Synthesis and biological evaluation of 1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted pyrazoles as inhibitors of transforming growth factor β type 1 receptor (ALK5)

Article abstract of DOI:10.1016/j.bmcl.2009.04.066

Two series of nitrogenous heterocycle compounds-1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted pyrazoles have been synthesized and evaluated for their ALK5 inhibitory activity and cytotoxicity in TGFβ-Smad2 assay and MTT assay, respectively. The

Full text of DOI:10.1016/j.bmcl.2009.04.066

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4868–4872
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 1,2,4-trisubstituted imidazoles and
1,3,5-trisubstituted pyrazoles as inhibitors of transforming growth factor b
type 1 receptor (ALK5)
*
Xingzhou Li, Lili Wang, Long Long, Junhai Xiao, Yuandong Hu, Song Li
Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of nitrogenous heterocycle compounds—1,2,4-trisubstituted imidazoles and 1,3,5-trisubsti-
tuted pyrazoles have been synthesized and evaluated for their ALK5 inhibitory activity and cytotoxicity
in TGFb-Smad2 assay and MTT assay, respectively. The ALK4/5/7 inhibitory activity of some compound
was also evaluated in ALK4/5/7 autophosphorylation assays. Compounds 6c and 14c showed relatively
potent ALK5 inhibition while weak cytotoxicity. At the same time, compounds 6c and 14c display rela-
tively better ALK5 selectivity versus ALK4/ALK7 (nearly 10-fold) compared with SB431542, a well known
ALK5 inhibitor. Compound 6g2 proved to be a moderately selective ALK4 inhibitor versus ALK5 and ALK7
(>10-fold). The binding mode of 14c generated by flexible docking study shows that 14c fits well into the
site cavity of ALK5 by forming several tight interactions.
Received 22 January 2009
Revised 14 April 2009
Accepted 16 April 2009
Available online 20 April 2009
Keywords:
Transforming growth factor bata
ALK5
Kinase inhibitor
Ó 2009 Published by Elsevier Ltd.
Progressive fibrosis in the major organs such as kidney, liver,
lung, heart, and skin is both a major cause of suffering and death
and an important contributor to the cost of health care. Transform-
ing growth factor b (TGFb) has been postulated to play a central
role in pathological fibrosis.1–5 The complex function of TGFb is
dependent on the activation of two highly conserved single
trans-membrane serine/threonine kinases, the type I receptor
[TbRI or activin-like kinase 5 (ALK5)] and type II receptor (TbRII),
respectively. Upon TGFb binding, TbRII phosphorylates threonine
residues in the GS region of the ligand-occupied TbRI. The activated
TbRI in turn phosphorylates Smad2/Smad3 proteins at the C-termi-
nal SSXS-motif, thereby causing Smad2/Smad3 dissociation from
the receptor and heteromeric complex formation with Smad4.
Then the Smad complexes translocate to the nucleus and regulate
gene respouns.6 Therefore, it becomes evident that inhibition of
phosphorylation of Smad2/Smad3 by ALK5 could reduce TGFb-in-
duced pathological fibrosis.
In additional to the central core modifications, new type of substi-
tute group were also investigated. It is particular interest to us
whether novel type of substitute groups other than the 2-pyridyl
group, which is necessary to traditional ALK5 inhibitor, may work
as well or better. So we attempted to replace the pyridine moiety
with 2-fluorophenyl, 2-methoxyphenyl, 2-hydroxylphenyl, thia-
zole-2-yl or 2-trifluoromethylphenyl. Likewise, replacement of
the benzo[1,3]dioxol-5-yl and 4-fluorophenyl with other group,
such as 3,4-dimethoxyphenyl, 3,4-dihydroxyphenyl, benzo[1,4]-
dioxol-5-yl and 3-fluoro-4-methoxyphenyl, were also practiced
to enrich the versatility of the substituent. The present Letter de-
scribes our efforts in this field, and a limited exploration of the
SAR of the synthesized compounds was also discussed.
The 1,3,5-tri-substituted pyrazoles were prepared as shown in
Scheme 1. Treatment of the solution of substituted benzaldehyde
(1) and 4-acetylbenzonitril (2) in EtOH in the presence of NaOH
catalysts gave substituted chalcones (3) in good yield.9 Reaction
The 2-pyridyl substituted nitrogenous heterocycle small mole-
cules (e.g., SB-431542, SB-505124, as shown in Fig. 1) are a novel
class of selective inhibitors of ALK5. SB-431542 and SB-505124
selectively inhibit the in vitro phosphorylation of immobilized
Smad3 with an IC50 of 94 nM, and 47 nM, respectively.7,8 To devel-
op novel type of ALK5 inhibitor, we have designed and synthesized
a series of 1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted
pyrazoles to mimic the structure of SB-431542 and SB-505124.
O
O
H
N
O
H
N
O
O
NH2
N
N
N
N
SB505124
SB431542
* Corresponding author. Tel./fax: +86 10 66931250.
E-mail address: lis@nci.bmi.ac.cn (S. Li).
Figure 1. The structure of SB431542 and SB505124.
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.04.066

X. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4868–4872
4869
O
carboxamide by treatmentwith KOH powder in t-BuOH under reflux
to afford compounds 14.
O
O
b
+
H
a
c
To investigate whether these potential inhibitors could inhibit
TGFb-induced downstream transcriptional activation to ALK5 sig-
naling, cell based TGFb-Smad2 assay (Bioimage) was used for this
analysis (Tables 1 and 2).15 In order to explore the kinase inhibition
activities against ALK5 and its two close relative ALK4 and ALK7,
their ALK4/5/7 inhibition activities were also evaluated by kinase
based autophosphorylation assay16 (Table 3). In addition to this,
the cytotoxicities of some active compounds were also determined
in MTT assay in HLF cell (Tables 1 and 2).17 A standard compound,
SB431542, was used for the calibration or comparison of result in
all above assays.
Exploration of the SAR of our compounds revealed that the en-
zyme potency of our compounds did not match well correlate with
the observed cellular activities. Good alk5 inhibition activity was
observed with the 1,3,5-trisubstituted pyrazoles in kinase based
autophosphorylation assay (Table 3), while better overall cellular
activity was observed with the 1,2,4-trisubstituted imidazoles in
cell based TGFb-Smad2 assay (Table 2). These finding suggest that
the 1,2,4-trisubstituted imidazoles may have better cellular per-
meability than the 1,3,5-trisubstituted pyrazoles.
R1
R1
1
2
CN
CN
3
CN
R1
R1
CN
N
N
N
N
R2
R2
4
5
R1
R3
N
N
R2
d
5
5
6 R3=amidecarboxy
7 R3=carboxy
e
f
8 R3=hydroxymethyl
6g R1=3,4-methylenedioxyl
R2=2-methoxylphenyl,
R3=amidocarboxy
g
6g2 R1=3,4-dihydroxy
R2=2-hydroxphenyl,
R3=amidocarboxy
Replacement of the pyridine-2-yl group with 6-methyl-pyri-
dine-2-yl and 2-fluoro-phenyl led to improvement of activity.
Compound 14c displayed significant ALK5 inhibition (69% inhibi-
Scheme 1. Reagents and conditions: (a) NaOH,EtOH; (b) 2-fluorophenylhydazin,
EtOH, reflux; (c) MnO2/SiO2, CH2Cl2; (d) KOH, t-BuOH, reflux; (e) NaOH, EtOH/H2O,
reflux; (f) LiAlH4, THF; (g) BBr3/CH2Cl2.
tion at 1
(76% inhibition at 1
even better ALK5 inhibitory activity (53.9% at 0.1
l
M, Table 2), which is compared to that of SB431542
M), in cell base assay. Compound 6c showed
M and 91.2% at
M and 75.1% at
M) in kinase based autophosphorylation assay. Introduction of
l
l
1
1
l
l
M, Table 3) than that of SB431542 (14.6% at 0.1 l
of substituted chalcones (3) and arylhydrazine or hydrochloric
arylhydrazine in ethanol produced the 1,3,5-trisubstituted pyrazo-
thiazole-2-yl, 2-trifluoromethyl-phenyl or 2-methoxyphenyl to re-
place the pyridine-2-yl group causes a loss of activity of kinase
inhibition. Replacement benzo[1,3]dioxol-5-yl with 4-fluoro-
phenyl, benzo[1,4]dioxol-6-yl or 3-fluoro-4-methoxyphenyl re-
sulted in a decline of activity, while the 3,4-dimethoxyphenyl
retained the activity. Interestingly, analogue 6g2 with 3,4-dihy-
droxyphenyl and 2-hydroxyxyphenyl substitute displayed relative
good ALK5 inhibition. The SAR of R3 observed in our work were the
same as reported.18 4-amidocarboxphenyl containing analogues
displayed the most potent activity compared with analogues con-
taining 4-cyanophenyl, 4-carboxyphenyl or 4-hydroxymethylphe-
nyl and t-butyl.
It is worth of mentioning that the ALK5 inhibition of SB431542
is very close to the ALK4 inhibition and ALK7 inhibition according
to kinase based assay as shown in Table 3. This observation is in
line with the report by Gareth8. Compounds 6c and 14c showed
relatively better ALK5 selectivity versus ALK4/ALK7 (nearly 10-
fold) compared with SB431542. Some compounds showed rela-
tively good ALK4 or ALK7 inhibitory activity against ALK5. For
example, compounds 6a, 5c, 6h, 6g2, 13c and 14a maintained even
better ALK4 inhibitory activities than that of SB431542. Herein,
compound 6g2 proved to be a moderately selective ALK4 inhibitor
versus ALK5 and ALK7 (>10-fold). On the other hand, compounds
5c, 6c, 6h and 14d possessed good ALK7 autophosphorylation
inhibitory activity. It should be noted that the kinase domains of
ALK4 and ALK5 are 89.3% identical, and those of ALK5 and ALK7
are 82.4% identical.8 Therefore, it is very difficult for small mole-
cule inhibitors to distinguish between these closely related family
members.
lins (4)10, which can be oxidated to corresponding 1,3,5-tri-substi-
11
tuted-pyrazoles (5) by MnO2/SiO2.
Conversion of the nitrile
functionality of 5 to carboxamide was accomplished by treatment
with KOH powder in t-BuOH under reflux to afford compounds 6.12
While under the condition of NaOH/ethanol/water, the nitrile
group can be converted to carboxyl giving compound 7. The car-
boxyl was reduced to hydroxymethyl by treatement with LiAlH4
to give compounds 8. Treatment of 6g with BBr3 in CH2Cl2 offered
the trihydroxyl substituted product 6g213
The 1,2,4-trisubstituted imidazoles were prepared as shown in
Scheme 2. Coupling of substituted cyanobenzene (9) with substi-
tuted aniline (10) in a mixture of THF in the presence of NaN(SiMe3)2
gave the intermediates substituted amidines(11).14 The amidines 11
condensed with 4-bromoacetylbenzonitrile or 1-bromo-3,3-di-
methyl-butan-2-one offered the corresponding 1,2,4-trisubstituted
imidazoles (12 or 13). The nitrile group of 13 can be converted to
NH
NH2
N
a
CN
R2
+
R2
R1
R1
9
10
11
R1
b
N
R4
N
R2
Besides, nearly all our compounds exhibited weaker cytotoxic-
ity against human lung fibroblast (HLF) compared with SB431542
at concentration of 30 lM.
To account for the SAR of our compounds, we built a docking
model of ALK5/14c complex based on the X-ray structure of
ALK5 complexed with LY580276 (PDB entry 1RW8).19,20 As demon-
strated in Figure 2, Compound 14c is well superimposed over the
12 R3=t-Bu
13 R3=cyanophenyl
c
14 R3=amidocarboxyphenyl
Scheme 2. Reagents and conditions: (a) NaN(SiMe3)2,THF; (b) 4-bro-
moacetylbenzonitrile or 1-bromo-3,3-dimethyl-butan-2-one, NaHCO3, i-PrOH; (c)
KOH, t-BuOH, reflux.

4870
X. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4868–4872
Table 1
The synthesized 1,3,5-trisubstituted pyrazoles and their ALK5 inhibition activity as well as cytotoxicity
R1
R3
N
R2
N
Compounds
R1
R2
R3
ALK5 inhibition (%, 1
l
M)a
Cytotoxicity (30 l
M)d
5a
6a
5b
Benzo[1,3]dioxol-5-yl
Pyridin-2-yl
4-Cyanophenyl
4-Amidocarboxphenyl
4-Cyanophenyl
NAb
15.4
9.0
NDc
26.7
ND
2-Fluoro-phenyl
6b
7b
4-Amidocarboxphenyl
4-Carboxyphenyl
26.0
9.6
ND
5.4
8b
5c
6c
6d
6e
6f
6g
5i
4-Hydroxymethylphenyl
4-Cyanophenyl
NA
ND
15.4
5.6
ND
ND
ND
ND
ND
6-Methyl-pyridin-2-yl
32.0
45.3
NA
NA
NA
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Cyanophenyl
Thiazole-2-yl
2-Trifluoromethyl phenyl
Phenyl
2-Methoxyphenyl
Pyridin-2-yl
NA
NA
3, 4-Dimethoxy phenyl
6i
4-Amidocarboxphenyl
4-Cyanophenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Cyanophenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
14.7
8.3
33.9
NA
6.0
19.0
NA
NA
NA
24.4
21.0
5.3
ND
5h
6h
6j
5k
6k
2-Fluoro-phenyl
19.4
ND
57.5
0.52
23.7
ND
Pyridin-2-yl
2-Fluoro-phenyl
Benzo[1,3]dioxol-5-y
6l
4-Fluorophenyl
Pyridin-2-yl
6m
6m2
6n
6g2
SB431542
2-Methoxyphenyl
2-Hydroxyphenyl
2-Fluoro-phenyl
2-Hydroxyphenyl
ND
19.54
48.9
55.0
3,4-Dihydroxy-phenyl
24.2(0.1
lM), 82.5(1 lM)
a
Activity is given as the mean of triplicated determinations relative to control incubations with DMSO vehicle.
NA = no significant inhibition.
ND = not detected.
b
c
d
Determined by using the MTT method.
Table 2
The synthesized 1,2,4-trisubstituted-imidazoles and their ALK5 inhibition activity as well as cytotoxicity
R1
N
R3
N
R2
d
Compounds
R1
R2
R3
ALK5 inhibitiona (%, 1
lM)
Cytotoxicity (30
l
M)
14a
14b
13c
14c
12c
13d
14d
12d
13e
14e
12e
13f
14f
12f
14g
13g
SB431542
Benzo[1,3]dioxol-5-yl
Benzo[1,4]dioxol-5-yl
Benzo[1,3]dioxol-5-yl
Pyridin-2-yl
6-Methyl-pyridin-2-yl
4-Amidocarboxphenyl
4-Amidocarboxphenyl
4-Cyanophenyl
4-Amidocarboxphenyl
t-Bu
4-Cyanophenyl
4-Amidocarboxphenyl
t-Bu
4-Cyanophenyl
4-Amidocarboxphenyl
t-Bu
4-Cyanophenyl
4-Amidocarboxphenyl
t-Bu
20.9
8.1
21.6
21.0
4.0
14.9
7.3
41.4
69.0
24.7
NAb
37.8
NA
21.1
28.2
NA
10.5
10.8
NA
15.8
42.4
4-Fluorophenyl
20.2
19.1
59.0
28.0
0.7
Benzo[1,4]dioxol-6-yl
3-Fluoro-4-methoxyphenyl
Benzo[1,3]dioxol-5-yl
NDc
57.0
9.1
22.1
ND
12.3
55.0
2-Fluoro-phenyl
4-Cyanophenyl
4-Amidocarboxphenyl
24.2(0.1 lM), 82.5(1 lM)
a
Activity is given as the mean of triplicated determinations relative to control incubations with DMSO vehicle.
NA = no significant inhibition.
ND = not detected.
b
c
d
Determined by using the MTT method.
X-ray pose of LY580276, occupying the binding site of ATP and the
nearby ‘selectivity pocket’. The benzo[1,3]dioxol-5-yl moiety of
14c interacts with the backbone of amino acids (residues 281–
283) that links the N and C terminal domains of the kinase (hinge
region), a region normally occupied by the adenine ring of ATP. One
hydrogen bond exists between the O atom at the 1-position of the

X. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4868–4872
4871
Table 3
ited more potent ALK5 inhibition activity than the corresponding
pyridin-2-yl analogues may contribute to this additional contact.
The phenyl ring at the 4 position of imidazole makes hydrophobic
interaction with Phe216, Val219 and Ala350. The amino group of
the carboxamide interacts with Asp351 and Asn338 by hydrogen
bonds. Another hydrogen bond is also formed between the car-
bonyl group of the carboxamide and Lys335. Conclusively, the
binding model of 14c generated by flexible docking studies shows
that the structure of ligand fits well onto the binding cavity of ALK5
by forming tight interactions.
In this Letter, two series of nitrogenous heterocycle com-
pounds—1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted
pyrazoles have been synthesized and evaluated for their ALK5
inhibitory activity in TGFb-Smad2 assay and cytotoxicity assay,
respectively. The ALK4/5/7 inhibitory activity of some active com-
pounds was also evaluated in ALK4/5/7 autophosphorylation assay.
Some compounds showed moderate to high inhibition against
ALK5, wherein compounds 6c and 14c showed relatively good
ALK5 inhibition activities while weak cytotoxicity. At the same
time, compounds 6c and 14c display relatively better ALK5 selec-
tivity versus ALK4/ALK7 (nearly 10-fold) compared with
SB431542. Compound 6g2 proved to be a moderately selective
ALK4 inhibitor versus ALK5 and ALK7 (>10-fold). It is worth men-
tioning that 2-fluoro-phenyl was found to be a more favorite sub-
stituent compared with the pyridine-2-yl, which is necessary to
the classic ALK5 inhibitors.
ALK4/5/7 Inhibition activities of selected compounds determined by phosphorylation
assay
Compounds
Inhibition (%)
ALK5
ALK4
ALK7
0.1
l
M
1
l
M
0.1
lM
1
l
M
0.1
l
M
1 lM
SB431542
6a
6b
5c
6c
25.7
70.0
15.5
53.6
39.4
32.1
3.2
73.8
65.6
45.5
15.6
25.0
28.5
13.3
69.4
74.7
50.7
77.7
66.8
70.4
41.9
93.7
73.7
76.9
29.8
34.5
49.3
36.6
14.6
6.9
9.9
24.6
53.9
6.2
30.7
8.8
5.3
2.5
32.7
0.4
3.5
75.1
17.2
24.5
45.2
91.2
42.8
55.4
30.3
11.7
21.0
67.2
15.5
12.7
14.1
8.9
54.0
57.3
39.1
73.4
64.0
65.8
57.4
56.5
54.8
44.6
41.3
35.1
25.0
63.6
35.5
22.7
31.4
4.2
6h
6k
5.3
50.9
34.8
24.5
33.9
4.5
16.2
13.4
30.5
6g2
14a
13c
14c
13g
14e
14d
2.7
Acknowledgment
This work was supported by a grant from National Nature Sci-
ence Fundation of China No. 30873135).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.066.
References and notes
1. Atamas, S. P.; White, B. Curr. Opin. Rheumatol. 2003, 15, 772.
2. Cutroneo, K. R. J. Cell. Biochem. 2003, 90, 1.
3. Kissin, E. Y.; Korn, J. H. Rheum. Dis. Clin. North. Am. 2003, 29, 351.
4. Laping, N. J. Curr. Opin. Pharmacol. 2003, 3, 204.
5. Robson, M. C. Surg. Clin. North. Am. 2003, 83, 557.
6. Massagu, E. Annu. Rev. Biochem. 1998, 67, 753.
7. Stacey, D. B.; Christopher, M.; Nicholas, J. L.; Anita, B. R. Mol. Pharmacol. 2004,
65, 744.
8. Gareth, J. I.; Francisco, J. N.; James, F. C., et al Mol. Pharmacol. 2002, 62, 65.
9. Sompong, W.; William, S. M. Synthesis 1980, 647.
Figure 2. The binding mode of 14c (rendered in capped stick and colored by atom
type) observed in the docking model, in comparison with the X-ray pose of
LY580276 (brown stick). Key amino acid residues within the ALK5-binding site are
represented in line form. The trapped water in the X-ray structure is shown in red
and white sphere. Green dotted lines are hydrogen bond interactions among 14c,
the amino acid residues (His283, Lys232, Asp351, Asn338, Lys335, Glu245, Tyr249)
and the trapped water.
10. Gladstone, W. A. F.; Norman, R. O. J. Chem. Soc. C 1966, 1536.
11. Liu, K. T.; Shih, M. H.; Huang, H. W. Synthesis 1988, 715.
12. Hall, J. H.; Gisler, M. J. Org. Chem. 1976, 41, 3769.
benzo[1,3]dioxol-5-yl and the backbone of His283. An analogous
hydrogen bond acceptor-donor pair occurs in many other kinase
crystal structures and is also seen for N1 of adenine in ATP. In addi-
tion, residues Val219, His283, Ala230, and Leu340 make hydropho-
bic interaction with the benzene ring of benzo[1,3] dioxol-5-yl. The
N3 (1) of the imidazole scaffold interacts with the side chain of
Lys232 through a hydrogen bond. The 6-methyl pyridine ring is in-
13. Demuyuck, M.; Clercq, P. D.; Vandewalle, M. J. Org. Chem. 1979, 44, 4863.
14. John, J. T.; David, L. B.; Jeffery, S. C. J. Med. Chem. 2000, 43, 775.
15. Cellular assays for measuring anti-TGF-b activity of ALK5 inhibitors. The
activity of compounds weretested in EGFP-SMAD2 Assay (Bioimage, the assay
is based on RedistributionTM technology to quantitate the intracellular
translocation of an EGFP-SMAD2 fusion protein in a stably transfected CHO
cell line. Following activation with TGF-b, EGFP-SMAD2 fusion protein hetero).
To test anti-TGF-b activity of compounds, the cells were seeded in 96 well
serted into the so called ‘selectivity pocket’ formed between
aC
microplates at a concentration of 20,000 cells per well in 100 lL of serum-
containing medium. The microplates were then placed for 24 h in a cell
incubator at 37 °C, 5% CO2 atm. The compounds dissolved in DMSO were then
added at different concentrations (final concentration of DMSO 0.1%) for
30 min prior to the addition of recombiation TGF-b(3 ng/mL). After an hour
incubation, the cells images wereacquired by Incell Analyzer 1000(GE
Healthy). The data analysis using the Trafficking-Analysis Module (incell
Analyzer 1000 Workstation version 1.4). Inhibition of compounds on TGF-b-
SMAD2 were expressed as the inhibitory activity on ALK5.
and b1–b4, making contacts with the following cluster of residues:
Ala230, Lys232, Leu260, Leu278, Val279 and Ser280. The hydrogen
bond to a water molecule hold in place by additional hydrogen
bonds to Asp 351, Glu245 and Tyr249 is also maintained in this
model, which is commonly observed in inhibitor containing pyri-
dyl moiety in this position.21,22 This pyridine N1–H2O can explain
why a hydrogen bond acceptor group in this position is beneficial.
The methyl group of the 6-methyl pyridyl moiety insert into a
small hydrophobic cave formed by the side chain of Leu278,
Tyr249 and Phe262. The 6-methyl-pyridin-2-yl analogues exhib-
16. Inhibition on ALK4/5/7 at molecular level. The kinase domain of ALK4/5/7 were
cloned by PCR and expressed in baculovirus/Sf9 cells system. The protein 6-His
tagged in the C terminus and purified by affinity chormatography using a Ni2+
column, and the obtained materials were used to assess compound activity in
autophosphorylation assay. Purified ALK4/5/7 3 lg/mL was incubated with

4872
X. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4868–4872
different concentrations of compounds for 30 min at 37 °C, respectively. The
reaction was then initiated by addition of 10–8 mol/L ATP and 10 g/mL
Smad3. After 3 h at 30 °C, phosphorylation was directly quantified by
determining the consumed ATP using The ENLITENÒ ATP Assay
System(Promega).
0.001 kcal molÀ1 ÅÀ1
. The ligand was saved in the Tripos Mol2 format.
l
Receptor preparation: The X-ray crystal coordinate of ALK5 complexed with
LY580276 (PDB entry 1RW8) was retrieved from the Protein Data Bank (PDB),
and all crystallographic water molecules were removed expect the one
(HOH20, atom ID 2460) involved in H-bond with ligand inside the binding
pocket. A correct atom assignment for Asn, Gln, and His residues was done, and
hydrogen atoms were added. Partial atomic charges were computed using the
Amber7FF02 force field. All heavy atoms were then fixed, and hydrogen atoms
were minimized using the Amber7FF02 force field and a constant dielectric of
1, terminating at a gradient of 0.001 kcal molÀ1 ÅÀ1. The receptor was saved in
the Tripos Mol2 format. Docking: The docking experiments were performed
with GOLD 3.1. Parameters were maintained as standard default GOLD 3.1 settings
with the following exceptions. The active site was defined as all amino acid
residues enclosed within 6.5 Å radius sphere centered by LY580276. The side
chain of Lys232, Asp351 was set to be flexible. The water state of HOH20 (atom
ID 2460) was set to ‘on’ and its orientaion was set ‘spin’. Two hydrogen-bond
constraint was added during calculations, guiding the O atom at the 1-position
of the benzo[1,3]dioxol-5-yl and the N atom of the pyridine to interact with the
backbone NH of His-283 and one proton of the water, respectively. Ten poses
docking were saved, and the top-scoring pose was taken to be the favored pose.
21. Singh, J.; Chuaqui, C. E.; Boriack-Sjodin, P. A., et al Bio. Med. Chem. Lett. 2003, 13,
4355.
17. HLF (Human Embryo Lung Fibrio lisis) Cell proliferation and cytotoxicity was
determined by MTT assay. Approximately 3000 cells/well were seeded in 96-
well plates (Coster) and incubated for 24 h before treatment with 0.1–5 mmol/
L adenosine. The initial number of viable HLF cells at the time of treatment,
termed t = 0, was then determined to correct for differences in starting cell
number between experiments and to monitor changes in cell number over
time. At the indicated times, MTT tetrazolium salt with phenazine
methosulfate were added directly to the culture media and the cells were
allowed to incubate for 2–3 h. Mitochondrial dehydrogenases of viable cells
convert MTT into a color-dense formazan. All data presented in the present
Letter were obtained from six independent experiments.
18. James, F. C.; Joelle, L. B.; James, A. F., et al J. Med. Chem. 2002, 45, 999.
19. Sawyer, J. S.; Douglas, W. B.; Karen, S. B., et al Bio. Med. Chem. Lett. 2004, 14,
3581.
20. The ligand and receptor preparation were performed with the SYBYL 6.9
software package (Tripos, Inc., St. Louis, MO). Ligand preparation: The structure
of the ligand (14c) was prepared in MOL2 format using the sketcher module of
SYBYL 6.9, Gasteiger–Huckel charges were assigned to the ligand atoms, and
22. Sawyer, J. S.; Anderson, B. D.; Beight, D. W., et al J. Med. Chem. 2003, 46, 3953.
then energyminimized until converged to
a maximum derivative of