Structure-activity relationship (SAR) studies of numerous opioid ligands have shown that introduction of a methyl or ethyl group on the tertiary amino group at position 17 of the epoxymorphinan skeleton generally results in a mu opioid receptor (MOR) agonist while introduction of a cyclopropylmethyl group typically leads to an antagonist. Furthermore, it has been shown that introduction of heterocyclic ring systems at position 6 can favor antagonism. However, it was reported that 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2'-indolyl)acetamido]morphinan (INTA), which bears a cyclopropylmethyl group at position 17 and an indole ring at position 6, acted as a MOR agonist. We herein report a SAR study on INTA with a series of its complementary derivatives to understand how introduction of an indole moiety with α or β linkage at position 6 of the epoxymorphinan skeleton may influence ligand function. Interestingly, one of INTA derivatives, compound 15 (NAN) was identified as a MOR antagonist both in vitro and in vivo. Molecular modeling studies revealed that INTA and NAN may interact with different domains of the MOR allosteric binding site. In addition, INTA may interact with W293 and N150 residues found in the orthosteric site to stabilize MOR activation conformation while NAN does not. These results suggest that INTA and NAN may be bitopic ligands and the type of allosteric interactions with the MOR influence their functional activity. These insights along with our enriched comprehension of the "message-address" concept will to benefit future ligand design.

Here, we described the structural modification of previously identified μ opioid receptor (MOR) antagonist NAN, a 6α-N-7′-indolyl substituted naltrexamine derivative, and its 6β-N-2′-indolyl substituted analogue INTA by adopting the concept of “bivalent bioisostere”. Three newly prepared opioid ligands, 25 (NBF), 31, and 38, were identified as potent MOR antagonists both in vitro and in vivo. Moreover, these three compounds significantly antagonized DAMGO-induced intracellular calcium flux and displayed varying degrees of inhibition on cAMP production. Furthermore, NBF produced much less significant withdrawal effects than naloxone in morphine-pelleted mice. Molecular modeling studies revealed that these bivalent bioisosteres may adopt similar binding modes in the MOR and the “address” portions of them may have negative or positive allosteric modulation effects on the function of their “message” portions compared with NAN and INTA. Collectively, our successful application of the “bivalent bioisostere concept” identified a promising lead to develop novel therapeutic agents toward opioid use disorder treatments.

The opioid crisis is a significant public health issue with more than 115 people dying from opioid overdose per day in the United States. The aim of the present study was to characterize the in vitro and in vivo pharmacological effects of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(indole-7-carboxamido)morphinan (NAN), a μ opioid receptor (MOR) ligand that may be a potential candidate for opioid use disorder treatment that produces less withdrawal signs than naltrexone. The efficacy of NAN was compared to varying efficacy ligands at the MOR, and determined at the δ opioid receptor (DOR) and κ opioid receptor (KOR). NAN was identified as a low efficacy partial agonist for G-protein activation at the MOR and DOR, but had relatively high efficacy at the KOR. In contrast to high efficacy MOR agonists, NAN did not induce MOR internalization, downregulation, or desensitization, but it antagonized agonist-induced MOR internalization and stimulation of intracellular Ca2+ release. Opioid withdrawal studies conducted using morphine-pelleted mice demonstrated that NAN precipitated significantly less withdrawal signs than naltrexone at similar doses. Furthermore, NAN failed to produce fentanyl-like discriminative stimulus effects in rats up to doses that produced dose- and time-dependent antagonism of fentanyl. Overall, these results provide converging lines of evidence that NAN functions mainly as a MOR antagonist and support further consideration of NAN as a candidate medication for opioid use disorder treatment.

Evidence has shown that downstream signaling by mu opioid receptor (MOR) agonists that recruit β-arrestin2 may lead to the development of tolerance. Also, it has been suggested that opioid receptor desensitization and cyclic AMP overshoot contributes to the development of tolerance and occurrence of withdrawal, respectively. Therefore, studies were conducted with 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ), a MOR selective partial agonist discovered in our laboratory, to characterize its effect on β-arrestin2 recruitment and precipitation of a cyclic AMP overshoot. DAMGO, a MOR full agonist dose-dependently increased β-arrestin2 association with the MOR, whereas NAQ did not. Moreover, NAQ displayed significant, concentration-dependent antagonism of DAMGO-induced β-arrestin2 recruitment. After prolonged morphine treatment of mMOR-CHO cells, there was a significant overshoot of cAMP upon exposure to naloxone, but not NAQ. Moreover, prolonged incubation of mMOR-CHO cells with NAQ did not result in desensitization nor downregulation of the MOR. In functional studies comparing NAQ with nalbuphine in the cAMP inhibition, Ca flux and [S]GTPγS binding assays, NAQ did not show agonism in the Ca flux assay but showed partial agonism in the cAMP and [S]GTPγS assays. Also, NAQ significantly antagonized DAMGO-induced intracellular Ca increase. In conclusion, NAQ is a low efficacy MOR modulator that lacks β-arrestin2 recruitment function and does not induce cellular hallmarks of MOR adaptation and fails to precipitate a cellular manifestation of withdrawal in cells pretreated with morphine. These characteristics are desirable if NAQ is pursued for opioid abuse treatment development.

μ opioid receptor (MOR) agonists have been widely applied for treating moderate to severe pain. However, numerous adverse effects have been associated with their application, including opioid-induced constipation (OIC), respiratory depression, and addiction. On the basis of previous work in our laboratory, NAP, a 6β-N-4′-pyridyl substituted naltrexamine derivative, was identified as a peripheral MOR antagonist that may be used to treat OIC. To further explore its structure–activity relationship, a new series of NAP derivatives were designed, synthesized, and biologically evaluated. Among these derivatives, NFP and NYP significantly antagonized the antinociception effect of morphine. Whereas NAP acted mainly peripherally, its derivatives NFP and NYP actually can act centrally. Furthermore, NFP produced significantly lesser withdrawal symptoms than naloxone at similar doses. These results suggest that NFP has the potential to be a lead compound to treat opioid abuse and addiction.