Syntheses of phenylpyrazolodiazepin-7-ones as conformationally rigid analogs of aminopyrazole amide scaffold and their antiproliferative effects on cancer cells

Article abstract of DOI:10.1016/j.bmc.2011.09.042

Recently, we have reported the syntheses and antiproliferative activities of N-(5-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl amide derivatives on melanoma cells. As a continuous work for antiproliferative agents in melanoma, here we report the synthesis of conformationally rigid analogs, phenyl-6,8- dihydropyrazolo[3,4-b][1,4]diazepin-7(1H)-one derivatives 7a-g, 8a-o and their antiproliferative activities against A375P melanoma cell line and U937 hematopoietic cell line. Most compounds showed competitive antiproliferative activities to sorafenib, the reference standard. Among them, N-(3-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl) -4-chloro-3-(trifluoro methyl)benzamide-amino-1-(4-methoxybenzyl)-1H-pyrazol-4- yl)-5-(3-(4-chloro-3-(trifluoromethyl) phenyl) ureido)-2-methylbenzamide (7b) exhibited potent activities (GI₅₀ = 0.43 μM and 0.06 μM) on both cell lines. It has been further confirmed to be a potent and selective Raf kinases inhibitor and also mild inhibitor of PI3Kα.

Full text of DOI:10.1016/j.bmc.2011.09.042

Bioorganic & Medicinal Chemistry 19 (2011) 6760–6767
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses of phenylpyrazolodiazepin-7-ones as conformationally rigid
analogs of aminopyrazole amide scaffold and their antiproliferative effects
on cancer cells
⇑
Hyangmi Kim, Minjung Kim, Junghun Lee, Hana Yu, Jung-Mi Hah
Department of Pharmacy, College of Pharmacy, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan Kyeonggi-do 426-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, we have reported the syntheses and antiproliferative activities of N-(5-amino-1-(4-methoxyben-
zyl)-1H-pyrazol-4-yl amide derivatives on melanoma cells. As a continuous work for antiproliferative
agents in melanoma, here we report the synthesis of conformationally rigid analogs, phenyl-6,8-dihydro-
pyrazolo[3,4-b][1,4]diazepin-7(1H)-one derivatives7a–g, 8a–o and their antiproliferative activities against
A375P melanoma cell line and U937 hematopoietic cell line. Most compounds showed competitive
antiproliferative activities to sorafenib, the reference standard. Among them, N-(3-(1-benzyl-7-oxo-1,6,
7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl)-4-chloro-3-(trifluoro methyl)benzamide-amino-
1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-(3-(4-chloro-3-(trifluoromethyl) phenyl) ureido)-2-methylb-
Received 24 August 2011
Revised 22 September 2011
Accepted 23 September 2011
Available online 1 October 2011
Keywords:
Dihydropyrazolodiazepinone
Antiproliferative activity
Cancer cell line
enzamide (7b) exhibited potent activities (GI50 = 0.43
lM and 0.06 lM) on both cell lines. It has been
further confirmed to be a potent and selective Raf kinases inhibitor and also mild inhibitor of PI3K
a.
Kinase inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
Kinase selectivity
1. Introduction
that there are more than one targets and inhibitors necessary for
this set of disease.9
The irregulation of kinase activity has been considered as a
major mechanism of growth and survival of cancer cells. Therefore,
there are significant amounts of efforts to develop selective and po-
tent kinase inhibitors and approximately 30 distinct kinase targets
are being developed as in Phase I clinical trial to date. Among
which, kinases in Ras/Raf/MEK/ERK and phosphatidylinositol
3-kinase (PI3K)/Akt signaling cascades are the most commonly
up-regulated in human cancers.1–4 It is more important that these
two signaling cascades cooperate to promote transformed cancer
cell survival. Regulation through crosstalk and feedback between
the Ras/Raf/MEK/ERK and PI3K/Akt pathways have been demon-
strated, indicating the potential of interruption of more than one
signaling pathways in cancer therapy.2,5,6
The activating mutations in KRAS are found in more than 30% of
all human tumors and mutations of B-Raf in 7% of human can-
cers7,8 with particularly high frequency in melanoma (50–70%),
ovarian (35%), thyroid (30%), and colorectal (10%) cancers. Thus
targeting this pathway could have broad therapeutic effects9 and
many small molecule ATP-competitive B-Raf (V600E) kinase inhib-
itors have been developed and found to have potent antitumor
effects on mutant B-Raf (V600E) tumors. Surprisingly, however,
they were not potent against RAS mutant tumor models implying
Recently, there are reports that the potent B-Raf (V600E) inhib-
itor could also trigger the C-Raf activation sequence by independent
mechanism,10 which can lead to enhanced growth of tumors.
Therefore, it is proposed that the inhibition of both B-Raf (V600E)
and C-Raf should be attained at the same time and we have found
several series of compounds fitting this criteria.11,12
As shown in Figure 1, we started from imidazolopyrazole bicy-
clic ring scaffold11 based on binding mode of sorafenib, which is a
potent inhibitor of preactivated C-Raf, V600E B-Raf holding a un-
ique binding mode, namely type II inhibitor.13 Afterwards, we
found imidazolopyrazole bicyclic ring is a novel effective chemo-
type for both Raf kinases, and to develop more effective hydrogen
bonding and hydrophobic interaction, we modified the bicyclic
ring into aminopyrazole amide scaffold.12 We found that they were
all potent and selective inhibitors of B-Raf (V600E) and C-Raf as
well.
In our docking studies (Fig. 2), the aminopyrazole amide scaf-
fold is supposed to have two hydrogen bonds in the hinge region
and one intramolecular hydrogen bond between the hydrogen of
the 5-amino group and the carbonyl oxygen in the 4-amide group
in binding mode. Therefore, we decided to continue this work by
introducing a seven-membered ring mimicking intramolecular
hydrogen bond next to pyrazole ring as a namely ‘conformation-
restricted analogs’. The scaffold evolution of Raf-inhibitors are
shown in Figure 1, and here we report the syntheses of designed
⇑
Corresponding author. Tel.: +82 31 400 5803; fax: +82 31 400 5958.
E-mail address: jhah@hanyang.ac.kr (J.-M. Hah).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.042

H. Kim et al. / Bioorg. Med. Chem. 19 (2011) 6760–6767
6761
aromatic acids under EDCI/HOBt conditions to give amide (7a–7g)
analogs and also directly reacted with aromatic isocynate to give
urea (8a–8o) analogs.
O
Cl
O
Sorafenib
N
N
H
N
H
CF3
3. Results and discussion
N
H
O
O
The N-[1-benzyl-5-dihydropyrazolodiazepin-7(1H)-one]phenyl
amide derivatives 7a–g and N-[1-benzyl-5-dihydropyrazolodiaze-
pin-7(1H)-one]phenyl urea derivatives 8a–o were evaluated for
their antiproliferative activities against human melanoma cell line,
A375P and human hematopoietic cell line, U937. A375P cell line is
a well known melanoma cell line, which is an excellent index for
V600E B-Raf inhibition, and U937 cell line was selected since it
has been known to be a good model to study the Raf/MEK/ERK
and PI3K/Akt signaling pathways as well.
H
N
Imidazolopyrazole
R2
N
N
H
H
N
N
N
R1
O
O
H
N
Aminopyrazole amide
Pyrazolodiazepinone
N
N
N
R2
N
H
H
O
R1
HN
H
N
Table 1 shows the antiproliferative activities14 of N-[1-benzyl-
5-dihydropyrazolodiazepin-7(1H)-one]phenyl amide derivatives
linked with various aromatic tail groups by amide bond. Since this
scaffold has been designed as conformationally rigid analogs of
aminopyrazoleamide series, we focused on the connection in the
middle phenyl ring (m-/p-) with known hydrophobic tails. As
shown in Table 1, no growth inhibition was observed for com-
pounds 7a, 7d, and 7e on both cell lines, which indicates whether
the connection in the middle phenyl ring is m- or p-, the preference
of substitution pattern of hydrophobic tail benzene is 3-, 4-disub-
stitution not 3-, 5-disubstitution in amide derivatives. Compound
7b showed the best activities in this series toward both cell lines.
Interestingly, when the middle connection is changed to p-(7c), it
loses its activity on A375P, but it was tolerated in U937 cells. This
may indicate that 7c is not an effective inhibitor of V600E B-Raf in
A375P, but potent enough to have an effect on survival signaling in
U937 cell, probably impacting on different molecular target. This
tendency has also been found the same in case of 7f and 7g.
Table 2 shows the antiproliferative activity of N-[1-benzyl-5-
dihydropyrazolodiazepin-7(1H)-one]phenyl urea derivatives 8a–o.
Considering that aminopyrazole amide compounds are more active
in their urea derivatives and also the fact that these are conforma-
tionally rigid analogs, we expected urea analogs to be more potent.
But it was not generally true with the same hydrophobic tails
(7b > 8a on both cell lines, 7c > 8b on U937). However, we found that
compound8h, 8i, 8n and 8o with simpler mono-substitution on ben-
zene tail showed good efficacy and potency on both cell lines. It
seems to be better than the reference compound, sorafenib. Notably,
replacement of benzyl group in pyrazole moiety (R1) with PMB (p-
methoxybenzyl) tolerated in their activity, but removal of this group
led to significant decrease or total loss of activity (R1 = H; 8d, 8j, 8m),
which highlight the importance of physicochemical property in cel-
lular potency.
N
N
H
N
H
R2
N
R1
HN
O
Figure 1. Evolution of Raf inhibitors from Sorafenib to 1-benzyl-5-
dihydropyrazolodiazepin-7(1H)-one derivatives.
Figure 2. Docking structures of designed pyrazolodiazepinone scaffold (7b, bold,
green) overlaid with amino-1H-pyrazole amide derivatives (thin, cyan)12 in B-Raf
V600E (1uwj).
pyrazolodiazepin-7-one analogs and their antiproliferative effects
on two cancer cell lines.
2. Chemistry
The approach taken towards syntheses of the novel 1-benzyl-5-
dihydropyrazolodiazepin-7(1H)-one analogs is based on formation
of a bicyclic key intermediate pyrazolodiazepinone, 5 and is out-
lined in Scheme 1. The pyrazolodiazepinone was synthesized by
two sequential steps; acid-catalyzed imine formation and amide
coupling of diamino pyrazole moiety (4)11 and corresponding ethyl
b-keto nitrophenylpropano ester (3). The ethyl b-keto nitrophenyl-
propano ester (3) was synthesized by thermal ethanolysis of
5-nitrobenzoyl-2, 2-dimethyl-1, 3-dioxane-4, 6-dione (2), which
was adduct of nitrobenzoyl chloride (1) and Meldrum’s acid.
1-benzyl (or para-methoxy benzyl) -1H-pyrazole-4,5-diamine
(4) was reacted with ethyl b-keto nitrophenylpropano ester (3),
under acidic conditions, to smoothly afford ethyl 3-(5-amino-1-
benzyl-1H-pyrazol-4-ylimino)-3-nitrophenyl propanoate, and this
enamine was cyclized using sodium methoxide in dry methanol
in situ to furnish the core scaffold, 5.
Shown in Table 3, the representative compound 7b was
screened on selected 30 different kinases panel at a single dose
concentration of 10
has an excellent selectivity profile. While this compound has effec-
tively inhibited V600E B-Raf, C-Raf, and PI3K , the inhibition ex-
l
M15 and it was revealed that the compound
a
erted in most other kinases tested activity was below 20%.
Furthermore, we evaluated in vitro enzymatic inhibition of two
compounds, 7b and 8a toward V600E B-Raf, C-Raf, and PI3K
shown in Table 4, compound 7a has good to mild potency on three
kinases (0.655 M, 0.96 M, 21.5 M, respectively), however, 8a
a. As
l
l
l
has only good potency on C-Raf (0.667 lM).
4. Conclusions
A series of N-[1-benzyl-5-dihydropyrazolodiazepin-7(1H)-one]-
phenyl derivatives 7a–7g and 8a–8o, designed as conformationally
rigid analogs of aminopyrazole amide has been synthesized and
evaluated their antiproliferative activities against A375P human
Then, the nitro group in 5 was selectively reduced to amino
group using iron and ammonium chloride and linked with various

6762
H. Kim et al. / Bioorg. Med. Chem. 19 (2011) 6760–6767
NH2
NH2
O
O
O
O
O
O
O
N
N
N
N
(iii)
(iv)
(i)
(ii)
NO2
R1
4
O
N
O
Cl
R1
HN
O2N
O2N
O2N
O
1a: p- NO2
2
1b: m- NO2
3
5
O
O
N
N
N
N
N
N
N
R3
N
R2
NH2
N
H
N
H
(v)
(vi)
H
N
N
R1
R1
R1
HN
HN
HN
(vii)
O
O
6
O
7
8
Scheme 1. Reagents and conditions: (i) Meldrum’s acid, DMAP, MC, -15°C, 4 h; (ii) EtOH, reflux, overnight; (iii) 4, AcOH, MC, rt, 30 min; (iv) NaOMe/MeOH, overnight; (v) Fe,
NH4Cl,THF:H2O (1:1), 60 °C, 3 h; (vi) R2-CO2H, HOBt, EDCI, TEA, DMF, rt; (vii) R3-NCO, THF, rt.
cell line, together with hematopoietic U937 cell lines. Many com-
pounds in this scaffold showed potent antiproliferative activities,
and furthermore, one of the best compound 7b has been confirmed
as a potent and selective Raf kinases and also mild inhibitor of
ent temperature, the solvent was removed in vacuo, the pure prod-
uct 3 (3 g, 12.6 mmol, 74%) was obtained. Further purification was
not necessary at this step. (1H NMR (400 MHz, CDCl3) 12.61 (1 H,
s), 8.61 (1 H, s), 8.30 (1 H, d, J = 8.20 Hz), 8.09 (1 H, d, J = 7.82 Hz),
7.61 (1H, t, J = 8.0 Hz), 5.76 (1 H, s), 4.29 (2 H, q, J = 7.1 Hz), 1.35 (3
H, t, J = 7.1 Hz).
PI3Ka, suggesting that the N-[1-benzyl-5-dihydropyrazolodiaze-
pin-7(1H)-one]phenyl derivatives could serve as a promising scaf-
fold for new therapeutics of cancer, having absolutely impressive
kinase profiling.
5.1.3. Synthesis of 4, 5-Diamino-1-(4-methoxy-benzyl)pyrazole
(4)
To a suspension of 1-(4-methoxybenzyl)-4-nitroso-1H-pyrazol-
5-amine (100 mg, 0.43 mmol) in EtOH (0.86 ml), was added 35%
HCl (0.22 ml, 2.15 mmol), then the mixture was stirred at 80 °C.
5. Materials and methods
5.1. Chemistry general
A
solution of SnCl2Á2H2O (213.4 mg, 0.95 mmol) in EtOH
(0.25 ml) was added to dissolved mixture over 15 min. Stirring
was continued for 30 min and the clear solution was cooled to
room temperature, then poured into ice. The pH was made slightly
basic (pH 7 ꢀ 8) by addition of saturated aqueous sodium bicar-
bonate before being extracted with organic solvent (IPA/
CHCl3 = 4:1). The organic phase is thoroughly washed with brine,
dried over sodium sulfate. Evaporation of the solvent leaves crude
100 mg of 4,5-diamino-1-(4-methoxy-benzyl) pyrazole 4, which
gives one spot on TLC. (1H NMR (400 MHz, CDCl3) 7.15 (1 H, s),
7.11 (2 H, d, J = 8.55 Hz), 6.85 (2 H, d, J = 8.64 Hz), 5.11 (2 H, s),
3.76 (3 H, s).
All chemicals (reagent grade) used were purchased from Aldrich
(USA). Separation of the compounds by column chromatography
was carried out with silica gel 60 (200–300 mesh ASTM, E. Merck,
Germany). The quantity of silica gel used was 50–100 times the
weight charged on the column. Thin layer chromatography (TLC)
was run on the silica gel coated aluminum sheets (Silica Gel 60
GF254, E. Merck, Germany) and visualized in ultraviolet (UV) light
(254 nm). 1H NMR and 13C NMR spectra were recorded on Brucker
model digital AVANCE III 400 MHz spectrometer at 25 °C, using tet-
ramethylsilane (TMS) as the internal standard. High-resolution MS
(HR/MS) experiments were conducted with a Finnigan LTQ Orbi-
trap mass spectrometry (Thermo Fisher Scientific Inc., MA, USA)
operated in positive-ion electrospray mode.
5.1.4. Synthesis of 5-(3-aminophenyl)-1-benzyl-6,8-
dihydropyrazolo[3,4-b][1,4]diazepin-7(1H)-one (6)
To a solution of 3 (252 mg, 1.06 mmol) and 4,5-diamino-1-(4-
methoxy-benzyl) pyrazole (300 mg, 1.6 mmol) in methylene
chloride (2 ml) was added acetic acid (0.3 mL, 5.3 mmol) drop-
wise. The mixture was stirred for about 30 min and the solvent
was removed in vacuo. To this imine adduct, 25% NaOMe/MeOH
solution was added and stirred at 50 °C for 24 h. The reaction
mixture was then concentrated under vacuum and the crude
product was transferred to the next step without purification.
To a suspension of crude 5 (350 mg) in THF/H2O = 1:1 solution
(4 ml), wad added Fe (245 mg, 4.39 mmol) and NH4Cl (238 mg,
4.45 mmol) was added and heated to 60 °C. Stirring was contin-
ued for 4 h, and then the clear solution was cooled room tem-
perature. The pH is made slightly basic (pH 7 ꢀ 8) by addition
of saturated aqueous sodium bicarbonate before being extracted
with ethyl acetate. The organic phase was thoroughly washed
with brine, dried over sodium sulfate. Evaporation of the solvent
leaves oil, and purification of column chromatography with
5.1.1. Synthesis of 2,2-dimethyl-5-(3-nitrobenzoyl)-1,3-
dioxane-4,6-dione (2)
To a solution of Meldrum’s acid (2.6 g, 17.96 mmol) and
N,N-dimethylamino pyridine (4.4 g, 35.9 mmol) in methylene chlr-
oride (70 ml) was added nitrobenzoyl chloride (5 g, 26.94 mmol)
dropwise at À15 °C. Then the mixture was stirred at the same tem-
perature for 4 h. Methylene chloride wad added (50 ml) to the mix-
ture, and the mixture was washed with brine, dried over Na2SO4
and evaporated to dryness. Purification of column chromatography
with methylene chlroide/methanol = 10:1 yielded
2
(5 g,
17.05 mmol, 94%) as solid. 1H NMR (400 MHz, CDCl3) 8.50 (1 H,
s), 8.44 (1 H, d, J = 8.25 Hz), 7.96 (1 H, d, J = 7.76 Hz), 7.67 (1 H, t,
J = 8.03 Hz), 2.63 (1 H, s), 1.86 (6 H, s).
5.1.2. Synthesis of ethyl 3-(3-nitrophenyl)-3-oxopropanoate (3)
2 (5 g, 17.05 mmol) was dissolved in absolute ethanol (40 ml)
and heated to reflux (100 °C) for 15 h. After cooling down to ambi-

H. Kim et al. / Bioorg. Med. Chem. 19 (2011) 6760–6767
6763
Table 1
Antiproliferative activity of N-[1-benzyl-5-dihydropyrazolodiazepin-7(1H)-one]phenyl amide derivatives 7a–g
O
N
N
N
H
R2
N
R1
HN
O
7
R2
Compd
Substit-ution
R1
A375PGI50
(lM)
U937 GI50 (lM)
O
N
7a
m-
Bn
>30
>30
CF
3
CF
3
7b
7c
m-
p-
Bn
Bn
0.43
13.1
0.06
0.06
Cl
CF
3
Cl
N
N
7d
m-
p-
Bn
Bn
>30
>30
CF
3
N
N
7e
7f
>30
>30
CF
3
m-
p-
Bn
Bn
2.91
1.75
N
N
O
CF3
7g
16.4
5.58
0.21
2.85
O
CF
3
Sorafenib
hexane/ethyl acetate = 1:1 to afford compound
6
(250 mg,
(1H, s), 7.30–7.36 (4H, m), 7.14 (2H, d, J = 6.24), 5.35 (2H, s), 3.88
(2H, t, J = 4.71), 3.58 (2H, s), 3.29 (2H, t, J = 4.79); HRMS calcd for
69%). 1H NMR (400 MHz, CDCl3) 7.84 (2 H, d, J = 8.66 Hz), 7.75
(1 H, bs), 7.72 (1 H, s), 7.33–7.25 (5 H, m), 7.12 (2 H, d,
J = 6.37 Hz), 6.69 (2 H, d, J = 8.63 Hz), 5.32 (2 H, s), 3.95 (2 H,
s), 3.53 (2 H, s).
C31H28F3N6O3 m/z 589.2175 Found 589.2166.
5.1.5.2. N -(3-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-
b][1,4]diazepin-5-yl)phenyl)-4-chloro-3-(trifluoromethyl)benz-
amide (7b). 1H NMR (400 MHz, CDCl3) d 8.2 (1H, s), 7.99–8.06 (3H,
m), 7.96 (1H, s), 7.90 (1H, s), 7.73–7.77 (3H, m), 7.66 (1H, d, J = 8.26),
7.32–7.35 (3H, m), 7.15 (2H, d, J = 6.7 Hz), 5.35 (2H, s), 3.26 (2H, s);
HRMS calcd for C27H20ClF3N5O2 m/z 538.1258 Found 538.1247.
5.1.5. General syntheses of N-(3-(1-benzyl-7-oxo-1,6,7,8-
tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl) amide (7)
A solution of 6 compound (10 mg, 0.028 mmol), substituted
benzoic acid (0.028 mmol), HOBt (6.8 mg, 0.05 mmol), EDCI
5.1.5.3. N-(4-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b]-
[1,4]diazepin-5-yl)phenyl)-3-chloro-4-(trifluoromethyl)benz-
amide (7c). 1H NMR (400 MHz, DMSO-d6) 11.40 (1 H, s), 10.73 (1
H, s), 8.41 (1 H, s), 8.28 (1 H, d, J = 7.35 Hz), 8.03 (2 H, d,
J = 8.80 Hz), 7.94 (1 H, d, J = 8.34 Hz), 7.90 (1 H, d, J = 8.68 Hz),
7.71 (1 H, s), 7.36 (2 H, t), 7.29 (1 H, d, J = 7.08 Hz), 7.15 (1 H, d,
J = 7.88 Hz), 5.75 (2 H, s), 5.37 (2 H, s), 3.48 (2 H, s); HRMS calcd
for C27H20ClF3N5O2 m/z 538.1258 Found 538.1273.
(8.05 mg, 0.04 mmol) and TEA (10 lL, 0.07 mmol) in DMF
(0.5 mL) was stirred at room temperature overnight. The reaction
mixture was diluted with ethyl acetate and washed with saturated
aqueous sodium bicarbonate. The organic layer dried over Na2SO4.
Purification of column chromatography with Hexane/ethyl ace-
tate = 1:1 to afford compound 7.
5.1.5.1. N-(3-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b]-
[1,4]diazepin-5-yl)phenyl)-3-morpholino-5-(trifluoromethyl)-
5.1.5.4. N -(3-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[4,3-
b][1,4]diazepin-5-yl)phenyl)-3-(4-methyl-1H-imidazol-1-yl)-5-
(trifluoromethyl)benzamide (7d). 1H NMR (400 MHz, DMSO-
benzamide (7a). 1H NMR (400 MHz, CDCl3)
d
8.04 (2H, d,
J = 8.68), 7.94–7.97 (2H, m), 7.74–7.77 (3H, m), 7.61 (1H, s), 7.44

6764
H. Kim et al. / Bioorg. Med. Chem. 19 (2011) 6760–6767
Table 2
Antiproliferative activity of N-[1-benzyl-5-dihydropyrazolodiazepin-7(1H)-one]phenyl urea derivatives 8a–o
O
N
R3
N
N
H
N
H
N
R1
HN
O
8
Compd
Substit-ution
R1
R2
A375P (GI50
4.45
(l
M)
U937 GI50
7.31
(lM)
CF
Cl
3
3
3
3
8a
m-
Bn
CF
8b
8c
8d
p-
p-
p-
Bn
1.19
3.93
>30
8.68
7.29
>30
Cl
CF
PMB
H
Cl
CF
Cl
N
N
8e
8f
p-
Bn
>30
>30
>30
CF
3
O
N
m-
PMB
3.32
CF
3
Cl
8g
8h
p-
Bn
2.07
0.91
0.67
0.63
Cl
m-
PMB
8i
8j
m-
m-
PMB
H
0.64
>30
0.58
>30
Cl
Cl
Cl
8k
8l
m-
PMB
PMB
0.78
3.86
6.48
14.9
Cl
Cl
m-
Cl
Cl
8m
8n
m-
m-
m-
H
>30
>30
Cl
CF
3
3
PMB
PMB
0.25
0.28
8o
0.77
5.58
0.11
2.85
CF
Sorafenib
d6) 10.47 (1 H, s), 9.54 (1 H, s), 8.32 (2 H, m), 8.16 (1 H, d,
J = 9.01 Hz), 8.01 (1 H, s), 7.55 (2 H, s), 7.52 (1 H, s), 7.44
(1 H, s), 7.15 (2 H, d, J = 8.22 Hz), 6.88 (2 H, d, J = 8.588
Hz), 5.13 (2 H, s), 5.05 (2 H, s), 3.72 (3 H, s), 2.34 (3 H, s);
HRMS calcd for
584.2073.
C31H245F3N7O2 m/z 584.2022 Found

H. Kim et al. / Bioorg. Med. Chem. 19 (2011) 6760–6767
6765
Table 3
5.1.5.7. N -(4-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-
b][1,4]diazepin-5-yl)phenyl)-4-morpholino-3-(trifluoro-
Percentages of enzymatic inhibitions by compound 7b (10 lM) on selected Protein
Kinases
methyl)benzamide (7g). 1H NMR (400 MHz, DMSO-d6) 10.57
(1H, s), 8.27 (1H, s), 8.02 (2H, d, J = 8.85), 7.91 (2H, d, J = 8.91),
7.65–7.71 (3H, m), 7.15 (2H, d, J = 7.16), 6.27–7.36 (4H, m), 5.34
(2H, s), 3.74 (2H, t, J = 4.34), 3.53 (2H, s), 2.97 (2H, t, J = 4.35);
HRMS calcd for C31H27F3N6O3 m/z 589.2175 Found 589.2141.
Kinase
Inhibition (%)
Staurosporine IC50 (nM)
ABL1
AKT1
ALK
Aurora A
B-Raf (V599E)
CDK1/cyclin B
CDK2/cyclin E
c-Kit
c-MET
c-Src
EGFR
ERK2/MAPK1
FAK/PTK2
FGFR2
FGFR3
FLT3
FMS
GSK3b
IGF1R
JAK3
JNK1
JNK3
KDR/VEGFR2
LCK
LYN
0
0
0
105.90
4.95
3.00
6.5
85.6
7.0
0.9
3.6
1.3
4.3
1.6
0
0
0
0
19.3
0.5
17.8
1.9
0
2.28
4.33a
1.30
5.1.6. General syntheses of N-(3-(1-benzyl-7-oxo-1,6,7,8-
tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl) urea
(8a–8o)
A mixture of 6 and substituted isocyanate in THF was stirred at
room temperature overnight. The reaction mixture was cooled, ex-
tracted with CH2Cl2. The organic layer was washed with water and
brine. After drying over anhydrous MgSO4, and the mixture was
evaporated under reduced pressure to give a residue. The residue
was purified by silica gel chromatography.
1.88
65.19
153.00
3.78
188.30
3948.00
17.15
3.36
14.71
1.01
3.13
5.1.6.1. 1-(3-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b]
[1,4]diazepin-5-yl)phenyl)-3-(4-chloro-3-(trifluoro-
methyl)phenyl)urea (8a). 1H NMR (400 MHz, DMSO-d6) 11.45
(1H, s), 9.20 (1H, s), 9.13 (1H, s), 8.12 (2H, s), 7.74 (1H, s), 7.59–
7.74 (4H, m), 7.26–7.45 (4H, m), 7.15 (2H, d, J = 2.40), 5.38 (2H,
s), 3.53 (2H, s); HRMS calcd for C27H21ClF3N6O2 m/z 553.1367
Found 553.1386.
9.23
85.33
< 1.0
1746.00
6745b
13.73
4.49
0
0
0
5.6
4.6
0
1.30
8.07
MEK1
mTOR/FRAP1
P38a/MAPK14
0
0
6796c
14.3d
4.40e
317.90
4.93a
442.50
<1.0
5.1.6.2. 1-(4-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b]-
[1,4]diazepin-5-yl)phenyl)-3-(4-chloro-3-(trifluoro-
PI3K
a
75.2
0
92.9
4.4
0
methyl)phenyl)urea (8b). 1H NMR (400 MHz, CDCl3) 8.21 (1H, s),
8.05 (2H, d, J = 8.73), 8.00 (1H, d, J = 8.30), 7.92 (1H, s), 7.78 (1H, s),
7.74 (2H, d, J = 8.78), 7.66 (1H, d, J = 8.33), 7.42–7.32 (4H, m), 7.14
(2H, d, J = 7.86), 5.37 (2H, s), 3.65 (2H, s); HRMS calcd for m/z
PLK1
C-Raf
RON/MST1R
ROS/ROS1
SYK
0
<1.0
TRKB
0.2
<1.0
C27H21ClF3N6O2 553.1367 Found 553.1352.
a
b
c
Data of GW5074.16
Data of JNKi VIII.17
Data of LY294002.18
Data of SB202190.19
Data of PI103.20
5.1.6.3. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(1-(4-met-
hoxybenzyl)-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b][1,4]diaze-
pin-5-yl)phenyl)urea (8c). 1H NMR (400 MHz, CDCl3) 8.11 (1 H,
s), 7.90 (1 H, d, J = 8.44 Hz), 7.83 (1 H, d, J = 8.64 Hz), 7.72 (1 H, d,
J = 8.57 Hz), 7.67 (1 H, d, J = 2.13 Hz), 7.55 (1 H, d, J = 9.10 Hz),
7.39–7.31 (2 H, m), 7.10 (2 H, t, J = 8.25 Hz), 6.84 (2 H, d,
J = 8.57 Hz), 6.68 (1 H, d, J = 8.66 Hz), 5.26 (2 H, s), 3.76 (3 H, s),
3.48 (2 H, d, J = 5.66 Hz); HRMS calcd for m/z C28H23ClF3N6O3
583.1472 Found 583.1418.
d
e
Table 4
Enzymatic activities of selected compounds
IC50 (nM)
B-Raf V600E
C-Raf
PI3Ka
5.1.6.4. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(7-oxo-1,6,
7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl)urea
(8d). 1H NMR (400 MHz, DMSO-d6) 12.86 (1 H, s), 10.62 (1 H, s),
9.44 (1 H, s), 9.33 (1 H, s,), 8.13 (1 H, s), 7.96 (1 H, s), 7.93 (2 H,
d, J = 8.74 Hz), 7.67–7.60 (4 H, m), 3.48 (2 H, s); HRMS calcd for
7b
8a
655.2
NA
960.9
667.8
2150
ND
5.1.5.5. N -(4-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[4,3-
b][1,4]diazepin-5-yl)phenyl)-3-(4-methyl-1H-imidazol-1-yl)-5-
(trifluoromethyl)benzamide (7e). 1H NMR (400 MHz, DMSO-d6)
11.21 (1 H, s), 9.02 (1 H, s), 8.99 (1 H, s), 8.10 (1 H, s), 7.72 (2 H, d,
J = 20.4 Hz), 7.60 (1 H, s), 7.36 (1 H, d, J = 11.22 Hz),, 7.32 (2 H, d,
J = 7.72 Hz), 7.27 (1 H, d, J = 7.34 Hz), 7.11 (2 H, d, J = 6.84 Hz),
6.97 (1 H, s), 6.92 (1 H, s), 6.80 (1 H, s), 6.59 (2 H, d, J = 8.61 Hz),
5.87 (1 H, s), 5.73 (2 H, s), 3.47 (2 H, s), 2.15 (3 H, s); HRMS calcd
for C31H25F3N7O2 m/z 584.2022 Found 584.2092.
C20H15ClF3N6O2 m/z 463.0897 Found 463.0849.
5.1.6.5. 1-(4-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b]-
[1,4]diazepin-5-yl)phenyl)-3-(3-(4-methyl-1H-imidazol-1-yl)-5-
(trifluoromethyl)phenyl)urea (8e). 1H NMR (400 MHz, DMSO-
d6) 9.11 (1 H, s), 8.99 (1 H, s), 8.01 (1 H, s), 7.73 (1 H, s), 7.72 (2
H, d, J = 16 Hz), 7.36 (1 H, d, J = 11.22 Hz), 7.32 (2 H, d, J = 7.7 Hz),
7.21 (1 H, d, J = 7.6 Hz), 7.11 (2 H, d, J = 6.84 Hz), 6.97 (1 H, s),
6.92 (1 H, s), 6.80 (1 H, s), 6.59 (2 H, d, J = 8.61 Hz), 5.73 (2 H, s),
3.47 (2 H, s), 2.15 (3 H, s); HRMS calcd for m/z C31H26F3N8O2
599.2131 Found 599.2132.
5.1.5.6. N -(3-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-
b][1,4]diazepin-5-yl)phenyl)-4-morpholino-3-(trifluoro-
methyl)benzamide (7f). 1H NMR (400 MHz, CDCl3) 8.16 (1 H, d)
8.08 (1H, s), 8.05 (1 H, s), 8.01 (1 H, s), 7.98 (1 H, s), 7.76 (1 H, s),
7.75 (1 H, s), 7.46 (1 H, t, J = 8.0 Hz), 7.39 (2 H, d, J = 8.43 Hz),
7.35–7.31 (3 H, m), 7.14 (2 H, d, J = 7.78 Hz), 5.41 (2 H, s), 3.86 (4
H, m), 3.48 (2 H, s), 3.01 (4 H, m); HRMS calcd for C31H28F3N6O3
m/z 589.2175 Found 589.2145.
5.1.6.6. 1-(3-(1-(4-methoxybenzyl)-7-oxo-1,6,7,8-tetrahydropy-
razolo[3,4-b][1,4]diazepin-5-yl)phenyl)-3-(3-morpholino-5-(tri-
fluoromethyl)phenyl)urea (8f). 1H NMR (400 MHz, CDCl3) 8.16
(1 H, d) 8.08 (1H, s), 8.05 (1 H, s), 8.01 (1 H, s), 7.98 (1 H, s), 7.76
(1 H, s), 7.75 (1 H, s), 7.46 (1 H, t, J = 8.0 Hz), 7.39 (2 H, d,

6766
H. Kim et al. / Bioorg. Med. Chem. 19 (2011) 6760–6767
5.1.6.14. 1-(3-(1-(4-methoxybenzyl)-7-oxo-1,6,7,8-tetrahydro-
pyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl)-3-(3-(trifluoro-
methyl)phenyl)urea (8n). 1H NMR (400 MHz, CDCl3) 8.13 (1 H,
s), 8.11 (1 H, s), 7.90 (1 H, d, J = 8.44 Hz), 7.83 (1 H, d,
J = 8.64 Hz), 7.72 (1 H, d, J = 8.57 Hz), 7.67 (1 H, t, J = 2.13 Hz),
7.62 (1 H, s), 7.55 (1 H, d, J = 9.10 Hz), 7.39–7.31 (4 H, m),
7.10 (2 H, t, J = 8.25 Hz), 6.84 (2 H, d, J = 8.57 Hz), 6.68 (1 H, d,
J = 8.66 Hz), 5.26 (2 H, s), 3.76 (3 H, s), 3.48 (2 H, d, J = 5.7
Hz; HRMS calcd for C28 H24F3N6O3 m/z 549.1862 Found
549.1819.
J = 8.43 Hz), 7.14 (1 H, d, J = 7.78 Hz), 7.10 (2 H, d, J = 8.25 Hz), 6.84
(2 H, d, J = 8.26 Hz), 5.41 (2 H, s), 3.86 (4 H, m), 4.76 (3 H, s), 3.48 (2
H, s), 3.01 (4 H, m); HRMS calcd for m/z C32H31F3N7O4 634.2390
Found 634.2358.
5.1.6.7.
1-(4-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-
b][1,4]diazepin-5-yl)phenyl)-3-(3-chlorophenyl)urea (8g). 1H
NMR (400 MHz, CDCl3) 7.93 (1 H, d, J = 8.34 Hz), 7.75 (1 H, s),
7.55 (1 H, s), 7.44–7.30 (5 H, m), 7.29–7.21 (2 H, d, J = 7.08 Hz),
7.15 (2 H, d, J = 7.88 Hz), 5.27 (2 H, s), 3.48 (2 H, s); HRMS calcd
for C26H22ClN6O2 m/z 485.1493 Found 485.1494.
5.1.6.15. 1-(3-(1-(4-methoxybenzyl)-7-oxo-1,6,7,8-tetrahydro-
pyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl)-3-(4-(trifluoro-
methyl)phenyl)urea (8o). 1H NMR (400 MHz, CDCl3) 8.11 (1 H,
s), 7.90 (1 H, d, J = 8.44 Hz), 7.83 (1 H, d, J = 8.64 Hz), 7.72 (1 H, d,
J = 8.57 Hz), 7.67 (1 H, t, J = 2.13 Hz), 7.62 (2 H, s), 7.61 (2 H, s),
7.55 (1 H, d, J = 9.10 Hz), 7.39–7.31 (2 H, m), 7.10 (2 H, d,
J = 8.25 Hz), 6.84 (2 H, d, J = 8.57 Hz), 6.68 (1 H, d, J = 8.66 Hz),
5.26 (2 H, s), 3.76 (3 H, s), 3.48 (2 H, d, J = 5.7 Hz); HRMS calcd
for C28 H24F3N6O3 m/z 549.1862 Found 549.1806.
5.1.6.8. 1-(3-chlorophenyl)-3-(3-(1-(4-methoxybenzyl)-7-oxo-
1,6,7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)pheny-
l)urea (8h). 1H NMR (400 MHz, CDCl3) 8.12 (1 H, s), 8.01 (1 H, s),
7.90 (1 H, d, J = 2.5 Hz), 7.88 (1 H, d, J = 2.5 Hz), 7.76 (1 H, s), 7.71 (2
H, s), 7.46 (1 H, t, J = 8.0 Hz), 7.39 (2 H, d, J = 8.43 Hz), 7.35–7.31 (3
H, m), 7.14 (2 H, d, J = 7.78 Hz), 7.10 (2 H, d, J = 8.26 Hz), 6.84 (2H,
d, J = 8.25 Hz), 3.75 (3 H, s), 3.48 (2 H, s); HRMS calcd for m/z
C27H24ClN6O3 515.1598 Found 515.1575.
5.1.6.9. 1-(4-chlorophenyl)-3-(3-(1-(4-methoxybenzyl)-7-oxo-
1,6,7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)pheny-
l)urea (8i). 1H NMR (400 MHz, CDCl3) 8.12 (1 H, s), 8.01 (1 H, s),
7.90 (1 H, d, J = 2.5 Hz), 7.88 (1 H, d, J = 2.5 Hz), 7.76 (1 H, s), 7.75
(2 H, s), 7.46 (1 H, t, J = 8.0 Hz), 7.39 (2 H, d, J = 8.43 Hz), 7.35–
7.31 (3 H, m), 7.14 (2 H, d, J = 7.8 Hz), 3.74 (2 H, s), 3.48 (2 H, s);
HRMS calcd for C27H24ClN6O3 m/z 515.1598 Found 515..1559.
5.2. Antiproliferative activity
A375P cells were purchased from American Type Culture Col-
lection (ATCC, Rockville, MD, US) and maintained in DMEM
medium (Welgene, Daegu, Korea) supplemented with 10% FBS
(Welgene) and 1% penicillin/streptomycin (Welgene) in
a
humidified atmosphere with 5% CO2 at 37 °C. A375P cells were
taken from culture substrate with 0.05% trypsin-0.02% EDTA
and plated at a density of 5 Â 103 cells/well in 96 well plates
and then incubated at 37 °C for 24 h in a humidified atmosphere
with 5% CO2 prior to treatment of various concentration (three-
fold serial dilution, 12 points) of test compounds. The A357P cell
viability was assessed by the conventional 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction as-
say. MTT assays were carried out with CellTiter 96Ò (Promega)
according to the manufacturer’s instructions. The absorbance at
590 nm was recorded using EnVision 2103 (Perkin Elmer; Bos-
ton, MA, US). The IC50 was calculated using GraphPad Prism
4.0 software.
U937 cells were purchased from American Type Culture Collec-
tion (ATCC, Rockville, MD, US) and maintained in RPMI 1640 med-
ium (Welgene, Daegu, Korea) supplemented with 10% FBS
(Welgene), 1% penicillin/streptomycin (Welgene) and 25 mM
HEPES (Welgene) in a humidified atmosphere with 5% CO2 at
37 °C. U937 cells were taken from culture substrate and plated
at a density of 5 Â 103 cells/well in 96 well plates and then incu-
bated at 37 °C for 24 h in a humidified atmosphere with 5% CO2
prior to treatment of various concentration (threefold serial
dilution, 12 points) of test compounds. The U937 cell viability
was assessed by the conventional 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) reduction assay. MTT as-
says were carried out with Thiazolyl Blue Tetrazolium Bromide
(SIGMA) according to the manufacturer’s instructions. The absor-
bance at 570 nm was recorded using Multiskan EX (Thermo;
Waltham, MA, US). The IC50 was calculated using GraphPad Prism
4.0 software.
5.1.6.10. 1-(4-chlorophenyl)-3-(3-(7-oxo-1,6,7,8-tetrahydropy-
razolo[3,4-b][1,4]diazepin-5-yl)phenyl)urea
(8j). 1H
NMR
(400 MHz, DMSO-d6) 13.7 (1 H, s), 10.82 (1 H, s), 9.18 (1 H, s),
9.08 (1 H, s), 8.12 (1 H, s), 8.01 (1 H, s), 7.90 (1 H, d, J = 2.5 Hz),
7.88 (1 H, d, J = 2.5 Hz), 7.76 (1 H, s), 7.75 (2 H, s), 7.46 (1 H, t,
J = 8.0 Hz), 7.39 (2 H, d, J = 8.43 Hz), 7.35–7.31 (3 H, m), 7.14 (2
H, d, J = 7.78 Hz), 3.48 (2 H, s); HRMS calcd for m/z C19H16ClN6O2
395.1023 Found 395.1094.
5.1.6.11. 1-(3,5-dichlorophenyl)-3-(3-(1-(4-methoxybenzyl)-7-
oxo-1,6,7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)pheny-
l)urea (8k). 1H NMR (400 MHz, CDCl3) 8.10 (1 H, s), 7.90 (1 H, d,
J = 8.44 Hz), 7.87 (2 H, d, J = 8.2 Hz), 7.78 (1 H, d, J = 8.1 Hz), 7.60 (2
H, s), 7.43 (1 H, s), 7.39–7.31 (2 H, m), 7.15 (1 H, s), 7.10 (2 H, d,
J = 8.25 Hz), 6.84 (2 H, d, J = 8.57 Hz), 6.68 (1 H, d, J = 8.66 Hz),
5.26 (2 H, s), 3.76 (3 H, s), 3.48 (2 H, s); HRMS calcd for m/z
C27H23Cl2N6O3 549.1209 Found 549.1295.
5.1.6.12. 1-(3,4-dichlorophenyl)-3-(3-(1-(4-methoxybenzyl)-7-
oxo-1,6,7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)pheny-
l)urea (8l). 1H NMR (400 MHz, CDCl3) 8.13 (1 H, s), 8.11 (1 H, s),
7.90 (1 H, d, J = 8.44 Hz), 7.83 (1 H, d, J = 8.64 Hz), 7.78 (1 H, d,
J = 8.1 Hz), 7.67 (1 H, t, J = 2.13 Hz), 7.60 (2 H, s), 7.51 (1 H, d,
J = 5.8 Hz), 7.39–7.31 (4 H, m), 7.15 (1 H, s), 7.10 (2 H, d,
J = 8.25 Hz), 6.84 (2 H, d, J = 8.57 Hz), 6.68 (1 H, d, J = 8.66 Hz),
5.26 (2 H, s), 3.76 (3 H, s), 3.48 (2 H, d, J = 5.66 Hz); HRMS calcd
for m/z C27H23Cl2N6O3 549.1209 Found 549.1281.
5.1.6.13. 1-(3,4-dichlorophenyl)-3-(3-(7-oxo-1,6,7,8-tetrahydro-
pyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl)urea (8m). 1H NMR
(400 MHz, DMSO-d6) 13.7 (1H, s), 10.82 (1 H, s), 9.18 (1 H, s),
9.08 (1 H, s), 8.16 (1 H, s), 8.01 (1 H, s), 7.90 (1 H, d, J = 2.6 Hz),
7.88 (1 H, d, J = 2.6 Hz), 7.76 (1 H, s), 7.54 (1 H, s), 7.46 (1 H, t,
J = 8.0 Hz), 7.39 (2 H, d, J = 8.43 Hz), 7.35–7.31 (3 H, m), 7.14 (2
H, d, J = 7.78 Hz), 3.49 (2 H, s); HRMS calcd for C19H15Cl2N6O2 m/z
429.0634 Found 429.0603.
5.3. Docking simulations
Molecular docking of compound 7b into 3D X-ray structure of
V600E B-Raf (PDB code: 1uwj) was carried out using the Glide
(Schrodinger software package Version 9.1).

H. Kim et al. / Bioorg. Med. Chem. 19 (2011) 6760–6767
6767
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5.4. Selected kinase profiling
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We used Reaction Biology Corp. Kinase HotSpotSM service
(http://www.reactionbiology.com) for screening of 7b, and IC50
ProfilerExpress for IC50 measurement. Assay protocol: In a final
reaction volume of 25
25 mM Tris pH 7.5, 0.02 mM EGTA, 0.66 mg/mL myelin basic pro-
tein, 10 mM Mg Acetate and [
33P-ATP] (specific activity approx.
lL, c-Raf (h) (5–10 mU) is incubated with
c-
500 cpm/pmol, concentration as required). The reaction is initiated
13. Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2, 358.
by the addition of the Mg–ATP mix. After incubation for 40 min at
14. A375P cells were purchased from American Type Culture Collection (ATCC,
Rockville, MD, US) and maintained in DMEM medium (Welgene, Daegu, Korea)
supplemented with 10% FBS (Welgene) and 1% penicillin/streptomycin
(Welgene) in a humidified atmosphere with 5% CO2 at 37 °C. A375P cells
were taken from culture substrate with 0.05% trypsin-0.02% EDTA and plated
at a density of 5 Â 103 cells/well in 96 well plates and then incubated at 37 °C
for 24 h in a humidified atmosphere with 5% CO2 prior to treatment of various
concentration (threefold serial dilution, 12 points) of test compounds. The
A357P cell viability was assessed by the conventional 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. MTT assays
were carried out with CellTiter 96Ò (Promega) according to the manufacturer’s
instructions. The absorbance at 590 nm was recorded using EnVision 2103
(Perkin Elmer; Boston, MA, US). The IC50 was calculated using GraphPad Prism
4.0 software.
room temperature, the reaction is stopped by the addition of 5
lL
of a 3% phosphoric acid solution. 10 L of the reaction is then spot-
l
ted onto a P30 filtermat and washed three times for 5 min in
75 mM phosphoric acid and once in methanol prior to drying and
scintillation counting.
Acknowledgment
This work was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2009-0087992; J.M.H.).
15. We used Reaction Biology Corp. Kinase HotSpotSM service (http://
www.reactionbiology.com) for screening of 7b, and IC50ProfilerExpress for
IC50 measurement. Assay protocol: In a final reaction volume of 25
(5–10 mU) is incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA, 0.66 mg/mL
myelin basic protein, 10 mM Mg Acetate and [
33P-ATP] (specific activity
lL, c-Raf (h)
c
-
References and notes
approx. 500 cpm/pmol, concentration as required). The reaction is initiated by
the addition of the Mg–ATP mix. After incubation for 40 min at room
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phosphoric acid solution. 10 L of the reaction is then spotted onto a P30
l
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