Design, synthesis and investigation of potential anti-inflammatory activity of O-alkyl and O-benzyl hesperetin derivatives

Article abstract of DOI:10.1016/j.intimp.2018.05.009

Hesperetin has been known to exert several activities such as anti-oxidant, antitumor and anti-inflammatory. To find hesperetin derivatives showing better activity, sixteen novel hesperetin derivatives were designed and synthesized. The new obtained compo

Full text of DOI:10.1016/j.intimp.2018.05.009

InternationalImmunopharmacology61(2018)82–91
Contents lists available at ScienceDirect
International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp
Design, synthesis and investigation of potential anti-inflammatory activity of
O-alkyl and O-benzyl hesperetin derivatives
Ai-Ling Huanga,b,c, Yi-Long Zhanga,b,c,1, Hai-Wen Dinga,b,c, Bo Lia,b,c, Cheng Huanga,b,c
,
⁎
Xiao-Ming Menga,b,c, Jun Lia,b,c,
a
The Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, People's Republic of China
The Key Laboratory of Anti-inflammatory of Immune medicines, Ministry of Education, People's Republic of China
Institute for Liver Diseases of Anhui Medical University, People's Republic of China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hesperetin derivatives
Synthesis
Anti-inflammatory activity
NF-κB
Structure-activity relationships
Hesperetin has been known to exert several activities such as anti-oxidant, antitumor and anti-inflammatory. To
find hesperetin derivatives showing better activity, sixteen novel hesperetin derivatives were designed and
synthesized. The new obtained compounds were investigated for their anti-inflammatory activity by inhibiting
interleukin-1β (IL-1β), interleukin-6 (IL-6) and production of nitric oxide (NO) in mouse RAW264.7 macro-
phages, and the structure-activity relationship of them was discussed. Among them, the compound 1l, 2c de-
monstrated more effective inhibitory activity of IL-1β and IL-6, meanwhile, the compound 1l showed the best
inhibition of NO production. The results of NO inhibition study were basically accord with the molecular
docking results of inducible nitric oxide synthase (iNOS). Furthermore, the expression of LPS-induced iNOS and
components of NF-κB signaling pathway were reduced by compound 1l. Our results suggest that the inhibitory
effect of compound 1l on LPS-stimulated inflammatory mediator production in RAW 264.7 cells is associated
with the suppression of NF-κB signaling pathway and inhibition of iNOS protein and iNOS activity. From in vivo
study, it was also observed that compound 1l had hepato-protective and anti-inflammatory effects in CCl4-
induced acute liver injury mouse models.
Molecular docking
1. Introduction
[5,7], anti-tumor and anti-cardiovascular [8,9], antioxidant, anti-in-
flammatory [10] and antihypertensive [11] activities. The biological
In Chinese Traditional Medicine, Citrus is the fruit peel of the
Rutaceae plants that produce oranges (Citrus reticulata Blanco) and its
cultivars. Hesperetin and hesperedin are the primary active contents of
citrus fruits. [1] Flavonoids represent a class of polyphenolic com-
pounds found in plants, such as fruit, vegetables, tea and soybeans,
which are generally stored as glycosides because they are more stable
than their aglycones [2,3]. However, due to their poor bioavailability,
glycosides are hydrolyzed to aglycones to be used [4]. Hesperedin is a
flavanone glycoside, consisting of an aglycone (hesperetin) and an at-
tached disaccharide (rutinose) [5]. As a result, hesperedin has a lower
bioavailability than hesperetin because of the rutinoside moiety at-
tached to the flavonoid and orally ingested hesperedin is hydrolyzed to
hesperetin in the gastrointestinal tract and conjugated during absorp-
tion [6].
activities depend remarkably on compounds' structural characteristics
such as conjugations, degree of hydroxylation and substitutions [12].
Because hesperetin belongs to polyphenols, various functional groups
can be substituted for its phenyl groups [2]. Notably, a small alterna-
tion in the chemical structure of flavonoids may lead to significant
changes in biological activities. We have previously studied that 5, 7,
3′-triacetyl hesperetin could suppress adjuvant-induced arthritis (AA) in
rats through modulating JAK2/STAT3 pathway, while 7, 3′-dimethoxy
hesperetin inhibited inflammation by inducing synovial apoptosis in AA
rats [13]. Then we found hesperetin derivative-7 inhibited the activa-
tion and proliferation of PDGF-BB-induced HSC-T6 and attenuated liver
fibrosis through targeting the Wnt/β-catenin signaling pathway [14],
while hesperetin derivative 11 suppressed hepatic stellate cell activa-
tion and proliferation by targeting PTEN/AKT pathway [15]. Based on
this, we tried to prepare compounds derivatized at 7-position, which
may facilitate the design of more potent hesperetin derivatives as anti-
Hesperetin and its derivatives have been found to possess a wide
range of pharmacological properties, including anti-hyperglycemic
⁎
Corresponding author at: School of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, People's Republic of China.
E-mail addresses: huangcheng812@ahmu.edu.cn (C. Huang), lj@ahmu.edu.cn (J. Li).
This author contributes equally to the first author
1
https://doi.org/10.1016/j.intimp.2018.05.009
Received 7 February 2018; Received in revised form 17 April 2018; Accepted 14 May 2018
1567-5769/©2018ElsevierB.V.Allrightsreserved.

A.-L. Huang et al.
InternationalImmunopharmacology61(2018)82–91
inflammatory leads.
Table 1, most of these compounds are not significantly cytotoxic.
Thereby, further studies for the anti-inflammatory activity with the
compounds were observed at the test concentration of 10 μM.
In the inflammation processes, the expression of cytokines or med-
iators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β),
and interleukin-6 (IL-6), as well as nitric oxide synthase are induced in
immune cells by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). The
suppression of cytokines production is an indicator of anti-in-
flammatory agents. NO plays an important physiological role as a de-
fense molecule in the immune system, while the excess production of
NO by macrophages contributes to numerous pathological processes
[16]. Inducible nitric oxide synthase (iNOS), which is responsible for
the overproduction of NO, has been involved in a number of in-
flammatory diseases. Therefore, selective inhibition of iNOS would be a
valid approach and prime target for the reduction of inflammation [17].
On the other hand, NF-κB represents a group of structurally related and
evolutionarily conserved proteins that are responsible for the regulation
of the expression of several genes involved in inflammatory response
[18,19]. NF-κB is a major transcription factor that modulates the ex-
pression of pro-inflammatory proteins (e.g., iNOS) and pro-in-
flammatory cytokines (e.g., TNF-α and IL-1β) induced by LPS. Several
studies have indicated the contribution of components of the NF-κB
signaling pathways in the pathogenesis of chronic inflammatory disease
[20].
Herein, we report on synthesis and inhibitory activity on expression
of IL-1β and IL-6, iNOS and consequent production of NO, a re-
presentative mediator of inflammatory responses, in the LPS stimulated
RAW 264.7 macrophage cell line of novel hesperetin derivatives.
Furthermore, we investigated the suppression of NF-κB signaling
pathway by the candidate. For the in vivo study, the potential candidate
demonstrated a remarkable hepato-protective effect in CCl4-induced
acute liver injury as expected. In addition, according to our speculation,
we did a supplementary molecular docking between compounds and
iNOS at the end of the study, which finally found that the docking
scores were essentially correspond with the in vitro study. The further
investigation is in progress. The goal of this study was to find hesperetin
derivatives showing better biological activity than hesperetin itself.
Analysis of the structure-activity relationship of the derivatives may
provide a way to develop novel anti-inflammatory drug candidates.
2.2.2. Inhibitory activity of NO production
Macrophages produce various pro-inflammatory mediators, in-
cluding the short-lived free radical NO. Moreover, LPS is one of the
most powerful activators of macrophages and stimulates NO produc-
tion. First of all, the compounds had been confirmed to have no cyto-
toxic activity toward RAW264.7 cells at the concentration of 10 μM.
Thereby, the inhibition of NO production by compounds was not at-
tributable to cytotoxic effects at the test concentration. The synthetic
hesperetin derivatives were evaluated for inhibitory effects on the
production of NO in LPS stimulated RAW264.7 macrophage cells. When
the cells were treated with LPS (1 μg/mL) for 22 h, the NO production
was markedly stimulated from the basal level of 1.095
19.908 0.898 μM. The inhibitory activity of the compounds is
0.056 μM to
summarized in Table 2. LPS stimulation resulted in a marked induction
of NO production as compared to untreated cells. In addition, pre-
treatment with some of these compounds prior to LPS stimulation sig-
nificantly reduced NO production. Among them, compound 1f, 1h, 1i
and 1k showed great activities, while compound 1 l exhibited the best
inhibitory effect. Meanwhile, compound 1l inhibited production of NO
in 37.30% at the concentration of 10 μM.
2.2.3. Inhibitory activity of IL-1β and IL-6
The anti-inflammatory activity of hesperetin and its derivatives
were also evaluated by their inhibitory effect against LPS-induced IL-1β
and IL-6 release in the RAW264.7 macrophages. The results are sum-
marized in Fig. 1 and expressed as percent of LPS control. IL-1β and IL-6
secretion was significantly inhibited by most of the synthetic com-
pounds at the concentration of 10 μM. The inhibition rates were from
40.12% to 91.72% and from 45.94% to 89.37%, respectively. Half of
the synthetic hesperetin derivatives such as compound 1h, 1k, 1l, 2a,
2b and 2c, had greater inhibitory effects than hesperetin itself. Though
the activities of some compounds were somewhat low in comparison
with hesperetin, the remainder tested compounds significantly in-
hibited LPS-induced expression of both IL-1β and IL-6. What's more,
compound 1l can efficiently block the LPS-induced production of dif-
ferent cytokines in macrophages, which is in good agreement with our
observations of its activity in NO inhibition. Therefore, compound 1l
was selected for further pharmacological evaluations.
2. Result and discussion
2.1. Chemistry
The preparation of a series of novel hesperetin derivatives was
carried out as illustrated in Scheme 1. The compounds differed in the
substitution of the 7-hydroxyl. The derivatives of the title compounds
were prepared by reacting with different brominated aryl and alkyl to
obtain 1a–1m and 2a–2c. The choice of the reaction is at a hydroxyl
group, which means at the 7-position or/and 3′-position. During the
stirring, appropriate and slight K2CO3 was intermittently added to the
solution so that the nucleophilic substitution could act or only act at the
7-position as far as possible. The structures of compounds were con-
firmed by elemental and spectral analyses. Analytical and spectral data
(1H NMR, 13C NMR and mass spectra) of the synthesized compounds
were in full agreement with the proposed structures.
2.2.4. Analysis of structure-activity relationship
Wen-Jun Jiang et al. [21] have discussed that the suppression of NO
production of flavonoids is related to the overall topology of the mo-
lecules as well as the electrostatic property of the B ring. However, the
impact on the biological activity of the 7-position could be observed
according to Table 2. Comparing compound 1m with other benzyl
substituted hesperetin derivatives, the presence of an electron-with-
drawing group on the benzyl group is important for the inhibition of NO
production. Compounds with the cyanobenzyl group or tri-
fluoromethylbenzyl group are generally better inhibitors than those of
the fluorobenzyl group or methylbenzyl group. That is, compounds that
introduce an electron-withdrawing group on the 7-benzyl ring generally
have higher activities than compounds substituted with an electron
donor group. The result can also be obtained from the inhibitory effect
of IL-1β and IL-6 release shown in Fig. 1. Besides, the carbon in the
cyano group and the fluorine in the trifluoromethyl group have lone
pair electrons, which theoretically can form hydrogen bonds to enhance
the binding capacity of the compound to receptors. Furthermore, from
the comparison between electron-withdrawing groups, it can be found
that the attachment of the group to the benzyl group also affects the
biological activities. Compound 1h (meta-position), 1k (meta-position),
and 1l (para-position) remarkably suppressed the NO production and
the release of IL-1β and IL-6 at the same time. In contrast, compound
2.2. In vitro pharmacology
2.2.1. Cytotoxicity and cell viability test
MTT assay was carried out to evaluate cytotoxicity and cell viability
of hesperetin derivatives. The cytotoxicity tests were performed at the
concentration of 400 μM, 200 μM, 100 μM, 50 μM, and 25 μM, and the
results are shown in Table 1. According to the IC50 values, the con-
centrations of the cell viability tests were designed at 40 μM, 20 μM,
10 μM, and 5 μM. The results clearly demonstrate that after the con-
centration of 20 μM treatment, the cell viability of several compounds
are < 90%. Nevertheless, at the concentration of 10 μM as shown in
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InternationalImmunopharmacology61(2018)82–91
OH
O
OH
O
OH
HO
HO
OH
H2
O
HO
O
O
C
O
O
O
O
O
A
HO
OH
OH
OH
OH
hesperitin
hesperidin
1a: R1=H, R2=H, R3=F
1b: R1=H, R2=F, R3=H
1c: R1=F, R2=H, R3=H
1d: R1=H, R2=H, R3=CH3
1e: R1=H, R2=CH3, R3=H
1f: R1=CH3, R2=H, R3=H
1g: R1=H, R2=H, R3=CN
1h: R1=H, R2=CN, R3=H
1i: R1=CN, R2=H, R3=H
1j: R1=H, R2=H, R3=CF3
1k: R1=H, R2=CF3, R3=H
1l: R1=CF3, R2=H, R3=H
1m: R1=H, R2=H, R3=H
Ar
OH
R1
O
O
O
R2
R3
OH
O
1a-1m
B
OH
O
2a: R4=C2H5
O
O
O
R4
2b: R4=C3H7
2c: R4=CH2CH=CH2
OH
Scheme 1. Synthesis of hesperetin derivatives 1a–1m and 2a–2c. Reagents and conditions: (A) 96% H2SO4, MeOH, 80 °C, reflux. (B) DMF, K2CO3, Bromide, 20 °C.
Table 1
Table 2
IC50 value and cell viability of RAW 264.7 cells at the compound concentration
of 20 μM and 10 μM.
NO production in LPS-treated RAW264.7 cells at the
compound concentration of 10 μM.
Compound
IC50(μM)
%cell viability in 20 μM
%cell viability in 10 μM
Compound
NO production(μM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
2a
2b
2c
185.66
287.28
245.13
291.05
216.10
266.44
249.38
208.75
131.60
120.14
128.01
148.87
177.35
229.34
158.73
158.45
1.96
4.34
1.33
8.13
5.97
2.99
5.28
8.58
2.34
2.27
1.14
1.44
1.14
4.00
2.64
3.98
83.08
83.96
82.77
92.11
89.70
98.43
87.71
100.95
95.08
103.02
79.63
80.31
89.95
112.59
92.93
98.32
4.51
0.81
2.71
2.23
0.36
1.96
3.18
0.81
1.20
1.09
1.54
0.09
0.23
2.06
1.13
2.78
89.45
90.89
89.29
102.33
91.00
102.50
98.50
101.62
102.52
98.41
88.34
92.85
92.05
106.47
91.93
96.94
2.11
1.78
0.99
5.78
0.15
3.84
4.05
0.97
1.78
0.46
2.59
3.86
2.91
0.34
1.10
2.20
LPS
DMSO
Hesperetin
19.908
1.095
0.898
0.056
0.099*
0.836
18.056
19.702
16.184
17.803
15.666
17.612
15.418
16.364
14.107
14.986
18.086
14.465
12.892
19.39
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
2a
2b
2c
0.108**
0.218*
0.295**
0.183*
0.193**
0.113**
0.143***
0.191***
0.277*
0.316***
0.261****
0.193
18.132
17.729
19.911
0.145*
1.178*
0.267
The results are presented as the means SD of three different experiments.
2a, 2b, and 2c, which had potential inhibitory effect of IL-1β and IL-6
release, demonstrated no NO inhibition. The mechanism of the two
different series of derivatives needs further investigation.
The LPS and DMSO group were pretreated with 1‰ DMSO
solution without compound. The test compounds (10 μM)
were 2 h prior to LPS stimulation. Data are presented as
mean
compared to vehicle. The difference was considered sta-
tistically significant when (*) < 0.05, (**) < 0.01,
(***) < 0.001, (****) < 0.0001.
SD (n = 3) and analyzed using Dunnett-t-test
2.2.5. Effect of compound 1l on iNOS and NF-κB pathway
p
High levels of NO are considered to be a biomarker for in-
flammatory disorders and a useful target for the procurement of anti-
inflammatory agents [22]. The overproduction of NO can attribute to
the overexpression of iNOS which is significant in inflammatory pro-
cesses. The iNOS protein level was markedly increased in LPS-induced
RAW264.7 cells (Fig. 2), but co-treatment with compound 1l sup-
pressed the iNOS protein level in a concentration-dependent manner.
This finding suggests that the inhibition of NO production by compound
1l may be correlated with its suppressive effect on LPS-induced iNOS
protein.
The translocation of NF-κB to the nucleus is preceded by the phos-
phorylation and proteolytic degradation of IκBα by IκB kinases (IKKs)
[23]. In general, in un-stimulated cells, NF-κB subunits (p50 and/or
p65) form a complex with an inhibitory factor, IκBα, in the cytosol and
thus are inactivated. [22] When stimulated by pro-inflammatory
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InternationalImmunopharmacology61(2018)82–91
Fig. 1. Hesperetin and synthetic compounds inhibited
LPS-induced IL-1β and IL-6 secretion in RAW 264.7
macrophages. Cells were pretreated with synthetic com-
pounds (10 μM) for 2 h, then treated with LPS (1 μg/ml)
for 22 h. The results are expressed as percent of LPS
100
75
50
25
0
control. Data are presented as mean
SD (n = 3) and
analyzed using Dunnett-t-test. The difference was con-
sidered statistically significant when
p (*) < 0.05,
(**) < 0.01, (***) < 0.001.
B
100
75
50
25
0
p65
ꢄꢅꢀ
ꢃꢀ
ꢂꢀ
ꢁꢀ
ꢀ
p-p65
iNOS
*
IκBα
β-actin
**
***
LPS
(1μg/ml)
Compound 1l
(μM)
β-actin
LPS
ͬ
+
-
+
+
+
5
+
-
20 10
2.5
ͬ
+
-
+
+
+
5
+
(1μg/ml)
Compound 1l
(μM)
-
20 10
2.5
D
F
E
ꢄꢅꢀ
ꢄꢅꢀ
ꢃꢀ
ꢂꢀ
ꢁꢀ
ꢀ
ꢄꢅꢀ
ꢃꢀ
ꢂꢀ
ꢁꢀ
ꢀ
ꢊꢊ
ꢃꢀ
ꢂꢀ
ꢁꢀ
ꢀ
*
**
ꢊ
*
**
***
***
Fig. 2. The effects of compound 1l on iNOS, NF-κB and its related proteins. RAW 264.7 cells were pretreated with various concentrations of compound 1l for 2 h and
then stimulated with LPS (1 μg/mL) for 1 h (p-p65) or 22 h (iNOS, p65, IκBα). Proteins were analyzed using western blotting.(B) Quantitative analysis of iNOS
expression, β-actin was used as loading control; (D) Quantitative analysis of p65 expression, β-actin was used as loading control; (E) Quantitative analysis of IκBα
expression, β-actin was used as loading control; (F) Quantitative analysis of p-p65 expression, β-actin was used as loading control. Data are presented as means
(n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus the LPS (treated with LPS only) group.
SD
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InternationalImmunopharmacology61(2018)82–91
Fig. 3. Hepato-protective effects of compound 1l on mice with CCl4-induced acute liver injury model, n = 8 mice per group. (A) Representative images from C57BL/
6J mice fresh livers in Control, CCl4, compound 1l treatment groups and Silymarin treatment group. (B)Pathology observation of mice liver tissues sections stained
with hematoxylin and eosin (H & E). Representative views from each group are presented (×10). Low: compound 1l (50 mg/kg) + CCl4; Middle: compound 1l
(100 mg/kg) + CCl4; High: compound 1l (200 mg/kg) + CCl4.
signaling, IκBα is phosphorylated by IKKs and then subject to ubiquitin-
mediated degradation. In NF-κB signaling, IκB degradation frees NF-κB
p65 subunit and allows it to translocate to the nucleus, followed by
turning on transcription of inflammatory genes [24]. As shown in
Fig. 2, LPS induced the IκBα degradation and the phosphorylation of
p65. A pretreatment with compound 1l significantly reversed LPS-in-
duced degradation of IκBα, further showed dose-dependent inhibition
against LPS-induced IκBα degradation. A similar result was also ob-
served in inhibition on p65 phosphorylation in a dose-dependent
manner. Thus, compound 1 l seems to exert anti-inflammatory actions
partly via inhibiting NF-κB pathway.
We have previously demonstrated that several hesperetin deriva-
tives affected iNOS protein and Notch signaling [6]. These results in-
dicate a mechanistic difference among active anti-inflammatory he-
speretin derivatives, although they are derived from the same structural
lead. This suggests that the structural modification increases and even
changes or has different possible molecular targets. It is really worthy to
be further investigated in the future for the chemical and structural
features influencing the biological mechanism.
restored to some extent compared with the control group. High-dose
compound 1l (200 mg/kg) yielded more effective rescue, almost com-
parable to positive control group.
2.3.2. Effects of compound 1l on hepatic markers and cytokine production
in the CCl4-induced acute liver injury model
ALT, AST, are liver enzyme markers, and elevated levels of these
markers in serum indicate the loss of hepatocyte structural integrity and
liver injury [25]. Compared their levels with the control group, AST,
ALT levels were significantly increased in mice in the CCl4 group
(p < 0.001),as shown in Fig. 4A and B. However, mice treated with
compound 1l (50 mg/kg, 100 mg/kg, 200 mg/kg) and Silymarin
(200 mg/kg) markedly attenuated the serum levels of ALT and AST in a
dose-dependent manner. Moreover, results of ELISA assay demon-
strated that compound 1l pretreatment significantly attenuated pro-
inflammatory cytokines IL-1β and IL-6 secretion levels in a concentra-
tion-dependent manner (Shown in Fig. 4C and D). Additionally, the
compound 1l treatment group (200 mg/kg) showed similar hepato-
protective and anti-inflammatory effects compared with Silymarin
(200 mg/kg) group. All the features indicate that compound 1l has
significant effects on hepato-protective and suppresses the in-
flammatory response in CCl4-induced acute liver injury.
2.3. In vivo anti-inflammatory activity
2.3.1. Effects of compound 1l on histopathological changes in the liver
tissue of CCl4-induced acute liver injury mice
2.4. Molecular docking
In Fig. 3, histopathological study was performed to investigate the
effect of compound 1l on mice with CCl4- induced acute liver injury.
Mice liver tissues sections stained with hematoxylin and eosin (H & E)
showed that CCl4 (1%) injection caused remarkable acute liver injury
exhibited hepatocyte necrosis, irregularly arranged, vacuole formation,
loss of cellular boundaries and inflammatory cells infiltration compared
with the control group. Notably, after the compound 1l of different
concentrations (50 mg/kg, 100 mg/kg, 200 mg/kg) and Silymarin
(200 mg/kg) treatment, the histological features of hepatocytes were
Molecular docking simulations have become an important tool for
understanding the interaction mode and the structure-activity re-
lationships of ligands with receptors [1]. Indeed, targeting the iNOS has
been proposed as an anti-inflammatory therapeutic strategy since high
concentrations of NO are essential in inflammation and related pro-
cesses [26]. It has been reported that the anti-inflammatory effect of
lots of natural products and their derivatives was associated with the
active site of iNOS. Although our results have verified the iNOS
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Fig. 4. Effects of compound 1l on hepatic mar-
kers and cytokine production in the CCl4-in-
duced acute liver injury model. (A) Effect of
different concentrations of compound 1l and
Silymarin on serum ALT level of each group. (B)
Effect of different concentrations of compound
1l and Silymarin on serum AST level of each
group. (C) Effect of different concentrations of
compound 1l and Silymarin on serum IL-1β
level of each group. (D) Effect of different con-
centrations of compound 1l and Silymarin on
serum IL-6 level of each group. Data represent
the mean
experiments.
S.D. for at least three independent
*p < 0.05, **p < 0.01,
***p < 0.001 versus the CCl4 (treated with CCl4
only)
group.
#p < 0.05,
##p < 0.01,
###p < 0.001 versus the control group. Low:
compound 1l (50 mg/kg) + CCl4; middle: com-
pound 1l (100 mg/kg) + CCl4; high: compound
1l (200 mg/kg) + CCl4.
Fig. 5. Molecular docking study of 1l binding to iNOS protein. (A)Docking model of 1l in the active site of iNOS. (B)2D interaction diagram of compound 1l and
iNOS.
expression was reduced through NF-κB signaling pathway, the Quantity
of iNOS expression after compound pre-treated was still high enough
compared to vehicle. The significant NO inhibitory activities of the
hesperetin derivatives prompted us to explore the interactions of these
bioactive compounds with iNOS protein (PDB code: 3EAI) [27] by
molecular docking studies using Discovery Studio 2017 R2. The results
were unexpected and showed that the LibDockScore of the compounds
were approximately in good accordance with the activity of NO in-
hibitory effects. As shown in Fig. 5, it revealed that compound 1l binds
similarly in the iNOS active site heme pocket with the co-crystal
inhibitor ligand and showed a higher LibDockScore (175.714) than the
co-crystal inhibitor (150.933). Additionally, the ether group of the
hesperetin moiety established a conventional hydrogen bond with the
hydroxyl group of the main chain of ARG382. It seemed that the pre-
sence of the 7-benzyl substituted hesperetin backbone led to the for-
mation of the π-alkyl interaction with the amino acid residue PRO344,
ARG375 and the π-anion interaction with the amino acid residue
ASP376, HEM901, which were important to the binding site. The tri-
fluoromethyl group established a π-donor hydrogen bond with GLU371,
PRO461 and formed a halogen interaction with THR370. The results of
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molecular docking indicate that the possible mechanism of NO inhibi-
tion of these bioactive hesperetin derivatives is to interact with the
iNOS protein by targeting residues of the active cavities of iNOS. Fur-
ther bioactive studies of the high LibDockScore compounds corre-
sponding to the inflammatory activity are warranted, and further stu-
dies are currently underway in our laboratory. It reminds us that the
anti-inflammatory activity of hesperitin derivatives may be the result of
a Multi-target effect.
drying with anhydrous sodium sulfate, the solution was filtered through
absorbent cotton. The precipitate obtained by removing the solvent
under reduced pressure was purified by column chromatography (SiO2)
using trichloromethane/petroleum ether (1:1–5) as eluent. The purified
precipitate was recrystallized from dichloromethane/petroleum ether.
4.2.1. (S)-7-[(2-fluorobenzyl)oxy]-5-hydroxy-2-(3-hydroxy-4-
methoxyphenyl)chroman-4-one (1a)
Synthesis of 1a followed the general procedure in 72% yield as a
white to off-white crystalline solid: mp: 159–163 °C; 1H NMR (400 MHz,
CDCl3): δ 12.00 (s, 1H, 5-OH), 7.44 (td, J = 7.5, 1.7 Hz, 1H, ArH), 7.33
(tdd, J = 7.3, 5.3, 1.8 Hz, 1H, ArH), 7.17 (td, J = 7.5, 1.1 Hz, 1H, ArH),
7.12–7.06 (m, 1H, ArH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H), 6.93 (dd,
J = 8.3, 2.0 Hz, 1H, 6′-H), 6.88 (d, J = 8.3 Hz, 1H, 5′-H), 6.16 (d,
J = 2.3 Hz, 1H, 8-H), 6.13 (d, J = 2.3 Hz, 1H, 6-H), 5.33 (dd, J = 12.9,
3.0 Hz, 1H, 2-H), 5.13 (s, 2H, ArCH2O), 3.92 (s, 3H, OCH3), 3.08 (dd,
J = 17.2, 12.9 Hz, 1H, 3-H), 2.79 (dd, J = 17.2, 3.1 Hz, 1H, 3-H); TOF-
HRMS: m/z [M + H]+calcd for C23H20FO6: 411.1238; found: 411.1240.
3. Conclusion
In summary, a series of hesperetin derivatives were successfully
synthesized with high overall yield (65–80%). Their anti-inflammatory
activity was evaluated by inhibiting IL-1β, IL-6 and production of NO in
mouse RAW264.7 macrophages. Several compounds showed potent
inhibitory activity toward LPS-induced NO production as well as similar
inhibitory activity on IL-1β and IL-6. Compound 1l and 2c appeared to
be most potent among the synthesized compounds. In addition, com-
pound 1l significantly suppressed cytokines produced. The present
study suggests that the suppression of iNOS and NF-κB pathway is, at
least in part, related to the inhibitory activity of these hesperetin de-
rivatives. Besides, the results of molecular docking study verify the anti-
inflammatory effect of compound 1l and may reveal the Multi-target
effect of hesperetin derivatives. From the in vivo study, compound 1l
had an anti-inflammatory effect in the CCl4-induced acute liver injury
model by inhibiting the expression of cytokines in serum, including IL-
1β and IL-6. Moreover, it was also observed that compound 1l atte-
nuated the levels of ALT, AST and liver histopathologic changes. It
shows a promising anti-inflammatory activity which could be beneficial
for use in the treatment of inflammatory diseases. Based on the struc-
ture-activity relationship of above-mentioned derivatives, established
in this study, electron-withdrawing group attached on the A ring was
crucial for the needed biological activities.
4.2.2. (S)-7-[(3-fluorobenzyl)oxy]-5-hydroxy-2-(3-hydroxy-4-
methoxyphenyl)chroman-4-one (1b)
Synthesis of 1b followed the general procedure in 75% yield as a
faint yellow powder: mp: 149–155 °C; 1H NMR (400 MHz, CDCl3): δ
12.00 (s, 1H, 5-OH), 7.35 (td, J = 8.0, 5.8 Hz, 1H, ArH), 7.15 (dd,
J = 7.6, 0.6 Hz, 1H, ArH), 7.13–7.08 (m, 1H, ArH), 7.06–6.99 (m, 2H,
ArH, 2′-H), 6.92 (dd, J = 8.4, 1.9 Hz, 1H, 6′-H), 6.88 (d, J = 8.3 Hz, 1H,
5′-H), 6.12 (d, J = 2.3 Hz, 1H, 8-H), 6.10 (d, J = 2.3 Hz, 1H, 6-H), 5.33
(dd, J = 12.9, 3.0 Hz, 1H, 2-H), 5.06 (s, 2H, ArCH2O), 3.92 (s, 3H,
OCH3), 3.08 (dd, J = 17.2, 12.9 Hz, 1H, 3-H), 2.79 (dd, J = 17.2,
3.1 Hz, 1H, 3-H); TOF-HRMS: m/z [M + H]+calcd for C23H20FO6:
411.1238; found: 411.1239.
4.2.3. (S)-7-[(4-fluorobenzyl)oxy]-5-hydroxy-2-(3-hydroxy-4-
methoxyphenyl)chroman-4-one (1c)
4. Experimental section
Synthesis of 1c followed the general procedure in 78% yield as a
faint yellow crystalline solid: mp: 167–168 °C; 1H NMR (400 MHz,
CDCl3): δ 12.00 (s, 1H, 5-OH), 7.40–7.33 (m, 2H, ArH), 7.10–7.05 (m,
2H),7.04 (d, J = 2.1 Hz, 1H, 2′-H), 6.92 (dd, J = 8.4, 2.0 Hz, 1H, 6′-H),
6.88 (d, J = 8.3 Hz, 1H, 5′-H), 6.12 (d, J = 2.3 Hz, 1H, 8-H), 6.09 (d,
J = 2.3 Hz, 1H, 6-H), 5.33 (dd, J = 12.9, 3.0 Hz, 1H, 2-H), 5.02 (s, 2H,
ArCH2O), 3.92 (s, 3H, OCH3), 3.07 (dd, J = 17.2, 12.9 Hz, 1H, 3-H),
2.79 (dd, J = 17.2, 3.1 Hz, 1H, 3-H); TOF-HRMS: m/z [M + H]+calcd
for C23H20FO6: 411.1238; found: 411.1235.
4.1. Chemistry
Reagents and starting materials were obtained from commercial
suppliers and were used without purification. The reactions were
monitored by thin-layer chromatography (TLC) on precoated silica
GF254 plates. Column chromatography was done on silica gel 200
(Sorbent technologies). 1H and 13C NMR spectra were recorded on a
Brucker AM-300 (300 MHz) spectrometer with CDCl3 or DMSO‑d6 as
the solvent and TMS as the internal standard. Mass spectra data was
obtained on an Agilent 6220 Accurate-Mass time-of-flight liquid chro-
matography/mass spectrometer (TOF LC/MS). Chemical names were
generated using ChemDraw Ultra (CambridgeSoft, version16.0).
Melting points were measured on a XT-4 microscopic thermometer
(Beijing Tech Instrument Co., Ltd., Beijing, China) without calibration.
Chemical shifts were reported in parts per million (d) units relative to
the solvent peak. The 1H NMR data were reported as peak multi-
plicities: s for singlet; d for doublet; t for triplet; and m for multiplet.
Coupling constants were recorded in hertz.
4.2.4. (S)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-[(2-
methylbenzyl)oxy]chroman-4-one (1d)
Synthesis of 1d followed the general procedure in 74% yield as an
off-white powder: mp: 137–141 °C; 1H NMR (400 MHz, CDCl3): δ 12.01
(s, 1H, 5-OH), 7.38–7.33 (m, 1H, ArH), 7.29–7.24 (m, 1H, ArH),
7.24–7.19 (m, 2H, ArH), 7.05 (d, J = 2.1 Hz, 1H), 6.93 (dd, J = 8.4,
2.0 Hz, 1H, 6′-H), 6.88 (d, J = 8.3 Hz, 1H, 5′-H), 6.16 (d, J = 2.3 Hz,
1H, 8-H), 6.13 (d, J = 2.3 Hz, 1H, 6-H), 5.33 (dd, J = 12.9, 3.0 Hz, 1H,
2-H), 5.04 (s, 2H, ArCH2O), 3.92 (s, 3H, OCH3), 3.08 (dd, J = 17.2,
13.0 Hz, 1H, 3-H), 2.79 (dd, J = 17.2, 3.0 Hz, 1H, 3-H), 2.34 (s, 3H,
ArCH3); TOF-HRMS: m/z [M + H]+calcd for C24H23O6: 407.1489;
found: 407.1494.
4.2. General method for the preparation of hesperetin derivatives
K2CO3 (172.76 mg, 1.25 mmol) and the appropriate substituted
bromine (4–10 mmol) were added, under stirring, to a solution of he-
speretin (604 mg, 2 mmol) in anhydrous DMF (15 mL). Another 1 mmol
of K2CO3 was intermittently added to the solution to keep the pH as a
weak alkaline. The mixture was reacted at room temperature for 4–6 h
under atmospheric conditions. The progress of the reaction was mon-
itored by TLC. The reaction mixture poured into ice cold water then
acidified by dilute hydrochloric acid and extracted with ethyl acetate,
then washed three times with saturated aqueous NaCl solution. After
4.2.5. (S)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-[(3-
methylbenzyl)oxy]chroman-4-one (1e)
Synthesis of 1e followed the general procedure in 67% yield as a
white fluffy solid: mp: 118–125 °C; 1H NMR (400 MHz, CDCl3): δ 12.00
(s, 1H, 5-OH), 7.29–7.24 (m, 1H, ArH), 7.21–7.12 (m, 3H, ArH), 7.04
(d, J = 2.0 Hz, 1H, 2′-H), 6.92 (dd, J = 8.5, 2.0 Hz, 1H, 6′-H), 6.88 (d,
J = 8.3 Hz, 1H, 5′-H), 6.14 (d, J = 2.3 Hz, 1H, 8-H), 6.11 (d,
J = 2.3 Hz, 1H, 6-H), 5.31 (dt, J = 5.1, 2.5 Hz, 1H, 2-H), 5.02 (s, 2H,
ArCH2O), 3.91 (s, 3H, OCH3), 3.07 (dd, J = 17.2, 12.9 Hz, 1H, 3-H),
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2.78 (dd, J = 17.2, 3.1 Hz, 1H, 3-H), 2.37 (s, 3H, ArCH3); TOF-HRMS:
m/z [M + H]+calcd for C24H23O6: 407.1489; found: 407.1497.
4.2.11. (S)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-{[3-
(trifluoromethyl)benzyl]oxy}chroman-4-one (1k)
Synthesis of 1k followed the general procedure in 78% yield as a
white fluffy solid: mp: 141–143 °C; 1H NMR (400 MHz, CDCl3): δ 12.01
(s, 1H, 5-OH), 7.68–7.48 (m, 4H, ArH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H),
6.93 (dd, J = 8.4, 1.9 Hz, 1H, 6′-H), 6.88 (d, J = 8.3 Hz, 1H, 5′-H), 6.14
(d, J = 2.3 Hz, 1H, 8-H), 6.11 (d, J = 2.3 Hz, 1H, 6-H), 5.34 (dd,
J = 12.9, 3.0 Hz, 1H, 2-H), 5.11 (s, 2H, ArCH2O), 3.92 (s, 3H, OCH3),
3.08 (dd, J = 17.2, 12.9 Hz, 1H, 3-H), 2.80 (dd, J = 17.2, 3.1 Hz, 1H, 3-
H); TOF-HRMS: m/z [M + H]+calcd for C24H20F3O6: 461.1206; found:
461.1210.
4.2.6. (S)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-[(4-
methylbenzyl)oxy]chroman-4-one (1f)
Synthesis of 1f followed the general procedure in 73% yield as a
white fluffy solid: mp: 153–155 °C; 1H NMR (400 MHz, CDCl3): δ 12.00
(s, 1H, 5-OH), 7.27 (d, J = 8.0 Hz, 2H, ArH), 7.19 (d, J = 7.8 Hz, 2H,
ArH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H), 6.92 (dd, J = 8.5, 2.2 Hz, 1H, 6′-
H), 6.87 (d, J = 8.3 Hz, 1H, 5′-H), 6.13 (d, J = 2.3 Hz, 1H, 8-H), 6.10
(d, J = 2.3 Hz, 1H, 6-H), 5.31 (dd, J = 12.9, 3.0 Hz, 1H, 2-H), 5.02 (s,
2H, ArCH2O), 3.91 (s, 3H, OCH3), 3.06 (dd, J = 17.2, 12.9 Hz, 1H, 3-
H), 2.77 (dd, J = 17.2, 3.1 Hz, 1H, 3-H), 2.36 (s, 3H, ArCH3); TOF-
HRMS: m/z [M + H]+calcd for C24H23O6: 407.1489; found: 407.1492.
4.2.12. (S)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-{[4-
(trifluoromethyl)benzyl]oxy}chroman-4-one (1l)
Synthesis of 1l followed the general procedure in 77% yield as a
white powder: mp: 141–146 °C; 1H NMR (400 MHz, CDCl3): δ 12.01 (s,
1H, 5-OH), 7.65 (d, J = 8.1 Hz, 2H, ArH), 7.50 (d, J = 8.0 Hz, 2H,
ArH), 7.04 (d, J = 2.0 Hz, 1H, 2′-H), 6.92 (dd, J = 8.4, 2.0 Hz, 1H, 6′-
H), 6.88 (d, J = 8.3 Hz, 1H, 5′-H), 6.12 (d, J = 2.3 Hz, 1H, 8-H), 6.10
(d, J = 2.3 Hz, 1H, 6-H), 5.33 (dd, J = 12.9, 3.0 Hz, 1H, 2-H), 5.13 (s,
2H, ArCH2O), 3.91 (s, 3H, OCH3), 3.08 (dd, J = 17.2, 12.9 Hz, 1H, 3-
H), 2.79 (dd, J = 17.2, 3.1 Hz, 1H, 3-H); TOF-HRMS: m/z
[M + H]+calcd for C24H20F3O6: 461.1206; found: 461.1210.
4.2.7. (S)-2-{[(5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-
oxochroman-7-yl)oxy]methyl}benzonitrile (1g)
Synthesis of 1 g followed the general procedure in 65% yield as a
white powder: mp: 173–178 °C; 1H NMR (400 MHz, CDCl3): δ 12.00 (s,
1H, 5-OH), 7.75–7.68 (m, 1H, ArH), 7.67–7.59 (m, 2H, ArH), 7.50–7.43
(m, 1H, ArH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H), 6.93 (dd, J = 8.5, 2.0 Hz,
1H, 6′-H), 6.89 (d, J = 8.3 Hz, 1H, 5′-H), 6.16 (d, J = 2.3 Hz, 1H, 8-H),
6.14 (d, J = 2.3 Hz, 1H, 6-H), 5.34 (dd, J = 12.9, 3.0 Hz, 1H, 2-H), 5.26
(s, 2H, ArCH2O), 3.92 (s, 3H, OCH3), 3.09 (dd, J = 17.2, 12.9 Hz, 1H, 3-
H), 2.80 (dd, J = 17.2, 3.1 Hz, 1H, 3-H); TOF-HRMS: m/z
[M + H]+calcd for C24H20NO6: 418.1285; found: 418.1285.
4.2.13. (S)-7-(benzyloxy)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)
chroman-4-one (1m)
Synthesis of 1m followed the general procedure in 71% yield as a
white fluffy solid: mp: 142–144 °C; 1H NMR (400 MHz, CDCl3): δ 12.00
(s, 1H, 5-OH), 7.41–7.30 (m, 5H, ArH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H),
6.92 (dd, J = 8.5, 2.2 Hz, 1H, 6′-H), 6.88 (d, J = 8.3 Hz, 1H, 5′-H), 6.14
(d, J = 2.3 Hz, 1H, 8-H), 6.11 (d, J = 2.3 Hz, 1H, 6-H), 5.32 (dd,
J = 12.9, 3.0 Hz, 1H, 2-H), 5.07 (s, 2H, ArCH2O), 3.91 (s, 3H, OCH3),
3.07 (dd, J = 17.2, 12.9 Hz, 1H, 3-H), 2.78 (dd, J = 17.2, 3.1 Hz, 1H, 3-
H); TOF-HRMS: m/z [M + H]+calcd for C23H21O6: 393.1333; found:
393.1331.
4.2.8. (S)-3-{[(5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-
oxochroman-7-yl)oxy]methylbenzonitrile (1h)
Synthesis of 1h followed the general procedure in 67% yield as an
off-white powder: mp: 200–205 °C; 1H NMR (400 MHz, [D6]DMSO): δ
12.10 (s, 1H, 5-OH), 9.11 (s, 1H, 3′-OH), 7.91 (br, 1H, ArH), 7.83 (d,
J = 7.7 Hz, 1H, ArH), 7.79 (d, J = 7.9 Hz, 1H, ArH), 7.63 (t, J = 7.8 Hz,
1H, ArH), 6.96–6.94 (m, 2H, 2′-H, 5′-H), 6.89 (dd, J = 8.3, 2.1 Hz, 1H,
6′-H), 6.22 (d, J = 2.3 Hz, 1H, 8-H), 6.20 (d, J = 2.3 Hz, 1H, 6-H), 5.50
(dd, J = 12.4, 3.0 Hz, 1H, 2-H), 5.25 (s, 2H, ArCH2O), 3.79 (s, 3H,
OCH3), 3.28 (dd, J = 17.2, 12.4 Hz, 1H, 3-H), 2.77 (dd, J = 17.2,
3.2 Hz, 1H, 3-H); TOF-HRMS: m/z [M + H]+calcd for C24H20NO6:
418.1285; found: 418.1284.
4.2.14. (S)-7-ethoxy-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-
4-one (2a)
Synthesis of 2a followed the general procedure in 66% yield as an
white fragmentary solid: mp: 145–151 °C; 1H NMR (400 MHz, CDCl3): δ
12.00 (s, 1H, 5-OH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H), 6.93 (dd, J = 8.2,
1.9 Hz, 1H, 5′-H), 6.88 (d, J = 8.3 Hz, 1H, 4′-H),6.05 (d, J = 2.3 Hz,
1H, 8-H), 6.03 (d, J = 2.3 Hz, 1H, 6-H), 5.32 (dd, J = 12.9, 3.0 Hz, 1H,
2-H), 4.04 (q, J = 7.0 Hz, 2H, O), 4.04 (q, J = 7.0 Hz, 2H, OCH2C), 3.92
(s, 3H, OCH3), 3.07 (dd, J = 17.2, 12.9 Hz, 1H, 3-H), 2.78 (dd,
J = 17.1, 3.1 Hz, 1H, 3-H), 1.40 (t, J = 7.0 Hz, 3H, OCCH3); TOF-
HRMS: m/z [M + H]+calcd for C18H19O6: 331.1176; found: 331.1178.
4.2.9. (S)-4-{[(5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-
oxochroman-7-yl)oxy]methyl}benzonitrile (1i)
Synthesis of 1i followed the general procedure in 70% yield as a
white crystalline solid: mp: 165–166 °C; 1H NMR (400 MHz, CDCl3): δ
12.00 (s, 1H, 5-OH), 7.70–7.66 (m, 2H, ArH), 7.52–7.47 (m, 2H, ArH),
7.04 (d, J = 2.0 Hz, 1H, 2′-H), 6.92 (dd, J = 8.5, 1.9 Hz, 1H, 6′-H), 6.88
(d, J = 8.3 Hz, 1H, 5′-H), 6.11 (d, J = 2.3 Hz, 1H, 8-H), 6.08 (d,
J = 2.3 Hz, 1H, 6-H), 5.33 (dd, J = 12.9, 3.0 Hz, 1H, 2-H), 5.13 (s, 2H,
ArCH2O), 3.92 (s, 3H, OCH3), 3.08 (dd, J = 17.2, 12.9 Hz, 1H, 3-H),
2.80 (dd, J = 17.2, 3.1 Hz, 1H, 3-H); TOF-HRMS: m/z [M + H]+calcd
for C24H20NO6: 418.1285; found: 418.1287.
4.2.15. (S)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-
propoxychroman-4-one (2b)
Synthesis of 2b followed the general procedure in 75% yield as a
yellow crystalline solid: mp: 129–130 °C; 1H NMR (400 MHz, CDCl3): δ
12.00 (s, 1H, 5-OH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H), 6.93 (dd, J = 8.4,
2.0 Hz, 1H, 5′-H), 6.88 (d, J = 8.3 Hz, 1H, 4′-H), 6.05 (d, J = 2.3 Hz,
1H, 8-H), 6.03 (d, J = 2.3 Hz, 1H, 8-H), 5.32 (dd, J = 12.9, 3.0 Hz, 1H,
2-H), 3.92 (t, J = 6.6 Hz, 2H, OCH2), 3.91(s, 3H, OCH3), 3.06 (dd,
J = 17.2, 12.9 Hz, 1H, 3-H), 2.78 (dd, J = 17.1, 3.1 Hz, 1H, 3-H),
1.84–1.73 (m, 2H, OCCH2C), 1.01 (t, J = 7.4 Hz, 3H, OCCCH3); TOF-
HRMS: m/z [M + H]+calcd for C19H21O6: 345.1333; found: 345.1331.
4.2.10. (S)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-{[2-
(trifluoromethyl)benzyl]oxy}chroman-4-one (1j)
Synthesis of 1j followed the general procedure in 80% yield as a
faint yellow crystalline solid: mp: 129–131 °C; 1H NMR (400 MHz,
CDCl3): δ 12.00 (s, 1H, 5-OH), 7.70 (d, J = 7.8 Hz, 1H, ArH), 7.66 (d,
J = 7.8 Hz, 1H, ArH), 7.58 (t, J = 7.6 Hz, 1H, ArH), 7.45 (d, J = 7.7 Hz,
1H, ArH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H), 6.93 (dd, J = 8.4, 1.9 Hz, 1H,
6′-H), 6.88 (d, J = 8.3 Hz, 1H, 5′-H), 6.14 (d, J = 2.3 Hz, 1H, 8-H), 6.12
(d, J = 2.3 Hz, 1H, 6-H), 5.33 (dd, J = 13.0, 3.0 Hz, 1H, 2-H), 5.27 (s,
2H, ArCH2O), 3.92 (s, 3H, OCH3), 3.08 (dd, J = 17.2, 13.0 Hz, 1H, 3-
H), 2.79 (dd, J = 17.2, 3.0 Hz, 1H, 3-H); TOF-HRMS: m/z [M + H]+
calcd for C24H20F3O6: 461.1206; found: 461.1210.
4.2.16. (S)-7-(allyloxy)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)
chroman-4-one (2c)
Synthesis of 2c followed the general procedure in 65% yield as an
off-white crystalline solid: mp: 130–135 °C; 1H NMR (400 MHz, CDCl3):
δ 12.00 (s, 1H, 5-OH), 7.04 (d, J = 2.1 Hz, 1H, 2′-H), 6.93 (dd, J = 8.3,
2.1 Hz, 1H, 6′-H), 6.88 (d, J = 8.3 Hz, 5H, 5′-H), 6.07 (d, J = 2.3 Hz,
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1H, 8-H), 6.05 (d, J = 2.3 Hz, 1H, 6-H), 6.04–5.95 (m, 1H, OCCH]C),
5.40 (do, J = 17.3, 1.6 Hz, 1H, OCC]CH), 5.35–5.28 (m, 2H,
OCC]CH, 2-H), 4.54 (dt, J = 5.3, 1.5 Hz, 2H, OCH2C]C), 3.92 (s, 3H,
OCH3), 3.07 (dd, J = 17.2, 12.9 Hz, 1H, 3-H), 2.78 (dd, J = 17.2,
3.1 Hz, 1H, 3-H); TOF-HRMS: m/z [M + H]+calcd for C19H19O6:
343.1176; found: 343.1174.
replaced and cells were then incubated with or without 1 μg/mL LPS in
the presence or absence of the test compounds (10 μM) for 24 h. The
supernatant were collected and stored at −80 °C prior to analysis. The
amount of nitrite released in the culture supernatant was measured
using the Gris reagent (0.1% naphthylethylenediamine and 1% sulfa-
nilamide in 5% H3PO4 solution, Bigtime). Nitrite levels in the samples
were calculated from a standard curve with a known concentration of
sodium nitrite, and the absorbance of the samples was then measured at
540 nm using a microplate reader.
4.3. Biological assays and experimental procedures
4.3.1. Animal and treatment
C57BL/6 mice (weighting about 18–22 g each) were obtained from
the Experimental Animal Center, Anhui Medical University. All the
animal experimental and procedures were approved by the Ethic
Committee and the Animal Experimental Committee of Anhui Medical
University. Mice were reviewed and performed under the permission of
the Animal Experiments Guidelines and Animal Care of Chinese
Academy of Sciences. The mice were housed in micro isolator cages and
received food and water ad libitum. For all experiments, C57BL/6 mice
were randomly divided into six groups (n = 8 per group): normal
group, model group, compound 1l treatment group (50 mg/kg, 100 mg/
kg, 200 mg/kg) [28], and Silymarin group (200 mg/kg) [29]. Com-
pound 1l treatment group, CCl4 group and Silymarin group were in-
jected intraperitoneally with 1% CCl4 dissolved in olive oil (10 ml/kg in
oil) to induce acute liver injury model [30], in addition, compound 1l
treatment groups and Silymarin group were given via gavage different
concentrations of compound 1l and Silymarin at the same volume for
7 days before. Compounds dissolved in 0.5% sodium carboxymethyl
cellulose (CMCeNa) distilled water solution for gavage administration.
Control group was given via gavage for 0.5% CMC-Na only and injected
intraperitoneally the same volume of vehicle (olive oil). Mouse serum
was collected stored at −80 °C for the detection of inflammatory fac-
tors. The biggest lobe of the liver was excised and sectioned from each
C57BL/6 mice and fixed with 4% paraformaldehyde for at least 24 h
then embedded in paraffin. The pathological changes of liver was ob-
served and photographed by Olympus BX-51 microscope after 4 am
thick sections stained with hematoxylin and eosin (H & E) with a
standard procedure.
4.3.5. Measurement of cytokines
The cell culture supernatants were collected and the IL-1β and IL-6
levels were determined by enzyme-linked immunosorbent assay
(ELISA) (Ela science, China) following the manufacturer's protocol. The
IL-1β and IL-6 levels in Mouse serum were measured by the same
method. Two independent experiments were performed in triplicate
and a representative set of data (means standard deviations) was
used to determine the activity of each compound. The absorbance of the
samples was then measured at 450 nm using a microplate reader. The
inhibition rate of each production was calculated as inhibition rate
(%) = [1 − (sample optical density − blank optical density) / (control
optical density − blank optical density)] × 100.
4.3.6. ALT/AST activity assay
Serum extracted from C57BL/6 mice was stored at −80 °C. The
serum levels of Alanine aminotransferase (ALT) and Aspartate amino-
transferase (AST) were assayed by using ALT and AST activity assay kits
(Jincheng, Wuhan, China). The absorbance at 510 nm was obtained
with a microplate reader.
4.3.7. Western blot analysis
After culture supernatants were harvested, cells were collected and
lysed with RIPA lysis buffer (Bigtime, China). Cell lysates were cen-
trifuged at 1.2 k rpm for 30 min at 4 °C. The protein concentrations in
cell lysates were determined by using the BCA protein assay kit
(Bigtime, China). Equal amounts of protein were separated using
SDS–PAGE. Proteins were transferred to PVDF membranes (Millipore,
USA) and subsequently blocked in 5% skim milk in Tries-buffered saline
(TBS, Booster, China) containing 1% tween 20 for 1 h at room tem-
perature. After incubation with the appropriate primary antibody for a
night, membranes were incubated for 1 h at room temperature with a
secondary antibody (ZSGB-BIO, China). Following three washes in
TBST, immunoreactivity bands were visualized using the ECL-chemi-
luminescent kit (ECL-plus, Thermos Scientific). Primary antibodies used
were: iNOS, IκBα, p65 and p-p65 from Abeam and actin from ZSGB-
BIO.
4.3.2. Cell culture
RAW 264.7 cells, purchased from Cell Bank of Chinese Academy of
Sciences (Shanghai, China), were cultured in DMEM (Cyclone, USA),
supplemented with 10% fetal bovine serum (Gibson), 100 units/mL of
penicillin (Bigtime, China), 100 mg/mL of streptomycin (Bigtime,
China). Cell cultures were maintained at 37 °C in a humidified atmo-
sphere containing 5% CO2.
4.3.3. MTT cytotoxicity assay and cell viability
Cytotoxicity and cell viability was determined by 3-(4, 5-di-
methylthiazol-2-yl)-2, 5-diphenylthetrazolium bromide (MTT, Sigma,
USA) assay. Briefly, cells were inoculated at a density of 5 × 103 cells/
well into 96-well plate and cultured at 37 °C for 12 h. Cultured cells
were treated with vehicle (control) and various concentrations of
compound for 24 h. After incubation, 20 mL MTT solution (5 mg/mL in
PBS) was added to each well and incubated for another 4 h. Media were
removed and dimethyl sulfoxide (DMSO) was added to dissolve purple
crystals. Then the absorbance at 490 nm was measured using a micro-
plate reader (Synergy HTX, Biotech, USA). The values (IC50) were cal-
culated by SPSS 16.0. The cell viability was calculated from the plotted
results using untreated cells at 100%.
4.4. Molecular docking
To explore the potential interacting mode of the isolated compounds
with the iNOS protein (PDB code: 3EAI) [27], a molecular modeling
study was performed using the docking program named Lib Dock, a
software package in Discovery Studio 2017 R2. The molecular docking
procedure was performed to eliminate any bond length and bond angle
biases, so the ligands (compounds 1a–1m and 2a–2c) were subjected to
a full minimization prior to docking. The binding affinities (LibDock-
Score) in Discovery Studio 2017 R2 were used to evaluate the inter-
actions between iNOS and the ligands. The standard 3D structures of all
compounds for molecular docking were constructed by ChemDrew 16.0
software.
4.3.4. Measurement of NO levels
NO has
a
short half-life and is oxidized to stable nitrite.
4.5. Statistical analysis
Consequently, to evaluate the inhibitory activity of the test compounds
on LPS-induced NO production, RAW 264.7 cells were plated at a
density of 2 × 105 per well in a 6-well culture plate and incubated in a
37 °C humidified incubator (5% CO2) for 12 h. Then the media were
All experiments were repeated at least three times. Data are pre-
sented as the means standard deviation (SD) for the indicated
number of independently performed experiments. The significance of
90

A.-L. Huang et al.
InternationalImmunopharmacology61(2018)82–91
differences between drug-treated groups compared to vehicle was de-
termined by one way ANOVA using SPSS v16.0. The difference was
considered statistically significant when p(*) < 0.05, (**) < 0.01,
(***) < 0.001.
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Conflict of interest statement
The authors declare no conflict of interest.
Acknowledgements
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Financial support by the National Natural Science Foundation of
China (No. 81473268), Higher Education Natural Science Foundation
of Anhui Province (KJ2016A364) and National Students' project for
innovation and entrepreneurship training program (201610366036) is
gratefully acknowledged.
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