The development of a novel transforming growth factor-β (TGF-β) inhibitor that disrupts ligand-receptor interactions

Article abstract of DOI:10.1016/j.ejmech.2020.112042

Transforming growth factor-β (TGF-β) plays an important role in regulating epithelial to mesenchymal transition (EMT) and the TGF-β signaling pathway is a potential target for therapeutic intervention in the development of many diseases, such as fibrosis and cancer. Most currently available inhibitors of TGF-β signaling function as TGF-β receptor I (TβR-I) kinase inhibitors, however, such kinase inhibitors often lack specificity. In the present study, we targeted the extracellular protein binding domain of the TGF-β receptor II (TβR-II) to interfere with the protein-protein interactions (PPIs) between TGF-β and its receptors. One compound, CJJ300, inhibited TGF-β signaling by disrupting the formation of the TGF-β-TβR-I-TβR-II signaling complex. Treatment of A549 cells with CJJ300 resulted in the inhibition of downstream signaling events such as the phosphorylation of key factors along the TGF-β pathway and the induction of EMT markers. Concomitant with these effects, CJJ300 significantly inhibited cell migration. The present study describes for the first time a designed molecule that can regulate TGF-β-induced signaling and EMT by interfering with the PPIs required for the formation of the TGF-β signaling complex. Therefore, CJJ300 can be an important lead compound with which to study TGF-β signaling and to design more potent TGF-β signaling antagonists.

Full text of DOI:10.1016/j.ejmech.2020.112042

Journal Pre-proof
The development of a novel transforming growth factor-β (TGF-β) inhibitor that
disrupts ligand-receptor interactions
Han Wu, Yu Sun, Wee Lin Wong, Jiajia Cui, Jingyang Li, Xuefu You, Lee Fah Yap,
Yu Huang, Wei Hong, Xinyi Yang, Ian Paterson, Hao Wang
PII:
S0223-5234(20)30009-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112042
EJMECH 112042
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 November 2019
Revised Date: 5 January 2020
Accepted Date: 6 January 2020
Please cite this article as: H. Wu, Y. Sun, W.L. Wong, J. Cui, J. Li, X. You, L.F. Yap, Y. Huang, W.
Hong, X. Yang, I. Paterson, H. Wang, The development of a novel transforming growth factor-β (TGF-β)
inhibitor that disrupts ligand-receptor interactions, European Journal of Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.ejmech.2020.112042.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. 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.
© 2020 Published by Elsevier Masson SAS.

The development of a novel transforming growth factor-β
(TGF-β) inhibitor that disrupts ligand-receptor interactions
.
Han Wu#a, Yu Sun#b, Wee Lin Wong#c, Jiajia Cui#d, Jingyang Lie, Xuefu
Youb, Lee Fah Yapc, Yu Huanga, Wei Hong*f, g, Xinyi Yang*b, Ian
Paterson*c, Hao Wang*a, h, i
a. School of Pharmacy, Ningxia Medical University, Yinchuan 750004, China
b. Laboratory of Pharmacology/Beijing Key Laboratory of Antimicrobial Agents,
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China
c. Department of Oral and Craniofacial Sciences and oral Cancer Research and
Coordinating Centre, Faculty of Dentistry, University of Malaya, 50603 Kuala
Lumpur, Malaysia.
d. Department of Pharmacy，Shanxi Provincial Hospital of Tuberculosis Prevention
and Treatment，Xi’an 710100，China
e. Department of Pharmacy, Logistics University of Chinese People's Armed Police
Forces, Tianjin 300309, China
f. School of Chemistry and Chemical Engineering, North Minzu University,
Yinchuan 750021, China
g. Key Laboratory of Chemical Engineering and Technology, State Ethnic Affairs
Commission, North Minzu University, Yinchuan 750021, China
h. School of Pharmacy, Minzu University of China, Beijing, 100081, China
i. Key Laboratory of Ethnomedicine (MINZU University of China), Ministry of
Education, Beijing, 100081, China
*Wei Hong: hongwei136@hotmail.com
*Hao Wang: paxhw@yahoo.co.uk
*Xinyi Yang: yangxinyi1976@hotmail.com
* Ian Paterson: ipaterson@um.edu.my

# Han Wu, Yu Sun, Wee Lin Wong, Jiajia Cui contributed equally
Abstract:
Transforming growth factor-β (TGF-β) plays an important role in regulating epithelial
to mesenchymal transition (EMT) and the TGF-β signaling pathway is a potential
target for therapeutic intervention in the development of many diseases, such as
fibrosis and cancer. Most currently available inhibitors of TGF-β signaling function as
TGF-β receptor I (TβR-I) kinase inhibitors, however, such kinase inhibitors often lack
specificity. In the present study, we targeted the extracellular protein binding domain
of the TGF-β receptor II (TβR-II) to interfere with the protein-protein interactions
(PPIs) between TGF-β and its receptors. One compound, CJJ300, inhibited TGF-β
signaling by disrupting the formation of the TGF-β-TβR-I-TβR-II signaling complex.
Treatment of A549 cells with CJJ300 resulted in the inhibition of downstream
signaling events such as the phosphorylation of key factors along the TGF-β pathway
and the induction of EMT markers. Concomitant with these effects, CJJ300
significantly inhibited cell migration. The present study describes for the first time a
designed molecule that can regulate TGF-β-induced signaling and EMT by interfering
with the PPIs required for the formation of the TGF-β signaling complex. Therefore,
CJJ300 can be an important lead compound with which to study TGF-β signaling and
to design more potent TGF-β signaling antagonists.
Keywords: Transforming growth factor-β inhibitor; TGF-β inhibitor; protein-protein
interactions; epithelial to mesenchymal transition
1. Introduction
Epithelial–mesenchymal transition (EMT) is a well-recognized biologic process, by
which epithelial cells lose their intercellular adhesion and gain the migratory and
invasive characteristics of mesenchymal cells [1], [2]. EMT has long been shown to

contribute to a number of disease pathologies, such as the development of fibrosis in
multiple organ systems (skin, heart, liver, kidney, and lung), cardiovascular disease
and cancer, the development of drugs to inhibit or reverse the pathophysiological
EMT process is expected to be beneficial for the treatment of these diseases [3], [4].
Transforming growth factor-β (TGF-β) plays a key role in the EMT process due to its
pleiotropic functions in activating EMT-related intracellular signal transduction,
promoting the expression of mesenchymal biomarkers and enhancing cell motility.
Therefore, TGF-β signaling has been increasingly recognized as a target for
therapeutic intervention in EMT-related pathologies [5].
TGF-β binds to two receptors on the cell surface, namely TGF-β receptor I and II
(TβR-I and TβR-II receptively), which are transmembrane serine/threonine kinases,
containing extracellular binding domains for protein-protein interactions and
intracellular kinase domains. TGF-β signal transduction is initiated by the binding of
TGF-β to the TβR-II extracellular domain, followed by the binding of TβR-I to the
complex. After the formation of the TGF-β-TβR-II-TβR-I heterotrimeric complex, the
intracellular domain of TβR-I is phosphorylated and downstream signaling is initiated.
Firstly, receptor-specific (R) Smads (Smad2 and Smad3) are phosphorylated and form
oligomers with Smad4 and this complex then translocates to the nucleus to regulate
the transcription of various target genes [6]. In additional to the canonical Smad
pathway, TGFβ can activate the Smad-independent pathways, including PI3K/AKT
and ERK/MAPK pathways, which can also contribute to EMT [5] .
Theoretically, there are two potential ways to inhibit the TGF-β signaling pathway,
namely, by inhibiting the receptor kinases and by interrupting ligand-receptor
complex formation via disrupting protein-protein interactions (PPIs). Currently most
of the known TGF-β signaling inhibitors, such as Galunisertib (LY2157229) [7],
SB-431542 [8], SB-505124 [9], SD-208 [10], and R-268712 [11] (Figure 1), were
developed to target the TβR-I kinase. However, such ATP binding site inhibitors
usually lack specificity and can cause various side effects and, therefore, new ways to

inhibit TGF-β signaling would be beneficial.
Fig 1, Structures of known TGF-β signaling pathway inhibitors, NCI-48454
(NCI declared) and NCI-48455 (confirmed).
Previously, we used virtual screening and molecular docking to identify compounds,
NCI-48454 and NCI-48455 (Fig 1), which were obtained from National Cancer
Institute (NCI) USA and strongly inhibited TGF-β signaling, as measured by
TGF-β-dependent luciferase assays in vitro [12]. According to the previous paper, the
chemical structures declared by NCI of these two compounds were not correct, in
which the structure of NCI-48454 was not determined and only NCI-48455 was
confirmed as Fig 1 shows [12]. We were not able to conclusively determine whether
NCI-48455 inhibited the signaling pathway by binding to the kinase domain of TβR-I
or by disturbing the protein-protein interactions (PPIs) that mediate ligand-receptor
binding. PPIs are essential components of most cellular processes and such
interactions are believed to serve as potential drug targets. Therefore, designing small
molecules to disturb the TGF-β-receptor complex formation would be important in
terms of potential drug discovery and also for the understanding of the regulation of
PPIs. However, in general, it is challenging to disrupt PPIs using small organic
compounds. The interacting surfaces involved in these PPIs are much larger than

small molecules, and the protein surfaces are usually flat, which do not contain deep
pockets that indicate the ligand binding sites. In the current study, a group of novel
molecules were designed and synthesized. Among them, one molecule, CJJ300, was
shown to disturb protein-protein interactions and prevent TGF-β receptor dimerization.
CJJ300 functioned as a signaling antagonist because the compound inhibited the
phosphorylation of intracellular mediators in the downstream TGF-β signaling
pathways. Further, CJJ300 suppressed the expression of markers of EMT, which was
in consistent with CJJ300-mediated inhibition of cell migration in scratch-wound
healing and transwell assays.
2. Results and Discussion
2.1. Molecular Design
As shown in Fig 2A and 2B, the crystal structure of the complex of TGF-β bound to
the extracellular domain of TβR-II (PDB ID 1KTZ) clearly shows a direct binding site
on TβR-II, which is composed of Leu27, Phe30, Asp32, Arg34, Ile50 and Ile53. We
hypothesized this PPI may be disturbed if small molecules occupy this binding site. In
the current study, a group of compounds were designed (Table 1), which contained
triangular shaped hydrophobic cores according to the hydrophobic region of TβR-II
binding site (colored in orange in Fig. 2C). Molecular docking studies suggested that
the molecule (represented by CJJ300，Fig 2C and 2D) can form strong hydrophobic
interactions with TβR-II, and especially form π-π interactions with Phe30. CJJ300 can
also form three potential hydrogen bonds with Ser52, Ile53 and Glu55. The molecular
docking showed that NCI-48455 formed strong π-π interactions with Phe30 of TβR-II,
and also formed two hydrogen bonds with Ser52 and Glu55 (Fig 2E) [12]. By
comparing the docking results of CJJ300 and NCI-48455, similar binding poses were
obtained, but one extra hydrogen bond of CJJ300 may increase its binding affinities.
Based on the docking results, we can reasonably predict that such new compounds
may occupy the protein binding site on TβR-II to disturb the PPI, which may inhibit
downstream TGF-β signaling. However, it must be addressed that, currently, we do

not have direct evidences to indicate the binding site of NCI-48455 or CJJ300.
Fig 2. The molecular design of analogs of NCI-48455. (A) The TGF-β-TβR-II
complex. TGF-β was represented by surface and TβR-II was represented by cartoon
tube. (B) The Leu27, Phe30, Asp32, Arg34, Ile50 and Ile53 on TβR-II (sticks) form
direct interactions with TGF-β (surface). (C) The binding of CJJ300 (ball-stick model)
on the TβR-II (surface). The interactions between CJJ300 (D) or NCI48455 (E)
(ball-stick model) and the key residues of TβR-II (stick model). In the surface
representation, the basic residues were colored in blue, the acidic residues in red, the
polar residues in green and the nonpolar residues in orange. In the ball-stick or stick
model, oxygen atoms were colored in red, nitrogen atoms were colored in blue and
carbon atoms were colored in purple (for protein residues) or in cyan (for CJJ300 or
NCI-48455).

2.2. Chemistry
We synthesized a series of phenyl amine derivatives 4a-g and benzyl amine
derivatives 9a-c as illustrated in Scheme
1
and 2. In Scheme 1,
was generated by
1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene
2
bromomethylation of 1,3,5-trimethylbenzene 1 with paraformaldehyde and a solution
of HBr in AcOH in the presence of zinc bromide as reported previously [13].
Treatment of compound 2 with the substituted anilines 3a-g in the presence of
potassium carbonate in acetonitrile or DMF generated substituted
1,3,5-tris(phenylaminomethyl)-2,4,6-trimethylbenzene derivatives 4a-g, which were
purified by recrystallization or chromatography. Among the substituted anilines,
compounds 3a, 3b, 3c, 3f were commercially available, and compounds 3d, 3e were
synthesized according the reported method [14]. As shown in Scheme 1, Compound
3g was synthesized from 3-nitrobenzoic acid 5 by amidation and reduction. Treatment
of compound 5 with ammonium carbonate and N,N-diisopropylethylamine (DIPEA)
in
the
presence
of
hydroxybenzotriazole
(HOBt)
and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), which were used as
carboxylate activator, produced 3-nitrobenzamide 6. Then the amide underwent
reduction with Pd/C and hydrazine hydrate in isopropyl alcohol to give compound 3g.

Scheme 1. Reagents and conditions: (a) (HCHO)n, ZnBr, HBr/AcOH, AcOH; (b)
3(a-g), K2CO3, CH3CN or DMF; (c) HOBt, EDCI, DIPEA, (NH4)2CO3, dry CH3CN;
(d) Pd/C, N2H4·H2O, isopropyl alcohol.
In Scheme 2, starting from compound 2, 2,4,6-trimethyl-1,3,5-benzenetricarbaldehyde
7 was prepared by three steps, nucleophilic substitution, hydrolysis and oxidation in
one pot. Treatment of compound 2 with potassium acetate in DMF generated benzyl
acetate, which can be hydrolyzed by potassium hydrate and sequentially oxidized by
PCC to give compound 7. And then compound 7 underwent reductive amination with
appropriate benzylamine 8a-c in the presence of sodium borohydride to give
substituted 1,3,5-tris(benzylaminomethyl)-2,4,6-trimethylbenzene 9a-c. Among the
substituted benzylamine, compounds 8a was commercially available, and compounds
8b-c were synthesized. As shown in Scheme 2, starting from compounds 10a-b,
compounds 8b-c were prepared by three steps reaction. Treatment of compounds
10a-b with N-bromosuccinimide (NBS) and triphenylphosphine in the presence of
benzoyl peroxide (BPO) generated benzyl bromide, which were reacted directly with
phthalimide potassium in DMF to give compounds 11a-b. Then phthalimide
compounds underwent Gabriel synthesis with hydrazine hydrate to give compounds
8b-c.

Scheme 2. Reagents and conditions: (a) CH3COOK, DMF; (b) KOH, H2O; (c) PCC,
dry DCM; (d) 8(a-c), NaBH4, CH3OH; (e) NBS, Ph3P, BPO, dry DCM; (f)
phthalimide potassium, DMF; (g) N2H4·H2O, C2H5OH.

2.3. Preliminary screening for TGF-β signaling inhibitors
As shown in Table 1, the abilities of ten compounds to inhibit TGF-β signaling were
initially measured by using mink lung epithelial cells (MLEC) stably transfected with
a TGF-β-responsive plasminogen activator inhibitor-1 promoter-luciferase construct
[15]. Briefly, MLECs were stably transfected with an expression construct containing
a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Upon the
addition of TGF-β, a dose-dependent increase in luciferase activity is induced, which
can
be
used
to
identify
inhibitory.
In
parallel
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assays were
carried out to identify any compounds that caused marked cytotoxicity; 7 of the 10
compounds did not cause significant cell death up to 50 µM. Of these 7 compounds,
CJJ300, CJJ302, CJJ323 and CJJ329 inhibited TGF-β-induced luciferase activity
without causing significant cytotoxicity, indicating that these four compounds can
inhibit TGF-β signaling. Among these 4 compounds, the most potent inhibitor was
CJJ300, with an IC50 of 5.3 µM.

Table 1. The initial screening of TGF-β signaling pathway inhibitors.
Compound Compound
R
Cytotoxicity Inhibition
of
No.
ID
at 50 µM
TGF-β1-induced
luciferase IC50 (μM)a
5.3 µM
4a
4b
4c
CJJ-300
CJJ-323
CJJ-296
No
No
No
>50.0 µM
No
significant
significant
significant
inhibition
No
4d
4e
CJJ-320
CJJ-313
No
No
inhibition
No
inhibition
31.9µM
4f
CJJ-302
CJJ-329
No
No
4g
38.1µM
9a
9b
9c
CJJ-273
LJY-1
LJY-2
Yes
Yes
Yes
N/Tb
N/T
N/T
NCI-48455
[12]
No
18.0 µM
amean of 2 independent experiments; bN/T = Not Tested.

However, as this luciferase assay provides only an indirect measure of the TGF-β
signaling pathway, it is not possible to ascertain whether the compounds inhibited the
formation of TβR-I-TβR-II dimers or inhibited downstream components of the
pathway, such as receptor kinases. Therefore, we next investigated whether these 4
compounds could disrupt the TGF-β1-induced formation of TβR-I-TβR-II dimers,
using a commercially available cell-based dimerization assay (DiscoverX, USA).
Only CJJ300 was able to inhibit receptor dimerization (IC50 23.6 ± 5.8 µM; Figure 3),
indicating that CJJ300 inhibited the TGF-β pathway by disturbing the formation of the
TGF-β1-TβR-I-TβR-II complex, whilst the other compounds affected other
components of the signaling pathway. A known TβR-I kinase inhibitor, SB431542,
was used as a negative control and this compound did not affect receptor dimerization
(data not shown).
Fig 3. Antagonist dose-response curve to show CJJ300 inhibiting the formation of
TβR-I-TβR-II dimers. Cells were pretreated with increasing concentrations of CJJ300
for 30 minutes before stimulation with the TGF-β1. The potency of the antagonism
was calculated as a percentage of the maximum effect induced by the EC80
concentration of TGF-β1 (set at 100%). Basal activity of the cells was set at 0%. A
representative curve is shown. The results to demonstrate CJJ300 inhibition of dimer
formation (IC50 23.6± 5.8 µM) were obtained from four independent experiments

performed in triplicate.
2.4. The effect of CJJ300 on downstream TGF-β signaling and the induction of
EMT
To confirm that the TGF-β signaling pathway would be deregulated by disturbing
TβR-I-TβR-II dimerization, the effect of CJJ300 on critical TGF-β signaling
mediators such as phosphorylated Smad2/3, Akt and Erk1/2 (P-Smad2/Smad3, P-Akt
and P-Erk1/2 respectively) in human A549 lung epithelial cells was examined by
Western blotting. Initially, the potential cytotoxic effect of CJJ300 against A549 lung
epithelial cells was examined by using MTT assays and the results showed that
treatment of cells with CJJ300 (20 - 80 µM) for up to 48h, did not result in any
cytotoxicity (data not shown). As shown in Fig 4A-D, the treatment of A549 cells
with TGF-β1 (5ng/mL) for 4h led to a significant increase in the levels of
P-Smad2/Smad3, P-Erk1/2 and P-Akt, while pretreatment of cells with CJJ300 (40
µM) significantly attenuated these responses. When compared with the control group,
treatment of cells with CJJ300 alone had little effect (P>0.05) on the phosphorylation
level of the mediators in the cells except for P-Smad (P<0.05), which may be due to
the inhibition of the endogenous TGF-β activity by CJJ300. Taken together, these
results demonstrated that CJJ300 inhibited the TGF-β1-induced activation of
intracellular Smad and non-Smad signaling pathways.

Fig 4. CJJ-300 inhibits TGF-β1-induced signaling and EMT in A549 cells. (A)
Western blot results of P-Smad2/3, P-Erk1/2 and P-Akt proteins in A549 cells treated
with TGF-β1 (4 h) alone or in combination with CJJ-300 (40 µM). (B-D) Expression
levels of P-Smad2/3, P-Erk1/2 and P-Akt relative to internal control β-actin. (E)
Western blot results of the indicated EMT-related proteins in A549 cells treated with
TGF-β1 alone or combined with CJJ-300 (20,40,80 µM). (F-H) Expression levels of
Fibronectin, MMP-2 and α-SMA relative to internal control β-actin . #P<0.05,
##P<0.01 vs. the control group; *P<0.05, **P<0.01, ***P<0.001,****P<0.0001 vs.
the TGF-β1 group.
EMT stimulated by TGF-β1 enables epithelial cells to acquire a more mesenchymal
phenotype via the induction of EMT. One hallmark of EMT is the increased
production of extracellular matrix (ECM) and pro-fibrotic components, such as
fibronectin, alpha-smooth muscle actin (α-SMA) and matrix metalloproteinases
(MMPs), which enables transitioned cells to exhibit invasive and motile phenotypes.
Therefore, the effect of CJJ300 on the expression of EMT-associated proteins,
including fibronectin, α-SMA and MMP-2, in A549 cells in response to TGF-β1 was
evaluated by Western blotting. As shown in Fig 4E-H, the levels of these proteins
were significantly increased in response to TGF-β1 stimulation, and were effectively
down regulated by the pretreatment of CJJ300 in a concentration-dependent manner.
2.5. CJJ-300 suppresses TGF-β induced cell migration in A549 cells
To investigate the effects of CJJ300 on TGF-β1-induced migration in A549 cells,
scratch wound healing and transwell assays were performed to determine the rate of
wound closure and the number of migrating cells, respectively.
In the scratch wound healing assay the percentages of scratched area at 24 h and 48 h
compared to that of 0 h were used to indicate the rate of cell migration. As shown in
the Fig 5A-C, the treatment of TGF-β1 promoted the scratched wound closure of
A549 cells. Such TGF-β1 induced cell migration were obviously delayed by the
pretreatment of CJJ-300 at 40 or 80 µM at 24 h, and the retardation effect of CJJ-300
on the migration of the cells were further observed in a dose-depended manners at 48

h.
Similar results were obtained in transwell migration assays. The number of cells
passing through the membrane was significantly increased by TGF-β1 treatment.
CJJ-300 (20, 40, 80 µM) significantly reduced the number of migrated cells in a
concentration-dependent manner.

Fig 5. CJJ-300 suppressed TGF-β induced cell migration in A549 cells. (A) Effect of
CJJ-300 on TGF-β1-induced A549 cell migration in a scratch-wound healing-based
migration assay. (B) The scratched wound closure of cells at 24 hours in the
scratch-wound healing assay. (C) The scratched wound closure of cells at 48 hours in
the scratch-wound healing assay. (D) Effect of CJJ-300 on TGF-β1-induced A549 cell
migration in a transwell-based migration assay. (E) Number of migration cells in each
group in the transwell assay. ###P<0.001, ####P<0.0001 vs. the control group;
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the TGF-β1 group.
3. Conclusion
In the present study, a group of NCI-48455 structural analogs were designed and
synthesized. One compound, CJJ300, was shown to inhibit TGF-β signaling by
interfering with the formation of TβR-I-TβR-II dimers. Downstream signaling was
subsequently inhibited because TGF-β1-induced the phosphorylation of Smad2/3, Akt
and Erk1/2, was inhibited by CJJ300. Similarly, the up-regulation in expression of the
EMT markers fibronectin, α-SMA and MMP-2 was also attenuated by CJJ300 and
this resulted in an inhibition of cell migration. The mechanism of action of CJJ300 is
different from most known TGF-β signaling inhibitors, such as SB431542, which is a
typical TGF-β receptor I kinase inhibitor. To the best of our knowledge, the current
study is the first report of an organic molecule that can inhibit TGF-β1-induced EMT
by disturbing the protein-protein interactions of the TGF-β receptors. Therefore,
CJJ300 can be an important lead compound with which to study the TGF-β signaling
pathway and to develop TGF-β antagonists.
4. Experimental
4.1. Molecular docking
The TβR-II extracellular domain was separated from the crystal structure of the

complex of TGF-β-TβR-II extracellular domain (PDB ID 1KTZ) as the receptor for
the molecular docking study. The active site (composed by Leu27, Phe30, Asp32,
Arg34, Ile50 and Ile53) was identified by the residues that directly contact with
TGF-β. GOLD (v 5.2; Genetic Optimization for Ligand Docking) was performed for
molecular docking [16]. In the current study, each compound was docked 10 times,
starting each time from a different random population of ligand orientations. The
default automatic genetic algorithm parameter settings was used, and the GoldScore
was performed as the ranking score. All torsion angles in each compound were
allowed to rotate freely.
4.2. Chemistry
All the required chemicals were purchased from commercial sources and used without
further purification. TLC was carried out using silica gel 60 pre-coated aluminium
plates (0.20mm thickness) from Macherey-Nagel, with visualization by UV light
(254nm). Flash chromatography was performed on silica gel (particle size 80-100 µm)
1
from Fischer Chemicals. H and 13C NMR spectra were obtained from Bruker
AVANCE III 400 spectrometers. The chemical shifts, given as δ values, are quoted in
1
13
parts per million (ppm); H and C NMR chemical shifts were measured relative to
internal tetramethylsilane; apparent coupling constants (absolute values), J, are
measured in Hertz and multiplicities quoted as singlet (s), doublet (d), triplet (t),
quartet (q) or combinations thereof as appropriate. HRMS spectra were obtained on a
Thermo Q-Exactive Orbitrap mass spectrometer. IR spectra were obtained from the
solid phase using Bruker Tensor 27 spectrometer. Melting points were determined on
a WRS-1B apparatus without corrected.
4.2.1. 2,4,6-Tribromomethyl-1,3,5-trimethylbenzene (2)
To a mixture of 1,3,5-trimethylbenzene (0.02 mol, 2.40 g), paraformaldehyde (0.13
mol, 3.95 g) and zinc bromide (0.02 mol, 4.50 g) in acetic acid (100 mL) was added a
solution of HBr in acetic acid (14 mL). The reaction mixture was stirred at 95°C

overnight, and then poured into water (20 mL). The resulting precipitation was
filtered and dried to give compound 2 as a white solid (7.59 g, 95%); m.p.
186.1-187.0°C (lit., 186°C) [13]; IR (KBr): ν 3007 (=CH), 1565 (C=C), 1446 (C=C),
572 (C-Br); 1HNMR (400MHz, CDCl3): δ 4.58 (s, 6H, CH2), 2.47 (s, 9H, CH3)。
4.2.2. 3-Nitrobenzamide (6)
HOBT (3.27 g, 24.20 mmol) and EDCI (4.64 g, 24.20 mmol) was added to a stirred
solution of 3-nitrobenzoic acid (2.69 g, 16.12 mmol) and DIPEA (10.99 mL, 64.60
mmol) in CH3CN (100 mL) at room temperature. After stirring for 10mins,
ammonium carbonate (9.29 g, 96.80 mmol) was added and the resulting solution
stirred at room temperature overnight. The reaction mixture was concentrated and the
residue was suspended in H2O (200 mL). The resulting suspension was sonicated for
10mins, then the solid filtered to give compound 6 as a white solid (1.82 g, 70%), m.p.
142°C (lit., 141-143°C) [17]; IR (KBr): ν 3344, 3177, 1688, 1622, 1527, 1350 cm−1;
1HNMR (400MHz, CDCl3) δ 8.68 (t, J = 1.9, 1H, Ar-H), 8.42 (ddd, J1 = 1.0, J2 = 2.2,
J3 = 8.2, 1H, Ar-H); 8.23 (dt, J1 = 1.3, J2 = 7.7, 1H, Ar-H), 7.70 (t, J = 8.0, 1H, Ar-H),
6.29 (brs, 1H, CONH2), 6.01 (brs, 1H, CONH2); 13CNMR (100MHz, CDCl3): δ 166.7
(C=O), 148.3 (C), 134.9 (C), 133.5 (CH), 130.0 (CH), 126.6 (CH), 122.3 (CH).
4.2.3. 3-Aminobenzamide (3g)
3-Nitrobenzamide 6 (1.34 g, 8.09 mmol) was dissolved in isopropanol (13 mL) and
Pd/C (215.10 mg) and 80% hydrazine hydrate (1.01 mL, 16.19 mmol) added to the
reaction mixture at 80°C. The resulting solution was stirred at 80°C for 1h and the
reaction mixture filtered, then the filtrate concentrated to give compound 3g as a
white solid (0.98 g, 89%); m.p. 116ºC (lit., 117-117.5ºC) [18]; IR (KBr): ν 3430, 3465,
1
3382, 3197, 1689, 1579, 1466 cm−1; HNMR (400MHz, DMSO-d6): δ 7.71 (brs, 1H,
CONH2), 7.12 (brs, 1H, CONH2), 7.07-7.04 (m, 2H, Ar-H), 6.98 (d, J = 7.7, 1H,
Ar-H), 6.67 (d, J = 7.7, 1H, Ar-H), 5.18 (s, 2H, NH2); 13CNMR (100MHz, DMSO-d6):
δ 169.2 (C=O), 149.0 (C), 135.7 (C), 129.0 (CH), 116.9 (CH), 115.1 (CH), 113.6
(CH); ES-MS 137.1 (M + H)+; HRMS calcd for C7H9N2O+ 137.0715, found

137.0717.
4.2.4. General preparation of compound 4a-g
To a mixture of 1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene (1.0 mmol equiv)
and K2CO3 (3.2 mmol equiv) in CH3CN or DMF (30 mL) was added appropriate
aniline 3a-g (4.0 mmol equiv). The reaction mixture was stirred at 85°C for 36 h, and
then poured into water (30 mL). The precipitate was filtered and purified by
recrystallization or chromatography.
4.2.4.1. 1,3,5-Tris(phenylaminomethyl)-2,4,6-trimethylbenzene (4a, CJJ-300)
Compound 4a was synthesized from compound 2 and 3a. The crude compound was
purified by recrystallization with CHCl3/C2H5OH. White solid (427 mg, 79%); m.p.
1
206-208°C; IR (KBr): ν 3405, 1600, 1499 cm−1; HNMR (400MHz, CDCl3): δ
7.30-7.26 (m, 6H, Ar-H), 6.80 (t, J = 7.3, 3H, Ar-H), 6.74 (d, J = 7.9, 6H, Ar-H), 4.31
(s, 6H, CH2), 2.47 (s, 9H, CH3); 13CNMR (100MHz, CDCl3): δ 148.1 (C), 137.1 (C),
134.0 (C), 129.4 (CH), 117.7 (CH), 112.7 (CH), 43.4 (CH2), 15.8 (CH3); ES-MS
436.3 (M + H)+; HRMS calcd for C30H34N3 436.2753, found 436.2754.
+
4.2.4.2. 1,3,5-Tris[(4-acetylphenyl)aminomethyl]-2,4,6-trimethylbenzene (4b,
CJJ-323)
Compound 4b was synthesized from compound 2 and 3b. The crude compound was
purified by recrystallization with CHCl3/C2H5OH. White solid (392 mg, 56%);
1
m.p.197.9-199.5°C; IR (KBr): ν 3341, 1657, 1595, 1526 cm−1; HNMR (400MHz,
CDCl3): δ 7.88 (d, J = 8.2, 6H, Ar-H), 6.69 (d, J = 8.3, 6H, Ar-H), 4.36 (s, 6H, CH2),
2.53 (s, 9H, CH3CO), 2.43 (s, 9H, CH3); 13CNMR (100MHz, CDCl3): δ 196.3 (C=O),
151.5 (C), 137.5 (C), 133.2 (C), 130.9 (CH), 127.4 (C), 111.6 (CH), 43.0 (CH2), 26.1
(CH3), 16.0 (CH3); ES-MS 584.3 (M + Na)+; HRMS calcd for C36H39N3O3Na+

584.2889, found 584.2890.
4.2.4.3. 1,3,5-Tris[(3-acetylphenyl)aminomethyl]-2,4,6-trimethylbenzene (4c,
CJJ-296)
Compound 4c was synthesized from compound 2 and 3c. The crude compound was
purified by recrystallization with CHCl3/C2H5OH. Pale white solid (608 mg, 89%);
1
m.p.186.8-187.6°C; IR (KBr): ν 3376, 1677, 1601, 1483, 1320 cm−1; HNMR
(400MHz, CDCl3): δ 7.40-7.26 (m, 9H, Ar-H), 6.90 (dt, J1 = 7.0, J2 = 2.3, 3H, Ar-H),
4.34 (s, 6H, CH2), 2.61 (s, 9H, CH3CO), 2.44 (s, 9H, CH3); 13CNMR (100MHz,
CDCl3): δ 198.7 (C=O), 148.4 (C), 138.3 (C), 137.2 (C), 133.7 (CH), 129.4 (CH),
118.2 (C), 117.7 (CH), 110.9 (CH), 43.4 (CH2), 26.8 (CH3), 15.8 (CH3); ES-MS 562.3
(M + H)+; HRMS calcd for C36H39N3O3 562.3070, found 562.3070.
+
4.2.4.4.
1,3,5-Tris[[4-(methyloxycarbonyl)phenyl]aminomethyl]-2,4,6-trimethylbenzene
(4d, CJJ-320)
Compound 4d was synthesized from compound 2 and 3d. The crude compound was
purified by column chromatography on silica gel using EtOAc/ Petroleum ether (1/1,
v/v) as eluent. Yellow solid (336 mg, 45%); m.p.143.5-144.9°C; IR (KBr): ν 3362,
1
1698, 1602, 1526 cm−1; HNMR (400MHz, CDCl3): δ 7.92 (d, J = 8.5, 6H, Ar-H),
6.67 (d, J = 8.5, 6H, Ar-H), 4.33 (s, 6H, CH2), 3.88 (s, 9H, CH3O), 2.42 (s, 9H, CH3);
13CNMR (100MHz, CDCl3): δ 167.2 (C=O), 151.5 (C), 137.4 (C), 133.3 (C), 131.6
(CH), 118.9 (C), 111.5 (CH), 51.7 (CH3), 43.0 (CH2), 15.9 (CH3); ES-MS 632.3 (M +
Na)+; HRMS calcd for C36H39N3O6Na+ 632.2737, found 632.2739.
4.2.4.5.
1,3,5-Tris[[3-(methyloxycarbonyl)phenyl]aminomethyl]-2,4,6-trimethylbenzene
(4e, CJJ-313)

Compound 4e was synthesized from compound 2 and 3e. The crude compound was
purified by recrystallization with CH3OH. White solid (327 mg, 43%); m.p.
159.6-162.1°C; IR (KBr): ν 3373, 1710, 1602, 1511 cm−1; 1HNMR (400MHz, CDCl3):
δ 7.45 (d, J = 7.6, 3H, Ar-H), 7.40 (s, 3H, Ar-H), 7.32-7.28 (m, 3H, Ar-H), 6.91 (d, J =
13
7.5, 3H, Ar-H), 4.33 (s, 6H, CH2), 3.93 (s, 9H, CH3O), 2.44 (s, 9H, CH3); CNMR
(100MHz, CDCl3): δ 167.5 (C=O), 147.9 (C), 137.2 (C), 133.6 (C), 131.2 (CH), 129.3
(CH), 119.0 (C), 117.5 (CH), 113.1 (CH), 52.1 (CH3), 43.6 (CH2), 15.9 (CH3); ES-MS
+
610.3 (M + H)+; HRMS calcd for C36H40N3O6 610.2917, found 610.2910.
4.2.4.6
1,3,5-Tris[[4-(aminocarbonyl)phenyl]aminomethyl]-2,4,6-trimethylbenzene (4f,
CJJ302)
Compound 4f was synthesized from compound 2 and 3f. The crude compound was
purified by recrystallization with C2H5OH/H2O. White solid (513 mg, 73%); m.p.
1
249.5°C; IR (KBr): ν 3401, 3320, 3190, 1648, 1604, 1524 cm−1; HNMR (400MHz,
DMSO-d6): δ 7.69 (d, J = 8.3, 6H, Ar-H), 7.59 (brs, 3H, CONH2), 6.90 (brs, 3H,
CONH2), 6.73 (d, J = 8.4, 6H, Ar-H), 5.86 (brs, 3H, NH), 4.23 (s, 6H, CH2), 2.35 (s,
9H, CH3); 13CNMR (100MHz, DMSO-d6): δ 168.5 (C=O), 151.8 (C), 137.0 (C),
133.5 (C), 129.5 (CH), 121.7 (C), 111.4 (CH), 42.8 (CH2), 16.1 (CH3); ES-MS 632.3
(M + Na)+; HRMS calcd for C36H39N3O6Na+ 632.2737, found 632.2739.
4.2.4.7.
1,3,5-Tris[[3-(aminocarbonyl)phenyl]aminomethyl]-2,4,6-trimethylbenzene
(4g, CJJ-329)
Compound 4g was synthesized from compound 2 and 3g. The crude compound was
purified by recrystallization with C2H5OH/H2O. White solid (685 mg, 97%); m.p.
1
177.0-178.7°C; IR (KBr): ν 3402, 3346, 3187, 1652, 1604, 1581 cm−1; HNMR

(400MHz, DMSO-d6): δ 7.83 (s, 3H, Ar-H), 7.23-7.14 (m, 9H, Ar-H and NH2), 7.09
(d, J = 7.5, 3H, Ar-H), 6.89 (dd, J = 7.9, 2.4, 3H, Ar-H), 5.45 (t, J = 4.4, 3H, NH),
4.21 (d, J = 4.4, 6H, CH2), 2.38 (s, 9H, CH3); 13CNMR (100MHz, DMSO-d6): δ 169.2
(C=O), 149.4 (C), 136.9 (C), 135.6 (C), 133.7 (CH), 129.0 (C), 116.0 (CH), 115.5
(CH), 111.1 (CH), 43.2 (CH2), 16.0 (CH3); ES-MS 565.3 (M + H)+; HRMS calcd for
+
C33H37N6O3 565.2927, found 565.2923.
4.2.5. 2,4,6-trimethyl-1,3,5-benzenetricarbaldehyde (7)
To a solution of 1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene (8.0 g, 20.06 mmol)
in DMF (70 mL) was added KOAc (11.8 g, 120.33 mol). The reaction mixture was
heated to 70°C and stirred mechanically for 19 h. After cooling down, the precipitate
was filtered and the filtrate poured into CH
2
Cl (300 mL). The organic layer was
2
washed with brine then with H2O, dried with Na2SO4, filtered and concentrated to
give the crude product as a light yellow solid (6.62 g, 98%), which was used as such
in the next step.
To a solution of crude 1,3,5-tris(acetoxymethyl)-2,4,6-trimethybenzene (6.0 g, 17.85
mmol) in H2O (100 mL) was added KOH (10.81 g, 192.77 mol). The reaction mixture
was heated to reflux for 24 h. After cooling down, the precipitate was filtered and
washed with H2O, acetone to give the crude product as a light yellow solid (2.95g,
79%), which was used as such in the next step.
To a solution of PCC (20.81 g, 96.54 mmol) in dry CH2Cl2 (300 mL) was added celite
(8.0 g) and 4Å molecular sieves (8.0 g), followed by crude 1,3,5-tris(hydroxymethyl)-
2,4,6-trimethybenzene (2.9 g, 13.79 mmol). The reaction mixture was stirred
vigorously at room temperature for 2 d, filtered, and washed with CH2Cl2. The filtrate
was concentrate and the residue purified by column chromatography on silica gel
using EtOAc/Petro (1/9, v/v) to give compound 7 as a white solid (1.0 g, 34%). m.p.
1
174°C (dec) (lit., 169-170°C (dec)) [19], IR (KBr): ν 1692, 1557, 1448 cm−1; H

NMR (400MHz, CDCl3): δ 10.65 (s, 3H, CHO), 2.67 (s, 9H, CH3); 13C NMR
(100MHz, CDCl3): δ 194.2 (C=O), 143.1 (C), 135.0 (C), 16.2 (CH3).
4.2.6. General preparation of compound 8b-c
To a solution of methyl 4-methylbenzoate or methyl 3-methylbenzoate (69 mmol) in
CCl4 (70 mL) was added NBS (76 mmol) and benzoyl peroxide (2.3 mmol) and the
resulting solution was refluxed for 6h under lamp. After cooling to room temperature,
the precipitate was filtered. The filtrate was washed with sat. NaHCO3, dried with
Na2SO4 and concentrated to give yellow oil. This material was added to DMF (60 mL)
and potassium phthalimide (124 mmol) was added. The reaction mixture was stirred
at 90°C for 10 h and poured into H2O (600 mL). The precipitate was filtered and dried
to give a light yellow solid. This intermediate was added into C2H5OH (200 mL) and
80% hydrazine hydrate (207 mmol) was added. The mixture was refluxed for 9h.
After cooling to room temperature, the precipitate was filtered and the filtrate was
concentrated. The residue was purified by column chromatography on silica gel using
CH2Cl2/CH3OH (100/1, v/v).
4.2.6.1. Methyl 4-(aminomethyl)benzoate (8b)
Compound 8b was synthesized from compound 10a. Light yellow solid (6.9 g, 61%);
1
m.p. 104-106°C; IR (KBr): ν 3367, 1723, 1568, 1475 cm−1; HNMR (400MHz,
DMSO-d6): δ 8.02 (d, J = 8.3 Hz, 2H, Ar-H), 7.40 (d, J = 8.4 Hz, 2H, Ar-H), 3.95 (s,
2H, CH2N), 3.93 (s, 3H, CH3O), 1.67 (brs, 2H, NH2); 13CNMR (100MHz, DMSO-d6):
δ 167.0 (C=O), 148.3 (C), 129.9 (CH), 128.7 (C), 126.9 (CH), 52.0 (CH2), 46.1 (CH3);
+
ES-MS 166.1 (M + H)+; HRMS calcd for C9H12NO2 166.0868, found 166.0870.
4.2.6.2. Methyl 3-(aminomethyl)benzoate (8c)
Compound 8c was synthesized from compound 10b. Light yellow solid (6.8 g, 60%),

1
m.p. 93-95°C; IR (KBr): ν 3320, 1725, 1597, 1468 cm−1; HNMR (400MHz,
DMSO-d6): δ 8.02 (s, 1H, Ar-H), 7.93 (d, J = 7.8, 1H, Ar-H), 7.54 (d, J = 7.5, 1H,
Ar-H), 7.42 (t, J = 7.7, 1H, Ar-H), 3.95 (s, 2H, CH2N), 3.93 (s, 3H, CH3O), 2.48 (brs,
2H, NH2); 13CNMR (100MHz, DMSO-d6): δ 167.1 (C=O), 142.7 (C), 131.9 (CH),
130.4 (C), 128.6 (CH), 128.3 (CH), 128.2 (CH), 52.1 (CH2), 45.9 (CH3); ES-MS
+
166.1 (M + H)+; HRMS calcd for C9H12NO2 166.0868, found 166.0867.
4.2.7. General preparation of compound 9a-c
To a solution of appropriate benzylamine (3.3 mmol equiv) in CH3OH (50 mL) was
added 2,4,6-trimethyl-1,3,5-benzenetricarbaldehyde (1.0 mmol equiv) and the
resulting solution was refluxed for 1h. After cooling to 0°C, sodium borohydride (3.5
mmol equiv) was added to the reaction mixture and refluxed for 1h. The reaction
solution was then quenched with H2O (2.0 mL), extracted with CH
Na SO , filtered and concentrated. The residue was purified by column
chromatography on silica gel.
2
Cl and dried with
2
2
4
4.2.7.1. 1,3,5-Tris(benzylaminomethyl)-2,4,6-trimethylbenzene (9a, CJJ-273)
Compound 9a was synthesized from compound 7 and 8a. The crude product was
purified by column chromatography on silica gel using EtOAc/CH3OH (40/1, v/v) as
eluent to compound 9a as a yellow solid (780mg, 67%); m.p. 48-50°C; IR (KBr): ν
1
3404, 1601, 1494 cm−1; HNMR (400MHz, CDCl3): δ 7.44-7.33 (m, 12H, Ar-H),
7.32-7.26 (m, 3H, Ar-H), 3.91 (s, 6H, NCH2), 3.75 (s, 6H, NCH2), 2.33 (s, 9H, CH3);
13CNMR (100MHz, CDCl3): δ 140.4 (C), 135.5 (C), 134.9 (C), 128.4 (CH), 128.3
(CH), 127.0 (CH), 54.3 (CH2), 48.0 (CH2), 15.4 (CH3); ES-MS 478.3 (M + H)+;
+
HRMS calcd for C33H40N3 478.3222, found 478.3223.
4.2.7.2.
1,3,5-Tris[[4-(methyloxycarbonyl)benzyl]aminomethyl]-2,4,6-trimethylbenzene
(9b, LJY-1)

Compound 9b was synthesized from compound 7 and 8b. The crude product was
purified by column chromatography on silica gel using EtOAc as eluent to compound
9c as a yellow solid (1.10 g, 69%); m.p. 113-115°C; IR (KBr): ν 3309, 1709, 1609,
1505 cm−1; 1HNMR (400MHz, CDCl3): δ 8.03 (d, J = 8.2, 6H, Ar-H), 7.48 (d, J = 8.2,
6H, Ar-H), 3.94 (s, 15H, NCH2 and CH3O), 3.73 (s, 6H, NCH2), 2.32 (s, 9H, CH3);
13CNMR (100MHz, CDCl3): δ 167.1 (C=O), 145.6 (C), 135.6 (C), 134.7 (C), 129.7
(CH), 129.0 (C), 128.2 (CH), 54.0 (CH2), 52.1 (CH3), 48.0 (CH2), 15.5 (CH3);
+
ES-MS 652.3 (M + H)+; HRMS calcd for C39H46N3O6 652.3387, found 652.3384.
4.2.7.3.
1,3,5-Tris[[3-(methyloxycarbonyl)benzyl]aminomethyl]-2,4,6-trimethylbenzene
(9c, LJY-2)
Compound 9c was synthesized from compound 7 and 8c. The crude product was
purified by column chromatography on silica gel using EtOAc as eluent to compound
1
9c as yellow oil (1.47 g, 71%); IR (KBr): ν 3347, 1719, 1608, 1534 cm−1; HNMR
(400MHz, CDCl3): δ 8.06 (s, 3H, Ar-H), 7.95 (d, J = 7.7, 3H, Ar-H), 7.62 (d, J = 7.7,
3H, Ar-H), 7.43 (t, J = 7.7, 3H, Ar-H), 3.95 (s, 6H, NCH2), 3.94 (s, 9H, CH3O), 3.75
(s, 6H, NCH2), 2.32 (s, 9H, CH3); 13CNMR (100MHz, CDCl3): δ 167.2 (C=O), 140.7
(C), 135.6 (C), 134.7 (C), 132.9 (CH), 130.2 (C), 129.3 (CH), 128.5 (CH), 128.3 (CH),
53.9 (CH2), 52.1 (CH3), 48.0 (CH2), 15.5 (CH3); ES-MS 652.3 (M + H)+; HRMS
+
calcd for C39H46N3O6 652.3387, found 652.3389.
4.3. Cell lines and cell culture
Mink lung epithelial cells (MLEC) stably transfected with a TGF-β-responsive
plasminogen activator inhibitor-1 promoter-luciferase construct were a kind gift from
Dr D Rifkin. The human lung epithelial cell line A549 was obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were

cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine
serum (FBS), plus 1% penicillin and streptomycin (for A549 cells) or 200µg/mL
G418 (for MLEC), in a 37°C humidified environment containing 5% CO2.
4.4. Cells viability and toxicity assay
The cells were then treated with the test compounds, prior to the addition of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT; Calbiochem,
Merck Millipore; 2 mg/mL). Following 4 hours of incubation with MTT, 10% sodium
dodecyl sulphate (SDS) in 1 mM hydrochloric acid were added into the wells to
dissolve the blue crystal. After overnight incubation, cell viability was assessed by
measuring the absorbance at 575 nm with a reference wavelength of 650 nm. In the
subsequent luciferase assays, dimerization assays and migration assays, the effects of
CJJ300 on the cell viability were examined in parallel to exclude the compound
induced cell death.
4.5. In-vitro screening for inhibition of TGF-β signaling
The ability of selected compounds to inhibit TGF-β signaling was assayed using mink
lung epithelial cells stably transfected with a TGF-β-responsive plasminogen activator
inhibitor-1 promoter-luciferase construct [15]. Briefly, cells were seeded in 96 well
plates at a density of 1 × 104 cells per well in DMEM with 10% FBS, and allowed to
adhere to the surface for a period of 6 hours. The medium was removed and the cells
were rinsed with DMEM containing 1% FBS. The cells were then incubated with the
test compounds at various concentrations for 30 minutes before 0.05 ng/mL of
TGF-β1 (Peprotech) were added to the cells for an incubation of 16 hours in DMEM
containing 1% FBS. All the test compounds were first dissolved in DMSO to form 50
mmol/L stock solutions and then further diluted with DMEM containing 1% FBS to
desired working concentrations. The final concentration of DMSO was maintained at
0.1%. After 18 hours of incubation, the media were removed and the cells were