A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects

Article abstract of DOI:10.1111/bph.12013

Background and Purpose: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. Experimental Approach: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. Key Results: Of the new compounds, O-7460 exhibited the highest potency (IC₅₀ = 690 nM) against the human recombinant DAGLα, and selectivity (IC₅₀ > 10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥10 μM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB₁ or CB₂ cannabinoid receptors (K_i > 10 μM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 μM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg^-1, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. Conclusions and Implications: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

Full text of DOI:10.1111/bph.12013

DOI:10.1111/bph.12013
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Themed Section: Cannabinoids 2012, Part Two
Correspondence
Tiziana Bisogno or Vincenzo
Di Marzo, Endocannabinoid
Research Group, Institute of
Biomolecular Chemistry,
Consiglio Nazionale delle
Ricerche, Via Campi Flegrei 34,
Comprensorio Olivetti, Pozzuoli
(NA) 80078, Italy. E-mail:
tbisogno@icb.cnr.it;
RESEARCH PAPER
A novel fluorophosphonate
inhibitor of the biosynthesis
of the endocannabinoid
vdimarzo@icb.cnr.it
----------------------------------------------------------------
Keywords
inhibitor; diacylglycerol;
cannabinoid;
2-arachidonoylglycerol; serine
lipase; anti-obesity effects
----------------------------------------------------------------
2-arachidonoylglycerol with
potential anti-obesity effects
Tiziana Bisogno1,7, Anu Mahadevan2, Roberto Coccurello3,
Jae Won Chang4, Marco Allarà1, Yugang Chen2, Giacomo Giacovazzo3,
Aron Lichtman5, Benjamin Cravatt4, Anna Moles3,6 and
Vincenzo Di Marzo1
Received
18 April 2012
Revised
3 August 2012
Accepted
13 August 2012
1Endocannabinoid Research Group, Institute of Biomolecular Chemistry, C.N.R., Pozzuoli, Italy,
2Organix Inc., Woburn, MA, USA, 3Cell Biology and Neurobiology Institute (IBCN)/IRCCS
Fondazione Santa Lucia, Roma, Italy, 4The Skaggs Institute for Chemical Biology and
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA,
5Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond,
VA, USA, 6Genomnia srl Via Nerviano, Milano, Italy, and 7Department of Experimental
Medicine and Biochemical Science, University TorVergat, Rome, Italy
BACKGROUND AND PURPOSE
The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG)
via DAG lipases (DAGL) a and b is just starting to be considered as a novel and promising source of pharmaceuticals for the
treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects.
EXPERIMENTAL APPROACH
Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various
enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice.
KEY RESULTS
Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLa,
and selectivity (IC50 > 10 mM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid
amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at
concentrations Ն10 mM, and showed that this compound has only one major ‘off-target’, that is, the serine hydrolase
KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 mM).
In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 mM) reduced 2-AG levels. When administered
to mice, O-7460 dose-dependently (0–12 mg·kg-1, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period,
and, subsequently, slightly but significantly reduced body weight.
CONCLUSIONS AND IMPLICATIONS
O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in
vivo under physiological as well as pathological conditions.
LINKED ARTICLES
This article is part of a themed section on Cannabinoids. To view the other articles in this section visit
http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8
© 2012 The Authors
British Journal of Pharmacology © 2012 The British Pharmacological Society
784 British Journal of Pharmacology (2013) 169 784–793

A novel inhibitor of endocannabinoid 2-AG biosynthesis
BJP
Abbreviations
2-AG, 2-arachidonoylglycerol; CB1, cannabinoid receptor subtype 1; CB2, cannabinoid receptor subtype 2; COS-7,
monkey kidney fibroblast cell line; CP-55940, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl) phenyl]-4-(3-
hydroxypropyl)cyclohexan-1-ol; DAG, diacylglycerol; DAGL, diacylglycerol lipase; FAAH, fatty acid amide hydrolase;
HFD, high-fat diet; MAGL, monoacylglycerol lipase; N18TG2, mouse neuroblastoma cell line; O-3640, octadec-9-enoic
acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester; O-3841, octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-
methyl-phosphinoyloxy)-ethyl ester; O-5596, octadec-9-enoic acid 1-tert-butoxymethyl-2(fluoro-methyl-
phosphinoyloxy)-ethyl ester; O-7344, (S)-1-methoxy-5-oxopentan-2-yl oleate; O-7458, 1-((fluoro(methyl)
phosphoryl)oxy)-3-(penthyloxy)propan-2-yl oleate; O-7459, 1-ethoxy-3-((fluoro(methyl)phosphoryl)oxy)propan-2-yl
oleate; O-7460, 1-((fluoro(methyl)phosphoryl)oxy)-3-isopropoxypropan-2-yl oleate; OMDM188, N-formyl-L-isoleucine-
(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]-methyl]dodecyl ester; WIN55312-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-
morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl methanone mesylate
for these proteins is available. We have previously described
the development of two fluorophosphonate inhibitors of
Introduction
DAGLa, O-3640 and O-3841, with excellent selectivity for
The
endocannabinoid
2-arachidonoylglycerol
(2-AG)
DAGLa over the other proteins of the endocannabinoid
system tested (Bisogno et al., 2006). These compounds have
been successfully used in vitro but, unfortunately, they are not
suitable for systemic use in vivo due to their lack of stability
and poor permeability through the plasma membrane, which
have been only partially solved by the design, a few years
later, of O-5596, a more stable and slightly more potent and
cell membrane permeable DAGL inhibitor (Figure 1) (Bisogno
et al., 2009a). Interestingly, O-5596 was shown to inhibit a
typical endocannabinoid-mediated effect in vivo, since its
administration to mice reduced selectively the intake of pal-
atable food (Bisogno et al., 2009a). Here we report three new
fluorophosphonate compounds, that is O-7458, O-7459 and
O-7460 (Figure 1), and their in vitro and in vivo pharmacologi-
cal characterization as potential selective inhibitors of
DAGLa.
(Mechoulam et al., 1995; Sugiura et al., 1995) exerts its physi-
ological action by binding and functionally activating the
two cannabinoid receptor subtypes identified to date, CB1
and CB2 (Pertwee, 2008). The tissue levels of 2-AG, as well as
those of the other proposed endocannabinoid, anandamide,
undergo remodelling during either acute or chronic patho-
logical conditions. The general strategy of these changes is to
afford protection, usually by aiming at restoring physiologi-
cal homeostasis (Di Marzo, 2008). However, endocannabi-
noid plasticity can lose specificity and contribute to the
symptoms or progression of certain diseases. In this case, the
pharmacological reduction of 2-AG tissue levels, by blocking
its biosynthesis, might be therapeutically valuable. Obesity,
liver fibrosis, and Alzheimer’s and Parkinson’s disease are
only some of the pathological conditions that might benefit
from a reduction in endocannabinoid tone (Di Marzo, 2008).
In fact, the use of inhibitors of 2-AG formation might have
pharmacological consequences and therapeutic applications
similar to those of ‘neutral’ CB1 receptor antagonists, which
have been suggested to produce fewer side effects than CB1
receptor inverse agonists (Janero, 2012), such as rimonabant
(Ward and Raffa, 2011). The latter compound was in clinical
development until 2008 and even reached the market as an
anti-obesity drug before being discontinued due to poten-
tially serious psychiatric effects. Indeed, obesity is character-
ized by pathologically elevated endocannabinoid levels in
both the CNS and peripheral tissues involved in energy
homeostasis, and rimonabant, as well as other CB1 inverse
agonists, proved very effective at reducing body weight (BW)
and several parameters of the metabolic syndrome, although
these compounds were not devoid of undesired adverse side
effects (Di Marzo and Després, 2009).
Methods
Nomenclature
Drug and receptor nomenclature have been used according to
BJP’s ‘Guide to receptors and channels’ (Alexander et al.,
2011).
Synthesis of O-7460
Compound O-7460 was synthesized from commercially
available 1-chloro-3-isopropoxypropan-2-ol (1) in four steps
in an overall yield of 8% (see Supporting Information
Figure S1). Coupling of compound 1 with oleic acid in the
presence of EDCI/DMAP gave compound 2 (84%), which on
treatment with NaI/dimethyl methylphosphonate in a sealed
tube at 135°C for 2 days yielded pure compound 3 (30%) after
chromatography. Demethylation of 3 with TMSBr followed
by chromatography yielded compound 4 (80%), which on
treatment with methyl DAST followed by chromatography
yielded pure target compound O-7460 (40%). Similar proce-
dures were applied for the synthesis of O-7458 and O-7459,
and will be described, together with the synthesis of O-7344,
in a separate report in a specialist journal.
The endocannabinoid 2-AG is biosynthesized from dia-
cylglycerol (DAG) precursors by the action of specific sn-1
selective DAG lipases (DAGL). Two sn-1 DAGL isozymes
(DAGLa and DAGLb) have been cloned and enzymatically
characterized, and at least one of them, DAGLa, is localized
to the postsynaptic dendritic spines establishing synapses
with CB1-expressing axons, supporting the proposed role for
2-AG as a retrograde signal mediating depolarization-induced
suppression of neurotransmission and heterosynaptic plastic-
ity (Bisogno et al., 2003; Ludányi et al. 2011). Since 2-AG
biosynthetic enzymes have been identified only recently, still
relatively little information on possible selective inhibitors
DAGLa assay
DAGL activity was assessed as previously reported
(Bisogno et al., 2003) by using membrane preparations
British Journal of Pharmacology (2013) 169 784–793 785

T Bisogno et al.
BJP
Figure 1
Chemical structures of new and already known fluorophosphonate inhibitors of 2-AG biosynthesis by DAGLa.
(70 mg of protein) obtained from COS-7 cells overexpress-
ing the human recombinant DAGLa, and 1-[14C]oleoyl-2-
arachidonoylglycerol [1.0 mCi·mmol–1, 25 mM, synthesized as
reported previously (Bisogno et al., 2003)] as substrate in the
presence of vehicle or increasing concentrations of test com-
pounds in Tris–HCl buffer, pH = 7 for 20 min. After the
incubation, lipids were extracted with two volumes of CHCl3/
CH3OH (2:1, v · v-1). The organic extracts, lyophilized under
vacuum, were purified by using TLC on silica on polypropyl-
ene plates eluted in CHCl3/CH3OH/NH4OH (85:15:0.1, v · v-1)
as the eluting solvent. Bands corresponding to [14C]-oleic acid
(40 Ci·mmol-1, St. Louis, MO, USA) diluted with non-
radiolabelled 2-AG (Cayman Chemicals) to 20 mM, as previ-
ously reported (Bisogno et al., 2009b), in the presence of
vehicle or increasing concentrations of test compounds. After
the incubation, the amount of [3H]-glycerol produced was
measured by scintillation counting of the aqueous phase after
extraction of the incubation mixture with 2 volumes of
CHCl3/MeOH 1:1 (v · v-1).
CB1 and CB2 binding assays
Membranes from HEK-293 cells transfected with either the
human recombinant CB1 or CB2 cDNA (Perkin-Elmer, Monza,
Italy) were incubated with increasing concentrations of the
test compounds and [3H]-CP-55940 (KD = 0.12 nM for CB1 and
KD = 0.18 nM for CB2) as the high affinity ligand, as described
by the manufacturer (Perkin-Elmer). Displacement curves
were generated by incubating drugs with a fixed concentra-
tion of [3H]-CP-55940 (0.4 nM for CB1 and 0.53 nM for CB2
binding assay). Non-specific binding was determined using
1 mM WIN55312-2 (Tocris, Bristol, UK).
were cut and their radioactivity was measured with
a
b-counter. In order to investigate the irreversibility of the
inhibitory action of O-7460, the most active inhibitor tested
in this study, membrane preparations obtained from COS-7
cells overexpressing the human recombinant DAGLa were
incubated in the presence of vehicle or 0.5 or 5 mM concen-
trations of the compound in Tris–HCl buffer, pH = 7 for
20 min. After incubation, DAGLa membrane preparations
were washed three times with either Tris–HCl buffer, 50 mM
pH = 7 (buffer A) or Tris–HCl buffer, 50 mM pH = 7 containing
0.03% BSA (buffer B), using centrifugations at 10 000¥ g. The
DAGLa membrane preparations were than resuspended in
Tris–HCl buffer, 50 mM pH = 7, and DAGL activity was
re-assessed as reported above in the presence of 1-[14C]-oleoyl-
2-arachidonoylglycerol.
Fatty acid amide hydrolase (FAAH) assay
The effect of compounds on the enzymatic hydrolysis of
[14C]-anandamide (2.4 mM, 5.0 mCi·mmol-1) was studied as
previously reported (Cascio et al., 2004) by using membranes
prepared from rat brain, incubated with increasing concen-
trations of test compounds in 50 mM Tris–HCl, pH = 9, for
30 min at 37°C. [14C]-Ethanolamine produced from [14C]-
anandamide hydrolysis was measured by scintillation count-
ing of the aqueous phase after the extraction of the
incubation mixture with two volumes of CHCl3/CH3OH (1:1,
v · v-1).
Monoacylglycerol lipase (MAGL) assay
The 10 000 g cytosolic fraction (100 mg of protein) and
human recombinant MAGL (0.05 mg, Cayman Chemicals,
Ann Arbor, MI, USA) were incubated in Tris–HCl 50 mM, at
pH 7.0 at 37°C for 20 min, with 2-arachidonoyl-[3H]-glycerol
786 British Journal of Pharmacology (2013) 169 784–793

A novel inhibitor of endocannabinoid 2-AG biosynthesis
BJP
lected at baseline before drug administration (0 min) and
14 h after O-7460 administration. Each measurement was
made by a Mettler ML802E precision balance (Toledo, OH,
USA) providing an accuracy of 0.01g.
Affinity-based protein profiling assay
The screening of possible ‘off-target’ serine hydrolases for
O-7458, O-7459 and O-7460 was performed with mouse
brain proteomes (Long et al., 2009). Proteomes (mouse mem-
brane fractions) (50 mL, 1.0 mg·mL-1 total protein concentra-
tion) were pre-incubated with varying concentrations of
inhibitors at 37°C. After 30 min, fluorophosphonate rhodam-
ine (1.0 mL, 50 mM in DMSO) was added, and the mixture was
incubated for another 30 min at room temperature. Reactions
were quenched with SDS loading buffer (35 mL) and run on
SDS-PAGE, and visualized in gel with a Hitachi FMBio lle
flatbed fluorescence scanner (MiraiBio, San Francisco, CA,
USA). The selective MAGL inhibitor, JZL184, was used as a
positive control for MAGL inhibition.
Effects of O-7460 on motor activity and body
temperature in mice
Seven mice maintained on the same dual diet regimen (either
4RF21 or HFD) were administered O-7460 i.p. (1 mL 100 g-1
BW) at 0 mg·kg-1 (n = 3) and 12 mg·kg-1 (n = 4), and then
individually placed into activity chambers equipped with
infrared photo sensors (InfraMot system® by TSE Systems, Bad
Homburg, Germany) suitable for measuring, over time, the
animal’s horizontal and vertical spatial movement. Motor
activity was recorded for a total time of 60 min. Body tem-
perature was measured 30 min after drug administration with
a digital thermometer (model BAT7001H) connected to a
thermostat rectal probe (model RET-3) both produced by
Physitemp Instruments, Inc. (Clifton, NJ, USA).
Effect of O-7460 on endocannabinoid levels
in intact N18TG2 cells
Confluent N18TG2 cells were stimulated for 20 min at 37°C
with ionomycin (3 mM) with or without O-7460 (10 mM).
After stimulation, cells plus medium were extracted with
CHCl3/CH3OH 2:1 (v · v-1). Each extract was purified by open
bed chromatography and 2-AG was quantified by LC-MS as
previously reported (Marsicano et al., 2002).
Effects of HFD exposure with or without
O-7460 administration on 2-AG levels in
hypothalamus, liver and white adipose
tissue (WAT)
Animals
Seven mice were habituated (10 days) to the transition from
the standard 4RF21 diet to the HFD (D12451) while another
six mice were maintained on regular diet. After an additional
10 days of dual diet regimen, seven mice were administered
either O-7460 (12 mg·kg-1; n = 4) or vehicle (0 mg·kg-1; n = 3)
whereas the other six mice (4RF21, n = 3 and HFD, n = 3) were
left drug-free. At day 10, 60 min after O-7460 administration,
both groups were killed and tissue samples of WAT, liver and
hypothalamus were collected. WAT (epididymal), liver and
brain were quickly removed and flash-frozen in liquid
nitrogen. For hypothalami extraction, each brain was fixed
vertically on the freeze plate of a freezing microtome. Micro-
punches of both hemispheres were obtained from brain slices
(coronal sections) no thicker than 300 mm. A probe stainless
steel needle of 1.3 mm (i.d.) was used for tissue punching.
The coordinates were taken according to the atlas of Franklin
and Paxinos (1998) as follows: three slices from -1.28 to
-2.12 mm (coronal sections from Bregma, to include lateral,
ventromedial hypothalamus and arcuate nucleus). Tissues
were stored at -80°C up to the day of analysis. Tissues were
homogenized in 5 vol of chloroform/methanol/Tris–HCl
50 mM (2:1:1) containing 100 pmol of d5-2-AG and extracted
three times with chloroform. Each extract was purified by
open bed chromatography and 2-AG was quantified by
LC-MS as previously reported (Marsicano et al., 2002).
Seven-week-old male C57BL/6N inbred mice (n = 16, weigh-
ing 20 Ϯ 0.5 g on arrival) were purchased from Charles River
(Calco, CO, Italy). Mice were housed in individual cages (26.7
¥ 20.7 ¥ 14 cm), which were kept in a room at 22–24°C with
a 50–60% relative humidity, 12 h light/12 h dark cycle, and
maintained with standard diet (diet no. 4RF21; Mucedola
s.r.l., Milan, Italy) and water available ad libitum. Animal
husbandry and experiments were carried out in accordance
with the Council Directive of the European Community (86/
EEC) of the Italian D.L. 116 (27 January 1992) and approved
by veterinarian supervision. Before starting the evaluation of
the effects of O-7460 administration on high-fat diet (HFD)
intake, mice were habituated for 10 days to the transition
from the standard 4RF21 diet to a HFD dietary regimen con-
taining 45% fat (6% of soybean oil and 39% of lard, kcal) and
providing about 4.73 kcal·g-1 of energy level (diet no.
D12451; Research Diets, New Brunswick, NJ, USA). Daily HFD
intake and BW were monitored during the habituation phase
(data not shown). All studies involving animals are reported
in accordance with the ARRIVE guidelines for reporting
experiments involving animals (Kilkenny et al., 2010;
McGrath et al., 2010).
Effects of O-7460 on HFD intake and BW
in mice
O-7460 was dissolved in ethanol : saline (1:1) solution and
administered i.p. at the following doses, 0 mg·kg-1 (n = 6),
6 mg·kg-1 (n = 5), and 12 mg·kg-1 (n = 5), in a volume of 1 mL
100 g-1 BW. Animals were not deprived of food and the dif-
ferent doses of O-7460 were administered just before the 12 h
dark cycle (19:00 h) and HFD intake assessed at the following
time intervals: 30 min, 60 min and 14 h after O-7460 treat-
ment. HFD consumption was measured as the difference
between the initial quantity of food allotted (g) in each cage
and the weight of the remaining food 30– 60 min and 14 h
after O-7460 administration, respectively. BW was also col-
Data analysis
IC50 values, means Ϯ SEM of n = 3 experiments, were obtained
using GraphPad Prism4® (GraphPad Software, Inc., La Jolla,
CA, USA). Ki values were calculated by applying the Cheng–
Prusoff equation to the IC50 values (obtained using GraphPad
Prism4). Results from the in vitro studies and 2-AG content
were analysed by ANOVA followed by the Bonferroni post hoc
test. Results from in vivo studies were analysed by ANOVA
followed by Tukey’s honestly significant difference (HSD) as
post hoc test. Differences between means were deemed to be
significant when P < 0.05.
British Journal of Pharmacology (2013) 169 784–793 787

T Bisogno et al.
BJP
Results
Effects of compounds on human DAGLa
The possible inhibitory effect of compounds on DAGL was
tested in COS-7 cells overexpressing human DAGLa. Com-
pound O-7344 exhibited no significant inhibitory activity up
to 10 mM. Under the same conditions, O-7458 exhibited
a moderate inhibitory action on DAGLa (IC50 = 3.26 Ϯ
0.09 mM), whereas O-7459 and O-7460 exhibited higher
potency as DAGLa inhibitors with IC50 values of 1.17 Ϯ
0.16 mM and 0.69 Ϯ 0.087 mM, respectively (Figure 2A–C and
Table 1).
Moreover, O-7460 inhibited DAGLa in a seemingly irre-
versible way since its inhibitory effect persisted after repeated
washing of the DAGLa membrane preparations with buffer A
or buffer B. In fact, the % of DAGLa inhibition by O-7460 at
the concentrations of 0.5 and 5 mM changed from 24 Ϯ 5 to
24 Ϯ 8 and 13 Ϯ 6 and from 80 Ϯ 8 to 70 Ϯ 7 and 66 Ϯ 5 after
no washing or three washings with buffer A or buffer B respec-
tively. Data are means Ϯ SEM, n = 3.
Effect of compounds on rat brain FAAH
The effect of the compounds on FAAH was studied by using
membrane preparations from rat brain. None of the com-
pounds exerted any significant inhibition of anandamide
hydrolysis up to a concentration of 50 mM (Table 1).
Effects of compounds on MAGL
The effect of the compounds on MAGL was studied using
cytosolic fractions of COS-7 cells, or, for O-7460 only, on
human recombinant MAGL, and by using 2-arachidonoyl-
[3H]-glycerol as substrate. None of the compounds exhibited
any significant inhibitory activity in any of the enzymatic
preparations up to 10 mM (Table 1).
Effect of compounds in an affinity-based
protein profiling assay
As shown in Figure 3, and as denoted by the progressive
reduction in intensity of the labelling of some proteins with
increasing concentrations of the compounds, when tested in
this assay, O-7458, O-7459 and O-7460 were found to ‘hit’
particularly two types of serine hydrolases: MAGL (bands at
33–35 kDa), but only at the highest concentration tested
(10 mM), and KIAA 1363 (bands at ~45 kDa), even at the
0.1 mM concentration with an estimated potency of 0.5 < IC50
< 1 mM.
᭤
Figure 2
Concentration–response curves for inhibition of DAGLa activity by
O-7460 (A), O-7459 (B) and O-7458 (C). The effect of increasing
concentrations of drugs is expressed as percentage of inhibition, and
IC50 values (reported in Table 1) were obtained with GraphPad
Prism4. Data are means of n = 3 determinations. (D) Ionomycin-
induced formation of 2-AG in intact N18TG2 cells. The effect of
O-7460 (10 mM) is reported as percentage of the maximum effect
observed with ionomycin 3 mM plus vehicle. Data are means Ϯ SEM
of n = 3 determinations (means were compared by ANOVA followed by
the Bonferroni test. *P < 0.05).
788 British Journal of Pharmacology (2013) 169 784–793

A novel inhibitor of endocannabinoid 2-AG biosynthesis
BJP
Table 1
Screening of the new compounds on enzymes of the endocannabinoid system
DAGLa human
recombinant
(IC50 mM)
MAGL COS
cell cytosol
(IC50 mM)
Human
recombinant
MAGL
FAAH rat brain
membranes
(IC50 mM)
CB1 human
recombinant
(Ki mM)
CB2 human
recombinant
(Ki mM)
O-7344
O-7458
O-7459
O-7460
JZL184
>10
>10
n.t.
n.t.
n.t.
>10
n.t.
n.t.
n.t
>50
>10
1.21 Ϯ 0.20
3.26 Ϯ 0.09
1.17 Ϯ 0.16
0.69 Ϯ 0.09
n.t.
>10
>50
>10
>10
>10
>50
>10
>10
>10
>50
>10
>10
0.12 Ϯ 0.08
n.t.
n.t.
n.t.
n.t.
URB597
WIN55212-2
n.t.
0.05 Ϯ 0.02
n.t
n.t.
n.t.
n.t
n.t
0.00267 Ϯ 0.0004
0.001 Ϯ 0.00029
Data are means Ϯ SEM of n = 3 experiments (see Figure 2). Ki values were calculated by applying the Cheng–Prusoff equation to the IC50
values (obtained using GraphPad Prism4).
n.t., not tested.
Figure 3
Affinity-based proteomic profiling assay of O-7460, O-7459 and O-7458 using the mouse brain proteome. MAGL, KIAA1363, FAAH, ABHD6 and
ABHD12 bands are shown by arrows. The effect of the selective MAGL inhibitor JZL184 is also shown. Con: control incubation with no inhibitor.
These results are representative of three separate experiments.
Binding affinity for human recombinant CB1
and CB2
Experiments in intact cells
The effect of O-7460 (10 mM, corresponding approximately to
an IC80 concentration of the compound) on 2-AG formation
stimulated by ionomycin was studied in intact N18TG2 cells
where the enzymatic formation of 2-AG has been previously
reported (Bisogno et al., 2003). As reported in Figure 2D,
O-7460 decreased the ionomycin-induced formation of 2-AG
in intact cells (down to 33 Ϯ 10% of the maximum effect
observed with ionomycin 3 mM).
Radioligand competition binding studies were performed by
using membranes from HEK-293 cells transfected with
human recombinant CB1 or CB2 receptors. These data are
summarized in Table 1. None of the compounds exhibited
any significant affinity for cannabinoid receptors up to 10 mM
except for compound O-7344, which showed some affinity
for CB2 receptors with a Ki of 1.21 Ϯ 0.20 mM.
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T Bisogno et al.
BJP
Repeated-measures ANOVA carried out on BW (g) 0 min
(baseline) and 14 h after O-7460 administration did not
reveal any significant treatment [F(2,13) = 0.12; n.s.] or time
[F(1,13) = 1.52; n.s.] effect, although a significant treatment ¥
time interaction was found [F(2,13) = 80.37; P < 0.0001]. Post
hoc analysis (Figure 4) revealed the progressive enhancement
of BW only for vehicle-treated animals (0 min and 14 h;
O-7460, 0 mg·kg-1, Tukey’s HSD, P < 0.05), whereas the inter-
mediate dose (O-7460, 6 mg·kg-1) did not affect the increase
in BW. However (Figure 4), in contrast to the time-dependent
increase in BW observed in vehicle-treated animals, the
administration of the highest dose (O-7460, 12 mg·kg-1) sig-
nificantly counteracted the BW increase.
Effects of O-7460 on motor activity and body
temperature in mice
In order to exclude the possibility that the effect of O-7460 on
food intake could be due to overt changes in the normal
behaviour of the mice, we investigated both motor activity
and body temperature after O-7460 administration
(12 mg·kg-1, i.p.) in HFD mice. Motor activity was recorded
for a total time of 60 min and O-7460 administration exhib-
ited no effect on total locomotor activities (1717 Ϯ 720.22
and 1618 Ϯ 719.22 counts in vehicle- and O-7460-treated
mice, respectively). Furthermore, O-7460 did not modify
body temperature in HFD mice (36.76 Ϯ 0.2°C in vehicle and
36.85 Ϯ 0.18°C in treated mice). Data are means Ϯ SEM of
n = 3 or 4 mice.
Effects of HFD exposure or O-7460
administration on 2-AG levels in the
hypothalamus, liver and WAT
We next wanted to assess whether the effect of O-7460
observed on food intake and body weight in HFD mice cor-
related with a reduction in 2-AG levels at the both central and
peripheral levels. In the hypothalamus, 10 days of HFD
caused an up-regulation of 2-AG levels (from 3.50 Ϯ 0.59 to
5.03 Ϯ 0.71 pmol·mg-1 tissue in standard diet and HFD,
respectively), which was significantly counteracted after
60 min from O-7460 (12 mg·kg-1) i.p. injection (5.06 Ϯ 0.004
to 3.42 Ϯ 0.65 pmol·mg-1 tissue in vehicle- and O-7460-
treated HFD mice respectively). Furthermore, when periph-
eral tissues were analysed for 2-AG content, no differences
were observed in the WAT, whereas the 2-AG concentration
in liver was modified, as in the hypothalamus. In fact, 2-AG
levels in the liver were up-regulated after 10 days of HFD
(from 3.39 Ϯ 0.25 to 6.41 Ϯ 1.25 pmol·mg-1 tissue in standard
diet and HFD respectively), and were significantly reduced
after 60 min from O-7460 (12 mg·kg-1) i.p. injection (6.22 Ϯ
1.42 to 3.46 Ϯ 0.03 pmol·mg-1 tissue in vehicle- and O-7460-
treated mice respectively). Data are means Ϯ SEM of n = 3 or
4 mice, with P < 0.05 for all the differences described above,
as assessed by one-way ANOVA followed by the Bonferroni test.
Figure 4
Effects of O-7460 on HFD intake (A) and body weight (B) in mice.
HFD cumulative food intake (HFD-I) and body weight (BW) evolution
were analysed by separate two-way ANOVAs with drug (three doses)
and three or two time points as HFD-I (30 min, 60 min and 14 h) and
BW (0 min and 14 h) factors respectively. A P-value below 0.05 was
considered statistically significant as compared with vehicle group
(HFD-I) or baseline BW. Post hoc analyses were carried out by Tukey’s
HSD test comparisons. All analyses were executed by STATISTICA
version 8.0 (data analysis software system, StatSoft, Inc., Tulsa, OK,
USA, 2007).
Effects of O-7460 on HFD intake and BW
in mice
Repeated-measures ANOVA performed on total HFD intake (g)
30 min, 60 min and 14 h after O-7460 administration
revealed a significant main effect of treatment [F(2,13) =
81.76; P < 0.0001] and time [F(2,26) = 833.83; P < 0.0001], as
well as a significant treatment ¥ time interaction [F(4,26) =
43.70; P < 0.0001]. The administration of O-7460 (Figure 4)
induced a time and dose-dependent decrease in HFD intake as
compared with vehicle-treated mice: 30 and 60 min, O-7460
6 mg·kg-1, Tukey’s HSD n.s.; 14 h, 6 mg·kg-1, Tukey’s HSD, P <
0.05; 30 min, 60 min and 14 h, O-7460 12 mg·kg-1, Tukey’s
HSD, P < 0.05.
Discussion
We have reported here the synthesis and pharmacological
activity of O-7460, a novel potent and relatively selective
inhibitor of the biosynthesis of the endocannabinoid 2-AG,
790 British Journal of Pharmacology (2013) 169 784–793

A novel inhibitor of endocannabinoid 2-AG biosynthesis
BJP
and have confirmed, through the use of this compound in
mice fed with HFD, that inhibition of DAGLa might repre-
sent a new strategy to counteract hyperphagia and obesity.
We have previously described a series of fluorophosphonate
inhibitors of 2-AG biosynthesis by DAGLa (Bisogno et al.,
2006), and showed more recently that the substitution, in the
most potent of these compounds, that is, O-3841, of its meth-
oxyl moiety with a tert-butoxyl group yielded O-5596, a
relatively more stable and cell membrane permeant DAGLa
inhibitor, with some inhibitory activity in vivo against palat-
able versus normal chow intake in mice (Bisogno et al.,
2009a). However, compounds obtained by replacing all H
atoms of the methoxyl moiety of O-3841 had not been inves-
tigated yet. Hence, in this study we synthesized three new
compounds, O-7459, O-7460, O-7458, in which one or two H
atoms of O-3841 were replaced with one or two methyl
groups or an n-butane group respectively. Furthermore, we
also synthesized O-7344, that is, an O-3841 analogue without
the phosphonate moiety, which we reasoned would allow us
to establish the importance of this group for inhibitory activ-
ity. Indeed, among these new compounds, O-7344 exhibited
no DAGLa inhibitory activity up to 10 mM, confirming that
the fluoro-methyl-phosphinoyloxy group present in the
chemical structure of both O-3841 and its tert-butoxymethyl
derivative, O-5596 (Bisogno et al., 2006; 2009a), is strictly
required for these compounds to covalently inhibit this
enzyme. Accordingly, O-7459 and O-7460 inhibited DAGLa
and were selective for CB1 and CB2 receptors as well as for
FAAH and MAGL, but were less potent than the previously
reported O-5596. Indeed, the rank order of potency against
DAGLa of this previous compound and the new compounds
reported here was O-7459 < O-7460 < O-5596, and hence
increased with the increasing number of methyl groups and,
therefore, with the steric hindrance of the substituent. Thus,
replacement of all the H atoms in the methoxyl moiety of
O-3841 with methyl groups is required to increase the
potency of this phosphonate derivative of oleic acid as a
DAGLa inhibitor. On the other hand, the substitution of its
methoxyl moiety with a pentyloxy group, as in O-7458,
yielded a less-potent inhibitor of the enzyme, again suggest-
ing that a reduction in the steric hindrance, due in this case
to the presence of a linear C5-aliphatic chain instead of a
tert-butyl group, has a negative effect on its potency against
DAGLa. Importantly, and as expected from this class of com-
pounds, O-7460 inhibited DAGLa in a seemingly irreversible
way, because its effect could not be removed by repeated
washing of the DAGLa membrane preparations.
comparable to those needed to inhibit DAGLa, and the func-
tion of which in conditions other than cancer and cholesteryl
ester hydrolysis is still poorly understood (Chang et al., 2011).
The assay also confirmed that O-7460 inhibited MAGL only
at concentrations Ն10 mM, and showed that O-7459, which is
slightly less potent as an inhibitor of DAGLa than O-7460, is
also less potent as a KIAA1363 inhibitor, but perhaps slightly
more potent at inhibiting MAGL, whereas O-7458 was com-
pletely inactive at inhibiting this latter enzyme.
We also showed here that O-7460, like other DAGLa
inhibitors, inhibits 2-AG de novo biosynthesis in intact cells.
In mouse neuroblastoma N18TG2 cells stimulated with iono-
mycin, a nearly maximally effective (IC80) concentration of
the compound was found to cause a reduction in 2-AG levels
comparable to that induced, under identical experimental
conditions, by a IC80 concentration of the more potent
OMDM188 (Ortar et al., 2008), and stronger than that
induced by a IC80 concentration of O-5596 (Bisogno et al.,
2009a), perhaps suggesting that plasma membrane perme-
ability of a phosphonate DAGLa inhibitor like O-7460 might
be lower than that of a non-phosphonate inhibitor such as
OMDM188, but higher than that of the other phosphonate,
O-5596. On the other hand, OMDM188 was developed start-
ing from tetrahydrolipstatin, a lipase inhibitor with low
reported permeability through the gastrointestinal tract,
which limits its bioavailability after oral administration (Ball-
inger, 2000). Hence, the necessity to develop DAGL inhibitors
belonging to other chemical classes but still suitable for in
vivo use.
When administered i.p. to mice, O-7460 dose-
dependently inhibited the intake of HFD, as one would
expect from a ‘suppressor’ of central endocannabinoid tone,
and it did so at doses comparable to those previously shown
to be necessary for CB1 receptor inverse agonists to suppress
food intake when administered i.p. In fact, rimonabant and
AM251 (Simiand et al., 1998; Mathes et al., 2008), two widely
used CB1 inverse agonists, as well as O-5596 (Bisogno et al.,
2009a), an inhibitor of 2-AG biosynthesis, have been previ-
ously reported to reduce the intake of palatable food in mice.
Perhaps more relevant to our present results, a HFD-induced
increase in hypothalamic 2-AG levels, and the subsequent
overactivation of CB1, were reported to be responsible for
mouse preference for HFD, because the intake of this diet was
suppressed by a neutral CB1 antagonist, O-2050 (Higuchi
et al., 2010; 2011). Here, we not only confirmed that such an
increase in hypothalamic 2-AG concentrations also occurred
in our HFD mice, but showed that the high-fat dietary
regimen also elevates the levels of this endocannabinoid in
the liver. Importantly, concomitant with its effect on food
intake, O-7460 also reduced 2-AG levels in both the hypotha-
lamus and liver. Thus, it also likely that in the mice used in
our study, which were habituated to HFD consumption for
several days, up-regulated hypothalamic 2-AG levels contrib-
ute to stronger HFD consumption and preference, and that
O-7460 reduces the consumption of the HFD both 30 and
60 min after administration, as well as over a period of 14 h,
by producing an acute inhibition of DAGLa, an enzyme
abundantly expressed in the hypothalamus (Suárez et al.,
2011).
We showed here that the most potent compound devel-
oped in this study, O-7460, is ~20-fold selective towards both
human and primate (COS-7) MAGL, rat FAAH, and human
CB1 and CB2 receptors [please note that some linear aliphatic
methyl-fluoro-phosphonates were also previously found to
potently and irreversibly bind to CB1 receptors (Fernando and
Pertwee, 1997; Martin et al., 2000)]. However, possibly the
best way to rule out ‘off targets’ for newly developed inhibi-
tors of serine hydrolases is through the use of affinity-based
proteomic profiling (Long et al., 2009), which we performed
here using a mouse brain proteome. We show that O-7460, up
to a concentration of 10 mM, performed rather well in this
screen as we could recognize only one other serine hydrolase,
KIAA1363, which this compound inhibited at concentrations
We also showed here, for the first time, that acute DAGLa
inhibition causes a reduction in BW. In fact, mice treated
British Journal of Pharmacology (2013) 169 784–793 791

T Bisogno et al.
BJP
with the highest dose of O-7460, unlike mice treated with
vehicle, not only did not gain weight over the observation
period, but even experienced a small but statistically signifi-
cant reduction in BW. It is likely that this latter effect is due
to the reduction of HFD consumption described above,
although other possible mechanisms cannot be ruled out. In
fact, very recently, 2-AG was suggested to participate in the
regulation of the activity of forebrain neural circuits impli-
cated in the control of energy dissipation, based on the obser-
vation that transgenic mice overexpressing MAGL in
forebrain neurons are resistant to BW gain if fed with a HFD
(Jung et al., 2012). Interestingly, although the effects of
chronically reduced brain 2-AG levels in a transgenic mouse
following congenital up-regulation of 2-AG degradation in
forebrain neurons, and the transient reduction of whole body
2-AG levels following acute systemic inhibition of 2-AG bio-
synthesis, may not necessarily yield similar results, MAGL-
overexpressing mice did not eat less HFD than their wild-type
littermates (Jung et al., 2012). This may indicate that the
effects of O-7460 on HFD consumption and BW, described
here, were also exerted at the peripheral level, as suggested
also by our present finding of reduced hepatic 2-AG levels in
HFD mice following administration of the compound.
In conclusion, we have reported here the synthesis and
pharmacological characterization in vitro and in vivo of
O-7460, a novel selective inhibitor of DAGLa, belonging to
the class of fluorophosphonates. Our data suggest that
O-7460 might be considered a useful pharmacological tool to
further investigate the role played by 2-AG in physiological
and pathological conditions, and particularly in the frame-
work of endocannabinoid function in the control of food
intake and energy homeostasis. Since DAGLa inhibitors have
been shown to only counteract the stimulated biosynthesis of
2-AG, with no effect on the basal levels of this endocannabi-
noid (Bisogno et al., 2003; 2006; present in vitro data; see
Alger and Kim, 2011, for review), these compounds, like
‘neutral’ CB1 agonists, and unlike CB1 ‘inverse agonists’,
might reduce endocannabinoid tone only when and where
this is pathologically altered. They might, therefore, be used
to treat conditions determined by pathologically elevated
2-AG levels, with fewer side effects than compounds like
rimonabant and taranabant (Di Marzo and Després, 2009).
References
Alexander SPH, Mathie A, Peters JA (2011). Guide to receptors and
channels (GRAC), 5th edition. Br J Pharmacol 164 (Suppl. 1):
S1–S324.
Alger BE, Kim J (2011). Supply and demand for endocannabinoids.
Trends Neurosci 34: 304–315.
Ballinger A (2000). Orlistat in the treatment of obesity. Expert Opin
Pharmacother 1: 841–847.
Bisogno T, Howell F, Williams G, Minassi A, Cascio MG, Ligresti A
et al. (2003). Cloning of the first sn-1-DAG lipases points to the
spatial and temporal regulation of endocannabinoid signaling in
the brain. J Cell Biol 163: 463–468.
Bisogno T, Cascio MG, Saha B, Mahadevan A, Urbani P, Minassi A
et al. (2006). Development of the first potent and specific inhibitors
of endocannabinoid biosynthesis. Biochim Biophys Acta 176:
205–212.
Bisogno T, Burston JJ, Rai R, Allarà M, Saha B, Mahadevan A et al.
(2009a). Synthesis and pharmacological activity of a potent
inhibitor of the biosynthesis of the endocannabinoid
2-arachidonoylglycerol. ChemMedChem 4: 946–950.
Bisogno T, Ortar G, Petrosino S, Morera E, Palazzo E, Nalli M
et al. (2009b). Development of a potent inhibitor of
2-arachidonoylglycerol hydrolysis with antinociceptive activity in
vivo. Biochim Biophys Acta 1791: 53–60.
Cascio MG, Minassi A, Ligresti A, Appendino G, Burstein S,
Di Marzo V (2004). A structure-activity relationship study on
N-arachidonoyl-amino acids as possible endogenous inhibitors of
fatty acid amide hydrolase. Biochem Biophys Res Commun 314:
192–196.
Chang JW, Nomura DK, Cravatt BF (2011). A potent and selective
inhibitor of KIAA1363/AADACL1 that impairs prostate cancer
pathogenesis. Chem Biol 18: 476–484.
Di Marzo V (2008). Targeting the endocannabinoid system: to
enhance or reduce? Nat Rev Drug Discov 7: 438–455.
Di Marzo V, Després JP (2009). CB1 antagonists for obesity – what
lessons have we learned from rimonabant? Nat Rev Endocrinol 5:
633–638.
Fernando SR, Pertwee RG (1997). Evidence that methyl arachidonyl
fluorophosphonate is an irreversible cannabinoid receptor
antagonist. Br J Pharmacol 121: 1716–1720.
Franklin K, Paxinos G (1998). In: the mouse brain in stereotaxic
co-ordinates. Academic Press: San Diego.
Higuchi S, Irie K, Mishima S, Araki M, Ohji M, Shirakawa A et al.
(2010). The cannabinoid 1-receptor silent antagonist O-2050
attenuates preference for high-fat diet and activated astrocytes in
mice. J Pharmacol Sci 112: 369–372.
Acknowledgements
We thank Dr. Fabiana Piscitelli, Institute of Biomolecular
Chemistry, C.N.R. Pozzuoli (Napoli), Italy, for valuable help
and advice.
We are grateful to the NIH for partly funding this study
(Grant No. DA009789).
Higuchi S, Ohji M, Araki M, Furuta R, Katsuki M, Yamaguchi R et al.
(2011). Increment of hypothalamic 2-arachidonoylglycerol induces
the preference for a high-fat diet via activation of cannabinoid 1
receptors. Behav Brain Res 216: 477–480.
Janero DR (2012). Cannabinoid-1 receptor (CB1R) blockers as
medicines: beyond obesity and cardiometabolic disorders to
substance abuse/drug addiction with CB1R neutral antagonists.
Expert Opin Emerg Drugs 17: 17–29.
Conflict of interest
Jung KM, Clapper JR, Fu J, D’Agostino G, Guijarro A,
Thongkham D et al. (2012). 2-arachidonoylglycerol signaling in
forebrain regulates systemic energy metabolism. Cell Metab 15:
299–310.
The authors state no conflict of interest.
792 British Journal of Pharmacology (2013) 169 784–793

A novel inhibitor of endocannabinoid 2-AG biosynthesis
BJP
Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010).
NC3Rs Reporting Guidelines Working Group. Br J Pharmacol
160:1577-1579.
Ortar G, Bisogno T, Ligresti A, Morera E, Nalli M, Di Marzo V
(2008). Tetrahydrolipstatin analogues as modulators of
endocannabinoid 2-arachidonoylglycerol metabolism. J Med Chem
51: 6970–6979.
Long JZ, Li W, Booker L, Burston JJ, Kinsey SG, Schlosburg JE et al.
(2009). Selective blockade of 2-arachidonoylglycerol hydrolysis
produces cannabinoid behavioral effects. Nat Chem Biol 5: 37–44.
Pertwee RG (2008). Ligands that target cannabinoid receptors in the
brain: from THC to anandamide and beyond. Addict Biol 13:
147–159.
Ludányi A, Hu SS, Yamazaki M, Tanimura A, Piomelli D,
Watanabe M et al. (2011). Complementary synaptic distribution
of enzymes responsible for synthesis and inactivation of the
endocannabinoid 2-arachidonoylglycerol in the human
hippocampus. Neuroscience 174: 50–63.
Simiand J, Keane M, Keane PE, Soubrié P (1998). SR 141716, a CB1
cannabinoid receptor antagonist, selectively reduces sweet food
intake in marmoset. Behav Pharmacol 9: 179–181.
Suárez J, Ortíz O, Puente N, Bermúdez-Silva FJ, Blanco E,
Fernández-Llebrez P et al. (2011). Distribution of diacylglycerol
lipase alpha, an endocannabinoid synthesizing enzyme, in the rat
forebrain. Neuroscience 192: 112–131.
McGrath J, Drummond G, Kilkenny C, Wainwright C (2010).
Guidelines for reporting experiments involving animals: the
ARRIVE guidelines. Br J Pharmacol 160: 1573–1576.
Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K et al.
(1995). 2-Arachidonoylglycerol: a possible endogenous cannabinoid
receptor ligand in brain. Biochem Biophys Res Commun 215:
89–97.
Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G,
Cascio MG et al. (2002). The endogenous cannabinoid system
controls extinction of aversive memories. Nature 418: 530–534.
Ward SJ, Raffa RB (2011). Rimonabant redux and strategies to
improve the future outlook of CB1 receptor neutral-
antagonist/inverse-agonist therapies. Obesity (Silver Spring) 19:
1325–1334.
Martin BR, Beletskaya I, Patrick G, Jefferson R, Winckler R,
Deutsch DG et al. (2000). Cannabinoid properties of
methylfluorophosphonate analogs. J Pharmacol Exp Ther 294:
1209–1218.
Mathes CM, Ferrara M, Rowland NE (2008). Cannabinoid-1 receptor
antagonists reduce caloric intake by decreasing palatable diet
selection in a novel dessert protocol in female rats. Am J Physiol
Regul Integr Comp Physiol 295: R67–R75.
Supporting information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE,
Schatz AR et al. (1995). Compton, identification of an endogenous
2-monoglyceride, present in canine gut, that binds to cannabinoid
receptors. Biochem Pharmacol 50: 83–90.
Figure S1 Synthesis, purification and spectroscopic charac-
terization of O-7460.
British Journal of Pharmacology (2013) 169 784–793 793