Synthesis and discovery of new compounds bearing coumarin scaffold for the treatment of pulmonary fibrosis

Article abstract of DOI:10.1016/j.ejmech.2019.111790

Idiopathic pulmonary fibrosis, characterized by excess accumulation of extracellular matrix, involved in many chronic diseases or injuries, threatens human health greatly. We have reported a series of compounds bearing coumarin scaffold which potently inhibited TGF-β-induced total collagen accumulation in NRK-49F cell line and migration of macrophages. Compound 9d also suppressed the TGF-β-induced protein expression of COL1A1, α-SMA, and p-Smad3 in vitro. Meanwhile, 9d at a dose of 100 mg/kg/day through oral administrations for 4 weeks effectively alleviated infiltration of inflammatory cells in lung tissue and fibrotic degree in bleomycin-induced pulmonary fibrosis model, which may related to its inhibition of TGF-β/Smad3 pathway and anti-inflammation efficacy. In addition, 9d demonstrated decent bioavailability (F = 39.88%) and suitable eliminated half-life time (T_1/2 = 13.09 h), suggesting that 9d could be a potential drug candidate for the treatment of fibrotic diseases.

Full text of DOI:10.1016/j.ejmech.2019.111790

Journal Pre-proof
Synthesis and discovery of new compounds bearing coumarin scaffold for the
treatment of pulmonary fibrosis
Dexin Deng, Heying Pei, Tingxuan Lan, Jiali Zhu, Minghai Tang, Linlin Xue, Zhuang
Yang, Shoujun Zheng, Haoyu Ye, Lijuan Chen
PII:
S0223-5234(19)30942-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.111790
EJMECH 111790
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 August 2019
Revised Date: 7 October 2019
Accepted Date: 14 October 2019
Please cite this article as: D. Deng, H. Pei, T. Lan, J. Zhu, M. Tang, L. Xue, Z. Yang, S. Zheng, H.
Ye, L. Chen, Synthesis and discovery of new compounds bearing coumarin scaffold for the treatment
of pulmonary fibrosis, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
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Graphical Abstract

Synthesis and discovery of new compounds bearing coumarin
scaffold for the treatment of pulmonary fibrosis
a, 1
a, 1
a
a
a
Dexin Deng , Heying Pei , Tingxuan Lan , Jiali Zhu , Minghai Tang , Linlin
a
a
a
a
a, *
Xue , Zhuang Yang , Shoujun Zheng , Haoyu Ye , Lijuan Chen
a. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University and Collaborative Innovation Center, Chengdu, 610041, China
*
Corresponding authors. Lab of Natural Product Drugs, State Key Laboratory of
Biotherapy and Cancer Center, West China Medical School, West China Hospital,
Sichuan University, Chengdu 610041, PR China.
1
Both authors contributed equally to this study.
Abstract:
Idiopathic pulmonary fibrosis, characterized by excess accumulation of
extracellular matrix, involved in many chronic diseases or injuries, threatens human
health greatly. We have reported a series of compounds bearing coumarin scaffold
which potently inhibited TGF-β-induced total collagen accumulation in NRK-49F
cell line and migration of macrophages. Compound 9d also suppressed the
TGF-β-induced protein expression of COL1A1, α-SMA, and p-Smad3 in vitro.
Meanwhile, 9d at a dose of 100 mg/kg/day through oral administrations for 4 weeks
effectively alleviated infiltration of inflammatory cells in lung tissue and fibrotic

degree in bleomycin-induced pulmonary fibrosis model, which may related to its
inhibition of TGF-β/Smad3 pathway and anti-inflammation efficacy. In addition, 9d
demonstrated decent bioavailability (F = 39.88%) and suitable eliminated half-life
time (T1/2 = 13.09 h), suggesting that 9d could be a potential drug candidate for the
treatment of fibrotic diseases.
Keywords:
Coumarins; Collagen accumulation; Anti-fibrosis; Anti-inflammatory;
Pulmonary fibrosis.
1
. Introduction
Idiopathic pulmonary fibrosis (IPF), characterized by exaggerated angiogenesis,
fibro proliferation, and deposition of extracellular matrix, is a fatal type of fibrotic
disease occurred in lung [1, 2]. Progressive scarring or fibrosis is confined to the
interstitium of the lungs, leading to loss of lung function and eventual death [3-6].
Inflammation, due to the systemic and local responses of living tissues towards injury,
also plays an important role in the development of fibrosis. Tissue damage recruits a
variety of immunocytes and activates a series of pathologic responses, leading from
tissue damage to inflammation, inflammation to fibrosis and fibrosis to function
dysfunction [7, 8]. Meanwhile, migration of macrophages (a kind of inflammatory
cell) driven by fibroblasts has shown to play a key part in evoking inflammatory

responses during tissue injury, leading to further fibrillation [9-11]. In a word, fibrosis
is aggravated by the feedback loop between fibroblasts activation and inflammation.
Figure 1. Structure of Pirfnidone, Nintedanib and other compounds bearing coumarin scaffold
At present, there are very few small molecule compounds approved for the
treatment of fibrotic diseases. So far, only Pirfenidone (1) and Nintedanib (2) have
been approved by FDA to treat for IPF. The major antifibrotic mechanism of
Pirfenidone is considered to inhibit the TGF-β pathway, and proinflammatory
cytokines such as TNF-α, IL-6, and IFN-γ have also been found to be inhibited [12].
Nintedanib, known as a small-molecule kinase inhibitor, mainly targets three
receptor classes (VGFR, PDGFR, FGFR) involved in blood vessel formation and
angiogenesis [13-15]. However, oral bioavailability of Nintedanib is approximately
1
2% in rats and 13% to 24% in monkeys [16]. Additional, some side effects have
been reported during the medication, such as diarrhea, nausea, and abdominal pain

[
16]. Hence, low bioavailability and adverse side effects derived by multiple targets
actually limit Nintedanib in further clinical treatment.
Coumarins, widely found in nature, known as a family of nature-occurring
lactones first isolated from Tonka beans in 1820. Over 1300 coumarins have been
identified, being reported to have multiple biological activities [17] treating diverse
ailments. Warfarin (3), a coumarins’ derivative, known as a traditional anticoagulant,
has been tested its treatment in idiopathic pulmonary fibrosis (Anticoagulant
Effectiveness in Idiopathic Pulmonary Fibrosis, ACE-IPF) trial [18] for the
prothrombotic state is 4 times higher in patients with IPF [19]. However, this
program has been terminated because of increased deaths in the Warfarin group.
Despite these unfavorable reports, using a new anticoagulant therapeutic strategy still
is a hopefully strategy to improve patient’s fibrotic states. Compound 4
(4-(1-piperazinyl) coumarins derivatives) also was proved to be a potent inhibitor of
human platelet aggregation (IC =37 nM) in vitro [20]. XLF-III-43 (5), a novel
5
0
coumarin-aspirin compound, inhibits the expressions of CML-AGE, TGF-β1, CTGF,
fibronectin and collagen IV in kidneys [21]. Coumarin derivatives or analogues are
also potential candidates for development of anti-inflammatory drugs [22].
Compound 6 possess obviously anti-inflammatory and analgesic activity in
Carrageenin-induced paw edema in rats [23].
Encouraged by the anticoagulant, anti- platelet aggregation, anti-fibrosis and
anti-inflammatory of coumarins, we designed a series of novel compounds using
hybriding coumarin skeleton (Figure 2) and hydrophobic group of Nintedanib to

improve the oral bioavailability and decrease the side effects. The anti-fibrosis and
anti-inflammatory mechanism involved in fibrosis of the optimized compounds were
also investigated both in vitro and in vivo.
Figure 2. Design a novel series of compounds with anti-fibrotic effects by hybriding coumarin
skeleton and hydrophobic group of Nintedanib.
2
. Results and discussion
2
.1 Chemistry
At the beginning, we consider the effect of the different hydrophobic group to its
anti-fibrotic biological activity. Compounds 5a-5e were prepared by the synthesis
method outlined in Scheme 1. 4-hydroxycoumarin (1) was reacted with TABA (
Tetrabutylammonium bromide) and P O in acetonitrile refluxing at 90 °C for 4 h,
2
5
giving the important intermediate 2 that allowed completely reaction with benzene-1,
-diamine to offer 3 in a good yield. 3 was treated with chloroacetyl chloride in dry
4

DMF at 0-5°C to provide 4, which was introduced with a variety of aliphatic amine
via substitution reaction to give the final compounds 5a-5e.
Scheme 1. Reagents and conditions: (a) TABA, P O , acetonitrile, 90 °C, 4 h. (b) Et N, EtOH,
2
5
3
7
0 °C, 2 h, rt, 4 h. (c) Chloroacetyl chloride, Et N, DMF, 0 °C, 3 h. (d) DMF, KI, rt,3 h. (e)
3
o
K CO , acetone, 65 C, overnight. (f) Pd/C, H , MeOH, rt, 3 h. (g) NaH, diethyl carbonate, 0℃ for
2
3
2
1
4
h, then heated to 100℃ for further 3 h. (h) Sodium nitrate, concentrated sulphuric acid, 0℃ for
h.
Then we initially researched the relationship between the position of the
substituents in coumarins and its anti-fibrotic effects. Methoxy was added to the C-8
of coumarin to give 5k-5o. 1-(2-hydroxy-4-methoxyphenyl)ethan-1-one) was reacted
with diethyl carbonate and sodium hydride in diethyl carbonate at 0-5 °C for 1 h, then
refluxed at 100 °C for 3 h to obtain Intermediate 2b. The following processes were
described as 5a-5e, eventually giving target compounds 5k-5o. To search the SAR of

electron-withdrawing substituent such as fluorine-containing and nitro group, other
two kinds of analogues 5f-5j and 5p-5t were synthesized also according to Scheme 1,
which was same as the procedures of 5a-5e.
Finally, in consideration of bioisosteres, we’d like to explore the importance of
linker N atom, which were subsequently substituted by O atom listed in Scheme 1.
Intermediate 6a (6b) was obtained by 2a (2b) and 4-nitrophenol refluxed in acetone
overnight. The following processes were described as 5a-5e. Eventually, we got new
series compounds 9a-9j.
2
2
.2. Biology
.2.1 Inhibition of total collagen accumulation on NRK-49F cells
Excessive extracellular matrix (ECM), which is mainly composed of collagen, is
a pathological marker of fibrotic disease, so detection of collagen synthesis is an
effective indicator for evaluating fibrotic diseases. According to our previous
research and other reports, rat fibroblast cells (NRK-49F) induced by TGF-β would
produce much extracellular collagen that similar to the feature of fibrosis in vivo
[
24-26].Thus, it has been recognized an effective and convenient method for
anti-fibrotic screening model in vitro.
Compounds 5a-5t were preliminarily screened for their effects on inhibiting
total collagen accumulation induced by TGF-β in the NRK-49F cell line at a
concentration of 10 µM and the cell survival rate was calculated by MTT assay in
NRK-49F cells for toxicity. Nintedanib was chosen as positive control.

Table 1 Collagen accumulation inhibition and cell survival rate of 5a-5t.
Inhibition%
10 µM
Survival%
Inhibition%
Survival%
Entry
R
Entry
R
@
@ 10 µM
@ 10 µM
@ 10 µM
N
N
26.82 ± 4.84
41.83 ± 3.22
80.21 ± 2.30
75.21 ± 7.07
12.43 ± 2.18
36.84 ± 2.82
34.54 ± 3.68
21.90 ± 2.13
11.33 ± 1.87
33.30 ± 6.33
87.54 ± 3.25
75.24 ± 1.21
91.48 ± 1.52
43.63 ± 1.96
62.04 ± 1.37
78.06 ± 1.50
72.35 ± 3.52
88.47 ± 4.22
67.70 ± 2.52
74.27 ± 3.84
5k
5l
N
N
80.20 ± 2.10
72.92 ± 1.27
72.71 ± 2.30
74.47 ± 1.63
15.72±3.46
22.26 ± 1.39
31.67 ± 2.34
28.73 ± 1.40
15.37 ± 0.84
17.90 ± 1.53
87.22 ± 2.37
89.13 ± 4.54
90.11 ± 2.31
41.33 ± 1.48
52.05 ± 2.17
32.06 ± 3.17
23.11 ± 2.65
17.82 ± 1.44
20.34 ± 2.60
45.32 ± 2.38
5
5
a
b
5
c
5m
5n
5o
5p
5q
5r
5s
5
d
5
e
N
N
N
N
5
f
5
5
g
h
5
i
5
j
5t
Nintedanib
9
5.47 ± 2.65
40.02 ± 2.46

Inhibitory against TGF-β-induced total collagen accumulation in NRK-49F cells at a
concentration of 10 µM. Cell survival rate is calculated by MTT assay. The results are the means
±
SD of at least three independent experiments.
As shown in Table 1, compound 5c possessed the good inhibition of collagen
accumulation with weaker toxicity in NRK-49F cells among 5a-5e. Meanwhile,
methoxy introducing to the C-8 position of coumarin was taken into account to give
compounds 5k-5o. The collagen inhibition of these compounds generally superior to
5
a-5b and have little effect on toxicity, indicating that electron-donating ability of the
substituents at C-8 may contribute to its bioactivity. Compounds 5d and 5n owned
piperidyl terminal also have good inhibition, but they showed toxicity in vitro to
some extent. As compounds bearing penta cyclic ring such as pyrrolidine (5e and 5o)
have little inhibitory activity. Compounds which have substitution of F atom on
benzene ring were designed initially to improve biological activity. In fact, although
the inhibitions of 5f-5j were not improved, they have little effects on toxicity
compared to 5a-5d. However, compounds owned electron-hogging substituents at
C-6 generally (5p-5t) have neither inhibition of collagen accumulation nor low
toxicity.
Though Nintedanib showed excellent inhibition of collagen accumulation in
NRK-49F cells, it was proved to inhibit collagen accumulation via killing cells,
which is consistent with the observed phenomenon under the microscope (Figure 3).
To reduce toxicity of compounds, bioisosteres, a principle and method widely used in

drug design, was then applied to subsequent compound designs to get compounds
a-9e. Specifically, the linker N atom was subsequently substituted by O atom and
9
hopefully to reduce its toxicity. As we expected, all compounds in Table 2 have little
effect on cell proliferation outstandingly compared to 5a-5t. Methoxy group at C-8
position has no influence on cell proliferation, which means the linker atom O plays
an important role in reducing the toxicity of compounds. Only compounds 9d and 9g
show best biological activity among them.
Table 2 Collagen accumulation inhibition and cell survival rate of 9a-9j
Inhibition%
10 µM
Survival%
Inhibition%
Survival%
Entry
R
Entry
R
@
@ 10 µM
@ 10 µM
@ 10 µM
N
N
24.04 ± 1.63
34.63 ± 2.37
75.32 ± 3.24
85.14 ± 1.32
18.80 ± 3.48
9f
9g
9h
9i
N
N
9
9
a
83.57 ± 3.26
97.05 ± 3.18
98.02 ± 2.43
93.62 ± 1.94
82.94 ± 2.36
65.36 ± 2.66
78.33 ± 2.17
32.60 ± 2.64
73.46 ± 2.54
8.07 ± 1.25
95.61 ± 5.37
96.54 ± 2.58
90.47 ± 8.99
90.43 ± 2.51
93.34 ± 3.29
40.02 ± 2.46
b
9
c
9
d
9
e
9j
Nintedanib
9
5.47 ± 2.65

Inhibitory against TGF-β-induced total collagen accumulation in NRK-49F cells at a
concentration of 10 µM. Cell survival rate is calculated by MTT assay. The results are the means
±
SD of at least three independent experiments.
The terminal of hydrophobic group affects the inhibition activity to some extent.
Compounds owned different structures which distinguished their biological activity
even they have the same hydrophobic group at terminal. However, compounds owned
pyrrolidine (5e, 5o, 9e, 9j) both have little inhibitory activity to collagen
accumulation. Modifications with electron-withdrawing group seemed unpopular.
More important, substitution of the linker N atom with O atom was successfully to
improve its inhibition to collagen accumulation while reducing compounds toxicity.
By means of a series of chemical modifications, we got four highly active and
low toxic compounds: 5c, 5k, 9d, and 9g. Taken together, the results above
encouraged us to further measure IC of the four compounds. Their screening data
5
0
were depicted in Table 3.
Table 3. IC values against TGF-β-induced total collagen accumulation in NRK-49F cells.
5
0
Compds
IC (µM)
Compds
5k
IC (µM)
5
0
50
5c
3.96 ± 0.31
4.31 ± 0.35
1.10 ± 0.13
4.35 ± 0.31
3.71 ± 0.22
9d
9g
Nintedanib
The results are the means ± SD of at least three independent experiments.

Figure 3. Picro-Sirius red (PSR) staining for the change of TGF-β-induced total collagen
accumulation in NRK-49F cells.
The visible mode of extracellular collagen deposition was established upon
Picro-Sirius red (PSR) staining to test inhibitory activity of 5c, 5k, 9d, and 9g against
TGF-β-induced total collagen accumulation. As shown in Figure 3, treatment with 5c,
5
k, 9d, and 9g alleviated the total collagen accumulation in a dose-dependent manner.
It was proved that the four compounds do have potential effect on inhibiting the
production of collagen in vitro.
2
.2.2 Inhibition by fibroblasts-driven macrophage migration in vitro.
Macrophages play an important role in the occurrence and progression of
fibrosis [27]. Macrophages are recruited to the lesions, and then differentiate to
proliferate to form local macrophages, which infiltrate and secrete differently.

Inflammatory mediators cause aggravation of inflammation and promote fibrosis, and
migration of macrophages is particularly important for the above processes. We
further developed in vitro model to study fibroblasts-induced macrophage migration
and activation.
Figure 4. Effect of compounds on fibroblasts-driven macrophage migration in vitro.
Representative images and Quantitative analysis (expressed as % macrophage migration) of
DAPI stained migrated mouse RAW macrophages (seeded on the upper chamber on the transwell
inserts) in response to non-cell treated control medium (control), conditioned medium from
NIN3T3 cells that are treated with TGF-β (5 ng/ml) with or without 1 µM different compounds in
the lower chamber.
As shown in Figure 4, the four compounds 5c, 5k, 9d and 9g had different
degrees of inhibition on macrophage migration at a concentration of 1 µM compared
to the control group treated with only TGF-β, with compound 9d being the most
active. Macrophage migration is important for the formation of local macrophages,

secretion of cytokines, promotion of local inflammatory response, and aggravation of
fibrosis. This experiment further confirmed that the four compounds have
anti-inflammatory and anti-fibrotic effects in vitro. In summary, compound 9d was
selected as a potential drug candidates for follow-up research.
2
.2.3 9d suppressed the TGF-β1-induced protein expression of COL1A1, α-SMA, and
p-Smad3 in vitro.
Figure 5. Protein expression levels of COL1A1, α-SMA, and Smad3 were probed through
western blot. NRK-49F cells were treated with/without TGF-β (5 ng/mL) and compound 9d (20
and 40 µΜ) for 24 h. The expression of α-SMA, CoL1A1 were examined by Western blot.
NRK-49F cells were treated with/without TGF-β (5 ng/mL) and compound 9d (20 and 40 µΜ)
for 30min, the expression of p-Smad3 was examined by Western blot. β-Actin was used as a
loading control.

Collagen type I alpha 1 (COL1A1), α-smooth muscle actin (α-SMA)
overexpressed in fibrotic diseases, which have been commonly considered as fibrotic
markers. To further investigate the anti-fibrotic activity of compound 9d, their
abilities of suppressing protein expression of COL1A1, α-SMA in vitro were
investigated. TGF-β binds to its receptor and recruits downstream Smad3 proteins to
cause the differentiation of quiescent fibroblasts into ECM-secreting myofibroblasts.
Therefore, TGF-β/Smad3 pathway has been the target of many attempts in fibrosis
[
28]. It was obviously in Figure 5 to observe that 9d could decrease TGF-β-induced
COL1A1 and α-SMA expression in NRK-49F cells, which means ECM deposition
was also reduced. In addition, much lower phosphorylation levels of Smad3 indicted
that 9d perhaps inhibited TGF-β/Smad3 pathway to ameliorate fibrotic degree.
2
.2.4 Pharmacokinetic experiment
In rat pharmacokinetic study (Table 4), 9d exhibited reasonable maximum
concentration (Cmax = 74.73 µg/L), acceptable oral clearance rate (Cl = 6.45 L/h/kg),
favorable half-life (t1/2 = 13.09 h) and good bioavailability (F = 39.88%) at an oral
dose of 10 mg/kg, which is much better than the oral bioavailability of Nintedanib
(F= 12% in rats).
Table 4. Pharmacokinetic parameters for compounds 9d in SD rat
9
d
Compound
intravenous injection
oral administration
10
dose (mg/kg)
5

Cmax (µg/L)
tmax (h)
316.44
2.68
74.73
0.63
t1/2 (h)
7.42
13.09
1104.95
39.88
AUC0-t (µg/L*h)
F%
1385.24
Data were mean concentrations in rat plasma (n = 5).
2
.2.5 Pulmonary fibrosis model induced by bleomycin in vivo
Pulmonary fibrosis is a long-term, progressive pulmonary disease accompanied
with excessive deposition of extracellular matrix, which was composed of collagen，
fibronectin and other inflammatory cells [29]. To search anti-fibrotic potencies of 9d
in vivo, bleomycin induced lung fibrosis model was employed [30, 31]. Nintedanib
was administered orally respectively at the dose of 50 mg/kg/day after the bleomycin
injection as a positive medicine. Compound 9d also was administered orally
respectively at the dose of 50 and 100 mg/kg/day.
Hydroxyproline is one of the biomarkers of collagen, so it was measured on the
1
1
4th and 28th day of administration. Hydroxyproline contents in model group at day
4 and 28 shows significantly increased compared to sham group, which means
bleomycin induced lung fibrosis model was successful established. Nintedanib
group distinguish with sham group at day 28, and long-term medication may reduce
the degree of fibrosis. The in vivo effect of inhibiting fibrosis between Compound 9d
and Nintedanib group is not obvious at the dose of 50 mg/kg/day. However,
Compound 9d at the dose of 100 mg/kg/day indicated a better therapeutic effect
because of its lower hydroxyproline content (Figure 6A). Additionally, the survival

rate of 9d at both doses (50 mg/kg/day and 100 mg/kg/day) was about 90%, which
was better than Nintedanib group and model group, suggesting 9d possessed lower
toxicity and better efficacy in the treatment of pulmonary fibrosis.
Figure 6. In vivo effects of compounds 9d on bleomycin-induced pulmonary fibrosis in mice.
C57BL/6 male mice were intratracheal instillated with BLM (2 U/kg) to induce pulmonary
fibrosis. Compounds 9d and Nintedanib were given orally once daily after BLM treatment and
lungs were harvested at day 14 and day 28 for the following analysis (n = 10 per group).
(A)Hydroxyproline contents in lung tissues of each group were measured at day 14 and 28 as
described in the methods (*P < 0.05, **P < 0.01). (B-F) 28 days after BLM instillation, lung

tissues of each group mice were measured by hematoxylin-eosin (H&E) (100X & 400X). (↓)
Bronchiolar epithelial cell shedding; (↓) Bronchioles and perivascular inflammatory cell
infiltration; (↓) Bronchial epithelial vacuolar degeneration. (G-I) Inflammatory cells accounted
for the white blood cell in broncho alveolar lavage fluid. (*p < 0.05) (G) Total white blood cells
in each group after administration of 28 days. (H) Neutrophils in WBC and (I) Lymphocytes in
WBC. (J) Structure of compound 9d.
The sections of the sham group displayed clear lung structures that the bronchial
ciliated epithelium was neatly arranged, and the bronchial and perivascular interstitial
were hardly edematous. In contrast, the alveolar structures were destroyed in the
model group. It is clear to observe the degeneration and necrosis of bronchial
epithelial cells (Figure 6C, ↓ & ↓). There are a number of inflammatory cell infiltration
in the alveolar interstitial, mainly lymphocytes and neutrophils (↓). Compound
treatments (Nintedanib group and 9d group) delayed the reduction of the number of
alveoli and destruction of alveolar structures. In the vicinity of the pulmonary artery,
the inflammatory cell infiltration in the drug treatment group was also reduced,
especially in 9d group at 100 mg/kg (Figure 6F).
Inflammation aggravating fibrosis in airway, and the airway wall is mainly
dominated by macrophages and lymphocytes, while macrophages and neutrophils
play important roles in the lumen. Inflammatory cells such as leukocytes,
lymphocytes and neutrophils were documented to act importantly in response to lung
fibrotic reactions. To evaluate effects of 9d on the airway inflammation in mice, we

checked the amount of inflammatory cells in broncho alveolar lavage fluid (BALF)
in day 28, which also determined the degree of inflammation caused by bleomycin.
Compared with the sham group (Figure 6G-I), the number of total white blood cells
9
in model group is 0.80 × 10 /L that consist of 13.41% for neutrophils, 62.28% for
lymphocytes. 9d can greatly reduce the percentage of inflammatory cells in broncho
9
alveolar lavage fluid at 100 mg/kg (0.30 × 10 /L for total WBC number, 15.49% for
neutrophils, and 44.71% for lymphocytes). Meantime, cell composition of
9
Nintedanib group at 50 mg/kg (0.41 × 10 /L for total WBC number, 19.70% for
neutrophils, and 37.55% for lymphocytes) was similar to compound 9d group at 100
mg/kg, which suggest that 9d could effectively reduce inflammatory cell infiltration
in BALF at high dose.
To sum up, compound 9d showed low toxicity and high survival rate in mice in
a 28-day dosing regimen. Additional, 9d obviously reduced collagen deposition in
lung tissue and ameliorate inflammatory condition by decrease total number of WBC
and other inflammatory cells (especially lymphocytes) in BALF in the lung fibrosis
model in vivo. Therefore, compound 9d can be used as a potential effective
compound for anti-fibrosis in vivo by oral administration.
3
. Conclusion
In this study, 30 novel compounds bearing coumarin scaffold were synthesized
and evaluated for their anti-fibrosis, anti-migration of macrophages in vitro. Among
them, compound 9d showed potent anti-fibrosis abilities in inhibiting TGF-β-induced

total collagen accumulation in NRK-49F cells with low toxicity and migration of
macrophages. 9d was also observed to suppress the TGF-β1-induced protein
expression of COL1A1, α-SMA, and p-Smad3 in vitro by western blot analysis,
which indicted TGF-β/Smad3 signaling pathway was inhibited to reduce ECM. In
addition, compound 9d attenuated bleomycin-induced pulmonary fibrosis in mice by
reducing hydroxyproline content and inflammatory cell infiltration in lung tissue,
which may related to its inhibition of TGF-β pathway and anti-inflammatory.
Considering of its extensive structural sources, good pharmacokinetic characteristics
(F = 39.88%), anti-fibrotic treatment and low toxicity in vivo, compound 9d could be
a promising, potential, orally active candidate for the treatment of fibrotic disease.
4
. Experimental
4
.1 Chemistry.
All the chemical solvents and reagents used in this study were analytically pure
without further purification and commercially available. TLC was performed on 0.20
mm silica gel 60 F254 plates (Qingdao Ocean Chemical Factory, Shandong, China).
Visualization of spots on TLC plates was done by UV light and I . NMR data were
2
1
13
measured for H at 400 MHz and for C at 101 MHz on a Bruker Avance 400
spectrometer (Bruker Company, Germany) using TMS as an internal standard. Mass
spectra (MS) were obtained by Q-TOF Priemier mass spectrometer (Micromass,
Manchester, UK).
Procedure for synthesis of compound 1b

A solution of 1-(2-hydroxy-4-methoxyphenyl)ethan-1-one(1.0 equiv) in
appropriate amount of diethylcarbonate was added dropwise to a oven-dried round
bottom flask containing sodium hydride (5.0 equiv) in equal amount of
diethylcarbonate at 0°C. After complete addition, the reaction mixture was stirred at
0
°C for 30 min and refluxed for three more hours at 100°C. After the reaction was
over monitored by TLC ,the reaction mixture was cooled to room temperature and ice
water was added slowly to quench the reaction mixture. Then use a large amount of
ether to wash away diethylcarbonate. After that, the aqueous phase was acidized to
pH 3-4 with diluted hydrochloric acid solution to form white solid. Finally the pure
light white precipitate was collected by filtration, washed with water, and dried in
1
vacuum to obtain 1b. H NMR (400 MHz, DMSO-d6) δ: 12.37 (s, 1H), 7.72 (d, J =
8
.6 Hz, 1H), 6.93 (m, 2H), 5.47 (s, 1H), 3.86 (s, 3H). MS (ESI), m/z: 193.04 [M +
+
H] .
Procedure for synthesis of compound 1c
The sodium nitrate (1.0 equiv) in concentrated sulfuric acid stired at 0°C, 1a (1.0
equiv) being added. Kept the temperature stirring for 1 h. Ended reaction via TLC
monitoring. Slowly added crushed ice under low temperature, then filtrated and dried
1
under vacuum to obtain 1c. H NMR (400 MHz, DMSO-d6) δ: 8.53 (d, J = 2.6 Hz,
1
H), 8.45 (dd, J = 9.1, 2.7 Hz, 1H), 7.63 (dd, J = 17.8, 8.2 Hz, 1H), 5.71 (s, 1H). MS
+
(
ESI), m/z: 208.02 [M + H] .
General procedure for synthesis of compounds 2a ,2b and 2c

An appropriate amount of 1a(1b, 1c) (1.0 equiv) was dissolved in freshly
distilled acetonitrile in a oven-dried round bottom flask at room temperature. TBAB
(1.5 equiv) was added randomly in one time. Then the reaction was moved to heating
and refluxed at 100°C. P O (2.4 equiv) was added in batches. Kept the temperature
2
5
for more than four hours and monitored the reaction by TLC. Then the solvent was
removed in vacuum. Added little amount of water to the oil-like compound. Then the
aqueous layer was extracted with ethyl acetate three times. The combined organic
layer was washed with brine and dried over Na SO Finally, the solvent was removed
2
4.
by evaporation to obtain light yellow solid 2a(2b, 2c).
1
4
-bromo-2H-chromen-2-one (2a). H NMR (400 MHz, DMSO-d ) δ: 6.9 (s, 1H),
6
+
7
.29 (m, 1H), 7.4 (m, 2H), 7.80 (m, 1H). MS (ESI), m/z: 224.95 [M + H] .
1
4
-bromo-7-methoxy-2H-chromen-2-one (2b). H NMR (400 MHz, DMSO) δ
7
.72 – 7.67 (m, 1H), 7.02 (dd, J = 7.5, 2.2 Hz, 2H), 6.87 (s, 1H), 3.88 (s, 3H). MS
+
(
ESI), m/z: 254.94 [M + H] .
1
4
-bromo-6-nitro-2H-chromen-2-one (2c). H NMR (400 MHz, DMSO) δ 8.63 (d,
.
J = 2.9 Hz, 1H), 8.34 (dd, J = 9.0, 2.9 Hz, 1H), 7.43 (d, J = 9.0 Hz, 1H) 7.43 (d, J =
+
9
.0 Hz, 1H). MS (ESI), m/z: 269.03 [M + H] .
General procedure for synthesis of compounds 3a, 3b, 3c and 3d
An appropriate amount of 2a(2b, 2c) (1.0 equiv) and p-phenylenediamine or
-fluorobenzene-1,4-diamine (1.1 equiv) were dissolved in ethanol in a oven-dried
2
round bottom flask. Et N (1.0 equiv) was added to the reaction mixture. The reaction
3
mixture was kept at 70°C for 2 h and then stired at room temperature for further 4 h.

The crude product was recrystallized from ethanol to obtain a white solid 3a(3b, 3c,
d).
3
1
4
-((4-aminophenyl)amino)-2H-chromen-2-one (3a). H NMR (400 MHz,
DMSO-d ) δ: 9.06 (s, 1H), 8.21 (d, J = 7.5 Hz, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.35 (m,
6
2
H), 6.99 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.5 Hz, 2H), 5.23 (s, 2H), 5.00 (s, 1H). MS
+
(
ESI), m/z: 253.09 [M + H] .
1
4
-((4-amino-3-fluorophenyl)amino)-2H-chromen-2-one (3b). H NMR (400
MHz, DMSO-d ) δ: 9.11 (s, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H),
6
7
5
.37 (dd, J = 10.9, 8.3 Hz, 2H), 7.04 (d, J = 12.0 Hz, 1H), 6.87 (m, 2H), 5.28 (s, 2H),
+
.07 (s, 1H). MS (ESI), m/z: 271.08 [M + H] .
1
4
-((4-aminophenyl)amino)-7-methoxy-2H-chromen-2-one (3c). H NMR (400
MHz, DMSO-d ) δ: 1H NMR (400 MHz, DMSO) δ 8.96 (s, 1H), 8.11 (d, J = 9.0 Hz,
6
1
H), 6.96 (m, 3H), 6.90 (d, J = 2.5 Hz, 1H), 6.64 (m, 2H), 5.21 (s, 2H), 4.87 (s, 1H),
+
3
.86 (s, 3H). MS (ESI), m/z: 283.10 [M + H] .
1
4
-((4-aminophenyl)amino)-6-nitro-2H-chromen-2-one (3d). H NMR (400 MHz,
DMSO-d ) δ: 9.52 (s, 1H), 9.28 (d, J = 2.1 Hz, 1H), 8.45 (dd, J = 9.1, 2.2 Hz, 1H),
6
7
2
.57 (d, J = 9.1 Hz, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 5.27 (s,
+
H), 5.12 (s, 1H). MS (ESI), m/z: 298.07 [M + H] .
General procedure for synthesis of compound 6
A solution of 2a (1.0 equiv), p-nitropheno (1.5 equiv) and K CO (1.8 equiv) in
2
3
acetone in a oven-dried round bottom flask was heated to 65°C and stirred for 24 h.
After the reaction was over monitored by TLC, the solvent was removed by

evaporation to get a kind of yellow solid. Water was added into the solid and then
extracted with ethyl acetate three times, washed with brine and dried over Na SO4.
2
The solvent was removed in vacuum to get crude yellow solid and then the residue
was purified by recrystallization from ethanol to obtain a light yellow product 6.
1
4
-(4-nitrophenoxy)-2H-chromen-2-one (6a). H NMR (400 MHz, DMSO-d ) δ:
6
8
.41 (m, 2H), 8.01 (dd, J = 7.9, 1.4 Hz, 1H), 7.77 (m, 1H), 7.68 (m, 2H), 7.48 (m,
+
2
H), 5.53 (s, 1H). MS (ESI), m/z: 284.05 [M + H] .
1
7
-methoxy-4-(4-nitrophenoxy)-2H-chromen-2-one (6b). H NMR (400 MHz,
DMSO-d ) δ: 8.39 (m, 2H), 7.90 (d, J = 8.8 Hz, 1H), 7.65 (m, 2H), 7.11 (d, J = 2.4
6
Hz, 1H), 7.05 (dd, J = 8.8, 2.4 Hz, 1H), 5.35 (s, 1H), 3.90 (s, 3H). MS (ESI), m/z:
+
3
14.02 [M + H] .
General procedure for synthesis of compound 7
Compound 6 was dissolved in methanol in a oven-dried three-neck round
bottom flask with little amount of palladium as a catalyst and hydrogenated for over
three hours at room temperature. After the reaction was over, the catalyst was filtered
off and the solvent was removed by evaporation to get a brown solid which was then
dried at 70 °C under vacuum to obtain 7.
1
4
-(4-aminophenoxy)-2H-chromen-2-one (7a). H NMR (400 MHz, DMSO-d ) δ:
6
8
2
2
.01 (d, J = 7.0 Hz, 1H), 7.74 (dd, J = 11.4, 4.2 Hz, 1H), 7.45 (dd, J = 12.7, 7.9 Hz,
H), 6.98 (d, J = 8.7 Hz, 2H), 6.70 (m, 2H), 5.26 (s, 2H), 5.18 (s, 1H). MS (ESI), m/z:
+
54.07 [M + H] .

1
4
-(4-aminophenoxy)-7-methoxy-2H-chromen-2-one (7b). H NMR (400 MHz,
DMSO-d ) δ: 7.90 (d, J = 8.8 Hz, 1H), 7.02 (m, 2H), 7.01 (m, 2H), 6.61 (m, 2H),
6
+
5
.24 (s, 2H), 5.01 (s, 1H), 3.95 (m, 3H). MS (ESI), m/z: 284.08 [M + H] .
General procedure for synthesis of compounds 4a, 4b, 4c, 4d and 8a, 8b.
Chloroacetyl chloride (1.2 equiv) was added dropwise to a mixture of the
appropriate aromatic amine (1.0 equiv) and triethylamine (1.5 equiv) in freshly
distilled DMF in a oven-dried round bottom flask at 0-5°C for 1 h and at room
temperature for further 2 h. It was then poured into crushed ice to form a white solid
which was filtered, washed with little amount of ethanol and dried under vacuum to
obtain 4a, 4b, 4c, 4d, 8, respectively.
1
2
-chloro-N-(4-((2-oxo-2H-chromen-4-yl)amino)phenyl)acetamide (4a). H NMR
(
400 MHz, DMSO-d ) δ: 10.44 (s, 1H), 9.28 (s, 1H), 8.24 (d, J = 7.7 Hz, 1H), 7.68
6
+
(
m, 3H), 7.38 (m, 4H), 5.24 (s, 1H), 4.28 (s, 2H). MS (ESI), m/z: 329.06 [M + H] .
2-chloro-N-(2-fluoro-4-((2-oxo-2H-chromen-4-yl)amino)phenyl)acetamide (4b).
1
H NMR (400 MHz, DMSO-d ) δ: 10.17 (s, 1H), 9.33 (s, 1H), 8.21 (d, J = 7.6 Hz,
6
1
H), 7.96 (t, J = 8.7 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.39 (m, 3H), 7.24 (d, J = 8.8
+
Hz, 1H), 5.41 (s, 1H), 4.37 (s, 2H). MS (ESI), m/z: 347.05 [M + H] .
2-chloro-N-(4-((7-methoxy-2-oxo-2H-chromen-4-yl)amino)phenyl)acetamide
1
(
4c). H NMR (400 MHz, DMSO-d ) δ: 10.42 (s, 1H), 9.19 (s, 1H), 8.14 (d, J = 9.0
6
Hz, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 6.99 (dd, J = 8.9, 2.5 Hz,
1
H), 6.94 (d, J = 2.5 Hz, 1H), 5.11 (s, 1H), 4.27 (s, 2H), 3.87 (s, 3H). MS (ESI), m/z:
+
3
59.07 [M + H] .

2-chloro-N-(4-((6-nitro-2-oxo-2H-chromen-4-yl)amino)phenyl)acetamide (4d).
1
H NMR (400 MHz, DMSO-d ) δ: 10.45 (s, 1H), 9.74 (s, 1H), 9.31 (d, J = 2.5 Hz,
6
1
7
H), 8.48 (dd, J = 9.1, 2.6 Hz, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 9.1 Hz, 1H),
+
.37 (d, J = 8.8 Hz, 2H), 5.35 (s, 1H), 4.28 (s, 2H). MS (ESI), m/z: 374.05 [M + H] .
1
2
-chloro-N-(4-((2-oxo-2H-chromen-4-yl)oxy)phenyl)acetamide (8a). H NMR
(
400 MHz, DMSO-d ) δ: 10.59 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.76 (dd, J = 16.1,
6
8
3
.5 Hz, 3H), 7.47 (m, 2H), 7.35 (m, 2H), 5.22 (s, 1H), 4.31 (s, 2H). MS (ESI), m/z:
+
30.05 [M + H] .
2-chloro-N-(4-((7-methoxy-2-oxo-2H-chromen-4-yl)oxy)phenyl)acetamide (8b).
1
H NMR (400 MHz, DMSO-d ) δ: 10.49 (s, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.75 (d, J
6
=
8.8 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 7.05 (m, 2H), 5.05 (s, 1H), 4.29 (s, 2H), 3.89
+
(
s, 3H). MS (ESI), m/z: 360.06 [M + H] .
General procedure for synthesis of the final products by means of substitution
5a-5t and 9a-9j).
(
Different acetyl chloride derivatives (1.0 equiv) with catalytic equivalent KI was
dissolved in freshly distilled DMF in a oven-dried round bottom flask. Different
aliphatic amines (3.0 equiv) was added dropwise. The reaction mixture was stirred at
room temperature for 3 h. After the reaction monitored by TLC was over, the reaction
mixture was poured into water, extracted with ethyl acetate three times, washed with
brine and dried over Na SO The solvent was removed by evaporation. The crude
2
4.
residue was purified by chromatography on silica gel to obtain the final products.

2
-(4-methylpiperazin-1-yl)-N-(4-((2-oxo-2H-chromen-4-yl)amino)phenyl)
aceta-mide (5a).
a is a white solid, yield 80%. Melting point: 283.7 ± 2.26 ℃ . H NMR (400
MHz, DMSO) δ 9.92 (s, 1H), 9.41 (d, J = 6.5 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.77
1
5
(dd, J = 14.6, 8.7 Hz, 2H), 7.66 (t, J = 7.3 Hz, 1H), 7.35 (dd, J = 12.7, 10.5, 5.9 Hz,
1
3
4
H), 5.21 (s, 1H), 3.15 (s, 2H), 2.55 (s, 4H), 2.42 (s, 4H), 2.20 (s, 3H). C NMR
(101 MHz, DMSO) δ 168.33, 161.97, 153.82, 153.23, 136.99, 133.77, 132.85, 126.26,
1
24.08, 123.25, 120.93, 117.50, 114.89, 84.45, 60.60, 53.33, 51.66, 50.00. MS (ESI),
+
m/z: 393.18 [M + H]
2-morpholino-N-(4-((2-oxo-2H-chromen-4-yl)amino)phenyl)acetamide(5b).
1
5
b is a white solid, yield 89%. Melting point: 244.25 ± 3.05 ℃ . H NMR (400
MHz, DMSO) δ 9.87 (s, 1H), 9.27 (s, 1H), 8.24 (d, J = 7.3 Hz, 1H), 7.75 (d, J = 8.8
Hz, 2H), 7.66 (m, 1H), 7.39 (dd, J = 13.1, 7.8 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 5.21
1
3
(
s, 1H), 3.65 (m, 4H), 3.15 (s, 2H), 2.53 (m, 4H). C NMR (101 MHz, DMSO) δ
1
1
68.61, 161.91, 153.87, 153.21, 137.11, 133.71, 132.79, 126.23, 124.03, 123.20,
20.91, 117.51, 114.94, 100.00, 84.46, 66.57, 62.54, 53.67. MS (ESI), m/z: 380.15
+
[
M + H]
2
-((S)-3-methylpiperidin-1-yl)-N-(4-((2-oxo-2H-chromen-4-yl)amino)phenyl)
aceta-mide(5c).
c is a white solid, yield 89%. Melting point: 225.15 ± 1.34 ℃ . H NMR (400
1
5
MHz, DMSO) δ 9.78 (s, 1H), 9.27 (s, 1H), 8.24 (d, J = 7.2 Hz, 1H), 7.76 (d, J = 8.8
Hz, 2H), 7.70 – 7.63 (m, 1H), 7.39 (dd, J = 13.1, 7.8 Hz, 2H), 7.32 (d, J = 8.8 Hz,

2
–
1
H), 5.22 (s, 1H), 3.10 (s, 2H), 2.79 (t, J = 8.8 Hz, 2H), 2.08 (d, J = 3.5 Hz, 1H), 1.79
1
3
1.52 (m, 5H), 0.85 (d, J = 6.3 Hz, 4H). C NMR (101 MHz, DMSO) δ 169.12,
61.91, 153.87, 153.21, 137.10, 133.66, 132.80, 126.25, 124.03, 123.20, 120.77,
117.51, 114.94, 84.45, 62.87, 61.88, 54.03, 40.65, 40.44, 40.23, 40.02, 39.81, 39.60,
+
3
9.40, 32.58, 30.98, 25.37, 19.91. MS (ESI), m/z: 392.19 [M + H]
N-(4-((2-oxo-2H-chromen-4-yl)amino)phenyl)-2-(piperidin-1-yl)acetamide(5d).
1
5
d is a white solid, yield 89%. Melting point: 242.3 ± 1.98℃ . H NMR (400
MHz, DMSO) δ 9.79 (s, 1H), 9.27 (s, 1H), 8.24 (d, J = 7.1 Hz, 1H), 7.75 (d, J = 8.8
Hz, 2H), 7.65 (m, 1H), 7.39 (dd, J = 13.1, 7.8 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 5.21
1
3
(
(
s, 1H), 3.09 (s, 2H), 2.49 (s, 4H), 1.58 (d, J = 4.8 Hz, 4H), 1.41 (s, 2H). C NMR
101 MHz, DMSO) δ 169.13, 161.91, 153.87, 153.21, 137.10, 133.66, 132.79, 126.24,
1
24.03, 123.21, 120.80, 117.50, 114.94, 84.45, 63.16, 54.58, 25.94, 24.04. MS (ESI),
+
m/z: 378.19 [M + H]
N-(4-((2-oxo-2H-chromen-4-yl)amino)phenyl)-2-(pyrrolidin-1-yl)acetamide
(5e).
1
5
e is a white solid, yield 76%. Melting point: 248.65 ± 1.63℃ . H NMR (400
MHz, DMSO) δ 9.83 (s, 1H), 9.26 (s, 1H), 8.24 (d, J = 7.1 Hz, 1H), 7.76 (d, J = 8.8
Hz, 2H), 7.66 (m, 1H), 7.39 (dd, J = 13.0, 7.9 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 5.21
1
3
(
s, 1H), 3.26 (s, 2H), 2.60 (d, J = 5.5 Hz, 4H), 1.76 (m, 4H). C NMR (101 MHz,
DMSO) δ 169.34, 161.91, 153.87, 153.22, 137.27, 133.58, 132.79, 126.21, 124.03,
1
3
23.20, 120.86, 117.51, 114.94, 84.43, 60.05, 54.19, 36.06, 23.96. MS (ESI), m/z:
+
64.16 [M + H]