Synthesis and anti-rheumatoid arthritis activities of 3-(4-aminophenyl)-coumarin derivatives

Article abstract of DOI:10.1080/14756366.2021.1873978

Rheumatoid arthritis is a chronic systemic disease characterised by an unknown aetiology of inflammatory synovitis. A large number of studies have shown that synoviocytes show tumour-like dysplasia in the pathological process of RA, and the changes in the expression of related cytokines are closely related to the pathogenesis of RA. In this thesis, a series of novel 3-(4-aminophenyl) coumarins containing different substituents were synthesised to find new coumarin anti-inflammatory drugs for the treatment of rheumatoid arthritis. The results of preliminary activity screening showed that compound 5e had the strongest inhibitory activity on the proliferation of fibroid synovial cells, and it also had inhibitory effect on RA-related cytokines IL-1, IL-6, and TNF-α. The preliminary mechanism study showed that compound 5e could inhibit the activation of NF-κB and MAPKs signal pathway. The anti-inflammatory activity of compound 5ein?vivo was further determined in the rat joint inflammation model.

Full text of DOI:10.1080/14756366.2021.1873978

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and anti-rheumatoid arthritis activities
of 3-(4-aminophenyl)-coumarin derivatives
Yuhang Miao, Jie Yang, Yinling Yun, Jie Sun & Xiaojing Wang
To cite this article: Yuhang Miao, Jie Yang, Yinling Yun, Jie Sun & Xiaojing Wang
(2021) Synthesis and anti-rheumatoid arthritis activities of 3-(4-aminophenyl)-coumarin
derivatives, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 450-461, DOI:
10.1080/14756366.2021.1873978
To link to this article: https://doi.org/10.1080/14756366.2021.1873978
© 2021 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 08 Feb 2021.
Submit your article to this journal
Article views: 550
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 450–461
https://doi.org/10.1080/14756366.2021.1873978
RESEARCH PAPER
Synthesis and anti-rheumatoid arthritis activities of 3-(4-aminophenyl)-coumarin
derivatives
Yuhang Miao, Jie Yang, Yinling Yun, Jie Sun
and Xiaojing Wang
Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, China
ABSTRACT
ARTICLE HISTORY
Received 12 November 2020
Revised 9 December 2020
Accepted 5 January 2021
Rheumatoid arthritis is a chronic systemic disease characterised by an unknown aetiology of inflammatory
synovitis. A large number of studies have shown that synoviocytes show tumour-like dysplasia in the
pathological process of RA, and the changes in the expression of related cytokines are closely related to
the pathogenesis of RA. In this thesis, a series of novel 3-(4-aminophenyl) coumarins containing different
substituents were synthesised to find new coumarin anti-inflammatory drugs for the treatment of rheuma-
toid arthritis. The results of preliminary activity screening showed that compound 5e had the strongest
inhibitory activity on the proliferation of fibroid synovial cells, and it also had inhibitory effect on RA-
related cytokines IL-1, IL-6, and TNF-a. The preliminary mechanism study showed that compound 5e could
inhibit the activation of NF-jB and MAPKs signal pathway. The anti-inflammatory activity of compound 5e
in vivo was further determined in the rat joint inflammation model.
KEYWORDS
3-(4-Aminophenyl)-couma-
rin; rheumatoid arthritis;
RA-FLSs proliferation
inhibition; anti-inflammation
Introduction
Coumarin (called Benzopyran one or o-hydroxycinnamic acid-8-
lactone), which is composed of fused benzene and a-pyranone
ring, is a kind of rich phenolic derivatives15,16. Coumarins have a
variety of biological activities including anti-tumour17, antibacter-
ial18, antioxidant19, anti-Alzheimer’s disease20, and anti-inflamma-
tion21. There have been many reports on the anti-inflammatory
activity of coumarin derivatives, and the relationship between
structure and activity of (SAR) has also been explored, it was
found that the introduction of the electron-rich hydrophobic
group into the 3-position of coumarin nucleus could enhance the
anti-inflammatory activity22,23. The therapeutic effect of coumarin
on RA has been reported before24. In recent years, many coumarin
derivatives obtained from natural products related to the treat-
ment of RA have been found, such as Daphning, a coumarin
derivative extracted from natural product Daphne odora Var. It
has a good therapeutic effect on autoimmune arthritis25. Synthetic
coumarin derivatives are also widely used as in anti-RA. It has
been found that scavenging reactive oxygen species, inhibiting
the activation of NF-jB and the synthesis of TNF-a can effectively
treat RA26,27; Multi-target drugs (such as NSAIDs, CORMs) and car-
bonic anhydrase inhibitors (CAI) can be used as hybrid com-
pounds to treat RA through synergy, which is an important
research direction for anti-RA drugs in the future28,29. Carbonic
anhydrase (CAs) isoforms are overexpressed in the synovium of
RA patients and there is the interaction between various CAs sub-
types and arthritis-like diseases, so carbonic anhydrase inhibitors
have been paid more and more attention in the development of
anti-RA drugs30. Coumarin derivative is a kind of suicided CAIs,
which is hydrolysed by esterase CA activity and binds to the
active site of carbonic anhydrase, so coumarin has a unique and
new carbonic anhydrase inhibition mechanism31,32. 3-(4-amino-
phenyl)-coumarin derivatives are derived from 3-arylcoumarin, and
Rheumatoid arthritis (RA) is the most common chronic inflamma-
tory joint disease and a progressive autoimmune disease1. The
main pathological feature of RA is the recurrent chronic inflamma-
tion in the synovial tissue of the joint, which gradually infiltrates
the cartilage and subchondral bone in the joint, and significantly
affects the joints, organs, and systems, and eventually leads to
irreversible joint deformities and dysfunction2. At present, the
research on the pathogenesis of RA is still a top priority3. During
the pathological process of RA, synovial cells exhibit tumour-like
abnormal proliferation, and invade into cartilage and bone, pro-
ducing a variety of cytokines, leading to chronic inflammation of
cartilage and bone4. There are two types of synovial cells: macro-
phage-like type A cells, with filamentous pseudopodia, invagin-
ation of serosa, vesicles, mitochondria, lysosomes, cytoplasmic
fibres, and Golgi bodies, which have phagocytic function5; type B
fibroblast-like synoviocytes (FLSs), which have a high concentra-
tion of endoplasmic reticulum structure. RA-FLS is an important
effector cell in the course of RA. The over proliferation of RA-FLSs
will lead to damage of joint and chronic persistent inflamma-
tion6–9. Therefore, inhibiting the proliferation of RA-FLSs is an
important therapeutic strategy in the clinical treatment of RA.
There are four main types of drugs for clinical treatment of RA:
non-steroidal anti-inflammatory drugs such as aspirin10, gluco-
corticoid drugs such as Prednisone11, disease-altering drugs such
as methotrexate (MTX)12, and biological agents such as TNF-a
inhibitors13,14. Most of these RA treatment drugs have many side
effects, and the biological agents are relatively expensive, leading
to a huge burden on patients and delay of disease treatment.
Therefore, it is imperative to develop new anti-RA drugs with high
efficiency, low cost, and few side effects.
CONTACT Jie Sun
sunjie310@126.com; Xiaojing Wang
xiaojing6@gmail.com
Institute of Materia Medica, Shandong First Medical University & Shandong
Academy of Medical Sciences, No. 6699, Qingdao Road, Huaiyin District, Jinan 250000, China
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
451
the positional isomer structure of 3-arylcoumarin is similar to that synthesised from substituted p-amino-phenylacetic acid and o-
hydroxybenzaldehyde by Perkin reaction and acidified with hydro-
chloric acid to give 3-(4-aminophenyl)-coumarin intermediate 3. In
the next step, a series of substituted benzoyl chlorides were syn-
thesised from benzoic acid. The target compound was obtained
by amide condensation of 3 with substituted benzoyl chloride in
the mixed solution of pyridine and acetone (Table 1, 4a–4q). The
synthetic route of 3-(4-aminophenyl)-coumarin derivatives with
heterocycles is shown in Scheme 3. Hydroxy benzotriazole (HOBt)
and dicyclohexylcarbodiimide (DCC) were added to the anhydrous
toluene solution containing compound A-E. Then the intermediate
3 was put into the reaction solution, and the reaction mixture was
stirred at room temperature for 8–16 h to obtain 5a–5h34.
of flavonoids and isoflavones. From the structure-activity relation-
ship analysis, the electron-rich hydrophobic group introduced at
position 3 can enhance the anti-inflammatory activity, and the
hydroxyl group at position 7 or 8 can interact with the receptor
through an intermolecular hydrogen bond. Joint inflammation can
lead to elevated ROS, which can erode bones and joints and cause
pain. The persistent pain behaviour in RA is related to the increase
of inflammatory mediators in ROS. Flavonoids have the effect of
anti-oxidation and relieving pain33. Therefore, we speculate that 3-
(4-aminophenyl)-coumarin derivatives may also have antioxidant
imbalance activity. We tend to explore the anti-RA activity of 3-(4-
aminophenyl)-coumarin derivatives through structural extension,
which provides a new type of coumarin structure for the treat-
ment of RA, but also a new direction for structural modification of
coumarin compounds (Scheme 1).
Biology
Effect of compounds on proliferation of RA-FLSs
Rheumatoid arthritis fibroblast-like synovial cells (RA-FLSs) are the
main effector cells of RA, which exhibit abnormal activation and
proliferation in synovial lesions. and mediate joint destruction and
synovitis during RA pathological process35. Recent studies have
found that RA-FLS cells activated in the chronic inflammatory
environment have unique morphology and show characteristics
similar to tumour cells, so the inhibition of RA-FLS cell prolifer-
Results and discussion
Synthesis of novel 3-(4-aminophenyl)-coumarin compounds
containing various substituents
The 3-(4-aminophenyl)-coumarin derivatives were synthesised
using the synthesis method previously reported by our group, and
the synthesis route is shown in Scheme 2. The intermediate 2 was ation is very important for the treatment of RA36. We first tested
Scheme 1. Structural derivation of 3-(4-aminophenyl)-coumarins.
Scheme 2. The synthetic route of 3-(4-aminophenyl)-coumarins 3 and target product 4. Reagents and conditions: (a) acetic anhydride, Et3N, 115 ꢀC. (b) HCl ethanol.
(c) COCl2, DCM, reflux. (d) 3-(4-aminophenyl)-coumarin 3, acetone, pyridine, and RT.

452
Y. MIAO ET AL.
RA-FLS proliferation inhibitory activities of 25 3-(4-aminophenyl) derivatives exhibited an inhibitory effect on RA-FLS proliferation.
coumarin derivatives using MTT assay with MTX as a reference
compound. As shown in Table 2, some of the coumarin
Among these compounds, heterocyclic substituted compound 5e,
with substitution of side-chain phenyl by naphthalene has the
strongest activities (IC50 ¼ 1.78 0.02 mM), which was even stron-
ger than MTX (IC50 ¼ 5.00 0.04 mM). When R1 is hydrogen and R2
Table 1. Structures of compounds 4a–4q, 5a–5h.
Product
R1
R2
R3
R4
R5
R6
R7
Yield (%)
is hydroxyl substitution, the activities of compound 4e (IC50
¼
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
OCH3
H
OCH3
H
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
F
H
H
H
CH3
Cl
H
H
CH3
H
H
H
H
CH3
H
H
Cl
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
70
58
77
85
81
72
80
72
75
78
68
70
87
82
77
62
58
70
75
78
82
65
72
65
72
5.60 0.29 mM) and 4g (IC50 ¼ 9.13 0.02 mM) are comparable to
that of MTX. We, therefore, infer that R4 or R5 chlorination is sig-
nificantly beneficial to the activity of RA-FLS proliferation inhib-
ition. The activity of compound 4p (IC50 ¼ 7.61 0.13 mM) is
CH3
CH2Cl
H
H
H
Cl
H
H
F
CF3
CH2Cl
H
H
H
H
CH2Cl
H
H
H
H
H
H
CH3
F
H
H
H
H
H
H
H
H
better than 4l (IC50
¼
15.71 0.11 mM) and 4o (IC50
¼
16.57 0.04 mM), suggesting that the RA-FLS inhibitory activity of
R2 hydroxyl substituted compounds is better than that of R1
methoxy substituted compounds (Figure 1).
4j
4k
4l
4m
4n
4o
4p
4q
5a
5b
5c
5d
5e
5f
Effect of compounds 4e, 4p, and 5e expression levels of RA-related
cytokines
H
H
H
OCH3
Cl
H
Cytokines are critical local protein mediators involved in almost all
important biological processes, including cell growth, activation,
and differentiation, inflammation, immunity37. It is well known
that the imbalance between the activities of pro-inflammatory
and anti-inflammatory cytokines in rheumatoid joints is conducive
to the induction of autoimmunity and chronic inflammation,
resulting in a joint injury. Therefore, the expression of related
cytokines is closely related to the pathogenesis of RA38. Cytokines
OH
OH
H
OH
H
OH
H
H
5g
5h
H
Scheme 3. General synthetic route to compounds 5a–5h. Reagents and conditions: (e) 3-(4-aminophenyl)-coumarin 3, DCC, HOBt, toluene, and RT.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
453
Table 2. Effect of compounds on proliferation of RA-FLSs. (mean SD, n ¼ 3)
IC50 value (mM)
IC50 value (mM)
RA-FLSs cell proliferation inhibitory activity
Compound
RA-FLSs cell proliferation inhibitory activity
Compound
4a
4b
4c
4d
4e
4f
4g
4h
4i
10.55 0.28
13.93 0.02
>50
4n
4o
4p
4q
5a
5b
5c
5d
5e
5f
5g
5h
>50
16.57 0.04
7.61 0.13
17.39 0.33
>50
>50
5.60 0.29
18.59 0.04
9.13 0.02
>50
8.79 0.19
>50
>50
15.71 0.11
>50
>50
13.07 0.02
19.83 0.03
1.78 0.02
10.40 0.32
24.72 0.03
11.74 0.29
5.00 0.04
4j
4k
4l
4m
Methotrexate
Figure 1. The cell proliferation inhibitory activity of compounds 4e, 4p, and 5e.
respond comprehensively to a variety of stimuli in the process of mainly plays an anti-inflammatory role39. IL-1 is a prototype proin-
immunity and inflammation, involving both pro-inflammatory and flammatory cytokine and has two forms, IL-1a and IL-1b, with
anti-inflammatory activities. Cytokines such as IL-1 and tumour indistinguishable biological activities in most studies40. IL-1 affects
necrosis factor (TNF) have pro-inflammatory activity, while IL-10 almost all cell types and usually acts synergistically with another

454
Y. MIAO ET AL.
pro-inflammatory cytokine, TNF. Although IL-1 is well known to the secretion of IL-1a, IL-1b, and TNF-a. Compound 5e exhibited
stronger activity than Compounds 4e and 4p (Figure 2).
up-regulate host defence and act as an immune adjuvant, it is
also a highly inflammatory cytokine41. Inhibition of IL-1 was first
applied for the treatment of RA. IL-1a is characterised by its imme-
diate effect after release without further maturation. By binding to
IL-1R1, IL-1a can trigger inflammation through induction of IL-1b
activation and production of other cytokines, such as TNF or IL-6.
IL-1b is the most functional member of the IL-1 family and high
levels of IL-1b are found in synovium and fluid of RA patients42.
As another important intercellular messenger, TNF-a has drawn
more and more interest in the development of therapeutic meth-
ods for RA in recent years. It is generally believed that IL-1 plays a
key role in joint destruction, while TNF-a is also involved in the
systemic inflammatory process43. Numerous studies on synovial
tissue of RA have demonstrated that TNF-a is a potential thera-
peutic target for RA, so the discovery of novel TNF-a inhibitors is
RA-FLS cells were cultured at 37 ꢀC for 24 h, then the cells were
divided into four groups: blank control group, model group (LPS),
LPS þ compound group, and LPS þ MTX group. Different concen-
trations of compounds and 1.0 mg/mL MTX, were added to incu-
bate for 48 h, and then LPS was added to stimulate the secretion
of proinflammatory cytokines at the final concentration of 5 mg/
mL. After 24 h of incubation, pro-inflammatory cytokines including
IL-1 a, IL-1 b, and TNF-a were measured by ELISA method.
Multiple cytokine IL-10 is an effective inhibitor in immune cells
including macrophages, T cells and NK cells, and it can block the
synthesis and activation of cytokines in these cells. Different from
pro-inflammatory factors IL-1 and TNF-a, IL-I0 has effective anti-
inflammatory properties46,47. We examined the effects of 4e, 4p,
and 5e on cytokine IL-10. The results showed that all the deriva-
tives could increase the amount of IL-10, which preliminarily indi-
cated that the derivatives were beneficial to the fight against RA
disease (Figure 3).
of great significance for treatment of RA44,45
Compounds 4e (IC50
.
¼
5.60 0.29 mM), 4p (IC50 ¼ 7.61
0.13 mM), and 5e (IC50 ¼ 1.78 0.02 mM) showed an excellent
inhibitory effect on RA-FLS proliferation. We next used these opti-
mised compounds to further explore their anti-RA activities and
related mechanisms. We first detected the effect of these com-
pounds on the contents of cytokine IL-1a, IL-1b, and TNF-a
released by RA-FLSs with stimulation LPS. The results showed that
We also detected the effect of these compounds on the con-
tent of IL-10 released by RA-FLSs stimulated by LPS, and the
results showed that 4e, 4p, and 5e could significantly increase the
content of IL-10 released by RA-FLSs. These results indicate that
compounds 4e, 4p, and 5e could inhibit the production of proin-
flammatory cytokines and enhance the production of anti-inflam-
the three compounds could concentration-dependently reduce matory cytokines, therefore exert anti-inflammatory functions.
Figure 2. Effect of compounds 4e, 4p, and 5e on secretion of IL-1a, IL-1b, and TNF-a.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
455
Figure 3. Effect of compounds 4e, 4p, and 5e on secretion of cytokine IL-10.
Compound 5e has the strongest activity among them, which
might be a potential anti-RA drug candiate48.
Effect of compound 5e on luciferase activity of NF-jB
The activation of a series of signal pathways is a typical feature of
chronic synovitis in RA. These signal pathways induce phosphoryl-
ation of cytoplasmic proteins in synovial cells, and phosphoryl-
ation of transcription factors and nuclear proteins such as NF-
jB49, thus promoting cell proliferation and activation. On the
other hand, inflammatory factors such as TNF-a, IL-1a, and IL-1b in
turn regulate cell membrane receptors and activate the mitogen-
activated protein kinase (MAPK) signal pathway. Therefore, investi-
gation of the above signalling events is of great significance for
the in-depth understanding of the pathogenesis of RA and the
development of new drugs for treatment. NF-jB-Luc luciferase
reporter gene plasmid is a reporter gene used to detect the tran-
scriptional activity of NF-jB. The enhanced activity of NF-jB stimu-
lated by LPS leads to a significant increase in the expression of
inflammatory proteins. We next treated RA-FLSs with compound
5e and tested the luciferase activity of NF-jB. Similar to MTX,
compound 5e can significantly reduce the activity of NF-jB, which
is of great significance in inhibiting inflammatory factors and
reducing inflammation (Figure 4).
Figure 4. Inhibitory effect of compound 5e on luciferase activity of NF-jB. RA-
FLS cells were cultured at 37 ꢀC for 24 h, then NF-jB luciferase plasmid and con-
trol sea kidney plasmid were transfected for 48 h. Different concentrations of
compound 5e and LPS (final concentration: 5 lg/mL) were added to stimulate
the secretion of proinflammatory cytokines. After incubation for 48 h, the cells
were collected and the luciferase activity of NF-jB pathway was detected by
dual luciferase system. #p < 0.05, ##p < 0.01, ###p < 0.001, compared with model
ꢁꢁꢁꢁ
group;
p < 0.0001 compared with ctrl group.
results showed that 80–90% of the rats showed paw swelling
within 14–28 days (Figure 6, model group). The model rats were
treated with compound 5e of different concentrations once a
day from day 14 to day 28. MTX was used as a positive control.
The experimental results are shown in Figure 6. After successfully
establishing the model, we tested the pain threshold and motor
function of RA rats. The reflex latency (paw withdraw thermal
latency, PWTL) of rat foot contraction induced by thermal stimu-
lation was used as an index of hindlimb pain threshold by the
hot plate method. As shown in Figure 7, the PWTL of the model
group was shorter than that of the control group (p < 0.001).
After the intervention of compound 5e in the low-dose group
and high-dose group, the results showed that the PWTL of RA
rats was prolonged, and the prolongation of PWTL was positively
correlated with the drug concentration55. It is proved that com-
pound 5e reduces thermal hyperalgesia in RA rats and has an
analgesic effect on RA rats. In the motor function test, the left
gait width and hind limb gait width of RA rats before and after
treatment were measured by gait analysis. Compared with the
control group, the left stride length of the model group was
shorter, and the hindlimb stance width was wider, and the motor
function of rats was blocked (p < 0.001). After treatment with
compound 5e in low-dose group and high-dose group, it was
found that compound 5e could improve the exercise ability of
rats (Figure 8).
Effect of compound 5e on MAPK signal pathway
MAPK mediates important pathways in the eukaryotic signal trans-
duction network, which plays a key role in regulating gene expres-
sion and promoting disease development50,51. In mammals, five
different MAPK-mediated signal pathways regulate cell growth
and differentiation. JNK and p38 MAPK signalling pathways play
an important role in stress responses such as inflammation and
apoptosis52. We next treated RA-FLSs with compound 5e and
tested the changes in MAPK-mediated signal pathways. The
results showed that LPS stimulation significantly increased the lev-
els of pho-extracellular signal-regulated kinases (ERK)1/2 and pho-
p38, and compound 5e could significantly reduce the levels of
pho-ERK1/2 and pho-p38. These results indicate that compound
5e can effectively suppress the phosphorylation and activation of
ERK1/2 and P38 in the MAPK signal pathway (Figure 5), which is
well known to cause suppression of NF-jB activity. In contrast,
MTX only suppressed the phosphorylation of ERK1/2 but not
the p38.
Anti-RA activity of compound 5e in vivo
To establish an arthritis model, we used Freund’s complete adju-
vant to induce arthritis in rats via injection of CFA into the left
We then treated RA rats with compound 5e. In normal rats,
hind paw of rats53,54. After injection of CFA into SD rats, the no inflammation pathological features were observed, the

456
Y. MIAO ET AL.
Figure 5. Inhibitory effect of compound 5e on MAPK signal pathway. RA-FLS cells were cultured at 37 ꢀC for 24h. The cells were preincubated with compound 5e of
different concentrations for 1 h, and then treated with LPS (final concentration: 5 lg/mL) for 72 h. Collected cells were used for Western blotting analysis of anti-ERK1/
####
2, anti-pho-ERK1/2, anti-p38 and anti-pho-p38 antibodies. ##p < 0.01,
p < 0.0001 compared with model group;
ꢁꢁꢁꢁ
p < 0.0001 compared with ctrl group.
Figure 6. Establishment and treatment of CFA rat model of RA. Normal control group (Ctrl, saline), model group (Model, saline), high dose compound 5e (60mg/kg)
group, low dose compound 5e (30mg/kg) group, positive control MTX (30mg/kg) group. (n ¼ 5 per group).
surface of cartilage was smooth and free of cracks, and the
cells were evenly distributed (Figure 9(A)). In the arthritis model
group, obvious inflammation pathological features were
observed, including disorder of chondrocytes and vacuolisation
of some cells, which indicate decreased activity or died cells,
and destroyed cartilage surface (Figure 9(B-1,B-2)). After treat-
ment with 30 mg/kg of compound 5e, the inflammation
was significantly improved, the vacuole of chondrocytes
decreased, and the destruction of the cartilage surface was
recovered (Figure 9(C)). After treatment with 60 mg/kg of com-
pound 5e, almost no inflammatory cell infiltration was
observed, the surface of cartilage tended to be smooth, indicat-
ing a better therapeutic effect than low dose compound 5e
(Figure 9(D)).
Figure 7. The analgesic effect of compound 5e on rats was detected by the
###
ꢁꢁꢁ
method of thermal pain threshold of rat claws. p < 0.001,
p < 0.001. (n ¼ 5
per group).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
457
Figure 8. Motor function test of SD rats. Place the rat in the plexiglass chamber of the treadmill (25cm long and 5 cm wide), set the treadmill speed to 18 cm/s, the
shutter speed to take pictures: 40 ms, and record 5 s for each animal. The motor function of RA rats was explained by the motor parameters of left stride length and
#
###
hindlimb stance width. p < 0.05, ##p < 0.01, p < 0.001, compared with model group,
p < 0.001 compared with ctrl group. (n ¼ 5 per group).
ꢁꢁꢁ
Figure 9. Compound 5e recovers RA histopathological features of rats. (A) Normal control group (Ctrl, normal saline), (B-1, B-2) model group (model, normal saline),
(C) low-dose compound 5e group (30mg/kg), (D) high-dose compound 5e Group (60mg/kg), and (E) positive control MTX (30mg/kg). (n ¼ 5 per group).

458
Y. MIAO ET AL.
proinflammatory cytokines. After a 24 h incubation, the cell culture
medium of each group was collected, and the cell fragments were
removed by centrifugation at 10,000 ꢂ g for 5 min at 4 ꢀC. The
proinflammatory cytokines including IL-1a, IL-1b, TNF-a, and IL-10
were tested using ELISA assay by following the manufacturer’s
instructions.
Conclusion
In this study, we screened the anti-RA activity of a series of 3-(4-
aminophenyl)-coumarins synthesised by our team. Compound 5e
had the strongest inhibitory effect on the proliferation of RA-FLSS.
We further discussed the anti-inflammatory activity of compound
5e and its related mechanism. We found that compound 5e could
inhibit the expression of inflammation-related cytokines (IL-1 a, IL-
1 b, and TNF-a) induced by lipopolysaccharide. The preliminary
mechanism study found that compound 5e has an effect on
inflammation-related signal transduction, which can inhibit the
activity of nuclear factor-mitogen-activated protein B (NF-jB)
through mitogen-activated protein kinase (MAPK) signal pathway.
Finally, the rat arthritis model established by Freund’s complete
adjuvant further proved that compound 5e had anti-RA activity
in vivo. To sum up, compound 5e may be a potential protective
compound for the treatment of RA.
Test of luciferase activity of NF-jB
RA-FLS cells were seeded in 24-well plate (1 ꢂ 105/well) and cul-
tured at 37 ꢀC for 24 h, then NF-jB luciferase plasmid and control
renilla plasmid were transfected for 48 h. Different concentrations
of compound 5e and LPS (final concentration: 5 mg/mL) were
added to stimulate the secretion of proinflammatory cytokines.
After a 48-h incubation later, the cells were collected and the luci-
ferase activity of NF-jB pathway was detected using the double
luciferase system.
Experimental
Test of MAPK signal pathway
RA-FLS cells (3 ꢂ 105/well/2 mL) were seed in 6-well plate cultured
at 37 ꢀC and for 24 h. The cells were pre-incubated with different
concentrations of compound 5e for 1 h, and then were treated
with LPS (final concentration: 5 mg/mL) for 72 h. The cells were col-
lected for Western blotting analysis using anti-ERK1/2, anti-pho-
ERK1/2, anti-p38, and anti-pho-p38 antibodies.
Materials and methods
Materials
Methotrexate, manufacturer: MCE. Bio Legend LEGEND plex TM
multiplex bead-based assay (# 740007), brand: Biolegend; manu-
facturer: Biolegend Corporation of the United States. Saline, manu-
facturer: Guangdong Litai Pharmaceutical Co., Ltd. PBS powder,
manufacturer: American SIGMA Company.
Western blotting
Cells were lysed in 20 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40,
10 mM tetrasodium pyrophosphate, 100 mM NaF, 17.5 mM 1-glyc-
erophosphate buffer supplemented with Complete Mini Protease
Inhibitor Cocktail tablets (Roche Applied Sciences, Indianapolis,
IN). Samples separated by SDS-PAGE were transferred to nitrocel-
lulose membranes, blocked with 5% bovine serum albumin (w/v)
at room temperature for 1 h, and incubated with primary antibod-
ies (1:1000 dilution) at 4 ꢀC overnight. After incubation with sec-
ondary antibody (1:3000 dilution) at room temperature for 1 h, the
membranes were developed with chemiluminescence ECL reagent
(Lumi Gold, Signa Gen Laboratories, Rockville, MD) and exposed
to Hyper film MP (GE Healthcare Life Science, Pittsburgh, PA).
Cells and cell culture
RA-FLS cells were obtained from Beijing Luyuan Byrd
Biotechnology Co., Ltd., and the 3rd–5th generations were used in
the experiments. The cells were cultured with high glucose
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with
10% foetal bovine serum (FBS), 100 units/mL penicillin, 100 mg/mL
streptomycin, and 2 mM glutamine. All cells were incubated in a
humidified atmosphere of 95% air and 5% CO2 at 37 ꢀC.
MTT assay
RA-FLS cells were inoculated in a 96-well plate (5 ꢂ 10ꢃ3/well/
100 mL), incubated for 24 h, and then treated with different con-
centrations of synthesised derivatives (0.1, 0.3, 1, 3, 10, 30, 100,
and 300 mM) or 1.0 mg/mL MTX for 72 h. The culture medium, was
then removed and the cells were incubated with 5 mg/mL MTT at
37 ꢀC for 4 h, and 100 mL DMSO was added to each well to dis-
solve the formed methylazan The absorbance (OD value) was
measured at 570 nm using a plate reader. The cell proliferation
inhibition rate was calculated by following the formula: inhibition
rate ¼ (OD value of the control group-OD value of the treated
groups)/OD value of the control group ꢂ 100%. The IC50 value
(mM) was calculated according to the curve.
Animals
SD rats weighing 180–220 g were obtained from Jinan Peng Yue
Experimental Animal Co., Ltd. (license No.: SCXK (Lu) 2014 Mel
0007), Co., Ltd. The animals were kept in captivity under standard
laboratory conditions and ingested standard pellet feed and water
at will. All experiments involving live animals and their care are
conducted in strict accordance with the “National Care and use of
Experimental Animals” of the State Animal Research Bureau
(China) and the “regulations on the Administration of
Experimental Animals” of the Life Science Research Centre of
Shandong first Medical University. The experiment was approved
by the Animal Protection and use Committee of Shandong first
Medical University. Make every effort to reduce the suffering of
animals and reduce the number of animals used.
Test of cytokines using enzyme-linked immunosorbent
assay (ELISA)
The 3rd–5th passage RA-FLS cells were inoculated in 48-well
plates (1.5 ꢂ 106/well) and cultured at 37 ꢀC for 24 h, and then
cells were divided into four groups: blank control group, model Establishment and treatment of adjuvant arthritis in rats
group (LPS), LPS þ compound group and LPS þ MTX group.
The rat adjuvant arthritis model was first established by Freund in
Different concentrations of compounds and 1.0 mg/mL MTX were the 1950s. Which is simple and selective and is a widely used
added and incubated for 48 h, and then LPS was added at a final immune inflammation model56,57
.
8-week-old Sprague–Dawley
concentration of 5 mg/mL to stimulate the secretion of (SD) rats, weighing 180–220 g, were purchased from Jinan Peng

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
459
Yue Experimental Animal Breeding Co., Ltd., Jinan, China. The
Freund’s complete adjuvant (CFA) was purchased from Sigma-
Aldrich (item number and specification: F5881-10 mL) and 0.1 mL
CFA was injected into the left hind paw of rats to induce inflam-
mation. Arthritis was evaluated by observing the changes in paw
swelling every week. After the rat adjuvant arthritis model was
successfully established, SD rats were divided into the following
groups: normal control group (Ctrl, saline), model group (Model,
saline), high dose compound 5e (60 mg/kg) group, low dose com-
pound 5e (30 mg/kg) group, positive control MTX (30 mg/kg)
group, (n ¼ 5 per group). The compounds solutions (1 mL) or
saline were orally administered to rats once a day for four weeks.
Then the mice were euthanized using carbon dioxide (CO2) and
the knee joints were harvested and analysed.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was financially supported by Science and Shandong
Provincial Natural Science Foundation [ZR2018LH021], the
Innovation Project of Shandong Academy of Medical Sciences and
Academic promotion programmer of Shandong First Medical
University [2019LJ003].
ORCID
Jie Sun
http://orcid.org/0000-0003-0552-9061
Heat pain threshold determination
The thermal pain threshold of rats was measured by rat claw ther-
mal radiation stimulation pain metre (7371 plantar test, Ugo
Basile, Italy). The rats are in a state of free activity, adjust the
instrument stimulation intensity (IR ¼ 72), put the left hindlimb
on the instrument, and measure the paw recovery time of rats.
The reflex latency (paw withdrawal thermal latency, PWTL) of foot
contraction induced by thermal stimulation at the bottom of rat
foot is the thermal pain threshold.
References
1. Smolen JS, Aletaha D, Koeller M, et al. New therapies
for treatment of rheumatoid arthritis. Lancet 2007;370:
1861–74.
2. Mcinnes IB, Schett G. Pathogenetic insights from the
treatment of rheumatoid arthritis. Lancet 2017;389:
2328–37.
3. Firestein GS. Evolving concepts of rheumatoid arthritis.
Nature 2003;423:356–61.
4. Schett G, Gravallese E. Bone erosion in rheumatoid arthritis:
mechanisms, diagnosis and treatment. Nat Rev Rheumatol
2012;8:656–64.
5. Bartok B, Firestein GS. Fibroblast-like synoviocytes: key
effector cells in rheumatoid arthritis. Immunol Rev 2010;233:
233–55.
6. Bugatti S, Codullo V, Caporali R, Montecucco C. B cells in
rheumatoid arthritis. Autoimmun Rev 2007;7:137–42.
7. Marston B, Palanichamy A, Anolik JH. B cells in the patho-
genesis and treatment of rheumatoid arthritis. Curr Opin
Rheumatol 2010;22:307–15.
Rat motor function test
R
The automatic gait analysis system (DigiGaitV, Mouse Specific,
Inc.)) was used to test the rats. The rats were placed in the plexi-
glass chamber of the treadmill (25 cm long and 5 cm wide). The
plastic rear bumper of the rats was used to tap the rats to keep
them in the video shooting range. The treadmill speed was set
to 18 cm/s. The shutter speed was 40 ms, and each animal
recorded 5 s. DigiGait software automatically recognises paw
prints and analyses gait parameters such as step size and
step width.
8. Weyand CM, Seyler TM, Goronzy JJ. B cells in rheumatoid
synovitis. Arthritis Res Ther 2005;7:S9.
9. Noss EH, Brenner MB. The role and therapeutic implications
of fibroblast-like synoviocytes in inflammation and cartilage
erosion in rheumatoid arthritis. Immunol Rev 2008;223:
252–70.
10. Wienecke T. Paracetamol versus nonsteroidal anti-inflamma-
tory drugs for rheumatoid arthritis. Cochrane DB Syst Rev
2004;1:CD003789.
11. Spies CM, Bijlsma JWJ, Burmester G-R, Buttgereit F.
Pharmacology of glucocorticoids in rheumatoid arthritis.
Curr Opin Pharmacol 2010;10:302–7.
Immunohistochemistry (IH)
The rat knee joints were fixed in phosphate-buffered formalin,
embed in paraffin, cut in 4 mm thickness, and applied to slides.
The slides were deparaffinized in xylenes using three changes for
5 min each and hydrated gradually through graded alcohols:
100% ethanol twice for 10 min each, 95% ethanol twice for
10 min each, and then deionised water for 1 min with stirring.
For antigen unmasking, slides were placed in a container, cov-
ered with 10 mM sodium citrate buffer, pH 6.0, and heated in a
convection steamer for 1 h. The slides were washed in deionised
water three times for 2 min each, blocked with 5% normal goat
blocking serum for 30 min, and then stained with haematoxylin
and eosin (HE). The slides were analysed and photographed
using a micromirror.
ꢀ
12. Kremer JM, Alarcon GS, Lightfoot RW, et al. Methotrexate for
rheumatoid arthritis. Suggested guidelines for monitoring
liver toxicity. American College of Rheumatology. Arthritis
Rheum 1994;37:316–28.
ꢁ
13. Atzeni F, Benucci M, Sallı S, et al. Different effects of bio-
logical drugs in rheumatoid arthritis. Autoimmun Rev 2013;
12:575–9.
14. Feldmann M, Maini RN. Anti-TNF alpha therapy of rheuma-
toid arthritis: what have we learned? Annu Rev Immunol
2001;19:163–96.
15. Peng X-M, Damu GLV, Zhou C-H. Current developments of
coumarin compounds in medicinal chemistry. Curr Pharm
Des 2013;19:3884–930.
Statistics
t-Test and analysis of variance (ANOVA) were performed using
StatView (SAS Institute, Cary, NC). The data shown are representa-
tives of at least two independent experiments with similar results,
and the data points represent the mean of at least triplicate meas-
urements with error bars corresponding to standard deviation.
p < 0.05 was considered significantly different.

460
Y. MIAO ET AL.
16. Fylaktakidou KC, Hadjipavlou-Litina DJ, Litinas KE, Nicolaides
DN. Natural and synthetic coumarin derivatives with anti-
treatment of rheumatoid arthritis. J Med Chem 2017;60:
1159–70.
inflammatory/ antioxidant activities. Curr Pharm Des 2004; 33. Di Cesare Mannelli L, Bani D, Bencini A, et al. Therapeutic
effects of the superoxide dismutase mimetic compound
MnIIMe2DO2A on experimental articular pain in rats.
Mediators Inflammation 2013;2013:905360.
10:3813–33.
17. Menezes JC, Diederich M. Translational role of natural cou-
marins and their derivatives as anticancer agents. Future
Med Chem 2019;11:1057–82.
18. Wei Y, Peng W, Wang D, Hao SH, et al. Design, synthesis,
antifungal activity, and 3D-QSAR of coumarin derivatives. J
Pestic Sci 2018;43:88–95.
34. Hu Y-H, Yang J, Zhang Y, et al. Synthesis and biological
evaluation of 3-(4-aminophenyl)-coumarin derivatives as
potential anti-Alzheimer’s disease agents. J Enzyme Inhib
Med Chem 2019;34:1083–92.
19. Kostova I, Bhatia S, Grigorov P, et al. Coumarins as antioxi-
dants. Curr Med Chem 2011;18:3929–51.
20. Najafi Z, Mahdavi M, Saeedi M, et al. Novel tacrine-coumarin
hybrids linked to 1,2,3-triazole as anti-Alzheimer’s com-
pounds: in vitro and in vivo biological evaluation and dock-
ing study. Bioorg Chem 2019;83:303–16.
21. Kontogiorgis CA, Hadjipavlou-Litina DJ. Synthesis and antiin-
flammatory activity of coumarin derivatives. J Med Chem
2005;48:6400–8.
22. Arora RK, Kaur N, Bansal Y, Bansal G. Novel coumarin-benzi-
midazole derivatives as antioxidants and safer anti-inflam-
matory agents. Acta Pharm Sin B 2014;4:368–75.
23. Bansal Y, Sethi P, Bansal G. Coumarin: a potential nucleus
for anti-inflammatory molecules. Med Chem Res 2013;22:
3049–60.
35. Liu Y, Pan YF, Xue Y-Q, et al. uPAR promotes tumor-like bio-
logic behaviors of fibroblast-like synoviocytes through PI3K/
Akt signaling pathway in patients with rheumatoid arthritis.
Cell Mol Immunol 2018;15:171–81.
36. Firestein GS, Zvaifler NJ. How important are T cells in
chronic rheumatoid synovitis?: II. T cell-independent mecha-
nisms from beginning to end. Arthritis Rheum 2002;46:
298–308.
37. Feldmann M, Brennan FM, Maini RN. Role of cytokines in
rheumatoid arthritis. Annu Rev Immunol 1996;14:397–440.
38. McInnes IB, Schett G. Cytokines in the pathogenesis of
rheumatoid arthritis. Nat Rev Immunol 2007;7:429–42.
39. Maini RN, Taylor PC. Anti-cytokine therapy for rheumatoid
arthritis. Annu Rev Med 2000;51:207–29.
40. Dinarello CA. Interleukin-1. Cytokine Growth Factor Rev
1997;8:253–65.
41. Dinarello CA. Biologic basis for interleukin-1 in disease.
Blood 1996;87:2095–147.
24. Pan R, Gao XH, Li Y, et al. Anti-arthritic effect of
scopoletin, a coumarin compound occurring in Erycibe
obtusifolia Benth stems, is associated with decreased angio-
genesis in synovium. Fundam Clin Pharmacol 2010;24: 42. Schett G, Dayer J-M, Manger B. Interleukin-1 function and
477–90.
role in rheumatic disease. Nat Rev Rheumatol 2016;12:
14–24.
25. Yao R, Fu Y, Li S, et al. Regulatory effect of daphnetin, a
coumarin extracted from Daphne odora, on the balance of
Treg and Th17 in collagen-induced arthritis. Eur J Pharmacol
2011;670:286–94.
26. Cheng JF, Chen M, Wallace D, et al. Discovery and
structure-activity relationship of coumarin derivatives
as TNF-alpha inhibitors. Bioorg Med Chem Lett 2004;14:
2411–5.
27. Sahu D, Raghav SK, Gautam H, Das HR. A novel coumarin
derivative, 8-methoxy chromen-2-one alleviates collagen
induced arthritis by down regulating nitric oxide, NFjB and
proinflammatory cytokines. Int Immunopharmacol 2015;29:
891–900.
28. Berrino E, Milazzo L, Micheli L, et al. Synthesis and evalu-
ation of carbonic anhydrase inhibitors with carbon monox-
ide releasing properties for the management of rheumatoid
arthritis. J Med Chem 2019;62:7233–49.
43. Genovese MC, Becker J-C, Schiff M, et al. Abatacept for
rheumatoid arthritis refractory to tumor necrosis factor
alpha inhibition. N Engl J Med 2005;353:1114–23.
44. Kollias G, Douni E, Kassiotis G, Kontoyiannis D. On the
role of tumor necrosis factor and receptors in models of
multiorgan failure, rheumatoid arthritis, multiple sclerosis
and inflammatory bowel disease. Immunol Rev 1999;169:
175–94.
45. Palladino MA, Bahjat FR, Theodorakis EA, Moldawer LL. Anti-
TNF-alpha therapies: the next generation. Nat Rev Drug
Discov 2003;2:736–46.
46. Katsikis PD, Chu CQ, Brennan FM, et al. Immunoregulatory
role of interleukin 10 in rheumatoid arthritis. J Exp Med
1994;179:1517–27.
47. Moore KW, O’Garra A, de Waal Malefyt R, et al. Interleukin-
10. Annu Rev Immunol 1993;11:165–90.
29. Bua S, Lucarini L, Micheli L, et al. Bioisosteric development
of multitarget nonsteroidal anti-inflammatory drug-
48. Bissantz C, Kuhn B, Stahl M. A medicinal chemist’s guide to
molecular interactions. J Med Chem 2010;53:5061–84.
49. Baker RG, Hayden MS, Ghosh S. NF-jB, inflammation, and
metabolic disease. Cell Metab 2011;13:11–22.
50. Lin X, Wang M, Zhang J, Xu R. p38 MAPK: a potential target
of chronic pain. Curr Med Chem 2014;21:4405–18.
carbonic
anhydrases
inhibitor
hybrids
for
the
management of rheumatoid arthritis. J Med Chem 2020;63:
2325–42.
30. Menicatti M, Pallecchi M, Bua S, et al. Resolution of co-elut-
ing isomers of anti-inflammatory drugs conjugated to car- 51. Tian W, Zhang Z, Cohen DM. MAPK signaling and the kid-
bonic anhydrase inhibitors from plasma in liquid
chromatography by energy-resolved tandem mass spec-
trometry. J Enzyme Inhib Med Chem 2018;33:671–9.
ney. Am J Physiol Renal Physiol 2000;279:F593–604.
52. Dent P, Yacoub A, Fisher PB, et al. MAPK pathways in radi-
ation responses. Oncogene 2003;22:5885–96.
31. Supuran CT. Coumarin carbonic anhydrase inhibitors from 53. He Y-H, Zhou J, Wang Y-S, et al. Anti-inflammatory and
natural sources.
1462–70.
32. Bua S, Di Cesare Mannelli L, Vullo D, et al. Design and syn-
J
Enzyme Inhib Med Chem 2020;35:
anti-oxidative effects of cherries on Freund’s adjuvant-
induced arthritis in rats. Scandinavian J Rheumatol 2006;35:
356–8.
thesis of novel nonsteroidal anti-inflammatory drugs and 54. Guo W-Y, Chen L-Z, Shen B-N, et al. Synthesis and in vitro
carbonic anhydrase inhibitors hybrids (NSAIDs-CAIs) for the and in vivo anti-inflammatory activity of novel

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
461
4-ferrocenylchroman-2-one derivatives. J Enzyme Inhib Med 56. Wang S, Wang Y, Liu X, et al. Anti-inflammatory and anti-
Chem 2019;34:1678–89.
arthritic effects of taraxasterol on adjuvant-induced arthritis
55. Maresca M, Micheli L, Cinci L, et al. Pain relieving
in rats. J Ethnopharmacol 2016;187:42–8.
and protective effects of Astragalus hydroalcoholic extract 57. Wang X, He X, Zhang C-F, et al. Anti-arthritic effect of ber-
in rat arthritis models.
J
Pharm Pharmacol 2017;69:
berine on adjuvant-induced rheumatoid arthritis in rats.
1858–70.
Biomed Pharmacother 2017;89:887–93.