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2 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 1 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Epoxyeicosatrienoic acids
(EETs) are endothelium-derived eicosanoids that activate
potassium channels, hyperpolarize the membrane, and cause relaxation.
We tested 19 analogs of 14,15-EET on vascular tone to determine the
structural features required for activity. 14,15-EET relaxed bovine
coronary arterial rings in a concentration-related manner
(ED50 = 10
6 M). Changing the carboxyl to
an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester
and 14,15-EET-methylsulfonimide retained full activity. Shortening the
distance between the carboxyl and epoxy groups reduced the agonist
potency and activity. Removal of all three double bonds decreased
potency. An analog with a 
8 double bond had full activity and
potency. However, the analogs with only a 5 or 11 double bond had
reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen
resulted in loss of activity. 14(S),15(R)-EET was
more potent than 14(R),15(S)-EET, and
14,15-(cis)-EET was more potent than
14,15-(trans)-EET. These studies indicate that the
structural features of 14,15-EET required for relaxation of the bovine
coronary artery include a carbon-1 acidic group, a 8 double bond,
and a 14(S),15(R)-(cis)-epoxy group.
endothelium-derived hyperpolarizing factor; cytochrome P-450; arachidonic acid; epoxyeicosatrienoic acid
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ENDOTHELIAL CELLS MEDIATE relaxation of vascular smooth muscle through the release of a number of soluble mediators such as nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF) (6, 19, 31). The identity of EDHF remains controversial, with studies that implicate metabolites of arachidonic acid, potassium ion, and hydrogen peroxide (3, 8, 15, 23, 30, 34, 37). In the coronary artery, a number of laboratories have shown that EDHF is an epoxyeicosatrienoic acid (EET), a cytochrome P-450 metabolite of arachidonic acid (3, 15, 18, 20, 21, 23, 39). The coronary endothelium synthesizes the EETs, and agonists such as acetylcholine and bradykinin stimulate their release (3, 32, 39, 40). The acetylcholine- and bradykinin-stimulated, endothelium-dependent relaxation and hyperpolarization of coronary smooth muscle are blocked by inhibitors of cytochrome P-450, an EET antagonist, and inhibitors of calcium-activated potassium (KCa) channels (3, 15, 20, 23). The EETs open large-conductance KCa channels in smooth muscle cells, which causes hyperpolarization of coronary smooth muscle and relaxation of the coronary artery (3, 35, 39). Thus EETs mimic the effects of EDHF. The activation of the KCa channel by the EETs involves a guanine nucleotide-binding protein, most likely Gs (16, 21, 27).
Although the EETs represent important mediators of coronary vascular tone, it is not known which structural component(s) of the EET molecule is necessary for vasorelaxation. Previous studies indicate that all four regioisomeric EETs, 14,15-, 11,12-, 8,9-, and 5,6-EET, relax bovine and canine coronary arteries equally (3, 39, 41). Thus the location of the epoxy group is not critical for vasodilation in these arteries. In contrast, 5,6-EET was more active than the other regioisomeric EETs in relaxing the rat tail artery, rabbit and pig cerebral arteries, and rat mesenteric artery (4, 9, 26, 36). The EETs are hydrolyzed by epoxide hydrolase to dihydroxyeicosatrienoic acids (DHETs) (46, 50). In some studies, the DHETs relax coronary arteries and are equipotent to the EETs (33, 45). The relaxations to DHETs are blocked by inhibitors of KCa channels. In other studies, the DHETs were not active (4) or were less active than the EET (2).
In the present study, we synthesized 19 analogs of 14,15-EET (Fig.
1) and tested the analogs for their
ability to relax the bovine coronary artery. The analogs were
specifically made with changes in the epoxy group, the carboxyl group,
carbon chain length, and the double bonds to determine the
contributions of these structures to vasorelaxation. As in previous
studies (33, 45), we found that 14,15-DHET relaxed the
bovine coronary artery but was approximately fivefold less potent than
14,15-EET. These studies indicate the importance of the carboxyl group,
the epoxy group, the carboxyl-to-epoxy group distance, and double bonds
in the relaxant effect of 14,15-EET.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Vascular Reactivity of Bovine Coronary Arteries
Bovine hearts were purchased from a local slaughterhouse, and the left anterior descending coronary artery was dissected and cleaned of connective tissue. Vessels of 1 mm diameter were cut into rings of 3 mm width as previously described (3, 27, 39). Vessels were stored in Krebs buffer consisting of (in mM) 119 NaCl, 4.8 KCl, 24 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, 11 glucose, 0.02 EDTA, and 3.2 CaCl2. The vessels were suspended from a pair of stainless steel hooks in a 6-ml water-jacketed organ chamber. The organ chamber was filled with Krebs buffer and bubbled with 95% O2-5% CO2 at 37°C. One hook was anchored to a steel rod and the other hook to a force transducer (model FT-03C; Grass Instruments, West Warwick, RI). Tension of the vessel was measured by an ETH-400 bridge amplifier, and the data were acquired with a MacLab 8e analog-to-digital converter and MacLab software version 3.5.6 (AD Instruments, Milford, MA) and stored on a Macintosh computer for subsequent data analysis.Basal tension was set at the length-tension maximum of 3.5 g and equilibrated for 1.5 h. KCl (40 mM) was added to the chamber until reproducible maximal contractions were maintained. U-46619 (10-20 nM), a thromboxane receptor agonist, was used to precontract the vessels from basal tension to between 50% and 90% of the maximal KCl contraction. Cumulative additions of 14,15-EET, 14,15-DHET, or analogs of 14,15-EET were added to the chamber. Between concentration-response curves, the chambers were rinsed with fresh Krebs buffer, 40 mM KCl was administered to determine the maximum contraction, and the vessels were rinsed. Consecutive concentration-response curves were performed with 14,15-EET followed by a concentration-response curve to a 14,15-EET analog. The experiment was always repeated with the order of the agonists reversed. In control experiments with consecutive concentration-response curves to 14,15-EET, the second concentration-response curve with 14,15-EET was identical to the first. Tension was represented as percent relaxation where 100% relaxation was basal pre-U-46619 tension.
Syntheses of 14,15-EET Analogs
General. All reactions were conducted under an argon atmosphere unless otherwise stated. 14,15-EET (7, 17), 14,15-EET-thiirane (11, 12), 14,15-EET-aziridine (11, 12), 14,15-epoxyeicosanoic acid [14,15-EEA (51)], 14,15-(trans)-EET (24), 8,9-epoxybutadecaenoic acid [8,9-EBDE; (10)], and 14,15-EET-methylsulfonimide [14,15-EET-SI (5)] were prepared according to published procedures.
Preparation of cis-14,15-oxidoeicosa-5(Z)-enoic acid
(8,9, 11,12-tetrahydro-14,15-EET).
cis-14,15-Oxidoeicosa-5(Z)-enoic acid
(8,9,11,12-tetrahydro-14,15-EET) (14,15-EE-5-ZE; acid 10)
was prepared as follows. n-BuLi (0.66 ml, 1.6 M hexane
solution, 1.05 mmol) was added dropwise to a stirring, 
40°C
solution of 1-(tert-butyldiphenylsilyloxy)hex-5-yne (acetylene 1; Ref. 29) (320 mg, 0.96 mmol) in
anhydrous tetrahydrofuran (THF)-hexamethylphosphoramide
(HMPA) (4:1, 5 ml) (scheme 1; Fig. 2). After 2 h, a solution of
2-(7-bromoheptyloxy)tetrahydropyran (bromide 13; Ref.
29) (291 mg, 1.05 mmol) in anhydrous THF (3 ml) was slowly
cannulated into the reaction mixture, which was then allowed to warm
gradually to room temperature over 2 h. On quenching with
saturated aqueous NH4Cl (5 ml), the aqueous layer was
extracted with ethyl acetate (2 × 50 ml) and the combined organic
extracts were washed with H2O (50 ml) and brine (50 ml), dried over Na2SO4, and evaporated in vacuo.
Purification of the residue by SiO2 column chromatography
with 5% ethyl acetate (EtOAc)-hexane as eluant afforded diol
2 (310 mg, 65%) as a pale yellow oil. The properties of
the product are as follows. TLC: EtOAc-hexane (15:85),
Rf ~ 0.64; 1H NMR [400 MHz,
CDCl3-tetramethylsilane (TMS)]: 
1.08 (s, 9 H), 1.29-1.41 (m, 6 H), 1.42-1.77 (m, 12 H), 1.78-1.88 (m, 2 H), 2.11-2.16 (m, 4 H), 3.34-3.39 (m, 1 H), 3.46-3.50
(m, 1 H), 3.67 (t, J = 6.0 Hz, 2 H), 3.71-3.75 (m, 1 H),
3.83-3.88 (m, 1 H), 4.58 (apparent t, J = 2.8 Hz, 1 H),
7.35-7.42 (m, 6 H), 7.66 (dd, J = 1.6, 7.6 Hz, 4 H).
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1.04 (s, 9 H), 1.32-1.39 (m, 6 H), 1.41-1.49 (m, 2 H), 1.52-1.60 (m, 4 H), 1.62-1.68 (m, 2 H), 2.11-2.17 (m, 4 H), 3.59-3.65 (m, 2 H), 3.67 (t, J = 6.4 Hz, 2 H), 7.35-7.41 (m, 6 H), 7.66 (dd, J = 1.6, 7.6 Hz,
4 H).
CBr4 (124 mg, 0.37 mmol) and PPh3 (98 mg, 0.37 mmol) were added sequentially to a 0°C solution of alcohol
3 (140 mg, 0.31 mmol) in CH2Cl2 (5 ml)
under argon. After stirring for 1 h, all volatiles were removed in
vacuo and the residue was purified by SiO2 column
chromatography with 2% EtOAc-hexane as eluant, furnishing bromide 4 (148 mg, 80%) as a colorless oil. The properties
of the product are as follows. TLC: EtOAc-hexane (1:9),
Rf ~ 0.58; 1H NMR (400 MHz,
CDCl3-TMS): 1.04 (s, 9 H), 1.32-1.48 (m, 8 H), 1.55-1.61 (m, 2 H), 1.62-1.68 (m, 2 H), 1.84 (apparent
quintet, J = 7.4 Hz, 2 H), 2.11-2.17 (m, 4 H), 3.38 (t,
J = 6.4 Hz, 2 H), 3.67 (t, J = 6.4 Hz, 2 H),
7.35-7.41 (m, 6 H), 7.66 (dd, J = 1.6, 7.6 Hz, 4 H).
Alkylation of bromide 4 using 1-heptyne as described for the
preparation of diol 2 gave rise to bis-acetylene
5 (70% yield). The properties of the product are as follows. TLC:
EtOAc-hexane (1:9), Rf ~ 0.64; 1H NMR
(400 MHz, CDCl3-TMS): 0.90 (t, J = 7.2 Hz, 3 H),
1.06 (s, 9 H), 1.25-1.40 (m, 10 H), 1.40-1.72 (m, 10 H),
2.10-2.20 (m, 8 H), 3.67 (t, J = 6.4 Hz, 2 H), 7.35-7.48
(m, 6 H), 7.66 (dd, J = 1.6, 7.6 Hz, 4 H).
Partial reduction of bis-acetylene 5 was achieved via
NaBH4 (1 mg, 0.02 mmol) addition to a stirring suspension
of Ni(OAc)2 (3 mg, 0.01 mmol) in EtOH (10 ml) at room
temperature under an argon atmosphere. After 30 min, neat
ethylenediamine (1.64 µl, 0.024 mmol) was introduced, followed 10 min
later by an ethanolic solution (2 ml) of bis-acetylene 5 (130 mg, 0.24 mmol). The reaction was purged with H2 and
maintained under a H2 atmosphere for the next 1 h with
a H2-filled balloon. The reaction mixture was diluted with
ether (20 ml) and filtered through a silica gel pad, and the filtrate
was evaporated in vacuo, yielding pure diene 6 (119 mg,
91%). The properties of the product are as follows. TLC: EtOAc-hexane (1:9), Rf ~ 0.66; 1H NMR (400 MHz,
CDCl3-TMS): 0.90 (t, J = 7.2 Hz, 3 H), 1.06 (s, 9 H), 1.25-1.40 (m, 16 H), 1.39-1.45 (m, 2 H), 1.56-1.61
(m, 2 H), 1.94-2.05 (m, 2 H), 3.65 (t, J = 6.4 Hz, 2 H),
5.30-5.40 (m, 4 H), 7.35-7.44 (m, 6 H), 7.66 (dd, J = 1.6, 7.6 Hz, 4 H).
Tetra-n-butylammonium fluoride (1.11 ml, 1.11 mmol, 1.0 M
solution in THF) was added to a room temperature solution of
diene 6 (119 mg, 0.22 mmol) in dry THF (4 ml). After 3 h, all volatiles were evaporated in vacuo and the crude product was
dissolved in EtOAc (25 ml), washed with water (2 × 50 ml) and
brine (20 ml), and dried over Na2SO4. Silica
gel column chromatography (20% EtOAc-hexane) of the residue, obtained
after evaporation, furnished alcohol 7 (56.3 mg) in 97%
yield. The properties of the product are as follows. TLC: EtOAc-hexane
(3:7), Rf ~ 0.35; 1H NMR (400 MHz,
CDCl3-TMS): 0.88 (t, J = 7.2 Hz, 3 H),
1.25-1.40 (m, 18 H), 1.54-1.62 (m, 2 H), 1.94-2.05 (m, 8 H), 3.64 (t, J = 6.4 Hz, 2 H), 5.28-5.42 (m, 4 H).
Alcohol 7 (56 mg, 0.217 mmol) was slowly added to a stirring
0°C solution of Jones reagent [1 ml; prepared by addition of 0.53 ml
of concentrated H2SO4 to a solution of
CrO3 (0.677 g) in 2.5 ml of H2O cooled to
10°C] in acetone (3 ml). After 15 min, the reaction was
quenched with isopropanol (5 ml) and filtered to remove precipitated
chromium salts and the filtrate was evaporated in vacuo. The residue
was dissolved in EtOAc (10 ml), washed with water (2 × 20 ml) and
brine (20 ml), dried over Na2SO4, concentrated in vacuo, and purified by SiO2 preparative (P) TLC to give
acid 8 (48 mg, 80%) as a colorless, viscous oil.
The properties of the product are as follows. TLC: EtOAc-hexane (1:1)
Rf ~ 0.4; 1H NMR (400 MHz,
CDCl3-TMS): 0.88 (t, J = 6.8 Hz, 3 H),
1.10-1.20 (m, 16 H), 1.69 (apparent quintet, J = 7.6 Hz, 2 H), 1.95-2.10 (m, 6 H), 2.10 (apparent quintet, J = 7.2 Hz, 2 H), 2.36 (t, J = 7.4 Hz, 2 H), 5.29-5.46 (m, 4 H).
A mixture of acid 8 (50 mg, 0.16 mmol) and 1,1'-carbonyl
diimidazole (Im2CO; 41 mg, 0.25 mmol) in anhydrous
CH2Cl2 (20 ml) was stirred for 40 min at room
temperature and then transferred via cannula to a stirring 0°C, 3.5 M
H2O2 solution in ether (5 ml, 17.5 mmol)
containing a catalytic amount of lithium imidazole. After 5 min, the
reaction mixture was diluted with CH2Cl2 (10 ml) followed by powdered KH2PO4 (1.28 mmol, 174 mg). After an additional 5-min stirring, the resultant suspension was
filtered through a cotton plug into a flask containing anhydrous
Na2SO4 (1 g) in CH2Cl2
(20 ml). The reaction mixture was stored at room temperature under an
argon atmosphere for 12 h, filtered, and washed with brine until
the aqueous layer tested negative with starch-I2 paper for
hydrogen peroxide. The organic layer was evaporated in vacuo, and the
residue was dissolved in Et2O-MeOH (3:1, 10 ml) to which
excess ethereal CH2N2 was added at 0°C. After
30 min, all volatiles were removed in vacuo and the residue was
purified via SiO2 column chromatography to provide
methyl ester 9 (38 mg, 68%) as a colorless oil along with
recovered methyl ester 8 (5 mg). The properties of the
product are as follows. TLC: EtOAc-hexane (3:7), Rf ~ 0.61; 1H NMR (400 MHz, CDCl3-TMS): 0.90 (t, J = 6.8 Hz, 3 H), 1.22-1.59 (m, 20 H), 1.69 (apparent
quintet, J = 7.6 Hz, 2 H), 1.95-2.10 (m, 4 H), 2.31 (t,
J = 7.6 Hz, 2 H), 2.87-2.93 (m, 2 H), 5.25-5.46 (m, 2 H).
Ester 9 (50 mg, 0.15 mmol) was saponified by stirring with
LiOH (0.45 ml of 1.0 M aqueous sol, 0.45 mmol) at room temperature in
THF-H2O (5:1, 5 ml) for 12 h. The reaction mixture was
then acidified with 1.0 M aqueous oxalic acid to pH 4 and extracted with ethyl acetate (2 × 40 ml). The combined organic extracts were dried (Na2SO4) and concentrated in vacuo
to give acid 10 (45 mg, 95%) as a pale yellow oil. The
properties of the product are as follows. TLC: EtOAc-hexane (3:7),
Rf ~ 0.21; 1H NMR (400 MHz,
CDCl3-TMS): 0.91 (t, J = 7.0 Hz, 3 H),
1.23-1.58 (m, 20 H), 1.76 (apparent quintet, J = 7.6 Hz, 2 H), 2.03 (dd, J = 6.8, 6.8 Hz, 2 H), 2.15 (dd, J = 7.2, 7.2 Hz, 2 H), 2.35 (dd, J = 7.2, 7.2 Hz, 2 H), 2.86-2.91 (m, 2 H), 5.27-5.68 (m, 2 H).
Preparation of N-methylsulfonimide.
Acid 10 (45 mg, 0.14 mmol) and
N-hydroxysuccinimide (NHS; 18 mg, 0.154 mmol) were mixed and
azeotropically dried with anhydrous benzene (scheme 1). The
mixture was dissolved in dry THF (10 ml) to which
1,3-dicyclohexylcarbodiimide (DCC; 32 mg, 0.156 mmol) was added all at
once. After being stirred at room temperature for 12 h, all
volatiles were moved in vacuo and the residue was purified by
SiO2 column chromatography to give NHS-ester 11 (47 mg, 75%) as a colorless gum. The properties of the product are as
follows. TLC: EtOAc-hexane (3:7), Rf ~ 0.25;
1H NMR (400 MHz, CDCl3-TMS): 0.91 (t,
J = 7.0 Hz, 3 H), 1.23-1.58 (m, 20 H), 1.76 (apparent
quintet, J = 7.6 Hz, 2 H), 2.02 (dd, J = 6.8, 6.8 Hz, 2 H),
2.15 (dd, J = 7.2, 7.2 Hz, 2 H), 2.60 (dd, J = 7.2, 7.2 Hz, 2 H), 2.82 (bs, 4 H), 2.86-2.91 (m, 2 H), 5.27-5.48 (m, 2 H).
0.91(t, J = 7.0 Hz, 3 H),
1.23-1.59 (m, 20 H), 1.76 (apparent quintet, J = 7.6 Hz, 2 H), 2.02 (dd, J = 6.8, 6.8 Hz, 2 H), 2.13 (dd, J = 7.2, 7.2 Hz, 2 H), 2.33 (dd, J = 7.2, 7.2 Hz, 2 H), 2.86-2.91 (m, 2 H), 3.29 (s, 3 H), 5.27-5.48 (m, 2 H).
Preparation of cis-14,15-oxidoeicosa-8(Z)-enoic acid
(5,6, 11,12-tetrahydro-14,15-EET).
cis-14,15-Oxidoeicosa-8(Z)-enoic acid
(5,6,11,12-tetrahydro-14,15-EET) (14,15-EE-8-ZE; acid 20)
was prepared as follows. Seventy percent m-chloroperbenzoic
acid (m-CPBA; 990 mg, 5.72 mmol) was added in portions to a
0°C solution of undec-5-en-1-o1 (14; Ref. 22)
(650 mg, 3.80 mmol) in CH2Cl2 (20 ml)
(scheme 2; Fig. 3). After
being stirred for 3 h, the reaction mixture was diluted with
CH2Cl2 (100 ml), washed with saturated aqueous
NaHCO3 (2 × 50 ml), water (50 ml), and brine (50 ml),
dried (Na2SO4), and concentrated in vacuo. The
residue was purified by column chromatography over silica gel to afford
epoxide 15 (640 mg, 90%) as a colorless syrup. The
properties of the product are as follows. TLC: EtOAc-hexane (3:7),
Rf ~ 0.24; 1H NMR (300 MHz,
CDCl3-TMS): 0.89 (t, J = 7.0 Hz, 3 H),
1.21-1.68 (m, 14 H), 1.99 (bs, 1 H), 2.89-2.97 (m, 2 H), 3.68 (dd, J = 6.1, 6.1 Hz, 2 H); 13C NMR (75 MHz,
CDCl3-TMS): 13.98, 22.59, 22.97, 26.26, 27.51, 27.74, 31.70, 32.39, 57.29, 57.41, 62.31.
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0.89 (t, J = 7.0 Hz, 3 H), 1.21-1.68 (m, 12 H), 1.84-1.99 (m, 2 H), 2.89-2.97
(m, 2 H), 3.41 (dd, J = 6.0, 6.0 Hz, 2 H).
Following the protocol used to couple acetylene 1 with
bromide 13 (see scheme 1), bromide 16 was alkylated with 1-(tert-butyldiphenylsilyloxy)non-8-yne (21; Ref. 38) to give 17 (77%). The
properties of the product are as follows. TLC: EtOAc-hexane (1:9),
Rf ~ 0.28; 1H NMR (300 MHz,
CDCl3-TMS): 0.89 (t, J = 7.0 Hz, 3 H), 1.04 (s, 9 H), 1.24-1.59 (m, 24 H), 2.08-2.19 (m, 4 H), 2.89-2.97
(m, 2 H), 3.65 (dd, J = 6.6, 6.6 Hz, 2 H), 7.26-7.41 (m, 6 H), 7.62-7.71 (m, 4 H); 13C NMR (75 MHz,
CDCl3-TMS): 4.19, 18.89, 18.93, 19.39, 22.78, 25.86, 25.99, 26.47, 27.05, 27.59, 27.96, 29.04, 29.07, 29.13, 29.28, 31.91, 32.70, 57.20, 57.33, 64.11, 79.86, 80.71, 127.74, 129.66, 134.32, 135.74.
The desilylation of 17 to alcohol 18 (90%) was
carried out as described for the conversion of diene 6 to
alcohol 7 (see scheme 1). The properties of the
product are as follows. TLC: EtOAc-hexane (3:7), Rf ~ 0.22; 1H NMR (400 MHz, CDCl3-TMS): 0.89 (t, J = 7.0 Hz, 3 H), 1.24-1.62 (m, 24 H), 2.10-2.22 (m,
4 H), 2.88-2.94 (m, 2 H), 3.63 (t, J = 6.4 Hz, 2 H).
The P-2 Ni hydrogenation of alcohol 18 generating
cis-olefin 19 (91%) was carried out as described
for the conversion of bis-acetylene 5 to diene 6 (see scheme 1). The properties of the product are as
follows. TLC: EtOAc-hexane (1:4), Rf ~ 0.26;
1H NMR (400 MHz, CDCl3-TMS): 0.90 (t,
J = 7.0 Hz, 3 H), 1.24-1.60 (m, 24 H), 1.95-2.10 (m, 4 H), 2.86-2.96 (m, 2 H), 3.62 (t, J = 6.7 Hz, 2 H),
5.29-5.41 (m, 2 H).
Pyridinium dichromate (PDC; 1.39 g, 3.70 mmol) was added to a
continuously stirred room temperature solution of alcohol 19 (230 mg, 0.74 mmol) in dry N,N-dimethylformamide
(10 ml). After 16 h, the reaction mixture then was
diluted with EtOAc (20 ml), washed with water (3 × 20 ml) and
brine (20 ml), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography to give acid 20 (160 mg, 67%). The
properties of the product are as follows. TLC: EtOAc-hexane (1:1),
Rf ~ 0.20; 1H NMR (400 MHz,
CDCl3-TMS): 0.90 (t, J = 7.0 Hz, 3 H),
1.24-1.55 (m, 20 H), 1.56-1.66 (m, 2 H), 1.89-2.01 (m, 4 H), 2.30 (t, J = 7.6 Hz, 2 H), 2.86-2.95 (m, 2 H), 3.67 (s, 3 H), 5.30-5.40 (m, 2 H); 13C NMR (75 MHz,
CDCl3-TMS): 14.20, 22.81, 25.12, 26.45, 26.50, 27.31, 27.36, 27.95, 27.99, 29.11, 29.26, 29.72, 29.81, 31.94, 34.29, 51.66, 57.38, 57.46, 129.73, 130.25, 174.51.
Preparation of cis-14,15-oxidoeicosa-11(Z)-enoic acid
(5,6, 8,9-tetrahydro-14,15-EET).
cis-14,15-Oxidoeicosa-11(Z)-enoic acid
(5,6,8,9-tetrahydro-14,15-EET) (14,15-EE-11-ZE; 28) was
prepared as follows. Epoxy-alcohol 23 was prepared from
oct-2-en-1-o1 (22; Refs. 43 and
49) (90%) (scheme 3; Fig.
4) as described for the conversion of
undec-5-en-1-o1 14 to epoxide 15 (see
scheme 2). The properties of the product are as follows.
TLC: EtOAc-hexane (1:4), Rf ~ 0.34; 1H
NMR (400 MHz, CDCl3-TMS): 0.92 (t, J = 7.2 Hz, 3 H), 1.22-1.62 (m, 8 H), 3.01-3.10 (m, 1 H), 3.18-3.20
(m, 1 H), 3.62-3.71 (m, 1 H), 3.82-3.91 (m, 1 H).
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0.92 (t, J = 7.2 Hz, 3 H),
1.29-1.38 (m, 3 H), 1.46-1.60 (m, 5 H), 3.06-3.10 (m, 1 H), 3.24-3.31 (m, 2 H), 3.43-3.55 (m, 1 H); 13C
NMR (300 MHz, CDCl3-TMS): 13.93, 22.49, 26.14, 27.26, 29.07, 31.58, 55.60, 58.81.
Epoxy-bromide 24 was alkylated with
1-(tert-butyldiphenylsilyloxy)dodec-11-yne (29;
Ref. 38) to give 25 (76%) as described for the
conversion of bromide 16 to 17 (see scheme
2). The properties of the product are as follows. TLC:
EtOAc-hexane (1:19), Rf ~ 0.38; 1H NMR
(400 MHz, CDCl3-TMS): 0.92 (t, J = 7.2 Hz, 3 H),
1.02 (s, 9 H), 1.20-1.58 (m, 2 H), 2.12-2.28 (m, 3 H),
2.53-2.61 (m, 1 H), 2.92-3.21 (m, 2 H), 3.65 (t, J = 6.3 Hz, 2 H), 7.35-7.42 (m, 4 H), 7.65-7.69 (m, 6 H).
The P-2 Ni hydrogenation of epoxy-silyl ether 25 generating
cis-olefin 26 (90%) was carried out as described for the
conversion of bis-acetylene 5 to diene 6 (see
scheme 1). The properties of the product are as follows.
TLC: EtOAc-hexane (1:19), Rf ~ 0.4; 1H
NMR (400 MHz, CDCl3-TMS): 0.92 (t, J = 7.2 Hz, 3 H), 1.09 (s, 9 H), 1.21-1.61 (m, 24 H), 2.05 (q, J = 8.0 Hz,
2 H), 2.20 (q, J = 7.6 Hz, 1 H), 2.39 (q, J = 6.8 Hz, 1 H),
2.93-2.97 (m, 2 H), 3.66 (t, J = 8.0 Hz, 2 H), 5.40-5.57
(m, 2 H), 7.36-7.43 (m, 6 H), 7.68-7.72 (m, 4 H).
The desilylation of 26 to alcohol 27 (80%) was
carried out as described for the conversion of diene 6 to
alcohol 7 (see scheme 1). The properties of the
product are as follows. TLC: EtOAc-hexane (1:4), Rf ~ 0.24; 1H NMR (400 MHz, CDCl3-TMS): 0.91 (t, J = 7.6 Hz, 3 H), 1.21-1.59 (m, 24 H), 2.01-2.07 (m,
2 H), 2.14-2.22 (m, 1 H), 2.34-2.42 (m, 1 H), 2.91-2.94
(m, 2 H), 3.64 (t, J = 8.0 Hz, 2 H), 5.40-5.54 (m, 2 H);
13C NMR (75 MHz, CDCl3-TMS): 14.10, 22.26, 25.87, 26.30, 26.38, 27.53, 27.82, 29.37, 29.55, 29.60, 29.65, 29.70, 31.84, 32.88, 56.71, 57.37, 62.95, 123.87, 132.78.
The PDC oxidation of alcohol 27 to acid 28 (64%)
was carried out as described for the conversion of alcohol
19 to acid 20 (see scheme 2). The properties
of the product are as follows. TLC: EtOAc-hexane (1:1),
Rf ~ 0.38; 1H NMR (400 MHz,
CDCl3-TMS): 0.90 (t, J = 6.7 Hz, 3 H),
1.28-1.64 (m, 22 H), 2.01-2.06 (m, 2 H), 2.15-2.22 (m, 1 H), 2.32-2.41 (m, 3 H), 2.92-2.96 (m, 2 H), 5.38-5.55
(m, 4 H); 13C NMR (75 MHz, CDCl3): 14.20, 22.79, 24.88, 26.38, 26.46, 27.62, 27.91, 29.24, 29.42, 29.43, 29.57,
29.64, 29.74, 31.93, 34.30, 56.84, 57.52, 123.99, 132.88, 180.20.
Preparation of cis-14,15-oxidoeicosa-5(Z),11(Z)-dienoic
acid (8,9-tetrahydro-14,15-EET).
cis-14,15-Oxidoeicosa-5(Z),11(Z)-dienoic
acid (8,9-tetrahydro-14,15-EET) (14,15-EE-5,11-ZD; acid 36)
was prepared as follows. The alkylation of epoxy-bromide 24 with acetylene 1 to give 30 (76%) (scheme
4; Fig. 5) was carried out as
described for the conversion of bromide 16 to 17 (see scheme 2). The properties of the product are as
follows. TLC: EtOAc-hexane (1:9), Rf ~ 0.42; 1H NMR (400 MHz, CDCl3-TMS): 0.90 (t,
J = 7.3 Hz, 3 H), 1.06 (s, 9 H), 1.33-1.36 (m, 4 H), 1.48 (m,
8 H), 2.16-2.26 (m, 3 H), 2.53-2.59 (m, 1 H), 2.94-2.96
(m, 1 H), 3.08-3.12 (m, 1 H), 3.68 (t, J = 5.8 Hz, 2 H),
7.36-7.43 (m, 6 H), 7.66-7.68 (m, 4 H).
|
0.90 (t, J = 7.0 Hz, 3 H), 1.31-1.69 (m, 12 H), 2.19-2.29 (m,
3 H), 2.51-2.57 (m, 1 H), 2.93-2.97 (m, 1 H), 3.08-3.12
(m, 1 H), 3.67 (t, J = 6.0 Hz, 2 H).
Bromide 32 was obtained in 80% yield from alcohol
31 as described for the conversion of epoxide 15 to
bromide 16 (see scheme 2). The properties of the
product are as follows. TLC: EtOAc-hexane (1:4), Rf ~ 0.62; 1H NMR (400 MHz, CDCl3-TMS): 0.90 (t, J = 7.0 Hz, 3 H), 1.30-1.35 (m, 4 H), 1.46-1.55 (m,
4 H), 1.61-1.67 (m, 2 H), 1.93-2.00 (m, 2 H), 2.18-2.28
(m, 3 H), 2.50-2.57 (m, 1 H), 2.93-2.97 (m, 1 H),
3.07-3.11 (m, 1 H), 3.42 (t, J = 6.7 Hz, 2 H).
The alkylation of epoxy-bromide 32 with acetylene
1 to give bis-acetylene 33 (90%) was carried out as
described for the conversion of bromide 16 to 17 (see scheme 2). The properties of the product are as
follows. TLC: EtOAc-hexane (1:9), Rf ~ 0.42;
1H NMR (400 MHz, CDCl3-TMS): 0.91 (t,
J = 7.3 Hz, 3 H), 1.06 (s, 9 H), 1.33-1.36 (m, 4 H),
1.49-1.67 (m, 14 H), 2.14-2.27 (m, 7 H), 2.53-2.58 (m, 1 H), 2.94-2.97 (m, 1 H), 3.09-3.14 (m, 1 H), 3.69 (t, J = 6.1 Hz, 2 H), 7.36-7.43 (m, 6 H), 7.67-7.69 (m, 4 H).
The P-2 Ni hydrogenation of bis-acetylene 33 generating
cis,cis-diene 34 (80%) was carried out as described for the
conversion of bis-acetylene 5 to diene 6 (see
scheme 1). The properties of the product are as follows.
TLC: EtOAc-hexane (1:9), Rf ~ 0.44; 1H
NMR (400 MHz, CDCl3-TMS): 0.91 (t, J = 7.2 Hz, 3 H), 1.26-1.61 (m, 18 H), 2.01-2.06 (m, 6 H), 2.15-2.22
(m, 1 H), 2.34-2.39 (m, 1 H), 2.92-2.94 (m, 2 H), 3.66 (t,
J = 6.4 Hz, 2 H), 5.33-5.46 (m, 4 H), 7.36-7.42 (m, 6 H), 7.66-7.68 (m, 4 H).
The desilylation of cis,cis-diene 34 to alcohol
35 (80%) was carried out as described for the conversion of
diene 6 to alcohol 7 (see scheme 1).
The properties of the product are as follows. TLC: EtOAc-hexane (1:4),
Rf ~ 0.24; 1H NMR (300 MHz,
CDCl3-TMS): 0.90 (t, J = 7.2 Hz, 3 H),
1.31-1.68 (m, 18 H), 2.13-2.22 (m, 8 H), 2.90-2.96 (m, 2 H), 3.64 (t, J = 6.0 Hz, 2 H), 5.32-5.58 (m, 4 H);
13C NMR (75 MHz, CDCl3-TMS): 14.21, 22.80, 26.04, 26.43, 26.48, 27.13, 27.27, 27.52, 29.94, 29.35, 29.50, 31.95, 32.58, 56.78, 57.47, 63.10, 124.17, 129.76, 130.26, 132.72.
The PDC oxidation of alcohol 35 to acid 36 (64%)
was carried out as described for the conversion of alcohol
19 to acid 20 (see scheme 2). The properties
of the product are as follows. 1H NMR (400 MHz,
CDCl3-TMS): 0.90 (t, J = 7.0 Hz, 3 H),
1.32-1.71 (m, 14 H), 2.01-2.39 (m, 12 H), 2.92-2.96 (m,
2 H), 5.29-5.51 (m, 4 H); 13C NMR (75 MHz,
CDCl3-TMS): 14.20, 22.79, 24.80, 26.38, 26.47, 26.63, 27.28, 27.50, 27.90, 29.35, 29.45, 31.93, 33.55, 56.88, 57.58, 124.15, 128.60, 131.20, 132.72, 179.62.
Preparation of trans-14,15-EET.
trans-14,15-EET (38) was prepared as follows.
n-BuLi (0.1 ml, 2.5 M solution in THF) was added to a
solution of diphenylphosphine (46 mg, 0.25 mmol) in THF (3 ml) at 0°C
and stirred for 2 h at 23°C (scheme 5; Fig.
6). A solution of 14,15-EET (20 mg,
0.0625 mmol) in THF (3 ml) was added over 5 min (7). After
2 h, freshly distilled MeI (35 mg, 0.25 mmol) was added to the
reaction mixture, which was then allowed to stand for 30 min (the color
of the reaction mixture turns white from red). The contents were
diluted with EtOAc (10 ml), washed with H2O (10 ml) and
brine (10 ml), dried (Na2SO4), and concentrated
in vacuo. The residue was purified by SiO2 PTLC to give
37 (14 mg, 74%) as a colorless oil. The properties of the
product are as follows. TLC: EtOAc-hexane (3:7), Rf ~ 0.31; 1H NMR (400 MHz, CDCl3): 0.88 (t,
J = 6.4 Hz, 3 H), 1.22-1.38 (m, 6 H), 1.71 (quintet, J = 7.4 Hz, 2 H), 1.98 (dd, J = 7.0, 7.0 Hz, 2 H), 2.13 (dd, J = 7.0, 7.0 Hz, 2 H), 2.36 (apparent t, J = 7.4 Hz, 2 H),
2.74-2.82 (m, 6 H), 5.32-5.44 (m, 8 H).
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0.92 (t, J = 7.0 Hz, 3 H), 1.26-1.57 (m, 8 H), 1.71 (apparent
quintet, J = 7.3 Hz, 2 H), 2.11 (dd, J = 7.2, 7.2 Hz, 2 H),
2.25-2.31 (m, 1 H), 2.37 (dd, J = 7.2, 7.2 Hz, 2 H),
2.42-2.49 (m, 1 H), 2.75-2.84 (m, 6 H), 5.32-5.58 (m, 6 H).
Preparation of cis-10,11-oxidoheptadeca-4(Z),7(Z)-dienoic acid
(10,11-epoxyheptadecadienoic acid).
cis-10,11-Oxidoheptadeca-4(Z),7(Z)-dienoic
acid (10,11-epoxyheptadecadienoic acid) (10,11-EHDD; 42) was
prepared as follows. Pb(OAc)4 (1.06 g, 2.4 mmol) was added
in three portions over 15 min to a stirring 40°C solution of
diol 39 (Ref. 17; 450 mg, 1.2 mmol) in
CH2Cl2 (10 ml) (scheme 6; Fig.
7). After 0.5 h, the reaction
mixture was warmed to room temperature and then passed through a small
pad of silica gel with CH2Cl2 (250 ml) as
eluent. The eluent was dried over Na2SO4 and
concentrated in vacuo to give the corresponding aldehyde as a labile,
colorless liquid (350 mg) that was used immediately without further
purification.
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0.89 (t, J = 6.8 Hz, 3 H), 1.25-1.39 (m, 6 H), 2.05 (q, J = 7.2 Hz, 2 H), 2.36 (q, J = 8 Hz, 2 H), 2.79-2.87
(m, 4 H), 3.66 (q, J = 6.4 Hz, 3 H), 5.30-5.44 (m, 5 H)
5.52-5.58 (m, 1 H).
Methanesulfonyl(mesyl)chloride (154 mg, 1.35 mmol) was added dropwise
with stirring to alcohol 40 (230 mg, 0.96 mmol) in
CH2Cl2 (10 ml) followed by triethylamine (202 mg, 2.0 mmol). After 6 h, the reaction mixture was washed with
water (2 × 50 ml) and brine (50 ml) and dried, and all volatiles
were removed in vacuo. SiO2 chromatography (EtOAc-hexanes,
5:95) of the residue afforded the corresponding mesylate (290 mg, 91%)
as a colorless oil. The properties of the product are as follows.
1H NMR (CDCl3, 400 MHz): 0.89 (t, J = 7.2 Hz, 3H), 1.27-1.37 (m, 6 H), 2.05 (q, J = 6.8 Hz, 2 H)
2.54 (q, J = 6.8 Hz, 2 H), 2.79-2.85 (m, 4 H), 4.22 (t,
J = 6.8 Hz, 2 H), 5.30-5.44 (m, 5 H), 5.33-5.60 (m,
1 H).
The preceding mesylate (290 mg, 0.96 mmol) was stirred with KCN (56 mg,
1.1 mmol) in DMSO (10 ml) at room temperature for 12 h. The
reaction mixture was diluted with water (20 ml) and extracted with
ether (2 × 50 ml). The combined organic extracts were washed with
water (2 × 10 ml) and brine (10 ml) and dried over
Na2SO4, and the solvent was evaporated in
vacuo. SiO2 chromatography (EtOAc-hexanes, 1:99) of the
residue afforded the corresponding cyanide (240 mg, 90%) as a
colorless oil. The properties of the product are as follows.
1H NMR (CDCl3, 400 MHz): 0.89 (t, J = 7.2 Hz, 3 H), 1.25-1.38 (m, 6H), 2.05 (q, J = 8.0 Hz, 2 H),
2.36-2.46 (m, 4 H), 2.79-2.86 (m, 4 H), 5.32-5.41 (m, 5 H), 5.52-5.06 (m, 1 H); 13C NMR (CDCl3, 75 MHz): 14.15, 17.56, 22.65, 23.36, 25.71, 27.30, 29.37, 31.58, 119.36, 125.53, 127.21, 127.37, 129.12, 130.76, 131.57.
Diisobutylaluminum hydride (DIBAL-H; 980 µl, 1.0 M solution in
toluene, 1.5 mmol) was added to a 78°C solution of the above cyanide (240 mg, 1.00 mmol) in CH2Cl2 (10 ml).
The reaction mixture was slowly warmed to 40°C. After 0.5 h,
the reaction was quenched with MeOH (100 µl, 3.0 mmol) and then
warmed to room temperature. The mixture was diluted with ether (150 ml), washed with water (2 × 50 ml) and brine (50 ml), dried over
Na2SO4, and concentrated in vacuo.
SiO2 chromatography (EtOAc-hexanes, 5:95) of the residue afforded aldehyde 41 (187 mg, 83%) as a colorless oil. The
properties of the product are as follows. 1H NMR
(CDCl3, 400 MHz): 0.88 (t, J = 7.2 Hz, 3 H),
1.26-1.39 (m, 6 H), 2.05-2.12 (m, 2 H), 2.41-2.43 (m,
2H), 2.45-2.57 (m, 2 H), 2.81-2.93 (m, 4 H), 5.31-5.45
(m, 4 H).
Aldehyde 41 (185 mg, 0.80 mmol) in acetone (20 ml) was
slowly added to a 20°C solution of Jones reagent (2.4 ml of 1.0 M
solution, 2.4 mmol). After 0.5 h, the reaction was quenched with
isopropanol (2 ml, 2.4 mmol), concentrated in vacuo, and extracted with
ether (2 × 100 ml). The combined ethereal extracts were washed
with water (2 × 50 ml) and brine (50 ml), dried over
Na2SO4, and concentrated in vacuo.
SiO2 chromatography (EtOAc-hexanes, 5:95) of the residue afforded the corresponding acid (165 mg, 84%) as a colorless oil. The
properties of the product are as follows. 1H NMR (400 MHz,
CDCl3): 0.88 (t, J = 6.4 Hz, 3 H),
1.25-1.37(m, 6 H), 2.05 (q, J = 8.0 Hz, 2 H), 2.79-2.85
(m, 4 H), 5.31-5.52 (m, 6 H): 13C NMR
(CDCl3, 75 MHz): 14.29, 22.71, 22.79, 25.79, 25.84, 27.43, 29.54, 29.92, 31.73, 34.14, 127.67, 127.70, 127.88, 128.93, 129.91, 130.75, 179.23.
Im2CO3 (136 mg, 0.84 mmol) was added to the
above acid (140 mg, 0.56 mmol) in CH2Cl2 (10 ml) at room temperature. After 0.75 h, the reaction mixture was
cannulated into a stirring 0°C solution of anhydrous
H2O2 (18 ml, 3.5 M solution in ether, 63 mmol)
containing lithium imidazolide (10 mg, 0.056 mmol). After 5 min, the
reaction mixture was diluted with CH2Cl2 (20 ml) and KH2PO4 (600 mg, 5.0 mmol) was added.
After another 10-min interval at room temperature, the mixture was
filtered through a plug of glass wool into a degassed suspension of
Na2SO4 (20 g) in CH2Cl2
(30 ml). After 12 h in the dark, the reaction mixture was
filtered, diluted with EtOAc (100 ml), washed free of peroxide, dried
over Na2SO4, and concentrated in vacuo. The
residue was dissolved in Et2O-MeOH (9:1) and treated with
excess CH2N2 for 0.5 h. Removal of all
volatiles in vacuo and SiO2 chromatography
(Et2O-hexanes, 5:95) of the residue afforded the
corresponding 10,11-epoxide methyl ester (52 mg, 67%) as a colorless
oil. The properties of the product are as follows. 1H NMR
(400 MHz, CDCl3): 0.89 (t, J = 7.2 Hz, 3 H),
1.25-1.36 (m, 6 H), 2.02-2.07 (m, 1 H), 2.17-2.43 (m, 7 H), 2.82-2.84 (m, 2 H), 2.91-2.96 (m, 2 H), 3.67 (s, 3 H),
5.37-5.52 (m, 4 H), mass spectrometry [matrix-assisted laser
desorption ionization (MALDI), 
-cyano-4-hydroxycinnamic acid
matrix]: M+ (280.25), M + Na+ (300.37).
Saponification of the above ester (20 mg, 0.075 mmol) in
THF-H2O (5 ml, 4:1) used LiOH (230 µl of 1 M solution in
water, 0.22 mmol) for 8 h. The reaction mixture was acidified with
oxalic acid (128 µl of 1 M aqueous solution), washed with water
(2 × 10 ml), dried over Na2SO4, and
concentrated in vacuo to afford acid 42 (18 mg, 94%) as a
colorless oil. The properties of the product are as follows.
1H NMR (400 MHz, CDCl3): 0.88 (t, J = 7.2 Hz, 3 H), 1.25-1.37 (m, 6 H), 2.01-2.06 (m, 1 H),
2.17-2.43 (m, 7 H), 2.80-2.82 (m, 2 H), 2.91-2.96 (m, 2 H), 5.36-5.53 (m, 4 H).
Preparation of 12,13-epoxy-octadec-6(Z),9(Z)-dienoic acid.
12,13-Epoxy-octadec-6(Z),9(Z)-dienoic acid
(12,13-EODD) was made from 
-linolenic acid (Nu Chek Prep, Elysian,
MN) by internal epoxidation as described for the conversion of
37 to 38. The epoxide was obtained in 70%
overall yield as a colorless oil. The properties of the product are as
follows. 1H NMR (CDCl3, 400 MHz): 0.90 (t,
J = 7.0 Hz, 3 H), 1.24-1.58 (m, 10 H),
1.62-1.74 (m, 2 H), 2.02-2.14 (m, 2 H),
2.16-2.28 (m, 2 H), 2.32-2.46 (m, 2 H), 2.80 (t,
J = 6.4 Hz, 2 H), 2.90-3.00 (m, 2 H),
5.32-5.56 (m, 4 H).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Vascular Relaxations to 14,15-EET and 14,15-EET Analogs
The structures and names of the 19 analogs of 14,15-EET that were tested for agonist activity are shown in Fig. 1. 14,15-EET relaxed the U-46619-precontracted bovine coronary artery in a concentration-related manner as previously described (Figs. 8-10). Similar relaxation responses were obtained with 14,15-, 11,12-, 8,9-, and 5,6-EET in the bovine and canine coronary arteries (3, 39, 41). The four regioisomers were equipotent. The sensitivity of the vessels to 14,15-EET varied slightly from heart to heart over the 3-yr period of the study. For this reason, analogs were compared with 14,15-EET in each experiment. We initially compared the effect of analogs with changes in the carbon-1 carboxyl group, 14,15-EET-SI, 14,15-epoxyeicosatrienol (14,15-EET-OH), 14,15-epoxyeicosa-11(Z)-enol (14,15-EE-11-ZE-OH), and 14,15-EET methyl ester (14,15-EET-Me). The distance between the carboxyl and epoxy groups was reduced by two carbons with 12,13-epoxyoctadecadienoic acid (12,13-EODD), four carbons with 10,11-epoxydexadecadienoic acid (10,11-EHDD), and six carbons with 8,9-EBDE. 14,15-EET-SI relaxed the coronary artery and was equipotent and equally active with 14,15-EET (Fig. 8A). 14,15-EET-Me also relaxed the coronary artery and was equipotent with 14,15-EET (Fig. 8C). The maximal activity of 14,15-EET-Me was slightly greater than that of 14,15-EET. In contrast, 14,15-EET-OH and 14,15-EE-11-ZE-OH were less active and less potent than 14,15-EET (Fig. 8B). They relaxed the vessels slightly at the highest concentration tested, 105 M. Shortening the carbon chain
length to 14 (8,9-EBDE), 16 (10,11-EHDD), or 18 (12,13-EODD) carbons
resulted in a loss of potency and maximal activity (Fig.
8D). The sulfonimide group is commonly used as a substitute
for carboxyl groups because it has a similar pKa (1). Thus it is not surprising that 14,15-EET-SI had
activity and potency similar to those of 14,15-EET. The conversion of
the carboxyl to an alcohol resulted in a large loss in activity,
indicating the importance of an acid group at carbon-1. The potency of
the methyl ester was surprising but may represent conversion of
14,15-EET-Me to 14,15-EET by vascular cells (2, 42).
Changing the relationship between the epoxy and carboxyl group by
shortening the carbon chain also decreased the activity, indicating the
importance of the distance of 12 carbons between these groups.
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In the second series of analogs, one, two, or three of the double bonds
were eliminated. EET analogs with a single double bond varied in
activity (Fig. 9A). The
14,15-EEZE with a 8 double bond was as potent and as active as
14,15-EET. In contrast, 14,15-EEZE analogs with the 5 or 11
double bonds were less potent than 14,15-EET or 14,15-EE-8-ZE. The
concentration-response curve to 14,15-EE-5-ZE was shifted to the right
~10-fold compared with 14,15-EET. These data indicate that the 8
double bond is essential for full agonist potency, whereas the 5 and
11 double bonds are not as critical for potency. The concentration
response curve for 14,15-EE-5,11-ZD was shifted significantly to the
right of the curve for 14,15-EET but retained the same maximal activity (Fig. 9B). This further indicates the importance of the 8
double bond. The saturated analog without double bonds (14,15-EEA)
relaxed the vessels in a concentration-related manner, but the
concentration-response curves were shifted to the right of the curve
for 14,15-EET (Fig. 9C). Thus a loss of double bonds
decreased the potency of the EET but did not eliminate activity. The
presence of the 8 double bond is necessary for full agonist potency.
The third series of analogs changed the epoxy group. The oxygen of the epoxy group was changed to a sulfur (14,15-EET-thiirane) or nitrogen (14,15-EET-aziridine). Neither analog caused relaxation, indicating the importance of the oxygen (Fig. 10A). 14,15-DHET, the hydrolysis product of 14,15-EET, has two adjacent alcohols. 14,15-DHET relaxed the bovine coronary artery as previously indicated in the canine coronary artery (Fig. 10A; Refs. 33, 45). However, in our studies, the concentration-response curve for 14,15-DHET was shifted approximately fivefold to the right of the curve for 14,15-EET with no change in maximal effect. Thus the hydrolysis of the epoxy to a vicinal diol results in loss of potency but retention of full agonist activity. The 14(S),15(R)-EET isomer was more potent than the 14(R),15(S)-EET isomer (Fig. 10B). The concentration-response curve for 14(R),15(S)-EET was shifted to the right ~10-fold of the curve for 14(S),15(R)-EET. Thus the response is stereoselective. Finally, the 14,15-(cis)-isomer was more potent than the 14,15-(trans)-isomer (Fig. 10C). These studies indicate that a 14(S),15(R)-epoxy oxygen in the cis configuration is required for full agonist potency.
Figure 11 shows the chemical structure
and ball and stick molecular model of the active vasodilator. It
contains the carbon-1 carboxyl, the 8 double bond, 20 carbons, and
the 14(S),15(R)-(cis)-epoxide that are
required for full agonist potency and activity. It is 14(S),15(R)-(cis)-epoxyeicosa-8-enoic
acid.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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EETs are synthesized by the vascular endothelium and are participants in the endothelium-dependent relaxations to bradykinin and acetylcholine (3, 15, 18, 20, 23, 39). They open KCa channels on vascular smooth muscle. This results in membrane hyperpolarization and vasodilation. The activation of KCa channels by EETs requires a G protein (16, 21, 27); however, it is not known whether a receptor is involved. Binding sites for 14,15-EET have been described in macrophages but not in smooth muscle (47, 48). Our laboratory (28) showed that 11,12-EET stimulates the endogenous ADP-ribosylation of the G protein Gs, resulting in activation of KCa channels. These findings may indicate that EETs can increase KCa channel activity, but it is unclear whether this involves a receptor-mediated mechanism. Because the EETs relax coronary arteries and open KCa channels in nanomolar concentrations, it is likely that the EETs have a binding site(s) and the binding event is amplified to produce the biological response. In the present study, we examined the structure-activity relationships between a number of 14,15-EET analogs to determine which portion of the 14,15-EET molecule was necessary for vasodilation.
All four regioisomeric EETs relax bovine and canine coronary arteries,
indicating that the position of the epoxy group on the arachidonic acid
backbone does not influence this action (3, 39, 41).
Arteries from different vascular beds differ in the EET regioisomer
that causes relaxation. For example, 11,12-EET, but not 14,15-EET,
relaxed the rat renal artery (52). Only 5,6-EET relaxed
the rat tail artery (4). At the present time, it is not
clear whether these observations in the coronary artery indicate that
vascular smooth muscle cells have four EET, regioisomer-specific receptors, four binding proteins that activate endogenous ADP ribosylation, or that the position of the epoxy group is not a structural requirement for activity. Whether the EETs act through G
protein-coupled receptors, directly on a G protein, or through endogenous ADP ribosylation, the present study indicates that there are
specific structural requirements for 14,15-EET to cause vasorelaxation
of bovine coronary arteries. Changing the carboxy group at carbon-1 to
an alcohol, shortening the distance between the carboxyl and epoxy
groups, or conversion of the oxygen of the 14,15-epoxide to a thiirane
sulfur or aziridine nitrogen results in loss of activity. For full
agonist potency, the epoxide must be a
S,R-stereoisomer in the cis-epoxide
configuration. Removal of the double bonds decreases the potency but
does not necessarily result in complete loss of activity. Some double
bonds were more critical than others. There was little difference
between the loss of the 8 double bonds and the loss of the 5,
8, and 11 double bonds. 14,15-EE-5-ZE, 14,15-EE-11-ZE, and
14,15-EEA had reduced activity. The 8 double bond is required for
full agonist potency. Thus the 5 and 11 double bonds do not seem
as important as the 8 double bond. These data indicate that two
double bonds can be removed without affecting potency or activity.
Interestingly, the EEZE and EEZD analogs would be less susceptible to
autooxidation and therefore more stable for physiological studies.
These findings indicate that the presence of the negatively charged
carboxyl and oxygen of the cis-epoxide are essential for
activity. The cis-double bond must convey rigidity and a
specific conformation in the carbon backbone that is required for full potency.
Fang and co-workers (14) showed that 11,12-EET is
metabolized in porcine aortic smooth muscle cells by a combination of 
-oxidation and hydrolysis of the epoxide by epoxide hydrolase. The
major metabolites were 11,12-DHET and 7,8-dihydroxy-hexadecadienoic acid (7,8-DHHD). Human skin fibroblast metabolized 11,12-EET by -oxidation to 9,10-EODD and 7,8-EHDD and 14,15-EET to 10,11-EHDD and
12,13-EODD (13). 11,12-DHET and 7,8-DHHD relaxed
the porcine coronary artery, but a quantitative comparison to 11,12-EET
was not made. The other metabolites were not tested for activity. We
found that 10,11-EHDD and 12,13-EODD were less potent than 14,15-EET,
indicating that -oxidation represents a pathway of 14,15-EET inactivation.
Interestingly, 14,15-DHET relaxed the bovine coronary artery; however, the DHET was fivefold less potent than 14,15-EET. This finding indicates that the vicinal diol can partially substitute for the epoxide group. This finding confirms published studies that 14,15-DHET is a potent vasorelaxant in coronary arteries and microvessels (14, 33). In recent studies, we (2, 3) and others (23, 33) reported that the relaxations to 14,15-DHET were blocked by increasing the extracellular potassium concentration from 4.8 to 20 mM and by inhibitors of KCa channels. 14,15-DHET activated KCa channels in cell-attached patches but was ~10-fold less potent than 14,15-EET (2, 3). Like 14,15-EET, 14,15-DHET failed to open KCa channels in inside-out patches unless GTP was added to the bathing solution (16, 21, 27). Thus 14,15-DHET appears to have the same mechanism of action as 14,15-EET, with both requiring a G protein for KCa channel activation.
Interestingly, 14,15-EET-Me was as active as 14,15-EET in relaxing coronary arteries. This finding was surprising because results with other analogs suggested the need for a negatively charged group at carbon-1. These differences could be reconciled by conversion of the methyl ester to the free acid. Previous studies indicate that long-chain fatty acid methyl esters are taken up by cells and hydrolyzed to their fatty acids (25, 42). In support of this possibility, we have found that [14C]14,15-EET-Me is metabolized to 14,15-DHET and 14,15-DHET-Me and to a lesser extent to 14,15-EET by coronary arteries (2). These biochemical studies indicate that the coronary artery contains an epoxide hydrolase to convert the EET to DHET and esterases to convert the methyl esters to the free acids. These data are consistent with the free acid of 14,15-DHET and, to a lesser extent, 14,15-EET mediating the action of 14,15-EET-Me. Because the 14,15-DHET was fivefold less potent than 14,15-EET in causing relaxation, it is surprising that 14,15-EET-Me was as active as 14,15-EET if 14,15-DHET mediates its effect. This discrepancy is understandable if EETs and DHETs act intracellularly. The EETs and DHETs would not enter the cell as easily as the EET-Me, so less would get to the site of action. In addition, the free acids of the EET and DHET are incorporated into membrane phospholipids and may reduce the amount of EET/DHET reaching the site of action (44). Previous studies from our lab support this possibility. When [14C]EETs were incubated with coronary arteries for 30 min, <10% of the EET was converted to the DHET (39). This finding suggests that the EET does not have access to the intracellular soluble epoxide hydrolase. In contrast, 14,15-[14C]EET-Me was almost completely converted to 14,15-DHET or 14,15-DHET-Me after 10 min (2). These findings support an intracellular site of action of the 14,15-EET and 14,15-DHET.
In summary, there are specific structural requirements for 14,15-EET to
cause relaxation of the bovine coronary artery. These requirements
include a negatively charged carboxyl group at carbon-1 separated by 12 carbons from a 14(S),15(R)-cis-epoxy
oxygen. The 8 cis-double bond is necessary for full
agonist potency. The vicinal diol of 14,15-DHET may partially
substitute for the 14,15-epoxy group. From these data, inferences can
be made about the essential elements for the 14,15-EET receptor/binding
site in the bovine coronary artery. There must be 1) an
ionic binding site that interacts with the carboxylic acid that is
ionized at physiological pH; 2) p-p bonding
between the 8 double bond and aromatic amino acids such as a
phenylalanine or tyrosine; 3) hydrogen bonding with the
epoxide in which the oxygen functions as an acceptor; and 4)
a shape-specific channel or cleft for the epoxide that best accommodates the bend or kink formed by the cis-epoxide
rather than the more linear chain formed by the
trans-epoxide.
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ACKNOWLEDGEMENTS |
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The authors thank Gretchen Barg for secretarial assistance and Erik Edwards for technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-51055 and GM-31278 and the Robert A. Welch Foundation.
Address for reprint requests and other correspondence: W. B. Campbell, Dept. of Pharmacology and Toxicology, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: wbcamp{at}mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 19, 2002;10.1152/ajpheart.00831.2001
Received 21 September 2001; accepted in final form 3 September 2002.
