Synthesis and biological evaluation of bivalent cannabinoid receptor ligands based on hCB2R selective benzimidazoles reveal unexpected intrinsic properties

doi:10.1016/j.bmc.2014.06.008

Bioorganic & Medicinal Chemistry

Volume 22, Issue 15, 1 August 2014, Pages 3938-3946

https://doi.org/10.1016/j.bmc.2014.06.008 Get rights and content

Abstract

The design of bivalent ligands targeting G protein-coupled receptors (GPCRs) often leads to the development of new, highly selective and potent compounds. To date, no bivalent ligands for the human cannabinoid receptor type 2 (hCB₂R) of the endocannabinoid system (ECS) are described. Therefore, two sets of homobivalent ligands containing as parent structure the hCB₂R selective agonist 13a and coupled at different attachment positions were synthesized. Changes of the parent structure at these positions have a crucial effect on the potency and efficacy of the ligands. However, we discovered that bivalency has an influence on the effect at both cannabinoid receptors. Moreover, we found out that the spacer length and the attachment position altered the efficacy of the bivalent ligands at the receptors by turning agonists into antagonists and inverse agonists.

Graphical abstract

Introduction

Since the identification of (−)-trans-Δ⁹-tetrahydrocannabinol as the major bioactive component in Cannabis sativa L. by Gaoni and Mechoulam in 1964,1, 2 the biological system behind these physiological effects of natural cannabinoids was intensively studied. Moreover, the endocannabinoid system (ECS) was discovered, which consists of the endogenous ligands (endocannabinoids like anandamide or 2-arachidonoyl glycerol), the synthesis and deactivation of these ligands, the cannabinoid receptors and the intracellular signaling pathways activated by the endogenous ligands at the receptors. Till now, many ligands targeting the ECS were synthesized and two subtypes of cannabinoid receptors in humans were identified and characterized as G protein-coupled receptors (GPCRs), the human cannabinoid receptor type 1 (hCB₁R) and human cannabinoid receptor type 2 (hCB₂R).3, 4, 5, 6

In the past decades, for many GPCRs bivalent ligands were designed, which consist of two ligands connected by a spacer. If both parent ligands (pharmacophores) are identical, then the bivalent ligand is called homobivalent, otherwise heterobivalent. For parent ligands, which are potent and selective to one GPCR subtype, and linked at a preferred position by an appropriate spacer with regards to chemical structure and length, it can be possible to achieve bivalent ligands with a high affinity for their receptor subtypes.7, 8 If these bivalent ligands bridge two neighboring, orthosteric binding sites (i.e., the main binding site of the endogenous ligands) of two interacting GPCRs (homo- or heterodimers) or bind at orthosteric and allosteric binding sites (i.e., sites distinct from the orthosteric one, but able to influence binding of orthosteric ligands) of one GPCR, depends on the spacer structure and length, and this is often not clearly distinguishable.7, 8, 9, 10

However, so far it is possible to get bivalent ligands, which bind to a receptor subtype with a higher potency and selectivity than the original parent ligand or the corresponding univalent compound, which only consists of the pharmacophore connected with a spacer, for example, for the μ-opioid receptor11, 12, 13, 14 or dopamine D₂ receptor.15, 16, 17, 18, 19 This suggests that bivalency can have a positive effect on the binding properties and furthermore can lead to new options in the research and study of receptor subtypes, but also in the development of new drugs, for example, hexoprenaline (a bivalent norepinephrine coupled with a hexane spacer) as a selective β₂ adrenergic receptor agonist applied in the treatment of asthma (Fig. 1).20, 21

For the cannabinoid receptors the first ‘bivalent’ cannabinoids were synthesized by connecting cannabinoids with an ethane bridge in 1981.²² Afterwards, the next bivalent ligands were selective for the hCB₁R by linking of the known, selective hCB₁R inverse agonist drug rimonabant at the carboxamide with different spacers instead of piperidine (Fig. 1).23, 24 Hereby, a suitable spacer structure consisted of two hydrophobic alkylene chains at a secondary or tertiary amine. Their optimal spacer length for the highest receptor affinity was 15 atoms.²³ Moreover, heterobivalent ligands targeting the CB₁R and μ-opioid receptor were developed by coupling of modified rimonabant with the known, selective μ-opioid receptor agonist α-oxymorphamine.²⁵ But, no bivalent ligands for the CB₂R have been reported to date.

Therefore, we designed bivalent ligands for the hCB₂R. An appropriate hCB₂ ligand may be the highly selective and potent hCB₂R agonist 13a, which was developed by AstraZeneca and published in 2008 (Fig. 2).²⁶ Assumed attachment positions were the para-ethoxy group of the benzyl substituent in position 2, the N-alkyl substituent in position 1 and the amide at the carboxyl group in position 5 of the benzimidazole (shown in Fig. 2). At these positions, it is feasible to introduce a spacer by the synthetic way without significant changes at the ligand’s chemical structure.

The para-ethoxy group is a determining factor for the efficacy of the pharmacophore as an agonist²⁷ and therefore this position is not a suitable connection position to evaluate the effect of bivalency on ligand binding. At the nitrogen of the imidazole branched, cyclic or aromatic substituents were introduced. Beside these hydrophobic side chains, ligands with basic substituents had also been synthesized.²⁶ In order to prove that a linear substituent can be tolerated there and consequently this position is suited for linear spacers, a modified ligand 13b with a linear alkyl chain at the imidazole was synthesized and tested. Then the parent structure was coupled at the imidazole with different spacers to achieve a set of bivalent and univalent ligands (17a–b, 21a–c). On position 5 of the benzimidazole, different carboxamides with linear and large hydrophobic amines have been synthesized already.²⁶ In addition, other functional groups like amido, ido, sulfonamido or sulfonyl had been introduced.26, 28, 29, 30, 31 Therefore, we presumed that the amide is also an appropriate position to link the parent ligand. It was connected with different spacers at the amide to achieve a second set of bivalent and univalent ligands (26a–b, 31a–b). We presumed that these bivalent compounds for both attachment positions can selectively target the hCB₂R and may be new pharmacological probes to investigate this receptor subtype. Additionally, the methyl ester at position 5 of the benzimidazole (13c) was synthesized to find out, whether the ester function is tolerated, and thus bivalent ligands with an ester coupling might be promising.¹²

Short (7–9 atoms) and long (21 atoms) spacers were utilized to investigate the influence of spacer length on potency and efficacy. These spacers had the same structure as the suitable spacers for bivalent, hCB₁R selective inverse agonists with a tertiary amine.²³ The symmetric, bivalent spacers consists of two alkylene chains combined through methylamine. For the univalent spacers we used the ‘half’ bivalent spacers, the alkylene chain is linked to dimethylamine. We consider these univalent ligands were necessary to study the influence of bivalency on the effect of the ligands at the receptors and to distinguish this effect of bivalent from the univalent ones, which we assumed to differ from the parent ligand.

The potency and efficacy of all compounds at the hCB₂R and hCB₁R were determined in a functional steady-state GTPase assay as previously described.32, 33, 34

Section snippets

Chemistry

The spacers were synthesized via two strategies (Scheme 1): The first possibility is the substitution of the bromide from an ω-bromoalkylnitrile with methylamine to get the corresponding tertiary amine 1. The cyano groups were then reduced by tetrahydrofuran-borane to the primary amines of the bivalent spacer 5c. For longer spacers, the ω-bromoalkylnitrile has to be synthesized by a reaction of α,ω-dibromoalkane with sodium cyanide. For spacers with ten carbon atoms on each side, however, the

Conclusion

In summary, two series of homobivalent ligands with different attachment points containing the parent ligand (hCB₂R selective agonist 13a) and their corresponding univalent ligands were designed and synthesized to target the hCB₂R. Biological evaluation of these compounds in a functional steady-state GTPase assay demonstrates that changes of the parent structure at the imidazole or amide have a crucial effect on the potency and efficacy at the hCB₂R and hCB₁R. They still showed a micromolar

General methods

Unless indicated otherwise, all chemicals and solvents were purchased from commercial suppliers and used without further purification. THF was dried by reflux over sodium overnight and freshly distilled before use. For reactions with dry, freshly distilled THF oven-dried glassware was used. Reactions over argon or nitrogen atmosphere were carried out by filling the reaction apparatuses by a gas flow of the corresponding, commercially available gas and afterwards closing the filled reaction

Acknowledgment

M. Decker gratefully acknowledges the German Research Foundation (DFG) for financial support (DFG DE 1546/4-1).

Appreciation is expressed to Professor S. Elz for supporting the synthesis in his chemical laboratories, and to M. Beer-Krön, M. Flemming and S. Pockes for support in performance of the steady-state GTPase assay.

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Synthesis and biological evaluation of bivalent cannabinoid receptor ligands based on hCB2R selective benzimidazoles reveal unexpected intrinsic properties

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Conclusion

General methods

Acknowledgment

Best Pract. Res. Clin. Endocrinol.

Prog. Neuro-Psychopharmacol.

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Life Sci.

Life Sci.

Life Sci.

J. Am. Chem. Soc.

J. Am. Chem. Soc.

Pharmacol. Rev.

Alcohol Alcohol.

J. Med. Chem.

ChemMedChem

Angew. Chem., Int. Ed.

Synthesis and biological evaluation of bivalent cannabinoid receptor ligands based on hCB₂R selective benzimidazoles reveal unexpected intrinsic properties