Synthesis of 15N-, 13C-, and 2H-labeled methanandamide analogs

Article abstract of DOI:10.1002/jlcr.650

Four isotopically labeled, metabolically stable analogs of arachidonylethanolamide (anandamide), an endogenous cannabinoid ligand, were synthesized via a five-step reaction sequence starting from arachidonic acid. These stable methanandamide derivatives will serve as probes for studying the conformational properties of anandamide in model membrane systems using solid-state NMR spectroscopy. The synthetic methods described can be applied to the preparations of other anandamide analogs with isotopic labeling in different positions of the molecule, which could be utilized in biochemical and pharmacological experiments. Copyright

Full text of DOI:10.1002/jlcr.650

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 115–129.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.650
Research Article
15
13
2
Synthesis of N-, C-, and H-labeled
methanandamide analogs
1
1
1
Fen-Mei Yao , Sonya L. Palmer , Atmaram D. Khanolkar ,
1
Xiaoyu Tian , Jianxin Guo and Alexandros Makriyannis
1
1,2,3,
*
1
Department of Pharmaceutical Sciences, University of Connecticut, Storrs,
CT 06269, USA
2
Department of Molecular and Cell Biology, University of Connecticut, Storrs,
CT 06269, USA
3
Center for Drug Discovery, University of Connecticut, Storrs, CT 06269, USA
Summary
Four isotopically labeled, metabolically stable analogs of arachidony-
lethanolamide (anandamide), an endogenous cannabinoid ligand, were
synthesized via a five-step reaction sequence starting from arachidonic
acid. These stable methanandamide derivatives will serve as probes for
studying the conformational properties of anandamide in model membrane
systems using solid-state NMR spectroscopy. The synthetic methods
described can be applied to the preparations of other anandamide
analogs with isotopic labeling in different positions of the molecule, which
could be utilized in biochemical and pharmacological experiments. Copyright
#
2002 John Wiley & Sons, Ltd.
Key Words: anandamide; isotopically labeled; conformation
*Correspondence to: A. Makriyannis, Department of Pharmaceutical Sciences, Center for Drug
Discovery, University of Connecticut, Storrs, CT 06269, USA.
E-mail: makriyan@uconnvm.uconn.edu
Contract/grant sponsor: National Institute on Drug Abuse; contract/grant number: DA03801,
DA07215, and DA9158
Copyright # 2002 John Wiley & Sons, Ltd.
Received 29 March 2002
Revised 20 May 2002
Accepted 6 August 2002

1
16
F.-M. YAO ET AL.
Introduction
Anandamide (arachidonylethanolamide, AEA, Table 1) is an endo-
genous cannabinoid receptor ligand initially isolated from porcine brain
in 1992. Like other cannabimimetic agents, anandamide was shown to
modulate cAMP levels and inhibit the electrically evoked twitch
1
response of the mouse vas deferens. Anandamide was also shown
to produce the four characteristic in vivo effects of cannabimimetics,
which include analgesia, hypothermia, hypoactivity, and catalepsy;
9
however, it was found to be somewhat less potent than (-)-D -
9
tetrahydrocannabinol (D -THC, Table 1), the most widely known
2
; 3; 4
prototypical cannabinoid.
The structure of anandamide differs greatly from the tricyclic
9
structure of the plant-derived classical cannabinoids, such as D -THC.
This has stimulated vigorous research in order to explain their
pharmacological similarity and provide a reasonable structural basis
for comparison of these dissimilar cannabimimetic ligands. The
currently known cannabinoid receptors, CB1 and CB2, have not yet
been structurally characterized and only limited information is available
regarding their active sites. Also, no crystallographic data is available
outlining the receptor-bond ligand. For this reason, the information on
the conformational properties of this highly flexible molecule and the
conformation it adopts when bound to the receptor site can greatly help
in understanding the molecular features involved in anandamide–
cannabinoid receptor interaction.5 Recently, molecular dynamics
calculations were used to characterize the conformational mobility of
a variety of anandamide analogs. The studies involving anandamide and
its analogs identified an energetically favored loop-like conformation,
6
which can be structurally correlated to the classical cannabinoids. Also,
Welsh et al. used constrained molecular dynamics simulations on
Table 1 Natural and Endogenous Cannabinoids
O
OH
N
H
O
(-)- 9-THC
Anandamide
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

SYNTHESIS OF LABELED METHANANDAMIDE ANALOGS
117
anandamide, in combination with a systematic distance comparison
search, to reveal a specific low-energy conformer whose spatial
disposition of the pharmacophoric elements closely matches that of
9
7
hexahydrocannabinol, a synthetic D -THC analog. Finally, Reggio
et al. have used conformational memories to explore the conformational
space of anandamide in both polar and nonpolar environments and
found that anandamide in water favors hybrid extended/U-shaped
conformations, whereas, in chloroform, the extended (angle-iron)
5
conformation is predominant. Thus, the computational work on the
conformational properties of anandamide revealed substantially differ-
ent results depending on the method being used. These discrepancies
argued in favor of an experimental approach for studying the
conformational properties of anandamide in solution, in the membrane
bilayer and at the receptor active site. In our laboratory, we have
designed solid-state NMR experiments to study the conformational
properties of anandamide in a membrane environment. Such work
necessitated the availability of anandamide and related analogs
specifically labeled with 13C; 15N, and H stable isotopes. Complemen-
2
8
tary to our earlier work focusing on labeled anandamide analogs, this
paper discusses the synthesis of metabolically stable methanandamide
derivatives (11a–d) for the use in pharmacological and NMR experi-
%
ments. The methods reported here are also easily applicable to the
synthesis of other isotopically labeled anandamides and related analogs
of potential usefulness in pharmacological or biochemical experiments.
Results and discussion
Preparation of the title compound was carried out as outlined in
Schemes 1 and 2. The entry point for the synthesis of the 13C-20 labeled
analogs was the known intramolecular self-epoxidation of arachidonic
9
acid developed by Corey et al. to form the 14,15-epoxide regiospeci-
fically. This was followed by reconstruction of the double bond via the
Wittig reaction using a 13C-labeled phosphonium salt. Thus, arachi-
donic acid was treated with carbonyldiimidazole and the resulting
arachidonyl imidazolide was reacted with a large excess of ethereal
hydrogen peroxide at 08C to afford the corresponding epoxy acid, which
was esterified with carbonyldiimidazole/methanol to give the epoxy
ester 2. The epoxide was opened under acidic conditions to the diol and
%
then oxidized to the unstable aldehyde ester 4. The o-labeled
%
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

1
18
F.-M. YAO ET AL.
O
O
OMe
OH
a
b
1
2
O
O
O
OMe
OMe
OH
c
O
4
3
OH
H
Scheme 1. (a): (i) COðimidazoleÞ , 1.05eq; (ii) 5:4 MH2O2-ether; (iii) CO
2
ðimidazoleÞ , MeOH, CH Cl , (b) 10% HCIO -ether, and (c) PbðOAcÞ -CH
Cl2; ꢀ30 C
2
2
2
4
4
2
8
8
Scheme 2. (a) TBDMSCI/imidazole in THF, 0 C-RT, (b) RLi, CuCN, ether,
8
8
ꢀ
20 C-RT, (c) PPh3Br2-CH2Cl2, RT, (d) PPh3, in a sealed vial, 110 C; 12 h,
8
8
(
e): (i) n-BuLi-THF, ꢀ78 C; (ii) 4 in THF, ꢀ78 C-RT, (f) 2-amino-propanol,
8
13
NaCN (Cat.), MeOH, in a sealed vial, 50 C, overnight. 11a R ¼ꢀ CH3;
X ¼ N; Y ¼ CH; 11b R ¼ ꢀ CH3; X ¼ N; Y ¼ CH; 11c R ¼ ꢀC H3;
X ¼ N; Y ¼ CH; 11d R ¼ ꢀCH ; X ¼ N; Y ¼ CH
1
5
13
2
1
3
3
phosphonium salt 9 was prepared in a straightforward method outlined
%
in Scheme 2 starting with 5-bromopentanol. Its hydroxyl was protected
with a t-butyldimethylsilyl group before it was subjected to a cross-
13
coupling reaction with isotopically labeled methyl lithium ðLiCD =Li
3
CH Þ to introduce the isotope-labeled C-20 methyl.
3
The Wittig reaction between the ylides derived from 9 and 4 led to the
%
desired cis-isomer exclusively.1
0; 11
The o-labeled arachidonate methyl
ester was subjected to differently labeled 2-amino-propanol isotopomers
%
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

SYNTHESIS OF LABELED METHANANDAMIDE ANALOGS
119
in coupling reactions catalyzed by sodium cyanide in methanol to give
the desired labeled compounds. The head group 15N-, 13C-labeled
analogs were synthesized by direct amidation of arachidonate methyl
15
ester with dl- N-2-aminopropanol (12) obtained from the reduction of
the corresponding amino acid with LiAlH4.12
Conclusion
Four isotopically labeled anandamide analogs, 11a–d, were synthesized.
Their structures were confirmed by HRMS and NMR spectroscopy.
13
1
5
2
Evidence for the N; C, and H isotopic labeling was provided by the
1
respective high-field H-NMR spectra (Figures 1–4). These compounds
are currently being used to study the conformation of anandamide in
the membrane bilayer using REDOR NMR experiments.13 Preliminary
experimental results to be reported elsewhere show that anandamide in
the membrane bilayer adopts an extended conformation in which the
individual allylic fragments are orthogonal to each other.
Experimental
1H-NMR spectra were recorded on a Bruker AMX-500 MHz spectro-
meter using TMS as the internal standard in CDCl . All chemical shifts
3
are reported in ppm. Mass spectra were obtained on a ZAB-SE mass
spectrometer via FAB. Arachidonic acid was purchased from Nu-Chek-
13
Prep, Inc. The 2- C-aminopropanol was obtained from Cambridge
Isotope Laboratories, Inc.
Methyl 14,15-epoxyeicosa-cis-5,8,11-trienoate (2)
0
1
; 1 -Carbonyl-diimidazole ð1:96 g; 12:09 mmolÞ in 15 ml dry methylene
chloride was treated with arachidonic acid ð3:5 g; 11:51 mmolÞ under
argon with stirring at an ambient temperature. After 40 min of stirring,
the resulting arachidonyl imidazolide solution was added dropwise over
a 5 min period to ethereal hydroperoxide ð4:56 M; 80 mlÞ at 08C.
KHSO ð22:73 g; 166:94 mmolÞ was added to the reaction when half
4
the imidazolide had been added. The mixture was stirred for an
additional 5 min, filtered, washed with water and brine, and dried over
Na SO . Evaporation under reduced pressure afforded 3:64 g of a
2
4
mixture of arachidonic acid and the peroxy acid.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

1
20
F.-M. YAO ET AL.
1
0
0
Figure 1. H-NMR spectrum of 2 -methylanandamide for the C-20, C2 -methyl
1
15
and N-protons. The 15N-labeled compounds exhibit a J H- N ¼ 89:1 Hz: (A)
molecule without isotopic labeling and (B) molecule with C-20 and N-double
1
3
15
labeling
0
The above epoxide acid ð3:64 gÞ was added to 1; 1 -carbonyldiimida-
zole ð3:73 g; 23:02 mmolÞ in 25 ml of dry methylene chloride with
stirring under argon at ambient temperature. After stirring for 30 min,
methanol ð4:2 ml; 103:62 mmolÞ was added, and the reaction was
stirred for 4 h at ambient temperature (monitored by TLC, 30% ethyl
acetate in petroleum ether). The volatiles were evaporated on a rotary
evaporator and the residue partitioned between ether and water. The
combined ether layers were washed with brine and dried ðNa SO Þ. The
2
4
residue upon evaporation in a vacuum ð4:5 gÞ was purified by
chromatography on silica gel (10% ethyl acetate–petroleum ether) to
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

SYNTHESIS OF LABELED METHANANDAMIDE ANALOGS
121
1
0
0
Figure 2. H-NMR spectra of 2 -methylanandamide for the C-20 and C2 -methyl
1
13
protons. The 1H-spectrum for C-20 labeled compounds exhibit a J H-
1
3
C ¼ 124:5 Hz: (A) molecule without isotopic labeling and (B) molecule with
C-20 labeling
1
3
afford 1:2 g of methyl arachidonate and 2:2 g of the title epoxy ester
1
(
57%, based on arachidonic acid): Oil, H-NMR ðCDCl Þ: d5:47
3
(m, 2H, 11, 12-vinyl H), 5.36 (m, 4H, 5, 6, 8, 9-vinyl H), 3.66 (s, 3H,
OCH ), 2.94 (m, 2H, 13-CH ); 2.84 (t, 2H, J ¼ 5:9 Hz, 10-CH ), 2.80 (t,
3
2
2
2
H, J ¼ 5:5 Hz, 7-CH ), 2.40 (m, 1H, 14-CHO), 2.32 (2H, t,
2
J ¼ 7:5 Hz, 2-CH ), 2.24 (m, 1H, 15-CHO), 2.10 (m, 2H, 4-CH ),
2
2
1
.70 (m, 2H, J ¼ 7:5 Hz 3-CH ), 1.51 (br.m, 2H, 16-CH ), 1.33 (br. m,
2
2
6
H, 1719-CH ), 0.90 (t, 3H, J ¼ 7:1 Hz, 20-CH ).
2
3
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

1
22
F.-M. YAO ET AL.
1
0
0
Figure 3. H-NMR spectra of anandamide for the Cl , C2 -methylene protons. In
1
13
0
0
the H spectrum of the C-l labeled anandamide, the frequency due to the H-l
1
13
protons is a doublet of triplets J H- C ¼ 125:1 Hz: (A) molecule without
isotopic labeling and (B) molecule with 13C-2 labeling
0
Methyl 14,15-dihydroxyeicosa-cis-5,8,11-trienoate (3)
To a stirred solution of the above epoxy ester ð2:2 g; 6:59 mmolÞ in
5
0 ml of ether at 08C was added 20 ml of cold 10% aqueous HClO4
dropwise. The ice bath was removed, and the reaction mixture was
stirred at ambient temperature until hydrolysis appeared to be complete
ðꢁ 6 hÞ by TLC (10% ethyl acetate in petroleum ether). The reaction
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

SYNTHESIS OF LABELED METHANANDAMIDE ANALOGS
123
1
0
0
Figure 4. H-NMR spectra of 2 -methylanandamide for the C2 -methyl and
2
2
0-methyl protons. H-labeling at the C-20 carbon results in the disappearance of
1
the terminal methyl H frequencies: (A) molecule without isotopic labeling and
2
(
B) molecule with H -20 double labeling
3
mixture was diluted with ether and the ether layer was washed with
aqueous NaHCO and brine. After drying ðNa SO Þ, ether was removed
3
2
4
and the residue was chromatographed on silica gel (30% ethyl acetate–
1
petroleum ether) to afford 1:5 g of the diol (65%) as an oil, H-NMR
ðCDCl Þ: d 5.5 (m, 2H, 11, 12-vinyl H), 5.36 (m, 4H, 5, 6, 8, 9-vinyl H),
3
3
.67 (s, 3H, OCH ), 3.46 (m, 2H, 14, 15-CHO), 2.82 (m, 4H, 7, 10-CH ),
3 2
2
.32 (t, overlap, 4H, J ¼ 7:4 Hz, 2, 13-CH ), 2.20 (m, 2H, OH), 2.09 (m,
2
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

1
24
F.-M. YAO ET AL.
2
1
H, 4-CH ), 1.69 (q, 2H, J ¼ 7:4 Hz, 3-CH ), 1.49 (m, 2H, 16-CH ),
2
2
2
.31 (br. M, 6H, 17-19-CH ), 0.89 (m, 3H, 20-CH ).
2
3
Methyl 14-oxotetradeca-cis-5, 8,11-trienoate (4)
To a stirred solution of the diol ð500 mg; 1:42 mmolÞ in 5 ml of
anhydrous CH Cl at ꢀ308C was added dropwise to a solution of lead
2
2
tetraacetate ð692 mg; 1:56 mmolÞ in 5 ml of CH Cl and the resulting
2
2
mixture was stirred for 30 min then quenched with ethylene glycol.
Solvent was removed under reduced pressure and the residue was
purified by flash chromatography on silica gel ðCH Cl Þ to afford 230
2
2
mg (64%) the title aldehyde.
1
3
2
Bromohexane-1- C and Bromohexane-1-C H
3
5
-t-Butyl dimethylsilyloxyl-1-bromo-pentane (6)
To a stirred solution of 5-bromo-pentanol (9) ð2:2 g; 13:7 mmolÞ and
imidazole ð3:75 g; 53:7 mmolÞ in 5 ml of anhydrous DMF at 08C was
added a solution of t-butyldimethylchlorosilane ð4:57 g; 30:3 mmolÞ in
5
ml of anhydrous DMF under argon. TLC showed the reaction to be
complete in 1 h (5% ethyl acetate–petroleum ether). The reaction
mixture was poured into ice water, and extracted with ether. The
combined organic phase was washed with brine and dried ðNa SO Þ.
2
4
After evaporation on a rotary evaporator, the residue was purified by
chromatography (3% ethyl acetate–petroleum ether) to afford 3:29 g
1
(
85%) of the title compound: H-NMR ðCDCl Þd3:61 (2H, t,
3
J ¼ 5:8 Hz; CH OÞ; 3.41 (2H, t, J ¼ 6:7 Hz; CH Br), 1.87 (3H, q,
2
2
J ¼ 7:0 Hz, 1.49 (4H, m, CH CH CH O), 0.89 (9H, s, tBu), 0.05
2
2
2
(6H, s, Me ).
2
1
3
2
1
-t-Butyl dimethylsilyl-6- C (or C H )-hexanol (7)
3
13
CH Li
3
To a stirred suspension of lithium ð366:8 mg; 52:8 mmolÞ in 15 ml
13
anhydrous ether was added CH I ð5:0 g; 35:2 mmolÞ dropwise while
3
keeping the reaction mixture gently refluxing. After the addition was
complete, the reaction mixture was refluxed for 2 h.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

SYNTHESIS OF LABELED METHANANDAMIDE ANALOGS
125
A flask charged with CuCN ð1:578 g; 17:615 mmolÞ was gently
heated with a heat gun under vacuum to remove residual moisture,
and then flushed with argon. After cooling to room temperature
under argon, the solid was suspended in 5 ml of anhydrous THF
13
and was allowed to cool to ꢀ788C. Freshly prepared CH Li
3
(or commercially available CD Li) was added via cannula using a
3
positive pressure of argon. After the addition was complete, the
mixture was warmed to 08C and stirred for 5 min at this temperature.
The dark green opaque mixture was then cooled to ꢀ788C and a
solution of 6 ð3:12 g; 10:7 mmolÞ in 25 ml of anhydrous THF was
added via cannula. The resulting mixture was warmed to ꢀ208C,
stirred for 4 h, then allowed to warm to room temperature and
stirred overnight. The reaction was quenched with NH :H O–NH Cl
3
2
4
aqueous solution (1:2), extracted with ether, washed with brine,
and dried ðNa SO Þ. After purification by chromatography on
2
4
silica gel (5% ether:petroleum ether) affording 1:43 g (60%) of the
1
13
title product: H-NMR ðCDCl Þ ( C-labeled) d3:59 (2H, t, J ¼ 6:5 Hz,
3
1
-CH ), 1.49 (2H, m, 2-CH )), 1.281.07 (6H, m, 3-and 5-CH ), 0.88
2 2 2
13
(
(
(
3H, dt, J ¼ 124:5 and 6:5 Hz, - CH ) 0.81 (9H, s, tBuSi), 0.04
6H, s, Me Si). ( H -labeled) d3:59 (2H, t, J ¼ 6:5 Hz, 1-CH ), 1.49
2H, p, J ¼ 6:5 Hz, 2-CH ) 1.30 (6H, m, 3- and 5-CH ), 0.81 (9H, s,
3
2
2
3
2
2
2
tBuSi), 0.04 (6H, s, Me Si).
2
13
2
Bromohexane-6- C (or C H ) (8)
3
To a stirred suspension of triphenyldibromo-phosphorane ð2:18 g;
:95 mmolÞ in anhydrous methylene chloride at room temperature
4
was added a solution of 4 ð827 mg; 3:81 mmolÞ in anhydrous
methylene chloride under argon. The mixture was stirred at room
temperature for 30 min and then diluted with ether and
filtered through a pad of silica gel topped with celite. The solvent
was removed by distillation under atmospheric pressure to give
1
the expected product as an oil (900 mg, 69%): H-NMR ðCDCl Þ
3
13
(
C-labeled):
d3:31
(2H,
t,
J ¼ 6:8 Hz,
1-CH ),
2
1.75
(
2H, p, J ¼ 7:3 Hz, 2-CH ), 1.33 (2H, m, 3-CH ), 1.20 (4H, m,
2
2
4
,
5-CH ), 0.88 (3H, dt, J ¼ 124:5 and 6:8 Hz; 13CH ).
2
3
(
D -labeled) ðCDCl Þ d3:31 (2H, t, J ¼ 6:8 Hz, 1-CH ), 1.75
3
3
2
(
2H, p, J ¼ 7:3 Hz, 2-CH ), 1.33 (2H, m, 3-CH ), 1.20 (4H, m,
2
2
4
, 5-CH ).
2
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

1
26
F.-M. YAO ET AL.
13
2
1
-Triphenylphosphoniumyl-hexane bromide-6- C (or 6-C H ) (9)
3
A solution of 8 ð800 mg; 4:81 mmolÞ, and triphenylphosphine ð1:299 g
4:95 mmolÞ in 5 ml of acetonitrile was heated in a sealed Reacti-vial
;
with stirring at 1108C for 48 h. After evaporation of acetonitrile under
reduced pressure and the excess of triphenylphosphine was removed by
triturating with ether leaving 1:8 g of the title phosphonium salt 9.
1
13
%
H-NMR ( C -labeled in methanol-d ): d7:907:61 (5H, m, tri-Ph), 3.46
4
(
2H, m, 1-CH ), 1.71 (2H, m), 1.60 (2H, q, J ¼ 7:0 Hz), 1.35(4H, m),
2
13
0
.93 (3H, dt, J ¼ 126 and 6:8 Hz, - CH ). (D -labeled in CDCl ): 7.90–
3
3
3
7
.61 (15H, m, 3Ph H), 3.75 (2H, m, 1-CH ), 1.62 (4H, m, 2, 3-CH ), 1.21
2
2
(4H, m, 4, 5-CH ).
2
13
2
Methyl-20- C-eicosa-cis-5,8,11, 14-tetraenoate (or 20-C H ) (10)
3
A solution of the phosphonium salt (9) (832 mg; 1:95 mmol) in
%
anhydrous THF under argon was cooled to ꢀ768C and treated with
n-BuLi (1:06 ml; 2 M in hexane, 2:11 mmol) with stirring. The resulting
deep red solution was stirred at ꢀ768C for 30 min, and the freshly
prepared aldehyde ð4; 1:62 mmolÞ in anhydrous THF ð3 mlÞ was added
dropwise. The mixture was stirred for 1 h at ꢀ768C and then allowed to
warm to room temperature gradually over 3 h. Adding a phosphate
buffer (pH 7) quenched the reaction, and the volatiles were evaporated
under reduced pressure. The residue was dissolved in ethyl ether, and
the solution was washed with water, brine and dried ðNa SO Þ.
2
4
Evaporation of the ether and chromatography of the residual oil on
silica gel (5% diethyl ether–petroleum ether) afforded 100 mg of the title
1
compound (30%, based on the diol ester). H-NMR ðCDCl Þ,
3
(13
C-labeled) d5:38 (8H, m, 5, 6, 8, 9, 11, 12, 14, 15-vinyl H), 3.67
3H, s, OCH ), 2.82 (6H, m, 7, 10, 13-CH ), 2.32 (2H, t, J ¼ 7:5 Hz,
(
3
2
2
1
2
1
-CH ), 2.132.03 (4H, m, 4, 16-CH ), 1.71 (2H, p, J ¼ 7:5 Hz, 3-CH ),
2
2
2
.371.29 (6H, 1719-CH ), 0.89 (3H, dt, J ¼ 124:5 and 6:8 Hz,
2
13
2
0- CH ). ( H -labeled): 5.38 (8H, m, 5, 6, 8, 9, 11, 12, 14,
3
3
5-vinyl H), 3.67 (3H, S, -OCH ), 2.82 (6H, m, 7, 10, 13-CH ), 2.32
3
2
(
2H, t, J ¼ 7:4 Hz, 2-CH ), 2.13–2.03 (4H, 4, 16-CH ), 1.71 (2H,
2
2
p, J ¼ 7:4 Hz, 3-CH ), 1.28 (6H, m, 1719-CH ).
2
2
15
dl- N-2-amino-propanol (12)
15
A solution of dl- N-alanine ð200 mg; 2:245 mmolÞ in 10 ml of
anhydrous THF was cooled to 08C and 4:5 ml of a 1:0 M solution of
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

SYNTHESIS OF LABELED METHANANDAMIDE ANALOGS
127
LiAlH in THF added dropwise. The reaction mixture was stirred at
4
0
8C to room temperature for 5 h. TLC showed it was complete. The
reaction was quenched with aqueous NaHCO , extracted with ethyl
3
acetate, dried over Na SO to afford 150 mg of the titled aminoalcohol
2
4
after evaporation, which was used in the next step without purification.
13
15
0
0
dl-20- C- N-(eicosa-cis-5,8,11,14-tetraenoyl)-1 -hydroxy-2 -
propyl-amine (11a)
A solution of 10 ð40 mg; 0:125 mmolÞ, sodium cyanide ð0:90 mg;
15
0
:0184 mmolÞ, and dl- N-amino -propanol (150 mg, crude) in
methanol in a sealed Reacti-vial was heated to 508C with oil bath and
stirred overnight. The solvent was evaporated on a rotary evaporator
and the residue was purified by chromatography (5% methanol in
1
CH Cl ) affording 20 mg (43.9%) of the title compound: H-NMR
2
2
ðCDCl Þ: d5:58 (1H, dd, J ¼ 89:1 and 7:2 Hz; 15N-H), 5.42–5.32 (8H,
3
m, 5, 6, 8, 9, 11, 12, 14, 15-vinyl H), 4.07 (1H, m, 15N -CH-), 3.66 (1H,
0
0
m, 2 -CH-OH), 3.53(1H, m, 2 -CH-OH), 2.84–2.74 (6H, m, 7, 10,
1
3-CH ), 2.19 (2H, t, J ¼ 7:5 Hz, 2-CH ), 2.13 (2H, m, 4/16-CH ),
2
2
2
2
.05 (2H, m, 16/4-CH ), 1.72 (2H, p, J ¼ 7:5 Hz, 3-CH ), 1.34 (6H,
2
2
15
m, 17,19-CH ), 1.17 (3H, dd, J ¼ 6:8 and 2:8 Hz; NHCCH ), 0.89
2
3
13
þ
(3H, dt, J ¼ 124:5 and 6:8 Hz, 20- CH ). FAB-MS: MH calc.
3
3
64.3216; found 364.3213.
13
0
0
dl-20- C-(eicosa-cis-5,8,11,14-tetraenoyl)-1 -hydroxy-2 -
propylamine (11b)
1
H-NMR ðCDCl Þ: 5.65 (1H, br.m., N–H), 5.42–5.32 (8H, m,
3
5
,6,8,9,11,12,14,15-vinyl-H), 4.06 (1H, m, NCH-), 3.65 (1H, m,
0
-CHO), 3.52 (1H, m, -CH O), 2.97 (1H, br.m, -OH), 2.84–2.74 (6h, m,
7
, 10, 13-CH ), 2.19 (2H, t, J ¼ 7:5 Hz, 2-CH ), 2.13 (2H, m, 4/16-
2
2
CH ), 2.05 (2H, m, 16/4-CH ), 1.72 (2H, p, J ¼ 7:5 Hz, 3-CH ), 1.38–
2
2
2
1
.27 (6H, m, 17-19-CH ), 1.17 (3H, d, J ¼ 6:9 Hz; NCCH ), 0.89 (3H,
2
3
þ
13
dt, J ¼ 124:5 and 6:8 Hz, 20- CH ). FAB-HRMS: MH , 363.3090;
calc. MH : 363.3092.
3
þ
2
0
0
2
propylamine (11c)
0-C H -(eicosa-cis-5,8,11,14-tetraenoyl)-1 -hydroxy-2 -
3
1
H-NMR ðCDCl Þ: 5.56 (1H, br.m, N-H), 5.42–5.32 (8H, m, 5, 6, 8, 9,
3
1
1, 12, 14, 15-vinyl H), 4.06 (1H, m, N-CH), 3.65 (1H, dd, J ¼ 10:8 and
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

1
28
F.-M. YAO ET AL.
0
0
0
3
2
2
:2 Hz, 2 -CHO), 3.52 (1H, dd, J ¼ 10:8 and 6:2 Hz, 2 -CH O),
.85–2.75 (6H, m, 7, 10, 13-CH ), 2.19 (2H, t, J ¼ 7:5 Hz, 2-CH ),
.13 (2H, m, 4/16-CH ), 2.05 (2H, m, 16/4-CH ), 1.72 (2H,
2
2
2
2
p, J ¼ 7:5 Hz, 3-CH ), 1.38–1.27 (6H, m, 17–19-CH ), 1.17 (3H,
2
2
þ
d, J ¼ 6:9 Hz, N-CCH ). FAB-MS: MH calc. 365.3789; found
3
3
65.3786.
2
0-13C-(eicosa-cis-5,8,11,14-tetraenoyl) ethanolamine (11d)
1
H-NMR ðCDCl Þ: 6.18 (1H, m, CON-H), 5.45–5.28 (8H, m, 5, 6, 8, 9,
3
1
1, 12, 14, 15-vinyl H), 3.70 (2H, dt, J ¼ 125:1 and 5:1 Hz, CON-
1
3
0
CH ), 3.35 (2H, m, 2 -CH O), 2.85–2.77 (6H, m, 7, 10, 13-CH ), 2.20
2
2
2
(
2H, t, J ¼ 7:5 Hz, 2-CH ), 2.11–1.99 (4H, m, 4, 16-CH ), 1.70 (2H, p,
2
2
J ¼ 7:5 Hz, 3-CH ), 1.28 (6H, m, 1719-CH ), 0.87 (3H, m, 20-CH ).
2
2
3
þ
FAB-HRMS: MH calc. 363.3092; found 363.3089.
Acknowledgements
This research was supported by grants DA03801, DA07215 and
DA9158 from the National Institute on Drug Abuse. The authors
would also like to thank Drs. Spiro Pavlopoulos, Gerard Leclerc, and
Chen Li for their valuable contributions to this paper.
References
1
2
3
4
5
6
. Devane WA, Dysarz FA, Johnson RM, Melvin LS, Howlett AC. Science
1
992; 258: 1946–1949.
. Smith PB, Compton DR, Welch SP, Razdan RK, Mechoulam R, Martin
BR. J Pharmacol Exp Ther 1994; 270: 219–227.
. Varga K, Lake K, Martin BR, Kunos G. Eur J Pharmacol 1995; 278:
2
79–283.
. Welch SP, Dunlow LD, Patrick GS, Razdan RK. J Pharmacol Exp Ther
995; 273: 1235–1244.
. Barnett-Norris J, Guarnier F, Hurst DP, Reggio PH. J Med Chem 1998;
1: 4861–4872.
1
4
. Thomas BF, Adams IB, Mascarella SW, Martin BR, Razdan RK. J Med
Chem 1996; 39: 472–482.
7
. Tong W, Collantes ER, Welsh WJ. J Med Chem 1998; 41: 4207–4215.
. Xie X-Q, Yang H, Choi G, Lin S, Makriyannis A. J Label Compd
Radiopharm 2002; 45: 775–784.
8
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129

SYNTHESIS OF LABELED METHANANDAMIDE ANALOGS
129
9
. Corey EJ, Niwa H, Falch JR. J Am Chem Soc 1979; 101: 1586–1587.
1
0. Ryan WJ, Banner WK, Wiley JL, Martin BR, Razdan RK. J Med Chem
997; 40: 3617–3625.
1. Labelle M, Falgueyret JP, Riendeau D, Rokach J. Tetrahedron 1990; 46:
301–6310.
1
1
6
1
1
2. Kulkarni SU, Patil VK. Heterocycles 1992; 18: 163–167.
3. Garbow JR, Breslav M, Antohi O, Naider F. Biochemistry 1994; 33:
1
0094–10099.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 115–129