Novel morphine framework derivatives

Abstract

Morphine derivative comprise a modification of the framework of the molecule, wherein the derivative possesses novel bioactivity profiles that are different from morphine, and can address pain without an associated untoward effect, such as respiratory depression, addiction, etc., or may be used to treat pain without an addictive side effect or respiratory depression, or may be applied as an alternative to naloxone for addressing opioid overdose.

Description

Novel morphine framework derivatives

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 555,003, filed Feb 17, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0003] Government Support Clause

[0004] This invention was made with government support under grant numbers GM130345 and DA057790 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] Introduction

[0006] The isolation of morphine (1) from opium poppy by the pharmacist Sertürner in the early 19thcentury transformed the medicinal chemistry landscape.1Today, morphine is by all accounts the most famous alkaloid and forms the basis for some of the most prescribed medicines.2Morphine is an agonist of the µ-opioid receptor (MOR),3activation of which leads to a number of downstream signaling events that result in potent analgesia. This important biological activity is unfortunately accompanied by a number of side effects including addiction, constipation and, most importantly, lowered respiratory activity.4,5Morphine analogs that retain the analgesic activity but lack the respiratory depression and addiction liabilities are important targets in the pain management field.5In addition, given the associated challenges of the opioid epidemic,6and in particular overdose deaths resulting from highly potent opioids such as fentanyl and derivatives,7MOR antagonists that are longer acting than naloxone (2),8are actively being sought.

[0007] Given its captivating chemical structure (first proposed almost a century ago) 9 andassociated biological activity, it is not surprising that the syntheses of morphine and related opioids have been pursued for over 70 years.10Because morphine is routinely isolated from Papaver somniferum, one of the goals of past syntheses was not to necessarily solve a supply problem, but rather, to test existing strategies and methods for chemical synthesis against this target and to also identify synthesis pathways to access variants that cannot be accessed through biosynthesis.11Ultimately, these chemical synthesis endeavors have led to morphine derivatives possessing a range of functions and the realization of multiple opioid receptor subtypes. Extensive semi-synthetic efforts have provided a number of compounds with subtype and / or functional selectivity as agonists at the opioid receptors. For example, the naturally occurringmethyl ether of morphine (i.e., codeine, 3; an antitussive that functions at opioid and non-opioid receptors), the pain reliever oxycodone, as well as the antagonists nalorphine (5), and the clinically employed naloxone (2), naltrexone, and nalmefene are easily accessed through semi- synthesis (Figure 1a).12Many of the early derivatives of morphine that were prepared through chemical synthesis were accessed by effecting peripheral changes on the morphine framework. For example, the highly addictive derivative heroin (4) can be synthesized through a double acetylation of the hydroxy groups on the periphery of the morphine core, whereas naltrindole(6), 13 a delta opioid receptor (DOR) selective antagonist that facilitated structuralcharacterization of the inactive state of that receptor,14is synthesized from the ketone group on naltrexone using a Fischer indole synthesis.15Other derivatives include a kappa opioid receptor (KOR) selective antagonist norBNI16and KOR agonist, MP110417which was used to elucidate the active state of the KOR.18Other semi-synthetic derivatives of morphine include the MOR antagonist β-funaltrexamine (β-FNA).

[0008] Surprisingly, very few synthesis campaigns have been undertaken to vary the core atoms that comprise the cyclic framework of morphine.19Recent efforts on derivatizing morphine and related opioids have focused on rational design fueled by advances in molecular recognition, examining co-crystal structures or docking with theMany of the non-covalent interactions that are established by ligands with the MOR that lead to strong binding are established by atoms that reside on the periphery of the morphine framework, save for the tertiary amine group in the piperidine D-ring and O-atom in the dihydro benzofuran (i.e., E-ring) moiety (Figure 1b). The importance of the tertiary amine, the protonated form of which forms a salt bridge with an aspartate residue on transmembrane 3 (TM3) (ASP1473.32in the murine MOR (mMOR) or ASP1493.32in the human MOR (hMOR)), is critical to binding to the MOR (Figure 1c).23 ,24(Superscript numbers after residues refer to their Ballesteros-Weinstein numbering, which indicates the TM and proximity to the most conserved residue within that TM).25On the other hand, the interaction of the E-ring O-atom with the MOR has been less highlighted. A co- crystal structure by Kobilka and coworkers of β-funaltrexamine bound in the mMOR unveiled a key H-bond interaction of TYR1483.33with the E-ring O-atom.26Rothman and coworkers have shown that mutating TYR1483.33(in the mMOR; TYR1503.33in hMOR) to a phenylalanine (which would remove this key H-bond interaction) leads to a 2–7 fold lower binding of several µ-selective agonists.27Recent structures have shed additional light on the role TYR1483.33plays in the binding of opioids across other chemical templates as well. TYR1483.33is located next to the conserved residue ASP1473.32on TM3. In the crystal structure of the mMOR bound to the morphinan agonist BU72,28engages a water molecule to form a hydrogen bonding network between BU72, LYS2335.39and HIS2976.52. An analogous interaction was observed inmolecular dynamics simulations of the cryo-EM structure of the hMOR bound to a peptidic enkephalin derived agonist, DAMGO.29In the cryo-EM structure of hMOR bound to morphine, TYR1503.33is observed to interact significantly with the small molecule ligand (Figure 1c). In the same study, mutation of TYR1503.33to an alanine was found to significantly decrease the binding affinity of morphine as well as the chemically distinct templates fentanyl, PZM21 and SR1701821 at the hMOR. Furthermore, in the cryo-EM co-structure of the mMOR bound to thefentanyl analog lofentanil, 30 it was observed that lofentanil’s carbomethoxy moiety waspositioned betweenand TRP3187.35. Overall, this combination of evidence from multiple structural studies strongly suggests that TYR1483.33in the mMOR either directly or indirectly interacts with MOR ligands and loss of TYR1483.33interactions can significantly affect the potency of multiple MOR ligands.22,28,30 In part, the effect of removing the E-ring O-atom in morphine can also be inferred from function studies with levorphanol and dextrorphan (7 and 8, respectively in Figure 1b). Levorphanol is a MOR analgesic31whereas dextrorphan has antitussive and hallucinogenic properties.32However, in addition to lacking the E-ring O-atom and C-ring double bond, 7 and 8 also lack the C-ring hydroxy group. As such, the function of 7 and 8 cannot be attributed to a single difference in structure as compared to morphine.

[0009] We postulated that by preparing a variant of morphine where the E-ring O-atom has been replaced with a methylene group, we could retain the conformation of the morphine framework but also remove the key interaction with TYR1483.33in the mMOR (TYR1503.33in hMOR) to study whether the resulting molecule, 9, which we have dubbed “carbamorphine”, would possess analgesic activity (Figure 1d) with reduced side effects. While the (–)-enantiomer of morphine is a potent agonist of the MOR, the (+)-enantiomer is known to be inactive.33On the other hand, levorphanol and dextrorphan, which are enantiomers, are known to have different activity (Figure 1b). Therefore, we also investigated what effect a change from O®CH2in the morphine skeleton (i.e., morphine to carbamorphine) would have on the activity of the associated enantiomers (Figure 1b). Here, we show that through a single heavy atom change in the morphine framework (i.e., O®CH2in the E-ring) to access carbamorphine, each enantiomer of 9 is active but possesses a different pharmacological profile. Indeed, (–)-9 and (+)-9 each bind to the MOR but in different ways according to our docking (Figure 1e) and molecular dynamic simulation studies. Notably, in contrast to (+) and (–)-morphine, the enantiomers of carbamorphine retain binding affinity and functional activity at the MOR in cell lines, and intracerebroventricular (i.c.v.) administration of (+)-carbamorphine demonstrated antinociception in vivo in the mouse 55 °C warm-water tail-withdrawal (WWTW). More importantly, in sharp contrast to (–)-morphine, mice administered i.c.v. with (+)-carbamorphinedisplay reduced respiratory depression and conditioned place preference. Overall, we demonstrate that changing a single heavy atom (O®CH2) in the core framework of morphine results in differences in both in vitro as well as in vivo biological activity. The observed differences in biological activity profile as a result of changes in structure enables the synthesis of other morphine analogs useful in combating the various challenges associated with the use of opioids.

[0010] Summary of the Invention

[0011] The invention provides morphinans and / or derivatives of morphine featuring modification of the framework of the molecule. These derivatives possess novel bioactivity profiles that are different from morphine, and can address pain without the associated untoward effects of respiratory depression, addiction, etc. The compositions may be used to treat pain without addictive side effects and respiratory depression. The compositions may also be applied as an alternative to naloxone for addressing opioid overdose (e.g., from fentanyl).

[0012] In aspects and embodiments, the invention provides:

[0013] 1. A carbamorphinan compound comprising an O-to-CH2exchange in the E-ring of the corresponding morphinan which comprises an epoxy group between carbons 4 and 5 (i.e., 4,5α- epoxy), thereby forming the E ring, including a pharmaceutically acceptable salt, hydrate and stereoisomer thereof.

[0014] 2. A carbamorphinan compound of claim 1, comprising a ring structure:

[0016] wherein rings B and C may independently comprise an unsaturation, such as the lower right bond of the C ring, as in morphine, including a pharmaceutically acceptable salt, hydrate and stereoisomer thereof.

[0017] 3. A carbamorphinan compound of claim 1, comprising a structure:

[0018]

[0019]

[0020] R1 is selected from R, OR, OCOR, D, F, Cl, Br, CN, NRR;

[0021] R2 is selected from R, OR, OCOR, O, D, F, Cl, Br, CN, NRR;

[0022] R3 is selected from R, OR, OCOR, D, F, Cl, Br, CN, NRR, including 2-3,3- dimethylbutan-2-ol, and 2-pentan-2-ol;

[0023] R4 is selected from R, OR, OCOR, D, F, Cl, Br, CN, NRR;

[0024] R5 is R, including 2-propylenyl, cyclopropylmethyl, cyclobutylmethyl;

[0025] R is H, D, F, Cl, Br, CN, OH, OMe, OAc, NH2, or substituted or unsubstituted C1-C4 hydrocarbyl, including alkyl, alkenyl or alkynyl, such as methyl, ethyl, propyl, ethylenyl, propylenyl, etc., wherein substituents are selected from D, F, Cl, Br, CN, OH, OMe, OAc, NH2, and unsubstituted C1-C4 hydrocarbyl;

[0026] wherein R2 and R3 or R2 and R4 maybe joined to form a ring, including a pharmaceutically acceptable salt, hydrate and stereoisomer thereof.

[0027] 4. A carbamorphinan compound of claim 3, wherein:

[0028] R1 is OH, OMe or OAc;

[0029] R2 is OH, O or OAc;

[0030] R3 is H;

[0031] R4 is H, OH or OMe; and

[0032] R5 is H or Me.

[0033] 5. A carbamorphinan compound of claim 1, comprising a structure selected from Table 1:

[0034] 6. A carbamorphinan compound of claim 1, wherein the morphinan is selected from morphine, naloxone, naloxegol, codeine, hydromorphone, oxymorphone, hydrocodone, oxycodone, dihydrocodeine, nicocodeine, heroin (diacetyl morphine), nalorphine, naltrindole, and thebaine (paramorphine), or from buprenorphine, diamorphine, ethylmorphine, dihydromorphine, desomorphine, pholcodine.

[0035] 7. A carbamorphinan compound of claim 1, 2, 3, 4, 5 or 6, comprising a bioactivity profile different from the corresponding morphinan, including pain relief without an associated respiratory depression or addiction.

[0036] 8. A pharmaceutical composition comprising a compound of claim 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt, a hydrate or a stereoisomer thereof, and a pharmaceutically acceptable carrier or excipient, in a pharmaceutically acceptable unit dosage.

[0037] 9. A method to treat or modulate a physiological condition comprising administering to a person in need thereof a compound of claim 1, 2, 3, 4, 5 or 6, such as for use an analgesic or MOR antagonist.

[0038] 10. A method of claim 9, further comprising the antecedent step of detecting or diagnosing a condition indicating the need thereof, and the subsequent step of detecting a resultant modulation of the condition

[0039] 11. A method of using a compound of claim 1, 2, 3, 4, 5, or 6 as an alternative to naloxone for addressing opioid overdose (e.g., from fentanyl).

[0040] 12. Use of a compound of claim 1, 2, 3, 4, 5 or 6 in the manufacture of a medicament for modulating a condition, such as for use an analgesic or MOR antagonist, preferably to treat pain without addictive side effects and respiratory depression.

[0041] 13. A compound of claim 1, 2, 3, 4, 5 or 6 for use in modulating a condition, preferably to treat pain without addictive side effects and respiratory depression.

[0042] 14. A method comprising total synthesis of a carbamorphinan of claim 1, 2, 3, 4, 5 or 6.

[0043] 15. A method of claim 14, comprising an intramolecular inverse electron-demand Diels−Alder cycloaddition and a stereoselective radical Giese addition to construct a quaternary carbon center.

[0044] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0045] Brief Description of the Drawings

[0046] Figs. 1A-E. Selected examples of morphinans and -E-ring modification in morphinans. (a) Selected examples of morphinans. (b) Examples of core structure modifications in morphinans. (c) Cryo-EM structure of human MOR (hMOR) bound to morphine. (d) This work: Single atom-exchange in the core of morphine derivatives. (e) Docking pose of (+) and (–)-9 at the hMOR.

[0047] Figs. 2A-E. Synthesis of (±)-carbamorphine and (±)-carbanalorphine. (a) Retrosynthesis of (±)-carbamorphine (±-9). (b) Reaction sequence for the synthesis of carbamorphine (±-9). (c) Giese-type radical addition reactions toward 19 using a range of radical precursors. (d) Observed rearrangement during the demethylation reaction from carbacodeine (25). (e) Reaction sequence for the synthesis of carbanalorphine (±-10)

[0048] Description of Particular Embodiments of the Invention

[0049] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0050] The term "alkyl" refers to a hydrocarbon group selected from linear and branched saturated hydrocarbon groups of 1-18, or 1-12, or 1-6 carbon atoms. Examples of the alkyl group include methyl, ethyl,1-propyl or n-propyl ("n-Pr"), 2-propyl or isopropyl ("i-Pr"), 1-butyl or n-butyl ("n-Bu"), 2-methyl-1-propyl or isobutyl ("i-Bu"), 1-methylpropyl or s-butyl ("s-Bu"), and 1,1-dimethylethyl or t-butyl ("t-Bu"). Other examples of the alkyl group include 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3- pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl groups.

[0051] Lower alkyl means 1-8, preferably 1-6, more preferably 1-4 carbon atoms; lower alkenyl or alkynyl means 2-8, 2-6 or 2-4 carbon atoms.

[0052] The term "alkenyl" refers to a hydrocarbon group selected from linear and branched hydrocarbon groups comprising at least one C=C double bond and of 2-18, or 2-12, or 2-6 carbon atoms. Examples of the alkenyl group may be selected from ethenyl or vinyl, prop-1- enyl, prop-2-enyl, 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2- methylbuta-1,3-diene, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, and hexa-1,3-dienyl groups.

[0053] The term "alkynyl" refers to a hydrocarbon group selected from linear and branched hydrocarbon group, comprising at least one C≡C triple bond and of 2-18, or 2-12, or 2-6 carbon atoms. Examples of the alkynyl group include ethynyl, 1-propynyl, 2-propynyl (propargyl), 1- butynyl, 2-butynyl, and 3-butynyl groups.

[0054] The term "cycloalkyl" refers to a hydrocarbon group selected from saturated and partially unsaturated cyclic hydrocarbon groups, comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups. For example, the cycloalkyl group may be of 3-12, or 3-8, or 3-6 carbon atoms. Even further for example, the cycloalkyl group may be a monocyclic group of 3-12, or 3- 8, or 3-6 carbon atoms. Examples of the monocyclic cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl groups. Examples of the bicyclic cycloalkyl groups include those having 7-12 ring atoms arranged as a bicycle ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ring systems, or as a bridged bicyclic ring selected from bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. The ring may be saturated or have at least one double bond (i.e. partially unsaturated), but is not fully conjugated, and is not aromatic, as aromatic is defined herein.

[0055] The term “aryl” herein refers to a group selected from:5- and 6-membered carbocyclic aromatic rings, for example, phenyl; bicyclic ring systems such as 7-12 membered bicyclic ring systems wherein at least one ring is carbocyclic and aromatic, selected, for example, from naphthalene, indane, and 1,2,3,4-tetrahydroquinoline; and tricyclic ring systems such as 10-15 membered tricyclic ring systems wherein at least one ring is carbocyclic and aromatic, for example, fluorene.

[0056] For example, the aryl group is selected from 5- and 6-membered carbocyclic aromatic rings fused to a 5- to 7-membered cycloalkyl or heterocyclic ring optionally comprising at leastone heteroatom selected from N, O, and S, provided that the point of attachment is at the carbocyclic aromatic ring when the carbocyclic aromatic ring is fused with a heterocyclic ring, and the point of attachment can be at the carbocyclic aromatic ring or at the cycloalkyl group when the carbocyclic aromatic ring is fused with a cycloalkyl group. Bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. Bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in "-yl" by removal of one hydrogen atom from the carbon atom with the free valence are named by adding "-idene" to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene. Aryl, however, does not encompass or overlap with heteroaryl, separately defined below. Hence, if one or more carbocyclic aromatic rings are fused with a heterocyclic aromatic ring, the resulting ring system is heteroaryl, not aryl, as defined herein.

[0057] The term "halogen" or “halo” refers to F, Cl, Br or I.

[0058] The term "heteroalkyl" refers to alkyl comprising at least one heteroatom.

[0059] The term "heteroaryl" refers to a group selected from:

[0060] 5- to 7-membered aromatic, monocyclic rings comprising 1, 2, 3 or 4 heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon;

[0061] 8- to 12-membered bicyclic rings comprising 1, 2, 3 or 4 heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in the aromatic ring; and

[0062] 11- to 14-membered tricyclic rings comprising 1, 2, 3 or 4 heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in an aromatic ring.

[0063] For example, the heteroaryl group includes a 5- to 7-membered heterocyclic aromatic ring fused to a 5- to 7-membered cycloalkyl ring. For such fused, bicyclic heteroaryl ring systems wherein only one of the rings comprises at least one heteroatom, the point of attachment may be at the heteroaromatic ring or at the cycloalkyl ring.

[0064] When the total number of S and O atoms in the heteroaryl group exceeds 1, those heteroatoms are not adjacent to one another. In some embodiments, the total number of S and O atoms in the heteroaryl group is not more than 2. In some embodiments, the total number of S and O atoms in the aromatic heterocycle is not more than 1.

[0065] Examples of the heteroaryl group include, but are not limited to, (as numbered from the linkage position assigned priority 1) pyridyl (such as 2-pyridyl, 3-pyridyl, or 4-pyridyl), cinnolinyl, pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl, imidazopyridinyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl,thiadiazolyl, tetrazolyl, thienyl,triazinyl,benzothienyl, furyl, benzofuryl, benzoimidazolyl, indolyl, isoindolyl, indolinyl, phthalazinyl, pyrazinyl, pyridazinyl, pyrrolyl, triazolyl, quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridinyl (such as 1H-pyrrolo[2,3-b]pyridin-5-yl), pyrazolopyridinyl (such as1H- pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl (such as benzo[d]oxazol-6-yl), pteridinyl, purinyl, 1- oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl, 1-thia-2,3-diazolyl, 1-thia-2,4-diazolyl, 1-thia-2,5-diazolyl, 1-thia-3,4-diazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, benzothiazolyl (such as benzo[d]thiazol-6-yl), indazolyl (such as 1H-indazol-5-yl) and 5,6,7,8-tetrahydroisoquinoline.

[0066] The term "heterocyclic" or "heterocycle" or "heterocyclyl" refers to a ring selected from 4- to 12-membered monocyclic, bicyclic and tricyclic, saturated and partially unsaturated rings comprising at least one carbon atoms in addition to 1, 2, 3 or 4 heteroatoms, selected from oxygen, sulfur, and nitrogen. “Heterocycle” also refers to a 5- to 7-membered heterocyclic ring comprising at least one heteroatom selected from N, O, and S fused with 5-, 6-, and / or 7- membered cycloalkyl, carbocyclic aromatic or heteroaromatic ring, provided that the point of attachment is at the heterocyclic ring when the heterocyclic ring is fused with a carbocyclic aromatic or a heteroaromatic ring, and that the point of attachment can be at the cycloalkyl or heterocyclic ring when the heterocyclic ring is fused with cycloalkyl.

[0067] “Heterocycle” also refers to an aliphatic spirocyclic ring comprising at least one heteroatom selected from N, O, and S, provided that the point of attachment is at the heterocyclic ring. The rings may be saturated or have at least one double bond (i.e. partially unsaturated). The heterocycle may be substituted with oxo. The point of the attachment may be carbon or heteroatom in the heterocyclic ring. A heterocyle is not a heteroaryl as defined herein.

[0068] Examples of the heterocycle include, but not limited to, (as numbered from the linkage position assigned priority 1) 1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl, 2,3-pyrazolidinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2,5-piperazinyl, pyranyl, 2- morpholinyl, 3-morpholinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2- dithietanyl, 1,3-dithietanyl, dihydropyridinyl, tetrahydropyridinyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, 1,4-oxathianyl, 1,4-dioxepanyl, 1,4-oxathiepanyl, 1,4-oxaazepanyl, 1,4-dithiepanyl, 1,4-thiazepanyl and 1,4- diazepane 1,4-dithianyl, 1,4-azathianyl, oxazepinyl, diazepinyl, thiazepinyl, dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H- pyranyl, 1,4-dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrazolidinylimidazolinyl, pyrimidinonyl, 1,1-dioxo-thiomorpholinyl, 3-azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl and azabicyclo[2.2.2]hexanyl. Substituted heterocycle also includes ring systems substituted with one or more oxo moieties, such as piperidinyl N-oxide, morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and 1, 1-dioxo-1- thiomorpholinyl.

[0069] Substituents are selected from: halogen, -R', -OR', =O, =NR', =N-OR', -NR'R", -SR', - SiR'R"R'", -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'- C(O)NR"R'", -NR'-SO2NR'", -NR"CO2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH- C(NH2)=NR', -S(O)R', -SO2R', -SO2NR'R", -NR"SO2R, -CN and -NO2, -N3, -CH(Ph)2, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, in a number ranging from zero to three, with those groups having zero, one or two substituents being particularly preferred. R', R" and R'" each independently refer to hydrogen, unsubstituted (C1-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with one to three halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- or 7-membered ring. Hence, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl, "alkyl” includes groups such as trihaloalkyl (e.g., -CF3and -CH2CF3), and when the aryl group is 1,2,3,4-tetrahydronaphthalene, it may be substituted with a substituted or unsubstituted (C3-C7)spirocycloalkyl group. The (C3- C7)spirocycloalkyl group may be substituted in the same manner as defined herein for "cycloalkyl".

[0070] Preferred substituents are selected from: halogen, -R', -OR', =O, -NR'R", -SR', - SiR'R"R'", -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR"CO2R', - NR'-SO2NR"R'", -S(O)R', -SO2R', -SO2NR'R", -NR"SO2R, -CN and -NO2, perfluoro(C1- C4)alkoxy and perfluoro(C1-C4)alkyl, where R' and R" are as defined above.

[0071] The term "fused ring" herein refers to a polycyclic ring system, e.g., a bicyclic or tricyclic ring system, in whcih two rings share only two ring atoms and one bond in common. Examples of fused rings may comprise a fused bicyclic cycloalkyl ring such as those having from 7 to 12 ring atoms arranged as a bicyclic ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ring systems as mentioned above; a fused bicylclic aryl ring such as 7 to 12 membered bicyclic aryl ring systems as mentioned above, a fused tricyclic aryl ring such as 10 to 15 membered tricyclic aryl ring systems mentioned above; a fused bicyclic heteroaryl ring such as 8- to 12-membered bicyclic heteroaryl rings as mentioned above, a fused tricyclic heteroaryl ring such as 11- to 14-membered tricyclic heteroaryl rings as mentioned above; and a fused bicyclic or tricyclic heterocyclyl ring as mentioned above.

[0072] The compounds may contain an asymmetric center and may thus exist as enantiomers. Where the compounds possess two or more asymmetric centers, they may additionally exist asdiastereomers. Enantiomers and diastereomers fall within the broader class of stereoisomers. All such possible stereoisomers as substantially pure resolved enantiomers, racemic mixtures thereof, as well as mixtures of diastereomers are intended to be included. All stereoisomers of the compounds and / or pharmaceutically acceptable salts thereof are intended to be included. Unless specifically mentioned otherwise, reference to one isomer applies to any of the possible isomers. Whenever the isomeric composition is unspecified, all possible isomers are included.

[0073] The compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds, such as deuterium, e.g. – CD3, CD2H or CDH2in place of methyl. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the invention, whether radioactive or not, are intended to be encompassed within the scope of the invention.

[0074] The term “substantially pure” means that the target stereoisomer contains no more than 35%, such as no more than 30%, further such as no more than 25%, even further such as no more than 20%, by weight of any other stereoisomer(s). In some embodiments, the term “substantially pure” means that the target stereoisomer contains no more than 10%, for example, no more than 5%, such as no more than 1%, by weight of any other stereoisomer(s).

[0075] When compounds contain olefin double bonds, unless specified otherwise, such double bonds are meant to include both E and Z geometric isomers.

[0076] Some of the compounds may exist with different points of attachment of hydrogen, referred to as tautomers. For example, compounds including carbonyl -CH2C(O)- groups (keto forms) may undergo tautomerism to form hydroxyl -CH=C(OH)- groups (enol forms). Both keto and enol forms, individually as well as mixtures thereof, are also intended to be included where applicable.

[0077] It may be advantageous to separate reaction products from one another and / or from starting materials. The desired products of each step or series of steps is separated and / or purified (hereinafter separated) to the desired degree of homogeneity by the techniques common in the art. Typically such separations involve multiphase extraction, crystallization from a solvent or solvent mixture, distillation, sublimation, or chromatography. Chromatography can involve any number of methods including, for example: reverse-phase and normal phase; size exclusion; ion exchange; high, medium and low pressure liquid chromatography methods and apparatus; small scale analytical; simulated moving bed ("SMB") and preparative thin or thick layer chromatography, as well as techniques of small scale thin layer and flash chromatography. One skilled in the art will apply techniques most likely to achieve the desired separation.

[0078] Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as by chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereoisomers to the corresponding pure enantiomers. Enantiomers can also be separated by use of a chiral HPLC column.

[0079] A single stereoisomer, e.g., a substantially pure enantiomer, may be obtained by resolution of the racemic mixture using a method such as formation of diastereomers using optically active resolving agents. Racemic mixtures of chiral compounds of the invention can be separated and isolated by any suitable method, including: (1) formation of ionic, diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers, and conversion to the pure stereoisomers, and (3) separation of the substantially pure or enriched stereoisomers directly under chiral conditions.

[0080] “Pharmaceutically acceptable salts” include, but are not limited to salts with inorganic acids, selected, for example, from hydrochlorates, phosphates, diphosphates, hydrobromates, sulfates, sulfinates, and nitrates; as well as salts with organic acids, selected, for example, from malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfonates, p- toluenesulfonates, 2-hydroxyethylsulfonates, benzoates, salicylates, stearates, alkanoates such as acetate, and salts with HOOC-(CH2)n-COOH, wherein n is selected from 0 to 4. Similarly, examples of pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium.

[0081] In addition, if a compound is obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, such as a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used without undue experimentation to prepare non-toxic pharmaceutically acceptable addition salts.

[0082] “Treating,” “treat,” or "treatment" refers to administering at least one compound and / or at least one stereoisomer thereof, and / or at least one pharmaceutically acceptable salt thereof to a subject in recognized need thereof.

[0083] An "effective amount" refers to an amount of at least one compound and / or at least onestereoisomer thereof, and / or at least one pharmaceutically acceptable salt thereof effective to "treat" a disease or disorder in a subject, and that will elicit, to some significant extent, the biological or medical response of a tissue, system, animal or human that is being sought, such as when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.

[0084] The term "at least one substituent" includes, for example, from 1 to 4, such as from 1 to 3, further as 1 or 2, substituents. For example, "at least one substituent R" herein includes from 1 to 4, such as from 1 to 3, further as 1 or 2, substituents selected from the list of R as described herein.

[0085] The subject compounds and stereoisomers thereof, and pharmaceutically acceptable salts thereof may be employed alone or in combination with at least one other therapeutic agent for treatment. In some embodiments, the compounds, stereoisomers thereof, and pharmaceutically acceptable salts thereof can be used in combination with at least one additional therapeutic agent. The compound and / or one pharmaceutically acceptable salt disclosed herein may be administered with the at least one other therapeutic agent in a single dosage form or as a separate dosage form. When administered as a separate dosage form, the at least one other therapeutic agent may be administered prior to, at the same time as, or following administration of the compound and / or one pharmaceutically acceptable salt disclosed herein.

[0086] Also provided is a composition comprising a subject compound and stereoisomers thereof, and pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier.

[0087] The composition comprising a subject compound and stereoisomers thereof, and pharmaceutically acceptable salts thereof can be administered in various known manners, such as orally, topically, rectally, parenterally, by inhalation spray, or via an implanted reservoir, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The compositions disclosed herein may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art.

[0088] The subject compounds and stereoisomers thereof, and pharmaceutically acceptable salts thereof can be administered orally in solid dosage forms, such as capsules, tablets, troches, dragées, granules and powders, or in liquid dosage forms, such as elixirs, syrups, emulsions,dispersions, and suspensions. The subject compounds and stereoisomers thereof, and pharmaceutically acceptable salts thereof disclosed herein can also be administered parenterally, in sterile liquid dosage forms, such as dispersions, suspensions or solutions. Other dosages forms that can also be used to administer the subject compounds and stereoisomers thereof, and pharmaceutically acceptable salts thereof disclosed herein as an ointment, cream, drops, transdermal patch or powder for topical administration, as an ophthalmic solution or suspension formation, i.e., eye drops, for ocular administration, as an aerosol spray or powder composition for inhalation or intranasal administration, or as a cream, ointment, spray or suppository for rectal or vaginal administration.

[0089] Gelatin capsules containing the compound and / or the at least one pharmaceutically acceptable salt thereof disclosed herein and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like, can also be used. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of time. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

[0090] Liquid dosage forms for oral administration can further comprise at least one agent selected from coloring and flavoring agents to increase patient acceptance.

[0091] In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene gycols can be examples of suitable carriers for parenteral solutions. Solutions for parenteral administration may comprise a water soluble salt of the at least one compound describe herein, at least one suitable stabilizing agent, and if necessary, at least one buffer substance. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, can be examples of suitable stabilizing agents. Citric acid and its salts and sodium EDTA can also be used as examples of suitable stabilizing agents. In addition, parenteral solutions can further comprise at least one preservative, selected, for example, from benzalkonium chloride, methyl- and propylparaben, and chlorobutanol.

[0092] A pharmaceutically acceptable carrier is, for example, selected from carriers that are compatible with active ingredients of the composition (and in some embodiments, capable of stabilizing the active ingredients) and not deleterious to the subject to be treated. For example, solubilizing agents, such as cyclodextrins (which can form specific, more soluble complexes with the at least one compound and / or at least one pharmaceutically acceptable salt disclosed herein), can be utilized as pharmaceutical excipients for delivery of the active ingredients.Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and pigments such as D&C Yellow # 10. Suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences, A. Osol, and other reference texts in the art.

[0093] For administration by inhalation, the subject compounds and stereoisomers thereof, and pharmaceutically acceptable salts thereof may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulisers. The subject compounds and stereoisomers thereof, and pharmaceutically acceptable salts thereof may also be delivered as powders, which may be formulated and the powder composition may be inhaled with the aid of an insufflation powder inhaler device. One exemplary delivery system for inhalation can be metered dose inhalation (MDI) aerosol, which may be formulated as a suspension or solution of a subject compound and stereoisomers thereof, and pharmaceutically acceptable salts thereof disclosed herein in at least one suitable propellant, selected, for example, from fluorocarbons and hydrocarbons.

[0094] For ocular administration, an ophthalmic preparation may be formulated with an appropriate weight percentage of a solution or suspension of the subject compound and stereoisomers thereof, and pharmaceutically acceptable salts thereof in an appropriate ophthalmic vehicle, such that the subject compound and stereoisomers thereof, and at least one pharmaceutically acceptable salts thereof is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye.

[0095] Useful pharmaceutical dosage-forms for administration of the subject compounds and stereoisomers thereof, and pharmaceutically acceptable salts thereof disclosed herein include, but are not limited to, hard and soft gelatin capsules, tablets, parenteral injectables, and oral suspensions.

[0096] The dosage administered will be dependent on factors, such as the age, health and weight of the recipient, the extent of disease, type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. In general, a daily dosage of the active ingredient can vary, for example, from 0.1 to 2000 milligrams per day. For example, 10- 500 milligrams once or multiple times per day may be effective to obtain the desired results.

[0097] In some embodiments, a large number of unit capsules can be prepared by filling standard two-piece hard gelatin capsules each with, for example, 100 milligrams of the subject compound and stereoisomers thereof, and pharmaceutically acceptable salt thereof disclosed herein in powder, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate.

[0098] In some embodiments, a mixture of the compound, stereoisomers thereof, and pharmaceutically acceptable salts thereof a digestible oil such as soybean oil, cottonseed oil or olive oil can be prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules are washed and dried.

[0099] In some embodiments, a large number of tablets can be prepared by conventional procedures so that the dosage unit comprises, for example, 100 milligrams of the compound, stereoisomers thereof, and pharmaceutically acceptable salts thereof, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

[0100] In some embodiments, a parenteral composition suitable for administration by injection can be prepared by stirring 1.5% by weight of the compound and / or at least an enantiomer, a diastereomer, or pharmaceutically acceptable salt thereof disclosed herein in 10% by volume propylene glycol. The solution is made to the expected volume with water for injection and sterilized.

[0101] In some embodiment, an aqueous suspension can be prepared for oral administration. For example, each 5 milliliters of an aqueous suspension comprising 100 milligrams of finely divided compound, stereoisomers thereof, and pharmaceutically acceptable salts thereof, 100 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0 grams of sorbitol solution, U.S.P., and 0.025 milliliters of vanillin can be used.

[0102] The same dosage forms can generally be used when the compound, stereoisomers thereof, and pharmaceutically acceptable salts thereof are administered stepwise or in conjunction with at least one other therapeutic agent. When drugs are administered in physical combination, the dosage form and administration route should be selected depending on the compatibility of the combined drugs. Thus the term coadministration is understood to include the administration of at least two agents concomitantly or sequentially, or alternatively as a fixed dose combination of the at least two active components.

[0103] The compounds, stereoisomers thereof, and pharmaceutically acceptable salt thereof disclosed herein can be administered as the sole active ingredient or in combination with at least one second active ingredient.

[0104] The subject compounds are incorporated into pharmaceutical compositions or formulations. The compositions will contain pharmaceutically acceptable diluents and / or carriers, i. e. diluents or carriers that are physiologically compatible and substantially free from pathogenic impurities. Suitable excipients or carriers and methods for preparing administrablecompositions are known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, Mack Publishing Co, NJ (1991). The compositions may also be in the form of controlled release or sustained release compositions as known in the art. For many applications the subject compounds are administered for morning / daytime dosing, with off period at night.

[0105] The subject compounds may be used per se, or in the form of their pharmaceutically acceptable salts, such as hydrochlorides, hydrobromides, acetates, sulfates, citrates, carbonates, trifluoroacetates and the like. When compounds contain relatively acidic functionalities, salts can be obtained by addition of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or the like. When compounds contain relatively basic functionalities, salts can be obtained by addition of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic,benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like.

[0106] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid, and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of this invention.

[0107] In addition to salt forms, this invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be more bioavailable by oral administration than the parent drug. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. A wide variety ofprodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. An example, without limitation, of a prodrug would be a compound of the present invention which is administered as an ester (the "prodrug"), but then is metabolically hydrolyzed to the carboxylic acid, the active entity.

[0108] Certain compounds of the invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the invention.

[0109] Some of the subject compounds possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.

[0110] The compounds are generally administered in a "therapeutically effective amount", i.e. the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.

[0111] The contacting is generally effected by administering to the subject an effective amount of one or more compounds having a formula herein, including the various embodiments described above. Generally administration is adjusted to achieve a therapeutic dosage of about 0.1 to 50, preferably 0.5 to 10, more preferably 1 to 10 mg / kg, though optimal dosages are compound specific, and generally empirically determined for each compound.

[0112] The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules, lozenges or the like in the case of solid compositions. In such compositions, the mimetic is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form. Unit dosage formulations are preferably about of 5, 10, 25, 50, 100, 250,500, or 1,000 mg per unit. In a particular embodiment, unit dosage forms are packaged in a multipack adapted for sequential use, such as blisterpack comprising sheets of at least 6, 9 or 12 unit dosage forms.

[0113] Examples: Total Synthesis and Biological Activity of Carbamorphinans: O-to-CH2Replacement in the E-Ring of the Morphine Core Structure

[0114] Abstract

[0115] Morphine is a µ-opioid receptor (MOR) agonist and potent analgesic. However, it displays several side effects including respiratory depression and addiction. Here, we show that a single heavy atom replacement in the morphine core structure (O to CH2exchange in the E- ring) prepared through a 15-step total synthesis, displays a distinct pharmacological profile. The total synthesis features an intramolecular inverse electron-demand Diels−Alder cycloaddition and a stereoselective Giese radical addition to construct a quaternary carbon center. Unlike morphine, where only the (–)-morphine enantiomer binds the MOR, both enantiomers of this “carba” variant, which we have named carbamorphine, possess activity as agonists of the MOR. Cell-based functional assays show that (+)-carbamorphine shows reduced G-protein as well as β-arrestin efficacy at the MOR. In mouse behavioral assays, (+)-carbamorphine exhibits MOR- selective antinociception while showing reduced respiratory depression and a lack of conditioned place preference at supratherapeutic doses. Overall, through a net “single atom” change (i.e., O to CH2) in the morphine framework, different pharmacological profiles have been realized. This example provided a basis for additional syntheses of analogous morphine analogs that incorporate atom changes in the core framework.

[0116] Synthesis of Carbamorphine

[0117] Our approach to carbamorphine benefitted significantly from our established synthesis of cephanolide natural products such as 11,34,35which share a related tetracyclic framework. Indanone 15 was targeted as a precursor to carbamorphine. An intramolecular cycloaddition in 15 would lead to pentacycle 17, which could be advanced to carbamorphine by installing the bridging piperidine ring of morphine. At the outset, we recognized that applying the strategy for the preparation of 11 and related congeners might not directly translate to the preparation of 9 given the different substitution on the A-ring (OMe in place of the Me group) that could significantly influence the electronics of the system.

[0118] Our synthesis commenced with commercially available indanone 12,36which was site- selectively brominated to give 13 using N-bromosuccinimide and a catalytic amount of iodine.37The para-OMe group likely guides the selectivity in this step. A Stille coupling38of bromide 13 with vinyltributyltin then set the stage for a reductive Heck reaction39with pyrone triflate 14 to give adduct 15. This sequence was found to be superior to an initial Heck reaction followed byreduction of the resulting styrene. Upon subjecting 15 to trimethylsilyl triflate under soft enolization conditions,40pentacycle 16 was obtained in excellent yield, presumably through a formal [4+2] cycloaddition of the pyrone group with an in situ generated enol. Cleavage of the resulting TMS ether gave tertiary alcohol 17, which was converted to a chloride (see 18) using anhydrous HCl generated in situ from acetyl chloride and methanol. To install the propyl amine substituent required to form the piperidine E-ring, addition of a tertiary radical generated from chloride 18 to acrylonitrile was pursued. Success was realized using a modification of the conditions of Lin (Cp2TiCl2, Zn, Et3N•HCl),41which gave 19 in 91% yield (entry 1). This result emerged only after exploring a range of radical precursors and conditions for the Giese-type42conjugate addition (Figure 2c). These included the direct use of tertiary alcohol 17 (entry 2)43, photoredox approaches using the corresponding bromide (entry 3)44or oxalate (entry 4),45or a reductive generation of the tertiary radical from the methyl oxalate (entry 5).46Notably, the C–C bond formation from 18 proceeds with a high level of diastereoselectivity to give 19 with addition from the convex face of the pentacycle. With nitrile 19 in hand, hydration of the nitrile group was achieved using the conditions of Chang and Lee47to give a carboxamide intermediate (not shown). At the high temperatures that were necessary for this reaction (110 °C), spontaneous decarboxylation of the β,γ-Δ-lactone moiety occurred to give the diene that is shown in 20. Hoffmann rearrangement of the primary amide group using phenyliodine(III) diacetate (PIDA) gave carbamate 20 following trapping of the intermediate isocyanate with methanol. At this stage, following the precedent of Fukuyama,48singlet oxygen addition acrossthe diene gave an endoperoxide, which upon treatment with tr -hydroxyenone (21) through a Kornblum–De LaMarre reaction.49Elimination of the tertiary hydroxy group in 21 was achieved using Martin’s sulfurane50,51to give dienone 22. Upon treatment of 23 with borontrifluoride etherate (BF3•OEt2), 1,6-conjugate addition ensued to give a carba-neopinone derivative which bears β,γ-unsaturation. This particular alkene group was isomerized to the α,β-unsaturated compound (a carbacodeinone derivative) by adding HCl to the crude reaction mixture to give a β-chloroketone,52,53followed by elimination of the generated secondary chloride group to give 24 using diazabicycloundecene (DBU). X-Ray crystallographic analysis of a single crystal of 24 provided unambiguous support for its structure. At this stage, cleavage of the phenol methyl ether was accomplished using borontrifluoride dimethylsulfide complex.54A global reduction of the methylcarbamate and enone carbonyl groups using lithium aluminum hydride (LiAlH4) then yielded carbamorphine (9) in 40% over the latter two steps. This endgame emerged only after an extensive survey of different synthesis pathways. For example, LiAlH4reduction of 24 yielded carbacodeine (25). However, cleaving the methylether of 25 led to rearrangement of the allylic alcohol to give bromide 26. This demethylationsequence is known to work well for the conversion of codeine (3) to morphine (1), presumably because binding of the boron to the dihydrobenzofuran oxygen (see Figure 2d) prevents the rearrangement that is observed for 26. The structure of 9 is unambiguously supported by a single-crystal X-ray crystallographic analysis. Overall, the synthesis of carbamorphine (±-9) was achieved in a total of 15 steps from commercially available indanone 12 in 3.0% overall yield. This synthesis compares favorably to past syntheses of morphine and provided the amounts of material necessary to support the function studies that are described herein. In addition, the outlined synthetic plan has formed the basis for the preparation of the carba-analogue of the µ- opioid antagonist nalorphine (10, Figure 2e). Specifically, starting from dienone 22, 1,6- conjugate addition of the methylcarbamate group followed by the addition of HCl yielded secondary chloride 23. Cleavage of the methylcarbamoyl group using trimethylsilyliodide (TMSI) unveiled the secondary amine (27), which was allylated with allylbromide in the presence of triethylamine. Under these conditions, the secondary chloride was eliminated to yield enone 28. As was the case in the endgame for the preparation of carbamorphine, cleavage of the methyl group of the phenol methyl ether using BF3•SMe2and reduction of the enone carbonyl group with LiAlH4gave carbanalorphine (10). Access to carbamorphine and carbanalorphine in racemic form set the stage for our preliminary studies as described below. In addition, we have employed supercritical fluid chromatography (SFC) chiral separation to resolve the enantiomers of 9 and 10. Thus, racemic syntheses provided access to each enantiomeric series, permitting a study of the impact of the absolute stereochemistry on the biological activity of these compounds.

[0119] Biological Activity of Carbamorphine

[0120] We first tested the binding affinity of (±)-carbamorphine, (±)-9, to 42 targets at the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) facility55using a 10 µM stock solution. As expected, (±)-9 interacts with the opioid receptors. Therefore, secondary screening was carried out at the MOR, KOR and DOR and the affinity of (±)-9 to these receptors was compared to standard controls. Racemic carbamorphine, (±)-9, (IC50= 29 nM) showed affinity similar to (–)-morphine, i.e., (–)-1, (IC50= 11 nM) at the MOR but showed poor affinity at KOR and DOR. The binding of the enantiomers of both carbamorphine (9) and carbanalorphine (10) to the MOR were then characterized at the NIMH–PDSP. It has been previously established that (+)-morphine {i.e., (+)-1} has no affinity at the MOR.34a(–)- Carbamorphine {(–)-9} binds the MOR with an IC50= 17 nM, whereas (–)-carbanalorphine {(–)- 10} has an IC50= 8.6 nM, which is similar to (–)-morphine {(–)-1; IC50= 4.3 nM}. In contrast to (+)-morphine {(+)-1}, (+)-carbamorphine {(+)-9} showed affinity at MOR (IC50= 78 nM)whereas (+)-carbanalorphine {(+)-10} had relatively poor affinity (IC50= 4.1 µM) albeit higher than for (+)-morphine {(+)-1} which did not bind the receptor (IC50>10 µM).

[0121] We next probed the racemic mixtures and enantiomers of carbamorphine (9) and carbanalorphine (10) for functional activity at all opioid subtypes using bioluminescence resonance energy transfer (BRET)-based assays at all opioid subtypes.21 Consistent with their binding at the MOR, both (–)-carbamorphine {(–)-9} as well as (+)-carbamorphine {(+)-9} showed agonist activity at Gi1-MOR. The potency of (–)-carbamorphine {(–)-9; IC50= 7.4 nM} was comparable to that of (–)-morphine {(–)-1; IC50= 6.8 nM} whereas (+)-carbamorphine {(+)-9; IC50= 52 nM} was less active. When tested for subtype selectivity, both (–)- carbamorphine {(–)-9} and (+)-carbamorphine {(+)-9} were selective for the MOR. Moderate activity was also observed at the KOR and both agents showed no agonist activity at the DOR.

[0122] In a Gi1and β-arrestin2 antagonist mode assay, it was determined that (–)- carbanalorphine {(–)-10} was able to almost fully block DAMGO activity at high concentrations, though with lower potency than naloxone; a remaining activity of <20% is potentially very low partial activity. On the other hand, high concentrations of (+)- carbanalorphine {(+)-10} only blocked around 35% of DAMGO Gi1and 55% of DAMGO β- arrestin2 response, relative to naloxone antagonist activity, this is perhaps explained by its poor affinity for the receptor. In binding studies, (+)-carbanalorphine {(+)-10} did not fully displace DAMGO, indicating that the remaining activity in antagonist mode is most likely resulting from the DAMGO response.

[0123] Since MOR signals through five other Gα-subtypes (i.e., Gi2, Gi3, GoA, GoBand Gz), along with our recent work, suggests that Gi / o / z isoform bias may be an avenue to design novel opioids,56we profiled the carbamorphines as well as morphine using the TRUPATH assay,56,57which measures heterotrimeric G protein dissociation. No differences in activity were observed between (–)-carbamorphine {(–)-9}, (–)-morphine {(–)-1} and (+)-carbamorphine {(+)-9} in this assay. All three activate all Gα-subtypes.

[0124] Recently, a correlation between low efficacy partial agonist activity and a decoupling of respiratory depression from antinociception has been proposed.56,58,59,60,61Given the lower efficacy for (+)-carbamorphine {(+)-9} at 10 µM compared to (–)-morphine {(–)-1}, we next evaluated the efficacy data at 10 µM for (+)-carbamorphine {(+)-9} and (–)-morphine {(–)-1} in a less amplified Nb39 assay.62Analogous to the BRET based Gi1assay, (+)-carbamorphine {(+)- 9} had lower efficacy compared to (–)-morphine {(–)-1}. These results also show that efficacy profiles for G-protein signaling at MOR for (+)-carbamorphine {(+)-9} are different from (–)- morphine {(–)-1}.

[0125] Opioid-agonist induced β-arrestin recruitment has been proposed as one possible mediator of tolerance and the duration of analgesia.63,64Therefore, we profiled the carbamorphines at both β-arrestin subtypes. No β-arrestin-1 recruitment was observed for either morphine or the carbamorphines. In contrast, (–)-morphine {(–)-1} showed weak recruitment of β-arrestin-2 (Emaxof 31%) while racemic carbamorphine and its enantiomers {(±)-9, (+)-9 and (– )-9, respectively} all showed lowered efficacy (less than 20%) — therefore, potencies could not be determined. Similar to the G-protein signaling, (+)-carbamorphine {(+)-9} had lower intrinsic efficacy relative to (–)-morphine {(–)-1} at the βarrestin-2 pathway. An amplified TANGO assay also showed similar results.

[0126] We next evaluated (+)-carbamorphine {(+)-9} in mice to determine if the opioid activity observed in cell lines would translate in vivo to a more physiological system. To limit pharmacokinetic and blood-brain-barrier (PK / BBB) penetration issues, we administered (+)- carbamorphine {(+)-9} through a supraspinal, intracerebroventricular (i.c.v.) route. Using the established 55oC warm water tail withdrawal assay (WWTW)56administration of 30 nmol and 100 nmol doses of (+)-carbamorphine {(+)-9} produced significant antinociception compared to vehicle control. The antinociceptive efficacy at a 30 nmol dose for (+)-carbamorphine was comparable to (–)-morphine {(–)-1} tested at a 10 nmol, i.c.v., dose.

[0127] To test for opioid receptor subtype selectivity, we examined (+)-carbamorphine {(+)-9} (30 nmol, i.c.v.) in MOR- and DOR-knockout mice and wild-type mice pretreated for 24 h with the KOR selective antagonist (norbinaltorphimine; norBNI).13 The antinociception activity of (+)-carbamorphine {(+)-9} was significantly reduced only in MOR KO mice (F(3,28)=32.5, p < 0.0001; One-way ANOVA w / Dunnett’s post hoc test), demonstrating antinociception mediated by the MOR and not by the KOR or DOR, consistent with the subtype selectivity observed in vitro with cell line binding and functional assays.

[0128] Next, respiratory depression was tested for (+)-carbamorphine {(+)-9} (300 nmol, i.c.v.) and (–)-morphine {(–)-1} (30 nmol, i.c.v.). When compared to vehicle controls, (–)-morphine {(–)-1} produced significant respiratory depression (time x treatment, F(15,330)=2.68; p = 0.0007; Two-way RM ANOVA w / Tukey post-hoc test) lasting over 40 min, at a dose consistent with our previously published work56whereas supratherapeutic doses of (+)-carbamorphine {(+)-9} did not significantly alter breath rate over the 2 h testing period (p = 0.2 or greater, with p = 0.06 at 100-120 min; not significant; Tukey’s post hoc test).

[0129] Finally, as (–)-morphine is known to have behavioral reinforcing properties, we evaluated abuse liability (+)-carbamorphine {(+)-9} with the established conditioned place preference (CPP) model in mice. Mice place-conditioned with (–)-morphine (30 nmol / d, i.c.v.) demonstrated significant CPP (conditioning x treatment, F(3,76)=3.23, p = 0.03; Two-Way RMANOVA w / Sidak post hoc test). In contrast, mice place conditioned with (+)-carbamorphine {(+)-9} at therapeutic (30 nmol / d, i.c.v.) or even supratherapeutic (300 nmol / d, i.c.v.) doses did not show significant differences from preconditioning preferences (p = 0.52 and 0.61, respectively; Sidak’s post hoc test).21,56Collectively, these mouse testing results demonstrate (+)-carbamorphine {(+)-9} showed distinct atypical MOR agonist-like pharmacological properties as compared to the prototypical MOR agonist morphine.

[0130] Molecular Docking and Simulations

[0131] To gain insight into the interaction between (+)-carbamorphine {(+)-9}, (–)- carbamorphine {(–)-9} and the MOR, molecular docking and molecular dynamics simulations (MDs) were conducted. (+)-Carbamorphine {(+)-9} and (–)-carbamorphine {(–)-9} were first docked into the cryo-EM structure that was solved for the hMOR bound to (–)-morphine {(–)- 1}.22The likely poses of (+)-carbamorphine {(+)-9} and (–)-carbamorphine {(–)-9} were determined based on their binding energy and proximity to conserved residue ASP1493.32. The more interesting of these poses was for (+)-carbamorphine {(+)-9}, which occupied the same binding pocket as (–)-morphine {(–)-1}, with a slight rotation of the ligand, and with its protonated tertiary amine group 3.4 Å from ASP1493.32.

[0132] To further explore this preferred binding pose, MDs were generated from the MOR- (–)- morphine {(–)-1} cryo-EM structure, and the docked pose of (+)-carbamorphine {(+)-9} in the MOR. The receptor was modeled in a lipid bilayer solvated in water and NaCl. Each 1 μs simulation was initiated from the minimized structure using a random seed to ensure independent replicates, three replicates were run for each (–)-morphine {(–)-1} and (+)- carbamorphine {(+)-9}. During the simulations, both ligands displayed stability with very little root mean squared deviation (RMSD) fluctuation compared to the initial frames ( though (+)- carbamorphine {(+)-9} did show some movement away from key residue D1493.32. A native contact analysis was applied to the simulations to determine the residues within 4 Å of the ligand and to give a % of the trajectory that these ligands were within 4 Å. Several key differences in the interaction profile of the ligands were determined, (–)-morphine {(–)-1} made additional contacts with TM5 and TM6 (K2355.39, H2996.52, V3026.55and I3247.39) whilst (+)- carbamorphine {(+)-9} had a sustained proximity to F1543.37in TM3, which was not observed for (–)-morphine {(–)-1}. In the (+)-carbamorphine {(+)-9} simulation, F1543.37had a conformation distinct from the morphine simulation and the inactive-state MOR. The position of M1533.36was also distinct for the (+)-carbamorphine {(+)-9} simulation, in the (–)-morphine {(– )-1} simulation M1533.36remained in an inactive-like position,26whilst in the (+)- carbamorphine {(+)-9} simulation, M1533.36was positioned away from the ligand and the contact time was decrease.

[0133] The residue interaction profile for (–)-morphine during the simulation largely overlaps with the morphine-bound cryo-EM structure as well as previous mutagenesis studies, and other computational studies,65,66,67,68which highlight interactions with D1493.32, Y1503.33, M1533.36, V2385.42, I2986.51and H2996.52. These simulations indicate that (+)-carbamorphine {(+)-9} likely binds differently in the MOR compared to (–)-morphine {(–)-1}, leading to a distinct interaction profile. Residues within the binding pocket also showed movements for the (+)-carbamorphine {(+)-9} simulation that were not observed for (–)-morphine {(–)-1}. The different conformation adopted by (+)-carbamorphine {(+)-9} along with the different contacts with residues in the MOR could be contributing to its different signaling profile compared to (–)-morphine {(–)-1}. In particular, the lack of contact between (+)-carbamorphine {(+)-9} with specific residues in TM5 / 6 could drive the decreased efficacy and potency observed in the in vitro signaling assays. However, cryo-EM structures of ligand MOR complexes are required in order to draw concrete conclusions regarding binding.

[0134] Overall, access to the enantiomers of carbamorphine and carbanalorphine presented opportunities to study the function of these previously unknown compounds. The bioactivity profile for (+)-carbamorphine {(+)-9} is especially intriguing given the lack of MOR activity for (+)-morphine {(+)-1}. While the initially envisioned removal of the interaction with TYR1483.33(mMOR) or TYR1503.33(hMOR) was anticipated to result in lowered potency of binding, the present data demonstrate instead different pharmacological trends, possibly due to additional changes in interactions with residues in the MOR (as supported by our docking and MD simulation studies). Single core heavy atom changes to the morphine framework and the corresponding impact on function have not been a focus of prior work on these molecules. The studies reported here set the stage for a comprehensive analysis of the core framework of morphine; one heavy atom at a time. For example, a net O®NR or O®S replacement of the E- ring O-atom is likely to yield several unanticipated functional outcomes as has been found in other natural product families.69These and related studies are the basis of ongoing investigations in our laboratories.

[0135] In summary, we have synthesized morphine analogs that bear a carbon atom in place of the oxygen atom in the E-ring, including “carba” analogs of the agonist morphine and the antagonist nalorphine. Pharmacological characterization of these compounds shows that they possess different pharmacological profiles at the MOR. For example, both enantiomers of carbamorphine show affinity and activity whereas only (–)-morphine is opioid active. In functional assays at both G-protein as well as β-arrestin pathways, (+)-carbamorphine {(+)-9} showed lower efficacy compared to (–)-morphine {(–)-1}, which could explain its distinct in vivo profile58,59,60,61,70of MOR dependent antinociception with reduced respiratory depressionand conditioned place preference. In summary, a total-synthesis-enabled O®CH2replacement in morphine has led to the development of safer functionally selective opioids.

[0136] References1R. Schmitz, Pharm. Hist.27, 61-74 (1985).2M. L. Barnett, et al. N. Engl. J. Med. 376, 663-673 (2017)3H. W. D. Matthes, et al. Nature 383, 819-823 (1996).4A. Kliewer, et al. Nat. Commun. 10, 1-11 (2019).5A. Kliewer, et al. Br. J. Pharmacol. 177, 2923-2931 (2020).6B. R. Varga, et al. Br. J. Pharmacol. 180, 975-993 (2023).7S. DeWeerdt, Tracing the US opioid crisis to its roots. Nature 573, 10-12 (2019).8P. Skolnick, Pharmacol. Ther. 233, 108019 (2022).9S. H. Ngai, et al. Anesthesiology 44, 398-401 (1976).10J. M. Gulland, R et al. Mem. Proc. Manchester Lit. Phil. Soc. 69, 79-86 (1925).11U. Rinner, et al. Top. Curr. Chem. 309, 33-66 (2012).12S. Galanie, et al. Science 349, 1095-1100 (2015).13S. Berenyi, et al. Curr. Med. Chem. 16, 3215-3242 (2009).14P. S. Portoghese, et al. Eur. J. Pharmacol. 146, 185-186 (1988).15S. Granier, et al. Nature 485, 400-404 (2012).16B. Robinson, Chem. Rev. 63, 373-401 (1963).17P. S. Portoghese, et al. . Life Sci. 40, 1287-1292 (1987).18K. M. Wassum, et al. ACS Chem. Neurosci. 6, 11-13 (2015).19T. Che, et al. Cell 172, 55-67 (2018).20C. Wicks, et al. Morphine alkaloids: History, biology, and synthesis. Alkaloids Chem. Biol. 86, 1-89 (2021).21K. Kanematsu, et al. Curr. Med. Chem. Nerv. Syst. Agents 1, 1-25 (2001).22R. Uprety, et al. Elife 10, 1-58 (2021).23Y. Zhuang, et al. Cell 185, 4361-4375 (2022).24X. Cui, et al. Eur. J. Pharmacol. 702, 309-315 (2013).25G. W. Pasternak, et al. Pharmacol. Rev. 65, 1257-1317 (2013).26D. B. Bylund, et al. Pharmacol. Rev. 46, 121-136 (1994).27A. Manglik, et al. Nature 485, 321-326 (2012).28H. Xu, et al. Synapse 32, 23-28 (1999).29W. Huang, et al. Nature 524, 315-321 (2015).30A. Koehl, et al. Nature 558, 547-552 (2018).31Q. Qu, et al. Nat. Chem. Biol. 19, 423-430 (2023).32V. Le Rouzic, et al. Anesth. Analg. 128, 365-373 (2019).33A. W. Sromek, et al. ACS Chem. Neurosci. 5, 93-99 (2014).34(a) I. Iijima, et al. J. Org. Chem. 43, 1462-1463 (1978) (b) I. Iijima, et al. J. Med. Chem. 21, 398-400 (1978) (c) H.-e. Wu, et al. Eur. J. Pharmacol. 571, 145-151 (2007) (d) E. E. Codd, et al. J. Pharmacol. Exp. Ther. 274, 1263-1270 (1995) (e) For a case where opioid enantiomers are known to have similar activity, see: G. Weltrowska, et al. J. Pept. Sci. 111, e24078 (2019).35M. Haider, et al. J. Am. Chem. Soc. 143, 2710-2715 (2021).36G. Sennari, et al. . J. Am. Chem. Soc. 144, 19173-19185 (2022).37CAS :13336-31-738P. K. Pramanick, et al. Tetrahedron 73, 7105-7114 (2017).39B. J. K. Stille,. Angew. Chem. Int. Ed. 25, 508-524 (1986).40L. J. Oxtoby, et al. Trends Chem. 1, 572-587 (2019).41S. Murata, et al. J. Am. Chem. Soc. 102, 3248-3249 (1980).42X. Wu, et al. J. Am. Chem. Soc. 140, 14836-14843 (2018).43B. Giese,. Angew. Chem. Int. Ed. 22, 753-764 (1983).44H. Xie, et al. J. Am. Chem. Soc. 142, 16787-16794 (2020)45Y. Sun, et al. Angew. Chem. Int. Ed. 52, 9201-9204 (2013).46C. C. Nawrat, et al. J. Am. Chem. Soc. 137, 11270-11273 (2015).47Y. Ye, et al. Org. Lett. 22, 2070-2075 (2020).48J. Lee, et al. Org. Lett. 11, 5598-5601 (2009).49H. Umihara, et al. Chem. Eur. J. 23, 6993-6995 (2017).50N. Kornblum, et al. J. Am. Chem. Soc. 73, 880-881 (1951).51J. C. Martin, R. J. Arhart, J. A. Franz, E. F. Perozzi, L. Kaplan, J. Org. Synth. 57, 22-26 (1977).52S. S. Pooppanal, et al. . Synlett 5, 850-851 (2009).53R. B. Barber, et al. J. Med. Chem. 19, 1175-1180 (1976).54S. Chu, et al. . Angew. Chem. Int. Ed. 55, 14306-14309 (2016).55K. Tejinder, K. Rupesh Chhaganbhai, S. Aakarshm, WO2020 / 188583, A1 (2020).56https: / / pdsp.unc.edu / pdspweb / 57A. Faouzi, et al. Nature 613, 767-774 (2023).58R. H. J. Olsen, et al. Nat. Chem. Biol. 16, 841-849 (2020).59S. Bhowmik, et al. Nat. Commun. 12, 3858 (2021).60A. Gillis, et al. . Sci. Signal. 14, 670-688 (2021).61B. Paul, et al. , Opioid Signaling and Design of Analgesics, 1st ed. Elsevier Inc. 195 (2023).62(a) N. Ramos-Gonzalez, et al. . ACS Cent. Sci. 10, 1490-1503 (2024).63M. Stoeber, et al. Elife 9, 1-20 (2020).64C. L. Schmid, et al. Cell 171, 1165-1175 (2017).65L. He, et al. Neuropsychopharmacology 46, 2241-2249 (2021).66A. Kapoor, et al. Sci. Rep. 7, 1-15 (2017).67S. Liao, et al. Life Sciences 269, 119026 (2021).68A. Ricarte, et al.. J. Chem. Inf. Model. 61, 1251-1274 (2021). 69S. Fukuyoshi, et al. 2D6.1, 2, 10, 14A, 51, and 62. PLoS One 11, 1-16 (2016).70(a) D. L. Boger, et al. Eur. J. Med. Chem. 208, 112829 (2020).71Chakraborty, J. F. et al.. J. Med. Chem. 64, 13873-13892 (2021). 72 Pramanick, P. K., et al. Tetrahedron 73, 7105–7114 (2017). 73 Haider, M., et al. J. Am. Chem. Soc. 143, 2710–2715 (2021). 74 Faouzi, A., et al. Nature 613, 767–774 (2023). 75 Chakraborty, S., et al. J. Med. Chem. 64, 16553–16572 (2021). 76 Olsen, R. H. J., et al. Nat. Chem. Biol. 16, 841–849 (2020). 77 Chakraborty, S., et al. J. Med. Chem. 64, 13873–13892 (2021). 78 DiBerto, J. F.; et al. Methods in molecular biology (Clifton, N.J.) 2022, 2525, 185. 79 Eberhardt, J., et al. J. Chem. Inf. Model.61, 3891–3898 (2021). 80 Aldrich, J. V., et al. Br. J. Pharmacol. 171, 3212–3222 (2014). 81 Váradi, A., et al. J. Med. Chem. 59, 8381–8397 (2016). 82 McLaughlin, J. P., et al. J. Pharmacol. Exp. Ther.289, 304–311 (1999). 83 Li, Y., et al. Molecules 28, (2023). 84 Scherrer, K. H., et al. Pharmaceuticals 16, (2023). 85 Uprety, R., et al. Elife, 10, 1–58 (2021). 86 Cirino, T. J., et al. Front. Pharmacol.10, 678 (2019). 87 Zhuang, Y., et al. Cell 185, 4361–4375 (2022). 88 Meng, E. C., et al. Protein Sci. 32, 1–13 (2023). 89 Trott, O. et al. J. Comput. Chem.31, 455–461 (2010). 90 Pettersen, E. F., et al.. Protein Sci.30, 70–82 (2021). 91 Jo, S., et al. Journal of Computational Chemistry, 29, 1859–1865 (2008). 92 Tian, C., et al. J. Chem. Theory Comput, 16, 528–552 (2019). 93 Dickson, C., et al. J. Chem. Theory Comput, 18, 1726–1736 (2022). 94 Case, D. et al. Amber Tools. J. Chem. Inf. Model., 63, 6183–6191(2023).95Roe, D., et al. J. Chem. Theory Comput, 9, 3084–3095 (2013).

[0137] Supporting Information

[0138] Preparation of bromide 13

[0139] This procedure was adapted from Yao et al.71To a solution of NBS (8.23 g, 46.2 mmol, 1.5 equiv) and 12 in ACS grade MeCN (100 mL) at 23 °C was added I2(0.78 g, 3.1 mmol, 0.1 equiv). The resulting dark orange suspension was stirred, covered by aluminum foil, for 18 h at 23 °C under N2. The reaction mixture was then quenched by the addition of sat. aq. Na2S2O3(250 mL) and the resulting mixture was stirred for an additional 1h. The organic and aqueous layers were separated, and the aqueous phase was extracted with CH2Cl2(3 x 200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash column chromatography (DCM to 4% MeOH / DCM) affording 13 (6.66 g, 90% yield) as a white solid.

[0140] TLC (50% EtOAc / hexanes): Rf= 0.8 (p-anisaldehyde, yellow)

[0141] 1H NMR (400 MHz, CDCl3): δ 7.46 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.20 – 2.87 (m, 2H), 2.87 – 2.58 (m, 2H).

[0142] 13C NMR (126 MHz, CDCl3): δ 204.3, 156.4, 146.9, 135.1, 133.2, 115.8, 108.9, 77.4, 77.2, 76.9, 55.8, 36.9, 21.7.

[0143] HRMS (EI+) m / z calc’d for C12H9O2Br [M]+: 239.9786, found: 239.9789

[0144] Stille coupling for S1

[0145] To a 2.0 L round-bottomed flask was added 13 (27.0 g, 112mmol, 1.0 equiv) and Pd(PPh3)4(6.47g, 5.60 mmol, 5 mol%), and the flask was evacuated and backfilled with N2three times. Toluene (1 L) was added into the 2 L round-bottomed flask under N2, followed by the addition of tributyl(vinyl)tin (36.1 mL, 123 mmol, 1.1 equiv), and the reaction mixture was stirred at 100 ºC for 16 h. The solution was then allowed to cool to room temperature and concentrated in vacuo. The crude product was purified by flash column chromatography [KF / silica (1:9, w / w), CH2Cl2] affording S1 (17.6 g, 84% yield) as a white solid.

[0146] TLC (50% EtOAc / hexanes): Rf= 0.8 (UV / p-anisaldehyde, purple.

[0147] 1H NMR (500 MHz, CDCl3) δ 7.76 (dd, J = 17.7, 11.0 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 5.65 (dd, J = 17.7, 1.4 Hz, 1H), 5.23 (dd, J = 11.0, 1.4 Hz, 1H), 3.84 (s, 3H), 2.94 – 2.88 (m, 2H), 2.63 – 2.57 (m, 2H).

[0148] 13C NMR (126 MHz, CDCl3) δ 208.2, 156.5, 144.3, 134.1, 132.1, 129.7, 125.0, 115.1, 114.8, 55.7, 36.9, 22.0.

[0149] HRMS (EI+) m / z calc’d for C12H12O2[M]+: 188.0837, found: 188.0837

[0150] Reductive Heck reaction to form 15

[0151] To a 2 L round-bottomed flask was added S1 (14.7 g, 78.1 mmol, 1.0 equiv), pyranone 3-triflate 14 (28.6 g, 117 mmol, 1.5 equiv), HCO2Na (13.3 g, 195 mmol, 2.5 equiv) and Pd(PPh3)4(4.51 g, 5.60 mmol, 5 mol%). The flask was evacuated and backfilled with N2three times, followed by the addition of THF (781 mL) into the flask under N2, and the reaction mixture was stirred at 60 ºC for 24 h. After checking the conversion of the reaction by1H NMR, additional Pd(PPh3)4(2.26 g, 1.95 mmol, 2.5 mol%) and pyranone 3-triflate 14 (9.53 g, 39.1 mmol, 0.5 equiv) were added into the mixture. The resulting mixture was then stirred at 60 ºC for an additional 24 h, before adding Pd(PPh3)4(2.26 g, 1.95 mmol, 2.5 mol%) and pyranone 3- triflate 14 (4.75 g, 19.6 mmol, 0.25 equiv) again. The resulting solution was stirred for an additional 48 h and subsequently allowed to cool to room temperature and concentrated in vacuo. The crude product was purified by flash column chromatography (CH2Cl2to 2% MeOH / CH2Cl2), followed by trituration with EtOAc (100 mL; conducted twice) affording 15 (17.9 g, 81% yield) as a white solid.

[0152] *Pyranone 3-triflate 14 was prepared according to our previous protocol.72

[0153] TLC (50% EtOAc / hexanes): Rf= 0.6 (UV / p-anisaldehyde, orange)

[0154] 1H NMR (500 MHz, CDCl3) δ 7.38 (d, J = 4.9 Hz, 1H), 7.16 (d, J = 6.5 Hz, 1H), 7.10 (d, J = 5.8 Hz, 1H), 6.92 (dd, J = 8.1, 1.9 Hz, 2H), 6.17 – 6.10 (m, 1H), 3.88 (s, 3H), 3.21 (t, J = 7.9 Hz, 2H), 3.03 – 2.96 (m, 2H), 2.74 – 2.63 (m, 4H).

[0155] 13C NMR (126 MHz, CDCl3) δ 208.1, 163.1, 155.5, 149.5, 145.2, 138.9, 135.2, 132.9, 129.9, 129.7, 114.6, 106.4, 55.6, 36.8, 32.1, 29.6, 22.2.

[0156] HRMS (ESI+) m / z calc’d for C17H17O4[M+H]+: 285.1121, found: 285.1121

[0157] Intramolecular [4+2] cycloaddition reaction to form 16

[0158] Pyrone-indanone adduct 15 (11.2 g, 39.4 mmol, 1.0 equiv) was dissolved in 360 mL of dry DCM. DIPEA (15.8 mL, 90.6 mmol, 2.3 equiv) was added, at which point the mixture turns deep yellow. The yellow solution was cooled to 0 ̊C and TMSOTf (15.7 mL, 86.7 mmol, 2.2 equiv) was added dropwise. Upon completion of addition, the resulting dark red solution was stirred for an additional 5 min at 0 C̊ before being allowed to warm to room temperature and then stirred for an additional 24 h. After full conversion of the starting material (as judged by TLC), the mixture was cooled to 0̊C. The reaction mixture was quenched by dropwise addition of sat. aq. NaHCO3solution and extracted with DCM four times, dried over Na2SO4, filtered, and concentrated by rotary evaporation to give a dark red solid residue. A SiO2-plug filtration (DCM eluent) yielded essentially pure product 16 (8.44 g, 60% yield, >20:1 d.r.) as a yellow solid.

[0159] TLC (50% EtOAc / hexanes): Rf= 0.6 (UV / p-anisaldehyde, purple)

[0160] 1H NMR (400 MHz, CDCl3) δ 6.94 (d, J = 8.2 Hz, 1H), 6.69 (d, J = 7.9 Hz, 1H), 6.08 (dd, J = 7.6, 4.7 Hz, 1H), 5.46 (dd, J = 7.6, 2.1 Hz, 1H), 5.21 (td, J = 4.8, 2.1 Hz, 1H), 3.79 (s, 3H), 3.32 (dd, J = 17.4, 9.3 Hz, 1H), 3.22 – 3.02 (m, 2H), 2.87 (ddd, J = 15.0, 10.5, 4.0 Hz, 1H), 2.74 (ddd, J = 16.8, 9.7, 4.2 Hz, 1H), 2.16 (dd, J = 17.3, 3.0 Hz, 1H), 2.04 (ddd, J = 13.7, 9.6, 3.9 Hz, 1H), -0.11 (s, 9H).

[0161] 13C NMR (151 MHz, CDCl3) δ 173.2, 154.3, 144.0, 137.2, 129.3, 128.2, 127.6, 127.2, 111.4, 85.6, 77.3, 55.4, 54.5, 50.5, 30.8, 22.9, 21.1, 1.0 (3C).

[0162] HRMS (ESI+) m / z calc’d for C20H25O4Si [M+H]+: 357.1517, found: 357.1520.

[0163] Melting Point: 110–115 ºC

[0164] Chlorination to form 18

[0165] To a solution of 16 (7.41 g, 20.79 mmol, 1.0 equiv) in MeOH (208 mL) at 0 ºC was added dropwise an aqueous HCl solution (6.0 M, 173 mL) under air. The resulting mixture was stirred at 23 °C for 20 min until the formation of a white solid was observed. The resulting mixture was then diluted with CH2Cl2, and the organic and aqueous phase were separated. Theaqueous phase was extracted with CH2Cl2four times, and the combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was used for the next reaction without further purification.

[0166] The flask containing the crude alcohol was evacuated and backfilled with N2three times. Acetyl chloride (240 mL) was added into the flask under N2, which was then cooled to 0 ºC. To the resulting mixture was then carefully added MeOH (200 mL) to generate HCl in-situ, and the resulting mixture was warmed to 23 °C and stirred for 2.5 h. After this period, the mixture was carefully concentrated in vacuo, and crude material was purified without work-up. Purification by flash column chromatography (20% EtOAc / Hexane) afforded 18 (3.37 g, 54% yield, >20:1 d.r.) as a white solid.

[0167] TLC (50% EtOAc / hexanes): Rf= 0.7 (UV / p-anisaldehyde, purple)

[0168] 1H NMR (600 MHz, CDCl3) δ 6.99 (d, J = 8.1 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.18 (dd, J = 7.6, 4.9 Hz, 1H), 5.51 (dd, J = 7.6, 2.1 Hz, 1H), 5.24 (td, J = 4.9, 2.1 Hz, 1H), 3.79 (s, 3H), 3.71 (ddd, J = 9.2, 4.8, 2.8 Hz, 1H), 3.47 (dd, J = 17.4, 9.2 Hz, 1H), 3.24 (ddd, J = 16.2, 12.1, 5.0 Hz, 1H), 3.00 (ddd, J = 14.5, 11.4, 3.2 Hz, 1H), 2.83 (ddd, J = 16.7, 10.0, 3.1 Hz, 1H), 2.34 (dd, J = 17.3, 2.8 Hz, 1H), 2.14 (ddd, J = 14.6, 10.0, 5.0 Hz, 1H).

[0169] 13C NMR (151 MHz, CDCl3) δ 171.6, 154.4, 143.2, 137.7, 129.1, 128.7, 128.0, 126.1, 111.9, 77.2, 76.5, 55.5, 54.1, 31.4, 22.5, 22.5.

[0170] HRMS (ESI+) m / z calc’d for C17H16O3Cl [M+H]+: 303.0782, found: 303.0786

[0171] Melting Point: 135–140 ºC

[0172] Giese-type radical addition from chloride 18 to 19

[0173] To a 100 mL round-bottomed flask was added Zn (713.0 mg, 10.9 mmol, 3.0 equiv), Cp2TiCl2, (1.36 g, 5.45 mmol, 1.5 equiv) and Et3N•HCl (2.50 g, 18.2 mmol, 5.0 equiv). The flask was evacuated and backfilled with N2three times, followed by the addition of EtOAc (33.0 mL) into the flask under N2. The resulting mixture was then stirred at 23 °C for 10 min, after which acrylonitrile (0.81 mL, 12.3 mmol, 3.4 equiv) and 18 (1.10 g, 3.63 mmol, 1.0 equiv) in EtOAc (17 mL) were added to the mixture. The resulting mixture was stirred at 23 °C for 18 h, and then quenched with H2O. The organic and aqueous phase were separated. The aqueous phase was extracted with EtOAc three times, and the combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by short silica plug (EtOAc eluent) affording 19 (1.12 g, 91% yield, >20:1 d.r.) as a white solid.

[0174] TLC (50% EtOAc / hexanes): Rf= 0.5 (UV / p-anisaldehyde, green)

[0175] 1H NMR (600 MHz, CDCl3) δ 6.94 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 6.12 (dd, J = 7.6, 5.0 Hz, 1H), 5.42 (dd, J = 7.6, 2.0 Hz, 1H), 5.18 (td, J = 5.0, 2.0 Hz, 1H), 3.76 (s, 3H), 3.19 (dd, J = 17.4, 9.4 Hz, 1H), 3.12 – 3.06 (m, 1H), 3.02 (ddd, J = 17.1, 11.4, 5.0 Hz, 1H), 2.85 (ddd, J = 17.1, 10.3, 3.2 Hz, 1H), 2.70 (ddd, J = 14.7, 11.4, 3.2 Hz, 1H), 2.37 (dd, J = 17.5, 2.8 Hz, 1H), 2.29 – 2.11 (m, 3H), 2.09 – 2.02 (m, 1H), 1.85 – 1.76 (m, 1H).

[0176] 13C NMR (151 MHz, CDCl3) δ 173.4, 154.5, 143.6, 139.2, 129.4, 128.0, 127.9, 125.6, 119.1, 110.5, 77.2, 55.3, 54.8, 52.4, 48.4, 31.8, 30.8, 22.7, 22.6, 14.0.

[0177] HRMS (ESI+) m / z calc’d for C20H19O3NNa [M+Na]+: 344.1257, found: 344.1256

[0178] Melting Point: 180–185 ºC

[0179] Other attempts at the Giese-type radical addition toward 19 using a range of radical precursors.

[0180] Hydration of nitrile 19 and Hoffman rearrangement sequence to form 20

[0181] Acetaldoxime (3.48 g, 59.0 mmol, 5.0 equiv) was added to a solution of 19 (3.79 g, 11.8 mmol, 1.0 equiv) and Rh(PPh3)3Cl (1.64 g, 1.77 mmol, 0.15 equiv) in toluene (118 mL) under N2. The resulting mixture was stirred at 110 ºC for 17 h. The reaction mixture was then cooled to 23 °C, concentrated in vacuo, and diluted with CH2Cl2(120 mL) and water. The organic and aqueous phase were separated, and the aqueous phase was extracted with CH2Cl2four times. After the organic phase was concentrated in vacuo, the obtained crude product was used in the next reaction without further purification.

[0182] The flask containing the crude amide was evacuated and backfilled with N2three times before MeOH (118 mL) was added into the flask. KOH powder (2.32 g, 41.3 mmol, 3.50 equiv) was added to the mixture which was then cooled to 0 ºC. PIDA (5.70 g, 17.7 mmol, 1.50 equiv) was added and the resulting mixture was warmed to 23 °C and stirred under N2for 1 h. After this period, the mixture was carefully quenched with aqueous NH4Cl and concentrated in vacuo to remove MeOH. The solution was diluted with CH2Cl2, the organic and aqueous phase were separated, and the aqueous phase was extracted with CH2Cl2three times. The crude material was purified by flash column chromatography (hexanes to 25% EtOAc / hexanes) affording 20 (3.09 g, 81% yield) as a white solid.

[0183] TLC (50% EtOAc / hexanes): Rf= 0.8 (UV / p-anisaldehyde, purple)

[0184] 1H NMR (600 MHz, CDCl3) δ 6.90 (d, J = 8.3 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 5.63 – 5.57 (m, 1H), 5.53 – 5.46 (m, 2H), 4.72 (br, 1H), 3.79 (s, 3H), 3.65 (s, 3H), 3.48 – 3.41 (m, 2H), 3.30 (br, 2H), 3.10 (ddd, J = 15.7, 10.5, 4.7 Hz, 1H), 2.91 (t, J = 12.9 Hz, 1H), 2.82 (ddd, J = 16.3, 9.5, 3.5 Hz, 1H), 2.77 – 2.71 (m, 1H), 2.40 (ddd, J = 14.8, 9.5, 4.7 Hz, 1H), 2.21 – 2.12 (m, 1H), 1.58 – 1.50 (m, 1H).

[0185] 13C NMR (151 MHz, CDCl3) δ 157.1, 155.1, 150.0, 139.3, 127.8, 127.3, 126.1, 121.0, 116.9, 108.8, 55.4, 52.1, 50.5, 44.0, 39.7, 38.7, 38.4, 27.4, 26.6.

[0186] HRMS (ESI+) m / z calc’d for C20H23NO3Na [M+Na]+: 348.1570, found: 348.1570

[0187] Singlet oxygen addition across diene 20 followed by Kornblum–De LaMarre reaction to give 21

[0188] To a solution of 20 (3.04 g, 9.34 mmol, 1.0 equiv) in CH2Cl2(934 mL) was added methylene blue (59.8 mg, 0.187 mmol, 2 mol%). The resulting mixture was then irradiated with a sun-lamp with bubbling of O2into the solution at 0 ºC for 1 h. After this period, Et3N (40.0 mL, 287 mmol, 30.7 equiv) was added to the mixture and it was stirred at 23 °C for 4 h. The resulting mixture was then passed through a short plug of SiO2(eluted with DCM and EtOAc) and after concentration of the eluents, the crude material was purified by flash column chromatography (hexanes to 50% EtOAc / hexanes to 90% EtOAc / hexanes) affording 21 (2.34 g, 70% yield) as a white solid.

[0189] TLC (50% EtOAc / hexanes): Rf= 0.15 (UV / p-anisaldehyde, Blue)

[0190] 1H NMR (400 MHz, CDCl3) δ 6.85 (d, J = 8.3 Hz, 1H), 6.68 – 6.58 (m, 2H), 5.90 (d, J = 10.3 Hz, 1H), 4.92 (br, 1H), 3.76 (s, 3H), 3.64 (s, 3H), 3.55 (br, 1H), 3.33 (d, J = 16.3 Hz, 1H), 3.28 – 3.14 (m, 2H), 3.10 (d, J = 6.4 Hz, 1H), 2.95 (dd, J = 17.0, 7.0 Hz, 1H), 2.66 – 2.42 (m, 2H), 2.09 (ddd, J = 14.3, 9.4, 4.8 Hz, 1H), 2.04 – 1.95 (m, 1H), 1.95 – 1.82 (m, 1H).

[0191] 13C NMR (151 MHz, CDCl3) δ 201.7, 157.6, 154.5, 150.3, 147.1, 131.0, 126.9, 126.8, 123.7, 110.6, 70.2, 55.5, 52.7, 52.3, 51.6, 39.4, 36.3, 32.9, 32.7, 24.7.

[0192] HRMS (ESI+) m / z calc’d for C20H23NO5Na [M+Na]+: 380.1468, found: 380.1466

[0193] Dehydration with Martin’s sulfurane to give 22

[0194] To a solution of 21 (486 mg, 1.36 mmol, 1.0 equiv) in dry CHCl3(13.6 mL) was carefully added, dropwise, a solution of Martin’s sulfurane (1.00 g, 1.50 mmol, 1.10 equiv) in dry CHCl3(2.99 mL, 0.50 M) under N2. (Note: The product is sensitive to the use of an excess amount of Martin’s sulfurane. Using more than 1.5 equiv of Martin’s sulfurane caused decomposition of the product.) The reaction mixture was stirred for 15 min., and then the resulting mixture was passed through a short plug of SiO2(eluted with EtOAc). After concentration of the eluents, the crude material was purified by flash column chromatography (hexanes to 30% EtOAc / hexanes to 60% EtOAc / hexanes) affording 22 (340 mg, 74% yield) as a yellow solid.

[0195] TLC (50% EtOAc / hexanes): Rf= 0.25 (UV / p-anisaldehyde, blown)

[0196] 1H NMR (500 MHz, CDCl3) δ 7.13 (d, J = 9.9 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 6.30 (dd, J = 6.8, 2.4 Hz, 1H), 5.96 (d, J = 10.1 Hz, 1H), 4.52 (br, 1H), 3.80 (s, 3H), 3.61 (s, 3H), 3.58 – 3.30 (m, 4H), 3.28 – 3.23 (m, 2H), 3.17 – 3.08 (m, 1H), 2.06 – 1.94 (m, 1H), 1.77 – 1.67 (m, 1H).

[0197] 13C NMR (151 MHz, CDCl3) δ 199.2, 157.0, 155.0, 147.8, 143.5, 141.5, 131.5, 126.8, 126.6, 126.3, 124.7, 109.3, 55.6, 53.2, 52.2, 50.8, 40.7, 38.1, 31.9, 30.8.

[0198] HRMS (ESI+) m / z calc’d for C20H22O4N [M+H]+: 340.1543, found: 340.154

[0199] 1,6-Conjugate amine addition to convert enone 22 to 24

[0200] To a solution of 22 (240 mg, 0.71 mmol, 1.0 equiv) in dichloroethane (14.1 mL) was slowly added BF3•OEt2(448 μL, 3.54 mmol, 5.0 equiv) under N2at 0 ºC. The reaction mixture was then allowed to warm to 23 °C and stirred for 16 h. After this period, 4M HCl in dioxane (3.0 mL, 12.0 mmol, 17.0 equiv) was added to the reaction mixture which was stirred at 23 °C for an additional 2 h. Then, the reaction mixture was quenched by the addition of sat. aq. NaHCO3solution. The organic and aqueous phase were separated, and the aqueous phase was extracted with CH2Cl2three times. After the organic phase was concentrated in vacuo, the obtained crude product was used for the next reaction without further purification.

[0201] The flask containing crude material was evacuated and backfilled with N2three times, and THF (14.0 mL) was added into the flask. To the mixture was added dropwise DBU (426 μL, 2.83 mmol, 4.00 equiv), and the resulting mixture was stirred at 23 °C under N2for 10 min. After this period, the crude mixture was passed through a short plug of SiO2(eluted with EtOAc). After concentration of the eluents, essentially pure 24 (230 mg, 99% yield) was obtained as a yellow solid. The product was isolated as an approximately 6:4 mixture of rotamers.

[0202] TLC (50% EtOAc / hexanes): Rf= 0.5 (UV / p-anisaldehyde, blown)

[0203] 1H NMR (600 MHz, CDCl3) δ 6.89 – 6.84 (m, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.59 (d, J = 10.0 Hz, 0.6H, major rotamer), 6.55 (d, J = 10.0 Hz, 0.4H, minor rotamer), 5.94 (dd, J = 10.2, 2.9 Hz, 1H), 4.97 (br, 0.6H, major rotamer), 4.80 (br, 0.4H, minor rotamer), 4.13 – 4.06 (m, 0.4H, minor rotamer), 4.00 – 3.92 (m, 0.6H, major rotamer), 3.77 (s, 3H), 3.73 (s, 3H), 3.34 (s, 0.4H, minor rotamer), 3.32 (s, 0.6H, major rotamer), 3.29 – 3.20 (m, 1H), 2.98 – 2.93 (m, 1H), 2.89 – 2.73 (m, 4H), 1.88 – 1.80 (m, 1H), 1.71 – 1.62 (m, 1H).

[0204] 13C NMR (151 MHz, CDCl3, peaks for major rotamer tabulated) δ 200.7, 156.2, 154.7, 146.6, 142.8, 132.3, 127.3, 126.3, 124.6, 110.6, 55.4, 52.9, 50.6, 44.9, 40.8, 38.9, 34.2, 32.4, 30.0, 29.7.

[0205] HRMS (ESI+) m / z calc’d for C20H21NO4Na [M+Na]+: 362.1363, found: 362.1362.

[0206] Melting Point: 180 ºC (decomposed)

[0207] Demethylation and reduction to form carbamorphine (9)

[0208] To a solution of 24 (111 mg, 0.71 mmol, 1.0 equiv) in dichloroethane (4.0 mL) was slowly added BF3•SMe2(343 μL, 3.26 mmol, 10.0 equiv) under N2at 0 ºC. The reaction mixture was then allowed to warm to 23 °C and stirred for 16 h. After this period, the reaction mixture was quenched by the addition of 10% aqueous solution of NH4OH (w / w). The organic and aqueous phases were separated, and the aqueous phase was extracted with CH2Cl2 / MeOH (10 vol% MeOH) four times. The combined organic layers were dried over Na2SO4, and after the organic phase was concentrated in vacuo, the obtained crude product was used for the next reaction without further purification. The flask containing the crude material was evacuated and backfilled with N2three times, and THF (4.0 mL) was added into the flask. To the mixture was added dropwise a 2M LiAlH4solution in THF (814 μL, 1.63 mmol, 5.00 equiv) at 0 ºC, and the resulting mixture was stirred at 60 ºC under N2for 45 min. After this period, the resulting mixture was cooled to 0 ºC and Na2SO4•10H2O (600 mg) was added in several portions to quench the remaining LiAlH4. The mixture was then filtered through a glass filter and the filtrate was concentrated. The crude material was purified by flash column chromatography (2% DCM / MeOH with 1% NH4OH to 10% DCM / MeOH with 1% NH4OH) affording 9 (37.7 mg, 41% yield) as a white solid.

[0209] TLC (10% MeOH / CH2Cl2): Rf= 0.1 (UV / p-anisaldehyde, pink)

[0210] 1H NMR (700 MHz, CDCl3) δ 6.75 (d, J = 8.2 Hz, 1H), 6.52 (d, J = 8.0 Hz, 1H), 5.63 (dt, J = 10.0, 2.8 Hz, 1H), 5.42 (dt, J = 10.0, 2.5 Hz, 1H), 4.35 (dq, J = 9.3, 2.7 Hz, 1H), 3.30 (dd, J = 5.7, 3.2 Hz, 1H), 3.25 (d, J = 16.1 Hz, 1H), 3.01 (d, J = 18.2 Hz, 1H), 2.93 (dd, J = 16.0, 7.2 Hz, 1H), 2.76 (t, J = 8.3 Hz, 1H), 2.64 – 2.60 (m, 1H), 2.56 (dd, J = 11.3, 4.0 Hz, 1H), 2.43 (s, 3H), 2.33 (dd, J = 18.3, 5.9 Hz, 1H), 2.24 (td, J = 12.3, 3.5 Hz, 1H), 1.89 (td, J = 12.6, 5.0 Hz, 1H), 1.67 (d, J = 12.7 Hz, 1H).

[0211] 13C NMR (151 MHz, CDCl3) δ 150.2, 146.0, 133.4, 129.0, 127.1, 126.2, 125.8, 114.3, 65.8, 59.2, 50.5, 47.8, 44.8, 43.1, 41.6, 36.0, 26.3, 21.2.

[0212] HRMS (ESI+) m / z calc’d for C18H22NO2[M+H]+: 284.1645, found: 284.1642.

[0213] Rearrangement reaction during demethylation with BBr3to form 26

[0214] To a solution of 24 (9.50 mg, 0.028 mmol, 1.0 equiv) in THF (0.5 mL) was added a 2M LiAlH4solution in THF (70.0 μL, 0.14 mmol, 5.0 equiv) at 0 ºC, and the resulting mixture was stirred at 60 ºC under N2for 90 min. After this period, the resulting mixture was cooled to 0 ºC and Na2SO4•10H2O (150 mg) was added in several portions to quench the remaining LiAlH4. The mixture was then filtered through a glass filter, and the filtrate was concentrated to afford essentially pure product 25, which was used in the next step without further purification.

[0215] To a solution of 25 (3.0 mg, 0.010 mmol, 1.0 equiv) in dichloromethane (0.20 mL) was slowly added BBr3in dichloromethane (1M, 50.4 μL, 0.504 mmol, 5.0 equiv) under N2at 0 ºC. The reaction mixture was then allowed to warm to 23 °C. and stirred for 1.5 h. After this period, the reaction mixture was quenched by the addition of 10% NH4OH aqueous solution. The organic and aqueous phase were separated, and the aqueous phase was extracted with 10% CH2Cl2 / MeOH five times. The resulting residue was purified by thin layer preparative TLC (DCM / MeOH = 5:1), yielding 26 (2.20 mg, 63% yield) as a white solid.

[0216] TLC (10% MeOH / CH2Cl2): Rf= 0.3 (UV / p-anisaldehyde, pink)

[0217] 1H NMR (500 MHz, CDCl3) δ 6.84 (d, J = 7.6 Hz, 1H), 6.58 (d, J = 8.2 Hz, 1H), 5.78 (dt, J = 10.4, 2.0 Hz, 1H), 5.54 – 5.47 (m, 1H), 4.15 – 4.09 (m, 1H), 3.55 (dd, J = 5.9, 2.8 Hz, 1H), 3.19 (dd, J = 15.8, 7.6 Hz, 1H), 3.09 – 3.00 (m, 1H), 2.92 – 2.86 (m, 1H), 2.60 – 2.46 (m, 4H), 2.45 (s, 3H), 2.19 (td, J = 12.4, 3.4 Hz, 1H), 1.88 (td, J = 12.6, 4.9 Hz, 1H), 1.57 – 1.51 (m, 1H).

[0218] 13C NMR (151 MHz, CDCl3) δ 150.9, 143.6, 131.9, 130.1, 126.6, 126.1, 124.5, 114.7, 56.8, 49.3, 48.7, 47.3, 45.8, 45.6, 43.1, 35.4, 32.4, 20.2.

[0219] HRMS (ESI+) m / z calc’d for C18H21NOBr [M+H]+: 346.0801, found: 346.0805

[0220] Cleavage of carbamate and allylation for 28

[0221] To a solution of 22 (150 mg, 0.442 mmol, 1.0 equiv) in DCE (8.8 mL) was slowly added BF3•OEt2(280 μL, 2.21 mmol, 5.0 equiv) under N2at 0 ºC. The reaction mixture was then allowed to warm to 23 °C and stirred for 15 h. After this period, 4M HCl in dioxane (1.0 mL, 2.2 mmol, 9.1 equiv) was added to the reaction mixture and it was stirred at 23 °C for an additional 2 h. The reaction mixture was then quenched by the addition of saturated NaHCO3aqueous solution. The organic and aqueous phase were separated, and the aqueous phase was extractedwith CH2Cl2three times. After the organic phase was concentrated in vacuo, the obtained crude product was used in the next reaction without further purification.

[0222] The flask containing the crude material was evacuated and backfilled with N2three times, and CHCl3(7.0 mL) was added into the flask. TMSI (300 μL, 2.21 mmol, 5.0 equiv) was added dropwise and the resulting mixture was stirred at 60 ºC under N2for 15 h. After this period, the resulting mixture was quenched by the addition of MeOH at 23 °C and the crude mixture was concentrated in vacuo. The obtained crude product (27) was used for the next reaction without further purification.

[0223] The flask containing crude material 27 was evacuated and backfilled with N2three times, and MeCN (7.0 mL) was added into the flask. To the mixture was added dropwise Et3N (616 μL, 4.42 mmol, 10.0 equiv), and allyl bromide (230 μL, 2.65 mmol, 6.0 equiv). The reaction mixture was then heated at 55 ºC and stirred for 1 h at this temperature. After this period, the mixture was concentrated in vacuo, and the crude material was purified by flash column chromatography (2% DCM / MeOH to 4% DCM / MeOH) affording 28 (72.7 mg, 51% yield) as a brown oil.

[0224] TLC (10% MeOH / CH2Cl2): Rf= 0.6 (UV / KMnO4, yellow)

[0225] 1H NMR (400 MHz, CDCl3) δ 6.86 (d, J = 8.5 Hz, 1H), 6.61 (d, J = 8.2 Hz, 1H), 6.57 (dd, J = 10.2, 2.1 Hz, 1H), 5.97 – 5.78 (m, 2H), 5.38 – 5.12 (m, 2H), 3.77 (s, 3H), 3.70 – 3.60 (m, 1H), 3.46 (t, J = 4.2 Hz, 1H), 3.34 – 3.13 (m, 4H), 3.11 – 2.98 (m, 2H), 2.84 (d, J = 5.9 Hz, 1H), 2.67 (dd, J = 11.1, 3.9 Hz, 1H), 2.40 – 2.29 (m, 1H), 2.17 (td, J = 12.2, 3.5 Hz, 1H), 1.94 (td, J = 12.4, 4.9 Hz, 1H).

[0226] 13C NMR (151 MHz, CDCl3) δ 201.5, 154.4, 148.6, 144.2, 135.9, 131.7, 127.2, 125.9, 125.7, 117.7, 110.2, 58.4, 56.7, 56.0, 55.5, 45.9, 45.1, 42.0, 34.4, 32.5, 21.8.

[0227] HRMS (ESI+) m / z calc’d for C21H24NO2[M+H]+: 322.1802, found: 322.1800.

[0228] Demethylation and reduction to form carbanalorphine (10)

[0229] To a solution of 28 (7.2 mg, 0.02 mmol, 1.0 equiv) in dichloroethane (0.60 mL) was slowly added BF3•SMe2(23.6 μL, 0.22 mmol, 10.0 equiv) under N2at 0 ºC. The reaction mixture was then allowed to warm to 23 °C and stirred for 20 h. After this period, the reaction mixture was quenched by the addition of a 10% aqueous solution of NH4OH (w / w). The organic and aqueous phase were separated, and the aqueous phase was extracted with 10% CH2Cl2 / MeOH four times. The combined organic layers were dried over Na2SO4, and after theorganic phase was concentrated in vacuo, the obtained crude product was used for the next reaction without further purification.

[0230] The vial containing crude material was evacuated and backfilled with N2three times, and THF (0.80 mL) was added into the vial. To the mixture was add dropwise a 2M LiAlH4solution in THF (56.1 μL, 0.11 mmol, 5.00 equiv) at 0 ºC, and the resulting mixture was stirred at 23 °C under N2for 2.5 h. After this period, the resulting mixture was cooled to 0 ºC and Na2SO4•10H2O (100 mg) was added in several portions to quench the remaining LiAlH4. The mixture was then filtered using a glass filter and the filtrate was concentrated. The crude material was purified by amine-coated silica gel flash column chromatography (2% DCM / MeOH to 10% DCM / MeOH) to afford 10 (4.0 mg, 58% yield) as a white solid.

[0231] TLC (10% MeOH / CH2Cl2): Rf= 0.1 (UV / p-anisaldehyde, pink)

[0232] 1H NMR (600 MHz, CDCl3) δ 6.75 (d, J = 8.2 Hz, 1H), 6.53 (d, J = 8.1 Hz, 1H), 5.90 (ddt, J = 16.9, 10.1, 6.5 Hz, 1H), 5.63 (dt, J = 9.9, 2.8 Hz, 1H), 5.42 (dt, J = 9.9, 2.5 Hz, 1H), 5.23 (dq, J = 17.1, 1.6 Hz, 1H), 5.18 – 5.11 (m, 1H), 4.35 (dq, J = 9.3, 2.6 Hz, 1H), 3.40 (dd, J = 5.9, 3.3 Hz, 1H), 3.24 (d, J = 16.0 Hz, 1H), 3.24 – 3.15 (m, 2H), 2.94 (d, J = 17.6 Hz, 1H), 2.93 (dd, J = 16.8, 7.1 Hz, 1H), 2.77 (t, J = 8.3 Hz, 1H), 2.64 (ddd, J = 12.1, 5.0, 1.8 Hz, 1H), 2.60 (t, J = 2.8 Hz, 1H), 2.31 (dd, J = 18.3, 5.7 Hz, 1H), 2.20 (td, J = 12.3, 3.5 Hz, 1H), 1.88 (td, J = 12.6, 4.9 Hz, 1H), 1.66 (d, J = 11.1 Hz, 1H).

[0233] 13C NMR (151 MHz, CDCl3) δ 150.0, 146.2, 136.1, 133.3, 129.2, 127.0, 126.4, 125.8, 117.6, 114.2, 65.9, 58.5, 56.8, 50.6, 46.0, 45.4, 41.6, 36.0, 26.3, 21.8.

[0234] HRMS (ESI+) m / z calc’d for C20H24NO2[M+H]+: 310.1802, found: 310.1800

[0235] Evaluation of Biological Activity

[0236] Drugs and Materials

[0237] The sources of the reagents and solvents for compound synthesis are as reported previously.73All other drugs used in testing were purchased from Sigma-Aldrich, Gibco and Invitrogen from Thermo Fisher Scientific (St. Louis, MO, USA).

[0238] Radioligand Binding Assays (General Protocol)74

[0239] The membranes were prepared from either stably expressing cell lines or cells transfected with the receptor of interest. Cells were washed, harvested, and lysed, and crude membrane fractions were then washed and pelleted. Receptor expression was quantified using Bradford assay and saturation binding with a reference radioligand in order to determine total protein and GPCR expression, respectively. Membranes were then stored at –80 ºC prior to use in competition binding experiments. For competition binding, membranes were thawed then briefly triturated before being diluted and added to well plates at a known final receptor concentration. Hot and cold ligands were then added and incubated with shaking. Afterincubation, the wells were then harvested onto filter mats soaked with 0.3% polyethyleneimine, which were then washed with buffer and dried. Scintillation reagent was then applied to the mats, which were then counted using a luminescence detector.

[0240] Radioligand binding bioassay data was generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2018-00023-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. BRET based assays (TRUPATH and β-arrestin signaling)75,76,77

[0241] To measure G proteins dissociation or β-arrestin recruitment, 2.5 million cells were plated in 10 cm dishes and incubated overnight at 37 ºC. The next day, cells were co-transfected, using Lipofectamine 2000 following vendor protocol. Human MOR, Gα-RLuc8, Gβ, Gγ-GFP2 plasmids (2.5 μg per construct) were transfected at a ratio of 1:1:1:1 for G protein dissociation and a 1:5 DNA ratio of Rluc tagged MOR, Venus-tagged N-terminal β-arrestin (1 or 2) was used for β-arrestin recruitment. The following day, transfected cells were plated into a 384-well Poly- D-lysine coated plate with DMEM supplemented with 1% dialyzed FBS at a density of 20,000 cells per well and incubated overnight at 37 ºC. On the day of the assay, the media was removed, and cells were rinsed with 30 μL of assay buffer (1×Hank’s balanced salt solution (HBSS), 20 mM HEPES, pH 7.4), followed by the addition of the 30 μL substrate buffer (7.85 μM coelenterazine 400a for G-protein or 7.85 μM coelenterazine h for β-arrestin in assay buffer) for 5 min in the dark at room temperature. After a 5 min equilibration period, the cells were treated with 15 μL of drug (3X drug concentration in assay buffer with 0.3% bovine serum albumin) for an additional 5 min in the dark at room temperature. The plates were then read four times in a BioTek Synergy Neo Alpha plate reader using 395 nm (RLuc8-coelenterazine 400a) and 510 nm (GFP2) emission filters and the measurements from the 10 min read were used for G protein analyses. For β-arrestin, the plates were read six times using 480 (RLuc8-coelenterazine h) and 530 nm (YFP2) emission filters and the measurements from the 15 min read were used for β- arrestin recruitment analyses. The ratio of GFP2 / RLuc8 for G protein and eYFP / RLuc for β- arrestin were calculated per well in quadruplicates and plotted as a function of drug concentration, normalized to % control agonist stimulation and analyzed using ‘‘log(agonist) vs. response’’ in GraphPad Prism 10.0.

[0242] Antagonist assays (Gα and β-arrestin signaling)

[0243] Cells were prepared as previously described. On the day of the assay, the media was removed, and cells were rinsed with 30 μL of assay buffer (20 mM HEPES, 1X HBSS, pH 7.4), followed by the addition of the 30 μL substrate buffer (7.85 μM coelenterazine 400 a for G- protein or 7.85 μM coelenterazine h for β-arrestin in assay buffer). After the addition ofsubstrate buffer, the cells were treated with 15 μL of drug / antagonist (3X drug concentration in assay buffer with 0.3% bovine serum albumin) and kept in the dark for 15 min at room temperature. After incubation, cells were treated with 15 μL of agonist DAMGO (4X drug concentration of EC80 of agonist in assay buffer with 0.3% bovine serum albumin) and the plate was kept in the dark for 5 additional minutes. The plates were then read four times in a BioTek Synergy Neo Alpha plate reader using 395 nm (RLuc8-coelenterazine 400a) and 510 nm (GFP2) emission filters and the measurements from the 10 min read were used for G protein analyses. For β-arrestin the plates were read six times using 480 (RLuc8-coelenterazine h) and 530 nm (YFP2) emission filters, and the measurements from the 15 min read were used. The ratio of GFP2 / RLuc8 for G protein and eYFP / RLuc for β-arrestin were calculated per well and plotted as a function of drug concentration, it was normalized to % agonist control stimulation and analyzed using ‘‘log(agonist) vs. response’’ in GraphPad Prism 10.0.

[0244] Tango arrestin recruitment assay

[0245] The µOR Tango constructs were designed and assays were performed as previously described. HTLA cells expressing TEV fused-β-Arrestin2 were transfected with the µOR Tango construct. The next day, cells were plated in DMEM supplemented with 1% dialyzed FBS in poly-L-lysine coated 384-well white clear bottom cell culture plates at a density of 10,000- 15,000 cells / well in a total of 40 ml. The cells were incubated for at least 6 h before receiving drug stimulation. Drug solutions were prepared in drug buffer (20 mM HEPES, 1X HBSS, 0.3% BSA, pH 7.4) at 3X and added to cells (20 ml per well) for overnight incubation. Plates were decanted and received 20 mL per well of drug buffer (20 mM HEPES, 1X HBSS) followed by addition of 10 mL of drug solution (3 wells per condition) for 15 min in the dark at room temperature. The next day, media and drug solutions were removed and 20 ml per well of BrightGlo reagent (purchased from Promega, after 1:20 dilution) was added. The plate was incubated for 20 min at room temperature in the dark before being counted using a luminescence counter. Results (relative luminescence units) were plotted as a function of drug concentration, normalized to % agonist control stimulation, and analyzed using ‘‘log(agonist) vs. response’’ in GraphPad Prism 10.0.

[0246] Nb39 assay76

[0247] HEK293T cells were transiently transfected with MOR-Rluc8 and Nb39-Venus in a 1:3 ratio, after 16 hrs cells were plated onto a poly-D-lysine coated 384-well plate in DMEM supplemented with 1% dialyzed FBS at 20,000 cells / well in 40 µl. After a further 24 hrs, cells were washed with 20 µl assay buffer (1X HBSS, 20 mM HEPES) then incubated with 20 µl per well coelenterazine h (5 µM, in assay buffer) for 5 min in the dark. Drugs were diluted to 30 µM in drug buffer (assay buffer + 0.3 g / ml BSA), 10 µl was added onto the plate (final concentrationof 10 µM in the well) and incubated for 5 min in the dark. The plate was then immediately read for luminescence at 480 nm and Venus emission at 530 nm on a BioTek Synergy Neo plate reader. The ratio of Venus / Rluc8 signal was calculated, Nb39 recruitment was calculated by subtracting the mean ratio of the wells that received DMSO vehicle control.

[0248] In vivo assays

[0249] Animals and Drug Administration

[0250] Male C57BL / 6J mice weighing 24–30 g were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Additionally, adult male, mu-opioid receptor gene-disrupted “knockout” (MOR KO; B6.129S2-Oprm1tm1Kff / J) and delta-opioid receptor gene-disrupted “knockout” (DOR KO; B6.129S2-Oprd1tm1Kff / J) mice were obtained from colonies at the University of Florida, established from homozygous breeding progenitor pairs obtained from Jackson Labs. All knockout mice are on the C57BL / 6J background for at least 13 generations. All mice were used at an age of 8–12 weeks old, and all mice were opioid naïve when tested. All mice were housed in groups of five until testing and maintained on a 12 h light / dark cycle with Purina rodent chow and water available ad libitum, in accordance with the 2011 National Institute of Health Guide for the Care and Use of Laboratory Animals. These mice were kept at a constant temperature of 22 ± 2 ºC, and relative humidity was maintained at 48–52%. All animal studies reported adhere to the ARRIVE guidelines.78All procedures were preapproved by the Institutional Animal Care and Use Committee (University of Florida). Upon the completion of testing, all mice were euthanized by inhalation of carbon dioxide, followed by cervical dislocation as a secondary measure, as recommended by the American Veterinary Medical Association.

[0251] For intracerebroventricular (i.c.v.) administration, compounds were dissolved in dimethyl sulfoxide (DMSO), followed by addition of sterile saline (0.9%) so that the final vehicle was 5% DMSO and 95% saline, and the i.c.v. administration was performed in a fixed volume of 5 μL as described previously.79Briefly, mice were anesthetized using isoflurane (0.4%). A small (3 mm) incision was made in the scalp, and a 10 μL Hamilton syringe fitted with a 27-gauge needle used to administer drug into the right lateral ventricle at the following coordinates: 2 mm caudal to bregma, 2 mm lateral to sagittal suture, and 2 mm in depth. Tail- withdrawal data points are the means of 8 mice unless otherwise indicated, with SEM shown by error bars. Note that the concentration of DMSO used was not observed to have antinociceptive or behavioral effects in previous use. All solutions for animal administration were prepared fresh daily.

[0252] Antinociception

[0253] The 55 °C warm-water tail-withdrawal assay was conducted in mice as a measure of acute thermal antinociception as described previously.74,80,81Briefly, each mouse was initially tested for baseline tail-withdrawal latency prior to drug administration, with the latency of the mouse to withdraw its tail from the water taken as the endpoint. Following drug administration, the latency for each mouse to withdraw the tail was measured every 10 min until latency returned to the baseline value. A maximum response (cut-off) time of 15 s was utilized to prevent tissue damage. If the mouse failed to display a tail-withdrawal response within 15 s, the tail was removed from the water and the animal was assigned a maximal antinociceptive score of 100%. Data are reported as percent antinociception, calculated by the equation: % antinociception = 100 × [(test latency−baseline latency) / (15−baseline latency)]. This was utilized to account for innate variability between mice. Compounds were administered intracerebroventricular route (icv) and the analgesic action of compounds was assessed at the peak effect.

[0254] Opioid receptor involvement in the agonist activity of carbamorphine was determined by testing in MOR KO and DOR KO mice as utilized recently82. The potential kappa-opioid receptor agonist activity of carbamorphine was instead determined by pretreating mice with a single dose of nor-BNI (10 mg / kg, i.p.) 24 h in advance of administration of carbamorphine, as utilized previously.83

[0255] Respiratory Depression

[0256] Respiration rates were monitored using the automated, computer controlled Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments, Columbus, OH) as described previously.84,85Awake, freely moving adult male mice (C57BL6 / J wild-type, µOR KO) were habituated in closed, sealed individual apparatus cages (23.5 cm x 11 / 5 cm x 13 cm) for 60 min before testing. A baseline for each animal was obtained over the 60-min period before drug injection, and testing began immediately post-injection. Vehicle, saline, morphine (30 nmol, icv), or (+)-carbamorphine (300 nmol, icv) were administered (icv) and five min later mice were confined to the CLAMS testing cages for 100 min. Using a pressure transducer built into the sealed CLAMS cage, the respiration rate (breaths / min) of each occupant mouse was measured. Data are expressed as percent of vehicle control response.

[0257] Conditioned Place Preference

[0258] Mice were conditioned with a counterbalanced place conditioning paradigm using similar timing as detailed previously.74,76Groups of C57BL / 6J mice (n = 18–24) freely explored a three-compartment apparatus for 30 min. The amount of time subjects spent in each compartment was measured over the 30 min testing period. Prior to place conditioning, the animals did not demonstrate significant differences in their time spent exploring the left vs rightcompartments. During each of the next 2 days, mice were administered vehicle (0.9% saline) and consistently confined in a randomly assigned outer compartment for 40 min, half of each group in the right chamber, half in the left chamber. Four hours later, mice were administered drugs morphine (30 nmol, icv), (+)-carbamorphine (30 nmol, icv), (+)-carbamophine (300 nmol, icv) or vehicle or saline and were placed to the opposite compartment for 40 min. Conditioned place preference data are presented as the difference in time spent in drug and vehicle associated chambers.

[0259] Statistical Analysis

[0260] Percent antinociception was used to determine between-group effects and to allow comparison to baseline latency in tail-withdrawal experiments. Significant differences in antinociception were analyzed by two-way ANOVA, with significant results further analyzed with Tukey’s multiple comparison post hoc test (Prism 10.0 software; GraphPad, La Jolla, CA, USA). All data are presented as mean ± SEM, with significance set at p < 0.05.

[0261] Computational Work

[0262] Ligand and Receptor Preparation

[0263] The cryo-EM structure of the morphine-bound MOR-Gi (PDB: 8EFD)86complex was obtained from the protein databank, the G protein heterotrimer, ligand and excess molecules were removed in ChimeraX.87The ligand was built using ChimeraX from its SMILES code, ChimeraX was used to protonate the ligand.

[0264] Molecular Docking

[0265] Molecular docking was carried out using AutoDock Vina.78,88The structure of morphine bound to MOR (PDB: 8EF6)86was obtained from the protein databank, the G proteins, ligand, and excess molecules were first removed, then polar only hydrogens were added using Autodock Tools. A search space was defined using Autodock Tools, focusing on the orthosteric binding site, the box was 20 x 24 x 22 Å. Torsions in the ligands remained and exhaustiveness was set to 100, the ligands were docked into MOR using Autodock Vina’s Iterated Local Search global optimizer algorithm.78The docked poses were visualized in UCSFnine potential poses were given, and the most likely pose was determined using two criteria: docking affinity and proximity between the ligand’s protonated nitrogen and the conserved residue ASP1493.32(distance of < 4 Å required). Images were generated and residues within 4 Å of the docked ligands were defined using UCSF ChimeraX .

[0266] Molecular Dynamics simulations

[0267] The ligand-receptor complex for the docked (+)-carbamorphine and MOR and the cryo- EM of (–)-morphine bound to MOR were inserted into a bilayer using CHARMM-GUI,90the bilayer for both systems had a composition of 1:5:5 for cholesterol:POPC:POPE. The ligand-receptor-bilayer was placed in a simulation box and solvated with TIP3P water and 0.15 M NaCl, the initial dimensions of the box were 90 x 90 x 80 Å for both the (–)-morphine and (+)- carbamorphine systems. Coordinate and topology files were generated using LEaP. Both systems (MOR with (–)-morphine and MOR with (+)-carbamorphine) were prepared as follow: the system was minimized first for 1000 steps with periodic boundary conditions constant and without pressure controls, followed by minimization over 10,000 steps. The system was then heated to 100 K over 2500 steps with the ligand, receptor and bilayer restrained, followed by heating from 100 K to 310 K over 50,000 steps with the ligand, receptor and bilayer restrained, under constant pressure. The system was then equilibrated with short MD trajectories in three steps, first 1 ns of MD was run with the ligand and the protein backbone retrained, then a 1 ns was run with the alpha carbons restrained, finally a 50 ns trajectory was run without restraints, this equilibrated the systems periodic boundary conditions. From this equilibrated system three replicates of 1 μs MD were initiated, using a wallclock seed to ensure that the replicates were independent. The trajectories were generated under the ff19SB91and Lipid2192AMBER forcefields,93the simulations had 2 fs timesteps and were written out every 100 ps. Temperature was controlled using the Langevin thermostat, pressure was controlled using Monte Carlo barostat, SHAKE algorithm was applied to all bonds involving a hydrogen. The three replicates for each system were combined to give 3 μs of simulation time, analysis was performed on the 3 μs datasets using CPPTRAJ,94all images were made using UCSF ChimeraX.

[0268] Supplemental References1Pramanick, P. K., et al. Tetrahedron 73, 7105–7114 (2017).2Haider, M., et al. J. Am. Chem. Soc. 143, 2710–2715 (2021).3Faouzi, A., et al. Nature 613, 767–774 (2023).4Chakraborty, S., et al. J. Med. Chem. 64, 16553–16572 (2021).5Olsen, R. H. J., et al. Nat. Chem. Biol. 16, 841–849 (2020).6Chakraborty, S., et al. J. Med. Chem. 64, 13873–13892 (2021).7DiBerto, J. F.; et al. Methods in molecular biology (Clifton, N.J.) 2022, 2525, 185.8Eberhardt, J., et al. J. Chem. Inf. Model. 61, 3891–3898 (2021).9Aldrich, J. V., et al. Br. J. Pharmacol. 171, 3212–3222 (2014).10Váradi, A., et al. J. Med. Chem. 59, 8381–8397 (2016).11McLaughlin, J. P., et al. J. Pharmacol. Exp. Ther. 289, 304–311 (1999).12Li, Y., et al. Molecules 28, (2023).13Scherrer, K. H., et al. Pharmaceuticals 16, (2023).14Uprety, R., et al. Elife, 10, 1–58 (2021).15Cirino, T. J., et al. Front. Pharmacol. 10, 678 (2019).Zhuang, Y., et al. Cell 185, 4361–4375 (2022). Meng, E. C., et al. Protein Sci. 32, 1–13 (2023). Trott, O. et al. J. Comput. Chem. 31, 455–461 (2010). Pettersen, E. F., et al.. Protein Sci. 30, 70–82 (2021). Jo, S., et al. Journal of Computational Chemistry, 29, 1859–1865 (2008). Tian, C., et al. J. Chem. Theory Comput, 16, 528–552 (2019). Dickson, C., et al. J. Chem. Theory Comput, 18, 1726–1736 (2022). Case, D. et al. Amber Tools. J. Chem. Inf. Model., 63, 6183–6191(2023). Roe, D., et al. J. Chem. Theory Comput, 9, 3084–3095 (2013).

Claims

CLAIMS 1. A carbamorphinan compound comprising an O-to-CH2exchange in the E-ring of the corresponding morphinan which comprises an epoxy group between carbons 4 and 5 (i.e., 4,5α- epoxy), thereby forming the E ring, including a pharmaceutically acceptable salt, hydrate and stereoisomer thereof.

2. A carbamorphinan compound of claim 1, comprising a ring structure:wherein rings B and C may independently comprise an unsaturation, such as the lower right bond of the C ring, as in morphine, including a pharmaceutically acceptable salt, hydrate and stereoisomer thereof.

3. A carbamorphinan compound of claim 1, comprising a structure:wherein: R1 is selected from R, OR, OCOR, D, F, Cl, Br, CN, NRR; R2 is selected from R, OR, OCOR, O, D, F, Cl, Br, CN, NRR; R3 is selected from R, OR, OCOR, D, F, Cl, Br, CN, NRR, including 2-3,3- dimethylbutan-2-ol, and 2-pentan-2-ol;R4 is selected from R, OR, OCOR, D, F, Cl, Br, CN, NRR; R5 is R, including 2-propylenyl, cyclopropylmethyl, cyclobutylmethyl; R is H, D, F, Cl, Br, CN, OH, OMe, OAc, NH2, or substituted or unsubstituted C1-C4 hydrocarbyl, including alkyl, alkenyl or alkynyl, such as methyl, ethyl, propyl, ethylenyl, propylenyl, etc., wherein substituents are selected from D, F, Cl, Br, CN, OH, OMe, OAc, NH2, and unsubstituted C1-C4 hydrocarbyl; wherein R2 and R3 or R2 and R4 maybe joined to form a ring, including a pharmaceutically acceptable salt, hydrate and stereoisomer thereof.

4. A carbamorphinan compound of claim 3, wherein: R1 is OH, OMe or OAc; R2 is OH, O or OAc; R3 is H; R4 is H, OH or OMe; and R5 is H or Me.

5. A carbamorphinan compound of claim 1, comprising a structure selected from:

6. A carbamorphinan compound of claim 1, wherein the morphinan is selected from morphine, naloxone, naloxegol, codeine, hydromorphone, oxymorphone, hydrocodone, oxycodone, dihydrocodeine, nicocodeine, heroin (diacetyl morphine), nalorphine, naltrindole, and thebaine (paramorphine), or from buprenorphine, diamorphine, ethylmorphine, dihydromorphine, desomorphine, pholcodine.

7. A carbamorphinan compound of claim 1, 2, 3, 4, 5 or 6, comprising a bioactivity profile different from the corresponding morphinan, including pain relief without an associated respiratory depression or addiction.

8. A pharmaceutical composition comprising a compound of claim 1, 2, 3, 4, 5 or 6, or a pharmaceutically acceptable salt, a hydrate or a stereoisomer thereof, and a pharmaceutically acceptable carrier or excipient, in a pharmaceutically acceptable unit dosage.

9. A method to treat or modulate a physiological condition comprising administering to a person in need thereof a compound of claim 1, 2, 3, 4, 5 or 6, such as for use an analgesic or MOR antagonist.

10. A method of claim 9, further comprising the antecedent step of detecting or diagnosing a condition indicating the need thereof, and the subsequent step of detecting a resultant modulation of the condition 11. A method of using a compound of claim 1, 2, 3, 4, 5, or 6 as an alternative to naloxone for addressing opioid overdose (e.g., from fentanyl).

12. Use of a compound of claim 1, 2, 3, 4, 5 or 6 in the manufacture of a medicament for modulating a condition, such as for use an analgesic or MOR antagonist, preferably to treat pain without addictive side effects and respiratory depression.

13. A compound of claim 1, 2, 3, 4, 5 or 6 for use in modulating a condition, preferably to treat pain without addictive side effects and respiratory depression.

14. A method comprising total synthesis of a carbamorphinan of claim 1, 2, 3, 4, 5 or 6.

15. A method of claim 14, comprising an intramolecular inverse electron-demand Diels−Alder cycloaddition and a stereoselective radical Giese addition to construct a quaternary carbon center.

Images