All of chemicals used in this work were obtained from Merck and used without additional purification. Compounds 2 were prepared from Meldrum’s acid and the corresponding Oxo-compounds by known methods [6] and identified by 1H NMR spectroscopy. Electrothermal 9100 apparatus was used for measuring the melting points. IR spectra (KBr) were obtained on Shimadzu IR-460 spectrometer. 1H NMR and 13C NMR spectra were obtained using the BRUKER DRX-500 AVANCE spectrometer at 500 and 126 MHz, respectively, in CDCl3 employing TMS as internal standard. Mass spectra were recorded on a Finnigan-MAT-8430EI-MS mass spectrometer, at an ionization potential of 70 eV, in m/z (rel. %). Elemental analyses for C, H and N were performed using a Heraeus CHN-O-Rapid analyzer.
A typical procedure for the synthesis bis-spiro compounds 4
A magnetically stirred solution of isatin 1 (2 mmol) and secondary amine 2 (2 mmol) in MeCN (5 mL) was stirred for 30 min, then alkylidene Meldrum’s acid 3 (2 mmol) was added. Then, the reaction mixture was stirred for 3 h. After completion of reaction (monitored by TLC), the solvent was evaporated and the residue was purified by flash column chromatography (Merck silica gel 230-400 mesh) using n-hexane-EtOAc (7:1) as an eluent to give pure title compound 4.
1',2'',2'',3',3'-Pentamethyldispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4a)
Yellow powder, mp 165-167 °C, 66 mg, yield 92%. IR (KBr) (νmax/cm-1): 3439, 1732, 1624, 1456 and 1230 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.25(3 H, s, Me), 1.39 (3 H, s, Me), 1.62 (3 H, s, Me), 1.78 (3 H, s, Me), 2.58 (3 H, s, Me), 3.72 (1 H, d, 2J = 3.9 Hz, CH ), 3.93 (1 H, d, 2J = 3.9 Hz, CH),7.42 (1 H, t, 3JHH = 7.6Hz, CH ), 7.72 (1 H, t, 3JHH = 7.6 Hz,CH ), 7.90 (1 H, d, 3J = 7.6 Hz, CH ), 8.05 (1H, s, NH ), 8.19 (1H, d, 3J = 7.6 Hz,CH ) ppm. 13C NMR (125.7 MHz, CDCl3):δ 22.9 (Me), 25.2 (Me), 27.9 (Me), 30.1 (Me), 40.4 (Me), 44.3 (C), 58.2 (CH2), 65.1 (C), 75.8 (C), 107.2 (C), 113.8 (CH), 120.8 (CH), 124.9 (C), 131.3 (CH), 133.8 (CH), 146.8 (C), 162.0 (C=O), 163.2 (C=O), 178.2 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 358 (M+, 10), 147 (48), 58 (100). Anal. Calcd for C19H22N2O5 (358.39): C, 63.67; H, 6.19; N, 7.82. Found: C, 63.85; H, 6.22; N, 7.86%.
1'-Ethyl-2'',2'',3',3',5'-pentamethyldispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4b)
Yellow powder, mp 172-174 °C, 67 mg, yield 87%. IR (KBr) (νmax/cm-1): 34.27, 1732, 1626, 1585 and 1227 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.12 (3 H, t, 3JHH = 7.6 Hz, Me), 1.28 (3 H, s, Me), 1.44 (3 H, s, Me), 1.57 (3 H, d, 3JHH = 7.4 Hz, Me), 1.69 (3 H, s, Me), 1.85 (3 H, s, Me ), 2.58- 2.72 (1H, m, CH), 2.98- 3.12 (1H, m, CH), 3.68 (1H, q, 3JHH = 7.4 Hz, CH), 7.12 (1H, d, 3JHH = 7.7 Hz, CH), 7.42 (1H, t, 3JHH = 7.7 Hz, CH), 7.58 (1H, t, 3J = 7.6 Hz, CH), 7.82 ( 1H, d, 3J = 7.6 Hz, CH), 8.38 ( 1H, s, NH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 15.8 (Me), 16.9 (Me), 22.9 (Me), 25.2 (Me), 27.1 (Me), 29.2 (Me), 42.6 (C), 45.2 (Me), 55.4 (CH2), 67.8 (C), 73.2 (C), 105.2 (C), 112.8 (CH), 118.9 (CH), 124.2 (C), 131.2 (CH), 132.8 (CH), 145.2 (C), 160.9 (C=O), 161.8 (C=O), 177.6 (C=O) ppm. ( MS (EI, 70 eV): m/z (%) = 386 (M+, 10), 121 (100), 76 (54). Anal. Calcd for C21H26 N2O5 (386.44): C, 65.27; H, 6.78; N, 7.25. Found: C, 65.43; H, 6.76; N, 7.28%.
1'-Isopropyl-2'',2'',3',3',5',5'-hexamethyldispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4c)
Yellow powder, mp 179- 181 °C, 70 mg, yield 85%. IR (KBr) (νmax/cm-1): 3446, 1719, 1628, 1597 and 1257 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.12 (3 H, s, Me), 1.28 (6 H, d, 3JHH = 7.4 Hz, 2 Me), 1.38 (3 H, s, Me), 1.52 (3 H, s, Me), 1.68 (3 H, s, Me), 1.78 (3 H, s, Me), 1.89 (3 H, s, Me), 3.52- 3.68 (1 H, m, CH), 6.88 (1 H, t, 3JHH = 7.5 Hz, CH), 7.02 (1 H, t, 3JHH = 7.5 Hz, CH), 7.23 (1 H, d, 3J= 7.5 Hz, CH), 7.42 (1 H, s, NH), 7.62 (1 H, d, 3J = 7.5 Hz, CH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 21.9 (2 Me), 22.9 (Me), 23.8 (Me), 24.2 (Me), 25.9 (Me), 28.2 (Me), 30.0 (Me), 42.2 (C), 48.2 (CH), 70.1 (C), 71.2 (C), 72.8 (C), 105.2 (C), 113.2 (CH), 118.8 (CH), 122.8 (C), 130.0 (CH), 131.2 (CH), 145.0 (C), 162.2 (C=O), 164.2 (C=O), 176.2 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 414 (M+, 15), 390 (68), 55 (100). Anal. Calcd for C23H30N2O5 (414.49): C, 66.65; H, 7.30; N, 6.76. Found: C, 66.86; H, 7.33; N, 6.78%.
2',2',2'',2''-Tetramethyl-5',6',7',7a'-tetrahydro-2'H-dispiro[indoline-3,3'-pyrrolizine-1',5''-[1,3]dioxane]-2,4'',6''-trione (4d)
Yellow powder, mp 187- 189 °C, 64 mg, yield 83%. IR (KBr) (νmax/cm-1): 3450, 1735, 1625, 1508 and 1265 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.27 (3 H, s, Me), 1.41 (3 H, s, Me), 1.58 (3 H, s, Me), 1.72 (3 H, s, Me), 1.79- 1.92 (2 H, m, CH2), 2.22- 2.35 (2 H, m, CH2), 2.82- 2.93 (2 H, m, NCH2 ), 3.52- 3.69 (1 H, m, CH), 7.44 (1 H, d, 3JHH = 7.6 Hz, CH), 7.62 (1 H, t, 3JHH = 7.7 Hz, CH), 7.84 (1 H, t, 3J= 7.6 Hz, CH), 7.98 (1 H, d, 3J= 7.6 Hz, CH), 8.22 (1 H, s, NH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 21.2 (Me), 22.8 (Me), 24.8 (Me), 25.2 (Me), 26.8 (Me), 28.3 (CH2), 29.2(CH2) ,48.3 (CH2), 54.2 (C), 59.3 (C), 63.8 (CH), 75.2 (C), 107.9 (C), 113.2 (CH), 117.5 (CH), 118.9 (C), 120.0 (CH), 123.8 (CH) 134.3 (C), 161.5(C=O), 164.8 (C=O), 176.2 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 384 (M+, 15), 147 (86), 58 (100). Anal. Calcd for C21H24N2O5 (384.43): C, 65.61; H, 6.29; N, 7.29. Found: C, 65.88; H, 6.32; N, 7.33% .
2',2',2'',2''-Tetramethyl-6',7',8',8a'-tetrahydro-2'H,5'H-dispiro[indoline-3,3'-indolizine-1',5''-[1,3]dioxane]-2,4'',6''-trione (4e)
Yellow powder, mp 201-203 °C, 66 mg, yield 83%. IR (KBr) (νmax/cm-1): 3452, 1729, 1628, 1585 and 1266 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.08 (3 H, s, Me), 1.27 (3 H, s, Me), 1.41 (3 H, s, Me), 1.47- 1.62 (2 H, m, CH2), 1.82 (3 H, s, Me), 1.92- 2.03 (2 H, m, CH2), 2.19- 2.38 (2 H, m, CH2), 2.82-2.97 (2 H, m, NCH2), 3.55- 3.68 (1 H, m, CH), 7.54(1 H, d, 3JHH = 7.6 Hz, CH), 7.72 (1 H, t, 3JHH = 7.6 Hz, CH), 7.87(1 H, t, 3J= 7.6 Hz, CH), 7.98(1 H,d, 3J= 7.6 Hz, CH), 8.15( 1H, s, NH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 21.9 (Me), 22.9 (Me), 23.8 (Me), 24.8 (CH2), 26.2 (Me), 27.8 (CH2), 29.8 (CH2), 42.2 (C), 45.8 (CH2), 59.2 (CH), 66.9 (C), 71.8 (C), 105.2 (C), 114.2 (CH), 119.2 (CH), 123.8 (C),130.0 (CH), 131.9 (CH), 143.2 (C), 161.5 (C=O), 163.5 (C=O), 176.2 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 398 (M+, 15), 147 (86), 43 (100). Anal. Calcd for C22H26 N2 O5 (398.45): C, 66.32; H, 6.58; N, 7.03. Found: C, 66.53; H, 6.61; N, 7.05%.
1',2'',2'',3'-Tetramethyldispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4f)
Yellow powder, mp 154- 156 °C, 65 mg, yield 95%. IR (KBr) (νmax/cm-1): 3448, 1716, 1663, 1547, 1473 and 1228 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.38 (3 H, d, 3JHH = 7.4 Hz, Me), 1.53 (3 H, s, Me), 1.69 (3 H, s, Me), 2.32 (3 H, s, Me), 3.48 (1 H, d, 2J = 4.5 Hz, CH), 3.67 (1 H, d, 2J = 4. 5 Hz, CH), 4.49 (1 H, q , 3JHH = 7.4 Hz, CH), 7.46 (1 H, t , 3JHH = 7.6 Hz, CH), 7.72 (1 H, t , 3JHH = 7.6 Hz, CH), 7.89 (1 H, d, 3J = 7.6 Hz, CH), 8.05 (1 H, s, NH), 8.20 (1 H,d, , 3J = 7.6 Hz, CH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 13.8 (Me), 27.9 (Me), 29.3 (Me), 39.1 (Me), 40.0 (CH), 57.8 (CH2), 61.9 (C), 73.0 (C), 106.8 (C), 114.2 (CH), 120.0 (CH), 124.8 (C), 130.1 (CH), 132.2 (CH), 149.2 (C), 159.7 (C=O), 161.0 (C=O), 177.3 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 344 (M+, 10), 55 (100), 45 (84). Anal. Calcd for C18H20N2O5 (344.36): C, 62.78; H, 5.85; N, 8.13. Found: C, 62.96; H, 5.88; N, 8.16%.
1',2'',2''-Trimethyl-3'-phenyldispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4g)
Yellow powder, mp 182-184 °C, 75 mg, yield 92%. IR (KBr) (νmax/cm-1): 3422, 1710, 1635, 1460, 1213 and 1092 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.42 (3 H, s, Me), 1.70 (3 H, s, Me), 2.42 (3 H, s, Me), 3.79 (1 H, d, 2J = 4. 2 Hz, CH), 3.94 (1 H, d, 2J = 4. 2 Hz, CH), 5.28 (1 H, s, CH), 6.94 (2 H, d, 3JHH = 7.8 Hz, 2 CH), 7.02 (1 H, d, 3JHH = 7.6 Hz, CH), 7.04 (2 H, t, 3J = 7.8 Hz, 2 CH), 7.16 (1 H, t, 3J = 7.8 Hz, CH), 7.34 (1 H, d, 3J = 7.6 Hz, CH), 7.36 (2 H, t, 3J = 7.6 Hz, 2 CH), 7.68 (1 H, s, NH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 27.2 (Me), 29.8 (Me), 38.2 (Me), 48.5 (CH), 61.2 (CH2), 63.9 (C), 73.5 (C), 106.8 (C), 113.4 (CH), 119.2 (CH), 123.2 (CH), 128.2 (2 CH), 130.1 (2 CH), 131.8 (CH), 132.5 (CH), 33.8 (C), 145.0 (C), 149.5 (C), 159.9 (C=O), 161.8 (C=O), 176.8 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 406 (M+, 10), 77 (100), 58 (48). Anal. Calcd for C23H22 N2 O5 (406.43): C, 67.97; H, 5.46; N, 6.89. Found: C, 68.12; H, 5.44; N, 6.93%.
2'-(4-Methoxyphenyl)-2'',2''-dimethyl-5',6',7',7a'-tetrahydro-2'H-dispiro[indoline-3,3'-pyrrolizine-1',5''-[1,3]dioxane]-2,4'',6''-trione (4h)
Yellow powder, mp 216-218 °C, 75 mg, yield 90%. IR (KBr) (νmax/cm-1): 3442, 17.27, 1630, 1444 and 1113 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.22 (3 H, s, Me), 1.59 (3 H, s, Me), 1.76- 1.92 (2 H, m, CH2), 2.24- 2.44 (2 H, m, CH2), 2.82- 2.94 (2 H, m, NCH2), 3.48- 3.59 (1 H, m, CH), 3.87 (3 H, s, MeO), 5.32 (1 H, s, CH), 6.99 (2 H, d, 3JHH = 7.8 Hz, 2 CH), 6.98 (1 H,d, 3JHH = 7.6 Hz, CH), 7.09 (1 H, t, 3J = 7.6 Hz, CH), 7.12 (2 H, d, 3J = 7.8 Hz, 2CH), 7.24(1 H, d, 3J = 7.6 Hz, CH), 7.39(1 H, t, 3J = 7.6 Hz, CH), 7.75(1 H, s, NH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 23.5 (CH2), 28.2 (Me), 29.3 (CH2), 30.1 (Me), 48.3 (CH), 52.2 (CH2), 54.8 (MeO), 65.2 (C), 66.8 (CH), 71.9 (C), 63.8 (CH), 104.5 (C), 113.2 (2CH), 113.9 (CH), 120.0 (CH), 124.2 (C), 125.8 (C), 131.0 (CH), 131.9 (2 CH), 132.2 (CH), 133.9 (C), 144.2 (C), 158.0 (C), 160.0 (C=O), 163.2 (C=O), 177.8 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 462 (M+, 15), 390 (74), 55 (100). Anal. Calcd for C26H26N2O6 (462.49): C, 67.52; H, 5.67; N, 6.06. Found: C, 67.43; H,5.68; N, 6.09%.
2'',2'',3',3',5'-Pentamethyl-1'-(4-nitrophenyl)dispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4i)
Orange powder, mp 225- 227 °C, 72 mg, yield 75%. IR (KBr) (νmax/cm-1): 3442, 1710, 1628, 1439 and 1219 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.34 (3 H, s, Me), 1.46 (3 H, s, Me), 1.59 (3 H, s, Me), 1.64 (3 H, s, Me), 1.92 (3 H, d, 3JHH = 7.4 Hz, Me), 3.95 (1 H, q, 3JHH = 7.4 Hz, CH), 6.94 (2 H, d, 3JHH = 7.8 Hz, 2 CH), 7.02 (1 H, d, 3JHH = 7.6 Hz, CH), 7.12 (1 H, t, 3J = 7.6 Hz, CH), 7.34 (1 H, t, 3J = 7.6 Hz, CH), 7.48 (1 H, d, 3J = 7.6 Hz, CH), 7.98 (1 H, s, NH), 8.17 (2H, d, 3J = 7.8 Hz, 2 CH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 11.8 (Me), 23.8 (Me), 25.2 (Me), 28.2 (Me), 30.3 (Me), 42.8 (C), 57.8 (CH), 69.2 (C), 71.0 (C), 104.5 (C), 114.2 (2 CH), 115.8 (CH), 119.5 (CH), 124.5 (C), 126.2 (2 CH), 131.0 (CH), 132.6 (CH), 137.8 (C), 145.8 (C), 149.8 (C), 161.2 (C=O), 163.0 (C=O), 179.0 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 479 (M+, 20), 147 (52), 58 (100). Anal. Calcd for C25H25 N3 O7 (479.48): C, 62.62; H, 5.26; N, 8.76. Found: C, 62.45; H, 5.28; N, 8.79%.
2'',2'',3'-Trimethyl-1'-(4-nitrophenyl)dispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4j)
Orange powder, mp 219- 221 °C, 70 mg, yield 78%. IR (KBr) (νmax/cm-1): 3439, 1722, 1661, 1440 and 1123 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.48 (3 H, d, 3JHH = 7.4 Hz, Me), 1.60 (3 H, s, Me), 1.89 (3 H, s, Me), 3.32 (1H, d, 2JHH= 3.8 Hz, CH), 3.62 (1 H, d, 2JHH = 3.8 Hz, CH), 4.28 (1 H, q, 3JHH = 7.4 Hz, CH), 7.12 (2 H, d, 3JHH = 7.7 Hz, 2 CH), 7.23 (1 H, t, 3JHH = 7.6 Hz, CH), 7.38 (1 H, d, 3J = 7.6 Hz, CH), 7.56 (1 H, t, 3J = 7.6 Hz, CH), 7.89 (1 H, d, 3J = 7.6 Hz, CH), 8.02 (1 H, s, NH), 8.28 (2H, d, 3J = 7.7 Hz, 2 CH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 13.9 (Me), 27.9 (Me), 30.0 (Me), 42.8 (CH), 55.9 (CH2), 65.1 (C), 69.1 (C), 107.9 (C), 112.8 (2CH), 114.6 (CH), 119.2 (CH), 123.2 (C), 127.2 (2CH), 129.7 (CH), 132.2 (CH), 137.5 (C), 145.2 (C), 148.9 (C), 161.07 (C=O), 163.4 (C=O), 178.5 (C=O) ppm. MS (EI, 70 eV): m/z (%)= 451 (M+, 15), 131 (100), 76 (48). Anal. Calcd for C23H21 N3 O7 (451.43): C, 61.19; H, 4.69; N, 9.31. Found: C, 61.35; H, 4.72; N, 9.33%.
1'-(4-Methoxyphenyl)-2'',2'',3',3'-tetramethyldispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4k)
Pale Orange powder, mp 234-236 °C, 74 mg, yield 82%. IR (KBr) (νmax/cm-1): 3442, 1742, 1646, 1459 and 10.87 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.12 (3 H, s, Me), 1.22 (3 H, s, Me), 1.42 (3 H, s, Me), 1.62 (3 H, s, Me), 3.78 (3 H, s, MeO), 3.86 (1 H, d, 2JHH = 3.5 Hz, CH), 4.01 (1 H, d, 2JHH = 3.5 Hz, CH), 7.02 (2 H, d, 3JHH = 7.8 Hz, 2 CH), 7.13 (1 H, t, 3JHH = 7.5 Hz, CH), 7.38 (2 H, d, 3J = 7.8 Hz, 2 CH), 7.52 (1 H, t, 3J = 7.5 Hz, CH), 7.68 (1 H, d, 3J = 7.5 Hz, CH), 7.79 (1 H, d, 3J = 7.5 Hz, CH), 8.18 (1 H, s, NH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 23.9 (Me), 25.2 (Me), 26.8 (Me), 29.2 (Me), 44.8 (C), 53.9 (CH2), 55.8 (MeO), 64.2 (C), 72.2 (C), 105.8 (C), 112.8 (CH), 115.3 (2 CH), 120.8 (CH), 123.9 (C), 124.8 (2 CH), 129.5 (CH), 132.2 (CH), 141.2 (C), 144.8 (C), 155.9 (C), 161.2 (C=O), 162.1 (C=O), 176.2 (C=O) ppm. MS (EI, 70 eV): m/z (%)= 450 (M+, 15), 121 (100), 43 (68). Anal. Calcd for C25H26 N2 O6 (450.48): C, 66.65; H, 5.82; N, 6.22. Found: C, 66.79; H, 5.80; N, 6.23%.
1'-(4-Methoxyphenyl)-2'',2'',3'-trimethyldispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4l)
Pale Orange powder, mp 228- 230 °C, 74 mg, yield 89%. IR (KBr) (νmax/cm-1): 3424, 1738, 1631, 1452 and 1210 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.49 (3 H, d, 3JHH = 7.4 Hz,Me), 1.63 (3 H, s, Me), 1.89 (3 H, s, Me), 3.28 (1 H,d, 2JHH = 3.7 Hz, CH), 3.48 (1 H, d, 2JHH = 3.7 Hz, CH), 3.92 (3 H, s, MeO), 4.46 (1 H, q, 3JHH = 7.4 Hz, CH), 6.98 (2 H, d, 3JHH = 7.7 Hz, 2CH), 7.18 (1 H, t, 3JHH = 7.6 Hz, CH), 7.38 (1 H, t, 3J = 7.6 Hz, CH), 7.59 (2 H, d, 3J = 7.7 Hz, 2CH), 7.72 (1 H, d, 3J = 7.6 Hz, CH), 7.98 (1 H, d, 3J = 7.6 Hz, CH ), 8.06 (1 H, s,NH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 14.1 (Me), 28.0 (Me), 29.2 (Me), 39.2 (Me), 40.1 (CH), 55.5 (MeO), 57.8 (CH2), 61.8 (C), 73.0 (C), 106.9 (C), 114.2 (CH), 116.1 (2 CH), 121.2 (CH), 124.9 (C), 127.9 (2 CH), 129.2 (CH), 132.1 (CH), 138.2 (C), 146.5 (C), 156.2 (C), 161.2 (C=O), 162.8 (C=O), 177.2 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 436 (M+, 10), 76 (100), 59 (48). (100). Anal. Calcd for C24H24 N2 O6 (436.46): C, 66.04; H, 5.54; N, 6.42. Found: C, 66.14; H, 5.55; N, 6.45%.
2'',2'',3',3'-Tetramethyl-1'-(p-tolyl)dispiro[indoline-3,2'-pyrrolidine-4',5''-[1,3]dioxane]-2,4'',6''-trione (4m)
Pale orange powder, mp 243- 245 °C, 69 mg, yield 80%. IR (KBr) (νmax/cm-1): 3437, 1744, 1634, 15.73, 14.39 and 12.15 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.18 (3 H, s, Me), 1.39 (3 H, s, Me), 1.56 (3 H, s, Me), 1.68 (3 H, s, Me), 2.33 (3 H, s, Me), 3.84 (1 H, d, 2JHH = 4.2 Hz, CH), 4.12 (1 H, d, 2JHH = 3.5 Hz, CH), 6.79 (2 H, d, 3JHH = 7.7 Hz, 2CH), 7.08 (1 H, t, 3JHH = 7.6 Hz, CH), 7.15 (1 H, d, 3J = 7.6 Hz, 2CH), 7.34 (1 H, t, 3J = 7.6 Hz, CH), 7.48 (2 H, d, 3J = 7.7 Hz, 2CH), 7.68 (1 H, d, 3J = 7.6 Hz, CH), 7.80 (1 H, s,NH) ppm. 13C NMR (125.7 MHz, CDCl3):δ 20.02 (Me), 22.7 (Me), 25.8 (Me), 27.8 (Me), 30.02 (Me), 42.8 (C), 56.2 (CH2), 66.8 (C), 71.9 (C), 106.8 (C), 113.2 (CH), 115.2 (2CH), 117.7 (CH), 124.1 (C), 127.9 (C), 129.2(CH), 130.02 (2 CH), 132.2 (CH), 145.2 (C), 147.9 (C), 162.1 (C=O), 163.8 (C=O), 176.2 (C=O) ppm. MS (EI, 70 eV): m/z (%) = 434 (M+, 15), 131 (64), 77 (100). Anal. Calcd for C25H26N2 O5 (434.48): C, 69.11; H, 6.03; N, 6.45. Found: C, 69.26; H, 6.02; N, 6.48%.
Study of antioxidant activities of bis-spiro compounds utilizing DPPH radical trapping test
The DPPH radical scavenging experiment was utilized for investigation of some generated Spiro heterocyclic compounds antioxidant ability such as 4a-4d like the method reported by Shimada et. al. [14].To obtain this object, various concentrations (200–1000 ppm) of bis-spiro products 4a-4d were added to equal volume of DPPH methanolic solution (1 mmol/L). The mixture was stirred for 30 min at environmental temperature and putted in a dark room after this time and the absorbance of mixture was recorded at 517 nm. The Spiro heterocyclic compounds 4a-4d was replaced with standard type of methanol (3 mL). In this procedure, Butylated hydroxyl toluene (BHT) and 2-tertbutylhydroquinone (TBHQ) are standard antioxidants. The percentage of inhibition for the radical of DPPH is calculated by utilizing Yen and Duh [15] formula.
Study of reducing abilities of bis-spiro compounds 4a-4d
The ability of iron (III) reduced by the spiro heterocyclic compounds 4a-4d was investigated utilizing Yildirim et al. method [10]. For this object, the samples (1 mL), potassium ferricyanide (K3Fe(CN)6 2.5 mL, 10 g/L) and buffer of phosphate (2.5 mL, 0.2 mol/L, pH 6.6) were combined together and maintained for 30 min at 50 oC. Then, trichloroacetic acid (2.5 mL, 10% w/v) was added to the previous solution and centrifuged for 10 min. Finally, the supernatant (2.5 mL), distilled water (2.5 mL) and FeCl3 (0.5 mL, 1 g/L) mixed together and the absorbance of samples was measured at 700 nm. The higher absorbance of sample show higher reducing power of it. For accuracy of calculating, each measuring was carried out in three times. One way study of variance (ANOVA) that was used for data analyzing of compounds is running the SPSS software version 18.0 that proved difference of samples and control. Duncan multiple range experiments was employed for separation mean with the importance quantity of 95% (P < 0.05).
Antibacterial activities study of bis-spiro compounds 4
Gram-positive and Gram-negative bacteria along with the disk diffusion method were employed for analysis of antibacterial effect of prepared spiro heterocyclic compounds against two bacteria. All of bacteria were prepared from the Persian type culture collection (PTCC), Tehran, Iran. The prepared bacteria cultured for 16 to 24 h at 37°C via getting bacteria to turbidity equivalent to McFarland Standard No. 0.5. Two standards such as Streptomycin and Gentamicin with a concentration 40 𝜇g/mL were utilized against bacteria. The bacteria were cultured for 16 to 24 h at 37°C for preparing the turbidity equivalent bacteria to McFarland Standard No. 0.5. The bacterial suspension was produced according to the turbidity of the 0.5 McFarland (about 1.5 × 108 CFU/mL) standards and cultured with a sterile swab on Mueller Hinton agar. For valuation of their antibacterial activity of spiro heterocyclic compounds with concentration of 25 µg/ml were poured on sterile blank disks. The plates were incubated in an incubator at 37 °C for 24 h. The inhibition zone diameter was measured and compared to with the standard.