An epoxyisoprostane is a major regulator of endothelial cell function

Article abstract of DOI:10.1021/jm400959q

The goal of these studies was to determine the effect of 5,6-epoxyisoprostane, EI, on human aortic endothelial cells (HAEC). EI can form as a phospholipase product of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine, PEIPC, a proinflammatory molecule that accumulates in sites of inflammation where phospholipases are also increased. To determine the effect of EI on HAEC, we synthesized several stereoisomers of EI using a convergent approach from the individual optically pure building blocks, the epoxyaldehydes 5 and 6 and the bromoenones 14 and 16. The desired stereoisomer of EI can be prepared from these materials in only six operations, and thus, large amounts of the product can be obtained. The trans/trans isomers had the most potent activity, suggesting specificity in the interaction of EI with the cell surface. EI has potent anti-inflammatory effects in HAEC. EI strongly inhibits the production of MCP-1, a major monocyte chemotactic factor, and either decreases or minimally increases the levels of 10 proinflammatory molecules increased by PEIPC. EI also strongly down-regulates the inflammatory effects of IL-1β, a major inflammatory cytokine. Thus EI, a hydrolytic product of PEIPC, has potent anti-inflammatory function.

Full text of DOI:10.1021/jm400959q

Article
pubs.acs.org/jmc
An Epoxyisoprostane Is a Major Regulator of Endothelial Cell
Function
Wei Zhong,†,⊥ James R. Springstead,‡,⊥ Ramea Al-Mubarak,§ Sangderk Lee,‡ Rongsong Li,∥
Benjamin Emert,‡ Judith A. Berliner,‡ and Michael E. Jung*,†
‡
†Department of Chemistry and Biochemistry and Department of Medicine, University of California, Los Angeles, 405 Hilgard
Avenue, Los Angeles, California 90095, United States
§Department of Chemical and Paper Engineering, Western Michigan University, 4601 Campus Drive, Kalamazoo, Michigan 49008,
United States
∥Department of Biomedical Engineering and Division of Cardiovascular Medicine, University of Southern California, Los Angeles,
California 90089, United States
S
* Supporting Information
ABSTRACT: The goal of these studies was to determine the
effect of 5,6-epoxyisoprostane, EI, on human aortic endothelial
cells (HAEC). EI can form as a phospholipase product of 1-
palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocho-
line, PEIPC, a proinflammatory molecule that accumulates in
sites of inflammation where phospholipases are also increased. To
determine the effect of EI on HAEC, we synthesized several
stereoisomers of EI using a convergent approach from the individual optically pure building blocks, the epoxyaldehydes 5 and 6
and the bromoenones 14 and 16. The desired stereoisomer of EI can be prepared from these materials in only six operations, and
thus, large amounts of the product can be obtained. The trans/trans isomers had the most potent activity, suggesting specificity
in the interaction of EI with the cell surface. EI has potent anti-inflammatory effects in HAEC. EI strongly inhibits the production
of MCP-1, a major monocyte chemotactic factor, and either decreases or minimally increases the levels of 10 proinflammatory
molecules increased by PEIPC. EI also strongly down-regulates the inflammatory effects of IL-1β, a major inflammatory cytokine.
Thus EI, a hydrolytic product of PEIPC, has potent anti-inflammatory function.
hydrolyze PEIPC11 increase in atherosclerotic lesions and can
INTRODUCTION
■
release EI. In order to facilitate studies of the action of 5,6-
epoxyisoprostane, EI, on HAEC and to facilitate the synthesis
of PEIPC, we developed an efficient strategy for the synthesis
of a series of epoxyisoprostane (EI) analogues. We previously
demonstrated that four regioisomers of PEIPC can be formed
during oxidation.9 However, the isomer where the epoxide
group was located in the 5,6-position was the most active and
most readily formed during PAPC oxidation.9 Therefore, we
synthesized four optically pure stereoisomers of this EI for
testing in our studies. Their ability to regulate HAEC function
was then examined. We present evidence that EI has many
functions in common with PEIPC and may be a useful analogue
to identify pathways regulated by both agents. However, EI
treatment of HAEC results in lower production of inflamma-
tory cytokines than PEIPC, actually decreases constitutive
levels of MCP-1, and inhibits the action of IL-1β, a cytokine
highly increased in atherosclerotic lesions.
In vitro lipid oxidation products of 1-palmitoyl-2-arachidonoyl-
sn-phosphatidylcholine (PAPC) have been shown to accumu-
late in oxidized lipoproteins, the membranes of cells exposed to
oxidative stress, and in apoptotic and necrotic cells.1−3 In vivo
they have been shown to accumulate in atherosclerotic lesions
and other sites of chronic inflammation.2,4−6 One of the
oxidation products that accumulates is 1-palmitoyl-2-(5,6-
epoxyisoprostane E2)-sn-glycero-3-phosphocholine, PEIPC.4
Using microarray analysis of human aortic endothelial cells
(HAEC) from 147 donors, our group has shown that a 4 h
treatment with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-
3-phosphocholine (Ox-PAPC) regulates more than 1000 genes.
PEIPC regulates 80% of these genes. Some of the pathways
activated are protective against cell death including increases in
unfolded protein response (UPR), inhibition of DNA synthesis,
and an increase in antioxidant genes.7 PEIPC and Ox-PAPC
also have proinflammatory effects, increasing the binding of
monocytes to HAEC and increasing the production of
chemotactic factors that cause monocytes to enter the vessel
wall.8−10 PEIPC is the most active oxidation product of Ox-
PAPC with regard to effects on HAEC inflammatory molecules,
being active in the low micromolar range.7 Previous studies
have demonstrated that phospholipases (PLA2) that can
Received: June 26, 2013
Published: October 13, 2013
© 2013 American Chemical Society
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RESULTS
Scheme 2. Synthesis of Pure Bromoenones 14 and 16
■
Synthesis of EI Analogues. On the basis of our previous
work on the synthesis of PEIPC,12,13 we designed a more
efficient synthetic route to produce EI4 4 and its analogues
EI1−EI3 (1−3, Figure 1). Since the p-methoxybenzyl (PMB)
Figure 1. Structures of EI analogues.
ether was the key protecting group, because it was removable
under nonbasic conditions, we decided to install it at an earlier
stage than previously described. Then our previous developed
triply convergent coupling strategy could be followed to achieve
the synthesis of the EI analogues.
The components for the key coupling were prepared as
follows. The optically pure epoxyaldehydes 5 and 6 were
prepared by our previous synthetic route via initial selective
protection of pentane-1,5-diol as the monosilyl ether (Scheme
1), followed by Swern oxidation to the aldehyde, Horner−
very selectively and gave, after flash column chromatography,
only the cis-isomer 10 in 94% yield as a mixture of enantiomers.
Enzymatic kinetic resolution of the racemic mono-PMB ether
of cyclopent-1-ene-3,5-diol 10 was achieved using lipase AK
with vinyl acetate in methyl ethyl ketone (MEK) as solvent.
This enzyme-catalyzed transesterification produced both
enantiomeric products in excellent yield and optical purity,
namely, the 3R,5S-5-p-methoxybenzyloxycyclopent-1-en-3-ol
11 (47% yield, >99% ee) and the 3S,5R-5-p-methoxybenzylox-
ycyclopent-1-en-3-ol acetate 12 (48% yield, >97% ee). Thus,
this is an improvement of our previous route13 to produce such
important, optically active oxygenated cyclopentene systems.
The enzyme can be reused several times without loss of
efficiency or selectivity. As far as we can tell, this is the first time
an enzymatic kinetic resolution using lipase AK has been
employed to separate a racemic mixture of the mono-PMB
ether of cyclopent-1-ene-3,5-diol derivatives, although several
other ethers have been used.17 The conversion of the
enantiomerically enriched cyclopentenol 11 to the desired S-
2-bromo-4-arylmethoxycyclopentenone 14 was achieved in two
steps, via oxidation to the enone 13 with pyridinium
chlorochromate (PCC, 89%) followed by bromination in the
presence of triethylamine to give 14 in 80% yield. The
enantiomeric R-2-bromo-4-arylmethoxycyclopentenone 16 was
prepared in three steps from 12 via deprotection of the acetate
and PCC oxidation to give the enone 15 in 87% yield followed
by bromination in the presence of base to give 16 in 80% yield.
With these two building blocks 14 and 16 in hand, we
examined various processes, e.g., a silyl group transfer reaction,
to introduce substituents on the enone. The silyl group transfer
of the enone 14 with the silyl ketene acetal 17 produced an
inseparable 1:1 mixture of the two silyl enol ether esters 18
(Scheme 3). The analogous reaction catalyzed by either HgI2
and SmI2 gave lower yields (<20%) than the reaction using
TiCl4, which gave the highest yield (77%).18 Selective reduction
of the ester of 18 to the aldehyde with diisobutylaluminum
hydride (DIBAL) at −78 °C allowed for the easy chromato-
graphic separation of the trans and cis disubstituted bromoenol
Scheme 1. Synthesis of Epoxyaldehydes 5 and 6
Wadsworth−Emmons reaction to give the E-enoate, and
reduction to give the E-allylic alcohol. Subjection of this
alcohol to Sharpless asymmetric epoxidation, with either L- or
D-dialkyl tartrate, and final oxidation with Dess−Martin
periodinane gave the optically pure epoxides 5 and 6.13
The second key components were the desired optically pure
intermediates 14 and 16, which were prepared in an efficient,
high-yielding six- or seven-step sequence (Scheme 2). The
route featured an enzymatic resolution of the mono-PMB-
protected 1,4-cyclopentenediol and La(OTf)3-promoted PMB
ether formation. The commercially available, inexpensive
furfuryl alcohol 7 was converted into 4-hydroxycyclopentenone
8 in 46% yield by refluxing under mildly acidic conditions (pH
4.1).14 Even though the yield was somewhat low, the ready
availability of the starting materials and ease of synthesis made
this the preferable method for preparing 8. Our initial attempts
to install the PMB ether protecting group using traditional
methods of base and PMB chloride or bromide resulted in low
yields (<40%). However, treatment of 4-hydroxycyclopente-
none 8 with freshly prepared p-methoxybenzyl trichloroaceti-
midate in the presence of a catalytic amount of lanthanum
triflate15 in toluene at 21 °C gave the desired product 9 in 90%
yield. Luche reduction16 of the α,β-unsaturated ketone of 9
using sodium borohydride and cerium(III) chloride proceeded
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analogous set of reactions on the other four aldehydes, 20, 21,
and 22, gave the expected EI analogues, 2, 3, and 4.
Scheme 3. Synthesis of Aldehydes 19−22
Since the two trans enantiomers, 3 and 4, showed very
promising biological results (see later), we reexamined the
synthetic route in an attempt to improve the selectivity for the
trans isomer (Scheme 5). Our initial attempts to use a
vinylcuprate reagent19,20 for the 1,4-addition resulted in the
formation of a 2:1 ratio of the desired trans isomer to the cis
isomer. However, we also isolated up to 30% of an unexpected
byproduct, namely, the compound in which the vinyl bromide
had been replaced by a TBS group. Thus, treatment of 14 with
vinylmagnesium bromide in the presence of copper iodide and
TBSCl in THF/HMPA gave 44% of the desired trans
compound 26 and 22% of the cis compound 27 along with
30% of the silylated silyl enol ether 28 (Table 1). Addition of
lithium chloride gave no improvement, while changing the
solvent to diethyl ether raised the ratio of trans to cis to 3:1 but
still produced the unusual product. We reasoned that the
byproduct was perhaps produced because of some free radical
species formed during the preparation of the cuprate reagent.
Therefore, we decided to add some styrene as a radical
scavenger prior to the addition of the enone and the trapping
with TBSCl. As shown in Table 1, by increasing the amounts of
the additive styrene, we were able to completely prevent the
formation of the byproduct 28. Thus, the use of 2 equiv of
styrene produced an inseparable 4:1 mixture of the trans and cis
disubstituted bromoenol ethers 26 and 27 with none of the
unusual product 28 observed. Although these compounds
could not be separated at this stage, the corresponding
aldehydes, 19 and 20, could be easily separated, namely, after
hydroboration−oxidation of the vinyl group followed by Dess−
Martin periodinane oxidation of the resulting alcohol. By using
the optimized conditions were we able to prepare the desired
trans aldehyde 20 from 14 in 63% yield over three steps. By use
of this and our previously established synthetic route, the two
trans enantiomers of EI, 3 and 4, were synthesized in
reasonable quantities from 20 and 22 using the epoxyaldehydes
6 and 5, respectively (20 and 6 gave 3, and 22 and 5 gave 4).
Finally PEIPC4 was prepared by the method described in detail
in our earlier paper,8 namely, the coupling of lyso-PC with the
PMB ether of EI4 followed by removal of the PMB ether with
DDQ.
ethers 19 and 20. Starting with the enantiomeric enone 16, an
identical series of reactions (silyl transfer and reduction) gave
the two separable aldehydes 21 and 22 in similar yields.
With this protocol established, we were able to prepare
sufficient quantities of the building blocks 19, 20, 21, and 22
for completion of the synthesis. We then followed our
previously developed triply convergent coupling strategy to
afford the four EI analogues (Scheme 4).13 Thus, we first
Scheme 4. Synthesis of EI Analogue 1
Comparison of the Biological Effects of EI Stereo-
isomers. With these EI analogues in hand, we used qPCR to
examine their activity in regulating genes from several pathways
previously identified as regulated in HAEC by Ox-PAPC.7
Treatment of HAECs with 3 μM EI1, EI2, EI3, or EI4 induced
expression of IL-8, HO-1, ATF-3, and HSP1A1, representative
genes from the inflammatory, oxidative stress, unfolded protein
response, and stress response pathways (Figure 2). However,
EI3 and EI4 induced these genes to a greater extent.
Furthermore, EI4 induced these genes to a slightly greater
extent than EI3 over the course of several experiments.
Comparison of the Effect of EI3 and EI4 with the
Effect of PEIPC4. Since the fatty acid EI4 appeared to be most
active on the regulation of these important hub genes (Figure
2), we synthesized the PEIPC isomer with EI4 in the sn-2
position of the molecule (PEIPC4) (Figure 3A). Using qPCR,
we then compared the ability of EI3/4 with the effects of
PEIPC4 in regulating four important genes (Figure 3B). Since
the fatty acid EI4 appeared to be most active on the regulation
of these important hub genes (Figure 2), we synthesized the
PEIPC isomer with EI4 in the sn-2 position of the molecule
carried out an E-selective olefination via a Wittig reaction to
convert the aldehyde, e.g., 19, to the desired Z-alkene, e.g., 23,
in yields ranging from 70% to 76%. The coupling of the
cyclopentenyl bromide with the epoxyaldehyde 5 (or 6) was
carried out by first forming the alkenyllithium species by
treatment of the bromide with tert-butyllithium, followed by
addition of the epoxyaldehyde to give the allylic alcohol.
Treatment of this intermediate with aqueous formic acid
effected hydrolysis of the silyl enol ether with concomitant
dehydration and deprotection of the primary silyl ether to give
the desired PMB-protected primary alcohol, e.g., 24, a process
that occurred in 40−46% yield. A two-step oxidation of the
primary alcohol to the acid (Dess−Martin periodinane and
then chlorite oxidation) furnished the PMB ether of EI, e.g., 25,
in yields of 60−65% for the two steps. Final oxidative cleavage
of the PMB ether using dichlorodicyanoquinone (DDQ) in a
mixed solvent system (chloroform and pH 7 buffer) afforded
the desired EI analogues, e.g., 1, in 71−76% yields. The
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Scheme 5. Improved Synthesis of Aldehyde 20
Table 1. 1,4-Addition of Vinylmagnesium Bromide to Enone 14
entry
additive
solvent
temp (°C)
26/27/28
1
2
3
4
5
6
none
LiCl
none
THF
THF
Et2O
Et2O
Et2O
Et2O
−78
−78
−100
−100
−100
−100
44:22:30
40:21:28
48:16:30
64:16:15
68:17:6
1 equiv of styrene
1.5 equiv of styrene
2 equiv of styrene
72:18:0
(PEIPC4) and compared its activity with EI3 and EI4. The
PEIPC isomer 4 was prepared by the coupling of the
components 22 and 5 by the method we have already
described.13 HAECs were treated for 4 h with EI3, EI4, and
PEIPC4, and qPCR was performed. HO-1 and ATF-3 were
induced by all three lipids though these were more strongly
regulated by EI3/4 (Figure 3B). The most striking difference
was seen in the effects of EI3/4 and PEIPC4 on MCP-1 gene
regulation. Both EI3 and EI4 strongly reduced MCP-1 mRNA
levels, while PEIPC4 strongly up-regulated MCP-1. We also
tested the effect of EI4 on the protein levels of IL-8 and MCP-
1, both of which have been shown to play a role in
atherosclerosis. HAECs were treated for 4 h with varying
levels of EI4, medium was collected, and levels of protein were
measured by ELISA (Figure 4). We observed that IL-8 protein
was modestly increased at low levels of EI and actually
decreased at higher EI concentrations. MCP-1 protein was
strongly decreased as had been shown for MCP-1 message.
Treatment of HAEC with EI Results in a Lower
Induction of Inflammatory Genes than Treatment with
PEIPC. We performed microarray analysis comparing the genes
regulated by EI4 to the genes regulated by mixed isomers of
PEIPC (Figure 5 and Supporting Information Table 1). Mixed
isomers of PEIPC obtained from Ox-PAPC (subsequently
referred to as PEIPC) were employed in this array analysis so
that data could be compared to our previously published
studies using mixed isomers. There was an approximately 50%
Figure 2. Comparison of the effect of the four EI isomers on gene
regulation in HAECs. Duplicate wells of cultured HAECs were treated
for 4 h in medium containing 1% FBS with or without 3 μM EI isomer
1, 2, 3, or 4. Cells were scraped, and IL-8, HO-1, ATF-3, and MCP-1
mRNA levels were measured with qPCR. GAPDH was used to
normalize mRNA levels, and fold changes between untreated and
treated cells were obtained. Representative experiment with three
replicate wells is shown.
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Figure 3. Comparison of gene regulation by EI4 present in a phospholipid (PEIPC4) and as a free fatty acid (EI4). (A) Full MS spectra of
synthesized PEIPC4 (i.e., PEIPC containing EI isomer 4). (B) Duplicate wells of HAECs were treated for 4 h without or with EI3, EI4, or PEIPC4.
Cells were collected and selected gene expression was measured by PCR as described for Figure 2. Representative experiment with three replicate
wells is shown.
Figure 4. Dose−response effect of EI treatment on IL-8 and MCP-1 protein. HAEC triplicate wells were treated for 4 h with or without the
indicated doses of EI. Medium was collected, and concentrations were measured. Student t test was performed for significance in difference with
control samples with (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001.
a
Table 2. Inflammatory Genes Regulated by EI
gene
FC EI
FC PEIPC
MCP-1
CXCL1
NfKB1A
CXCL6
IL-8
0.2
13
1.3
4.8
4.0
1.4
17
0.79
0.59
3.0
VCAM-1
E-selectin
ICAM-1
IL-6
0.27
0.21
0.75
0.6
1.2
b
2.38
2.3
2.2
4.2
RelB
0.8
a
The microarrays shown in Figure 5 were used to examine the effect of
EI and PEIPC on inflammatory genes most correlated with MCP-1
levels. E-selectin levels from PEIPC4 qPCR results.
Figure 5. Microarray analysis: Venn diagram of genes regulated by 6
μM PEIPC vs 3 μM EI4 following a 4 h treatment. Data are filtered for
p ≤ 0.001 and fold change of 1.2 by lipids compared to untreated cells.
b
overlap in genes regulated by PEIPC and EI4. Both lipids
similarly regulated genes associated with oxidative stress, UPR,
and cell migration. However, there was a difference in the
regulation of inflammatory genes by PEIPC and EI4 (Table 2).
Compared to untreated cells, treatment of cells for 4 h with
PEIPC increased expression of 10 major inflammatory genes,
while EI either inhibited or only minimally increased expression
of these genes.
hypothesized that EI4 might inhibit the effect of a major
proinflammatory cytokine, IL-1β, which accumulates in
atherosclerotic lesions. The data obtained indicate that
exposure to 3 μM EI4 for 1 h followed by co-treatment with
EI4 and IL-1β for 4 h led to a strong inhibition of the IL-1β
increase in MCP-1 mRNA (Figure 6A). Levels of MCP-1
protein were also strongly decreased by pre- and co-treatment
with EI4 (Figure 6B). Message levels of a group of
proinflammatory genes induced by IL-1β were also decreased
by pretreatment with EI4 (Table 3).
EI4 Inhibits the Inflammatory Effects of IL-1β. On the
basis of the effects of EI4 on inflammatory molecules, we
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regulated by at least 1.2-fold in HAECs. Protective pathways,
including UPR and oxidative stress, were regulated by both
molecules, suggesting that EI will be useful in identifying these
common pathways. However, there were differences in the
effects of EI4 and PEIPC on some inflammatory molecules. In
contrast to our published PEIPC data which showed a positive
linear dose−response on IL-8 and MCP-1 protein,9 EI actually
dose dependently decreased the constitutive levels of MCP-1
and showed a bell shaped curve and a relatively low induction
of IL-8. Compared to PEIPC, EI also showed a lower increase
in message levels of eight other inflammatory molecules (Table
2). The difference in induction by EI and PEIPC was less for
some molecules such as IL-6, and other inflammatory
molecules were equally affected by both lipids (data not
shown). Importantly, we have shown for the first time that IL-
1β induction of a number of inflammatory mediators was also
inhibited by EI (Table 3). IL-1β is a cytokine that accumulates
in mice atherosclerotic lesions, and knockout of IL-1β in mice
decreases atherosclerosis.21 Furthermore, levels of IL-1β in
human plasma are increased in atherosclerosis and are elevated
in human lesions.22−24 Two clinical trials using an inhibitor of
IL-1β action have been initiated.
The data on effects of PEIPC and EI on inflammatory
molecules in HAEC would suggest that PEIPC is more
proinflammatory. In HAEC, in specific experimental conditions,
this does appear to be the case. However, even in HAEC,
PEIPC at low concentrations decreases breakdown of the
endothelial barrier (increased breakdown is seen in inflamma-
tion), whereas at higher concentrations PEIPC stimulates
barrier breakdown.25 In dendritic cells PEIPC strongly inhibits
the proinflammatory action of bacterial lipids.26,27 While the
effect of PEIPC is more complex, the effect of EI is mainly anti-
inflammatory in both HAEC and dendritic cells. Taken
together, these data suggest that both EI and PEIPC can be
anti-inflammatory at some concentrations in some cell types. It
is not yet known what determines the differences in effects on
inflammatory molecules by the two lipids in different cell types.
One possibility is that in dendritic cells PEIPC is more rapidly
degraded to EI by phospholipases. In depth kinetic studies will
be important to resolve these issues.
Figure 6. (A) MCP-1 mRNA regulation by IL-1β in the presence or
absence of EI. HAECs were treated for 1 h with or without 3 μM EI
and then for 4 h with or without IL-1β in the presence of EI. Cells
were collected and PCR performed. (B) Levels of MCP-1 protein in
medium of cells treated as shown in (A). (A) shows a representative
experiment of three replicate wells. In (B), IL1β and IL1β + EI showed
a significant difference in protein value, with p value as indicated.
Table 3. Proinflammatory Genes Induced by IL-1β and
a
Induction Reduced by EI
gene name
IL1β fold change
% reduction by EI
MCP-1
ICAM-1
VCAM-1
E-selectin
IL-6
25.98 1.97
41.21 8.89
259.21 79.16
205.03 29.24
206.05 15.31
121.97 4.90
86.43 2.35
79.6
52.3
92.1
69.5
76.0
53.4
87.6
CXCL1
CXCL6
a
Cells were treated as for Figure 6, and PCR was performed.
DISCUSSION
■
Studies from our group suggested the importance of the EI
group in the action of Ox-PAPC and PEIPC.9−11 Thus, we
reasoned that EI might have a similar effect to PEIPC and
would be a molecule that was easier to synthesize, more stable,
and easier to work with that would allow us to easily compare
the effects of the stereoisomers of the active group. We
therefore developed a rapid method for the synthesis of EI.
This method features lanthanide-promoted PMB ether
formation, enzymatic kinetic resolution of the mono-PMB
protected cyclopent-1-ene-3,5-diol, a TiCl4 catalyzed silyl
transfer of a silyl ketene acetal, and the 1,4-addition of a
vinylcuprate reagent using styrene as a radical scavenger to
avoid the formation of an unusual byproduct. We had
previously shown that there were at least four regioisomers of
PEIPC in Ox-PAPC and that the 5,6-epoxide isomer was the
most active.9 Therefore, in these studies we developed a simple
method to produce several stereoisomers of EI with the epoxide
group in the 5,6-position. Isomers 1 and 2 had much less
activity than 3 and 4 (Figure 2), suggesting considerable
specificity in the interaction of EI with the cell surface. It seems
like the trans arrangement of the hydroxyl and the adjacent 2Z-
octenyl chains is required for high activity, since the cis isomers
show much lower activity. However, we have no good
explanation for why that is the case except that that
arrangement must allow the trans isomers to interact better
with their biological target.
The phospholipase that might be responsible for EI release in
vivo has not been determined. However, we previously showed
that treatment of PEIPC with snake venom PLA2 decreased the
ability of PEIPC to induce monocyte binding.11 At the time we
interpreted this finding to suggest that EI was inactive.
However, in light of the present findings, it is likely that EI
actually inhibits monocyte binding. It has been shown that
28
several mammalian PLA2, Lp-PLA2 and sPLA2-5,29 are
increased in atherosclerotic lesions. Ox-PAPC treatment of
endothelial cells and exposure to bacterial lipids in dendritic
cells lead to increased levels of Lp-PLA2 message.7,26 Lp-PLA2
has been shown to release F2 prostaglandins from phospholi-
pids30 and may have the capacity to release isoprostanes, such
as EI, from the phospholipid backbone. The ability of
isoprostanes to be released from phospholipids is well
documented and involves more than one PLA2.31 In
preliminary experiments we have shown an increase in EI in
Ox-PAPC treated HAEC perhaps caused by the increase in
PLA2’s seen in these cells.
We showed differences in the effect of EI4 and PEIPC4 using
qPCR (Figure 3). To gain a more in depth comparison
between the effects of EI4 and PEIPC, we performed
microarray analysis (Figure 5 and Supporting Information
Table 1). Although EI contained the most active part of PEIPC,
there was only a 50% overlap between PEIPC and EI in genes
CONCLUSION
In summary, we have developed a new and simple method to
synthesize four stereoisomers of EI and shown that isomer 4 is
■
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Chemistry. General. All reactions were carried out under an argon
atmosphere unless otherwise specified. Tetrahydrofuran (THF),
diethyl ether, toluene, and benzene were distilled from benzoquinone
ketyl radical under an argon atmosphere. Dichloromethane (DCM)
and triethylamine (TEA) were distilled from calcium hydride under an
argon atmosphere. All other solvents or reagents were purified
the most active in regulating endothelial cell function. Using
this synthetic EI, we have demonstrated a major difference in
the actions of the EI isoprostane and EI esterified to a
phospholipid. We identified EI as a potent anti-inflammatory
molecule in HAEC. During the preparation of this manuscript,
Egger et al. published another method to prepare EI and
evaluated the effects of EI (and mainly its dehydration product)
in reducing secretion of proinflammatory cytokines IL-6 and
IL-12 by dendritic cells in response to a TLR4 agonist.27 Taken
together, the data from Egger et al. and the data presented here
on IL-1β suggest that EI is a regulator of both acute and
chronic inflammation acting on two major inflammatory cell
types, endothelial cells and dendritic cells. These data suggest
that EI may be a useful anti-inflammatory agent.
1
according to literature procedures. H NMR spectra were recorded at
200 and 400 MHz and are reported relative to deuterated solvent
1
signals. Data for H NMR spectra are reported as follows: chemical
shift (δ ppm), multiplicity, coupling constant (Hz), and integration.
Splitting patterns are designated as follows: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br, broad. 13C NMR spectra were
recorded at 50 and 100 MHz. Data for 13C NMR spectra are reported
in terms of chemical shift. The chemical shifts are reported in parts per
million (ppm, δ). Thin-layer chromatography (TLC) was carried out
using precoated silica gel sheets. Visual detection was performed using
ceric ammonium nitrate or p-anisaldehyde stains. Flash chromatog-
raphy was performed using SilicaFlash P60 (60 Å, 40−63 μm) silica
gel with compressed air. The purity of the compounds was assayed by
high field proton and carbon NMR and was ≥95% except for mixtures
of isomers (diastereomers) as pointed out in the text.
EXPERIMENTAL SECTION
■
Biology. Human Aortic Endothelial Cell Culture. HAECs were
isolated as described previously. HAECs were cultured in VEC
complete medium (VEC technologies), and medium was changed to
10% FBS (Thermo Scientific) in M199 medium (Mediatech)
overnight before use in experiments.
(RS)-4-(4-Methoxyphenylmethoxy)cyclopent-2-enone, 9. To a
solution of 4-hydroxycyclopentenone 8 (13 g, 132.5 mmol) and
freshly prepared 4-methoxybenzyl trichloroacetimidate (19) (55.3 g,
195.7 mmol) in toluene (1 L) was added lanthanide tris-
(trifluoromethanesulfonate) (3.88 g, 6.62 mmol) at 21 °C. The
reaction mixture was stirred for 5 min and then concentrated to
dryness at 40 °C in vacuo. The residue was purified by flash column
chromatography over silica gel (hexane/ethyl acetate, 3:1, v/v) to
afford the desired product 9 (26.1 g, 119.6 mmol, 90%) as a colorless
Measurement of EI and PEIPC Regulation of Genes Using PCR.
HAECs were treated with 3 μM EI1-EI4 or 3 μM PEIPC 4 in M199
medium containing 1% FBS for 4 h. Cells were washed with PBS and
lysed, RNA was extracted, and qPCR analysis was performed,
measuring GAPDH, IL-8, HO-1, ATF-3, MCP-1, and/or HSP1A1
mRNA levels. GAPDH levels showed minimal change. IL-8, HO-1,
ATF-3, MCP-1, and HSP1A1 levels were normalized to GAPDH
levels. The primer sequences used for qPCR were the following:
GAPDH, forward 5′-CCT CAA GAT CAT CAG CAA TGC CTC
CT-3′, reverse 5′-GGT CAT GAG TCC TTC CAC GAT ACC AA-
3′; HO-1, forward 5′-ATA GAT GTG GTA CAG GGA GGC CAT
CA-3′, reverse 5′-GGC AGA GAA TGC TGA GTT CAT GAG GA-
3′; IL-8, forward 5′-ACC ACA CTG CGC CAA CAC AGA AAT-3′,
reverse 5′-TCC AGA CAG AGC TCT CTT CCA TCA GA-3′; ATF-
3, forward 5′-TTG CAG AGC TAA GCA GTC GTG GTA-3′, reverse
5′-ATG GTT CTC TGC TGC TGG GAT TCT-3′; MCP-1, forward
5′-TGC TCA TAG CAG CCA CCT TCA TTC-3′, reverse 5′- GAC
ACT TGC TGC TGG TGA TTC TTC; HSP1A1, forward 5′-AAC
CAC TTC GTG GAG GAG TTC AAG-3′, reverse 5′-TAG AAG
TCG ATG CCC TCA AAC AGG-3′.
1
oil: Rf = 0.48 (hexane/ethyl acetate, 2:1, v/v); H NMR (CDCl3, 200
MHz) δ 7.52 (dd, J = 5.7, 2.3 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 6.81
(d, J = 8.8 Hz, 2H), 6.18 (dd, J = 5.8, 1.2 Hz, 1H), 4.70−4.64 (m, 1H),
4.50 (d, J = 11.2 Hz, 1H), 4.47 (d, J = 11.2 Hz, 1H), 3.73 (s, 3H), 2.59
(dd, J = 18.4, 6.0 Hz, 1H), 2.26 (dd, J = 18.4, 2.2 Hz, 1H); 13C NMR
(CDCl3, 50 MHz) δ 205.9, 161.1, 159.3, 135.4, 129.4, 113.8 (3 C’s),
76.4, 71.4, 55.1, 41.6; ESI-HRMS found 241.0838 [M + Na]+, calcd for
C13H14O3Na 241.0841.
( )-(1S,4R)-4-(4-Methoxyphenylmethoxy)cyclopent-2-enol, 10.
To a solution of (RS)-4-(4-methoxyphenylmethoxy)cyclopent-2-
enone, 9 (14.8 g, 67.8 mmol), and cerous chloride heptahydrate
(27.8 g, 74.6 mmol) in methanol (180 mL) was added sodium
borohydride (1.92 g, 50.8 mmol) portionwise at −78 °C. The reaction
mixture was stirred for 1 h and then concentrated to dryness in vacuo.
The residue was diluted with ethyl acetate (500 mL) and then washed
with aqueous saturated ammonium chloride solution (500 mL) and
brine (500 mL). The organic layer was dried over MgSO4, filtered, and
then concentrated to dryness in vacuo. The residue was purified by
flash column chromatography over silica gel (hexane/ethyl acetate,
2:1, v/v) to afford the desired product 10 (14.0 g, 63.6 mmol, 94%) as
Microarray Analysis. Duplicate wells of HAECs were treated with
1% M199 medium with or without 3 μM EI4 (or PEIPC4) for 4 h, and
RNA was extracted. For microarray analysis, RNA was prepared for
hybridization to Illumina arrays, measuring 45 000 probes, using a
standard protocol described previously.7 Data were analyzed using the
Genome Studio software package. During data analysis, data were
analyzed using background subtraction, quantile normalization, and
t h e c o n t e n t d e s c r i p t o r fi l e “ H u m a n H T -
12_V4_0_R1_15002873_B.bgx” in the Genome Studio files. The
data were filtered for results with the detection p value less than 0.001
for the EI/PEIPC RNA measurement and for the control measure-
ment. The fold changes in probes were calculated as the average probe
level for each treatment divided by the average probe level for control
treatment and converted to ln values to analyze for gene regulation.
Up-regulated/down-regulated genes were identified as those that
changed at least 1.2-fold from control values in the presence of lipids.
Cell Treatment for Analysis of EI4 Effects on IL-8 and MCP-1
Proteins and Effects of EI on IL-1β Induction of MCP-1 Message and
Protein. For dose−response studies, HAECs were untreated or treated
with increasing concentrations of EI for 4 h in 199 medium containing
1% FBS. Medium was collected, and levels of IL-8 and MCP-1 were
determined using ELISA kits from RD Systems. To determine the
effect of EI on IL-1β message and protein, cells were pretreated for 1 h
with 3 μM EI4, then treated with 20 ng/mL IL-1β for 4 h. For qPCR,
cells were collected as above and MCP-1 message was determined. For
MCP-1 protein measurements, medium was collected and protein
determined using ELISA.
1
a colorless oil: Rf = 0.25 (hexane/ethyl acetate, 2:1, v/v); H NMR
(CDCl3, 200 MHz) δ 7.18 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz,
2H), 5.89 (bs, 2H), 4.49−4.46 (bm, 1H), 4.36 (d, J = 11.4 Hz, 1H),
4.33 (dd, J = 7.0, 4.4 Hz, 1H), 4.32 (d, J = 11.4 Hz, 1H), 3.71 (s, 3H),
3.42−3.39 (bm, 1H), 2.55 (ddd, J = 14.0, 7.2, 7.2 Hz, 1H), 1.54 (ddd, J
= 14.0, 4.4, 4.4 Hz, 1H); 13C NMR (CDCl3, 50 MHz) δ 158.9, 137.0,
133.4, 130.1, 129.2, 113.5, 81.0, 74.3, 70.3, 54.9, 40.5; ESI-HRMS
found 243.0989 [M + Na]+, calcd for C13H16O3Na 243.0997.
(1R,4S)-4-(4-Methoxyphenylmethoxy)cyclopent-2-enol, 11, and
(1S,4R)-1-Acetyloxy-4-(4-methoxyphenylmethoxy)cyclopent-2-ene,
12. To a solution of racemic (1S,4R)-4-(4-methoxyphenylmethoxy)-
cyclopent-2-enol, 10 (11.8 g, 53.6 mmol) in methyl ethyl ketone (2.5
L) were added vinyl acetate (450 mL), water (2.5 mL), and lipase AK
(Amano Enzyme U.S.A. Co., Ltd., 35 g). The reaction mixture was
stirred at 21 °C for 20 h and then filtered. The enzyme cake was
washed with methyl ethyl ketone (3 × 50 mL) and then air-dried for
future use. The filtrates were concentrated to dryness in vacuo. The
residue was purified by flash column chromatography over silica gel
(hexane/ethyl acetate, 5:1, v/v) to afford the desired products 11
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Journal of Medicinal Chemistry
Article
(7.13 g, 25.6 mmol, 48%, 97% ee) and 12 (5.58 g, 25.3 mmol, 47%,
99% ee) as colorless oils.
Compound 11: The NMR and MS data were the same as for the
racemic compound 10.
methoxyphenylmethoxy)cyclopent-2-enone product (22.3 g, 75.1
mmol, 80%) as a pale yellow oil.
1(S)-2-Bromo-4-(4-methoxyphenylmethoxy)cyclopent-2-enone,
14. This compound was synthesized according to the general
procedure for the preparation of compound 14 and 16. Rf = 0.60
Compound 12: Rf = 0.70 (hexane/ethyl acetate, 2:1, v/v); 1H NMR
(CDCl3, 400 MHz) δ 7.25 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz,
2H), 6.09 (ddd, J = 5.6, 1.6, 1.3 Hz, 1H), 5.97 (ddd, J = 5.6, 1.7, 1.4
Hz, 1H), 5.48 (m, 1H), 4.50 (d, J = 11.4 Hz, 1H), 4.46 (d, J = 11.4 Hz,
1H), 4.50−4.44 (m, 1H), 3.78 (s, 3H), 2.75 (dd, J = 14.2, 7.3, 7.3 Hz,
1H), 2.03 (s, 3H), 1.75 (ddd, J = 14.2, 4.4, 4.4 Hz, 1H); 13C NMR
(CDCl3, 100 MHz) δ 170.8, 159.2, 136.2, 132.7, 130.4, 129.3, 113.8,
80.9, 70.7, 55.2, 37.7, 37.6, 21.1; ESI-HRMS found 301.1044 [M +
Na]+, calcd for C15H18O4Na 301.1052.
1
(hexane/ethyl acetate, 2:1, v/v); H NMR (CDCl3, 400 MHz) δ 7.67
(d, J = 2.4 Hz, 1H), 7.26 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H),
4.67 (ddd, J = 8.0, 2.6, 2.4 Hz, 1H), 4.58 (d, J = 11.3 Hz, 1H), 4.55 (d,
J = 11.3 Hz, 1H), 3.86 (s, 3H), 2.82 (dd, J = 18.4, 6.0 Hz, 1H), 2.47
(dd, J = 18.4, 2.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 197.8,
159.7, 158.7, 129.6, 129.1, 128.9, 114.1, 75.0, 71.6, 55.3, 40.6; ESI-
HRMS found 318.9942 [M + Na]+, calcd for C13H13BrO3Na
318.9946.
(R)-2-Bromo-4-(4-methoxyphenylmethoxy)cyclopent-2-enone,
16. This compound was synthesized according to the general
procedure for the preparation of compounds 14 and 16. The NMR
and MS data were the same as those for compound 14.
To a solution of compound 11 (163 mg, 0.74 mmol) in pyridine (3
mL) were added acetic anhydride (0.3 mL) and 4-(dimethylamino)-
pyridine (DMAP, 5 mg). The reaction mixture was stirred at 21 °C
overnight and then diluted with diethyl ether (20 mL). The resulting
organic layer was washed with 1 N aqueous HCl (3 × 20 mL),
saturated NaHCO3 (20 mL), and brine (20 mL). The organic layer
was dried over MgSO4, filtered, and then concentrated to dryness in
vacuo. The residue was purified by flash column chromatography over
silica gel (hexane/ethyl acetate, 5:1, v/v) to afford the desired product
(201 mg, 0.72 mmol, 97%) as a colorless oil. The NMR and MS data
were the same as those for compound 12. The enantiomeric excess
was determined by chiral GC.
(1S,4R)-4-(4-Methoxyphenylmethoxy)cyclopent-2-enol. To a sol-
ution of 1-acetyloxy-4-(4-methoxyphenylmethoxy)cyclopent-2-ene, 12
(30 g, 0.108 mol), in methanol (200 mL) was added potassium
carbonate (16.39 g, 0.119 mol). The reaction mixture was stirred at 21
°C overnight and then concentrated at to dryness in vacuo. The
residue was diluted with dichloromethane (500 mL) and then washed
with 5% aqueous HCl (500 mL) and brine (500 mL). The organic
layer was dried over MgSO4, filtered, and then concentrated to dryness
in vacuo. The residue was purified by flash column chromatography
over silica gel (hexane/ethyl acetate, 2:1, v/v) to afford the (1S,4R) 4-
(4-methoxyphenylmethoxy)cyclopent-2-enol (23.3 g, 0.106 mol, 98%)
as a colorless oil. The NMR and MS data were the same as the
compound 10.
General Procedure for the Preparation of Compounds 13 and
15. To a solution of 4-(4-methoxyphenylmethoxy)cyclopent-2-enol
(5.51 g, 25.0 mmol) in dichloromethane (50 mL) was added a mixture
of pyridinium chlorochromate (7.55 g, 35.0 mmol) and Celite (7.55 g)
portionwise at 0 °C. The reaction mixture was stirred overnight at 21
°C. Isopropyl alcohol (60 mL) was then added and the mixture stirred
for 30 min. The solution was filtered through a pad of Celite and silica
gel (4 cm), washed with ethyl acetate (2 × 60 mL), and then
concentrated to dryness in vacuo. The residue was purified by flash
column chromatography over silica gel (hexane/ethyl acetate, 2:1, v/v)
to afford the 4-(4-methoxyphenylmethoxy)cyclopent-2-enone product
13 or 15 (4.85 g, 22.2 mmol, 89%) as a colorless oil.
(S)-4-(4-Methoxyphenylmethoxy)cyclopent-2-enone, 13. This
compound was synthesized according to the general procedure for
the preparation of compounds 13 and 15. The NMR and MS data
were the same as those for compound 9.
(R)-4-(4-Methoxyphenylmethoxy)cyclopent-2-enone, 15. This
compound was synthesized according to the general procedure for
the preparation of compound 13 and 15. The NMR and MS data were
the same as those for compound 9.
General Procedure for the Preparation of Compounds 14 and
16. To a solution of 4-(4-methoxyphenylmethoxy)cyclopent-2-enone
(20.5 g, 93.9 mmol) in dichloromethane (240 mL) was added bromine
(4.81 mL, 93.9 mmol) dropwise at 0 °C. The reaction mixture was
stirred for 5 min, and then triethylamine (17 mL, 122 mmol) was
added. The resulting mixture was stirred for 30 min at 21 °C and then
poured into water (200 mL). The aqueous layer was extracted with
dichloromethane (3 × 200 mL). The combined organic layers were
dried over MgSO4, filtered, and then concentrated to dryness in vacuo.
The residue was purified by flash column chromatography over silica
gel (hexane/ethyl acetate, 4:1, v/v) to afford the 2-bromo-4-(4-
General Procedure for the Preparation of Compound 18 (and Its
Enantiomer) by the Titanium Tetrachloride Catalyzed Silyl Transfer
Reaction. To a solution of 2-bromo-4-(4-methoxyphenylmethoxy)-
cyclopent-2-enone 14 (or 16) (5.63 g, 18.9 mmol) in dichloromethane
(280 mL) was added 1 M TiCl4 in dichloromethane (1.89 mL, 1.89
mmol) dropwise at −78 °C. The resulting mixture was stirred at −78
°C for 30 min, and then freshly prepared ethyl (tert-
butyldimethylsilyl)ketene acetal 17 (9.1 g, 45.0 mmol) was added
dropwise over 30 min. The reaction mixture was stirred at −78 °C for
1 h and then quenched with triethylamine (4 mL). The solution was
slowly warmed to 21 °C and then concentrated to dryness in vacuo.
The residue was purified by flash column chromatography over silica
gel (hexane/ethyl acetate, 10:1, v/v) to afford the desired product 18
(7.7 g, 15.7 mmol, 83%) as a pale yellow oil.
(1RS,5S)-Ethyl 2-Bromo-3-[(1,1-dimethylethyl)dimethylsilyloxy]-
5-(4-methoxyphenylmethoxy)cyclopent-2-ene-1-acetate, 18. Rf =
0.48 (hexane/ethyl acetate, 10:1, v/v); 1H NMR (CDCl3, 400 MHz) δ
7.23 (m, 4H), 6.85 (m, 4H), 4.48−4.36 (m, 6H), 4.49 (d, J = 11.3 Hz,
1H), 4.44 (d, J = 11.3 Hz, 1H), 4.39 (d, J = 11.3 Hz, 1H), 4.35 (d, J =
11.3 Hz, 1H), 4.29 (ddd, J = 7.2, 5.2, 5.2 Hz, 1H), 4.14−4.04 (m, 5H),
3.79 (s, 6H), 2.70−2.20 (m, 8H), 1.26 (t, J = 7.2 Hz, 3H), 1.19 (t, J =
7.2 Hz, 3H), 0.95 (s, 9H), 0.948 (s, 9H), 0.20−0.15 (m, 12H); ESI-
HRMS found 521.1317 [M + Na]+, calcd for C23H35BrO5SiNa
521.1335.
Enantiomer of 18. This compound was synthesized according to
the general procedure for the preparation of compound 18. The NMR
and MS data were the same as those for compound 18.
General Procedure for the Preparation of 2-[2-Bromo-3-[(1,1-
dimethylethyl)dimethylsilyloxy]-5-(4-methoxyphenylmethoxy)-
cyclopent-2-ene]acetaldehyde 19, 20, 21, and 22. Method A. To a
solution of the starting material 18 (or its enantiomer) (7.7 g, 15.7
mmol) in dichloromethane (260 mL) was added 1 M DIBAL in
dichloromethane (16.5 mL, 16.5 mmol) dropwise at −78 °C. The
reaction mixture was stirred at −78 °C for 15 min and then quenched
with saturated aqueous Rochelle’s salt (260 mL). The solution was
slowly warmed to 21 °C and stirred for 1 h. The organic layer was
separated and extracted with dichloromethane (3 × 200 mL). The
combined organic layers were dried over Na2SO4, filtered, and then
concentrated to dryness in vacuo. The residue was purified by flash
column chromatography over silica gel (pentane/diethyl ether, 20:1,
v/v) to afford first the cis aldehyde 19 or 21 (2.92 g, 6.4 mmol, 41%)
and then the trans aldehyde 20 or 22 (2.78 g, 6.1 mmol, 39%) as pale
yellow oils.
Method B. To a solution of CuI (13.34 g, 70 mmol) in diethyl ether
(400 mL) at −78 °C was added dropwise 1 M vinylmagnesium
bromide in THF (140 mL). The reaction mixture was stirred at −20
°C for 20 min and then cooled to −78 °C. Freshly distilled styrene
(8.03 mL, 70 mmol dried over CaH2) was added and stirred at −78 °C
for 20 min. The reaction mixture was cooled to −100 °C. A solution of
2-bromo-4-(4-methoxyphenylmethoxy)cyclopent-2-enone 14 or 16
(10.4 g, 35 mmol) in diethyl ether (70 mL) was added dropwise and
stirred at −100 °C for 1 h. TBSCl (21.1 g, 140 mmol) in diethyl ether
(35 mL) and HMPA (98.6 mL, 567 mmol) were subsequently added.
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The reaction mixture was allowed to warm to 21 °C and stirred
overnight. The solution was quenched with a pH 7 buffer (500 mL),
filtered through a pad of Celite, and then washed with diethyl ether (3
× 100 mL). The organic layer was washed with water (500 mL) and
brine (500 mL). The organic layer was dried over Na2SO4, filtered,
and then concentrated to dryness in vacuo. The residue was purified
by flash column chromatography over silica gel (hexane/ethyl acetate,
10:1, v/v) to afford a separable trans product (28.9 g, 65.8 mmol,
94%) as a pale yellow oil. Rf = 0.68 (hexane/ethyl acetate, 6:1, v/v);
1H NMR (CDCl3, 400 MHz) δ 7.26 (d, J = 8.8 Hz, 2H), 6.89 (d, J =
8.8 Hz, 2H), 5.69 (ddd, J = 18.6, 10.0, 8.4 Hz, 1H), 5.23 (d, J = 18.6
Hz, 1H), 5.19 (d, J = 10.0 Hz, 1H), 4.53 (d, J = 11.4 Hz, 1H), 4.45 (d,
J = 11.4 Hz, 1H), 3.97 (ddd, J = 7.4, 4.0, 4.0 Hz, 1H), 3.81 (s, 3H),
3.38 (m, 1H), 2.61 (dd, J = 15.9, 7.6 Hz, 1H), 2.36 (ddd, J = 15.9, 4.0,
1.2 Hz, 1H), 0.97 (s, 9H), 0.21 (s, 3H), 0.20 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ 159.3, 148.5, 138.6, 130.1, 129.3, 117.1, 113.8,
96.8, 80.1, 70.7, 57.0, 55.3, 39.3, 25.6, 18.1, −3.9; ESI-HRMS found
461.1116 [M + Na]+, calcd for C21H31BrO3SiNa 461.1124. No
undesired byproduct was formed. Rf = 0.70 (hexane/ethyl acetate, 6:1,
v/v); 1H NMR (CDCl3, 400 MHz) δ 7.25 (d, J = 8.4 Hz, 2H), 6.88 (d,
J = 8.4 Hz, 2H), 5.68 (ddd, J = 18.3, 10.0, 8.0 Hz, 1H), 5.05 (dd, J =
18.3, 1.5 Hz, 1H), 5.95 (dd, J = 10.0, 1.5 Hz, 1H), 4.68 (s, 1H), 4.53
(d, J = 11.6 Hz, 1H), 4.42 (d, J = 11.6 Hz, 1H), 3.80 (s, 3H), 3.82−
3.70 (m, 1H), 3.38 (bd, J = 8.4 Hz, 1H), 2.71 (ddd, J = 16.5, 6.3, 0.6
Hz, 1H), 2.32 (dd, J = 16.5, 2.0 Hz, 1H), 0.94 (s, 9H), 0.89 (s, 9H),
0.16 (s, 3H), 0.12 (s, 3H), 0.09 (s, 3H), 0.01 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ 161.7, 159.1, 141.7, 130.9, 129.1, 127.5, 114.2,
113.7, 113.6, 109.7, 83.1, 70.2, 64.7, 56.6, 55.3, 41.5, 27.3, 26.0, 18.4,
−3.1, −3.2, −4.3, −4.5, −5.2; ESI-HRMS found 497.2878 [M + Na]+,
calcd for C27H46O3Si2Na 497.2883.
To the vinyl bromide (9.95 g, 22.6 mmol) was added dropwise 0.5
M 9-BBN in THF (90.4 mL, 45.2 mmol) at 0 °C. The reaction
mixture was then allowed to warm to 21 °C and stirred for 1.5 h. The
solution was cooled to −78 °C, and a mixture of H2O2 (15 mL) and 3
N aqueous NaOH (15 mL) was added slowly. The reaction mixture
was stirred for 1 h at 21 °C and then quenched with a pH 7 buffer (1
L). The aqueous layer was extracted with diethyl ether (3 × 500 mL).
The combined organic layers were washed with brine (500 mL), dried
over Na2SO4, filtered, and then concentrated to dryness in vacuo. The
residue was purified by flash column chromatography over silica gel
(hexane/ethyl acetate, 3:1, v/v) to afford an inseparable mixture of the
desired cis and trans alcohol products (9.5 g, 20.8 mmol, 92%) as pale
yellow oils. Rf = 0.35 (hexane/ethyl acetate, 3:1, v/v); 1H NMR
(CDCl3, 400 MHz) δ 7.26 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz,
2H), 4.48 (d, J = 11.2 Hz, 1H), 4.40 (d, J = 11.2 Hz, 1H), 3.97 (ddd, J
= 7.2, 5.2, 5.2 Hz, 1H), 3.80 (s, 3H), 3.70 (t, J = 6.1 Hz, 1H), 2.81 (m,
1H), 2.60 (ddd, J = 15.2, 7.4, 1.8 Hz, 1H), 2.37 (ddd, J = 15.2, 5.2, 1.6
Hz, 1H), 2.07 (m, 1H), 1.46 (m, 1H), 0.96 (s, 9H), 0.20 (s, 3H), 0.19
(s, 3H); 13C NMR (CDCl3, 100 MHz) δ 159.5, 147.2, 129.6, 129.4,
128.6, 113.9, 98.5, 80.4, 70.8, 60.9, 55.3, 49.2, 38.9, 35.8, 27.4, 25.5,
22.7, 18.1, −3.9; ESI-HRMS found 479.1221 [M + Na]+, calcd for
C21H33BrO4SiNa 479.1229.
To a solution of the alcohol (4.19 g, 9.18 mmol) in dichloro-
methane (46 mL) at 0 °C was added solid NaHCO3 (3.86 g, 45.9
mmol) followed by Dess−Martin periodinane (4.67 g, 11.0 mmol)
portionwise. The reaction mixture was allowed to warm to 21 °C and
stirred for 1 h. The solution was quenched with saturated aqueous
NaHCO3 (40 mL) and saturated aqueous Na2S2O3 (40 mL). The
aqueous layer was extracted with dichloromethane (3 × 50 mL). The
combined organic layers were dried over Na2SO4, filtered, and then
concentrated to dryness in vacuo. The residue was purified by flash
column chromatography over silica gel (pentane/diethyl ether, 20:1,
v/v) to afford first the cis aldehyde 19 or 21 (0.65 g, 1.43 mmol, 16%)
and then the trans aldehyde 20 or 22 (2.68 g, 5.90 mmol, 64%) as pale
yellow oils.
δ 9.81 (dd, J = 1.4, 1.4 Hz, 1H), 7.18 (d, J = 8.7 Hz, 2H), 6.86 (d, J =
8.7 Hz, 2H), 4.40 (d, J = 11.2 Hz, 1H), 4.31 (d, J = 11.2 Hz, 1H), 4.28
(ddd, J = 12.3, 7.3, 4.9 Hz, 1H), 3.80 (s, 3H), 3.42 (m, 1H), 2.75 (ddd,
J = 17.2, 9.4, 1.8 Hz, 1H), 2.55 (ddd, J = 17.2, 4.1, 1.1 Hz, 1H), 2.49
(ddd, J = 15.6, 7.2, 0.5 Hz, 1H), 2.36 (ddd, J = 15.6, 5.0, 2.0 Hz, 1H),
0.96 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H); 13C NMR (CDCl3, 100 MHz)
δ 201.8, 159.3, 148.3, 129.9, 129.3, 113.8, 97.2, 74.9, 71.5, 55.3, 45.3,
41.9, 38.4, 25.5, 18.1, −3.9; ESI-HRMS found 477.1064 [M + Na]+,
calcd for C21H31BrO4SiNa 477.1073.
2-[(1S,5R)-2-Bromo-3-[(1,1-dimethylethyl)dimethylsilyloxy]-5-(4-
methoxyphenylmethoxy)cyclopent-2-eneacetaldehyde 20 and 2-
[(1R,5S)-2-Bromo-3-[(1,1-dimethylethyl)dimethylsilyloxy]-5-(4-
methoxyphenylmethoxy)cyclopent-2-eneacetaldehyde 22. Rf
=
1
0.38 (hexane/ethyl acetate, 4:1, v/v); H NMR (CDCl3, 400 MHz)
δ 9.81 (dd, J = 2.4, 1.8 Hz, 1H), 7.25 (d, J = 8.7 Hz, 2H), 6.88 (d, J =
8.7 Hz, 2H), 4.46 (d, J = 11.9 Hz, 1H), 4.44 (d, J = 11.9 Hz, 1H), 3.86
(ddd, J = 7.5, 3.8, 3.8 Hz, 1H), 3.80 (s, 3H), 3.24 (m, 1 H), 2.73 (ddd,
J = 16.5, 4.6, 1.7 Hz, 1H), 2.59 (ddd, J = 15.8, 7.3, 1.9 Hz, 1H), 2.41
(ddd, J = 16.5, 8.5, 2.6 Hz, 1H), 2.34 (ddd, J = 15.8, 4.0, 1.2 Hz, 1H),
0.96 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H); 13C NMR (CDCl3, 100 MHz)
δ 201.5, 159.3, 148.6, 129.9, 129.4, 113.9, 96.4, 79.3, 70.8, 55.3, 47.4,
46.4, 39.2, 25.5, 18.1, −3.9; ESI-HRMS found 477.1062 [M + Na]+,
calcd for C21H31BrO4SiNa 477.1073.
General Procedure for the Preparation of 2-Bromo-3-[(1,1-
dimethylethyl)dimethylsilyloxy]-4-(4-methoxyphenylmethoxy)-3-
((Z)-oct-2-enyl)cyclopent-1-ene, 23. To a solution of hexyltriphenyl-
phosphonium bromide (5.29 g, 12. Four mmol) in THF (24 mL) at 0
°C was added 1 M NaHMDS in THF (11.8 mL, 11.8 mmol). The
reaction mixture was stirred at 0 °C for 1 h and then cooled to −78
°C. A solution of the aldehyde 19 (2.67 g, 5.9 mmol) in THF (12 mL)
was added dropwise and then stirred at −78 °C for 1 h. The solution
was quenched with a mixture of triethylamine, water, and diethyl ether
(64 mL, 1:2:1, v/v/v) and then allowed to warm to 21 °C. The
aqueous layer was extracted with diethyl ether (3 x50 mL). The
combined organic layers were washed with brine (100 mL), dried over
Na2SO4, filtered, and then concentrated to dryness in vacuo. The
residue was purified by flash column chromatography over silica gel
(hexane/ethyl acetate, 6:1, v/v) to afford the desired Z-alkene, e.g., 23
(2.35 g, 4.48 mmol, 76%), as a pale yellow oil.
(3S,4S)-2-Bromo-1-[(1,1-dimethylethyl)dimethylsilyloxy]-4-(4-me-
thoxyphenylmethoxy)-3-((Z)-oct-2-enyl)cyclopent-1-ene, 23, and
Its (3R,4R)-Enantiomer. Rf = 0.64 (hexane/ethyl acetate, 4:1, v/v);
1H NMR (CDCl3, 400 MHz) δ 7.25 (d, J = 8.4 Hz, 2H), 6.87 (d, J =
8.8 Hz, 2H), 5.52 (m, 1H), 5.41 (m, 1H), 4.46 (d, J = 11.2 Hz, 1H),
4.42 (d, J = 11.2 Hz, 1H), 4.27 (ddd, J = 7.2, 7.2, 7.2 Hz, 1H), 3.82 (s,
3H), 2.83 (bddd, J = 6.4, 6.4, 6.4 Hz, 1H), 2.42 (m, 4H), 2.07 (m,
2H), 1.35 (m, 6H), 1.01 (s, 9H), 0.91 (t, J = 6.8 Hz, 3H), 0.23 (s, 3H),
0.19 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 159.3, 147.8, 131.3,
130.4, 129.2, 129.1, 127.4, 113.8, 98.9, 71.4, 55.2, 49.3, 38.6, 31.7, 29.5,
27.5, 25.9, 25.6, 22.7, 18.1, 14.2, −3.9; ESI-HRMS found 545.2058 [M
+ Na]+, calcd for C27H43BrO3SiNa 545.2063.
(3S,4R)-2-Bromo-3-[(1,1-dimethylethyl)dimethylsilyloxy]-4-(4-me-
thoxyphenylmethoxy)-3-((Z)-oct-2-enyl)cyclopent-1-ene and Its
1
(3R,4S)-Enantiomer. Rf = 0.61 (hexane/ethyl acetate, 4:1, v/v); H
NMR (CDCl3, 400 MHz) δ 7.25 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.8
Hz, 2H), 5.48 (m, 1H), 5.40 (m, 1H), 4.44 (d, J = 11.6 Hz, 1H), 4.40
(d, J = 11.6 Hz, 1H), 3.85 (ddd, J = 7.2, 2.8, 2.8 Hz, 1H), 3.80 (s, 3H),
2.80 (m, 1H), 2.56 (ddd, J = 16.0, 7.2, 2.0 Hz, 1H), 2.44 (ddd, J =
14.8, 7.6, 4.0 Hz, 1H), 2.31 (ddd, J = 16.0, 3.2, 0.7 Hz, 1H), 2.09 (ddd,
J = 14.8, 7.2, 7.2 Hz, 1H), 2.06 (m, 2H), 1.36−1.27 (m, 6H), 1.04 (s,
9H), 0.95 (t, J = 6.8 Hz, 3H), 0.27 (s, 6H); 13C NMR (CDCl3, 100
MHz) δ 159.3, 147.9, 132.3, 130.4, 129.2, 125.8, 113.8, 98.6, 78.2,
70.3, 55.2, 52.3, 39.7, 31.7, 29.5, 29.3, 27.5, 25.7, 22.7, 18.2, 14.2, −3.9;
ESI-HRMS found 545.2056 [M + Na]+, calcd for C27H43BrO3SiNa
545.2063.
General Procedure for the Preparation of E-2-((3-(4-
Hydroxybutyl)oxiran-2-yl)methylene)-4-(4-methoxyphenyl-
methoxy)-3-((Z)-oct-2-enyl)cyclopentanone, 24. To a solution of 2-
bromo-3-[(1,1-dimethylethyl)dimethylsilyloxy]-4-(4-methoxyphenyl-
methoxy)-3-((Z)-oct-2-enyl)cyclo-pent-1-ene, e.g., 23 (1.27 g, 2.43
mmol), in diethyl ether (28 mL) was added 1.7 M tert-butyllithium in
2-[(1S,5S)-2-Bromo-3-[(1,1-dimethylethyl)dimethylsilyloxy]-5-(4-
methoxyphenylmethoxy)cyclopent-2-eneacetaldehyde 19 and 2-
[(1R,5R)-2-Bromo-3-[(1,1-dimethylethyl)dimethylsilyloxy]-5-(4-
methoxyphenylmethoxy)cyclopent-2-eneacetaldehyde 21. Rf
0.42 (hexane/ethyl acetate, 4:1, v/v); H NMR (CDCl3, 400 MHz)
=
1
8529
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Journal of Medicinal Chemistry
Article
pentane (3.14 mL, 5.32 mmol) at −78 °C. The reaction mixture was
stirred at −78 °C for 1 h, and then a solution of 3-(4-[(1,1-
dimethylethyl)dimethylsilyloxy]butyl)oxirane-2-carboxaldehyde, e.g., 5
(0.92 g, 3.63 mmol), in diethyl ether (7 mL) was added dropwise. The
reaction mixture was stirred at −78 °C for 2 h and then quenched with
saturated aqueous NH4Cl (15 mL). The aqueous layer was extracted
with diethyl ether (2 × 50 mL). The combined organic layers were
washed with brine (100 mL), dried over Na2SO4, filtered, and then
concentrated to dryness in vacuo. The residue was purified by flash
column chromatography over silica gel (hexane/ethyl acetate, 5:1, v/v)
to afford the desired diastereomeric alcohols (1.36 g, 1.95 mmol, 80%)
as a pale yellow oil. To the mixture of the alcohol (1.36 g, 1.95 mmol)
was added a mixture of THF/formic acid/water (20 mL, 6:3:1, v/v/v).
The reaction mixture was stirred at 21 °C for 1 h and then quenched
with a pH 7 buffer (30 mL). The mixture was extracted with diethyl
ether (3 × 30 mL). The combined organic layers were washed with
saturated aqueous NaHCO3 (250 mL), brine (100 mL), dried over
Na2SO4, filtered, and then concentrated to dryness in vacuo. The
residue was purified by flash column chromatography over silica gel
(hexane/ethyl acetate, 2:1, v/v) to afford the desired enone product,
e.g., 24 (0.79 g, 1.13 mmol, 58%), as a colorless yellow oil.
(m, 1H), 1.95 (m, 2H), 1.82 (m, 3H), 1.51 (m, 1H), 1.28 (m, 7H),
0.86 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 201.6, 201.5,
159.4, 144.1, 133.6, 132.9, 129.8, 129.3, 126.2, 113.9, 75.1, 71.1, 59.9,
55.3, 55.0, 43.2, 42.8, 41.7, 31.5, 30.9, 29.3, 27.3, 25.7, 22.5, 18.4, 14.0;
ESI-HRMS found 477.2611 [M + Na]+, calcd for C28H38O5Na
477.2617.
4-[(2R,3R)/(2S,3S)-3-((E)-((2R,3R)/(2S,3S)-3-(4-Methoxyphenylme-
thoxy)-2-((Z)-oct-2-enyl)-5-oxocyclopentylidene]methyl)oxiran-2-
yl]butanal (Trans). Rf = 0.60 (hexane/ethyl acetate, 1:1, v/v); 1H
NMR (CDCl3, 400 MHz) δ 9.74 (s, 1H), 7.18 (d, J = 8.4 Hz, 2H),
6.84 (d, J = 8.8 Hz, 2H), 6.19 (d, J = 8.8 Hz, 1H), 5.48 (m, 1H), 5.36
(m, 1H), 4.41 (s, 2H), 3.96 (m, 1H), 3.75 (s, 3H), 3.22 (m, 2H), 2.92
(m, 1H), 2.51 (m, 4H), 2.19 (ddd, J = 14.0, 8.0, 8.0 Hz, 1H), 2.07
(ddd, J = 14.0, 7.0, 7.0 Hz, 1H), 1.94 (m, 2H), 1.78 (m, 3H), 1.51 (m,
1H), 1.28 (m, 6H), 0.86 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100
MHz) δ 203.8, 201.6, 159.2, 148.9, 133.7, 133.2, 129.9, 129.2, 125.4,
113.8, 77.5, 70.1, 59.9, 55.2, 54.9, 45.4, 43.2, 42.5, 31.5, 31.4, 31.0,
29.2, 27.4, 22.5, 18.4, 14.0; ESI-HRMS found 477.2613 [M + Na]+,
calcd for C28H38O5Na 477.2617.
General Procedure for the Preparation of 4-[3-(E)-(3-(4-
Methoxyphenylmethoxy)-2-((Z)-oct-2-enyl)-5-oxocyclopentylidene)-
methyl]oxiran-2-yl]butanoic Acid, 25. To a solution of 4-[3-((E)-(3-
( 4 - m e t h o x y p h e n y l m e t h o x y ) - 2 - ( ( Z ) - o c t - 2 - e n y l ) - 5 -
oxocyclopentylidene]methyl)oxiran-2-yl]butanal (143 mg, 0.32 mmol)
in tert-butyl alcohol (4.8 mL) and 2-methyl-2-butene (2.88 mL) was
added a solution of 80% NaClO2 (215 mg, 1.89 mmol) and
NaH2PO4.H2O (262 mg, 1.89 mmol) in water (4.8 mL) at 21 °C.
The reaction mixture was stirred at 21 °C for 1 h and then quenched
with water (10 mL). The aqueous layer was extracted with diethyl
ether (3 × 20 mL). The combined organic layers were dried over
Na2SO4, filtered, and then concentrated to dryness in vacuo. The
residue was purified by flash column chromatography over silica gel
(dichloromethane/methanol, 20:1, v/v) to afford the desired acid (123
mg, 0.26 mmol, 82%) as a colorless oil.
(3S,4S,E)-2-(((2R,3S)/(2S,3R)-3-(4-Hydroxybutyl)oxiran-2-yl)-
methylene)-4-(4-methoxyphenylmethoxy)-3-((Z)-oct-2-enyl)-
cyclopentanone (Cis) 24 and Its (3R,4R)-Enantiomer. Rf = 0.48
1
(hexane/ethyl acetate, 1:1, v/v); H NMR (CDCl3, 400 MHz) δ 7.27
(d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.11 (d, J = 9.2 Hz, 1H),
5.54−5.41 (m, 2H), 4.54 (d, J = 11.4 Hz, 1H), 4.49 (d, J = 11.4 Hz,
1H), 4.16 (m, 1H), 3.78 (s, 3H), 3.63 (t, J = 6.0 Hz 2H), 3.33 (m,
1H), 3.24 (dd, J = 7.2, 2.0 Hz, 1H), 2.91 (m, 1H), 2.67−2.49 (m, 3H),
2.18 (m, 1H), 1.97 (m, 2H), 1.73−1.50 (m, 6H), 1.32−1.25 (m, 7H),
0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 201.7, 159.4,
143.9, 134.0, 132.9, 129.8, 129.3, 126.2, 113.9, 76.8, 62.4, 60.4, 55.3,
42.8, 41.7, 32.3, 31.5, 29.3, 27.3, 25.7, 22.5, 22.2, 14.2; ESI-HRMS
found 479.2771 [M + Na]+, calcd for C28H40O5Na 479.2773.
4-[(2R,3S)/(2S,3R)-3-((E)-((2R,3R)/(2S,3S)-3-(4-Methoxyphenylme-
thoxy)-2-((Z)-oct-2-enyl)-5-oxocyclopentylidene)methyl]oxiran-2-
yl]butanoic Acid (Cis), 25, and Its Enantiomer. Rf = 0.36 (hexane/
ethyl acetate, 1:1, v/v); 1H NMR (CDCl3, 400 MHz) δ 7.28 (d, J = 8.8
Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.13 (dd, J = 9.3, 1.6 Hz, 1H), 5.46
(m, 1H), 5.33 (m, 1H), 4.56 (d, J = 11.3 Hz, 1H), 4.50 (d, J = 11.3 Hz,
1H), 4.19 (ddd, J = 9.6, 7.3, 7.3 Hz, 1H), 3.79 (s, 3H), 3.32 (m, 1H),
3.23 (dd, J = 7.2, 2.0 Hz, 1H), 2.98 (m, 1H), 2.66−2.41 (m, 5H), 2.09
(m, 1H), 1.95 (m, 2H), 1.82 (m, 2H), 1.52 (m, 1H), 1.28 (m, 7H),
0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 201.7, 178.2,
159.4, 144.1, 133.7, 133.0, 129.8, 129.3, 126.2, 113.9, 75.1, 71.1, 59.9,
55.3, 55.0, 42.8, 41.7, 33.3, 31.5, 30.9, 29.3, 27.3, 25.7, 22.5, 21.0, 14.0;
ESI-HRMS found 469.2586 [M − H]−, calcd for C28H37O6 469.2590.
4-[(2R,3R)/(2S,3S)-3-(E)-((2R,3R)/(2S,3S)-3-(4-Methoxyphenylme-
thoxy)-2-((Z)-oct-2-enyl)-5-oxocyclopentylidene)methyl]oxiran-2-
yl]butanoic Acid (Trans). Rf = 0.35 (hexane/ethyl acetate, 1:1, v/v);
1H NMR (CDCl3, 400 MHz) δ 10.1 (b, 1H), 7.21 (d, J = 8.4 Hz, 2H),
6.86 (d, J = 8.4 Hz, 2H), 6.23 (dd, J = 8.4, 1.6 Hz, 1H), 5.51 (m, 1H),
5.36 (m, 1H), 4.44 (s, 2H), 3.99 (m, 1H), 3.80 (s, 3H), 3.26 (m, 2H),
2.97 (m, 1H), 2.54−2.41 (m, 4H), 2.20 (ddd, J = 14.0, 7.6, 7.6 Hz,
1H), 2.09 (ddd, J = 14.0, 7.2, 7.2 Hz, 1H), 1.97 (m, 2H), 1.81 (m,
3H), 1.59 (m, 1H), 1.30 (m, 7H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR
(CDCl3, 100 MHz) δ 204.0, 178.7, 159.3, 142.9, 133.8, 133.3, 129.9,
129.3, 125.4, 113.9, 77.5, 70.1, 59.9, 55.3, 55.0, 45.5, 42.5, 31.5, 31.4,
31.1, 30.3, 29.2, 27.3, 22.5, 21.1, 14.0; ESI-HRMS found 469.2582 [M
− H]−, calcd for C28H37O6 469.2590.
General Procedure for the Preparation of 4-[3-((E)-(3-Hydroxy)-2-
((Z)-oct-2-enyl)-5-oxocyclopentylidene)methyl)oxiran-2-yl]butanoic
Acid, EI. To a solution of 4-[3-(E)-(3-(4-methoxyphenylmethoxy)-2-
((Z)-oct-2-enyl)-5-oxocyclopentylidene)methyl]oxiran-2-yl]butanoic
acid (137 mg, 0.29 mmol) in dichloromethane (7.3 mL) and pH 7
buffer (3.65 mL) was added DDQ (100 mg, 0.44 mmol) at 21 °C. The
reaction mixture was stirred for 2 h at 21 °C and then filtered through
a pad of Celite. The filtrates were washed with brine (5 mL). The
aqueous layer was extracted with dichloromethane (2 × 5 mL). The
combined organic layers were dried over Na2SO4, filtered, and then
concentrated to dryness in vacuo. The residue was purified by flash
(3R,4R,E)/(3S,4S,E)-2-(((2R,3R)/(2S,3S)-3-(4-Hydroxybutyl)oxiran-
2-yl)methylene)-4-(4-methoxyphenylmethoxy)-3-((Z)-oct-2-enyl)-
cyclopentanone (Trans). Rf = 0.46 (hexane/ethyl acetate, 1:1, v/v);
1H NMR (CDCl3, 400 MHz) δ 7.20 (d, J = 8.4 Hz, 2H), 6.85 (d, J =
8.8 Hz, 2H), 6.23 (dd, J = 8.8, 1.6 Hz, 1H), 5.49 (m, 1H), 5.37 (m,
1H), 4.42 (s, 2H), 3.98 (m, 1H), 3.77 (s, 3H), 3.62 (t, J = 6.4 Hz, 2H),
3.24 (m, 2H), 2.95 (m, 1H), 2.53 (m, 2H), 2.19 (ddd, J = 14.4, 7.6, 7.6
Hz, 1H), 2.08 (ddd, J = 15.2, 7.2, 7.2 Hz, 1H), 1.94 (m, 3H), 1.72 (m,
1H), 1.58 (m, 5H), 1.29 (m, 6H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR
(CDCl3, 100 MHz) δ 204.0, 159.3, 142.7, 134.1, 133.2, 129.9, 129.2,
125.4, 113.8, 70.1, 62.4, 60.3, 55.3, 55.2, 45.5, 42.5, 32.3, 31.6, 31.5,
31.4, 29.2, 29.1, 27.4, 22.5, 22.2, 14.0; ESI-HRMS found 479.2768 [M
+ Na]+, calcd for C28H40O5Na 479.2773.
General procedure for the preparation of 4-[3-((E)-(3-(4-
m e t h o x y p h e n y l m e t h o x y ) - 2 - ( ( Z ) - o c t - 2 - e n y l ) - 5 -
oxocyclopentylidene]methyl)oxiran-2-yl]butanal. To a solution of
E-2-((3-(4-hydroxybutyl)oxiran-2-yl)methylene)-4-(4-methoxyphenyl-
methoxy)-3-((Z)-oct-2-enyl)cyclopentanone (166 mg, 0.36 mmol) in
dichloromethane (10 mL) at 0 °C was added solid NaHCO3 (154 mg)
followed by Dess−Martin periodinane (186 mg, 0.44 mmol)
portionwise. The reaction mixture was allowed to warm to 21 °C
and stirred for 1 h. The solution was quenched with saturated aqueous
NaHCO3 (10 mL) and saturated aqueous Na2S2O3 (10 mL). The
aqueous layer was extracted with dichloromethane (3 × 20 mL). The
combined organic layers were dried over Na2SO4, filtered, and then
concentrated to dryness in vacuo. The residue was purified by flash
column chromatography over silica gel (hexane/ethyl acetate, 3:1, v/v)
to afford the desired aldehyde (140 mg, 0.31 mmol, 86%) as a
colorless yellow oil.
4-[(2R,3S)/(2S,3R)-3-((E)-((2R,3R)/(2S,3S)-3-(4-Methoxyphenylme-
thoxy)-2-((Z)-oct-2-enyl)-5-oxocyclopentylidene]methyl)oxiran-2-
1
yl]butanal (Cis). Rf = 0.62 (hexane/ethyl acetate, 1:1, v/v); H NMR
(CDCl3, 400 MHz) δ 9.78 (t, J = 1.36 Hz, 1H), 7.27 (d, J = 8.8 Hz,
2H), 6.88 (d, J = 8.8 Hz, 2H), 6.12 (dd, J = 9.3, 1.6 Hz, 1H), 5.46 (m,
1H), 5.32 (m, 1H), 4.55 (d, J = 11.3 Hz, 1H), 4.49 (d, J = 11.3 Hz,
1H), 4.18 (ddd, J = 14.4, 7.4, 7.4 Hz, 1H), 3.78 (s, 3H), 3.32 (m, 1H),
3.22 (dd, J = 9.3, 2.0 Hz, 1H), 2.94 (m, 1H), 2.64−2.45 (m, 4H), 2.08
8530
dx.doi.org/10.1021/jm400959q | J. Med. Chem. 2013, 56, 8521−8532

Journal of Medicinal Chemistry
Article
column chromatography over silica gel (dichloromethane/methanol,
20:1, v/v) to afford the desired EI (77 mg, 0.22 mmol, 76%) as a pale
yellow solid.
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4-[(2R,3S)/(2S,3R)-3-((E)-((2R,3R)/(2S,3S)-3-Hydroxy-2-((Z)-oct-2-
enyl)-5-oxocyclopentylidene)methyl]oxiran-2-yl]butanoic Acid (cis-
1
EI), 1 and 2. Rf = 0.36 (hexane/ethyl acetate, 1:1, v/v); H NMR
(CDCl3, 400 MHz) δ 6.17 (d, J = 9.0 Hz, 1H), 5.50 (m, 1H), 5.38 (m,
1H), 4.54 (m, 1H), 3.25 (m, 2H), 2.98 (m, 1H), 2.64 (m, 2H), 2.44
(m, 3H), 2.16 (m, 1H), 1.95 (m, 2H), 1.80 (m, 3H), 1.58 (m, 1H),
1.33−1.25 (m, 7H), 0.86 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100
MHz) δ 201.7, 178.2, 144.1, 133.2, 126.0, 118.8, 69.1, 59.9, 55.3, 44.8,
44.4, 33.3, 31.5, 31.2, 29.3, 27.4, 25.2, 22.5, 21.0, 14.1; ESI-HRMS
found 349.2011 [M − H]−, calcd for C20H29O5 349.2015.
4-[(2R,3R)/(2S,3S)-3-(E)-((2R,3R)/(2S,3S)-3-Hydroxy-2-((Z)-oct-2-
enyl)-5-oxocyclopentylidenemethyl]oxiran-2-yl]butanoic Acid
1
(trans-EI), 3 and 4. Rf = 0.35 (hexane/ethyl acetate, 1:1, v/v); H
NMR (CDCl3, 500 MHz) δ 6.25 (d, J = 9.0 Hz, 1H), 5.50 (m, 1H),
5.36 (m, 1H), 4.36 (bd, J = 5.0 Hz, 1H), 3.29 (m, 1H), 3.11 (m, 1H),
2.98 (m, 1H), 2.64 (m, 2H), 2.44 (m, 2H), 2.26 (m, 1H), 2.11 (m,
1H), 1.95 (m, 2H), 1.81 (m, 3H), 1.59 (m, 1H), 1.32 (m, 7H), 0.86 (t,
J = 6.8 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ 204.1, 178.3, 142.6,
134.4, 133.3, 125.3, 71.3, 60.0, 55.0, 48.9, 45.1, 33.3, 31.5, 31.3, 30.9,
29.2, 27.3, 22.5, 21.0, 14.0; ESI-HRMS found 349.2018 [M − H]−,
calcd for C20H29O5 349.2015.
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ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectra for all new compounds and an Excel file
containing metadata and normalized array data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(8) Berliner, J. A.; Watson, A. D. A role for oxidized phospholipids in
atherosclerosis. N. Engl. J. Med. 2005, 353, 9−11.
AUTHOR INFORMATION
■
(9) Subbanagounder, G.; Wong, J. W.; Lee, H.; Faull, K. F.; Miller,
E.; Witztum, J. L.; Berliner, J. A. Epoxyisoprostane and epoxycyclo-
pentenone phospholipids regulate monocyte chemotactic protein-1
and interleukin-8 synthesis. Formation of these oxidized phospholipids
in response to interleukin-1beta. J. Biol. Chem. 2002, 277, 7271−7281.
(10) Cole, A. L.; Subbanagounder, G.; Mukhopadhyay, S.; Berliner, J.
A.; Vora, D. K. Oxidized phospholipid-induced endothelial cell/
monocyte interaction is mediated by a cAMP-dependent R-Ras/PI3-
kinase pathway. Arterioscler., Thromb., Vasc. Biol. 2003, 23, 1384−1390.
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identification of a novel pro-inflammatory epoxyisoprostane phospho-
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Corresponding Author
*Phone: 310-825-7954. Fax: 310-206-3722. E-mail: jung@
chem.ucla.edu. Address: Department of Chemistry and
Biochemistry, 607 Charles E. Young Drive East, Box 951569,
Los Angeles, CA 90095-1569, United States.
Notes
The authors declare no competing financial interest.
⊥W.Z. and J.R.S. are co-first-authors.
ACKNOWLEDGMENTS
■
This work was supported by Ruth L. Kirschstein National
Research Service Award T32-HL-69766 (J.R.S.) and National
Institutes of Health Grants HL-30568 (J.R.S and J.A.B.), HL-
064731 (W.Z., J.R.S., J.A.B., and M.E.J.), and 1K99-HL-105577
(S.L.), and Western Michigan internal research funding (R.A.-
M.).
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ABBREVIATIONS USED
■
DDQ, dichlorodicyanoquinone; DIBAL, diisobutylaluminum
hydride; HAEC, human aortic endothelial cell; IL-1β,
interleukin 1β; IL-8, interleukin 8; LDL, low-density lip-
oprotein; MCP-1, monocyte chemotactic protein 1; MEK,
methyl ethyl ketone; MM-LDL, minimally modified low-
density lipoprotein; Ox-PAPC, oxidized 1-palmitoyl-2-arach-
idonoyl-sn-glycero-3-phosphocholine; PCC, pyridinium chlor-
ochromate; PEIPC, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-
sn-glycero-3-phosphocholine; PMB, p-methoxybenzyl; EI,
epoxyisoprostane; PGE2, prostaglandin E2; UPR, unfolded
protein response
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