Benzopyran and Phenylpyrazole Derivatives as Akt Inhibitors
General procedure for the synthesis of compounds
5a–c, 6a, b, and 7
J = 1.5 Hz, 1H), 8.49 (dd, J = 4.7, 1.6 Hz, 1H), 8.22 (dt,
J = 7.9, 1.9 Hz, 1H), 7.39 (dd, J = 7.9, 4.8 Hz, 1H), 7.33
(s, 1H), 6.35 (s, 1H), 4.00 (s, 3H), 3.88 (s, 3H), 3.85 (s,
2H), 3.58 (s, 4H), 1.74–1.65 (m, 4H), 1.55 (s, 2H). ESI-MS:
m/z [M + H]+ 395.
To a solution of 14 (or 16) (0.1 mmol) in 2 mL dioxane
were added corresponding amine (0.1 mmol) and parafor-
maldehyde (0.3 mmol), and the mixture was stirred at
60 °C overnight. After cooling to room temperature, the
mixture was poured into water (5 mL) and extracted with
ethyl acetate (3 9 5 mL). The combined organic layers
were dried over anhydrous Na2SO4, concentrated and
purified by column chromatography on silica gel using
petroleum ether-ethyl acetate to afford the desired prod-
uct.
2-((Benzyl(methyl)amino)methyl)-3,5-dimethoxy-6-
(5-(pyridin-3-yl)-1H-pyrazol-3-yl)phenol (6b)
Reagent: 14b (29.7 mg, 0.1 mmol), N-methylbenzylamine
(12.1 mg, 0.1 mmol), paraformaldehyde (84 mg, 0.3 mmol).
The product was obtained as white solid (45%), m.p.
171–173 °C. 1H NMR (500 MHz, acetone-d6) d 12.34
(brs, 1H), 9.09 (s, 1H), 8.48 (dd, J = 4.7, 1.5 Hz, 1H),
8.21 (dt, J = 7.9, 1.9 Hz, 1H), 7.44–7.36 (m, 5H), 7.35 (s,
1H), 7.32 (t, J = 7.0 Hz, 1H), 6.40 (s, 1H), 4.02 (s, 3H),
3.91 (s, 5H), 3.74 (s, 2H), 2.29 (s, 3H). 13C NMR
(101 MHz, DMSO) d 157.81, 157.68, 157.48, 157.41,
150.41, 150.02, 129.33, 128.47, 127.54, 119.40, 103.90,
99.18, 87.18, 60.00, 55.66, 55.60, 45.59. ESI-MS: m/z
[M + H]+ 431.
3,5-Dimethoxy-2-(piperidin-1-ylmethyl)-6-(5-
(pyridin-4-yl)-1H-pyrazol-3-yl) phenol (5a)
Reagent: 14a (29.7 mg, 0.1 mmol), piperidine (8.5 mg,
0.1 mmol), paraformaldehyde (84 mg, 0.3 mmol). The
product was obtained as white solid (55%), m.p. 178–
1
180 °C. H NMR (500 MHz, CDCl3) d 8.62 (d, J = 5.9 Hz,
2H), 7.80 (d, J = 6.1 Hz, 2H), 7.34 (s, 1H), 6.11 (s, 1H),
3.98 (s, 3H), 3.84 (s, 3H), 3.81 (s, 2H), 1.75–1.61 (m, 4H),
0.93–0.80 (m, 6H). ESI-MS: m/z [M + H]+ 395.
2,4-bis((benzyl(methyl)amino)methyl)-6-(5-(pyridin-
4-yl)-1H-pyrazol-3-yl) benzene-1,3-diol 7
2-((Cyclohexyl(methyl)amino)methyl)-3,5-
dimethoxy-6-(5-(pyridin-4-yl)-1H-pyrazol-3-yl)
phenol (5b)
Reagent: 16 (25.4 mg, 0.1 mmol), N-methylbenzylamine
(24.2 mg, 0.2 mmol), paraformaldehyde (112 mg, 0.4 mmol).
The product was obtained as
a white solid (39%),
Reagent: 14b (29.7 mg, 0.1 mmol), N-methylcyclohexyl-
amine (11.3 mg, 0.1 mmol), paraformaldehyde (84 mg,
0.3 mmol). The product was obtained as white solid
(52%), m.p. 165–166 °C. 1H NMR (500 MHz, CDCl3) d
8.63 (d, J = 6.0 Hz, 2H), 7.81 (d, J = 4.8 Hz, 2H), 7.34 (s,
1H), 6.10 (s, 1H), 4.00 (s, 3H), 3.94 (s, 2H), 3.85 (s, 3H),
2.65–2.68 (m, 1H), 2.35 (s, 3H), 1.93–1.97 (m, 2H), 1.85–
1.89 (m, 2H), 1.68–1.72 (m, 1H), 1.44–1.36 (m, 2H), 1.16–
1.12 (m, 1H), 0.83 -0.88 (m, 2H). ESI-MS: m/z [M + H]+
423.
m.p. 110–113 °C. 1H NMR (500 MHz, CDCl3) d 8.66
(d, J = 5.8 Hz, 2H), 7.78 (d, J = 6.0 Hz, 1H), 7.46–7.30
(m, 11H), 6.87 (s, 1H), 4.00 (s, 2H), 3.79 (s, 4H), 3.64
(s, 2H), 2.35 (s, 3H), 2.27 (s, 3H). ESI-MS: m/z [M + H]+
520.
Akt1 inhibitory activity assay
in vitro kinase assays were carried out using HTScanꢀ
PKB/Akt1 Kinase Assay kit (Cell Signaling Technology).
Active recombinant Akt1 kinase (GST fusion protein, 4 ng)
in 8 lL of 2.59 kinase buffer [62.5 mM Tris–HCl (pH 7.5),
25 mM MgCl2, 12.5 mM b-glycerophosphate, 0.25 mM
Na3VO4, 5 mM dithiothreitol (DTT)] was mixed with 2 lL of
dimethyl sulfoxide (DMSO) vehicle or each of the com-
pound (indicated concentrations), incubated at room tem-
perature for 5 min, and 10 lL of ATP/substrate cocktail
(20 mM ATP, 3 mM eNOS served as substrate) was added.
After incubation at room temperature for 30 min, add
20 lL of 50 mM ethylenediaminetetraacetic acid (EDTA)
(pH 8.0) and terminate the reaction. Then, PKB/Akt1
kinase activity was analyzed according to the manufac-
turer’s instructions.
2-((Benzyl(methyl)amino)methyl)-3,5-dimethoxy-
6-(5-(pyridin-4-yl)-1H-pyrazol-3-yl)phenol (5c)
Reagent: 14c (29.7 mg, 0.1 mmol), N-methylbenzylamine
(12.1 mg, 0.1 mmol), paraformaldehyde (84 mg, 0.3 mmol).
The product was obtained as white solid (44%), m.p.
182–185 °C. 1H NMR (500 MHz, CDCl3)
d 8.63 (d,
J = 5.6 Hz, 2H), 7.81 (d, J = 5.6 Hz, 2H), 7.39–7.29
(m, 5H), 7.26 (s, 1H), 6.14 (s, 1H), 3.99 (s, 3H), 3.89
(s, 2H), 3.86 (s, 3H), 2.28 (s, 3H). ESI-MS: m/z [M + H]+
431.
3,5-Dimethoxy-2-(piperidin-1-ylmethyl)-6-(5-
(pyridin-3-yl)-1H-pyrazol-3-yl) phenol (6a)
Cytotoxic activity assay
Reagent: 14b (29.7 mg, 0.1 mmol), piperidine (8.5 mg,
0.1 mmol), paraformaldehyde (84 mg, 0.3 mmol). The
product was obtained as white solid (59%), m.p. 183–
The cytotoxic activity of tested compounds in PC3, OV-
CAR-8, HepG2, and HL-60 cells was measured using the
MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
185 °C. 1H NMR (500 MHz, acetone-d6)
d
9.11 (d,
bromide, a tetrazole) assay (23). Cells were seeded in
Chem Biol Drug Des 2014
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