Synthesis and biological evaluation of 2-pyridyl-substituted pyrazoles and imidazoles as transforming growth factor-β type 1 receptor kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.05.032

A series of 2-pyridyl-substituted pyrazoles (16a-d, 17, 18, and 28a-e) and imidazoles (22 and 23) has been synthesized and evaluated for their ALK5 inhibitory activity in cell-based luciferase reporter assays. Among them, 3-(3-(6-methylpyridin-2-yl)-4-(qu

Full text of DOI:10.1016/j.bmcl.2010.05.032

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4228–4232
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 2-pyridyl-substituted pyrazoles and
imidazoles as transforming growth factor-b type 1 receptor kinase inhibitors
*
Purushottam M. Dewang, Dae-Kee Kim
College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2010
Revised 6 May 2010
Accepted 12 May 2010
Available online 15 May 2010
A series of 2-pyridyl-substituted pyrazoles (16a–d, 17, 18, and 28a–e) and imidazoles (22 and 23) has
been synthesized and evaluated for their ALK5 inhibitory activity in cell-based luciferase reporter assays.
Among them, 3-(3-(6-methylpyridin-2-yl)-4-(quinolin-6-yl)-1H-pyrazole-1-carbothioamido)benzamide
(28c) showed 96% and 93% inhibition at 0.1
transfected with p3TP-luc reporter construct and ARE-luc reporter construct, respectively.
Ó 2010 Elsevier Ltd. All rights reserved.
lM in luciferase reporter assays using HaCaT cells transiently
Keywords:
TGF-b
ALK5 inhibitor
Cell-based luciferase reporter assays
Preceding its purification and cloning in the 1980s transforming
growth factor (TGF)-b1 has become a subject of intense investiga-
tion, initially by virtue of its potential to direct the diverse pheno-
typic changes in normal cells and later due to the finding that it
could inhibit growth in epithelial and immune cells.1 TGF-b1 repre-
sentstheprototypicmemberoftheTGF-bsuperfamily. TGF-bs signal
through a heterotetrameric receptor complex that consists of two
‘type II’ and two ‘type I’ transmembrane serine/threonine kinase
receptors. The signaling cascade is initiated by the binding of ligand
tothe constitutively active type II receptorwhichfurther recruitsthe
type I receptor, also named as activin-like kinase 5 (ALK5), into the
complex. Subsequently, ALK5 is phosphorylated in the juxtamem-
brane GS domain by the type II receptor thereby stimulating its ki-
nase activity.2 The activated ALK5 propagates the signals through
phosphorylation ofreceptor-regulatedSmads thatinturnformcom-
plexes with the common-mediator Smads. These Smad complexes,
when delivered to the nucleus regulate the expression of several
hundred genes involved in cell differentiation, proliferation, apopto-
sis, migration, and extracellular matrix production.3
525334),13 4 (GW6604),14 5 (SD-208),15 and 6 (LY580276)16 are
in various stages of clinical development (Fig. 1). These inhibitors
have indicated palliative effects on diseases modulated by TGF-
b.11,17–19
We have reported a number of 2-pyridyl-substituted heterocy-
cles as ALK5 inhibitors such as IN-1166 (7), IN-1130 (8), and
IN-1233 (9) and found that insertion of
a methylene, an
aminomethylene, or a methyleneamino linkage between a central
five-membered heterocyclic ring and a phenyl group markedly
increased ALK5 inhibitory activity and selectivity.20–30 The com-
pound 8, one of our preclinical candidates, has shown activity as
a suppressor of fibrogenic process of unilateral ureteral obstruction
in rats underscoring the potential clinical benefits in the treatment
of renal fibrosis.27 This compound reduced not only TGF-b signal-
ing in the CNS but also the onset and severity of paralytic disease.28
Additionally, it has been observed that the injection of 8 into the
tunica albuginea in rats lessened tunical fibrosis and corrected pe-
nile curvature.29 Recent studies demonstrated that 9 effectively
prevented the development and progression of pulmonary arterial
hypotension in the monocrotaline rat model through the inhibition
of TGF-b signaling.30
A novel class of ALK5 inhibitors that possess a carbothioamide
linkage between a pyrazole ring and a phenyl group has been re-
ported by Tojo et al.31 One of the representative compounds, A-
83-01 (10), from this carbothioamide series exhibited significant
inhibition of the transcriptional activity induced by ALK5. The
compound 10 is more potent ALK5 inhibitor than 1 and was found
to be more efficacious to prevent phosphorylation of Smad2/3 and
the growth inhibition induced by TGF-b.31
TGF-b plays a pivotal role in the initiation and progression of
fibrosis in a variety of organ systems such as kidney,4 heart,5 lung,6
and liver.7 Perturbation of TGF-b signaling has been implicated in
various human diseases including cancer,8 pancreatic diseases,9
and hematological malignancies.10
One of the strategies used to inhibit TGF-b signaling is to block
the catalytic activity of ALK5 by the use of small molecule inhibi-
tors. Due to growing interest of pharmaceutical industries, several
inhibitors such as
1 2 3 (SB-
(SB-431542),11 (SB-505124),12
It has been established by us that a carbonitrile- or carboxam-
ide-substituted phenyl substitution on a central five-membered
* Corresponding author. Tel.: + 82 2 3277 3025; fax: +82 2 3277 2467.
E-mail address: dkkim@ewha.ac.kr (D.-K. Kim).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.032

P. M. Dewang, D.-K. Kim / Bioorg. Med. Chem. Lett. 20 (2010) 4228–4232
4229
N
F
N
O
HN
N
O
O
N
N
N
N
N
O
N
N
O
N
NH
N
H
N
H
N
N
H
NH2
N
N
N
N
Cl
1
2
(SB-431542)
N
(SB-505124)
3
4
(GW6604)
(SB-525334)
5
(SD-208)
F
N
N
N
CN
O
O
S
N
N
N
HN
N
N
NH2
N
NH2
N
N
HN
N
N
H
N
H
N
H
N
N
N
N
N
6 (LY580276)
7 (IN-1166)
8 (IN-1130)
Figure 1. ALK5 inhibitors under development.
9 (IN-1233)
10 (A-83-01)
heterocyclic ring in ALK5 inhibitors increased activity and selectiv-
ity.20,23–27 On the basis of these results, a new series of chimeric
molecules 16a–d and 28a–e possessing a carbonitrile- or carbox-
amide-substituted phenyl substituent on a pyrazole and an imid-
azole ring, derived from 9 and 10, were designed as target
molecules (Fig. 2). Since the 6-quinolinyl moiety of 9 was proved
to be one of the most suitable warhead group,24 we replaced the
4-quinolinyl moiety on 16a–d with the 6-quinolinyl moiety
(28a–d) for comparison.
To compare the influence of the carbothioamide linkage of 16a
and 16c, their counterpart derivatives 17 and 18 having the methy-
lene linkage were prepared. We also prepared the imidazole deriva-
tives 22 and 23 along with the pyrazoles 17 and 18 to investigate the
effect of the central heterocycles in ALK5 inhibition. Expectedly, this
strategy was successful and provided us with more active ALK5
inhibitors than 10. Here, we are now reporting the valuable struc-
ture–activity relationships of these new chimeric molecules.
The 4-quinolinyl pyrazole derivatives 16a–d were prepared as
shown in Scheme 1.
N
O
a
b
+
N
O
O
N
N
13
11
12
(70%)
S C
N
R1
N
N
15a
d
S
R1
NH
N
N
c
N
HN
N
N
14 (68%)
16
16a : R1 = m-CN (74%)
16b : R1 = p-CN (79%)
16c : R1 = m-CONH2 (78%)
16d : R1 = p-CONH2 (80%)
Treatment of lepidine 11 with sodium bis(trimethylsilyl)amide
in anhydrous THF at ꢀ60 °C followed by reaction with ethyl 6-
methylpicolinate (12) gave 1-(6-methylpyridin-2-yl)-2-(quinolin-
4-yl)ethanone (13) in 70% yield.32 Cyclization of the ethanone 13
by reaction with dimethylformamide dimethylacetal and hydra-
zine monohydrate in the presence of acetic acid in DMF provided
Scheme 1. Reagents and conditions: (a) sodium bis(trimethylsilyl)amide, anhy-
drous THF, ꢀ60 °C, Ar atmosphere; (b) DMF?DMA, acetic acid, DMF, N2H4ꢁH2O; (c)
3- or 4-isothiocyanatobenzonitrile (15a or 15b), 3- or 4-isothiocyanatobenzamide
(15c or 15d), NaH, anhydrous THF.
the pyrazole 14 in 68% yield.32 The pyrazole 14 was alkylated with
carbonitrile- or carboxamide-substituted phenyl isothiocynates
15a–d using NaH in anhydrous THF to afford the triaryl-substi-
tuted derivatives 16a–d with a carbothioamide linkage between
pyrazole and phenyl moiety in 74–80% yields. The cyanophenyl
isothiocynates 15a and 15b were commercially available, and the
carboxamide-substituted phenyl isothiocynates 15c and 15d were
prepared33 in 78% and 82% yields, respectively, from the reaction of
meta- or para-aminobenzamide with thiophosgene in the presence
of NaHCO3 in water and chloroform.
N
N
S
R
N
N
N
HN
N
R
N
N
16a−d
17, 18
N
The 4-quinolinyl pyrazole derivatives 17 and 18 having a meth-
ylene linkage were prepared as shown in Scheme 2.
N
S
N
R
N
The pyrazole 14 was alkylated using
a-bromo-m-tolunitrile to
N
HN
afford the carbonitrile derivative 17 in 75% yield. Hydrolysis of
the nitrile 17 with 28% H2O2 and 1 N NaOH solution gave the car-
boxamide 18 in 90% yield.
N
H
N
R
N
To synthesize the 4-quinolinyl imidazole derivatives 22 and 23,
22, 23
28a−e
Figure 2. Target compounds.
the ketone 13 was converted to the
a-oximinoketone 19 which
was obtained as a mixture of oxime regioisomers (Scheme 3).

4230
P. M. Dewang, D.-K. Kim / Bioorg. Med. Chem. Lett. 20 (2010) 4228–4232
N
N
N
O
Br
+
a
b
N
O
a
N
Cl
14
+
N
N
CN
CN
N
24
25
26 (75%)
17 (75%)
N
N
S
N
c
R1
N
NH
b
N
HN
N
N
N
N
N
O
N
28
27
NH2
(73%)
28a : R1 = m-CN (70%)
1
18
(90%)
28b
28c
: R = p-CN (76%)
1
: R = m-CONH2 (83%)
28d : R1 = p-CONH2 (81%)
28e : R1 = H (81%)
Scheme 2. Reagents and conditions: (a) NaH, anhydrous THF; (b) 28% H2O2, 1 N
NaOH, EtOH, 40 °C.
Scheme 4. Reagents and conditions: (a) palladium acetate, 2-dicyclohexylphos-
phino-20-(N,N-dimethylamino)biphenyl, potassium tert-butoxide, anhydrous THF,
80 °C, Ar atmosphere; (b) DMF?DMA, acetic acid, DMF, N2H4ꢁH2O; (c) 15a–d or
isothiocyanatobenzene (15e), NaH, anhydrous THF.
The resulted oximinoketone 19 was coupled with m-cyanophenyl-
acetaldehyde (20) and ammonium acetate to give the N-hydroxy
imidazole 21 in 59% yield. The imidazole 21 was reduced to 22
by reaction with triethyl phosphite34 in 50% yield, and then the
carbonitrile group of 22 was hydrolyzed to give the carboxamide
23 in 86% yield.
moter. When cotransfected with the forkhead activin signal trans-
ducer FAST-1, ARE-luc construct is induced by TGF-b- or activin-
activated Smad2/4 complexes.39 The SBE-luc reporter construct
contains four tandem copies of the CAGA Smad-binding element
cloned upstream of the adenovirus major late promoter (MLP).40
All the pyrazole derivatives having a carbothioamide linkage,
The 6-quinolinyl moiety of 9 was introduced into the scaffold of
10 using the synthetic strategy shown in Scheme 4.
The 6-chloroquinoline (24) was coupled with 2-acetyl-6-meth-
ylpyridine (25) using palladium catalyzed coupling reaction to ob-
tain the ketone 26 in 75% yield.35,36 This ketone was further
cyclized by the treatment with dimethylformamide dimethylacetal
and hydrazine monohydrate to give the pyrazole 27 in 73% yield,
which was further alkylated with phenyl isothiocynates 15a–d or
isothiocyanatobenzene (15e) to afford the target compounds
28a–e in 70–83% yields.
16a–d and 28a–e, displayed more than 85% inhibition at 0.1 lM
in p3TP-luciferase reporter assay.
As we expected, introduction of a carboxamide-substituent at
the meta-position on the phenyl ring increased ALK5 inhibitory
activity, thus, showing that 16c (p3TP-luciferase, 94%; ARE-lucifer-
ase, 74%) and 28c (p3TP-luciferase, 96%; ARE-luciferase, 93%) were
more inhibitory than the unsubstituted 10 (p3TP-luciferase, 87%;
ARE-luciferase, 60%) and 28e (p3TP-luciferase, 88%; ARE-luciferase,
88%), respectively. The pyrazole derivatives having a carbothioa-
mide linkage, 16a (p3TP-luciferase, 92%; ARE-luciferase, 64%;
SBE-luciferase, 76%) and 16c (p3TP-luciferase, 94%; ARE-luciferase,
74%; SBE-luciferase, 77%), displayed much higher ALK5 inhibition
compared to the corresponding derivatives having a methylene
linkage, 17 (p3TP-luciferase, 48%; ARE-luciferase, 11%; SBE-lucifer-
ase, 24%) and 18 (p3TP-luciferase, 43%; ARE-luciferase, 1%; SBE-
luciferase, 14%).
To evaluate TGF-b-induced downstream transcriptional activa-
tion to ALK5 signaling, cell-based luciferase activity of 16a–d, 17,
18, 22, 23, and 28a–e was measured using HaCaT cells transiently
transfected with three different luciferase reporter genes
(p3TP-luciferase reporter, ARE-luciferase reporter, and SBE-lucifer-
ase reporter) at a concentration of 0.1 l
M (Table 1).37 The p3TP-luc
reporter construct contains three AP-1 binding elements and the
plasminogen-activator inhibitor-1 (PAI-1) promoter.38 The ARE-
luc reporter construct consists of three copies of activin response
element sequence derived from the Mix.2 homeobox gene pro-
Among the compounds having a methylene linkage, the pyra-
zole derivatives 17 and 18 were more potent than the respective
imidazole derivatives 22 (p3TP-luciferase, 9%; ARE-luciferase,
15%; SBE-luciferase, 5%) and 23 (p3TP-luciferase, 2%; ARE-lucifer-
ase, 2%; SBE-luciferase, 10%). The compounds 28a–e possessing a
6-quinolinyl moiety as a warhead group on the central pyrazole
ring exhibited higher ALK5 inhibitory activity than the 16a–d pos-
sessing a 4-quinolinyl moiety. This implies that the 6-quinolinyl of
9 is a better functionality as a warhead group in these kinds of
ALK5 inhibitors than the 4-quinolinyl of 10.
H
N
OH
N
N
O
CN
20
b
N
O
a
13
OH
N
CN
N
N
19(78%)
21 (59%)
N
The most potent compound 28c (p3TP-luciferase, 96%;
ARE-luciferase, 93%; SBE-luciferase, 80%) possessing a carboxam-
ide-substituent at the meta-position on the phenyl ring and a
6-quinolinyl moiety was chosen, and its ALK5 inhibitory activity
was compared with 2, 3, 8, and 10 at four different concentrations
N
N
N
c
d
N
H
O
N
H
CN
N
N
NH2
(0.01, 0.05, 0.1, and 0.5 lM) using HaCaT cells transiently transfec-
22(50%)
23 (86%)
ted with p3TP-luciferase reporter construct. As shown in Figure 3,
28c inhibited ALK5 in a dose-dependent manner, and it was more
potent than 2, 3, 8, and 10 at all four concentrations tested.
Scheme 3. Reagents and conditions: (a) NaNO2, 2 N HCl, EtOH; (b) NH4Ac, tert-
butyl methyl ether/MeOH; (c) P(OEt)3, DMF; (d) 28% H2O2, 1 N NaOH, EtOH, 40 °C.

P. M. Dewang, D.-K. Kim / Bioorg. Med. Chem. Lett. 20 (2010) 4228–4232
4231
Table 1
Inhibitory activity of trisubstituted imidazole and pyrazole derivatives 15, 17, 18, 21, 22 and 27
N
N
N
N
S
N
R
S
N
R
N
N
N
HN
N
H
N
HN
N
N
R
R
N
N
N
16a−d
17, 18
22, 23
28a−e
Compd
R
Activitya (% control)
ARE-luciferaseb
p3TP-luciferaseb
SBE-luciferaseb
20
100 10
Mockc
TGF-b
3
0
6
2
2
100 23
100 48
16a
16b
16c
16d
17
18
22
23
28a
28b
28c
28d
m-CN
p-CN
8
11
6
2
3
3
9
36
39
26 11
42 14
89 10
99
85 29
98 14
14
12
7
12
12
93
54
6
6
24
24
23
25
76
86 11
95 15
90 15
21
21
20
22
22
56
54
4
4
4
2
9
m-CONH2
p-CONH2
CN
CONH2
CN
CONH2
m-CN
p-CN
15
52 27
57 20
91 43
98
5
6
4
6
12 11
35
39
20 13
13
7
6
1
1
1
1
7
8
0
1
2
2
0
1
1
1
1
2
6
9
m-CONH2
p-CONH2
H
28e
2 (SB-505124)
3 (SB-525334)
8 (IN-1130)
10 (A-83-01)
5
8
43 41
40
42 20
28
3
5
5
a
b
c
Activity is given as means SD of three independent experiments run in triplicate relative to control incubation with DMSO vehicle.
Luciferase activity was determined at a concentration of 0.1 M of inhibitor.
Luciferase activity was determined without treatment of TGF-b and inhibitor.
l
erase reporter assays using HaCaT cells transiently transfected
with p3TP-luciferase reporter construct, ARE-luciferase reporter
construct, and SBE-luciferase reporter construct, respectively.
Acknowledgments
This work was supported by a grant from Ministry of Com-
merce, Industry and Energy, Korea (10027900).
References and notes
1. Pardali, K.; Moustakas, A. Biochim. Biophys. Acta. 2007, 1775, 21.
2. Massague, J. Nat. Rev. Mol. Cell Biol. 2000, 1, 169.
3. Derynck, R.; Zhang, Y. E. Nature 2003, 425, 577.
4. Wang, W.; Koka, V.; Lan, H. Y. Nephrology 2005, 10, 48.
5. Lim, H.; Zhu, Y. Z. Cell. Mol. Life Sci. 2006, 63, 2584.
6. Gu, L.; Zhu, Y.-j.; Yang, X.; Guo, Z.-J.; Xu, W.-b.; Tian, X.-l. Acta Pharmacol. Sin.
2007, 28, 382.
7. Shek, F. W.; Benyon, R. C. Eur. J. Gastroenterol. Hepatol. 2004, 16, 123.
8. Bierie, B.; Moses, H. L. Nat. Rev. Cancer 2006, 6, 506.
9. Rane, S. G.; Lee, J.-H.; Lin, H.-M. Cytokine Growth Factor Rev. 2006, 17, 107.
10. Dong, M.; Blobe, G. C. Blood 2006, 107, 4589.
11. Halder, S. K.; Beauchamp, R. D.; Datta, P. K. Neoplasia 2005, 7, 509.
12. Byfield, S. D.; Major, C.; Laping, N. J.; Roberts, A. B. Mol. Pharmacol. 2004, 65,
744.
Figure 3. Effect of 28c 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.
13. Laping, N. J.; Everitt, J. I.; Frazier, K. S.; Burgert, M.; Portis, M. J.; Cadacio, C.;
Gold, L. I.; Walker, C. L. Clin. Cancer Res. 2007, 13, 3087.
14. 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. Br. J. Pharmacol.
2005, 145, 166.
15. Ge, R.; Rajeev, V.; Ray, P.; Lattime, E.; Rittling, S.; Medicherla, S.; Protter, A.;
Murphy, A.; Chakravarty, J.; Dugar, S.; Schreiner, G.; Barnard, N.; Reiss, M. Clin.
Cancer Res. 2006, 12, 4315.
16. Sawyer, J. S.; Beight, D. W.; Britt, K. S.; Anderson, B. D.; Campbell, R. M.;
Goodson, T., Jr.; Herron, D. K.; Li, H.-Y.; McMillen, W. T.; Mort, N.; Parsons, S.;
Smith, E. C. R.; Wagner, J. R.; Yan, L.; Zhanga, F.; Yingling, J. M. Bioorg. Med.
Chem. Lett. 2004, 14, 3581.
In this Letter, a series of 2-pyridyl-substituted pyrazoles and
imidazoles containing a carbothioamide or a methylene linkage
between a phenyl moiety and a central 5-membered heterocycle
was synthesized and evaluated for their ALK5 inhibitory activity
in cell-based luciferase reporter assays. The structure–activity rela-
tionships in this series of compounds have been established and
discussed. The most potent compound in this series, 28c, inhibited
TGF-b-induced ALK5 activity 96%, 93%, and 80% at 0.1 lM in lucif-

4232
P. M. Dewang, D.-K. Kim / Bioorg. Med. Chem. Lett. 20 (2010) 4228–4232
17. Peng, S.-B.; Yan, L.; Xia, X.; Watkins, S. A.; Brooks, H. B.; Beight, D.; Herron, D.
K.; Jones, M. L.; Lampe, J. W.; McMillen, W. T.; Mort, N.; Sawyer, J. S.; Yingling, J.
M. Biochemistry 2005, 44, 2293.
18. Subramanian, G.; Schwarz, R. E.; Higgins, L.; McEnroe, G.; Chakravarty, S.;
Dugar, S.; Reiss, M. Cancer Res. 2004, 64, 5200.
19. 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. H.;
Wick, W.; Higgins, L. S.; Weller, M. Cancer Res. 2004, 64, 7954.
20. Kim, D.-K.; Jung, S. H.; Lee, H. S.; Dewang, P. M. Eur. J. Med. Chem. 2009, 44, 568.
21. Kim, Y. W.; Kim, Y. K.; Kim, D.-K.; Sheen, Y. Y. Xenobiotica 2008, 38, 451.
22. Kim, Y. W.; Kim, Y. K.; Lee, J. Y.; Chang, K. T.; Lee, H. J.; Kim, D.-K.; Sheen, Y. Y.
Xenobiotica 2008, 38, 325.
23. Kim, D.-K.; Choi, J. H.; An, Y. J.; Lee, H. S. Bioorg. Med. Chem. Lett. 2008, 18, 2122.
24. Kim, D. K.; Jang, Y.; Lee, H. S.; Park, H. J.; Yoo, J. J. Med. Chem. 2007, 50, 3143.
25. Kim, D.-K.; Kim, J.; Park, H.-J. Bioorg. Med. Chem. 2004, 12, 2013.
26. Kim, D.-K.; Kim, J.; Park, H.-J. Bioorg. Med. Chem. Lett. 2004, 14, 2401.
27. Moon, J.-A.; Kim, H.-T.; Cho, I.-S.; Sheen, Y. Y.; Kim, D.-K. Kidney Int. 2006, 70, 1234.
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.
29. Ryu, J.-K.; Piao, S.; Shin, H.-Y.; Choi, M. J.; Zhang, L. W.; Jin, H.-R.; Kim, W. J.;
Han, J.-Y.; Hong, S. S.; Park, S. H.; Lee, S.-J.; Kim, I.-H.; Lee, C. R.; Kim, D.-K.;
Mamura, M.; Kim, S.-J.; Suh, J.-K. J. Sex. Med. 2009, 6, 1284.
Smith, W.; Krysa, G.; Boullay, V.; deGouville, A. C.; Huet, S.; Hartley, D. J. Med.
Chem. 2004, 47, 4494.
33. Nikulin, V. I.; Rakov, I. M.; De Los Angeles, J. E.; Mehta, R. C.; Boyd, L. S. Y.;
Feller, D. R.; Miller, D. D. Bioorg. Med. Chem. 2006, 14, 1684.
34. Laufer, S. A.; Wagner, G. K.; Kotschenreuther, D. A.; Albrecht, W. J. Med. Chem.
2003, 46, 3230.
35. Blumberg, L. C.; Munchhof, M. J. World Patent WO 04/026863, 2004, p 72.
36. Blumberg, L. C.; Munchhof, M. J.; Shavnya, A. U.S. Pat. Appl. Publ. US 2004/
0176390, 2004, p 35.
37. Cellular assays to measure anti-TGF-b activity of ALK5 inhibitors: biological
activity of the test compounds was determined by measuring their ability to
inhibit TGF-b-induced p3TP-luciferase reporter activity, ARE-luciferase
reporter activity, and SBE-luciferase reporter activity in HaCaT cells. HaCaT
cells were seeded at concentrations of 5 ꢂ 104 in 24-well plates. The next day,
when they reach approximately 90% confluence, cells were transfected with
0.1
lg of p3TP-Luc reporter construct, ARE-Luc reporter construct, or SBE-Luc
reporter construct and 0.1
lg of b-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-b for 18–24 h. Cell lysates were harvested according to the
manufacturer’s instruction, and luminescence was measured by
luminometer VICTOR (Perkin–Elmer Life).
a
30. Long, L.; Crosby, A.; Yang, X.; Southwood, M.; Upton, P. D.; Kim, D.-K.; Morrell,
N. W. Circulation 2009, 119, 566.
38. Wrana, J. L.; Attisano, L.; Carcamo, J.; Zentella, A.; Doody, J.; Laiho, M.; Wang, X.
F.; Massague, J. Cell 1992, 71, 1003.
31. Tojo, M.; Hamashima, Y.; Hanyu, A.; Kajimoto, T.; Saitoh, M.; Miyazono, K.;
Node, M.; Imamura, T. Cancer Sci. 2005, 96, 791.
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.;
39. Liu, F.; Pouponnot, C.; Massague, J. Genes Dev. 1997, 11, 3157.
40. Dennler, S.; Itoh, S.; Vivien, D.; ten Dijke, P.; Huet, S.; Gauthier, J. M. EMBO J.
1998, 17, 3091.