Design, synthesis and biological evaluation of novel arachidonic acid derivatives as highly potent and selective endocannabinoid transporter inhibitors

Article abstract of DOI:10.1021/jm015545y

In the present work, we have designed and synthesized a series of arachidonic acid derivatives of general structure I which have been characterized as highly potent and selective inhibitors of anandamide transporter (IC₅₀ = 24-0.8 μM, K_i > 1000-5000 nM for CB₁ and CB₂ cannabinoid receptors and vanilloid VR₁ receptor). Among them, N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide deserves special attention as being the most potent endocannabinoid transporter inhibitor (IC₅₀ = 0.8 μM) described to date.

Full text of DOI:10.1021/jm015545y

J . Med. Chem. 2001, 44, 4505-4508
4505
cellular proliferation, among others. This broad spec-
trum of action makes the ECS an important therapeutic
target for the treatment of diverse pathologies,12,13
including asthma, pain, multiple sclerosis, malignant
gliomas, and neurodegenerative diseases.
Design , Syn th esis a n d Biologica l
Eva lu a tion of Novel Ar a ch id on ic Acid
Der iva tives a s High ly P oten t a n d
Selective En d oca n n a bin oid Tr a n sp or ter
In h ibitor s
Recent findings suggest that in mammalian cells an
increased level of endocannabinoids can be obtained by
inhibiting its uptake and/or degradation, raising the
possibility of producing local cannabimimetic effects
without directly activating cannabinoid receptors with
classic agonists and therefore avoiding their associated
undesirable side effects. On this basis, such synthetic
inhibitors may be of potential therapeutic value for the
treatment of disorders characterized by a low endocan-
nabinoid activity and where direct agonists have proven
to be effective, yet produce other undesirable effects.14
In particular, the therapeutic utility of such uptake
inhibitors has been considered for the treatment of
diverse pathologies as Huntington’s chorea13 or multiple
sclerosis.15 However, the number of compounds with a
demonstrated capacity to increase the endocannabinoid
tonus by blocking the mechanism of termination of their
biological activity is limited.16-20 Among them, the N-(4-
hydroxyphenyl)arachidonamide (AM404) is a unique
compound that has been extensively studied due to its
ability to inhibit the transporter in a potent and selec-
tive way,7 although recent results have revealed an
agonist activity of AM404 at vanilloid VR1 receptors.20-22
Additionally, the lack of molecular characterization of
the transporter and the scarce structure-affinity rela-
tionship (SAFIR) studies reported to date16,17 make
studies regarding the structural features of the carrier
involved in the recognition and translocation of sub-
strates an important area of research aimed at provid-
ing new potential therapeutic agents for the treatment
of hypofunctionalities of the ECS. The intracellular fate
of these uptake inhibitors is an additional point to be
taken into consideration due to the possibility of pro-
ducing toxic metabolites through enzymatic hydrolysis.
For example, it has been described that some amino-
phenols, which could be produced as possible metabo-
lites of AM404 hydrolysis, generate methemoglobin both
in vivo and in vitro.23 Thus, there remains a need for
the design of selective inhibitors.
Mar´ıa L. Lo´pez-Rodr´ıguez,*,§ Alma Viso,§
Silvia Ortega-Gutie´rrez,§ Isabel Lastres-Becker,‡
Sara Gonza´lez,‡ J avier Ferna´ndez-Ruiz,‡ and
J ose´ A. Ramos‡
Departamento de Quı´mica Orga´nica I, Facultad de Ciencias
Quı´micas, and Departamento de Bioquı´mica y Biologı´a
Molecular III, Facultad de Medicina, Universidad
Complutense, 28040 Madrid, Spain
Received J une 19, 2001
Abstr a ct: In the present work, we have designed and syn-
thesized a series of arachidonic acid derivatives of general
structure I which have been characterized as highly potent
and selective inhibitors of anandamide transporter (IC50 ) 24-
0.8 µM, Ki > 1000-5000 nM for CB1 and CB2 cannabinoid
receptors and vanilloid VR1 receptor). Among them, N-(3-
furylmethyl)eicosa-5,8,11,14-tetraenamide deserves special at-
tention as being the most potent endocannabinoid transporter
inhibitor (IC50 ) 0.8 µM) described to date.
In tr od u ction . The different elements of the endog-
enous cannabinoid system (ECS) constitute a variety of
pharmacological targets for the broad group of com-
pounds generally termed as cannabinoids. Included
among these elements are two types of G-protein-
1
coupled membrane receptors (the central CB1 and the
2
peripheral CB2 ), the endogenous ligands anandamide,3
2-arachidonoylglycerol,4 and the recently reported
2-arachidonyl glyceryl ether,5 as well as a mechanism
for the termination of biological activity of these com-
pounds composed of a carrier-mediated transport system
(ANT)6-8 and a hydrolyzing enzyme, named fatty acid
amide hydrolase (FAAH).9 Cannabinoid agonists include
both exogenous active molecules as well as endocan-
nabinoids. Exogenous agonists are usually classified as
classical cannabinoids (Cannabis sativa derived com-
pounds as, for example, ∆9-THC and their analogues),
nonclassical cannabinoids (which lack the characteristic
tricyclic structure of classical ones, as, for instance,
CP55940), and aminoalkylindoles (WIN552122 being
the most representative), whereas endogenous cannab-
inoids belong to the eicosanoid class. Among the an-
tagonists, diarylpyrazoles merit special mention as
being the most widely used compounds.10
In this line, we have designed and synthesized a
series of arachidonic acid derivatives of general struc-
ture I which have proved to be highly potent and
selective inhibitors of anandamide reuptake24 (Table 1).
In these derivatives we have analyzed the effect of the
replacement of ethanolamine moiety of anandamide
with a fragment containing a five-membered ring with
one heteroatom. Thus, we have evaluated the influence
of several factors such as aromaticity, position of the
arachidonic chain, as well as the nature of the hetero-
atom. Also, we have compared amides with its isosteric
corresponding esters in order to assess the ability of the
carrier to uptake both types of endocannabinoids. In
particular, compound 9 deserves special attention as
being the most active derivative with an IC50 value of
0.8 µM, which makes it the most potent uptake inhibitor
described to date.
The ECS seems to be involved in the regulation of a
wide variety of central and peripheral processes11 such
as antinociception, brain development, retrograde neu-
ronal communication, memory, appetite, psychomotor
control, cardiovascular and immune regulation, and
* Corresponding author. Phone: 34-91-3944239. Fax: 34-91-
3944103. E-mail: mluzlr@quim.ucm.es.
§ Departamento de Qu´ımica Orga´nica I, Universidad Complutense.
‡ Departamento de Bioqu´ımica y Biolog´ıa Molecular III, Universidad
Complutense.
10.1021/jm015545y CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/17/2001

4506 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Ta ble 1. Inhibition of ANT and Binding Data of Compounds I
Letters
Sch em e 1. Synthesis of Compounds of General
Structure Ia
a
Reagents: (a) oxalyl chloride, DMF, CH2Cl2, rt; (b) HX-CH2-
HM, HM ) heterocyclic moiety.
amination in 28% ammonium hydroxide solution using
Raney nickel as catalyst.26
All new compounds were assessed for their ability to
inhibit [3H]anandamide uptake in human lymphoma
U937 cells, for their affinity for CB1 and CB2 cannab-
inoid receptors in radioligand binding assays using [3H]-
WIN552122 in rat cerebellum membranes and [3H]-
CP55940 in HEK293EBNA human CB2 receptor trans-
fected cells, respectively, as well as for their affinity for
the vanilloid VR1 receptor in radioligand binding assays
using [3H]Rexiniferatoxin ([3H]RTX) in rat spinal cord
membranes (Table 1). For comparison purposes and as
reference values, we have also included results obtained
for AM404, anandamide, RTX, and for the two proto-
typical cannabinoid ligands WIN552122 and CP55940.
Most of compounds I display an excellent ability to
inhibit anandamide reuptake with IC50 values in the
low micromolar range as well as selectivity for the
transporter, as deduced from their affinity constants for
both CB1 and CB2 receptors (Ki > 1000 nM) as well as
for VR1 receptors (Ki > 5000 nM). Well-characterized
cannabinoid10 or vanilloid ligands such as WIN552122
and CP55940 or RTX, respectively, exhibit Ki values in
the low nanomolar or even in the picomolar range, as
shown in Table 1.
In particular, special attention should be paid to
compounds 6 and 9 which exhibit the best IC50 values
(3 and 0.8 µM, respectively).
The compounds described here allow us to ascertain
the effect of the heterocyclic moiety on the transporter
affinity as well as affinity for cannabinoid receptors.
With respect to the ANT, the most important points can
be summarized as follows:
(i) The position of the arachidonic chain attached to
the C-2 or the C-3 of the heterocyclic ring seems to exert
a different influence depending on the heterocycle. Thus,
in case of the furan ring it does not seem to play a
relevant role in affinity for the transporter when esters
are considered (IC50 (1) ) 24 µM; IC50 (2) ) 14 µM),
whereas in amides (compound 8 vs 9) this change
improves the affinity more than 6 times. Furthermore,
this substitution pattern has a crucial influence in the
affinity of thienyl derivatives (compound 5 vs 6) as well
as in the behavior of tetrahydrofuryl derivatives (com-
pound 3 vs 4).
(ii) Regarding esters, when the arachidonic chain is
attached to the 3-position of the heterocyclic ring,
replacement of the furan with a thiophene ring improves
more than 4 times the affinity for the transporter (IC50
(2) ) 14 µM; IC50 (6) ) 3 µM), whereas when the
arachidonic chain is at the C-2 position, the same
isosteric change implies a complete loss of affinity
(compound 1 vs 5). However, in the case of amides, the
inhibitory potency is comparable regardless of the
a
Inhibition of anandamide transport was determined using
human lymphoma U937 cells and [3H]anandamide. Affinity of
b
compounds for the CB1 receptor was evaluated using rat cerebel-
lum membranes and [3H]WIN552122. c Affinity of compounds for
the CB2 receptor was assayed using [3H]CP55940 in HEK293EBNA
human CB2 receptor transfected cells. Ki and IC50 values were
obtained from two or three independent experiments, respectively,
carried out in triplicate and are expressed as the mean ( standard
d
error. This compound presented a strange profile which did not
fit the sigmoid dose-response, characteristic of the rest of the
compounds. e Affinity of compounds for the VR1 receptor was
evaluated using rat spinal cord membranes and [3H]RTX. f These
compounds exhibited a partial capacity to displace [3H]RTX (Ki <
5000 nM).
The present study explores the structural features
involved in the recognition of substrates for the en-
docannabinoid transporter in a SAFIR study focused on
the influence exerted by the heterocyclic moiety on the
affinity for the carrier.
R esu lt s a n d Discu ssion . The synthesis of the
amides and esters of general structure I listed in Table
1 is detailed in Scheme 1. In general, these compounds
were prepared from arachidonic acid by treatment of
the acyl chloride25 with the appropriate amine or
alcohol. The noncommercial amines were prepared from
the corresponding aldehydes by catalytic reductive

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4507
heteroatom of the heterocycle, as deduced from IC50
values for compounds 7, 8, and 11.
slight increase of Ca2+ could trigger the release of a
potent ligand of VR1 (as anandamide, for example)
which would be responsible for the activation of all VR1
receptors and therefore for the great increase in the
intracellular levels of Ca2+, which are measured in
agonist assays.
The capacity of analogues 2 and 8 to displace [3H]-
RTX at its binding sites and also to inhibit the ANT
further support the existence of a partially common
pharmacophore between these two targets.20
(iii) With respect to the aromaticity of the heterocyclic
moiety, when the arachidonic chain is attached to the
C-2 position, the equipotent IC50 values observed for
compounds 1 vs 3 and 8 vs 10 indicate that the presence
of an aromatic fragment is not essential for affinity.
However, when the arachidonic chain is attached to the
C-3 position (compound 2 vs 4), aromaticity becomes an
important factor in the inhibitory trend observed, which
does not fit the sigmoid dose-response typical of the
rest of compounds. This rare behavior may reveal the
existence of different mechanisms of interaction or
recognition between the transporter and its substrates
or involve some other explanation, such as an additional
interaction with the new specific 2-arachidonoylglycerol
transporter recently described and which is not inhib-
ited by AM404.27
From all the above considerations, it should be
stressed that the small variations in the heterocyclic
moiety reported here have allowed us to obtain different
selectivities and affinities in the various test systems.
Also, affinity values of compound 7 at CB1 and CB2
cannabinoid receptors and the affinity value of com-
pound 9 at CB2 seem to suggest that the receptors and
the transporter share some of the requirements involved
in recognition and binding of substrates but also that
they indicate the existence of significant differences
which could be used to develop future ligands with
enhanced affinity, efficacy, and selectivity for the vari-
ous proteins that constitute the ECS. Furthermore, the
fact that compound 9, the most potent derivative in the
transporter, seems to bind with moderate affinity only
to CB2 raises the possibility of selectively activating this
receptor and simultaneously inhibiting the metabolism
of endocannabinoids, with all the interesting and at-
tractive therapeutic applications that this implies in
search of new agents capable of increasing the physi-
ological levels of anandamide but devoid of non desired
psychoactive effects of CB1 ligands.
With all these points taken into consideration, further
research is still required to elucidate the nature of the
interaction of the new compounds with biological targets
such as the FAAH and VR1 to determine the extent of
their common ligand recognition properties, and this is
currently under investigation in our laboratory.
In conclusion, in the present work we have synthe-
sized new arachidonic acid derivatives which act as
potent inhibitors of anandamide reuptake with IC50
values similar or even better than previously reported
inhibitors (AM404). Particularly, special attention should
be paid to compound 9 whose excellent IC50 value (IC50
) 0.8 µM) makes it a valuable candidate for future
pharmacological studies.
(iv) In general, carboxamide and carboxylate groups
are able to compete with [3H]anandamide for transport.
Comparison between furan esters and amides suggests
that the presence of the NH group enhances the affinity
for the carrier, as shown from the IC50 values for
analogues 3 vs 10 (about 2-fold), 1 vs 8 (5-fold), and
especially 2 vs 9 (nearly 20-fold). Regarding the thienyl
derivative 5, the substitution of the ester for the amide
bond allows recovery of its inhibitory ability, leading to
11 (IC50 ) 5.7 µM).
(v) With respect to cannabinoid receptors, most of
compounds analyzed here turned out to have little
affinity for both CB1 and CB2 (Ki > 1000 nM). However,
it should be pointed out that the presence of N-methyl
pyrrole fragment dramatically increases affinity for both
cannabinoid receptors as deduced from the Ki values
obtained for 7.
(vi) Regarding the interaction of compounds with
vanilloid VR1 receptors, the data obtained here show
that most of the compounds synthesized are essentially
unable to bind to VR1 (Ki > 5000 nM), especially when
comparing these data with the Ki values of high affinity
VR1 ligands such as RTX (Kd ) 25 pM,28 which is in
accordance with the value determined in our experi-
ment). The fact that AM404 appears to be devoid of
affinity for VR1 receptors seems to be in apparent
contradiction with previously published results19-21
which describe this inhibitor as a potent agonist at VR1
receptors. However, it should be taken into account that
these studies reflect a measure of agonist activity by
evaluating the Ca2+ influx within the cell and that this
action is determined using concentrations of AM404 or
of the other compounds tested as potential agonists in
the micromolar range (3-10 µM).20-22 Another impor-
tant point that deserves consideration is the difference
existing between the model most widely used (cells
transfected with and overexpressing hVR1)20-22 and the
one described here (rat spinal cord membranes). Finally,
it is also possible that an alternative mechanism could
explain these different observations since neither the
interaction between both systems (VR1 and ANT) nor
the physiological functions, endogenous ligands, and
transduction mechanisms of vanilloid receptor have
been completely elucidated. Thus, perhaps compounds
with low affinity for the VR1 receptors (such as AM404)
provoke a slight Ca2+ influx within the cell. Then, this
Results of these studies may help to develop insights
in this emerging area of interest in order to further
explore the molecular features involved in the recogni-
tion and/or translocation of substrates. Ultimately, new
agents may be identified (such as compound 9) which
could be useful not only as tools to improve our
understanding of the ECS but also as novel therapeutic
approaches for the treatment and cure of a broad range
of pathological dysfunctions.
Ack n ow led gm en t. This work was supported by
grants from DGESIC (PB97-0282) and CAM-PRI (08.5/
0029/98). The authors thank MECD for a predoctoral
grant to S.O.G.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and NMR data are available free of charge via the
Internet at http://pubs.acs.org.

4508 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Letters
Guzma´n, M. Antitumoral Action of Cannabinoids: Involvement
of Sustained Ceramide Accumulation and Extracellular Signal-
Regulated Kinase Activation. Nat. Med. 2000, 6, 313-319. (d)
Pertwee, R. G. Pharmacology of Cannabinoid Receptor Ligands.
Curr. Med. Chem. 1999, 6, 635-664.
Refer en ces
(1) Matsuda, L. A.; Lolait, S. J .; Brownstein, M. J .; Young, A. C.;
Bonner, T. I. Structure of a Cannabinoid Receptor and Func-
tional Expression of the Cloned cDNA. Nature 1990, 346, 561-
564.
(2) Munro, S.; Thomas, K. L.; Abu-Shaar, M. Molecular Character-
ization of a Peripheral Receptor for Cannabinoids. Nature 1993,
365, 61-65.
(13) Lastres-Becker, I.; Hansen, H. H.; Berrendero, F.; de Miguel,
R.; Pe´rez-Rosado, A.; Manzanares, J .; Ramos, J . A.; Ferna´ndez-
Ruiz, J . J . Loss of Cannabinoid CB1 Receptors and Alleviation
of Motor Hyperactivity and Neurochemical Deficits by Endocan-
nabinoid Uptake Inhibition in a Rat Model of Huntington’s
Disease. Neuroscience In press.
(14) Piomelli, D.; Giuffrida, A.; Calignano, A.; Rodr´ıguez de Fonseca,
F. The Endocannabinoid System as a Target for Therapeutic
Drugs. Trends Pharmacol. Sci. 2000, 21, 218-224.
(15) Baker, D.; Pryce, G.; Croxford, J . L.; Brown, P.; Pertwee, R. G.;
Makriyannis, A.; Khanolkar, A.; Layward, L.; Fezza, F.; Bisogno,
(3) Devane, W. A.; Hanusˇ, L.; Breuer, A.; Pertwee, R. G.; Stevenson,
L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.;
Mechoulam, R. Isolation and Structure of a Brain Constituent
That Binds to the Cannabinoid Receptor. Science 1992, 258,
1946-1949.
(4) Sugiura, T.; Kondo, S.; Sukagawa, A.; Nakane, S.; Shinoda, A.;
Itoh, K.; Yamashita, A.; Waku, K. 2-Arachidonoylglycerol:
Possible Endogenous Cannabinoid Receptor Ligand in Brain.
Biochem. Biophys. Res. Commun. 1995, 215, 89-97.
a
T.; Di Marzo, V. Endocannabinoids Control Spasticity in
a
Multiple Sclerosis Model. FASEB J . 2001, 15, 300-302.
(5) Hanusˇ, L.; Abu-Lafi, S.; Fride, E.; Breuer, A.; Vogel, Z.; Shalev,
D. E.; Kustanovich, I.; Mechoulam, R. 2-Arachidonyl Glyceryl
Ether, an Endogenous Agonist of the Cannabinoid CB1 Receptor.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3662-3665.
(16) Piomelli, D.; Beltramo, M.; Glasnapp, S.; Lin, S. Y.; Goutopoulos,
A.; Xie, X.-Q.; Makriyannis, A. Structural Determinants for
Recognition and Translocation by the Anandamide Transporter.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5802-5807.
(6) Hillard, C. J .; Edgemond, W. S.; J arrahian, A.; Campbell, W. B.
Accumulation of N-Arachidonoylehanolamine (Anandamide) into
Cerebellar Granule Cells Occurs via Facilitated Diffusion. J .
Neurochem. 1997, 69, 631-638.
(7) Beltramo, M.; Stella, N.; Calignano, A.; Lin, S. Y.; Makriyannis,
A.; Piomelli, D. Functional Role of High-Affinity Anandamide
Transport, as Revealed by Selective Inhibition. Science 1997,
277, 1094-1097.
(8) Hillard, C. J .; J arrahian, A. The Movement of N-arachidonoyle-
thanolamine (Anandamide) across Cellular Membranes. Chem.
Phys. Lipids 2000, 108, 123-134.
(9) Ueda, N.; Puffenbarger, R. A.; Yamamoto, S.; Deutsch, D. G.
The Fatty Acid Amide Hydrolase (FAAH). Chem. Phys. Lipids
2000, 108, 107-121.
(10) Pertwee, R. G. Cannabinoid Receptor Ligands: Clinical and
Neuropharmacological Considerations, Relevant to Future Drug
Discovery and Development. Expert Opin. Invest. Drugs 2000,
9, 1-19.
(11) (a) Calignano, A.; La Rana, G.; Giuffrida, A.; Piomelli, D. Control
of Pain Initiation by Endogenous Cannabinoids. Nature 1998,
394, 277-281. (b) Walker, J . M.; Hohmann, A. G.; Martin, W.
J .; Strangman, N. M.; Huang, S. M.; Tsou, K. The Neurobiology
of Cannabinoid Analgesia. Life Sci. 1999, 65, 665-673. (c)
Ferna´ndez-Ruiz, J .; Berrendero, F.; Herna´ndez, M. L.; Ramos,
J . A. The Endogenous Cannabinoid System and Brain Develop-
ment. Trends Neurosci. 2000, 23, 14-20. (d) Wilson, R. I.; Nicoll,
R. A.; Endogenous Cannabinoids Mediate Retrograde Signaling
at Hippocampal Synapses. Nature 2001, 410, 588-592. (e)
Hampson, R. E.; Deadwyler, S. A. Cannabinoids, Hippocampal
Function and Memory. Life Sci. 1999, 65, 715-723. (f) Di Marzo,
V.; Goparaju, S. K.; Wang, L.; Liu, J .; Ba´tkai, S.; J a´rai, Z.; Fezza,
F.; Miura, G. I.; Palmiter, R. D.; Sugiura, T.; Kunos, G. Leptin-
Regulated Endocannabinoids Are Involved in Maintaining Food
Intake. Nature 2001, 410, 822-825. (g) Giuffrida, A.; Piomelli,
D. The Endocannabinoid System: a Physiological Perspective
on its Role in Psychomotor Control. Chem. Phys. Lipids 2000,
108, 151-158. (h) De Petrocellis, L.; Melck, D.; Bisogno, T.; Di
Marzo, V. Endocannabinoids and Fatty Acid Amides in Cancer,
Inflammation and Related Disorders. Chem. Phys. Lipids 2000,
108, 191-209.
(17) J arrahian, A.; Manna, S.; Edgemond, W. S.; Campbell, W. B.;
Hillard, C. J . Structure-Activity Relationships Among N-
Arachidonylehanolamine (Anandamide) Head Group Analogues
for the Anandamide Transporter. J . Neurochem. 2000, 74, 2597-
2606.
(18) Muthian, S.; Nithipatikom, K.; Campbell, W. B.; Hillard, C. J .
Synthesis and Characterization of a Fluorescent Substrate for
the N-Arachidonoylethanolamine (Anandamide) Transmem-
brane Carrier. J . Pharmacol. Exp. Ther. 2000, 293, 289-295.
(19) Bisogno, T.; Melck, D.; Bobrov, M. Y.; Gretskaya, N. M.;
Bezuglov, V. V.; De Petrocellis, L.; Di Marzo, V. N-acyl-
dopamines: Novel Synthetic CB1 Cannabinoid-Receptor Ligands
and Inhibitors of Anandamide Inactivation with Cannabimimetic
Activity in vitro and in vivo. Biochem. J . 2000, 351, 817-824.
(20) De Petrocellis, L.; Bisogno, T.; Davis, J . B.; Pertwee, R. G.; Di
Marzo, V. Overlap between the Ligand Recognition Properties
of the Anandamide Transporter and the VR1 Vanilloid Recep-
tor: Inhibitors of Anandamide Uptake with Negligible Capsai-
cin-Like Activity. FEBS Lett. 2000, 483, 52-56.
(21) Zygmunt, P. M.; Chuang, H.-h.; Movahed, P.; J ulius, D.; Ho¨ges-
ta¨tt, E. D. The Anandamide Transport Inhibitor AM404 Acti-
vates Vanilloid Receptors. Eur. J . Pharmacol. 2000, 396, 39-
42.
(22) Smart, D.; J erman, J . C. Anandamide: an Endogenous Activator
of the Vanilloid Receptor. Trends Pharmacol. Sci. 2000, 21, 134.
(23) Klaassen, C. D. Casarett & Doull’s Toxicology The Basic Science
of Poisons, 5th ed.; McGraw-Hill Companies, Inc.: New York,
1996; p 345.
(24) Lo´pez-Rodr´ıguez, M. L.; Viso, A.; Ortega-Gutie´rrez, S.; Lastres-
Becker, I.; Gonza´lez, S.; Ferna´ndez-Ruiz, J . J .; Ramos, J . A.
PCT/ES01/00305, 2001.
(25) Corey, E. J .; Cashman, J . R.; Kantner, S. S.; Wright, S. W.
Rationally Designed, Potent Competitive Inhibitors of Leuko-
triene Biosynthesis. J . Am. Chem. Soc. 1984, 106, 1503-1504.
(26) Hudlicky´, M. Reductions in Organic Chemistry, 2nd ed.; Ameri-
can Chemical Society: Washington, DC, 1996; pp 187-190.
(27) Maccarrone, M.; Bari, M.; Menichelli, A.; Giuliani, E.; Del
Principe, D.; Finazzi-Agro`, A. Human Platelets Bind and De-
grade 2-Arachidonoylglycerol, Which Activates These Cells
Through a Cannabinoid Receptor. Eur. J . Biochem. 2001, 268,
819-825.
(28) Szallasi, A.; B´ıro´, T.; Szabo´, T.; Modarres, S.; Petersen, M.;
Klusch, A.; Blumberg, P. M.; Krause, J . E.; Sterner, O. A
Nonpungent Triprenyl Phenol of Fungal Origin, Scutigeral,
Stimulates Rat Dorsal Root Ganglion Neurons Via Interaction
at Vanilloid Receptors. Br. J . Pharmacol. 1999, 126, 1351-1358.
(12) (a) Calignano, A.; Ka´tona, I.; De´sarnaud, F.; Giuffrida, A.; La
Rana, G.; Mackie, K.; Freund, T. F.; Piomelli, D. Bidirectional
Control of Airway Responsiveness by Endogenous Cannabinoids.
Nature 2000, 408, 96-101. (b) Baker, D.; Pryce, G.; Croxford, J .
L.; Brown, P.; Pertwee, R. G.; Huffman, J . W.; Layward, L.
Cannabinoids Control Spasticity and Tremor in
Sclerosis Model. Nature 2000, 404, 84-87. (c) Galve-Roperh, I.;
Sa´nchez, C.; Corte´s, M. L.; Go´mez del Pulgar, T.; Izquierdo, M.;
a Multiple
J M015545Y