Synthesis, cannabinoid receptor activity, and enzymatic stability of reversed amide derivatives of arachidonoyl ethanolamide

Article abstract of DOI:10.1016/j.bmc.2006.03.051

Retroanandamide (2f) and its 10 analogues (1a-e, 2a-e) were synthesized and evaluated for the cannabinoid receptor activation by a [³⁵S]GTPγS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing huma

Full text of DOI:10.1016/j.bmc.2006.03.051

Bioorganic & Medicinal Chemistry 14 (2006) 5252–5258
Synthesis, cannabinoid receptor activity, and enzymatic stability of
reversed amide derivatives of arachidonoyl ethanolamide
Teija Parkkari,a,* Juha R. Savinainen,b Katri H. Raitio,a Susanna M. Saario,a
Laura Matilainen,a Tuomas Sirvio¨,a Jarmo T. Laitinen,b Tapio Nevalainen,a
Riku Niemia and Tomi Ja¨rvinena
aDepartment of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
bDepartment of Physiology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
Received 13 June 2005; revised 23 February 2006; accepted 31 March 2006
Available online 27 April 2006
Abstract—Retroanandamide (2f) and its 10 analogues (1a–e, 2a–e) were synthesized and evaluated for the cannabinoid receptor acti-
vation by a [35S]GTPcS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing human
CB2 receptors. The primary goal of the study was to develop cannabinoid receptor agonists having improved enzymatic stability
compared to endogenous N-arachidonoyl ethanolamide (AEA). Furthermore, by reversing the amide bond of AEA, the formation
of arachidonic acid would be prevented. Finally, an effect of the carbonyl carbon position on the cannabinoid receptor activity was
explored by synthesizing retroanandamide analogues having different chain lengths (1a–e, C19; 2a–f, C20). All the synthesized com-
pounds, except 2c, behaved as partial agonists for the both cannabinoid receptors. In rat brain homogenate, the reversed amides
possessed significantly higher stability against FAAH induced degradation than AEA. Therefore, the reversed amide analogues
of AEA may serve as enzymatically stable structural basis for the drug design based on the endogenous cannabinoids.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
disorders, like multiple sclerosis, Huntington’s disease,
Parkinson’s disease, and ischemic stroke, are of great
interest at the moment.5–12
During the past 10 years the endogenous cannabinoid
system (ECS) has shown its potential as a target for the
drug discovery.1 The quantity and the distribution of
the cannabinoid receptors in the central nervous system
(CNS) as well as in the peripheral tissues have suggested
that the ECS has a significant role in certain physiologi-
cal events. It is well known that the CB1 receptor partic-
ipates, for example, in the appetite stimulation, nausea
suppression, cognition and memory, pain perception,
and the regulation of intra ocular pressure.2–6 Few
countries have already accepted cannabinoids (Mari-
nolꢁ, Cesametꢁ) for the treatment of chemotherapy
related nausea and the appetite stimulation of AIDS pa-
tients, but several other therapeutic applications have
also been suggested, such as pain, certain types of cancer,
asthma, glaucoma, and especially various CNS
So far, the most potent CB1 ligand in our experimental
setting assessing CB1 receptor-dependent G protein
activity has been the classical cannabinoid, HU-210
(pEC50 SEM = 8.3 0.1, unpublished data). The most
effective ligand is, however, the endocannabinoid 2-AG
(Emax = 620 5, % Basal SEM),13 which focused our
interest in the molecular structure of endocannabinoids
and their derivatives. Unfortunately, 2-AG’s susceptibil-
ity for acid, base or heat promoted acyl migration makes
it a difficult compound to synthesize and handle, and
therefore, the other major endocannabinoid, arachido-
noyl ethanolamide (AEA), and its derivatives have been
synthesized and studied more extensively. Hillard
reported that AEA acts as a partial agonist in a
[35S]GTPcS binding assay with the rat cerebellar mem-
branes, nevertheless, being more potent than 2-AG.14
The study of Savinainen et al.13 confirmed the partial
agonist behavior of AEA, but in their experimental
setting 2-AG was clearly more potent than AEA
(Table 1).13,15
Keywords: Cannabinoid; AEA; Reversed amide; Synthesis; CB1; CB2;
35S]GTPcS binding assay; Enzymatic stability.
[
*
Corresponding author. Tel.: +358 17 162431; fax: +358 17 162456;
e-mail: Teija.Parkkari@uku.fi
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.051

T. Parkkari et al. / Bioorg. Med. Chem. 14 (2006) 5252–5258
Table 1. Comparison of efficacy (Emax) and potency (pEC50) values of
5253
2. Chemistry
2-AG, AEA, 1a–e (C19 series), and 2a–f (C20 series) at rat cerebellar
membranes (means SEM, n = 3)
Norarachidonyl amides 1a–e (C19 series) were prepared
by the method published by Nazih and Heissler (Scheme
1).20 This method allows one-pot conversion of t-butyl
carbamates to amides. It is based on a cleavage of a t-
butoxycarbonyl group with hydrogen iodide generated
in situ by reaction of acyl halide with methanol and
on an acylation of an intermediate amine with excess
acyl halide in the presence of di-isopropylethylamine
(DIPEA). The synthesis of the key intermediate nor-
arachidonyl N-t-butylcarbamate 5 was started by con-
verting arachidonic acid to acyl azide with diphenyl
phosphorylazide (DPPA).21 The acyl azide undergoes
the Curtius rearrangement to norarachidonyl isocyanate
4 upon heating. Compound 5 is finally achieved by
refluxing the isocyanate 4 with t-butanol for several
days. Scheme 2 summarizes the synthesis method for
3-(t-butyldiphenylsilyloxy)-propionyl chloride 14 used
in the synthesis of the final product 1c.
Compound
CB1 activation
Relative Emax
(% 2-AG Emax SEM)
Potency
(ꢀlog EC50 SEM)
2-AG
AEA
1a
100
6.0 0.0
5.3 0.1
5.4 0.1
5.4 0.2
4.4 0.1
5.3 0.3
6.1 0.1
5.7 0.1
5.6 0.1
NA
67
45
36
46
2
4
3
6
1b
1c
1d
1e
38 12
37
49
4
5
2
2a
2b
39
2c
2d
NA
34
1
4
7
5.8 0.2
5.6 0.3
4.9 0.1
2e
2f
40
55
NA, no detectable CB1 activation at 10ꢀ4 M concentration.
The data represent means ( SEM) of [35S]GTPcS binding as per-
centage of the maximal response evoked by 2-AG and are from at least
three independent experiments performed in duplicate.
Synthesis methods for compounds 2a–f (C20 series) are
summarized in Schemes 3 and 4. Synthesis of a key
intermediate, arachidonyl amine 8, was performed by
converting commercially available arachidonyl alcohol
to arachidonyl azide 7 via more reactive mesylate and
further reducing the azide to amine with LiAlH4
(LAH).19 Compounds 2a–b and 2e–d were synthesized
from arachidonyl amine and appropriate acid halides.
Retroanandamide 2f was synthesized as previously de-
scribed.19 Compound 2c was prepared by coupling cis/
trans-2-phenyl-5-carboxy-1,3-dioxane 18 (Scheme 4)22
Although AEA and 2-AG are good ligands for the can-
nabinoid receptors, and they have been shown to act as
therapeutic agents in several in vitro and in vivo models,
they are enzymatically degraded too easily to serve as
good probes for the cannabinoid research, not to men-
tion as pharmaceuticals. There have been several at-
tempts to stabilize the structure of AEA. Branching of
an ethanolamine moiety stabilizes the amide bond; espe-
cially an addition of a methyl group adjacent to nitrogen
((R)-methanandamide) increases the enzymatic stability
as well as activity in vivo.16,17 Attachment of a methyl
or dimethyl group on the a-position of AEA also
increases the enzymatic stability and thereby the biolog-
ical activity both in vitro and in vivo.17,18 Nevertheless,
the branching of the chain generates a chiral center to
the molecule and it has been observed that enantiomers
have differences in their CB1 receptor binding. Thus, to
achieve reliable binding data, pure enantiomers must be
provided, which is always an extra challenge for the syn-
thesis. Makriyannis et al. observed indirectly that rever-
sal of the carbonyl and NH in the amido group provides
high metabolic stability.19 However, this retroananda-
mide (2f, Scheme 3) showed weaker CB1 affinity than
AEA; Ki values being 61 nM for AEA and 115 nM for
retroanandamide when fatty acid amide hydrolase
(FAAH) was inhibited by phenyl methylsulfonyl
fluoride (PMSF). In contrast, when PMSF was not used,
the Ki values were 5810 and 134 nM, respectively, indi-
O
N
O
a, b
c
OH
3
4
H
H
N
N
R
O
d
O
O
1
5
R
a) CH2CH3
d
b) CH2CH2CH3
c) CH2CH2OH
d) CH2CH2Cl
H
N
O
1e
Scheme 1. Reagents: (a) DPPA, Et3N, benzene; (b) toluene, D; (c)
t-BuOH; (d) i—RCOCl, NaI, acetonitrile, MeOH; ii—DIPEA.
cating the increased enzymatic
retroanandamide.
stability
of
O
O
O
Ph
Si
b
a
O
O
O
HO
O
Ph
11
In the present study, we have synthesized various re-
versed amides to study their cannabinoid receptor
activity at rat cerebellar membranes and human
CB2 (hCB2) receptors. In addition, we have deter-
mined their enzymatic stability using rat brain
homogenate. Finally, we have also investigated the
chain length effect (C19 vs C20) on the cannabinoid
receptor activity.
12
10
O
Ph
Si
O
Ph
Si
c
d
O
Cl
Ph
O
OH
Ph
14
13
Scheme 2. Reagents: (a) NaOH, MeOH; (b) TBDPSCl, imidazole,
THF; (c) 1M LiOH, MeOH, H2O; (d) (COCl)2, benzene.

5254
T. Parkkari et al. / Bioorg. Med. Chem. 14 (2006) 5252–5258
FAAH is blocked with an enzyme inhibitor (MAFP).
a, b
c
OH
N3
However, compounds appear to have comparable
potency to AEA in the reversed amide series where
hydroxyethyl group is replaced with alkyl chain (1a–b,
1d–e and 2a–b, 2d–e); the results are in a good agree-
ment with findings of Pinto et al. and Sheskin et al.
who observed that by increasing the lipophilicity, the
CB1 receptor affinity can be improved.23,24
7
6
O
e
NH2
d
N
H
OH
2f
8
O
N
H
f
2e
It has been shown that naturally occurring ananda-
mides, such as (22:4, n-6) and (20:4, n-6), that is,
AEA, possess similar affinities for the CB1 receptor.23
Reports about reversed amides with different chain
lengths or different degree of double bonds have not
been published. In our reversed amide series, a longer
chain length (i.e., C20) is preferred although the differ-
ences are not very significant.
O
O
O
N
R
Ph
N
H
H
g
O
2
R
9
a) CH2CH3
b) CH2CH2CH3
c) CH(CH2OH)2
d) CH2CH2Cl
Scheme 3. Reagents: (a) MsCl, pyridine; (b) NaN3; (c) LAH; (d)
RCOCl, Et3N; (e) Al(CH3)3, b-propiolactone, CH2Cl2; (f) 18, EDCl,
DMAP, CH2Cl2; (g) HCl, MeOH.
3.1.2. CB2 receptor activation. The CB2 receptor activi-
ties for the compounds 1a–e (C19 series) and 2a–f (C20
series) were determined at the human CB2 (hCB2)
receptor expressed in Chinese hamster ovary (CHO)
cells as previously described.25 As shown in Table 2,
all the studied compounds behaved as partial agonists
and had comparable efficacy and potency values to
AEA. The most interesting finding in the both C19 and
C20 series was that compounds with butanoyl tail (1b,
2b) possess potencies that are equal to that of 2-AG
(7.4 0.1). In addition, compound 2b is relatively effica-
cious. Although, Makriyannis et al. reported that ret-
roanandamide has only weak affinity for the CB2
receptor,19 our findings suggest that reversed amides
are able to activate both subtypes of cannabinoid
receptors.
O
O
O
O
O
O
OH
OH
O
O
O
O
a
b
16
15
O
O
O
O
O
HO
HO
c
HO
O
18
O
17
Scheme 4. Reagents and condition: (a) benzaldehyde, toluene, H2SO4;
(b) i—KOH, EtOH; ii—HCl; (c) Et3N, D.
3.2. Enzymatic stability of reversed amide derivatives
with arachidonyl amine using N0-(3-dimethylaminopro-
pyl)-N-ethylcarbodiimide hydrochloride (EDCl) and
4-dimethylaminopyridine (DMAP) as coupling reagents.
The desired product was obtained from compound 9 by
removing the benzylidene acetal protective group with
concd HCl/MeOH solution.
One of the main goals of this study was to stabilize the
amide linkage of endogenous AEA against enzymatic
Table 2. Comparison of efficacy (relative Emax) and potency
(ꢀlogEC50) values of AEA, 1a–e (C19 series), and 2a–f (C20 series) at
CHO-hCB2 cells
Compound
CB2 activation
3. Results and discussion
Relative Emax
(% HU-210 Emax SEM)
Potency
3.1. [35S]GTPcS membrane binding studies
(ꢀlogEC50 SEM)
HU-210
AEA
1a
100
9.8 0.125
6.5 0.2
6.7 0.1
7.1 0.2
5.8 0.3
6.6 0.5
6.7 0.2
7.1 0.2
7.0 0.2
ND
48
54
53
5
3
4
3.1.1. CB1 receptor activation. The ability of the synthe-
sized compounds to activate the CB1 receptor was deter-
mined by the [35S]GTPcS membrane binding assay as
previously described.13 Comparison of the efficacy and
the potency values are presented in Table 1. All the syn-
thesized compounds, except for 2c, showed dose-depen-
dent CB1 activity, and their responses at 5 · 10ꢀ5 M
were reversed by the CB1 receptor antagonist, AM251
at 10ꢀ6 M concentration (data not shown).
1b
1c
54 15
59 10
1d
1e
55
64
4
3
3
2a
2b
73
2c
2d
ND
64
4
4
7
6.8 0.2
6.7 0.2
6.6 0.2
2e
2f
67
75
In the [35S]GTPcS binding assay, the reversed amides, as
well as AEA,13,15 acted as partial agonists. As the previ-
ous affinity studies of retroanandamide suggested,19
AEA has higher Emax value than 1c and 2f (retroa-
nandamide) in the study conditions where function of
ND, not determined.
The data represent means ( SEM) of [35S]GTPcS binding as per-
centage of the maximal response evoked by HU-210 and are from at
least three independent experiments performed in duplicate.

T. Parkkari et al. / Bioorg. Med. Chem. 14 (2006) 5252–5258
5255
hydrolysis. The stability of retroanandamide (2f) has not
been studied, but it has been hypothesized to be more
stable against FAAH mediated degradation than
AEA, based on the results of affinity studies with and
without a FAAH inhibitor.19 The stability studies were
performed by incubating the synthesized compounds
1a, 1c, 1e, 2b, 2e, and 2f in the rat brain homogenate
for 90 min (37 ꢂC, pH 7.4), taking the samples at time
points 0 and 90 min, analyzing the samples by HPLC,
and comparing the results to those using AEA. All the
synthesized compounds were stable (no degradation
during 90 min) in the rat brain homogenate, whereas
AEA was almost fully degraded in 90 min. The degrada-
tion of AEA in the rat brain homogenate was inhibited
also in the presence of a potent FAAH inhibitor
URB597. URB597 was able to inhibit the AEA degra-
dation with an IC50 value of 3.8 0.6 nM (n = 3) which
is comparable to the previously reported results, and it
confirms that in the rat brain homogenate AEA is main-
ly metabolized by FAAH.26
m = multiplet. Coupling constants are reported in Hz
and letter J indicates J if not otherwise noted. ESI-
3
MS spectra were acquired using a LCQ ion trap mass
spectrometer equipped with an electrospray ionization
source (Finnigan MAT, San Jose, CA, USA). Elemental
analyses for C, H, and N were performed on a Thermo-
Quest CE Instruments EA1110-CHNS-O elemental
analysator (ThermoQuest, Italy). TLC was performed
using silica gel (60 F254) coated aluminum sheets
(Merck, Germany). Arachidonic acid and arachidonyl
alcohol were purchased from Nu-Chek-Prep, Inc.
4.2. Norarachidonyl N-t-butylcarbamate (5)
Arachidonic acid (500 mg, 1.64 mmol) and dry Et3N
(0.34 ml, 2.46 mmol) were dissolved in dry benzene
(6 ml) and stirred for 10 min. DPPA (0.53 ml,
2.46 mmol) was added, and stirring was continued at
RT for 2 h. Benzene was evaporated, the residue was
dissolved in petroleum ether/EtOAc 10:1 and filtered
through the pad of silica gel. Solvents were evaporated
and the oily residue was dissolved in dry toluene
(3 ml). t-BuOH (0.31 ml, 3.28 mmol) in dry toluene
(3 ml) was added, and the reaction mixture was stirred
at 65 ꢂC for 5 days. The reaction mixture was cooled
to rt, water (20 ml) and EtOAc (20 ml) were added,
and the solution was washed with brine. The organic
layers were combined, dried over Na2SO4, and evapo-
rated. The crude product was purified by flash chroma-
tography eluting with 2.5% EtOAc in petroleum ether.
Evaporation of solvents yielded 240 mg of yellowish oily
product (39%). Rf = 0.45 (petroleum ether/EtOAc 6:1).
1H NMR: d 0.89 (t, J = 7.0, 3H), 1.26–1.40 (m, 6H),
1.44 (s, 9H), 1.56 (q, J = 7.4, 2H), 2.04–2.12 (m, 4H),
2.80–2.85 (m, 6H), 3.12–3.13 (m, 2H), 4.51 (br s, 1H),
5.31–5.43 (m, 8H).
3.3. Conclusions
We have developed a synthetic method for arachido-
noyl ethanolamide (AEA) derivatives having the chain
length of C19, synthesized reversed amide analogues of
AEA, measured their activities via the rat brain CB1
and human recombinant CB2 receptors, and deter-
mined their metabolic stability in the rat brain homog-
enate. The reversal of the amide bond of AEA
resulted in the weaker CB1 activity properties, but
both efficacy and potency values were reverted or even
enhanced by removing the hydroxyl group and there-
fore making the compound more lipophilic. In con-
trast, all the reversed amides studied had similar or
improved activity properties for the CB2 receptor
compared to AEA. Finally, the stability studies con-
ducted with rat brain homogenate showed that the re-
versed amides remain intact in the studied conditions,
whereas AEA is almost fully degraded. In conclusion,
we have shown that by reversing the amide bond of
AEA it is possible to develop cannabinoid receptor
agonists with good CB1 and CB2 activity, and a struc-
ture which is stable against FAAH or other enzyme
mediated metabolism.
4.3. General synthesis procedure for the compounds 1a–b
and 1d–e
An appropriate acid halide (4 equiv) was added into the
mixture of arachidonyl N-t-butylcarbamate (5)
(1 equiv), MeOH (2 equiv), and NaI (2 equiv) in dry
ACN (10 ml/mmol). The reaction mixture was stirred
for 0.5 h. The yellow solution was cooled on an ice-bath
and DIPEA (4 equiv) was added. Stirring was continued
at rt for 1 h. 10% HCl was added and the solution was
extracted with ether. The organic layer was washed with
satd NaHCO3 and evaporated. The crude product was
purified by flash chromatography eluting with petro-
leum ether/EtOAc 5:1.
4. Experimental
4.1. General
1H NMR and 13C NMR were recorded on a Bruker
Avance 500 spectrometer operating on 500.1 and
125.8 MHz, respectively. CDCl3 was used as a solvent
if not otherwise noted, and tetramethylsilane (TMS)
was used as an internal standard. The spectra were pro-
cessed from the recorded FID files with MestRe-C soft-
4.3.1. N-Propionyl norarachidonyl amide (1a). Colorless
oil (65%). Rf = 0.59 (petroleum ether/EtOAc 1:1) 1H
NMR: d 0.89 (t, J = 7.0, 3H), 1.15 (t, J = 7.6, 3H),
1.26–1.40 (m, 6H), 1.59 (q, J = 7.2, 2H), 2.03–2.13
(m, 4H), 2.2 (q, J = 7.6, 2H), 2.80–2.85 (m, 6H), 3.25–
3.29 (m, 2H), 5.31–5.43 (m, 8H). 13C NMR: d 9.9,
14.1, 22.6, 24.7, 25.7 (2C), 27.3, 29.3, 29.6, 29.8, 31.6,
39.2, 127.6, 127.8, 128.1, 128.3, 128.7 (2C), 129.1,
130.6, 173.6. ESI-MS [M+H] 332.2. Elemental analysis.
`
`
ware (version 2.3a, Departemento Quımica Organica,
Universidade de Santiago de Compostela, Spain).
Chemical shifts (d) are reported in parts per million
(ppm) downfield from TMS. Following abbreviations
are used: s = singlet, d = doublet, t = triplet, br
s = broad singlet, qv = quartet, q = quintet, st = sextet,
1
Calculated for C22H37NOꢁ H2O: C, 78.28%; H, 11.25%;
3
N, 4.15%. Found: C, 78.30%; H, 11.48%; N, 4.07%.

5256
T. Parkkari et al. / Bioorg. Med. Chem. 14 (2006) 5252–5258
4.3.2. N-Butanoyl norarachidonyl amide (1b). Colorless
oil (52%). Rf = 0.60 (petroleum ether/EtOAc 1:1). 1H
NMR: d 0.89 (t, J = 7.0, 3H), 0.95 (t, J = 7.3, 3H),
1.26–1.39 (m, 6H), 1.58 (q, J = 7.3, 2H), 1.66 (st,
J = 7.4, 2H), 2.03–2.15 (m, 6H), 2.80–2.58 (m, 6H),
3.25–3.29 (m, 2H), 5.31–5.43 (m, 8H), 5.51 (br s, 1H).
13C NMR: d 13.8, 14.1, 19.2, 22.6, 24.7, 25.6, 25.7,
27.3, 29.3, 29.6, 31.5, 38.8, 39.2, 127.6, 127.8, 128.1,
128.3, 128.7 (2C), 129.1, 130.5, 172.9. ESI-MS [M+H]
was added slowly. After the addition, stirring was con-
tinued at rt for 5 h. The reaction mixture was poured
into ice-cold water and extracted with ether/hexane
1:1. The water layer was acidified with 0.5 M KHSO4
and extracted several times with ether/hexane 1:1. The
organic layers were combined, washed with water, dried
over Na2SO4, and evaporated. The crude product was
recrystallized from hexane. Yield 1.78 g (68%).
Rf = 0.14 (petroleum ether/EtOAc 9:1). 1H NMR: d
1.04 (s, 9H), 3.05 (t, J = 5.9, 2H), 3.97 (t, J = 5.9, 2H),
7.36–7.46 (m, 6H), 7.64–7.67 (m, 4H).
346.2.
Elemental
1
analysis.
Calculated
for
C23H39NOꢁ H2O: C, 77.91%; H, 11.37%; N, 3.95%.
2
Found: C, 77.50%; H, 11.43%; N, 3.99%.
4.3.7. N-(3-Hydroxypropionyl)norarachidonyl amide (1c).
3-(t-Butyldiphenylsilyloxy)-propionic acid (13) was dis-
solved in benzene (12 ml) and oxalyl chloride (1.2 ml)
and few drops of DMF were added. The mixture was
stirred at rt for 1.5 h, after which unreacted oxalyl chlo-
ride and benzene were distilled off. Benzene (12 ml) was
added and the distillation was repeated. 3-(t-Butyldiphe-
nylsilyloxy)-propionylchloride (14) (291 mg, 0.84 mmol)
was added into the mixture of arachidonyl N-t-butylcar-
bamate (5) (80 mg, 0.21 mmol), MeOH (0.018 ml,
0.42 mmol), and NaI (60 mg, 0.042 mmol) in dry ACN
(2 ml). The reaction mixture was stirred for 0.5 h. The
yellow solution was cooled on an ice-bath and DIPEA
(0.14 ml, 0.84 mmol) was added. Stirring was continued
at rt for 1 h. 10% HCl was added and the solution was
extracted with ether. The organic layer was washed with
satd NaHCO3 and evaporated. The crude product was
purified by flash chromatography eluting with petro-
leum ether/EtOAc 5:1. Evaporation of solvents yielded
107 mg of oily product (86%). Rf = 0.22 (petroleum
ether/EtOAc 6:1). N-[(3-t-butyldiphenylsilyloxy) propio-
nyl] norarachidonyl amide (107 mg, 0.18 mmol) and
Bu4N+Fꢀ (86 mg, 0.274 mmol) in dry THF (3 ml) were
stirred at rt for 2 h. The solvent was evaporated, and
the crude product was purified by flash chromatography
eluting 33–100% EtOAc in petroleum ether. Reaction
proceeded quantitatively. Rf = 0.10 (petroleum ether/
EtOAc 1:1). 1H NMR: d 0.89 (t, J = 7.0, 3H), 1.26–
1.39 (m, 6H), 1.60 (q, J = 7.3, 2H), 2.06 (q, J = 7.1,
2H), 2.12 (q, J = 7.1, 2H), 2.41 (t, J = 5.4, 2H), 2.80–
2.85 (m, 6H), 3.26–3.30 (m, 2H), 3.87 (t, J = 5.4, 2H),
5.31–5.43 (m, 8H), 5.93 (br s, 1H). 13C NMR d 14.01,
22.6, 24.7, 25.6, 25.7, 27.2, 29.3, 29.5, 31.5, 38.0, 39.1,
59.0, 127.5, 127.8, 128.1, 128.4, 128.7, 128.8, 128.9,
4.3.3. N-(3-Chloropropionyl)norarachidonyl amide (1d).
Yellowish oil (43%). Rf = 0.49 (petroleum ether/EtOAc
1:1). H NMR: d 0.89 (t, J = 6.9, 3H), 1.26–1.39 (m,
6H), 1.61 (q, J = 7.4, 2H), 2.04–2.15 (m, 4H), 2.60 (t,
J = 6.4, 2H), 2.80–2.85 (m, 6H), 3.30 (q, J = 6.9, 2H),
3.81 (t, J = 6.5, 2H), 5.32–5.44 (m, 8H), 5.60 (br s,
1H). 13C NMR: d 14.1, 22.6, 24.6, 25.6, 25.7, 27.2,
29.3, 29.4, 31.5, 39.4, 39.8, 40.3, 127.5, 127.8, 128.1,
128.4, 128.7, 128.8, 129.0, 130.5, 169.3. ESI-MS
[M+H+] 366.0.
1
4.3.4. N-(Cyclopropanecarbonyl) norarachidonyl amide
(1e). Yellowish oil (99%). Rf = 0.15 (petroleum ether/
EtOAc 6:1). 1H NMR: d 0.69–0.73 (m, 2H), 0.89 (t,
J = 6.9, 3H), 0.94–0.97 (m, 2H), 1.26–1.39 (m, 7H),
1.59 (q, J = 7.4, 2H), 2.03–2.14 (m, 4H), 2.79–2.87 (m,
6H), 3.28 (q, J = 7.0, 2H), 5.31–5.43 (m, 8H), 5.68 (br
s, 1H). 13C NMR: d 7.0, 14.1, 14.8, 22.6, 24.7, 25.6,
25.7, 27.2, 29.3, 29.6, 31.5, 39.4, 71.5, 127.5, 127.8,
128.2, 128.3, 128.6, 128.7, 129.1, 130.5, 173.4. ESI-MS
[M+H+] 344.1. Elemental analysis. Calculated for
1
C23H37NOꢁ H2O: C, 79.99%; H, 10.86%; N, 4.0%.
10
Found: C, 79.83%; H, 10.88%; N, 4.11%.
4.3.5. 3-Hydroxypropionylmethylester (11). b-Propiolac-
tone (5 g, 69 mmol) was added dropwise into the pre-
cooled solution of NaOH (150 mg) in MeOH (50 ml).
After the addition, stirring was continued on an ice-bath
for 10 min. The solution was neutralized with 1 M HCl,
and most of the solvent was evaporated. The remaining
solution was extracted with EtOAc, the organic layers
were combined, washed with brine, dried over Na2SO4,
and evaporated. The crude product was purified by
1
vacuum distillation (12 mbar, 67–72 ꢂC). H NMR: d
2.57–2.59 (m, 3H), 3.72 (s, 3H), 3.87 (q, J = 5.9, 2H).
130.6, 172.4. ESI-MS [M+H] 348.1. Elemental analysis.
1
Calculated for C22H37NO2ꢁ H2O: C, 74.11%; H,
10.74%; N, 3.93%. Found: C2, 74.47%; H, 10.77%; N,
3.76%.
4.3.6. 3-(t-Butyldiphenylsilyloxy)-propionic acid (13). The
mixture of 3-hydroxypropionylmethylester (10) (1 g,
9.6 mmol),
t-butyldiphenylchlorosilane
(3.17 g,
4.3.8. Arachidonylamine (8). Methanesulfonylchloride
(0.16 ml, 2.07 mmol) was added slowly into the precool-
ed solution of arachidonyl alcohol (400 mg, 1.38 mmol)
in dry pyridine (8 ml). The reaction mixture was stirred
on an ice-bath for 5 h, after which the solution was
poured into ice-cold water and extracted with ether.
The organic layers were combined, washed with 0.5 M
H2SO4 and satd NaHCO3, dried over Na2SO4, and
evaporated. The residue was dissolved in dry DMF
(10 ml) and NaN3 (449 mg, 6.9 mmol) in dry DMF
(30 ml) was added dropwise. After the addition, the
reaction mixture was stirred at 90 ꢂC overnight. The
11.5 mmol), and imidazole (1.96 g, 28.8 mmol) in dry
THF (18 ml) was stirred at rt for 3 h. The reaction mix-
ture was washed with satd NaHCO3, organic layers were
combined, dried over Na2SO4, and evaporated. The
crude product was purified by flash chromatography
eluting with 2.5% EtOAc in petroleum ether. Evapora-
tion of solvents yielded 2.78 g of desired product
(85%). Rf = 0.38 (petroleum ether/EtOAc 9:1). 3-(t-butyl-
diphenylsilyloxy)-propionylmethylester (11) was dis-
solved in MeOH/H2O (100 ml/20 ml), the solution was
cooled on an ice-bath, and LiOH (1.02 g, 24 mmol)

T. Parkkari et al. / Bioorg. Med. Chem. 14 (2006) 5252–5258
5257
1
solution was cooled to rt, poured into ice-cold water and
extracted with ether. The organic layers were combined,
dried over Na2SO4, and evaporated. The crude product
was purified by flash chromatography eluting with 1%
EtOAc in petroleum ether. The evaporation of solvents
yielded 350 mg of colorless oil (80%). 1H NMR d 0.89 (t,
J = 7.0, 3H), 1.26–1.39 (m, 6H), 1.42–1.49 (m, 2H),
1.59–1.35 (m, 2H), 2.04–2.13 (m, 4H), 2.80–2.85 (m,
6H), 3.27 (t, J = 6.9, 2H), 5.31–5.43 (m, 8H). Arachido-
nylazide (7) (105 mg, 0.33 mmol) in diethyl ether (3 ml)
was added to the stirred solution of LAH (12.5 mg,
0.33 mmol) in dry THF (0.35 ml). The reaction mixture
was refluxed for 4 h, cooled to rt and diethyl ether was
added. The mixture was filtered and evaporated. The
crude product was purified by flash chromatography
eluting with 10–100% MeOH in DCM. Evaporation of
solvents yielded 60 mg of oily product (64%). 1H
NMR: d 0.89 (t, J = 6.9, 3H), 1.26–1.51 (m, 10H), 1.81
(br s, 2H), 2.07 (m, 4H), 2.69–2.72 (m, 2H), 2.80–2.84
(m, 6H), 5.31–5.43 (m, 8H).
4:1). H NMR: d 0.89 (t, J = 6.9, 3H), 1.26–1.44 (m,
8H), 1.54 (q, J = 7.2, 2H), 2.03–2.12 (m, 4H), 2.60 (t,
J = 6.6, 2H), 2.80–2.85 (m, 6H), 3.30 (q, J = 7.1, 2H),
3.81 (t, J = 6.4, 2H), 5.31–5.43 (m, 8H), 5.59 (br s,
1H). 13C NMR: d 14.1, 22.6, 25.7, 26.8 (2C), 27.2,
29.2, 29.3, 31.5, 39.6, 39.8, 40.3, 127.6, 127.9, 128.2,
128.3 (2C), 128.6, 129.6, 130.5, 169.3. ESI-MS [M+H+]
380.0.
4.4.4. N-Cyclopropanecarbonyl arachidonylamide (2e).
Yellowish oil (82%). Rf = 0.14 (petroleum ether/EtOAc
1
4:1). H NMR: d 0.69–0.73 (m, 2H), 0.89 (t, J = 6.8,
3H), 0.94–0.97 (m, 2H), 1.26–1.44 (m, 9H), 1.49–1.56
(m, 2H), 2.04–2.12 (m, 4H), 2.80–2.87 (m, 6H), 3.27
(q, J = 6.9, 2H), 5.31–5.43 (m, 8H), 5.58 (br s, 1H).
13C NMR: d 7.0, 14.1, 14.8, 22.6, 25.7, 26.9 (2C), 27.2,
29.3, 29.4, 29.7, 31.5, 39.6, 127.6, 127.9, 128.2, 128.3
(2C), 128.6, 129.7, 130.5, 173.4. ESI-MS [M+H+] 358.1.
4.4.5. N-(2-Hydroxymethyl-3-hydroxy-propanoyl)arachi-
donylamide (2c). Arachidonylamine (8) (120 mg,
0.41 mmol), EDCl (159 mg, 0.83 mmol), and DMAP
(15 mg, 0.12 mmol) were dissolved in dry CH2Cl2
(8 ml) and stirred at rt for 20 h. The solvent was evapo-
rated and the residue was dissolved in EtOAc. The solu-
tion was washed with water and brine, dried over
Na2SO4, and evaporated. The crude product was puri-
fied by flash chromatography eluting with petroleum
ether/EtOAc 4:1. The evaporation of solvents yielded
4.4. General synthesis procedure for the compounds 2a–b
and 2d–e
Propionyl chloride (0.06 ml, 0.70 mmol) in dry DCM
(2 ml) was added into the precooled solution of arachi-
donylamine (8) (100 mg, 0.35 mmol) and Et3N
(0.10 ml, 0.70 mmol) in dry CH2Cl2 (2 ml). After the
addition, the cooling bath was removed and stirring
was continued at rt for 2.5 h. CH2Cl2 was added and
the solution was washed with water and brine. The
organic layers were combined, dried over Na2SO4 and
evaporated. The crude product was purified by flash
chromatography eluting with petroleum ether/EtOAc
4:1.
1
50 mg of white waxy product (25%). H NMR: d 0.89
(t, J = 7.0, 3H), 1.26–1.42 (m, 8H), 1.50–1.55 (m, 2H),
2.04–2.12 (m, 4H), 2.80–2.89 (m, 7 H), 3.25 (q, J = 7.3,
2H), 4.13–4.17 (m, 2H), 4.31–4.34 (m, 2H), 5.31–5.43
(m, 8H), 5.51 (s, 1H), 5.53 (br s, 1H), 7.33–7.39 (m,
3H), 7.46–7.48 (m, 2H). Rf = 0.33 (petroleum ether/
EtOAc 6:1). Compound 9 was dissolved in concd HCl/
MeOH (3 ml/7 ml) and the mixture was stirred at rt
for 3 h. The solvents were evaporated, and the crude
product was purified with flash chromatography eluting
with petroleum ether/EtOAc 1:2. Evaporation of sol-
vents yielded 40 mg of yellowish oil (98%). Rf = 0.2
4.4.1. N-Propionyl arachidonylamide (2a). Yellowish oil
(27%). 1H NMR: d 0.89 (t, J = 6.9, 3H), 1.15 (t,
J = 7.6, 3H), 1.26–1.42 (m, 8H), 1.49–1.55 (m, 2H),
2.03–2.17 (m, 4H), 2.20 (q, J = 7.5, 2H), 2.81–2.58 (m,
6H), 3.25 (t, J = 7.0, 2H), 5.31–5.43 (m, 8H), 5.50 (s,
1H). 13C NMR: d 10.0, 14.1, 22.6, 25.7, 26.9 (2C),
27.2, 29.3 (2C), 29.7, 29.8, 31.5, 39.4, 127.6, 127.9,
128.2, 128.3 (2C), 128.6, 129.7, 130.5, 173.8. ESI-MS
[M+H+] 346.2. Elemental analysis. Calculated for
1
(DCM/MeOH 9:1). H NMR d: 0.89 (t, J = 6.8, 3H),
1.26–1.43 (m, 8H), 1.54 (q, J = 7.4, 2H), 2.03–2.11
(m, 4H), 2.48–2.52 (m, 1H), 2.81–2.85 (m, 6H), 3.20 (s,
2H), 3.27 (q, J = 6.9, 2H), 3.84–3.93 (m, 4H), 5.31–
5.43 (m, 8H), 6.64 (s, 1H). 13H NMR: d 14.1, 22.6,
25.7, 26.8, 26.9, 27.2, 29.1, 29.3, 29.7, 31.5, 39.3, 49.1,
62.0, 127.6, 127.9, 128.2, 128.3, 128.4, 128.6, 129.6,
130.5, 173.9. ESI-MS [M+H+] 392.2. Elemental analysis.
1
C23H39NOꢁ H2O: C, 78.58%; H, 11.37%; N, 3.98%.
3
Found: C, 78.17%; H, 11.54%; N, 3.70%.
4.4.2. N-Butanoyl arachidonylamide (2b). Yellowish oil
(83%). Rf = 0.12 (petroleum ether/EtOAc 4:1). 1H
NMR: d 0.89 (t, J = 6.9, 3H), 0.95 (t, J = 7.4, 3H),
1.26–1.43 (m, 8H), 1.49–1.55 (m, 2H), 1.66 (q, J = 7.4,
2H), 2.04–2.11 (m, 4H), 2.13 (t, J = 7.5, 2H), 2.80–2.85
(m, 6H), 3.25 (q, J = 7.1, 2H), 5.31–5.43 (m, 8H). 13C
NMR: d 13.8, 14.1, 19.2, 22.6, 25.7 (2C), 26.9 (2C),
27.2, 29.3, 29.4, 31.5, 38.8, 39.4, 127.6, 127.9, 128.2,
128.3 (2C), 128.6, 129.7, 130.5, 172.9. ESI-MS [M+H+]
360.2. Elemental analysis. Calculated for C24H41NO:
C, 80.16%; H, 11.49%; N, 3.90%. Found: C, 80.49%;
H, 11.77%; N, 3.81%.
1
Calculated for C24H41NO3ꢁ H2O: C, 73.28%; H,
10
10.56%; N, 3.56%. Found: C, 73.24%; H, 11.00%; N,
3.24%.
4.4.6. N-(3-Hydroxypropionyl)arachidonylamide (2f).
Arachidonylamine (8) (142 mg, 0.49 mmol) and 2M
Al(CH3)3 (in heptane, 0.25 ml, 0.49 mmol) were dis-
solved in dry CH2Cl2 (6 ml) and stirred at rt for
20 min. b-propiolactone (0.03 ml, 0.49 mmol) was added
and the reaction mixture was refluxed for 4 h. The solu-
tion was cooled to rt, 1M HCl was added, and the mix-
ture was extracted with CH2Cl2. Organic layers were
combined, dried over Na2SO4, and evaporated. The
crude product was purified by flash chromatography
4.4.3. N-(3-Chloropropionyl)arachidonylamide (2d). Yel-
lowish oil (69%). Rf = 0.18 (petroleum ether/EtOAc

5258
T. Parkkari et al. / Bioorg. Med. Chem. 14 (2006) 5252–5258
eluting with EtOAc. Evaporation of solvents yielded
70 mg of colorless oil (40%). 1H NMR: d 0.89 (t,
J = 6.9, 3H), 1.26–1.43 (m, 8H), 1.43 (q, J = 7.5, 2H),
2.04–2.12 (m, 4H), 2.42 (t, J = 5.4, 2H), 2.80–2.85 (m,
6H), 3.27 (q, J = 6.7, 2H), 3.88 (t, J = 5.4, 2H), 5.31–
5.43 (m, 8H), 5.80 (s, 1H). 13C NMR: d 14.5, 23.0,
26.0, 27.2, 27.3, 27.6, 29.6, 29.7, 32.0, 38.3, 40.0, 59.4,
127.9, 128.3, 128.6, 128.6, 128.7, 129.0, 130.0, 131.0,
172.8. ESI-MS 362.2. Elemental analysis. Calculated
help. The study was supported by the grants from the
National Technology Agency of Finland and Academy
of Finland (Grant No. 107300).
References and notes
1. Di Marzo, V.; Bifulco, M.; De Petrocellis, L. Nat. Rev.
Drug Discov. 2004, 3, 771–784.
2. Berry, E. M.; Mechoulam, R. Pharmacol. Ther. 2002, 95,
185–190.
3. Martin, B. R.; Wiley, J. L. J. Support Oncol. 2004, 2, 305–
314, discussion 314–306.
4. Davies, S. N.; Pertwee, R. G.; Riedel, G. Neuropharma-
cology 2002, 42, 993–1007.
1
for C23H39NO2ꢁ H2O: C, 75.16%; H, 10.88%; N,
3.81%. Found: C3, 75.22%; H, 10.99%; N, 3.62%.
4.5. Stability study
Eight-week-old Wistar rats (200 g weight) were decapi-
tated, whole brains minus cerebellum were dissected
and homogenized in one times the volume (v/w) of ice-
cold 0.1 M potassium phosphate buffer (pH 7.4) with a
Potter-Elvehjem homogenizer (Heidolph). The homoge-
nate was centrifuged at 10,000g for 20 min at 4 ꢂC and
the resulting supernatant was used as a source of FAAH
activity. The protein concentration of the supernatant
(7.2 mg/ml) was determined by the method of Bradford
with bovine serum albumin as a standard (Bradford
1976). Aliquots of the supernatant were stored at
ꢀ70 ꢂC until use. The experiments were initiated by a
preincubation of a mixture (1380 ll, 37 ꢂC, 10 min) con-
taining 929 lg of a rat brain homogenate, 81 mM potas-
sium phosphate buffer (pH 7.4), and 0.5% (w/v) BSA.
The actual experiment was carried out by taking
1188 ll of the preincubated brain homogenate protein,
adding 12 ll of studied compound (5 mM in ethanol, fi-
nal volume 1200 ll), and incubating the mixture for
90 min at 37 ꢂC. The final incubation mixture contained
800 lg rat brain homogenate protein, 80 mM potassium
phosphate buffer (pH 7.4), 0.5% (w/v) BSA, and 50 lM
of studied compound. At time points 0 and 90 min,
300 ll samples were taken and 600 ll of cold acetonitrile
was added in order to stop the enzymatic reaction. The
samples were centrifuged at 23,700g for 4 min at rt and
the supernatant was analyzed for remaining compound
by HPLC. The analytical HPLC system used in the sta-
bility studies consisted of a Merck Hitachi L-7100
pump, D-7000 interface module, L-7455 diode-array
detector (190–800 nm, set at 211 nm), and L-7250 pro-
grammable autosampler. The separations were per-
formed with Zorbax SB-C18 end capped reversed-
phase column (4.6 mm · 150 mm, 5 lm). The injection
volume was 50 lL. A mobile phase of 28% phosphate
buffer (30 mM, pH 3.0) in acetonitrile was used at a flow
rate of 2 ml/min. Retention times for AEA, 1a, 1c, 1e,
2b, 2e, and 2f were 4.5, 10.5, 4.9, 10.0, 17.2, 14.7, and
6.5 min, respectively.
5. Walker, J. M.; Huang, S. M. Pharmacol. Ther. 2002, 95,
127–135.
6. Jarvinen, T.; Pate, D. W.; Laine, K. Pharmacol. Ther.
2002, 95, 203–220.
7. De Petrocellis, L.; Melck, D.; Bisogno, T.; Di Marzo, V.
Chem. Phys. Lipids 2000, 108, 191–209.
8. Williamson, E. M.; Evans, F. J. Drugs 2000, 60, 1303–
1314.
9. Pertwee, R. G. Pharmacol. Ther. 2002, 95, 165–174.
10. Lastres-Becker, I.; De Miguel, R.; Fernandez-Ruiz, J. J.
Curr. Drug. Targets CNS Neurol. Disord. 2003, 2, 335–
347.
11. Sieradzan, K. A.; Fox, S. H.; Hill, M.; Dick, J. P.;
Crossman, A. R.; Brotchie, J. M. Neurology 2001, 57,
2108–2111.
12. Croxford, J. L. CNS Drugs 2003, 17, 179–202.
13. Savinainen, J. R.; Saario, S. M.; Niemi, R.; Jarvinen, T.;
Laitinen, J. T. Br. J. Pharmacol. 2003, 140, 1451–1459.
14. Hillard, C. J. Prostaglandins Other Lipid Mediat. 2000, 61,
3–18.
15. Savinainen, J. R.; Jarvinen, T.; Laine, K.; Laitinen, J. T.
Br. J. Pharmcol. 2001, 134, 664–672.
16. Abadji, V.; Lin, S.; Taha, G.; Griffin, G.; Stevenson, L. A.;
Pertwee, R. G.; Makriyannis, A. J. Med. Chem. 1994, 37,
1889–1893.
17. Adams, I. B.; Ryan, W.; Singer, M.; Razdan, R. K.;
Compton, D. R.; Martin, B. R. Life Sci. 1995, 56, 2041–
2048.
18. Adams, I. B.; Ryan, W.; Singer, M.; Thomas, B. F.;
Compton, D. R.; Razdan, R. K.; Martin, B. R.
J. Pharmacol. Exp. Ther. 1995, 273, 1172–1181.
19. Lin, S.; Khanolkar, A. D.; Fan, P.; Goutopoulos, A.; Qin,
C.; Papahadjis, D.; Makriyannis, A. J. Med. Chem. 1998,
41, 5353–5361.
20. Nazih, A.; Heissler, D. Synthesis 2002, 2, 203–206.
21. Ng, E. W.; Aung, M. M.; Abood, M. E.; Martin, B. R.;
Razdan, R. K. J. Med. Chem. 1999, 42, 1975–1981.
´
´ ´ ´
22. Juaristi, E.; Dıaz, F.; Cuellar, G.; Jimenez-Vazquez, H. A.
J. Org. Chem. 1997, 62, 4029–4035.
23. Sheskin, T.; Hanus, L.; Slager, J.; Vogel, Z.; Mechoulam,
R. J. Med. Chem. 1997, 40, 659–667.
24. Pinto, J. C.; Potie, F.; Rice, K. C.; Boring, D.; Johnson,
M. R.; Evans, D. M.; Wilken, G. H.; Cantrell, C. H.;
Howlett, A. C. Mol. Pharmacol. 1994, 46, 516–522.
25. Savinainen, J. R.; Kokkola, T.; Salo, O. M.; Poso, A.;
Jarvinen, T.; Laitinen, J. T. Br. J. Pharmacol. 2005, 145,
636–645.
Acknowledgments
26. Mor, M.; Rivara, S.; Lodola, A.; Plazzi, P. V.; Tarzia, G.;
Duranti, A.; Tontini, A.; Piersanti, G.; Kathuria, S.;
Piomelli, D. J. Med. Chem. 2004, 47, 4998–5008.
The authors are grateful to Mrs. Miia Reponen, Mrs.
Tiina Koivunen, Ms. Kristiina Huttunen, Mrs. Minna
Glad, and Mrs. Helly Rissanen for their skilful technical