Discovery of a novel inhibitor of nitric oxide production with potential therapeutic effect on acute inflammation

Article abstract of DOI:10.1016/j.bmcl.2021.128106

Inflammation as a host's excessive immune response to stimulation, is involved in the development of numerous diseases. To discover novel anti-inflammatory agents and based on our previous synthetic work on marine natural product Chrysamide B, it and a series of derivatives were synthesized and evaluated for their anti-inflammatory activity on inhibition of LPS-induced NO production. Then the preliminary structure–activity relationships were conducted. Among them, Chrysamide B is the most potent anti-inflammatory agent with low cytotoxicity and strong inhibition on the production of NO (IC₅₀ = 0.010 μM) and the activity of iNOS (IC₅₀ = 0.082 μM) in LPS-stimulated RAW 264.7 cells. Primary studies suggested that the mechanism of action may be that it interfered the formation of active dimeric iNOS but not affected transcription and translation. Furthermore, its good performance of anti-inflammatory effect on LPS-induced multiple inflammatory cytokines production, carrageenan-induced paw edema, and endotoxin-induced septic mice, was observed. We believe that these findings would provide an idea for the further modification and research of these analogs in the future.

Full text of DOI:10.1016/j.bmcl.2021.128106

Bioorg. Med. Chem. Lett. 44 (2021) 128106
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel inhibitor of nitric oxide production with potential
therapeutic effect on acute inflammation
,
,
,
Long-Qing Zhub 1, Xiao-Hong Fanb 1, Jun-Fang Lib 1, Jin-Hong Chenb, Yan Liangb,
Xiao-Ling Hub, Shu-Meng Mab, Xiang-Yong Haod, Tao Shib *, Zhen Wanga
,
,b,c,*
a School of Pharmaceutical Science, University of South China, Hengyang 421001, China
b School of Pharmacy, Lanzhou University, Lanzhou 730000, China
c State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
d Department of General Surgery, Gansu Provincial Hospital, Lanzhou 730000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Inflammation as a host’s excessive immune response to stimulation, is involved in the development of numerous
diseases. To discover novel anti-inflammatory agents and based on our previous synthetic work on marine
natural product Chrysamide B, it and a series of derivatives were synthesized and evaluated for their anti-
inflammatory activity on inhibition of LPS-induced NO production. Then the preliminary structure–activity re-
lationships were conducted. Among them, Chrysamide B is the most potent anti-inflammatory agent with low
Anti-inflammatory activity
Nitric oxide
Structure-activity relationship
Chrysamide B
cytotoxicity and strong inhibition on the production of NO (IC50 = 0.010
μ
M) and the activity of iNOS (IC50
=
0.082
μM) in LPS-stimulated RAW 264.7 cells. Primary studies suggested that the mechanism of action may be
that it interfered the formation of active dimeric iNOS but not affected transcription and translation. Further-
more, its good performance of anti-inflammatory effect on LPS-induced multiple inflammatory cytokines pro-
duction, carrageenan-induced paw edema, and endotoxin-induced septic mice, was observed. We believe that
these findings would provide an idea for the further modification and research of these analogs in the future.
Inflammation is an overactive immune response to the harmful
stimuli. In the inflammatory response, activated macrophages produce
large amounts of inflammatory factors such as interleukin-6 (IL-6),
earliest and most widely studied inhibitors are imidazole analogues,
including clotrimazole, miconazole,9 PPA250,10 BBS-2,11,12 BBS-4,13
and KLYP961,14,15 which could coordinate actively to the heme iron and
occupy the active site of L-arginine or H4B to further disturb the for-
mation of dimeric iNOS. Also, there are two non-imidazole inhibitors of
iNOS dimerization, including FR260330,16 and novel pyrazolo[4,3-d]
pyrimidine derivative D27,17 reported by Nagpal L et al. and Shi J B
et al., respectively. However, most of the imidazole-based NOS in-
hibitors have shown inhibition on cytochrome P450s, resulting in the
potential of drug-drug interactions.18 Moreover, selective inhibition on
different isoforms of NOS has always been the other challenge for the
development of iNOS inhibitors.19 In view of the importance of NO in
the process of inflammation and the research status on these inhibitors
of dimeric iNOS, it is imperative to find novel anti-inflammatory agents
disturbing the formation of dimeric iNOS.
tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO) to induce
tissue injury at the inflammatory site.1–2 Endogenous free radical nitric
oxide (NO) is produced in the process of oxidating L-arginine to L-
citrulline catalyzed by over-expressed inducible nitric oxide synthase
(iNOS).3–4 Studies have shown that excessive NO production has been
involved in developing inflammation and the pathological process of a
variety of inflammatory diseases.5–6 INOS is a kind of dimeric enzyme,
whose monomer is in-active and the formation of its homologous dimers
is the decisive factor of NO production.7–8 Therefore, interfering with
the dimerization of the monomer may block the biological effects of
inducible nitric oxide synthase and further reduce the NO production,
which is of great significance for the development of novel anti-
inflammatory agent.
In recent years, marine natural products have been an essential
source of bioactive lead compounds due to their unique skeletons and
abundant functional groups, especially in anti-inflammation.20–21 As
Only several molecules that can disturb NOS dimer’s stability or
block NOS dimerization have been researched in the past decades. The
* Corresponding authors at: School of Pharmaceutical Science, University of South China, Hengyang 421001, China (Z. Wang).
E-mail addresses: shit18@lzu.edu.cn (T. Shi), zhenw@lzu.edu.cn (Z. Wang).
1
These authors made equal contributions to this work.
https://doi.org/10.1016/j.bmcl.2021.128106
Received 7 February 2021; Received in revised form 28 April 2021; Accepted 8 May 2021
Available online 13 May 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

L.-Q. Zhu et al.
Bioorganic & Medicinal Chemistry Letters 44 (2021) 128106
shown in Fig. 1, Chrysamide B, characterized as a novel dimeric nitro-
phenyl trans-epoxyamide structure, was isolated from the deeper sea
fungi Penicillium chrysogenum SCSIO4100124. Although the structure of
Chrysamide B possessing a stereochemistry-rich framework, symmetri-
cal dimer skeleton, and dense functional groups is novel, its anti-
inflammatory effect against the production of interleukin-17 (IL-17)
was weak.22 The low abundance, the difficulty of separation, and the
narrow range of activity screening of marine natural products are the
essential reasons that limit the discovery of their effective biological
activity.
subsequent amide reduction reactions. Carboxylic acid side chain g, h,
and piperazine core m, n can be directly purchased. All the specific
synthesis processes could be found in the experiment methods sections.
Nitric oxide (NO), as a vital gas signaling molecular, is a unique
transmitter of acute or chronic inflammation.24 It released from the cells
into the culture medium is present in nitrite, which can be measured
with Griess reagent.25 Therefore, the inhibitory efficacy of nitric oxide
production of Chrysamide B and its derivatives was detected by Griess
assay, as shown in Table 1. The result revealed that Chrysamide B
treatment in LPS-stimulated RAW 264.7 cells had a powerful inhibitory
However, synthetic chemistry and various derivatizations are effec-
tive methods for quantifying natural products and discovering their
biological activities. Aiming to explore a wider range of anti-
inflammatory activity of Chrysamide B and derivatives, we synthe-
sized them and evaluated their inhibition on LPS-induced NO produc-
tion. Then the mechanism of action of the most potential Chrysamide B
was investigated, and we found Chrysamide B is possibly an inhibitor of
dimerization of iNOS. Furthermore, good performance of Chrysamide B
administration on anti-inflammatory activity in LPS-induced multiple
inflammatory cytokines productions, carrageenan-induced paw edema
model, and LPS-induced septic mice model was observed.
effect on NO release (IC50 = 0.10 μM). Subsequently, the structur-
e–activity relationships for Chrysamide B derivatives on LPS-induced
NO production were conducted. The details are as follows: the role of
the three-dimensional structure was verified by reversing or eliminating
A-ring or B-ring’s chirality. Compared with Chrysamide B, the inhibitory
effect on NO production by derivatives (compounds 2, IC50 = 16.36 μM;
3, IC50 = 35.98 μM; 4, IC50 = 15.08 μM) that reversed the chirality of A
and B rings declined to some extent, whereas the inhibitory effect on NO
production by the derivatives eliminating the methyl group at A-ring
(compound 5, IC50 > 40
μM) or the chiral epoxy group (compound 6,
IC50 > 40 M; 7, IC50 > 40 μM; 8, IC50 > 40 μM) disappeared. These
μ
First of all, combined with the structural characteristics of Chrys-
amide B, we modified it mainly from the following three aspects through
adopting principles of functional group replacement and skeleton
transform: a) altering the chirality at A-ring and B-ring to check the
importance of three-dimensional structure; b) degrading the dimeric
structure to check the importance of dimeric skeleton; c) changing the
substituents at C-ring to check the importance of these substituents
(Fig. 1). Based on our previous synthetic strategy,23 the optimized syn-
thetic routes of Chrysamide B and its derivatives were depicted in
Scheme 1, which featured a simple and convergent assembly of the
carboxylic acid chain (c, f, g or h) and chiral piperazine core (l, m or n).
Among which, the carboxylic acid side chain c can be synthesized from
starting material a after one step Wittig reaction and one step ester
hydrolysis reaction. Besides, the carboxylic acid side chain f can be
obtained from starting material a through one-step Wittig reaction, one-
step ester reduction reaction, and three-step continuous oxidation re-
action. The chiral piperazine core i can be derived from D-or L-alanine by
their methyl esterification derivatives and Boc protection derivatives
through intermolecular and intramolecular condensation well as
results suggested that the three-dimensional structure of Chrysamide B
(compound 1) is significant for maintaining its nitric oxide inhibitory
activity. Besides, the importance of the dimeric skeleton was demon-
strated by monomer derivative (compound 9, IC50 > 40 μM), which
barely had an inhibitory effect.
Based on the above results, with a retention of the chirality both at A-
ring and B-ring as well as the dimeric structure of Chrysamide B, we
synthesized another series of Chrysamide B derivative where the nitro
substituent was replaced with various groups or its substituent position
was changed, to test the effect of substituent groups at C-ring of
Chrysamide B. Compared with Chrysamide B, the inhibitory activity of
derivative with a nitro-meta substitution (compound 12, IC50 = 3.12
μ
M) against NO production was reduced by about 30 times,
whereas other derivatives, including substituent-free (compound 10,
IC50 > 40 μM), nitro-ortho substitution (compound 11, IC50 > 40 μM),
weak electron-drawing bromo-para substitution (compound 13, IC50
40 M), and electron-donating methyl-para substitution (compound 14,
IC50 > 40 M), at C-ring did not contribute to an inhibitory effect on NO
production. The results showed that their inhibitory activity order of NO
>
μ
μ
Fig. 1. Structure of Chrysamide B and modification strategy of its derivatives.
2

L.-Q. Zhu et al.
Bioorganic & Medicinal Chemistry Letters 44 (2021) 128106
Scheme 1. The synthetic routes of Chrysamide B and its derivatives. Reagents and conditions: (I) ethyl 2-(diethoxyphosphoryl) propanoate, NaH, THF, 0 ◦C ~ indoor
temperature, 10 h; (II) KOH, MeOH, indoor temperature, 0.5 h; (III) Diisobutyl aluminum hydride DIBAL-H, DCM, ꢀ 78 ◦C, 3 h; (Ⅳ) Ti(OiPr)4, (-)-DIPT or (+)-DIPT, t-
BuOOH, 4 Å MS, DCM, ꢀ 35 ◦C, 2 h; (Ⅴ) Dess-Martin Periodinane, DCM, indoor temperature, 1 h; then 2-methyl-2-butene, NaOCl2, NaH2PO4, t-BuOH/H2O, 0 ◦C to
indoor temperature, 2 h; (VI) EDC • HCl, HOBT, Et3N, DMF, indoor temperature, 8 h; (VII) TFA, DCM, 30 ◦C, 6 h; morpholine, t-BuOH, indoor temperature, 24 h;
(VIII) LiAlH4, THF, reflux, 8 h. (IX) BOP-Cl, Et3N, DCM, indoor temperature, overnight.
Table 1
Inhibitory effect of all compounds on NO production.
Compd.
Inhibition [a]
Compd.
Inhibition [a]
Compd.
Cytotoxicity [b]
Compd.
Cytotoxicity [b]
IC50
(
μM)
IC50
(
μM)
IC50
(μ
M)
IC50 (μM)
1
2
3
4
5
6
7
8
0.10 ± 0.0023
16.36 ± 1.81
35.98 ± 3.23
15.08 ± 1.62
>40
9
>40
>40
>40
1
2
3
4
5
6
7
8
>100
>100
>100
>100
9
>100
>100
>100
>100
>100
>100
>100
10
11
12
13
14
SMT
10
11
12
13
14
SMT
3.12 ± 0.22
>40
39.23 ± 3.45
>100
>40
>40
>40
9.62 ± 0.95
>100
>40
>100
RAW 264.7 cells were stimulated using 1 μg/mL LPS and treated with selected compounds at different concentrations for 24 h. Compound 1 represents Chrysamide B.
[a]: The NO levels in cell supernatant with the form of nitrite were detected by Griess assay. [b]: The cellular viability was detected by MTT assay. All data are the mean
± SD (n = 3).
production was p-NO2 > m-NO2 > o-NO2≈non-substituent ≈ p-Br≈ p-
Me, which illustrated the significance of the existence and the suitable
substituent position of strong electron-withdrawing group on C-ring. In
a word, these results revealed the preliminary structure–activity re-
lationships of these compounds that the three-dimensional structure,
dimeric skeleton, and the existence as well as the suitable substituent
position of the strong electron-withdrawing group on C-ring of Chrys-
amide B are important for maintaining the inhibitory effect on NO
production (Fig. S1).
efficacy on NO production into consideration, the most potential
Chrysamide B (compound 1) was chosen as the targeted compound for
further research.
The formation of NO is catalyzed by active iNOS, and subsequently is
involved in the inflammatory response along with various inflammatory
mediators.3,8,26 The NO levels in cells were positively related to the
activity of nitric oxide synthase (NOS). Therefore, the NO production in
cells was examined by an inducible nitric oxide synthase detection kit.17
As shown in Fig. 2A and 2B, in the LPS-stimulated RAW 264.7 cells,
different concentrations of Chrysamide B treatment obviously reduced
Moreover, to check whether the presented anti-inflammatory effect
correlated with the cell viability, the MTT method was used to measure
the cytotoxicity of Chrysamide B and its derivatives in RAW 264.7 cells.
As shown in Table 1, after treated by different concentrations of com-
pounds, the IC50 value of all compounds inhibited the growth of RAW
264.7 cells were>100 uM, except for compound 5. According to the
Griess assay data, there are only compound 1, 2 3, 4, and 12 possessing a
certain anti-inflammatory activity of inhibition on NO production
among all compounds. Combined with the MTT results, it suggested that
the anti-inflammatory activity of compound 1, 2 3, 4, and 12 is possibly
not related to their cytotoxicity. Taking the cytotoxicity and inhibitory
the intracellular NO levels with the IC50 value (0.082
the nanomole level, which are much lower than that of SMT (6.25
the inhibitor of iNOS. Moreover, there was a relatively good correlation
between IC50 of NO inhibition in the supernatant (0.10 M) and in cells
μ
M) reaching at
μ
M),
μ
(0.082 μM), which suggested that inhibition on NO may result from
inhibition on the activity of iNOS by Chrysamide B treatment.
To further ensure the inhibitory effect of Chrysamide B on NO pro-
duction, NO-sensitive fluorescence probe DAF-FM DA was used to detect
the NO levels in RAW 264.7 cells,17 as shown in Fig. 2C. After probe
DAF-FM DA loaded into cells, there was an intensive green fluorescence
3

L.-Q. Zhu et al.
Bioorganic & Medicinal Chemistry Letters 44 (2021) 128106
Fig. 2. Inhibition of Chrysamide B or SMT on the activity of iNOS. (A and B) The cellular NO levels were detected by an inducible nitric oxide synthase detection kit.
(C) The fluorescence intensity of the probe after reaction with NO. Data are the mean ± SD (n = 3). ##P < 0.01 vs control group; *P < 0.05, **P < 0.01 vs the LPS-
stimulated group.
in the LPS-stimulated group, compared with the control group.
Conversely, Chrysamide B treatment significantly decreased the fluo-
rescence intensity in RAW 264.7 cells and it worked stronger than the
inhibitor of inducible nitric oxide SMT, which indicated the inhibition of
Chrysamide B treatment on NO produdction in cells, to some extent.
The fact that Chrysamide B treatment significantly inhibited the NO
production and the activity of iNOS in LPS-stimulated RAW 264.7 cells,
has been mentioned above. To illustrate the mechanism of inhibition on
iNOS by Chrysamide B treatment, a western blot experiment on dena-
tured iNOS protein was analyzed. As shown in Fig. 3A and 3B, Chrys-
amide B treatment at various concentrations had barely influence on the
expression of denatured iNOS protein (total iNOS protein), consistent
with the inhibitor of inducible nitric oxide SMT. Under inflammatory
conditions, inducible nitric oxide synthase is first regulated by upstream
transcription factors for transcription and translation, then dimerization
occurs to form active nitric oxide synthase to perform its catalytic
function.27 Therefore, we guessed that the mechanism of action of
Chrysamide B might be related to the dimerization of iNOS, but not at
the transcription level. Next, the western blot experiment on non-
denatured protein was used to further analyze the ratio of dimer to
monomer of iNOS, after treated with Chrysamide B treatment in LPS-
stimulated RAW 264.7 cells. As displayed in Fig. 3A and 3C, unlike
the inhibitor of inducible nitric oxide SMT which did not affect the
expression or dimerization of iNOS, Chrysamide B treatment prevented
the formation of dimeric iNOS in a dosage-dependent manner. This
result further suggested that the mechanism of action of Chrysamide B
on the iNOS is likely to through interfering with the formation or sta-
bility of the active dimeric iNOS, but not affecting its transcription and
translation.
264.7 cells with different concentrations of Chrysamide B caused the
reduced levels of the pro-inflammatory cytokines, including TNF-α, IL-6,
IL-1β, to some extent. (Fig. 4A, 4B, and 4C). Although there is no
obvious dosage-dependence for the inhibitory effects, these data also
demonstrated that the targeted compound Chrysamide B exited a certain
anti-inflammatory activity in vitro.
To determine the anti-inflammatory effect of Chrysamide B in vivo,
carrageenan-induced paw edema, which is a widely used model of non-
specific localized inflammation,29 was applied. After evaluating the
acute toxicity of Chrysamide B, which showed that the LD50 was over
2000 mg/kg in oral administration, the relatively secure dose, including
12.5, 25, and 50 mg/kg, was determined to evaluate the anti-
inflammatory effect of Chrysamide B in the carrageenan-induced paw
edema model. As shown in Fig. 5A, the paw edema volume of rats was
dramatically increased after carrageenan injection, and oral adminis-
tration of Chrysamide B at the dosage of 25 mg/kg and 50 mg/kg
markedly attenuated the swelling degree at 3, 6, and 9 h. Of note, at 9 h,
25 mg/kg and 50 mg/kg of Chrysamide B treatment could induce
approximately 30% reduction in paw swelling rate of rats. To be specific,
as the dosage of Chrysamide B was 25 mg/kg, the paw swelling degree of
rats was decreased to 31.09%, as the dosage of Chrysamide B was 50
mg/kg, the paw swelling degree of rats was decreased to 27.87 %.
Moreover, in the second phase of carrageenan-induced paw edema,
PGE2 as one of the most important pro-inflammatory mediators30
amplified in the hind paw leading to pain and inflammation, was
obviously reduced by Chrysamide B (Fig. 5B). Besides, the pathological
examination by H&E staining also indicated that Chrysamide B
remarkably ameliorated the inflammatory cell infiltration compared
with the model group (Fig. 5C).
The inflammatory response is mainly through the recruitment of
macrophages to the inflammatory site, and produces a large number of
pro-inflammatory factors, leading to overactive inflammatory response
and cause body damage.28 Our results revealed that incubation of RAW
To further investigate the anti-inflammatory effect of Chrysamide B
treatment in vivo, another acute inflammation model, LPS-induced
septic model31 was used. After evaluating the acute toxicity of Chrys-
amide B, which showed that the MTD was over 100 mg/kg in
Fig. 3. (A) Western blot analysis of the expression of total iNOS protein and the ratio of iNOS dimer to monomer. (B and C) Histograms display the density ratios of
iNOS monomer to β-actin or iNOS dimer to monomer. Data are the mean ± SD (n = 3). ##P < 0.01, ###P < 0.001vs the control group; *P < 0.05, **P < 0.01 vs the
LPS-stimulated group.
4

L.-Q. Zhu et al.
Bioorganic & Medicinal Chemistry Letters 44 (2021) 128106
Fig. 4. (A to C). Effect of Chrysamide B on TNF-
α
, TL-6, and IL-1β levels measured by ELISA. Data are the mean ± SD (n = 3). ###P < 0.001 vs the control group; *P
< 0.05, **P < 0.01 vs. the LPS-stimulated group.
Fig. 5. (A to C) Anti-inflammatory effects of Chrysamide B on carrageenan-induced paw edema in SD rats. (A). The swelling degree of rat right hind paw after treated
by Chrysamide B or Meloxicam. Data are the mean ± SE (n = 9). (B). The levels of PGE2 in the rat right hind paw. Data are the mean ± SE (n = 6). (C). The H&E
staining images of the rat right hind paw. The scar bar is 100
model group.
μ
m. ## P < 0.01, ### P < 0.001 vs the vehicle group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs the
intraperitoneal administration, the relatively secure dose-25 mg/kg was
determined to evaluate its anti-inflammatory effect. To be specific, in
the sepsis model induced by LPS at a lethal dose (35 mg/kg), Chrys-
amide B treatment (25 mg/kg, administration 30 min before LPS in-
jection) obviously increased median survival by 12 h (Fig. 6A),
compared with model group. Furthermore, it presented a more signifi-
cant therapeutic efficacy by administrating 25 mg/kg Chrysamide B 30
min before and 4 h after LPS administration, which prolonged the me-
dian survival by 24.5 h (Fig. 6B).
As for the detection of cytokines in serum,31 mice in the treatment or
model group were intraperitoneally injected with 25 mg/kg Chrysamide
B or an equal volume of blank solution 30 min before 15 mg/kg LPS
administration. Other mice in the vehicle group were only intraperito-
neally injected with an equal volume of blank solution at the same time.
5

L.-Q. Zhu et al.
Bioorganic & Medicinal Chemistry Letters 44 (2021) 128106
Fig. 6. (A to B) Chrysamide B treatment dramatically extends the survival time of LPS (35 mg/kg) induced C57 mice. (C to E) Chrysamide B treatment decreased the
levels of IL-1β and IL-6 in serum (C and D) and amends lung injury (E) of LPS (15 mg/kg) induced C57 mice. Data are mean ± SE (n = 6). ###P < 0.001 vs the vehicle
group, *P < 0.05, **P < 0.01 vs the model group. The scar bar is 100 μm.
Serum taken from the above groups demonstrated a significant decrease
of cytokines, including IL-1β and IL-6, in the mouse treated by Chrys-
amide B (Fig. 6C and 6D). After the blood collection, mice in the above
groups was sacrificed after anesthetized to perform pathological obser-
vation on the lung using H&E staining. The result showed that pre-
treatment of mice with Chrysamide B effectively reduced airspace
inflammation and amended the structure of pulmonary lobules in LPS-
induced septic mice (Fig. 6E).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
To conclude, we have synthesized Chrysamide B and a series of de-
rivatives, then evaluated their anti-inflammatory activity by targeting
the NO signaling pathway. In subsequent studies, we found that the most
potential Chrysamide B can obviously inhibit the overproduction of NO
This work was financially supported by the Development Project of
Lanzhou Science and Technology Bureau (2020-XG-34), Recruitment
Program of Global Experts (1000 Talents Plan), Gansu Province Science
Foundation for Distinguished Young Scholars (20JR5RA304), and
Fundamental Research Funds for the Central Universities (lzujbky-2019-
ct08).
(IC50 = 0.10 μM) and the activation of iNOS (IC50 = 0.082 μM) through
interfering with the formation of dimeric iNOS. Additionally, pre-
liminary structure–activity relationship study indicated that the three-
dimensional structure, dimeric skeleton, and the existence as well as
the suitable substituent position of strong electron-withdrawing group
on C-ring of Chrysamide B are important for maintaining its inhibitory
effect on NO production, which will provide an idea for further modi-
fication of these compounds. Furthermore, Chrysamide B treatment
showed good performance of anti-inflammatory effect on LPS-induced
multiple inflammatory cytokines production and two acute inflamma-
tory models including carrageenan-induced paw edema and LPS-
induced septic mice.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128106.
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