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

Article abstract of DOI:10.1016/j.bmc.2010.04.071

A series of 4(5)-(6-methylpyridin-2-yl)imidazoles 16-19 and -pyrazoles 22-29, 33, and 34 have been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in cell-based luciferase reporter assays. The 6-quinolinyl imidazole analogs 16 and 18 inhibited ALK5 phosphorylation with IC₅₀ values of 0.026 and 0.034 μM, respectively. In a luciferase reporter assay using HaCaT cells transiently transfected with p3TP-luc reporter construct, 18 displayed 66% inhibition at 0.05 μM, while competitor compounds 2 and 3 showed 44% inhibition. The binding mode of 18 generated by flexible docking studies with ALK5:18 complex shows that it fits well into the active site cavity of ALK5 by forming broad and tight interactions.

Full text of DOI:10.1016/j.bmc.2010.04.071

Bioorganic & Medicinal Chemistry 18 (2010) 4459–4467
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 4(5)-(6-methylpyridin-2-yl)imidazoles
and -pyrazoles as transforming growth factor-b type 1 receptor kinase inhibitors
a
a
a
a
Dae-Kee Kim a, , Yeon-Im Lee , Yeon Woo Lee , Purushottam M. Dewang , Yhun Yhong Sheen ,
*
Yeo Woon Kim a, Hyun-Ju Park b, Jakyung Yoo b, Ho Soon Lee c, Yong-Kook Kim c
a College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Republic of Korea
b College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
c In2Gen Co., Ltd, 608 Daerung Posttower II Building, 182-13 Guro-dong, Guro-gu, Seoul 152-050, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4(5)-(6-methylpyridin-2-yl)imidazoles 16–19 and -pyrazoles 22–29, 33, and 34 have been
synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in cell-based lucif-
erase reporter assays. The 6-quinolinyl imidazole analogs 16 and 18 inhibited ALK5 phosphorylation with
Received 4 March 2010
Revised 20 April 2010
Accepted 21 April 2010
Available online 28 April 2010
IC50 values of 0.026 and 0.034
lM, respectively. In a luciferase reporter assay using HaCaT cells tran-
siently transfected with p3TP-luc reporter construct, 18 displayed 66% inhibition at 0.05
l
M, while com-
petitor compounds 2 and 3 showed 44% inhibition. The binding mode of 18 generated by flexible docking
studies with ALK5:18 complex shows that it fits well into the active site cavity of ALK5 by forming broad
and tight interactions.
Keywords:
TGF-b
ALK5 inhibitor
Cell-based luciferase reporter assays
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
strated an association of TGF-b with the initiation and progres-
sion of fibrosis in a variety of organ systems such as kidney,4
Transforming growth factor-b (TGF-b) is the superfamily of
proteins which includes TGF-b1, TGF-b2, and TGF-b3. These cyto-
kines are pleiotropic modulators of cell proliferation and differ-
entiation, wound healing, extracellular matrix production, and
immunosuppression.1 Other members of this superfamily include
activins, inhibins, bone morphogenetic proteins, growth and dif-
ferentiation factors, and Mullerian inhibiting substance. TGF-b1
represents the prototypic member of the TGF-b superfamily.
TGF-b1 transduces signals through two highly conserved trans-
membrane serine/threonine kinases, the type I (ALK5) and type
II TGF-b receptors. The ligand induced oligomerization promotes
the type II receptor to hyperphosphorylate serine/threonine resi-
dues in the GS region of the ALK5.2 This phosphorylation leads to
activation of the ALK5 by creating a binding site for Smad pro-
teins. The activated ALK5 in turn phosphorylates Smad2 and
Smad3 proteins thereby causing their 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
transcription of extracellular matrix components and inhibitors
of matrix-degrading proteases.3 Numerous studies have demon-
heart,5 lung,6 and liver.7 TGF-b signaling and over-expression of
TGF-b receptors are linked to various human diseases including
cancer,8 pancreatic diseases,9 and hematological malignancies.10
Inhibition of the catalytic activity of ALK5 by the use of small
molecule inhibitors is one of the strategies used to normalize
TGF-b signaling. Several ALK5 inhibitors such as
1 (SB-
431542),11 2 (SB-505124),12 3 (SB-525334),13 4 (GW6604),14
5
(SD-208),15,16 and 6 (LY580276),17 are under preclinical develop-
ment (Fig. 1). These and other similar inhibitors have become a
subject of intense investigations.11,18–21
We have also prepared the 2-pyridinyl-substituted tria-
zoles,22,23 imidazoles such as 7 (IN-1166),24,25 and thiazoles26 hav-
ing
a carbonitrile-, carboxamide-, or sulfonamide-substituted
phenyl or benzyl moiety as ALK5 inhibitors and found that both
introduction of a carbonitrile, carboxamide, or sulfonamide group
at meta- or para-position in the phenyl ring and incorporation of
a methylene, a methyleneamino, or an aminomethylene linkage
between a central heterocyclic ring and a phenyl ring significantly
increased ALK5 inhibitory activity. Among them, 8 (IN-1130) effec-
tively suppressed renal fibrosis induced by unilateral ureteral
obstruction (UUO) in rats27 and ameliorated experimental autoim-
mune encephalomyelitis (EAE) in SBE-luc and GFAP-luc mice
28
immunized with MOG35–55
.
Additionally, it has been observed
that the injection of 8 into the tunica albuginea in rats lessened
* Corresponding author. Tel.: +82 2 3277 3025; fax: +82 2 3277 2467.
E-mail address: dkkim@ewha.ac.kr (D.-K. Kim).
tunical fibrosis and corrected penile curvature.29
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.071

4460
D.-K. Kim et al. / Bioorg. Med. Chem. 18 (2010) 4459–4467
O
N
O
N
O
N
N
N
O
O
N
N
H
NH
N
H
N
H
NH
2
N
N
N
N
N
1 (SB-431542)
2 (SB-505124)
3 (SB-525334)
4 (GW6604)
F
F
N
N
CN
O
N
N
Cl
N
N
HN
N
NH
N
2
N
N
N
N
N
H
N
H
NH
N
N
N
N
5 (SD-208)
6 (LY580276)
7 (IN-1166)
8 (IN-1130)
Figure 1. ALK5 inhibitors under development.
est to us whether replacement of the 6-quinoxalinyl of 8 with a dif-
ferent heteroaryl moiety could reduce metabolic hydroxylation.
Since the N-1 in the 6-quinoxalinyl of 8 is essential for hydro-
gen-bond interaction in the ATP binding site, we attempted to re-
N
N
O
HO
N
NH
2
N
H
place the 6-quinoxalinyl of
8 with a 6-quinoliny or 1,5-
naphthyridin-2-yl moiety. We prepared the imidazole derivatives
16–19 and the pyrazole derivatives 22–29, 33, and 34 to examine
the effect of the central heterocyclic ring and optimal positioning
of a carbonitrile- or carboxamide-substituted benzyl moiety on
ALK5 inhibition.
N
9
In pharmacokinetic studies with 8, a major metabolite of 8 was
detected in the systemic circulation of rat and mouse and was puri-
fied and tentatively characterized as 3-((4-(2-hydroxyquinoxalin-
6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)benzam-
ide or 3-((4-(3-hydroxyquinoxalin-6-yl)-5-(6-methylpyridin-2-yl)-
1H-imidazol-2-yl)methyl)benzamide) (9) based on its 1H NMR
spectra and LC-MS/MS spectra.30 Metabolic hydroxylation of 8 at
the 2- or 3-position of the 6-quinoxaliny moiety significantly
reduced its biological activity. Therefore, it was of particular inter-
2. Results and discussion
2.1. Chemistry
The 4(5)-(6-methylpyridin-2-yl)imidazoles 16–19 were
prepared as shown in Scheme 1. Treatment of 1031 and 1132 with
N
N
N
OH
CN
N
O
N
X
X
X
b
a
OH
N
O
N
N
N
14 : X = CH (39%)
15 : X = N (35%)
10 : X = CH
11
12 : X = CH (87%)
13 : X = N (81%)
: X = N
O
N
N
CN
N
NH
N
2
X
X
c
d
N
H
N
H
N
N
18
16 : X = CH (61%)
17 : X = N (59%)
: X = CH (69%)
19 : X = N (90%)
Scheme 1. Reagents and conditions: (a) NaNO2, 18% HCl, 0 °C then rt, 30 min; (b) 3-cyanophenylacetaldehyde, NH4OAc, AcOH, 110 °C, 16 h; (c) triethyl phosphite, anhydrous
DMF, 110 °C, 16 h; (d) 28% H2O2, 1 N NaOH, 95% EtOH, 60 °C, 2 h.

D.-K. Kim et al. / Bioorg. Med. Chem. 18 (2010) 4459–4467
4461
NaNO2 in 18% HCl solution at room temperature gave the
respective hydroxyimino ketones 12 and 13. Cyclization of these
compounds with 3-cyanophenylacetaldehyde in the presence of
NH4Ac in AcOH afforded the hydroxyl imidazoles 14 and 15 in
39% and 35% yields, respectively. Dehydroxylation of the resulting
imidazoles with triethyl phosphite in anhydrous DMF at 110 °C
produced 1H-imidazoles 16 and 17 in 61% and 59% yields. Conver-
sion of the benzonitriles 16 and 17 to the carboxamide derivatives
18 and 19 was accomplished by treatment of 28% H2O2 and 1 N
NaOH in 95% ethanol.
The counterpart pyrazole derivatives 22–29 were prepared as
shown in Scheme 2. Treatment of the ketones 10 and 11 with
N,N-dimethylformamide dimethyl acetal (DMFÁDMA) in the pres-
ence of AcOH in DMF, followed by reaction with hydrazine mono-
hydrate, produced the pyrazoles 2031 and 21.32 Alkylation of 20 or
of Cs2CO3 gave the two positional isomers 22 and 23 or 24 and 25,
respectively. The positional isomers were separated by flash col-
umn chromatography and identified by NOE experiments. In NOE
experiments, irradiation of the benzylic methylene protons of the
derivatives 22 and 24 gave enhancement for proton H-5 in pyra-
zole ring, while irradiation of the similar methylene protons for
compounds 23 and 25 gave enhancement of methyl protons at-
tached at 6-position of pyridyl ring, confirming the respective posi-
tions of benzylic substitutions. Then the nitrile group was
hydrolyzed to carboxamide as mentioned above for Scheme 1.
The 6-quinolinyl substituted pyrazole isomers 33 and 34 were
prepared as shown in Scheme 3. Commercially available quino-
line-6-carboxylic acid was converted to Weinreb amide 3033 in a
quantitative yield using peptide coupling conditions. The amide
30 was coupled with an anion of 2,6-lutidine generated by treat-
ment with n-BuLi to obtain the ketone 31 in 64% yield. The ketone
21 with a-bromo-m-tolunitrile in anhydrous DMF in the presence
NC
N
N
N
X
b
X
X
10
or
a
NH
N
N
CN
N
N
N
+
11
N
N
N
23
: X = CH (40%)
20 : X = CH (76%)
21 : X = N (72%)
22 : X = CH (59%)
24 : X = N (68%)
25 : X = N (24%)
c
c
NH
2
O
N
N
N
X
O
N
X
N
NH
2
N
N
N
27
26 : X = CH (70%)
28 : X = N (92%)
: X = CH (80%)
29 : X = N (93%)
Scheme 2. Reagents and conditions: (a) (i) DMFÁDMA, AcOH, DMF, rt, 1 h; (ii) N2H4ÁH2O, 50 °C, 2 h; (b)
1 N NaOH, 95% EtOH, 60 °C, 1 h.
a-bromo-m-tolunitrile, Cs2CO3, anhydrous DMF, rt, 2 h; (c) 28% H2O2,
N
N
N
O
a
N
b
OH
O
O
O
N
30
(99.7%)
31
(64% )
R
N
N
N
N
d
c
NH
N
N
N
33 : R = CN (79%)
34
32 (44% )
e
: R = CONH (89% )
2
Scheme 3. Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, PYBOP, 1-hydroxybenzotriazole (HOBt), Et3N, anhydrous DMF, rt, 16 h; (b) 2,6-lutidine,
n-BuLi, anhydrous THF, Ar atmosphere, À78 °C for 10 min then rt; (c) (i) DMFÁDMA, THF, 48 h; (ii) N2H4ÁH2O, EtOH, rt, 12 h; (d)
a-bromo-m-tolunitrile, Cs2CO3, anhydrous
DMF, 80 °C, 12 h; (e) 28% H2O2, 1 N NaOH, 95% EtOH, 60 °C, 1 h.

4462
D.-K. Kim et al. / Bioorg. Med. Chem. 18 (2010) 4459–4467
31 was cyclized to the desired pyrazole 32 which was alkylated
with -bromo-m-tolunitrile to give the benzonitrile 33 using the
than 8, and compound 18 showed the similar level of inhibitory
activity to that of 8. These two compounds 16 and 18 displayed
a
conditions mentioned for Scheme 2. Conversion of the nitrile func-
tionality of the compound 33 to carboxamide was accomplished as
mentioned above to give the pyrazole 34.
much higher ALK5 inhibitory activity than 2 (IC50 = 0.169
3 (IC50 = 0.162 M).
lM) and
l
To evaluate TGF-b-induced downstream transcriptional activa-
tion to ALK5 signaling, cell-based luciferase activity of 16–19,
2.2. Biological evaluation
To investigate whether these inhibitors 16–19, 22–29, 33, and
34 could inhibit ALK5, a kinase assay was performed using the puri-
fied human ALK5 kinase domain produced in Sf9 insect cells.
Among them, 1-benzyl-5-(pyridin-2-yl)pyrazole derivatives 23,
25, 27, and 29 displayed no significant ALK5 inhibitory activity up
3TP-Luc luciferase assay
300000
18
8
2
3
250000
200000
150000
100000
50000
0
to a concentration of 10 lM (Table 1). The compounds possessing
a 6-quinolinyl moiety showed higher ALK5 inhibitory activity than
the compounds possessing a 1,5-naphthyridin-2-yl moiety. In the
case of imidazole derivatives, the 6-quinolinyl analogs 16
(IC50 = 0.026
more potent than the corresponding 1,5-naphthyridin-2-yl analogs
17 (IC50 = 2.25 M) and 19 (IC50 = 2.60 M). However, the
lM) and 18 (IC50 = 0.034 lM) are 87- and 76-fold
l
l
difference in inhibitory activity with respect change in pyrazole
derivatives was less profound. The 6-quinolinyl analogs 22
(IC50 = 0.231
fold higher ALK5 inhibitory activity than the 1,5-naphthyridin-2-
yl analogs 24 (IC50 = 0.318 M) and 28 (IC50 = 0.255 M). In the 6-
lM) and 26 (IC50 = 0.131 lM) displayed 1.4- and 1.9-
mock
TGF-β1
0.5
0.1
0.05
0.01 (μM)
l
l
quinolinyl analogs, the imidazole derivatives 16 and 18 exhibited
higher inhibitory activity than their counterpart pyrazole deriva-
Figure 2. Effect of 18 on the activity of TGF-b-induced ALK5. HaCaT cells were
transiently transfected with p3TP-luc reporter construct. Luciferase activity was
determined in the presence of different concentrations of each compound and is
given as the mean SD of three independent experiments run in triplicate relative
to control.
tives 22, 23 and 33 (IC50 = 0.034
lM), and 26, 27 and 34
(IC50 = 0.061 M), respectively. In this series, the most potent com-
l
pound 16 is approximately 1.4-fold more potent ALK5 inhibitor
Table 1
Inhibitory activity of imidazoles 16–19 and pyrazoles 22–29, 33, and 34 on ALK5
R
R
N
N
N
N
R
N
N
X
X
X
N
N
N
R
N
H
N
N
N
N
N
N
16−19
22,24, 26, 28
23,25,27,29
33, 34
Luciferase activityb,c (% control)
SBE-luc
a
Compd
X
R
IC50 (lM)
p3TP-luc
ARE-luc
Mock
TGF-b
16
17
18
19
22
23
24
25
26
27
28
29
33
34
2
2
16
100 37
3
3
0
100 13
100 32
13
87 38
22
CH
N
CH
N
CH
CH
N
CN
CN
CONH2
CONH2
CN
CN
CN
CN
CONH2
CONH2
CONH2
CONH2
CN
0.026
2.25
0.034
2.60
4
1
16
74
22
74 31
38 12
95 11
29 16
94
18
106 24
30
105 30
74 54
80 28
22
26
16
1
7
5
4
104 26
7
1
8
74 16
15
114 33
21
132 31
74 23
35 19
86 58
38 13
86 24
22 11
87 52
43 20
85 85
63 36
50 15
47 24
55 31
0.231
4
>10
0.318
>10
0.131
>10
0.255
>10
0.034
0.061
0.169
0.162
0.036
2
N
4
0
CH
CH
N
9
2
147 42
20
95 12
49 13
87 28
26
29
6
4
9
N
CONH2
3
4
2
1
5
3
3
8
21
9
a
ALK5 was expressed in Sf9 insect cells as human recombinant GST-fusion protein by means of the baculovirus expression system. A proprietary radioisotopic protein
kinase assay (33PanQinase activity assay) was performed at ProQinase GmbH (Freiburg, Germany) using casein as a substrate.
b
Activity is given as the mean SD of three independent experiments run in triplicate relative to control incubations with DMSO vehicle.
Luciferase activity was determined at a concentration of 0.1 lM of inhibitor.
c

D.-K. Kim et al. / Bioorg. Med. Chem. 18 (2010) 4459–4467
4463
Figure 3. (A) Docked pose of 18 in the active site of ALK5. Bound 1,5-naphthyridine inhibitor (yellow) and a trapped water molecule (red sphere) in the X-ray structure (PDB
id: 1VJY) are shown for comparison. Hydrogen bonding interactions between 18 and key amino acid residues within the binding site are represented in yellow dotted lines
(<2.5 Å). (B) Lipophilic potential surface map of the ATP-binding pocket of ALK5 is demonstrated in the docking model of 18. Lipophilicity increases from blue (hydrophilic) to
brown (lipophilic).
observed in the X-ray structures of ALK5 complexed with other
22–29, 33, and 34 was measured using HaCaT cells transiently
inhibitors.32,38,39 It was reported that Asp351 is usually located
transfected with three different luciferase reporter genes, p3TP-
close to bound inhibitors in the inactivated conformation of
luciferase reporter,34 SBE-luciferase reporter,35 and ARE-luciferase
ALK5.32 The H-bond between imidazole NH of 18 and Asp351
reporter36 at a concentration of 0.1
lM (Table 1). Similar to kinase
may contribute to hold the loop containing Asp351 and stabilize
inactive conformation of ALK5. The 6-methylpyridin-2-yl moiety
occupies the hydrophobic pocket containing Ser280, which is crit-
ical for the selectivity of 18 for ALK5 over p38 MAP kinase.24,32,39
This model also implies that the pyridinyl nitrogen would form a
H-bond with water molecule, a center of H-bond network in the
binding pocket, which is crystallographically determined in other
inhibitors containing pyridinyl moiety at this position.17,32 The
nitrogen atom of the 6-quinolinyl group forms a H-bond with
backbone amide NH of His283 in the hinge region of the kinase,
originally a binding pocket for the adenine ring of ATP, and it is an-
other major interaction of inhibitor with the binding site.32,38 In
addition, the carboxamide group attached to the benzyl ring con-
assay, 1-benzyl-5-(pyridin-2-yl)pyrazole derivatives 23, 25, 27,
and 29 showed no ALK5 inhibitory activity, and the luciferase
activity of most compounds consisted with that of kinase assay.
However, the pyrazole 34 (IC50 = 0.061
lM) is 2.1-fold more
inhibitory than the pyrazole 26 (IC50 = 0.131
lM) in a kinase as-
say, the former displayed much lower inhibition than the latter
in luciferase assays suggesting that 26 has better cellular perme-
ability than 34. Consistent with the kinase assay, 16 showed the
most potent luciferase inhibitory activity, and 18 showed the sim-
ilar level of inhibitory activity to 8. Compound 18 (14.3 mg/mL)
has twofold higher aqueous solubility than compound 16
(7.1 mg/mL) in buffered solution (pH 1.2), hence it was chosen
for advanced studies.
The ALK5 inhibitory activity of 18 was compared with those of
2, 3, and 8 at four different concentrations (0.01, 0.05, 0.1, and
0.5 lM) using HaCaT cells transiently transfected with p3TP-luc re-
porter construct. As shown in Figure 2, 18 inhibited ALK5 in a dose-
dependent manner and was equipotent to 8 and more potent than
tacts the a-helix next to the hinge region and forms H-bonds with
backbone NH of Ser287 and carboxyl side chain of Asp290. The
other active carbonitrile derivative 16 exhibited the binding mode
very similar to that of 18, except that the CN group forms H-bond
only with side chain OH of Ser 287, not with Asp290 (results not
shown). The present docking model demonstrates a possible broad
and tight interaction of 18 with the left side of ALK5 active site
ranging from Ser280 to Asp290, and this may be related to the
excellent activity of 18. Conclusively, the binding mode of 18 gen-
erated by docking studies supports the strong and selective activity
of this compound and provides the insights for further modifica-
tion to develop analogs with more desirable biological activity.
2 and 3. Compound 18 showed 66% inhibition at 0.05
competitor compounds 2 and 3 showed 44% inhibition.
lM, while
The selectivity of 18 for ALK5 versus other kinases was evalu-
ated using a panel of 156 kinases (ProQinase GmbH (Germany)).
Compound 18 inhibited ALK5 phosphorylation with an IC50 of
0.034
lM. Except p38
a
(IC50 = 0.54
lM), PKC
l
(IC50 = 0.65
lM),
VEGFR-1 (IC50 = 2.1
lM), VEGFR-2 (IC50 = 2.2
lM), VEGFR-3
(IC50 = 0.94 lM), and PDGFRa (IC50 = 3.0 lM), 18 was at least
2.4. Metabolism
100-fold more selective for ALK5 than the rest of 149 kinases.37
To compare the metabolic stability of 8 and 18, these com-
pounds were incubated with nine human CYP supersomes™ in
the presence of an NADPH generating system (Table 2).
2.3. Molecular modeling
To examine the binding mode of 18 in the active site of ALK5,
we built a docking model of ALK5:18 complex based on the X-
ray structure of ALK5 co-crystallized with 1,5-naphthyridine inhib-
itor32 (PDB id: 1VJY). As shown in Figure 3, 18 fits well both in the
cavity for ATP and the hinge region (residues 281–283) that link
the N- and C-terminal domains of kinase.32 The central imidazole
ring and its substituents form several interactions with amino acid
residues in the binding pocket, facilitating binding of the ligand
deep into the active site. The NH of imidazole ring forms a hydro-
gen bond (H-bond) with Asp351, which is one of key interactions
N
O
NH
HO
N
2
N
H
N
35

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D.-K. Kim et al. / Bioorg. Med. Chem. 18 (2010) 4459–4467
Table 2
Comparison between compound 8 metabolite (9) and compound 18 metabolite (35) by supersomal CYP
Metabolitea,b (peak area)
CYP isozymes
CYP1A2
CYP2A6
CYP2B6
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP2E1
CYP3A4
9
35
NDc
ND
ND
ND
ND
ND
33.40 1.68
1.99 0.38
ND
ND
12.37 0.58
11.31 0.70
15.39 1.89
ND
ND
ND
17.00 1.75
10.6 1.2
a
120
lM of 8 or 18 was incubated with nine human CYP supersomes™ in the presence of an NADPH generating system. HPLC analysis was performed as described in
Section 4.
b
Data represent the mean SD (n = 3).
ND, not detected.
c
A major metabolite of 18 was isolated, purified and tentatively
chromatography (MPLC) was performed using Merck Silica Gel
60 (230–400 mesh) with a YFLC-540 ceramic pump (Yamagen).
characterized as 35 based on its 1H NMR spectra and LC-MS/MS
spectra. Because standards for metabolites of both compounds 8
and 18 were not available, amounts of metabolite 9 and 35 were
expressed as peak area comparing with those of compound 8 and
18 of which concentrations were known. When amounts of major
metabolites of compounds 8 (9) and 18 (35) were compared,
metabolite 35 was much less (30% of 9) than 9, which indicated
that both N-1 and N-4 at compound 8 were important to be metab-
olized to 9 (Table 2). Compound 8 seems to be metabolized by
CYP2C8, CYP3A4, CYP2C19, and CYP2D6 whereas compound 18
seems to be metabolized mainly by CYP3A4 and CYP2C19 and min-
imally by CYP2C8. In other words, CYP2D6 metabolize only com-
pound 8 suggesting that N-1 might need for CYP2D6 metabolism
as well as maximal metabolism by CYP2C8.
4.2. Chemistry
4.2.1. General procedure for the preparation of the 2-
(hydroxyimino)-1-(6-methylpyridin-2-yl)ethanones 12 and 13
A solution of 10 or 11 (0.58 mmol) in aqueous HCl (18%, 2.3 mL)
was cooled to 0 °C using an ice-NaCl bath. To this solution was
added sodium nitrite (0.70 mmol), while the reaction temperature
was maintained at 0 °C during this addition. After addition was
complete, the ice-NaCl bath was removed, and the reaction mix-
ture was allowed to warm to room temperature and stirred for
an additional 30 min. The reaction mixture was neutralized with
6 N NaOH solution, and the precipitates were filtered, washed with
water, and dried in vacuo to afford the title compound 12 or 13 as a
solid.
3. Conclusion
4.2.1.1. 2-(Hydroxyimino)-1-(6-methylpyridin-2-yl)-2-(quino-
lin-6-yl)ethanone (mixture of E & Z form) (12). Yield 87%; mp
223 °C; 1H NMR (DMSO-d6) d 2.42 (s, 3H), 2.49 (s, 3H), 7.46 (d,
1H, J = 7.6 Hz), 7.53 (dd, 1H, J = 8.2, 4.4 Hz), 7.56–7.59 (m, 2H),
7.72 (d, 1H, J = 7.6 Hz), 7.81 (d, 1H, J = 2.0 Hz), 7.87–7.90 (m, 2H),
7.95–8.01 (m, 2H), 8.06–8.10 (m, 2H), 8.12 (d, 1H, J = 2.0 Hz),
8.15 (d, 1H, J = 1.6 Hz), 8.39 (dd, 1H, J = 8.4, 1.6 Hz), 8.46 (dd, 1H,
J = 8.4, 1.6 Hz), 8.91 (ddd, 1H, J = 4.4, 2.0, 1.6 Hz), 8.96 (ddd, 1H,
J = 4.4, 2.0, 1.6 Hz), 11.7 (s, 1H), 12.8 (s, 1H); IR (CHCl3)
3411 cmÀ1; MS (ESI) m/z 292.11 (MH+).
In this report, a series of 4(5)-(6-methylpyridin-2-yl)imidazoles
and -pyrazoles have been synthesized and evaluated for their ALK5
inhibitory activity in an enzyme assay and in cell-based luciferase
reporter assays. The structure–activity relationships in this series
of compounds have been established and discussed. The imidazole
analogs 16 and 18 having a 6-quinolinyl moiety showed the most
significant ALK5 inhibitory activity in the series of compounds that
is much higher than those of 2 and 3 and is equipotent to 8. The
in vitro metabolism study of compounds 8 and 18 in human CYP
supersomes™ revealed that the 6-quinolinyl analog 18 is more sta-
ble than the 6-quinoxalinyl analog 8. A docking model of ALK5:18
complex shows that 18 fits well into the active site cavity of ALK5.
Recent studies demonstrated that 18 effectively prevented the
development and progression of pulmonary arterial hypertension
in the monocrotaline rat model through the inhibition of TGF-b sig-
naling40 and also prevented granulation tissue formation after bare
metallic stent placement in a rat urethral model.41
4.2.1.2.
2-(Hydroxyimino)-1-(6-methylpyridin-2-yl)-2-(1,5-
naphthyridin-2-yl)ethanone (13). Yield 81%; mp 216À219 °C; 1H
NMR (DMSO-d6) d 2.36 (s, 3H), 7.52 (dd, 1H, J = 6.0, 2.4 Hz), 7.70
(dd, 1H, J = 8.4, 4.4 Hz), 7.93À7.98 (m, 2H), 8.15 (dt, 1H, J = 8.4,
0.8 Hz), 8.34 (d, 1H, J = 9.2 Hz), 8.50 (dd, 1H, J = 9.2, 0.6 Hz), 8.97
(dd, 1H, J = 4.0, 1.6 Hz), 12.19 (s, 1H); IR (MeOH) 3401,
1498 cmÀ1; MS (ESI) m/z 293.11 (MH+).
On the basis of these results, the regulatory preclinical studies
of 18 are currently under way.
4.2.2. General procedure for the preparation of the 3-((1-
hydroxy-4-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-
benzonitriles 14 and 15
4. Experimental
A mixture of NH4OAc (9.40 mmol), 12 or 13 (0.47 mmol), and 3-
cyanophenylacetaldehyde (0.94 mmol) in AcOH (5 mL) was heated
at 110 °C for 16 h. The reaction mixture was cooled to room tem-
perature and neutralized with NH4OH to pH ꢀ7. The mixture
was extracted with CH2Cl2 (40 mL Â 3), and the combined organic
extracts were dried over anhydrous Na2SO4, filtered, and evapo-
rated to dryness under reduced pressure. The residue was purified
by MPLC on silica gel with CH2Cl2/MeOH as eluent to afford the ti-
tle compound 14 or 15 as a solid.
4.1. General methods
1H NMR spectra were recorded on a Varian Unity 400 spectro-
photometer. The chemical shifts are reported in parts per million
(ppm) relative to internal tetramethylsilane in CDCl3, CD3OD, or
DMSO-d6. Infrared spectra were recorded on a FT-infrared spec-
trometer (Bio-Rad). Electrospray ionization mass spectra (ESIMS)
were obtained on a Q-Tof2 mass spectrometer (Micromass). All
melting points were taken in Pyrex capillaries using an electrother-
mal digital melting point apparatus (Buchi) and are not corrected.
Analytical thin-layer chromatography (TLC) was performed on
Merck Silica Gel 60F-254 glass plates. Medium-pressure liquid
4.2.2.1. 3-((1-Hydroxy-4-(6-methylpyridin-2-yl)-5-(quinolin-6-
yl)-1H-imidazol-2-yl)methyl)benzonitrile (14). Yield 39%; mp
135 °C; 1H NMR (CDCl3) d 2.43 (s, 3H), 4.24 (s, 2H), 7.04 (d, 1H,

D.-K. Kim et al. / Bioorg. Med. Chem. 18 (2010) 4459–4467
4465
J = 7.6 Hz), 7.16 (d, 1H, J = 8.4 Hz), 7.31–7.38 (m, 2H), 7.49 (d, 2H,
J = 7.6 Hz), 7.52–7.56 (m, 1H), 7.64 (s, 1H), 7.69 (d, 1H, J = 8.0 Hz),
7.84 (d, 1H, J = 8.8 Hz), 7.98 (s, 1H), 8.05 (d, 1H, J = 8.0 Hz), 8.71
(s, 1H); IR (CHCl3) 3400, 3024, 2938, 2230 cmÀ1; MS (ESI) m/z
418.17 (MH+).
s, 1H), 7.03 (d, 1H, J = 7.6 Hz), 7.31 (d, 1H, J = 8.0 Hz), 7.37 (t, 1H,
J = 7.6 Hz), 7.43 (dd, 1H, J = 8.0, 4.4 Hz), 7.45–7.52 (m, 2H), 7.74
(d, 1H, J = 7.6 Hz), 7.89 (dd, 1H, J = 8.8, 2.0 Hz), 8.05 (s, 1H), 8.11
(d, 1H, J = 8.8 Hz), 8.16–8.18 (m, 2H), 8.93 (dd, 1H, J = 4.4,
1.6 Hz); IR (CHCl3) 3433, 1636 cmÀ1; MS (ESI) m/z 420.18 (MH+).
Anal. Calcd for C26H21N5O: C, 74.44; H, 5.05; N, 16.70. Found: C,
74.39; H, 5.15; N, 16.54.
4.2.2.2.
3-((1-Hydroxy-5-(6-methylpyridin-2-yl)-4-(1,5-naph-
thyridin-2-yl)-1H-imidazol-2-yl)methyl)benzonitrile (15). Yield
35%; mp 120 °C; 1H NMR (CDCl3) d 2.59 (s, 3H), 4.27 (s, 2H), 7.15
(d, 1H, J = 6.8 Hz), 7.37 (t, 1H, J = 8.0 Hz), 7.49 (dt, 1H, J = 8.0,
1.6 Hz), 7.60–7.66 (m, 2H), 7.69 (t, 1H, J = 7.6 Hz), 7.72 (s, 1H),
7.78 (d, 1H, J = 7.6 Hz), 8.23 (d, 1H, J = 8.4 Hz), 8.32 (dd, 1H,
J = 9.2, 0.4 Hz), 8.79 (d, 1H, J = 9.2 Hz), 8.90 (dd, 1H, J = 4.4,
1.6 Hz); IR (CH2Cl2) 3406, 2229 cmÀ1; MS (ESI) m/z 419.14 (MH+).
4.2.4.2. 3-((5-(6-Methylpyridin-2-yl)-4-(1,5-naphthyridin-2-yl)-
1H-imidazol-2-yl)methyl)benzamide
(19). Yield
90%;
mp
229 °C; 1H NMR (CDCl3) d 2.60 (s, 3H), 4.31 (s, 2H), 5.72 (br s,
1H), 6.31 (br s, 1H), 7.16 (d, 1H, J = 7.2 Hz), 7.38 (t, 1H,
J = 7.6 Hz), 7.59 (d, 1H, J = 7.6 Hz), 7.64 (dd, 1H, J = 8.4, 4.4 Hz),
7.67–7.73 (m, 2H), 7.78 (d, 1H, J = 7.6 Hz), 7.87 (s, 1H), 8.23 (d,
1H, J = 8.8 Hz), 8.32 (d, 1H, J = 9.2 Hz), 8.74 (d, 1H, J = 9.2 Hz),
8.92 (d, 1H, J = 2.8 Hz); IR (CH2Cl2) 3299, 3180, 1655 cmÀ1; MS
(ESI) m/z 421.17 (MH+). Anal. Calcd for C25H20N6O: C, 71.41; H,
4.79; N, 19.99. Found: C, 71.35; H, 4.77; N, 19.74.
4.2.3. General procedure for the preparation of the 3-((4-(6-
methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)benzonitrile 16
and 17
To a stirred solution of 14 or 15 (0.14 mmol) in anhydrous DMF
(1.5 mL) was added triethyl phosphite (0.21 mmol) at room tem-
perature. The mixture was heated at 110 °C for 16 h and then
cooled to room temperature. The reaction mixture was diluted
with EtOAc (100 mL) and washed with water (10 mL Â 8) and brine
(20 mL). The organic layer was dried over anhydrous Na2SO4, fil-
tered, and evaporated to dryness under reduced pressure. The res-
idue was purified by MPLC on silica gel with CH2Cl2/MeOH as
eluent to afford the title compound 16 or 17 as a solid.
4.2.4.3. 3-((3-(6-Methylpyridin-2-yl)-4-(quinolin-6-yl)-1H-pyra-
zol-1-yl)methyl)benzamide (26). Yield 70%; mp 198.5 °C; 1H NMR
(CD3OD) d 2.44 (s, 3H), 5.54 (s, 2H), 7.26 (dd, 1H, J = 7.8, 0.6 Hz),
7.38 (dd, 1H, J = 7.2, 0.4 Hz), 7.48–7.51 (m, 2H), 7.60 (m, 1H),
7.67 (dd, 1H, J = 8.8, 2.0 Hz), 7.70 (t, 1H, J = 7.8 Hz), 7.84 (dt, 1H,
J = 8.0, 2.0 Hz), 7.87 (d, 1H, J = 2.0 Hz), 7.90 (d, 1H, J = 8.8 Hz),
7.94–7.95 (m, 1H), 8.17 (s, 1H), 8.22 (dd, 1H, J = 8.0, 0.8 Hz), 8.78
(dd, 1H, J = 4.4, 1.8 Hz); IR (CHCl3) 3357, 3180, 1670 cmÀ1; MS
(ESI) m/z 420.18 (MH+). Anal. Calcd for C26H21N5O: C, 74.44; H,
5.05; N, 16.70. Found: C, 74.22; H, 5.25; N, 16.64.
4.2.3.1. 3-((4-(6-Methylpyridin-2-yl)-5-(quinolin-6-yl)-1H-imi-
dazol-2-yl)methyl)benzonitrile (16). Yield 61%; mp 114 °C; 1H
NMR (CDCl3) d 2.52 (s, 3H), 4.25 (s, 2H), 7.00 (d, 1H, J = 7.2 Hz),
7.30 (d, 1H, J = 8.0 Hz), 7.41–7.46 (m, 3H), 7.55 (d, 1H, J = 7.6 Hz),
7.61 (d, 1H, J = 7.6 Hz), 7.65 (s, 1H), 7.95 (dd, 1H, J = 8.8, 2.0 Hz),
8.13 (d, 1H, J = 8.8 Hz), 8.19 (m, 2H), 8.93 (dd, 1H, J = 4.4, 1.6 Hz);
IR (CHCl3) 3405, 3180, 2230 cmÀ1; MS (ESI) m/z 402.17 (MH+).
Anal. Calcd for C26H19N5: C, 77.79; H, 4.77; N, 17.44. Found: C,
77.58; H, 4.81; N, 17.40.
4.2.4.4. 3-((5-(6-Methylpyridin-2-yl)-4-(quinolin-6-yl)-1H-pyra-
zol-1-yl)methyl)benzamide (27). Yield 80%; mp 97 °C; 1H NMR
(CDCl3) d 2.67 (s, 3H), 5.62 (s, 2H), 6.90 (d, 1H, J = 8.0 Hz), 7.18
(d, 1H, J = 7.6 Hz), 7.34–7.36 (m, 2H), 7.46 (t, 2H, J = 7.6 Hz), 7.55
(dd, 1H, J = 7.2, 1.6 Hz), 7.67–7.69 (m, 2H), 7.76 (s, 1H), 7.85 (s,
1H), 8.09 (br s, 1H), 8.16 (br s, 1H), 8.87 (dd, 1H, J = 4.8, 1.8 Hz);
IR (CHCl3) 3426, 1664 cmÀ1; MS (ESI) m/z 420.18 (MH+). Anal.
Calcd for C26H21N5O: C, 74.44; H, 5.05; N, 16.70. Found: C, 74.65;
H, 5.01; N, 16.49.
4.2.3.2. 3-((5-(6-Methylpyridin-2-yl)-4-(1,5-naphthyridin-2-yl)-
1H-imidazol-2-yl)methyl)benzonitrile (17). Yield 59%; mp
148 °C; 1H NMR (CDCl3) d 2.62 (s, 3H), 4.29 (s, 2H), 7.17 (d, 1H,
J = 8.0 Hz), 7.40 (t, 1H, J = 8.0 Hz), 7.51 (dt, 1H, J = 7.6, 1.2 Hz),
7.63–7.68 (m, 2H), 7.72 (t, 1H, J = 7.6 Hz, overlapped), 7.74 (s,
1H), 7.81 (d, 1H, J = 7.6 Hz), 8.27 (d, 1H, J = 8.4 Hz), 8.35 (d, 1H,
J = 9.2 Hz), 8.81 (d, 1H, J = 9.2 Hz), 8.92 (dd, 1H, J = 4.0, 1.6 Hz); IR
(CH2Cl2) 2228 cmÀ1; MS (ESI) m/z 403.16 (MH+). Anal. Calcd for
C25H18N6: C, 74.61; H, 4.51; N, 20.88. Found: C, 74.47; H, 4.67; N,
20.68.
4.2.4.5. 3-((3-(6-Methylpyridin-2-yl)-4-(1,5-naphthyridin-2-yl)-
1H-pyrazol-1-yl)methyl)benzamide
(28). Yield
92%;
mp
86À88 °C; 1H NMR (CDCl3) d 2.49 (s, 3H), 5.41 (s, 2H), 6.07 (br s,
1H), 6.83 (br s, 1H), 7.12 (d, 1H, J = 7.6 Hz), 7.36 (t, 1H,
J = 7.6 Hz), 7.47 (d, 1H, J = 6.8 Hz), 7.49 (d, 1H, J = 7.6 Hz),
7.53À7.61 (m, 2H), 7.72 (d, 1H, J = 9.2 Hz), 7.75 (d, 1H, J = 7.6 Hz),
7.87 (s, 1H), 8.12 (s, 1H), 8.16 (d, 1H, J = 9.2 Hz), 8.26 (m, 1H),
8.86 (dd, 1H, J = 4.4, 1.6 Hz); IR (CH2Cl2) 3341, 3191, 1671 cmÀ1
;
MS (ESI) m/z 421.17 (MH+). Anal. Calcd for C25H20N6O: C, 71.41;
4.2.4. General procedure for the conversion of carbonitrile
derivatives 16, 17, 22–25, and 33 to carboxamide derivatives 18,
19, 26–29, and 34
H, 4.79; N, 19.99. Found: C, 71.28; H, 4.92; N, 19.74.
4.2.4.6. 3-((5-(6-Methylpyridin-2-yl)-4-(1,5-naphthyridin-2-yl)-
A stirred solution of carbonitrile compound 16, 17, 22–25 or 33
(0.06 mmol), 1 N NaOH (0.21 mL, 0.21 mmol), and 28% H2O2
(0.008 mL, 0.06 mmol) in 95% EtOH (2 mL) was heated at 60 °C
for 2 h. The reaction mixture was cooled to 0 °C and neutralized
with 2 N HCl (0.1 mL) to pH ꢀ8. The mixture was extracted with
CHCl3 (30 mL Â 3), and the combined organic extracts were dried
over anhydrous Na2SO4, filtered, and evaporated to dryness under
reduced pressure. The residue was purified by MPLC on silica gel
with CHCl3/MeOH or CH2Cl2/MeOH as eluent to afford the title
compound 18, 19, 26–29, or 34 as a solid.
1H-pyrazol-1-yl)methyl)benzamide
(29). Yield
93%;
mp
88À90 °C; 1H NMR (CDCl3) d 2.66 (s, 3H), 5.52 (s, 2H), 5.96 (br s,
1H), 6.37 (br s, 1H), 7.07 (d, 1H, J = 7.6 Hz), 7.22 (d, 1H,
J = 8.0 Hz), 7.25–7.31 (m, 2H), 7.36 (d, 1H, J = 8.8 Hz), 7.50–7.58
(m, 2H), 7.62 (s, 1H), 7.66 (dt, 1H, J = 6.4, 2.2 Hz), 8.16 (d, 1H,
J = 8.8 Hz), 8.19 (s, 1H), 8.22 (d, 1H, J = 8.0 Hz), 8.85 (dd, 1H,
J = 4.4, 1.6 Hz); IR (CH2Cl2) 3352, 3191, 1671 cmÀ1; MS (ESI) m/z
421.17 (MH+). Anal. Calcd for C25H20N6O: C, 71.41; H, 4.79; N,
19.99. Found: C, 71.35; H, 4.87; N, 19.79.
4.2.4.7. 3-((4-(6-Methylpyridin-2-yl)-3-(quinolin-6-yl)-1H-pyra-
zol-1-yl)methyl)benzamide (34). Yield 89%; mp 96 °C; 1H NMR
(CDCl3) d 2.66 (s, 3H), 5.46 (s, 2H), 6.99 (d, 1H, J = 7.6 Hz), 7.07
(d, 1H, J = 8.0 Hz), 7.26 (1H, overlapped with residual CHCl3), 7.41
4.2.4.1. 3-((4-(6-Methylpyridin-2-yl)-5-(quinolin-6-yl)-1H-imi-
dazol-2-yl)methyl)benzamide (18). Yield 69%; mp 151.5 °C; 1H
NMR (CDCl3) d 2.60 (s, 3H), 4.29 (s, 2H), 5.55 (br s, 1H), 6.56 (br

4466
D.-K. Kim et al. / Bioorg. Med. Chem. 18 (2010) 4459–4467
(dd, 1H, J = 8.4, 4.4 Hz), 7.45 (t, 2H, J = 7.8 Hz), 7.57 (d, 1H,
J = 8.0 Hz), 7.83 (d, 2H, J = 8.0 Hz), 7.94 (br s, 1H), 8.07–8.09 (m,
2H), 8.14 (d, 1H, J = 8.0 Hz), 8.92 (dd, 1H, J = 4.2, 1.8 Hz); IR (CHCl3)
3367, 3196, 1674 cmÀ1; MS (ESI) m/z 420.18 (MH+). Anal. Calcd for
C26H21N5O: C, 74.44; H, 5.05; N, 16.70. Found: C, 74.69; H, 4.87; N,
16.44.
4.2.6.4. 3-((5-(6-Methylpyridin-2-yl)-4-(1,5-naphthyridin-2-yl)-
1H-pyrazol-1-yl)methyl)benzonitrile (25). Yield 23%; mp 149–
151 °C; 1H NMR (CDCl3) d 2.66 (s, 3H), 5.55 (s, 2H), 7.08 (d, 1H,
J = 7.6 Hz), 7.23 (d, 1H, J = 7.6 Hz), 7.35 (dd, 1H, J = 7.6, 7.6 Hz),
7.40 (d, 1H, J = 8.8 Hz), 7.43À7.47 (m, 2H), 7.50 (dt, 1H, J = 7.6,
1.6 Hz), 7.54 (t, 1H, J = 7.6 Hz), 7.59 (dd, 1H, J = 8.8, 4.0 Hz), 8.20
(s, 1H), 8.21 (d, 1H, J = 8.8 Hz), 8.26 (d, 1H, J = 8.8 Hz), 8.87 (dd,
1H, J = 4.0, 1.6 Hz); IR (CH2Cl2) 2230 cmÀ1; MS (ESI) m/z 403.16
(MH+). Anal. Calcd for C25H18N6: C, 74.61; H, 4.51; N, 20.88. Found:
C, 74.43; H, 4.77; N, 20.74.
4.2.5. General procedure for the preparation of the 3-(6-
methylpyridin-2-yl)-1H-pyrazoles 20 and 21
To a stirred solution of 10 or 11 (0.33 mmol) in anhydrous DMF
(3 mL) were added AcOH (0.84 mmol) and DMFÁDMA (0.70 mmol).
After the mixture was stirred for 1 h at room temperature, hydra-
zine monohydrate (5.14 mmol) was added, and the mixture was
heated at 50 °C for 2 h. The cooled reaction mixture was poured
into water (10 mL) and extracted with EtOAc (100 mL). The organic
layer was washed with water (10 mL Â 8) and saturated aqueous
NaHCO3 (10 mL), dried over anhydrous MgSO4, filtered, and evap-
orated to dryness under reduced pressure. The residue was purified
by MPLC on silica gel with CH2Cl2/MeOH as eluent to afford the
compounds 20 or 21 as a solid.
4.2.6.5. 3-((4-(6-Methylpyridin-2-yl)-3-(quinolin-6-yl)-1H-pyra-
zol-1-yl)methyl)benzonitrile (33). Yield 79%; mp 70 °C; 1H NMR
(CDCl3) d 2.61 (s, 3H), 5.45 (s, 2H), 6.98 (d, 1H, J = 7.6 Hz), 7.04
(d, 1H, J = 8.0 Hz), 7.26 (1H, overlapped with residual CHCl3),
7.41–7.45 (m, 2H), 7.49–7.53 (m, 1H), 7.61–7.65 (m, 3H), 7.86
(dd, 1H, J = 8.8, 1.6 Hz), 8.09–8.11 (m, 2H), 8.16 (d, 1H, J = 7.6 Hz),
8.93 (dd, 1H, J = 4.4, 1.6 Hz); IR (CHCl3) 3399, 2231 cmÀ1; MS
(ESI) m/z 402 (MH+). Anal. Calcd for C26H19N5: C, 77.79; H, 4.77;
N, 17.44. Found: C, 77.93; H, 4.56; N, 17.23.
4.2.6. General procedure for the alkylation of 20, 21, and 32
A solution of pyrazole 20, 21, or 32 (0.69 mmol) in anhydrous
DMF (4 mL) was added dropwise over 15 min to a stirred suspen-
sion of Cs2CO3 (0.75 mmol) in anhydrous DMF (1 mL) at room tem-
4.2.7. 2-(6-Methylpyridin-2-yl)-1-(quinolin-6-yl)ethanone (31)
To a stirred solution of 2,6-lutidine (2.33 mmol) in anhydrous
THF (8 mL) at À78 °C under Ar was treated dropwise with n-BuLi
(2.5 M solution in hexane, 3.50 mmol). After 10 min, a solution of
N-methoxy-N-methylquinoline-6-carboxamide (2.80 mmol) in
anhydrous THF (7 mL) was added dropwise to the reaction mix-
ture, and the solution was allowed to warm to room temperature.
The solution was quenched with saturated aqueous NH4Cl solution
(7 mL) and extracted with EtOAc (300 mL). The organic layer was
washed with H2O (30 mL Â 2) and brine (30 mL), dried over anhy-
drous MgSO4, filtered, and evaporated to dryness under reduced
pressure. The residue was purified by MPLC on silica gel with
CH2Cl2/MeOH (19:1 (v/v)) as eluent to afford the compound 31
(389 mg, 64%) as a solid. Mp 87 °C; 1H NMR (CDCl3) d 2.57 (s,
3H), 6.23 (s, 1H), 6.85 (d, 1H, J = 7.6 Hz), 6.95 (d, 1H, J = 8.0 Hz),
7.46 (dd, 1H, J = 8.0, 4.2 Hz), 7.55 (t, 1H, J = 7.8 Hz), 8.09–8.16 (m,
2H), 8.23 (dd, 1H, J = 8.0, 1.6 Hz), 8.26–8.29 (m, 1H), 8.37 (d, 1H,
J = 1.6 Hz), 8.92 (dd, 1H, J = 4.2, 1.8 Hz); IR (CHCl3) 3432,
1690 cmÀ1; MS (ESI) m/z 263 (MH+).
perature. The mixture was stirred for 15 min, and to it,
a-bromo-
m-tolunitrile (0.69 mmol) was added. After 2 h (for 33, 12 h at
80 °C), the reaction mixture was diluted with EtOAc (120 mL),
washed with water (12 mL Â 8) and brine (12 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 with CH2Cl2/MeOH as eluent to afford the title
compound 22–25 or 33 as a solid.
4.2.6.1. 3-((3-(6-Methylpyridin-2-yl)-4-(quinolin-6-yl)-1H-pyra-
zol-1-yl)methyl)benzonitrile (22). Yield 59%; mp 76 °C; 1H NMR
(CDCl3) d 2.52 (s, 3H), 5.49 (s, 2H), 7.10 (dd, 1H, J = 8.0, 0.4 Hz),
7.31 (dd, 1H, J = 7.6, 0.4 Hz), 7.41 (dd, 1H, J = 8.0, 4.4 Hz), 7.48–
7.54 (m, 2H), 7.57–7.60 (m, 1H), 7.63–7.65 (m, 3H), 7.68 (dd, 1H,
J = 8.8, 2.0 Hz), 7.86 (d, 1H, J = 2.0 Hz), 8.05 (d, 1H, J = 8.8 Hz),
8.11 (d, 1H, J = 8.0 Hz), 8.88 (dd, 1H, J = 4.4, 1.6 Hz); IR (CHCl3)
3384, 3061, 2927, 2231 cmÀ1; MS (ESI) m/z 402 (MH+). Anal. Calcd
for C26H19N5: C, 77.79; H, 4.77; N, 17.44. Found: C, 77.45; H, 4.97;
N, 17.24.
4.2.8. 6-(4-(6-Methylpyridin-2-yl)-1H-pyrazol-3-yl)quinoline
(32)
To a stirred solution of 31 (1.37 mmol) in THF (10 mL) was
added N,N-dimethylformamide dimethyl acetal (10.98 mmol).
After 48 h, the mixture was concentrated in vacuo. The residue
was dissolved in EtOH (10 mL), and to it, hydrazine monohydrate
(32.94 mmol) was added. The mixture was stirred for 12 h at room
temperature and concentrated. The residue was diluted with
CH2Cl2 (200 mL) and washed with H2O (40 mL). The CH2Cl2 solu-
tion was dried over anhydrous MgSO4, filtered, and evaporated to
dryness under reduced pressure. The residue was purified by MPLC
on silica gel with CH2Cl2/MeOH (15:1 (v/v)) as eluent to afford the
compound 32 (173 mg, 44%) as a solid. Mp 95 °C; 1H NMR (CDCl3) d
2.55 (s, 3H), 7.01 (dd, 2H, J = 6.8, 6.4 Hz), 7.42 (m, 2H), 7.85 (dd, 1H,
J = 8.8, 1.6 Hz), 8.08 (t, 3H, J = 4.4 Hz), 8.13 (dd, 1H, J = 8.4, 0.8 Hz),
8.92 (dd, 1H, J = 4.4, 1.6 Hz); IR (CHCl3) 3384, 3151, 2927 cmÀ1; MS
(ESI) m/z 287 (MH+).
4.2.6.2. 3-((5-(6-Methylpyridin-2-yl)-4-(quinolin-6-yl)-1H-pyra-
zol-1-yl)methyl)benzonitrile (23). Yield 40%; mp 83 °C; 1H NMR
(CDCl3) d 2.65 (s, 3H), 5.64 (s, 2H), 6.88 (dd, 1H, J = 8.0, 0.4 Hz),
7.17 (dd, 1H, J = 7.6, 0.4 Hz), 7.37–7.45 (m, 3H), 7.46–7.48 (m,
2H), 7.51–7.54 (m, 2H), 7.75 (d, 1H, J = 2.0 Hz), 7.85 (s, 1H), 8.04
(d, 1H, J = 8.8 Hz), 8.11 (d, 1H, J = 8.0 Hz), 8.87 (dd, 1H, J = 4.4,
1.6 Hz); IR (CHCl3) 3443, 3067, 2230 cmÀ1; MS (ESI) m/z 402
(MH+). Anal. Calcd for C26H19N5: C, 77.79; H, 4.77; N, 17.44. Found:
C, 77.58; H, 4.87; N, 17.23.
4.2.6.3. 3-((3-(6-Methylpyridin-2-yl)-4-(1,5-naphthyridin-2-yl)-
1H-pyrazol-1-yl)methyl)benzonitrile (24). Yield 68%; mp 49–
50 °C; 1H NMR (CDCl3) d 2.47 (s, 3H), 5.42 (s, 2H), 7.11 (d, 1H,
J = 7.6 Hz), 7.42 (t, 1H, J = 7.6 Hz), 7.48 (d, 1H, J = 7.6 Hz), 7.53–
7.63 (m, 5H), 7.75 (d, 1H, J = 8.8 Hz), 8.10 (s, 1H), 8.16 (d, 1H,
J = 8.8 Hz), 8.26 (d, 1H, J = 8.4 Hz), 8.85 (dd, 1H, J = 4.4, 1.6 Hz); IR
(CH2Cl2) 2231 cmÀ1; MS (ESI) m/z 403.13 (MH+). Anal. Calcd for
C25H18N6: C, 74.61; H, 4.51; N, 20.88. Found: C, 74.79; H, 4.35; N,
20.62.
4.3. Docking experiments
Protein modeling and docking experiments have been per-
formed with the SYBYL 8.0 software package (Tripos, Inc., St. Louis,
MO, USA)42 based on Linux CentOS 4.0.

D.-K. Kim et al. / Bioorg. Med. Chem. 18 (2010) 4459–4467
4467
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4.3.1. Preparation of molecular structures
The structure of 18 was prepared in MOL2 format using the
sketcher module and Gasteiger-Huckel charges were assigned to
the ligand atoms. The structure of 18 was optimized by energy
minimization until a convergence value of 0.001 kcal/(Å mol),
and molecular dynamics using simulated annealing method. The
conformer database for 18 was prepared by random selection of
50 conformers from molecular dynamics output.
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4.3.2. Preparation of target protein structure and flexible
docking
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Goodson, T., Jr.; Herron, D. K.; Li, H.-Y.; McMillen, W. T.; Mort, N.; Parsons, S.;
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The X-ray coordinate of ALK5 complexed with 1,5-naphthyri-
dine inhibitor (PDB id: 1VJY)32 was retrieved from the PDB, and
all crystallographic water molecules were removed except one in-
volved in H-bond with ligand inside the binding pocket. After the
hydrogen atoms and atomic charges were added to the receptor,
side chain amides of ALK5 were fixed. The active site was defined
as all the amino acid residues enclosed within 6.5 Å radius sphere
centered by the bound 1,5-naphthyridine inhibitor. The docking
was performed using the default parameters of the FlexX programs
implanted in the SYBYL 8.0, and subsequent scoring for FlexX solu-
tion was conducted by a consensus scoring function (CScore).
One of the conformers having the highest consensus score
(CScore = 5) was selected and complexed with ALK5, resulting in
a final model shown in Figure 3.
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Compound 8 or 18 (120 lM) was incubated with recombinant
28. Luo, J.; Ho, P. P.; Buckwalter, M. S.; Hsu, T.; Lee, L. Y.; Zhang, H.; Kim, D.-K.; Kim,
S.-J.; Gambhir, S. S.; Steinman, L.; Wyss-Coray, T. J. Clin. Invest. 2007, 117, 3306.
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Han, J.-Y.; Hong, S. S.; Park, S. H.; Lee, S.-J.; Kim, I.-H.; Lee, C. R.; Kim, D.-K.;
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31. Blumberg, L. C.; Munchhof, M. J.; Shavnya, A. U.S. Pat. Appl. Publ. US 2004/
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32. Gellibert, F.; Woolven, J.; Fouchet, M. H.; Mathews, N.; Goodland, H.;
Lovegrove, V.; Laroze, A.; Nguyen, V. L.; Sautet, S.; Wang, R.; Janson, C.;
Smith, W.; Krysa, G.; Boullay, V.; deGouville, A. C.; Huet, S.; Hartley, D. J. Med.
Chem. 2004, 47, 4494.
human CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, or 3A4
(1 mg protein/mL supersomes™), and the NADPH generating sys-
tem (0.065 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/mL of
glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium
chloride) in potassium phosphate buffer (pH 7.4, 100 mM) for
30 min at 37 °C. The reaction was then quenched with three vol-
umes of MeOH, chilled, and centrifuged. The supernatants were di-
rectly assayed by HPLC with UV detection. The reaction mixture
without the NADPH generating system was used as negative con-
trol. HPLC chromatographic separation was carried out with a
Shiseido Capcell Pak UG120 C18 column (250 Â 4.6 mm, particle
33. Cheng, H.; Cui, J. J.; Hoffman, J. E.; Jia, L.; Johnson, M. C.; Kania, R. S.; Le, P. T. Q.;
Nambu, M. D.; Pairish, M. A.; Shen, H.; Tran-Dube, M. B. U.S. Pat. Appl. Publ. US
2007/0265272 A1, 2007, p 40.
size 5 lM, Shiseido, Tokyo, Japan) using the Hewlett Packard HP
1100 system (Waldbrone, Germany). The ratio of mobile phase A
to B (mobile phase A, CH3CN:TFA = 100:0.1 (v/v); mobile phase B,
H2O:TFA = 100:0.1 (v/v)) was 5–95% in the beginning and gradually
changed in a linear manner to 90–10% at 20 min after running at a
flow rate of 1 mL/min. The column effluent was monitored by an
UV detector at 240 nm. All the procedures were performed at room
temperature. The detection limit of compounds 8 and 18 in sam-
34. Wrana, J. L.; Attisano, L.; Carcamo, J.; Zentella, A.; Doody, J.; Laiho, M.; Wang, X.
F.; Massague, J. Cell 1992, 71, 1003.
35. Dennler, S.; Itoh, S.; Vivien, D.; ten Dijke, P.; Huet, S.; Gauthier, J. M. EMBO J.
1998, 17, 3091.
36. Liu, F.; Pouponnot, C.; Massague, J. Genes Dev. 1997, 11, 3157.
37. Tested 149 kinases: ABL1, ACK1, ACV-R1, AKT1, AKT2, AKT3, ALK, AMPK-
a1fl,
AMPK- 1 tr, ARK5, Aurora-A, Aurora-B, Aurora-C, AXL, BLK, BMX, B-RAF VE, B-
a
RAF wt, BRK, BTK, CDC42BPB, CDK1/CycB, CDK1/CycE, CDK2/CycA, CDK2/CycE,
CDK3/CycE, CDK4/CycD1, CDK4/CycD3, CDK5/p25NCK, CDK5/p35NCK, CDK6/
CycD1, CDK7/CycH/Mat1, CDK9/CycT, CHK1, CHK2, CK1-a1, CK2-a1, CK2-a2,
CLK1, COT, CSF1R, CSK, DAPK1, DAPK2, DAPK3, DYRK1A, EGF-R, EPHA1, EPHA2,
EPHA3, EPHA4, EPHB1, EPHB2, EPHB3, EPHB4, ERBB2, ERBB4, FAK, FER, FGF-R1,
ples was 0.25 lM, and the retention times of metabolites 9 and
35 were 8.7 and 8.0 min, respectively.
FGF-R3, FGF-R4, FGR, FLT3, FRK, FYN, GRK3, GSK3-b, HCK, IGF1-R, IKK-b, IKK-e,
INS-R, IRAK1, IRAK4, ITK, JAK2 (m), JAK3, JNK1, JNK3, KIT, LCK, LYN,
MAPKAPK5, MARK1, MARK2, MARK3, MATK, MEK1 SESE, MET, MKK6 SDTD,
MST1, MST4, MUSK, MYLK2, NEK2, NEK6, NLK, p38b, PAK1, PAK2, PAK3, PBK,
Acknowledgment
This work was supported by a grant from Ministry of Com-
merce, Industry and Energy, Korea (10027900).
PDGFRb, PDK1, PIM1, PIM2, PKCa, PKCb1, PKCb2, PKCd, PKCe, PKCg (m), PKCc,
PKC , PKCh, PKCf, PLK1, PLK3, PRK1, PRKX, PYK2, RAF-1, RET, ROCK1, ROCK2,
i
RSK1, S6K, SAK, SGK1, SGK3, SNARK, SNK, SRC, SRPK1, SRPK2, SYK, TBK1, TIE2,
TRK-A, TRK-B, TSF1, TSF2, TTK, TYRO3, VRK1, WEE1, YES, ZAP70.
38. Gellibert, F.; deGouville, A. C.; Woolven, J.; Mathews, N.; Nguyen, V. L.; Bertho-
Ruault, C.; Patikis, A.; Grygielko, E. T.; Laping, N. J.; Huet, S. J. Med. Chem. 2006,
49, 2210.
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