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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

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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 28.1H NMR spectrum of 2d

Figure 29.1H NMR spectrum of 2e

Figure 30.1H NMR spectrum of 2f



Figure 3313C NMR spectrum of 2c




Figure 37. IR spectrum of 2a

Figure 38. IR spectrum of 2b

Figure 39. IR spectrum of 2c


















Figure 5713C NMR spectrum of 3c




Figure 61. IR spectrum of 3a

Figure 62. IR spectrum of 3b

Figure 63. IR spectrum of 3c





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





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



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


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


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 37.IR spectrum of2a


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



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



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 46.Mass spectrum of 2d

Figure47.Mass spectrum of 2e

Figure 48.Mass spectrum of 2f

Figure49.1H NMR spectrum of 3a




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



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


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



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


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