Discovery of a highly potent anti-inflammatory epoxyisoprostane-derived lactone

Article abstract of DOI:10.1021/ja509892u

Epoxyisoprostanes EI (1) and EC (2) are effective inhibitors of the secretion of proinflammatory cytokines IL-6 and IL-12. In detailed studies toward the investigation of the molecular mode of action of these structures, a highly potent lactone (3) derived from 1 was identified. The known isoprostanoids 1 and 2 are most likely precursors of 3, the product of facile intramolecular reaction between the epoxide with the carboxylic acid in 2.

Full text of DOI:10.1021/ja509892u

Communication
pubs.acs.org/JACS
Discovery of a Highly Potent Anti-inflammatory Epoxyisoprostane-
Derived Lactone
Julian Egger,† Peter Bretscher,‡ Stefan Freigang,‡ Manfred Kopf,‡ and Erick M. Carreira*,†
†Laboratory of Organic Chemistry, ETH Zurich, HCI H335, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland
‡Institute for Integrative Biology, ETH Zurich, HPL-G 12, Schafmattstrasse 27, 8093 Zurich, Switzerland
S
* Supporting Information
underscore the role of the enone as the key structural feature
ABSTRACT: Epoxyisoprostanes EI (1) and EC (2) are
effective inhibitors of the secretion of proinflammatory
cytokines IL-6 and IL-12. In detailed studies toward the
investigation of the molecular mode of action of these
structures, a highly potent lactone (3) derived from 1 was
identified. The known isoprostanoids 1 and 2 are most
likely precursors of 3, the product of facile intramolecular
reaction between the epoxide with the carboxylic acid in 2.
responsible for biological activity as an anti-inflammatory agent.
The bioactivity of cyclopentenone (iso-)prostanes is versatile
and resides both in interesting receptor-based mechanisms on
their properties as potent electrophiles.4−6 For the latter, OxPLs
have been shown to undergo conjugate addition reactions with
common nucleophiles, such as thiols found in intracellular
domains.7 This plays an important role in the oxidative stress
response pathway, in which electrophiles such as eicosanoid
enones bind to the cytoplasmic Keap1 complex. Upon binding,
transcription factor Nrf2 is released from the complex, which
then translocates into the nucleus, turning on the expression of
antioxidant proteins.8 In this context, the epoxyisoprostanes are
especially intriguing because, in addition to electrophilic enones,
they include an embedded allylic epoxide. The latter is expected
to be highly susceptible to nucleophilic ring-opening reactions.
In this respect, in previous work, treatment of an EI-derived ether
with pentyl amine was observed to lead to an amino alcohol
resulting from nucleophilic ring opening of the allylic epoxide
(Scheme 2). This led to the proposal that similar reactions of
xidized phospholipids (OxPLs) comprise a collection of
Obiomolecules that have gained increasing attention as
important, highly potent regulators of various physiological
functions in higher organisms.1 Among the different classes of
OxPLs, the epoxyisoprostanes, such as EI (1) and EC (2) are
particularly interesting, as their phosphatidylcholine-bound
versions have been shown to play a pivotal role in the early
development of atherosclerosis and other inflammatory
conditions.2 Recently, we disclosed a synthesis of epoxyisopros-
tanes 1 and 2 and, in accompanying biological studies, noted
their unprecedented anti-inflammatory effects. This was manifest
as the observed reduced secretion of proinflammatory cytokines
IL-6 and IL-12 in bone-marrow-derived dendritic cells
(BMDCs).3 Additionally, we demonstrated that free acids 1
and 2 are more potent than their phospholipid-bound
derivatives; significantly, 2 was shown to be more potent than 1.
Over the course of studies aimed at understanding the
chemistry and biology of 1 and 2, we noted the ready formation
of lactone 3 from 2 under physiologically relevant conditions
(Scheme 1). Herein, we report the discovery of EC-derived
lactone 3 as the most highly potent anti-inflammatory compound
in the series as inhibitors of proinflammatory cytokines IL-6 and
IL-12 secretion. We also document a collection of studies that
Scheme 2. Previous Model with Electrophilic Epoxide as Key
to Biological Activity
lysines would account for the molecular mode of action for 1 and
2.9 However, it is important to note that these previous studies
were conducted with an ether function at C1 rather than the
native carboxylic acid.
The synthesis to prepare 1 and 2 provides convenient access to
a wide range of derivatives of isoprostanoids. We surmised that
these could serve as probes with which to examine the mode of
action of this class of oxidized lipids. Because our initial
investigations specifically underscored the high potency of
dienone 2, we sought to identify the origin of activity with
enhanced resolution.
Scheme 1. EI (1), EC (2), and EC-Derived Lactone 3 and
Their Relative Activity as Inhibitors of Proinflammatory
Cytokines IL-6 and IL-12 Secretion
In our initial investigations, for the purposes of calibration, we
compared the dose-dependent potency of 2 with known “anti-
Received: September 25, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja509892u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
inflammatory prostaglandin” 15d-PGJ2 (4, Figure 1a)4a and
noted the superior potency of 2 in reduced secretion of
a
Scheme 3. Synthesis of EC Analogues 5 and 6
a
Reagents and conditions: (a) i-Pr2NLi, 9, THF, −78 °C; then
TMSCHN2, C6H6/MeOH, rt; (b) MsCl, Et3N, CH2Cl2, −78 °C; then
Al2O3, CH2Cl2, rt; (c) Novozyme, buffer pH 7/THF (4:1), 46% over 3
steps; (d) LiHMDS, 11, THF, −78 °C; (e) MsCl, Et3N, CH2Cl2, −78
°C; then Al2O3, CH2Cl2, rt, 55% over 2 steps; (f) Novozyme, buffer
pH 7/THF (4:1), 78%.
Figure 1. Comparison of the activity of prostaglandin 15d-PGJ2 (4) and
EC (2) to inhibit secretion of proinflammatory cytokines IL-6 and IL-12
in BMDCs. (a) Effects at varying doses of 4 and 2. (b) Structural
comparison of 4 and 2. (c) Effects of 4 and 2 at a concentration of 2 μM
compared to the solvent control.
enone 8 and epoxyaldehyde 9 that is prepared by organocatalytic
enantioselective epoxidation of (E)-oct-2-enal10 afforded methyl
ester 10, which was transformed into acid 5 by enzymatic
saponification. Synthesis of 6 was achieved in a similar fashion:
aldol condensation of cyclopentenone 12 with (E)-enal 11
furnished methyl ester 13 that in turn was converted into
trienone 6.
proinflammatory cytokines IL-6 and IL-12 in BMDCs as it
becomes even more obvious when compared at single
concentration points (e.g., 2.0 μM, Figure 1c). This finding
compelled us to compare and contrast the structures of the two
lipids (Figure 1b), especially with respect to the influence of the
Cα side chain. In this respect, 2 and 4 incorporate electrophilic
endo- and exocyclic enones (red stars), but differ by the absence
of the epoxide in the latter and the disposition of the carboxylic
acid (Cα vs Cβ side chains, blue boxes). In our initial synthesis
studies, we noted that, under mild acidic conditions, such as on
silica gel, 2 undergoes isomerization into lactone 3 through a 6-
exo-tet cyclization process (Scheme 1). This observation sparked
our interest to investigate the contribution of the structure of the
Cα side chain to anti-inflammatory effects and, specifically, to
assess whether lactone 3 was active. If the latter were shown to be
active, then the biological activity of this series of compounds
would not be due to the electrophilic epoxide.
Preparation of 3 and 7 commenced with 2 (Scheme 4).
Lactone 3 had been observed to form as a side product during
a
Scheme 4. Synthesis of EC Analogues 3 and 7
a
Reagents and conditions: (a) SiO2, CHCl3, rt, 65%; (b) K2CO3,
MeOH, rt; (c) Novozyme, buffer pH 7/THF (4:1), 30% over 2 steps.
column chromatographic purification of 2 on silica gel.11
Accordingly, 2 was dissolved in a slurry of silica gel in CHCl3
to afford 3 in 65% yield. Direct hydrolysis of lactone 3 to 7 under
alkaline conditions proved difficult as a consequence of the
instability of the product. Instead, a two-step procedure was
enacted in which the lactone was first opened in alkaline
methanol to furnish the corresponding methyl ester, prior to
enzymatic hydrolysis to yield diol 7.
To assess the activity of lactone 3, a set of EC analogues 5−7
with 3 was synthesized to systematically probe the influence of
the Cα side chain (Figure 2). Compound 5 results from
Parent compounds 2 and 4 along with the various analogues
synthesized were examined in assays to probe reduction in the
secretion of IL-6 and IL-12 in a dose-dependent manner (Figure
3). All compounds were active as inhibitors, albeit with a range of
efficiencies. Importantly, none of 5−7 were more active than 2.
Figure 2. Set of EC analogues prepared and investigated.
switching the position of the carboxylic acid from the Cα to the
Cβ side chain, so that the epoxide and the carboxylic acid are no
longer proximal and thereby unlikely to participate in macro-
lactonization. Trienone 6 displays a 1,6-dienone similar to that
found in 4. Comparison of the potency of structures 5 and 6 with
parent structures 2 and 4 could give insight into the effects of a
1,6-dienone versus a 1,4-(5,6-epoxy)enone on the electro-
philicity of the oxidized lipid. Diol acid 7 was investigated to
determine whether lactone 3 is a precursor to a more active diol
seco-acid derivative.
Synthesis of 5 begins with cyclopentenone 8 (Scheme 3).
Compound 8 is an intermediate in the synthesis of 4 and was
prepared in analogy to 12.3 Stepwise aldol condensation of
Figure 3. Comparison of the activity of prostaglandin 15d-PGJ2 (4), EC
(2), and analogues 3 and 5−7 in inhibiting secretion of proinflammatory
cytokines IL-6 and IL-12 in BMDCs.
B
dx.doi.org/10.1021/ja509892u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
a
Analogue 5 was more active than 4 and trienone 6. This result
gives insight into the inductive effect of the epoxide compared to
the corresponding γ,δ-enone, where it becomes evident that the
presence of the epoxide enhances the potency of the compounds.
However, since 5 was also less active than parent 2, the epoxide
cannot be solely responsible for the superior potency of 2.
Examination of lactone 3 and diol 7 led to a surprising
observation. Although seco-acid 7 is less active than 2, lactone 3
elicits the strongest effect of all compounds examined to date in
inhibiting the secretion of proinflammatory IL-6 and IL-12.
The large concentration intervals used in the assays illustrated
in Figure 3 resulted in only three assessment data points for
lactone 3 before the cytotoxic concentration was reached. To
examine the activity of 2 and 3 at greater resolution, a second
titration series with smaller concentration intervals was
conducted (Figure 4). The observed results show that 3 is
Scheme 5. Synthesis of the EC Analogues 15, 19, and 21
a
Reagents and conditions: (a) LiHMDS, THF, −78 °C; then methyl
7-oxoheptanoate; (b) MsCl, Et3N, CH2Cl2, −78 °C; then Al2O3,
CH2Cl2, rt, 57% (2 steps); (c) Novozyme, buffer pH 7/THF (4:1), rt,
77%; (d) LiHMDS, THF, −78 °C; then 17; (e) MsCl, Et3N, CH2Cl2,
−78 °C; then Al2O3, CH2Cl2, rt, 64% (2 steps); (f) [(PPh3)CuH]6
(0.175 equiv), water, C6H6, rt, 47%; (g) Novozyme, buffer pH 7/THF,
rt, 56%; (h) [(PPh3)CuH]6 (0.35 equiv), water, C6H6, rt, 65%; (i)
Novozyme, buffer pH 7/THF, rt, 51%. Blue boxes indicate alterations
with respect to EC (2).
a
Scheme 6. Synthesis of EC-Analog 26
a
Reagents and conditions: (a) t-BuOOH, DBU, THF, 0 °C, 74%; (b)
Novozyme, buffer pH 7/THF (4:1), rt, 74%; (c) [(PPh3)CuH]6 (0.28
equiv), water, C6H6, rt, 74%, dr = 10:1; (d) SmI2, THF−MeOH (7:3),
−90 °C, 86%; (e) MsCl, Et3N, CH2Cl2, 90%; (f) Novozyme, buffer
pH 7/THF (4:1), rt, 65%. Blue boxes indicate alterations with respect
to EC (2).
Figure 4. Titration assays of the highly potent compounds EC (2) and
lactone 3 to establish their activity in inhibiting secretion of
proinflammatory cytokines IL-6 and IL-12 in BMDCs. (a) Effects at
varying doses of 2 and 3. (b) Effects of 2 and 3 at a common
concentration of 0.53 μM compared to the solvent control.
more potent than 2 even at very lowest concentrations, which is
surprising in light of the prior proposal suggesting the epoxide as
the reactive site.
6). The higher reactivity of the endocyclic enone rendered the
selective reduction of the exocyclic enone difficult. A method
published by Evans and Fu for the selective reduction of (E)-
enones, leaving (Z)- or endocyclic enones intact, was not
successful.13 Thus, we decided to exploit the increased reactivity
of the endocyclic enone for the preparation of 26. The endocyclic
enone in dienone 16 was epoxidized selectively under
nucleophilic conditions to provide epoxyketone 22. Exposure
of 22 to Stryker’s reagent gave reduced epoxide 23 that, upon
reductive epoxide opening with SmI2, delivered β-hydroxy
ketone 24.14 Mesylation of the β-hydroxy resulted in
concomitant elimination and regeneration of the endocyclic
enone to yield methyl ester 25, which was further elaborated into
26.
The new analogues obtained were tested together with parent
2 for their ability to reduce IL-6 and IL-12 secretion (Figure 5).
Cyclopentanone 21 showed no biological activity, which
indicates that the presence of an enone in the molecule is crucial
for the investigated bioactivity. Compound 19, which no longer
contained an endocyclic enone, exhibited drastically diminished
potency compared to 2. In contrast, 26, which is lacking the
exocyclic enone, still displays considerable potency, although it is
less active than the parent compound 2. If no epoxide is present
in the molecule, as in 15, the potency of the molecule is reduced
and comparable to that of 26.
Having established the superiority and unexpected activity of
lactone 3, we subsequently set out to identify the electrophilic
site in 2 necessary for bioactivity in the assays. Analogue
structures 15, 19, 21, and 26 were prepared from cyclopentenone
12, as shown in Schemes 5 and 6. Dienone 15, lacking an
epoxide, was accessed by aldol addition with methyl 7-
oxoheptanoate, followed by elimination to generate enone 14.
Hydrolysis of methyl ester 14 to free acid 15 was carried out
enzymatically using Novozyme. Dienone 16, which incorporates
an epoxide, served as precursor for 19 and 21. In our previous
synthetic studies to 3, we observed that the endocyclic enone in 2
reacted selectively in the presence of 1 equiv of nucleophile,
especially when the nucleophile is sterically demanding.
Consequently, Stryker’s reagent [(PPh3)CuH]6 was used to
conduct enone reduction.12 Selective conjugate reduction of the
endocyclic enone was accomplished when 0.175 equiv of the
hexameric reducing complex was employed to obtain methyl
ester 18. With 0.35 equiv, both enones were reduced to furnish
methyl ester 20. Enzymatic hydrolysis of 18 and 20 afforded free
acids 19 and 21.
Analogue 26, in which the exocyclic enone was reduced
selectively, proved to be more challenging to prepare (Scheme
C
dx.doi.org/10.1021/ja509892u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
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́
,
The observation that 2 undergoes ready conversion to lactone
3 has provided key insights into the chemistry of this class of
isoprostanes. Importantly, the greater activity of the lactone,
which lacks an epoxide, underscores that this plays a minor role
as an electrophilic site for decreasing IL-6 and IL-12 secretion.
Additional investigations of structures in which the endocyclic
enone is absent destroy the molecule’s capability for cytokine
inhibition completely, revealing that the endocyclic enone is
crucial for biological activity. Prior investigations by us revealed
that 1 readily undergoes elimination in aqueous media,3 and
indeed, it is well worth noting that analysis of the NMR spectra
first reported for 1 reveals the presence of signals consistent with
2.2a When combined with the results from this study, it leads to a
hypothesis that lactone 3, as the most active agent, elicits the
observed anti-inflammatory effects. It can be speculated that
highly potent lactone 3 is formed to some degree under
physiological conditions from 2. Lactone 3 is not only the
chemically most stable compound from the series 1−3 but, in
turn, might also constitute a longer lived version of free acid 2
being stabilized against β-oxidation and therefore exhibiting
higher activity in vivo.15 This explanation could account for the
significant difference in biological activity between 4 and 2, as
only the latter is able to form the lactone. The results set the stage
for additional challenging experiments involving detection of the
various structures intracelullarly in real time. Additionally, it
provides new leads for the development of anti-inflammatory
therapeutics.
́
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ASSOCIATED CONTENT
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S
* Supporting Information
Experimental procedures and characterization data for all
reactions and products, including H and 13C NMR spectra.
1
Bretscher, P.; Egger, J.; Shamshiev, A.; Trotzmuller, M.; Kofeler, H.;
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This material is available free of charge via the Internet at http://
pubs.acs.org.
Carreira, E. M.; Kopf, M.; Freigang, S. Manuscript submitted.
AUTHOR INFORMATION
Corresponding Author
carreira@org.chem.ethz.ch
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to the ETH Zurich for generous support through
grant ETH-18 09-1.
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dx.doi.org/10.1021/ja509892u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX