Synthesis and biological evaluation of novel 2-pyridinyl-[1,2,3]triazoles as inhibitors of transforming growth factor β1 type 1 receptor

Article abstract of DOI:10.1016/j.bmcl.2004.03.024

A series of 2-pyridinyl-[1,2,3]triazoles have been synthesized and evaluated for their ALK5 inhibitory activity in the luciferase reporter assays. Compound 8d showed significant ALK5 inhibition (SBE-luciferase activity, 25%; p3TP-luciferase activity, 17%) at a concentration of 5μM that is comparable to that of SB-431542 (SBE-luciferase activity, 21%; p3TP-luciferase activity, 12%), but weak p38α MAP kinase inhibition (13%) at a concentration of 10μM that is much lower than that of SB-431542 (54%).

Full text of DOI:10.1016/j.bmcl.2004.03.024

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2401–2405
Synthesis and biological evaluation of novel
2-pyridinyl-[1,2,3]triazoles as inhibitors of transforming growth
factor b1 type 1 receptor
Dae-Kee Kim,a,* Joonseop Kima and Hyun-Ju Parkb
aCollege of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, South Korea
bCollege of Pharmacy, Sungkyunkwan University, Suwon, Kyungki-do 440-746, South Korea
Received 5 February 2004; revised 10 March 2004; accepted 10 March 2004
Abstract—A series of 2-pyridinyl-[1,2,3]triazoles have been synthesized and evaluated for their ALK5 inhibitory activity in the
luciferase reporter assays. Compound 8d showed significant ALK5 inhibition (SBE-luciferase activity, 25%; p3TP-luciferase activity,
17%) at a concentration of 5 lM that is comparable to that of SB-431542 (SBE-luciferase activity, 21%; p3TP-luciferase activity,
12%), but weak p38a MAP kinase inhibition (13%) at a concentration of 10 lM that is much lower than that of SB-431542 (54%).
Ó 2004 Elsevier Ltd. All rights reserved.
Chronic tissue fibrosis in the major organs such as
kidney, liver, lung, heart, and skin present major medi-
cal problems ranging from disfigurement to progressive
disability and death. Much attention is focused on the
roles of the many cytokines and growth factors, among
them, transforming growth factor b1 (TGF-b1) plays a
central role in excessive accumulation of extracellular
matrix by both stimulating the expression of matrix
components such as collagens, fibronectin, and
matrix proteoglycans and inducing inhibitors of matrix
degrading metalloproteinases and plasminogen-activa-
tor inhibitor.1 TGF-b1 transduces signals through two
highly conserved single transmembrane serine/threonine
kinases, the type I and type II TGF-b receptors (TbR-I
and TbR-II, respectively).2 TbR-II activates TbR-I
upon formation of the ligand–receptor complex by hy-
perphosphorylating serine/threonine residues in the GS
region of the TbR-I or activin-like kinase (ALK5),
which creates a binding site for Smad proteins. The
activated TbR-I in turn phosphorylates Smad2/Smad3
proteins at the C-terminal SSXS-motif thereby causing
dissociation from the receptor and heteromeric complex
formation with Smad4. Smad complexes translocate to
the nucleus, assemble with specific DNA-binding
co-factors and co-modulators to finally activate tran-
scription of extracellular matrix components, and
inhibitors of matrix-degrading proteases.2 Therefore, it
becomes evident that inhibition of ALK5 phosphoryl-
ation of Smad2/Smad3 could reduce TGF-b1-induced
excessive accumulation of extracellular matrix. The 4-
pyridyl substituted triarylimidazoles are known to be
selective inhibitors of p38 MAP kinase since a 40-nitro-
gen atom is involved in a required hydrogen bond to the
ATP binding site of p38 MAP kinase.3 Callahan et al.
have recently reported that the 2-pyridyl substituted
triarylimidazoles are selective inhibitors of ALK5 over
p38 MAP kinase, suggesting that there may be an
alternative ATP binding site available to ALK5 inhibi-
tors.4 SB-431542, one of the leading 2-pyridyl substi-
tuted triarylimidazole, inhibits the in vitro
phosphorylation of immobilized Smad3 with an IC50 of
94 nM.4 It inhibits also the activin type 1 receptor ALK4
and the nodal type 1 receptor ALK7, which are very
highly related to ALK5 in their kinase domains, but has
no effect on the other ALK family members that rec-
ognize bone morphogenetic proteins, on components of
the ERK, JNK, or p38 MAP kinase pathways, or on
components of the signaling pathways activated in
response to serum.5 The 4(5)-(2-pyridinyl)-[1,2,3]triazoles
having no substituent, a methyl or an ethyl substituent
at the 2-position of the triazole ring have recently been
claimed as selective ALK5 inhibitors, but their specific
ALK5 inhibitory activity has not been disclosed.6 On
the basis of these findings, we have synthesized a series
Keywords: 2-Pyridinyl-[1,2,3]triazoles; Inhibitors; TGF-b1 type 1 rec-
eptor; ALK5.
* Corresponding author. Tel.: +82-2-3277-3025; fax: +82-2-3277-2467;
e-mail: dkkim@ewha.ac.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.024

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D.-K. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2401–2405
of 4-aryl-5-(2-pyridinyl)-2-(4-substituted-benzyl)-[1,2,3]-
triazoles 7a–f and 8a–f and 4-aryl-5-(2-pyridinyl)-2-(4-
substituted-phenyl)-[1,2,3]triazoles 10a–b, 10d–f, 11a–b,
and 11d–f, and evaluated their ALK5 inhibitory activity
in the luciferase reporter assays.
a mixture of THF and TMEDA in the presence of CuI
and Pd(PPh3)4 catalysts gave the disubstituted acetylene
intermediates 4a and 4c–f in good yields. Reaction of 1-
benzo[1,3]dioxol-5-yl-2-(6-methylpyridin-2-yl)ethanone
(3)9 with trifluoromethanesulfonic anhydride and N,N-
diisopropylethylamine in CH2Cl2 produced the disub-
stituted acetylene 4b in 70% yield. Cycloaddition of the
acetylenes 4a–f with TMSN3 in DMF afforded the
[1,2,3]-triazoles 5a–f in 38–71% yields.6 Alkylation of
5a–f with 4-cyanobenzyl bromide (6) using NaH as a
base and n-Bu4NI in THF yielded 2-(4-cyanobenzyl)-
The 4-aryl-5-(2-pyridinyl)-[1,2,3]triazoles 5a–f were
prepared as shown in Scheme 1. Coupling of 5-eth-
ynylbenzo[1,3]dioxolane (1a),7 6-ethynylquinoxaline
(1b),8 or 1-ethynyl-4-methoxybenzene (1c) with either 2-
bromopyridine (2a) or 2-bromo-6-methylpyridine (2b) in
Br
R1
N
H
R2
1a—c
1a: R1 = benzo[1,3]dioxol-5-yl
2a—b
2a: R2 = H
2b: R2 = Me
1b: R1 = quinoxalin-6-yl
a
1c: R1 = 4-methoxyphenyl
R1
N
R1
NH
N
N
N
R2
4a—f
R2
O
5a—f
b
O
O
5a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (69%)
5b: R1 = benzo[1,3]dioxol-5-yl, R2 = Me (71%)
5c: R1 = quinoxalin-6-yl, R2 = H (64%)
5d: R1 = quinoxalin-6-yl, R2 = Me (68%)
5e: R1 = 4-methoxyphenyl, R2 = H (38%)
5f: R1 = 4-methoxyphenyl, R2 = Me (42%)
4a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (69%)
4b: R1 = benzo[1,3]dioxol-5-yl, R2 = Me (70%)
4c: R1 = quinoxalin-6-yl, R2 = H (>99%)
4d: R1 = quinoxalin-6-yl, R2 = Me (80%)
4e: R1 = 4-methoxyphenyl, R2 = H (88%)
4f: R1 = 4-methoxyphenyl, R2 = Me (88%)
N
3
Scheme 1. Reagents and conditions: (a) CuI, Pd(PPh3)4, TMEDA, THF, 55 °C, 10 h; (b) (CF3SO2)2O, N,N-diisopropylethylamine, CH2Cl2, reflux,
overnight; (c) TMSN3, DMF, reflux, 14 h.
R1
N
N
R1
N
N
N
N
b
N
N
Br
NH2
CN
R2
R2
O
8a—f
7a—f
o
8a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (71%)
8b: R1 = benzo[1,3]dioxol-5-yl, R2 = Me (83%)
8c: R1 = quinoxalin-6-yl, R2 = H (77%)
8d: R1 = quinoxalin-6-yl, R2 = Me (62%)
8e: R1 = 4-methoxyphenyl, R2 = H (71%)
8f: R1 = 4-methoxyphenyl, R2 = Me (71%)
7a: R1 = benzo[1,3]di xol-5-yl, R2 = H (71%)
7b: R1 = benzo[1,3]dioxol-5-yl, R2 = Me (72%)
7c: R1 = quinoxalin-6-yl, R2 = H (75%)
7d: R1 = quinoxalin-6-yl, R2 = Me (61%)
7e: R1 = 4-methoxyphenyl, R2 = H (71%)
7f: R1 = 4-methoxyphenyl, R2 = Me (72%)
CN
6
a
R1
N
N
R1
N
N
O
5a—f
N
CN
N
b
NH2
N
N
R2
R2
c
11a—b, 11d—f
10a—b, 10d—f
Br
11a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (90%)
11b: R1 = benzo[1,3]dioxol-5-yl, R2 = Me (58%)
11d: R1 = quinoxalin-6-yl, R2 = Me (56%)
11e: R1 = 4-methoxyphenyl, R2 = H (88%)
11f: R1 = 4-methoxyphenyl, R2 = Me (87%)
10a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (23%)
10b: R1 = benzo[1,3]dioxol-5-yl, R2 = Me (31%)
10d: R1 = quinoxalin-6-yl, R2 = Me (10%)
10e: R1 = 4-methoxyphenyl, R2 = H (21%)
10f: R1 = 4-methoxyphenyl, R2 = Me (13%)
CN
9
Scheme 2. Reagents and conditions: (a) 60% NaH, n-Bu4NI, THF, rt, 1 h; (b) 28% H2O2, 6 N NaOH, 95% EtOH, 55 °C, 3 h; (c) K2CO3, DMSO,
150 °C, 14 h.

D.-K. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2401–2405
2403
triazoles 7a–f as major positional isomers (61–75%
yields). Conversion of the nitrile functionality of the
compounds 7a–f to carboxamide was accomplished by
treatment with 28% H2O2 and 6 N NaOH solution to
afford the triazoles 8a–f in 62–83% yields. Alternatively,
compounds 5a–f were tried to alkylate with 4-bro-
mobenzonitrile (9) with K2CO3 in DMSO at 150 °C.
Although 5c failed to give the desired alkylated product
under this condition, other 2-(4-cyanophenyl)triazoles
10a–b and 10d–f could be obtained in rather low yields
(10–31%). The carboxamides 11a–b and 11d–f were
produced from 10a–b and 10d–f in 56–90% yields in the
same reaction condition as for 8a–f (Scheme 2).
reporter genes, SBE-Luc reporter construct containing
four tandem copies of the CAGA Smad binding element
cloned upstream of the adenovirus major late promoter
(MLP)10 and p3TP-Lux reporter construct containing
three AP-1 binding elements and the plasminogen acti-
vator inhibitor-1 (PAI-1) promoter11 were used for this
analysis. HepG2 cells were transiently transfected with
either SBE-Luc reporter construct or p3TP-Lux reporter
construct. Immediately after transfection, cells were
treated with DMSO alone or 5 lM of inhibitors dis-
solved in DMSO followed by TGF-b (5 ng/mL). After
24 h incubation, luciferase activity in cell lysates was
determined by using a luciferase assay system (Promega)
(Table 1).
To investigate whether these potential inhibitors could
inhibit TGF-b-induced downstream transcriptional
activation to ALK5 signaling, two different luciferase
Of the 4-aryl-5-(2-pyridinyl)-2-(4-substituted-benzyl)-
[1,2,3]triazoles 7a–f and 8a–f, the benzo[1,3]dioxolyl
Table 1. Effect of 2-pyridinyl-[1,2,3]triazoles on the activity of TGF-b-induced ALK5 and p38a MAP kinase
R1
R1
N
N
N
N
N
R3
N
N
N
R3
R2
10a—b, 10d—f, 11a—b, 11d—f
Activity (% control)a
R2
7a—f, 8a—f
Compound
R1
R2
R3
SBE-luciferaseb
p3TP-luciferaseb
p38a MAP kinasec
7a
7b
Benzo[1,3]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
Quinoxalin-6-yl
H
CN
CN
28
25
62
27
70
53
68
26
60
25
68
40
84
103
51
76
78
60
27
33
79
49
18
25
35
16
98
55
39
18
46
17
60
28
60
109
37
110
75
39
21
17
61
33
85
89
81
92
102
98
82
80
80
87
96
94
91
105
95
102
99
82
70
93
96
96
Me
H
7c
7d
CN
CN
Quinoxalin-6-yl
Me
H
7e
4-Methoxyphenyl
4-Methoxyphenyl
Benzo[1,3]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
Quinoxalin-6-yl
CN
CN
7f
8a
Me
H
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CN
8b
8c
Me
H
8d
8e
Quinoxalin-6-yl
Me
H
4-Methoxyphenyl
4-Methoxyphenyl
Benzo[1,3]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
Quinoxalin-6-yl
8f
Me
H
10a
10b
10d
10e
10f
11a
11b
11d
11e
11f
Me
Me
H
CN
CN
4-Methoxyphenyl
4-Methoxyphenyl
Benzo[1,3]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
Quinoxalin-6-yl
CN
CN
Me
H
CONH2
CONH2
CONH2
CONH2
CONH2
Me
Me
H
4-Methoxyphenyl
4-Methoxyphenyl
Me
O
O
N
SB-431542
21
12
46
CONH2
N
H
N
a Activity is given as the mean of triplicate determinations relative to control incubations with DMSO vehicle.
b Luciferase activity was determined with 5 lM of inhibitor.
c Kinase activity was determined with 10 lM of inhibitor.

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D.-K. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2401–2405
analogues 7a (SBE-luciferase activity, 28%; p3TP-lucif-
erase activity, 18%), 7b (SBE-luciferase activity, 25%;
p3TP-luciferase activity, 25%), and 8b (SBE-luciferase
activity, 26%; p3TP-luciferase activity, 18%), and the
quinoxalinyl analogues 7d (SBE-luciferase activity, 27%;
p3TP-luciferase activity, 16%) and 8d (SBE-luciferase
activity, 25%; p3TP-luciferase activity, 17%) displayed
significant ALK5 inhibition at 5 lM compared to that of
control, that is comparable to that of SB-431542 (SBE-
luciferase activity, 21%; p3TP-luciferase activity, 12%).
However, the 4-methoxyphenyl analogues 7e, 7f, 8e, and
8f showed much lower ALK5 inhibition compared to
the benzo[1,3]dioxolyl analogues and the quinoxalinyl
analogues. Introduction of a methyl substituent at the 6-
position of the pyridine ring moiety markedly increased
their ALK5 inhibitory activity (7c vs 7d; 7e vs 7f; 8a vs
8b; 8c vs 8d; 8e vs 8f). Although compound 7a showed
much higher ALK5 inhibition compared to 8a, most of
the carbonitrile analogues showed the similar level of
ALK5 inhibition compared to their corresponding car-
boxamide derivatives (7b vs 8b; 7c vs 8c; 7d vs 8d).
Among the 4-aryl-5-(2-pyridinyl)-2-(4-substituted-phen-
yl)-[1,2,3]triazoles 10a–b, 10d–f, 11a–b, and 11d–f, the
benzo[1,3]dioxolyl analogue 11b (SBE-luciferase activ-
ity, 27%; p3TP-luciferase activity, 21%) and the qui-
noxalinyl analogue 11d (SBE-luciferase activity, 33%;
p3TP-luciferase activity, 17%) displayed significant
ALK5 inhibition, but inhibitory activity of most this
series of compounds were lower than those of the cor-
responding 2-(4-substituted-benzyl) series of com-
pounds. The most potent compound 8d was chosen and
its ALK5 inhibition was determined at different con-
centrations using HepG2 cells transiently transfected
with SBE-Luc reporter construct. As shown in Figure 1,
8d inhibited ALK5 in a dose-dependent manner (SBE-
luciferase activity: 0.05 lM, 93%; 0.1 lM, 70%; 1.0 lM,
31%; 5.0 lM, 24%). P38a MAP kinase is the only kinase
to be significantly affected in vitro on a panel of 24
kinases unrelated to the ALKs by SB-431542.5 There-
fore, in vitro kinase assay on this enzyme was performed
in the presence of 10 lM of inhibitors. SB-431542 dis-
Figure 1. Effect of 8d on the activity of TGF-b-induced ALK5. HepG2
cells were transiently transfected with SBE-Luc reporter construct.
Luciferase activity was determined in the presence of different con-
centrations of 8d and is given as the mean of triplicate determinations
relative to control incubations with DMSO vehicle.
played 54% inhibition at this concentration, however,
the most inhibitory compound 11b showed 30% inhibi-
tion, and the rest of compounds including 8d (13%) also
exhibited weak or no measurable inhibition for this
enzyme (<20%), thus showing high selectivity (Table 1).
To examine a possible binding mode of 8d at the active
site of ALK5, we built a docking model of ALK5:8d
complex using the flexible docking algorithms (Flexi-
Dock and FlexX).12 As demonstrated in Figure 2, the
inhibitor 8d lies in the same binding pocket of ALK5 as
is occupied by NPC-30345 in the X-ray structure.13 All
three substituents attached to the central triazole ring of
8d form various hydrogen bonds (H-bonds) with amino
acid residues in the binding pocket. The carboxamide
group of phenyl substituent forms H-bonds with back-
bone amide of Ile211 and Ser287. The nitrogen atom in
the 2-pyridinyl moiety forms strong H-bond with the
Figure 2. The binding mode of 8d observed in the docking model. The amino acid residues within the ALK5-binding site are represented in line form.
Carbon atoms of amino acids are colored in magenta. The ligand is rendered in capped stick. Yellow dotted lines are hydrogen bonding interactions
ꢀ
(<2.5 A) between 8d and the amino acid residues (Ile211, Lys232, Ser280, and Ser287).

D.-K. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2401–2405
2405
6. Gaster, L. M.; Harling, J. D.; Heer, J. P.; Heightman,
T. D.; Payne, A. H. PCT Int. Appl. WO 02/40476 A1.
7. Bridges, A. J.; Lee, A.; Schwartz, C. E.; Towle, M. J.;
Littlefield, B. A. Bioorg. Med. Chem. 1993, 1, 403.
8. Gaster, L. M. PCT Int. Appl. WO 02/055077 A1.
9. Lee, W.-C.; Sun, L.; Shan, F.; Chuaqui, C.; Zheng, Z.;
Petter, R. C. PCT Int. Appl. WO 03/087304 A2.
10. Dennler, S.; Itoh, S.; Vivien, D.; ten Dijke, P.; Huet, S.;
Gauthier, J. M. EMBO J. 1998, 17, 3091.
11. Wrana, J. L.; Attisano, L.; Carcamo, J.; Zentella, A.;
positively charged amino protons of the Lys232 side
chain. Finally, a nitrogen atom of quinoxalinyl ring
forms H-bond with the OH of Ser280. In addition,
quinoxalinyl ring is in close contact with Leu278, and
the 6-methyl group of 2-pyridinyl moiety occupies a
hydrophobic cavity consisted of Leu278 and Phe262,
possibly forming hydrophobic interactions. Taking into
account that Leu278 and Ser280 residues are poorly
conserved in other kinases,14 the observed interactions
between these residues and quinoxalinyl ring and 6-
methyl group of 2-pyridinyl moiety of 8d would play a
crucial role in selective inhibition of ALK5 activity. The
binding mode of 8d generated by flexible docking studies
revealed that the structure of ligand is compatible with
the (NPC-30345)-binding cavity of ALK5,13 and sup-
ported the selective activity of 8d for ALK5.
ꢁ
Doody, J.; Laiho, M.; Wang, X. F.; Massague, J. Cell
1992, 71, 1003.
12. Preparation of 3D––molecular structure and conformational
search: The structure of the ligand (8d) was prepared in.
MOL2 format using the sketcher module of Sybyl 6.9
(SYBYL molecular modeling software, Tripos Inc.: St.
Louis, MO, USA, 2003), Gasteiger–Huckel charges were
assigned to the ligand atoms, and then energy-
minimized until converged to a maximum derivative of
0.001 kcal molꢀ1
ꢀ1. To obtain a global minimum energy
ꢀ
On the basis of these results and its excellent aqueous
solubility data (8d, >100 mg/mL; SB-431542, <1 mg/mL
in 1 N HCl), compound 8d has been selected as a pre-
clinical candidate, and further toxicological and phar-
macological evaluation is currently undergoing in our
laboratory. These data will be published in due course in
elsewhere.
A
conformation of 8d, molecular dynamics was run with a
simulated annealing protocol. The calculation followed
the temperature protocol beginning at 700 K and gradu-
ally cooling down to 200 K for 1000 fs and was run for five
cycles. The dynamics result was analyzed and 200
conformers were randomly selected. The selected con-
formers were briefly minimized and the final coordinates
were saved into a database.
Docking: The X-ray crystal structure of ALK5:NPC-30345
complex (pdb entry ¼ 1IAS)13 was retrieved from the
Protein Data Bank (PDB). The active site was defined as
Acknowledgements
ꢀ
all the amino acid residues enclosed within 6.5 A radius
This work was supported by a grant from KISTEP,
Korea (M1-0310-43-0000).
sphere centered by the bound ligand, NPC-30345. For the
docking of the conformer library of 8d into the target
active site, the FlexX docking and subsequent scoring were
performed using the default parameters of the FlexX
References and notes
program implanted in the Sybyl 6.9. Final score for FlexX
solutions per conformer was calculated by a standard
scoring function, and used for ranking. Top-ranked
conformer of 8d complexed with ALK5 was selected,
and then FlexiDock was run to build a final optimized
model. The binding site was defined as all residues within
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6.5 A distance from 8d. (All basic amino acid residues
ꢁ
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lining the binding site were positively charged.) Rotatable
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bonds, were allowed conformational flexibility in the
docking process, while the backbone and remaining bonds
were held rigid. In the initial FlexX-docked structure of
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within the distance that could form H-bonds with 8d.
Therefore, FlexiDock was performed with the introduc-
tion of constraints for formation of hydrogen bond
between selected donor–acceptor atoms (i.e., donor in
protein: Ser280 and Ser287) in the active site. Flexidock
provided nearly 20 (maximum number of generations to
allow: 30,000) solutions for each docking experiment.
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Figure 2.
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