New arylpyrazoline-coumarins: Synthesis and anti-inflammatory activity

Article abstract of DOI:10.1016/j.ejmech.2017.06.044

To develop new anti-inflammatory agents with improved pharmaceutical profiles, a series of new phenyl-pyrazoline-coumarin derivatives (4a～4m) were designed and synthesized. Compounds 4a and 4b were determined by X-ray crystallography. All of the compounds have been screened for their anti-inflammatory activity characterized by evaluating their inhibition against LPS-induced IL-6 release. Among them, compound 4m showed the highest anti-inflammatory activity with inhibiting IL-6, TNF-α and nitric oxide (NO) production lipopolysaccharide (LPS)-stimulated. The further study showed that title compound 4m could significantly suppress expressions of nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and the productions of IL-6, TNF-α, NO through NF-κB/MAPK signaling pathway. The anti-inflammatory activity of compound 4m was determined by carrageenan induced paw edema. Furthermore in vivo evaluation results indicated that compound 4m could inhibit AA-induced rat ankle joints.

Full text of DOI:10.1016/j.ejmech.2017.06.044

European Journal of Medicinal Chemistry 138 (2017) 170e181
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
New arylpyrazoline-coumarins: Synthesis and anti-inflammatory
activity
Liu Zeng Chen a, Wei Wei Sun a, Li Bo a, Jie Quan Wang a, Cheng Xiu a, Wen Jian Tang a,
, b, *
Jing Bo Shi a, Hai Pin Zhou b, Xin Hua Liu a
a Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei
230032, PR China
b School of Material Science Chemical Engineering, ChuZhou University, ChuZhou 239000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To develop new anti-inflammatory agents with improved pharmaceutical profiles, a series of new
phenyl-pyrazoline-coumarin derivatives (4a~4m) were designed and synthesized. Compounds 4a and 4b
were determined by X-ray crystallography. All of the compounds have been screened for their anti-
inflammatory activity characterized by evaluating their inhibition against LPS-induced IL-6 release.
Received 5 April 2017
Received in revised form
21 June 2017
Accepted 23 June 2017
Available online 26 June 2017
Among them, compound 4m showed the highest anti-inflammatory activity with inhibiting IL-6, TNF-
a
and nitric oxide (NO) production lipopolysaccharide (LPS)-stimulated. The further study showed that
title compound 4m could significantly suppress expressions of nitric oxide synthase (iNOS),
cyclooxygenase-2 (COX-2) and the productions of IL-6, TNF-a, NO through NF-kB/MAPK signaling
pathway. The anti-inflammatory activity of compound 4m was determined by carrageenan induced paw
edema. Furthermore in vivo evaluation results indicated that compound 4m could inhibit AA-induced rat
ankle joints.
Keywords:
Coumarin
Pyrazoline
Synthesis
Anti-inflammatory
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
(containing JNK, ERK and P38), were quite significant in the regu-
lation of inflammation because of mediating the production of NO,
Inflammation is a protective attempt of host defense against
injury or infection. However, when inflammation becomes chronic,
it has been proven tissue damage and may lead to diseases, such as
atherosclerosis and arthritis [1]. It has been reported that Tak1 is
traditionally accepted as the primary LPS receptor and critical for
the inflammatory response to LPS. Upon stimulation with LPS, Tak1
initiates series of signaling cascades that result in activation of NF-
TNF-a, IL-6, IL-b and other inflammatory mediators [3e6].
Five membered rings such as pyrazoline and pyrazole are priv-
ileged structures used in anticancer [7e11] and anti-inflammatory
activity [12e15]. Celecoxib, which is formed of diaryl moiety con-
taining central pyrazole ring, is one of the most important
extremely marketed COX-2 selective drugs. Based on this diaryl
five-member heterocyclic skeleton, containing diphenyl-
oxadiazole/thiadiazole hybrids, a lot of novel compounds with
high anti-inflammatory activity were designed and synthesized
k
B and mitogen-activated protein kinases (MAPK) to induce the
release of pro-inflammatory cytokines [2]. Recent studies found
that inhibition of Tak1 could decrease the expressions of IL-6, IL-1
and TNF- , which were consistent with inflammatory. So, the Tak1
signal pathway may be a target in the treatment of inflammatory
disease. NF- B, downstream protein of Tak1, which is a key acti-
vator in the inflammatory processes, it turns on transcriptions of
inflammatory genes including TNF- and IL-6. Meanwhile, MAPK
b
[16,17]. Curcumin analogs (containing a,b-unsaturated ketone
a
moiety), precursors of pyrazolines, show wide anti-inflammatory
activity [18e22]. In addition, compounds containing coumarin
scaffold are largely present in natural products and display a variety
of biological activities, namely those associated with activation
Nrf2/ARE pathway [23] and anti-inflammatory activity [24e26]. A
large number of coumarin derivatives have been prepared by
k
a
suppressing the DNA binding ability of NF-kB [27]. Motivated by
above findings (Fig. 1), a series of aryl-pyrazoline-coumarin de-
rivatives were synthesized and evaluated for their anti-
inflammatory activity.(see:Fig. 2)
* Corresponding author. Anhui Province Key Laboratory of Major Autoimmune
Diseases, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical
University, Hefei 230032, PR China.
E-mail address: xhliuhx@163.com (X.H. Liu).
http://dx.doi.org/10.1016/j.ejmech.2017.06.044
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
171
Fig. 1. The general design strategy.
Fig. 2. Possible molecular basis for the anti-inflammatory property.
Based on Tak1, we investigated anti-inflammatory activity and
preliminary molecular mechanisms using both cell and animal
models in this study. First, we used mouse macrophage-like cell
(RAW264.7), which can be stimulated with LPS to mimic a status of
infection and inflammation, to investigate the inhibition and mo-
lecular mechanisms of compounds on the productions of iNOS/NO,
Fig. 3. ORTEP drawing of compounds 4a and 4b.
C
30H24F3N3O3, Triclinic, space group p-1; a ¼ 8.518 (4), b ¼ 11.528
TNF-a and IL-6. Immunofluorescence and western blot experi-
(6), c ¼ 13.779 (7) (Å);
a
¼ 107.561 (4),
b
¼ 92.912 (5),
g
¼ 101.897
(5) (ꢀ), V ¼ 1252.9 (11) nm3, T ¼ 293 (2) K, Z ¼ 2, Dc ¼ 1.409 g/cm3,
F(000) ¼ 552, Reflections collected/unique ¼ 9057/4584, Data/re-
straints/parameters ¼ 4584/0/352, Goodness of fit on F2 ¼ 1.048,
Fine, R1 ¼ 0.0619, wR (F2) ¼ 0.1199. Crystallographic data (excluding
structure factors) for the structures have been deposited into the
Cambridge Crystallographic Data Center (Registered No. CCDC-
861074 and 861073).
ments will also be performed in an attempt to illustrate above
mechanisms. Finally, we confirmed the anti-inflammatory effects
in vivo by carrageenan method.
2. Results and discussion
2.1. Crystal structure of compounds 4a and 4b
The structure of compounds 4a and 4b were determined by X-
ray crystallography (Fig. 3). Crystallographical data 4a:
2.2. Initial evaluation release of TNF-
a and IL-6
C
26H24F3N3O3, Triclinic, space group p-1; a ¼ 8.976 (7), b ¼ 10.537
The pro-inflammatory mediators (IL-6 and TNF-a) play impor-
(8), c ¼ 15.453 (7) (Å);
a
¼ 101.639 (8),
b
¼ 103.663 (9),
g
¼ 99.998
tant roles in inflammation-related diseases [4,28]. Hence, in the
preliminary anti-inflammatory activity screening studies, the
enzyme-linked immunosorbent assay (ELISA) was used to screen
(9) (ꢀ), V ¼ 1352.9 (18) nm3, T ¼ 293 (2) K, Z ¼ 2, Dc ¼ 1.300 g/cm3,
F(000) ¼ 556, Reflections collected/unique ¼ 9813/4950, Data/re-
straints/parameters ¼ 4950/0/345, Goodness of fit on F2 ¼ 1.013,
Fine, R1 ¼ 0.0749, wR (F2) ¼ 0.1445. Crystallographical data 4b:
the inhibition of all synthetic compounds (4a~4m) toward TNF-
a
and IL-6 release in RAW 264.7 mouse macrophages LPS-induced.

172
L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
The ability (% inhibition) of the tested compounds to reduce
proinflammatory cytokines IL-6 and TNF- was summarized in
Fig. 4 (A and B). Among them, compounds 4f, 4i and 4m exhibited
similar or better TNF- , IL-6 inhibitory activity than other com-
pounds. Particularly, compound 4m exhibited the most potent
inhibitory activity and it's TNF- , IL-6 inhibition rate was 40.38%,
41.17% at the concentration of 10 M. When the concentration of
compound 4m is 5 Me20 M, the IC50 values of TNF- and IL-6
were observed for 16.83 M and 14.18 M, respectively. These re-
(Fig. 8A). LPS could cause a significant phosphorylation of MAPK
(p38, JNK and ERK) when stimulated for 30 min (Fig. 8B, C and 8D).
Compound 4m could concentration-dependently diminish P38
phosphorylation (Fig. 8B), but had little effect on phosphorylation
of JNK or ERK (Fig. 8C and D). These results strongly suggested tha
inhibition of Tak1 and P38 phosphorylation might be responsible
for achieving LPS-induced inflammatory response. Furthermore,
a
a
a
m
m
m
a
compound 4m also could concentration-dependently (2.5
mM,
m
m
5
m
M and 10 M) inhibit P38 phosphorylation LPS-induced (Fig. 8B).
m
sults could be very useful for our next study.
These results suggested that the anti-inflammatory activity of
compound 4m might be associated with its negative effects against
2.3. Compound 4m inhibits of NO production
activation of Tak1/P38/NF-kB.
The pro-inflammatory mediator NO plays an important role in
inflammation-related diseases, also related to expressions of iNOS
and COX-2 [1]. Thus, the inhibitory effects of compound 4m on LPS-
mediated expressions of iNOS and COX-2 were analyzed by West-
2.6. Anti-inflammatory activity in vivo
2.6.1. Carrageenan induced paw edema
The anti-inflammatory activity of some synthesized compounds
was determined by carrageenan induced paw edema method [17].
The results were expressed as% inhibition of oedema over the un-
treated control group. The results were shown in Table 1. Based on
the data, we concluded that the types of substituent had greater
effect on anti-inflammatory activity, ring substitution is more
beneficial to activity (Compound 4i). Fuethermore, introducing of
flavone moiety, afforded compound 4m with a remarkable increase
in anti-inflammatory activity.
ern blot. The cell viability experiment was performed at 10e40
concentration and there was no significantly cytotoxic of com-
pound 4m at the concentration up to 40 M (Fig. 5A). As shown in
Fig. 5A, the LPS (1 g/mL) stimulation for 24 h could markedly
mM
m
m
augment NO production, compound 4m concentration-
dependently suppressed LPS-induced NO generation. In accor-
dance with the inhibitory effect on NO production, compound 4m
was also concentration-dependently reduced expressions of iNOS
(130 kDa) and COX-2 (72 kDa) (Fig. 5B) LPS-induced. This results
preliminary demonstrated that title compound could prevent in-
flammatory response in RAW264.7 cells LPS-induced.
2.6.2. Pathological changes of AA-induced rat ankle joints
In order to confirm the activity of compound 4m in vivo, effect
on pathological changes of AA-induced rat ankle joints was
detected [29]. Photomicrographs of knee joints sections stained
with H&E showed severity of synovial hyperplasia, bone or carti-
lage destruction and inflammatory cells infiltration analyses were
performed to investigate whether compound 4m relieved the his-
tological changes in knee joint (Fig. 9). The results of histological
analysis showed inflammation or bone destruction was not
observed in sections from sham rats. However, AA knee joints
showed many pathological features such as synovial hyperplasia
and inflammatory cell infiltration (Fig. 9B). AA rats treated with
compound 4m (45 mg/kg) exhibited moderate synovial hyperplasia
and cartilage damage, although slight inflammatory cells infiltra-
tion remained (Fig. 9D). In a word, compound 4m could markedly
inhibit bone destruction, synovial hyperplasia and inflammatory
cells infiltration in a dose-dependent manner (Fig. 9C and E).
2.4. Compound 4m suppresses NF-kB activation
NF-
flammatory cytokines [5]. When NF-
B protein phosphorylated and degradated frees NF-k
k
B is one of the principal factors for the production proin-
B activation LPS-stimulated,
B p65 sub-
k
Ik
unit from sequestration, allowing it to translocate to the nucleus,
bind to target promoters, and turn on transcriptions of inflamma-
tory genes including TNF-
pound 4m on I B protein phosphorylation and degradation (Fig. 6)
were screened. Meanwhile it effects of NF- B p65 translocation
a and IL-6. Herein, the effects of com-
k
k
from cytoplasm to nuclei was analyzed by Western blot and
immunofluorescence. Immunofluorescence analysis (Fig. 7)
^
showed that NF-eB subunit P65 was almost exclusively observed in
the cytoplasm in the un-stimulated cells. After stimulation with LPS
(1
nucleus. Nuclear localization of p65 was significantly reduced by
Bay-117082 (20 M). Similar result was also found with compound
mg/mL) for 3 h, most cytoplasmic p65 was translocated into the
3. Conclusions
m
4m at all concentrations examined. Consistent with these findings,
immunoblot showed that the level of p65 was significantly
increased in nuclear after LPS-induction.
In summary, based on finding efficient compounds with anti-
inflammatory activity, thirteen pyrazoline coumarin derivatives
were synthesized. The results of initial evaluation showed that
some compounds exhibited better TNF-
a and IL-6 inhibitory ac-
2.5. Compound 4m inhibits P38 signaling activation
tivity. Among them, 3-(1-(2-(3-(4-methoxyphenyl)-4-oxo-4H-
chromen-7-yloxy)acetyl)-5-(4-(trifluoromethyl)phenyl)-4,5-
dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one (Compound 4m)
showed the highest anti-inflammatory activity with inhibition IL-6
MAPK were quite significant in the regulation of inflammation
because of their crucial roles in the mediation of the production of
NO, TNF-
a
, IL-6, IL-
b
and other inflammatory mediators. In addi-
and TNF-
tion indicated that title compound could significantly suppress
expressions of iNOS, COX-2 and productions of IL-6, TNF- , NO
through NF- B/MAPK signaling pathway in concentration
a. The preliminary mechanism of anti-inflammatory ac-
tion, Tak1 is traditionally accepted as the primary LPS receptor and
has been reported as critical for the inflammatory response to LPS.
Upon stimulation with LPS, Tak1 initiates a series of signaling cas-
a
k
a
cades result in activating NF-
pro-inflammatory cytokines [6]. Recent studies found that inhibi-
tion of Tak1 could decrease the expressions of IL-6, IL-1 and TNF-a.
So we investigated whether compound 4m inhibited expression of
Tak1 by Western blot. The results confirmed that P-Tak1 expression
was strongly up-regulated in LPS-induced RAW264.7 cells, which
was concentration-dependently reversed with compound 4m
k
B and MAPK to induce the release of
dependent manner.
4. Experimental section
4.1. Chemistry
b
The reactions were monitored by thin layer chromatography

L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
173
Fig. 4. Initial evaluation release of TNF-
RAW264.7 cells were precultured for 24 h, the cells were then treated with the indicated concentrations of compounds for 1 h, and then exposed to 1
of TNF- and IL-6 in the culture medium were measured by ELISA. (A and B) Cells were treated with 10
compound 4m. TNF- and IL-6 levels in the medium were determined with an ELISA kit (eBioScience, Inc.) according to the manufacturer's instructions. The total amount of the
inflammatory factor in the medium was normalized to the total protein quantity of the viable cell pellets.
a and IL-6.
mg/mL LPS for 24 h. The levels
a
mM compounds. (C) Cells were pretreated with different concentrations of
a

Fig. 5. Compound 4m inhibited LPS-induced inflammatory response.
Treated with compound 4m, RAW 264.7 cells were stimulated with LPS (1
m
g/mL) for 24 h. Cell viability was evaluated using the MTT assay. NO production was measured using
M concentration and there
M; Quantitative analysis of NO production; (B) Quantitative analysis of iNOS and COX-2 expressions.
-actin was used as loading control; ###p < 0.001 compared with unstimulated cells, ***p < 0.001, *p < 0.05 compared with LPS-stimulated cells; Data were obtained by at least
three independent experiments, and each was performed in duplicate.
nitrite and nitrate assay. iNOS and COX-2 expressions were detected by Western blot analysis. (A) Cell viability experiment was performed at 12.5e50
was no significantly cytotoxic of compound 4m at the concentration up to 40
m
m
b

L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
175
Fig. 6. Compound 4m suppressed activation of NF-
kB.
Treated with compound 4m (2.5e10 M), RAW 264.7 cells were stimulated with LPS (1
m
mg/mL) for 30 min. P-IKB IKB, p-p65, P65 and
b-actin were detected with their antibodies,
respectively. The induction fold of the phosphorylated kinase was calculated as the intensity of the treatment relative to that of control normalized to a-tubulin by densitometry. Bay
11-7082 is the NF- B inhibitor. ###p < 0.001 compared with unstimulated cells, ***p < 0.001, **p < 0.01, *p < 0.05 compared with LPS-stimulated cells; The blots shown are the
k
examples of three separate experiments.
(TLC) on pre-coated silica GF254 plates. Melting points are uncor-
rected. 1H NMR spectra was recorded on a Brucker AM-400
(400 MHz) spectrometer with CDCl3 and Acetone-d6 as the sol-
vent. Elemental analyses were performed on a CHN-O-Rapid in-
strument and were within 0.4% of the theoretical values.
4.2. General procedure for preparation of compounds 4a~4m
3-(1-(2-bromoacetyl)-5-(2 or 4-(trifluoromethyl)phenyl)-4,5-
dihydro-1H-pyrazol-3- yl)-2H-chromen-2-one (compound 3)
(10 mmol) was dissolved in acetone (20 mL), 7-hydroxy-3-(4-
methoxyphenyl)-4H-chromen-4-one or amine (15 mmol) was
added at room temperature, piperazine (15 mmol) was added, the

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L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
Fig. 7. Compound 4m exhibited potent inhibitory effect on nuclear translocation of P65.
Compound 4m exhibited potent inhibitory effect on nuclear translocation of P65 in LPS-stimulated RAW264.7 cells. Cells were treated with compound 4m for 1 h, followed by
stimulation with LPS for 3 h. The subcellular localization of P65 was determined by immunofluorescence.
mixture was allowed to stand at 40e50 ꢀC for 4e6 h. The product
was collected by filtration and the residue was purified by chro-
matography (dichloromethane/methanol, v/v ¼ 65:1) to give title
compounds 4a~4m (Scheme 1) as colorless solids
H), 8.71 (s, 1H, coumarin, 4-H); 13C NMR (125 MHz, CDCl3): 22.38
(tetra-hydroquinoline, 3-C), 28.07 (tetra-hydroquinoline, 4-C),
44.47 (pyrazole, 4-C), 50.84 (tetra-hydroquinoline, 2-C), 53.53 (N-
CH2-carbonyl), 57.48 (pyrazole, 5-C), 151.07 (coumarin, O-C), 154.27
(pyrazole, 3-C), 159.04 (coumarin, C¼O), 168.05 (pyrazolyl, -C¼O).
Anal. Calcd for: C30H24F3N3O3: C, 67.79; H, 4.55; N, 7.91%. Found: C,
68.08; H, 4.19; N, 8.29%.
4a: Mp 186-187 ꢀC, colorless crystals, yield, 81%; 1H NMR
(400 MHz, CDCl3): 1.44 (s, 2H, piperidine, 4-H), 1.63 (brs, 4H,
Piperidine, 3-H and 5-H), 2.62 (brs, 4H, piperidine, 2-H and 6-H),
3.28 (dd, 1H, J ¼ 19.5 and 6.8 Hz, pyrazole, 4-Ha), 3.67 (d, 1H,
J ¼ 17.1 Hz, COCH2), 3.81 (d, 1H, J ¼ 16.7 Hz, COCH2), 3.95 (dd, 1H,
J ¼ 19.4, 12.0 Hz, pyrazole, 4-Hb), 5.94 (dd, 1H, J ¼ 11.8, 4.9 Hz,
pyrazole, 5-H), 7.15e7.68 (m, 8H, Ar-H), 8.41 (s, 1H, coumarin, 4-H);
13C NMR (125 MHz, CDCl3): 24.14 (piperidine 4-C), 26.07 (piperi-
dine 3-C and 5-C), 44.36 (pyrazole, 4-C), 54.84 (piperidine 3-C and
5-C), 58.48 (N-CH2-carbonyl), 60.14 (pyrazole, 5-C), 150.45
(coumarin, O-C), 154.21 (pyrazole, 3-C), 159.06 (coumarin, C¼O),
168.04 (pyrazolyl-C¼O). Anal. Calcd for: C26H24F3N3O3: C, 64.59; H,
5.00; N, 8.69%. Found: C, 64.80; H, 4.71; N, 9.04%.
4c: Mp 143-144 ꢀC, colorless crystals, yield, 73%; 1H NMR
(600 MHz, DMSO-d6)
d 8.48 (s, 1H, coumarin, C4-H), 7.78e7.71 (m,
1H), 7.65 (m, 2H), 7.57e7.59 (m, 1H), 7.43e7.40 (m, 2H), 7.33e7.32
(m, 1H), 7.28e7.29 (m, 1H), 5.57 (dd, J ¼ 12.1, 5.1 Hz, 1H, pyrazole, 5-
H), 4.03e3.96 (m, 2H), 3.86e3.81 (m, 1H), 3.20e3.07 (m, 2H), 2.48
(s, 3H), 0.97 (d, J ¼ 6.4 Hz, 3H). 13C NMR (151 MHz, DMSO-d6)
d
¼ 170.27 (pyrazolyl-C¼O), 158.67 (coumarin, C¼O), 154.31, 151.38,
147.65, 142.87, 133.94, 130.23, 128.90, 127.32, 127.30, 126.53, 126.02,
125.73, 124.22, 119.63, 119.47, 116.90, 60.17, 53.42, 52.55, 44.19,
41.67, 21.49 (CH3). Anal. Calcd for: C24H22F3N3O3: C, 63.01; H, 4.85;
N, 9.19%.> Found: C, 62.89; H, 5.12; N, 8.88%.
4b: Mp 199-200 ꢀC, colorless crystals, yield, 79%; 1H NMR
(500 MHz, Acetone-d6): 1.90e1.97 (m, 2H, tetra-hydroquinoline, 3-
H), 2.72 (t, 2H, J ¼ 6.3 Hz, tetra-hydroquinoline, 4-H), 3.36e3.44 (m,
2H, tetra-hydroquinoline, 2-H), 3.48 (dd, 1H, J ¼ 11.4 and 5.7 Hz,
pyrazole, 4-Ha), 4.10 (dd, 1H, J ¼ 18.8 and 12.1 Hz, pyrazole, 4-Hb),
4.51 (d, 1H, J ¼ 17.8 Hz, COCH2), 4.69 (d, 1H, J ¼ 17.7 Hz, COCH2), 5.74
(dd, 1H, J ¼ 12.2 and 5.2 Hz, pyrazole, 5-H), 6.47e7.87 (m, 12H, Ar-
4d: Mp 200-202 ꢀC, colorless crystals, yield, 77%; 1H NMR
(400 MHz, CDCl3): 1.13 (s, 9H, 3CH3), 1.88 (brs, 1H, NH), 3.30 (dd,1H,
J ¼ 19.4 and 5.2 Hz, pyrazole, 4-Ha), 3.83e3.99 (m, 3H, COCH2 and
pyrazole, 4-Hb), 5.61 (dd, 1H, J ¼ 11.9 and 4.8 Hz, pyrazole, 5-H),
7.14e7.66 (m, 8H, Ar-H), 8.45 (s, 1H, coumarin, 4-H); 13C NMR
(125 MHz, CDCl3): 28.99 (CH3), 44.51 (pyrazole, 4-C), 45.08 (N-CH2-

L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
177
Fig. 8. Compound 4m suppressed activation of MAPK signaling pathway.
RAW264.7 cells were treated with compound 4m (2.5 Me10 M) and LPS (1
m
m
mg/mL) for 30 min. The levels of P-Tak1/Tak1, P-P38/P38, P-ERK/ERK, P-JNK/JNK and
b-actin proteins,
and their phosphorylated forms were analyzed using western blotting. The results were showed as means SD (n ¼ 3) of at least three independent experiments. ###p < 0.001
compared with the control group; *p < 0.05, **p < 0.01, ***p < 0.001 compare with LPS-stimulated.

178
L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
8.44 (s, 1H, coumarin, 4-H). 13C NMR (125 MHz, CDCl3): 17.33 (CH2),
45.02 (pyrazole, 4-C), 45.33 (N-CH2-carbonyl), 99.89 (CH), 134.03
(CH2-N), 151.95 (coumarin, O-C), 154.20 (pyrazole, 3-C), 159.34
(coumarin, C¼O), 171.22 (pyrazolyl-C¼O). Anal. Calcd for:
Table 1
The anti-inflammatory activity at different time intervals and ulcer indices.
Compounds
% Of edema inhibition (% mean SEM)
1 h
3 h
5 h
C
27H24F3N3O3: C, 65.45; H, 4.88; N, 8.48%. Found: C, 65.11; H, 5.17;
4c
4f
4i
4m
Diclofenac
Celecoxib
17 0.35
28 0.70
32 0.61
49 0.55
41 0.51
55 0.75
20 0.41
49 1.50
41 0.80
60 0.49
77 1.33
59 1.28
13 0.65
45 0.99
36 0.71
59 0.88
48 1.89
61 1.30
N, 8.80%.
4g: Mp 152-153 ꢀC, colorless crystals, yield, 79%; 1H NMR
(500 MHz, CDCl3): 0.88e0.90 (m, 12H, 4CH3), 1.71e1.79 (m, 2H,
2CH), 2.38e2.45 (m, 4H, 2CH2), 3.27 (dd, 1H, J ¼ 19.2 and 5.8 Hz,
pyrazole, 4-Ha), 3.72 (d, 1H, J ¼ 16.0 Hz, COCH2), 3.93 (dd, 1H,
J ¼ 18.9 and 12.3 Hz, pyrazole, 4-Hb), 4.00 (d, 1H, J ¼ 16.0 Hz,
COCH2), 5.93 (dd, 1H, J ¼ 12.3 and 5.8 Hz, pyrazole, 5-H), 7.14e7.68
(m, 8H, Ar-H), 8.43 (s, 1H, coumarin, 4-H). 13C NMR (125 MHz,
Values are expressed as the mean S.D (n ¼ 5).
Control: 0.5% sodium CMC solution in distilled water (10 mL kgꢁ1, p.o.).
Compounds and positive drugs were administered at a dose of 0.03 mmol kgꢁ1, p.o.
in 0.5% sodium CMC solution.
Fig. 9. Effect of compound 4m on pathological changes of AA-induced rat ankle joints by HE staining (magnification ꢂ 100).
Effect of compound 4m on pathological changes of AA-induced rat ankle joints by HE staining (magnification ꢂ 100).
(A) Normal; (B) AA; (C) Aspirin 50 mg/kg; (D) Compound 4 m 45 mg/kg; (E) Compound 4 m 90 mg/kg.
AA, adjuvant arthritis; Aspirin used as the positive control.
carbonyl), 50.35 (N-tertiary-C), 57.52 (pyrazole, 5-C), 151.17
(coumarin, O-C), 154.26 (pyrazole, 3-C), 159.10 (coumarin, C¼O),
170.20 (pyrazolyl-C¼O). Anal. Calcd for: C25H24F3N3O3: C, 63.69; H,
5.13; N, 8.91%. Found: C, 64.02; H, 5.10; N, 9.22%.
CDCl3): 9.87 (CH3), 18.92 (CH3), 28.77 (CH2), 45.33 (pyrazole, 4-C),
45.62 (N-CH2-carbonyl), 55.10 (CH), 151.60 (coumarin, O-C), 154.67
(pyrazole, 3-C), 160.10 (coumarin, C¼O), 171.00 (pyrazolyl-C¼O).
Anal. Calcd for: C29H32F3N3O3: C, 66.02; H, 6.11; N, 7.96%. Found: C,
65.80; H, 5.94; N, 8.11%.
4e: Mp 231-232 ꢀC, colorless crystals, yield, 70%; 1H NMR
(400 MHz, DMSO-d6): 3.02 (dd, 1H, J ¼ 18.4 and 5.2 Hz, pyrazole, 4-
Ha), 3.76 (dd, 1H, J ¼ 18.6 and 12.3 Hz, pyrazole, 4-Hb), 3.84 (d, 1H,
J ¼ 16.6 Hz, COCH2), 3.98 (s,1H, NH), 4.15 (d,1H, J ¼ 16.6 Hz, COCH2),
5.64 (dd, 1H, J ¼ 12.3 and 5.5 Hz, pyrazole, 5-H), 6.09e6.40 (m, 2H,
Furfuryl), 7.15 (d, 1H, J ¼ 8.1 Hz, furan, 3-H), 7.19 (t, 1H, J ¼ 7.2 Hz,
furan, 4-H), 7.26 (d, 1H, J ¼ 8.0 Hz, furan, 5-H), 7.44e7.75 (m, 8H, Ar-
H), 8.46 (s, 1H, coumarin, 4-H). 13C NMR (125 MHz, CDCl3): 44.98
(pyrazole, 4-C), 45.17 (N-CH2-carbonyl), 51.08 (CH2), 51.97 (CH2-
Furan), 57.65 (pyrazole, 5-C), Furan-C (107.11, 110.79, 143.00,
149.10), 151.80 (coumarin, O-C), 154.45 (pyrazole, 3-C), 159.02
(coumarin, C¼O), 170.89 (pyrazolyl-C¼O). Anal. Calcd for:
4h: Mp 118-119 ꢀC, colorless crystals, yield, 80%; 1H NMR
(400 MHz, DMSO-d6): 0.78 (t, 6H, J ¼ 19.4 Hz, 2CH3), 0.92e2.47 (m,
16H, 8CH2), 3.06 (dd, 1H, J ¼ 18.3 and 4.6 Hz, pyrazole, 4-Ha), 3.63
(dd, 1H, J ¼ 16.3 and 10.3 Hz, pyrazole, 4-Hb), 3.85e3.91 (m, 2H,
COCH2), 5.72 (dd, 1H, J ¼ 12.0 and 4.6 Hz, pyrazole, 5-H), 7.14e7.82
(m, 8H, Ar-H), 8.55 (s, 1H, coumarin, 4-H). 13C NMR (125 MHz,
CDCl3): 13.61 (CH3), 22.04 (CH2), 26.05 (CH2), 30.11 (CH2), 45.11
(pyrazole, 4-C), 45.91 (N-CH2-carbonyl), 151.14 (coumarin, O-C),
154.40 (pyrazole, 3-C), 160.31 (coumarin, C¼O), 171.22 (pyrazolyl-
C¼O). Anal. Calcd for: C31H36F3N3O3: C, 67.01; H, 6.53; N, 7.56%.
Found: C, 66.91; H, 6.26; N, 7.40%.
C
26H20F3N3O4: C, 63.03; H, 4.07; N, 8.48%. Found: C, 63.41; H, 3.79;
N, 8.20%.
4f: Mp 120-121 ꢀC, colorless crystals, yield, 65%; 1H NMR
4i: Mp 204-205 ꢀC, colorless crystals, yield, 82%; 1H NMR
(400 MHz, CDCl3): 1.45e1.64 (m, 6H, piperidine, 3-H, 4-H and 5-H),
2.64 (brs, 4H, piperidine, 2-H and 6-H), 3.40 (dd, 1H, J ¼ 19.1 and
5.1 Hz,pyrazole, 4-Ha), 3.72 (m, 2H, COCH2), 3.98 (dd, 1H, J ¼ 19.2
and 12.2 Hz, pyrazole, 4-Hb), 5.62 (dd, 1H, J ¼ 12.2 and 5.1 Hz,
pyrazole, 5-H), 7.32e7.65 (m, 8H, Ar-H), 8.45 (s, 1H, coumarin, 4-H);
13C NMR (125 MHz, CDCl3): 22.39 (piperidine 4-C), 28.09 (piperi-
dine 3-C and 5-C), 43.88 (pyrazole, 4-C), 50.80 (piperidine 3-C and
(400 MHz, CDCl3) 3.29 (dd, 1H, J ¼ 19.1 and 5.5 Hz, pyrazole, 4-Ha),
3.38 (d, 4H, J ¼ 4.2 Hz, 2 ¼ CH2), 3.79e3.85 (m, 2H, 2CH ¼ ), 3.94
(dd,1H, J ¼ 19.0 and 12.2 Hz, pyrazole, 4-Hb), 4.40 (d,1H, J ¼ 17.5 Hz,
COCH2), 4.83 (d, 1H, J ¼ 17.5 Hz, COCH2), 5.21 (m, 4H, 2NCH2), 5.93
(dd, 1H, J ¼ 11.9 and 5.0 Hz, pyrazole, 5-H), 7.05e7.26 (m, 8H, Ar-H),

L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
179
Scheme 1. Synthesis of compounds 4a~4m. Reagent and conditions: (A) piperazine, 20e30 ꢀC, 20 min (B) 2 or 4-(trifluoromethyl) benzaldehyde, piperidine, butanol, reflux, 14 h
(C). NH2-NH2. H2O, C2H5OH, BrCH2COOH, 4-Nitrobenzenesulfonyl chloride 40e60 ꢀC, 2 h (D) flavone or amine, 40e50 ꢀC, 4e6 h.
5-C), 53.49 (N-CH2-carbonyl), 60.58 (pyrazole, 5-C), 151.35
(coumarin, O-C), 154.30 (pyrazole, 3-C), 159.25 (coumarin, C¼O),
168.44 (pyrazolyl-C¼O). Anal. Calcd for: C26H24F3N3O3: C, 64.59; H,
5.00; N, 8.69%. Found: C, 64.22; H, 5.33; N, 8.72%.
(151 MHz, DMSO-d6)
d
¼ 167.89, 158.33, 153.97, 151.69, 146.97,
145.57, 142.84, 133.59, 129.81, 129.11, 128.49, 128.28, 127.15, 126.87,
126.05, 125.52, 125.43, 123.71, 122.22, 119.27, 119.11, 116.57, 116.06,
110.63, 59.95, 52.89, 50.35, 43.82, 27.92, 22.24. Anal. Calcd for:
4j: Mp 205-206 ꢀC, colorless crystals, yield, 82%; 1H NMR
C
30H24F3N3O3: C, 67.79; H, 4.55; N, 7.91%. Found: C, 68.12; H, 4.88;
N, 8.27%.
4k: Mp 64-66 ꢀC, colorless crystals, yield, 80%; 1H NMR
(600 MHz, DMSO-d6)
d
8.71 (s, 1H, C4-H), 7.90 (d, J ¼ 6.9 Hz, 1H),
7.71e7.74 (M, 3H), 7.53e7.42 (m, 4H), 6.94 (t, J ¼ 7.5 Hz, 1H), 6.89 (d,
J ¼ 7.2 Hz, 1H), 6.50 (t, J ¼ 7.2 Hz, 1H), 6.43 (d, J ¼ 8.2 Hz, 1H), 5.69
(dd, J ¼ 12.0, 5.0 Hz, 1H, pyrazole, 5-H), 4.71 (d, J ¼ 17.9 Hz, 1H), 4.51
(d, J ¼ 17.9 Hz, 1H), 4.01 (dd, J ¼ 18.6, 12.1 Hz, 1H, pyrazole, 4-Ha),
3.45e3.41 (m, 1H), 3.37e3.34 (m, 1H), 3.28 (dd, J ¼ 18.6, 5.1 Hz, 1H,
pyrazole, 4-Hb), 2.71 (t, J ¼ 6.1 Hz, 2H), 1.96e1.84 (m, 2H). 13C NMR
(600 MHz, DMSO-d6) 8.63 (s, 1H), 7.89 (d, J ¼ 7.7 Hz, 1H), 7.77e7.69
(m, 3H), 7.52e7.40 (m, 4H), 7.04e6.99 (m, 1H), 5.67 (dd, J ¼ 12.1,
4.9 Hz, 1H), 3.96 (dd, J ¼ 18.5, 12.2 Hz, 1H), 3.86 (d, J ¼ 15.9 Hz, 1H),
3.69 (d, J ¼ 15.9 Hz, 1H), 3.24 (dd, J ¼ 18.5, 4.9 Hz, 1H), 1.47e1.37 (m,
4H), 1.30e1.18 (m, 12H), 0.84 (t, J ¼ 6.7 Hz, 6H). 13C NMR (151 MHz,

180
L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
DMSO-d6)
d
¼ 163.20, 159.06, 158.26, 153.90, 151.11, 142.45, 133.51,
absorbance at 570 nm was measured by a microplate reader
(MQX200, Bio-Tek, USA) [31].
131.24, 129.76, 126.83, 126.01, 125.38, 120.04, 119.38, 119.07, 118.63,
116.96, 116.54, 59.58, 54.48, 54.29, 31.68, 27.67, 26.92, 22.58, 14.32.
Anal. Calcd for: C33H40F3N3O3: C, 67.91; H, 6.91; N, 7.20%. Found: C,
68.16; H, 7.00; N, 6.84%.
4.6. Assay for NO production
4l: Mp 217-219 ꢀC, colorless crystals, yield, 82%; 1H NMR
(400 MHz, CDCl3): 2.44 (t, 4H, J ¼ 5.1 Hz, morpholine, 2-H and 6-H),
3.61e3.69 (m, 4H, morpholine, 3-H and 5-H), 3.35 (dd, 1H, J ¼ 19.2
and 5.4 Hz, pyrazole, 4-Ha), 3.62 (d, 1H, J ¼ 17.2 Hz, COCH2), 3.75 (d,
1H, J ¼ 16.6 Hz, COCH2), 3.90 (dd, 1H, J ¼ 18.8 and 12.0 Hz, pyrazole,
4-Hb), 5.65 (dd, 1H, J ¼ 12.0 and 5.2 Hz, pyrazole, 5-H), 7.22e7.57
(m, 8H, Ar-H), 8.46 (s, 1H, coumarin, 4-H); 13C NMR (125 MHz,
CDCl3): 43.75 (pyrazole, 4-C), 53.61 (morpholine, 3-C and 5-C),
67.01 (morpholine, 2-C and 6-C), 60.59 ((N-CH2-carbonyl)), 60.88
(pyrazole, 5-C), 151.65 (coumarin, O-C), 154.19 (pyrazole, 3-C),
160.10 (coumarin, C¼O), 168.46 (pyrazolyl-C¼O). Anal. Calcd for:
NO production was quantified by nitrite accumulation in the
culture medium using the Griess reaction. Briefly, RAW264.7 cells
were pretreated with compounds for 1 h, and then stimulated with
or without LPS (1 mg/mL) for 24 h. The isolated supernatants were
mixed with an equal volume of Griess reagent (Beyotime Biotech-
nology, China). NaNO2 was used to generate a standard curve, and
nitrite production was determined by measuring the optical den-
sity at 540 nm by a microplate reader (MQX200, Bio-Tek, USA) [31].
4.7. Measurement of cytokine production
C
25H22F3N3O4: C, 61.85; H, 4.57; N, 8.66%. Found: C, 62.12; H, 4.60;
N, 8.94%.
4m: Mp 232-234 ꢀC, yield, 80%; 1H NMR (400 MHz, CDCl3): 3.52
Cytokine production was measured by Enzyme-Linked
Immuno-Sorbent Assay (ELISA). In brief, RAW264.7 macrophage
cells (7 ꢂ 104 cells/well) were plated in 24-well plates. After incu-
bation for 24 h, cells were starved by being cultured in serum-free
medium for another 2.5 h to eliminate FBS influence. The cells were
(dd, 1H, J ¼ 19.3 and 5.2 Hz, pyrazole, 4-Ha), 3.84 (s, 3H, OCH3), 4.07
(dd, 1H, J ¼ 19.3 and 12.1 Hz, pyrazole, 4-Hb), 5.26 (q, 2H,
J ¼ 15.8 Hz, COCH2O), 5.67 (dd, 1H, J ¼ 12.0 and 5.1 Hz, pyrazole, 5-
H), 6.86 (d, 1H, J ¼ 2.3 Hz, isoflavones, 8-H), 6.96 (d, 2H, J ¼ 8.8 Hz,
isoflavones, 30, 50-H), 7.06 (dd, 1H, J ¼ 8.9 and 2.3 Hz, isoflavones, 6-
H), 7.36e7.40 (m, 4H, Ar-H), 7.49 (d, 2H, J ¼ 8.7 Hz, isoflavones, 20,
60-H), 7.59e7.66 (m, 4H, Ar-H), 7.89 (s, 1H, isoflavones, 2-H), 8.22 (d,
1H, J ¼ 9.0 Hz, isoflavones, 5-H), 8.47 (s, 1H, coumarin, 4-H). 13C
then treated with compounds for 1 h before exposure to 1
LPS for 24 h. The culture medium was used to assay the cytokine
production with mouse ELISA kit (TNF- : DY410-05; IL-6: DY406-
mg/mL
a
05) according to manufacturer's instructions [31].
4.8. Western blotting
NMR (151 MHz, DMSO-d6)
d
¼ 175.01, 165.07, 162.87, 161.26, 159.44,
158.42, 157.57, 154.04, 153.93, 152.70, 146.53, 143.13, 133.74, 130.49,
129.85, 128.62, 128.41, 127.38, 127.04, 126.09, 125.50, 124.44,123.80,
123.71, 119.15, 119.04, 118.30, 116.63, 115.29, 114.06, 102.06, 66.42,
60.04, 55.59, 43.88. Anal. Calcd for: C37H25F3N2O7: C, 66.67; H, 3.78;
N, 4.20%. Found: C, 67.02; H, 4.15; N, 3.81%.
Western blotting assay was performed as described previously.
Briefly, RAW264.7 cells (2 ꢂ 106 cells/well) were cultured in 6 cm
dishes for 24 h, cells were starved by being cultured in serum-free
medium for another 2.5 h to eliminate influence of FBS. The cells
were then treated with or without compounds for 1 h before
exposure to LPS for the indicated times. The cells were lysed in
4.3. Crystallographic studies
240 mL RIPA cell lysis buffer (Beyotime china), and incubated on ice
for 30 min. Approximate 40 mg of proteins were run on 10%
SDSPAGE and then transferred to PVDF membrane (GE Healthcare,
UK). The blotted membrane was incubated with specific primary
antibody for overnight at 4 ꢀC and further incubated for 1 h with
HRP-conjugated secondary antibody [29].
A colorless single crystal of compounds 4a and 4b were chosen
for X-ray diffraction analysis performed on a BRUCKER SMART
APEX-CCD diffractometer equipped with
chromatic MoKa radiation (
¼ 0.71073 A). A total reflections were
collected in the range of 1.46< scan mode
< 25.50ꢀ by using a
with independent ones, of which I > 2 (I) were observed and used
a graphite mono-
l
q
j-u
s
4.9. Immunofluorescence analysis
in the succeeding refinements. The data set was corrected by
SADABS program; the structures were solved by direct methods
with SHELXS-97 and refined by full-matrix least-squares method
on F2 [30].
RAW264.7 cells were grown on glass coverslip in six well plates,
fixed with 4% paraformaldehyde (w/v) for 20 min at room tem-
perature and blocked for 1 h with 5% BSA in TBS containing 0.1%
Triton X-100. Then, the cells were incubated with a primary anti-
body, followed by Alexa Fluor 488-labeledgoatanti-rabbit IgG. After
a wash step, they were stained with DAPI and the images were
acquired.
4.4. Cell culture
Murine monocyte-macrophage RAW264.7 cells maintained in
Dulbecco's modified Eagle's medium (DMEM, Hyclone, Miami, FL,
USA) supplemented with 10% fetal bovine serum (Beyotime), 100
units/mL penicillin, and 100 mg/mL streptomycin and incubated at
37 ꢀC in a humidified atmosphere containing 5% CO2. Mouse peri-
toneal macrophages purchased from BeNa Culture Collection
Company.
4.10. Animals
Male Sprague-Dawley (SD) rats weighing 180-200 g were ob-
tained from Animal Department of Anhui Medical University. After
one week acclimatization, rats were randomly divided into five
groups (8 rats per group): normal, AA (adjuvant arthritis), 4 m (45,
90 mg/kg) and aspirin (50 mg/kg). All experiments and animal care
procedures were approved by the Animal Resource Center of Anhui
Medical University in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. Complete
Freund's adjuvant (CFA) was prepared by suspending heat-killed M.
butyricium in sterile paraffin oil (10 mg/mL). Rat AIA was induced
by a single intradermal injection of 0.1 mL CFA into the right hind
paw. The normal group inject with saline. 4 m or APC in 0.5%
4.5. Cell viability assay
Cell cytotoxicity was evaluated by MTT assay as previous re-
ported. The medium was changed before the assay. MTT dissolved
in phosphate buffered saline (PBS) and was added to the culture
medium to reach a final concentration of 0.5 mg/mL. After incu-
bation at 37 ꢀC for 4 h, the culture media containing MTT were
removed, and then DMSO was added into each well and the

L.Z. Chen et al. / European Journal of Medicinal Chemistry 138 (2017) 170e181
181
carboxymethylcellulose sodium (CMC-Na) solution at concentra-
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Supporting information
CCDC-861074 and 861073 (compounds 4a and 4b) contains the
supplementary crystallographic data for this paper. These data can
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conts/retrieving.html (or from the CCDC,12 Union Road, Cambridge
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ac.uk).
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Appendix A. Supplementary data
[30] G.M. Sheldrick, SHELXTL-97, Program for Crystal Structure Solution and
€
€
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