Synthesis of tritium labelled 3(R)-HETE and 3(R),18(R/S)-DiHETE through a common synthetic route

Article abstract of DOI:10.1002/jlcr.791

An efficient procedure for the synthesis of 3-hydroxyoxylipins labelled with tritium on all double bond positions is reported. The synthetic scheme involves a joint route for the formation of tetraacetylenic precursors followed by stereoselective reductio

Full text of DOI:10.1002/jlcr.791

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 11–17.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.791
Research Article
Synthesis of tritium labelled 3(R)-HETE and
3(R),18(R/S)-DiHETE through a common
synthetic route
Natalya V. Groza1, Igor V. Ivanov1, Stepan G. Romanov1,
Valery P. Shevchenko2, Nikolai F. Myasoedov2, Santosh Nigam3,* and
Galina I. Myagkova1
1 M.V. Lomonosov State Academy of Fine Chemical Technology, Pr. Vernadskogo 86,
119571 Moscow, Russian Federation
2 Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Square 2,
123182 Moscow, Russian Federation
3 Eicosanoid Research Division, Department of Gynecology, University Medical Centre
Benjamin Franklin, Free University Berlin, Hindenburgdamm 30,
D-12200 Berlin, Germany
Summary
An efficient procedure for the synthesis of 3-hydroxyoxylipins labelled with tritium on
all double bond positions is reported. The synthetic scheme involves a joint route for
the formation of tetraacetylenic precursors followed by stereoselective reduction of the
triple bonds either with hydrogen or tritium. The final tritiated products
were obtained with specific activities ranging from 1.65 to 1.80 Ci/mmol.
Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: tritium labelled fatty acids; 3(R)-hydroxy-oxylipins; 3(R)-HETE;
3(R),18(R/S)-DiHETE
Introduction
3(R)-Hydroxy fatty acid derivatives of AA, a group of biologically active
commonly named 3(R)-hydroxy-oxylipins, were identified in some fungal
species (Dipodascopsis uninucleata and Mucor genevensis)1–3 when exogenous
*Correspondence to: S. Nigam, Eicosanoid Research Division, Department of Gynecology, University
Medical Centre Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, D-12200 Berlin,
Germany. E-mail: nigam@zedat.fu-berlin.de
Contract/grant sponsor: FRG; contract/grant number: Ni-1/77643
Contract/grant sponsor: RFBR; contract/grant number: 02-03-33138
Copyright # 2003 John Wiley & Sons, Ltd.
Received 18 August 2003
Revised 2 October 2003
Accepted 5 October 2003

12
N. V. GROZA ET AL.
AA was utilized as a substrate. 3(R)-hydroxy-oxylipins have been shown to be
potent microbial growth factors4,5 and to play a crucial role in the
morphogenesis of Candida albicans.4 Moreover, 3-HETE was found to be a
strong proinflammatory lipid mediator, which modulates several human
neutrophil functions, such as chemotaxis and phagocytosis. Recently, a novel
AA metabolite – 3,18-DiHETE – has been identified in Candida albicans.4
Moreover, its formation was strongly suppresed by salicylic acid and its
acetylated derivative. Despite progress in the research of 3(R)-hydroxy-
oxylipins, a number of open issues on their role in intracellular signal
transduction pathways needs to be clarified. The site of the formation of 3,18-
DiHETE still remains to be established, and whether its formation affects the
pattern of endogenous 3(R)-hydroxy-oxylipins, and if the various oxylipins
reveal different biological activities. The utilization of radiolabelled analogues
will facilitate the detection of small changes in metabolic products. Hence,
efficient synthetic methods for their preparation are required to understand the
extensive biological role of 3(R)-hydroxy-oxylipins in mammalian cells. In the
present study we describe simple and effective method for the synthesis of
3(R)-HETE, and 3(R),18(R/S)-DiHETE, selectively labelled with tritium on
all double bond positions.
Experimental
1H and 13C NMR spectra for compounds 5–7 were recorded either on a
Bruker MSL 200 or Bruker MSL 300 spectrometers in CDCl3 as solvent.
Chemical shifts are referenced to tetramethylsilane as an internal standard for
1H NMR or to the deuterium lock signal of CDCl3 (d13C=77.19 ppm). HPLC
analysis was carried out on a Shimadzu LC-10Avp liquid chromatograph
connected to a SPD-10Advp UV detector. RP-HPLC analysis was performed
on a Nucleosil C18-column; 250 Â 4 mm, 5 mm particle size (Machery-Nagel,
.
Duren, Germany) with different solvent systems: MeOH/H2O (95:5, by vol.)
and a flow rate of 1 ml/min were used for analysis of compounds 6, 8 and
MeOH/H2O/Ac (85:15:0.1, by vol.) for acid 2 and MeOH/H2O/Ac (75:25:0.1,
by vol.) for dihydroxy acids 7 and 9. Preparative HPLC was carried out on a
Lichrospher 100 RP18 column; 250 Â 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 ESI MS analysis a Finnigan MAT-900XL mass-spectrometer
was used. 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 preformed Silica Gel 60 F254 sheets (Merck,
Darmstadt, Germany). 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 an atmosphere of dry argon.
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF TRITIUM LABELLED 3(R)-HETE AND 3(R),18(R/S)-DIHETE
13
[5,6,8,9,11,12,14,15-3H]-(3R,5Z,8Z,11Z,14Z)-3-Hydroxyeicosa-5,8,11,14-tet-
raenoic acid (2)
A solution of 1 (20 mg, 0.06 mmol) in benzene (1 ml), Lindlar’s catalyst (35 mg)
and quinoline (0.03 ml) were placed in the reaction ampoule and the mixture
was frozen with liquid nitrogen. After evacuation the ampoule was filled with a
hydrogen–tritium mixture, containing 1% of tritium, up to a pressure of
400 hPa. The reaction mixture was stirred for 1 h at room temperature 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 Â 1 ml). The combined filtrates were concentrated under reduced pressure
and the residue was evaporated twice with methanol (2 Â 2 ml) to remove
labile tritium. The crude product was purified by preparative HPLC followed
by saponification using LiOH (7 mg, 0.3 mmol) in water–methanol solution.
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 Â 5 ml),
combined organic extracts were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. The crude residue was purified by filtration
over silica gel (hexane/Et2O, 1:3) to yield 64% of acid 2 with a specific
radioactivity of 1.65–1.75 Ci/mmol. The radiochemical purity of [3H-8]-2 was
found to be 95%.
Methyl (3R,18R/S)-3-Hydroxy-18-(benzoyloxy)eicosa-5,8,11,14-tetraynoate (5)
In an argon filled previously dried, round-bottomed flask equipped with
magnetic stirrer anhydrous K2CO3 (108 mg, 0.78 mmol), NaI (156 mg,
1.04 mmol) and CuI (198 mg, 1.04 mmol) were introduced followed by DMF
(7 ml). Methyl 3(R)-hydroxy-5-hexynoate (4) (74 mg, 0.52 mmol) was added at
once to the suspension followed by rac-3-(benzoyloxy)-14-bromotetradeca-
6,9,12-triyne (3) (200 mg, 0.52 mmol) each in DMF (2 ml). The resulting
suspension was vigorously stirred overnight at room temperature. The mixture
was then quenched with saturated aqueous solution of NH4Cl (200 ml) and the
lipophilic products were extracted with Et2O (3 Â 80 ml). The combined
organic extracts were washed with a saturated aqueous solution of NaCl
(2 Â 100 ml) and concentrated under reduced pressure. The crude residue was
purified by silica gel flash chromatography (hexane/Et2O, 1:2) under argon to
give pure 5 as a yellow oil. Yield of 5: 187 mg (80%). TLC: Rf=0.29 (hexane/
1
Et2O, 1:3). H NMR (200 MHz, CDCl3): d 8.01 (m, 2H, o-Bz), 7.52 (m, 1H,
p-Bz), 7.42 (m, 2H, m-Bz), 5.10 (m, 1H, 18-CH), 4.12 (m, 1H, 3-CH), 3.65 (s, 3H,
OCH3), 3.10 (m, 4H) and 3.02 (m, 2H, 7-,10- and 13-CH2), 2.65 (dd, 1H,
J1=3.8 Hz, J2=16.5 Hz) and 2.50 (dd, 1H, J1=8.8 Hz, J2=16.5 Hz, 2-CH2),
2.39 (m, 2H, 4-CH2), 2.22 (m, 2H, 16-CH2), 1.85 (m, 2H, 17-CH2), 1.67
Copyright # 2003 John Wiley & Sons, Ltd.
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N. V. GROZA ET AL.
(m, 2H, 19-CH2), 0.92 (t, 3H, J=7.6 Hz, CH3). 13C NMR (50 MHz, CDCl3): d
172.67, 166.39, 132.83, 131.07, 129.74 (2C), 128.46 (2C), 82.08, 80.02, 76.38,
75.42 (2C), 75.31, 74.76, 74.50, 74.32, 67.01, 51.75, 40.40, 33.04, 27.13, 27.06,
15.41, 9.93 (3C), 9.56.
Methyl (3R,18R/S,5Z,8Z,11Z,14Z)-3-Hydroxy-18-(benzoyloxy)eicosa-5,8,11,
14-tetraenoate (6)
A suspension of Lindlar’s catalyst (374 mg) in dry benzene (10 ml) was
saturated with H2 at room temperature and then cooled to 108C. The ester 5
(187 mg, 0.42 mmol) in benzene (20 ml) and quinoline (0.37 ml) were added to
the catalyst suspension under a stream of argon. The argon was subsequently
exchanged with H2 and the reaction mixture was stirred for 1 h at 108C. H2
uptake was measured with a gas burette. After the H2 absorption was
complete, the mixture was filtered and the filtrate was washed with HCl (2 M,
2 Â 50 ml). Evaporation of the solvent yielded a crude residue, which was
purified by preparative RP-HPLC (MeOH/H2O, 95:5) to afford 243 mg (64%)
of pure 6. TLC: Rf=0.39 (Et2O/hexane, 3:1). RP-HPLC: Rt=4.25 min
1
(MeOH/H2O/AcOH, 95:5:0.1). H NMR (200 MHz, CDCl3): d 8.01 (m, 2H,
o-Bz), 7.52 (m, 1H, o-Bz), 7.42 (m, 2H, o-Bz), 5.21–5.41 (m, 8H, CH=CH),
5.10 (m, 1H, 18-CH), 4.05 (m, 1H, 3-CH), 3.65 (s, 3H, OCH3), 2.70 (m, 6H, 7-,
10-, 13-CH2), 2.42 (m, 2H, 2-CH2), 2.32 (m, 2H, 4-CH2), 2.16 (m, 2H, 16-
CH2), 1.60–1.80 (m, 6H, 17- and 19-CH2), 0.90 (t, 3H, J=6.8 Hz, CH3). 13C
NMR (50 MHz, CDCl3): d 173.04, 166.46, 132.76, 131.29 (2C), 129.71 (2C),
129.62, 129.45, 128.64, 128.57, 128.46 (2C), 128.24, 128.06, 125.11, 75.98,
68.18, 51.68, 40.75, 34.77, 33.89, 27.31, 26.03, 25.84, 23.53, 9.63. ESI MS m/z:
477 [M+Na]+.
(3R,18R/S,5Z,8Z,11Z,14Z)-3,18-Dihydroxyeicosa-5,8,11,14-tetraenoic acid (7)
An aqueous solution of NaOH (0.33 M, 10 ml) was added under argon to a
solution of ester 6 (102 mg, 0.22 mmol) in MeOH (15 ml). The mixture was
stirred for 40 h at room temperature, MeOH was removed by evaporation, and
the pH was adjusted carefully to 5.0 using HCl (1 M). Lipophilic products
were then extracted with Et2O (3 Â 40 ml), the ethereal extracts dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The products
were purified by flash chromatography (Et2O/hexane, 5:1) to yield 59 mg
(79%) of 7. RP-HPLC: Rt=8.15 min MeOH/H2O/AcOH (75:25:0.1, by vol.).
TLC: Rf=0.21 (CHCl3/MeOH, 9:1). 1H NMR (300 MHz, CDCl3): d 5.55 (m,
1H) and 5.30–5.40 (m, 7H, CH=CH), 4.05 (m, 1H, 3-CH), 3.55 (m, 1H, 18-
CH), 2.85 (m, 6H, 7-, 10-, 13-CH2), 2.56 (dd, 1H, J1=3.8, J2=16.5) and 2.45
(dd, 1H, J1=7.9, J2=16.5, 2-CH2), 2.31 (m, 2H, 4-CH2), 2.14 (m, 2H,
16-CH2), 1.41–1.59 (m, 4H, 17- and 19-CH2), 0.87 (t, 3H, J=6.8 Hz, CH3).
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF TRITIUM LABELLED 3(R)-HETE AND 3(R),18(R/S)-DIHETE
15
13C NMR (75 MHz, CDCl3): d 177.20, 131.39, 129.62, 128.38 (3C), 127.93,
127.68, 124.55, 73.30, 67.88, 40.14, 36.30, 34.39, 30.15, 25.87, 25.72, 25.67,
23.55, 9.85.
[5,6,8,9,11,12,14,15-3H]-(3R,18R/S,5Z,8Z,11Z,14Z)-3-Hydroxy-18-(benzoy-
loxy)eicosa-5,8,11,14-tetraenoic acid (8)
Compound 8 was prepared from methyl (3R,18R/S)-3-hydroxy-18-(benzoy-
loxy)eicosa-5,8,11,14-tetraynoate (5) (20 mg, 0.045 mmol) using Lindlar’s
catalyst (40 mg) and quinoline (0.4 ml) in a similar manner as described for
the tritiation of tetraacetylene 1. The crude product was purified by RP-HPLC
(MeOH/H2O, 95:5) to afford 56% of [3H8]-8, specific radioactivity 1.70–
1.80 Ci/mmol. Compound 8 was found to be identical with its non-labeled
analogue 6 as revealed by analytical RP-HPLC using a solvent system
(MeOH/H2O/AcOH, 95:5:0.1).
[5,6,8,9,11,12,14,15-3H]-(3R,18R/S,5Z,8Z,11Z,14Z)-3,18-Dihydroxyeicosa-
5,8,11,14-tetraenoic acid (9)
An aqueous solution (3 ml) of NaOH (15 mg, 0.378 mmol) was added to a
solution of 8 (43 mCi, 0.025 mmol) in methanol (12 ml) under an argon
atmosphere. The resulting mixture was stirred for 30 h at room temperature.
After the reaction was complete, methanol was removed by evaporation. The
residue was acidified using HCl (1 M), the products were extracted with Et2O
(4 Â 2 ml) and the combined organic extracts were concentrated under reduced
pressure. The crude residue was filtered over silica gel (CHCl3/MeOH, 9:1) to
give 86% of 9 with a radiochemical purity of 97%; the specific activity of 1.70–
1.80 Ci/mmol remained unchanged.
Results and discussion
Acetylenic functions are widely used to build up tetraenoic systems selectively
labelled with tritium on all double bond positions. Recently, we have reported
a fast and efficient method for the preparation of 3(R)-HETE via its
tetraacetylenic precursor.6 Here we extend its application to the synthesis of
tritium labelled 3(R)-HETE (2). As illustrated in Scheme 1, stereospecific
OH
OH
3H
3H 3H
3H
COOH
COOCH3
a,b
64%
3H
3H 3H
3H
2
1
Scheme 1. (a) T2/Lindlar’s catalyst, quinoline, benzene, 400 hPa and (b) LiOH,
MeOH, rt
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 11–17

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N. V. GROZA ET AL.
tritiation (hydrogen–tritium mixture, 1% of tritium) of the triple bonds of
methyl (R)-3-hydroxyeicosa-5,8,11,14-tetraynoate6 (1) using Lindlar’s catalyst
and quinoline (catalyst/substrate ratio, 1.4:1) in benzene afforded [3H8]-2 with
an overall yield of 64% in two steps and a specific radioactivity of 1.65–
1.75 Ci/mmol.
As illustrated in Scheme 2, [3H8]-3(R),18(R/S)-DiHETE (9) can be obtained
in a similar manner starting from tetraacetylene 5. Copper(I) mediated
cross-coupling7 of methyl 3(R)-hydroxy-5-hexynoate6 (4) and rac-3-(benzoy-
loxy)-14-bromotetradeca-6,9,12-triyne8 (3) resulted in methyl (3R,18R/S)-3-
hydroxy-18-(benzoyloxy)eicosa-5,8,11,14-tetraynoate (5) in 80% yield. In
order to optimize the conditions for preparation of [3H8]-(9) we first
synthesised the unlabelled analogue 7. Stereospecific hydrogenation of the
triple bonds of 5 using Lindlar’s catalyst and quinoline (catalyst/substrate
ratio, 1.2:1) in benzene afforded, after reverse-phase HPLC methyl (3R,18R/
S,5Z,8Z,11Z,14Z)-3-hydroxy-18-(benzoyloxy)eicosa-5,8,11,14-tetraenoate (6)
in 64% yield. Interestingly, the increase in the amount of catalyst (catalyst/
substrate ratio, 2:1) led to a significant decrease in the reaction time without
any increase in the formation of over hydrogenated products. Simultaneous
deprotection of both, carboxy and hydroxy groups of 6, using NaOH in
MeOH-H2O solution proceeded well to give 7 in 56% yield. Tritiation of 5 was
OBz
Br
3
a
80%
OH
COOCH3
OBz
5
c
b
56%
64%
OH
OH
3H
3H 3H
3H
COOR1
COOR1
3H
3H 3H
3H
OR2
OR2
6 R1=CH3, R2=Bz
7 R1=R2=H
8 R1=CH3, R2=Bz
9 R1=R2=H
79%
d
86% d
Scheme 2. (a) 4, Cul, Nal, K2CO3, DMF, rt; (b) H2/Lindlar’s catalyst,
quinoline, benzene, 440 hPa; (c) T2/Lindlar’s catalyst, quinoline, benzene and
(d) NaOH, MeOH, rt
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF TRITIUM LABELLED 3(R)-HETE AND 3(R),18(R/S)-DIHETE
17
performed in benzene with a method used for the synthesis 2. Subsequent
deprotection yielded 36.5 mCi of 9 with a specific radioactivity of 1.70–1.80 Ci/
mmol and a radiochemical purity of more than 97%.
In conclusion, we describe herein an effective method for the synthe-
sis of [3H8]-3(R)-HETE and [3H8]-3(R),18(R/S)-DiHETE through a common
synthetic route, which can also be applied to the preparation of analogues with
high specific activity.
Acknowledgements
The authors wish to thank the VW-foundation, Hannover, FRG (Ni-1/77643)
and RFBR, Russia (02-03-33138) for partial financial support.
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Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 11–17