Novel selective and metabolically stable inhibitors of anandamide cellular uptake

Article abstract of DOI:10.1016/S0006-2952(03)00109-6

Novel aromatic analogues of N-oleoylethanolamine and N-arachidonoylethanolamine (anandamide, AEA) were synthesized and, based on the capability of similar compounds to interact with proteins of the endocannabinoid and endovanilloid signaling systems, were

Full text of DOI:10.1016/S0006-2952(03)00109-6

Biochemical Pharmacology 65 (2003) 1473–1481
Novel selective and metabolically stable inhibitors of
anandamide cellular uptake
Giorgio Ortara,b, Alessia Ligrestib,c, Luciano De Petrocellisc,d,
Enrico Moreraa,b, Vincenzo Di Marzob,c,*
a
`
Dipartimento di Studi Farmaceutici, Universita ‘La Sapienza’, P.le A. Moro 5, 00185 Roma, Italy
bInstitute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Italy
cEndocannabinoid Research Group, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Italy
dInstitute of Cybernetics, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Italy
Received 13 December 2002; accepted 13 February 2003
Abstract
Novel aromatic analogues of N-oleoylethanolamine and N-arachidonoylethanolamine (anandamide, AEA) were synthesized and, based
on the capability of similar compounds to interact with proteins of the endocannabinoid and endovanilloid signaling systems, were tested
on: (i) cannabinoid CB1 and CB2 receptors; (ii) vanilloid VR1 receptors; (iii) anandamide cellular uptake (ACU); and (iv) the fatty acid
amide hydrolase (FAAH). The (R)- and, particularly, the (S)-10-(4-hydroxybenzyl) derivatives of N-oleoylethanolamine and AEA
(OMDM-1, OMDM-2, OMDM-3, and OMDM-4) inhibited to a varied extent ACU in RBL-2H3 cells (Ki ranging between 2.4 and
17.7 mM), the oleoyl analogues (OMDM-1 and OMDM-2, Ki 2.4 and 3.0 mM, respectively) being 6- to 7-fold more potent than the
arachidonoyl analogues (OMDM-3 and OMDM-4). These four compounds exhibited: (i) poor affinity for either CB1 (Ki ꢀ 5 mM) or CB2
(Ki > 10 mM) receptors in rat brain and spleen membranes, respectively; (ii) almost no activity at vanilloid receptors in the intracellular
calcium assay carried out with intact cells over-expressing the human VR1 (ec50 ꢀ 10 mM); and (iii) no activity as inhibitors of FAAH in
N18TG2 cell membranes (Ki > 50 mM). The oleoyl- and arachidonoyl-N0-(4-hydroxy-3-methoxybenzyl)hydrazines (OMDM-5 and
OMDM-6), inhibited ACU (Ki 4.8 and 7.0 mM, respectively), and were more potent as VR1 agonists (EC50 75 and 50 nM, respectively),
weakly active as CB1 receptor ligands (Ki 4.9 and 3.2 mM, respectively), and inactive as CB2 ligands (Ki > 5 mM) as well as on FAAH
(Ki ꢀ 40 mM). In conclusion, we report two novel potent and selective inhibitors of ACU (OMDM-1 and OMDM-2) and one ‘‘hybrid’’
agonist of CB1 and VR1 receptors (OMDM-6). Unlike other compounds of the same type, OMDM-1, OMDM-2, and OMDM-6 were very
stable to enzymatic hydrolysis by rat brain homogenates.
# 2003 Elsevier Science Inc. All rights reserved.
Keywords: Endocannabinoid; Cannabinoid; Receptor; Transport; VR1; FAAH
1. Introduction
Endocannabinoids are by definition endogenous com-
pounds capable of binding to, and functionally activating,
the two cannabinoid CB1 and CB2 receptors for marijuana’s
active principle D9-tetrahydrocannabinol [1,2]. Four pro-
totypical endocannabinoids have been described to date:
* Corresponding author. Tel.: þ39-081-8675093;
fax: þ39-081-8041770.
E-mail address: vdimarzo@icmib.na.cnr.it (V. Di Marzo).
Abbreviations: AEA, N-arachidonoylethanolamine; 2-AG, 2-arachido-
noylglycerol; 2-AGE, 2-arachidonylglyceryl ether; OEA, N-oleoylethano-
AEA (anandamide) [3], 2-arachidonoylglycerol (2-AG)
[4,5], 2-arachidonylglyceryl ether (2-AGE, noladin ether)
[6], and O-arachidonoylethanolamine (virodhamine) [7].
A function as local mediators has been proposed for
the endocannabinoids [1], which requires for these sub-
stances the presence of specific biosynthetic and metabolic
lamine; ACU, anandamide cellular uptake; FAAH, fatty acid amide
hydrolase; CB1, cannabinoid receptor type 1; CB2, cannabinoid receptor
type 1; VR1, vanilloid receptor type 1; OLDA, N-oleoyldopamine; NADA,
N-arachidonoyldopamine; NATA, N-arachidonoyltyramine; arvanil,
N0-(4-hydroxy-3-methoxybenzyl)arachidonoylamine; olvanil, N0-(4-hydro-
xy-3-methoxybenzyl)oleoylamine; HEK-293 cells, human embryonic
kidney-293 cells; RBL-2H3 cells, rat basophilic leukemia-2H3 cells.
0006-2952/03/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0006-2952(03)00109-6

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G. Ortar et al. / Biochemical Pharmacology 65 (2003) 1473–1481
mechanisms subject to regulation during physiological and
pathological conditions. Although it is now clear that AEA
and 2-AG are produced ‘‘on demand’’ from the enzymatic
hydrolysis of (phospho)-glyceride precursors, the proteins
mediating these processes have not been fully character-
ized yet ([8] for a recent review). Nothing is known yet on
the biosynthetic mechanisms of 2-AGE and virodhamine.
Endocannabinoid inactivation is instead better under-
stood. AEA and 2-AG are inactivated via a three-step
mechanism including: (i) cellular uptake from the extra-
cellular matrix, which appears to occur via diffusion
through the cell membrane, facilitated by one or more
transporters (see [9] for a recent review); (ii) enzymatic
hydrolysis, which is catalyzed by FAAH [10,11] and, in the
case of 2-AG, also by monoacylglycerol lipases [12,13];
(iii) re-esterification of the hydrolysis products into mem-
brane phospholipids. 2-AGE, which cannot be hydrolyzed
enzymatically, is taken up by cells via the same mechanism
as AEA and 2-AG and is partly metabolized via direct
esterification into membrane phospholipids [14], a process
used to inactivate also part of 2-AG [15]. Nothing is yet
known on the inactivation of virodhamine.
than those required to inhibit AEA transport into the cell
[26,27,31]. These compounds behave as ‘‘hybrid’’ agonists
of VR1 receptors and of cannabinoid CB1 receptors, the
latter of which they activate either directly or ‘‘indirectly’’,
i.e. by enhancing the endogenous levels of AEA and 2-AG
through inhibition of their inactivation [29]. On the other
hand, two other AEA aromatic congeners, VDM11 and
VDM13, have been developed with inhibitory activity on
ACU comparable to AM404 and negligible activity at VR1
[26]. One compound, UCM-707, was found to inhibit ACU
at sub-micromolar concentrations and to be inactive on
VR1 [32]. However, VDM11 and AM404 are not very
stable to enzymatic hydrolysis by rat brain homogenates,1
while the metabolic stability of UCM-707 and its effect on
FAAH have not been investigated.
With the aim of obtaining novel metabolically stable
ACU inhibitors selective vs. both VR1/cannabinoid recep-
tors and FAAH, in the present study we have synthesized
novel aromatic analogues (Fig. 1) of AEA and of its con-
gener, N-oleoylethanolamine (OEA), which inhibits AEA
uptake [33]). We report the finding of two novel selective
ACU inhibitors and of one potential ‘‘hybrid’’ agonist of
CB1 and VR1 receptors.
Unlike the enzymes for AEA and 2-AG hydrolysis, the
putative endocannabinoid transporter has not been cloned
yet, and only indirect evidence points to its existence [9]. It
was suggested that this protein facilitates endocannabinoid
diffusion through the cell membrane via a saturable,
energy-independent and selective mechanism, which, by
being dependent on the gradient of solute concentrations
across the cell membrane, relies on the rate of intracellular
hydrolysis [16]. Accordingly, several inhibitors of ACU are
also FAAH inhibitors [17] and vice versa [16]. However,
nitric oxide donors were found to significantly enhance the
rate of ACU, but not the activity of FAAH [18,19], and
substances capable of inhibiting AEA and 2-AG cellular
uptake without influencing FAAH have also been devel-
oped [20,21], including AEA analogues with variously
derivatized phenyl groups instead of the ethanolamine
‘‘head’’. Furthermore, 2-AGE, which cannot be hydro-
lyzed by FAAH, is still rapidly taken up by cells [14],
thus suggesting that ACU is mediated by a mechanism not
relying uniquely on FAAH.
Another molecular target for AEA, considered by some
as part of the endocannabinoid system [22], is VR1, a
capsaicin-sensitive, non-selective cation channel acting
as a molecular transducer for inflammatory and thermal
pain in sensory neurons, and expressed also in the brain
[23,24]. It has been observed that several substances acting
as ACU inhibitors, and in particular the capsaicin-like
N-acylvanillylamines, are also potent VR1 agonists
[20,21,25–28]. In particular, N0-(4-hydroxy-3-methoxy-
benzyl)arachidonoylamine (arvanil) is one of the most
potent ACU inhibitors and VR1 agonists described to date
[26]. Another aromatic AEA analogue, and widely used
ACU inhibitor, N-arachidonoyl-(4-hydroxyphenyl)amine
(AM404) [29,30], activates VR1 at concentrations lower
2. Materials and methods
2.1. Synthesis of compounds
Amides OMDM-1 to OMDM-4 (Fig. 1) were synthe-
sized in 74–82% yields by treatment of (S)- or (R)-tyrosinol
with oleic or arachidonic acid using 1-hydroxybenzotria-
zole (HOBt)–N-ethyl-N0-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDC) as the carboxylate
activator. Briefly, to a solution of fatty acid in DMF at
08, HOBt (1.05 Eq.) and EDC (1.05 Eq.) were added under
stirring. After 15 min at 08 and 0.5 hr at room temperature,
(S)- or (R)-tyrosinol hydrochloride (1 Eq.) and Et3N (1 Eq.)
were added and the mixture was stirred at room tempera-
ture overnight. The mixture was diluted with brine and
extracted with AcOEt. The organic phase was washed with
10% citric acid solution, saturated NaHCO3, and brine,
dried (Na2SO4), and evaporated under reduced pressure.
The residue was purified on silica gel by using CH2Cl2/
AcOEt ¼ 6/4 as eluent. OMDM-1 (C27H45NO3, 431.7)
was characterized as follows: m.p. 88–898; [a]D À138
(CHCl3, c 1.0); IR (KBr) 3432, 3325, 2928, 2856, 1651,
1515, 1234, 1201 cmÀ1; H NMR (300 MHz, CDCl3) d
1
0.87 (3H, t, J ¼ 6:5 Hz), 1.26 (20H, m), 1.52–1.56 (2H,
m), 1.96–2.00 (4H, m), 2.12 (2H, t, J ¼ 7:6 Hz), 2.65–2.78
(2H, m), 3.12 (1H, br s), 3.47 (1H, dd, J ¼ 11:1, 5.1 Hz),
3.56 (1H, dd, J ¼ 11:1, 4.0 Hz), 4.07 (1H, m), 5.26–5.37
(2H, m), 6.38 (1H, d, J ¼ 8:4 Hz), 6.70 (2H, d,
J ¼ 8:4 Hz), 6.97 (2H, d, J ¼ 8:4 Hz), 8.05 (1H, br s);
1 T. Bisogno and V. Di Marzo, unpublished data.

G. Ortar et al. / Biochemical Pharmacology 65 (2003) 1473–1481
1475
Fig. 1. Chemical structures of the six novel compounds described in this study and of N-oleoyldopamine (OLDA), N-arachidonoyltyramine (NATA), N-
arachidonoyldopamine (NADA), olvanil, and arvanil, shown as a comparison.
13C NMR (75 MHz, CDCl3) d 14.09, 22.66, 25.72, 27.17,
27.21, 29.15, 29.30, 29.50, 29.74, 31.87, 36.15, 36.77,
52.73, 52.82, 63.63, 115.34, 128.46, 129.55, 129.81,
129.97, 155.05, 174.19. OMDM-2 was characterized as
follows: m.p. 88–898; [a]D þ138 (CHCl3, c 1.0); other data
were identical to OMDM-1. OMDM-3 (C29H43NO3,
453.7, wax) was characterized as follows: [a]D À178
(CHCl3, c 1.0); IR (CHCl3) 3433, 3331, 2930, 2870,
129.94, 130.32, 154.91, 173.90. OMDM-4 was character-
ized as follows: [a]D þ188 (CHCl3, c 1.0); other data were
identical to OMDM-1. Acyl hydrazines, OMDM-5 and
OMDM-6 (Fig. 1), were prepared in 43 and 48% overall
yields, respectively, by reduction with Et3SiH/CF3CO2H
[32] of the corresponding acyl hydrazones, in turn obtained
by condensation between vanillin hydrazone [34] and oleic
or arachidonic acid using the isobutyl chloroformate
method [35]. In detail, to a solution of fatty acid in dry
CH2Cl2 at 08 under N2, Et3N (1.4 Eq.) and i-BuOCOCl (1.2
Eq.) were added. The mixture was stirred for 1 hr at 08. A
solution of vanillin hydrazone (1.5 Eq.) and Et3N (1.2 Eq.)
in dry DMF was then added and the mixture was stirred for
4 hr at room temperature. The mixture was diluted with
brine and extracted with AcOEt. The organic phase was
washed twice with brine, dried (NaSO4), and evaporated
under reduced pressure. To a solution of the crude acyl
hydrazone in dry CH2Cl2 at 08, CF3CO2H (20 Eq.) and
Et3SiH (3 Eq.) were added. The solution was stirred for
1656, 1615, 1515, 1456, 1261 cmÀ1
;
1H NMR
(300 MHz, CDCl3) d 0.88 (3H, t, J ¼ 6:9 Hz), 1.25–
1.37 (6H, m), 1.64 (2H, quintet, J ¼ 7:5 Hz), 2.04 (4H,
q, J ¼ 6:9 Hz), 2.15 (2H, t, J ¼ 7:6 Hz), 2.71–2.83 (8H,
m), 3.49 (1H, m), 3.52 (1H, dd, J ¼ 11:1, 4.9 Hz), 3.61
(1H, dd, J ¼ 11:1, 3.3 Hz), 4.12 (1H, m), 5.26–5.41 (8H,
m), 5.98 (1H, d, J ¼ 7:8 Hz), 6.71 (2H, d, J ¼ 8:4 Hz),
6.97 (2H, d, J ¼ 8:4 Hz), 7.65 (1H, br s); 13C NMR
(75 MHz, CDCl3) d 14.07, 22.54, 25.47, 25.60, 26.53,
27.18, 29.27, 31.46, 36.12, 36.21, 52.96, 63.76, 115.44,
127.30, 127.62, 127.91, 128.07, 128.42, 128.49, 128.67,

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G. Ortar et al. / Biochemical Pharmacology 65 (2003) 1473–1481
4 hr at 08, brought to pH ꢁ8 with 10% NaHCO3 solution
and extracted with CH2Cl2. The organic phase was washed
with brine, dried (Na2SO4), and evaporated under reduced
pressure. The residue was purified on silica gel using
CH2Cl2/AcOEt ¼ 1/1 as eluent. OMDM-5 (C26H44N2O3,
432.6) was characterized as follows: m.p. 78–818; IR
(KBr) 3544, 3440, 2929, 2856, 1667, 1613, 1516, 1465,
On the day of the experiment the cells (50,000–60,000 per
well) were loaded for 2 hr at 258 with 4 mM Fluo-3 methy-
lester (Molecular Probes) in DMSO containing 0.04%
Pluoronic. After the loading, cells were washed with Tyr-
ode’s pH 7.4, trypsinized, resuspended in Tyrode’s, and
transferred to the cuvette of the fluorescence detector (Per-
kin-Elmer LS50B) under continuous stirring. Experiments
were carried out by measuring cell fluorescence at 258
(lEX ¼ 488 nm, lEM ¼ 540 nm) before and after the addi-
tion of the test compounds at various concentrations. Data
are expressed as the concentration exerting a half-maximal
1272, 1223 cmÀ1; H NMR (300 MHz, CDCl3) d 0.87
1
(3H, br t), 1.26–1.28 (20H, m), 1.59–1.63 (2H, m), 1.97–
2.04 (4H, m), 2.09 (2H, t, J ¼ 7:5 Hz), 3.85 (3H, s), 3.88
(2H, s), 5.30–5.34 (2H, m), 5.88 (2H, br s), 6.79 (1H, dd,
J ¼ 8:4, 1.8 Hz), 6.83 (1H, d, J ¼ 8:4 Hz), 6.87 (1H, d,
J ¼ 1:8 Hz), 7.03 (1H, br s); 13C NMR (75 MHz, CDCl3) d
14.10, 22.66, 25.52, 27.14, 27.20, 29.19, 29.29, 29.49,
29.67, 31.87, 34.68, 55.74, 55.82, 111.31, 114.16, 121.83,
129.12, 129.49, 129.82, 145.00, 146.41, 172.43. OMDM-6
(C28H42N2O3, 454.7, wax) was characterized as follows:
IR (CHCl3) 3544, 3439, 2930, 2858, 1668, 1612, 1515,
effect (EC ). The efficacy of the effect was determined by
50
comparing it to the analogous effect observed with 4 mM
ionomycin.
2.4. ACU assay
The effect of compounds on the uptake of [14C]AEA by
intact RBL-2H3 cells was studied by using 5.0 mM
(20,000 cpm) of [14C]AEA as described previously [34].
Cells were incubated with [14C]AEA for 5 min at 378, in
the presence or absence of varying concentrations of the
inhibitors. Residual [14C]AEA in the incubation media
after extraction with CHCl3/CH3OH 2:1 (by vol.), deter-
mined by scintillation counting of the lyophilized organic
phase, was used as a measure of the AEA that was taken up
by cells. Previous studies [36] had shown that, after a 5 min
incubation, the amount of [14C]AEA disappeared from the
medium of RBL-2H3 cells is found mostly (>90%) as
unmetabolized [14C]AEA in the cell extract. Non-specific
binding of [14C]AEA to cells and plastic dishes was
determined in the presence of 100 mM AEA and was never
higher than 30%. Data are expressed as the Ki, calculated
using the Cheng–Prusoff equation from the concentration
1463, 1272, 1234 cmÀ1; H NMR (300 MHz, CDCl3) d
1
0.88 (3H, t, J ¼ 6:8 Hz), 1.25–1.37 (6H, m), 1.70
(2H, quintet, J ¼ 7:5 Hz), 2.01–2.13 (6H, m), 2.76–2.84
(6H, m), 3.85 (3H, s), 3.88 (2H, s), 5.29–5.41 (8H, m), 5.90
(2H, br s), 6.79 (1H, dd, J ¼ 1:8, 8.1 Hz), 6.84 (1H, d,
J ¼ 8:1 Hz), 6.86 (1H, d, J ¼ 1:8 Hz), 6.99 (1H, br s); 13
C
NMR (75 MHz, CDCl3) d 14.06, 22.54, 25.30, 25.61,
26.57, 27.19, 29.28, 31.47, 33.98, 55.73, 55.83, 111.27,
114.15, 121.80, 127.31, 127.62, 127.87, 128.08, 128.41,
128.65, 128.75, 129.10, 130.31, 144.99, 146.40. VDM11,
N-arachidonoyltyramine (NATA), N-arachidonoyldopa-
mine (NADA), N-oleoyldopamine (OLDA), arvanil,
N0-(4-hydroxy-3-methoxybenzyl)oleoylamine (olvanil),
and AM404were synthesizedin 70–80% yieldsasdescribed
previously [26]. All compounds were purified to homoge-
neity (>99%) and characterized on the basis of their 1H and
13C NMR and IR spectra and elemental analyses.
exerting 50% inhibition of AEA uptake (IC ).
50
2.2. Cells
2.5. CB1 receptor binding assay
Rat basophilic leukemia-2H3 cells (RBL-2H3 cells) and
mouse N18TG2 neuroblastoma cells were purchased from
DSMZ and grown according to the specifications of the
manufacturer [36,37].
Displacement assays for CB1 receptors were carried out
by using [3H]SR141716A (0.4 nM, 55 Ci/mmol, Amer-
sham) as the high-affinity ligand, and the filtration technique
described previously [20], on membrane preparations
(0.4 mg per tube) from frozen male CD rat brains (Charles
River), and in the presence of 100 mM PMSF. Specific
binding was calculated with 1 mM SR141716A and was
84.0%. Data are expressed as the Ki, calculated using the
Cheng–Prusoff equation from the concentration exerting
2.3. Cytosolic calcium concentration ([Ca2þ]i) assay
Human embryonic kidney-293 cells (HEK-293) over-
expressing the human VR1 receptor were a kind gift from
John Davis (SmithKline Beecham). Cells were grown as
monolayers in minimum essential medium supplemented
with non-essential amino acids, 10% fetal calf serum, and
0.2 mM glutamine, and maintained under 95%/5% O2/CO2
at 378. The effect of the substances on [Ca2þ]i was deter-
mined by using Fluo-3, a selective intracellular fluorescent
probe for Ca2þ. One day prior to experiments, cells were
transferred into 6-well dishes coated with poly-L-lysine
(Sigma) and grown in the culture medium mentioned earlier.
50% inhibition of [3H]SR141716A specific binding (IC ).
50
2.6. CB2 receptor binding assay
Displacement assays for CB2 receptors were carried
out by using [3H]WIN55,212 (0.8 nM, 43 Ci/mmol,
Amersham) as the high-affinity ligand, and the filtration
technique described previously [20], on membrane prepara-
tions (0.05 mg per tube) from frozen male CD rat spleens

G. Ortar et al. / Biochemical Pharmacology 65 (2003) 1473–1481
1477
(Charles River). Specific binding was calculated with 1 mM
SR144528 and was 69.4%.
determined by densitometry scanning. Under these condi-
tions, the Rf of OMDM-1, OMDM-2, and OMDM-6 and of
long chain fatty acids were 0.62, 0.64, 0.86, and 0.34,
respectively. Control incubations were carried out either
with boiled homogenates or with [14C]AEA as the sub-
strate, under the same conditions.
2.7. FAAH assay
The effect of compounds on the enzymatic hydrolysis of
AEA was studied as described previously [36], using mem-
branes prepared from mouse neuroblastoma N18TG2 cells,
incubated with the test compounds and [14C]AEA (10 mM,
40,000 cpm) in 50 mM Tris–HCl, pH 9, for 30 min at 378.
[14C]Ethanolamine produced from [14C]AEA hydrolysis
was measured by scintillation counting of the aqueous
phase after extraction of the incubation mixture with 2
vol. of CHCl3/CH3OH 2:1 (by vol.). Data are expressed
as the Ki, calculated using the Cheng–Prusoff equation from
the concentration exerting 50% inhibition of [14C]AEA
3. Results
The effect of the novel compounds on the uptake of
[14C]AEA by intact RBL-2H3 cells, where ACU has
been well characterized [36], is shown in Fig. 2. In these
cells, ACU has been reported to exhibit an apparent Km
for AEA ranging from 9.3 to 33 mM [9,36]. All compounds
inhibited the uptake, OMDM-1, OMDM-2, OMDM-5, and
OMDM-6 being significantly more potent than OLDA,
OMDM-3, and OMDM-4 (Table 1). The previously
described NATA was also a relatively potent inhibitor
(Table 1).
hydrolysis (IC ).
50
2.8. Enzymatic hydrolysis of compounds
Whole homogenates of rat (CD male) brain in 50 mM
Tris–HCl, pH 7.4 (1 mg protein) were incubated with 1 mg
of each compound in 1 mL of 50 mM Tris–HCl, pH 7.4, for
30 min at 378. After the incubation, the reaction was
stopped by adding 2 mL CHCl3/CH3OH 2:1 (by vol.)
and mixing. The organic phase was then purified by thin
layer chromatography on analytical silica gel thin layer
chromatography plates (Merck), using CHCl3/CH3OH/
NH4OH 85:15:1 (by vol.), in order to separate the starting
compound from the long chain fatty acid (oleic or arachi-
donic acid) potentially produced from its hydrolysis. Spots
were visualized by exposure to iodine vapors followed
by charring with cerium sulfate. Intensity of bands was
The novel compounds were able, to some extent, to
displace [3H]SR141716A from its specific CB1 binding
sites in rat brain membranes (Fig. 3). However, only
OMDM-6 and OLDA exhibited some notable affinity
for these binding sites, whereas OMDM-1 was almost
inactive (Table 1). Using this same assay, the Kd for
unlabeled SR141716A is 0.4 nM [20]. Since NADA,
OLDA, arvanil, olvanil, and other aromatic AEA deriva-
tives were previously found to be inactive on CB2 receptors
from rat spleen [20,21,28], the six novel compounds
were tested in the same preparation only at two concentra-
tions (1 and 10 mM). None of the compound did
appreciably displace [3H]WIN55,212 binding from rat
Fig. 2. Effect of the novel compounds on anandamide cellular uptake. The effect of increasing concentrations of the six compounds on the uptake of
14C]anandamide by intact RBL-2H3 cells is shown. Data are means of three independent experiments carried out in duplicate. Standard deviations are not
shown for the sake of clarity and were never higher than 15% of the means.
[

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G. Ortar et al. / Biochemical Pharmacology 65 (2003) 1473–1481
Table 1
Summary of the effects of the novel compounds on anandamide cellular uptake (ACU) in RBL-2H3 cells, the fatty acid amide hydrolase (FAAH) in N18TG2
cell membranes, on cannabinoid CB1 receptors in rat brain membranes, and on human VR1 in HEK cells
ACU (Ki, mM)
FAAH (Ki, mM)
CB1 (Ki, mM)
VR1 (EC50, mM)
VR1 (maximum effect)
OMDM-1
OMDM-2
OMDM-3
OMDM-4
OLDA
2.4 Æ 0.7
3.0 Æ 0.6
16.6 Æ 0.9
17.7 Æ 1.5
14.1 Æ 2.5
17.3 Æ 7.3a
7.4 Æ 2.6
4.8 Æ 0.5
7.0 Æ 0.6
7.3 Æ 1.6a
2.9 Æ 0.6a
>50
12.1 Æ 1.3
5.1 Æ 0.3
6.1 Æ 0.4
4.9 Æ 0.2
1.6 Æ 0.4
0.5 Æ 0.2
0.6c
>10
16.0 Æ 2.0
31.0 Æ 3.2
15.0 Æ 2.2
27.5 Æ 2.8
62.1 Æ 5.5
73.1 Æ 6.6
18.0 Æ 3.7
67.3 Æ 3.5
72.0 Æ 3.1
67.2 Æ 9.1a
75.4 Æ 4.7a
>50
10.0 Æ 0.5
>10
>10
>50
>50
>25
0.036 Æ 0.009
0.063 Æ 0.011
>10
NADA
NATA
22.0 Æ 5.0b
11.0 Æ 2.1
>50
OMDM-5
OMDM-6
Olvanil
4.9 Æ 0.3
3.2 Æ 0.2
10.1 Æ 0.5
2.6 Æ 0.6
0.075 Æ 0.006
0.050 Æ 0.004
0.0005 Æ 0.0003a
0.0005 Æ 0.0002a
40.1 Æ 4.2
>50d
Arvanil
32.0 Æ 5.0d
The effect on VR1 is expressed as both EC50 (potency) and as the effect at 10 mM concentration (as percent of the effect of 4 mM ionomycin) (efficacy).
Data are means Æ SD of three independent experiments carried out in duplicate. Little, if any, displacement of [3H]WIN55,212 from rat spleen membranes by
any of the six novel compounds (tested at either 1 or 10 mM) was found (Ki > 10 mM, data not shown). Abbreviations used: OLDA, N-oleoyldopamine;
NADA, N-arachidonoyldopamine; NATA, N-arachidonoyltyramine; arvanil, N0-(4-hydroxy-3-methoxybenzyl)arachidonoylamine; olvanil, N0-(4-hydroxy-3-
methoxybenzyl)oleoylamine.
a Data were calculated from the corresponding IC from Ref. [26].
50
b Data were calculated from the corresponding IC from Ref. [41].
50
c Data were calculated from the corresponding IC from Ref. [44].
50
d Data were calculated from the corresponding IC from Ref. [20].
50
spleen membranes, except for OMDM-5 and OMDM-6
(67:5 Æ 2:1 and 66:9 Æ 2:4% displacement at 10 mM,
means Æ SEM, N ¼ 3) (data not shown).
Of the novel compounds tested on VR1-mediated
increase of [Ca2þ]i in HEK cells over-expressing with the
human VR1, only OMDM-5 and OMDM-6 exerted agonist
When the novel compounds were tested on [14C]AEA
hydrolysis by N18TG2 cell membranes, which express
high levels of FAAH with an apparent Km for AEA of
15 mM [37], none was found to exert an appreciable
inhibitory activity, except for OMDM-6 at very high
concentrations (Table 1). The previously described NATA
was instead a good inhibitor (Table 1).
activity (Fig. 4), with potency (EC ) and efficacy (maximal
50
effect expressed as percent of the effect of ionomycin)
similar to that of the prototypical VR1 agonist, capsaicin
(Table 1). The two compounds were inactive in HEK cells
that had not been transfected with human VR1 (not shown).
Finally, when VDM11, AM404, OMDM-1, OMDM-2,
and OMDM-6 were incubated with rat brain whole
Fig. 3. Effect of the novel compounds on cannabinoid CB1 receptors. The effect of increasing concentrations of the six compounds on the amount of
[3H]SR141716A bound to rat brain membranes is shown. Data are means of three independent experiments in duplicate. Standard deviations are not shown
for the sake of clarity and were never higher than 15% of the means.

G. Ortar et al. / Biochemical Pharmacology 65 (2003) 1473–1481
1479
VDM11 and VDM13 [26], which are almost inactive at
VR1 and FAAH but, in the case of the former compound at
least, not very stable to enzymatic hydrolysis.
These observations prompted the synthesis of the (R)- and
(S)-10-(4-hydroxybenzyl) analogues of OEA (OMDM-1 and
OMDM-2) and AEA (OMDM-3 and OMDM-4), and of the
oleoyl- and arachidonoyl-N0-(4-hydroxy-3-methoxyben-
zyl)hydrazines (OMDM-5 and OMDM-6). In fact, alkyla-
tion of the 10-carbon atom, as in (R)-methanandamide [39],
or modification of the amide region, as in ‘‘retro-ananda-
mide’’, dramatically enhance the metabolic stability of
AEA (see [40] for review). Furthermore, the synthesis of
OMDM-1, OMDM-2, OMDM-3, and OMDM-4 was also
suggested by the previous findings that: (i) the chemically
related NADA [41,42], also inhibits ACU and is stable to
enzymatic hydrolysis, although it potently activates VR1
(Table 1); and (ii) elimination of the 3-hydroxy group in
NADA, as in NATA, abolishes activity at VR1 (see below)2.
The synthesis of OMDM-5 and OMDM-6, on the other
hand, was also prompted by the fact that two closely related
compounds, arvanil and olvanil, are more stable to enzy-
matic hydrolysis than AEA and OEA, and are potent,
although non-selective, inhibitors of ACU [20,26].
We found that, in the 10-(4-hydroxybenzyl) series,
only the derivatives of OEA potently inhibited ACU
(Ki < 10 mM). Furthermore, the 10-(S)-enantiomers
(OMDM-1 and OMDM-3) were slightly (although not
significantly) more potent than the 10-(R)-enantiomers
(OMDM-2 and OMDM-4), as previously shown also for
the 10-methyl analogues of AEA (methanandamides)
[39,40]. Conversely, none of the four compounds exhibited
high affinity for CB1 receptors, although it is interesting to
note that, as previously reported again for the methanan-
damides [39,40], and opposite to the situation observed
here with ACU, the 10-(S)-enantiomers were weaker
ligands than the 10-(R)-enantiomers. Finally, very little,
if any, activity at CB2 and VR1 receptors, or at FAAH, was
found for the four compounds. Therefore, OMDM-1 and
OMDM-2 can be considered among the most potent and
selective inhibitors of ACU reported so far since they are:
(i) more potent than VDM11 and VDM13, when these
latter compounds are tested under the same conditions
(Ki 9.0 and 9.7 mM, respectively, [26]), (ii) more selective
than arvanil (Ki 2.9 mM, Table 1), and both more potent and
more selective than AM404 (Ki 6.5), two compounds that
are also VR1 agonists; and (iii) possibly at least as potent as
UCM-707 (ic50 ¼ 0:8 mM, [32]), which was originally
tested under conditions (i.e. using a concentration of
radiolabeled AEA substrate 50 times lower than the one
Fig. 4. Effect of the novel compounds on VR1 receptors. The effect of
increasing concentrations of the six compounds on the intracellular
calcium concentration in HEK cells over-expressing the human VR1 is
shown and expressed as percent of the effect of ionomycin (4 mM). Data
are means of three independent experiments carried out in duplicate.
Standard deviations are not shown for the sake of clarity and were never
higher than 15% of the means.
homogenates under conditions sufficient to hydrolyze
about 40% of [14C]AEA, hydrolysis to the corresponding
fatty acids could be observed only with the former two
compounds (i.e. 15:8 Æ 1:2, 18:3 Æ 1:3, <5, <5, and <5%
of the starting compound was hydrolyzed, respectively,
means Æ SEM, N ¼ 3Þ.
4. Discussion
In this study, we synthesized novel analogues of AEA
and OEA and tested them on some of the best characterized
proteins of the endocannabinoid system, with the aim of
developing new potent, selective, and metabolically stable
inhibitors of ACU. When deciding what types of modifica-
tions had to be introduced into the chemical structure of
AEA and OEA, several previous observations needed to be
taken into account. First, the most potent ACU inhibitors
developed to date have been obtained by introducing into
AEA an aromatic group instead of the ethanolamine
‘‘head’’ (see [38] for a recent review). This is possibly
due to strong interaction with the AMT binding site via
aromatic stacking [17]. Secondly, it must be remembered
that, to date, the major cause of the lack of selectivity of
ACU inhibitors has been their capability of interacting with
either VR1 receptors or FAAH, or both [38]. Therefore,
changes previously shown to diminish the affinity of
aromatic long chain fatty acid amides for these two pro-
teins, such as modification of their phenol or amide moi-
eties, respectively, had to be introduced. For example,
alkylation of the amide nitrogen in arvanil produced an
ACU inhibitor selective vs. both FAAH and VR1, O-2093,
which, however, might interact also with an as yet
unknown brain receptor [21,22]. Changes on the phenol
moiety, on the other hand, resulted in two ACU inhibitors,
used here) likely to yield lower IC values than those
50
obtainable using our procedure, and was never assayed on
FAAH. Moreover, compared to VDM11 and AM404, both
OMDM-1 and OMDM-2 are extremely stable to enzymatic
hydrolysis in rat brain whole homogenates, while the
2 V. Di Marzo, unpublished data.

1480
G. Ortar et al. / Biochemical Pharmacology 65 (2003) 1473–1481
metabolic stability of UCM-707 has not been examined
[32].
cally stable, and relatively potent inhibitors of ACU. The
future use as pharmacological tools for the study of the
patho-physiological role of endocannabinoids can be pre-
dicted for these two compounds, since they represent a
further improvement over two previously developed and
widely used inhibitors of ACU, AM404 and VDM11.
Furthermore, we have described a further potential mem-
ber of the family of ‘‘hybrid’’ CB1/VR1 agonists [20,28],
whose multiple potential therapeutic uses are being
increasingly suggested by recent reports [43].
It is worthwhile noting how the chemical modifications
introduced into OMDM-1 and OMDM-2 with respect to
OLDA (i.e. a missing 3-hydroxy group in the phenol
moiety and the presence of the 10-hydroxymethyl group)
confer to these compounds a more potent activity as ACU
inhibitors, while abolishing the VR1 agonist activity and
strongly decreasing the CB1 ligand activity (Table 1). Also,
notable is the fact that the presence of the 10-hydroxy-
methyl group alone, disregardingly of its stereochemistry,
reduces the potency at the AMT of the arachidonoyl
analogue, NATA, which is significantly more potent than
both OMDM-3 and OMDM-4 (Table 1). NATA, however,
although inactive at VR1, significantly inhibits FAAH.
The fact that OMDM-1 and OMDM-2 inhibited the
AMT and bound at CB1 at low and medium micromolar
concentrations, respectively, should not seen as poor selec-
tivity for the putative AEA transporter vs. the receptors. In
fact, the Ki of the two compounds in the two assays should
be matched against the affinity constants of the putative
AEA transporter and of CB1 for their natural ligands
(which are in the middle micromolar and nanomolar ranges
of concentrations, respectively), as well as with the con-
centrations normally required to compounds to produce
pharmacological actions via these two targets. Thus, a Ki of
5–10 mM is normally sufficient for ACU inhibitors to
enhance AEA CB1-mediated effects both in vivo and in
vitro, but not to CB1 ligands to cause CB1-mediated effects.
The two hydrazides, OMDM-5 and OMDM-6, which can
be considered close analogues of olvanil and arvanil,
respectively, inhibited ACU with potency lower or compar-
able to that of these latter compounds and to VDM11 and
AM404. However, OMDM-5 and OMDM-6 stimulated
VR1 activity as efficaciously and potently as capsaicin,
and, particularly OMDM-6, exhibited higher affinity for
CB1 receptors than the other five compounds examined in
this study. Neither compound exhibited high affinity for
CB2 receptors in rat spleen membranes (Ki > 5 mM). There-
fore, the introduction of a further NH group in the two
N-acylvanillylamines appears to: (i) preserve or slightly
improve the ACU inhibitory activity, (ii) decrease the VR1
agonist activity, and (iii) preserve or slightly enhance the
CB1 ligand activity. Like arvanil (see Table 1), OMDM-6
can be considered a direct ‘‘hybrid’’ agonist at VR1 and CB1
receptors [20,28]. Although its capability to activate CB1
receptors directly still needs to be assessed in functional
assays (see [21], for example), OMDM-6, as evidenced
previously for AM404 [29], is likely to enhance endocan-
nabinoid levels by inhibiting endocannabinoid degradation,
thereby leading to ‘‘indirect’’ activation of cannabinoid
receptors. Furthermore, OMDM-6 appears to be more
metabolically stable than both arvanil and AM404, and
hence is likely to have a longer half-life in vivo.
Acknowledgments
The authors are grateful to Dr. Tiziana Bisogno for her
support during the enzymatic hydrolysis experiments and
Dr. John Davis, SmithKline Beecham, for the gift of HEK
cells over-expressing hVR1. Supported by MURST (Grant
3933 to V.D.M.).
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