A structure/activity relationship study on arvanil, an endocannabinoid and vanilloid hybrid

Article abstract of DOI:10.1124/jpet.300.3.984

Arvanil, a structural "hybrid" between the endogenous cannabinoid CB₁ receptor ligand anandamide and capsaicin, is a potent agonist for the capsaicin receptor VR₁ (vanilloid receptor type 1), inhibits the anandamide membrane transpor

Full text of DOI:10.1124/jpet.300.3.984

0022-3565/02/3003-984–991$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 300:984–991, 2002
Vol. 300, No. 3
4497/965636
Printed in U.S.A.
A Structure/Activity Relationship Study on Arvanil, an
Endocannabinoid and Vanilloid Hybrid
VINCENZO DI MARZO, GRAEME GRIFFIN, LUCIANO DE PETROCELLIS, INES BRANDI, TIZIANA BISOGNO,
WILLIAM WILLIAMS, MARK C. GRIER, SANJITHA KULASEGRAM, ANU MAHADEVAN, RAJ K. RAZDAN, and
BILLY R. MARTIN
Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Pozzuoli (Napoli), Italy (V.D.M., I.B., T.B.); and Istituto di Cibernetica,
Consiglio Nazionale delle Ricerche, Comprensorio Olivetti, Pozzuoli (Napoli), Italy (L.D.P.); Department of Pharmacology and Toxicology,
Medical School of Virginia, Virginia Commonwealth University, Richmond, Virginia (G.G., B.R.M.); and Organix Inc., Woburn, Massachusetts
(W.W., M.C.G., S.K., A.M., R.K.R.)
Received August 30, 2001; accepted November 15, 2001
This article is available online at http://jpet.aspetjournals.org
ABSTRACT
Arvanil, a structural “hybrid” between the endogenous canna- 4-hydroxy group decreased the activity on VR1 and AMT, but
binoid CB1 receptor ligand anandamide and capsaicin, is a not the affinity for CB1 receptors, and increased the capability
potent agonist for the capsaicin receptor VR1 (vanilloid receptor to inhibit FAAH. The urea or thiourea analogs retained activity at
type 1), inhibits the anandamide membrane transporter (AMT), VR1 and AMT but exhibited little affinity for CB1 receptors. The
and induces cannabimimetic responses in mice. Novel arvanil urea analog was a potent FAAH inhibitor (IC50 ϭ 2.0 ␮M). A
derivatives prepared by N-methylation, replacement of the water-soluble analog of arvanil, O-2142, was as active on VR1,
amide with urea and thiourea moieties, and manipulation of the much less active on AMT and CB1, and more potent on FAAH.
vanillyl group were evaluated for their ability to bind/activate All compounds induced a response in the mouse “tetrad”,
CB1 receptors, activate VR1 receptors, inhibit the AMT and fatty particularly those with EC50 Ͻ10 nM on VR1. However, the
acid amide hydrolase (FAAH), and produce cannabimimetic most potent compound, N-NЈ-di-(3-chloro-4-hydroxy)benzyl-
effects in mice. The compounds did not stimulate the CB1 arachidonamide (O-2093, ED50 ϳ0.04 mg/kg), did not activate
receptor. Methylation of the amide group decreased the activity VR1 or CB1 receptors. Our findings suggest that VR1 and/or as
at VR1, AMT, and FAAH. On the aromatic ring, the substitution yet uncharacterized receptors produce cannabimimetic re-
of the 3-methoxy group with a chlorine atom or the lack of the sponses in mice in vivo.
Recent studies have revealed a certain overlap among the 2000). More recently, despite the fact that similar structural
binding sites for fatty acid derivatives of the cannabinoid CB1
receptor (for review, see Pertwee, 1997), the vanilloid recep-
tor type 1 (VR1) for capsaicin (Caterina et al., 1997; for
review, see Szallasi and Blumberg, 1999), and the membrane
transporter for the endocannabinoid arachidonoylethanol-
amide (AEA; Devane et al., 1992; for review, see Di Marzo et
al., 2000a). In particular, it was shown (Di Marzo et al., 1998)
that some long-chain derivatives of capsaicin, such as olvanil
(Dray, 1992), weakly bind to and activate the CB1 receptor
and competitively inhibit the AEA membrane transporter
(AMT), whereas AEA was found to act as a full, albeit weak,
agonist of VR1 receptors (Zygmunt et al., 1999; Smart et al.,
prerequisites are necessary for long-chain fatty acid deriva-
tives to interact with both the human VR1 and the AMT,
selective VR1 agonists and AMT competitive inhibitors could
be developed (De Petrocellis et al., 2000). CB1/VR1 hybrid
agonists were designed as possible analgesic, anti-inflamma-
tory and antitumor agents (Melck et al., 1999; Di Marzo et
al., 2000b, 2001b). For one of these hybrids, named arvanil
(Fig. 1), the chemical modification of the fatty acid chain,
and, particularly, the introduction of two methyl groups on
the C-16 and of a bromine group instead of the methyl group
on the C-20, led to a compound, O-1861 (Fig. 1), with nearly
the same activity on VR1 and CB1 receptors and high potency
in the mouse tetrad of behavioral tests of cannabimimetic
activity in vivo (Di Marzo et al., 2001b). These tests consist
of: 1) inhibition of spontaneous activity in an open field, 2)
rectal hypothermia, 3) analgesia in the tail-flick test, and 4)
Supported by Grant 3933 (to V.D.M.) from Ministero dell’Universita´ e della
Ricerca Scientifica e Technologica and National Institute on Drug Abuse
(DA-03672 and DA-08904 and DA-09789 to R.K.R.).
ABBREVIATIONS: VR1, vanilloid receptor type 1; hVR1, human VR1; CB1, cannabinoid receptor type 1; AMT, anandamide membrane transporter;
FAAH, fatty acid amide hydrolase; AEA, arachidonoylethanolamide (anandamide); BSA, bovine serum albumin; [35S]GTP␥S, guanosine 5Ј-O -(3-
35
[
S]thiotriphosphate); HEK, human embryonic kidney; %MPE, percent maximal possible effect; CP55940, (Ϫ)-cis-3-[2-hydroxy-4-(1,1-dimeth-
ylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclo-hesanol; SR141716A, N-piperidino-5-(4-chlorophenyl)1-(2,4-dichloro-phenyl)-4-methylpyrazole-
3-carboxamide; AM404, N-(4-hydroxyphenyl)-arachidonamide; VDM13, N-(5-methoxy-tryptomin)-arachidonamide.
984

Novel Arvanil Analogs
985
zaldehyde/CH3NH2⅐HCl/methanol/NaCNBH4 for the former and
3-chloro-4-hydroxybenzaldehyde/ammonium acetate/NaCNBH4/
methanol/mol.sieves 3A° for the latter. O-2093 was synthesized
from the appropriate amine, which was formed as a by-product
during the reductive amination of 3-chloro-4-hydroxybenzalde-
hyde described above. The urea analog O-1987 was prepared from
arachidonic acid, in a one-pot reaction, via its isocyanate followed
by treatment with 4-hydroxy-3-methoxybenzylamine HCl using
our published procedure (Ng et al., 1999). The thiourea O-2095
was synthesized by treatment of vanillyl isothiocyanate with
norarachidonylamine (synthesized from arachidonyl isocyanate
(Ng et al., 1999) and 2-trimethylsilylethanol/80°C/16 h/followed by
deprotection with CF3COOH at 0°C). Similarly, the thiourea
O-2109 was prepared using 3-chloro-4-hydroxybenzylisothiocya-
nate (prepared from 3-chloro-4-hydroxybenzylamine using the
same procedure as described for vanillyl isothiocyanate). O-2142
was synthesized from arvanil and 4-morpholinobutyric acid in
methylene chloride/N,NЈ-dicyclohexylcarbodiimide using our pro-
cedure (Razdan et al., 1976). All compounds were characterized on
the basis of their [1H] nuclear magnetic resonance spectra (run on
a Jeol Eclipse 300 MHz) and elemental analyses.
Fig. 1. Chemical structures of arvanil and O-1861, another hybrid ago-
nists of CB1/VR1 receptors. The chemical moieties targeted by chemical
changes in this study are shown by arrows.
Guanosine 5Ј-O-(3-[35 S]thiotriphosphate) ([35S]GTP␥S) was pur-
chased from PerkinElmer Life Sciences (Boston, MA). [3H]CP55940
was purchased from PerkinElmer Life Sciences. GDP and GTP␥S
were purchased from Roche Molecular Biochemicals (Summerville,
NJ). All other reagent grade chemicals and enzymes were obtained
from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pitts-
burgh, PA).
immobility on a ring. Although none of these behavioral
assays taken alone is indicative of a particular class of com-
pounds, a positive response in all four tests is considered to
be diagnostic of cannabimimetic activity (Martin et al., 1991).
However, it was found recently that capsaicin, which does
not activate CB1 receptors (Di Marzo et al., 1998), can also
elicit a response in the mouse tetrad tests and that this
natural product can induce immobility and hypolocomotion
in rats by acting on vanilloid receptors (Di Marzo et al.,
2000b; 2001c). Hence, the possible interference from VR1
receptors in a cannabimimetic response in these tests de-
serves further investigation, particularly in view of the fact
that AEA and some of its analogs (e.g., methanandamide;
Zygmunt et al., 1999; Ralevic et al., 2000) might activate both
CB1 and VR1 receptors at similar concentrations (for review,
see Di Marzo et al., 2001a).
No study so far has addressed the question of whether the
chemical modification of the amide and aromatic moieties of
arvanil leads to CB1/VR1 hybrid activators. Therefore, the
activity of eight novel compounds, obtained from arvanil by
modifying these two regions, was analyzed here on: 1) CB1
and VR1 receptors; 2) the AMT (Hillard and Jarrahian,
2000); and 3) the fatty acid amide hydrolase (FAAH). The
latter enzyme is responsible for AEA hydrolysis (Cravatt et
al., 1996; Ueda et al., 2000) and drives in part the activity of
the AMT in intact cells (Deutsch et al., 2001). We obtained
four new potent VR1 agonists that were 450- to 19,000-fold
selective over CB1 receptors. When tested in the mouse tet-
rad, these four compounds exhibited potent cannabimimetic
activity. Additionally, a compound inactive at both CB1 and
VR1 receptors was very potent in the in vivo mouse model.
We propose that non-CB1 receptors are also important in
determining high activity in the mouse tetrad tests.
Agonist-Stimulated [35S]GTP␥S Binding Assays. The hip-
pocampus of young adult rats, dissected on ice, was used for these
assays since this brain area exhibits a more efficacious coupling of
CB1 receptors to G-proteins than whole brain. Each hippocampus
was homogenized with a Tissumizer (Tekmar, Cincinnati, OH) in
cold membrane buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2
mM EGTA, 100 mM NaCl, pH 7.7) and centrifuged at 42,000g for 20
min at 4°C. Pellets were resuspended in membrane buffer, then
centrifuged again at 42,000g for 20 min at 4°C. Pellets from the
second centrifugation were homogenized in membrane buffer and
stored at Ϫ80°C. Frozen membranes were thawed and diluted in
membrane buffer, homogenized, and preincubated for 10 min at 30°C
in 0.004 units/ml adenosine deaminase (240 units/mg of protein;
Sigma Chemical Co.) to remove endogenous adenosine, then assayed
for protein content before addition to assay tubes. Assays were con-
ducted at 30°C for 1 h in membrane buffer, including 10 ␮g of
membrane protein with 0.1% (w/v) bovine serum albumin (BSA), 10
␮M GDP, and 0.1 nM [35S]GTP␥S in a final volume of 0.5 ml.
Nonspecific binding was determined in the absence of agonists and in
the presence of 30 ␮M unlabeled GTP␥S. Reactions were terminated
by rapid filtration under vacuum through Whatman GF/B glass fiber
filters, followed by three washes with cold Tris-HCl buffer, pH 7.4.
Bound radioactivity was determined by liquid scintillation spectro-
photometry at 95% efficiency for [35S] in 4 ml of BudgetSolve scin-
tillation fluid (Sigma-RBI, Natick, MA). Net agonist-stimulated
[
35S]GTP␥S binding values were calculated by subtracting basal
binding values (obtained in the absence of agonist) from agonist-
stimulated values (obtained in the presence of agonist). Data anal-
yses (including agonist concentration effect and competition curves)
were conducted by iterative nonlinear regression using Prism for
Windows (GraphPad Software, San Diego, CA) to obtain EC50, Emax
,
and Ki values. Significant stimulation of [35S]GTP␥S binding was
determined by analysis of variance followed by Dunnett’s test at the
p Ͻ 0.05 level to compare each concentration of ligand with basal
Materials and Methods
Synthesis and Chemicals. Arachidonyl analogs O-1986, binding. Data are expressed as means Ϯ S.E. of experiments per-
O-1988, and O-2094 were synthesized by treatment of the appro- formed in triplicate in membranes from at least three different
priate amines with the acid chloride of arachidonic acid as de-
scribed by us previously (Dasse et al., 2000). The amines used for
hippocampi.
CB1 Receptor Binding Assays. All experiments were performed
O-1988 and O-2094 were prepared by reductive amination proce- with whole brain membranes rather than hippocampal membranes,
dures (Abdel-Magid et al., 1996) using 3-methoxy-4-hydroxyben- and preparation of these membranes was the same as for the hip-

986
Di Marzo et al.
pocampus. Binding was initiated by the addition of 75 ␮g of whole
brain membranes to siliconized tubes containing [3H]CP55940 (1
nM), competing ligand (concentrations from 0.001–30 ␮M), 0.5%
(w/v) BSA, and a sufficient volume of buffer (membrane buffer minus
sodium chloride) to bring the total volume to 0.5 ml. The addition of
2 ␮M unlabeled CP55940 was used to assess nonspecific binding.
Membranes were then incubated at 30°C for 60 min. The reaction
was terminated by addition of ice-cold wash buffer (50 mM Tris-HCl,
0.5% BSA, pH 7.4) followed by rapid filtration under vacuum
through Whatman GF/B glass-fiber filters using a 96-well harvester
(Brandell, Gaithersburg, MD). The tubes were washed twice with 2
ml of ice-cold wash buffer, and the filters were rinsed twice with 4 ml
of wash buffer. Filters were placed into 7-ml plastic scintillation
vials, and 5 ml of BudgetSolve scintillation fluid was added. Bound
radioactivity was determined by liquid scintillation spectrophotom-
etry at 45% efficiency for [3H].
Pharmacological Effects in Mice. Cannabinoids were dissolved
in a 1:1:18 mixture of ethanol, Emulphor (North American Chemi-
cals, Cranbury, NJ), and saline for i.v. administration. The analogs
were administered to mice by tail-vein injection and evaluated for
their ability to produce hypomotility, hypothermia, and antinocicep-
tion. These pharmacological measures were determined in the same
mouse at a time when maximal activity was present. To measure
locomotor activity, mice were placed into individual photocell activity
chambers (11 ϫ 6.5 inches) 5 min after injection. Spontaneous activ-
ity was measured during the next 10-min period, and the number of
interruptions of 16 photocell beams per chamber was recorded. An-
tinociception was determined using the tail-flick reaction time to a
heat stimulus. Before vehicle or drug administration, the baseline
latency period (2–3 s) was determined. Tail-flick latency was as-
sessed once more 20 min after the injection, and the differences in
control and test latencies were calculated. A 10-s maximum latency
was used. Antinociception was expressed as %MPE as described
below. As for hypothermia, rectal temperature was determined prior
to vehicle or drug administration with a telethermometer (Yellow
Springs Instrument Co., Yellow Springs, OH) and a thermistor probe
(model YSI 400; Markson LabSales Inc., Hillsboro, OR) inserted at a
depth of 2 mm. Rectal temperature was measured again 30 min after
the injection, and the difference between pre- and postinjection val-
ues was calculated.
Cytosolic Calcium Concentration ([Ca2؉]i) Assay. Over-ex-
pression of human VR1 cDNA into human embryonic kidney (HEK)
293 cells was carried out as described previously (Hayes et al., 2000).
Cells were grown as monolayers in minimum essential medium
supplemented with nonessential amino acids, 10% fetal calf serum,
and 0.2 mM glutamine and maintained under 95% O2/5% CO2 at
37°C. The effect of the substances on [Ca2ϩ
] was determined by
i
using Fluo-3 (Molecular Probes, Eugene, OR), a selective intracellu-
lar fluorescent probe for Ca2ϩ (De Petrocellis et al., 2000; Smart et
al., 2000). Cells were transferred into six-well dishes coated with
poly-L-lysine (Sigma) 1 day prior to experiments and grown in the
culture medium mentioned above. On the day of the experiment, the
cells (50–60,000/well) were loaded for 2 h at 25°C with 4 ␮M Fluo-3
methylester in dimethyl sulfoxide containing 0.04% Pluoronic. After
loading, cells were washed with Tyrode’s solution, pH ϭ 7.4,
trypsinized, resuspended in Tyrode’s solution, and transferred to the
cuvette of the fluorescence detector (PerkinElmer LS50B) under
continuous stirring. Experiments were carried out by measuring cell
fluorescence at 25°C (␭EX ϭ 488 nm, ␭EM ϭ 540 nm) before and after
the addition of the test compounds at various concentrations. Data
are expressed as the concentration exerting a half-maximal effect
(EC50). The efficacy of the effect was determined by comparing it to
the analogous effect observed with 4 ␮M ionomycin.
Data Analysis. For production of hypomotility and hypothermia,
the data were expressed as percentage of control activity and change
in temperature, respectively. Antinociception was calculated as fol-
lows.
(test latency Ϫ control latency)
% MPE ϭ
ϫ 100
ͫ
ͬ
(10s Ϫ control latency)
At least six animals were treated with each dose so that dose-
response relationships could be determined for each analog. ED50
values were determined from least-squares unweighted linear re-
gression analysis of the log dose-response plots. Maximal effects for
all compounds combined on spontaneous activity, temperature, an-
tinociception, and catalepsy were, respectively, 90% inhibition,
Ϫ5°C, 100% MPE, and 60% immobility. Thus, the ED50 values indi-
cate response levels of 45% inhibition, Ϫ2.5°C, 50% MPE, and 30%
immobility.
Anandamide Membrane Transport Assay. The effect of com-
pounds on the uptake of [14C]AEA by rat basophilic leukemia (RBL-
2H3) cells was studied by using 3.6 ␮M (10,000 cpm) of [14C]AEA as
described previously (Bisogno et al., 1997). Cells were incubated with
Results
[
14C]AEA for 5 min at 37°C, in the presence or absence of varying
All compounds were found to exhibit low CB1 receptor
affinity and little efficacy for stimulating G-protein coupling
(Table 1). The methylation of the amide in arvanil (O-1988)
led to dramatic decreases in both the affinity and functional
activity for CB1 receptors. Deletion of the p-hydroxy-group on
the aromatic moiety (O-1986) resulted in similar decreases,
although some affinity for CB1 receptors was retained. Sub-
stitution of an m-chloro for the m-methoxy in arvanil led to
O-2094, a compound that had reasonable CB1 receptor affin-
concentrations of the inhibitors. Residual [14C]AEA in the incubation
medium after extraction with CHCl3/CH3OH 2:1 (by volume), deter-
mined by scintillation counting of the lyophilized organic phase, was
used as a measure of the AEA that was taken up by cells (De
Petrocellis et al., 2000). Previous studies (Bisogno et al., 1997) had
shown that after a 5-min incubation the amount of [14C]AEA that
disappeared from the medium of RBL-2H3 cells is found mostly
(Ͼ90%) as unmetabolized [14C]AEA in the cell extract. Nonspecific
binding of [14C]AEA to cells and plastic dishes was determined in the
presence of 100 ␮M AEA and was never higher than 30%. Data are ity and stimulated GTP␥S binding [Emaxϭ 15% stimulation
expressed as the concentration exerting 50% inhibition of AEA up-
take (IC50) calculated by GraphPad.
(8–20), EC50 ϭ 131 nM (5–1900), reversed by 2 nM
SR141716A] slightly less potently than arvanil. When a sec-
ond 3-chloro-(4-hydroxy)benzyl group was substituted on the
nitrogen, the resulting analog (O-2093) exerted a significant
inhibition of GTP␥S binding, which was not sensitive to the
CB1 antagonist SR141716A (2 nM, data not shown) or to the
FAAH inhibitor phenylmethylsulfonyl fluoride (50 ␮M, data
not shown).
Conversion of the amide group in arvanil to a urea in
O-1987 decreased both CB1 affinity and GTP␥S binding ac-
tivity. When the urea was changed to a thiourea (O-2095),
CB1 receptor affinity was reduced further. The introduction
Fatty Acid Amide Hydrolase Assay. The effect of compounds
on the enzymatic hydrolysis of AEA was studied as described previ-
ously (Di Marzo et al., 2001b), using membranes prepared from
frozen brains of CD rats (Charles River, France), incubated with the
test compounds and [14C]AEA (9 ␮M) in 50 mM Tris-HCl, pH 9, for
30 min at 37°C. [14C]Ethanolamine produced from [14C]AEA hydro-
lysis was measured by scintillation counting of the aqueous phase
after extraction of the incubation mixture with 2 volumes of
CHCl3/CH3OH 2:1 (by volume). Data are expressed as the concen-
tration exerting 50% inhibition of AEA uptake (IC50), calculated
by GraphPad.

Novel Arvanil Analogs
987
TABLE 1
Evaluation of arvanil analogs at CB1 receptors, VR1 receptors, AEA transport, and FAAHa
AEA Transport
Compound
CB1 Affinity Ki
nM
GTP␥S Binding (Emax
)
hVR1 Potency (EC50
nM
)
FAAH (IC50)
(IC50
)
␮M
␮M
250 Ϫ 2600b
182b
24b
0.5 Ϯ 0.2b
(75.4 Ϯ 4.7)b
3.6b
32.0b
2829 Ϯ 175
0
25.0 Ϯ 3.9 (69.1 Ϯ 5.2)
10.0 Ϯ 2.1
Ͼ50
484 Ϯ 17
274 Ϯ 19
0
63.0 Ϯ 10.1 (68.0 Ϯ 4.3)
10.0 Ϯ 1.4 (63.5 Ϯ 5.3)
27.3 Ϯ 3.5
19.0 Ϯ 2.1
18.2 Ϯ 2.8
4.5 Ϯ 0.7
15 (8 to 20)
1290 Ϯ 140
Ϫ30 (Ϫ11.3 to Ϫ39.9)
Ͼ50,000 (26.9 Ϯ 4.2)
11.5 Ϯ 1.3
Ͼ50
1718 Ϯ 200
8626 Ϯ 130
0
0
0.7 Ϯ 0.2 (80.6 Ϯ 8.3)
0.4 Ϯ 0.1 (72.1 Ϯ 4.8)
19.3 Ϯ 3.3
7.5 Ϯ 1.8
2.0 Ϯ 0.4
Ͼ50
1801 Ϯ 204
483 Ϯ 63
0
0
4.0 Ϯ 1.1
3.8 Ϯ 0.7
Ͼ50
0.6 Ϯ 0.2 (86.6 Ϯ 8.2)
30.0 Ϯ 4.3
9.1 Ϯ 1.7
a Affinity for CB1 receptors was measured with the Ki (nM) for the displacement of [3H]CP55940 from whole rat brain membranes. Efficacy at these as well as other
G-protein-coupled receptors was measured as the capability of stimulating the binding of [35S]GTP␥S to rat hippocampal membranes. Potency (nM) and efficacy (maximal
effect as percentage of the stimulation of the effect of 4 ␮M capsaicin) at human VR1 receptors were measured as the capability of enhancing [Ca2ϩ]i via HEK-hVR1 cells.
Data for VR1 and CB1 are means Ϯ S.D. of n ϭ 3. For GTP␥S binding, data are shown as means and range of n ϭ 3. The Emax and EC50 of the potent CB1 agonist WIN55,212-2
in the GTP␥S assay were 51% and 82 nM, respectively. For the previously described CB1/VR1 hybrid agonist, O-1861 (Fig. 1) Emax and EC50 were 32% and 70 nM, respectively.
The capability of the compounds to interact with the AMT and FAAH was assessed by determining their inhibitory effect on [14C]AEA uptake by RBL-2H3 cells, where the
AMT has been well characterized, and on [14C]AEA hydrolysis by rat brain membranes, where FAAH is quite abundant and uniquely responsible for anandamide
degradation. The inhibitory effects are expressed as IC50 and represent means Ϯ S.D., n ϭ 3.
b Values determined in previous studies (Melck et al., 1999; Di Marzo et al., 2000c; De Petrocellis et al., 2000).
All the novel compounds except one were quite potent and
of a chlorine atom in O-2095, which yielded O-2109, the
efficacious in the functional assay of VR1 activity performed
thiourea analog of O-2094, increased the activity of this com-
in this study (Table 1), where the capability of increasing the
pound in the CB1 binding assay but did not restore the
activity in the GTP␥S binding assay (Table 1). Finally, intro-
duction of a 4Ј-morpholinobutyryl group on the p-hydroxy-
benzyl group of arvanil, which yielded the water soluble
[Ca2ϩ]i was measured in HEK cells over-expressing the hVR1
receptor (De Petrocellis et al., 2000). In agreement with pre-
vious studies carried out with capsaicin (Walpole et al.,
1993a,b), the substitution of the m-methoxy group with a
chlorine atom (O-2094) and the methylation of the amide
group (O-1988) in arvanil decreased its potency at hVR1 by
25- to 50-fold. Introduction of a second N-(3-chloro-4-hy-
droxy)-benzyl group (O-2093) abolished the activity at VR1.
Introduction of an ␣ amide group (O-1987), and further sub-
stitution of the carbonyl for a CϭS group (O-2095) did not
alter arvanil efficacy/potency at VR1. The importance of the
p-hydroxybenzyl group for the functional activation of these
compound O-2142, again did not greatly influence the affin-
ity for CB1 receptors. This might be the result of the hydro-
lysis of the ester bond during the binding assay (see also
below). However, it should be noted that: 1) O-2142 was
inactive in the GTP␥S binding assay, which is also performed
with membrane preparations, and 2) a similar chemical mod-
ification in (R)-methanandamide decreased its affinity for
CB1 receptors about 20-fold (from 20–426 nM, data not
shown).

988
Di Marzo et al.
receptors is underlined by the observation that O-1986 was pounds were not significant (Table 1). However, if one ex-
more than 100-fold less potent than arvanil, whereas the role cludes O-2142, whose activity at VR1 might have been due to
of the m-methoxy group in the correct interaction with VR1 hydrolysis to arvanil, one of the compounds (O-1986) with the
was confirmed by the finding that O-2109 was 10-fold less lowest activity on the AMT exhibited also low potency at VR1,
potent than O-2095 (Table 1). Finally, and surprisingly, whereas O-2095 and O-2109 were quite potent as both VR1
O-2142 was as potent as arvanil in inducing a VR1-mediated agonists and AMT inhibitors. In fact, the IC50 of the widely
increase of [Ca2ϩ]i in HEK-hVR1 cells. Since 1) this com- used AMT inhibitor, AM404 (Khanolkar and Makriyannis,
pound did not appear to be a good substrate for the AMT (see 1999) was 8.1 Ϯ 2.6 ␮M under our conditions. Another ex-
below), 2) AMT mediated facilitated transport into HEK- ception to the rule was O-2093, whose inhibitory activity on
hVR1 cells is important to observe high potency at hVR1 (Di AEA uptake (IC50 ϭ 11.5 Ϯ 1.3 ␮M) was not surprising since
Marzo et al., 2001a), and 3) the p-hydroxy group is funda- the AMT, unlike VR1, has been shown to bind also to amides
mental for interaction with vanilloid receptors (see above and of arachidonic acids with very hindering aromatic groups
Walpole et al., 1993b), this finding suggests that O-2142 is (Jarrahian et al., 2000). As a consequence of these findings,
hydrolyzed by cells prior to its interaction with VR1.
only one AMT inhibitor (O-2093) with activity on VR1 much
The order of potency of the novel compounds as AMT lower than that observed for AM404 in previous studies
inhibitors (O-2109 Ͼ O-2095 Ͼ O-1988 Ͼ O-2093 Ͼ O-2094 ϭ (EC50 ϭ 26–32 nM; De Petrocellis et al., 2000) was found in
O1987 Ͼ O-1986 Ͼ O-2142) was slightly different from the this study. This suggests that a way of obtaining AMT inhib-
order of potency at hVR1 (O-2095 Ն O-2142 Ն O-1987 Ͼ itors selective versus VR1 is to condense arachidonic acid
O-2109 Ͼ O-2094 Ͼ O-1988 Ͼ O-1986 Ͼ O-2093), although in with hindering aromatic moieties, as in the case of O-2093,
most cases the differences between the activities of the com- VDM13, and, possibly, other previously described arachido-
TABLE 2
Effect of arvanil derivatives in the mouse tetrad of behavioral tests
The ED50 (milligrams per kilogram, i.v.) dose for inhibition of sector crossings (spontaneous activity) in an open field, antinociception (delay in seconds in the tail-flick
response), decrease of rectal temperature in degrees Celsius, and induction of time spent in immobility on a ring are shown. Data are means (and ranges) of n Ն six animals.
Compound
Spontaneous Activity
T.F.
Rectal Temperature
R.I.
Average
5.98
3.11
(1.54–6.29)
7.98
(5.17–12.34)
8.74
(5.61–13.60)
4.08
(2.99–5.56)
4.74
(3.17–7.09)
6.15
6.15
6.13
5.80
1.68
0.04
(4.63–8.16)
(3.22–11.73)
(4.79–7.83)
0.52
(0.38–0.71)
0.49
(0.35–0.67)
3.77
(1.21–11.76)
1.93
(1.56–2.39)
0.05
(0.04–0.07)
0.04
(0.03–0.05)
0.04
(0.02–0.06)
0.04
(0.03–0.05)
0.06
(0.04–0.08)
0.12
(0.08–0.17)
0.08
(0.03–0.20)
0.09
(0.06–0.12)
0.09
0.02
0.08
0.03
0.08
0.05
0.15
(0.02–0.03)
(0.06–0.10)
(0.02–0.07)
(0.05–0.12)
0.02
(0.02–0.03)
0.09
(0.06–0.12)
0.33
(0.02–5.06)
0.15
(0.10–0.23)
0.20
(0.16–0.24)
0.19
(0.15–0.25)
0.11
(0.07–0.17)
0.17
(0.13–0.22)
0.17
T. F., delay in seconds in the tail-flick response; R.I., induction of time spent in immobility on a ring.

Novel Arvanil Analogs
989
TABLE 3
Lack of effect by SR141716A (3 mg/kg, administered i.v. 10 min prior to compounds) on the hypolocomotor and antinociceptive effects of select
arvanil derivatives in mice
No inhibitory effect by SR141716A was observed on the hypothermia induced by O-2093 and O-2094 under similar conditions (not shown). Data are means Ϯ S.E. of n Ն
six animals.
Spontaneous Activity (Beam Interruptions)
Vehicle ϩ Compound SR141716A ϩ Compound
Tail-Flick Latency (%MPE)
Dose
Vehicle ϩ Compound
SR141716A ϩ Compound
mg/kg
O-1988
O-2093
0
3
10
0
0.03
0.056
0.1
0
1224 Ϯ 94
766 Ϯ 157
179 Ϯ 34
1288 Ϯ 87
1198 Ϯ 102
529 Ϯ 110
120 ϩ 35
1095 Ϯ 93
83 Ϯ 17
1188 Ϯ 142
689 Ϯ 68
117 Ϯ 17
1452 Ϯ 171
1461 Ϯ 131
71 Ϯ 25
264ϩ155
2034 Ϯ 182
116 Ϯ 21
52 Ϯ 19
4 Ϯ 2
54 Ϯ 9
100
4 Ϯ 1
46 Ϯ 15
73 Ϯ 10
94ϩ5
9 Ϯ 2
94 Ϯ 5
100
21 Ϯ 3
67 Ϯ 15
100
31 Ϯ 3
51 Ϯ 5
97 Ϯ 3
94ϩ6
19 Ϯ 5
100
96 Ϯ 4
O-2094
1
3
118 Ϯ 16
nate derivatives (De Petrocellis et al., 2000; Jarrahian et al., the effect of O-2094 (either 1 or 3 mg/kg, i.v.) and of O-2093
2000). (0.03, 0.056, and 0.1 mg/kg, i.v.) in the spontaneous activity,
Unlike arvanil and its derivatives obtained through the tail-flick, and rectal temperature tests, and of O-1988 (either
modification of the aliphatic moiety (Melck et al., 1999; Di 3 or 10 mg/kg, i.v.) in the spontaneous activity and tail-flick
Marzo et al., 2001b), analogs obtained from the substitution tests were not affected by a 10 min pretreatment of mice with
of the m-methoxy group for a chlorine atom (O-2094) or from SR141716A (3 mg/kg, i.v.) (Table 3 and data not shown).
the introduction of an amide ␣ to the carbonyl (O-1987) are
potent FAAH inhibitors. Also, elimination or derivatization
of the p-hydroxy group, as in O-1986 and O-2142, respec-
Discussion
tively, slightly increases the affinity for FAAH. Given the
None of the novel arvanil analogs described here bound to
esterase activity of FAAH (Goparaju et al., 1998), it is possi- the CB1 receptor with high affinity. However, some general
ble that the enzyme recognizes O-2142 as a better substrate conclusions emerge. Since O-2094 exhibited an affinity for
due to the presence of the ester, rather than the amide, bond. CB1 receptors similar to that previously observed for arvanil,
Conversely, modification of one of those chemical moieties it is possible to conclude that the presence of an m-methoxy
that were previously shown to confer to AEA derivatives the group in the latter molecule is not crucial for the functional
capability of interacting with the enzyme, e.g., the carbonyl interaction with the central cannabinoid receptor. Con-
group (Lang et al., 1999), as in O-2095 versus O-1987, abol- versely, the derivatization of the amide in arvanil, as in
ished inhibitory activity (Table 1). Our data also indicate that O-1988 and O-2093, and the lack of the para-hydroxy group
the carbonyl group is such a necessary requisite for interac- on the aromatic moiety, as in O-1986, led to dramatic
tion with FAAH that its elimination in O-1987 cannot be changes in both the affinity for, and functional activity at,
compensated for by the presence of the m-chlorine atom in CB1 receptors. In fact, previous studies showed that a
the vanillyl moiety (O-2109). Another important requisite is secondary amide group in AEA derivatives is fundamental
the presence of a secondary or primary amide (Lang et al., for interaction with cannabinoid receptors (for reviews, see
1999; Boger et al., 2000), and in fact O-1988 and O-2093 were Khanolkar and Makriyannis, 1999; Martin et al., 1999).
even less potent inhibitors of FAAH activity than arvanil These findings are also in agreement with previous data
(Table 1). Finally, the finding that O-1988, O-2095, and showing that the carbonyl function in AEA is important for
O-2109 are all much more potent as AMT than as FAAH the interaction with CB1 binding site (Khanolkar and
inhibitors confirms that AEA transport into cells is not Makriyannis, 1999).
uniquely driven by FAAH activity (Day et al., 2001; Deutsch
Although a certain overlap in the ligand recognition prop-
et al., 2001), because substances that inhibit this process erties between CB1 and VR1 receptors can be observed, the
without significantly affecting AEA hydrolysis can be found. chemical requisites for the optimal interaction of arvanil
Of the eight novel compounds tested in this study, and with analogs with the binding sites within each receptor class are
only one exception (O-2093), only those with a threshold different. In particular, the p-hydroxy and m-methoxy groups
potency at VR1 receptors of 10 nM exhibited very strong on the vanillyl moiety are important for the interaction with
activity (average ED50 Ͻ 1 mg/kg) in the mouse tetrad of VR1 but not so much with CB1 receptors. Conversely, a
tests (Table 2). Usually, a positive response (inhibition of carbonyl function on C-1 and a methylene group on C-2 in
locomotor activity, induction of immobility, antinociception, arvanil are important to achieve high affinity for CB1 recep-
and hypothermia) in all four tests is considered highly indic- tors but can be substituted for CϭS and NH groups, respec-
ative of cannabimimetic activity (Martin et al., 1991). Yet, tively, without modifying the efficacy at VR1. On the other
none of the arvanil analogs tested here bound with very high hand, it must be noted that the presence of a 20-carbon atom
affinity to CB1 receptors or exhibited high efficacy in the polyunsaturated chain (Melck et al., 1999; De Petrocellis et
GTP␥S binding assay. Conversely, they displayed very high al., 2000) and a secondary amide group (this study) are
potency/efficacy at human VR1 receptors, although their important for an optimal interaction with both receptor
EC50 values for VR1 activation did not appear to correlate classes.
linearly with the ED50 values observed in vivo. At any rate,
Although the overlap between the AMT and VR1 ligand

990
Di Marzo et al.
recognition properties is supported to a great extent by the studied here, four were potent VR1 agonists with 450- to
present findings, rather surprising data emerged here on the 19,000-fold selectivity versus CB1 receptors. More impor-
capability of some of the novel analogs to inhibit FAAH. In tantly, we have provided unprecedented evidence that a
general, it can be concluded from our findings that, despite strong cannabimimetic response in the mouse tetrad of neu-
the relatively high potency of arvanil as an AMT inhibitor, robehavioral tests in vivo can also be indicative of potent
the types of modifications of the amide and aromatic moieties activity at VR1 as well as at yet-to-be characterized non-CB1,
made here on arvanil do not confer to this compound any non-VR1 brain receptors. This latter finding should be taken
further selectivity for the AMT versus VR1 or FAAH.
into account when interpreting pharmacological data ob-
The observation that: 1) capsaicin exhibits a certain albeit tained with this widely used paradigm of cannabinoid activ-
more limited activity in some of the tetrad tests (Di Marzo et ity.
al., 2000b), 2) arvanil analogs are very potent in this mouse
model (Di Marzo et al., 2001b), and 3) an 18-carbon atom
Acknowledgments
unsaturated capsaicin analog, livanil, inhibits locomotor ac-
tivity in rats (Di Marzo et al., 2001c), might suggest that
activation of VR1 is also involved in inducing cannabimimetic
responses in these assays. This suggestion is strengthened by
our present observation that the novel compounds with very
high potency at hVR1 are also the most potent in the mouse
tetrad. Since AEA also activates VR1 receptors with a po-
tency that may depend on several regulatory factors (Di
Marzo et al., 2001a), it is possible that these sites also par-
ticipate in AEA actions in the mouse tetrad tests, actions that
cannot be reversed by a CB1 receptor antagonist (Adams et
al., 1998). Another possible explanation is that non-CB1,
non-VR1 cannabinoid receptors (for example, see Di Marzo et
al., 2000c; Breivogel et al., 2001) are involved in the effects of
arvanil-related compounds and, to some extent, of AEA in
these four behavioral assays. In fact, several pharmacological
actions of arvanil do not appear to be sensitive to effective
doses of the CB1 antagonist SR141716A or of the VR1 antag-
onist capsazepine (Di Marzo et al., 2000b; V. Di Marzo, un-
published data). In support to this hypothesis, we have found
here that: 1) the effects in some of the tetrad tests of three
compounds with low, intermediate, and high potency, i.e.,
O-1988, O-2094, and O-2093, respectively, were not antago-
nized by SR141716A, and 2) O-2093 was one of the most
potent compounds ever found in the mouse tetrad (average
ED50 ϳ0.04 mg/kg) and yet it exhibited very little affinity for
CB1 receptors and almost no potency/efficacy at hVR1 recep-
tors in vitro. The inhibitory effect by O-2093 of endocannabi-
noid uptake, with a possible subsequent increase of endoge-
nous cannabinoid tonic activity in the tetrad tests, is unlikely
to explain O-2093 high potency in vivo. In fact, other equipo-
tent AMT and/or FAAH inhibitors in this study (e.g., O-1988
or O-2094) were active in the mouse tetrad only at 50- to
100-fold higher doses, and furthermore, a putative “indirect”
activation of CB1 receptors by these compounds would have
been blocked by SR141716A. The behavioral effects of
O-2093, therefore, might be mediated by non-CB1, non-VR1
sites of action specific for arvanil-like compounds, possibly
via an inverse agonist effect on G-protein-coupled receptors,
since O-2093 was found to inhibit GTP␥S binding to hippocam-
pal membranes in a manner insensitive to SR141716A. Al-
though the in vivo activity of O-2093 was not blocked by
SR141716A, an effect for this compound as a prodrug at either
CB1 or VR1 receptors still cannot be excluded.
We are grateful to Aniello Schiano Moriello for technical assis-
tance.
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Address correspondence to: Dr. Vincenzo Di Marzo, Endocannabinoid Re-
search Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle
Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, Fabbricato 70, 80078
Pozzuoli (Napoli), Italy. E-mail: vdimarzo@icmib.na.cnr.it