Evaluation of Dual 5-Lipoxygenase/Microsomal Prostaglandin E2 Synthase-1 Inhibitory Effect of Natural and Synthetic Acronychia-Type Isoprenylated Acetophenones

Article abstract of DOI:10.1021/acs.jnatprod.6b01008

Among the pathways responsible for the development of inflammatory responses, the cyclooxygenase and lipoxygenase pathways are among the most important ones. Two key enzymes, namely, 5-LO and mPGES-1, are involved in the biosynthesis of leukotrienes and prostaglandins, respectively, which are considered attractive therapeutic targets, so their dual inhibition might be an effective strategy to control inflammatory deregulation. Several natural products have been identified as 5-LO inhibitors, with some also being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone dimers from Acronychia pedunculata have been identified for their dual inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of this family of natural products, the synthesis and biological evaluation of analogues are presented. The results show the ability of the natural and synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The potency of the most active compound (10) has been evaluated in vivo in an acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton used as a positive control.

Full text of DOI:10.1021/acs.jnatprod.6b01008

Article
pubs.acs.org/jnp
Evaluation of Dual 5‑Lipoxygenase/Microsomal Prostaglandin E2
Synthase‑1 Inhibitory Effect of Natural and Synthetic Acronychia-
Type Isoprenylated Acetophenones
Alexandra Svouraki,† Ulrike Garscha,‡ Eirini Kouloura,† Simona Pace,‡ Carlo Pergola,‡ Verena Krauth,‡
Antonietta Rossi,§ Lidia Sautebin,§ Maria Halabalaki,† Oliver Werz,*,‡ Nicolas Gaboriaud-Kolar,†
and Alexios-Leandros Skaltsounis*,†
†Department of Pharmacognosy and Natural Products Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis
Zografou, GR-15771, Athens, Greece
‡Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743 Jena, Germany
§Department of Pharmacy, University of Naples Federico II, Naples, Italy
S
* Supporting Information
ABSTRACT: Among the pathways responsible for the development of
inflammatory responses, the cyclooxygenase and lipoxygenase pathways are
among the most important ones. Two key enzymes, namely, 5-LO and mPGES-
1, are involved in the biosynthesis of leukotrienes and prostaglandins,
respectively, which are considered attractive therapeutic targets, so their dual
inhibition might be an effective strategy to control inflammatory deregulation.
Several natural products have been identified as 5-LO inhibitors, with some also
being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone
dimers from Acronychia pedunculata have been identified for their dual
inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of
this family of natural products, the synthesis and biological evaluation of
analogues are presented. The results show the ability of the natural and
synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The
potency of the most active compound (10) has been evaluated in vivo in an
acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton
used as a positive control.
nflammation is the immune response of the human body
when faced with any tissue injury. Deregulation of the
treatment.6 Despite continuous efforts for developing 5-LO
inhibitors,7 no LT biosynthesis inhibitor other than zileuton has
been marketed due to toxic side effects or to lack of efficacy in
vivo. Recent data suggest that anti-inflammatory drugs with
dual or multiple targets possess higher efficacy accompanied by
reduced severity of side effects.2 In this context, initially, dual
inhibition of 5-LO and COX led to greater anti-inflammatory
efficiency and lower gastric toxicity.8 More recently, dual
inhibitors of 5-LO and microsomal prostaglandin E2 synthase-1
(mPGES-1) were proposed as a promising class of anti-
inflammatory agents that hamper cardiovascular toxicity that
was observed previously.9
Among the identified 5-LO inhibitors, several classes of
natural products such as flavonoids (epi-gallocatechin, Figure
1), phenols (curcumin, Figure 1), coumarins, and terpenoids
have been highlighted as promising molecules.10 Several of
these natural inhibitors possess unsaturated aliphatic chains
I
inflammatory process may lead to severe chronic inflammation-
related diseases such as asthma, rheumatism, atherosclerosis, or
even cancer. There are several pro-inflammatory mediators that
include eicosanoids, which are lipid mediators produced from
arachidonic acid (AA).1 Two main enzymatic routes are
involved in eicosanoid biosynthesis, namely, the cyclooxygenase
(COX) pathway and the 5-lipoxygenase (5-LO) pathway.2
While the first one triggers the biosynthesis of prostaglandins
(PGs) including PGE2 linked with pain, fever, and arthritis, the
5-LO pathway is involved with the biosynthesis of leukotrienes
(LTs). LTs have potent pro-inflammatory properties, and
elevated concentrations of LTB4 and cysteinyl-leukotrienes
(Cys-LTs) have been detected in asthmatic subjects.3,4 5-LO is
a nonheme iron-containing enzyme that catalyzes the first two
steps in the biosynthesis of LTs from AA.5 Suppression of LT
production through inhibition of 5-LO is thus considered an
attractive strategy for the development of therapeutics,
highlighting 5-LO as a drug target. Zileuton, a hydroxyurea
derivative with a benzothiophene core, is the only 5-LO
inhibitor introduced to the market and is prescribed for asthma
Special Issue: Special Issue in Honor of Phil Crews
Received: November 4, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b01008
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Natural product inhibitors of 5-LO and mPEGS-1/5-LO.
Figure 2. Acronychia-type acetophenones from A. pedunculata.
(prenyl, geranyl, or isoprenyl), such as hyperforin or garcinol
(Figure 1). More recently, indirubin was highlighted as a new
chemotype for inhibition of 5-LO through unique binding in
the ATP pocket of the enzyme.11 Finally, phloroglucinol-
derived molecules were shown to inhibit 5-LO as well, such as
myrtucommulone A isolated from Myrtus communis, hyperforin
isolated from Hypericum perforatum,12,13 and arzanol isolated
from Helichrysum italicum (Figure 1).6,14,15 Interestingly,
myrtucommulone A, hyperforin,12 and arzanol also inhibit
mPGES-1,15,16 and myrtucommulone A was characterized as
the first natural product that inhibits both mPGES-1 and 5-
LO.1 To date, a number of natural compounds and synthetic
analogues have indicated advantageous structural features based
on SAR studies with enhanced potency as dual mPGES-1 and
5-LO inhibitors,17 such as benzoquinones with aliphatic
chains,18 prenylated chalcones, and flavonoids.12,15 Overall,
most of the identified natural products acting as 5-LO
inhibitors are phenolic substructures or phloroglucinol-based
molecules possessing prenylated or modified prenylated chains
with possible aliphatic connections between each phlorogluci-
nol moiety, clearly indicating a structural feature for the
development of this type of inhibition.
Within the context of a continuation of work aimed at
discovering novel natural dual mPGES-1/5-LO inhibitors, the
trunk bark of Acronychia pedunculata (L.) Miq., a tree growing
in Southeast Asia, was selected to be investigated.19 The genus
B
DOI: 10.1021/acs.jnatprod.6b01008
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
Acronychia, belonging to the Rutaceae family, is composed of 44
species, some of which have a long traditional use in Asian
ethnomedicine, notably for treating microbial or viral
infections.18 Moreover, the aerial parts of certain species are
used as food ingredients, whereas the leaves and flowers are
incorporated in cosmetic preparations.18 Regarding A. peduncu-
lata, there are limited data in the literature regarding its
beneficial health effects, but some reports refer to its traditional
use against chronic inflammatory diseases such as asthma and
rheumatism.20,21
Scheme 1. Synthesis of Phloroglucinol Monomers
Alkaloids, triterpenoids, and acetophenones are the charac-
teristic chemical classes of the genus Acronychia. Particularly,
acetophenones encompass certain structural features typical of
this genus, and they are named commonly Acronychia-type
acetophenones, while most of them have been recently
reported as new natural products from A. pedunculata (Figure
2).19 Structurewise, this class of compounds can be divided into
monomers (one phloroglucinol unit)22,23 and fully substituted
dimers (two phloroglucinol units).19,20 The occurrence of
isoprenyl and modified isoprenyl chains represents an integral
part of this chemical group. They commonly incorporate an
additional ring biosynthetically originated from the ring closure
of an isoprenyl side chain (Figure 2). Depending on the
orientation of the fusion relative to the isobutyl chain,
Acronychia-type acetophenones occur as A-type (ring closure
on the phenol ortho to the chain) and B-type (ring closure on
the phenol para to the chain). Taken together, based on the
occurrence of this structural motif with the long traditional use
of A. pedunculata for the treatment of inflammation-related
diseases, it becomes clear that Acronychia-type acetophenones
could serve as a promising scaffold for 5-LO or dual 5-LO and
mPEGS-1 inhibition.
In order to gain better insight into the structure−activity
relationships (SAR) of Acronychia-type acetophenones, various
mono- and dimeric analogues of these were synthesized.
Among them, the first synthesis of demethylacrovestone was
carried out. All synthetic analogues obtained, together with the
isolated natural Acronychia-type acetophenones, were evaluated
for mPGES-1 and 5-LO inhibition. Finally, the potency of the
most active molecule (10) has been evaluated in vivo using a
mouse model of acute inflammation (i.e., zymosan-induced
peritonitis).
a
Reagents and conditions: (a) BF3-Et2O, 1,4-dioxane, rt, 12 h; (b)
geranyl bromide, K2CO3, acetone, rt, 18 h; (c) K2CO3, acetone, 40 °C,
20 h; (d) CH2Cl2, 40 °C, 24 h.
In the case of monomers possessing additional rings such as a
furan or pyran moiety, numerous methodologies have been
developed for obtaining these naturally widespread hetero-
cycles. The furan moiety has been introduced on the
acetophenone skeleton (11, 22% yield, Scheme 1) following
a procedure that successfully provided furan-containing
acronycine analogues.24 For the synthesis of a pyran-ring-
containing monomer, a versatile methodology involving 3-
methyl-2-butenal25 has been preferred and led successfully to
compound 12 with moderate yield (34%) with the main
byproduct possessing two rings also being produced.25
Synthesis of Phloroglucinol Dimers. The naturally
occurring Acronychia-type acetophenone dimers can be further
subdivided into homo- and heterodimers, with the latter far
exceeding the former in number.19,20,26 The only homodimer
isolated so far is demethylacrovestone (14), which lacks a
methoxy group compared to the model compound acrovestone
(1), constituting a totally symmetrical dimer.26 Having no SAR
data concerning the biological activity of compound 14 and
with this being the only representative of a homodimer
produced by A. pedunculata, the chemical development of such
molecules was considered challenging. In addition, guided by
the goal to generate a library of Acronychia-type acetophenones
analogues, the synthesis of demethylacrovestone along with a
new type of homodimers was undertaken. The presence of both
natural monomethylated and nonmethylated monomers
possessing 3,3-dimethylpropanone patterns22 led to the
hypothesis of putative biosynthetic formation of the dimers
eventually through the connection of the aforementioned
monomers with an acylphloroglucinol unit, thus representing
the starting point of the synthetic strategy. Acronychia-type
acetophenones could be disconnected in the isobutyl chain to
give the two monomers and an isobutyl carbocation. A work
reporting the synthesis of an electron-rich dirayl alkane using a
Friedel−Craft reaction with aldehydes27 allowed envisaging the
access of the desired dimers following this approach. As a proof
of concept, the acetophenone 8 has been engaged in a Friedel−
Craft reaction with isovaleraldehyde in the presence of
aluminum chloride in refluxing anhydrous toluene (Scheme
2). The nonsubstituted homodimer 13 connected with an
isobutyl chain has been successfully obtained although with
RESULTS AND DISCUSSION
■
Synthesis of Phloroglucinol Monomers. The first goal
was the synthesis of phloroglucinol monomers, not only in
order to evaluate their activity but also to serve as building
blocks for the synthesis of dimers. In total, five monomers have
been produced with different substitution patterns on the core
of acetyl phloroglucinol, i.e., prenyl, geranyl, pyran, and furan
(Scheme 1). However, due to the difficulty in achieving high
percentages of stereoselectivity as a result of the equivalent
reactivity of each carbon in the phloroglucinol skeleton, the
yields obtained were low to moderate. Nevertheless, the
synthesis of the monoprenylated acetophenone 9 was achieved
in satisfactory yield using a procedure involving 2-methyl-3-
buten-2-ol and BF3-Et2O (Scheme 1). This method represents
a practical alternative to the traditional strategy involving prenyl
bromide in the presence of potassium carbonate, which
generally leads to several products including C-diprenylated
and O-prenylated phloroglucinols. Compound 10 has been
obtained following a classical approach involving the use of
geranyl bromide (Scheme 1).
C
DOI: 10.1021/acs.jnatprod.6b01008
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 2. Synthesis of Phloroglucinol Homodimers
moderate yield (Scheme 2). On the basis of this strategy, a
small assembly of homodimers has been obtained including
demethylacrovestone (14), synthesized here for the first time,
as well as different analogues possessing geranyl (15), pyran
(16), or furan (17) rings (Scheme 2). Compound 14 and its
analogues were evaluated for their activity against 5-LO and
mPGES-1.
Evaluation of 5-LO and mPEGS-1 Inhibition. Inhibition
of 5-LO activity was first evaluated by means of a cell-free assay
using isolated human recombinant 5-LO, which allows the
identification of direct interference of the test compound with
the target enzyme, and IC50 values have been calculated for
both natural and synthetic compounds displaying 5-LO activity
below 50% at 10 μM. Zileuton was used as a positive control.
The results are summarized in Table 1.
Figure 3. Characteristics of 5-LO inhibition by compounds 1 and 10.
(A) Inhibition of 5-LO and mPGES1 activity by compounds 1 and 10.
(B) Effects of compound 10 on the activity of human recombinant 5-
LO at different AA concentrations. (C) Inhibition of 5-LO by
compound 10 is reversible (wash-out experiments). Zil = positive
control, zileuton. Data in (A) and (C) are given as means SEM, n =
3−4. Data in (B) are means of a representative experiment out of three
independent experiments. **, p < 0.01; ***, p < 0.001; versus 100%
control.
Table 1. Evaluation of Natural and Synthetic Acronychia-
Type Acetophenones toward 5-LO
poor membrane permeation into the cell, but other
mechanisms may contribute as well. All the natural products
were found to be also good inhibitors of mPEGS-1 in a cell-free
assay (Table 1), supporting the Acronychia-type acetophenones
as a new class of dual 5-LO/mPEGS-1 inhibitors. The synthetic
dimers 13−17 presented good inhibitory activities toward 5-
LO, particularly in the cell-free assays. However, as for the
natural heterodimers, their activity in the cell-based assays was
decreased, except for compound 15. Surprisingly, demethyla-
crovestone (14) was found to be inactive despite several
repetitions of the assay for unexplained reasons, while the
unsubstituted dimer 13 was found to be potent in both the cell-
free assay and the cell-based assay. Concerning the inhibition of
mPEGS-1, monomers 10 and 11 and dimers 13 and 16
displayed good inhibitory activity, suggesting that they also act
as dual 5-LO/mPEGS-1 inhibitors. Overall, these results may
reflect a similar mode of action between the natural Acronychia-
type acetophenones and the synthetic homodimers.
a
5-LO
mPEGS-1
cell -free IC50
intact cells IC50
cell-free IC50
compound
(μM)
(μM)
(μM)
1
2.7 0.2
2.3 0.6
>10
1.1 0.02
4.2 0.2
2.7 0.1
1.9 0.2
1.1 0.1
1.3 0.2
1.1 0.1
>10
2
6
0.6
3
7.3 1.8
5.3 0.7
5.0 0.6
4
2
0.5
5
>10
4.5
1
6
5.3 1.3
2.5 0.3
7.3 0.7
7
6.3
2
b
b
9
n.d.
n.d.
10
1.0 0.1
1.7 0.1
>10
1.4 0.3
6.3 0.3
>10
3.5 0.1
1.0 0.1
>10
13
14
15
3.6 0.4
>10
>10
5.34 1.6
>10
16
5
3
The position and nature of additional rings (3−6) had a
considerable impact on the inhibitory efficiency. The pyran-
containing products (3 and 4) were more active, and A-type
Acronychia-type acetophenones improved the potency of the
molecules (4 and 6) compared to B-type (3 and 5).
Demethylacronyline (9) was inactive in cell-free assays.
However, introduction of an extended unsaturated chain,
such as a geranyl moiety, led to potent inhibition of 5-LO in the
cell-free assay, as exemplified by compound 10 (IC50 = 1.0 μM)
(Figure 3A).
Compound 10 has recently been identified as a soybean 15-
lipoxygenase inhibitor.28 The soybean 15-LO assay is used as a
preliminary study of human 15-LO, due to the similarities in
their structure and mechanism.29 Additionally, human 5-LO
and 15-LO possess a high level of similarity, especially in the
area of the catalytic site.30,31 The main differences rely on the
orientation of helix α-2 affecting the position of Phe177 and
Tyr181 (for 5-LO) and Leu181 (for 15-LO-1) residues,30
17
2.4
1
>10
1.2 0.1
b
zileuton
MK886
0.8 0.1
1.7 0.7
n.d
b
b
n.d.
n.d.
2.5 0.5
a
b
Data are given as means SEM n = 3−4. n.d.: not determined.
All the natural compounds displayed a medium to high
inhibition of 5-LO in cell-free assays. Specifically, acrovestone
(1) (Figure 3A), acropyranol A (4), and acrovestenol (7)
inhibited 5-LO potently, with an IC50 around 2 μM,
comparable to the positive control zileuton. Apart from
acrovestone (1), all the natural metabolites showed no 5-LO
inhibitory activity in the cell-based assays used (Table 1). These
cell-based assays were performed using activated human
neutrophils, which allows the analysis of the interference of
the test compounds with 5-LO in a cellular (biological)
environment. The loss of potency of the majority of
compounds in the cell-based assay might be explained by
D
DOI: 10.1021/acs.jnatprod.6b01008
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
which may direct the carbon of AA to be oxidized (C-5, C-12,
or C-15). In silico studies have revealed previously the
interaction of a geranyl acylphloroglucinol derivative with
soybean 15-LO;28 the molecule interacts deeply inside the
catalytic cavity with His518, His513, and Glu716, three
important residues coordinating the active-site iron. In this
case, Phe177 and Leu181 were directed outward, opening a
large access allowing the phloroglucinol derivatives to enter the
active site. In the present case, in the absence of a cocrystal
structure and the low reliability of docking calculations (only a
modified structure of 5-LO affecting the size of catalytic site is
available30), few positive conclusions could be proposed.
Nevertheless, in the case of 5-LO, Phe177, and Tyr181 in
being more inward and in closing off the access to the catalytic
site,30 interactions of compound 10 deeply inside the cavity
could be discarded. Therefore, the hypothesis of π−π
interactions of the phloroglucinol moiety with one of the
aforementioned residues at the entrance of the active site and
insertion of the geranyl chain inside the binding cavity could be
proposed. To support this hypothesis, a competition assay was
performed between compound 10 and the 5-LO substrate AA
at different concentrations (Figure 3B). This revealed that 10 is
not a true competitor of AA binding, but based on wash-out
experiments (10-fold dilution), 10 inhibits 5-LO in a reversible
manner (Figure 3C), partly confirming the hypothesis. Up to
now, the precise mode of inhibition of 5-LO by homo- or
heteroacetophenone dimers remained elusive. Nevertheless, the
extension of the unsaturated chain on acylphloroglucinol
appears as a vector to develop new chemical entities, and the
evaluation of such compounds would certainly provide new
insights in terms of both SAR and mechanism of action.
In Vivo Evaluation in a Mouse Model. Next, the effects
of the most interesting compound, 10, was evaluated in vivo
using a mouse model of acute inflammation, namely, zymosan-
induced peritonitis. Zileuton and indomethacin were used as
controls. The ip pretreatment of mice with 10 (10 mg/kg, 30
min before zymosan injection) inhibited the LTC4 levels (38%)
and cell infiltration (32%) in the peritoneal exudates, 30 min
and 4 h after zymosan injection, respectively. No repression of
PGE2 production was observed (Table 2), indicating that
compound 10 preferentially acted in vivo as a 5-LO inhibitor
rather than a dual 5-LO/mPEGS-1 inhibitor as suggested by
the in vitro assays.
and mPEGS-1 in the development of chronic inflammatory
diseases and the traditional use of A. pedunculata to treat such
diseases connect traditional medicine, plant metabolites, and
biological targets. Furthermore, synthetic compounds derived
from the natural metabolites and specifically homodimers were
found to possess a similar activity profile compared to natural
products, while the access to monomers revealed their 5-LO
inhibitory activity, highlighting interesting structural features.
Among the derivatives investigated, demethylacrovestone (14)
was synthesized for the first time, whereas a simple synthetic
route to the homodimers was proposed. Using a mouse model
of acute inflammation, one of the synthetic compounds,
geranylphloroglucinol (10), displayed a potent anti-inflamma-
tory response comparable to the known 5-LO inhibitor
zileuton, a drug used for asthma treatment. Acronychia-type
acetophenones and acrovestone (as an active natural product)
represent a new class of 5-LO inhibitors and a new chemotype
for the development of more potent agents. Further studies
should be carried out for the evaluation of additional
acetophenones of this type including both monomers and
dimers, as well as for the investigation of additional mechanisms
of action.
EXPERIMENTAL SECTION
■
Natural Acetophenones. All seven natural acronychia-type
acetophenones reported in this study (Figure 1) have been previously
isolated from the trunk bark of Acronychia pedunculata and
unambiguously identified by spectroscopic methods.19 The purity of
all compounds was determined by HPLC-DAD and 1H NMR
spectroscopy and determined to be >95%.
Chemistry. All chemicals were purchased from Aldrich Chemical
Co. Microwave-assisted reactions were performed in a multimode
Milestone Start E apparatus (Milestone, Italy). Melting points were
recorded on a Buchi apparatus and are uncorrected. FT-IR spectra
̈
were recorded on a PerkinElmer RX1 FT-IR spectrometer. UV spectra
were recorded on a Shimadzu UV-160A UV−visible recording
spectrophotometer. NMR spectra were recorded on Bruker Avance
600 spectrometer (Karlsruhe, Germany) (1H 600 MHz, 13C 150
MHz); chemical shifts are expressed in ppm downfield from
tetramethylsilane. HRMS spectra were recorded on an LTQ-Orbitrap
spectrometer (ThermoScientific, Bremen, Germany). Column chro-
matography was conducted using flash silica gel 60 (40−63 μm) from
Merck. Purity of the compounds has been determined by HPLC-DAD
(Thermo Fisher Scientific) and was above 95%.
1-(2,4,6-Trihydroxy-3-(3-methylbut-2-en-1-yl)phenyl)ethanone
(9). 2,4,6-Trihydroxyacetophenone (8) (100 mg, 0.54 mmol) was
dissolved in 5 mL of 1,4-dioxane. To this solution were added 61.75
μL of 2-methyl-3-buten-2-ol (0.54 mmol) and 17 μL of BF3-Et2O
(0.14 mmol), and the mixture was stirred for 12 h at room
temperature. The solvent was evaporated, and the resulting residue
was purified using silica gel column chromatography with CH2Cl2/
EtOAc (94:6) as solvent system. This led to 26.9 mg of 9 being
collected as a yellow solid (20% yield): 1H NMR (CDCl3, 600 MHz) δ
5.91 (1H, s, H-5), 5.23 (1H, t, J = 6.0 Hz, H-2), 3.42 (2H, d, J = 6.0
Hz, H-1′), 2.68 (3H, s, CH3CO), 1.37 (6H, s, H-4′, H-5′); 13C NMR
(CDCl3, 150 MHz) δ 203.9 (C, CH3CO−Ar), 162.2 (C, C-6), 161.3
(C, C-4), 161.1 (C, C-2), 136.2 (C, C-3′), 121.4 (CH, C-2′), 105.2
(C, C-3), 103.3 (C, C-1), 96.5 (CH, C-5), 32.9 (CH3, CH3CO-Ar),
21.7 (CH3, C-4′, C-5′), 21.4 (CH2, C-1′); (+)-HRESIMS m/z
259.1102 [M + Na]+ (calcd for C13H16O4Na, 259.0946).32
(E)-1-(3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)-
ethanone (10). 2,4,6-Trihydroxyacetophenone (8) (50 mg, 0.17
mmol) was dissolved in 10 mL of acetone, and then were added 82.9
mg (0.37 mmol) of K2CO3 and 57 μL (0.17 mmol) of geranyl
bromide. After 18 h at room temperature, the reaction was quenched
with the addition of 2 N HCl. The solvent was evaporated and the
residue was extracted with EtOAc and water. The organic fraction was
The study has revealed that Acronychia-type acetophenones
are natural inhibitors of 5-LO, and some also displayed dual 5-
LO/mPEGS-1 inhibition in vitro. The involvement of 5-LO
Table 2. Effect of Compound 10 on the Production of Pro-
inflammatory Metabolites and Cell Infiltration
LTC4 (ng/
inflammatory cells
PGE2 (ng/
a
b
c
c
treatment
vehicle (2%
mL)
(× 106)
mL)
92.2 12.8
7.3 0.72
10.5 1.34
DMSO)
e
10 (10 mg/kg)
57.3 7.1
65 2.41
5.00 0.69
11.3 1.5
d
d
zileuton (10
mg/kg)
n.d.
n.d.
d
f
f
indomethacin (5
mg/kg)
n.d
2.08 1.02
3.52 1.06
a
Male mice (n = 6−8, each group) were treated ip 30 min before ip
b
injection of zymosan. LTC4 and PGE2 levels were evaluated 30 min
after injection with zymosan. Cell infiltration was evaluated 4 h after
zymosan injection. n.d.: not determined. p < 0.05 versus vehicle
(Student’s t test). p < 0.01 versus vehicle (Student’s t test).
c
d
e
f
E
DOI: 10.1021/acs.jnatprod.6b01008
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
dried on Na2SO4, filtered, and evaporated. The crude product was
purified with silica gel column chromatography using CH2Cl2/EtOAc
(92:8). In total, 35.1 mg of 10 as a yellow solid was collected (68%
yield): 1H NMR (CDCl3, 600 MHz) δ 5.91 (1H, s, H-5), 5.32 (1H, t, J
= 6.3 Hz, H-2′), 5.06 (1H, t, J = 6.6 Hz, H-7′), 3.51 (4H, d, J = 7.2 Hz,
H-1′, H-6′), 2.81 (3H, s, CH3CO), 1.34 (5H, s, H-4′, H-5′), 1.33 (6H,
s, H-9′, H-10′); 13C NMR (CDCl3, 150 MHz) δ 204.2 (C, CH3CO−
Ar), 160.8 (C, C-6), 159.1 (C, C-4), 158.2 (C, C-2), 136.6 (C, C-3′),
135.9 (C, C-8′), 123.3 (CH, C-7′), 121.5 (CH, C-2′), 108.7 (CH, C-
5), 106.5 (C, C-3), 104.7 (C, C-1), 30.8 (CH3, CH3CO-Ar), 28.5
(CH2, C-5′), 28.40 (CH3, C-4′), 21.3 (CH2, C-1′), 21.2 (CH2, C-6′),
20.3 (CH3, C-9′, C-10′); (+)-HRESIMS m/z 327.1727 [M + Na]+
(calcd for C18H24O4Na, 327.1572).33
Demethylacrovestone (14). 2,4,6-Trihydroxy-3-prenylacetophe-
none (9) (30 mg, 0.12 mmol) was dissolved in 5 mL of toluene. A
1.7 mg amount of anhydrous AlCl3 (0.012 mmol) and 6.8 μL of
isovaleraldehyde (0.06 mmol) were then added. The reaction was
irradiated at 300 W (T = 111 °C) for 1.5 h. The solvent was
evaporated, and the residue was extracted with CH2Cl2 and a saturated
aqueous solution of NaCl. The organic phases were gathered, dried
over NaSO4, filtered, and evaporated. The crude product was purified
on a silica gel column (CH2Cl2/cyclohexane, 1:1). In total, 3.1 mg of
compound 14 was collected as a yellow solid (5% yield): mp 149−150
°C; UV (MeOH) λmax (log ε) 347 (1.55), 303 (4.89), 244 (6.19), 209
(4.93) nm; IR (Nujol) νmax 3320, 3137, 2919, 2724, 1610, 1519, 1458,
1
1376, 1318, 1242, 1173, 988, 940, 845 cm−1; H NMR (CDCl3, 600
1-(4,6-Dihydroxy-2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran-5-
yl)ethanone (11). 2,4,6-Trihydroxyacetophenone (8) (50 mg, 0.27
mmol) was dissolved in 3 mL of acetone, and 74 mg (0.54 mmol) of
K2CO3 and 60.7 μL (0.41 mmol) of 1,4-dibromo-2-methylbut-2-ene34
were added. The mixture was heated at 40 °C for 20 h. The solvent
was evaporated, and the residue was extracted with EtOAc and water.
After gathering, the organic phase was dried on Na2SO4, filtered, and
evaporated. Compound 11 was purified with a silica gel column using
as solvent system CH2Cl2/EtOAc (96:4). Altogether, 14.1 mg of 11
MHz) δ 5.28 (2H, t, J = 6.9 Hz, H-2′), 4.69 (1H, brs, H-1″), 3.47 (4H,
d, J = 6.0 Hz, H-1′), 2.78 (6H, s, CH3CO), 2.15 (2H, s, H-2″), 1.65
(1H, brs, H-3″), 1.34 (12H, s, H-4′, H-5′), 0.87 (6H, brs, H-4″, H-5″);
13C NMR (CDCl3, 150 MHz) δ 201.1 (C, CH3CO−Ar), 161.3 (C, C-
6), 161.0 (C, C-4), 160.5 (C, C-2), 136.6 (C, C-3′), 121.5 (CH, C-2′),
109.1 (C, C-5), 107.2 (C, C-3), 103.4 (C, C-1), 37.1 (CH2, C-2″),
30.4 (CH3, CH3CO-Ar), 27.8 (CH, C-1″), 25.5 (CH, C-3″), 21.7
(CH2, C-1′), 20.9 (CH3, C-4′, C-5′), 20.5 (CH3, C-4″,C-5″);
(+)-HRESIMS m/z 563.2599 [M + Na]+ (calcd for C31H40O8Na,
563.2621).
1
was collected as a yellow solid (22% yield): H NMR (CDCl3, 600
MHz) δ 5.92 (1H, s, H-5), 5.62 (1H, brs, H-2′), 5.03 (2H, s, H-5′),
3.40 (2H, brs, H-1′), 2.74 (3H, s, H-4′), 2.68 (3H, s, CH3CO); 13C
NMR (CDCl3, 150 MHz) δ 203,1 (C, CH3CO−Ar), 161.3 (C, C-6),
159.9 (C, C-4), 152.5 (C, C-2), 144.5 (C, C-3′), 120.8 (CH, C-2′),
111.8 (CH2, C-5′), 109.2 (CH, C-5), 106.5 (C, C-3), 101.1 (C, C-1),
32.8 (CH3, C-4′), 32.5 (CH3, CH3CO−Ar), 20.6 (CH2, C-1′);
(−)-HRESIMS m/z 233.1133 [M − H]− (calcd for C13H13O4,
233.0892).34
1-(5,7-Dihydroxy-2,2-dimethyl-2H-chromen-8-yl)ethanone (12).
2,4,6-Trihydroxyacetophenone (8) (50 mg, 0.27 mmol) was dissolved
in 5 mL of CH2Cl2, and 16.4 μL of 3-methyl-2-butenal (0.17 mmol)
was added. The reaction mixture was heated to 40 °C for 24 h. After
completion of the reaction, the solvent was evaporated and the residue
was extracted with EtOAc and water. The organic fractions were dried
on Na2SO4, filtered, and evaporated. The crude product was subjected
to purification on a silica gel column, and 13.4 mg of pure compound
12 was collected as a yellow solid (34% yield): 1H NMR (CDCl3, 600
MHz) δ 6.64 (1H, d, J = 10.02 Hz, H-1′), 5.92 (1H, s, H-5), 5.43 (1H,
d, J = 10.02 Hz, H-2′), 2.67 (3H, s, CH3CO), 1.41 (6H, brs, H-4′, H-
5′); 13C NMR (CDCl3, 150 MHz) δ 204.1 (C, CH3CO−Ar), 160.1
(C, C-6), 159.8 (C, C-4), 152,5 (C, C-2), 124.5 (CH, C-2′), 116.8
(CH, C-1′), 109.1 (CH, C-5), 104.5 (C, C-1), 103.8 (C, C-2), 102.6
(C, C-3), 77.1 (C, C-3′), 32.4 (CH3, CH3CO−Ar), 28.4 (CH3, C-4′,
C-5′); (−)-HRESIMS m/z 233.1132 [M − H]− (calcd for C13H13O4,
233.0892).25
1,1′-((3-Methylbutane-1,1-diyl)bis(2,4,6-trihydroxy-3,1-
phenylene))diethanone (13). 2,4,6-Trihydroxyacetophenone (8) (1.0
g, 5.4 mmol) was dissolved in 15 mL of toluene, and 72 mg (0.54
mmol) of anhydrous AlCl3 was added. The reaction mixture was
stirred for 15 min at room temperature. Then isovaleraldehyde (250
μL, 2.7 mmol) was added, and the mixture was heated under reflux for
48 h. The solvent was evaporated, the residue was extracted with
CH2Cl2 and a saturated aqueous solution of NaCl, and the organic
phase was dried over Na2SO4, filtered, and evaporated. The crude
product was purified with a silica gel column (CH2Cl2/MeOH, 97:3).
Altogether, 287 mg of compound 13 was collected as an orange solid
(13% yield): mp 149−150 °C; UV (MeOH) λmax (log ε) 289 (3.92),
236 (4.52) nm; IR (Nujol) νmax 3320, 2919, 2720, 1619, 1520, 1455,
1382, 1268, 1155, 988, 935, 850 cm−1; 1H NMR (CDCl3, 600 MHz) δ
5.91 (2H, brs, H-3), 4.64 (1H, t, J = 7.5 Hz, H-1′), 2.68 (6H, s,
CH3CO), 2.18 (2H, brs, H-2′), 1.41 (1H, m, H-3′), 0.87 (6H, d, J =
7.1 Hz, H-4′, H-5′); 13C NMR (CDCl3, 150 MHz) δ 203.8 (C,
CH3CO−Ar), 163.1 (C, C-4), 162.6 (C, C-6), 159.1 (C, C-2), 109.0
(C, C-5), 104.5 (C, C-1), 96.3 (CH, C-3), 38.9 (CH2, C-2′), 32.1
(CH3, CH3CO−Ar), 27.4 (CH, C-1′), 26.5 (CH, C-3′), 22.1 (CH3, C-
4′, C-5′); (−)-HRESIMS m/z 403.1390 [M − H]− (calcd for
C21H23O8, 403.1398).
1-(3-(1-(3-Acetyl-5-((E)-3,7-dimethylocta-2,6-dien-1-yl)-2,4,6-tri-
hydroxyphenyl)-3-methylbutyl)-5-((Z)-3,7-dimethylocta-2,6-dien-1-
yl)-2,4,6-trihydroxyphenyl)ethanone (15). 2,4,6-Trihydroxy-3-
geranylacetophenone (10) (182 mg, 0.60 mmol) was dissolved in 5
mL of toluene, and after 15 min of stirring, 23 μL of isovaleraldehyde
(0.30 mmol) and 8 mg of anhydrous AlCl3 (0.06 mmol) were added.
The reaction mixture was heated under reflux for 48 h. The solvent
was evaporated, and the residue was extracted with CH2Cl2 and
saturated aqueous NaCl solution. The organic phase was dried over
NaSO4, filtered, and evaporated. The crude product was purified on a
silica gel column (CH2Cl2/cyclohexane, 6:4). In total, 15 mg of
compound 15 was collected as a yellow solid (11% yield): mp 147−
148 °C; UV (MeOH) λmax (log ε) 346 (1.42), 303 (4.27), 245 (4.83)
nm; IR (Nujol) νmax 3246, 2919, 2850, 1619, 1594, 1420, 1360, 1281,
1165, 957, 841 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.32 (2H, t, J =
6.6 Hz, H-2′), 5.06 (2H, t, J = 6.6 Hz, H-7′), 4.66 (1H, brs, H-1″),
3.51 (8H, d, J = 7.2 Hz, H-1′, H-6′), 2.81 (6H, s, CH3CO), 2.17 (2H,
brs, H-2″), 1.78 (1H, m, H-3″), 1.34 (12H, s, H-9′, H-10′), 1.33 (10H,
s, H-4′, H-5′), 0.88 (6H, brs, H-4″, H-5″); 13C NMR (CDCl3, 150
MHz) δ 204.2 (C, CH3CO−Ar), 160.8 (C, C-6), 159.1 (C, C-4),
158.2 (C, C-2), 136.6 (C, C-3′), 135.9 (C, C-8′), 123.3 (CH, C-7′),
121.55 (CH, C-2′), 108.7 (C, C-5), 106.1 (C, C-3), 104.7 (C, C-1),
38.8 (CH2, C-2″), 30.7 (CH3, CH3CO−Ar), 29.2 (C, C-3″), 28.5
(CH2, C-5′), 28.4 (CH3, C-4′), 27.6 (CH, C-1′), 26.2 (CH, C-3″),
21.3 (CH2, C-1′), 21.2 (CH2, C-6′), 21.6 (CH3, C-4″, C-5″), 20.4
(CH3, C-9′, C-10′); (+)-HRESIMS m/z 699.3865 [M + Na]+ (calcd
for C41H56O8Na, 699.3873).
1,1′-(5,5′-(3-Methylbutane-1,1-diyl)bis(4,6-dihydroxy-2-(prop-1-
en-2-yl)-2,3-dihydrobenzofuran-7,5-diyl))diethanone (16). 2-Iso-
prenyl-5-acetyl-4,6-dihydroxy-2,3-dihydrobenzofuran (11) (250 mg,
1.07 mmol) was dissolved in 10 mL of toluene, and 43.5 μL of
isovaleraldehyde (0.53 mmol) and 14.3 mg of anhydrous AlCl3 (0.11
mmol) were added. The mixture was heated under reflux for 48 h. The
solvent was evaporated, and the residue was extracted with CH2Cl2
and saturated aqueous NaCl solution. The organic phase was dried
over Na2SO4, filtered, and evaporated. The crude product was purified
with column chromatography (CH2Cl2/cyclohexane, 6:4). A 15 mg
amount of compound 16 was obtained as a yellow oil (10% yield): mp
150−151 °C; UV (MeOH) λmax (log ε) 293 (3.91), 237 (4.45), 204
(4.75) nm; IR (Nujol) νmax 3340, 2960, 2919, 1610, 1584, 1450, 1370,
1255, 1203, 1158, 970, 891, 810 cm−1; 1H NMR (CDCl3, 600 MHz) δ
0.88 (6H, d, J = 7.1 Hz, H-4″, H-5″), 1.42 (1H, brs, H-3″); 2.49 (2H,
brs, H-2″), 2.68 (6H, s, CH3CO), 2.72 (6H, s, H-4′), 3.40 (4H, brs, H-
1′), 4.70 (1H, brs, H-1″); 5.03 (4H, s, H-5′), 5.62 (2H, brs, H-2′); 13C
NMR (CDCl3, 150 MHz) δ 202.1 (C, CH3CO−Ar), 161.3 (C, C-6),
159.9 (C, C-4), 152.5 (C, C-2), 144.5 (C, C-3′), 120.8 (CH, C-2′),
111.8 (CH2, C-5′), 109.2 (C, C-5), 106.5 (C, C-3), 101.1 (C, C-1),
F
DOI: 10.1021/acs.jnatprod.6b01008
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
38.1 (CH2, C-2″), 32.8 (CH3, C-4′), 32.5 (CH3, CH3CO-Ar), 27.4
(CH, C-1″), 26.2 (CH, C-3″), 22.1 (CH3, C-4″, C-5″), 20.6 (CH2, C-
1′); (−)-HRESIMS m/z 535.2329 [M − H]− (calcd for C31H35O8,
535.2337).
preincubated with each test compound as indicated. After 10 min at 4
°C, samples were prewarmed for 30 s at 37 °C, and 2 mM CaCl2 plus
20 μM AA were added to start 5-LO product formation. The reaction
was stopped after 10 min at 37 °C by addition of 1 mL of ice-cold
methanol, and the formed metabolites were analyzed by RP-HPLC as
described before.35 5-LO products include the all-trans isomers of
LTB4 and 5-H(p)ETE.
1,1′-(6,6′-(3-Methylbutane-1,1-diyl)bis(5,7-dihydroxy-2,2-dimeth-
yl-2H-chromene-8,6-diyl))diethanone (17). (5,5′-Isobutyl)-
diacetophenone (13) (35 mg, 0.087 mmol) was dissolved in
dichloromethane, 16.7 μL of 3-methyl-2-butenal (0.174 mmol) was
added, and the mixture was heated under reflux for 24 h. The solvent
was evaporated, and the residue was purified using silica gel column
chromatography (CH2Cl2/cyclohexane, 95:5); 5 mg of 17 was
collected as a yellow solid (12% yield): mp 149−150 °C; UV
(MeOH) λmax (log ε) 292 (3.32), 249 (3.29) nm; IR (Nujol) νmax
3320, 2967, 2921, 1616, 1582, 1443, 1366, 1256, 1209, 1152, 964, 877,
807 cm−1; 1H NMR (CDCl3, 600 MHz) δ 6.64 (2H, d, J = 10.02 Hz,
H-2′), 5.44 (2H, d, J = 10.02 Hz, H-1′), 4.67 (1H, t, J = 7.0 Hz, H-1″),
2.67 (6H, s, CH3CO), 2.30 (2H, m, H-2″), 1.41 (12H, s, H-4′, H-5′),
1.22 (1H, brs, H-3″), 0.84 (6H, d, J = 7.1 Hz, H-4″, H-5″); 13C NMR
(CDCl3, 150 MHz) δ 203.7 (C, CH3CO−Ar), 160.1 (C, C-6), 159.8
(C, C-4), 152.7 (C, C-2), 124.8 (CH, C-2′), 117.3 (CH, C-1′), 109.1
(C, C-5), 104.5 (C, C-1), 102.6 (C, C-3), 77.1 (C, C-3′), 33.9 (CH2,
C-2″), 32.4 (CH3, CH3CO−Ar), 28.4 (CH3, C-4′, C-5′), 27.6 (CH, C-
1″), 27.4 (CH, C-3″), 20.6 (CH3, C-4″, C-5″); (−)-HRESIMS m/z
535.2339 [M − H]− (calcd for C31H35O8, 535.2337).
Cells and Cell Isolation. Human peripheral blood was taken from
fasted (12 h) healthy donors that had not taken any anti-inflammatory
drugs during the last 10 days, by venipuncture in heparinized tubes (16
IE heparin/mL blood). The blood was centrifuged at 4000g for 20 min
at room temperature for preparation of leukocyte concentrates
(University Hospital, Jena, Germany). The protocols for experiments
with human leukocytes were approved by the ethical commission of
the Friedrich-Schiller-University Jena (No. 4025-02/14). All methods
were performed in accordance with the relevant guidelines and
regulations. Leukocyte concentrates were subjected to dextran
sedimentation and centrifugation on Nycoprep cushions (PAA
Laboratories, Linz, Austria). Contaminating erythrocytes of pelleted
neutrophils were lysed by hypotonic lysis, and neutrophils were
washed twice in ice-cold PBS and finally resuspended in PBS pH 7.4
containing 1 mg/mL glucose and 1 mM CaCl2 (PGC buffer) (purity
>96−97%). For analysis of acute cytotoxicity of test compounds
during preincubation periods (routinely 15 min), the viability of
neutrophils was analyzed by light microscopy and trypan blue
exclusion. None of the compounds (up to 10 μM) caused a cytotoxic
effect within 30 min of neutrophil incubation (not shown).
Determination of 5-LO Activity in Intact Cells. For
determination of 5-LO products in intact neutrophils, 5 × 106 cells
were resuspended in 1 mL of PGC buffer, preincubated for 15 min at
37 °C with a test compound or vehicle (0.3% DMSO), and incubated
for 10 min at 37 °C with Ca2+-ionophore A23187 (2.5 μM) with 20
μM AA. After 10 min at 37 °C, the reaction was stopped on ice by
addition of 1 mL of methanol. A 30 μL portion of 1 N HCL, 500 μL of
PBS, and 200 ng of prostaglandin B1 were added, and the samples
were subjected to solid-phase extraction on C18 columns (100 mg,
UCT, Bristol, PA, USA). 5-LO products (LTB4, trans-isomers, 5-
H(p)ETE) were quantified by HPLC, and quantities calculated on the
basis of the internal standard, prostanglandin B1. Cysteinyl-
leukotrienes C4, D4, and E4 were not detected, since their amounts
were below the detection limit, and oxidation products of LTB4 were
not determined.
Expression, Purification, and Cell-Free Activity Determina-
tion of 5-LO. Escherichia coli MV1190 was transformed with pT3-5-
LO plasmid, and recombinant 5-LO protein was expressed at 27 °C as
described previously.35 Cells were lysed in 50 mM triethanolamine/
HCl at pH 8.0, 5 mM EDTA, soybean trypsin inhibitor (60 μg/mL), 1
mM phenylmethanesulfonyl fluoride, and lysozyme (500 μg/mL),
homogenized by sonication (3 × 15 s), and centrifuged at 40000g for
20 min at 4 °C. The 40000g supernatant (S40) was applied to an ATP-
agarose column to partially purify 5-LO, as described previously.35
Aliquots of semipurified 5-LO were diluted with ice-cold PBS
containing 1 mM EDTA, and 1 mM ATP was added. Samples were
Preparation of mPGES-1 and Cell-Free mPGES-1 Activity
Assay. Preparations of A549 cells and the determination of mPGES-1
activity were performed as described previously.36 In brief, cells were
treated with 1 ng/mL interleukin-1β for 48 h at 37 °C, 5% CO2. Cells
were harvested and sonicated, and the homogenate was subjected to
differential centrifugation at 10000g for 10 min and 174000g for 1 h at
4 °C. The pellet (microsomal fraction) was resuspended in 1 mL of
homogenization buffer (0.1 M potassium phosphate buffer, pH 7.4, 1
mM phenylmethanesulfonyl fluoride, 60 μg/mL soybean trypsin
inhibitor, 1 μg/mL leupeptin, 2.5 mM glutathione, and 250 mM
sucrose), and the total protein concentration was determined.
Microsomal membranes were diluted in potassium phosphate buffer
(0.1 M, pH 7.4) containing 2.5 mM glutathione. Test compounds or
vehicle was added, and after 15 min at 4 °C, a reaction (100 μL total
volume) was initiated by addition of 20 μM prostaglandin H2. After 1
min at 4 °C, the reaction was terminated using stop solution (100 μL;
40 mM FeCl2, 80 mM citric acid, and 10 μM 11β-prostagladin E2 as
internal standard). Prostaglandin E2 was separated by solid-phase
extraction and analyzed by RP-HPLC, as described previously.36
Animals and Zymosan-Induced Peritonitis. Male CD-1 mice
(8−9 weeks old, Charles River, Calco, Italy) were housed in a
controlled environment (21
2 °C) and provided with standard
rodent chow and water. All animals were allowed to acclimate for 4
days prior to experiments and were subjected to a 12 h light−12 h dark
schedule. Experiments were conducted during the light phase. The
experimental protocol was approved by the Animal Care Committee
of the University of Naples Federico II, in compliance with Italian
regulations on protection of animals used for experimental and other
scientific purpose (Ministerial Decree 116/92) as well as with the
European Economic Community regulations (Official Journal of
European Community L 358/1 12/18/1986). Compound 10, zileuton,
or indomethacin at the indicated dose or vehicle (0.5 mL of 0.9%
saline solution containing 2% DMSO) was given ip 30 min before
zymosan ip injection (0.5 mL of suspension of 2 mg/mL in 0.9% w/v
saline). Mice were sacrificed by inhalation of CO2 at the indicated
time, followed by a peritoneal lavage with 3 mL of cold PBS. Exudates
were collected, the cells in the exudates were counted, and leukotrienes
C4 and prostaglandin E2 were measured by enzyme immunoassay
(Cayman Chemical).37
Statistics. Data are expressed as means
SE. IC50 values were
graphically calculated from averaged measurements at four or five
different concentrations of the compounds using SigmaPlot 9.0 (Systat
Software Inc., San Jose, CA, USA). Statistical evaluation of the data
was performed by one-way ANOVA, followed by a Bonferroni or
Tukey−Kramer post hoc test for multiple comparisons, respectively. A
p value < 0.05 (*) was considered significant.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b01008.
NMR spectra of the synthesized compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*Tel: (+49) 3641949801. Fax: (+49) 3641949802. E-mail:
oliver.werz@uni-jena.de (O. Werz).
*Tel: (+30) 2107274598. Fax: (+30) 2107274594. E-mail:
skaltsounis@pharm.uoa.gr (A.-L. Skaltsounis).
G
DOI: 10.1021/acs.jnatprod.6b01008
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(23) Kozaki, S.; Takenaka, Y.; Mizushina, Y.; Yamaura, T.; Tanahashi,
T. J. Nat. Med. 2014, 68, 421−426.
ORCID
Nicolas Gaboriaud-Kolar: 0000-0001-9307-1921
Notes
The authors declare no competing financial interest.
(24) Boutefnouchet, S.; Gaboriaud-Kolar, N.; Minh, N. T.; Depauw,
́ ́
S.; David-Cordonnier, M.-H.; Pfeiffer, B.; Leonce, S.; Pierre, A.;
Tillequin, F.; Lallemand, M.-C.; Michel, S. J. Med. Chem. 2008, 51,
7287−7297.
(25) Adler, M. J.; Baldwin, S. W. Tetrahedron Lett. 2009, 50, 5075−
ACKNOWLEDGMENTS
■
5079.
This work was supported by project NATPROT (No. 3207)
funded by the Greek Secreteriat for Research and Technology
and action ARISTEIA II. We thank Dr. M. Litaudon (Institut
de Chimie des Substances Naturelles, CNRS) and Dr. K.
Awang and Prof. H. A. Hadi (both of the University of Malaya)
for the plant collection. The authors also thank the Deutsche
Forschungsgemeinschaft (DFG) within the SFB1127 (Chem-
ical Mediators in Complex Biosystems) for funding.
(26) De Silva, L. B.; Herath, W. M.; Liyanage, C.; Kumar, V.; Uddin
Ahmad, V.; Sultana, A. Phytochemistry 1991, 30, 1709−1710.
(27) Jaratjaroonphong, J.; Sathalalai, S.; Techasauvapak, P.;
Reutrakul, V. Tetrahedron Lett. 2009, 50, 6012−6015.
(28) Ng, C. H.; Rullah, K.; Aluwi, M. F.; Abas, F.; Lam, K. W.; Ismail,
I. S.; Narayanaswamy, R.; Jamaludin, F.; Shaari, K. Molecules 2014, 19,
11645−11659.
(29) Wecksler, A. T.; Garcia, N. K.; Holman, T. R. Bioorg. Med.
Chem. 2009, 17, 6534−6539.
(30) Gilbert, N. C.; Bartlett, S. G.; Waight, M. T.; Neau, D. B.;
Boeglin, W. E.; Brash, A. R.; Newcomer, M. E. Science 2011, 331, 217−
219.
DEDICATION
■
Dedicated to Professor Phil Crews, of the University of
California, Santa Cruz, for his pioneering work on bioactive
natural products.
(31) Gilbert, N. C.; Rui, Z.; Neau, D. B.; Waight, M. T.; Bartlett, S.
G.; Boeglin, W. E.; Brash, A. R.; Newcomer, M. E. FASEB J. 2012, 26,
3222−3229.
(32) Diller, R. A.; Riepl, H. M.; Rose, O.; Frias, C.; Henze, G.;
Prokop, A. Chem. Biodiversity 2005, 2, 1331−1337.
(33) Jung, D. H.; Lee, Y. R.; Kim, S. H. Helv. Chim. Acta 2010, 93,
635−647.
REFERENCES
■
(1) Medzhitov, R. Nature 2008, 454, 428−435.
(2) Koeberle, A.; Werz, O. Drug Discovery Today 2014, 19, 1871−
1882.
(34) Bigi, F.; Casiraghi, G.; Casnati, G.; Sartori, G. Tetrahedron 1983,
39, 169−174.
(3) Peters-Golden, M.; Henderson, W. R. N. Engl. J. Med. 2007, 357,
1841−1854.
(35) Fischer, L.; Szellas, D.; Radmark, O.; Steinhilber, D.; Werz, O.
̊
FASEB J. 2003, 17, 949−951.
(4) Montuschi, P. Mini-Rev. Med. Chem. 2008, 8, 647−656.
(36) Koeberle, A.; Siemoneit, U.; Buhring, U.; Northoff, H.; Laufer,
S.; Albrecht, W.; Werz, O. J. Pharmacol. Exp. Ther. 2008, 326, 975−
̈
(5) Radmark, O.; Werz, O.; Steinhilber, D.; Samuelsson, B. Biochim.
̊
Biophys. Acta, Mol. Cell Biol. Lipids 2015, 1851, 331−339.
(6) Pergola, C.; Werz, O. Expert Opin. Ther. Pat. 2010, 20, 355−375.
(7) Steinhilber, D.; Hofmann, B. Basic Clin. Pharmacol. Toxicol. 2014,
114, 70−77.
982.
(37) Rossi, A.; Pergola, C.; Pace, S.; Radmark, O.; Werz, O.;
̊
Sautebin, L. Pharmacol. Res. 2014, 87, 1−7.
(8) Laufer, S. A.; Augustin, J.; Dannhardt, G.; Kiefer, W. J. Med.
Chem. 1994, 37, 1894−1897.
(9) Koeberle, A.; Zettl, H.; Greiner, C.; Wurglics, M.; Schubert-
Zsilavecz, M.; Werz, O. J. Med. Chem. 2008, 51, 8068−8076.
(10) Werz, O. Planta Med. 2007, 73, 1331−1357.
(11) Pergola, C.; Gaboriaud-Kolar, N.; Jestadt, N.; Konig, S.;
̈
̈
Kritsanida, M.; Schaible, A. M.; Li, H.; Garscha, U.; Weinigel, C.; Barz,
D.; Albring, K. F.; Huber, O.; Skaltsounis, A. L.; Werz, O. J. Med.
Chem. 2014, 57, 3715−3723.
(12) Koeberle, A.; Rossi, A.; Bauer, J.; Dehm, F.; Verotta, L.;
Northoff, H.; Sautebin, L.; Werz, O. Front. Pharmacol. 2011,
2.10.3389/fphar.2011.00007
(13) Albert, D.; Zundorf, I.; Dingermann, T.; Muller, W. E.;
̈
̈
Steinhilber, D.; Werz, O. Biochem. Pharmacol. 2002, 64, 1767−1775.
(14) Feißt, C.; Franke, L.; Appendino, G.; Werz, O. J. Pharmacol.
Exp. Ther. 2005, 315, 389−396.
(15) Bauer, J.; Koeberle, A.; Dehm, F.; Pollastro, F.; Appendino, G.;
Northoff, H.; Rossi, A.; Sautebin, L.; Werz, O. Biochem. Pharmacol.
2011, 81, 259−268.
(16) Koeberle, A.; Pollastro, F.; Northoff, H.; Werz, O. Br. J.
Pharmacol. 2009, 156, 952−961.
(17) Koeberle, A.; Werz, O. Biochem. Pharmacol. 2015, 98, 1−15.
(18) Epifano, F.; Fiorito, S.; Genovese, S. Phytochemistry 2013, 95,
12−18.
(19) Kouloura, E.; Halabalaki, M.; Lallemand, M.-C.; Nam, S.; Jove,
R.; Litaudon, M.; Awang, K.; Hadi, H. A.; Skaltsounis, A.-L. J. Nat.
Prod. 2012, 75, 1270−1276.
(20) Wu, T.-S.; Wang, M.-L.; Jong, T.-T.; McPhail, A. T.; McPhail,
D. R.; Lee, K.-H. J. Nat. Prod. 1989, 52, 1284−1289.
(21) Lesueur, D.; De Rocca Serra, D.; Bighelli, A.; Minh Hoi, T.; Huy
Thai, T.; Casanova, J. Nat. Prod. Res. 2008, 22, 393−398.
(22) Su, C.-R.; Kuo, P.-C.; Wang, M.-L.; Liou, M.-J.; Damu, A. G.;
Wu, T.-S. J. Nat. Prod. 2003, 66, 990−993.
H
DOI: 10.1021/acs.jnatprod.6b01008
J. Nat. Prod. XXXX, XXX, XXX−XXX