Synthesis of tritium labelled arachidonic acid amide and ester derivatives with dopamine, serotonin, vanillylamine, and ethyleneglycol moieties

Article abstract of DOI:10.1002/jlcr.663

Tritium labelled arachidonic acid amides with dopamine, serotonin, vanillylamine and the ethyleneglycol ester moieties with high specific activity (120 Ci/ mmol) and yield (70-90%) were prepared from tritiated arachidonic acid by condensation with the cor

Full text of DOI:10.1002/jlcr.663

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 187–193.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.663
Research Article
Synthesis of tritium labelled arachidonic acid
amide and ester derivatives with dopamine,
serotonin, vanillylamine, and ethyleneglycol
moieties
V.P. Shevchenko1, I.Yu. Nagaev1, N.F. Myasoedov1, I.A. Yudushkin2,
N.M. Gretskaya2, M.Yu. Bobrov2 and V.V. Bezuglov2,*
1 Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova,
Moscow 123182, Russia
2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy
of Sciences, ul. Miklukho–Maklaya 16/10, Moscow 117437, Russia
Summary
Tritium labelled arachidonic acid amides with dopamine, serotonin, vanillyl-
amine and the ethyleneglycol ester moieties with high specific activity (120 Ci/
mmol) and yield (70–90%) were prepared from tritiated arachidonic acid by
condensation with the corresponding amines and alcohol via mixed anhydrides
or acyl fluorides. The labelled compounds were used for studying their uptake
by mouse spleen lymphocytes. From the data obtained it was suggested that
arachidonic acid amides permeate the membrane by means of passive
diffusion, while transmembrane transport of arachidonoylethyleneglycol seems
to be driven by the concentration gradient, maintained by hydrolytic enzymes.
The compounds synthesized by the reported methods can also be used in
receptor binding studies and in the oxidative metabolism of fatty acids amides
and esters. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: tritium; labelled compounds; fatty acid amides; endocannabi-
noids; transport
*Correspondence to: V.V. Bezuglov, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, ul. Miklukho–Maklaya 16/10, Moscow 117437, Russia,
E-mail: vvbez@oxylipin.ibch.ru
Contract/grant sponsor: Russian Foundation for Basic Research; Contract/grant number: 99-04-
48514 and # 00-04-81087
Copyright # 2002 John Wiley & Sons, Ltd.
Received 22 May 2002
Revised 2 August 2002
Accepted 28 August 2002

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V. P. SHEVCHENKO ET AL.
Introduction
Studies over the last decade have demonstrated that arachidonic and
other polyenoic fatty acid amides and esters are potent lipid
bioregulators involved in the regulation of memory, emotions,
nociception, sleep, motor activity, immune and neuroendocrine status
(for comprehensive reviews on the subject see1–3). Recently, we reported
that polyenoic fatty acid derivatives with biogenic amines (dopamine,
serotonin, tyramine and others) and alcohols (ethyleneglycol and some
nitroderivatives) also have a wide spectrum of biological activity. Thus,
arachidonic and eicosapentaenoic acid amides with dopamine and
serotonin possess certain anti-aggregatory and cytoprotective proper-
ties, being able to inhibit arachidonic acid- and ADP-induced platelet
aggregation and to protect early sea urchin embryos from the cytotoxic
action of dopamine and serotonin antagonists.4 Several polyenoic fatty
acid amides with dopamine5 as well as arachidonoylethyleneglycol and
its nitroester6 are potent cannabimimetic compounds, and arachido-
noylserotonin proved to be a selective, non-covalent inhibitor of fatty
acid amide hydrolase7 – a key enzyme in endocannabinoid’ metabolism
– and interfere with lipoxygenases of plant and animal origin4,6,8
.
Besides, a series of vanillylamine N-acyl derivatives, analogues of the
pungent constituent of hot pepper Capsicum sp., capsaicin, were
recently shown to bind vanilloid (VR1) and cannabinoid (CB1)
receptors and interfere with anandamide facilitated uptake.9,10
However, most of the data on the biochemical effects of fatty acid
amides and esters were obtained from the competitive binding studies
using the Cheng–Prusoff equation and have the drawback of indirect
determination of the receptor affinity and effective dose (influence of the
ligand depletion, cooperativity, dissimilar affinity to ligands, irreversible
binding, etc.). Furthermore, due to the lack of individual isotopically
labelled compounds, little is known about their own transport through
the plasma membrane. Here, we report the synthesis and application of
tritium labelled arachidonoyl dopamine (1), serotonin (2), vanillylamine
(3) and ethyleneglycol (4) derivatives (Figure 1) and studies of their
uptake by mouse spleen lymphocytes.
Results and discussion
The starting compound for the synthesis}[5,6,8,9,11,12,14,15-3H8]
arachidonic acid – was prepared by selective hydrogenation of
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Figure 1. Chemical structures of labelled amides and ester of arachidonic acid.
1 – [3H]arachidonoyl-3-hydroxytyramine, 2 – [3H]arachidonoyl-5-hydroxytrypt-
amine, 3 – [3H]arachidonoyl-4-hydroxy-3-methoxybenzylamine, 4 – [3H]arachi-
donoylethyleneglycol
5,8,11,14-eicosatetraynoic acid with gaseous tritium over Lindlar
catalyst according to a procedure described earlier for the correspond-
ing methyl ester.11 Although direct tritiation of free eicosatetraynoic
acid to labelled arachidonic acid proceeds with a lower yield than in the
case of its methyl ester, the approach utilizes an acetylenic precursor
stored in the carboxylic form, thus avoiding two additional steps:
methylation of the starting compound and subsequent saponification of
the hydrogenation product.
The synthesis of the amides and ester of labelled arachidonic acid was
performed using the mixed anhydride and carbonyl fluorides techni-
ques, respectively. Unlabelled compounds are usually prepared from
milligram and above quantities of arachidonic acid with equivalents of
other reagents. The reaction conditions allow the straightforward
separation and chromatographic isolation of the final product. In the
case of labelled arachidonic acid, a huge excess of the unlabelled
reagents was applied to ensure its full conversion into target
compounds. To avoid loss of labelled compounds during water workup,
which is usually applied to eliminate most of the reagents,12 the reaction
mixture was directly separated by HPLC followed by evaporation to
dryness and dissolution of the residue in methanol. Due to a
considerable difference between the retention time of the labelled
amides or ester and other reagents, and since the column load did not
exceed 1 mg, the target compounds were isolated with a radiochemical
purity of more than 98% within a single run.
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V. P. SHEVCHENKO ET AL.
The compounds thus prepared were used to study their transport into
mouse splenic cells. Amides (1–3) undergo rapid (t1/251 min) uptake by
mouse splenocytes (Figure. 2A); the amount of the label in the lipid
extracts of cells after 15 min incubation averages 8–9% of the total
radioactivity of the samples and does not depend on temperature for all
amides (data not shown).
The ester derivative (4), which was previously demonstrated to be
exhaustively hydrolysed in mouse splenocytes by a PMSF- and pHMB-
insensitive lipase-like activity, also accumulates in the chloroform:-
methanol extracts (10% of total activity after 15 min). The time course
curve does not reach the plateau during the 15 min incubation period
(Figure. 2B).
The results suggest that arachidonic acid amides (1–3) permeate the
membrane by means of passive diffusion. Moderate permeability of the
membrane for these compounds might be due to the presence of polar
hydrophilic functionalities in their structure. Transmembrane transport
of arachidonoylethyleneglycol (4) seems to be driven by the concentra-
tion gradient, maintained by hydrolytic enzymes and subsequent drain
of arachidonic acid by remodelling into other lipid species.
Experimental
Materials
Catalysts, solvents and other reagents were purchased from Fluka
(Buchs, Switzerland). Preparative purification of the labelled com-
pounds was performed using a Gilson high-performance liquid
Figure 2. Time course of arachidonoylserotonin (2) – a and arachidonoylethylene-
glycol (4) – b uptake by mouse spleen lymphocytes
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SYNTHESIS OF TRITIUM LABELLED
191
chromatography system (France) equipped with a radioactivity detector
and a variable wavelength UV detector.
Synthesis of labelled arachidonic acid: This was performed as
described,11 with minor modifications. The solution of 5,8,11,14-
eicosatetraynoic acid in dioxane was exposed to gaseous tritium in the
presence of Lindlar catalyst (acetylenic acid-catalyst-quinoline ratio
1:1:2, mg/mg/m1) for 2 h at room temperature, the pressure of gaseous
tritium was 400 hPa. The final product was isolated by HPLC11 with a
specific radioactivity of 170–180Ci/mmol, yield 20–25%.
Synthesis of labelled amides: Unlabelled arachidonic acid amides (1–3)
were synthesized as described earlier.5 Corresponding labelled com-
pounds were prepared as follows. A solution of [3H]arachidonic acid in
acetonitrile (3mCi, 120 Ci/mmol) was treated with a 100-fold excess of
isobutylchloroformate and NEt3. The reaction was carried out at 238C in
an argon atmosphere; reaction time is indicated in Table 1. The reaction
mixture was evaporated under an argon stream and treated with 0.1 ml of
the corresponding amine in DMF (100-fold excess of unlabelled over
labelled compounds; 238C, argon atmosphere). The reaction mixture was
further evaporated under an argon stream, diluted in the eluent, and
loaded onto the Kromasil C18 column (7mm, 4 Â 150 mm).
Synthesis of labelled ester: The ester derivative, arachidonoylethyle-
neglycol (4), was synthesized by the treatment of [3H]arachidonic acid
solution in acetonitrile (3 mCi, 120 Ci/mmol) with a solution of cyanuric
fluoride and pyridine in acetonitrile (100-fold excess of non-labelled
over labelled compounds; 238C, argon atmosphere; reaction time
indicated in Table 1). Then 0.1 ml of ethyleneglycol and a pyridine
Table 1. Reaction conditions for the synthesis of labelled amides (1–3) and ester
(4) and corresponding HPLC retention times
Compound
Reaction time (h)
Retention Yield,
timea
(min)
(%)
Stage 1 reagents: Stage 2 reagents:
isobutylchloroformate corresponding amines
(1–3) or cyanuric
fluoride (4)
(1–3) or ethyleneglycol (4)
1
2
3
4
2
2
2
4
20
20
20
48
9.21
8.49
9.86
91
83
90
73
12.67
a HPLC, see Experimental.
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V. P. SHEVCHENKO ET AL.
solution in acetonitrile was added to the reaction mixture; the solvent
was evaporated under an argon stream, and the residue dissolved in the
eluent and loaded onto the column. The corresponding unlabelled ester
was prepared according to the instruction given in Reference [6].
The compounds were purified by HPLC in MeOH:H2O:AcOH:CF3-
COOH (90:10:0.1:0.01, v:v:v:v); elution rate 1 ml/min. The peaks were
assigned by comparison of the retention times of the respective
standards, prepared from ‘cold’ arachidonic acid (see Table 1).
Isolation of mouse spleen lymphocytes: Female CBA mice (18–25 g),
maintained at 228C with free access to food and water were sacrificed by
means of cervical dislocation. Spleens were isolated and dissected in a
Petri dish in a small amount of Hanks’ balanced salt solution (HBSS).
The cell suspension was filtered through a fine mesh (6.25Â 10À4 mm2)
nylon sieve, and erythrocytes were lysed by addition of 0.83% NH4Cl
solution to the suspension (6:1, v:v), followed by 15min centrifugation at
300 Â g at 48C. The supernatant was discarded, and the pellet was
resuspended in 10ml of 0.25 M sucrose solution containing 3 mM MgCl2.
Centrifugation was repeated, and the resulting pellet was washed in
HBSS twice. Cells were counted in a cytometer; their viability was
assessed using a Trypan blue dye exclusion test and exceeded 95–97%.
Incubation: Mouse spleen lymphocytes (2 Â 107 cells/ml) were
incubated in HBSS for 1–15 min at 48 or 378C with the compounds
under study (ꢀ2 nM; 0.11–0.22 mCi per sample). The reaction was
terminated by rapid (10–15 s) centrifugation at 8000 Â g, the pellet
washed with 0.2% BSA solution in HBSS and extracted with an
acidified mixture of chloroform:methanol (2:1, v:v). The radioactivity of
lipid extracts was measured by liquid scintillation counting in
‘Universol’ scintillation cocktail (ICN Biomedicals) (counting efficiency
ꢀ40%) using Beckman LS9800 (Beckman) or Packard 2100TR
(Packard BioSystems) counters.
Statistical analysis and data presentation: Statistical analysis of the
temperature dependence of the uptake was performed using student’s
paired two-tailed t-test (p50.05). Points on the charts represent
mean Æ S.D. of the respective samples (n=3).
Conclusions
Tritium labelled compounds of high specific activity closely related to
endocannabinoids were synthesized by means of a one pot method.
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SYNTHESIS OF TRITIUM LABELLED
193
These compounds can be used as tracers in studies of transport, receptor
interaction and metabolism of the corresponding amides and ester of
arachidonic acid.
Acknowledgements
The partial financial support of the Russian Foundation for Basic
Research (RFBR Grant Nos. 99-04-48514 and 00-04-81087) is
gratefully acknowledged.
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