N-morpholino- and N-diethyl-analogues of palmitoylethanolamide increase the sensitivity of transfected human vanilloid receptors to activation by anandamide without affecting fatty acid amidohydrolase activity

Article abstract of DOI:10.1016/S0968-0896(02)00567-9

The abilities of 19 analogues of palmitoylethanolamide and two analogues of oleoylethanolamide to affect the Ca²⁺ influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) in response to anandamide (AEA) have been investigated using a FLIPR assay and a bovine serum albumin-containing assay medium. Only palmitoylethanolamide produced any effect in the absence of AEA. The ability of palmitoylethanolamide to potentiate the response to AEA was retained when the N-CH₂CH₂OH group was replaced by N-CH₂CH₂Cl,whereas replacement with N-alkyl substituents [from -H up to -(CH₂)₁₂CH₃] resulted either in a reduction or in a complete loss of this activity. The tertiary amide N-(CH₂CH₃)₂ (19) and N-morpholino (20) analogues of palmitoylethanolamide potentiated the response to 1 μM AEA to a greater degree than the parent compound, whereas the N-(CH₃)₂ analogue was inactive. 19 and 20 produced leftward shifts in the dose-response curve for AEA activation of Ca²⁺ influx into hVR1-HEK293 cells. EC₅₀ values for AEA to produce Ca²⁺ influx into hVR1-HEK293 cells were 1.1, 1.1, 0.54 and 0.36 μM in the presence of 0, 1, 3 and 10 μM 19, respectively. The corresponding values for 20 were 1.5, 1.3, 0.77 and 0.17 μM, respectively. The compounds did not affect the dose-response curves to capsaicin. The ability of oleoylethanolamide to potentiate AEA is retained by the N-CH₂CH₃ and N-CH(CH₃)₂ analogues (22 and 23, respectively). 22 and 23 produced a small (～25%) inhibition of the binding of [³H]-CP55,940 and [³H]-WIN 55,212-2 to CB₁ and CB₂ receptors, respectively, expressed in CHO cells. The compounds inhibited the metabolism of 2 μM [³H]-AEA by rat brain fatty acid amidohydrolase with IC₅₀ values of 5.6 and 11 μM, respectively. In contrast, 19 and 20 were without effect on either binding to CB receptors or fatty acid amidohydrolase activity. Minor reductions in the accumulation of 10 μM [³H]-AEA into C6 glioma cells were seen at 10 μM concentrations of 19 and 20. It is concluded that 19 and 20 selectively enhance AEA effects upon VR1 receptors without potentially confounding effects upon CB receptors or fatty acid amidohydrolase activity.

Full text of DOI:10.1016/S0968-0896(02)00567-9

Bioorganic & Medicinal Chemistry 11 (2003) 817–825
N-Morpholino- and N-Diethyl-analogues of
Palmitoylethanolamide Increase the Sensitivity of Transfected
Human Vanilloid Receptors to Activation by Anandamide Without
Affecting Fatty Acid Amidohydrolase Activity
a
a
b
Severine Vandevoorde, Didier M. Lambert, Darren Smart,
´
c
c,
Kent-Olov Jonsson and Christopher J. Fowler *
aUnite´ de Chimie pharmaceutique et de Radiopharmacie, Universite´ catholique de Louvain, Avenue Mounier, 73,
UCL-CMFA 73.40, B-1200 Brussels, Belgium
b
Neurology CEDD, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
cDepartment of Pharmacology and Clinical Neuroscience, Umea˚ University, SE-901 87 Umea˚, Sweden
Received 1 October 2002; accepted 5 November 2002
Abstract—The abilities of 19 analogues of palmitoylethanolamide and two analogues of oleoylethanolamide to affect the Ca2+
influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) in response to anand-
amide (AEA) have been investigated using a FLIPR assay and a bovine serum albumin-containing assay medium. Only palmitoy-
lethanolamide produced any effect in the absence of AEA. The ability of palmitoylethanolamide to potentiate the response to AEA
was retained when the N-CH
H up to –(CH ] resulted either in a reduction or in a complete loss of this activity. The tertiary amide N-(CH
and N-morpholino (20) analogues of palmitoylethanolamide potentiated the response to 1 mM AEA to a greater degree than the
parent compound, whereas the N-(CH analogue was inactive. 19 and 20 produced leftward shifts in the dose–response curve for
2
CH
2
OH group was replaced by N-CH
2
CH
2
Cl,whereas replacement with N-alkyl substituents [from
–
2
)
12CH
3
2
CH (19)
3 2
)
3 2
)
2
+
2+
AEA activation of Ca influx into hVR1-HEK293 cells. EC50 values for AEA to produce Ca influx into hVR1-HEK293 cells
were 1.1, 1.1, 0.54 and 0.36 mM in the presence of 0, 1, 3 and 10 mM 19, respectively. The corresponding values for 20 were 1.5, 1.3,
0
.77 and 0.17 mM, respectively. The compounds did not affect the dose–response curves to capsaicin. The ability of oleoylethano-
lamide to potentiate AEA is retained by the N-CH CH and N-CH(CH analogues (22 and 23, respectively). 22 and 23 produced
and CB receptors, respectively,
2
3
3 2
)
3
3
a small (ꢀ25%) inhibition of the binding of [ H]-CP55,940 and [ H]-WIN 55,212-2 to CB
1
2
3
expressed in CHO cells. The compounds inhibited the metabolism of 2 mM [ H]-AEA by rat brain fatty acid amidohydrolase with
IC50 values of 5.6 and 11 mM, respectively. In contrast, 19 and 20 were without effect on either binding to CB receptors or fatty acid
3
amidohydrolase activity. Minor reductions in the accumulation of 10 mM [ H]-AEA into C6 glioma cells were seen at 10 mM con-
centrations of 19 and 20. It is concluded that 19 and 20 selectively enhance AEA effects upon VR1 receptors without potentially
confounding effects upon CB receptors or fatty acid amidohydrolase activity.
2003 Elsevier Science Ltd. All rights reserved.
#
Introduction
ethanolamide, AEA).3,4 A number of experiments have
indicated that the binding site(s) for capsaicin and ana-
2
,5
Vanilloid receptors (VR) are ligand-gated ion channels
that are located on sensory nerve terminals and play an
ndamide, in contrast to that for protons, are located
In hVR1-HEK cells transfected with
6ꢁ9
intracellularly.
1
important role in nociception. VR can be activated not
only by heat, protons, vanilloids like capsaicin and resi-
VR1 receptors, AEA acts as a full agonist, producing a
rapid influx of calcium that is blocked by the VR
antagonist capsazepine, and is not seen when untrans-
2
niferatoxin, but also by the endogenous cannabinoid
receptor (CB) agonist anandamide (arachidonoyl-
4
fected HEK cells are used. AEA is removed from the
medium by a process of cellular accumulation followed
by hydrolysis by fatty acid amidohydrolase (FAAH) to
1
0,11
form arachidonic acid. Consistent with the intra-
cellular localization of the AEA activation site on VR,
*Corresponding author. Fax: +46-90-785-2752; e-mail: cf@pharm.
umu.se
0
968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00567-9

818
S. Vandevoorde et al. / Bioorg. Med. Chem. 11 (2003) 817–825
FAAH inhibitors enhance, and inhibitors of AEA
2
even more dramatic in this respect, reducing the EC
50
+
15
accumulation block, respectively, the Ca response to
8
value for AEA more than 8-fold. This property of
long chain N-acyl ethanolamines may be an important
regulator of the actions of AEA upon vanilloid recep-
tors under severe inflammatory conditions, given that
exogenous AEA in hVR1-HEK cells. These cellular
processes are shown schematically in Figure 1.
1
7
Although it has been established without doubt that
AEA activates VR, it is not clear as to whether or not
this compound is an endovanilloid, since the potency
required for VR activation is usually rather higher than
their synthesis is increased under such conditions.
Although the abilities of the N-acyl ethanolamine
homologues of 1 to affect the VR response to AEA in
1
2
15
needed for activation of CB receptors. In vivo, how-
ever, AEA is synthesised on demand together with other
N-acyl ethanolamines such as palmitoylethanolamide
hVR1-HEK cells have been fully investigated, little is
known as to whether variations in the ethanolamine side
chain of 1 affect its properties in this regard. The only
compounds so far evaluated, where the N-ethanolamine
(1), (structure shown in Fig. 2); indeed, in most tissues,
the concentrations of these other N-acyl ethanolamines
side chain was replaced by either a primary NH ,(2)
2
1
3
are much higher than that of AEA. Addition of these
compounds potentiate VR1-mediated effects of AEA
N-ethylamine (4), N-butylamine (6), N-isopropylamine
(8), N-cyclohexylamine (15) (structures shown in Fig. 3)
or by trifluoromethyl ketone groups, were without
1
4,15
both for cells transfected with the human receptor,
and, in the case of 1, in perivascular sensory nerves
naturally expressing the receptor.16 For 1 the EC value
2
+
obvious effect either per se or upon the Ca -response
15
to AEA. In the present study, we have investigated the
5
0
2
+
for the ability of AEA to produce a Ca -influx into
1
abilities of 14 additional analogues of 1 and 2 analogues
of oleoylethanolamide to potentiate the response to
AEA in hVR1-HEK cells. The most interesting com-
pounds have in addition been characterised for their
ability to interact with CB receptors, FAAH and the
AEA accumulation process.
4
hVR1-HEK cells was decreased by a factor of two.
The shorter chain homologue lauroylethanolamide was
Figure 1. Schematic representation of the action of anandamide at
VR1. After intracellular accumulation (2), the compound acts intra-
cellularly upon VR1 (1) to produce an influx of calcium. The intracel-
lular anandamide is metabolised to arachidonic acid by fatty acid
amidohydrolase (3). These three sites are thus potential targets
whereby novel compounds can potentiate the response of the vanilloid
receptor to anandamide.
Figure 3. Structures of primary (2), secondary (3–17), tertiary (18–20)
amides deriving from palmitoylethanolamide. Structures of oleoyl-
derivatives (22,23).
Figure 2. Structures of palmitoylethanolamide (1) and oleoylethanol-
amide (21).

S. Vandevoorde et al. / Bioorg. Med. Chem. 11 (2003) 817–825
819
Figure 4. Effects of a series of analogues of 1 upon the Ca2+-influx in hVR1-HEK293 cells in the absence (‘basal’) and presence of 1 mM AEA. The
compounds were used at a concentration of 10 mM. Shown are meansꢂSE mean, n=3–4. The solid bars indicate the mean response to 1 mM AEA in
the absence of the test compounds assayed concomitantly. The meansꢂSE mean values corresponding to these two solid bars are 30ꢂ2.65 and
15 {
{{
4
8ꢂ9.6%. Compounds listed with an asterisk indicate data from Smart et al. p<0.05, p<0.01 versus the corresponding response to 1 mM AEA
in the absence of the test compound, two-tailed t-test.
Results
[from –H up to –(CH ) CH ] and N-cyclic substituents
2 12 3
(
-cyclopropyl, -cyclohexyl, and -naphthyl) resulted
either in a reduction or in a complete loss of this activity
Fig. 4).
Effects of amide analogues of palmitoylethanolamide
upon AEA-induced activation of VR1 receptors in hVR1-
HEK293 cells
(
A series of primary and secondary amide analogues
were tested at a concentration of 10 mM to determine
their effects upon Ca influx into hVR1-HEK293 cells
Three tertiary amide analogues of 1 were tested (Fig. 4).
The N-(CH CH ) and N-morpholino analogues (19
2
3 2
2
+
and 20, respectively) produced a large potentiation of
the response to 1 mM AEA, whereas the N-(CH ) ana-
(Fig. 4). Experiments were conducted to see whether the
3
2
compounds alone produced a calcium response (‘basal’)
and to determine whether they potentiated the response
to a submaximal concentration (1 mM) of AEA. The
FLIPR assay methodology required the presence of
bovine serum albumin to prevent binding of the AEA to
the assay plates. Bovine serum albumin avidly binds
AEA and thus affects the observed potency of AEA at
logue (18) did not. 20 was also tested in the second
experimental series (i.e., with the higher response to
AEA alone) and gave similar values (5.4ꢂ1.3 and
89ꢂ3.3% of maximal response for basal and +AEA
conditions, respectively).
Two secondary amide analogues of oleoylethanolamide
(21) were also tested (Fig. 5). Both the N-CH CH and
1
4
VR1. However, the AEA concentration used here
(
2
3
1 mM) is close to its EC50 value (ꢀ1.3 mM, see below)
N-CH(CH ) analogues (22 and 23, respectively) of 21
3 2
potentiated the response to AEA.
under the present assay conditions, and is thus on the
steepest part of the dose-response curve, and therefore
highly sensitive to potentiating effects. The compounds
were tested in two separate experimental series. In the
first series, AEA gave a response of 30ꢂ2.7% of maxi-
mal, whereas in the second series, the response was
slightly higher (48ꢂ10%). This difference simply reflects
the fact that the concentration of AEA is on the steepest
part of the dose–response curve. In consequence, the
appropriate AEA controls are shown for each com-
pound in the figure. The analogues previously pub-
1
5
lished are also shown in the figure for comparative
purposes.
Of all the analogues tested, only palmitoylethanolamide
(1) possessed any large intrinsic activity per se at the
concentration of 10 mM. In addition, among the pri-
mary and secondary amide analogues, the only com-
pound which completely retained the ability of 1 to
potentiate the response to AEA was the compound
where the N-CH CH OH group was replaced by
Figure 5. Effects of two secondary amide analogues of 21 upon the
2+
Ca -influx in hVR1-HEK293 cells in the absence (‘basal’) and pre-
sence of 1 mM AEA. The compounds were used at a concentration of
10 mM. Shown are meansꢂSE mean, n=3–4. For further details, see
legend to Figure 4.
2
2
N-CH CH Cl (12). Replacement with N-alkyl substituents
2
2

820
S. Vandevoorde et al. / Bioorg. Med. Chem. 11 (2003) 817–825
Comparison of the effects of 19 and 20 upon the Ca2+
influx response to AEA and capsaicin in hVR1-HEK293
cells
the binding to the receptors, and 20 produced a small
(15.5ꢂ0.3%) inhibition of the binding to CB receptors.
1
22 and 23 produced a modest inhibition (20–28%) of
the binding. Thus the analogues behave like their
corresponding-ethanolamines with respect to effects at
Dose–response curves for the stimulation of Ca2+ influx
in hVR1-HEK293 cells by AEA and capsaicin are
shown in Fig. 6. 19 and 20 produced leftward shifts in
the dose-response curve for AEA activation of Ca2+
influx into hVR1-HEK293 cells (Fig. 6A and B). EC
1
8
CB receptors.
Interaction of 19, 20, 22 and 23 with FAAH
5
0
2
+
values for AEA to produce Ca
influx into hVR1-
The ability of the compounds to inhibit the FAAH-cat-
3
HEK293 cells were 1.1, 1.1, 0.54 and 0.36 mM in the
presence of 0, 1, 3 and 10 mM 19, respectively. The cor-
responding values for 20 were 1.5, 1.3, 0.77 and
0.17 mM, respectively. The compounds did not affect the
dose–response curves to capsaicin (Fig. 6C and D).
alysed metabolism of [ H]-AEA (2 mM) was investigated
using rat brain membranes as enzyme source (Fig. 7A).
3
Table 1. Effects of 19, 20, 22 and 23 upon the binding of [ H]-
3
1 2
CP55,940 and [ H]-WIN 55,212-2 to CB and CB receptors, respec-
tively, expressed in CHO cells
Compd (10 mM)
% Inhibition of specific binding
Cell line: CHO-CB CHO-CB
Interaction of 19, 20, 22 and 23 with CB1 and CB2
receptors
1
2
3
3
Ligand: [ H]-CP 55,940
[ H]-WIN 55212,2
The ability of 19, 20, 22 and 23 (10 mM) to inhibit the
3
binding of [ H]-CP55,940 to CB receptors and of
3
19
20
3.3ꢂ0.3
15.5ꢂ0.3
27.5ꢂ0.3
27.3ꢂ0.5
2.7ꢂ0.1
4.2ꢂ0.3
1
[ H]-WIN55,212-2 to CB receptors expressed on CHO
2
cells is shown in Table 1. The data are shown as % of
2
2
2
3
20.1ꢂ0.3
23.1ꢂ0.3
specific inhibition of the radioligand. 19 did not affect
Figure 6. Effects of 19 (A,C) and 20 (B,D) upon the Ca2+-influx in hVR1-HEK293 cells produced by (A,B) AEA and (C,D) capsaicin. Data are
means of four experiments. In (A) and (B) the SE means are shown (when not enclosed by the symbols) for the sake of clarity only for the curves for
0, 3 and 10 mM compounds; In (C) the SE means are shown only for the curves for 0 and 1 mM 19; and in (D) the SE means are shown only for the
curves for 1 and 10 mM 20.

S. Vandevoorde et al. / Bioorg. Med. Chem. 11 (2003) 817–825
821
3
Figure 7. Panel A: Effects of 19, 20, 22 and 23 upon the FAAH-catalysed metabolism of 2 mM [ H]-AEA in rat brain homogenates. Panel B: Effects
3
of 19 and 20 upon the uptake of 10 mM [ H]-AEA into C6 glioma cells. Data are means n=3–5, with SE mean values shown when not enclosed by
ꢃ
the symbols or columns. In panel (B), the effects upon uptake at 4 C were not tested for 3 and 30 mM concentrations of 19 and 20.
3
to affect the VR-mediated Ca2+-influx in response to
AEA has been investigated in hVR1-HEK293 cells. In
contrast to the modest partial agonist effect of 1 seen in
2
2 and 23 inhibited [ H]-AEA metabolism with IC50
values of 5.6 and 11 mM, respectively. The IC values
refer to the inhibitable fraction of the [ H]-AEA meta-
bolism (100 and 58% of the total for 22 and 23, respec-
tively). In contrast, 19 and 20 did not inhibit [ H]-AEA
metabolism even at a concentration of 100 mM.
5
0
3
4
these cells, none of the compounds produced dramatic
3
VR agonist effects in the absence of AEA. The most
interesting finding in this respect is that the N-phenyl
derivative 16 was without observable efficacy, since the
N-(3-methoxy-4-hydroxyphenyl) analogue of 1, palva-
nil, is as effective and considerably more potent than
3
Effect of 19 and 20 upon [ H]-AEA accumulation into
C6 glioma cells
1
9
AEA as a VR agonist in hVR1-HEK293 cells. The
lack of efficacy of 21 (and indeed the efficacy per se of 1)
contrasts with the situation for VR in mesenteric arter-
The effects of 19 and 20 upon the uptake of 10 mM
3
[
H]-AEA into C6 glioma cells are shown in Figure 7B.
ꢃ
16
1
9 was a weak inhibitor of uptake at 37 C, producing
ies. However, this responsiveness is presumably rela-
1
0ꢂ8% and 39ꢂ7% inhibition at concentrations of 10
ted to a number of factors, not the least being the level
of receptor expression. More important for the present
study are the reports that 1 not only potentiates AEA
and 100 mM, respectively. 20 was a more potent uptake
inhibitor (IC50 value 49 mM), although at a concentration
of 10 mM, the inhibition of uptake was only 23ꢂ4%.
1
4,15
responsiveness at VR in transfected cells,
physiological systems naturally expressing VR.1
but also in
6
The structural requirements for the ethyl side chain with
respect to the potentiation of the VR response to a
submaximal concentration of AEA are rather different
to that for an effect per se. Whist the hydroxyl group of
Discussion
In the present study, the abilities of a series of palmitoyl-
ethanolamide (1) and oleoylethanolamide (21) analogues

822
S. Vandevoorde et al. / Bioorg. Med. Chem. 11 (2003) 817–825
1
can be replaced by a chloride group without loss of
In conclusion, the present study has demonstrated that the
2
+
efficacy, replacement with a methyl or ethyl group
removes the ability to potentiate AEA. Among the
analogues of 1, the two compounds showing the most
marked potentiation of the response to AEA were 19
and 20. This potentiation was due to a leftward shift in
the dose–response curve for AEA. A corresponding
leftward shift for the capsaicin dose–response curve was
not seen, indicating a selective effect upon the response
to AEA. This result is similar to that reported pre-
Ca influx response mediated by VR1 receptors in hVR1-
HEK293 cells can be potentiated not only by N-acyl
ethanolamines, but by the N-morpholinyl and N-diethyl
analogues of 1. These analogues lack potential confound-
ing effects on FAAH and upon CB and CB receptors,
1
2
and may be useful agents, by enhancing the effects of exo-
genously added AEA upon VR1 receptors, to explore the
physiological properties of this ‘endovanilloid’.
1
4
15
viously for 1 and lauroylethanolamide, although the
leftward shift for 1 was less marked than for 19 and
lauroylethanolamide.
Experimental
Compounds
A direct effect on the VR to increase the sensitivity of
the receptor to AEA is the simplest explanation of the
present data (and supported by previous findings with
3
3
Anandamide [ethanolamine-1- H] ([ H]-AEA; specific
ꢁ
1
activity 30–60 Ci mmol ) was obtained from American
Radiolabeled Chemicals, Inc. (St. Louis, MO, USA).
1
4
1
). However, other modes of action should be con-
sidered. In theory, compounds can produce a potentia-
3
[ H]-WIN 55,212-2 (specific activity 1.68 TBq/mmol,
ꢁ1
2
+
3
tion of Ca -influx response to AEA by preventing the
breakdown of AEA and thereby increasing its available
concentration. FAAH inhibitors potentiate the effects
45.5 Ci mmol ) and [ H]-CP55,940 (specific activity
ꢁ
1
3.737 TBq/mmol, 101 Ci mmol ) were obtained from
New England Nuclear, Boston, MA, USA. The synth-
eses of the compounds tested, with the exception of the
four compounds described below, have either been
8
,20
16
of AEA at VR receptors in some,
but not all
model systems. However, 19 and 20 do not affect
FAAH, which would rule out this mode of action, at
least for these compounds. Nevertheless, FAAH inhi-
bition can in theory contribute to the potentiating
effects of the 21 analogues 22 and 23, since these com-
pounds inhibited FAAH with IC50 values of 5.6 and
1
8,29,30
reported previously
or will be reported elsewhere.
Capsaicin, non-radioactive AEA (for the VR1 experi-
ments), HU-210 [(6aR)-trans-3-(1,1-dimethylheptyl)-
6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-
dibenzo [b,d]pyran-9-methanol] was purchased from
Tocris (Bristol, UK) and non-radioactive WIN 55,212-2
mesylate [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpho-
1
1 mM, respectively. For comparison, 21 inhibited
21
FAAH (by acting as an alternative substrate ) with an
IC50 value, under the same assay conditions, of
4.7 mM.
N-CH CH (4) and N-CH(CH ) (8) compounds in the
linylmethyl)
pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-
1
8
The corresponding N-ethanolamine (1),
naphthalenylmethanone] was purchased from RBI
(Sigma-RBI, Boornem, Belgium). Non-radioactive
AEA (for the FAAH and AEA accumulation experi-
ments), was purchased from Cayman Chemical Com-
pany (Ann Arbor, MI, USA). Fatty acid free bovine
serum albumin, polyethyleneimine and phenylmethyl-
sulfonyl fluoride were obtained from the Sigma Chemi-
cal Co. (St Louis, MO, USA). Cell culture media were
obtained from Gibco/Life Technologies.
2
3
3 2
palmitoyl-series were also FAAH inhibitors with rather
similar potencies (IC50 values 5.1, 3.6 and 13 mM) (ref
1
8 and unpublished data).
An alternative explanation would be that the com-
pounds are able to increase the sensitivity of the VR1 to
AEA by increasing its rate of cellular accumulation,
given that the activation site for AEA is located intra-
8
,9
cellularly. Although no molecular characterization of
an anandamide membrane transporter has yet been
reported and the detailed mechanism of cellular AEA
0
Synthesis
of
N,N -diethylhexadecanamide
(20,
palmitoyl(diethyl)amide), 4-(1-oxohexadecyl) morpholine
21, palmitoylmorpholinamide), N-ethyloleamide (22,
oleoylethylamide) and N-(1-methylethyl)oleamide (23,
(
2
2ꢁ24
accumulation is a matter of debate,
the accumu-
lation process can certainly be modulated either to be
less active (by inhibiting FAAH, since removal of AEA
oleoylisopropylamide)
2
5,26
intracellularly drives the uptake process
2
) or more
). However,
In all four cases, the same protocol was used. In a two-
neck flask, 100 mmol (unless otherwise stated) of di-
ethylamine (for 20), morpholine (for 21), ethylamine
(for 22, 5.85 g of a 70% solution in water) or iso-
propylamine (for 23) were poured into 10 mL of dry
methylene chloride. The solutions was cooled in an ice
bath and magnetically stirred after which 10 mmol of
either palmitoyl chloride (20 and 21) or oleoyl chloride
(22 and 23) was added dropwise. The reaction mixture
was stirred for 12 h at room temperature and then washed
with 5% sodium bicarbonate solution, 1 M HCl and
7,28
active (by the addition of NO donors
this mechanism does not account for the properties of
9 and 20, since they have minor effects on the accu-
1
mulation of AEA, at least into C6 glioma cells, at the
concentrations required to affect the VR response to
AEA. In this respect, it is at first sight surprising that 21
produces a potentiation of the VR response to AEA,
2
2
given the report that the concentration of this com-
pound used here reduces the accumulation of AEA into
cerebellar granule cells by 61%. However, the concen-
3
tration of [ H]AEA used in those experiments was very
low (0.2 nM), and at a higher AEA concentration,
brine. The organic layer was dried over MgSO and after
4
filtration and solvent was evaporated under reduced
pressure. Yields, chromatographic and spectroscopic
data for the compounds are summarised below.
1
C6 cells.
0 mM 21 does not affect the accumulation of AEA into
18

S. Vandevoorde et al. / Bioorg. Med. Chem. 11 (2003) 817–825
823
0
CA 57303-21-6. Yield: 2.3 g (74%) of a yellowish oil.
N,N -Diethylhexadecanamide 20 (palmitoyl(diethyl)amide).
Rat C6 glioma cells (passage range 59–60), obtained
from the American Type Culture Collection, MD, USA,
ꢁ
1
2
ꢃ
were grown in 75 cm culturing flasks at 37 C, 5% CO
2
TLC (chloroform/methanol 6:4 vv ) R =0.82; spectro-
f
1
scopic data: H NMR (d-CDCl ) d (ppm) 0.87 (t,
in air at normal atmospheric pressure. The cells were
cultured in Ham’s F10 medium, supplemented with
3
J=6 Hz, 3H); 1.09–1.16 (m, 6H); 1.22–1.41 (m, 26H);
1
3
-1
2
.27 (t, J=7 Hz, 2H); 3.33 (q, J=12 Hz, 4H); C NMR
10% foetal bovine serum and 100 units mL penicillin
+100 mg mL streptomycin.
-1
(
d-CDCl ) d (ppm) 13.18; 14.14; 14.49 (CH ); 22.79;
3
3
2
1
5.58; 29.64; 29.81; 32.04; 33.17; 40.11; 42.03 (CH );
2
+
.
72.11 (C¼O); mass spectrometry [M ]=312; IR n
Measurement of [Ca2+]i using the FLIPR technology
ꢁ
1
(
cm ): 1646 (C¼O).
4
The method of Smart et al. was used. hVR1-HEK293
3
ꢁ1
4-(1-Oxohexadecyl)morpholine 21 (palmitoylmorpholin-
amide). CA 5299-68-3. Yield: 1.7 g (52%) of a white
cells (density 25 ꢅ 10 cells well ) were seeded into
black walled clear-base 96-well plates (Costar UK) and
cultured overnight in MEM, supplemented as described
above. The cells were then incubated with MEM con-
taining the cytoplasmic calcium indicator, Fluo-3AM
ꢃ
solid. Mp: 38–39 C (uncorrected); TLC (chloroform/
1
ꢁ1
methanol 6:4 vv ) R =0.9; spectroscopic data:
f
H
NMR (d-CDCl ) d (ppm) 0.86 (t, J=6 Hz, 3H); 1.22–
3
ꢃ
1
.43 (m, 26H); 2.29 (t, J=7 Hz, 2H); 3.43–3.46 (m, 4H);
1
(4 mM; Teflabs, Austin, TX, USA) at 25 C for 120 min.
3
3
.62–3.65 (m, 4H); C NMR (d-CDCl ) d (ppm) 14.12
After washing the plates four times with Tyrode’s med-
ium containing 0.1% bovine serum albumin, they were
placed into a FLIPR (Fluorometric Imaging Plate
Reader, Molecular Devices, UK). Cell fluorescence
(lEX=488 nm, lEM=540 nm) was monitored before
and after the addition of various compounds in the
presence or absence of 1 mM AEA. Incubations were
routinely run over three min. The calcium responses
generally appeared within a few seconds of injection of
the anandamide and peaked at about 30 s. Responses
were measured as peak fluorescence intensity (FI) minus
basal FI, and results are expressed as a percentage of the
maximum response obtained with either anandamide or
capsaicin, as appropriate. The observed potency of
AEA is highly dependent upon the assay conditions, in
particular upon the concentration of bovine serum
3
(
2
CH ); 22.71; 25.02; 25.31; 29.39; 29.47; 29.52; 29.55;
9.69; 31.73; 31.96; 33.14; 41.92; 46.13; 66.72; 66.99
3
+
ꢄ
(
CH ); 171.89 (C¼O); mass spectrometry [M ]=326;
2
ꢁ
1
IR n (cm ): 1649 (C¼O).
N-Ethyloleamide 22 (oleoylethylamide). CA 85075-82-7.
Yield: 2.5 g (82%) of a yellowish oil. TLC (ethyl acetate/
ꢁ1
1
methanol 8:2 vv ) R =0.76; spectroscopic data : H
f
NMR (d-CDCl ) d (ppm) 0.87 (t, J=7 Hz, 3H); 1.12 (t,
3
J=7 Hz, 3H); 1.21–1.42 (m, 22H); 1.98–2.01 (m, 4H);
2
2
2
3
.14 (t, J=7 Hz, 2H); 3.25–3.28 (m, 2H); 5.31–5.35 (m,
1
3
H); C NMR (d-CDCl ) d (ppm) 14.10; 14.94 (CH );
3
3
2.71; 25.82; 27.23; 29.31; 29.56; 29.76; 31.89; 34.29;
5.58; 36.87 (CH ); 129.78; 129.98 (CH); 172.94 (C¼O);
2
+
ꢁ1
mass spectrometry [M ]=310; IR n (cm ): 3288 (NH);
1
4
1
644 (C¼O).
albumin. However, bovine serum albumin is required
in the FLIPR assay to prevent binding of the AEA to
the plastic wells. Thus, it is important to stress that the
screening concentration of AEA was chosen to give a
response of about 30–50% max, thus allowing for
potentiation to be seen.
N-(1-methylethyl)oleamide 23 (oleoylisopropylamide).
CA 10574-01-3. Yield: 2.42 g (75%) of a yellowish oil.
ꢁ
1
TLC (ethyl acetate/methanol 8:2 vv ) R =0.82; spec-
f
1
troscopic data: H NMR (d-CDCl ) d (ppm) 0.87(t,
3
J=7 Hz, 3H) ; 1.12 (t, J=3 Hz, 6H); 1.21–1.42 (m, 22H);
1
2
.98–2.02 (m, 2H); 2.11 (t, J=7 Hz, 2H); 3.25–3.28 (m,
3
Radioligand binding experiments
1
H); 4.07–4.09 (m, 1H); 5.31–5.34 (m, 2H); C NMR
(
2
4
d-CDCl ) d (ppm) 14.16; 14.54 (CH ); 22.71; 22.90;
Membranes (40 mg) from the CB receptor-transfected
CHO cells were incubated at 30 C with 1 nM
3
3
ꢃ
[ H]-CP55,940 (CHO-CB ) or 1 nM [ H]-WIN 55,212-2
5.88; 27.23; 29.37; 29.56; 29.82; 31.96; 37.07 (CH2);
1.21; 129.78; 130.04 (CH); 172.23 (C¼O); mass spectro-
3
3
1
+
.
ꢁ1
metry [M ]=324; IR n (cm ): 3285 (NH); 1641 (C¼O).
(CHO-CB ) for 1 h in 50 mM Tris–HCl with MgCl and
2
2
EDTA (pH 7.4) in the presence of 50 mM phenyl-
methylsulfonylfluoride. Non-specific binding was deter-
mined in parallel samples using either 10 mM HU-210
(CHO-CB ) or 10 mM WIN 55,212-2 (CHO-CB ), as
Cell culture
hVR1-HEK293 cells, grown as monolayers in minimum
essential medium (MEM) supplemented with non-
essential amino acids, 10% foetal bovine serum, and
1
2
appropriate. After incubation, membrane suspensions
were rapidly filtered through 0.5% polyethyleneimine-
pretreated GF/B glass fibre filters (Whatman), and the
radioactivity trapped on the filters was measured by
liquid scintillation spectroscopy. Assays were conducted
in quadruplicates.
0
O /CO at 37 C. Cells were passaged every 3–4 days
.2 mM l-glutamine, were maintained under 95%/5%
ꢃ
and the highest passage number used was 15.
2
2
Human CB -(‘CHO-CB ’) and CB -(‘CHO-CB ’)
1
1
2
2
transfected CHO cells (passage range 11–13 and 12–15,
respectively), kindly donated by Drs. Nokin and
Detheux (Euroscreen, Belgium) were cultured in Ham’s
F12 medium containing 10% fetal bovine serum,
Assay of FAAH activity
3
1
The assay of Omeir et al., adapted to the tritiated
3
2
substrate was used. Frozen adult rat brains (minus
cerebellum) that had been stored at ꢁ70 C were thawed
ꢃ
and homogenized at 4 C in 20 mM HEPES buffer, pH
100 mg/mL streptomycin, 100 U/mL penicillin and
00 mg/mL G418.
ꢃ
2

824
S. Vandevoorde et al. / Bioorg. Med. Chem. 11 (2003) 817–825
7.0, with 1 mM MgCl . The homogenates were cen-
trifuged twice (36,000 g, 20 min, 4 C). The tissue pellets
uptake experiments. This study was supported by grants
from the Swedish Research Council (Grant no. 12158),
Konung Gustav V’s and Drottning Victorias Founda-
tion, the Swedish Asthma and Allergy Association’s
Research Foundation, the Swedish Psoriasis Associ-
ation, and the Research Funds of the Medical Odonto-
˚
logical Faculty, Umea University. The chemical work
was supported by a grant from the Belgian National
Fund for Scientific Research and an FSR grant from the
2
ꢃ
were resuspended in homogenisation buffer and incu-
ꢃ
36,000 g, 20 min, 4 C), the membranes were re-sus-
bated at 37 C for 15 min. After further centrifugation
(
ꢃ
pended in 50 mM Tris–HCl buffer, pH 7.4, containing
ꢃ
1
mM EDTA and 3 mM MgCl , and stored at ꢁ70 C
2
until used for assay. Upon assay, membranes (ꢀ15 mg
ꢁ
1
protein assay ), test compounds or ethanol carrier,
[ H]-AEA and assay buffer (10 mM Tris–HCl, 1 mM
EDTA, 1% (w/v) fatty acid-free bovine serum albumin,
3
Universite
´
catholique de Louvain.
ꢃ
pH 7.6) were incubated for 10 min at 37 C (final assay
volume of 200 mL). Blanks contained homogenization
buffer instead of the membranes. The reactions were
stopped by placing the tubes in ice followed by addition
of chloroform/methanol (1:1 v/v, 400 mL). Samples were
thoroughly mixed, the phases were separated by centri-
fugation and aliquots (200 mL) of the methanol/buffer
phase containing the [ H]-ethanolamine product were
removed and analyzed for radioactivity by liquid
scintillation spectroscopy with quench correction.
References and Notes
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4
8
2
2
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´
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ꢁ1
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concomitantly (12ꢂ0.9 and 15ꢂ6.6% of the total tri-
1
1
ꢃ
tium retained at 37 and 4 C, respectively).
1
Determination of pI50 values
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