Unique analogues of anandamide: Arachidonyl ethers and carbamates and norarachidonyl carbamates and ureas

Article abstract of DOI:10.1021/jm980711w

To examine the effect of changing the amide bond of anandamide (5, AN) to a less hydrolyzable moiety, analogues la-11, 2a-2c, 3a-3c, and 4a-4h were synthesized from commercially available arachidonyl alcohol or arachidonic acid and tested for their pharmacological activity. Arachidonyl ethers 1a-1k were obtained through the coupling of the arachidonyl mesylate (6) (generated from the mesylation of arachidonyl alcohol) with the appropriate alcohol in potassium hydroxide. Arachidonyl ether 11 was obtained through the phase- transfer coupling of arachidonyl alcohol with 2-(2-iodoethoxy)tetrahydropyran (which was generated from its bromide) followed by cleavage of the tetrahydropyran group with Dowex resin. Arachidonyl carbamates 2a-2c were obtained through the coupling of arachidonyl alcohol with the appropriate isocyanates. Norarachidonyl carbamates 3a-3c and ureas 4a-4h were obtained through the coupling of the norarachidonyl isocyanate (generated from arachidonic acid using diphenyl phosphorazidate and triethylamine upon heating) with the appropriate alcohols and amines, respectively. AN analogues 1-3 have shown poor binding affinities to the CB1 receptor and fail to produce significant pharmacological effect at doses up to 30 mg/kg. Several ether analogues 1 were also evaluated in the CB2 binding assay and were found to be of low affinity. However, norarachidonyl urea analogues 4 have shown generally good binding affinities to the CB1 receptor (K(i) = 55-746 nM) and pharmacological activity with AN-like profiles. The most potent analogue of this series is the 2-fluoroethyl analogue 4f which binds 2 times better than AN and was more active in several mouse behavioral assays. It was also observed that urea analogues 4a and 4g, which have weak binding affinities to the CB1 receptor (K(i)=436 and 347 nM, respectively), produced surprisingly potent pharmacological activity. These urea analogues have also shown hydrolytic stability toward the amidase enzymes, responsible for the primary degradation pathway of anandamide, in binding affinity assays in the absence of the enzyme inhibitor PMSF.

Full text of DOI:10.1021/jm980711w

J . Med. Chem. 1999, 42, 1975-1981
1975
Un iqu e An a logu es of An a n d a m id e: Ar a ch id on yl Eth er s a n d Ca r ba m a tes a n d
Nor a r a ch id on yl Ca r ba m a tes a n d Ur ea s
Edward W. Ng, Mie Mie Aung,† Mary E. Abood,† Billy R. Martin,† and Raj K. Razdan*
Organix, Inc., 240 Salem Street, Woburn, Massachusetts 01801, and Department of Pharmacology and Toxicology,
Medical College of Virginia, Richmond, Virginia 23298
Received December 18, 1998
To examine the effect of changing the amide bond of anandamide (5, AN) to a less hydrolyzable
moiety, analogues 1a -1l, 2a -2c, 3a -3c, and 4a -4h were synthesized from commercially
available arachidonyl alcohol or arachidonic acid and tested for their pharmacological activity.
Arachidonyl ethers 1a -1k were obtained through the coupling of the arachidonyl mesylate
(6) (generated from the mesylation of arachidonyl alcohol) with the appropriate alcohol in
potassium hydroxide. Arachidonyl ether 1l was obtained through the phase-transfer coupling
of arachidonyl alcohol with 2-(2-iodoethoxy)tetrahydropyran (which was generated from its
bromide) followed by cleavage of the tetrahydropyran group with Dowex resin. Arachidonyl
carbamates 2a -2c were obtained through the coupling of arachidonyl alcohol with the
appropriate isocyanates. Norarachidonyl carbamates 3a -3c and ureas 4a -4h were obtained
through the coupling of the norarachidonyl isocyanate (generated from arachidonic acid using
diphenyl phosphorazidate and triethylamine upon heating) with the appropriate alcohols and
amines, respectively. AN analogues 1-3 have shown poor binding affinities to the CB1 receptor
and fail to produce significant pharmacological effect at doses up to 30 mg/kg. Several ether
analogues 1 were also evaluated in the CB2 binding assay and were found to be of low affinity.
However, norarachidonyl urea analogues 4 have shown generally good binding affinities to
the CB1 receptor (Ki ) 55-746 nM) and pharmacological activity with AN-like profiles. The
most potent analogue of this series is the 2-fluoroethyl analogue 4f which binds 2 times better
than AN and was more active in several mouse behavioral assays. It was also observed that
urea analogues 4a and 4g, which have weak binding affinities to the CB1 receptor (Ki ) 436
and 347 nM, respectively), produced surprisingly potent pharmacological activity. These urea
analogues have also shown hydrolytic stability toward the amidase enzymes, responsible for
the primary degradation pathway of anandamide, in binding affinity assays in the absence of
the enzyme inhibitor PMSF.
In tr od u ction
of PMSF, a KD value of 5400 ( 1600 nM was observed
as a result of metabolic degradation. The hydrolyzable
amide moiety was the focus of study for increasing
stability and thus longer activity. Altering the head-
group of AN13-17 met with some success in generating
less hydrolyzable amide analogues. Introducing a sub-
stituent at the 2-position of the arachidonyl moiety to
sterically hinder access to the amide bond also produced
analogues which were more stable than AN.12,14,18-20
Since the discovery of anandamide (arachidonyletha-
nolamide, 5, AN) as a putative endogenous ligand which
competitively binds to the central cannabinoid receptor
(CB1),1 it has been established that AN exhibits can-
nabimimetic effects similar to those of ∆9-tetrahydro-
cannabinol (∆9-THC).2-4 AN has a faster onset and
shorter duration and is less potent than ∆9-THC in the
pharmacological tests of hypoactivity, analgesia, hypo-
thermia, and catalepsy. In addition, like the THCs, it
inhibits forskolin-stimulated adenylate cyclase activ-
ity5,6 as well as calcium currents as a partial agonist in
N18 neuroblastoma cells.7,8
Despite its faster onset than ∆9-THC, its short dura-
tion is attributed to its susceptibility to rapid enzymatic
hydrolysis of the amide bond by amidases.9-11 This
anandamide amidase has been shown to be sensitive
to serine protease inhibitors such as phenylmethane-
sulfonyl fluoride, PMSF.10 Binding studies using a
filtration assay demonstrated that AN displaces [3H]-
CP-55940 from rat whole brain P2 membrane prepara-
tions with a KD value of 89 ( 10 nM in the presence of
the enzyme inhibitor PMSF.12 However, in the absence
The availability of more stable AN analogues to
amidase hydrolysis will prove useful for future in vitro
and in vivo studies aimed at delineating their mode of
action in the brain. To design novel AN analogues less
susceptible to enzymatic hydrolysis, we have replaced
the amide bond in AN by a nonhydrolyzable moiety. We
employed groups which are chemically somewhat un-
reactive, are isosteric, and yet can participate in hydro-
gen bonding, a phenomenon which is very important in
ligand-receptor interaction for the CB1 receptor. These
analogues were designed to incorporate headgroups of
amide analogues which have previously shown good
binding affinities.12-18 With this background, we syn-
thesized the various arachidonyl ethers 1 and carbam-
ates 2 and norarachidonyl carbamates 3 and ureas 4
that are presented in this paper.
* To whom inquiries about this paper should be directed.
† Medical College of Virginia.
10.1021/jm980711w CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/28/1999

1976 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Ng et al.
Sch em e 1a
Ch em istr y
Arachidonyl ethers 1a -1k were prepared by the
alcoholic KOH reaction of arachidonyl mesylate (6) in
the appropriate alcohol at 50 °C (Scheme 1). The
arachidonyl mesylate was prepared by treatment of the
commercially available arachidonyl alcohol with meth-
anesulfonyl chloride in the presence of triethylamine in
methylene chloride. These ether analogues were syn-
thesized in 73-96% yields. Arachidonyl ether 1l was
prepared by the phase-transfer21 coupling of arachidonyl
alcohol with THP-protected 2-iodoethanol (prepared
from the corresponding 2-bromo analogue) using tet-
rabutylammonium bisulfate (TBAB) in 50% aqueous
NaOH heated at 55 °C (Scheme 1). The THP group of
ether 7 is then cleaved off in the presence of Dowex resin
in methanol to give the hydroxyethyl analogue.
The arachidonyl carbamates 2a -2c were prepared by
coupling arachidonyl alcohol with the appropriate alkyl
isocyanate in benzene at 65 °C (Scheme 2). These
carbamate analogues were synthesized in 89-98%
yields.
a
(a) MsCl, Et3N, CH2Cl2, 0 °C-rt; (b) KOH, R′OH, 50 °C; (c)
NaI, acetone, reflux; (d) TBAB, 50% NaOH/H2O, CH2Cl2, 50 °C;
(e) Dowex 50W-XB, MeOH, rt.
Sch em e 2
Sch em e 3
The norarachidonyl carbamates 3a -3c and ureas
4a -4h were prepared by coupling norarachidonyl iso-
cyanate with the appropriate alcohol or amine, respec-
tively, in benzene at 65 °C. The norarachidonyl iso-
cyanate was prepared by treatment of the commercially
available arachidonic acid with diphenyl phosphorazi-
date in the presence of triethylamine in benzene to
generate the acyl azide which undergoes a Curtius
rearrangement22,23 upon heating (Scheme 3). These
syntheses were carried out in one-pot reactions and
afforded the carbamates in 36-64% yields and the ureas
in 55-80% yields.
that further testing of these analogues could not be
justified and was not carried out. In addition, analogues
1b-1f and 1i-1l were evaluated in the CB2 receptor
binding assay, and all failed to produce significant
displacement at 10 µM.
The arachidonyl carbamate analogues 2a -2c (Table
1) were devised to study the presence of an oxygen
heteroatom at the 2-position of arachidonic acid. Al-
though these carbamates are two atoms longer than AN,
we regard this of limited significance. The reason for
this is that previously reported results on docosatetrae-
nylethanolamide, with 2-fold lower affinity as well as
being two carbon atoms longer than AN, has shown
similar pharmacological activity as AN.25,26 In addition,
work from our laboratories27 and Seltzman et al.28 had
shown that increasing the terminal chain length of the
arachidonyl moiety led to very potent and pharmaco-
logically active AN analogues. With this rationale we
synthesized the carbamate analogues 2a -2c, but they
all showed poor binding affinity for the receptor (Ki )
2200, 995, and 471 nM, respectively).
Resu lts a n d Discu ssion
The arachidonyl ether analogues 1a -1l (Table 1)
were devised such that the carbonyl of AN was reduced
to a methylene and the amide nitrogen was replaced
by an oxygen; i.e., the trans-amide bond has been
replaced by an oxymethylene ether linkage. The geom-
etry of the amide bond remains unaffected; this ap-
proach has been successfully applied in renin inhibi-
tors.24 Whereas the oxymethylene moiety is not suscepti-
ble to enzymatic hydrolysis, its ability to hydrogen bond
is reduced. However, all the arachidonyl ether analogues
1a -1l possessed relatively low affinity to the CB1
receptor, displacing [3H]CP-55,940 with Ki values rang-
ing from 578 to 4260 nM, which show poor competitive
binding at the cannabinoid receptor CB1. In vivo testing
of some of these analogues supported the correlation of
poor in vitro CB1 binding to poor in vivo activity (data
not shown). The low pharmacological activities indicated
The norarachidonyl carbamate and urea analogues
3a -3c and 4a -4h , respectively (Table 2), were designed
such that the carbon at the 2-position of AN is replaced

Unique Analogues of Anandamide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1977
Ta ble 1. Arachidonyl Ether (1a -1l) and Carbamate (2a -2c)
in hypothermia, ED50 0.85 mg/kg; and 4 times more
active in catalepsy, ED50 4.48 mg/kg). The other ana-
logues, in general, showed pharmacological activities
which correlated well with their binding affinities.
However, the methyl analogue 4a and the hydroxyethyl
analogue 4g were the two exceptions. Both exhibited
weak binding affinities (Ki ) 436 and 347 nM, respec-
tively) but showed potent pharmacological activity.
Analogue 4a was more potent than AN in both the
spontaneous activity (1.8-fold) and ring immobility (4.2-
fold) tests and was 12-fold more active in the hypo-
thermia test. Similarly, analogue 4g showed robust
pharmacological activity and was more potent that AN
in all tests.
It is noteworthy that like the SAR of AN,13,14 the
propyl and isopropyl urea analogues 4d and 4c were
both more active in binding affinity than the corre-
sponding hydroxyethyl analogue 4g. However, in phar-
macological activity, the hydroxyethyl analogue 4g was
more potent in spontaneous activity, tail flick, and
immobility than both 4c and 4d . In hypothermia, 4g
was more potent than 4d but was one-half as potent as
4c. The potent activities of 4a and 4g cannot be
explained at this time but may be due to different
pharmacokinetics of these analogues.
Analoguesa
analogue
R
Ki (nM)
5
AN
CH3
CH2CH3
CH(CH3)2
89 ( 10
4260 ( 628
2200 ( 561
871 ( 33
1080 ( 193
1310 ( 154
2480 ( 656
578 ( 38
1a
1b
1c
1d
1e
1f
1g
1h
1i
CH2CH2CH3
CH(CH3)CH2CH3
CH2CH(CH3)2
CH2CH(CH2)2
CH2CH2CH2CH3
CH2CHdCH2
CH2CtCH
1710 ( 451
1170 ( 287
1580 ( 263
885 ( 79
1j
1k
1l
CH2CH2F
CH2CH2OH
797 ( 25
2a
2b
2c
CONHCH2CH3
CONHCH(CH3)2
CONHCH2CH2CH3
2200 ( 257
995 ( 208
471 ( 44
a
Ki’s (nM) were determined in the presence of PMSF and are
expressed as means of (SE for at least three experiments.
by a nitrogen. Isosterically, the chain length on the
arachidonyl part is increased by only one atom. This
alteration also introduced a hydrogen-bonding site at
the nitrogen contained within the arachidonyl part of
AN. The norarachidonyl carbamate analogues 3a -3c
also showed weak binding affinities (Ki ) 218, 1230, and
331 nM, respectively). In in vivo tests, they showed only
marginal activity in the spontaneous activity test and
were inactive in the tail flick test at a dose of 30 mg/kg.
The norarachidonyl urea analogues 4a -4h showed
greater activity than the carbamate analogues. These
ureas were more potent in binding affinities (Ki values
of 55-746 nM) and showed more robust pharmacologi-
cal effects and AN-like profiles. The most active ana-
logue of this series was the norarachidonyl 2-fluoroethyl
urea 4f. In binding studies, it was twice as active (Ki )
55 ( 8 nM) as AN, and in in vivo study its activity was
equal to or greater than that of AN (5 times more active
in spontaneous activity, ED50 3.5 mg/kg; equiactive in
antinociception, ED50 6.4 mg/kg; 31 times more active
The susceptibility of these urea analogues toward
enzyme hydrolysis was also tested. The in vitro binding
assays for selected active analogues were studied in the
absence of the enzyme inhibitor PMSF. The binding
affinities were either unchanged (Ki ) 52 ( 9 nM for
the 2-fluoroethyl analogue) or halved (Ki ) 170 ( 21
and 204 ( 13 nM for the propyl and isopropyl analogues,
respectively) in these studies. These results show that
these urea analogues are indeed stable toward enzyme
hydrolysis.
Con clu sion s
A variety of novel ether, carbamate, and urea ana-
logues of anandamide was synthesized, and their bind-
ing affinity and pharmacological activity in the mouse
tetrad tests were determined. Only the urea analogues
exhibited good binding affinity (4f was twice as potent
Ta ble 2. Norarachidonyl Carbamate (3a -3c) and Urea (4a -4h ) Analoguesa
ED50 (mg/kg)
TF RT
analogue
X-R
Ki (nM) (PMSF)
Ki (nM)
SA
RI
5
AN
O-CH2CH3
O-CH(CH3)2
O-CH2CH2F
NH-CH3
NH-CH2CH3
NH-CH(CH3)2
NH-CH2CH2CH3
NH-CH2CH(CH3)2
NH-CH2CH2F
NH-CH2CH2OH
NH-CH2CH2OCH3
89 ( 10
218 ( 10
1230 ( 149
331 ( 47
436 ( 91
156 ( 40
131 ( 5
5400 ( 1600
ND
ND
ND
ND
17.9
36%b
35%b
18%b
9.9
6.2
5%b
3%b
20%b
12.3
3.3
26.5
ND
ND
ND
2.2
16.2
1.28
3.4
19.1
ND
ND
ND
4.51
19.9
38.0
5.4
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
ND
2.8
5.8
204 ( 13
170 ( 21
ND
52 ( 9
ND
14.0
8.9
91 ( 7
7.2
335 ( 46
55 ( 8
347 ( 33
746 ( 22
59%b
3.5
26%b
6.4
ND
ND
4.48
3.9
0.85
2.6
3.1
4.0
ND
69%b
32%b
-2.8 °Cb
3%b
a
Ki’s (nM) were determined in the presence and absence of PMSF and are expressed as means of (SE for at least three experiments.
The pharmacological measures included inhibition of spontaneous activity (SA), antinociception as measured by tail flick response (TF),
hypothermia as changes in rectal temperature (RT), and ring immobility (RI). ND, not determined. Activity at 30 mg/kg dosage.
b

1978 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Ng et al.
Gen er a l P r oced u r e for th e Syn th eses of Ar a ch id on yl
Eth er s. To a solution of arachidonyl mesylate (160 mg, 0.44
mmol) in an appropriate alcohol (8 mL) was added freshly
ground potassium hydroxide (73 mg, 1.30 mmol), and the
mixture was heated to 50 °C overnight. The mixture was cooled
to room temperature, quenched with water, and extracted with
ethyl acetate. The organic extracts were washed with water
and brine and dried over magnesium sulfate. The filtrate was
concentrated in vacuo, and the crude product was purified by
silica gel column chromatography eluting with 1% EtOAc/
hexanes.
as AN) and showed robust pharmacological activity. It
was also found that the urea analogues were relatively
more stable to amidase hydrolysis than AN since their
binding affinities were marginally affected when deter-
mined in the presence or absence of the enzyme inhibi-
tor PMSF.
The present studies also contribute to the known SAR
of the ethanolamido headgroup in AN. The known SAR
is quite strict and indicates that (i) monosubstitution
of the amide is a requirement for activity; (ii) substitu-
tion by an alkyl, fluoroalkyl, or hydroxyalkyl moiety
increases activity with a two or three carbon chain being
optimal; (iii) branching of the chain (methyl is optimal)
enhances stability and in some cases binding affinity;
and (iv) substitution of the hydroxyl in AN by a methyl
ether, phenyl ether, or phosphate derivative of AN
decreases activity, while introduction of an amino or a
carboxyl group eliminates activity.12-15,18
A comparison of the biological activities of carbamate
and urea analogues in the present study provides
further insights on the interactions between the CB1
receptor and AN. It shows that the presence of an
additional hydrogen-donating nitrogen atom, as in
ureas, contributes to better binding affinity and phar-
macological activity. This enhancement suggests that
hydrogen bonding with the receptor is involved at this
site, which is in complete agreement with the known
SAR and emphasizes its role in the interaction with the
CB1 receptor. All this information could prove valuable
in the design of novel and metabolically stable analogues
of AN.
Ar a ch id on yl m eth yl eth er (1a ): pale-yellow oil, 93%; 1H
NMR (100 MHz, CDCl3) δ 0.89 (bt, 3 H), 1.28-1.58 (m, 10 H),
2.04 (m, 4 H), 2.78 (m, 6 H), 3.32 (s, 3 H), 3.37 (t, J ) 5.69 Hz,
2 H), 5.37 (m, 8 H); 13C NMR (100 MHz, CDCl3) δ 13.99, 22.53,
25.60 (3), 26.14, 26.99, 27.18, 29.23, 29.29, 31.48, 58.44, 72.68,
127.53, 127.92 (2), 127.98, 128.32, 128.47, 129.91, 130.37;
HRMS calcd for C21H37O (MH+) 305.2844, found 305.2826.
Anal. (C21H36O‚0.1CHCl3‚0.2H2O) C, H.
1
Ar a ch id on yl eth yl eth er (1b): yellow oil, 96%; H NMR
(100 MHz, CDCl3) δ 0.89 (bt, 3 H), 1.20 (t, J ) 7.00 Hz, 3 H),
1.26-1.64 (m, 10 H), 2.06 (m, 4 H), 2.84 (m, 6 H), 3.41 (t, J )
6.39 Hz, 4 H), 3.47 (q, J ) 14.0, 7.02 Hz, 2 H), 5.37 (m, 8 H);
13C NMR (100 MHz, CDCl3) δ 13.95, 15.14, 22.50, 25.57 (3),
26.20, 26.98, 27.14, 29.26, 29.38, 31.45, 65.97, 70.44, 127.50,
127.84 (2), 127.92, 128.30, 128.41, 129.92, 130.31; HRMS calcd
for C22H38O (M) 318.2922, found 318.2952. Anal. (C22H38O‚
0.8CHCl3‚0.2H2O) C, H.
Ar a ch id on yl 1-m eth yleth yl eth er (1c): yellow oil, 88%;
1H NMR (100 MHz, CDCl3) δ 0.89 (bt, 3 H), 1.15 (d, J ) 6.13
Hz, 3 H), 1.28-1.55 (m, 10 H), 2.05 (m, 4 H), 2.82 (m, 6 H),
3.40 (t, J ) 6.36 Hz, 2 H), 3.46 (sept., J ) 6.12 Hz, 1 H), 5.37
(m, 8 H); 13C NMR (100 MHz, CDCl3) δ 14.02, 22.14 (2), 22.56,
25.63 (3), 26.32, 27.05, 27.21, 29.32, 29.81, 31.51, 67.99, 71.25,
127.56, 127.86, 127.92, 128.01, 128.41, 128.50, 130.10, 130.42;
HRMS calcd for C23H41O (MH+) 333.3157, found 333.3131.
Anal. (C23H40O‚0.1 CHCl3‚0.2 H2O) C, H.
Exp er im en ta l Section
Ar a ch id on yl p r op yl eth er (1d ): colorless oil, 92%; 1H
NMR (100 MHz, CDCl3) δ 0.88 (m, 6 H), 1.28-1.69 (m, 12 H),
2.07 (m, 4 H), 2.79 (m, 4 H), 2.79 (m, 6 H), 3.36 (t, J ) 6.63
Hz, 2 H), 3.41 (t, J ) 6.09 Hz, 2 H), 5.37 (m, 8 H); 13C NMR
(100 MHz, CDCl3) δ 10.54, 13.97, 22.52, 22.91, 25.59 (2), 26.22,
27.00, 27.17, 29.28, 29.37, 31.48, 70.63, 72.51, 127.52, 127.86
(2), 127.96, 128.34, 128.44, 129.99, 130.34; HRMS calcd for
1H and 13C NMR spectra were recorded on either a Bruker
100, a J EOL Eclipse 300, or a Varian XL400 spectrophotom-
eter using CDCl3 as the solvent with trimethylsilane as an
internal standard. Thin-layer chromatography (TLC) was
carried out on Baker Si 250F plates and was developed upon
treatment with phosphomolybdic acid (PMA). Flash column
chromatography was carried out on EM Science silica gel 60.
Elemental analyses were performed by Atlantic Microlab, Inc.,
Atlanta, GA, and were found to be within (0.4% of calculated
values for the elements shown, unless otherwise noted. Mass
spectrometry was performed by the Chemistry Department at
Boston University, Boston, MA.
C
23H41O (MH+) 333.3157, found 333.3175. Anal. (C23H40O‚
0.1CHCl3) C, H.
Ar a ch id on yl (1-m eth ylp r op yl) eth er (1e): yellow oil,
77%; 1H NMR (100 MHz, CDCl3) δ 0.89 (t, J ) 7.00 Hz, 6 H),
1.11 (d, J ) 6.15 Hz, 3 H), 1.26-1.66 (m, 12 H), 2.07 (m, 4 H),
2.78 (m, 6 H), 3.38 (m, 1 H), 3.38 (t, J ) 6.15 Hz, 2 H), 5.37
(m, 8 H); 13C NMR (100 MHz, CDCl3) δ 9.80, 14.00, 19.23,
22.54, 25.60 (2), 26.32, 27.04, 27.19, 29.24, 29.29, 29.81, 31.49,
68.22, 76.56, 127.54, 127.81, 127.89, 127.97, 128.46, 130.08,
130.38; HRMS calcd for C24H43O (MH+) 347.3314, found,
347.3252. Anal. (C24H42O‚0.1CHCl3) C, H.
All final compounds were oils. The purity of the products
on which the high-resolution mass spectral data are reported
1
was determined by H and 13C NMR spectroscopy as well as
TLC. The presence of chloroform or water in the target
compounds was confirmed by 1H and 13C NMR. For TLC
solvent system, 10% EtOAc/hexanes was used for ethers 1a -
1k ; 20% EtOAc/hexanes was used for ether 1l and carbamates
2a -2c and 3a -3c; 50% EtOAc/hexanes was used for ureas
4a -4f and 4h ; and EtOAc was used for urea 4g.
Ar a ch id on yl (2-m eth ylp r op yl) eth er (1f): pale-yellow
1
oil, 94%; H NMR (100 MHz, CDCl3) δ 0.89 (m, 3 H), 0.89 (d,
J ) 6.64 Hz, 6 H), 1.28-1.57 (m, 11 H), 2.05 (m, 4 H), 2.79
(m, 6 H), 3.16 (d, J ) 6.64 Hz, 2 H), 3.40 (t, J ) 6.02 Hz, 2 H),
5.57 (m, 8 H); 13C NMR (100 MHz, CDCl3) δ 14.01, 19.37 (2),
22.54, 25.62 (3), 26.26, 27.02, 27.20, 28.44, 29.33, 29.37, 31.50,
70.81, 77.83, 127.55, 127.88 (2), 127.98, 128.37, 128.46, 130.04,
130.37; HRMS calcd for C24H43O (MH+) 347.3314, found
347.3291. Anal. (C24H42O‚0.3H2O) C, H.
Ar a ch id on yl Mesyla te (6). To a solution of arachidonyl
alcohol (400 mg, 1.38 mmol) and triethylamine (0.58 mL, 4.14
mmol) in dichloromethane (20 mL) at 0 °C was added meth-
anesulfonyl chloride (0.16 mL, 2.07 mmol). The mixture was
warmed to room temperature and stirred for an additional 2
h. The mixture was quenched with water and extracted with
ethyl acetate. The organic extracts were washed with water
and brine and dried over magnesium sulfate. The filtrate was
concentrated in vacuo, and the crude product was purified by
silica gel column chromatography eluting with 15% EtOAc/
hexanes to give a colorless oil (512 mg, 97%): 1H NMR (100
MHz, CDCl3) δ 0.88 (bt, 3 H), 1.18-1.76 (m, 10 H), 2.03-2.14
(m, 4 H), 2.80 (m, 6 H), 2.99 (s, 3 H), 4.22 (t, J ) 6.27 Hz, 2
H), 5.36 (m, 8 H); 13C NMR (300 MHz, CDCl3) δ 14.16, 22.65,
25.48, 25.71, 26.59, 27.29, 28.75, 29.39, 31.58, 37.42, 70.03,
127.59, 127.90, 128.17, 128.31, 128.66, 128.74, 129.20, 130.55.
Ar a ch id on yl cyclop r op ylm eth yl eth er (1g): yellow oil,
1
84%; H NMR (100 MHz, CDCl3) δ 0.20 (m, 2 H), 0.53 (m, 2
H), 0.89 (bt, 3 H), 1.28-1.68 (m, 11 H), 2.04 (m, 4 H), 2.76 (m,
6 H), 3.24 (d, J ) 6.81 Hz, 2 H), 3.43 (t, J ) 6.36 Hz, 2 H),
5.37 (m, 8 H); 13C NMR (100 MHz, CDCl3) δ 2.87 (2), 10.61,
13.97, 22.50, 25.57 (3), 26.19, 26.97, 27.14, 29.26, 29.35, 31.45,
70.48, 75.45, 127.50, 127.84 (2), 127.93, 128.32, 128.41, 129.95,
130.32; HRMS calcd for C24H41O (MH+) 345.3157, found
345.3173. Anal. (C24H40O‚0.3 H2O) C, H.
Ar a ch id on yl bu tyl eth er (1h ): yellow oil, 94%; 1H NMR

Unique Analogues of Anandamide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1979
(100 MHz, CDCl3) δ 0.90 (m, 6 H), 1.25-1.61 (m, 14 H), 2.07
(m, 4 H), 2.79 (m, 6 H), 3.40 (t, J ) 6.33 Hz, 4 H), 5.37 (m, 8
H); 13C NMR (100 MHz, CDCl3) δ 13.86, 13.98, 19.35, 22.53,
25.61 (3), 26.23, 27.01, 27.19, 29.29, 29.39, 31.49, 31.86, 70.62,
70.68, 127.53, 127.87 (2), 127.97, 128.35, 128.46, 130.00,
130.36; HRMS calcd for C24H43O (MH+) 347.3314, found
347.3313. Anal. (C24H42O‚0.3CHCl3) C, H.
mixture was cooled to room temperature, quenched with water,
and extracted with ethyl acetate. The organic extracts were
washed with water and brine and dried over magnesium
sulfate. The filtrate was concentrated in vacuo, and the crude
product was purified by silica gel column chromatography
eluting with 10% EtOAc/hexanes.
Ar a ch id on yl N-eth ylca r ba m a te (2a ): pale-yellow oil,
89%; 1H NMR (300 MHz, CDCl3) δ 0.88 (t, J ) 6.88 Hz, 3 H),
1.12 (t, J ) 7.30 Hz, 3 H), 1.25-1.37 (m, 6 H), 1.43 (m, 2 H),
1.62 (m, 2 H), 2.08 (m, 4 H), 2.82 (m, 6 H), 3.20 (quint., 2 H),
4.04 (t, J ) 6.47 Hz, 2 H), 4.60 (bs, 1 H), 5.35 (m, 8 H); 13C
NMR (300 MHz, CDCl3) δ 14.14, 15.38, 22.65, 25.71 (3), 26.00,
26.91, 27.29, 28.77, 29.39, 31.59, 35.86, 64.72, 127.61, 127.97,
128.19, 128.25, 128.36, 128.62, 129.78, 130.55, 156.71; HRMS
calcd for C23H40NO2 (MH+) 362.3059, found 362.3083. Anal.
(C23H39NO2‚4.0H2O) C, N; H: calcd, 10.92; found, 9.04.
Ar a ch id on yl a llyl eth er (1i): pale-yellow oil, 73%; 1H
NMR (100 MHz, CDCl3) δ 0.89 (bt, 3 H), 1.26-1.59 (m, 10 H),
2.06 (m, 4 H), 2.79 (m, 6 H), 3.43 (t, J ) 6.32 Hz, 2 H), 3.96
(td, J ) 5.49, 1.36 Hz, 2 H), 5.10-5.23 (m, 2 H), 5.37 (m, 8 H),
5.74-6.12 (m, 1 H); 13C NMR (100 MHz, CDCl3) δ 13.96, 22.49,
25.56 (3), 26.16, 26.95, 27.14, 29.28 (2), 31.44, 70.14, 116.43,
127.49, 127.85 (2), 127.92, 128.28, 128.40, 129.87, 130.29,
135.01; HRMS calcd for C23H39O (MH+) 331.3000, found
331.29780. Anal. (C23H38O‚0.1CHCl3) C, H.
Ar a ch id on yl p r op a r gyl eth er (1j): colorless oil, 66%; 1H
NMR (100 MHz, CDCl3) δ 0.89 (bt, 3 H), 1.26-1.63 (m, 8 H),
2.07 (m, 6 H), 2.41 (t, J ) 2.38 Hz, 1 H), 2.60 (m, 6 H), 3.52 (t,
J ) 6.26 Hz, 2 H), 4.13 (d, J ) 2.39 Hz, 2 H), 5.37 (m, 8 H);
13C NMR (100 MHz, CDCl3) δ 13.99, 22.51, 25.59 (3), 26.05,
26.89, 27.16, 29.04, 29.26, 31.47, 57.93, 69.93, 73.99, 79.96,
127.51, 127.86, 127.98 (2), 128.29, 128.46, 129.83, 130.38;
HRMS calcd for C23H37O (MH+) 329.2844, found 329.2818.
Anal. (C23H36O) C, H.
Ar a ch id on yl N-(1-m eth yleth yl)ca r ba m a te (2b): pale-
yellow oil, 94%; 1H NMR (300 MHz, CDCl3) δ 0.88 (t, J ) 6.88
Hz, 3 H), 1.14 (d, J ) 6.33 Hz, 6 H), 1.26-1.30 (m, 8 H), 1.38
(m, 2 H), 1.63 (m, 2 H), 2.08 (m, 4 H), 2.82 (m, 6 H), 3.79 (bm,
1 H), 4.03 (t, J ) 6.33 Hz, 2 H), 4.46 (bs, 1 H), 5.36 (m, 8 H);
13C NMR (300 MHz, CDCl3) δ 14.14, 22.65, 23.16 (2), 25.71
(3), 26.02, 26.92, 27.29, 28.77, 29.40, 31.59, 43.03, 64.62,
127.62, 127.97, 128.25 (2), 128.37, 128.63, 129.80, 130.55,
156.00; HRMS calcd for C24H42NO2 (MH+) 376.3215, found
376.3229. Anal. (C24H41NO2‚3.8H2O) C, N; H: calcd, 11.03;
found, 9.44.
Ar a ch id on yl 2-flu or oeth yl eth er (1k ): colorless oil, 94%;
1H NMR (100 MHz, CDCl3) δ 0.89 (bt, 3 H), 1.28-1.63 (m, 8
H), 2.11 (m, 6 H), 2.78 (m, 6 H), 3.48 (t, J ) 3.85 Hz, 1 H),
3.50 (t, J ) 6.15 Hz, 2 H), 3.81 (t, J ) 4.21 Hz, 1 H), 4.31 (t,
Ar a ch id on yl N-p r op ylca r ba m a te (2c): pale-yellow oil,
1
98%; H NMR (300 MHz, CDCl3) δ 0.89 (m, 6 H), 1.28-1.34
J ) 4.20 Hz, 1 H), 4.79 (t, J ) 4.21 Hz, 1 H), 5.37 (m, 8 H); 13
C
(m, 8 H), 1.47 (m, 2 H), 1.61 (m, 2 H), 2.06 (m, 4 H), 2.81 (m,
6 H), 3.12 (m, 2 H), 4.04 (t, J ) 6.47 Hz, 2 H), 4.65 (bs, 1 H),
5.36 (m, 8 H); 13C NMR (300 MHz, CDCl3) δ 11.27, 14.13, 22.64,
23.32, 25.71 (3), 26.00, 26.91, 27.29, 28.77, 29.39, 31.58, 42.76,
64.74, 127.61, 127.97, 128.19, 128.25, 128.36, 128.61, 129.78,
130.55, 156.85; HRMS calcd for C24H42NO2 (MH+) 376.3215,
found 376.3197. Anal. (C24H41NO2‚4.0H2O) C, N; H: calcd,
11.03; found, 9.35.
NMR (100 MHz, CDCl3) δ 13.98, 22.50, 25.57 (3), 26.04, 26.92,
27.15, 29.20, 29.26, 31.45, 69.41, 70.19, 71.39, 79.65, 86.37,
127.50, 127.84, 127.96 (2), 128.29, 128.44, 129.85, 130.35;
HRMS calcd for C22H37OF (M) 336.2828, found 336.2831. Anal.
(C22H37OF‚0.2CHCl3‚0.3H2O) C, H.
Ar a ch id on yl 2-Hyd r oxyeth yl Eth er (1l). To a solution
of 2-(2-bromoethoxy)tetrahydro-2H-pyran (1.00 g, 4.73 mmol)
in acetone (20 mL) was added sodium iodide (1.44 g, 9.57
mmol). The mixture was refluxed for 3 h and then filtered to
remove the solid. The filtrate was then concentrated in vacuo
to isolate the 2-(2-iodoethoxy)tetrahydro-2H-pyran (69% yield)
which was used for the next step.
Gen er a l P r oced u r e for th e Syn th eses of Nor a r a ch i-
d on yl Ca r ba m a tes a n d Ur ea s. To a solution of arachidonic
acid (150 mg, 0.49 mmol) in benzene (5 mL) were added
triethylamine (0.07 mL, 0.49 mmol) and diphenyl phospho-
razidate (DPPA) (0.11 mL, 0.49 mmol), and the mixture was
heated to 65 °C for 2 h. An excess of the alcohol or amine (for
the corresponding carbamate or urea, respectively) was added
via syringe, and the mixture was stirred at 65 °C overnight.
The mixture was cooled to room temperature, quenched with
water, and extracted with ethyl acetate. The organic extracts
were washed with water and brine and dried over magnesium
sulfate. The filtrate was concentrated in vacuo, and the crude
product was purified by silica gel column chromatography
eluting with 10% EtOAc/hexanes for compounds 3a -3c, 50%
EtOAc/hexanes for compounds 4a -4f and 4h , and 75% EtOAc/
hexanes for compound 4g.
Eth yl N-n or a r a ch id on ylca r ba m a te (3a ): yellow oil,
64%; 1H NMR (100 MHz, CDCl3) δ 0.88 (bt, 3 H), 1.23 (t, J )
7.11 Hz, 3 H), 1.28-1.37 (m, 6 H), 1.56 (quint., J ) 6.90 Hz,
2 H), 2.08 (m, 4 H), 2.81 (m, 6 H), 3.17 (q, J ) 13.4, 6.63 Hz,
2 H), 4.10 (q, J ) 14.2, 7.09 Hz, 2 H), 4.60 (bs, 1 H), 5.37 (m,
8 H); 13C NMR (100 MHz, CDCl3) δ 13.93, 14.54, 22.45, 24.36,
25.52 (3), 27.11, 29.20, 29.78, 31.41, 40.51, 60.53, 127.44,
127.72, 128.02, 128.13, 128.48, 128.52, 128.93, 130.35, 156.57;
HRMS calcd for C22H38NO2 (MH+) 348.2902, found 348.2920.
Anal. (C22H37NO2‚0.3H2O) C, H, N.
To a mixture of arachidonyl alcohol (73 mg, 0.25 mmol), 2-(2-
iodoethoxy)tetrahydro-2H-pyran (0.64 g, 2.5 mmol), and tet-
rabutylammonium bisulfate (TBAB) (85 mg, 0.25 mmol) was
added a solution of sodium hydroxide (70 mg, 2.41 mmol) in
water (0.14 mL). The mixture was heated to 55 °C for 3 days;
however, the reaction remained incomplete. The mixture was
cooled to room temperature, quenched with water, and ex-
tracted with ethyl acetate. The organic extracts were washed
with water and brine and dried over magnesium sulfate. The
filtrate was removed in vacuo, and the crude product was
purified by silica gel column chromatography. The arachidonyl
2-ethoxytetrahydro-2H-pyranyl ether (7) was isolated as a
beige oil (44 mg, 41% yield, 41% conversion). The arachidonyl
2-ethoxytetrahydro-2H-pyranyl ether was then treated with
Dowex 50W-X8 (4 mg, 10 wt %) in methanol (1 mL) at room
temperature overnight to cleave the THP protecting group. The
mixture was filtered to remove the resin and concentrated in
vacuo to give the crude product. The crude product was
purified by silica gel column chromatography eluting with 10%
EtOAc/hexanes to give a yellow oil (20 mg, 73%): 1H NMR
(100 MHz, CDCl3) δ 0.88 (bt, 3 H), 1.25-1.68 (m, 8 H), 2.07
(m, 6 H), 2.79 (m, 6 H), 3.49 (m, 4 H), 3.71 (m, 2 H), 5.37 (m,
8 H); 13C NMR (100 MHz, CDCl3) δ 14.02, 22.53, 25.61 (3),
26.10, 26.96, 27.18, 29.24, 29.28, 31.48, 61.79, 71.18, 71.77,
127.52, 127.87, 128.04 (2), 128.29, 128.50, 129.84, 130.42;
HRMS calcd for C22H39O2 (MH+), 335.2950; found, 335.2940.
Anal. (C22H38O2‚0.8CHCl3) C, H.
1-Meth yleth yl N-n or a r a ch id on ylca r ba m a te (3b): pale-
1
yellow oil, 36%; H NMR (100 MHz, CDCl3) δ 0.89 (bt, 3 H),
1.22 (d, J ) 6.25 Hz, 6 H), 1.28-1.38 (m, 6 H), 1.56 (quint., J
) 6.90 Hz, 2 H), 2.08 (m, 4 H), 2.81 (m, 6 H), 3.16 (q, J ) 13.6,
6.41 Hz, 2 H), 4.55 (bs, 1 H), 4.90 (sept., J ) 6.23 Hz, 1 H),
5.37 (m, 8 H); 13C NMR (100 MHz, CDCl3) δ 14.01, 22.14 (2),
22.53, 24.44, 25.59 (3), 27.17, 29.28, 29.87, 31.47, 40.52, 67.83,
127.50, 127.80, 128.10, 128.19, 128.57 (2), 129.02, 130.44,
156.27; HRMS calcd for C23H40NO2 (MH+) 362.3059, found
362.3047. Anal. (C23H39NO2‚0.3H2O) C, H, N.
Gen er a l P r oced u r e for th e Syn th eses of Ar a ch id on yl
Ca r ba m a tes. To a solution of arachidonyl alcohol (100 mg,
0.35 mmol) in benzene (5 mL) heated at 65 °C for 2 h was
added an excess of the alkyl isocyanate (1.03 mmol) via
syringe, and the mixture was stirred at 65 °C overnight. The
2-F lu or oeth yl N-n or a r a ch id on ylca r ba m a te (3c): yel-

1980 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Ng et al.
4.82 Hz, 2 H), 4.37 (bs, 1 H), 5.35 (m, 9 H), 5.59 (bt, 1 H); 13
NMR (300 MHz, CDCl3) δ 14.14, 22.64, 24.66, 25.69 (3), 27.29,
29.39, 30.20, 31.58, 40.19, 43.20, 62.90, 127.57, 127.88, 128.19,
128.37, 128.71 (2), 129.15, 130.60, 159.93; LRMS (CI) found
for C22H39N2O2 (MH+) 363.2. Anal. (C22H38N2O2‚1.2H2O) C, N;
H: calcd, 10.60; found, 10.04.
1-Nor a r a ch id on yl-3-(2′-m eth oxyeth yl)u r ea (4h ): yellow
oil, 67%; 1H NMR (300 MHz, CDCl3) δ 0.87 (t, J ) 6.88 Hz, 3
H), 1.24-1.37 (m, 6 H), 1.55 (quint., 2 H), 2.07 (m, 4 H), 2.81
(m, 6 H), 3.15 (q, J ) 6.67, 13.3 Hz, 2 H), 3.35 (m, 5 H), 3.44
(t, J ) 4.95 Hz, 2 H), 4.77 (bt, 1 H), 4.87 (bt, 1 H), 5.36 (m, 8
H); 13C NMR (300 MHz, CDCl3) δ 14.15, 22.65, 24.68, 25.71
(3), 27.29, 29.39, 30.18, 31.59, 40.23, 40.49, 58.81, 72.49,
127.59, 127.91, 128.31 (2), 128.61, 128.68, 129.27, 130.59,
158.58. Anal. (C23H40N2O2‚0.5H2O) C, H, N.
P h a r m a cology. 1. Dr u g P r ep a r a tion a n d Ad m in istr a -
tion . For binding assays, compounds were prepared as 1 mg/
mL stock solutions in absolute ethanol and were stored at -20
°C. For behavioral assays, drugs were dissolved in a 1:1:18
mixture of ethanol, emulphor (GAF Corp., Linden, NJ ), and
saline (0.9% NaCl) and were administered intravenously (iv)
in the mouse tail vein in volumes of 0.1 mL/10 g of body weight.
2. Bin d in g Assa ys. Radioligand binding to P2 membrane
preparations were performed as described elsewhere.12,29 Etha-
nolic 1 mg/mL stock solutions of anandamide analogues were
diluted in buffer (50 mM Tris-HCl, 1 mM EDTA, 3 mM MgCl2,
and 5 mg/mL bovine serum albumin) without evaporation of
the ethanol (final concentration not exceeding 0.4%). Analogue
concentrations ranging from 1 nM to 10 µM and a 1 nM
concentration of [3H]CP-55,940 were used. Binding was initi-
ated with the addition of 100-150 µg of membrane preparation
protein. Nonspecific binding was determined in the presence
of 1 µM CP-55,940. The binding experiments were performed
either with or without the amidase inhibitor phenylmethane-
sulfonyl fluoride (PMSF; 50 µM). After 1 h incubation at 30
°C, the reaction was terminated with the addition of 2 mL of
ice-cold buffer (50 mM Tris-HCl, 1 mg/mL bovine serum
albumin) followed by rapid filtration through PEI-treated
filters. The assays were performed in triplicate, and the results
represent the combined data from three to six independent
experiments.
3. Beh a vior a l Eva lu a tion s. Mice were acclimated to the
laboratory overnight. Depression of locomotor activity and
antinociception, as determined by the tail flick (TF) response
to heat stimulus,30 were measured in the same animal. Control
tail flick latencies of 2-4 s were measured for each animal
with a standard tail flick apparatus prior to drug or vehicle
administration. Four minutes following an iv injection of either
vehicle or drug, mice were tested for tail flick response.
Immediately thereafter, the mice were placed into individual
photocell activity cages (11 × 6.5 in.) for assessment of
spontaneous activity. For the next 10 min the total number of
beam interruptions in the 16 photocell beams/cage was re-
corded using a Digiscan Animal Activity Monitor (Omnitech
Electronics Inc., Columbus, OH). Spontaneous activity was
expressed as percent of control activity. Antinociception was
expressed as the percent maximum possible effect (MPE) using
a 10-s maximum test latency as described earlier.30 Each dose
tested in the antinociception and hypomotility assays repre-
sents one group of animals (6 mice/group). Cannabinoid-
induced hypothermia and immobility were determined in a
separate group of animals. Prior to vehicle or drug administra-
tion, rectal temperature was determined by a thermistor probe
(inserted 25 mm) and a telethermometer (Yellow Springs
Instrument Co., Yellow Springs, OH). Four minutes after the
iv injection of the drug, mice were tested for body temperature.
The difference between pre- and postinjection rectal temper-
atures was calculated. Immediately after measure of body
temperature, the mice were placed on a 5.5-cm ring attached
at a height of 16 cm to a ring stand, and the amount of time
the animals remained motionless during a 5-min period was
recorded.31 The time that each animal remained motionless
on the ring was divided by 300 s and multiplied by 100 to
obtain a percent immobility rating.
low oil, 60%;1H NMR (400 MHz, CDCl3) δ 0.87 (t, J ) 6.74
Hz, 3 H), 1.25-1.36 (m, 6 H), 1.57 (m, 2 H), 2.04 (q, J ) 14.1,
7.03 Hz, 2 H), 2.09 (q, J ) 14.1, 7.03 Hz, 2 H), 2.81 (m, 6 H),
3.18 (q, J ) 13.5, 7.03 Hz, 2 H), 4.25 (t, J ) 3.81 Hz, 1 H),
4.32 (t, J ) 3.81 Hz, 1 H), 4.50 (t, J ) 4.10 Hz, 1 H), 4.62 (t,
J ) 4.10 Hz, 1 H), 4.76 (bs, 1 H), 5.38 (m, 8 H); HRMS calcd
C
for C22H37NO2F (MH+) 366.2808, found 366.2798. Anal. (C22H36
-
NO2F‚0.1CHCl3) C, H, N.
1-Nor a r a ch id on yl-3-m eth ylu r ea (4a ): yellow oil, 77%;
1H NMR (300 MHz, CDCl3) δ 0.87 (t, J ) 6.88 Hz, 3 H), 1.24-
1.37 (m, 6 H), 1.56 (quint., 2 H), 2.07 (m, 4 H), 2.75 (d, J )
4.95 Hz, 3 H), 2.81 (m, 6 H), 3.16 (q, J ) 6.60 Hz, 2 H), 4.65
(bm, 2 H), 5.36 (m, 8 H); 13C NMR (300 MHz, CDCl3) δ 14.15,
22.64, 24.68, 25.71 (3), 27.29 (2), 29.39, 30.22, 31.59, 40.29,
127.58, 127.90, 128.23, 128.32, 128.70 (2), 129.28, 130.61,
159.09; HRMS calcd for C21H37N2O (MH+) 333.2906, found
333.2912. Anal. (C21H36N2O‚1.3H2O) C, N; H: calcd, 11.18;
found, 10.26.
1-Nor a r a ch id on yl-3-eth ylu r ea (4b): yellow oil, 76%; 1H
NMR (300 MHz, CDCl3) δ 0.88 (t, J ) 6.88 Hz, 3 H), 1.12 (t,
J ) 7.30 Hz, 3 H), 1.24-1.34 (m, 7 H), 1.56 (quint., 1 H), 2.07
(m, 4 H), 2.80 (m, 6 H), 3.17 (m, 4 H), 4.37 (bt, 1 H), 4.43 (bt,
1 H), 5.37 (m, 8 H); 13C NMR (300 MHz, CDCl3) δ 14.15, 15.58,
22.65, 24.70, 25.71 (3), 27.30, 29.39, 30.20, 31.59, 35.41, 40.26,
127.58, 127.90, 128.23, 128.32, 128.70 (2), 129.28, 130.61,
158.50. Anal. (C22H38N2O‚0.9H2O) C, N; H: calcd, 11.05; found,
10.60.
1-Nor a r a ch id on yl-3-(1′-m eth yleth yl)u r ea (4c): yellow
oil, 80%; 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J ) 6.78 Hz, 3
H), 1.12 (d, J ) 6.59 Hz, 6 H), 1.26-1.37 (m, 6 H), 1.56 (quint.,
J ) 7.32 Hz, 2 H), 2.03 (q, J ) 13.9, 6.96 Hz, 2 H), 2.09 (q, J
) 12.6, 6.78 Hz, 2 H), 2.80 (m, 6 H), 3.14 (q, 2 H), 3.82 (m, 1
H), 4.01 (bs, 1 H), 4.18 (bs, 1 H), 5.38 (m, 8 H); 13C NMR (300
MHz, CDCl3) δ 14.03, 22.51, 23.43 (2), 24.58, 25.54, 25.57 (2),
27.16, 29.27, 30.11, 31.45, 40.02, 42.07, 127.44, 127.76, 128.10,
128.19, 128.49, 128.57, 129.15, 130.46, 157.82; HRMS calcd
for C23H41N2O (MH+) 361.3219, found 361.3188. Anal. (C23H40
-
N2O‚0.1CHCl3‚0.2H2O) C, H, N.
1-Nor a r a ch id on yl-3-p r op ylu r ea (4d ): orange oil, 80%;
1H NMR (400 MHz, CDCl3) δ 0.87 (t, J ) 6.78 Hz, 3 H), 0.90
(t, J ) 7.32 Hz, 3 H), 1.26-1.36 (m, 6 H), 1.49 (quint., J )
7.51 Hz, 2 H), 1.56 (quint., J ) 7.32 Hz, 2 H), 2.03 (q, J )
14.1, 6.78 Hz, 2 H), 2.10 (q, J ) 12.6, 7.14 Hz, 2 H), 2.80 (m,
6 H), 3.10 (t, J ) 7.14 Hz, 2 H), 3.16 (t, J ) 6.96 Hz, 2 H), 4.28
(bs, 2 H), 5.36 (m, 8 H); 13C NMR (300 MHz, CDCl3) δ 11.34,
14.05, 22.54, 23.44, 24.59, 25.58 (3), 27.17, 29.28, 30.15, 31.47,
40.08, 42.21, 127.45, 127.79, 128.13, 128.20, 128.49, 128.59,
129.18, 130.48, 158.53; HRMS calcd for C23H41N2O (MH+)
361.3219, found 361.3186. Anal. (C23H40N2O‚0.2CHCl3‚0.2H2O)
C, H, N.
1-Nor a r a ch id on yl-3-(2′-m eth ylp r op yl)u r ea (4e): orange
oil, 58%; 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J ) 7.03 Hz, 3
H), 0.89 (d, J ) 7.03 Hz, 6 H), 1.25-1.35 (m, 6 H), 1.56 (quint.,
J ) 7.33 Hz, 2 H), 1.72 (sept., J ) 6.74 Hz, 1 H), 2.03 (q, J )
13.8, 6.74 Hz, 2 H), 2.10 (q, J ) 12.9, 7.03 Hz, 2 H), 2.80 (m,
6 H), 2.96 (bt, 2 H), 3.16 (bt, 2 H), 4.25 (bs, 2 H), 5.36 (m, 8
H); HRMS calcd for C24H43N2O (MH+) 375.3375, found 375.3364.
Anal. (C24H42N2O‚0.3H2O) C, H, N.
1-Nor a r a ch id on yl-3-(2′-flu or oeth yl)u r ea (4f): yellow oil,
55%;1H NMR (400 MHz, CDCl3) δ 0.87 (t, J ) 6.74 Hz, 3 H),
1.23-1.37 (m, 6 H), 1.56 (quint., J ) 7.33 Hz, 2 H), 2.03 (q, J
) 13.8, 6.74 Hz, 2 H), 2.10 (q, J ) 13.8, 6.74 Hz, 2 H), 2.81
(m, 6 H), 3.16 (bm, 2 H), 3.46 (q, 1 H), 3.53 (q, 1 H), 4.34 (bs,
1 H), 4.41 (t, J ) 4.69 Hz, 1 H), 4.53 (t, J ) 4.69 Hz, 1 H), 4.59
(bs, 1 H), 5.38 (m, 8 H); 13C NMR (300 MHz, CDCl3) δ 14.08,
22.59, 24.57, 25.65 (3), 27.24, 29.34, 29.99, 31.53, 40.31, [40.78,
41.03], [82.60, 84.79], 127.51, 127.83, 128.14, 128.30, 128.72
(2), 129.07, 130.56, 157.74; HRMS calcd for C22H38N2OF (MH+)
365.2968, found 365.2970. Anal. (C22H37N2OF‚0.4CHCl3) C, H,
N.
1-Nor a r a ch id on yl-3-(2′-h yd r oxyeth yl)u r ea (4g): yellow
oil, 70%; 1H NMR (300 MHz, CDCl3) δ 0.86 (t, J ) 6.74 Hz, 3
H), 1.29 (m, 6 H), 1.53 (quint., 2 H), 2.05 (m, 4 H), 2.80 (m, 6
H), 3.12 (m, 2 H), 3.26 (q, J ) 5.23, 10.4 Hz, 2 H), 3.62 (t, J )

Unique Analogues of Anandamide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1981
4. Da ta An a lysis. IC50 values were converted to Ki values.32
Statistical evaluation of parallelism between displacement
curves generated in the presence and absence of PMSF was
performed using ALLFIT.33 Dose-dependent relationships were
determined for each analogue in the pharmacological assays.
Percent effect was determined based upon the maximal effects
that are produced by ∆9-THC and anandamide which are 90,
100, and 60% for spontaneous activity, antinociception, and
ring immobility, respectively. The percent effect for hypoth-
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damide (-3.0 °C) rather than that produced by ∆9-THC (-6.0
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Ack n ow led gm en t. Thanks is given to NIDA Grants
DA-08904, DA-09789, DA-03672, and DA-05274 for the
support of this work.
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