Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/AP-1 Signaling Pathway and Cyclooxygenases and Lipoxygenase

Article abstract of DOI:10.1021/acs.jnatprod.9b00081

Stilbenoids are important components of foods (e.g., peanuts, grapes, various edible berries), beverages (wine, white tea), and medicinal plants. Many publications have described the anti-inflammatory potential of stilbenoids, including the widely known trans-resveratrol and its analogues. However, comparatively little information is available regarding the activity of their prenylated derivatives. One new prenylated stilbenoid (2) was isolated from Artocarpus altilis and characterized structurally based on 1D and 2D NMR analysis and HRMS. Three other prenylated stilbenoids were prepared synthetically (9-11). Their antiphlogistic potential was determined by testing them together with known natural prenylated stilbenoids from Macaranga siamensis and Artocarpus heterophyllus in both cell-free and cell assays. The inhibition of 5-lipoxygenase (5-LOX) was also shown by simulated molecular docking for the most active stilbenoids in order to elucidate the mode of interaction between these compounds and the enzyme. Their effects on the pro-inflammatory nuclear factor-κB (NF-κB) and the activator protein 1 (AP-1) signaling pathway were also analyzed. The THP1-XBlue-MD2-CD14 cell line was used as a model for determining their anti-inflammatory potential, and lipopolysaccharide (LPS) stimulation of Toll-like receptor 4 induced a signaling cascade leading to the activation of NF-κB/AP-1. The ability of prenylated stilbenoids to attenuate the production of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was further evaluated using LPS-stimulated THP-1 macrophages.

Full text of DOI:10.1021/acs.jnatprod.9b00081

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/
AP‑1 Signaling Pathway and Cyclooxygenases and Lipoxygenase
†
,▲
,†,▲
⊥
‡
‡
,§
Jan Hose
̌
k,
Veronika Lelak
́
ova,
́
*
Pavel Bobal
́
,
Hana Pízo Marketa Gazdova,*
̌
́ ́ ́
va,
̌
Premysl Landa, Veronika Temml,
§
⊥,∥
⊥
¶
Milan Malaník, Karolina Jakubczyk, Ondre
Daniela Schuster,
Michal Oravec, and Karel Smejkal*
̌
j Vesely,
́
⬡
▽
▽
○
△
Vilailak Prachyawarakorn, Phanruethai Pailee, Gang Ren,
Filip Zpurny,
́
□
,§
̌
†
‡
§
Department of Molecular Biology and Pharmaceutical Biotechnology, Department of Chemical Drugs, and Department of
Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeh
Brno, Czech Republic
́
̌
o trída 1946/1, 61242
⊥
Laboratory of Plant Biotechnologies, Institute of Experimental Botany, Czech Academy of Sciences, Rozvojova
Czech Republic
́
263, 16502 Prague,
∥
Department of Quality of Agricultural Products, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life
ka
Department of Pharmacy/Pharmacognosy and Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 80-82,
Sciences Prague, Kamyc
́
́
129, 16521 Prague 6−Suchdol, Czech Republic
¶
6020 Innsbruck, Austria
⬡
Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Paracelsus Medical University Salzburg,
Strubergasse 21, 2020 Salzburg, Austria
▽
Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Laksi, Bangkok 10210, Thailand
○
Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional
Chinese Medicine, Nanchang 330004, People’s Republic of China
△
Botanical Garden Teplice, J. Suka 1388/18, 41501 Teplice, Czech Republic
□
Global Change Research Institute of the Czech Academy of Sciences, Bel
̌
idla 986/4a, 60300 Brno, Czech Republic
*
S Supporting Information
ABSTRACT: Stilbenoids are important components of foods
e.g., peanuts, grapes, various edible berries), beverages (wine,
(
white tea), and medicinal plants. Many publications have
described the anti-inflammatory potential of stilbenoids, includ-
ing the widely known trans-resveratrol and its analogues.
However, comparatively little information is available regarding
the activity of their prenylated derivatives. One new prenylated
stilbenoid (2) was isolated from Artocarpus altilis and
characterized structurally based on 1D and 2D NMR analysis
and HRMS. Three other prenylated stilbenoids were prepared
synthetically (9−11). Their antiphlogistic potential was deter-
mined by testing them together with known natural prenylated
stilbenoids from Macaranga siamensis and Artocarpus heterophyllus
in both cell-free and cell assays. The inhibition of 5-lipoxygenase
(
5-LOX) was also shown by simulated molecular docking for the most active stilbenoids in order to elucidate the mode of
interaction between these compounds and the enzyme. Their effects on the pro-inflammatory nuclear factor-κB (NF-κB) and
the activator protein 1 (AP-1) signaling pathway were also analyzed. The THP1-XBlue-MD2-CD14 cell line was used as a
model for determining their anti-inflammatory potential, and lipopolysaccharide (LPS) stimulation of Toll-like receptor 4
induced a signaling cascade leading to the activation of NF-κB/AP-1. The ability of prenylated stilbenoids to attenuate the
production of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was further evaluated
using LPS-stimulated THP-1 macrophages.
nflammation of tissues occurs when the immune system is
1
I
activated to combat an infection, an irritation, or a wound.
Immune-system cells generate and release a variety of
mediators that affect the development of the inflammatory
Received: January 27, 2019
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00081
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Article
response. Macrophages, which play an important role in acute
as well as in chronic inflammation, produce a number of
endogenous mediators, including the vasoactive amines
histamine and serotonin, metabolites of arachidonic acid, the
complement system, the kinin system, the fibrinolytic system,
platelet-activating factor (PAF), neuropeptides, hydrolytic
enzymes, adhesion molecules, reactive oxygen species (ROS),
other C-prenylated analogues could facilitate the comparison
of influence of structural aspects on tested biological activity.
RESULTS AND DISCUSSION
■
Isolation of Compounds and Elucidation of Struc-
tures. Eleven prenylated stilbenoids (1−11) were tested for
their anti-inflammatory potential. Eight of these (1−8) occur
in Nature. Compound 1 was obtained from A. heterophyllus,
and compound 2 from A. altilis. The isolation and
identification of compounds 3−8 from M. siamensis has been
2
,3
and inflammatory cytokines. Studies describing the bio-
activity of prenylated phenols have shown that they may
2
potently affect the production of these substances.
14,21
Prenylated phenolics are an interesting class of plant
described previously,
and reported here are the syntheses
2
secondary metabolites, because their structures combine the
of the new compounds 9−11.
common structural features of a phenolic skeleton with
terpenoid side chains, most commonly isopentenyl and geranyl
2
,4
units. Prenylated phenols are classified into several groups,
2
,5,6
according to the type of phenolic skeleton.
The 1,2-
diphenylethylene core characteristic for stilbenoids is formed
in plants through the shikimate pathway via cinnamoyl-CoA or
p-coumaroyl-CoA, and the reaction is catalyzed by stilbene
7
synthase. The prenyl unit is synthesized via the isoprenoid
mevalonate or deoxyxylulose pathway, and prenylation is
4
,7
mediated by stilbenoid-specific prenyltransferase. Many of
the plants containing prenylated phenolic constituents that are
used in traditional medicine show a variety of biological effects,
such as anti-inflammatory, antioxidant, antimicrobial, or
2
,4,8
cytotoxic activities.
The many possible structural combi-
nations of a prenyl group with a phenolic skeleton seem to be
especially auspicious for achieving such beneficial biological
9
activities. Moreover, the prenyl moiety could facilitate the
permeation of cell membranes and thus enhance bioavail-
1
0,11
ability.
Study comparing the pharmacokinetics of two
The new compound 2 was isolated by column chromatog-
raphy on silica gel and reversed-phase semipreparative HPLC
in the form of a pale yellow, amorphous powder. Its molecular
formula (C H O ) was determined by HRMS, which gave a
prenylated stilbenes, trans-arachidin-1 and trans-arachidin-3,
with their nonprenylated analogues, trans-piceatannol and
trans-resveratrol, respectively, suggested that improved bio-
availability of prenylated stilbenes could be achieved by their
1
9
18
3
−
11
pseudomolecular ion peak at m/z 293.1198 [M − H] , calcd
slower metabolism.
1
2
93.1178. The H NMR spectrum showed signals for trans-
Although stilbenoids have been described to be relatively
widespread in food and medicinal plants, the number of studies
describing the distribution of prenylated stilbenoids in the
plant kingdom is limited to date. Like prenylated flavonoids
and benzofurans, prenylated stilbenoids are typically isolated
from plants of the Moraceae family, such as Morus or
olefinic protons at δ 6.81 and 6.98 ppm (each 1H, d, J = 16.0
H
Hz) and six aromatic protons [δ 6.44 (1H, s), 6.60 (1H, ov),
H
6.84 (2H, d, J = 8.5 Hz), and 7.37 ppm (2H, d, J = 8.5 Hz)],
suggesting the presence of a trans-stilbenoid structure.
Furthermore, two cis-olefinic protons at δH 5.60 and 6.63
ppm (each 1H, d, J = 10.0 Hz) and two methyl groups at δH
1
2,13
Artocarpus species.
They have also been found in several
1
2
.44 ppm (6H, s) were observed, indicating the presence of a
,2-dimethylpyran group attached to one of the stilbenoid
14,15
Macaranga species, belonging to the family Euphorbiaceae.
Three species from Fabaceae, Arachis hypogea, Cajanus cajan,
and Lonchocarpus chiricanus, have also been described as
sources of prenylated stilbenoids.
reported their isolation from various exotic propolis samples.
16
rings. That the 2,2-dimethylpyran unit is attached to ring A of
the stilbenoid at positions C-2 and C-3 was deduced from the
heteronuclear multiple bond correlation (HMBC) spectrum.
17,18
Some studies have
19
The proton at δ 6.63 ppm (C-1″) correlated with a carbon
H
Recently, the in vitro anti-inflammatory potential of several
nonprenylated stilbenoids was described. The results indicate
that stilbenoids are agents able to target several inflammatory
NMR signal at δ 153.0 ppm (C-3) and the quaternary carbon
C
signal at δ 75.9 ppm (C-3″), suggesting a linkage to oxygen.
C
Accordingly, 2 was assigned as 7-[(1E)-2-(4-hydroxyphenyl)-
ethenyl]-2,2-dimethyl-2H-1-benzopyran-5-ol and was de-
scribed as obtained for the first time from a natural source.
Synthesis of Prenylated Stilbenoids. Prenylated stilbe-
noids 9−11 were prepared from 1-[(E)-2-(3-bromo-4-
methoxyphenyl)ethenyl]-3,5-dimethoxybenzene via direct lith-
iation followed by the addition of prenyl bromide in the
2
0
pathways. The activities of prenylated stilbenoids have been
16
15
reported as mostly antimicrobial and cytotoxic, but due to
their structural similarity to prenylated flavonoids, it is assumed
that they may also have anti-inflammatory activity. However,
the anti-inflammatory potential of prenylated stilbenoids has
not been reported yet. To fill this gap, the in vitro anti-
inflammatory effects of several prenylated stilbenoids obtained
from M. siamensis, A. altilis, and A. heterophyllus were evaluated.
Because the number of prenylated stilbenoids in plants is
limited, a method using prenylation was developed to extend
the series of compounds tested. Moreover, the synthesis of
22
presence of a cuprous bromide dimethyl sulfide complex.
The bromostilbenoid used for these syntheses was itself
synthesized from commercially available 3,5-dimethoxybenzoic
acid by standard methods. The carboxylic acid function was
reduced selectively using Red−Al and chlorinated with thionyl
B
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Scheme 1. Synthesis of a trans-3′-Bromo-3,5,4′-trimethoxystilbene Intermediate
Scheme 2. Prenylation Method Yielding a Variety of Products
2
3
chloride. The phosphonate derivative was prepared by the
Michaelis−Arbuzov method using triethylphosphite, because it
produced a better yield and purity than the Michaelis−Becker
Table 1. Calculated IC50 Values for COX-1, COX-2, and 5-
LOX and the COX-1/COX-2 Selectivity Ratio for Selected
Stilbenoid Compounds and Corresponding Standards, as
Determined from Serial Measurements in Triplicate
24
procedure with diethyl phosphite used in previous work. The
bromostilbenoid was prepared from the phosphonate inter-
mediate by the base-catalyzed Horner−Wadsworth−Emmons
reaction with 3-bromo-4-methoxybenzaldehyde (Scheme 1).
Three compounds were isolated following the prenylation
step: the expected trans-isomer of the prenylated stilbenoid
11), its cis-isomer (10), and a product of allylic rearrangement
9) (Scheme 2). The structures of all isolated compounds were
determined by spectroscopic methods, as described in the
Experimental Section.
compound/ COX-1 IC50
COX-2 IC
COX-1/
COX-2 ratio
5-LOX IC
[μM]
50
50
assay
[μM]
[μM]
25
2
4
6
7
8
13.3 ± 7.59
12.6 ± 4.98
0.4 ± 0.03
0.8 ± 0.41
0.9 ± 0.43
6.9 ± 2.74
>20
2.4 ± 0.44
inactive
0.9 ± 0.31
1.1 ± 0.54
0.9 ± 0.59
35.5 ± 12.57
5.7 ± 3.67
4.7 ± 1.39
14.7 ± 5.55
2.0 ± 1.11
0.36
0.07
0.17
0.06
3.4
(
(
ibuprofen
zileuton
0.1 ± 0.06
Inhibition of COX and LOX. Compounds 1−11 were
tested for their ability to inhibit the catalytic activity of
cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and
inhibitors. They inhibit activity of the inducible COX-2
isoform to a similar extent as the activity of constitutive
COX-1. COX-1 plays a cytoprotective role in the gastro-
intestinal system, and its suppression can result in erosions,
5
-lipoxygenase (5-LOX) in vitro in a cell-free assay using
human recombinant enzymes. Primary screening of activity
was performed at a concentration of 20 μM. Based on the
results of this screening, IC50 values were calculated for the
most promising compounds. Several of the test compounds
showed some activity against COX-1 and/or COX-2.
Compounds 6, 7, and 8 were the most active; the
submicromolar IC50 values for their anti-COX-1 effect were
greater than that of ibuprofen, used as a positive control (Table
). However, these compounds were much more selective for
COX-1 than for COX-2 (COX-1/COX-2 selectivity ratio
.06−0.17). Commonly used nonsteroidal anti-inflammatory
drugs (e.g., ibuprofen or naproxen) are nonselective COX
26
ulcers, bleeding, and perforation. Therefore, selective COX-2
inhibitors (with their side effects) were developed to avoid
such complications. However, selective inhibition of COX-1
could be useful as well, because COX-1-dependent prosta-
glandin synthesis is involved in pathological processes such as
atherosclerosis, endothelial dysfunction, neuroinflammation,
preterm labor, pain, and cancer. Therefore, the development of
1
27−29
selective COX-1 inhibitors was initiated.
0
The ability of certain stilbenoids to inhibit COX enzymes is
well known. However, information on the anti-COX activity of
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Figure 1. (A) Compound 6 shown in the crystal structure of 5-LOX. It binds to the residues Ile167, Thr403, Ile404, and Ala405, which act as
hydrogen bond interaction partners to the para-hydroxy group and to Tyr81, Tyr100, Glu622, and His624, forming interactions with one of the
meta-hydroxy groups on the other phenyl moiety. (B) Compound 3 in 5-LOX, forming an interaction between Ile404 and the methoxy group,
while the hydroxylated chromane interacts with Tyr100 and Tyr81. (C) Compound 7 in 5-LOX, which also binds to Tyr81, Thr403, and Ile404.
Red arrows signify interactions with a hydrogen bond acceptor; green arrows with a hydrogen bond donor. Yellow spheres represent hydrophobic
contact with the binding pocket.
prenylated stilbenoids is scarce. One study showed the
propensity of the prenylated stilbenoid carexane to decrease
the most active compound, docked with a score of 41.76. It
interacted with the two major anchor sites within the binding
pocket (see Figure 1A). One of these was formed by Ile167,
Thr403, Ile404, and Ala405, which serve as partners for
hydrogen bonding to the para-hydroxy group. The second site
has formed by Tyr81, Tyr100, Glu622, and His624, which
interact with one or the other meta-hydroxy group on the other
phenyl moiety. The other highly active compound, 8, was
found to bind to these two sites with a score of 39.82.
Compound 7, which only differs from 6 by having a methoxy
group instead of hydroxy substituent, also followed this
binding pattern, but with a significantly lower score of 27.27
(Figure 1C). This suggests that the hydroxy group fits into the
binding site slightly better. The inactive compound 4 would be
turned around in the binding pocket and hence did not interact
with either of the two sites. Compound 3 formed key
interactions and docked with a score of 39.72 (see Figure 1B).
Compound 5 is inverted and did not form any key interactions.
Cell Viability Testing. Before the anti-inflammatory
potential of a compound in a cellular system may be assessed,
its cytotoxicity should be determined. The THP-1 cell line was
used to evaluate the effect of each compound on cell viability.
The chemopreventive and cytotoxic potential of the test
stilbenoids obtained from M. siamensis for a panel of cell lines
30
the COX-2 expression in HspB-transfected AGS cells, and
31
three prenylated stilbenoids from A. dadah were selectively
active for COX-1 when compared to COX-2. Prenylation of a
stilbenoid does not seem to strongly increase anti-COX
activity, but the activities of prenylated stilbenoids 6, 7, and 8
against COX-1 are noteworthy. Further modifications of the
prenyl moiety, like O-cyclization, diminished the anti-COX/
LOX activity, as evident from the comparison of the activities
of 4 and 8 (Table 1). One report describing the activities of
trans-resveratrol, pterostilbene, and piceatannol against COX-
32
1
, COX-2, and 5-LOX has been presented, and another
20
relevant publication has appeared recently. The activities of
these nonprenylated stilbenoids were similar to those of the
prenylated stilbenoids described herein, with the exception of
pinostilbene, which showed a greater selectivity for COX-2
than for COX-1. Prenylated stilbenoids 2 and 6−8 showed also
promising anti-5-LOX effects (Table 1).
Molecular Docking Studies. To rationalize the activity of
the prenylated stilbenoids observed in vitro against LOX,
docking studies of selected compounds (2−8) were performed
to elucidate their interaction with the target enzyme. The
prenylated stilbenoids were docked into an allosteric binding
site of 5-LOX located between the catalytic and the membrane
binding domains that was identified for AA-noncompetitive
14
have been reported. Lipophilic substituents that increase the
ability of a compound to penetrate the cellular membranes and
interact with the internal cell environment may be responsible
33
inhibitors of a similar size by a previous study. Compound 6,
D
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3
4,35
for cytotoxic effects.
For previously untested compounds, a
lipopolysaccharide (LPS)-stimulated activation of NF-κB/AP-
1 was evaluated using THP1-XBlue-MD2-CD14 cells. The
stimulation of TLR4 receptors using this cell line by LPS
activates signaling cascades that lead to an increase in both NF-
κB and AP-1 transcription factors and the resulting production
of the reporter protein SEAP, the activity of which may be
WST-1 assay was performed, which measures cell viability on
the THP-1 model of human leukemic monocytes, and IC50
concentrations were determined (Table 2). Compounds 3, 4,
Table 2. Effects of Compounds 1−11 on Cell Viability,
Expressed as IC Values Calculated for THP-1 Cell Line
a
20
measured with a QUANTI-Blue assay. Most of the test
5
0
compounds attenuated the activities of NF-κB and AP-1 as
much as or even more than prednisone, a widely used anti-
inflammatory drug (Figure 2). Stilbenoids 6, 7, and 9 were the
most active compounds, and their inhibitory effects on the
activity of NF-κB/AP-1 were statistically significant and greater
compound
IC50 [μM]
compound
IC50 [μM]
1
2
3
4
5
6
>10
>10
1.8 ± 1.1
3.3 ± 1.0
4.9 ± 1.9
12.8 ± 2.4
7
8
9
10
11
2.7 ± 1.1
3.9 ± 1.1
>10
>10
>10
(
p < 0.0001) than that of prednisone (p < 0.001). Marked
activity, comparable to the effect of prednisone, was
demonstrated also by compound 2, newly isolated from A.
altilis, and the synthetic compound 10. The activity of
prenylated stilbenoids in mediating the activation of NF-κB/
AP-1 is reported herein for the first time.
a
The data are expressed as the means ± SE for serial measurements in
triplicate mode using at least five-point concentration curves.
The effects of compounds 1−11 on the secretion of pro-
inflammatory cytokines IL-1β and TNF-α, which are tran-
scriptionally controlled by NF-κB and AP-1, were assessed
using a phorbol myristate acetate (PMA)-differentiated human
macrophage model. The release of these two cytokines is
characteristic of certain chronic inflammatory disorders, such
as rheumatoid arthritis. The influence of compounds 1−11
on the LPS-triggered inflammatory-like response of THP-1
macrophages was evaluated using ELISA. The test compounds
were more effective against the secretion of TNF-α, where
their activity correlated with the effect observed on NF-κB/AP-
1, rather than IL-1β (Figures 3 and 4). Compounds 2 and 9
reduced the secretion of TNF-α as much as or more than the
reference drug prednisone. However, neither of these
compounds suppressed the release of IL-1β. Conversely,
compound 6 suppressed the secretion of IL-1β, but had only
a moderate effect on TNF-α. Compounds 5, 6, 7, and 10
5
, 7, and 8 affected the viability of THP-1 cells; their IC50
values were less than 10 μM. On the other hand, compounds 1
and 2 and synthetic stilbenoids 9, 10, and 11, with IC values
greater than 10 μM, showed relatively safer profiles. No
detailed structure−activity relationships were deduced, but
prenylation increased the cytotoxicity based on a comparison
of these results with those obtained for nonprenylated
stilbenoid analogues. Subsequent experiments showed that
at nontoxic concentration the test stilbenoids interacted well
with the intracellular environment of cells and affected their
metabolism. These assays formed the basis of a series of
bioactivity assays to evaluate the anti-inflammatory potentials
of compounds 1−11. The nontoxic concentration of 1 μM was
used for all compounds except 3, for which the concentration
employed was 0.1 μM.
5
0
36
20
Anti-inflammatory Potential in Cell-Based Models.
The ability of prenylated stilbenoids to attenuate the
Figure 2. Effects of compounds 1−11 on NF-κB/AP-1 activity. THP-1-XBlue-MD2-CD14 cells were pretreated for 1 h with the test compounds (1
μM dissolved in DMSO, 0.1 μM for compound 3), prednisone (P; 1 μM dissolved in DMSO), or the vehicle (DMSO) only (V and C).
Subsequently, LPS (1 μg/mL) was added [except for the control cells (C)] to trigger the activation of NF-κB/AP-1. After 24 h, the activity of NF-
κB/AP-1 was measured spectrophotometrically. The results are expressed as the means ± SE for five independent experiments measured in
triplicate (* indicates a significant difference in comparison with the vehicle-treated cells (V), p < 0.05, *** indicates p < 0.001, and ****
corresponds to p < 0.0001).
E
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Figure 3. Effects of compounds 1−11 on the secretion of TNF-α. THP-1 macrophages were pretreated with the test compounds [1 μM (0.1 μM
for compound 3), dissolved in DMSO], prednisone (P; 1 μM, dissolved in DMSO), or the vehicle (DMSO) only (V and C) for 1 h. Subsequently,
LPS (1 μg/mL) was added [except for the control cells (C)] to trigger the secretion of the pro-inflammatory cytokine TNF-α. After 24 h, the
amount of TNF-α was evaluated by ELISA. The results are expressed as the means ± SE for four independent experiments measured in triplicate
(** indicates a significant difference in comparison with the vehicle-treated cells (V), p < 0.01, *** indicates p < 0.001).
Figure 4. Effects of compounds 1−11 on the secretion of IL-1β. THP-1 macrophages were pretreated with the test compounds [1 μM (0.1 μM for
compound 3), dissolved in DMSO], prednisone (P; 1 μM, dissolved in DMSO), or the vehicle (DMSO) only (V and C) for 1 h. Subsequently,
LPS (1 μg/mL) was added [except for the control cells (C)] to trigger the secretion of the pro-inflammatory cytokine IL-1β. After 24 h, the
amount of IL-1β was evaluated by ELISA. The results are expressed as the means ± SE for three independent experiments measured in triplicate
(** indicates a significant difference in comparison with the vehicle-treated cells (V), p < 0.01).
inhibited the activity of NF-κB/AP-1, but reduced the
secretion of TNF-α only slightly. Synthetic compounds 10
and 11 moderately inhibited the secretion of IL-1β.
Compound 1 slightly increased the secretion of both pro-
inflammatory cytokines, what was congruent with its ability to
activate NF-κB/AP-1. Interestingly, compound 3 decreased the
activity of NF-κB/AP-1 moderately as well as the secretion of
TNF-α, but slightly increased that of IL-1β. This effect might
result from a further interaction of this compound with a post-
transcription or post-translation regulatory mechanism for
both. It is worth noting that because of its cytotoxic effect,
compound 3 was tested at a concentration of only 0.1 μM.
Hence, its observed effect on the production of TNF-α and IL-
1β could be caused by an unknown mechanism that also causes
the toxicity of this compound (Table 2).
Compound 2, which attenuated the activity of NF-κB/AP-1
and markedly reduced the subsequent NF-κB-controlled
secretion of TNF-α, was selected for more detailed analysis
of the events that occur upstream. The activation of NF-κB is
mediated by the phosphorylation of its inhibitory kappa B
37
(IκB) protein, which subsequently degrades. Activated NF-
κB subsequently translocates from the cytosol into the nucleus,
where it binds to DNA and controls the transcription of genes
implicated in inflammation, including those for TNF-α and IL-
F
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β. The activation of AP-1 is up-regulated by mitogen-
Article
1
potential and suggest that these compounds deserve additional
attention as potential multitargeting anti-inflammatory com-
pounds. However, more studies concerning the prenylation
and modification of the prenyl moiety would be done to
elucidate the exact influence of such modifications on anti-
inflammatory activity.
activated protein kinases (MAPKs). MAPK cascades, repre-
38
sented by the kinases p38, ERK, and JNK, can be triggered
by phosphorylation events in their activation loops during
inflammation or in response to extracellular stimuli, such as
LPS. Moreover, MAP kinases can participate in the regulation
of NF-κB transcriptional activity. Stilbenoid 2 showed an
inhibitory effect on NF-κB by reducing the degradation of
IκBα (Figure 5). Unlike the nonprenylated stilbenoids
EXPERIMENTAL SECTION
■
General Experimental Procedures. The reagents and solvents
used for isolation and synthesis were purchased from Sigma-Aldrich
and used as received, if not stated otherwise. Anhydrous solvents and
reagents were distilled prior to use. Melting points were measured
using a Boetius apparatus (Franz Kustner Nachf. KG) and were not
̈
̈
corrected. IR spectra were recorded on a SmartMIRacle ATR ZnSe
for a Nicolet Impact 410 FT-IR spectrometer (Thermo Scientific
Corp.). 1D and 2D NMR spectra were obtained on a Bruker Avance
III 400 Ultrashield spectrometer with a superconducting 400 MHz
magnet system or a JEOL ECZR-400 MHz instrument (JEOL Corp.).
All of the NMR measurements were done at 25 °C. Chemical shifts δ
are reported in parts per million (ppm), and J values in Hz. NMR
spectra were acquired in CD OD, CDCl , or DMSO-d with
3
3
6
tetramethylsilane (TMS) as an internal standard. The signal of
TMS or the residual solvent signals of CD OD, CDCl , or DMSO-d
3
3
6
were used for reference. Norell StandartSeries 5 mm NMR tubes were
utilized. MS analysis was performed on a Mariner ESITOF
spectrometer or an LTQ Orbitrap XL high-resolution mass
spectrometer (Thermo Fisher Scientific). Mass spectra were obtained
using electrospray ionization (ESI) in the positive- or negative-ion
mode. A solution of each test compound was prepared in a mixture of
methanol and water (50/50 v/v) with 0.2% ammonia and injected
directly into the spectrometer. The isolation process, progress of the
reactions, and purity of intermediates were monitored using TLC
plates with normal-phase silica gel 60 F254 (Merck). HPLC analysis
was performed using a DionexUltiMate 3000 liquid chromatography
pump coupled to a DionexUltiMate 3000 diode array detector. Merck
Kieselgel 60 silica gel (70−230 mesh particle size) was used for
column chromatography. All fractions were screened on silica gel thin-
layer chromatography (TLC) plates (225 μm thickness, 60 A silica gel
medium) with the compounds detected using fluorescent light.
Plant Material. The wood and bark of Artocarpus altilis were
collected from the Botanical Garden Teplice, Teplice, Czech
Republic. The original plant in Botanical Garden Teplice was
acquired as A. altilis from the Thai company Meyer & Mongkol in
Figure 5. Effects of stilbenoid 2 on the LPS-stimulated NF-κB and
IκBα signaling cascade using the THP1-XBlue-MD2-CD14 cell line.
The cells were pretreated with the compound (1 μM) or the vehicle
(
DMSO) only. After 1 h of incubation, the inflammatory response
was triggered by LPS [except for the control cells (CTRL)]. The level
of proteins was detected in cell lysates by Western blot and
immunodetection after 30 min. Each blot represents the results of
three independent experiments.
piceatannol, pinostilbene, and thunalbene, which diminish
41
2
002. The plant was identified based on Flora Malesiana by Dr.
the activity of NF-κB/AP-1 by reducing the phosphorylation of
Filip Zpurny (Botanical Garden Teplice), and a voucher specimen (A.
́
20
MAPKs p38, ERK1/2, and JNK, compound 2 did not affect
the pathways of MAPKs. It is indicated that 2 specifically
inhibits the NF-κB pathway via attenuation of IκB degradation.
This ability is relatively rare among natural compounds, and it
was described, for example, for the diterpenoid incensole
acetate, isolated from Boswellia resin, a well-known anti-
altilis/2013) has been deposited in the herbarium of the Faculty of
Pharmacy, University of Veterinary and Pharmaceutical Sciences
Brno.
Extraction and Isolation. The wood (both heartwood and
sapwood) and bark from A. altilis was air-dried until the weight was
constant (2287 g) and coarsely ground. Extraction was performed by
maceration in 95% ethanol (3 × 7 L) at room temperature. The
ethanolic extract was concentrated under vacuum at 40 °C using a
rotary evaporator to afford a residue of 70.86 g. This residue was
suspended in 80% MeOH and partitioned with petroleum ether, to
produce a petroleum ether portion (26.84 g). The 80% MeOH layer
39
inflammatory natural drug. Observed effects could be caused
by inhibition of either the IκB kinase (IKK) cascade or
40
proteasome, which is responsible for IκB clearance. However,
any other mechanisms of action could not be excluded.
Prenylated stilbenoids showed promising anti-inflammatory
potential. Both natural and synthetic compounds with this
structure have shown this type of activity. A synthetic method
for achieving prenylation with satisfying yields would certainly
lead to the discovery of many more active substances. The
direct inhibition of the activity of NF-κB/AP-1 and/or the
IκBα signaling cascade, the inhibition of the secretions of
TNF-α and IL-1β, and the inhibition of the COX and LOX
enzymes, including interactions with the binding pocket of 5-
LOX, could contribute to the overall effects of these
compounds. The present study has shown that prenylation
can favorably affect activity and increase anti-inflammatory
was then concentrated under vacuum, redissolved in CHCl , and
3
partitioned successively with H O and EtOAc to afford CHCl (14.2
2
3
g), H O (26.65 g), and EtOAc (3.16 g) portions, respectively. The
2
chloroform-soluble part was selected for subsequent column
chromatography on silica gel (elution with C H −EtOAc in different
6
6
proportions). All necessary personal protective equipment and
principles of personal hygiene were used, as stated in the Safety
Data Sheet. All work with C H was carried out in a laboratory hood
6
6
with permanent air extraction. Column chromatography was followed
by TLC screening and purification using preparative HPLC on an RP-
Amide column (Ascentis RP-amide 250 × 10 mm, particle size 5 μm,
Supelco) to yield compound 2 (for a scheme of separation see the
Supporting Information).
G
DOI: 10.1021/acs.jnatprod.9b00081
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
7
-[(1E)-2-(4-Hydroxyphenyl)ethenyl]-2,2-dimethyl-2H-1-benzo-
dimethoxybenzylphosphonate and 3.45 g (0.0161 mol) of 3-bromo-4-
methoxybenzaldehyde in dry THF (20 mL) to a solution of 1.77 g
(0.0161 mol) of sodium tert-pentoxide in dry THF (100 mL), with
the reaction mixture refluxed and stirred under an inert atmosphere.
The reaction was monitored by TLC (ethyl acetate−hexane, 1:2).
Water was (20 mL) added, and the mixture was extracted into ethyl
acetate three times (3 × 50 mL). The organic layer was washed with
1
pyran-5-ol (2): pale yellow, amorphous powder; H NMR (MeOD-
d4, 400 MHz, 303 K) δH 7.37 (2H, d, J = 8.5 Hz, H-2′, H-6′), 6.98
(
1H, d, J = 16.0 Hz, H-7′), 6.84 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.81
1H, d, J = 16.0 Hz, H-7), 6.63 (1H, d, J = 10.0 Hz, H-1″), 6.60 (1H,
(
ov, H-6), 6.44 (1H, s, H-4), 5.60 (1H, d, J = 10.0 Hz, H-2″), 1.44
13
(
4
1
(
2
(
6H, s, CH ); C NMR (MeOD-d , 101 MHz, 303 K) δ 154.9 (C-
3 4 C
′), 153.0 (C-3), 150.0 (C-5), 130.0 (C-1′), 128.9 (C-7′, C-2″),
27.8 (C-2′, C-6′), 126.0 (C-7), 125.7 (C-2), 116.1 (C-1″), 115.4
brine (50 mL) and dried over anhydrous MgSO . The solvent was
4
evaporated under reduced pressure. Recrystallization from ethanol
C-3′, C-5′), 108.2 (C-1), 106.7 (C-6), 105.5 (C-4), 75.9 (C-3″),
gave 2.87 g (51%) of trans-3′-bromo-3,4′,5-trimethoxystilbene as
−
1
4
8.1 (C-4″, C-5″); HRESIMS (negative) m/z 293.1198 [M − H]
colorless crystals: H NMR (400 MHz, CDCl ) δ 7.74 (1H, d, J =
3
3
,4
3
calcd for C H O , 293.1178).
2.0 Hz), 7.39 (1H, dd, J = 8.6, 1.9 Hz), 6.95 (1H, d, J = 16.2 Hz),
6.88 (1H, d, J = 16.2 Hz), 6.87 (1H, d, J = 8.6 Hz), 6.65 (2H, d, J =
2.2 Hz), 6.40 (1H, t, J = 2.2 Hz), 3.92 (3H, s), 3.84 (6H, s);
19
18
3
3
3
4
Compound 1 from A. heterophyllus and compounds 3−8 from M.
siamensis were isolated and identified according to published
procedures.
4
13
C
14,21
NMR (101 MHz, CDCl ) δ 161.0, 155.4, 139.2, 131.4, 131.0, 127.9,
3
Synthesis of Compounds 9−11. The synthesis of compounds
−11 involved first synthesizing the reactants required (see Scheme
). To prepare 3,5-dimethoxybenzyl alcohol, 27.7 g (0.0892 mol) of
0% Red−Al was added dropwise to a mixture of 5.00 g (0.0274 mol)
127.2, 126.9, 112.1, 111.9, 104.4, 99.9, 56.3, 55.3.
2
2
9
1
6
Prenylation Procedure. A 2.30 g (6.5861 mmol) aliquot of
trans 3′-bromo-3,5,4′-trimethoxystilbene was dissolved in dry THF
(100 mL) and cooled to −80 °C. Exactly 4.0 mL (9.8792 mmol) of a
2.5 M solution of n-butyllithium was added dropwise, and the solution
was stirred for 1 h at −80 °C. The reaction mixture was then warmed
to −40 °C, 0.68 g (3.2931 mmol) of CuBr·DMS at −40 °C was added
all at once, and the mixture was stirred at this temperature for 1 h.
The reaction mixture was again cooled to −80 °C, with 1.47 g
(9.8792 mmol) of prenyl bromide added dropwise, and the resulting
mixture was then stirred at this temperature for 1 h. HPLC analysis
showed that the reaction yielded 7.64% trans-3′-(2-methylbut-3-en-2-
yl)-3,4′,5-trimethoxystilbene 9, 10.26% cis-3′-prenyl-3,4′,5-trimethox-
ystilbene 10, 63.42% trans-3′-prenyl-3,5,4′-trimethoxystilbene (11),
and 18.69% trans-3, 5,4′-trimethoxystilbene. A saturated solution of
ammonium chloride (50 mL) was slowly added to the reaction
mixture, which was then extracted three times with diethyl ether (3 ×
100 mL). The organic layer was washed with 1 M HCl (50 mL) and
of 3,5-dimethoxybenzoic acid in 100 mL of dry toluene. The reaction
mixture was held at 80 °C and stirred for 1.5 h under an atmosphere
of argon. The reaction was monitored by TLC (ethyl acetate−hexane,
2
:3). When the starting material had disappeared, the mixture was
cooled to 0 °C and 75 mL of water was added slowly. The reaction
mixture was then acidified with 1 M HCl and extracted three times
with toluene (3 × 100 mL). The organic layer was separated, washed
twice with water, and dried with anhydrous MgSO . The solvent was
4
evaporated under reduced pressure to produce 3.79 g (82%) of white
1
crystals of 3,5-dimethoxybenzyl alcohol: H NMR (400 MHz,
4
4
DMSO-d ) δ 6.48 (2H, d, J = 2.3 Hz), 6.34 (1H, t, J = 2.3 Hz),
6
3
3
13
5
.17 (1H, t, J = 6.0 Hz), 4.43 (2H, d, J = 6.0 Hz), 3.72 (6H, s);
C
NMR (101 MHz, DMSO-d ) δ 160.3, 145.2, 103.9, 98.5, 62.8, 55.0.
6
25
3,5-Dimethoxybenzyl chloride was produced by dissolving 3.79 g
(0.0225 mol) of 3,5-dimethoxybenzyl alcohol in dry diethyl ether (70
dried over anhydrous MgSO . The solvent was evaporated under
4
mL), cooling this mixture in an ice bath, and adding dimethylforma-
reduced pressure, and the crude product was purified using a short
silica gel column (10% ethyl acetate in hexane) to produce 1.46 g
(67%) of a mixture of products.
mide (DMF) (1 mL), followed by a mixture of SOCl [5.36 g (0.0451
2
mol)] in dry diethyl ether (10 mL). The reaction mixture at 25 °C
was stirred overnight under a condenser equipped with a calcium
chloride closure to exclude moisture. The reaction was monitored by
TLC (ethyl acetate−hexane, 2:3). When the starting material had
disappeared, the mixture was cooled and water (50 mL) was added
slowly. It was then extracted three times with diethyl ether (3 × 50
mL). The organic layer was washed with water (50 mL) and then
This mixture of products was dissolved in MeOH and separated by
semipreparative HPLC using a Dionex UltiMate 3000 instrument
with an Ascentis RP-amide (250 mm × 10 mm, particle size 5 μm)
column (Supelco) and gradient elution with a MeOH and H O
2
mixture. The flow rate was 5 mL/min, the column temperature was
40 °C, and the separation was monitored using UV detection at 254
and 350 nm. The mobile phase was removed using a rotavapor
followed by lyophilization. A yellowish precipitate was obtained.
trans-3′-(2-Methylbut-3-en-2-yl)-3,5,4′-trimethoxystilbene (9):
brine (50 mL) and dried with anhydrous MgSO . The solvent was
4
evaporated under reduced pressure, and the product was purified by
column chromatography (ethyl acetate−hexane, 2:3) to yield 3.08 g
1
1
4
(
73%) of yellowish crystals of 3,5-dimethoxybenzyl chloride:
H
H NMR (400 MHz, CDCl ) δ 7.44 (1H, d, J = 2.3 Hz,), 7.38
3
4
4
3,4
3
3
NMR (400 MHz, CDCl ) δ 6.55 (2H, d, J = 2.3 Hz), 6.40 (1H, t, J
2.3 Hz), 4.53 (2H, s), 3.81 (6H, s); C NMR (101 MHz, CDCl ) δ
(1H, dd, J = 8.2, 2.3 Hz), 7.06 (1H, d, J = 16.0 Hz), 6.90 (1H, d, J
3
1
3
3
4
=
= 16.0 Hz), 6.88 (1H, d, J = 8.2 Hz), 6.67 (2H, d, J = 2.3 Hz,), 6.38
4 3,3
3
1
60.9, 139.5, 106.4, 100.4, 55.4, 46.3.
Diethyl 3,5-dimethoxybenzylphosphonate was prepared by
mixing 2.70 g (0.0145 mol) of 3,5-dimethoxybenzyl chloride, 1.00 g
0.01445 mol) of triethyl phosphite, and 0.20 g of potassium iodide
(1H, t, J = 2.1 Hz), 6.23 (1H, dd, J = 17.4, 11.0 Hz), 5.02−4.94
2
5
13
(1H, m), 3.86−3.80 (9H, m), 1.50 (6H, s); C NMR (101 MHz,
CDCl ) δ 161.0, 158.2, 147.9, 139.8, 136.7, 129.3, 126.3, 125.8, 125.6,
3
−
1
(
112.1, 110.0, 109.8, 104.3, 99.6, 55.4, 55.2, 40.6, 27.2; IR νmax (cm )
2928, 1593, 1499, 1460, 1253, 1204, 1155; HRMS m/z [M + H]+
and stirring for 3 h at 130 °C under a condenser equipped with a
calcium chloride closure to exclude moisture. The same amounts of
triethyl phosphite and potassium iodide were again added, and the
mixture was again stirred for 3 h at 130 °C. The reaction was
monitored by TLC (ethyl acetate−hexane, 2:3). The reaction mixture
was cooled, water (10 mL) was added, and the mixture was extracted
three times with ethyl acetate (3 × 10 mL). The organic layer was
339.1961 (calcd for C22
H O 339.1955).
27 3
1
cis-3′-Prenyl-3,5,4′-trimethoxystilbene (10): H NMR (400 MHz,
3
CDCl ) δ 7.12−7.05 (2H, m), 6.72 (1H, d, J = 8.2 Hz), 6.52 (1H, d,
4 3
3
3
J = 11.9 Hz), 6.45 (2H, d, J = 2.3 Hz) 6.42 (1H, d, J = 11.9 Hz),
4
6.32 (1H, t, J = 2.3 Hz), 5.20−5.13 (1H, m), 3.81−3.78 (3H, m),
3
3.67 (6H, s), 3.21 (2H, d, J = 7.3 Hz), 1.68 (3H, s), 1.62 (3H, s);
1
3
washed with brine (10 mL) and dried over anhydrous MgSO . The
C NMR (101 MHz, CDCl ) δ 160.5, 158.5, 139.7, 132.4, 130.5,
4
3
solvent was evaporated under reduced pressure to produce 4.10 g
130.1, 129.6, 129.2, 128.3, 127.7, 122.2, 109.8, 106.5, 104.3, 99.6,
−
1
(
98%) of yellow oil, diethyl 3,5-dimethoxybenzylphosphonate, which
55.4, 55.2, 28.2, 25.7, 17.59; IR νmax (cm ) 2930, 1589, 1498, 1453,
1332, 1250, 1202, 1152, 1121, 1063, 1031, 835, 782; HRMS m/z [M
1
was used in the next step without purification: H NMR (400 MHz,
4
4
+
CDCl ) δ 6.45 (2H, t, J = 2.3 Hz,), 6.35 (1H, q, J = 2.1 Hz), 4.07−
+ H] 339.1960 (calcd for C H O 339.1955).
3
22 27
3
2
trans-3′-Prenyl-3,5,4′-trimethoxystilbene (11): 1H NMR (400
3
.99 (4H, m), 3.77 (6H, s), 3.09 (2H, d, J = 22.0 Hz), 1.26 (6H, t,
PH
3
13
3
J = 7.1 Hz); C NMR (101 MHz, CDCl ) δ 160.7, 133.5, 107.8,
MHz, CDCl ) δ 7.36−7.29 (2H, m), 7.04 (1H, d, J = 16.0 Hz), 6.90
3
3
3
3
4
9
9.0, 62.2, 55.2, 33.9, 16.4.
trans-3′-Bromo-3,5,4′-trimethoxystilbene was prepared by drop-
(1H, d, J = 16.0 Hz), 6.84 (1H, d, J = 8.2 Hz), 6.67 (2H, d, J = 2.3
24
4
Hz,), 6.38 (1H, t, J = 2.3 Hz), 5.38−5.30 (1H, m), 3.90−3.81 (9H,
m), 3.35 (2H, d, J = 7.3 Hz), 1.78 (3H, s), 1.75 (3H, s); C NMR
3
13
wise addition of a solution of 4.63 g (0.0161 mol) of diethyl 3,5-
H
DOI: 10.1021/acs.jnatprod.9b00081
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
101 MHz, CDCl ) δ 160.9, 157.2, 139.8, 132.6, 130.3, 129.5, 129.1,
and the chemiluminescence was measured using a Syngene PXi4
imaging system (Cambridge, UK).
3
1
1
1
3
27.6, 126.2, 125.4, 122.3, 110.3, 104.2, 99.5, 55.4, 55.3, 28.5, 25.8,
−
1
7.8; IR νmax (cm ) 2931, 1593, 1500, 1459, 1253, 1204, 1154, 1066,
COX-1 and COX-2 Assays. The inhibition of COX enzymes was
+
20
032, 860, 827; HRMS m/z [M + H] 339.1955 (calcd for C H O
determined in the same manner as described previously. Samples
2
2
27
3
39.1955).
were dissolved in DMSO, pure DMSO was used as blank, and
ibuprofen was used as positive control. COX-1 from ram seminal
vesicles and human recombinant COX-2 (Sigma-Aldrich) were used,
Cell Culture and Differentiation to Macrophages. The THP-
human monocytic leukemia cell line and THP-1-XBlue-MD2-CD14
1
cells were purchased from the ECACC (Salisbury, UK) and Invivogen
San Diego, CA, USA), respectively. Both cell lines were cultured in
RPMI 1640 medium (Biosera, France) supplemented with antibiotics
100 U/mL penicillin and 100 mg/mL streptomycin (Biosera)] and
0% fetal bovine serum (HyClone, Logan, UT, USA) at 37 °C. To
and the concentration of PGE
2
created in catalytic reaction from
(
arachidonic acid was quantified with the aid of a prostaglandin E
2
ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA), followed by a
measurement of OD at 405 nm using a Tecan Infinite M200
̈
microplate reader (Tecan Group, Mannedorf, Switzerland). The
results were expressed as a percentage of inhibition compared to a
blank.
[
1
differentiate the THP-1 monocytes to macrophages, cells were split
into 96-well microtiter plates at a concentration of 5 × 10 cells/well
and stimulated by phorbol myristate acetate, as described
5
5
-LOX Assay. The inhibition of human recombinant 5-LOX
35,42
enzyme (Cayman Chemicals, Ann Arbor, MI, USA) was assessed as
the potential to reduce the synthesis of leukotriene B (LTB ) created
previously
with some modifications.
Cell Viability Testing. Undifferentiated floating THP-1 cells were
4
4
20
5
in catalytic reaction from arachidonic acid. As described previously,
transferred into serum-free medium at a density of 5 × 10 cells/well
samples were dissolved in DMSO, pure DMSO was used as blank, and
zileuton (Farmak a.s., Olomouc, Czech Republic) was used as positive
control. LTB was quantified using a leukotriene B ELISA kit (Enzo
in 96-well plates. After 2 h, the test compounds dissolved in DMSO
were added at increasing concentrations (0.125−20 μM). Measure-
ments were taken after a 24 h incubation with each of the test
compounds. Cell viability was measured using the Cell Proliferation
Reagent kit WST-1 (Roche Diagnostics, Basel, Switzerland),
according to the manufacturer’s instructions. The resultant viability
curves were used to calculate the IC50 values using four parameters
logistic (4PL) analysis.
4
4
Life Sciences); measurements and analyses were the same as for the
COX assays.
Molecular Docking. The docking simulations on 5-LOX were
carried out using the program Gold Suite 5.2 (CCDC, www.ccdc.cam.
ac.uk, 2015). Docking poses and protein−ligand interactions were
visualized in Ligandscout 4.1 (Inte:Ligand, http://www.inteligand.
com/, 2017). The docking simulation for 5-LOX was carried out on a
Evaluation of Cytokine Secretion. Differentiated THP-1
macrophages were pretreated for 1 h with test compounds 1, 2, and
4
44
structure based on pdb entry 3O8Y, which was mutated virtually to
−11 at a nontoxic concentration of 1 μM (0.1 μM for compound 3)
wild-type 5-LOX and energetically minimized in Discovery Studio 4.0
or 1 μM prednisone dissolved in DMSO or DMSO alone (the
vehicle). An inflammatory-like response was triggered by adding 1
μg/mL of LPS isolated from Escherichia coli 0111:B4 (Sigma-Aldrich).
The control cells were cultured without LPS treatment. After 24 h, the
medium was collected and the concentration of TNF-α was measured
using a human TNFα ELISA kit (Diaclone, Besanc o̧ n, France),
according to the manufacturer’s instructions. Experiments were
performed in triplicates and repeated at least three times.
(
Biovia, San Diego, CA, USA, 2015). The binding site was defined
within an 8 Å radius around CG2 of ILE167. ChemScore Kinase was
selected as the configuration template, and CHEMPLP as the scoring
function.
Statistical Evaluation. Statistical analyses were carried out using
GraphPad Prism 6.01 software (San Diego, CA, USA). The data were
graphed as the means ± SEM. Comparison between groups was made
using a one-way ANOVA test, followed by a Bonferroni’s multiple
comparisons post hoc test.
Determination of the NF-κB/AP-1 Activity. The activity of NF-
κB/AP-1 was evaluated using the THP-1-XBlue-MD2-CD14 cell line
as described previously, with slight modifications. Briefly, cells (5 ×
1
43
5
ASSOCIATED CONTENT
0 /well in 96-well plates) were pretreated with test compounds or
■
prednisone at a concentration of 1 μM (0.1 μM for compound 3).
After a 1 h incubation, cells were stimulated by LPS (1 μg/mL). After
2
Quanti-Blue reagent (Invivogen, San Diego, CA, USA) according to
the standard protocol, and measurement of OD at 655 nm was
performed in a Fluostar Omega microplate reader (BMG Labtech,
Ortenberg, Germany) and compared with a vehicle (100%).
Signaling Pathway Analysis. The level of IκB and mitogen-
activated protein (MAP) kinases p38, ERK1/2, and JNK was
determined on the THP-1-XBlue-MD2-CD14 cell line, in a similar
way to that described previously. Briefly, cells were pretreated with
test compound 2 at a concentration of 1 μM and stimulated with 1
μg/mL LPS 1 h later. After 30 min, the cells were collected and cell
lysates were prepared. The equal amounts of proteins (9 μg) were
separated in 12% PAGE gel, transferred to PVDF membranes,
blocked in 5% bovine serum albumin, and incubated with primary
mouse anti-IκB-α antibody and primary rabbit anti-phospho-SAPK/
JNK, anti-p44/42 MAPK (ERK1/2), anti-phospho-p44/42 MAPK
S
*
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00081.
4 h of incubation, the activity of NF-κB/AP-1 was determined using
Figures illustrating the NMR spectra (Figures S1−S14)
(PDF)
AUTHOR INFORMATION
20
■
Corresponding Authors
*E-mail: veronika.lelakova@gmail.com. Tel: +420773105951.
*
*
E-mail: Gazdova.Marketa@seznam.cz. Tel: +420541562834.
E-mail: karel.mejkal@post.cz. Tel: +420724243643.
ORCID
Pavel Bobal: 0000-0002-8429-3810
́
(
(
ERK1/2), anti-p38 MAPK, and anti-phospho-p38 MAPK antibodies
Cell Signaling, Danvers, MA, USA; product nos. 4814, 4668, 4695,
̌
Premysl Landa: 0000-0003-0364-2247
Daniela Schuster: 0000-0002-9933-8938
Gang Ren: 0000-0001-9836-4424
4
1
370, 9212, and 4511, respectively) at dilutions of 1:500 (anti-IκB-α),
:2000 (anti-phospho-p44/42 MAPK), or 1:1000 (the other
̌
Karel Smejkal: 0000-0002-4336-7924
antibodies). Two other primary mouse antibodies were used, anti-β-
actin (Abcam, Cambridge, UK; product No. 8226) at a dilution of
Author Contributions
J.H. and V.L. contributed equally.
1
:5000 and anti-SAPK/JNK (Sigma-Aldrich, product no.
▲
SAB4200176) at a dilution of 1:1000. Appropriate secondary IgG
antibodies (1:2000; Sigma-Aldrich, products Nos. A0168 and A0545)
were applied to the membranes, followed by ECL reagent (Bio-Rad),
Notes
The authors declare no competing financial interest.
I
DOI: 10.1021/acs.jnatprod.9b00081
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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28) Liedtke, A. J.; Crews, B. C.; Daniel, C. M.; Blobaum, A. L.;
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