Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic acid phenylamide (CP-272871) for the CB1 cannabinoid receptor

doi:10.1016/S0006-2952(00)00447-0

Biochemical Pharmacology

Volume 60, Issue 9, 1 November 2000, Pages 1315-1323

https://doi.org/10.1016/S0006-2952(00)00447-0 Get rights and content

Abstract

Two subtypes of cannabinoid receptors are currently recognized, CB₁, found in brain and neuronal cells, and CB₂, found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB₁ receptor. CP-272871 competed for binding of the cannabinoid agonist ³H-labeled (−)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([³H]CP-55940) at the CB₁ receptor in rat brain membranes with a K_d value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A). CP-272871 also competed for binding with the aminoalkylindole agonist ³H-labeled (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl)methanone ([³H]WIN-55212–2), as well as the aryl pyrazole antagonist [³H]SR141716A. Inverse agonist as well as antagonist properties were observed for both SR141716A and CP-272871 in signal transduction assays in biological preparations in which the CB₁ receptor is endogenously expressed. SR141716A augmented secretin-stimulated cyclic AMP (cAMP) accumulation in intact N18TG2 neuroblastoma cells, and this response was reversed by the agonist desacetyllevonantradol. CP-272871 antagonized desacetyllevonantradol-mediated inhibition of adenylyl cyclase in N18TG2 membranes, and increased adenylyl cyclase activity in the absence of agonist. SR141716A and CP-272871 antagonized desacetyllevonantradol-stimulated ³⁵S-labeled guanosine-5′-O-(γ-thio)-triphosphate ([³⁵S]GTPγS) binding to brain membrane G-proteins, and decreased basal [³⁵S]GTPγS binding to G-proteins. K⁺ enhanced CP-272871 and SR141716A inverse agonist activity compared with Na⁺ or NMDG⁺ in the assay. These results demonstrated that the aryl pyrazoles SR141716A and CP-272871 behave as antagonists and as inverse agonists in G-protein-mediated signal transduction in preparations of endogenously expressed CB₁ receptors.

Section snippets

Materials

Reagent grade chemicals obtained from the Sigma Chemical Co. were used for these studies. DALN, CP-272871, and SR141716A for these studies were gifts of Pfizer, Inc. CHO cells expressing the human CB₁ receptor (CHO-HCR) and control untransfected CHO cells were provided by Dr. M. Parmentier at the Université Libre de Bruxelles [14].

The P2 membranes from rat brains were prepared using the method of Devane et al.[15], and the human tonsil membranes were prepared employing the same method, except

CP-272871 binding to cannabinoid receptors

The ability of CP-272871 to bind the CB₁ receptors in rat brain membranes was established using the CB₁ agonists [³H]CP-55940 and [³H]WIN-55212–2. CP-272871 displaced [³H]CP-55940 with a K_i = 57 nM and [³H]WIN-55212–2 with a K_i = 92 nM (Fig. 2). Heterologous competition of [³H]CP-55940 demonstrated that SR141716A has a K_i = 3.1 nM. CP-272871 displaced [³H]SR141716A with a K_i of 39 nM using these experimental conditions. These heterologous competition data demonstrated that CP-272871 binds to

Discussion

This study has introduced CP-272871 as a CB₁ cannabinoid receptor antagonist. CP-272871 exhibited a lower affinity for the CB₂ cannabinoid receptor, with relative selectivity for the CB₁ receptor of 2-fold. CP-272871 also exhibited competitive inhibitory properties against the cannabinoid agonist DALN in activation of G-proteins ([³⁵S]GTPγS binding) and the adenylyl cyclase signal transduction pathway.

The present study describes inverse agonist properties of the aryl pyrazole antagonists

Acknowledgements

These studies were supported, in part, by National Institute on Drug Abuse Grants R01-DA03690, DA06312, and K05–00182 to A. C. H and F30-DA05806 to J. P. M

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^∗: Current address: Proctor and Gamble Health Care Research Center, Mason, OH 45040.

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Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic acid phenylamide (CP-272871) for the CB1 cannabinoid receptor

Abstract

Section snippets

Materials

CP-272871 binding to cannabinoid receptors

Discussion

Acknowledgements

Biochem Pharmacol

FEBS Lett

J Biol Chem

Eur J Pharmacol

Trends Pharmacol Sci

Trends Pharmacol Sci

Trends Pharmacol Sci

Anal Biochem

Life Sci

Trends Pharmacol Sci

Biochem Biophys Res Commun

Biochem Biophys Res Commun

Biochem Pharmacol

FEBS Lett

Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic acid phenylamide (CP-272871) for the CB₁ cannabinoid receptor