OPTO-opioids for light-induced, local, reversible peripherally restricted analgesia

Abstract

The present invention is directed to a compound of Formula (I): where, A, R, R1, R2, Y, l, and n are as defined herein. The present invention is also directed to the use of these compounds for the treatment of pain, respiratory 5 depression, or mood or substance use disorders.

Description

-1- OPTO-OPIOIDS FOR LIGHT-INDUCED, LOCAL, REVERSIBLE PERIPHERALLY RESTRICTED ANALGESIA

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No.63 / 662,089, filed June 20, 2024, which is hereby incorporated by reference in its 5 entirety.

[0002] This invention was made with government support under R33 DA051529 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD

[0003] The present invention relates to opto-opioids for light-induced, local, reversible 10 peripherally restricted analgesia. BACKGROUND

[0004] The MOR, a Gi / o-coupled family A G protein-coupled receptor (GPCR), is the major target of opioid analgesics, including morphine (FIG.1A), oxycodone, and fentanyl (Che and Roth, “Molecular Basis of Opioid Receptor Signaling,” Cell 186:5203–5219 (2023)). The 15 MOR is expressed broadly and has been found to contribute to various forms of analgesia in both the central and peripheral nervous system (Matthes et al., “Loss of Morphine-induced Analgesia, Reward Effect and Withdrawal Symptoms in Mice Lacking the Mu-opioid-receptor Gene,” Nature 383:819–823 (1996)). The effectiveness of opioids for the treatment of pain has led to the desire for new opioid-based strategies that can maintain analgesic properties while minimizing 20 adverse side effects. This has included extensive efforts to develop G protein-biased and / or low intrinsic efficacy agonists (Manglik et al., “Structure-based Discovery of Opioid Analgesics With Reduced Side Effects,” Nature 537:185–190 (2016); Faouzi et al., “Structure-based Design of Bitopic Ligands for the µ-opioid Receptor,” Nature 613:767–774 (2023); Chakraborty et al., “A Novel Mitragynine Analog with Low-Efficacy Mu Opioid Receptor Agonism Displays 25 Antinociception with Attenuated Adverse Effects,” J. Med. Chem.64:13873–13892 (2021)). Alternatively, given that many opioid-driven side effects, including respiratory depression and addiction, are centrally mediated in the brain, peripherally-restricted opioids have emerged as an attractive alternative approach (Iwaszkiewicz et al., “Targeting Peripheral Opioid Receptors to Promote Analgesic and Anti-inflammatory Actions,” Front. Pharmacol .4:132 (2013); Martínez 30 and Abalo, “Peripherally Acting Opioid Analgesics and Peripherally-induced Analgesia,” Behav. Pharmacol.31:136–158 (2020)). However, gastrointestinal side effects and peripheral tolerance occur outside of the central nervous system (Benyamin et al., “Opioid Complications and Side 316084209v2-2- Effects,” Pain Physician 11:S105–20 (2008)), highlighting the limitations of this approach and emphasizing the need for more advanced targeting mechanisms.

[0005] Photopharmacology, the use of light-sensitive ligands, represents a powerful approach to enable spatiotemporally precise control of drug action (Hüll et al., “In Vivo 5 Photopharmacology,” Chem. Rev.118:10710–10747 (2018); Kobauri et al., “Rational Design in Photopharmacology with Molecular Photoswitches,” Angew. Chem. Int. Ed. Engl. 62:e202300681 (2023)), and may be a viable strategy for local opioid-driven pain relief. Recently reported photopharmacological designs for in vivo optical control of opioid agonists have employed caged approaches that irreversibly release ligands in response to illumination 10 (McClain et al., “In vivo Photopharmacology With Light-activated Opioid Drugs,” Neuron 1113926–3940.e10 (2023); Ma et al., “In vivo Photopharmacology With a Caged Mu Opioid Receptor Agonist Drives Rapid Changes in Behavior,” Nat. Methods 20:682–685 (2023); López- Cano et al., “Remote Local Photoactivation of Morphine Produces Analgesia Without Opioid- related Adverse Effects,” Br. J. Pharmacol.180:958–974 (2023)). In contrast, photochromic 15 ligands, which employ reversible photoswitchable moieties, such as azobenzenes, may provide a higher degree of spatiotemporal control to enable reversible and repeatable control of ligand action. To date, reversibly photoswitchable azobenzene-based opioid agonists using fentanyl scaffolds have exhibited tonic MOR activation in the relaxed trans state, with decreased activation in the excited cis form (Schönberger and Trauner, “A Photochromic Agonist for μ- 20 opioid Receptors,” Angew Chem Int Ed Engl 53:3264–3267 (2014); Lahmy et al. “Photochromic Fentanyl Derivatives for Controlled μ-Opioid Receptor Activation,” Chemistry 28:e202201515 (2022); Lahmy et al., “Development of Photoswitchable Tethered Ligands that Target the μ- Opioid Receptor,” ChemMedChem 18:e202300228 (2023)).

[0006] The present disclosure is directed to overcoming these and other deficiencies in 25 the art. SUMMARY

[0007] A first aspect of the present disclosure relates to a compound of Formula (I):where 30is a double bond with an unspecified configuration; 316084209v2-3- is optional and, if present, is a single bond;is selected from the group consisting of monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl, wherein monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl can be optionally substituted from 1 to 6 times 5 with a substituent selected independently at each occurrence thereof from the group consisting of H, D, halogen, OC1-6alkyl, OC1-6alkyl, NH2, NHC1-6alkyl, and N(C1-6alkyl)2;is selected from the group consisting of monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl, wherein monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl can be optionally substituted from 1 to 6 times 10 with a substituent selected independently at each occurrence thereof from the group consisting of H, D, halogen, OC1-6 alkyl, OC1-6 alkyl, NH2, NHC1-6 alkyl, and N(C1-6 alkyl)2; A is a morphinan or a fragment thereof; R is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; R1is H, C1-6alkyl, or -X1-X2-X3-X4-X5-X6-X7-X8-X9; 15 R2is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; X1is C1-6alkylene; X2is -NH-C(O)- or -C(O)NH-; X3is C1-6alkylene; X4is –(OCH2CH2)m- or –(OCH2CH2)m-OCH2–; 20 X5is -NH-C(O)- or -C(O)NH-; X6is a bond, C1-6 alkylene, or –(CH2CH2O)k-C1-12 alkylene–; X7is absent or -C(O)-NH-; X8is a bond or C1-6alkylene; X9is a Tag; 25 Y is NH, CH2, O, S, or N(C1-6alkyl); k is 1-10; l is 0 or 1; n is 0, 1, 2, or 3; and m is 1-50, 30 or an isomer thereof, an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.

[0008] A second aspect of the present disclosure relates to a compound of Formula (I´): 316084209v2-4-whereis a double bond with an unspecified configuration; A is a morphinan or a fragment thereof; 5 R1is H or C1-6alkyl; R2is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; X1is C1-6alkylene; X2is -NH-C(O)-; X3is C1-6alkylene; 10 X4is –(OCH2CH2)m-; X5is -NH-C(O)-; X6is C1-6alkylene; X7is absent or -C(O)-NH-; X8is a bond or C1-6alkylene; 15 X9is a Tag; n is 0, 1, 2, or 3; and m is 1-50, or an isomer thereof, an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof. 20

[0009] A third aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound according to the first or second aspect of the present disclosure and a pharmaceutically acceptable carrier.

[0010] A fourth aspect of the present disclosure relates to a method of treating pain in a subject. This method comprises administering to the subject in need thereof the compound 25 according to the first or second aspect of the present disclosure.

[0011] A fifth aspect of the present disclosure relates to a method of treating a mood or substance use disorder in a subject. This method comprises administering to the subject in need thereof the compound according to the first or second aspect of the present disclosure.

[0012] A sixth aspect of the present disclosure relates to a method of treating a 30 respiratory depression in a subject. This method comprises administering to the subject in need thereof the compound according to the first or second aspect of the present disclosure. 316084209v2-5-

[0013] A seventh aspect of the present disclosure relates to a method of activating a mu- opioid receptor (MOR). This method comprises contacting a MOR with the compound according to the first or second aspect of the present disclosure under conditions effective to activate the MOR. 5

[0014] An eighth aspect of the present disclosure relates to a method of activating a kappa-opioid receptor and / or delta-opioid receptor. This method comprises contacting a kappa- opioid receptor and / or delta-opioid receptor with the compound according to the first or second aspect of the present disclosure under conditions effective to activate kappa-opioid receptor and / or delta-opioid receptor. 10

[0015] A ninth aspect of the present disclosure relates to a method of preventing and / or treating a condition where it is desired to activate MOR. This method comprises administering to the subject in need thereof the compound according to the first or second aspect of the present disclosure.

[0016] A tenth aspect of the present disclosure relates to a method of preventing and / or 15 treating a condition where it is desired to activate kappa-opioid receptor and / or delta-opioid receptor. This method comprises administering to the subject in need thereof the compound according to the first or second aspect of the present disclosure.

[0017] A major challenge in the design of reversible, photoswitchable ligands for GPCRs, including MORs, is a limited understanding of the molecular basis of ligand efficacy. In 20 principle, the development of efficacy photoswitches (Müller et al., “Ideal Efficacy Photoswitches for TRPC4 / 5 Channels Harness High Potency for Spatiotemporally-resolved Control of TRPC Function in Live Tissues,” bioRxiv 2024.07.12.602451 (2024), which is hereby incorporated by reference in its entirety), ligands which change their maximal efficiency of target activation with light, would provide powerful tools for dissecting the structural and 25 biophysical basis of GPCR agonism. Disclosed herein, a structure-guided approach used to develop a family of azobenzene-conjugated “azo-morphines”. Subtle tweaks in linker length and attachment geometry produced dramatic differences, with one variant “AM-3” showing a large difference in efficacy between trans and cis following illumination with blue versus UV light. Patch clamp electrophysiology experiments revealed the ability of AM-3 to provide reversible, 30 repeatable, and bistable control of MOR-driven G protein signaling in live cells. The basis of this light-dependent effect on ligand efficacy was probed by determining cryo-EM structures of cis- AM-3 and trans-AM-3 bound to MOR, revealing that a distinct azobenzene moiety pose is associated with high versus low efficacy agonism. Application of AM-3 in vivo in mice enabled local, light-dependent analgesic action in both acute and chronic pain with reduced central and 316084209v2-6- peripheral side effects. Together these features provided a new approach to opioid pharmacology and opened the door for further design and application of targeted, reversible GPCR photopharmacology.

[0018] The mu-opioid receptor (MOR) is a major target for the treatment of pain. 5 However, opioids are prone to side effects which limit their effectiveness as analgesics and can lead to opioid use disorders or, even, lethal overdose. The systemic administration of opioid agonists makes it both very difficult to decipher their underlying circuit mechanisms of action and to limit drug action to specific receptor subpopulations to isolate therapeutic effects from adverse side effects. A reversibly photoswitchable morphinan agonist termed “azo-morphine-3” 10 (AM-3) was designed, synthesized, and characterized. AM-3 interconverted from low to high efficacy in response to different wavelengths of light to enable optical control of MOR signaling. Cryo-EM structures of the low efficacy “trans” and high efficacy “cis” states of AM-3 bound to the MOR revealed distinct binding modes of the photoswitchable azobenzene moiety, each inducing unique structural dynamics, providing insight into the molecular basis of agonist 15 efficacy. In mice, AM-3 drove reversible and repeatable optical control of anti-nociception with a reduced side effect profile owing to its restriction to the periphery and its ability to be locally activated at the site of pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGs.1A-1F show structure-guided design and synthesis of azo-morphines. FIG. 20 1A shows chemical structures of morphinan ligands. FIG.1B (left) shows alignment of structures showing binding poses of morphine (PDB: 8EF6), BU-72 (PDB: 5C1M), and fentanyl (PDB: 8EF5). FIG.1B (right) shows chemical structures of BU-72 and fentanyl with rings that extend into the secondary subpocket (shaded). FIG.1C shows chemical structures of tested azo- morphine variants. FIG.1D shows a synthetic scheme for the synthesis of AM-3. FIG.1E 25 shows isomerization of AM-3 between trans and cis isomers. FIG.1F shows UV / vis spectroscopy following different illumination conditions. Inset in FIG.1F shows reversible photoswitching of AM-3 following alternating 460 nm and 365 nm illumination.

[0020] FIGs.2A-2C show a data processing pipeline for the cis-AM-3 a mu-opioid receptor (MOR):Gi complex. FIG.2A shows a workflow depicting cryo-EM processing the cis- 30 AM-3 MOR:Gi complex with representative micrograph and 2D classes. Data processing was performed in cryoSPARC (Punjani et al., “cryoSPARC: Algorithms for Rapid Unsupervised Cryo-EM Structure Determination,” Nat. Methods 14:290–296 (2017), which is hereby incorporated by reference in its entirety) using established workflows. Following motion 316084209v2-7- correction, CTF estimation, and particle picking, the dataset was cleaned using 2D classification. Ab initio reconstruction was performed to obtain a 3D volume, which was subjected to non- uniform and local refinement resulting in a density map with unambiguous density for cis-AM-3. 3D Variability Analysis (3DVA) was performed on the final particle set to determine 5 conformational dynamics within the MOR:Gi complex. FIGs.2B-2C show data quality of the final reconstruction, shown as a gold-standard Fourier shell correlation plot (masked and unmasked) (FIG.2B) and angular sampling distribution (FIG.2C).

[0021] FIGs.3A-3C show a data processing pipeline for the trans-AM-3 MOR:Gi complex. FIG.3A shows a workflow depicting cryo-EM processing the trans-AM-3 MOR:Gi 10 complex with representative micrograph and 2D classes. Data processing was performed in cryoSPARC (Punjani et al., “cryoSPARC: Algorithms for Rapid Unsupervised Cryo-EM Structure Determination,” Nat. Methods 14:290–296 (2017), which is hereby incorporated by reference in its entirety). Following motion correction, CTF estimation, and particle picking, the dataset was cleaned using 2D classification and 3D ab initio reconstruction. Initial refinements 15 revealed strong density for the naltrexone scaffold but dynamic density for the trans-azobenzene group. Two rounds of 3DVA were performed to select particles contributing to frames with improved trans-azobenzene density, followed by focused refinements. FIGs.3B-3C show data quality of the final reconstruction, shown as a gold-standard Fourier shell correlation plot (masked and unmasked) (FIG.3B) and angular sampling distribution (FIG.3C). 20

[0022] FIGs.4A-4H show cryo-EM reconstructions of cis-AM-3 and trans-AM-3 in complex with MOR:G protein heterotrimer. FIGs.4A-4B show cryo-EM reconstructions of the MOR:Gi complex bound to cis-AM-3 (FIG.4A) and trans-AM-3 (FIG.4B), highlighting individual ligand densities (grey mesh). FIG.4C shows superimposed structures showing that the Gαi αN helix is tilted 6° towards the cis-AM-3 bound MOR, relative to trans-AM-3, 25 suggesting stronger receptor-Gαi engagement. FIG.4D shows an overview of structural rearrangements in TM2 and ECL2, as a result of AM-3 binding in the cis or trans state. FIGs. 4E-4F show detailed comparisons of the MOR orthosteric pocket from the side (FIG.4E) and top (FIG.4F) views. The terminal benzene of trans-AM-3 penetrates deeper towards TM2, resulting in differences in rotamer conformations of Q2.60and N2.63. FIGs.4G-4H show 3D 30 variability analysis of the cis-AM-3 (FIG.4G) and trans-AM-3 (FIG.4H) bound MOR:Gi complexes. Vectors (arrows) represent trajectories of motion along principal component 0 that are larger than 1 Å, highlighting distinct conformational dynamics between the cis-AM-3 and trans-AM-3 bound MOR:Gi complexes. 316084209v2-8-

[0023] FIGs.5A-5F show alleviation of chronic neuropathic pain with reduced side effects via AM-3. FIG.5A is a schematic showing spared nerve injury (SNI) model of chronic neuropathic pain followed by radiant heat test. FIG.5B shows a time course showing hyperalgesic effect of SNI followed by light-controlled analgesia via AM-3 following hindpaw 5 injection. FIG.5C shows a time course showing analgesic effect of pre-illuminated cis-AM-3 and loperamide compared to trans-AM-3 and vehicle following tail vein injection. FIG.5D shows a time course showing in vivo photoactivation of AM-3 following tail vein injection of trans-AM-3. FIG.5E shows a summary AUC analysis of analgesic effect of loperamide and AM-3 following pre-illumination (“cis-AM-3”) or in vivo photoswitching over the course of 90 10 min post-injection. FIG.5F shows a summary of accumulated faecal boli in SNI mice for all drug treatments during the 90 min time course used to assess hyperalgesia. All data in this figure are for the paw on the ipsilateral side relative to SNI. All data are shown as mean ± s.e.m. with n> 5 mice / condition. For FIGS.5E-5F, points represent individual mice. ∗p>0.05; ∗∗p<0.01; ∗∗∗p<0.001 vs vehicle (FIG.5B) or as depicted by lines (FIGS.5E-5F).2-Way ANOVA was 15 used for FIG.5B and 1-way ANOVA was used for (FIGS.5E-5F).

[0024] FIG.6 shows molecular docking analysis of azo-morphines. Docking results of AM1-4 analogs in cis- and trans isoforms to the MOR. The cis-azo isomer is shown by one conformation, consistently reproduced in 15 parallel docking simulations. The trans-azo isomer is shown by several examples of top-scoring conformations with similar scores, observed in 20 docking simulations. The receptor models are shown in grey, with residues of the pocket in stick and surface presentations. The ligands are shown as sticks.

[0025] FIGs.7A-7C show photophysical characterization of azo-morphines. FIG.7A shows UV / vis spectra showing similar light sensitivity across azo-morphine variants with maximal cis content following 360 nm illumination and maximal trans content following >460 25 nm illumination. FIG.7B shows time course of thermal relaxation to the trans state following 365 nm illumination to drive cis enrichment. FIG.7C shows photostationary state measurements of relative cis and trans proportions of AM-3 following illumination with different wavelengths. Data in FIG.7A-7B were collected in PBS:DMSO = 9:1. Data in FIG.7C was collected in acetonitrile / H2O mixture. 30

[0026] FIGs.8A-8H show that AM-3 is a photochromic MOR agonist which enables reversible efficacy switching. FIG.8A shows representative patch clamp electrophysiology trace showing GIRK currents induced by trans or cis-AM-3 relative to DAMGO in cells expressing the MOR. Light grey=460 nm illumination; Dark grey=365 nm illumination. FIG.8B shows a summary graph (box plot shows median and error bars show min and max) of relative activation 316084209v2-9- by cis (365 nm illumination) versus trans (460 nm illumination) across azo-morphine variants at 100 nM. * p<0.05; ** p<0.001; 1-way ANOVA with multiple comparisons. FIG.8C shows representative trace showing bistable, reversible, and repeatable photoactivation of the MOR by AM-3. FIGs.8D-8G show dose-response curves showing the difference between cis (365 nm 5 illumination) and trans (460 nm illumination or dark-adapted) forms of AM-3 across assays that measure orthosteric binding (FIG.8D), G protein activation (FIG.8E), GIRK current activation (FIG.8F), and receptor internalization (FIG.8G). FIG.8H shows a summary of AM-3 pharmacological data.

[0027] FIGs.9A-9D show further comparative electrophysiological analysis of azo- 10 morphines. FIGs.9A-9C show representative GIRK current traces and FIG.9D shows a summary graph showing different light sensitivities of azo-morphine variants on the rat MOR. In FIG.9D, each pair of points represents an individual cell and 100 nM azo-morphine was used.

[0028] FIGs.10A-10B show analysis of wavelength-dependence of AM-3 photo- activation. FIGs.10A-10B show a representative GIRK current trace (FIG.10A) and a 15 summary bar graph (FIG.10B) showing wavelength-dependence of AM-3 photoactivation. Each point represents an individual cell.

[0029] FIGs.11A-11B show AM-3 photoactivation of mouse and human mu-opioid receptor. FIG.11A shows a representative GIRK current trace showing light dependent AM-3 activation of the human MOR. FIG.11B shows a summary graph showing stronger activation of 20 mouse and human MOR via cis-AM-3. In FIG.11B, each pair of points represents an individual cell and current values are normalized to the response to 1 µM DAMGO.

[0030] FIGs.12A-12G show further analysis of dose-dependence of AM-3 MOR activation. FIG.12A shows a representative GIRK current trace showing photoactivation of MOR via 1 µM AM-3. FIG.12B is a summary graph showing relative activation of MOR by cis 25 and trans-AM-3 across a range of concentrations. FIG.12C shows representative images of cells showing that cis-AM-3 and DAMGO produce substantially more intracellular puncta (arrowheads) compared to trans-AM-3 or no ligand treatment. Cells were labeled with BG- Alexa-546 to visualize SNAP-tagged MOR and then treated with ligands for 30 minutes prior to imaging. FIG.12D shows ONE-GO G protein BRET Dose response curves for DAMGO and30 morphine showing relative potency and efficacy compared to cis and trans-AM-3. FIGs.12E- 12G are summary bar graphs showing relative to maximal response to ligands across assays. All data are shown as mean ± s.e.m. Points represent independent experimental days for FIG.12E and FIG.12B and individual cells for FIGs.12B and 12F. ∗p<0.05; ∗∗∗p<0.001 vs vehicle.1- way ANOVA was used. 316084209v2-10-

[0031] FIGs.13A-13B show lack of light-dependence of activation of KOR and DOR by AM3. FIGs.13A-13B show ONE-GO G protein BRET dose response curves showing similar activation of KOR (FIG.13A) and DOR (FIG.13B) by cis versus trans-AM-3. All data are shown as mean ± s.e.m. 5

[0032] FIGs.14A-14J show structural comparison of MOR bound to AM-3, fentanyl, and alvimopan. FIGs.14A-14B show model overlays of cis- and trans-AM-3 bound MOR to the active state fentanyl-bound (PDB:8EF5) (FIG.14A), and inactive state alvimopan-bound MOR (PDB:7UL4) (FIG.14B). FIG.14C shows alignments of cis- and trans-AM-3 to morphine (PDB:8EF6) and β-FNA (PDB:4DKL) highlighting the position of the morphinan 10 tertiary amine. Deeper penetration into the orthosteric site correlates with ligand efficacy. FIGs. 14D-14J show detailed comparisons of the cis- and trans-AM-3 bound orthosteric site of MOR to fentanyl (FIGs.14D, 14F, 14G, 14J) and alvimopan (FIGs.14E, 14H, 14F, 14J) showing overlapping occupation of the subpocket through the terminal azo-benzene of AM-3, and the phenyl and benzyl groups of fentanyl and alvimopan, respectively. 15

[0033] FIGs.15A-15F show that AM-3 enables optical control of peripheral antinociception. FIG.15A is a schematic showing timing of pre-illuminated i.pl. drug administration prior to the Radiant Heat Test. FIG.15B shows time course indicating that cis- AM-3, but not trans-AM-3, increases the PWL and has a comparable time course to morphine. FIG.15C shows area under the curve (AUC) analysis of cumulative analgesic effect of i.pl. cis- 20 AM-3, trans-AM-3, morphine, and vehicle over 45 min post drug administrations. FIG.15D shows that i.pl. pretreatment with naloxone 10 min prior to i.pl. administration of pre-illuminated cis-AM-3 completely blocked the antinociceptive effect. FIG.15E shows that PWL increases after hind paw exposure to 365 nm light (shaded bar). FIG.15F shows locomotor activity indicating that, unlike morphine, pre-illuminated cis-AM-3 does not promote hyperlocomotion 25 in the open field test. All data are shown as mean ± s.e.m. with n> 5 mice / condition; for FIG. 15C points represent individual mice; ∗p>0.05; ∗∗p<0.01; ∗∗∗p<0.001 vs vehicle (FIGs.15C, 15E, 15F) or saline / vehicle (FIG.15D).1-way ANOVA was used for (FIG.15C) and 2-way ANOVA was used for (FIGs.15D-15F).

[0034] FIGs.16A-16F show dose-dependence of antinociceptive effects of AM-3. FIG. 30 16A shows a time course of PWL of cis-AM-3 across doses. FIGs.16B-16C show summary bar graphs of area under curve showing analgesic effect of cis-AM-3 and morphine in the ipsilateral, injected paw (FIG.16B), but not the contralateral paw (FIG.16C). FIG.16D shows a time course of PWL of trans-AM-3 across doses. FIGs.16E-16F show summary bar graphs of area under curve showing analgesic effect of trans-AM-3 and morphine in the ipsilateral, injected 316084209v2-11- paw (FIG.16E), but not the contralateral paw (FIG.16F). All data are shown as mean ± s.e.m. with n>6 mice / condition (FIGs.16A-16C) and n>5 (FIGs.16D-16F); for FIGs.16B, 16C, 16E, 16F points represent individual mice; ∗∗p<0.01; ∗∗∗p<0.001 vs vehicle.1-way ANOVA was used for FIGs.16B, 16C, 16E, 16F. 5

[0035] FIGs.17A-17B show photo-deactivation of AM-3 in vivo. FIGs.17A-17B show a schematic (FIG.17A) and a time course (FIG.17B) indicating reversal of the PWL effect of cis-AM-3 via 460 nm illumination to drive conversion to trans-AM-3. All data are shown as mean ± s.e.m. with n>5 mice / condition; ∗p<0.05; ∗∗p<0.01; vs blue light illumination.2-way gANOVA was used for FIG.17B. 10

[0036] FIGs.18A-18B show lack of effect of intra-venous AM-3 in the tail flick test. FIGs.18A-18B show a schematic (FIG.18A) and a time course (FIG.18B) indicating lack of spinal analgesic effect of 2.5, 5, and 10 mg / kg i.v. cis-AM-3 in the tail flick test. All data are shown as mean ± s.e.m. with n>4 mice / condition; ∗∗p<0.01; ∗∗∗p<0.001 vs vehicle.2-way ANOVA was used for FIG.18B. 15

[0037] FIGs.19A-19C show lack of effect of AM-3 in the contralateral paw in the SNI neuropathic pain model. FIGs.19A-19C show time courses inndicating lack of antihyperalgesic effect of AM-3 in the contralateral paw in SNI mice following ipsilateral hindpaw (FIG.19A) or tail vein injection (FIGs.19B-19C). All data are shown as mean ± s.e.m. with n>5 mice / condition. 20

[0038] FIG.20 shows the results of a variable temperature NMR experiment conducted to confirm the existence of rotamers for AM-3. The1H NMR spectra taken at various temperatures are overlayed. DETAILED DESCRIPTION N

[0039] A first aspect of the present disclosure relates to a compound of Formula (I):25whereis a double bond with an unspecified configuration; is optional and, if present, is a single bond; 316084209v2-12-is selected from the group consisting of monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl, wherein monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl can be optionally substituted from 1 to 6 times with a substituent selected independently at each occurrence thereof from the group consisting of 5 H, D, halogen, OC1-6alkyl, OC1-6alkyl, NH2, NHC1-6alkyl, and N(C1-6alkyl)2;is selected from the group consisting of monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl, wherein monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl can be optionally substituted from 1 to 6 times with a substituent selected independently at each occurrence thereof from the group consisting of 10 H, D, halogen, OC1-6alkyl, OC1-6alkyl, NH2, NHC1-6alkyl, and N(C1-6alkyl)2; A is a morphinan or a fragment thereof; R is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; R1is H, C1-6alkyl, or -X1-X2-X3-X4-X5-X6-X7-X8-X9; R2is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; 15 X1is C1-6alkylene; X2is -NH-C(O)- or -C(O)NH-; X3is C1-6alkylene; X4is –(OCH2CH2)m- or –(OCH2CH2)m-OCH2–; X5is -NH-C(O)- or -C(O)NH-; 20 X6is a bond, C1-6alkylene, or –(CH2CH2O)k-C1-12alkylene–; X7is absent or -C(O)-NH-; X8is a bond or C1-6alkylene; X9is a Tag; Y is NH, CH2, O, S, or N(C1-6alkyl); 25 k is 1-10; l is 0 or 1; n is 0, 1, 2, or 3; and m is 1-50, or an isomer thereof, an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate 30 thereof, or a prodrug thereof.

[0040] As used above, and throughout the description herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings. If not defined 316084209v2-13- otherwise herein, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this technology belongs. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. 5

[0041] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0042] The terms “comprising,” “comprises,” and “comprised of” as used herein are synonymous with “including,” “includes,” or “containing,” “contains,” and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. 10

[0043] The terms “comprising,” “comprises,” and “comprised of” also encompass the term “consisting of.” The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, un-recited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The 15 transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed subject matter. In some embodiments or claims where the term comprising is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of” or “consisting essentially of.” 20

[0044] Terms of degree such as “substantially,” “about,” and “approximately” and the symbol as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±0.1% (and up to ±1%, ±5%, or ±10%) of the modified term if this deviation would not negate the meaning of the word it modifies. Unless otherwise clear from 25 context, all numerical values provided herein are modified by the term about. All numerical values provided herein that are modified by terms of degree set forth in this paragraph (e.g.,are also explicitly disclosed without the term of degree. For example, “about 1%” is also explicitly disclosed as “1%”.

[0045] The term “and / or” as used herein means that the listed items are present, or used, 30 individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

[0046] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. Any listed range can be easily recognized as sufficiently describing and enabling the same range being 316084209v2-14- broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be 5 subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0047] The term “alkyl” means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 12 carbon atoms in the chain. Particular alkyl groups have 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as 10 methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl. The term “alkylene” refers to a divalent group formed from an alkane by removal of two hydrogen atoms. Exemplary alkylene groups include, but are not limited to, methylene, ethylene, propylene, and other divalent groups derived from the alkanes described above. 15

[0048] The term “cycloalkyl” means a non-aromatic mono- or multicyclic ring system of about 3 to about 8 carbon atoms, preferably of about 5 to about 7 carbon atoms. Exemplary monocyclic cycloalkyls include cyclopentyl, cyclohexyl, cycloheptyl, and the like.

[0049] The term “aryl” means an aromatic monocyclic or multicyclic ring system of 6 to about 14 carbon atoms, preferably of 6 to about 10 carbon atoms. Representative aryl groups 20 include phenyl and naphthyl.

[0050] The term “heteroaryl” means an aromatic monocyclic or multicyclic ring system of about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is / are element(s) other than carbon, for example, nitrogen, oxygen, or sulfur. In the case of multicyclic ring system, only one of the rings needs to be 25 aromatic for the ring system to be defined as “Heteroaryl”. Preferred heteroaryls contain about 5 to 6 ring atoms. The prefix aza, oxa, thia, or thio before heteroaryl means that at least a nitrogen, oxygen, or sulfur atom, respectively, is present as a ring atom. A nitrogen atom of a heteroaryl is optionally oxidized to the corresponding N-oxide. Representative heteroaryls include pyridyl, 2- oxo-pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, furanyl, pyrrolyl, thiophenyl, 30 pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, indolyl, isoindolyl, benzofuranyl, benzothiophenyl, indolinyl, 2- oxoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, indazolyl, benzimidazolyl, benzooxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, benzotriazolyl, benzo[1,3]dioxolyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, pthalazinyl, quinoxalinyl, 316084209v2-15- 2,3-dihydro-benzo[1,4]dioxinyl, benzo[1,2,3]triazinyl, benzo[1,2,4]triazinyl, 4H-chromenyl, indolizinyl, quinolizinyl, 6aH-thieno[2,3-d]imidazolyl, 1H-pyrrolo[2,3-b]pyridinyl, imidazo[1,2- a]pyridinyl, pyrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, [1,2,4]triazolo[1,5- a]pyridinyl, thieno[2,3-b]furanyl, thieno[2,3-b]pyridinyl, thieno[3,2-b]pyridinyl, furo[2,3- 5 b]pyridinyl, furo[3,2-b]pyridinyl, thieno[3,2-d]pyrimidinyl, furo[3,2-d]pyrimidinyl, thieno[2,3- b]pyrazinyl, imidazo[1,2-a]pyrazinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, 6,7-dihydro- 4H-pyrazolo[5,1-c][1,4]oxazinyl, 2-oxo-2,3-dihydrobenzo[d]oxazolyl, 3,3-dimethyl-2- oxoindolinyl, 2-oxo-2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, benzo[c][1,2,5]oxadiazolyl, benzo[c][1,2,5]thiadiazolyl, 3,4-dihydro-2H-benzo[b][1,4]oxazinyl, 5,6,7,8-tetrahydro-10 [1,2,4]triazolo[4,3-a]pyrazinyl, [1,2,4]triazolo[4,3-a]pyrazinyl, 3-oxo-[1,2,4]triazolo[4,3- a]pyridin-2(3H)-yl, and the like.

[0051] The term “monocyclic” used herein indicates a molecular structure having one ring.

[0052] The term “polycyclic” or “multi-cyclic” used herein indicates a molecular 15 structure having two or more rings, including, but not limited to, fused, bridged, or spiro rings.

[0053] The term “phenyl” means a phenyl group as shown below.

[0054] The term “halogen” means fluoro, chloro, bromo, or iodo.

[0055] The term “substituted” or “substitution” of an atom means that one or more 20 hydrogen on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded.

[0056] “Unsubstituted” atoms bear all of the hydrogen atoms dictated by their valency. When a substituent is keto (i.e., =O), then two hydrogens on the atom are replaced. Combinations of substituents and / or variables are permissible only if such combinations result in 25 stable compounds; by “stable compound” or “stable structure” is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0057] The term “optionally substituted” is used to indicate that a group may have a substituent at each substitutable atom of the group (including more than one substituent on a 30 single atom), provided that the designated atom's normal valency is not exceeded and the identity of each substituent is independent of the others. Up to three H atoms in each residue are replaced with alkyl, halogen, haloalkyl, hydroxy, loweralkoxy, carboxy, carboalkoxy (also referred to as alkoxycarbonyl), carboxamido (also referred to as alkylaminocarbonyl), cyano, carbonyl, nitro, 316084209v2-16- amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, or heteroaryloxy.

[0058] The term “method of treating” means amelioration or relief from the symptoms and / or effects associated with the disorders described herein. As used herein, reference to 5 “treatment” of a patient is intended to include prophylaxis.

[0059] The term “compounds of the present disclosure”, and equivalent expressions, are meant to embrace compounds of general Formula (I), Formula (I´), Formula (I´´), Formula (Ia), Formula (I´a), Formula (Ib), Formula (I´b), Formula (Ic), Formula (I´c), Formula (Id), and Formula (Ie) as hereinbefore described, which expression includes the prodrugs, the 10 pharmaceutically acceptable salts, and the solvates, e.g. hydrates, where the context so permits. Similarly, reference to intermediates, whether or not they themselves are claimed, is meant to embrace their salts, and solvates, where the context so permits. For the sake of clarity, particular instances when the context so permits are sometimes indicated in the text, but these instances are purely illustrative and it is not intended to exclude other instances when the context so permits. 15

[0060] The term “pharmaceutically acceptable salts” means the relatively non-toxic, inorganic, and organic acid addition salts, and base addition salts, of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt 20 thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates, malonates, salicylates, propionates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methane- 25 sulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinateslaurylsulphonate salts, and the like (see, for example, Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 66:1-9 (1977) and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, which are hereby incorporated by reference in their entirety). Base addition salts can also be prepared by 30 separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include, for example, sodium 316084209v2-17- hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low 5 toxicity and acceptability for medical use, such as ammonia, ethylenediamine, N-methyl- glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylarnine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, 10 tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, dicyclohexylamine, and the like.

[0061] The term “pharmaceutically acceptable prodrugs” as used herein means those prodrugs of the compounds useful according to the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower 15 animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term “prodrug” means compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. Functional groups which may be rapidly 20 transformed, by metabolic cleavage, in vivo form a class of groups reactive with the carboxyl group of the compounds of this invention. They include, but are not limited to, such groups as alkanoyl (such as acetyl, propionyl, butyryl, and the like), unsubstituted and substituted aroyl (such as benzoyl and substituted benzoyl), alkoxycarbonyl (such as ethoxycarbonyl), trialkylsilyl (such as trimethyl- and triethysilyl), monoesters formed with dicarboxylic acids (such as 25 succinyl), and the like. Because of the ease with which the metabolically cleavable groups of the compounds useful according to this invention are cleaved in vivo, the compounds bearing such groups act as pro-drugs. he compounds bearing the metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of enhanced solubility and / or rate of absorption conferred upon the parent compound by virtue of the presence of the 30 metabolically cleavable group. A thorough discussion of prodrugs is provided in the following: Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985); Methods in Enzymology, K. Widder et al, Ed., Academic Press, 42, p.309-396 (1985); A Textbook of Drug Design and Development, Krogsgaard-Larsen and H. Bundgaard, ed., Chapter 5; "Design and Applications of Prodrugs" p.113-191 (1991); Advanced Drug Delivery Reviews, H. Bundgard, 8, p.1-38 (1992); J. Pharm. 316084209v2-18- Sci., 77:285 (1988); Nakeya et al, Chem. Pharm. Bull., 32:692 (1984); Higuchi et al., “Pro-drugs as Novel Delivery Systems,” Vol.14 of the A.C.S. Symposium Series, and Bioreversible Carriers in Drug Design, Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press (1987), which are incorporated herein by reference in their entirety. Examples of prodrugs 5 include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the invention.

[0062] The term “solvate” refers to a compound of Formula (I), Formula (I´), Formula (I´´), Formula (Ia), Formula (I´a), Formula (Ib), Formula (I´b), Formula (Ic), Formula (I´c), Formula (Id), and Formula (Ie) in the solid state, wherein molecules of a suitable solvent are 10 incorporated in the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is 15 typically dried or azeotroped under ambient conditions.

[0063] The term “therapeutically effective amounts” is meant to describe an amount of compound of the present invention effective to produce the desired therapeutic effect. Such amounts generally vary according to a number of factors well within the purview of ordinarily skilled artisans given the description provided herein to determine and account for. These 20 include, without limitation: the particular subject, as well as its age, weight, height, general physical condition, and medical history; the particular compound used, as well as the carrier in which it is formulated and the route of administration selected for it; and the nature and severity of the condition being treated.

[0064] The term “pharmaceutical composition” means a composition comprising a 25 compound of Formula (I), Formula (I´), Formula (I´´), Formula (Ia), Formula (I´a), Formula (Ib), Formula (I´b), Formula (Ic), Formula (I´c), Formula (Id), and Formula (Ie) and at least one component comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial 30 agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms. Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar—agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial 316084209v2-19- and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. Examples of 5 suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, 10 talc, as well as high molecular weight polyethylene glycols.

[0065] The term “pharmaceutically acceptable” means it is, within the scope of sound medical judgement, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. 15

[0066] The term “pharmaceutically acceptable dosage forms” means dosage forms of the compound of the invention, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid preparations for injections, including liposome preparations. Techniques and formulations generally may be found in 20 Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition.

[0067] Compounds described herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. This technology is meant to include all such possible isomers, as well as mixtures thereof, including racemic and 25 optically pure forms. Optically active (R)- and (S)-, (-)- and (+)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. 30

[0068] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a11C- or13C- or14C -enriched carbon are within the scope of this invention. 316084209v2-20-

[0069] This technology also envisions the “quaternization” of any basic nitrogen- containing groups of the compounds disclosed herein. The basic nitrogen can be quaternized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates 5 including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0070] In the characterization of some of the substituents, it is recited that certain 10 substituents may combine to form rings. Unless stated otherwise, it is intended that such rings may exhibit various degrees of unsaturation (from fully saturated to fully unsaturated), may include heteroatoms and may be substituted with lower alkyl or alkoxy.

[0071] The term “morphinan” refers to a chemical compound that forms the core structure of a class of alkaloids known as opioids. As used herein “morphinan” can encompass 15 both opioid agonists and opioid antagonists. Morphinan’s are tetracyclic compounds, meaning they contain four interconnected rings. Morphinan derivatives include well-known substances such as morphine, codeine, naltrexone, and other opioid agonists and antagonists. Morphinan derivatives include compounds that exhibit a range of pharmacological activities, notably analgesic properties, due to their interaction with opioid receptors in the central nervous system. 20 These compounds interact with opioid receptors to produce effects such as pain relief, sedation, and euphoria.

[0072] A second aspect of the present disclosure relates to a compound of Formula (I´):where 25is a double bond with an unspecified configuration; A is a morphinan or a fragment thereof; R1is H or C1-6alkyl; R2is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; X1is C1-6alkylene; 30 X2is -NH-C(O)-; X3is C1-6alkylene; 316084209v2-21- X4is –(OCH2CH2)m-; X5is -NH-C(O)-; X6is C1-6alkylene; X7is absent or -C(O)-NH-; 5 X8is a bond or C1-6alkylene; X9is a Tag; n is 0, 1, 2, or 3; and m is 1-50, or an isomer thereof, an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate 10 thereof, or a prodrug thereof.

[0073] In some embodiments, the morphinan is a morphine-related compound (agonist or antagonist). In some embodiments, A is a naltrexone derivative. ,,15 whereis the point of attachment of A to the corresponding nitrogen atom of the structure of Formula (I); R3is selected from the group consisting of H, C1-12alkyl, and C3-6cycloalkyl, wherein C1-12alkyl can be optionally substituted with C3-6cycloalkyl, NH2, or; 20 R4is selected from the group consisting of H, OH, C1-6alkyl, and OC1-6alkyl; R5is selected from the group consisting of H, OH, C1-6alkyl, and OC1-6alkyl; or R4and R5combine to form -CH2- or -O- group. 316084209v2-22-whereis the point of attachment of A to the corresponding nitrogen atom of the 5 structure of Formula (I); R4is selected from the group consisting of H, OH, C1-6alkyl, and OC1-6alkyl; R5is selected from the group consisting of H, OH, C1-6alkyl, and OC1-6alkyl; or R4and R5combine to form -CH2- or -O- group.

[0076] In some embodiments, R1is Me. 10

[0077] In some embodiments, the Tag is a SNAP-Tag or a derivative thereof, Halo-tag or a derivative thereof, or improved Tadross Halo Tag or a derivative thereof.

[0079] In some embodiments, the compound is a cis-isomer. In other embodiments, the 15 compound is a trans-isomer.

[0080] In some embodiments, the compound has the Formula (I´´):

[0081] In some embodiments, the compound has the Formula (Ia): 316084209v2-23-where R3is selected from the group consisting of H, C1-12alkyl, and C3-6cycloalkyl, wherein C1-12alkyl can be optionally substituted with C3-6cycloalkyl, NH2, or. 5

[0082] In some embodiments, the compound has the Formula (I´a):where R3is selected from the group consisting of H, C1-12alkyl, and C3-6cycloalkyl, wherein C1-12alkyl can be optionally substituted with C3-6cycloalkyl, NH2, or. 10

[0083] In some embodiments, the compound has the Formula (Ib):where p is 1, 2, 3, 4, or 5.

[0084] In some embodiments, the compound has the Formula (I´b): 316084209v2-24-where p is 1, 2, 3, 4, or 5.

[0085] In some embodiments, the compound has the Formula (Ic):5 (Ic), where k is 1-45.

[0086] In some embodiments, the compound has the Formula (I´c):(I´c), where k is 1-45.

[0087] In some embodiments, the compound has the Formula (Id): 316084209v2-25-where k is 1-45.

[0088] In some embodiments, the compound has the Formula (Ie): 316084209v2-26-where k is 1-45.

[0089] In some embodiments,is, without limitation, phenyl, naphthyl, biphenyl, or pyrazolyl. 5

[0090] In some embodiments,is, without limitation, phenyl, naphthyl, biphenyl, or pyrazolyl.

[0091] In some embodiments, the compound is, without limitation,, 316084209v2-27- 5, 316084209v2-28- 5, 316084209v2-29- , , , 5, 316084209v2-30- ,, 316084209v2-31-, 316084209v2-32- H, 316084209v2-33-316084209v2-34-, or an isomer thereof.

[0092] In some embodiments, the compound of Formula (I) is, without limitation, 5316084209v2-35- 5316084209v2-36-316084209v2-37-316084209v2-38-, 316084209v2-39- HO, 316084209v2-40-, or an isomer thereof.

[0093] In some embodiments, the compound of Formula (I) is, without limitation, 5, 316084209v2-41- , 5 ,, 316084209v2-42- ,, 5 or an isomer thereof.

[0094] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound according to any aspect of the present disclosure and a pharmaceutically acceptable carrier.

[0095] Another aspect of the present disclosure relates to a method of treating pain in a 10 subject. This method comprises administering to the subject in need thereof the compound according to any aspect of the present disclosure.

[0096] In some embodiments, the pain is medium to severe pain, visceral pain, chronic pain, cancer pain, migraine, inflammatory pain, acute pain, neuropathic pain, allodynia, or hyperalgesia. In some embodiments, the pain is a severe pain. 15

[0097] Another aspect of the present disclosure relates to a method of treating a mood or substance use disorder in a subject. This method comprises administering to the subject in need thereof the compound according to any aspect of the present disclosure.

[0098] In some embodiments, the mood disorder is a depression or anxiety. In some embodiments, the depression is a major depression. In some embodiments, the substance use 20 disorder is an addiction. 316084209v2-43-

[0099] Another aspect of the present disclosure relates to a method of treating a respiratory depression in a subject. This method comprises administering to the subject in need thereof the compound according to any aspect of the present disclosure.

[0100] Another aspect of the present disclosure relates to a method of activating a mu- 5 opioid receptor (MOR). This method comprises contacting a MOR with the compound according to any aspect of the present disclosure under conditions effective to activate the MOR.

[0101] Another aspect of the present disclosure relates to a method of activating a kappa- opioid receptor and / or delta-opioid receptor. This method comprises contacting a kappa-opioid receptor and / or delta-opioid receptor with the compound according to any aspect of the present 10 disclosure under conditions effective to activate kappa-opioid receptor and / or delta-opioid receptor.

[0102] Another aspect of the present disclosure relates to a method of preventing and / or treating a condition where it is desired to activate MOR. This method comprises administering to the subject in need thereof the compound according to any aspect of the present disclosure. 15

[0103] Another aspect of the present disclosure relates to a method of preventing and / or treating a condition where it is desired to activate kappa-opioid receptor and / or delta-opioid receptor. This method comprises administering to the subject in need thereof the compound according to any aspect of the present disclosure.

[0104] In some embodiments, the condition is an anxiety disorder, obsessive-compulsive 20 disorder (OCD), or stress.

[0105] In some embodiments of the methods disclosed herein, the compound is administered as a trans-isomer.

[0106] In some embodiments, the method further comprises converting the trans-isomer into a cis-isomer after the administering. In some embodiments, the converting is carried out by 25 exposing the trans-isomer to a one photon or two photon light from about 300 to about 1500 nm under conditions effective to convert the trans-isomer into a cis-isomer. For example, in some embodiments, the converting is carried by exposing the trans-isomer to a one photon or two photon light from about 300 to about 500 nm, from about 330 nm to about 400 nm, from about 350 nm to about 380 nm, from about 355 nm to about 375 nm, from about 360 nm to about 370 30 nm, from about 300 to about 750 nm, from about 300 to about 1000 nm, from about 300 to about 1250 nm, from about 400 to about 500 nm, from about 410 to about 490 nm, from about 420 to about 480 nm, from about 430 to about 480 nm, from about 440 to about 480 nm, from about 450 nm to about 470 nm, from about 460 nm to about 470 nm, from about 400 to about 750 nm, from about 400 to about 1000 nm, from about 400 to about 1250 nm, from about 400 to about 316084209v2-44- 1500 nm, from about 500 to about 750 nm, from about 500 to about 1000 nm, from about 500 to about 1250 nm, from about 500 to about 1500 nm, from about 600 to about 750 nm, from about 600 to about 1000 nm, from about 600 to about 1250 nm, from about 600 to about 1500 nm, from about 700 to about 750 nm, from about 700 to about 1000 nm, from about 700 to about 5 1250 nm, from about 700 to about 1500 nm, from about 800 to about 1000 nm, from about 800 to about 1250 nm, from about 800 to about 1500 nm, from about 900 to about 1000 nm, from about 900 to about 1250 nm, from about 900 to about 1500 nm, from about 1000 to about 1250 nm, from about 1000 to about 1500 nm, or from about 1250 to about 1500 nm. For example, in some embodiments, the converting is carried by exposing the trans-isomer to a one photon or 10 two photon light of about 325 nm, about 330 nm, about 335 nm, about 340 nm, about 345 nm, about 350 nm, about 355 nm, about 360 nm, about 365 nm, about 370 nm, about 375 nm, about 380 nm, about 385 nm, about 390 nm, about 395 nm, about 400 nm, about 405 nm, about 410 nm, about 415 nm, about 420 nm, about 425 nm, about 430 nm, about 435 nm, about 440 nm, about 445 nm, about 450 nm, about 455 nm, about 460 nm, about 465 nm, about 470 nm, about 15 475 nm, about 480 nm, about 485 nm, about 490 nm, about 495 nm, about 500 nm, about 505 nm, or about 510 nm.

[0107] In some embodiments, the method further comprises converting the cis-isomer into a trans-isomer.

[0108] In some embodiments, the converting is carried out in vivo. 20

[0109] In some embodiments, the administering does not create an addiction or dependence.

[0110] In some embodiments, the administering does not create a respiratory depression.

[0111] In some embodiments, the administering does not cause constipation.

[0112] In some embodiments, the administering does not cause tolerance. 25

[0113] In practicing the method of the present disclosure, compounds suitable for treating a subject can be administered using any method standard in the art. The compounds, in their appropriate delivery form, can be administered orally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or intranasally. The compositions of the present disclosure may be administered alone or with suitable pharmaceutical carriers, and can be in 30 solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.

[0114] The compounds of the present disclosure may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or it may be enclosed in hard or soft shell capsules, or it may be compressed into tablets, or they may be incorporated directly with the food of the diet. The compounds of the present disclosure may also be administered in a 316084209v2-45- time release manner incorporated within such devices as time-release capsules or nanotubes. Such devices afford flexibility relative to time and dosage. For oral therapeutic administration, the compounds of the present disclosure may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and 5 preparations should contain at least 0.1% of the agent, although lower concentrations may be effective and indeed optimal. The percentage of the compound in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of the compounds of the present disclosure in such therapeutically useful compositions is such that a suitable dosage will be obtained. 10

[0115] Also specifically contemplated are oral dosage forms of the compounds of the present disclosure. The compounds may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is 15 the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline (Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts,” In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp.367- 20 383 (1981), which are hereby incorporated by reference in their entirety). Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

[0116] The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a 25 disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, sucralose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

[0117] The compounds of the present disclosure may also be administered parenterally. 30 Solutions or suspensions of the agent can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as 316084209v2-46- propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0118] The pharmaceutical forms suitable for injectable use include sterile aqueous 5 solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, 10 water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0119] When it is desirable to deliver compounds of the present disclosure systemically, they may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in 15 ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0120] Intraperitoneal or intrathecal administration of the compounds of the present disclosure can also be achieved using infusion pump devices such as those described by 20 Medtronic, Northridge, CA. Such devices allow continuous infusion of desired compounds avoiding multiple injections and multiple manipulations.

[0121] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or 25 ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0122] The percentage of active ingredient in the compositions of the present disclosure may be varied, it being necessary that it should constitute a proportion such that a suitable dosage shall be obtained. Obviously, several unit dosage forms may be administered at about the same time. The dose employed will be determined by the physician, and depends upon the 30 desired therapeutic effect, the route of administration and the duration of the treatment, and the condition of the patient. In the adult, the doses are generally from about 0.01 to about 100 mg / kg body weight, preferably about 0.01 to about 10 mg / kg body weight per day by inhalation, from about 0.01 to about 100 mg / kg body weight, preferably 0.1 to 70 mg / kg body weight, more especially 0.1 to 10 mg / kg body weight per day by oral administration, and from about 0.01 to 316084209v2-47- about 50 mg / kg body weight, preferably 0.01 to 10 mg / kg body weight per day by intravenous administration. In each particular case, the doses will be determined in accordance with the factors distinctive to the subject to be treated, such as age, weight, general state of health, and other characteristics which can influence the efficacy of the medicinal product. 5

[0123] The compounds according to the present disclosure may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. 10 Generally, the active product may be administered orally 1 to 4 times per day. It goes without saying that, for other patients, it will be necessary to prescribe not more than one or two doses per day.

[0124] The above disclosure is general. A more specific description is provided below in the following examples. The examples are described solely for the purpose of illustration and are 15 not intended to limit the scope of the present application. Changes in form and substitution of equivalents are contemplated as circumstances suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. EXAMPLES 20

[0125] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof. Materials and Methods Molecular Docking

[0126] The active-state BU-72-bound structure of the µ-opioid receptor (PDB ID: 5C1M) 25 (Huang et al., “Structural Insights Into µ-opioid Receptor Activation,” Nature 524:315–321 (2015), which is hereby incorporated by reference in its entirety) was retrieved from the Protein Data Bank. The G-protein mimetic camelid antibody fragment was removed, retaining the receptor subunit, three crystallographic water molecules (wa* 8, 25, and 46), and the crystallized BU-72. Residues 52–63 at the N-terminus were deleted. Hydrogen atoms were added and 30 optimized, and side-chain conformations were refined to improve structural accuracy. The inactive-state, beta-FNA-bound structure of the µ-opioid receptor (PDB ID: 4DKL) (Manglik et al., “Crystal Structure of the µ-opioid Receptor Bound to a Morphinan Antagonist,” Nature 316084209v2-48- 485:321–326 (2012), which is hereby incorporated by reference in its entirety) was similarly retrieved and processed retaining the receptor subunit, two crystallographic water molecules (wa* 18 and 19), and the crystallized ligand. Residues 263–269, which are part of the T4 lysozyme (T4L) fusion protein, were deleted. As with the active-state receptor, hydrogen atoms 5 were added and optimized, and side-chain conformations were refined.

[0127] Prior to docking, the azo-morphine (AM-1, AM-2, AM-3, AM-4) in both cis and trans isomers of the azo group underwent chiral definition and formal charge assignment. Two- dimensional molecular representations were converted into three-dimensional models, which were energy optimized using the MMFF94 force field (Halgren, T. A., “Potential Energy 10 Functions,” Curr Opin Struct Biol 5:205–210 (1995), which is hereby incorporated by reference in its entirety). A biased-probability Monte Carlo (BPMC) optimization approach was applied for docking simulations, where compound conformations in internal coordinates were sampled using pre-calculated grid energy potentials of the receptor (Totrov and Abagyan, “Flexible Protein-ligand Docking by Global Energy Optimization in Internal Coordinates,” Proteins Suppl. 15 1:215–220 (1997), which is hereby incorporated by reference in its entirety). The grid potentials accounted for receptor limited flexibility through “soft” van der Waals potentials, while maintaining the receptor's overall conformational state.

[0128] All-atom docking simulations were conducted with flexible models of azo- morphines in cis and trans isomers, with effort value of 5. To avoid major compound 20 displacement for the suboptimal AM-x isomers, the common morphinan substructure from the naltrexone binding pose was used as a template, where the ligand atoms were tethered to the corresponding common substructure template atoms by soft harmonic restraints. The docking box was defined to encompass the extracellular half of the receptor. A minimum of 15 independent docking runs, each generating three top scoring conformations, were performed 25 starting from random ligand conformations. The best ten docking results were analyzed for consistency by comparing ligand poses. Top-scoring docking solutions were refined iteratively using energy minimization and Monte Carlo sampling. Refinements focused on ligand conformations and receptor side chains within a 5 Å radius of the binding site. All molecular modeling operations were performed in the ICM-Pro v3.9-2b molecular modeling and drug 30 discovery suite (Molsoft LLC). Synthesis Methods and Chemicals

[0129] Unless otherwise stated, all reactions were performed with magnetic stirring under a positive pressure of nitrogen or argon gas. Solvents and reagents were used as received 316084209v2-49- from commercial sources (Sigma-Aldrich, Alfa Aesar, Acros Organics, Strem Chemicals, Combi-Blocks, Oakwood, Toronto Research Chemicals). Solvents were purchased from Fisher and Acros Organics. The anhydrous solvents were either purified using a Pure Process Technologies solvent purification system or purchased from Acros Organics and used as 5 received. Thin Layer Chromatography (TLC) and Column Chromatography

[0130] Reactions were monitored by thin-layer chromatography (TLC) using silica gel F254pre-coated glass plates (Merck) and visualized by exposure to ultraviolet light (λ = 254 nm) or by staining with aqueous potassium permanganate (KMnO4) solution (7.5 g KMnO4, 50 g 10 K2CO3, 6.25 mL aqueous 10% NaOH, 1000 mL distilled H2O) or aqueous acidic ceric ammonium molybdate (IV) (CAM) solution (2.0 g Ce(NH4)4(SO4)4·2 H2O, 48 g (NH4)6Mo7O24·4 H2O, 60 mL concentrated sulfuric acid, 940 mL distilled H2O) followed by heating with a heat gun (150–600 °C). Column chromatography was performed using silica gel (pore size, 60 Å; 40 to 63 μm; Merck KGaA) using a Teledyne ISCO CombiFlash EZ Prep flash 15 purification system. Nuclear Magnetic Resonance (NMR) Spectroscopy

[0131] Proton (1H) and carbon (13C) nuclear magnetic resonance spectra were recorded on a Bruker NEO 400 MHz and Bruker NEO 600 MHz. Proton chemical shifts are expressed in parts per million (ppm, δ scale) and referenced to residual undeuterated solvent signals. Carbon 20 chemical shifts are expressed in parts per million (ppm, δ scale) and referenced to the central carbon resonance of the solvent. The reported data is represented as follows: chemical shift in parts per million (ppm, δ scale) (multiplicity, coupling constants J in Hz, integration intensity). Abbreviations used for analysis of multiplets are as follows: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), p (pentet), h (hextet), and m (multiplet) or combinations thereof. 25 NMR spectroscopy was performed on a Bruker AVIII-500 MHz spectrometer equipped with a BBO probe and using a BCUII for variable temperature. NMR spectra were acquired at 25 °C unless stated otherwise. Spectra analysis was conducted with the software MestReNova. The compounds that contain a photoswitchable moiety were thermally relaxed to the dark-adapted state by wrapping the NMR tubes with aluminum foil and placing them in a hot water bath 30 overnight before taking an NMR. Determination of Photophysical Properties UV-Vis Spectroscopy

[0132] Ultraviolet-visible (UV-vis) spectra were recorded on the Varian Cary 60 UV- Visible spectrophotometer using Brand disposable UV cuvettes (850 μL, 10 mm light path) by 316084209v2-50- Brandtech Scientific Inc. Samples were stored and prepared under red light to avoid the formation of the (Z) isomers. Stock solutions (10 mM) were prepared in the dark and diluted with DMSO and PBS to a final concentration of 50 μM for measurement. Wavelength Scan 5

[0133] Light at different wavelengths was provided by an Optoscan Monochromator with an Optosource (75 mW lamp), which was controlled through a program written in Matlab. UV- vis spectra of Azo-morphines were recorded following irradiation with different wavelengths for 5 min using a monochromator. Measurement was started from the dark-adapted state followed by 550 nm irradiation and incrementally decreased the wavelength. The wavelength scan of Azo- 10 morphines were measured in phosphate-buffered saline (PBS) with 10% DMSO. Thermal Relaxation, Photocycling, and PSS

[0134] Thermal relaxation was measured by preirradiating Azo-morphines with 365-nm light and observing the absorption at 320 nm over 36-72 hours at 37°C in PBS with 10% DMSO in tightly sealed cuvettes. The reversible switching and photochemical stability of Azo- 15 morphines were demonstrated in PBS with 10% DMSO, cycling the irradiation of the monochromator between 365 nm and 460 nm. PSSs (photostationary state) were obtained from the internal UV-vis detector of the liquid chromatography–mass spectrometry (LC-MS) by irradiating the sample with different wavelengths for 5 mins before injection. The absorption peaks at isosbestic point for the trans and cis isomers were integrated to determine their ratios on 20 LCMS. Synthetic Procedures and Characterization Synthesis of (4R,4aS,7R,7aR,12bS)-7-(Benzyl(methyl)amino)-3- (cyclopropylmethyl)-1,2,3,4,5,6,7,7a-octahydro-4aH-4,12-methanobenzofuro[3,2- e]isoquinoline-4a,9-diol (S-1) 25

[0135] In a flame-dried 10 mL round bottom flask, naltrexone hydrochloride (113.4 mg, 0.3 mmol, 1.0 eq.), N-methylbenzylamine (155 µL, 1.2 mmol, 4.0 eq.), benzoic acid (55 mg, 0.45 mmol, 1.5 eq.), and p-TsOH·H2O (11.4 mg, 0.06 mmol, 0.2 eq.) were dissolved in anhydrous benzene (6 mL, 0.05 M). The solution was refluxed in the presence of a Dean-Stark 30 apparatus for 48 hours at 140 °C. Upon completion of the reaction, the reaction mixture was 316084209v2-51- concentrated in vacuo. The residue was dissolved in anhydrous MeOH (3 mL) and cooled to −20 °C. A freshly prepared solution of NaBH3CN (56.6 mg, 0.9 mmol, 3.0 eq.) in anhydrous MeOH (3 mL) was then added. The reaction mixture was allowed to slowly warm up to rt for 4 hours and stir at rt for another 14 hours. Upon completion of the reaction, the reaction mixture was 5 diluted with EtOAc (30 mL), transferred to a separatory funnel, and the organic phase was washed with sat. NaHCO3(aq) (2x 20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified on silica gel (gradient from 0% to 5% MeOH in CH2Cl2with 1% ammonia, 7M in methanol) to afford the desired tertiary amine (102 mg, 0.23 mmol, 76%) as a white foamy solid. 10

[0136] 1H NMR (400 MHz, CDCl3) δ 7.38 – 7.26 (m, 4H), 7.25 – 7.17 (m, 1H), 6.64 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 8.1 Hz, 1H), 5.30 (s, 1H), 5.03 (s, 1H), 4.69 (d, J = 8.0 Hz, 1H), 3.80 (d, J = 13.5 Hz, 1H), 3.58 (d, J = 13.6 Hz, 1H), 3.08 – 2.91 (m, 2H), 2.66 – 2.48 (m, 3H), 2.36 (d, J = 7.3 Hz, 5H), 2.23 (td, J = 12.4, 4.9 Hz, 1H), 2.10 (td, J = 11.9, 3.6 Hz, 1H), 1.95 (qd, J = 13.5, 3.4 Hz, 1H), 1.66 – 1.53 (m, 2H), 1.49 – 1.40 (m, 1H), 1.30 (td, J = 13.2, 3.1 Hz, 1H), 15 0.90 – 0.76 (m, 1H), 0.58 – 0.45 (m, 2H), 0.17 – 0.06 (m, 2H).13C NMR (101 MHz, CDCl3) δ 142.4, 139.8, 139.6, 131.9, 128.8, 128.3, 127.0, 124.8, 118.7, 116.6, 91.4, 70.5, 63.5, 62.6, 59.3, 58.9, 48.0, 44.2, 38.2, 30.9, 30.7, 22.8, 17.9, 9.6, 4.0, 4.0. The characterizations match the data that has been reported previously (St. Onge et al., “Systematic Structure–Activity Relationship Study of Nalfurafine Analogues toward Development of Potentially Nonaddictive Pain 20 Management Treatments,” J. Med. Chem.67(11): 9552-9574 (2024), which is hereby incorporated by reference in its entirety). Synthesis of (4R,4aS,7R,7aR,12bS)-3-(Cyclopropylmethyl)-7-(methylamino)- 1,2,3,4,5,6,7,7a-octahydro-4aH-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (1)Me 1 25

[0137] To a flame-dried 5 mL round bottom flask, S-1 (78 mg, 0.175 mmol, 1.0 eq.) and Pd / C 10% (37 mg, 0.035 mmol, 0.2 eq.) was added. The atmosphere was exchanged to argon, and a 1:1 ratio of i-PrOH:AcOH was added (1.75 mL respectively, 3.5 mL total, 0.1 M). The solution was then purged three times with H2(1 atm), and two hydrogen balloons were used for the reaction. The reaction mixture was stirred at room temperature until complete conversion and 30 the progress of the reaction was monitored with LCMS. Upon the completion of the reaction, the 316084209v2-52- atmosphere was changed back to argon. The reaction mixture was vacuum-filtered over celite and rinsed with i-PrOH and CH2Cl2. The filtrate was concentrated in vacuo. The crude product was purified on silica gel (gradient from 5% to 15% MeOH in CH2Cl2with 1% ammonia, 7M in methanol) to afford the desired secondary amine (60 mg, 0.17 mmol, 97%) as a pale yellow 5 solid.

[0138] 1H NMR (400 MHz, CDCl3) δ 6.65 (d, J = 8.1 Hz, 1H), 6.54 (d, J = 8.1 Hz, 1H), 4.51 (d, J = 7.5 Hz, 1H), 3.10 – 2.88 (m, 2H), 2.59 (ddt, J = 18.5, 14.1, 5.1 Hz, 3H), 2.47 (s, 3H), 2.35 (d, J = 6.5 Hz, 2H), 2.27 – 2.07 (m, 2H), 1.97 – 1.82 (m, 1H), 1.71 – 1.57 (m, 2H), 1.45 – 1.33 (m, 2H), 0.89 – 0.76 (m, 1H), 0.51 (dt, J = 8.2, 3.1 Hz, 2H), 0.17 – 0.06 (m, 2H).13C NMR 10 (101 MHz, CDCl3) δ 142.2, 140.9, 131.5, 123.5, 119.4, 118.3, 91.3, 70.5, 62.6, 59.3, 59.3, 47.5, 44.2, 30.9, 30.5, 30.5, 22.8, 21.7, 9.6, 4.0, 3.9. The characterizations match the data that has been reported previously (St. Onge et al., “Systematic Structure–Activity Relationship Study of Nalfurafine Analogues toward Development of Potentially Nonaddictive Pain Management Treatments,” J. Med. Chem.67(11): 9552-9574 (2024), which is hereby incorporated by 15 reference in its entirety). Synthesis of 3-(Phenyldiazenyl)benzoic Acid (S-2)

[0139] In a 50 mL round bottom flask, 3-aminobenzoic acid (411 mg, 3.0 mmol, 1.0 eq.) and nitrosobenzene (418 mg, 3.9 mmol, 1.3 eq.) were suspended in AcOH (12 mL). The reaction 20 mixture was stirred vigorously (800 rpm) for 96 hours at room temperature. After the completion of the reaction, celite was added to the flask and the solution was concentrated in vacuo. The crude mixture was then loaded on silica gel and purified by flash chromatography (0-5% MeOH in EtOAc) to afford the desired azobenzene (594 mg, 2.626 mmol, 88%) as an orange solid.

[0140] 1H NMR (600 MHz, CDCl3) δ 8.67 (d, J = 1.9 Hz, 1H), 8.24 (dt, J = 7.7, 1.4 Hz, 25 1H), 8.17 (dt, J = 7.9, 1.5 Hz, 1H), 7.99 – 7.94 (m, 2H), 7.65 (t, J = 7.8 Hz, 1H), 7.58 – 7.48 (m, 3H).13C NMR (151 MHz, CDCl3) δ 171.2, 152.8, 152.6, 132.3, 131.7, 130.5, 129.5, 129.3, 127.8, 124.9, 123.2. The characterizations match the data that has been reported previously (Hartrampf et al., “Development of a Photoswitchable Antagonist of NMDA Receptors,” Tetrahedron 73(33):4905-4912 (2017), which is hereby incorporated by reference in its entirety). 30 Synthesis of 2-(4-(Phenyldiazenyl)phenyl)acetic Acid (2) 316084209v2-53-

[0141] In a 50 mL round bottom flask, 4-aminophenylacetic acid (454 mg, 3.0 mmol, 1.0 eq.) and nitrosobenzene (418 mg, 3.9 mmol, 1.3 eq.) were suspended in AcOH (12 mL). The reaction mixture was stirred vigorously (800 rpm) for 96 hours at room temperature. After the 5 completion of the reaction, celite was added to the flask and the solution was concentrated in vacuo. The crude mixture was then loaded on silica gel and purified by flash chromatography (0- 5% MeOH in EtOAc) to afford the desired azobenzene (334 mg, 1.390 mmol, 46%) as an orange solid.

[0142] 1H NMR (600 MHz, CDCl3) δ 7.91 (td, J = 5.9, 3.1 Hz, 4H), 7.55 – 7.43 (m, 5H), 10 3.76 (s, 2H).13C NMR (151 MHz, CDCl3) δ 174.8, 152.8, 152.0, 136.4, 131.2, 130.3, 129.2, 123.3, 123.0, 40.6. The characterizations match the data that has been reported previously (Kawaguchi et al., “Development of Peptide-Based Sirtuin Defatty-Acylase Inhibitors Identified by the Fluorescence Probe, SFP3, That Can Efficiently Measure Defatty-Acylase Activity of Sirtuin,” J. Med. Chem.62(11): 5434-5452 (2019), which is hereby incorporated by reference in 15 its entirety). Synthesis of 2-(4-(Phenyldiazenyl)phenyl)acetic Acid (S-3)

[0143] In a 50 mL round bottom flask, 3-aminophenylacetic acid (454 mg, 3.0 mmol, 1.0 eq.) and nitrosobenzene (418 mg, 3.9 mmol, 1.3 eq.) were suspended in AcOH (12 mL). The 20 reaction mixture was stirred vigorously (800 rpm) for 96 hours at room temperature. After the completion of the reaction, celite was added to the flask and the solution was concentrated in vacuo. The crude mixture was then loaded on silica gel and purified by flash chromatography (0- 5% MeOH in EtOAc) to afford the desired azobenzene (625 mg, 2.601 mmol, 87%) as an orange solid. 25

[0144] 1H NMR (600 MHz, CDCl3) δ 7.94 – 7.89 (m, 2H), 7.88 – 7.84 (m, 2H), 7.55 – 7.45 (m, 4H), 7.41 (dt, J = 7.6, 1.5 Hz, 1H), 3.78 (s, 2H).13C NMR (151 MHz, CDCl3) δ 176.3, 153.0, 152.7, 134.5, 132.0, 131.3, 129.5, 129.2, 123.6, 123.0, 122.5, 40.8. HRMS (ESI): m / z [M- H]- calcd for C14H11N2O2: 239.082601, found: 239.082519. 316084209v2-54- Synthesis of N-((4R,4aS,7R,7aR,12bS)-3-(Cyclopropylmethyl)-4a,9- dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)- N-methyl-4-((E)-phenyldiazenyl)benzamide (AM-1)5

[0145] In a flame-dried 5 mL round bottom flask, naltrexone-derived amine (1) (14 mg, 0.040 mmol, 1.0 eq.), 4-(phenylazo)benzoic acid (11 mg, 0.048 mmol, 1.2 eq.), and COMU coupling reagent (19 mg, 0.044 mmol, 1.1 eq.) were dissolved in anhydrous DMF (0.40 mL, 0.1 M). The reaction was stirred under argon at 0 °C, and anhydrous DIPEA (15 µL, 0.088 mmol, 2.2 eq.) was added. The reaction mixture was stirred at 0 °C for 1 hour before the cooling bath 10 was removed, and the solution was allowed to warm up and stirred at room temperature for another 16 hours. Upon the completion of the reaction, the mixture was diluted with EtOAc:Et2O 1:1 (20 mL) and transferred to a separatory funnel.0.1 M NaHCO3(10 mL) was added to the separatory funnel, and layers were separated. The aqueous layer was extracted with EtOAc (2x20 mL). The combined organic layer was extracted with 10% LiCl solution (2x20 mL) and brine 15 (1x20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified on silica gel (gradient from 0% to 10% MeOH in CH2Cl2with 1% ammonia, 7M in methanol) to afford the desired AM-1 (24.6 mg, 0.033 mmol, 82%) as an orange oil. For long-term storage, the compound was kept as a trifluoroacetic acid or a formic acid salt. Trifluoroacetic acid or formic acid (1.3 eq) in acetonitrile (99:1 MeCN:TFA / formic 20 acid) was added to the compound and concentrated in vacuo to afford the salt in order to avoid the formation of N-oxides. The dilution of acid was necessary to avoid the decomposition of the product.

[0146] 1H NMR (500 MHz, CDCl3, exists as ~8:2 rotamers, only resonance of major rotamer shown) δ 7.92 – 7.88 (m, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.63 – 7.50 (m, 5H), 6.66 (d, J = 25 8.2 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 4.92 (d, J = 8.1 Hz, 1H), 3.81 (d, J = 5.7 Hz, 1H), 3.53 (ddd, J = 12.8, 8.1, 4.4 Hz, 1H), 3.24 (q, J = 6.8 Hz, 1H), 3.17 (s, 3H), 3.11 – 3.03 (m, 2H), 2.98 (dd, J = 19.6, 6.0 Hz, 1H), 2.88 – 2.80 (m, 1H), 2.66 – 2.53 (m, 2H), 2.35 – 2.22 (m, 1H), 1.74 – 1.66 (m, 1H), 1.54 – 1.45 (m, 1H), 1.34 – 1.20 (m, 2H), 1.07 – 1.00 (m, 1H), 0.74 (dtt, J = 39.2, 8.6, 4.1 Hz, 2H), 0.46 (ddp, J = 17.3, 9.4, 5.4 Hz, 2H).13C NMR (151 MHz, MeOD, exists as 316084209v2-55- ~8:2 rotamers, only resonance of major rotamer shown) δ 174.2, 154.0, 153.9, 142.7, 142.5, 139.7, 132.7, 130.3, 129.1, 128.8, 124.0, 123.9, 123.9, 120.8, 118.9, 88.7, 71.3, 67.6, 66.9, 63.8, 61.8, 59.3, 38.6, 31.4, 29.0, 23.8, 23.5, 15.4, 5.4, 3.7. The NMR of this compound at room temperature was a rotameric mixture. HRMS (ESI): m / z [M+H]+calcd for C34H36N4O4: 5 565.280932, found: 565.283392. Rf(4% MeOH in CH2Cl2with 1% ammonia, 7M in methanol): 0.47; stains yellow with KMnO4; detected by UV. Synthesis of N-((4R,4aS,7R,7aR,12bS)-3-(Cyclopropylmethyl)-4a,9- dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)- N-methyl-3-((E)-phenyldiazenyl)benzamide (AM-2) 10

[0147] In a flame-dried 5 mL round bottom flask, naltrexone-derived amine (1) (14 mg, 0.040 mmol, 1.0 eq.), 3-(phenylazo)benzoic acid (S-2) (11 mg, 0.048 mmol, 1.2 eq.), and COMU coupling reagent (19 mg, 0.044 mmol, 1.1 eq.) were dissolved in anhydrous DMF (0.40 mL, 0.1 M). The reaction was stirred under argon at 0 °C, and anhydrous DIPEA (15 µL, 0.088 mmol, 15 2.2 eq.) was added. The reaction mixture was stirred at 0 °C for 1 hour before the cooling bath was removed, and the solution was allowed to warm up and stirred at room temperature for another 16 hours. Upon the completion of the reaction, the mixture was diluted with EtOAc:Et2O 1:1 (20 mL) and transferred to a separatory funnel.0.1 M NaHCO3(10 mL) was added to the separatory funnel, and layers were separated. The aqueous layer was extracted with EtOAc (2x20 20 mL). The combined organic layer was extracted with 10% LiCl solution (2x20 mL) and brine (1x20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified on silica gel (gradient from 0% to 10% MeOH in CH2Cl2with 1% ammonia, 7M in MeOH) to afford the desired AM-2 (17.4 mg, 0.031 mmol, 77%) as an orange oil. For long-term storage, the compound was kept as a trifluoroacetic acid or a formic acid salt. 25 Trifluoroacetic acid or formic acid (1.3 eq) in acetonitrile (99:1 MeCN:TFA / formic acid) was added to the compound and concentrated in vacuo to afford the salt in order to avoid the formation of N-oxides. The dilution of acid was necessary to avoid the decomposition of the product.

[0148] 1H NMR (500 MHz, MeOD, exists as a mixture of rotamers, only resonance of 30 major rotamer shown) δ 8.51 (br s, 0H, formate salt peak), 7.90 (dt, J = 6.4, 1.7 Hz, 2H), 7.83 – 316084209v2-56- 7.79 (m, 2H), 7.53 (tt, J = 21.6, 7.9 Hz, 5H), 7.3 – 7.1 (m, toluene), 7.3 – 7.1 (m, toluene), 6.60 (d, J = 8.2 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 4.91 (d, J = 8.1 Hz, 1H), 3.78 (d, J = 5.7 Hz, 1H), 3.55 (ddd, J = 12.7, 8.0, 4.4 Hz, 1H), 3.24 – 3.17 (m, 4H), 3.12 – 2.91 (m, 2H), 2.82 (dd, J = 13.4, 7.5 Hz, 1H), 2.58 (d, J = 8.1 Hz, 2H), 2.32 (s, toluene), 2.35 – 2.23 (m, 1H), 1.68 (t, J = 5 14.8 Hz, 2H), 1.55 (dd, J = 23.0, 11.2 Hz, 1H), 1.35 – 1.14 (m, 2H), 1.07 (d, J = 46.8 Hz, 1H), 0.72 (d, J = 34.8 Hz, 2H), 0.57 – 0.32 (m, 2H).13C NMR (126 MHz, MeOD, exists as a mixture of rotamers, only resonance of major rotamer shown) δ 174.1, 153.8, 153.7, 142.8, 138.4, 132.7, 130.7, 130.5, 130.3, 129.9, 129.2, 126.3, 124.4, 124.0, 121.5, 121.0, 119.4, 88.4, 71.2, 69.5, 64.0, 61.6, 58.9, 56.2, 47.8, 31.3, 29.1, 24.1, 23.2, 7.0, 6.0, 3.4. The NMR of this compound at 10 room temperature was a rotameric mixture. HRMS (ESI): m / z [M+H]+calcd for C34H36N4O4: 565.280932, found: 565.283586. Rf(4% MeOH in CH2Cl2with 1% ammonia, 7M in methanol): 0.41; stains yellow with KMnO4; detected by UV. Synthesis of N-((4R,4aS,7R,7aR,12bS)-3-(Cyclopropylmethyl)-4a,9- dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)- 15 N-methyl-2-(4-((E)-phenyldiazenyl)phenyl)acetamide (AM-3)AM-3

[0149] In a flame-dried 5 mL round bottom flask, naltrexone-derived amine (1) (14 mg, 0.040 mmol, 1.0 eq.), azobenzene (2) (11 mg, 0.048 mmol, 1.2 eq.), and COMU coupling reagent (19 mg, 0.044 mmol, 1.1 eq.) were dissolved in anhydrous DMF (0.40 mL, 0.1 M). The 20 reaction was stirred under argon at 0 °C, and anhydrous DIPEA (15 µL, 0.088 mmol, 2.2 eq.) was added. The reaction mixture was stirred at 0 °C for 1 hour before the cooling bath was removed, and the solution was allowed to warm up and stirred at room temperature for another 16 hours Upon the completion of the reaction, the mixture was diluted with EtOAc:Et2O 1:1 (20 mL) and transferred to a separatory funnel.0.1 M NaHCO3(10 mL) was added to the separatory 25 funnel, and layers were separated. The aqueous layer was extracted with EtOAc (2x20 mL). The combined organic layer was extracted with 10% LiCl solution (2x20 mL) and brine (1x20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified on silica gel (gradient from 0% to 10% MeOH in CH2Cl2with 1% ammonia, 7M in MeOH) to afford the desired opto-opioid (18.0 mg, 0.031 mmol, 78%) as an orange oil. For 30 long-term storage, the compound was kept as a trifluoroacetic acid or a formic acid salt. 316084209v2-57- Trifluoroacetic acid or formic acid (1.3 eq) in acetonitrile (99:1 MeCN:TFA / formic acid) was added to the compound and concentrated in vacuo to afford the salt in order to avoid the formation of N-oxides. The dilution of acid was necessary to avoid the decomposition of the product. It has been confirmed that neither TFA nor formate salt has influence on biological 5 activity.

[0150] 1H NMR (500 MHz, DMSO, exists as 8:2 rotamers, * denotes minor rotamer) δ 8.22 (br s, 0H, formate salt peak), 7.91 – 7.82 (m, 2H+3H*), 7.77 – 7.70 (m, 2H), 7.63 – 7.51 (m, 3H+3H*), 7.45 (d, J = 8.1 Hz, 2H*), 7.10 (d, J = 8.3 Hz, 2H), 6.72 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H*), 6.52 (d, J = 8.1 Hz, 1H*), 4.71 (d, J = 8.2 Hz, 1H*), 10 4.62 (d, J = 8.0 Hz, 1H), 3.90 – 3.78 (m, 2H*), 3.70 (q, J = 15.7 Hz, 2H), 3.57 (ddd, J = 12.2, 7.7, 3.3 Hz, 1H+1H*), 3.16 – 3.05 (m, 2H*), 3.02 (h, J = 5.7 Hz, 1H), 2.98 (d, J = 3.3 Hz, 1H), 2.81 (s, 3H), 2.74 (s, 3H*), 2.69 – 2.54 (m, 1H+2H*), 2.54 – 2.46 (m, 1H), 2.36 (qt, J = 12.7, 7.0 Hz, 2H+2H*), 2.20 (qd, J = 12.6, 5.1 Hz, 1H+1H*), 2.00 (qd, J = 11.9, 3.4 Hz, 2H), 1.50 (d, J = 13.2 Hz, 1H*), 1.40 (dt, J = 13.2, 3.2 Hz, 1H), 1.35 – 1.28 (m, 1H), 1.27 – 1.21 (m, 2H*), 1.18 – 15 1.09 (m, 1H+1H*), 1.03 (dt, J = 12.6, 3.6 Hz, 1H), 0.82 (dd, J = 9.5, 4.3 Hz, 1H+1H*), 0.46 (dtt, J = 11.9, 7.6, 3.5 Hz, 2H+2H*), 0.17 – 0.09 (m, 2H+2H*).13C NMR (126 MHz, DMSO, exists as 8:2 rotamers, * denotes minor rotamer) δ 170.0, 152.0*, 151.9, 150.5*, 150.4, 142.1*, 141.5, 140.9, 140.7*, 140.1, 139.8*, 131.8, 131.4, 131.3*, 130.2*, 130.0, 129.4, 123.7, 122.5, 122.3, 119.2, 117.1, 88.5, 87.7*, 69.5*, 69.4, 65.9, 61.7*, 61.5, 58.3, 57.6, 47.2, 47.0*, 43.7, 37.9, 30.6, 20 29.7, 29.6*, 27.8, 22.4*, 22.2, 21.4*, 9.0, 3.7, 3.5. The NMR of this compound at room temperature is a rotameric mixture; a variable temperature NMR was conducted to confirm the existence of rotamers (FIG.20). HRMS (ESI): m / z [M+H]+calcd for C35H38N4O4: 579.2971, found: 579.2982. Rf(4% MeOH in CH2Cl2with 1% ammonia, 7M in methanol): 0.29; stains yellow with KMnO4; detected by UV. 25 Synthesis of N-((4R,4aS,7R,7aR,12bS)-3-(Cyclopropylmethyl)-4a,9- dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)- N-methyl-2-(3-((E)-phenyldiazenyl)phenyl)acetamide (AM-4)

[0151] In a flame-dried 5 mL round bottom flask, naltrexone-derived amine (1) (9.0 mg, 30 0.025 mmol, 1.0 eq.), azobenzene (S-4) (7.9 mg, 0.033 mmol, 1.3 eq.), and COMU coupling 316084209v2-58- reagent (13 mg, 0.030 mmol, 1.2 eq.) were dissolved in anhydrous DMF (0.25 mL, 0.1 M). The reaction was stirred under argon at 0 °C, and anhydrous DIPEA (10 µL, 0.056 mmol, 2.2 eq.) was added. The reaction mixture was stirred at 0 °C for 1 hour before the cooling bath was removed, and the solution was allowed to warm up and stirred at room temperature for another 5 16 hours. Upon the completion of the reaction, the mixture was diluted with EtOAc:Et2O 1:1 (20 mL) and transferred to a separatory funnel.0.1 M NaHCO3(10 mL) was added to the separatory funnel, and layers were separated. The aqueous layer was extracted with EtOAc (2x20 mL). The combined organic layer was extracted with 10% LiCl solution (2x20 mL) and brine (1x20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude product 10 was purified on silica gel (gradient from 0% to 10% MeOH in CH2Cl2with 1% ammonia, 7M in MeOH) to afford the desired AM-4 (10.3 mg, 0.018 mmol, 59%) as an orange oil. For long-term storage, the compound was kept as a trifluoroacetic acid or a formic acid salt. Trifluoroacetic acid or formic acid (1.3 eq) in acetonitrile (99:1 MeCN:TFA / formic acid) was added to the compound and concentrated in vacuo to afford the salt in order to avoid the formation of N- 15 oxides. The dilution of acid was necessary to avoid the decomposition of the product.

[0152] 1H NMR (600 MHz, MeOD, exists as 8:2 rotamers, * denotes minor rotamer) δ 7.89 (dt, J = 7.8, 1.9 Hz, 2H), 7.84 – 7.79 (m, 2H*), 7.71 (dq, J = 7.7, 2.8 Hz, 2H*), 7.71 – 7.66 (m, 1H), 7.62 – 7.49 (m, 3H+3H*), 7.44 (d, J = 7.7 Hz, 1H*), 7.35 (td, J = 7.8, 1.8 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.83 (dd, J = 8.1, 1.9 Hz, 1H), 6.79 – 6.74 (m, 20 1H*), 6.64 (d, J = 7.8 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H*), 4.64 (dd, J = 8.2, 1.8 Hz, 1H), 4.33 (qd, J = 7.2, 1.9 Hz, 2H*), 3.86 (dd, J = 15.4, 1.9 Hz, 1H), 3.76 (d, J = 15.4 Hz, 1H), 3.67 (ddt, J = 11.4, 6.4, 2.9 Hz, 1H+1H*), 3.23 – 3.18 (m, 3H*), 3.15 – 3.07 (m, 1H+1H*), 3.02 (d, J = 18.5 Hz, 1H), 2.92 (d, J = 1.8 Hz, 3H), 2.84 (d, J = 1.9 Hz, 3H*), 2.72 (td, J = 15.1, 5.9 Hz, 1H+1H*), 2.49 (dt, J = 14.9, 7.2 Hz, 2H), 2.39 (dd, J = 12.9, 6.6 Hz, 1H+1H*), 2.28 (td, J = 12.5, 4.8 Hz, 25 1H+1H*), 2.18 (dt, J = 13.1, 6.5 Hz, 1H+1H*), 1.98 (qd, J = 12.9, 2.7 Hz, 1H+1H*), 1.46 (dd, J = 12.1, 3.4 Hz, 1H+1H*), 1.38 – 1.25 (m, 1H+1H*), 0.99 (td, J = 13.5, 3.2 Hz, 1H+1H*), 0.92 – 0.82 (m, 1H+1H*), 0.74 (dd, J = 13.0, 3.7 Hz, 1H), 0.61 – 0.47 (m, 2H), 0.14 (t, J = 4.0 Hz, 2H). 13C NMR (126 MHz, MeOD, exists as 8:2 rotamers, only resonance of major rotamer shown) δ 173.9, 154.3, 154.0, 143.2, 142.4, 138.2, 132.9, 132.4, 132.1, 130.5, 130.3, 125.3, 123.8, 123.8, 30 122.2, 121.0, 119.0, 101.4, 90.2, 71.5, 63.7, 60.1, 60.0, 45.5, 42.7, 31.7, 31.0, 28.8, 23.7, 23.4, 9.8, 4.5, 4.1. *Note: the NMR of this compound at room temperature is a rotameric mixture. HRMS (ESI): m / z [M+H]+calcd for C35H38N4O4: 579.296582, found: 579.297980. Rf(4% MeOH in CH2Cl2with 1% ammonia, 7M in methanol): 0.27; stains yellow with KMnO4; detected by UV. 316084209v2-59- Molecular Biology

[0153] The rat SNAP-MOR (N-terminal HA followed by SNAP-tag followed by full length MOR) was previously described (Xiang et al., “Control of Gα Signaling Dynamics and GPCR Cross-talk by GRKs,” Sci Adv 8:eabq3363 (2022), which is hereby incorporated by 5 reference in its entirety). SNAP-tagged human MOR (cloned from addgene #66464), SNAP- tagged human KOR (Addgene #66462), SNAP-tagged human DOR (Addgene #66461), and SNAP-tagged mouse MOR (cloned from SSF-MOR, a gift from the Von Zastrow lab) were produced using Gibson Assembly Cloning Kits (NEB) by placing a SNAP tag between the signal sequence / FLAG tag and the N-terminus of the receptor. For SNAP-DOR, SNAP-KOR, and 10 SNAP-MOR (human), the mGluR5 signal sequence was added and the C-terminal TANGO domain was removed. The previously described GIRK1-F137S hometetramerization mutant (Vivaudou et al., “Probing the G-protein Regulation of GIRK1 and GIRK4, the Two Subunits of the KACh Channel, Using Functional Homomeric Mutants,” Journal of Biological Chemistry 272:31553–31560 (1997), which is hereby incorporated by reference in its entirety) was used for 15 patch clamp recordings and the ONE-Go Gαi3 biosensor (Addgene kit #1000000224) was used for BRET measurements. For recombinant protein expression, a full length human MOR construct was subcloned into a modified pFastBac1 vector with an N-terminal HA-signal peptide, FLAG-tag, 10xHis-tag and thermostabilized b562RIL (BRIL) and a C-terminal TwinStrep tag and an additional 10xHis-tag. Both N- and C-terminal tags are cleavable by 20 PreScission sites (pFastBac1-BRIL-hMOR). Cell Culture and Transfection

[0154] HEK 293 and HEK 293T cells purchased from ATCC (CRL-1573) and tested routinely for mycoplasma were used. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS) and maintained at 25 37°C in a 5% CO2 humidified incubator. For patch clamp and live cell imaging experiments, cells were seeded 24 hours prior to transfection on poly-L-lysine-coated glass coverslips (18 mm) in a 12-well plate. For BRET measurements, cells were seeded on a 10 cm non-treated tissue culture dish. Lipofectamine 2000 (Invitrogen) was used for transfection of DNA plasmids. For whole-cell patch clamp, cells were plated at low density and transfected with 0.7 μg SNAP- 30 tagged mouse, rat, or human MOR, 0.7 μg GIRK1, and 0.15 μg tdTomato (as a transfection marker) per well. For cell surface labeling and internalization assays, cells were transfected with 0.35 μg rat SNAP-tagged MOR per well. For BRET measurements, cells were transfected with the following plasmids with 1 µg of the ONE-GO Gαi3 biosensor (1 µg) and 5 µg of SNAP- rMOR per dish. 316084209v2-60- Surface Labeling Internalization Assay and Widefield Fluorescence Imaging

[0155] A previously described surface labeling-based internalization assay was used (Xiang et al., “Control of Gα Signaling Dynamics and GPCR Cross-talk by GRKs,” Sci Adv 8:eabq3363 (2022); Abreu et al., “Mechanisms of Differential Desensitization of Metabotropic 5 Glutamate Receptors,” Cell Rep 35:109050 (2021), which are hereby incorporated by reference in their entirety).24–36 hours post-transfection, coverslips containing cells expressing SNAP- rMOR were washed three times in 1.5 mL of extracellular (EX) solution composed of (in mM): 135 NaCl, 5.4 KCl, 10 HEPES, 2 CaCl2, and 1 MgCl2 (pH 7.4) and incubated in either no drug (media, control), 1 µM DAMGO (positive control), or cis or trans AM-3 (1 nM to 1 µM), in EX 10 for 40-45 min at 37 degrees. AM-3 photoactivation was performed using a handheld 365 nm UV Flashlight Torch Light (HQRP 365 nm 12 LED). Cells were washed three times prior to labeling by incubation in 1 µM BG-Alexa-546 (New England BioLabs) in EX for 20 min at room temperature. Following fluorophore labeling, live cells were washed and imaged on an inverted microscope (Olympus IX83) with a 60x 1.49 NA objective. Alexa-546 was excited using a 560 15 nm laser and snapshots were acquired to measure the mean fluorescence intensity of labeled surface receptors using ImageJ. Fluorescence was normalized to the average fluorescence of the no drug control. Raw fluorescence values were normalized to 1 µM DAMGO for side-by-side comparison to the electrophysiology. The decrease in surface fluorescence was calculated as 100 * (1 - normalized surface fluorescence). Data was calculated across at least three experimental 20 days per condition.

[0156] To observe the internalization of SNAP-tagged surface receptors, cells were incubated in 1 µM BG-Alexa-546 for 30-45 min at 37°C in EX. To induce internalization of SNAP-tagged receptors, cells were incubated with agonist in EX solution for 30 min at 37°C and imaged. Alexa-546 was excited with a 560 nm laser and snapshots of randomized fields were 25 acquired. The basal condition was run side-by-side with drug conditions with the same allotted time but in EX solution. Patch Clamp Electrophysiology

[0157] Whole-cell patch-clamp recordings were performed as previously described (Gutzeit et al., “A Fine-tuned Azobenzene for Enhanced Photopharmacology In vivo,” Cell 30 Chem Biol 28:1648–1663.e16 (2021), which is hereby incorporated by reference in its entirety). 24-36 hours after transfection in a high potassium extracellular solution containing (mM): 120 KCl, 25 NaCl, 10 HEPES, 2 CaCl2, and 1 MgCl2 (pH 7.4). Solutions were delivered to a recording chamber using a gravity-driven perfusion system with exchange times of ~1 s. Cells were voltage-clamped at −60 mV using an Axopatch 200B amplifier (Molecular Devices). Patch 316084209v2-61- pipettes with resistances of 3-8 MΩ were filled with an intracellular solution containing (in mM): 140 KCl, 10 HEPES, 3 Na2ATP, 0.2 Na2GTP, 5 EGTA, 3 MgCl2(pH 7.4). Currents were sampled at 10 kHz and filtered using a low-pass Bessel (8-pole) at 2 kHz. Illumination was applied to the entire field of view using a CoolLED pE-4000 through a 40x objective. Light 5 intensity in the sample plane was 1-2 mW / mm². pCLAMP software was used for data acquisition, control of illumination, and data analysis. All responses were quantified relative to 1 μM DAMGO responses. Recordings were analyzed using Clampfit (Molecular Devices) and Prism (GraphPad) software. Data was calculated across at least three experimental days per condition. 10 ONE-GO BRET Biosensor Measurements

[0158] 8 hours after transfection, media was replaced and cells were harvested 24–36 hours later. Cells were centrifuged for 5 min at 550 x g, and resuspended in BRET buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.37 mM NaH2PO4, 20 mM HEPES, pH 7.4, 0.1% glucose) at a concentration of approximately 1 million cells / mL. Forty to fifty thousand 15 cells were added to a white opaque 96-well plate (Corning) and mixed with NanoLuciferase substrate Nano-Glo (Promega) for 2 min before measuring luminescence in a Promega GloMax Discover plate reader at 28°C. Luminescence was measured at 450 BP (±10 nm) and 540 SP (±10 nm), and the BRET signal was calculated as the ratio between the emission intensity at 540 nm divided by the emission intensity at 450 nm. Reagents were added to the wells during live 20 measurements by hand. AM-3 was preactivated with a handheld UV Flashlight Torch Light (HQRP 365 nm 12 LED). BRET data are presented as the difference from baseline BRET signal (ΔBRET) by subtracting the average of the signal pre-stimulation from all data points. The BRET signal (540 nm luminescence / 450 nm luminescence) was measured every minute for 3-5 min with a signal integration time of 0.3 s for each measurement. Where indicated, the EC50and 25 Emaxvalues were determined using a 3-parameter sigmoidal curve-fit in Prism (GraphPad). Data was calculated across at least three experimental days per condition. MOR Expression and Purification

[0159] Recombinant expression of the pFastBac-BRIL-MOR construct bound to either cis or trans AM-3 was performed in Spodoptera frugiperda (Sf9) insect cells using the Bac-to- 30 Bac expression system (Gibco). Briefly, 1% (v / v) baculovirus was transduced into Sf9 cells at a density of 4 x 106cells / mL in ESF921 medium, containing 1% (v / v) production boost additive. 10 µM naloxone was maintained in the insect cell culture to improve receptor surface expression. Following incubation for 48 hours at 27 °C, cells were harvested, and resuspended in a hypotonic buffer containing 10 mM HEPES (pH 7.5), 10 mM MgCl₂, 20 mM KCl, and an in- 316084209v2-62- house protease inhibitor cocktail (2 mM AEBSF, 14 μM E-64, 1 μM leupeptin, and 0.3 μM aprotinin). The cell suspension was lysed using a glass homogenizer and then subjected to further homogenization in a hypertonic buffer (hypotonic buffer supplemented with 1 M NaCl). The resulting mixture was centrifuged at 175,000 x g for 45 minutes at 4°C. This process was 5 repeated twice, with the final two washes supplemented with 10 μM of either cis or trans AM-3. Following the washes, membranes were diluted in buffer containing 40 mM HEPES (pH 7.5), 100 mM NaCl, 10  μM AM-3, 10% glycerol, 0.5% (w / v) LMNG (Anatrace), 0.05% (w / v) CHS (Anatrace), and 2 mg / mL iodoacetamide, and incubated at 4°C for 14 hours. The insoluble fraction was cleared by centrifugation at 175,000 g for 1 hour. MOR was purified using Strep- 10 Tactin®XT 4Flow® resin (IBA), in buffer containing 40  mM HEPES pH 7.5, 100  mM NaCl, 5 mM MgCl2, 3 mM CaCl2, 0.1 mM TCEP, 0.001% (w / v) LMNG, 0.0001% (w / v) CHS, 5% glycerol and 10 μM AM-3, supplemented with 50 mM Biotin (IBA). Expression and Purification of Heterotrimeric G Proteins

[0160] Wild-type Gαi1 was co-expressed with Gβ1 and Gγ2 subunits in Sf9 cells at a 15 density of 2 × 10⁶ cells / mL in ESF921 medium, using P2 baculovirus at a multiplicity of infection (MOI) ratio of 10:5. After 48 hours of incubation at 27°C, cells were harvested by centrifugation, washed with ice-cold PBS, and stored at −80°C until further processing. Purification of Gαi(wt)βγ / Gαi(DN)βγ heterotrimer was performed following established protocols. 20

[0161] Briefly, cells were thawed, homogenized once in hypotonic buffer supplemented with 10 µM GDP and 5 mM β-mercaptoethanol, and centrifuged at 100,000 x g. The pellet was then solubilized in a buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 1% sodium cholate, 0.05% DDM, 5 mM MgCl₂, 5 mM β-mercaptoethanol, 15 mM imidazole, 10 µM GDP, and protease inhibitor for 90 minutes at 4°C. Insoluble material was removed by centrifugation at 25 150,000 x g for 45 minutes at 4°C. The supernatant was applied to Ni-NTA resin in the same buffer, and the protein was eluted with 300 mM imidazole. The eluate was concentrated using a 50-kDa cutoff concentrator (Amicon) and further purified via anion exchange chromatography on a 1 mL HiTrap Q FF column (Cytiva). Cryo-EM Sample Preparation and Data Collection 30

[0162] MOR, purified in the presence of either 10 µM cis or trans AM-3, was incubated with Gαiβγ at a 1:1.5 molar ratio for 2 hours at room temperature. Following the formation of complexes, GDP hydrolysis was catalyzed via the addition of 2 units of apyrase (NEB) and a further incubation for 2 hours. Uncomplexed G protein was separated by an additional purification step with M2 anti-FLAG resin (Sigma Aldrich). The resulting eluate was subjected 316084209v2-63- to size exclusion chromatography using a Superdex 20010 / 300 column in 40  mM HEPES pH 7.5, 100  mM NaCl, 3 mM MgCl2, 0.1 mM TCEP, 5 μM AM-3, 0.00075% LMNG, 0.000075% CHS and 0.00025% GDN buffer. Peak fractions were concentrated and immediately deployed for downstream cryo-EM studies. MOR:Gi-wt complexes were concentrated to ~1 mg / mL using 5 a 50-kDa cutoff concentrator (Amicon). Immediately after, 3 µL of the purified complex was applied to glow-discharged UltrAuFoil 1.2 / 1.3300-mesh grids. Grids were blotted for 1-2 seconds in 95% relative humidity at 4°C, then rapidly vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Cryo-EM data collection was performed on a Titan Krios (Thermo Fisher) at 300 keV, using an aberration-free image shift (AFIS) data collection scheme, and a K3 10 direct-electron detector coupled with a BioQuantum energy filter (Gatan), set to a 20 eV slit width. Four images per hole were acquired with EPU data acquisition software (version 2.0). Each image was captured with a total exposure time of 1.8 seconds, a cumulative dose of 51 e− / Ų (cis-AM-3) and 55 e− / Ų (trans-AM-3), and a defocus range of −1 µm to −3 µm. Single Particle Cryo-EM Image Processing 15

[0163] All data processing was performed using the software package cryoSPARC (Punjani et al., “cryoSPARC: Algorithms for Rapid Unsupervised Cryo-EM Structure Determination,” Nat. Methods 14:290–296 (2017), which is hereby incorporated by reference in its entirety) (v4.5.3; Structura Biotechnology) (FIGs.2A-2C and 3A-3C, Table 1). A total of 13,418 and 28,180 micrographs were collected for cis-AM-3 and trans-AM-3 respectively. 20 Motion correction was performed on raw micrographs using Patch Motion Correction, followed by CTF estimation. Micrographs with CTF estimates worse than 3.5 Å were excluded from further processing. Automated particle picking was performed using a reference-free blob picker, and particles were extracted with a box size of 128 pixels (bin=4, 512 pixels uncropped box size), followed by three rounds of 2D classification. Ab initio reconstruction was performed 25 using a “clean” stack of 1,142,073 particles for cis-, and 1,329,432 particles for trans-AM-3, resulting in a 3D volume with clearly visible densities for all components of the MOR-Gi complex (FIGs.2A-2C and 3A-3C). Particles were then re-extracted using a box size of 256 pixels (bin=2, 512 pixels uncropped box size) for cis- and 384 pixels (bin=1.33, 512 pixels uncropped box size) for trans-, followed by additional 2D classification. Final particle sets 30 comprising 580,551 particles for cis- and 628,564 particles for trans-AM-3 were deployed for further downstream processing. 316084209v2-64- Table 1. Cryo-EM statistics316084209v2-65-

[0164] For the cis-AM-3 MOR:Gi complex, the initial ab initio reconstruction was subjected to multiple rounds of non-uniform refinement (low pass filter = 10 Å), interspersed with local refinements resulting in a density map with resolution estimates of 3 Å. Next, focused 5 refinements were performed using manually created masks around the TMD, masking the micelle (‘Mask 1’) and heterotrimeric G protein (‘Mask 2’), yielding focused maps with resolutions between 2.9 Å - 3.1 Å. These were combined in Chimera, for subsequent model 316084209v2-66- building and refinement. Finally, 3D Variability Analysis (Sounier et al., “Propagation of Conformational Changes During μ-opioid Receptor Activation,” Nature 524:375–378 (2015), which is hereby incorporated by reference in its entirety) (FIGs.4G and 2A-2C) was performed on the final particle set (filter resolution = 3.3 Å), and the resulting principal components of 5 motion were visualized in chimera.

[0165] Processing of the trans-AM-3 MOR-Gi density map followed initial non-uniform and local refinement steps. While these refinements produced maps with strong and unambiguous density for the naltrexone scaffold, the trans-azobenzene group appeared highly dynamic. To address this, the particle stack underwent two rounds of 3D variability analysis 10 (Sounier et al., “Propagation of Conformational Changes During μ-opioid Receptor Activation,” Nature 524:375–378 (2015), which is hereby incorporated by reference in its entirety) (filter resolution = 3.3 Å), focusing on particles contributing to frames displaying prominent density for the trans-azobenzene group. The first round of 3DVA was performed on the stack of 628,564 particles. Frames 5–14 from principal component (PC) 2 were selected, resulting in a subset of15 511,627 particles. Ab initio reconstruction was performed on this subset, followed by non- uniform and local refinements. These steps resulted in the appearance of the trans-azobenzene group, however it remained insufficiently resolved. A second round of 3DVA (Sounier et al., “Propagation of Conformational Changes During μ-opioid Receptor Activation,” Nature 524:375–378 (2015), which is hereby incorporated by reference in its entirety) was subsequently 20 conducted, and frames 9–13 from PC2 were selected, reducing the particle set to 284,756 particles (FIGs.4H and 3A-3C). Ab initio reconstruction, followed by non-uniform and local refinements, yielded maps with unambiguous density for the trans-azobenzene group.

[0166] Finally, focused refinements were performed using manually created masks. Mask 1 targeted the transmembrane domain (TMD) while excluding the micelle, and Mask 2 did 25 the same for the heterotrimeric G protein (FIGs.4H and 3A-3C). The focused maps were finally combined in Chimera for subsequent model building and refinement. Radioligand Binding

[0167] Radioligand competition assays were performed using membrane fractions prepared from HEK293F cells transiently expressing wild-type MOR. To prepare membranes, 30 cells transfected with 1 µg / mL plasmid were first harvested and resuspended in hypotonic buffer (10  mM HEPES pH 7.5, 10  mM MgCl2, 20  mM KCl supplemented with 2  mM AEBSF, 14  μM E-64, 1  μM leupeptin, and 0.3  μM aprotinin). Resuspended cells were then dounce homogenized and centrifuged at 175,000 g in two rounds to clear soluble fractions. Protein concentration of the membrane pellets was typically determined to be 4 mg / mL using a Bradford 316084209v2-67- assay (Pierce), and aliquots were flash frozen in liquid nitrogen, and stored at -80°C until further use.

[0168] Competition binding assays were setup in 96-well plates containing membrane fractions diluted to 0.15 mg / mL, along with [3H]-Naltrexone at 2 nM, and an AM-3 dose (25 µM 5 - 6.25 pM), all prepared in binding buffer (10 mM HEPES, 10 mM MgCl2, 20 mM KCl, 0.1% BSA, and 100 µM Bacitracin). Competition reactions were incubated for 2 hours in the dark, and terminated by vacuum filtration onto cold 0.3 % PEI soaked GF / A filters, followed by three rounds of washing with cold 50 mM HEPES (pH 7.50). Counts were read using a Microbeta2 plate reader (PerkinElmer) for one minute per well. Results were analyzed in GraphPad Prism 10 10.1.1, and the inhibitor constant (Ki) was determined using the one site - Fit Kimodel equation: logEC50=log(10^logKi*(1+RadioligandNM / HotKdNM)) (Eq.1) Y=Bottom + (Top-Bottom) / (1+10^(X-LogEC50)) (Eq.2) Mice

[0169] All experiments used male C57BL / 6J (Jackson Laboratory; 20–30 g) or CD-115 (Charles River, 25–40 g) mice, between 8 and 13 weeks old, housed 4 or 5 per cage under a 12- hour light–dark cycle (lights off at 19:00 and on at 7:00) with ad libitum access to food and water. Mice were kept at a constant temperature (20–24 °C) and relative humidity (40–50%). C57BL / 6J mice were used for tests following local intraplantar administrations, whereas CD-1 mice were used for tests following systemic intravenous (tail vein) administrations. 20 In vivo AM-3 and Light Exposure Protocols

[0170] AM-3 was dissolved in a vehicle consisting of 0.36, 1.44, or 2.88% DMSO in sterile 0.9% NaCl for intraplantar (i.pl.) injections. Morphine sulfate (Sigma-Aldrich, Inc., St. Louis, MO, US) was dissolved in the vehicle and injected i.pl. at 5 µg / 5 µL or intravenously (i.v., tail vein) at 5 mg / kg. Naloxone hydrochloride (20 µg / 5 µL, i.pl.; Tocris, Minneapolis, MN, 25 US) was dissolved in sterile 0.9% NaCl. All drugs were i.pl. injected in a final volume of 5 µL. For intravenous (i.v.) tail vein injections, AM-3, loperamide (1 mg / kg; Tocris, Minneapolis, MN, US), or morphine (5 mg / kg) was dissolved in 10% DMSO in 0.9% NaCl. The injected volume was 5 ml / kg as a bolus injection.

[0171] For pre-illumination experiments, AM-3 or vehicle was exposed to 365 nm UV 30 light (EA-140, 4 Watt, 120 V, 60 Hz, 0.2 mA; Spectro-UV, Farmingdale, NY, US) for 10 minutes prior to injections, under dark conditions using a far-red lamp (BlockBlueLight, North Charleston, SC, US). In vivo photoswitching experiments involved post-injection exposure of mice to 365 nm UV light for a total of 5 minutes (single or repeated exposure) or to blue 465 nm light (10 Hz, 10 ms pulses, 68 pulses per sequence, 90 sequences; Doric Lenses, Quebec, 316084209v2-68- Canada) for a total of 3 minutes or as otherwise specified. All injections and light exposures were conducted under dark conditions using a far-red lamp. For in vivo photoswitching with alternating UV and blue light illumination (FIG.5B), mice were exposed to 460 nm light for 2 minutes, tested 4 minutes after switching the light off, then re-exposed to 365 nm light for 3 5 minutes, retested after 4 minutes, and the cycle was repeated.

[0172] For structural studies, purification of MOR in the presence of cis-AM-3 or trans- AM-3 was performed in the dark or in ambient light, respectively. In each case, AM-3 was exposed to either 360 nm UV or blue 465 nm light for 30 minutes prior to addition, followed by periodic exposure of the sample, to the respective light. 10 Measurement of Analgesia and Pain Models Hargreaves Thermal Plantar Test

[0173] Mice were habituated to a Hargreaves apparatus (Ugo Basile, Varese, Italy) on tempered glass maintained at ~28°C for 90–120 minutes per day over 2 days before behavioral testing. On test days, thermal nociceptive thresholds were recorded following habituation by 15 measuring paw withdrawal latencies (PWL) to an infrared light source (800–1200 nm) focused on the plantar surface of each hind paw. After determining baseline PWLs, mice received vehicle or drug administrations. For opioid blockade, mice were pre-injected with naloxone or saline 10 minutes prior to AM-3 or vehicle administration. Recorded withdrawal behaviors included paw withdrawal, licking, biting, or shaking of the targeted hind paw. Measurements were repeated 1– 20 3 times per hind paw, with at least 2-minute intervals, and averaged for each paw. The intensity of the light source was adjusted to produce baseline responses averaging ~6 seconds under both acute and post-SNI (spared nerve injury) surgery conditions, with a cut-off of 20 seconds to avoid tissue damage. To reduce experimental variability due to potential increases in paw skin temperature during photoswitching experiments (Dirig et al., “Characterization of Variables 25 Defining Hindpaw Withdrawal Latency Evoked by Radiant Thermal Stimuli,” J Neurosci Methods 76:183–191 (1997); Wu et al., “Effects of Baseline Skin Temperature on Pain Ratings to Suprathreshold Temperature-controlled Stimuli,” Pain 90:151–156 (2001), which are hereby incorporated by reference in their entirety), glass temperature was monitored to prevent increases exceeding +1–1.5°C. 30 Tail Flick Test

[0174] Analgesia was assessed using the Ugo Basile 37360 tail flick apparatus. For baseline measurements, mice were gently wrapped, and the lower one-third of the tail was positioned over the sensor emitting radiant heat via an infrared light source (800–1200 nm). The response latency was measured by the tail flick, with a maximal latency of 20 seconds set to 316084209v2-69- avoid tissue damage. Following baseline recording, mice received an i.v. injection of vehicle or cis-AM-3 at doses of 2.5, 5, or 10 mg / kg (pre-illuminated). Analgesic response was tested every 5 minutes for 60 minutes, followed by assessments at 90 and 95 minutes post-administration. Spared Nerve Injury Model 5

[0175] Spared nerve injury was performed according to the method of Decosterd and Woolf, “Spared Nerve Injury: An Animal Model of Persistent Peripheral Neuropathic Pain,” Pain 87:149–158 (2000), which is hereby incorporated by reference in its entirety. Under isoflurane anesthesia, the sciatic nerve was exposed, identifying the 3 peripheral branches (sural, common peroneal, and tibial nerves), and both tibial and common peroneal nerves were ligated 10 and transected together. Animals recovered for 3 weeks after surgery. Thermal hyperalgesia was absent in healthy (pre-surgery) animals, and the paw withdrawal threshold in mice before SNI (pre-surgery) was very close to the set cut-offs. Locomotor Activity

[0176] Activity was measured in a 50 cm x 50 cm black paved open field arena with 35 15 cm high walls. Locomotor activity was measured at 5-minute intervals, with cumulative counts taken for data analysis; counts represented the total number of beam breaks per 5-minute increment, capturing all movements, including running and turning behaviors (Wang et al., “Knockout of the Vesicular Monoamine Transporter 2 Gene Results in Neonatal Death and Supersensitivity to Cocaine and Amphetamine,” Neuron 19:1285–1296 (1997); Xu et al., “Mice 20 Lacking the Norepinephrine Transporter are Supersensitive to Psychostimulants,” Nat Neurosci 3:465–471 (2000), which are hereby incorporated by reference in their entirety). To evaluate the effects of AM-3 or morphine on locomotor behavior, mice were habituated in the activity monitor for 120 minutes. Immediately after the habituation period, mice were injected with drug or vehicle and returned to the monitor, where locomotor activity was recorded for the following 25 90 minutes. All analyses were conducted using EthoVision software (v.17.5, Noldus, Leesburg, VA, US). Gastrointestinal Motility

[0177] The constipative effect of AM-3 and loperamide was assessed by measuring total accumulated fecal boli, as previously described (Raehal et al., “Morphine Side Effects in Beta- 30 arrestin 2 Knockout Mice,” J Pharmacol Exp Ther 314:1195–1201 (2005), which is hereby incorporated by reference in its entirety). Briefly, SNI mice were injected with drugs or vehicle and placed in a Plexiglass chamber (5 cm × 8 cm × 8 cm) positioned on a mesh screen. Mice had ad libitum access to food and water before testing. Fecal boli were collected and weighed 95 minutes post-administration. 316084209v2-70- P-Glycoprotein Transporter Substrate Analysis

[0178] The permeability test was performed as previously described (Feng et al., “In vitro P-glycoprotein Assays to Predict the In vivo Interactions of P-glycoprotein With Drugs in the Central Nervous System,” Drug Metab Dispos 36:268–275 (2008); Feng et al., “Validation 5 of Human MDR1-MDCK and BCRP-MDCK Cell Lines to Improve the Prediction of Brain Penetration,” J Pharm Sci 108:2476–2483 (2019), which are hereby incorporated by reference in their entirety). MDCKII-MDR1 cells were seeded at 1.56 × 10⁶ cells / mL in 96-well HTS Transwell plates (50 µL per well) and cultured at 37 °C, 5% CO₂, and 95% relative humidity for 3–8 days, with medium replaced every other day. Before experiments, plates were washed twice 10 with pre-warmed HBSS (10 mM HEPES, pH 7.4) and incubated at 37 °C for 30 minutes. Test compounds (10 µM) were prepared by diluting 2 mM DMSO stock solutions in HBSS (final DMSO concentration: 0.5%). For apical-to-basolateral (A→B) transport, 125 µL of the compound solution was added to the apical compartment, with 50 µL immediately transferred to acetonitrile containing internal standards (IS: 100 nM ketoprofen, 200 nM labetalol, and 100 nM 15 tolbutamide). For basolateral-to-apical (B→A) transport, 285 µL of the compound solution was added to the basolateral compartment, and 50 µL was sampled similarly. Receiver compartments were filled with transport buffer (1 μM for control compound or 5 μM for test compound diluted in 0.5% DMSO in 10 mM HEPES, pH 7.4). Plates were incubated at 37 °C for 2 hours. Post- incubation, 50 µL samples were collected from donor and receiver compartments, mixed with 20 acetonitrile containing IS, vortexed (10 min), and centrifuged (3,220 g, 40 min). Supernatants (100 µL) were diluted with ultra-pure water for LC-MS / MS analysis. Data Analyses and Statistics

[0179] All results are presented as mean ± s.e.m. In vivo data were tested for normality and sphericity and a Greenhouse Geisser correction was applied where appropriate. Assumption 25 of Normality was tested either with the Shapiro–Wilk test or with a Q-Q plot. Two-way ANOVAs were used with treatment and time as variables. Tuckey’s was used for post hoc comparisons of groups. GraphPad Prism 9.5 was used for data treatment and statistical analysis. Data Availability

[0180] The final cryo-EM maps for MOR:Gαi:cis-AM-3 and MOR:Gαi:trans-AM-3 30 have been deposited in the Electron Microscopy Data Bank under accession code: EMD-48249 and EMD-48250. Corresponding atomic coordinates have been deposited in the PDB under accession code: 9GMC and 9GMD. 316084209v2-71- Example 1 – Design and Synthesis of Azo-Morphines

[0181] To produce potential photoswitchable morphinan agonists, agonist-bound MOR X-ray crystallography (Manglik et al., “Crystal Structure of the µ-opioid Receptor Bound to a Morphinan Antagonist,” Nature 485:321–326 (2012); Huang et al., “Structural Insights Into µ- 5 opioid Receptor Activation,” Nature 524:315–321 (2015), which are hereby incorporated by reference in their entirety) and cryo-EM structures (Faouzi et al., “Structure-based Design of Bitopic Ligands for the µ-opioid Receptor,” Nature 613:767–774 (2023); Koehl et al., “Structure of the µ-opioid Receptor-Gi Protein Complex,” Nature 558:547–552 (2018); Zhuang et al., “Molecular Recognition of morphine and Fentanyl by the Human μ-opioid Mreceptor,” Cell 10 1854361–4375.e19 (2022); Wang et al., “Structures of the Entire Human Opioid Receptor Family,” Cell 186413–427.e17 (2023); Qu et al., “Insights Into Distinct Signaling Profiles of the µOR Activated by Diverse Agonists,” Nat. Chem. Biol.19:423–430 (2023), which are hereby incorporated by reference in their entirety) were examined and the presence of a secondary binding subpocket was noted that accommodates aromatic moieties of a variety of agonists 15 including fentanyl and BU-72 (FIG.1B). It was reasoned based on the orientation of morphinans in G protein-bound structures (Manglik et al., “Crystal Structure of the µ-opioid Receptor Bound to a Morphinan Antagonist,” Nature 485:321–326 (2012); Huang et al., “Structural Insights Into µ-opioid Receptor Activation,” Nature 524:315–321 (2015); Zhuang et al., “Molecular Recognition of morphine and Fentanyl by the Human μ-opioid Mreceptor,” Cell 1854361– 20 4375.e19 (2022), which are hereby incorporated by reference in their entirety), that chemical extension from the morphinan core may enable differential engagement of this subpocket via bent cis and extended trans azobenzene configurations. Notably, the agonist IBNtx employs a naltrexone-like core with a similar extension (FIG.1A), which enables mode-switching of the common scaffold from an antagonist to an agonist (Majumdar et al., “Truncated G protein- 25 coupled Mu Opioid Receptor MOR-1 Splice Variants are Targets for Highly Potent Opioid Analgesics Lacking Side Effects,” Proc Natl Acad Sci U S A 108:19778–19783 (2011); Majmudar et al., “Probing the Isoprenylcysteine Carboxyl Methyltransferase (Icmt) Binding Pocket: Sulfonamide Modified Farnesyl Cysteine (SMFC) Analogs as Icmt Inhibitors,” Bioorg Med Chem Lett 21:2616–2620 (2011); Majumdar et al., “Synthesis and Evaluation of Aryl- 30 naloxamide Opiate Analgesics Targeting Truncated Exon 11-associated μ Opioid Receptor (MOR-1) Splice Variants,” J Med Chem 55:6352–6362 (2012), which are hereby incorporated by reference in their entirety). A small library of “azo-morphine” compounds was thus designed using a naltrexone scaffold and variable length linkers to an azobenzene conjugated in the meta or para position (FIG.1C). Molecular docking analysis supported the hypothesis that such 316084209v2-72- compounds can bind with the morphinan group in the primary binding pocket and azobenzenes at positions toward the extracellular face of the receptor. Intriguingly, while the azobenzenes consistently occupied the secondary subpocket in cis, in trans various positions were predicted with similar binding scores (FIG.6). 5

[0182] Synthesis of the azo-morphines started with reductive amination of naltrexone with methyl benzyl amine, followed by debenzylation, to yield the known secondary amine 1 (FIG.1D) (St Onge et al., “Systematic Structure-Activity Relationship Study of Nalfurafine Analogues toward Development of Potentially Nonaddictive Pain Management Treatments,” J Med Chem 67:9552–9574 (2024), which is hereby incorporated by reference in its entirety).10 Amide coupling with a variety of azobenzene carboxylic acids, such as 2, then yielded the azo- morphines, specifically AM-3. Photophysical characterization showed that AM-3 behaved as a regular azobenzene, with maximum cis content achieved at 360 nm (trans:cis = 28:72) and maximum trans content at 460 nm (trans:cis = 84:16) (FIGs.1E-1F and 7A-7C). Thermal relaxation to the trans form was found to be slow (t1 / 2= > 24 h in PBS:DMSO = 9:1) (FIG.7B). 15 Similar photophysical properties were observed across all azo-morphine variants tested (FIGs. 7A-7B). Example 2 – Functional Characterization of Azo-Morphines

[0183] Compounds AM-1, AM-2, AM-3, and AM-4 were tested using patch clamp electrophysiology in HEK 293T cells expressing the rat MOR and the G protein-coupled inward 20 rectifier potassium channel (GIRK) as a reporter of G protein activation. Each ligand was applied at 100 nM in the presence of 460 nm light to maintain the maximal trans state and then light was switched between 365 nm and 460 nm to toggle the ligand between primarily cis and primarily trans, respectively. Following at least two rounds of switching, the azo-morphine was removed and the full agonist DAMGO was applied to enable normalization of the current amplitudes 25 induced by trans versus cis azo-morphine (FIGs.8A and 9A-9D). The four ligands showed strikingly different behavior. AM-1 produced clear activation in trans, which was maintained but not altered by photoswitching to the cis state. In contrast, AM-2, AM-3, and AM-4 all showed some activation in trans followed by enhanced activation in cis. However, the extent of trans activation was substantially less with AM-3, pointing to a more pronounced difference in the 30 ability of cis and trans forms of the ligand to activate the receptor. FIG.8B shows a summary of the cis / trans activation ratio across all four ligands, highlighting the enhanced photoswitching of AM-3 relative to AM-2 and AM-4. Based on these results, AM-3 was focused on as the most promising azo-morphine variant. 316084209v2-73-

[0184] AM-3 produced robust photocurrents in response to 365 nm that were repeatable and were maintained in the dark prior to 460 nm illumination (FIG.8C). This bistability was consistent with the UV / Vis spectroscopy results (FIGs.7A-7C) and enabled sustained MOR activation without the need for ongoing, potentially harmful UV illumination. The spectral 5 properties of AM-3 were characterized and it was found that longer wavelengths than 365 nm were unable to efficiently drive photocurrents (FIGs.10A-10B). Importantly, comparable cis photo-activation via AM-3 was observed for rat, mouse, and human MOR subtypes (FIGs.11A- 11B).

[0185] To better understand the mechanism of the enhanced activity in cis versus trans, a 10 series of dose-response titrations of AM-3 in either form were performed. Radioligand binding experiments, using cis-AM-3 and trans-AM-3 against tritiated [3H]-Naltrexone, showed robust competition, with an estimated Kiof 35 nM and 16 nM, respectively (FIG.8D). To detect MOR- mediated G protein activation, the ONE-GO G^^^^i3 bioluminescence resonance energy transfer (BRET) sensor was used, which detects agonist-evoked GPCR-mediated release of G^^^^i3-GTP 15 (Janicot et al., “Direct Interrogation of Context-dependent GPCR Activity With a Universal Biosensor Platform,” Cell 187:1527–1546.e25 (2024), which is hereby incorporated by reference in its entirety) (FIG.8E). Both trans-AM-3 and cis-AM-3 produced clear responses of comparable potencies (EC50,cis=199 nM; EC50,trans=293 nM), but the maximal activation evoked by cis-AM-3 was substantially higher (Emax,cis=0.59; Emax, trans=0.36), suggesting that AM-3 20 served as an efficacy photoswitch. To evaluate the dose-dependence of downstream signaling activity initiated by AM-3, the photoswitching patch clamp GIRK current experiment was performed across a range of concentrations to produce a dose-response curve for cis and trans (FIGs.12A-12B). cis-AM-3 showed a higher potency (EC50,cis=27 nM; EC50,trans=121 nM) and a ~3 fold increase in Emax (Emax,cis=0.73; Emax, trans=0.25) (FIG.8F), further supporting the 25 efficacy switch model. Finally, MOR internalization was measured as a means of assessing the ability of AM-3 to initiate receptor desensitization and downregulation. Using a SNAP-tag based surface labeling assay, it was found that extended treatment with both cis-AM-3 and trans-AM- 3 can drive MOR internalization, but with increased potency (EC50,cis=1.7 nM; EC50,trans=12 nM) and efficacy for cis (Emax,cis=0.85; Emax, trans=0.32) (FIG.8G). Live cell imaging confirmed that 30 cis-AM-3 produced substantially more intracellular puncta from surface-labeled SNAP-tagged MOR compared to trans-AM-3 (FIG.12C). Notably, cis-AM-3 showed a lower Emax than DAMGO but was comparable to morphine across assays (FIG.12D-12G). Altogether, these data showed that both forms of AM-3 served as partial MOR agonists, but that the cis form contained a modestly higher potency and a substantially enhanced efficacy relative to trans (FIG.8H). 316084209v2-74- Finally, the two other major opioid receptor subtypes were tested and it was found that AM-3 potently activates both the kappa and delta opioid receptors, but without any light dependence (FIGs.13A-13B). 5 Example 3 – Structural Basis of AM-3 Efficacy Switching

[0186] To probe the structural basis of the distinct efficacies of cis and trans-AM-3, single particle cryo-EM experiments of the ternary complex consisting of either cis-AM-3 or trans-AM-3, wild type human MOR, wild type human Gαi1 and Gβ1 and Gγ2 were performed. High resolution reconstructions of the complex with the compound in either state were obtained,10 with an overall estimated resolution of 3.2 Å (cis-AM-3) and 3.1 Å (trans-AM-3) (FIGs.4A- 4B, Table 1). For cis- the consensus refinement was followed by local refinement of the receptor and G protein heterotrimer separately and merged into a composite map, which was used for modeling and further structural analysis (FIGs.2A-2C). For trans-AM-3, additional classification was performed using 3D Variability Analysis (Sounier et al., “Propagation of 15 Conformational Changes During μ-opioid Receptor Activation,” Nature 524:375–378 (2015), which is hereby incorporated by reference in its entirety), to select for subsets of particles with improved density for the AM-3 azobenzene group (FIGs.3A-3C). The refined models closely resembled previously published active-state MOR structures (FIGs.14A-14B). Comparing cis- AM-3 and trans-AM-3 structures to the highest resolution MOR structure without the use of the 20 receptor:G protein complex-stabilizing scFv16 (Mitragynine pseudoindoxyl MP, PDB ID: 7T2G), the receptors align well, with an overall RMSD of 0.79 Å and 0.62 Å for cis- and trans- AM-3, respectively. General structural hallmarks of GPCR activation, including the DR3.50Y, CW6.48xP6.50, P5.50-I3.40-F6.44and NP7.50xxY7.53motifs (values indicate Ballesteros–Weinstein numbering for GPCRs (Ballesteros and Weinstein, “Integrated Methods for the Construction of 25 Three-dimensional Models and Computational Probing of Structure-function Relations in G Protein-coupled Receptors,” in Methods in Neurosciences (ed. Sealfon, S. C.) 25:366–428 (Academic Press, 1995), which is hereby incorporated by reference in its entirety)), are identical to those derived from structures with prototypical agonists DAMGO, fentanyl and morphine (PDB ID: 8EFQ, 8EF5, 8EF6 (Zhuang et al., “Molecular Recognition of morphine and Fentanyl 30 by the Human μ-opioid Mreceptor,” Cell 1854361–4375.e19 (2022), which is hereby incorporated by reference in its entirety). The interface between the nucleotide-free G protein and MOR are also very similar, representing a canonical MOR:Gi1 coupling state. One noteworthy difference between cis- and trans-AM-3 bound states is the relative angle between the N-terminus of G^^^^i, which is approximately 6 degree tilted towards the receptor, suggesting a 316084209v2-75- closer engagement of the receptor with the G protein in the cis state, which could support its higher efficacy (FIG.4C).

[0187] Initial attempts of structure refinement for both datasets produced maps with strong and unambiguous density for the naltrexone scaffold. While a complete ligand density for 5 cis-azobenzene was readily obtained (FIGs.2A-2C), the trans-azobenzene group initially appeared somewhat dynamic, and an unambiguous ligand density was only obtained after extensive classification attempts (FIGs.3A-3C). For both cis- and trans-AM-3 the azobenzene moiety protruded into the secondary subpocket, located between TM1 and TM2, analogous to fentanyl and other synthetic opioids (Zhuang et al., “Molecular Recognition of morphine and 10 Fentanyl by the Human μ-opioid Mreceptor,” Cell 1854361–4375.e19 (2022); Qu et al., “Insights Into Distinct Signaling Profiles of the µOR Activated by Diverse Agonists,” Nat. Chem. Biol.19:423–430 (2023), which are hereby incorporated by reference in their entirety) (FIGs.4A, 4D-F). The naltrexone scaffold is located in the canonical, orthosteric binding pocket, with a similar pose to the previously published structure of the irreversible morphinan 15 antagonist beta-FNA (PDB ID: 4DKL) (Manglik et al., “Crystal Structure of the µ-opioid Receptor Bound to a Morphinan Antagonist,” Nature 485:321–326 (2012), which is hereby incorporated by reference in its entirety) (FIG.14C). Previous work has suggested that the morphinan scaffold of agonists sits deeper in the orthosteric binding pocket, compared to antagonists (Zhuang et al., “Molecular Recognition of morphine and Fentanyl by the Human μ- 20 opioid Mreceptor,” Cell 1854361–4375.e19 (2022); Kolinski and Filipek, “Molecular Dynamics of μ opioid Receptor Complexes With Agonists and Antagonists,” Open Struct. Biol. J.2:8–20 (2008); Sutcliffe et al., “Drug Binding Poses Relate Structure with Efficacy in the μ Opioid Receptor,” J Mol Biol 429:1840–1851 (2017), which are hereby incorporated by reference in their entirety). Using the tertiary amine in beta-FNA as a reference point, the naltrexone moiety 25 of trans-AM-3 sits 0.6 Å deeper, cis-AM-3 by 0.9 Å, and morphine by 1.1 Å, which, while subtle, shows a correlation with the efficacy of the respective ligand (FIG.14C).

[0188] The terminal benzene ring of cis-AM-3 shows hydrophobic interactions with side-chains Q2.60, N2.63, W23.50, V3.28and I3.29, analogous to fentanyl-like scaffolds shown previously (Zhuang et al., “Molecular Recognition of morphine and Fentanyl by the Human μ- 30 opioid Mreceptor,” Cell 1854361–4375.e19 (2022); Qu et al., “Insights Into Distinct Signaling Profiles of the µOR Activated by Diverse Agonists,” Nat. Chem. Biol.19:423–430 (2023), which are hereby incorporated by reference in their entirety). The trans-AM-3 structure occupies the same overall binding pocket, but due to isomerization around the azo group, the terminal benzene moiety protrudes 2.5 Å further towards TM2 (FIGs.4A-4F). This results in a modest 316084209v2-76- rearrangement of the extracellular half of TM2 moving towards TM1 by approximately 1.5 Å. In addition to this global change, differences in rotamer conformations of Q2.60, N2.63, Y2.64were observed, all of which are proximal to the azo-benzene groups, and have been previously linked to ligand efficacy (Qu et al., “Insights Into Distinct Signaling Profiles of the µOR Activated by 5 Diverse Agonists,” Nat. Chem. Biol.19:423–430 (2023), which is hereby incorporated by reference in its entirety) (FIGs.4D-4F and 14D-14J). Interestingly, occupation of the fentanyl subpocket in MOR by itself is not clearly predictive of agonist efficacy. For instance, the benzyl moiety of alvimopan, a non-morphinan MOR-specific antagonist, also penetrates this subpocket, and extends similarly towards TM2 (Robertson et al., “Structure Determination of Inactive-state 10 GPCRs With a Universal Nanobody,” Nat. Struct. Mol. Biol.29:1188–1195 (2022), which is hereby incorporated by reference in its entirety). However, the precise location of the phenyl group of fentanyl more closely matches cis-AM-3, while trans-AM-3 penetrates the subpocket similarly to alvimopan (FIGs.14D-14J). Additionally, in trans-AM-3, both the linker and the proximal benzene protrude towards extracellular loop 2 (ECL2), which is similarly occupied by 15 carboxylic acid in alvimopan (FIG.14E). Both beta-strands in ECL2 point away from the core of the receptor by approximately 1.8 Å in cis, compared to the trans structure, in which the beta hair-pin in ECL2 is stabilized by an additional interaction between R213 and D218 (FIG.4D). ECL2 repositioning has previously been seen upon agonist binding suggesting that this interaction may contribute to ligand efficacy (Sounier et al., “Propagation of Conformational 20 Changes During μ-opioid Receptor Activation,” Nature 524:375–378 (2015), which is hereby incorporated by reference in its entirety). Finally, while distinct binding poses of the ligands at MOR in their respective states were observed, in light of both the docking results and the exclusion of a substantial particle subset by 3D Variability Analysis for the trans-AM-3 structure, it cannot be ruled out that either alternative conformations of azobenzene in the bound 25 ligand, or binding kinetics, contribute to the reduced efficacy of trans-AM-3.

[0189] In addition to a standard cryo-EM data processing pipeline, 3D Variability Analysis (3DVA) was performed to visualize structural dynamics within the respective datasets (Punjani and Fleet, “3D Variability Analysis: Resolving Continuous Flexibility and Discrete Heterogeneity From Single Particle Cryo-EM,” J. Struct. Biol.213:107702 (2021), which is 30 hereby incorporated by reference in its entirety). Strikingly, while the average conformation obtained from structural refinement only showed modest differences between cis- and trans- AM-3, substantial differences were observed in terms of conformational dynamics (FIG.4G and 4H). Most notably, 3DVA from the cis dataset shows a twisting motion of the receptor, relative to the G protein, which is routinely observed in nucleotide free GPCR:G protein complex 316084209v2-77- structures (Punjani and Fleet, “3D Variability Analysis: Resolving Continuous Flexibility and Discrete Heterogeneity From Single Particle Cryo-EM,” J. Struct. Biol.213:107702 (2021); Marino and Schertler, “A Set of Common Movements Within GPCR-G-protein Complexes From Variability Analysis of Dryo-EM Datasets,” J. Struct. Biol.213:107699 (2021); Zhang et 5 al., “Evolving Cryo-EM Structural Approaches for GPCR Drug Discovery,” Structure 29:963– 974.e6 (2021), which are hereby incorporated by reference in their entirety). The twisting motion in the cis structure is accompanied by a correlated motion within the receptor, mainly driven by a rotation of the intracellular portion of TM7 / helix 8, which coincides with the appearance of a density for the C-terminal loop of the Gai subunit. Analogous findings from MD simulations 10 (Zhuang et al., “Molecular Recognition of morphine and Fentanyl by the Human μ-opioid Mreceptor,” Cell 1854361–4375.e19 (2022); Qu et al., “Insights Into Distinct Signaling Profiles of the µOR Activated by Diverse Agonists,” Nat. Chem. Biol.19:423–430 (2023), which are hereby incorporated by reference in their entirety), as well as NMR studies (Sounier et al., “Propagation of Conformational Changes During μ-opioid Receptor Activation,” Nature 15 524:375–378 (2015), which is hereby incorporated by reference in its entirety), were previously described, where the degree of this rotation correlated with the efficacy of an agonist at MOR. On the other hand, 3DVA for the trans dataset shows a relatively rigid receptor, while a surprising degree of dynamics within the G protein and its displacement relative to the receptor was observed. It is noteworthy that similar movements across all three determined principal 20 components was observed, suggesting that these dynamics are dominant within the respective datasets. Qualitatively, these dynamic movements within the ternary complex suggest a less stable interaction between MOR and the G protein in the trans state, while cis results in a more tightly coupled ternary complex. 25 Example 4 – In vivo Local Control of Pain with AM-3

[0190] Motivated by the robust efficacy switching observed in cultured cells, it was speculated that AM-3 may be employed in vivo for light-dependent antinociception in mice. As the MOR is strongly expressed in heat-sensitive nociceptors (Scherrer et al., “Dissociation of the Opioid Receptor Mechanisms That Control Mechanical and Heat Pain,” Cell 137:1148–1159 30 (2009); Wang et al., “Functional Divergence of Delta and Mu Opioid Receptor Organization in CNS Pain Circuits,” Neuron 98:90–108.e5 (2018), which are hereby incorporated by reference in their entirety), the Hargreaves test was used as an assay of thermal pain sensitivity in the hind paw. Either relaxed trans-AM-3 or pre-illuminated cis-AM-3 were injected directly into the plantar surface of the hind paw (i.pl.) (FIG.15A) and the paw withdrawal latency (PWL) was 316084209v2-78- measured over 45 minutes. cis-AM-3 produced an increase in PWL that peaked at 20 minutes, was dose-dependent, and was observed in the injected paw but not the contralateral paw (FIGs. 15B and 16A-16C). At the same i.pl. injection dose of 5 μg / 5 μL, morphine and cis-AM-3 showed comparable effects (FIG.15C and 16B). However, across all doses tested, trans-AM-3 5 showed no effect relative to vehicle (FIGs.15B-15C and 16D-16F). This suggests that the modest efficacy observed for trans-AM-3 in cultured cells (FIGs.8E-8G) is insufficient to drive a behavioral effect in this assay. The antinociceptive effect of cis-AM-3 was blocked by pre- injection of the MOR antagonist naloxone (20 μg / 5 μL), confirming opioid receptor-dependence (FIG.15D). 10

[0191] It was next tested if AM-3 can be locally activated to its cis form in vivo to initiate antinociception. trans-AM-3 was i.pl. injected and 365 nm light illumination through the bottom of the behavioral chamber was used to photoswitch to the cis form, which led to a large increase in PWL (FIG.15E). In contrast, UV light had no effect in control mice that received a vehicle injection (FIG.15E). It was further shown that in vivo optical deactivation of AM-315 blocks its analgesic effect, as 465 nm light illumination following i.pl. injection of the pre- activated cis form of AM-3 prevented the observed increase in PWL in the absence of light illumination (FIGs.17A-17B). Together these experiments demonstrate the ability of the antinociceptive activity of AM-3 to be controlled in vivo with light.

[0192] It was next asked if AM-3 can accumulate in the central nervous system (brain 20 and spine) as this is a key determinant of its ability to drive centrally-mediated side effects. This was first tested using the tail-flick assay, in which the latency to the tail reflex from the heat source is driven by MOR populations in the spine and brain (Irwin et al., “The Effects of Morphine, Methadone and Meperidine on Some Reflex Responses of Spinal Animals to Nociceptive Stimulation,” J. Pharmacol. Exp. Ther.101:132 (1951); Goodchild et al., 25 “Supraspinal and Spinal Cord Opioid Receptors are Responsible for Antinociception Following Intrathecal Morphine Injections,” European Journal of Anaesthesiology | EJA 21:179 (2004), which are hereby incorporated by reference in their entirety). Whereas systemic (intravenous, i.v.) tail-vein injection of morphine produced a robust increase in tail-flick latency, no tested doses of cis-AM-3 had an effect (FIGs.18A-18B). In addition, cis-AM-3 did not produce 30 hyperlocomotion in the open field test (FIG.15F), a well-established acute effect of morphine mediated by mesolimbic dopaminergic systems in the brain related to reinforcement processes (Matthes et al., “Loss of Morphine-induced Analgesia, Reward Effect and Withdrawal Symptoms in Mice Lacking the Mu-opioid-receptor Gene,” Nature 383:819–823 (1996); Di Chiara and Imperato, “Drugs Abused by Humans Preferentially Increase Synaptic Dopamine 316084209v2-79- Concentrations in the Mesolimbic System of Freely Moving Rats,” Proc. Natl. Acad. Sci. U S A 85:5274–5278 (1988), which are hereby incorporated by reference in their entirety). These data show that AM-3 does not have a central effect, which would enable it to serve as a peripherally restricted analgesic. To further test this, an in vitro permeability assay was conducted to evaluate 5 AM-3 brain penetration assessing the role of drug transporters in its uptake into the brain. Both cis and trans forms of AM-3 exhibited an efflux ratio of ~7 (Table 2), indicating that it is a substrate for the P-glycoprotein efflux transporter, a multidrug-resistance membrane protein expressed at the blood-brain-barrier (BBB) that actively pumps various foreign substances out of cells (Schinkel, A. H., “P-Glycoprotein, a Gatekeeper in the Blood-brain Barrier,” Adv. Drug 10 Deliv. Rev.36:179–194 (1999), which is hereby incorporated by reference in its entirety). These findings align with the behavioral data which show that AM-3 does not produce centrally- mediated effects. Table 2. Permeability Analysis of AM-3 in a MDCKII-MDR1 Cell Line Assay15 The efflux ratios for cis- and trans-AM-3 were compared to standard compound Metoprolol (non-substrate for efflux transporters) and Digoxin (substrate for efflux transporters) were used as controls to validate the assay. Apparent permeability (Papp) was calculated for drug transport assays using the equation: Papp= {VA / (Area x time)} x [drug]acceptor / [durg]initial, donor(Eq.3) 20 where Pappis apparent permeability (cm / s x 10-6), VAis the volume (in mL) in the acceptor well, Area is the surface area of the membrane (0.143 cm2for Transwell-96 Well Permeable Supports) time is the total transport time in seconds. Efflux ratio was determined using the following equation: Efflux Ratio = Papp(B-A) / Papp(A-B) (Eq.4) 25 where Papp(B-A) indicates the apparent permeability coefficient in basolateral to apical direction, and Papp(A-B) indicates the apparent permeability coefficient in apical to basolateral direction. Mass balance (% recovery) was determined using the following equation: Recovery% = { [drug]acceptorx VA+ [drug]donorx VD / [drug]initial, donorx VD} x 100 (Eq.5) 316084209v2-80- where VAand VDare the acceptor and donor well volumes, respectively (0.235 mL for A→B flux, 0.075 mL for B→A flux).

[0193] Finally, the ability of AM-3 to alleviate chronic pain symptoms was assessed 5 using the spared nerve injury (SNI) model of neuropathic pain (FIG.5B). Three weeks post- surgery, mice developed thermal hyperalgesia in the ipsilateral paw (FIGs.5B-5D) and systemic AM-3 administration reversed this condition (FIGs.5B-5D and 19A-19C). Injection of pre- illuminated cis-AM-3 into the paw on the injured side relieved the thermal hyperalgesia and alternating illumination with 460 nm and 365 nm light enabled reversible control of this 10 analgesic effect (FIGs.5B and 19C), demonstrating the ability of AM-3 to undergo multiple rounds of photoswitching in vivo.

[0194] To test if systemic AM-3 application is also effective in the SNI model, either relaxed trans-AM-3 or pre-illuminated cis-AM-3 was i.v. injected at the dose of 5 mg / kg, and the PWL was measured over 90 minutes. cis-AM-3, but not trans-AM-3, reversed the thermal 15 hyperalgesia of the ipsilateral paw without affecting the contralateral one (FIGs.5C and 19B). As a control the peripherally restricted MOR agonist loperamide was used (Chung et al., “Analgesic Properties of Loperamide Differ Following Systemic and Local Administration to Rats After Spinal Nerve Injury,” Eur. J. Pain 16:1021–1032 (2012); DeHaven-Hudkins et al., “Loperamide (ADL 2-1294), an Opioid Antihyperalgesic Agent With Peripheral Selectivity,” J 20 Pharmacol. Exp. Ther.289:494–502 (1999), which are hereby incorporated by reference in their entirety), which showed a similar effect to cis-AM-3 (FIG.5C and 19B). It was next asked if AM-3 may be locally activated to its cis form in vivo following systemic treatment to promote a local anti-hyperalgesic effect.10 mg / kg trans-AM-3 was i.v. injected and locally photoactivated to the cis form with 365 nm light illumination of the paw, which rapidly increased PWL that 25 returned to baseline within 30 min (FIG.5D). Subsequent 365 nm exposures produced similar anti-hyperalgesic effects, supporting the ability of repeated photoactivation of AM-3 to successfully provide robust analgesia.

[0195] To assess the ability of local, optically-targeted peripheral opioid action to drive analgesia with reduced gastrointestinal (GI) side effects, constipation was assessed by measuring 30 faecal boli accumulation over 95 min and comparing it to loperamide (Hurwitz et al., “Loperamide Effects on Hepatobiliary Function, Intestinal Transit and Analgesia in Mice,” Life Sci.54:1687–1698 (1994); Tan-No et al., “Development of Tolerance to the Inhibitory Effect of Loperamide on Gastrointestinal Transit in Mice,” Eur. J. Pharm. Sci.20:357–363 (2003), which are hereby incorporated by reference in their entirety). Strikingly, despite a similar net analgesic 316084209v2-81- effect for loperamide, systemic cis-AM-3, and local photoactivation of AM-3 (FIG.5E), only loperamide or systemic cis-AM-3 produced substantial constipation (FIG.5F). Together these data support the potential of AM-3 to enable local analgesia as a means of limiting both central and peripheral GI side effects. 5 Example 5 – Discussion of Examples 1-4

[0196] Peripheral opioids are promising compounds for effective analgesia with a better safety profile than systemic opioids (Martínez and Abalo, “Peripherally Acting Opioid Analgesics and Peripherally-induced Analgesia,” Behav. Pharmacol .31:136–158 (2020); 10 Tegeder et al., “Peripheral Opioid Analgesia in Experimental Human Pain Models,” Brain 126:1092–1102 (2003), which are hereby incorporated by reference in their entirety). While chemical strategies have been reported to achieve peripheral restriction, herein both chemical modification and light were used to target peripheral opioid analgesia, providing further precision to minimize off-target effects. AM-3 showed minimal accumulation in the brain, 15 minimal activity in the relaxed trans state, and robust analgesic effects in the active cis state following illumination. Importantly, and in contrast to caged ligand approaches (McClain et al., “In vivo Photopharmacology With Light-activated Opioid Drugs,” Neuron 1113926–3940.e10 (2023), which is hereby incorporated by reference in its entirety), AM-3 can be reversibly and repeatedly photo-activated and photo-deactivated, providing a high degree of spatiotemporal 20 control both for basic and clinical application. Thework described above complements previous non-opioid azobenzene-based photopharmacological approaches to preclinical pain modulation (Landra-Willm et al., “A Photoswitchable Inhibitor of TREK Channels Controls Pain in Wild- type Intact Freely Moving Animals,” Nature Communications 14:1–12 (2023); Morstein et al., “Optical Control of Sphingosine-1-phosphate Formation and Function,” Nat. Chem. Bio.l 25 15:623–631 (2019); Mourot et al., “Rapid Optical Control of Nociception With an Ion-channel Photoswitch,” Nat. Methods 9:396–402 (2012), which are hereby incorporated by reference in their entirety), supporting this novel mode of drug action as a promising alternative to traditional analgesic drugs.

[0197] Herein, two ways of applying AM-3 as a local analgesic are described: first, AM- 30 3 may be “pre-activated” by illuminating prior to local application, as demonstrated with hindpaw injection. Local administration has the advantage of delivering high drug concentration at the site of injection and has previously been harnessed for topical opioid formulations (Krajnik et al., “Potential Uses of Topical Opioids in Palliative Care--Report of 6 Cases,” Pain 80:121– 125 (1999); Gutierrez et al., “Topical Opioid Use in Dermatologic Disease: A Systematic 316084209v2-82- Review,” Dermatol. Ther.34:e15150 (2021), which are hereby incorporated by reference in their entirety). In this mode of application, visible light illumination or thermal relaxation of azobenzenes can drive deactivation of AM-3 to further limit sustained or systemic MOR activation compared to classical opioids. Second, AM-3 can be applied systemically in its 5 inactive trans form and locally activated via targeted illumination. While many peripheral sites are optically accessible, implant-based light delivery techniques may be required depending on the application. Notably, AM-3 should be well-suited for probing MOR signaling in the brain using optogenetic light fiber techniques following local delivery (Frank, J.A., “Optofluidic Neural Interfaces for In vivo Photopharmacology,” Current Opinion in Pharmacology 10 63:102195 (2022), which is hereby incorporated by reference in its entirety).

[0198] AM-3 serves as an efficacy switch such that cis and trans forms show comparable binding affinities for the MOR but cis is more effective as an agonist. While affinity switches represent the traditional mode of photochromic GPCR ligands (Wijtmans et al., “Optical Control of Class A G Protein-coupled Receptors With Photoswitchable Ligands,” Curr. Opin. 15 Pharmacol.63:102192 (2022); Hauwert et al., “A Photoswitchable Agonist for the Histamine H Receptor, a Prototypic Family A G-Protein-Coupled Receptor,” Angew. Chem. Int. Ed. Engl. 58:4531–4535 (2019); Morstein et al., “Photoswitchable Serotonins for Optical Control of the 5- HT Receptor,” Angew. Chem. Int. Ed. Engl.61:e202117094 (2022), which are hereby incorporated by reference in their entirety), they have emerged as an alternative mechanism 20 (Gómez-Santacana et al., “A Toolbox of Molecular Photoswitches to Modulate the CXCR3 Chemokine Receptor With Light,” Beilstein J. Org. Chem.15:2509–2523 (2019); Gerwe et al., “Frontispiece: Enlightening the ‘spirit molecule’: Photomodulation of the 5‐HT2A Receptor by a Light‐controllable N,N‐dimethyltryptamine Derivative,” Angew. Chem. Int. Ed. Engl.61(26): e202282661 (2022); Westphal et al., “Synthesis of Photoswitchable Δ-Tetrahydrocannabinol 25 Derivatives Enables Optical Control of Cannabinoid Receptor 1 Signaling,” J. Am. Chem. Soc. 139:18206–18212 (2017), which is hereby incorporated by reference in its entirety). Efficacy switches offer a clear advantage in terms of therapeutic window. Since optical illumination does not lead to 100% population of either cis or trans states, competition between the two forms will occur under most conditions. If cis had a substantially higher affinity than trans, at high doses 30 strong effects would be seen following visible light illumination due to the 10-20% of molecules in the cis state which would outcompete the 80-90% in trans. Interestingly, despite clear partial agonism of trans-AM-3 in cultured cell experiments, no or minimal effects of trans-AM-3 were observed in vivo. A simple and plausible explanation is that the high expression seen in heterologous studies leads to an overestimation of partial agonism compared to in vivo, 316084209v2-83- endogenous systems which may have a higher threshold for a biologically impactful activation. The absence of noticeable activity of trans-AM-3 in vivo could also be due, at least in part, to a pharmacokinetic effect. Trans-AM-3 is more lipophilic than its cis isomer and therefore more prone to be sequestered, e.g., by serum albumin (Wenskowsky et al., “Resolving Binding Events 5 on the Multifunctional Human Serum Albumin,” ChemMedChem 15:738–743 (2020), which is hereby incorporated by reference in its entirety), and thus may be less bioavailable in the blood circulatory system.

[0199] Cryo-EM structures of both cis- and trans-AM-3 bound to the MOR reported herein provide a structural explanation for the efficacy differences between these states. In both 10 forms, the naltrexone moiety binds in the canonical major pocket with a similar pose to prior structures. However, the azobenzene extension occupies the fentanyl subpocket in both cis and trans, with substantial differences which likely reposition key transmembrane helices, ultimately modulating conformational dynamics within the ternary complex, which results in low or high efficacy activation. Similarly to IBNtxA (Majumdar et al., “Synthesis and Evaluation of Aryl- 15 naloxamide Opiate Analgesics Targeting Truncated Exon 11-associated μ Opioid Receptor (MOR-1) Splice Variants,” J. Med. Chem.55:6352–6362 (2012), which is hereby incorporated by reference in its entirety), AM-3 is able to serve as an agonist despite the naltrexone antagonist scaffold, demonstrating the steep sensitivity of the MOR to subtle changes in ligand composition. The functional and structural data support a simple model where the morphinan 20 core serves to produce binding affinity while efficacy is controlled by the precise pose of the ligand in extracellular subpockets. The structural analysis provides a framework for rational engineering of AM-3 variants with improved properties including red-shifted activation spectra, accelerated thermal relaxation, fine-tuned efficacy, opioid receptor subtype selectivity, and, possibly, G protein or arrestin bias. 25

[0200] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. 30 316084209v2

Claims

-84- WHAT IS CLAIMED:

1. A compound of Formula (I):5 whereinis a double bond with an unspecified configuration; is optional and, if present, is a single bond;is selected from the group consisting of monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl, wherein monocyclic aryl, bicyclic aryl, 10 monocyclic heteroaryl, and bicyclic heteroaryl can be optionally substituted from 1 to 6 times with a substituent selected independently at each occurrence thereof from the group consisting of H, D, halogen, OC1-6alkyl, OC1-6alkyl, NH2, NHC1-6alkyl, and N(C1-6alkyl)2;is selected from the group consisting of monocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclic heteroaryl, wherein monocyclic aryl, bicyclic aryl, 15 monocyclic heteroaryl, and bicyclic heteroaryl can be optionally substituted from 1 to 6 times with a substituent selected independently at each occurrence thereof from the group consisting of H, D, halogen, OC1-6alkyl, OC1-6alkyl, NH2, NHC1-6alkyl, and N(C1-6alkyl)2; A is a morphinan or a fragment thereof; R is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; 20 R1is H, C1-6alkyl, or -X1-X2-X3-X4-X5-X6-X7-X8-X9; R2is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; X1is C1-6alkylene; X2is -NH-C(O)- or -C(O)NH-; X3is C1-6alkylene; 25 X4is –(OCH2CH2)m- or –(OCH2CH2)m-OCH2–; X5is -NH-C(O)- or -C(O)NH-; X6is a bond, C1-6alkylene, or –(CH2CH2O)k-C1-12alkylene–; X7is absent or -C(O)-NH-; 316084209v2-85- X8is a bond or C1-6alkylene; X9is a Tag; Y is NH, CH2, O, S, or N(C1-6alkyl); k is 1-10; 5 l is 0 or 1; n is 0, 1, 2, or 3; and m is 1-50, or an isomer thereof, an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof. 10 2. A compound of Formula (I´):whereinis a double bond with an unspecified configuration; 15 A is a morphinan or a fragment thereof; R1is H or C1-6alkyl; R2is H or -X1-X2-X3-X4-X5-X6-X7-X8-X9; X1is C1-6alkylene; X2is -NH-C(O)-; 20 X3is C1-6alkylene; X4is –(OCH2CH2)m-; X5is -NH-C(O)-; X6is C1-6alkylene; X7is absent or -C(O)-NH-; 25 X8is a bond or C1-6alkylene; X9is a Tag; n is 0, 1, 2, or 3; and m is 1-50, or an isomer thereof, an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate 30 thereof, or a prodrug thereof. 316084209v2-86- 3. The compound according to claim 1 or claim 2, wherein the morphinan is a morphine-related compound (agonist or antagonist). 5 4. The compound according to claim 1 or claim 2, wherein A is a naltrexone derivative.

5. The compound according to claim 1, wherein A is selected from the group consisting of 10, whereinis the point of attachment of A to the corresponding nitrogen atom of the structure of Formula (I); 15 R3is selected from the group consisting of H, C1-12alkyl, and C3-6cycloalkyl, wherein C1-12alkyl can be optionally substituted with C3-6cycloalkyl, NH2, or; R4is selected from the group consisting of H, OH, C1-6alkyl, and OC1-6alkyl; R5is selected from the group consisting of H, OH, C1-6alkyl, and OC1-6alkyl; or R4and R5combine to form -CH2- or -O- group. 20 6. The compound according to claim 1, wherein A is selected from the group consisting of 316084209v2-87-whereinis the point of attachment of A to the corresponding nitrogen atom of the 5 structure of Formula (I); R4is selected from the group consisting of H, OH, C1-6alkyl, and OC1-6alkyl; R5is selected from the group consisting of H, OH, C1-6alkyl, and OC1-6alkyl; or R4and R5combine to form -CH2- or -O- group. 10 7. The compound according to any one of claims 1-6, wherein R1is Me.

8. The compound according to any one of claims 1-7, wherein the Tag is a SNAP-Tag or a derivative thereof, Halo-tag or a derivative thereof, or improved Tadross Halo Tag or a derivative thereof. 15The compound according to claim 8, wherein the Tag is. 20 10. The compound according to any one of claims 1-9, wherein the compound is a cis-isomer. 316084209v2-88- 11. The compound according to any one of claims 1-9, wherein the compound is a trans-isomer. 5 12. The compound according to claim 1, which has the Formula (I´´):

13. The compound according to claim 1, which has the Formula (Ia):10 wherein R3is selected from the group consisting of H, C1-12alkyl, and C3-6cycloalkyl, wherein C1-12alkyl can be optionally substituted with C3-6cycloalkyl, NH2, or.

13. The compound according to claim 2, which has the Formula (I´a): 15wherein R3is selected from the group consisting of H, C1-12alkyl, and C3-6cycloalkyl, wherein C1-12alkyl can be optionally substituted with C3-6cycloalkyl, NH2, or. 316084209v2-89- 14. The compound according to claim 1, which has the Formula (Ib):wherein p is 1, 2, 3, 4, or 5. 5wherein p is 1, 2, 3, 4, or 5. 10 16. The compound according to claim 1, which has the Formula (Ic)(Ic), wherein k is 1-45. 15 17. The compound according to claim 2, which has the Formula (I´c): 316084209v2-90-(I´c), wherein k is 1-45. 5 18. The compound according to claim 1, which has the Formula (Id)wherein k is 1-45. 10 19. The compound according to claim 1, which has the Formula (Ie): 316084209v2-91-wherein k is 1-45.

20. The compound according to claim 1, whereinis selected from the 5 group consisting of phenyl, naphthyl, biphenyl, and pyrazolyl.

21. The compound according to claim 1, whereinis selected from the group consisting of phenyl, naphthyl, biphenyl, and pyrazolyl. 10 22. The compound according to claim 1, wherein the compound of Formula (I) is selected from the group consisting of: 316084209v2-92- 5, 316084209v2-93- 5, 316084209v2-94- , , , , 5, 316084209v2-95- ,, 316084209v2-96-, 316084209v2-97- HO, 316084209v2-98- HO316084209v2-99-, or an isomer thereof.

23. The compound according to claim 1, wherein the compound of Formula 5316084209v2-100- 5316084209v2-101-316084209v2-102-316084209v2-103-, 316084209v2-104- HO, 316084209v2-105-, or an isomer thereof. 5 24. The compound according to claim 2, wherein the compound of Formula (I) is selected from the group consisting of: 316084209v2-106- 5, 316084209v2-107- , , , 5, or an isomer thereof.

25. A pharmaceutical composition comprising a therapeutically effective 10 amount of the compound according to any one of claims 1-24 and a pharmaceutically acceptable carrier.

26. A method of treating pain in a subject, said method comprising: 316084209v2-108- administering to the subject in need thereof the compound according to any one of claims 1-24.

27. The method of claim 26, wherein the pain is medium to severe pain, 5 visceral pain, chronic pain, cancer pain, migraine, inflammatory pain, acute pain, neuropathic pain, allodynia, or hyperalgesia.

28. The method of claim 27, wherein the pain is a severe pain. 10 29. A method of treating a mood or substance use disorder in a subject, said method comprising: administering to the subject in need thereof the compound according to any one of claims 1-24. 15 30. The method of claim 29, wherein the mood disorder is a depression or anxiety.

31. The method of claim 30, wherein the depression is a major depression. 20 32. The method of claim 29, wherein the substance use disorder is an addiction.

33. A method of treating a respiratory depression in a subject, said method comprising: 25 administering to the subject in need thereof the compound according to any one of claims 1-24.

34. A method of activating a mu-opioid receptor (MOR), said method comprising: 30 contacting a MOR with the compound according to any one of claims 1-24 under conditions effective to activate the MOR.

35. A method of activating a kappa-opioid receptor and / or delta-opioid receptor, said method comprising: 316084209v2-109- contacting a kappa-opioid receptor and / or delta-opioid receptor with the compound according to any one of claims 1-24 under conditions effective to activate kappa-opioid receptor and / or delta-opioid receptor. 5 36. A method of preventing and / or treating a condition where it is desired to activate MOR, said method comprising: administering to the subject in need thereof the compound according to any one of claims 1-24. 10 37. A method of preventing and / or treating a condition where it is desired to activate kappa-opioid receptor and / or delta-opioid receptor, said method comprising: administering to the subject in need thereof the compound according to any one of claims 1-24. 15 38. The method of claim 29, wherein the condition is an anxiety disorder, obsessive-compulsive disorder (OCD), or stress.

39. The method of any one of claims 26-38, wherein the compound is administered as a trans-isomer. 20 40. The method of claim 39, further comprising: converting said trans-isomer into a cis-isomer after said administering.

41. The method of claim 40, further comprising: 25 converting said cis-isomer into a trans-isomer.

42. The method of claim 40, wherein said converting is carried out by exposing the trans-isomer to a one photon or two photon light from about 300 to about 1500 nm under conditions effective to convert the trans-isomer into a cis-isomer. 30 43. The method of claim 42, wherein said exposing is carried out at a wavelength of one photon or two photon light from about 355 nm to about 375 nm. 316084209v2-110- 44. The method of claim 42, wherein said exposing is carried out at a wavelength of one photon or two photon light of about 365 nm.

45. The method of claim 42, wherein said exposing is carried out at a 5 wavelength of one photon or two photon light from about 450 nm to about 470 nm.

46. The method of claim 42, wherein said exposing is carried out at a wavelength of one photon or two photon light of about 460 nm. 10 47. The method of claim 40 or 41, wherein said converting is carried out in vivo.

48. The method of any one of claims 26-47, wherein said administering does not create an addiction or dependence. 15 49. The method of any one of claims 26-47, wherein said administering does not create a respiratory depression.

50. The method of any one of claims 26-47, wherein said administering does 20 not cause constipation.

51. The method of any one of claims 26-47, wherein said administering does not cause tolerance. 25 316084209v2

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