ars.els-cdn.com · web viewethyl acetate layer was washed with water and dried over mgso 4. ......

Supplementary Material

Pim kinase inhibitory and antiproliferative activity of a novel series of meridianin C

derivatives

Authors: Kunal N. More, Hyo Weon Jang, Victor S. Hong, and Jinho Lee

Synthesis

5-Bromo-1-tosyl-1H-indole (2)

To a solution of 5-bromoindole (2.0 g, 10 mmol) in N,N-dimethylformamide (DMF, 10

mL) was added 490 mg (20 mmol) of sodium hydride under N2 atmosphere. After stirring for

1 hr at RT, the reaction mixture was cooled to 0 ºC and was added 2.14 g (11.2 mmol) of

toluenesulfonyl chloride. The reaction mixture was stirred at same temperature for 3 hr. After

removal of solvent under reduced pressure, the residue was dissolved in ethyl acetate. Ethyl

acetate layer was washed with water and dried over MgSO4. After filtration, removal of

solvent under reduced provided 5-bromo-1-tosyl-1H-indole in quantitative yield (3.90 g); 1H

NMR (CDCl3, 400MHz) 7.85 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 2

Hz, 1H), 7.56 (d, J = 3.6 Hz, 1H), 7.39 (dd, J = 2 Hz, 8.8 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H),

6.58 (d, J = 3.6 Hz, 1H), 2.34 (s, 3H).

1-(5-Bromo-1-tosyl-1H-indole-3-yl)ethanone (3)

To a suspension of aluminium chloride (4.44 g, 33.4 mmol) in carbon disulfide (20 mL)

was added 1.18 mL (16.7 mmol) of acetyl chloride. After stirring for 1 hr at RT, was added

carbon disulfide solution of 5-bromo-1-tosyl-1H-indole (3.90 g, 11.1 mmol) to the reaction

mixture and stirred for 2 hr at RT. The reaction mixture was poured into 150 mL of ice cold

water. After removal of volatiles under reduced pressure, the reaction product was extracted

with ethyl acetate. Ethyl acetate layer was dried over MgSO4 and filtered. Removal of solvent

under reduced pressure provided compound 2 in 96 % yield (4.20 g); 1H NMR (CDCl3, 400

MHz) 8.49 (d, J = 4 Hz, 1H), 8.17 (s, 1H), 7.8 (t, J = 8.6 Hz, 3H), 7.46 (dd, J = 2 Hz, 8.8

Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 2.55 (s, 3H), 2.38 (s, 3H).

유기합성실험실, 01/15/14,

1-(5-Bromo-1-tosyl-1H-indole-3-yl)-3-(dimethylamino)prop-2-en-1-one (4)

To Ar gas purged DMF (2 mL) in microwave reaction vessel were added compound 2

(2.00 g, 5.09 mmol) and N,N-dimethylformamide diethylacetal (1.30 mL,7.64 mmol). The

reaction mixture was heated in the microwave reactor at power 100 W and 110 °C for 10 min.

After solvents were removed under reduced pressure, the residue was loaded directly on the

flash column for purification. The purification by flash chromatography eluted with

dichloromethane: ethyl acetate (DCM:EA, 9:1) mixture provided the compound 3 (1.73 g) in

76% yield; 1H NMR (CDCl3, 400 MHz) 8.53 (d, J = 2 Hz, 1H), 8.05 (s, 1H), 7.80~7.75 (m,

4H), 7.41 (dd, J = 2 Hz, 8.8 Hz, 1H), 7.24 (d, J = 8.4 Hz, 2H), 5.55 (d, J = 12.4 Hz, 1H), 3.16

(br, 3H), 2.95 (br, 3H), 2.35 (s, 3H).

Meridianin C:

To 2-methoxyethanol (2.5 mL) in microwave reaction vessel were added compound 4 (100

mg, 0.224 mmol), K2CO3 (0.062 g, 0.45 mmol), and guanidine carbonate (0.081 g, 0.45

mmol). The reaction mixture was heated in the microwave reactor at power 100 W and 150

°C for 10 min. After removal of solvent, the residue was purified by flash column

chromatography to obtain Meridianin C; 1H NMR (400 MHz, DMSO-d6): δ 11.86 (1H, s),

8.76 (1H, d, J = 1.6 Hz), 8.26 (1H, s), 8.11 (1H, d, J = 5.2 Hz), 7.41 (1H, d, J = 8.4 Hz), 7.29

(1H, dd, J1 = 8.8 Hz, J2 = 1.6 Hz), 7.00 (1H, d, J = 5.2 Hz), 6.49 (2H, s).13C NMR (100 MHz,

DMSO-d6): δ 164.02, 162.68, 157.61, 136.18, 130.04, 127.48, 125.07, 124.98, 114.25,

113.79, 113.71, 105.66. ESI-MS: m/z: 289 (M+H)+.

3-(Dimethylamino)-1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole-

3-yl)prop-2-en-1-one (5)

To Ar gas purged 1,4-dioxane (2 mL) in microwave reaction vessel were added compound

4 (1.00 g, 2.24 mmol), bis(pinacolato)diboron (0.624 g, 2.46 mmol), potassium acetate (0.658

g, 0.067 mmol), and PdCl2(dppf) (0.049 g, 0.067 mmol). The reaction mixture was heated in

the microwave reactor at power 100 W and 110 °C for 10 min. After solvents were removed

under reduced pressure, the residue was loaded directly on the flash column for purification.

The purification by flash chromatography eluted with DCM:EA (3:1) mixture provided the

compound 5 (620 mg) in 57% yield; 1HNMR (CDCl3, 400 MHz) 8.81 (s, 1H), 8.06 (s, 1H)

7.93 (dd, J = 0.8 Hz, 8.4 Hz, 1H), 7.81~7.77 (m, 4H), 7.22 (d, J = 8.4 Hz, 2H), 5.60 (d, J =

12.4 Hz, 1H), 3.17 (br, 3H), 2.96 (br, 3H), 2.35 (s, 3H) 1.33 (s, 12H).

General procedure for synthesis of 2-chloro-6-alkoxypyrazine (Procedure A)

2-Chloro-6-ethoxypyrazine

To THF (2 mL) in microwave reaction vessel were added ethanol (0.059 mL, 1.0 mmol)

and sodium hydride (32 mg, 1.3 mmol). After stirring for 30 min at room temperature, 2,6-

dichloropyrazine (100 mg, 0.671 mmol) was added to the reaction mixture. The reaction

mixture was heated in the microwave reactor at power 100 W and 50 °C for 10 min. After

solvents were removed under reduced pressure, the residue was dissolved in ethyl acetate.

Organic layer was washed with water and dried over MgSO4. Filtration and removal of

solvent of the filtrate provided 97 mg of title compound in 91% yield; 1H NMR (400 MHz,

CDCl3): δ 8.13 (1H, s), 8.11 (1H, s), 4.40 (2H, q, J = 7.06 Hz), 1.43 (3H, t, J = 7.0 Hz).

2-(6-Chloropyrazin-2-yloxy)-N,N-dimethylethanamine (Procedure A)

Obtained quantitatively; 1H NMR (400 MHz, CDCl3): δ 8.19 (1H, s), 8.14 (1H, s), 4.44

(2H, t, J = 5.4 Hz), 2.73 (2H, t, J = 5.6 Hz), 2.34 (6H, s).

2-(6-Chloropyrazin-2-yloxy)-N,N-diethylethanamine (Procedure A)

Obtained 99 % yield; 1H NMR (400 MHz, CDCl3): δ 8.14 (1H, s), 8.13 (1H, s), 4.41 (2H,

t, J = 6.0 Hz), 2.86 (2H, t, J = 6.0 Hz), 2.63 (4H, q, J = 7.2 Hz), 1.06 (6H, t, J = 7.2 Hz).

3-(6-chloropyrazin-2-yloxy)-N,N-dimethylpropan-1-amine (Procedure A)


(2H, t, J = 6.4 Hz), 2.44 (2H, t, J = 7.4 Hz), 2.26 (6H, s), 1.97 (2H, quint, J = 6.9 Hz).

3-(6-chloropyrazin-2-yloxy)-N,N-diethylpropan-1-amine (Procedure A)


(2H, t, J = 6.4 Hz), 2.64-2.54 (6H, m), 1.95 (2H, quint, J = 6.9 Hz), 1.04 (6H, t, J = 7.2 Hz).

General procedure for synthesis of 2-chloro-6-aminopyrazine (Procedure B)

N1-(6-Chloropyrazin-2-yl)-N2,N2-dimethylethane-1,2-diamine

To a solution of N,N-dimethylethylenediamine (0.192 mL, 1.75 mmol) in DMF (5mL) was

added K2CO3 (0.278 g, 2.01 mmol). After stirring for 30 min at RT, 2,6-dichloropyrazine

(0.200 g, 1.34 mmol) was added to the reaction mixture. After stirring overnight at RT,

solvent was removed under reduced pressure. After filtering off the precipitate formed by

treatment the residue with DCM:methanol (95:5) mixture, removal of solvent of the filtrate

provided the compound 225 mg in 84% yield; 1H NMR (400 MHz, CDCl3): δ 7.77 (1H, s),

7.76 (1H, s), 5.51 (1H, br), 3.39 (2H, q, J = 5.46 Hz), 2.54 (2H, t, J = 5.8 Hz), 2.26 (6H, s).

N1-(6-Chloropyrazin-2-yl)-N2,N2-diethylethane-1,2-diamine (Procedure B)

Obtained 34% yield; 1H NMR (400 MHz, CDCl3): δ 7.75 (1H, s), 7.73 (1H, s), 5.60 (1H,

br), 3.33 (2H, q, J = 5.6 Hz), 2.64 (2H, t, J = 6.0 Hz), 2.53 (4H, q, J = 7.06 Hz), 1.0 (6H, t, J

= 7.2 Hz).

N1-(6-Chloropyrazin-2-yl)-N3,N3-dimethylpropane-1,3-diamine (Procedure B)

Obtained 69% yield; 1H NMR (400 MHz, CDCl3): δ 7.72 (2H, s), 6.53(1H, br), 3.43 (2H,

q, J = 5.86 Hz), 2.43 (2H, t, J = 6.2 Hz), 2.25 (6H, s), 1.77 (2H, quint, J = 6.3 Hz).

N1-(6-Chloropyrazin-2-yl)-N3,N3-diethylpropane-1,3-diamine (Procedure B)

Obtained 64% yield; 1H NMR (400 MHz, CDCl3): δ 7.62 (2H, s), 7.14 (1H, br), 3.37 (2H,

q, J = 5.6 Hz), 2.53-2.44 (6H, m), 1.69 (2H, quint, J = 6.1 Hz), 0.99 (6H, t, J = 7.0 Hz).

N1-(6-Chloropyrazin-2-yl)-N1,N2,N2-trimethylethane-1,2-diamine (Procedure B)

Obtained 69% yield; 1H NMR (400 MHz, CDCl3): δ 7.85 (1H, s), 7.74 (1H, s), 3.64 (2H, t,

J = 7.0 Hz), 3.1 (3H, s), 2.49 (2H, t, J = 7.0 Hz), 2.28 (6H, s).

General Procedure for Suzuki coupling (Procedure C, 6a to 6j)

3-(Dimethylamino)-1-(5-(6-ethoxypyrazin-2-yl)-1-tosyl-1H-indol-3-yl)prop-2-en-1-one

(6a)

To Ar gas purged 1,4-dioxane:ethanol (1:1.5) mixture (2.5 mL) in microwave reaction

vessel were added compound 5 (0.10 g, 0.20 mmol), 2-chloro-6-ethoxypyrazine (35 mg, 0.22

mmol), bis(triphenylphosphine) palladium (II) dichloride (5.0 mg, 0.0061 mmol), 2M

solution of potassium carbonate (0.50 mL, 1.0 mmol). The reaction mixture was heated in the

microwave reactor at power 100 W and 110 °C for 10 min. After removal of solvent, the

residue was semi-purified by filtering using short silica column and used for the next reaction

without further purification.

3-(Dimethylamino)-1-(5-(6-(2-(dimethylamino)ethoxy)pyrazin-2-yl)-1-tosyl-1H-indol-3-

yl)prop-2-en-1-one (6b)

Obtained quantitatively by procedure C.

1-(5-(6-(2-(diethylamino)ethoxy)pyrazin-2-yl)-1-tosyl-1H-indol-3-yl)-3

(dimethylamino)prop-2-en-1-one (6c)


3-(Dimethylamino)-1-(5-(6-(3-(dimethylamino)propoxy)pyrazin-2-yl)-1-tosyl-1H-indol-

3-yl)prop-2-en-1-one (6d)


1-(5-(6-(3-(Diethylamino)propoxy)pyrazin-2-yl)-1-tosyl-1H-indol-3-yl)-3-

(dimethylamino)prop-2-en-1-one (6e)


3-(Dimethylamino)-1-(5-(6-(2-(dimethylamino)ethylamino)pyrazin-2-yl)-1-tosyl-1H-

indol-3-yl)prop-2-en-1-one (6f)

Obtained 96% yield (procedure C).

1-(5-(6-(2-(Diethylamino)ethylamino)pyrazin-2-yl)-1-tosyl-1H-indol-3-yl)-3-

(dimethylamino)prop-2-en-1-one (6g)


3-(Dimethylamino)-1-(5-(6-(3-(dimethylamino)propylamino)pyrazin-2-yl)-1-tosyl-1H-

indol-3-yl)prop-2-en-1-one (6h)


1-(5-(6-(3-(Diethylamino)propylamino)pyrazin-2-yl)-1-tosyl-1H-indol-3-yl)-3-

(dimethylamino)prop-2-en-1-one (6i)


3-(Dimethylamino)-1-(5-(6-((2-(dimethylamino)ethyl)(methyl)amino)pyrazin-2-yl)-1-

tosyl-1H-indol-3-yl)prop-2-en-1-one (6j)


General procedure for synthesis of compounds 7a to 7j (Procedure D)

4-(5-(6-ethoxypyrazin-2-yl)-1H-indol-3-yl)pyrimidin-2-amine (7a)

To Ar gas purged 2-methoxyethanol (2.5 mL) in microwave reaction vessel were added

compound 6a (0.14 g, 0.29 mmol), K2CO3 (0.079 g, 0.57 mmol), and guanidine carbonate

(0.103 g, 0.57 mmol). The reaction mixture was heated in the microwave reactor at power

100 W and 150 °C for 10 min. After removal of solvent, the residue was purified by flash

column chromatography using eluent (DCM:MeOH 95:5) to provide the compound 7a in 59

% overall yield in two steps from compound 5; 1H NMR (400 MHz, DMSO-d6) δ 11.88 (1H,

d, J = 2.0 Hz), 9.27 (1H, d, J = 1.2 Hz), 9.00 (1H, s), 8.28 (1H, d, J = 2.8 Hz), 8.17 (1H, s),

8.14 (1H, d, J = 5.2 Hz), 8.04 (1H, dd, J1 = 8.6 Hz, J2 = 1.8 Hz), 7.57 (1H, d, J = 8.4 Hz), 7.06

(1H, d, J = 5.2 Hz), 6.59 (2H, br), 4.52 (2H, q, J = 6.9 Hz), 1.42 (3H, t, J = 7.0 Hz); 13C NMR

(100 MHz, DMSO-d6) δ 164.06, 162.89, 159.32, 157.62, 149.97, 138.48, 133.47, 132.30,

129.91, 128.62, 126.08, 121.58, 121.35, 114.88, 112.91, 105.79, 62.04, 14.89; ESI-MS m/z =

333 (M+H)+.

4-(5-(6-(2-(Dimethylamino)ethoxy)pyrazin-2-yl)-1H-indol-3-yl)pyrimidin-2-amine (7b)

Obtained 82% overall yield in two steps from compound 5; 1H NMR (400 MHz, DMSO-

d6+CDCl3) δ 11.83 (1H, s), 9.28 (1H, s), 8.97 (1H, s), 8.22 (1H, d, J = 2.4 Hz), 8.13 (1H, s),


(1H, d, J = 5.2 Hz), 6.54 (2H, br), 4.57 (2H, t, J = 5.8 Hz), 2.71 (2H, t, J = 5.8 Hz), 2.26 (6H,

s); 13C NMR (100 MHz, DMSO-d6+CDCl3) δ 164.04, 162.89, 159.19, 157.47, 149.86,

138.47, 133.46, 132.25, 129.71, 128.52, 126.07, 121.45, 121.37, 114.89, 112.80, 105.74,

63.75, 57.87, 45.98; ESI-MS m/z = 376 (M+H)+.

4-(5-(6-(2-(Diethylamino)ethoxy)pyrazin-2-yl)-1H-indol-3-yl)pyrimidin-2-amine (7c)


d6) δ 11.90 (1H, d, J = 2.0 Hz), 9.30 (1H, s), 9.01 (1H, s), 8.30 (1H, d, J = 2.4 Hz), 8.18 (1H,

s), 8.14 (1H, d, J = 5.2 Hz), 8.05 (1H, dd, J1 = 8.4 Hz, J2 = 1.6 Hz), 7.58 (1H, d, J = 8.8 Hz),

7.08 (1H, d, J = 5.2 Hz), 6.60 (2H, br), 4.54 (2H, t, J = 6.4 Hz), 2.85 (2H, t, J = 6.4 Hz), 2.58

(4H, q, J = 7.06 Hz), 0.97 (6H, t, J = 7.2Hz); 13C NMR (100 MHz, DMSO-d6) δ 164.06,

162.88, 159.30, 157.61, 149.88, 138.48, 133.49, 132.38, 129.93, 128.55, 126.09, 121.51,

121.38, 114.89, 112.89, 105.78, 64.35, 51.27, 47.47, 12.32; ESI-MS m/z = 404 (M+H)+.

4-(5-(6-(3-(Dimethylamino)propoxy)pyrazin-2-yl)-1H-indol-3-yl)pyrimidin-2-amine (7d)


d6) δ 11.91 (1H, s), 9.29 (1H, d, J = 0.8 Hz), 9.01 (1H, s), 8.30 (1H, d, J = 1.6 Hz), 8.19 (1H,

s), 8.15 (1H, d, J = 5.2 Hz), 8.04 (1H, dd, J1 = 8.8 Hz, J2 = 1.6 Hz), 7.59 (1H, d, J = 8.4 Hz),

7.08 (1H, d, J = 5.2 Hz), 6.60 (2H, br), 4.50 (2H, t, J = 6.6 Hz), 2.40 (2H, t, J = 7.2 Hz), 2.16

(6H, s), 1.96 (2H, quint, J = 6.9 Hz); 13C NMR (100 MHz, DMSO-d6) δ 164.06, 162.88,

159.44, 157.62, 149.94, 138.47, 133.48, 132.33, 129.92, 128.58, 126.08, 121.55, 121.35,

114.88, 112.91, 105.80, 64.64, 56.19, 45.70, 27.04; ESI-MS m/z = 390 (M+H)+.

4-(5-(6-(3-(Diethylamino)propoxy)pyrazin-2-yl)-1H-indol-3-yl)pyrimidin-2-amine(7e)


d6+CDCl3) δ 11.85 (1H, s), 9.27 (1H, s), 9.00 (1H, s), 8.25 (1H, d, J = 2.4 Hz), 8.14 (1H, s),


(1H, d, J = 5.2 Hz), 6.58 (2H, br), 4.50 (2H, t, J = 6.4 Hz), 2.57 (2H, t, J = 7.0 Hz), 2.49 (4H,

q, J = 7.0 Hz), 1.93 (2H, quint, J = 6.7 Hz), 0.96 (6H, t, J = 7.0 Hz); 13C NMR (100 MHz,

DMSO-d6+CDCl3) δ 164.03, 162.87, 159.43, 157.50, 149.92, 138.46, 133.40, 132.20, 129.72,

128.61, 126.04, 121.51, 121.26, 114.88, 112.80, 105.75, 64.60, 49.19, 46.81, 26.73, 12.20;

ESI-MS m/z = 418 (M+H)+.

N1-(6-(3-(2-Aminopyrimidin-4-yl)-1H-indol-5-yl)pyrazin-2-yl)-N2,N2 dimethylethane-

1,2-diamine (7f)


d6+CDCl3) δ 11.50 (1H, s), 9.13 (1H, s), 8.32 (1H, s), 8.10 (1H, d, J = 5.2 Hz), 7.96 (1H, d, J

= 2.4 Hz ), 7.87 (1H, dd, J1 = 8.6 Hz, J2 = 1.4 Hz), 7.80 (1H, s), 7.45 (1H, d, J = 8.4 Hz), 6.93

(1H, d, J = 5.2 Hz), 6.47 (1H, t, J = 5.2 Hz), 6.04 (2H, br), 3.54 (2H, q, J = 6.13 Hz), 2.57

(2H, t, J = 6.4 Hz), 2.26 (6H, s); 13C NMR (100 MHz, DMSO-d6+CDCl3) δ 163.76, 162.99,

157.27, 154.28, 150.09, 138.10, 130.46, 129.98, 128.60, 128.18, 125.85, 121.21, 120.67,

114.87, 112.25, 106.13, 58.25, 45.55, 38.66; ESI-MS m/z = 375 (M+H)+.

N1-(6-(3-(2-aminopyrimidin-4-yl)-1H-indol-5-yl)pyrazin-2-yl)-N2,N2-diethylethane-1,2-

diamine (7g)


d6) δ 11.83 (1H, s), 9.18 (1H, s), 8.46 (1H, s), 8.25 (1H, d, J = 2.8 Hz), 8.14 (1H, d, J = 5.2

Hz ), 7.99 (1H, dd, J1 = 8.8 Hz, J2 = 1.6 Hz), 7.86 (1H, s), 7.52 (1H, d, J = 8.4 Hz), 7.06 (1H,

d, J = 5.2 Hz), 6.97 (1H, t, J = 5.6 Hz), 6.55 (2H, br), 3.49 (2H, q, J = 6.66 Hz), 2.67 (2H, t, J

= 6.8 Hz), 2.58 (2H, q, J = 7.06Hz), 0.98 (6H, t, J = 7.0 Hz); 13C NMR (100 MHz, DMSO-d6)

δ 166.95, 166.47, 164.05, 162.96, 157.57, 154.54, 149.92, 138.15, 129.93, 129.62, 128.04,

125.93, 121.39, 120.74, 114.83, 112.49, 105.87, 51.66, 47.19, 38.86, 12.13; ESI-MS m/z =

403 (M+H)+.

N1-(6-(3-(2-aminopyrimidin-4-yl)-1H-indol-5-yl)pyrazin-2-yl)-N3,N3-dimethylpropane-

1,3-diamine (7h)



Hz ), 7.98 (1H, dd, J1 = 8.6 Hz, J2 = 1.4 Hz), 7.84 (1H, s), 7.53 (1H, d, J = 8.4 Hz), 7.10-7.06

(2H, m), 6.53 (2H, br), 3.45-3.42 (2H, m), 2.40 (2H, t, J = 7.2 Hz), 2.20 (6H, s), 1.79 (2H,

quint, J = 7.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ 164.04, 162.96, 157.58, 154.63, 149.99,

138.15, 129.98, 129.62, 128.00, 125.91, 121.45, 120.70, 114.80, 112.54, 105.88, 57.27,

45.51, 38.92, 27.14; ESI-MS m/z = 389 (M+H)+.

N1-(6-(3-(2-aminopyrimidin-4-yl)-1H-indol-5-yl)pyrazin-2-yl)-N3,N3-diethylpropane-1,3-

diamine (7i)

Obtained 53 % overall yield in two steps from compound 5; 1H NMR (400 MHz, DMSO-


Hz ), 7.98 (1H, dd, J1 = 8.6 Hz, J2 = 1.4 Hz), 7.82 (1H, s), 7.52 (1H, d, J = 8.8 Hz), 7.05 (2H,

d, J = 5.2 Hz), 6.50 (2H, br), 2.48-2.44 (4H, m), 1.74 (2H, quint, J = 6.9 Hz), 0.94 (6H, t , J =

7.2 Hz); 13C NMR (100 MHz, DMSO-d6): δ 164.05, 162.95, 157.58, 154.66, 149.97, 138.16,

130.00, 129.60, 127.97, 125.90, 121.45, 120.64, 114.81, 112.52, 105.87, 50.64, 46.75, 26.84,

12.12; ESI-MS m/z = 417 (M+H)+.

N1-(6-(3-(2-aminopyrimidin-4-yl)-1H-indol-5-yl)pyrazin-2-yl)-N1,N2,N2 trimethylethane-

1,2-diamine(7j)


d6) δ 11.86 (1H, s), 9.22 (1H, s), 8.58 (1H, s), 8.25 (1H, s), 8.13 (1H, d, J = 5.6 Hz ), 8.01-

7.98 (2H, m), 7.53 (1H, d, J = 8.8 Hz), 7.05 (1H, d, J = 5.2 Hz), 6.58 (2H, br), 3.78 (2H, t, J

= 6.8 Hz), 3.14 (3H, s), 2.50-2.48 (2H, m), 2.21 (6H, s); 13C NMR (100 MHz, DMSO-d6) δ

164.09, 162.98, 157.56, 153.47, 149.63, 138.26, 129.72, 128.12, 127.45, 125.98, 121.32,

120.90, 114.84, 112.63, 105.83, 56.44, 47.36, 46.02, 36.21; ESI-MS m/z = 389 (M+H)+.

(E)-3-(dimethylamino)-1-(5-(6-(2-(dimethylamino)ethylamino)pyrazin-2-yl)-1H-indol-3-

yl)prop-2-en-1-one (7k)

Obtained 33% overall yield in two steps from compound 5; 1H NMR (400 MHz, CDCl3) δ

9.06 (1H, s), 8.33 (1H, s), 7.82-7.73 (4H, m), 7.42 (1H, d, J = 8.4 Hz), 5.60 (1H, d, J = 12.4

Hz), 5.44 (1H, br), 3.45 (2H, br), 2.90 (6H, Br), 2.51 (2H, t, J = 5.6 Hz), 2.24 (6H, s); ESI-

MS m/z = 379 (M+H)+.

3-(Dimethylamino)-1-(5-(6-(2-(dimethylamino)ethoxy)pyrazin-2-yl)-1H-indol-3-yl)prop-

2-en-1-one (7l)

Obtained 30% overall yield in two steps from compound 5 ;1H NMR (400 MHz, DMSO-

d6) δ 11.83 (1H, s), 9.05 (1H, s), 8.73 (1H, s), 8.24 (1H, d, J = 2.8 Hz), 8.17 (1H, s), 7.92 (1H,

dd, J1 = 8.4 Hz, J2 = 1.6 Hz), 7.60-7.52 (2H, m), 5.81 (1H, d, J = 12.4 Hz), 4.54 (2H, t, J = 5.6

Hz), 2.98 (6H, br), 2.73 (2H, t, J = 5.6 Hz), 2.26 (6H, s); 13C NMR (100MHz, DMSO-d6) δ

183.84, 159.37, 151.78, 150.17, 137.97, 133.19, 132.47, 131.81, 128.68, 127.03, 121.40,

121.32, 119.10, 112.69, 93.67, 63.72, 57.88, 45.92; ESI-MS m/z = 379 (M+H)+.

1-(5-(6-(2-(dimethylamino)ethylamino)pyrazin-2-yl)-1H-indol-3-yl)ethanone(7m)

1H NMR (400 MHz, DMSO-d6) δ 12.06 (1H, s), 8.86 (1H, d, J = 0.8 Hz), 8.37 (1H, s),

8.21 (1H, s), 7.91 (1H, dd, J1 = 8.8 Hz, J2 = 1.6 Hz), 7.88 (1H, s), 7.55 (1H, d, J = 8.4 Hz),

6.99 (1H, t, J=5.2 Hz), 3.50 (2H, q, J = 6.13 Hz), 2.50 (2H, s), 2.49 (3H, s), 2.24 (6H, s);13C

NMR (100MHz, DMSO-d6) δ 193.13, 154.65, 149.91, 137.74, 135.72, 131.30, 127.72,

126.06, 122.04, 120.19, 117.72, 112.71, 58.55, 45.81, 38.55, 27.79; ESI-MS m/z = 324

(M+H)+.

Cyclopropyl(5-(6-(2-(dimethylamino)ethylamino)pyrazin-2-yl)-1H-indol-3-

yl)methanone(7n)

1H NMR (400 MHz, DMSO-d6) δ 12.10 (1H, s), 8.88 (1H, d, J = 1.2 Hz), 8.58 (1H, s),

8.21 (1H, s), 7.92 (1H, dd, J1 = 8.8 Hz, J2 = 1.6 Hz), 7.89 (1H, s), 7.56 (1H, d, J = 8.4 Hz),

6.97 (1H, t, J=5.4 Hz), 3.49 (2H, q, J = 6.13 Hz), 2.76 (1H, Sep, J=4.13 Hz), 2.52-2.51 (2H,

m), 2.23 (6H, s); 13C NMR (100MHz, DMSO-d6) δ 194.76, 154.64, 149.90, 137.71, 135.18,

131.27, 127.70, 126.03, 122.09, 120.30, 118.05, 112.69, 58.51, 45.77, 38.55, 17.68, 9.74;

ESI-MS m/z = 350 (M+H)+.

(5-(6-(2-(dimethylamino)ethoxy)pyrazin-2-yl)-1H-indol-3-yl)(phenyl)methanone(7o)

1H NMR (400 MHz, DMSO-d6) δ 12.25 (1H, s), 8.98 (1H, s), 8.78 (1H, s), 8.20 (1H, s),

8.05-8.03 (2H, m), 7.82 (2H, d, J=7.2 Hz), 7.65 (1H, d, J = 8.4 Hz), 7.62 (1H, d, J = 7.6 Hz),

7.56 (2H, t, J=7.2 Hz), 4.55 (2H, t, J=5.8 Hz), 2.74 (2H, t, J= 5.6 Hz), 2.27 (6H, s); 13C NMR

(100MHz, DMSO-d6) δ 190.50, 159.40, 149.71, 140.77, 138.15, 137.29, 133.34, 132.92,

131.73, 130.06, 128.94, 128.89, 127.16, 122.58, 120.75, 116.03, 113.28, 63.83, 57.84, 45.89;

ESI-MS m/z = 387 (M+H)+.

Biochemical Assay

The potency of the compounds in this study was measured using a fluorescence

polarization assay method. Enzyme, substrate, and ATP were prepared in kinase reaction

buffer containing 10 mM Tris-HCl (pH 7.2), 10 mM MgCl2, 0.05% NaN3, 0.01% Triton X-

100 and 2 mM DTT. In 384-well black flat bottom polystyrene plates, the IMAP PIM kinase

assays were formatted using 10 L reaction volumes consisting of 2.5 L compound, 2.5 L

PIM1 (1 nM) or PIM2 (1 nM) or PIM3 (1 nM), 2.5 L ATP and 2.5 L 5-FAM-labeled BAD

peptide (100 nM). The final concentrations of ATP were 30 M, 5 M and 20 M for PIM1,

PIM2 and PIM3, respectively. Following 90 min incubation at room temperature, IMAP

binding reagent (Molecular Devices, solution containing 75% Buffer A: 25% Buffer B and a

1 in 600 dilution of beads) was added to each well to stop the reaction. After incubation for a

2-hr at room temperature, the fluorescence polarization was measured on an Infinity F200

plate reader (Tecan) at an excitation wavelength of 485 nm and an emission wavelength of

530 nm. The data were then fitted to a 4-parameter logistic equation shown below and IC50s

were determined using GraphPad Prism (GraphPad Software, Inc., La Jolla).

Y=Bottom+(Top−Bottom)

1+10(logIC 50−X )∗Hillslope

Cell culture conditions and viability assays

MV4-11 (human acute myelocytic leukemia cell line) cells purchased from the American

Type Culture Collection (ATCC, Manassas, VA) were grown in Iscove’s Modified Dulbecco’s

Medium (IMDM, ATCC, Manassas, VA) containing 10% fetal bovine serum (FBS, Gibco-

BRL, Grand Island, NY), 100 U/mL penicillin and 100 g/mL streptomycin at 37°C in a 5%

CO2 humidified atmosphere. K562 (human erythromyeloblastoid leukemia cell line) and

Jurkat clone E6-1 (human acute T cell leukemia) cells were purchased from Korean Cell Line

Bank (KCBL, Seoul, Korea). K562 and Jurkat cells were grown in RPMI 1640 (Gibco-BRL,

Grand Island, NY), supplemented with 10% heat inactivated FBS, 100 U/mL penicillin and

100 g/mL streptomycin. Cell viability was measured using the CellTiter 96R AQUES One

Solution Cell Proliferation Assay (MTS) from Promega (Madison, WI). MV4-11 and Jurkat

cells were seeded in a 96-well plate at the density 200,000 cells per well. K562 cells were

seeded at 10,000 cells per well. The next day, tested drugs at indicated concentrations were

added and incubated at 37°C. After 24 h, 20 L of MTS solution was added to each well, and

the plates were incubated at 37°C for 4 h. The absorbance of each well was measured at 490

nm with a microplate reader (SPECTRA max 340PC, Molecular Devices, Sunnyvale, CA).

Spectra:1H-NMR of 7a

13C-NMR of 7a

Mass Spectrum of 7a

1H-NMR of 7b

13C-NMR of 7b

Mass Spectrum of 7b

1H-NMR of 7c

13C-NMR of 7c

Mass Spectrum of 7c

1H-NMR of 7d

13C-NMR of 7d

Mass Spectrum of 7d

1H-NMR of 7e

13C-NMR of 7e

Mass Spectrum of 7e

1H-NMR of 7f

13C-NMR of 7f

Mass Spectrum of 7f

1H-NMR of 7g

13C-NMR of 7g

Mass Spectrum of 7g

1H-NMR of 7h

13C-NMR of 7h

Mass Spectrum of 7h

1H-NMR of 7i

13C-NMR of 7i

Mass Spectrum of 7i

1H-NMR of 7j

13C-NMR of 7j

Mass Spectrum of 7j

1H-NMR of 7k

13C-NMR of 7k

Mass Spectrum of 7k

1H-NMR of 7l

Mass Spectrum of 7l

1H-NMR of 7m

13C-NMR of 7m

Mass Spectrum of 7m

1H-NMR of 7n

13C-NMR of 7n

Mass Spectrum of 7n

1H-NMR of 7o

13C-NMR of 7o

Mass Spectrum of 7o

1H NMR of Meridianin C

13C NMR of Meridianin C

Mass spectrum of Meridianin C

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