It has been the objective of medicinal chemists and pharmacologists to synthesize and screen novel opioids to find ligands which would retain the analgesic potency of currently available agonists, such as morphine, with a much improved side effect and abuse liability profile. However, such a compound has remained elusive. One approach has been to synthesize ligands which were designed to bridge two opioid receptors, or dimers (2
The concept of G-protein coupled receptor (GPCR) dimerization has evolved tremendously, since its inception over 20 years ago (4
). Studies have demonstrated that opioid receptor dimerization alters signaling (5
), trafficking (5
), and internalization (10
). It also has been suggested that opioid receptor heterodimers could explain pharmacological subtypes (3
), such as δ1
which can be observed, in vivo, with the receptor-selective agonists such as [D-Pen2
]enkephalin (DPDPE) and ([D-Ala2
]-Deltorphin II (Deltorphin II) respectively, yet, these same subtypes have not been found utilizing cloning methods.
As opioid receptor dimerization has become recognized as an important phenomenon, many ligands have been synthesized to directly target these complexed receptors, and are generally termed bivalent ligands. Several key considerations in the design of these bivalent ligands are: to choose a monomeric ligand with the desired pharmacological profile, which also has the necessary connecting sites; and to find an appropriate spacer with the correct length and character to allow for bridging of the receptors (11
Previously, our laboratories have reported on a series of ligands for the μ and κ opioid receptors. One monovalent ligand, of particular interest, has been (−)MCL-101, a N-cyclobutylmethyl analogue of cyclorphan (1
). In radioligand binding assays, (−)MCL-101 displayed high affinity for both the μ and κ receptors. (−)MCL-101 was also shown to be a full agonist at the κ receptor and displayed mixed agonist/antagonist properties at the μ receptor, as measured by the [35
S]GTPγS assay (1
) (–). In vivo, (−)MCL-101 acted as a κ agonist with μ agonist/antagonist properties (1
Ki Inhibition values of μ, δ, and κ opioid binding to CHO membranes by MCL-101, (−)(−)MCL-145, (−)(−)MCL-144, (+)(−)MCL-193, and (+)(+)MCL-192
Agonist and antagonist properties of MCL-101, (−)(−)MCL-145, (−)(−) MCL-144, (+)(−) MCL-193, and (+)(+)MCL-192 in stimulating [35S]GTPγS binding mediated by the μ opioid receptor
The characterization of this compound was important, as it now serves as the pharmacologically active (−) pharmacophore for a series of novel bivalent ligands. Within the bivalent ligand series, we previously reported on (−)(−)MCL-145, a bivalent compound with two (−)MCL-101 pharmacophores bridged by a conformationally constrained fumaryl ester (2
) (). In both radioligand binding and [35
S]GTPγS assays, (−)(−)MCL-145 displayed virtually identical pharmacological properties to (−)MCL-101 (1
) (–). In vivo, (−)(−)MCL-145 was a κ agonist and μ partial agonist, similar to (−)MCL-101 (1
Chemical structures of the monovalent pharmacophore, (−)MCL-101, (+)MCL-101 and the bivalent ligands, (−)(−)MCL-145, (−)(−)MCL-144, (+)(−)MCL-193 and (+)(+)MCL-192.
Further studies have yielded two isomeric, bivalent ligands, (−)(−)MCL-144 and (+)(−) MCL-193 (). Both compounds are linked by a 10-carbon chain ester, and the active enantiomer for both ligands is (−)MCL-101. In radioligand binding assays, (−)(−)MCL-144 displayed higher affinity for both the μ and κ receptors (Ki
= 0.09 nM and 0.05 nM, respectively) compared to (+)(−)MCL-193 (Ki
= 2.2 nM and 1.2 nM, respectively) (2
) (). For comparison, the bivalent ligand, (+)(+)MCL-192 (11
) (), was also studied in the radioligand binding assay, and displayed poor affinity for the μ, κ, and δ receptors, respectively (Ki
= 130 nM, 700 nM, and 130 nM) (2
) (). These data demonstrated that the compounds with (−) pharmacophores: (−)MCL-101, (−)(−) MCL-145, and (−)(−)MCL-144, had high affinities for the μ and κ receptors, and were all similar to each other. (+)(−) MCL-193, with only one active pharmacophore, had lower affinity than the others, but displayed markedly higher affinities than bis((+)N-cyclobutylmethylmorphinan-3-yl)sebacoylate (MCL-192), which contains the two (+)(+) isomers of (−)MCL-101. Structurally, (−)(−)MCL-145 and (−)(−)MCL-144 are similar, with two active enantionmers of (−)MCL-101. The only difference between these two compounds is the spacer length. (−)(−)MCL-145 is bridged by a conformationally constrained fumaryl ester, while (−)(−)MCL-144 is bridged by a flexible10-carbon chain.
The efficacy of (−)(−)MCL-144 and (+)(−)MCL-193 for stimulating [35
S]GTPγS binding mediated by the κ opioid receptor was similar, as both produced maximal stimulation (Emax
) values of 60–70%. (−)(−)MCL-144 was more efficacious at the μ receptor for stimulating [35
S]GTPγS binding producing an Emax
value of 50%, than (+)(−)MCL-193 which produced an Emax
value of 28% (–). Both compounds were μ antagonists, in this assay, with maximal inhibition (Imax
) values of 60–70% (2
) (–). (+)(+)MCL-192 was not tested in the [35
S]GTPγS assay due to its low binding affinities. In this functional assay, (−)MCL-101, (−)(−)MCL-145, (−)(−)MCL-144, and (+)(−) MCL-193 all acted as κ agonists, and μ agonist/antagonists. All produced similar Emax
values at the κ receptor while (+)(−)MCL-193 displayed lower efficacy than the other three at the μ receptor.
Agonist and antagonist properties of MCL-101, (−)(−)MCL-145, (−)(−) MCL-144, (+)(−) MCL-193, and (+)(+)MCL-192 in stimulating [35S]GTPγS binding mediated by the κ opioid receptor
The aim of the current study was to explore whether the two isomeric bivalent ligands, (−)(−)MCL-144 and (+)(−)MCL-193 would have appreciable pharmacological differences after central administration, in vivo, utilizing mouse antinociceptive assays. Additionally, (−)(−)MCL-145 and (−)(−)MCL-144 were compared. These two compounds differ only in their spacer length, yet display distinct differences in their pharmacological properties. Preliminary metabolism studies were used to address some of these differences.