Synthesis and characterization of a peripherally restricted CB1 cannabinoid antagonist, URB447, that reduces feeding and body-weight gain in mice

Article abstract of DOI:10.1016/j.bmcl.2008.12.059

Cannabinoid CB₁ receptor antagonists reduce body weight in rodents and humans, but their clinical utility as anti-obesity agents is limited by centrally mediated side effects. Here, we describe the first mixed CB₁ antagonist/CB₂

Full text of DOI:10.1016/j.bmcl.2008.12.059

Bioorganic & Medicinal Chemistry Letters 19 (2009) 639–643
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of a peripherally restricted CB1 cannabinoid
antagonist, URB447, that reduces feeding and body-weight gain in mice q
Jesse LoVerme a, Andrea Duranti b, Andrea Tontini b, Gilberto Spadoni b, Marco Mor c, Silvia Rivara c,
a,e,
Nephi Stella d, Cong Xu d, Giorgio Tarzia b, , Daniele Piomelli
*
*
a Department of Pharmacology, 360 MedScience II, University of California, Irvine, CA 92697-4625, USA
b Istituto di Chimica Farmaceutica e Tossicologica, Università degli Studi di Urbino ‘‘Carlo Bo”, Piazza del Rinascimento 6, Urbino 61029, Italy
c Dipartimento Farmaceutico, Università degli Studi di Parma, Viale G. P. Usberti 27/A, Parma 43100, Italy
d Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA
e Department of Drug Discovery and Development, Italian Institute of Technology, via Morego 30, I-16163 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Cannabinoid CB1 receptor antagonists reduce body weight in rodents and humans, but their clinical util-
ity as anti-obesity agents is limited by centrally mediated side effects. Here, we describe the first mixed
CB1 antagonist/CB2 agonist, URB447 ([4-amino-1-(4-chlorobenzyl)-2-methyl-5-phenyl-1H-pyrrol-3-
yl](phenyl)methanone), which lowers food intake and body-weight gain in mice without entering the
brain or antagonizing central CB1-dependent responses. URB447 may provide a useful pharmacological
tool for investigating the cannabinoid system, and might serve as a starting point for developing clinically
viable CB1 antagonists devoid of central side effects.
Received 28 October 2008
Revised 12 December 2008
Accepted 14 December 2008
Available online 24 December 2008
Keywords:
URB447
Peripheral cannabinoid antagonism
Anti-obesity agents
Pyrrolic CB-antagonists
Ó 2009 Elsevier Ltd. All rights reserved.
Cannabinoid type I (CB1) receptors play essential roles in the
control of energy balance. Their activation by agonist drugs stimu-
lates feeding in both rodents and humans,2 while CB1 receptor
blockade counters these effects.3 Clinical studies have shown that
selective CB1 antagonists such as rimonabant or taranabant lower
body weight and improve waist circumference, high-density lipo-
protein cholesterol, triglycerides and insulin sensitivity in over-
weight and obese patients.4 These actions are accompanied,
however, by centrally mediated adverse events, including nausea,
anxiety, and depression, which limit the clinical utility of CB1
antagonism as an anti-obesity strategy.4
CB1 antagonists are thought to reduce food intake by occupying
receptors located in brain circuits involved in the regulation of
feeding and motivated behaviors.5 Emerging evidence indicates,
however, that CB1 receptors present outside the central nervous
system (CNS) also contribute to this response.6 Consistent with a
peripheral site of action for these drugs, the neurotoxin capsaicin
abolishes the anorexic effects of systemically administered rimo-
nabant in rats, while rimonabant fails to alter feeding behavior
when is administered centrally to mice.7 Anatomical data indicate
that CB1 receptors are present on vagal sensory afferent neurons,8
where their expression is regulated by the feeding state.9 Thus,
peripheral CB1 receptors may represent an important mechanism
by which CB1 antagonists regulate food intake and energy balance.
Despite the large number of CB1 receptor antagonists/inverse
agonists developed thus far,10 compounds characterized by limited
access to the CNS are still lacking. A triazole derivative, LH-21 (5-
(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-tria-
zole), was reported to be a neutral CB1 antagonist with no brain
penetrability,11 but its plasma-to-brain concentration ratio has
been recently reported to be near 1:1 in rats after systemic
administration.12
In the present study, we describe a compound that mimics the
anorexic and anti-obesity effects of the prototypical CB1 antago-
nist, rimonabant, without antagonizing centrally mediated CB1-re-
sponses. This compound, URB447, belongs to a class of pyrrole-
based cannabimimetics previously described by us.13 We observed
that the introduction of a p-chlorobenzyl group onto position 1 and
of a 1-naphthoyl group onto position 3 of a 2,5-dimethylpyrrole
scaffold afforded a compound that displayed partial agonism at
CB1 receptor. Moreover, position 4 resulted tolerant to substitu-
tion, at least by a bromine atom. To develop antagonists from this
pyrrole-based chemical scaffold we introduced a phenyl ring in po-
sition 5, to mimic the substituted 5-phenyl ring on the pyrazole
q
See Ref. 1.
* Corresponding authors. Tel.: +39 0722 303325; fax: +39 0722 303313 (G.T.),
tel.: +1 949 824 6180; fax: +1 949 824 6305 (D.P.).
E-mail addresses: giorgio.tarzia@uniurb.it (G. Tarzia), piomelli@uci.edu (D.
Piomelli).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.059

640
J. LoVerme et al. / Bioorg. Med. Chem. Lett. 19 (2009) 639–643
scaffold of rimonabant and SR144528, two selective CB1 and CB2
antagonists, respectively. The group at position 3 and the substitu-
ent at position 4 were then modulated to test the effect of lipophil-
icity variation on receptor binding affinity. Compounds 3a–c, 4, 5b,
8a and 8b (Table 1) were thus synthesized and tested for their abil-
ity to displace binding of the CB agonist [3H]WIN55,212-2 from rat
cerebellar membranes (which mostly contain CB1 subtype) and
from CHO-K1 cells overexpressing human CB2 receptors.
The exploration started from the 3-benzoyl derivative 8a, a
weak CB2 ligand, in which a polar fragment was introduced either
replacing the ketone function by an amide (3a–c, 4) or with an
amino group in position 4 (8b, URB447). The amide derivatives
showed interesting affinity data for the adamantyl derivative 3c,
but, differently from what observed in the previous series of pyr-
roles,13 introduction of a bromine in position 4 resulted in a signif-
icant decrease in CB1 receptor binding affinity. Interestingly, the 3-
benzoyl-4-amino derivative, URB447, showed significant affinity
for both receptor subtypes, indicating that the polar amino group
can produce favorable interactions at the two binding sites. Finally,
the role of the N1-p-chlorobenzyl group is confirmed by the inac-
tivity of compound 5b.
The synthesis of compounds 3a–c started with the alkylation of
2-methyl-5-phenyl-1H-pyrrol-3-carboxylic acid ethyl ester (1)14
,
followed by hydrolysis of the ester group of 2 and amidation via
acyl chloride. Compound 4 was obtained by bromination of 3c with
N-bromosuccinimide (Scheme 1). To prepare 8b (URB447), (4-ami-
no-2-methyl-5-phenylpyrrol-3-yl)(phenyl)methanone (5b)15 was
converted to the corresponding imine (6) by condensation with
4-chlorobenzaldehyde and p-toluensulphonic acid in the presence
of toluene. The imine was N1-alkylated with 4-chlorobenzylchlo-
ride and hydrolyzed to afford the desired compound16 (Scheme
2). Compound 8a was synthesized by alkylation of (2-methyl-5-
phenyl-1H-pyrrol-3-yl)(phenyl)methanone (5a)17 (Scheme 2).
In rat cerebellar membrane preparations, URB447 (100 nM–
30
WIN55,212-2 (1
WIN-5512-2, URB447 (100 nM–10
GTP S binding (data not shown), suggesting that the compound
l
M) inhibited GTP
M) (EC50 = 4.9 0.8
M) failed to inhibit basal
c
S
binding induced by the CB agonist
l
lM). In the absence of
l
c
acts as neutral antagonist at CB1 receptors.
In Hek-293 cells stably expressing mouse CB2 receptors,
URB447 (1
lM) inhibited cAMP accumulation induced by the b-
adrenergic agonist isoproterenol (10
l
M) (cAMP levels in pmol/
well: basal, 18 2.4; URB447, 5.9 1.2; isoproterenol, 56.2 6; iso-
proterenol plus URB447, 14.3 1.2; n = 4). Similar results were ob-
tained with a higher concentration of URB447 (10 lM, data not
Table 1
a,c
b,c
Structural formulas and IC50 (nM) values for rat CB1 or human CB2 receptors
shown). The results suggest that URB447 acts as an agonist at
CB2 receptors.
O
O
R2
R2
To determine the effects of URB447 on food intake, we admin-
istered the drug (5 or 20 mg-kgÀ1, i.p.) or its vehicle to Swiss mice
30 min before dark onset and monitored feeding for 24 h. Vehicle
or a low-dose of URB447 (5 mg-kgÀ1, i.p.) had no effect on food in-
take, whereas a high-dose of URB447 (20 mg-kgÀ1, i.p.) drastically
reduced total food consumption (Fig. 1A). This effect was mimicked
by rimonabant (20 mg-kgÀ1, i.p.) (Fig. 1B). The reduction in food in-
take was not likely to be caused by non-specific behavioral effects,
as URB447 (20 mg-kgÀ1, i.p.) did not alter motor activity (data not
shown). The results indicate that URB447 reduces free feeding in
mice.
To identify potential sites of action of URB447, we measured
drug levels in various tissues after systemic administration
(20 mg-kgÀ1, i.p.). Plasma URB447 levels peaked 30 min after i.p.
injection (Cmax 596 117 nM) and maximal tissue levels were
reached 15 min post-injection in liver (Cmax 4.3 0.7 nmol/g) and
white adipose fat (Cmax 42 12.2 nmol/g). Strikingly, URB447 was
not detected in brain tissue at any time after administration
(detection limit in the assay was 10 pmol/g) (data not shown).
These findings suggest that URB447 does not penetrate the brain,
R1
R1
N
N
H
Cl
5b
3a-c,4,8a,8b
Compound
R1
R2
CB1 (nM)
CB2 (nM)
3a
3b
3c
4
5b
8a
NH-1-piperidinyl
NH-2-norbornyl
NH-2-adamantyl
NH-2-adamantyl
C6H5
H
H
H
Br
NH2
H
NH2
>1000
>1000
70 10
>1000
>1000
>1000
313 72
>1000
420 18
11
4
123 29
>1000
498 32
41 23
C6H5
C6H5
8b URB447
Inhibition of [3H]WIN55,212-2 binding:
a
Rat cerebellar membranes.
b
CHO-K1 cells overexpressing human CB2 receptors. Values are means of four
experiments; standard deviation is given.
IC50 values of rimonabant are 5.2 1.8 nM18 (Ki = 1.98 0.36 nM)19 for CB1 and
c
>1000 nM for CB2.20
O
O
O
LiOH/H2O, MeOH, H2O
(COCl)2, DMF
RNH2, TEA, CH2Cl2
O
NaH, DMF
N
p-ClPhCH2Cl
N
H
Cl
1
2
O
O
Br
R
R
3a R = NH-1-piperidinyl
3b R = NH-2-norbornyl
3c R = NH-2-adamantyl
NBS, dioxane, AcOH
N
N
Cl
Cl
4
Scheme 1.

J. LoVerme et al. / Bioorg. Med. Chem. Lett. 19 (2009) 639–643
p-ClPh
641
O
O
N
R
p-ClPhCHO
N
N
PTSA, toluene
H
H
5a,b
6
NaH
DMF
NaH
DMF
p-ClPhCH2Cl
p-ClPhCH2Cl
O
p-ClPh
O
R
N
HCl 10%
DMF
N
N
Cl
Cl
8a,b
7
a R = H, b R = NH2
Scheme 2.
and that its anorexic effects result from the engagement of periph-
eral CB receptors. To further test this possibility, we asked if
URB447 selectively prevents peripheral effects of CB agonists.
The stable anandamide analogue (R)-methanandamide inhibits
nocifensive behavior in the formalin plantar test (Fig. 2A) through a
peripheral mechanism.21 Pretreatment with either URB447
Figure 2. (A) Effects of vehicle, URB447 (URB, 20 mg-kgÀ1, i.p.) or rimonabant (RIM,
2 mg-kgÀ1, i.p.) administered 30 min before intraplantar formalin co-injected with
or without (R)-methanandamide (mAEA, 50 lg, i.p.) on first (0–15 min) and second
(15–45 min) phase formalin-evoked nociception in Swiss mice (n = 8–13). (B)
Effects of a 30 min pretreatment (injection #1) of vehicle, URB447 (20 mg-kgÀ1, i.p.)
or rimonabant (2 mg-kgÀ1, i.p.) on WIN-55,212 (3 mg-kgÀ1, i.p., injection #2) –
induced hypothermia (n = 5). (C) Effects of vehicle, URB447 (URB, 20 mg-kgÀ1, i.p.)
or rimonabant (RIM, 5 mg-kgÀ1
WIN55,212-2 (5 mg-kgÀ1
,
i.p.) administered 30 min before vehicle or
,
i.p.) on cataleptic behavior measured 30 min after
WIN55,212-2 treatment (n = 6). Results were analyzed using a 1-way or 2-way
repeated measures ANOVA followed by Bonferroni post hoc tests as appropriate.
*P < 0.05, **P < 0.01 vs vehicle; P < 0.01 vs mAEA. Error bars represent SEM.
(20 mg-kgÀ1 i.p.) or rimonabant (2 mg-kgÀ1
, , i.p.) completely
blocked the analgesic effects of methanandamide (Fig. 2A), sug-
gesting that URB447 inhibits peripheral CB1-mediated analgesia.
We next induced centrally mediated hypothermia in Swiss mice
by administering a single dose of the CB agonist WIN55,212-2
(3 mg-kgÀ1, i.p.). 30 min after WIN55,212-2 administration the
mice developed maximal hypothermia, which was attenuated by
pretreatment with rimonabant (2 mg-kgÀ1, i.p.), but not URB447
(20 mg-kgÀ1, i.p.) (Fig. 2B). To investigate the effects of URB447
on catalepsy, another central effect of CB1 agonists, we adminis-
tered URB447 (20 mg-kgÀ1, i.p.) or rimonabant (5 mg-kgÀ1, i.p.)
30 min prior to a single injection of vehicle or WIN55,212-2
(5 mg-kgÀ1) and scored cataleptic behavior 30 min later. Mice trea-
ted with WIN55,212-2 exhibited a significant increase in the time
spent cataleptic in the ring catalepsy test (Fig. 2C). This response
Figure 1. Effects of vehicle (open circles), URB447 (URB) or rimonabant (RIM) (each
at 5 mg-kgÀ1, closed diamonds, or 20 mg-kgÀ1, closed circles, i.p.) on cumulative
food intake in male Swiss mice (n = 12). Results were analyzed using a 2-way
repeated measures ANOVA followed by
a Bonferroni post hoc test. *P < 0.05,
**P < 0.01 vs vehicle. Error bars represent SEM.

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J. LoVerme et al. / Bioorg. Med. Chem. Lett. 19 (2009) 639–643
failed to inhibit WIN55,212-2 induced hypothermia or catalepsy.
The ability of URB447 to reduce food intake suggests that this
CNS-impermeant antagonist produces anorexia by blocking CB1
receptors in peripheral organs such as the gastrointestinal tract,
in which endocannabinoids may act as local orexigenic hor-
mones.7,9 We cannot exclude the possibility, however, that CB2
receptor activation might contribute to the effects of URB447. In-
deed, the CB2 agonist JWH133 was shown to improve glucose tol-
erance after a glucose load26 and ameliorate liver disease27
,
suggesting a possible role of CB2 in metabolic regulation. Irrespec-
tive of these speculations, our findings are clinically relevant be-
cause rimonabant and other brain-permeant CB1 antagonist exert
serious psychiatric side effects, which limit their clinical usefulness
in anti-obesity therapy.
Acknowledgments
The Italian Ministry of Instruction, University and Research,
Universities of Urbino ‘‘Carlo Bo” and Parma, and the Center of
Drug Discovery at the University of California, Irvine supported this
work. The authors thank Fariba Oveisi for technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.12.059.
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Figure 3. Effects of vehicle, URB447 (URB, 20 mg-kgÀ1, i.p.) or rimonabant (RIM,
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was unaffected by URB447, but was reversed by pretreatment with
rimonabant (Fig. 2C). These findings indicate that URB447 selec-
tively prevents peripherally mediated CB responses in mice.
To test whether URB447 inhibits body-weight gain, we treated
genetically obese ob/ob mice once daily with URB447, rimonabant
(each at 20 mg-kgÀ1, i.p.) or vehicle for 2 weeks. In keeping with
our previous results (Fig. 1), we found that URB447 produced a sig-
nificant reduction in the amount of food consumed throughout the
treatment period (Fig. 3A). This effect was accompanied by a signif-
icant attenuation of body-weight gain (Fig. 3B) and was similar in
magnitude to that of rimonabant (Fig. 3A and 3B).
Contrary to the present findings, URB447 would be expected to
cross the blood–brain barrier by passive diffusion,22 considering
that its molecular weight (MW = 401), lipophilicity (estimated
LogP = 6.39) and polar surface area (PSA = 48.02) are too similar
to those of rimonabant (MW = 464, LogP = 6.01, PSA = 50.1).23 The
unexpected lack of CNS penetration for URB447 may then be due
to high clearance by efflux transporters (e.g., P-gp, MRP1-6 or
BCRP)24 or by rapid metabolism by P450 cyrochromes in the
astrocytes.25
16. To a solution of 7 (0.261 g, 0.5 mmol) in DMF (2.5 mL), 10% HCl (1.3 mL) was
added slowly (exothermic reaction) at room temperature. After stirring at
room temperature for 6 h, the mixture was poured onto 2 N Na2CO3 and
extracted (CH2Cl2). The organic layer was dried (Na2SO4) and concentrated.
Purification of the residue by column chromatography (cyclohexane EtOAc
8:2) and recrystallization gave URB447 as a yellow solid. Yield 66% (0.132 g).
Mp 128–130 °C (EtOAc-petroleum ether). MS (EI): m/z 400 (M+), 275 (100). 1H
NMR (CDCl3): d 1.82 (s, 3H); 4.29 (br s, 2H); 4.97 (s, 2H); 6.84 (d, 2H); 7.24–
7.51 (m, 10H); 7.68 (m, 2H) ppm. IR (nujol): 3444, 3359, 1605, 1595 cmÀ1
.
In conclusion, in the present study we have identified URB447
as the first peripherally restricted mixed CB1 antagonist/CB2
agonist.
We have shown that URB447 reduces food intake and body-
weight gain in mice with an efficacy comparable to that of the
standard of reference rimonabant. Furthermore, URB447 reversed
the analgesic effect of (R)-methanandamide without entering the
brain or antagonizing CB1 receptors in the CNS. Indeed, URB447
Anal. calcd for C25H21ClN2O (400.90): C, 73.84; H, 5.48; N, 6.62. Found: C,
74.03; H, 5.21; N, 6.90.
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