A convergent synthesis of (17R,5Z,8Z,11Z,14Z)-17-hydroxyeicosa-5,8,11,14-tetraenoic acid analogues and their tritiated derivatives

Article abstract of DOI:10.1016/j.tet.2003.08.043

17(R)-OH-AA and 14,15-dehydro-17(R)-OH-AA were synthesized from a common tetraacetylenic precursor and their azide derivatives were obtained in moderate yields via the corresponding p-toluenesulfonates. Since the azido group remained stable during tritiat

Full text of DOI:10.1016/j.tet.2003.08.043

Tetrahedron 59 (2003) 8091–8097
A convergent synthesis of
(17R,5Z,8Z,11Z,14Z)-17-hydroxyeicosa-5,8,11,14-tetraenoic acid
analogues and their tritiated derivatives
a
c
d
Igor V. Ivanov,a,b Stepan G. Romanov, Valery P. Shevchenko, Elena A. Rozhkova,
Mikhail A. Maslov,a Nataliya V. Groza,a Nikolai F. Myasoedov,c Hartmut Kuhnb
and Galina I. Myagkovaa
,
*
aM.V. Lomonosov State Academy of Fine Chemical Technology, Chemistry and Technology of Fine Organic Compounds,
Vernadskogo Pr. 86, Moscow 119571, Russian Federation
b
´
Institute of Biochemistry, University Clinics Charite, Humboldt University, Berlin D-10117, Germany
cInstitute of Molecular Genetics, Kurchatov Square 2, Moscow 123182, Russian Federation
dDepartment of Chemistry, Princeton University, Princeton, NJ 08544, USA
Received 21 June 2003; revised 23 July 2003; accepted 22 August 2003
Abstract—17(R)-OH-AA and 14,15-dehydro-17(R)-OH-AA were synthesized from a common tetraacetylenic precursor and their azide
derivatives were obtained in moderate yields via the corresponding p-toluenesulfonates. Since the azido group remained stable during
tritiation procedure on Lindlar’s catalyst in benzene, both 14,15-dehydro-17(S)-N3-AA and 14,15-dehydro-17(R)-OH-AA constitute
useful intermediates in the synthesis of radio-labelled 17(S)-N3-AA and 17(R)-OH-AA. In contrast, reduction of azide in methanol afforded
17(S)-NH2-AA with 95% yield.
q 2003 Published by Elsevier Ltd.
1. Introduction
tion leading to both, photosensitive azido and primary
amino derivatives.8 Azido fatty acid can be used as active
site affinity labels for fatty acid oxygenases, such as
lipoxygenase- and/or cyclooxygenase isoforms.
Lipoxygenases (LOXs) constitute a heterogeneous family of
lipid peroxydizing enzymes, which catalyze oxygenation of
polyunsaturated fatty acids to their corresponding hydro-
peroxy derivatives.1 LOXs were isolated from various
sources and some isoforms have been characterized
comprehensively with respect to their enzymatic and
molecular biological properties.2–4 Although the X-ray
structures of some LOX-isoforms have recently been
solved5,6 there are a number of open questions on the
structural biology of these enzymes. Studying the structural
basis for the specificity of the LOX we recently observed
that hydroxylated derivatives of arachidonic acid (v/v-4-
OH-AA) may constitute valuable tools to investigate the
mechanism of the oxygenation process. Preliminary experi-
ments suggested that a hydroxy group in close proximity to
the methyl terminus of the fatty acid chain may impact the
oxygenation characteristics of these substrates7 and we
observed a pronounced oxygen dependence of the reaction
rate even at normoxic oxygen tensions. The presence of an
OH-group in a hydrocarbon chain allows simple derivatiza-
An enantiospecific procedure has already been reported for
synthesis of 17-OH-AA.9 However, this method may not be
applicable for the preparation of tritiated compounds, unless
an inefficient isotope exchange procedure is employed. To
obtain preparative amounts of radio-labelled 17(R)-OH-AA
with high specific radioactivity a convergent and stereo-
selective synthesis is still required. This paper reports an
efficient method for the synthesis of (17R,5Z,8Z,11Z,14Z)-17-
hydroxyeicosa-5,8,11,14-tetraenoic acid (17(R)-OH-AA),
(17S,5Z,8Z,11Z,14Z)-17-azidoeicosa-5,8,11,14-tetraenoic
(17(S)-N3-AA), (17S,5Z,8Z,11Z,14Z)-17-aminoeicosa-
5,8,11,14-tetraenoic acids (17(S)-NH2-AA) and their tritiated
analogues. The procedure is illustrated for 17(R)-OH-AA
(Scheme 1), but it works equally well for the S-enantiomer.
2. Results and discussion
The most crucial point of our synthetic strategy was to
minimize number of manipulations with radioactive
material. Thus, radioactive labeling should be performed
Keywords: coupling reactions; eicosanoids; azides; labelling.
*
Corresponding author. Tel.: þ7-95-434-8544; fax: þ7-95-434-86-78;
e-mail: ivanov.mitht@mail.ru
0040–4020/$ - see front matter q 2003 Published by Elsevier Ltd.
doi:10.1016/j.tet.2003.08.043

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I. V. Ivanov et al. / Tetrahedron 59 (2003) 8091–8097
Scheme 1. (a) NaI, acetone, 658C, 78 h; (b) CuI, EtMgBr, THF-HMPA, 2458C, 65 h; (c) BzCl, Py, benzene, rt, 10 h; (d) n-Bu4NF, THF, rt, 2 h; (e) 6, CuI, NaI,
K2CO3, DMF, rt; (f) CBr4, PPh3, CH2Cl2, rt, 1 h; (g) 9, CuI, NaI, K2CO3, DMF, rt then HPLC; (h) H2 or tritium/Lindlar’s catalyst, quinoline, benzene, 108C; (i)
NaOH, MeOH–H2O, rt; (j)TsCl, CH2Cl2, Py, rt, 24 h; (k) NaN3, DMF, 75–808C, 2.5 h; (l) T2/Lindlar’s catalyst, benzene; (m) H2/Lindlar’s catalyst, MeOH.
in the synthesis as late as possible. Since acetylenic
functions permit the incorporation of tritium (partial
tritiation) to yield labeled compounds with high specific
activity, the acetylenic approach was selected as method
of choice. To construct the tetraacetylenic precursor 10
(Scheme 1) we elected three building blocks: (R)-4-
(benzoyloxy)hept-1-yn (5), 7-bromo-2,5-heptadiyne-1-ol
(6)7 and methyl 5-hexynoate (9). Heptyne 5 was obtained
as indicated in Scheme 1 starting from (S)-epichlorohydrin
(1). Oxirane ring opening was achieved by the Yamaguchi
method as described previously for the R-enantiomer10
affording (S)-1-chloro-5-(trimethylsilyl)pent-4-yne-2-ol (2).
Conversion of the chloride 2 to corresponding iodide 3 and
chain elongation using Et2CuMgBr resulted in (R)-1-
(trimethylsilyl)hept-1-yne-4-ol (3). Protection of OH-group
followed by desilylation (TBAF, THF) afforded 5 in an
overall yield of 42% for four-step procedure. Copper(I)
mediated cross-coupling11 of methyl 5-hexynoate (9) and
propargyl bromide 8 obtained from acetylene 5 and bromo
alcohol 6 resulted in methyl (R)-17-(benzoyloxy)eicosa-
5,8,11,14-tetraynoate (10) in 81% yield. Stereospecific
hydrogenation of the skipped triple bonds of 10 using
Lindlar’s catalyst (catalyst/substrate ratio, 1.2:1) afforded
both methyl (R,5Z,8Z,11Z,14Z)-17-(benzoyloxy)eicosa-
5,8,11,14-tetraenoate (12) and its 14,15-dehydro analogue
13. Recently, we have shown that the close neighborhood of
a benzoate residue to an acetylene moiety may impact its
hydrogenation efficiency,7 which may be due to a steric
hindrance of the accessibility for the Pd-catalyst. For the
tetraacetylene 10 this also seems to be the case. Increase in
the catalyst concentration (catalyst/substrate ratio, 2:1)
diminished the formation of 13 to trace amounts. Com-
pounds 12 and 13 were separated by preparative reverse
phase HPLC (MeOH/H2O, 95:5). The structural identifi-
cation of 13 as methyl (R,5Z,8Z,11Z)-17-(benzoyloxy)-
eicosa-5,8,11-trien-14-ynoate was based on both mass
spectral data and NMR spectroscopy. Two dimensional
1H/13C heteronuclear multiple-bond correlation (HMBC)
spectra were used to confirm the position of the triple bond
(Fig. 1). We observed correlation between neighboring
atoms (C-15/H2-16) and remote ones (between others C-15/
H-17, C-15/H2-13). Moreover, three separate cross peaks
indicated the additional correlations: C-14/H2-13, C-14/H-12
and C-14/H2-16. Simultaneous deprotection of both,

I. V. Ivanov et al. / Tetrahedron 59 (2003) 8091–8097
8093
ation of 16 and 20 in methanol14,15 afforded corresponding
amino acids 17 and 21 with good yields.
3. Conclusion
In conclusion, a novel convergent synthetic procedure
is reported for enantio-selective synthesis of 17-OH-AA,
17-NH2-AA and 17-N3-AA. This method can be used to
prepare the corresponding tritiated derivatives, which
constitute valuable tools for mechanistic studies of eicosa-
noid biosynthesis.
4. Experimental
4.1. General
1H and 13C NMR spectra were recorded either on a Brucker
MSL 200, Brucker MSL 300 or Brucker MSL 500
spectrophotometers in CDCl3 as solvent. Chemical shifts
are referenced to tetramethylsilane as an internal standard
Figure 1. Fragment of 2D 1H/13C heteronuclear multiple-bond correlation
(HMBC) spectra for compound 13.
1
for H NMR or to the deuterium lock signal of CDCl3
(d 13C¼77.19 ppm). IR spectra were recorded on Shimadzu
IR-435. HPLC analysis was carried out on a Shimadzu
LC-10Avp liquid chromatograph connected to SPD-10Advp
UV detector. RP-HPLC analysis was performed on a
Nucleosil C18-column; 250£4 mm, 5 mm particle size
(Machery-Nagel, Du¨ren, Germany) with different solvent
systems: MeOH/H2O (95:5, by vol.) and a flow rate of
1 mL/min were used for analysis of compound 12, 13.
MeOH/H2O/Ac (85:15:0.1, by vol.) and a flow rate of 1 mL/
min were used for other compounds. Preparative HPLC was
carried out on a Lichrospher 100 RP18 column;
250£22.5 mm, 10 mm particle size (Knauer, Berlin,
Germany) with MeOH/H2O/AcOH (95:5, by vol.) and a
flow rate of 10 mL/min. For EIMS analysis a Shimadzu
QP-2000 system was used. High resolution mass spectra
were recorded either on a MAT 711 mass spectrometer
(Finigan MAT) or VG Autospec instrument. Column
chromatography was carried out on Silica Gel 60 (Merck,
Darmstadt, Germany particle size ranging from 70–230
mesh). For thin-layer chromatography we employed
precasted Silica Gel 60 F254 sheets (Merck, Darmstadt,
Germany). THF was freshly distilled from sodium/benzo-
phenone ketyl, and HMPA was dried over CaH2. All
solvents and reagents used were of extra pure grade and
purchased from Merck, Aldrich or Across (Germany). Prior
to use all glassware and syringes were dried at 1408C
overnight and all reactions were carried out under
atmosphere of dry argon.
carboxy and hydroxy groups of 12 and 13, using NaOH in
MeOH–H2O solution proceeded well to give the free acids
14 and 15 in 86 and 75% yields, respectively. Tritiation of
10 was performed in benzene with a method used before12
with slight modifications. Subsequent deprotection afforded
11 with high specific activity (175–180 Ci/mmol) and a
radiochemical purity of more than 98%.
Conversion of the hydroxy compounds 14 and 15 to the
corresponding p-toluenesulfonates followed by tosylate
substitution with azide in DMF13 afforded the (S)-azido
acids 16 and 18 in moderate yields, 42 and 37%,
respectively. In contrast to the tosylate pathway, the
synthesis via bromides retained the R-configuration of the
final product. However, substitution of bromide during
the reaction with NaN3 was incomplete and we recovered
about 30% of the starting material. The corresponding
(R)-azides were prepared from 17(S)-OH-AA, itself
obtained by exactly the same procedure starting with
commercially available (R)-epichlorohydrin. Mitsunobu
inversion of 17(R)-OH-AA9 may also serve as alternative
way to obtain the S-isomer.
The high specific activity of [5,6,8,9,11,12,14,15-3H8]-
17(R)-OH-AA (11), which is required for biological
applications, complicates its extensive derivatization in
the preparative scale. Hence, the monoacetylenes 15 and 18
were used as key intermediates for partial tritiation of the
acetylene moiety at the final step. To optimize the tritiation
procedure we first performed model experiments on
hydrogenation of 15 and 18 and the analytical data of
hydrogenated compound 15 appeared to be identical with
that of obtained previously for 17(R)-OH-AA (14).
Importantly, hydrogenation of azide 18 using Lindlar’s
catalyst in dry benzene (catalyst/substrate ratio, 2:1)
proceeded without sizeable azide reduction. Tritiation was
carried out by the same procedure as hydrogenation to yield
[14,15-3H]-17(R)-OH-AA (19) and [14,15-3H]-17(S)-N3-
AA (20) with a high specific activities of 45 Ci/mmol and
40 Ci/mmol, respectively. In contrast, catalytic hydrogen-
4.1.1. (S)-1-Iodo-5-(trimethylsilyl)pent-4-yne-2-ol (3). A
mixture of 2 (2.54 g, 13.31 mmol) and NaI (5.99 g,
39.95 mmol) in dry acetone (15 mL) was stirred at 658C
for 78 h. After acetone was evaporated, Et2O (60 mL) was
added to the residue. The resulting mixture was washed with
H2O (2£50 mL) and the organic layer was dried over
Na2SO4. After evaporation under reduced pressure, the
crude residue was purified on silica gel (hexane/Et2O, 3:1)
to yield 3 as a colorless oil, yield: 3.02 g (80%).
[a]2D0¼þ18.3 (c 0.7, acetone). TLC: Rf¼0.42 (hexane/
Et2O, 1:1). IR (neat)/cm21: 3600–3200 (OH), 2170 (CuC),

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I. V. Ivanov et al. / Tetrahedron 59 (2003) 8091–8097
1
1273 (C–O), 1245, 839 (Si(CH3)3), 590 (C–I). H NMR
(300 MHz, CDCl3): d 3.71 (m, 1H, 2-CH), 3.43 (dd, 1H,
J¼3.7, 10.2 Hz) and 3.32 (dd, 1H, J¼6.1, 10.2 Hz, 1-CH2),
2.61 (dd, 1H, J¼5.8, 16.9 Hz) and 3.52 (dd, 1H, J¼6.2,
16.9 Hz, 3-CH2), 2.24 (s,1H, OH), 0.09 (m, 9H, (CH3)3Si).
13C NMR (50 MHz, CDCl3): d 101.61, 88.49, 69.89, 48.36,
12.67, 0.31 (3C). Anal. calcd for C8H15IOSi: C, 34.05; H,
5.36. Found: C, 34.14; H, 5.49.
18.77, 14.77. Anal. calcd for C14H16O2: C, 77.75; H, 7.46.
Found: C, 77.99; H, 7.34.
4.1.4. (R)-11-(Benzoyloxy)tetradeca-2,5,8-triyne-1-ol (7).
To a suspension of previously dried salts CuI (1.12 g,
5.90 mmol), NaI (0.885 g, 5.90 mmol) and K2CO3 (0.610 g,
4.42 mmol) in DMF (25 mL) were added bromide 6
(0.548 g, 2.93 mmol) and acetylene 5 (0.637 g, 2.95 mmol)
under argon atmosphere. After stirring overnight at rt, the
mixture was quenched with satd aq. NH4Cl (100 mL) and
the products were extracted with Et2O (4£100 mL). The
combined organic extracts were washed with satd aq. NaCl
(2£50 mL), dried over Na2SO4 and the solvent was
evaporated under reduced pressure. The residue was
purified by silica gel flash chromatography (hexane/Et2O,
1:2) to afford 0.680 g (72%) of pure acetylene alcohol 7.
TLC: Rf¼0.30 (hexane/Et2O, 1:2). IR (neat)/cm21: 3200–
3600 (OH), 2240, 2170 (CuC), 1725 (CvO), 1115 (C–O),
4.1.2. (R)-1-(Trimethylsilyl)hept-1-yne-4-ol (4). To a
suspension of CuI (3.39 g, 17.82 mmol) in THF (80 mL)
and HMPA (10 mL), which was cooled to 2108C, a solution
of EtMgBr (33.30 mL, 35.64 mmol, 1.07 M) in THF was
added with a syringe. The mixture was stirred for 45 min at
25–08C till homogenous dark solution formed. After the
mixture was called to 2508C, a solution of iodide 3 (2.01 g,
7.13 mmol) in THF (10 mL) was added. The resulting
mixture was kept with a stirring at 2458C for 1.5 h, then
was quenched with satd aq. NH4Cl, acidified with HCl
(1 M) to pH 5.0 and the organic products were extracted
with Et2O (3£60 mL). The combined ethereal extracts were
washed with satd aq. NaCl, dried over Na2SO4 and then
concentrated under vacuum. Purification by silica gel
chromatography (hexane/Et2O 2:1) gave 1.15 g (88%) of
pure 4. [a]2D0¼þ21.8 (c 0.9, acetone). TLC: Rf¼0.39
(hexane/Et2O, 1:1). IR (neat)/cm21: 3600–3200 (OH),
1
709 (Ph). H NMR (200 MHz, CDCl3): d 8.05 (m, 2H,
o-Bz), 7.49 (m, 3H, (mþp)-Bz), 5.15 (m, 1H, 11-CH), 4.17
(m, 2H, 1-CH2), 3.09 (m, 4H, 4- and 7-CH2), 2.52 (m, 2H,
10-CH2), 1.75 (m, 2H, 12-CH2), 1.37 (m, 2H, 13-CH2), 0.88
(t, 3H, J¼6.8 Hz, CH3). 13C NMR (50 MHz, CDCl3): d
166.29, 132.91, 130.82, 129.79 (2C), 128.43 (2C), 80.06,
79.07, 76.39, 76.28, 75.25, 74.11, 72.78, 51.21, 35.63,
24.56, 18.65, 13.98, 9.90 (2C). Anal. calcd for C21H22O3: C,
78.23; H, 6.88. Found: C, 78.01; H, 6.76.
1
2240 (CuC), 1245, 840 (Si(CH3)3). H NMR (300 MHz,
CDCl3): d 3.74 (m, 1H, 4-CH), 2.45 (dd, 1H, J¼4.7,
16.9 Hz) and 2.30 (dd, 1H, J¼7.1, 16.9 Hz, 3-CH2), 1.92 (s,
1H, OH), 1.30–1.49 (m, 4H, 5- and 6-CH2), 0.92 (t, 3H,
J¼6.8 Hz, CH3), 0.1 (m, 9H, (CH3)3Si). 13C NMR
(50 MHz, CDCl3) d: 103.66, 87.78, 69.92, 38.72, 29.20,
19.02 14.17, 0.31 (3C). Anal. calcd for C10H20OSi C, 65.15;
H, 10.94. Found: C, 65.34; H, 11.15.
4.1.5. (R)-4-(Benzoyloxy)-14-bromotetradeca-6,9,12-
triyne (8). To
a solution of alcohol 7 (600 mg,
1.86 mmol) and CBr4 (925 mg, 2.79 mmol) in CH2Cl2
(30 mL) was added a solution of PPh3 (732 mg, 2.79 mmol)
in CH2Cl2 (15 mL) at 108C. The reaction mixture was
stirred for 1 h at rt, then quenched with MeOH (2 mL) and
volatile components were removed under vacuum. Column
chromatography on silica gel (hexane/Et2O, 1:1) afforded
pure 8. Yield: 616 mg (86%). TLC: Rf¼0.69 (hexane/Et2O,
1:2). IR (neat)/cm21: 2240, 2170 (CuC), 1725 (CvO),
4.1.3. (R)-4-(Benzoyloxy)hept-1-yn (5). A solution of BzCl
(0.762 mL, 6.56 mmol) in benzene (15 mL) was added to a
solution of 4 (0.805 g, 4.37 mmol) in benzene (30 mL) and
pyridine (20 mL). The mixture was stirred overnight at rt,
then acidified with H2SO4 (1 M, 50 mL). Reaction products
were extracted with Et2O (2£50 mL) and the extracts were
concentrated under reduced pressure. The residue was
filtrated through a silica gel column (hexane/Et2O, 4:1). The
product which showed an Rf¼0.60 in silica gel thin layer
chromatography (hexane/Et2O, 1:1) was dissolved in THF
(50 mL) and the silyl group was removed by addition of
n-Bu4NF (2.37 g, 7.53 mmol) within 2 h at rt. The mixture
was quenched with H2O (100 mL), organic layer was
separated and lipophilic products were extracted from the
water phase with Et2O (2£40 mL). The combined organic
layers were washed with satd aq. NaCl (70 mL), dried over
Na2SO4 and concentrated under vacuum. Silica gel
chromatography using gradient elution with hexane/Et2O
(from 1 to 10% Et2O) gave pure 5 as a colourless oil in a
yield 0.745 g (79%). [a]D20¼þ29.2 (c 0.8, acetone). TLC:
Rf¼0.55 (hexane/Et2O, 1:1). IR (neat)/cm21: 3272 (CuC),
1
1115 (C–O), 710 (Ph), 598 (C-Br). H NMR (200 MHz,
CDCl3): d 8.05 (m, 2H, o-Bz), 7.49 (m, 3H, (mþp)-Bz),
5.12 (m, 1H, 4-CH), 3.89 (m, 2H, 14-CH2), 3.18 (m, 2H,
8-CH2), 3.10 (m, 2H, 11-CH2), 2.54 (m, 2H, 5-CH2), 1.75
(m, 2H, 3-CH2), 1.41 (m, 2H, 2-CH2), 0.92 (t, 3H,
J¼6.8 Hz, CH3). 13C NMR (50 MHz, CDCl3): d 166.29,
132.91, 130.85, 129.78 (2C), 128.43 (2C), 81.49, 79.11,
76.13 (2C), 75.76, 75.54, 72.71, 35.63, 24.56, 18.68, 14.56,
14.01, 9.89 (2C). Anal. calcd for C21H21O2Br: C, 65.46; H,
5.49. Found: C, 65.26; H, 5.35.
4.1.6. Methyl (R)-17-(benzoyloxy)eicosa-5,8,11,14-tetra-
ynoate (10). In an Ar filled previously dried round-
bottomed flask equipped with magnetic stirrer anhydrous
K2CO3 (261 mg, 1.89 mmol), NaI (378 mg, 2.52 mmol) and
CuI (480 mg, 2.52 mmol) were suspended in DMF (15 mL).
Methyl 5-hexynoate (9) (159 mg, 1.26 mmol) was added at
once to the suspension followed by bromide 8 (490 mg,
1.27 mmol). The reaction mixture was vigorously stirred
overnight at rt, then quenched with satd aq. NH4Cl
(200 mL). The lipophilic products were extracted with
Et2O (4£100 mL). The combined organic extracts were
washed with satd aq. NaCl (2£150 mL). After drying over
Na2SO4 the ethereal solution was concentrated in vacuum.
The crude residue was purified by silica gel flash
1
1725 (CvO), 1273 (C–O), 710 (Ph). H NMR (200 MHz,
CDCl3): d 8.09 (m, 2H, o-Bz), 7.54 (m, 1H, p-Bz), 7.40 (m,
2H, m-Bz), 5.19 (m, 1H, 4-CH), 2.59 (m, 2H, 3-CH2), 1.89
(t, 1H, J¼2.7 Hz, 1-CH), 1.75–1.82 (m, 2H, 5-CH2), 1.35–
1.45 (m, 2H, 6-CH2), 0.89 (t, 3H, J¼6.8 Hz, CH3). 13C
NMR (50 MHz, CDCl3): d 166.26, 133.07, 132.06, 129.88
(2C), 128.55, 127.30, 79.96, 72.39, 70.63, 35.51, 24.36,

I. V. Ivanov et al. / Tetrahedron 59 (2003) 8091–8097
8095
chromatography (hexane/Et2O, 3:1) under Ar to give pure
10 as yellow oil. Yield of 10: 439 mg (81%). TLC: Rf¼0.42
(hexane/Et2O, 1:1). IR (neat)/cm21: 2240 (CuC), 1740,
tetraenoic acid (14). An aqueous solution (15 mL) of
NaOH (164 mg, 4.10 mmol) was added to a solution of 12
(180 mg, 0.41 mmol) in methanol (40 mL) under argon
atmosphere. The resulting mixture was stirred for 30 h at rt.
After the reaction was completed, methanol was removed
by evaporation under reduced pressure and the residue was
carefully acidified to pH 4.0 using HCl (1 M). The lipophilic
products were extracted with Et2O (3£40 mL), combined
organic extracts were dried over Na2SO4 and concentrated
in vacuum. The crude residue was purified by silica gel
column chromatography (hexane/Et2O, 1:3) to give 14 in
113 mg (86%). [a]2D0¼þ4.3 (c 0.6, acetone). TLC: Rf¼0.31
(hexane/Et2O, 1:3). Analytical RP-HPLC: Rt¼6.07 min. 1H
NMR (300 MHz, CDCl3): d 5.50–5.60 (m, 2H, CH¼CH),
5.29–5.45 (m, 6H, CH¼CH),3.65 (m, 1H, 17-CH), 2.82 (m,
6H, 7-, 10- and 13-CH2), 2.33 (m, 2H, J¼7.11 Hz, 2-CH2),
2.24 (m, 2H, 16-CH2), 2.10 (m, 2H, 4-CH2), 1.68 (m,
2H, 3-CH2), 1.41 (m, 4H, 18- and 19-CH2), 0.89 (t, 3H,
J¼6.8 Hz, CH3). 13C NMR (75 MHz, CDCl3): d 178.03,
131.55, 129.16, 128.99, 128.48, 128.39, 128.11, 128.05,
125.70, 71.60, 38.94, 35.40, 33.30, 26.58, 25.97, 25.87,
25.84, 24.65, 19.08, 14.23. EIMS m/z (%): 320 (6.95) [M]þ,
302 (15.94) [M2H2O]þ, 259 (8.08) [M2H2O–C3H9]þ.
FABMS m/z: 319 [M2H]2. EIHRMS calcd for C20H32O3
[M]þ: 320.2351. Found: 320.2364.
1
1725 (CvO), 1115 (C–O), 710 (Ph). H NMR (200 MHz,
CDCl3): d 8.01 (m, 2H, o-Bz), 7.48 (m, 3H, (mþp)-Bz),
5.15 (m, 1H, 17-CH), 3.65 (s, 3H, OCH3), 3.19 (m, 6H, 7-,
10- and 13-CH2), 2.65 (dt, 2H, J¼6.0, 2.1 Hz, 16-CH2), 2.40
(t, 2H, J¼7.2 Hz, 2-CH2), 2.21 (tt, 2H, J¼6.8, 1.7 Hz,
4-CH2), 1.75 (m, 4H, 3- and 18-CH2), 1.41 (m, 2H, 19-CH2),
0.90 (t, 3H, J¼6.8 Hz, CH3). 13C NMR (50 MHz, CDCl3): d
173.42, 166.17, 132.77, 130.75, 129.95 (2C), 128.25 (2C),
81.28, 79.18 (2C), 77.47, 76.01 (2C), 75.51, 73.97, 72.36,
51.48, 35.04, 32.95, 24.02, 23.91, 18.26, 14.07, 13.95, 9.83
(3C).
4.1.7. Methyl (17R,5Z,8Z,11Z,14Z)-17-(benzoyloxy)-
eicosa-5,8,11,14-tetraenoate (12) and methyl
(17R,5Z,8Z,11Z)-17-(benzoyloxy)eicosa-5,8,11-trien-14-
ynoate (13). The suspension of Lindlar’s catalyst (500 mg)
in dry benzene (15 mL) was saturated with H2 in a 100-mL
Erlenmeyer flask at rt and cooled to 108C. Then a solution of
10 (400 mg, 0.93 mmol) in benzene (25 mL) and quinoline
(0.40 mL) were added to the catalyst under a stream of Ar.
The Ar was exchanged with H2 and the mixture was stirred
for 1 h at 108C. After the absorption of hydrogen was
completed the solution was filtered, washed with HCl (2 M,
2£30 mL) and the solvent was evaporated. The crude
residue was filtered over silica gel (hexan/Et2O, 2:1) and the
products formed were separated by preparative RP-HPLC
(solvent system MeOH/H2O, 95:5) to yield 192 mg (47%)
of pure tetraenoate 12 and 113 mg (27%) of 14,15-
dehydroanalogue 13. Data for 12: [a]2D0¼þ17.2 (c 0.5,
acetone). TLC: Rf¼0.53 (hexane/Et2O, 1:1). Analytical
4.1.9. (17R,5Z,8Z,11Z)-17-Hydroxyeicosa-5,8,11-trien-
14-ynoic acid (15). Free acid 15 was prepared by analogues
procedure as described for 14 from methyl (R,5Z,8Z,11Z)-
17-(benzoyloxy)eicosa-5,8,11-trien-14-ynoate (13) (209 mg,
0.25 mmol) and LiOH (105 mg, 2.50 mmol). Yield of pure
15: 59.6 mg (75%). [a]2D0¼þ5.0 (c 0.6, acetone). TLC:
Rf¼0.31 (hexane/Et2O, 1:3). Analytical RP-HPLC:
1
1
RP-HPLC: Rt¼6.58 min. H NMR (300 MHz, CDCl3): d
Rt¼4.98 min. H NMR (300 MHz, CDCl3): d 5.30–5.45
8.01 (m, 2H, o-Bz), 7.52 (m, 1H, p-Bz), 7.40 (m, 2H, m-Bz),
5.42–5.46 (m, 2H, CH¼CH), 5.29–5.39 (m, 6H, CH¼CH),
5.15 (m, 1H, 17-CH), 3.61 (s, 3H, OCH3), 2.79 (m, 6H, 7-,
10- and 13-CH2), 2.46 (m, 2H, 16-CH2), 2.30 (t, 2H,
J¼7.21 Hz, 2-CH2), 2.07 (m, 2H, 4-CH2), 1.62–1.70 (m,
4H, 3- and 18-CH2), 1.35 (m, 2H, 19-CH2), 0.89 (t, 3H,
J¼6.81 Hz, CH3). 13C NMR (50 MHz, CDCl3): d 173.22,
166.41, 132.92, 131.07 (2C), 130.71, 129.73 (2C), 129.13,
128.99, 128.46 (2C), 128.40, 128.24, 128.17, 124.95 (2C),
74.40, 51.65, 36.08, 32.27, 26.71, 25.92, 25.78 (2C), 24.94,
18.89, 14.16. EIMS m/z (%): 316 (2.74) [M-BzCOO]þ.
Anal. calcd for C28H38O4: C, 76.68; H, 8.73. Found: C,
76.44; H, 8.91. Data for 13: [a]2D0¼þ26.0 (c 0.5, acetone).
TLC: Rf¼0.53 (hexane/Et2O, 1:1). Analytical RP-HPLC:
Rt¼5.91 min. 1H NMR (500 MHz, CDCl3): d 8.02 (m, 2H,
o-Bz), 7.52 (m, 1H, p-Bz), 7.41 (m, 2H, m-Bz), 5.30–5.45
(m, 6H, CH¼CH), 5.14 (m, 1H, 17-CH), 3.64 (s, 3H,
OCH3), 2.89 (m, 2H, 13-CH2), 2.78 (m, 6H, 7-and 10-CH2),
2.53 (m, 2H, 16-CH2), 2.29 (t, 2H, J¼7.21 Hz, 2-CH2), 2.07
(m, 2H, 4-CH2), 1.76 (m, 2H, 18-CH2), 1.69 (m, 2H, 3-CH2),
1.41 (m, 2H, 19-CH2), 0.93 (t, 3H, J¼6.81 Hz, CH3). 13C
NMR (125 MHz, CDCl3): d 174.18, 166.27, 132.97, 130.77,
129.81 (2C), 129.43, 129.18, 128.92, 128.70, 128.46 (2C),
127.73, 125.30, 80.58, 75.59, 72.92, 51.62, 35.55, 33.61,
26.73, 25.77, 25.66, 24.95, 24.65, 18.72, 17.37, 14.10.
EIMS m/z (%): 314 (1.35) [M2BzCOO]þ. Anal. calcd for
C28H36O4: C, 77.03; H, 8.31. Found: C, 76.84; H, 8.43.
(m, 6H, CH¼CH), 3.70 (m, 1H, 17-CH), 2.94 (m, 2H, 10-
CH2), 2.81 (m, 4H, 7- and 13-CH2), 2.34 (m, 2H, J¼7.11 Hz,
2-CH2), 2.22–2.45 (m, 2H, 16-CH2), 2.10 (m, 2H, 4-CH2),
1.68 (m, 2H, 3-CH2), 1.41 (m, 4H, 18- and 19-CH2), 0.89 (t,
3H, J¼6.8 Hz, CH3). 13C NMR (75 MHz, CDCl3): d 177.94,
129.77, 129.03 (2C), 128.72, 127.63, 125.07, 81.35, 76.34,
70.21, 38.51, 33.22, 27.86, 26.63, 25.82, 25.72, 24.67,
19.02, 17.36, 14.18. ESIHRMS calcd for C20H30O3Na
341.2093 [MþNa]þ. Found 341.2079.
4.1.10. [5,6,8,9,11,12,14,15-3H]-(17R,5Z,8Z,11Z,14Z)-17-
Hydroxyeicosa-5,8,11,14-tetraenoic acid (11). A solution
of 10 (5 mg, 0.012 mmol) in benzene (1 mL), Lindlar’s
catalyst (10 mg) and quinoline (0.10 mL) were placed in the
reaction ampule and the mixture was frozen with liquid
nitrogen. After evacuation the ampule was filled with
gaseous tritium up to a pressure 400 hPa. The reaction
mixture was stirred for 1 h at rt and then frozen again.
Unreacted tritium gas was removed by applying vacuum.
The catalyst was removed by filtration over silica gel and
washed with benzene (3£1 mL). The combined filtrates
were concentrated under reduced pressure and the residue
was evaporated two times with methanol (2£2 mL) to
remove labile tritium. The crude product was purified by
preparative HPLC followed by deprotection according to
procedure described for preparation of compound 14. Yield
of 11: 1.16 Ci (57%) with specific radioactivity 175 Ci/
mmol. The radiochemical purity of [3H-8]-11 was found to
be 95%. Analytical RP-HPLC: Rt¼6.07 min.
4.1.8. (17R,5Z,8Z,11Z,14Z)-17-Hydroxyeicosa-5,8,11,14-

8096
I. V. Ivanov et al. / Tetrahedron 59 (2003) 8091–8097
4.1.11. (17S,5Z,8Z,11Z,14Z)-17-Azidoeicosa-5,8,11,14-
tetraenoic acid (16). A mixture of 17-HETE (14) (42 mg,
0.13 mmol), p-toluenesulphonyl chloride (95 mg, 0.5 mmol)
in CH2Cl2 (2 mL) and anhydrous pyridine (3 mL) was
stirred for 24 h at rt and the reaction was monitored by
HPLC using MeOH/H2O/AcOH (85:15:0.1, by vol.) as
solvent system. After the reaction was completed the
mixture was poured into diluted HCl (1 M, 50 mL), and
extracted with Et2O (3£40 mL). Combined organic extracts
were additionally washed with HCl (1 M, 50 mL), water
(50 mL) and dried over Na2SO4. The crude residue was
purified by silical gel column chromatography (hexane/
Et2O, 1:1) to give 37 mg of the product with Rt¼7.98 min,
as it was shown by RP-HPLC. The latter was then stirred
with sodium azide (20 mg, 0.31 mmol) in DMF (3 mL) for
2.5 h at 75–808C. The reaction mixture was quenched with
satd aq. NH4Cl (20 mL) and the products were extracted
with Et2O (3£30 mL). The combined organic extracts were
washed with satd aq. NaCl (20 mL), dried over Na2SO4 and
the solvent was evaporated under reduced pressure. The
residue was purified by silica gel flash chromatography
(hexane/Et2O, 1:1) to afford pure 16 with overall yield
19 mg (42%). [a]2D0¼243.6 (c 0.5, acetone). Analytical
was stirred additionally for 1 h in tritium atmosphere.
Unreacted tritium gas was removed by applying vacuum.
The catalyst was filtered off over silica gel and washed with
benzene (3£1 mL). The combined filtrates were concen-
trated under reduced pressure and the residue was
evaporated two times with methanol (2£2 mL) to remove
labile tritium. The crude products were purified by HPLC to
give either 630 mCi (90%) of 19 (specific activity 45 Ci/
mmol) or 490 mCi (85%) of 20 (specific activity 40 Ci/
mmol). Analytical RP-HPLC for 19: Rt¼6.07 min. Analyti-
cal RP-HPLC for 20: Rt¼13.5 min.
4.2.1. (17S,5Z,8Z,11Z,14Z)-17-Aminoeicosa-5,8,11,14-
tetraenoic acid (17). In a three-neck flask was placed a
solution of 16 (5.0 mg, 0.014 mmol) in methanol (3 mL).
The flask was purged with argon and Lindlar catalyst
(10 mg) was added to the solution. The argon was
exchanged with hydrogen and the mixture was stirred for
1 h at rt. After the reaction was completed the catalyst was
removed by filration over Celite and washed with methanol
(3 mL). Combined filtrates were concentrated in vacuum.
Yield of 17: 4.4 mg (95%). [a]2D0¼245.7 (c 0.7, acetone).
1
TLC: Rf¼0.17 (CHCl3/MeOH, 4:1 with 0.1% NH3). H
1
RP-HPLC: Rt¼13.5 min. H NMR (300 MHz, CDCl3): d
NMR (300 MHz, CD3OD): d 5.30–5.50 (m, 8H, CH¼CH),
3.30 (m, 1H, 17-CH), 2.81 (m, 6H, 7-, 10- and 13-CH2),
2.30 (m, 4H, 2- and 16-CH2), 2.09 (m, 2H, 4-CH2), 1.65 (m,
2H, 3-CH2), 1.41 (m, 4H, 18- and 19-CH2), 0.89 (t, 3H,
J¼6.8 Hz, CH3). 13C NMR (75 MHz, CDCl3): d 132.20,
129.61, 128.78, 128.61, 128.54, 127.78, 127.46, 124.51,
51.12, 35.85, 35.73, 27.30, 26.05 (4C), 25.83, 19.00, 14.17.
ESIMS m/z (%): 319 (100) [MþH]þ, 341 (20) [MþNa]þ.
5.30–5.50 (m, 8H, CH¼CH), 3.30 (m, 1H, 17-CH), 2.81
(m, 6H, 7-, 10- and 13-CH2), 2.30 (m, 4H, 2- and 16-CH2),
2.09 (m, 2H, 4-CH2), 1.65 (m, 2H, 3-CH2), 1.41 (m, 4H, 18-
and 19-CH2), 0.89 (t, 3H, J¼6.8 Hz, CH3). 13C NMR
(75 MHz, CDCl3): d 178.90, 131.06, 129.22, 128.92,
128.59, 128.49, 128.20, 127.98, 125.32, 62.73, 36.30,
32.50, 26.78, 25.97, 25.81 (3C), 25.02, 19.57, 14.03.
ESIHRMS calcd for C20H31N3O2Na [MþNa]þ: 368.2314.
Found: 368.2316.
4.2.2. [14,15-3H]-(17S,5Z,8Z,11Z,14Z)-17-Aminoeicosa-
5,8,11,14-tetraenoic acid (21). Radio-labelled acid 21
was prepared by entyrely analogous procedure as described
for 17 from 90 mCi (specific radioactivity 40 Ci/mmol).
Yield of 21 was 90%, while specific radioactivity remained
unchanged.
4.1.12. (17S,5Z,8Z,11Z)-17-Azidoeicosa-5,8,11-trien-14-
ynoic acid (18). Azido acid 18 was prepared from acid 15
(40 mg, 0.126 mmol) by an analogous procedure as
described for tetraene 16. Yield of pure 18: 16 mg (37%).
[a]2D0¼228.5 (c 0.4, acetone). Analytical RP-HPLC:
1
Rt¼9.64 min. H NMR (300 MHz, CDCl3): d 5.30–5.50
(m, 6H, CH¼CH), 3.38 (m, 1H, 17-CH), 2.95 (m, 2H) and
2.79(m, 4H, 7-, 10- and13-CH2), 2.35(m, 4H, 2-and 16-CH2),
2.09 (m, 2H, 4-CH2), 1.70 (m, 2H, 3-CH2), 1.41 (m, 4H, 18-
and 19-CH2), 0.92 (t, 3H, J¼6.8 Hz, CH3). 13C NMR
(75 MHz, CDCl3): d 178.87, 129.64, 129.10, 129.00,
128.69, 127.71, 124.99, 81.18, 75.84, 61.31, 35.83, 33.25,
26.62, 25.79, 25.69, 25.34, 24.67, 19.41, 17.39, 13.97.
ESIHRMS calcd for C20H29N3O2Na [MþNa]þ: 366.2157.
Found: 366.2160.
Acknowledgements
The financial support of the Russian Ministry of Industry,
Science and Technology (Grant MK-2503.2003.03), the
Russian Ministry of Education and Alexander von
Humboldt Foundation is acknowledged.
4.2. General procedure for tritiation of (17R,5Z,8Z,11Z)-
17-hydroxyeicosa-5,8,11-trien-14-ynoic acid (15) and
(17R,5Z,8Z,11Z)-17-azidoeicosa-5,8,11-trien-14-ynoic
acid (18)
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