4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2-yl)imidazole and -pyrazole derivatives as potent and selective inhibitors of transforming growth factor-β type i receptor kinase

Article abstract of DOI:10.1016/j.bmc.2014.03.022

A series of 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2- yl)imidazoles and -pyrazoles 14a-c, 15a-c, 16a, 16b, 19a-d, 21a, and 21b has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. Among them, the pyrazole derivative 21b inhibited ALK5 phosphorylation with an IC₅₀ value of 0.018 μM and showed 95% inhibition at 0.03 μM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct. The 21b showed a high selectivity index of 284 against p38α MAP kinase. The binding pose of 21b generated by docking analysis reveals that it fits well into the ATP binding cavity of ALK5 by forming several hydrogen bond interactions.

Full text of DOI:10.1016/j.bmc.2014.03.022

Bioorganic & Medicinal Chemistry 22 (2014) 2724–2732
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2-yl)
imidazole and -pyrazole derivatives as potent and selective
inhibitors of transforming growth factor-b type I receptor kinase
Cheng Hua Jin a, Maddeboina Krishnaiah a, Domalapally Sreenu a, Vura Bala Subrahmanyam a,
Hyun-Ju Park b, So-Jung Park b, Yhun Yhong Sheen a, Dae-Kee Kim a,
⇑
a Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Republic of Korea
b School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of 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 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2-yl)imidazoles and -pyrazoles
14a–c, 15a–c, 16a, 16b, 19a–d, 21a, and 21b has been synthesized and evaluated for their ALK5 inhibitory
activity in an enzyme assay and in a cell-based luciferase reporter assay. Among them, the pyrazole deriv-
Received 30 December 2013
Revised 6 February 2014
Accepted 11 March 2014
Available online 21 March 2014
ative 21b inhibited ALK5 phosphorylation with an IC50 value of 0.018
0.03 M in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter
construct. The 21b showed a high selectivity index of 284 against p38 MAP kinase. The binding pose of
lM and showed 95% inhibition at
l
a
Keywords:
TGF-b
ALK5 inhibitor
Fibrosis
21b generated by docking analysis reveals that it fits well into the ATP binding cavity of ALK5 by forming
several hydrogen bond interactions.
Ó 2014 Elsevier Ltd. All rights reserved.
Cancer
Kinase assay
Cell-based luciferase reporter assay
Docking
1. Introduction
several small molecule ATP-competitive ALK5 inhibitors such as 1
(SB-505154),10 2 (GW6604),11 3 (SD-208),12 and 4 (LY-2157299)13
The transforming growth factor-b (TGF-b) is the most potent
and ubiquitous immunosuppressive cytokine. TGF-b transduces
signals through two distinct serine/threonine kinase receptors,
the type I and type II receptors.1 Following the binding of TGF-b
to the constitutively active type II receptor, the type I receptor, also
called as activin receptor-like kinase 5 (ALK5), is phosphorylated,
which further phosphorylates Smad2/Smad3 proteins. Phosphory-
lated Smad2/Smad3 proteins form a heteromeric complex with
Smad4 to translocate into the nuclei, where they regulate the tar-
get gene transcription involved in cell differentiation, proliferation,
apoptosis, migration, and extracellular matrix production.2 TGF-b
plays a critical role in the initiation and progression of fibrosis in
various organ systems such as kidney,3 heart,4 lung,5 and liver.6
Deregulation of TGF-b signaling has been also implicated in various
human diseases including cancer,7 pancreatic diseases,8 and hema-
tological malignancies.9 ALK5 inhibitors specifically inhibit Smad
pathway by occupying the ATP binding site of ALK5 kinase domain,
which phosphorylates Smad2 and Smad3. It has been reported that
(Fig. 1) inhibited autophosphorylation of ALK5 and TGF-b-induced
transcription of matrix genes in reporter assays at sub-micromolar
concentrations. In animal models, 2 and 3 effectively suppressed
progressive fibrosis in liver and lung, respectively, and 3 and 4
strongly inhibited growth and invasiveness of cancer cells. The
clinical candidate 4 was proven to be safe and well tolerated in
cancer patients in a Phase I clinical trial14 and entered Phase II clin-
ical trials against hepatocellular carcinoma, glioma, glioblastoma,
and pancreatic cancer.
We have prepared a number of the 2-pyridyl-substituted
five-membered heterocycles as ALK5 inhibitors and found that
insertion of a small linker such as methylene, methyleneamino, ami-
nomethylene, thioamido, methyleneamido, or methylenethioamido
between a central five-membered heterocyclic ring and a phenyl
ring significantly increased ALK5 inhibitory activity and selectiv-
ity.15–27 Introduction of a substituent such as carbonitrile or
carboxamide group at meta- or para-position or fluro atom at
ortho-position on the phenyl ring also significantly enhanced ALK5
inhibitory activity and selectivity. These structural modifications
led to the discovery of two preclinical candidates, 5 (IN-1130) and
6 (IN-1233) (Fig. 1).
⇑
Corresponding author. Tel.: +82 2 3277 3025; fax: +82 2 3277 2467.
E-mail address: dkkim@ewha.ac.kr (D.-K. Kim).
http://dx.doi.org/10.1016/j.bmc.2014.03.022
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

C. H. Jin et al. / Bioorg. Med. Chem. 22 (2014) 2724–2732
2725
O
N
NH2
N
O
HN
N
N
N
O
N
F
N
N
N
N
NH
N
H
N
N
N
N
Cl
1
4
(LY-2157299)
(SB-505124)
2
(GW6604)
3 (SD-208)
N
N
N
O
O
NH2
NH2
N
N
N
H
N
H
N
N
5 (IN-1130)
6 (IN-1233)
Figure 1. ATP-competitive ALK5 inhibitors under development.
moiety was found to be difficult due to the presence of two adjacent
nitrogen atoms.38
N
O
HO
N
NH
N
2
2. Results and discussion
2.1. Chemistry
N
H
N
7
The 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-
2-yl)imidazole derivatives 14a–c and 15a–c were prepared as
shown in Scheme 1. Oxidation of 839 with 48% HBr in DMSO gave
the diketone 940 in 82% yield. The diketone 9 was condensed with
2-(1,3-dioxoisoindolin-2-yl)acetaldehyde (10)41 and NH4OAc in a
mixture of MTBE and MeOH to afford the imidazole 11 in 67% yield.
Treatment of 11 with hydrazine monohydrate in EtOH produced
(4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-
imidazol-2-yl)methanamine (12) in 64% yield. The amine 12 was
coupled with an appropriate acid chloride 13a, 13b, or 13c in the
presence of triethylamine in CH2Cl2 to produce the target com-
pounds 14a–c in 71–91% yields. Thionation of the amides 14a–c
with Lawesson’s reagent in DME afforded the corresponding carbo-
thioamides 15a–c in 68–92% yields. Conversion of the carbonitrile
functionality in 14c and 15c to the corresponding carboxamides
was achieved by treatment with 28% H2O2 and 1 N NaOH in abso-
lute EtOH to give 16a and 16b in 25% and 19% yields, respectively
(Scheme 2).
The 1-substituted 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-3(5)-(6-
methylpyridin-2-yl)pyrazoles were prepared as shown in Scheme 3.
Treatment of the monoketone 8 with N,N-dimethylformamide
dimethyl acetal (DMFꢀDMA) and followed by cyclization with
hydrazine monohydrate in absolute EtOH gave 6-(3-(6-methylpyri-
din-2-yl)-1H-pyrazol-4-yl)-[1,2,4]triazolo[1,5-a]pyridine (17)42 in
80% yield. The pyrazole 17 was alkylated with 2-chloro-N-phenyl-
acetamide (18a),43 2-chloro-N-(2-fluorophenyl)acetamide (18b),44
or 2-chloro-N-(3-cyanophenyl)acetamide (18c)45 in the presence
of NaH and NaI in anhydrous DMF to yield the target compounds
19a–c and their positional isomers 20a–c in 67–78% and 3–7%
yields, respectively. The positional isomers were separated by
MPLC, and their structures were confirmed by NOE experiments.
In NOE experiments, irradiation of the methylene protons of the
19c at d 5.21 gave an enhancement of the proton H-5 in the pyrazole
ring at d 8.28, while irradiation of the methylene protons of the 20c
at d 5.27 gave no enhancement of the proton H-5 in the pyrazole
Figure 2. Structure of a major metabolite of 5 in rat and mouse.
The 5 demonstrated its pronounced activity as a suppressor of
fibrogenic process of unilateral ureteral obstruction (UUO) in
mice28 and rats,29 ameliorated experimental autoimmune enceph-
alomyelitis, a mouse model for multiple sclerosis,30 reduced
tunical fibrosis and corrected penile curvature in rats,31 and en-
hanced CTL response in cancer mice.32 Another preclinical candi-
date 6 effectively prevented development and progression of
pulmonary arterial hypertension in a monocrotaline rat model
through inhibition of TGF-b signaling,33 prevented granulation tis-
sue formation in a rat urethral model and neointimal hyperplasia
in a rat common iliac artery model after bare metallic stent
placement,34,35 and suppressed Smad2/Smad3 phosphorylation,
plasminogen-activator inhibitor-1 (PAI-1) expression, as well as
collagens and
a-smooth muscle actin (a-SMA) in a mouse ozone-
induced airway fibrosis model.36
To develop a novel potent and selective ALK5 inhibitor, in this
report, we have synthesized a series of 4-([1,2,4]triazolo[1,5-a]
pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazole derivatives pos-
sessing a methyleneamido or methylenethioamido linkage, 14a–c,
15a–c, 16a, and 16b and compared their ALK5 inhibitory activity
with those of the 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-3-(6-meth-
ylpyridin-2-yl)pyrazole derivatives 19a–d, 21a, and 21b.
In pharmacokinetic studies of 5 in rodents, the major metabolite
of 5 was isolated in the systemic circulation and identified
tentatively as either 3-((4-(2-hydroxyquinoxalin-6-yl)-5-(6-methyl
pyridin-2-yl)-1H-imidazol-2-yl)methyl)benzamide or 3-((4-(3-hy
droxyquinoxalin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)
methyl)benzamide (7) (Fig. 2).37 To overcome metabolic oxidation
of the quinoxalin-6-yl moiety of 5 and of the quinolin-6-yl moiety
of 6, we adopted a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety in the
target molecules since metabolic oxidation of 2-position of this

2726
C. H. Jin et al. / Bioorg. Med. Chem. 22 (2014) 2724–2732
O
OHC
N
N
N
N
N
N
N
O
N
N
N
O
O
N
10
N
O
a
O
N
H
b
O
N
N
N
11
(67%)
8
9 (82%)
R
N
N
N
N
N
N
HN
NH2
N
N
d
c
O
N
H
N
H
O
N
N
Cl
R
12
(64%)
14a: R = H (82%)
13a
: R = H
14b
14c
: R = o-F (91%)
13b: R = o-F
13c
: R = m-CN (71%)
: R = m-CN
R
N
N
N
HN
S
N
e
N
H
N
15a: R = H (92%)
15b:
15c:
R = o-F (71%)
R = m-CN (68%)
Scheme 1. Reagents and conditions: (a) 48% HBr in H2O, DMSO, 60–70 °C, 2 h; (b) 2-(1,3-dioxoisoindolin-2-yl)acetaldehyde (10), NH4OAc, MTBE, MeOH, 50 °C, 10 h; (c)
N2H4ꢀH2O, EtOH, reflux, 5 h; (d) triethylamine, CH2Cl2, 0 °C to rt, 1 h; (e) Lawesson’s reagent, anhydrous DME, 85 °C, 12 h.
pyridin-6-yl)-5-(6-methylpyridin-2-yl)pyrazole derivatives 20a–c
have not been tested since these derivatives were expected to
display much lower inhibitory activity compared to the correspond-
ing positional isomers, 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-3-(6-
methylpyridin-2-yl)pyrazole derivatives 19a–c based on our
previous findings.24 The 50% inhibitory concentrations (IC50) of all
the target compounds against ALK5 were in the range of 0.018–
CONH2
N
N
N
HN
N
a
X
14c, 15c
N
H
N
0.106 lM as shown in Table 1. Among them, the pyrazole derivative
21b possessing a methylenethioamido linkage and a m-CN substitu-
ent in the phenyl ring showed the most potent ALK5 inhibition with
16a
: X = O (25%)
16b: X = S (19%)
Scheme 2. Reagents and conditions: (a)1 N NaOH, 28% H2O2, EtOH, rt, 12 h.
IC50 of 0.018 lM. In the imidazole derivatives, replacement of a
methyleneamido linkage with a methylenethioamido linkage re-
sulted in an increase in the ALK5 inhibitory activity. Thus, the
15a–c and 16b possessing a methylenethioamido linkage between
a central imidazole ring and a phenyl ring showed more potent
ALK5 inhibitory activity than the corresponding methyleneamido
ring at d 7.96, confirming the respective positions of alkylation.
Hydrolysis of the carbonitrile 19c with 28% H2O2 and 1 N NaOH
gave the corresponding carboxamide 19d in 75% yield. Compounds
19b and 19c were thionated in the same reaction condition as de-
scribed in Scheme 1 to give the corresponding carbothioamides
21a and 21b in 43% and 78% yields, respectively. Attempted hydro-
lysis of the carbonitrile 21b with 28% H2O2 and 1 N NaOH resulted
in the desulfurization, thus providing 19c (16%) and 19d (14%) in-
stead of the desired carboxamide possessing a thiocarboxamide
linkage.
linkage compounds 14a–c and 16a. The 16b (IC50 = 0.022
lM) hav-
ing a m-CONH2 substituent and the 15c (IC50 = 0.036 M) having a
l
m-CN substituent in the phenyl ring displayed 4.8-fold and 2.5-fold
more potent ALK5 inhibition than the corresponding compounds,
16a (IC50 = 0.106
unsubstituted 15a (IC50 = 0.048
having a o-F substituent were also slightly more potent than the
respective 14a (IC50 = 0.057 M) and 14b (IC50 = 0.037 M). Intro-
lM) and 14c (IC50 = 0.090
l
M), respectively. The
lM) and the 15b (IC50 = 0.027
lM)
2.2. ALK5 inhibitory activity in an enzyme assay and in a cell-
based luciferase reporter assay
l
l
duction of a o-F, m-CN or m-CONH2 substituent in the phenyl ring
was beneficial in ALK5 inhibition in the compounds 15a–c and
16b possessing a methylenethioamido linkage, showing that the
15b, 15c, and 16b were 1.8-fold, 1.3-fold, and 2.2-fold more potent
than 15a, respectively. In the pyrazole derivatives, replacement of a
methyleneamido linkage with a methylenethioamido linkage also
To investigate whether these potential inhibitors 14a–c, 15a–c,
16a, 16b, 19a–d, 21a, and 21b could inhibit ALK5, a kinase assay
was performed using the purified human ALK5 kinase domain
produced in Sf9 insect cells (Table 1). The 4-([1,2,4]triazolo[1,5-a]

C. H. Jin et al. / Bioorg. Med. Chem. 22 (2014) 2724–2732
2727
N
N
b
N
a
NH
8
H
N
N
Cl
N
R
O
18a
: R = H
17 (80%)
18b: R = o-F
18c
: R = m-CN
N
N
N
N
H
N
N
N
N
N
N
R
+
N
O
O
N
N
HN
R
20a
19a
: R =H (3%)
: R =H (78%)
19b: R = o-F (68%)
19c
20b: R = o-F (5%)
20c
: R = m-CN (7%)
: R = m-CN (67%)
c
19d: R = m-CONH2 (75%)
N
N
H
N
N
N
d
R
19b, 19c
N
S
N
21a
: R = o-F (43%)
21b: R = m-CN (78%)
Scheme 3. Reagents and conditions: (a) (i) DMFꢀDMA, 90 °C 4 h; (ii) N2H4ꢀH2O, EtOH, reflux, 3 h; (b) NaH, NaI (cat), anhydrous DMF, rt, 30 min; (c) 1 N NaOH, 28% H2O2, EtOH,
rt, 2 h; (d) Lawesson’s reagent, anhydrous DME, 85 °C, 12 h.
increased ALK5 inhibitory potency. Thus, the 21a (IC50 = 0.029
having a o-F substituent and 21b (IC50 = 0.018 M) having a m-CN
substituent were 2.9-fold and 1.5-fold more potent than the corre-
sponding 19b (IC50 = 0.085 M) and 19c (IC50 = 0.027 M), respec-
l
M)
2.3. p38
p38
a
MAP kinase assay
l
a
MAP kinase was chosen to examine the selectivity profile
l
l
of this series of compounds because its kinase domain is known to
be one of the most homologous to that of ALK5.47 All the com-
pounds possessing a methyleneamido linkage, 14a–c, 16a and
tively. Comparing the role of central heterocycles, imidazole and
pyrazole on ALK5 inhibition is one of our concern in this study.
Although the imidazole derivatives 14a and 14b were more inhibi-
tory than the corresponding pyrazole derivatives 19a and 19b, the
imidazole derivatives 14c, 16a, and 15c were less inhibitory than
the corresponding pyrazole derivatives 19c, 19d, and 21b, respec-
tively. And, the imidazole derivative 15b and the corresponding pyr-
azole derivative 21a showed the similar level of inhibition.
Therefore, it is hard to conclude that which heterocycle is an optimal
one for ALK5 inhibition in this series of compounds. The most potent
21b was 3.0-fold more potent than the competitor 1 (IC50 = 0.054
19a–d did not inhibit p38
of >10 M, whereas the imidazole derivatives possessing
methylenethioamido linkage, 15a–c and 16b inhibited it more
efficiently with IC50 of 1.05–3.77 M. The 21b displaying the most
a MAP kinase efficiently, showing IC50
l
a
l
potent ALK5 inhibitory activity in this series of compounds showed
a selectivity index of 284 that is much higher than those of 1 (11)
and 5 (28).
2.4. Docking model of 21b complex with ALK5
lM) and equipotent to 5 (IC50 = 0.017
lM) in ALK5 inhibition.
To evaluate TGF-b-induced downstream transcriptional activa-
To investigate the docking pose of 21b which showed the most
potent ALK5 inhibition in this series of compounds, we conducted
Surflex-Dock docking of 21b into the X-ray structure of ALK5 (PDB
id: 1VJY.pdb). We compared docking poses of 200 conformers of
21b with the X-ray pose of native ligand 1,5-naphthyrine inhibi-
tor.48 As shown in Figure 3, 21b (white carbon) fits into active site
of ALK5 and is superimposed over the X-ray pose of 1,5-naphthy-
rine inhibitor (yellow carbon). The triazolo-pyridine ring of 21b
contacts the hinge region of ALK5 forming hydrogen bond with
the backbone NH of His 283. The hydrogen bonding interaction
of ALK5 inhibitor and His 283 was reported to be important for
inhibitory activity in the previous study.24 The central pyrazole
ring N of 21b forms hydrogen bond with protonated side chain
amine group of Lys232. The cyano group of phenyl ring interacts
with Ser 287 via hydrogen bonding. As shown in Figure 3B, 21b fits
well into cavity of active site. Our docking model presented good
agreement with previous results showing the interactions between
tion to ALK5 signaling, cell-based luciferase activity of the target
compounds was measured using HaCaT cells permanently trans-
fected with p3TP-luciferase reporter construct at a concentration
of 0.03 lM (Table 1). The p3TP-luciferase reporter construct
contains three Ap-1 binding elements and the PAI-1 promoter.46
Contrary to the kinase assay, the imidazole and pyrazole deriva-
tives possessing a methyleneamido linkage, 14a (78% inhibition),
14b (57% inhibition) and 19b (68% inhibition) were more inhibi-
tory than the corresponding 15a (22% inhibition), 15b (no inhibi-
tion) and 21a (23% inhibition) possessing a methylenethioamido
linkage in the cell-based luciferase assay, suggesting that
a
methylenethioamido linkage contributes to the lower cellular
permeability compared to a methyleneamido linkage. The pyrazole
derivative 21b was the most inhibitory, showing 95% inhibition
that is much higher than that of 1 (22% inhibition) and equipotent
to that of 5 (95% inhibition).

2728
C. H. Jin et al. / Bioorg. Med. Chem. 22 (2014) 2724–2732
Table 1
Inhibitory activity of 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2-yl)imidazoles and -pyrazoles 14a–c, 15a–c, 16a, 16b, 19a–d, 21a, and 21b on ALK5 and p38
a
N
N
N
N
H
N
R
H
N
N
N
N
N
R
X
N
N
H
X
N
N
14a-c, 15a-c, 16a, 16b
19a-d, 21a, 21b
Compound
X
R
IC50
(
lM)
Selectivity indexc
p3TP-luciferase activity
(% control)d,e
ALK5a
p38ab
Mock
TGF-b
14a
14b
14c
16a
15a
15b
15c
16b
19a
19b
19c
19d
21a
21b
1 (SB-505124)
5 (IN-1130)
3
0.3
100 2.5
22 2.1
43 2.5
35 4.7
82 9.1
78 9.6
110 6.4
23 2.9
80 5.5
26 2.4
32 3.3
14 1.0
90 5.4
77 4.4
O
O
O
O
S
S
S
S
O
O
O
O
S
H
o-F
m-CN
m-CONH2
H
0.057
0.037
0.090
0.106
0.048
0.027
0.036
0.022
0.078
0.085
0.027
0.036
0.029
0.018
0.054
0.017
>10.0
>10.0
>10.0
>10.0
3.77
1.05
1.49
2.83
>10.0
>10.0
>10.0
>10.0
5.21
5.11
0.59
0.48
>175
>270
>111
>94
79
o-F
39
41
m-CN
m-CONH2
H
129
>128
>118
>370
>278
180
284
11
o-F
m-CN
m-CONH2
o-F
S
m-CN
5
0.2
78 0.5
0.4
28
5
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 0.03
Activity is given as the mean SD of three independent experiments run in triplicate relative to control incubations with DMSO vehicle.
d
e
l
M of inhibitor.
Figure 3. Binding pose of 21b in the active site of ALK5. (A) Superimposed over the X-ray pose of 1,5-naphthyrine inhibitor (yellow carbon). Key amino acid residues (grey
carbon) within the binding site are represented in line form, and docking pose of 21b is capped stick (white carbon). Red dashed lines are hydrogen bonding interactions
(<2.8 Å). (B) The active site of ALK5 is shown as MOLCAD49 lipophilic potential surface map in the docking model of 21b. Lipophilicity increases from blue (hydrophilic) to
brown (lipophilic).
ALK5 inhibitor and key amino acid residues in the active site of
ALK5. It also supported that cyano group may be important role
in improving ALK5 inhibition.
a methyleneamido linkage or a methylenethioamido linkage
between an imidazole or a pyrazole and a phenyl ring has been
synthesized and evaluated for their ALK5 inhibitory activity in an
enzyme assay and in a cell-based luciferase reporter assay. It has
been proved that incorporation of a [1,2,4]triazolo[1,5-a]pyridin-
6-yl moiety and a methylenethioamido linkage at the 4- and
1-positions of the pyrazole ring, respectively, and a m-CN substitu-
ent in the phenyl ring significantly increased both ALK5 inhibitory
3. Conclusion
In this report, a series of 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-
5(3)-(6-methylpyridin-2-yl)imidazoles and -pyrazoles possessing

C. H. Jin et al. / Bioorg. Med. Chem. 22 (2014) 2724–2732
2729
activity and selectivity. The most potent compound 21b inhibited
ALK5 phosphorylation with IC50 of 0.018 M and showed 95% inhi-
bition at 0.03 M in a luciferase reporter assay using HaCaT cells
permanently transfected with p3TP-luc reporter construct. The
selectivity index of 21b against p38 MAP kinase is 284 that is
(s, 3H), 4.00 (s, 2H), 7.01 (d, 1H, J = 7.6 Hz), 7.19 (d, 1H,
J = 7.6 Hz), 7.46 (t, 1H, J = 7.6 Hz), 7.69 (dd, 1H, J = 9.2, 0.8 Hz),
7.77 (dd, 1H, J = 9.2, 1.6 Hz), 8.30 (s, 1H), 8.96 (br s, 1H); 13C
NMR (100 MHz, CDCl3/CD3OD) d 24.3, 37.7, 115.7, 118.6, 122.3,
122.4, 127.7, 127.8, 131.9, 137.3, 137.4, 145.7, 147.5, 149.5,
153.9, 158.7; HRMS-ESI: m/z [M+Na]+ calcd for C16H15N7Na:
328.1281, found 328.1295.
l
l
a
much higher than those of 1 (11) and 5 (28).
4. Experimental section
4.1.3. N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-
methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)benzamide (14a)
A stirred solution of 12 (0.19 mmol) in CH2Cl2 was cooled to
0 °C in an ice bath, and triethylamine (0.29 mmol) was added.
The mixture was stirred for 20 min, and to it, benzoyl chloride
(13a) (0.23 mmol) was added. The reaction mixture was allowed
to warm to room temperature and stirred for 1 h. The reaction
mixture was evaporated to dryness under reduced pressure. The
residue was purified by MPLC on silica gel using MeOH/CH2Cl2
(1:20) as eluent to afford the titled compound as a light yellow so-
lid. Yield 82%; mp 235–237 °C; purity by HPLC: 99.4% (20% aceto-
4.1. Chemistry
1H NMR and 13C NMR spectra were recorded on a varian Unity
400 spectrophotometer 400 MHz and 100 MHz, respectively.
Chemical shifts are reported in parts per million (ppm) relative
to internal tetramethylsilane in CDCl3, CDCl3/CD3OD, or DMSO-d6.
Infrared spectra were recorded on a FT-infrared spectrometer
(Bio-Rad). High resolution mass spectra electrospray ionization
(HRMS-ESI) was obtained on an Agilent technologies 6220 TOF
LC/MS spectrometer. All melting points were taken in Pyrex capil-
laries using an electrothermal digital melting point apparatus
(Büchi) and were not corrected. The purity of the tested com-
pounds was determined using an Agilent 1100 series HPLC system
nitrile); IR (KBr) 3167, 3061, 1649, 1529 cmꢂ1 1H NMR (400 MHz,
;
CDCl3) d 2.53 (s, 3H), 4.79 (d, 2H, J = 6.0 Hz), 7.01 (d, 1H, J = 7.6 Hz),
7.18 (d, 1H, J = 8.0 Hz), 7.36 (td, 2H, J = 7.6, 0.8 Hz), 7.42–7.48 (m,
2H), 7.66 (dd, 1H, J = 9.2, 0.8 Hz), 7.71 (dd, 1H, J = 9.2, 0.6 Hz),
7.82 (dd, 2H, J = 8.4, 1.2 Hz), 8.03 (t, 1H, J = 6.0 Hz), 8.32 (s, 1H),
8.94 (dd, 1H, J = 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3/CD3OD) d
24.1, 36.4, 115.9, 118.7, 120.7, 122.8, 127.5 (2C), 128.2, 128.7
(2C), 129.9, 130.5, 131.8, 132.2, 137.6, 146.2, 147.9, 149.6, 154.1,
158.8, 169.0; HRMS-ESI: m/z [M+H]+ calcd for C23H20N7O:
410.1724, found 410.1738.
using a C18 column (Capcell-pak C18 MG 5
lM, 3.0 ꢁ 250 mm), and
that for all the compounds was found to be >98%. 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 (230–400 mesh)
with an YFLC-540 ceramic pump (Yamagen). All chemicals and sol-
vents were purchased from Aldrich or TCI Laboratory Chemicals.
4.1.1. 2-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-
methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)isoindoline-1,3-
dione (11)
4.1.4. N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-
methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-
fluorobenzamide (14b)
To a stirred solution of 9 (0.38 mmol) in MeOH (2 mL), NH4OAc
This compound was prepared according to the same procedure
for 14a, using 12 and 2-fluorobenzoyl chloride (13b), and the res-
idue was purified by MPLC on silica gel using MeOH/CH2Cl2 (1:20)
as eluent to give the titled compound as a white solid. Yield 91%;
mp 219–221 °C; purity by HPLC: 99.5% (20% acetonitrile); IR
(3.76 mmol),
2-(1,3-dioxoisoindolin-2-yl)acetaldehyde
(10)
(0.56 mmol), and MTBE (2 mL) were added at room temperature.
The mixture was heated at 50 °C for 10 h and then cooled to room
temperature. The pH of the reaction mixture was adjusted to 8
with saturated aqueous NaHCO3 solution. The reaction mixture
was extracted with EtOAc (2 ꢁ 35 mL), and the combined organic
layer was washed with water (10 mL) and brine (10 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 MeOH/CH2Cl2 (1:20) as eluent
to give the titled compound as a yellow solid. Yield 67%; mp 262–
(KBr) 3209, 3078, 1648, 1572, 1530 cmꢂ1
;
1H NMR (400 MHz,
2.51 (s, 3H), 4.68 (br s, 2H), 7.01 (d, 1H,
CDCl3/CD3OD)
d
J = 7.6 Hz), 7.09 (ddd, 1H, J = 12.0, 8.4, 0.8 Hz), 7.17–7.22 (m, 2H),
7.41–7.48 (m, 2H), 7.68 (dd, 1H, J = 9.2, 0.8 Hz), 7.77 (dd, 1H,
J = 9.2, 1.6 Hz), 7.96 (td, 1H, J = 7.8, 1.6 Hz), 8.29 (s, 1H), 9.00 (br
s, 1H); 13C NMR (100 MHz, CDCl3/CD3OD) d 24.2, 37.0, 115.8,
116.3 (d, J = 23.4 Hz), 118.5, 120.7 (d, J = 11.7 Hz), 121.6, 122.4,
124.8 (d, J = 3.7 Hz), 128.0, 129.9, 131.4, 131.5, 131.8, 133.9 (d,
J = 9.6 Hz), 137.4, 145.9, 148.3, 149.6, 154.0, 158.7, 160.7 (d,
J = 248.3 Hz), 165.0 (d, J = 2.9 Hz); HRMS-ESI: m/z [M+H]+ calcd
for C23H19FN7O: 428.1630, found 428.1647.
263 °C; IR (KBr) 3076, 2928, 1716, 1391 cmꢂ1 1H NMR (400 MHz,
;
CDCl3/CD3OD) d 2.51 (s, 3H), 5.00 (s, 2H), 7.00 (d, 1H, J = 7.6 Hz),
7.10 (d, 1H, J = 8.0 Hz), 7.42 (t, 1H, J = 7.8 Hz), 7.67 (d, 1H,
J = 9.2 Hz), 7.69–7.72 (m, 2H), 7.74 (dd, 1H, J = 9.2, 1.6 Hz), 7.83–
7.86 (m, 2H), 8.28 (s, 1H), 8.87 (br s, 1H); 13C NMR (100 MHz,
CDCl3) d 24.6, 35.5, 116.3, 117.7, 122.2, 122.8, 123.9 (2C), 128.0,
128.9, 131.7, 132.1 (2C), 134.2, 134.6 (2C), 137.3, 143.3, 147.7,
150.0, 154.5, 159.1, 168.1 (2C); HRMS-ESI: m/z [M+H]+ calcd for
4.1.5. N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methyl
pyridin-2-yl)-1H-imidazol-2-yl)methyl)-3-cyanobenzamide
(14c)
C
24H18N7O2: 436.1516, found 436.1530.
This compound was prepared according to the same procedure
for 14a, using 12 and 3-cyanobenzoyl chloride (13c), and the resi-
due was purified by MPLC on silica gel using MeOH/CH2Cl2 (1:20)
as eluent to give the titled compound as a white solid. Yield 71%;
mp 125–126 °C; purity by HPLC: 99.9% (18% acetonitrile); IR
4.1.2. (4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-
methylpyridin-2-yl)-1H-imidazol-2-yl)methanamine (12)
To a stirred solution of 11 (1.12 mmol) in absolute EtOH
(20 mL), N2H4ꢀH2O (11.19 mmol) was added at room temperature.
The mixture was refluxed for 5 h and then cooled to room temper-
ature. The reaction mixture was treated with acetone (5 mL) to
quench excess hydrazine and evaporated to dryness under reduced
pressure. The residue was purified by MPLC on silica gel using
MeOH/CH2Cl2/NH4OH (10:100:1) to afford the titled compound
as a yellow solid. Yield 64%; mp 67–68 °C; IR (KBr) 3354, 3181,
(KBr) 3246, 3073, 2234, 1650, 1543 cmꢂ1 1H NMR (400 MHz,
;
CDCl3/CD3OD) d 2.52 (s, 3H), 4.62 (s, 2H), 7.03 (d, 1H, J = 7.6 Hz),
7.16 (br s, 1H), 7.47 (t, 1H, J = 7.8 Hz), 7.53 (td, 1H, J = 7.8,
0.8 Hz), 7.69 (dd, 1H, J = 9.2, 0.8 Hz), 7.72–7.75 (m, 2H), 8.10
(ddd, 1H, J = 8.0, 1.6, 1.2 Hz), 8.19 (ddd, 1H, J = 2.0, 1.6, 0.4 Hz),
8.30 (s, 1H), 8.95 (br s, 1H); 13C NMR (100 MHz, CDCl3/CD3OD) d
24.2, 36.8, 112.8, 115.9, 118.1, 118.5, 121.3, 122.6, 128.0, 129.6,
129.9, 131.3, 131.5, 131.7, 131.8, 134.7, 135.1, 137.5, 145.9,
3086, 1576, 1549 cmꢂ1 1H NMR (400 MHz, CDCl3/CD3OD) d 2.52
;

2730
C. H. Jin et al. / Bioorg. Med. Chem. 22 (2014) 2724–2732
148.1, 149.6, 154.1, 158.8, 166.5; HRMS-ESI: m/z [M+H]+ calcd for
24H19N8O: 435.1676, found 435.1686.
mixture was neutralized with 1 N HCl to pH 7–8. The mixture
was extracted with CH2Cl2 (3 ꢁ 20 mL), and the combined organic
extracts were washed with water (20 mL) and brine (20 mL). The
CH2Cl2 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 (1:10) as eluent
to afford the titled compound as a yellow solid. Yield 25%; mp 177–
179 °C; purity by HPLC: 98.8% (13% acetonitrile); IR (KBr) 3351,
C
4.1.6. N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methyl
pyridin-2-yl)-1H-imidazol-2-yl)methyl)benzothioamide (15a)
A stirred mixture of 14a (0.16 mmol), Lawesson’s reagent
(0.16 mmol), and anhydrous DME (5 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/
CH2Cl2 (1:20) as eluent to give the titled compound as a white so-
lid. Yield 92%; mp 232–234 °C; purity by HPLC: 99.1% (25% aceto-
nitrile); IR (KBr) 3199, 3072, 2971, 1594, 1252, 1181 cmꢂ1; 1H NMR
(400 MHz, CDCl3) d 2.57 (s, 3H), 5.23 (d, 2H, J = 5.6 Hz), 7.04 (d, 1H,
J = 8.0 Hz), 7.18 (d, 1H, J = 7.6 Hz), 7.34 (t, 2H, J = 7.8 Hz), 7.41–7.45
(m, 1H), 7.46 (t, 1H, overlapped, J = 7.8 Hz), 7.67 (dd, 1H, J = 9.2,
1.6 Hz), 7.71 (d, 1H, J = 9.2 Hz), 7.81 (d, 2H, J = 7.6 Hz), 8.35 (s,
1H), 8.90 (br s, 1H), 9.31 (br s, 1H); 13C NMR (100 MHz, CDCl3) d
24.1, 43.0, 116.7, 118.5, 120.3, 123.0, 127.4 (2C), 128.3, 128.6
(2C), 129.0, 131.1, 131.9, 138.1, 140.2, 141.4, 145.3, 147.1, 150.0,
154.7, 158.7, 200.4; HRMS-ESI: m/z [M+H]+ calcd for C23H20N7S:
426.1495, found 426.1514.
3132, 2917, 1677, 1542 cmꢂ1 1H NMR (400 MHz, CDCl3/CD3OD)
;
d 2.53 (s, 3H), 4.66 (s, 2H), 7.04 (d, 1H, J = 8.0 Hz), 7.12 (br d, 1H,
J = 7.6 Hz), 7.48 (m, 1H), 7.51 (t, 1H, overlapped, J = 7.8 Hz), 7.71
(d, 1H, J = 9.2 Hz), 7.75 (dd, 1H, J = 9.2, 1.6 Hz), 8.00–8.05 (m, 2H),
8.31–8.32 (m, 2H), 8.94 (br s, 1H); 13C NMR (100 MHz, DMSO-d6)
d 23.6, 36.5, 115.1, 118.8, 122.2, 126.9, 128.4, 128.5, 130.2, 130.3,
131.7, 132.3, 132.4, 133.9, 134.4, 134.9, 137.8, 146.4, 148.5,
149.2, 154.4, 157.3, 166.0, 167.4; HRMS-ESI: m/z [M+H]+calcd for
C24H21N8O2: 453.1782, found 453.1799.
4.1.10. 3-(((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-
methylpyridin-2-yl)-1H-imidazol-2-
yl)methyl)carbamothioyl)benzamide (16b)
This compound was prepared according to the same procedure
for 16a using 15c as the starting material. Yield 19%; mp 217–
219 °C; purity by HPLC: 98.2% (18% acetonitrile); IR (KBr) 3464,
4.1.7. N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methyl
pyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluorobenzothioa
mide (15b)
3383, 3152, 2921, 1668 cmꢂ1 1H NMR (400 MHz, CDCl3/CD3OD)
;
d 2.54 (s, 3H), 5.12 (s, 2H), 7.05 (d, 1H, J = 7.6 Hz), 7.17 (d, 1H,
J = 7.6 Hz), 7.43–7.50 (m, 2H), 7.71–7.76 (m, 2H), 7.94 (dt, 1H,
J = 7.6, 1.6 Hz), 8.07 (ddd, 1H, J = 8.0, 2.0, 1.2 Hz), 8.23 (t, 1H,
J = 1.6 Hz), 8.32 (s, 1H), 8.94 (br s, 1H); 13C NMR (100 MHz, CDCl3/
CD3OD) d 22.6, 41.4, 116.0, 116.7, 119.5, 124.3, 124.8, 128.5, 128.8,
129.1, 129.4, 131.1, 131.4, 132.2, 132.8, 133.1, 139.3, 139.8, 145.4,
149.9, 154.5, 157.9, 169.7, 199.4; HRMS-ESI: m/z [M+H]+ calcd for
This compound was prepared according to the same procedure
for 15a using 14b as the starting material. Yield 71%; mp 364–
365 °C; purity by HPLC: 98.6% (25% acetonitrile); IR (KBr) 3180,
3071, 2969, 1595, 1252, 1182 cmꢂ1 1H NMR (400 MHz, CDCl3) d
;
2.58 (s, 3H), 5.24 (d, 2H, J = 5.2 Hz), 6.70 (br s, 1H), 7.05 (d, 1H,
J = 8.0 Hz), 7.08 (dd, 1H, J = 8.0, 1.2 Hz), 7.17 (td, 1H, J = 8.0,
1.2 Hz), 7.19 (br d, 1H, J = 8.0 Hz), 7.38–7.43 (m, 1H), 7.47 (t, 1H,
J = 7.8 Hz), 7.71 (d, 1H, J = 9.2 Hz), 7.75 (d, 1H, J = 9.2 Hz), 8.09 (t,
1H, J = 7.8 Hz), 8.37 (s, 1H), 8.92 (s, 1H), 9.45 (br s, 1H); 13C NMR
(100 MHz, CDCl3) d 24.1, 42.8, 115.8, 116.0 (d, J = 22.7 Hz), 118.5,
121.3, 122.6, 124.4 (d, J = 2.9 Hz), 128.0, 128.8 (d, J = 11.7 Hz),
129.7, 131.3, 131.8, 132.2, 132.4 (J = 8.8 Hz), 137.6, 144.6, 147.9,
149.5, 153.9, 157.6 (d, J = 249.7 Hz), 158.7, 195.0; HRMS-ESI: m/z
[M+H]+ calcd for C23H19FN7S: 444.1401, found 444.1401.
C24H21N8OS: 469.1554, found 469.1559.
4.1.11. 2-(4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-3-(6-methyl
pyridin-2-yl)-1H-pyrazol-1-yl)-N-phenylacetamide (19a) and 2-
(4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-
1H-pyrazol-1-yl)-N-phenylacetamide (20a)
To a stirred solution of 6-(3-(6-methylpyridin-2-yl)-1H-pyra-
zol-4-yl)-[1,2,4]triazolo[1,5-a]pyridine (17) (0.72 mmol) in
anhydrous DMF (6 ml), a catalytic amount of NaI, 2-chloro-N-
phenylacetamide (18a) (1.08 mmol), and NaH (1.08 mmol) were
added. The mixture was stirred at room temperature for 30 min
and then evaporated to dryness under reduced pressure. The resi-
due was purified by MPLC on silica gel using MeOH/CHCl3 (1:40) as
eluent to give the two positional isomers 19a and 20a as white sol-
ids. Compound 19a: Yield 78%; mp 120–122 °C; purity by HPLC:
99.8% (21% acetonitrile); IR (KBr) 3276, 2985, 1684, 1600,
4.1.8. N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-
methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-3-
cyanobenzothioamide (15c)
This compound was prepared according to the same procedure
for 15a using 14c as the starting material. Yield 68%; mp 188–
189 °C; purity by HPLC: 99.1% (25% acetonitrile); IR (KBr) 3188,
3018, 2976, 2233, 1534, 1252, 1174 cmꢂ1 1H NMR (400 MHz,
;
CDCl3/CD3OD) d 2.55 (s, 3H), 5.10 (s, 2H), 7.07 (d, 1H, J = 7.6 Hz),
7.19 (d, 1H, J = 7.6 Hz), 7.49 (td, 1H, overlapped, J = 8.0, 0.4 Hz),
7.50 (t, 1H, overlapped, J = 7.6 Hz), 7.71 (ddd, 1H, J = 8.0, 1.6,
1.2 Hz), 7.74 (m, 2H), 8.08 (ddd, 1H, J = 8.0, 2.0, 1.2 Hz), 8.18 (dd,
1H, J = 2.0, 1.6 Hz), 8.33 (s, 1H), 8.95 (t, 1H, J = 1.6 Hz); 13C NMR
(100 MHz, CDCl3/CD3OD) d 23.9, 42.6, 112.4, 116.0, 118.1, 118.7,
120.4, 122.9, 128.2, 129.3, 129.6, 130.6, 131.5, 131.6, 131.7,
134.3, 137.9, 141.4, 144.7, 147.4, 149.6, 154.1, 158.6, 197.5;
HRMS-ESI: m/z [M+H]+ calcd for C24H19N8S: 451.1448, found
451.1465.
1558 cmꢂ1 1H NMR (400 MHz, CDCl3) d 2.52 (s, 3H), 5.08 (s, 2H),
;
7.11–7.15 (m, 1H), 7.17 (d, 1H, J = 7.6 Hz), 7.29–7.33 (m, 2H),
7.49–7.52 (m, 2H), 7.57 (d, 1H, overlapped, J = 7.6 Hz), 7.58 (dd,
1H,overlapped, J = 9.2, 1.6 Hz), 7.67 (t, 1H, J = 7.8 Hz), 7.70 (dd,
1H, J = 9.2, 0.8 Hz), 7.79 (s, 1H), 8.35 (s, 1H), 8.68 (br s, 1H), 8.88
(dd, 1H, J = 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3) d 24.4, 56.1,
115.5, 118.1, 119.9, 120.1, 120.3 (2C), 123.1, 125.1, 127.7, 129.2
(2C), 130.6, 132.5, 132.8, 137.2, 149.7, 150.3, 150.9, 154.3, 158.6,
164.5; HRMS-ESI: m/z [M+H]+ calcd for C23H20N7O: 410.1724,
found 410.1729. Compound 20a: Yield 3%; mp 187–188 °C; IR
(KBr) 3275, 2924, 1684, 1599, 1555 cmꢂ1 1H NMR (400 MHz,
;
4.1.9. N0-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-
methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)isophthalamide
(16a)
A stirred solution of 14c (0.07 mmol) in EtOH (3 mL) and 1 N
NaOH (0.26 mmol) was cooled to 0 °C, and to it, 28% H2O2
(0.08 mmol) was added dropwise. The mixture was allowed to
warm to room temperature and stirred for 12 h. The reaction
CDCl3) d 2.68 (s, 3H), 5.04 (s, 2H), 7.07 (d, 1H, J = 7.6 Hz), 7.11 (br
t, 1H, J = 7.2 Hz), 7.27 (d, 1H, J = 7.6 Hz), 7.31–7.35 (m, 2H), 7.38
(dd, 1H, J = 9.2, 1.6 Hz), 7.57–7.63 (m, 3H), 7.70 (dd, 1H, J = 9.2,
0.8 Hz), 7.84 (s, 1H), 8.33 (s, 1H), 8.53 (dd, 1H, J = 1.6, 0.8 Hz),
9.67 (br s, 1H); 13C NMR (100 MHz, CDCl3) d 24.6, 55.5, 116.9,
117.6, 120.0 (2C), 120.2, 123.0, 124.2, 124.6, 126.8, 129.2 (2C),
131.4, 137.9, 138.0, 139.7, 139.9, 147.2, 149.7, 154.4, 159.8,

C. H. Jin et al. / Bioorg. Med. Chem. 22 (2014) 2724–2732
2731
165.3; HRMS-ESI: m/z [M+H]+ calcd for C23H20N7O: 410.1724,
146.5, 149.2, 154.0, 159.3, 165.8; HRMS-ESI: m/z [M+H]+ calcd
found 410.1732.
for C24H19N8O: 435.1676, found 435.1676.
4.1.12. 2-(4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-3-(6-
methylpyridin-2-yl)-1H-pyrazol-1-yl)-N-(2-fluorophenyl)
acetamide (19b) and 2-(4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-
(6-methylpyridin-2-yl)-1H-pyrazol-1-yl)-N-(2-fluorophenyl)
acetamide (20b)
4.1.14. 3-(2-(4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-3-(6-
methylpyridin-2-yl)-1H-pyrazol-1-yl)acetamido)benzamide
(19d)
This compound was prepared according to the same procedure
for 16a using 19c as the starting material. Yield 75%; mp 274–
275 °C; purity by HPLC: 99.8% (15% acetonitrile); IR (KBr) 3325,
These compounds were prepared according to the same
procedure for 19a and 20a using 17 and 2-chloro-N-(2-fluoro-
phenyl)acetamide (18b), and the residue was purified by MPLC
on silica gel using MeOH/CHCl3 (1:40) as eluent to give the titled
compounds as white solids. Compound 19b: Yield 68%; mp 212–
214 °C; purity by HPLC: 98.4% (21% acetonitrile); IR (KBr) 3275,
3167, 2928, 1668, 1563 cmꢂ1 1H NMR (400 MHz, DMSO-d6) d
;
2.36 (s, 3H), 5.18 (s, 2H), 7.21 (d, 1H, J = 7.6 Hz), 7.35 (br s, 1H),
7.41 (t, 1H, J = 8.0 Hz), 7.58 (br d, 1H, J = 8.0 Hz), 7.63 (br d, 1H,
J = 7.6 Hz), 7.71–7.75 (m, 2H), 7.78–7.81 (m, 2H), 7.95 (br s, 1H),
8.08 (t, 1H, J = 1.6 Hz), 8.28 (s, 1H), 8.48 (s, 1H), 9.27 (dd, 1H,
J = 1.6, 1.2 Hz), 10.58 (br s, 1H); 13C NMR (100 MHz, DMSO-d6) d
23.7, 54.9, 114.5, 116.3, 118.9, 119.1, 120.5, 121.9, 122.1, 122.5,
127.2, 128.7, 133.0, 133.6, 135.2, 137.1, 138.5, 147.5, 148.7,
151.4, 153.9, 156.8, 165.2, 167.7; HRMS-ESI: m/z [M+H]+ calcd
for C24H21N8O2: 453.1782, found 453.1804.
2997, 1695, 1620, 1547 cmꢂ1 1H NMR (400 MHz, CDCl3) d 2.46
;
(s, 3H), 5.08 (s, 2H), 7.06–7.09 (m, 2H), 7.12–7.15 (m, 2H), 7.63
(dd, 1H, J = 9.2, 1.6 Hz), 7.65–7.71 (m, 3H), 7.77 (s, 1H), 8.30 (t,
1H, J = 8.0 Hz), 8.35 (s, 1H), 8.93 (s, 1H), 9.07 (br s, 1H); 13C NMR
(100 MHz, CDCl3) d 24.4, 56.1, 115.2 (d, J = 21.7 Hz), 115.3, 118.4,
119.6 (d, J = 1.4 Hz), 120.1, 121.9, 123.0, 124.8 (d, J = 2.8 Hz),
125.3 (d, J = 7.7 Hz), 125.8 (d, J = 10.6 Hz), 127.9, 132.5, 133.1,
137.2, 149.7, 150.7, 150.9, 152.8 (d, J = 243.5 Hz), 154.3, 158.3,
164.5; HRMS-ESI: m/z [M+K]+ calcd for C23H18FKN7O: 466.1188,
found 466.1204. Compound 20b: Yield 5%; mp 166–168 °C; IR
4.1.15. 2-(4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-3-(6-
methylpyridin-2-yl)-1H-pyrazol-1-yl)-N-(2-fluorophenyl)
ethanethioamide (21a)
This compound was prepared according to the same procedure
for 15a using 19b as the starting material. Yield 43%; mp 194–
196 °C; purity by HPLC: 99.4% (23% acetonitrile); IR (KBr) 3207,
(KBr) 3270, 2930, 1698, 1544 cmꢂ1 1H NMR (400 MHz, CDCl3) d
;
2.64 (s, 3H), 5.14 (s, 2H), 7.03–7.07 (m, 3H), 7.11–7.15 (m, 1H),
7.23 (dd, 1H, J = 7.6, 0.4 Hz), 7.38 (dd, 1H, J = 9.2, 2.0 Hz), 7.57 (t,
1H, J = 7.8 Hz), 7.70 (dd, 1H, J = 9.2, 0.8 Hz), 7.84 (s, 1H), 8.33 (s,
1H), 8.36 (t, 1H, J = 8.0 Hz), 8.55 (dd, 1H, J = 1.6, 0.8 Hz), 9.63 (br
2924, 1562, 1431, 1406, 1189 cmꢂ1 1H NMR (400 MHz, CDCl3) d
;
2.69 (s, 3H), 5.52 (s, 2H), 7.08–7.13 (m, 1H), 7.15–7.19 (m, 1H),
7.21–7.25 (m, 1H), 7.29 (d, 1H, J = 8.0 Hz), 7.57 (dd, 1H, J = 9.2,
1.6 Hz), 7.61 (d, 1H, J = 7.6 Hz), 7.75 (d, 1H, J = 9.2 Hz), 7.80 (t,
1H, J = 8.0 Hz), 7.86 (s, 1H), 8.32 (br d, 1H, J = 8.0 Hz), 8.37 (s,
1H), 8.83 (s, 1H), 10.87 (br s, 1H); 13C NMR (100 MHz, CDCl3) d
24.2, 63.8, 115.2, 115.6 (d, J = 19.0 Hz), 118.3, 119.7, 119.9, 123.1,
124.1 (d, J = 3.6 Hz), 124.2, 126.7 (d, J = 10.3 Hz), 127.8, 127.9 (d,
J = 8.1 Hz), 132.3, 132.9, 137.4, 149.6, 150.2, 150.5, 154.1, 154.4
(d, J = 246.8 Hz), 158.1, 193.4; HRMS-ESI: m/z [M+H]+ calcd for C23-
H19FN7S: 444.1401, found 444.1412.
s, 1H); 13C NMR (100 MHz, CDCl3)
d 24.3, 55.4, 115.0 (d,
J = 18.3 Hz), 116.7, 117.6, 120.4, 122.0, 122.7, 124.4, 124.7 (d,
J = 2.9 Hz), 124.8, 126.3 (d, J = 11.0 Hz), 126.9, 131.7, 137.9, 139.6,
139.9, 146.9, 149.4, 152.7 (d, J = 243.2 Hz), 154.1, 159.8, 165.7;
HRMS-ESI: m/z [M+K]+ calcd for C23H18FKN7O: 466.1188, found
466.1209.
4.1.13. 2-(4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-3-(6-
methylpyridin-2-yl)-1H-pyrazol-1-yl)-N-(3-cyanophenyl)
acetamide (19c) and 2-(4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-
(6-methylpyridin-2-yl)-1H-pyrazol-1-yl)-N-(3-cyanophenyl)
acetamide (20c)
4.1.16. 2-(4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-3-(6-methyl
pyridin-2-yl)-1H-pyrazol-1-yl)-N-(3-
cyanophenyl)ethanethioamide (21b)
These compounds were prepared according to the same proce-
dure for 19a and 20a using 17 and 2-chloro-N-(3-cyano-
phenyl)acetamide (18c), and the residue was purified by MPLC
on silica gel using MeOH/CHCl2 (1:20) as eluent to give the titled
compounds as white solids. Compound 19c: Yield 67%; mp 253–
255 °C; purity by HPLC: 99.8% (22% acetonitrile); IR (KBr) 3242,
This compound was prepared according to the same procedure
for 15a using 19c as the starting material. Yield 78%; mp 240–
242 °C; purity by HPLC: 98.9% (24% acetonitrile); IR (KBr) 3270,
2232, 1565, 1186 cmꢂ1 1H NMR (400 MHz, CDCl3) d 2.75 (s, 3H),
;
5.52 (s, 2H), 7.32 (d, 1H, J = 7.6 Hz), 7.45–7.49 (m, 2H), 7.50–7.53
(m, 2H), 7.76 (dd, 1H, J = 9.2, 0.8 Hz), 7.81 (t, 1H, J = 8.0 Hz), 7.88
(s, 1H), 8.05–8.08 (m, 1H), 8.27 (t, 1H, J = 1.6 Hz), 8.38 (s, 1H),
8.77 (dd, 1H, J = 1.6, 0.8 Hz), 11.30 (br s, 1H); 13C NMR (100 MHz,
CDCl3/CD3OD) d 23.8, 63.3, 112.4, 115.1, 117.0, 118.1, 120.3,
120.7, 123.1, 126.5, 127.1, 127.8, 129.7, 130.0, 132.4, 133.0,
137.3, 139.6, 149.0, 149.2, 150.5, 153.3, 158.4, 194.9; HRMS-ESI:
m/z [M+H]+ calcd for C24H19N8S: 451.1448, found 451.1466.
3058, 2226, 1688, 1589 cmꢂ1 1H NMR (400 MHz, DMSO-d6) d
;
2.35 (s, 3H), 5.21 (s, 2H), 7.20 (d, 1H, J = 7.6 Hz), 7.57–7.60 (m,
2H), 7.62 (d, 1H, J = 8.0 Hz), 7.73 (t, 1H, overlapped, J = 7.8 Hz),
7.74 (dd, 1H, overlapped, J = 9.2, 1.6 Hz), 7.80 (dd, 1H, J = 9.2,
0.8 Hz), 7.83–7.86 (m, 1H), 8.10 (d, 1H, J = 2.4 Hz), 8.28 (s, 1H),
8.48 (s, 1H), 9.27 (dd, 1H, J = 1.6, 0.8 Hz), 10.81 (br s, 1H); 13C
NMR (100 MHz, DMSO-d6) d 23.7, 54.9, 111.7, 114.5, 116.4,
118.6, 119.1, 120.4, 122.0, 122.1, 123.9, 127.2, 127.3, 130.4,
133.0, 133.6, 137.1, 139.3, 147.6, 148.7, 151.4, 153.9, 156.8,
165.8; HRMS-ESI: m/z [M+H]+ calcd for C24H19N8O: 435.1676,
found 435.1691. Compound 20c: Yield 7%; mp 146–147 °C; IR
4.2. Luciferase reporter assay
Biological activity of the test compounds was determined by
measuring their ability to inhibit TGF-b-induced p3TP-luciferase
reporter activity in HaCaT stable cells transfected with p3TP-Lux.
HaCaT cells were seeded at concentrations of 3 ꢁ 104 in 96-well
plates. The next day, when they reach approximately 90% conflu-
ency, 0.03 lM of ALK5 inhibitors and 2 ng/mL of TGF-b were added
to the cells. After 24 h, cell lysates were harvested using Luciferase
assay kit (Promega) according to the manufacturer’s instruction,
(KBr) 3267, 2232, 1672, 1588 cmꢂ1 1H NMR (400 MHz, DMSO-
;
d6) d 2.46 (s, 3H), 5.27 (s, 2H), 7.16 (d, 1H, J = 7.6 Hz), 7.26 (d, 1H,
J = 7.6 Hz), 7.39 (dd, 1H, J = 9.2, 1.6 Hz), 7.52–7.55 (m, 2H), 7.67
(t, 1H, J = 7.6 Hz), 7.72–7.75 (m 1H), 7.78 (dd, 1H, J = 9.2, 0.8 Hz),
7.96 (s, 1H), 7.99 (dd, 1H, J = 2.0, 1.2 Hz), 8.49 (s, 1H), 8.98 (dd,
1H, J = 1.6, 0.8 Hz), 10.63 (br s, 1H); 13C NMR (100 MHz, CDCl3) d
24.1, 55.4, 113.2, 116.8, 118.1, 118.6, 120.2, 123.0, 123.7, 124.1,
124.9, 126.9, 127.9, 130.1, 131.7, 138.7, 139.0, 139.2, 139.9,
and luminescence was measured by
a luminometer Micro
Lumat Plus (Berthold). Activity is given as the mean SD of three

2732
C. H. Jin et al. / Bioorg. Med. Chem. 22 (2014) 2724–2732
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4.3. Docking experiments
All performances of computational works were carried out on
the Tripos Sybyl-X 2.0 (Tripos Inc, St Louis, MO, USA) molecular
modeling package with CentOS Linux 5.4. operating system.
4.3.1. Preparation of ligand and receptor
14. Calvo-Aller, E.; Baselga, J.; Glatt, S.; Claverly, A.; Lahn, M.; Arteaga, C. L.;
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7,407,958 B2, 2008.
Structure of compound 21b was prepared using sketch modules
and saved as MOL2 format. All hydrogen atoms and Gasteiger–Hüc-
kel charges were added to atoms. To optimize the structure, energy
minimization was performed with the standard tripos force field
with convergence to maximum derivatives of 0.001 kcal molꢂ1 Åꢂ1
.
For the docking performance, the complex of ALK5 with 1,5-naph-
thyrine inhibitor (PDB id: 1VJY)48 which was retrieved from Protein
Data Bank (PDB) (http://www.rcsb.org/), was optimized using
structure preparation tool embedded in biopolymer module. All
water molecules were removed, and native ligand was extracted
from the X-ray structure. Hydrogen atoms were added, and then
Kollman-all charge was assigned to all the atoms in the target pro-
tein. This modified ALK5 structure was saved as mol2 format and
used as a receptor for docking study.
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Y.; Son, J.-Y.; Sheen, Y. Y.; Kim, D.-K. Bioorg. Med. Chem. 2011, 19, 2633.
24. Jin, C. H.; Sreenu, D.; Krishnaiah, M.; Subrahmanyam, V. B.; Rao, K. S.; Mohan, A.
V. N.; Park, C.-Y.; Son, J.-Y.; Son, D.-H.; Park, H.-J.; Sheen, Y. Y.; Kim, D.-K. Eur. J.
Med. Chem. 2011, 46, 3917.
25. Jin, C. H.; Krishnaiah, M.; Sreenu, D.; Subrahmanyam, V. B.; Rao, K. S.; Mohan, A.
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2011, 21, 6049.
26. Kim, D.-K.; Sheen, Y. Y. Jin, C. H.; Park, C.-Y.; Sreenu, D.; Rao, K. S.; Krishnaiah,
M.; Subrahmanyam, V. B. U.S. Patent 8,080,568 B1, 2011.
27. Krishnaiah, M.; Jin, C. H.; Sreenu, D.; Subrahmanyam, V. B.; Rao, K. S.; Son,
D.-H.; Park, H.-J.; Kim, S. W.; Sheen, Y. Y.; Kim, D.-K. Eur. J. Med. Chem.
2012, 57, 74.
28. Galarreta, C. I.; Thornhill, B. A.; Forbes, M. S.; Simpkins, L. N.; Kim, D.-K.;
Chevalier, R. L. Am. J. Physiol. Renal Physiol. 2013, 304, F481.
29. Moon, J.-A.; Kim, H.-T.; Cho, I.-S.; Sheen, Y. Y.; Kim, D.-K. Kidney Int. 2006, 70,
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30. Luo, J.; Ho, P. P.; Buckwalter, M. S.; Hsu, T.; Lee, L. Y.; Zhang, H.; Kim, D.-K.; Kim,
S.-J.; Gambhir, S. S.; Steinman, L.; Wyss-Coray, T. J. Clin. Invest. 2007, 117, 3306.
31. Ryu, J.-K.; Piao, S.; Shin, H.-Y.; Choi, M. J.; Zhang, L. W.; Jin, H.-R.; Kim, W. J.;
Han, J.-Y.; Hong, S. S.; Park, S. H.; Lee, S.-J.; Kim, I.-H.; Lee, C. R.; Kim, D.-K.;
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4.3.2. Molecular docking
To examine docking pose of 21b, the compound was docked
into the active site of ALK5 (PDB id: 1VJY) using Surflex-Dock
embedded in Tripos Sybyl X 2.0 software package. Surflex-Dock
uses an idealized ligand binding site called protomol to which
putative ligands are aligned. The protomol was constructed based
on the extracted native ligand from the X-ray structure, aligned
in the same coordinate space as the receptor ALK5. Two parame-
ters, such as threshold to determine buried volume of protomol
and bloat value to inflate the protomol, were given as 0.5 and 0,
respectively. Docking was performed using default Surflex-Dock
parameters, and maximum number of poses per ligand was set
as 200 conformers. Binding affinity of each docking pose was calcu-
lated by consensus scoring module CScore embedded in Sybyl X
2.0. To select the most reliable docking pose, not only docking
scores (Surflex-Dock score and consensus score) were considered,
but also important interactions between the ligand and ALK5 ac-
tive site were examined by visual inspection. One of the poses with
the highest Surflex-Dock score (ꢂlogKd = 6.98) and consensus
score (CScore = 5) was selected as the docking model shown in
Figure 3.
Acknowledgment
This work was supported by RP-Grant 2011 of Ewha Womans
University.
43. Pier, G. B.; Delia, P.; Mojgan, A. T.; Francesca, F.; Giulia, S.; Stefania, B.; Romeo,
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.022.
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