Supplementary Data
Synthesis of novel pyridopyridazin-3(2H)-one derivatives and evaluation of their cytotoxic activity against MCF-7 cells
Periasamy Selvakumar,1 Sathiah Thennarasu,1and Asit Baran Mandal2
1Organic Chemistry Laboratory, 2Chemical Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai-600 020, India.
Corresponding author. Tel.: +91-44-24913289; fax: +91 44 24912150;
E-mail: [email protected]
Corresponding author. Tel.: +91 44 24910846/0897; fax: +91 44 24912150;
E-mail: [email protected]
Table of contents Page
I. Experimental and spectral data S5-S9
II. Procedure for MTT assay S10
III. List of Figures S11-S46
Figure 1.1H NMR spectrum of 1a
Figure 2.1H NMR spectrum of 1b
Figure 3.1H NMR spectrum of 1c
Figure 4.1H NMR spectrum of1d
Figure 5.1H NMR spectrum of1e
Figure 6.1H NMR spectrum of1f
Figure 7.13C NMR spectrum of 1a
Figure 8.13C NMR spectrum of 1b
Figure 9.13C NMR spectrum of 1c
Figure 10.13C NMR spectrum of 1d
Figure 11.13C NMR spectrum of 1e
Figure 12.13C NMR spectrum of 1f
Figure 13.IR spectrum of 1a
Figure 14.IR spectrum of 1b
Figure 15.IR spectrum of 1c
Figure 16. IR spectrum of 1d
Figure 17. IR spectrum of 1e
Figure 18. IR spectrum of 1f
Figure 19. Mass spectrum of 1a
Figure 20. Mass spectrum of 1b
Figure 21. Mass spectrum of 1c
Figure 22. Mass spectrum of 1d
Figure 23. Mass spectrum of 1e
Figure 24. Mass spectrum of 1f
Figure 25.1H NMR spectrum of 2a
Figure 26.1H NMR spectrum of 2b
Figure 27.1H NMR spectrum of 2c
Figure 28.1H NMR spectrum of 2d
Figure 29.1H NMR spectrum of 2e
Figure 30.1H NMR spectrum of 2f
Figure 31.13C NMR spectrum of 2a
Figure 32.13C NMR spectrum of 2b
Figure 3313C NMR spectrum of 2c
Figure 34.13C NMR spectrum of 2d
Figure 35.13C NMR spectrum of 2e
Figure 36.13C NMR spectrum of 2f
Figure 37. IR spectrum of 2a
Figure 38. IR spectrum of 2b
Figure 39. IR spectrum of 2c
Figure 40. IR spectrum of 2d
Figure 41. IR spectrum of 2e
Figure 42. IR spectrum of 2f
Figure 43. Mass spectrum of 2a
Figure 44. Mass spectrum of 2b
Figure 45. Mass spectrum of 2c
Figure 46. Mass spectrum of 2d
Figure 47. Mass spectrum of 2e
Figure 48. Mass spectrum of 2f
Figure 49.1H NMR spectrum of 3a
Figure 50.1H NMR spectrum of 3b
Figure 51.1H NMR spectrum of 3c
Figure 52.1H NMR spectrum of 3d
Figure 53.1H NMR spectrum of 3e
Figure 54.1H NMR spectrum of 3f
Figure 55.13C NMR spectrum of 3a
Figure 56.13C NMR spectrum of 3b
Figure 5713C NMR spectrum of 3c
Figure 58.13C NMR spectrum of 3d
Figure 59.13C NMR spectrum of 3e
Figure 60.13C NMR spectrum of 3f
Figure 61. IR spectrum of 3a
Figure 62. IR spectrum of 3b
Figure 63. IR spectrum of 3c
Figure 64. IR spectrum of 3d
Figure 65. IR spectrum of 3e
Figure 66. IR spectrum of 3f
Figure 67. Mass spectrum of 3a
Figure 68. Mass spectrum of 3b
Figure 69. Mass spectrum of 3c
Figure 70. Mass spectrum of 3d
Figure 71. Mass spectrum of 3e
Figure 72. Mass spectrum of 3e
A. Experimental and spectral data
General procedure for the synthesis of 4-oxo-2,6-diphenylpiperidin-3-yl-acetate derivatives (1a-f): 4-oxo-2,6-diphenylpiperidin-3-yl-acetate (1a) was synthesized as described elsewhere23
with a slight modification. Methanol was used as the solvent and glacial acetic acid (70 mol%) was used as the catalyst. In a typical experiment, ammonium acetate (3.85 g, 50 mmol) was dissolved in methanol (60 mL) and then benzaldehyde (9.0 mL, 100 mmol) was added and warmed over a water-bath for 5 min. Ethyl levulinate (7.0 mL, 50 mmol) and glacial acetic acid (2.0 mL, 35 mmol) were added and heated at ~60 C over a water-bath for 2 h and then left aside at room temperature. The pale-yellow precipitate formed was crystallized from methanol.
Ethyl 2-(4-oxo-2,6-diphenylpiperidin-3-yl)acetate (1a): Colourless needles; m.p 114–116 oC; yield 77%; IR (KBr cm-1): 3425, 2980, 1723, 1654, 1497, 1462, 1404, 1324, 1224, 1178, 1094, 1019, 918, 758, 700; 1H NMR (500 MHz, CDCl3): δ ppm 1.18 (t , 3H, J = 6.8 Hz), 2.04 (dd, 1H, J = 3.5, 16.5 Hz), 2.09 (Bs, amine NH), 2.55 (dd, 1H, J = 8.5, 17 Hz), 2.65 (dd, 1H, J = 3, 14 Hz), 2.81 (dd - t, 1H, J = 12, 13 Hz), 3.18–3.23 (m, 1H), 3.82 (d, 1H, J = 10.7 Hz), 3.96–4.04 (m, 2H), 4.11 (dd, 1H, J = 3, 11.5 Hz), 7.27–7.37 (m, 6H Ar protons), 7.45–7.47 (m, 4H Ar protons); 13C NMR (125 MHz, CDCl3):δ ppm 14.1, 30.6, 50.7, 53.7, 60.6, 61.5, 66.3, 126.6, 127.9, 128.0, 128.5, 128.8, 128.8, 140.9, 142.5, 172.3, 207.8; MS m/z = 338 [MH]+.
Ethyl 2-(4-oxo-2,6-bis(4-methylphenyl)piperidin-3-yl)acetate (1b): Colourless liquid, yield 67%, IR (KBr cm-1): 3412, 3315, 2976, 2924, 2819, 1721, 1632, 1508, 1433, 1315, 1164, 1029, 931, 810. 1H NMR (500 MHz, CDCl3): δ ppm 1.15 - 1.18 (m, 3H); ); 2.02 (dd, 1H, J = 2.5, 17 Hz); 2.06 (bs, amine NH); 2.32 (s, 6H) 2.53 (dd, 1H, J = 8.5, 17 Hz); 2.60 (d, 1H, J = 13 Hz); 2.76 (dd - t, 1H, J = 11.5, 13 Hz); 3.14 – 3.18 (m, 1H); 3.74 (d, 1H, J = 10.5 Hz); 3.96 – 4.06 (3H merged); 7.12 – 7.16 (m, 4H Ar protons); 7.30 -7.34 (m, 4H Ar protons). 13C NMR (125 MHz, CDCl3): δ ppm 14.2, 21.23, 21.26, 30.6, 50.8, 53.8, 60.5, 61.3, 66.0, 126.5, 127.8, 129.4, 137.6, 138.1, 139.8, 172.3, 207.8; MS m/z = 366 [MH]+.
Ethyl 2-(4-oxo-2,6-bis(4-methoxyphenyl)piperidin-3-yl)acetate (1c): Colourless spongy needles; m.p 110 - 112oC; yield 66%; IR (KBr cm-1): 3414, 3304, 2920, 2840, 1724, 1611, 1511, 1452, 1374, 1309, 1243, 1169, 1029, 832, 650; 1H NMR (500 MHz, CDCl3):δ ppm 1.18 (t, 3H, J = 7 Hz), 2.02 (dd, 1H, J = 4.5, 17 Hz), 2.02 (bs, amine NH), 2.54 (dd, 1H, J = 9, 16.5 Hz), 2.60 (dd, 1H, J = 3, 14 Hz), 2.76 (dd - t, 1H, J = 12, 13 Hz), 3.13 – 3.17 (m, 1H), 3.74 (d, 1H, J = 11 Hz), 3.794 (s, 3H), 3.797 (s, 3H), 3.97–4.06 (3H merged), 6.87 (d, 4H, J = 8.5 Hz Ar protons), 7.35–7.38 (m, 4H Ar protons); 13C NMR (125 MHz, CDCl3): δ ppm14.1, 30.6, 50.8, 54.0, 55.3, 60.5, 60.9, 65.7, 114.0, 127.7, 128.9, 133.2, 134.9, 159.2, 159.5, 172.4, 208.0; MS m/z = 398 [MH]+.
Ethyl 2-(4-oxo-2,6-bis(4-chlorophenyl)piperidin-3-yl)acetate (1d): Colourless spongy solid; m.p 121 - 123oC; yield 71%; IR (KBr cm-1): 3417, 3324, 2980, 2920, 1722, 1488, 1378, 1318, 1219, 1163, 1089, 1017, 826; 1H NMR (500 MHz, CDCl3): δ ppm1.19 (t, 3H, J = 7 Hz), 2.03 (dd, 3H, J = 4, 17 Hz), 2.10 (bs, amine NH), 2.53 (dd, 1H, J = 7.5, 16.5 Hz), 2.61 (dd, 1H, J = 3, 13.5 Hz), 2.72 (dd - t, 1H, J = 12, 13 Hz), 3.09–3.14 (m, 1H), 3.82 (d, 1H, J = 11 Hz), 3.97–4.04 (m, 2H), 4.08 (dd, 1H, J = 3, 12.5 Hz), 7.31–7.34 (m, 4H Ar protons), 7.38–7.42 (m, 4H Ar protons); 13C NMR (125 MHz, CDCl3): δ ppm14.1, 30.3, 50.5, 53.6, 60.71, 60.79, 65.4, 128.0, 129.0, 129.0, 129.2, 133.7, 134.3, 139.3, 140.9, 172.0, 206.7; MS m/z = 406 [MH]+.
Ethyl 2-(4-oxo-2,6-bis(4-bromophenyl)piperidin-3-yl)acetate (1e): Colourless spongy solid; m.p 135 - 137oC; yield 69%; IR (KBr cm-1): 3421, 3320, 2979, 2919, 1722, 1484, 1378, 1317, 1220, 1163, 1013, 824; 1H NMR (500 MHz, CDCl3): δ ppm 1.18 (t, 3H, J = 7 Hz), 2.02 (dd, 1H, J = 4, 17 Hz), 2.09 (bs, amine NH), 2.52 (dd, 1H, J = 8.5, 17 Hz), 2.60 (dd, 1H, J = 3, 13 Hz), 2.70 (dd - t, 1H, J = 12, 13 Hz), 3.07–3.12 (m, 1H), 3.80 (d, 1H, J = 9.5 Hz), 3.97–4.02 (m, 2H), 4.06 (dd, 1H, J = 2, 11.5 Hz), 7.31–7.35 (m, 4H Ar protons), 7.46 – 7.49 (m, 4H Ar protons); 13C NMR (125 MHz, CDCl3): δ ppm 14.1, 30.3, 50.3, 53.6, 60.7, 60.8, 65.4, 121.8, 122.4, 128.3, 129.6, 132.0, 132.0, 139.8, 141.4, 172.0, 206.6; MS m/z = 495 [MH]+.
Ethyl 2-(4-oxo-2,6-bis(3-nitrophenyl)piperidin-3-yl)acetate (1f): Colourless crystals; m.p 148-150oC; yield 64%; IR (KBr cm-1): 3407, 3299, 3080, 2983, 2924, 2856, 1714, 1526, 1472, 1349, 1274, 1228, 1154, 1085, 1021, 909, 799; 1H NMR (500 MHz, CDCl3): δ ppm 1.19 (t, 3H, J = 6.5), 2.12 (dd, 1H, J = 3.5, 16.5), 2.30 (bs, amine NH), 2.56 (dd, 1H, J = 7.5, 16.5 Hz), 2.73 (dd, 1H, J = 3, 14 Hz), 2.82 (dd - t, 1H, J = 12.5, 13 Hz), 3.17–3.22 (m, 1H), 3.97–4.04 (m, 2H), 4.11 (d, 1H, J = 11 Hz), 4.31 (dd, 1H, J = 3.5, 11.5), 7.56–7.62 (m, 2H Ar protons), 7.88 (d, 1H, J = 7.5 Hz Ar protons), 7.83 (d, 1H, J = 7.5 Hz Ar protons), 8.21 (d, 1H, J = 7.5 Hz Ar protons), 8.18 (d, 1H, J = 8.5 Hz Ar protons), 8.36–8.38 (m, 2H, Ar protons); 13C NMR (125 MHz, CDCl3): δ ppm14.1, 30.2, 50.0, 53.2, 60.5, 60.8, 65.0, 121.7, 123.0, 123.3, 123.7, 130.0, 130.1, 132.9, 134.1, 142.6, 144.1, 148.5, 148.6, 171.6, 205.2; MS m/z = 428 [MH]+.
General procedure for the synthesis of 5,7-diarylpyrido[4,3-c]pyridazin-3(2H)-one derivatives (2a-2f): To a solution of appropriate ethyl 4-oxo-2,6-diphenylpiperidin-3-yl-acetates (337 mg, 1.0 mmol) in ethanol was added hydrazine hydrate (0.053 mL, 1.2 mmol) and the resultant solution was refluxed for 17 h. The solvent was evaporated and the residue obtained was extracted with chloroform (320 mL). The combined organic layer was dried over sodium sulphate, concentrated under reduced pressure and the solid obtained was purified by crystallization using methanol.
4,4a,5,6,7,8-hexahydro-5,7-diphenylpyrido[4,3-c]pyridazin-3(2H)-one (2a): Colourless solid; mp 153-154oC; yield 82; IR (KBr cm-1): 3371, 3281, 3025, 2925, 2848, 1685, 1514, 1416, 1334, 1106, 1038, 823, 759; 1H NMR (500 MHz, CDCl3): δ ppm 2.10 (bs, 1H, amine NH), 2.17-2.28 (m, 2H), 2.51-2.56 (m, 1H), 2.71-2.76 (m, 2H merged), 3.63 (d, 1H, J = 9.7 Hz), 3.9 (d, 1H, J = 11.4Hz), 7.26-7.35 (m, 6H Ar protons), 7.41 – 7.44 (m, 4H Ar protons), 8.77 (bs, 1H, amide NH); 13C NMR (125 MHz, CDCl3): δ ppm 29.7, 40.5, 41.7, 60.9, 68.3, 126.7, 127.6, 127.9, 128.6, 128.7, 128.9, 140.5, 142.9, 152.8, 166.3; MS m/z = 306 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(4-methylphenyl)pyrido[4,3-c]pyridazin-3(2H)-one (2b): Colourless solid; mp 183-185oC; yield 72; IR (KBr cm-1): 3303, 3210, 3087, 2944, 2831, 1665, 1611, 1511, 1458, 1303, 1367, 1241, 1175, 1107, 1031, 834;1H NMR (500 MHz, CDCl3): δ ppm 2.07 (bs, 1H, amine NH), 2.18–2.34 (m, 2H), 2.36 (s, 3H), 2.37 (s, 3H), 2.54 (dd, 1H, J = 15, 12Hz), 2.72-2.78 (m, 2H merged), 3.62 (d, 1H, J = 9.5 Hz), 3.96 (dd, 1H, J = 12, 3 Hz), 7.18 (d, 4H, J = 6.5 Hz Ar protons), 7.32–7.35 (m, 4H Ar protons), 8.66 (bs, 1H, amide NH); 13C NMR (125 MHz, CDCl3): δ ppm 21.12, 21.14, 29.6, 40.4, 41.7, 60.6, 68.0, 126.4, 127.4, 129.3, 129.4, 137.51, 137.55, 138.2, 140.0, 152.9, 166.2; MS m/z = 334 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(4-methoxyphenyl)pyrido[4,3-c]pyridazin-3(2H)-one (2c): Colourless solid; mp 188-190oC; yield 76.7; IR (KBr cm-1): 3318, 3222, 3090, 2920, 2849, 1664, 1439, 1414, 1370, 1305, 1230, 1174, 1091, 1014, 828; 1H NMR (500 MHz, CDCl3): δ ppm 2.04 (bs, 1H, amine NH), 2.17-2.33 (m, 2H), 2.52 (dd, 1H, J = 15.5, 11.5 Hz), 2.69-2.75 (m, 2H merged), 3.59 (d, 1H, J = 9.5 Hz), 3.81 (s, 3H), 3.82 (s, 3H), 3.93 (dd, 1H, J = 11.5, 3 Hz), 6.88-6.90 (m, 4HAr protons), 7.34–7.38 (m, 4H Ar protons), 8.66 (bs, 1H, amide NH); 13C NMR (125 MHz, CDCl3): δ ppm29.6, 40.6, 41.7, 55.30, 55.34, 60.3, 67.7, 114.0, 114.1, 127.6, 128.6, 132.6, 135.1, 152.9, 159.1, 159.6, 166.2; MS m/z = 366 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(4-chlorophenyl)pyrido[4,3-c]pyridazin-3(2H)-one (2d): Colourless solid, mp 185-187oC, yield 79, IR (KBr cm-1): 3425, 3322, 3225, 3086, 2931, 2367, 2339, 1663, 1525, 1478, 1353, 1211, 1166; 1H NMR (500 MHz, CDCl3): δ ppm 2.07 (bs, 1H, amine NH), 2.16-2.30 (m, 2H), 2.48 (dd, 1H, J = 15, 11.5 Hz), 2.67-2.75 (m, 2H merged), 3.63 (d, 1H, J = 9.5 Hz), 3.96 (dd, 1H, J = 11.5, 3 Hz), 7.32-7.39 (m, 8HAr protons), 8.75 (bs, 1H, amide NH); 13C NMR (125 MHz, CDCl3): δ ppm29.5, 40.4, 41.6, 60.1, 67.4, 127.9, 128.9, 129.0, 133.6, 134.4, 138.8, 141.1, 151.6, 165.8; MS m/z = 374 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(4-bromophenyl)pyrido[4,3-c]pyridazin-3(2H)-one (2e): Brown solid, mp 133-135oC, yield 75, IR (KBr cm-1): 3236, 3097, 2922, 2854, 1674, 1485, 1439, 1412, 1345, 1305, 1230, 1102, 1070, 1008, 825; 1H NMR (500 MHz, CDCl3): δ ppm 2.07 (bs, 1H, amine NH), 2.17-2.31 (m, 2H), 2.48 (dd, 1H, J = 15, 12 Hz), 2.67-2.75 (m, 2H merged), 3.62 (d, 1H, J = 10 Hz), 3.95 (dd, 1H, J = 11.5, 3 Hz), 7.31 (d, 4H, J = 8.5 Hz Ar protons), 7.48–
7.53 (m, 4H Ar protons), 8.67 (bs, 1H, amide NH); 13C NMR (125 MHz, CDCl3): δ ppm 29.5, 40.4, 41.5, 60.2, 67.5, 121.7, 122.5, 128.2, 129.2, 131.8, 132.0, 139.2, 141.6, 151.5, 165.7; MS m/z = 463 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(3-nitrophenyl)pyrido[4,3-c]pyridazin-3(2H)-one (2f): Pale Brown solid, mp 248-250oC, yield 69, IR (KBr cm-1): 3417, 3322, 3227, 3091, 2926, 2853, 1665, 1523, 1353, 1453, 808, 735, 688; 1H NMR (500 MHz, CDCl3): δ ppm 1.89(dd, 1H, J = 9, 17.5Hz), 2.32 (dd, 1H, J = 13.5, 17.5 Hz), 2.42–2.48 (m, 1H), 2.70 (dd, 1H, J = 14.5, 3 Hz), 2.81–2.87 (m, 1H), 3.90 (d, 1H, J = 10 Hz), 4.11 (d, 1H, J = 10.5 Hz), 7.64-7.70 (m, 2H Ar protons), 7.93–7.97 (m, 2H Ar protons), 8.16 (2 set of dd, 2H, J = 8, 1.5 Hz 8.5, 2 Hz Ar protons), 8.34–8.39 (m, 2H Ar protons), 10.6 (bs, 1H, amide NH); 13C NMR (125 MHz, CDCl3): δ ppm29.7, 39.3, 40.8, 59.0, 66.0, 121.8, 122.6, 123.1, 123.2, 130.3, 134.2, 135.2, 144.0, 146.1, 148.2, 148.3, 150.6, 165.8.Ms m/z = 395.34 [M+]
General procedure for the synthesis of 2-phenyl-5,7-diarylpyrido[4,3-c]pyridazin-3(2H)-one derivatives(3a -3f): A mixture of ethyl 4-oxo-2,6-diphenylpiperidin-3-yl-acetates (337 mg, 1.0 mmol) in dry toluene were added phenyl hydrazine (0.118 mL, 1.2 mmol) and trifluoroacetic acid (0.0155 mL, 0.20 mmol), and the solution was refluxed under nitrogen atmosphere for 2 days. After the reaction was complete the solvent was evaporated under reduced pressure, and the residue obtained was extracted with chloroform (320 mL). The combined organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure and the residue obtained was purified using column chromatography (neutral aluminium oxide, 150-200 mesh; eluent: 7% ethyl acetate in hexane).
4,4a,5,6,7,8-hexahydro-2,5,7-triphenylpyrido[4,3-c]pyridazin-3(2H)-one (3a): Pale brown solid; mp 140-143oC; yield 79; IR (KBr cm-1): 3304, 3030, 2960, 2887, 2806, 1680, 1595, 1492 1454, 1326, 1305, 1227, 1160, 1107, 752; 1H NMR (500 MHz, CDCl3): δ ppm 2.18 (bs, 1H, amine NH), 2.43-2.54 (m, 2H), 2.66 (dd, 1H, J = 15, 11.5 Hz), 2.90-2.96 (m, 2H merged), 3.73 (d, 1H, J = 9.5 Hz), 4.07 (dd, 1H, J = 11.5, 2.5 Hz), 7.27-7.33 (m, 2H Ar protons), 7.34–7.44 (m, 7H Ar protons), 7.48–7.52 (m, 6H Ar protons); 13C NMR (125 MHz, CDCl3): δ ppm 31.3, 41.0, 41.7, 60.8, 68.4, 124.9, 126.5, 126.7, 127.6, 127.9, 128.6, 128.7, 128.8, 140.6, 140.9, 142.9, 153.6, 164.3; MS m/z = 382 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(4-methylphenyl)-2-phenylpyrido[4,3-c]pyridazin-3(2H)-one (3b): Pale brown solid;mp 166-167oC; yield 72; IR (KBr cm-1): 3320, 3021, 2919, 2878, 2815, 1688, 1594, 1492 1321, 1299, 1228, 1155, 1105, 821, 756; 1H NMR (500 MHz, CDCl3): δ ppm 2.08 (bs, 1H, amine NH), 2.38 (s, 3H), 2.39 (s, 3H), 2.41-2.51 (m, 2H), 2.64 (dd, 1H, J = 15, 12 Hz), 2.87-2.91 (m, 2H merged), 3.69 (d, 1H, J = 9.5 Hz), 4.03 (dd, 1H, J = 11.5, 3 Hz), 7.19-7.22 (m, 4H, Ar protons), 7.28–7.29 (m, 1H, Ar protons), 7.36–7.38 (m, 4H, Ar protons), 7.40–7.43 (m, 2H, Ar protons), 7.51 (d, 2H, J = 8 Hz Ar protons); 13C NMR (125 MHz, CDCl3): δ
ppm 21.14, 21.16, 31.3, 41.0, 41.8, 60.6, 68.1, 124.9, 126.4, 126.6, 127.4, 128.6, 129.3, 129.5, 137.5, 137.6, 140.0, 141.0, 153.9, 164.4; MS m/z = 410 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(4-methoxyphenyl)-2-phenylpyrido[4,3-c]pyridazin-3(2H)-one (3c): Pink solid, mp 128-130oC;yield 74;IR (KBr cm-1): 3415, 3306, 2954, 2832, 1682, 1606, 1506, 1449, 1302, 1242, 1175, 1105, 927, 833, 752; 1H NMR (500 MHz, CDCl3): δ ppm1.99 (bs, 1H, amine NH), 2.39-2.54 (m, 2H), 2.62 (dd, 1H, J = 14.5, 12 Hz), 2.84-2.88 (m, 2H merged), 3.66 (d, 1H, J = 9.5 Hz), 3.82 (s, 3H), 3.84 (s, 3H), 4.00 (dd, 1H, J = 12, 3 Hz), 6.90-6.93 (m, 4H Ar protons), 7.27–7.28 (m, 1H,Ar proton), 7.38–7.43 ( m, 6H,Ar protons), 7.50 (d, 2H, J = 8 Hz Ar protons); 13C NMR (125 MHz, CDCl3): δ ppm 31.3, 41.1, 41.8, 55.30, 55.38, 60.3, 67.8, 114.0, 114.2, 124.9, 126.6, 127.6, 128.6, 141.0, 153.9, 159.2, 159.7, 164.4; MS m/z = 442 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(4-chlorophenyl)-2-phenylpyrido[4,3-c]pyridazin-3(2H)-one (3d): Pale brown solid; mp 113-115oC; yield 77; IR (KBr cm-1): 3315, 3061, 2853, 2810, 1676, 1593, 1491, 1442, 1413, 1359, 1329, 1304, 1227, 1202, 1158, 1093, 1014, 830; 1H NMR (500 MHz, CDCl3): δ ppm 2.07 (bs, 1H, amine NH), 2.39-2.51 (m, 2H), 2.57 (dd, 1H, J = 15, 12 Hz), 2.81-2.88 (m, 2H merged), 3.69 (d, 1H, J = 10 Hz), 4.02 (dd, 1H, J = 12, 3 Hz), 7.27-7.29 (m, 1H Ar protons), 7.34–7.44 (m, 10H Ar protons), 7.48 ( d, 2H, J = 8.5 Hz Ar protons); 13C NMR (125 MHz, CDCl3): δ ppm 31.3, 40.9, 41.7, 60.1, 67.5, 124.9, 126.8, 127.9, 128.6, 128.9, 128.9, 129.1, 133.6, 134.4, 138.9, 140.8, 141.2, 152.6, 163.9; MS m/z = 450 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(4-bromophenyl)-2-phenylpyrido[4,3-c]pyridazin-3(2H)-one (3e): Pale brown solid;mp 101-102oC;yield 71.6; IR (KBr cm-1): 3300, 3041, 2923, 2814, 1679, 1593, 1488, 1410, 1328, 1305, 1227, 1201, 1159, 1106, 1069, 827; 1H NMR (500 MHz, CDCl3): δ ppm2.16 (bs, 1H, amine NH), 2.36-2.48 (m, 2H), 2.53 (dd, 1H, J = 15.5, 11.5 Hz), 2.78-2.84 (m, 2H merged), 3.65 (d, 1H, J = 10 Hz), 3.98 (dd, 1H, J = 12.5, 3 Hz), 7.23-7.26 (m, 1H Ar protons), 7.31–7.39 (m, 6HAr protons), 7.43–7.52 (m, 6H Ar protons); 13C NMR (125 MHz, CDCl3): δ ppm31.2, 41.0, 41.7, 60.2, 67.6, 121.8, 122.6, 125.0, 126.9, 128.3, 131.9, 132.1, 139.4, 140.8, 141.7, 152.6, 164.0; MS m/z = 539 [MH]+.
4,4a,5,6,7,8-hexahydro-5,7-bis(3-nitrophenyl)-2-phenylpyrido[4,3-c]pyridazin-3(2H)-one (3f): Brown solid; m.p 190-191oC; yield 67; IR ( KBr cm-1 ): 3418, 3085, 2922, 2852, 1679, 1596, 1522, 1345, 1103, 908, 810; 1H NMR ( 500 MHz, CDCl3 ) : δ ppm2.04 ( Bs, amine NH ), 2.42–2.52 ( m, 2H ), 2.64 ( dd, 1H, J = 11.5, 15.5 Hz ), 2.91–2.97 ( m, 2H merged ), 3.88 ( d, 1H, J = 10 Hz), 4.19 (dd, 1H, J = 12.5, 3 Hz ), 7.24–7.27 (m, 1H Ar protons), 7.35–7.40 (m, 2H Ar protons), 7.44 (d, 2H, J = 8 HzAr protons), 7.55–7.62 (m, 2H Ar protons), 7.81–7.85 (m, 2H Ar protons), 8.18 (d, 1H, J = 8 Hz Ar protons), 8.23 (d, 1H, J = 9 Hz Ar protons), 8.35–8.39 (m, 2H Ar protons); 13C NMR ( 125 MHz, CDCl3 ) : δ ppm31.1, 40.8, 41.4, 60.1, 67.3, 121.8, 122.7, 123.3, 123.9, 125.0, 127.0, 128.8, 130.0, 130.2, 132.9, 133.9, 140.7, 142.3, 144.4, 148.6, 148.7, 151.3, 163.5. Ms m/z = 471.42 [M+]
B. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay28-29
Human breast adenocarcinoma (MCF-7) cell culture was procured from National Centre for Cell Sciences (NCCS), Pune, India.The cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS), penicillin (100 IU/ml), streptomycin (100μg/ml) and amphotericin-B (5 μg/ml) in a humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were trypsinized with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 flat bottles and the studies were carried out in 96-well microtitre plates.
Cells were plated in 96-well flat bottom microtitre plate at a density of 1 × 10 4 cells per well and cultured for 24 h at 37C in 5% CO2 atmosphere to allow cell adhesion. After 24 h, when partial monolayer was formed, medium was removed and cells were treated with different concentrations of standard drug (Doxorubicin) and test compounds. Microscopic examination was carried out and observations recorded every 24 h. After the treatment, the solutions in the wells were discarded and 50 μl of the freshly prepared MTT solution (2 mg/mL PBS) was added to each well. The plates were gently shaken and incubated for 3 h at 37⁰C in 5% CO2
atmosphere. After 3 h, the supernatant was removed and the formazan crystals formed in the cells were solubilized by adding iso-propanol (50 μL). Finally, the amountof formazan formed in different wells was measured from its absorbance at 540 nm on a microplate reader (Bio-Tek, EL X -800 MS. The concentration of drug required to kill 50% of cells in exponentially growing cultures after).a 48 h exposure to the drug (IC50) values was calculated from the plot of A540versus concentration of test sample.
C. List of Figures
Figure 1.1H NMR spectrum of 1a
Figure 2.1H NMR spectrum of 1b
Figure 3.1H NMR spectrum of 1c
Figure 4.1H NMR spectrum of 1d
Figure 5.1H NMR spectrum of 1e
Figure 6.1H NMR spectrum of 1f
Figure 7.13C NMR spectrum of 1a
Figure 8.13C NMR spectrum of 1b
Figure 9.13C NMR spectrum of 1c
Figure 10.13C NMR spectrum of 1d
Figure 11.13C NMR spectrum of 1e
Figure 12.13C NMR spectrum of 1f
Figure 13.IR spectrum of 1a
4000.0 3000 2000 1500 1000 400.0
0.0
10
20
30
40
50
60
70
80
90
100.0
cm-1
%T
3425.58
2980.462369.86
1723.20
1654.831497.30
1462.301404.90
1324.011224.52
1178.561094.60
1019.02
918.30
858.55
758.17
700.95
654.10
614.40
511.28
481.34
454.49
Figure 14.IR spectrum of 1b
Figure 15.IR spectrum of 1c
meo-pip_1_1_1Name
selva By organic Date tuesday, July 31 2012Description
4000 4503500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1243.61cm-11724.36cm-1
1169.79cm-11511.53cm-1
1029.48cm-11309.80cm-1
1452.57cm-1
1611.73cm-1 832.05cm-11374.35cm-1
2920.52cm-1 1116.26cm-13304.35cm-1
2840.54cm-1
3414.68cm-1
650.34cm-1
537.88cm-11894.10cm-1
2047.66cm-1923.32cm-1
579.57cm-1
Methyl pip_002_1_1_1Name
Psk By organic Date Monday, August 13 2012Description
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1721.69cm-1 1164.00cm-1
1208.73cm-12976.71cm-1 2924.58cm-1 1433.89cm-1
1315.27cm-1
1029.54cm-13315.76cm-1
810.95cm-12819.30cm-13412.26cm-1
1632.96cm-1
1508.53cm-1
931.87cm-1 504.34cm-1
647.25cm-1
1903.75cm-1
Figure 16.IR spectrum of 1d
Figure 17.IR spectrum of 1e
Br-pip_1_1_1Name
selva By organic Date Tuesday, July 31 2012Description
4000 4503500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1722.16cm-1
1220.72cm-11163.71cm-1
1317.76cm-11484.47cm-1
1013.25cm-1
1378.22cm-1
1072.20cm-1824.30cm-1
1636.84cm-1
3421.56cm-1
3320.76cm-1
1906.75cm-1
2979.19cm-1
2919.41cm-1
630.88cm-1715.40cm-1
509.97cm-1
cl-pip_1_1_1Name
selva By organic Date Tuesday, July 31 2012Description
4000 4503500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1722.78cm-1
1219.73cm-11163.11cm-1
1089.97cm-1
1318.52cm-11488.14cm-1
1378.83cm-1
1017.65cm-1826.72cm-1
943.35cm-11905.79cm-1
2980.14cm-1
637.23cm-1
2920.03cm-1
717.66cm-1
3324.57cm-1
686.42cm-1
3417.36cm-1
512.50cm-1
Figure 18.IR spectrum of 1f
3NO-pip_1_1_1Name
selva By organic Date Tuesday, July 31 2012Description
4000 4503500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1349.47cm-1
1526.56cm-11714.20cm-1
1228.43cm-11154.31cm-1
697.31cm-11274.36cm-1
1472.94cm-1
1085.87cm-1
1021.49cm-1
799.17cm-1
739.90cm-1859.08cm-1
909.19cm-1
3299.56cm-1
2983.51cm-1
3080.20cm-1
2856.67cm-1
2924.06cm-13407.05cm-1
612.52cm-1499.58cm-1
Figure 19.Mass spectrum of 1a
Figure 20.Mass spectrum of 1b
Figure 21.Mass spectrum of 1c
Figure 22.Mass spectrum of 1d
Figure 23.Mass spectrum of 1e
Figure 24.Mass spectrum of 1f
Figure25.1H NMR spectrum of 2a
Figure 26.1H NMR spectrum of 2b
Figure 27.1H NMR spectrum of 2c
Figure 28.1H NMR spectrum of 2d
Figure 29.1H NMR spectrum of 2e
Figure 30.1H NMR spectrum of 2f
Figure 31.13C NMR spectrum of 2a
Figure 32.13C NMR spectrum of 2b
Figure 33.13C NMR spectrum of 2c
Figure 34.13C NMR spectrum of 2d
Figure 35.13C NMR spectrum of 2e
Figure 36.13C NMR spectrum of 2f
Figure 37.IR spectrum of2a
Figure 38.IR spectrum of 2b
4000.0 3000 2000 1500 1000 400.0
0.0
10
20
30
40
50
60
70
80
90
100.0
cm-1
%T
3303.93
3210.70
3087.382944.38
2831.68
2543.97
2370.01
2340.35
2274.042054.55
1889.32
1665.95
1611.97
1511.72
1458.54
1367.81
1303.711241.26
1175.47
1107.291031.92
963.59921.84
834.40
806.56
757.51
641.46
611.27
542.22489.92
4000.0 3000 2000 1500 1000 400.0
0.0
10
20
30
40
50
60
70
80
90
100.0
cm-1
%T
3371.06
3281.83
3025.63
2925.702848.54
2372.17
1685.47
1514.171416.14
1334.781106.32
1038.48823.01
759.78613.98
Figure 39.IR spectrum of 2c
Figure 40. IR spectrum of 2d
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1674.04cm-1825.86cm-1
1485.23cm-11345.60cm-1
1305.46cm-1
1008.95cm-1
1070.16cm-1
1412.15cm-1758.74cm-1
1439.52cm-1
1102.71cm-1
1230.48cm-1
3236.42cm-1
599.45cm-1
1038.13cm-1
2922.93cm-1 714.64cm-1
901.59cm-1
3097.89cm-1 2854.20cm-1
964.82cm-1
527.57cm-1
631.22cm-1
487.82cm-11904.09cm-1
3407.2
2810.4
4000.0 3000 2000 1500 1000.0
3.2
10
20
30
40
50
60
70
80
90
100.0
cm-1
%T
3425.213322.79
3225.793086.86
2931.09
2367.85
2339.18
1920.611843.68
1663.811525.00
1478.69
1353.37
1238.16
1211.79
1166.65
1097.83
1065.92
1030.72
4000.0 3000 2000 1500 1000 400.0
0.0
10
20
30
40
50
60
70
80
90
100.0
cm-1
%T
3318.53
3222.59
3090.00
2953.12
2920.16
2849.13
2374.312341.66
2276.671901.23
1664.59
1489.86
1439.55
1414.54
1370.92
1305.75
1230.93
1174.27
1091.49
1014.21
964.82
918.57
828.78
791.20
755.80
714.40
634.16
602.51
572.30
531.60
492.21
463.97413.84
1Tue_1_1_1_1_1Name
Sample 001 By organic Date Tuesday, October 16 2012Description
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1523.77cm-1
1353.79cm-1
1665.00cm-1
1642.80cm-1
735.20cm-11309.95cm-1
688.48cm-1
808.82cm-11477.58cm-1
3227.13cm-1
3322.72cm-1 766.99cm-11094.91cm-1
3417.46cm-1
1421.05cm-11236.25cm-13091.46cm-1 1736.26cm-1
2926.43cm-11066.07cm-1
1161.67cm-1
1030.40cm-1857.71cm-1
2853.64cm-1600.36cm-1906.57cm-1
643.12cm-1
964.91cm-12345.34cm-1 473.15cm-1
542.70cm-1
NH
HN
NO
O2N NO2
Figure 41.IR spectrum of 2e
Figure 42.IR spectrum of 2f
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1%
T
1674.04cm-1825.86cm-1
1485.23cm-11345.60cm-1
1305.46cm-1
1008.95cm-1
1070.16cm-1
1412.15cm-1758.74cm-1
1439.52cm-1
1102.71cm-1
1230.48cm-1
3236.42cm-1
599.45cm-1
1038.13cm-1
2922.93cm-1 714.64cm-1
901.59cm-1
3097.89cm-1 2854.20cm-1
964.82cm-1
527.57cm-1
631.22cm-1
487.82cm-11904.09cm-1
3407.2
2810.4
Figure 43.Mass spectrum of 2a
Figure 44.Mass spectrum of 2b
Figure 45.Mass spectrum of 2c
Figure 46.Mass spectrum of 2d
Figure47.Mass spectrum of 2e
Figure 48.Mass spectrum of 2f
Figure49.1H NMR spectrum of 3a
Figure 50.1H NMR spectrum of 3b
Figure 51.1H NMR spectrum of 3c
Figure 52.1H NMR spectrum of 3d
Figure 53.1H NMR spectrum of 3e
Figure 54.1H NMR spectrum of 3f
Figure 55.13C NMR spectrum of 3a
Figure 56.13C NMR spectrum of 3b
Figure 57.13C NMR spectrum of 3c
Figure 58.13C NMR spectrum of 3d
Figure 59.13C NMR spectrum of 3e
Figure 60.13C NMR spectrum of 3f
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1680.40cm-1 1305.91cm-1
697.09cm-1
1326.10cm-1 752.51cm-1
1492.25cm-11107.19cm-1
1454.38cm-1
1595.21cm-11227.10cm-1
1069.57cm-1 1030.22cm-11160.09cm-1
927.37cm-1663.89cm-1
970.99cm-1
1813.01cm-11880.93cm-1
1949.89cm-1
604.62cm-1
3030.92cm-1
2806.17cm-1
2344.62cm-12887.48cm-1
572.44cm-1
542.78cm-1
2960.19cm-1
3304.85cm-1
3780.46cm-1
3430
Figure 61.IR spectrum of 3a
Figure 62.IR spectrum of 3b
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1242.80cm-1
1682.43cm-1
1506.04cm-11302.17cm-1
1031.18cm-11175.10cm-1
1606.17cm-1
1105.36cm-11449.12cm-1 833.40cm-1
752.95cm-1
691.92cm-1
927.85cm-1
1885.66cm-1 644.12cm-1
2832.12cm-1
2348.71cm-1
2954.06cm-1
561.09cm-1
3306.46cm-1
3415.82cm-1
3l_1_1_1Name
psk By organic Date Friday, July 06 2012Description
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1688.86cm-1 1299.00cm-11321.01cm-1
756.11cm-11492.12cm-1 1105.21cm-1821.93cm-1
1228.35cm-11442.33cm-1
1594.43cm-1 693.19cm-11065.26cm-1
1030.78cm-1
1155.25cm-1
652.46cm-1
553.30cm-1
902.71cm-1
931.29cm-1
968.85cm-1
1805.62cm-11907.17cm-1
2815.03cm-1
2878.60cm-1
510.66cm-12919.61cm-1
3021.18cm-1
595.81cm-1
3320.55cm-1
3414.3
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1%
T
1680.40cm-1 1305.91cm-1
697.09cm-1
1326.10cm-1 752.51cm-1
1492.25cm-11107.19cm-1
1454.38cm-1
1595.21cm-11227.10cm-1
1069.57cm-1 1030.22cm-11160.09cm-1
927.37cm-1663.89cm-1
970.99cm-1
1813.01cm-11880.93cm-1
1949.89cm-1
604.62cm-1
3030.92cm-1
2806.17cm-1
2344.62cm-12887.48cm-1
572.44cm-1
542.78cm-1
2960.19cm-1
3304.85cm-1
3780.46cm-1
3430
Figure 63.IR spectrum of 3c
Figure64.IR spectrum of 3d
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1679.28cm-11488.04cm-1
1328.14cm-11305.41cm-1
827.50cm-11069.64cm-1755.60cm-1
1009.22cm-1
1106.07cm-1
1593.04cm-1
1227.54cm-1
1410.88cm-1
1201.27cm-1
694.36cm-1
1159.89cm-1
901.68cm-1
932.26cm-1
970.95cm-1
1902.23cm-1
620.43cm-1
538.19cm-12923.12cm-1
2814.41cm-1
3041.85cm-1
581.34cm-1
3300.26cm-1
2853.3
phpcl-2_001_1_1_1Name
56.7 %TCursor
Sample 005 By organic Date Wednesday, June 27 2012Description
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1676.18cm-1
1491.05cm-11329.26cm-1
1304.88cm-11093.77cm-1
1359.34cm-1
830.33cm-1 752.68cm-1
1593.84cm-11014.68cm-1
1227.07cm-1694.96cm-1
1413.45cm-1
1442.81cm-11202.49cm-1
1158.74cm-1540.05cm-1
903.28cm-1
932.05cm-1
633.98cm-1
971.26cm-1
1901.10cm-1
2853.24cm-1
3061.95cm-13315.95cm-1
583.87cm-1
3421.30cm-1
422.45cm-1
2810.4
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1%
T1242.80cm-1
1682.43cm-1
1506.04cm-11302.17cm-1
1031.18cm-11175.10cm-1
1606.17cm-1
1105.36cm-11449.12cm-1 833.40cm-1
752.95cm-1
691.92cm-1
927.85cm-1
1885.66cm-1 644.12cm-1
2832.12cm-1
2348.71cm-1
2954.06cm-1
561.09cm-1
3306.46cm-1
3415.82cm-1
Figure 65.IR spectrum of 3e
Figure 66.IR spectrum of 3f
phenylNO2_1_1_1Name
Psk By organic Date Monday, August 13 2012Description
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1345.76cm-11522.63cm-1
1679.21cm-1
1103.27cm-11596.29cm-1
694.37cm-1
738.52cm-1
810.03cm-1
3418.73cm-1908.22cm-1
2922.08cm-1
2852.62cm-1
3085.58cm-1
4000 4003500 3000 2500 2000 1500 1000 500
100
0
10
20
30
40
50
60
70
80
90
cm-1
%T
1679.28cm-11488.04cm-1
1328.14cm-11305.41cm-1
827.50cm-11069.64cm-1755.60cm-1
1009.22cm-1
1106.07cm-1
1593.04cm-1
1227.54cm-1
1410.88cm-1
1201.27cm-1
694.36cm-1
1159.89cm-1
901.68cm-1
932.26cm-1
970.95cm-1
1902.23cm-1
620.43cm-1
538.19cm-12923.12cm-1
2814.41cm-1
3041.85cm-1
581.34cm-1
3300.26cm-1
2853.3
Figure 67.Mass spectrum of3a
Figure 68.Mass spectrum of 3b
Figure 69.Mass spectrum of 3c
Figure 70. Mass spectrum of 3d
Figure 71. Mass spectrum of 3e
Figure 72. Mass spectrum of 3f