SAR studies on curcumin's pro-inflammatory targets: Discovery of prenylated pyrazolocurcuminoids as potent and selective novel inhibitors of 5-lipoxygenase

Article abstract of DOI:10.1021/jm500308c

The anticarcinogenic and anti-inflammatory properties of curcumin have been extensively investigated, identifying prostaglandin E₂ synthase (mPGES)-1 and 5-lipoxygenase (5-LO), key enzymes linking inflammation with cancer, as high affinity targets. A comparative structure-activity study revealed three modifications dissecting mPGES-1/5-LO inhibition, namely (i) truncation of the acidic, enolized dicarbonyl moiety and/or replacement by pyrazole, (ii) hydrogenation of the interaryl linker, and (iii) (dihydro)prenylation. The prenylated pyrazole analogue 11 selectively inhibited 5-LO, outperforming curcumin by a factor of up to 50, and impaired zymosan-induced mouse peritonitis along with reduced 5-LO product levels. Other pro-inflammatory targets of curcumin (i.e., mPGES-1, cyclooxygenases, 12/15-LOs, nuclear factor-κB, nuclear factor-erythroid 2-related factor-2, and signal transducer and activator of transcription 3) were hardly affected by 11. The strict structural requirements for mPGES-1 and 5-LO inhibition strongly suggest that specific interactions rather than redox or membrane effects underlie the inhibition of mPGES-1 and 5-LO by curcumin.

Full text of DOI:10.1021/jm500308c

Article
pubs.acs.org/jmc
SAR Studies on Curcumin’s Pro-inflammatory Targets: Discovery
of Prenylated Pyrazolocurcuminoids as Potent and Selective
Novel Inhibitors of 5‑Lipoxygenase
Andreas Koeberle,*,† Eduardo Munoz,‡ Giovanni B. Appendino,§ Alberto Minassi,§ Simona Pace,†,∥
̃
Antonietta Rossi,∥ Christina Weinigel,⊥ Dagmar Barz,⊥ Lidia Sautebin,∥ Diego Caprioglio,§
Juan A. Collado,‡ and Oliver Werz†
†Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University Jena, Philosophenweg 14, 07743 Jena, Germany
‡Instituto Maimon
Cordoba, Avda Menendez Pidal s/n, 14004 Cor
́
ides de Investigacion
́
Biomed
́
ica de Cor
́
doba (IMIBIC), Hospital Universitario Reina Sofía, Universidad de
́
́
doba, Spain
§Dipartimento di Scienze del Farmaco, Alimentari, Farmaceutiche e Farmacologiche, Universita
Largo Donegani 2, 28100 Novara, Italy
̀
del Piemonte Orientale,
∥Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano 46, 80131 Naples, Italy
⊥Institute of Transfusion Medicine, University Hospital Jena, Bachstrasse 18, 07743 Jena, Germany
S
* Supporting Information
ABSTRACT: The anticarcinogenic and anti-inflammatory
properties of curcumin have been extensively investigated,
identifying prostaglandin E2 synthase (mPGES)-1 and 5-
lipoxygenase (5-LO), key enzymes linking inflammation with
cancer, as high affinity targets. A comparative structure−
activity study revealed three modifications dissecting mPGES-
1/5-LO inhibition, namely (i) truncation of the acidic, enolized
dicarbonyl moiety and/or replacement by pyrazole, (ii) hydro-
genation of the interaryl linker, and (iii) (dihydro)prenylation.
The prenylated pyrazole analogue 11 selectively inhibited 5-LO,
outperforming curcumin by a factor of up to 50, and impaired zymosan-induced mouse peritonitis along with reduced 5-LO product
levels. Other pro-inflammatory targets of curcumin (i.e., mPGES-1, cyclooxygenases, 12/15-LOs, nuclear factor-κB, nuclear factor-
erythroid 2-related factor-2, and signal transducer and activator of transcription 3) were hardly affected by 11. The strict structural
requirements for mPGES-1 and 5-LO inhibition strongly suggest that specific interactions rather than redox or membrane effects
underlie the inhibition of mPGES-1 and 5-LO by curcumin.
cyclooxygenase (COX)-1 (IC50 = 25−50 μM),9,10 and protein
INTRODUCTION
■
kinase C (IC50 = 15 μM).11 A host of other targets respond to
The diarylheptanoid curcumin (diferuloylmethane, 1, Table 1)
is a major ingredient of turmeric (Curcuma longa L.), and is
curcumin at concentrations that can be achieved only topically
in the gastrointestinal tract (up to 20 μmol/kg) but that are
used in folk and herbal medicine for the treatment of a host of
conditions, mainly in the realm of inflammation and infection.1
unlikely to be reached in plasma or in organs after oral
application.11 The substantial dissection between the pharma-
Small scale human clinical trials have suggested anti-
inflammatory and anticancer properties for curcumin,2 fostering
codynamic and the pharmacokinetic profiles of curcumin, and
the lack of solid clinical documentation of activity, make it
an intense research activity on this dietary compound. There is
difficult to evaluate the potential of this dietary compound in
a general agreement that curcumin exerts anticancer and anti-
inflammatory effects by interfering with multiple targets.
medicine.12 We and others have recently identified curcumin as
a potent dual inhibitor of microsomal prostaglandin E2 synthase
However, only few of them are sensitive to the submicromolar
(mPGES)-1 and 5-lipoxygenase (5-LO) that are key enzymes at
concentrations of this compound that can be achieved by
supplementation with various formulations of curcumin.3 Thus,
the interface of inflammation and cancer. The submicromolar
IC50 values (0.2−0.7 μM)9,13 are in the range of the plasma
curcumin activates the redox-regulated transcription factor
nuclear factor-erythroid 2-related factor-2 (Nrf2, at 30 μM),4
concentrations of curcumin conjugates observed upon oral
administration of various formulations3,14 or megadosages of
interferes with nuclear factor (NF)-κB, p53 and signal
transducer and activator of transcription (STAT)3 signaling
(NF-κB, IC50 ≈ 20 μM; p53, at 10 μM; STAT3 ≈ 10−25 μM),5−7
Received: February 26, 2014
and inhibits histone acetyltransferases (IC50 = 20−40 μM),8
© XXXX American Chemical Society
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the unformulated natural product.15 Metabolic curcumin
conjugates are in general devoid of anti-inflammatory activity,2
but mPGES-1 and 5-LO are strongly expressed in inflamed and
tumor tissues,11,12 where accumulation of free curcumin has
been observed as a result of the local upregulation of
deconjugating enzymes.16 mPGES-1 and 5-LO catalyze the
formation of the potent pro-inflammatory lipid mediators
prostaglandin (PG)E2 and leukotrienes (LTs) that not only
promote inflammation, allergy, pain, and fever but also possess
pro-tumorigenic properties.17,18 For their biosynthesis, arach-
idonic acid is first released from phospholipids by phospholi-
pases A2 and then transformed by COX and 5-LO to PGH2 and
LTA4, respectively.17,19 PGH2 is further converted by mPGES-1
to PGE2,17 while LTA4 is hydrolyzed to the chemotactic LTB4
or metabolized to cysteinyl-LTs, which induce bronchocon-
striction and elevate vascular permeability.19 Pharmacological
strategies to intervene with LTs are based on antagonists of the
G-protein-coupled LT receptors as well as on inhibitors of 5-
LO (i.e., zileuton), which are used in asthma therapy.20
Moreover, preclinical studies suggest anti-LT therapy being
effective for the treatment of cardiovascular disease and
cancer.19,20 The high potency of the dietary diarylheptanoid
curcumin to inhibit mPGES-1 and 5-LO together with its
selective accumulation in inflamed and tumor tissues qualify
these inducible enzymes as primary targets, providing a
potential pharmacodynamic and pharmacokinetic basis for the
“smart” mechanism of action of curcumin, which lacks
significant toxicity and side effects unlike most anti-inflamma-
tory agents.
A large number of curcumin derivatives have been synthesized
and assayed on diverse molecular targets with often relatively flat
SARs.21 Surprisingly, only few studies focused on eicosanoid
biosynthesis, despite its critical role in the biological profile of
curcumin. These studies addressed the direct inhibition of
mammalian COX-1/2,10,22 mPGES-1,22 5-LO,23 and 12-LO24 as
well as the inducible (and NF-κB-dependent) expression of
COX-2.25,26 The limited number of analogues investigated and the
focus on few read-out systems make it difficult to draw
comprehensive predictions about the regulation of the eicosanoid
profile. To address this limitation, we have investigated the effect
of curcumin and a series of analogues on various enzymes of
eicosanoid biosynthesis (mPGES-1, 5-, 12-, and 15-LO, COX-1,
COX-2) as well as on inducible transcription factors, such as NF-
κB, STAT3 and Nrf2, that are involved in inflammatory and
carcinogenic processes. We present evidence that the activity of
curcumin on mPGES-1 and 5-LO can be fundamentally dissected
and, in the case of 5-LO, significantly improved by structural
modification, suggesting that specific interactions govern the
interference of curcumin with its high-affinity targets.
Scheme 1. Synthesis of the Novel Curcuminoids 11−13
potency of curcumin against these two targets suggests the
involvement of specific molecular interactions. To evaluate this
hypothesis, we investigated if, and how, chemical modification
could affect inhibition of mPGES-1 and 5-LO by curcumin in cell
free systems. To this aim, we focused on (i) the 1,3-diketone motif
(truncation and replacement with a pyrazole ring), (ii) the
interaryl linker (reduction), and (iii) the aryl substitution pattern
of the natural product (introduction of substituents ortho to the
free hydroxyl). The biological profile of curcuminoids is, indeed,
critically sensitive to these maneuvers,18 and aryl fluorination and
prenylation were selected for their potential to alter the
pharmacokinetic properties of phenolics.31,32 The role of
curcumin’s interaryl linker was evaluated by comparing the activity
of curcumin with that of compounds 2−4, the archetypal
members of the classes of truncated (C5-) curcuminoids and
tetrahydrocurcuminoids, respectively. Because of the complete
tautomerization of the dicarbonyl system at physiological pH (i.e.,
pH 7.2 to 7.4), curcumin is a weak acid33 like arachidonic acid and
PGH2, the natural substrates of 5-LO and mPGES-1, respectively.
The monoketonic C5-curcuminoid 2 is not enolizable but was
almost equipotent to curcumin in terms of inhibiting the activity of
purified 5-LO, while the diketonic C5-curcuminoid 3 and
tetrahydrocurcumin 4 were 5−6-fold less effective (Table 1), the
latter in accordance with a previous finding.9 However, all three
modifications were detrimental for inhibition of mPGES-1,
because compounds 2−4 did not, or only slightly, suppress the
activity of this enzyme up to 10 μM (Table 1). Thus, shortening
the interaryl linker of curcumin substantially dissects mPGES-1
from 5-LO inhibition, even if the enolizable 1,3-dicarbonyl system
is preserved. Nonspecific antioxidant activity does not seem to be
involved in the inhibition of mPGES-1 or 5-LO because
all compounds were poor radical scavengers (EC50 > 10 μM,
Table 1); ascorbic acid (16; used as positive control) worked as
expected.
RESULTS AND DISCUSSION
■
Chemistry. The fluorinated analogues 12 and 13 (Table 1)
were prepared from 4-fluorovanillin (18)27 by condensation
with the boric complex of acetylaceton (Pabon reaction)28 or
with acetone under acidic conditions.29 The prenylated pyrazole
derivative 11 was obtained by reacting its corresponding
prenylcurcuminoid (7)29 with hydrazine hydrate (Scheme 1).30
All other compounds were prepared according to literature (see
Experimental Section).
The pyrazole analogue of curcumin 5 has previously been
described as potent inhibitor of LT formation by activated rat
neutrophils, and 5-LO was suggested as a molecular target.23
Replacement of the 1,3-dicarbonyl system of curcuminoids with
a pyrazole ring retains the hydrogen donor and acceptor
properties of the enolized 1,3-dicarbonyl system of curcumin.
Because this element is critical for the interaction with mPGES-1,
we speculated that pyrazole analogues of curcumin might combine
Structure−Activity Relationships. In agreement with
previous findings,9,13 curcumin (1) was found to be an equipotent
inhibitor of 5-LO and mPGES-1 in cell-free activity assays with
IC50 values of 0.5 and 0.6 μM, respectively (Table 1). The high
B
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Table 1. Stability of Curcumin and Structural Analogues and Effects on mPGES-1, 5-LO, and Antioxidant Activity
a
IC50 values (μM) and residual activity (% of control) are given as mean SEM of single determinations obtained in three to four independent
b
c
experiments. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001; student t-test. No inhibition/no effect. Residual activity at 10 μM compound
concentration. Not determined. Residual activity at 50 μM compound concentration. Residual amount of compound (% of control).
d
f
g
superior inhibition of 5-LO with decreased inhibition of
mPGES-1. In fact, both the pyrazole analogues of curcumin
(5) and C5-curcumin (6) potently interfered with purified
human recombinant 5-LO, outperforming curcumin by a factor
of 6 and 7, respectively (Table 1), while inhibition of mPGES-1
was considerably reduced (IC50 ≥ 10 μM).
C
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Table 2. Effect of Curcumin and Structural Analogues on COX-1, COX-2, 12-LO, and 15-LO
a
COX-1
COX-2
neutrophils
a
a
enzyme
platelets
enzyme
12-LO
15-LO
b
c
a
1
ni
8.1 0.5
ni
65.0 4.4**,
70.1 3.5**,
60.6 5.5**,
ni
a
a
2
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
3
ni
ni
a
4
ni
87.4 10.1
ni
a
c
5
82.3 5.3
ni
7.3 2.4
ni
c
6
ni
ni
ni
ni
ni
ni
ni
7.7 1.7
ni
7
80.2 26.1
ni
ni
89.7 3.3
a
8
82.7 16.0
ni
76.4 4.1*,
ni
ni
ni
ni
ni
ni
nd
a
9
ni
ni
75.7 1.9***,
a
10
78.7 8.7
ni
64.7 9.7
a
11
ni
ni
87.2 7.8
ni
68.7 3.2**,
69.7 5.3*,
67.7 5.7*,
c
a
12
ni
ni
7.5 0.1
a
a
a
13
65.2 3.2**,
5.9 3.1***,
d
indomethacin
25.0 2.8***
43.0 2.4***
nd
a
b
c
Residual activities (% of control) at a compound concentration of 10 μM. No inhibition. IC50 values are given as mean SEM of single
d
determinations obtained in three independent experiments. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001; student t-test. Not determined.
To further explore the structural determinants for mPGES-1
and 5-LO inhibition and to modulate the pharmacodynamic
and pharmacokinetic profile of the resulting analogues, we
introduced either a fluorine- or a (dihydro)prenyl-group on the
aromatic ring. Distinct fluorinated curcuminoids have been
described to inhibit COX-1 and COX-2 more potently than
curcumin,25 and we speculated that fluorination of curcumin,
apart from slowing its metabolism, might also affect its
interaction with 5-LO and mPGES-1. Prenylated curcuminoids
are unknown as natural products,34 but prenylation is a classic
strategy to improve the bioavailability of phenolics,35 which
is of particular interest due to the poor oral absorption of
curcumin. Prenyl-groups are frequently found in phenolic 5-LO
and/or mPGES-1 inhibitors.36−40 Here, prenylation (7 and 11)
was clearly detrimental for the inhibition of mPGES-1 but
had little effect on the inhibition of purified 5-LO by either
curcumin, C5-curcumin (2), or the pyrazole analogue 5 (Table 1).
In contrast, prenylation unexpectedly turned the poorly active
analogue C5-curcuminoid 2 into the moderate mPGES-1 inhibitor
10, and dihydroprenylation either reduced (9) or improved (8)
inhibition of purified 5-LO depending on the parent compound
(curcumin or tetrahydrocurcumin 4, Table 1). Opposing SARs
were also observed when fluorine was introduced on the aromatic
ring of either curcumin (yielding 12) or C5-curcumin (2, yielding
13). While the curcumin derivative 12 inhibited mPGES-1 less
potently than the parent compound, both inhibition of mPGES-1
and purified 5-LO was enhanced by fluorination of C5-curcumin
(13; Table 1). The 5-LO reference drug 14 (N-[(E)-3-(3-
phenoxyphenyl)prop-2-enyl]acetohydroxamic acid; BWA4C)41
and the mPGES-1 inhibitor 15 (2-(2-chlorophenyl)-1H-
phenanthro[9,10-d]-imidazole; MD52)42 were used as control
(Table 1). Taken together, our SAR study has identified three
complementary strategies to dissect mPGES-1 and 5-LO inhibition
by curcumin, namely (i) truncation of the dicarbonyl system and/
or its replacement with a pyrazole ring, (ii) hydrogenation of the
interaryl linker, and (iii) (dihydro)prenylation.
increased both by introduction of fluorine- (12, 13) or
(dihydro)prenyl-groups (7−11) on the aromatic ring and by
disruption of the α,β-unsaturated 1,3-diketone motif through
either hydration (4, 8), truncation (2, 10, 13), or replacement by
pyrazole (5, 6, 11) (Table 1).
Effect of Curcuminoids on Key Enzymes of Eicosanoid
Formation and Selected Molecular Targets of Curcumin.
Studying the interference of multitarget compounds like
curcumin with specific metabolic pathways requires a compre-
hensive evaluation of the related target profile. For the synthesis
of eicosanoids, particularly COX and LO isoenzymes have to be
taken into account because curcumin was previously described as
a direct but weak inhibitor of COX-1, COX-2, and 12-LO
(IC50 ≥ 16 μM).10,13,24,25 In line with these studies, we found
that curcumin and its derivatives (up to 10 μM) did not, or only
slightly, inhibit COX-1 and -2 as well as 12- and 15-LO activities
in cell-free assays, in intact human platelets (COX-1), and/or in
activated human neutrophils (5-, 12-, 15-LO) (Table 2). Weak
inhibition of COX-1 in intact platelets was particularly evident
for the fluorinated curcumin 12 (IC50 ≥ 7.5 μM), and 12-LO
product formation was considerably suppressed by the pyrazole
analogues 5 and 6 (IC50 ≥ 7.3 μM). Conversely, prenylation of 5
to generate 11 strongly reduced the potency to inhibit 12-LO.
These observations show that the chemical modification of
curcumin can generate selective inhibitors of 5-LO.
Next, we investigated the effect of curcuminoids on
previously proposed molecular targets (i.e., NF-κB and
STAT3 signaling and Nrf2 activation) not directly linked to
the production of eicosanoid but capable of affecting the
expression of genes involved in inflammation and carcino-
genesis. SARs toward NF-κB and Nrf2 (a transcription factor
regulating antioxidant response genes) were previously ex-
plored for the curcumin derivatives 2, 4, 7, 8, and 10.29 In this
context, prenylation (7) and hydrogenation (4) were detrimental
for anti-NF-κB activity, while truncation to C5-curcuminoids (2)
had an opposite effect.29 Here, we show that these modifications
influence STAT3 signaling and Nrf2 activation in the same way
(Table 3). The electrophilic compound 229 thus represents a
potent NF-κB/STAT3 dual inhibitor with potential antitumoral
activity in addition to being a potent inhibitor of 5-LO (Table 1)
and activator of Nrf2 (Table 3). On the other hand, the inclusion
of pyrazole (5 and 6) increased the potency of curcuminoids to
Curcumin is not stable in aqueous solutions at physiological
pH and rapidly degrades mainly into trans-6-(4′-hydroxy-3′-
methoxyphenyl)-2,4-dioxo-5-hexenal as previously shown43 and
confirmed in the present study by reversed phase liquid
chromatography ESI mass spectrometry (Table 1 and Supporting
Information Figure S1). The stability of curcuminoids is strongly
D
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Table 3. Effect of Curcumin and Structural Analogues on NF-κB, Nrf2 and STAT3 Transcriptional Activities and Cell Viability
a
cell viability
a
a
b
NF-κB
STAT3
Nrf2
LNCaP
PBMC
1
20.9 2.6
1.8 1.7***
28.3 3.2
>50
33.9 2.3
6.0 2.4***
35.7 7.9
>50
9.9 5.9
0.9 0.4
negative
negative
negative
negative
negative
negative
negative
negative
8.6 4.1
3.1 0.9
36.0 3.8
10.0 3.4***
>50
34.8 5.0
34.7 7.1
>50
2
3
4
>50
>50
5
29.8 3.5
30.0 8.3
>50
16.2 9.9**
18.2 2.7**
>50
>50
>50
6
>50
>50
7
>50
>50
8
33.4 3.6
32.3 2.6
>50
>50
>50
>50
10
11
12
13
22.4 6.6*
>50
43.8 6.4
>50
>50
>50
>50
15.6 8.4**
19.4 7.4**
>50
>50
16.8 0.5**
25.1 4.8*
>50
a
IC50 values (μM) are given as mean SEM of single determinations obtained in four independent experiments. (*) P < 0.05, (**) P < 0.01, (***)
b
P < 0.001 compared to curcumin; student t-test. EC50 values (μM) give the concentration of half-maximal activity compared to tert-
butylhydroquinone (20 μM).
Figure 1. Effect of curcuminoids on eicosanoid formation in activated human monocytes. Lipopolysaccharide-primed monocytes were preincubated
with vehicle (DMSO), curcumin (“open triangle”) or 11 (“closed circle”) for 15 min. Eicosanoid formation was initiated by 2.5 μM A23187 plus
20 μM arachidonic acid. Data are expressed as means SEM of single determinations obtained in two to three independent experiments. *P < 0.05,
**P < 0.01, ***P < 0.001 vs vehicle control; ANOVA + Tukey HSD posthoc tests.
inhibit 5-LO 5−7-fold (Table 1) but neither activated Nrf2 nor
induced cytotoxicity (Table 3). The effect of 5 and 6 on NF-κB
and STAT3 signaling was comparable or even significantly
enhanced compared to curcumin. In contrast, the prenylated C7
curcuminoids 7 and 11 (derived from curcumin and pyrazole 5,
respectively) neither interfered with NF-κB and STAT3 signal-
ing nor activated Nrf2 or were cytotoxic (Table 3). Although
compounds targeting NF-κB and STAT3 pathways are being
developed mainly for the treatment of cancer, it is expected that
chronic inhibition of these pathways could induce side effects such
as immunosuppression and/or loss of homeostatic renewal cells.
In the same sense, Nrf2 was shown to promote the formation and
chemoresistance of solid cancers,44 thereby acting as proto-
oncogene. Altogether, our results highlight compound 11 as an
outstanding selective 5-LO inhibitor.
Activity of Curcuminoids on 5-LO Product Formation
in Intact Cells. Compounds that inhibit 5-LO in cell-free
assays are not necessarily active in intact cells.36 Hence, we
investigated the effect of curcumin and its derivatives on
5-LO product formation in stimulated human neutrophils and
E
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monocytes. 5-LO products include LTB4, its all-trans isomers,
5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 5,12-
dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-DiHETE).
Although SARs were similar for inhibition of purified 5-LO
and neutrophil 5-LO activity within this series of curcuminoids,
suppression of cellular 5-LO activity was, in general, less marked
than under cell-free conditions but still in the (sub)micromolar
range (Table 1). Exceptions are (dihydro)prenylated curcumin 7
and 9 and the fluorinated curcuminoids 12 and 13, which were
considerably less potent in intact cells (Table 1). The most
efficient inhibitors of 5-LO within our series were the pyrazoles 5,
6, and 11, as expected from the data obtained in the cell-free assay.
These compounds were 2−52-fold more potent in suppressing
5-LO product formation than curcumin both in activated
neutrophils (Table 1) and monocytes (Figure 1, Supporting
Information Figures S2 and S3; pyrazoles, IC50 = 0.013−1.9 μM;
curcumin, IC50 = 0.6−3.8 μM). Remarkably, the prenylated
C5-curcuminoid 10 was 2-fold superior to curcumin in inhibiting
neutrophil 5-LO product formation despite being equipotent in
the cell free assay (Table 1).
Table 4. Effect of Compound 11 on Zymosan-Induced
Peritonitis in Mice
cysteinyl-LTs
cell
MPO
c
b
c
c
(LTC4)
LTB4
[pg/mL]
infiltration
activity
a
treatment
vehicle
[ng/mL]
[106/mL]
[U/mL]
123 10
682 112 7.64 0.51 1.23 0.13
11
10
92%
1
d
267 92
1.91 0.35 0.75 0.18
75%*** 39%
61%*
e
17 (MK-886)
nd
nd
258 48
4.54 0.33 0.72 0.13
41%*** 41%*
63%**
a
Male mice (n = 5−11 for each experimental group, with age of
8−9 weeks) were treated ip with 10 mg/kg compound 11, 1 mg/kg
compound 17, or vehicle (2% DMSO) 30 min before zymosan
b
injection. Analysis was performed 30 min after zymosan injection.
c
Analysis was performed 4 h after zymosan injection. Values are
d
given as mean SEM of single determinations per mouse. Italics:
percent inhibition of vehicle control. (*) P < 0.05, (**) P < 0.01,
e
(***) P < 0.001; student t-test. Not determined.
Effect of Curcuminoids on the Eicosanoid Profile of
Activated Human Monocytes. Structural modification of
curcumin might not only influence the interaction with existing
targets but also create novel ones, especially because eicosanoid
formation is a complex process involving multiple isoenzymes,
regulatory loops, and compensatory mechanisms.45 To recognize
additional high-affinity targets of pyrazolocurcuminoids, the most
potent 5-LO inhibitors of this series, we monitored the spectrum
of eicosanoids produced by activated human monocytes using
ultraperformance liquid chromatography-coupled ESI tandem
mass spectrometry (UPLC-MS/MS). At low physiologically
relevant concentrations, curcumin preferentially suppressed the
formation of 5-LO products, but also the formation of PGE2,
PGF2α, PGD2, thromboxane (Tx)B2, 11-HETE, and 15-HETE
was markedly reduced (IC50 =1.2−5.4 μM; Figure 1). The
generation of PGE2 (and other prostanoids) was also inhibited by
the nonprenylated pyrazole analogues 5 and 6 (Supporting
Information Figures S2 and S3), which is surprising as they exhibit
clearly impaired inhibitory potency against mPGES-1 (Table 1). In
contrast, the prenylated pyrazole 11 exclusively interfered with the
synthesis of 5-LO products (IC50 = 0.013−0.4 μM) up to 10 μM
(Figure 1), suggesting that prenyl substitution is critical for the
pronounced selectivity of 11. Specific inhibitors of 5-LO (14,
BWA4C), COX-1/2 (indomethacin), and mPGES-1 (15, MD52)
were used as control and inhibited eicosanoid biosynthesis as
expected (Supporting Information Figure S4).
Efficiency of the Prenylated Pyrazolocurcuminoid 11
in Zymosan-Induced Peritonitis in Mice. The prenylated
pyrazolocurcuminoid 11 is the most selective inhibitor of 5-LO
in cellular systems within this series. Thus, we investigated its
effectiveness in an in vivo model of acute inflammation, namely
zymosan-induced mouse peritonitis, which is critically dependent
on the formation of LTs and other lipid mediators.46 Compound
11 (10 mg/kg, ip) suppressed the levels of cysteinyl-LTs (LTC4
derived from resident peritoneal macrophages in the early phase47)
and LTB4 (derived predominantly from infiltrating neutrophils in
the late phase47) as well as cell infiltration and exudate myeloper-
oxidase (MPO; a neutrophil marker) activity comparable or even
superior to the well-recognized LT synthesis inhibitor 17 (3-(3-
(tert-butylthio)-1-(4-chlorobenzyl)-5-isopropyl-1H-indol-2-yl)-2,2-
dimethylpropanoic acid; MK-886, 1 mg/kg, ip)48 (Table 4).
Taken together, despite its pleiotropic bioactivity profile,
curcumin can selectively modulate eicosanoid biosynthesis at
physiologically relevant concentrations with distinct SARs
revealing prenylated pyrazol derivatives, exemplified by 11, as
selective 5-LO inhibitors with promising pharmacological
profile.
CONCLUSION
■
Our study provides evidence that curcumin interacts with its
high-affinity targets mPGES-1 and 5-LO in a specific manner,
while a flat scenario of SARs was observed for the low affinity
targets COX-1/2 and 12/15-LOs. Inhibition of mPGES-1 was
critically dependent on the 1,3-diketone motif, possibly because
an acidic enol proton is required. More structural variations were
tolerated for inhibition of 5-LO (as in 2, 7, 8, 10, 12, and 13),
with the nonenolizable pyrazole analogues (5, 6, and 11)
showing remarkably enhanced inhibitory activity. Furthermore,
the effect of (dihydro)prenylation, as in 7−11, was strongly
dependent on the length and the nature of the aryl−aryl linker,
with significant differences in terms of mPGES-1 and 5-LO
inhibition. Pyrazole derivatives of curcumin have previously
been reported as potent inhibitors of LT biosynthesis in rat
basophil leukocytes (IC50 = 1 μM) and in a rat model of acute
inflammation.23 In principle, this activity could be due to a
direct inhibition of 5-LO, an impaired activation of 5-LO, a
reduced substrate supply to 5-LO, or a combination of these
effects. Our data clearly show that these compounds are direct
inhibitors of 5-LO, essentially devoid of significant activity on
mPGES-1 and, when prenylated, with a remarkable selectivity
for 5-LO within eicosanoid biosynthesis and among pro-
inflammatory targets of curcumin. More than 100 different
targets have been identified for curcumin so far, including
druggable transcription factors, receptors, and enzymes.15 The
(partially redundant) interference of curcumin with multiple
cellular networks might explain its “cure-all” efficacy in cellular
and (pre)clinical studies. However, a growing number of
negative clinical trials49 suggest that this pleiotropic profile
could be difficult to translate clinically, not only because of the
limited bioavailability of curcumin but also due to the potential
cacophonic response associated with its interaction with such a
large array of targets. By showing that specific interactions, and
not simply membrane effects or antioxidant activities, underlie
the capacity of curcumin to directly modulate the production
of inflammatory eicosanoids, we suggest that this compound
F
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Journal of Medicinal Chemistry
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(235 μL, 100 μL/mmol) were sequentially added. The reaction was
stirred at 40 °C overnight and quenched with the addition of brine and
extraction with EtOAc. The crude product was purified by gas column
chromatography on silica gel (petrol ether/EtOAc 6:4 as eluent) to
might represent a privileged structure for the selective
modulation of distinct druggable macromolecules as well as
for obtaining more potent and target-focused leads (such as the
novel prenylated pyrazole derivative 11).
1
afford 13 as an orange solid (275 mg, 65%); mp 190 °C. H NMR
(300 MHz, CDCl3) δ: 7.60 (2H, d, J = 15.6 Hz), 7.06 (2H, dt, J =
10.8, 1.8 Hz), 6.90 (2H, d, J = 15.6 Hz), 6.91 (2H, d, J = 1,8 Hz), 3.97
(6H, s). 13C NMR (acetone-d6, 75 MHz) δ 187.7, 152.9, 149.8, 149.6,
149.6, 139.8, 139.8, 137.2, 137.0, 126.3, 126.2, 124.5, 109.3, 109.0,
107.6, 56.1. (+) ESI-MS m/z 385 [M + Na]+. HR-ESI-MS m/z
385.0879 (calcd for C19H16F2O5Na, 385.0863).
EXPERIMENTAL SECTION
■
Solvents and Reagents. Curcumin was purchased from Sigma-
Aldrich (Deisenhofen, Germany). Compounds 2,35 3,50 4,29 5,30 6,30
7,29 8,29 9,29 and 1029 were prepared according to literature. 1H
(300 MHz) and 13C (75 MHz) NMR spectra were measured on a Jeol
Eclipse 300 spectrometer. Chemical shifts were referenced to the
residual solvent signal (CDCl3, δH = 7.26; acetone-d6, δC = 77.0).
Low- and high-resolution ESI-MS spectra were obtained on a LTQ
OrbitrapXL mass spectrometer (Thermo Scientific). Silica gel 60
Blood Cell Isolation and Cultivation of Cell Lines. Human
platelets, neutrophils, and monocytes were freshly isolated from
peripheral blood obtained at the Institute for Transfusion Medicine of
the University Hospital Jena (Germany) as described.51 Venous blood
from healthy adult donors was subjected to centrifugation (4000g/
20 min/20 °C) to prepare leukocyte concentrates. Cells were promptly
isolated by dextran sedimentation and centrifugation on Nycoprep
cushions (PAA, Coelbe, Germany). The pellet was resuspended,
erythrocytes were lysed under hypotonic conditions, and neutrophils
were recovered by centrifugation. For the isolation of platelets, the
supernatant obtained after centrifugation on Nycoprep cushions
(platelet-rich plasma) was mixed with PBS pH 5.9 (3:2 v/v) and
centrifuged (2100g, 15 min). The pellet was washed with PBS pH 5.9/
0.9% NaCl (1:1, v/v). Monocytes were isolated from the peripheral
blood mononuclear cell (PBMC) fraction by adherence to culture flasks
(Greiner, Nuertingen, Germany). Washed PBMCs were seeded at 2 ×
107 cells/mL in RPMI 1640 medium containing L-glutamine (2 mM)
and penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively;
monocyte medium). Adherent cells were collected after 1.5 h at 37 °C
and 5% CO2. The purity of the monocyte preparation was >85% as
defined by forward- and side-light scatter properties and detection of the
CD14 surface molecule by flow cytometry (BD FACS Calibur,
Heidelberg, Germany). Washed cells were finally suspended in PBS
pH 7.4 plus 1 mM CaCl2 (platelets) containing 1 mg/mL glucose
(neutrophils) or monocyte medium supplemented with FCS (5%, v/v;
monocytes). The experimental protocol was approved by the ethics
committee of the University of Jena.
Human lung adenocarcinoma epithelial A549 cells were cultured in
DMEM/high glucose (4.5 g/L) medium supplemented with heat-
inactivated FCS (10%, v/v), penicillin (100 U/mL), and streptomycin
(100 μg/mL) at 37 °C and 5% CO2. Confluent cells were detached
using 1× trypsin/EDTA and reseeded at 2 × 106 cells in 20 mL
medium in 175 cm2 flasks.
HaCaT-ARE-luc and LNCaP (human prostate carcinoma) cell lines
were cultured in DMEM containing 10% FCS, L-glutamine (2 mM),
penicillin (50 U/mL), and streptomycin (100 μg/mL). 5.1 cells were
cultured in RPMI 1640 containing 10% FCS, penicillin (50 U/mL),
and streptomycin (100 μg/mL).
Preparation of mPGES-1. A549 cells were treated with
interleukin-1β (2 ng/mL) at 37 °C and 5% CO2 for 48 h to induce
mPGES-1. Harvested cells were frozen in liquid nitrogen and then
taken up in ice-cold homogenization buffer (0.1 M potassium
phosphate buffer pH 7.4, 1 mM phenylmethanesulphonyl fluoride,
60 μg/mL soybean trypsin inhibitor, 1 μg/mL leupeptin, 2.5 mM
glutathione, and 250 mM sucrose). After incubation for 15 min, cells
were sonicated on ice (3 × 20 s) and centrifuged at 10000g for 10 min.
The microsomal fraction (pellet) was obtained by centrifugation of the
supernatant at 174000g for 1 h at 4 °C. The pellet was resuspended in
homogenization buffer and diluted in potassium phosphate buffer
(0.1 M, pH 7.4) containing 2.5 mM glutathione to a concentration of
25−50 μg total protein/mL.
Determination of mPGES-1 Activity. mPGES-1 activity in
microsomes of A549 cells was determined as described.52 Microsomes
of interleukin-1β-treated A549 cells, used as source of mPGES-1, were pre-
incubated with the test compounds (dissolved in DMSO) for 15 min
at 4 °C. The reaction was started by addition of PGH2 (20 μM, final
concentration; reaction volume, 100 μL) and stopped after 1 min by
addition of stop solution (100 μL; 40 mM FeCl2, 80 mM citric acid, and
(70−230 mesh) was purchased from Macherey-Nagel (Duren,
̈
Germany). Reactions were monitored by TLC on Merck 60 F254
(0.25 mm) plates that were visualized by UV inspection and/or
staining with 5% H2SO4 in ethanol and heating. Organic phases were
dried with Na2SO4 before evaporation. The purity (95% or higher) of
all final products evaluated for reactivity and bioactivity was assessed
by analytical HPLC (isocratic elution, 70% acetonitrile/30% water
containing 1% formic acid), with UV detection at 240 nm on a JASCO
Herculite apparatus equipped with a Phenomenex Luna C18 column.
All test compounds were dissolved in DMSO, stored in the dark at
−20 °C, and freezing/thawing cycles were kept to a minimum. The
mPGES-1 inhibitor 15 (MD52) was kindly provided by Dr. M.
Schubert-Zsilavecz (University of Frankfurt, Germany). Materials
used: DMEM/high glucose (4.5 g/L) medium, penicillin, streptomy-
cin, trypsin/EDTA solution, PAA (Coelbe, Germany); PGH2, Larodan
(Malmo, Sweden); 11β-PGE , PGB , human recombinant COX-2,
̈
2
1
ovine COX-1, Cayman Chemical (Ann Arbor, MI). Solvents, 14
(BWA4C) and all other chemicals were obtained from Sigma-Aldrich
(Deisenhofen, Germany) unless stated otherwise.
4,4′-(1E,1′E)-2,2′-(1H-Pyrazole-3,5-diyl)bis(ethene-2,1-diyl)-
bis(2-methoxy-6-(3-methylbut-2-enyl)phenol) (11). To a stirred
solution of 7 (200 mg, 0.39 mmol, 1 equiv) in AcOH (1 mL),
hydrazine monohydrate (44 μL, 0.897 mmol, 2.3 equiv) was added.
The reaction was stirred overnight at 60 °C, and then quenched by
addition of brine and extraction with EtOAc. After evaporation, the
residue was triturated with ether to afford 157 mg (80%) of an orange
foam. 1H NMR (300 MHz, CDCl3) δ 7.01 (2H, d, J = 16.4 Hz), 7.00
(4H, br m), 6.82 (2H, d, J = 16.3 Hz), 6.54 (1H, s), 5.33 (2H, t, J = 5.8
Hz), 3.90 (3H, s), 3.89 (3H, s), 3.35 (4H, d, J = 7.0 Hz), 2.10 (6H, br
s), 2.09 (6H, br s). 13C NMR (acetone-d6, 75 MHz) δ 146.7, 144.0,
133.0, 132.1, 131.3, 128.1, 127.7, 122.2, 122.1, 121.6, 117.7, 114.5,
110.2, 105.9, 56.1, 28.1, 25.9, 17.9. (+) ESI-MS m/z 523 [M + Na]+.
HR-ESI-MS m/z 523.2784 (calcd for C31H36N2O4Na 523.2573).
5,5′-Difluorocurcumin (12). To a stirred solution of 3-fluoro-4-
hydroxy-5-methoxybenzaldehyde (540 mg, 3.2 mmol, 1 equiv) in dry
DMF (3.3 mL), B2O3 (225 mg, 3.2 mmol, 1 equiv), 2,4-pentandione
(173 μL, 1.6 mmol, 0.5 equiv) and B(OCH3)3 (355 μL, 3.2 mmol,
1 equiv) were sequentially added. The reaction was stirred at 90 °C,
and n-butylamine (71 μL) was added over a period of 2.5 h. After 1 h,
the reaction was cooled to 60 °C, and 5% AcOH (8 mL) was added.
The mixture was stirred at 60 °C for 3 h, during which a precipitate
was formed. After filtration and washing with water, the crude product
was purified by gas column chromatography on silica gel (petrol ether/
EtOAc 7:3 as eluent) to afford 5,5′-difluorocurcumin (12) as an
orange solid (324 mg, 50%); mp 195 °C. 1H NMR (300 MHz,
CDCl3) δ 7.54 (2H, d, J = 15.7 Hz), 7.00 (2H, dd, J = 10.8, 1.8 Hz,),
6.85 (2H, br t, J = 1.8 Hz), 6.46 (2H, d, J = 15.7 Hz), 5.79 (s, 1H),
3.96 (s, 6H). 13C NMR (acetone-d6, 75 MHz) δ 183.5, 153.0, 149.8,
149.6, 149.6, 139.8, 139.8, 137.1, 136.9, 126.4, 126.2, 122.9, 109.1,
108.8, 107.5, 101.5, 56.0. (+) ESI-MS m/z 427 [M + Na]+. HR-ESI-MS
m/z 427.0951 (calcd for C21H18F2O6Na 427.0969).
(1E,4E)-1,5-Bis(3-fluoro-4-hydroxy-5-methoxyphenyl)penta-
1,4-dien-3-one (13). To a stirred solution of 3-fluoro-4-hydroxy-5-
methoxybenzaldehyde (400 mg, 2.35 mmol, 1 equiv) in EtOH
(2.5 mL), acetone (85 μL, 1.17 mmol, 0.5 equiv), and HCl (37%)
G
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10 μM of 11β-PGE2 as internal standard). PGE2 was extracted and
analyzed by reversed phase-HPLC as described.52
stimulated with the compounds for 6 h. To study STAT3 signaling,
LNCaP cells were transiently transfected with the pTATA-TK-Luc
plasmid that contains four copies of the STAT-binding site. Transient
transfections were performed with Rotifect (Carl Roth GmbH,
Karlsruhe, Germany) according to the manufacturer’s instructions,
and 24 h after transfection, cells were preincubated with increasing
concentrations of the compounds and then treated with interleukin-6
(10 ng/mL) for another 12 h. After stimulation of the transfected cell
lines, cells were washed twice with PBS pH 7.4 and lysed in 25 mM
Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100,
and 7% glycerol for 15 min at room temperature in a horizontal shaker.
Following centrifugation, the supernatant was used to measure luciferase
activity using an Autolumat LB 9510 (Berthold) following the instructions
of the luciferase assay kit (Promega, Madison, WI, USA).
Expression and Purification of Human Recombinant 5-LO.
Escherichia coli Bl21 (DE3) cells were transformed with pT3−5LO
plasmid, lysed in 50 mM triethanolamine/HCl pH 8.0 plus EDTA
(5 mM), soybean trypsin inhibitor (60 μg/mL), phenylmethanesul-
phonyl fluoride (1 mM), dithiothreitol (1 mM), and lysozyme
(1 mg/mL) and then sonified (3 × 15 s). The homogenate was
centrifuged at 10000g for 15 min, and the remaining supernatant at
40000g for 70 min at 4 °C. 5-LO in the supernatant was partially
purified by affinity chromatography on an ATP-agarose column as
described.53 Semipurified 5-LO was diluted in PBS containing EDTA
(1 mM) and ATP (1 mM) and immediately used for activity assays.
Activity Assay for Human Recombinant 5-LO. Human
recombinant 5-LO was preincubated with the test compounds for
10 min at 4 °C and prewarmed for 30 s at 37 °C. 5-LO product formation
was initiated by addition of 2 mM CaCl2 and 20 μM arachidonic acid.
After 10 min at 37 °C, the reaction was terminated by addition of 1 mL of
ice-cold methanol. Formed 5-LO metabolites (all-trans isomers of LTB4
and 5-H(P)ETE) were analyzed by RP-HPLC as described.40
Determination of Radical Scavenging Activity. Compounds
were dissolved in ethanol and combined with an equal volume of
100 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) in ethanol (final
volume: 200 μL). After incubation for 30 min, absorbance was
measured at 520 nm using a Multiskan Spectrum microplate reader
(Thermo Scientific) as described.46
Determination of the Stability of Curcuminoids in Aqueous
Solution. Curcuminoids (2 μM) in 10 mM aqueous ammonium
bicarbonate pH 7.9 were incubated for 30 min at 37 °C. Degradation
was stopped by acidification to pH 3.3 with an equal volume of ice-
cold methanol plus 0.14% formic acid. Curcuminoids and curcumin’s
main degradation product trans-6-(4′-hydroxy-3′-methoxyphenyl)-2,4-
dioxo-5-hexenal were immediately quantified by reversed phase liquid
chromatography-coupled mass spectrometry.
Cytotoxicity Assays. LNCaP cells (104 cells/well) and human
PBMC (2 × 105/well) were seeded in 96-well plates (LNCaP, 200 μL;
monocytes, 100 μL) and incubated with the compounds for 24 h.
MTT (5 mg/mL; LNCaP, 100 μL; monocytes, 20 μL) was added to
each well, and cells were incubated for 4 h at 37 °C and 5% CO2 in
darkness. The formazan product was solubilized with SDS (10%, w/v
in 20 mM HCl) for monocytes and after removal of the supernatant by
addition of DMSO (100 μL) for LNCaP cells. Finally, the absorbance
was measured at 550 nm using a TriStar LB 941 Microplate Reader
(Berthold Technologies, GmbH & Co. KG) and at 570 nm using a
Multiskan Spectrum microplate reader (Thermo Scientific), respec-
tively. The absorbance of untreated cells was taken as 100% viability to
calculate cytotoxic IC50 values.
Analysis of the Eicosanoid Profile of Activated Monocytes.
Freshly isolated monocytes (5 × 106/6 well plate) were stimulated
with lipopolysaccharide (1 μg/mL) for 24 h at 37 °C and 5% CO2.
Cells were washed, preincubated with the test compounds for 15 min,
and treated with 2.5 μM Ca2+-ionophore A23187 plus 20 μM
arachidonic acid for 30 min. Then, the reaction was stopped and
eicosanoids extracted as described.56
Determination of 5-LO Product Formation in Neutrophils.
Freshly isolated neutrophils (1 × 107/mL) were preincubated with the
test compounds for 15 min at 37 °C. Then, 5-LO product formation
was started by addition of 2.5 μM Ca2+-ionophore A23187 plus 20 μM
arachidonic acid. The reaction was stopped after 10 min at 37 °C with
1 mL of methanol. Major 5-LO metabolites (LTB4 and its all-trans
isomers and 5-H(P)ETE) were extracted and analyzed by HPLC as
described.54 Cysteinyl-LTs C4, D4, and E4 and oxidation products of
LTB4 were not determined.
Determination of COX-1 Activity in Washed Platelets.
Freshly isolated platelets (108/mL) were preincubated with the test
compounds for 5 min at room temperature and prewarmed at 37 °C
for 1 min. Formation of 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrie-
noic acid (12-HHT; which is nonenzymatically formed from
COX-derived PGH2) was started by addition of 5 μM arachidonic
acid. After incubation for 5 min at 37 °C, 12-HHT was extracted and
analyzed by RP-HPLC as described.51
Activity Assays of Isolated COX-1 and -2. Purified ovine COX-
1 (50 units) or human recombinant COX-2 (20 units) were
preincubated with the test compounds in reaction buffer (1 mL;
100 mM Tris buffer pH 8, 5 mM glutathione, 5 μM hemoglobin, and
100 μM EDTA) for 5 min at 4 °C followed by 1 min at 37 °C.
Arachidonic acid (COX-1:5 μM; COX-2:2 μM) was added to initiate
COX product formation. COX-derived 12-HHT was extracted after
5 min and analyzed by HPLC as described.51
Determination of NF-κB, STAT3, and Nrf2 Transcriptional
Activities. Anti-NF-κB activity was investigated in 5.1 cells, a
validated in vitro model to study TNFα-induced NF-κB activation.55
5.1 cells were stimulated with TNFα (20 ng/mL) in the presence or
absence of the compounds for 6 h. To study the activity of the
compounds on the Nrf2 pathway, we generated the HaCaT-ARE-Luc
cell line. Nqo1 Antioxidant Response Element (ARE)-Luc reporter
plasmid and pPGK-Puro plasmid were cotransfected into HaCaT cells.
After antibiotic selection, the cells were cloned by limiting dilution,
and several positive clones responding to tert-butylhydroquinone were
pooled to generate the HaCaT-ARE-Luc cell line. Cells were
Reversed Phase Liquid Chromatography and Mass Spec-
trometry. Eicosanoids were separated on an Acquity UPLC BEH
C18 column (1.7 μm, 2.1 mm × 50 mm, Waters, Milford, MA) using
an Acquity UPLC system (Waters, Milford, MA, USA) as previously
described.56 In brief, chromatography was performed at a flow rate of
0.8 mL/min and a column temperature of 45 °C. The solvents for the
mobile phase were acetonitrile (A) and water/acetonitrile (90/10; B),
both acidified with 0.07% (v/v) formic acid. Isocratic elution at A/B =
30/70 was performed for 2 min and followed by a linear gradient to
A/B = 70/30 within 5 min. In variation, curcuminoids and trans-6-
(4′-hydroxy-3′-methoxyphenyl)-2,4-dioxo-5-hexenal were analyzed at
a flow rate of 0.7 mL/min using a gradient from A/B = 30/70 to 80/
20 within 3 min and 100% B for 1 min. The retention times are
summarized in Supporting Information Table S1.
The chromatography system was coupled to a QTRAP 5500 mass
spectrometer (AB Sciex, Darmstadt, Germany) equipped with an
electrospray ionization source. For eicosanoid analysis, parameters
were adjusted as described.56 Identification of eicosanoids was based
on the detection of specific fragment ions through multiple reaction
monitoring. Monitored transitions and their collision energies are
given in Supporting Information Table S2. The transition first
mentioned (“transition 1”) was used for quantification. Curcuminoids
and trans-6-(4′-hydroxy-3′-methoxyphenyl)-2,4-dioxo-5-hexenal were
analyzed by full scans in the negative (1−8, 10−13) and/or positive
ion mode (1−3, 6, 9, 11). The ion spray voltage was set to 4500 V in
the negative and 5500 V in the positive ion mode, the heated capillary
temperature to 500 °C, the sheath gas and auxiliary gas pressure to
50−60 psi, and the declustering potential to 50 V in the negative and
190 V in the positive ion mode. Automatic peak integration was
performed with Analyst 1.6 software (AB Sciex, Darmstadt, Germany)
using IntelliQuan default settings. For eicosanoid analysis, data were
normalized on the internal standard PGB1 and are given as relative
intensities. The reported method was optimized to compare eicosanoid
profiles between samples and not for absolute quantification.
Zymosan-Induced Peritonitis in Mice. Male CD-1 mice (33−39 g,
Charles River Laboratories, Calco, Italy) were housed in a controlled
H
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environment (21 2 °C) and provided with standard rodent chow
and water. 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. Animal care
was 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 E.C. L 358/1 12/18/1986). The animal studies were
approved by the local ethical committee of the University of Naples
Federico II on 27th of February 2014 (2014/0018760).
Mouse peritonitis was induced and parameters analyzed as
previously described.46 In brief, compounds 11 (10 mg/kg), 17
(1 mg/kg), or vehicle (0.5 mL of 0.9% saline solution containing 2%
DMSO) were given ip 30 min before zymosan ip injection (0.5 mL of
a 2 mg/mL suspension in 0.9% w/v saline). Mice were killed by
inhalation of CO2 at the indicated time points followed by a peritoneal
lavage with 3 mL of cold PBS pH 7.4. Exudates were collected, and
cells were counted with a light microscope in a Burker’s chamber after
vital trypan blue staining. After centrifugation of the exudates (18000g,
5 min, 4 °C), the amounts of cysteinyl-LTs and LTB4 were analyzed in
the supernatant by enzyme immunoassay (Enzo Life Sciences GmbH,
ABBREVIATIONS USED
■
COX, cyclooxygenase; H(P)ETE, hydro(pero)xy-6-trans-
8,11,14-cis-eicosatetraenoic acid; 12-HHT, 12(S)-hydroxy-5-
cis-8,10-trans-heptadecatrienoic acid; LO, lipoxygenase; LT,
leukotriene; MPO, myeloperoxidase; NF-κB, nuclear factor-κB;
Nrf2, nuclear factor-erythroid 2-related factor-2; mPGES,
microsomal prostaglandin E2 synthase; PG, prostaglandin;
PBMC, peripheral blood mononuclear cells; STAT3, signal
transducer and activator of transcription 3
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Lorrach, Germany) according to manufacturer’s instructions. For
̈
determining MPO activity, the pelleted cells were disrupted, the
homogenate centrifuged, and the supernatant (20 μL) mixed with
0.167 mg/mL of o-dianisidine and 0.0005% hydrogen peroxide in PBS
pH 7.4 (200 μL) as described.46 The change of absorbance was
monitored in kinetic mode using an iMark microplate absorbance
reader (Biorad). Levels of MPO were determined from a calibration
curve using human neutrophil MPO as reference standard and are
expressed as units MPO per mL (U/mL).
Statistics. Data are expressed as mean standard error (SEM).
Statistical evaluation of the data was performed by one-way ANOVAs
for independent or correlated samples followed by Tukey HSD
posthoc tests or by Student’s t test for paired and correlated samples. P
values <0.05 were considered statistically significant. All statistical
calculations were performed using GraphPad InStat 3.10 (GraphPad
Software Inc., La Jolla, CA, USA). IC50 values were determined by graphical
analysis using SigmaPlot 12.0 (Systat Software Inc., San Jose, USA).
ASSOCIATED CONTENT
■
S
* Supporting Information
Retention times of curcuminoids, conditions for UPLC-MS/
MS analysis of eicosanoids, stability of curcumin as well as
effect of compound 5 and 6, and control inhibitors on the
eicosanoid profile of activated human monocytes. This material
is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49-3641-949815. Fax: +49-3641-949802. E-mail:
andreas.koeberle@uni-jena.de.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Katrin Fischer, Baerbel Schmalwasser, Heidi Traber,
and Petra Wiecha for expert technical assistance. Work in the
Novara laboratories was supported by MIUR (project
2009RMW3Z5: Metodologie Sintetiche per la Generazione di
̀
Diversita Molecolare di Rilevanza Biologica).
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