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
1
Organic 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: thennarasu@gmail.com
Corresponding author. Tel.: +91 44 24910846/0897; fax: +91 44 24912150;
E-mail: abmandal@hotmail.com
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 (320 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), 2.172.28 (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):
-1
Colourless solid; mp 183-185oC; yield 72; IR (KBr cm ): 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):
-1
o
Colourless solid; mp 188-190 C; yield 76.7; IR (KBr cm ): 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):
-1
o
Colourless solid, mp 185-187 C, yield 79, IR (KBr cm ): 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):
-1
o
Brown solid, mp 133-135 C, yield 75, IR (KBr cm ): 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 (320 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), 7.197.22 (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 × 104 cells per well and
cultured for 24 h at 37C 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
100.0
90
80
454.49
70
614.40
481.34
60
%T
511.28
50
858.55
40
918.30
758.17
1019.02
1094.60
1178.56
30
3425.58
700.95
654.10
20
2369.86
10
2980.46
1723.20
1497.30
1654.83
1324.01
1462.30 1224.52
1404.90
0.0
4000.0
3000
2000
1500
1000
400.0
cm-1
Figure 13.IR spectrum of 1a
100
90
80
70
%T
60
50
1 9 0 3 .7 5 c m
- 1
6 4 7 . 2 5 c m
40
931.87cm-1
- 1
504.34cm-1
30
1315.27cm-1
20
1508.53cm-1
3412.26cm-1
10
2819.30cm-1
810.95cm-1
1208.73cm-1
3315.76cm-1
0
4000
3500
2976.71cm-1
2924.58cm-1
3000
2500
1632.96cm-1
1433.89cm-1
1721.69cm-1
2000
1500
cm-1
Name
Methyl pip_002_1_1_1
Description
Psk By organic Date Monday, August 13 2012
Figure 14.IR spectrum of 1b
1029.54cm-1
1164.00cm-1
1000
500 400
100
90
80
579.57cm-1
70
923.32cm-1
1894.10cm-1
2047.66cm-1
537.88cm-1
60
650.34cm-1
%T
3414.68cm-1
50
2840.54cm-1
3304.35cm-1
40
1116.26cm-1
2920.52cm-1
1611.73cm-1
30
1374.35cm-1
832.05cm-1
1452.57cm-1
1309.80cm-1
1 0 2 9 .4 8 c m -1
1511.53cm-1
20
1169.79cm-1
10
1724.36cm-1
1243.61cm-1
0
4000
3500
3000
2500
2000
1500
1000
500 450
cm-1
Name
meo-pip_1_1_1
Description
selva By organic Date tuesday, July 31 2012
Figure 15.IR spectrum of 1c
100
90
80
70
60
%T
512.50cm-1
50
717.66cm-1
686.42cm-1
637.23cm-1
40
3417.36cm-1
1905.79cm-1
943.35cm-1
2920.03cm-1
30
3324.57cm-1
2980.14cm-1
826.72cm-1
1318.52cm-1 1017.65cm-1
1488.14cm-1
1163.11cm-1
1219.73cm-1
1378.83cm-1 1089.97cm-1
20
10
1722.78cm-1
0
4000
3500
3000
2500
2000
1500
cm-1
Name
cl-pip_1_1_1
Description
selva By organic Date Tuesday, July 31 2012
Figure 16.IR spectrum of 1d
1000
500 450
100
90
80
70
509.97cm-1
60
7 1 5 .4 0 c m -1
%T
630.88cm-1
50
1906.75cm-1
40
2919.41cm-1
3320.76cm-1
1636.84cm-1
2979.19cm-1
1 0 7 2 .2 0 c m -1
30
824.30cm-1
3421.56cm-1
1317.76cm-1 1013.25cm-1
1484.47cm-1
1163.71cm-1
1220.72cm-1
20
1378.22cm-1
10
1722.16cm-1
0
4000
3500
3000
2500
2000
1500
1000
500 450
cm-1
Name
Description
Br-pip_1_1_1 selva By organic Date Tuesday, July 31 2012
Figure 17.IR spectrum of 1e
100
90
80
499.58cm-1
612.52cm-1
70
2856.67cm-1
60
909.19cm-1
%T
3080.20cm-1
50
8 5 9 .0 8 c m -1
2924.06cm-1
3407.05cm-1
7 3 9 . 9 0 c m
40
- 1
799.17cm-1
2983.51cm-1
1085.87cm-1
1274.36cm-1
1021.49cm-1
1472.94cm-1
3299.56cm-1
30
697.31cm-1
1228.43cm-1
1154.31cm-1
20
10
1526.56cm-1
1714.20cm-1
1349.47cm-1
0
4000
3500
3000
2500
2000
1500
cm-1
Name
Description
3NO-pip_1_1_1 selva By organic Date Tuesday, July 31 2012
Figure 18.IR spectrum of 1f
1000
500 450
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
100.0
90
80
70
%T
60
3025.63
2848.54
2925.70
50
40
3281.83
2372.17
30
1514.17
1416.14
20
10
759.78
613.98
1106.32
1334.78
1038.48
3371.06
1685.47
823.01
0.0
4000.0
3000
2000
1500
1000
400.0
cm-1
Figure 37.IR spectrum of2a
100.0
90
2340.35
80
1889.32
70
2274.04
2543.97 2054.55
60
%T
2370.01
1303.71
1241.26
50
641.46
963.59
921.84
40
1458.54
30
3303.93
2831.68
20
10
611.27
1031.92
489.92
1107.29
757.51 542.22
1511.72
3087.38
2944.38
1665.95
834.40
3210.70
1367.81
1611.97
1175.47
806.56
0.0
4000.0
3000
2000
1500
cm-1
Figure 38.IR spectrum of 2b
1000
400.0
100.0
572.30
964.82
1174.27
90
463.97
2341.66
80
413.84
1439.55
1901.23
2374.31
2276.67
70
634.16
492.21
918.57
2849.13
60
1230.93
%T
2953.12
714.40
602.51
50
2920.16
3318.53
40
1014.21
1414.54
3090.00
791.20
1305.75
531.60
30
1370.92
1489.86
3222.59
755.80
1091.49
20
828.78
10
1664.59
0.0
4000.0
1500
2000
3000
400.0
1000
cm-1
Figure 39.IR spectrum of 2c
100.0
90
1920.61
1843.68
1065.92
80
1211.79
70
2339.18
60
2367.85
1166.65
50
%T
1478.69
40
1238.16
1097.83
2931.09
1030.72
30
20
3425.21
3322.79
3086.86
3225.79
1663.81
10
1525.00 1353.37
3.2
3000
2000
cm-1
4000.0
Figure 40. IR spectrum of 2d
1500
1000.0
100
90
80
70
%T
60
50
1038.13cm-1
2810.4
964.82cm-1
901.59cm-1
487.82cm-1
1 9 0 4 .0 9 c m -1
527.57cm-1
40
631.22cm-1
3407.2
30
2854.20cm-1
3097.89cm-1
2922.93cm-1
599.45cm-1
1008.95cm-1
714.64cm-1
1230.48cm-1
1439.52cm -1
20
1412.15cm-1
3236.42cm-1
1102.71cm-1
758.74cm-1
1305.46cm-1
10
1485.23cm-1
1345.60cm-1
1070.16cm-1825.86cm-1
1674.04cm-1
0
0.00
4000
3500
3000
2500
2000
1500
1000
500 400
cm-1
Name
Description
hpbr_1_1_1 Sample 004 By organic Date Wednesday, June 27 2012
Figure 41.IR spectrum of 2e
100
90
80
2345.34cm-1
70
3322.72cm-1
60
%T
2853.64cm-1
3091.46cm-1
2926.43cm-1
1736.26cm-1
3417.46cm-1
473.15cm-1
542.70cm-1
964.91cm-1
906.57cm-1
600.36cm-1
643.12cm-1
1030.40cm-1
857.71cm-1
1236.25cm-1
1421.05cm-1
1094.91cm-1
766.99cm-1
1066.07cm-1
1477.58cm-1
1161.67cm-1
808.82cm-1
735.20cm-1
1309.95cm-1
688.48cm-1
3227.13cm-1
50
1642.80cm-1
40
1665.00cm-1
1353.79cm-1
30
1523.77cm-1
20
10
0
4000
3500
3000
2500
2000
1500
cm-1
Name
Description
1Tue_1_1_1_1_1 Sample 001 By organic Date Tuesday, October 16 2012
Figure 42.IR spectrum of 2f
1000
500 400
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
100
90
3780.46cm-1
80
70
%T
60
50
3304.85cm-1
542.78cm-1
3430
572.44cm-1
2960.19cm-1
40
2344.62cm-1
604.62cm-1
2887.48cm-1
1813.01cm-1
1880.93cm-1
1949.89cm-1
2806.17cm-1
30
663.89cm-1
927.37cm-1
3030.92cm-1
1595.21cm-1
970.99cm-1
20
1227.10cm-1
1069.57cm-1
1454.38cm-1
1030.22cm-1
1160.09cm-1
697.09cm-1
1107.19cm-1
10
1492.25cm-1
1305.91cm-1
1326.10cm-1
1680.40cm-1
0
4000
3500
3000
2500
2000
1500
752.51cm-1
1000
500 400
cm-1
Name
Description
3k_1_1_1 psk By organic Date Friday, July 06 2012
Figure 61.IR spectrum of 3a
100
90
80
70
60
%T
968.85cm-1
50
3414.3
931.29cm-1
3320.55cm-1
40
510.66cm-1
2919.61cm-1
595.81cm-1
2815.03cm-1
30
1805.62cm-1
1907.17cm-1
3021.18cm-1
2878.60cm-1
553.30cm-1
652.46cm-1
1065.26cm-1
20
1594.43cm-1
1228.35cm-1
1442.33cm-1
10
1492.12cm-1
3500
3000
2500
2000
693.19cm-1
902.71cm-1
1155.25cm-1
1105.21cm-1
821.93cm-1
756.11cm-1
1299.00cm-1
1321.01cm-1
1688.86cm-1
0
4000
1030.78cm-1
1500
cm-1
Name
Description
3l_1_1_1 psk By organic Date Friday, July 06 2012
Figure 62.IR spectrum of 3b
1000
500 400
100
90
80
70
%T
60
50
40
561.09cm-1
3415.82cm-1
2348.71cm-1
30
2954.06cm-1
644.12cm-1
1885.66cm-1
3306.46cm-1
691.92cm-1
2832.12cm-1
20
1606.17cm-1
752.95cm-1
1105.36cm-1
1449.12cm-1
10
833.40cm-1
927.85cm-1
1682.43cm-1
1302.17cm-1
1506.04cm-1
0
4000
3500
3000
2500
2000
1175.10cm-1
1031.18cm-1
1242.80cm-1
1500
1000
500 400
cm-1
Name
Description
3m_1_1_1_1_1 psk By organic Date Friday, July 06 2012
Figure 63.IR spectrum of 3c
100
90
80
422.45cm-1
70
932.05cm-1
60
%T
2810.4
50
1442.81cm-1
2853.24cm-1
583.87cm-1
971.26cm-1
1202.49cm-1
1413.45cm-1
1158.74cm-1
540.05cm-1
1901.10cm-1
40
903.28cm-1
633.98cm-1
3421.30cm-1
30
3315.95cm-1 3061.95cm-1
1227.07cm-1
1014.68cm-1
694.96cm-1
1593.84cm-1
20
830.33cm-1
752.68cm-1
1359.34cm-1
10
1491.05cm-1
0
4000.00
4000
1304.88cm-1
1329.26cm-1 1093.77cm-1
1676.18cm-1
3500
3000
2500
2000
1500
cm-1
Name
Cursor
Description
phpcl-2_001_1_1_1 56.7 %T Sample 005 By organic Date Wednesday, June 27 2012
Figure64.IR spectrum of 3d
1000
500 400
100
90
80
70
%T
60
50
970.95cm-1
2853.3
40
1305.41cm-1
1328.14cm-1
30
581.34cm-1
932.26cm-1
1159.89cm-1
2923.12cm-1
3041.85cm-1
1902.23cm-1
538.19cm-1
1201.27cm-1
620.43cm-1
20
3300.26cm-1
2814.41cm-1
1593.04cm -1
10
694.36cm-1
1009.22cm-1
1410.88cm-1
1069.64cm-1
901.68cm-1
827.50cm-1
1679.28cm-1
1488.04cm-1
0
4000
3500
3000
2500
755.60cm-1
2000
1227.54cm-1 1106.07cm-1
1500
1000
500 400
cm-1
Name
phpbr_1_1_1
Description
Sample 006 By organic Date Wednesday, June 27 2012
Figure 65.IR spectrum of 3e
100
90
80
70
%T
60
50
3085.58cm-1
40
2852.62cm-1
3418.73cm-1
908.22cm-1
2922.08cm-1
30
810.03cm-1
1596.29cm-1
1103.27cm-1
7
20
3
8
.
5
2
c
m
-
1
694.37cm-1
1679.21cm-1
10
1 3 4 5 .7 6 c m -1
1522.63cm-1
0
4000
3500
3000
2500
2000
1500
cm-1
Name
phenylNO2_1_1_1
Description
Psk By organic Date Monday, August 13 2012
Figure 66.IR spectrum of 3f
1000
500 400
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