Design, synthesis, and biological evaluation of novel 2-pyridinyl-[1,2,4] triazoles as inhibitors of transforming growth factor β1 type 1 receptor

Article abstract of DOI:10.1016/j.bmc.2004.03.004

A series of 2-pyridinyl-[1,2,4]triazoles have been synthesized and evaluated for their ALK5 inhibitory activity in the luciferase reporter assays. Compound 12b showed significant ALK5 inhibition (SBE-Luciferase, 73%; p3TP-Luciferase, 85%) at a concentration of 5μM that is comparable to that of SB-431542 (SBE-Luciferase, 79%; p3TP-Luciferase, 88%), but weak p38α MAP kinase inhibition (4%) at a concentration of 10μM that is much lower than that of SB-431542 (54%). The binding mode of 12b generated by flexible docking studies revealed that the structure of 12b is a good fit into the (NPC-30345)-binding cavity of ALK5.

Full text of DOI:10.1016/j.bmc.2004.03.004

Bioorganic & Medicinal Chemistry 12 (2004) 2013–2020
Design, synthesis, and biological evaluation of novel 2-pyridinyl-
[1,2,4]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 30 January 2004; revised 2 March 2004; accepted 2 March 2004
Abstract—A series of 2-pyridinyl-[1,2,4]triazoles have been synthesized and evaluated for their ALK5 inhibitory activity in the
luciferase reporter assays. Compound 12b showed significant ALK5 inhibition (SBE-Luciferase, 73%; p3TP-Luciferase, 85%) at a
concentration of 5 lM that is comparable to that of SB-431542 (SBE-Luciferase, 79%; p3TP-Luciferase, 88%), but weak p38a MAP
kinase inhibition (4%) at a concentration of 10 lM that is much lower than that of SB-431542 (54%). The binding mode of 12b
generated by flexible docking studies revealed that the structure of 12b is a good fit into the (NPC-30345)-binding cavity of ALK5.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Smad4. Smad complexes translocate to the nucleus,
assemble with specific DNA-binding co-factors and
co-modulators to finally activate transcription of extracel-
lular matrix components and inhibitors of matrix-
degrading proteases.2 Therefore, it becomes evident that
inhibition of ALK5 phosphorylation of Smad2/Smad3
could reduce TGF-b1-induced excessive accumulation
of extracellular matrix. The 4-pyridyl substituted tri-
arylimidazoles are known to be selective inhibitors of
p38 MAP kinase since a 40-nitrogen 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 inhibitors.4 SB-431542, one of
the leading 2-pyridyl substituted 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 recognize bone morphoge-
netic proteins, on components of the ERK, JNK, or p38
MAP kinase pathways, or on components of the sig-
naling pathways activated in response to serum.5 On the
basis of these findings, we have synthesized a series of
2-pyridinyl-[1,2,4]triazoles 6a–f, 7a–f, 11a–d, and 12a–d,
and evaluated their ALK5 inhibitory activity in the
luciferase reporter assays.
Progressive tissue fibrosis in the major organs such as
kidney, liver, lung, heart, and skin can lead to significant
organ dysfunction and resulting patient morbidity and
mortality. Many cytokines and growth factors are
involved in fibrogenesis, 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 metallopro-
teinases and plasminogen-activator 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, respec-
tively).2 Upon ligand induced oligomerization, TbR-II
hyperphosphorylates serine/threonine residues in the GS
region of the TbR-I or activin-like kinase 5(ALK5),
which leads to activation of TbR-I by creating a binding
site for Smad proteins. The activated TbR-I in turn
phosphorylates Smad2/Smad3 proteins at the C-termi-
nal SSXS-motif thereby causing dissociation from the
receptor and heteromeric complex formation with
Keywords: 2-Pyridinyl-[1,2,4]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
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.004

2014
D.-K. Kim et al. / Bioorg. Med. Chem. 12 (2004) 2013–2020
2. Results and discussion
the compounds 6a–f to carboxamide was accomplished
by treatment with 28% H2O2 and 6 N NaOH solution to
afford the triazoles 7a–f in 64–84% yields.
The 1-(2-pyridinyl)-[1,2,4]triazoles 6a–f and 7a–f and the
5-(2-pyridinyl)-[1,2,4]triazoles 11a–d and 12a–d were
prepared as shown in Schemes 1 and 2, respectively.
The imidate 2 was reacted with picolinoyl chloride (8a)
or 6-methylpicolinoyl chloride (8b) in toluene in the
presence of Et3N to give N-picolinoylimidates 9a–b,
which were subsequently treated with benzo[1,3]dioxol-
5-ylhydrazine (10a) or 4-methoxyphenylhydrazine (10b)
to afford the 5-(2-pyridinyl)-[1,2,4]triazoles 11a–d in 23–
27% yields. The carboxamides 12a–d were produced
from 11a–d in 72–78% yields in the same reaction con-
dition shown at Scheme 1. The unknown hydrazine 10a
was prepared by diazotization of 3,4-(methylene-
dioxy)aniline (13) with NaNO2 and concentrated HCl
followed by reduction of the resulting diazonium salt
Treatment of 1,4-dicyanobenzene (1) with NaOMe in
MeOH gave 4-cyanobenzimidic acid methyl ester (2)6 in
79% yield. Coupling of the imidate 2 with the aroyl
chlorides 3a–c7 in CH2Cl2 in the presence of Et3N and a
catalytic amount of DMAP followed by cyclization
of the resulting unstable N-aroylimidates 4a–c with
2-hydrazinopyridine (5a) or 2-hydrazino-6-methylpyri-
dine (5b)8 according to the method of Baccar and Bar-
rans9 yielded the 1-(2-pyridinyl)-[1,2,4]triazols 6a–f in
23–35% yields. Conversion of the nitrile functionality of
O
R1COCl
HN
a
3a—c
R1
N
NC
CN
CN
CN
b
MeO
MeO
2 (79%)
1
3a: R1 = benzo[1,3]dioxol-5-yl
3b: R1 = quinoxalin-6-yl
4a: R1 = benzo[1,3]dioxol-5-yl
4b: R1 = quinoxalin-6-yl
3c: R1 = 4-methoxyphenyl
4c: R1 = 4-methoxyphenyl
R1
N
R1
N
N
N
d
c
CN
CONH2
N
N
N
N
R2
N
5a—b
NHNH2
R2
R2
7a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (79%)
7b: R1 = benzo[1,3]dioxol-5yl, R2 = Me (77%)
7c: R1 = quinoxalin-6-yl, R2 = H (70%)
7d: R1 = quinoxalin-6-yl, R2 = Me (64%)
7e: R1 = 4-methoxyphenyl, R2 = H (81%)
7f: R1 = 4-methoxyphenyl, R2 = Me (84%)
6a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (35%)
6b: R1 = benzo[1,3]dioxol-5yl, R2 = Me (26%)
6c: R1 = quinoxalin-6-yl, R2 = H (28%)
6d: R1 = quinoxalin-6-yl, R2 = Me (23%)
6e: R1 = 4-methoxyphenyl, R2 = H (25%)
6f: R1 = 4-methoxyphenyl, R2 = Me (32%)
5a: R2 = H
5b: R2 = Me
Scheme 1. Reagents and conditions: (a) NaOMe (1.0 equiv), MeOH, rt, 22 h; (b) R1COCl (3a–c) (1.0 equiv), Et3N (1.1 equiv), DMAP (cat.), CH2Cl2,
30 ꢁC, 6 h; (c) 5a–b (1.0 equiv), rt, 6 h; (d) 28% H2O2, 6 N NaOH, 95% EtOH, 55 ꢁC, 3 h.
R1NHNH2
10a—b
b
MeO
R2
N
8a—b
a
COCl
HN
CN
CN
N
O
MeO
N
R2
2
8a: R2 = H
8b: R2 = Me
9a: R2 = H
9b: R2 = Me
10a: R1 = benzo[1,3]dioxol-5-yl
10b: R1 = 4-methoxyphenyl
R1
R1
N
N
N
N
N
c
CN
CONH2
N
N
N
R2
11a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (25%)
11b: R1 = benzo[1,3]dioxol-5-yl, R2 = Me (27%)
11c: R1 = 4-methoxyphenyl, R2 = H (25%)
11d: R1 = 4-methoxyphenyl, R2 = Me (23%)
R2
12a: R1 = benzo[1,3]dioxol-5-yl, R2 = H (75%)
12b: R1 = benzo[1,3]dioxol-5-yl, R2 = Me (74%)
12c: R1 = 4-methoxyphenyl, R2 = H (72%)
12d: R1 = 4-methoxyphenyl, R2 = Me (78%)
Scheme 2. Reagents and conditions: (a) 8a–b (1.2 equiv), Et3N (2.0 equiv), toluene, 40 ꢁC, 12 h; (b) R1NHNH2 (10a–b) (1.0 equiv), CH2Cl2, rt, 6 h;
(c) 28% H2O2, 6 N NaOH, 95% EtOH, 55 ꢁC, 3 h.

D.-K. Kim et al. / Bioorg. Med. Chem. 12 (2004) 2013–2020
2015
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).
O
O
O
O
a
NH2
NHNH2
10a (26%)
13
Scheme 3. Reagents and conditions: (a) (i) NaNO2 (2.5 equiv), concd
HCl (10 equiv), H2O, 0 ꢁC, 30 min, (ii) SnCl2Æ2H2O (2.3 equiv), concd
HCl, 15 ꢁC.
Among the 1-(2-pyridinyl)-[1,2,4]triazoles, the benzo-
[1,3]dioxolyl analogue 7b (SBE-Luciferase, 54%; p3TP-
Luciferase, 67%) and the quinoxalinyl analogue 7d
(SBE-Luciferase, 45%; p3TP-Luciferase, 60%) displayed
modest ALK5 inhibition at 5 lM compared to that of
control. Of the 5-(2-pyridinyl)-[1,2,4]triazoles, the
benzo[1,3]dioxolyl analogue 11b displayed modest
ALK5 inhibition (SBE-Luciferase, 52%; p3TP-Lucifer-
ase, 59%), but its carboxamide derivative 12b showed
significantly improved ALK5 inhibitory activity
(SBE-Luciferase, 73%; p3TP-Luciferase, 85%) that is
comparable to that of SB-431542 (SBE-Luciferase, 79%;
p3TP-Luciferase, 88%). P38a MAP kinase is the only
kinase to be significantly affected in vitro on a panel of
with SnCl2Æ2H2O in concentrated HCl in 26% yield
(Scheme 3).
To investigate whether these potential inhibitors could
inhibit TGF-b-induced downstream transcriptional
activation to ALK5 signaling, two different luciferase
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
Table 1. Effect of 2-pyridinyl-[1,2,4]triazoles on the activity of TGF-b-induced ALK5 and p38a MAP kinase
R1
R1
N
N
N
N
N
R3
R3
N
N
N
R2
R2
6a—f, 7a—f
11a—d, 12a—d
Compound
R1
R2
R3
Activity (% control)a
SBE-Luciferaseb
p3TP-Luciferaseb p38a MAP Kinasec
6a
6b
Benzo[1,3]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
Quinoxalin-6-yl
H
CN
CN
86
77
88
87
66
105
75
46
95
55
93
94
93
48
91
62
77
27
86
56
21
80
99
83
91
74
64
79
33
55
40
67
43
72
41
75
53
76
15
78
34
12
98
97
Me
H
6c
6d
CN
CN
102
105
95
Quinoxalin-6-yl
Me
H
6e
4-Methoxyphenyl
4-Methoxyphenyl
Benzo[1,3]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
Quinoxalin-6-yl
CN
CN
6f
7a
Me
H
99
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CN
103
106
108
101
100
110
96
7b
7c
Me
H
7d
7e
Quinoxalin-6-yl
Me
H
4-Methoxyphenyl
4-Methoxyphenyl
Benzo[1,3]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
4-Methoxyphenyl
4-Methoxyphenyl
Benzo[1,3]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
4-Methoxyphenyl
4-Methoxyphenyl
7f
Me
H
11a
11b
11c
11d
12a
12b
12c
12d
Me
H
CN
CN
94
98
Me
H
CN
106
98
CONH2
CONH2
CONH2
CONH2
Me
H
96
95
Me
102
46
O
O
N
CONH2
N
H
N
SB-431542
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.

2016
D.-K. Kim et al. / Bioorg. Med. Chem. 12 (2004) 2013–2020
24 kinases unrelated to the ALKs by SB-431542.5
Therefore, in vitro kinase assay on this enzyme was
performed in the presence of 10 lM of inhibitors. All the
2-pyridinyl-[1,2,4]triazoles tested exhibited weak or no
measurable inhibition for p38a MAP kinase (<10%),
thus showing high selectivity (Table 1).
Previous study suggested that key features of triaryl-
imidazole ALK5 inhibitors are the 2-pyridinyl moiety
and a substituent containing terminal hydrogen bond
donor group (carboxylic acid or carboxamide).4 The X-
ray structure of ALK5 complexed with inhibitor NPC-
30345 revealed that the inhibitor occupies not only the
space for ATP but also the backside of the ATP binding
pocket by contacting Leu278 and Ser280.12 This region
is poorly conserved in other kinases, which commonly
have larger residue at the position equivalent to Ser280
of ALK5. Therefore, it was suggested that the interac-
tion with Ser280 residue gives the specificity of NPC-
30345 for ALK5.12 To obtain an insight into the selec-
tive ALK5 inhibition of 12b, we built a docking model
of ALK5:12b complex using the flexible docking algo-
rithms (FlexiDock and FlexX). As demonstrated in
Figure 1, the inhibitor 12b lies in the same binding
pocket of ALK5 as is occupied by NPC-30345 in the
X-ray structure.12 All three substituents attached to the
central triazole ring of 12b form various hydrogen bonds
(H-bonds) with amino acid residues in the binding
pocket, and the 6-methyl group of pyridine moiety is
positioned close to the hydrophobic residues (Leu278,
Trp249, and Phe262), possibly forming a hydrophobic
interaction (Fig. 1B). The carboxamide group of phenyl
substituent form H-bonds with backbone amide of
Ile211 and Ser287. Two oxygen atoms of 3,4-(methyl-
enedioxy)phenyl ring form strong H-bonds with the
positively charged amino protons of the Lys232 side
chain. Finally, the nitrogen atom in the 2-pyridinyl
moiety forms H-bond with the OH of Ser280, which is
critical for the selectivity of 12b for ALK5 over p38a
MAP kinase. Considering that Thr106 of p38 MAP ki-
nase is located at the position equivalent to Ser280 of
ALK5,13 the interaction between methyl-substituted
2-pyridinyl moiety and Thr106 would not be formed in
the binding pocket of p38 MAP kinase due to the steric
clash. In addition, the interaction between 6-methyl
substituent of 2-pyridinyl and the hydrophobic residues
(Leu278, Trp249, and Phe262) would also play a crucial
role in selective inhibition of ALK5 activity. As shown
in Table 1, the attachment of methyl group at the R2
position of the compounds consistently improves the
ALK5 inhibitory activity. The docking model suggests
that further modification of the R2 position with a
substituent, which induces p–p stacking interactions
with the aromatic amino acid residues (Trp249 and
Phe262) may increase the activity.
Figure 1. (A) Secondary structure of ALK5 docked with 12b. The
binding pocket for the ligand is demonstrated by Connolly surface
(purple). The ligand is rendered in capped stick. Oxygen atoms of the
ligand are red, and nitrogen atoms are blue. (B) The binding mode of
12b 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. Yellow dotted lines are hydrogen
ꢀ
bonding interactions (<2.5 A).
logical evaluation is currently undergoing in our labo-
ratory.
3. Experimental
1H NMR and 13C NMR spectra were recorded on a
Varian Unity 400 spectrophotometer. The chemical
1
shifts for H NMR spectra are reported in parts per
million (ppm) relative to internal tetramethylsilane in
CDCl3 or CD3OD. When CDCl3 or DMSO-d6 was used
as solvent for 13C NMR spectra, it served as the internal
standard at d 77.0 or 39.5, respectively. Electrospray
ionization mass spectra (ESI-MS) were obtained on a
Q-Tof2 mass spectrometer (Micromass, Manchester,
UK). Analytical thin-layer chromatography (TLC) was
performed on Merck silica gel 60F-254 glass plates.
Medium-pressure chromatography (MPLC) was per-
formed using Merck silica gel 60 (230–400 mesh) with a
VSP-2200 ceramic pump (Eyela). Elemental analyses
were performed on a Carlo Erba 1106 elemental ana-
lyzer.
Conclusively, the binding mode of 12b generated by
flexible docking studies revealed that the structure of
the molecule is a good fit into the (NPC-30345)-bind-
ing cavity of ALK5, and well elucidated the selective
activity of 12b for ALK5. On the basis of these
results, compound 12b has been selected as a preclinical
candidate, and further toxicological and pharmaco-

D.-K. Kim et al. / Bioorg. Med. Chem. 12 (2004) 2013–2020
2017
3.1. General procedure for the preparation of the 1-(2-
pyridinyl)-[1,2,4]triazols 6a–f
3.1.4. 4-[1-(6-Methylpyridin-2-yl)-5-quinoxalin-6-yl-1H-
[1,2,4]triazol-3-yl]benzonitrile (6d). Yield 23%; H NMR
1
(CDCl3) d 2.38 (s, 3H), 7.25 (d, 1H, J ¼ 8:0 Hz), 7.54 (d,
1H, J ¼ 8:0 Hz), 7.78 (m, 3H), 8.05 (dd, 1H, J ¼ 8:8 Hz,
2.0 Hz), 8.13 (d, 1H, J ¼ 8:8 Hz), 8.38 (m, 3H), 8.90 (dd,
2H, J ¼ 5:2 Hz, 2.0 Hz); 13C NMR (CDCl3) d 24.09,
113.21, 115.81, 118.97, 124.20, 127.41, 129.52, 130.37,
130.84, 130.87, 132.70, 134.85, 139.47, 142.52, 143.61,
146.00, 146.23, 149.92, 154.79, 158.87, 160.67; MS (EIS)
m=z 390.16 (MHþ). Anal. Calcd for C23H15N7: C, 70.94;
H, 3.88; N, 25.18. Found: C, 71.23; H, 3.54; N, 24.87.
To a stirred solution of 4-cyanobenzimidic acid methyl
ester (2) (4.00 mmol) in CH2Cl2 (25 mL) at room tem-
perature were added aroyl chloride 3a–c (4.00 mmol),
Et3N (4.40 mmol) and a catalytic amount of DMAP
(0.40 mmol). The mixture was warmed to 30 ꢁC and
stirred for 6 h, and to it, 2-hydrazinopyridine (5a) or
2-hydrazino-6-methylpyridine (5b) (4.00 mmol) was
added. The reaction mixture was stirred for an addi-
tional 6 h at room temperature, and to it, water (30 mL)
was added. The organic layer was separated, and the
aqueous layer was extracted with CH2Cl2 (2 · 25 mL).
The combined CH2Cl2 solution was washed with brine
(50 mL), dried (anhydrous Na2SO4), filtered, and evap-
orated to dryness under reduced pressure. The residue
was purified by MPLC on silica gel using a mixture of
Et2O and CH2Cl2 as eluent to afford the titled com-
pound 6a–f as a solid.
3.1.5. 4-[5-(4-Methoxyphenyl)-1-pyridin-2-yl-1H-[1,2,4]tri-
azol-3-yl]benzonitrile (6e). Yield 25%; 1H NMR (CDCl3)
d 3.84 (s, 3H), 6.87 (m, 2H), 7.37(ddd, 1H, J ¼ 7:6 Hz,
5.2 Hz, 1.2 Hz), 7.48 (m, 2H), 7.53 (d, 1H, J ¼ 7:6 Hz),
7.72 (m, 2H), 7.85 (td, 1H, J ¼ 7:2 Hz, 2.0 Hz), 8.32 (dd,
2H, J ¼ 6:8 Hz, 2.0 Hz), 8.51 (dd, 1H, J ¼ 5:2 Hz,
2.0 Hz); 13C NMR (CDCl3) d 55.58, 113.01, 114.16,
116.29, 119.06, 119.79, 120.42, 124.33, 127.17, 127.38,
130.86, 132.63, 135.15, 139.09, 149.37, 151.12, 156.15,
160.49, 161.40; MS (EIS) m=z 354.18 (MHþ). Anal.
Calcd for C21H15N5O: C, 71.38; H, 4.28; N, 19.82.
Found: C, 71.25; H, 4.35; N, 19.75.
3.1.1. 4-(5-Benzo[1,3]dioxol-5-yl-1-pyridin-2-yl-1H-[1,2,4]-
triazol-3-yl)benzonitrile (6a). Yield 35%; 1H NMR
(CDCl3) d 5.99 (s, 2H), 6.79 (d, 1H, J ¼ 8:4 Hz), 7.03 (d,
1H, J ¼ 1:2 Hz), 7.06 (dd, 1H, J ¼ 8:4 Hz, 1.2 Hz), 7.39
(dd, 1H, J ¼ 8:0 Hz, 4.8 Hz), 7.58 (d, 1H, J ¼ 8:4 Hz),
7.73 (d, 2H, J ¼ 8:4 Hz), 7.88 (td, 1H, J ¼ 8:0 Hz,
1.6 Hz), 8.33 (d, 2H, J ¼ 8:4 Hz), 8.52 (dd, 1H,
J ¼ 4:8 Hz, 1.6 Hz); 13C NMR (CDCl3) 101.84, 108.68,
109.59, 119.05, 119.79, 124.10, 124.43, 127.36, 132.66,
139.18, 149.39; MS (EIS) m=z 368.10 (MHþ). Anal.
Calcd for C21H13N5O2: C, 68.66; H, 3.57; N, 19.06.
Found: C, 68.82; H, 3.40; N, 18.88.
3.1.6. 4-[5-(4-Methoxyphenyl)-1-(6-methylpyridin-2-yl)-
1H-[1,2,4]triazol-3-yl]benzonitrile (6f). Yield 32%; 1H
NMR (CDCl3) d 2.51 (s, 3H), 3.80 (s, 3H), 6.85 (dt, 2H,
J ¼ 8:8 Hz, 2.4 Hz), 7.23 (t, 2H, J ¼ 7:2 Hz), 7.49 (dt,
2H, J ¼ 8:8 Hz, 2.4 Hz), 7.70 (m, 3H), 8.32 (dt, 2H,
J ¼ 8:4 Hz, 2.0 Hz); 13C NMR (CDCl3) d 24.37, 55.57,
112.93, 113.99, 116.96, 119.08, 119.79, 120.49, 124.04,
127.41, 130.92, 132.59, 135.28, 139.14, 150.38, 156.11,
159.20, 160.40, 161.35; MS (EIS) m=z 368.16 (MHþ).
Anal. Calcd for C22H17N5O: C, 71.92; H, 4.66; N, 19.06.
Found: C, 71.74; H, 4.85; N, 18.89.
3.1.2.
4-[5-Benzo[1,3]dioxol-5-yl-1-(6-methylpyridin-2-
yl)-1H-[1,2,4]triazol-3-yl]benzonitrile (6b). Yield 26%;
1H NMR (CDCl3) d 2.54 (s, 3H), 5.99 (s, 2H), 6.77 (dd,
1H, J ¼ 8:4 Hz, 0.8 Hz), 7.05 (s, 1H), 7.07 (dd, 1H,
J ¼ 8:4 Hz, 0.8 Hz), 7.26 (t, 2H, J ¼ 7:6 Hz), 7.73 (m,
3H), 8.32 (d, 2H, J ¼ 8:0 Hz); 13C NMR (CDCl3)
d 24.40, 101.79, 108.51, 109.69, 113.00, 116.99, 119.06,
121.82, 124.15, 124.39, 127.39, 132.62, 135.15, 139.20,
147.85, 149.54, 150.23, 159.25, 160.36; MS (EIS) m=z
382.12 (MHþ). Anal. Calcd for C22H15N5O2: C, 69.28;
H, 3.96; N, 18.36. Found: C, 69.01; H, 4.05; N, 18.24.
3.2. General procedure for the preparation of the 1-(2-
pyridinyl)-[1,2,4]triazols 7a–f
To a stirred solution of 6a–f (0.25 mmol) in 95% EtOH
(10 mL) at room temperature were added 28% H2O2
(1.15 mmol) and 6 N NaOH (0.09 mmol) solution. The
mixture was warmed to 55 ꢁC and stirred for 3 h, and to
it, 1 N HCl solution was added to adjust to pH 7 at 0 ꢁC.
The ethanol solvent was evaporated off under reduced
pressure, and the residue was dissolved in CH2Cl2
(40 mL). The CH2Cl2 solution was washed with brine
(20 mL), dried (anhydrous Na2SO4), filtered, and evap-
orated to dryness under reduced pressure. The residue
was purified by MPLC on silica gel using a mixture of
MeOH and CH2Cl2 as eluent to afford the titled com-
pound 7a–f as a solid.
3.1.3. 4-(1-Pyridin-2-yl-5-quinoxalin-6-yl-1H-[1,2,4]tria-
zol-3-yl)benzonitrile (6c). Yield 28%; H NMR (CDCl3)
1
d 7.41 (m, 1H), 7.79 (d, 2H, J ¼ 8:0 Hz), 7.83 (dd, 1H,
J ¼ 8:0 Hz, 1.6 Hz), 7.96 (td, 1H, J ¼ 8:0 Hz, 1.6 Hz),
8.04 (dd, 1H, J ¼ 8:0 Hz, 1.6 Hz), 8.16 (d, 1H,
J ¼ 8:0 Hz), 8.35 (d, 1H, J ¼ 1:6 Hz), 8.40 (m, 3H), 8.90
(dd, 2H, J ¼ 8:0 Hz, 1.6 Hz); 13C NMR (CDCl3) d
113.33, 118.91, 118.96, 124.60, 127.41, 129.86, 130.29,
130.64, 130.84, 132.76, 134.77, 139.46, 142.61, 143.68,
146.04, 146.28, 149.01, 150.72, 154.90, 160.84; MS (EIS)
m=z 376.12 (MHþ). Anal. Calcd for C22H13N7: C, 70.39;
H, 3.49; N, 26.12. Found: C, 70.30; H, 3.53; N, 26.00.
3.2.1. 4-(5-Benzo[1,3]dioxol-5-yl-1-pyridin-2-yl-1H-[1,2,4]-
triazol-3-yl)benzamide (7a). Yield 79%; 1H NMR
(CD3OD) d 6.04 (s, 2H), 6.85 (d, 1H, J ¼ 8:4 Hz), 7.04
(s, 1H), 7.07 (dd, 1H, J ¼ 8:0 Hz, 1.6 Hz), 7.54 (dd, 1H,
J ¼ 8:0 Hz, 4.8 Hz), 7.73 (d, 1H, J ¼ 8:4 Hz), 8.04 (m,

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D.-K. Kim et al. / Bioorg. Med. Chem. 12 (2004) 2013–2020
3H), 8.27 (d, 2H, J ¼ 8:4 Hz), 8.52 (dd, 1H, J ¼ 4:4 Hz,
0.8 Hz); 13C NMR (CD3OD) d 100.99, 107.27, 108.14,
119.23, 120.54, 122.86, 123.82, 125.46, 127.12, 132.55,
133.86, 138.67, 146.97, 147.97, 148.71, 149.82, 154.75,
159.75, 161.88, 162.18, 162.47, 168.94; MS (EIS) m=z
386.10 (MHþ). Anal. Calcd for C21H15N5O3: C, 65.45;
H, 3.92; N, 18.17. Found: C, 65.38; H, 4.13; N, 18.01.
3.2.6. 4-[5-(4-Methoxyphenyl)-1-(6-methylpyridin-2-yl)-
1H-[1,2,4]triazol-3-yl]benzamide (7f). Yield 84%; 1H
NMR (CD3OD) d 2.48 (s, 3H), 3.78 (s, 3H), 4.32 (br d,
2H), 6.85 (d, 2H, J ¼ 7:6 Hz), 7.42 (d, 2H, J ¼ 7:2 Hz),
7.73 (t, 1H, J ¼ 6:4 Hz), 7.90 (d, 2H, J ¼ 7:6 Hz), 8.20
(d, 2H, J ¼ 7:6 Hz); 13C NMR (CD3OD) d 24.18, 55.75,
114.43, 117.65, 120.16, 124.80, 127.23, 128.47, 131.15,
134.08, 134.74, 139.89, 150.31, 156.43, 159.61, 161.34,
161.87, 170.95; MS (EIS) m=z 386.18 (MHþ). Anal.
Calcd for C22H19N5O2: C, 68.56; H, 4.97; N, 18.17.
Found: C, 68.78, H, 4.62; N, 17.98.
3.2.2.
4-[5-Benzo[1,3]dioxol-5-yl-1-(6-methylpyridin-2-
1
yl)-1H-[1,2,4]triazol-3-yl]benzamide (7b). Yield 77%; H
NMR (CDCl3) d 2.53 (s, 3H), 5.85 (br s, 1H), 5.98 (s,
2H), 6.14 (br s, 1H), 6.77 (d, 1H, J ¼ 8:4 Hz), 7.07 (m,
2H), 7.23 (d, 1H, J ¼ 8:0 Hz), 7.27 (d, 1H, J ¼ 8:0 Hz),
7.71 (t, 1H, J ¼ 8:0 Hz), 7.88 (d, 2H, J ¼ 8:4 Hz), 8.29
(d, 2H, J ¼ 8:4 Hz); 13C NMR (CDCl3) d 24.39, 101.74,
108.48, 109.74, 117.00, 122.02, 124.01, 124.15, 127.11,
127.88, 134.05, 134.32, 139.15, 147.79, 149.41, 150.32,
155.64, 159.15, 161.04, 169.23; MS (EIS) m=z 400.12
(MHþ). Anal. Calcd for C22H17N5O3: C, 66.16; H, 4.29;
N, 17.53. Found: C, 66.43; H, 4.21; N, 17.39.
3.3. Benzo[1,3]dioxol-5-ylhydrazine (10a)
To a stirred solution of benzo[1,3]dioxol-5-ylamine
(4.08 g, 30 mmol) in H2O (20 mL) and concentrated HCl
(31 mL, 300 mmol) at 0 ꢁC was added NaNO2 (2.47 g,
36 mmol) dissolved in H2O (8 mL), and the mixture was
stirred for 30 min. The reaction mixture was poured into
a stirred solution of SnCl2Æ2H2O (15.5 g, 68 mmol) in
concentrated HCl (38 mL) at 15 ꢁC, and stirred for an
additional 30 min. The precipitates were filtered, washed
with H2O, and suspended in H2O (80 mL) and 6 N
NaOH (10 mL) solution. The suspension was stirred for
30 min at room temperature and filtered, and the filtered
solution was extracted with CH2Cl2 (200 mL). The
CH2Cl2 solution was washed with brine (50 mL), dried
(anhydrous MgSO4), filtered, and evaporated to dryness
under reduced pressure. The residue was purified by
MPLC on silica gel using a mixture of MeOH and
CH2Cl2 (5:95) as eluent to afford 10a (1.15 g, 26%) as a
3.2.3. 4-(1-Pyridin-2-yl-5-quinoxalin-6-yl-1H-[1,2,4]tria-
zol-3-yl)benzamide (7c). Yield 70%; H NMR (CDCl3)
1
d 5.71 (br s, 1H), 6.11 (br s, 1H), 7.36 (m, 1H), 7.81 (d,
1H, J ¼ 8:4 Hz), 7.92 (m, 3H), 8.02 (dd, 1H, J ¼ 8:8 Hz,
2.0 Hz), 8.12 (d, 1H, J ¼ 8:8 Hz), 8.34 (m, 4H), 8.86 (dd,
2H, J ¼ 6:0 Hz, 2.0 Hz); 13C NMR (CDCl3) d 118.93,
124.46, 127.16, 128.04, 129.82, 130.54, 130.78, 130.81,
134.00, 134.32, 146.00, 146.21, 148.97, 161.55, 168.97;
MS (EIS) m=z 394.16 (MHþ). Anal. Calcd for
C22H15N7O: C, 67.17; H, 3.84; N, 24.92. Found: C,
66.92; H, 3.92; N, 24.83.
1
solid: H NMR (CDCl3) d 3.50 (br s, 2H), 5.00 (br s,
1H), 5.86 (s, 2H), 6.24 (dd, 1H, J ¼ 8:4 Hz, 2.4 Hz), 6.44
(d, 1H, J ¼ 2:4 Hz), 6.67 (d, 1H, J ¼ 8:4 Hz); 13C NMR
(CDCl3) d 95.79, 101.06, 104.12, 108.62, 147.29; MS
(EIS) m=z 153.04 (MHþ). Anal. Calcd for C7H8N2O2: C,
55.26; H, 5.30; N, 18.41. Found: C, 55.03; H, 5.35; N, 18.32.
3.2.4. 4-[1-(6-Methylpyridin-2-yl)-5-quinoxalin-6-yl-1H-
[1,2,4]triazol-3-yl]benzamide (7d). Yield 64%; H NMR
1
(CDCl3) d 2.31 (s, 3H), 5.71 (br s, 1H), 6.13 (br s, 1H),
7.17 (d, 1H, J ¼ 7:6 Hz), 7.49 (d, 1H, J ¼ 8:4 Hz) 7.72
(d, 1H, J ¼ 7:6 Hz), 7.88 (d, 2H, J ¼ 8:4 Hz), 8.00 (dd,
1H, J ¼ 8:4 Hz, 2.0 Hz), 8.07 (d, 1H, J ¼ 8:4 Hz), 8.31
(m, 3H), 8.83 (dd, 2H, J ¼ 4:4 Hz, 1.6 Hz); 13C NMR
(DMSO-d6) d 23.21, 115.42, 123.58, 125.72, 127.78,
128.56, 129.67, 129.79, 130.01, 132.43, 134.96, 139.30,
141.52, 142.59, 145.88, 146.08, 149.22, 153.71, 157.49,
160.29, 167.62; MS (EIS) m=z 408.20 (MHþ). Anal.
Calcd for C23H17N7O: C, 67.80; H, 4.21; N, 24.06.
Found: C, 68.12; H, 4.04; N, 23.72.
3.4. General procedure for the preparation of the 5-(2-
pyridinyl)-[1,2,4]triazoles 11a–d
To a stirred solution of 2 (0.56 mmol) in toluene (4 mL)
at room temperature were added picolinoyl chloride (8a)
or 6-methylpicolinoyl chloride (8b) (0.67 mmol) and
Et3N (1.12 mmol). The mixture was warmed to 40 ꢁC,
stirred for 12 h, and filtered. The filtered solution was
evaporated to dryness, and the residue was dissolved in
CH2Cl2 (4 mL), and to it, benzo[1,3]dioxol-5-ylhydr-
azine (10a) or 4-methoxyphenylhydrazine (10b)
(0.56 mmol) was added. The mixture was stirred for 6 h
at room temperature and evaporated to dryness, and the
residue was purified by MPLC on silica gel using a
mixture of EtOAc and hexane as eluent to afford the
titled compound 11a–d as a solid.
3.2.5. 4-[5-(4-Methoxyphenyl)-1-pyridin-2-yl-1H-[1,2,4]-
triazol-3-yl]benzamide (7e). Yield 81%; 1H NMR
(CDCl3) d 3.84 (s, 3H), 5.71 (br s, 1H), 6.16 (br s, 1H),
6.90 (dt, 2H, J ¼ 8:8 Hz, 2.0 Hz), 7.39 (ddd, 1H,
J ¼ 7:4 Hz, 5.0 Hz, 1.0 Hz), 7.53 (dt, 2H, J ¼ 8:8 Hz,
2.0 Hz), 7.58 (d, 1H, J ¼ 8:0 Hz), 7.88 (td, 1H,
J ¼ 7:2 Hz, 2.0 Hz), 7.91 (d, 2H, J ¼ 8:8 Hz), 8.34 (dt,
1H, J ¼ 8:0 Hz, 2.0 Hz), 8.53 (m, 1H); 13C NMR
(CDCl3) d 55.58, 114.13, 119.80, 119.79, 120.66, 124.17,
127.10, 127.90, 130.88, 134.07, 134.34, 139.04, 149.23,
155.94, 161.19, 161.30, 169.10; MS (EIS) m=z 372.16
(MHþ). Anal. Calcd for C21H17N5O2: C, 67.91; H, 4.61;
N, 18.86. Found: C, 67.73; H, 4.85; N, 18.83.
3.4.1. 4-(1-Benzo[1,3]dioxol-5-yl-5-pyridin-2-yl-1H-[1,2,4]-
triazol-3-yl)benzonitrile (11a). Yield 25%; 1H NMR
(CDCl3) d 6.00 (s, 2H), 6.76 (dd, 1H, J ¼ 8:2 Hz,
1.4 Hz), 6.83 (m, 1H), 6.89 (dd, 1H, J ¼ 5:4 Hz, 1.8 Hz),
7.29 (m, 1H), 7.70 (m, 1H), 7.75 (m, 1H), 7.87 (m, 1H),

D.-K. Kim et al. / Bioorg. Med. Chem. 12 (2004) 2013–2020
2019
8.07 (m, 1H), 8.28 (m, 2H), 8.51 (m, 1H); MS (EIS) m=z
368.10 (MHþ). Anal. Calcd for C21H13N5O2: C, 68.66;
H, 3.57; N, 19.06. Found: C, 68.93; H, 3.40; N, 18.83.
107.57, 108.05, 109.31, 119.97, 121.64, 124.38, 127.02,
127.91, 133.95, 134.37, 137.10, 146.62, 158.83, 160.79;
MS (EIS) m=z 400.14 (MHþ). Anal. Calcd for
C22H17N5O3: C, 66.16; H, 4.29; N, 17.53. Found: C,
65.88; H, 4.43; N, 17.35.
3.4.2.
4-[1-Benzo[1,3]dioxol-5-yl-5-(6-methylpyridin-2-
yl)-1H-[1,2,4]triazol-3-yl]benzonitrile (11b). Yield 27%;
1H NMR (CDCl3) d 2.41 (s, 3H), 6.02 (s, 2H), 6.78 (dd,
1H, J ¼ 8:4 Hz, 1.6 Hz), 6.85 (m, 1H), 6.92 (d, 1H,
J ¼ 1:6 Hz), 7.16 (m, 1H), 7.62 (m, 2H), 7.71 (m, 2H),
8.31 (m, 2H); MS (EIS) m=z 382.12 (MHþ). Anal. Calcd
for C22H15N5O2: C, 69.28; H, 3.96; N, 18.36. Found: C,
69.00; H, 4.23; N, 18.29.
3.5.3. 4-[1-(4-Methoxyphenyl)-5-pyridin-2-yl-1H-[1,2,4]-
triazol-3-yl]benzamide (12c). Yield 72%; 1H NMR
(CDCl3) d 3.86 (s, 3H), 5.70 (br s, 1H), 6.18 (br s, 1H),
6.94 (m, 2H), 7.32 (m, 1H), 7.37 (m, 2H), 7.77 (td, 1H,
J ¼ 8:0 Hz, 2.0 Hz), 7.90 (m, 3H), 8.34 (d, 2H,
J ¼ 8:0 Hz), 8.55 (m, 1H); MS (EIS) m=z 372.16 (MHþ).
Anal. Calcd for C21H17N5O2: C, 67.91; H, 4.61; N,
18.86. Found: C, 68.25; H, 4.38; N, 18.64.
3.4.3. 4-[1-(4-Methoxyphenyl)-5-pyridin-2-yl-1H-[1,2,4]-
triazol-3-yl]benzonitrile (11c). Yield 25%; 1H NMR
(CDCl3) d 3.82 (s, 3H), 6.91 (m, 2H), 7.29 (m, 1H), 7.32
(m, 2H), 7.71 (d, 2H, J ¼ 8:4 Hz), 7.76 (dd, 1H,
J ¼ 7:8 Hz, 1.2 Hz), 7.84 (d, 1H, J ¼ 7:8 Hz), 8.32 (d,
2H, J ¼ 8:4 Hz), 8.52 (d, 1H, J ¼ 4:4 Hz); 13C NMR
(CDCl3) d 55.77, 112.89, 114.38, 119.04, 124.62, 124.75,
127.02, 127.23, 131.72, 132.65, 135.16, 136.99, 147.34,
149.81, 153.98, 160.07, 160.13; MS (EIS) m=z 354.20
(MHþ). Anal. Calcd for C21H15N5O: C, 71.38; H, 4.28;
N, 19.82. Found: C, 71.52; H, 4.02; N, 19.69.
3.5.4. 4-[1-(4-Methoxyphenyl)-5-(6-methylpyridin-2-yl)-
1H-[1,2,4]triazol-3-yl]benzamide (12d). Yield 78%; 1H
NMR (CDCl3) d 2.39 (s, 3H), 3.82 (s, 3H), 5.80 (br s,
1H), 6.19 (br s, 1H), 6.89 (m, 2H), 7.13 (dd, 1H,
J ¼ 6:2 Hz, 2.6 Hz), 7.34 (m, 2H), 7.59 (m, 2H), 7.87 (d,
2H, J ¼ 8:4 Hz), 8.29 (d, 2H, J ¼ 8:4 Hz); 13C NMR
(CDCl3) d 24.50, 55.80, 114.11, 121.61, 124.28, 126.98,
127.27, 127.88, 132.00, 133.93, 134.42, 137.03, 146.71,
154.05, 158.80, 159.96, 160.77, 169.21; MS (EIS) m=z
386.19 (MHþ). Anal. Calcd for C22H19N5O2: C, 68.56;
H, 4.97; N, 18.17. Found: C, 68.50; H, 5.12; N, 17.98.
3.4.4. 4-[1-(4-Methoxyphenyl)-5-(6-methylpyridin-2-yl)-
1
1H-[1,2,4]triazol-3-yl]benzonitrile (11d). Yield 23%; H
3.6. P38 kinase inhibition assay
NMR (CDCl3) d 2.40 (s, 3H), 3.83 (s, 3H), 6.91 (m, 2H),
7.15 (dd, 1H, J ¼ 6:6 Hz, 1.8 Hz), 7.33 (m, 2H), 7.60 (m,
2H), 7.72 (d, 2H, J ¼ 8:8 Hz), 8.33 (d, 2H, J ¼ 8:8 Hz);
MS (EIS) m=z 368.16 (MHþ). Anal. Calcd for
C22H17N5O: C, 71.92; H, 4.66; N, 19.06. Found: C,
72.23; H, 4.44; N, 18.98.
P38a kinase assay was performed according to the
instruction manual of assay kit provided by the manu-
facturer (Upstate Biotechnology). Briefly, to activate
MAPKAP kinase-2, 10 lL of reaction mixture con-
tained 200 ng of inactive MAPKAP kinase-2, 0.06 unit
of purified p38a kinase and 2 lL of magnesium/ATP
cocktail (75 mM MgCl2/500 lM cold ATP) were mixed
by vortexing and incubated for 15 min at 30 ꢁC with
agitation. Before activation reaction was started, inhib-
itors dissolved in DMSO were added to the reaction
tube with 10 lM of final concentration. After activation,
10 lCi [c-32P]ATP and 10 lL of 0.86 mM MAPKAP
kinase-2 substrate were added, and the mixture was
incubated for 10 min at 30 ꢁC with agitation. Then,
40 lL of reaction mixture was transferred into P81
phosphocellulose paper. After extensively washing the
paper three times with 40 mL of 0.75% phosphoric acid,
the bound radioactivity was determined by liquid scin-
tillation counter.
3.5. General procedure for the preparation of the 5-(2-
pyridinyl)-[1,2,4]triazoles 12a–d is the same as for the
compounds 7a–f
3.5.1. 4-(1-Benzo[1,3]dioxol-5-yl-5-pyridin-2-yl-1H-[1,2,4]-
triazol-3-yl)benzamide (12a). Yield 75%; 1H NMR
(CDCl3) d 5.81 (br s, 1H), 6.02 (s, 2H), 6.17 (br s, 1H),
6.78 (d, 1H, J ¼ 8:0 Hz), 6.85 (dd, 1H, J ¼ 8:0 Hz,
2.0 Hz), 6.92 (d, 1H, J ¼ 2:0 Hz), 7.29 (ddd, 1H,
J ¼ 8:0 Hz, 4.8 Hz, 1.2 Hz), 7.77 (td, 1H, J ¼ 8:0 Hz,
1.6 Hz), 7.88 (m, 3H), 8.29 (d, 2H, J ¼ 8:4 Hz), 8.53 (m,
1H); 13C NMR (CDCl3) d 102.19, 107.32, 108.21,
119.71, 124.66, 124.71, 127.00, 127.94, 132.85, 134.06,
134.26, 137.00, 147.46, 148.11, 148.26, 149.79, 153.86,
160.84, 169.19; MS (EIS) m=z 386.12 (MHþ). Anal.
Calcd for C21H15N5O3: C, 65.45; H, 3.92; N, 18.17.
Found: C, 65.30; H, 4.13; N, 17.88.
3.7. Flexible docking
The FlexiDock module in Sybyl 6.9 (SYBYL molecular
modeling software, Tripos Inc.: St. Louis, MO, USA,
2003) was used to dock 12b onto the active site of
ALK5. To ensure similar starting geometry of the ligand
in the binding site of known inhibitor NPC-30345, the
crystal structure of ALK5:NPC-30345 complex (pdb
entry ¼ 1IAS)12 was used as a reference. (Since the chemi-
cal structure of NPC-30345 is not revealed in the refined
X-ray structure, the electron density for NPC-30345
3.5.2.
4-[1-Benzo[1,3]dioxol-5-yl-5-(6-methylpyridin-2-
yl)-1H-[1,2,4]triazol-3-yl]benzamide (12b). Yield 74%;
1H NMR (CDCl3) d 2.46 (s, 3H), 5.60 (br s, 1H), 6.05 (s,
2H), 6.10 (br s, 1H), 6.82 (d, 1H, J ¼ 8:0 Hz), 6.90 (dd,
1H, J ¼ 8:0 Hz, 1.6 Hz), 6.98 (d, 1H, J ¼ 1:6 Hz), 7.19
(m, 1H), 7.66 (m, 2H), 7.91 (d, 2H, J ¼ 8:0 Hz), 8.33 (d,
2H, J ¼ 8:0 Hz); 13C NMR (CDCl3) d 24.57, 102.13,

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D.-K. Kim et al. / Bioorg. Med. Chem. 12 (2004) 2013–2020
was viewed with molecular graphics program O14
and used to locate the docking position for 12b.) In the
initial docked structure of ALK5:12b complex, Lys232
and Ser287 are positioned within the distance that could
form H-bonds with the acceptor atoms of 12b. The
References and notes
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(b) Branton, M. H.; Kopp, J. B. Microbes Infect. 1999, 1,
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2001, 99, 308; (d) Giri, S. N. Annu. Rev. Pharmacol.
Toxicol. 2003, 43, 73.
ꢀ
binding site was defined as all residues within 6.5 A
distance from 12b, and all basic amino acid residues
lining the binding site were positively charged. Rotat-
able bonds of these residues, primarily the side chain
single bonds, were allowed conformational flexibility in
the docking process, while the backbone and remaining
bonds were held rigid. Docking was performed with the
introduction of constraints for formation of hydrogen
bond between selected donor–acceptor atoms (i.e.,
donor in protein: Lys232 and Ser287) in the active site.
FlexiDock provided nearly 20 (maximum number of
generations to allow: 30,000) solutions for each docking
experiment. Each of these structures was minimized to
eliminate bad electronic and/or steric contacts, and the
structure with lowest energy was selected. The selected
structure of ALK5:12b complex was used as a receptor
for further flexible docking of 12b to build a final opti-
mized model. The FlexX docking and subsequent scor-
ing were performed using the default parameters of the
ꢁ
2. (a) Massague, J. Annu. Rev. Biochem. 1998, 67, 753; (b)
Lutz, M.; Knaus, P. Cell. Signal. 2002, 14, 977.
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S.; Cobb, M. H.; Young, P. R.; Abdel-Meguid, S.; Adams,
J. L.; Goldsmith, E. J. Structure (London) 1998, 6, 1117;
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N. J. J. Med. Chem. 2002, 45, 999.
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ꢁ
FlexX program implanted in the Sybyl. For the docking
of 12b into the target active site, the main settings are
1000 solutions per iteration during the incremental
construction algorithm and a maximum protein–ligand
3
ꢀ
atom–atom overlap of 2.5 A. Final scores for all FlexX
solutions (up to 1000) per compound were calculated by
a standard scoring function, and used for database
ranking. Top-ranked conformer of 12b complexed with
ALK5 was selected as a final model shown in Figure 1.
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Acknowledgements
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€
13. Yakymovych, I.; Engstrom, U.; Grimsby, S.; Heldin, C.-H.;
This work was supported by the Ewha Womans
University Research Grant of 2002 and a grant from
KISTEP, Korea (M1-0310-43-0000).
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