Synthesis of 15N and 13C selectively labeled anandamide

Article abstract of DOI:10.1002/jlcr.601

Anandamide (Figure 1a) (arachidonyl ethanolamide, AEA), (5Z,8Z, 11Z, 14Z)-N-(2-hydroxyethyl)-5,8,11,14-Eicosatetraenamide, is an endogenous cannabinoid ligand possessing important biological activity. The conformation of AEA in its native receptor binding

Full text of DOI:10.1002/jlcr.601

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 775–784.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.601
Research Article
15
13
Synthesis of N and C selectively labeled
anandamide
,
1,2
1
1
2
Xiang-Qun Xie* , Hongqin Yang , Greg Choi , Sunyuan Lin and
1,2
Alexandros Makriyannis
1
Institute of Materials Science, University of Connecticut, Storrs,
CT 06269, USA
2
Department of Pharmaceutical Science and Center for Drug Discovery,
University of Connecticut, Storrs, CT 06269, USA
Summary
Anandamide (Figure 1a) (arachidonyl ethanolamide, AEA), (5Z,8Z,11Z,
1
4Z)-N-(2-hydroxyethyl)-5,8,11,14-Eicosatetraenamide, is an endogenous can-
nabinoid ligand possessing important biological activity. The conformation of
AEA in its native receptor binding environment is particularly of interest for
pharmaceutical research and biochemistry in general. Here we report a novel
1
5
13
synthetic pathway, which selectively introduces N and C isotopes into the
anandamide molecule. This isotopically labeled AEA can be studied
conformationally in its native binding condition via solid state NMR. These
synthetic procedures can also be adapted to produce radioactive ligands for
receptor binding assays and other structural studies. Copyright # 2002 John
Wiley & Sons, Ltd.
Key Words: anandamide; stable isotope labeled; NMR; cannabinoid ligand
*Correspondence to: X.-Q. Xie, U-3136, Institute of Materials Science, Department of
Pharmaceutical Science, School of Pharmacy, University of Connecticut, Storrs, CT 06269-3136,
USA. E-mail: xie@uconn.edu
Contract/grant sponsor: Pfizer Company
Contract/grant sponsor: NIH NIDA; contract/grant number:DA11510
Copyright # 2002 John Wiley & Sons, Ltd.
Received 1 October 2001
Revised 14 February 2002
Accepted 11 April 2002

7
76
X.-Q. XIE ET AL.
Introduction
One of the most significant advances in recent cannabinoid (CB)
research is the discovery of anandamide (arachidonyl ethanolamide,
AEA, Figure 1a) as the first cannabinoid endogenous ligand, and the
1
confirmation of the existence of the cannabinoid system in mammals. It
is known that AEA binds to the cannabinoid receptor and induces
9
cannabimimetic activity comparable to (-)-D -tetrahydrocannabinol
9
(
D -THC, Figure 1b), the active constituent in marijuana.
Questions have been raised as to how such a structurally different
9
molecule has similar pharmacological effects as that of D -THC. Also of
interest is its native binding conformation(s), as well as the pharmaco-
logical implications of AEA as a lead compound for novel CB ligand
2
,3
development programs. Thus, intensive efforts have been made to
understand AEA’s biological properties and its interaction with
cannabinoid receptors. Although various computer-generated AEA
4
ꢀ7
conformations have been reported,
the in vivo conformation of
receptor-bound AEA still remains unknown. Furthermore, these
theoretical models are considered to be inconclusive due to the lack of
experimental evidence.
8
Xie et al. have recently reported the extended ‘pseudo-helical shape’
conformation of AEA based on the NMR Comparative Molecular
Field Analysis (CoMFA) studies. Despite such efforts, solution NMR
provided only limited conformational data due to the flexible nature of
AEA and its rapid chain motion in solution. However, these limitations
of liquid phase NMR can be overcome by using solid state NMR
9
techniques with a combination of isotopically labeled ligands.
O
9
3
7
10
8
7
OH
1
1
N
H
2
10a
10
*
2
3
13
HO
6a
20
1
8
O
(CH ) CH
2 4 3
4
9
9
(
a) AEA; Anandamide (Endogenous ligands)
(b) ∆ -THC; (-)-∆ tetrahydrocannabinol
(
Active constituent in marijuana)
9
9
Figure 1. Structures of anandamide (AEA) and (-)-D -tetrahydrocannabinol (D -
THC)
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 775–784

SYNTHESIS OF LABELED ANANDAMIDE
777
REDOR (rotational echo double resonance) experiments are
1
3
15
2
designed to observe isotopically labeled (e.g. C, N, or H, as
necessary) nuclei, and to allow direct distance measurements between
strategically labeled positions to obtain structural information at the
molecular level.
In this article, we report the synthesis of double-labeled AEA, with
13
N at the amine head group, and C in the terminal methyl group.
1
5
This labeled compound is designed for solid state NMR spectroscopy in
order to obtain experimental data on AEA in both free and membrane-
bound states. These data are then used to verify computer predicted
models. Furthermore, this synthesis can be modified to produce
radioactive ligands for receptor affinity binding assays or other similar
structural studies.
Results and discussion
The synthetic pathway of double-labeled AEA is summarized in
Figure 2. The synthesis started from the preparation of aldehyde 4 by
1
0,11
a previously reported method.
an ethereal solution of hydrogen peroxide (ether/H O ) was prepared
In the preparation of the epoxide 2,
2
2
1
0
based on the Seltzman method. From experimental trials (unpublished
data) we found that the amount of sodium sulfate added affects the
concentration of H O and the subsequent epoxidation, since an excess
2
2
1
2
of sodium sulfate tends to form complexes with H O . Therefore, the
2
2
amount of sodium sulfate added should be carefully controlled in order
to obtain reproducible yields.
5
-Bromopentanol was prepared by the cleavage of tetrahydropyran 5
with boron tribromide, and the hydroxyl group was protected using
1
tetrahydropyranyl mixed acetyl function, yielding bromide 6. The
3
Grignard reagent, the organocopper (I) intermediate, was prepared
13
from 6, and coupled with C iodomethane in the presence of dilithium
13
0
tetrachlorocuprate in THF at 08C, to give 2-(6 - C-hexyloxy) tetra-
hydropyran 7. Treatment of compound 7 with dibromotriphenyl-
14
15
phosphorane yielded the bromide 8,
which was then refluxed
with triphenylphosphine in xylenes to produce the phosphonium
bromide 9.
Coupling of compounds 4 and 9 was achieved by the ylide formation
of 9 with n-butyllithium/hexanes in THF at ꢀ788C, then linkage with
aldehyde 4 through a Wittig reaction to produce 20- C labeled methyl
1
3
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 775–784

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78
X.-Q. XIE ET AL.
1)H O /CO(IM)
O
O
(
2
2
2
HClO4
OCH3
OH
(
2)CH OH/ CO(IM)2
1
3
O
2
O
O
Pd (OAc) 4
OCH3
OCH3
CHO
4
3
HO
OH
(
(
1) BBr3
2) dihydrofuran / H
(1) Mg
O
O
Br
O
O
O
2) 13CH3 I / Li2CuCl4
13CH
3
(
5
6
7
Ph 3P . Br2
(
1) BuLi
2) 4
Ph 3P
.
PPh 3
Br
13CH3
H313
Br
C
(
8
9
O
O
OCH3
13CH3
LiOH . H2O
OH
13CH3
oxalyl chloride
1
0
11
O
O
H215NCH2CH2OH
OH
Cl
13CH3
15N
13CH3
1
2
13
1
3
15
Figure 2. Procedures for the synthesis of the 20- C, N-labeled AEA molecule
1
3
arachidonate 10. Ester 10 was then hydrolyzed to give 20- C
arachidonic acid, 11, and further transformed to its acid chloride, 12.
13
15
Finally, the double-labeled C, N AEA 13 was obtained by reacting
15
compound 12 with excess N ethanolamine, prepared according to a
16
previously reported procedure.
1
5
NMR analysis of the end product (Figure 3) confirmed the N and
C enrichment at the ethanolamine head group and terminal methyl
1
3
group, respectively. Figure 3 shows a stacked plot of the isotope labeled
AEA (Figure 3A) and the unlabeled AEA (Figure 3B). The labeled
1
AEA shows a doublet-of-triplet peak pattern for the NH group at
5
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 775–784

SYNTHESIS OF LABELED ANANDAMIDE
779
O
1'
3
7
15
N
1
2
13CH
1
0
13
H
2'
3
18
2
0-13CH3
15
N-H
(A)
(B)
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
ppm
1
Figure 3. A stacked plot of high-resolution 500 MHz H NMR spectra of the
1
5
13
N, C-labeled AEA molecule (A), and the unlabeled compound (B)
1
5
1
1
1
.84 ppm (dt, 2H, J N- H=90 Hz, J H- H=6 Hz). The NH chemical
5
shift difference between the enriched and non-enriched AEA com-
pounds is due to the concentration-dependent property of the NH
1
3
proton. The methyl group was not fully C labeled, thus showing a
triplet-of-triplet peak pattern instead of the expected doublet-of-triplet
1
3
1
1
1
peak pattern (dt, 3H, J C- H=124 Hz, J H- H=7 Hz). This is
13
attributed to deterioration of the C-enriched reagent CH I, which
3
showed a similar NMR spectral pattern for the methyl peak (data not
shown).
The synthetic procedure reported above can also be adapted to
14
3
produce the corresponding H or
C radioactive ligands for
3
14
receptor binding assays. Radioactive ( C and H) forms of AEA or
-arachidonylglycerol (2-AG) have been used as radioactive substrates,
2
1
e.g. [1,2- C]-AEA and [5,6,8,9,11,12,14,15- H]-AEA, for a variety
4
17
3
18
1
of radioenzymic assays, occurrence and metabolism studies, transport
9
20
and hydrolysis investigations, and cellular uptake and enzymatic
21,22
hydrolysis work.
The labeling approach reported here provides an
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 775–784

7
80
X.-Q. XIE ET AL.
alternate approach to the labeled endogenous cannabinoid ligand for
use in biophysical studies.
Experimental
Arachidonic acid was purchased from Nu-Chek Prep, Inc. (Elysian,
MN). All other chemicals and chromatographic materials were
purchased from Aldrich Chemical Co. (Milwaukee, WI). Solution
NMR spectra were obtained on a Bruker AVANCE DMX500
Spectrometer using tetramethylsilane as an internal reference. All
chemical reactions involved were conducted under dry N gas to exclude
2
moisture and prevent possible oxidation.
The double-labeled AEA 13 was prepared as illustrated in Figure 2,
following literature procedures for the synthesis of the unlabeled
material, with appropriate modification.
Methyl 14-oxotetradeca-cis-5,8,11-trienoate 4
The synthesis started from arachidonic acid 1, and involved three steps,
10
as described by Seltzman. In the preparation of the epoxide 2, an
ethereal solution of hydrogen peroxide (ether/H O ) was prepared
2
2
10
based on the reported method, and sodium sulfate was used to remove
the water in the ethereal solution. Since sodium sulfate tends to form
12
complexes with H O , the amount of sodium sulfate added affects the
2
2
concentration of H O and the subsequent epoxidation. Therefore, the
2
2
proper amount (ethereal H O /Na SO , 100:6 v/w) of sodium sulfate
2
2
2
4
was experimentally determined to obtain reproducible TLC results for
the aldehyde conversion. The aldehyde product 4 was immediately used
in the following reaction.
0
13
-(6 - C-hexyloxy)tetrahydropyran 7
2
%
0
The preparation of Grignard reagent is as follows: A solution of 2-(5 -
bromopentyloxy)tetrahydropyran 6 (2 g, 7.97 mmol) in dry THF (10 ml)
was added dropwise under gentle reflux conditions to a mixture of
magnesium turnings (0.243 g, 10.0 mmol), dry THF (5 ml), a crystal of
iodine and 6 (0.51 g, 2.03 mmol). Thereafter the reaction mixture was
heated under reflux for 5 h until most of the magnesium had dissolved.
The reaction was stopped and cooled to room temperature.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 775–784

SYNTHESIS OF LABELED ANANDAMIDE
781
1
3
C Labeled iodomethane (1.42 g, 10.0 mmol), the Grignard solution
prepared above (equivalent to 10.0 mmol) and anhydrous THF (20 ml)
were combined in a 100 ml round-bottomed flask which was flushed
with nitrogen and sealed with a rubber serum cap. The solution was
cooled in an ice bath. A solution of dilithium tetrachlorocuprate in THF
(
0.1 M, 0.5 ml) was added. The reaction mixture was stirred for 3 h at
8C, then poured into 100 g of crushed ice and extracted with hexanes
3 ꢁ 10 ml). The extract was dried over Na SO . Removal of the solvent
0
(
2
4
in vacuo gave 1.2 g of the crude product, which was purified by silica gel
column chromatography (30 g SiO , 2% ethyl acetate/hexane) to obtain
2
1
0
.98 g of pure product in 52% yield. H NMR (CDCl ): d 0.91 (dt, 3H, J
3
1
3
1
1
1
C- H=124 Hz, J H- H=7 Hz); 1.29–1.34 (m, 6H); 1.53–1.61 (m, 6H);
.77 (m, 2H); 2.45 (m, 2H); 2.78 (m, 2H); 4.58 (t, 1H).
1
13
- C-bromohexane 8
6
%
-(6 - C) Hexyloxytetrahydropyran 7 (1 g, 5.35 mmol) was dissolved in
0
13
2
anhydrous CH Cl (20 ml) and dibromotriphenylphosphorane (2.30 g,
2
2
5
.45 mmol) was added, followed by 3 ml of CH Cl for rinsing. The
2 2
mixture was stirred at room temperature for 20 h, washed with 5%
sodium bicarbonate (20 ml) and brine (2 ꢁ 20 ml), and dried over
Na SO . After evaporation of the solvent in vacuo, the residue was
2
4
extracted with hexanes (3 ꢁ 20 ml). The extract was concentrated on a
rotary evaporator. Further purification of the crude product was
accomplished by column chromatography (15 g SiO , petroleum ether)
2
1
to afford 380 mg of 8 in 43% yield. H NMR (CDCl ): d 0.92 (dt, 3H, J
3
1
3
1
1
1
C- H=124 Hz, J H- H=7 Hz); 1.28–1.33 (m, 4H); 1.43 (m, 2H); 1.86
m, 2H); 3.41 (t, 3H).
(
1
- C-hexyltriphenylphosphonium bromide 9
3
6
%
13
A solution of 6- C-bromohexane 8 (187 mg, 1.1 mmol) and triphenyl-
phosphine (288 mg, 1.1 mmol) in xylenes (3 ml) was heated at reflux
overnight. While the reaction was still warm, the condenser was
removed and a stream of nitrogen was blown over the reaction until the
solvent evaporated. After cooling, the excess triphenylphosphine was
removed by repeated washing and decanting with hexanes/benzene (1/1,
5
ml). The solvent was decanted and the procedure was repeated. The
remaining solvent was removed in vacuo and the residue was heated in a
vacuum oven overnight at 85–908C to give 385 mg of pale yellow gum in
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 775–784

7
82
X.-Q. XIE ET AL.
8
2% yield. The material was used directly in the Wittig reaction,
without characterization.
1
0- C-methyl arachidonate 10
3
2
A solution of the Wittig reagent was prepared by the dropwise addition
of a solution of n-butyllithium in hexanes (2.5 M, 0.5 ml) to a solution of
9
in THF (5 ml) at –788C and stirred at that temperature for 0.5 h. The
aldehyde 4 (prepared from 383 mg of the diol 3) dissolved in dry THF
5 ml) was added dropwise to the above Wittig reagent. The reaction
(
mixture was stirred, allowing it to warm to room temperature over 2.5 h.
The reaction was quenched with brine, extracted with ethyl acetate
(
3 ꢁ 10 ml) and dried over Na SO , evaporated in vacuo to give crude
2
4
product which was then chromatographed on silica gel (10 g SiO2,
hexanes/ethyl acetate 5:1) to produce 198 mg of the pure product in
1
5
8% yield (based on the diol 4 used). H NMR: d 0.90 (dt, 3H, J
1
3
1
1
1
C- H=124 Hz, J H- H=7 Hz); 1.26–1.37 (m, 6H); 1.71 (m, 2H);
.02–2.10 (m, 4 H); 2.31 (t, 2H); 2.77–2.84 (m, 6H); 3.67 (s, 3H);
.36–5.42 (m, 8H).
2
5
13
0- C-arachidonic acid 11
2
13
To a solution of 20- C-methyl arachidonate 10 (198 mg, 0.62 mmol) in
methanol (18 ml) and water (6 ml) was added LiOH ꢂ H O (100 mg,
2
2
.4 mmol) and the reaction was heated at 608C overnight. After cooling,
the reaction was diluted with H O (20 ml) and ether (20 ml). The layers
2
were separated and the aqueous layer was acidified with 1 N HCl and
saturated with NaCl. The aqueous layer was extracted with ether
(3 ꢁ 10 ml), and the combined extract was washed with brine (5 ml) and
dried over Na SO . The ether was removed in vacuo to afford 148 mg of
4
2
oil in 78% yield. The product was treated twice with fresh benzene
5 ꢁ 2 ml) and evaporated to remove moisture, and used directly in the
following reactions.
(
1
5
13
N, 20- C-anandamide 13
13
To a solution of 20- C-arachidonic acid 11 (98 mg, 0.32 mmol) in
anhydrous benzene (10 ml) cooled to 08C was added oxalyl chloride
(0.06 ml, 0.69 mmol) and a drop of DMF. The reaction mixture was
slowly brought to room temperature while stirring for 2 h. The solvent
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 775–784

SYNTHESIS OF LABELED ANANDAMIDE
783
was removed in vacuo and the residue was treated twice with fresh
benzene (2 ꢁ 10 ml) and evaporated to remove moisture. The crude acid
chloride 12 was used in the following reaction without further
purification.
1
5
15
A solution of N labeled ethanolamine was prepared by treating
ethanolamine hydrochloride (337 mg, 3.42 mmol) with triethylamine
N
(
0.48 ml, 3.45 mmol) at 08C in anhydrous CH Cl (20 ml) and stirred for
2
2
3
0 min.
The crude acid chloride 12 was dissolved in anhydrous CH Cl (6 ml)
2
2
1
5
and was added dropwise to the solution of N ethanolamine at 08C.
The reaction was stirred at 08C for 30 min, brine (20 ml) was added and
then CH Cl (20 ml). The aqueous layer was extracted with CH Cl
2
2
2
2
(
3 ꢁ 10 ml). The organic phase was combined and dried over Na SO .
2
4
After evaporation of the solvent in vacuo, the residue was chromato-
graphed on a column (SiO 5 g, hexanes/ethyl acetate 1:5) to afford
2
1
13
1
4
6 mg of 13 in 41% yield. H NMR: d 0.90 (dt, 3H, J C- H=124 Hz, J
1
1
H- H=7 Hz); 1.25–1.37 (m, 6H); 1.73 (m, 2H); 2.05 (m, 2H); 2.12 (m,
H); 2.81–2.85 (m, 6H); 3.43 (m, 2H); 3.72 (m, 2H); 5.33–5.41 (m, 8H);
1
.84 (dt, 2H, J N- H=90 Hz, J H- H=6 Hz).
2
15
1
1
5
Acknowledgements
We would like to acknowledge J. K. Kawakami and Qian Liu for
their professional discussions. We would like to thank Pfizer Company
for strong support on this project. This project was supported by a grant
from the NIH NIDA (DA11510).
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J Label Compd Radiopharm 2002; 45: 775–784