WO2020226628A1 - Opioid receptor antagonist prodrugs for treating opioid dependence - Google Patents

Abstract

Provided herein are prodrugs of opioid receptor antagonists such as nalmefene, pharmaceutical compositions comprising said compounds, and methods for using said compounds for the treatment of behavioral disorders.

Description

OPIOID RECEPTOR ANTAGONIST PRODRUGS

FOR TREATING OPIOID DEPENDENCE

BACKGROUND

[0001] A need exists in the medicinal arts for compositions and methods for the modulation of opioid receptor activity in the course of treating behavioral disorders.

BRIEF SUMMARY OF THE INVENTION

[0002] Provided herein are prodrugs of opioid receptor antagonists such as nalmefene and

naltrexone, pharmaceutical compositions comprising said compounds, and methods for using said compounds for the treatment of behavioral disorders.

[0003] Some compounds of the invention have superior properties. For example, some compounds of the invention have superior stabilities in oil based pharmaceutical compositions such as sesame oil or cottonseed oil.

[0004] Some compounds of the invention have better pharmacokinetic activities in vivo (for

example, rat or dog), e.g., extended half-life.

[0005] Some compounds of the invention have better safety in vivo (for example, rat or dog), e.g., diminished injection site reactions.

[0006] Some compounds of the invention have superior stability either neat or in oil based

pharmaceutical compositions.

[0007] In one aspect, provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (I),

wherein,

X is CH₂;

R is -C(O)OC₇-C₂₀ alkyl.

[0008] One embodiment provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of any one of Formula (I), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof.

[0009] Provided herein is a method of treating opioid dependence in a patient in need thereof

comprising administering a pharmaceutical composition comprising a compound of Formula (I), or a compound disclosed in Table 1, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0010] One embodiment provides a method of treating a patient wherein the therapeutic effect of a long acting opioid antagonist depot can be overcome in a patient by administering an opioid based analgesic.

[0011] One embodiment provides a method of treating opioid dependence in a patient in need

thereof, wherein the patient receives a first injection of an injectable formulation comprising a compound of any one of Formula (I), or a compound disclosed in Table 1, or a

pharmaceutically acceptable salt thereof, wherein said first injection provides a

therapeutically relevant plasma concentration for about 1 week, about 2 weeks, about 3 weeks or about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months or at least about 6 months, followed by a second injection of an injectable formulation comprising a compound of any one of Formula (I), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, wherein said second injection provides a therapeutically relevant plasma concentration for at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months or at least about 6 months.

[0012] One embodiment provides a method of treating opioid dependence in a patient in need

thereof, wherein the patient receives a first injection of an injectable formulation of naltrexone loaded PLGA microspheres that provides a therapeutically relevant plasma concentration for about 4 weeks, followed by one or more injections of an injectable formulation comprising a compound of any one of Formula (I), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, that provides a therapeutically relevant plasma concentration for about 2 months, about 3 months, about 4 months, or about 5 months or more.

[0013] One embodiment provides a method of treating opioid dependence in a patient in need

thereof, wherein the patient receives one or more injections of an injectable formulation comprising at least one compound of any one of Formula (I), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, wherein the patient has been previously treated with opioid agonists or partial agonists, such as buprenorphine or methadone, and the patients are now transitioning to discontinuation from such agonist or partial agonist treatment.

[0014] One embodiment provides a method of treating opioid dependence in a patient in need

thereof, wherein the patient receives one or more injections of an injectable formulation comprising at least one compound of any one of Formula (I), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, wherein the patient is recently addicted and naive to prior medication assisted treatment, or wherein the patient has recently discontinued opioid pain medication, are at risk of future opioid drug abuse, and are in need of prevention of future opioid drug abuse via antagonist treatment.

INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference for the specific purposes identified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings below.

The patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] Fig. 1 provides the nuclear magnetic resonance spectrum of Example 3 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl undecyl carbonate;

[0018] Fig. 2 provides the nuclear magnetic resonance spectrum of Example 6 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate;

[0019] Fig. 3 provides the nuclear magnetic resonance spectrum of Example 17 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl octyl carbonate;

[0020] Fig. 4 provides the nuclear magnetic resonance spectrum of Example 18 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate;

[0021] Fig. 5 provides the nuclear magnetic resonance spectrum of Example 19 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate; [0022] Fig. 6 provides the nuclear magnetic resonance spectrum of Example 28 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl icosyl carbonate;

[0023] Fig. 7 provides the nuclear magnetic resonance spectrum of Example 30 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl tridecyl carbonate;

[0024] Fig. 8 provides the nuclear magnetic resonance spectrum of Example 31 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl tetradecyl carbonate;

[0025] Fig. 9 provides the nuclear magnetic resonance spectrum of Example 32 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl pentadecyl carbonate;

[0026] Fig. 10 provides the nuclear magnetic resonance spectrum of Example 33 (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl octadecyl carbonate;

DETAILED DESCRIPTION OF THE INVENTION

[0027] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "the cell" includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and

subcombinations of ranges and specific embodiments therein are intended to be included.

The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary between 1% and 15% of the stated number or numerical range. The term

"comprising" (and related terms such as "comprise" or "comprises" or "having" or

"including") is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, "consist of' or "consist essentially of" the described features. Definitions

[0028] As used in the specification and appended claims, unless specified to the contrary, the

following terms have the meaning indicated below.

[0029] "Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to fifteen carbon atoms ( e.g ., Ci-C₁₅ alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms ( e.g ., C₁-C₁₃ alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., Ci-Ce alkyl). In other embodiments, an alkyl comprises one to five carbon atoms (e.g., C₁-C₅ alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (e.g., C₁-C₄ alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (e.g., C₁-C₃ alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (e.g., C₁-C₂ alkyl). In other embodiments, an alkyl comprises one carbon atom (e.g., Ci alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C₅-C₁₅ alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., Cs-Cs alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (e.g., C₂-C₅ alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (e.g., C₃-C₅ alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl («-propyl), 1- methylethyl (iso-propyl), 1-butyl («-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso butyl), 1,1-dimethylethyl (ieri-butyl), 1 -pentyl («-pentyl). The alkyl is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethyls ilanyl, -OR^a, -SR^a, -OC(0)-R^a, -N(R^a)₂, -C(0)R^a, -C(O)OR^a, -C(0)N(R^a)₂, -N(R^a)C(O)OR^a, -OC(0)-N(R^a)₂, -N(R^a)C(0)R^a, - N(R^a)S(0)_tR^a (where t is 1 or 2), -S(0)_tOR^a (where t is 1 or 2), -S(0)_tR^a (where t is 1 or 2) and -S(0)_tN(R^a)₂ (where t is 1 or 2) where each R^a is independently hydrogen, alkyl

(optionally substituted with halogen, hydroxy, methoxy, or trifluoro methyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoro methyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoro methyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoro methyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoro methyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoro methyl),

heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or

trifluoro methyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoro methyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoro methyl).

[0030] The compounds disclosed herein, in some embodiments, contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as ( R )- or (5)-. Unless stated otherwise, it is intended that all stereoisomeric forms of the compounds disclosed herein are contemplated by this disclosure. When the compounds described herein contain alkene double bonds, and unless specified otherwise, it is intended that this disclosure includes both E and Z geometric isomers ( e.g ., cis or trans.) Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. The term “geometric isomer” refers to Z? or Z geometric isomers (e.g., cis or trans) of an alkene double bond. The term“positional isomer” refers to structural isomers around a central ring, such as ortho-, meta-, and para- isomers around a benzene ring.

[0031] A "tautomer" refers to a molecule wherein a proton shift from one atom of a molecule to another atom of the same molecule is possible. The compounds presented herein, in certain embodiments, exist as tautomers. In circumstances where tautomerization is possible, a chemical equilibrium of the tautomers will exist. The exact ratio of the tautomers depends on several factors, including physical state, temperature, solvent, and pH. Some examples of tautomeric equilibrium include:

[0032] The compounds disclosed herein, in some embodiments, are used in different enriched

isotopic forms, e.g., enriched in the content of H. In one particular embodiment, the compound is deuterated in at least one position. Such deuterated forms can be made by the procedure described in U.S. Patent Nos. 5,846,514 and 6,334,997. As described in U.S. Patent Nos. 5,846,514 and 6,334,997, deuteration can improve the metabolic stability and or efficacy, thus increasing the duration of action of drugs.

[0033] In another embodiment, the compounds disclosed herein have some or all of the Ή atoms replaced with H atoms. The methods of synthesis for deuterium-containing compounds are known in the art and include, by way of non-limiting example only, the following synthetic methods.

[0034] Deuterium substituted compounds are synthesized using various methods such as described in: Dean, Dennis C.; Editor. Recent Advances in the Synthesis and Applications of

Radiolabeled Compounds for Drug Discovery and Development. [In: Curr., Pharm. Des., 2000; 6(10)] 2000, 110 pp; George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans, E. Anthony. Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1- 2), 9-32.

[0035] Deuterated starting materials are readily available and are subjected to the synthetic methods described herein to provide for the synthesis of deuterium-containing compounds. Large numbers of deuterium-containing reagents and building blocks are available commercially from chemical vendors, such as Aldrich Chemical Co.

[0036] Deuterium-transfer reagents suitable for use in nucleophilic substitution reactions, such as iodomethane-d3 (CD3I), are readily available and may be employed to transfer a deuterium- substituted carbon atom under nucleophilic substitution reaction conditions to the reaction substrate. The use of CD3I is illustrated, by way of example only, in the reaction schemes below.

[0037] Deuterium-transfer reagents, such as lithium aluminum deuteride (L1AID4), are employed to transfer deuterium under reducing conditions to the reaction substrate. The use of LiAlD4 is illustrated, by way of example only, in the reaction schemes below.

[0038] Deuterium gas and palladium catalyst are employed to reduce unsaturated carbon-carbon linkages and to perform a reductive substitution of aryl carbon-halogen bonds as illustrated, by way of example only, in the reaction schemes below.

[0039] In one embodiment, the compounds disclosed herein contain one deuterium atom. In another embodiment, the compounds disclosed herein contain two deuterium atoms. In another embodiment, the compounds disclosed herein contain three deuterium atoms. In another embodiment, the compounds disclosed herein contain four deuterium atoms. In another embodiment, the compounds disclosed herein contain five deuterium atoms. In another embodiment, the compounds disclosed herein contain six deuterium atoms. In another embodiment, the compounds disclosed herein contain more than six deuterium atoms. In another embodiment, the compound disclosed herein is fully substituted with deuterium atoms and contains no non-exchangeable *11 hydrogen atoms. In one embodiment, the level of deuterium incorporation is determined by synthetic methods in which a deuterated synthetic building block is used as a starting material.

[0040] "Pharmaceutically acceptable salt" includes both acid and base addition salts. A

pharmaceutically acceptable salt of any one of the opioid receptor antagonist prodrug compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0041] "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the

biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydro iodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoro acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S.M. et al,

"Pharmaceutical Salts," Journal of Pharmaceutical Science, 66: 1-19 (1997)). Acid addition salts of basic compounds are, in some embodiments, prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.

[0042] "Pharmaceutically acceptable base addition salt" refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts are, in some embodiments, formed with metals or amines, such as alkali and alkaline earth metals or organic amines.

Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine,

2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, /V,/V-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline,

/V-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, /V-ethylpiperidine, polyamine resins and the like. See Berge et al., supra.

[0043] As used herein,“treatment” or“treating,” or“palliating” or“ameliorating” are used

interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By“therapeutic benefit” is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient is still afflicted with the underlying disorder. For prophylactic benefit, the compositions are, in some embodiments, administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease has not been made.

Opioid Receptor Pharmacology

[0044] The opioid receptors, m, d, k, and the opioid- like receptor ORL-1 belong to the super family of G-protein coupled receptors (GPCRs) that possess seven helical trans-membrane spanning domains in their architecture. The majority of research efforts focused upon this group of proteins has been directed toward the m receptor since it mediates the actions of both the opiate and opioid analgesics such as morphine and fentanyl, respectively. However, over the years it has become increasingly clear that the entire family of proteins is actively involved in a host of biological processes. Furthermore, the advent of selective antagonists has demonstrated that pharmacotherapeutic opportunities exist via both negative and positive modulation of this receptor family.

[0045] The m (mu, OP₃ or MOP) receptor was originally defined and characterized

pharmacologically by Martin, Kosterlitz and their colleagues on the basis of its high affinity for, and sensitivity to, morphine (Martin el al. The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog J. Pharmacol. Exp. Ther. (1976), 197: 517-532; Kosterlitz, et al. Endogenous opioid peptides: multiple agonists and receptors, Nature (1977) 267: 495-499). The endogenous opioids, [Met⁵] -enkephalin, [Leu⁵] -enkephalin, extended forms of [Met⁵] -enkephalin including metorphamide and B AM- 18, b-endorphin, and truncated forms of dynorphin (e.g. dynorphin-(l-9) and shorter dynorphin peptides), also have affinities for m receptors that are consistent with a possible role for each of these peptides as natural ligands for this receptor type, although these endogenous peptides are not selective for m receptors. Two putative natural ligands, endomorphin-1 and -2, that appear to mediate their effects exclusively through the m opioid receptor, also have been reported to be present in brain although no gene, precursor protein, or other mechanism for their endogenous synthesis has been identified.

[0046] The m receptors are distributed throughout the neuraxis. The highest m receptor densities are found in the thalamus, caudate putamen, neocortex, nucleus accumbens, amygdala, interpeduncular complex, and inferior and superior colliculi (Watson et al. Autoradiographic differentiation of mu, delta and kappa receptors in the rat forebrain and midbrain, J. Neurosci. (1987), 7: 2445-2464). The m receptors, as well as d and k receptors, are also present in the superficial layers of the dorsal horn of spinal cord. A moderate density of m receptors is found in periaqueductal gray and raphe nuclei. These brain regions have a well-established role in pain and analgesia. Other physiological functions regulated by m receptors include respiratory and cardiovascular functions, intestinal transit, feeding, mood, thermoregulation, hormone secretion and immune functions.

[0047] The d (delta, OPi or DOP) opioid receptor was defined using the mouse vas deferens

preparation and the enkephalins are generally considered the preferred endogenous ligands. The d receptors are discretely distributed in the central nervous system (CNS), with a prominent gradient of receptor density from high levels in forebrain structures to relatively low levels in most hindbrain regions. The highest densities are found in olfactory bulb, neocortex, caudate putamen, nucleus accumbens, and amygdala (Watson et al.

Autoradiographic differentiation of mu, delta and kappa receptors in the rat forebrain and midbrain, J. Neurosci. (1987), 7: 2445-2464). The thalamus and hypothalamus have a moderate density of d receptors; in more caudal regions the interpeduncular nucleus and pontine nuclei show high binding in rat, but much lower levels in mouse (Kitchen et al.

Quantitative autoradiographic mapping of mu, delta and kappa-opioid receptors in knockout mice lacking the mu-opioid receptor gene, Brain Res. (1997), 778: 73-88). In the spinal cord, d receptors are present in dorsal horn where they play a role in mediating the analgesic effects of d agonists.

[0048] The k (kappa, OP2 or KOP) opioid receptor was first proposed on the basis of in vivo studies in dogs with ketocyclazocine and related drugs (Martin et al. The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog J. Pharmacol. Exp. Ther. (1976), 197: 517-532). Subsequent studies have confirmed the presence of this receptor type in other species including guinea pig, a species that was preferred for many of the early studies on kappa opioid receptors. Dynorphins A and B and a- neoendorphin appear to be the endogenous ligands for opioid k receptors, although shorter peptides derived from prodynorphin have comparable affinities at m and k receptors. The k receptors are located predominantly in the cerebral cortex, nucleus accumbens, claustrum and hypothalamus of rat and mouse (Kitchen et al. Quantitative autoradiographic mapping of mu, delta and kappa-opioid receptors in knockout mice lacking the mu-opioid receptor gene,

Brain Res. (1997), 778: 73-88; Watson et al. Autoradiographic differentiation of mu, delta and kappa receptors in the rat forebrain and midbrain, J. Neurosci. (1987), 7: 2445-2464), and have been implicated in the regulation of nociception, diuresis, feeding, neuroendocrine and immune system functions (Dhawan et al. International Union of Pharmacology. XII. Classification for opioid receptors, Pharmacol. Rev. (1996), 48: 567-592).

[0049] ORL1 receptors (also called nociceptin, or orphaninFQ receptors) are the youngest members of the opioid receptor family. Agonist-induced internalization of ORL1 is rapid and concentration dependent. Agonist challenge also reduces the ability of ORL1 to couple to inhibition of forskolin- stimulated cAMP production, suggesting that ORL1 undergoes similar desensitization mechanisms as compared with the other three opioid receptors subtypes.

[0050] The structure of the ORL1 receptor indicates that it has evolved as part of the opioid receptor family. Sequence comparisons with m, k, and d receptors, and with other similar G protein- coupled receptors ( e.g . of the SOM receptor family), indicate that the ORL1 receptor is more closely related to opioid receptors than to other types of G protein-coupled receptors (Birgul, et al. Reverse Physiology in drosophila: identification of a novel allato statin- like

neuropeptide and its cognate receptor structurally related to the mammalian

somatostatin/galanin/opio id receptor family. EMBO J. (1999), 18: 5892-5900). Additionally, agonists at ORL1 receptors induce activation of the same set of transduction pathways activated by m, k, and d receptors, and the endogenous ligand, ORL1, shares considerable sequence homology with dynorphin A and, to a lesser extent, with the enkephalins. Thus, the ORL1 receptor and its endogenous ligand are closely related in an evolutionary sense to the m, K, and d receptors.

[0051] Despite the evidence of evolutionary and functional homology, the ORL1 receptor is not an opioid receptor from a pharmacological perspective. The effects of activation of this receptor are not obviously 'opiate-like' with respect to pain perception. The ORL1 receptor has negligible affinity for naloxone and for most other antagonists at m, k or d receptors. The ORL1 receptor is, however, expressed in many functional systems in which endogenous opioids play a regulatory role. Although the functions of ORL1 are not yet fully understood, regulatory functions for ORL1 parallel to but not identical to those of the endogenous opioid peptides seem very probable. Despite these functional differences, the subcommittee finds the structural relationship between the ORL1 receptor and m, d and k receptors compelling.

[0052] ORL1 receptor regulation, while increasingly studied, is still in the infant stages of

understanding when compared to the other three opioid receptor subtypes. To date few site- directed mutagenesis studies have been conducted, and receptor regulation in primary neurons, dorsal root ganglion, or dorsal horn neurons remains unknown. [0053] An integral part of the effort to characterize the opioid receptor system has been the discovery of potent, pure antagonists of opioid receptors. Nalmefene (la) and naltrexone (lb), both competitive antagonists at m, d, and k opioid receptors, were used as

pharmacological tools to identify and characterize opioid systems.

[0054] Nalmefene is an opioid receptor antagonist that has been available for several years as

Revex® injection for use in reversing opioid effects and for opioid overdose. Nalmefene is also described in literature for the treatment of substance abuse disorders such as alcohol dependence and abuse, and impulse control disorders such as pathological gambling and addiction to shopping. It is marketed as Selincro in Europe as an on demand oral pill for alcohol abuse. It has the IUPAC name 17-cyclopropylmethyl-4,5a-epoxy-6- methylenemorphinan-3,14-diol and has the structure provided in Formula (1A).

[0055] Naltrexone is an opioid receptor antagonist used primarily in the management of alcohol dependence and opioid dependence. It is marketed in the generic form as its hydrochloride salt, naltrexone hydrochloride under the trade names Revia® and Depade® in the form of 50mg film coated tablets. Once monthly extended release naltrexone, marketed in the United States as Vivitrol, has gained wide acceptance in opioid use disorder due to increased patient adherence. Naltrexone has the IUPAC name 17-(cyclopropylmethyl)-4,5a-epoxy-3, 14- dihydro xymorphinan-6-one and has the structure provided in Formula (1B)

[0056] Low doses of naltrexone have also been investigated in patients with multiple sclerosis, autism, active Crohn's disease, AIDS, rheumatoid arthritis, celiac disease, certain forms of cancer, and autoimmune diseases. Opioids act as cytokines, the principal communication signallers of the immune system, creating immunomodulatory effects through opioid receptors on immune cells. Very low doses of naltrexone were shown to boost the immune system and helps to fight against diseases characterized by inadequate immune function. [0057] In terms of pharmacology, naltrexone blocks the effects of opioids by its highly competitive binding at the m-opioid receptors. Being a competitive antagonist, the suppression of an opiate's agonistic, euphorigenic effect can be overcome. However, clinical studies have indicated that naltrexone in an oral dosage of approximately 50 mg is able to block the pharmacological effects of up to 25 mg of intravenously administered heroin for periods as long as twenty four hours.

[0058] The mechanism of action of naltrexone in the treatment of alcoholism is not understood

although involvement of the endogenous opioid system is suggested by preclinical data. Opioid antagonists have been shown to reduce alcohol consumption by animals,

and naltrexone has shown efficacy in maintaining abstinence in clinical studies in humans.

Opioid Receptor Antagonists Prodrugs

[0059] Although using nalmefene and naltrexone in the treatment of alcohol dependence and opioid dependence provides a great benefit to the society, the problem with these drugs is that they have very short period of action. Thus, for example, well absorbed orally (approximately 96% of an oral dose is absorbed from the gastrointestinal tract), naltrexone is subject to significant first pass metabolism with oral bio availability estimates ranging from 5% to 40%. The activity of naltrexone is believed to be as a result of both naltrexone and its 6-b-naltrexol metabolite. Two other minor metabolites are 2 - hydrox- 3y - methoxy- 6- -naltrexol and 2- hydroxy-3-methyl-naltrexone. Peak plasma levels of both naltrexone and 6-b-naltexol occur within one hour after oral dosing; mean elimination half-life values for naltrexone and 6-b- naltrexol are four and thirteen hours respectively. Even for long acting naltrexone injections, clinicians indicate that patients discontinue treatment too early. Therefore, a need exists for ultra-long acting opioid antagonists in the treatment of substance abuse disorder.

[0060] One of the solutions to overcome the problem of short period of action of nalmefene and naltrexone is to use prodrugs which provide a long, sustained, and controlled release of nalmefene and naltrexone opioid receptor antagonists upon administration into the body.

[0061] As used in this disclosure, the term "prodrug" is meant to indicate a compound that is

converted under physiological conditions to nalmefene or naltrexone. A prodrug, in some embodiments, is inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis. Thus, the term "prodrug" refers to a precursor compound that is pharmaceutically acceptable, and in some embodiments, is devoid of the pharmacological properties of nalmefene or naltrexone. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). [0062] A discussion of prodrugs is provided in Higuchi, T., et al., "Pro-drugs as Novel Delivery

Systems," A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0063] The term "prodrug" is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject.

Prodrugs of nalmefene or naltrexone, as described herein, are prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved to the parent active compound. Prodrugs include compounds wherein a hydroxy group is bonded to any group that, when the prodrug of the active compound is administered to a mammalian subject, cleaves to form a free hydroxy group.

[0064] Provided herein are prodrugs of opioid receptor antagonists nalmefene and naltrexone.

[0065] In one aspect, provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (I),

wherein,

X is CH₂;

R is -C(O)OC₇-C₂₀ alkyl.

[0066] In some embodiments, R is -C(O)OC₁₂-C₁₃ alkyl. In some embodiments, R is -C(O)OC₁₂- C₁₄ alkyl. In some embodiments, R is -C(O)OC₁₂-C₁₅ alkyl. In some embodiments, R is -C(O)OC₁₂-C₁₆ alkyl. In some embodiments, R is -C(O)OC₁₂-C₁₇ alkyl. In some embodiments, R is -C(O)OC₁₂-C₁₈ alkyl. In some embodiments, R is -C(O)OC₁₂-C₁₉ alkyl. In some embodiments, R is -C(O)OC₁₂-C₂₀ alkyl. In some embodiments, R is -C(O)OC₁₃- Ci alkyl. In some embodiments, R is -C(O)OC₁₃-C₁₅ alkyl. In some embodiments, R is - C(O)OC₁₃-C₁₆ alkyl. In some embodiments, R is -C(O)OC₁₃-C₁₇ alkyl. In some

embodiments, R is

-C(O)OC₁₃-C₁₈ alkyl. In some embodiments, R is -C(O)OC₁₃-C₁₉ alkyl. In some embodiments, R is -C(O)OC₁₃-C₂₀ alkyl. In some embodiments, R is -C(O)OC₁₄-C₁₅ alkyl. In some embodiments, R is -C(O)OC₁₄-C₁₆ alkyl. In some embodiments, R is -C(O)OC₁₄- Ci alkyl. In some embodiments, R is -C(O)OC₁₄-C₁₈ alkyl. In some embodiments, R is - C(O)OC₁₄-C₁₉ alkyl. In some embodiments, R is -C(O)OC₁₄-C₂₀ alkyl. In some embodiments, R is

-C(O)OC₁₅-C₁₆ alkyl. In some embodiments, R is -C(O)OC₁₅-C₁₇ alkyl. In some embodiments, R is -C(O)OC₁₅-C₁₈ alkyl. In some embodiments, R is -C(O)OC₁₅-C₁₉ alkyl. In some embodiments, R is -C(O)OC₁₅-C₂₀ alkyl. In some embodiments, R is -C(O)OC₁₆- Ci7 alkyl. In some embodiments, R is -C(O)OC₁₆-C₁₈ alkyl. In some embodiments, R is - C(0)0C₁₆-Ci9 alkyl. In some embodiments, R is -C(O)OC₁₆-C20 alkyl. In some

embodiments, R is

-C(O)OC₁₇-C₁₈ alkyl. In some embodiments, R is -C(O)OC₁₇-C₁₉ alkyl. In some embodiments, R is -C(O)OCi7-C20 alkyl. In some embodiments, R is -C(O)OC₁₈-C₁₉ alkyl. In some embodiments, R is -C(O)OC₁₈-C₂₀ alkyl. In some embodiments, R is -C(O)OC₁₉- C20 alkyl. In some embodiments, R is -C(O)OC₁₂ alkyl. In some embodiments, R is - C(O)OC₁₃ alkyl. In some embodiments, R is -C(O)OC₁₄ alkyl. In some embodiments, R is - C(O)OC₁₅ alkyl. In some embodiments, R is -C(O)OC₁₆ alkyl. In some embodiments, R is - C(O)OC₁₇ alkyl. In some embodiments, R is -C(O)OC₁₈ alkyl. In some embodiments, R is - C(O)OC₁₉ alkyl. In some embodiments, R is -C(O)OC₂₀ alkyl.

[0067] In some embodiments, the opioid receptor antagonist prodrug compound described herein has a structure provided in Table 1.

[0068] In some embodiments, the opioid receptor antagonist prodrug compound described herein has a structure provided in Table 2.

TABLE 2

Preparation of Compounds

[0069] The compounds used in the reactions described herein are made according to organic

synthesis techniques known to those skilled in this art, starting from commercially available chemicals and/or from compounds described in the chemical literature. "Commercially available chemicals" are obtained from standard commercial sources including Acros Organics (Pittsburgh, PA), Aldrich Chemical (Milwaukee, WI, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park, UK), Avocado Research (Lancashire, U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester, PA), Crescent Chemical Co. (Hauppauge, NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, PA), Fisons Chemicals (Leicestershire,

UK), Frontier Scientific (Logan, UT), ICN Biomedicals, Inc. (Costa Mesa, CA), Key Organics (Cornwall, U.K.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. Ltd.

(Cornwall, U.K.), Parish Chemical Co. (Orem, UT), Pfaltz & Bauer, Inc. (Waterbury, CN), Polyorganix (Houston, TX), Pierce Chemical Co. (Rockford, IL), Riedel de Haen AG

(Hanover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland, OR), Trans World Chemicals, Inc. (Rockville, MD), and Wako Chemicals USA, Inc. (Richmond, VA).

[0070] Suitable reference books and treatise that detail the synthesis of reactants useful in the

preparation of compounds described herein, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A.

Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, "Heterocyclic Chemistry", 2nd Ed., John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure", 4th Ed., Wiley-Interscience, New York, 1992. Additional suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds described herein, or provide references to articles that describe the preparation, include for example, Fuhrhop, J. and Penzlin G. "Organic Synthesis: Concepts, Methods, Starting Materials", Second, Revised and Enlarged Edition (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman, R.V. "Organic Chemistry, An Intermediate Text" (1996) Oxford University Press, ISBN 0-19-509618-5; Larock, R. C. "Comprehensive Organic Transformations: A Guide to Functional Group Preparations" 2nd Edition (1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J. "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure" 4th Edition (1992) John Wiley & Sons, ISBN: 0-471-60180-2; Otera, J. (editor) "Modern Carbonyl Chemistry" (2000) Wiley- VCH, ISBN: 3-527-29871-1; Patai, S. "Patai's 1992 Guide to the Chemistry of Functional Groups" (1992) Interscience ISBN: 0-471-93022-9; Solomons, T. W. G. "Organic Chemistry" 7th Edition (2000) John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, J.C., "Intermediate Organic Chemistry" 2nd Edition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; "Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann's Encyclopedia" (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8 volumes; "Organic Reactions" (1942-2000) John Wiley & Sons, in over 55 volumes; and "Chemistry of Functional Groups" John Wiley & Sons, in 73 volumes.

[0071] Specific and analogous reactants are optionally identified through the indices of known

chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (contact the American Chemical Society, Washington, D.C. for more details). Chemicals that are known but not commercially available in catalogs are optionally prepared by custom chemical synthesis houses, where many of the standard chemical supply houses ( e.g ., those listed above) provide custom synthesis services. A reference for the preparation and selection of pharmaceutical salts of the opioid receptor antagonist prodrug compounds described herein is P. H. Stahl & C. G. Wermuth "Handbook of Pharmaceutical Salts",

Verlag Helvetica Chimica Acta, Zurich, 2002.

[0072] Nalmefene can be produced from naltrexone by the Wittig reaction. The Wittig reaction is a well-known method within the art for the synthetic preparation of olefins (Georg Wittig, Ulrich Schollkopf (1954).“ Ober Triphenyl-phosphin-methylene als olefinbildende

Reagenzien 7”. Chemische Berichte 87: 1318), and has been widely used in organic synthesis.

[0073] The procedure in the Wittig reaction can be divided into two steps. In the first step, a

phosphorus ylide is prepared by treating a suitable phosphonium salt with a base. In the second step the ylide is reacted with a substrate containing a carbonyl group to give the desired alkene.

[0074] The present invention discloses a method for preparing nalmefene from naltrexone in a

Wittig reaction wherein tetrahydrofuran (THF) is used both in the formation of a phosphorus ylide and in the subsequent reaction between the ylide and naltrexone.

[0075] In a method according to the present invention the Wittig reaction may be performed by

mixing a methyltriphenylphosphonium salt with tetrahydrofuran (THF) and a suitable base to afford the ylide methylene triphenylphosphorane:

[0076] The preformed ylide is subsequently reacted‘in situ’ with naltrexone to give nalmefene and triphenylphosphine oxide (TPPO):

[0077] Accordingly, one embodiment of the present invention relates to a method for preparing

nalmefene using the basic principles of the Wittig reaction, the method comprising the steps of

a) preparing a phosphorus ylide, such as a methylene phosphorus ylide, such as methylene triphenylphosphorane, by mixing a suitable phosphonium salt, such as a methyl triphenyl phosphonium salt, with THF and a suitable base, and b) adding a mixture comprising naltrexone and THF to the mixture obtained in step a) in order to obtain nalmefene.

[0078] In step a), one or more of the phenyl groups in the phosphorus ylide can be substituted with groups that do not interfere with the Wittig reaction, e.g., C₁-C₄ alkyl, C₁-C₄ alkoxy, or halogen.

[0079] It is envisaged that steps a) and b) may be performed simultaneously in the same vessel or step a) and b) may be performed sequentially.

[0080] In a preferred embodiment, the phosphonium salt used in step a) is a

methyltriphenylphosphonium halide, such as the chloride, bromide or iodide, and more preferably is methyltriphenylphosphonium bromide (MTPPB).

[0081] The phosphonium salt, preferably MTPPB, is usually suspended in THF in an excess relative to naltrexone added in step b). Typical molar ratio ranges are from about 1:1 to about 4:1, more preferably about 3:1 (3.1:1), of methyltriphenylphosphonium salt relative to naltrexone. [0082] The amount of THF relative to methyltriphenylphosphonium salt, preferably MTPPB, used in step a) is about (v/w) 1:1 to about 4:1, preferably about 2:1.

[0083] The methyltriphenylphosphonium salt is treated with a base, such as, alkoxide bases (e.g., KO-t-Bu), amide bases (e.g., diisopropylamide), hydroxide bases (e.g., NaOH), or NaH, in order to obtain the ylide as a reagent for step b). In a preferred embodiment, the base is used in an equimolar (or 1.1 equimolar) quantity relative to the methyltriphenylphosphonium salt.

[0084] Relative to Naltrexone, the molar ratio of the base used in step a) is from about 1:1 to about 4:1, preferably about 3:1 (3.4:1).

[0085] The resulting mixture obtained in step a) is suitably stirred for at least 1 hour, e.g., for about two hours.

[0086] In step b), naltrexone as anhydrous solid or as an anhydrous solution in THF is added to the mixture comprising the ylide obtained in a).

[0087] In a preferred embodiment, an anhydrous solution of naltrexone in THF is added to the pre formed ylide. The amount (v/w) of THF relative to naltrexone may range from about 2: 1 to about 6:1, such as about 3:1 to about 5:1, or about 4:1.

[0088] The mixture obtained in step b), comprising Naltrexone, is then suitably stirred for at least 1 hour, such as from about 2 to about 16 hours, from about 2 to about 10 hours or from about 2 to about 7 hours, in order to complete the conversion of naltrexone into nalmefene. Typically, it is substantially complete within 5-7 hours, for example, 6 hours.

[0089] The overall reaction [ i.e . step a) and step b)] may be performed at a temperature in the range from about 5 to about 50° C., such as between 20 and 25° C.

[0090] Separation of nalmefene from the phosphine oxide by-products (such as TPPO) formed

during the Wittig reaction and during the work-up needs to be performed in order to obtain pure nalmefene. The method of the present invention is therefore also advantageous in that it has been especially adapted to:

1) remove efficiently and selectively the phosphorus oxide by-products (TPPO and related compounds); and

2) permit the isolation of the product directly from the reaction mixture and to transform it into the desired pharmaceutical salt form (i.e. nalmefene HC1) in a single step.

[0091] Therefore, there is no need to carry out a separate salt formation step (as is often the case in the prior art methods) which results in loss of end product (nalmefene HC1).

[0092] Furthermore, the isolation of nalmefene as the hydrochloride salt instead of the free base is convenient from an operational point of view, as the hydrochloride salt is the desired pharmaceutical salt form. It has also been found that the chemical purity is highly improved in the salt formation by the method of the present invention. In fact, the impurities remain selectively dissolved in the mother liquors, thus allowing isolation of the product in a highly pure form.

[0093] Since the Wittig reaction and the salt formation steps are combined in the method of the

present invention, the yield of nalmefene HC1 from naltrexone is excellent.

[0094] The invention therefore also relates to a method for isolating nalmefene obtained in step b) above, which method comprise the steps of;

c) removing THF by concentrating the reaction mixture from step b) above to obtain a residue;

d) diluting the residue obtained from step c) with an aqueous solution comprising ammonium chloride (NH₄CI);

e) adding an organic solvent (e.g., ethyl acetate (EtOAc) and/or dichloromethane) to the solution obtained from step d);

f) separating the organic phase obtained in step e),

g) optionally washing the organic phase obtained in f) with water and separating the organic phase,

h) concentrating the organic phase obtained in step f) or g) under vacuum to remove volatiles,

i) diluting the residue obtained in step h) with one or more suitable organic solvents (e.g., dichloromethane),

j) adding hydrogen chloride (HC1) to the mixture obtained in step i),

k) isolating the resulting solid,

l) optionally, re- slurrying the solid obtained in step i) in one or more appropriate solvents (e.g., dichloromethane and/or acetone) and isolating the solid, and

m) optionally drying the final solid.

[0095] The final residue obtained in step h) may be dissolved in a suitable organic solvent (step i). A suitable solvent is one which can keep triphenylphosphine oxide and related phosphine oxides in solution, which permits the preparation of the hydrochloride salt of nalmefene, as well as its precipitation. Suitable solvents include halogenated hydrocarbons, alcohols, ethers, ketones, esters and aromatic hydrocarbons. Preferred solvents are acetone, ethyl acetate,

THF, 2-propanol, toluene or dichloromethane, or a combination thereof. Preferably, dichloromethane is used. [0096] The organic solution is then treated with hydrogen chloride (HC1) to precipitate nalmefene as the hydrochloride salt (step j). The acid can be added as a gas or as concentrated aqueous solution of hydrochloric acid. When using hydrochloric acid, the concentration of HC1 is usually from about 30 to about 37% in water, more preferably about 37% in water.

[0097] The hydrochloride salt formation is carried out at a temperature in the range of from about 0 to about 40° C., preferably 20-30° C., under vigorous stirring.

[0098] The product crystallizes out during the addition of the acid. Phosphine oxides might be

entrapped in the crystalline product, and therefore it is convenient to maintain the suspension under stirring for at least 1 hour, such as between about 1 hour and about 5 hours, or between about 1 hour and about 3 hours.

[0099] The resulting solid may then be isolated e.g. by filtering off and washing the product (step k) with appropriate solvents, such as halogenated hydrocarbons, alcohols, ethers, ketones, esters or aromatic hydrocarbons. Preferred solvents are acetone, ethyl acetate, THF, 2-propanol, toluene or dichloromethane or a combination thereof. Preferably, dichloromethane is used.

[00100] If necessary the product may be re-slurried (step 1) in an appropriate solvent chosen from the solvents listed above in order to further remove the phosphine oxide by-products, and the nalmefene hydrochloride may then be filtered off and washed with appropriate solvents, as mentioned above. A preferred solvent for this last step is dichloromethane. The product may finally be dried e.g. under vacuum.

[00101] Nalmefene HC1 obtained according to the method of the present invention can be transformed into a form more suitable for pharmaceutical formulation, such as the dihydrate. Nalmefene HC1 prepared by the above-described Wittig process may be transformed into nalmefene HC1 dihydrate by recrystallization from aqueous solution.

[00102] A further aspect of the present invention thus relates to a method for obtaining nalmefene HC1 dihydrate, which method may comprise the steps of:

(1) mixing nalmefene hydrochloride, obtained in step k, 1 or m as described above, and water,

(2) heating the mixture to obtain a substantially homogenous solution,

(3) optionally removing volatiles from the solution obtained in step (2),

(4) cooling the solution obtained in step (2) or (3) and then seeding the solution with nalmefene HC1, and

(5) isolating the resulting solid.

[00103] In the present invention, the term“substantially homogenous solution” is intended to mean a liquid mixture free of visible undissolved material. [00104] The amount of aqueous solution, such as water, which is used in step 1) may range from about 0.9 ml to about 4 ml water per gram nalmefene hydrochloride, such as from about 1 ml to about 2 ml water per gram nalmefene hydrochloride, or about 1.5 ml water per gram nalmefene hydrochloride.

[00105] The suspension may be heated until a substantially homogenous solution is obtained. The heating in step 2) may be performed to obtain a temperature of from about 50° C. to about 100° C., such as from about 50° C. to about 90° C., or from about 70° C. to about 85° C.

[00106] Partial vacuum may then be applied to remove traces of organic volatiles, if present, in step

3).

[00107] The solution obtained from either step 2) or step 3) may optionally be filtered ( e.g . through a 0.65 pm cartridge) to remove foreign matter before proceeding to step 4).

[00108] In step 4), the solution may be cooled to a temperature between 40° C. to 60° C., such as between 40° C. and about 50° C., and seeded with Nalmefene HC1. Preferably Nalmefene HC1 dihydrate is used as seeding material.

[00109] In the present invention, the term“seeding” is intended to mean the addition of a small

amount of crystalline solid in order to initiate the precipitation of the product.

[00110] The amount of seed crystals added in step 4) may be from about 1/2000 (w/w) of seed crystal of nalmefene HCl/nalmefene HC1 added in step 1), such as from about 1/1000 (w/w) of seed crystal or 1/200 of seed crystal of Nalmefene HCl/Nalmefene HC1 added in step 1).

[00111] Rapid cooling and vigorous stirring prevent the crystals that are already formed from growing further, and help to achieve a product with a well-defined, narrow size range and relatively small particle size. The cooling from seeding temperature to isolation temperature may be performed over a period of a few hours, preferably within 1 hour. The seeded mixture obtained in step 4) may thus suitably be subjected to a fast cooling procedure which comprises the steps of:

(4') further cooling of the mixture to a temperature of about 0-5° C. over a time period of about 45 minutes or more, and

(4") maintaining the resulting mixture at a temperature of about 0-5° C. for about 45 minutes or more,

before isolating the formed solid according to step 5).

[00112] The solid formed in step 5) may be isolated at a temperature within the range of about 0-20° C., more preferably in the range of 0-5° C., in order to minimize the solubility of the product in water and to increase the yield. The solid may be isolated by filtration and washed with a suitable solvent. Suitable solvents for washing include water, mixtures of water and organic solvents, and pure organic solvents. Preferably, water is used, and in a further embodiment pre-cooled water is preferred. When organic solvents are used, Class 2 or 3 solvents are preferred, more particularly acetone.

[00113] The product may suitably be dried under vacuum at a temperature below 40° C., more

preferably at a temperature in the range 25-35° C.

[00114] The product obtained will typically be at least 98% chemically pure, such as at least 99% chemically pure, or at least 99.5% chemically pure. The term chemically pure in this context has its normal meaning within the art, and chemical purity may be determined by e.g. HPLC.

[00115] The present invention also relates to a pharmaceutical composition comprising nalmefene hydrochloride obtained by the present method. The pharmaceutical composition may further comprise at least one pharmaceutically acceptable excipient, carrier and/or diluent, and may be in a solid dosage form, such as a tablet, for oral administration.

Pharmaceutical Compositions

[00116] In certain embodiments, the opioid receptor antagonist prodrug compound as described

herein is administered as a pure chemical. In other embodiments, the opioid receptor antagonist prodrug compound described herein is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21^st Ed. Mack Pub. Co., Easton, PA (2005)).

[00117] Provided herein is a pharmaceutical composition comprising at least one opioid receptor antagonist prodrug compound, or a stereoisomer, pharmaceutically acceptable salt, hydrate, solvate, or N-oxide thereof, together with one or more pharmaceutically acceptable carriers. The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient ( i.e the subject) of the composition.

[00118] One embodiment provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of any one of Formula (I), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof.

[00119] In certain embodiments, the opioid receptor antagonist prodrug compound as described by any one of Formula (I), or a compound disclosed in Table 1, is substantially pure, in that it contains less than about 5%, or less than about 1%, or less than about 0.1%, of other organic small molecules, such as unreacted intermediates or synthesis by-products that are created, for example, in one or more of the steps of a synthesis method.

[00120] Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. In some embodiments, suitable nontoxic solid carriers are used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. (See, e.g., Remington: The Science and Practice of Pharmacy (Gennaro, 21^st Ed. Mack Pub. Co.,

Easton, PA (2005)).

[00121] In some embodiments, the opioid receptor antagonist prodrug compound as described by any one of Formula (I), or a compound disclosed in Table 1, is formulated for administration by injection. In some instances, the injection formulation is an aqueous formulation. In some instances, the injection formulation is a non-aqueous formulation. In some instances, the injection formulation is an oil-based formulation, such as sesame oil, cottonseed oil, or the like.

[00122] The dose of the composition comprising at least one opioid receptor antagonist prodrug

compound as described herein differ, depending upon the patient's (e.g., human) condition, that is, general health status, age, and other factors.

[00123] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity. Optimal doses are generally determined using experimental models and/or clinical trials. The optimal dose depends upon the body mass, weight, or blood volume of the patient.

Dosing and Therapeutic Regimens

[00124] In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic applications. In some embodiments, the pharmaceutical composition is administered once per day, twice per day, three times per day, four times per day or more.

The pharmaceutical composition is administered daily, every day, every alternate day, two days a week, three days a week, four days a week, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or other greater or lesser intervening frequency; also, it could be dosed once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once yearly, or with greater or lesser than aforementioned interval frequency. The pharmaceutical composition is administered for at least 1 week, 2 weeks, 1 month, 2 months,

3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[00125] In the case wherein the patient’s status does not improve, upon the physician’s discretion the administration of the composition is given continuously; alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time ( i.e ., a“drug holiday”). In some instances, the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days,

5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, 365 days, or 366 days. The dose reduction during a drug holiday is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,

45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[00126] Once improvement of the patient's conditions has occurred, a maintenance dose is

administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be adjusted, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.

[00127] In some embodiments, the amount of given opioid receptor antagonist prodrug compound varies depending upon factors such as the particular compound, the severity of the disease, the identity ( e.g ., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[00128] In some embodiments, the amount of given opioid receptor antagonist prodrug compound will typically be in the range of about 0.02 mg to about 5000 mg per dose. (Note: all prodrug mass quantities are expressed in base moiety equivalents). In some embodiments, the amount of given opioid receptor antagonist prodrug compound is in the range of about 1 mg to about 5000 mg per dose. In some embodiments, the amount of given opioid receptor antagonist prodrug compound is in the range of about 10 mg to about 1600 mg per dose. The desired dose may conveniently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[00129] In some embodiments, the daily dosages appropriate for the opioid receptor antagonist

prodrug compound described herein are from about 0.01 mg/kg to about 30 mg/kg. In one embodiment, the daily dosages are from about 0.1 mg/kg to about 165 mg/kg. An indicated daily dosage in the larger mammal, including, but not limited to, humans, is in the range from about 0.5 mg to about 1000 mg, conveniently administered in a single dose or in divided doses. Suitable unit dosage forms for intramuscular administration include from about 1 to about 5000 mg active ingredient. In one embodiment, the unit dosage is about 10 mg, about 50 mg, about, 100 mg, about 200 mg, about 500 mg, about 1000 mg, about 2000 mg, about 2500 mg, about 4000 mg, or about 5000 mg.

[00130] The foregoing ranges are merely suggestive, as the number of variables in regard to an

individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages may be altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

Treatment of Behavioral Disorders

[00131] In some embodiments, described herein is a method of treating one or more medical

conditions in a subject in need thereof, comprising administering to the subject in need thereof an opioid receptor antagonist compound described herein.

[00132] In some embodiments, the medical condition is selected from the group comprising opioid dependence, alcohol dependence, drug addiction, polydrug addiction and pain.

[00133] In some embodiments, described herein is an opioid receptor antagonist compound for use in reduction of opioid consumption in a patient with opioid dependence.

[00134] In some embodiments, described herein is an opioid receptor antagonist compound for use in reduction of alcohol consumption in a patient with alcohol dependence, pathological gambling shopping addiction or other diseases of compulsive behavior.

[00135] Provided herein is a method of treating opioid dependence in a patient in need thereof

comprising administering a pharmaceutical composition comprising a compound of Formula (I), or a compound disclosed in Table 1, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Provided herein is the method wherein the

pharmaceutical composition is administered orally. Provided herein is the method wherein the pharmaceutical composition is administered by injection. Provided herein is the method wherein the pharmaceutical composition is administered by intramuscular injection. Provided herein is the method wherein the intramuscular injection is a depot injection. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of 2 days to 3 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 2 days. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 4 days. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 7 days. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 10 days. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 1 week. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 2 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 3 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 4 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 5 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 6 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 1 month. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 2 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 3 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 4 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 5 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 6 months or greater.

[00136] Other embodiments and uses will be apparent to one skilled in the art in light of the present disclosures. The following examples are provided merely as illustrative of various embodiments and shall not be construed to limit the invention in any way. EXAMPLES

I. Chemical Synthesis

[00137] Unless otherwise noted, reagents and solvents were used as received from commercial

suppliers. Anhydrous solvents and oven-dried glassware were used for synthetic

transformations sensitive to moisture and/or oxygen. Yields were not optimized. Reaction times are approximate and were not optimized. Column chromatography and thin layer chromatography (TLC) were performed on silica gel unless otherwise noted. Spectra are given in ppm (d) and coupling constants, J are reported in Hertz. For proton spectra the solvent peak was used as the reference peak.

[00138] In some embodiments, opioid receptor antagonists prodrug compounds disclosed herein are synthesized according to the following examples.

[00139] General Scheme 1 for the Synthesis of Nalmefene Prodrugs.

Scheme 1

[00140] Scheme 2 for the Synthesis of Nalmefene from Naltrexone.

Scheme 2

[00141] To a solution of methyl/ triphenyl)phosphonium;bromide (162.18 g, 454.01 mmol, 3.1 eq) in THF (600 mL) was added KOtBu (55.88 g, 497.95 mmol, 3.4 eq) at 20 °C under N₂. The mixture was stirred at 20 °C for 1 h. To the mixture was added naltrexone (compound lb, 50 g, 146.46 mmol, 1 eq) at 20 °C under N₂. The reaction mixture was stirred at 20 °C for 6 h. Then the reaction mixture was concentrated under reduced pressure to remove THF. The residue was diluted with NH₄CI (cone., 800 mL) and extracted with EtOAc 1000 mL (500 mL * 2). The organic phase was washed with H₂O 1000 mL (500 mL * 2). The organic phase was concentrated under vacuum and the residue was diluted with dichloromethane (400 mL) to give a clear solution. Concentrated aqueous hydrochloric acid (HC1 37%, 80 mL) was added to the mixture. The suspension was filtered and the filter cake was washed with dichloromethane (300 mL) and acetone (300 mL). The solid was concentrated under reduced pressure to give pure product. Nalmefene (compound la, 50 g, 133.02 mmol, -88% yield, -97% purity, HC1 salt) was obtained as a white solid. M+H⁺ = 340.2 (LCMS).

[00142] Examples 1 and 2 are intentionally left blank.

[00143] Example 3: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl undecyl carbonate

[00144] To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13- hexahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (15 g, 39.91 mmol, 1 eq, HC1) in DCM (150 mL) was added TEA (12.11 g, 119.72 mmol, 16.66 mL, 3 eq ) in one portion at 25°C under N2.The mixture was stirred at 25 °C for 30 min, To a mixture of (4- nitrophenyl) undecyl carbonate (26.93 g, 79.81 mmol, 2 eq) in DCM (150 mL) , then add to the former mixture, the mixture was stirred at 25 °C for 12 h. The mixture was diluted with ¾0 (800 mL), extracted with DCM (300 mL*3). The organic phase was washed with brine (300 mL), dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=40/l to l/l).The residue was further purified by prep-HPLC, MeOH as solvent, select conventional reverse phase separation as method, separation system is TFA. NaHCCL was added to adjust pH to about 8, the aqueous phase was extracted with ethyl acetate (200 mL*3).The combined organic phase was washed with brine (500 mL), dried with anhydrous Na2S04, filtered and concentrated in vacuum. The compound [(3R,4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a, 13-hexahydro- 1H-4, 12- methanobenzofuro[3,2-e]isoquinoline-9-yl] undecyl carbonate (11.40 g, 21.14 mmol, 52.97% yield) was obtained as a yellow oil. M+H⁺ = 538.3 (LCMS). NMR (400 MHz, CDCI₃): see Fig. 1.

[00145] Examples 4 and 5 are intentionally left blank.

[00146] Example 6: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.5 g;¹H NMR (400 MHz, CDCI3): see Fig. 2. Briefly, to a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-¹H-4,12- methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (2.5 g, 6.65 mmol, 1 eq , HC1) in DCM (10 mL) was added TEA (2.02 g, 19.95 mmol, 2.78 mL, 3 eq) and dodecyl carbonochloridate (2.48 g, 9.98 mmol, 1.5 eq). The mixture was stirred at -10°C for 1 hour and then warmed to 25°C for 4 hours under N2. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Si02, petroleum ether/ethyl acetate=5/l to 1:1. The compound [(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2- e]isoquinolin-9-yl dodecyl carbonate] was 98.570% pure and obtained as a yellow oil (1.5 g, 40.56% yield).

[00147] Examples 7-16 are intentionally left blank.

[00148] Example 17: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl octyl carbonate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs and was obtained as an oil. 1.5 g; *H NMR (400 MHz, CDCI3): see Fig. 3.

[00149] Example 18: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs and was obtained as an oil. 1.5 g; H NMR (400 MHz, CDCI3): see Fig. 4.

[00150] Example 19: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.8 g; H NMR (400 MHz, CDCI₃): see Fig. 5. Briefly, to a solution of (3R, 4aS, 7aS, 12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-lH-4,12- methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (5 g, 13.30 mmol, 1 eq ) in DCM (50 mL), cooled to -10°C, TEA (4.04g, 39.91 mmol, 5.55 mL, 3 eq) and hexadecyl carbonochloridate (8.11 g, 26.60 mmol, 2 eq) was added. Then, the mixture was stirred at 25 °C for 5 hours under N2 atmosphere. The reaction mixture was extracted with ¾0 (80 mLxl) and DCM (80 mLx2). The combined organice phase was washed with brine (60 mLx3), dried with anhydrous Na₂SO₄, filtered and concentration in vacuum. The reside and compound was purified by column chromatography (Si02, petroleum ether/ethyl acetate=10/l to 1:1) The residue was purified by prep-HPLC (TFA condition: column - Phenomenex lune C18 250c50mmx 10mm; mobile phase - [water(0.1% TFA)-CAN]; B% 65-95%, 20 minutes). NaHCO₃ was added to adjust pH to 8, and then extracted with EtOAc (20 mLx3). The organic layer was evaporated under reduced pressure to get the final product. The compound [4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro- lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate] was 99.723% pure and was obtained as a white solid (1.8g, 12.33% yield).

[00151] Examples 20-27 are intentionally left blank.

[00152] Example 28: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl icosyl carbonate

To a mixture of icosyl (4-nitrophenyl) carbonate (9.87 g, 21.28 mmol, 4 eq) in DCM (40 mL) was added TEA (538.40 mg, 5.32 mmol, 740.58 uL, 1 eq) and (3R,4aS,7aS,12bS)-3- (cyclopropylmethyl)-7-methylene-2,4,5,6,7a, 13-hexahydro- 1H-4, 12-methanobenzofuro[3,2- e]isoquinoline-4a,9-diol (2 g, 5.32 mmol, 1 eq , HC1) in one portion atl5 °C under N2. The mixture was stirred at 15 °C for 12 hr. The reaction mixture was extracted with H2O mL (20mL * 2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC.

Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-y1] icosyl carbonate (1.6 g, 2.35 mmol, 44.25% yield) was obtained as a white solid.₁H NMR (400 MHz, CDCI₃): see Fig. 6.

[00153] Example 29 is intentionally left blank.

[00154] Example 30: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl tridecyl carbonate

To a mixture of (4-nitrophenyl) tridecyl carbonate (5.83 g, 15.96 mmol, 2 eq) in DCM (50 mL) was added TEA (2.42 g, 23.94 mmol, 3.33 mL, 3 eq) and (3R,4aS,7aS,12bS)-3- (cyclopropylmethyl)-7-methylene-2,4,5,6,7a, 13-hexahydro- 1H-4, 12-methanobenzofuro[3,2- e]isoquinoline-4a,9-diol (3 g, 7.98 mmol, 1 eq, HC1) in one portion at 15 °C under N2.The mixture was stirred at 15 °C for 12 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=l/0 to 3:1). [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a- hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2- e]isoquinoline-9-yl] tridecyl carbonate (2.3 g, 4.07 mmol, 50.94% yield) was obtained as a colorless oil. NMR (400 MHz, CDCl₃): see Fig. 7.

[00155] Example 31: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl tetradecyl carbonate

To a solution of (3R, 4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2, 4, 5, 6, 7a, 13- hexahydro- 1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3 g, 7.98 mmol, 1 eq, HC1) in DCM (20 mL) was added TEA (1.62 g, 15.96 mmol, 2.22 mL, 2 eq) and tetradecyl carbonochloridate (2.21 g, 7.98 mmol, 1 eq). The mixture was stirred at 15 °C for 12 hr. The mixture was concentrated under reduced pressure. The residue was mixed with ¾0 (80mL) and extracted with DCM (80mL*3). The combined organic phase was washed with saturated NaHCO₃ solution (60mL*2) and brine (60mL*3), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=4/l to 0:1). Compound [(3R,4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2, 4, 5, 6, 7a, 13-hexahydro- 1H-4, 12- methanobenzofuro[3,2-e]isoquinoline-9-yl] tetradecyl carbonate (2 g, 3.41 mmol, 42.79% yield) was obtained as a colorless oil. M+H⁺ = 580.4 (LCMS).

NMR (400 MHz, CDC1₃): see Fig. 8.

[00156] Example 32: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl pentadecyl carbonate

To a mixture of (4-nitrophenyl) pentadecyl carbonate (6.28 g, 15.96 mmol, 2 eq) in DCM (30 mL) was added TEA (2.42 g, 23.94 mmol, 3.33 mL, 3 eq) and [(3R,4aS,7aS,12bS)-3- (cyclopropylmethyl)-7-methylene-2,4,5,6,7a, 13-hexahydro- 1H-4, 12-methanobenzofuro[3,2- e]isoquinoline-4a,9-diol (3 g, 7.98 mmol, 1 eq , HC1) in one portion at 15 °C under N2. The mixture was stirred at 15 °C for 12 hr. The residue was purified by column chromatography (Si0₂, Petroleum ether/Ethyl acetate=1/0 to 2:1). Compound [(3R,4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a-hydroxy-7-methylene-2, 4, 5, 6, 7a, 13-hexahydro- 1H-4, 12- methanobenzofuro[3,2-e]isoquinoline-9-y1] pentadecyl carbonate (2.6 g, 1.80 mmol, 22.49% yield) was obtained as a white solid. M+H⁺ = 594.3 (LCMS). NMR (400 MHz, CDCl₃): see Fig. 9.

[00157] Example 33: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a-octahydro-lH-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl octadecyl carbonate

To a solution of (3R, 4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2, 4, 5, 6, 7a, 13- hexahydro- lH-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (2 g, 5.32 mmol, 1 eq , HC1) in DCM (30 mL) was added TEA (1.62 g, 15.96 mmol, 2.22 mL, 3 eq) and (4- nitrophenyl) octadecyl carbonate (3.48 g, 7.98 mmol, 1.5 eq). The mixture was stirred at 15 °C for 12 hr. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (Si02, Petroleum ether/Ethyl acetate=10/l to 1:1) and then by pre-HPLC. Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7- methylene-2, 4, 5, 6, 7a, 13-hexahydro- lH-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl] octadecyl carbonate (0.8 g, 1.22 mmol, 22.93% yield) was obtained as a yellow oil. M+H⁺ = 636.5 (LCMS). NMR (400 MHz, CDC1₃): see Fig. 10.

II. Biological Evaluation

Example 1: Plasma and liver S9 Fraction stability assay

[00158] Plasma stability determination of the test compounds in rat, dog, cynomolgus monkey and human plasma is performed using HPLC-MS. For rat, incubations are carried out in 96-well polypropylene plates in 5 aliquots of 70 mL each (one for each time point). Test compounds (10 mM, final solvent concentration 1 %) are incubated at 37 °C. Five time points are analyzed (0, 15, 120, 480 and 1440 min). For dog, monkey and human, test compounds (2 mM, final solvent concentration 1%) were also incubated at 37°C and analyzed at five time points (0, 10, 30, 60 and 120 min). All incubations are performed in duplicates. The samples are analyzed by HPLC-MS. The percentage of parent compound remaining after incubation in plasma is determined. Nalmefene dodecanoate and nalmefene palmitate were previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202). Results are provided in Table 4a-d.

Table 4a - Rat Plasma Stability

Table 4b - Dog Plasma stability

Table 4c - Monkey Plasma stability

Table 4d - Human Plasma stability

[00159] Liver S9 fraction stability determination of the test compounds in dog, cynomolgus monkey and human is performed using HPLC-MS. Test compound (2 mM, 0.1% DMSO, 1%

Methanol final concentration) was assessed for stability in a 50 mΐ phosphate buffer containing 1.0 mg/ml S9 protein from each of the three species and 5 mM D-saccharic acid-1, 4-lactone. Samples were incubated at 37°C for 60 minutes and the % compound remaining was assessed.

Table 4e - Liver S9 Fraction Stability

Example 2: Opioid Receptor Binding Assay

[00160] Receptor binding assays were performed to assess the ability of compounds to inhibit binding to radiolabeled ligand. First, the IC50 values were determined for select compounds for all 3 opioid receptor subtypes (DOR, MOR and KOR) and compared these values to that of the parent molecule, Nalmefene. The general observation is that prodrug derivatization greatly reduces the binding affinity to the opioid receptors, in some cases by several orders of magnitude.

Apparatus

Unifilter-96 GF/C filter plates, Perkin Elmer (Cat#6005174)

96 well conical polypropylene plates, Agilent (Cat#5042-385)

TopSeal-A sealing film, Perkin Elmer (Cat#6005250)

TopCount NXT HTS, (PerkinElmer)

MicroBeta (PerkinElmer)

Cell harvest C961961, (Perkin Elmer)

Reagents

The stable cell lines were established and prepared cell membrane obtained using these cell lines.

³H-diprenophrine (PerkinElmer, Cat: NET1121250UC, Lot: 2143599)

³H-DAMGO (PerkinElmer, Cat: NET902250UC, Lot: 2139100)

³H-DADLE (PerkinElmer, Cat: NET648250UC, Lot: 2060549) Tris base (Sigma, Cat: T6066-1KG), prepare 1M stock and adjust pH to 7.4. 0.5M EDTA (Invitrogen, Cat: 15575-038)

1M MgCl₂ (Sigma, Cat: M1028- 100ml)

PEI (Poly ethyleneimine) (Sigma, Cat: P3143)

Microscint 20 cocktail (PerkinElmer, Cat: 6013329)

Naltrindole (Sigma, Cat; N115)

(±)trans-U-50488 (Sigma, Cat: D8040)

DAMGO (Sigma, Cat: E7384)

Assay Buffer

Wash Buffer

Methods

1) Membrane and Radio ligand Preparation

2) Compound Preparation

3) Assay procedure

1) Transfer 1 mΐ of specified concentration compound to assay plate according to the plate map for nonspecific binding. Transfer 1 mΐ of DMSO to assay plate according to plate map for total binding.

2) Follow the plate map. Dispense 99m1 of membrane stocks into the plate.

3) Add 100 mΐ of radio ligand.

4) Seal the plates. Incubate at RT for 1 hour.

5) Soak the Unifilter-96 GF/C filter plates with 50 mΐ of 0.3% PEI per well for at least 0.5 hour at room temperature. 6) When binding assays are completed, filter the reaction mixture through GF/C plates using Perkin Elmer Filtermate Harvester, and then wash each plate for 4 times with cold wash buffer.

7) Dry the filter plates for 1 hour at 50 degrees.

8) After drying, seal the bottom of the filter plate wells using Perkin Elmer Unifilter-96 backing seal tape. Add 50 ml of Perkin Elmer Microscint 20 cocktail.

Seal top of filter plates with Perkin Elmer TopSeal-A sealing film.

9) Count H trapped on filter using Perkin Elmer MicroBeta2 Reader second day.

10) Analyze the data with GraphPad Prism 5. Calculate the "Inhibition [% Control]" using the equation: %Inh = (1-Background subtracted Assay value/Background subtracted HC value)* 100.

Results

Table 5a

Table 5b

Table 5c

Example 3: Solubility Determination

[00161] A known amount of test substance (~40mg) was weighed into the vial, 100 pL of oil was added and heated to 60 °C and then system was slurried to reach equilibrium. More oil was added until clear solution was obtained or the solubility was <50mg/mL. Then the clear solution was placed at room temperature (25 °C) for 24 h to confirm whether there was solid precipitation. Extra oil was added into the vial once compound precipitated out and then the system was re-equilibrated at 1000 rpm at room temperature (25 °C). Final concentration was determined by HPLC method as described below in Table 6a and 6b.

Table 6a

Table 6b

The HPLC method for Compounds 6, 12-20, and 36-43 is provided in Table 7.

Table 7

The HPLC method for Compounds 10, 21-23, 53, 55, 56, nalmefene, and naltrexone is provided in Table 8.

Table 8

The HPLC method for Compounds 3-5, 8, 24-25, 26-34, 44-51, 54, 55, 57, 59, and 60 is provided in Table 9.

Table 9

Table 10

* Data from compound 8 initial failed batch due to non-optimized synthesis * * Data from compound 8 second batch after successfully optimized synthesis

Example 4: Stability Determination of Drug Product

[00162] Compounds were resuspended in oil vehicles, stored at room temperature for the indicated time period and assessed by HPLC. Data is presented as absolute percentage loss normalized to 30 days. Nalmefene dodecanoate were previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202).

Table 11

Table 12

Table 13

Table 14

Example 4: Stability Determination of Drug Substance

[00163] Compounds were stored at room temperature for the indicated time period and assessed by HPLC. Data is presented as absolute percentage loss normalized to 30 days. Nalmefene dodecanoate were previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202).

Table 15

Table 16

Table 17 - HPLC method for compounds 10, 11, 21, 36, and 53

Table 18 - HPLC method for compounds 14, 19 and 55

Table 19 - HPLC method for compounds 1, 3-9, 15, 17, 18, 23-27, 29, 31, 32, 34, 35, 44- 48, 57, and 59

Table 20

Example 5: Physical Characterization of solid state drug substance

[00164] The analysis of the physical characteristics of drug substances that were in a solid state was conducted using polarized light microscopy (PLM), X-ray powder diffractometer (XRPD) assessment, Differential Scanning Calorimetry (DSC) and Thermal Gravimetric Analysis (TGA). For PLM, samples were dispersed in immersion oil and were observed using an ocular lens (lOx) and objective lens (20x) under crossed polarizers. For XRPD, samples were run on a diffractometer using the following method: Tube - Cu: K-alpha (l=1 .54179 Å); Generator - Voltage 40 kV, Current 40 mA; Scan scope - 3 to 40°; sample rotation speed - 15 rpm; scanning rate - 10 deg/min. For DSC, ~1 mg of sample was tested using a crimped aluminum pan and covered by a lid with a hole, heated from room temperature to 300°C at a speed of 10°C/minute. For TGA, 2-5 mg of sample was placed in an open platinum pan and heated from room temperature to 300°C at a rate of 10°C/minute. Nalmefene palmitate were previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202).

Table 21 - XRPD, TGA and DSC results

Example 5: Polymorph Screening of Solid state drug substances

[00165] In order to identify stable polymorph forms of solid state drug substances, approximately 50 mg of compound (nalmefene or naltrexone equivalnets) was weighed into vials. Next, 500 pL of the indicated solvents was added and the suspension was stirred at 700 rpm, 50°C for 72 hours. For samples in suspension, solids were separated by centrifuge (10 minutes, 14000 rpm) and dried in vacuum oven at 30°C overnight. For samples in solution, solids were generated by evaporation (stir bar removed and covered with aluminum foil with pinholes, then dried in vacuum oven at 30°C overnight). Dried solids were characterized by XRPD, TGA and DSC. Results are presented in Table 23. Table 23

III. Preparation of Pharmaceutical Dosage Forms

Example 1: Oral capsule

[00166] The active ingredient is a compound of Table 1, or a pharmaceutically acceptable salt thereof.

A capsule for oral administration is prepared by mixing 1-1000 mg of active ingredient with starch or other suitable powder blend. The mixture is incorporated into an oral dosage unit such as a hard gelatin capsule, which is suitable for oral administration.

Example 2: Solution for injection

[00167] The active ingredient is a compound of Table 1, or a pharmaceutically acceptable salt thereof, and is formulated as a solution in sesame oil, cottonseed oil, castor oil or other

pharmaceutically acceptable lipophilic excipient, preferably at a concentration of greater than 100 mg/mL. The resulting solution is administered by intramuscular injection.

Compounds were resuspended to 1 mL at the indicated concentrations (in mg/ml base equivalents) by mixing with magnetic stirring (1000 rpm) at 60°C until a homogeneous clear solution was achieved, then cooled down to room temperature and stored protected from light. Appearance of oil formulations was observed and recorded at room temperature (25°C) at initial, 2 hours, and 24 hours. Samples for“Assay” measurements were taken at initial, 2 hours and 24 hours post resuspension and subjected to HPLC analysis where actual concentration was based on a standard curve (Assay = Concentration measured by

HPLC)/Actual concentration(by weight) x 100%). Purity was calculated at indicated time points based on the percentage of area under the curve of the main peak from the HPLC spectrogram. Syringability was assessed by drawing through a 21 Gauge needle. Some indicated samples were assessed for Appearance, Assay and Purity after 7 months in

40°C/75% Relative Humidity. Data are presented in Table 21.

Table 24

Table 25A

Table 25B

Table 26A

Table 26B

Table 26C

IV. Pharmacokinetic Evaluation

Example 1: Rat pharmacokinetic studies

Purpose

[00168] The purpose of this study is to determine the pharmacokinetics of test compounds in plasma, following intramuscular administration to male Sprague Dawley Rats (n=3, unless otherwise specified).

Acclimation/ Quarantine

[00169] Animals are assessed as to their general health and acclimated for at least 3 days before being placed on study.

Animal Husbandry

[00170] Animals are housed during acclimation and individually housed during the study. The animal room environment was controlled (target conditions: temperature 18 to 26°C, relative humidity 30 to 70%, 12 hours artificial light and 12 hours dark). Temperature and relative humidity were monitored daily. Water was provided to the animals ad libitum.

Animal Body Weights and Clinical Observation

[00171] Body weights were determined before selection to the study and on the day of dose

administration. Weight monitoring was done every week.

Detailed clinical observation including behavior and activity, reflection, respiration, skin and fur, facial feature, genitourinary system, and other gross lesions was performed on the dosing day and at each sample collection time point.

Dose Administration

[00172] The dose formulation of 400 mg base equivalents/ ml in sesame oil + 1% benzyl alcohol

(unless otherwise specified) was administered by intramuscular injection. The dose volume was determined by the animals' body weight determined on the morning of dosing day.

Sample Collection

[00173] Each blood collection (about 0.2 mL per time point) was performed from jugular vein

puncture of each animal into pre-chilled plastic microcentrifuge tubes containing 5 pL of 160 mg/mL sodium fluoride/potassium oxalate (NaF/KO=l/3) with 5%PMSF(100mM in ethanol) as stabilizer and 4 pF of EDTA-K2 as anti-coagulant and placed on wet ice until

centrifugation.

Plasma Processing

[00174] Each collected blood sample was centrifuged for 4 minutes at 4°C and 10000rpm for plasma collection. Plasma was collected and transferred into a pre-labeled PP tube in dry ice at each time point and precipitated immediately using ACN at a ratio of 1:4 (plasma: ACN). Centrifuged again (10 minutes, 12000rpm) and obtain the supernatant.

After terminal collection, all supernatant was stored at approximately -80°C until bioanalysis.

Bioanalytical Method and Sample Analysis

[00175] LC-MS/MS methods for the quantitative determination of test compound in biological matrix were developed. A calibration curve with 8 non- zero calibration standards were applied for the method including LLOQ (0.05 ng/ml). The sample analysis was performed concurrently with a set of calibration standards and two sets of QC samples using the LC-MS/MS method.

Data Analysis

[00176] Plasma concentration versus time data was analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C_max, T_max, T_½, AUC_(0-t), AUC_(o-inf), MRT_(0.t), MRT_(0-t) and graphs of plasma concentration versus time profile were prepared.

[00177] The dose for nalmefene dodecanoate was determined by allometric scaling to rat from dog doses as previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196- 202). Terminal half life was determined for active metabolite of select compounds, and is used for estimating duration above minimally effective plasma concentration for the active metabolite.

Table 27

No adverse affect on body weight or clinical observations were noted in any rats across all studies.

[00178] Time vs nalmefene concentration data for nalmefene HCL in 1 mg/ml at 0.80 mg/kg is provided in Table 28a.

Table 28a

*Not detected

[00179] Time vs nalmefene concentration data for compound 59 (nalmefene dodecanoate) in 86 mg/ml concentration at 17 mg/kg is provided in Table 28b.

Table 28b

[00180] Time vs nalmefene concentration data for compound 6 at 80 mg/kg is provided in Table 29.

Table 29

[00181] Time vs nalmefene concentration data for compound 6 at 123 mg/kg is provided in Table 30.

Table 30

[00182] Time vs nalmefene concentration data for compound 6 at 165 mg/kg is provided in Table 31.

Table 31

[00183] Time vs nalmefene concentration data for compound 15 at 80 mg/kg is provided in Table 32.

Table 32

[00184] Time vs nalmefene concentration data for compound 15 at 123 mg/kg is provided in Table

33.

Table 33

[00185] Time vs nalmefene concentration data for compound 15 at 165 mg/kg is provided in Table

34.

Table 34

[00186] Time vs nalmefene concentration data for compound 17 at 200 mg/kg is provided in Table

35.

Table 35

[00187] Time vs nalmefene concentration data for compound 18 at 80 mg/kg is provided in Table 36.

Table 36

[00188] Time vs nalmefene concentration data for compound 18 at 123 mg/kg is provided in Table

37. Table 37

[00189] Time vs nalmefene concentration data for compound 18 at 200 mg/kg is provided in Table

38.

Table 38

[00190] Time vs naltrexone concentration data for compound 23 at 200 mg/kg is provided in Table

39.

Table 39

[00191] Time vs nalmefene concentration data for compound 24 at 80 mg/kg is provided in Table 40.

Table 40

[00192] Time vs nalmefene concentration data for compound 24 at 123 mg/kg is provided in Table

41.

Table 41

[00193] Time vs nalmefene concentration data for compound 24 at 165 mg/kg is provided in Table

42.

Table 42

[00194] Time vs naltrexone concentration data for compound 29 at 165 mg/kg (at 400 mg/ml in sesame oil) is provided in Table 43a.

Table 43 a

[00195] Time vs naltrexone concentration data for compound 29 (at 300 mg/ ml in cottonseed oil, n=2) at 165 mg/kg is provided in Table 43b.

Table 43b

Example 2: Dog pharmacokinetic studies

Purpose

[00196] The purpose of this study is to determine the pharmacokinetics of test compounds in plasma, following deep intramuscular administration to Beagle dogs (n=3, unless otherwise specified).

Acclimation/ Quarantine

[00197] Animals are assessed as to their general health and acclimated for at least 5 days before being placed on study. Animal Husbandry

Animals are pair housed during acclimation and individually housed during the study. The room(s) will be controlled and monitored for relative humidity (targeted mean range 40% to 70%, and any excursion from this range for more than 3 hours will be documented as a deviation) and temperature (targeted mean range 18°to 26°C, and any excursion from this range will be documented as a deviation) with 10 to 20 air changes/hour. The room will be on a 12-hour light/dark cycle except when interruptions are necessitated by study

activities. Animals will be fed twice daily. Stock dogs will be fed approximately 220 grams of Certified Dog Diet daily (Beijing Keao Xieli Feed Co., Ltd. Beijing, P. R. China). These amounts can be adjusted as necessary based on food consumption of the group or an individual body weight changes of the group or an individual and/or changes in the certified diet. Reverse osmosis (RO) water is available to all animals, ad libitum. RO water is analyzed every three months and every batch of feed is analyzed before using. Enrichment toys are provided.

[00198] Animal Body Weights and Clinical Observation

[00199] Body weights were determined before selection to the study and on the day of dose

administration. Weight monitoring was done every week.

Detailed clinical observation including behavior and activity, reflection, respiration, skin and fur, facial feature, genitourinary system, and other gross lesions was performed on the dosing day and at each sample collection time point.

Dose Administration

[00200] The dose formulation (concentration - 400 mg base equivalents/ml in sesame oil + 1% benzyl alchol, unless otherwise specified) was administered via deep intramuscularly (unless otherwise specified). The injection vehicle was also dosed via deep intramuscular route (unless otherwise specified) on contralateral site of each animal at study initiation. The animals were sedated with Propofol at 6mg/kg via IV administration. Following sedation hair was carefully removed from around the injection site and the area gently cleaned. Care will be taken to avoid irritating skin during shaving and cleaning the injection site. Then dogs will be dosed with deep IM administration. At least 2.5 cm depth from the surface into the central aspect of the quadriceps or biceps femoris muscle, by angling the needle toward the femur. If the needle hits the femur, simply draws back slightly and then inject. The dose volume will be determined by the animals' body weight collected on the morning of dosing day. For repeated administration, the injection sites may be rotated to minimize tissue injury. [00201] Sample Collection

Blood samples were collected from a peripheral vessel from restrained, non-sedated animals per sampling time point.

Approximately 0.8 mL blood will be collected at each time point. All blood samples will be transferred into pre-chilled plastic microcentrifuge tubes containing 20 pL of 160 mg/mL sodium fluoride/potassium oxalate (NaF/KO=l/3) with 5%PMSF(100mM in ethanol) as stabilizer and 16 pL of EDTA-K2 (0.5M) as anti-coagulant and placed on wet ice until centrifugation.

Each collected blood will be in the wet-ice before centrifuge.

Plasma Processing

[00202] Each collected blood sample was centrifuged for 4 minutes at 4°C and lOOOOrpm for plasma collection. Plasma was collected and transferred into a pre-labeled PP tube in dry ice at each time point and precipitated immediately using ACN at a ratio of 1:4 (plasma: ACN). Centrifuged again (10 minutes, 12000rpm) and obtain the supernatant.

After terminal collection, all supernatant was stored at approximately -80°C F until bioanalysis.

Bioanalytical Method and Sample Analysis

[00203] LC-MS/MS methods for the quantitative determination of test compound in biological matrix were developed. A calibration curve with 8 non- zero calibration standards were applied for the method including LLOQ (0.05 ng/ml). The sample analysis was performed concurrently with a set of calibration standards and two sets of QC samples using the LC-MS/MS method.

Data Analysis

[00204] Plasma concentration versus time data was analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C_max, T_max, T_½, AUQ_(0-t), AUC_(o-inf), MRT_(0.t), MR_(o-inf) and graphs of plasma concentration versus time profile were prepared.

Table 44

Time vs nalmefene concentration data for compound 6 at 30 mg/kg is provided in Table 45.

Table 45*

*study is ongoing

Time vs nalmefene concentration data for compound 6 at 48 mg/kg (shallow IM injection) is provided in Table 46.

Table 46

Time vs nalmefene concentration data for compound 6 at 48 mg/kg (deep IM injection; redosed in dogs from Table 46) is provided in Table 47a. Table 47a*

*study is ongoing

Time vs nalmefene concentration data for compound 6 at 48 mg/kg (deep IM injection; single dose in naive dogs n=2) is provided in Table 47b.

Table 47b*

* study is ongoing Time vs nalmefene concentration data for compound 6 at 96 mg/kg is provided in Table 48.

Table 48*

* study is ongoing

Time vs nalmefene concentration data for compound 15 at 30 mg/kg is provided in Table 49.

Table 49*

* study is ongoing

Time vs nalmefene concentration data for compound 15 at 48 mg/kg is provided in Table 50a.

Table 50a*

* study is ongoing

Time vs nalmefene concentration data for compound 15 at 48 mg/kg (repeat of study from Table 50a) is provided in Table 50b.

Table 50b*

* study is ongoing Time vs nalmefene concentration data for compound 15 at 96 mg/kg is provided in Table 51.

Table 51*

* study is ongoing

Time vs nalmefene concentration data for compound 18 at 48 mg/kg is provided in Table 52.

Table 52

Time vs nalmefene concentration data for compound 24 at 48 mg/kg (shallow IM injection) is provided in Table 53.

Table 53

Time vs nalmefene concentration data for compound 24 at 48 mg/kg (deep IM injection) is provided in Table 54.

Table 54

Time vs nalmefene concentration data for compound 45 at 30 mg/kg is provided in Table 55. Table 55*

*study is ongoing

Time vs nalmefene concentration data for compound 45 at 48 mg/kg is provided in Table 56.

Table 56*

*study is ongoing

Time vs nalmefene concentration data for compound 45 at 96 mg/kg is provided in Table 57.

Table 57*

*study is ongoing

Time vs naltrexone concentration data for compound 7 at 24 mg/kg is provided in Table 58.

Table 58*

*study is ongoing

Time vs naltrexone concentration data for compound 7 at 48 mg/kg (n=2) is provided in Table 59.

Table 59*

*study is ongoing

Time vs naltrexone concentration data for compound 8 at 48 mg/kg is provided in Table 60.

Table 60*

*study is ongoing

Time vs nalmefene concentration data for compound 1 at 48 mg/kg is provided in Table 61.

Table 61*

*study is ongoing Time vs nalmefene concentration data for compound 3 at 48 mg/kg is provided in Table 62.

Table 62

Time vs naltrexone concentration data for compound 4 at 48 mg/kg is provided in Table 63.

Table 63

** ND = none detected

Time vs naltrexone concentration data for compound 5 at 48 mg/kg (n=2) is provided in Table 64. Table 64

Time vs naltrexone concentration data for compound 35 at 48 mg/kg is provided in Table 65.

Table 65

Clinical observations for dogs treated with compound 6 at 30 mg/kg are provided in Table 66.

Clinical observations for dogs treated with compound 6 at 48 mg/kg (Shallow IM) are provided in Table 67.

Clinical observations for dogs treated with compound 6 at 48 mg/kg (Deep IM; redosed in dogs from Table 67) are provided in Table 68a.

Clinical observations for dogs treated with compound 6 at 48 mg/kg (deep IM injection; single dose in naive dogs n=2) are provided in Table

68b.

Clinical observations for dogs treated with compound 6 at 96 mg/kg are provided in Table 69.

Clinical observations for dogs treated with compound 15 at 30 mg/kg are provided in Table 70.

Clinical observations for dogs treated with compound 15 at 48 mg/kg are provided in Table 71a.

Clinical observations for dogs treated with compound 15 at 48 mg/kg are provided in Table 71b.

Clinical observations for dogs treated with compound 15 at 96 mg/kg are provided in Table 72.

Clinical observations for dogs treated with compound 18 at 48 mg/kg are provided in Table 73.

Clinical observations for dogs treated with compound 24 at 48 mg/kg (DeepIM) are provided in Table 75.

Clinical observations for dogs treated with compound 45 at 30 mg/kg are provided in Table

76.

Clinical observations for dogs treated with compound 45 at 48 mg/kg are provided in Table

77.

Clinical observations for dogs treated with compound 45 at 96 mg/kg are provided in Table

78.

Clinical observations for dogs treated with compound 7 at 24 mg/kg are provided in Table 79.

Clinical observations for dogs treated with compound 7 at 48 mg/kg are provided in Table 80.

Clinical observations for dogs treated with compound 8 at 48 mg/kg are provided in Table 81.

Clinical observations for dogs treated with compound 1 at 48 mg/kg are provided in Table 82.

Clinical observations for dogs treated with compound 3 at 48 mg/kg are provided in Table 83.

Clinical observations for dogs treated with compound 4 at 48 mg/kg are provided in Table 84.

Clinical observations for dogs treated with compound 5 at 48 mg/kg are provided in Table 85.

Clinical observations for dogs treated with compound 35 at 48 mg/kg are provided in Table

86.

Claims

CLAIMS We Claim:

1. A compound, or pharmaceutically acceptable salt thereof, wherein the compound is (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro- 1H-4, 12-methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate.

2. A pharmaceutical composition comprising a compound of claim 1, or

pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipient is cottonseed oil.

4. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipient is sesame oil.

5. A method of treating opioid dependence in a patient in need thereof comprising

administering a pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

6. The method of claim 5, wherein the pharmaceutical composition is administered by injection.

7. The method of claim 6, wherein the pharmaceutical composition is administered by intramuscular injection.

8. The method of claim 7, wherein the intramuscular injection is a depot injection.

9. The method of claim 8, wherein the depot injection provides a therapeutically

effective concentration for a period of about 2 days to at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.

10. The method of claim 8, wherein the depot injection provides a therapeutically

effective concentration for a period of about at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.

11. The method of claim 8 or 9, wherein the depot injection provides therapeutically

effective antagonism of a dose of an opioid in a subject after at least one depot injection, and the degree of antagonism is determined at one or more timepoints, from 6 hours to about 4 weeks, 2 months, 3 months, 4 months, 5 months or 6 months after said depot injection.

12. The method of claim 11, wherein the opioid is a natural or synthetic opioid selected from fentanyl, carfentanil, heroin, morphine, codeine, hydrocodone, oxycodone, hydromorphone or combinations thereof.

13. The method of any one of claims 6-12, wherein the composition for injection

comprises at least one pharmaceutically acceptable oil vehicle, at least one pharmaceutically acceptable excipient, and the compound of claim 1, or

pharmaceutically acceptable salt thereof, is present in a concentration ranging from about 0.5 mg/mL to about 600 mg/mL.

14. The method of claim 13, wherein the pharmaceutically acceptable oil vehicle is

cottonseed oil.

15. The method of claim 13, wherein the pharmaceutically acceptable oil vehicle is

sesame oil.

16. The method of any one of claims 13-15, wherein the at least one pharmaceutically acceptable oil vehicle, and the at least one pharmaceutically acceptable excipient are the same.

17. The method of claim 13, wherein the at least one pharmaceutically acceptable oil vehicle, and the at least one pharmaceutically acceptable excipient are both cottonseed oil.

18. The method of claim 13, wherein the at least one pharmaceutically acceptable oil vehicle, and the at least one pharmaceutically acceptable excipient are both sesame oil.