Preparation of Sulphated Ceria
Hydrous cerium oxide was prepared by the hydrolysis of cerium (III) nitrate hexahydrate with 1:1 ammonia. Cerium (III) nitrate was dissolved in double distilled water. To this clear solution, dilute (1:1) aqueous ammonia was added drop-wise from a burette with vigorous stirring until the pH of the solution reached 8.
The solution was boiled for 15 min and allowed to stand overnight. The mother liquor was then decanted and the precipitate was washed several times with distilled water till it is completely free of nitrate ions which was confirmed by brown ring test. The precipitate was filtered and dried overnight at 383 K for 16 h. The hydroxide obtained was sieved to get particles of 75-100 μm mesh size and immersed in (1:1) H2SO4 solution (2 mL/g) and subjected to stirring for 4 h. Excess water was evaporated and the resulting sample was oven dried at 383 K for 16 h, calcined at 823 K for 5 h and stored in vacuum desiccator.
General procedure for Microwave synthesis of 4-amino-5-phenyl-4 h-1,2,4-triazole-3-thiol (2)
A mixture of methylbenzoate (1 mmol) and hydrazine hydrate (1 mmol) in 20 mL ethanol was irradiated in microwave at 700 W in a specially designed Teflon vessel containing lead acetate, until all the starting material was consumed (1-2 min, as monitored by TLC). To the above mixture (0.006 mmol) of KOH, CS2 (1 mmol) was added and further irradiated at 700 W for 1 min. Finally, hydrazine hydrate (2 mmol) was added drop wise to the above mixture and continued the irradiation at 700 W until a white solid appeared at the bottom (2-3 min). The lead acetate worked as a trap for H2S that was evolved during reaction. The solid obtained was dissolved in water (15-20 mL) and acidified with conc. HCl. The separated solid was filtered, dried and recrystallized to obtain pure 4-amino-5-phenyl-4 h-1,2,4-triazole-3-thiol. Yield 78%, m.p. 232-234 °C; IR (KBr) γ/cm−1: 3310.07 (NH2 stretch), 3071.36 (aromatic CH stretch), 1472.38 (tautomeric C = S). 1H NMR: (400 MHz, DMSO-d6). δ:7.6-7.5 (m, 2H, ArH), 7.34-7.2 (m, 3H, ArH), 5.14 (s, 2H, NH2).
General procedure for the synthesis of 6-substituted-3-phenyl-(1,2,4)-triazolo(3,4-b)(1,3,4-thiadiazole (4a-4 h) by using SCe
To a mixture of 4-amino-5-phenyl-4 h-1,2,4-triazole-3-thiol (1 mmol) and (3a-h) (1 mmol) in DMF (10 mL), SCe (20 mol%) and POCl3 (0.1 mmol) were added. The reaction mixture was refluxed for 10 h. Completion of the reaction was monitored by TLC and the catalyst was filtered and washed with water. Solvent was removed under reduced pressure and crushed ice was added to the concentrated mass. The pH of reaction mixture was adjusted to 8.0 using K2CO3 and KOH. The solid obtained was separated by filtration, washed with excess water, dried and recrystallized using appropriate solvent.
General procedure for the synthesis of 2-hydroxy-3,5-diiodo-N-(3-phenyl-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)benzamide (5a) and 2-hydroxy-5-iodo-N-(3-phenyl-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)benzamide (5b)
To 3a (1 eq) in DMF, EDC (1.1 eq) and HOBt (1.1 eq) was added and stirred at room temperature for 30 min. It was followed by the addition of amine (2) and stirred for 2 h. After completion of the reaction, it was diluted with water and the obtained solid was filtered and re-crystallized in appropriate solvent.
2,4-Diiodo-6-(3-phenyl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazol-6yl)phenol (4a, DTP)
Yellow colored solid; 1H NMR (400 MHz, DMSO-d6) 8.37-8.35 (d, 2H), 8.26 (s, 1H), 7.85 (s, 1H), 7.69-7.63 (m, 2H), 7.54-7.52 (d, 1H), 4.92 (s, 1H); 13C NMR (DMSO-d6); 165.53, 154.53, 149.29, 148.83, 140.98, 137.51, 133.83, 132.45, 129.11, 128.64, 123.10, 122.44, 120.72, 96.18, 85.11; HRMS Calcd 568.840; Found: 568.840 (M + Na)+; Anal. Calcd for C15H8I2N4OS: C, 32.99; H, 1.48; N, 10.26; Found: C, 33.00; H, 1.49; N, 10.28.
6-(4-(1H-Imidazol-1-yl)phenyl)-3-phenyl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazole (4b)
Pale yellow colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.46-8.44 (d, 2H), 7.81-7.77 (m, 2H), 7.53-7.49 (m, 3H), 7.39-7.34 (m, 3H), 7.27-7.24 (m, 2H); 13C NMR (DMSO-d6); 161.55, 149.29, 148.53, 140.98, 137.18, 137.11, 133.83, 132.48, 131.97, 129.11, 128.64, 128.18, 123.10, 122.43, 120.27; LCMS (MM:ES + APCI) 345.2 (M + H)+. Anal. Calcd for C18H12N6S: C, 62.77; H, 3.51; N, 24.40; Found: C, 62.79; H, 3.53; N, 24.43.
4-Iodo-2-(3-phenyl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazol-6-yl)phenol (4c, ITP)
Yellow colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.44-8.42 (d, 2H), 8.08-8.06 (d, 2H), 8.02-8.00 (m, 1H), 7.95-7.91 (m, 1H), 7.71 (s, 1H), 7.16-7.14 (d, 1H), 4.92 (s, 1H); 13C NMR (DMSO-d6) δ: 164.19, 159.73, 152.02, 147.46, 138.26, 133.27, 131.64, 129.40, 127.70, 124.93, 120.48, 119.82, 118.66, 88.23; HRMS Calcd 442.943; Found: 442.943 (M + Na)+; Anal. Calcd for C15H9IN4OS: C, 42.87; H, 2.16; N, 13.33; Found: C, 42.89; H, 2.17; N, 13.35.
6-(((R)-Tetrahydro-2H-pyran-2-yl)(phenyl)methyl)-3-phenyl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazole (4d)
White colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.25-8.16 (d, 2H), 8.06 (m, 1H), 7.78-7.76 (m, 1H), 7.62-7.60 (m, 1H), 7.27-7.15 (m, 4H), 4.58-4.53 (m, 2H), 3.88-3.84 (m, 2H), 1.78-1.73 (m, 4H), 1.50-1.45 (m, 2H); 13C NMR (DMSO-d6) δ: 164.56, 149.30, 143.93, 141.04, 137.49, 132.82, 132.41, 130.23, 129.10, 128.10, 120.70, 80.11, 71.09, 43.59, 30.41, 30.33, 21.48; LCMS (MM:ES + APCI) 377.2 (M + H)+; Anal. Calcd for C21H20N4OS: C, 67.00; H, 5.35; N, 14.88; Found: C, 67.02; H, 5.37; N, 14.90.
2-(3-Phenyl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazol-6yl)-1-p-tolylethanone (4e)
White colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.43-8.41 (m, 2H), 8.03-7.99 (m, 3H), 7.92 (m, 1H), 7.69 (m, 1H), 7.40-7.38 (m, 2H), 4.1 (s, 2H), 2.42 (m, 3H); 13C NMR (DMSO-d6) δ:192.83, 164.18, 159.42, 151.99, 146.87, 137.47, 132.28, 130.26, 125.66, 123.38, 121.01, 120.89, 48.13, 21.13; HRMS Calcd 357.078; Found: 357.078 (M + Na)+. Anal. Calcd for C18H14N4OS: C, 64.65; H, 4.22; N, 16.75; Found: C, 64.67; H, 4.25; N, 16.77.
6-(3-4-Dimethoxybenzyl)-3-phenyl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazole (4f)
Yellow colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.2 (d, 2H), 7.6-7.4 (m, 3H), 7.0 (s, 1H), 6.9 (d, 2H), 4.4 (s, 2H), 3.8 (s, 6H); LCMS (MM:ES + APCI) 353.2 (M + H)+; Anal.Calcd for C18H16N4O2S: C, 61.35; H, 4.58; N, 15.90; Found: C, 61.39; H 4.59; N, 15.93.
3-(3-Phenyl--[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazol-6-yl-)phenol (4 g)
White colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.32-8.31 (m, 2H), 8.13 (s, 1H), 7.94-7.87 (m, 3H), 7.65-7.59 (m, 2H), 7.46 (m, 1H), 4.91 (s, 1H); LCMS (MM:ES + APCI) 295.2 (M + H)+; Anal. Calcd for C15H10N4OS: C, 61.21; H, 3.42; N, 19.04; Found: C, 61.23; H, 3.44; N, 19.07.
3-Phenyl-6-styryl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazole (4 h)
White colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.25-8.22(d, 2H), 7.92-7.87 (m, 2H), 7.73-7.55 (m, 4H), 7.32-7.26 (m, 2H), 6.45-6.42 (m, 2H); 13C NMR (DMSO-d6) δ: 164.84, 159.58, 153.39, 145.42, 139.89, 131.08, 131.04, 130.53, 130.42, 130.31, 129.09, 125.84, 125.47, 118.08, 116.18, 115.90; HRMS Calcd 327.067; Found: 327.067 (M + Na)+; Anal. Calcd for C17H12N4S: C, 67.O8; H, 3.97; N, 18.41; Found: C, 67.09; H, 3.99; N, 18.44.
2-Hydroxy-3,5-diiodo-N-(3-phenyl-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)benzamide (5a, HTP)
Pale yellow colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 14.64 (s, NH), 12.30 (s, NH), 8.48 (s, 1H), 8.40 (s, 1H), 8.24-8.15 (m, 3H), 7.81-7.78 (m, 2H), 4.73 (s, 1H); 13C NMR (DMSO-d6) δ:181.47, 173.23, 153.47, 147.94, 145.12, 136.38, 134.36, 131.13, 129.09, 128.78, 128.21, 126.02, 125.58, 90.79, 72.33; HRMS Calcd 586.851; Found: 586.851 (M + Na)+; Anal.Calcd for C15H10I2N4O2S: C, 31.94; H, 1.79; N, 9.93; Found: C, 31.96; H, 1.81; N, 9.93.
2-Hydroxy-5-iodo-N-(3-phenyl-5-thioxo-1H-1,2,4-triazol-4(5H)-yl)benzamide (5b)
Pale yellow colored solid; 1H NMR (400 MHz, DMSO-d6) δ: 12.5 (s, NH), 8.5 (s, 1H), 8.4 (m, 1H), 8.1 (m,1H), 7.8 (m, 3H), 7.6 (m,1H), 4.6 (s, 1H); LCMS (MM:ES + APCI) 438.4 (M + H)+; Anal. Calcd for C15H11IN4O2S: C, 41.11; H, 2.53; N, 12.78; Found: C, 41.12; H, 2.56; N, 12.80.
Spectral data of the compounds are presented in Additional file
1: Figure S1.
Colorimetric heparanase assay
The assay, carried out in 96 well microplates, measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage, colorimetrically using the tetrazolium salt WST-1 [
31]. Briefly, assay solutions (100 μL) are composed of 40 mM sodium acetate buffer (pH 5.0) and 100 mM fondaparinux (Arixtra) with or without increasing concentrations of inhibitor. Recombinant heparanase was added to a final concentration of 140 pM, to start the assay. The plates are incubated at 37 °C for 18 h and the reaction is stopped by the addition of 100 μL solution containing 1.69 mM 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) in 0.1 M NaOH. The plates are developed at 60 °C for 60 min, and the absorbance is measured at 584 nm. In each plate, a standard curve constructed with D-galactose as the reducing sugar standard is prepared in the same buffer and volume over the range of 2–100 μM [
31].
ECM degradation heparanase assay
The semi-quantitative heparanase assay was performed as described previously [
32,
33]. Briefly, metabolically sulfate [
35S] labeled ECM deposited by cultured endothelial cells and coating the surface of 35 mm tissue culture dishes [
33], is incubated (3 h, 37 °C, pH 6.0, 1 mL final volume) with recombinant human heparanase (200 ng/mL) in the absence and presence of candidate small molecules. The ECM was also incubated (24 h, 37 °C, pH 6.0) with cell lysates (200 μg protein/dish) prepared by 3 cycles of freeze and thaw in reaction buffer, as described [
32]. To evaluate the occurrence of proteoglycan degradation, the incubation medium is collected and applied for gel filtration on Sepharose 6B columns (0.9 × 30 cm). Fractions (0.2 mL) are eluted with PBS and counted for radioactivity. The excluded volume (Vo) is marked by blue dextran and the total included volume (Vt) by phenol red. Degradation fragments of HS side chains are eluted from Sepharose 6B at 0.5 < Kav < 0.8 (fractions 12-25) [
32].
In vitro cytotoxicity assay
The antiproliferative effect of the compounds against LLC (Lewis lung carcinoma) and HepG2 (hepatocellular carcinoma) cells was determined by the MTT dye uptake method as described previously [
34‐
36]. Briefly, cells (2.5 × 10
4/mL) were incubated in triplicate in a 96-well plate, in the presence of varying concentrations of test compounds at a volume of 0.2 mL, for different time intervals at 37 °C. Thereafter, 20 μL MTT solution (5 mg/mL in PBS) was added to each well. After 2 h incubation at 37 °C, 0.1 mL lysis buffer (20% SDS, 50% dimethylformamide) was added and incubated for 1 h at 37 °C, and the optical density (OD) at 570 nm was measured using a plate reader.