Synthesis and discovery of ω-3 polyunsaturated fatty acid- alkanolamine (PUFA-AA) derivatives as anti-inflammatory agents targeting Nur77

Article abstract of DOI:10.1016/j.bioorg.2020.104456

In this work, three series of ω-3 polyunsaturated fatty acid-alkanolamine derivatives (PUFA-AAs) were synthesized, characterized and their anti-inflammatory activity in vivo was evaluated. Compounds 4a, 4f, and 4k exhibited marked anti-inflammatory activity in LPS-stimulated RAW 264.7 cells. The most promising compound 4k dose-dependently suppressed the cytokines with IC₅₀ values in the low micromolar range. Further, 4k exhibited potential in vitro Nur77-binding affinity (K_d = 6.99 × 10^?6 M) which is consistent with the result of docking studies. Next, the anti-inflammatory mechanism of 4k was found to be through NF-κB signal pathway in a Nur77-dependent manner. Moreover, we also observed 4k significantly inhibited LPS-induced expression of cytokines (IL-6, TNF-α, and IL-1β) through suppressing NF-κB activation and attenuated LPS-induced inflammation in mouse acute lung injury (ALI) model. In conclusion, the study strongly suggests that the PUFA-AA derivatives can be particularly as new Nur77 mediators for further treatment in inflammatory diseases.

Full text of DOI:10.1016/j.bioorg.2020.104456

Journal Pre-proofs
Synthesis and discovery of ω-3 polyunsaturated fatty acid- alkanolamine (PU-
FA-AA) derivatives as anti-inflammatory agents targeting Nur77
Hua Fang, Jianyu Zhang, Mingtao Ao, Fengming He, Weizhu Chen, Yuqing
Qian, Yuxiang Zhang, Yang Xu, Meijuan Fang
PII:
S0045-2068(20)31754-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.104456
YBIOO 104456
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
22 July 2020
7 September 2020
1 November 2020
Please cite this article as: H. Fang, J. Zhang, M. Ao, F. He, W. Chen, Y. Qian, Y. Zhang, Y. Xu, M. Fang,
Synthesis and discovery of ω-3 polyunsaturated fatty acid- alkanolamine (PUFA-AA) derivatives as anti-
inflammatory agents targeting Nur77, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.104456
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Synthesis and discovery of ω-3 polyunsaturated fatty acid-
alkanolamine (PUFA-AA) derivatives as anti-inflammatory
agents targeting Nur77
Hua Fang a,#, Jianyu Zhangb,#, Mingtao Aob, Fengming Heb, Weizhu Chena, Yuqing
Qianb, Yuxiang Zhangb, Yang Xub,**, Meijuan Fangb,*
a Third Institute of Oceanography, Ministry of Natural Resources, Technical Innovation Center
for Utilization of Marine Biological Resources, Ministry of Natural Resources, Xiamen, 361005,
China.
b
Fujian Provincial Key Laboratory of Innovative Drug Target Research and State Key
Laboratory of Cellular Stress Biology, School of Pharmaceutical Sciences, Xiamen University,
Xiamen 361102, China.
#These authors contributed equally to this paper.
*Corresponding author: Meijuan Fang, School of Pharmaceutical Sciences, Xiamen University,
South Xiang-An Road, Xiamen, 361102, China. Phone: +86-592-2189868; Fax: +86-592-2189868.
Email: fangmj@xmu.edu.cn.
**Corresponding author: Yang Xu, School of Pharmaceutical Sciences, Xiamen University, South
Xiang-An Road, Xiamen, 361102, China. Phone: +86-592-2189868; Fax: +86-592-2189868. Email:
xu_yang@xmu.edu.cn.

Synthesis and discovery of ω-3 polyunsaturated fatty acid-
alkanolamine (PUFA-AA) derivatives as anti-inflammatory
agents targeting Nur77
Hua Fang a,#, Jianyu Zhangb,#, Mingtao Aob, Fengming Heb, Weizhu Chena, Yuqing
Qianb, Yuxiang Zhangb, Yang Xub,*, Meijuan Fangb,*
a Third Institute of Oceanography, Ministry of Natural Resources, Technical Innovation
Center for Utilization of Marine Biological Resources, Ministry of Natural Resources,
Xiamen, 361005, China.
b Fujian Provincial Key Laboratory of Innovative Drug Target Research and State Key
Laboratory of Cellular Stress Biology, School of Pharmaceutical Sciences, Xiamen
University, Xiamen 361102, China.
E-mail: fangmj@xmu.edu.cn; xu_yang@xmu.edu.cn

Abstract
In this work, three series of ω-3 polyunsaturated fatty acid-alkanolamine
derivatives (PUFA-AAs) were synthesized, characterized and their anti-inflammatory
activity in vivo was evaluated. Compounds 4a, 4f, and 4k exhibited marked
anti-inflammatory activity in LPS-stimulated RAW 264.7 cells. The most promising
compound 4k dose-dependently suppressed the cytokines with IC50 values in the low
micromolar range. Further, 4k exhibited potential in vitro Nur77-binding affinity (Kd =
6.99×10−6 M) which is consistent with the result of docking studies. Next, the
anti-inflammatory mechanism of 4k was found to be through NF-B signal pathway in
a Nur77-dependent manner. Moreover, we also observed 4k significantly inhibited
LPS-induced expression of cytokines (IL-6, TNF-α, and IL-1) through suppressing
NF-B activation and attenuated LPS-induced inflammation in mouse acute lung injury
(ALI) model. In conclusion, the study strongly suggests that the PUFA-AA derivatives
can be particularly as new Nur77 mediators for further treatment in inflammatory
diseases.
Keywords: Nur77; anti-inflammatory activity; ω-3 polyunsaturated fatty acid; molecular
docking; acute lung injury.

1. INTRODUCTION
Inflammation is the basic defense mechanism of the immune system, which can
protect the body from harmful stimuli such as pathogens and toxins. Resolution of
inflammation is necessary to re-establish homeostasis after injury or infection.
Excessive inflammatory responses that fail to undergo resolution may lead to chronic
inflammation associated with many diseases [1]. The critical role of inflammatory
processes in health and disease has long been recognized, yet the detailed molecular
mechanisms and biological events that regulate the progression and resolution of
inflammation remain of interest. Acute inflammation in the body is less dangerous and
can cause edema and intracellular flow due to changes in vascular permeability and
local hemodynamics, whereas chronic inflammation can lead to diseases such as asthma,
rheumatoid arthritis, and cancer [2]. In some cases, tumorigenesis and cancer
progression are associated with inflammatory processes caused by pathogen infection
[3]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are important in the treatment of
pain and inflammation, but long-term use of NSAIDs quickly leads to a high incidence
of gastrointestinal adverse events [4]. Although many compounds have been found to
possess anti-inflammatory activity [5-11], developing anti-inflammatory agents with the
new structure and novel mechanism of action is still an urgent need.
Nur77 (also known as TR3, NGFIB, and NR4A1), as a member of the orphan
nuclear receptor 4A (NR4A) subfamily, is an immediate-early response gene that plays
a key role in hyper-cellular responses to multiple stimuli, such as mitogens, cytokines,
stress, metabolic, and apoptotic signals [12-14]. Recent studies have highlighted the role
of Nur77 as a significant regulator of the inflammatory response [15-19]. In human and
mouse macrophages, inflammatory stimuli such as tumor necrosis factor-α (TNF-α),
Toll-like receptor ligands, and lipopolysaccharide (LPS) can induce the expression of
Nur77 which plays an important role in inflammatory responses [20, 21]. For example,
the elevation of Nur77 expression was shown to lead to the reduction of expression of
several cytokines and chemokines in macrophages in response to LPS or tumor necrosis
factor stimulation [3]. In contrast, Nur77 deficiency elevated the production of
inflammatory mediators, such as interleukin 1 beta (IL-1), interleukin-6 (IL-6), TNF-α,
and nitric oxide (NO), in two mouse models of sepsis [22]. Several studies have

reported the molecular mechanisms of Nur77 mediating anti-inflammatory [3, 23]. A
genomic analysis using in vitro modeling of monocyte adherence identified that Nur77
can attenuate the activation of the transcription factor nuclear factor κB (NF-κB) like
IκBα, an anti-inflammatory modulator [24]. NF-κB is inactive in the cytoplasm of
resting cells but it is activated and plays a critical role in inflammation [25]. Nur77
overexpression represses the activation of the inflammatory response in front of several
stimuli, by preventing the NF-κB interaction with its promoter [26]. Recently, it has
been reported that Nur77 phosphorylation by p38 hampers its inhibition of NF-κB in
LPS-stimulated
RAW264.7
cells,
while
PDNPA
(n-pentyl
2-[3,5-dihydroxy-2-(1-nonanoyl)-phenyl]acetate) can impede the interaction between
Nur77 and p38α, prevent the p38α phosphorylation of Nur77, and show strong
anti-inflammatory [22]. Besides, the cross-talk between Nur77 and the inflammatory
response can be mediated through other transrepression mechanisms. For instance,
Nur77 overexpression inhibits NF-κB nuclear translocation via the induction of IκBα
expression at the transcriptional level [27]. Also, it was demonstrated that the
subcellular location of Nur77 from the nucleus to mitochondria was involved in
Nur77-dependent anti-inflammatory inducing by celastrol and its analogs [28, 29].
Taken together, Nur77 is a potent therapeutic target for inflammatory disease and the
discovery of anti-inflammatory inhibitors against Nur77 is an effective approach for the
treatment of various inflammatory diseases.
Polyunsaturated fatty acids (PUFAs) are fatty acids containing two or more double
bonds. PUFAs are classified as omega-3 (ω-3) and omega-6 (ω-6) based on the location
of the last double bond relative to the terminal methyl end of the molecule. The ω-6/ω-3
content of cell and organelle membranes and lipid membrane microdomains strongly
influence membrane function and numerous cellular processes such as cell survival [30].
Nowadays, the importance of ω-3 PUFAs has been documented for many health
benefits, not only resolving inflammation and prevention of infection, curing cancer,
developing brain, also preventing cardiovascular disease (CVD) [31-33]. Published
findings provide evidence suggested that ω-3 PUFAs might alleviate inflammatory
diseases through several mechanisms, such as activation of G-protein-coupled receptor
110 (GPR110) [34], inhibition of the sterol regulatory element-binding protein (SREBP)

[35], plasma membrane remodeling of lymphocytes [36], oxygenation of
endocannabinoids [31] and reduce lipid accumulation in adipose tissues [37].
Interestingly, a recent metabolomics study identified that PUFAs, including arachidonic
acid (ARA) and docosahexaenoic acid (DHA), could bind to the ligand-binding domain
(LBD) of the anti-inflammatory modulator Nur77, and Nur77-LBD might undergo
conformational changes to mediate the small-molecule binding [38]. However, there is
no literature at present of elucidating how PUFAs interact with Nur77-LBD. Besides,
although the anti-inflammatory actions of PUFAs are well established, few studies have
been conducted to evaluate the anti-inflammatory activity of PUFAs, especially the
Nur77-dependent anti-inflammatory activity [39]. DHA, eicosapentaenoic acid (EPA),
and docosapentaenoic acid (DPA) are the three major ω-3 PUFAs enriched in marine
organisms, e.g. fish, shrimp, algae, and so on. Herein, we reported the synthesis and
anti-inflammatory activities of a novel class of DHA/EPA/DPA derivatives that were
created by covalently linking moieties alkanolamines to these ω-3 fatty acids (Figure 1).
Furthermore, we evaluated the anti-inflammatory properties and in vivo ALI treatment
effects of 4k, one DPA derivative.
2. Result and Discussion
2.1. Chemistry
Design of the target compounds. Figure 1 shows several potent binders of Nur77
and drug design conception of target compounds [22, 38, 40-42]. These potent binders
of Nur77 including ARA and DHA have a hydrophilic head (blue) and a hydrophobic
tail (black). DHA, EPA, and DPA, as three major ω-3 PUFAs, have many health
benefits including resolving inflammation and prevention of infection. Besides,
docosahexaenoyl ethanolamide (DHA-EA) was found endogenously in the human brain
and retina, previous studies focus on cannabinoid receptor activation. Because they are
multifunctional molecules, it is necessary to acknowledge that ω-3 PUFA-AAs could
activate other receptors [43, 44]. Herein, in continuation to extend anti-inflammatory
agents targeting Nur77, ω-3 PUFA-AA derivatives with the unsaturated fatty chains as
the hydrophobic moiety and the hydroxyl group of alkanolamines as a hydrophilic head

were designed and screened for their anti-inflammatory activities in this study.
Insert Figure 1 here.
Figure 1. Several potent binders of Nur77 and drug design conception
Synthesis of the target compound. The synthetic procedure employed to obtain the
target compounds 4a-4o is depicted in Scheme 1. cis-4,7,10,13,16,19-Docosahexaenoic
acid methyl ester (ω-3 DHA-ME, 1a) and cis-7,10,13,16,19-docosapentaenoic acid
methyl ester (ω-3 DPA-ME, 3a) were prepared from the reactant of algae oil and
methanol in the presence of alkali, while cis-5,8,11,14,17-eicosapentaenoic acid methyl
ester (ω-3 EPA-ME, 2a) from fish oil. The starting materials 1a/2a/3a and
ethanolamine/3-amino-1-propanol/4-amino-1-butanol/5-amino-1-pentanol/6-amino-1-he
o
xanol were reacted in a free solvent at 80 C for 24h to yield corresponding target
compounds. All target compounds gave satisfactory analytical and spectroscopic data
1
including H NMR, 13C NMR, IR, and HRMS, which were in accordance with their
depicted structures (Scheme 1).
Insert Scheme 1 here.
Scheme 1. General procedure for the synthesis of compounds 4a-4o
2.2. Biological evaluation
2.2.1 Initial screening of target compounds against LPS-induced inflammation
The cytotoxic evaluation. The cytotoxicity of target compounds was evaluated in
mouse RAW264.7 macrophages by the MTT assay to investigate the possible
correlation between inflammatory inhibitory activity and cell viability. As shown in
Figure S1, the relative cell viabilities of the treated cells were all more than 60% and

ten of the target compounds at 10 M showed no significant cytotoxicity in RAW264.7
cells. So target compounds at 10 M were further used to evaluate anti-inflammatory
activity.
Initial Evaluation against LPS-induced NO production. Pro-inflammatory
macrophages produce large amounts of NO, a molecule used to both attack pathogens
and remodel intracellular metabolism [21]. To test whether our synthesized compounds
inhibited the inflammatory mediator NO production, we used Griess reagent to detect
the level of LPS-induced NO release in RAW264.7 cells treated with target compounds.
The ability of the tested compounds to reduce pro-inflammatory cytokines NO was
summarized in Figure 2A. LPS treatment caused a great increase of NO release
compared to control, while compounds 4l, 4m, 4d, 4n, 4f, 4a, and 4k could significantly
alleviate the increase of LPS-induced NO release. Particularly, compounds 4a and 4k
exhibited the most potent inhibitory activity and their NO inhibition rate was 83.33%
and 84.92% at the concentration of 10 M, respectively.
Insert Figure 2 here.
Figure 2. In-vitro inhibition effect of synthesized compounds 4a-4o against nitric oxide
(A), TNF-α (B), IL-6 (C), and IL-1β (D) productions induced by LPS in RAW 264.7
cells. RAW 264.7 cells were pretreated with or without compounds (10 μM) for 2 h, and
then cultured in the presence or absence of LPS (1 μg/mL) for 24 h. The NO levels in
the medium were determined by the Griess assay. The inflammatory genes expression
of TNF-α, IL-6, and IL-1β were determined by qPCR. The results were shown as means
± SD (n = 3) of at least three independent experiments. #p < 0.05, ##p < 0.01, ###p <
0.001 compared with the control group; *p < 0.05, **p < 0.01, ***p < 0.001 compare
with only LPS-stimulated group.
Initial Evaluation against LPS-Induced pro-inflammatory cytokine Release. TNF-α,
IL-6, and IL-1 are important pro-inflammatory mediators that play a crucial role in
promoting the inflammatory process. Herein, the qPCR and Western Blot were used to

test the inhibitory effect of all synthetic compounds (4a-4o) on the mRNA and protein
expressions of LPS-induced IL-1β, TNF-α, and IL-6 in RAW 264.7 cells. Compounds
4a and 4k remarkably reduced the LPS-induced up-regulation of the mRNAs of these
cytokines (Figure 2B-2D). Moreover, 4k also significantly downregulated the protein
expression of IL-1β and TNF-α (Figure 3).
Insert Figure 3 here.
Figure 3. The suppression of synthesized compounds 4a-4o on the protein expression
of pro-inflammatory cytokines such as TNF- and IL-1 RAW 264.7 mouse
macrophages were pretreated with compounds (10 μM) for 7.5 h, followed by
incubation with LPS (1 μg/mL) for 30 min. The cell lysates were subjected to
immunoblotting assay for TNF-α, IL-1β, and β-actin.
Initial SARs of PUFA-AA derivatives for the anti-inflammatory activity. The
preliminary screening studies on anti-inflammatory activity indicated: (1) When
ethanolamine (n=2) was introduced into PUFAs, there was a significant increase in
anti-inflammatory activity. However, the increase in the number of carbon atoms in
alkanolamine group led to an obvious decrease in anti-inflammatory activity, especially
among DHA-AAs. We observed that 4a, 4f, and 4k with ethanolamine moiety had the
best anti-inflammatory activities among DHA-AAs, EPA-AAs, and DPA-AAs
derivatives, respectively. (2) Except for 4a, other DHA-AA derivatives showed lower
anti-inflammatory activities than EPA-AA and DPA-AA derivatives, implying that
–(CH=CH–CH2)5– was better than –(CH=CH–CH2)6– for anti-inflammatory activity. (3)
Generally, among the three series of PUFA-AA derivatives, DPA-AA derivatives with 5
-CH2- at the carbon of the carboxyl group displayed the best inhibitory effect on the
production of NO and pro-inflammatory cytokines in LPS-pretreated RAW 246.7 cells.
It indicates that a long flexible linker between the polyallyl moiety and the carboxyl
group of PUFA-AA is helpful for anti-inflammatory activity. To further explore SAR

information, we would like to perform the modification of the double bond of PUFA in
future work.
Based on the results of initial screening, we found that compound 4k at 10 M
showed superior inhibitory efficacies on all pro-inflammatory cytokine productions.
Herein, compound 4k was selected for further screening. As 4k displayed slight toxic
for macrophages with the cell growth inhibitory rate of 30.2%. We first examined the
non-toxic concentrations of 4k being used to evaluate anti-inflammatory activity. As
shown in S1B, 4k below 10 M showed no significant cytotoxicity in RAW264.7 cells,
and the relative cell viabilities of the dosing test cells exceeded 75% at 5 M. Therefore,
the non-toxic concentrations (5 M, 2.5 M, and 1.25 M) were further used to
evaluate the anti-inflammatory activity of 4k.
2.2.2 Compound 4k dose-dependently inhibits the productions of pro-inflammatory
cytokines
To confirm the anti-inflammatory effect of compound 4k, the NO, TNF-, and IL-6
levels in the medium were measured by ELISA assay and the mRNA and protein
expression levels of pro-inflammatory cytokines in RAW264.7 cells were determined
by qPCR and Western Blot, respectively. As shown in Figure 4A-4C, 4k exhibited a
dose-dependent inhibition of the LPS-induced releases of NO, TNF-α, and IL-6 at the
concentrations ranging from 1.25 μM to 10 μM. Besides, compounds 4k
dose-dependently down-regulated the mRNA and protein expression levels of
pro-inflammatory cytokines such as IL-1, IL-6, and TNF- at the concentrations of
1.25, 2.5, 5, and 10 μM (Figure 4D-4G). These data indicate that 4k may take excellent
inhibitory effects on pro-inflammatory mediators and 4k at 5 μM exhibited similar
anti-inflammatory activity with the reference dexamethasone at 5 μM.
Insert Figure 4 here.
Figure 4. Compound 4k dose-dependently inhibits NO, TNF-, IL-6, and IL-1
productions in LPS-stimulated RAW264.7 cells. (A) The NO levels in the medium. (B)

The TNF- levels in medium C The IL-6 levels in the medium. (D) The mRNA
expression levels of IL-1 in RAW264.7 cells E The mRNA expression levels of
IL-6 in RAW264.7 cells. (F) The protein expression levels of TNF- and IL-1 in
RAW264.7 cells. RAW264.7 cells were plated for 24 h and then challenged with LPS
(1 μg/mL) with or without 4k at different concentrations, NO levels in the medium were
determined by the Griess assay and the levels of TNF-α and IL-6 levels in the medium
were determined by ELISA. Besides, the mRNA expression levels of IL-1β, and IL-6
and the protein expression levels of TNF- and IL-1 in RAW264.7 cells were
determined by qPCR and Western Blot, respectively. Data are expressed as fold change
relative to control values (samples treated with LPS alone), mean ± SEM (n ≥ 3).
Student's t-test was used for the statistical analysis, #p < 0.05, ##p < 0.01, ###p < 0.001
compared with the control group; *p < 0.05 and **p < 0.01 vs. LPS alone-stimulated
group.
2.2.3 Compound 4k can bind to Nur77-LBD and take anti-inflammatory effects in a
Nur77-dependent manner.
4k binds to Nur77. In recent years, Nur77 has been confirmed as an important target
for the development of anti-new inflammatory drugs [28]. Our prepared compounds
have very similar structural features with several Nur77 binders previously reported.
Compound 4k exhibited good anti-inflammatory activity in LPS-treated RAW264.7. So,
we used Surface Plasmon Resonance (SPR)-based assay and molecular docking study to
evaluate the binding affinity of 4k with Nur77-LBD. The SPR results showed that 4k
dose-dependently bound to Nur77-LBD with a Kd (equilibrium dissociation constant)
value of 6.99×10−6 M, and exhibited slow association/dissection kinetics to interact with
Nur77-LBD (Figure 5A). To further understand how ω-3 PUFAs and their derivatives
bind to Nur77-LBD, we performed molecular docking studies between the selected
compounds (DHA, 4a, and 4k) and Nur77-LBD using induced-fit docking (IFD)
program of Schrödinger software (Version 2019-1). The IFD docking scores of DHA,
4a, and 4k with Nur77-LBD (PDB: 4JGV) were -7.35, -4.50, and -9.40, respectively,
while the native ligand THPN had an IFD docking score of -4.43 (Table S1). The

docking results indicated that ω-3 PUFAs and their derivatives had potential binding
affinities with Nur77-LBD. The THPN-bound structure obtained using the IFD program
was set as the reference for comparison. As depicted in Figure 5B-5D, 4k bound to a
region that overlapped with THPN binding region in the ligand-binding pocket of
Nur77-LBD, the long fatty chains of 4k and THPN were oriented in the same direction
and produced hydrophobic interactions with the hydrophobic residues in the
ligand-binding pocket. Besides, the alkanolamine moiety of 4k was located in the same
position as the 3,4,5-trihydroxyphenyl moiety of THPN. However, the alkanolamine
moiety of 4k formed two hydrogen bonds which interacted with the side chain hydroxyl
group of residues THR513 (distance: 1.8 Å) and the backbone carbonyl oxygen of
LEU509 (distance: 2.1 Å), while the 3,4,5-trihydroxyphenyl moiety of THPN only
formed a hydrogen bond interaction with THR513 but no hydrogen bond interaction
with LEU509. In addition, DHA and 4a binding to Nur77 had similar conformation
with THPN and 4k (Figure S2-S6), but only 4k formed an additional hydrogen-bond(s)
with LEU509. Compared with the native ligand THPN (
= -49.89 kcal/mol), DHA
= -99.51
∆퐺푏푖푛푑
( = -55.71 kcal/mol), and 4a (
∆퐺푏푖푛푑
= -55.31 kcal/mol), 4k (
∆퐺푏푖푛푑
∆퐺푏푖푛푑
kcal/mol) showed a higher binding affinity to Nur77 (Table S1). These results might
explain the better potency of 4k compared to DHA and 4a in the anti-inflammatory
assay.
Insert Figure 5 here.
Figure 5. Compound 4k binds to Nur77-LBD. (A) The physical binding affinity of 4k
to Nur77-LBD by SPR assay. (B) Comparison of THPN (blue) and 4k (purple) binding
to Nur77-LBD (PDB ID: 4JGV). (C) The IFD binding model of THPN in the
ligand-binding pocket (LBP) of Nur77-LBD. (D) The IFD binding model of 4k in the
LBP of Nur77-LBD.
4k may suppress NF-κB activity and take anti-inflammatory effects by
targeting Nur77. It is well documented that Nur77 exerts an anti-inflammatory effect

through inhibiting NF-κB activation in LPS-induced inflammation and NF-κB activation
is accompanied by phosphorylation and degradation of IκB . Our above
experiment results indicate that 4k is both a good anti-inflammatory compound and a
potent Nur77 ligand. However, the anti-inflammatory mechanism of 4k remained
elusive. Herein, in consideration of the subsequent in vivo anti-inflammatory assay, we
first checked the levels of the pro-inflammatory cytokines (IL-1 and TNF-) and the
key NF-κB pathway-related proteins p-IKKα/β in human lung cancer H460 cells treated
with or without 4k. As shown in Figure 6A, the rise in the production of p-IKKα/β and
pro-inflammatory molecules (IL-1 and TNF-) in LPS-treated H460 cells implied that
LPS activated the NF-κB pathway and induced inflammation, while 4k could
dose-dependently decrease p-IKKα/β, IL-1, and TNF- indicating that 4k effectively
inhibited NF-kB activity and, as a result, dampened LPS-induced inflammation. To
further confirm that the anti-inflammatory activity of 4k was Nur77-related, we
established Nur77 knockdown cells (Nur77 KD-H460 cells) through the transfection of
Nur77 shRNA into H460 cells. The wild-type and Nur77-KD H460 cells were
pretreated with LPS and then treated with or without 4k, the levels of p-IKKα/β and
IκBα were then analyzed by Western Blot and visually represented by grayscale
analysis (Figure 6B). It showed that LPS induced Nur77 expression both in wild and
Nur77-KD H460 cells. It is consistent with previous studies. Meanwhile, we observed
that p-IKKα/β had a significant decrease and IκBα had a significant increase in
LPS-stimulated wild H460 cells with 4k pretreatment, indicating that 4k inhibited
NF-B activity. However, 4k did not significantly change p-IKKα/β and IκBα levels in
the LPS-treated Nur77 KD-H460 cells. These data suggest that Nur77 is required for
compound 4k inhibition of LPS-induced macrophage cytokine and 4k is a promising
anti-inflammatory candidate for the treatment of inflammatory diseases.
Insert Figure 6 here.
Figure 6. Compound 4k Nur77-dependently inhibits LPS-induced inflammation
through NF-B signaling pathway in human normal lung H460 cells (A) Effects of
compound 4k on LPS-induced TNF-α, IL-1β and p-IKKα/β protein expression in H460

cells. (B) The protein expression levels of IκBα and p-IKKα/β in wild-type and
Nur77-/-H460 cells treated with or without LPS and compound 4k. Data are expressed
as fold change relative to control values (samples treated with LPS alone), mean ± SEM
(n ≥ 3). Student's t-test was used for the statistical analysis, #p < 0.05, ##p < 0.01, ###p
< 0.001 compared with the control group; *p < 0.05 and **p < 0.01 vs. LPS
alone-stimulated group.
2.2.4 In vivo anti-inflammatory activity of 4k
Acute inflammatory diseases remain the main cause of death in intensive care units
worldwide. ALI is a critical illness that is primarily driven by inflammation [46-48].
Today, steroidal and nonsteroidal anti-inflammatory drugs are therapeutically limited in
the treatment of ALI [49-51]. Besides, numerous pharmacological therapies for
established ALI including corticosteroids and steroids have failed to show benefit in
multicenter clinical trials. Therefore, today more and more researchers are focusing on
the development of new agents for ALI treatment [52-54].
Importantly, recent studies have reported that Nur77 is involved in the
pathogenesis of lung diseases including ALI and pulmonary fibrosis and Nur77 can be
used as a therapeutic target for lung inflammation and diseases [23, 55, 56]. Besides,
previous studies confirmed that ω-3 fatty acids had anti-inflammatory activity and could
improve the clinical outcomes of patients undergoing ALI [57]. Interestingly, the
Nur77-targeting compound 4k could attenuate LPS-induced inflammation in wild
human lung H460 cells [28]. Therefore, we induced ALI in mice by intratracheal LPS
instillation to further evaluate in vivo anti-inflammatory activity of 4k. Dexamethasone
was used as a positive control, and DMSO was used as a vehicle. It was found that
cytokines IL-6 and TNF- in mice bronchial alveolar lavage fluid (BALF) had a
marked increase after LPS instillation, while the increase was significantly inhibited by
dexamethasone or 4k treatment (Figure 7A and 7B). Besides, in LPS-induced lung
tissues, IκBα exhibited an increase with dexamethasone or 4k pretreatment,
companying with a decrease of TNF- and IL-1 Figure 7C  These data suggested
4k effectively attenuated pulmonary inflammation. Moreover, we performed

hematoxylin and eosin (H&E) staining of lung tissues to evaluate the protective effect
of 4k on ALI (Figure 7D). H&E staining showed the normal structure of lung tissue in
control mice, without histopathologic changes under a light microscope. In the LPS
group, the lung tissues exhibited marked pathologic changes, such as inflammatory
infiltration, thickened alveolar septa, interstitial edema, and lung tissue destruction.
These LPS-induced pathological changes were significantly reversed by treatment with
4k or dexamethasone. Collectively, these results indicated that 4k could potently protect
against pulmonary inflammation in vivo, and it could potentially be used for treating
ALI and other inflammatory injures.
Insert Figure 7 here.
Figure 7. Compound 4k attenuates LPS-induced ALI in mice. (A) and (B) Concentrations of IL-6
and TNF-α were determined by ELISA assay in BALF samples. (C) Effects of compound 4k on
LPS-induced IκBα, TNF-α, and IL-1β protein expression in ALI lung tissue. Data were shown as
mean ± SEM, n = 6. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the control group; *p < 0.05,
**p < 0.01, ***p < 0.001 vs LPS vehicle. (D) Hematoxylin and eosin (H&E) staining (Microscope
magnification: 100*) showing reduced pathological changes in lung tissues from mice treated with
4k compared to LPS challenged mice.
3. Conclusion
In summary, 15 PUFA-AA derivatives were synthesized as anti-inflammatory
agents targeting Nur77. The in vitro anti-inflammatory activity was screened, the
majority of PUFA-AA derivatives exhibited anti-inflammatory effects to different
extents. The preliminary SAR studies show that the introduction of aminoethanol into
polyunsaturated fatty acids (PUFAs) (e.g. 4a, 4k, and 4f) could increase
anti-inflammatory activity. Specifically, the most potent compound 4k was selected to
further study the mechanism. First, 4k was found could dose-dependently suppress
LPS-induced production of cytokines (TNF-, IL-6, and IL-1) and inhibit NO
production in RAW 246.7. Next, SPR assay and docking studies showed that 4k had a

moderate binding affinity with Nur77. Moreover, we found 4k significantly attenuated
LPS-induced inflammation through the blocking of NF-κB activation in a
Nur77-dependent manner. Finally, in vivo studies in LPS-stimulated ALI mice showed
that pretreatment with active compound 4k led to a remarkable reduction of cytokines
(IL-6, TNF- or IL- and IκBα levels in BALF or lung tissues and pulmonary
histopathological changes. Therefore, the Nur77-targeting compound 4k is a promising
potential anti-inflammatory candidate for the treatment of inflammatory diseases such
as ALI.
4. Experimental section
Chemistry. Algae oil was purchased from Xiamen Kingdomway Group Company
(Fujian, China). Other reagents were purchased and used without further purification
unless otherwise indicated. The reactions were monitored by thin-layer chromatography
(TLC). All NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer,
operating at 400 MHz for 1H, and 100 MHz for 13C, tetramethylsilane (TMS) was used
as an internal reference for 1H and 13C chemical shifts, and DMSO-d6/CDCl3 were used
as the solvent. High-resolution mass spectra were determined on Micromass-LCT
Premier Time of Flight (TOF) mass spectrometer (Waters, USA). HPLC was performed
on Hanbon Nu3010C to collect different retention time for various target compounds.
The infrared spectra were determined by A Bruker ALPHA Fourier-transform infrared
(FTIR) spectrometer (Bruker Optics Inc., Germany).
Biology. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was
purchased from Beyotime (Beyotime, Shanghai, China). Lipopolysaccharide (LPS) was
purchased from Sigma (Sigma, St. Louis, MO, USA). Standard molecules including
EPA, DPA, and DHAwere purchased from Tokyo Chemical Industry (TCI, Tokyo,
Japan). Griess reagent was purchased from Beyotime (Beyotime, Shanghai, China). The
mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit and mouse TNF-α
ELISA kit were purchased from DAKEWE (DAKEWE, China). Trizol-reagent, the
two-step M-MLV, and the Platinum SYBR Green qPCR SuperMix-UDG kit were
purchased from YEASEN (YEASEN, Shanghai, China). IκBα, IL-1β, and TNF-α mAb

were purchased from Abcam (Abcam, Cambridge, UK), p-IKKα/β mAb was purchased
from Cell Signaling Technology (CST, Boston, USA). Other reagents used in this study
were purchased from the domestic market and were all analytically pure used without
further purification. The absorbance of the cell samples for cell viability assay was
measured in a microplate reader (Thermo Fisher Scientific, MA, USA). The relative
RNA amounts were calculated with the ΔΔCt method by Agilent AriaMx PCR (Agilent
USA) instrument. BIAcore-T200 (GE, USA) was used to evaluate the binding affinity
of 4k with Nur77-LBD. The lung sections stained with hematoxylin and eosin were
observed by light microscopy (Nikon, Japan).
4.2 Preparation of DHA- ME (1a), EPA- ME (2a), and DPA- ME (3a)
According to the slightly modified literature method, algae oil or fish oil (100 g) was
dissolved in 125 mL methanol in a 250 mL round bottom flask equipped with a reflux
condenser, then, 50 mL 20% NaOH (aq) was added to the mixture, and the reaction was
o
stirred for 5 h at 75 C. At the end of the reaction, the organic layer was separated and
dried in vacuo [58]. The initial compounds 1a, 2a, and 3a with the purity of >95% were
obtained through the following process and directly used for the next reaction. First,
1a/3a and 2a were enriched by molecular distillation (1.0×10-3 mbar, 65 oC, the content
of 1a/3a increased from 37.08 to 75.59% and 2a increased from 12.28 to 35.62%,
respectively). Then, the product is purified by preparative HPLC (C18, methanol/H2O =
90:10, tR for 1a, 2a, 3a was 10.2, 8.2, and 13.0 min, respectively). (Scheme 1)
4.3. General procedure for synthesis of the target compounds (4a-4o)
A
mixture of 1a/2a/3a (3 mmol) and ethanolamine/3-amino-1-propanol/
4-amino-1-butanol/5-amino-1-pentanol/6-amino-1-hexanol (9 mmol) were added in 20
mL round bottom flask. The reaction mixture was stirred for 24 h at 80 oC, which was
purified by silica gel column chromatography (petroleum ether/ethyl acetate 1:1) to give
the target compounds (4a-4o) as pale yellow oil in yield of 57-78%. (Scheme 1)
cis-Docosa-4,7,10,13,16,19-hexaenoic acid (2-hydroxy-ethyl)-amide (4a). Pale
1
yellow oil, yield 78%, HPLC purity: 98.3% (tR = 7.06 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 7.76 (br, 1H, NH), 5.38-5.27 (m, 12H, 6 -CH=CH-), 4.61 (br, 1H,

OH), 3.40-3.32 (m, 2H, CH2OH), 3.11-3.08 (m, 2H, NHCH2), 2.83-2.77 (m, 10H,
5CH2), 2.26-2.23 (m, 2H, CH2), 2.13-1.99 (m, 4H, 2CH2), 0.94-0.86 (m, 3H, CH3).13C
NMR (100 MHz, DMSO-d6, ppm): δ 172.00 , 132.01, 129.42, 128.59, 128.56, 128.48,
128.37, 128.35, 128.32, 128.21, 128.16, 127.41, 60.44, 41.91, 35.63, 29.15, 25.68,
25.63, 25.58, 23.59, 20.50, 14.56. IR (KBr, cm-1): ṽ 3308 (N-H), 3028 (HC=CH), 1638
(C=O), 1548, 1406, 1268, 1069, 916, 668. HRMS (TOF-MS, +): m/z [M+H]+ calculated
+
for C24H38NO2 372.2897, found 372.2886.
cis-Docosa-4,7,10,13,16,19-hexaenoic acid (3-hydroxy-propyl)-amide (4b). Pale
1
yellow oil, yield 69%, HPLC purity: 95.4% (tR = 7.14 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 7.73 (br, 1H, NH), 5.35-5.26 (m, 12H, 6 -CH=CH-), 4.37 (br, 1H,
OH), 3.40-3.37 (m, 2H, CH2OH), 3.08-3.04 (m, 2H, NHCH2), 2.81-2.76 (m, 10H,
5CH2), 2.26-2.21 (m, 2H, CH2), 2.09-1.99 (m, 4H, 2CH2), 1.54-1.48 (m, 2H, CH2),
0.93-0.91 (m, 3H, CH3). 13C NMR (100 MHz, DMSO-d6, ppm): δ 171.84, 131.99,
129.36, 128.57, 128.53, 128.50, 128.35, 128.34, 128.30, 128.19, 128.15, 127.39, 58.86,
36.10, 35.68, 32.93, 25.66, 25.61, 25.56, 23.62, 20.48, 14.54. IR (KBr, cm-1): ṽ 3283
(N-H), 3011 (HC=CH), 1644 (C=O), 1532, 1447, 1069, 803, 729. HRMS (TOF-MS, +):
+
m/z [M+H]+ calculated for C25H40NO2 386.3054, found 386.3046.
cis-Docosa-4,7,10,13,16,19-hexaenoic acid (4-hydroxy-butyl)-amide (4c). Pale
1
yellow oil, yield 63%, HPLC purity: 98.4% (tR = 6.95 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 7.72 (br, 1H, NH), 5.38-5.24 (m, 12H, 6 -CH=CH-), 4.34 (br, 1H,
OH), 3.37-3.35 (m, 2H, CH2OH), 3.01-3.00 (m, 2H, NHCH2), 2.81-2.77 (m, 10H,
5CH2), 2.26-2.21 (m, 2H, CH2), 2.09-2.00 (m, 4H, 2CH2), 1.39-1.37 (m, 4H, 2CH2),
0.93-0.89 (m, 3H, CH3). 13C NMR (100 MHz, DMSO-d6, ppm): δ 171.61, 131.99,
129.39, 128.57, 128.54, 128.47, 128.35, 128.33, 128.30, 128.19, 128.14, 127.98, 127.39,
60.89, 38.81, 35.71, 30.37, 26.30, 25.66, 25.61, 25.56, 23.64, 20.48, 14.54. IR (KBr,
cm-1): ṽ 3298 (N-H), 3012 (HC=CH), 1647 (C=O), 1549, 1445, 1268, 1062, 918, 712.
+
HRMS (TOF-MS, +): m/z [M+H]+ calculated for C26H42NO2 400.3210, found
400.3202.
cis-Docosa-4,7,10,13,16,19-hexaenoic acid (5-hydroxy-pentyl)-amide (4d). Pale
1
yellow oil, yield 67%, HPLC purity: 95.3% (tR = 7.17 min), H NMR (400 MHz,

DMSO-d6, ppm): δ 7.72 (br, 1H, NH), 5.34-5.23 (m, 12H, 6 -CH=CH-), 4.32 (br, 1H,
OH), 3.38-3.33 (m, 2H, CH2OH), 3.02-2.97 (m, 2H, NHCH2), 2.81-2.77 (m, 10H,
5CH2), 2.21-2.23 (m, 2H, CH2), 2.09-1.99 (m, 4H, 2CH2), 1.42-1.32 (m, 4H, 2CH2),
1.28-1.22 (m, 2H, CH2), 0.93-0.89 (m, 3H, CH3). 13C NMR (100 MHz, DMSO-d6,
ppm): δ 171.60, 131.99, 129.38, 129.06, 128.68, 128.56, 128.54, 128.47, 128.35, 128.32,
128.30, 128.17, 128.14, 127.39, 61.10, 38.85, 35.70, 32.68, 31.74, 29.53, 29.15, 25.66,
25.61, 25.56, 23.65, 23.42, 22.76, 22.53, 20.48, 14.54. IR (KBr, cm-1): ṽ 3291 (N-H),
3010 (HC=CH), 1645 (C=O), 1541, 1450, 1265, 1060, 922, 749. HRMS (TOF-MS, +):
+
m/z [M+H]+ calculated for C27H44NO2 414.3367, found 414.3362.
cis-Docosa-4,7,10,13,16,19-hexaenoic acid (6-hydroxy-hexyl)-amide (4e). Pale
1
yellow oil, yield 76%, HPLC purity: 95.5% (tR = 7.38 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 7.72 (br, 1H, NH), 5.34-5.25 (m, 12H, 6 -CH=CH-), 4.31 (br, 1H,
OH), 3.38-3.35 (m, 2H, CH2OH), 3.01-2.97 (m, 2H, NHCH2), 2.81-2.77 (m, 10H,
5CH2), 2.26-2.21 (m, 2H, CH2), 2.08-2.00 (m, 4H, 2CH2), 1.40-1.35 (m, 4H, 2CH2),
1.33-1.22 (m, 4H, 2CH2), 0.93-0.89 (m, 3H, CH3). 13C NMR (100 MHz, DMSO-d6,
ppm): δ 171.60, 131.99, 129.37, 128.56, 128.54, 128.47, 128.34, 128.32, 128.29, 128.17,
128.14, 127.39, 61.11, 38.87, 35.70, 32.95, 29.68, 26.79, 25.70, 25.65, 25.62, 25.56,
23.66, 20.48, 14.55. IR (KBr, cm-1): ṽ 3305 (N-H), 3010 (HC=CH), 1612 (C=O), 1487,
+
1228, 1072, 750. HRMS (TOF-MS, +): m/z [M+H]+ calculated for C28H46NO2
428.3523, found 428.3520.
cis-Eicosa-5,8,11,14,17-pentaenoic acid (2-hydroxy-ethyl)-amide (4f). Pale yellow
1
oil, yield 70%, HPLC purity: 99.7% (tR = 5.80 min), H NMR (400 MHz, DMSO-d6,
ppm): δ 7.81 (br, 1H, NH), 5.36-5.31 (m, 10H, 5 -CH=CH-), 4.63 (br, 1H, OH),
3.38-3.35 (m, 2H, CH2OH), 3.10-3.08 (m, 2H, NHCH2), 2.83-2.78 (m, 6H, 3CH2),
13
2.09-1.99 (m, 6H, 3CH2), 1.54-1.43 (m, 4H, 2CH2), 0.92-0.87 (m, 3H, CH3). C NMR
(100 MHz, DMSO-d6, ppm): δ 172.47, 132.02, 130.09, 129.93, 128.59, 128.54, 128.44,
128.37, 128.33, 128.21, 128.16, 127.41, 60.46, 41.86, 35.83, 35.28, 31.59, 29.55, 29.48,
29.14, 27.06, 26.76, 25.73, 25.67, 25.58, 422.54, 20.50, 14.57. IR (KBr, cm-1): ṽ 3297
(N-H), 3016 (HC=CH), 1645 (C=O), 1538, 1454, 1232, 10679, 972, 727. HRMS
+
(TOF-MS, +): m/z [M+H]+ calculated for C22H36NO2 346.2741, found 346.2735.

cis-Eicosa-5,8,11,14,17-pentaenoic acid (3-hydroxy-propyl)-amide (4g). Pale
1
yellow oil, yield 57%, HPLC purity: 99.7% (tR = 5.90 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 7.75 (br, 1H, NH), 5.40-5.27 (m, 10H, 5 -CH=CH-), 4.42 (br, 1H,
OH), 3.41-3.37 (m, 2H, CH2OH), 3.10-3.05 (m, 2H, NHCH2), 2.84-2.78 (m, 8H, 4CH2),
13
2.07-1.99 (m, 6H, 3CH2), 1.58-1.49 (m, 4H, 2CH2), 0.93-0.91 (m, 3H, CH3). C NMR
(100 MHz, DMSO-d6, ppm): δ 172.34, 132.02, 129.91, 128.59, 128.52, 128.46, 128.37,
128.32, 128.22, 128.16, 127.41, 58.89, 36.08, 35.32, 32.96, 26.74, 25.79, 25.67, 25.58,
20.51, 14.58. IR (KBr, cm-1): ṽ 3298 (N-H), 3011 (HC=CH), 1645 (C=O), 1546, 1445,
+
1263, 1070, 969, 711. HRMS (TOF-MS, +): m/z [M+H]+ calculated for C23H38NO2
360.2897, found 360.2891.
cis-Eicosa-5,8,11,14,17-pentaenoic acid (4-hydroxy-butyl)-amide (4h). Pale
yellow oil, yield 73%, HPLC purity: 99.6% (tR = 5.92 min), 1H NMR (400 MHz, CDCl3,
ppm): δ 5.71 (br, 1H, NH), 5.45-5.32 (m, 10H, 5 -CH=CH-), 3.71-3.68 (m, 2H,
CH2OH), 3.33-3.28 (m, 2H, NHCH2), 2.86-2.82 (m, 8H, 4CH2), 2.21-2.06 (m, 6H,
3CH2), 1.96 (br, 1H, OH), 1.77-1.69 (m, 2H, CH2), 1.62-1.60 (m, 4H, 2CH2), 0.99-0.97
13
(m, 3H, CH3). C NMR (100 MHz, CDCl3, ppm): δ 173.06, 132.09, 129.16, 128.73,
128.61, 128.29, 128.24, 128.17, 128.09, 127.87, 127.00, 62.38, 39.20, 36.18, 29.67,
26.69, 26.34, 25.65, 25.57, 25.55, 20.57, 14.28. IR (KBr, cm-1): ṽ 3298 (N-H), 3012
(HC=CH), 1646 (C=O), 1550, 1446, 1266, 1061, 920, 711. HRMS (TOF-MS, +): m/z
+
[M+H]+ calculated for C24H40NO2 374.3054, found 374.3045.
cis-Eicosa-5,8,11,14,17-pentaenoic acid (5-hydroxy-pentyl)-amide (4i). Pale
yellow oil, yield 62%, HPLC purity: 95.1% (tR = 6.07 min), 1H NMR (400 MHz, CDCl3,
ppm): δ 5.61 (br, 1H, NH), 5.43-5.34 (m, 10H, 5 -CH=CH-), 3.67-3.64 (m, 2H,
CH2OH), 3.29-3.24 (m, 2H, NHCH2), 2.86-2.81 (m, 8H, 4CH2), 2.20-2.10 (m, 6H,
3CH2), 1.92 (br, 1H, OH), 1.74-1.70 (m, 2H, CH2), 1.62-1.52 (m, 4H, 2CH2), 1.44-1.39
13
(m, 2H, CH2), 0.99-0.97 (m, 3H, CH3). C NMR (100 MHz, CDCl3, ppm): δ 172.96,
132.08, 129.18, 128.71, 128.60, 128.29, 128.24, 128.16, 128.09, 127.86, 127.00, 62.54,
39.33, 36.17, 32.17, 29.70, 29.44, 26.69, 25.64, 25.57, 25.55, 23.04, 20.57, 14.28. IR
(KBr, cm-1): ṽ 3296 (N-H), 3012 (HC=CH), 1646 (C=O), 1550, 1447, 1261, 1060, 713.
+
HRMS (TOF-MS, +): m/z [M+H]+ calculated for C25H42NO2 388.3210, found

388.3203.
cis-Eicosa-5,8,11,14,17-pentaenoic acid (6-hydroxy-hexyl)-amide (4j). Pale yellow
oil, yield 73%, HPLC purity: 96.1% (tR = 7.41 min), 1H NMR (400 MHz, CDCl3, ppm):
δ 5.54 (br, 1H, NH), 5.43-5.34 (m, 10H, 5 -CH=CH-), 3.66-3.63 (m, 2H, CH2OH),
3.26-3.23 (m, 2H, NHCH2), 2.87-2.81 (m, 8H, 4CH2), 2.19-2.07 (m, 6H, 3CH2), 1.83
(br, 1H, OH), 1.74-1.71 (m, 2H, CH2), 1.59-1.50 (m, 4H, 2CH2), 1.41-1.35 (m, 4H,
13
2CH2), 0.99-0.97 (m, 3H, CH3). C NMR (100 MHz, CDCl3, ppm): δ 172.89, 132.08,
129.19, 128.71, 128.60, 128.29, 128.24, 128.16, 128.09, 127.86, 127.00, 62.63, 39.28,
36.18, 32.53, 29.67, 26.69, 26.49, 25.64, 25.58, 25.55, 25.27, 20.57, 14.28. IR (KBr,
cm-1): ṽ 3288 (N-H), 3010 (HC=CH), 1644 (C=O), 1542, 1451, 1262, 1060, 925, 749.
+
HRMS (TOF-MS, +): m/z [M+H]+ calculated for C26H44NO2 402.3367, found
402.3363.
cis-Docosa-7,10,13,16,19-pentaenoic acid (2-hydroxy-ethyl)-amide (4k). Pale
1
yellow oil, yield 65%, HPLC purity: 99.4% (tR = 8.31 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 6.21 (br, 1H, NH), 5.46-5.31 (m, 10H, 5 -CH=CH-), 3.72-3.70 (m,
2H, CH2OH), 3.42-3.39 (m, 2H, NHCH2), 2.85-2.82 (m, 6H, 3CH2), 2.81 (s, 1H, OH),
2.45-2.40 (m, 2H, CH2), 2.30-2.26 (m, 2H, CH2), 2.09-2.04 (m, 2H, CH2), 1.40-1.26 (m,
13
8H, 2CH2), 0.90-0.88 (m, 3H, CH3). C NMR (100 MHz, DMSO-d6, ppm): δ 173.78,
130.52, 129.47, 128.63, 128.37, 128.06, 128.02, 127.99, 127.84, 127.53, 62.20, 42.40,
36.31, 31.51, 29.31, 27.22, 25.64, 25.60, 23.39, 22.57, 14.07. IR (KBr, cm-1): ṽ 3295
(N-H), 3081 (HC=CH), 1646 (C=O), 1550, 1442, 1268, 1062, 710. HRMS (TOF-MS,
+
+): m/z [M+H]+ calculated for C24H40NO2 374.3054, found 374.3036.
cis-Docosa-7,10,13,16,19-pentaenoic acid (3-hydroxy-propyl)-amide (4l). Pale
1
yellow oil, yield 77%, HPLC purity: 96.3% (tR = 8.85 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 6.03 (br, 1H, NH), 5.40-5.39 (m, 10H, 5 -CH=CH-), 3.66-3.60 (m,
2H, CH2OH), 3.44-3.40 (m, 2H, NHCH2), 2.89-2.79 (m, 8H, 4CH2), 2.46-2.39 (m, 2H,
CH2), 2.31-2.27 (m, 2H, CH2), 2.10-2.02 (m, 2H, CH2), 1.97 (br, 1H, OH), 1.72-1.64 (m,
2H, CH2), 1.41-1.25 (m, 6H, 3CH2), 0.92-0.90 (m, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6, ppm): δ 173.77, 130.52, 129.52, 128.63, 128.36, 128.04, 127.83, 127.52,
59.16, 36.40, 32.30, 31.51, 29.32, 27.24, 25.64, 23.45, 22.57, 14.09. IR (KBr, cm-1): ṽ

3298 (N-H), 3012 (HC=CH), 1645 (C=O), 1540, 1447, 1268, 1070, 750. HRMS
+
(TOF-MS, +): m/z [M+H]+ calculated for C25H42NO2 388.3210, found 388.3204.
cis-Docosa-7,10,13,16,19-pentaenoic acid (4-hydroxy-butyl)-amide (4m). Pale
1
yellow oil, yield 75%, HPLC purity: 98.5% (tR = 8.38 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 5.75 (br, 1H, NH), 5.45-5.34 (m, 10H, 5 -CH=CH-), 3.71-3.68 (m,
2H, CH2OH), 3.31-3.28 (m, 2H, NHCH2), 2.86-2.81 (m, 8H, 4CH2), 2.43-2.40 (m, 2H,
CH2), 2.26-2.22 (m, 2H, CH2), 2.10-2.06 (m, 2H, CH2), 2.01 (br, 1H, OH), 1.63-1.60 (m,
13
4H, 2CH2), 1.39-1.27 (m, 6H, 3CH2), 0.92-0.89 (m, 3H, CH3). C NMR (100 MHz,
DMSO-d6, ppm): δ 172.54, 130.53, 129.34, 128.63, 128.36, 128.33, 128.26, 128.05,
127.85, 127.53, 62.37, 39.24, 36.55, 31.52, 29.67, 29.33, 27.23, 26.31, 25.65, 25.62,
23.47, 22.58, 14.08. IR (KBr, cm-1): ṽ 3287 (N-H), 3011 (HC=CH), 1645 (C=O), 1539,
1450, 1263, 1064, 925, 728. HRMS (TOF-MS, +): m/z [M+H]+ calculated for
+
C26H44NO2 402.3367, found 402.3361.
cis-Docosa-7,10,13,16,19-pentaenoic acid (5-hydroxy-pentyl)-amide (4n). Pale
1
yellow oil, yield 68%, HPLC purity: 98.0% (tR = 8.69 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 5.71 (br, 1H, NH), 5.44-5.33 (m, 10H, 5 -CH=CH-), 3.66-3.61 (m,
2H, CH2OH), 3.28-3.23 (m, 2H, NHCH2), 2.98 (br, 1H, OH), 2.85-2.81 (m, 6H, 3CH2),
2.44-2.39 (m, 2H, CH2), 2.25-2.21 (m, 2H, CH2), 2.09-2.04 (m, 2H, CH2), 1.61-1.50 (m,
13
8H, 4CH2), 1.44-1.31 (m, 6H, 3CH2), 0.91-0.88 (m, 3H, CH3). C NMR (100 MHz,
DMSO-d6, ppm): δ 172.60, 130.52, 129.32, 128.62, 128.35, 128.32, 128.26, 128.04,
127.84, 127.52, 62.50, 39.39, 36.50, 32.12, 31.51, 29.36, 29.31, 27.22, 25.64, 25.61,
23.46, 23.02, 22.57, 14.07. IR (KBr, cm-1): ṽ 3299 (N-H), 3011 (HC=CH), 1645 (C=O),
1547, 1448, 1264, 1180, 729. HRMS (TOF-MS, +): m/z [M+H]+ calculated for
+
C27H46NO2 416.3523, found 416.3519.
cis-Docosa-7,10,13,16,19-pentaenoic acid (6-hydroxy-hexyl)-amide (4o). Pale
1
yellow oil, yield 58%, HPLC purity: 97.8% (tR = 9.04 min), H NMR (400 MHz,
DMSO-d6, ppm): δ 5.57 (br, 1H, NH), 5.44-5.34 (m, 10H, 5 -CH=CH-), 3.66-3.63 (m,
2H, CH2OH), 3.28-3.23 (m, 2H, NHCH2), 2.86-2.81 (m, 8H, 4CH2), 2.45-2.39 (m, 2H,
CH2), 2.25-2.21 (m, 2H, CH2), 2.10-2.04 (m, 2H, CH2), 1.84 (br, 1H, OH), 1.59-1.50 (m,
13
4H, 2CH2), 1.43-1.29 (m, 10H, 5CH2), 0.92-0.88 (m, 3H, CH3). C NMR (100 MHz,

DMSO-d6, ppm): δ 172.38, 130.52, 129.31, 128.62, 128.35, 128.31, 128.05, 127.84,
127.53, 62.64, 39.33, 36.55, 32.53, 31.52, 29.63, 29.32, 27.22, 26.50, 25.64, 25.62,
25.28, 23.48, 22.58, 14.08. IR (KBr, cm-1): ṽ 3298 (N-H), 3009 (HC=CH), 1610 (C=O),
1585, 1263, 1076, 931, 752. HRMS (TOF-MS, +): m/z [M+H]+ calculated for
+
C28H48NO2 430.3680, found 430.3676.
4.4 Cell lines and cell culture
Mouse RAW 264.7 macrophages and Human large cell lung cancer cells (H460)
were obtained from the American Type Culture Collection (ATCC, USA) and were
incubated in DMEM media (Hyclone, Logan, Utah, USA) supplemented with 10% FBS,
100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C with 5% CO2.
4.5 Cell viability assay
Cell cytotoxicity was evaluated by methyl thiazolyl tetrazolium (MTT) assay. Cells
were inoculated at 4.5 × 103 cells per well in a 96-well plate. After cultured for 24 h,
compounds at different concentrations were added into the cell culture medium in
DMSO solution with the final concentration of DMSO is 0.1%, and the
compound-treated cells were cultured for 24 h at 37 °C with 5% CO2. Then 20 μL of 5
mg/mL MTT reagent was added into the cells and incubated for 4 h. After 4 h, cell
culture was removed and then 150 mL DMSO was added to dissolve the formazan. The
optical density was measured at 490 nm (OD490). Cell viability was calculated from
three independent experiments. The density of formazan formed in the blank group was
set as 100% of viability.
Cell viability (%) = compound (OD490)/blank (OD490) × 100%.
Blank: cultured with fresh medium only. Compound: treated with compounds or LPS.
4.6 Assay for NO production
RAW 264.7 cells were inoculated at 1 × 105 cells per well in a 24-well plate and
cultured for 20 h. The cells were then pre-treated with different concentrations
compounds which were prepared in a serum-free medium for 2 h before stimulation
with LPS (1 μg/mL). After LPS-stimulated for 24 h, the NO production was determined

by detecting the nitrite level using Griess reagent (Beyotime, Shanghai, China)
according to the manufacturer's instructions, then measured absorbance of the samples
at 570 nm (OD570) in a microplate reader (Thermo Fisher Scientific, MA, USA).
4.7 Real-Time Quantitative PCR
RAW 264.7 cells were inoculated at 1 × 105 cells per well in a 24-well plate and
cultured for 20 h. The cells were then pre-treated with different concentrations
compounds which were prepared in a serum-free medium for 2 h before stimulation
with LPS (1 μg/mL). After LPS-stimulated for 24 h, cells were homogenized in
TRIZOL kit for extraction of RNA according to each manufacturer’s protocol. Both
reverse transcription and quantitative PCR were carried out using a two-step M-MLV
Platinum SYBR Green qPCR SuperMix-UDG kit. (YEASEN, Shanghai, China)
Eppendorf Mastercycler ep realplex detection system was used for qPCR analysis. The
primers of genes including TNF-α, IL-6, IL-1β, and GAPDH were synthesized by
BAIJIN (BAIJIN, Xiamen, China). The primer sequences used are shown in Table1.
The amount of each gene was determined and normalized by the amount of GAPDH.
4.8 Western Blot Analysis
RAW264.7 cells were inoculated at 1 × 105 cells per well in a 24-well plate and
cultured for 20 h. The cells were pretreated with different concentrations of different
compounds for 7.5h, and then cultured in the presence or absence of LPS (1 μg/mL) for
0.5 h. After treatment, cells were washed with ice-cold PBS three times and lysed in
RIPA buffer (Sangon, Shanghai, China). A Bradford protein assay was used to calculate
the concentration of total protein in each sample. A routine Western blot operation was
conducted to detect TNF-α (Abcam, Cambridge, UK), IL-1β (Abcam, Cambridge, UK),
p-IKKα/β (CST, Boston, USA), and IκBα (Abcam, Cambridge, UK) expression, and
normalized by the amount of β-actin or α-tubulin (CST, Boston, USA).
4.9 Determination of TNF-α and IL-6 in medium
RAW264.7 cells were inoculated at 1 × 105 cells per well in a 24-well plate and
cultured for 20 h. The cells were then pre-treated with different concentrations
compounds which were prepared in a serum-free medium for 2 h before stimulation

with LPS (1 μg/mL) for 24 h. After treatment of cells with compounds and LPS, the
TNF-α and IL-6 levels in the medium were determined with an enzyme-linked
immunosorbent assay (ELISA) kit (DAKEWE, China) according to the manufacturer’s
instructions. The total amount of the inflammatory factor in the media was normalized
to the total protein quantity of the viable cell pellets.
4.10 Protein expression and purification
The human Nur77-LBD was cloned as an N-terminal histidine-tagged fusion protein
in the pET15b expression vector and overproduced in Escherichia coli BL21 DE3 strain.
Briefly, cells were harvested and sonicated, and the extract was incubated with the
His60 Ni Superflow resin.
4.11 Surface Plasmon Resonance (SPR) Analysis
After coupling 5 μg purified ligand-binding domain (LBD) of Nur77 (Nur77-LBD)
protein to CM5 of Biacore, compound 4k (10 μM) with known anti-inflammatory
activity were screened for its binding to Nur77-LBD by Biacore T200. The identified
compound was tested again with a gradient concentration of 2.097 μM, 2.621 μM, 3.277
μM, 4.09 μM, 5.12 μM 6.4 μM, 8.0 μM, and 10 μM injected through flow cells
immobilized with Nur77-LBD. The binding kinetics between Nur77-LBD and
compounds were analyzed at RT by Biacore T200. A CM5 sensor chip (GE Healthcare)
was chemically activated by injecting 100 µL of N-ethyl-N0-3-(diethylaminopropyl)
carbodiimide (EDC) (200 mM) and N-hydroxysuccinimide (NHS) (50 mM) (v/v 1:1).
Purified Nur77-LBD dissolved in 10 mM sodium acetate (pH 4.5) and binding to the
preactivated CM5 chip using amine coupling. The remaining ester groups were blocked
by 1 M ethanolamine-HCl (pH 9.5). The amount of immobilized Nur77-LBD was
detected by mass concentration dependent changes in the refractive index on the
sensorchip surface, and corresponded to about 10,000 resonance units (RU). A serial
concentration of compounds was added at a flow rate of 20 µL/min. When the data
collection was finished in each cycle, the sensor surface was washed with glycine-HCl
(10 mM, pH 2.5).
4.12 Molecular docking simulation

The molecular docking simulation was performed to better understand the binding
mode of compound DHA, 4a, and 4k with Nur77. Molecular docking between selected
compounds and Nur77-LBD was performed using Schrödinger software (Version
2019-1). The complex structure of Nur77-LBD (PDB ID: 4JGV) was used as a mode
structure for molecular docking study. The protein prepared using the Protein
Preparation Wizard of Schrödinger [59]. In this process, the force field applied OPLS3e
and the root mean square deviation (RMSD) of the atom was specified as 0.30 Å, bond
orders were assigned, unwanted waters were deleted and missing atoms were added.
The selected compounds including DHA, 4a, and 4k were prepared using the LigPrep
suit of Schrödinger with default settings and then bound to Nur77-LBD using the
induced fit docking (IFD) protocol [60, 61]. To validate the docking method, the native
ligand THPN was first re-docked to Nur77-LBD. The re-docking pose of THPN
obtained using the IFD protocol had an RMSD of 1.0352 referring to the conformation
of the crystal structure. The MM/GBSA (Prime MMGBSA v3.000) was used to
calculate the absolute binding free energy of complexes from the docking result [62].
All of the docking studies were used the induced fit docking calculation protocol with
default settings. Schrödinger's Maestro (Version 11.9) was used as the primary
graphical user interface for the visualization of the crystal structure and docking results.
The interaction analysis between molecule and protein complex employed by PyMol
and the graphics of the binding site were generated using PyMol, version 2.3.0
(Open-Source PyMOLTM by Schrödinger) [63].
4.13 Mouse models
Male C57BL/6 mice of 20-25 g were obtained from the Animal Centre of Xiamen
University (Xiamen, China). All animal care and experimental procedures were
approved by the Animal Policy and Welfare Committee.
The LPS-induced ALImodel [64, 65]. The ALI was induced in mice by intratracheal
LPS instillation to evaluate the protective ability of 4k against LPS-induced acute
inflammation in vivo. mice were randomly divided into groups as follows: control (six
mice received vehicle of 0.9% saline), LPS (six mice received LPS alone),
dexamethasone, or 4k (each of the two compounds was administered to a group of six

mice at 10 mg/kg, 20 mg/kg, 50 mg/kg). The active compounds were given daily via
intraperitoneal injection consecutively for one week. The mice were euthanized with
chloral hydrate 6 h after intratracheal injection of LPS at 6 mg/kg. Broncho alveolar
lavage fluid (BALF), blood, and lung tissues were collected for further analysis.
4.14 Histopathologic examination of lung
The superior lobe of the right lung was collected and fixed in 4% paraformaldehyde,
then embedded in paraffin and cut into 5 μm sections. The sections were stained with
hematoxylin and eosin using standard protocol for light microscopy examination.
4.15 Statistical Analysis.
All assays were performed in triplicate. The results were expressed as mean ± SEM
(standard deviation). Other data were analyzed by the SPSS statistical software, version
18.0 (SPSS Inc., USA). ANOVA with Tukey’s post-test (One-way ANOVA for
comparisons between groups) was used to compare values among different
experimental groups using the GraphPad program. For experiments with only two
groups, Student’s t-test was used as specified in the figure legends. p < 0.05 was
considered statistically significant (*), p < 0.01 as highly significant (**), p < 0.001 as
extremely significant (***), and ns as not significant.
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
This work was supported by grants from the Natural Science Foundation of Fujian
Province of China (No. 2018J01132 and 2019J01011672), the Fundamental Research
Funds for the Central Universities (No. 20720180051), and the Marine Economy
Innovation Development Area Demonstration Project of Beihai (Bhsfs003, Bhsfs009).
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