Design, synthesis, and antifibrosis evaluation of 4-(benzo-[c][1,2,5]thiadiazol-5-yl)-3(5)-(6-methyl- pyridin-2-yl)pyrazole and 3(5)-(6-methylpyridin- 2-yl)-4-(thieno-[3,2,-c]pyridin-2-yl)pyrazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.07.013

Six series of 4-(benzo[c][1,2,5]thiadiazol-5-yl)-3(5)-(6-methylpyridin-2-yl)- pyrazoles 18a–d, 19a–d, 22a–d and 3(5)-(6-methylpyridin-2-yl)-4-(thieno[3,2,-c]- pyridin-2-yl)pyrazoles 20a–d, 21a–d, 23c, 23d have been synthesized and evaluated for their activin receptor-like kinase 5 (ALK5) and p38α mitogen activated protein (MAP) kinase inhibitory activities in enzymatic assays. Among these compounds, the most active compound, 22c, inhibited ALK5 phosphorylation with an IC₅₀ value of 0.030 μM in the enzymatic assay. Compound 22c showed four-fold more potent activity against ALK5 kinase than the clinical candidate, compound LY-2157299. The selectivity index of 22c against p38α MAP kinase is 235, which is much higher than that of LY-2157299 (4) and equally selective to that of EW-7197 (218). Compound 22c effectively suppressed protein and mRNA expression of collagen I and α-SMA in TGF-β-induced LX-2 human hepatic stellate cell (HSC), this result shows that compound 22c has the ability to inhibit the activation of HSC. Compound 22c is expected to be a preclinical candidate for the treatment of hepatic fibrosis.

Full text of DOI:10.1016/j.ejmech.2019.07.013

Accepted Manuscript
Design, synthesis, and antifibrosis evaluation of 4-(benzo-[c][1,2,5]thiadiazol-5-
yl)-3(5)-(6-methyl- pyridin-2-yl)pyrazole and 3(5)-(6-methylpyridin- 2-yl)-4-(thieno-
[3,2,-c]pyridin-2-yl)pyrazole derivatives
Wen-Jing Zhu, Ben-Wen Cui, Hui Min Wang, Ji-Xing Nan, Hu-Ri Piao, Li-Hua Lian,
Cheng Hua Jin
PII:
S0223-5234(19)30634-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.013
EJMECH 11510
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 March 2019
Revised Date: 4 July 2019
Accepted Date: 4 July 2019
Please cite this article as: W.-J. Zhu, B.-W. Cui, H.M. Wang, J.-X. Nan, H.-R. Piao, L.-H. Lian, C.H.
Jin, Design, synthesis, and antifibrosis evaluation of 4-(benzo-[c][1,2,5]thiadiazol-5-yl)-3(5)-(6-methyl-
pyridin-2-yl)pyrazole and 3(5)-(6-methylpyridin- 2-yl)-4-(thieno-[3,2,-c]pyridin-2-yl)pyrazole derivatives,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.013.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Design, Synthesis, and Antifibrosis Evaluation of
4
-(benzo-[c][1,2,5]thiadiazol-5-yl)-3(5)-(6-methyl-
pyridin-2-yl)pyrazole and 3(5)-(6-methylpyridin-
-yl)-4-(thieno-[3,2,-c]pyridin-2-yl)pyrazole
2
Derivatives
a,1
a,1
a
a
a
Wen-Jing Zhu , Ben-Wen Cui , Hui Min Wang , Ji-Xing Nan , Hu-Ri Piao ,
a,∗
a,b,∗
Li-Hua Lian , Cheng Hua Jin
a
College of Pharmacy, Yanbian University, Yanji 133002, PR China
b
Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules,
Ministry of Education, Molecular Medicine Research Center, Yanbian University, Yanji
1
33002, PR China.
∗
Corresponding authors.
E-mail addresses: lhlian@ybu.edu.cn (L.H. Lian), jinchenghua@ybu.edu.cn (C.H. Jin).
1
These authors contributed equally to this work.

ACCEPTED MANUSCRIPT
Abstract
Six series of 4-(benzo[c][1,2,5]thiadiazol-5-yl)-3(5)-(6-methylpyridin-2-yl)-
pyrazoles 18a–d, 19a–d, 22a–d and 3(5)-(6-methylpyridin-2-yl)-4-(thieno[3,2,-c]-
pyridin-2-yl)pyrazoles 20a–d, 21a–d, 23c, 23d have been synthesized and evaluated
for their activin receptor-like kinase 5 (ALK5) and p38α mitogen activated protein
(MAP) kinase inhibitory activities in enzymatic assays. Among these compounds, the
most active compound, 22c, inhibited ALK5 phosphorylation with an IC value of
50
0
.030 µM in the enzymatic assay. Compound 22c showed four-fold more potent
activity against ALK5 kinase than the clinical candidate, compound LY-2157299.
The selectivity index of 22c against p38α MAP kinase is 235, which is much higher
than that of LY-2157299 (4) and equally selective to that of EW-7197 (218).
Compound 22c effectively suppressed protein and mRNA expression of collagen I
and α-SMA in TGF-β-induced LX-2 human hepatic stellate cell (HSC), this result
shows that compound 22c has the ability to inhibit the activation of HSC. Compound
2
2c is expected to be a preclinical candidate for the treatment of hepatic fibrosis.
Keywords: ALK5; TGF-β; pyrazole; antifibrosis; HSC

ACCEPTED MANUSCRIPT
1
. Introduction
Hepatic fibrosis is a pathophysiological process caused by pathogenic factors.
Liver fibrosis occurs during the repair and healing process of any liver injury. Hepatic
fibrosis is the pathological result of chronic hepatitis, which eventually leads to
cirrhosis and even hepatocellular carcinoma [1]. The progression of hepatic fibrosis is
characterized by excessive deposition of extracellular matrix (ECM) [2]. Static
hepatic stellate cells (HSCs) are activated and transdifferentiated by various stimuli,
and then a large amount of ECM, generated by HSCs, is deposited in the fibrous
tissue [3]. During the process of HSC activation, ECM increases in both quantity and
quality, and the excess ECM results in imbalanced ECM degradation and production.
Activated HSCs play an important role in hepatic fibrosis, so inhibition of HSC
proliferation and activation is an ideal treatment for hepatic fibrosis [4]. During
chronic liver injury, static HSCs are activated to the myofibroblast-like phenotype,
characterized by high expression of α-smooth muscle actin (α-SMA), and excessive
deposition of extracellular matrix, including collagen I [5]. Activated HSCs produce
inflammatory cytokines or chemokines, such as IL-1β, tumor necrosis factor-α
(TNF-α) or transforming growth factor-β (TGF-β) [6].
The transforming growth factor-β (TGF-β) including TGF-β1, TGF-β2, TGF-β3,
activins, inhibins, and bone morphogenetic proteins belongs to the cytokine
superfamily, which provides multi-directional adjustment and contains a variety of
functional proteins. The TGF-β members have been found in species ranging from
worms and insects to mammals, and they regulate cellular functions such as cell
proliferation, differentiation, adhesion, migration, and apoptosis [7]. The signal
transduction of TGF-β has two distinct types of single-membrane serine/threonine
receptor kinases, which are termed the type I receptor or activin receptor-like kinase
5
(ALK5) and the type II receptor, respectively [8]. ALK5 is activated by the
combination of TGF-β and the type II receptor, stimulating its kinase activity. Smad2
and Smad3 proteins are phosphorylated by the activated ALK5 and they then bind to
Smad4 to form a heteromeric complex, which is then delivered into the nuclei. The

ACCEPTED MANUSCRIPT
activated ALK5 participates in the regulation of the expression of several hundred
genes by forming of Smad complexes [9]. TGF-β is present in various organs, such as
the kidneys [10], lungs [11], heart [12], and liver [13], and plays a very important role
in the initiation and development of fibrosis. Moreover, overexpression of TGF-β
signals leads to the development of a variety of human diseases, including
hematological malignancy [14], breast cancer [15], and pancreatic diseases [16].
Therefore, the TGF-β signaling pathway is a potential target for the prevention and
treatment of fibrosis/cancer, and the direct inhibition of ALK5 has the potential to
treat TGF-β induced fibrosis/cancer.
A variety of small-molecule compounds with ALK5 inhibitory activity have been
reported by several research groups recently, such as 1 (SB-505124)[17], 2
(SD-208)[18], 3 (LY-2157299)[19–22], 4 (EW-7197)[23], 6 [24, 25] and 7 [26, 27].
Among these, compounds 3 (Phase II) and 4 (Phase II) are under evaluation in
clinical trials as antitumor agents (Fig. 1). Compound 2 effectively retarded
progressive fibrosis in the liver, kidneys and lungs, 2 and 3 also strongly inhibited
growth and invasiveness of cancer cells in animal models.
We recently reported that
a
series of compounds containing
[
1,2,4]triazolo[1,5-a]pyridin-6-yl moiety have potent ALK5 inhibitory activity in an
enzymatic assay [23, 28]. In these studies, it was found that these compounds show
high selectivity for p38α MAP kinase, which has one of the most homologous ATP
binding pockets to that of ALK5. Among these, compound 4 showed highly
selectivity towards 320 other human kinases and no adverse effect in the safety
pharmacology studies of it in dogs and rats [23]. Park et al. reported that compound 4
decreased the expression of collagen I, α-SMA, fibronectin, 4-hydroxy-2,3-nonenal,
and integrins in the livers of CCl4 mice and BDL rats, and in the lungs of BLM rats
and mice. They also extended the lifespan of CCl4 mice, BDL rats, and BLM mice
[
29]. Although recent studies have shown that many mediators are critical to the
initiation and progression of fibrosis, the relationship among these mediators remains
unclear [30].

ACCEPTED MANUSCRIPT
It has been reported that a series of compounds possessing a thioamide linkage
between a pyrazole ring and a phenyl ring showed inhibitory activity against ALK5.
Among them, A-83-01 exhibited significant inhibition of the transcriptional activity
induced by ALK5 kinase [31]. Whereas it was found that the thioamide linkage was
fairly unstable and slowly degraded to a free pyrazole ring during long-term storage.
Based on previous work and inquiry into the literature, we tried to substitute the
thioamide linkage with a chemically stable thioamidomethylene linkage and designed
compounds 22a–d, 23c, and 23d. To compare the influence of the
thioamidomethylene linkage in 22a–d, 23c, and 23d on ALK5 inhibitory activity,
their counterpart derivatives 18a–d, 19a–d, 20a–d and 21a–d possessing an
amidomethylene linkage, were also designed. Previously, it has been reported that the
nitrogen atom of the 6-quinolinyl moiety in compound 8 (IN-1233) forms a H-bond
with the backbone amidyl NH of His283 in the hinge region of the kinase, which
occupies the binding pocket for the adenine ring of ATP [32]. Therefore, we
introduced a methoxy group as an electron-donating group at the 4-position of the
6
-quinolinyl moiety for the purpose of improving their activity, but the activity of
these compounds was not good [32]. We hypothesized that this may be because of the
steric hindrance of the methoxy group causing the nitrogen atom in the 6-quinolinyl
moiety not to bind to the key amino acids. To improve the capability of the nitrogen
atom of 6-quinolinyl moiety as
a
H-bond acceptor, we introduced
benzo[c][1,2,5]thiadiazol-5-yl and thieno[3,2-c]pyridin-2-yl groups, which have less
steric hindrance at the 4-position of pyrazole moiety in compound 5. (Fig. 2)
2
2
. Results and Discussion
.1. Chemistry
The requisite thieno[3,2-c]pyridine-2-carbaldehyde (14b) was prepared as shown
in Scheme 1. The synthesis of 2,2-dimethoxy-N-(thiophen-2-ylmethyl)ethanamine
11) was carried out by reductive amination of the commercially available
,2-dimethoxyethanamine (9) and thiophene-2-carboxaldehyde (10) at 85°C in the
presence of catalytic amount of para-toluenesulfonic acid monohydrate (PTSA) and
(
2

ACCEPTED MANUSCRIPT
followed
by
treatment
with
sodium
borohydride.
N-(2,2-Dimethoxyethyl)-4-methyl-N-(thiophen-2-ylmethyl)-benzenesulfonamide (12)
was then conveniently synthesized by condensation of 11 with para-toluenesulfonyl
chloride in pyridine, which was subsequently cyclized to give thieno[3,2-c]pyridine
o
(
13)[33] in two steps in 21% yield. Lithiation of 13 with n-BuLi at -78 C in
tetrahydrofuran
N,N-dimethylformamide (DMF) afforded the aldehyde 14b in 42% yield [34].
The 2-(4-substituted-3-(6-methylpyridin-2-yl)-1H-pyrazol-1-yl)-N-
(THF),
followed
by
treatment
with
anhydrous
phenylacetamides (18a–d and 20a–d) were synthesized as shown in Scheme 2. The
target compounds 18b–d, and 20b–d each possesses a substituent, either o-F, m-F or
m-CN in the phenyl ring because these were previously found to be most salutary for
ALK5
benzo[c][1,2,5]thiadiazole-5-carbaldehyde (14a) and 14b were coupled with diphenyl
6-methylpyridin-2-yl)(phenylamino)methylphosphonate[35] in a mixture of THF
inhibitory
activity
and
selectivity
[23].
The
(
and i-PrOH in the presence of Cs CO at room temperature, followed by treatment
2
3
with
1
N
HCl
to
afford
the
corresponding
ketones
and
2
1
8
-(benzo[c][1,2,5]thiadiazol-5-yl)-1-(6-methylpyridin-2-yl)ethanone (15a)
-(6-methylpyridin-2-yl)-2-(thieno[3,2-c]pyridin-2-yl)ethanone (15b) in 68% and
6% yields, respectively. Treatment of 15a and 15b with N,N-dimethylformamide
o
o
dimethyl acetal (DMF•DMA) in THF or DMF at 45 C or 90 C, followed by
cyclization with hydrazine monohydrate in absolute EtOH produced the pyrazoles
1
6a and 16b in 57% and 43% yields[36], respectively. The pyrazoles 16a and 16b
were alkylated with substituted phenylacetamides 17a–d [37, 38] in the presence of
NaH in anhydrous DMF to yield the target compounds 18a–d, 20a–d and their
positional isomers 19a–d and 21a–d in 28%–85% and 9%–15% yields, respectively.
The positional isomers were separated by column chromatography and their
structures were confirmed by nuclear overhauser enhancement (NOE) experiments.
In the NOE experiments, irradiation of the methylene protons of compound 18b at δ
5
.12 gave an enhancement of the H-5 proton in the pyrazole ring at δ 7.83, while

ACCEPTED MANUSCRIPT
irradiation of the methylene protons of compound 19b at δ 5.18 gave no enhancement
of the proton H-5 in the pyrazole ring at δ 7.94, confirming the respective alkylation
positions.
Thionation of the 18a–d, 20c, and 20d with Lawesson's reagent in anhydrous
o
DME at 85 C produced the thioamides 22a–d, 23c, and 23d in 27%–80% yields
(Scheme 3). Compared with benzo[c][1,2,5]thiadiazol-5-yl series of compounds
1
8a–d, the conversion rate of thieno[3,2-c]pyridin-2-yl series of compounds 20a and
0b was very low, and there were problems with separation and purification which
2
only allowed us to synthesize two compounds, 23c and 23d. As we expected, all the
synthesized target compounds were fairly stable during long-term storage at room
temperature.
2
.2. ALK5 inhibitory activity in an enzymatic assay
To investigate whether these compounds (18a–d, 19a–d, 20a–d, 21a–d, 22a–d,
3c and 23d) could inhibit ALK5, a preliminary screening kinase assay was
2
performed using the purified human ALK5 kinase domain produced in Sf9 insect
cells with compounds at 10 µM. All the compounds possessing
a
benzo[c][1,2,5]thiadiazol-5-yl moiety (18a–d, 19a–d, and 22a–d) and
thieno[3,2-c]pyridin-2-yl moiety (20a–d and 21a–d) showed significant ALK5
inhibition (46%–100%). The amides 18a–d (98%–99%) and 20a–d (98%–100%)
showed more potent ALK5 inhibition than their respective positional isomers, 19a–d
(52%–98%) and 21a–d (46%–91%), respectively. We selected p38α MAP kinase to
determine the selectivity profile of this series of compounds because its kinase
domain is very similar to that of ALK5 [39]. All target compounds showed low or
moderate inhibition (0%–66%) against p38α MAP kinase, even at their maximum
concentration of 10 µM, which indicates selectivity for ALK5.
To evaluate the ALK5 inhibitory activities and selectivity of the compounds
possessing benzo[c][1,2,5]thiadiazol-5-yl and thieno[3,2,-c]pyridin-2-yl moiety, the
amides (18a–d and 20a–d) and the thioamides (22a–d, 23c and 23d) were selected in
view of the residual activity assay, and their half maximal inhibitory concentration

ACCEPTED MANUSCRIPT
(
IC ) values were measured. Compounds 3 (LY-2157299) and 4 (EW-7197) were
5
0
used as positive controls. We envisioned that the reduction of steric hindrance at the
-position of pyrazole moiety in compound 5 may improve the binding ability of the
4
nitrogen atom to ALK5 related proteins, and further increase its ALK5 inhibitory
activity. As we expected, the results showed that the introduction of the
benzo[c][1,2,5]thiadiazol-5-yl and thieno[3,2-c]pyridin-2-yl group at the 4-position
of the pyrazole moiety in compound 5 seemed to be accommodated favorably into
the ATP binding pocket of ALK5. All the compounds possessing a
benzo[c][1,2,5]thiadiazol-5-yl
moiety
(18a–d
and
22a–d)
and
thieno[3,2-c]pyridin-2-yl moiety (20a–d, 23c and 23d) showed significant ALK5
inhibitory activity (IC50 = 0.030–0.312 µM) as shown in Table 1. Among the
pyrazoles (18a–d and 22a–d) possessing the benzo[c][1,2,5]thiadiazol-5-yl moiety,
introduction of fluoro- or carbonitrile-substituent at the ortho- or meta-position of the
phenyl ring decreased ALK5 inhibitory activity, except for compound 22c (IC50
=
0
.030 µM), specifically, 18b–d (IC = 0.087–0.168 µM), 22b (IC = 0.055 µM) and
50 50
2
2d (IC50 = 0.116 µM) showed less inhibitory activity than the unsubstituted
compounds 18a (IC50 = 0.030 µM) and 20a (IC50 = 0.042 µM). Apart from the
unsubstituted compounds 18a and 22a, all compounds (22b–d, IC50 = 0.042–0.116
µM) possessing a thioamidomethylene linkage showed more potent ALK5 inhibitory
activity than the corresponding compounds (18b–d, IC50 = 0.087–0.168 µM)
possessing an amidomethylene linkage. Among the pyrazoles (20a–d, 23c and 23d)
possessing the thieno[3,2,-c]pyridin-2-yl moiety, introduction of a fluoro- or
carbonitrile-substituent at the ortho- or meta-position on the phenyl ring also
decreased ALK5 inhibitory activity, specifically, 20b–d (IC50 = 0.119–0.312 µM),
2
3c (IC50 = 0.125 µM) and 23d (IC50 = 0.078 µM) showed less inhibitory activity
than the unsubstituted compound 20a (IC = 0.042 µM). In these series, compounds
5
0
2
3c (IC50 = 0.125 µM) and 23d (IC50 = 0.078 µM), which possess a
thioamidomethylene linkage, showed more potent ALK5 inhibitory activity than the
corresponding compounds 20c (IC50 = 0.119 µM) and 20d (IC50 = 0.119 µM)

ACCEPTED MANUSCRIPT
possessing an amidomethylene linkage. The above result showed that the 18 and 22
series compounds (18a–d and 22a–d) with a benzo[c][1,2,5]thiadiazol-5-yl moiety
showed more potent ALK5 inhibition than the corresponding 20 and 23 series (20a–d,
2
3c and 23d) with a thieno[3,2,-c]pyridin-2-yl moiety. Compounds 18a and 22c
showed the most potent ALK5 inhibitory activity in these four series of compounds,
and they were four-fold more potent than the positive control compound 3 (IC50
.119 µM) and three-fold less potent than 4 (IC50 = 0.010 µM). Furthermore, the
=
0
compounds 18a–d and 22a–d which possessed a benzo[c][1,2,5]thiadiazol-5-yl
moiety, and 20a–d, 23c and 23d which possessed a thieno[3,2,-c]pyridin-2-yl moiety
did not inhibit p38α MAP kinase efficiently, showing IC of >3.80 µM. Compound
5
0
1
8a was the most selective in the four series (18a–d, 20a–d, 22a–d, 23c and 23d),
showing a selectivity index of >333, which is much higher than that of 3 (4).
2
.4. Docking model of 20c and 22c complex with ALK5
To explain the observed ALK5 inhibition activity and the mechanism-of-action of
the compounds shown in Table 1, we examined the binding modes of two
representative ligands, 20c and 22c using the semi-flexible molecular docking
program DS LibDock [40]. Docking analyses were performed using the recently
reported X-ray structure of ALK5 complexed to a pyrazole ALK5 inhibitor (PDB:
1
RWB) [41]. As shown in Figure 3, the pyridine ring and thiophene ring of the
thieno[3,2,-c]pyridin-2-yl moiety in 20c formed a Pi-alkyl bond with Lys337. The
NH group on the side chain of 20c formed a hydrogen bond with the side chain
carboxylic acid of Glu245. The phenyl ring of the side chain in 20c formed a Pi-alkyl
bond with Lys232. Leu340 formed Pi-alkyl and alkyl bond network with the central
pyrazole ring, pyridine ring and methyl group at the 5-position in 20c. The free amine
of His283, a residue previously reported to be important for inhibitory activity,
formed a sulfur-X bond with the sulfur atom of benzo[c][1,2,5]thiadiazol-5-yl moiety
in 22c, and its imidazole ring formed a stacked Pi-Pi bond with the thiadiazole ring in
2
2c [1]. The two rings of the benzo[c][1,2,5]thiadiazol-5-yl moiety in 22c formed a
Pi-alkyl bond with Leu340. Lys232 formed a Pi-alkyl bond with the pyridine ring in

ACCEPTED MANUSCRIPT
2
2c and the benzene ring of Tyr249 formed a Pi-alkyl bond with the methyl group of
the pyridine ring in 22c. The NH group of the side chain in 22c formed a hydrogen
bond with the carbonyl group of Lys337 and Asn338, and also with the carboxylic
acid of the side chain in Asp351. The phenyl ring of the side chain in 22c formed a
Pi-alkyl bond with Lys337. Not only did the calculated binding energy scores
(
LibDock Score) of these two compounds indicate that 22c (-134.27 kcal/mol)
formed more stable complexes with ALK5 than 20c (-130.73 kcal/mol), but also that
compound 22c (His283, Leu340, Lys232, Tyr249, Asp351, Asn338 and Lys337)
formed more bonding interactions with previously reported key amino acids than
compound 20c (Lys337, Glu245, Lys232 and Leu340) (Figure 3A and 3B) [1, 23, 42].
Especially, compound 20c did not form any bond with the most important amino acid,
His283, and the thieno[3,2,-c]pyridin-2-yl moiety of compound 20c was stretched to
the outside of the binding pocket. The docking analysis indicated that these six series
of compounds act with ALK5 kinase in this way, and the most active compound 22c
could more favorably fit into the binding site of ALK5 kinase than compound 20c,
which also illustrates that it may be a potent ALK5 inhibitor.
2
.5. Effect of compound 20a and 22c on the viability
To observe the cytotoxicity of the synthesized compounds, the compounds with the
highest activity, 22c and 20a, were selected among the compounds possessing a
benzo[c][1,2,5]thiadiazol-5-yl moiety and thieno[3,2-c]pyridin-2-yl moiety. The
cytotoxicity of these two compounds was determined by MTT assay in LX-2 cells at
4
different concentrations (0, 4, 20, 100 µM). Compound 20a significantly inhibited
the viability of LX-2 cells in a dose-dependent manner after 3 h incubation, while
compound 22c showed little cytotoxicity.
2
.6. Targeting TGF-β signaling suppresses the fibrogenic phenotype of LX-2 cells
Progression of liver fibrosis is characterized by excessive deposition of ECM, with
primary indicators being collagen I and α-SMA. An increase of ECM deposition
including collagen I and α-SMA in TGF-β activated LX-2 cells was confirmed by
immunofluorescence staining (Fig. 5A and 5B). The LX-2 cell line is immortalized

ACCEPTED MANUSCRIPT
human HSC. To verify that TGF-β signaling could modulate the fibrogenic
phenotype of HSCs, we pretreated inhibitors of ALK5 to the TGF-β activated LX-2
cells. As we expected, the fluorescence intensity of collagen I and α-SMA was
suppressed by compound 20a and 22c in a dose-dependent manner. Compound 22c
was better than LY-2157299, a potent TGFβ receptor I (TβRI) inhibitor, as the
positive control, at inhibiting the expression of collagen I and α-SMA by TGF-β,
while compound 20a was similar to that of the positive control. These results suggest
that TGF-β regulates HSC activation and the inhibitory effects of compounds 20a and
2
2
2c on TGF-β signaling may help block of HSC activation.
.7. Effect of compounds 20a and 20c on liver fibrosis marker in TGF-β
activated LX-2 cells
To examine the anti-fibrotic effect of compounds 20a (J-1063) and 22c (J-1048),
we determined protein expression of collagen I and α-SMA by western blotting.
TGF-β treatment increased the protein expression of liver fibrosis markers, such as
collagen I and α-SMA, while pre-treatment with J-1048 and J-1063 prior to TGF-β
stimulation inhibited the expression of collagen I and α-SMA (Fig. 6A and 6B). The
decrease scope of collagen I was much more significant than that of α-SMA with
pretreatment with J-1048 and J-1063. Our data confirmed the inhibition of ECM
production by J-1048 may be related with decreased HSC activation.
We also measured the mRNA levels of collagen I and α-SMA in TGF-β induced
LX-2 cells to gain insight into the inhibitory effect of J-1063 and J-1048 on HSC
activation and EMT in fibrosis by RT-PCR [43]. After TGF-β treatment, a significant
increase of mRNA expression of both collagen I and α-SMA was observed, relative
to those of normal cells (Fig. 6A and 6C). Pretreatment with compounds J-1048 and
J-1063 significantly inhibited the mRNA expressions of collagen I and α-SMA,
compared with those in TGF-β-activated LX-2 cells. The above results indicate that
J-1048 and J-1063 could regulate the expression of collagen I and α-SMA to block
the development of liver fibrosis.
3
. Conclusion

ACCEPTED MANUSCRIPT
In this report, six series of 4-(benzo[c][1,2,5]thiadiazol-5-yl)-3(5)-(6-
methylpyridin-2-yl)pyrazoles 18a–d, 20a–d, 22a–d and 3(5)-(6-methylpyridin-2-yl)-
4
-(thieno[3,2,-c]pyridin-2-yl)pyrazoles 19a–d, 21a–d, 23c and 23d have been
synthesized and evaluated for ALK5 and p38α MAP kinase inhibitory activities in
enzymatic assays. We discovered that the insertion of a benzo[c][1,2,5]thiadiazol-5-yl
and thieno[3,2,-c]pyridin-2-yl moiety at 4-position of pyrazole ring significantly
increased ALK5 kinase inhibition activity. The most potent compound, 18a and 22c,
inhibited ALK5 phosphorylation with an IC50 value of 0.030 µM in the enzymatic
assay. The selectivity index of 22c against p38α MAP kinase was 235, which was 59
times higher than that of 3 (4) and equally selective to that of 4 (218). Compounds
2
2c and 20a efficiently inhibited TGF-β induced HSC activation in the
immunocytochemistry staining, western blotting and RT-PCR assay, demonstrating
that it has high therapeutic potential for fibrosis treatment. To verify the mechanism
and SAR of the target compounds, a molecular docking study was performed and
good binding interactions were observed. The result of cytotoxicity assay and
antifibrosis assay suggest that compound 22c may be valuable as a potential
preclinical candidate for research.
4
. Experimental section
4
.1. Chemistry
All commercially available reagents and solvents were purchased from Aladdin
(Shanghai, China), and used without further purification. In general, all reactions
were performed under normal atmosphere and at room temperature unless otherwise
noted. Melting points were measured in open glass capillaries tube in an electrical
melting point (B-540, Büchi) and are uncorrected. All reactions were monitored for
completion by TLC using Merck silica gel 60 F254 glass plates. Spots were detected
1
by viewing under UV lamps (254 nm). H NMR spectra were recorded on Bruker
1
3
NMR spectrometers at 300 MHz and C NMR spectra were recorded on Bruker
NMR spectrometers at 125 MHz, respectively, tetramethylsilane (TMS) was used as
internal standard. Chemical shifts were signified in ppm (δ) relative to internal TMS

ACCEPTED MANUSCRIPT
in CDCl , CDCl /CD OD, or DMSO-d and coupling constants (J) are in hertz (Hz).
3
3
3
6
High resolution mass spectra electrospray ionization (HRMS-ESI) was obtained on a
Thermo Scientific LTQ Orbitrap XL spectrometer (Theromo Fisher Scientific, USA).
The purity of the tested compounds was determined using an Agilent 1260 series
HPLC system using a C column (packing ODS HG 5 µM, 4.6 × 250 mm), and that
1
8
for all the compounds was found to be >95%.
4
.1.1 Dimethoxy-N-(thiophen-2-ylmethyl)ethanamine (11)
Thiophene-2-carboxaldehyde (10, 4.48 g, 40 mmol ) was dissolved in anhydrous
ethanol (40 mL) and treated with 2,2-dimethoxyethanamine (compound 9, 6.3 g, 60
mmol) and para-toluenesulfonic acid hydrate (PTSA) (0.1 g, catalytic amount), and
then refluxed at 85°C for 4 h. The reaction mixture was allowed to cool to 0°C and
sodium borohydride (1.51 g, 40 mmol) was added in small portions, and then it
stirred at the room temperature for 4 h. The mixture was evaporated to dryness in
vacuo. The residue was dissolved in ethyl acetate (60 mL) and washed with saturated
sodium bicarbonate (20 mL), water (20 mL) and brine (20 mL). The organic layer
was dried with anhydrous sodium sulfate, filtered, and evaporated to dryness under
reduced pressure. The residue was purified by silica gel column chromatography
(Petroleum ether/Ethyl acetate, 6:1) as eluent to give the titled compound 11 as a
colorless liquid.
1
Yield 60%; H NMR (300 MHz, CDCl ) δ 7.26 (t, 1H, J = 6.0 Hz), 7.12 (br s, 1H),
3
7
2
4
.03 (d, 1H, J = 6.0 Hz), 4.48 (t, 1H, J = 6.0 Hz), 3.81 (s, 2H), 3.35 (s, 6H), 2.74 (d,
H, J = 6.0 Hz).
.1.2 N-(2,2-Dimethoxyethyl)-4-methyl-N-(thiophen-2-ylmethyl)benzenesulfonamide
（
12
）
Compound 11 (3 g, 14.9 mmol) was dissolved in anhydrous dichloromethane (20
mL) and pyridine (5 mL) was added to the solution. To it, para-toluenesulfonyl
chloride (3.41 g, 17.9 mmol) was added portionwise at 0°C. The reaction was stirred
at the room temperature for 4 h. The reaction mixture was washed with 1 N HCl (9
mL), water (5 mL) and saturated sodium bicarbonate (5 mL). The organic layer was

ACCEPTED MANUSCRIPT
dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under
reduced pressure. The residue was purified by silica gel column chromatography
(Petroleum ether/Ethyl acetate, 4:1) as eluent to give the titled compound 12 as a
white solid.
1
Yield 69%; H NMR (300 MHz, CDCl ) δ 7.70 (d, 2H, J =9.0 Hz), 7.29 (d, 2H, J =
3
9
2
4
.0 Hz), 7.21 (t, 1H, J = 6.0 Hz), 7.04 (br s, 1H), 6.84 (d, 1H, J = 6.0 Hz), 4.48 (s,
H), 4.38 (t, 1H, J=6.0 Hz), 3.30 (s, 6H), 3.23 (d, 2H, J = 6.0 Hz), 2.43 (s, 3H).
.1.3 Thieno[3,2-c]pyridine（13）
Compound 12 (6.1 g, 17.2 mmol) was dissolved in anhydrous dioxane (9 mL) and
treated with concentrated hydrochloric acid (9 mL). The mixture was heated to reflux
for 17 h. After cooled to room temperature, the reaction mixture was diluted with
water (9 mL) and brought to pH 7 with 2 N NaOH in an ice bath. The mixture was
extracted with ethyl acetate (50 mL), and the organic layer was washed with saturated
sodium bicarbonate (20 mL), water (20 mL) and saturated sodium chloride (20 mL).
The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated
to dryness under reduced pressure. The residue was purified by silica gel column
chromatography (Petroleum ether/Ethyl acetate, 3:1) as eluent to give the titled
compound 13 as a tan solid.
1
Yield 30%; H NMR (300 MHz, CDCl ) δ 9.12 (s, 1H), 8.44 (d, 1H, J = 6.0 Hz), 7.80
3
(d, 1H, J = 6.0 Hz), 7.49 (d, 1H, J = 6.0 Hz), 7.43 (d, 1H, J = 6.0 Hz).
4
.1.4 Thieno[3,2-c]pyridine-2-carbaldehyde (14b)
To a stirred solution of 13 (3 g, 22.22 mmol) in dry tetrahydrofuran (THF, 90 mL)
o
at -78 C, n-BuLi (1.6 M in hexane, 16 mL) was added dropwise and stirred at that
temperature for 2 h. Anhydrous dimethylformamide (DMF, 2 g, 27.77 mmol) was
added dropwise to the solution and the solution was stirred for an additional 3 h. The
o
mixture was treated with water at -78 C and allowed to warm to room temperature.
The mixture was extracted with ethyl acetate (200 mL) and the organic phase was
dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under
reduced pressure. The residue was purified by silica gel column chromatography

ACCEPTED MANUSCRIPT
(Petroleum ether/Ethyl acetate, 1:1) as eluent to give the titled compound 14b as a
yellowish solid.
1
Yield 42%; H NMR (300 MHz, CDCl ) δ 10.17 (s, 1H), 9.31 (s, 1H), 8.60 (d, 1H, J
3
=
6.0 Hz), 8.19 (s, 1H), 7.90 (d, 1H, J = 6.0 Hz).
.1.5 General procedure for
-(6-methylpyridin-2-yl)-2-(thieno[3,2-c]pyridin-2-yl)ethanone (15a) and 2-(benzo[c]
4
1
the
preparation
of
[
1,2,5]thiadiazol-5-yl)-1-(6-methylpyridin-2-yl)ethanone (15b)
To a stirred solution of 14a or 14b (18.40 mmol) in a mixture of THF (40 mL) and
i-PrOH (10 mL), diphenyl (6-methylpyridin-2-yl)(phenylamino)methylphosphonate
20.24 mmol) and Cs CO (24.65 mmol) were added. The mixture was stirred at
(
2
3
room temperature for 8 h, and to it, 1 N HCl (74 mL) was added dropwise about 20
min. The reaction mixture was diluted with tert-butyl methyl ether (MTBE, 40 mL).
The aqueous layer was separated, and the organic layer was extracted with 1 N HCl
(3 × 100 mL). The combined aqueous layer was neutralized with 2 N KOH (pH 7–8)
o
at 0 C. The precipitates were collected by filtration using a Buchner funnel and dried
in a vacuum oven to gain the compound 15a or 15b as a yellow solid.
4
.1.5.1 1-(6-Methylpyridin-2-yl)-2-(thieno[3,2-c]pyridin-2-yl)ethanone (15a)
1
Yield 68%; H NMR (300 MHz, CDCl ) δ 7.96–7.93 (m, 2H), 7.87 (d, 1H, J =9.0
3
Hz), 7.72 (t, 1H, J =9.0 Hz), 7.61 (d, 1H, J = 9.0 Hz), 7.36 (d, 1H, J = 9.0 Hz), 4.74
(s, 2H), 2.67 (s, 3H).
4
.1.5.2 2-(Benzo[c][1,2,5]thiadiazol-5-yl)-1-(6-methylpyridin-2-yl)ethanone(15b)
1
Yield 86%; H NMR (300 MHz, CDCl ) δ 8.99 (s, 1H), 8.38 (d, 1H, J = 6.0 Hz), 7.91
3
(d, 1H, J = 9.0 Hz), 7.74 (t, 1H, J = 9.0 Hz), 7.71 (d, 1H, J = 6.0 Hz), 7.38 (d, 1H, J =
9
.0 Hz), 7.31 (s, 1H), 4.87 (s, 2H), 2.68 (s, 3H).
4
.1.6 General procedure of 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)thieno[3,2-
c]pyridine
yl)benzo[c][1,2,5]thiadiazole (16b)
To a stirred solution of 15a or 15b (11.19 mmol) in anhydrous THF (30 mL) or
DMF (30 mL), N,N-dimethylformamide dimethyl acetal (DMF DMA, 5 mL) was
(16a)
and
5-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-
•

ACCEPTED MANUSCRIPT
o
o
added. The mixture was heated at 45 C or 90 C for 4 h. After cooled to room
temperature, the reaction mixture was evaporated to dryness under reduced pressure.
The residue was dissolved in EtOH (30 mL), and to it, hydrazine monohydrate (20
mL) was added. The mixture was heated at reflux temperature for 4 h, then cooled to
room temperature, and evaporated to dryness under reduced pressure. The residue
was diluted with ethyl acetate (50 mL) and washed with water (20 mL) and brine (20
mL). The organic layer was dried over anhydrous Na SO , filtered, and evaporated to
2
4
dryness under reduced pressure. The residue was purified by the column of silica gel
using CH Cl /MeOH (20:1) as eluent to gain the titled compound 16a or 16b as a
2
2
semi-solid.
4
.1.6.1 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)thieno[3,2-c]pyridine (16a)
1
Yield 86%; H NMR (300 MHz, CDCl ) δ 8.07 (s, 1H), 8.00 (d, 1H, J = 9.0 Hz), 7.80
3
(s, 1H), 7.64 (dd, 1H, J = 9.0, 3.0 Hz), 7.50 (t, 1H, J =7.5 Hz), 7.21 (d, 1H, J = 6.0
Hz), 7.14 (d, 1H, J = 9.0 Hz), 2.64 (s, 3H).
4
.1.6.2 5-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)benzo[c][1,2,5]thiadiazole(16b)
1
Yield 43%; H NMR (300 MHz, CDCl ) δ 9.05 (s, 1H), 8.45 (d, 1H, J = 6.0 Hz),
3
7
7
4
.78–7.75 (m, 2H), 7.52 (t, 1H, J = 7.5 Hz), 7.45 (s, 1H), 7.39 (d, 1H, J =6.0 Hz),
.13 (d, 1H, J = 9.0 Hz), 2.60 (s, 3H).
.1.7 General procedure of 2-(4-substituted-3-(6-methylpyridin-2-yl)-1H-pyrazol-1-
yl)-N-phenylacetamide
(18a–d
and
20a–d)
and
2-(4-substituted-5-(6-
methylpyridin-2-yl)-1H-pyrazol-1-yl)-N-phenylacetamide (19a–d and 21a–d)
To a solution of pyrazole 16a or 16b (0.35 mmol) in anhydrous DMF (5 mL), a
catalytic
amount
of
sodium
iodide,
NaH
(0.42
mmol),
and
2
-chloro-N-phenylacetamides 17a–d (0.42 mmol) were added. The mixture was
stirred at room temperature for 2 h and then evaporated to dryness under reduced
pressure. The residue was purified by the silica gel column using CH Cl /MeOH as
2
2
eluent to give the two positional isomers 18a–d, 20a–d and 19a–d, 21a–d as light
yellow solids.
4
.1.7.1 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-1H-pyrazol-

ACCEPTED MANUSCRIPT
1
-yl)-N-phenylacetamide (18a)
o
1
Yield 72%; mp 190–192 C; purity by HPLC: 100% (25% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 9.03 (s, 1H, NH), 7.99 (s, 1H) , 7.90 (d, 1H, J = 9.0 Hz), 7.85 (s,
3
1
H), 7.62 (t, 1H, J = 7.5 Hz), 7.56–7.52 (m, 3H), 7.40 (d, 1H, J = 9.0 Hz), 7.30 (d,
H, J = 9.0 Hz), 7.18 (d, 1H, J = 6.0 Hz), 7.10 (t, 1H, J =6.0 Hz), 5.16 (s, 2H), 2.55
2
13
(
s, 3H); C NMR (125 MHz, CDCl ) δ 164.52, 158.64, 155.09, 154.02, 150.84,
3
1
50.22, 137.15, 136.85, 134.15, 132.32, 132.23, 128.95 (2C), 124.80, 122.85, 121.54,
+
1
20.50, 120.33, 120.18 (2C), 119.86, 53.44, 24.31; HRMS-ESI (m/z): [M+H] calcd
for C H N OS 427.13356, found 427.13354.
2
3
19
6
4
.1.7.2 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-1H-pyrazol-
1
-yl)-N-(2-fluorophenyl)acetamide (18b)
o
1
Yield 64%; mp 160–162 C; purity by HPLC: 99.9% (35% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 9.07 (s, 1H, NH), 8.28 (t, 1H, J = 7.5 Hz), 8.04 (s, 1H), 7.90 (d,
3
1
H, J = 9.0 Hz), 7.83 (s, 1H), 7.68–7.56 (m, 3H), 7.17–7.05 (m, 4H), 5.10 (s, 2H),
13
2
.49 (s, 3H); C NMR (125 MHz, CDCl ) δ 164.49, 158.37, 155.09, 154.07, 152.68
3
J = 243.75 Hz), 150.87, 150.70, 136.84, 134.27, 132.58, 132.28, 125.67 (d, J = 11.25
Hz), 125.08 (d, J = 7.5 Hz), 124.59 (d, J = 3.75 Hz), 122.75, 121.84, 121.78, 120.14,
+
1
19.90, 114.97 (d, J = 18.75 Hz), 53.44, 24.27; HRMS-ESI (m/z): [M+H] calcd for
C H FN OS 445.12413, found 445.12405.
23
18
6
4
1
.1.7.3 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-1H-pyrazol-
-yl)-N-(3-fluorophenyl)acetamide (18c)
o
1
Yield 85%; mp 172–174 C; purity by HPLC: 100% (35% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 9.44 (br s, 1H, NH), 7.99 (s, 1H), 7.88–7.84 (m, 2H), 7.71 (s,
3
1
2
1
1
1
H), 7.56 (br s, 2H), 7.45–7.36 (m, 2H), 7.17 (br s, 2H), 6.77 (br s, 1H), 5.19 (s, 2H),
1
3
.52 (s, 3H); C NMR (125 MHz, CDCl ) δ 163.53, 161.82 (d, J = 243.75 Hz),
3
57.70, 154.07, 153.02, 149.69, 149.23, 137.72 (d, J = 11.25 Hz), 135.89, 133.02,
31.40, 131.10, 128.96 (d, J = 8.75 Hz), 121.93, 120.48, 119.63, 119.40, 118.91,
14.29, 110.39 (d, J = 21.25 Hz), 106.51 (d, J = 26.25 Hz), 54.88, 23.24; HRMS-ESI
+
(
m/z): [M+H] calcd for C H FN OS 445.12413, found 445.12396.
23
18
6

ACCEPTED MANUSCRIPT
4
.1.7.4 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-1H-pyrazol-
1
-yl)-N-(3-cyanophenyl)acetamide (18d)
o
1
Yield 54%; mp 191–192 C; purity by HPLC: 99.1% (35% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 10.22 (s, 1H, NH), 7.98–7.86 (m, 4H), 7.59–7.52 (m, 3H),
3
1
3
7
.32–7.28 (m, 3H), 7.17 (d, 1H, J = 9.0 Hz), 5.28 (s, 2H), 2.51 (s, 3H); C NMR
(
125 MHz, CDCl ) δ 164.86, 158.79, 155.10, 154.06, 150.55, 138.36, 137.08, 133.93,
3
1
1
32.75, 131.92, 131.12, 129.66, 128.88, 127.88, 123.88, 123.12, 122.88, 121.02,
+
20.69, 120.04, 118.41, 112.82, 53.43, 24.11; HRMS-ESI (m/z): [M+H] calcd for
C H N OS 452.12881, found 452.12842.
24
18
7
4
.1.7.5 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-pyrazol-1-
yl)-N-phenylacetamide (19a)
1
Yield 15%; H NMR (300 MHz, CDCl ) δ 9.87 (s, 1H, NH), 7.92–7.87 (m, 3H),
3
7
.62–7.60 (m, 3H), 7.44 (d, 1H, J = 9.0 Hz), 7.31 (t, 2H, J = 9.0 Hz), 7.16–7.10 (m,
+
3
H), 5.10 (s, 2H), 2.74 (s, 3H); HRMS-ESI (m/z): [M+H] calcd for C H N OS
2
3
19
6
4
27.13356, found 427.13348.
4
.1.7.6 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-pyrazol-
1
- yl)-N-(2-fluorophenyl)acetamide (19b)
o
1
Yield 14%; mp 150–152 C; H NMR (300 MHz, CDCl ) δ 9.72 (s, 1H, NH) , 8.38 (t,
3
1
7
H, J = 9.0 Hz), 7.92–7.89 (m, 3H), 7.54 (t, 1H, J = 9.0 Hz), 7.44 (d, 1H, J = 9.0 Hz),
.24 (d, 1H, J = 9.0 Hz), 7.16–7.04 (m, 4H), 5.15 (s, 2H), 2.66 (s, 3H); HRMS-ESI
+
(
m/z): [M+H] calcd for C H FN OS 445.12413, found 445.12390.
23
18
6
4
1
.1.7.7 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-pyrazol-
-yl)-N-(3-fluorophenyl)acetamide (19c)
o
1
Yield 14%; mp 144–145 C; H NMR (300 MHz, CDCl ) δ 10.13 (s, 1H, NH),
3
7
.92–7.89 (m, 3H), 7.60–7.55 (m, 2H), 7.43 (dd, 1H, J = 9.0, 3.0 Hz), 7.30–7.25 (m,
4
H), 7.11 (d, 1H, J = 9.0 Hz), 6.84–6.79 (m, 1H), 5.04 (s, 2H), 2.71 (s, 3H);
+
HRMS-ESI (m/z): [M+H] calcd for C H FN OS 445.12413, found 445.12302.
2
3
18
6
4
1
.1.7.8 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-pyrazol-
-yl)-N-(3-cyanophenyl)acetamide (19d)

ACCEPTED MANUSCRIPT
1
Yield 9%; H NMR (300 MHz, CDCl ) δ 9.72 (s, 1H, NH), 8.02 (s, 1H), 7.93 (d, 1H,
3
J = 9.0 Hz), 7.87 (s, 1H), 7.83 (s, 1H), 7.61–7.51 (m, 3H), 7.37–7.31 (m, 3H), 7.18 (d,
+
1
H, J = 9.0 Hz), 5.21 (s, 2H), 2.51 (s, 3H); HRMS-ESI (m/z): [M+H] calcd for
C H N OS 452.12881, found 452.12860.
24
18
7
4
1
.1.7.9 2-(3-(6-Methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-yl)-1H-pyrazol-
-yl)-N-phenylacetamide (20a)
o
1
Yield 27%; mp 177–179 C; purity by HPLC: 96.1% (23% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 8.99 (s, 1H, NH), 8.62 (s, 1H), 8.41 (d, 1H, J = 6.0 Hz), 7.89 (s,
3
1
6
7
H), 7.71 (d, 1H, J = 6.0 Hz), 7.66 (t, 1H, J = 7.5 Hz), 7.58 (s, 1H), 7.52 (d, 1H, J =
.0 Hz), 7.47 (d, 2H, J = 9.0 Hz), 7.30 (t, 2H, J = 7.5 Hz), 7.22 (d, 1H, J = 9.0 Hz),
1
3
.13 (t, 1H, J = 7.5 Hz), 5.07 (s, 2H), 2.64 (s, 3H); C NMR (125 MHz,
CDCl /CD OD) δ 164.69, 158.40, 150.53, 149.37, 147.44, 144.43, 141.46, 137.39,
3
3
1
1
37.11, 136.72, 136.23, 132.45, 128.86 (2C), 124.76, 123.22, 120.99, 120.29 (2C),
+
20.07, 117.15, 114.91, 55.47, 23.93; HRMS-ESI (m/z): [M+H] calcd for
C H 0N OS 426.13831, found 426.13809.
24
2
5
4
.1.7.10 N-(2-Fluorophenyl)-2-(3-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-
yl)-1H-pyrazol-1-yl)acetamide (20b)
o
1
Yield 49%; mp 172–174 C; purity by HPLC: 98.2% (23% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 9.49 (s, 1H, NH), 8.97 (s, 1H), 8.62 (d, 1H, J = 6.0 Hz), 8.59 (s,
3
1
H), 8.40 (d, 1H, J = 6.0 Hz), 7.85 (d, 1H, J = 6.0 Hz), 7.72 (t, 2H, J = 9.0 Hz), 7.53
(t, 1H, J = 7.5 Hz), 7.22 (br s, 1H), 7.06 (t, 1H, J = 7.5 Hz), 6.95 (t, 1H, J = 9.0 Hz),
1
3
6
1
1
3
1
.62 (d, 1H, J = 6.0 Hz), 5.29 (s, 2H), 2.29 (s, 3H); C NMR (125 MHz, CDCl ) δ
3
64.36, 158.31, 152.80 (d, J = 243.75 Hz), 150.93, 150.79, 147.10, 145.36, 142.53,
36.99, 135.55, 132.64, 131.51 (d, J = 7.5 Hz), 125.24 (d, J = 7.5 Hz), 124.70 (d, J =
.75 Hz), 123.11, 121.93, 121.14, 120.56 (d, J = 11.25 Hz), 120.37, 116.80, 115.84,
+
15.08 (d, J = 18.75 Hz), 55.93, 24.36; HRMS-ESI (m/z): [M+H] calcd for
C H FN OS 444.12889, found 444.12900.
24
19
5
4
.1.7.11 N-(3-Fluorophenyl)-2-(3-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-
yl)-1H-pyrazol-1-yl)acetamide (20c)

ACCEPTED MANUSCRIPT
o
1
Yield 29%; mp 182–184 C; purity by HPLC: 96.8% (23% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 9.03 (s, 1H, NH), 8.98 (s, 1H), 8.41 (d, 1H, J = 6.0 Hz), 7.88 (s,
3
1
1
9
H), 7.71 (dd, 1H, J = 12.0, 6.0 Hz), 7.64 (d, 1H, J = 6.0 Hz), 7.55 (s, 1H), 7.50 (d,
H, J = 9.0 Hz), 7.44 (d, 1H, J = 9.0 Hz), 7.22 (d, 1H, J = 9.0 Hz), 7.10 (d, 2H, J =
1
3
.0 Hz), 6.80 (t, 1H, J = 7.5 Hz), 5.07 (s, 2H), 2.63 (s, 3H); C NMR (125 MHz,
CDCl /CD OD) δ 164.84, 162.82 (d, J = 242.5 Hz), 158.41, 150.48, 149.33, 147.46,
3
3
1
44.38, 141.35 (d, J = 12.5 Hz), 139.12, 137.12, 136.71, 136.24, 132.48, 129.99 (d, J
=
8.75 Hz), 123.25, 121.02, 120.03, 117.16, 115.47, 114.92, 111.27 (d, J = 21.25 Hz),
+
1
07.53 (d, J = 26.25 Hz), 55.34, 23.88; HRMS-ESI (m/z): [M+H] calcd for
C H FN OS 444.12889, found 444.12891.
24
19
5
4
.1.7.12 N-(3-Cyanophenyl)-2-(3-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-
yl)-1H-pyrazol-1-yl)acetamide(20d)
o
1
Yield 52%; mp 177–178 C; purity by HPLC: 98.7% (23% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 8.99 (s, 1H, NH), 8.62 (s, 1H), 8.41 (d, 1H, J = 6.0 Hz), 7.89 (s,
3
1
9
7
H), 7.71 (d, 1H, J = 6.0 Hz), 7.66 (t, 1H, J = 7.5 Hz), 7.59 (s, 1H), 7.53 (d, 1H, J =
.0 Hz), 7.47 (d, 2H, J = 9.0 Hz), 7.31 (d, 1H, J = 6.0 Hz), 7.22 (d, 1H, J = 9.0 Hz),
1
3
.13 (t, 1H, J = 7.5 Hz), 5.06 (s, 2H), 2.64 (s, 3H); C NMR (125 MHz,
CDCl /CD OD) δ 164.61, 158.46, 150.57, 149.52, 147.37, 144.63, 141.56, 137.37,
3
3
1
1
37.08, 136.72, 136.09, 132.46, 131.20, 128.92, 124.82, 123.22, 120.98, 120.92,
+
20.45, 120.34, 120.24, 117.11, 115.05, 55.65, 24.07; HRMS-ESI (m/z): [M+H]
calcd for C H N OS 451.13356, found 451.13348.
2
5
19
6
4
1
.1.7.13
2-(5-(6-Methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-yl)-1H-imidazol-
-yl)-N-phenylacetamide (21a)
1
Yield 9%; H NMR (300 MHz, CDCl ) δ 9.75 (s, 1H, NH), 8.97 (s, 1H), 8.41 (br s,
3
1
H), 7.90 (s, 1H), 7.69 (br s, 2H), 7.59 (d, 2H, J = 6.0 Hz), 7.33–7.23 (m, 5H), 7.11
+
(
br s, 1H), 4.99 (s, 2H), 2.69 (s, 3H); HRMS-ESI (m/z): [M+H] calcd for
C H N OS 426.13831, found 426.13846.
24
20
5
4
.1.7.14 N-(2-Fluorophenyl)-2-(5-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-
yl)-1H-imidazol-1-yl)acetamide (21b)

ACCEPTED MANUSCRIPT
1
Yield 13%; H NMR (300 MHz, CDCl ) δ 9.66 (s, 1H, NH), 8.97 (s, 1H), 8.41–8.33
3
(m, 2H), 7.90 (s, 1H), 7.69 (d, 1H, J = 6.0 Hz), 7.64 (d, 1H, J = 9.0 Hz), 7.32–7.25
+
(m, 2H), 7.16–7.04 (m, 4H), 5.09 (s, 2H), 2.65 (s, 3H); HRMS-ESI (m/z): [M+H]
calcd for C H FN OS 444.12889, found 444.12878.
2
4
19
5
4
.1.7.15 N-(3-Fluorophenyl)-2-(5-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-
yl)-1H-imidazol-1-yl)acetamide (21c)
1
Yield 11%; H NMR (300 MHz, CDCl ) δ 10.06 (s, 1H, NH), 8.96 (s, 1H), 8.39 (d,
3
1
H, J = 6.0 Hz), 7.89 (s, 1H), 7.72–7.68 (m, 2H), 7.54 (d, 1H, J = 9.0 Hz), 7.33 (t, 2H,
J = 7.5 Hz), 7.25–7.19 (m, 3H), 6.79 (br s, 1H), 4.97 (s, 2H), 2.68 (s, 3H);
+
HRMS-ESI (m/z): [M+H] calcd for C H FN OS 444.12889, found 444.12885.
2
4
19
5
4
.1.7.16 N-(3-Cyanophenyl)-2-(5-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-
yl)-1H-imidazol-1-yl)acetamide (21d)
1
Yield 5%; H NMR (300 MHz, CDCl ) δ 10.39 (s, 1H, NH), 8.96 (s, 1H), 8.39 (d, 1H,
3
J = 6.0 Hz), 7.98 (s, 1H), 7.89 (s, 1H), 7.83 (dd, 1H, J = 9.0, 3.0 Hz), 7.74–7.68 (m,
3
H), 7.44–7.33 (m, 3H), 7.23 (d, 1H, J = 6.0 Hz), 4.99 (s, 2H), 2.67 (s, 3H);
+
HRMS-ESI (m/z): [M+H] calcd for C H N OS+ (M+H)+ 451.13356, found
2
5
19
6
4
4
2
2
51.13361.
.1.8
General
procedure
for
the
preparation
of
-(4-(benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-
or
-(3-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-yl)-1H-pyrazol-1-yl)-N-phenyl
ethanethioamide (22a–d, 23c, and 23d )
A stirred mixture of 22a–d, 23c, and 23d (0.96 mmol), Lawesson's reagent (0.96
o
mmol), and anhydrous DME (3 mL) in a dry sealed tube was heated at 85 C for 8–12
h. After cooled to room temperature, the solvent was evaporated to dryness under
reduced pressure, and the residue was purified by silica gel column chromatography
to gain the titled compound 22a–d, 23c, and 23d as a light yellow solid.
4
.1.8.1 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-1H-pyrazol-
1
-yl)-N-phenylethanethioamide (22a)

ACCEPTED MANUSCRIPT
o
1
Yield 80%; mp 167–169 C; purity by HPLC: 98.6% (40% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 10.69 (s, 1H, NH), 8.01 (s, 1H), 7.92 (d, 1H, J = 9.0 Hz), 7.88
3
(s, 1H), 7.77 (d, 2H, J = 9.0 Hz), 7.64 (t, 1H, J = 9.0 Hz), 7.56 (dd, 1H, J = 9.0, 3.0
Hz), 7.42 (d, 1H, J = 6.0 Hz), 7.37 (t, 2H, J = 7.5 Hz), 7.25 (d, 1H, J = 6.0 Hz), 7.20
1
3
(
d, 1H, J = 9.0 Hz), 5.50 (s, 2H), 2.57 (s, 3H); C NMR (125 MHz, CDCl ) δ 192.72,
3
1
1
2
4
58.70, 155.08, 154.05, 150.67, 150.59, 138.23, 136.84, 134.01, 132.25, 132.22,
28.87 (2C), 127.01, 123.07 (2C), 122.91, 121.62, 120.52, 120.24, 119.99, 63.73,
+
4.34; HRMS-ESI (m/z): [M+H] calcd for C H N S 443.11071, found 443.11069.
2
3
19
6 2
.1.8.2
2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-1H-
pyrazol-1-yl)-N-(2-fluorophenyl)acetamide (22b)
o
1
Yield 66%; mp 156–157 C; purity by HPLC: 100% (40% acetonitrile); H NMR (300
MHz, CDCl ) δ 10.80 (s, 1H, NH), 8.37 (br s, 1H), 8.01 (s, 1H), 7.93 (d, 1H, J = 9.0
3
Hz), 7.89 (s, 1H), 7.74 (br s, 1H), 7.56 (dd, 2H, J = 15.0, 6.0 Hz), 7.26–7.21 (m, 3H),
1
3
7
1
1
1
.19–7.07 (m, 2H), 5.52 (s, 2H), 2.65 (s, 3H); C NMR (125 MHz, CDCl ) δ 193.47,
3
58.36, 155.37 (d, J = 247.5 Hz), 155.07, 154.08, 150.67, 136.98, 134.13, 132.49,
32.08, 127.74 (d, J = 7.5 Hz), 126.64 (d, J = 10.0 Hz), 124.08, 124.05, 124.01,
22.88, 121.97, 120.22, 119.99, 115.48 (d, J = 18.75 Hz), 63.79, 24.19; HRMS-ESI
+
(
m/z): [M+H] calcd for C H FN S 461.10129, found 461.10101.
23
18
6 2
4
1
.1.8.3 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-1H-pyrazol-
-yl)-N-(3-fluorophenyl)ethanethioamide (22c)
o
1
Yield 64%; mp 134–136 C; purity by HPLC: 99.8% (40% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 11.18 (s, 1H, NH), 8.00–7.87 (m, 5H), 7.73–7.49 (m, 3H),
3
1
3
7
.37–7.26 (m, 2fH), 6.91 (t, 1H, J = 7.5 Hz), 5.50 (s, 2H), 2.63 (s, 3H); C NMR
(
125 MHz, CDCl ) δ 192.89, 162.42 (d, J = 243.75 Hz), 158.66, 155.07, 154.05,
3
1
50.42, 150.01, 139.86 (d, J = 11.25 Hz), 137.13, 133.91, 132.32, 132.07, 129.91 (d,
J = 8.75 Hz), 123.08, 121.49, 120.71, 120.45, 120.02, 118.34 (d, J = 2.5 Hz),
1
13.58 (d, J = 21.25 Hz), 110.02 (d, J = 26.25 Hz), 63.89, 24.13; HRMS-ESI (m/z):
+
[
M+H] calcd for C H FN S 461.10129, found 461.10126.
2
3
18
6 2
4
.1.8.4 2-(4-(Benzo[c][1,2,5]thiadiazol-5-yl)-3-(6-methylpyridin-2-yl)-1H-pyrazol-

ACCEPTED MANUSCRIPT
1
-yl)-N-(3-cyanophenyl)ethanethioamide (22d)
o
1
Yield 60%; mp 190–192 C; purity by HPLC: 98.8% (40% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 11.46 (s, 1H, NH), 8.27 (s, 1H), 8.00–7.91 (m, 4H), 7.71 (dd,
3
2
H, J = 6.0, 3.0 Hz), 7.54–7.41 (m, 3H), 6.97 (br s, 1H), 5.49 (s, 2H), 2.68 (s, 3H);
1
3
C NMR (125 MHz, CDCl ) δ 193.63, 158.71, 155.06, 154.09, 150.02, 139.23,
3
1
1
37.51, 133.65, 132.55, 131.86, 130.88, 130.01, 129.63, 127.16, 125.98, 123.39,
21.52, 121.02, 120.71, 120.16, 118.09, 112.79, 63.73, 24.47; HRMS (ESI) m/z
+
[
M+H] calcd for C H N S +(M+H)+ 468.10596,found 468.10571.
2
4
18
7 2
4
.1.8.5
N-(3-Fluorophenyl)-2-(3-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-
yl)-1H-pyrazol-1-yl)ethanethioamide (23c)
o
1
Yield 33%; mp 154–156 C; purity by HPLC: 95.5% (32% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 10.76 (s, 1H, NH), 9.06 (s, 1H), 8.43 (br s, 1H), 7.94 (s, 1H),
3
7
7
.80–7.76 (m, 2H), 7.65 (br s, 1H), 7.56 (br s, 1H), 7.52 (br s, 1H), 7.37 (br s, 1H),
.21 (d, 1H, J = 9.0 Hz), 6.92 (br s, 1H), 6.72 (br s, 1H), 5.44 (s, 2H), 2.62 (s, 3H);
+
HRMS (ESI) m/z [M+H] calcd for C H FN S 460.10604, found 460.10626.
2
4
19
5 2
4
.1.8.6
N-(3-Cyanophenyl)-2-(3-(6-methylpyridin-2-yl)-4-(thieno[3,2-c]pyridin-2-
yl)-1H-pyrazol-1-yl)ethanethioamide (23d)
o
1
Yield 27%; mp 142–144 C; purity by HPLC: 96.6% (23% acetonitrile); H NMR
(
300 MHz, CDCl ) δ 10.44 (s, 1H, NH), 9.01 (s, 1H), 8.41 (br s, 1H), 7.93 (s, 1H),
3
7
.72–7.63 (m, 4H), 7.60 (s, 1H), 7.52 (d, 1H, J = 6.0 Hz), 7.37 (t, 2H, J = 9.0 Hz),
+
7
.22 (d, 1H, J = 9.0 Hz), 5.44 (s, 2H), 2.63 (s, 3H); HRMS (ESI) m/z [M+H] calcd
for C H N S 467.11071, found 467.11078.
2
5
19
6 2
4
.2. Kinase assay
All kinase experiments were completed by the ProQinase (Germany). All protein
kinases were expressed in Sf9 insect cells or in E.coli as recombinant GST-fusion
proteins or His-tagged proteins, either as full-length or enzymatically active
3
3
®
fragments. A radiometric protein kinase assay ( PanQinase activity assay) was used
for measuring the kinase activity of the two protein kinases. All kinase assays were
TM
performed in 96-well FlashPlates from PerkinElmer (Boston, MA, USA) in 50 µL
reaction volumes. The reaction cocktail was pipetted in four steps in the following

ACCEPTED MANUSCRIPT
order: 20 µL of assay buffer (standard buffer), 5 µL of ATP solution (in H O), 5 µL of
2
test compound (in 10% DMSO), 20 µL enzyme/substrate mix.
The assay for all protein kinases contained 70 mM HEPES-NaOH pH 7.5, 3 mM
MgCl , 3 mM MnCl , 3 µM Na-orthovanadate, 1.2 mM DTT, 50 µg/mL PEG20000,
2
2
33
ATP, [γ- P]-ATP, protein kinase, and substrate.
The reaction cocktail was incubated at 30°C for 60 minutes. The reaction was
halted with 50 µL of 2% (v/v) H PO , plates were aspirated and washed two times
3
4
33
with 200 µL 0.9% (w/v) NaCl. Incorporation of Pi was established with a
microplate scintillation counter (Microbeta, Wallac). All assays were performed with
a BeckmanCoulter/SAGIAN™ Core System.
4
.3 Docking Study
All molecular computation studies were carried out using Discovery Studio 2017
(Accelrys, San Diego, USA). The X-ray crystal structure of ALK5 complexed with
5
,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhibitor was obtained from protein data bank,
respectively. (PDB: 1RW8) The water molecules and complexed ligand in protein
were removed and the protein was prepared by adding hydrogen and correcting
incomplete residues using Clean Protein tool of DS, then the protein was refined with
CHARMm. The structures of 20c and 22c were sketched in 2D and converted into
3
D using the DS molecule editor. Automated docking studies were carried out to
investigate the binding mode of compound 20c and 22c in the crystal structure of
,6-dihydro-4H-pyrrolo[1,2-b]pyrazole utilizing DS LibDock protocol. The pose
5
with the top LibDockScore was chosen for analyzing the binding features of
compounds 20c and 22c with ALK5.
4
.4 Cell culture
LX-2 is a human immortalized HSCs cell line, retaining features of HSCs. LX-2
cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 100 units/ml penicillin G plus 100 mg/ml
streptomycin at 37 °C and 5% CO2. The cultures were passaged every two or three
days.
4
.5 MTT assay
Cell viability was measured using the 3-(4, 5-dimethylthiazol-2-

ACCEPTED MANUSCRIPT
yl)-2,5-diphenylterazoliun bromide (MTT) assay. LX-2 cells were seeded in a
9
6-well plate as a density of 1 × 104 cells/well. After treatment, MTT was added, and
then incubated additional 3 h. The medium containing MTT was suctioned out and
replaced with DMSO. The reduction of MTT to purple formazan was detected using
a microplate reader at 540 nm.
4
.6 Immunocytochemistry staining assay [44]
LX-2 Cells were fixed with 4% paraformaldehyde and then permeabilized
treatment in 0.1% TritonX-100, followed by blocking with 10% goat serum in PBS.
Cells were incubated in the primary antibody at 4°C for overnight and followed by
FITC-conjugated secondary antibody incubation. Cells were mounted with
fluoroshield mounting medium with DAPI (Abcam) and then visualized an Olympus
TM
BX53 fluorescence microscope or Olympus FluoView
FV10i Confocal Laser
Scanning Biological Microscope.
4
.7 Western Blotting Analysis.
The protein extracted from LX-2 cells was used for Western blotting analysis.
Equivalent proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). After gel electrophoresis, protein was transferred onto
a polyvinylidene fluoride (PVDF) membrane (GE, Freiburg, Germany). Then the
membrane was blocked by 5% skim milk for 1 h at room temperature and incubated
with specific primary antibodies at 4°C overnight, and then incubated with
HRP-conjugated secondary antibody for 1 h at room temperature and visualized by
ECL detection reagent (Bio-Rad, Hercules, CA, USA). Each membrane was stripped
and cultivated with GAPDH antibody as a loading control. Quantity One software
(Bio-Rad, Hercules, CA, USA) was used to qualify the band intensities.
4
.8 Cell mRNA Isolation and RT-PCR.
Total RNA was extracted from LX-2 cells by using the RNA extraction kit
(Promega, Madison, WI, USA). Isolated RNA was reverse-transcribed into
complementary DNA (cDNA). And RT-PCR was performed by using primers
specific for the genes as shown in Table 2. The PCR products were loaded in agarose
gel electrophoresis and stained with ethidium bromide. The transcript level was

ACCEPTED MANUSCRIPT
normalized with GAPDH mRNA in the same samples.
Acknowledgments
This work was supported by the National Science Foundation of China (No.
8
1560557 and 81560597) and the Education Department of Jilin Province Scientific
Research Fund Project (No. 2016-283). We thank James Murray, Ph.D. from Edanz
Group (www.edanzediting.com) for editing a draft of this manuscript.
Conflict of Interest
The authors have declared no conflict of interest.

ACCEPTED MANUSCRIPT
References
[
1] Y. Yong, T. Bai, Y.L. Yao, D.Q. Zhang, Y.L. Wu, L.H. Lian, J.X. Nan, Upregulation of
SIRT1-AMPK by thymoquinone in hepatic stellate cells ameliorates liver injury, Toxicol.
Lett. 262 (2016) 80–91. https://doi.org/10.1016/j.toxlet.2016.09.014.
[
2] Y. Wang, J. Gao, D. Zhang, J. Zhang, J. Ma, J. Jiang, New insights into the antifibrotic
effects of sorafenib on hepatic stellate cells and liver fibrosis, J. Hepatol. 53 (2010)
1
32–144. https://doi.org/10.1016/j.jhep.2010.02.027.
3] M. Pinzani, F. Marra, V. Carloni, Signal transduction in hepatic stellate cells, Liver 18
1998) 2–13. https://doi.org/10.1111/j.1600-0676.1998.tb00120.x.
[
[
(
4] S.J. Park, H.Y. Sohn, J. Yoon, S.I. Park, Down-regulation of FoxO-dependent c-FLIP
expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells, Cell.
Signal. 21 (2009) 1495–1503. https://doi.org/10.1016/j.cellsig.2009.05.008.
5] R. Bataller, D.A. Brenner, Liver fibrosis, J. Clin. Invest. 115 (2005) 209–218.
https://doi.org/10.1172/JCI24282.
[
[
6] S. Robert, T. Gicquel, A. Bodin, V. Lagente, E. Boichot, Characterization of the
mmp/timp imbalance and collagen production induced by IL-1 beta or TNF-alpha release
from human hepatic stellate cells, PLoS One 11 (2016) e0153118.
https://doi.org/10.1371/journal.pone.0153118.
[
7] C.H. Jin, D. Sreenu, M. Krishnaiah, V.B. Subrahmanyam, K.S. Rao, A.V.N. Mohan, C.Y.
Park, J.Y. Son, D.H. Son, H.J. Park, Y.Y. Sheen, D.K. Kim, Synthesis and biological
evaluation of
as transforming growth factor-β type 1 receptor kinase inhibitors, Eur. J. Med. Chem. 46
2011) 3917–3925. https://doi.org/10.1016/j.ejmech.2011.05.063.
8] C.H. Heldin, K. Miyazono, P. Ten Dijke, TGF-beta signalling from cell membrane to
nucleus through SMAD proteins, Nature 390 (1997) 465–471.
https://doi.org/10.1038/37284.
1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles
(
[
[
[
9] R. Derynck, Y.E. Zhang, Smad-dependent and Smad-independent pathways in TGF-beta
family signaling, Nature 425 (2003) 577–584. https://doi.org/10.1038/nature02006.
10] W. Wang, V. Koka, H.Y. Lan, Transforming growth factor-beta and Smad signaling in
kidney
diseases,
Nephrology
10
(2005)
48–56.

ACCEPTED MANUSCRIPT
https://doi.org/10.1111/j.1440-1797.2005.00334.x.
[
[
[
11] L. Gu, Y.J. Zhu, X. Yang, Z.J. Guo, W.B. Xu, X.L. Tian, Effect of TGF-beta/smad
signaling pathway on lung myofibroblast differentiation, Acta Pharmacol. Sin. 28 (2007)
3
82–391. Doi: 10.1111/j.1745-7254.2007.00468.x.
12] H. Lim, Y.Z. Zhu, Role of transforming growth factor-beta in the progression of heart
failure, Cell. Mol. Life Sci. 63 (2006) 2584–2596.
https://doi.org/10.1007/s00018-006-6085-8.
13] F.W. Shek, R.C. Benyon, How can transforming growth factor beta be targeted usefully
to combat liver fibrosis? Eur. J. Gastroenterol. Hepatol. 16 (2004) 123–126. DOI:
1
0.1097/01.meg.0000085544.79233.93.
14] M. Dong, G.C. Blobe, Role of transforming growth factor-β in hematologic
malignancies, Blood 107 (2006) 4589–4596.
https://doi.org/10.1182/blood-2005-10-4169.
[
[
15] V. Inanović, N. Todorović-Raković, M. Demajo, Z. Nešković-Konstantinović, V. Subota,
O. Ivanišević-Milovanović, Nikolić-Vukosavljević, Elevated plasma levels of
transforming growth factor-beta 1 (TGF-beta 1) in patients with advanced breast cancer:
association with disease progression, Eur. J. Cancer. 39 (2003) 454–461.
https://doi.org/10.1016/S0959-8049(02)00502-6.
[
[
[
16] S.G. Rane, J.H. Lee, H.M. Lin, Transforming growth factor-β pathway: role in pancreas
development and pancreatic disease, Cytokine. Growth. Factor. Rev. 17 (2006) 107–119.
https://doi.org/10.1016/j.cytogfr.2005.09.003.
17] S.D. Byfield, C. Major, N.J. Laping, A.B. Roberts, SB-505124 is a selective inhibitor of
transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7, Mol.
Pharmacol. 65 (2004) 744–752. https://doi.org/10.1124/mol.65.3.744.
18] M. Uhl, S. Aulwurm, J. Wischhusen, M. Weiler, J.Y. Ma, R. Almirez, R. Mangadu,Y.W.
Liu, M. Platten, U. Herrlinger, A. Murphy, D.A. Wong, W. Wick, L.S. Higgins, M.
Weller, SD-208, a novel transforming growth factor beta receptor I kinase inhibitor,
inhibits growth and invasiveness and enhances immunogenicity of murine and human
glioma cells in vitro and in vivo, Cancer Res. 64 (2004) 7954–7961.
DOI:10.1158/0008-5472.CAN-04-1013.
[
19] L. Bueno, D.P. de Alwis, C. Pitou, J. Yingling, M. Lahn, S. Glatt, I. Troconiz,