Synthesis, biological evaluation and molecular modelling of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles as ALK5 inhibitors

Article abstract of DOI:10.1080/14756366.2020.1734799

A series of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles, 7a–c, 11a–h, and 16a–h has been synthesised and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. Incorporation of a quinoxal

Full text of DOI:10.1080/14756366.2020.1734799

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis, biological evaluation and molecular
modelling of 2,4-disubstituted-5-(6-alkylpyridin-2-
yl)-1H-imidazoles as ALK5 inhibitors
Myoung-Soon Park, Hyun-Ju Park, Young Jae An, Joon Hun Choi, Geunyoung
Cha, Hwa Jeong Lee, So-Jung Park, Purushottam M. Dewang & Dae-Kee Kim
To cite this article: Myoung-Soon Park, Hyun-Ju Park, Young Jae An, Joon Hun Choi, Geunyoung
Cha, Hwa Jeong Lee, So-Jung Park, Purushottam M. Dewang & Dae-Kee Kim (2020) Synthesis,
biological evaluation and molecular modelling of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-
imidazoles as ALK5 inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
702-712, DOI: 10.1080/14756366.2020.1734799
To link to this article: https://doi.org/10.1080/14756366.2020.1734799
© 2020 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 702–712
https://doi.org/10.1080/14756366.2020.1734799
RESEARCH PAPER
Synthesis, biological evaluation and molecular modelling of 2,4-disubstituted-5-
(6-alkylpyridin-2-yl)-1H-imidazoles as ALK5 inhibitors
Myoung-Soon Parka, Hyun-Ju Parkb, Young Jae Ana, Joon Hun Choia, Geunyoung Chaa, Hwa Jeong Leea,
So-Jung Parkb, Purushottam M. Dewanga and Dae-Kee Kima
b
aGraduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, South Korea; School of Pharmacy,
Sungkyunkwan University, Suwon, South Korea
ABSTRACT
ARTICLE HISTORY
Received 20 January 2020
Revised 18 February 2020
Accepted 19 February 2020
A series of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles, 7a–c, 11a–h, and 16a–h has been syn-
thesised and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase
reporter assay. Incorporation of a quinoxalin-6-yl moiety and a methylene linker at the 4- and 2-position
of the imidazole ring, respectively, and a m-CONH2 substituent in the phenyl ring generated a highly
potent and selective ALK5 inhibitor 11e. Docking model of ALK5 in complex with 11e showed that it fit-
ted well in the ATP-binding pocket with favourable interactions.
KEYWORDS
2,4-Disubstituted-5-(6-alkyl-
pyridin-2-yl)-1H-imidazoles;
ALK5 inhibition; cancer
immunotherapeutic
agent; docking
1. Introduction
cancer, multiple myeloma, etc.16. Another ALK5 inhibitor,
LY3200882 (3) has entered a phase 1 clinical trial16 (Figure 1).
Certain imidazo[2,1-b][1,3,4]thiadiazoles and 2,3,4-substituted
5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazoles have been
Transforming growth factor-b (TGF-b) is one of the most potent
immunosuppressive cytokines in the tumour microenvironment1.
Elevated serum levels of TGF-b commonly observed in patients
with advanced colorectal cancer2, breast cancer3,4, bladder carcin-
oma5,6, prostate cancer7,8, malignant melanoma9, pancreatic duc-
tal adenocarcinoma10, and hepatocellular carcinoma11 have been
strongly associated with tumour progression and poor clinical out-
come. Overexpression of TGF-b receptors has been implicated in
cancer12. In patients with advanced hepatocellular carcinoma
treated with the first clinically available ALK5 inhibitor, galunisertib
(1)13, an approximately two-fold longer overall survival was
observed in patients having a TGF-b1 response compared to
patients who did not have a TGF-b1 response14. Therefore, TGF-b
signalling pathway is an attractive target for development of can-
cer immunotherapeutic agents.
TGF-b signals through two distinct serine/threonine kinase
receptors, the type I (activin receptor-like kinase 5 (ALK5)) and
type II receptors. Small-molecule ALK5 inhibitors specifically inhibit
the Smad pathway by competing with ATP at the hydrophobic
ATP binding pocket of ALK5 kinase domain, which is essential for
the phosphorylation of its substrates, Smad2/Smad3 proteins.
Galunisertib progressed to phase 2/3 clinical trials against pancre-
atic carcinoma, glioblastoma, hepatocellular carcinoma, and mye-
lodysplastic syndrome13, however, Eli Lilly discontinued further
clinical development of galunisertib in 2017. Recently, we devel-
oped an ALK5 inhibitor, vactosertib (2)15, and it has progressed to
reported to possess ALK5 inhibitory activity17,18
.
Vactosertib exhibited subnanomolar ALK5 inhibitory activity in
a kinase assay and in a cell-based luciferase reporter assay15, high
selectivity against a panel of 320 protein kinases including p38a15,
moderate oral bioavailability in rats15, and high efficacy in animal
models of cancer19–21 and fibrosis22–26. In this report, we exam-
ined whether structural modification of vactosertib could increase
its subnanomolar ALK5 inhibitory activity, thus further increasing
its selectivity. For this purpose, we replaced a [1,2,4]triazolo[1,5-
a]pyridin-6-yl moiety of vactosertib with either a benzo[1,3]dioxol-
5-yl or a quinoxalin-6-yl moiety and inserted either a methylene,
an ethylene, or a propylene linker instead of a methyleneamino
linker to optimise the distance between a central imidazole ring
and a phenyl ring.
2. Materials and methods
2.1. Chemistry
1H NMR spectra were recorded on a Varian Unity-Inova 400 MHz
instrument. The chemical shifts are reported in parts per million
(ppm). For 1H NMR spectra, CDCl3 was used as solvent, and it
served as the internal standard at d 7.26. Infra-red spectra were
recorded on a FT-infra-red spectrometer (Bio-Rad). Electrospray
phase 1b/2a clinical trials either alone or in combination with ionisation mass spectra (ESIMS) were obtained on a Q-Tof2 mass
pembrolizumab, durvalumab, or pomalidomide against myelodys- spectrometer (Micromass). Elemental analyses (C, H, and N) were
plastic syndrome, non-small cell lung cancer, gastric cancer, colon used to determine the purity of all tested compounds, and the
CONTACT Dae-Kee Kim
dkkim@ewha.ac.kr
Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil,
Seodaemun-gu, Seoul 03760, South Korea
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
703
Figure 1. Small-molecule ATP-competitive ALK5 inhibitors in clinical trials.
results were within 0.4% of the calculated values (Carlo Erba (m, 4 H, 2 CH2), 2.47 (s, 3 H, CH3), 2.07 (m, 2 H, CH2); IR (CHCl3)
2228, 1573 cmꢀ1; MS (EIS) m/z 423.14 (MHþ).
1106 elemental analyzer). Analytical thin-layer chromatography
(TLC) was performed on Merck silica gel 60 F-254 glass plates.
Medium-pressure liquid chromatography (MPLC) was performed
using Merck silica gel 60 (230–400 mesh) with a YFLC-540 ceramic
2.1.2. General procedure for the preparation of the 4-(benzo[1,3]-
dioxol-5-yl)-5-(6-methylpyridin-2-yl)-1H-imidazoles 7a–c
pump (Yamagen).
A stirred solution of 6a–c (0.17 mmol), 6 N NaOH (0.04 mmol), and
28% H2O2 (0.59 mmol) in 95% EtOH (4 mL) was heated at 55 ꢁC for
3 h. The reaction mixture was cooled to 0 ꢁC and neutralised with
1 N HCl to pHꢂ8. The mixture was extracted with CH2Cl2 (30 mL),
and the organic solution was washed with water (15 mL) and
brine (15 mL), dried over anhydrous Na2SO4, filtered, and evapo-
rated 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 titled compounds 7a–c as a solid.
2.1.1. General procedure for the preparation of the 4-(benzo[1,3]-
dioxol-5-yl)-5–(6-methylpyridin-2-yl)-1H-imidazoles 6a–c
To a stirred solution of 1-(benzo[1,3]dioxol-5-yl)ꢀ2-(6-methylpyri-
din-2-yl)ethane-1,2-dione (4) (0.19 mmol) in AcOH (3 mL) were
added NH4OAc (1.11 mmol) and 4-(2-oxoethyl)benzonitrile (5a), 4-
(3-oxopropyl)benzonitrile (5b), or 4-(4-oxobutyl)benzonitrile (5c)
(0.19 mmol), and the mixture was heated at 120 ꢁC for 3 h. The pH
of the cooled reaction mixture was adjusted to pHꢂ8 at 0 ꢁC with
28% NH4OH solution in water, and the reaction mixture was
extracted with CH2Cl2 (10 mL). The CH2Cl2 solution was washed
with water (5 mL) and brine (5 mL), 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 6a–c as a solid.
2.1.2.1. 4-((4-(Benzo[d][1,3]dioxol-5-yl)-5-(6-methylpyridin-2-yl)-1H-
imidazol-2-yl)methyl)benzamide (7a). Yield 55%; mp 248 ꢀ 250 ꢁC;
1H NMR (CDCl3) d 11.70 (br s, 1 H, NH), 7.45 (d, 2 H, 2 phenyl), 7.39
(dd, 1 H, pyridyl), 7.27 (d, 1 H, pyridyl), 7.10 (d, 2 H, 2 phenyl), 7.06
(overlapped, 1 H, pyridyl), 7.05 (s, 1 H, piperonyl), 6.87 (d, 1 H,
piperonyl), 6.80 (d, 1 H, piperonyl), 6.39 (br s, 1 H, CONH), 5.95 (s,
2 H, OCH2O), 5.78 (br s, 1 H, CONH), 4.02 (s, 2 H, CH2), 2.27 (s, 3 H,
CH3); IR (CHCl3) 3191, 1661 cmꢀ1; MS (EIS) m/z 413.11 (MHþ). Anal.
Calcd for C24H20N4O3: C, 69.89; H, 4.89; N, 13.58. Found: C, 69.78;
H, 4.95; N, 13.48.
2.1.1.1. 4-((4-(Benzo[d][1,3]dioxol-5-yl)-5-(6-methylpyridin-2-yl)-1H-
imidazol-2-yl)methyl)benzonitrile (6a). Yield 30%; mp 176 ꢀ 179 ꢁC;
1H NMR (CDCl3) d 11.40 (br s, 1 H, NH), 7.45 (d, 2 H, 2 phenyl), 7.44
(overlapped, 1 H, pyridyl), 7.33 (d, 1 H, pyridyl), 7.25 (d, 2 H, 2 phe-
nyl), 7.10 (d, 1 H, pyridyl), 7.07 (s, 1 H, piperonyl), 6.93 (d, 1 H,
piperonyl), 6.84 (d, 1 H, piperonyl), 6.00 (s, 2 H, OCH2O), 4.09 (s,
2 H, CH2), 2.34 (s, 3 H, CH3); IR (CHCl3) 2229, 1574 cmꢀ1; MS (EIS)
m/z 395.13 (MHþ).
2.1.2.2. 4-(2-(4-(Benzo[d][1,3]dioxol-5-yl)-5-(6-methylpyridin-2-yl)-
1H-imidazol-2-yl)ethyl)benzamide (7b). Yield 49%; mp 189ꢀ192 ꢁC;
1H NMR (CDCl3) d 10.85 (br s, 1 H, NH) 7.66 (d, 2 H, 2 phenyl), 7.42
(t, 1 H, pyridyl), 7.30 (d, 1 H, pyridyl), 7.19 (d, 2 H, 2 phenyl), 7.07
(d, overlapped, 1 H, pyridyl), 7.05 (s, 1 H, piperonyl), 6.93 (d, 1 H,
piperonyl), 6.82 (d, 1 H, piperonyl), 6.37 (br s, 1 H, CONH), 5.99 (s,
2 H, OCH2O), 5.69 (br s, 1 H, CONH), 3.04 (m, 2 H, CH2), 3.01 (m,
2 H, CH2), 2.45 (s, 3 H, CH3); IR (CHCl3) 3441, 1653 cmꢀ1; MS (EIS)
m/z 427.10 (MHþ). Anal. Calcd for C25H22N4O3: C, 70.41; H, 5.20; N,
13.14. Found: C, 70.23; H, 5.28; N, 12.98.
2.1.1.2. 4-(2-(4-(Benzo[d][1,3]dioxol-5-yl)-5-(6-methylpyridin-2-yl)-
1H-imidazol-2-yl)ethyl)benzonitrile (6b). Yield 49%; mp 94 ꢀ 96 ꢁC;
1H NMR (CDCl3) d 10.53 (br s, 1 H, NH), 7.56 (d, 2 H, 2 phenyl), 7.43
(dd, 1 H, pyridyl), 7.31 (d, overlapped, 1 H, pyridyl) 7.29 (d, 2 H, 2
phenyl), 7.07 (d, 1 H, pyridyl), 7.04 (d, 1 H, piperonyl), 6.94 (d, 1 H,
piperonyl), 6.84 (d, 1 H, piperonyl), 6.00 (s, 2 H, OCH2O), 3.13 (m,
2 H, CH2), 3.05 (m, 2 H, CH2), 2.48 (s, 3 H, CH3); IR (CHCl3) 3375,
2229, 1573 cmꢀ1; MS (EIS) m/z 409.13 (MHþ).
2.1.2.3. 4-(3-(4-(Benzo[d][1,3]dioxol-5-yl)-5-(6-methylpyridin-2-yl)-
1H-imidazol-2-yl)propyl)benzamide
(7c).
Yield
52%;
mp
1
178ꢀ180 ꢁC; H NMR (CDCl3) d 10.60 (br s, 1 H, NH) 7.68 (d, 2 H, 2
phenyl), 7.41 (dd, 1 H, pyridyl), 7.30 (d, 1 H, pyridyl), 7.20 (d, 2 H, 2
phenyl), 7.10ꢀ7.05 (m, 2 H, 1 pyridyl and 1 piperonyl), 6.92 (d, 1 H,
piperonyl), 6.82 (d, 1 H, piperonyl), 6.25 (br s, 1 H, CONH), 5.98 (s,
2 H, OCH2O), 5.75 (br s, 1 H, CONH), 2.73 (m, 4 H, 2 CH2), 2.49 (s,
3 H, CH3), 2.07 (m, 2 H, CH2); IR (CHCl3) 3183, 1660 cmꢀ1; MS (EIS)
m/z 441.12 (MHþ). Anal. Calcd for C26H24N4O3: C, 70.89; H, 5.49; N,
2.1.1.3. 4-(3-(4-(Benzo[d][1,3]dioxol-5-yl)-5-(6-methylpyridin-2-yl)-
1H-imidazol-2-yl)propyl)benzonitrile
(6c).
Yield
36%;
mp
60 ꢀ 62 ꢁC; 1H NMR (CDCl3) d 10.63 (br s, 1 H, NH), 7.51 (d, 2 H, 2
phenyl), 7.42 (dd, 1 H, pyridyl), 7.30 (d, 1 H, pyridyl), 7.24 (d, 2 H, 2
phenyl), 7.10 ꢀ 7.04 (m, 2 H, 1 pyridyl and 1 piperonyl), 6.93 (d,
1 H, piperonyl), 6.82 (d, 1 H, piperonyl), 5.98 (s, 2 H, OCH2O), 2.74 12.72. Found: 70.63; H, 5.52; N, 12.66.

704
M.-S. PARK ET AL.
2.1.3. General procedure for the preparation of the 5-(6-alkylpyri- 2.1.3.6. 3-((5-(6-Ethylpyridin-2-yl)-1-hydroxy-4-(quinoxalin-6-yl)-1H-
imidazol-2-yl)methyl)benzonitrile (10f). Yield 71%; mp 195–196 ꢁC;
din-2-yl)-1-hydroxy-4-(quinoxalin-6-yl)-1H-imidazoles 10a–h
1H NMR (CDCl3) d 8.85 (m, 2 H, 2 quinoxalinyl), 8.36 (d, 1 H, qui-
To a stirred solution of 9a–d (0.23 mmol) in t-BuOMe (2.5 mL)
noxalinyl), 8.17 (d, 1 H, quinoxalinyl), 8.07 (dd, 1 H, quinoxalinyl),
7.75 (s, 1 H, phenyl), 7.70 (d, 1 H, phenyl), 7.54 (m, 2 H, 2 phenyl),
7.42 (t, 1 H, pyridyl), 7.38 (d, 1 H, pyridyl), 7.07 (d, 1 H, pyridyl), 4.29
(s, 2 H, CH2), 2.90 (q, 2 H, CH2), 1.38 (t, 3 H, CH3); IR (CHCl3) 2973,
2231, 1572 cmꢀ1; MS (EIS) m/z 433.24 (MHþ).
were added either 5a or 3-(2-oxoethyl)benzonitrile (5d)
(0.69 mmol) and NH4OAc (1.15 mmol) dissolved in MeOH (1.2 mL),
and the mixture was stirred at room temperature overnight under
argon atmosphere. The pH of the reaction mixture was adjusted
to pHꢂ8 at 0 ꢁC with saturated NaHCO3 solution. The reaction
mixture was partitioned between CH2Cl2 (40 mL) and water
(40 mL). The aqueous layer was extracted with CH2Cl2 (15 mL ꢃ 3). 2.1.3.7. 3-((1-Hydroxy-5-(6-isopropylpyridin-2-yl)-4-(quinoxalin-6-
The combined organic solution was dried over anhydrous Na2SO4,
filtered, and evaporated to dryness under reduced pressure. The
yl)-1H-imidazol-2-yl)methyl)benzonitrile (10g). Yield 59%; mp
146–147 ꢁC; 1H NMR (CDCl3) d 8.86 (m, 2 H, 2 quinoxalinyl), 8.36
residue was purified by MPLC on silica gel with CH2Cl2/MeOH as (d, 1 H, quinoxalinyl), 8.18 (d, 1 H, quinoxalinyl), 8.08 (dd, 1 H, qui-
noxalinyl), 7.76 (m, 1 H, phenyl), 7.71 (m, 1 H, phenyl), 7.54 (m, 2 H,
2 phenyl), 7.43 (t, 1 H, pyridyl), 7.38 (dd, 1 H, pyridyl), 7.10 (dd, 1 H,
pyridyl), 4.30 (s, 2 H, CH2), 3.14 (heptet, 1 H, CH), 1.39 (s, 3 H, CH3),
1.37 (s, 3 H, CH3); IR (CHCl3) 2967, 2231, 1597, 1572 cmꢀ1; MS (EIS)
m/z 447.22 (MHþ).
eluent to afford the titled compounds 10a–h as a solid.
2.1.3.1. 4-((1-Hydroxy-5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-
1H-imidazol-2-yl)methyl)benzonitrile (10a). Yield 40%; mp
200–203 ꢁC; 1H NMR (CDCl3) d 8.86 (m, 2 H, 2 quinoxalinyl), 8.35
(d, 1 H, quinoxalinyl), 8.17 (d, 1 H, quinoxalinyl), 8.06 (dd, 1 H, qui-
noxalinyl), 7.62 (m, 2 H, 2 phenyl), 7.56 (m, 2 H, 2 phenyl), 7.52 (t,
1 H, pyridyl), 7.36 (d, 1 H, pyridyl), 7.06 (d, 1 H, pyridyl), 4.31 (s, 2 H,
CH2), 2.61 (s, 3 H, CH3); IR (CHCl3) 3075, 2228, 1600, 1572 cmꢀ1; MS
(EIS) m/z 419.23 (MHþ).
2.1.3.8. 3-((5-(6-n-Butylpyridin-2-yl)-1-hydroxy-4-(quinoxalin-6-yl)-
1H-imidazol-2-yl)methyl)benzonitrile (10h). Yield 67%; mp
164–166 ꢁC; 1H NMR (CDCl3) d 8.85 (m, 2 H, 2 quinoxalinyl), 8.35
(d, 1 H, quinoxalinyl), 8.17 (d, 1 H, quinoxalinyl), 8.07 (dd, 1 H, qui-
noxalinyl), 7.75 (s, 1 H, phenyl), 7.70 (m, 1 H, phenyl), 7.53 (m, 2 H,
2 phenyl), 7.42 (t, 1 H, pyridyl), 7.37 (d, 1 H, pyridyl), 7.05 (d, 1 H,
pyridyl), 4.29 (s, 2 H, CH2), 2.85 (t, 2 H, CH2), 1.76 (m, 2 H, CH2), 1.43
(m, 2 H, CH2), 0.98 (t, 3 H, CH3); IR (CHCl3) 2957, 2230, 1599,
1572 cmꢀ1; MS (EIS) m/z 461.27 (MHþ).
2.1.3.2. 4-((5-(6-Ethylpyridin-2-yl)-1-hydroxy-4-(quinoxalin-6-yl)-1H-
imidazol-2-yl)methyl)benzonitrile (10b). Yield 37%; mp 84–85 ꢁC;
1H NMR (CDCl3) d 8.86 (m, 2 H, 2 quinoxalinyl), 8.36 (d, 1 H, qui-
noxalinyl), 8.17 (d, 1 H, quinoxalinyl), 8.07 (dd, 1 H, quinoxalinyl),
7.62 (m, 2 H, 2 phenyl), 7.57 (m, 2 H, 2 phenyl), 7.54 (t, 1 H, pyr-
idyl), 7.37 (d, 1 H, pyridyl), 7.08 (d, 1 H, pyridyl), 4.32 (s, 2 H, CH2),
2.90 (q, 2 H, CH2), 1.38 (t, 3 H, CH3); IR (CHCl3) 2229, 1599,
1572 cmꢀ1; MS (EIS) m/z 433.18 (MHþ).
2.1.4. General procedure for the preparation of the 5-(6-alkylpyri-
din-2-yl)-4-(quinoxalin-6-yl)-1H-imidazoles 11a–h
To a stirred solution of 10a–h (0.63 mmol) in a mixture of EtOH
(16 mL) and DMSO (4 mL) at room temperature were added 28%
H2O2 (6.62 mmol) and 6 N NaOH (0.47 mmol). The mixture was
warmed to 55 ꢁC and stirred overnight, and to it, 1 N HCl solution
was added to adjust to pHꢂ8 at 0 ꢁC. The ethanol solvent was
evaporated off under reduced pressure, and the residue was parti-
tioned between CH2Cl2 (30 mL) and H2O (50 mL). The aqueous
layer was saturated with NaCl and extracted with CH2Cl2 (30 mL ꢃ
3). The combined organic solution was washed with brine (30 mL),
dried over anhydrous Na2SO4, filtered, and evaporated to dryness
under reduced pressure. The residue was dissolved in anhydrous
DMF (20 mL) and treated with triethyl phosphite (2.39 mmol). The
mixture was heated at 110 ꢁC for 3 days, cooled to room tempera-
ture, and evaporated to dryness under reduced pressure. The reac-
tion mixture was partitioned between CH2Cl2 (30 mL) and water
(50 mL), and the aqueous layer was extracted with CH2Cl2 (30 mL
ꢃ 2). The combined organic solution was washed with saturated
NaHCO3 solution (40 mL) and brine (50 mL), dried over anhydrous
Na2SO4, filtered, and evaporated to dryness under reduced pres-
sure. The residue was purified by MPLC on silica gel with CH2Cl2/
MeOH as eluent to afford the titled compounds 11a–h as a solid.
2.1.3.3. 4-((1-Hydroxy-5-(6-isopropylpyridin-2-yl)-4-(quinoxalin-6-
yl)-1H-imidazol-2-yl)methyl)benzonitrile (10c). Yield 46%; mp
178–179 ꢁC; 1H NMR (CDCl3) d 8.86 (m, 2 H, 2 quinoxalinyl), 8.36
(d, 1 H, quinoxalinyl), 8.17 (d, 1 H, quinoxalinyl), 8.07 (dd, 1 H, qui-
noxalinyl), 7.62 (m, 2 H, 2 phenyl), 7.57 (m, 2 H, 2 phenyl), 7.55 (t,
1 H, pyridyl), 7.37 (d, 1 H, pyridyl), 7.09 (d, 1 H, pyridyl), 4.32 (s, 2 H,
CH2), 3.13 (heptet, 1 H, CH), 1.38 (s, 3 H, CH3), 1.37 (s, 3 H, CH3); IR
(CHCl3) 2229, 1598, 1571 cmꢀ1; MS (EIS) m/z 447.22 (MHþ).
2.1.3.4. 4-((5-(6-n-Butylpyridin-2-yl)-1-hydroxy-4-(quinoxalin-6-yl)-
1H-imidazol-2-yl)methyl)benzonitrile (10d). Yield 51%; mp
77–78 ꢁC; 1H NMR (CDCl3) d 8.86 (m, 2 H, 2 quinoxalinyl), 8.35 (d,
1 H, quinoxalinyl), 8.17 (d, 1 H, quinoxalinyl), 8.06 (dd, 1 H, quinoxa-
linyl), 7.62 (m, 2 H, 2 phenyl), 7.57 (m, 2 H, 2 phenyl), 7.53 (t, 1 H,
pyridyl), 7.36 (dd, 1 H, pyridyl), 7.06 (d, 1 H, pyridyl), 4.31 (s, 2 H,
CH2), 2.85 (t, 2 H, CH2), 1.76 (m, 2 H, CH2), 1.42 (m, 2 H CH2), 0.98
(t, 3 H, CH3); IR (CHCl3) 3402, 2228, 1608, 1572 cmꢀ1; MS (EIS) m/z
461.20 (MHþ).
2.1.3.5. 3-((1-Hydroxy-5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-
1H-imidazol-2-yl)methyl)benzonitrile (10e). Yield 77%; mp
210–212 ꢁC; 1H NMR (CDCl3) d 8.86 (m, 2 H, 2 quinoxalinyl), 8.36
(d, 1 H, quinoxalinyl), 8.18 (d, 1 H, quinoxalinyl), 8.08 (dd, 1 H, qui-
noxalinyl), 7.76 (s, 1 H, phenyl), 7.70 (d, 1 H, phenyl), 7.53 (m, 1 H,
phenyl), 7.51 (d, 1 H, phenyl), 7.43 (t, 1 H, pyridyl), 7.37 (d, 1 H, pyr-
idyl), 7.07 (d, 1 H, pyridyl), 4.29 (s, 2 H, CH2), 2.63 (s, 3 H, CH3); IR
(CHCl3) 3052, 2231, 1575 cmꢀ1; MS (EIS) m/z 419.20 (MHþ).
2.1.4.1. 4-((5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-
2-yl)methyl)benzamide (11a). Yield 36%; mp 227–229 ꢁC; 1H NMR
(CDCl3) d 12.01 (br s, 1 H, NH), 8.83 (m, 2 H, 2 quinoxalinyl), 8.38 (s,
1 H, quinoxalinyl), 8.15 (dd, 2 H, 2 quinoxalinyl), 7.55 (d, 2 H, 2 phe-
nyl), 7.42 (dd, 1 H, pyridyl), 7.33 (d, 1 H, pyridyl), 7.21 (d, 2 H, 2
phenyl), 6.95 (d, 1 H, pyridyl), 6.62 (br s, 1 H, CONH), 5.83 (br s, 1 H,
CONH), 4.13 (s, 2 H, CH2), 2.29 (s, 3 H, CH3); IR (CHCl3) 3185, 1665,
1616, 1572 cmꢀ1; MS (EIS) m/z 421.14 (MHþ). Anal. Calcd for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
705
C25H20N6O: C, 71.41; H, 4.79; N, 19.99. Found: C, 71.44; H, 4.65; quinoxalinyl), 8.11 (m, 2 H, 2 quinoxalinyl), 7.71 (s, 1 H, phenyl),
N, 19.87.
7.65 (m, 1 H, phenyl), 7.50 (d, 1 H, phenyl), 7.46 (t, 1 H, pyridyl),
7.36 (t, 1 H, phenyl), 7.35 (overlapped, 1 H, pyridyl), 7.02 (dd, 1 H,
pyridyl), 6.37 (br s, 1 H, CONH), 5.70 (br s, 1 H, CONH), 4.22 (s, 2 H,
CH2), 2.99 (heptet, 1 H, CH), 1.24 (s, 3 H, CH3), 1.22 (s, 3 H, CH3); IR
(CHCl3) 3184, 1665, 1574 cmꢀ1; MS (EIS) m/z 449.25 (MHþ). Anal.
Calcd for C27H24N6O: C, 72.30; H, 5.39; N, 18.74. Found: C, 72.03; H,
5.52; N, 18.67.
2.1.4.2. 4-((5-(6-Ethylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-
2-yl)methyl)benzamide (11 b). Yield 44%; mp 218–219 ꢁC; 1H NMR
(CDCl3) d 11.62 (br s, 1 H, NH), 8.83 (s, 2 H, 2 quinoxalinyl), 8.39 (s,
1 H, quinoxalinyl), 8.15 (dd, 2 H, quinoxalinyl), 7.59 (d, 2 H, 2 phe-
nyl), 7.45 (t, 1 H, pyridyl), 7.33 (d, 1 H, pyridyl), 7.26 (d, 2 H, 2 phe-
nyl), 6.99 (d, 1 H, pyridyl), 6.45 (br s, 1 H, CONH), 5.83 (br s, 1 H,
CONH), 4.17 (s, 2 H, CH2), 2.65 (q, 2 H, CH2), 1.09 (t, 3 H, CH3); IR
(CHCl3) 3179, 1663, 1615, 1571 cmꢀ1; MS (EIS) m/z 435.19 (MHþ).
Anal. Calcd for C26H22N6O: C, 71.87; H, 5.10; N, 19.34. Found: C,
71.57; H, 5.28; N, 19.12.
2.1.4.8. 3-((5-(6-n-Butylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
zol-2-yl)methyl)benzamide (11h). Yield 27%; mp 110ꢀ111 ꢁC; 1H
NMR (CDCl3) d 8.83 (m, 2 H, 2 quinoxalinyl), 8.41 (s, 1 H, quinoxa-
linyl), 8.13 (s, 2 H, 2 quinoxalinyl), 7.81 (s, 1 H, phenyl), 7.70 (d, 1 H,
phenyl), 7.55 (d, 1 H, phenyl), 7.42 (m, 2 H, phenyl and pyridyl),
7.32 (br d, 1 H, pyridyl), 6.98 (d, 1 H, pyridyl), 6.20 (br s, 1 H,
CONH), 5.60 (br s, 1 H, CONH), 4.27 (s, 2 H, CH2), 2.77 (t, 2 H), 1.63
(m, 2 H, CH2), 1.38 (m, 2 H, CH2), 0.93 (t, 3 H, CH3); IR (CHCl3) 3183,
1666, 1576 cmꢀ1; MS (EIS) m/z 463.25 (MHþ). Anal. Calcd for
C28H26N6O: C, 72.71; H, 5.67; N, 18.17. Found: C, 72.89; H, 5.51;
N, 18.03.
2.1.4.3. 4-((5-(6-Isopropylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
zol-2-yl)methyl)benzamide (11c). Yield 35%; mp 151–152 ꢁC; 1H
NMR (CDCl3) d 10.33 (br s, 1 H, NH), 8.84 (m, 2 H, 2 quinoxalinyl),
8.42 (s, 1 H, quinoxalinyl), 8.15 (s, 2 H, 2 quinoxalinyl), 7.76 (m, 2 H,
2 phenyl), 7.44 (m, 3 H, 2 phenyl and 1 pyridyl), 7.33 (d, 1 H pyr-
idyl), 7.01 (d, 1 H, pyridyl), 6.10 (br s, 1 H, CONH), 5.60 (br s, 1 H,
CONH), 4.28 (s, 2 H, CH2), 3.01 (heptet, 1 H, CH), 1.27 (s, 3 H, CH3),
1.25 (s, 3 H, CH3); IR (CHCl3) 3183, 1664, 1615, 1571 cmꢀ1; MS (EIS)
m/z 449.23 (MHþ). Anal. Calcd for C27H24N6O: C, 72.30; H, 5.39; N,
18.74. Found: C, 72.25; H, 5.45; N, 18.61.
2.1.5. General procedure for the preparation of the 4-(3-oxopro-
pyl)benzamide (14a) and 3-(3-oxopropyl)benzamide (14b)
To a stirred solution of 4-(2-(1,3-dioxolan-2-yl)ethyl)benzonitrile
(12a) (1.50 g, 7.34 mmol) in MeOH (50 mL) at room temperature
were added 28% H2O2 (25.70 mmol) and 6 N NaOH (7.34 mmol).
The mixture was warmed to 55 ꢁC and stirred for 2 h, and to it,
1 N HCl solution was added to adjust to pHꢂ8 at 0 ꢁC. The MeOH
was evaporated off under reduced pressure, and the residue was
extracted with CH2Cl2 (30 mL ꢃ 3). The organic solution was
washed with brine (30 mL), dried over anhydrous Na2SO4, filtered,
and evaporated to dryness under reduced pressure. The residue
was purified by MPLC on silica gel with MeOH/CH2Cl2 (1:19, then
1:9 (v/v)) as eluent to give 1.58 g (97%) of 4-(2-(1,3-dioxolan-2-yl)e-
thyl)benzamide (13a) as a solid. To a stirred solution of 13a
(0.50 g, 2.26 mmol) in THF (22 mL) was added 1 N HCl solution
(20 mL) at room temperature. The mixture was heated under
reflux for 1 h and cooled to room temperature. After saturation
with NaCl, the reaction mixture was extracted with CHCl3 (20 mL
ꢃ 5). The combined organic solution was dried over anhydrous
Na2SO4, filtered, and evaporated under reduced pressure to give
0.40 g (98%) of 4-(3-oxopropyl)benzamide (14a) as a solid which
was used to the next step without further purification.
2.1.4.4. 4-((5-(6-n-Butylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
zol-2-yl)methyl)benzamide (11d). Yield 42%; mp 170–171 ꢁC; 1H
NMR (CDCl3) d 11.47 (br s, 1 H, NH), 8.84 (s, 2 H, 2 quinoxalinyl),
8.40 (s, 1 H, quinoxalinyl), 8.16 (m, 2 H, 2 quinoxalinyl), 7.61 (d, 2 H,
2 phenyl), 7.43 (t, 1 H, pyridyl), 7.33 (d, 1 H, pyridyl), 7.28 (d, 2 H, 2
phenyl), 6.97 (d, 1 H, pyridyl), 6.35 (br s, 1 H, CONH), 5.68 (br s, 1 H,
CONH), 4.19 (s, 2 H, CH2), 2.64 (t, 2 H, CH2), 1.45 (m, 2 H, CH2), 1.24
(m, 2 H, CH2), 0.81 (t, 3 H, CH3); IR (CHCl3) 3182, 1661, 1616,
1570 cmꢀ1; MS (EIS) m/z 463.24 (MHþ). Anal. Calcd for C28H26N6O:
C, 72.71; H, 5.67; N, 18.17. Found: C, 72.83; H, 5.56; N, 18.02.
2.1.4.5. 3-((5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-
1
2-yl)methyl)benzamide (11e). Yield 23%; mp 181 ꢀ 182 ꢁC; H NMR
(CDCl3) d 8.79 (s, 2 H, 2 quinoxalinyl), 8.34 (s, 1 H, quinoxalinyl),
8.06 (s, 2 H, 2 quinoxalinyl), 7.71 (s, 1 H, phenyl), 7.55 (d, 1 H, phe-
nyl), 7.41 (t, 1 H, pyridyl), 7.34 (d, 1 H, pyridyl), 7.29 (d, 1 H, phenyl),
7.19 (t, 1 H, phenyl), 6.96 (d, 1 H, pyridyl), 6.83 (br s, 1 H, CONH),
6.30 (br s, 1 H, CONH), 4.13 (s, 2 H, CH2), 2.36 (s, 3 H, CH3); IR
(CHCl3) 3196, 1674, 1658 cmꢀ1; MS (EIS) m/z 421.23 (MHþ). Anal.
Calcd for C25H20N6O: C, 71.41; H, 4.79; N, 19.99. Found: C, 71.26; H,
4.92; N, 19.85.
The 3-(3-oxopropyl)benzamide (14 b) was prepared by the
same procedure as for 14a.
2.1.4.6. 3-((5-(6-Ethylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-
1
2-yl)methyl)benzamide (11f). Yield 26%; mp 125 ꢀ 126 ꢁC; H NMR
2.1.6. General procedure for the preparation of the 5-(6-alkylpyri-
(CDCl3) d 8.84 (m, 2 H, 2 quinoxalinyl), 8.40 (s, 1 H, quinoxalinyl), din-2-yl)-4-(quinoxalin-6-yl)-1H-imidazoles 16a–h
8.13 (d, 2 H, 2 quinoxalinyl), 7.85 (s, 1 H, phenyl), 7.72 (m, 1 H, phe- To a stirred solution of 15a–d (3.79 mmol) in a mixture of t-
nyl), 7.57 (m, 1 H, phenyl), 7.44 (m, 2 H, phenyl and pyridyl), 7.34
(br d, 1 H, pyridyl), 7.00 (dd, 1 H, pyridyl), 6.20 (br s, 1 H, CONH),
5.60 (br s, 1 H, CONH), 4.28 (s, 2 H, CH2), 2.80 (q, 2 H, CH2), 1.28 (t,
3 H, CH3); IR (CHCl3) 3407, 1670, 1657 cmꢀ1; MS (EIS) m/z 435.22
(MHþ). Anal. Calcd for C26H22N6O: C, 71.87; H, 5.10; N, 19.34.
Found: C, 71.89; H, 5.15; N, 19.24.
BuOMe (35 mL) and MeOH (25 mL) were added either 14a or 14b
(5.69 mmol) and NH4OAc (18.95 mmol), and the mixture was
stirred at 30 ꢁC overnight under argon atmosphere. The pH of the
reaction mixture was adjusted to pHꢂ8 at 0 ꢁC with saturated
NaHCO3 solution. After removal of solvent, the reaction mixture
was extracted with CH2Cl2 (25 mL ꢃ 3), and the organic solution
was dried over anhydrous Na2SO4, filtered and evaporated to dry-
ness under reduced pressure. The residue was purified by MPLC
on silica gel with CH2Cl2/MeOH as eluent to afford the titled com-
2.1.4.7. 3-((5-(6-Isopropylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
zol-2-yl)methyl)benzamide (11g). Yield 29%; mp 114ꢀ115 ꢁC; 1H
NMR (CDCl3)
d 8.81 (m, 2 H, 2 quinoxalinyl), 8.37 (s, 1 H, pounds 16a–h as a solid.

706
M.-S. PARK ET AL.
2.1.6.1. 4-(2-(5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida- (CHCl3) 3317, 1657, 1581 cmꢀ1; MS (EIS) m/z 449.26 (MHþ). Anal.
1
zol-2-yl)ethyl)benzamide (16a). Yield 66%; mp 145–147 ꢁC; H NMR
(CDCl3) d 8.82 (m, 2 H, 2 quinoxalinyl), 8.36 (s, 1 H, quinoxalinyl),
8.09 (d, 2 H, 2 quinoxalinyl), 7.64 (d, 2 H, 2 phenyl), 7.43 (t, 1 H,
pyridyl), 7.32 (d, 1 H, pyridyl), 7.17 (d, 2 H, 2 phenyl), 6.99 (d, 1 H,
pyridyl), 6.58 (br s, 1 H, CONH), 6.09 (br s, 1 H, CONH), 3.06 (s, 4 H,
Calcd for C27H24N6O: C, 72.30; H, 5.39; N, 18.74. Found: C, 72.44; H,
5.25; N, 18.58.
2.1.6.7. 3-(2-(5-(6-Isopropylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imi-
dazol-2-yl)ethyl)benzamide (16g). Yield 20%; mp 116ꢀ119 ꢁC; 1H
NMR (CDCl3) d 8.82 (s, 2 H, 2 quinoxalinyl), 8.37 (s, 1 H, quinoxa-
linyl), 8.10 (m, 2 H, 2 quinoxalinyl), 7.69 (t, 1 H, phenyl), 7.62 (m,
1 H, phenyl), 7.46 (t, 1 H, pyridyl), 7.34 (m, 2 H, phenyl and pyridyl),
7.31 (t, 1 H, phenyl), 7.02 (dd, 1 H, pyridyl), 6.50 (br s, 1 H, CONH),
6.00 (br s, 1 H, CONH), 3.13 (s, 4 H, 2 CH2), 3.00 (heptet, 1 H, CH),
1.25 (s, 3 H, CH3), 1.24 (s, 3 H, CH3); IR (CHCl3) 3427, 1658 cmꢀ1; MS
(EIS) m/z 463.26 (MHþ). Anal. Calcd for C28H26N6O: C, 72.71; H,
5.67; N, 18.17. Found: C, 72.86; H, 5.46; N, 18.23.
2 CH2), 2.45 (s, 3 H, CH3); IR (CHCl3) 3191, 1674, 1615, 1572 cmꢀ1
;
MS (EIS) m/z 435.19 (MHþ). Anal. Calcd for C26H22N6O: C, 71.87; H,
5.10; N, 19.34. Found: C, 71.65; H, 5.23; N, 19.30.
2.1.6.2.
4-(2-(5-(6-Ethylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
1
zol-2-yl)ethyl)benzamide (16b). Yield 63%; mp 123–125 ꢁC; H NMR
(CDCl3) d 11.40 (br s, 1 H, NH), 8.82 (m, 2 H, 2 quinoxalinyl), 8.37 (s,
1 H, quinoxalinyl), 8.11 (m, 2 H, 2 quinoxalinyl), 7.64 (d, 2 H, 2 phe-
nyl), 7.47 (t, 1 H, pyridyl), 7.35 (br d, 1 H, pyridyl), 7.19 (d, 2 H, 2
phenyl), 7.01 (d, 1 H, pyridyl), 6.50 (br s, 1 H, CONH), 5.90 (br s, 1 H,
CONH), 3.08 (s, 4 H, 2 CH2), 2.74 (q, 2 H, CH2), 1.19 (t, 3 H, CH3); IR
(CHCl3) 3418, 1666, 1570 cmꢀ1; MS (EIS) m/z 449.20 (MHþ). Anal.
Calcd for C27H24N6O: C, 72.30; H, 5.39; N, 18.74. Found: C, 72.55; H,
5.26; N, 18.61.
2.1.6.8. 3-(2-(5-(6-n-Butylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
zol-2-yl)ethyl)benzamide (16h). Yield 18%; mp 115ꢀ118 ꢁC; 1H
NMR (CDCl3) d 8.79 (s, 2 H, 2 quinoxalinyl), 8.32 (t, 1 H, quinoxa-
linyl), 8.05 (m, 2 H, 2 quinoxalinyl), 7.56 (m, 1 H, phenyl), 7.52 (s,
1 H, phenyl), 7.47 (t, 1 H, pyridyl), 7.34 (d, 1 H, pyridyl), 7.23 (over-
lapped, 1 H, phenyl), 7.19 (t, 1 H, phenyl), 7.00 (dd, 1 H, pyridyl),
6.90 (br s, 1 H, CONH), 6.23 (br s, 1 H, CONH), 3.04 (m, 2 H, CH2),
2.94 (m, 2 H, CH2), 2.66 (t, 2 H, CH2), 1.48 (m, 2 H, CH2), 1.23 (m,
2 H, CH2), 0.78 (t, 3 H, CH3); IR (CHCl3) 1657 cmꢀ1; MS (EIS) m/z
477.30 (MHþ). Anal. Calcd for C29H28N6O: C, 73.09; H, 5.92; N,
17.63. Found: C, 72.88; H, 6.15; N, 17.55.
2.1.6.3. 4-(2-(5-(6-Isopropylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imi-
dazol-2-yl)ethyl)benzamide (16c). Yield 71%; mp 113–115 ꢁC; 1H
NMR (CDCl3) d 10.75 (br s, 1 H, NH), 8.83 (m, 2 H, 2 quinoxalinyl),
8.38 (s, 1 H, quinoxalinyl), 8.12 (m, 2 H, 2 quinoxalinyl), 7.70 (m,
2 H, 2 phenyl), 7.46 (t, 1 H, pyridyl), 7.34 (br s, 1 H, pyridyl), 7.27 (d,
2 H, 2 phenyl), 7.02 (d, 1 H, pyridyl), 6.33 (br s, 1 H, CONH), 5.85 (br
s, 1 H, CONH), 3.14 (m, 4 H, 2 CH2), 3.00 (heptet, 1 H, CH), 1.26 (s,
3 H, CH3), 1.24 (s, 3 H, CH3); IR (CHCl3) 3446, 1652, 1626 cmꢀ1; MS
(EIS) m/z 463.21 (MHþ). Anal. Calcd for C28H26N6O: C, 72.71; H,
5.67; N, 18.17. Found: C, 72.53; H, 5.82; N, 18.11.
2.2. Luciferase reporter assay
To establish HaCaT (3TP-luc) stable cells, cells were seeded on six-
well plates. Cells were allowed to adhere overnight and then
transfected with the p3TP-luc (neo) expression plasmid using PEI
reagent (Sigma Aldrich). Transfected cells were cultured for four
weeks in the presence of G418 (500 mg/mL). Several single clones
were isolated and measured luciferase activity. The clone showing
response to TGF-b1 treatment was used for reporter assay. HaCaT
(3TP-luc) stable cells were seeded at 2.5 ꢃ 104 cells/well in 96-well
plate and were allowed to adhere overnight. Cells were concomi-
tantly treated with TGF-b1 (2 ng/mL) and indicated concentrations
of ALK5 inhibitors in 0.2% FBS medium and incubated for 24 h at
37 ꢁC in 5% CO2. Cell lysates were prepared using Luciferase Assay
System (Promega) according to the manufacturer’s instruction,
and luminescence was measured by a luminometer, Micro Lumat
Plus (Berthold, Germany).
2.1.6.4. 4-(2-(5-(6-n-Butylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
1
zol-2-yl)ethyl)benzamide (16d). Yield 57%; mp 100–102 ꢁC; H NMR
(CDCl3) d 11.55 (br s, 1 H, NH), 8.83 (m, 2 H, 2 quinoxalinyl), 8.36 (s,
1 H, quinoxalinyl), 8.11 (m, 2 H, 2 quinoxalinyl), 7.62 (d, 2 H, 2 phe-
nyl), 7.46 (t, 1 H, pyridyl), 7.36 (br d, 1 H, pyridyl), 7.18 (d, 2 H, 2
phenyl), 7.00 (dd, 1 H, pyridyl), 6.50 (br s, 1 H, CONH), 5.80 (br s,
1 H, CONH), 3.07 (s, 4 H, 2 CH2), 2.69 (t, 2 H, CH2), 1.54 (m, 2 H,
CH2), 1.27 (m, 2 H, CH2), 0.82 (t, 3 H, CH3); IR (CHCl3) 3411, 1657,
1621, 1569 cmꢀ1; MS (EIS) m/z 477.23 (MHþ). Anal. Calcd for
C29H28N6O: C, 73.09; H, 5.92; N, 17.63. Found: C, 72.98; H, 5.85;
N, 17.71.
2.1.6.5. 3-(2-(5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
zol-2-yl)ethyl)benzamide (16e). Yield 16%; mp 120ꢀ121 ꢁC; 1H
NMR (CDCl3) d 8.82 (m, 2 H, 2 quinoxalinyl), 8.35 (s, 1 H, quinoxa-
linyl), 8.08 (s, 2 H, 2 quinoxalinyl), 7.62 (m, 2 H, 2 phenyl), 7.43 (t,
1 H, pyridyl), 7.32 (m, 2 H, phenyl and pyridyl), 7.29 (d, 1 H, phenyl),
6.99 (d, 1 H, pyridyl), 6.63 (br s, 1 H, CONH), 6.10 (br s, 1 H, CONH),
3.08 (m, 4 H, 2 CH2), 2.46 (s, 3 H, CH3); IR (CHCl3) 3192, 1658,
1581 cmꢀ1; MS (EIS) m/z 435.20 (MHþ). Anal. Calcd for C26H22N6O:
C, 71.87; H, 5.10; N, 19.34. Found: C, 71.53; H, 5.35; N, 19.21.
2.3. Cell permeability assay
R
Caco-2 cells were seeded in TranswellV polycarbonate filter at a
density of 8 ꢃ 104 cells/filter and cultured for 21 days. Culture
medium was removed from both apical (AP) and basolateral (BL)
chambers of transwell, and the wells were rinsed three times
with PBS. AP buffer (HBSS, pH 6.5) containing 10 mM MES and BL
buffer (HBSS, pH 7.4) containing 10 mM HEPES were loaded in AP
(500 mL) and BL (1500 mL) chambers, respectively, followed by incu-
bation for 30 min at 37 ꢁC. Then, test compound (100 mM)
was added to the AP side and incubated for 2 h at 37 ꢁC. After
the incubation, BL buffer was collected and analysed using an
2.1.6.6.
3-(2-(5-(6-Ethylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imida-
zol-2-yl)ethyl)benzamide (16f). Yield 16%; mp 119ꢀ121 ꢁC; 1H
NMR (CDCl3) d 8.80 (s, 2 H, 2 quinoxalinyl), 8.34 (t, 1 H, quinoxa-
linyl), 8.06 (s, 2 H, 2 quinoxalinyl), 7.59 (m, 2 H, 2 phenyl), 7.47 (t,
1 H, pyridyl), 7.33 (d, 1 H, pyridyl), 7.23 (t, 1 H, phenyl), 7.01 (dd,
1 H, pyridyl), 6.80 (br s, 1 H, CONH), 6.25 (br s, 1 H, CONH), 3.06 (m,
UV spectrophotometer at
a
maximum wavelength (wave-
2 H, CH2), 3.01 (m, 2 H, CH2), 2.71 (q, 2 H, CH2), 1.16 (t, 3 H, CH3); IR length 225–357 nm).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
707
2.4. Docking study
All computational works were performed on the Sybyl-X 2.1.1
(Tripos Inc., St Louis, MO, USA) molecular modelling package with
CentOS Linux 5.4. operating system127.
2.4.1. Preparation of ligands and receptor
The 11e was prepared with sketch module embedded in Sybyl
package and saved as mol2 format. After sketching the molecule,
€
Gasteiger-Huckel charges were assigned to all atoms. To optimise
the ligand, energy minimisation was conducted by the standard
tripos force field with convergence to maximum derivatives of
0.001 kcal molꢀ1ꢄÅꢀ1. The X-ray structure of ALK5 complexed with
5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhibitor was used as
a
receptor for docking (PDB id: 1RW8)28. Receptor structure was
retrieved from PDB (http://www.rcsb.org/) and optimised using
structure preparation tool embedded in biopolymer module.
Native ligand was extracted and all water molecules except key
water molecule for water-mediated hydrogen bond network were
deleted from the complex structure.
2.4.2. Molecular docking
To examine the binding poses of 11e, docking study was con-
ducted using Surflex-Dock3 embedded in Tripos Sybyl X 2.1.1 soft-
ware package. For docking performance, the active site was
assigned as a protomol generated by using the native ligand in
the X-ray structure. Flexible docking was carried out by using
default parameter values (threshold ¼ 0.5 and bloat ¼ 0), produc-
ing 200 conformers as maximum number of poses per ligand.
Binding affinity of each docking pose of ligand was calculated by
Surflex-dock score and consensus scoring function (CScore). The
total Surflex-Dock score was expressed as –logKd to represent
binding affinities. To build the best docking model, key interac-
tions between candidate compound and active site were investi-
gated by visual inspection.
Scheme 1. Reagents and conditions: (a) NH4OAc, AcOH, 120 ꢁC, 3 h; (b) 28%
H2O2, 6 N NaOH, EtOH, 55 ꢁC, 3 h.
The 11a–h were prepared as shown in Scheme 2. The 2-(6-
alkylpyridin-2-yl)-1-(quinoxalin-6-yl)ethanones 8a–d33 were treated
with NaNO2 in 5 N HCl to give the 2-(6-alkylpyridin-2-yl)-2-(hydrox-
yimino)-1-(quinoxalin-6-yl)ethanones 9a–d in 88–99% yields.
Condensation of 9a–d with either 5a or 3-(2-oxoethyl)benzonitrile
(5d)34 and NH4OAc in a mixture of t-BuOMe and MeOH at room
temperature afforded the 5-(6-alkylpyridin-2-yl)-1-hydroxy-4-(qui-
noxalin-6-yl)-1H-imidazoles 10a–h in 37–77% yields. Conversion of
the carbonitrile group of 10a–h to the carboxamide group and
subsequent dehydroxylation with triethyl phosphite in anhydrous
DMF at 110 ꢁC for 3 days gave the target compounds 11a–h in
23–44% yields.
3. Results and discussion
3.1. Chemistry
The requisite aldehydes, 4–(3-oxopropyl)benzamide (14a) and
3-(3-oxopropyl)benzamide (14b) for the synthesis of the target
compounds 16a–h were prepared as shown in Scheme 3.
Selective hydrolysis of the carbonitrile group of 12a31 and
12b35 to the carboxamide group followed by acidic hydrolysis of
1,3-dioxolanyl protecting group of 13a and 13 b afforded 14a and
14 b in almost quantitative yield. The 2-(6-alkylpyridin-2-yl)-1-(qui-
noxalin-6-yl)ethane-1,2-diones 15a–d33 was condensed with either
14a or 14 b and NH4OAc in a mixture of t-BuOMe and MeOH to
obtain the 4-carboxamide analogues 16a–d in modest yields
(57–71%) and the 3-carboxamide analogues 16e–h in lower yields
(16–20%) (Scheme 4).
To develop a potent, selective, and orally bioavailable ALK5 inhibi-
tor, as our starting point, we designed three target molecules, 4-
((4-(benzo[1,3]dioxol-5-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-
yl)methyl)benzamide (7a) and its ethyl and n-propyl analogues,
7b and 7c to optimise the distance between the two pharmaco-
phores, a central imidazole ring and a phenyl ring.
The 7a–c were prepared as shown in Scheme 1. The 1-(ben-
zo[1,3]dioxol-5-yl)-2-(6-methylpyridin-2-yl)ethane-1,2-dione
(4)29
was condensed with 4-(2-oxoethyl)benzonitrile (5a)30, 4-(3-oxopro-
pyl)benzonitrile (5b)31, or 4-(4-oxobutyl)benzonitrile (5c)32 and
NH4OAc in AcOH at 120 ꢁC to produce the 2-((4-cyanophenyl)alky-
l)imidazoles 6a–c in 30–49% yields. Transformation of the carboni-
trile group of 6a–c to the corresponding carboxamide group was
accomplished by treatment with 28% H2O2 and 6 N NaOH in EtOH
at 55 ꢁC to provide the 7a–c in 49–55% yields.
3.2. ALK5 inhibitory activity in an enzyme assay and in a cell-
based luciferase reporter assay
To evaluate whether these potential inhibitors 7a–c, 11a–h, and
16a–h could inhibit ALK5, a kinase assay was performed using the
purified human ALK5 kinase domain produced in Sf9 insect cells
and casein as a substrate (Table 1). The ALK5 inhibitory activity of
7b (IC50 ¼ 0.093 mM) was 2.4-fold and 3.6-fold higher than those
of 7a (IC50 ¼ 0.224 mM) and 7c (IC50 ¼ 0.338 mM), respectively, in a
kinase assay (Table 1). In a cell-based luciferase reporter assay
Since a methylene or an ethylene linker was proved to be
more beneficial than a n-propylene linker in ALK5 inhibition in
both a kinase assay and a cell-based luciferase reporter assay, we
next synthesised a series of 5-(6-alkylpyridin-2-yl)-4-(quinoxalin-6-
yl)-1H-imidazoles, 11a–h and 16a–h, to compare the impact of a
benzo[1,3]dioxol-5-yl moiety and
ALK5 inhibition.
a quinoxalin-6-yl moiety in

708
M.-S. PARK ET AL.
Scheme 2. Reagents and conditions: (a) NaNO2, 5 N HCl, rt, 1 h; (b) 5a or 3-(2-oxoethyl)benzonitrile (5d), NH4OAc, t-BuOMe/MeOH, rt, overnight, Ar atmosphere; (c) (i)
28% H2O2, 6 N NaOH, EtOH, DMSO, 55ꢁC, overnight; (ii) triethyl phosphite, anhydrous DMF, 110 ꢁC, 72 h.
Scheme 3. Reagents and conditions: (a) 28% H2O2, 6 N NaOH, MeOH, 55 ꢁC, 2 h; (b) 1 N HCl, THF, reflux, 1 h.
using HaCaT (3TP-luc) stable cells, 7c (40% inhibition) was much
less inhibitory than 7a (72% inhibition) and 7b (74% inhibition) at
a concentration of 0.1 mM.
Replacement of a benzo[1,3]dioxol-5-yl moiety in 7a and 7b
with a quinoxalin-6-yl moiety increased ALK5 inhibitory activity,
thus, the corresponding analogues, 11a (IC50 ¼ 0.088 mM) and 16a
(IC50 ¼ 0.030 mM), were 2.5-fold and 3.1-fold more potent than 7a
and 7b, respectively, in a kinase assay (Table 1). Similar to kinase
assay, 11a (84% inhibition) and 16a (85% inhibition) were much
more inhibiting than 7a and 7b in a luciferase reporter assay. It
was previously demonstrated that a Me substituent at the six-pos-
ition of the pyridine ring in the pyridyl-substituted ALK5 inhibitors
significantly increased ALK5 inhibitory activity36–38. Therefore, we
examined the effect of bulkier alkyl groups such as Et, i-Pr, and n-
Bu in ALK5 inhibition. The 6-ethylpyridyl analogues 11b, 11f, 16b,
and 16f were 1.3-, 2.1-, 1.4-, and 1.4-fold less potent than the
Scheme 4. Reagents and conditions: (a) 14a or 14 b, NH4OAc, t-BuOMe/MeOH,
30 ꢁC, overnight, Ar atmosphere.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
709
Table 1. ALK5 inhibitory activity and Caco-2 cell permeability of 2,4-disubsti- Table 2. ALK5 and p38a inhibitory activity of 11e, 1, and 2.
tubed-5–(6-alkylpyridin-2-yl)-1H-imidazoles 7a–c, 11a–h, and 16a–h.
IC50 for
ALK5
IC50 for
p38a
IC50 for
Selectivity
indexd
p3TP-luciferase
Compound
(lM)a,b
(lM)b,c
(lM)e,f
11e
0.013
0.086
0.013
0.288
0.320
1.775
22
4
137
0.0196
>0.1
0.0165
Galunisertib (1)
Vactosertib (2)
R
aA proprietary radioisotopic protein kinase assay (33PanQinaseV Activity Assay)
was performed at ProQinase GmbH (Freiburg, Germany). The assay contained
70 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 lM Na-orthovanadate,
1.2 mM DTT, 50 lg/mL PEG20000, 1 lM [c-33P]-ATP (approximately 6 ꢃ 105 cpm
per well). Five ng/50 lL of ALK5 and 1000 ng/50 lL of GSK3 (14–27) as a sub-
strate were used per well.
p3TP-luciferase
activityd
(% control)
Caco-2
permeability
(%)
a,b
IC50
bMean of duplicates.
Compound
R
CONH2
n
(lM)
R
cA proprietary radioisotopic protein kinase assay (33PanQinaseV Activity Assay)
Mock
TGF-b
7a
7b
7c
2
0e
was performed at ProQinase GmbH (Freiburg, Germany) using ATF2 as
100 5.0
28 7.1
26 7.9
60 9.5
16 4.5
33 21.3
39 11.4
85 32.5
a substrate.
35 0.7f
23 3.0
32 1.1
25 1.3
26 3.5
30 9.0
41 5.3
56 26.8
38 13.8
34 15.6
n.d.g
45 5.3
25 3.7
23 3.1
24 1.9
21 9.5
dIC50 for p38a/IC50 for ALK5.
eHaCaT (3TP-luc) stable cells were used.
fMean of triplicates.
1
2
3
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0.224
0.093
0.338
0.088
0.112
2.195
2.700
0.036c
0.075
1.250
1.038
0.030
0.041
0.653
2.383
0.047
0.066
0.593
2.875
11a
11b
11c
11d
11e
11f
11g
11h
16a
16b
16c
16d
16e
16f
Me
Et
i-Pr
p-CONH2
p-CONH2
p-CONH2
showing IC50 values of <0.05 mM in a kinase assay, 11e (IC50
¼
n-Bu p-CONH2
Me
Et
0.036 mM), 16a, 16 b (IC50 ¼ 0.041 mM), and 16e (IC50 ¼ 0.047 mM)
exhibited much higher inhibitory activity in a luciferase reporter
assay, also (11e: 92%, 16a: 85%, 16b: 84%, 16e: 93%).
m-CONH2
m-CONH2
m-CONH2
8 5.8
17 3.7
69 6.7
83 21.1
15 8.2
16 8.4
85 38.9
76 5.7
i-Pr
n-Bu m-CONH2
Me
Et
Among this series of compounds, 11e possessing the most
potent ALK5 inhibitory activity and the highest permeability in
Caco-2 cells was selected, and its ALK5 inhibitory activity was
compared with those of potential competitors, galunisertib and
vactosertib in a kinase assay and in a luciferase reporter assay. In
a kinase assay, 11e (IC50 ¼ 0.013 mM) showed the same level of
potency to that of vactosertib (IC50 ¼ 0.013 mM) and 6.6-fold
higher potency compared to that of galunisertib (IC50 ¼ 0.086 mM)
(Table 2). Luciferase activity of HaCaT (3TP-luc) cells was increased
by 65-fold after treatment of TGF-b1 (2 ng/mL), and 11e and vac-
tosertib displayed the similar level of inhibition on the TGF-b1-
induced luciferase reporter activity with IC50 values of 0.0196 mM
and 0.0165 mM, respectively. Similar to a kinase assay, galunisertib
displayed much lower inhibition (IC50 >0.1 mM) compared to 11e
and vactosertib (Table 2).
The kinase domain of p38a is known to be one of the most
homologous to that of ALK539, therefore, this enzyme was chosen
to compare selectivity of 11e, galunisertib, and vactosertib. In a
p38a kinase assay, IC50 values of 11e, galunisertib, and vactosertib
were 0.288 mM, 0.320 mM, and 1.775 mM, respectively, thus, their
selectivity indices (IC50 for p38a/IC50 for ALK5) were 22, 4, and
137, respectively. Although 11e was 5.5-fold more selective
against p38a than galunisertib, it was 6.2-fold less selective than
vactosertib.
p-CONH2
p-CONH2
p-CONH2
i-Pr
n-Bu p-CONH2
Me
Et
m-CONH2
m-CONH2
m-CONH2
7 0.7
56 14.6
48 41.8
92 10.4
6 4.1
0
0
16g
16h
i-Pr
n-Bu m-CONH2
aALK5 was expressed in Sf9 insect cells as human recombinant GST-fusion pro-
tein by means of the vaculovirus expression system. A Proprietary radioisotopic
R
protein kinase assay (33PanQinaseV Activity Assay) was performed at ProQinase
GmbH (Freiburg, Germany). The assay contained 60 mM HEPES-NaOH, pH 7.5,
3 mM MgCl2, 3 mM MnCl2, 3 lM Na-orthovanadate, 1.2 mM DTT, 50 lg/mL
PEG20000, 1 lM [c-33P]-ATP (approximately 5 ꢃ 105 cpm per well). One hundred
ng/50 lL of ALK5 and 1000 ng/50 lL of casein as a substrate were used per well.
bSinglicate.
cMean of triplicates.
dHaCaT (p3TP-luc) stable cells were used. Luciferase activity was determined at a
concentration of 0.1 lM of inhibitor.
eThe mean SD of three independent experiments run in triplicates relative to
control incubations with DMSO vehicle.
fThe mean SD of quadruplicates.
gNot determined.
corresponding 6-methylpyridyl analogues 11a, 11e, 16a, and 16e,
respectively. However, the 6-i-propylpyridyl analogues (11c, 11g,
16c, and 16g) and 6-n-butylpyridyl analogues (11d, 11h, 16d, and
16h) displayed 13–35-fold and 29–71-fold lower inhibitory activity
compared to the respective 6-methylpyridyl analogues, indicating
that a bulkier group than Et group cannot be accommodated
favourably into the ATP binding pocket of ALK5. Regarding the
length of a linker, an ethylene linker was generally more beneficial
than a methylene linker in the ALK5 inhibition as shown in 7a
and 7b, thus, 16a, 16b, 16c, and 16g were 2.9-, 2.7-, 3.4-, and 2.1-
fold more inhibiting than the respective 11a, 11b, 11c, and 11g.
Compounds 16d–f showed the similar level of potency to that of
11d–f, respectively. The position of a carboxamide group in the
phenyl ring also influenced ALK5 inhibition. The m-CONH2 ana-
logues 11e–h were 1.5–2.6-fold more inhibiting than the respect-
ive p-CONH2 analogues 11a–d in a series of compounds having a
methylene linker, whereas the p-CONH2 analogues having an
ethylene linker, 16a and 16 b, were 1.6-fold more inhibiting than
3.3. Caco-2 cell permeability assay
To estimate oral absorption of target compounds, their permeabil-
ity in a Caco-2 monolayer was evaluated at a concentration of
100 mM (Table 1). The 11e showed the highest permeability (56%),
but 16f (6%), 16g (0%), and 16h (0%) showed very limited or no
permeation in this assay, demonstrating that even simple struc-
tural modification markedly affected the permeability in this series
of compounds.
3.4. Kinase profiling assay
Considering the ALK5 inhibitory activity in both a kinase assay
and a luciferase reporter assay and the Caco-2 cell permeability,
the corresponding m-CONH2 analogues 16e and 16f. Compounds 11e was selected as a candidate for preliminary kinase profiling.

710
M.-S. PARK ET AL.
Figure 2. Inhibitory profile of 11e in 28 protein kinase assays.
Figure 3. Docked pose of 11e in the active site of ALK5 (PDBid:1RW8). (A, B) 11e is (magenta carbon atoms) superimposed over the X-ray pose of native ligand (grey
carbon atoms). The active site of ALK5 is shown as MOLCAD lipophilic potential surface map (A), and lipophilicity increases from blue (hydrophilic) to brown (lipho-
philic). Grey capped sticks represent key amino acid residues within the binding site, and the backbone of ALK5 is shown as ribbon. The bound water molecule in the
X-ray structure is represented by ball and stick. (C) Intermolecular interaction between 11e and ALK5. Grey capped sticks represent key amino acid residues in the
active site. Red dashed lines are hydrogen bonding interactions (<3.0 Å).
Because ALK5 is a serine/threonine kinase, we chose 19 serine/ between amino acid residues in the active site and 11e, in com-
parison with the X-ray pose of native ligand (3-(4-fluorophenyl)-2-
(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole)28. As
shown in Figure 3(A,B), 11e fits well into cavity of active site, with
quinoxaline ring and methyl group of 6-methylpyridine ring occu-
pying the hydrophobic pockets. Hydrogen bond interactions
between 11e and ALK5 are exhibited in Figure 3(C). The quinoxa-
line ring nitrogen acts as a hydrogen bond acceptor and interacts
with NH of His283 in the backbone of hinge region of ALK5. The
6-methylpyridine ring nitrogen forms a water-mediated hydrogen
bond network with the backbone NH of Asp351, and the side
chain of both Tyr249 and Glu245. The imidazole ring NH of 11e is
also involved in the water-mediated hydrogen bond network. The
NH2 group of carboxamide in the meta position of phenyl ring
interacts with the side chain of Asn338 by forming hydrogen
bond, which cannot be formed for the compounds, 11a–d having
the carboxamide group in the para position. Our docking model
for the most active compound 11e well supports the key interac-
threonine kinases including ALK5 and an ALK family kinase, ACV-
R1 (ALK5, ACV-R1, Aurora-A, ARK5, B-Raf, CK2a1, COT, DAPK1,
IRAK4, JNK3, MAPKAPK5, MST4, NEK2, NLK, PAK1, PIM1, PRK1, S6K,
SGK1) and 9 tyrosine kinases (ABL1, CSK, FGF-R1, FLT3, IGF1-R,
PDGFR-a, SRC, VEGF-R1, ZAP70). Selectivity profiling of 11e using
a panel of 28 protein kinases showed selectivity indices of >100
against all the kinases tested (ProQinase GmbH (Freiburg,
Germany)) (Figure 2).
3.5. Docking model of ALK5 in complex with 11e
To determine the binding pose of 11e in the active site of ALK5,
we executed docking modelling with flexible molecular docking
programme Surflex-dock40. We analysed the result of docking con-
sidering Surflex-dock docking score (–logKd) and consensus score
(obtained from CScore module27), and selected the poses of 11e
with high scores (–logKd ꢅ7 and CScore ꢅ3). To select the best
docked pose among them, we also identified key interactions tions for ALK5 inhibition which was previously reported28.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
711
5. Eder IE, Stenzl A, Hobisch A, et al. Transforming growth fac-
tors-b1 and b2 in serum and urine from patients with blad-
der carcinoma. J Urol 1996;156:953–7.
6. Shariat SF, Kim JH, Andrews B, et al. Preoperative plasma
levels of transforming growth factor b1 strongly predict clin-
ical outcome in patients with bladder carcinoma. Cancer
2001;92:2985–92.
7. Adler HL, McCurdy MA, Kattan MW, et al. Elevated levels of
circulating interleukin-6 and transforming growth factor-b1
in patients with metastatic prostatic carcinoma. J Urol 1999;
161:182–7.
8. Shariat SF, Shalev M, Menesses-Diaz A, et al. Preoperative
plasma levels of transforming growth factor beta1 (TGF-b1)
strongly predict progression in patients undergoing radical
prostatectomy. J Clin Oncol 2001;19:2856–64.
4. Conclusions
In this report, a series of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-
1H-imidazoles, 7a–c, 11a–h, and 16a–h has been synthesised and
evaluated for their ALK5 inhibitory activity in an enzyme assay
and in a cell-based luciferase reporter assay. The structure–activity
relationships in this series of compounds revealed that an ethyl-
ene linker at the two-position of the imidazole ring was the most
beneficial in ALK5 inhibitory activity. Replacement of a benzo[1,3]-
dioxol-5-yl moiety at the four-position of the imidazole ring with a
quinoxalin-6-yl moiety markedly increased ALK5 inhibitory activity.
Regarding the alkyl substituent at the six-position of the pyridine
ring, the compounds having a methyl or an ethyl substituent dis-
played much higher inhibitory activity than the compounds hav-
ing an i-propyl or a n-butyl substituent. The m-CONH2 analogues
were more inhibiting than the p-CONH2 analogues in compounds
having a methylene linker, whereas the p-CONH2 analogues were
more inhibiting than the m-CONH2 analogues in compounds hav-
ing an ethylene linker. In a cell permeability assay using Caco-2
monolayer, 11e showed the highest permeability in this series of
compounds. The 11e was equipotent to vactosertib, but much
more potent than galunisertib in an ALK5 kinase assay and in a
cell-based luciferase reporter assay. Although 11e was 5.5-fold
more selective against p38a than galunisertib, it was 6.2-fold less
selective than vactosertib. Therefore, it can be concluded that
combination of replacement of a [1,2,4]triazolo[1,5-a]pyridin-6-yl
moiety with a quinoxalin-6-yl moiety, insertion of a methylene
linker instead of a methyleneamino linker, and a m-CONH2 sub-
stituent in the phenyl ring in vactosertib maintained its high ALK5
inhibitory activity, but decreased its selectivity against p38a.
Selectivity profiling of 11e using a panel of 28 protein kinases
showed that it is highly selective for ALK5. Our docking results
demonstrate that 11e fits well in the ATP-binding pocket of ALK5
with favourable intermolecular interactions.
€
9. Krasagakis K, Tholke D, Farthmann B, et al. Elevated plasma
levels of transforming growth factor (TGF)-b1 and TGF-b2 in
patients with disseminated malignant melanoma. Br
J
Cancer 1998;77:1492–4.
10. Zhao J, Liang Y, Yin Q, et al. Clinical and prognostic signifi-
cance of serum transforming growth factor-beta1 levels in
patients with pancreatic ductal adenocarcinoma. Braz J Med
Biol Res 2016;49:e5485.
11. Lin TH, Shao YY, Chan SY, et al. High serum transforming
growth factor-b1 levels predict outcome in hepatocellular
carcinoma patients treated with sorafenib. Clin Cancer Res
2015;21:3678–84.
12. Bierie B, Moses HL. Tumour microenvironment: TGFbeta: the
molecular Jekyll and Hyde of cancer. Nat Rev Cancer 2006;6:
506–20.
13. Herbertz S, Sawyer JS, Stauber AJ, et al. Clinical develop-
ment of galunisertib (LY2157299 monohydrate), a small mol-
ecule inhibitor of transforming growth factor-beta signaling
pathway. Drug Des Devel Ther 2015;9:4479–99.
14. Faivre S, Santoro A, Kelley RK, et al. Novel transforming
growth factor beta receptor I kinase inhibitor galunisertib
(LY2157299) in advanced hepatocellular carcinoma. Liver Int
2019;39:1468–77.
Disclosure statement
No potential conflict of interest was reported by the author(s).
15. Jin CH, Krishnaiah M, Sreenu D, et al. Discovery of
N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-
imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): a highly potent,
selective, and orally bioavailable inhibitor of TGF-b type I
receptor kinase as cancer immunotherapeutic/antifibrotic
agent. J Med Chem 2014;57:4213–38.
Funding
This work was supported by the Ministry of Commerce, Industry
and Energy, Korea under grant [numbers M1-0310-43-0001 and
M1-0310-43-0002].
16. Batlle E, Massague J. Transforming growth factor-beta sig-
naling in immunity and cancer. Immunity 2019;50:924–40.
17. Patel HM, Sing B, Bhardwaj V, et al. Design, synthesis and
evaluation of small molecule imidazo[2,1-b][1,3,4]thiadiazoles
as inhibitors of transforming growth factor-b type-I receptor
kinase (ALK5). Eur J Med Chem 2015;93:599–613.
18. Reznickova E, Tenora L, Pospisilova P, et al. ALK5 kinase
inhibitory activity and synthesis of 2,3,4-substituted 5,5-
dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazoles. Eur J Med
Chem 2017;127:632–42.
References
1. Pickup M, Novitskiy S, Moses HL. The roles of TGFb in the
tumour microenvironment. Nat Rev Cancer 2013;13:788–99.
2. Tsushima H, Kawata S, Tamura S, et al. High levels of trans-
forming growth factor b1 in patients with colorectal cancer:
association with disease progression. Gastroenterology 1996;
110:375–82.
3. Ivanovic V, Todorovic-Rakovic N, Demajo M, et al. Elevated
plasma levels of transforming growth factor- b1 (TGF- b1) in
patients with advanced breast cancer: association with dis-
ease progression. Eur J Cancer 2003;39:454–61.
4. Dave H, Shah M, Trivedi S, Shukla S. Prognostic utility of cir-
culating transforming growth factor beta 1 in breast cancer
patients. Int J Biol Markers 2012;27:53–9.
19. Yoon JH, Jung SM, Park SH, et al. Activin receptor-like kin-
ase5 inhibition suppresses mouse melanoma by ubiquitin
degradation of Smad4, thereby derepressing eomesodermin
in cytotoxic T lymphocytes. EMBO Mol Med 2013;5:1720–39.
20. Son JY, Park SY, Kim SJ, et al. EW-7197, a novel ALK-5 kinase
inhibitor, potently inhibits breast to lung metastasis. Mol
Cancer Ther 2014;13:1704–16.

712
M.-S. PARK ET AL.
21. Naka K, Ishihara K, Jomen Y, et al. Novel oral transforming 32. Diulgheroff N, Pirkes M, Pontiroli A, Villa M. Processes for
preparing intermediates of pemetrexed. Patent WO 2008/
021385 A2; 2008.
growth factor-b signaling inhibitor EW-7197 eradicates CML-
initiating cells. Cancer Sci 2016;107:140–8.
33. Kim D-K, Jung SH, Lee HS, Dewang PM. Synthesis and bio-
logical evaluation of benzenesulfonamide-substituted 4-(6-
alkylpyridin-2-yl)-5-(quinoxalin-6-yl)imidazoles as transform-
ing growth factor-b type 1 receptor kinase inhibitors. Eur J
Med Chem 2009;44:568–76.
34. Pasternak A, Dejesus RK, Zhu Y, et al. Inhibitors of the renal
outer medullary potassium channel. Patent WO 2012/
058134 A1; 2012.
22. Park SA, Kim MJ, Park SY, et al. EW-7197 inhibits hepatic,
renal, and pulmonary fibrosis by blocking TGF-b/Smad and
ROS signaling. Cell Mol Life Sci 2015;72:2023–39.
23. Kim MJ, Park SA, Kim CH, et al. TGF-b type I receptor kinase
inhibitor EW-7197 suppresses cholestatic liver fibrosis by
inhibiting HIF1a-induced epithelial mesenchymal transition.
Cell Physiol Biochem 2016;38:571–88.
24. Jun EJ, Park JH, Tsauo J, et al. EW-7197, an activin-like kinase
5 inhibitor, suppresses granulation tissue after stent place-
ment in rat esophagus. Gastrointest Endosc 2017;86:219–28.
25. Han K, Park JH, Yang SG, et al. EW-7197 eluting nano-fiber
covered self-expandable metallic stent to prevent granula-
tion tissue formation in a canine urethral model. PLoS One
2018;13:e0192430.
26. Tsauo J, Song HY, Choi EY, et al. EW-7197, an oral transform-
ing growth factor b type I receptor kinase inhibitor, for pre-
venting peritoneal adhesion formation in a rat model.
Surgery 2018;164:1100–8.
ꢀ
35. Fleury-Bregeot N, Oehlrich D, Rombouts F, Molander GA.
Suzuki-Miyaura cross-coupling of potassium dioxolanylethyl-
trifluoroborate and aryl/heteroaryl chlorides. Org Lett 2013;
15:1536–9.
36. Byfield SD, Major C, Laping NJ, Roberts AB. SB-505124 is a
selective inhibitor of transforming growth factor-beta type I
receptors ALK4, ALK5, and ALK7. Mol Pharmacol 2004;65:
744–52.
37. Gellibert F, Woolven J, Fouchet MH, et al. Identification of
1,5-naphthyridine derivatives as a novel series of potent and
selective TGF-beta type I receptor inhibitors. J Med Chem
2004;47:4494–506.
38. Kim D-K, Jang Y, Lee HS, et al. Synthesis and biological
evaluation of 4(5)-(6-alkylpyridin-2-yl)imidazoles as trans-
forming growth factor-b type 1 receptor kinase inhibitors. J
Med Chem 2007;50:3143–7.
39. Eyers PA, Craxton M, Morrice N, et al. Conversion of SB
203580-insensitive MAP kinase family members to drug-sen-
sitive forms by a single amino-acid substitution. Chem Biol
1998;5:321–8.
27. Sybyl-X 2.1.1. SYBYL molecular modeling software. St. Louis
(MO): Tripos Inc.; 2013.
28. Sawyer JS, Beight DW, Britt KS, et al. Synthesis and activity
of new aryl- and heteroaryl-substituted 5,6-dihydro-4H-pyr-
rolo[1,2-b]pyrazole inhibitors of the transforming growth fac-
tor-b type I receptor kinase domain. Bioorg Med Chem Lett
2004;14:3581–4.
29. Gaster LM, Hadley MS, Harling JD, et al. Pyridinylimidazoles.
Patent WO 01/62756 A1; 2001.
30. Powers JC, Boduszek B, Oleksyszyn J, Basic alpha-aminoalkyl-
phosphonate derivatives. Patent US 5,686,419 A; 1997.
31. Kelly SM, Liquid crystals. Patent US 5,204,018 A; 1993.
40. Spitzer R, Jain AN. Surflex-Dock: Docking benchmarks and
real-world application. J Comput Aided Mol Des 2012;26:
687–99.