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

Cannabinoid CB 1 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 CB 1 antagonist/CB 2 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 CB 1 -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 CB 1 antagonists devoid of central side effects. CB-antagonists Cannabinoid type I (CB 1 ) receptors play essential roles in the control of energy balance. Their activation by agonist drugs stimulates feeding in both rodents and humans, 2 while CB 1 receptor blockade counters these effects. 3 Clinical studies have shown that selective CB 1 antagonists such as rimonabant or taranabant lower body weight and improve waist circumference, high-density lipoprotein cholesterol, triglycerides and insulin sensitivity in overweight 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 CB 1 antagonism as an anti-obesity strategy. 4 CB 1 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 CB 1 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 rimonabant in rats, while rimonabant fails to alter feeding behavior when is administered centrally to mice. 7 Anatomical data indicate that CB 1 receptors are present on vagal sensory afferent neurons, 8 where their expression is regulated by the feeding state. 9 Thus, peripheral CB 1 receptors may represent an important mechanism by which CB 1 antagonists regulate food intake and energy balance.
Despite the large number of CB 1 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 CB 1 antagonist with no brain penetrability, 11 but its plasma-tobrain 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 CB 1 antagonist, rimonabant, without antagonizing centrally mediated CB 1 -responses. This compound, URB447, belongs to a class of pyrrole-based cannabimimetics previously described by us. 13 We observed that the introduction of a pchlorobenzyl group onto position 1 and of a 1-naphthoyl group onto position 3 of a 2,5dimethylpyrrole scaffold afforded a compound that displayed partial agonism at CB 1 receptor. Moreover, position 4 resulted tolerant to substitution, at least by a bromine atom. To develop antagonists from this pyrrole-based chemical scaffold we introduced a phenyl ring in position 5, to mimic the substituted 5-phenyl ring on the pyrazole scaffold of rimonabant and SR144528, two selective CB 1 and CB 2 antagonists, respectively. The group at position 3 and the substituent at position 4 were then modulated to test the effect of lipophilicity variation on receptor binding affinity. Compounds 3a-c, 4, 5b, 8a and 8b (Table 1) were thus synthesized and tested for their ability to displace binding of the CB agonist [ 3 H]WIN55,212-2 from rat cerebellar membranes (which mostly contain CB 1 subtype) and from CHO-K1 cells overexpressing human CB 2 receptors.
The exploration started from the 3-benzoyl derivative 8a, a weak CB 2 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 pyrroles, 13 introduction of a bromine in position 4 resulted in a significant decrease in CB 1 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 N 1 -p-chlorobenzyl group is confirmed by the inactivity of compound 5b.
To determine the effects of URB447 on food intake, we administered the drug (5 or 20 mgkg −1 , 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-kg −1 , i.p.) had no effect on food intake, whereas a high-dose of URB447 (20 mg-kg −1 , i.p.) drastically reduced total food consumption (Fig. 1A). This effect was mimicked by rimonabant (20 mg-kg −1 , i.p.) (Fig.  1B). The reduction in food intake was not likely to be caused by non-specific behavioral effects, as URB447 (20 mg-kg −1 , 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-kg −1 , i.p.). Plasma URB447 levels peaked 30 min after i.p. injection (C max 596 ± 117 nM) and maximal tissue levels were reached 15 min postinjection in liver (C max 4.3 ± 0.7 nmol/g) and white adipose fat (C max 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, and that its anorexic effects result from the engagement of peripheral CB receptors. To further test this possibility, we asked if URB447 selectively prevents peripheral effects of CB agonists.
To test whether URB447 inhibits body-weight gain, we treated genetically obese ob/ob mice once daily with URB447, rimonabant (each at 20 mg-kg −1 , i.p.) or vehicle for 2 weeks. In keeping with our previous results (Fig. 1), we found that URB447 produced a significant reduction in the amount of food consumed throughout the treatment period (Fig. 3A). This effect was accompanied by a significant 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 (M W = 401), lipophilicity (estimated LogP = 6.39) and polar surface area (PSA = 48.02) are too similar to those of rimonabant (M W = 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 In conclusion, in the present study we have identified URB447 as the first peripherally restricted mixed CB 1 antagonist/CB 2 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 CB 1 receptors in the CNS. Indeed, URB447 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 CB 1 receptors in peripheral organs such as the gastrointestinal tract, in which endocannabinoids may act as local orexigenic hormones. 7,9 We cannot exclude the possibility, however, that CB 2 receptor activation might contribute to the effects of URB447. Indeed, the CB 2 agonist JWH133 was shown to improve glucose tolerance after a glucose load 26 and ameliorate liver disease 27 , suggesting a possible role of CB 2 in metabolic regulation. Irrespective of these speculations, our findings are clinically relevant because rimonabant and other brain-permeant CB 1 antagonist exert serious psychiatric side effects, which limit their clinical usefulness in antiobesity therapy.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.   Effects of vehicle, URB447 (URB, 20 mg-kg −1 , i.p.) or rimonabant (RIM, 20mg-kg −1 , i.p.) administered daily for 2 weeks on (A) food intake or (B) body weight (expressed as % change from day 0) in ob/ob mice (n = 8). Results were analyzed using a 2-way repeated measures ANOVA followed by a Bonferroni post hoc test. **P < 0.01 vs vehicle. Error bars represent SEM.