Synthesis of 10,11-dihydro-12-oxo-LTB4, a key biochemical intermediate

Article abstract of DOI:10.1021/jo9614957

The first total synthesis of the 5(S)-hydroxy-10,11-dihydro-12-oxo-6(Z),8(E),14(Z)-eicosatrienoic acid (10,11-dihydro-12-oxo-LTB₄) (3) is reported. This compound is a key pivotal intermediate in the biotransformation of LTB₄ by the so-called 'LTB₄ reductase pathway'.

Full text of DOI:10.1021/jo9614957

J . Org. Chem. 1997, 62, 325-330
325
Syn th esis of 10,11-Dih yd r o-12-oxo-LTB4, a Key Bioch em ica l
In ter m ed ia te
Subhash P. Khanapure, Steven S. Wang, William S. Powell,† and J oshua Rokach*
Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology,
150 W. University Boulevard, Melbourne, Florida 32901 and Meakins-Christie Laboratories,
McGill University, 3626 St-Urbain Street, Montreal, Quebec H2X 2P2, Canada
Received August 2, 1996X
The first total synthesis of the 5(S)-hydroxy-10,11-dihydro-12-oxo-6(Z),8(E),14(Z)-eicosatrienoic acid
(10,11-dihydro-12-oxo-LTB4) (3) is reported. This compound is a key pivotal intermediate in the
biotransformation of LTB4 by the so-called “LTB4 reductase pathway”.
Leukotriene B4 (LTB4) is a metabolite of arachidonic
acid which is synthesized by neutrophils and other
inflammatory cells.1 Several total syntheses of this key
mediator have appeared over the years.2 LTB4 is a potent
stimulator of neutrophils,3 monocytes,4 and lymphocytes.5
It stimulates the expression of integrins6 on neutrophils
and promotes their adhesion to the vascular endothe-
lium7 and migration to tissues8 in response to proinflam-
matory stimuli. Because of its potent biological effects,
it is very important to understand the pathways respon-
sible for the metabolism of LTB4 as well as the biological
activities of its metabolites. In common with other fatty
acids, LTB4 is metabolized by omega-oxidation9,10 and
â-oxidation.11 ω-oxidation results in substantial loss of
its biological activity,12 whereas one of the initial products
of â-oxidation, 3(S)-hydroxy-LTB4, still retains substan-
tial biological activity.13
We have identified an additional pathway for the
metabolism of LTB4 in neutrophils resulting in the
formation of 10,11-dihydro metabolites14 (Scheme 1). The
10,11-double bond of LTB4 is not reduced directly, but
first requires oxidation of the 12-hydroxyl group by a
microsomal 12-hydroxyeicosanoid dehydrogenase to give
12-oxo-LTB415 (Scheme 1). This is followed by reduction
of the 10,11-double bond by a cytosolic olefin reductase
to give 10,11-dihydro-12-oxo-LTB4 (3) (Scheme 1), which
is the initial stable product of this pathway isolated after
15
incubation of intact neutrophils with LTB4, and is the
main focus of this paper. The 12-oxo group of the
dihydro-oxo compound can then be reduced to either 12-
16
(R)- or 12(S)-10,11-dihydro-LTB4. Complete identity of
the very unstable metabolite 12-oxo-LTB4 has been
established by comparison with an authentic synthetic
sample.17 In addition, using synthetic 6-trans-12-oxo-
LTB4, we have identified variable amounts of 6-trans-
12-oxo-LTB4 during metabolism in porcine leukocytes.17
The 12-keto reductase appeared to be stereospecific
during LTB4 metabolism by subcellular fractions of
porcine PMNL resulting in formation of 10,11-dihydro-
LTB4 (4) and not in the formation of 10,11-dihydro-12-
epi-LTB4 (5). The 10,11-dihydro-LTB4 metabolite (4) was
formed initially and the 12-epi isomer (5) was only
detected following longer incubation times.18 However,
both dihydro epimers were found during LTB4 metabo-
lism by human monocytes,19 mesangial cells,20 and hu-
man lung.21 In another study, both epimers of 10,11-
dihydro-LTB4 were detected following a 24-h incubation
of LTB4 with keratinocytes.22 Additional time-course
studies in keratinocytes are necessary to determine if
both isomers are formed directly from 10,11-dihydro-12-
oxo-LTB4 (Scheme 1) or if only one isomer is initially
formed, followed by the appearance of the 12-epi-isomer.
† McGill University.
X Abstract published in Advance ACS Abstracts, December 15, 1996.
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S0022-3263(96)01495-8 CCC: $14.00 © 1997 American Chemical Society

326 J . Org. Chem., Vol. 62, No. 2, 1997
Khanapure et al.
Sch em e 1
Sch em e 2
We cannot completely eliminate the possibility that direct
enzymatic epimerization at the 12-hydroxy position
results in the production of the epimeric metabolite 5
(Scheme 1). 12(S)-HETE is metabolized in a similar
fashion by neutrophils to give 12-oxo-ETE (7), 10,11-
dihydro-12-oxo-ETE (i.e. 12-oxo-ETrE) (8), and the 12-
(R) and 12(S) isomers of 10,11-dihydro-12-HETE (i.e. 12-
HETrE) (9 and 10).18,23,24
Preliminary evidence suggested that 12-oxo-LTB425 and
the R and S isomers of 10,11-dihydro-LTB426,27 all possess
lower biological activities than LTB4. 12(R)-HETrE (9),
on the other hand, was found to be a potent proinflam-
matory agent.28,29 As can be seen from Scheme 2, 12(R)-
HETrE (9) is probably formed through the intermediacy
of 12-oxo-ETE (7), and we have previously described the
total synthesis of 12-oxo-ETE (7) and studied its prelimi-
nary biological and biochemical profile.24,30 In order to
study the biological activity of 10,11-dihydro-12-oxo-LTB4
(3) and to investigate its metabolism to 10,11-dihydro-
LTB4 (4) and its 12-epi isomer (5), it was important to
have the authentic, chemically synthesized compound.
This paper describes the first total synthesis of 10,11-
dihydro-12-oxo-LTB4 (3) along with its 6,7-trans isomer
(27). Scheme 3 describes the steps involved. The syn-
thesis starts with the octenyl dithiane 12 prepared from
nonenol 11 in a two-step one-pot reaction.17 Alkylation
of octenyl dithiane 12 with 2-(2-bromoethyl)-1,3-dioxolane
gave dioxolane 13 in 94% yield. Aldehyde 14 was
obtained in 74% yield by the hydrolysis of the dioxolane
13. Wittig reaction of the aldehyde 14 with commercial
trimethyl phosphonoacetate, using LDA as base, pro-
duced a mixture of E- and Z-isomers 16 and 15 in 65%
and 21% yields, respectively. The Z-isomer 15, upon
treatment with iodine, was converted to the E-isomer 16.
Thus the E-isomer 16 was obtained in effective 86% yield.
We have also developed conditions which minimize the
formation of the Z-isomer 15. The treatment of trimethyl
phosphonoacetate with sodium hydride at room temper-
ature followed by addition of aldehyde 14 afforded
E-isomer 16 in 73% yield with the Z-isomer 15 being
formed in 2.5% yield. Both procedures are described in
the Experimental Section. The reduction of the ester 16
with DIBAL-H was clean and gave the alcohol 17 in
nearly quantitative yield. Bromination of the alcohol 17
with CBr4 and DIPHOS furnished bromide 18. Due to
the high reactivity of the allyl bromide 18 and presence
of sulfur in the molecule, the bromide 18 was unstable.
We prepared the bromide 18 and immediately treated it
with Ph3P to generate the phosphonium salt 19 which
was purified by flash column chromatography to give
pure 19 in 72% yield. The benzoate aldehyde 20, which
contains the chiral component of the 12-oxo-10,11-dihy-
dro-LTB4 molecule, was prepared by two different routes,
as described before. One uses 2-deoxy-D-ribose as start-
ing material,2c,d,17 the other starts with D-arabinose.2g,17
The Wittig coupling of the phosphonium salt 19 with
aldehyde 20 followed by purification gave an 87:17 Z and
E mixture of 21 and 24 in 89.6% yield. The separation
of the individual isomers was carried out by normal phase
HPLC. Hydrolysis of the benzoate 21 with sodium
methoxide in methanol gave the hydroxy ester 22 in 75%
yield. Hydroxy ester 22 was treated with 1 M LiOH in
THF/H2O to give, after purification by column chroma-
tography, the hydroxy acid 23 in 93% yield. Finally,
deprotection of the dithio acid 23 with [bis(trifluoroace-
toxy)iodo]benzene31,17 gave the 10,11-dihydro-12-oxo-
LTB4 3 in 79% yield. The 6,7-trans isomer 24, isolated
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Synthesis of 10,11-Dihydro-12-oxo-LTB4
J . Org. Chem., Vol. 62, No. 2, 1997 327
Sch em e 3
Sch em e 4
dithiane 35 with allyl halides (vide infra). In an attempt
to improve on this reaction, we used the 3-nonynyl
bromide 29 in the alkylation reaction (Scheme 4a).24 This
approach, which worked well, led also to the formation
in varying degrees (2-5%) of the trans product. The
purification of the desired dithiane 12 proved tedious, as
the trans product 31 is very close to the cis on TLC. We
attributed the formation of the trans product 31 to a free-
radical-induced isomerization. The free radicals are
presumably formed by hydrogenolysis of the sulfur-
carbon bond during hydrogenation. The addition of a
free-radical inhibitor such as 4-hydroxy-TEMPO reduces
substantially the amount of trans product 31 formed, but
does not eliminate it entirely.
It is worth mentioning that we had initially approached
the synthesis of aldehyde 14 by another route shown in
Scheme 5a. It is interesting to note that the main
product of the reaction between 29 and 35 is the allene
42, not the desired 36. Intermediates 40 and 41 repre-
sent an attempt to rationalize the formation of the allene
product 42. This reaction is similar to the known
alkylation of formyl dithiane 35 with allyl halides32,33 in
which the nucleophilic attack occurs at carbon 3 of the
allyl halides. Although we were able to prepare 37 by
formylation of 12,17 this approach was dropped because
the reduction of 38 to 39 using hydride reducing agents
did not work as well as some of our model compounds.
We compared the effects of 10,11-dihydro-12-oxo-LTB4
on calcium mobilization and chemotaxis in neutrophils
to those of LTB4 and a number of its metabolites.34
Although 10,11-dihydro-12-oxo-LTB4 stimulated both
calcium mobilization (EC50, 400 nM) and chemotaxis
(EC50, ca. 20 uM), it was considerably less active than
LTB4. Introduction of a 12-oxo group or reduction of the
10,11-double bond each reduced the biological activity of
LTB4 by about 100-fold.34 When these two modifications
were combined in 10,11-dihydro-12-oxo-LTB4, there was
a further 10- to 100-fold reduction in potency.34 The 6,7-
trans isomer of 10,11-dihydro-12-oxo-LTB4 displayed
substantially lower potency than the 6,7-cis compound,
in the Wittig reaction, was converted to the 10,11-
dihydro-6,7-trans-12-oxo-LTB4 27, as shown in Scheme
3b.
Before we settled for the very convenient preparation
of 12 in a two-step one-pot procedure, we developed
conditions to alkylate dithiane 28 as shown in Scheme
4a,b. Alkylation of dithiane 28 with the allyl bromide
32 afforded the desired compound in 57% yield. Two
byproducts 33 and 34 were isolated. The formation of
byproduct 34 is reminiscent of the alkylation of formyl
(32) Wilson, S. R.; Mathew, J . Synthesis 1980, 625.
(33) Young, C. G.; J ames, B. R.; Rettig, S. J . Can. J . Chem. 1985,
63, 1985.
(34) Powell, W. S.; Rokach, J .; Khanapure, S. P.; Manna, S.; Hashefi,
M.; Gravel, S.; MacLeod, R. J .; Falck, J . R.; Bhatt, R. K. J . Pharm.
Exp. Ther. 1996, 276, 728.

328 J . Org. Chem., Vol. 62, No. 2, 1997
Khanapure et al.
(m, 2 H), 3.95 (m, 2 H), 4.95 (t, J ) 4.5 Hz, 1 H), 5.42-5.60
(m, 2 H); 13C NMR (CDCl3) δ 14.22, 22.74, 25.39, 26.20 (2 ×
C), 27.89, 29.18, 29.36, 31.72, 32.01, 36.48, 52.98, 65.12 (2 ×
C), 104.37, 123.06, 133.62.
Sch em e 5
4,4-(Tr im eth ylen ed ith io)d od ec-6(Z)-en a l (14). A solu-
tion of 13 (3.1 g, 9.1 mmol) in THF (40 mL) and 2 M HCl (30
mL) was refluxed for 6 h. The reaction mixture was cooled to
room temperature, diluted with water (100 mL), neutralized
with NaHCO3 and extracted with diethyl ether (2 × 100 mL).
The combined organic extract washed with water (2 × 50 mL)
and brine (1 × 50 mL), dried over anhydrous Na2SO4, and
filtered, and the solvent was evaporated in vacuo. The crude
product was obtained as a slightly yellow oil, which was
purified by flash column chromatography using 5% ethyl
acetate in hexane to give the aldehyde 14 (2.21 g, 85%). 1H
NMR (CDCl3) δ 0.87 (t, J ) 6.8 Hz, 3 H), 1.21-1.40 (m, 6 H),
1.85-2.00 (m, 2 H), 2.05 (q, J ) 7.2 Hz, 2 H), 2.27 (t, J ) 7.2
Hz, 2 H), 2.57 (d, J ) 6.9 Hz, 2 H), 2.64 (t, J ) 7.1 Hz, 2 H),
2.70-2.78 (m, 2 H), 2.82-2.90 (m, 2 H), 5.41-5.50 (m, 1 H)
5.51-5.62 (m, 1 H), 9.82 (s, 1H); 13C NMR (CDCl3) δ 14.26,
22.75, 25.15, 26.26 (2 × C), 27.92, 29.37, 30.44, 31.75, 36.92,
39.82, 52.62, 122.72, 133.98, 201.31.
Meth yl 6,6-(Tr im eth ylen ed ith io)-2(E),8(Z)-tetr a d eca -
d ien oa te (16). P r oced u r e a : Sodium hydride 60% disper-
sion (21 mg, 0.555 mmol) was placed in a flame-dried flask
and washed with dry hexane (1 mL) under argon atmosphere.
Anhydrous benzene (2 mL) was then added and, while stirring,
trimethyl phosphonoacetate (134.7 mg, 0.185 mmol) was added
dropwise under argon atmosphere. The white suspension
formed was stirred at room temperature for 15 min, and then
aldehyde 14 (53 mg, 0.185 mmol) in benzene (1 mL) was added
dropwise at room temperature. The reaction mixture was
stirred at room temperature for 1 h and then quenched with
saturated aqueous ammonium chloride, diluted with benzene
(5 mL), and acidified with 10% HCl. The benzene layer was
separated and the aqueous layer extracted with benzene (10
mL). The combined extract was washed with water (3 × 5
mL) and dried over Na2SO4 and the solvent evaporated at
reduced pressure to give the crude product, which was purified
by flash column chromatography over silica gel using ethyl
acetate/hexane (3:97) as an eluant. The less polar compound
(1.6 mg), isolated in 2.5% yield, was characterized as the
Z-isomer 15. 1H NMR (CDCl3) δ 0.86 (t, J ) 6.6 Hz, 3 H),
1.3-1.41 (m, 6 H), 1.90-2.12 (m, 6 H), 2.61 (d, J ) 6.8 Hz, 2
H), 2.73-2.82 (m, 4 H), 2.83-2.92 (m, 4 H), 3.69 (s, 3 H), 5.45-
5.60 (m, 2 H), 5.78 (d, J ) 11.4 Hz, 1 H), 6.95 (dt, J ) 11.5
and 3.7 Hz, 1 H); 13C NMR (CDCl3) δ 14.18, 22.79, 24.96, 25.42,
26.34 (2 × C), 27.96, 29.45, 31.79, 36.51, 37.36, 51.32, 53.18,
120.17, 123.16, 133.79, 149.31, 167.79. The more polar
compound, obtained in 73% yield, was the desired ester 16.
1H NMR (CDCl3) δ 0.86 (t, J ) 6.7 Hz, 3 H), 1.22-1.35 (m, 6
H), 1.91-2.05 (m, 6 H), 2.34(q, J ) 7.2 Hz, 2 H), 2.60 (d, J )
6.8 Hz, 2 H), 2.78-2.81 (m, 4 H), 3.70 (s, 3 H), 5.40-5.47 (m,
1 H), 5.51-5.61 (m, 1 H), 5.81(d, J ) 15.7 Hz, 1 H), 6.95 (dt,
J ) 15.7 and 6.8 Hz, 1 H); 13C NMR (CDCl3) δ 14.18, 22.71,
25.29, 26.26 (2 × C), 27.63, 27.92, 29.36, 31.72, 36.51 (2 × C),
51.57, 52.85, 121.41, 122.99, 133.75, 148.64, 167.09.
P r oced u r e b: To a cooled (-78 °C), stirred solution of
trimethyl phosphonoacetate (375 mg, 1.59 mmol) in THF (4
mL) was added LDA (1.5 M, 0.848 mL) (1.27 mmol) dropwise
under argon. The reaction mixture was stirred at -78 °C for
15 min, and then a solution of aldehyde 14 (303 mg, 1.06 mmol)
in THF (2 mL) was added dropwise. The reaction mixture was
stirred at -78 °C for 1 h, slowly allowed to warm to room
temperature, and stirred at room temperature overnight. The
resulting mixture was poured on saturated aqueous am-
monium chloride solution (20 mL) and extracted with diethyl
ether (2 × 50 mL). The combined organic layers were washed
with water (2 × 25 mL) and brine (1 × 25 mL), dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. The
product was purified by flash column chromatography using
5% EtOAc in hexane to give the less polar product 15 (75 mg,
21%) as a colorless thick oil. 1H NMR (CDCl3) δ 0.86 (t, J )
6.6 Hz, 3 H), 1.3-1.41 (m, 6 H), 1.90-2.12 (m, 6 H), 2.61 (d, J
) 6.8 Hz, 2 H), 2.73-2.82 (m, 4 H), 2.83-2.92 (m, 4 H), 3.69
(s, 3 H), 5.45-5.60 (m, 2 H), 5.78 (d, J ) 11.4 Hz, 1 H), 6.95
as might be expected from the relatively low potencies
of 6,7-trans isomers of LTB4.
35,36
Exp er im en ta l Section
Rea gen ts a n d Meth od s. Unless stated otherwise, all
reagents and chemicals were obtained from commercial sources
and used without further purification.
1H NMR spectra were recorded on a 360 MHz spectrometer
with tetramethylsilane as an internal standard, J values are
given in hertz.
All reactions were carried out under an inert (nitrogen or
argon) atmosphere with dry, freshly distilled solvents under
anhydrous conditions unless otherwise noted. Yields refer to
chromatographically and spectroscopically (1H NMR) homo-
geneous materials.
1-(2,4-Dioxola n yl)-4,4-(tr im eth ylen ed ith io)d od ec-6(Z)-
en a l (13). To a cooled (-78 °C) stirred solution of 2-octenyl
dithiane 12 (2.3 g, 10 mmol) in THF (40 mL) was added n-BuLi
(1.6 M, 7.5 mL, 12 mmol). The reaction mixture was stirred
for 30 min at -78 °C and then slowly allowed to warm to -10
°C and stirred for 3 h at -10 °C. The reaction mixture was
cooled to -78 °C, and HMPA (5 mL) was added dropwise. The
resulting mixture was stirred further at -78 °C for 10 min,
and then 2-(2-bromoethyl)-1,3-dioxolane (2.728 g, 15 mmol) in
THF (4 mL) was added and allowed to warm to room temper-
ature during 1 h. Workup consisted of the addition of
saturated aqueous ammonium chloride solution (100 mL),
extraction of the aqueous layer with diethyl ether (2 × 100
mL), washing the combined organic layers with brine (2 × 50
mL), drying over anhydrous Na2SO4, filtration, and concentra-
tion in vacuo. The crude product was obtained as a slightly
yellow oil, which was purified by flash column chromatography
using 10% ethyl acetate in hexane to give pure 13 (3.1 g, 94%).
1H NMR (CDCl3) δ 0.87 (t, J ) 6.9 Hz, 3 H), 1.27-1.40 (m, 6
H), 1.83 (m, 2 H), 1.87-2.0 (m, 2 H), 2.04 (m, 4 H), 2.57 (d, J
) 6.7 Hz, 2 H), 2.71-2.80 (m, 2 H), 2.82-2.92 (m, 2 H), 3.84
(35) Lewis, R. A.; Goetzl, E. J .; Drazen, J . M.; Soter, N. A.; Austen,
K. F.; Corey, E. J . J . Exp. Med. 1981, 154, 1243.
(36) Sirois, P.; Roy, S.; Beraldin, G.; Vallerand, P.; Borgeat, P.
Prostaglandins 1982, 24, 405.

Synthesis of 10,11-Dihydro-12-oxo-LTB4
J . Org. Chem., Vol. 62, No. 2, 1997 329
(dt, J ) 11.5 and 3.7 Hz, 1 H); 13C NMR (CDCl3) δ 14.18, 22.79,
24.96, 25.42, 26.34 (2 × C), 27.96, 29.45, 31.79, 36.51, 37.36,
51.32, 53.18, 120.17, 123.16, 133.79, 149.31, 167.79. The more
polar product 16, 236 mg, was isolated in 65% yield. The less
polar Z-isomer 15 was dissolved in methylene chloride (5 mL),
a catalytic amount of iodine was added, and the solution was
stirred at room temperature for 2 days. It was converted to
the (E)-ester 16. Thus the (E)-ester 16 was obtained in total
86% yield. 1H NMR (CDCl3) δ 0.86 (t, J ) 6.7 Hz, 3 H), 1.22-
1.35 (m, 6 H), 1.91-2.05 (m, 6 H), 2.34 (q, J ) 7.2 Hz, 2 H),
2.60 (d, J ) 6.8 Hz, 2 H), 2.78-2.81 (m, 4 H), 3.70 (s, 3 H),
5.40-5.47 (m, 1 H), 5.51-5.61 (m, 1 H), 5.81(d, J ) 15.7 Hz,
1 H), 6.95 (dt, J ) 15.7 and 6.8 Hz, 1 H); 13C NMR (CDCl3) δ
14.18, 22.71, 25.29, 26.26 (2 × C), 27.63, 27.92, 29.36, 31.72,
36.51 (2 × C), 51.57, 52.85, 121.41, 122.99, 133.75, 148.64,
167.09.
6,6-(Tr im e t h yle n e d it h io)-2(E ),8(Z)-t e t r a d e ca d ie n -
1-ol (17). To a -78 °C cooled, stirred solution of ester (16)
(830 mg, 2.42 mmol) in methylene chloride (80 mL) was slowly
added DIBAL-H (5.3 mmol) dropwise under argon. The
reaction mixture was stirred for 1 h at -78 °C and then
quenched by the addition of methanol (1 mL), poured over cold
water, acidified, and extracted with methylene chloride (2 ×
100 mL). The combined organic layers were washed with brine
(2 × 50 mL), dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The product was purified by flash
column chromatography (4:1 EtOAc/hexane) to afford the
alcohol 17 (0.690 mg, 91%) as a colorless thick oil. 1H NMR
(CDCl3) δ 0.85 (t, J ) 6.7 Hz, 3 H), 1.20-1.4 (m, 6 H), 1.95
(m, 4 H), 2.05 (m, 2 H), 2.2 (m, 2 H), 2.62 (d, J ) 6.7 Hz, 2 H),
2.70 (m, 4 H), 4.13 (s, 2 H), 5.41-5.50 (m, 1 H), 5.51-5.59 (m,
1 H), 5.6-5.73 (m, 2 H); 13C NMR (CDCl3) δ 14.25, 22.77, 25.47,
26.31 (2 × C), 27.41, 27.97, 29.43, 31.78, 36.36, 37.88, 53.12,
63.88, 123.36, 129.70, 132.37, 133.56.
[6,6-(Tr im eth ylen edith io)-2(E),8(Z)-tetr adecadien -1-yl]-
tr ip h en ylp h osp h on iu m Br om id e (19). To a cooled (0-5
°C), stirred solution of alcohol 17 (640 mg, 2.03 mmol) and
1,2-bis(diphenylphosphino)ethane (800 mg, 2.03 mmol) in dry
CH2Cl2 (5 mL) was slowly added carbon tetrabromide (1.01 g,
3.06 mmol) under argon. The reaction mixture was stirred
for 10 min at 0-5 °C and then diluted with hexane (200 mL),
and the resulting solution of the labile bromide 18 was quickly
filtered through Celite. The filtrate was evaporated at reduced
pressure, the residue was dissolved in dry acetonitrile (20 mL),
and triphenylphosphine (800 mg, 3.06 mmol) was added. The
reaction mixture was stirred for 24 h at room temperature and
then concentrated in vacuo. The phosphonium salt was
purified by flash column chromatography with 19:1 methylene
chloride/methanol to afford the pure phosphonium salt (930
mg, 72%) (overall yield from alcohol 17) as a colorless fluffy
solid. 1H NMR (CDCl3) δ 0.85 (t, J ) 6.7 Hz, 3 H), 1.2 (m, 6
H), 1.6-1.7 (m, 2 H),1.80-2.0 (m, 4 H), 2.0-2.1 (m, 2 H), 2.5
(d, J ) 6.7 Hz, 2 H), 2.70 (m, 4 H), 4.6-4.7 (m, 2 H), 5.20-
5.40 (m, 2 H), 5.40-5.50 (m, 1 H), 6.8-6.95 (m, 1 H), 7.6-7.7
(m, 6 H), 7.7-7.9 (m, 9 H); 13C NMR (CDCl3) δ 14.21, 22.66,
25.26, 26.21 (2 × C), 27.86, 28.25, 29.31, 31.65, 36.33, 52.83,
114.87, 117.78, 118.72, 123.20, 130.59, 133.47, 134.06, 134.17,
135.23, 147.87.
Meth yl 5(S)-(Ben zoyloxy)-12,12-(tr im eth ylen ed ith io)-
6(Z),8(E),14(Z)-icosa tr ien oa te (21). To a cooled (-93 °C),
stirred solution of the phosphonium salt 19 (384 mg, 0.6 mmol)
in THF (16 mL) and HMPA (4 mL) was added lithium
hexamethyldisilazide (1 M, 0.55 mL, 0.55 mmol) dropwise
under argon. After stirring for 2 min, aldehyde 20 (131 mg,
0.5 mmol) in THF (2 mL) was added to the resulting red
solution. The reaction mixture was stirred for 30 min at -93
°C, and at -78 °C for 2 h, and then allowed to warm slowly to
0 °C. It was then quenched by the addition of saturated
aqueous ammonium chloride solution (50 mL) and extracted
with diethyl ether (3 × 50 mL). The combined extract was
washed with cold water (3 × 25 mL), dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure to
afford the crude product which was purified by flash column
chromatography to give an 83:17 mixture of Z- and E-isomers
(244 mg, 89.6%). The separation of the Z- and E-isomers was
carried out by NP HPLC [µ-poracil silica, 7.8 × 300 mm;
solvent system 8% EtOAc in hexane, flow rate 4 mL/min
(retention time for Z-isomer 8.7 min, and retention time for
E-isomer 10.85 min). The NP HPLC separation gave pure
Z-isomer 21 (154 mg, 47%), 1H NMR (CDCl3) δ 0.88 (t, J )
6.9 Hz, 3 H), 1.22-1.41 (m, 6 H), 1.67-2.03 (m, 8 H), 2.09 (q,
J ) 6.9 Hz, 2 H), 2.30 (m, 2 H), 2.37 (t J ) 7.0 Hz, 2 H), 2.63
(d, J ) 6.8 Hz, 2 H), 2.74- 2.92 (m, 4 H), 3.67 (s, 3 H), 5.34 (t,
J ) 10.1 Hz, 1 H), 5.46-5.63 (m, 2 H), 5.77 (dt, J ) 15.0, 7.0
Hz, 1 H), 5.90 (m, 1 H), 6.11 (t, J ) 11.0 Hz, 1 H), 6.52 (dd, J
) 14.6, 11.5 Hz, 1 H), 7.43 (t, J ) 7.4 Hz, 2 H), 7.55 (t, J ) 7.4
Hz, 1 H), 8.04 (dd, J ) 7.2 and 1.3 Hz, 1 H); 13C NMR (CDCl3)
δ 14.23, 20.79, 22.74, 25.43, 26.28 (2 × C), 27.93, 28.24, 29.40,
31.74, 33.90, 34.53, 36.46, 37.69, 51.70, 53.14, 70.89, 123.24,
125.78, 126.81, 128.78 (2 × C), 129.78 (2 × C), 130.73, 132.22,
133.00, 133.60, 137.27, 134.43, 166.04, 173.81; HREIMS calcd
(C31H44S2O4, M+) 544.2670, obsd 544.2693. Pure E-isomer 24,
32 mg (9.8%), was obtained, 1H NMR (CDCl3) δ 0.88 (t, J )
6.9 Hz, 3 H), 1.23-1.40 (m, 6 H), 1.69-1.88 (m, 4 H), 1.92-
2.00 (m, 4 H), 2.06 (q, J ) 7.1 Hz, 2 H), 2.2-2.28 (m, 2 H),
2.37 (t, J ) 6.8 Hz, 2 H), 2.63 (d, J ) 6.7 Hz, 2 H), 2.82 (m, 4
H), 3.68 (s, 3 H), 5.42-5.65 (m, 4 H), 5.76 (dt, J ) 15.1, 6.8
Hz, 1 H), 6.04 (dd, J ) 15.2, 10.6 Hz, 1 H), 6.30 (dd, J ) 15.0,
10.5 Hz, 1 H), 7.45 (t, J ) 7.4 Hz, 2 H), 7.56 (t, J ) 7.4 Hz, 1
H), 8.05 (dd, J ) 7.1 and 1.2 Hz, 1 H); 13C NMR (CDCl3) δ
14.25, 20.87, 22.77, 25.45, 26.30 , 27.92 (2 × C), 29.42, 31.77,
33.89, 34.21, 36.41, 37.70, 51.74, 53.10, 74.83, 123.26, 128.53
(2 × C), 128.89, 129.80, 129.84, 130.75 (2 × C), 132.22, 133.07,
133.41, 137.61, 135.51, 166.02, 173.89
Meth yl 5(S)-Hyd r oxy-12,12-(tr im eth ylen ed ith io)-6(Z),
8(E),14(Z)-icosa tr ien oa te (22). Sodium methoxide (1 mL,
25% wt in methanol) was added to a solution of benzoate ester
21 (36 mg, 0.066 mmol) in methanol (1.5 mL) at 0 °C and then
the reaction mixture was stirred for 10 min at room temper-
ature. After acidification with aqueous 5% KH2PO4 buffer (pH
4.3), the organic material was extracted with ethyl acetate (3
× 10 mL). The combined ethyl acetate extract was washed
with cold water (1 × 10 mL) and brine (1 × 10 mL), dried over
anhydrous Na2SO4, and filtered and the solvent evaporated
under reduced pressure to afford the crude hydroxy methyl
ester product 22 which was purified by column chromatogra-
phy using ethyl acetate hexane (20:80) to give pure 22 (27 mg,
93% yield). 1H NMR (CDCl3) δ 0.89 (t, J ) 6.9 Hz, 3 H), 1.25-
1.43 (m, 6 H), 1.45-1.80 (m, 4H), 1.93-2.02 (m, 4 H), 2.08 (q,
J ) 6.9 Hz, 2 H), 2.22-2.33 (m, 2 H), 2.37 (t, J ) 6.9 Hz, 2 H),
2.65 (d, J ) 6.8 Hz, 2 H), 2.84 (m, 4 H), 3.68 (s, 3 H), 4.58 (m,
1 H), 5.30 (t, J ) 10.1 Hz, 1 H), 5.45-5.53 (m, 1 H), 5.55-5.65
(m, 1 H), 5.75 (dt, J ) 14.9, 6.9 Hz, 1 H), 6.03 (t, J ) 10.1, 1
H), 6.35 (dd, J ) 14.7, 10.4 Hz, 1 H). 13C NMR (CDCl3) δ 14.03,
20.76, 22.53, 25.19, 26.06, 27.73, 27.90, 29.18, 31.53, 33.81,
33.16, 36.69, 37.56, 51.51, 52.85, 67.44, 123.04, 125.42, 130.47,
131.38, 133.39, 136.22, 173.95.
5 (S )-H y d r o x y -1 2 ,1 2 -(t r i m e t h y l e n e d i t h i o )-6 (Z ),
8(E),10(E),14(Z)-eicosa tr ien oic Acid (23). A solution of the
dithio compound 22 (22 mg) in THF (2.3 mL) and 1 M LiOH
(750 µL) was stirred at room temperature overnight. It was
acidified with 1 M HCl and extracted with ethyl acetate (3 ×
10 mL). The combined ethyl acetate extract was washed with
cold water, dried over anhydrous Na2SO4, and filtered and the
solvent evaporated under reduced pressure to afford the
hydroxy acid, which was purified by flash column chromatog-
raphy over silica gel using 1:9 MeOH/CH2Cl2 to give the pure
hydroxy acid 23 (19 mg, 88%). 1H NMR (CDCl3) δ 0.89 (t, J )
6.8 Hz, 3 H), 1.23-1.42 (m, 7 H), 1.50-1.70 (m, 3 H), 1.96 (m,
4 H), 2.08 (q, J ) 7.1 Hz, 2 H), 2.41 (t, J ) 7.1 Hz, 2 H), 2.65
(d, J ) 6.8 Hz, 2 H), 2.84 (m, 4H), 4.60 (m, 1 H), 5.31 (t, J )
10.4 Hz, 1 H), 5.45-5.53 (m, 1 H), 5.54-5.63 (m, 1 H), 5.75
(dt, J ) 14.3, 6.8 Hz, 1 H), 6.04 (t, J ) 11.1 Hz, 1 H), 6.35 (dd,
J ) 14.5, 11.7 Hz, 1 H); 13C NMR (CDCl3) δ 14.00, 20.57, 22.30,
25.21, 26.09 (2 × C), 27.76, 27.92, 29.21, 31.56, 33.59, 36.15,
36.53, 37.53, 52.70, 67.52, 123.05, 125.39, 130.58, 133.38,
131.25, 133.44, 136.35, 178.93. Electrospray MS calcd
[C23H38S2O3 + Na, (M + Na)+] 449, obsd 449.
5(S)-Hydr oxy-12,12-(tr im eth ylen edith io)-6(Z),8(E),14(Z)-
eicosa tr ien oic Acid (10,11-d ih yd r o-12-oxo-LTB4) (3). A
solution of dithio acid 23 (5.8 mg, 0.0136 mmol) in methanol/
H2O (9:1, 1.5 mL) and [bis(trifluoroacetoxy)iodo] benzene (15

330 J . Org. Chem., Vol. 62, No. 2, 1997
Khanapure et al.
mg, 0.0348 mmol) was stirred at room temperature for 2 min.
The reaction mixture was diluted with water (20 mL) and
extracted with ethyl ether (3 × 20 mL). The combined extract
was washed with cold water (7 × 10 mL) and brine (1 × 10
mL), dried over anhydrous Na2SO4, and filtered and the
solvent evaporated under reduced pressure to afford the crude
product 3 which was purified by flash column chromatography
over silica gel using 1:19 MeOH/CH2Cl2 to give the pure 10,-
11-dihydro-12-oxo-LTB4 (3) (3.6 mg, 79%). 1H NMR (C6D6) δ
0.95 (t, J ) 7.2 Hz, 3 H), 1.25-1.75 (m, 10 H), 1.98 (q, J ) 7.0
Hz, 2 H), 2.15 (m, 4 H), 2.35 (q, J ) 7.2 Hz, 2 H), 2.92 (d, J )
6.8 Hz, 2 H), 4.69 (q, J ) 7.1 Hz, 1 H), 5.31 (t, J ) 10.1 Hz, 1
H), 5.55-5.75 (m, 3 H), 5.98 (t, J ) 11.1 Hz, 1 H), 6.42 (dd, J
) 11.9, 4.0 Hz, 1 H). Electrospray MS calcd [(C20H32O4 + Na)
- H2O, (M + Na)+ - H2O] 341, obsd 341.
Meth yl 5(S)-Hyd r oxy-12,12-(tr im eth ylen ed ith io)-6(E),
8(E),14(Z)-eicosa tr ien oa te (25). 5(S)-(Benzoyloxy)-12,12-
(trimethylenedithio)-6(E),8(E),14(Z)-eicosatrienoate (24) was
hydrolyzed to 25 in nearly quantitative yield, as described for
the preparation of 22. 1H NMR (C6D6) δ 0.88 (t, J ) 6.9 Hz,
3 H), 1.23-1.40 (m, 6 H), 1.50-1.60 (m, 2 H), 1.62-1.75 (m, 2
H), 1.92-2.00 (m, 4 H), 2.06 (q, J ) 7.0 Hz, 2 H), 2.24 (m, 2
H), 2.35 (t, J ) 7.4 Hz, 2 H), 2.64 (d, J ) 5.7 Hz, 2 H), 2.83 (m,
4 H), 3.67 (s, 3 H), 4.12 (m, 1 H), 5.42-5.50 (m, 1 H), 5.55-
5.62 (m, 2 H), 5.69 (dt, J ) 14.2, 6.8 Hz, 1 H), 6.05 (dd, J )
15.0, 10.4 Hz, 1 H), 6.17 (dd, J ) 15.1, 10.6 Hz, 1 H). 13C NMR
(CDCl3) δ 14.03, 20.79, 22.53, 25.21, 26.05 (2 × C), 27.06, 27.73,
29.19, 31.77, 36.09, 36.49, 37.56, 51.50, 52.85, 72.22 123.04,
129.84, 130.88, 133.36, 133.60, 134.25, 174.00.
described for the preparation of 23. 1H NMR (CDCl3) δ 0.89
(t, J ) 6.7 Hz, 3 H), 1.23-1.43 (m, 6 H), 1.51-1.80 (m, 4 H),
2.07 (m, 2 H), 2.25 (m, 2 H), 2.38 (t, J ) 7.1 Hz, 2 H), 2.64 (d,
J ) 6.5 Hz, 2 H), 2.83 (m, 4 H), 4.15 (m, 1 H), 5.42-5.52 (m,
1 H), 5.52-5.63 (m, 2 H), 5.70 (dt, J ) 14.3, 6.5 Hz, 1 H), 6.05
(dd, J ) 14.8, 10.5 Hz, 1 H), 6.18 (dd, J ) 14.9, 10.4 Hz, 1 H);
13C NMR (CDCl3) δ 14.04, 20.56, 22.54, 25.21, 26.05 (2 × C),
27.73, 29.19, 31.54, 33.64, 36.10, 36.30, 37.54, 52.86, 72.31,
123.04, 129.80, 131.03, 133.38, 133.39, 134.39, 178.90.
5(S)-Hydr oxy-12-oxo-6(E),8(E),14(Z)-eicosatr ien oic Acid
(10,11-d ih yd r o-6,7-tr a n s-12-oxo-LTB4) (27). Deprotection
of the dithio group in 26, as described for the preparation of
3, produced 27 in 77% yield. 1H NMR (C6D6) δ 0.89 (t, J )
6.9 Hz, 3 H), 1.15-1.70 (m, 12 H), 1.95 (q, J ) 7.0 Hz, 2 H),
2.10 (m, 4 H), 2.30 (q, J ) 7.1 Hz, 2 H), 2.86 (d, J ) 7.0 Hz, 2
H), 3.84 (m, 1 H), 5.4-5.70 (m, 4 H), 5.98 (dd, J ) 14.6, 10.4
Hz, 1 H), 6.08 (dd, J ) 14.4, 10.5 Hz, 1 H).
Ack n ow led gm en t. We acknowledge the National
Institutes of Health for support under Grant DK-44730
(J .R.), the National Science Foundation for an AMX-
360 NMR instrument (Grant CHE-9013145), and the
Medical Research Council of Canada for support under
grant MT-6254 (W.S.P.).
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H and 13C
NMR spectral data for new compounds described herein (25
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
5(S )-H yd r oxy-12,12-(t r im e t h yle n e d it h io)-6(E ),8(E ),
10(E),14(Z)-eicosa tr ien oic Acid (26). Methyl-5(S)-hydroxy-
12,12-(trimethylenedithio)-6(E),8(E),14(Z)-eicosatrienoate (25)
was hydrolyzed to the hydroxy acid 26 in 94% yield as
J O9614957