General experimental methods
Reichert Thermovar apparatus was used to recording the melting points which was uncorrected. Perkin-Elmer model 1720 FTIR spectrometer was used to recording the Infra-red spectra. Bruker AC-300 or DPX-300 spectrometer (500 MHz 1H) (126 MHz 13C) was used to investigate the Nuclear magnetic resonance. The values of δppm (chemical shifts) were registered compared to TMS as a standard reference. The coupling constants (J values) are offered in Hz. TLC holding aluminum silica gel 60 F245 was used to checking The advance of reaction completion. IR, 1H NMR, 13C NMR, Elemental analyses were measured at the National Research Center, Egypt.
Synthesis of azid 3 and 4 derivatives:
Sulfanilamide derivatives 1 and 2 (0.007 mol) was dissolved in conc. acetic acid (18 mL) and distilled water (18 mL), then cooled in an ice bath until reach the temperature to (0-5) °C, then Sodium nitrite solution (0.47g, 0.007 mol in (5mL) water) was added very slowly to the mixture and stirred for (10 min.). Finally, the solution of sodium azide (0.45 g, 0.007 mol in (25mL) distilled water) was added drop-by-drop to the reaction mixture and stirred for (30 min.). The solid product was filtered off and washed with distilled water to obtain compounds 3 and 4.
4-azido-N-butylbenzenesulfonamide (3).
Yield: 88%; m.p. 47-49 °C; IR (KBr) cm−1, ν: 3203 (NH), 2907 (CH-aliph.), 2101 (N=N=N), 1330, 1158 (SO2). 1H-NMR (500 MHz, DMSO-d6) δ/ppm: 0.79 (t, 3H, J = 7.3 Hz, CH3), 1.17-1.26 (m, 2H, CH2), 1.28-1.38 (m, 2H, CH2), 2.71 (t, 2H, J = 7.0 Hz, CH2), 7.31 (d, 2H, J = 8.6 Hz, Ar-H), 7.56 (br. s, 1H, NH), 7.79 (d, 2H, J = 8.6 Hz, Ar-H). 13C-NMR (125 MHz, DMSO-d6) δ/ppm: 13.94, 19.74, 31.57, 42.72, 120.23, 129.03, 137.48, 144.00. Analysis calcd. for C10H14N4O2S (254.31): C, 47.23; H, 5.55; N, 22.03. Found: C, 47.18; H, 6.01; N, 21.93.%.
4-azido-N-cyclohexylbenzenesulfonamide (4).
Yield: 85%; m.p. 65-67 °C; IR (KBr) cm−1, ν: 3223 (NH), 2890 (CH-aliph.), 2100 (N=N=N), 1337, 1168 (SO2). 1H-NMR (500 MHz, DMSO-d6) δ/ppm: 0.84-0.86 (m, 2H, CH2), 1.16-1.19 (m, 4H, 2CH2), 1.21-1.25 (m, 2H, CH2), 1.31-1.34 (m, 2H, CH2), 2.68-2.72 (m, 1H, N-CH2), 7.31 (d, 2H, J = 8.6 Hz, Ar-H), 7.55 (br. s, 1H, NH), 8.78 (d, 2H, J = 8.6 Hz, Ar-H). 13C-NMR (125 MHz, DMSO-d6) δ/ppm: 13.87, 22.05, 25.95, 28.86, 31.25, 42.44, 119.65, 128.47, 136.98, 143.40. Analysis calcd. for C12H16N4O2S (280.35): C, 51.41; H, 5.75; N, 19.99. Found: C, 51.49; H, 5.65; N, 20.04.%.
Click Reaction 6-9 derivatives:
To a well stirred mixture of the acetylated acetylenic glacto, xylo-pyranosyl sugar (3 mmol) and the azido benzenesulfonamide derivatives 3 and 4 (2 mmol) in THF–H2O (2:1; 25 mL) was added CuSO4·5H2O (0.4 mmol) then add quickly Na-ascorbate (0.4 mmol) and 3-4 drops of diisopropylethylamine at 0 °C. The reaction mixture was stirred at 60 °C for 12 h (TLC, petroleum ether–EtOAc, 4:1). the organic layer was separated after adding ethyl acetate (30 mL), washed with water (2 x 40 mL) and dried. The residue was purified by column chromatography (petroleum ether – EtOAc, 4:1) to obtain the acetylated glycosyl triazole derivatives 6-9.
N-butyl-4-(4-([2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl]oxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (6).
Yield: 77%; m.p. yellowish foam; IR (KBr) cm−1, ν: 3203 (NH), 2907 (CH-aliph.), 1740 (C=O), 1330, 1158 (SO2). 1H-NMR (500 MHz, CDCl3) δ/ppm: 0.86 (t, 3H, J = 7.2 Hz, CH3), 1.28-1.32 (m, 2H, CH2), 1.44-1.50 (m, 2H, CH2), 1.99, 2.01, 2.03, 2.15 (4s, 12H, CH3CO), 2.99 (t, 2H, J = 7.0 Hz, CH2), 4.06-4.11 (m, 2H, H-6′′,H-6'), 4.33 (t, 1H, J = 6.6 Hz, H-5′), 5.05 (dd, 1H, J = 10.6, 3.6 Hz, H-4′), 5.32 (s, 2H, CH2), 5.41 (d, 1H, J = 3.2 Hz, H-2′), 5.49 (d, 1H, H-3′), 6.36 (d, 1H, J = 2.4 Hz, H-1′), 7.26 (br. s, 1H, NH), 7.83 (d, 1H, J = 8.3 Hz, Ar-H), 7.92 (d,1H, J = 8.3 Hz, Ar-H), 8.03 (d, 2H, J = 7.5 Hz, Ar-H), 8.13 (s, 1H, triazole). 13C NMR (126 MHz, CDCl3) δ 13.32, 19.46, 20.31, 20.36, 20.41, 20.64, 31.34, 42.78, 61.10, 66.30, 67.17, 67.27, 68.57, 70.61, 89.44, 100.37, 119.26, 120.49, 128.85, 140.25, 168.85, 169.77, 169.95, 170.22. Analysis calcd. for C27H36N4O12S (640.66): C, 50.62; H, 5.66; N, 8.75. Found: C, 50.54; H, 5.73; N, 8.65.%.
N-cyclohexyl-4-(4-([2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl]oxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide(7).
Yield: 75%; m.p. yellowish foam; IR (KBr) cm−1, ν: 3225 (NH), 2881 (CH-aliph.), 1750 (C=O), 1340, 1170 (SO2). 1H-NMR (500 MHz, CDCl3) δ/ppm: 0.82-0.86 (m, 2H, CH2), 1.20-1.26 (m, 6H, 3CH2), 1.46-1.48 (m, 2H, CH2), 1.99, 2.00, 2.02, 2.14 (4s, 12H, CH3CO), 2.96-2.98 (m, 1H, N-CH), 4.06-4.09 (m, 2H, H-6′′,H-6'), 4.33 (t, 1H, J = 6.7 Hz, H-5′), 5.03-5.06 (m, 1H, H-4′), 5.31 (s, 2H, CH2), 5.40 (t, 1H, J = 2.6 Hz, H-2′), 5.48 (d, 1H, J = 1.9 Hz, H-3′), 6.36 (d, 1H, J = 2.5 Hz, H-1′), 7.26 (br. s, 1H, NH), 7.91 (d, 2H, J = 8.2 Hz, Ar-H), 8.02 (d, 2H, J = 8.3 Hz, Ar-H), 8.16 (s, 1H, triazole). 13C NMR (126 MHz, CDCl3) δ 13.96, 20.41, 20.47, 20.52, 20.75, 22.50, 26.44, 29.33, 31.70, 43.23, 61.17, 66.38, 67.27, 67.35, 68.66, 70.71, 89.57, 100.53, 120.52, 128.92, 140.38, 168.90, 169.83, 170.08, 170.29. Analysis calcd. for C29H38N4O12S (666.70): C, 52.25; H, 5.75; N, 8.40. Found: C, 52.36; H, 5.80; N, 8.31.%.
N-butyl-4-(4-([2,3,4-tri-O-acetyl-β-D-xylopyranosyl]oxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (8).
Yield: 80%; m.p. yellowish foam; IR (KBr) cm−1, ν: 3208 (NH), 2900 (CH-aliph.), 1737 (C=O), 1331, 1155 (SO2). 1H-NMR (500 MHz, CDCl3) δ/ppm: 0.85 (t, 3H, J = 8.1 Hz, CH3), 1.28-1.47 (m, 4H, CH2), 2.01, 2.04, 2.07 (3s, 9H, CH3CO), 2.98 (t, 2H, CH2), 3.41-3.85 (m, 1H, H-5′′), 4.18-4.34 (m, 1H, H-5′), 4.71-4.73 (m, 1H, H-4′), 4.96 (s, 2H, CH2), 4.99-5.09 (m, 2H, H-2′, H-3′), 5.18 (d, 1H, H-1′), 7.27 (br. s, 1H, NH), 7.92-8.02 (m, 4H, Ar-H), 8.19 (s, 1H, triazole). 13C NMR (126 MHz, CDCl3) δ 13.44, 19.60, 20.59, 20.64, 25.50, 29.55, 31.48, 42.87, 42.93, 55.67, 62.08, 62.25, 67.82, 68.78, 70.80, 71.28, 98.28, 100.00, 120.50, 128.52, 128.86, 139.47, 140.35, 169.65, 169.94, 169.99. Analysis calcd. for C24H32N4O10S (568.60): C, 50.70; H, 5.67; N, 9.85. Found: C, 50.66; H, 5.73; N, 9.92.%.
N-cyclohexyl-4-(4-([2,3,4-tri-O-acetyl-β-D-xylopyranosyl]oxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (9).
Yield: 79%; m.p. yellowish foam; IR (KBr) cm−1, ν: 3244 (NH), 2894 (CH-aliph.), 1756 (C=O), 1341, 1173 (SO2). 1H-NMR (500 MHz, CDCl3) δ/ppm: 0.84-0.87 (m, 2H, CH2), 1.21-1.25 (m, 6H, 3CH2), 1.45-1.50 (m, 2H, CH2), 2.03, 2.04, 2.05 (3s, 9H, CH3CO), 2.97-3.00 (m, 1H, N-CH), 3.39-3.44 (m, 1H, H-5′′), 4.11-4.18 (m, 1H, H-5′), 4.32 (d, 1H, J = 3.3 Hz, H-4′), 4.69-4.75 (m, 1H, H-2′), 4.95 (s, 2H, CH2), 5.01-5.03 (m, 1H, H-3′), 5.18 (d, 1H, J = 4.0 Hz, H-1′), 7.25 (br. s, 1H, NH), 7.93 (d, 2H, J = 7.7 Hz, Ar-H), 8.04 (d, 2H, J = 7.7 Hz, Ar-H), 8.13 (s, 1H, triazole). 13C NMR (126 MHz, CDCl3) δ 14.14, 20.79, 22.70, 26.61, 29.13, 29.52, 29.74, 31.90, 43.45, 55.71, 62.14, 68.93, 70.51, 70.95, 71.30, 75.37, 98.38, 100.27, 120.76, 129.12, 169.65, 170.09. Analysis calcd. for C26H34N4O10S (594.64): C, 52.52; H, 5.76; N, 9.42. Found: C, 52.43; H, 5.69; N, 9.50.%.
Synthesis of Deacetylated O-Glycosides (10-13)
This reaction was achieved by adding a saturated methanolic ammonia solution (15 mL) at 0 oC for 20 min. to the acetylated 1,2,3-triazole glycoside 6-9 which dissolved in methanol. then stirried at room temperature for 8 h., until the completion of the deacetylation process (TLC, petroleum ether–hexane, 2:1), By using the rotatory, the solvent was evaporated under reduced pressure at 40 oC to obtain a yellowish powder residue, which crystallized from ethanol to yield compound 10-13.
N-butyl-4-(4-([β-D-Galactopyranosyl]oxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (10).
Yield: 60%; m.p. 100-102°C; IR (KBr) cm−1, ν: 3455–3425 (OH), 3182 (NH), 2917 (CH-aliph.), 1331, 1159 (SO2). 1H-NMR (500 MHz, DMSO-D6) δ/ppm: 0.79 (t, 3H, CH3), 1.21-1.37 (m, 2H, CH2), 1.75-1.83 (m, 2H, CH2), 2.63-2.78 (m, 2H, CH2), 3.35-3.36 (m, 1H, H-6′′), 3.38-3.42 (m, 2H, H-6', H-5′), 3.46 (d, 1H, H-4′), 3.55-3.56 (m, 2H, H-3′, H-2′), 3.65-3.69 (m, 1H, 1OH), 3.78-3.82 (m, 1H, OH), 4.28 (s, 2H, CH2), 4.73-4.78 (m, 1H, OH), 4.92-4.94 (m, 1H, OH), 5.37 (d, 1H, H-1′), 7.73 (br. s, 1H, NH), 7.98-8.03 (m, 2H, Ar-H), 8.14 (d, 2H, Ar-H), 8.95 (s, 1H, triazole). 13C NMR (126 MHz, DMSO-D6) δ 13.47, 19.24, 31.14, 42.31, 60.72, 61.43, 68.36, 70.65, 73.48, 75.46, 103.01, 120.48, 122.86, 128.46, 139.05, 140.45, 145.67. Analysis calcd. for C19H28N4O8S (472.51): C, 48.30; H, 5.97; N, 11.86. Found: C, 48.21; H, 6.06; N, 11.91.%.
N-cyclohexyl-4-(4-([β-D-Galactopyranosyl]oxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (11).
Yield: 59%; m.p. 118-120°C; IR (KBr) cm−1, ν: 3451–3435 (OH), 3187 (NH), 2927 (CH-aliph.), 1333, 1158 (SO2). 1H-NMR (500 MHz, DMSO-D6) δ/ppm: 0.82-0.84 (m, 2H, CH2), 1.16-1.18 (m, 2H, CH2), 1.22-1.25 (m, 2H, CH2), 1.34-1.37 (m, 2H, CH2), 1.75-1.78 (m, 2H, CH2), 2.76-2.79 (m, 1H, N-CH), 3.45-3.46 (m, 1H, H-6′′), 3.54-3.57 (m, 1H, H-6'), 3.64-3.71 (m, 2H, H-5', H-4′), 3.76-3.81 (m, 1H, H-3′), 3.90-3.98 (m, 1H, H-2'), 4.27-4.28 (m, 1H, 1OH), 4.37-4.44 (m, 1H, OH), 4.62-4.68 (m, 1H, OH), 4.73-4.77 (m, 1H, OH), 4.91 (s, 2H, CH2), 5.37 (d, 1H, H-1′), 7.72 (br. s, 1H, NH), 7.98-8.02 (m, 2H, Ar-H), 8.12-8.16 (m, 2H, Ar-H), 8.94 (s, 1H, triazole). 13C NMR (126 MHz, DMSO-D6) δ 13.93, 22.10, 26.04, 28.94, 31.30, 42.60, 60.72, 61.43, 68.35, 70.65, 73.49, 75.46, 103.02, 120.37, 122.78, 128.46, 139.05, 140.48, 145.67. Analysis calcd. for C21H30N4O8S (498.55): C, 50.59; H, 6.07; N, 11.24. Found: C, 50.67; H, 5.99; N, 11.29.%.
N-butyl-4-(4-([β-D-Xylopyranosyl]oxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (12).
Yield: 55%; m.p. 150-152°C; IR (KBr) cm−1, ν: 3449–3439 (OH), 3183 (NH), 2932 (CH-aliph.), 1346, 1174 (SO2). 1H-NMR (500 MHz, DMSO-D6) δ/ppm: 0.79 (t, 3H, CH3), 1.21-1.27 (m, 2H, CH2), 1.33-1.38 (m, 2H, CH2), 2.77-2.80 (m, 2H, CH2), 3.01-3.03 (m, 1H, H-5′′), 3.08-3.14 (m, 2H, H-5', H-4′), 3.42-3.47 (m, 1H, H-3′), 3.74-3.77 (m, 1H, H-2′), 4.29-4.31 (m, 1H, 1OH), 4.70-4.72 (m, 1H, OH), 4.87-4.90 (m, 1H, OH), 4.99 (s, 2H, CH2), 5.06 (d, 1H, J = 5.1 Hz, H-1′), 7.72 (br. s, 1H, NH), 7.99 (d, 2H, J = 8.4 Hz, Ar-H), 8.14 (d, 2H, J = 8.3 Hz, Ar-H), 8.93 (s, 1H, triazole). 13C NMR (126 MHz, DMSO-D6) δ 13.43, 19.20, 31.10, 42.26, 61.23, 65.80, 69.61, 73.28, 76.56, 102.91, 120.47, 122.68, 128.41, 138.98, 140.42, 145.36. Analysis calcd. for C18H26N4O7S (442.49): C, 48.86; H, 5.92; N, 12.66. Found: C, 48.78; H, 6.00; N, 12.59.%.
N-cyclohexyl-4-(4-([β-D-Xylopyranosyl]oxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (13).
Yield: 58%; m.p. 160-162°C; IR (KBr) cm−1, ν: 3442–3436 (OH), 3185 (NH), 2922 (CH-aliph.), 1335, 1168 (SO2). 1H-NMR (500 MHz, DMSO-D6) δ/ppm: 0.81-0.84 (m, 2H, CH2), 1.16-1.24 (m, 6H, 3CH2), 1.34-1.38 (m, 2H, CH2), 2.76-2.79 (m, 1H, N-CH), 3.00-3.04 (m, 1H, H-5′′), 3.08-3.14 (m, 2H, H-5', H-4′), 3.29-3.32 (m, 1H, H-3′), 3.74-3.78 (m, 1H, H-2′), 4.29-4.31 (m, 1H, 1OH), 4.70-4.72 (m, 1H, OH), 4.87-4.90 (m, 1H, OH), 4.99 (s, 2H, CH2), 5.07 (d, 1H, J = 5.0 Hz, H-1′), 7.71 (br. s, 1H, NH), 7.99 (d, 2H, J = 8.5 Hz, Ar-H), 8.14 (d, 2H, J = 8.3 Hz, Ar-H), 8.93 (s, 1H, triazole). 13C NMR (126 MHz, DMSO-D6) δ 13.92, 22.10, 26.04, 28.94, 31.30, 42.59, 61.27, 65.83, 69.63, 73.29, 76.58, 102.95, 120.36, 122.59, 128.43, 139.00, 140.48, 145.40. Analysis calcd. for C20H28N4O7S (468.53): C, 51.27; H, 6.02; N, 11.96. Found: C, 51.19; H, 5.92; N, 12.03.%.
Cytotoxic activity
The newly synthesised sulfonamide-based derivatives 3, 4 and 6–13 were estimated for their cytotoxic activities in vitro using the MTT assay method, following the published procedure56–58, using human lung A-549, liver HepG-2, breast MCF-7, colorectal HCT-116 cancer cell lines, and human retinal pigment epithelial normal RPE-1 cell line. The supplemental file contained extra details.
Inhibitory assessment against VEGFR-2 and carbonic anhydrase isoforms hCA IX and hCA XII activities
The promising sulfonamide-based derivatives 4, 7 and 9 were assessed in vitro for their inhibitory effect against VEGFR-2, hCA IX, and hCA XII activities, in complying with the previously outlined technique, using sorafenib and SLC-0111 as references59, 60. There was further information in the supplemental file.
Detection of cell cycle analysis and apoptosis of compound 9
The examination of cell cycle analysis and apoptosis was explained61 with the use of flow cytometry. Benzenesulfonamide-1,2,3-triazole-glycosides 9 was applied to MCF-7 cells for a duration of 24 hours, and the cells were then incubated at 37 °C. Further details were included in the supplemental file.
The effect of compound 9 on MCF-7 cell levels of Bax, Bcl-2 and p53
Bax, Bcl-2 and p53 levels in MCF-7 cells were clarified utilising the previously reported approach for the promising benzenesulfonamide-1,2,3-triazole-glycosides 9.
Molecular docking simulation
The rationalization of biological discovers has become easier with the assistance of computational docking simulation. The promising benzenesulfonamide-1,2,3-triazole-glycosides 7 and 9 were docked inside the active sites of VEGFR-2 and the carbonic anhydrase isoforms hCA IX and hCA XII (PDB codes: 4ASD, 3IAI, and 1JG0, respectively)59,66,67 through the use of MOE-Dock (Molecular Operating Environment) software version 2014.090164,65. Full descriptions are available in the supplementary material.