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Supporting Information

Synthesis and biological evaluation of oxindole linked indolyl-pyrimidine derivatives as potential cytotoxic agents

Santosh Kumar Prajaptia, Atulya Nagarsenkara, Sravanthi Gugillapua, Keshav Kumar Guptaa, Lingesh Allakondab, Manish Kumar Jeengarb, V. G. M. Naidub, Bathini Nagendra Babua,*

aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 500037, India.bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 500037, India.

*Corresponding Author: Tel.: +91 40 23073740; Fax: +91 40 23073751; E-mail address: [email protected]

Content

General experimental procedure and spectral data for compounds 2, 3, 5(a-j), 6(a-j), 7(a-

j) and 8(a-j)…...............................................................................................2-14

Experimental protocol for MTT assay, acridine orange assay, annexin binding assay, cell

cycle analysis………………………………………………….………………...14-16

1H and 13C NMR of compound 8e…………………………………………………….17

1

mailto:[email protected]

Experimental

Chemistry

General. All melting points were determined on a Stuart digital SMP-30 melting point

apparatus. IR spectra were recorded on a Perkin Elmer, FT-IR spectrometer using KBr discs. 1H

and 13C NMR spectra were recorded on either Bruker AVANCE-I 300 MHz NMR spectrometer,

Bruker AVANCE-II 300 MHz and Inova 500 and recorded in CDCl3, DMSO-d6 or DMSO-d6 +

CDCl3 solvents. Chemical shift were reported in parts per million (ppm) with respect to internal

standard tetramethylsilane (TMS). Mass spectra were obtained on Agilent technologies

LC/HRMS-TOF spectrometer. TLC experiments were performed on 0.2 mm Merck pre-coated

silica gel 60 F254 aluminum sheets and the spots were visualized under a UV lamp or by exposure

in iodine vapors. Column chromatography was performed using silica gel (60-120, 100-200

mesh) and the column was usually eluted with EtOAc/hexane.

Experimental procedure for the synthesis of 5-bromo-1H-indole-3-carbaldehyde (2)

To DMF (7 mL), phosphorus oxychloride (POCl3, 1 mL) was added dropwise at 0 °C. The

mixture was stirred for 20 min, then a solution of 5-bromoindole 1 (10 mmol), dissolved in DMF

(3 mL), was added dropwise. The mixture was stirred at 35°C for 1h and then heated at 75°C for

4h. On cooling, the mixture was poured into cold solution of sodium hydroxide to quench

residual POCl3, and the precipitated product 2 was collected, washed and dried.

Light yellow Solid; Yield: 95%; mp 190-192 °C; IR (KBr)νmax 3220, 3103, 2925, 1707, 1642,

1439, 1229, 1128, 885, 798, 781; 1H NMR (DMSO-d6, 300 MHz) δ 12.31 (brs, 1H), 9.93 (s, 1H),

8.35 (s, 1H), 8.22 (d, 1H, J = 1.70 Hz), 7.51 (d, J = 8.69 Hz, 1H), 7.41 (dd, J = 1.88, 8.69 Hz,

1H); TOF-HRMS (m/z) for C9H9BrNO, calculated 223.9711, observed 223.9704 [M+1] +

Experimental procedure for synthesis of 5-bromo-1-methyl-1H-indole-3-carbaldehyde (3)

5-bromoindole-3-carbaldehyde 2 (10 mmol) was dissolved in acetonitrile and to this powdered

NaOH (5 mmol) was added and stirred for 10 min. Methyl iodide (10 mmol) was added

dropwise to the reaction mixture. After 3h of stirring at room temperature, the solvent was

completely evaporated. It was extracted with ethyl acetate (3x20 ml) and dried over Na2SO4. The

combined organic layer was concentrated in vacuo to give a light yellow color solid purified by

recrystallization with diethyl ether.

2

Pale yellow solid; Yield: 96%; mp 122-124 °C; IR (KBr) νmax 3103, 2923, 2813, 1700, 1654.

1533, 1467, 1369, 1083, 799, 726; 1H NMR (DMSO-d6, 300 MHz) δ 9.88 (s, 1H), 8.33 (s, 1H),

8.23 (d, J = 1.70 Hz, 1H), 7.60 (d, J = 8.68 Hz, 1H), 7.48 (dd, J = 2.08, 8.87 Hz, 1H), 3.89 (s,

3H); TOF-HRMS (m/z) for C10H8BrNO, calculated 237.9862, observed 237.9855 [M+1] +

General experimental procedure for synthesis of Enaminones (5)

To a solution of acetophenone (1 mmol) in dry xylene (5 ml), was added a solution of DMF-

DMA (2 mmol) and refluxed for 48h at 120°C. After cooling, the solvent was evaporated and

residue was extracted with ethyl acetate (3x20ml). The combined organic layer was concentrated

in vacuo and purified by recrystallisation with diethyl ether.

(E)-3-(dimethylamino)-1-phenylprop-2-en-1-one (5a)

Pale yellow solid; Yield: 92%; mp 90-92 °C; IR (KBr) νmax 3266, 3068, 2924, 1630, 1536, 1446,

1403, 1300, 1131, 1073, 896, 799, 713; 1H NMR (DMSO-d6, 300 MHz) δ 7.89 (dd, J = 1.32,

7.55 Hz, 2H), 7.93 (d, J = 12.46 Hz, 1H), 7.46 (m, 3H), 5.74 (d, J = 12.26 Hz, 1H), 3.18 (s, 3H),

2.95 (s, 3H); TOF-HRMS (m/z) for C11H13NO, calculated 176.1070, observed 176.1701 [M+1] +

(E)-3-(dimethylamino)-1-(p-tolyl)prop-2-en-1-one (5b)

Pale yellow solid; Yield: 90%; mp 94-96 °C; IR (KBr) νmax 2918, 2806, 1638, 1578, 1537, 1434,

1356, 1277, 1111, 1055, 901, 768; 1H NMR (DMSO-d6, 300 MHz) δ 7.80 (d, J = 8.12 Hz, 2H),

7.70 (d, J = 12.28 Hz, 1H), 7.24 (d, J = 7.93 Hz, 2H), 5.82 (d, J = 12.28 Hz, 1H), 3.12 (s, 3H),

2.90 (s, 3H), 2.34 (s, 3H); TOF-HRMS (m/z) for C12H15NO, calculated 190.1226, observed

190.1218 [M+1] +

(E)-3-(dimethylamino)-1-(4-methoxyphenyl)prop-2-en-1-one (5c)

Brown solid; Yield: 85%; mp 73-75 °C; IR (KBr) νmax 2922, 2360, 1637, 1578, 1436, 1361, 1240,

1107, 901, 772; 1H NMR (DMSO-d6, 300 MHz) δ 7.89 (d, J = 8.49 Hz, 2H), 7.76 (d, J = 12.28

Hz, 1H), 6.92 (d, J = 8.69 Hz, 2H), 5.73 (d, J = 12.28 Hz, 1H), 3.85 (s, 3H), 3.07 (s, 6H); TOF-

HRMS (m/z) for C12H15NO2, calculated 206.1176, observed 206.1174 [M+1] +

(E)-3-(dimethylamino)-1-(4-hydroxyphenyl)prop-2-en-1-one (5d)

White solid; Yield: 80%; mp 102-104 °C; IR (KBr) νmax 3359, 2952, 2370, 1670, 1540, 1389,

1346, 1270, 1087, 901, 832; 1H NMR (DMSO-d6, 300 MHz) δ 7.84 (d, J = 7.89 Hz, 2H), 7.58 (d,

3

J = 12.3 Hz, 1H), 7.13 (d, J = 7.84 Hz, 2H), 6.83 (d, J = 12.28 Hz, 1H), 3.09 (s, 6H); TOF-

HRMS (m/z) for C11H13NO2, calculated 192.1019, observed 192.1012 [M+1] +

(E)-1-(4-chlorophenyl)-3-(dimethylamino)prop-2-en-1-one (5e)

Off white solid; Yield: 93%; mp 82-84 °C; IR (KBr) νmax 3193, 2924, 2361, 1577, 1466, 1236,

898, 748; 1H NMR (DMSO-d6, 300 MHz) δ 7.92 (d, J = 8.69 Hz, 2H), 7.74 (d, J = 12.28 Hz,

1H), 7.50 (d, J = 8.50 Hz, 2H), 5.83 (d, J = 12.09 Hz, 1H), 3.14 (s, 3H), 2.92 (s, 3H); TOF-

HRMS (m/z) for C11H12ClNO, calculated 210.0680, observed 210.0676 [M+1] +

(E)-3-(dimethylamino)-1-(4-fluorophenyl)prop-2-en-1-one (5f)

Brown solid; Yield: 95%; mp 74-76 °C; IR (KBr) νmax 2926, 1642, 1598, 1438, 1359, 1224,

1060, 902, 848, 778; 1H NMR (DMSO-d6, 300 MHz) δ 7.96 (dd, J = 5.67, 8.88 Hz, 2H), 7.74 (d,

J = 12.28 Hz, 1H), 7.19 (t, J = 8.88 Hz, 2H), 5.79 (d, J = 12.28 Hz, 1H), 3.17 (s, 3H), 2.94 (s,

3H); TOF-HRMS (m/z) for C11H12FNO, calculated 194.0976, observed 194.0970 [M+1] +

(E)-3-(dimethylamino)-1-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (5g)



J = 12.28 Hz, 1H), 7.68 (d, J = 7.93 Hz, 2H), 5.72 (d, J = 12.28 Hz, 1H), 3.21 (s, 3H), 2.97 (s,

3H); TOF-HRMS (m/z) for C12H12F3NO, calculated 244.0944, observed 244.0942 [M+1]+

(E)-1-(2,4-dichlorophenyl)-3-(dimethylamino)prop-2-en-1-one (5h)


1358, 1080, 899, 775; 1H NMR (DMSO-d6, 300 MHz) δ 7.47 (m, 4H), 5.33 (d, J = 12.65 Hz,

1H), 2.90 (s, 3H), 2.79 (s, 3H); TOF-HRMS (m/z) for C11H11Cl2NO, calculated 244.0290,

observed 244.0291 [M+1] +

(E)-3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one (5i)


1067, 901, 772; 1H NMR (DMSO-d6, 300 MHz) δ 9.06 (d, 1H, J = 1.51 Hz), 8.66 (dd, J = 1.51,

4.72 Hz, 1H), 8.23 (td, J = 1.89, 3.78 Hz, 1H), 7.78 (d, J = 12.09 Hz, 1H), 7.47 (m, 1H), 5.88 (d,

4

1H, J = 12.09 Hz), 3.16 (s, 3H), 2.93 (s, 3H); TOF-HRMS (m/z) for C10H12N2O, calculated

177.1022, observed 177.1016 [M+1] +

(E)-3-(dimethylamino)-1-(thiophen-2-yl)prop-2-en-1-one (5j)

Black solid; Yield: 94%; mp 106-108 °C; IR (KBr) νmax 3070, 2921, 2361, 1635, 1543,1409,

1352, 1247, 1113, 1065, 887, 763; 1H NMR (DMSO-d6, 300 MHz) δ 7.76 (dd, J = 1.13, 3.78

Hz, 1H), 7.73 (dd, J = 1.13, 4.91 Hz, 1H), 7.68 (d, J = 12.28 Hz, 1H), 7.15 (dd, J = 3.78, 4.91

Hz, 1H), 5.78 (d, J = 12.28 Hz, 1H), 3.13 (s, 3H), 2.89 (s, 3H); TOF-HRMS (m/z) for C9H11NOS,

calculated 182.0634, observed 182.0632 [M+1] +

General experimental procedure for synthesis of aminopyrimidines (6)

A mixture of compound 5 (1 mmol), guanidine nitrate (2 mmol), anhydrous K2CO3 (5 mmol) in

n-butanol (10 ml) was refluxed for 12 h. After cooling, the solution was poured into H2O (30 ml)

and then extracted with ethyl acetate (3x20 ml). The combined organic layer was concentrated in

vacuo and then purified by recrystallisation with diethyl ether.

4-phenylpyrimidin-2-amine (6a)


1343, 1210, 822, 766, 701; 1H NMR (DMSO-d6, 300 MHz) δ 8.34 (d, J = 5.29 Hz, 1H), 8.02 (m,

2H), 7.48 (m, 3H), 7.04 (d, 1H, J = 5.09 Hz), 5.68 (brs, 2H); 13C NMR (DMSO-d6, 75 MHz) δ

163.61, 163.48, 158.45, 136.85, 129.94, 128.19, 126.38, 105.69; TOF-HRMS (m/z) for C10H9N3,


4-(p-tolyl)pyrimidin-2-amine (6b)



J = 8.12 Hz, 2H), 7.29 (d, J = 8.12 Hz, 2H), 7.02 (d, J = 5.28 Hz, 1H), 5.44 (brs, 2H), 2.41 (s,

3H); 13C NMR (DMSO-d6, 75 MHz) δ 163.52, 163.38, 158.19, 139.76, 134.00, 128.79, 126.26,

105.36, 20.68; TOF-HRMS (m/z) for C11H11N3, calculated 186.1026, observed 186.1023 [M+1] +

4-(4-methoxyphenyl)pyrimidin-2-amine (6c)

Yellow solid; Yield: 89%; mp 193-95 °C; IR (KBr) νmax 3464, 3286, 3153, 2932, 2360, 1624,

1545, 1458, 1341, 1248, 1177, 1105, 1025, 806, 708; 1H NMR (DMSO-d6, 300 MHz) δ 8.28 (d,

5

J = 5.23 Hz, 1H), 8.00 (d, J = 8.80 Hz, 2H), 6.99 (m, 3H), 5.57 (brs, 2H), 3.87 (s, 3H); TOF-

HRMS (m/z) for C11H11N3O, calculated 202.0975, observed 202.0973 [M+1] +

4-(2-aminopyrimidin-4-yl)phenol (6d)

Light brown solid; Yield: 90%; mp 190-193 °C; IR (KBr) νmax 3392, 2924, 2361, 1579, 1236,

1111, 749; 1H NMR (DMSO-d6, 300 MHz) δ 13.83 (brs, 1H), 8.35 (d, J = 5.50 Hz, 1H), 7.78 (d,

J = 7.98 Hz, 1H), 7.35 (t, J = 7.15 Hz, 1H), 7.09 (d, J = 5.50 Hz, 1H), 6.95 (t, J = 8.80 Hz, 2H),

6.38 (brs, 2H); 13C NMR (DMSO-d6, 75 MHz) δ 164.08, 159.42, 132.34, 127.32, 118.52, 117.86,

117.01, 103.75; TOF-HRMS (m/z) for C10H9N3O, calculated 188.0818, observed 188.0818

[M+1] +

4-(4-chlorophenyl)pyrimidin-2-amine (6e)



8.07 (dd, J = 2.80, 8.41 Hz, 2H), 7.51 (dd, J = 3.73, 8.41 Hz, 2H), 7.08 (t, J = 4.67 Hz, 1H); 13C

NMR (DMSO-d6, 75 MHz) δ 163.71, 162.26, 159.31, 135.76, 135.19, 128.76, 128.66, 128.42,

105.65; TOF-HRMS (m/z) for C10H8ClN3, calculated 206.0480, observed 206.0477 [M+1] +

4-(4-fluorophenyl)pyrimidin-2-amine (6f)



8.06 (dd, J = 5.50, 9.08 Hz, 2H), 7.18 (t, J = 8.53 Hz, 2H), 7.00 (d, J = 5.23 Hz, 1H), 5.63 (brs,

2H); 13C NMR (DMSO-d6, 75 MHz) δ 162.37, 158.87, 130.28, 128.91, 115.52, 115.24, 114.02,

105.43; TOF-HRMS (m/z) for C10H8FN3, calculated 190.0775, observed 190.0768 [M+1] +

4-(4-(trifluoromethyl)phenyl)pyrimidin-2-amine (6g)

Light Brown solid; Yield: 95%; mp 180-182 °C; IR (KBr) νmax 3489, 3293, 3169, 2362, 1636,

1559, 1466, 1321, 1178, 1131, 1063, 863, 815, 685; 1H NMR (DMSO-d6, 300 MHz) δ 8.39 (d, J

= 5.29 Hz, 1H), 8.28 (d, J = 8.12 Hz, 2H), 7.88 (d, J = 8.31 Hz, 2H), 7.22 (d, J = 5.10 Hz, 1H),

6.82 (brs, 2H); 13C NMR (DMSO-d6, 75 MHz) δ 163.74, 161.93, 159.44, 140.82, 130.45, 127.35,

125.45, 122.20, 106.15; TOF-HRMS (m/z) for C11H8F3N3, calculated 240.0743, observed

240.0747 [M+1] +

4-(2,4-dichlorophenyl)pyrimidin-2-amine (6h)

6

Bright brown solid; Yield: 95%; mp 173-175 °C; IR (KBr) νmax 3070, 2919, 2360, 1634, 1543,

1515, 1409, 1352, 1247, 1113, 1065, 1012, 971, 887, 850, 780, 763; 1H NMR (DMSO-d6, 300

MHz) δ 8.34 (d, J = 5.10 Hz, 1H), 7.75 (d, J = 1.70 Hz, 1H), 7.59 (m, 2H), 6.82 (m, 3H); 13C

NMR (DMSO-d6, 75 MHz) δ 163.57, 163.15, 158.36, 136.30, 134.26, 132.16, 131.90, 129.32,

127.49, 109.99; TOF-HRMS (m/z) for C10H7Cl2N3, calculated 240.0090, observed 240.0088

[M+1] +

4-(pyridin-3-yl)pyrimidin-2-amine (6i)

Bown solid; Yield: 93%; mp 178-180 °C; IR (KBr) νmax 3328, 3152, 1655, 1553, 1474, 1344,

1217, 1110, 831, 798, 715, 655; 1H NMR (DMSO-d6, 400 MHz) δ 9.22 (s, 1H), 8.67 (d, J = 3.89

Hz, 1H), 8.38 (m, 2H), 7.51 (dd, J = 4.68, 7.79 Hz, 1H), 7.16 (d, J = 4.68 Hz, 1H), 6.67 (brs,

2H); 13C NMR (DMSO-d6, 75 MHz) δ 163.71, 161.49, 159.28, 151.04, 147.87, 134.06, 132.40,

123.67, 105.9; TOF-HRMS (m/z) for C9H8N4, calculated 173.0822, observed 173.0817 [M+1] +

4-(thiophen-2-yl)pyrimidin-2-amine (6j)


1339, 1286, 1050, 858, 814, 751, 716, 628; 1H NMR (DMSO-d6, 400 MHz) δ 8.24 (d, J = 4.68

Hz, 1H), 7.87 (d, J = 3.90 Hz, 1H), 7.72 (d, J = 4.68 Hz, 1H), 7.19 (t, J = 3.89 Hz, 1H), 7.06 (d, J

= 4.68 Hz, 1H), 6.66 (brs, 2H); 13C NMR (DMSO-d6, 75 MHz) δ 163.39, 158.82, 158.64, 142.76,

129.77, 128.32, 127.52, 104.23; TOF-HRMS (m/z) for C8H7N3S, calculated 178.0433, observed

178.0427 [M+1] +

General experimental procedure for the synthesis of compound 7

The pyrimidine amine 6 (1.1 mmol), CuI (1.0 mmol) and anhydrous Cs2CO3 (2.0 mmol) were

added to a round bottom flask alongwith magnetic stirbar and closed well with a septum. The

flask was evacuated and back filled with nitrogen gas three times. Dioxane (15 ml), aryl halide 3

(1.0 mmol) and DMEDA (1.0 mmol) were added by syringe at room temperature. The reaction

mixture was stirred at 80°C for 20 h under nitrogen atmosphere and then cooled to room

temperature. Concentrated ammonia (4 ml) was added, and the mixture was extracted with ethyl

acetate (3x20 ml). The combined organic layer was concentrated in vacuo, and the residue was

purified by column chromatography on silica gel.

1-methyl-5-((4-phenylpyrimidin-2-yl)amino)-1H-indole-3-carbaldehyde (7a)

7

Light brown solid; Yield: 64%; mp 191-193 °C; IR (KBr) νmax 3246, 2924, 1654, 1560, 1407,

1115, 805, 758, 727, 678, 628; 1H NMR (DMSO-d6, 300 MHz) δ 9.98 (s, 1H), 8.99 (m, 2H), 8.49

(d, J = 5.10 Hz, 1H), 8.26 (d, J = 6.61 Hz, 2H), 7.83 (s, 1H), 7.66 (dd, J = 2.08, 8.88 Hz, 1H),

7.57 (m, 3H), 7.37 (d, J = 8.69 Hz, 1H), 7.20 (d, J = 5.10 Hz, 1H), 3.90 (s, 3H); 13C NMR

(DMSO-d6, 75 MHz) δ 182.23, 162.28, 159.02, 157.13, 139.13, 135.39, 134.75, 132.05, 129.03,

127.21, 125.64, 123.63, 115.94, 115.74, 109.78, 108.55, 105.68, 32.03; TOF-HRMS (m/z) for

C20H16N4O, calculated 329.1397, observed 329.1388 [M+1] +

1-methyl-5-((4-(p-tolyl)pyrimidin-2-yl)amino)-1H-indole-3-carbaldehyde (7b)


1442, 1306, 1188, 1135, 1068, 808, 786, 634; 1H NMR (DMSO-d6, 300 MHz) δ 9.95 (s, 1H),

9.33 (brs, 1H), 9.05 (s, 1H), 8.46 (d, J = 5.10 Hz, 1H), 8.20 (d, J = 8.12 Hz, 2H), 7.96 (s, 1H),

7.63 (dd, J = 1.89, 8.68 Hz, 1H), 7.39 (t, J = 7.18 Hz, 3H), 7.21 (d, J = 5.29 Hz, 1H), 3.91 (s,

3H), 2.43 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 183.88, 163.24, 160.22, 158.82, 141.47,

140.62, 136.15, 133.77, 133.29, 129.39, 127.01, 124.72, 116.99, 116.75, 110.54, 106.89, 33.29,

20.89;TOF-HRMS (m/z) for C21H18N4O, calculated 343.1553, observed 343.1545 [M+1] +

5-((4-(4-methoxyphenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indole-3-carbaldehyde (7c)


1175, 1025, 804, 729; 1H NMR (DMSO-d6, 300 MHz) δ 9.91 (s, 1H), 9.62 (s, 1H), 8.99 (brs,

1H), 8.48 (d, J = 5.29 Hz, 1H), 8.34 (d, J = 8.69 Hz, 2H), 8.20 (s, 1H), 7.60 (dd, J = 1.70, 8.88

Hz, 1H), 7.52 (d, J = 8.69 Hz, 1H), 7.35 (d, J = 5.29 Hz, 1H), 7.12 (d, J = 8.88 Hz, 2H), 3.88 (s,

3H), 3.85 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 183.91, 162.91, 161.43, 160.16, 158.58,

141.49, 136.20, 133.26, 128.86, 128.72, 124.71, 116.98, 116.72, 114.09, 110.52, 106.44, 55.25,

33.29; TOF-HRMS (m/z) for C21H18N4O2, calculated 359.1503, observed 359.1496 [M+1] +

5-((4-(4-hydroxyphenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indole-3-carbaldehyde (7d)

Light yellow solid; Yield: 63%; mp 254-256°C; IR (KBr) νmax 3429, 3247, 2923, 1654, 1593,

1435, 1120, 818, 788, 759, 631; 1H NMR (DMSO-d6, 300 MHz) δ 13.06 (brs, 1H), 9.96 (s, 1H),

9.87 (s, 1H), 8.57 (d, 1H, J = 5.29 Hz), 8.37 (brs, 1H), 8.24 (s, 1H), 8.04 (d, J = 7.17 Hz, 1H),

7.67 (d, J = 8.49 Hz, 1H), 7.57 (d, J = 8.88 Hz, 1H), 7.49 (d, J = 5.47 Hz, 1H), 7.39 (t, J = 7.37

8

Hz, 1H), 6.95 (m, 2H), 3.89 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 183.98, 164.10, 159.71,

159.21, 158.38, 141.70, 134.89, 134.14, 132.79, 127.93, 124.77, 118.89, 117.97, 117.36, 116.78,

110.78, 106.16, 33.35; TOF-HRMS (m/z) for C20H16N4O2, calculated 345.1346, observed

345.1344 [M+1] +

5-((4-(4-chlorophenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indole-3-carbaldehyde (7e)


1403, 1250, 1085, 885, 797, 765, 726; 1H NMR (DMSO-d6, 300 MHz) δ 9.96 (s, 1H), 9.47 (brs,

1H), 9.14 (s, 1H), 8.50 (d, J = 3.59 Hz, 1H), 8.34 (d, J = 8.31 Hz, 2H), 7.98 (m, 1H), 7.56 (d, J =

8.12 Hz, 3H), 7.39 (d, J = 8.69 Hz, 1H), 7.25 (m, 1H), 3.91 (s, 3H); 13C NMR (DMSO-d6, 75

MHz) δ 183.77, 162.02, 160.15, 158.89, 141.17, 135.96, 135.59, 135.32, 133.25, 128.69, 128.64,

124.73, 117.05, 116.75, 110.68, 110.19, 106.87, 33.28; TOF-HRMS (m/z) for C20H15ClN4O,


5-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indole-3-carbaldehyde (7f)



9.71 (s, 1H), 9.03 (brs, 1H), 8.56 (d, J = 5.29 Hz, 1H), 8.45 (dd, J = 5.67, 8.69 Hz, 2H), 8.21 (s,

1H), 7.59 (dd, J = 1.89, 8.88 Hz, 1H), 7.52 (d, J = 8.88 Hz, 1H), 7.42 (m, 3H), 3.88 (s, 3H); 13C

NMR (DMSO-d6, 75 MHz) δ 183.99, 165.41, 162.14, 160.18, 159.09, 141.57, 136.05, 133.32,

133.06, 129.56, 124.70, 116.99, 116.75, 115.79, 115.51, 110.58, 106.99, 33.30; TOF-HRMS

(m/z) for C20H15FN4O, calculated 347.1303, observed 347.1294 [M+1] +

1-methyl-5-((4-(4-(trifluoromethyl)phenyl)pyrimidin-2-yl)amino)-1H-indole-3 carbaldehyde (7g)


1326, 1153, 1115, 1067, 797; 1H NMR (DMSO-d6, 300 MHz) δ 9.93 (s, 1H), 9.78 (s, 1H), 9.14

(brs, 1H), 8.61 (d, 2H, J = 5.10 Hz), 8.56 (s, 1H), 8.17 (s, 1H), 7.91 (d, J = 8.31 Hz, 2H), 7.58

(dd, J = 1.88, 8.88 Hz, 1H), 7.48 (d, J = 3.97 Hz, 1H), 7.45 (s, 1H), 3.90 (s, 3H); 13C NMR

(DMSO-d6, 75 MHz) δ 183.83, 161.61, 160.19, 159.25, 141.29, 140.36, 135.87, 133.27, 130.90,

130.47, 127.71, 125.43, 124.69, 117.03, 116.72, 110.65, 110.27, 107.48, 33.25; TOF-HRMS

(m/z) for C21H15F3N4O, calculated 397.1271, observed 397.1262 [M+1] +

5-((4-(2,4-dichlorophenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3-carbaldehyde (7h)

9

Light yellow solid; Yield: 55%; mp 192-194 °C; IR (KBr) νmax 3261, 2922, 1644, 1568, 1487,

1409, 802, 733; 1H NMR (DMSO-d6, 300 MHz) δ 9.96 (s, 1H), 8.73 (d, 1H, J = 1.65 Hz), 8.63

(brs, 1H), 8.50 (d, J = 4.95 Hz, 1H), 7.90 (d, J = 8.25 Hz, 1H), 7.77 (s, 1H), 7.65 (dd, J = 2.20,

8.80 Hz, 1H), 7.55 (d, J = 1.65 Hz, 1H), 7.50 (dd, J = 1.92, 5.78 Hz, 1H), 7.34 (d, J = 8.80 Hz,

1H), 7.12, (d, J = 4.95 Hz, 1H), 3.88 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 183.55, 162.49,

160.13, 158.05, 141.09, 135.61, 135.53, 134.62, 133.46, 132.60, 132.06, 129.36, 127.49, 124.61,

117.10, 116.84, 111.58, 111.18, 110.13, 33.21; TOF-HRMS (m/z) for C20H14Cl2N4O, calculated

397.0617, observed 397.0610 [M+1] +

1-methyl-5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-3-carbaldehyde (7i)


1484, 1405, 1301, 1065, 1022, 888, 789, 733, 699; 1H NMR (DMSO-d6, 300 MHz) δ 9.95 (s,

1H), 9.52 (brs, 1H), 9.39 (s, 1H), 9.03 (s, 1H), 8.71 (m, 2H), 8.54 (d, J = 4.91 Hz, 1H), 7.99 (m,

1H), 7.64 (dd, J = 1.51, 8.88 Hz, 1H), 7.57 (dd, J = 5.10, 7.18 Hz, 1H), 7.41 (d, J = 8.69 Hz,

1H), 7.32 (m, 1H), 3.91 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 183.62, 161.20, 160.22,

158.90, 150.96, 147.97, 140.93, 135.75, 134.42, 133.32, 132.25, 124.71, 123.54, 116.97, 116.91,

110.95, 110.06, 107.13, 33.22; TOF-HRMS (m/z) for C19H15N5O, calculated 330.1349, observed

330.1341 [M+1] +

1-methyl-5-((4-(thiophen-2-yl)pyrimidin-2-yl)amino)-1H-indole-3-carbaldehyde (7j)


1430, 1364, 1274, 1234, 1121, 1052, 898, 741, 698, 659; 1H NMR (DMSO-d6, 300 MHz) δ 9.91

(s, 1H), 9.52 (brs, 1H), 8.72 (s, 1H), 8.43 (d, J = 5.10 Hz, 1H), 8.08 (s, 1H), 7.97 (d, J = 3.59 Hz,

1H), 7.76 (dd, J = 1.88, 8.88 Hz, 1H), 7.68 (d, J = 5.10 Hz, 1H), 7.45 (d, J = 8.88 Hz, 1H), 7.22

(t, J = 4.91 Hz, 1H), 7.17 (d, J = 5.10 Hz, 1H), 3.90 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ

183.23, 159.84, 158.44, 158.04, 142.48, 140.33, 135.72, 133.26, 129.37, 128.64, 127.95, 127.26,

119.06, 116.97, 110.92, 109.73, 105.47, 33.14; TOF-HRMS (m/z) for C18H14N4OS, calculated

335.0961, observed 335.0952 [M+1] +

General experimental procedure for synthesis of 8

Compound 7 (1mmol) and oxindole (1.2mmol) were dissolved in 15 mL of ethanol and stirred

for 5 mins. To the reaction mixture, catalytic amount of piperidine was added and refluxed for

10

6h. The ethanol was evaporated from the reaction mixture and was extracted with ethyl acetate

(3x20 ml). The combined organic layers were dried over sodium sulphate and concentrated by

evaporating the solvent to give desired product 8.

(E)-3-((1-methyl-5-((4-phenylpyrimidin-2-yl)amino)-1H-indol-3-yl)methylene)indolin-2-one (8a)



9.42 (s, 1H), 8.98 (brs, 1H), 8.77 (s, 1H), 8.52 (brs, 1H), 8.19 (m, 2H), 7.94 (s, 1H), 7.58 (m,

5H), 7.39 (d, J = 8.50 Hz, 1H), 7.21 (d, J = 3.78 Hz, 1H), 7.15 (t, J = 7.55 Hz, 1H) 6.96 (dd, J =

7.37, 15.49 Hz, 2H), 3.93 (s, 3H); 13C NMR (CDCl3+DMSO-d6, 75 MHz) δ 166.68, 162.17,

137.54, 135.71, 135.58, 133.46, 130.85, 128.74, 127.34, 127.16, 126.21, 125.15, 124.83, 124.15,

124.00, 118.67, 116.83, 116.07, 115.12, 114.94, 108.84, 108.57, 107.42, 106.29, 105.76, 31.94;

TOF-HRMS (m/z) for C28H21N5O, calculated 444.1819, observed 444.1814 [M+1]+; UHPLC

Purity (98.06%).

(E)-3-((1-methyl-5-((4-(p-tolyl)pyrimidin-2-yl)amino)-1H-indol-3-yl)methylene)indolin-2-one

(8b)



(brs, 1H), 8.51 (m, 1H), 8.15 (m, 3H), 7.98 (m, 1H), 7.65 (m, 4H), 7.33 (d, J = 7.74 Hz, 1H),

7.27 (m, 1H), 7.15 (m, 1H), 6.98 (m, 2H), 3.98 (s, 3H), 2.42 (s, 3H); 13C NMR (DMSO-d6, 75

MHz) δ 169.69, 167.93, 163.26, 160.20, 158.29, 141.27, 140.12, 138.89, 136.88, 134.86, 132.09,

129.16, 126.79, 126.36, 125.49, 121.62, 120.47, 119.93, 117.41, 116.18, 109.93, 108.74, 108.08,

107.45, 106.67, 33.25, 20.81; TOF-HRMS (m/z) for C29H23N5O, calculated 458.1975, observed

458.1970 [M+1] +; UHPLC Purity (97.9%).

(E)-3-((5-((4-(4-methoxyphenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3

yl)methylene)indolin-2-one (8c)

Dark brown solid; Yield: 95%; mp 178-181 °C; IR (KBr) νmax 3122, 2923, 2853, 1683, 1603,

1488, 1347, 1249, 1173, 1095, 803, 744; 1H NMR (DMSO-d6, 300 MHz) δ 10.55 (s, 1H), 9.57

(s, 1H), 9.35 (s, 1H), 8.74 (brs, 1H), 8.50 (m, 1H), 8.25 (d, J = 7.55 Hz, 2H), 7.99 (s, 1H), 7.52

(s, 2H), 7.33 (s, 1H), 7.22 (m, 2H), 7.06 (d, J = 8.50 Hz, 2H) 6.94 (m, 2H), 3.94 (s, 3H), 3.75 (s,

11

3H); 13C NMR (DMSO-d6, 75 MHz) δ 161.33, 160.39, 158.65, 139.04, 137.11, 134.98, 132.35,

129.24, 128.34, 126.60, 126.08, 123.85, 121.95, 120.89, 120.33, 120.11, 118.12, 114.37, 114.17,

110.58, 110.17, 109.26, 108.91, 108.36, 106.65, 55.18, 33.51; TOF-HRMS (m/z) for C29H23N5O2,

calculated 474.1925, observed 474.1914 [M+1]+; UHPLC Purity (97.6%).

(E)-3-((5-((4-(4-hydroxyphenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3

yl)methylene)indolin-2-one (8d)


1489, 1351, 1296, 1194, 1116, 786, 753; 1H NMR (DMSO-d6, 300 MHz) δ 13.23 (brs, 1H),

10.54 (s, 1H), 9.89 (s, 1H), 9.39 (s, 1H), 8.59 (d, J = 5.29 Hz, 1H), 8.35 (brs, 1H), 8.04 (m, 2H),

7.76 (d, J = 7.37 Hz, 1H), 7.60 (m, 1H), 7.48 (m, 2H), 7.38 (t, J = 7.18 Hz, 1H), 7.15 (t, J = 7.74

Hz, 1H), 6.96 (dd, J = 7.55, 12.46 Hz, 3H), 6.87 (t, J = 5.85 Hz, 2H), 3.96 (s, 3H); 13C NMR

(DMSO-d6, 75 MHz) δ 168.02, 164.07, 159.84, 159.58, 158.90, 139.08, 137.39, 133.48, 133.35,

132.84, 128.96, 127.90, 126.67, 126.41, 125.58, 120.45, 118.95, 118.55, 118.41, 118.02, 117.43,

110.98, 110.36, 108.94, 105.86, 33.57; TOF-HRMS (m/z) for C28H21N5O2, calculated 460.1768,

observed 460.1762 [M+1]+; UHPLC Purity (97.93%).

(E)-3-((5-((4-(4-chlorophenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3-yl)methylene)indolin-

2-one (8e)



(s, 1H), 9.36 (s, 1H), 8.73 (brs, 1H), 8.59 (d, J = 4.91 Hz, 1H), 8.28 (d, J = 8.50 Hz, 2H), 7.98 (s,

1H), 7.62 (m, 5H), 7.41 (d, J = 5.10 Hz, 1H), 7.17 (t, J = 7.74 Hz, 1H), 6.98 (t, J = 7.55 Hz, 1H),

6.87 (d, J = 7.74, 1H), 3.94 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 167.95, 162.27, 160.43,

159.23, 139.04, 137.14, 135.84, 135.47, 134.75, 132.52, 128.91, 128.58, 126.60, 126.06, 125.50,

120.33, 118.16, 117.94, 116.78, 110.66, 110.19, 109.36, 108.99, 108.40, 107.36, 33.94; TOF-

HRMS (m/z) for C28H20ClN5O, calculated 478.1429, observed 478.1422 [M+1]+; UHPLC Purity

(96.74%).

(E)-3-((5-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3-yl)methylene)indolin-

2-one (8f)

12



(s, 1H), 9.35 (s, 1H), 8.70 (brs, 1H), 8.57 (d, J = 3.40 Hz, 1H), 8.31 (m, 2H), 7.98 (s, 1H), 7.62

(m,3H), 7.38 (m, 3H), 7.20 (m, 1H), 6.98 (m, 2H), 3.94 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ

167.91, 162.37, 160.46, 159.06, 139.02, 137.08, 134.76, 132.49, 129.07, 128.66, 126.57, 125.96,

125.46, 120.20, 118.14, 117.89, 116.79, 115.85, 115.57, 110.54, 110.14, 109.34, 108.93, 108.50,

107.20, 33.44; TOF-HRMS (m/z) for C28H20FN5O, calculated 462.1725, observed 462.1721

[M+1] +; UHPLC Purity (95.57%).

(E)-3-((1-methyl-5-((4-(4-(trifluoromethyl)phenyl)pyrimidin-2-yl)amino)-1H-indol-3-

yl)methylene)indolin-2-one (8g)

Orange solid; Yield: 92%; mp 288-290 °C; IR (KBr) νmax 3283, 3134, 2923, 1675, 1570, 1490,


(s, 1H), 8.70 (brs, 1H), 8.65 (d, 1H, J = 5.10 Hz), 8.46 (d, J = 8.12 Hz, 2H), 8.01 (s, 1H), 7.89 (d,

J = 8.31 Hz, 2H), 7.64 (d, J = 7.55 Hz, 1H), 7.53 (s, 2H), 7.48 (d, J = 5.10 Hz, 1H), 7.16 (t, J =

7.55 Hz, 1H), 6.92 (dd, J = 7.55, 16.24 Hz, 2H), 3.94 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ

167.96, 161.89, 160.57, 159.59, 140.87, 139.04, 137.19, 134.62, 132.62, 130.32, 128.69, 127.59,

127.48, 126.58, 125.70, 122.19, 120.24, 118.24, 117.87, 116.90, 110.64, 110.19, 109.38, 108.99,

108.71, 107.94, 33.49; TOF-HRMS (m/z) for C29H20F3N5O, calculated 512.1693, observed

512.1685 [M+1]+; UHPLC Purity (95.39%).

(E)-3-((5-((4-(2,4-dichlorophenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3-

yl)methylene)indolin-2-one (8h)

Orange solid; Yield: 90%; mp 267-269 °C; IR (KBr) νmax 3275, 3128, 2923, 1673, 1572, 1490,


9.34 (s, 1H), 8.75 (brs, 1H), 8.61 (d, J = 4.91 Hz, 1H), 7.87 (s, 1H), 7.82 (m, 2H), 7.61 (dd, J =

1.88, 8.31 Hz, 1H), 7.55 (m, 2H), 7.46 (dd, J = 1.13, 8.88 Hz, 1H), 7.19 (t, J = 7.55 Hz, 1H),

7.07 (m, 2H), 6.87 (d, J = 7.55 Hz, 1H), 3.92 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 167.92,

163.02, 160.20, 158.66, 139.05, 137.14, 136.22, 134.65, 132.49, 132.11, 129.61, 128.69, 127.74,

126.62, 125.99, 125.48, 120.42, 118.08, 117.68, 116.64, 111.70, 110.67, 110.16, 109.04, 108.20,

33.48; TOF-HRMS (m/z) for C28H19Cl2N5O, calculated 512.1039, observed 512.1038 [M+1]+;

UHPLC Purity (97.25%).

13

(E)-3-((1-methyl-5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indol-3-yl)methylene)indolin-2-

one (8i)



9.43 (brs, 1H), 9.38 (s, 1H), 8.76 (d, J = 4.53 Hz, 1H), 8.71 (s, 1H), 8.63 (d, J = 5.10 Hz, 1H),

8.57 (d, J = 8.12 Hz, 1H), 7.98 (s, 1H), 7.73 (d, J = 7.37 Hz, 1H), 7.57 (m, 3H), 7.48 (d, J = 5.10

Hz, 1H), 7.17 (t, J = 7.55 Hz, 1H), 6.99 (t, J = 7.37 Hz, 1H), 6.86 (d, J = 7.55 Hz, 1H) 3.94 (s,

3H); 13C NMR (DMSO-d6, 75 MHz) δ 169.73, 167.97, 161.45, 160.52, 159.38, 151.29, 148.08,

138.99, 137.17, 134.68, 134.26, 132.58, 128.62, 126.58, 125.94, 125.46, 123.81, 122.18, 120.42,

118.24, 116.87, 110.64, 110.19, 109.33, 108.88, 107.65, 33.48; TOF-HRMS (m/z) for

C27H20N6O, calculated 445.1771, observed 445.1768 [M+1]+; UHPLC Purity (95.4%).

(E)-3-((1-methyl-5-((4-(thiophen-2-yl)pyrimidin-2-yl)amino)-1H-indol-3-yl)methylene)indolin-2-

one (8j)


1345, 1192, 1115, 1060, 786, 703, 685; 1H NMR (DMSO-d6, 300 MHz) δ 10.57 (s, 1H), 9.69 (s,

1H), 9.37 (s, 1H), 8.85 (brs, 1H), 8.52 (d, J = 5.10 Hz, 1H), 8.06 (d, J = 3.02 Hz, 2H), 7.71 (d, J

= 4.91 Hz, 1H), 7.68 (d, J = 7.42 Hz, 1H), 7.55 (d, J = 8.69 Hz, 1H), 7.48 (d, J = 8.69 Hz, 1H),

7.36 (d, J = 5.10 Hz, 1H), 7.30 (dd, J = 3.78, 4.91 Hz, 1H) 7.18 (t, J = 7.37 Hz, 1H), 7.01 (t, J =

7.37 Hz, 1H), 6.88 (d, J = 7.42 Hz, 1H), 3.94 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 167.94,

160.01, 158.86, 158.38, 142.90, 139.07, 137.18, 134.96, 132.31, 129.95, 129.03, 128.68, 128.13,

126.65, 126.11, 125.57, 120.38, 118.13, 117.75, 116.34, 110.72, 110.25, 109.08, 107.52, 105.69,

33.49; TOF-HRMS (m/z) for C26H19N5OS, calculated 450.1383, observed 450.1374 [M+1]+;

UHPLC Purity (96.3%).

Biological evaluation

Cytotoxicity assay

Cytotoxicity of compound was determined by MTT assay based on mitochondrial reduction of

yellow MTT tetrazolium dye to a highly colored blue formazan product. 1x104 Cells (counted by

Trypan blue exclusion dye method) in 96- well plates were incubated with compounds with

series of concentrations for 48 h at 37 °C in corresponding medium with 10% FBS medium. The

14

above media was replaced with 90 µl of fresh serum free media and 10 µl of MTT reagent (5

mg/ml) and plates were incubated at 37 °C for 4 h, there after the above media was replaced with

200 µl of DMSO and incubated at 37 °C for 10 min. The absorbance at 570 nm was measured on

a spectrophotometer (spectra max, molecular devices) IC50 values were determined from plot: %

cell viabilty (from control) versus concentration.

Assessment of Cell Morphology

Cells (1x 106 cells /well) were grown in 6-well plates and treated with or without compound 8e at

concentrations 1 µM and 2.5 µM for 24 h. Morphological changes were observed with an

inverted phase contrast microscope (Model: Nikon,Japan) and photographs were taken with

digital camera (Nikon, Inc. Japan) at 100X magnification. The acridine orange/ethidium bromide

(AO/EB) staining procedure was followed to differentiate the live, apoptotic and necrotic cell as

reported previously. Briefly, treated or untreated cells were stained with acridine orange (10

µg/ml) and ethidium bromide (10 µg/ml) and analyzed under fluorescence microscope with

excitation (488 nm) and emission (550 nm) at 200X magnification.

Annexin V binding assay

It was performed by the previously reported method. Briefly, 1x106 cells were seeded in a 6 well

plate and treated with different concentrations of compound 8e for 24 h and stained with

Annexin V-FITC and propidium iodide according to manufacturer instructions using BD

Annexin V FITC Assay kit. Then cells were analyzed by flow cytometer BD FACSVerse™ (BD

Biosciences, USA). Apoptosis and necrosis were analyzed with quadrant statistics on propidium

iodide-negative cells, fluorescein positive cells and propidium iodide (PI)-positive cells,

respectively.

Cell cycle analysis

The distribution of DNA in the cell cycle was studied by flow cytometry. To determine the effect

of compound 8e on the cell cycle, cells were seeded in 6-well plates at a density of 1×105 cells/ml

for 24 h. After incubation, cells were treated with two concentrations of compound for 24 h in

PA-1 cell line. After treatment period, cells were collected, washed and fixed overnight in 70%

ethanol in PBS at -20 °C. Fixed cells were stained with cell cycle analysis reagent for 30 min at

15

37 °C in dark according to the manufacturer’s instruction, and about 10,000 events were

analysed on a flow cytometer BD FACSVerseTM.

1H and 13C NMR of compound 8e

16

1H NMR of Compound 8e

13C NMR of Compound 8e

17

ars.els-cdn.com€¦ · web viewall melting points were determined on a stuart digital smp-30...

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