Introduction

The structure–activity relationships of salvinorin A (1), a potent and selective κ (kappa) opioid, have been extensively investigated [1]. Modifications at C-2 have yielded compounds with increased potency and duration of action [2,3], and other compounds with unprecedented functional selectivity at the μ (mu) opioid receptor (μ-OR) [4]. However, modifications at other positions substantially reduce or eliminate affinity for opioid receptors [1]. In particular, most modifications of the furan ring tested to date dramatically reduce affinity for κ-OR [1], although small substituents at C-16 have little effect (Figure 1). Unexpectedly, epimerization at C-12, inverting the configuration of the furan ring, reduces efficacy in G-protein activation [5], but not in arrestin recruitment, resulting in functional selectivity (biased agonism) [6].

Figure 1: Binding affinities of salvinorin A (1) and furan derivatives for κ-OR [5]. Figure 1: Binding affinities of salvinorin A (1) and furan derivatives for κ-OR [5]. Jump to Figure 1

Extensive receptor mutagenesis data are available on the interactions of κ-OR with 1 and derivatives [1,7-9]. In Figure 2A, the crystal structure of κ-OR in complex with the selective antagonist JDTic is shown. Essential residues for high affinity binding of 1, where mutation reproducibly reduces affinity ≥10-fold, are shown with thick bonds for emphasis [7-9]. These key residues are all located in transmembrane helices (TMs) 2 and 7. The surfaces of these residues are contiguous, forming one face of a deep pocket between TMs 2, 3, and 7. Mutations of many other residues outside this pocket (TMs 1, 3, 5, 6, 7, and extracellular loop 2) failed to substantially reduce affinity for 1 [1,7-9]. Collectively, these results provide compelling evidence that 1 binds to this face of the binding pocket. The plausibility of this proposal is strengthened by the position of the hydroxyphenylpiperidine moiety of JDTic in the crystal structure (HPP, light blue). The HPP moiety and its N-substituent interact with five of the seven key residues for binding of 1: Val1082.53, Gln1152.60, Val1182.63, Ile3167.39, and Tyr3207.43 (superscripts refer to Ballesteros–Weinstein numbering) [7]. Intriguingly, 1 contains a substructure very similar to HPP (light blue in Figure 2B). Given that HPP is a near substructure of 1 and binds to these same key residues, it is tempting to speculate that the binding pose of 1 may be similar.

Figure 2: Crystal structure of κ-OR in complex with JDTic compared to naltrindole’s binding pose in δ-OR. A: Cross-eyed stereoview of the crystal structure of κ-OR (PDB 4DJH). All residues known to be required for high-affinity binding of 1 (mutation reproducibly reduces affinity ≥10-fold [7-9]) are shown with thick bonds and water-accessible surfaces. JDTic is shown in green with the hydroxyphenylpiperidine substructure (HPP) in light blue. Ionic H-bonds from JDTic to Asp1383.32 are shown in red. The binding pose of naltrindole (NTI, pink) to δ-OR is also superimposed (PDB 4EJ4). B: Structural similarities: atoms in 1 common to the HPP substructure of JDTic are shown in light blue; superimposable portions of naltrindole and JDTic are shown in pink. Figure 2: Crystal structure of κ-OR in complex with JDTic compared to naltrindole’s binding pose in δ-OR. A: ... Jump to Figure 2

By contrast, the morphinan naltrindole (shown in pink) binds to a different pocket of δ-OR among TMs 3, 6, and 7 [10]; β-funaltrexamine adopts a near-identical pose in complex with μ-OR (not shown) [11]. Interestingly, JDTic occupies this morphinan pocket as well. The tetrahydroisoquinoline moiety of JDTic adopts a pose almost identical to that of naltrindole; the superimposable atoms are shown in pink in Figure 2B [10]. JDTic is thus a bivalent ligand, containing two linked pharmacophores [12], also known as a linked multiple ligand [13]. Bivalent ligands can exhibit greatly increased potency and unusual functional selectivity [12]. Consistent with this, JDTic is extremely potent (K e < 40 pM) [14]; also, despite acting as an antagonist toward other κ-OR-mediated signaling pathways [7], JDTic reportedly activates JNK1 (MAPK8), causing extremely prolonged desensitization of κ-OR [15].

These two binding pockets are almost entirely separate, but overlap around Tyr3207.43. This residue interacts with JDTic [7], and the corresponding residue (Tyr7.43) also interacts with naltrindole in δ-OR [10] and β-funaltrexamine in μ-OR [11].

Interestingly, Tyr3207.43 and two adjacent residues (Val1082.53 and Ile3167.39) are also key residues for the binding of 1 [7]. Together, they surround the bottom of the binding pocket, strongly suggesting that an extremity of 1 occupies this space, overlapping with both the morphinan and hydroxyphenylpiperidine binding sites.

Immediately adjacent to these residues is Asp1383.32, a critical residue for binding of morphinans and related opioids. Asp3.32 forms strong ionic H-bonds (‘salt bridges’) to the basic nitrogen atoms found in almost all opioids, as in the crystal structures of JDTic (red in Figure 2A), naltrindole [10], β-funaltrexamine [11], and the NOP antagonist C-24 [16]. Beyond opioids, ionic H-bonds to Asp3.32 are conserved across biogenic amine receptors; indeed, this residue3.32 interacts with the ligand in almost all GPCR crystal structures reported to date [17].

In summary, 1 appears to bind adjacent to Asp1383.32, but not to interact with it strongly: mutation of this residue either has little effect or actually increases affinity [7-9]. Based on this proximity, Kane proposed appending a positively-charged moiety to 1, thereby creating an ionic H bond to Asp1383.32 that should increase affinity [18]. Like JDTic, this ligand would be bivalent. Kane also proposed linking to fragments of other κ-opioids such as arylacetamides, creating additional interactions. By this strategy, “designed multiple ligands” of several kinds could be obtained: linked, fused or merged [13].

A suitable attachment point and linker for this second moiety would also be required. Docking 1 to an early rhodopsin-based homology model of κ-OR, Kane proposed a binding pose qualitatively similar to that of the HPP moiety of JDTic, placing the furan ring near Tyr3207.43 [8,18]. Indeed, at the time we commenced our work, all published mutagenesis-based models placed the furan ring in contact with Tyr3207.43 [1]. This interaction is plausible in light of the structure–activity relationships of opioid antagonists. In JDTic, the N-substituent of the HPP moiety interacts with Tyr3207.43 [7], and N-furanylalkyl substitution of this scaffold yields potent opioid antagonists (Figure 3) [19]. Likewise, the N-substituents of naltrindole and β-funaltrexamine also interact with Tyr7.43 [10,11], and N-furanylmethyl substitution of morphinans and related scaffolds consistently yields potent opioid antagonists [20]. These convergent results firmly establish that residues adjacent to Asp3.32, including Tyr7.43, interact favorably with furan substituents; thus, models placing the furan ring of 1 in this region are plausible. The region appears to be quite tolerant to the position, orientation and substitution of the furan ring [20].

We therefore selected the furan ring as an attachment point for initial explorations of Kane’s bivalent ligand concept. Given the tolerance of 1 to modification at C-16 noted above, we chose to append H-bond donors (amino and hydroxy groups) at this position. Based on previous high-affinity ligands with a furanylmethyl substituent (Figure 3), we decided to use a short linker.

As an alternative approach, bivalent ligands with much longer linkers can bind simultaneously to both protomers in a receptor dimer, allowing selective targeting of specific receptor oligomers [21]. We also sought to provide a suitable attachment point for such linkers based on our previous studies of C-2 alkoxymethyl ethers [22,23], which have higher tolerance to alkyl chain extension [24] than other derivatives of 1 [1].