Synthesis and biological evaluation of 1-substituted-3(5)-(6-methylpyridin- 2-yl)-4-(quinoxalin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.05.063

A series of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl) pyrazoles 14a-d, 15a-d, 17a, 17b, 18a-d, 19a, and 19b has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporte

Full text of DOI:10.1016/j.ejmech.2011.05.063

European Journal of Medicinal Chemistry 46 (2011) 3917e3925
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of 1-substituted-3(5)-(6-methylpyridin-2-yl)-
4-(quinoxalin-6-yl)pyrazoles as transforming growth factor-
kinase inhibitors
b type 1 receptor
Cheng Hua Jin a, Domalapally Sreenu a, Maddeboina Krishnaiah a, Vura Bala Subrahmanyam a,
Kota Sudhakar Rao a, Annaji Venkata Nagendra Mohan a, Chul-Yong Park a, Jee-Yeon Son a, Do-Hyun Son b,
,
Hyun-Ju Park b, Yhun Yhong Sheen a, Dae-Kee Kim a
*
a College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, South Korea
b College of Pharmacy, Sungkyunkwan University, Suwon 440-746, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles 14aed, 15aed, 17a,
17b, 18aed, 19a, and 19b has been synthesized and evaluated for their ALK5 inhibitory activity in an
enzyme assay and in a cell-based luciferase reporter assay. The 2-[3-(6-methylpyridin-2-yl)-4-(quinox-
alin-6-yl)-1H-pyrazol-1-yl]-N-phenylethanethioamide (18a) inhibited ALK5 phosphorylation with an
Received 7 March 2011
Received in revised form
9 May 2011
Accepted 26 May 2011
Available online 21 June 2011
IC50 value of 0.013
cells permanently transfected with p3TP-luc reporter construct.
Ó 2011 Elsevier Masson SAS. All rights reserved.
mM and showed 80% inhibition at 0.1 mM in a luciferase reporter assay using HaCaT
Keywords:
TGF-b
ALK5 inhibitor
Fibrosis
Cancer
Kinase assay
Cell-based luciferase reporter assay
Docking
1. Introduction
binding of ligand to the constitutively active type II receptors on the
cell surface, which further recruits the type I receptors, also called
as activin receptor-like kinase 5 (ALK5), into the complex. Subse-
quently, ALK5 is phosphorylated in the juxtamembrane GS domain
by the type II receptors thereby creating a binding site for Smad
proteins and stimulating its kinase activity. The activated ALK5
phosphorylates Smad2 and Smad3 proteins thereby causing their
dissociation from the receptor and heteromeric complex formation
with Smad4. These Smad complexes translocate to the nucleus,
assemble with specific DNA-binding co-factors and co-modulators
to finally activate several hundred genes involved in cell differen-
tiation, proliferation, apoptosis, migration, and extracellular matrix
The transforming growth factor-
members, which include TGF-bs, activins, bone morphogenetic
b
(TGF-
b
)
superfamily
proteins (BMPs), growth and differentiation factors, and Mullerian
inhibiting substance are structurally related secreted cytokines
found in species ranging from worms and insects to mammals. A
wide range of cellular functions such as cell proliferation, differ-
entiation, adhesion, migration, and apoptosis are regulated by TGF-
b
superfamily members. The TGF-
isoforms, TGF- 1, TGF- 2, and TGF-
1 is the prototypic member of this
family of cytokines and is the major isoform. TGF- 1 transduces
b
s include the three major TGF-
b
b
b
b
3 which are expressed in
mammals. Among them, TGF-
b
b
production [4]. TGF-
progression of fibrosis in various organ systems such as kidney [5],
heart [6], lung [7], and liver [8]. Deregulation of TGF- signaling has
b1 plays a critical role in the initiation and
signals through a complex of two related but structurally and
functionally distinct serine/threonine kinase receptors, termed
type 1 and type II [1e3]. The signaling cascade is promoted by the
b
been also implicated in various human diseases including cancer
[9], pancreatic diseases [10], and hematological malignancies [11].
There are a number of reports that the therapeutic administration
of TGF-
soluble chimeric TGF-
b
binding proteins including the proteoglycan decorin [12],
receptors [13], and neutralizing antibodies
* Corresponding author. Tel.: þ82 2 3277 3025; fax: þ82 2 3277 2467.
E-mail address: dkkim@ewha.ac.kr (D.-K. Kim).
b
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.063

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C.H. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 3917e3925
[14] ameliorates experimental fibrosis. Recent studies have shown
that several small molecules ATP-competitive ALK5 inhibitors (Fig.1)
such as 1 (SB-505154) [15], 2 (SB-525334) [16], 3 (GW6604) [17], 4
(SD-208) [18,19], and 5 (LY-2157299) [20] inhibited autophosphor-
Therefore, in this report, we attempted to replace a thioamide
linkage with a chemically stable thioamidomethylene linkage, thus,
prepared the compounds 18aed as target molecules. To compare
the influence of the thioamidomethylene linkage of 18aed on ALK5
activity, their counterpart derivatives 14aed possessing the ami-
domethylene linkage were prepared. The compounds 17a and 17b
possessing the amidoethylene linkage were also prepared to opti-
mize the length between the pyrazole ring and a phenyl ring. A
docking model of ALK5:7 complex showed that the nitrogen atom
of the quinolin-6-yl moiety formed an H-bond with backbone
amide NH of His283 in the hinge region of the kinase, originally
a binding pocket for the adenine ring of ATP [27]. To examine
whether the capability of the N-1 of the quinoxalin-6-yl moiey as
an H-bond acceptor could be increased by aromatic substitution,
compounds having a 2,3-dimethylquinoxalin-6-yl moiety were
prepared for comparison (14b,14d,15b,15d,17b,18b,18d, and 19b).
ylation of ALK5 and TGF-b induced transcription of matrix genes in
receptor assays at sub micromolar concentrations. Among them 2, 3,
and 4 effectively retarded progressive fibrosis in kidney, liver and
lung, respectively, and 4 and 5 also strongly inhibited growth and
invasiveness of cancer cells in animal models.
We have prepared a number of the 2-pyridyl-substituted tri-
azoles, pyrazoles, imidazoles, and thiazoles as ALK5 inhibitors and
found that insertion of a methylene, a methyleneamino, or an
aminomethylene linkage between
a central five-membered
heterocyclic ring and a phenyl ring significantly increased ALK5
inhibitory activity and selectivity [21e29]. Among them, the
imidazole IN-1130 (6), one of our preclinical candidate, demon-
strated its remarkable activity as a suppressor of fibrogenic
process of unilateral ureteral obstruction in rats [30] and amelio-
2. Results and discussion
rated experimental autoimmune encephalomyelitis,
a mouse
model for multiple sclerosis, by inhibition of TGF- signaling [31].
b
2.1. Chemistry
It also reduced tunical fibrosis and corrected penile curvature in
rats [32], inhibited cancer metastasis in MMTV/c-Neu breast
cancer mice, and enhanced CTL response in cancer mice [33]. The
imidazole IN-1233 (7), another preclinical candidate, effectively
prevented development and progression of pulmonary arterial
hypertension in monocrotaline rat model through inhibition of
The 3-(6-methylpyridin-2-yl)-1-(phenylcarboxamidomethyl)-
4-(quinoxalin-6-yl)pyrazoles 14aed were synthesized as shown in
Scheme 1. The quinoxaline-6-carbaldehyde (10a) [37] and 2,3-
dimethylquinoxaline-6-carbaldehyde (10b) [37] were coupled
with
diphenyl
(6-methylpyridin-2-yl)(phenylamino)methyl-
TGF-b signaling [34] and reduced granulation tissue formation
phosphonate [38] in a mixture of THF and i-PrOH (4:1) in the
presence of Cs2CO3 at room temperature and followed by treatment
with 3 N HCl to afford the corresponding ketones 11a and 11b in
79% and 86% yields. Treatment of 11a and 11b with N,N-dime-
thylformamide dimethyl acetal (DMF$DMA) in DMF at 80 ꢀC in the
presence of AcOH and followed by cyclization with hydrazine
monohydrate in absolute EtOH produced the pyrazoles 12a [39]
and 12b in 79% and 75% yields. The pyrazoles 12a and 12b were
alkylated with 2-chloro-N-phenylacetamide (13a) [40] or 2-chloro-
N-(3-cyanophenyl)acetamide (13b) [41] in the presence of NaH in
anhydrous DMF to yield the target compounds 14aed and their
positional isomers 15aed in 41e70% and 4e10% yields, respectively.
The positional isomers were separated by MPLC, and their struc-
tures were confirmed by NOE experiments. In NOE experiments,
after bare metallic stent placement in a rat urethral model [35].
A novel class of ALK5 inhibitors possessing a thioamide linkage
between a pyrazole ring and a phenyl ring has been reported, and
among them, A-83-01 (8) exhibited significant inhibition of the
transcriptional activity induced by ALK5 [36]. We also recently
reported a new class of 2-pyridyl-substituted pyrazoles possessing
a thioamide linkage, and one of the compounds in this series, 3-[3-
(6-methylpyridin-2-yl)-4-(quinolin-6-yl)-1H-pyrazol-1-
carbothioamido]benzamide (9), showed more than 90% inhibition
at 0.1 mM in luciferase reporter assays [26]. Although insertion of
a thioamide linkage between a pyrazole ring and a phenyl ring
markedly increased ALK5 inhibitory activity as shown in previous
reports [26,36], the thioamide linkage was rather unstable and
slowly cleaved to generate a pyrazole ring on long-term storage.
irradiation of the methylene protons of the 14a at
d
5.14 gave an
7.86, while
enhancement of the proton H-5 in pyrazole ring at
d
Fig. 1. Small molecule ATP-competitive ALK5 inhibitors.

C.H. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 3917e3925
3919
Scheme 1. Reagents and conditions: (a) diphenyl (6-methylpyridin-2-yl)(phenylamino)methylphosphonate, Cs2CO3, THF: i-PrOH (4:1), rt, 16 h; (ii) 3 N HCl 1 h; (b) (i) DMF$DMA,
DMF, AcOH, 80 ꢀC, 2 h; (ii) N2H4$H2O, EtOH, reflux, 4 h; (c) NaI (cat.), NaH, anhydrous DMF, rt, 2 h.
irradiation of the methylene protons of the 15a at
enhancement of the proton H-5 in pyrazole ring at
firming the respective positions of alkylation.
d
5.06 gave no
7.95, con-
2.2. ALK5 inhibitory activity in an enzyme assay and in a cell-based
luciferase reporter assay
d
The 3-(6-methylpyridin-2-yl)-1-(phenylcarboxamidoethyl)-4-
(quinoxalin-6-yl)pyrazoles 17a and 17b were synthesized as shown
in Scheme 2. Reaction of aniline or 3-aminobenzonitrile with 3-
bromopropanoyl chloride in the presence of K2CO3 in CH2Cl2 gave
3-bromo-N-phenylpropanamide (16a) [42] and 3-bromo-N-(3-
cyanophenyl)propanamide (16b) in 58% and 70% yields, respec-
tively. Alkylation of the pyrazoles 12a and 12b with 16a or 16b in
the presence of Cs2CO3 in anhydrous DMF at 120 ꢀC afforded the
target compounds 17a and 17b in 66% and 41% yields, respectively.
In this case, the positional isomers of the 17a and 17b were not
isolated after column chromatography.
Thionation of the 14aed with Lawesson’s reagent in anhydrous
DME at reflux condition produced the thioamides 18aed in 24e68%
yields (Scheme 3). It was observed in the 1H NMR that the meth-
ylene protons attached to the N-1 in the thioamides 18aed were
shifted to the downfield by around ca. 0.4 ppm relative to those in
the amides 14aed.
Conversion of the nitrile functionality in compounds 18c and
18d to the corresponding carboxamide functionality was attempted
by treatment with 28% H2O2 and 1 N NaOH in EtOH at 40 ꢀC,
however, both hydrolysis and dethionation took place to generate
the 19a and 19b in 23% and 30% yields, respectively (Scheme 4). As
expected, all of the target compounds we prepared were quite
stable at room temperature on long-term storage.
To investigate whether these compounds 14aed, 15aed, 17a,
17b, 18aed, 19a, and 19b could inhibit ALK5, a kinase assay was
performed using the purified human ALK5 kinase domain produced
in Sf9 insect cells. All the compounds possessing a quinoxalin-6-yl
moiety (14a, 14c, 15a, 15c, 17a, 18a, 18c, and 19a) displayed potent
ALK5 inhibition (IC50 ¼ 0.013e0.737
mM), whereas those possessing
a 2,3-dimethylquinoxalin-6-yl moiety (14b, 14d, 15b, 15d, 17b, 18b,
18d, and 19b) displayed no significant ALK5 inhibitory activity up to
a concentration of 1 mM (Table 1). We envisioned that introduction
of electron-donating groups in the 6-quinoxalinyl moiety may
increase the capability of the N-1 in that moiety as an H-bond
acceptor, thus, potentiating ALK5 inhibitory activity. But, the result
showed that a 2,3-dimethylquinoxalin-6-yl moiety seemed to be
not accommodated favorably into the ATP binding pocket of ALK5.
The 3-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles 14a
(IC50 ¼ 0.038
m
M) and 14c (IC50 ¼ 0.153
mM) displayed 3.8-fold and
4.8-fold higher ALK5 inhibitory activity than their respective
positional isomers, 5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-
pyrazoles 15a (IC50 ¼ 0.143
m
M) and 15c (IC50 ¼ 0.737
mM). The
length of alkyl chain between the N-1 of the central pyrazole and
Scheme 3. Reagents and conditions: (a) Lawesson’s reagent, anhydrous DME, 85 ꢀC,
12 h.
Scheme 2. Reagents and conditions: (a) Cs2CO3, anhydrous DMF, 120 ꢀC, 2 h.

3920
C.H. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 3917e3925
ALK5 inhibitory activity. However, it is not the case in this series of
compounds. The unsubstituted pyrazole 14a was slightly more
potent than the respective carboxamide-substituted 19a
(IC50 ¼ 0.048
substituted 14c, and the unsubstituted 18a (IC50 ¼ 0.013
2.0-fold more potent than the respective carbonitrile-substituted
18c (IC50 ¼ 0.026 M). It was previously demonstrated by Tojo
mM) and 4.0-fold more potent than the carbonitrile-
mM) was
m
et al. [36] and us (unpublished work) that the pyrazoles possessing
a thioamido linkage between a pyrazole ring and a phenyl ring
were much more potent in ALK5 inhibition than those possessing
a respective carboxamido linkage. As expected, the pyrazoles 18a
and 18c possessing a thioamidomethylene linkage was 2.9-fold and
5.9-fold more potent than the corresponding 14a and 14c pos-
sessing an amidomethylene linkage. The pyrazole 18a showed the
most potent ALK5 inhibitory activity in this series of compounds. It
Scheme 4. Reagents and conditions: (a) 28% H2O2, 1 N NaOH, EtOH, 40 ꢀC, 4 h.
a phenyl ring also influenced the ALK5 inhibition. Thus, the pyr-
M) possessing an amidoethylene linkage
azole 17a (IC50 ¼ 0.082
m
was 2.2-fold less potent than the corresponding 14a possessing an
amidomethylene linkage. We have reported that introduction of
a carbonitrile or a carboxamide group at meta-position of the
phenyl or benzyl moiety in the 2-pyridinyl-substituted pyrazoles,
triazoles, imidazoles, and thiazoles significantly increased their
was slightly more potent than 6 (IC50 ¼ 0.017
more potent than 1 (IC50 ¼ 0.054 M).
To evaluate TGF- -induced downstream transcriptional activa-
tion to ALK5 signaling, cell-based luciferase activity of all target
mM) and 4.2-fold
m
b
Table 1
Inhibitory activity of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles 14aed, 15aed, 17a, 17b, 18aed, 19a, and 19b on ALK5.
Compound
R1
R2
X
IC50
(m
M)
Selectivity indexc
p3TP-luciferase activity (% control)d,e
ALK5a
p38ab
Mock
9 ꢁ 1
100 ꢁ 4
36 ꢁ 3
99 ꢁ 10
36 ꢁ 3
92 ꢁ 3
80 ꢁ 3
112 ꢁ 8
90 ꢁ 4
127 ꢁ 9
34 ꢁ 0
113 ꢁ 15
20 ꢁ 1
97 ꢁ 6
24 ꢁ 3
92 ꢁ 12
46 ꢁ 4
97 ꢁ 8
34 ꢁ 0
20 ꢁ 3
TGFe
14a
14b
14c
14d
15a
15b
15c
15d
17a
17b
18a
18b
18c
18d
19a
19b
b
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
H
H
CN
CN
H
O
O
O
O
0.038
>1.0
0.153
>1.0
0.143
>1.0
0.737
>1.0
0.082
>1.0
0.013
>1.0
0.026
>1.0
0.048
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
0.594
0.480
>26
>7
>7
H
CN
CN
H
CN
H
>1
>12
>77
>38
>21
S
S
S
S
O
O
H
CN
CN
CONH2
CONH2
CH3
1(SBe505124)
6 (IN-1130)
0.054
0.017
11
28
Elemental Analysis Data14a: C25H24N7O Found: C, 68.22; H, 5.60; N, 22.44. Requires: C, 68.48; H, 5.52; N, 22.36. 14b: C27H25N6O Found: C, 72.01; H, 5.68; N, 18.61. Requires: C,
72.14; H, 5.61; N, 18.70. 14c: C26H20N7O Found: C, 70.13; H, 4.44; N, 21.74. Requires: C, 69.94; H, 4.52; N, 21.96. 14d: C28H24N7O Found: C, 70.67; H, 5.22; N, 20.73. Requires: C,
70.87; H, 5.10; N, 20.66. 15a: C25H24N7O Found: C, 68.55; H, 5.36; N, 22.22. Requires: C, 68.48; H, 5.52; N, 22.36. 15b: C27H25N6O Found: C, 72.01; H, 5.77; N, 18.59. Requires: C,
72.14; H, 5.61; N, 18.70. 15c: C26H20N7O Found: C, 69.88; H, 4.58; N, 21.79. Requires: C, 69.94; H, 4.52; N, 21.96. 15d: C28H24N7O Found: C, 71.03; H, 4.88; N, 20.56.Requires: C,
70.87; H, 5.10; N, 20.66. 17a: C26H23N6O Found: C, 71.62; H, 5.52; N, 19.18.Requires: C, 71.71; H, 5.32; N, 19.30. 17b: C29H26N7O Found: C, 71.44; H, 5.28; N, 19.95. Requires: C,
71.29; H, 5.36; N, 20.07. 18a: C25H21N6S Found: C, 68.42; H,4.99; N,19.01. Requires: C, 68.63; H,4.84; N,19.21. 18b: C27H25N6S Found: C, 69.43; H, 5.63; N, 17.99. Requires: C,
69.65; H, 5.41; N, 18.05. 18c: C26H20N7S Found: C, 67.82; H, 4.12; N, 21.02. Requires: C, 67.51; H, 4.36; N, 21.20. 18d: C28H24N7S Found: C, 68.34; H, 5.14; N, 19.92. Requires: C,
68.55; H, 4.93; N, 19.99. 19a: C26H22N7OS Found: C, 64.77; H, 4.74; N, 20.63. Requires: C, 64.98; H, 4.61; N, 20.40. 19b: C28H26N7OS Found: C, 65.88; H, 5.31; N, 19.11. Requires:
C, 66.12; H, 5.15; N, 19.28.
a
ALK5 was expressed in Sf9 insect cells as human recombinant GST-fusion protein by means of the vaculovirus expression system. A proprietary radioisotopic protein
kinase assay (33PanQinaseÒ Activity Assay) was performed at ProQinase GmbH (Freiburg, Germany) using casein as a substrate.
b
p38a MAP kinase was expressed as untagged human recombinant protein in E. coli. The enzyme was purified by Ni-NTH-agarose (Qiagen). A proprietary radioisotopic
protein kinase assay (33PanQinaseÒ Activity Assay) was performed at ProQinase GmbH (Freiburg, Germany) using ATF2 as a substrate.
c
IC50 of p38
a
/IC50 of ALK5.
Luciferase activity was determined at a concentration of 0.1
Activity is given as the mean ꢁ SD of three independent experiments run in triplicate relative to control incubations with DMSO vehicle.
d
e
m
M of inhibitor.

C.H. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 3917e3925
3921
inhibitory activity up to the maximum concentration of 1
mM
tested. Compound 18a was found to be the most selective in this
series, showing the selectivity index of >77 that is higher than
those of 6 (28) and 1 (11).
2.4. Binding mode of 18a in the ALK5 active site
To examine whether compound 18a will bind into the same
binding pocket that is occupied by 9, we compared docking poses
between 18a and 9. As shown in Fig. 3, both 18a and 9 fit well into
the ALK5 active site, being superimposed over the X-ray pose of 1,5-
naphthyrine inhibitor [45] (yellow). The quinoxalinyl ring of 18a
and the corresponding 6-quinolinyl moiety of 9 occupy the pocket
for adenine ring of ATP, with forming a hydrogen bond backbone
NH of His283. The thioamide NH group of the linker in 18a forms an
H-bond with Lys337, one of the conserved lysines in the catalytic
domain of ALK5. Instead, the thioamide NH group of 9 is hydrogen-
bonded to Asp351, and the carboxamide-substituent forms addi-
tional H-bonds with Lys337 and Ser287. Although the calculated
binding energy scores (FlexX score) of both compounds show that
18a (ꢂ31.4 kcal/mol) forms less stable complex with ALK5 than 9
(ꢂ41.4 kcal/mol), 18a is more desirable than 9 in terms of chemical
stability.
Fig. 2. Effect of 18a on the activity of TGF-b-induced ALK5. HaCaT cells were perma-
nently transfected with p3TP-luciferase reporter construct. Luciferase activity was
determined in the presence of different concentrations of each compound and is given
as the mean ꢁ SD of three independent experiments run in triplicate relative to
control.
molecules was measured using HaCaT cells permanently trans-
fected with p3TP-luciferase reporter construct at a concentration of
0.1 mM (Table 1). The p3TP-luciferase reporter construct contains
three AP-1 binding elements and the plasminogen-activator
inhibitor-1 (PAI-1) promoter [43]. Similar to kinase assay, the
compounds possessing a 2,3-dimethylquinoxalin-6-yl moiety, 14b,
14d, 15b, 15d, 17b, 18b, 18d, and 19b showed no significant ALK5
inhibitory activity, and inhibition of the luciferase activity by most
compounds consisted with that of kinase assay. The pyrazole
derivative 18a was the most inhibitory, showing 80% inhibition that
is comparable to that of 6 (80%) and higher than that of 1 (66%).
The most potent compound 18a was chosen, and its ALK5
inhibitory activity was compared with those of 1 and 6 at five
3. Conclusion
In this report, a series of 1-substituted-3(5)-(6-methylpyridin-
2-yl)-4-(quinoxalin-6-yl)pyrazoles has been synthesized and eval-
uated for their ALK5 inhibitory activity in an enzyme assay and in
a cell-based luciferase reporter assay. The structureeactivity rela-
tionships in this series of compounds have been established and
discussed. The pyrazole derivative 18a displayed the most signifi-
cant ALK5 inhibitory activity in the series of compounds that is
much higher than that of 1 and is equipotent to 6. This series of
different concentrations (0.003, 0.01, 0.03, 0.05, and 0.1 mM) using
HaCaT cells permanently transfected with p3TP-luciferase reporter
construct. As shown in Fig. 2, 18a inhibited ALK5 in a dose-
dependent manner and was equipotent to 6 and more potent than 1.
compounds were selective for ALK5 inhibition than p38
a
MAP
MAP
kinase inhibition. The selectivity index of 18a against p38
a
kinase is >77 that is much higher than those of 1 (11) and 6 (28).
2.3. p38
a
MAP kinase assay
4. Experimental section
The kinase domain of p38
a
MAP kinase is known to be one of
4.1. Chemistry
the most homologous to that of ALK5 [44], therefore, it was chosen
to examine the selectivity profile of this series of compounds. All
the target molecules we prepared were devoid of p38a MAP kinase
1H and 13C NMR spectra were recorded on a varian Unity 400
spectrophotometer. The chemical shifts are reported in parts per
Fig. 3. Binding pose of 18a (A) and 9 (B) in the active site of ALK5, superimposed over the X-ray pose of 1,5-naphthyrine inhibitor (yellow carbon). Key amino acid residues within
the binding site are represented in line form, and the bound ligand is rendered in capped stick (grey carbon). Yellow dotted lines are hydrogen bonding interactions (<2.5 Å).

3922
C.H. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 3917e3925
million (ppm) relative to internal tetramethylsilane as an internal
standard. Infrared spectra were recorded on an FT-infrared
spectrometer (Bio-Rad). High resolution mass spectra electro
spray ionization (HRMS-ESI) was obtained on an Agilent tech-
nologies 6220 TOF LC/MS spectrometer. All melting points were
taken in Pyrex capillaries using an electrothermal digital melting
point apparatus (Buchi) and are not correct. Analytical thin-layer
chromatography (TLC) was performed on Merck silica gel 60F-
254 glass plates. Medium pressure liquid chromatography
(MPLC) was performed using Merck silica gel 60 (230e400 mesh)
with an YFLC-540 ceramic pump (Yamagen). All chemicals and
solvents were purchased from Aldrich or TCI Laboratory
Chemicals.
4.1.2.1. 6-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoxaline
(12a). Yield 79%; mp 105.4 ꢀC; IR (KBr) 3159 (NH) cmꢂ1 1H NMR
(CDCl3)
(t, 1H, J ¼ 7.6 Hz), 7.81 (s, 1H), 7.84 (dd, 1H, J ¼ 8.4, 2.0 Hz), 8.12 (d, 1H,
J ¼ 8.4 Hz), 8.19 (d,1H, J ¼ 1.6 Hz), 8.85 (d, 1H, J ¼ 2.0 Hz), 8.86 (d, 1H,
J ¼ 2.0 Hz).
;
d
2.59 (s, 3H), 7.10 (d,1H, J ¼ 7.6 Hz), 7.20 (d,1H, J ¼ 7.6 Hz), 7.45
4.1.2.2. 2,3-Dimethyl-6-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]ꢂ-1
quinoxaline (12b). Yield 75%; mp 119.2 ꢀC; IR (KBr) 3164 (NH) cm
;
1H NMR (CDCl3)
d 2.65 (s, 3H), 2.75 (s, 3H), 2.76 (s, 3H), 7.11 (d, 1H,
J ¼ 7.6 Hz), 7.20 (d,1H, J ¼ 7.6 Hz), 7.46 (t,1H, J ¼ 7.6 Hz), 7.70 (dd, 1H,
J ¼ 8.8, 2.0 Hz), 7.78 (s, 1H), 7.98 (d, 1H, J ¼ 8.8 Hz), 8.05 (d, 1H,
J ¼ 2.0 Hz)); HRMS-ESI m/z [M þ H]þ calcd. for C19H18N5: 316.1557,
found 316.1557.
4.1.1. General procedure for the preparation of 1-(6-methylpyridin-2-
yl)-2-(quinoxalin-6-yl)ethanone (11a) and 2-(2,3-dimethylquinoxalin
-6-yl)-1-(6-methylpyridin-2-yl)ethanone (11b)
4.1.3. General procedure for the preparation of 2-[3-(6-methylpyrid-
in-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-1-yl]-N-phenylacetamide
(14aed) and 2-[5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-
pyrazol-1-yl]-N-phenylacetamide (15aed)
To a stirred solution of 10a or 10b (18.97 mmol) in a mixture of
THF (40 mL) and i-PrOH (10 mL), diphenyl (6-methylpyridin-2-
yl)(phenylamino)methylphosphonate (18.97 mmol) and Cs2CO3
(24.65 mmol) were added. The mixture was stirred at room
temperature for 16 h, and to it, 3 N HCl (25 mL) was added dropwise
over a period of 5 min. The reaction mixture was diluted with MTBE
(30 mL). The aqueous layer was separated, and the organic layer
was extracted with 1 N HCl (2 ꢃ 50 mL). The combined aqueous
layer was neutralized with 50% aqueous KOH solution (pH 7〜8)
and extracted with EtOAc (3 ꢃ 100 mL). The EtOAc solution was
dried over anhydrous Na2SO4, filtered, and evaporated to dryness
under reduced pressure. The residue was purified by MPLC on silica
gel using EtOAc/hexane as eluent to give the titled compound 11a
or 11b as a light yellow solid.
To a solution of pyrazole 12a or 12b (0.348 mmol) in anhydrous
DMF (5 mL),
a catalytic amount of sodium iodide, NaH
(0.42 mmol), and 2-chloro-N-phenylacetamide 13a or 13b
(0.42 mmol) were added. The mixture was stirred at room
temperature for 2 h and then evaporated to dryness under
reduced pressure. The residue was purified by MPLC on silica gel
using MeOH/CHCl3 as eluent to give the two positional isomers
14aed and 15aed as white solids.
4.1.3.1. 2-[3-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-
1-y]-N-phenylacetamide (14a). Yield 41%; mp 182.3 ꢀC; IR (KBr)
3288, 1687, 1603, 1552 cmꢂ1 1H NMR (CDCl3)
; d 2.51 (s, 3H), 5.14
(s, 2H), 7.09 (br t, 1H, J ¼ 7.6 Hz), 7.15 (d, 1H, J ¼ 7.6 Hz), 7.28 (t,
2H, J ¼ 7.6 Hz), 7.39 (d, 1H, J ¼ 7.6 Hz), 7.49 (d, 2H, J ¼ 7.6 Hz),
7.58 (t, 1H, J ¼ 7.6 Hz), 7.76 (dd, 1H, J ¼ 8.8, 2.0 Hz), 7.86 (s, 1H),
8.01 (d, 1H, J ¼ 8.8 Hz), 8.11 (d, 1H, J ¼ 2.0 Hz), 8.81(d, 1H,
J ¼ 2.0 Hz), 8.83 (d, 1H, J ¼ 2.0 Hz), 8.90 (br s, 1H); 13C NMR
4.1.1.1. 1-(6-Methylpyridin-2-yl)-2-(quinoxalin-6-yl)ethanone (11a). Yi-
eld 79%; mp 128.6 ꢀC; IR (KBr) 1696 (CO) cmꢂ1; 1H NMR (CDCl3)
d 2.68
(s, 3H), 4.81 (s, 2H), 7.35 (dd, 1H, J ¼ 8.0, 0.8 Hz), 7.72 (t, 1H, J ¼ 7.8 Hz),
7.80 (dd, 1H, J ¼ 8.8, 1.6 Hz), 7.88 (dd, 1H, J ¼ 8.0, 0.8 Hz), 8.06 (d, 1H,
J ¼ 1.6 Hz), 8.07(d, 1H, J ¼ 8.8 Hz), 8.81 (d, 1H, J ¼ 2.0 Hz), 8.82 (d, 1H,
J ¼ 2.0 Hz); HRMS-ESI m/z [M þ H]þ calcd. for C16H14N3O: 264.1131,
found 264.1135.
(CDCl3)
d 24.49, 56.12, 120.35 (2C), 120.63, 121.76, 123.02, 124.90,
128.01, 128.36, 129.09 (2C), 132.08, 132.56, 134.76, 136.98, 137.36,
142.36, 143.24, 144.82, 145.40, 150.36, 151.08, 158.82, 164.76;
HRMS-ESI m/z [M þ NH4]þ calcd. for C25H24N7O: 438.2037, found
438.2028.
4.1.1.2. 2-(2,3-Dimethylquinoxalin-6-yl)-1-(6-methylpyridin-2-yl)et-
hanone (11b). Yield 86%; mp 195.5 ꢀC; IR (KBr) 1698 (CO) cmꢂ1; 1H
NMR (CDCl3)
d
2.67 (s, 3H), 2.73 (s, 6H), 4.76 (s, 2H), 7.33 (br d, 1H,
4.1.3.2. 2-[5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyraz-
J ¼ 7.6 Hz), 7.68 (dd, 1H, J ¼ 8.6, 1.8 Hz), 7.70 (t, 1H, J ¼ 7.6 Hz), 7.86
(br d, 1H, J ¼ 7.6 Hz), 7.94e7.96 (m, 2H)); HRMS-ESI m/z [M þ H]þ
calcd. for C18H18N3O: 292.1444, found 292.1454.
ol-1-yl]-N-phenylacetamide (15a). Yield 10%; mp 272.3 ꢀC; IR
(KBr) 3278, 1685, 1601 1553 cmꢂ1 1H NMR (CDCl3)
; d 2.71 (s, 3H),
5.06 (s, 2H), 7.10e7.13 (m, 2H), 7.28 (br d, 1H, J ¼ 7.6 Hz), 7.33 (br
t, 2H, J ¼ 8.0 Hz), 7.57e7.62 (m, 3H), 7.64 (dd, 1H, J ¼ 8.8, 2.0 Hz),
7.95 (s, 1H), 8.00 (d, 1H, J ¼ 2.0 Hz), 8.03 (d, 1H, J ¼ 8.8 Hz), 8.81
(d, 1H, J ¼ 2.0 Hz), 8.82 (d, 1H, J ¼ 2.0 Hz), 9.82 (br s, 1H); 13C
4.1.2. Generalprocedureforthepreparation of6-[3-(6-methylpyridin-
2-yl)-1H-pyrazol-4-yl]quinoxaline (12a) and 2,3-dimethyl-6-[3-(6-
methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoxaline (12b)
NMR (CDCl3)
d 24.53, 55.51, 120.01 (2C), 121.53, 123.35, 123.97,
To a stirred solution of 11a or 11b (2.66 mmol) in anhydrous DMF
(7 mL), AcOH (9.58 mmol) and N,N-dimethylformamide dimethyl
acetal (7.98 mmol) were added. The mixture was heated at 80 ꢀC for
2 h. After cooled to room temperature, the reaction mixture was
evaporated to dryness under reduced pressure. The residue was
dissolved in EtOH (10 mL), and to it, hydrazine monohydrate
(54.88 mmol) was added. The mixture was heated at reflux
temperature for 4 h, then cooled to room temperature, and evapo-
rated to dryness under reduced pressure. The residue was diluted
with CHCl3 (100 mL) and washed with water (30 mL) and brine
(30 mL). The CHCl3 solution was dried over anhydrous Na2SO4,
filtered, and evaporated to dryness under reduced pressure. The
residue was purified by MPLC on silica gel using MeOH/CH2Cl2 as
eluent to give the titled compound 12a or 12b as a light yellow foam.
124.47, 127.97, 129.16 (2C), 129.94, 131.03, 134.76, 137.82, 138.13,
139.45, 140.14, 142.30, 143.39, 144.93, 145.56, 147.72, 159.47,
165.59; HRMS-ESI m/z [M
438.2037, found 438.2019.
þ
NH4]þ calcd. for C25H24N7O:
4.1.3.3. 2-[4-(2,3-Dimethylquinoxalin-6-yl)-3-(6-methylpyridin-2-
yl)-1H-pyrazol-1-yl]-N-phenylacetamide (14b). Yield 70%; mp
227.9 ꢀC; IR (KBr) 3277, 1689, 1603, 1553 cmꢂ1 1H NMR (CDCl3)
;
d
2.53 (s, 3H), 2.72 (s, 3H), 2.74 (s, 3H), 5.08 (s, 2H), 7.12 (br t, 1H,
J ¼ 7.6 Hz), 7.15 (d, 1H, J ¼ 7.6 Hz), 7.28e7.32 (m, 2H), 7.34 (d,
1H, J ¼ 8.0 Hz), 7.47e7.50 (m, 2H), 7.55 (t, 1H, J ¼ 7.8 Hz), 7.63 (dd,
1H, J ¼ 8.8, 2.0 Hz), 7.80 (s, 1H), 7.88 (d, 1H, J ¼ 8.8 Hz), 7.98 (d, 1H,
J ¼ 2.0 Hz), 8.62 (br s, 1H); HRMS-ESI m/z [M þ H]þ calcd. for
C27H25N6O: 449.2084, found 449.2093.

C.H. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 3917e3925
3923
4.1.3.4. 2-[4-(2,3-Dimethylquinoxalin-6-yl)-5-(6-methylpyridin-2-
yl)-1H-pyrazol-1-yl]-N-phenylacetamide (15b). Yield 10%; mp
224.3 ꢀC; IR (KBr) 3275, 1684, 1600, 1554 cmꢂ1 1H NMR (CDCl3)
2.71 (s, 3H), 2.79 (s, 3H), 2.80 (s, 3H), 5.05 (s, 2H), 7.08e7.12 (m,
4.1.5. Generalprocedureforthe preparationof3-[3-(6-methylpyridin-
2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-1-yl]-N-phenylpropanamide
(17a) and N-(3-cyanophenyl)-3-[4-(2,3-dimethylquinoxalin-6-yl)-3-
(6-methylpyridin-2-yl)-1H-pyrazol-1-yl]propanamide (17b)
;
d
2H), 7.28 (d, 1H, J ¼ 8.0 Hz), 7.32 (t, 1H, J ¼ 8.0 Hz), 7.56 (dd, 1H,
J ¼ 8.8, 1.6 Hz), 7.58e7.62 (m, 3H), 7.93 (s, 1H), 8.00e8.02 (m, 2H),
9.86 (br s, 1H); HRMS-ESI m/z [M þ Na]þ calcd. for C27H24N6NaO:
471.1904, found 471.1920.
To a stirred mixture of 12a or 12b (0.174 mmol) and Cs2CO3
(0.23 mmol) in anhydrous DMF (3 mL), 3-bromo-N-phenyl-
propanamide (16a) or 3-bromo-N-(3-cyanophenyl)propanamide
(16b) (0.21 mmol) was added. The mixture was heated at 120 ꢀC for
2 h and then cooled to room temperature. The reaction mixture was
diluted with water (10 mL) and extracted with CHCl3 (2 ꢃ 40 mL).
The CHCl3 solution was dried over anhydrous Na2SO4, filtered, and
evaporated to dryness under reduced pressure. The residue was
purified by MPLC on silica gel using a mixture of MeOH/CHCl3 as
eluent to give the titled compound 17a or 17b as a white solid.
4.1.3.5. N-(3-Cyanophenyl)-2-[3-(6-methylpyridin-2-yl)-4-(quinox-
alin-6-yl)-1H-pyrazol-1-yl] acetamide (14c). Yield ꢂ152%; mp
280.6 ꢀC; IR (KBr) 3274, 2233, 1700, 1617, 1557 cm
;
1H NMR
(CDCl3)
d
2.53 (s, 3H), 5.17 (s, 2H), 7.18 (br d, 1H, J ¼ 8.0 Hz), 7.33 (br
d, 1H, J ¼ 7.6 Hz), 7.36e7.38 (m, 2H), 7.58 (t, 1H, J ¼ 7.8 Hz),
7.62e7.66 (m, 1H), 7.76 (dd, 1H, J ¼ 8.8, 2.0 Hz), 7.85 (s, 1H), 7.90 (br
s, 1H), 8.05 (d, 1H, J ¼ 8.8 Hz), 8.12 (d, 1H, J ¼ 2.0 Hz), 8.84 (d, 1H,
J ¼ 2.0 Hz), 8.85 (d, 1H, J ¼ 2.0 Hz), 9.55 (s, 1H); HRMS-ESI m/z
[M þ K]þ calcd. for C26H19KN7O: 484.1283, found 484.1303.
4.1.5.1. 3-[3-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-
1-yl]-N-phenylpropanamide (17a). Yield 66%; mp 221.2 ꢀC; IR (KBr)
3315, 1681, 1548, cmꢂ1 1H NMR (CDCl3)
; d 2.62 (s, 3H), 3.11 (t, 2H,
J ¼ 6.0 Hz), 4.66 (t, 2H, J ¼ 6.0 Hz), 7.05 (br t, 1H, J ¼ 7.6 Hz), 7.15 (d,
1H, J ¼ 7.6 Hz), 7.23e7.27 (m, 3H), 7.51 (d, 2H, J ¼ 8.0 Hz), 7.55 (t, 1H,
J ¼ 7.8 Hz), 7.64 (dd, 1H, J ¼ 8.8, 2.0 Hz), 7.78 (s, 1H), 7.97 (d, 1H,
J ¼ 8.8 Hz), 8.02 (d, 1H, J ¼ 2.0 Hz), 8.47 (br s, 1H), 8.79 (d, 1H,
4.1.3.6. N-(3-Cyanophenyl)-2-[5-(6-methylpyridin-2-yl)-4-(quinox-
alin-6-yl)-1H-pyrazol-1-yl] acetamide (15c). Yield 6%; mp 129.3 ꢀC;
IR (KBr) 3277, 2232, 1699, 1617, 1558 cmꢂ1; 1H NMR (CDCl3)
d 2.69 (s,
3H), 5.04 (s, 2H), 7.12 (d, 1H, J ¼ 7.6 Hz), 7.30 (d, 1H, J ¼ 7.6 Hz), 7.39
(dt, 1H, J ¼ 7.6, 1.6 Hz), 7.43 (t, 1H, J ¼ 7.8 Hz), 7.61 (t, 1H, J ¼ 7.8 Hz),
7.63 (dd, 1H, J ¼ 8.8, 2.0 Hz), 7.84 (ddd, 1H, J ¼ 8.0, 2.4,1.2 Hz), 7.95 (s,
1H), 8.00e8.05 (m, 3H), 8.82 (s, 2H), 10.47 (br s, 1H); HRMS-ESI m/z
[M þ H]þ calcd. for C26H20N7O: 446.1724, found 446.1720.
J ¼ 2.0 Hz), 8.80 (d, 1H, J ¼ 2.0 Hz); 13C NMR (CDCl3/CD3OD)
d 23.84,
37.41, 48.64, 119.98, 120.07 (2C), 121.13, 122.94, 124.17, 127.11,
128.59, 128.65 (2C), 131.56, 131.72, 135.11, 137.11, 137.94, 141.56,
142.73, 144.24, 145.02, 148.59, 151.06, 158.54, 169.25; HRMS-ESI m/z
[M þ H]þ calcd. for C26H23N6O: 435.1928, found 435.1921.
4.1.3.7. N-(3-Cyanophenyl)-2-[4-(2,3-dimethylquinoxalin-6-yl)-3-
(6-methylpyridin-2-yl)-1H-pyrazol-1-yl]acetamide (14d). Yield
60%; mp 196.3 ꢀC; IR (KBr) 3261, 2231, 1702, 1592, 1557 cmꢂ1; 1H
4.1.5.2. N-(3-Cyanophenyl)-3-[4-(2,3-dimethylquinoxalin-6-yl)-3-
(6-methylpyridin-2-yl)-1H-pyrazol-1-y]propanamide (17b).ꢂY1ield
41%; mp 207.1 ꢀC; IR (KBr) 3334, 2231, 1691, 1589, 1553 cm
;
1H
NMR (CDCl3)
d
2.54 (s, 3H), 2.73 (s, 3H), 2.74 (s, 3H), 5.16 (s, 2H),
NMR (CDCl3) d 2.62 (s, 3H), 2.71 (s, 3H), 2.72 (s, 3H), 3.11 (t, 2H,
7.15 (br d, 1H, J ¼ 7.6 Hz), 7.25 (br d, 1H, J ¼ 7.6 Hz), 7.36e7.39 (m,
2H), 7.53 (t, 1H, J ¼ 7.8 Hz), 7.60 (dd, 1H, J ¼ 8.4, 2.0 Hz), 7.62e7.65
(m, 1H), 7.80 (s, 1H), 7.89e7.92 (m, 2H), 7.98 (d, 1H, J ¼ 2.0 Hz),
9.63 (d, 1H, J ¼ 2.0 Hz); HRMS-ESI m/z [M þ H]þ calcd. for
C28H24N7O: 474.2037, found 474.2046.
J ¼ 5.8 Hz), 4.62 (t, 2H, J ¼ 5.8 Hz), 7.09 (d, 1H, J ¼ 7.6 Hz), 7.15 (d,
1H, J ¼ 7.6 Hz), 7.28e7.33 (m, 2H), 7.45 (t, 1H, J ¼ 7.6 Hz), 7.46 (dd,
1H, overlapped, J ¼ 8.8, 2.0 Hz), 7.67 (s, 1H), 7.77 (dt, 1H, J ¼ 7.6,
2.0 Hz), 7.85 (d, 1H, overlapped, J ¼ 8.8 Hz), 7.86 (d, 1H, over-
lapped, J ¼ 2.0 Hz), 7.89 (br s, 1H), 9.57 (s, 1H); HRMS-ESI m/z
[M þ H]þ calcd. for C29H26N7O: 488.2193, found 488.2208.
4.1.3.8. N-(3-Cyanophenyl)-2-[4-(2,3-dimethylquinoxalin-6-yl)-5-
(6-methylpyridin-2-yl)-1H-pyrazol-1-yl]acetamide
(15d).ꢂY1 ield
4.1.6. General procedure for the preparation of 2-[3-(6-
methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-1-yl]-N-
phenylethanethioamide 18aed
4%; mp 189.3 ꢀC; IR (KBr) 3281, 2232, 1699, 1591, 1558, cm
;
1H
NMR (CDCl3)
d 2.69 (s, 3H), 2.75 (s, 3H), 2.76 (s, 3H), 5.04 (s, 2H),
7.10 (br d, 1H, J ¼ 7.6 Hz), 7.28 (br d, 1H, J ¼ 7.6 Hz), 7.38 (dt, 1H,
J ¼ 8.0, 1.4 Hz), 7.43 (t, 1H, J ¼ 7.8 Hz), 7.52 (dd, 1H, J ¼ 8.8,
2.0 Hz), 7.59 (t, 1H, J ¼ 7.6 Hz), 7.83 (dt, 1H, J ¼ 8.0, 1.8 Hz), 7.91 (s,
1H), 7.93e7.95 (m, 2H), 8.03 (t, 1H, J ¼ 1.8 Hz), 10.54 (s, 1H);
HRMS-ESI m/z [M þ H]þ calcd. for C28H24N7O: 474.2037, found
474.2036.
A stirred mixture of 14aed (0.45 mmol), Lawesson’s reagent
(0.45 mmol), and anhydrous DME (10 mL) in a dry sealed tube was
heated at 85 ꢀC for 12 h. After cooled to room temperature, the
solvent was evaporated to dryness under reduced pressure, and the
residue was purified by MPLC on silica gel using MeOH/CHCl3 as
eluent to give the titled compound 18aed as a light yellow solid.
4.1.4. 3-Bromo-N-(3-cyanophenyl)propanamide (16b)
4.1.6.1. 2-[3-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-
3-Bromopropanoyl chloride (40.63 mmol) was added drop-
wise to a mixture of 3-aminobenzonitrile (33.86 mmol) and
anhydrous K2CO3 (40.63 mmol) in CH2Cl2 at room temperature.
The resulting mixture was heated at reflux temperature for 4 h,
then cooled to room temperature, and slowly poured into cold
water (120 mL). The aqueous solution was extracted with CH2Cl2
(2 ꢃ 100 mL), and the CH2Cl2 solution was dried over anhydrous
Na2SO4, filtered, and evaporated to dryness under reduced
pressure to give a white solid which was purified by crystalli-
zation from Et2O/hexane to give the titled compound 16b. Yield
1-yl]-N-phenylethanethioamide (18a). Yield 58%; mp 182 ꢀC; IR
(KBr) 3209, 1553, 1421, 1171 cmꢂ1 1H NMR (CDCl3)
; d 2.54 (s, 3H),
5.49 (s, 2H), 7.17 (d, 1H, J ¼ 7.6 Hz), 7.25 (tt, 1H, J ¼ 7.6, 1.6 Hz),
7.35e7.41 (m, 3H), 7.61 (t, 1H, J ¼ 7.6 Hz), 7.74e7.77 (m, 3H), 7.90 (s,
1H), 8.03 (d, 1H, J ¼ 8.8 Hz), 8.12 (d, 1H, J ¼ 2.0 Hz), 8.82 (d, 1H,
J ¼ 2.0 Hz), 8.83 (d, 1H, J ¼ 2.0 Hz), 10.61(s, 1H); 13C NMR (CDCl3)
d
24.50, 63.97, 120.55, 122.11, 123.24, 123.36 (2C), 127.28, 128.59,
129.13 (2C), 129.18, 132.12, 132.39, 134.53, 137.25, 138.41, 142.49,
143.28, 144.97, 145.50, 150.61, 150.76, 158.87, 192.94; HRMS-ESI m/z
[M þ H]þ calcd. for C25H21N6S: 437.1543, found 437.1553.
70%; mp 98.4 ꢀC; IR (KBr) 3222, 2233, 1676 cmꢂ1
(CDCl3)
;
1H NMR
d
2.99 (t, 2H, J ¼ 6.4 Hz), 3.70 (t, 2H, J ¼ 6.4 Hz), 7.39e7.45
4.1.6.2. 2-[4-(2,3-Dimethylquinoxalin-6-yl)-3-(6-methylpyridin-2-
(m, 2H), 7.75 (dt, 1H, J ¼ 7.6, 2.0 Hz), 7.94 (br s, 1H), 7.97 (br s,
1H); HRMS-ESI m/z [M þ H]þ calcd. for C10H10BrN2O: 252.9971,
found 252.9980.
yl)-1H-pyrazol-1-yl]-N-phenylethanethioamide (18b)ꢂ. 1Yield 24%;
mp 205.5 ꢀC; IR (KBr) 3266, 1554, 1494, 1160 cm
;
1H NMR
(CDCl3)
d 2.53 (s, 3H), 2.72 (s, 3H), 2.74 (s, 3H), 5.47 (s, 1H), 7.14

3924
C.H. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 3917e3925
(dd, 1H, J ¼ 7.6, 0.4 Hz), 7.23e7.27 (m, 1H), 7.32 (br d, 1H,
J ¼ 7.6 Hz), 7.35e7.40 (m, 2H), 7.54 (t, 1H, J ¼ 7.6 Hz), 7.61 (dd, 1H,
J ¼ 8.8, 2.0 Hz), 7.72e7.75 (m, 2H), 7.83 (d, 1H), 7.88 (d, 1H,
J ¼ 8.8 Hz), 7.98 (d, 1H, J ¼ 2.0 Hz), 10.54 (s, 1H); HRMS-ESI m/z
[M þ H]þ calcd. for C27H25N6S: 465.1856, found 465.1862.
reporter activity in HaCaT stable cells transfected with p3TP-Luc.
HaCaT cells were seeded at concentrations of 3 ꢃ 104 in 96-well
plates. The next day, when they reach approximately 90% con-
fluency, various concentrations of ALK5 inhibitors and 2 ng/mL of
TGF-b were added to the cells. After 24 h, cell lysates were har-
vested using Luciferase assay kit (Promega) according to the
manufacturer’s instruction, and luminescence was measured by
a luminometer Micro Lumat Plus (Berthold).
4.1.6.3. N-(3-Cyanophenyl)-2-[3-(6-methylpyridin-2-yl)-4-(quinox-
alin-6-yl)-1H-pyrazol-1-yl]ethanethioamide (18c). Yield 61%; mp
213.9 ꢀC; IR (KBr) 3271, 2232, 1431, 1186 cmꢂ1 1H NMR (CDCl3)
;
d
2.54 (s, 3H), 5.55 (s, 2H), 7.18 (d, 1H, J ¼ 7.6 Hz), 7.28 (d, 1H,
4.3. Docking experiments
J ¼ 8.0 Hz), 7.41 (t, 1H, J ¼ 8.0 Hz), 7.49 (dt, 1H, J ¼ 7.6, 1.2 Hz), 7.57
(t, 1H, J ¼ 7.8 Hz), 7.75 (dd, 1H, J ¼ 8.8, 2.0 Hz), 7.91 (s, 1H), 7.96
(ddd, 1H, J ¼ 8.0, 2.0, 1.2 Hz), 8.06 (d, 1H, J ¼ 8.8 Hz), 8.11 (d, 1H,
J ¼ 2.0 Hz), 8.25 (t, 1H, J ¼ 2.0 Hz), 8.84 (d, 1H, J ¼ 2.0 Hz), 8.85 (d,
1H, J ¼ 2.0 Hz), 11.50 (s, 1H); HRMS-ESI m/z [M þ H]þ calcd. for
C26H20N7S: 462.1495, found 462.1508.
Docking experiments have been performed with the Sybyl 8.1
software package (Tripos, Inc., St. Louis, MO, USA) based on CentOS
Linux 5.4.
4.3.1. Preparation of ligand database
The structures of ligands were prepared in MOL2 format using
the sketcher module, and Gasteiger-Huckel charges were assigned
to the ligand atoms. The structures of molecules were optimized by
energy minimization, and molecular dynamics using simulated
annealing method. The conformer library for each ligand was
prepared by random selection of 200 conformers from the molec-
ular dynamics output.
4.1.6.4. N-(3-Cyanophenyl)-2-[4-(2,3-dimethylquinoxalin-6-yl)-3-(6-
methylpyridin-2-yl)-1H-pyrazol-1-yl]ethanethioamide (18d). Yield
68%;mp219.4 ꢀC;IR(KBr) 3263, 2232,1588,1553,1163cmꢂ1; 1H NMR
(CDCl3) d 2.54 (s, 3H), 2.73 (s, 3H), 2.74 (s, 3H), 5.48 (s, 2H), 7.15 (d,1H,
J ¼ 7.6 Hz), 7.24 (d, 1H, J ¼ 7.6 Hz), 7.44 (t, 1H, J ¼ 8.0 Hz), 7.51 (dt, 1H,
overlapped, J ¼ 7.6,1.6 Hz), 7.52 (t,1H, overlapped, J ¼ 7.6Hz), 7.60 (dd,
1H, J ¼ 8.4, 2.0 Hz), 7.83 (s, 1H), 7.90 (d, 1H, J ¼ 8.4 Hz), 7.97 (d, 1H,
J ¼ 1.6 Hz), 8.00 (ddd, 1H, J ¼ 8.0, 2.4, 1.2 Hz), 8.25 (t, 1H, J ¼ 1.6 Hz),
11.27 (s, 1H); HRMS-ESI m/z [M þ H]þ calcd. for C28H24N7S: 490.1808,
found 490.1817.
4.3.2. Flexible docking
The X-ray coordinate of ALK5 in complex with 1,5-naphthyrine
inhibitor (PDB id: 1VJY) [45] was retrieved from the PDB, and all
crystallographic water molecules were removed. The active site
was defined as all the amino acid residues enclosed within 6.5 Å
radius sphere centered by the bound ligand. The docking and
subsequent scoring were performed using the default parameters
of the FlexX programs implanted in the Sybyl 8.1. For the docking of
ligand database, the main settings are 1000 solutions per iteration
during the incremental construction algorithm. Final scores for all
FlexX solutions were calculated by a consensus scoring function
(CScore), and used for database ranking. One of the poses with the
highest FlexX score and high consensus score (CScore ¼ 5 or 4) was
selected, and docked with ALK5.
4.1.7. General procedure for the preparation of 3-{2-[3-(6-methylpy-
ridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-1-yl]acetamido}benzam-
ide(19a)and 3-{2-[4-(2,3-dimethylquinoxalin-6-yl)-3-(6-methylpyr-
idin-2-yl)-1H-pyrazol-1-yl]acetamido}benzamide (19b)
A stirred solution of 18c or 18d (0.11 mmol), 1 N NaOH
(0.31 mmol), and 28% H2O2 (0.38 mmol) in absolute EtOH (3 mL)
was heated at 40 ꢀC for 4 h. The reaction mixture was cooled to 0 ꢀC
and neutralized with 1 N HCl to pH ꢂ8. The mixture was extracted
with 10% MeOH in CHCl3 (2 ꢃ 25 mL), and the combined organic
layer was dried over anhydrous Na2SO4, filtered, and evaporated to
dryness under reduced pressure. The residue was purified by MPLC
on silica gel using MeOH/CHCl3 as eluent to afford the titled
compound 19a or 19b as a white solid.
Acknowledgment
This work was supported by RP-Grant 2009 of Ewha Womans
University and a grant from Ministry of Education, Science and
Technology, Korea (20100029596).
4.1.7.1. 3-{2-[3-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyr-
azol-1-yl]acetamido}benzamide (19a). Yield 30%; mp 221.9 ꢀC; IR
(KBr) 3349, 3059, 1648, 1464, cmꢂ1; 1H NMR (CDCl3)
d 2.38 (s, 3H),
4.98 (s, 2H), 7.06 (d,1H, J ¼ 7.6 Hz), 7.18 (d,1H, J ¼ 7.6 Hz), 7.27 (t,1H,
J ¼ 7.6 Hz), 7.45e7.49 (m, 2H), 7.62 (dd, 1H, overlapped, J ¼ 8.8,
2.0 Hz), 7.63 (ddd, 1H, overlapped, J ¼ 8.0, 2.0, 0.8 Hz), 7.84 (t, 1H,
J ¼ 1.8 Hz), 7.86 (d, 1H, J ¼ 8.8 Hz), 7.90 (s, 1H), 7.92 (d, 1H,
J ¼ 2.0 Hz), 8.66 (d,1H, J ¼ 2.0 Hz), 8.67 (d,1H, J ¼ 2.0 Hz); HRMS-ESI
m/z [M þ H]þ calcd. for C26H22N7OS: 464.1829, found 464.1841.
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