Novel paeonol derivatives: Design, synthesis and anti-inflammatory activity in vitro and in vivo

Article abstract of DOI:10.1016/j.bioorg.2020.103735

Paeonol has been proved to have potential anti-inflammatory activity, but its clinical application is not extensive due to the poor anti-inflammatory activity (14.74% inhibitory activity at 20 μM). In order to discover novel lead compound with high anti-inflammatory activity, series of paeonol derivatives were designed and synthesized, their anti-inflammatory activities were screened in vitro and in vivo. Structure-activity relationships (SARs) have been fully concluded, and finally (E)-N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)-3-(3,4,5-trimet-hoxyphenyl)acrylamide (compound 11a) was found to be the best active compound with low toxicity, which showed 96.32% inhibitory activity at 20 μM and IC₅₀ value of 6.96 μM against LPS-induced over expression of nitric oxide (NO) in RAW 264.7 macrophages. Preliminary mechanism studies indicated that it could inhibit the expression of TLR4, resulting in inhibiting of NF-κB and MAPK pathways. Further studies have shown that compound 11a has obvious therapeutic effect against the adjuvant-induced rat arthritis model.

Full text of DOI:10.1016/j.bioorg.2020.103735

BioorganicChemistry98(2020)103735
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel paeonol derivatives: Design, synthesis and anti-inflammatory activity
in vitro and in vivo
Yang Sheng Hu, Xu Han, Pei Jing Yu, Ming Ming Jiao, Xin Hua Liu⁎, Jing Bo Shi⁎
School of Pharmacy, Anhui Medical University, Hefei 230032, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Paeonol has been proved to have potential anti-inflammatory activity, but its clinical application is not extensive
due to the poor anti-inflammatory activity (14.74% inhibitory activity at 20 μM). In order to discover novel lead
compound with high anti-inflammatory activity, series of paeonol derivatives were designed and synthesized,
their anti-inflammatory activities were screened in vitro and in vivo. Structure-activity relationships (SARs) have
been fully concluded, and finally (E)-N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)-3-(3,4,5-trimet-hoxyphenyl)
acrylamide (compound 11a) was found to be the best active compound with low toxicity, which showed 96.32%
inhibitory activity at 20 μM and IC50 value of 6.96 μM against LPS-induced over expression of nitric oxide (NO)
in RAW 264.7 macrophages. Preliminary mechanism studies indicated that it could inhibit the expression of
TLR4, resulting in inhibiting of NF-κB and MAPK pathways. Further studies have shown that compound 11a has
obvious therapeutic effect against the adjuvant-induced rat arthritis model.
Paeonol derivatives
Nitric oxide
Inhibit
Anti-inflammatory
1. Introduction
activity.
Rheumatoid arthritis (RA) is an autoimmune disease, which is
Paeonol, 1-(2-hydroxy-4-methoxyphenyl)ethan-1-one, the major
phenolic component of moutan cortex radicis, has been used as an anti-
inflammatory drug in China [1,2]. In addition, paeonol has also been
found to have other biological effects, such as antitumor [3,4], anti-
hepatitis B virus [5], antibacterial [6], antidiabetic [7] and antioxidant
[8] activity. Currently, paeonol has been used clinically as an anti-in-
flammatory drug approved by the CFDA (China Food and Drug Ad-
ministration), including various dosage forms, tablet, ointment, ad-
hesive and injection [2]. However, paeonol was not widely known in
the treatment of inflammatory diseases compared to other anti-in-
flammatory drugs in China, such as indometacin, celecoxib, asprin, etc.
Among various dosage forms, paeonol ointment is used for the treat-
ment of dermatitis. In order to expand paeonol use, we focus on this
moiety. Preliminary experimental studies show its anti-inflammatory
activity is extremely poor (14.74% inhibitory activity at 20 μM).
Herein, to improve its anti-inflammatory activity, it is of significance to
design and synthesize novel efficient paeonol-based derivatives. As a
classic drug design method, fragment-based drug design (FBDD) is one
of the way to obtain highly active compounds [9]. Based on paeonol
moiety, through structural optimizations (see 2.1 Design and 2.5
Structure-activity relationships), we will describe our efforts toward the
discovery of novel paeonol derivatives with high anti-inflammatory
characterized by chronic progressive inflammatory disease of joints and
surrounding tissues, eventually leads to irreversible joint damage [10].
It is widely accepted that the change of inflammatory cytokines con-
centration is related to the occurrence of RA [11]. More and more
evidence show that the content of pro-inflammatory factors in serum
and synovial fluid of RA patients is very high [12]. A large number of
inflammatory factors promote the entry of inflammatory cells into tis-
sues, aggravating the destruction of joints, which eventually leads to
the occurrence of arthritis [13]. At present, the conventional therapy
and biological therapy can only partially relieve RA [14]. For most
patients, there is still an urgent need to discover new drugs. It has been
reported that paeonol can trigger innate immune system by affecting
TLR4, and then inhibit the release of pro-inflammatory cytokines in-
duced by nuclear factor (NF-κB) and mitogen activated protein kinase
(MAPK) [15–17]. Meanwhile, paeonol can reverse the excessive pro-
duction of inducible nitric oxide synthase (iNOS) [18].
2. Results and discussion
2.1. Design
Huang et al. [19] etherified 2-hydroxyl group of paeonol with long-
⁎ Corresponding authors.
E-mail addresses: xhliuhx@163.com (X.H. Liu), sjbo616@126.com (J.B. Shi).
https://doi.org/10.1016/j.bioorg.2020.103735
Received 17 January 2020; Received in revised form 17 February 2020; Accepted 6 March 2020
Availableonline08March2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
Fig. 1. The process of design and analysis. Nimesulide (A) and diclofenac acid (B) contain a two-benzene ring scaffold linked by O or NH (red section) α, β-
unsaturated amide structure including compound C, D, E (blue section). (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
chain fatty alkane and found that some alkyl ether analogues of bro-
mopaeonol could improve the anti-inflammatory activity. In order to
observe whether the aromatic system has an effect on the activity, we
introduced a benzene ring through a short linker and performed a two-
step optimization (step 1, step 2) at first (Fig. 1).
2.4. NO release in RAW 264.7
It has been demonstrated that NO is well-known pro-inflammatory
mediator [24]. It has been found that excessive NO production plays an
important role in many inflammatory diseases [25]. The stimulation of
macrophages with LPS produces overexpressed pro-inflammatory cy-
tokines and pro-inflammatory mediators [26]. Here, all compounds
were examined their ability of inhibiting over expression NO. RAW
264.7 cells were pre-incubated with the compounds (20 µM) for 1 h,
then treated with LPS (0.5 µg/mL) for 24 h. Determination of NO
concentration in cell supernatant by Griess reagent. The results showed
that most of paeonol derivatives effectively suppressed LPS-induced NO
release in vitro (Tables 1–5). Most of compounds have improved in-
hibitory activity compared to paeonol at 20 µM. In particularly, com-
pound 11a exhibited the most potent activity with IC50 value of
6.96 µM (Fig. 3).
Among the many non-steroidal anti-inflammatory drugs, both ni-
mesulide (A) [20] and diclofenac acid (B) [21] were found to have a
two-benzene ring scaffold linked by O or NH, and therefore similar
structural fragments were introduced to paeonol moiety (step 3) in this
study. On the other hand, α, β-unsaturated ketone and unsaturated
amide moieties have been the common scaffolds with various biological
activities [22], So, on the basis of this and the summative SARs (C, D,
E), the final step (step 4) was carried out by adding α, β-unsaturated
moiety.
2.2. Chemistry
2.5. Structure-activity relationships (SARs)
The routes to synthesize the final compounds were depicted in
Scheme 1. Through a simple nucleophilic substitution, intermediates 1
and 4 were synthesized. The intermediate 1 was hydrolyzed, acidified,
and amidated to obtain the final compounds 3a-3l. The intermediate 4
was amidated to get the final compounds 6a-6m. Reaction of paeonol
with p-nitrofluorobenzene get intermediate 8 with high yield [23], then
nitro group is reduced with Pd/C as catalyst under H2 atmosphere to
obtain intermediate 9. Oxalyl chloride activates carboxylic acid to acyl
chloride intermediate, Intermediate 9 was subjected to the condensa-
tion to give compounds 10a-10e, 11a-11e. The detailed synthesis
procedures were listed in experimental section.
Firstly, compounds 3a-3l and their inhibitory rates (IR) were listed
in Table 1. Most of compounds showed weak inhibitory activity against
LPS-induced NO production at 20 μM (IRs were less than 23.33%).
Compared to paeonol, its derivatives had better anti-inflammatory ac-
tivity through introduction of aromatic system. On basis of this, in order
to improve the anti-inflammatory activity, we continued to optimize
them. As shown in Table 2, acetamide linker was replaced with ethy-
lamine and the order of amide was reversed, compounds 6a-6m were
designed and synthesized. The anti-inflammatory effect of these com-
pounds was greatly improved, especially compounds 6d, 6i with
75.94% and 63.29% inhibitory activity at 20 μM, respectively. From
Tables 1 and 2, it is easy to found that the anti-inflammatory activity
should be improved when the strong electron withdrawing group was
substituted on the aromatic ring, such as eCF3 (compounds 3b and 6d),
eOCH3 (compounds 3g and 6c), eF (compounds 3i and 6i).
2.3. Assessment of toxicity of active compounds
Before the determination of the anti-inflammatory activity in LPS-
induced RAW 264.7 cells, compounds’ cytotoxicity against RAW 264.7
cells was tested by MTT assay. As shown in Fig. 2. All compounds ex-
hibited no obvious toxicity against cell proliferation at 20 µM.
As we mention in the design section, fragment design was used for
further structural optimization. We initially designed, synthesized
2

Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
Scheme 1. Synthesis of title compounds, Reagents and conditions: (i) ethyl bromoacetate, K2CO3, acetone, rt; (ii) (1) 5% NaOH aq: ethanol (1:1), 65 °C. (2) 1 N
HCl, pH 3–4, rt; (iii) R-NH2 or 1-methylpiperazine or morpholine, HOBT, EDCI, Et3N, Dichloromethane, rt; (iv) tert-butyl (2-bromoethyl)carbamate, K2CO3, DMF,
80 °C; (v) HCl: ethanol (1:1), rt; (vi) R-COOH, HOBT, EDCI, Et3N, Dichloromethane, rt; (vii) 4-fluoronitrobenzene, K2CO3, DMSO, rt; (viii) H2, Pd/C, methanol, rt; (ix)
(1) R-COOH, oxalyl chloride, DMF (cat), Dichloromethane, rt. (2) compound 9, Et3N, Dichloromethane, 0 °C ~ rt.
Fig. 2. Cytotoxic evaluation of all compounds, the cytotoxic evaluation of compounds 3a-3l, 6a-6m, 7a-7e, 10a-10e and 11a-11e in RAW264.7 cells. Compounds at
concentration of 20 μM. Paeonol at concentration of 20 μM. The concentration of DMSO is 10-8 μM. Control: Untreated cells. The cell viability was evaluated by the
MTT assay. ***p < 0.001 compare with control group.
3

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BioorganicChemistry98(2020)103735
Table 1
Inhibitory activity against NO production of compounds 3a-3l.
Compd
R1
Inhibition rate (20 μM)
Compd
R1
Inhibition rate (20 μM)
3a
18.33
10.21
3h
3.37
6.38
3b
3c
23.33
6.74
5.21
3i
3j
20.22
21.67
14.23
12.11
7.23
3d
3e
5.32
10.21
10.32
3k
3l
17.98
19.10
10.23
10.20
21.66
3f
7.86
10.45
8.96
Paeonol
–
14.74
5.62
3g
20.22
compounds (7a-7e, 10a-10e) as shown in Tables 3 and 4. And the
difference is that one is two-benzene ring scaffold linked by O atom and
the other is α, β-unsaturated ketone structure. The anti-inflammatory
activity results demonstrated that compounds with two-benzene ring
scaffold linked by O atom had a slight increase, but, compounds’ ac-
tivity containing α, β-unsaturated ketone moiety was greatly improved.
Finally, we then tried to combine two-benzene ring scaffold and α, β-
unsaturated amide together to get compounds 11a-11e as shown in
Table 5. To our delight, the anti-inflammatory activity of these deri-
vatives has been greatly improved again. Among them, the IR of
compound 11a against NO reached 96.32%. Therefore, after several
rounds of optimization, lead compound 11a was initially selected for
further mechanism research.
2.6. Compound 11a suppresses LPS-induced iNOS and COX-2 activation
iNOS and COX-2 are well-known pro-inflammatory proteins
[27,28]. Many inflammatory diseases can active related cellular path-
ways and lead to high expressions of iNOS and COX-2. Therefore, in-
hibition of excessive expression of iNOS and COX-2 can attenuate the
Table 2
Inhibitory activity against NO production of compounds 6a-6m.
Compd
R2
Inhibition rate (20 μM)
Compd
R2
Inhibition rate (20 μM)
6a
32.12
31.64
48.10
5.62
6.21
5.32
6h
48.10
63.29
27.84
9.22
8.22
3.56
6b
6c
6i
6j
6d
6e
75.94
45.57
8.23
6.23
6k
6l
26.13
18.98
2.33
10.23
6f
31.64
60.75
3.20
6m
36.71
14.74
4.25
5.62
6g
10.24
Paeonol
–
4

Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
Table 3
Table 5
Inhibitory activity against NO production of compounds 10a-10e.
Inhibitory activity against NO production of compounds 11a-
11e.
R5
Inhibition rate (20 μM)
Compd
R4
Inhibition rate (20 μM)
Compd
10a
38.20
28.10
5.62
3.23
11a
96.32
5.32
10b
10c
11b
11c
89.66
28.73
3.12
5.63
25.84
4.15
10d
10e
20.22
15.50
6.32
6.33
11d
11e
94.38
56.32
6.32
8.23
Paeonol
–
14.74
5.62
Paeonol
–
14.74
5.62
Table 4
Inhibitory activity against NO production of compounds 7a-
7e.
[30].
Thus, we examined whether compound 11a inhibited phosphor-
ylation and degradation of IκB. RAW 264.7 cells were pretreated with
different concentration for 1 h and then treated with 0.5 mg/mL LPS for
30 min. The results showed that compound 11a significantly sup-
pressed LPS-induced phosphorylation and degradation of IκB. We fur-
ther examined the nuclear translocation of P65 by Western blot. We
found that compound 11a significantly suppressed LPS-induced P65
phosphorylation (Fig. 5). These results suggested that 11a might inhibit
NF-κB activation by blocking the LPS-induced phosphorylation of IκB
and P65.
Compd
R3
Inhibition rate (20 μM)
7a
88.76
3.25
7b
7c
32.58
44.94
5.62
6.23
2.8. Compound 11a inhibits LPS-induced ERΚ and P38 signaling activation
TLR4 is a member of the TLR family that is involved in innate im-
munity and inflammation response [31]. TLR4 is traditionally accepted
as the primary LPS receptor and has been reported as critical for the
inflammatory response to LPS [32]. The TLR4 mediated signaling plays
an important role in the activation of NF-κB and MAPΚ signaling in
LPS-induced macrophages [29]. When LPS stimulates cells, the NF-κB
and MAPK are activated through TIR4-MyD88-TAK1-dependent sig-
naling pathways, and then inflammatory cytokines and inflammatory
proteins are induced. TLR4 is an important upstream factor of the NF-
κB and MAPΚ signaling. Recent studies found that the inhibition of
TLR4 protein expression could suppress inflammation [27]. Therefore,
we studied whether compound 11a inhibited TLR4 expression by
Western blot. The results showed that TLR4 was up-regulated in LPS
induced RAW264.7 cells, and the expression of TLR4 was reversed in a
concentration dependent manner by pretreatment with compound 11a
(Fig. 6A).
7d
7e
57.30
17.98
2.15
4.25
Paeonol
–
14.74
5.62
severity of inflammatory diseases [28]. In the present study, compound
11a significantly suppressed LPS-induced protein expression, and it
could markedly augmente expressions of iNOS and COX-2 (Fig. 4). The
results indicated that compound 11a could prevent LPS induced in-
flammatory response in macrophages.
2.7. Compound 11a inhibits LPS-induced NF-κB signaling pathways
activation
MAPΚs are also known to participate in regulating inflammation
process. MAPΚ family is mainly composed of ERΚ, P38 and JNΚ sub-
family. When LPS-stimulated macrophages induce inflammation,
MAPKs protein will be phosphorylated, which will increase the trans-
location of the transcription factor AP-1 into the nucleus and bind to the
target promoter, thereby turning on the transcription of inflammatory
genes. Therefore, we detected the effect of compound 11a in MAPΚ
signaling pathway by Western blots. The results suggest that the anti-
inflammatory effect of compound 11a was mediated by blockade of
ERΚ and P38 phosphorylation signaling, but not JNΚ phosphorylation
signaling in LPS-induced RAW264.7 cells (Fig. 6B).
Accumulated data indicate that NF-κB transcription factor is one of
the principal factors for protein expression and cytokines secretion
mediated by LPS [29]. Transcription factor p65, which is one member
of NF-κB family, plays the most important role in signaling pathway. In
the control cells, NF-κB is inactivated in the cytoplasm by binding to
IκB. When a large number of inflammatory cytokines (such as LPS and
TNF-α) stimulate cells, IκB kinase (IKK) can be activated and then cause
IκB phosphorylation and degradation. At last, NF-κB enters the nucleus
and binds with the target gene to promote transcription and translation
5

Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
Fig. 3. Inhibitory activity against NO production
of compounds 11a, 11b, 11d, RAW264.7 cells
were pretreated with compounds (20, 10, 5,
2.5 μM) for 1 h, incubated with LPS (0.5 μg/mL)
for 24 h, NO production was measured using
Griess Reagent assay. Paeonol (20 μM) is a positive
compound. The concentration of DMSO is 10-8 μM
***p < 0.001, **p < 0.01, *p < 0.05 vs LPS
group.
intragastric administration of compound 11a once a day from day 14 to
28. Sinomenine was given intragastrically as the positive control.
In order to evaluate the in vivo effect of compound 11a, histo-
pathological analysis of the ankle joint was performed at the end of the
experiment. There was no inflammatory reaction in normal rats, and
the articular cavity was intact without any inflammatory cells (Fig. 7A).
AA rats exhibited extensive inflammation, inflammatory cells infiltra-
tion, synovial hyperplasia and bone or cartilage destruction (Fig. 7B).
AA rats treated with compound 11a (40 mg/kg) exhibited moderate
synovial hyperplasia, inflammatory cells infiltration and cartilage de-
struction (Fig. 7D). Compound 11a (80 mg/kg) and sinomenine
(80 mg/kg) ameliorated cartilage destruction and severe inflammatory
cell infiltration (Fig. 7C, E). Although sinomenine showed moderate
synovial hyperplasia, the articular cavity is intact. Compound 11a
(80 mg/kg) showed slightly synovial hyperplasia. In conclusion, the
results of histopathological evaluation showed that compound 11a had
anti-inflammatory effect on AA rats.
3. Conclusions
In summary, total of 40 new compounds were synthesized, char-
acterized and evaluated for their anti-inflammatory activities. After
several rounds SARs step by step, title compound 11a was discovered as
the most potent compound without obvious cytotoxicity. The pre-
liminary mechanism indicated that this compound could significantly
suppress LPS-induced expressions of iNOS, COX-2 and inhibit NO pro-
duction through NF-κB/MAPΚ signaling pathway in a concentration
dependent manner (Fig. 8). The further in vivo anti-inflammatory study
revealed that this compound effectively relieved the histological
changes in knee joints of AA rats. Therefore, this title compound is of
importance in the development of more efficient agents against anti-AA
in the future.
Fig. 4. Compound 11a inhibited the LPS-induced expression of iNOS and COX-
2 in RAW 264.7 cells, Cells were pre-treated with compound 11a at con-
centration of 10, 5 and 2.5 μM for 1 h, then stimulated with LPS (0.5 μg/mL) for
30 min. Bay 11–7082 (5 µM) is the NF-κB inhibitor. ###p < 0.001 compared
with LPS unstimulated cells, ***p
< 0.001, compared with LPS-stimulated
cells; the blots shown are the examples of three separate experiments.
4. Experimental section
2.9. In vivo activity of compound 11a
4.1. Chemistry
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune
disease characterized by joint swelling, synovitis, and progressive joint
injury of cartilage and bone. The complete Freund's adjuvant (CFA)-
induced arthritis (AA) is widely used in the study of RA because of its
similar pathogenesis. Complete Freund's adjuvant consists of in-
complete Freund's adjuvant and inactivated BCG vaccine (10 mg/mL).
Rat was induced by a single intradermal injection of 0.1 mL CFA into
the right hind paw. We observed that 70–80% of rats exhibited obvious
left hind pawhind paw swelling and body weight loss. Rats were given
All reagents and solvents were purchased from commercial sup-
pliers and used without further purification. Reactions were monitored
by thin-layer chromatography (TLC). 1H and 13C NMR spectral data
were recorded with a Bruker or Agilent, 400, 600 MHz spectrometer in
CDCl3 or DMSO‑d6 using tetramethylsilane (TMS) as the internal stan-
dard. High-resolution mass spectrometry (HRMS) was recorded on an
Agilent Technologies LC-TOF instrument. The purities of compound
11a were determined by monitoring at 300 nm and were confirmed to
be more than 95% (Supporting Information).
6

Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
Fig. 5. Compound 11a inhibited LPS-induced activation of NF-κB signaling pathway in RAW 264.7 cells. After pretreatment with compound 11a at concentration of
10, 5 and 2.5 μM for 1 h, cells were stimulated with LPS (0.5 µg/mL) for 30 min. Bay 11–7082 (5 µM) is the NF-κB inhibitor. ###p < 0.001 compared with LPS
unstimulated cells, ***p < 0.001, compared with LPS-stimulated cells.
4.2. The detailed synthesis procedures
was stirred for overnight. After completion of the reaction, as indicated
by TLC, the solvent was removed by rotary evaporation. Residue was
purified by flash chromatography to gave compound 3a (106 mg,
0.227 mmol, 62.17% yield) as white solid. 1H NMR (600 MHz,
DMSO‑d6) δ 10.51 (s, 1H), 7.83–7.75 (m, 3H), 7.37 (d, J = 8.6 Hz, 2H),
6.67 (d, J = 8.0 Hz, 2H), 4.88 (s, 2H), 3.83 (s, 3H), 2.60 (s, 3H). 13C
NMR (151 MHz, DMSO‑d6) δ 197.46, 166.79, 164.64, 159.46, 144.40,
137.95, 133.06, 122.17(2C), 121.17(2C), 120.80, 120.58 (d, JC-
4.2.1. General procedure for synthesis of compounds 3a-3l
K2CO3 (2.5 g, 18 mmol) was added to solution of paeonol (2 g,
12 mmol) in acetone (30 mL). Ethyl bromoacetate (6 g, 36 mmol) was
added dropwise. After the reaction was complete, as indicated by TLC,
the solvent was removed by rotary evaporation. Crude product was
dissolved in ethyl acetate (100 mL), then washed with water and brine
(100 mL). The organic layer was dried over anhydrous sodium sulfate,
filtered, and the filtrate concentrated under reduced pressure. Residue
was recrystallized with ethanol to obtain intermediate 1 as white
crystalline solid (2.8 g, yield 92%).
= 255.7 Hz), 107.14, 100.39, 68.23, 56.15, 31.32. HRMS (ESI): m/z
F
[M+Na]+ calcd for C18H16F3NO5Na: 406.0873; found: 406.0877.
Following the similar procedures as for compound 3a gave com-
pounds 3b-3l.
Intermediate 1 (1 g, 4 mmol) was dissolved in 5% NaOH aq/ethanol
(1:1, 30 mL) and stirred at 65 °C for 6 h. The mixture was acidified with
1 N HCl to pH 3–4. The precipitate was filtered, washed with water, and
dried to give intermediate 2 as white solid (800 mg, yield 90%).
Intermediate 2 (100 mg, 0.446 mmol), 4-(trifluoromethoxy)aniline
(79 mg, 0.446 mmol) was dissolved in dichloromethane (5 mL). HOBT
(90 mg, 0.669 mmol), EDCI (128 mg, 0.669 mmol) and Et3N (215 μL,
1.338 mmol) were added at room temperature. The reaction mixture
4.2.1.1. 2-(2-acetyl-5-methoxyphenoxy)-N-(4-(trifluoromethyl)phenyl)
acetamide (3b). White solid, 46.15% yield, m. p.:175–176 °C, 1H NMR
(600 MHz, DMSO‑d6) δ 10.66 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.78 (d,
J = 8.4 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H), 6.67 (d, J = 8.3 Hz, 2H),
4.92 (s, 2H), 3.83 (s, 3H), 2.60 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ
197.48, 167.23, 164.64, 159.43, 142.33, 133.07, 126.67 (d, JC-
= 3.8 Hz, 2C), 124.01 (d, JC-F = 47.4 Hz), 125.65, 120.78, 119.70
F
7

Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
Fig. 6. Compound 11a dose-dependently suppressed LPS-induced P38 and ERΚ activation. (A) Cells were pre-treated with compound 11a (10, 5, 2.5 µM) for 1 h,
then stimulated with LPS (0.5 μg/mL) for 24 h. TAK-242 (1 µM) is the TLR4 inhibitor. (B) Cells were treated with LPS (0.5 μg/mL) for 30 min. The expression of
phosphor and total proteins ERΚ, JNΚ and P38 were analyzed by Western blot. The results were showed as means
experiments. ###p < 0.001 compared with LPS unstimulated cells; ***p < 0.001 compare with LPS-stimulated cells.
SD (n = 3) of at least three independent
Fig. 7. Therapeutic effect of compound 11a on AA rats, (A) normal; (B) AA; (C) Sinomenine, 80 mg/kg; (D) compound 11a, 40 mg/kg; (E) compound 11a, 80 mg/kg.
Sinomenine was the positive control. Arrows indicate severe inflammatory sites.
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Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
Fig. 8. The possible anti-inflammatory mechanism of compound 11a.
(2C), 107.15, 100.39, 68.22, 56.16, 31.34. HRMS (ESI): m/z [M+Na]+
2H), 4.85 (s, 2H), 3.83 (s, 3H), 2.60 (s, 3H). 13C NMR (151 MHz,
DMSO‑d6) δ 197.41, 166.48, 164.62, 159.47, 138.76, 133.09, 129.31
(2C), 124.19, 120.74, 119.75 (2C), 107.07, 100.36, 68.26, 56.15,
31.27. HRMS (ESI): m/z [M+Na]+ calcd for C17H17NO4Na:
322.1050; found: 22.1050.
calcd for C18H16F3NO4Na: 390.0924; found: 390.0927.
4.2.1.2. 2-(2-acetyl-5-methoxyphenoxy)-N-(p-tolyl)acetamide
(3c). White solid, 58.82% yield, m. p.:159–160 °C, 1H NMR (600 MHz,
DMSO‑d6) δ 10.18 (s, 1H), 7.78 (dd, J = 9.3, 4.2 Hz, 1H), 7.59–7.53 (m,
2H), 7.15 (d, J = 8.0 Hz, 2H), 6.66 (dq, J = 4.1, 2.3 Hz, 2H), 4.83 (d,
J = 3.6 Hz, 2H), 3.83 (d, J = 3.7 Hz, 3H), 2.59 (d, J = 3.7 Hz, 3H),
2.26 (d, J = 3.6 Hz, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 197.40,
166.23, 164.61, 159.48, 136.26, 133.17, 133.08, 129.67(2C), 120.73,
119.74(2C), 107.06, 100.35, 68.26, 56.14, 31.26, 20.88. HRMS (ESI):
m/z [M+Na]+ calcd for C18H19NO4Na: 336.1206; found: 336.1209.
4.2.1.6. 2-(2-acetyl-5-methoxyphenoxy)-N-(4-methoxyphenyl)acetamide
(3g). White solid, 46.46% yield, m. p.:154–155 °C, 1H NMR (600 MHz,
DMSO‑d6) δ 10.18 (s, 1H), 7.87–7.70 (m, 1H), 7.65–7.54 (m, 2H),
6.94–6.87 (m, 2H), 6.66 (dd, J = 6.9, 2.3 Hz, 2H), 4.83 (s, 2H), 3.83 (s,
3H), 3.73 (s, 3H), 2.59 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 197.43,
165.99, 164.62, 159.52, 156.06, 133.07, 131.90, 121.31 (2C), 120.75,
114.48(2C), 107.07, 100.35, 68.28, 56.14, 55.65, 31.29. HRMS (ESI):
m/z [M+Na]+ calcd for C18H19NO5Na: 352.1155; found: 352.1156.
4.2.1.3. 2-(2-acetyl-5-methoxyphenoxy)-N-(m-tolyl)acetamide
(3d). White solid, 38.18% yield, m. p.:131–132 °C, 1H NMR (600 MHz,
DMSO‑d6) δ 10.21 (s, 1H), 7.82–7.73 (m, 1H), 7.54 (s, 1H), 7.48–7.41
(m, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 6.67 (d,
J = 7.6 Hz, 2H), 4.85 (s, 2H), 3.83 (s, 3H), 2.60 (s, 3H), 2.30 (s, 3H).
13C NMR (151 MHz, DMSO‑d6) δ 197.38, 166.39, 164.60, 159.47,
138.70, 138.54, 133.05, 129.12, 124.90, 120.78, 120.27, 116.97,
107.07, 100.34, 68.25, 56.15, 31.29, 21.61. HRMS (ESI): m/z [M
+Na]+ calcd for C18H19NO4Na: 336.1206; found: 336.1208.
4.2.1.7. 2-(2-acetyl-5-methoxyphenoxy)-N-(2-fluorophenyl)acetamide
(3h). White solid, 22.51% yield, m. p.:151–152 °C, 1H NMR (600 MHz,
DMSO‑d6) δ 10.00 (s, 1H), 7.90 (td, J = 7.6, 3.5 Hz, 1H), 7.75 (d,
J = 8.7 Hz, 1H), 7.29 (ddd, J = 10.7, 6.1, 2.4 Hz, 1H), 7.20 (ddt,
J = 10.0, 7.4, 3.8 Hz, 2H), 6.66 (d, J = 7.7 Hz, 2H), 4.94 (s, 2H), 3.83
(s, 3H), 2.58 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 196.99, 166.89,
164.52, 159.45, 154.29 (d, JC-F = 245.5 Hz), 132.76, 126.31 (d, JC-
= 7.5 Hz), 125.76 (d, JC-F = 11.7 Hz), 124.87 (d, JC-F = 3.5 Hz),
F
4.2.1.4. 2-(2-acetyl-5-methoxyphenoxy)-N-(o-tolyl)acetamide
(3e). White solid, 44.54% yield, m. p.:146–147 °C, 1H NMR (600 MHz,
DMSO‑d6) δ 9.65 (s, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.8 Hz,
1H), 7.24 (d, J = 7.2 Hz, 1H), 7.19 (dt, J = 7.8, 4.0 Hz, 1H), 7.12 (t,
J = 7.5 Hz, 1H), 6.71–6.63 (m, 2H), 4.90 (s, 2H), 3.84 (s, 3H), 2.58 (s,
3H), 2.22 (s, Hz, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 197.13, 166.56,
164.54, 159.45, 135.89, 132.94, 132.16, 130.83, 126.50, 126.00,
125.34, 120.82, 107.04, 100.19, 68.12, 56.14, 31.43, 18.10. HRMS
(ESI): m/z [M+Na]+ calcd for C18H19NO4Na: 336.1206; found:
336.1207.
124.74, 120.98, 116.01 (d, JC-F = 19.4 Hz), 107.14, 100.14, 68.01,
56.12, 31.48. HRMS (ESI): m/z [M+Na]+ calcd for C17H16FNO4Na:
340.0956; found: 340.0959.
4.2.1.8. 2-(2-acetyl-5-methoxyphenoxy)-N-(3-fluorophenyl)acetamide
(3i). White solid, 41.89% yield, m. p.:157–158 °C, 1H NMR (600 MHz,
DMSO‑d6) δ 10.48 (s, 1H), 7.81–7.75 (m, 1H), 7.65 (dt, J = 11.4,
2.3 Hz, 1H), 7.44–7.35 (m, 2H), 6.93 (td, J = 8.0, 2.8 Hz, 1H), 6.67 (d,
J = 7.1 Hz, 2H), 4.87 (s, 2H), 3.83 (s, 3H), 2.60 (s, 3H). 13C NMR
(151 MHz, DMSO‑d6)
δ 197.47, 166.91, 164.63, 162.61 (d, JC-
= 241.8 Hz), 159.41, 140.46 (d, JC-F = 11.1 Hz), 131.00 (d, JC-
= 9.6 Hz), 133.09, 120.75, 115.50, 110.68 (d, JC-F = 21.0 Hz),
F
F
4.2.1.5. 2-(2-acetyl-5-methoxyphenoxy)-N-phenylacetamide (3f). White
solid, 52.74% yield, m. p.:163–164 °C, 1H NMR (600 MHz, DMSO‑d6)
δ 10.27 (s, 1H), 7.81–7.76 (m, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.35 (t,
J = 7.9 Hz, 2H), 7.10 (t, J = 7.4 Hz, 1H), 6.66 (dd, J = 6.7, 2.6 Hz,
106.56 (d, JC-F = 26.1 Hz), 107.14, 100.40, 68.22, 56.16, 31.30. HRMS
(ESI): m/z [M+Na]+ calcd for C17H16FNO4Na: 340.0956; found:
340.0957.
9

Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
4.2.1.9. 2-(2-acetyl-5-methoxyphenoxy)-N-(4-fluorophenyl)acetamide
(3j). White solid, 36.65% yield, m. p.:155–156 °C, 1H NMR (600 MHz,
DMSO‑d6) δ 10.36 (s, 1H), 7.81–7.75 (m, 1H), 7.70 (dd, J = 9.0, 4.9 Hz,
2H), 7.19 (t, J = 8.9 Hz, 2H), 6.70–6.62 (m, 2H), 4.85 (s, 2H), 3.83 (s,
3H), 2.59 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 197.44, 166.44,
164.62, 159.46, 158.75 (d, JC-F = 240.3 Hz), 135.15 (d, JC-F = 2.5 Hz),
133.06, 121.59 (d, JC-F = 7.9 Hz, 2C), 120.80, 115.89 (d, JC-
3H).13C NMR (151 MHz, DMSO‑d6) δ 196.81, 164.90, 164.69, 160.48,
153.02, 152.76, 142.98, 132.19, 120.84, 114.24, 112.94, 106.76,
99.34, 98.22, 67.68, 57.00, 56.38, 56.33, 56.05, 39.00, 32.23. HRMS
(ESI): m/z [M+Na]+ calcd for C21H25NO7Na: 426.1523; found:
426.1520.
4.2.2.2. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-3,4,5-
= 22.4 Hz, 2C), 107.13, 100.41, 68.27, 56.15, 31.27. HRMS (ESI):
trimethoxybenzamide (6c). White solid, 40.92% yield, m.p.:
149–150 °C. 1H NMR (400 MHz, DMSO‑d6) δ 8.66 (t, J = 5.3 Hz,
1H), 7.65 (d, J = 8.7 Hz, 1H), 7.18 (s, 2H), 6.71 (d, J = 2.1 Hz, 1H),
6.60 (dd, J = 8.7, 2.1 Hz, 1H), 4.29 (t, J = 5.5 Hz, 2H), 3.83 (d,
J = 5.4 Hz, 9H), 3.75–3.69 (m, 5H), 2.50 (s, 3H). 13C NMR (151 MHz,
DMSO‑d6) δ 197.04, 166.48, 164.64, 160.40, 153.00 (2C), 140.47,
132.17, 129.94, 121.07, 106.72, 105.24 (2C), 99.46, 67.15, 60.50,
56.43 (2C), 56.06, 39.30, 32.30. HRMS (ESI): m/z [M+Na]+ calcd for
F
m/z [M+Na]+ calcd for C17H16FNO4Na: 340.0956; found: 340.0958.
4.2.1.10. 2-(2-acetyl-5-methoxyphenoxy)-1-(4-methylpiperazin-1-yl)
ethan-1-one (3k). oily liquid, 59.67% yield, 1H NMR (600 MHz,
DMSO‑d6) δ 7.65 (d, J = 8.7 Hz, 1H), 6.64–6.59 (m, 2H), 5.00 (s,
2H), 3.81 (s, 3H), 3.46 (t, J = 5.0 Hz, 4H), 2.58 (s, 3H), 2.30 (dt,
J = 41.9, 5.0 Hz, 4H), 2.18 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ
197.11, 165.71, 164.43, 159.96, 132.10, 121.25, 106.51, 100.13,
66.65, 56.05, 55.06, 54.68, 46.07, 44.39, 41.62, 32.23. HRMS (ESI):
m/z [M+Na]+ calcd for C16H22N2O4Na: 329.1472; found: 329.1472.
C
21H25NO7 Na: 426.1523; found: 426.1519.
4.2.2.3. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-4-(trifluoromethyl)
benzamide (6d). White solid, 54.31% yield, m.p.: 154–155 °C. 1H NMR
(400 MHz, DMSO‑d6) δ 8.94 (t, J = 5.3 Hz, 1H), 8.04 (d, J = 8.1 Hz,
2H), 7.86 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 8.7 Hz, 1H), 6.69 (d,
J = 2.3 Hz, 1H), 6.60 (dd, J = 8.8, 2.3 Hz, 1H), 4.30 (t, J = 5.5 Hz,
2H), 3.83 (s, 3H), 3.75 (q, J = 5.4 Hz, 2H), 2.47 (s, 3H). 13C NMR
(151 MHz, DMSO‑d6) δ 196.91, 165.91, 164.66, 160.42, 138.51,
132.16, 131.61 (d, JC-F = 31.6 Hz), 128.50 (2C), 125.79 (d, JC-
4.2.1.11. 2-(2-acetyl-5-methoxyphenoxy)-1-morpholinoethan-1-one
(3l). oily liquid, 55.52% yield, 1H NMR (600 MHz, DMSO‑d6) δ 7.66 (d,
J = 8.7 Hz, 1H), 6.64 (d, J = 2.3 Hz, 1H), 6.61 (dd, J = 8.7, 2.3 Hz,
1H), 5.02 (s, 2H), 3.81 (s, 3H), 3.60 (dt, J = 25.8, 4.7 Hz, 4H),
3.51–3.44 (m, 4H), 2.59 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ
197.13, 165.97, 164.44, 159.93, 132.12, 121.27, 106.47, 100.19,
66.56, 56.03, 45.04 (2C), 42.03 (2C), 32.21. HRMS (ESI): m/z [M
+Na]+ calcd for C15H19NO5Na: 316.1155; found: 316.1158.
= 3.6 Hz, 2C), 124.36 (q, JC-F = 272.5 Hz), 120.99, 106.72, 99.39,
F
67.06, 56.06, 39.42, 32.28. HRMS (ESI): m/z [M+Na]+ calcd for
C
19H18F3NO4 Na: 404.1080; found: 404.1084.
4.2.2. General procedure for synthesis of compounds 6a-6m, 7a-7e
K2CO3 (2.5 g, 18 mmol) was added to a stirring solution of the
paeonol (2 g, 12 mmol), tert-butyl (2-bromoethyl)carbamate (5.35 g,
24 mmol) in DMF (15 mL) at 80 °C. Upon completion by TLC, the re-
action mixture was cooled to room temperature, diluted with water,
and extracted with EtOAc (3 × 50 mL). The combined organic fractions
were washed with brine and dried over anhydrous sodium sulfate, fil-
tered and concentrated by evaporation under reduced pressure. Residue
was purified by flash chromatography gave intermediate 4 as white
solid powder (2.23 g, yield 83%).
4.2.2.4. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-2,4-dichlorobenzamide
(6e). White solid, 64.57% yield, m.p.: 125–127 °C. 1H NMR (400 MHz,
DMSO‑d6) δ 8.79 (t, J = 5.3 Hz, 1H), 7.67 (dd, J = 10.6, 5.3 Hz, 2H),
7.49 (dd, J = 8.2, 1.9 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 6.67 (d,
J = 2.2 Hz, 1H), 6.60 (dd, J = 8.7, 2.2 Hz, 1H), 4.25 (t, J = 5.2 Hz,
2H), 3.83 (s, 3H), 3.70 (q, J = 5.2 Hz, 2H), 2.52 (d, J = 4.4 Hz, 3H).
13C NMR (151 MHz, DMSO‑d6) δ 197.00, 166.18, 164.64, 160.44,
136.08, 134.95, 132.15, 131.58, 130.61, 129.57, 127.74, 121.00,
106.61, 99.34, 67.23, 56.07, 39.26, 32.51. HRMS (ESI): m/z [M
+Na]+ calcd for C18H17Cl2NO4 Na: 404.0427; found: 404.0431.
Intermediate 4 (2 g, 6.4 mmol) was dissolved in HCl/ethanol (1:1,
20 mL) and stirred at room temperature for 2 h. The solvent was then
removed under reduced pressure to afford crude intermediate 5 (1.26 g,
yield 90%) without further purification.
4.2.2.5. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-2-chlorobenzamide
(6f). White solid, 56.98% yield, m.p.: 111–112 °C. 1H NMR (400 MHz,
DMSO‑d6) δ 8.74 (t, J = 5.3 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H),
7.52–7.37 (m, 4H), 6.68 (d, J = 2.1 Hz, 1H), 6.61 (dd, J = 8.7, 2.1 Hz,
1H), 4.27 (t, J = 5.2 Hz, 2H), 3.84 (s, 3H), 3.71 (q, J = 5.2 Hz, 2H),
2.54 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 197.02, 167.06, 164.64,
160.48, 137.28, 132.15, 131.19, 130.31, 130.03, 129.21, 127.49,
121.03, 106.61, 99.34, 67.27, 56.07, 39.22, 32.51. HRMS (ESI): m/z
[M+Na]+ calcd for C18H18ClNO4 Na: 370.0817; found: 370.0820.
Intermediate 5 (100 mg, 0.479 mmol), benzoic acid (59 mg,
0.479 mmol) were dissolved in dichloromethane (5 mL). HOBT (90 mg,
0.669 mmol), EDCI (128 mg, 0.669 mmol) and Et3N (215 μL,
1.338 mmol) were added at room temperature. The reaction mixture
was stirred for overnight. After completion of the reaction, as indicated
by TLC, the solvent was removed by rotary evaporation. Residue was
purified by flash chromatography to gave compound 6a (76 mg,
0.224 mmol, yield 51.08%) as white solid. 1H NMR (400 MHz,
DMSO‑d6) δ 8.71 (t, J = 5.3 Hz, 1H), 7.87–7.81 (m, 2H), 7.64 (d,
J = 8.7 Hz, 1H), 7.56–7.43 (m, 3H), 6.69 (d, J = 2.2 Hz, 1H), 6.59 (dd,
J = 8.7, 2.2 Hz, 1H), 4.28 (t, J = 5.6 Hz, 2H), 3.83 (s, 3H), 3.73 (q,
J = 5.5 Hz, 2H), 2.47 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 196.95,
167.07, 164.66, 160.47, 134.78, 132.14, 131.65, 128.72 (2C), 127.57
(2C), 120.99, 106.72, 99.36, 67.19, 56.05, 39.25, 32.30. HRMS (ESI):
m/z [M+Na]+ calcd for C18H19NO4Na: 336.1206; found: 336.1209.
Following the similar procedures as for compound 6a gave com-
pounds 6b-6m, 7a-7e.
4.2.2.6. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-4-methylbenzamide
(6g). White solid, 46.40% yield, m.p.: 124–125 °C. 1H NMR (400 MHz,
DMSO‑d6) δ 8.63 (t, J = 5.2 Hz, 1H), 7.76 (d, J = 8.1 Hz, 2H), 7.65 (d,
J = 8.7 Hz, 1H), 7.27 (d, J = 8.1 Hz, 2H), 6.69 (d, J = 2.1 Hz, 1H),
6.60 (dd, J = 8.7, 2.1 Hz, 1H), 4.28 (t, J = 5.6 Hz, 2H), 3.83 (s, 3H),
3.72 (q, J = 5.5 Hz, 2H), 2.47 (s, 3H), 2.35 (s, 3H). 13C NMR (151 MHz,
DMSO‑d6) δ 196.95, 166.92, 164.66, 160.48, 141.52, 132.13, 131.96,
129.24 (2C), 127.59 (2C), 120.98, 106.73, 99.35, 67.20, 56.06, 39.18,
32.31, 21.37. HRMS (ESI): m/z [M+H]+ calcd for C19H22NO4:
350.1363; found: 350.1367.
4.2.2.1. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-2,4,5-
trimethoxybenzamide (6b). White solid, 25.67% yield, m.p.:
133–134 °C. 1H NMR (400 MHz, CDCl3) δ 8.37 (t, J = 5.6 Hz, 1H),
7.65 (d, J = 8.7 Hz, 1H), 7.46 (s, 1H), 6.75 (s, 1H), 6.68 (d, J = 2.2 Hz,
1H), 6.59 (dd, J = 8.7, 2.2 Hz, 1H), 4.25 (t, J = 5.4 Hz, 2H), 3.89–3.81
(m, 9H), 3.76 (q, J = 5.5 Hz, 2H), 3.72 (s, 3H), 2.51 (d, J = 2.0 Hz,
4.2.2.7. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-4-nitrobenzamide
(6h). White solid, 32.69% yield, m.p.: 162–164 °C. 1H NMR (400 MHz,
DMSO‑d6) δ 9.05 (t, J = 5.2 Hz, 1H), 8.33 (d, J = 8.7 Hz, 2H), 8.08 (d,
J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 1H), 6.70 (d, J = 2.1 Hz, 1H),
6.60 (dd, J = 8.7, 2.1 Hz, 1H), 4.31 (t, J = 5.4 Hz, 2H), 3.83 (s, 3H),
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Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
3.77 (q, J = 5.3 Hz, 2H), 2.47 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ
196.90, 165.45, 164.66, 160.40, 149.51, 140.37, 132.16, 129.10 (2C),
123.99 (2C), 121.00, 106.74, 99.41, 67.02, 56.08, 39.52, 32.28. HRMS
(ESI): m/z [M+Na]+ calcd for C18H18N2O6 Na: 381.1057; found:
381.1056.
125–127 °C. 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 8.7 Hz, 1H),
7.56 (d, J = 15.6 Hz, 1H), 6.93 (d, J = 4.4 Hz, 1H), 6.72 (s, 2H), 6.53
(dd, J = 8.8, 2.3 Hz, 1H), 6.43 (dd, J = 8.9, 6.5 Hz, 2H), 4.18 (t,
J = 5.1 Hz, 2H), 3.88–3.81 (m, 14H), 2.58 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 197.62, 166.14, 164.44, 159.87, 153.36 (2C), 141.09, 139.68,
132.80, 130.40, 121.23, 119.99, 105.89, 105.11 (2C), 99.99, 67.91,
60.88, 56.12(2C), 55.54, 38.92, 30.85. HRMS (ESI): m/z [M+Na]+
calcd for C23H27NO7Na: 452.1680; found: 452.1680.
4.2.2.8. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-3-chloro-4-
fluorobenzamide (6i). White solid, 32.07% yield, m.p.: 135–136 °C. 1H
NMR (400 MHz, DMSO‑d6) δ 8.84 (t, J = 5.1 Hz, 1H), 8.06 (dd,
J = 7.2, 2.1 Hz, 1H), 7.88 (ddd, J = 8.3, 4.7, 2.1 Hz, 1H), 7.65 (d,
J = 8.7 Hz, 1H), 7.54 (t, J = 8.9 Hz, 1H), 6.69 (d, J = 2.1 Hz, 1H),
6.60 (dd, J = 8.7, 2.1 Hz, 1H), 4.29 (t, J = 5.5 Hz, 2H), 3.83 (s, 3H),
3.73 (q, J = 5.4 Hz, 2H), 2.47 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ
196.90, 164.72 (d, JC-F = 20.5 Hz), 160.41, 160.19, 158.53, 132.33,
132.16, 130.06, 128.87 (d, JC-F = 8.2 Hz), 120.99, 120.06 (d, JC-
F = 18.1 Hz), 117.41 (d, JC-F = 21.1 Hz), 106.72, 99.39, 67.04, 56.06,
39.45, 32.28. HRMS (ESI): m/z [M+Na]+ calcd for C18H17ClFNO4Na:
388.0722; found: 388.0726.
4.2.2.14. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)cinnamamide
(7b). White solid, 44.02% yield, m.p.: 129–130 °C. 1H NMR (400 MHz,
CDCl3) δ 7.76 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 15.7 Hz, 1H), 7.50 (dd,
J = 7.3, 2.2 Hz, 2H), 7.38–7.32 (m, 3H), 7.00 (s, 1H), 6.54 (d,
J = 1.6 Hz, 1H), 6.52–6.49 (m, 1H), 6.45 (d, J = 2.3 Hz, 1H), 4.19 (t,
J = 5.2 Hz, 2H), 3.87–3.81 (m, 5H), 2.59 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 197.56, 166.16, 164.41, 159.86, 141.17, 134.86, 132.79,
129.61, 128.74 (2C), 127.82 (2C), 121.31, 120.62, 105.95, 100.09,
67.97, 55.54, 38.91, 30.79. HRMS (ESI): m/z [M+Na]+ calcd for
C
20H21NO4Na: 362.1363; found: 362.1367.
4.2.2.9. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)
cyclopropanecarboxamide (6j). White solid, 36.29% yield, m.p.:
138–140 °C. 1H NMR (400 MHz, DMSO‑d6) δ 8.32 (t, J = 5.2 Hz,
1H), 7.66 (d, J = 8.7 Hz, 1H), 6.65 (d, J = 2.1 Hz, 1H), 6.60 (dd,
J = 8.7, 2.1 Hz, 1H), 4.14 (t, J = 5.4 Hz, 2H), 3.83 (s, 3H), 3.53 (q,
J = 5.4 Hz, 2H), 2.51 (s, 3H), 1.62–1.53 (m, 1H), 0.71–0.61 (m, 4H).
13C NMR (151 MHz, DMSO‑d6) δ 196.97, 173.35, 164.66, 160.48,
132.14, 120.93, 106.73, 99.33, 67.63, 56.06, 38.67, 32.39, 13.98,
6.65(2C). HRMS (ESI): m/z [M+Na]+ calcd for C15H19NO4Na:
300.1206; found: 300.1209.
4.2.2.15. (E)-N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-3-(4-nitrophenyl)
acrylamide (7c). White solid, 30.75% yield, m.p.: 179–180 °C. 1H NMR
(400 MHz, DMSO‑d6) δ 8.53 (t, J = 5.3 Hz, 1H), 8.26 (d, J = 8.2 Hz,
2H), 7.84 (d, J = 8.4 Hz, 2H), 7.69–7.64 (m, 1H), 7.57 (d, J = 15.8 Hz,
1H), 6.87 (d, J = 15.8 Hz, 1H), 6.68 (d, J = 1.3 Hz, 1H), 6.63–6.58 (m,
1H), 4.23 (t, J = 5.2 Hz, 2H), 3.83 (s, 3H), 3.68 (dd, J = 10.4, 5.1 Hz,
2H), 2.51 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 196.90, 165.10,
164.68, 160.43, 147.98, 141.91, 137.07, 132.17, 129.04(2C), 126.64,
124.53(2C), 120.92, 106.78, 99.33, 67.48, 56.09, 38.94, 32.40. HRMS
(ESI): m/z [M+Na]+ calcd for C20H20N2O6Na: 407.1214; found:
407.1216.
4.2.2.10. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)furan-2-carboxamide
(6k). White solid, 55.18% yield, m.p.: 125–127 °C. 1H NMR (400 MHz,
DMSO‑d6) δ 8.59 (t, J = 5.5 Hz, 1H), 7.86–7.81 (m, 1H), 7.64 (d,
J = 8.7 Hz, 1H), 7.11 (d, J = 3.5 Hz, 1H), 6.67 (d, J = 2.1 Hz, 1H),
6.60 (ddd, J = 10.9, 6.1, 2.0 Hz, 2H), 4.24 (t, J = 5.6 Hz, 2H), 3.82 (s,
3H), 3.72–3.63 (m, 2H), 2.46 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ
196.96, 164.65, 160.39, 158.49, 148.27, 145.46, 132.14, 120.98,
113.88, 112.26, 106.78, 99.34, 67.13, 56.05, 38.42, 32.27. HRMS
(ESI): m/z [M+Na]+ calcd for C16H17NO5 Na: 326.0999; found:
326.0998.
4.2.2.16. (E)-N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-3-(furan-2-yl)
acrylamide (7d). White solid, 43.52% yield, m.p.: 105–107 °C. 1H NMR
(400 MHz, CDCl3) δ 7.79 (d, J = 8.7 Hz, 1H), 7.47–7.41 (m, 2H), 6.83
(s, 1H), 6.57–6.53 (m, 2H), 6.48–6.44 (m, 2H), 6.41 (d, J = 15.4 Hz,
1H), 4.20 (t, J = 5.2 Hz, 2H), 3.87–3.82 (m, 5H), 2.60 (s, 3H). 13C NMR
(151 MHz, DMSO‑d6) δ 196.93, 165.62, 164.67, 160.46, 151.35,
145.19, 132.15, 126.72, 120.92, 119.58, 114.26, 112.81, 106.78,
99.31, 67.55, 56.06, 38.83, 32.38. HRMS (ESI): m/z [M+Na]+ calcd
for C18H19NO5Na: 352.1155; found: 352.1159.
4.2.2.11. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)nicotinamide
(6l). White solid, 24.03% yield, m.p.: 108–110 °C. 1H NMR (400 MHz,
DMSO‑d6) δ 9.00 (d, J = 1.8 Hz, 1H), 8.92 (t, J = 5.1 Hz, 1H), 8.71 (d,
J = 3.8 Hz, 1H), 8.18 (dd, J = 7.9, 1.6 Hz, 1H), 7.64 (d, J = 8.7 Hz,
1H), 7.51 (dd, J = 7.9, 4.8 Hz, 1H), 6.69 (d, J = 2.1 Hz, 1H), 6.60 (dd,
J = 8.7, 2.1 Hz, 1H), 4.30 (t, J = 5.4 Hz, 2H), 3.83 (s, 3H), 3.75 (q,
J = 5.4 Hz, 2H), 2.47 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 197.57,
165.57, 164.36, 159.80, 151.89, 148.44, 135.29, 132.83, 129.88,
123.30, 121.42, 106.03, 100.83, 68.12, 55.53, 39.27, 30.10. HRMS
(ESI): m/z [M+Na]+ calcd for C17H18N2O4 Na: 337.1159; found:
337.1162.
4.2.2.17. (E)-N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-3-(3,5,6-
trimethylpyrazin-2-yl)acrylamide (7e). White solid, 29.85% yield, m.p.:
105–107 °C. 1H NMR (400 MHz, CDCl3) δ 7.83 (d, J = 14.9 Hz, 1H),
7.78 (d, J = 8.7 Hz, 1H), 7.10 (d, J = 14.9 Hz, 1H), 6.82 (s, 1H), 6.53
(dd, J = 8.8, 2.3 Hz, 1H), 6.44 (d, J = 2.3 Hz, 1H), 4.19 (t, J = 5.2 Hz,
2H), 3.89–3.82 (m, 5H), 2.59 (d, J = 4.3 Hz, 6H), 2.49 (d, J = 5.8 Hz,
6H). 13C NMR (151 MHz, CDCl3) δ 197.37, 165.86, 164.41, 159.85,
152.01, 149.50, 148.84, 143.00, 135.59, 132.80, 125.45, 121.25,
105.89, 99.79, 67.77, 55.52, 39.03, 31.02, 21.88, 21.63, 20.70.
HRMS (ESI): m/z [M+Na]+ calcd for C21H25N3O4Na: 406.1737;
found: 406.1742.
4.2.2.12. N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)furan-3-carboxamide
(6m). White solid, 52.42% yield, m.p.: 125–126 °C. 1H NMR (400 MHz,
DMSO‑d6) δ 8.41 (t, J = 5.3 Hz, 1H), 8.18 (s, 1H), 7.73 (t, J = 1.6 Hz,
1H), 7.65 (d, J = 8.7 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 6.69 (d,
J = 2.2 Hz, 1H), 6.60 (dd, J = 8.7, 2.2 Hz, 1H), 4.25 (t, J = 5.6 Hz,
2H), 3.83 (s, 3H), 3.67 (q, J = 5.5 Hz, 2H), 2.47 (s, 3H). 13C NMR
(151 MHz, DMSO‑d6) δ 196.98, 164.65, 162.42, 160.41, 145.54,
144.45, 132.15, 123.10, 121.00, 109.30, 106.77, 99.38, 67.19, 56.06,
38.65, 32.28. HRMS (ESI): m/z [M+H]+ calcd for C16H18NO5:
304.1179; found: 304.1178.
4.2.3. General procedure for synthesis of compounds 10a-10e, 11a-11e
K2CO3 (2.5 g, 18 mmol) was added to a stirring solution of the
paeonol (2 g, 12 mmol) and 1-fluoro-4-nitrobenzene (2 g, 14.4 mmol)
in DMSO (20 mL) at room temperature. The reaction mixture was
stirred for overnight and monitored by TLC. Upon completion, the re-
action mixture was diluted with water and extracted with EtOAc
(3 × 50 mL). The combined organic fractions were washed with brine
and dried over anhydrous sodium sulfate, filtered and concentrated by
evaporation under reduced pressure. Residue was purified by flash
chromatography to gave intermediate 8 as yellow oil liquid (2.96 g;
yield 86%).
4.2.2.13. (E)-N-(2-(2-acetyl-5-methoxyphenoxy)ethyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (7a). White solid, 48.47% yield, m.p.:
Intermediate 8 (2 g, 17.4 mmol) was dissolved in methanol (40 mL)
11

Y.S. Hu, et al.
BioorganicChemistry98(2020)103735
and 10% Pd-C (4% mmol) was added under H2 atmosphere. Monitored
by TLC and upon completion, the reaction mixture was filtered and
removed solvent by evaporation under reduced pressure, obtained
crude intermediate 9 as yellow solid without further purification.
P-nitrobenzoic acid (65 mg, 0.39 mmol), oxalyl chloride (100 μL,
1.17 mmol) and catalytic DMF were dissolved in DCM (5 mL), and the
mixture was stirred at 25 °C for 2 h. Then the mixture was concentrated
under vacuum and directly used in next step without any further pur-
ification. The obtained corresponding acyl chloride was dissolved in
DCM (2 mL) and the solution was added to the mixture of intermediate
9 (100 mg, 0.39 mmol) and Et3N (192 μL, 1.2 mmol) in DCM (6 mL) at
0 °C. The solution was allowed to stir at 0 °C to room temperature for
overnight. Reaction mixture was washed with brine. Organic solvent
was dried over anhydrous sodium sulfate, filtrated, concentrated by
evaporation under reduced pressure. Residue was purified by flash
chromatography to gave compound 10a (68 mg, 0.168 mmol, yield
43.12%). 1H NMR (600 MHz, CDCl3) δ 8.33 (d, J = 8.6 Hz, 2H),
8.11–8.08 (m, 1H), 8.06 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 8.9 Hz, 1H),
7.66 (d, J = 8.7 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 6.73–6.69 (m, 1H),
6.38 (d, J = 2.2 Hz, 1H), 3.77 (s, 3H), 2.60 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 196.82, 164.26, 158.29, 153.37, 149.87, 140.28, 133.24,
132.60, 128.21 (2C), 124.00 (2C), 123.17, 122.39 (2C), 119.54 (2C),
109.99, 109.43, 104.62, 55.58, 31.29. HRMS (ESI): m/z [M+Na]+
calcd for C22H18N2O6Na: 429.1057; found: 429.1055.
J = 8.9, 2.4 Hz, 1H), 6.37 (d, J = 2.3 Hz, 1H), 3.76 (s, 3H), 2.60 (s,
3H). 13C NMR (151 MHz, DMSO‑d6) δ 196.24, 164.36, 164.29, 158.46,
152.52, 152.30, 149.06, 135.82, 135.43, 132.58, 130.96, 123.91,
122.97, 122.67 (2C), 119.48 (2C), 109.94, 104.76, 56.16, 31.44.
HRMS (ESI): m/z [M+Na]+ calcd for C21H18N2O4Na: 385.1159;
found: 385.1156.
4.2.3.5. (E)-N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (11a). White solid, 49.35% yield, m.p.:
76–78 °C. 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.8 Hz, 1H), 7.80
(s, 1H), 7.66 (dd, J = 11.9, 8.6 Hz, 3H), 7.03–6.99 (m, 2H), 6.75 (s,
2H), 6.67 (dd, J = 8.9, 2.4 Hz, 1H), 6.52 (d, J = 15.4 Hz, 1H), 6.34 (d,
J = 2.4 Hz, 1H), 3.87 (d, J = 2.6 Hz, 3H), 3.86 (s, 6H), 3.74 (s, 3H),
2.61 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 196.25, 164.28, 163.92,
158.68, 153.56 (2C), 151.61, 140.75, 139.44, 136.11, 132.58, 130.72,
122.80, 121.87, 121.32(2C), 119.79 (2C), 109.73 (2C), 105.64, 104.52,
60.55, 56.36 (2C), 56.13, 31.52. HRMS (ESI): m/z [M+Na]+ calcd for
C27H27NO7Na: 500.1680; found: 500.1680.
4.2.3.6. N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)cinnamamide
(11b). White solid, 42.97% yield, m.p.: 128–129 °C. 1H NMR
(600 MHz,CDCl3) δ 8.20 (s, 1H), 7.91 (dd, J = 8.8, 1.2 Hz, 1H), 7.75
(d, J = 15.5 Hz, 1H), 7.69 (d, J = 7.9 Hz, 2H), 7.50–7.44 (m, 2H),
7.37–7.30 (m, 3H), 7.02–6.97 (m, 2H), 6.66 (d, J = 8.9 Hz, 1H), 6.63
(dd, J = 15.5, 1.1 Hz, 1H), 6.33 (s, 1H), 3.72 (s, 3H), 2.61 (s, 3H). 13C
NMR (151 MHz, DMSO‑d6) δ 196.25, 164.28, 163.84, 158.65, 151.69,
140.58, 136.00, 135.16, 132.58, 130.21, 129.45 (2C), 128.15 (2C),
122.81, 122.62, 121.41 (2C), 119.77 (2C), 109.78, 104.56, 56.14,
31.52. HRMS (ESI): m/z [M+Na]+ calcd for C24H21NO4Na: 410.1363;
found: 410.1366.
Following the similar procedures as for compound 10a gave com-
pounds 10b-10e, 11a-11e.
4.2.3.1. N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)-4-fluorobenzamide
(10b). White solid, 39.16% yield, m.p.: 154–155 °C. 1H NMR
(600 MHz, CDCl3) δ 8.03 (s, 1H), 7.91–7.88 (m, 3H), 7.64 (d,
J = 8.9 Hz, 2H), 7.15 (t, J = 8.6 Hz, 2H), 7.03 (d, J = 8.9 Hz, 2H),
6.68 (dd, J = 8.8, 2.4 Hz, 1H), 6.36 (d, J = 2.3 Hz, 1H), 3.75 (s, 3H),
2.60 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 196.99, δ 164.93 (d, JC-
F = 252.8 Hz), 164.69, 164.28, 158.63, 152.70, 134.01, 132.52, 130.94
(d, JC-F = 3.0 Hz), 129.42 (d, JC-F = 8.9 Hz, 2C), 122.98, 122.29 (2C),
119.58 (2C), 115.79 (d, JC-F = 22.0 Hz, 2C), 109.28, 104.29, 55.55,
31.33. HRMS (ESI): m/z [M+Na]+ calcd for C22H18FNO4Na: 402.1112;
found: 402.1111.
4.2.3.7. (E)-N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)-3-(4-nitrophenyl)
acrylamide (11c). White solid, 37.72% yield, m.p.: 186–187 °C. 1H
NMR (600 MHz, DMSO‑d6) δ 10.43 (s, 1H), 8.29 (d, J = 8.8 Hz, 2H),
7.89 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 8.9 Hz,
2H), 7.70 (d, J
= 15.7 Hz, 1H), 7.12–7.07 (m, 2H), 7.00 (d,
J = 15.8 Hz, 1H), 6.82 (dd, J = 8.9, 2.4 Hz, 1H), 6.38 (d,
J = 2.4 Hz, 1H), 3.74 (s, 3H), 2.51 (s, 3H). 13C NMR (151 MHz,
DMSO‑d6) δ 196.23, 164.28, 163.12, 158.53, 151.97, 148.11, 141.73,
138.12, 135.70, 132.59, 129.19 (2C), 126.93, 124.60 (2C), 122.86,
121.52 (2C), 119.74 (2C), 109.86, 104.68, 56.15, 31.50. HRMS (ESI):
m/z [M+Na]+ calcd for C24H20N2O6Na: 455.1214; found: 455.1211.
4.2.3.2. N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)benzamide
(10c). White solid, 30.24% yield, m.p.: 140–141 °C. 1H NMR
(600 MHz, CDCl3) δ 7.92–7.86 (m, 3H), 7.66 (d, J = 8.9 Hz, 2H),
7.58–7.46 (m, 3H), 7.05 (dt, J = 5.0, 3.1 Hz, 2H), 6.70–6.67 (m, 1H),
6.37 (d, J = 2.3 Hz, 1H), 3.75 (d, J = 1.3 Hz, 3H), 2.61 (d, J = 1.5 Hz,
3H). 13C NMR (151 MHz, DMSO‑d6) δ 196.23, 165.86, 164.29, 158.59,
152.00, 135.84, 135.35, 132.56, 131.95, 128.80, 128.04, 122.93,
122.61, 119.47, 109.86, 104.65, 56.15, 31.47. HRMS (ESI): m/z [M
+Na]+ calcd for C22H19NO4Na: 384.1206; found: 384.1209.
4.2.3.8. (E)-N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)-3-(furan-2-yl)
acrylamide (11d). White solid, 50.17% yield, m.p.: 134–136 °C. 1H
NMR (400 MHz, CDCl3 δ 7.91 (d, J = 8.8 Hz, 1H), 7.77 (s, 1H), 7.64 (d,
J = 8.5 Hz, 2H), 7.52 (d, J = 15.2 Hz, 1H), 7.44 (d, J = 1.5 Hz, 1H),
7.03–6.98 (m, 2H), 6.67 (dd, J = 8.8, 2.4 Hz, 1H), 6.58 (d, J = 3.4 Hz,
1H), 6.52–6.44 (m, 2H), 6.33 (d, J = 2.3 Hz, 1H), 3.73 (s, 3H), 2.61 (s,
3H). 13C NMR (151 MHz, DMSO‑d6) δ 196.25, 164.27, 163.72, 158.67,
151.64, 151.36, 145.56, 136.03, 132.56, 127.71, 122.80, 121.33 (2C),
119.78 (2C), 114.95, 112.99, 109.77 (2C), 104.50, 56.13, 31.51. HRMS
(ESI): m/z [M+Na]+ calcd for C22H19NO5Na: 400.1155; found:
400.1155.
4.2.3.3. N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)furan-2-carboxamide
(10d). White solid, 32.87% yield, m.p.: 108–109 °C. 1H NMR
(600 MHz, CDCl3) δ 8.11 (s, 1H), 7.90 (dd, J = 8.7, 7.2 Hz, 1H),
7.66 (d, J = 8.7 Hz, 2H), 7.52–7.48 (m, 1H), 7.25–7.22 (m, 1H),
7.07–7.00 (m, 2H), 6.71–6.65 (m, 1H), 6.56 (ddd, J = 12.7, 3.5, 1.7 Hz,
1H), 6.36 (d, J = 2.3 Hz, 1H), 5.29 (d, J = 6.5 Hz, 1H), 3.77–3.70 (m,
3H), 2.61–2.59 (m, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 196.22,
164.28, 158.50, 156.57, 152.12, 147.97, 146.06, 135.13, 132.55,
122.96, 122.64 (2C), 119.44 (2C), 115.07, 112.54, 109.89, 104.74,
56.16, 31.45. HRMS (ESI): m/z [M+Na]+ calcd for C20H17NO5Na:
374.0999; found: 374.1002.
4.2.3.9. (E)-N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)-3-(3,5,6-
trimethyl-1,4-dihydropyrazin-2-yl)acrylamide
(11e). White
solid,
49.37% yield, m.p.: 222–224 °C. 1H NMR (400 MHz, CDCl3) δ 7.92
(dd, J = 11.7, 10.8 Hz, 2H), 7.82 (s, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.22
(d, J = 14.7 Hz, 1H), 7.04–6.99 (m, 2H), 6.67 (dd, J = 8.8, 2.4 Hz,
1H), 6.34 (d, J = 2.4 Hz, 1H), 3.74 (s, 3H), 2.61 (s, 6H), 2.51 (d,
J = 10.2 Hz, 6H).13C NMR (151 MHz, DMSO‑d6) δ 196.23, 164.28,
163.48, 158.61, 152.54, 151.80, 149.89, 148.86, 142.79, 135.97,
135.18, 132.57, 127.07, 122.84, 121.38 (2C), 119.74 (2C), 109.81,
104.58, 56.14, 31.49, 22.02, 21.76, 20.82. HRMS (ESI): m/z [M+Na]+
calcd for C25H27N3O4Na: 454.1737; found: 454.1734.
4.2.3.4. N-(4-(2-acetyl-5-methoxyphenoxy)phenyl)nicotinamide
(10e). White solid, 30.79% yield, m.p.: 140–141 °C. 1H NMR
(600 MHz, CDCl3) δ 9.11 (s, 1H), 8.77 (d, J = 3.8 Hz, 1H), 8.22 (d,
J = 7.9 Hz, 1H), 8.11 (s, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.66 (d,
J = 8.8 Hz, 2H), 7.45 (s, 1H), 7.05 (d, J = 8.9 Hz, 2H), 6.70 (dd,
12

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BioorganicChemistry98(2020)103735
4.3. Cell culture
dehydrated according to gradient ethanol series, embedded in paraffin,
cut into 4 mm thick. The sections were stained with hematoxylin and
eosin (HE) for histopathological examination. Histological images were
obtained using the 3DHISTECH's Slide Converter (3DHISTECH,
Hungary).
Mouse peritoneal macrophages were obtained from the BeNa cul-
ture collection company. RAW264.7 cells were cultured in DMEM
(Hyclone, USA) with 10% FBS (Biological Industries, Israel, 100 U/mL
penicillin and 100 μg/mL streptomycin (Beyotime, China) at 37 °C in a
humid environment containing 5% CO2. All the cells used in the ex-
periment are in the logarithmic growth stage, and the number of cells in
the culture bottle is about 70%–80%.
4.8. Statistical analysis
The data were expressed as mean
SEM and analyzed statistically
by analysis of variance (ANOVE). p < 0.05 was considered statistically
4.4. Determination of NO release
significant. Data were expressed as mean
SEM and analyzed using
GraphPad Prism version 6.0 (GraphPad Software, USA). All experiment
data were repeated at least three times.
RAW264.7 cells were seeded into 48well plates (NEST, China) at a
density of 7 × 104 cells per well, and were used for experiments about
24 h later. RAW264.7 cells were pretreated with compounds (20 μM)
for 1 h and incubated with LPS (0.5 μg/mL) for 24 h. Cell supernatant
was collected for experiment. Measurement of NO production by Griess
reagent assay (Beyotime, China).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4.5. Cell viability assay (MTT)
Acknowledgment
RAW264.7 cells were pretreated with all compounds (20 μM) for
24 h. RAW264.7 cells were seeded into 96 well plate, each well with
1 × 104 cells, and kept at 37 °C, 5% CO2 for about 24 h. Discard the cell
culture medium and add the compounds to treat the cell for 24 h. After
MTT (sigma, USA, PBS solution at a concentration of 5 mg/mL) was
added and incubated at 37 °C for 4 h, the medium containing MTT was
removed , DMSO (150 μL) was added to each wells, Shaking for 10 min
and the absorbance at 492 nm was measured by a microplate reader
(MQX200, Bio-Tek, USA) [31].
This work was supported by the National Natural Science Funding
of China (21977001 and 21572003).
Appendix A. Supplementary material
The following files are available free. 1H NMR and 13C NMR spectra
and HRMS of all compounds.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103735.
4.6. Western blotting
References
RAW264.7 cells were seeded into 6-well plate with 4 × 106 cells or
2 × 106 cells per well and maintained at 37 °C in 5% CO2 about 24 h.
RAW264.7 cell were pretreated with compound 11a (10, 5, 2.5 μM) for
1 h, incubated with LPS (0.5 μg/mL) for 0.5 h or 24 h. The cells were
lysed in 400 uL RIPA cell lysis buffer (Contains PMSF and phosphatase
inhibitors, Beyotime china) and incubated on ice for 30 min. High speed
centrifugation for 10 min, proteins were run on 12% SDS-PAGE and
then transferred to PVDF membrane (GE Healthcare, UΚ). The blotted
membrane was incubated overnight at 4 °C with a specific primary
antibody p-iκB, iNOS (CST, USA), iκB, COX-2 (abcam, USA), ERΚ, p-
ERΚ, P38, p-P38, JNΚ, p-JNΚ (Immunoway, USA) The membranes
were washed in TBST (Beyotime, China), incubated with a 1:5000 di-
lution of HRP-conjugated secondary antibody (ZSGB-BIO, China) for
1 h at room temperature.
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