New synthesized polyoxygenated diarylheptanoids suppress lipopolysaccharide-induced neuroinflammation

Article abstract of DOI:10.1016/j.bbrc.2020.06.122

In neurodegenerative diseases, such as Alzheimer's disease, Huntington's disease, Parkinson's disease and multiple sclerosis, neuroinflammation induced by the microglial activation plays a crucial role. In effort to develop effective anti-neuroinflammatory compounds, different new linear polyoxygenated diarylheptanoids were synthesized. In LPS-triggered BV-2 microglial cells their ability to reduce the concentration of IL-6 and TNF-α pro-inflammatory cytokines was evaluated. Moreover, their effect on NF-κB and ATP citrate lyase (ACLY), a recently emerged target of metabolic reprogramming in inflammation, was assessed. Finally, we turned our attention to inflammatory mediators derived from the cleavage of citrate catalyzed by ACLY: prostaglandin E₂, nitric oxide and reactive oxygen species. All compounds showed null or minimal cytotoxicity; most of them had a great anti-neuroinflammatory activity. Diarylheptanoids 6b and 6c, bearing a halide atom and benzyl ether protective groups, exhibited the best effect since they blocked the secretion of all inflammatory mediators analyzed and reduced NF-κB and ACLY protein levels.

Full text of DOI:10.1016/j.bbrc.2020.06.122

Biochemical and Biophysical Research Communications 529 (2020) 1117e1123
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
New synthesized polyoxygenated diarylheptanoids suppress
lipopolysaccharide-induced neuroinflammation
Anna Santarsiero a, Antonella Bochicchio a, Maria Funicello a, Paolo Lupattelli a,
Sabine Choppin b, Françoise Colobert b, Gilles Hanquet b, Lucie Schiavo b,
,
,
Paolo Convertini a *, Lucia Chiummiento a **, Vittoria Infantino a
a Department of Science, University of Basilicata, Via dell’Ateneo Lucano 10, 85100, Potenza, Italy
Universite de Strasbourg, Universite de Haute-Alsace, CNRS, UMR 7042-LIMA, ECPM, 25 Rue Becquerel, Strasbourg, 67087, France
b
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
In neurodegenerative diseases, such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease
and multiple sclerosis, neuroinflammation induced by the microglial activation plays a crucial role. In
effort to develop effective anti-neuroinflammatory compounds, different new linear polyoxygenated
diarylheptanoids were synthesized. In LPS-triggered BV-2 microglial cells their ability to reduce the
Received 24 June 2020
Accepted 24 June 2020
Available online 1 August 2020
concentration of IL-6 and TNF-
a pro-inflammatory cytokines was evaluated. Moreover, their effect on NF-
Keywords:
Neuroinflammation
Microglia
Diarylheptanoids
Anti-neuroinflammatory activity
NF-kB
k
B and ATP citrate lyase (ACLY), a recently emerged target of metabolic reprogramming in inflammation,
was assessed. Finally, we turned our attention to inflammatory mediators derived from the cleavage of
citrate catalyzed by ACLY: prostaglandin E2, nitric oxide and reactive oxygen species. All compounds
showed null or minimal cytotoxicity; most of them had a great anti-neuroinflammatory activity. Dia-
rylheptanoids 6b and 6c, bearing a halide atom and benzyl ether protective groups, exhibited the best
effect since they blocked the secretion of all inflammatory mediators analyzed and reduced NF-kB and
ATP citrate lyase
ACLY protein levels.
© 2020 Elsevier Inc. All rights reserved.
1. Introduction
(e.g. TNF-
a
, IL-1
b
, IL-6), chemokines, prostaglandin E2 (PGE2),
reactive oxygen species (ROS), and reactive nitrogen species [2],
which elicit detrimental effects on adjacent neurons and further
exacerbate tissue inflammation and damage. By contrast, in M2
microglial cells the expression of anti-inflammatory mediators is
up-regulated and they contribute in neuroprotection and tissue
recovery and remodelling [3]. Therefore, neuroinflammation is a
double-edged sword because of its beneficial or harmful effects on
central nervous system, which depend on the type of microglia
activation and the duration of inflammatory response. In fact,
acute events scavenge harmful substances playing a protective
role for nervous tissue while chronic ones are always dangerous
due to the production and release of pro-inflammatory and
cytotoxic factors [4]. In neurodegenerative diseases the equilib-
rium between microglial surveillance and activation is disrupted
and growing evidence demonstrates that the control of microglia
activation could attenuate their severity [5]. Therefore, the dis-
covery of anti-neuroinflammatory compounds able to inhibit the
microglia activation could be helpful in neurodegenerative
disorders.
Chronic neuroinflammation has a pivotal role in the onset and
progression of a broad range of neurodegenerative diseases such
as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease
and multiple sclerosis [1]. Neuroinflammation is a consequence of
the activation of microglia, the resident immune cells of the
central nervous system. Microglial cells influence brain develop-
ment and constantly survey the microenvironment of paren-
chyma, but they also act as the first line of defence against injury
and invading pathogens. When the homeostasis is disrupted,
microglia becomes activated and undergoes morphological and
phenotypical changes into one of the two extremes of polariza-
tion: the classically activated M1 or alternatively activated M2
macrophages. M1 microglia secretes pro-inflammatory cytokines
* Corresponding author. Department of Science, Via dell’Ateneo Lucano 10,
85100, Potenza, Italy.
** Corresponding author.
E-mail addresses: paolo.convertini@uky.edu (P. Convertini), lucia.chiummiento@
unibas.it (L. Chiummiento).
Diarylheptanoids (C6-C7-C6), a class of oxygenated compounds
https://doi.org/10.1016/j.bbrc.2020.06.122
0006-291X/© 2020 Elsevier Inc. All rights reserved.

1118
A. Santarsiero et al. / Biochemical and Biophysical Research Communications 529 (2020) 1117e1123
with a 1,7-diphenylheptane skeleton, have been isolated from
wood, bark or leaves of many hardwoods including Betulaceae,
Leguminosae, Myricaceae and Proteaceae. They are increasingly
recognized as potential therapeutic agents for their wide spectrum
of biological and pharmacological activities such as anti-
inflammatory, antioxidant, antitumor, estrogenic, leishmanicidal,
melanogenesis, hepatoprotective, neuroprotective proprieties [6].
To this class of natural products belongs myricanol, a cyclic and not
symmetric diarylheptanoid extracted from Myrica species, and
curcumin, the main curcuminoid isolated from Curcuma longa
(Fig. 1). It has been shown that myricanol reduces the levels of the
microtubule-associated protein tau resulting in anti-Alzheimer’s
disease effect [7] and curcumin exerts neuroprotective and anti-
inflammatory activities [8e10]. In particular, curcumin inhibits
2. Material and methods
2.1. Cell culture, treatment and cell viability assay
The immortalized murine BV-2 microglial cell line was main-
tained in Roswell Park Memorial Institute 1640 Medium supple-
mented with 10% heat inactivated fetal bovine serum, 4 mM L-
Glutamine,100 U/ml penicillin and 100 m
g/ml streptomycin at 37 ꢀC
and 5% CO2. Where indicated BV-2 cells were activated with
200 ng/ml LPS from Salmonella enterica serotype typhimurium
(LPS, SigmaeAldrich, St. Louis, MO, United States). Cell viability was
evaluated by using CellTiter 96® Non-Radioactive Cell Proliferation
Assay (Promega, Madison, WI, USA) according to manufacturer’s
instructions. The details are given in Supplementary data.
PGE2, nitric oxide (NO), and tumor necrosis factor
a (TNF-a) release
following lipopolysaccharide (LPS) stimulation in RAW 264.7 mu-
rine macrophages [11] and switches the polarization of LPS-
activated BV2 microglial cells from M1 phenotype to a predomi-
nantly M2 phenotype [12].
Taking into consideration the biological importance of linear
diarylheptanoids in general, and myricanol and curcumin in
particular, we planned a straightforward access to a series of
linear diarylheptanoid derivatives using the cross-metathesis re-
action on readily available precursors 1a,b, 2a,b and 3 [13e15].
Terminal olefins 1e3 [14] were easily prepared and used to give
access to new linear diarylheptanoids and to the cyclic one,
myricanol. Herein some of these were differently protected,
halogenated and reduced. Eleven of the new synthesized diary-
2.2. Enzyme-linked immunosorbent assay (ELISA)
The concentration of pro-inflammatory cytokines IL-6 (Cat. nꢀ
32670069U1, ImmunoTools, Friesoythe, Germany) and TNF-a (Cat.
nꢀ 32673019U1, ImmunoTools) in cell culture media was measured
with ELISA kits following the manufacturer’s recommendations.
The levels of PGE2 were quantified by using DetectX® Prostaglandin
E2 High Sensitivity Immunoassay Kit (Arbor Assays, Ann Arbor, MI,
USA) as per manufacturer’s protocol.
2.3. ROS and NO detection
Reactive oxygen species and nitric oxide levels were measured
lheptanoids were tested on
a cell line model of neuro-
by using 6-Carboxy-20,70-Dichlorodihydrofluorescein Diacetate and
inflammation: LPS-triggered BV-2 cells. Their effect on different
mediators of inflammation was evaluated in comparison with
curcumin and celecoxib, a COX-2 selective non-steroidal anti-in-
flammatory drug (Fig. 1). Celecoxib, in a rat model with LPS-
triggered neuroinflammation, significantly decreased the rate of
4-Amino-5-Methylamino-20,70-Difluorofluorescein
Diacetate
(Thermo Fisher Scientific, San Jose, CA, USA), respectively, as pre-
viously described [17].
2.4. Western blotting
microglia activation and levels of IL-1
inflammatory cytokines [16]. Finally, the ability of the new syn-
thesized diarylheptanoids to reduce NF- B and ACLY e a meta-
bolic pro-inflammatory enzyme e was also investigated.
b and TNF-a pro-
Western blotting experiments were carried-out as reported
k
previously [18] using antieNFe
lyase (ab157098) or anti- eactin (ab8227) primary antibodies
(Abcam, Cambridge, MA). More details are reported in
kB/p65 (ab7970), anti-ATP citrate
b
Fig. 1. Diarylheptanoids, myricanol, curcumin and celecoxib structures.

A. Santarsiero et al. / Biochemical and Biophysical Research Communications 529 (2020) 1117e1123
1119
Supplementary data.
2b gave rise to compound 4c in high yield (82%) and cross
metathesis between 1a and 3 afforded alkene 4e in only 32% of
yield.
2.5. Statistical analysis
Subsequent transformations were performed in order to have
different protections and different functionalities (double bond,
ketone or halide) on the same parent scaffold. Therefore, the
acetylation of 4a by Ac2O in pyridine afforded 4d in a quantitative
yield, while the methylation of 4e furnished the allylketone 4f in
30% of yield.
Statistical significance of differences was determined by using
Student’s t-test. Results are shown as means SD of, at least, four
independent experiments. Differences were considered as signifi-
cant (*p < 0.05), very significant (**p < 0.01) and highly significant
(***p < 0.001).
Hydrogenation of the double-bond and concurrent debenzyla-
tion of 4a resulted in the quantitative formation of 5a which was
selectively protected on phenols as methyl ethers 5d. Chemo-
selective hydrogenation of 4c by o-NO2PhSO2Cl and N2H2 [25]
furnished 5b in high yield, preserving the benzyl protecting group
and the iodine atom of 4c. Oxidation of compound 5d using DMP in
DCM afforded the ketone 5e with 63% yield. Compounds 5a and 5b
were completely protected on phenol and carbinol functions as
tribenzylethers 6a and 6b using BnBr, NaH, NaI in DMF. Subse-
quently regioselective bromination of 6a furnished the mono-
brominated 6c and the di-brominated 6d in quantitative yield
when 1.2 or 2.4 equiv of NBS was used, respectively.
3. Results
3.1. Preparation of diarylheptanoids 4-6
The requisite allylphenols 1a and 1b were accessed in two steps
from commercially available 2,3-dimethoxyphenol [13]. Coupling
partners 2a, b and 3 were readily prepared from commercial
methyl 3-(4-benzyloxyphenyl)propanoate [14,15]. With the key
fragments 1a, 1b, 2a, 2b and 3 in hands, we attempted cross
metathesis (CM) reactions as depicted in Scheme 1 [14,19,20].
Hence, treatment of 1a (4 equiv.) with 2a or 2b (1 equiv.) in the
presence of G-II catalyst (5mol%) in dichloromethane from ꢁ78 ꢀ
C
3.2. Novel polyoxygenated diarylheptanoids do not affect microglial
cell viability
to room temperature provided diarylheptanoid 4a and 4c in a
gratifying 81% and 83% yield, respectively, if compared to the ex-
amples of CM at low temperature reported in literature [21].
Addition of G-II catalyst at ꢁ78 ꢀC was mandatory to obtain
good yields. Excess of partner 1a and its homocoupled were almost
fully recovered after chromatography.
BV-2 microglial cells were treated with curcumin, celecoxib or
the novel polyoxygenated diarylheptanoids at 0.01, 0.1 and 1 mM
and their cytotoxic effects were determined by MTT assay after 72 h
(Fig. S1). Curcumin and celecoxib as well as the new-synthesized
diarylheptanoids exhibited minimal or null toxic effect. We chose
Cross metathesis between 1b and 2a afforded coupling product
4b in only 35% yield without formation of homocoupling product
resulting from dimerization of 1b. While positive effect of free OH
(phenol or alcohol) on CM reactions, already reported in literature
[22e24], could explain the excellent yield obtained to produce 4a
(81% yield), an opposite behavior was observed in the preparation
of compound 4b (Scheme 1) despite the presence of two free OH on
the starting catechol 1b.
the lowest concentration (0.01
mM) as optimal concentration to
evaluate the anti-inflammatory activity.
3.3. Impact of treatment with the linear polyoxygenated
diarylheptanoids on IL-6, TNF-a and NF-kB
Probably the lower solubility of catechol 1b in DCM at ꢁ78 ꢀC
prevented the metathesis reaction which started only increasing
the temperature (20 ꢀC). No dimerization of 1b was observed or
after column chromatography. Moreover reaction between 1a and
When stimulated with lipopolysaccharide, the macrophages
secrete different mediators among which IL-6 and TNF- have a
a
primary role for the initiation and propagation of inflammation
[26]. Therefore, to probe the effect of the novel molecules on IL-6
Scheme 1. a) refs 13 and 14; b) Grubbs-II, DCM, ꢁ78 ꢀC to rt, 81% yield 4a, 35% yield 4b, 82% yield 4c, 32% yield 4e; c) Ac2O, Py, rt, 99%; d) methylation of 4e and 5a by MeI, K2CO3,
acetone, 30% yield 4f and 40% yield 5d; e) hydrogenation of 4a by H2, Pd/C, MeOH, rt, 1 h, 99% yield 5a; f) hydrogenation of 4c by o-NO2PhCl, N2H2$H2O, ACN, 0 ꢀC 91% yield 5c; g)
benzylation of 5a by BnBr, K2CO3, TBAI acetone, reflux, 99% yield 5c; h) DMP, DCM, 63%; i) benzylation of 5a and 5b by BnBr, NaH, NaI, DMF 75% yield 6a and 71% yield 6b; j)
bromination of 6a by NBS (1.1 equiv or 2.2 equiv.), TFA, ACN 99% yield 6c and 99% yield 6d.

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A. Santarsiero et al. / Biochemical and Biophysical Research Communications 529 (2020) 1117e1123
and TNF-
a
secretion, BV-2 cells were stimulated with LPS in the
curcumin (Fig. 2C). Molecules 4d and 4f were the less active;
however, they led to the reduction of about 30% in NF- B protein
levels with respect to BV-2 cells stimulated only with LPS (Fig. 2D).
presence or absence of each compound at the concentration of
0.01 M (Fig. 2). Twenty-four hours later, the levels of IL-6 and TNF-
were measured in cell culture media. LPS induced a significant
release of IL-6 (Fig. 2A; LPS vs. CONTROL, p < 0.001) and TNF-
k
m
a
a
3.4. Effect of the novel polyoxygenated diarylheptanoids on ACLY
expression and PGE2, NO and ROS production
(Fig. 2B; LPS vs. CONTROL, p < 0.001). Celecoxib resulted more
active than curcumin in lowering IL-6 levels compared to cells
activated only with LPS (Fig. 2A, ***p < 0.001). The majority of the
tested compounds was able to reduce the secretion of IL-6; ex-
ceptions were molecules 4a, 5a, 5c and 5d, which had no significant
effect (Fig. 2A). Interestingly, compound 4b was effective as cele-
coxib in lowering IL-6 levels, while compounds 5e, 6c and 6d had
an effect similar to curcumin. In the meantime, the diarylheptanoid
6d did not affect TNF-a release following LPS stimulation as well as
compounds 4b, 4d, 5a, 5d and 5e (Fig. 2B). The remaining mole-
cules 4a, 4f, 5c, 6b and 6c acted like celecoxib and curcumin in
letting TNF-
***p < 0.001).
Then we investigated the effect of the new synthesized com-
pounds on NF- B, a crucial transcription factor whose activation
leads to the induction of multiple genes regulating the immune and
the inflammatory responses [27]. Therefore, to verify if NF- B could
Since the new synthesized compounds reduced NF-
sion and ACLY up-regulation in LPS-induced macrophages is under
control of NF- B transcription factor [28], we evaluated the protein
kB expres-
k
levels of ACLY (Fig. 3A). Of note, ACLY is a metabolic enzyme -
recently identified as a new player of the immunometabolism -
which splits glucose-derived citrate into acetyl-CoA and oxaloace-
tate (OAA). Acetyl-CoA provides units for lipid elongation, including
arachidonic acid, precursor of prostaglandins. OAA leads to NADPH
production, necessary for NADPH oxidase and iNOS to generate ROS
and NO, respectively, in LPS-activated macrophages [29]. In BV-
2 cells primed with LPS, curcumin and celecoxib were able to
reduce ACLY expression levels (Fig. 3A). Among the new synthe-
sized diarylheptanoids, the molecule 6d was the most active, even
better than celecoxib, in lowering ACLY protein; on the contrary,
molecules 4b, 4d and 5e had no activity. The remaining compounds
were able to reduce the expression of ACLY (Fig. 3A).
a concentration down (Fig. 2B, compounds vs. LPS,
k
k
be a target for the polyoxygenated diarylheptanoids, LPS-triggered
BV-2 cells were treated with the novel compounds, curcumin, or
celecoxib. The protein levels of NF-
via Western blot. Surprisingly, all compounds were able to reduce
the expression of NF- B in the same manner as celecoxib and
Taking into consideration the inhibitory effect of ACLY by some
of novel diarylheptanoids, we decided to monitor the levels of in-
flammatory mediators linked to the citrate pathway: PGE2, NO and
ROS. When BV-2 microglial cells were activated with LPS we found
kB subunit p65 were quantified
k
Fig. 2. Effect of polyoxygenated diarylheptanoids on IL-6 and TNF-
a
release and NF-
k
B/p65 expression levels.
BV-2 cells were treated with DMSO (CONTROL) or 200 ng/ml LPS alone (LPS) or LPS plus 0.01
24 h IL-6 (A) and TNF- (B) concentrations were quantified. Means S.D. of four duplicate independent experiments are shown; differences between CONTROL and LPS were highly
significant (p < 0.001 Student’s t-test). Where indicated differences between LPS and cells treated with LPS plus compounds were significant (*p < 0.05, **p < 0.01, ***p < 0.001,
Student’s t-test). C) The expression levels of subunit p65 of NF- B were evaluated via western blotting. The intensity of immunolabelled protein bands depicted in C was quantified
by densitometric scanning and NF- B p65 levels were normalized against -actin to have NF- B p65 relative protein levels (D).
mM celecoxib, curcumin, 4a, 4b, 4d, 4f, 5a, 5c, 5d, 5e, 6b, 6c and 6d compounds. After
a
k
k
b
k

A. Santarsiero et al. / Biochemical and Biophysical Research Communications 529 (2020) 1117e1123
1121
Fig. 3. Effect of polyoxygenated diarylheptanoids on ACLY expression levels and PGE2, NO and ROS production.
BV-2 microglial cells were treated with or 200 ng/ml LPS alone (LPS) or LPS plus 0.01
specific antibodies, the expression levels of ACLY and -actin were detected (upper panel). The intensity of immunolabelled protein bands was analyzed by densitometric scanning
and ACLY levels were normalized against -actin to have ACLY relative protein levels (lower panel). B-C-D) PGE2 concentration was measured after 48 h of LPS treatment (B) while
mM celecoxib, curcumin, 4a, 4b, 4d, 4f, 5a, 5c, 5d, 5e, 6b, 6c and 6d compounds. A) By using
b
b
NO and ROS 24 h later (CeD). Means S.D. of four triplicate independent experiments are shown. In B-C-D differences between CONTROL and LPS were highly significant (p < 0.001,
Student’s t-test). Where indicated, differences between LPS and cells treated with LPS plus compounds were significant (*p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test).
a strong increase in PGE2 levels compared with untreated cells
(Fig. 3B, CONTROL vs. LPS p < 0.001). Celecoxib worked better than
curcumin in modulating the production of PGE2, bringing the levels
of PGE2 back to those of the untreated cells (Fig. 3B, CELECOXIB vs.
LPS ***p < 0.001). The majority of the tested diarylheptanoids acted
as curcumin with the exception of compound 4d, 4f, 5a and 5e.
Molecules 4f and 5a had no effect: the levels of PGE2 in BV-2 cells
were similar to cells treated only with LPS. On the contrary, com-
pound 4d was the only tested diarylheptanoid more active than
curcumin but less effective than celecoxib in reducing PGE2 levels
(Fig. 3B).
Then, efforts were dedicated to NO and ROS evaluation. Intense
production of both ROS and NO was observed in various diseases
[30] and can be correlated to neurodegenerative disorders [31,32].
Celecoxib resulted more active then curcumin in lowering NO and
ROS levels compared to cells activated only with LPS (Fig. 3CeD,
p < 0.001). The majority of the tested compounds had similar effect
of curcumin with the exception of 6b and 6c, the most active in the
reduction of NO with an effect similar to celecoxib (Figs. 3C, 6b, 6c
vs. LPS, ***p < 0.001). By contrary, all the tested polyoxygenated
diarylheptanoids, except 6d, showed a higher reduction of ROS
levels with respect to the leading curcumin. Compounds 4a, 4d, 5e
and 6b had the strongest inhibitor effect on LPS-induced ROS
production in BV-2 cells (Fig. 3D, compounds vs. LPS, ***p < 0.001).
Meanwhile, compound 5d was completely inactive: it even led to a
significant increase in NO (Fig. 3C, 6c vs. LPS, ***p < 0.001) as well
as in ROS (Fig. 3D, 6c vs. LPS, ***p < 0.001) levels.
4. Discussion
In our investigation, we reported the prospect of new eleven
linear polyoxygenated diarylheptanoids as a source of alternative
anti-neuroinflammatory compounds. To this end, they were syn-
thesized according to known methodology [14] and screened in
LPS-triggered BV-2 microglial cells. The tested diarylheptanoids
could be classified into three class: a) with double bond (4a, 4b, 4d
and 4f); b) without double bond (5a, 5c, 5d and 5e) and c) with one
or two halides on the aryl rings and completely protected on hy-
droxyl groups (6b, 6c and 6d). Our findings showed that the acet-
ylation of OHs had made more efficient compound 4d in lowering

1122
A. Santarsiero et al. / Biochemical and Biophysical Research Communications 529 (2020) 1117e1123
PGE2, NO and ROS levels with respect to 4a and 4b with one and
two free phenolic group, respectively. Moreover, the presence of
the carbonyl and the additional methyl ether in compound 4f made
it with similar activity of 4b in reducing NO and ROS production
and none inhibitory effect on PGE2 release. An opposite behaviour
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.bbrc.2020.06.122.
was observed in IL-6 and TNF-a secretion, where compound 4d was
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celecoxib and 4a had a strong inhibitory effect on TNF-a secretion.
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kB and ACLY protein
a
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Funding
This research was supported by Funding for Basic Research
(FFABR) from the Italian Ministry of Education, University and
Research and French government for LS PhD fellow.
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