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

Article abstract of DOI:10.1021/jm070129k

A series of 4(5)-(6-alkylpyridin-2-yl)imidazoles 13a-p, 17a, and 17b have been synthesized and evaluated for ALK5 inhibitory activity in an enzyme assay and in cell-based luciferase reporter assays. The quinoxalinyl analogue 13e inhibited ALK5 phosphoryla

Full text of DOI:10.1021/jm070129k

J. Med. Chem. 2007, 50, 3143-3147
3143
Synthesis and Biological Evaluation of 4(5)-(6-Alkylpyridin-2-yl)imidazoles as Transforming
Growth Factor-â Type 1 Receptor Kinase Inhibitors
Dae-Kee Kim,*,†,‡ Yoojeung Jang,† Ho Soon Lee,‡ Hyun-Ju Park,§ and Jakyung Yoo§
College of Pharmacy, Ewha Womans UniVersity, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Korea, In2Gen Co., Ltd.,
608 Daerung Posttower II Building, 182-13 Guro-dong, Guro-gu, Seoul 152-050, Korea, and College of Pharmacy, Sungkyunkwan UniVersity,
Suwon 440-746, Korea
ReceiVed February 2, 2007
A series of 4(5)-(6-alkylpyridin-2-yl)imidazoles 13a-p, 17a, and 17b have been synthesized and evaluated
for ALK5 inhibitory activity in an enzyme assay and in cell-based luciferase reporter assays. The quinoxalinyl
analogue 13e inhibited ALK5 phosphorylation with an IC50 of 0.012 µM and showed more than 90% inhibition
at 0.05 µM in a luciferase reporter assay using HaCaT cells transiently transfected with p3TP-luc reporter
construct. The binding mode of 13e generated by flexible docking studies shows that 13e fits well into the
active site cavity of ALK5 by forming several tight interactions.
Introduction
autophosphorylation of ALK5 and TGF-â-induced transcription
of matrix genes in reporter assays at submolar concentrations.
Among them, 3, 5, and SD-208 effectively retarded progressive
fibrosis in kidney, liver, and lung, respectively, and SD-208
also strongly inhibited growth and invasiveness of cancer cells
in animal models.
The transforming growth factor-â (TGF-âa) family has a
pivotal role in the regulation of a variety of physiological
processes. Three TGF-â isoforms (TGF-â1, TGF-â2, and TGF-
â3) are expressed in mammals, and each is encoded by a unique
gene and expressed in a tissue-specific manner. TGF-â1 is the
prototypic member of this family of cytokines that signals
through two highly conserved single transmembrane serine/
threonine kinases, the type I and type II TGF-â receptors (TâR-I
and TâR-II, respectively). Upon ligand-induced oligomerization,
TâR-II hyperphosphorylates serine/threonine residues in the GS
region of the TâR-I or activin receptor-like kinase 5 (ALK5),
which leads to activation of TâR-I by creating a binding site
for Smad2/Smad3 proteins. The activated ALK5 in turn phos-
phorylates Smad2/Smad3 proteins at the C-terminal SSXS-motif,
thereby causing dissociation from the receptor and heteromeric
complex formation with Smad4. These Smad complexes trans-
locate into the nucleus and assemble with specific DNA-binding
cofactors and co-modulators to finally regulate the transcription
of specific genes involved in cell growth, differentiation,
development, and the immune response.1 Deregulation of TGF-â
has been implicated in the pathogenesis of various diseases
including fibrosis, atherosclerosis, and cancer.1c,2 Numerous
studies have consistently indicated the role of TGF-â as a potent
fibrogenic cytokine evoking pathological fibrosis in various
organs such as kidney, liver, lung, heart, and skin.3 Attempts
to block the effects of TGF-â contributed to the development
of molecules that inhibit TGF-â binding to its receptor including
decorin,4 soluble chimeric TGF-â receptor,5 and neutralizing
antibodies.6 The extensive knowledge regarding TGF-â-medi-
ated ALK5-dependent signaling pathway as an initiating point
at the receptor level has highlighted the therapeutic potential
of TGF-â signaling antagonist. Recent studies have shown that
several small molecule ATP-competitive ALK5 inhibitors such
as 1 (SB-431542),7 2 (SB-505124),8 3 (SB-525334),9 4 (A-83-
01),10 5 (GW6604),11 6 (LY580276),12 and SD-20813 inhibited
To develop a more potent and selective ALK5 inhibitor, we
have synthesized a series of 4(5)-(6-alkylpyridin-2-yl)imidazoles
13a-p that have a carbonitrile- or carboxamide-substituted
phenylaminomethyl or phenylmethylamino moiety at the 2-posi-
tion of the imidazole ring. It was of particular interest to us
whether incorporation of a nitrogen atom between an imidazole
ring and a phenyl in this series of compounds may serve as an
additional binding site to the ATP binding site of ALK5.
Chemistry
A series of 4(5)-(6-alkylpyridin-2-yl)imidazoles 13a-p was
prepared as shown in Scheme 1. Treatment of 2,6-lutidine (7a)
and 6-ethyl-2-methylpyridine (7b) with lithium bis(trimethyl-
silyl)amide in anhydrous THF at -60 °C followed by reaction
with benzo[1,3]dioxole-5-carboxylic acid methyl ester (8a) gave
the 2-(6-alkylpyridin-2-yl)-1-(benzo[1,3]dioxol-5-yl)ethanones
9a14 and 9b in 76% and 36% yields, respectively. Alternatively,
the 2-(6-alkylpyridin-2-yl)-1-(quinoxalin-6-yl)ethanones 9c15 and
9d were prepared by treatment of 7a and 7b with n-BuLi and
Et2AlCl in anhydrous THF at -60 °C, followed by reaction
with quinoxaline-6-carbonyl chloride (8b) in 65% and 29%
yields, respectively.
* To whom correspondence should be addressed. Phone: (82) 2-3277-
3025. Fax: (82) 2-3277-2467. E-mail: dkkim@ewha.ac.kr.
† Ewha Womans University.
‡ In2Gen Co., Ltd.
§ Sungkyunkwan University.
a Abbreviations: TGF-â, transforming growth factor-â; ALK5, activin
receptor-like kinase 5.
10.1021/jm070129k CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/07/2007

3144 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Brief Articles
Scheme 1a
Table 1. Inhibitory Activity of 4(5)-(6-Alkylpyridin-2-yl)imidazoles
13a-p, 17a, and 17b on ALK5
luciferase activityb,c
(% control)
a
IC50
(µM) p3TP-luc SBE-luc ARE-luc
compd
R1
R2
R3
mock
TGF-â
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
17a
17b
1
5 ( 1 18 ( 3
2
100 ( 23 100 ( 11 100 ( 8
[B]d Me m-CN
[B] Me p-CN
0.046 14 ( 6
0.174 43 ( 3
0.073 21 ( 4
0.360 67 ( 12
0.012 7 ( 1
0.042 16 ( 3
0.119 33 ( 7
0.047 32 ( 9
36 ( 4
35 ( 5
33 ( 6
99 ( 12
25 ( 9
23 ( 7
62 ( 24
40 ( 7
54 ( 18
67 ( 2
59 ( 9
96
62 ( 6
123 ( 26
75 ( 6
a Reagents and conditions: (a) lithium bis(trimethylsilyl)amide,
benzo[1,3]dioxole-5-carboxylic acid methyl ester (8a), anhydrous THF, -60
°C, Ar atmosphere; (b) n-BuLi, Et2AlCl, quinoxaline-6-carbonyl chloride
(8b), anhydrous THF, -60 °C, Ar atmosphere; (c) HBr, DMSO, 60-70
°C, 2 h; (d) glyoxal dimethyl acetal, CH3CO2NH4, t-BuOMe/MeOH, rt, 3
h; (e) 1 N HCl, 70 °C, 3 h; (f) 3- or 4-aminobenzonitrile or 3- or
4-aminobenzamide, AcOH, 1,2-dichloroethane, rt, 1 h, then sodium triac-
etoxyborohydride, rt, overnight. b[B] represents benzo[1,3]dioxol-5-yl. c[Q]
represents quinoxalin-6-yl.
[B] Et
[B] Et
m-CN
p-CN
136 ( 17
13 ( 1
[Q]e Me m-CN
[Q] Me p-CN
55 ( 6
[Q] Et
[Q] Et
m-CN
p-CN
79 ( 8
61 ( 4
[B] Me m-CONH2 0.021 28 ( 10
[B] Me p-CONH2 0.262 55 ( 28
79 ( 11
119 ( 12
64 ( 3
[B] Et
[B] Et
m-CONH2 0.046 30 ( 2
p-CONH2 0.385 125
133 ( 11
99 ( 5
Scheme 2a
[Q] Me m-CONH2 0.010 63 ( 14
95 ( 33
[Q] Me p-CONH2 0.075 84 ( 8
101 ( 40 127 ( 12
[Q] Et
[Q] Et
m-CONH2 0.015 67 ( 13
p-CONH2 0.092 104 ( 7
0.024 33 ( 8
95 ( 23
112 ( 23 98 ( 8
52 ( 18
66 ( 9
68 ( 20
96 ( 10
95 ( 10
129 ( 15
128 ( 20
0.153 61 ( 13
1.542 69 ( 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. c Luciferase activity was determined at a concentration of 0.1 µM
of inhibitor. d [B] represents benzo[1,3]dioxol-5-yl. e [Q] represents qui-
noxalin-6-yl.
a Reagents and conditions: (a) N-bromosuccinimide, CH2Cl2, rt, 30 min;
(b) 1-acetylguanidine, anhydrous DMF, rt, 96 h; (c) H2SO4, MeOH, H2O,
reflux, 24 h; (d) 3- or 4-cyanobenzaldehyde, K2CO3, anhydrous MeOH, rt,
3 h, Ar atmosphere, then NaBH4, anhydrous THF, reflux, 15 h, Ar
atmosphere.
by reduction of the resulting imines with NaBH4 in anhydrous
THF afforded 17a or 17b in 44 or 43% yield, respectively.
Results and Discussion
Oxidation of the monoketones 9a-d with HBr in DMSO
afforded the diketones 10a-d in 50-92% yields. Condensation
of the diketones 10a-d with glyoxal dimethyl acetal and CH3-
CO2NH4 in t-BuOMe/MeOH produced 4(5)-(6-alkylpyridin-2-
yl)-2-(dimethoxymethyl)imidazoles 11a-d in 79-99% yields.
The acetal protecting group of the 11a-d was removed in an
acidic condition to generate the imidazole-2-carbaldehydes
12a-d in high yields. Coupling of the 12a-d with an
appropriate aminobenzonitrile or aminobenzaldehyde in the
presence of AcOH in CH2Cl2 followed by reduction of the
resulting unstable imines with sodium triacetoxyborohydride
afforded the 4(5)-(6-alkylpyridin-2-yl)imidazoles 13a-p in 21-
98% yields.
The 3- and 4-{[4-(benzo[1,3]dioxol-5-yl)-5-(6-methylpyridin-
2-yl)-1H-imidazol-2- ylamino]methyl}benzonitrile, 17a and 17b,
were prepared as shown in Scheme 2. Bromination of 9a with
N-bromosuccinimide in CH2Cl2 gave 1-(benzo[1,3]dioxol-5-yl)-
2-bromo-2-(6-methylpyridin-2-yl)ethanone (14)16 in 92% yield.
Cyclization of 14 with 1-acetylguanidine in anhydrous DMF
for 96 h yielded N-[4-(benzo[1,3]dioxol-5-yl)-5-(6-methylpy-
ridin-2-yl)-1H-imidazol-2-yl]acetamide (15) in 79% yield, which
was subsequently hydrolyzed in an acidic condition to give the
amine 16 in 89% yield. Coupling of 16 with 3- or 4-aminoben-
zonitrile in the presence of K2CO3 in anhydrous MeOH followed
To investigate whether these potential inhibitors 13a-p, 17a,
and 17b could inhibit ALK5, a kinase assay was performed
using the purified human ALK5 kinase domain produced in Sf9
insect cells (Table 1). Among them, the quinoxalinyl analogue
13e and its carboxamide derivative 13m showed the most potent
ALK5 inhibition with IC50 of 0.012 µM and 0.010 µM,
respectively, while 1 inhibited ALK5 with IC50 of 1.542 µM.
All of the quinoxalinyl analogues exhibited approximately 2-4-
fold more potent ALK5 inhibitory activity than the correspond-
ing benzo[1,3]dioxolyl analogues. The alkyl substituent at the
6-position of the pyriding ring also seemed to affect ALK5
inhibition, thus, 6-methylpyridyl analogues were equipotent or
slightly more potent than the corresponding 6-ethylpyridyl
analogues, except that the 6-methylpyridyl compound 13e was
even 10-fold more potent in ALK5 inhibition than the corre-
sponding 6-ethylpyridyl compound 13g (IC50 ) 0.119 µM).
Although it was previously demonstrated that introduction of a
methyl group at the 6-position of the pyriding ring in the pyridyl-
substituted imidazoles, thiazoles, and pyrazoles significantly
increased ALK5 inhibitory activity,7,8,17 a bulkier group than a
methyl group seems to reduce binding of those inhibitors in
the ATP binding site of ALK5. Compounds with either a
carbonitrile or a caboxamide substituent in the meta-position
displayed 3-5-fold or 6-12-fold more potent ALK5 inhibitory

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3145
activity than the corresponding compounds, which have sub-
stituents in the para-position. The position of a nitrogen atom
in the linker moiety also influenced ALK5 inhibition, thus, the
benzo[1,3]dioxolyl analogues 17a (IC50 ) 0.024 µM) having
NHCH2 as a linker showed approximately 2-fold more potent
ALK5 inhibition than the corresponding 13a (IC50 ) 0.046 µM)
having CH2NH as a linker. To evaluate TGF-â-induced
downstream transcriptional activation to ALK5 signaling, cell-
based luciferase activity of 13a-p, 17a, and 17b was measured
using HaCaT cells transiently transfected with three different
luciferase receptor genes, p3TP-luciferase reporter,18 SBE-
luciferase reporter,19 and ARE-luciferase reporter20 at a con-
centration of 0.1 µM (Table 1). The quinoxalinyl analogues 13e
(p3TP-luciferase, 93%; SBE-luciferase, 75%; ARE-luciferase,
87%) and 13f (p3TP-luciferase, 84%; SBE-luciferase, 77%;
ARE-luciferase, 45%), with a carbonitrile functionality in the
phenyl ring, displayed the most significant ALK5 inhibition at
0.1 µM compared to that of control, which is higher than that
of 1 (p3TP-luciferase, 31%; SBE-luciferase, 32%; ARE-
luciferase, 0%). Although the quinoxalinyl analogue 13m with
a carboxamide functionality in the phenyl ring was one of the
most potent ALK5 inhibitors in this series of compounds in
the kinase assay; it exhibited only marginal inhibition in the
cell-based luciferase assay (p3TP-luciferase, 37%; SBE-lu-
ciferase, 5%; ARE-luciferase, 1%). It was also observed that
introduction of a carbonitrile functionality in the phenyl ring
rather than a carboxamide functionality increased the ALK5
inhibitory activity in all the cases. These findings suggest that
the carbonitrile-containing analogues have better cellular perme-
ability than the carboxamide-containing analogues. The benzo-
[1,3]dioxolyl analogues with a carbonitrile functionality (13a,
13b, and 13d) showed lower ALK5 inhibition compared to the
quinoxalinyl analogues with a carbonitrile functionality (13e,
13f, and 13h), however, the benzo[1,3]dioxolyl analogues with
a carboxamide functionality (13i, 13j, and 13k) showed higher
ALK5 inhibition compared to the quinoxalinyl analogues with
a carboxamide functionality (13m, 13n, and 13o). As observed
in the kinase assay, the 6-methylpyridyl analogues (13a, 13b,
13e, 13f, and 13j) displayed higher ALK5 inhibitory activity
than the corresponding 6-ethylpyridyl analogues (13c, 13d, 13g,
13h, and 13l). Compounds with either a carbonitrile or a
caboxamide substituent in the meta-position, except 13g, showed
higher ALK5 inhibition compared to the corresponding com-
pounds that have either of those substituents in the para-position.
Although the ALK5 inhibitory activity of 13a and 13b was
lower than that of 17a and 17b in a kinase assay, in cell-based
luciferase reporter assays, 13a (p3TP-luciferase, 86%; SBE-
luciferase, 64%; ARE-luciferase, 38%) and 13b (p3TP-lu-
ciferase, 57%; SBE-luciferase, 65%; ARE-luciferase, 0%)
exhibited higher ALK5 inhibition than the corresponding 17a
(p3TP-luciferase, 67%; SBE-luciferase, 48%; ARE-luciferase ,
5%) and 17b (p3TP-luciferase, 39%; SBE-luciferase, 34%;
ARE-luciferase, 0%).
Figure 1. Effect of 13e and 13f on the activity of TGF-â-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.
Figure 2. Binding pose of 13e in the active site of ALK5, in
comparison with the X-ray pose of 1,5-naphthyrine inhibitor (orange
stick). A trapped water molecule in the X-ray structure is shown in the
green sphere. Key amino acid residues within the binding site are
represented in line form, and the docked 13e is rendered in capped
stick. Yellow dotted lines are hydrogen-bonding interactions (<2.5 Å).
The selectivity of 13e for ALK5 versus other kinases was
evaluated using a panel of 38 kinases (ProQinase GmbH
(Germany) and MDS Pharma Services (Taiwan)). Compound
13e inhibited ALK5 phosphorylation with an IC50 of 0.012 µM
and inhibited JNK2R2 by 79% at 1 µM. Except JNK2R2, p38R
(IC50 ) 1.03 µM), and PKCµ (IC50 ) 1.0 µM), 13e was at
least 100-fold more selective for ALK5 than the rest of 35
kinases (ABL1, AKT1, Ca2+/Calmodulin-Dep. II, Cdk1/Cyclin
B, Cdk2/Cyclin A, Cdk5/p35, CHK1, CK2R1, CSK, EGF
receptor, ERBB2, ERK1, GSK-3â, IKK-2(â), insulin receptor,
JNK1R1, JNK3, Lck, LynA, MAPKAPK5, MEK1, p38â, p38γ,
p38δ, PDK1, PKA (nonselective), PKCR, PKCâ1, PKCâ2,
PKCδ, PKCγ, PP60C-SRC, RPS6KB1(S6K), RPS6KB2, and
SGK1).
To account for strong and selective ALK5 inhibitory activity
of 13e, we built a docking model of the ALK5/13e complex
based on the X-ray crystal structure of ALK5 complexed with
1,5-naphthyrine inhibitor17 (PDB id: 1VJY). As demonstrated
in Figure 2, 13e is well superimposed over the X-ray pose of
1,5-naphthyrine compound (orange color) lying both in the
cavity for ATP and the backside of the ATP binding pocket
containing Ser280.17,21,22 The substituents attached to the central
imidazole ring of 13e form several hydrogen bonds (H-bonds)
with amino acid residues in the binding pocket, acting as
anchors, and helping the ligand bind deeper into active site
pockets. The cavity for quinoxaline ring includes the hinge
region (residues 281 to 283) of the kinase, normally occupied
The most potent compounds 13e and 13f in cell-based
luciferase reporter assays were chosen, and their ALK5 inhibi-
tory activity was compared with 1, 2, and 3 at four different
concentrations (0.01, 0.05, 0.1, and 0.5 µM) using HaCaT cells
transiently transfected with p3TP-luc reporter construct.
As shown in Figure 1, 13e and 13f inhibited ALK5 in a dose-
dependent manner, and both compounds were more potent than
1, 2, and 3 in all the four different concentrations. Compound
13e showed more than 90% inhibition at 0.05 µM, whereas 1
showed no inhibition, and 2 and 3 showed only 23% and 9%
inhibition, respectively.

3146 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Brief Articles
by the adenine ring of ATP,17 and the main interaction observed
in this region is a H-bond of quinoxaline ring nitrogen with
backbone NH of His283. The 6-methylpyridine moiety is
positioned in what is called the “selectivity pocket” of ALK5
containing Ser280, which is critical for the selectivity of 13e
for ALK5 over p38 MAP kinase.17,21,22 In case of p38 MAP
kinase, Thr106 residue lies in the equivalent position to Ser280
of ALK5. Therefore, a close contact between 6-methylpyridine
ring and Thr106 would not be formed in the binding pocket of
p38 MAP kinase due to the steric clash. In addition, the H-bond
between the pyridine nitrogen and the water molecule is also
maintained in this model, which is commonly observed in
inhibitors containing pyridyl moiety in this position.17,21 The
secondary amino group of the linker forms a bidentate H-bond
with residues, Asp351 and Asn338. If the secondary amino
group is protonated in the biological system, the H-bond will
be stronger and contribute to the increase in the binding
selectivity toward this site. The H-bond with Asp351 is observed
in the X-ray structures of ALK5 complexed with other inhibitors.
The reported data suggest that the loop containing Asp351 is
quite flexible, and usually Asp351 is located close to bound
inhibitors in the inactivated conformation of ALK5.17,21,22
Therefore, the amino linker group of 13e may contribute the
drug to stabilize inactive conformation of ALK5 by pinning
the loop containing Asp351 and Asn338. Conclusively, the
binding mode of 13e generated by flexible docking studies
shows that the structure of the ligand fits well into the binding
cavity of ALK5 by forming tight interactions and well elucidates
potent and selective activity of 13e for ALK5.
and the filtrate was concentrated under reduced pressure. The
residue was purified by MPLC on silica gel to afford the title
compound 9a or 9b.
General Procedure for the Preparation of the 2-(6-Alkylpy-
ridin-2-yl)-1-(quinoxalin-6-yl)ethanones 9c and 9d. A stirred
solution of pyridine 7a or 7b (13mmol) in anhydrous THF (100
mL) at -60 °C under an Ar atmosphere was treated dropwise with
a solution of n-BuLi in hexanes (2.0 M, 13 mmol). After 30 min,
a solution of Et2AlCl in hexanes (1.0 M, 14 mmol) was added
dropwise to the reaction mixture, and the reaction mixture was
allowed to warm to room temperature. The reaction mixture was
cooled to -60 °C and transferred via cannula to a stirred solution
of quinoxaline-6-carbonyl chloride (8b; 10.3 mmol) in anhydrous
THF (100 mL) at -60 °C. Stirring was continued for 20 min, and
the reaction mixture was quenched with saturated aqueous NH4Cl
solution. The mixture was filtered through a pad of Celite, and the
filtered residue was washed with EtOAc (100 mL). The combined
filtrate was concentrated under reduced pressure, and the residue
was purified by MPLC on silica gel to afford the title compound
9c or 9d as a solid.
General Procedure for the Preparation of the 1-Aryl-2-(6-
alkylpyridin-2-yl)ethane-1,2-diones 10a-d. A stirred suspension
of 9a-d (13 mmol) in DMSO was treated dropwise with HBr (48
wt % in water, 6 mL), and the mixture was heated to 60-70 °C.
After 2 h, the reaction mixture was cooled to 0 °C, poured onto
ice water, and brought to pH 10 with K2CO3. The mixture was
extrated with EtOAc (30 mL × 3), and the EtOAc solution was
dried over anhydrous Na2SO4, filtered, and evaporated under
reduced pressure. The residue was purified by MPLC on silica gel
to afford the title compound 10a-d as a solid.
General Procedure for the Preparation of the 4-Aryl-5-(6-
alkylpyridin-2-yl)-2-(dimethoxymethyl)-1H-imidazoles 11a-d.
Compound 10a-d (1.8 mmol) was dissolved in t-BuOMe (10 mL)
and treated with glyoxal dimethyl acetal (60 wt. % solution in water,
3.6 mmol). CH3CO2NH4 (4.5 mmol) in MeOH (5 mL) was added
to it, and the mixture was stirred at room temperature for 3 h. The
pH of the reaction was adjusted to pH 8 with saturated aqueous
NaHCO3 solution. The reaction mixture was partitioned between
CH2Cl2 (50 mL) and water (50 mL). The CH2Cl2 layer was
separated, dried over anhydrous MgSO4, filtered, and evaporated
to dryness under reduced pressure. The residue was purified by
MPLC on silica gel to afford the title compounds 11a and 11b as
an oil or 11c and 11d as a solid.
In this report, a series of 4(5)-(6-alkylpyridin-2-yl)imidazoles
has been synthesized and evaluated for 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. Compound 13e showed
the most significant ALK5 inhibitory activity in the series of
compounds that is much higher than those of 1, 2, and 3. A
docking model of ALK5/13e complex shows that 13e fits well
into the active site cavity of ALK5. The highly potent and
selective ALK5 inhibitor 13e might represent a valuable lead
for developing effective drugs to treat fibrosis and cancer.
General Procedure for the Preparation of the 4-Aryl-5-(6-
alkylpyridin-2-yl)-1H-imidazole-2-carbaldehydes 12a-d. Com-
pound 11a-d (0.59 mmol) was dissolved in 1 N HCl solution (4
mL), and the mixture was heated at 70 °C for 3 h. The cooled
reaction mixture was neutralized with saturated aqueous NaHCO3
solution and extracted with CH2Cl2 (50 mL × 3). The CH2Cl2
solution was dried over anhydrous MgSO4, filtered, and evaporated
to dryness under reduced pressure. The residue was purified by
MPLC on silica gel to afford the title compound 12a-d as a solid.
General Procedure for the Preparation of the 3- or 4-{[5-
(6-Alkylpyridin-2-yl)-4-aryl-1H-imidazol-2-ylmethyl]amino}-
benzonitriles 13a-h and the 3- or 4-{[5-(6-Alkylpyridin-2-yl)-
4-aryl-1H-imidazol-2-ylmethyl]amino}benzamides 13i-p. A
mixture of the imidazole-2-carbaldehyde 12a-d (0.56 mmol),
AcOH (0.56 mmol), and 3-aminobenzonitrile, 4-aminobenzonitrile,
3-aminobenzamide, or 4-aminobenzamide (0.84 mmol) in 1,2-
dichloroethane (10 mL) was stirred at room temperature for 1 h.
Sodium triacetoxyborohydride (0.84 mmol) was added to it, and
the mixture was stirred at room temperature overnight. The reaction
mixture was neutralized with 10% K2CO3 aqueous solution to pH
8 at 0 °C. The mixture was extracted with 20% MeOH in CHCl3
(50 mL × 5). The organic phase was dried over anhydrous Na2-
SO4, filtered, and evaporated 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 compound
13a-p as a solid.
Experimental Section
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
spectrometer (Bio-Rad). Electrospray ionization mass spectra (ESI-
MS) were obtained on a Q-Tof2 mass spectrometer (Micromass).
All melting points were taken in Pyrex capillaries using an
electrothermal digital melting point apparatus (Buchi) and are not
corrected. Analytical thin-layer chromatography (TLC) was per-
formed on Merck silica gel 60F-254 glass plates. Medium-pressure
liquid chromatography (MPLC) was performed using Merck silica
gel 60 (230-400 mesh) with a YFLC-540 ceramic pump (Yama-
gen). Elemental analyses were performed on a Carlo Erba 1106
elemental analyzer.
General Procedure for the Preparation of the 2-(6-Alkylpy-
ridin-2-yl)-1-(benzo[1,3]dioxol-5-yl)ethanones 9a and 9b. A
stirred solution of pyridine 7a or 7b (13 mmol) in anhydrous THF
(100 mL) at -60 °C under an Ar atmosphere was treated dropwise
with a solution of lithium bis(trimethylsilyl)amide in THF (1.0 M,
39 mmol), and then the solution was transferred via cannula to a
stirred solution of benzo[1,3]dioxole-5-carboxylic acid methyl ester
(8a; 10.3 mmol) in anhydrous THF (100 mL) at -60 °C. Stirring
was continued for 20 min, and the reaction mixture was quenched
with saturated aqueous NH4Cl solution. The mixture was filtered,
Cellular Assays to Measure Anti-TGF-â Activity of ALK5
Inhibitors. Biological activity of the test compounds was deter-

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3147
(8) Byfield, S. D.; Major, C.; Laping, N. J.; Roberts, A. B. SB-505124
is a Selective Inhibitor of Transforming Growth Factor-â Type I
Receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 2004, 65,
744-752.
(9) Grygielko, E. T.; Martin, W. M.; Tweed, C.; Thornton, P.; Harling,
J.; Brooks, D. P.; Laping, N. J. Inhibition of Gene Markers of Fibrosis
with a Novel Inhibitor of Transforming Growth Factor-â Type I
Receptor Kinase in Puromycin-Induced Nephritis. J. Pharmacol. Exp.
Ther. 2005, 313, 943-951.
mined by measuring their ability to inhibit TGF-â1-induced p3TP-
luciferase reporter activity, SBE-luciferase reporter activity, and
ARE-luciferase reporter activity in HaCaT cells. HaCaT cells were
seeded at concentrations of 5 × 104 in 24-well plates. And the next
day, when they reach approximately 90% confluence, cells were
transfected with 0.1 µg of p3TP-Luc reporter construct, SBE-Luc
reporter construct, or ARE-Luc reporter construct and 0.1 µg of
â-galactosidase, using lipofectamine 2000 (Invitrogen). At 24 h after
transfection, various concentrations of ALK5 inhibitors were added
to the cells. After 2 h, cells were treated with 5 ng/mL of TGF-â
for 18-24 h. Cell lysates were harvested according to the
manufacturer’s instruction, and luminescence was measured by a
luminometer VICTOR (Perkin-Elmer Life).
(10) Tojo, M.; Hamashima, Y.; Hanyu, A.; Kajimoto, T.; Saitoh, M.;
Miyazono, K.; Node, M.; Imamura, T. The ALK-5 Inhibitor A-83-
01 Inhibits Smad Signaling and Epithelial-to-Mesenchymal Transition
by Transforming Growth Factor-â. Cancer Sci. 2005, 96, 791-800.
(11) De Gouville, A.-C.; Boullay, V.; Krysa, G.; Pilot, J.; Brusq, J.-M.;
Loriolle, F.; Gauthier, J.-M.; Papworth, S. A.; Laroze, A.; Gellibert,
F.; Huet, S. Inhibition of TGF-â signaling by an ALK5 Inhibitor
Protects Rats from Dimethylnitrosamine-Induced Liver Fibrosis. Br.
J. Pharmacol. 2005, 145, 166-177.
(12) Sawyer, J. S.; Beight, D. W.; Britt, K. S.; Anderson, B. D.; Campbell,
R. M.; Goodson, Jr., T.; Herron, D. K.; Li, H.-Y.; McMillen, W. T.;
Mort, N.; Parsons, S.; Smith, E. C. R.; Wagner, J. R.; Yan, L.; Zhang,
F.; Yingling, J. M. Synthesis and Activity of New Aryl- and
Heteroaryl-Substituted 5,6-Dihydro-4H-pyrrolo[1,2-b]pyrazole Inhibi-
tors of the Transforming Growth Factor-â Type I Receptor Kinase
Domain. Bioorg. Med. Chem. Lett. 2004, 14, 3581-3584.
(13) (a) Chakravarty, S.; Dugar, S.; Higgins, L.; Kapoun, A. M.; Liu, D.
Y.; Schreiner, G. F.; Protter, A. A. Treatment of Fibroproliferative
Disorders Using TGF-â Inhibitors. World Patent WO 03/097615 A1,
2003. (b) Uhl, M.; Aulwurm, S.; Wischhusen, J.; Weiler, M.; Ma, J.
Y.; Almirez, R.; Mangadu, R.; Liu, Y.-W.; Platten, M.; Herrlinger,
U.; Murphy, A.; Wong, D. A.; Wick, W.; Higgins, L. S.; Weller, M.
SD-208, a Novel Transforming Growth Factor â Receptor I Kinase
Inhibitor, Inhibits Growth and Invasiveness and Enhances Immuno-
genicity of Murine and Human Glioma Cells In Vitro and In Vivo.
Cancer Res. 2004, 64, 7954-7961.
(14) Lee, W.-C.; Sun, L.; Shan, F.; Chuaqui, C.; Zheng, Z.; Petter, R. C.
Trisubstituted Heteroaryls and Methods of Making and Using the
Same. World Patent WO 03/087304 A2, 2003.
(15) Gaster, L. M.; Harling, J. D. Triarylimidazole Derivatives as Cytokine
Inhibitors. United States Patent Application US 2003/0149277 A1,
2003.
(16) Blumberg, L. C.; Munchhof, M. J.; Shavnya, A. Novel Fused
Heteroaromatic Compounds as Transforming Growth Factor (TGF)
Inhibitors. United States Patent Application US 2004/0176390 A1,
2004.
(17) 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.; de Gouville, A.-
C.; Huet, S.; Hartley, D. Identification of 1,5-Naphthyridine Deriva-
tives as a Novel Series of Potent and Selective TGF-â Type I
Receptor Inhibitors. J. Med. Chem. 2004, 47, 4494-4506.
(18) Wrana, J. L.; Attisano, L.; Carcamo, J.; Zentella, A.; Doody, J.; Laiho,
M.; Wang, X. F.; Massague, J. TGF beta Signals through a
Heteromeric Protein Kinase Receptor Complex. Cell 1992, 71, 1003-
1014.
Acknowledgment. This work was supported in part by the
grants from Ministry of Commerce, Industry and Energy, Korea
(M1-0310-43-0001 and M1-0310-43-0002), and from Research
Institute of Pharmaceutical Sciences, College of Pharmacy,
Ewha Womans University, Korea.
Supporting Information Available: Analytical data for new
compounds, synthetic procedures for compounds 15, 16, 17a, 17b,
and procedures of docking experiments. This material is available
free of charge via the Internet at http://pubs.acs.org.
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