Melting points were determined by open capillary method on STUART SMP3 instrument (BIBBY STERILIN, UK) and were uncorrected. IR spectra (KBr disc) were recorded on an Impact 410 FT-IR Spectrometer (Nicolet, USA). 1H and 13C NMR spectra were recorded on Bruker Avance Spectrometer AV500 (Bruker, Germany) at 500 MHz and 125 MHz, respectively, using CDCl3 as solvent and TMS as an internal standard; ESI-EI-HRMS and ESI/HR-mass spectra were recorded on Thermo Scientific Exactive Plus Orbitrap spectrometers (ThermoScientific, USA) in methanol using ESI method. The analytical thin-layer chromatography (TLC) was performed on silica gel 60WFS254 aluminum sheets (Merck, Germany) and was visualized with UV light. Chemical reagents in high purity were purchased from the Merck Chemical Company (in Viet Nam). All materials were of reagent grade for organic synthesis. N-(2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl)-thiosemicarbazide (3) was prepared from d-galactose monohydrate through its corresponding bromide derivative, followed its isocyanate according by using the similar procedure in previous literature [26]. All required for this work were prepared in in our lab. Substituted isatins 2b and 2c were prepared from isatin 1a by nitration and bromination reactions, respectively, according to reference [27]. Other substituted isatins 2d-2h were prepared from corresponding anilines according the literature procedures [28].
General methods for synthesis of substituted isatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazones (4a-4h)
A suspension mixture of N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazide 3 (1 mmol, 420 mg) and appropriate substituted isatin 1a,2b-2h (1 mmol) in 2 − 4 mL of absolute ethanol was added the appropriate ionic liquid triethylammonium acetate as catalyst (1 mmol%) under microwave irradiation for 30 − 40 min. Upon completion, monitored by TLC, the reaction mixture was cooled to room temperature; the color precipitate was filtered with suction. The crude product was recrystallized from 96% ethanol or mixture of toluene/96% ethanol (2:1 in volume) to afford the title compounds of substituted isatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl) thiosemicarbazones 4a-4h.
Isatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazone ( 4a )
White solid from isatin (1a, 1 mmol, 147 mg). MW irradiation time: 5 min. Yield: 405 mg (85%). M.p. 177 − 178°C (toluene-96% ethanol, 1:1 v/v); \({\left[\alpha \right]}_{D}^{25}\)+44.5 (c = 0.23, CHCl3). FT-IR (KBr) ν/cm−1: 3364, 3257 (NH), 1751 (C = O ester), 1621 (C = O lactam), 1373 (C = S), 1225, 1040 (COC ester); 1H NMR (DMSO-d6, 500 MHz): δ (ppm): 12.77 (s, 1H, NH thiosemicarbazone), 11.22 (s, 1H, NH isatin), 9.62 (d, J = 9.0 Hz, 1H, NH thiosemicarbazone), 7.66 (d, J = 7.5 Hz, 1H, H-4 isatin), 7.38 (t, J = 8.0 Hz, 1H, H-5 isatin), 7.12 (t, J = 7.5 Hz, 1H, H-6 isatin), 6.94 (d, J = 8.0 Hz, 1H, H-7 isatin), 5.94 (t, J = 9.0 Hz, 1H, H-1 galactopyranose), 5.42−5.41 (m, 1H, H-3 galactopyranose), 5.40−5.39 (m, 1H, H-2 galactopyranose), 4.38 (t, J = 6.5 Hz, 1H, H-5), 4.34−4.33 (m, 1H, H-4 galactopyranose), 4.07−4.05 (m, 2H, H-6a & H-H-6b galactopyranose), 2.15−1.94 (s, 12H, 4×CH3CO); 13C NMR (DMSO-d6, 125 MHz): δ (ppm): 179.1 (C = S), 170.0−169.4 (4×COCH3), 162.5 (C = O lactam), 142.7 (C-3 isatin), 133.3 (C-7 isatin), 131.7 (C-7 isatin), 122.3 (C-5 isatin), 121.5 (C-4 isatin), 119.7 (C-4 isatin), 111.1 (C-6 isatin), 82.2 (C-1 galactopyranose), 71.8 (C-3 galactopyranose), 70.7 (C-5 galactopyranose), 68.6 (C-2 galactopyranose), 67.5 (C-4 galactopyranose), 61.3 (C-6 galactopyranose), 20.5−20.3 (4×CH3CO); ESI-MS: C23H26N4O10S calcd. for M−H = 549.13 Da, found: m/z 549.23 (100%). Element. Anal.: C, 50.18; H, 4.76; N, 10.18%. Found: C, 50.45; H, 4.41; N, 10.45%.
5-Nitroisatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazone ( 4b )
Yellow solid from 5-nitroisatin (2b, 1 mmol, 192 mg). MW irradiation time: 6 min. Yield: 515 mg (85%). M.p. 166 − 168°C (toluene-96% ethanol, 1:1 v/v); \({\left[\alpha \right]}_{D}^{25}\)+45.4 (c = 0.22, CHCl3). FT-IR (KBr) ν/cm−1: 3620, 3305 (NH), 1714 (C = O ester), 1631 (C = O lactam), 1373 (C = S), 1223, 1043 (COC ester); 1H NMR (DMSO-d6, 500 MHz): δ (ppm): 12.59 (s, 1H, NH thiosemicarbazone), 11.85 (s, 1H, NH isatin), 10.05 (d, J = 9.0 Hz, 1H, NH thiosemicarbazone), 8.68 (d, J = 7.5 Hz, 1H, H-4 isatin), 8.28 (dd, J = 8.5, 2.5 Hz, 1H, H-7 isatin), 7.12 (t, J = 8.5 Hz, 1H, H-6 isatin), 6.01 (t, J = 8.5 Hz, 1H, H-1 galactopyranose), 5.43−5.42 (m, 1H, H-3 galactopyranose), 5.41 (t, J = 8.5 Hz, 1H, H-2 galactopyranose), 4.41 (t, J = 6.5 Hz, 1H, H-5), 4.34−4.33 (m, 1H, H-4 galactopyranose), 4.07−4.05 (m, 2H, H-6a & H-H-6b galactopyranose), 2.16−1.94 (s, 12H, 4×CH3CO); 13C NMR (DMSO-d6, 125 MHz): δ (ppm): 179.2 (C = S), 170.0−169.4 (4×COCH3), 162.9 (C = O lactam), 147.7 (C-3 isatin), 142.8 (C-7 isatin), 131.3 (C-7 isatin), 127.3 (C-5 isatin), 120.8 (C-4 isatin), 116.9 (C-4 isatin), 111.3 (C-6 isatin), 82.5 (C-1 galactopyranose), 72.0 (C-3 galactopyranose), 70.8 (C-5 galactopyranose), 68.6 (C-2 galactopyranose), 67.5 (C-4 galactopyranose), 61.4 (C-6 galactopyranose), 20.5−20.3 (4×CH3CO); ESI-MS: C23H25N5O12S, calcd. for M−H = 594.11 Da, found: m/z 594.12 (100%, M − H]+). Element. Anal.: C, 46.39; H, 4.23; N, 11.76%. Fond: C, 46.12; H, 4.51; N, 11.47%.
5,7-Dibromoisatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazone ( 4c )
Orange solid from 5,7-dibromoisatin (2c, 1 mmol, 305 mg). MW irradiation time: 7 min. Yield: 515 mg (73%). M.p. 210 − 212°C (toluene-96% ethanol, 1:1 v/v); \({\left[\alpha \right]}_{D}^{25}\)+38.6 (c = 0.20, CHCl3). FT-IR (KBr) ν/cm−1: 3468, 3213 (NH), 1743 (C = O ester), 1692 (C = O lactam), 1611 (C = N), 1373 (C = S), 1229, 1050 (COC ester); 1H NMR (DMSO-d6, 500 MHz): δ (ppm): 12.56 (s, 1H, NH thiosemicarbazone), 11.67 (s, 1H, NH isatin), 9.68 (d, J = 8.5 Hz, NH thiosemicarbazone), 7.92 (s, 1H, H-4 isatin), 6.81 (s, 1H, H-6 isatin), 5.95 (t, J = 9.0 Hz, 1H, H-1 galactopyranose), 5.39 (t, J = 9.0 Hz, 1H, H-2 galactopyranose), 5.33−5.32 (m, 1H, H-3 galactopyranose), 4.08−4.07 (m, 2H, H-6a & H-H-6b galactopyranose), 4.04 (d, J = 11.5 Hz, 1H, H-5), 3.83−3.82 (m, 1H, H-4 galactopyranose), 2.11−1.91 (s, 12H, 4×CH3CO); 13C NMR (DMSO-d6, 125 MHz): δ (ppm): 179.5 (C = S), 170.7−169.8 (4×COCH3), 162.8 (C = O lactam), 141.6 (C-3 isatin), 135.6 (C-7 isatin), 132.2 (C-7 isatin), 124.0 (C-5 isatin), 123.1 (C-4 isatin), 114.9 (C-4 isatin), 104.8 (C-6 isatin), 82.2 (C-1 galactopyranose), 76.4 (C-3 galactopyranose), 73.4 (C-5 galactopyranose), 70.9 (C-2 galactopyranose), 69.3 (C-4 galactopyranose), 67.6 (C-6 galactopyranose), 21.2−20.9 (4×CH3CO); ESI-MS: C23H2479Br2N4O10S/C23H2479Br81BrN4O10S/C23H2481Br2N5O10S, calcd. for M/M + 2/M+4 = 705.96/707.96/709.95 Da, found: m/z 705.12/707.00/709.03 [M + 4]+/[M + 2]+/[M]+. Element. Anal.: C, 39.00; H, 3.42; N, 7.91%. Found: C, 39.29; H, 3.14; N, 7.63%.
5-Chloroisatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazone ( 4d )
Pale-yellow solid from 5-chloroisatin (2d, 1 mmol, 181 mg). MW irradiation time: 6 min. Yield: 510 mg (87%). M.p. 202 − 203°C (toluene-96% ethanol, 1:1 v/v); \({\left[\alpha \right]}_{D}^{25}\)+45.3 (c = 0.22, CHCl3). FT-IR (KBr) ν/cm−1: 3558, 3259 (NH), 1709 (C = O ester), 1619 (C = N), 1373 (C = S), 1227, 1043 (COC ester); 1H NMR (DMSO-d6, 500 MHz): δ (ppm): 12.64 (s, 1H, NH thiosemicarbazone), 11.32 (s, 1H, NH isatin), 9.76 (d, J = 9.0 Hz, 1H, NH thiosemicarbazone), 7.88 (d, J = 1.5 Hz, 1H, H-4 isatin), 7.41 (dd, J = 8.5, 2.5 Hz, 1H, H-7 isatin), 6.95 (d, J = 8.5 Hz, 1H, H-6 isatin), 5.98 (t, J = 9.0 Hz, 1H, H-1 galactopyranose), 5.41−5.40 (m, 1H, H-3 galactopyranose), 5.35 (t, J = 9.0 Hz, 1H, H-2 galactopyranose), 5.34−5.33 (m, 1H, H-4 galactopyranose), 4.41 (t, J = 6.0 Hz, 1H, H-5), 4.08−4.06 (m, 2H, H-6a & H-H-6b galactopyranose), 2.17−1.95 (s, 12H, 4×CH3CO); 13C NMR (DMSO-d6, 125 MHz): δ (ppm): 179.0 (C = S), 169.9−169.3 (4×COCH3), 162.3 (C = O lactam), 141.3 (C-3 isatin), 132.0 (C-7 isatin), 130.9 (C-7 isatin), 126.6 (C-5 isatin), 121.7 (C-4 isatin), 121.1 (C-4 isatin), 112.6 (C-6 isatin), 82.3 (C-1 galactopyranose), 72.0 (C-3 galactopyranose), 70.7 (C-5 galactopyranose), 68.6 (C-2 galactopyranose), 67.5 (C-4 galactopyranose), 61.3 (C-6 galactopyranose), 20.4−20.2 (4×CH3CO) ESI-MS: C23H2535ClN4O10S/C23H2537ClN4O10S, calcd. for M/M + 2 = 584.10/586.09 Da, found: m/z 609.06/607.07 [M + 2+Na]+/[M + Na]+; 585.00/587.02 (100%/30%) ([M + H]+/[M + 2+H]+). Element. Anal.: C, 47.22; H, 4.31; N, 9.58%. Found: C, 47.49; H, 4.56; N, 9.25%.
5-Bromoisatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazone ( 4e )
Orange solid from 5-bromoisatin (2e, 1 mmol, 226 mg). MW irradiation time: 6 min. Yield: 460 mg (73%). M.p. 157 − 158°C (toluene-96% ethanol, 1:1 v/v); \({\left[\alpha \right]}_{D}^{25}\)+36.2 (c = 0.22, CHCl3). FT-IR (KBr) ν/cm−1: 3348, 3241 (NH), 1752 (C = O ester), 1702 (C = O lactam), 1615 (C = N), 1373 (C = S), 1228, 1049 (COC ester); 1H NMR (DMSO-d6, 500 MHz): δ (ppm): 12.63 (s, 1H, NH thiosemicarbazone), 11.34 (s, 1H, NH isatin), 9.79 (d, J = 9.0 Hz, 1H, NH thiosemicarbazone), 7.99 (s), Hz, 1H, H-4 isatin), 7.53 (dd, J = 8.5, 2.0 Hz, 1H, H-7 isatin), 6.89 (d, J = 8.5 Hz, 1H, H-6 isatin), 5.98 (t, J = 8.5 Hz, 1H, H-1 galactopyranose), 5.38−5.37 (m, 1H, H-3 galactopyranose), 5.36 (t, J = 8.5 Hz, 1H, H-2 galactopyranose), 5.34−5.33 (m, 1H, H-4 galactopyranose), 4.39 (t, J = 6.5 Hz, 1H, H-5), 4.07−4.06 (m, 2H, H-6a & H-H-6b galactopyranose), 2.16−1.94 (s, 12H, 4×CH3CO); 13C NMR (DMSO-d6, 125 MHz): δ (ppm): 179.1 (C = S), 170.0−169.4 (4×COCH3), 162.2 (C = O lactam), 141.7 (C-3 isatin), 133.7 (C-7 isatin), 131.9 (C-7 isatin), 123.8 (C-5 isatin), 122.1 (C-4 isatin), 114.2 (C-4 isatin), 113.1 (C-6 isatin), 82.4 (C-1 galactopyranose), 72.0 (C-3 galactopyranose), 70.7 (C-5 galactopyranose), 68.6 (C-2 galactopyranose), 67.5 (C-4 galactopyranose), 61.3 (C-6 galactopyranose), 20.5−20.3 (4×CH3CO); ESI-MS (+ MS): C23H2579BrN4O10S/C23H2581BrN4O10S, calcd. for M + H/M + 2+H = 628.05/630.05 Da, found: m/z 629.11/627.13 ([M + 2 + H]+/[M + H]+). Element. Anal.: C, 43.89; H, 4.00; N, 8.90%. Found: C, 43.61; H, 4.29; N, 8.65%.
5-Methylisatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazone ( 4f )
Pale-yellow solid from 5-methylisatin (2f, 1 mmol, 161 mg). MW irradiation time: 6 min. Yield: 435 mg (77%). M.p. 215 − 216°C (toluene-96% ethanol, 1:1 v/v); \({\left[\alpha \right]}_{D}^{25}\)+54.1 (c = 0.27, CHCl3). FT-IR (KBr) ν/cm−1: 3291, 3484 (NH), 1740 (C = O ester), 1700 (C = O lactam), 1620 (C = N), 1373 (C = S), 1224, 1045 (COC ester); 1H NMR (DMSO-d6, 500 MHz): δ (ppm): 12.80 (s, 1H, NH thiosemicarbazone), 11.12 (s, 1H, NH isatin), 9.57 (d, J = 9.0 Hz, 1H, NH thiosemicarbazone), 7.55 (s, 1H, H-4 isatin), 7.16 (d, J = 8.0 Hz, 1H, H-7 isatin), 6.79 (d, J = 8.0 Hz, 1H, H-6 isatin), 5.97 (t, J = 9.0 Hz, 1H, H-1 galactopyranose), 5.42−5.41 (m, 1H, H-2 galactopyranose), 5.38−5.37 (m, 1H, H-3 galactopyranose), 5.34 (d, J = 2.5 Hz, 1H, H-4 galactopyranose), 4.38 (t, J = 6.25 Hz, 1H, H-6a), 4.07 (d, J = 6.5 Hz, 1H, H-5), 4.06 (d, J = 6.5 Hz, 1H, H-H-6b galactopyranose), 2.16−1.94 (s, 12H, 4×CH3CO), 2.31 (s, 3H, 5 isatin-CH3); 13C NMR (DMSO-d6, 125 MHz): δ (ppm): 179.1 (C = S), 170.0−169.4 (4×COCH3), 162.6 (C = O lactam), 140.5 (C-5 isatin), 133.4 (C-7 isatin), 132.1 (C-3 isatin), 131.5 (C-6 isatin), 121.7 (C-4 isatin), 119.7 (C-7 isatin), 110.9 (C-4 isatin), 82.3 (C-1 galactopyranose), 71.9 (C-3 galactopyranose), 70.7 (C-5 galactopyranose), 68.7 (C-2 galactopyranose), 67.5 (C-4 galactopyranose), 61.3 (C-6 galactopyranose), 20.6−20.3 (4×CH3CO), 20.5 (5 isatin-CH3); ESI-MS: C24H28N4O10S, calcd. for M + H = 565.57 Da, found: m/z 587.00 ([M + Na]+), 565.02 ([M + H]+). Element. Anal.: C, 51.06; H, 5.00; N, 9.92%. Found: C, 51.37; H, 5.28; N, 9.65%.
5-Isopropylisatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazone ( 4g )
Pale-yellow solid from 5-isopropyllisatin (2g, 1 mmol, 189 mg). MW irradiation time: 7 min. Yield: 400 mg (65%). M.p. 151 − 152°C (toluene-96% ethanol, 1:1 v/v); \({\left[\alpha \right]}_{D}^{25}\)+59.2 (c = 0.26, CHCl3). FT-IR (KBr) ν/cm−1: 3600, 3343 (NH), 1700 (C = O ester), 1629 (C = N), 1373 (C = S), 1221, 1084 (COC ester); 1H NMR (DMSO-d6, 500 MHz): δ (ppm): 12.84 (s, 1H, NH thiosemicarbazone), 11.17 (s, 1H, NH isatin), 9.55 (d, J = 8.5 Hz, 1H, NH thiosemicarbazone), 7.61 (s) ( Hz, 1H, H-4 isatin), 7.41 (dd, J = 8.0, 1.5 Hz, 1H, H-7 isatin), 6.85 (d, J = 8.0 Hz, 1H, H-6 isatin), 5.94 (t, J = 9.0 Hz, 1H, H-1 galactopyranose), 5.43−5.42 (m, 1H, H-3 galactopyranose), 5.41 (t, J = 9.0 Hz, 1H, H-2 galactopyranose), 5.35−5.34 (m, 1H, H-4 galactopyranose), 4.38 (t, J = 6.0 Hz, 1H, H-5), 4.07 (d, J = 6.0 Hz, 1H, H-6a), 4.06−4.05 (m, 1H, H-H-6b galactopyranose), 2.90 [septet, J = 7Hz, 1H, 5 isatin-CH(CH3)2], 2.16−1.95 (s, 12H, 4×CH3CO), 1.22 [d, J = 7Hz, 6H, 5 isatin-CH(CH3)2]; 13C NMR (DMSO-d6, 125 MHz): δ (ppm): 179.0 (C = S), 170.0−169.3 (4×COCH3), 162.7 (C = O lactam), 142.8 (C-3 isatin), 140.8 (C-7 isatin), 133.6 (C-7 isatin), 129.7 (C-5 isatin), 119.6 (C-4 isatin), 119.2 (C-4 isatin), 111.0 (C-6 isatin), 82.3 (C-1 galactopyranose), 71.9 (C-3 galactopyranose), 70.7 (C-5 galactopyranose), 68.6 (C-2 galactopyranose), 67.6 (C-4 galactopyranose), 61.3 (C-6 galactopyranose), 33.2 [5 isatin-CH(CH3)2], 24.0−20.4 (4×CH3CO), 20.3 [5 isatin-CH(CH3)2]; ESI-MS: C26H32N4O10S, calcd. for M−H = 591.17 Da, found: m/z 591.11 ([M − H]+). Element. Anal.: C, 52.70; H, 5.44; N, 9.45%. Found: C, 52.63; H, 5.16; N, 9.18%.
7-Methylisatin N-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)thiosemicarbazone ( 4h )
Pale-yellow solid from 7-methylisatin (2h, 1 mmol, 189 mg). MW irradiation time: 7 min. Yield: 370 mg (66%). M.p. 226 − 227°C (toluene-96% ethanol, 1:1 v/v); \({\left[\alpha \right]}_{D}^{25}\)+57.2 (c = 0.27, CHCl3). FT-IR (KBr) ν/cm−1: 3489, 3284 (NH), 1745 (C = O ester), 1702 (C = O lactam), 1627 (C = N), 1373 (C = S), 1223, 1055 (COC ester); 1H NMR (DMSO-d6, 500 MHz): δ (ppm): 12.81 (s, 1H, NH thiosemicarbazone), 11.29 (s, 1H, NH isatin), 9.58 (d, J = 9.0 Hz, 1H, NH thiosemicarbazone), 7.59 (d, J = 7.5 Hz, 1H, H-4 isatin), 7.20 (d, J = 7.5 Hz, 1H, H-6 isatin), 7.03 (t, J = 7.5 Hz, 1H, H-5 isatin), 5.94 (t, J = 8.5 Hz, 1H, H-1 galactopyranose), 5.42−5.41 (m, 1H, H-3 galactopyranose), 5.38−5.37 (m, 1H, H-2 galactopyranose), 5.34−5.33 (m, 1H, H-4 galactopyranose), 4.38 (t, J = 6.5 Hz, 1H, H-5), 4.06−4.04 (m, 2H, H-6a & H-H-6b galactopyranose), 2.22 (s, 3H, 7 isatin-CH3), 2.15−1.94 (s, 12H, 4×CH3CO); 13C NMR (DMSO-d6, 125 MHz): δ (ppm): 179.1 (C = S), 170.0−169.4 (4×COCH3), 163.0 (C = O lactam), 141.3 (C-3 isatin), 133.7 (C-7 isatin), 132.9 (C-7 isatin), 122.3 (C-5 isatin), 120.6 (C-4 isatin), 119.4 (C-4 isatin), 118.9 (C-6 isatin), 82.2 (C-1 galactopyranose), 71.8 (C-3 galactopyranose), 70.7 (C-5 galactopyranose), 68.6 (C-2 galactopyranose), 67.5 (C-4 galactopyranose), 61.3 (C-6 galactopyranose), 20.5−20.3 (4×CH3CO), 15.8 (7 isatin-CH3); ESI-MS: C24H28N4O10S, calcd. for M + H = 565.57 Da, found: m/z 587.45 ([M + Na]+), 565.44 ([M + H]+). Element. Anal.: C, 51.06; H, 5.00; N, 9.92%. Found: C, 51.35; H, 5.27; N, 9.68%.
Commercially available assay kit was used for the determination of Topo IV decatenation IC50 values. Details of the materials, S. aureus Gyrase Topo IV assay buffer, dilution buffer,
enzyme (stored at − 80°C) and substrate kDNA (supplied at 100 ng/µL, stored at − 20°C or below). Serial dilutions of each compound were prepared using DMSO as solvent. IC50 values were determined at a final concentration of 100, 10, 1, and 0.1 µg/ml. Novobiocin and Ciprofloxacin were used as reference standards [40].