Structural requirements for binding of anandamide-type compounds to the brain cannabinoid receptor

Article abstract of DOI:10.1021/jm960752x

In order to establish the structural requirements for binding to the brain cannabinoid receptor (CB₁), we have synthesized numerous fatty acid amides, ethanolamides, and some related simple derivatives and have determined their K(i) values. A f

Full text of DOI:10.1021/jm960752x

J . Med. Chem. 1997, 40, 659-667
659
Str u ctu r a l Requ ir em en ts for Bin d in g of An a n d a m id e-Typ e Com p ou n d s to th e
Br a in Ca n n a bin oid Recep tor
Tzviel Sheskin,† Lumir Hanusˇ,† J oram Slager,† Zvi Vogel,‡ and Raphael Mechoulam*,†
Medical Faculty, Department of Natural Products, Hebrew University, J erusalem 91120, Israel, and Department of
Neurobiology, Weizmann Institute, Rehovot 76100, Israel
Received October 30, 1996X
In order to establish the structural requirements for binding to the brain cannabinoid receptor
(CB1), we have synthesized numerous fatty acid amides, ethanolamides, and some related simple
derivatives and have determined their Ki values. A few R-methyl- or R,R-dimethylarachi-
donoylalkylamides were also examined. In the 20:4, n-6 series, the unsubstituted amide is
inactive; N-monoalkylation, at least up to a branched pentyl group, leads to significant binding.
N,N-Dialkylation, with or without hydroxylation on one of the alkyl groups, leads to elimination
of activity. Hydroxylation of the N-monoalkyl group at the ω carbon atom retains activity. In
the 20:x, n-6 series, x has to be either 3 or 4; the presence of only two double bonds leads to
inactivation. In the n-3 series, the limited data reported suggest that the derived ethanolamides
are either inactive or less active than comparable compounds in the n-6 series. Alkylation or
dialkylation of the R carbon adjacent to the carbonyl group retains the level of binding in the
case of anandamide (compounds 48, 49); however, R-monomethylation or R,R-dimethylation of
N-propyl derivatives (50-53) potentiates binding and leads to the most active compounds seen
in the present work (Ki values of 6.9 ( 0.7 to 8.4 ( 1.1 nM). We have confirmed that the
presence of a chiral center on the N-alkyl substituent may lead to enantiomers which differ in
their levels of binding (compounds 54, 57 and 55, 56).
Several years ago we isolated from porcine brain
arachidonoylethanolamide (anandamide) (1), the first
known animal constituent that binds to the brain
cannabinoid receptor (CB1).1 Later we found two ad-
ditional active fatty acid ethanolamides: dihomo-γ-
linolenoylethanolamide (2) and docosatetraenoyletha-
nolamide (3).2-4 The activity of anandamide parallels
confirmed this observation (D. Piomelli, private com-
munication). The discrepancies reported may be due
to the differences in the extraction and purification
methods employed. Anandamide has also been detected
in rat testis.14b
In order to establish the structural requirements for
binding to CB1, we have synthesized numerous fatty
acid amides, ethanolamides, and some related simple
derivatives and have determined their Ki values in a
competition assay against the binding of [3H]HU-243,
a potent tricyclic cannabinoid.1 A few R-methyl or R,R-
dimethylarachidonoylalkylamides were also examined.
The monoalkyl, dialkyl and hydroxyalkyl amides were
prepared by reaction of the appropriate acyl chloride
with an alkyl-, dialkyl-, or hydroxyalkylamine, following
the procedure previously described.1 The phosphate (4)
was prepared by condensation of the N-hydroxysuccin-
imide ester of arachidonic acid (5) with O-phosphoryle-
thanolamine (Figure 1).15
The glycine derivative 6 was prepared by reaction of
arachidonoyl chloride with glycine in potassium hydrox-
ide solution. The carboxylic acid derivatives 7a and 7b
were prepared by condensation of 5 with L-serine and
D-serine, respectively.
to a large extent that of ∆9-tetrahydrocannabinol (∆9-
THC), the active constituent of Cannabis,5 including in
vitro inhibition of adenylylcylase6 and in vivo effects on
the hypothalamo-pituitary-adrenal axis,7 sedation,8
inhibition of working memory,9 reduction of blood pres-
sure,10 etc.11 The presence of anandamide in fresh pig
brain has been confirmed.12 Kempe et al. have reported
that it could not be detected in fresh rat brain.13
However Sugiura et al. have recently recorded its
presence in this tissue,14a and a further group has
* Address correspondence to: Prof. R. Mechoulam, Natural Products
Department, Medical Faculty, Hebrew University, Ein Kerem Campus,
J erusalem 91120, Israel. Fax: 972-2-6410740. E-mail: Mechou@yam-
suff.cc.huji.ac.il.
† Hebrew University.
‡ Weizmann Institute.
X Abstract published in Advance ACS Abstracts, February 1, 1997.
S0022-2623(96)00752-2 CCC: $14.00 © 1997 American Chemical Society

660 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Sheskin et al.
F igu r e 1. Synthesis of anandamide O-phosphate.
Several groups have published data on binding of
various anandamide derivatives to CB1.16-23 Some of
the published compounds were independently prepared
and evaluated by us. The results obtained are pre-
sented now. Binding data are notoriously sensitive to
experimental conditions, and it is difficult to compare
results from different laboratories except with regard
to trends in structure-activity changes. In the anan-
damide series such comparisons are further complicated
due to the facile hydrolysis of the amide bond. In some
of the early work very high Ki values (i.e., low binding)
were reported for the compounds tested.16 Later it was
found that the addition of the amidase inhibitor phe-
nylmethanesulfonyl fluoride (PMSF) prevents (or re-
duces) hydrolysis, and indeed, lower Ki values were
noted.24
lead to analogous results. We have reported that in the
open field ambulation test, which measures locomotor
activity, and in the ring test, which measures catalepsy,
anandamide, administered ip, was as active as ∆8-THC
(10).3a This isomer of ∆9-THC is generally only slightly
less active than ∆9-THC in most in vivo tests. However,
in the induction of hypothermia, and as an analgesic
(measured on the hot plate), anandamide was consider-
ably less active than ∆8-THC.3a Smith et al. have noted
that anandamide administered iv is 1.3-18 times less
potent than ∆9-THC in all behavioral assays.26 This
discrepancy may be due in part to the in vivo hydrolysis
of anandamide, a well established metabolic route.
Two binding assay methods are generally used. In
our original and later publications,1,2a we reported the
use of a centrifugation-based ligand binding assay with
rat synaptosomal membranes. However most other
groups have prefered a “filtration” method, which in the
absence of an amidase inhibitor apparently allows
enzymatic hydrolysis to take place and produces high
Ki values. In the centrifugation method, as used in our
laboratory, the addition of an amidase inhibitor does not
lower the Ki.
All compounds were tested for displacement of [3H]-
HU-243 (8), a highly potent probe prepared in our
laboratory, bound to brain synaptosomal membranes
(Tables 1-3).25
The above-mentioned results, as well as preliminary
unpublished in vivo data on a few of the compounds
discussed in this paper, indicate that in vivo and binding
SAR’s in the anandamide series do not necessarily
parallel each other. In a separate publication, we expect
to report data on in vivo SAR with anandamide-type
compounds.
Table 1 lists, in an increasing order of potency, the
synthetic arachidonoylamide derivatives prepared in our
laboratory and their Ki values. An upper limit of 1 µM
was arbitrarily set for binding potency. We do not
report the exact inhibition constant of an assayed
compound if it was above 1 µM, i.e., about 25 times less
potent than anandamide. Such compounds we consider
“inactive”.
Str u ctu r e-Activity Rela tion sh ip s (SAR)
Anandamide (20:4, n-6) (1) inhibited the specific
binding of [3H]HU-243 (8) to synaptosomal membranes
in a manner typical of competitive ligands, under the
conditions described in the Experimental Section, with
an inhibition constant (Ki) of 39.2 ( 5.7 nM. The
inhibition constants of the two other known additional
endogenous anandamides, namely dihomo-γ-linolenoyle-
thanolamide (anandamide 20:3, n-6) (2) and docosatet-
raenoylethanolamide (anandamide 22:4, n-6) (3) are
very close, 53.4 ( 5.5 and 34.4 ( 3.2 nM, respectively.2a,b
For comparison, the Ki of ∆9-THC (9) is 46 ( 3 nM.
These data indicate that the endogenous brain constitu-
ents and the main psychoactive Cannabis plant con-
stituent bind to the CB1 receptor at the same level of
potency. However a comparison of the in vivo activities
of anandamide (1) with those of ∆9-THC (9) does not
We were surprised to note that the hydroxyl group of
the alkanolamine moiety is not a requirement for
binding. This unexpected result has been independently
observed by Pinto et al.17 Arachidonoylethyl amide (11)
is slightly more active than anandamide (1). A short-

Binding of Anandamide-Type Compounds
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 661
Ta ble 1. Binding of Arachidonoyl Amides to the Brain Cannabinoid Receptor (CB1)
CONR1R2
compd
R1
R2
Ki (nM)
lit. Ki (nM)
7.3 (5.7-9.5)17
14
15
22
H
H
H
CH2CH2CH3
CH(CH3)2
CH2CH2CH2OH
11.7 ( 2.1
13.6 ( 1.1
29.9 ( 0.4
364 ( 9516
13 (11-17)17
189 ( 73 (with PMSF)22
11
1
12
25
H
H
H
H
CH2CH3
34.0 ( 2.7
39.2 ( 5.7
60.0 ( 7.4
85.2 ( 3.8
CH2CH2OH
CH3
CH2CH2OCH3
650 (470-880)17
1820 ( 280 (with PMSF)22
17
23
4
16
21
H
H
H
H
H
C(CH3)3
138.6 ( 9.4
161.8 ( 34.1
190.8 ( 11.1
235.7 ( 14.2
(R)-(-), 239.9 ( 63.8
(S)-(+), 377.2 ( 55.4
446.7 ( 40.3
497.4 ( 27.1
575.1 ( 35.3
>1000
CCH2(CH3)2OH
CH2CH2OP(dO)(OH)2
CH2CH2CH2CH3
CH(CH3)CH2CH3
30 (22-43)17
20
24
19
13
28
29
35
27
18
34
30
H
H
H
H
CCH2(CH3)2CH3
CH2CH2CH2CH2OH
CH2CH2CH(CH3)2
H
220 (190-260)17
1860 ( 300 (with PMSF)22
9600 ( 10016
CH3
CH3
CH2CH3
>1000
CH2CH3
>1000
H
H
H
H
CH(CH3)CH(OH)C6H5
CH2CH2N(CH2CH3)2
CH2CH2CH2CH2CH3
OH
>1000
>1000
>1000
>1000
CH3
CH2CH2OH
>10000
5240 ( 202519
4980 ( 376 (with PMSF)19
31
32
33
6
CH2CH3
CH2CH2CH3
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2COOH
>10000
>10000
>10000
H
H
>10000
7
CH(COOH)CH2OH
(a) D, >10000
(b) L, >10000
ened side chain as in arachidonoylmethylamide (12)
lowers activity, compared to that of 1, while removal of
the alkyl group, as in arachidonoyl amide (13),16 elimi-
nates binding. Activity increases with elongation of the
N-alkyl side chain, the Ki of arachidonoyl-n-propyl
amide (14) being 11.7 ( 2.1 nM and that of arachi-
donoylisopropyl amide (15), 13.6 ( 1.1 nM. However,
further elongation, with or without branching, sharply
reduces or eliminates binding (see compounds 16-21).
Another point of interest is that the straight chain
pentyl amide (18) is inactive, i.e., has a Ki value above
1 µM, while the branched chain pentyl amides (19-21)
were active with Ki values between ca. 235 and 575 nM.
The same regularity is observed in the butyl series. The
straight chain butyl derivative (16)17 has a Ki of 235.7
( 14.2 nM, while the branched butyl one (17) has a Ki
of 138.6 ( 9.4 nM. Only a small difference was observed
between the R-(-) enantiomer (21a ) with a Ki of 239.9
( 63.8 nM and the S-(+) enantiomer (21b) with a Ki of
377.2 ( 55.4 nM.
We noted the same regularity with regard to elonga-
tion of the side chain in the arachidonoylalkanolamide
series. Elongation of the alkanol chain, from ethanol,
as in 1, to propanol, leading to 22,16,17,22,23 causes a slight
increase in activity; however, the isobutyl alcohol 23 or
1-pentanol 2417,22,23 derivatives are considerably less
active.
Blocking of the hydroxyl group in anandamide as a
methyl ether (compound 25)17,22,23 decreased the activity
about 2-fold. Anandamide phosphate (4) was about 5
times less active than anandamide. This material was
prepared in view of the biogenesis of anandamide, which
is considered to be formed by the action of phospholipase
D on N-arachidonoylphosphatidylethanolamine (26).14,27
Assuming that the latter compound could also be
cleaved by phospholipase C, compound 4 would be
obtained. We have not observed 4 to be present in brain
(unpublished observations), and in view of the low
activity of 4 recorded now, we doubt whether phospho-
lipase C is involved in the formation of anandamide-
type compounds. However, we cannot rule out the
possibility that 4, once produced in the body, is quickly
modified by phosphatases to anandamide.
Disubstitution of the amide nitrogen, forming N,N-
dialkyl or N-alkyl, N-(2-hydroxyethyl) derivatives, led
to compounds that did not bind up to 1 µM (see 28-
33). Apparently a secondary amide grouping is es-
sential in the anandamide series to allow binding to
CB1.
Several serine amide derivatives of fatty acids have
been isolated from body tissues alongside the N-ethanol
derivatives.28 Hence, we synthesized the D- and L-serine

662 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Sheskin et al.
Ta ble 2. Binding of N-Acyl Ethanolamines to the Brain
brane receptor or transfected cells) or CB2 (transfected
cells). This is in contrast to the data published by Facci
et al. who found that 45 binds and activates CB2 in mast
cells.30 It is possible that in mast cells 45 binds to a
yet unidentified subtype of CB2.
Oleoyl amide (46) has been reported to be a “sleep
factor”.31 As cannabinoids are known to cause sedation
and promote sleep we assumed that 46 may cause its
activity via CB1 or CB2. We observed no binding.
Apparently any sleep promotion caused by 46 does not
take place via CB1 or CB2.
Cannabinoid Receptor (CB1)a
compd
acyl moiety
Ki (nM)
lit. Ki (nM)
598 ( 25422
3
2
22:4, n-6
34.4 ( 3.2
53.4 ( 5.5
20:3, n-6
20:5, n-3
22:6, n-3
20:2, n-6
18:3, n-6
20:1, n-9
18:4, n-3
20:3, n-3
18:2, n-6
16:0
42
43
36
37
44
40
39
38
45
41
47
162.3 ( 13.6 1470 ( 500 (with PMSF)22
324.1 ( 9.2
1500
12200 ( 50016
4600 ( 300
>1000
>41400 ( 600016
>10000 (with PMSF)22
>1000
>10000
>25000
no activity
no activity
no activity16
Eicosatetraynoylethanolamide (47) was prepared as
the parent acid is known to block prostaglandin syn-
thesis.32 However, 47 did not bind to CB1.
18:3, n-3
eicosatetraynoic no activity
acid
a
The acyl group is designated following the accepted shorthand
nomenclature for long-chain unsaturated fatty acids. Thus the
acyl moiety 20:4, n-6, in anandamide indicates the presence of a
20-carbon atom chain; four homoallylic double bonds, the first one
of which is on the sixth carbon atom, counting from the non-
carboxyl end of the acid.
amides of arachidonic acid (7a ,b). These compounds did
not bind to CB1. Likewise no binding was observed for
the related glycine amide of arachidonic acid (6).
Corey et al.29 have reported that the N-hydroxyl
derivative of arachidonoyl amide (34) is an inhibitor of
leukotriene synthesis. We found that this compound
does not displace the binding of [3H]HU-243 in concen-
trations up to 1 µM. The arachidonoyl amide of (()-
norephedrine (35) was also inactive.
It has been established that the major metabolic
degradation of anandamide is through hydrolysis, yield-
ing arachidonic acid and ethanolamine.33 We assumed
that the introduction of one or two alkyl groups R to
the carbonyl moiety may prevent, or at least reduce, the
rate of hydrolysis, which may be expressed as a lower
Ki value (assuming that during the binding assay
anandamide is partially hydrolyzed). In Table 3 we
present the Ki values of several R-mono- and R,R-
dimethylarachidonoyl amides. We first determined the
Ki values of R-monomethylanandamide (48) and R,R-
dimethylanandamide (49). Neither 48 nor 49 was
significantly more potent than anandamide (1). After
the completion of these determinations, a paper describ-
ing compounds 48 and 49 and their Ki values was
published.19 The R-monomethyl and R,R-dimethyl de-
rivatives of the n-propyl and isopropyl amides (50-53)
were the most potent compounds obtained by us thus
far, with Ki values in the range 6.9-8.4 nM.
Makriyannis et al. have reported the introduction of
an asymmetry center on the N-alkyl side of ananda-
mide, through placement of the hydroxyl group on the
secondary rather than primary carbon atom of the
ethanol side chain or by alkylation of the secondary
carbon atom.18 These changes led to a significant
variation in activity between the S and R compounds.
We have now produced the same type of derivatives of
R,R-dimethylarachidonic acid. We also observe signifi-
cant differences in binding: (55, R) and (54, S) are about
as active as anandamide, while their enantiomers (56,
S) and (57, R) are significantly less potent.
Table 2 presents the results obtained with a variety
of amides of fatty acids other than arachidonic acid. As
mentioned above, the endogenous anandamides (20:3,
n-6) (2) and (22:4, n-6) (3) bind to CB1 at the level of
anandamide (20:4, n-6) (1). However anandamide (20:
2, n-6) (36) is inactive. Apparently in the 20, n-6 series,
three or four double bonds are required for activity.
Anandamide (18:3, n-6) (37)16,20 and anandamide (18:
2, n-6) (38) are also inactive. Further work is required
to establish the lower limit of the length of the fatty
acid and the number of double bonds in them needed
for binding to CB1. We also looked into the n-3 series:
anandamides (20:3, n-3) (39), (18:4, n-3) (40), and (18:3
n-3) (41) are inactive. However anandamides (20:5, n-3)
(42)22,23 and (22:6, n-3) (43)16 retain activity (Ki, 162.3
( 13.6 nM and 324.1 ( 9.2 nM respectively). A definite
conclusion with regard to differences in activity in the
n-3 and n-6 series cannot be drawn as few compounds
that differ only in the positions of the double bonds (n-6
versus n-3) have been tested in both series (37 versus
41 and 2 versus 39). However the high activity of 2 as
compared to the inactivity of 39, as well as the general
low activity of those compounds in the n-3 series that
exhibit binding (42, 43), compared to related active
compounds in the n-6 series (for example 1 and 3) points
out that activity in the n-6 series is apparently higher
than in the n-3 series. An n-9 anandamide (20:1, n-9)
(44)20,22,23 was inactive.
In summary, we have found so far several structure-
activity regularities concerning binding of fatty acid
amides to CB1.
(1) In the 20:4, n-6 series, the unsubstituted amide
(13) is inactive; N-monoalkylation, at least up to a
branched pentyl group, leads to significant binding. The
following regularities in binding were noted for anan-
damide-type compounds with the indicated N-alkyl
moieties: n-C5H11 (18) < branched C5H11 (19, 20) < CH
(CH3)CH2CH2 (either R or S) (21) < n-C4H9 (16) <
C(CH3)3 (17) < CH3 (12) < C2H5 (11) < C (CH3)2 (15) <
n-C3H7 (14). The last two compounds were the most
Palmitoylethanolamide (anandamide, 16:0) (45)16,20
under our conditions did not bind to either CB1 (mem-

Binding of Anandamide-Type Compounds
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 663
Ta ble 3. Binding of R-Methyl and R,R-Dimethyl Arachidonoyl Amides to the Brain Cannabinoid Receptor (CB1)
CH3
R1
CONHR2
compd
R1
R2
Ki (nM)
6.9 ( 0.7
7.2 ( 0.1
7.4 ( 0.2
8.4 ( 1.1
25.5 ( 2.8
lit Ki (nM)
51
53
50
52
49
CH3
CH3
H
H
CH3
CH2CH2CH3
CH(CH3)2
CH2CH2CH3
CH(CH3)2
CH2CH2OH
47 ( 3 (with PMSF)19
41 ( 319
47 ( 2 (with PMSF)22
41 ( 322
55
48
CH3
H
CH(CH3)CH2OH
CH2CH2OH
(R)-(-), 31.1 ( 1.0
32.5 ( 5.1
53 ( 15 (with PMSF)19
137 ( 2019
53 ( 11 (with PMSF)22
137 ( 2022
54
57
56
CH3
CH3
CH3
CH2CH(OH)CH3
CH2CH(OH)CH3
CH(CH3)CH2OH
(S)-(+), 46.6 ( 2.2
(R)-(-), 153.9 ( 30.0
(S)-(+), 191.4 ( 24.5
All amines and amino alcohols were obtained from Aldrich
Chemical Co. Amino acids and fatty acids were obtained from
Sigma Chemical Co.
active in these homologous series, with Ki values about
three times lower than that of anandamide (20:4, n-6)
(1).
2. N,N-Dialkylation, with or without hydroxylation
on one of the alkyl groups, leads to elimination of
activity (compounds 28-33).
3. Hydroxylation of the N-monoalkyl group at the ω
carbon atom retains activity, as compared to the parent
N-alkyl group. However in most cases this activity is
slightly lower, at least in the relatively potent com-
pounds (cf. compound 22 versus 14; compound 1 versus
11; compound 23 versus 17). However this relationship
is not retained with the rather weak 24 (Ki 497.4 ( 27.1
µM) versus the inactive 18.
4. The methyl ether (25) and the phosphate (4) are
less active than the parent alcohol (1). The carboxyl
acid derivatives (6) and (7) are inactive, but these are
singular examples of these structural types.
5. In the 20:x, n-6 series, x has to be 3 or 4; two double
bonds only leads to inactivation.
Anandamides 1, 2, and 3 were prepared as described
previously.1
N-Hyd r oxysu ccin im id e Ester of Ar a ch id on ic Acid (5).
Arachidonic acid (440 mg, 1.44 mmol) was added to a solution
of N-hydroxysuccinimide (184 mg, 1.6 mmol) in dry ethyl
acetate (10 mL). After 5 min of stirring, a solution of
dicyclohexylcarbodiimide (330 mg, 1.6 mmol) in dry ethyl
acetate (2 mL) was added, and the reaction mixture was left
overnight at room temperature under a nitrogen atmosphere.
Dicyclohexylurea was filtered, and the crude material was
chromatographed on silica gel (eluting with chloroform) to give
440 mg (76%) as a colorless oil: 1H NMR (CDCl3) δ 5.32-5.43
(m, 8H), 2.82-2.84 (m, 10H), 2.62 (t, J ) 7.5 Hz, 2H), 2.19-
2.22 (m, 2H), 2.04-2.07 (m, 2H), 1.80-1.86 (m, 2H), 1.28-
1.38 (m, 6H), 0.89 (t, J ) 6.9 Hz, 3H). Anal. (C24H35NO4) C,
H, N.
An a n d a m id e O-P h osp h a te (4). A solution of N-hydrox-
ysuccinimide ester of arachidonic acid (5) (410 mg, 1.02 mmol)
in tetrahydrofuran (5 mL) was added to a solution of O-
phosphoethanolamine (360 mg, 2.55 mmol) and sodium bicar-
bonate (536 mg, 6.38 mmol) in water (3 mL). The reaction
mixture was left overnight at room temprature under a
nitrogen atmosphere, then it was acidified to pH 2 with 0.1 N
HCl, and the organic solvent was evaporated under reduced
pressure. After addition of water (50 mL), the product was
extracted with methylene chloride (4 × 50 mL) and dried
(MgSO4), and solvent was evaporated to give 265 mg (60%) as
a colorless oil: 1H NMR (CDCl3) δ 5.34-5.42 (m, 8H), 3.98 (br
s, 2H), 3.46 (br s, 2H), 2.77-2.81 (m, 6H), 2.26 (t, J ) 6.8 Hz,
2H), 2.00-2.06 (m, 4H), 1.60-1.69 (m, 2H), 1.29-1.42 (m, 6H),
0.88 (t, J ) 6.9 Hz, 3H). Anal. (C22H38NO5P) C, H, N.
N-Ar a ch id on oylglycin e (6). To a solution of arachidonic
acid (150 mg, 0.49 mmol) and N,N-dimethylformamide (38 µL,
0.49 mmol) in dry methylene chloride (5 mL) was added
dropwise oxalyl chloride (2.0 M solution in methylene chloride,
0.49 mL, 0.98 mmol) under nitrogen atmosphere. The reaction
mixture was stirred for 1 h and then the solvent was
evaporated under a nitrogen flow. The crude material in
methylene chloride (5 mL) was added to a solution of glycine
(110 mg, 1.46 mmol) and 2 N potassium hydroxide in an ice
bath. Then, the reaction mixture was stirred for 1 h, water
(10 mL) was added, and the mixture was acidified to pH 3
with 1 N HCl. The product was extracted with ether (3 × 50
mL) and dried (MgSO4), and solvent was evaporated under
reduced pressure. The residue was chromatographed on silica
gel (eluting with ether:petroleum ether, 40:6) to give 60 mg
(34%) as a colorless oil: 1H NMR (CDCl3) δ 6.25 (br s, 1H),
5.30-5.37 (m, 8H), 4.05 (d, J ) 5.1 Hz, 2H), 2.76-2.82 (m,
6. In the n-3 series, the limited data suggest that the
derived ethanolamides are either inactive or less active
than related compounds in the n-6 series.
7. Alkylation or dialkylation of the R carbon adjacent
to the carbonyl group retains the level of binding in the
case of anandamide (compounds 48, 49); however
R-monomethylation or R,R-dimethylation of N-propyl
derivatives (compounds 50-53) potentiated binding and
led to the most active compounds seen in the present
work (Ki values of 6.9 ( 0.7 to 8.4 ( 1.1 nM).
8. We have confirmed previous work that the pres-
ence of a chiral center on the N-alkyl substituent leads
to enantiomers with significantly different levels of
binding.
Exp er im en ta l Section
Ch em istr y. 1H NMR spectra were measured on a Varian
VXR-300S spectrophotometer using TMS as the internal
standard. All chemical shifts are reported in ppm. Specific
rotations were detected with a Perkin-Elmer 141 polarimeter.
Melting points (uncorrected) were determined on a Buchi 530
apparatus. Column chromatography was performed with ICN
silica 60A. Elemental analyses were obtained for all the newly
synthesized compounds and are (0.4% of the theoretical
values.

664 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Sheskin et al.
6H), 2.22 (t, J ) 7.8 Hz, 2H), 2.04-2.18 (m, 2H), 1.70-1.82
(m, 4H), 1.25-1.35 (m, 6H), 0.89 (t, J ) 7.1 Hz, 3H). Anal.
(C22H35NO3) C, H, N.
1.66-1.76 (m, 2H), 1.26-1.38 (m, 6H), 1.14 (d, J ) 6.6 Hz,
6H), 0.89 (t, J ) 6.9 Hz, 3H).
N-Bu tyl a r a ch id on oyl a m id e (16)17
was prepared from
arachidonic acid (100 mg, 0.33 mmol) and n-butylamine (0.33
mL, 3.3 mmol) as described for compound 11: yield, 70 mg
(56%) as a colorless oil; 1H NMR (CDCl3) δ 5.26-5.44 (m, 8H),
3.24 (q, J ) 6 Hz, 2H), 2.76-2.86 (m, 6H), 2.02-2.22 (m, 6H),
1.64-1.78 (m, 2H), 1.22-1.56 (m, 10H), 0.86-0.98 (m, 6H).
N-ter t-Bu tyl a r a ch id on oyl a m id e (17) was prepared from
arachidonic acid (100 mg, 0.33 mmol) and tert-butylamine (0.35
mL, 3.3 mmol) as described for compound 11: yield, 85 mg
(72%) as a colorless oil;
5.22 (br s, 1H), 2.76-2.84 (m, 6H), 2.02-2.16 (m, 6H), 1.62-
1.70 (m, 2H), 1.33 (s, 9H), 1.22-1.30 (m, 6H), 0.89 (t, J ) 6.9
Hz, 3H).
N-Am yl a r a ch id on oyl a m id e (18) was prepared from
arachidonic acid (100 mg, 0.33 mmol) and amylamine (0.38
mL, 3.3 mmol) as described for compound 11: yield, 90 mg
(73%) as a colorless oil; 1H NMR (CDCl3) δ 5.70 (br s, 1H),
5.23-5.33 (m, 8H), 3.15 (q, J ) 3 Hz, 2H), 2.71-2.77 (m, 6H),
1.97-2.16 (m, 6H), 1.58-1.68 (m, 2H), 1.18-1.42 (m, 12H),
0.79-0.85 (m, 6H). Anal. (C25H43NO) C, H, N.
N-Ar a ch id on oyl-D-ser in e (7a ) was prepared as described
by Shinitzky et al. for N-stearoylserine.34 A solution of D-serine
(190 mg, 1.8 mmol) in 20 mL of 0.1 M sodium carbonate and
0.1 M sodium bicarbonate was added to a solution of N-
hydroxysuccinimide ester of arachidonic acid (200 mg, 0.5
mmol) in tetrahydrofuran (20 mL). The reaction mixture was
stirred overnight at 35 °C, evaporated down to 20 mL, and
acidified to pH 1 with 1 N HCl. The product was extracted
with methylene chloride (2 × 30 mL) and dried (MgSO4), and
the solvent was evaporated under reduced pressure to give 118
mg (60%) as a colorless oil. 1H NMR (CD3OD) δ 5.30-5.43
(m, 8H), 4.49 (t, J ) 4.8 Hz, 1H), 3.80-3.88 (m, 2H), 2.80-
2.86 (m, 6H), 2.30 (t, J ) 6.6 Hz, 2H), 2.04-2.18 (m, 4H), 1.66-
1H NMR (CDCl3) δ 5.30-5.40 (m, 8H),
1.72 (m, 2H), 1.29-1.39 (m, 6H), 0.90 (t, J ) 6.9 Hz, 3H); [R]25
D
-8.9° (c ) 1, CHCl3). Anal. (C23H37NO4) C, H, N.
N-Ar a ch id on oyl-L-ser in e (7b) was prepared by the same
method as for arachidonoyl-D-serine (7a ) to give 107 mg (55%)
as a colorless oil: 1H NMR (CD3OD) δ 5.33-5.40 (m, 8H), 4.50
(t, J ) 4.8 Hz, 1H), 3.78-3.90 (m, 2H), 2.80-2.86 (m, 6H),
2.29 (t, J ) 6.6 Hz, 2H), 2.04-2.18 (m, 4H), 1.64-1.72 (m, 2H),
N-(3-Meth ylbu tyl) a r a ch id on oyl a m id e (19) was pre-
pared from arachidonic acid (100 mg, 0.33 mmol) and isoamy-
lamine (0.38 mL, 3.3 mmol) as described for compound 11:
yield, 87 mg (71%) as a colorless oil; 1H NMR (CDCl3) δ 5.30-
5.42 (m, 8H), 3.26 (q, J ) 3 Hz, 2H), 2.76-2.86 (m, 6H), 2.02-
2.20 (m, 6H), 1.68-1.78 (m, 2H), 1.54-1.64 (m, 1H), 1.26-
1.40 (m, 8H), 0.82-0.96 (m, 9H). Anal. (C25H43NO) C, H, N.
N-(1,1-Dim eth ylp r op yl) a r a ch id on oyl a m id e (20) was
prepared from arachidonic acid (100 mg, 0.33 mmol) and 1,1-
dimethylpropylamine (0.38mL, 3.3 mmol) as described for
1.29-1.39 (m, 6H), 0.90 (t, J ) 7.2 Hz, 3H); [R]25 +8.9° (c )
D
1, CHCl3). Anal. (C23H37NO4) C, H, N.
Gen er a l P r oced u r e for P r ep a r in g Com p ou n d s 11-25
a n d 27-35. The following procedure was used to prepare the
amide derivatives of arachidonic acid and the other fatty acid
amides. The preparation of N-ethyl arachidonoylamide (11)
is given as a representative example.
N-Eth yl Ar a ch id on oyl Am id e (11). To a solution of
arachidonic acid (100 mg, 0.33 mmol) and N,N-dimethylfor-
mamide (25 µL, 0.33 mmol) in dry methylene chloride (5 mL)
was added dropwise oxalyl chloride (2.0 M solution im meth-
ylene chloride, 0.33 mL, 0.66 mmol) under nitrogen atmo-
sphere. The reaction mixture was stirred for 1 h and then
the solvent was evaporated under nitrogen flow. The crude
material in methylene chloride (5 mL) was added to an ice-
cold solution of ethylamine (70 wt % solution in water, 0.2 mL,
3.55 mmol) in methylene chloride (5 mL). The reaction
mixture was stirred for 15 min, then it was washed with water
(3 × 20 mL) and dried (MgSO4), and solvent was evaporated
under reduced pressure. The residue was chromatographed
on silica gel (eluting with chloroform:petroleum ether, 40:6)
to give 80 mg (73%) as a colorless oil: 1H NMR (CDCl3) δ 5.31-
5.41 (m, 8H), 3.22-3.34 (m, 2H), 2.78-2.84 (m, 6H), 2.01-
2.18 (m, 6H), 1.68-1.78 (m, 4H), 1.22-1.40 (m, 6H), 1.13 (t, J
) 7.3 Hz, 3H), 0.88 (t, J ) 7.1 Hz, 3H). Anal. (C22H37NO) C,
H, N.
1
compound 11: yield, 71 mg (58%) as a colorless oil; H NMR
(CDCl3) δ 5.28-5.38 (m, 8H), 5.10 (br s, 1H), 2.67-2.82 (m,
6H), 2.04-2.12 (m, 6H), 1.66-1.76 (m, 4H), 1.24-1.32 (m,
12H), 0.81-0.90 (m, 6H). Anal. (C25H43NO) C, H, N.
N-((R-(-)-1-Meth ylp r op yl) a r a ch id on oyl a m id e (21a )
was prepared from arachidonic acid (100 mg, 0.33 mmol) and
(R)-(-)-sec-butylamine (0.33 mL, 3.3 mmol) as described for
1
compound 11: yield, 75 mg (63%) as a colorless oil; H NMR
(CDCl3) δ 5.30-5.42 (m, 8H), 5.20 (br s, 1H), 3.86-4.00 (m,
1H), 2.75-2.86 (m, 6H), 2.00-2.20 (m, 6H), 1.65-1.80 (m, 2H),
1.22-1.50 (m, 8H), 1.10 (d, J ) 7.5 Hz, 3H), 0.82-0.92 (m,
6H); [R]25D) -7.08° (c ) 1, EtOH). Anal. (C24H41NO) C, H,
N.
N-((S)-(+)-1-Meth ylp r op yl) a r a ch id on oyl a m id e (21b)
was prepared from arachidonic acid (100 mg, 0.33 mmol) and
(S)-(+)-sec-butylamine (0.33 mL, 3.3 mmol) as described for
1
compound 11: yield, 65 mg (55%) as a colorless oil; H NMR
N-Meth yl a r a ch id on oyl a m id e (12) was prepared from
arachidonic acid (100 mg, 0.33 mmol) and methylamine (40
wt % solution in water, 0.12 mL, 3.55 mmol) as described for
compound 11: yield, 70 mg (67%) as a colorless oil: 1H NMR
(CDCl3) δ 5.80 (br s, 1H) 5.30-5.40 (m, 8H), 2.78-2.85 (m,
9H), 2.03-2.19 (m, 6H), 1.66-1.76 (m, 2H), 1.25-1.34 (m, 6H),
0.88 (t, J ) 9 Hz, 3H). Anal. (C22H37NO) C, H, N.
(CDCl3) δ 5.30-5.42 (m, 8H), 5.22 (br s, 1H), 3.88-3.98 (m,
1H), 2.75-2.90 (m, 6H), 2.00-2.20 (m, 6H), 1.65-1.76 (m, 2H),
1.22-1.50 (m, 8H), 1.12 (d, J ) 7.5 Hz, 3H), 0.82-0.96 (m,
6H); [R]25 +7.08° (c ) 1, EtOH). Anal. (C24H41NO) C, H, N.
D
N-(3-Hyd r oxyp r op yl) a r a ch id on oyl a m id e (22)16,17,22,23
was prepared from arachidonic acid (100 mg, 0.33 mmol) and
3-amino-1-propanol (0.25 mL, 3.3 mmol) as described for
Ar a ch id on oyl a m id e (13)16,29 was prepared from arachi-
donic acid (100 mg, 0.33 mmol) and ammonium hydroxide (0.2
mL, 5.2 mmol) as described for compound 11: yield, 80 mg
(80%) as a colorless oil; 1H NMR (CDCl3) δ 5.82 (br s, 1H) 5.31-
5.42 (m, 8H), 2.79-2.85 (m, 6H), 2.23 (t, J ) 8.1 Hz, 2H), 2.04-
2.15 (m, 4H), 1.70-1.77 (m, 2H), 1.25-1.38 (m, 6H), 0.89 (t, J
) 6.8 Hz, 3H). Anal. (C22H33NO): C, H, N.
1
compound 11: yield, 81 mg (68%) as a colorless oil; H NMR
(CDCl3) δ 6.42 (br s, 1H), 5.22-5.38 (m, 8H), 3.90 (br s, 1H),
3.54 (t, J ) 5.5 Hz, 2H), 3.32 (q, J ) 6 Hz, 2H), 2.62-2.80 (m,
6H), 1.92-2.18 (m, 6H), 1.50-1.70 (m, 4H), 1.20-1.34 (m, 6H),
0.90 (t, J ) 7 Hz, 3H).
N-(1,1-Dim eth yl-2-h yd r oxyeth yl) a r a ch id on oyl a m id e
(23) was prepared from arachidonic acid (100 mg, 0.33 mmol)
and 2-amino-2-methyl-1-propanol (0.32 mL, 3.3 mmol) as
described for compound 11: yield, 60 mg (48%) as a colorless
oil; 1H NMR (CDCl3) δ 5.50 (br s, 1H), 5.28-5.40 (m, 8H), 3.57
(s, 2H), 2.78-2.90 (m, 6H), 2.02-2.20 (m, 6H), 1.62-1.74 (m,
2H), 1.20-1.40 (m, 12H), 0.89 (t, J ) 7.1 Hz, 3H). Anal
(C24H41NO2): C, H, N.
N-P r op yl a r a ch id on oyl a m id e (14)17 was prepared from
arachidonic acid (100 mg, 0.33 mmol) and n-propylamine (0.27
mL, 3.3 mmol) as described for compound 11: yield, 81 mg
(71%) as a colorless oil; 1H NMR (CDCl3) δ 5.62 (br s, 1H) 5.26-
5.38 (m, 8H), 3.13 (q, J ) 6 Hz, 2H), 2.62-2.80 (m, 6H), 2.10
(t, J ) 7.3 Hz, 2H), 1.96-2.06 (m, 4H), 1.56-1.66 (m, 2H),
1.38-1.48 (m, 2H), 1.20-1.32 (m, 6H), 0.79-0.86 (m, 6H).
N-(5-Hyd r oxyp en tyl) a r a ch id on oyl a m id e (24)17,22,23
was prepared from arachidonic acid (100 mg, 0.33 mmol) and
5-amino-1-pentanol (340 mg, 3.3 mmol) as described for
N-Isop r op yl a r a ch id on oyl a m id e (15) was prepared from
arachidonic acid (100 mg, 0.33 mmol) and isopropylamine
(0.28mL, 3.3 mmol) as described for compound 11: yield, 52
1
compound 11: yield, 78 mg (61%) as a colorless oil; H NMR
1
mg (46%) as a colorless oil; H NMR (CDCl3) δ 5.30-5.44 (m,
(CDCl3) δ 5.60 (br s, 1H), 5.22-5.42 (m, 8H), 3.64 (t, J ) 5.5
Hz, 2H), 3.20-3.32 (m, 2H), 2.76-2.89 (m, 6H), 2.00-2.22 (m,
8H), 4.02-4.12 (m, 1H), 2.76-2.86 (m, 6H), 2.02-2.16 (m, 6H),

Binding of Anandamide-Type Compounds
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 665
6H), 1.90 (br s, 1H), 1.20-1.80 (series of m, 14H), 0.89 (t, J )
7.1 Hz, 3H). Anal. (C25H43NO2): C, H, N.
(()-N-(1-Meth yl-2-h ydr oxy-2-ph en yleth yl) ar ach idon oyl
a m id e (35) was prepared from arachidonic acid (152 mg, 0.5
mmol), (()-phenylpropanolamine hydrochloride (938 mg, 5
mmol) and triethylamine (0.7 mL, 5 mmol) as described for
compound 11: yield, 146 mg (67%) as a colorless oil; 1H NMR
(CDCl3) δ 7.27-7.35 (m, 5H), 5.54 (br s, 1H), 5.30-5.41 (m,
8H), 4.84 (br s, 1H), 4.31-4.36 (m, 1H), 3.60 (br s, 1H), 2.78-
2.85 (m, 6H), 2.01-2.22 (m, 6H), 1.67-1.77 (m, 2H), 1.25-
1.37 (m, 6H), 1.01 (d, J ) 7.1 Hz, 3H), 0.89 (t, J ) 7.1 Hz,
3H). Anal. (C29H43NO2) C, H, N.
N-(2-Meth oxyeth yl) a r a ch id on oyl a m id e (25)17,22,23 was
prepared from arachidonic acid (100 mg, 0.33 mmol) and
methoxyethylamine (0.43 mL, 3.3 mmol) as described for
1
compound 11: yield, 75 mg (41%) as a colorless oil; H NMR
(CDCl3) δ 5.80 (br s, 1H), 5.30-5.42 (m, 8H), 3.45 (d, J ) 2.1
Hz, 4H), 3.35 (s, 3H), 2.75-2.85 (m, 6H), 2.00-2.20 (m, 6H),
1.62-1.80 (m, 2H), 1.20-1.40 (m, 6H), 0.89 (t, J ) 6.8 Hz, 3H).
N′-Ar a ch id on oyl-N′′-d ieth yleth ylen ed ia m in e (27) was
prepared from arachidonic acid (100 mg, 0.33 mmol) and N,N-
diethylenediamine (0.46 mL, 3.3 mmol) as described for
Com p ou n d s 36-47 were synthesized as described for
anandamide (1).1
1
An a n d a m id e (20:2, n -6) (36) was prepared from cis-11,-
14-eicosadienoic acid (100 mg, 0.32 mmol) and ethanolamine
(0.19 mL, 3.2 mmol) in 81% yield as a colorless oil: 1H NMR
(CDCl3) δ 5.90 (br s, 1H), 5.30-5.38 (m, 4H), 3.70 (t, J ) 4.4
Hz, 2H), 3.40-3.45 (m, 2H), 2.78 (t, J ) 5.4 Hz, 2H), 2.18 (t,
J ) 7.1 Hz, 2H), 2.00-2.12 (m, 2H), 1.50-1.70 (m, 8H), 1.30
(br s, 8H), 0.89 (t, J ) 7.5 Hz, 3H). Anal. (C22H37NO2) C, H,
N.
compound 11: yield, 93 mg (70%) as a colorless oil; H NMR
(CDCl3) δ 6.10 (br s, 1H), 5.26-5.42 (m, 8H), 3.22-3.30 (m,
2H), 2.76-2.90 (m, 6H), 2.50-2.62 (m, 6H), 2.00-2.20 (m, 6H),
1.66-1.80 (m, 2H), 1.20-1.40 (m, 6H), 1.02 (t, J ) 7.1 Hz, 6H),
0.89 (t, J ) 6.5 Hz, 3H). Anal. (C26H46N2O) C, H, N.
N,N-Dim eth yl a r a ch id on oyl a m id e (28) was prepared
from arachidonic acid (100 mg, 0.33 mmol) and dimethylamine
(40% aqueous solution, 0.17 mL, 1.4 mmol) as described for
An a n d a m id e (18:3, n -6) (37)16,20 was prepared from cis-
6,9,12-octadecatrienoic acid (100 mg, 0.36 mmol) and ethano-
lamine (0.22 mL, 3.6 mmol) in 79% yield as a colorless oil: 1H
NMR (CDCl3) δ 6.13 (br s, 1H), 5.29-5.41 (m, 6H), 3.71 (t, J
) 5.1 Hz, 2H), 3.41 (q, J ) 5.1 Hz, 2H), 2.80 (t, J ) 5.7 Hz,
4H), 2.20 (t, J ) 8.1 Hz, 2H), 2.00-2.12 (m, 4H), 1.60-1.70
(m, 4H), 1.31 (br s, 6H), 0.88 (t, J ) 7.5 Hz, 3H).
An a n d a m id e (18:2, n -6) (38) was prepared from cis-9,cis-
12-octadecadienoic acid (200 mg, 0.71 mmol) and ethanolamine
(0.43 mL, 7.1 mmol) in 84% yield as a colorless oil: 1H NMR
(CDCl3) δ 5.90 (br s, 1H), 5.30-5.39 (m, 4H), 3.72 (t, J ) 5.1
Hz, 2H), 3.42 (q, J ) 5.4 Hz, 2H), 2.76 (t, J ) 5.7 Hz, 2H),
2.20 (t, J ) 8.1 Hz, 2H), 2.01-2.06 (m, 4H), 1.60-1.70 (m, 2H),
1.30 (br s, 14H), 0.88 (t, J ) 7.2 Hz, 3H). Anal. (C20H37NO2)
C, H, N.
An a n d a m id e (20:3, n -3) (39) was prepared from cis-11,-
14,17-eicosatrienoic acid (100 mg, 0.33 mmol) and ethanola-
mine (0.20 mL, 3.3 mmol) in 50% yield as a colorless oil: 1H
NMR (CDCl3) δ 5.90 (br s, 1H), 5.30-5.39 (m, 4H), 3.72 (t, J
) 5.1 Hz, 2H), 3.42 (q, J ) 5.4 Hz, 2H), 2.80 (t, J ) 5.7 Hz,
4H), 2.20 (t, J ) 8.1 Hz, 2H), 2.01-2.12 (m, 4H), 1.56-1.67
(m, 4H), 1.26 (br s, 10H), 0.97 (t, J ) 7.5 Hz, 3H). Anal.
(C22H39NO2) C, H, N.
1
compound 11: yield, 65 mg (59%) as a colorless oil; H NMR
(CDCl3) δ 5.24-5.44 (m, 8H), 2.99 (s, 3H), 2.94 (s, 3H), 2.76-
2.86 (m, 6H), 2.31 (t, J ) 7 Hz, 2H), 2.00-2.18 (m, 4H), 1.66-
1.78 (m, 2H), 1.20-1.38 (m, 6H), 0.89 (t, J ) 7.1 Hz, 3H). Anal.
(C22H37NO) C, H, N.
N,N-Dieth yl a r a ch id on oyl a m id e (29) was prepared from
arachidonic acid (100 mg, 0.33 mmol) and diethylamine (0.34
mL, 3.3 mmol) as described for compound 11: yield, 78 mg
(66%) as a colorless oil; 1H NMR (CDCl3) δ 5.30-5.42 (m, 8H),
3.20-3.42 (m, 4H), 2.76-2.86 (m, 6H), 2.29 (t, J ) 7.1 Hz, 2H),
2.00-2.20 (m, 4H), 1.60-1.80 (m, 2H), 1.22-1.40 (m, 6H),
1.04-1.20 (m, 6H), 0.90 (t, J ) 6.1 Hz, 3H). Anal. (C24H41
NO) C, H, N.
-
N-Meth yl N-(2-h ydr oxyeth yl) ar ach idon oyl am ide (30)19
was prepared from arachidonic acid (152 mg, 0.5 mmol) and
2-(methylamino)ethanol (0.40 mL, 5 mmol) as described for
compound 11: yield, 136 mg (75%) as a colorless oil; 1H NMR
(CDCl3) δ 5.30-5.42 (m, 8H), 3.77 (t, J ) 5 Hz, 2H), 3.55 (t, J
) 4.6 Hz, 2H) 3.06 (s, 3H), 2.78-2.85 (m, 6H), 2.35 (t, J ) 7.8
Hz, 2H), 2.04-2.15 (m, 4H), 1.64-1.75 (m, 2H), 1.25-1.38 (m,
6H), 0.89 (t, J ) 6.8 Hz, 3H). Anal. (C23H39NO2) C, H, N.
N-Eth yl N-(2-h yd r oxyeth yl) a r a ch id on oyl a m id e (31)
was prepared from arachidonic acid (152 mg, 0.5 mmol) and
2-(ethylamino)ethanol (0.49 mL, 5 mmol) as described for
compound 11: yield, 156 mg (83%) as a colorless oil; 1H NMR
(CDCl3) δ 5.31-5.42 (m, 8H), 3.75 (t, J ) 4.6 Hz, 2H), 3.51 (t,
J ) 4.6 Hz, 2H), 3.34 (q, J ) 7.1 Hz, 2H), 2.79-2.86 (m, 6H),
2.35 (t, J ) 7.8 Hz, 2H), 2.04-2.15 (m, 4H), 1.64-1.77 (m, 2H),
1.25-1.40 (m, 6H), 1.19 (t, J ) 7.1 Hz, 3H), 0.89 (t, J ) 7.1
Hz, 3H). Anal. (C24H41NO2) C, H, N.
An a n d a m id e (18:4, n -3) (40) was prepared from cis-6,9,-
12,15-octadecatetraenoic acid (50 mg, 0.18 mmol) and etha-
nolamine (0.11 mL, 1.8 mmol) in 76% yield as a colorless oil:
1H NMR (CDCl3) δ 5.92 (br s, 1H), 5.33-5.40 (m, 8H), 3.72 (t,
J ) 5.1 Hz, 2H), 3.42 (q, J ) 5.2 Hz, 2H), 2.79-2.84 (m, 6H),
2.22 (t, J ) 7.8 Hz, 2H), 2.02-2.12 (m, 4H), 1.60-1.72 (m, 2H),
1.38-1.48 (m, 2H), 0.98 (t, J ) 7.5 Hz, 3H). Anal. (C20H33
NO2) C, H, N.
-
N-P r op yl N-(2-h yd r oxyeth yl) a r a ch id on oyl a m id e (32)
was prepared from arachidonic acid (152 mg, 0.5 mmol) and
2-(propylamino)ethanol (0.57 mL, 5 mmol) as described for
compound 11: yield, 119 mg (61%) as a colorless oil; 1H NMR
(CDCl3) δ 5.30-5.42 (m, 8H), 3.76 (t, J ) 4.9 Hz, 2H), 3.52 (t,
J ) 4.4 Hz, 2H), 3.24 (t, J ) 8.1 Hz, 2H), 2.79-2.86 (m, 6H),
2.35 (t, J ) 8.1 Hz, 2H), 2.02-2.20 (m, 4H), 1.54-1.76 (m, 4H),
1.25-1.38 (m, 6H), 0.86-0.89 (m, 6H). Anal. (C25H43NO2) C,
H, N.
An a n d a m id e (18:3, n -3) (41) was prepared from cis-9,12,-
15-octadecatrienoic acid (100 mg, 0.36 mmol) and ethanola-
mine (0.22 mL, 3.6 mmol) in 74% yield as a colorless oil: 1H
NMR (CDCl3) δ 6.13 (br s, 1H), 5.29-5.42 (m, 6H), 3.71 (t, J
) 5.1 Hz, 2H), 3.41 (q, J ) 5.1 Hz, 2H), 2.80 (t, J ) 5.7 Hz,
4H), 2.20 (t, J ) 8.1 Hz, 2H), 2.04-2.12 (m, 4H), 1.63 (t, J )
6.9 Hz, 2H), 1.31 (br s, 10H), 0.97 (t, J ) 7.5 Hz, 3H). Anal.
(C20H35NO2) C, H, N.
An a n d a m id e (20:5, n -3) (42)22,23 was prepared from cis-
5,8,11,14,17-eicosapentaenoic acid (50 mg, 0.16 mmol) and
ethanolamine (0.10 mL, 1.6 mmol) in 72% yield as a colorless
oil: 1H NMR (CDCl3) δ 5.90 (br s, 1H), 5.22-5.42 (m, 10H),
3.71 (t, J ) 5.1 Hz, 2H), 3.41 (q, J ) 4.9 Hz, 2H), 2.79-2.86
(m, 8H), 2.05-2.22 (m, 6H), 1.60-1.72 (m, 2H), 0.97 (t, J )
7.9 Hz, 3H).
N,N-(Di-2-h yd r oxyeth yl) a r a ch id on oyl a m id e (33) was
prepared from arachidonic acid (100 mg, 0.33 mmol) and
diethanolamine (0.32 mL, 3.3 mmol) as described for compound
1
11: yield, 58 mg (45%) as a colorless oil; H NMR (CDCl3) δ
5.30-5.42 (m, 8H), 3.83 (t, J ) 4.9 Hz, 2H), 3.77 (t, J ) 5.1
Hz, 2H), 3.54 (t, J ) 5.1 Hz, 2H), 3.49 (t, J ) 4.9 Hz, 2H),
2.77-2.86 (m, 6H), 2.40 (t, J ) 7.3 Hz, 2H), 2.02-2.16 (m, 4H),
1.69-1.76 (m, 2H), 1.25-1.38 (m, 6H), 0.88 (t, J ) 6.6 Hz, 3H).
Anal. (C24H41NO3) C, H, N.
An a n d a m id e (22:6, n -3) (43)16 was prepared from cis-4,7,-
10,13,16,19-docosahexaenoic acid (100 mg, 0.3 mmol) and
ethanolamine (0.18 mL, 3 mmol) in 65% yield as a colorless
oil: 1H NMR (CDCl3) δ 5.90 (br s, 1H), 5.28-5.42 (m, 12H),
3.72 (t, J ) 5.1 Hz, 2H), 3.41 (q, J ) 4.9 Hz, 2H), 2.77-2.88
(m, 10H), 2.42 (t, J ) 8.1 Hz, 2H), 2.22-2.30 (m, 2H), 2.02-
2.14 (m, 2H), 0.97 (t, J ) 7.8 Hz, 3H).
N-Hyd r oxy N-a r a ch id on oyl a m id e (34)29 was prepared
from arachidonic acid (200 mg, 0.66 mmol), hydroxylamine
hydrochloride (220 mg, 3.17 mmol) and triethylamine (0.44
mL, 3.17 mmol) as described for compound 11: yield, 103 mg
(49%) as a colorless oil; 1H NMR (CDCl3) δ 5.30-5.43 (m, 8H),
2.78-2.86 (m, 6H), 2.02-2.17 (m, 6H), 1.70-1.78 (m, 2H),
1.22-1.38 (m, 6H), 0.89 (t, J ) 6.9 Hz, 3H).
An a n d a m id e (20:1, n -9) (44)20,22,23 was prepared from cis-
11-eicosenoic acid (100 mg, 0.32 mmol) and ethanolamine (0.19
1
mL, 3.2 mmol) in 70% yield: mp 67-68 °C; H NMR (CDCl3)

666 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Sheskin et al.
δ 5.90 (br s, 1H), 5.32-5.36 (m, 2H), 3.70 (t, J ) 5.1 Hz, 2H),
3.40-3.42 (m, 2H), 2.18 (t, J ) 7.8 Hz, 2H), 1.96-2.02 (m, 4H),
mmol) and (S)-(+)-1-amino-2-propanol (0.16 mL, 2 mmol) in
57% yield, as a colorless oil: 1H NMR (CDCl3) δ 5.95 (br s,
1H), 5.30-5.42 (m, 8H), 3.92-3.96 (m, 1H), 3.40-3.50 (m, 1H),
3.02-3.23 (m, 1H), 2.70-2.95 (m, 7H), 2.20-2.28 (m, 1H), 2.00-
2.15 (m, 4H), 1.70-1.75 (m, 2H), 1.20-1.52 (series of m, 14H),
1.54-1.63 (m, 6H), 0.86 (t, J ) 6.1 Hz, 3H). Anal. (C22H43
-
NO2) C, H, N.
An a n d a m id e (16:0) (45)16,20 was prepared from palmitic
acid (100 mg, 0.39 mmol) and ethanolamine (0.23 mL, 3.9
mmol) in 79% yield: mp 100-101 °C; 1H NMR (CDCl3) δ 6.00
(br s, 1H), 3.72 (t, J ) 5.1 Hz, 2H), 3.41 (q, J ) 4.9 Hz, 2H),
2.22 (t, J ) 7.8 Hz, 2H), 1.56-1.80 (m, 4H), 1.00-1.40 (m,
22H), 0.86 (t, J ) 6.1 Hz, 3H).
0.89 (t, J ) 6.7 Hz, 3H). Anal. (C25H43NO2) C, H, N.
N-((R)-(-)-1-Met h yl-2-h yd r oxyet h yl)
r,r-d im et h yl-
a r a ch id on oyl a m id e (55) was prepared from arachidonic
acid (152 mg, 0.5 mmol) and (R)-(-)-2-amino-1-propanol (0.16
mL, 2 mmol) in 45% yield, as a colorless oil:
1H NMR (CDCl3)
Oleoyl a m id e (46)31 was prepared from oleic acid (882 mg,
3.12 mmol) and ammonium hydroxide (2.34 mL, 0.12 mol) in
δ 5.80 (br s, 1H), 5.30-5.42 (m, 8H), 4.01-4.12 (m, 1H), 3.60-
3.68 (m, 1H), 3.50-3.55 (m, 1H), 2.98-3.01 (m, 1H), 2.76-2.84
(m, 6H), 1.90-2.10 (m, 4H), 1.15-1.62 (series of m, 17H), 0.90
(t, J ) 7.1 Hz, 3H). Anal. (C25H43NO2) C, H, N.
N-((S)-(+)-1-Meth yl-2-h ydr oxyeth yl) r,r-dim eth ylar ach i-
d on oyl a m id e (56) was prepared from arachidonic acid (152
mg, 0.5 mmol) and (S)-(+)-2-amino-1-propanol (0.16 mL, 2
mmol) in 44% yield, as a colorless oil: 1H NMR (CDCl3) δ 5.80
(br s, 1H), 5.30-5.42 (m, 8H), 4.01-4.12 (m, 1H), 3.60-3.68
(m, 1H), 3.50-3.55 (m, 1H), 2.98-3.01 (m, 1H), 2.76-2.84 (m,
6H), 1.90-2.10 (m, 4H), 1.15-1.62 (series of m, 17H), 0.90 (t,
J ) 7.1 Hz, 3H). Anal. (C25H43NO2) C, H, N.
N-((R)-(-)-2-h ydr oxypr opyl) r,r-dim eth ylar ach idon oyl
a m id e (57) was prepared from arachidonic acid (152 mg, 0.5
mmol) and (R)-(-)-1-amino-2-propanol (0.16 mL, 2 mmol) in
63% yield, as a colorless oil: 1H NMR (CDCl3) δ 5.95 (br s,
1H), 5.30-5.42 (m, 8H), 3.92-3.96 (m, 1H), 3.40-3.50 (m, 1H),
3.02-3.23 (m, 1H), 2.70-2.95 (m, 7H), 2.20-2.28 (m, 1H), 2.00-
2.15 (m, 4H), 1.70-1.75 (m, 2H), 1.20-1.52 (series of m, 14H),
0.89 (t, J ) 6.7 Hz, 3H). Anal. (C25H43NO2) C, H, N.
Liga n d Bin d in g Assa y. The synaptosomal brain prepara-
tion was made from whole rat brain as described previously.1,25
Radiolabeled HU-24325 was stored at a concentration of 1 µM
(40.5 µCi/mmol) in the absolute ethanol at -20 °C. Unlabeled
drugs were stored as 10 mM and 100 µM ethanol solutions at
-20 °C. After evaporation of the ethanol, drug dilutions were
performed serially from 1 or 100 µM solutions in a vehicle of
5 mg/mL of fatty acid-free BSA (Sigma Chemical Co., St. Louis,
MO) in water. The radioligand was placed in a beaker, the
ethanol was evaporated, and then 0.2 mL of 50 mg/mL of BSA
was added, followed by 100 mL of TME buffer. This prepara-
tion was aliquoted in 0.8 mL volumes to siliconized microfuge
tubes (Sigma), followed by 0.1 mL of drug or vehicle and 0.1
mL of synaptosomal membrane preparation in TME buffer.
Thus, the final assay volume of 1 ml contained 45 mM Tris-
HCl, 2.7 mM MgCl2, 0.9 mM EDTA, 0.58 mg of BSA, 2.4-3.8
µg of protein of synaptosomal membrane preparation, 38-48
fmol of [3H]HU-243, and vehicle or drug.
1
60% yield: mp 74-75 °C; H NMR (CDCl3) δ 5.42 (br s, 2H),
5.32-5.36 (m, 2H), 2.22 (t, J ) 7.8 Hz, 2H), 1.98-2.02 (m, 6H),
1.62-1.66 (m, 4H), 1.27-1.31 (m, 16H), 0.88 (t, J ) 6.9 Hz,
3H).
cis-5,8,11,14-Eicosa tetr a yoyl eth a n ola m id e (47) was
prepared from cis-5,8,11,14-eicosatetrayoic acid (25 mg, 0.084
mmol) and ethanolamine (0.05 mL, 0.84 mmol) in 64% yield
as a colorless oil: 1H NMR (CDCl3) δ 6.15 (br s, 1H), 3.73 (t,
J ) 5.1 Hz, 2H), 3.42 (q, J ) 4.9 Hz, 2H), 3.06-3.22 (m, 6H),
2.36 (t, J ) 7.8 Hz, 2H), 2.20-2.24 (m, 2H), 2.10-2.18 (m, 2H),
1.78-1.88 (m, 2H), 1.40-1.56 (m, 2H), 1.20-1.38 (m, 4H), 0.90
(t, J ) 6.1 Hz, 3H). Anal. (C22H29NO2) C, H, N.
Com p ou n d s 48-57. R-Methylarachidonic acid was syn-
thesized from methyl arachidonate with 2 equivalents of LDA
(lithium diisopropylamine, prepared in situ) and methyl iodide
at -50 °C, as described by Adams et al.19 The R,R-dimethyl
arachidonic acid was synthesized by recycling the R-methy-
larachidonic acid through the same reaction a second time.
After hydrolysis of the ester with lithium hydroxide in 3:1
methanol:water, the acid derivatives were converted to the
corresponding amides by reaction with oxalyl chloride and the
appropriate amine.
r-Meth yl a n a n d a m id e (48)19,22 was prepared from arachi-
donic acid (152 mg, 0.5 mmol) and ethanolamine (0.15 mL,
2.5 mmol) in 82% yield, as a colorless oil: 1H NMR (CDCl3) δ
6.10 (br s, 1H), 5.22-5.40 (m, 8H), 3.75 (t, J ) 5.1 Hz, 2H),
3.42 (q, J ) 4.9 Hz, 2H), 2.74-2.86 (m, 6H), 2.20-2.30 (m,
1H), 2.00-2.12 (m, 4H), 1.68-1.80 (m, 1H), 1.20-1.50 (m, 6H),
1.16 (d, J ) 6.6 Hz, 3H), 0.89 (t, J ) 6.9 Hz, 3H).
r,r-Dim eth yl a n a n d a m id e (49)19,22,23 was prepared from
arachidonic acid (152 mg, 0.5 mmol) and ethanolamine (0.15
mL, 2.5 mmol) in 47% yield, as a colorless oil: 1H NMR (CDCl3)
δ 6.10 (br s, 1H), 5.30-5.42 (m, 8H), 3.75 (t, J ) 5.1 Hz, 2H),
3.42 (q, J ) 4.9 Hz, 2H), 2.78-2.86 (m, 6H), 2.00-2.10 (m,
4H), 1.50-1.62 (m, 4H), 1.24-1.40 (m, 4H), 1.22 (s, 6H), 0.90
(t, J ) 6.9 Hz, 3H).
Tubes were incubated at 30 °C for 90 min and then
centrifuged at 13 000 rpm for 6 min. After centrifugation,
samples of the supernatant were saved and counted. The
remaining supernatant was aspired off, and the tubes were
placed inverted on drying pins. After 20 min, the tips of the
tubes containing the pelleted membranes were removed with
a heated scalpel blade in a rig designed to ensure consistency.
The tips were then placed in Opti-Fluor, LSC-coctail (Packard,
A Canberra Co.) and vortexed, and after several hours,
radioactivity was determined as disintegrations per minute.
The radioligand adhering to the remaining part of the vial
after tip removal was also determined. Nonspecific binding
to the microfuge tip was assessed in tubes containing radio-
ligand and protein, which were not centrifuged. This value
was subtracted from the total binding of the cut tips to give
the total bound to the pelleted membranes. Specific binding
was defined as the difference between the total bound to the
pelleted membranes in the absence and presence of 50 nM
unlabeled HU-243 and was typically 70-80% of the total
bound. Assays were performed in triplicate, and experiments
were repeated three times.
N-P r op yl r-m eth yla r a ch id on oyl a m id e (50) was pre-
pared from arachidonic acid (152 mg, 0.5 mmol) and propy-
lamine (0.21 mL, 2.5 mmol) in 84% yield, as a colorless oil:
1H NMR (CDCl3) δ 5.20-5.45 (m, 9H), 3.18-3.25 (m, 2H),
2.70-2.86 (m, 6H), 2.00-2.20 (m, 5H), 1.70-1.80 (m, 1H),
1.40-1.60 (m, 4H), 1.10 (d, J ) 6.6 Hz, 3H), 0.86-0.95 (m,
6H). Anal (C24H41NO): C, H, N.
N-P r op yl r,r-d im eth yla r a ch id on oyl a m id e (51) was
prepared from arachidonic acid (152 mg, 0.5 mmol) and
propylamine (0.21 mL, 2.5 mmol) in 70% yield, as a colorless
oil: 1H NMR (CDCl3) δ 5.60 (br s, 1H), 5.30-5.40 (m, 8H),
3.20-3.23 (m, 2H), 2.79-2.84 (m, 6H), 2.00-2.10 (m, 4H), 1.20-
1.60 (series of m, 10H), 1.15 (s, 6H), 0.86-0.94 (m, 6H). Anal.
(C25H43NO) C, H, N.
N-Isop r op yl r-m eth yla r a ch id on oyl a m id e (52) was
prepared from arachidonic acid (152 mg, 0.5 mmol) and
isopropylamine (0.21 mL, 2.5 mmol) in 85% yield, as a colorless
oil: 1H NMR (CDCl3) δ 5.30-5.42 (m, 8H), 4.02-4.14 (m, 1H),
2.76-2.90 (m, 6H), 1.92-2.12 (m, 5H), 1.10-1.60 (series of m,
17H), 0.89 (t, J ) 6.9 Hz, 3H). Anal. (C24H41NO) C, H, N.
N-Isop r op yl r,r-d im eth yla r a ch id on oyl a m id e (53) was
prepared from arachidonic acid (152 mg, 0.5 mmol) and
isopropylamine (0.21 mL, 2.5 mmol) in 78% yield, as a colorless
oil: 1H NMR (CDCl3) δ 5.20-5.50 (m, 9H), 4.04-4.14 (m, 1H),
2.76-2.90 (m, 6H), 1.90-2.15 (m, 4H), 1.10-1.60 (series of m,
20H), 0.90 (t, J ) 6.9 Hz, 3H). Anal. (C25H43NO) C, H, N.
N-((S)-(+)-2-h yd r oxyp r op yl) r,r-d im eth yla r a ch id on oyl
a m id e (54) was prepared from arachidonic acid (152 mg, 0.5
Receptor binding assays with transfected COS-7 cells for
either CB1 or CB2 were performed as described in ref 35.
Ack n ow led gm en t. This work was supported by
NIDA Grants DA 9789 and DA 8169 (to R.M.) and a
grant by the Israel Academy of Sciences (to Z.V. and
R.M.).

Binding of Anandamide-Type Compounds
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shorthand designation. Thus the anandamide derived from
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