Synthesis and biological evaluation of 2-benzylamino-4(5)-(6-methylpyridin- 2-yl)-5(4)-([1,2,4]triazolo[1,5-a]-pyridin-6-yl)thiazoles as transforming growth factor-β type 1 receptor kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.09.011

A series of 2-benzylamino-4(5)-(6-methylpyridin-2-yl)-5(4)-([1,2,4] triazolo[1,5-a]pyridin-6-yl)thiazoles 12a-ab, 13a, 13b, and 18a-d has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The N-(3-fluorobenzyl)-4-(6- methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12b) inhibited ALK5 phosphorylation with an IC₅₀ value of 7.01 nM and showed 61% inhibition at 30 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.

Full text of DOI:10.1016/j.ejmech.2012.09.011

European Journal of Medicinal Chemistry 57 (2012) 74e84
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of 2-benzylamino-4(5)-(6-methylpyridin-
2-yl)-5(4)-([1,2,4]triazolo[1,5-a]-pyridin-6-yl)thiazoles as transforming growth
factor-b type 1 receptor kinase inhibitors
Maddeboina Krishnaiah a, Cheng Hua Jin a, Domalapally Sreenu a, Vura Bala Subrahmanyam a,
Kota Sudhakar Rao a, Do-Hyun Son b, Hyun-Ju Park b, Seung Won Kim a, 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 2-benzylamino-4(5)-(6-methylpyridin-2-yl)-5(4)-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazoles
12aeab, 13a, 13b, and 18aed has been synthesized and evaluated for their ALK5 inhibitory activity in an
enzyme assay and in a cell-based luciferase reporter assay. The N-(3-fluorobenzyl)-4-(6-methylpyridin-
2-yl)-5-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12b) inhibited ALK5 phosphorylation with an
IC50 value of 7.01 nM and showed 61% inhibition at 30 nM in a luciferase reporter assay using HaCaT cells
permanently transfected with p3TP-luc reporter construct.
Received 7 March 2012
Received in revised form
6 September 2012
Accepted 7 September 2012
Available online 20 September 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
TGF-b
ALK5 inhibitor
Fibrosis
Cancer
Kinase assay
Cell-based luciferase reporter assay
Docking
1. Introduction
Perturbation of TGF-b signaling has been also implicated in various
human diseases including cancer [7], pancreatic diseases [8], and
hematological malignancies [9]. Recent studies have shown that
The transforming growth factor-
b
(TGF-b) is the most potent
and ubiquitous profibrogenic cytokine. TGF-
b
transduces signals
blocking the TGF-b signaling pathway with several small molecule
through two distinct serine/threonine kinase receptors, the type I
and type II receptors [1]. Following the binding of ligand 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. Phosphorylated
Smad2/Smad3 proteins form a heteromeric complex with Smad4,
which translocates into the nucleus, assembles with specific DNA-
binding cofactors and co-modulators, and binds to the promoters of
ATP-competitive ALK5 inhibitors such as 1 (SB-505154) [10], 2
(GW6604) [11], 3 (SD-208) [12], and 4 (LY-2157299) [13] (Fig. 1)
inhibited autophosphorylation of ALK5 and TGF-
scription of matrix genes in reporter assays at sub-micromolar
concentrations. Among them, and effectively retarded
b-induced tran-
2
3
progressive fibrosis in liver and lung, respectively, and 3 and 4 also
strongly inhibited growth and invasiveness of cancer cells in animal
models.
TGF-
apoptosis, migration, and extracellular matrix production [2]. TGF-
plays a pivotal role in the development of fibrosis in various organ
b
target genes involved in cell differentiation, proliferation,
We have also reported a number of the 2-pyridyl-substituted
five-membered heterocycles as ALK5 inhibitors such as IN-1130 (5)
and IN-1233 (6) [14,15] (Fig. 1). One of our preclinical candidates, 5,
displayed its pronounced activity as a suppressor of fibrogenic
process of unilateral ureteral obstruction (UUO) in a rat renal
fibrosis model [16]. Another preclinical candidate, 6, effectively
prevented development and progression of pulmonary arterial
hypertension in a monocrotaline rat model [17].
b
systems such as kidney [3], heart [4], lung [5], and liver [6].
* Corresponding author. Tel.: þ82 2 3277 3025; fax: þ82 2 3277 2467.
E-mail address: dkkim@ewha.ac.kr (D.-K. Kim).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.011

M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
75
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
(SB-505124)
2
(LY-2157299)
(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)
Fig. 1. ALK5 inhibitors under development.
To develop a more potent and selective ALK5 inhibitor, we have
prepared a series of 2-benzylamino-4-(6-methylpyridin-2-yl)-5-
([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazoles 12aeab, 13a, and 13b
possessing an aminomethylene linkage between a thiazole ring and
a phenyl ring. It was of particular interest to us whether the amino
group at the linkage may serve as an additional binding group to
the ATP binding site of ALK5. The target molecules have a [1,2,4]
triazolo[1,5-a]pyridin-6-yl moiety rather than a quinoxalin-6-yl
moiety of 5 or a quinolin-6-yl moiety of 6 at the 5-position of the
thiazole and have various substituents such as F, Cl, Me, OMe, OCF3,
CN, and CONH2 in the phenyl ring. In pharmacokinetic studies, the
major metabolite of 5 was detected in the systemic circulation of rat
and mouse and was identified as 3-((4-(2-hydroxyquinoxalin-6-
yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)benzamide
or 3-((4-(3-hydroxyquinoxalin-6-yl)-5-(6-methylpyridin-2-yl)-1H-
imidazol-2-yl)methyl)benzamide [18].
To overcome metabolic oxidation of the quinoxalin-6-yl moiety
of 5, we adopted a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety since
metabolic oxidation of 2-position of this moiety is assumed to
be difficult due to the presence of two adjacent nitrogen atoms.
To examine the influence of the position of two hetero atoms
in a thiazole ring on ALK5 inhibition, we have prepared 2-
benzylamino-5-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]-
pyridin-6-yl)thiazoles 18aed, respective thiazole ring isosteres of
12a, 12g, 13a, and 12n for comparison.
pyridin-6-yl)thiazol-2-amine (10) in 82% yield. The 2-aminothiazole
10 was alkylated with an appropriately substituted benzyl bromide
(11aeab) in the presence of Cs2CO3 in DMF at 120 ꢀC to afford the
target compounds 12aeab in various yields (24e95%). Conversion
of the nitrile functionality in 12g and 12m to the corresponding
carboxamide was accomplished by treatment of 35% H2O2 and 1 N
NaOH in absolute ethanol to afford 13a and 13b in 60% and 71%
yields, respectively (Scheme 2).
Forcomparison, the 2-benzylamino-5-(6-methylpyridin-2-yl)-4-
([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazoles 18aed, the respective
thiazole ring isosteres of 12a, 12g, 13a, and 12n, were prepared as
shown in Scheme 3. The aldehyde 7 was treated with aniline and
diphenyl phosphite in i-PrOH at room temperature to afford
diphenyl ([1,2,4]triazolo[1,5-a]pyridin-6-yl(phenylamino)methyl)-
phosphonate (14) in 86% yield. Coupling of 14 with 6-
methylpyridine-2-carboxaldehyde in a mixture of THF and i-PrOH
(4:1) in the presence of Cs2CO3 and followed by treating with 3 N HCl
afforded the monoketone 15 in 83% yield. Bromination of 15 with
N-bromosuccinimide (NBS) in CH2Cl2 at 0 ꢀC gave the monobromo
ketone 16 in 81% yield. Condensation of 16 with thiourea in the
same reaction condition as described in Scheme 1 afforded 5-(6-
methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-
amine (17) in 67% yield. The 2-aminothiazole 17 was alkylated with
benzyl bromides (11a, 11g, and 11n) to afford the target compounds
18a, 18b, and 18d in 55%, 28%, and 49% yields, respectively. The
nitrile derivative 18b was converted to the carboxamide derivative
18c in 42% yield in the same reaction condition as mentioned in
Scheme 2.
2. Results and discussion
2.1. Chemistry
A series of 2-benzylamino-4-(6-methylpyridin-2-yl)-5-([1,2,4]-
triazolo[1,5-a]pyridin-6-yl)thiazoles 12aeab was prepared as
shown in Scheme 1. Coupling of the [1,2,4]triazolo[1,5-a]pyridine-
6-carbaldehyde (7) [19] with diphenyl (6-methylpyridin-2-
yl)(phenylamino)methylphosphonate [20] in a mixture of THF
and i-PrOH (4:1) in the presence of Cs2CO3 at room temperature
and followed by hydrolysis with 3 N HCl afforded the ketone 8
[21] in 90% yield. The ketone 8 was brominated with Br2 in 1,4-
dioxane at room temperature to give the monobromo ketone 9
in 83% yield. Condensation of 9 with thiourea in DMF at 120 ꢀC
2.2. ALK5 inhibitory activity in an enzyme assay and in a cell-based
luciferase reporter assay
To evaluate whether these potential inhibitors 12aeab, 13a, 13b,
and 18aed could inhibit ALK5, a kinase assay was performed using
the purified human ALK5 kinase domain produced in Sf9 insect
cells and casein as a substrate (Table 1). The ALK5 inhibitory activity
of the 2-(benzylamino)thiazole derivatives 12beab, 13a, and 13b
having various substituents such as F, Cl, Me, OMe, OCF3, CN, and
CONH2 in the phenyl ring was compared with that of the unsub-
stituted 2-(benzylamino)thiazole derivative 12a.
produced
4-(6-methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]-

76
M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
N
N
N
N
N
N
N
N
N
Br
O
N
N
S
N
c
b
a
NH2
N
O
CHO
N
N
7
N
8
(90%)
9 (83%)
10
(82%)
N
Br
R
R
N
S
N
11a-ab
NH
d
N
N
o
: R = o-Cl
p: R = o-Me
q: R = o-OCF3
12o:
12a: R = H (55%)
12b
R = o-Cl (67%)
a: R = H
: R = m-F
c: R = m-Cl
d: R = m-Me
12p: R = o-Me (56%)
12q: R = o-OCF3 (29%)
: R = m-F (89%)
b
12c: R = m-Cl (38%)
12d: R = m-Me (26%)
12e
: R = m-OMe (61%)
r
12r
: R = 2,3-F2
: R = 2,3-F2 (54%)
s: R = 3,4-F2
12s: R = 3,4-F2 (60%)
e
: R = m-OMe
t
12t
: R = 3,5-F2
12f: R = m-OCF3 (64%)
12g
: R = 3,5-F2 (87%)
f: R = m-OCF3
u: R = 2,3-Cl2
12u: R = 2,3-Cl2 (46%)
: R = m-CN (72%)
12h: R = p-F (95%)
12i
g
: R = m-CN
v
12v
: R = 3,4-Cl2
: R = 3,4-Cl2 (53%)
h: R = p-F
w: R = 3,5-Cl2
12w: R = 3,5-Cl2 (39%)
: R = p-Cl (57%)
12j: R = p-Me (46%)
12k
i
: R = p-Cl
x
12x
: R = 2,3-Me2
: R = 2,3-Me2 (38%)
j: R = p-Me
y: R = 3,5-Me2
12y: R = 3,5-Me2 (31%)
: R = p-OMe (41%)
12l: R = p-OCF3 (47%)
12m
k
: R = p-OMe
z
12z
: R = 2,3-(OMe)2
: R = 2,3-(OMe)2 (51%)
l: R = p-OCF3
aa: R = 3,4-(OMe)2
12aa: R = 3,4-(OMe)2 (57%)
12ab
: R = p-CN (78%)
12n: R = o-F (52%)
m:
R = p-CN
ab
: R = 3,5-(OMe)2
: R = 3,5-(OMe)2 (24%)
n: R = o-F
Scheme 1. Reagents and conditions: (a) (i) diphenyl (6-methylpyridin-2-yl)(phenylamino)methylphosphonate, Cs2CO3, THF/i-PrOH (4:1), rt, 16 h; (ii) 3 N HCl, rt, 1 h; (b) Br2,
1,4-dioxane, rt, 1 h; (c) thiourea, DMF, 120 ꢀC, 2 h; (d) Cs2CO3, DMF, 120 ꢀC, 2 h.
All of the 2-(benzylamino)thiazole derivatives except the p-OCF3
substituted compound 12l (IC50 ¼ 60.90 nM) exhibited more potent
ALK5inhibitoryactivity(IC50 ¼7.01e47.70 nM)than thecompetitor1
(IC50 ¼ 54.40 nM). Among the meta-substituted compounds 12beg,
the m-F substituted compound 12b (IC50 ¼ 7.01 nM) displayed 1.6-
fold more potent ALK5 inhibition than the unsubstituted
compound 12a (IC50 ¼ 11.30 nM). The m-CN substituted compound
(SAR) of this series of compounds clearlyshowed that introduction of
a fluoro atom at meta- or para-position in the phenyl ring was the
most beneficial in ALK5 inhibition. This SAR was extended to the di-
substituted derivatives, thus, the 3,4-F2 substituted compound 12s
(IC50
¼
7.43 nM) and 3,5-F2 substituted compound 12t
(IC50 ¼ 8.92 nM) displayed 1.5-fold and 1.3-fold more potent ALK5
inhibitory activity than 12a.
12g (IC50
¼
9.26 nM) and m-Cl substituted compound 12c
The position of hetero atoms in the central thiazole ring also
influenced the ALK5 inhibitory activity. Although, the m-CONH2
substituted compound 18c (IC50 ¼12.00 nM) showed the similarlevel
of potency to that of the respective positional isomer 13a
(IC50 ¼13.60nM), theunsubstitutedcompound18a (IC50 ¼ 24.50nM),
m-CN substituted compound 18b (IC50 ¼ 25.10 nM), and o-F
substituted compound 18d (IC50 ¼ 21.20 nM) were 2.2-fold, 2.7-fold,
and2.0-foldlessinhibitory thanthe corresponding positional isomers
12a, 12g, and 12n. Compound 12b showing the most potent ALK5
inhibitory activity in this series of compounds was 7.8-fold and 2.5-
fold more potent than the competitors 1 and 5 (IC50 ¼ 17.20 nM),
respectively.
(IC50 ¼ 10.20 nM) were slightly more potent than 12a, and the m-Me
substituted compound 12d (IC50 ¼11.20 nM) and m-OMe substituted
compound 12e (IC50 ¼ 11.40 nM) were equipotent to 12a. Among the
para-substituted compounds 12hem, the p-F substituted compound
12h (IC50
¼
8.61 nM) and p-Me substituted compound 12j
(IC50 ¼ 8.75 nM) were 1.3-fold more inhibitory in ALK5 inhibition
than 12a. Introduction of a substituent at the ortho position was
proved to be not beneficial, thus, the o-F substituted compound 12n
(IC50 ¼ 10.40 nM) only displayed the comparable ALK5 inhibitory
activity to that of the 12a. In overall, structureeactivity relationships
To examine TGF-b-induced downstream transcriptional activa-
N
tion to ALK5 signaling, cell-based luciferase activity of 12aeab, 13a,
13b, and 18aed was measured using HaCaT cells permanently
transfected with p3TP-luciferase reporter construct at a concen-
tration of 30 nM (Table 1). The p3TP-luciferase reporter construct
contains three AP-1 binding elements and the plasminogen-
activator inhibitor-1 (PAI-1) promoter [22]. Similar to kinase
assay, compound 12b displayed the most significant ALK5 inhibi-
tion (61%). Compounds 12g (60%), 12c (58%), 12h (57%), and 12t
(57%) exhibited the similar level of inhibition to that of 12a (58%),
while 1 and 5 exhibited 44% and 51% of inhibition in this assay.
R
N
N
S
N
a
NH
12g, 12m
N
13a
: R = m-CONH2 (60%)
13b: R = p-CONH2 (71%)
Scheme 2. Reagents and conditions: (a) 35% H2O2, 1 N NaOH, EtOH, rt, 4 h.

M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
77
N
N
N
N
N
N
N
N
O
N
O
H
N
N
a
c
b
N
N
PhO
CHO
Br
P
O
PhO
7
N
N
14 (86%)
16
(81%)
15 (83%)
N
N
N
N
N
N
S
N
N
e
d
NH2
NH
R
S
Br
R
N
N
11
a
: R = H
18a
17 (67%)
: R = H (55%)
: R = m-CN (28%)
g
: R = m-CN
18b
18c
18d
f
n: R = o-F
: R = m-CONH2 (42%)
: R = o-F (49%)
Scheme 3. Reagents and conditions: (a) aniline, diphenyl phosphite, i-PrOH, rt, 3 h; (b) (i) 6-methylpyridine-2-carboxaldehyde, Cs2CO3, THF/i-PrOH (4:1), rt, 12 h; (ii) 3 N HCl, rt,
1 h; (c) NBS, CH2Cl2, 0 ꢀC, 30 min; (d) thiourea, DMF, 120 ꢀC, 2 h; (e) Cs2CO3, DMF, 120 ꢀC, 2 h; (f) 35% H2O2, 1 N NaOH, EtOH, rt, 4 h.
2.3. Effect of 12b on the phosphorylation of Smad2
of >164. Compounds 12b and 12g showed selectivity indices of 504
and 408 that are much higher than those of 1 (11) and 5 (28).
To examine the effect of 12b on the phosphorylation of Smad2,
we have performed Western blot analysis of the phosphorylated
Smad2 protein in the NMuMG cells treated with either 12b or 1 in
3. Conclusion
the presence of TGF-
ylated Smad2 protein was rapidly increased by TGF-
NMuMG cells, whereas 12b treatment inhibited the TGF-
stimulated level of the phosphorylated Smad2. The comparison of
the inhibitory potency of 12b and 1 showed that 12b resulted in the
more potent inhibition than 1.
b
1. As shown in Fig. 2, the level of phosphor-
1 treatment in
1-
Compound 12g efficiently inhibited TGF-b1-induced Smad
b
signaling, epithelial-to-mesenchymal transition (EMT), cell
motility, and breast tumor metastasis to the lung in xenografted
Balb/c mice, demonstrating that this series of compounds has
a high therapeutic potential for cancer metastasis and fibrosis [26].
In this report, a series of 2-benzylamino-4(5)-(6-methylpyridin-
2-yl)-5(4)-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazoles has been
prepared 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 the [1,2,4]triazolo[1,5-a]pyridin-
6-yl moiety and benzyl moiety at the 4(5)- and 2-positions of the
thiazole ring, respectively, significantly increased both ALK5
inhibitory activity and selectivity. Introduction of fluoro atom at
meta- or para-position in the phenyl ring also increased ALK5
inhibitory activity. The most potent compound 12b inhibited ALK5
phosphorylation with IC50 value of 7.01 nM and showed 61% inhi-
bition at 30 nM in a luciferase reporter assay using HaCaT cells
permanently transfected with p3TP-luc reporter construct. The
b
2.4. Binding mode of 18a in the ALK5 active site
To rationalize the SAR shown in Table 1, we examined the
binding modes of three representative ligands including 12b, 12g,
and 18b. Docking analyses were conducted using the recently re-
ported X-ray structure of the kinase domain of human ALK5 in
complexed with quinazoline ALK5 inhibitor [23] (PDB id:
3GXL.pdb). As shown in Fig. 3, the [1,2,4]triazolo[1,5-a]pyridine
ring binds to the hinge region with forming a hydrogen bond (H-
bond) with the backbone NH of His 283. The methylpyridine ring
occupies the hydrophobic pocket, and pyridine nitrogen forms a H-
bond with Ser 280 which is critical for selectivity of ligands toward
selectivity index of 12b against p38
a MAP kinase is 504 that is
ALK5 over p38
a
MAP kinase where the corresponding residue is
much higher than those of 1 (11) and 5 (28).
Thr 106 [24]. For 12b and 12g, the nitrogen atom of thiazole core
forms a H-bond with Lys 232, a gatekeeper residue for the speci-
ficity pocket of ALK5. Furthermore, the amino group in the linker
forms a H-bond with side-chain carboxyl group of Asp 351, as we
expected. However, the binding conformation of 18b (a thiazole
ring isostere of 12g), is a little changed, and these two H-bonds with
Lys 232 and Asp 351 are missing (Fig. 3C).
4. Experimental section
4.1. Chemistry
1H and 13C NMR spectra were recorded on a Varian Unity 400
spectrophotometer. The chemical shifts are reported in parts per
million (ppm). For 1H NMR spectra, CDCl3 was used as solvent, and
it served as the internal standard at
CDCl3, CDCl3/CD3OD, or DMSO-d6 was used as solvent, and it served
as the internal standard at 77.23 (CDCl3, CDCl3/CD3OD) or 39.50
2.5. p38
a
MAP kinase assay
d
7.26. For 13C NMR spectra,
The kinase domain of p38
a
MAP kinase is known to be one of
d
the most homologous to that of ALK5 [25], therefore, it was chosen
to examine the selectivity profile of this series of compounds. All
(DMSO-d6). Infrared spectra were recorded on a FT-infrared spec-
trometer (Bio-Rad). All melting points were taken in Pyrex capil-
laries using an electrothermal digital melting point apparatus
(Buchi) and are not correct. High resolution mass spectra electro
the target compounds in this series did not inhibit p38
a MAP kinase
efficiently, showing IC50 values of >3280 nM and selectivity indices

78
M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
Table 1
Inhibitory activity of 2-benzylamino-4(5)-(6-methylpyridin-2-yl)-5(4)-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazoles 12aeab, 13a, 13b, and 18aed on ALK5 and p38
a
.
N
N
R
R
N
N
S
N
N
S
N
N
NH
NH
N
N
12a-ab, 13a, 13b
18a-d
Fig. 2. 12b inhibits TGF-
b-induced Smad2 phosphorylation. NMuMG cells were treated
Compound
Mock
R
IC50 (nM)
ALK5a
Selectivity
indexc
p3TP-luciferase
activityd,e
(% control)
with TGF- 1 (2 ng/mL) in the presence of 12b or 1 for 2 h. Total protein extracts from
b
p38ab
treated cell were immunoblotted with anti-Smad2, anti-phospho-Smad2, and anti-
GAPDH antibodies.
5 ꢃ 0
100 ꢃ 5
42 ꢃ 8
39 ꢃ 4
42 ꢃ 1
62 ꢃ 2
44 ꢃ 4
81 ꢃ 1
40 ꢃ 2
43 ꢃ 3
48 ꢃ 0
58 ꢃ 4
56 ꢃ 6
90 ꢃ 12
59 ꢃ 3
51 ꢃ 1
49 ꢃ 1
56 ꢃ 4
80 ꢃ 5
45 ꢃ 2
49 ꢃ 1
43 ꢃ 3
66 ꢃ 6
75 ꢃ 5
65 ꢃ 4
72 ꢃ 6
69 ꢃ 3
64 ꢃ 3
75 ꢃ 8
59 ꢃ 4
66 ꢃ 4
75 ꢃ 6
84 ꢃ 5
86 ꢃ 6
46 ꢃ 2
82 ꢃ 5
56 ꢃ 2
49 ꢃ 0
TGF-
12a
12b
12c
12d
12e
12f
12g
12h
12i
b
H
m-F
m-Cl
m-Me
m-OMe
m-OCF3
m-CN
p-F
p-Cl
11.30
7.01
5530
3530
3280
5370
>10,000
7390
489
504
322
479
>877
383
408
1069
>633
>1143
>412
>164
>325
>962
378
>592
>279
245
482
428
>292
>457
>662
>452
>455
>230
>210
484
455
>389
360
>398
>833
>472
11
Tokyo, Japan) with a solvent system consisting of an isocratic
solvent. The solvent contained 20e35% acetonitrile in distilled
water containing 0.1% trifluoroacetic acid and was delivered at
0.5 mL/min over 25 min. The column was maintained at 40 ꢀC
with a column oven (G1316A). All compounds were detected at
230 nm with a G1315A diode array detector (DAD). The HPLC
system was controlled by a ChemStation Software (Agilent). The
purity of the each compound was calculated from a surface inte-
gral of detected peaks and is described as percent (%). All
compounds were found to be >95% pure except for 12q with the
purity of 94.31%.
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 Chemical or
TCI Laboratory chemicals.
10.20
11.20
11.40
19.30
9.26
8.61
15.80
8.75
24.30
60.90
30.80
10.40
15.50
16.90
35.90
29.40
7.43
3780
9200
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
5860
>10,000
>10,000
7200
12j
12k
12l
p-Me
p-OMe
p-OCF3
p-CN
o-F
o-Cl
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12w
12x
12y
12z
12aa
12ab
13a
13b
18a
18b
18c
18d
1
o-Me
o-OCF3
2,3-F2
3,4-F2
3,5-F2
2,3-Cl2
3,4-Cl2
3,5-Cl2
2,3-Me2
3,5-Me2
2,3-(OMe)2
3,4-(OMe)2
3,5-(OMe)2
m-CONH2
p-CONH2
H
3580
3820
8.92
34.30
21.90
15.10
22.10
22.00
43.50
47.70
16.50
13.60
25.70
24.50
25.10
12.00
21.20
54.40
17.20
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
7990
6190
>10,000
8820
>10,000
>10,000
>10,000
594
4.1.1. 2-Bromo-1-(6-methylpyridin-2-yl)-2-([1,2,4]triazolo[1,5-a]-
pyridin-6-yl)ethanone (9)
To a stirred solution of 1-(6-methylpyridin-2-yl)-2-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)ethanone (8) (2.86 g, 11.34 mmol) in 1,4-
dioxane, bromine (0.64 mL, 12.47 mmol) was added. The mixture
was stirred at room temperature for 1 h and neutralized with
aqueous NaHCO3 solution. The reaction mixture was extracted with
EtOAc (3 ꢁ 100 mL), and the EtOAc solution was washed with water
(50 mL) and brine (50 mL). The 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 EtOAc/Hex (1:2) as eluent to afford the titled compound as
m-CN
m-CONH2
o-F
5
480
28
a
ALK5 was expressed in Sf9 insect cells as human recombinant GST-fusion
protein by means of the baculovirus expression system. A Proprietary radioiso-
topic protein kinase assay (33PanQinaseÒ Activity Assay) was performed at ProQi-
a brown syrup. Yield 83%; 1H NMR (400 MHz, CDCl3):
d 2.65 (s, 3H),
7.31 (s, 1H), 7.38 (d, 1H, J ¼ 7.6 Hz), 7.76 (t, 1H, overlapped,
J ¼ 7.8 Hz), 7.77 (dd, 1H, overlapped, J ¼ 9.2, 0.8 Hz), 7.88 (dd, 1H,
J ¼ 9.2, 1.6 Hz), 7.95 (br d, 1H, overlapped, J ¼ 8.0 Hz), 8.35 (s, 1H),
nase 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 NieNTHeagarose (Qiagen). A Proprietary
8.89 (dd, 1H, J ¼ 1.6, 0.8 Hz); IR (KBr) 3001, 1710, 1644, 1533 cmꢂ1
;
radioisotopic protein kinase assay (33PanQinaseÒ Activity Assay) was performed at
HRMS-ESI m/z [M þ H]þ calcd. for C14H12BrN4O: 331.0189, found
ProQinase GmbH (Freiburg, Germany) using ATF2 as a substrate.
c
IC50 of p38
a
/IC50 of ALK5.
331.0174.
d
e
Luciferase activity was determined at a concentration of 30 nM of inhibitor.
Activity is given as the mean ꢃ SD of three independent experiments run in
4.1.2. Diphenyl ([1,2,4]triazolo[1,5-a]pyridin-6-yl(phenylamino)-
methyl)phosphonate (14)
triplicate relative to control incubations with DMSO vehicle.
To a mixture of 7 (0.50 g, 3.40 mmol) and aniline (0.37 mL,
4.08 mmol) in i-PrOH (10 mL), diphenyl phosphite (1.04 mL,
5.44 mmol) was added. The mixture was stirred at room temper-
ature for 3 h and then evaporated to dryness under reduced pres-
sure. The residue was purified by MPLC on silica gel using EtOAc/
Hex (1:1) as eluent to afford the titled compound as a white solid.
spray ionization (HRMS-ESI) was obtained on an Agilent Technol-
ogies 6220 TOF LC/MS spectrometer.
The HPLC analyses were accomplished with an Agilent 1100
series HPLC system (Agilent, Germany) consisting of a solvent
degasser (G1322A degasser), a quaternary pump (G1311A liquid
chromatography), and an auto sampler injector (G1313A). Sepa-
rations were carried out using a reversed-phase C18 analytical
Yield 86%; 1H NMR (400 MHz, CDCl3):
d
4.96 (dd, 1H, J ¼ 10.4,
7.6 Hz), 5.22 (dd, 1H, J ¼ 24.8, 7.6 Hz), 6.63e6.66 (m, 2H), 6.78e6.82
column (Capcell-pak C18 MG120 5
m
m, 3.0 ꢁ 250 mm) (Shiseido,
(m, 1H), 6.99e7.02 (m, 2H), 7.08e7.11 (m, 2H), 7.13e7.19 (m, 4H),

M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
79
4.1.3. 2-(6-Methylpyridin-2-yl)-1-([1,2,4]triazolo[1,5-a]pyridin-6-
yl)ethanone (15)
To a stirred solution of 6-methylpyridine-2-carboxaldehyde
(3.18 g, 26.31 mmol) in a mixture of THF (120 mL) and i-PrOH
(30 mL), 14 (12.00 g, 26.31 mmol) and Cs2CO3 (11.11 g, 34.21 mmol)
were added. The mixture was stirred at room temperature for 16 h,
and to it, 3 N HCl (40 mL) was added dropwise over a period of
10 min. After 1 h, the reaction mixture was diluted with MTBE
(100 mL). The aqueous layer was separated, and the organic layer
was extracted with 1 N HCl (3 ꢁ 250 mL). The combined aqueous
layer was neutralized with 50% aqueous NaOH solution (pH 7e8).
The precipitates were collected by filtration using a Buchner fun-
nel and washed thoroughly with water (4 ꢁ 250 mL). The yellow
solid was dried in air for 48 h and dried for an additional 24 h in
a vacuum oven over P2O5 to give the titled compound as a yellow
sold. Yield 83%; 1H NMR (400 MHz, CDCl3): (enol form, 82%);
d 2.56
(s, 3H), 6.03 (s, 1H), 6.82 (d, 1H, J ¼ 7.6 Hz), 6.91 (d, 1H, J ¼ 8.0 Hz),
7.55 (t, 1H, overlapped, J ¼ 7.8 Hz), 7.74 (dd, 1H, J ¼ 9.6, 0.8 Hz), 7.94
(dd, 1H, J ¼ 9.6, 1.6 Hz), 8.37 (s, 1H), 9.13 (dd, 1H, J ¼ 1.6, 0.8 Hz),
16.38 (br s, 1H); IR (KBr) 3112, 3086, 2919, 1645 cmꢂ1 1H NMR
;
(400 MHz, CDCl3): (keto form, 18%); d 2.54 (s, 3H), 4.45 (s, 2H), 7.05
(d, 1H, J ¼ 7.6 Hz), 7.13 (d, 1H, J ¼ 7.6 Hz), 7.55 (t, 1H, overlapped,
J ¼ 7.6 Hz), 7.77 (d, 1H, J ¼ 9.6, 0.8 Hz), 8.16 (dd, 1H, J ¼ 9.6, 1.6 Hz),
8.45 (s, 1H), 9.59 (dd, 1H, J ¼ 1.6, 0.8 Hz); HRMS-ESI m/z [M þ H]þ
calcd. for C14H13N4O: 253.1084, found 253.1096.
4.1.4. 2-Bromo-2-(6-methylpyridin-2-yl)-1-([1,2,4]triazolo[1,5-a]-
pyridin-6-yl)ethanone (16)
To a stirred solution of 15 (5.40 g, 22.36 mmol) in CH2Cl2
(250 mL) at 0 ꢀC, N-bromosuccinimide (4.36 g, 24.54 mmol) was
added. After 30 min, the reaction mixture was quenched with 5%
aqueous Na2S2O3 solution and extracted with CHCl3 (4 ꢁ 150 mL).
The organic layer was washed with water (100 mL) and brine
(100 mL), dried over Na2SO4, filtered, and evaporated to dryness
under reduced pressure. The residue was purified by MPLC on silica
gel using EtOAc/Hex (2:1) as eluent to afford the titled compound as
yellow syrup. Yield 81%; 1H NMR (400 MHz, CDCl3):
d 2.52 (s, 3H),
6.33 (s,1H), 7.11 (d, 1H, J ¼ 7.6 Hz), 7.54 (d,1H, J ¼ 8.0 Hz), 7.67 (t,1H,
J ¼ 7.8 Hz), 7.78 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.12 (dd, 1H, J ¼ 9.2, 2.0 Hz),
8.45 (s, 1H), 9.49 (dd, 1H, J ¼ 2.0, 0.8 Hz); IR (KBr) 3075, 1708,
1631 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for C14H12BrN4O:
331.0189, found 331.0184.
4.1.5. General procedure for the preparation of the 4(5)-(6-methylpy-
ridin-2-yl)-5(4)-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-amines
10 and 17
A mixture of 9 or 16 (2.52 g, 7.61 mmol) and thiourea (1.22 g,
15.98 mmol) in DMF (20 mL) was heated at 120 ꢀC for 2 h and then
cooled to 0 ꢀC. To the mixture, cold water (100 mL) was added, and
the resulting precipitates were collected by filtration and washed
thoroughly with water to afford the titled compound as a brown
solid.
4.1.5.1. 4-(6-Methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyridin-6-
yl)thiazol-2-amine (10). Yield 82%; mp 279.0 ꢀC; 1H NMR (400 MHz,
Fig. 3. The top-scored docking poses of 12b (A), 12g (B), and 18b (C) generated by
Surflex-Dock program. The key amino acid residues in the ALK5 binding site are rep-
resented in line form. Yellow dotted lines are hydrogen bonding interactions (<2.5 Å).
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
CDCl3/CD3OD):
d
2.27 (s, 3H), 6.98 (d, 1H, J ¼ 7.6 Hz), 7.26 (d, 1H,
J ¼ 7.6 Hz), 7.36 (dd, 1H, J ¼ 9.2, 1.6 Hz), 7.47 (t, 1H, overlapped,
J ¼ 7.6 Hz), 7.49 (dd,1H, overlapped, J ¼ 9.2, 0.8 Hz), 8.18 (s,1H), 8.58
(dd, 1H, J ¼ 1.6, 0.8 Hz); IR (KBr) 3295, 3147, 2924, 1544 cmꢂ1
;
HRMS-ESI m/z [M þ H]þ calcd. for C15H13N6S: 309.0917, found
309.0913.
7.22e7.30 (m, 4H), 7.73 (d, 1H, J ¼ 9.2 Hz), 7.77 (td, 1H, J ¼ 9.2,
1.6 Hz), 8.32 (s, 1H), 8.77e8.78 (m, 1H); IR (KBr) 3313, 3054, 1597,
1492 cmꢂ1 HRMS-ESI m/z [M þ H]þ calcd. for C25H22N4O3P:
457.1424, found 457.1432.
4.1.5.2. 5-(6-Methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-
yl)thiazol-2-amine (17). Yield 67%; mp 275.3 ꢀC; 1H NMR (400 MHz,
CDCl3):
d 2.54 (s, 3H), 5.42 (br s, 2H), 6.98 (d, 1H, overlapped,

80
M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
J ¼ 7.6 Hz), 7.01 (d,1H, overlapped, J ¼ 7.6 Hz), 7.40 (t,1H, J ¼ 7.6 Hz),
7.68 (dd, 1H, J ¼ 9.6, 1.6 Hz), 7.73 (dd, 1H, J ¼ 9.2, 1.2 Hz), 8.36 (s, 1H),
(t, 1H, J ¼ 7.8 Hz), 7.62 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.73 (dd,
1H, J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3)
d 21.62, 24.42, 50.13,
8.90 (dd, 1H, J ¼ 1.6, 1.2 Hz); IR (KBr) 3316, 3134, 2923, 1516 cmꢂ1
;
115.46, 117.63, 120.28, 120.71, 122.90, 124.92, 128.49, 128.54, 128.96,
129.01, 133.13, 136.76, 137.15, 138.85, 145.79, 149.69, 151.79, 154.48,
158.34, 167.83; IR (KBr) 3225, 2981, 1553 cmꢂ1; HRMS-ESI m/z
[M þ H]þ calcd. for C23H21N6S: 413.1543, found 413.1538.
HRMS-ESI m/z [M þ H]þ calcd. for C15H13N6S: 309.0917, found
309.0913.
4.1.6. General procedure for the preparation of the 2-benzylamino-
4(5)-(6-methylpyridin-2-yl)-5(4)-([1,2,4]triazolo[1,5-a]pyridin-6-
yl)-thiazoles 12aeab, 18a, 18b, and 18d
4.1.6.5. N-(3-Methoxybenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12e). Yield 61%; mp
166.2 ꢀC; Purity by HPLC: 99.38% (30% acetonitrile); 1H NMR
A
stirred solution of 2-aminothiazole 10 or 17 (0.08 g,
0.27 mmol), an appropriately substituted benzyl bromide (11aeab)
(0.03 mL, 0.29 mmol), and Cs2CO3 (0.11 g, 0.35 mmol) in DMF (5 mL)
was heated at 120 ꢀC for 1 h and then cooled to room temperature.
The reaction mixture was diluted with water (25 mL) and extracted
with CHCl3 (3 ꢁ 60 mL). 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 (1:40) as eluent to afford the titled compounds
as a solid.
(400 MHz, CDCl3):
d
2.38 (s, 3H), 3.81 (s, 3H), 4.51 (d, 2H, J ¼ 4.4 Hz),
5.84 (br s, 1H), 6.86 (ddd, 1H, J ¼ 8.0, 2.4, 0.8 Hz), 6.95 (t, 1H,
J ¼ 2.0 Hz), 6.98 (ddd, 1H, J ¼ 7.6, 1.6, 1.2 Hz), 7.02e7.05 (m, 1H), 7.29
(t,1H, J ¼ 8.0 Hz), 7.48e7.50 (m, 2H), 7.54 (t, 1H, J ¼ 7.6 Hz), 7.61 (dd,
1H, J ¼ 9.2, 0.8 Hz), 8.31 (s, 1H), 8.73 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13C
NMR (100 MHz, CDCl3)
d 24.40, 49.92, 55.45, 113.36, 113.44, 115.31,
117.87, 119.98, 120.52, 120.77, 122.72, 128.36, 130.04, 133.20, 137.04,
138.81, 146.56, 149.61, 152.33, 154.39, 158.19, 160.17, 167.85; IR (KBr)
3220, 2928, 1552 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for
C23H21N6OS: 429.1492, found 429.1504.
4.1.6.1. N-Benzyl-4-(6-methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]-
pyridin-6-yl)thiazol-2-amine (12a). Yield 55%; mp 237.9 ꢀC; Purity
4.1.6.6. 4-(6-Methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyridin-6-
yl)-N-(3-(trifluoromethoxy)benzyl)thiazol-2-amine (12f). Yield 64%;
mp 145.7 ꢀC; Purity by HPLC: 97.18% (35% acetonitrile); 1H NMR
byHPLC: 96.65% (30%acetonitrile); 1H NMR (400 MHz, CDCl3):
d2.37
(s, 3H), 4.52 (s, 2H), 6.20 (br s, 1H), 7.03 (d, 1H, J ¼ 7.6), 7.31e7.41
(m, 5H), 7.46e7.49 (m, 2H), 7.53 (t, 1H, J ¼ 7.6 Hz), 7.60 (dd, 1H,
J ¼ 9.2, 0.8 Hz), 8.31 (s, 1H), 8.72 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13C NMR
(400 MHz, CDCl3):
d
2.37 (s, 3H), 4.59 (d, 2H, J ¼ 5.2 Hz), 5.67 (t, 1H,
J ¼ 5.2 Hz), 7.04 (ddd, 1H, J ¼ 7.6, 1.2, 0.4 Hz), 7.18 (br d, 1H,
J ¼ 7.6 Hz), 7.28 (br s, 1H), 7.36 (br d, 1H, J ¼ 8.0 Hz), 7.41 (t, 1H,
J ¼ 7.8 Hz), 7.49e7.51 (m, 2H), 7.55 (t, 1H, J ¼ 7.6 Hz), 7.62 (dd, 1H,
J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.74 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13C NMR
(100 MHz, CDCl3)
d 24.45, 50.04, 115.36, 117.92, 120.49, 120.77,
122.77,127.84 (2C),128.18,128.42,129.06 (2C),133.21,137.07,137.15,
146.53, 149.66, 152.28, 154.44, 158.26, 167.71; IR (KBr) 3221, 2924,
1553 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for C22H19N6S: 399.1386,
found 399.1389.
(100 MHz, DMSO-d6)
d 23.57, 46.79, 114.23, 117.24, 119.51, 119.92,
120.05, 120.13 (q, J ¼ 254.9 Hz), 120.26, 122.01, 126.65, 128.57,
130.36, 133.69, 137.03, 142.17, 145.08, 148.49, 148.90, 152.35, 154.22,
156.45, 165.86; IR (KBr) 3220, 3012, 1553, 1258 cmꢂ1; HRMS-ESI m/
z [M ꢂ H]þ calcd. for C23H16F3N6OS: 481.1064, found 481.1069.
4.1.6.2. N-(3-Fluorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12b). Yield 89%; mp
171.2 ꢀC; Purity by HPLC: 98.56% (30% acetonitrile); 1H NMR
(400 MHz, CDCl3):
d
2.35(s, 3H), 4.48 (s, 2H), 6.70(brs,1H), 6.95e6.98
4.1.6.7. 3-(((4-(6-Methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyr-
idin-6-yl)thiazol-2-yl)amino)methyl)benzonitrile (12g). Yield 72%;
mp 201.6 ꢀC; Purity by HPLC: 99.77% (30% acetonitrile); 1H NMR
(m, 1H), 7.01 (dd, 1H, J ¼ 7.6, 0.8 Hz), 7.05e7.08 (m, 1H), 7.12e7.15
(m, 1H), 7.28e7.33 (m, 1H), 7.45 (dd, 1H, J ¼ 9.2, 1.6 Hz), 7.46 (d, 1H,
J ¼ 1.6 Hz), 7.49 (t, 1H, overlapped, J ¼ 7.6 Hz), 7.59 (dd, 1H, J ¼ 9.2,
0.8 Hz), 8.29 (s, 1H), 8.70 (dd,1H, J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz,
(400 MHz, CDCl3): d 2.39 (s, 3H), 4.61 (s, 2H), 6.57 (br s, 1H), 7.06 (d,
1H, J ¼ 7.6 Hz), 7.46 (d, 1H, overlapped, J ¼ 8.0 Hz), 7.47e7.51 (m,
2H), 7.55 (t, 1H, J ¼ 7.8 Hz), 7.61 (dt, 1H, overlapped, J ¼ 7.6, 1.4 Hz),
7.63 (dd, 1H, J ¼ 9.2, 0.8 Hz), 7.66 (dd, 1H, J ¼ 8.0, 0.8 Hz), 7.71 (br s,
1H), 8.32 (s, 1H), 8.73 (dd, J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz,
CDCl3)
d
24.40, 49.30 (d, J ¼ 1.5 Hz), 114.59 (d, J ¼ 21.2 Hz), 114.98 (d,
J ¼ 21.2 Hz), 115.35, 120.43, 120.81, 122.79, 123.22 (d, J ¼ 2.9 Hz),
128.39, 129.82, 130.54 (d, J ¼ 8.1 Hz), 133.16, 137.07, 140.01
(d, J ¼ 6.6 Hz), 146.67, 149.64, 152.31, 154.42, 158.23, 163.25
(d, J ¼ 245.4 Hz), 167.67; IR (KBr) 3215, 3000, 1552 cmꢂ1; HRMS-ESI
m/z [M þ H]þ calcd. for C22H18FN6S: 417.1292, found 417.1284.
CDCl3)
d 24.35, 48.77, 113.04, 115.40, 118.37, 118.73, 120.28, 120.86,
122.87, 128.41, 129.76, 131.12, 131.62, 132.03, 133.10, 137.17, 139.36,
146.70, 149.62, 152.25, 154.41, 158.20, 167.39; IR (KBr) 3222, 3012,
2230, 1550 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for C23H18N7S:
424.1339, found 424.1341.
4.1.6.3. N-(3-Chlorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12c). Yield 38%; mp
191.3 ꢀC; Purity by HPLC: 99.23% (30% acetonitrile); 1H NMR
4.1.6.8. N-(4-Fluorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12h). Yield 95%; mp
205.0 ꢀC; Purity by HPLC: 98.88% (30% acetonitrile); 1H NMR
(400 MHz, CDCl3): d 2.37 (s, 3H), 4.53 (s, 2H), 5.94 (br s, 1H), 7.04 (d,
1H, J ¼ 7.6 Hz), 7.28e7.30 (m, 3H), 7.40 (br s, 1H), 7.48e7.50 (m, 2H),
7.54 (t, 1H, J ¼ 7.8 Hz), 7.61 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.73
(400 MHz, CDCl3):
d
2.38 (s, 3H), 4.52 (d, 2H, J ¼ 5.0 Hz), 5.78 (br s,
(dd, 1H, J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3/CD3OD)
d
23.55,
1H), 7.03e7.09 (m, 3H), 7.36e7.40 (m, 2H), 7.47e7.50 (m, 2H), 7.54
(t, 1H, J ¼ 7.6 Hz), 7.61 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.73 (dd,
49.12, 115.70, 118.18, 119.97, 121.03, 123.60, 125.90, 127.81, 128.21,
128.45,130.34,133.04,134.80,138.42,139.06,149.48,150.50,154.16,
155.13, 157.92, 168.17; IR (KBr) 3221, 2924, 1553 cmꢂ1; HRMS-ESI
m/z [M þ H]þ calcd. for C22H18ClN6S: 433.0997, found 433.0994.
1H, J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3/CD3OD)
d 24.07,
48.85, 115.05, 115.68 (2C, d, J ¼ 21.8 Hz), 117.38, 120.78, 120.84,
122.84, 128.09, 129.42 (2C, d, J ¼ 8.4 Hz), 133.07 (d, J ¼ 2.8 Hz),
133.39, 137.20, 146.62, 149.24, 152.19, 153.87, 158.10, 162.44 (d,
J ¼ 244.2 Hz),167.96; IR (KBr) 3217, 3001, 1553 cmꢂ1; HRMS-ESI m/z
[M þ H]þ calcd. for C22H18FN6S: 417.1292, found 417.1287.
4.1.6.4. N-(3-Methylbenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12d). Yield 26%; mp
191.3 ꢀC; Purity by HPLC: 98.53% (30% acetonitrile); 1H NMR
(400 MHz, CDCl3):
d
2.37 (s, 3H), 2.39 (s, 3H), 4.51 (d, 2H, J ¼ 5.6 Hz),
4.1.6.9. N-(4-Chlorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12i). Yield 57%; mp
248.3 ꢀC; Purity by HPLC: 99.43% (30% acetonitrile); 1H NMR
5.78 (br s, 1H), 7.04 (dd, 1H, J ¼ 7.6, 0.8 Hz), 7.14 (br d, 1H, J ¼ 7.6 Hz),
7.19e7.22 (m, 2H), 7.27 (t, 1H, J ¼ 7.4 Hz), 7.48e7.51 (m, 2H), 7.55

M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
81
(400 MHz, CDCl3):
d
2.37 (s, 3H), 4.50 (s, 2H), 6.19 (br s, 1H), 7.03 (br
(400 MHz, CDCl3): d 2.38 (s, 3H), 4.61 (s, 2H), 6.49 (brs,1H), 7.04e7.12
d, 1H, J ¼ 7.6 Hz), 7.33 (br s, 4H), 7.46 (d, 1H, J ¼ 8.0 Hz), 7.47 (dd, 1H,
J ¼ 9.2, 2.0 Hz), 7.52 (t, 1H, J ¼ 7.8 Hz), 7.61 (dd, 1H, J ¼ 9.2, 0.8 Hz),
8.31 (s,1H), 8.71 (dd,1H, J ¼ 2.0, 0.8 Hz); 13C NMR (100 MHz, DMSO-
(m, 2H), 7.15 (td, 1H, J ¼ 7.6 Hz, 1.2 Hz), 7.29e7.34 (m, 1H), 7.45e7.52
(m, 3H), 7.55 (t,1H, J ¼ 7.6Hz), 7.63 (d,1H, J ¼ 9.2 Hz), 8.33(s,1H), 8.75
(s, 1H); 13C NMR (100 MHz, CDCl3)
d 24.13, 44.62, 116.07
d6)
d
23.45, 46.72, 114.03, 117.01, 119.92, 120.13, 121.78, 128.10 (2C),
(d, J ¼ 20.6 Hz),116.99,119.88,121.28 (d, J ¼ 25.9 Hz),124.06,124.84,
125.20 (d, J ¼ 3.8 Hz), 129.73, 130.05 (d, J ¼ 3.1 Hz), 130.94, 131.02,
132.03, 138.26, 145.71, 150.28, 155.22, 155.68, 159.46, 161.02 (d,
J ¼ 246.5 Hz),168.55; IR (KBr) 3226, 2927,1554 cmꢂ1; HRMS-ESI m/z
[M þ H]þ calcd. for C22H18FN6S: 417.1292, found 417.1297.
128.22, 129.19 (2C), 131.70, 133.32, 136.83, 137.82, 144.95, 148.75,
152.21, 153.83, 156.23, 165.84; IR (KBr) 3229, 2928, 1553 cmꢂ1
;
HRMS-ESI m/z [M þ H]þ calcd. for C22H18ClN6S: 433.0997, found
433.0993.
4.1.6.10. N-(4-Methylbenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12j). Yield 46%; mp
238.3 ꢀC; Purity by HPLC: 98.93% (30% acetonitrile); 1H NMR
4.1.6.15. N-(2-Chlorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12o). Yield 67%; mp
174.4 ꢀC; Purity by HPLC: 98.18% (30% acetonitrile); 1H NMR
(400 MHz, CDCl3):
d
2.36 (s, 3H), 2.38 (s, 3H), 4.50 (d, 2H, J ¼ 5.2 Hz),
(400 MHz, CDCl3):
d
2.36 (s, 3H), 4.64 (d, 2H, J ¼ 4.4 Hz), 6.01 (br s,
5.78 (br s, 1H), 7.04 (dd, 1H, J ¼ 7.6, 0.8 Hz), 7.19 (d, 2H, J ¼ 8.0 Hz),
7.30 (d, 2H, J ¼ 8.0 Hz), 7.48e7.51 (m, 2H), 7.55 (t, 1H, J ¼ 7.8 Hz),
7.61 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.73 (dd, 1H, J ¼ 1.6,
1H), 7.03 (dd, 1H, J ¼ 7.6, 0.8 Hz), 7.25e7.30 (m, 2H), 7.39e7.42 (m,
1H), 7.47e7.52 (m, 3H), 7.54 (t, 1H, J ¼ 7.6 Hz), 7.61 (dd, 1H, J ¼ 9.2,
0.8 Hz), 8.31 (s, 1H), 8.73 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13C NMR
0.8 Hz); 13C NMR (100 MHz, CDCl3/CD3OD)
d
20.99, 23.86, 49.28,
(100 MHz, CDCl3/CD3OD) d 23.89, 47.21, 114.92, 117.32, 120.86,
114.86, 116.96, 120.86, 120.95, 122.81, 127.60 (2C), 127.91, 129.38
(2C), 133.43, 134.07, 137.22, 137.53, 146.53, 149.03, 152.15, 153.62,
157.99, 168.30; IR (KBr) 3221, 2923, 1551 cmꢂ1; HRMS-ESI m/z
[M þ H]þ calcd. for C23H21N6S: 413.1543, found 413.1543.
120.90, 122.86, 127.07, 128.03, 129.16, 129.41, 127.73, 133.46, 133.62,
134.75, 137.29, 146.61, 149.11, 152.13, 153.70, 157.98, 167.96; IR (KBr)
3221, 2968, 1553 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for
C
22H18ClN6S: 433.0997, found 433.1009.
4.1.6.11. N-(4-Methoxybenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]-
triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12k). Yield 41%; mp
219.0 ꢀC; Purity by HPLC: 98.30% (30% acetonitrile); 1H NMR
4.1.6.16. N-(2-Methylbenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12p). Yield 56%, mp
159.8 ꢀC; Purity by HPLC: 99.41% (30% acetonitrile); 1H NMR
(400 MHz, CDCl3):
d
2.38 (s, 3H), 3.82 (s, 3H), 4.47 (d, 2H, J ¼ 4.8 Hz),
(400 MHz, CDCl3): d 2.38 (s, 3H), 2.41 (s, 3H), 4.52 (d, 2H,
5.69 (br s, 1H), 6.91 (m, 2H), 7.04 (d, 1H, J ¼ 7.6 Hz), 7.33 (m, 2H),
7.48e7.50 (m, 2H), 7.54 (t, 1H, J ¼ 7.6 Hz), 7.61 (dd, 1H, J ¼ 9.2,
0.8 Hz), 8.32 (s, 1H), 8.73 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13C NMR
J ¼ 5.2 Hz), 5.58 (br s, 1H), 7.04 (dd, 1H, J ¼ 7.6, 0.8 Hz), 7.20e7.26
(m, 3H), 7.38 (dd, 1H, J ¼ 7.6, 1.6 Hz), 7.49e7.51 (m, 2H), 7.55 (t, 1H,
J ¼ 7.6 Hz), 7.62 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.74 (dd, 1H,
(100 MHz, CDCl3/CD3OD)
d
23.93, 49.03, 55.29, 114.13 (2C), 114.90,
J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3)
d 19.24, 24.40, 48.07,
115.23, 117.75, 120.65, 120.78, 122.63, 126.45, 128.25, 128.30,
128.43, 130.83, 133.23, 134.99, 136.60, 136.98, 146.89, 149.57,
117.03, 120.84, 120.92, 122.81, 127.95, 129.05 (2C), 129.18, 133.43,
137.20,146.52,149.08,152.17,153.68,158.02,159.23,168.18; IR (KBr)
3220, 2927, 1551, cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for
152.55, 154.34, 158.12, 167.55; IR (KBr) 3228, 2982, 1553 cmꢂ1
;
C
23H21N6OS: 429.1492, found 429.1510.
HRMS-ESI m/z [M þ H]þ calcd. for C23H21N6S: 413.1543, found
413.1541.
4.1.6.12. 4-(6-Methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyridin-6-
yl)-N-(4-(trifluoromethoxy)benzyl)thiazol-2-amine (12l). Yield 47%;
mp 177.0 ꢀC; Purity by HPLC: 99.29% (35% acetonitrile); 1H NMR
4.1.6.17. 4-(6-Methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyridin-6-
yl)-N-(2-(trifluoromethoxy)benzyl)thiazol-2-amine (12q). Yield 29%;
mp 185.4 ꢀC; Purity by HPLC: 94.31% (35% acetonitrile); 1H NMR
(400 MHz, CDCl3):
d
2.45 (s, 3H), 4.60 (d, 2H, J ¼ 3.6 Hz), 7.11 (d, 1H,
J ¼ 7.6 Hz), 7.24 (m, 2H), 7.43 (br d, 1H, J ¼ 7.6 Hz), 7.46 (m, 2H), 7.50
(dd, 1H, J ¼ 9.2, 1.6 Hz), 7.57 (t, 1H, J ¼ 7.6 Hz), 7.69 (dd, 1H, J ¼ 9.2,
0.8 Hz), 8.37 (s, 1H), 8.77 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13C NMR
(400 MHz, CDCl3):
d
2.37 (s, 3H), 4.65 (d, 2H, J ¼ 6.4 Hz), 5.68 (br s,
1H), 7.04 (dd, 1H, J ¼ 7.6, 1.0 Hz), 7.28e7.39 (m, 3H), 7.49 (dd, 1H,
J ¼ 9.2, 1.6 Hz), 7.51 (dd, 1H, J ¼ 7.8, 0.8 Hz), 7.53e7.58 (m, 2H), 7.61
(100 MHz, CDCl3)
d
24.37, 48.98, 115.33, 117.97, 120.44, 120.59 (q,
(dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.74 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13
C
J ¼ 255.6 Hz), 120.82, 121.41 (2C), 122.74, 128.30, 129.02 (2C),
133.08, 136.21, 137.01, 146.87, 148.88, 149.58, 152.45, 154.38, 158.23,
167.76; IR (KBr) 3220, 3017, 1552, 1260 cmꢂ1; HRMS-ESI m/z
[M ꢂ H]þ calcd. for C23H16F3N6OS: 481.1064, found 481.1067.
NMR (100 MHz, CDCl3/CD3OD) d 24.15, 44.12, 115.10, 117.69, 120.70,
120.71 (q, J ¼ 256.3 Hz), 120.76, 120.82, 122.79, 127.22, 128.21,
129.29, 129.57, 129.99, 133.38, 137.15, 146.76, 147.46, 149.35, 152.28,
154.02,158.08,167.82; IR (KBr) 3221, 3009, 1551,1255 cmꢂ1; HRMS-
ESI m/z [M ꢂ H]þ calcd. for C23H16F3N6OS: 481.1064, found
481.1068.
4.1.6.13. 4-(((4-(6-Methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyr-
idin-6-yl)thiazol-2-yl)amino)methyl)benzonitrile (12m). Yield 78%;
mp 225.0 ꢀC; Purity by HPLC: 99.05% (30% acetonitrile); 1H NMR
4.1.6.18. N-(2,3-Difluorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]-
triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12r). Yield 54%; mp
162.7 ꢀC; Purity by HPLC: 98.35% (30% acetonitrile); 1H NMR
(400 MHz, CDCl3): d 2.36 (s, 3H), 4.63 (s, 2H), 6.19 (br s, 1H), 7.03 (d,
1H, J ¼ 7.6 Hz), 7.45 (d, 1H, overlapped, J ¼ 8.0 Hz), 7.47 (dd, 1H,
overlapped, J ¼ 9.2, 1.6 Hz), 7.50e7.54 (m, 3H), 7.61 (dd, 1H, J ¼ 9.2,
(400 MHz, CDCl3):
d
2.36 (s, 3H), 4.66 (d, 2H, J ¼ 4.8 Hz), 5.72 (br s,
0.8 Hz), 7.66 (m, 2H), 8.31 (s, 1H), 8.72 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13
C
1H), 7.04 (dd, 1H, J ¼ 7.6, 1.2 Hz), 7.05e7.15 (m, 2H), 7.21e7.25 (m,
1H), 7.50 (dd, 1H, J ¼ 9.2, 2.0 Hz), 7.52 (br d, 1H, overlapped,
J ¼ 7.6 Hz), 7.55 (t, 1H, J ¼ 7.6 Hz), 7.62 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32
(s, 1H), 8.75 (dd, 1H, J ¼ 2.0, 0.8 Hz); 13C NMR (100 MHz, CDCl3/
NMR (100 MHz, CDCl3/CD3OD)
d 23.83, 48.51, 111.18, 114.96, 117.60,
118.71, 120.72, 120.89, 122.91, 128.05, 128.16 (2C), 132.52 (2C),
133.39, 137.35, 143.66, 146.44, 149.12, 152.00, 153.71, 157.94, 167.61;
IR (KBr) 3221, 2926, 2229, 1550 cmꢂ1; HRMS-ESI m/z [M ꢂ H]þ
calcd. for C23H16N7S: 422.1193, found 422.1192.
CD3OD)
d
23.84, 42.65, 114.95, 116.73 (d, J ¼ 16.8 Hz), 117.56, 120.81,
120.90, 122.90, 124.27 (dd, J ¼ 6.8, 4.6 Hz), 124.42 (t, J ¼ 3.1 Hz),
127.23 (d, J ¼ 11.4 Hz), 128.09, 133.48, 137.40, 146.43, 148.97 (dd,
J ¼ 246.4, 12.2 Hz), 149.15, 150.55 (dd, J ¼ 247.2, 12.2 Hz), 152.05,
153.73, 157.92, 167.62; IR (KBr) 3224, 3012, 1551 cmꢂ1; HRMS-ESI
m/z [M þ H]þ calcd. for C22H17F2N6S: 435.1198, found 435.1191.
4.1.6.14. N-(2-Fluorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12n). Yield 52%; mp
238.6 ꢀC; Purity by HPLC: 96.25% (30% acetonitrile); 1H NMR

82
M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
4.1.6.19. N-(3,4-Difluorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]-
triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12s). Yield 60%; mp
197.9 ꢀC; Purity by HPLC: 95.64% (30% acetonitrile); 1H NMR
137.40,141.31, 146.49, 149.28, 152.02, 153.91, 158.05, 167.62; IR (KBr)
3216, 2924, 1551 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for
C22H17Cl2N6S: 467.0607, found 467.0597.
(400 MHz, CDCl3): d 2.37 (s, 3H), 4.50 (s, 2H), 6.04 (br s, 1H), 7.04 (d,
1H, J ¼ 7.6 Hz), 7.11e7.16 (m, 2H), 7.19e7.25 (m, 1H), 7.47 (d, 1H,
overlapped, J ¼ 7.6 Hz), 7.48 (dd, 1H, J ¼ 9.2, 1.6 Hz), 7.54 (t, 1H,
J ¼ 7.6 Hz), 7.61 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.73 (dd, 1H,
4.1.6.24. N-(2,3-Dimethylbenzyl)-4-(6-methylpyridin-2-yl)-5-
([1,2,4]-triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12x). Yield 38%,
mp 210.6 ꢀC; Purity by HPLC: 99.17% (35% acetonitrile); 1H NMR
J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3/CD3OD)
d
23.82, 48.01,
(400 MHz, CDCl3): d 2.27 (s, 3H), 2.31 (s, 3H), 2.36 (s, 3H), 4.50 (s,
114.83, 116.44 (d, J ¼ 17.6 Hz), 117.34 (d, J ¼ 17.6 Hz), 120.83, 120.86,
122.80, 123.51 (t, J ¼ 3.1 Hz), 123.58, 127.95, 133.41, 134.84 (t,
J ¼ 4.6 Hz), 137.18, 146.59, 149.03, 149.72 (dd, J ¼ 246.5, 12.2 Hz),
150.35 (dd, J ¼ 247.2,12.2 Hz),152.16,153.61,157.95,167.75; IR (KBr)
3201, 2993, 1551 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for
2H), 5.80 (br s, 1H), 7.02 (dd, 1H, J ¼ 7.6, 2.4 Hz), 7.07e7.14 (m, 2H),
7.22 (br d, 1H, J ¼ 7.6 Hz), 7.47e7.54 (m, 3H), 7.60 (dd, 1H, J ¼ 9.2,
1.6 Hz), 8.28 (s,1H), 8.74 (dd,1H, J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz,
CDCl3)
d 15.23, 20.67, 24.44, 48.90, 115.31, 117.75, 120.58, 120.77,
122.73, 125.96, 126.86, 128.40, 130.19, 133.25, 134.73, 135.38, 137.06,
137.77, 146.62, 149.65, 152.34, 154.42, 158.21, 167.33; IR (KBr) 3228,
2972, 1551 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for C24H23N6S:
427.1699, found 427.1703.
C
22H17F2N6S: 435.1198, found 435.1193.
4.1.6.20. N-(3,5-Difluorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]-
triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12t). Yield 87%; mp
202.5 ꢀC; Purity by HPLC: 97.80% (30% acetonitrile); 1H NMR
4.1.6.25. N-(3,5-Dimethylbenzyl)-4-(6-methylpyridin-2-yl)-5-
([1,2,4]-triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12y). Yield 31%;
mp 225.2 ꢀC; Purity by HPLC: 97.68% (35% acetonitrile); 1H NMR
(400 MHz, CDCl3): d 2.37 (s, 3H), 4.56 (br s, 2H), 5.87 (br s, 1H), 6.75
(tt, 1H, J ¼ 9.0, 2.4 Hz), 6.93e6.96 (m, 2H), 7.04 (dd, 1H, J ¼ 7.6,
0.8 Hz), 7.49 (dd, 1H, J ¼ 7.6, 0.8 Hz), 7.50 (dd, 1H, J ¼ 9.2, 2.0 Hz),
7.55 (t, 1H, J ¼ 7.6 Hz), 7.62 (dd, 1H, J ¼ 9.2, 1.2 Hz), 8.32 (s, 1H), 8.74
(400 MHz, CDCl3):
d
2.31 (s, 6H), 2.38 (s, 3H), 4.44 (d, 2H, J ¼ 4.0 Hz),
5.93 (br s, 1H), 6.95 (s, 1H), 7.00 (s, 2H), 7.03 (dd, 1H, J ¼ 7.6, 0.8 Hz),
7.47e7.49 (m, 2H), 7.53 (t, 1H, J ¼ 7.6 Hz), 7.60 (dd, 1H, J ¼ 9.2,
0.8 Hz), 8.30 (s, 1H), 8.72 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13C NMR
(dd, 1H, J ¼ 2.0, 1.2 Hz); 13C NMR (100 MHz, CDCl3/CD3OD)
d 24.04,
48.44, 103.07 (t, J ¼ 25.2 Hz), 110.20 (2C, dd, J ¼ 18.3, 6.8 Hz), 115.03,
117.66, 120.71, 120.87, 122.83, 128.14, 133.39, 137.19, 141.97 (t,
J ¼ 8.8 Hz), 146.72, 149.23, 152.21, 153.87, 158.07, 163.28 (2C, dd,
J ¼ 247.2, 12.2 Hz), 167.74; IR (KBr) 3197, 3090, 1550 cmꢂ1; HRMS-
ESI m/z [M þ H]þ calcd. for C22H17F2N6S: 435.1198, found 435.1193.
(100 MHz, CDCl3)
d 21.50 (2C), 24.45, 50.18, 115.49, 117.63, 120.29,
120.71, 122.93, 125.65 (2C), 128.50, 129.92, 133.15, 136.64, 137.15,
138.77 (2C), 145.78, 149.72, 151.78, 154.51, 158.39, 167.79; IR (KBr)
3228, 3008, 1553 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for
C24H23N6S: 427.1699, found 427.1699.
4.1.6.21. N-(2,3-Dichlorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]-
triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12u). Yield 46%; mp
206.3 ꢀC; Purity by HPLC: 97.60% (35% acetonitrile); 1H NMR
4.1.6.26. N-(2,3-Dimethoxybenzyl)-4-(6-methylpyridin-2-yl)-5-
([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12z). Yield 51%;
mp 156.1 ꢀC; Purity by HPLC: 96.84% (29% acetonitrile); 1H NMR
(400 MHz, CDCl3):
d 2.35 (s, 3H), 4.63 (s, 2H), 6.32 (br s, 1H), 7.02
(dd, 1H, J ¼ 7.6, 0.8 Hz), 7.20 (t, 1H, J ¼ 8.0 Hz), 7.42 (d, 2H,
J ¼ 8.0 Hz), 7.45 (br d, 1H, J ¼ 8.0 Hz), 7.46 (dd, 1H, J ¼ 9.2, 2.0 Hz),
7.51 (t, 1H, J ¼ 7.6 Hz), 7.60 (dd, 1H, J ¼ 9.2, 1.2 Hz), 8.30 (s, 1H), 8.72
(400 MHz, CDCl3): d 2.35 (s, 3H), 3.87 (s, 3H), 3.91 (s, 3H),
4.53 (d, 2H, J ¼ 5.2 Hz), 5.98 (br s, 1H), 6.89 (dd, 1H, J ¼ 8.0, 2.0 Hz),
6.98e7.06 (m, 3H), 7.46e7.50 (m, 2H), 7.52 (t, 1H, J ¼ 7.8 Hz), 7.59
(dd, 1H, J ¼ 2.0, 0.8 Hz); 13C NMR (100 MHz, CDCl3/CD3OD)
d
24.13,
(dd, 1H, J ¼ 9.2, 0.8 Hz), 8.30 (s, 1H), 8.72 (dd, 1H, J ¼ 1.6, 0.8 Hz); 13
C
48.01, 115.28, 117.96, 120.45, 120.85, 122.94, 127.39, 127.61, 128.31,
129.95, 131.79, 133.24, 133.59, 137.21, 137.34, 146.37, 149.43, 151.96,
154.15, 158.09, 167.68; IR (KBr) 3221, 2924, 1549 cmꢂ1; HRMS-ESI
m/z [M þ H]þ calcd. for C22H17Cl2N6S: 467.0607, found 467.0604.
NMR (100 MHz, CDCl3) d 24.38, 45.36, 55.97, 60.96, 112.52, 115.17,
117.75, 120.70, 120.76, 121.13, 122.57, 124.29, 128.31, 131.02, 133.28,
136.97, 146.79, 147.39, 149.57, 152.56, 152.88, 154.33, 158.05, 167.74;
IR (KBr) 3228, 2930, 1553, 1481 cmꢂ1; HRMS-ESI m/z [M þ H]þ
calcd. for C24H23N6O2S: 459.1598, found 459.1616.
4.1.6.22. N-(3,4-Dichlorobenzyl)-4-(6-methylpyridin-2-yl)-5-
([1,2,4]-triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12v). Yield 53%;
mp 205.5 ꢀC; Purity by HPLC: 97.39% (35% acetonitrile); 1H NMR
4.1.6.27. N-(3,4-Dimethoxybenzyl)-4-(6-methylpyridin-2-yl)-5-
([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12aa). Yield 57%;
mp 157.5 ꢀC; Purity by HPLC: 98.10% (30% acetonitrile); 1H NMR
(400 MHz, CDCl3): d 2.37 (s, 3H), 4.52 (s, 2H), 6.00 (br s, 1H), 7.04 (d,
1H, J ¼ 7.6 Hz), 7.24 (dd, 1H, J ¼ 8.4, 2.0 Hz), 7.44 (d, 1H, J ¼ 8.4 Hz),
7.47 (br d, 1H, J ¼ 7.6 Hz), 7.48 (dd, 1H, J ¼ 9.2, 1.6 Hz), 7.50 (d, 1H,
overlapped, J ¼ 2.0 Hz), 7.54 (t, 1H, J ¼ 7.6 Hz), 7.62 (dd, 1H, J ¼ 9.2,
0.8 Hz), 8.32 (s,1H), 8.73 (dd,1H, J ¼ 2.0, 0.8 Hz), 13C NMR (100 MHz,
(400 MHz, CDCl3): d 2.36 (s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 4.41 (br s,
2H), 6.30 (br s, 1H), 6.82 (d, 1H, J ¼ 8.0 Hz), 6.88 (d, 1H, J ¼ 2.0 Hz),
6.91 (dd, 1H, J ¼ 8.0, 2.0 Hz), 7.01 (dd, 1H, J ¼ 7.6, 0.8 Hz), 7.44e7.47
(m, 2H), 7.50 (t, 1H, J ¼ 7.6 Hz), 7.59 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.29 (s,
CDCl3/CD3OD)
d
23.75, 29.68, 115.04, 117.68, 120.69, 120.96, 123.02,
1H), 8.70 (dd,1H, J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3)
d 24.39,
126.98, 128.09, 129.52, 130.66, 131.63, 132.74, 133.39, 137.56, 138.07,
146.17, 149.16, 151.82, 153.74, 157.92, 167.74; IR (KBr) 3221, 2924,
1549 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for C22H17Cl2N6S:
467.0607, found 467.0599.
49.77, 56.07 (2C),110.89,111.31,115.27, 117.84, 120.19,120.58,120.73,
122.64, 128.26, 129.63, 133.16, 136.96, 146.74, 148.90, 149.42, 149.55,
152.47, 154.34, 158.18, 167.78; IR (KBr) 3225, 2937, 1552 cmꢂ1
;
HRMS-ESI m/z [M þ H]þ calcd. for C24H23N6O2S: 459.1598, found
459.1619.
4.1.6.23. N-(3,5-Dichlorobenzyl)-4-(6-methylpyridin-2-yl)-5-
([1,2,4]-triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine
39%; mp 244.4 ꢀC; Purity by HPLC: 96.99% (35% acetonitrile); 1H
NMR (400 MHz, CDCl3): 2.37 (s, 3H), 4.52 (s, 2H), 6.09 (br s, 1H),
(12w). Yield
4.1.6.28. N-(3,5-Dimethoxybenzyl)-4-(6-methylpyridin-2-yl)-5-
([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12ab). Yield 24%;
mp 149.0 ꢀC; Purity by HPLC: 98.05% (30% acetonitrile); 1H NMR
d
7.04 (d, 1H, J ¼ 7.6 Hz), 7.28e7.31 (m, 3H), 7.48 (d, 1H, J ¼ 7.6 Hz),
7.49 (dd, 1H, J ¼ 9.2, 2.0 Hz), 7.54 (t, 1H, J ¼ 7.6 Hz), 7.62 (dd, 1H,
J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.74 (dd, 1H, J ¼ 2.0, 0.8 Hz); 13C NMR
(400 MHz, CDCl3):
d
2.38 (s, 3H), 3.79 (s, 6H), 4.47 (d, 2H, J ¼ 4.4 Hz),
5.90 (br s,1H), 6.40 (t,1H, J ¼ 2.4 Hz), 6.55 (d, 2H, J ¼ 2.4 Hz), 7.04 (d,
1H, J ¼ 7.6 Hz), 7.48 (br d,1H, J ¼ 7.6 Hz), 7.49 (dd,1H, J ¼ 9.2, 1.6 Hz),
7.53 (t, 1H, J ¼ 7.6 Hz), 7.61 (dd, 1H, J ¼ 9.2, 0.8 Hz), 8.32 (s, 1H), 8.73
(100 MHz, CDCl3/CD3OD)
d 24.00, 48.28, 115.13, 117.87, 120.66,
120.93, 122.96, 126.04 (2C), 127.93, 128.21, 133.41, 135.35 (2C),
(dd, 1H, J ¼ 1.6, 0.8 Hz); 13C NMR (100 MHz, CDCl3)
d 24.44, 50.16,

M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
83
55.62 (2C), 99.92, 105.71 (2C), 115.41, 117.98, 120.42, 120.74, 122.81,
128.46, 133.20, 137.10, 139.44, 146.34, 149.68, 152.13, 154.46, 158.29,
161.41 (2C), 167.72; IR (KBr) 3207, 2928, 1550 cmꢂ1; HRMS-ESI m/z
[M ꢂ H]þ calcd. for C24H21N6O2S: 457.1452, found 457.1457.
133.29, 133.86, 137.58, 138.10, 146.09, 149.22, 151.81, 153.86, 158.02,
168.03,170.30; IR (KBr) 3202, 2923,1667,1551 cmꢂ1; HRMS-ESI m/z
[M þ H]þ calcd. for C23H20N7OS: 442.1445, found 442.1453.
4.1.7.2. 4-(((4-(6-Methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyr-
idin-6-yl)thiazol-2-yl)amino)methyl)benzamide (13b). Yield 71%;
mp 214.3 ꢀC; Purity by HPLC: 98.50% (20% acetonitrile); 1H NMR
4.1.6.29. N-Benzyl-5-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-
a]-pyridin-6-yl)thiazol-2-amine (18a). Yield 55%; mp 177.4 ꢀC;
Purity by HPLC: 98.35% (30% acetonitrile); 1H NMR (400 MHz,
(400 MHz, CDCl3/CD3OD):
d 2.16 (s, 3H), 4.41 (s, 2H), 6.89 (d, 1H,
CDCl3):
d
2.51 (s, 3H), 4.51 (d, 2H, J ¼ 5.2 Hz), 6.19 (t, 1H, J ¼ 5.2 Hz),
J ¼ 8.0 Hz), 7.23e7.30 (m, 4H), 7.38 (dd, 1H, J ¼ 9.2, 1.2 Hz), 7.40 (t,
6.93 (d, 1H, J ¼ 8.0 Hz), 6.94 (d, 1H, J ¼ 7.6 Hz), 7.28e7.37 (m, 6H),
1H, J ¼ 7.6 Hz), 7.64 (m, 2H), 8.09 (s,1H), 8.47 (dd,1H, J ¼ 1.6,1.2 Hz);
7.63e7.68 (m, 2H), 8.33 (s, 1H), 8.88 (t, 1H, J ¼ 1.4 Hz); 13C NMR
13C NMR (100 MHz, CDCl3/CD3OD)
d 23.88, 48.81, 114.96, 117.31,
(100 MHz, CDCl3)
d
24.61, 50.05, 116.30, 118.90, 121.74, 122.70,
120.80, 120.91, 122.86, 127.61 (2C), 127.94 (2C), 127.98, 132.60,
133.37, 137.28, 141.85, 146.54, 149.10, 152.13, 153.71, 157.99, 167.97,
170.18; IR (KBr) 3203, 2922, 1666, 1551 cmꢂ1; HRMS-ESI m/z
[M ꢂ H]þ calcd. for C23H18N7OS: 440.1299, found 440.1298.
124.38, 127.80 (2C), 128.23, 129.02, 129.10 (2C), 131.53, 136.87,
137.00, 141.69, 150.20, 150.33, 154.73, 159.02, 168.99; IR (KBr) 3221,
2971, 1558, 1452 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for
C
22H19N6S: 399.1386, found 399.1398.
4.1.7.3. 3-(((5-(6-Methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-
6-yl)thiazol-2-yl)amino)methyl)benzamide (18c). This compound was
prepared according to the same procedure for 13a using 18b as the
starting material. Yield 42%; mp 258.4 ꢀC; Purity by HPLC: 96.94%
4.1.6.30. 3-(((5-(6-Methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyr-
idin-6-yl)thiazol-2-yl)amino)methyl)benzonitrile (18b). Yield 28%;
mp 187.6 ꢀC; Purity by HPLC: 97.24% (30% acetonitrile); 1H NMR
(400 MHz, CDCl3):
d
2.52 (s, 3H), 4.62 (d, 2H, J ¼ 4.8 Hz), 5.99 (br s,
(20% acetonitrile); 1H NMR (400 MHz, CDCl3):
d 2.49 (s, 3H), 4.53 (br
1H), 6.96 (d, 1H, J ¼ 7.6 Hz), 6.98 (d, 1H, J ¼ 7.6 Hz), 7.38 (t, 1H,
J ¼ 7.8 Hz), 7.48 (t, 1H, J ¼ 7.6 Hz), 7.59e7.65 (m, 2H), 7.67e7.70 (m,
3H), 8.35 (s, 1H), 8.89 (dd, 1H, J ¼ 1.6, 1.2 Hz); IR (KBr) 3226, 2925,
2231, 1558 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for C23H18N7S:
424.1339, found 424.1344.
s, 2H), 6.32 (br s, 1H), 6.53 (br s, 1H), 6.90 (d, 1H, J ¼ 8.0 Hz), 6.93 (d,
1H, J ¼ 7.6 Hz), 6.94 (br s, 1H, overlapped), 7.33 (t, 1H, overlapped,
J ¼ 7.8 Hz), 7.37 (d,1H, overlapped, J ¼ 8.0 Hz), 7.47 (d, 1H, J ¼ 7.6 Hz),
7.62 (m, 2H), 7.67 (d,1H, J ¼ 7.6 Hz), 7.87 (br s,1H), 8.31 (s,1H), 8.85 (t,
1H, J ¼ 1.2 Hz); 13C NMR (100 MHz, CDCl3/CD3OD)
d 24.16, 29.74,
115.72, 119.42, 121.82, 123.31, 123.49, 126.62, 126.94, 128.78, 129.02,
131.30, 132.01, 133.88, 137.04, 138.15, 142.06, 149.72, 150.26, 154.08,
4.1.6.31. N-(2-Fluorobenzyl)-5-(6-methylpyridin-2-yl)-4-([1,2,4]tri-
azolo[1,5-a]pyridin-6-yl)thiazol-2-amine (18d). Yield 49%; mp
229.9 ꢀC; Purity by HPLC: 98.08% (30% acetonitrile); 1H NMR
158.86, 169.11, 170.34; IR (KBr) 3352, 3213, 2924, 1668, 1557 cmꢂ1
;
HRMS-ESI m/z [M þ H]þ calcd. for C23H20N7OS: 442.1445, found
(400 MHz, CDCl3):
d
2.52 (s, 3H), 4.62 (d, 2H, J ¼ 5.6 Hz), 5.84 (br t,1H,
442.1453.
J¼ 5.6Hz), 6.95(d,1H,J ¼7.6Hz), 6.96(d,1H,J ¼7.6Hz), 7.06e7.11(m,
1H), 7.14 (td, 1H, J ¼ 7.6, 1.0 Hz), 7.27e7.31 (m, 1H), 7.37 (t, 1H,
J ¼ 7.6 Hz), 7.45 (td,1H, J ¼ 7.4,1.8 Hz), 7.65e7.70 (m, 2H), 8.35 (s,1H),
4.2. Luciferase reporter assay
8.90 (pseudo t, 1H, J ¼ 1.4 Hz); 13C NMR (100 MHz, CDCl3)
d
24.59,
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% con-
fluency, various concentrations of ALK5 inhibitors and 2 ng/mL of
43.74 (d, J ¼ 3.6 Hz), 115.70 (d, J ¼ 20.5 Hz), 116.05, 118.96, 121.56,
123.19, 124.35, 124.48 (d, J ¼ 14.7 Hz), 124.48 (d, J ¼ 3.0 Hz), 128.91,
129.75, 129.81 (d, J ¼ 3.0 Hz), 131.61, 136.78, 142.48, 150.07, 150.51,
154.58, 158.93, 161.10 (d, J ¼ 244.6 Hz), 168.82; IR (KBr) 3223, 2925,
1558 cmꢂ1; HRMS-ESI m/z [M þ H]þ calcd. for C22H18FN6S: 417.1292,
found 417.1302.
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).
4.1.7. General procedure for the preparation of the 3(4)-(((4(5)-(6-
methylpyridin-2-yl)-5(4)-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-
2-yl)amino)methyl)benzamide 13a, 13b, and 18c
To a stirred solution of 12g, 12m, or 18b (0.11 g, 0.26 mmol) in
absolute EtOH (3 mL), 1 N NaOH (0.78 mL, 0.78 mmol), and 35%
H2O2 (0.09 mL, 0.91 mmol) were added. The mixture was stirred at
room temperature for 3 h, cooled to 0 ꢀC, and then neutralized with
1 N HCl to pH 8. The reaction mixture was extracted with 10%
MeOH/CHCl3 (2 ꢁ 50 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 (1:25) as eluent to yield the titled compound
as a white solid.
4.3. Immunoblot assay
Recombinant human TGF-b1 was purchased from R&D Systems
(Minneapolis, MN, USA). Mouse mammary epithelial cells
(NMuMG) were maintained in DMEM supplemented with 10% fetal
bovine serum (FBS), insulin (10
mg/mL), penicillin (100 U/mL), and
streptomycin (100 g/mL, Invitrogen life technology, Grand Island,
m
NY, USA). Total cell lysates were prepared by RIPA buffer (50 mM
Tris, 0.5% sodium deoxycholate, 150 mM NaCl, and 81% NP-40)
containing protease inhibitor cocktail (Roche Diagnostics, Man-
nheim, Germany) and sodium orthovanadate (SigmaeAldrich, St.
Louis, Mo, USA) for 20 min on ice. The concentration of proteins was
determined by using Bradford assay (Bio-Rad, Hercules, CA, USA).
Equal amounts of proteins were electrophoresed in 10% SDS-PAGE
gel and transferred to a PVDF membrane (Millipore, Billerica, MA,
USA). The membranes were blocked in 5% nonfat dry milk solution
and then incubated for 1 h at room temperature with one of
following antibodies: anti-Smad2, anti-phospho-Smad2, and anti-
GAPDH (Cell Signaling Technology, Beverly, MA, USA) in Tris-
buffered saline. Following three washes in Tris-buffered saline,
4.1.7.1. 3-(((4-(6-Methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyr-
idin-6-yl)thiazol-2-yl)amino)methyl)benzamide (13a). Yield 60%;
mp 242.5 ꢀC; Purity by HPLC: 96.66% (20% acetonitrile); 1H NMR
(400 MHz, CDCl3): d 2.35 (s, 3H), 4.53 (s, 2H), 6.36 (br s,1H), 6.57 (br s,
1H), 6.98 (br s,1H), 7.01 (d,1H, J ¼ 7.6Hz), 7.38 (t,1H, J ¼ 7.8 Hz), 7.41e
7.44 (m, 2H), 7.48e7.52 (m, 2H), 7.58 (dd,1H, J ¼ 9.2, 0.8 Hz), 7.70 (br
d, 1H, J ¼ 8.0 Hz), 7.91 (br s, 1H), 8.29 (s, 1H), 8.67 (dd, 1H, J ¼ 2.0,
0.8 Hz); 13C NMR (100 MHz, DMSO-d6/CDCl3)
d 23.84, 29.74, 115.20,
117.57, 120.64, 120.99, 123.06, 126.65, 127.01, 128.10, 129.02, 131.35,

84
M. Krishnaiah et al. / European Journal of Medicinal Chemistry 57 (2012) 74e84
the membranes were incubated with either horseradish
peroxidase-conjugated goat anti-rabbit or goat anti-mouse anti-
bodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in Tris-
buffered saline for 1 h. Proteins were detected using an ECL kit
(Amersham Biosciences/GE Healthcare, Piscataway, NJ, USA).
Acknowledgments
This work was supported by a grant from Ministry of Education,
Science and Technology, Korea (20100029596).
Appendix A. Supplementary material
4.4. Docking experiments
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.09.011.
Docking experiments have been performed with the Sybyl 8.1
software package (Tripos, Inc., St. Louis, MO, USA) based on CentOS
Linux 5.4.
References
4.4.1. Preparation of ligand database
[1] C.H. Heldin, K. Miyazono, P. ten Dijke, Nature 390 (1997) 465e471.
[2] R. Derynck, Y.E. Zhang, Nature 425 (2003) 577e584.
[3] W. Wang, V. Koka, H.Y. Lan, Nephrology 10 (2005) 48e56.
[4] H. Lim, Y.Z. Zhu, Cell. Mol. Life Sci. 63 (2006) 2584e2596.
[5] L. Gu, Y.-J. Zhu, X. Yang, Z.-J. Guo, W.-B. Xu, X.-L. Tian, Acta Pharmacol. Sin. 28
(2007) 382e391.
[6] F.W. Shek, R.C. Benyon, Eur. J. Gastroenterol. Hepatol. 16 (2004) 123e126.
[7] B. Bierie, H.L. Moses, Nat. Rev. Cancer 6 (2006) 506e520.
[8] S.G. Rane, J.-H. Lee, H.-M. Lin, Cytokine Growth Factor Rev. 17 (2006) 107e
119.
The structures of ligands were prepared in MOL2 format using
the sketcher module, and GasteigereHuckel charges were assigned
to the ligand atoms. The structures of molecules were optimized by
energy minimization, and performed molecular dynamics using
simulated annealing method. The conformer library for each ligand
was prepared by random selection of 200 conformers from the
molecular dynamics output.
[9] M. Dong, G.C. Blobe, Blood 107 (2006) 4589e4596.
[10] S.D. Byfield, C. Major, N.J. Laping, A.B. Roberts, Mol. Pharmacol. 65 (2004)
744e752.
[11] A.-C. de Gouville, V. Boullay, G. Krysa, J. Pilot, J.-M. Brusq, F. Loriolle, J.-M. Gauthier,
S.A. Papworth, A. Laroze, F. Gellibert, S. Huet, Br. J. Pharmacol. 145 (2005) 166e
177.
[12] M. Uhl, S. Aulwurm, J. Wischhusen, M. Weiler, J.Y. Ma, R. Almirez, R. Mangadu,
Y.-W. Liu, M. Platten, U. Herrlinger, A. Murphy, D.A. Wong, W. Wick,
L.S. Higgins, M. Weller, Cancer Res. 64 (2004) 7954e7961.
[13] L. Bueno, D.P. de Alwis, C. Pitou, J. Yingling, M. Lahn, S. Glatt, I. Troconiz, Eur. J.
Cancer 44 (2008) 142e150.
[14] D.-K. Kim, Y.-J. Bang, H.-T. Kim, I.-S. Cho, M.-S. Park, Y.J. An, J.H. Choi, World
Patent WO 2005/103028 A1 (2005).
[15] C.H. Jin, D. Sreenu, M. Krishnaiah, V.B. Subrahmanyam, K.S. Rao, A.V.N. Mohan,
C.-Y. Park, J.-Y. Son, D.-H. Son, H.-J. Park, Y.Y. Sheen, D.-K. Kim, Eur. J. Med.
Chem. 46 (2011) 3917e3925.
[16] J.-A. Moon, H.-T. Kim, I.-S. Cho, Y.Y. Sheen, D.-K. Kim, Kidney Int. 70 (2006)
1234e1243.
[17] L. Long, A. Crosby, X. Yang, M. Southwood, P.D. Upton, D.-K. Kim, N.W. Morrell,
Circulation 119 (2009) 566e576.
[18] Y.W. Kim, Y.K. Kim, J.Y. Lee, K.T. Chang, H.J. Lee, D.-K. Kim, Y.Y. Sheen, Xen-
obiotica 38 (2008) 325e339.
[19] W.-C. Lee, L. Sun, C. Chuaqui, M. Cornebise, J. Singh, F. Shan, World Patent WO
2006026305 A1 (2006).
[20] N. Dodic, F. Donche, F.J. Gellibert, World Patent WO 2004111046 A2 (2004).
[21] W.-C. Lee, L. Sun, F. Shan, C. Chuaqui, Z. Zheng, R.C. Petter, World Patent WO
2003087304 A2 (2003).
[22] J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang,
J. Massague, Cell 71 (1992) 1003e1014.
[23] F. Gellibert, M.-H. Fouchet, V.-L. Nguyen, R. Wang, G. Krysa, A.-C. de Gouville,
S. Huet, N. Dodic, Bioorg. Med. Chem. Lett. 19 (2009) 2277e2281.
[24] I. Yakymovych, U. Engström, S. Grimsby, C.-H. Heldin, S. Souchelnytskyi,
Biochemistry 41 (2002) 11000e11007.
[25] P.A. Eyers, M. Craxton, N. Morrice, P. Cohen, M. Goedert, Chem. Biol. 5 (1998)
321e328.
4.4.2. FlexiDock and Surflex-Dock docking
Compounds (12b, 12g, and 18b) were docked into active site of
ALK5 (PDB id: 3GXL) by the FlexiDock module. The binding site was
defined around important residues (Lys 232, Ser 280, and Ser 287)
in the radius of 3 Å. Single bonds of these residues (Lys 232, Ser 280,
and Ser 287) and each compound were allowed to be flexible in
docking process, while the backbone and remaining bonds were
held rigid. The ligand was located within the distance that these
residues of ALK5 were capable of forming H-bonds. Through this
process, the results gained 20 (maximum number of generations to
allow: 30,000) solutions, and these structures were energy-
minimized to remove bad energy and steric position. Most stable
complex between ALK5 and each compound was selected as
a receptor structure for further docking experiments using Surflex-
Dock program. The Surflex-Dock program uses an idealized binding
site called protomol. The protomol was built from the hydrogen-
containing protein mol2 file. The construction was based on
protein residues that constitute the binding site using parameters
to produce a small and buried docking target (proto_thresh ¼ 0.5
and proto_bloat ¼ 0). We select 0.1 Å radius around each of ligands
to generate protomols. The docking and subsequent scoring were
performed using the default parameters of the Surflex-Dock
programs implanted in the Sybyl 8.1. Final scores for all Surflex-
Dock solutions were calculated by a consensus scoring function
(CScore), and used for database ranking. One of the poses with the
highest Surflex-Dock score (in ꢂlog (Kd) unit) and high consensus
score (CScore 5 or 4) was selected, and docked with ALK5.
[26] C.-Y. Park, D.-K. Kim, Y.Y. Sheen, Cancer Sci. 102 (2011) 1889e1896.