17(R),18(S)-Epoxyeicosatetraenoic acid, a potent eicosapentaenoic acid (EPA) derived regulator of cardiomyocyte contraction: Structure-activity relationships and stable analogues

Article abstract of DOI:10.1021/jm200132q

17(R),18(S)-Epoxyeicosatetraenoic acid [17(R),18(S)-EETeTr], a cytochrome P450 epoxygenase metabolite of eicosapentaenoic acid (EPA), exerts negative chronotropic effects and protects neonatal rat cardiomyocytes against Ca ²⁺-overload with EC₅₀ ≈ 1-2 nM. Structure-activity studies revealed that a cis-Δ^11,12- or Δ^14,15- olefin and a 17(R),18(S)-epoxide are minimal structural elements for antiarrhythmic activity whereas antagonist activity was often associated with the combination of a Δ^14,15-olefin and a 17(S),18(R)-epoxide. Compared with natural material, the agonist and antagonist analogues are chemically and metabolically more robust and several show promise as templates for future development of clinical candidates.

Full text of DOI:10.1021/jm200132q

ARTICLE
pubs.acs.org/jmc
17(R),18(S)-Epoxyeicosatetraenoic Acid, a Potent Eicosapentaenoic
Acid (EPA) Derived Regulator of Cardiomyocyte Contraction:
StructureꢀActivity Relationships and Stable Analogues
John R. Falck,*,† Gerd Wallukat,‡ Narender Puli,† Mohan Goli,† Cosima Arnold,‡ Anne Konkel,‡
Michael Rothe,§ Robert Fischer,|| Dominik N. M€uller,‡ and Wolf-Hagen Schunck*,‡
†Department of Biochemistry, University of Texas Southwestern, Dallas, Texas 75390, United States
‡Max Delbrueck Center for Molecular Medicine, Berlin, Germany
§Lipidomix GmbH, Berlin, Germany
Experimental and Clinical Research Center, Charitꢀe Medical Faculty, Berlin, Germany
S Supporting Information
b
ABSTRACT:
17(R),18(S)-Epoxyeicosatetraenoic acid [17(R),18(S)-EETeTr], a cytochrome P450 epoxygenase metabolite of eicosapentaenoic
acid (EPA), exerts negative chronotropic effects and protects neonatal rat cardiomyocytes against Ca2þ-overload with EC50 ≈ 1ꢀ2
nM. Structureꢀactivity studies revealed that a cis-Δ11,12- or Δ14,15-olefin and a 17(R),18(S)-epoxide are minimal structural elements
for antiarrhythmic activity whereas antagonist activity was often associated with the combination of a Δ14,15-olefin and a
17(S),18(R)-epoxide. Compared with natural material, the agonist and antagonist analogues are chemically and metabolically
more robust and several show promise as templates for future development of clinical candidates.
’ INTRODUCTION
18(S)-enantiomer 2.6b,c,7 A recent in vivo study revealed that
dietary ω-3 PUFA supplementation causes a profound shift in the
endogenous CYP-eicosanoid profile from AA- to EPA/DHA-
derived metabolites.8 In fact, 1 represented the predominant
epoxy metabolite in the heart, lung, kidney, and several other
organs of rats fed an EPA/DHA-rich diet, whereas this metabolite
was produced only in trace amounts when the animals received
normal chow rich in ω-6 PUFAs.8 There is also initial evidence for
the in vivo formation of EETeTrs in humans based on studies
showing the occurrence of these EPA metabolites and/or their
hydrolysis products in urine and plasma of volunteers ingesting
increased amounts of ω-3 PUFAs.9,10 Epoxide 2 is a potent
vasodilator and stimulates large-conductance Kþ (BK) channels
in rat cerebral arteries.11 The vasodilator effects of epoxides 1 and
The intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs)
such as eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) is associated with manifold health benefits,1 especially for
cardiovascular disease,2 e.g., lower triglyceride levels, ameliora-
tion of ischemic injury, and reduction of sudden cardiac death
resulting from fatal arrhythmia.3 However, the mechanism(s) of
action and the molecular specie(s) or metabolite(s) responsible
for the protective effects of the ω-3 PUFAs remain largely
obscure.4 A leading hypothesis is that EPA and its congeners
compete with arachidonic acid (AA) for enzymatic transforma-
tions and that the ω-3 PUFA metabolites have physiological
consequences that differ from those of AA.5
In concert with this proposal, a series of in vitro studies
demonstrated that many major cytochrome P450 (CYP) epoxy-
genase isoforms preferentially metabolize EPA to the 17,
18-epoxide 1 (17,18-EETeTr)6 and often favor the 17(R),
Received: February 6, 2011
Published: May 17, 2011
r
2011 American Chemical Society
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Table 1. Chronotropic Effects of 17,18-EETeTr and Analogues on NRCMsa
a All compounds were tested at a final concentration of 30 nM at 37 °C (control NRCM, 120ꢀ150 beats/min). b n = number of determinations.
2 are not blocked by the epoxyeicosatrienoic acid (EET) antago-
nist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE, 10 μM), suggest-
ing a recognition or binding site independent of the putative EET
receptor.12,13 In the human lung, 1 also hyperpolarizes and relaxes
pulmonary artery and bronchial smooth muscle cells and exerts
anti-inflammatory effects.14,15
ω-3 PUFAs.3a,18 In this model, EPA (3ꢀ10 μM) reduces the
contraction rate of the spontaneously beating heart cells; i.e., it
exerts a strong negative chronotropic effect. Furthermore, EPA
reduces the response of NRCMs to β-adrenergic agonists
and increases in calcium concentrations and terminates the
arrhythmias induced by these agents.18 We have shown pre-
viously that 1 and 2 (Table 1) exert the same effects as EPA on
NRCMs but in contrast are active in the low nanomolar range,
suggesting that these metabolites may function as mediators of
the antiarrhythmic mechanism of ω-3 PUFAs.8 In the present
study, we used the chronotropic effect on NRCMs as a bioassay
to evaluate the relative “antiarrhythmic potencies” of a series of
synthetic analogues based on the structures of the natural EPA
epoxy metabolites. This SAR study was performed (i) to identify
the structural determinants that mediate the unique biological
activities of 1, (ii) to find selective agonists19 and antagonists as
required for future mechanistic studies and for isolation of the
putative receptor, and (iii) to generate agonists with improved
chemical and metabolic stability as a first step toward the
development of novel antiarrhythmic drugs.
A major open question concerning the whole field of CYP-
eicosanoid research is the heretofore unidentified, primary targets
of CYP-eicosanoid action. Although some studies indicate direct
interactions with certain ion channels and transcription factors,
most of the observed CYP-eicosanoid actions apparently rely on
the ability of these metabolites to trigger distinct intracellular
signaling pathways. These pathways, depending upon the specific
CYP metabolite and physiological context, can involve protein
kinase A, guanine nucleotide binding proteins, tyrosine kinases,
mitogen-activated kinases, phosphoinositol-3 kinase, IkB kinase,
F-kinase, or the epidermal growth factor and a series of other well-
known key components of intracellular signaling.16 In general,
these signaling components become specifically engaged in path-
ways that are primarily elicited by membrane receptors. Recent
studies indeed revealed the presence of a high affinity EET-
binding site in U937 cells, which is probably a G-protein-coupled
receptor.17 These findings suggest that the various CYP-dependent
metabolites of ω-6- and ω-3-PUFAs may exert their specific func-
tions via a family of G-protein-coupled receptors analogous with
the cyclooxygenase- and lipoxygenase-generated eicosanoids.
Neonatal rat cardiomyocytes (NRCMs) have been introduced
as an in vitro model to analyze the antiarrhythmic mechanisms of
’ RESULTS AND DISCUSSION
While EPA required prolonged incubation and a ∼1000-fold
higher concentration, epoxide 2 was almost immediately effective
with EC50 ≈ 1ꢀ2 nM (Figure 1). Notably, the negative chrono-
tropic activity resides with the 17(R),18(S)-enantiomer 2 while its
antipode 3 had no significant effect (Table 1). The soluble epoxide
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Figure 1. Dose dependency of EPA epoxide 2 and analogues 13, 19, 21, and 22 on NRCM beating rates.
Table 2. Chronotropic Effects of Epoxide Bioisosteric Analogues on NRCMsa
a All compounds were tested at a final concentration of 30 nM at 37 °C (control NRCM, 120ꢀ150 beats/min). b n = number of determinations.
hydrolase (sEH) metabolite of 1, i.e., diol 4, was completely
inactive. To capitalize on these observations for the development
of more efficacious antiarrhythmic therapies,20 we initiated a
systematic structureꢀactivity relationship study to characterize this
pharmacophore and to simultaneously address the chemical and
metabolic labilities of 1 and 2 that restrict their wider utility.21,22
Thus, our first priority was the partial saturation of the polyenoic
backbone with the intention of strategically disrupting the three
1,4-dienyl combinations. This would obviate both autoxidation and
metabolism by lipoxygenases and cycloxygenases.4c Of the three
possible tetrahydro variants 5ꢀ7 that satisfied this criteria, only
7 was still competent to significantly reduce the beating rate.
The mono-olefinic series 8ꢀ11 was consistent with the prece
ding observations and clearly established that the presence of a
Δ
11,12-olefin, as found in analogue 10, confers strong negative
chronotropic activity. The fully saturated analogue 12 proved to be
largely inactive. As might be anticipated from the differences
between the 17,18-EETeTr enantiomers 2 and 3, analogue 13
was a powerful negative chronotrope whereas its mirror image,
analogue 14, had the opposite, albeit more modest activity. The
enantiomeric pair 15 and 16 presented a similar pharmacological
profile that suggests that the absolute configuration of the epoxide
can trump the influence of olefin position. Terminal hydroxylation
is a common degradation pathway for fatty acids and eicosanoids,23
so it was no surprise that this modification mostly abrogated
biological activity in the arrhythmia assay, e.g., 17 and 18.
In recognition of the central role of the sEH enzyme in the
metabolism of fatty epoxanoids,24 replacement of the epoxide with
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Figure 2. Antagonism of 10 by other analogues.
an achiral epoxide bioisostere21 was deemed crucial to the devel-
opment of any robust, long-lived analogues intended for in vivo
applications. Hence, we were delighted to find 19,21 a 1,3-
disubstituted urea version of 10, decisively suppressed the beating
rate (Table 2). In contrast to prior experience with 14,15-EET
analogues,21 the introduction of N-methyls onto the urea, i.e.,
analogue 20, strongly dampened the antiarrhythmic effect. 1,4-
Disubstituted oxamide 21 proved to be the most efficacious of all
the bioisosteres and, thus, was subjected to additional scrutiny.
Among these, the trans-olefinic and acetylenic versions 22 and 23,
respectively, were the most informative. The strict stereochemical
requirement for this portion of the molecule suggests that the
recognition or binding site contains a narrow pocket that best
accommodates the hairpin configuration of a cis-olefin. The
related amides 24ꢀ26 were disappointing; however, shifting the
amide functionality just one position away from the ω-terminus as
in 27 restored most of the negative chronotropic influence.
Repositioning the olefin to the Δ14,15-position consistently re-
versed or annulled any negative chronotropic behavior irrespective
of the epoxide surrogate: 28 vs 25, 29 vs 21, and 30 vs 19.
After identification of several potent analogues containing
both epoxide and bioisosteric replacements, it was of interest to
evaluate the dose response profile of select examples versus
17(R),18(S)-EETeTr (2) on NRCM beating rates. The weakly
active trans-olefinic analogue 22 was included as a control. As
evident in Figure 1, urea 19 and oxamide 21 outperformed 2 at
most concentrations. Above ∼5 nM, all of the test compounds
began to plateau except for analogue 22, which did not display
any significant efficacy until the low micromolar range.
Figure 3. Antagonism of negative chronotropes by 6.
resulted in any useful antagonism (Figure 2). While both share
a Δ14,15-olefin, this is not sufficient for antagonism, since
analogue 5 also contains a Δ14,15-olefin, yet was not distinguish-
able from vehicle.
While analogue 6 emerged as the most powerful antagonist in
the foregoing study, there was no certainty that it could function
as a universal antagonist against other negative chronotropes.
Consequently, NRCMs were preincubated with several of the
most efficacious negative chronotropes, inter alia, EPA, 1, 7, and
oxamide 21, and then challenged with an equimolar amount of 6
(Figure 3). In all cases, blockades of 90% or better were achieved.
Of particular note, antagonist 6 (30 nM) blocked not only the
effects of epoxide 1 (30 nM) and its synthetic agonists but also
that of EPA (3.3 μM), suggesting that EPA and its natural
metabolite act via the same mechanism.
To demonstrate that the chemical modifications described
above resulted in agonists with improved metabolic stability, we
analyzed the metabolism of 1, 12, 13, 19, and 21 by rat liver
homogenates. The results are shown in detail in the Supporting
Information. As expected, epoxide 1 was rapidly hydrolyzed to
vic-diol 4 and further attacked by epoxidation and hydroxylation
reactions. In total, about 80% of 1 was metabolized within
30 min. Analogue 21, carrying an oxamide moiety instead of
the epoxy group and only the functionally essential 11,12-double
bond, provided the current best solution to overcome the
metabolic instability of the natural compound. LCꢀMS analysis
indicated that analogue 21 was only slowly metabolized to
regioisomeric hydroxy metabolites, and more than 85% of the
original compound remained intact after 30 min of incubation
with liver homogenate (see Supporting Information).
In recognition of the ability of analogue 16 to block the
negative chronotropic effects of its antipode 15, we sought to
identify additional antagonists among the analogues in Table 1
that were themselves largely devoid of activity. To this end,
NRCMs were preincubated with the test antagonist (30 nM final
concentration) for 5 min at 37 °C before the addition of analogue
10 (30 nM final concentration). Only analogues 6 and 11
It was also of interest to understand the consequences, if any,
of the structural modifications on sEH activity. Other labo-
ratories25 have developed highly potent sEH inhibitors that
utilize ureas as the epoxide bioisostere. Similar functionality
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Scheme 1. Synthesis of 7, 8, 10, and 12a
Scheme 2. Synthesis of 21a
a Reagents and conditions: (a) n-BuLi, THF/HMPA (6:1), ꢀ78 to ꢀ10
°C, 2 h; Br(CH2)4OTHP, ꢀ78 °C to room temp, 3 h; room temp, 12 h,
66%; (b) n-Bu4NF, THF, room temp, 5 h, 80%; (c) P-2 Ni/(H2NCH2)2,
H2 (1 atm), EtOH, room temp, 99%; (d) Jones reagent, acetone, ꢀ40 to
ꢀ10 °C, 68%; (e) p-TSA, MeOH, room temp, 10 h, 83%; (f) DEAD,
Ph3P, THF, ꢀ20 °C, 30 min; (PhO)2P(O)N3, 0 °C, 6 h, 78%; (g) Ph3P,
THF/H2O, room temp, 8 h; (h) EDCI, HOBt, i-PrEt2N, MeNHC-
(O)C(O)OH, DMF, room temp, 12 h, 68% over two steps; (i) LiOH,
THF/H2O (4:1), room temp 10 h, 89%.
a Reagents and conditions: (a) n-BuLi (0.9 equiv), THF/HMPA (6:1),
ꢀ78 to ꢀ10 °C, 2 h; Br(CH2)4OTHP, ꢀ78 to room temp, 3 h; room
temp, 12 h, 65%; (b) n-BuLi, THF/HMPA (6:1), ꢀ78 to ꢀ10 °C, 2 h;
Br(CH2)4CHdCHCH2CH3, ꢀ78 °C to room temp, 3 h; room temp,
12 h, 65%; (c) p-TSA, MeOH, room temp, 4 h, 92%; (d) Jones reagent,
acetone, ꢀ40 to ꢀ10 °C, 88%;(e) p-TSA, MeOH, room temp, 10 h, 82%;
(f) m-CPBA, CH2Cl2, room temp, 2 h, 82%; (g) P-2 Ni/(H2NCH2)2, H2
(1 atm), EtOH, 98%; (h) (H2N)2, CuSO4, O2, EtOH, room temp, 12 h;
(i) LiOH, THF/H2O (4:1), room temp 10 h, 90ꢀ93%.
and selective epoxidation of the Δ17,18-olefin. The resultant
epoxide 34 smoothly led to dienoate 35 via semihydrogenation
over P-2 nickel/hydrogen gas. Random hydrogenation of 35
using diimide generated in situ gave rise to a chromatographically
separable mixture of unreacted 35, mono-olefins 36 and 37, and
tetrahydroepoxide 38. LiOH hydrolysis of the individual methyl
esters yielded the corresponding free acids 7, 8, 10, and 12.
Access to oxamide 21 (Scheme 2) began with alkylation of
12-(tert-butyldiphenylsilyloxy)dodec-1-yne29 (39) with 2-(4-
bromobutoxy)tetrahydro-2H-pyran.30 Subsequent desilylation
evolved 40, which was advanced to 41 via P-2 nickel mediated
semihydrogenation and Jones oxidation. Exposure of the product to
acidic methanol simultaneously esterified the carboxylic acid and
removed the THP ether. Replacement of the terminal alcohol with
azide using diphenylphosphoryl azide (DPPA) under Mitsunobu-
type conditions completed the transformation to 42. Sequential
Staudinger reduction, EDCI amidation with 2-(methylamino)-2-
oxoacetic acid,30 and hydrolysis delivered 21 and could be accom-
plished without the purification of intermediates.
appears in some of our 17,18-EETeTr agonists, e.g., 19 (Table 2).
Accordingly, the biological activity of our analogues might be
attributablein part or entirely to inhibition of sEH andsubsequent
accumulation of endogenous epoxy eicosanoids in the cardio-
myocytes. To test this possibility, we analyzed selected com-
pounds containing the urea (analogue 19) or oxamide (analogues
21 and 29) moieties for their capacity to inhibit sEH. Analogue 19
exerted weak dose-dependent sEH inhibition at 1 and 5 μM, i.e.,
concentrations 1000-fold higher than required for reducing the
contractility of cardiomyocytes (see Supporting Information and
Figure 2). Analogues 21 and 29 lacked any inhibitory effects up to
5 μM. Under the same conditions, AUDA (12-(3-adamantan-1-
yl-ureido)dodecanoic acid), a representative of the urea class of
highly potent sEH-inhibitors, almost completely blocked sEH
activity at 100 nM.25 AUDA itself had no effect on the sponta-
neous beating rate of cultured cardiomyocytes. However, AUDA
partially inhibited the negative chronotropic effect of 1 (see
Supporting Information and Figure 3). Furthermore, the com-
bined administration of 19 or 21 with 17,18-EETeTr did not
result in additive or synergistic effects (see Supporting Informa-
tion and Figure 3). Taken together, these results show that our
synthetic analogues do not or only weakly target sEH; hence, sEH
inhibition can be excluded as a significant contributor to their
effects on cardiomyocytes.
’ CONCLUSIONS
The potency and rapid onset of action by 17(R),18(S)-EETeTr
(2) are consistent with a prominent role for this cytochrome
P450-dependent eicosanoid in mediating the antiarrhythmic
effects of EPA. Structureꢀactivity correlations helped define
the essential pharmacophore and led to the development of
agonist and antagonist analogues that are more robust than
natural material and that provide a sound foundation for the
future development of potential clinical candidates. Additionally,
these data are also consistent with the existence of a specific ω-3
epoxanoid receptor, albeit other mechanisms of actions cannot
be excluded at present.
’ CHEMISTRY
Syntheses of 7, 8, 10, and 12 are summarized in Scheme 1 and
are representative of the methodology used to prepare the other
epoxide-containing analogues. Following literature precedent,26
commercial octa-1,7-diyne (31) was alkylated with a limiting
amount of 2-(4-bromobutoxy)tetrahydro-2H-pyran27 in THF/
HMPA to give 32. A second alkylation using 8-bromooct-3(Z)-
ene28 followed by acidic cleavage of the THP and Jones oxidation
furnished acid 33 that was then subjected to Fischer esterification
’ EXPERIMENTAL SECTION
General Procedures. Unless stated otherwise, yields refer to
purified products and are not optimized. Final compounds were judged
to be g95% pure by HPLC using a Zorbax Eclipse C18 (250 mm ꢁ
4.6 mm, Agilent) connected to an Agilent 1200 API/LCꢀMS
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instrument with acetonitrile/water combinations as solvent; enantio-
mers were also analyzed by chiral phase HPLC using a Chiralcel OJ-H
column (250 mm ꢁ 4.6 mm, Daicel) with hexane/isopropyl alcohol
combinations as solvent and judged to be g95% ee. All oxygen and/or
moisture sensitive reactions were performed under an argon atmosphere
using oven-dried glassware and anhydrous solvents. Anhydrous solvents
were freshly distilled from sodium benzophenone ketyl except for
CH2Cl2, which was distilled from CaH2. Extracts were dried over
anhydrous Na2SO4 and filtered prior to removal of all volatiles under
reduced pressure. Unless otherwise noted, commercially available
materials were used without purification. Flash chromatography (FC)
was performed using E Merck silica gel 60 (240ꢀ400 mesh). Thin layer
chromatography was performed using precoated plates purchased from
E. Merck (silica gel 60 PF254, 0.25 mm). Nuclear magnetic resonance
(NMR) spectra were recorded on a Varian 300, 400, or 500 spectro-
meter at operating frequencies of 300/400/500 MHz (1H) or 75/100/
125 MHz (13C). Nuclear magnetic resonance (NMR) splitting patterns
are described as singlet (s), doublet (d), triplet (t), quartet (q), and
broad (br); the values of chemical shifts (δ) are given in ppm relative to
was purified by SiO2 column chromatography using 15% EtOAc/
hexanes as eluent to give eicosa-17(Z)-en-5,11-diyn-1-ol (925 mg,
92%) as a colorless oil. TLC: 30% EtOAc/hexanes, Rf ≈ 0.35. 1H
NMR (400 MHz, CDCl3) δ 5.27ꢀ5.42 (m, 2H), 3.66 (t, J = 6.8 Hz,
2H), 2.00ꢀ2.19 (m, 12H), 1.43ꢀ1.72 (m, 12H), 0.95 (t, 3H, J = 7.7 Hz).
13C NMR (100 MHz, CDCl3) δ 132.56, 128.45, 80.42, 80.22, 62.69,
34.06, 32.51, 32.07, 28.39, 28.20, 27.73, 26.36, 25.51, 20.74, 18.73, 18.45,
18.16, 14.45. HRMS calcd for C20H33O [M þ 1]þ 289.2531, found
289.2534.
Jones reagent (5 mL of a 10 N aqueous solution) in acetone (10 mL)
was added slowly to a stirring, ꢀ40 °C solution of eicosa-17(Z)-ene-
5,11-diyn-1-ol (1.0 g, 3.47 mmol) in acetone (50 mL). After 1 h, the
reaction mixture was warmed to ꢀ10 °C and maintained at this
temperature for 3 h, then quenched with excess isopropanol. The green
chromium salts were removed by filtration, the filter cake was washed
with acetone, and the combined filtrates were concentrated in vacuo.
The residue was dissolved in EtOAc (100 mL), washed with water
(50 mL), and concentrated in vacuo. The residue was purified by SiO2
column chromatography, using 15% EtOAc/hexanes as eluent to give 33
(920 mg, 88%) as a colorless oil. TLC: 30% EtOAc/hexanes, Rf ≈ 0.35.
1H NMR (400 MHz, CDCl3) δ 5.24ꢀ5.41 (m, 2H), 2.41 (t, J = 6.9 Hz,
3H), 2.10ꢀ2.19 (m, 8H), 1.98ꢀ2.09 (m, 4H), 1.75ꢀ1.81 (m, 2H), 0.96
(t, J = 7.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 184.37, 132.54,
128.44, 84.43, 80.45, 80.26, 78.58, 34.08, 32.53, 32.09, 28.36, 28.23,
27.75, 26.38, 25.53, 20.76, 18.77, 18.48, 18.03, 14.59. HRMS calcd for
C20H31O2 [M þ 1]þ 303.2324, found 303.2324.
1
residual solvent (chloroform δ = 7.27 for H NMR or δ = 77.23 for
proton decoupled 13C NMR), and coupling constants (J) are given in
hertz (Hz). Melting points were determined using an OptiMelt
(Stanford Research Systems) and are uncorrected. The Michigan State
University Mass Spectroscopy Facility or Prof. Kasem Nithipatikom
(Medical College of Wisconsin) kindly provided high-resolution mass
spectral analysis results.
34. A solution of 33 (0.8 g, 2.63 mmol) and p-TSA (20 mg) in MeOH
(30 mL) was stirred at room temperature for 10 h, then concentrated in
vacuo. The residue was purified by SiO2 column chromatography, using
3% EtOAc/hexanes as eluent to give methyl eicos-17(Z)-en-5,11-
diynoate (682 mg, 82%) as a colorless oil. TLC: 10% EtOAc/hexanes,
Rf ≈ 0.60. 1H NMR (400 MHz, CDCl3) δ 5.27ꢀ5.42 (m, 2H), 3.67 (s,
3H), 2.43 (t, 2H, J = 7.6 Hz), 2.12ꢀ2.21 (m, 8H), 1.99ꢀ2.09 (m, 4H),
1.76ꢀ1.82 (m, 2H), 1.42ꢀ1.58 (m, 8H), 0.95 (t, 3H, J = 7.7 Hz). 13C
NMR (100 MHz, CDCl3) δ 184.30, 132.57, 128.48, 84.52, 80.46, 80.21,
79.97, 51.19, 33.09, 32.43, 32.19, 28.39, 28.27, 27.65, 26.36, 25.63, 20.73,
18.72, 18.49, 18.07, 13.76. HRMS calcd for C21H33O2 [M þ 1]þ
317.2481, found 317.2485.
m-CPBA (1.6 g, 4.76 mmol) was added in portions to a 0 °C solution
of methyl eicosa-17(Z)-en-5,11-diynoate (1.15 g, 3.66 mmol) in
CH2Cl2 (50 mL). After 2 h at room temperature, the reaction mixture
was diluted with CH2Cl2 (25 mL), washed with saturated aqueous
NaHCO3 (2 ꢁ 25 mL), brine (2 ꢁ 25 mL), water (50 mL), dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by SiO2 column chromatography, using 5% EtOAc/hexanes as
eluent to give 34 (990 mg, 82%) as a colorless oil. TLC: 10% EtOAc/
hexanes, Rf ≈ 0.3. 1H NMR (400 MHz, CDCl3) δ 3.67 (s, 3H),
2.84ꢀ2.94 (m, 2H), 2.42 (t, 2H, J = 7.3 Hz), 2.14ꢀ2.23 (m, 8H),
1.74ꢀ1.83 (m, 2H), 1.42ꢀ1.61 (m, 12H), 1.03 (t, 3H, J = 7.6 Hz). 13C
NMR (100 MHz, CDCl3) δ 184.21, 83.62, 80.35, 80.08, 62.96, 59.43,
52.32, 34.19, 32.28, 32.20, 29.17, 28.32, 28.25, 27.75, 26.43, 25.72, 20.75,
18.63, 18.28, 18.14, 14.53. HRMS calcd for C21H33O3 [M þ 1]þ
333.2430, found 333.2434.
32. n-BuLi (30 mL of 2.5 M solution in hexanes, 70 mmol) was added
dropwise to a ꢀ78 °C solution of 31 (9.0 g, 84.9 mmol; G F Smith, Inc.)
in dry THF/HMPA (350 mL, 6:1) under an argon atmosphere. After
30 min, the reaction mixture was warmed to ꢀ10 °C over 2 h and
maintained at this temperature for 20 min, then recooled to ꢀ78 °C. To
this was added a solution of 2-(4-bromobutoxy)tetrahydro-2H-pyran27
(15 g, 63.68 mmol) in dry THF (15 mL). The resulting mixture was
warmed to room temperature over 3 h, maintained at this temperature
for 12 h, then quenched with saturated aqueous NH4Cl (25 mL). After
20 min, the mixture was extracted with Et2O (2 ꢁ 125 mL). The
combined ethereal extracts were washed with water (2 ꢁ 100 mL), brine
(100 mL), dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by SiO2 column chromatography,
using 5% EtOAc/hexanes as eluent to give 32 (10.85 g, 65%) as a
colorless oil. TLC: 10% EtOAc/hexanes, Rf ≈ 0.6. 1H NMR (400 MHz,
CDCl3) δ 4.57 (t, J = 2.5 Hz, 1H), 3.82ꢀ3.87 (m, 1H), 3.70ꢀ3.77 (m,
1H), 3.46ꢀ3.51 (m, 1H), 3.36ꢀ3.42 (m, 1H), 2.14ꢀ2.20 (m, 6H), 1.93
(t, J = 2.5 Hz, 1H), 1.46ꢀ1.72 (m, 14H). 13C NMR (100 MHz, CDCl3)
δ 98.78, 84.23, 80.31, 79.82, 68.50, 67.07, 62.25, 30.80, 30.76, 28.98,
28.07, 27.58, 25.96, 25.57, 19.68, 18.64, 18.31. HRMS calcd for
C16H25O2 [M þ 1]þ 249.1855, found 249.1852.
33. Compound 32 (4.5 g, 17.2 mmol) was alkylated using n-BuLi
(8.3 mL of 2.5 M solution in hexanes, 20.65 mmol) and 8-bromooct-
3(Z)-ene31 (4.1 g, 21.5 mmol) in THF/HMPA (180 mL, 6:1) as
described above for the synthesis of 32 to give 2-[eicosa-17(Z)-en-
5,11-diynyloxy]tetrahydro-2H-pyran (4.15 g, 65%) as a colorless oil.
1
TLC: 10% EtOAc/hexanes, Rf ≈ 0.6. H NMR (400 MHz, CDCl3)
δ 5.26ꢀ5.41 (m, 2H), 4.58 (t, J = 2.5 Hz, 1H), 3.82ꢀ3.87 (m, 1H),
3.70ꢀ3.77 (m, 1H), 3.46ꢀ3.51 (m, 1H), 3.36ꢀ3.42 (m, 1H),
2.11ꢀ2.20 (m, 8H), 1.92ꢀ2.04 (m, 4H), 1.62ꢀ1.86 (m, 4H),
1.39ꢀ1.59 (m, 14H), 0.94 (t, J = 7.5 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 132.54, 128.42, 98.98, 80.47, 79.99, 68.56, 67.25, 62.49, 34.01,
32.49, 30.93, 29.12, 28.37, 28.21, 27.71, 26.34, 26.08, 25.69, 20.72, 19.83,
18.79, 18.46, 18.16, 14.53. HRMS calcd for C24H39O2 [M þ 1]þ
359.2950, found 359.2951.
35. NaBH4 (33 mg, 0.88 mmol) was added portionwise to a stirring,
room temperature solution of nickel(II) acetate tetrahydrate (190 mg,
0.76 mmol) in absolute ethanol (5 mL) under a hydrogen blanket
(1 atm). After 15 min, freshly distilled ethylenediamine (200 mg,
3.24 mmol) was added followed by 34 (250 mg, 0.75 mmol) in absolute
ethanol (5 mL) while maintaining the hydrogen blanket (1 atm). The
heterogeneous mixture was stirred at room temperature for 90 min, then
diluted with ether (40 mL) and filtered through a short pad of silica gel;
the filtration pad was washed with ether (3 ꢁ 10 mL). The combined
filtrates were dried over anhydrous Na2SO4 and concentrated in vacuo
to give 35 (246 mg, 98%) as a colorless oil sufficiently pure to be used
A solution of 2-[eicos-17(Z)-en-5,11-diynyloxy]tetrahydro-2H-pyr-
an (1.3 g, 3.49 mmol) and p-TSA (50 mg) in MeOH (50 mL) was stirred
at room temperature for 4 h, then concentrated in vacuo. The residue
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ARTICLE
directly in the next step. TLC: 20% EtOAc/hexanes, Rf ≈ 0.65. 1H NMR
(400 MHz, CDCl3) δ 5.27ꢀ5.42 (m, 4H), 3.66 (s, 3H), 2.83ꢀ2.93 (m,
2H), 2.30 (t, 2H, J = 7.3 Hz), 1.92ꢀ2.09 (m, 8H), 1.63ꢀ1.72 (m, 2H),
1.25ꢀ1.58 (m, 12H), 1.03 (t, 3H, J = 7.7 Hz). 13C NMR (100 MHz,
CDCl3) δ 174.29, 131.12, 130.20, 129.68, 128.64, 58.54, 57.45, 51.63,
33.62, 29.78, 29.53, 29.48, 27.79, 27.28, 26.71, 26.41, 25.05, 21.28, 10.80.
HRMS calcd for C21H37O3 [M þ 1]þ 337.2743, found 337.2741.
7. An aqueous solution of LiOH (1 mL, 2 M solution) was added to a
0 °C solution of 35 (250 mg, 0.74 mmol) in THF (8 mL) and deionized
H2O (2 mL). After being stirred at room temperature overnight, the
reaction mixture was cooled to 0 °C and the pH was adjusted to 4 with 1
M aqueous oxalic acid and extracted with ethyl acetate (2 ꢁ 20 mL). The
combined extracts were washed with water (30 mL), brine (25 mL),
dried over anhydrous Na2SO4, and concentrated in vacuo. The residue
was purified by SiO2 column chromatography, using 25% EtOAc/
hexanes as eluent to give 7 (222 mg, 93%) as a colorless oil. TLC:
30% EtOAc/hexanes, Rf ≈ 0.3. 1H NMR (400 MHz, CDCl3) δ
5.28ꢀ5.40 (m, 4H), 2.87ꢀ2.97 (m, 2H), 2.34 (t, 3H, J = 7.0 Hz),
1.97ꢀ2.12 (m, 8H), 1.63ꢀ1.74 (m, 2H), 1.30ꢀ1.60 (m, 12H), 1.02 (t,
3H, J = 7.4 Hz). 13C NMR (300 MHz, CDCl3) δ 180.06, 131.75, 130.03,
129.77, 128.66, 58.86, 57.87, 33.93, 29.93, 29.84, 29.81, 27.89, 27.68,
26.41, 26.36, 24.83, 21.26, 10.84. HRMS calcd for C20H35O3 [M þ 1]þ
323.4901, found 323.4901.
found 341.3056. Hydrolysis of 38 as described above gave 12 (94%) as
white solid, mp 62.1ꢀ62.5 °C. TLC, 30% EtOAc/hexanes, Rf ≈ 0.35. 1H
NMR (400 MHz, CDCl3) δ 2.86ꢀ2.94 (m, 2H), 2.34 (t, 2H, J =
7.3 Hz), 1.46ꢀ1.65 (m, 30H), 1.04 (t, 3H, J = 7.35 Hz). 13C NMR
(100 MHz, CDCl3) δ 180.04, 58.83, 57.47, 34.24, 30.06, 30.03, 29.92,
29.81, 29.66, 29.60, 29.46, 29.43, 29.25, 27.76, 27.43, 27.28, 26.41, 24.89,
21.27, 10.89. HRMS calcd for C20H39O3 [M þ 1]þ 327.5219, found
327.5216.
Synthesis of 40. Alkylation of 3929 with 2-(4-bromobutoxy)tetrahydro-
2H-pyran-2-yloxy32 as described for 32 produced tert-butyldiphenyl-[16-
(tetrahydro-2H-pyran-2-yloxy)hexadec-11-ynyloxy]silane (66%) as a
colorless oil which was used without further purification. TLC: 10% EtOAc/
hexane, Rf ≈ 0.5.
A solution of the above crude tert-butyldiphenyl-[16-(tetrahydro-2H-
pyran-2-yloxy)hexadec-11-ynyloxy]silane (6 g, 10.42 mmol) and tetra-
n-butylammonium fluoride (3.14 g, 12.5 mL of a 1 M solution in THF,
12.50 mmol) in THF (150 mL) was stirred at room temperature under
an argon atmosphere. After 5 h, the reaction mixture was quenched with
saturated aqueous NH4Cl (5 mL), washed with water (100 mL) and
brine (75 mL). The aqueous layer was back-extracted with ether (2 ꢁ
75 mL). The combined organic extracts were dried over Na2SO4,
concentrated under reduced pressure, and the residue was purified by
SiO2 column chromatography, using 5ꢀ10% EtOAc/hexanes as eluent
to give 40 (3.17 g, 80% overall) as a colorless oil. TLC: 40% EtOAc/
hexanes, Rf ≈ 0.4. 1H NMR (CDCl3, 300 MHz) δ 4.57ꢀ4.59 (m, 1H),
3.82ꢀ3.90 (m, 1H), 3.71ꢀ3.79 (m, 1H), 3.64 (t, 2H, J = 6.8 Hz),
3.46ꢀ3.53 (m, 1H), 3.36ꢀ3.44 (m, 1H), 2.10ꢀ2.22 (m, 4H),
1.20ꢀ1.80 (m, 26H). 13C NMR (100 MHz, CDCl3) δ 18.90, 18.95,
21.51, 25.01, 25.63, 25.82, 27.85, 29.03, 29.15, 29.44, 29.61, 29.72, 29.96,
30.42, 63.88, 64.57, 66.65, 80.20, 80.55, 108.24. HRMS calcd for
C21H39O3 [M þ 1]þ 339.2899, found 339.2897.
Syntheses of 8, 10, and 12. A stream of ethanol saturated air
was passed through a stirring solution of hydrazine hydrate (400 mg,
12 mmol, 20 equiv), 35 (200 mg, 0.60 mmol), and CuSO4 5H2O (10
3
mg) in ethanol (5 mL).26 After 12 h, the reaction mixture was passed
through a short pad of silica gel and the filter cake was washed with
dichloromethane (3 ꢁ 10 mL). The combined filtrates were dried over
anhydrous Na2SO4 and concentrated in vacuo. The residue was resolved
into its components by AgNO3-impregnated PTLC using 2% CH2Cl2/
benzene: Rf ≈ 0.2, 0.4, 0.55, and 0.85 for 35, 37, 36, and 38, respectively,
isolated in a ratio of 2:3:3:2, respectively.
Synthesis of 41. Semihydrogenation of 40 as described above gave
16-(tetrahydro-2H-pyran-2-yloxy)hexadec-11(Z)-en-1-ol (99%) as a
colorless oil. TLC: 20% EtOAc/hexane, Rf ≈ 0.30. 1H NMR (CDCl3,
300 MHz) δ 5.33ꢀ5.37 (m, 2H), 4.58 (m, 1H), 3.83ꢀ3.90 (m, 1H),
3.73ꢀ3.77 (m, 1H), 3.65 (t, 2H, J = 6.7 Hz), 3.46ꢀ3.53 (m, 1H),
3.34ꢀ3.44 (m, 1H), 1.97ꢀ2.09 (m, 4H), 1.20ꢀ1.83 (m, 26H). 13C
NMR (100 MHz, CDCl3) δ 20.70, 23.41, 23.99, 24.90, 24.96, 28.45,
28.47, 29.06, 29.37, 30.13, 30.23, 30.46, 30.51, 31.26, 32.44, 61.81, 62.37,
68.14, 107.45, 130.56, 131.83. HRMS calcd for C21H41O3 [M þ 1]þ
341.3056, found 341.0360.
Jones oxidation of the preceding 16-(tetrahydro-2H-pyran-2-ylox-
y)hexadec-11(Z)-en-1-ol as described above gave 41 (68%) as a color-
less oil. TLC: SiO2, 40% EtOAc/hexanes, Rf ≈ 0.40. 1H NMR (CDCl3,
300 MHz) δ 5.33ꢀ5.37 (m, 2H), 4.56ꢀ4.58 (m, 1H), 3.83ꢀ3.88 (m,
1H), 3.73ꢀ3.78 (m, 1H), 3.49ꢀ3.53 (m, 1H), 3.35ꢀ3.43 (m, 1H), 2.34
(t, J = 7.0 Hz, 2H), 1.97ꢀ2.09 (m, 4H), 1.20ꢀ1.84 (m, 24H). 13C NMR
(100 MHz, CDCl3) δ 19.54, 23.55, 25.71, 25.98, 26.41, 28.53, 28.67,
29.01, 29.14, 29.18, 29.22, 29.32, 30.04, 30.36, 32.56, 64.63, 67.89,
107.26, 130.85, 130.88, 180.48. HRMS calcd for C21H39O4 [M þ 1]þ
341.3056, found 341.0360.
36. 1H NMR (400 MHz, CDCl3) δ 5.27ꢀ5.42 (m, 2H), 3.66 (s, 3H),
2.84ꢀ2.92 (m, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.96ꢀ2.08 (m, 4H),
1.64ꢀ1.71 (m, 2H), 1.45ꢀ1.58 (m, 4H), 1.21ꢀ1.36 (m, 16H), 1.03 (t,
J = 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 174.45, 131.88, 128.63,
58.64, 57.87, 51.96, 33.88, 29.99, 29.86, 29.74, 29.46, 27.98, 27.76, 26.88,
26.72, 25.88, 21.32, 10.48. HRMS calcd for C21H39O3 [M þ 1]þ
339.2899, found 339.2896. Hydrolysis of 36 as described above gave 8
(92%) as a colorless oil: TLC, 30% EtOAc/hexanes, Rf ≈ 0.3. 1H NMR
(300 MHz, CDCl3) δ 5.27ꢀ5.43 (m, 2H), 2.85ꢀ2.93 (m, 2H), 2.34 (t,
J = 7.6 Hz, 2H), 1.95ꢀ2.11 (m, 4H), 1.64ꢀ1.72 (m, 2H), 1.49ꢀ1.60 (m,
4H), 1.22ꢀ1.36 (m, 16H), 1.03 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ 179.42, 131.54, 128.40, 60.08, 58.75, 57.73, 34.59, 31.86,
29.86, 29.74, 29.71, 29.45, 27.84, 27.42, 26.81, 26.64, 24.85, 21.28, 15.47,
10.81. HRMS calcd for C20H37O3 [M þ 1]þ 325.2743, found 325.2747.
37. 1H NMR (300 MHz, CDCl3) δ 5.25ꢀ5.35 (m, 2H), 3.61 (s, 3H),
2.79ꢀ2.89 (m, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.93ꢀ2.04 (m, 4H),
1.19ꢀ1.60 (m, 22H), 1.00 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 174.48, 130.41, 129.54, 58.54, 57.45, 51.62, 34.27, 29.92,
29.81, 29.67, 29.63, 29.47, 29.46, 29.34, 27.80, 27.42, 27.27, 26.42, 25.14,
10.82. HRMS calcd for C21H39O3 [M þ 1]þ 339.2899, found 339.2900.
Hydrolysis of 37 as described above gave 10 (92%) as a colorless oil:
TLC, SiO2, 30% EtOAc/hexanes, Rf ≈ 0.3. 1H NMR (300 MHz,
CDCl3) δ 5.28ꢀ5.40 (m, 2H), 2.84ꢀ2.94 (m, 2H), 2.31 (t, J =
7.6 Hz, 2H), 1.96ꢀ2.04 (m, 4H), 1.02ꢀ1.62 (m, 22H), 1.01 (t, 3H,
J = 7.4 Hz). 13C NMR (75 MHz, CDCl3) δ 180.10, 130.45, 129.57,
58.74, 57.67, 34.27, 29.92, 29.81, 29.66, 29.60, 29.46, 29.43, 29.25, 27.76,
27.43, 27.28, 26.41, 24.89, 21.27, 10.81. HRMS calcd for C20H37O3
[M þ 1]þ 325.2743, found 325.2745.
Synthesis of 42. A solution of 41 (2.1 g, 5.93 mmol) and p-TSA
(50 mg) in MeOH (30 mL) was stirred at room temperature for 10 h,
then concentrated in vacuo, and the residue was purified by SiO2 column
chromatography, using 15% EtOAc/hexanes as eluent to give methyl 16-
hydroxyhexadec-11(Z)-enoate (1.42 g, 83%) as a colorless oil. TLC:
20% EtOAc/hexanes, Rf ≈ 0.35. 1H NMR (CDCl3, 300 MHz) δ
5.33ꢀ5.37 (m, 2H), 3.65 (s, 3H), 3.63 (t, J = 7.3 Hz, 2H), 2.29 (t, J =
7.0 Hz, 2H), 1.97ꢀ2.08 (m, 4H), 1.21ꢀ1.64 (m, 18H). 13C NMR (75
MHz) δ 174.77, 130.30, 129.70, 62.95, 51.73, 34.28, 32.42, 29.64, 29.45,
29.17, 29.03, 27.29, 27.10, 27.08, 26.03, 25.07. HRMS calcd for
C17H33O3 [M þ 1]þ 285.2430, found 285.2434.
38. 1H NMR (400 MHz, CDCl3) δ 3.67 (s, 3H), 2.84ꢀ2.94 (m, 2H),
2.31 (t, 2H, J = 7.4 Hz), 1.42ꢀ1.65 (m, 6H), 1.22ꢀ1.34 (m, 24H), 1.04
(t, 3H, J = 7.3 Hz). HRMS calcd for C21H41O3 [M þ 1]þ 341.3056,
Diisopropyl azodicaboxylate (DIAD; 1.15 g, 5.70 mmol,) was added
dropwise to a ꢀ20 °C solution of triphenylphosphine (1.49 g, 5.70 mmol)
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Journal of Medicinal Chemistry
ARTICLE
in dry THF (30 mL) under an argon atmosphere. After the mixture was
stirred for 10 min, a solution of methyl 16-hydroxyhexadec-11(Z)-enoate
(1.35 g, 4.75 mmol) in anhydrous THF (5 mL) was added dropwise. After
30 min at ꢀ20 °C, the reaction mixture was warmed to 0 °C and
diphenylphosphoryl azide (DPPA, 1.38 g, 5.70 mmol) was added dropwise.
After the mixture was stirred at room temperature for 6 h, the reaction was
quenched with water (3 mL) and the mixture was diluted with ether
(50 mL) and washed with brine (40 mL). The aqueous layer was back-
extracted with ether (2 ꢁ 30 mL). The combined organic extracts were
dried over Na2SO4 and concentrated under reduced pressure. The residue
was purified by SiO2 column chromatography, using 5% EtOAc/hexanes as
eluent to give 42 (1.14 g, 78%) as a pale yellow oil. TLC: 10% EtOAc/
on the bottom of Falcon flasks (12.5 cm) in 2.0 mL of Halle SM 20-I
medium supplemented with 10% heat-inactivated fetal calf serum and
2 μM fluorodeoxyuridine. Spontaneously beating cell clusters occurred
after 5ꢀ7 days (120ꢀ150 beats/min, monitored at 37 °C using an
inverted microscope). The beating rates were determined for six to eight
individual clusters before and 5 min after addition of the test substance-
(s). On the basis of the difference between the basal and compound-
induced beating rate of the individual clusters, the chronotropic effects
(changes in beats/min) were calculated and are given as mean SE values;
n = 18ꢀ40 clusters originating from at least three independent NRCM
cultures. All test compounds were prepared as 1000-fold stock solutions
in ethanol and tested at a final concentration of 30 nM. The exception
was EPA that required a final concentration of 3.3 mM and 30 min of
preincubation.
1
hexanes, Rf ≈ 0.45. H NMR (300 MHz) δ 5.31ꢀ5.43 (m, 2H), 3.66
(s, 3H), 3.26 (t, J = 6.7 Hz, 2H), 2.30 (t, J = 7.1 Hz, 2H), 1.97ꢀ2.10 (m,
4H), 1.50ꢀ1.64 (m, 4H), 1.15ꢀ1.48 (m, 14H). 13C NMR (100 MHz) δ
174.32, 130.84, 129.01, 51.09, 51.05, 34.21, 32.58, 32.18, 29.86, 29.61, 29.59,
29.54, 29.42, 29.31, 27.40, 26.94, 25.23. IR (neat) 2985, 2954, 2845, 2106,
1754, 1250, 1104, 1029 cmꢀ1. HRMS calcd for C17H32N3O2 [M þ 1]þ
310.2495, found 310.2500.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures, analyti-
b
cal data, and NMR spectra for all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
Synthesis of 21. Triphenylphosphine (1.15 g., 4.41 mmol) was
added to a room temperature solution of 42 (1.05 g., 3.4 mmol) in THF
(25 mL). After 2 h, water (200 mL) was added and the stirring was
continued for another 8 h. The reaction mixture was then diluted with
EtOAc (20 mL), washed with water (20 mL) and brine (25 mL).
Aqueous layers were back-extracted with EtOAc (2 ꢁ 30 mL). The
combined organic extracts were dried over Na2SO4, concentrated under
reduced pressure, and further dried under high vacuum for 4 h. The
crude methyl 16-aminohexadec-11(Z)-enoate was used in the next step
without additional purification.
’ AUTHOR INFORMATION
Corresponding Author
*For J.R.F.: phone, 214-648-2406; fax, 214-648-6455; e-mail,
j.falck@utsouthwestern.edu. For W.-H.S.: phone, þ49-30 9406
3750; fax, þ49 30 9406 3760; e-mail, schunck@mde-berlin.de.
2-(Methylamino)-2-oxoacetic acid29 (79 mg, 0.77 mmol), 1-hydro-
xybenzotriazole (101 mg, 0.77 mmol; HOBt), and diisopropylethyla-
mine (105 mg, 0.77 mmol; DIPEA) were added to a stirring solution of
crude methyl 16-aminohexadec-11(Z)-enoate (180 mg, 0.64 mmol)
from above in anhydrous DMF (20 mL) under an argon atmosphere.
After 5 min, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (147 mg,
0.77 mmol; EDCI) was added as a solid. After being stirred for 12 h at
room temperature, the reaction mixture was diluted with EtOAc
(30 mL), washed with water (3 ꢁ 20 mL) and brine (20 mL). The
combined aqueous layers were back-extracted with EtOAc (3 ꢁ 30 mL).
The combined organic extracts were dried over Na2SO4, concentrated
under reduced pressure, and the residue was purified by SiO2 column
chromatography, using EtOAc as eluent to give methyl 16-(2-
(methylamino)-2-oxoacetamido)hexadec-11(Z)-enoate (160 mg, 68%).
TLC: 100% EtOAc, Rf ≈ 0.4. 1H NMR (CDCl3, 300 MHz) δ 7.45 (br s,
1H), 5.26ꢀ5.42 (m, 2H), 3.66 (s, 3H), 3.27ꢀ3.35 (m, 2H), 2.90 (d, 3H,
J = 5.2 Hz), 2.30 (t, 2H, J = 7.3 Hz), 1.96ꢀ2.08 (m, 4H), 1.24ꢀ1.66 (m,
18H). 13C NMR (CDCl3, 75 MHz) δ 174.60, 160.81, 159.94, 130.87,
129.08, 51.68, 39.79, 34.33, 29.91, 29.68, 29.63, 29.50, 29.46, 29.36, 29.02,
27.46, 27.08, 26.91, 26.40, 25.17. HRMS calcd for C20H36N2O4 [M þ
1]þ 368.2675, found 368.2675.
Methyl 16-(2-(methylamino)-2-oxoacetamido)hexadec-11(Z)-eno-
ate (150 mg, 0.40 mmol) was hydrolyzed using LiOH as described above
to afford 21 (126 mg, 89%) as a white powder, mp 110.2ꢀ110.6 °C.
TLC: 5% MeOH/CH2Cl2, Rf ≈ 0.4. 1H NMR(CDCl3, 300 MHz) δ 7.80
(br s, 1H), 7.66 (br s, 1H), 5.26ꢀ5.42 (m, 2H), 3.28ꢀ3.35 (m, 2H), 2.90
(s, 3H), 2.36 (t, 2H, J = 7.3 Hz), 1.97ꢀ2.08 (m, 4H), 1.51ꢀ1.64 (m, 4H),
1.22ꢀ1.42 (m, 14H). 13C NMR (CDCl3, 75 MHz) δ 177.98, 160.96,
159.93, 130.83, 129.22, 39.91, 33.91, 29.58, 29.25, 29.12, 29.01, 28.95,
27.21, 27.09, 26.93, 26.46, 24.89. HRMS calcd for C19H34N2O4 [M þ
1]þ 354.2519, found 354.2516.
’ ACKNOWLEDGMENT
Financial support was provided by the Robert A. Welch Founda-
tion (Grant GL625910) and NIH (Grants GM31278 and
DK38226) to J.R.F. and by the Deutsche Forschungsgemeinschaft
(Grant SCHU-822/5-1) to A.K., W.-H.S., and D.N.M. Prof. Kasem
Nithipatikom (Medical College of Wisconsin) kindly provided
high-resolution mass spectral analysis results.
’ ABBREVIATIONS USED
ω-3 PUFA, ω-3 polyunsaturated fatty acid; EPA, eicosa-5(Z),8-
(Z),11(Z),14(Z),17(Z)-pentaenoic acid; DHA, docosa-5(Z),8-
(Z),11(Z),14(Z),16(Z),19(Z)-hexaenoic acid; AA, arachidonic
acid or eicosa-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid; EETeTr,
epoxyeicosatetraenoic acid; NRCM, neonatal rat cardiomyo-
cytes; sEH, soluble epoxide hydrolase; EET, epoxyeicosatrie-
noic acid; EEZE, 14,15-epoxyeicosa-5(Z)-enoic acid
’ REFERENCES
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