MULTIPLE TARGETING OPIOID RECEPTOR LIGANDS AS NOVEL

ANALGESICS WITH MINIMUM ABUSE LIABILITY

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/400,059, filed August 23, 2022.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbers DA024022 and UG3DA050311 awarded by the National Institutes of Health/National Institute on Drug Abuse. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention generally relates to multi-opioid receptor ligands for pain relief without causing addiction. In particular, the invention provides opioid receptor ligands with conjoint targeting of at least mu opioid receptor (MOR) and kappa opioid receptor (KOR), to effectively treat pain without causing addiction and abuse.

State of Technology

Over 20% of people in the United States experience daily pain and approximately 10% of the patients who receive standard opioid treatment develop addiction to the medications. Despite vast efforts devoted to non-addictive analgesics from both academia and industry, there are currently no drugs that can replace the mainstay opioid analgesics in moderate-to- severe pain management, highlighting the urgent demand for more effective and safer pain medications.

It has been widely accepted that mu opioid receptor (MOR) activation produces analgesia as well as notorious abuse liability. So considerable attempts targeting different proteins or different pain mechanisms have been made and have led to many drug candidates. One example is the development of G protein-biased MOR agonists, which are based on the hypothesis that the abuse-related and respiration-depressing side effects result from P-arrestin rather than G-protein-mediated signaling pathways. However, in contrast to the original hypothesis, these “G-protein biased” MOR agonists, including the newly marketed TRV-130 (Oliceridine, Figure 1), still possess undesired abuse liability and cause respiratory depression.

There are three other members in the opioid receptor family: kappa opioid receptor (KOR), delta opioid receptor (DOR), and nociception/orphanin FQ peptide receptor (NOP), and activation of any of them may mediate analgesic effects. Although agonists of the KOR, DOR, and NOP also carry side effects, such as dysphoria, seizures, and altered renal function, respectively, none of them cause euphoria or severe dependence like MOR agonists. However, selective agonists solely targeting the KOR, DOR, or NOP have not achieved satisfactory clinical results.

Nalfurafine (NFU, Figure 1) is a potent MOR/KOR dual agonist which is used in Japan as an antipruritic for hemodialysis patients, has also showed potent analgesia in a variety of pain models. Other examples of clinically efficacious dual MOR/KOR agonists such as pentazocine, nalbuphine, and dihydroetorphine (Figure 1). However, these agonists still retain the limitations of MOR agonists including abuse liability and respiratory depression.

There is a pressing need to have available new compounds which effectively treat pain without causing symptoms of physical dependence that lead to abuse.

SUMMARY OF THE INVENTION

Discovery of analgesics devoid of abuse liability is critical to battle the opioid crisis in the US and elsewhere. The examples of the present disclosure describe a complementary structure- activity relationship (SAR) study that systematically explored multi-opioid receptor pharmacology of Nalfurafine (NFU) analogs. As a result, compounds have been developed which target more than one opioid receptor and minimize unwanted side-effects (such as addiction), while still achieving an effective therapeutic profile for pain relief.

Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof. It is an object of this invention to provide a compound of general Formula I:

Formula I where * represents an a or P configuration;

R ¹ is H or OH;

R ² is H or CH ₃ ;

R ³ is a heterocyclic system or where

X = O, NH or S;

X’, W, Y and Z are independently CH or N; and

Q includes a saturated or unsaturated, branched or unbranched, substituted or unsubstituted alkyl group having from 2 to 10 carbon atoms; with the caveat that the compound cannot be

where R ¹ is OH, R ² is CH3 and * is either alpha or beta; and pharmaceutically acceptable salts thereof.

In some aspects, the one or more carbon atoms of Q are independently substituted with one or more heteroatoms or heteroatomic groups selected from the group consisting of N, O, OH, SO, SO ₂ , COOH, N, NH, NH ₂ , NH ₃ ⁺ , PO, PO ₂ , PO3, and halogen.

In other aspects, Q includes a double bond, R ³ is a furan ring attached via C at position 3, and * is a or p. In yet other aspects, Q includes a single bond or or double bond, R ³ is a furan ring attached via C at popsition 2 or C at position 3; and * is a or p. In further additional aspects, the compound is and * is p.

Also provided is a method of preventing or treating pain in a subject in need thereof without causing addiction, comprising administering to the subject a therapeutically effective amount of at least one compound of general Formula I: where

* represents an a or P configuration;

R ¹ is H or OH;

R ² is H or CH ₃ ;

R ³ is a heterocyclic system or where

X = O, NH or S;

X’, W, Y and Z are independently CH or N; and

Q includes a saturated or unsaturated, branched or unbranched, substituted or unsubstituted alkyl group having from 2 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof; wherein administration of the at lease one compound does not cause addiction thereto.

In some aspects, the one or more carbon atoms of Q are independently substituted with one or more heteroatoms or heteroatomic groups selected from the group consisting of N, O, OH, SO, SO ₂ , COOH, N, NH, NH ₂ , NH ₃ ⁺ , PO, PO ₂ , PO ₃ , or halogen. In additional aspects, Q includes a double bond, R ³ is a furan ring attached via C at position 3, and * is a or p. In yet other aspects, Q includes a single bond or or double bond, R ³ is a furan ring attached via C at popsition 2 or C at position 3; and * is a or p. In some aspects, the at least one compound is and * is a.

In other aspects, the at least one compound is and * is p.

DESCRIPTION OF THE DRAWINGS

Figure 1. Prior art compounds that target one or more opioid receptors.

Figure 2. Molecular design of compounds according to the exemplary aspect of the disclosure described in Example 1.

Figure 3. Warm Water Tail Immersion Assays of compounds 9 to 16 at 10 mg/kg. (Mean values of %MPE for each group were presented and the error bar stands for SE (Standard

Error) Compared with vehicle, ns no significant difference at p < 0.05. #p < .1, **p < 0.01,

***p < 0.001, ****p < 0.0001.

Figure 4. Blocking the antinociceptive effects of NMF by selective KOR antagonist and DOR antagonist in warm water tail immersion assay, respectively. (nor-BNI and P-FNA were given s.c. at a dose of 10 mg/kg 24 h prior to 0.1 mg/kg NMF administration. NTI was injected s.c. at a dose of 15 mg/kg 30 minutes prior to 0.1 mg/kg NMF injection. Compared to 0.1 mg/kg NMF group: ****p < 0.0001. Compared to vehicle: #p < 0.05; : n.s. Error bars represent SE. -1-

Figure 5. Time course study of NMF and morphine. (All administration routes were s.c. compared to vehicle: *p<0.05, **p<0.01, ***p<0.001, *p<0.0001; Compared with the morphine group: ^AA p<0.01, ^AAA p<0.001, ^AAAA p<0.0001. Error bars represent SD.

Figure 6. Self-administration of NMF and fentanyl in rats. Rats were trained to self-administer (i.v.) 32 pg/kg/infusion heroin. All points represent the mean (± SEM) number of infusions for NMF doses (n=10; 5 males and 5 females) and fentanyl (n=5; 3 males and 2 females). Filled symbols: significantly different from saline at p <0.05.

Figure 7A-C. Drug tolerance and cross tolerance. (A) Experimental design. Mice were administered a vehicle, 10 mg/kg morphine, 0.1 mg/kg NMF or 0.5 mg/kg NMF twice daily for consecutive four days, (s.c.) (B) Antinociceptive effects of compounds were evaluated daily. Compared with DI (day 1), *p<0.05, ***p<0.001. (C) On day 5, vehicle, 10 mg/kg morphine or 0.1 mg/kg NMF was given to the morphine-treated group, after which the warmwater tail immersion assay was again performed. *p<0.05, **p<0.01. Error bars represent SD. Figure 8A-D. Weight change in mice received chronic administration of vehicle (8 A), NMF 0.1 mg/kg (8B), NMF 0.5 mg/kg (8C) or morphine (8D) compared to Day 1, *p<0.05, ****p<0.0001. Error bars represent SD.

Figure 9. Withdrawal and diarrhea symptoms precipitated by naloxone (NEX) in chronic analgesic-administration mice. (In comparison to morphine pellet group: *p<0.05, **p<0.01, ****p<0.0001. Percentage of diarrhea was calculated as the number of mice who had diarrhea divided by the total mouse number in each group. The severity of diarrhea was not quantified. Error bars represent SEM.

Figure 10A-C. The respiratory effects of NMF and morphine in mice. A) Respiratory frequency (breaths per min) change. B) Minute volume (mE/min) change. C) Tidal volume (ml) changes. Error bars represent SD.

Figure 11A-C. Spontaneous locomotor activity change after drug administration. A) distance (cm), B) ambulatory counts, C) Jump counts. Activity data was recorded for 30 min with the injection time as time zero. Compared to vehicle: ns, no significant difference at p<0.05. Error bars represent SD.

Figure 12. Metabolism study of NMF in human and rat liver S9 fractions. Error bars represent SD.

Figure 13. Summary of known nalfurafine SAR.

Figure 14. Newly designed opioid ligands to study SAR of NLF. 5 points of mutation are indicated.

Figure 15. Overall synthetic route to produce designed ligands.

Figure 16. Preliminary screen of synthesized ligands in the warm-water tail immersion assay using the single-dose agonism (10 mg/kg) protocol in male mice (n = 6/group). Ordinate: Control/comparator compounds (morphine, nalfurafine [NLF], NMF) and synthesized ligands 1 - 24. Abscissa: Percent maximum possible effect (MPE). All points represent the mean ± SEM. The compounds were divided into four main groups based on their MPE under these conditions MPE = 100% (black), MPE = 80 - 100 % (gray), MPE = 40 - 80 % (black and white checks), MPE = < 40 % (white checks). Compounds 6, 8, 14, and 16 were first published in Pagare, et al., Biol J Med Chem 2022, 65 (6), 5095-5112.

Figure 17. Time course study of synthesized ligands using the warm-water tail immersion assay in male mice (n = 6/group). Ordinate: Percent maximum possible effect. Abscissa: Time post subcutaneous injection of synthesized ligand in hours. All points represent the mean ± SEM. Black filled symbols denote significant difference relative to the pre-injection baseline measurement (-1 h), defined as p<0.05.

Figure 18. Receptor selectivity study of synthesized ligands using the warm-water tail immersion assay in male mice (n = 6/group). Ordinate: Percent maximum possible effect. Abscissa: Combination of synthesized ligands and opioid antagonists given. All points represent the mean ± SEM. *Denotes significant difference relative to the referenced test compound when given alone, defined as p<0.05.

Figure 19. Screen of synthesized ligands in the warm-water tail immersion assay using the single-dose antagonism (10 mg/kg) protocol in male mice (n = 6/group). Ordinate: Naloxone control and selected synthesized ligands. Abscissa: Percent maximum possible effect. All points represent the mean ± SEM. The compounds were divided into four main groups based on their MPE under these conditions MPE = 100% (black), MPE = 80 - 100 % (gray), MPE = 40 - 80 % (black and white checks), MPE = < 80 % (white checks).

Compounds 6, 8, 14, and 16 first published in Pagare, et al., Biol J Med Chem 2022, 65 (6), 5095-5112.

Figure 20A-I. Locomotor activity in male mice (n = 6/group) during the acclimation period (pre-injection; B - E) and post-injection of compounds (F - I). (A) Timeline for testing procedure. Abscissa: Compound and dose studied in mg/kg. (B and F) Ordinate: Total distance travelled during the 30 min session in centimeters. (C and G) Ordinate: Total ambulatory counts in the 30 min session. (D and H) Ordinate: Total vertical counts during the 30 min session. (E and I) Ordinate: Average speed during the 30 min session in centimeters/second. All bars represent the mean ± SEM. *Denotes significant difference relative to the vehicle condition (white bar), defined as p<0.05.

Figure 21A-C. Self-administration of fentanyl and test compounds in male and female rats (A: 2M,6F; B: 2M,6F; C: 3M,6F). Ordinate: Number of infusions earned under a FR5 schedule of reinforcement. Abscissa: Intravenous unit dose of compound infusion in pg/kg/inf with compound being fentanyl (A-C), nalfurafine (A), compound 21 (B) or compound 23 (NCF; C). Saline and “Fent” (Fentanyl) represent the mean ± SEM of all saline and 3.2 pg/kg/inf fentanyl days obtained as baselines between the testing days needed to acquire the respective dose-effect curves shown in A-C. All points represent the mean ± SEM. *Denotes significant difference relative to saline, defined as p<0.05.

Figure 22F. Tolerance (A - D) and Cross-Tolerance (E - F) as studied using a warm-water tail immersion assay in male mice (n = 6/group). (A) Graphical experimental procedure for tolerance assay. (B - D) Development of tolerance (or lack thereof) in morphine and vehicle groups as well as nalfurafine (NLF; B), compound 21 (C), compound 23 (NCF; D). Ordinate: percent maximum possible effect (MPE). Abscissa: Test day. All points represent the mean ± SEM. *Denotes significant difference relative to test day 1, defined as p<0.05. (E) Graphical experimental procedure for cross-tolerance assay. (F) Cross-tolerance (or lack thereof) of vehicle, morphine, nalfurafine (NLF), compound 21, and compound 23 (NCF) with morphine tolerant mice. Ordinate: percent maximum possible effect. Abscissa: Compound given on day 5 to test for cross-tolerance with morphine at specified doses. All points/bars represent the mean ± SEM. *Denotes significant difference relative to morphine-tolerant mice given morphine on day 5 (blue bar), defined as p<0.05.

Figure 23A-I. (A - 1) Weight fluctuation of male mice (n = 6/group) given vehicle, morphine, nalfurafine (NLF), compound 21, and compound 23 (NCF) throughout tolerance/ crosstolerance study. Ordinate: Weight change from day 1 in grams. Abscissa: Test day. Data is shown for each mouse (individual points) as well as the mean (horizontal lines) ± SEM. *Denotes significant difference relative to day 1, defined as p<0.05.

Figure 24A and B. Antagonist (naloxone 1 mg/kg)-induced withdrawal symptoms in male mice (n = 6/group). (A) Graphical experimental procedure (NLX: naloxone). (B) Ordinate: Mean count of observed withdrawal signs. Abscissa: Compounds given at specified doses in mg/kg. All points represent the mean ± SEM. *Denotes significant difference relative to the morphine (10 mg/kg) group, defined as p<0.05.

Figure 25A and B. Blood-brain barrier penetration study in male mice (n = 3/time point). Ordinate: Concentration of compound in brain (pg/g) and plasma (pg/mL). Abscissa: Time post subcutaneous injection of synthesized ligand in min. All points represent the mean ± SEM.

DETAILED DESCRIPTION

Provided herein are analgesic compounds that bind to at least two (more than one) of the opioid receptors mu opioid receptor (MOR), kappa opioid receptor (KOR), delta opioid receptor (DOR), and nociception/orphanin FQ peptide receptor (NOP). The compounds advantageously exhibit potent analgesic effects in subjects in need thereof without causing dependency and addiction.

DEFINITIONS

An ADMET study is the assessment of pharmacokinetics of a drug which stands for Absorption, Distribution, Metabolism, Excretion and Toxicity.

The hERG channel inhibition assay is a highly sensitive measurement which will identify compounds exhibiting cardiotoxicity related to hERG inhibition in vivo. However, not all compounds which inhibit hERG activity in vitro cause cardiotoxicity in vivo.

As used herein, a functional derivative (or functional analog) of a compound is a compound that has been or can be synthesized from another compound and differs therefrom by the replacement of 1-3 atoms or 1-3 functional groups of atoms (e.g., H replaced by methyl, ethyl, etc.; replacement of O by S or N, or vice versa; replacement of carboxyl, amide, etc. with an atom or a different functional group, and the like). Functional derivatives exhibit the same or highly similar chemical and/or biochemical properties and/or activities (e.g., physical, chemical, biochemical, and/or pharmacological properties/activities) as the compound from which they are derived, when the original compound from which they are derived and the functional derivative are tested under the same conditions. For example, the value may be higher or lower than the value of the property or activity of the original compound by about 50%-150% (e.g., about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150%). Methods of making and tests for identifying functional derivatives include those described in the Examples section of this disclosure.

THE COMPOUNDS

The compounds have the general Formula I:

Formula I where

* represents an alpha or beta configuration at carbon number 6 of the ring;

R ¹ is H or OH;

R ² is H or methyl (CH3);

R ³ is a heterocyclic system where

X = O, NH or S;

X’, W, Y and Z are independently CH or N; and

Q includes a saturated or unsaturated, branched or unbranched (i.e., straight chain, linear), substituted or unsubstituted alkyl group having from 2 to 10 carbon atoms; and pharmaceutically acceptable salts and functional derivatives thereof.

In some aspects, the compounds have a general formula as shown for Formula I and

Q includes a single, double (cis or trans) or triple bond, and C) i.e., a furan ring, and is attached to the rest of the molecule at the carbon atom at position 2 or 3, both of which are indicated on the ring; with the caveat that the compound per se cannot be where R ¹ is OH, R ² is CH3 and * is either alpha or beta.

In some aspects, one or more carbon atoms of Q are independently substituted with one or more heteroatoms or heteroatomic groups, including but not limited to: S, N, O, halogen (e.g., Cl, F, I), OH, SO, SO ₂ , COOH, N, NH, NH ₂ , NH ₃ ⁺ , PO, PO ₂ , PO3, etc. In the compounds which are descibed in Example 1, R ¹ is H or OH; R ² is H or CH3;

Q includes a double bond; R ³ is a furan ring attached via the C atom at position 3 of the ring; and both a and P oreintations at * were made and tested. It is noted that in Schemes 1 -3 presented in Example 1, the R1 group of Formula I is designated “R ” and the R ² group of Formula I is designated “R ’

In the compounds which are descibed in Example 2, R ¹ is H or OH; R ² is H or CH3; and Q includes a single bond or a double bond; R ³ = so that -Q-R ³ together are where the wavy line indicates where the -Q-R3 group attached to the C of the C=O group; and both a and P oreintations at * were made and tested.

It is noted that Tables 6-9 presented in Example 2, the “R3 ” group includes the carbonyl, unlike the R3 group of Formula I. .

A saturated carbon group or chain (alkane; aliphatic hydrocarbon) has only single covalent bonds between adjacent C atoms throughout the group, whether the group is a straight or branched chain. Exemplary univalent radicals of saturated carbon groups or chains included in the variable groups of Q include but are not limited to: methyl (CH3), ethyl (C2H5), propyl (C3H7), isopropyl, butyl (C4H9, including n-butyl, s-butyl, t-butyl), pentyl (C5H11), hexyl (CeHn), heptyl (C7H15), octyl (CsHn), nonyl (C9H19) and decyl (C10H21). The so-called “lower alkyls” comprising from 1-6 C atoms are also encompassed as are branched saturated alkane radicals including but are not limited to: isobutyl, isopentyl, neopentyl, various isohexyls, and the like.

An unsaturated or partially unsaturated carbon group or chain has one or more (at least one) covalent double bond between two adjacent carbon atoms (alkene); and/or at least one covalent triple bond(s) between two adjacent C atoms (alkyne). Exemplary alkene radicals having one double bond include but are not limited to: methenyl, ethenyl, propenyl, butenyl (e.g., 2-butenyl), pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc., and branched chains thereof. Exemplary alkyne radicals having one triple bond include but are not limited to: ethyne, propyne, butyne (e.g., 2-butynyl), pentyne, hexyne, heptyne, octyne, nonyne, etc. Di- and tri-alkene radicals comprising, respectively, 2 or 3 double bonds or more are also encompassed as are di- and tri-alkyne radicals comprising, respectively, 2 or 3 or more triple bonds. The term "substituted" refers to the addition of one or more substituents by replacement of a carbon atom or as an attachment to a carbon atom. In some embodiments, substituents are halogen, haloalkyl, alkyl, acyl, hydroxyalkyl, hydroxy, alkoxy, haloalkoxy, aminocarbonyl oxaalkyl, carboxy, cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino arylsulfonyl, arylsulfonylamino, and benzyloxy. Additional examples include: substituted alkyl, aryl, cycloalkyl, etc., wherein one or more H atoms are replaced with halogen, haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxy lower alkyl, carbonyl, phenyl, heteroaryl, benzenesulfonyl, hydroxy, lower alkoxy, haloalkoxy, oxaalkyl, carboxy, alkoxycarbonyl, alkoxycarbonylamino, aminocarbonyl (also known as carboxamido), alkylaminocarbonyl, cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, (alkyl)(aryl)aminoalkyl, alkylaminoalkyl (including cycloalkylaminoalkyl), dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy, mercapto, alkylthio, sulfoxide, sulfone, sulfonylamino, alkylsulfinyl, alkylsulfonyl, acylaminoalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl, heterocyclyl, heterocyclylalkyl, phenoxy, benzyloxy, heteroaryloxy, hydroxyimino, alkoxyimino, oxaalkyl, aminosulfonyl, trityl, amidino, guanidino, ureido, benzyloxyphenyl, and benzyloxy. "Oxo" is also included among the substituents referred to in "independently substituted". It will be appreciated by persons of skill in the art that, because oxo is a divalent radical, there are circumstances in which it will not be appropriate as a substituent (e.g., on phenyl). In one embodiment, 1, 2, or 3 hydrogen atoms are replaced with a specified radical.

Functional derivatives, stereoisomers, structural isomers, geometric isomers, tautomers, hydrates, solvates and pharmaceutically acceptable salts of each compound disclosed herein are also encompassed.

Exemplary compounds include but are not limited to: wherein * is p.

PHARMACEUTICAL COMPOSITIONS The compounds described herein are generally delivered (administered) as a pharmaceutical composition. Such pharmaceutical compositions generally comprise at least one of the disclosed compounds, i.e., one or more than one (a plurality) of the compounds (e.g., 2 or more such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) may be included in a single formulation. Accordingly, the present invention encompasses such formulations/compositions. The compositions generally include one or more substantially purified compounds as described herein, and a pharmacologically suitable (physiologically compatible) carrier. In some aspects, such compositions are prepared as liquid solutions or suspensions, or as solid forms such as tablets, pills, powders and the like. Solid forms suitable for solution in, or suspension in, liquids prior to administration are also contemplated (e.g., lyophilized forms of the compounds), as are emulsified preparations. In some aspects, the formulations are liquid and are aqueous or oil-based suspensions or solutions.

In some aspects, the active ingredients are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients, e.g., pharmaceutically acceptable salts. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, preservatives, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like are added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of compound in the formulations varies but is generally from about 1-99%. Still other suitable formulations for use in the present invention are found, for example in Remington's Pharmaceutical Sciences, 22nd ed. (2012; eds. Allen, Adejarem Desselle and Felton).

Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as Tween® 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

"Pharmaceutically acceptable salts" of the compounds refers to the relatively nontoxic, inorganic and organic acid addition salts and base addition salts of compounds of the present disclosure. In some aspects, these salts are prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-P-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, 1- 19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N- methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like. In some aspects, the salt is an HC1 (hydrochloride) salt.

The compounds may also be formulated for delayed, controlled, long-acting and/or sustained release. Those of skill in the art are aware of such formulations, including those described in published US patent applications US20210113469A1 US11179369B2, US 10463624B2, US 10624905B2, US 11090269B 1 and US 11110093B2, the complete contents of which are hereby incorporated by reference in entirety. For example, in some aspects, the formulation comprises one or more microparticles comprising one or more of the compounds, for example, pellets, beads, tablets, spheroids, liposomes, gels, or combinations of two or more of these. In some aspects, a pill or tablet is used for delivery, the tablet comprising a coating layer disposed on a core, the coating layer including at least one release rate controlling polymer and the core comprising the pharmacologically active compound(s) described herein, and the one or more microparticles disposed within the core. In various aspects, the compounds are disposed within a matrix (which may be or make up a microparticle, or a “core” as described above), e.g., a wax matrix, a polyethylene oxide matrix, hydroxypropyl methyl cellulose, and others that are known in the art. Such matrices, microparticles, outer layers and cores are preferably biodegradable.

ADMINISTRATION AND DOSING

The pharmaceutical compositions disclosed herein are administered in vivo by any suitable route including but not limited to: inoculation or injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, and the like), topical application (e.g. on areas such as eyes, skin, in ears or on afflictions such as wounds and burns) and by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosa, and the like). Other suitable means include but are not limited to: inhalation (e.g. as a mist or spray), orally (e.g. as a pill, capsule, tablet, liquid, etc.), intravaginally, intranasally, rectally, as eye drops, incorporated into dressings or bandages (e.g. lyophilized forms may be included directly in a dressing), as depot formulations, in implants or reservoirs for long term and/or chronic pain relief, etc. In preferred embodiments, the mode of administration is oral or by injection.

The compounds are administered using any type of dosing regimen that is suitable, generally as determined by a medical professional such as a physician or physician’s assistant. For example, the compounds may be administered from about 1-6 times per day, e.g., about 1, 2, 3, 4, 5, or 6 times per day, or even more frequently, especially in early phases of recovery from surgery, accidents, etc., when administration may be every 2-4 hours. In some aspects, administration is self-administration PRN (as needed) by the patient, e.g., using a self-dosing pump. Alternatively, if sustained release formulations are used, administration may be daily, or every 2, 3, 4, 5, or 6 days, or weekly, or every 7-10 days, or biweekly, or monthly.

A single dose is generally about 0.05 mg, 0.1 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 20 mg, 23 mg, 24 mg, 25 mg, 26 mg or 30 mg of the compound or more, including all decimal fractions in between these values, such as 4.1, 4.2, 4.3...4.8, 4.9, 5.0, for example. In some aspects, a single dose is about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 mg or more, such as about 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000 (or more, e.g., up to about 1500 or 2000 mg per dose) including all integers in between these values, such as 30, 31, 32, 33, 34, 35 and 125, 126, 127...148, 149, 150, as examples.

In addition, the compositions may be administered in conjunction with other treatment modalities such as substances that boost the immune system, various chemotherapeutic agents, antibiotic agents, other analgesic agents, and the like, or activities such as exercise, counseling, psychiatric care, electronic nerve signal blocking, etc. METHODS OF USE The compounds disclosed herein are used to prevent and/or treat pain, and include compounds of general Formula I:

Formula I

Where: * represents an a or P configuration; R ¹ is H or OH; R ² is H or CH3; R ³ is a heterocyclic system or where X = O, NH or S; X’, W, Y and Z are independently CH or N; and Q includes a saturated or unsaturated, branched or unbranched, substituted or unsubstituted alkyl group having from 2 to 10 carbon atoms; and pharmaceutically acceptable salts and functional derivatives thereof, without caveat. Administration of the compounds advantageously does not cause addiction thereto.

The compounds disclosed herein provide pain relief to subjects in need thereof, without the risk of addiction. As such, they replace current addictive opioid medicaments such as morphine and heroin. For example, patients with acute and/or chronic pain conditions are treated with the compounds, the conditions including but not limited to: acute pain, chronic pain, migraine, other headache disorders, osteoarthritis, diabetic neuropathy, chemotherapy -induced neuropathy, sickle-cell pain, post stroke pain, post-operative pain, pain during healing of scars, pain caused by burns, pain caused by spinal injury or disease, pain caused by broken bones and/or lacerations, pain during childbirth, and pain related to other neurological disorders, e.g. multiple sclerosis, opioid misuse, stimulant misuse, and the like.

Administration of the compounds may be used to treat existing pain or to prevent the occurrence of pain. For example, pain may exist as a result of an accident or surgery or disease and administration of the compounds treats (eliminates of lessens) the level of pain. Alternatively, if a situation will cause pain in the future, e.g., surgery, a developing disease, etc., the compounds can be administered to prevent the occurrence, or at least lessen, the level of pain when it would otherwise occur.

Those of skill in the art will recognize that while a preferred outcome of administration of the disclosed compounds is complete elimination of pain, in some cases much benefit can accrue without complete elimination but with simply lessening the level of pain to a tolerable level, e.g., a level at which the treated person is able to conduct daily activities, sleep comfortably, engage in physical therapy, engage in social activities, etc., to the best of his/her ability. Thus, by administering one or more compounds disclosed herein, symptoms of narcotic tolerance and/or dependence are prevented or at least lessened.

In some aspects, the present compounds are employed to “wean” persons addicted to an addictive narcotic from that drug to a compound disclosed herein. The person may or may not be in need of pain relief but is in any case dependent on the use of a narcotic to avoid and/or lessen symptoms of narcotic addiction. In these aspects, in the beginning, the addictive substance may be administered at a normal dose together with a small amount of a compound described herein, and over a period of time (e.g., several days or a week, or more), the amount of the addictive drug is decreased and the amount of the non-addictive compound disclosed herein is increased until the addictive drug can be eliminated with a minimum of side effects for the subject. Side effects of narcotic tolerance and/or addiction (or tolerance leading to addiction) that can be eliminated or lessened (decreased) include but are not limited to dysphoria, psychomimetisis, seizures, altered renal function, etc. Side effects of withdrawal from narcotics that can be eliminated of lessened (decreased) include but are not limited to muscle pain or aches, hunger or loss of appetite, fatigue, sweating, cravings, nausea, fever and/or chills, tremors, vivid dreams, flu-like symptoms, sweating, heart palpitations, and/or psychological withdrawal symptoms such as violent mood swings, anxiety and depression. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

In the description of the invention herein, it is understood that a word appearing in the singular encompasses its plural counterpart, and a word appearing in the plural encompasses its singular counterpart, unless implicitly or explicitly understood or stated otherwise. Furthermore, it is understood that for any given component or embodiment described herein, any of the possible candidates or alternatives listed for that component may generally be used individually or in combination with one another, unless implicitly or explicitly understood or stated otherwise. Moreover, it is to be appreciated that the figures, as shown herein, are not necessarily drawn to scale, wherein some of the elements may be drawn merely for clarity of the invention. Also, reference numerals may be repeated among the various figures to show corresponding or analogous elements. Additionally, it will be understood that any list of such candidates or alternatives is merely illustrative, not limiting, unless implicitly or explicitly understood or stated otherwise. In addition, unless otherwise indicated, numbers expressing quantities of ingredients, constituents, reaction conditions and so forth used in the specification and claims are to be understood as being modified by the term "about."

Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter presented herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the subject matter presented herein are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

All patents and publications mentioned in the specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

EXAMPLES

EXAMPLE 1. Characterization of a Potential KOR/DOR Dual Agonist with No Apparent Abuse Liability via a Complementary Structure-Activity Relationship Study on Nalfurafine Analogs

Discovery of analgesics devoid of abuse liability is critical to battle the opioid crisis in the US and elsewhere. In the process of understanding the structure-activity relationship of nalfurafine, we identified an analgesic agent, NMF, as a dual KOR/DOR agonist with minimum abuse liability. Characterizations, including primary in vitro ADMET studies (hERG toxicity, plasma protein binding, permeability and hepatic metabolism), and in vivo pharmacodynamic and toxicity profiling (time course, abuse liability, tolerance, withdrawal, respiratory depression, body weight, and locomotor activity) are described in this Example. RESULTS AND DISCUSSION

Molecular Design

The structural modifications of the NFU skeleton were focused on the 3-hydroxy (3- OH) group, C6 configuration, and amide nitrogen methylation. We hypothesized that i) 3- dehydroxylation may diminish MOR affinity, reducing the risk of abusive effects, but maintain KOR affinity; and ii) methylation on the amide nitrogen may improve the physiochemical properties of newly designed ligands. The design of different configurations of C6 give the side chain conformational flexibility in order to achieve a preferred binding mode in the target proteins. The SAR the 3-OH, C6 configuration, and amide nitrogen methylation locations are shown in Figure 2.

Chemical Syntheses

In the designed synthetic routes, two starting molecules, naltrexone and 3-dehydroxy naltrexone, were first needed to prepare the target compounds. Naltrexone was provided by NIDA, and 3-dehydroxy naltrexone was synthesized from naltrexone via activation and hydrogenation following the reported methods (Scheme 1) (Musliner, W. J. et al. J. Am. Chem. Soc. 1966, 88 (18), 4271-4273).

Eight enantiopure intermediates were required to prepare the target compounds. Part of the protocols to get these essential intermediate amines (1-8) were made available from previous studies from others and our lab (Nagase, H et al. Bioorganic Med. Chem. Lett. 2012, 22 (15), 5071-507;. Li, M. et al., Tetrahedron Lett. 2020, 61 (39), 152379-152381; Huang, B, Bioorganic Chemistry, 2021, 109, 104702-104712) and a systematic description for all synthetic routes are briefly discussed in the following. For preparing 1-8, at least one stereoselective synthetic route was adopted and established with reasonable yields. Taking advantage of the unique “T-shape” epoxymorphinan skeleton, the stereoselectivity was introduced by either Noyori catalysts (f, j, Scheme 1), which required no chiral ligands in this case, or a conformation-induced-steric -hindrance method (c-d, g-h, k-1, Scheme 1). For comparison, the method using asymmetric catalysts has advantages including simple one-step setup, higher yields, and carcinogen-free. On the other hand, the conformation-induced- steric- hindrance method (c-e) is more time-efficient for obtaining compounds 1 or 5.

Scheme 1. Stereoselective synthetic methods for intermediate amines (1-8). (a). DMF, K2CO3, l-phenyl-5-chlorotetrazole, r.t., 12 h. Yield: 85%. (b). H2, 45-50 psi, w.t. 30% Pd/C, AcOH, 50-55 °C, 10 h. Yield: 75%. (c). BnNH ₂ , PhH, PTSA, reflux, 10 h. (d). NaBH ₄ , EtOH, 4A MS, r.t. overnight. Yield of c-d: 26-28%. (e). H ₂ , 60-70 psi, MeOH, HC1, w.t. 28% Pd/C, r.t. 23 h. Yield: 43-83%. (f). TEA, MeCN, BnNH ₂ , HCOOH, 1% w.t. [Ru(p-cymene)Cl ₂ ]2, r.t. 40 or 70 h. Yield: 48-50%. (g). Bn ₂ NH, PhCOOH, Tol, PTSA, reflux, 18-30 h. (h). NaCNBH ₃ , EtOH, 4A MS, r.t., overnight. Yield of g-h: 30-57%. (i). H ₂ 60-70 psi, MeOH, HC1, w.t. 50% Pd/C, 70-72 h. Yield: 63-66%. (j). TEA, MeCN, CH ₃ NH ₃ «HC1, HCOOH, 2% wt. [Ru(p- cymene)Cl ₂ ]2, r.t. 24 h. Yield: 96-98% (k). BnNHCH ₃ , PhCOOH, PhH, PTSA, reflux, 24 h. (1). NaCNBH ₃ , EtOH, 4A MS, r.t., overnight, (m). H ₂ 60 psi, MeOH, HC1, 35% wt. Pd/C, r.t. 48 h. Yield of k-m: 19-20%.

Additionally, “one-pot” methods were also explored and established as alternative synthetic routes for the four N- methylated intermediate amines (Scheme 2). In such approaches, all starting materials were added together and allowed to reflux for 24 h. In both reactions, starting with naltrexone or 3-deydroxynaltrexone, both C6-epimers were generated while the 6/>-cpimcrs (4, 8) were the major products, respectively.

The reaction mechanism of “one-pot” reaction was proposed here (Scheme 2) for the first time. The coexistence of both chair (A) and boat (B) conformations of ring C is believed to account for the formation of both epimers. Once all starting materials were heated in methanol, amination by methylamine took place on the 6-position carbonyl group. The resulted iminiums could present in the form of a pair of stereoisomers, A and B’ (Scheme 2). In the case of A, the reducing agent cyanoborohydride favored the less hindered /-face (arrow), yielding compound 3 or 7, the 6a-epimers. On the other hand, a substantial A ^1,3 strain between iminium hydrogen and epoxy ether oxygen in B’ led to a conformational change of ring C, from chair to boat. Subsequently, the iminium (B) with a more stable ring C was generated, to which the hydride mainly approached from the less hindered a-face (arrow), giving the 6/?-epimer, 4 or 8.

The “one-pot” approach (Scheme 2) not only demands less effort in reaction setup and monitoring than the conformation-induced steric hindrance methods (Scheme 1), but also requires shorter reaction time. Hence, it is preferred to use a one-pot method to synthesize intermediates 4 and 8.

Scheme 2. One-pot method for methylated amines (3-4, 7-8) and proposed mechanisms, (n). NaBH ₃ CN, MeOH, CH ₃ NH ₃ «HC1, reflux, 24 h. Yields: 9% for 3 and 62% for 4; 35% for 7 and 50% for 8. After all intermediate amines were acquired, the amide formation using (E)-3-(furan- 3-yl)acryloyl chloride was conducted to prepare the final target compounds (compounds 9 to 16, Scheme 3). One extra step of 3-hydrolyzation (Scheme 3) was performed for compounds with the 3 -hydroxy group. Then all target compounds were converted into their hydrochloride salts. Compound 12 (nalfurafine, or NFU) was prepared and tested along through all the following studies.

1, * = a, R = OH, R’ = H 5, * = a, R = H, R’ = H 9, * = a, R = OH, R’ = H 13, * = a, R = H, R’ = H

2, * = p, R = OH, R’ = H 6, * = P, R = H, R’ = H 10, * = p, R = OH, R’ = H 14, * = p, R = H, R’ = H

3, * = a, R = OH, R’ = CH ₃ 7, * = a, R = H, R’ = CH ₃ 11, * = a, R = OH, R’ = CH ₃ 15, * = a, R = H, R’ = CH ₃

4, * = p, R = OH, R’ = CH ₃ 8, * = p, R = H, R’ = CH ₃ 12, * = p, R = OH, R’ = CH ₃ 16, * = p, R = H, R’ = CH ₃

Scheme 3. Preparation of target compounds 9-16. (o). DCM, TEA, (E)-3-(furan-3- yl)acryloyl chloride, 0°C to r.t., 3-5 h. (p). K2CO3, MeOH, 0°C to r.t., 16-48 h. (q). MeOH, methanolic HC1, 0°C to r.t., overnight. Reaction (p) was only conducted to 1, 3, 5, and 7. Overall yields: 10-50%.

In Vitro Binding and Functional Studies

Radioligand binding assays

Radioligand competitive binding assays are frequently performed to determine the binding affinity and selectivity of potential GPCR ligands. In this membrane-based assay, the binding affinity of the target compounds were determined based on their ability to compete and replace the corresponding radioligands at each receptor.

Table 1. Binding Affinity Results for compound 9 to 16 at the KOR, DOR, and MOR. Bindin Affinit K (nM) ± SEM Selecti it

Note: SEM stands for standard error of mean, calculated from at least three individual experiments.

As shown in Table 1, all compounds except 13 acted as high-affinity KOR ligands with sub- or single-digit nanomolar Ki values. Among them, compound 10, 11 and 12 possessed the highest affinity (Ki < 0.2 nM). While 9-12 showed relatively higher binding affinities compared with their 3-dehydroxy counterparts 13-16, respectively, 14-16 still carried sub- or single-digit nanomolar Ki. Hence, 3 -dehy droxygenation seemed not absolutely necessary for KOR binding, which supported our hypothesis. Comparing each C6-epimer pair, though C6 configuration seemed to have no consistent impact on KOR affinity, the dramatic affinity difference between 13 and 14 was interesting and might be useful in designing 3- dehydroxy KOR ligands. On the contrary, comparing the KOR binding affinity of 11, 12, and 16 with their des-methyl counterparts 9, 10, and 14 respectively, the role of amide methyl group seemed not as obvious in recognizing the KOR except for the case of compound 15 vs 13.

In contrast to KOR, the DOR binding affinity was dramatically reduced when the 3- OH group was removed (Table 1), i.e. compound 9 to 13 (Ki: 20.0 ± 3.3 to 77.6 ± 5.9 nM), 10 to 14 (Ki: 4.74 + 0.85 to 340 + 96 nM), 11 to 15 (Ki: 0.64 + 0.11 to 116 + 24 nM), and 12 to 16 (Ki: 116 + 20 to 1000 + 190 nM). Therefore, the 3-OH seemed essential for DOR binding. On the other hand, the impact of C6 configuration on DOR binding seemed not conclusive from these eight compounds. Comparing DOR binding affinity of the N- methylated compounds with their des-methyl counterparts, 10 with 12, 13 with 15, and 14 with 16, the compounds without A-mcthylation showed much lower Ki values, i.e. non- methylated amide seemed to be preferred for the DOR, except for the case of compounds 9 and 11.

The 3-hydroxy group played a critical role in MOR binding, that is, compounds 9 to 12 are potent MOR ligands with 0.3- 1.2 nM Ki values (Table 1), whereas the MOR binding affinity of their 3-dehydroxy counterparts, 13 to 16, were lowered by 20-170 folds, respectively. It seemed that 3 -dehydroxylation could diminish MOR affinity. The other two chemical features, however, C6 configuration and A-methylation seemed to have no evident impact on MOR binding.

In summary, 3 -dehydroxylation on the epoxymorphinan skeleton may diminish MOR and DOR affinity, but not that of KOR, which could help enhance the selectivity for the KOR over both the MOR and DOR (Table 1). iV-Methylation might be beneficial to KOR affinity but not to DOR binding.

[ ³⁵ S]-GTPyS binding assays

Functional assays are usually performed to determine the efficacy and potency of GPCR ligands. [ ³⁵ S]-GTPyS binding assay and calcium mobilization (flux) assay are two common and established functional assays used for evaluating opioid receptor ligands. The [ ³⁵ S]-GTPyS assay is applied to test the direct activation on a receptor level, the activation of which by the GPCR can be quantified by measuring the amount of the radiolabeled GTP analog bound to the cell membrane.

As shown in Table 2, compound 9 to 16 all acted as high-efficacy KOR agonists and seven out of eight were observed with > 90% efficacy. Among them, compound 10, 11 and 12 (NFU) are the most potent agonists with subnanomolar EC50 values. Impressively, compound 11 possessed picomolar level potency in the [ ³⁵ S]-GTPyS assay, almost four times more potent than NFU (12). Compounds 9, 14, 15, and 16 possessed single-to-double-digit nanomolar high potency, while the EC50 of 13 is at three-digit nanomolar level. Comparing the EC50 values of each pair of compounds (with or without 3-hydroxy group), 9 (15.3 ± 1.9 nM) vs 13 (135 ± 25 nM), 10 (0.36 ± 0.02 nM) vs 14 (46.0 ± 6.8 nM), 11 (0.075 ± 0.003 nM) vs 15 (2.52 ± 0.38 nM), and 12 (0.26 ± 0.02 nM) vs 16 (59.0 ± 7.1 nM), the ones with 3- hydroxy group always presented a higher potency. It seemed that, although not required for KOR binding affinity, 3-OH might reinforce KOR-activation. Then comparing the compounds with and without A-mcthylation, the methylated ones all possessed similar or lower EC50 values, hence, the methyl group on the amide nitrogen atom might be beneficial to KOR potency.

As shown in Table 2, all eight compounds acted as DOR full agonists except for NFU with a moderate-to-high efficacy activation. Among them, compound 11 is the most potent agonist with single-digit nanomolar EC50, followed by compounds 9 and 10 which showed double-digit nanomolar EC50 values. Similar to the binding affinity pattern, compound 9 to 12 (EC50: 83.6 ± 28.8, 24.8 ± 4.7, 2.68 ± 0.74, 141 ± 15 nM) showed much higher potency than their 3-dehydroxy counterparts 13 to 16 (EC50: 714 ± 87, 1500 ± 140, 242 ± 28, 2100 ± 400 nM), indicating that 3 -hydroxy group may be important for the potency of compounds as DOR agonists.

Table 2. [ ³⁵ S]-GTPyS Functional Assay Results of Compounds 9 to 16. ^a

(NMF) 0.003 0.74

12 0.26 + 65.7 +

(NFU) ’ ^{0H p} ’ ^CH3 0.02 ⁹⁵ ' ^{9 ± 4} ' ^{1 141 ± 15} 6.4 °- ^{51 ± 0} - ^{12 30} - ^{3 ± 3} - ⁷

135 +

13 -H a -H ₂₅ 103 + 2 714 + 87 102 + 8 283 + 49 22.8 + 2.1

46.0 + 1500 +

14 -H B -H 97.9 + 2.4 107 + 13 167 + 23 27.7 + 1.1

6.8 140

2.52 +

15 -H a -CH _{3 0 38} 98.4 + 2.4 242 + 28 108 + 7 132 + 10 55.8 + 2.9 a. All values are presented as mean ± SEM. b. %E _m ax = 100 was defined using 5 pM U50,488H. c. %E _m ax = 100 was defined using 5 pM SNC80. d. %E _m ax = 100 was defined using E _m ax of 3 pM DAMGO.

From the results of [ ³⁵ S]-GTPyS assay for MOR (Table 2), all compounds achieved low to moderate efficacies at the MOR, and the agonism potency (EC50) of compound 9 to 12 are 50-3500 times higher than those of their 3-dehydroxy counterparts, 13 to 16, respectively. It might be concluded that 3 -dehydroxylation, the MOR affinity-deteriorating factor, may also diminish agonist potency on the MOR. And as expected, a reasonably linear correlation between binding affinity and potency was found for each compound on all three receptors (not shown).

It was noticed that some previously reported compounds, e.g., compounds 11 and 12, showed somewhat varied binding affinity and functional potency compared with literature, which is probably due to the difference between membrane-based assays vs tissues-based ones.

Primary In Vivo Characterization

Warm-water tail immersion assays

Because the in vitro studies showed that all compounds acted as efficacious dual KOR/DOR agonists, all eight compounds were selected to be tested for their antinociceptive effects. Herein, this is the first report to evaluate this set of compounds as high-efficacy dual KOR/DOR agonists for their antinociceptive studies. The first in vivo approach we chose was a warm water immersion study. A single-dose (10 mg/kg) screening was conducted first in mice to see whether the compounds could exhibit antinociceptive effects in vivo at this initial dose (Figure 3).

In this assay, each compound was injected systemically (s.c.) to a group of six mice. After 20 minutes, the tail flick response times were determined. The longer duration their tails stayed in water before flicking, the higher antinociceptive effects the compound possessed. The antinociceptive effect was quantified using %MPE.

As shown in Figure 3, six out of eight compounds presented an antinociception effect in mice in the single-dose test. Surprisingly, no significant increase of %MPE was observed from compound 9 considering its even higher KOR and DOR agonist potency than compound 14 and 16 in vitro (Table 2). The absence of antinociception for compound 13 might mainly be due to its intrinsically low potency or unfavorable pharmacokinetics profile.

Afterwards, dose-response studies were carried out to evaluate the antinociception potency of six compounds. As shown in Table 3, all six compounds were more potent than the KOR agonist U50,488H. Moreover, four of them, compounds 11, 12, 15, and 16 have exhibited higher antinociception potency than the MOR agonist morphine. And the antinociceptive effects were believed to be mediated by both KOR and DOR activation while KOR may play a major role (Tables 2 and 3). Table 3. Antinociception Potency ED50 in Tail-flick Assays.

Compound ED50 (95% CL), mg/kg

Morphine 2.34 (1.57 - 3.50) ⁵⁴

Fentanyl 0.015 (0.013 - 0.018) ⁵⁵

U50,488H 8.84 (5.51 - 14.17) ⁵⁴

10 2.414 (1.720 - 3.388)

11 (NMF) 0.037 (0.014 - 0.096)

12 (NFU) 0.046 (0.016 - 0.126)

14 3.827 (1.224 -11.97)

15 0.341 (0.196 - 0.604)

16 1.610 (1.057 - 2.452)

In addition, one interesting observation from Table 3 was that the methylated compounds presented higher antinociception potency than their des-methyl counterparts. For example, compound 12 was 50 times more potent than compound 10, though they possessed a similar KOR and DOR potency in vitro. Meanwhile, compound 16 was more potent than compound 14 in the tail immersion assays even though it was the opposite case in the [ ³⁵ S]- GTPyS assay. Therefore, the amide N-methylation might play a critical role in increasing lipophilicity, which helped compounds 12 and 16 distribute through the body. As an electrondonating group, the methyl group was also likely to enhance the metabolic stability by making the amide and the acryloyl less susceptible to hydrolysis and Michael addition, respectively.

More importantly, compound 11 (NMF) possessed a comparable ED50 to NFU (12). Considering its more potent DOR agonism compared to NFU, a KOR/DOR dual activating mechanism may play a major role in its antinociception potency.

Antinociception mechanism studies

Observed as the most potent agonist in vitro and in vivo, NMF was selected for further pharmacology and pharmacokinetics characterizations. In vivo selectivity study was first carried out to verify the proposed KOR/DOR dual activation mechanism of the antinociceptive effects of NMF. Therefore, KOR-selective antagonist nor-BNI, DOR- selective antagonist NTI, and MOR-selective irreversible antagonist >-FNA were co- administered with NMF in warm-water tail immersion assays, respectively.

As shown in Figure 4, both nor-BNI and NTI were able to reduce the antinociception produced by 0.1 mg/kg NMF, the calculated ED90, profoundly as expected. Moreover, their blocking effects seemed addable as the combination of two antagonists completely abolished the antinociception effect of NMF in mice. In addition, the irreversible MOR antagonist 0- FNA was not able to block the antinociception of NMF antinociception effect at the dose tested, which indicated the possibility of minimum involvement of the MOR, which was likely to be caused by its low efficacy at the MOR (%Emax = 34.3). In comparison, NFU has been characterized as a selective agent to the MOR and KOR, but not the DOR. This is very intriguing since the only structural difference between these two compounds is their C6 chirality.

Time-course studies

In the warm water tail immersion time-course study, both morphine and NMF showed peak antinociceptive effects around 0.5 h. The onset of action for 10 mg/kg morphine and 0.1 mg/kg NMF seemed to require several minutes, while the antinociceptive effects of 0.5 mg/kg NMF was observed immediately after administration. The duration of antinociception was approximately 3 hours for 10 mg/kg morphine and 0.1 mg/kg of NMF, and the antinociceptive effects produced by 0.5 mg/kg NMF lasted up to 8 h and were significantly greater than morphine from 3 h (Figure 5). As a comparison, NFU showed 3-4 hours action time at 0.003 mg/kg in AA-induced abdominal constriction, less than 2 hours at 0.03 mg/kg in a hot plate studies, and about 1.5 hours at 0.1 nmol (i.t.) in warm water tail withdrawal studies.

In Vivo Pharmacodynamic and Toxicity Studies

Abuse liability studies via self-administration method

As discussed above, abuse liability is the main concern for all opioid analgesics. As shown in Figure 6, fentanyl functioned as a reinforcer and 3.2 pg/kg/infusion fentanyl maintained significantly higher rates of responding than saline. The present fentanyl results are consistent with previous results demonstrating intravenous fentanyl self-administration in rats. In contrast to fentanyl, no NMF dose, across a 100-fold range, reinforced the responding rates in rats. Though the effective analgesic doses of NMF in rats remain to be examined, the current results have further supported the low efficacy of NMF at the MOR in vivo, as concluded from the in vitro and in vivo studies. More importantly, as a non-reinforcer in rats, NMF would be predicted to have low-to-no abuse liability.

Tolerance potential

Chronic administration of analgesics may lead to tolerance, which has been widely observed for clinically used MOR agonists. In our study, both NMF and morphine groups developed antinociceptive tolerance on day 3 (Figure 7B and 7C), whereas NMF (0.1 mg/kg) retained a higher antinociceptive effect than morphine (10 mg/kg) in morphine- tolerated mice (Figure 7B and 7C) though the potency of NMF seems reduced under morphine-tolerated conditions. Similarly, NFU also retained its antinociceptive effect in the morphine-tolerated mice.

Weight loss

Alterations in weight have been observed in subjects chronically exposed to morphine. Recently, the underlying mechanism of metabolic influence from opioids is also being studied. Therefore, along with tolerance studies, weight changes were also recorded for a preliminary prediction of the effects of NMF on metabolism, and for informing animal care and potential patient care. Habituating in the same environment, the weight of mice in the vehicle group did not fluctuate significantly but the morphine group lost weight significantly after only one day. The chronic administration of 0.1 mg/kg NMF seemed to have a negligible influence in weight, whereas when the dose was increased to 0.5 mg/kg, weight loss was observed on Day 4. Therefore, compared to morphine, NMF was less likely to cause weight loss (Figure 8).

Withdrawal

Physical dependence usually occurs in subjects who repeatedly receive traditional opioid analgesics. Such dependence can always be visualized in morphine-dependent mice by injecting naloxone (NLX). NLX-precipitated withdrawal symptoms include wet dog shakes, paw tremors, jumps, and diarrhea. The mice who received 0.1 mg/kg, a dose resulting in maximum antinociception in the same mice, of NMF twice daily for consecutive four days showed no withdrawal symptoms at all after challenged with 1 mg/kg NLX. Also, the mice in the 0.5 mg/kg NMF group exhibited no jumps or signs of diarrhea after NLX challenge. Although 0.5 mg/kg NMF group showed a few wet dog shakes and paw tremors after NLX injection, all of the observed withdrawal symptoms were significantly fewer than those of morphine-dependent mice. Therefore, NMF administration results in less dependence, even at a very high dose, which is an advantage with respect to treating chronic pain. Moreover, if NMF (or NMF analogs) is to be used for its antinociceptive effects in patients who suffer from opioid use disorders and receive treatments which are MOR antagonists/partial agonists, the occurrence of withdrawal symptoms is unlikely to be a concern while the pain in these patients can be alleviated (Figures 7C and 9).

Respiratory depression

Agonism at the MOR can cause depressed respiration. As NMF exhibited partial MOR agonism in vitro, whole-body plethysmography was utilized to assess the possibility of NMF to induce respiratory depression. Overall, NMF (0.1 mg/kg) showed no significant effects on the respiration of tested mice except for insignificant decreases in frequency and minute volume which were observed in the first 10-15 mins after the administration. Therefore, the limited respiratory effects of NMF from this study agreed with the KOR-DOR dual agonist feature of the compound, but it should be also noted that higher doses of NMF might lead to MOR-mediated respiratory depression due to its partial agonism on the MOR (Figure 10A- C). To compare, NFU also did not induce respiratory depression at 0.001 mg/kg in monkeys.

Locomotor activity

Many KOR agonists have sedative side effects, which might be interpreted as “positive” results in pain-stimulated models, so evaluating the existence and degree of sedation NMF could elicit is important. On the other hand, hyperactivity has been observed in mice that received morphine injections. Therefore, locomotor activity tests were employed to evaluate locomotor effects of NMF in mice quantitively, serving both purposes mentioned above.

As shown (Figure 11), no significant activity-reducing effects were observed after injection of 0.1 mg/kg of NMF or 0.5 mg/kg of NMF. This indicated antinociceptive effects, rather than sedative effects, resulted from the higher %MPE value in the warm-water tail immersion studies. Additionally, although mice in the 0.1 mg/kg NMF group traveled more and made more jump and ambulatory attempts, no significant hyperactivity was elicited by NMF administration.

Primary In Vitro ADMET Studies on NMF

Cardiac toxicity

Inhibition of K _V 11.1 (simply denoted as hERG), a subunit of a potassium channel, can potentially prolong QT-interval and lead to a fatal tachyarrhythmia. This has made hERG a critical anti-target in drug development. Therefore, NMF was evaluated in an automated patch-clamp hERG inhibition assay. In this assay, the inhibition of tail current from a series of concentrations of NMF and a reference compound, E-4031, were tested concurrently using CH0-K1 cell line. The IC50 of E-4031 was 18 nM and NMF 6.7 pM, respectively. Taking the extraordinary analgesic potency into consideration, NMF is not likely to cause cardiac toxicity by blocking hERG when administered at therapeutic doses.

Plasma protein binding

Plasma protein binding (PPB) plays a significant role not only in chemical-induced toxicity but also in modulating drug concentration at the target sites, thereby influencing the in vivo efficacy. The ti/2-determining parameters, Cl and Vd, are also partially dependent on PPB. Therefore, PPB assessment is a necessary routine study in drug discovery and development. Additionally, interspecies difference in PPB, although not common, is sometimes encountered. In order to better predict human PK/PD from preclinical species, the PPB of NMF was tested using both human and rat plasma. NMF exhibited 68% and 76% PPB in human plasma and rat plasma, respectively (Table 4). Both PPBs were higher than those of morphine, but very much lower than alkaloids such as mitragynine and speciociliatine, which also function as opioid agonists and show 90-99% PPB.

Table 4. Plasma Protein Binding of NMF, NFU, and Morphine.

NMF NFU* Morphine plasma, human 68% 74% 35%** plasma, rat 76% 68% 85%*** a. NMF was tested at a concentration of 10 pM. b. Nalfurafine was tested at 2 nM.

*website located atwww.ema.europa.eu

**Klimas, R., Br. J. Anaesth. 2014, 113 (6), 935-944.

***Kimura, Y., In Vivo (Brooklyn). 2017, 31 (5), 811-817.

Permeability studies

In vitro caco-2 permeability assay is a commonly used and accepted surrogate for predicting human intestinal absorption. A compound with a greater-than-lOxlO ^-6 cm/s P _app has been conventionally considered as highly permeable, but the criteria and experiment condition are inconsistent through the literature. We evaluated NMF along with internal standards including propranolol, labetalol, and ranitidine, to more accurately predict the absorption features of NMF and facilitate comparisons (Table 5).

The absorptive permeability (A- B) of NMF was 10.1 xlO ^-6 cm/s, which was slightly lower than the highly permeable drugs, propranolol (%f _a = 100%) and labetalol (%f _a = 90%) (Table 5). Also, Papp (A-B) of NMF was 20 times higher than Ranitidine, which showed a 50% absorption rate. Therefore, NMF possesses moderate-to-high permeability and an absorption rate ranging from 50% to 90%.

On the other hand, NMF showed a secretive transport permeability of 31.8 xlO ^-6 cm/s, yielding an efflux ratio of 3.1. The efflux transport may pose a barrier for intestinal permeability, however according to the empirical rules summarized by Wang, J., et al. (Skolnik, J. W., Current Topics in Medicinal Chemistry. 2013, pp 1308-1316), this efflux ratio should not be a significant risk for reduced permeability.

Table 5. Permeability of NMF and model drugs.

A-B permeability (xlO ^-6 cm/s) B-A Permeability (x 10' ⁶ cm/s) %f _a *

NMF 10.1 + 0.4 31.8 + 0.8

Propranolol 23.8 + 0.4 25.6 + 0.7 100%

Labetalol 18.5 + 0.3 34.2 + 0.4 90%

Ranitidine 0.50 + 0.02 1.3 + 0.2 50% a. Test concentration of all compounds was 10 pM. b. %f _a : extent of absorption in humans. (Error bars represent SEM).

* Volpe, D. A., Clin. Res. Regul. Aff. 2007, 24 (1), 39-47.

Metabolism profiling

Satisfactory oral bioavailability has become critically important in drug development, in which not only permeability but also hepatic metabolic stability plays an important role. Hence, in vitro or ex vivo metabolism studies have become routine for preliminary examinations at an early discovery stage. For this reason, NMF was incubated in human and rat liver S9 fractions for rapid evaluation of overall liver metabolism in these two species. Assuming first-order kinetics (Figure 12), the half-life of NMF was calculated to be over 60 minutes in the human liver S9 fraction, but less than 15 minutes in the rat. Such relatively high stability in the human liver and low in the rat was also observed with other drugs including imipramine, propranolol, and clozapine, which were tested in parallel in this study (Table 6). In short, NMF has shown reasonable stability in human liver metabolism and relatively lower stability in rat liver. This should be considered for all future in vivo preclinical and translational studies.

Table 6. Half-lives of compounds in human and rat liver S9 fractions.

NMF imipramine propranolol clozapine terfenadine verapamil

TI/2 (human liver > 60 > 60 > 60 > 60 10 18

S9, min)

TI/2 (rat liver 10 11 13 10 14 10

S9, min)

CONCLUSIONS

Opioid analgesics have been on the frontline of pain management for many years while their abuse liability has been recognized since their introduction to the field. Through a complementary structure- activity relationship study on nalfurafine, the only KOR agonist approved clinically, we identified an exemplary analog, NMF, as a potent analgesic with minimum abuse liability by acting as a KOR/DOR dual agonist. Follow-up in vitro PK and in vivo PD studies demonstrated its advantageous properties. This chemical entity is a KOR/DOR dual agonist and is a novel analgesic with minimum abuse liability.

EXPERIMENTAL SECTION

Chemistry. General methods. Naltrexone (NTX) was obtained as a free base through the NIDA Drug Supply Program. Other reagents were purchased from commercial vendors (such as Sigma- Aldrich and Aidlab Chemicals) and used without further purification. Flash column chromatography was performed with silica gel columns (230-400 mesh, Merck). ’H (400 MHz) and ¹³ C (100 MHz) nuclear magnetic resonance (NMR) spectra were recorded with tetramethylsilane as the internal standard on a Bruker Ultrashield 400 Plus spectrometer. High resolution mass spectroscopy (HRMS) was performed on an Applied Bio Systems 3200 Q trap with a turbo V source for TurbolonSpray. HPLC analysis was done with a Varian ProStar 210 system on Microsorb-MV 100-5 C8/C18 column (250 mm x 4.6 mm) at 254 nm, eluting with acetonitrile/water (0.1% TFA) (85/15) at 1 mL/min over 30 min. Melting points were determined using OptiMelt automated melting point system (Fisher Scientific).

17-Cyclopropylmethyl-4,5a-epoxy-14-hydroxy-morphinan-6-on e (S2)

6a-Amino-17-cyclopropylmethyl-3, 14-dihydroxy-4, 5 (/-epoxy -morphinan dihydrochloride

(1) Conformation-induced Steric Hinderance Stereoselective Method: Naltrexone (1 g, 2.9 mmol), p-toluene sulfonic acid (PTSA, 60 mg, 0.3 mmol), and 170 mL anhydrous benzene were added to an oven-dried pear-shape round-bottom flask. After allowing the mixture to stir for 5 min, benzylamine (1.0 mL, 9.2 mmol) was added dropwise. Then a Dean-Stark apparatus was installed and the reaction was heated to reflux under N2 for 10 h. After solvent was evaporated to almost dryness and reaction mixture was cooled down to r.t., 20 mL anhydrous EtOH and 2 g 4 A MS were added and stirred for 5 min. Then NaBIL (117 mg, 3.1 mmol) was added and the reaction mixture was allowed to stir at r.t. overnight. The reaction mixture was filtered through celite and the filtrate was concentrated. Then the residue was washed with H2O and extracted with DCM (3 x 100 mL). The combined organic layers were dried over sodium sulfate and concentrated. Purification by flash column chromatography using DCM/MeOH/O.2% NH3 H2O (DCM: MeOH from 100:1 to 50:1) gave light yellow oil-like intermediate 6a-benzylamino-17-cyclopropylmethyl-3, 14-dihydroxy-4, 5<z-cpoxy- morphinan (370 mg, 0.8 mmol, 28%). Noyori catalyst Method: Naltrexone (1 g, 2.9 mmol) and 4.0 mL anhydrous acetonitrile were added to a 50 mL round-bottom flask. A muddy suspension was observed and immediately turned into clear yellow solution after anhydrous triethylamine (TEA, 2.2 mL, 15.6 mmol) was added. Then benzylamine (1.0 mL, 9.2 mmol) was added dropwise followed by dropwise addition of formic acid (1.5 mL, 39.0 mmol). After 10 min, dichloro(p-cymene)Ru(II)dimer (l lmg) was dissolved in 1.0 mL anhydrous acetonitrile and added dropwise. After reaction was allowed to stir for 70 h at r.t. the solvent was removed, pH was adjusted to 9 using NH3 H2O at the end of which no further salt formation was observed. Then, DCM (7 x 30 mL) was used to extract the product. Normal phase purification was performed on an NH3-H2O-basified column using DCM/MeOH/1% NH3 H2O (DCM: MeOH from 200:1 to 170:1) as the eluent. Pure oil-like intermediate 6a- benzylamino-17-cyclopropylmethyl-3, 14-dihydroxy-4, 5a-epoxy-morphinan was obtained (604 mg, 1.4 mmol, yield 48%). Then 6a-benzylamino-17-cyclopropylmethyl-3, 14- dihydroxy-4, 5a-epoxy-morphinan (220 mg, 0.5 mmol) was added to a hydrogenator bottle and 15 mL anhydrous methanol was used as the solvent. The pH of the reaction mixture was adjusted to approximately 2 using 0.4 mL concentrated hydrochloride. Then 10% palladium on carbon (62 mg) was transferred to the bottle and shaken well. Then the bottle was set on hydrogenator at r.t. and ran for 23 h. Then the catalyst was filtered off through celite and the filtrate was concentrated as the crude product. Cold mixture of methanol and isopropanol (1:9) was used to crystallize and light yellow powder was obtained (90 mg, 0.2 mmol, yield 43%). ’H NMR (400 MHz, MeOD) 6 6.78 (d, J = 8 Hz, 1H), 6.69 (d, J = 8 Hz, 1H), 3.81 (dt, Ji = 16 Hz, J ₂ = 4 HZ, 1H, TZ/a iai), 3.31-3.27 (m, 2H), 2.95-2.68 (m, 5 H), 2.52 (m, 1H), 1.88-1.82 (m, 1H), 1.75-1.72 (m, 1H), 1.59 (dd, Ji = 16 Hz, J ₂ = 8 Hz, 1H), 1.21-1.09 (m, 1H), 1.02 (m, 1H), 0.73- 0.68 (m, 1H), 0.36 (m, 1H). HRMS C ₂₀ H ₂₆ N ₂ O ₃ m/z calc. 342.1943, found [M+H] ⁺ 343.2022.

6f!-Amino-17-cyclopropylmethyl-3, 14-dihydroxy-4, 5 (/-epoxy -morphinan dihydrochloride (2) Naltrexone (500 mg, 1.4 mmol), benzoic acid (400 mg, 1.8 mmol), - toluene sulfonic acid (PTSA, 15 mg, 0.05 mmol), and 75 mL anhydrous toluene were added to a 250 mL oven- dried pear- shape round-bottom flask. After allowing the mixture to stir for 5 min, 20 mL anhydrous EtOH was added and the reaction mixture turned into a transparent solution. Then dibenzylamine (360 mg, 1.8 mmol) was added dropwise, followed by additional 25 mL anhydrous toluene. Next, a Dean-Stark apparatus was attached to the flask and reaction mixture was heated to reflux under N ₂ overnight, during which 20 mL liquid was carefully removed from Dean-Stark apparatus. On the following day, the solvent was evaporated to almost dryness and reaction mixture was cooled down to r.t.. Then 20 mL fresh anhydrous EtOH and 2 g 4A MS were added. After 5 min, NaCNBH ₃ (100 mg, 1.5 mmol) was added and the reaction was allowed to stir at r.t. overnight. Then the reaction mixture was filtered through celite and concentrated in vacuo. After water-washing and DCM-extraction, methanol recrystallization was employed to give the desired intermediate, 17 -cyclopropylmethyl- 6 i- dibenzylamino-3, 14-dihydroxy-4, 5a-epoxy-morphinan as fine white powder (390mg, 0.8mmol, yield 57%). Then 17-cyclopropylmcthyl-6/>-dibcnzylamino-3, 14-dihydroxy-4, 5a- epoxy-morphinan (400 mg, 0.7 mmol) was added to a hydrogenator bottle and dissolved in 40 mL anhydrous methanol, resulting in a clear yellow solution. The pH of the reaction mixture was adjusted to approximately 2 using 0.4 mL concentrated hydrochloride. Then, the catalyst, 10% palladium on carbon (200 mg), was transferred to the bottle and shaken well. After the bottle was set on Parr hydrogenator under 60 psi, the hydrogenation reaction was allowed to run at r.t. for three days. Then the catalysts were filtered off and the filtrate was concentrated to a yellow oil. A mixture of methanol and isopropanol (1:9 or 1:10) was used to recrystallize and yield a pure product (2, 210 mg, 0.5 mmol, yield 66%). ’H NMR (400 MHz, MeOD) 6 6.65 (d, J = 8 Hz, 1H), 6.61 (d, J = 8 Hz, 1H), 4.43 (d, J = 8 Hz, 1H), 3.58 (d, J = 4 Hz, 1H), 2.95-2.80 (m, 4H), 2.66-2.60 (m, 1H), 2.40 (d, J = 8 Hz, 1H), 1.97-1.90 (m, 1H), 1.87-1.85 (m, 3H)1.71-1.65 (m, 1H), 1.70-1.61 (m, 2H), 1.47-1.39 (m, 2H), 0.96-0.90 (m, 1H), 0.65- 0.56 (m, 2H), 0.31-0.27 (m, 2H). HRMS C20H26N2O3 m/z calc 342.1943, found [M-H ₂ 0+H] ⁺ 325.1908, [M+H] ⁺ 343.2014.

17-Cyclopropylmethyl-3, 14-dihydroxy-4,5a-epoxy-6a-methylaminomorphinan (3) Noyori catalyst method: Naltrexone (168 mg, 0.5 mmol) and methylamine hydrochloride (139 mg, 1.8 mmol) were added to a round-bottom flask with 1.3 mL anhydrous acetonitrile and stirred for 15 min. Anhydrous triethylamine (0.7 mL) was added dropwise followed by the addition of 0.4 mL formic acid. Dichloro(p-cymene)Ru(II)dimer (3 mg) was then dissolved in 0.5 mL anhydrous acetonitrile and added. After reaction was allowed to stir for 24 h at r.t., a large amount of white solid was precipitated out. Filtration was performed to give off-white solid and dark green filtrate. The solids were dried and obtained as the salt form of the desired product (3), which was neutralized by NH3 H2C) later. The filtrate was concentrated and adjusted to pH = 8-9 using NH3 H2O. Ethyl acetate was used for extraction and the organic layers were collected and concentrated. The products from the filtrate and the solids were combined (135 mg, 0.5 mmol, yield 98%). Note: If reaction mixture turned into a green suspension with little white precipitation, the solvent was removed directly and pH was adjusted to 8-9 using NH3 H2O. After extraction and concentration, the resulting residue was purified by flash silica column chromatography using DCM/MeOH/0.5%NH3-H2O (DCM: MeOH from 50: 1 to 30: 1). ¹ H NMR (400 MHz, MeOD) 6 6.45 (d, J = 8 Hz, 1H), 6.34 (d, J = 8 Hz, 1H), 4.53 (d, J = 4 Hz, 1H, H ₅ 2.97 (d, J = 8 Hz, 1H), 2.91 (d, J = 16 Hz, 1H), 2.87 (dt, Ji = 16 Hz, J ₂ = 4 Hz, 1H, H7), 2.54-2.50 (m, 1H), 2.45 (dd, Ji = 20 Hz, J ₂ = 8 Hz, 1H), 2.32 (s, 1H), 2.28-2.16 (m, 2H), 2.15- 2.06 (m, 2H), 1.56-1.35 (m, 3H), 1.31 (dd, Ji = 12 Hz, J2 = 10 Hz, 1H), 0.77-0.65 (m, 1H), 0.64, -0.57 (m, 1H), 0.41-0.33 (m, 2H), O.O3-O.O3 (m, 2H). HRMS C21H28N2O3 m/z calc 356.2100, found [M-H ₂ O-C4H ₆ +H] ⁺ 285.1613, [M- C ₄ H8+H] ⁺ 303.1722, [M-H ₂ 0+H] ⁺ 339.2078, [M+H] ⁺ 357.2190, [M+Na ⁺ ] ⁺ 379.2016.

17-Cyclopropylmethyl-3,14-dihydroxy-4,5a-epoxy-6[l-methyl aminomorphinan (4)

Naltrexone (450 mg, 1.3 mmol), methylamine hydrochloride (900 mg, 13.3 mmol), and 15 mL methanol was added in a round -bottom flask and stirred at r.t. After 5 min, NaCNBHs (130 mg, 2.1 mmol) was added and reaction mixture was heated to reflux for 20 h. After the reaction was completed, light yellowish solution was observed with some white precipitates at the flask bottom. After removal of the solvent, chloroform was added to dissolve the mixture and NaHCCL solution was added to adjust pH to approximately 9. Chloroform (3 x 70 mL) was used for extraction, after which organic layers were combined, dried over sodium sulfate, and evaporated to dryness. The purification was performed using flash column chromatography with EtOAc/MeOH/O.5% NH3 H2C) (EtOAc : MeOH from 20:1 to 12:1) to give the 6/?-compound 4 (280 mg, 0.8 mmol, yield 62%) and its 6a-epimer (3, 40 mg, yield 9%) (total yield 71%). ^X H NMR (400 MHz, CDCI3) 6 6.66 (d, J = 8 Hz, 1H), 6.54 (d, J = 8 Hz, 1H), 5.45 (broad s, 1H), 4.48 (d, J = 8 Hz, 1H), 3.05 (d, J = 8 Hz, 1H, Hsf 3.00 (d, J = 16 Hz, 1H), 2.62 (dd, Ji = 12 Hz, J ₂ = 4 Hz, 1H), 2.59-2.52 (m, 2H), 2.47 (s, 3H), 2.36 (d, J = 8 Hz, 1H), 2.22 (td, Ji = 12 Hz, J ₂ = 4 Hz, 1H), 2.13 (td, Ji = 12 Hz, J ₂ = 4 Hz, 1H), 1.92- 1.82 (m, 1H), 1.69-1.60 (m, 2H), 1.46-1.35 (m, 2H), 0.88-0.80 (m, 1H), 0.54-0.50 (m, 2H), 0.14-0.10 (m, 2H). HRMS C21H28N2O3 m/z calc 356.2100, found [M-H2O-NHCH3-C3H5- C ₃ H ₅ N+H] ⁺ 213.0765, [M-H ₂ O-NHCH3-C3H ₅ +H] ⁺ 267.1230, [M-H ₂ O-C4H ₆ +H] ⁺ 285.1573, [M-H ₂ O-NHCH ₃ +H] ⁺ 308.1620, [M-H ₂ 0+H] ⁺ 339.2047, [M+H] ⁺ 357.2159, [M+Na ⁺ ] ⁺ 379.1967.

6a-Amino-l 7-cyclopropylmethyl-4,5a-epoxy-14-hydroxy-morphinan dihydrochloride (5) Conformation-induced steric hinderance stereoselective method'. 3 -Dehydroxy-naltrexone (1.2 g, 3.8 mmol), PTSA (74 mg, 0.4 mmol) and 200 mL anhydrous benzene were added to a 500 mL oven-dried pear-shape round-bottom flask. After allowing the mixture to stir for 5 min, benzylamine (0.6 mL, 5.7 mmol) was added dropwise. Then a Dean-Stark apparatus was attached to the flask and the reaction was heated to reflux under N2 for 24 h, during which 20 mL liquid was cautiously removed from the bottom of Dean-Stark apparatus. Before reduction, solvent was evaporated to almost dryness and reaction mixture was cooled down to r.t. 40 mL anhydrous EtOH was added followed by 3 g 4 A MS and stirred for 5 min. NaBH4 (214 mg, 5.7 mmol) was then added and the reaction mixture was allowed to stir at r.t. overnight. After filtration through celite, the reaction mixture was concentrated, washed with H2O and extracted with DCM. Then the combined organic layers were dried over sodium sulfate and concentrated. Purification by flash column chromatography using DCM/NH3 H2O (NH3-H2O:DCM from 0 to 1%) gave intermediate 6a-benzylamino-17- cyclopropylmethyl-4,5a-epoxy-14-hydroxy-morphinan (390 mg, 1.0 mmol, yield 26%) and starting material S2 (250 mg, 0.8 mmol). Noyori catalyst method: 3 -Dehydroxy-naltrexone (1 g, 3.1 mmol) and 5.0 mL anhydrous acetonitrile were added to a 50 mL round-bottom flask, followed by addition of anhydrous TEA (2.2 mL, 15.6 mmol). Then, benzylamine (1.0 mL, 9.3 mmol) was added dropwise followed by dropwise addition of formic acid (1.5 mL, 40 mmol). After 10 min, dichloro(p-cymene)Ru(II)dimer (20 mg) was dissolved in 1.0 mL anhydrous acetonitrile and added dropwise. After reaction was allowed to stir for 40 h at r.t., the solvent was removed, pH was adjusted to 8-9 using NH3 H2O. Then, DCM (3 x 50 mL) was used to extract the product. After concentration, normal phase purification was performed on NH3-H2O-basified column using DCM/MeOH/1% NH3 H2O (DCM:MeOH from 200:1 to 170:1). Pure oil-like intermediate 6a-benzylamino-17-cyclopropylmethyl- 4,5a-epoxy-14-hydroxy-morphinan was obtained (730mg, 1.5mmol, yield 50%). Then 6a- Benzylamino-17-cyclopropylmethyl-4,5a-epoxy-14-hydroxy-morph inan (280 mg, 0.6 mmol) was added to a hydrogenator bottle and 23 mL anhydrous methanol was used as the solvent. The pH of the reaction mixture was adjusted to approximately 2 using 0.3 mL concentrated hydrochloride. Then, catalyst, 10% palladium on carbon (112 mg), was transferred to the bottle and shaken well. The bottle was set on a Parr hydrogenator at r.t. and ran for 24 h under 60 psi. When the reaction was completed, the catalyst was filtered off and the filtrate was concentrated to an oil-like crude product. Cold methanol (or methanol : isopropanol = 1:9) was used to crystallize and yellow powder was obtained (206 mg, 0.5 mmol, yield 83%). ’H NMR (400 MHz, DMSO- ₆ ) 6 9.02 (s, 1H), 8.48 (s, 1H), 7.20 (t, J = 8 Hz), 6.78 (d, J = 8 Hz), 6.70 (d, J = 8 Hz), 4.82 (d, J = 4 Hz, 1H), 4.04 (d, J = 8 Hz, 1H), 3.73 (s, 1H), 3.28-3.21 (m, 3H), 3.17-3.05 (m, 2H), 2.75-2.69 (m, 2H), 1.98-1.94 (m, 1H), 1.68 (d, J = 12 Hz, 2H), 1.44-1.40 (m, 1H), 1.11-1.09 (m, 1H), 0.94-0.85 (m, 1H), 0.70- 0.60 (m, 2H), 0.51-0.40 (m, 2H). ¹³ C NMR (101 MHz, DMSO- ₆ ) d 155.13, 131.44, 129.71, 127.69, 119.33, 109.01, 88.22, 69.30, 61.10, 56.66, 52.34, 46.03, 44.96, 28.67, 27.07, 23.62, 21.31, 5.68, 5.09, 2.63. HRMS C20H26N2O2 m/z calc 326.1994, found [M-H ₂ O-C4H7+H ⁺ ] ⁺ 255.1493, [M-H2O+HT 309.1959, [M+H ⁺ ] ⁺ 327.2069.

6f!-Amino-17-cyclopropylmethyl-4,5a-epoxy-14-hydroxy-morp hinan dihydrochloride (6) 3- Dehydroxy-naltrexone (600 mg, 1.8 mmol), benzoic acid (248 mg, 2.0 mmol), PTSA (30 mg, 0.1 mmol), 75 mL anhydrous toluene, and 20 mL anhydrous EtOH were added to a 250 mL round-bottom flask. After allowing the mixture to stir for 15 min, dibenzylamine (0.4 mL, 1.9 mmol) was added dropwise. Then, a Dean-Stark apparatus was applied to the flask and the reaction mixture was heated to reflux under N2 overnight. In the following morning, solvent was evaporated to almost dryness and reaction mixture was cooled down to r.t.. After 20 mL fresh anhydrous EtOH and 2.2 g 4A MS were added and stirred for 5 min, NaCNBHa (140 mg, 2.2 mmol) was added and the reaction was allowed to stir at r.t. overnight. In the workup, reaction mixture was first filtered through celite and the filtrate was concentrated in vacuo. The residue was washed with H2O and extracted with DCM (6 x 50 mL). Then, the combined organic layers were dried over sodium sulfate and concentrated to a dark yellow oil. Purification by flash column chromatography using DCM/MeOH/O.5% NH3 H2C) (MeOH:DCM from 0 to 1:100) gave the A-dibenzyl intermediate, 6/>-dibcnzylamino- 17- cyclopropylmethyl-4,5a-epoxy-14-hydroxy-morphinan (280 mg, 0.55 mmol, yield 30%). Next, the A-dibcnzyl intermediate was added to a hydrogenator bottle and methanol was used as the solvent. The pH of the reaction mixture was adjusted to approximately 2 using concentrated hydrochloride. Then, the catalyst, 10% palladium on carbon (90 mg), was transferred to the bottle and shaken well. The bottle was set on a hydrogenator under 60 psi at r.t. for three days. After filtration, filtrate was concentrated and washed by a small amount of methanol, resulting in the desired product 6 (yield 63%). ’H NMR (400 MHz, DMSO- d) 6 9.01 (s, 1H), 8.58 (s, 3H), 7.22 (t, J = 8 Hz, 1H), 6.87 (d, J = 8 Hz, 1H), 6.80 (d, J = 8Hz, 1H), 6.56 (s, 1H), 4.77 (d, J = 8 Hz, 1H), 4.13 (s, 1H), 4.00 (s, 1H), 3.39-3.34 (m, 2H), 3.19-3.12 (m, 4H), 3.05 (d, J = 8Hz, 1H), 2.93-2.88 (m, 1H), 2.79-2.66 (m, 1H), 2.48-2.44 (m, 1H), 2.02 (q, J = 12 Hz, 1H), 1.88 (d, J = 12 Hz, 1H), 1.76 (d, J = 12 Hz, 1H), 1.43 (d, J = 12Hz, 1H), 1.29 (t, J = 12Hz, 1H), 1.10-1.07 (m, 1H), 0.69-0.65 (m, 1H), 0.63-0.58 (m, 1H), 0.54- 0.50 (m, 1H), 0.43-0.39 (m, 1H). ¹³ C NMR (101 MHz, DMSO) 6 155.67, 132.08, 130.19, 128.34, 119.85, 109.51, 88.76, 69.84, 61.53, 57.20, 52.88, 49.04, 45.51, 29.24, 27.65, 24.19, 21.80, 6.33, 5.66, 3.21. HRMS C20H26N2O2 m/z calc 326.1994, found [M+H ⁺ ] ⁺ 327.2059.

17-Cyclopropylmethyl-4,5a-epoxy-14-hydroxy-6a-mehthylamin omorphinan (7) 3-

Dehydroxy-naltrexone (300 mg, 0.9 mmol) and methylamine hydrochloride (124 mg, 1.8 mmol) were added to a round-bottom flask with 1.0 mL anhydrous acetonitrile. Then, 0.8 mL anhydrous TEA was added followed by the dropwise addition of 0.4 mL formic acid and stirred for 30 min. Dichloro(p-cymene)Ru(II)dimer (8 mg) was then dissolved in 0.8 mL anhydrous acetonitrile and added dropwise to the reaction mixture. Reaction was allowed to stir for 24 h at r.t.. Next, white precipitate (free base of 7) was filtered off and filtrate was concentrated. The residual oil then was dissolved in ethyl acetate and adjusted to pH = 9 using NH3 H2O. Then, a small amount water was used to wash the organic layer and ethyl acetate (3 x 20 mL) was used for extraction. Next, the organic layers were concentrated to give light green oil-like residue. The combined product was purified by flash column chromatography (DCM/MeOH/l%NH3-H2O) to yield the pure product 7 (300 mg, 0.9 mmol, yield 96%). ’H NMR (400 MHz, CDCI3) 6 7.04 (t, J = 8 Hz, 1H), 6.61 (d, J = 8 Hz, 1H), 6.57 (d, J = 8 Hz, 1 H), 5.02 (s, 1H), 4.71 (d, 7 = 4 Hz, 1H), 3.11-3.05 (m, 3H), 2.71-2.61 (m, 2H), 2.53 (s, 3H), 2.41-2.32 (m, 2H), 2.27-2.21 (m, 2H), 1.77-1.54 (m, 3.5H), 1.44-1.37 (m, 1.5 H), 0.94-0.84 (m, 1H), 0.83-0.69 (m, 1H), 0.58-0.52 (m, 2H), 0.18-0.12 (m, 2H). ¹³ C NMR (101 MHz, CDC1 ₃ ) 6 159.73, 134.62, 130.03, 128.81, 117.79, 105.72, 88.80, 69.99, 62.16, 59.69, 55.35, 46.12, 43.09, 34.15, 33.51, 29.95, 23.53, 21.00, 9.40, 3.97, 3.80. HRMS m/z C ₂ iH28N ₂ O2m/z calc 340.2151, found [M-H ₂ 0+H] ⁺ 323.2100, [M+H] ⁺ 341.2211.

17-Cyclopropylmethyl-4,5(/.-epoxy-14-hydroxy-6f!-methylam inomorphinan (8)

Stereoselective method'. 3 -Dehydroxy -naltrexone (484 mg, 1.5 mmol), benzoic acid (454 mg, 3.7 mmol), PTS A (28 mg, 0.2 mmol), and 50 mL anhydrous benzene were added to an oven- dried pear-shape round-bottom flask. After allowing the mixture to stir for 15 min, 0.2 mL benzylmethylamine was added dropwise. Next, a Dean-Stark apparatus was attached to the flask and reaction mixture was heated to reflux under N2 for 24 h. On the next day, the solvent was evaporated to almost dryness and reaction mixture was cooled down to r.t. Then, 30 mL anhydrous EtOH was added followed by 2 g 4 A MS and stirred for 5 min. NaCNBHs (140 mg, 2.2 mmol) was then added and the reaction was allowed to stir at r.t. overnight. After the reduction was completed, the reaction mixture was filtered through Celite. The filtrate was concentrated, washed with H2O and extracted with DCM (3 x 70 mL). The combined organic layers were dried over sodium sulfate and concentrated, after which the resulted intermediate 6/?-A,A-benzylmethyl-3-dehydroxynaltrexamine (250 mg, 0.58 mmol) was added to a hydrogenator bottle and 20 mL methanol was used as the solvent. The pH of the solution was adjusted to approximately 2 using concentrated hydrochloride, and 10% palladium on carbon (78 mg) was transferred to the bottle and shaken well. After the hydrogenation was allowed to run under 60 psi at r.t. for two days, the catalyst was filtered off and the filtrate was concentrated. Cold methanol was used to crystalize and gave the final salt (97 mg, 0.3 mmol, overall yield 19%). One-pot method'. 3 -Dehydroxy-naltrexone (300 mg, 0.9 mmol), methylamine hydrochloride (622 mg, 9.2 mmol), NaCNBHa (75 mg, 1.2 mmol), and 10 mL anhydrous methanol were added to a round-bottom flask and stirred at r.t for 5 min. Then, reaction mixture was heated to reflux for 24 h. After methanol was removed, chloroform was added to dissolve the mixture and sodium bicarbonate solution was added to adjust pH to approximately 8. Chloroform was used for extraction and the resulting organic layers were combined, dried over sodium sulfate, and evaporated to dryness. The purification was conducted on normal phase silica column with EtOAc/MeOH/1% NH3 H2O (EtOAc:MeOH from 50:1 to 10:1) eluent system to give two desired compounds in approximately 50% yield for the beta-epimer (8, 157 mg) and 35% (7, 110 mg) yield for the alpha-epimer (total yield 85%). ¹ H NMR (400 MHz, CDC1 ₃ ) 67.07 (t, J =7.8 Hz, 1H), 6.67 (d, 7 = 8 Hz, 1H), 6.65 (d, J = 8 Hz, 1H ), 4.36 (d, J = 8 Hz, 1 H), 3.49 (s, 1H), 3.10-3.09 (d, J = 8 Hz, 1H), 3.07 (d, J = 16 Hz, 1H), 2.68-2.62 (m, 2H), 2.46 (s, 3H), 2.41-2.33 (td, 7; =12 Hz, J ₂ = 8 Hz, 1H) 2.37 (d, 7 = 8 Hz, 2H), 2.27-2.18 (td, Ji =12 Hz, 7 ₂ = 8 Hz, 1 H), 2.12-2.06 (td, Ji = 12 Hz, 7 ₂ = 4Hz, 1H), 1.75-1.69 (m, 2 H) 1.64-1.59 (m, 5H), 1.44-1.31 (m, 3H), 0.90-0.81 (m, 2H), 0.55-0.49 (m, 2H), 0.17-0.09 (m, 2H). HRMS C21H28N2O2 m/z calc 340.2151, found [M-H2O-CH3NH- C ₃ H ₅ +H] ⁺ 251.1309, [M-H ₂ O-CH ₃ NH+H] ⁺ 292.1698, [M-H ₂ 0+H] ⁺ 323.2121, [M+H] ⁺ 341.2230, [M+Na ⁺ ] ⁺ 363.2049, [2M+Na ⁺ ] ⁺ 703.4216.

General Procedures for the Final Compounds

Compounds 9 to 12 The corresponding intermediate amine (1-4) was dissolved in anhydrous DCM (0.1 M) and cooled down using an ice bath. Then, anhydrous TEA (4.0 equiv) was added dropwise. The reaction was allowed to stir for half an hour, followed by addition of (E')-3-(3-furanyl)-2-propenoyl chloride (3.5 equiv). After the starting material was consumed, the reaction was quenched using methanol and concentrated to dryness. After the crude product was redissolved in methanol, addition of K2CO3 was followed. The hydrolysis was completed after 14-50 h, and the purification was conducted using flash column chromatography (DCM/MeOH/l%NH3-H2O) gave pure products in form of free base. The free base was dissolved in anhydrous methanol, followed by addition of methanolic hydrochloride (1.3 equiv.) at O °C. The reaction was allowed to stir overnight and diethyl ether was added to precipitate the final hydrochloride salt (overall yield 10-30%).

Compounds 13 to 16 The corresponding intermediate amine (5-8) was dissolved in anhydrous DCM (0.1 M) and cooled down using an ice bath. Anhydrous TEA (4.0 equiv) was added dropwise. Reaction was allowed to stir for half an hour, followed by addition of (£)-3- (3-furanyl)-2-propenoyl chloride (2.0 equiv). After the starting material was consumed, the reaction was quenched using methanol and concentrated to dryness. Purification was done using flash column chromatography (DCM/MeOH/l%NH3-H2O) gave product in the form of free base. Then, the free base was dissolved in anhydrous methanol, followed by addition of methanolic hydrochloride (1.3 equiv.) at 0 °C. The reaction was allowed to stir overnight and then diethyl ether was added to precipitate the final hydrochloride salt (overall yield 20-50%). (2E )-N-[ 6a-l 7-( Cyclopropylmethyl)-3,14-dihydroxy-4,5a-epoxy-morphinan-6-yl] -3-(3- furanyl)-2-propenamide hydrochloride (9) The title compound was obtained following the general procedure for final compounds as a white solid. ’H NMR (400 MHz, DMSO-7d) 6 9.25 (s, 1H), 8.86 (broad s, 1H), 8.02 (s, 1H), 7.86 (d, J = 8 Hz, 1H), 7.74 (d, J = 4 Hz, 1H), 7.35 (d, 7 = 16 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.70 (d, J = 4 Hz, 1H), 6.57 (d, J = 8 Hz, 1H), 6.55(d, J = 16 Hz, 1H), 6.29 (s, 1H), 4.67(d, J = 4 Hz, 1H, H ₅ ), 4.54-4.47 (m, 1H), 3.91(d, J = 8 Hz, 1H, H ₇ axiai), 3.29-3.24 (m, 1H), 3.10-3.03 (m, 1H), 3.00-2.94 (m, 1H), 2.77- 2.67 (m, 1H), 2.48-2.44 (m, 1H), 1.94-1.85 (m, 1H), 1.64 (d, J = 12 Hz, 1H), 1.11-1.03 (m, 1H), 0.97-0.92 (m, 1H), 0.73-0.69 (m, 1H), 0.65-0.59 (m, 1H), 0.52-0.46 (m, 1H), 0.43-0.37 (m, 1H). ¹³ C NMR (101 MHz, DMSO-7 ₆ ) 6 165.00, 146.48, 145.30, 144.89, 139.31, 129.62, 129.23, 123.14, 122.57, 122.33, 119.60, 118.71, 107.98, 87.93, 69.82, 61.54, 57.51, 45.69, 45.65, 45.62, 30.69, 29.75, 23.95, 20.19, 6.16, 5.63, 3.04. HRMS m/z C27H ₃ oN ₂ 0 ₅ calc 462.2155, found [M+H] ⁺ 463.2245. Melting point: decomposed at 214.3 °C. HPLC: Rt = 7.106 min; purity: 98.2%.

(2E )-N-[ 6p~l 7-( Cyclopropylmethyl)-3,14-dihydroxy-4,5a-epoxy-morphinan-6-yl] -3-(3- furanyl)-2-propenamide hydrochloride (10) The title compound was obtained following the general procedure for final compounds as a white solid. ’H NMR (400 MHz, DMSO-7d) 6 9.29 (s, 1H), 8.82 (s, 1H), 8.33 (d, J = 8 Hz, 1H), 8.00 (s, 1H), 7.72 (s, 1H), 7.31 (d, J = 12 Hz, 1H), 6.73 (s, 1H), 6.72 (d, J = 8 Hz, 1H), 6.65 (d, J = 8 Hz, 1H), 6.31 (d, J = 12 Hz, 1H), 6.17 (s, 1H), 4.60 (d, J = 8 Hz, 1H, H ₅ ). 3.85 (d, J = 4 Hz, 1H), 3.56-3.47 (m, 1H), 3.35-3.30 (m, 2H), 3.09 (d, J = 8 Hz, 1H), 3.04 (d, J = 8 Hz, 1H), 2.86 (t, J = 12 Hz, 1H), 2.45-2.39 (m, 1H), 1.82-1.71 (m, 2H), 1.59-1.55 (m, 1H), 1.45 (d, J = 8 Hz, 1H), 1.37 (t, J = 12 Hz, 1H), 1.27-1.21 (m, 1H), 1.11-1.06 (m, 1H), 0.69-0.65 (m, 1H), 0.61-0.58 (m, 1H), 0.53-0.49 (m, 1H), 0.44-0.39 (m, 1H). ¹³ C NMR (101 MHz, DMSO- ₆ ) d 165.24, 145.28, 144.87, 142.62, 141.81, 130.13, 129.76, 123.03, 122.19, 121.05, 119.75, 118.44, 108.08, 90.47, 70.22, 62.22, 57.22, 51.25, 46.99, 46.10, 29.90, 27.82, 24.25, 23.50, 6.20, 5.57, 3.12. HRMS m/z C27H30N2O5 calc 462.2155, found [M+H] ⁺ 463.2222, [M+Na] ⁺ 485.2022. Melting point: decomposed at 231.5 °C. HPLC: Rt = 7.201 min; purity: 99.0%.

(2E)-N-[6a-17-(Cyclopropylmethyl)-3,14-dihydroxy-4,5a-epo xy-morphinan-6-yl]-3-(3- furanyl)-N-methyl-2-propenamide hydrochloride (11) The title compound was obtained following the general procedure for final compounds as a white solid. ¹ H NMR (400 MHz, DMSO-7 ₆ ) 6 9.32 (s, 1H), 8.83 (s, 1H), 8.06 (s, 1H), 7.73 (s, 1H), 7.45 (d, 7 = 16 Hz, 1H), 7.03 (s, 1H), 6.97 (d, 7 = 16 Hz, 1H), 6.73 (d, 7 = 8 Hz, 1H), 6.60 (d, 7 = 8 Hz, 1H), 6.27 (s, 1H), 5.04 (d, J = 12 Hz, 1H), 4.71 (d, J = 4 Hz, 1H, ft), 3.92 (d, J = 8 Hz, 1H), 3.37-3.27 (m, 2H), 3.14-3.09 (m, 1H), 3.05 (s, 1H), 2.94-2.88 (m, 2H), 2.74-2.68 (m, 1H), 2.46-2.43 (m, 1H), 1.99-1.91 (m, 1H), 1.64-1.56 (m, 2H), 1.44-1.41 (m, 1H), 1.24-1.06 (m, 2H), 0.71- 0.60 (m, 2H), 0.49-0.40 (m, 2H). ¹³ C NMR (101 MHz, DMSO- ₆ ) 6 166.42, 163.47, 146.17, 145.21, 145.11, 139.53, 132.45, 129.39, 123.61, 122.55, 119.81, 118.87, 118.58, 108.65, 89.47, 69.42, 61.59, 57.50, 49.44, 46.16, 45.71, 32.06, 30.58, 30.29, 23.89, 18.40, 6.17, 5.66, 3.05. HRMS C28H32N2O5 m/z calc 476.2311, found [M+H] ⁺ 477.2201. Melting point: decomposed at 231.5 °C. HPLC: Rt = 7.417 min; purity: 98.4%.

(2E )-N-[ 6p~l 7-( Cyclopropylmethyl)-3,14-dihydroxy-4,5a-epoxy-morphinan-6-yl] -3-(3- furanyl)-N-methyl-2-propenamide hydrochloride (12) The title compound was obtained following the general procedure for final compounds as a light yellow solid. ¹ H NMR (400 MHz, DMSO-ft) Abundance ratio of two tautomers is 2:3 = T1 : T2. 6 9.69 (s, 0.6 H, T2), 9.29 (s, 0.4H, Tl), 8.85 (s, 1H), 8.04 (s, 0.4 H, Tl), 7.92 (s, 0.6 H, T2), 7.73 (s, 0.4 H, Tl), 7.67 (s, 0.6 H, T2), 7.37 (d, 7 = 16 Hz, 0.4H, Tl), 7.23 (d, 7 = 16 Hz, 0.6H, T2), 7.01 (s, 0.4 H, Tl), 6.91 (d, 7 = 16 Hz, 0.4H, Tl), 6.85 (d, 7 = 8 Hz, 0.6H, T2), 6.71 (d, 7 = 8 Hz, 1H), 6.65 (d, 7 = 8 Hz, 0.4H, Tl), 6.63 (s, 0.6 H, T2), 6.50 (s, 0.6 H, T2), 6.40 (s, 0.4 H, Tl), 6.36 (d, 7 = 16 Hz, 0.6H, T2), 4.93 (d, 7 = 8 Hz, 0.4H, Tl, ft), 4.86 (d, 7 = 8 Hz, 0.6H, T2, ft), 4.20 (m, 0.4 H, Tl), 3.86 (m, 1H), 3.59 (m, 0.6 H, T2), 3.42- 3.38 (m, 2H), 3.16 (s, 1.2 H, Tl), 3.09-3.05 (m, 2H), 2.93 (s, 1.9 H, T2), 2.89-2.87 (m, 2H), 2.60-2.52 (m, 2H), 2.22-2.06 (m, 1H), 1.48-1.25 (m, 3H), 0.70-0.66 (m, 1H), 0.61-0.59 (m, 1H), 0.53-0.50 (m, 1H), 0.44-0.41 (m, 1H). ¹³ C NMR (101 MHz, DMSO-ft) 6 165.90, 144.46, 144.17, 142.09, 141.34, 132.01, 130.88, 123.07, 122.97, 118.97, 118.52, 117.80, 107.64, 69.69, 69.60, 61.34, 61.15, 56.61, 56.57, 46.38, 45.90, 45.84, 30.36, 30.21, 22.93, 22.48, 5.71, 5.11, 2.58. HRMS C ₂ 8H32N ₂ O ₅ m/z calc 476.2311, found [M+H] ⁺ 477.2394. Melting point: decomposed at 221.2 °C. HPLC: Rt = 7.563 min; purity: 98.9%.

(2E)-N-[6a-17-(Cyclopropylmethyl)-4,5a-epoxy-14-hydroxy-m orphinan-6-yl]-3-(3- furanyl)-2-propenamide hydrochloride (13) The title compound was obtained following the general procedure for final compounds as a white solid. ’H NMR (400 MHz, DMSO-d6) 6 8.91 (s, 1H), 8.02 (s, 1H), 7.94 (d, 7 = 8 Hz, 1H), 7.74 (s, 1H), 7.35 (d, 7 = 16 Hz, 1H), 7.18 (t, 7 = 8 Hz, 1H), 6.75 (d, 7 = 8 Hz, 1H), 6.68 (d, 7 = 8 Hz, 1H), 6.67 (s, 1H), 6.52 (d, 7 = 16 Hz, 1H), 6.34 (s, 1H), 4.68 (d, 7 = 4 Hz, 1H), 4.57-4.48 (m, 1H), 3.95 (d, 7 = 8 Hz, 1H), 3.47 (d, 7 = 20 Hz, 1H), 3.29-3.26 (m, 1H), 3.17 (dd, Ji = 20 Hz, 7 ₂ = 8 Hz, 1H), 3.14-2.94 (m, 1H), 2.76-2.70 (m, 1H), 2.46-2.41 (m, 1H), 1.95-1.86 (m, 1H), 1.65-1.53 (m, 1H), 1.47-1.38 (m, 1H), 1.08 (m, 1H), 0.99-0.84 (m, 1H), 0.72-0.59 (m, 2H), 0.52-0.38 (m, 1H). ¹³ C NMR (101 MHz, DMSO-7 ₆ ) 6 165.03, 159.77, 159.12, 145.32, 144.90, 133.15, 130.27, 129.65, 123.13, 122.25, 118.96, 107.95, 106.69, 69.74, 69.74, 61.39, 57.54, 45.49, 45.48, 44.93, 30.60, 29.63, 24.65, 20.05, 6.14, 5.64, 3.04. HRMS C27H30N2O4 m/z calc 446.2206, found [M+H] ⁺ 447.2285. HPLC: R _t = 8.514 min; purity: 98.8%.

(2E)-N-[6P-17-(Cyclopropylmethyl)-4,5a-epoxy-14-hydroxy-m orphinan-6-yl]-3-(3- furanyl)-2-propenamide hydrochloride (14) The title compound was obtained following the general procedure for final compounds as a white solid. ’H NMR (400 MHz, DMSO-7d) 6 8.90 (s, 1H), 8.36 (d, J = 8 Hz, 1H), 8.01 (s, 1H), 7.73 (s, 1H), 7.30 (d, J = 16 Hz, 1H), 7.19 (t, J = 8 Hz, 1H), 6.84 (d, J = 8 Hz, 1H), 6.75 (d, J = 8 Hz, 1H), 6.74 (s, 1H), 6.30 (d, 7 = 16 Hz, 1H), 6.26 (s, 1H), 4.61 (d, J = 8 Hz, 1H, H ₅ ), 3.91(d, J = 4 Hz, 1H), 3.56-3.42 (m, 2H), 3.19 (dd, J/ = 20 Hz, 7 ₂ = 8 Hz, 1H), 3.10-3.01 (m, 2H), 2.91-2.84 (m, 1H), 2.46-2.41 (m, 2H), 1.84-1.74 (m, 2H), 1.57-1.52 (m, 1H), 1.44 (d, 7 = 8 Hz, 1H), 1.40-1.33 (m, 1H), 1.11-1.06 (m, 1H), 0.73-0.66 (m, 1H), 0.63-0.57(m, 1H), 0.55-0.49 (m, 1H), 0.45-0.39 (m, 1H). ¹³ C NMR (101 MHz, DMSO-7 ₆ ) 6 165.17, 156.31, 145.30, 144.96, 131.69, 129.97, 129.85, 128.84, 123.01, 122.04, 119.24, 109.46, 108.07, 90.85, 70.13, 61.98, 57.16, 51.00, 46.72, 45.92, 45.41, 39.39, 29.93, 27.67, 24.17, 6.19, 5.59, 3.11. HRMS C27H30N2O4 m/z calc 446.2206, found [M+H] ⁺ 447.2296. Melting point: decomposed at 232.9 °C. HPLC: Rt = 7.657 min; purity: 97.9%.

(2E)-N-[6a-17-(Cyclopropylmethyl)-4,5a-epoxy-14-hydroxy-m orphinan-6-yl]-3-(3- furanyl)-N-methyl-2-propenamide hydrochloride (15) The title compound was obtained following the general procedure for final compounds as a white solid. ¹ H NMR (400 MHz, DMSO-7 ₆ ) 6 8.89 (s, 1H), 8.14 (s, 0.2H), 8.06 (s, 0.5H), 7.73 (s, 0.5H), 7.66 (s, 0.2H), 7.46 (d, 7 = 16 Hz, 1H), 7.20 (t, 7 = 8 Hz, 1H), 7.22-7.18 (m, 0.2H), 7.01 (s, 0.5H), 6.96 (d, 7 = 16 Hz, 1H), 6.78 (d, 7 = 8 Hz, 1H), 6.70 (d, 7 = 8 Hz, 1H), 6.71-6.69 (m, 0.5H), 6.34 (s, 1H), 5.10-4.98 (m, 1H), 4.72 (d, 7 = 4 Hz, 1H), 3.97-3.93 (m, 1H), 3.50-3.45 (m, 1H), 3.30-3.18 (m, 2H), 3.07-2.79 (m, 1H), 2.72-2.6 (m, 1H), 2.03-1.91 (m, 1H), 1.64-1.53 (m, 2H), 1.45- 1.42 (m, 1H), 1.24-1.05 (m, 3H), 0.73-0.61 (m, 2H), 0.52-0.40 (m, 1H). ¹³ C NMR (101 MHz, DMSO-7 ₆ ) 6 163.48, 145.14, 137.15, 133.22, 130.47, 128.03, 127.96, 127.37, 123.59, 122.58, 119.28, 118.79, 108.64, 96.41, 75.60, 69.38, 61.47, 61.39, 57.53, 45.45, 45.42, 39.59, 39.38, 30.40, 30.33, 30.28, 24.59, 6.15, 5.65, 3.06, 0.58. HRMS C28H32N2O4 m/z calc 460.2362, found [M+H] ⁺ 461.2417. Melting point: decomposed at 248.3 °C. HPLC: Rt = 8.705 min; purity: 99.0%.

( 2E )-N-[ 6p~l 7-( Cyclopropylmethyl)-4,5a-epoxy-14-hydroxy-morphinan-6-yl]-3-( 3- furany)-N-methyl-2-propenamide hydrochloride (16) The title compound was obtained following the general procedure for final compounds as a white solid. ¹ H NMR (400 MHz, CDC1 ₃ ) 67.57 (s, 1H), 7.50 (d, J = 16 Hz, 1H), 7.39 (s, 1H), 7.16 (t, J = 8 Hz, 1H ), 6.76 (d, J = 8 Hz, 1H), 6.70 (d, J = 8 Hz, 1H), 6.42 (s, 1H), 6.38 (d, 7 = 16 Hz, 1H), 4.58 (d, J = 8 Hz, 1H, H ₅ 3.72-3.66 (m, 1H), 3.14 (s, 1H), 3.12 (d, J = 12 Hz, 1H), 3.03 (s, 3H), 2.71 (d, J = 8 Hz, 1H), 2.67 (d, J = 4 Hz, 1H), 2.40 (d, J = 4 Hz, 2H), 2.28 (td, Ji = 8 Hz, J ₂ = 4 Hz, 2H), 2.11 (td, Ji = 12 Hz, J ₂ = 4 Hz, 1H), 1.71 (dt, Ji = 12 Hz, J ₂ = 4 Hz, 1H), 1.49-1.45 (m, 3H), 0.90-0.81 (m, 1H), 0.58-0.53 (m, 2H), 0.18-0.11 (m, 2H). ¹³ C NMR (101 MHz, DMSO-7 ₆ ) 6 163.48, 155.83, 145.21, 145.09, 144.99, 144.86, 131.19, 129.96, 129.84, 129.15, 123.42, 119.01, 109.51, 87.61, 70.14, 61.77, 61.65, 57.14, 46.69, 45.68, 45.65, 29.41, 27.40, 24.11, 21.11, 6.19, 5.60, 3.10. HRMS C28H32N2O4 m/z calc 460.2362, found [M+H] ⁺ 461.2414. Melting point: decomposed at 251.2 °C. HPLC: Rt = 8.251 min; purity: 97.3%.

In Vitro Biological Evaluations.

Competitive radioligand binding assay. [ ³ H]Naloxone was used to label the MOR and [ ³ H]diprenorphine was used to label the KOR and DOR. The Kd and B _m ax values of the MOR, KOR, and DOR were determined using 5 pM NTX, U50,488H and SNC80, respectively. The cells were centrifuged at 1000 g for 10 min and at 50,000 g for 10 min in membrane buffer. After homogenization in 4 mL TME buffer (50 mM Tris, 3 mM MgCh, and 0.2 mM EGTA, pH 7.4.), the Bradford assay was conducted to determine the concentration of the membrane protein. 300 L TME buffer and 50 pL radioligand solutions was added to all test-tubes. 50 pL of unlabeled ligands and testing compounds solutions were added to their respective testtubes. 50 pL Radioligand solutions were also to 3 scintillation vials containing 4 mL scintillation fluid as standard. Finally, 100 pl of membrane protein (30 pg/tube) was added to all the test-tubes to afford a total volume of 500 pl. All the test-tubes were vortexed, and incubated at 30 °C for 90 min. After incubation, the samples containing bound radioligands were filtered using a Brandel harvester. The filter papers containing filtered samples were then transferred into the scintillation vials filled with 4 mL of scintillation fluid. After 9 h, the samples were quantified using the liquid scintillation counter. Competition for bound radioligand was calculated using nonlinear regression analysis to determine the IC50 values with GraphPad 6.0 software. The Ki values were determined from the IC50 values using the Cheng-Prusoff equation. The assay was performed in duplicates and repeated at least three times.

[ ³⁵ S]-GTPyS binding assay. 10 pg of mMOR-CHO, mKOR-CHO or mDOR-CHO membrane protein was incubated with 20 pM GDP, 0.1 nM [ ³⁵ S]-GTPyS, assay buffer (TME + 100 mM NaCl), and varying concentrations of the testing compounds for 90 min in a 30 °C water bath. Nonspecific binding was determined with 20 pM unlabeled GTPyS. 3 pM DAMGO, 5 pM U50,488H or 5 pM SNC80 was included as maximally effective concentration of a full agonist for the MOR, KOR or DOR, respectively. Assay buffer was used for all the dilutions. 250 pL Assay buffer, 50 pL of GDP, 50 pL of cold GTPyS, testing compounds, and 50 pL [ ³⁵ S]-GTPyS were added to the test-tubes accordingly. 50 pL [ ³⁵ S]- GTPyS was also added to 2 scintillation vials as standards. Finally, 100 pL of membrane protein were added and all test tubes were incubated at 30 °C for 90 min. Similar work-up and quantification procedures were performed to the competitive radioligand binding assay. Percent DAMGO/U50,488H/SNC80-stimulated [ ³⁵ S]-GTPyS binding was defined as (net- stimulated binding by ligand/net- stimulated binding by 3 pM DAMGO/5 pM U50,488H/5 pM SNC80) x 100. The normalized data were subjected to nonlinear regression analysis to determine EC50 and E _m ax values using GraphPad 6.0 software. hERG (automated patch-lamp) toxicity. hERG toxicity study was performed in CHO-K1 cell line. The degree of inhibition (%) was obtained by measuring the tail current amplitude, which is induced by a one second test pulse to -40 mV after a two second pulse to +20 mV, before and after drug incubation (the difference current was normalized to the control). Concentration (log) response curves were fitted to a logistic equation (three parameters assuming complete block of the current at very high test compound concentrations) to generate estimates of the 50% inhibitory concentration (IC50). The concentration response relationship of the test compound was constructed from the percentage reductions of current amplitude by sequential concentrations.

Caco-2 permeability. The apparent permeability coefficient (Papp) of the test compound was calculated as follows:

V _R *CR,end 1

Papp(cm/s)= - * where VR is the volume of the receiver chamber. CR, _en d is the concentration of the test compound in the receiver chamber at the end time point, At is the incubation time and A is the surface area of the cell monolayer. CD, mid is the calculated mid-point concentration, which is the mean value of the donor concentration at time 0 and the concentration at the end time point. CR, id is the mid-point concentration in the receiver side. Concentrations of the test compound were expressed as peak areas of the test compound. Fluorescein was used as the cell monolayer integrity marker. Fluorescein permeability assessment (in the A-B direction at pH 7.4 on both sides) was performed after the permeability assay for the test compound. The cell monolayer had a fluorescein permeability of less than 1.5 x 10’ ⁶ cm/s.

Protein binding. Dialysis membranes were soaked in DI water, 30% ethanol and isotonic sodium phosphate buffer subsequently. Plasma was obtained by centrifuge from fresh blood. The dialysate side of the 96-well dialysis apparatus was loaded with 0.15 mL of phosphate buffer (0.05 M sodium phosphate in 0.07 M NaCl, pH 7.5). The same volume of plasma spiked with 10 mM test compound was pipetted into the sample side. After 8 h incubation at 37 °C, post-dialysis plasma and buffer volumes were recorded and 90 mL of phosphate buffer was added to every 10 mL of plasma, and then precipitated with two volumes of acetonitrile. The quantification data was collected using LC-MS and calculated as following:

Area _p -Areab

Protein binding (%) = 100

Area _p +Areab

Recovery (%) = - * 100

Area _c where Area _p = Peak area of analyte in protein matrix; Areab = Peak area of analyte in buffer; Area _c = Peak area of analyte in control sample

Hepatic metabolism S9 fraction incubation 0.1 pM of NMF or reference compounds was tested in human liver S9 plus 1 mM UDPGA or rat liver S9 plus 1 mM UDPGA, respectively. At time 0, 15, 30, 45 and 60 minutes of incubation, the concentration of each compound was determined using LC-MS. After the experiment, metabolic stability, expressed as percent of the parent compound remaining, was calculated by comparing the peak area of the compound at the time point relative to that at time 0. The half-life (T1/2) was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming the first-order kinetics. In Vivo Studies. Animals. Mice: 5-8 Week 25-35 g male Swiss Webster mice (Envigo Laboratories, Frederick, MD, USA) were housed in cages (5 maximal per cage) in animal care quarters and maintained at 22 ± 2 °C on a 12 h light-dark cycle, except for the mice used for respiration measurement who were maintained in the reversed light-dark cycle. Food (standard chow) and water were available ad libitum. The mice were brought to the lab (22 ± 2 °C, 12 h light/dark cycle) and allowed 18 h to recover from transport. All studies used at least six mice for each group, and withdrawal studies were performed in the respective mice that were used in tolerance studies. Rats: A total of 10 rats (5 males and 5 females) Sprague- Dawley rats were acquired at approximately 8-10 weeks of age (Envigo Laboratories, Frederick, MD, USA) and surgically implanted with custom-made jugular catheters and vascular access ports (Instech, Plymouth Meeting, PA, USA) as described in detail elsewhere. Rats were singly housed in a temperature and humidity-controlled vivarium that was maintained on a 12 h light/dark cycle. Water and food (Teklad Rat Diet, Envigo) were provided ad libitum in the home cage. Protocols and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Commonwealth University Medical Center and comply with the recommendations of the IASP (International Association for the Study of Pain).

Warm-water immersion assay. The tail-flick test was performed using a water bath with the temperature maintained at 56 ± 0.1 °C. The distal one-third of the tail was immersed perpendicularly in water, and the mouse rapidly flicked the tail from the bath at the first sign of discomfort. The duration of time the tail remained in the water bath was counted as the baseline latency. Untreated mice with baseline latency ranging from 2 to 4 seconds were used. Test latency was obtained 20 min later after injection (s.c.). A 10-second maximum cutoff latency was used to prevent any tissue damage. Antinociception was quantified as the percentage of maximal possible effect (%MPE), which was calculated as %MPE= [(test latency - control latency )/( 10 - control latency)] x 100. Each compound was tested in a group of six mice. In dose-response studies, each dose of each compound was tested in a group of six mice. The doses were designed to give %MPE from 80%-20% for an accurate ED50. ED50 values were calculated using the least squares linear regression analysis followed by calculation of 95% confidence interval by the Bliss method. One-way ANOVA followed by the post-hoc Dunnett test was performed to assess the significance using Prism 8.0 software (GraphPad Software, San Diego, CA). Drug self-administration. Heroin hydrochloride and fentanyl hydrochloride were provided by the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD). Modular operant chambers located in sound-attenuating cubicles (Med Associates, St. Albans, VT) were equipped with two retractable levers and LED lights. Intravenous (i.v.) compound solutions were delivered as described previously elsewhere. After each behavioral session, catheters were flushed with gentamicin (0.4 mg) and catheter patency was verified at the end of each experiment by instantaneous muscle tone loss following methohexital (0.5 mg) administration. Rats were initially trained to respond for i.v. heroin (32 pg/kg/infusion) under a fixed ratio (FR) 5/time out 20 s schedule of reinforcement during daily 2 h sessions. Each session began with a non-contingent infusion of the available heroin dose followed by a 60 s time out. The response period was signaled by the extension of only the right lever and illumination of the right green stimulus light. Following each response requirement completion, the lever was retracted, the green light was extinguished, and a heroin dose was infused. This schedule was in effect until the number of heroin infusions earned per session was within 20% of the running mean for three consecutive sessions. Subsequently, saline was substituted for heroin every other session (i.e., SDSDS; S, saline; D, drug) until the number of saline infusions earned was at least 75% lower than the number of heroin infusions earned during the preceding heroin session for two consecutive alternations. The same experimental program was utilized during the saline substitution sessions, using the same infusion duration as a 32 pg/kg/infusion of heroin of 5 s per 300g of rat weight. Once training criteria were met, test sessions were inserted into the sequence (i.e., DTSTD or STDTS; T, test) to evaluate responses over a range of NMF doses (1-100 pg/kg/infusion). Fentanyl doses (0.32-10 pg/kg/infusion) were also determined as a positive control. Saline and each unit NMF and fentanyl dose was tested once in each rat using a counterbalanced dosing order. The primary dependent measure for the NMF and fentanyl self-administration studies was the number of infusions earned per session and these data were plotted as a function of drug dose. Data were analyzed using a one-way repeated-measures ANOVA followed with Dunnet’s post-hoc tests using Prism 9.0 software.

Toxicity, tolerance and cross tolerance. Vehicle, 0.1 mg/kg NMF, 0.5 mg/kg NMF, and 10 mg/kg morphine was given (s.c.) to four groups of mice twice a day. Weight was measured before drug administration every day and warm water tail immersion was performed to each mouse 20 min after the first injection in the day. On day 5, vehicle, 10 mg/kg morphine, and 0.1 mg/kg NMF, were given to the mice who received morphine injections for 4 days continuously, respectively. Then cross tolerance was evaluated using warm water tail immersion experiment. Weight change and tolerance data were analyzed using t-test, i.e., data collected from day 2-4 was compared to day 1, and cross tolerance data was analyzed using one-way ANOVA followed with Dunnet’s post-hoc tests using Prism 8.0 software.

Withdrawal studies, morphine-dependent mice: 75 mg morphine pellets were implanted as described previously elsewhere. In brief, mice were anesthetized with 2.5% isoflurane, neck was shaved and cleaned with povidone-iodine, and then a 1-cm horizontal incision was made at the base of the neck. A 75-mg morphine pellet was inserted in the space before closing the site with Clay Adams Brand, MikRon AutoClip 9-mm wound clips (BD Diagnostics, Sparks, MD). The animals were allowed to recover in their home cages where they remained throughout the experiment. NMF-dependent mice: 0.1 mg/kg NMF or 0.5 mg/kg NMF was given subcutaneously to mice every 12 hours for four consecutive days. On day 5, 1 mg/kg of naloxone was administered to NMF-treated and morphine-pelleted mice, 3 minutes after which the numbers of paw tremors, wet dog shake, and jumping were counted for a period of 20 minutes for each mouse. And the occurrence of diarrhea was recorded as 0 or 1 for each mouse at the end of each withdrawal-observation experiment. Data was analyzed using one-way ANOVA followed with Dunnet’s post-hoc tests using Prism 8.0 software.

Measurement of respiration. Respiration was measured in freely moving mice using plethysmography chambers (EMKA Technologies, France) supplied with a 5% CO2 in air mixture (BOC Gas Supplies, UK) as described previously. Mice were habituated to plethysmograph chambers for 15 min before the experimentation. A 5-min baseline respiration period was recorded prior to challenge with any compound. Rate and depth of respiration were recorded and averaged over 1 min periods and converted to minute volume (rate x tidal volume). Tidal volume was calculated from the raw inspiration and expiration data ⁹¹ . Data were normalized as percentage of baseline and analyzed using a one-way repeated-measures ANOVA followed with Dunnet’s post-hoc tests using Prism 9.0 software.

Locomotor activities.Activity chambers (Med Associates, St. Albans, VT) were used for locomotor activity study. Each individual chamber has closeable doors and a ventilation system. The interior of the chamber consists of a 27 x 27 cm Plexiglas enclosure that is wired with photo-beam cells connected to a computer console that counts the activity of the animal. Mice were habituated to the chamber for 20 minutes 24 h before the experiment. On the day of experiment, mice were injected with a desired dose of the compound subcutaneously and placed in the chambers immediately. Ambulatory counts, jumps, distance traveled, and average speed were monitored and recorded for 30 minutes. Data were analyzed using a oneway repeated-measures ANOVA followed with Dunnet’s post-hoc tests using Prism 9.0 software.

ABBREVIATIONS AND ACROYNMS

CHO, Chinese hamster ovary; CL, clearance; CNS, central nervous system; DDI, drug-drug interaction; DOR, delta opioid receptor; FDA, Food and Drug Administration; KOR, kappa opioid receptor; MOR, mu opioid receptor; MPE, maximum possible effects; P _app : apparent permeability coefficient; PD: pharmacodynamics; PK: pharmacokinetics; PPB: plasma protein binding; Vd, volume of distribution.

EXAMPLE 2. Systematic Structure-Activity Relationship Study of Nalfurafine and Characterization of Developed Analogs

Despite the availability of numerous approved pain medications, it is crucial to acknowledge that the current array of FDA-approved options falls short in adequately addressing pain in some scenarios, consequently fueling the problem of opioid misuse. Thus, it is imperative to develop novel, non-addictive pain medications. Towards addressing this, nalfurafine was chosen as a lead due to its intriguing pharmacological profile and mild side effects. Thus, nalfurafine’ s structure-activity relationships were systematically studied through the design, synthesis, and evaluation of twenty-four analogs. Subsequently, these analogs were subjected to cellular and behavioral pharmacological testing in a tier wise manner to evaluate binding affinity, selectivity, efficacy, and functional activity at the three canonical opioid receptors, in vivo opioid agonism, dose-response, time-course, receptor selectivity, potential for tolerance, dependence, abuse, drug distribution, weight fluctuations with chronic treatment and effects on locomotor activity.

Rational molecular design was conducted with NLF as the lead resulting in 64 designed ligands. The design was based around variation of five key components of the NLF molecular structure (Figure 13.) 1) Compounds either maintain or lack the 3-hydroxy group present on NLF. This functional group may be crucial for recognition of binding sites of different opioid receptors, and combined with the C6 conformation may govern selectivity, as demonstrated by us and others. 2) Compounds vary in their conformational arrangement, taking either the 6a or 6P isomeric form; this may test for a more favorable selectivity profile. 3) The amide nitrogen atom is secondary or tertiary via presence of a methyl group. This may verify whether the N-methyl is important for KOR agonist activity. Additionally, it may influence the hydrophobicity of the ligands as well. 4) The linker between the furan and amide carbon atoms is either conjugated via a trans double bond or saturated (i.e., single bond). This enables assessment of the degree of rigidity that is tolerated and/or flexibility that is needed for binding. 5) The furan ring attachment point is varied between the 2’ and 3’ position; this variation probes the binding pocket for the optimal binding interaction. Taken together, these variations result in changes in affinity, efficacy, and biophysical properties, thus expanding upon the SAR profile and potentially yielding a new lead compound for further investigation. Of note, the 17-cyclopropylmethyl and 14-hydroxy group were not varied because their modification has been previously found to decrease KOR selectivity and binding affinity.

2.2 Chemical Synthesis

Syntheses of the 24 designed ligands contain multiple divergent and convergent methods as depicted in Figure 15. If the final compound will not bear a 3-hydroxyl group, 3 -dehy doxy naltrexone must first be synthesized. Naltrexone and 3-dehydoxynaltrexone, as the two starting materials containing the classical epoxymorphinan skeleton, were then subjected to four different reductive amination methods to yield eight different naltrexamines: namely, 6a-naltrexamine, 6a-3-dehydroxynaltrexamine, 6P-naltrexamine, 6P-3-dehydroxynaltrexamine, 6a-N-methyl-naltrexamine, 6a-N-methyl-3- dehydroxynaltrexamine, 6P-N-methyl-naltrexamine, and 6P-N-methyl-3- dehydroxynaltrexamine. Primary amines were then directly coupled to carboxylic acids. Secondary amines, however, must be coupled to acid chlorides. Due to the lack of commercial availability of this series of acid chloride, these compounds were prepared prior to coupling. These two distinct coupling conditions resulted in two groups of compounds: one that can be presumed to have a substituted 3-OH position (due to the presence of 3-OH in the starting naltrexamine) and one that cannot have a substituted 3-OH position (due to the absence of 3-OH in the starting naltrexamine) not dependent on the coupling mechanism used. Compounds that had substituted 3-hydroxyl positions were then hydrolyzed and all final, free base compounds were purified by column chromatography. After purification, target compounds were transferred into their respective hydrochloride salt forms. Final compounds were characterized by 1H NMR, 13C NMR, HRMS, HPLC and MP. 2.2.1 Physiochemical Property Prediction

The Advanced Chemistry Development Inc. (ACD) Percepta® software was used to predict the physiochemical properties (e.g., cLogP, cpKa, cLogD and TPSA) of designed ligands to assess their drug-likeness. This software aids in screening of compounds using generally accepted guidelines for drug discovery such as Lipinski’s rule of five for oral bioavailability. This rule of five postulates that ligands should have a LogP less than five, less than five hydrogen bond donors, less than ten hydrogen bond acceptors, and a molecular weight less than 500 g/mol. According to Table 6, the designed ligands meet all of these criteria. Additionally, while LogP describes the partition coefficient of uncharged molecules, LogD is calculated at a specific pH (often the physiological pH of 7.4) to account for ionizable molecules. The optimal LogD for drug molecules may be between 1-3, and all of the compounds fall within this range. For CNS permeability, base pKa values must fall below 10.5 which is demonstrated in the results that are further supportive of potential for oral delivery as pKa values between 2-3 and 7-8 are promising for absorption in the stomach and intestines, respectively. TPSA is a predictor of drug absorption with values over 140 A indicating poor absorption which these compounds are not subject to. Of note, while there are slight, rather predictable differences among the compounds (e.g., increased lipophilicity in the presence of the R2 methyl group [1 vs. 2; 3 vs. 4 etc.]), the overall similarity of structural features makes it difficult to exclude synthesis of any compounds solely based on these predictions.

Table 6. Physiochemical property predictions made with ACD Percepta ®.

Mol. Wt.: Molecular weight (g/mol); cLogP: calculated partition coefficient; cLogD: calculated distribution constant (at physiological pH); cpKa: calculated negative logarithm of the acid dissociation constant; HBD: hydrogen bond donors; HBA: hydrogen bond acceptors; TPSA: topological polar surface area

2.3 In Vitro Binding Affinity and Functional Activity Studies

2.3.1 Radioligand Binding Assays

Competitive radioligand binding assays are a standard characterization method to determine the binding affinity and selectivity of synthesized ligands for the opioid receptors, namely MOR, KOR, and DOR. These assays were carried out according to previously published methods. As demonstrated in Table 7, the synthesized ligands have a range of binding affinities for the opioid receptors. Half of the compounds (1, 5, 6, 7, 8, 13, 15, 16, 17, 21, 23 (NCF), and 24) have subnanomolar binding for the KOR. Additionally, half of the compounds have subnanomolar binding for the MOR (5, 6, 7, 8, 13, 14, 15, 16, 21, 22, 23 (NCF), and 24), while three compounds (5, 13, and 21) have subnanomolar binding for the DOR.

Table 7. Affinity and selectivity for the KOR, MOR, and DOR as determined by radioligand binding assays using CHO cell membrane homogenate.

Compd. Compd. Variations Radioligand Binding Affinity (K., Selectivity nM) Ratio

NMF ²⁸ OH CH ₃ a a 0.12 + 0.47+ 0.64+ 4.10 5.57

0.02 0.06 0.11

NLF ²⁸ OH CH ₃ a 0.19+ 0.72+ 115.9+ 3.76 603.7

0.01 0.09 20.9

1 H CH ₃ a b 0.48+ 7.74+ 48.58+ 16.22 101.85

0.07 0.69 6.51

2 H H a b 161.4+ 54.52+ 153.9+ 0.34 0.95

11.8 2.01 26.5

3 H CH ₃ p b 34.48 + 43.81 + 890.8+ 1.27 25.84

6.27 2.85 52.5

4 H H p b 8.37+ 17.26+ 749.4+ 2.06 89.51

1.44 1.68 59.1

5 OH CH ₃ a b 0.13 + 0.18+ 0.59+ 1.40 4.53

0.02 0.02 0.06

6 OH H a b 0.78+ 0.44+ 3.54+ 0.56 4.54

0.04 0.10 0.50

7 OH CH ₃ p b 0.28+ 0.53 + 8.42+ 1.88 29.98

0.05 0.06 0.90

8 OH H p b 0.47+ 0.21 + 32.70+ 0.45 69.57

0.03 0.01 3.47

9 H CH ₃ a c 1.00+ 14.14+ 46.82+ 14.10 46.68

24 OH H p d 0.17 + 0.30 + 12.84 + 1.74 74.64

0.02 0.02 2.37

Based on the radioligand binding assay, multiple structure-activity relationship (SAR) trends can be either observed or reinforced. The first of these is that KOR binding affinity is increased with the presence of the 3-hydroxyl group (Table 7). At the onset of the study, the importance of the 3-hydroxyl group for binding affinity was still debatable. Eight structurally related compounds suggested that the presence of the 3-hydroxyl group is not absolutely necessary for strong KOR binding affinity as 5 of the 8 compounds had subnanomolar binding affinity with two of them being 3 -dehydroxy compounds. However, the trend that KOR binding affinity is increased with the presence of a 3-hydoxyl group is more strongly supported in head-to-head comparisons found in Table 7 due to three separate R3 sidechain variations. Further, it is interesting that in all three sets for comparison, the binding affinity difference is much smaller for the first pair of compounds in the set (i.e., 1 vs. 5). This is intriguing because for each of these pairs, the amine portion of the compound is the same, and the only difference is the C6 sidechain. The second trend is that all compounds and only compounds with a 3- hydroxyl group have subnanomolar binding affinity for the MOR (Table 7). This perfectly aligns with published SAR regarding the presence of the 3-OH and MOR binding affinity. A third SAR trend is that DOR binding affinity seems to be dependent on 3-OH presence (Table 7) In all pairwise comparisons, binding affinity is stronger in the presence of the 3-hydroxyl. However, it is of note that binding affinity is weaker than the other 3-OH compounds when the amide bears 3-OH, is secondary, and is of P conformation and stronger when it bears 3- OH, -CH3, and a conformation. Additionally, there is no apparent effect of the methyl group, the a/p configuration, or the R3 sidechain on KOR or MOR binding affinity while P isomers seemed to have decreased DOR binding affinity (Table 7).

2.3.2 [35S]-GTPyS Functional Assays

The [35S]-GTPyS -binding assay is a functional assay used to assess the in vitro potency and efficacy of synthesized ligands. [ ³⁵ S]-GTPyS binding assays were used to determine whether ligands function as agonists, partial agonists, antagonists, or inverse agonists for the KOR, MOR, and DOR. Their efficacy was related to that of the respective full agonist control following previously established protocol. As displayed in Table 8, most synthesized ligands are full KOR agonists with partial MOR agonism and a range of DOR agonism. More specifically, all compounds have KOR efficacy >80% except compound 2, and all compounds have MOR efficacy <40% except compounds 8 and 16. Compounds that have DOR efficacy >80% include 2, 10, 17, 19, 20, 21 and 23 (NCF).

Table 8. Functional activity at the KOR, MOR, and DOR as determined by [ ³⁵ S]-GTPyS assays using CHO cell membrane homogenate. Efficacy values (Emax) are represented as percent of U50-488, DAMGO, and SCN80 full agonist controls for KOR, MOR, and DOR, respectively.

Compd. Compd. Variations [ ³⁵ S]-GTPyS Functional Activity

0.00 3.02 0.03 0.53 0.74 10.6

NLF ²⁸ OH CH ₃ p a 0.26+ 95.95+ 0.52+ 30.24+ 141.4+ 65.68+

0.03 4.13 0.12 3.73 14.8 6.42

1 H CH ₃ a b 6.50+ 97.51+ 20.03+ 11.53+ 137.3+ 56.44+

1.11 4.80 3.49 0.81 26.5 2.60

2 H H a b 1014+ 66.06+ 199.7+ 8.80+ 429.4+ 93.13+

176 5.20 63.1 0.55 62.6 1.57

3 H CH ₃ b 245.6+ 86.42+ 223.6+ 13.77+ 5783+ 66.86+

29.2 0.63 44.9 1.86 829 3.52

4 H H p b 117.1+ 81.39+ 185.2+ 14.19+ 1687+ 55.78+

21.2 7.72 38.0 1.27 116 2.39

5 OH CH ₃ a b 0.04+ 105.9+ 0.37+ 17.95+ 0.75+ 68.31+

0.01 2.2 0.05 1.81 0.09 1.00

6 OH H a b 6.11+ 80.32+ 2.31+ 27.62+ 4.26+ 76.87+

0.83 4.73 0.87 1.96 0.76 5.40

7 OH CH ₃ p b 0.99+ 101.8+ 4.30+ 12.20+ 19.17+ 51.44+

0.04 1.05 0.47 0.92 1.62 2.78

8 OH H p b 0.90+ 95.91+ 0.63+ 42.61+ 14.29+ 46.59+

0.10 1.43 0.12 2.61 1.27 4.06

9 H CH ₃ a c 18.42+ 91.38+ 76.50+ 11.22+ 211.8+ 70.51+

1.05 1.46 48.47 1.55 33.8 5.90

10 H H a c 441.8+ 348.3+ 278.1+ 10.21+ 348.3+ 85.65+

29.9 40.2 106.8 1.33 40.2 3.74

11 H CH ₃ p C 145.9+ 109.3+ 5625+ 22.95+ 1105+ 61.71+

27.1 5.9 2130 2.39 108 4.27

12 H H p c 367.5+ 86.83+ 104.4+ 10.84+ 2395+ 70.98+

31.8 0.69 24.2 1.13 200 6.10

13 OH CH ₃ a c 0.04+ 100.6+ 0.31+ 15.26+ 0.66+ 63.07+

24 OH H p d 0.54+ 98.53+ 6.76+ 7.33+ 13.07+ 42.81+

0.11 0.60 1.99 0.65 2.09 4.01

Multiple S AR trends can either be observed or reinforced based on the functional activity data. First of which is that the methyl group seems to be important for KOR agonism. In almost all cases, the KOR efficacy is higher when the methyl group is present (Table 8). The two exceptions to this are compounds 10 and 20. This helps to bring clarity to the literature inconsistencies surrounding this trend. The second trend is that compounds with the 3- dehydroxy, secondary amide, a configuration combination and a saturated side chain may result in higher DOR efficacy. Finally, the exceptions to the generation of ligands with MOR efficacy values under 40 are limited to those bearing the 3-OH, desmethyl, P configuration, and saturated R3 sidechain combination (Table 8; compounds 8 and 16).

2.4 Preliminary In Vivo Characterization

Warm-water tail immersion (WWTI) assays are often employed to rapidly characterize opioid receptor ligands; this is a technique that we have used for many years. Briefly, to assess potential antinociception, an animal’s tail (in this case a mouse) is dipped into a warm-water bath. The mouse then flicks its tail to remove the thermal stimulus. This establishes a baseline latency for withdrawal. Administration of synthesized ligands as pretreatments allows determination of whether the compound has agonist activity similar to known control agonists (single-dose agonism paradigm) while pretreatment to known agonists allows determination of potential antagonist effects (single-dose antagonism paradigm) similar to that of known control antagonists. These assays elucidate information about in vivo functional activity, potency, time course, and opioid receptor involvement.

2.4.1 Step 1: Single-Dose Agonism

First, a WWTI assay using the single-dose agonism protocol was used as a preliminary in vivo screen. In this assay, morphine, as a positive control, standardly shows a maximum possible effect (MPE) of 100% indicating that the mouse’s tail remained in the warm-water bath for the maximal amount of time (10 s). Figure 16 shows that the MPE elicited by a single dose of 10 mg/kg in the WWTI assay varied greatly by test compound with the results divided into categories based on MPE values. Of note, antinociceptive effects indicated here cannot be interpreted as compounds acting as partial agonists because their response in this assay is dose-dependent and dose was not an independent variable, these compounds may simply be less potent. Nonetheless, compounds with MPEs from 80-100% include 1, 7, 13, 15, 17, 21, 22, 23 (NCF), and 24. This is mainly in agreement with the most promising compounds identified by the in vitro binding assay which were 1, 5, 7, 8, 13, 15, 16, 21, 23 [NCF], and 24 (overlapping compounds underlined).

2.4.2 Step 2: Dose-Response

The single-dose agonism paradigm of the WWTI assay was also used to determine the in vivo potency of the synthesized ligands that displayed MPEs between 80-100% in the single-dose agonism screen. Here, the dose of the novel ligand given as a pretreatment was an independent variable rather than a fixed dose. Table 9 shows that the test compounds have roughly a 1,000-fold range of in vivo potencies. Further, all but two compounds (17 and 22) were more potent than morphine of which six compounds had potencies <1 mg/kg while one compound (23 [NCF]) is approximately equipotent to fentanyl. Finally, compounds 21 and 23 (NCF) are approximately equipotent to more potent than NLF.

Interestingly, seven out of the nine compounds that were selected for dose-dependence study bear the R2 methyl group. The remaining two aid in forming the conclusion that presence of this methyl group may be important for in vivo antinociceptive potency via comparison to their methyl-bearing counterparts (22 vs 21; 24 vs. 23 [NCF]). This is additionally in agreement with a previously published study of structurally similar compounds.

Table 9. Potency determination of selected synthesized ligands using a warm-water tail immersion assay in male mice (n = 6/group).

Morphine ¹²⁴ - - 3.9 [3.3 - 4.6]

Fentanyl ¹²⁴ - - - - 0.015 [0.013 - 0.018]

Nalfurafine OH CH p a 0.046 [0.016 - 0.126] (NEF) ²⁸

NMF ²⁸ OH CH ₃ a a 0.037 [0.014 - 0.096]

1 H CH ₃ a b 0.553 [0.353 - 0.866] 3.094 [1.576 - 6.073]

7 OH CH ₃ p b 0.443 [0.293 - 0.669] 3.297 [1.674 - 6.493]

13 OH CH ₃ a c 0.123 [0.039 - 0.384] 2.182 [1.296 - 3.673]

15 OH CH ₃ p C 0.251 [0.170 - 0.371] 3.105 [1.280 - 7.531]

17 H CH ₃ a d 7.796 [0.394 - 2,484.127 [0.072 -

154.348] 85,616,662.29]

21 OH CH ₃ a d 0.051 [0.035 - 0.075] 0.234 [0.163 - 0.336]

22 OH H a d 4.077 [1.041 - 15.968] 182.680 [1.246 -

26,781.913]

23 (NCF) OH CH ₃ p d 0.010 [0.007 - 0.015] 0.108 [0.064 - 0.182]

24 OH H p d 3.322 [0.545 - 20.251] 475.589 [0.047 -

4,841,934.455]

2.4.3 Step 3: Time Course To determine the time course of the synthesized ligands in the WWTI assay, the singledose agonism paradigm was used wherein the ligand is administered s.c. one time and the time lapse in relation to the injection is an independent variable following published protocol. Shown in Figure 17, 0.1 mg/kg NLF resulted in significant antinociceptive effects immediately after injection that lasted for 1 h and were significant once more 4 h post- injection with a return to baseline 9 h post-injection. This is consistent with previous studies which showed: 1) an onset of sedative effects 10 min post i.v. infusion of 0.001 mg/kg with significant sedation lasting for 2 h but a visual decrease in behavior remaining for 4 h (study end) in rhesus monkeys; 2) 0.032 mg/kg NLF results in a significant decrease in intracranial self-stimulation (ICSS) 10 min post i.p. injection with a peak effect at 30 min post-injection and a return to baseline 24 h post-injection; 3) low dose (0.002 mg/kg, i.t.) NLF showed significant antinociception from 5-60 min after injection with non-significant effects lasting out to 150 min. Significant antinociception was produced by morphine 30 min to 1 h postinjection with effects near-baseline by 3 h and back to baseline by 5 h.

Compounds 21 and 23 (NCF) both show antinociceptive effects by 30 min postinjection. While those of compound 21 are significant for 2 h and those of compound 23 (NCF) are significant for 5 h, neither compound returns to baseline by 10 h post-injection. Thus, 0.1 mg/kg compounds 21 and 23 (NCF) show longer durations of action than both 0.1 mg/kg NLF and 10 mg/kg morphine in the WWTI assay. They appear to have similar onsets in this assay at approximately 30 min with a slightly delayed peak for morphine (1 h post-injection). However, this assay is not sufficient to make detailed conclusions comparing onset of action due to the frequency of tail immersions used.

2.4.4 Step 4: Opioid Receptor Selectivity

The WWTI assay was additionally used to assess which opioid receptors are responsible for the in vivo effects of ligands. The receptor selectivity study is a modification of the single-dose antagonism paradigm wherein known selective opioid receptor antagonists are pretreatments to synthesized ligands following previously published protocol. More specifically, the selective and irreversible MOR antagonist P-funaltrexamine (P-FNA), the selective KOR antagonist nor-binaltorphimine (nor-BNI), and the selective DOR antagonist naltrindole (NTI) were used. These compounds at these doses would be hypothesized to inhibit the antinociceptive effects mediated by their respective receptors in the WWTI assay.

Figure 18 shows that the MPE of NLF is significantly decreased by both NTI and nor- BNI indicating DOR and KOR involvement, respectively. Interestingly the combination of NTI and nor-BNI pretreatment did not result in a more drastic decrease than either antagonist alone. However, the high variance in this data point makes it difficult to draw any conclusions. The selectivity of NLF at the opioid receptors has been previously assessed and was repeated herein as an internal control to ensure alignment with previously reported data. In previous reports, 10 mg/kg nor-BNI s.c. pretreatment antagonized the sedative and antinociceptive effects of low dose (0.01 mg/kg) but not high dose (0.03 mg/kg) NLF and 20 mg/kg nor-BNI s.c. pretreatment antagonized antinociceptive effects of 0.001 - 0.01 mg/kg NLF.36, 37 In addition, 3 mg/kg NTI was not able to antagonize the antinociceptive effects of NLF.36 In the current study, nor-BNI when given at the same 10 mg/kg dose s.c. was able to antagonize a higher dose of NLF (0.1 mg/kg). One potential explanation for the difference in these results is that the study that reported a lack of antagonism at the 0.03 mg/kg dose was performed in a different species. As a result, the dose at which nor-BNI is no longer effective may be different in mice. Further, while NTI is able to antagonize the antinociceptive effects of NLF in the current study and not in the previous one, this is most likely due to the 5-fold difference in the NTI dose administered.

As for compound 21, the antinociceptive effects are antagonized by both nor-BNI and NTI while pretreatment with both nor-BNI and NTI seems to compound the decrease in compound 21 MPE. This indicates that the in vivo effects of compound 21 are primarily KOR and DOR mediated. Compound 23 (NCF) on the other hand is antagonized by NTI but shows only a slight, non-significant decrease in antinociception with nor-BNI pretreatment while pretreatment with both NTI and nor-BNI seems to compound antagonistic effects. This result that 10 mg/kg s.c. nor-BNI is not effective at antagonizing antinociceptive effects of a higher dose of compound 23 (NCF; 0.1 mg/kg) seems to be in agreement with the previously discussed finding for NLF performed in monkeys. Additionally, these results are in agreement with a previous study on a structurally related compound.

2.4.5 Step 5: Single-Dose Antagonism

In studying the SAR of NLF it is important to recognize any small structural differences that could result in an agonist to antagonist change. Thus, ligands not producing antinociceptive activity in the single-dose agonism screen at 56°C were further studied to monitor their potential to block morphine’ s effects using published procedures. As the positive control, naloxone is able to sufficiently antagonize morphine’s effects in this assay. However, none of the other compounds administered acted as antagonists (Figure 19). There are many potential explanations for this, making speculation difficult without a complete dose-response curve for these compounds in both agonism and antagonism mode. It may be that these compounds have very low potencies which would be in agreement with their in vitro results. 2.5 Further In Vivo Characterization

2.5.1 Locomotor Activity

A well-known liability of KOR agonists is hypolocomotion. This hypolocomotion could result in false positives in assays of evoked antinociception such as WWTI. Thus, a locomotor activity assay was used to assess this endpoint in accordance with previous studies. Mice followed the testing schedule shown in Figure20A. In brief, the day prior to testing, mice were placed in the open field chamber for acclimation. On test day, they were injected with the synthesized ligand or vehicle (double-distilled water, negative control) and immediately placed back into the open field chamber. Photo beam breaks then record the mouse’s locomotion over a 30 min test period.

Figure 20B-E shows the distance travelled (cm), ambulatory counts, vertical counts and average speed (cm/s) during the acclimation period of the locomotor activity assay while Figure 20F-I displays these same endpoints after compound injection. Overall, distances, counts and speed are reduced in the testing sessions as compared to their corresponding acclimation periods. This is attributable to the novelty-induced hyperlocomotion of the open field chamber during the acclimation period as expected.

Further, there are no statistical differences from vehicle except for vertical counts in the 0.5 mg/kg compound 23 (NCF) group. However, there is a dose-dependent trend for decreased distance travelled, ambulatory counts, vertical counts, and average speed with compound 23 (NCF) administration. Thus, a post hoc power analysis was done using GPower3.1 to determine the achieved power with these studies. While a power of 0.8 is commonly accepted, the achieved power was roughly 0.3 meaning that there is a 70% chance that statistical significance would not be detected even if it is there. This is most likely attributable to the high variance that can be observed in the vehicle and compound 21 groups. A way to overcome this would be to add more mice to the study. However, approximately 120 mice (n=20/group) would be needed to achieve a power of 0.8 which is cost, time and resource prohibitive. Thus, the conclusion drawn would be that there is a trend for a dose-dependent decrease in locomotor activity with compound 23 (NCF) administration. In addition, there are slight dose-dependent trends for decreased locomotion with NLF administration. This is in agreement with published studies that NLF does not result in hypolocomotion at low doses but does at higher doses. In addition, high variance in the compound 21 group across all endpoints both in the acclimation period and after compound administration makes it hard to conclude that this compound does not have any effects on locomotion; this is similar to a previously reported structurally related compound. If there is less hypolocomotion produced by compound 21, it could be due to the synergistic effect of the KOR and DOR allowing for antinociception at non-sedative doses as hypothesized. However, any diminished hypolocomotion may be more likely attributable to a potency difference as compound 23 (NCF) is approximately 5x as potent than NLF or compound 21. If this is the case, at more equivalent low doses, compound 23 (NCF) would be hypothesized to similarly not engender hypolocomotion while at equivalent high doses compound 21 and NLF would.

2.6 In Vivo Pharmacodynamic and Toxicity Studies

2.6.1 Abuse Liability via a Self- Administration Model

The potential for abuse liability is a concern with the development of novel opioid ligands. To address this, self-administration assays were conducted in rats under a single-lever operant procedure with only slight variations of published methods. In brief, rats were implanted with indwelling catheters as per published methods and trained to respond for drug (i.e., fentanyl) on an FR5 schedule of reinforcement. Then, the fentanyl-containing syringe that delivers i.v. infusions with the aid of a syringe pump, was switched out on and off with saline until rats were able to reliably distinguish the difference between fentanyl and saline according to established criteria. Once this criterion was met, the novel ligand (or fentanyl at different doses as a positive control) was inserted into the rotation as “test days”. Behavioral sessions occurred daily, in 2 h blocks from approximately 9:30 AM - 11:30 AM.

As a positive control, the reinforcing nature of fentanyl was determined using the selfadministration procedure. Figure 21 shows that in this procedure, fentanyl yields the expected inverted U-shaped dose-effect function. These results are in alignment with a previous study that used fentanyl at the same doses as well as the same self-administration procedure. In addition, they are in concordance with years of self-administration research. Supportive of a lack of abuse liability, nalfurafine did not function as a reinforcer in a dose range similar to that of fentanyl. This is congruent with previous studies of nalfurafine in rats, rhesus monkeys and humans. Further, these results are similar to those of a previous study in which the same doses of nalfurafine were used and the number of infusions at each dose was found to be significantly lower than that of saline. Additionally, neither compound 21 nor compound 23 (NCF) show significant differences from the number of earned infusions during a saline baseline. This is consistent with prior studies that interrogate the potential abuse liability of compounds that carry KOR agonist activity. One interpretational limit to this is that the long duration of these compounds may result in earned infusions spaced further than the 2 h behavioral session can detect (i.e., rate-limiting effects).

2.6.2 Tolerance Potential

The potential for the development of tolerance is a liability for pain medications, particularly those that are opioidergic. Thus, an assay was used to assess the potential for the development of tolerance with chronic treatment of the synthesized ligands. As shown in Figure 22A, the synthesized ligands as well as morphine (positive control) and vehicle (negative control) were administered two times per day for four days with injections spaced 12 h apart. Efficacy was assessed after the second injection of the day using the WWTI assay. Morphine is known to result in tolerance due its effects at the MOR, it is hypothesized that administration of a ligand with a distinct mechanism of action to mice that are morphine- tolerant may result in a return of antinociceptive effects. This would manifest as an increased withdrawal latency or percent maximum possible effect (MPE) when compared to morphine- tolerant mice in the WWTI assay. Thus, synthesized ligands were administered to morphine- tolerant mice on day 5 and the MPE reassessed (Figure 22E).

As shown in Figure 22B-D, statistically significant tolerance developed in the morphine group by day 4. However, at this point roughly 70% of the maximum possible effect was maintained. Thus, it was not very drastic and retention of antinociception at this point does not allow for much potential recovery of antinociceptive effects with administration of a second compound (cross-tolerance). This is similar to previously reported effects using the same protocol wherein day 4 antinociception in the morphine group was approximately 50%. Under the same conditions, mice given compound 21 (0.1, 0.5 mg/kg) and compound 23 (NCF; 0.5 mg/kg) developed tolerance on day 3 while mice that received compound 23 (NCF; 0.1 mg/kg) developed tolerance on day 4. This dose-response difference may indicate involvement of a second receptor responsible for antinociceptive effects and these results are consistent with previously reported studies. Figure 22F displays the results of the cross -tolerance portion of the assay. Here, it seems that cross-tolerance is present with lower dose KOR agonists with dual-activity (e.g., 0.1 mg/kg compound 21, 0.1 mg/kg NLF, and 0.05 mg/kg compound 23 [NCF]) but is overcome with higher dose KOR agonists with dual-activity (e.g., 0.1 mg/kg compound 23 [NCF] ; 5x more potent than compound 21 and NLF) . Additionally, the level of antinociception reached when compound 23 (NCF; 0.1 mg/kg) was given to morphine-tolerant mice was lower than that when the same dose was given to non-morphine-tolerant mice, similar to previous studies.

2.6.3 Weight Fluctuation

Attention to potential weight fluctuations when novel ligands that may be administered chronically, in particular those with opioid receptor activity, is important. This is because a liability of mu opioid agonists with chronic use is weight loss. Further, weights recorded daily during the chronic treatments in the tolerance and cross-tolerance studies discussed previously facilitate assessment of this endpoint without the inclusion of any additional animals.

The weight fluctuations observed throughout these studies are documented in Figure 23. As shown in Figure 23A-B, there were no significant weight fluctuations for vehicle treated mice (negative control) while the morphine treated mice lost weight over the course of the five-day study (positive control). While there are no significant changes for many of the compound doses, there are increases on day 3 and days 3 and 5 in 0.5 mg/kg NLF and 0.1 mg/kg compound 23 (NCF), respectively. As per current understanding, this weight gain is not readily attributable to the KOR or DOR. Further, weight gain with chronic administration of compounds with similar structures did not result in weight increases in other studies. Due to the presence of a vehicle group as a negative control and the lack of observation in other groups, it is hard to attribute these increases due to normal growth. In addition, the weight increase is not present at the higher dose of compound 23 (NCF; 0.5 mg/kg) indicating that it is not a result of sedation. Thus, while the cause is unclear, the small but significant changes may be due to the variability in the mice.

2.6.4 Withdrawal

Chronic drug administration may result in physical dependence and spontaneous withdrawal is difficult to study in vivo. Thus, antagonist-induced withdrawal studies are a common, efficient, and translationally relevant experimental approach. A modification of our procedure (to more closely resemble NLF studies) was used to determine the propensity of -12- synthesized ligands to produce dependence. As shown in Figure 24A-C, mice were injected with the synthesized ligand or morphine positive control two times per day for four days with injections spaced 12 h apart to potentially result in dependence on the ligand. On day five, the non-selective MOR/KOR antagonist naloxone (1 mg/kg) was administered to precipitate any potential withdrawal symptoms and 3 min after injection, mice were observed for the effects of jumps, front paw tremors, “wet dog shakes” and diarrhea for a period of 20 min. Notably, jumps and diarrhea are not included on Figure 24B because they were not observed in any of the groups. While the absence of these two typical preclinical indications of withdrawal in mice may initially result in suspicion surrounding dependence formation, previously discussed tolerance and weight fluctuation studies indicated that this dosing regimen of morphine produces significant, albeit slight, tolerance. However, this tolerance may not have been significant enough to produce robust dependence and thus precipitate all typical withdrawal signs.

Regardless, NLF seemed to produce less withdrawal on the wet dog shakes and front paw tremors endpoints than morphine. However, this difference was non-significant. While a different dosing regimen is followed, this is in alignment with published results that NLF results in slight dependency and withdrawal. In contrast, compounds 21 and 23 (NCF) [0.1 and 0.5 mg/kg; 0.05, 0.1, 0.5 mg/kg, respectively] resulted in significantly less wet dog shakes when compared to morphine. Additionally, compounds 21 and 23 (NCF) [0.1 and 0.5 mg/kg; 0.1 mg/kg, respectively] resulted in significantly less paw tremoring than morphine. These results are consistent with a previously reported, structurally related compound. Overall, compounds 21 and 23 (NCF) appear to precipitate less withdrawal (as an indicator of dependence formation) than both morphine and NLF, which is a positive indication for any future development as therapeutics with indications of pain management wherein chronic treatment would be involved.

2.6.5 Drug Distribution

Drug distribution studies can be used to assess the ability of a synthesized ligand to penetrate the blood-brain barrier. Briefly, following previously published methods, mice were injected with the synthesized ligand while brain and blood samples were collected at several time points and further analyzed via LC-MS/MS to allow visualization of any progressive distribution.

Figure 25A-F shows that compounds 21 and 23 (NCF) were present in the plasma as early as 5 min after their s.c. injection, at which point the plasma concentration was the highest for both compounds. In addition, the brain-to-plasma concentration ratio progressively increased from 5 to 60 min for both compounds 21 and 23 (NCF). This indicates that the compounds not only penetrated the BBB, but also that they were accumulating in the CNS over the time points tested. Additionally, it implies that they were not rapidly effluxed. Further, the initial spike in plasma concentration is expected considering pharmacokinetic principles wherein there is a high plasma concentration initially after injection followed by drug distribution and metabolism.

3 Conclusion

Despite the multitude of existing options available for the treatment of pain, they often fall short in providing adequate relief. This shortcoming is one driver of the opioid crisis. Given this challenge, this research project aimed to design a non-addictive pain medication. Nalfurafine is an approved antipruritic agent in Japan with additional intriguing pharmacological results with regard to pain was chosen as a lead for further optimization through systematic structure-activity relationship (SAR) studies. Modifications to the structure of nalfurafine at five positions yielded a series of twenty-four analogs. These twenty- four analogs were obtained in 5-6 steps each.

Analyzing these analogs through radioligand binding and functional assays for the MOR, KOR, and DOR as well as warm-water tail immersion studies in male mice allowed identification and further confirmation of 3 main SAR trends: (1) the 3-hydroxyl group increases KOR binding affinity, is essential for MOR binding affinity, and additionally increases DOR binding affinity, (2) KOR in vitro efficacy is higher with R2 methyl group presence, and (3) the R2 methyl group may increase in vivo antinociceptive potency. The designed ligands ranged in both binding affinity and functional activity for three canonical opioid receptors. There were twelve ligands with subnanomolar binding affinity for KOR, twelve ligands with subnanomolar binding affinity for MOR, and three ligands with subnanomolar binding affinity for DOR. All ligands (except one) had KOR efficacy >80% and all ligands except two had MOR efficacy <40% while seven ligands had DOR efficacy >80%.

The behavioral pharmacology of these ligands was further assessed in a progressive, tier- wise manner. Nine of the twenty-four compounds have maximum possible effects (MPEs) over 80% in the warm-water tail immersion assay indicating potential antinociception. When nine compounds displaying under 40% MPE in the agonism screening were tested for potential antagonism for SAR purposes, none of the compounds were able to antagonize the effects of morphine. Based on these results, the nine compounds displaying over 80% MPE in the agonism screen were further analyzed for their antinociceptive potency; seven compounds were more potent than morphine of which six compounds had potencies <1 mg/kg. Taking their in vitro and preliminary in vivo results together, two compounds (compounds 21 and 23 [NCF]) were selected for further studies. Both compounds have durations of action longer than nalfurafine and morphine in the warm-water tail immersion assay. Further, pretreatment with selective opioid receptor antagonists prior to the warm-water tail immersion study revealed that compound 21 and NCF in vivo effects were primarily KOR and DOR mediated. In addition, these two compounds precipitated less withdrawal than morphine following naloxone challenge in mice while future studies may consider modifying the schedule of morphine pretreatment with subcutaneous injections to ensure that morphine dependence is reliably produced. Drug distribution studies in mice indicated that the compounds penetrate the blood-brain barrier with increasing brain/plasma concentration ratios over time. Single-lever operant drug self-administration studies in rats indicate that neither compound had reinforcing efficacy greater than that of saline at tested doses.

Compound 21 and compound 23 (NCF) were tested toward potential identification of a pain management treatment. To assess the potential for the development of tolerance to these compounds with chronic treatment, compounds were given as subcutaneous injections for four days and mice developed tolerance in the warm-water tail immersion assay on days 3 and 4 for each of the compounds, respectively: furthermore, compound 21 showed cross-tolerance with morphine. Taken together, this may indicate that tolerance develops more rapidly at the KOR than at the MOR and that repeated MOR agonist administration sensitizes the KOR. However, further studies are warranted to examine this more closely. While chronic administration of the compounds yields tolerance, it does not result in the weight loss characteristic of opioid dependence. In addition, a locomotor activity assay was used to monitor the potential for sedative side-effects due to KOR agonism in mice; apart from vertical counts when NCF was given at 0.5 mg/kg, there was a non- significant trend for a decrease in locomotor activity as monitored by distance travelled, ambulatory counts, vertical counts, and average speed. Across the dose range tested, compound 21 did not result in the same decrease in locomotor activity seen with NCF. To address this potential for sedation as well as the previously noted limitation of using an escape model, studies may consider further probing the therapeutic window using a model that instead assesses the compound’s ability to alleviate pain-induced depression of normal behaviors. Altogether, compound 21 shows potential as an acute treatment for pain management in patients that are not morphine dependent.

4 Experimental Section

4.1 Chemical Synthesis

4.1.1 Reactions and Instrumentation

All reactions that involved moisture- sensitive reagents were conducted in oven-dried glassware under inert atmosphere maintained by nitrogen with anhydrous solvents. Chemicals and solvents were obtained from either Sigma-Aldrich or Combi-Blocks. Naltrexone was obtained as a free base through the NIDA Drug Supply Program. 1H NMR spectra were recorded at ambient temperature using a Bruker UltraShield Plus 400 MHz NMR (13C run at 100 MHz) spectrometer with IconNMR automation and B-ACS 60 autosampler and referenced to the solvent peak. Chemical shifts are expressed in ppm and coupling constants, J, are in hertz (Hz). Peak multiplicities are abbreviated as singlet, s; doublet, d; triplet, t; quartet, q; pentet, p; and multiplet, m; with two letters next to each other representing the first of the second e.g., dd, doublet of doublets. Mass spectrometry analysis was performed on a PerkinElmer AxION 2 TOF MS. Purity as determined by high pressure liquid chromatography was resolved using a Varian ProStar 210. Melting points were determined using an OptiMelt automated melting point apparatus. Flash column chromatography was performed with silica gel columns (230-400 mesh, Merck).

4.1.2 General Procedures

General Procedure 1 : Acyl Chloride Formation

The acid (3 equiv.*) was dissolved in anhydrous CH2CI2 (2 mF) in a pre-dried 50 mL round-bottom flask and stirred overnight with 4 A molecular sieves to pre-dry. The next morning, a N2 balloon was added followed by oxalyl chloride (4 equiv.) and catalytic dimethylformamide (2 drops). The reaction was allowed to stir at room temperature for two h, after which time a very small portion was removed dried thoroughly in vacuo and compared to the starting material via 1H NMR. When NMR revealed completion, the solvent was removed in vacuo (with molecular sieves and stir bar remaining inside the round-bottom flask). The acid chloride (dried contents of the flask) was then used immediately for the amidation of a secondary amine in general procedure 2 without further purification. *The base of 1 equiv. is the amine in General Procedure 2.

General Procedure 2: Amidation of a Secondary Amine

Acid chloride (as previously prepared according to general procedure 1 was dissolved in anhydrous CH2CI2 (2 mL) without further purification and 4 A molecular sieves were added on an ice water bath under N2 atmosphere. The amine (1 equiv.) was added in one portion followed by the dropwise addition of triethylamine (4 equiv.). The reaction was allowed to come to room temperature with stirring. After approximately 3 h it was checked by TLC (30: 1 CH2CI2: MeOH W/NH3 H2O) which revealed completion. The reaction mixture was then filtered through celite and concentrated in vacuo. If R1=H, purification was performed by flash column chromatography with CthCh/MeOH (1% NH3 H2O) as the eluent and purified compounds were then subjected to General Procedure 5. If R1=OH, compounds were used directly in General Procedure 4 without further purification.

General Procedure 3: For Amidation of a Primary Amine

Carboxylic acid (1.5 equiv.) was dissolved in anhydrous dimethylformamide (2 mL) in a pre-dried 50 mL round-bottom flask and stirred with 4 A molecular sieves overnight to pre-dry. In the morning a N2 balloon was added followed by the application of an ice-water bath and the addition of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 2 equiv.) and hydroxybenzotriazole (HOBt, 2 equiv. + 20%). Triethylamine (5 equiv.) was then added dropwise and fresh 4 A molecular sieves were added to the reaction. The reaction was allowed to come to room temperature while stirring for 2 h after which time the amine (1 equiv.) was added and the reaction was left to stir monitoring by TLC (30:1 CH2Ch:MeOH w/NFh-FhO) until completion was reached. At this time, it was filtered over celite and concentrated in vacuo. Purification of 3 -dehydroxy compounds was performed by flash column chromatography with CFLCh/MeOH (1% NH3 H2O) as the eluent. Purified compounds were then subjected to General Procedure 5. Compounds originating from starting materials with a 3-OH group were used directly in General Procedure 4 without further purification.

General Procedure 4: Deprotection of 3-OH (Only When R1 = OH)

The presumed disubstituted intermediate was dissolved in anhydrous methanol (3 mL) under N2 atmosphere and an ice-water bath was applied. Potassium carbonate (10 equiv.) was added in one portion and the reaction was allowed to come to room temperature with stirring. After approximately 2 days to one week when TLC (30:1 CH2Ch:MeOH w/ NH3 H2O) indicated completion, the reaction was filtered over celite and concentrated in vacuo. Purification was performed by flash column chromatography with CtkCh/MeOH (1% NH3 H2O) as the eluent. Purified free bases were then subjected to General Procedure 5.

General Procedure 4: For Deprotection of 3 -OH (Employed Only When R1 = OH)

The presumed disubstituted intermediate was dissolved in anhydrous methanol (3 mL) under N2 atmosphere and an ice-water bath was applied. Potassium carbonate (10 equiv.) was added in one portion and the reaction was allowed to come to room temperature with stirring. After approximately 2 days to one week when TLC (30:1 CH2Ch:MeOH w/ NH3 H2O) indicated completion, the reaction was filtered over celite and concentrated in vacuo. Purification was performed by flash column chromatography with CH2Ch/MeOH (1% NH3 H2O) as the eluent. Purified free bases were then subjected to General Procedure 5. General Procedure 5: Hydrolysis Reaction

The final compound as a free base (1 equiv.) was dissolved in anhydrous methanol under N2 atmosphere and HC1 in methanol solution (2 equiv.) was added dropwise after application of an ice-water bath. After stirring for 30 min, 15 mL of diethyl ether was added, which precipitated out a fine powder in most cases. After 2 h, the powder was filtered by vacuum and fully dried.

4.1.3 Final Compounds 17-Cyclopropylmethyl-4,5a-epoxy-6a-[3’-(furan-3”-yl) N-methylpropanamido]-14p- hydroxymorphinan hydrochloride (1)

The title compound was prepared following general procedures 1, 2, and 5 as a lightyellow solid in 26.4% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.87 (br s, 1H, exchangeable), 7.56 (s, 1H), 7.47 (s, 1H), 7.19 (t, J=7.8 Hz, 1H), 6.78 (d, J=7.7 Hz, 1H), 6.69 (d, J=7.9 Hz, 1H). 6.44 (s, 1H), 6.31 (s, 1H, exchangeable), 5.08-4.95 (m, 1H), 4.65 (d, J=3.0 Hz, 1H), 3.96 (d, J=6.2 Hz, 1H), 3.47 (d, J=20.2, 2H), 3.24 (d, J=7.0 Hz, 1H), 3.07- 2.97 (m, 3H), 2.81 (d, J=37.8 Hz, 3H), 2.74-2.58 (m, 5H), 2.46 (m, 1H), 1.96 (dd, J=16.8, 8.6 Hz, 1H), 1.65-1.51 (m, 2H), 1.04 (t, J=8 Hz, 1H), 1.10-1.08 (m, 1H), 0.74—0.67 (m, 1H), 0.66-0.58 (m, 1H), 0.54-0.46 (m, 1H), 0.45-0.37 (m, 1H). 13C NMR (100 MHz, DMSO-d6) 6 171.7, 159.2, 142.8, 139.0, 132.6, 129.9, 127.5, 124.2, 118.6, 111.3, 105.9, 89.2, 68.9, 61.0, 57.1, 48.5, 48.4, 45.0, 44.9, 33.5, 31.2, 29.8, 24.1, 19.8, 17.6, 5.6, 5.1, 2.5. HRMS calcd. for C28H34N2O4 (m/z) 463.2491, found [M+H]+ (m/z) 463.2578, MMA=2.81 ppm. Mp 185.7 °C, % Purity 97.02 (RT 6.91 min).

17-Cyclopropylmethyl-4,5a-epoxy-6a-[3 ’-(furan-3”-yl)propan amido]- 14P- hydroxymorphinan hydrochloride (2)

The title compound was prepared following general procedures 3 and 5 as a lightyellow solid in 52.5% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.87 (br s, 1H, exchangeable), 7.79 (d, J=7.9 Hz, 1H, exchangeable), 7.54 (s, 1H), 7.43 (s, 1H), 7.17 (t, J=7.7 Hz, 1H), 6.74 (d, J=7.6 Hz, 1H), 6.67 (d, J=7.9 Hz, 1H), 6.38 (s, 1H, exchangeable), 6.28 (br s, 1H), 4.61 (d, J=3.5 Hz, 1H), 4.46-4.37 (m, 1H), 3.92 (d, J=6.4 Hz, 1H), 3.48-3.43 (m, 2H), 3.31-3.26 (m, 1H), 3.15 (dd, J=20.1, 6.9 Hz, 1H), 3.05 (d, J=12.3 Hz, 1H), 2.95 (m, 1H), 2.76-2.67 (m, 1H), 2.62 (t, J=7.5 Hz, 2H), 2.39 (t, J=7.4 Hz, 2H), 1.87 (dd, J=16.6, 8.4 Hz, 1H), 1.61 (d, J=12.0 Hz, 1H), 1.38 (m, 2H), 1.07 (br s, 1H), 0.87 (t, J=11.3 Hz, 1H), 0.74- 0.58 (m, 2H), 0.53-0.38 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 171.0, 159.3, 142.9, 138.9, 132.6, 129.7, 127.5, 124.1, 118.4, 111.2, 106.2, 87.6, 69.2, 60.9, 57.0, 45.0, 44.8, 44.4, 35.3, 30.1, 29.1, 24.1, 20.4, 19.5, 5.6, 5.1, 2.5. HRMS calcd. for C27H32N2O4 (m/z) 449.2435, found [M+H]+ (m/z) 449.2419, MMA=3.56 ppm. Mp 175.3 °C, % Purity 95.68 (RT 5.77 min). 17-Cyclopropylmethyl-4,5a-epoxy-6P-[3’-(furan-3”-yl)-N-m ethylpropanamido]-14p- hydroxymorphinan hydrochloride (3)

The title compound was prepared following general procedures 1, 2, and 5 as a yellow solid in 47.6% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.83 (br s, 1H, exchangeable), 7.52 (d, J=14.7 Hz, 1H), 7.37 (d, J=49.1 Hz, 1H), 7.22-7.15 (m, 1H), 6.89- 6.81 (m, 1H), 6.72 (dd, J=19.1, 7.3 Hz, 1H), 6.41 (s, 1H), 6.24 (s, 0.5H, exchangeable), 5.75 (s, 0.5H, exchangeable), 4.82 (t, J=9.0 Hz, 1H), 3.89 (s, 1H), 3.48-3.35 (m, 2H), 3.18-3.14 (m, 1H), 3.07-3.04 (m, 1H), 2.98 (s, 1H), 2.89 (s, 1H), 2.84 (s, 2H), 2.63-2.52 (m, 3H), 2.47-2.40 (m, 1H), 2.31 (t, J=7.4 Hz, 1H), 2.08 (t, J=16.5 Hz, 1H), 1.73 (d, J=12.2 Hz, 1H), 1.40 (dd, J=20.7, 10.8 Hz, 3H), 1.30-1.16 (m, 1H), 1.08 (d, J=3.9 Hz, 1H), 0.72-0.57 (m, 2H), 0.47 (dd, J=33.3, 3.3 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) 6 171.7, 171.3, 155.9, 155.3, 142.8, 138.8, 131.3, 129.5, 128.5, 124.1, 119.2, 118.6, 111.1, 87.9, 69.5, 61.1, 56.6, 54.8, 46.1, 45.1, 32.5, 30.2, 26.9, 23.6, 20.0, 5.7, 5.1, 2.6. HRMS calcd. For C28H34N2O4 (m/z) 463.2591, found [M+H]+ (m/z) 463.2580, MMA=2.37 ppm. Mp 197.1 °C, % Purity 96.61 (RT 6.44 min).

17-Cyclopropylmethyl-4,5a-epoxy-6P-[3 ’-(furan-3”-yl)propan amido]- 14P- hydroxymorphinan hydrochloride (4)

The title compound was prepared following general procedures 3 and 5 as a light- -19- yellow solid in 40.5% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.85 (br s, 1H, exchangeable), 8.13 (d, J=8.1 Hz, 1H, exchangeable), 7.55 (t, J=1.6 Hz, 1H), 7.42 (s, 1H), 7.18 (t, J=7.8 Hz, 1H), 6.83 (d, J=7.8 Hz, 1H), 6.74 (d, J=7.9 Hz, 1H), 6.36 (s, 1H), 6.17 (br s, 1H, exchangeable), 4.53 (d, J=7.9 Hz, 1H), 3.86 (d, J=4.8 Hz, 1H), 3.46-3.41 (m, 3H), 3.20-3.14 (m, 1H), 3.02 (d, J=7.1 Hz, 1H), 2.88-2.80 (m, 1H), 2.60 (t, J=7.4 Hz, 2H), 2.41 (d, J=8.8 Hz, 2H), 2.34-2.28 (m, 2H), 1.76-1.64 (m, 2H), 1.44 (t, J=10.4 Hz, 2H), 1.33 (m, 1H), 1.09 (t, J=7.0 Hz, 1H), 0.72-0.65 (m, 1H), 0.63-0.55 (m, 1H), 0.49 (dt, J=9.3, 4.6 Hz, 1H), 0.42 (dt, J=9.2, 4.5 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) 6 171.0, 155.8, 142.9, 138.9, 131.2, 129.5, 128.3, 124.0, 118.7, 111.2, 108.9, 90.3, 69.6, 61.6, 56.7, 50.4, 46.2, 44.9, 35.9, 29.4, 27.2, 23.7, 23.6, 20.3, 5.7, 5.1, 2.6. HRMS calcd. for C27H32N2O4 (m/z) 449.2435, found [M+H]+ (m/z) 449.2415, MMA=4.45 ppm. Mp 205.8 °C, % Purity 95.49 (RT 5.74 min).

17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6a-[3’- (furan-3”-yl)-N- methylpropanamido] morphinan hydrochloride (5)

The title compound was prepared following general procedures 1, 2, 4, and 5 as a white solid in 24.8% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.30 (s, 1H, exchangeable), 8.80 (br s, 1H, exchangeable), 7.56 (d, J=1.6 Hz, 1H), 7.48 (s, 1H), 6.72 (d, J=8.1 Hz, 1H), 6.59 (d, J=8.1 Hz, 1H), 6.45 (s, 1H), 6.21 (s, 1H, exchangeable), 5.04-4.94 (m, 1H), 4.63 (d, J=3.9 Hz, 1H), 3.89 (d, J=6.4 Hz, 1H), 3.31-3.23 (m, 2H), 3.12 (d, J=6.7 Hz, 1H), 3.04-3.02 (m, 1H), 2.92 (s, 1H), 2.85 (d, J=36.0 Hz, 3H), 2.72-2.60 (m, 5H), 2.45-2.41 (m, 1H), 1.97-1.88 (m, 1H), 1.65-1.53 (m, 2H), 1.34 (t, J=10.7 Hz, 1H), 1.15-1.08 (m, 1H), 1.05 (s, 1H), 0.72-0.59 (m, 2H), 0.50-0.38 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 171.6, 142.9, 139.1, 124.3, 122.0, 119.3, 118.3, 118.1, 117.9, 111.5, 111.4, 110.2, 68.9, 68.8, 61.2, 57.0, 48.5, 45.6, 45.0, 33.5, 31.2, 29.9, 29.8, 23.4, 19.9, 5.7, 4.9, 2.5. HRMS calcd. for C28H34N2O5 (m/z) 479.2540, found [M+H]+ (m/z) 479.2523, MMA=3.55 ppm. Mp 297.4 °C, % Purity 97.62 (RT 5.84 min).

17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6P-[3’- (furan-3”-yl)-N- methylpropanamido] morphinan hydrochloride (7)

The title compound was prepared following general procedures 1, 2, 4, and 5 as a white solid in 53.1% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.54 (d, J=112 Hz, 1H, exchangeable), 8.79 (br s, 1H, exchangeable), 7.50 (dt, J=21.8, 1.5 Hz, 1H), 7.38 (d, J=58.0 Hz, 1H), 6.77-6.62 (m, 2H), 6.39 (d, J=22.8 Hz, 1H, exchangeable), 6.28 (s, 1H), 4.80 (dd, J=28.O, 8.2 Hz, 1H), 4.10 (s, 0.3 H), 3.84 (d, J=5.7 Hz, 1H), 3.47 (ddd, J=12.4, 8.0, 4.2 Hz, 0.7H), 3.41-3.33 (m, 1H), 3.08-3.01 (m, 2H), 2.98 (s, 1H), 2.87 (d, J=11.8 Hz, 1H), 2.83 (s, 2H), 2.75-2.60 (m, 1H), 2.57 (d, J=8.0 Hz, 1H), 2.47-2.42 (m, 2H), 2.34-2.26 (m, 1H), 2.12-2.03 (m, 1H), 1.70 (d, J=13.5 Hz, 1H), 1.51-0.97 (m, 6H), 0.71-0.65 (m, 1H), 0.62-0.56 (m, 1H), 0.53-0.47 (m, 1H), 0.44-0.38 (m, 1H). 13C NMR (100 MHz, DMSO-d6) 6 171.5, 142.7, 141.5, 138.9, 129.9, 124.1, 120.7, 119.8, 117.8, 111.5, 111.2, 87.6, 69.6, 61.3, 56.9,

56.6, 46.4, 45.8, 32.3, 30.3, 27.9, 27.0, 22.9, 21.7, 20.1, 5.7, 5.1, 2.6. HRMS calcd. for C28H34N2O5 (m/z) 479.2540, found [M+H]+ (m/z) 479.2516, MMA=5.01 ppm. Mp 229.2 °C, % Purity 96.78 (RT 5.75 min).

17-Cyclopropylmethyl-4,5 a-epoxy-6a- [3 ’ -(furan-2”-yl)N-methylpropanamido] - 14p- hydroxymorphinan hydrochloride (9)

The title compound was prepared following general procedures 1, 2, and 5 as a yellow solid in 16% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.86 (br s, 1H, exchangeable), 7.50 (s, 1H), 7.18 (t, J=7.8 Hz, 1H), 6.77 (d, J=7.7 Hz, 1H), 6.69 (t, J=8.1 Hz, 1H), 6.35-6.28 (m, 2H), 6.12 (s, 1H, exchangeable), 5.03-4.79 (m, 1H), 4.63-4.47 (m, 1H), 3.94 (d, J=6.3 Hz, 1H), 3.48-3.43 (m, 2H), 3.28-3.16 (m, 2H), 3.07-2.88 (m , 3H), 2.85 (s, 3H), 2.83-2.65 (m, 4H), 2.44 (d, J=8.7 Hz, 1H), 2.01-1.89 (m, 1H), 1.65-1.49 (m, 2H), 1.34 (t, J=9.9 Hz, 1H), 1.08 (d, J=7.0 Hz, 2H), 0.74-0.57 (m, 2H), 0.52-0.37 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 171.2, 159.2, 154.9, 141.2, 132.7, 130.0, 127.5, 118.7, 110.4, 105.9, 105.1, 89.3, 68.9, 61.0, 57.0, 48.5, 45.0, 44.9, 31.4, 31.2, 29.9, 29.8, 24.1, 23.0, 17.6, 5.7, 5.2,

2.6. HRMS calcd. for C28H34N2O4 (m/z) 463.2591, found [M+H]+ (m/z) 463.2578, MMA=2.81 ppm. Mp 170.6 °C, % Purity 95.24 (RT 8.82 min). 17-Cyclopropylmethyl-4,5a-epoxy-6a-[3 ’-(furan-2”-yl)propan amido]- 14P- hydroxy morphinan hydrochloride (10)

The title compound was prepared following general procedures 3 and 5 as a white solid in 38.9% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.87 (br s, 1H, exchangeable), 7.87 (d, J=8.1 Hz, 1H, exchangeable), 7.49 (d, J=l.l Hz, 1H), 7.17 (t, J=7.8 Hz, 1H), 6.74 (d, J=7.7 Hz, 1H), 6.67 (d, J=7.9 Hz, 1H), 6.34 (dd, J=3.0, 1.9 Hz, 1H), 6.27 (br s, 1H, exchangeable), 6.09 (d, J=3.1 Hz, 1H), 4.60 (d, J=3.7 Hz, 1H), 4.43 (ddd, J=16.7, 8.0, 4.0 Hz, 1H), 3.91 (d, J=6.7 Hz, 1H), 3.48-3.43 (m, 1H), 3.29-3.24 (m, 1H), 3.19-3.12 (m, 1H), 3.04 (d, J=11.2 Hz, 1H), 2.98-2.92 (m, 1H), 2.83 (t, J=7.5 Hz, 2H), 2.68 (dd, J=14.1, 7.7 Hz, 1H), 2.46 (t, J=7.6 Hz, 3H), 1.86 (dt, J=15.5, 9.5 Hz, 1H), 1.61 (d, J=10.7 Hz, 1H), 1.41-1.35 (m, 2H), 1.09 (t, J=7.0 Hz, 1H), 0.93-0.82 (m, 1H), 0.73-0.57 (m, 2H), 0.52-0.37 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 170.6, 159.3, 154.8, 141.3, 132.6, 129.8, 127.6, 118.5, 110.4, 106.3, 105.1, 87.6, 69.2, 60.9, 57.0, 45.0, 44.8, 44.4, 33.2, 30.1, 29.1, 24.2, 23.5, 19.5, 5.7, 5.2, 2.6. HRMS calcd. for C27H32N2O4 (m/z) 449.2435, found [M+H]+ (m/z) 449.2419, MMA=3.56 ppm. Mpl88.8 °C, dec. % Purity 97.83 (RT 6.61 min).

17-Cyclopropylmethyl-4,5a-epoxy-6P-[3’-(furan-2”-yl)- N-methylpropanamido]-14p- hydroxymorphinan hydrochloride (11)

The title compound was prepared following general procedures 1, 2, and 5 as a yellow solid in 20.3% yield (average). Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.83 (s, 1H, exchangeable), 7.48 (d, J = 9.2 Hz, 1H), 7.21 - 7.14 (m, 1H), 6.85 (dd, J = 14.7, 7.7 Hz, 1H), 6.70 (t, J = 8.1 Hz, 1H), 6.41 - 6.29 (m, 2H), 6.12 - 5.95 (m, 1H, exchangeable), 4.82 (t, J = 9.3 Hz, 1H), 3.88 (s, 1H), 3.43 (s, 3H), 3.26 (s, 1H), 3.21 - 3.11 (m, 2H), 3.06 (dd, J = 13.7, 8.4 Hz, 2H), 2.98 (s, 1H), 2.84 (s, 2H), 2.82 - 2.62 (m, 3H), 2.41 (dd, J = 17.2, 8.7 Hz, 2H), 2.08 (s, 1H), 1.73 (d, J = 12.1 Hz, 1H), 1.43 (d, J = 12.3 Hz, 2H), 1.19 (t, J = 7.3 Hz, 1H), 0.73 - 0.55 (m, 2H), 0.52 - 0.39 (m, 2H).13C NMR (100 MHz, DMSO-d6) 6 154.9, 141.1, 129.5, 119.2, 110.2, 105.1, 104.9, 99.5, 87.2, 73.6, 69.6, 69.5, 64.9, 61.2, 56.6, 46.2, 45.5, 45.1, 30.2, 27.9, 23.6, 23.1, 15.1, 8.5, 5.7, 5.1, 2.6, 0.1. HRMS calcd. For C28H34N2O4 (m/z) 463.2591, found [M+H]+ (m/z) 463.2570, MMA=4.53 ppm. Mp 172.8 °C, % Purity 97.38 (RT 5.60 min).

17-Cyclopropylmethyl-4,5a-epoxy-6P-[3 ’-(furan-2”-yl)propan amido]- 14P- hydroxymorphinan hydrochloride (12)

The title compound was prepared following general procedures 3 and 5 as a lightyellow solid in 22.1% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.86 (br s, 1H, exchangeable), 8.18 (d, J=7.9 Hz, 1H, exchangeable), 7.49 (s, 1H), 7.17 (t, J=7.8 Hz, 1H), 6.83 (d, J=7.7 Hz, 1H), 6.74 (d, J=7.8 Hz, 1H), 6.34 (d, J=1.5 Hz, 1H), 1H), 6.19 (br s, 1H, exchangeable), 6.08 (d, J=2.4 Hz, 1H), 4.54 (d, J=7.9 Hz, 1H), 3.87 (d, J=5.0 Hz, 1H), 3.48-3.37 (m, 3H), 3.17 (dd, J=19.7, 5.7 Hz, 1H), 3.03 (d, J=6.7 Hz, 1H), 2.81 (t, J=7.4 Hz, 3H), 2.40 (t, J=7.6 Hz, 4H), 1.71 (d, J=12.5 Hz, 2H), 1.51-1.38 (m, 2H), 1.32 (t, J=13.5 Hz, 1H), 1.09 (t, J=6.9 Hz, 1H), 0.64 (ddd, J=33.9, 8.1, 4.6 Hz, 2H), 0.46 (ddd, J=35.6, 8.7, 4.3 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) 6 170.5, 155.8, 154.6, 141.2, 131.2, 129.5, 128.3, 118.7, 110.3, 108.9, 105.0, 90.3, 69.6, 64.9, 61.5, 56.7, 50.4, 46.2, 44.9, 33.7, 29.3, 27.2, 23.6, 23.4, 5.7, 5.1, 2.6. HRMS calcd. for C27H32N2O4 (m/z) 449.2435, found [M+H]+ (m/z) 449.2434, MMA=0.22 ppm. Mp 185.3 °C, % Purity 96.15 (RT 6.14 min). 17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6a-[3’-(fu ran-2”-yl)-N- methylpropanamido] morphinan hydrochloride (13)

The title compound was prepared following general procedures 1, 2, 4 and 5 as a white solid in 25.2% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.29 (s, 1H, exchangeable), 8.80 (br s, 1H, exchangeable), 7.52 (t, J=5.8 Hz, 1H), 6.72 (d, J=8.1 Hz, 1H), 6.58 (d, J=8.2 Hz, 1H), 6.43 (s, 0.2H, exchangeable), 6.36 (dd, J=5.8, 2.8 Hz, 1H), 6.22 (s, 0.8H, exchangeable), 6.12 (d, J=2.9 Hz, 1H), 4.98 (dt, J=7.1, 3.2 Hz, 1H), 4.69 (dd, J=61.5, 3.0 Hz, 1H), 3.90 (d, J=6.4 Hz, 1H), 3.43-3.32 (m, 1H), 3.29-3.22 (m, 1H), 3.15-3.06 (m, 1H), 3.06-3.00 (m, 1H), 2.92 (d, J=5.4 Hz, 1H), 2.90 (s, 2.5H), 2.86 (d, J=7.9 Hz, 2H), 2.81 (s, 0.5H), 2.71-2.66 (m, 2H), 2.46-2.38 (m, 1H), 1.92 (dt, J=13.5, 9.4 Hz, 1H), 1.64-1.52 (m, 2H), 1.48-1.20 (m, 2H), 1.18-1.02 (m, 2H), 0.65 (ddd, J=28.8, 7.9, 4.4 Hz, 2H), 0.50-0.37 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 171.1, 154.9, 145.7, 141.2, 139.0, 128.8, 122.0, 119.3,

118.1, 110.4, 105.1, 89.0, 68.9, 61.1, 57.0, 48.5, 45.6, 45.2, 31.4, 31.2, 30.0, 29.8, 23.4, 23.0, 17.8, 5.6, 5.1, 2.5. HRMS calcd. For C28H34N2O5 (m/z) 479.2540, found [M+H]+ (m/z) 479.2529, MMA=2.30 ppm. Mp 284.5 °C, % Purity 98.88 (RT 6.25 min). 17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6a-[3’-(fu ran-2”- yl)propanamido]morphinan hydrochloride (14)

The title compound was prepared following general procedures 3, 4 and 5 as a white solid in 47.3% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.15 (s, 1H, exchangeable), 8.79 (br s, 1H, exchangeable), 7.71 (d, J=7.5 Hz, 1H, exchangeable), 7.49 (s, 1H), 6.71 (d, J=8.1 Hz, 1H), 6.56 (d, J=8.1 Hz, 1H), 6.33 (s, 1H), 6.19 (s, 1H, exchangeable), 6.10 (s, 1H), 4.59 (s, 1H), 4.41 (s, 1H), 3.87 (d, J=5.3 Hz, 1H), 3.36 (d, J=7.5 Hz, 1H), 3.26 (s, 2H), 3.04 (d, J=13.8, Hz, 2H), 2.94 (s, 1H), 2.84 (t, J=7.1 Hz, 2H), 2.75-2.68 (m, 1H), 2.46 (s, 1H), 1.88-1.79 (m, 1H), 1.61 (d, J=13.2 Hz, 1H), 1.45-1.34 (m, 2H), 1.11-1.05 (m, 2H), 0.97-0.87 (m, 1H), 0.68-0.61 (m, 2H), 0.47-0.40 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 170.6, 154.8, 146.0, 141.4, 138.7, 128.8, 122.2, 119.3, 118.2, 110.5, 105.2, 87.6, 69.4, 65.0,

61.1, 57.1, 55.0, 45.2, 33.4, 30.2, 29.2, 23.6, 19.7, 15.3, 5.7, 5.3, 2.6. HRMS calcd. for C27H32N2O5 (m/z) 465.2384, found [M+H]+ (m/z) 465.2384, MMA=0 ppm. Mp 199.6 °C, % Purity 99.01 (RT 6.13 min). 17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6P-[3’-(fu ran-2”-yl)-N- methylpropanamido] morphinan hydrochloride (15) The title compound was prepared following general procedures 1, 2, 4 and 5 as a white solid in 23.4% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.35 (d, J=82.5 Hz, 1H, exchangeable), 8.78 (br s, 1H, exchangeable), 7.45 (d, J=32.5 Hz, 1H), 6.76-6.62 (m, 2H), 6.34 (s, 1H, exchangeable), 6.28 (dd, J=7.5, 5.5 Hz, 1H), 6.12-5.98 (m, 1H), 4.80 (dd, J=31.1, 8.0 Hz, 1H), 4.10-4.03 (m, 0.3H), 3.83 (s, 1H), 3.54-3.46 (m, 0.7H), 3.42-3.34 (m, 1H), 3.04 (d, J=12.0 Hz, 2H), 2.98 (s, 1H), 2.89 (s, 1H), 2.83 (s, 2H), 2.81-2.53 (m, 4H), 2.47-

2.38 (m, 2H), 2.12-2.03 (m, 1H), 1.71 (d, J=13.6 Hz, 1H), 1.42 (dt, J=26.6, 13.3 Hz, 3H), 1.22 (d, J=13.1 Hz, 1H), 1.10-1.13 (m, 1H), 0.72-0.66 (m, 1H), 0.59 (dd, J=7.6, 3.9 Hz, 1H), 0.54- 0.47 (m, 1H), 0.44-0.38 (m, 1H). 13C NMR (100 MHz, DMSO-d6) 6 171.0, 154.9, 141.6, 141.4, 141.1, 140.9, 129.9, 120.6, 119.8, 117.8, 110.3, 105.2, 104.8, 87.6, 69.6, 61.3, 57.0, 56.7, 46.4, 45.8, 30.0, 28.0, 23.2, 22.9, 21.7, 5.7, 5.1, 2.6. HRMS calcd. For C28H34N2O5 (m/z) 479.2540, found [M+H]+ (m/z) 479.2528, MMA=2.50 ppm. Mp 237.0 °C, % Purity 99.18 (RT 5.95 min).

17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6P-[3’- (furan-2”- yl)propanamido]morphinan hydrochloride (16)

The title compound was prepared following general procedures 3, 4 and 5 as a white solid in 51.7% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.32 (s, 1H, exchangeable), 8.81 (br s, 1H, exchangeable), 8.19 (d, J=7.8 Hz, 1H, exchangeable), 7.50 (s, 1H), 6.71 (d, J=8.1 Hz, 1H), 6.63 (d, J=8.1 Hz, 1H), 6.34 (d, J=1.5 Hz, 1H), 6.12 (s, 1H, exchangeable), 6.09 (d, J=2.1 Hz, 1H), 4.54 (d, J=7.8 Hz, 1H), 3.82 (d, J=5.0 Hz, 1H), 3.42-

3.38 (m, 1H), 3.29 (s, 1H), 3.04 (dd, J=22.5, 8.5 Hz, 2H), 2.82 (t, J=7.1 Hz, 3H), 2.47-2.35 (m, 4H), 1.74-1.63 (m, 2H), 1.54-1.47 (m, 1H), 1.43 (d, J=10.1 Hz, 1H), 1.33 (t, J=13.2 Hz, 1H), 1.10-1.01 (m, 1H), 0.70-0.55 (m, 2H), 0.45 (ddd, J=38.1, 9.0, 4.5 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) 6 154.7, 142.1, 141.4, 141.2, 129.7, 120.7, 119.4, 117.9, 110.5, 105.2, 89.9, 69.7, 61.7, 56.7, 55.0, 50.6, 48.6, 46.5, 45.6, 33.8, 29.3, 27.4, 23.5, 23.0, 5.8, 5.2, 2.7. HRMS calcd. for C27H32N2O5 (m/z) 465.2384, found [M+H]+ (m/z) 465.2367, MMA=3.65 ppm. Mp 248.9 °C, % Purity 98.88 (RT 5.85 min). 17-Cyclopropylmethyl-4,5a-epoxy-6a-[(2E)-3’-(furan-2”-yl )N-methylprop-2- enamido]- 14p-hydroxymorphinan hydrochloride (17)

The title compound was prepared following general procedures 1, 2 and 5 as a white solid in 27.3% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.88 (br s, 1H, exchangeable), 7.81 (s, 1H), 7.37 (d, J=15.2 Hz, 1H), 7.20 (t, J=7.7 Hz, 1H), 6.89 (dd, J=9.0, 6.2 Hz, 2H), 6.78 (d, J=7.6 Hz, 1H), 6.70 (d, J=7.8 Hz, 1H), 6.63 (s, 1H, exchangeable), 6.45 (d, J=95.2 Hz, 1H), 5.09 (d, J=14.1 Hz, 0.7H), 4.78 (s, 0.3H), 4.72 (d, J=2.1 Hz, 1H), 3.96 (d, J=5.6 Hz, 1H), 3.50-3.45 (m, 1H), 3.28-3.13 (m, 2H), 3.05 (d, J=11.0 Hz, 1H), 3.00 (s, 2H), 2.98-2.92 (m, 1H), 2.86 (s, 1H), 2.70 (d, J=12.7 Hz, 1H), 2.02-1.92 (m, 1H), 1.68-1.05 (m, 6H), 0.74-0.59 (m, 2H), 0.52-0.38 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 165.6, 159.2, 151.2, 145.0, 132.7, 130.0, 128.9, 127.5, 118.7, 115.7, 114.2, 112.6, 105.9, 89.2, 68.9, 61.0, 57.0, 49.0, 45.0, 31.5, 29.9, 29.8, 24.1, 17.6, 5.7, 5.2, 2.6, 0.1. HRMS calcd. for C28H35N2O4 (m/z) 461.2435, found [M+H]+ (m/z) 461.2413, MMA=4.77 ppm. Mp 268.8 °C, % Purity 97.53 (RT 8.21 min).

17-Cyclopropylmethyl-4,5a-epoxy-6a-[(2E)-3’-(furan-2” -yl)prop-2-enamido]-14p- hydroxy morphinan hydrochloride (18)

The title compound was prepared following general procedures 3 and 5 as a lightyellow solid in 58.2% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.89 (br s, 1H, exchangeable), 8.11 (d, J=8.1 Hz, 1H, exchangeable), 7.78 (s, 1H), 7.25 (d, J=15.6 Hz, 1H), 7.18 (t, J=7.8 Hz, 1H), 6.78-6.74 (m, 2H), 6.68 (d, J=7.9 Hz, 1H), 6.62-6.55 (m, 2H), 6.31 (br s, 1H, exchangeable), 4.68 (d, J=3.7 Hz, 1H), 4.58-4.47 (m, 1H), 3.93 (d, J=6.7 Hz, 1H), 3.49-3.44 (m, 2H), 3.21-3.14 (m, 1H), 3.06 (d, J=11.9 Hz, 1H), 2.99-2.94 (m, 1H), 2.71 (dd, J=22.8, 9.9 Hz, 1H), 2.45 (d, J=4.8 Hz, 1H), 1.94-1.84 (m, 1H), 1.63 (d, J=11.1 Hz, 1H), 1.50-1.38 (m, 2H), 1.09 (s, 1H), 0.98-0.89 (m, 1H), 0.66 (ddd, J=17.2, 8.2, 4.5 Hz, 2H), 0.52- 0.39 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 164.3, 159.3, 151.0, 144.8, 132.7, 129.8, 127.6, 126.2, 119.5, 118.5, 113.7, 112.4, 106.2, 87.5, 69.3, 64.9, 57.0, 45.1, 45.0, 44.4, 30.1, 29.1, 24.2, 19.5, 5.7, 5.2, 2.6. HRMS calcd. for C27H30N2O4 (m/z) 447.2278, found [M+H]+ (m/z) 447.2258, MMA=4.47 ppm. Mp 226.9 °C, % Purity 95.35 (RT 7.06 min). 17-Cyclopropylmethyl-4,5a-epoxy-6P-[(2E)-3’-(furan-2”-yl )-N-methylprop- 2-enamido]- 14p-hydroxymorphinan hydrochloride (19)

The title compound was prepared following general procedures 1, 2 and 5 as a white solid in 42% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.89 (s, 1H, exchangeable), 8.11 (d, J = 8.0 Hz, 1H), 7.78 (s, 1H), 7.25 (d, J = 15.6 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 6.82 - 6.72 (m, 2H), 6.68 (d, J = 7.9 Hz, 1H), 6.62 - 6.53 (m, 2H), 6.32 (s, 1H, exchangeable), 4.68 (d, J = 3.7 Hz, 1H), 4.52 (td, J = 8.6, 4.1 Hz, 1H), 3.94 (d, J = 6.6 Hz, 1H), 3.49 (s, 3H), 3.32 - 3.23 (m, 1H), 3.21 - 2.89 (m, 5H), 2.78 - 2.65 (m, 1H), 1.94 - 1.82 (m, 1H), 1.63 (d, J = 12.1 Hz, 1H), 1.49 - 1.37 (m, 2H), 1.02 - 0.85 (m, 1H), 0.72 - 0.60 (m, 2H), 0.52 - 0.32 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 164.3, 159.3, 151.0, 144.8,

132.6. 129.8. 127.5. 126.2. 119.5. 118.4. 113.7. 112.4. 106.2, 87.5, 69.2, 64.9, 60.9, 57.0, 45.1, 44.4, 30.1, 29.1, 24.1, 19.5, 15.1, 5.6, 5.1, 2.5. HRMS calcd. For C28H32N2O4 (m/z) 461.2434, found [M+H]+ (m/z) 461.2449, MMA=3.04 ppm. Mp 233.4 °C, % Purity 97.24 (RT 7.49 min).

17-Cyclopropylmethyl-4,5a-epoxy-6P-[(2E)-3’-(furan-2” -yl)prop-2-enamido]-14p- hydroxymorphinan hydrochloride (20)

The title compound was prepared following general procedures 3 and 5 as a white solid in 26.4% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 8.88 (br s, 1H, exchangeable), 8.45 (d, J=8.2 Hz, 1H, exchangeable), 7.78 (s, 1H), 7.25-7.14 (m, 2H), 6.85 (d, J=7.7 Hz, 1H), 6.77 (t, J=6.1 Hz, 2H), 6.59 (d, J=1.9 Hz, 1H), 6.39 (d, J=15.6 Hz, 1H), 6.24 (br s, 1H, exchangeable), 4.58 (d, J=7.9 Hz, 1H), 3.89 (d, J=4.5 Hz, 1H), 3.58-3.45 (m, 2H), 3.19 (dd, J=20.1, 5.5 Hz, 1H), 3.05 (d, J=6.9 Hz, 1H), 2.90-2.83 (m, 1H), 2.44 (d, J=8.4 Hz, 2H), 1.77 (t, J=13.8 Hz, 2H), 1.59-1.52 (m, 1H), 1.45 (d, J=8.8 Hz, 1H), 1.42-1.20 (m, 2H), 1.09 (t, J=7.0 Hz, 1H), 0.72-0.65 (m, 1H), 0.60 (dd, J=8.0, 4.4 Hz, 1H), 0.51 (dd, J=9.3, 4.6 Hz, 1H), 0.46-0.39 (m, 1H). 13C NMR (100 MHz, DMSO-d6) 6 164.4, 155.8, 150.8, 144.8, 131.2, 129.5, 128.3, 126.4, 119.2, 113.9, 112.4, 109.0, 90.3, 69.6, 64.9, 61.5, 56.7, 50.6,

46.2, 44.9, 29.4, 27.2, 23.7, 15.1, 5.7, 5.1, 2.6. HRMS calcd. for C27H31N2O4 (m/z) 448.2357, found [M+H]+ (m/z) 447.2268, MMA=2.24 ppm. Mp 195.8 °C, % Purity 95.19 (RT 6.16 min).

17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6a-[(2E)- 3’-(furan-2”-yl)-N- methylprop-2-enamido] morphinan hydrochloride (21)

The title compound was prepared following general procedures 1, 2, 4 and 5 as a white solid in 65.5% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.32 (s, 1H, exchangeable), 8.84 (br s, 1H, exchangeable), 7.81 (s, 1H), 7.37 (d, J=15.3 Hz,lH), 6.95-6.84 (m, 2H), 6.73 (d, J=8.1 Hz, 1H), 6.66-6.57 (m, 2H), 6.42 (d, J=101.5 Hz, 1H, exchangeable), 5.05 (d, J=13.9 Hz, 1H), 4.71 (d, J=3.3 Hz, 1H), 3.93 (d, J=6.3 Hz, 1H), 3.40-3.34 (m, 1H), 3.26 (dd, J=10.8, 4.2 Hz, 1H), 3.17-3.08 (m, 1H), 3.04 (s, 3H), 2.92 (d, J=14.0 Hz, 2H), 2.69 (d, J=12.8 Hz, 1H), 2.45 (d, J=12.9 Hz, 1H), 2.01-1.90 (m, 1H), 1.66-1.53 (m, 2H), 1.41 (t, J=10.3 Hz, 1H), 1.23-1.15 (m, 1H), 1.10-1.02 (m, 1H), 0.73-0.66 (m, 1H), 0.62 (dd, J=8.3, 4.4 Hz, 1H), 0.53-0.45 (m, 1H), 0.43-0.35 (m, 1H). 13C NMR (100 MHz, DMSO-d6) 6 165.6,

151.2, 145.7, 145.0, 139.0, 128.9, 122.1, 119.4, 118.1, 115.8, 114.2, 112.6, 89.0, 69.0, 61.1, 57.0, 49.1, 45.7, 45.2, 31.6, 30.1, 29.8, 23.4, 17.9, 15.0, 5.7, 5.2, 2.6. HRMS calcd. For C28H32N2O5 (m/z) 477.2384, found [M+H]+ (m/z) 477.2361, MMA=4.82 ppm. Mp 299.6 °C, % Purity 99.16 (RT 6.38 min).

17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6a-[(2E)- 3’-(furan-2”-yl)prop-2- enamido]morphinan hydrochloride (22)

The title compound was prepared following general procedures 3, 4 and 5 as a white solid in 81.6% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.21 (s, 1H, exchangeable), 8.83 (br s, 1H, exchangeable), 8.05 (d, J=7.9 Hz, 1H, exchangeable), 7.78 (s, 1H), 7.25 (d, J=15.6 Hz, 1H), 6.78 (d, J=3.3 Hz, 1H), 6.72 (d, J=8.1 Hz, 1H), 6.62-6.55 (m, 3H), 6.26 (s, 1H, exchangeable), 4.67 (d, J=3.8 Hz, 1H), 4.53-4.46 (m, 1H), 3.89 (d, J=6.7 Hz, 1H), 3.26 (d, J=11.6 Hz, 1H), 3.11-2.88 (m, 4H), 2.72 (d, J=12.2 Hz, 1H), 2.45 (dd, J=13.4, 5.0 Hz, 1H), 1.92-1.84 (m, 1H), 1.63 (d, J=11.3 Hz, 1H), 1.43 (dd, J=15.4, 9.3 Hz, 2H), 1.08- 0.95 (m, 2H), 0.71-0.59 (m, 2H), 0.50-0.38 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 164.3, 151.0, 146.0, 144.8, 138.8, 128.7, 126.2, 122.1, 119.5, 119.1, 118.2, 113.7, 112.4, 87.3,

69.3, 61.1, 57.0, 45.3, 45.2, 30.2, 29.2, 23.5, 23.4, 19.6, 5.7, 5.2, 2.5. HRMS calcd. for C27H30N2O5 (m/z) 463.2227, found [M+H]+ (m/z) 463.2245, MMA=3.89 ppm. Mp 254.7 °C, % Purity 96.69 (RT 6.06 min). 17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6P-[(2E)-3� �-(furan-2”-yl)-N- methylprop-2-enamido] morphinan hydrochloride (23)

The title compound was prepared following general procedures 1, 2, 4 and 5 as a white solid in 39.8% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.34 (d, J=55.6 Hz, 1H, exchangeable), 8.79 (br s, 1H, exchangeable), 7.74 (d, J=49.8 Hz, 1H), 7.22 (d, J=39.6 Hz, 1H), 6.88-6.43 (m, 5H), 6.34 (d, J=29.0 Hz, 1H, exchangeable), 4.86 (dd, J=28.9, 8.1 Hz, 1H), 4.19 (s, 0.5H), 3.84 (s, 1H), 3.62 (s, 1H), 3.36 (s, 1H) 3.27 (d, J=2.5 Hz, 1H), 3.15 (s, 1.5H), 3.07 (dd, J=19.5, 5.1 Hz, 2H), 2.93 (s, 1.5H), 2.88 (d, J=7.7 Hz, 1H), 2.45 (s, 1H), 2.12 (dd, J=23.5, 10.6 Hz, 1H), 1.73 (d, J=11.9 Hz, 1H), 1.51-1.00 (m, 5H), 0.72-0.56 (m, 2H), 0.53-0.39 (m, 2H). 13C NMR (100 MHz, DMSO-d6) 6 166.8, 166.0, 151.7, 145.4, 145.0,

142.3, 130.3, 129.3, 128.5, 120.9, 120.3, 118.3, 117.2, 114.6, 112.6, 70.1, 61.8, 57.1, 46.9, 46.4, 30.9, 27.6, 23.4, 22.5, 21.2, 6.2, 5.6, 3.1. HRMS calcd. For C28H32N2O5 (m/z) 477.2384, found [M+H]+ (m/z) 477.2377, MMA=1.47 ppm. Mp 265.8 °C, % Purity 95.94 (RT 6.28 min). 17-Cyclopropylmethyl-3,14p-dihydroxy-4,5a-epoxy-6P-[(2E)-3� �-(furan-2”-yl)prop-2- enamido]morphinan hydrochloride (24)

The title compound was prepared following general procedures 3, 4 and 5 as a white solid in 39.6% yield. Hydrochloride salt: 1H NMR (400 MHz, DMSO-d6) 6 9.33 (s, 1H, exchangeable), 8.82 (br s, 1H, exchangeable), 8.45 (d, J=7.8 Hz, 1H, exchangeable), 7.78 (s, 1H), 7.22 (d, J=15.5 Hz, 1H), 6.78 (s, 1H), 6.72 (d, J=8.1 Hz, 1H), 6.65 (d, J=8.1 Hz, 1H), 6.59 (s, 1H), 6.40 (d, J=15.6 Hz, 1H), 6.17 (s, 1H, exchangeable), 4.58 (d, J=7.8 Hz, 1H), 3.84 (d, J=4.3 Hz, 1H), 3.52 (d, J=6.5 Hz, 1H), 3.06 (dd, J=24.9, 6.2 Hz, 3H), 2.82 (dd, J=36.0, 27.7 Hz, 2H), 2.46-2.36 (m, 2H), 1.80-1.67 (m, 2H), 1.59 (d, J=10.3 Hz, 1H), 1.48-1.34 (m, 2H), 1.11-1.05 (m, 1H), 0.67 (s, 1H), 0.59 (s, 1H), 0.50 (s, 1H), 0.41 (s, 1H). 13C NMR (100 MHz, DMSO-d6) 6 164.4, 150.9, 144.8, 142.0, 141.3, 129.6, 126.3, 120.6, 119.3, 119.3, 117.9, 113.9, 112.4, 89.9, 69.7, 61.7, 56.7, 50.8, 46.5, 45.6, 29.4, 27.3, 23.7, 23.0, 5.7, 5.1, 2.6. HRMS calcd. for C27H30N2O5 (m/z) 463.2227, found [M+H]+ (m/z) 463.2218, MMA=1.94 ppm. Mp 293.2 °C, % Purity 95.07 (RT 5.90 min).

4.2 Biological Methods

4.2.1 Cell Lines

Monoclonal mouse opioid receptor (denoted “m”, mKOR and mMOR) and monoclonal human opioid receptor (denoted “h”, hDOR) stably expressed in Chinese hamster ovary (CHO) cell lines were used. Cell lines heterologously expressed each of the cloned receptors to provide a reliable source of one opioid receptor subtype to be studied. This method also yields a higher receptor density, which provides for optimal signal-to-noise ratios in assays.86 Cell membrane homogenate prepared from these cell lines was used in in vitro assays.

Radioligand Binding Assay

Competitive radioligand binding assays were employed to assess the binding affinity and selectivity of designed compounds (1-24) for the KOR, MOR, and DOR. [ ³ H]naloxone ([ ³ H]NLX) was used to label the MOR and [ ³ H]diprenorphine ([ ³ H]DPN) was used to label the KOR and DOR. A saturation assay was previously conducted to determine the Kd and Bmax values for [ ³ H]NLX at MOR and [ ³ H]DPN at KOR and DOR. A fixed concentration of membrane protein (30 pg) was then incubated with the corresponding radioligand in the presence of varying concentrations of designed compound in TME buffer (50 mM Tris, 3 mM MgCh, and 0.2 mM EGTA, pH 7.7) for 1.5 h at 30°C. The bound radioligand was then separated by filtration using a Brandel harvester to determine total binding. Non-specific binding was determined by adding an excess of unlabeled competitive ligand: 5 pM U50- 488, naltrexone, and SCN80 for the KOR, MOR, and DOR respectively. Specific (i.e., opioid receptor-related) binding was defined as the difference between total binding and non-specific binding. Data from these assays allowed determination of IC50 and Hill Slope. The Cheng-Prushoff equation (K_i= [IC ] _50/(l+([L*]yK_d ) ) was then used to calculate the equilibrium dissociation constant of each compound (i.e., Ki).118 All assays were conducted in duplicate and repeated at least three times. Results were reported as mean ± SEM.

[ ³⁵ S]-GTPyS Functional Assay

[ ³⁵ S]-GTPyS binding assays were used to determine the functional activity of compounds for the KOR, MOR, and DOR (i.e., agonist, partial agonist, antagonist, or inverse agonist). This was done by defining a compound’ s efficacy in relation to that of the full agonist control for that receptor; that is U-50488, DAMGO, and DPDPE for the KOR, MOR and DOR, respectively. Membrane proteins (10 pg of mKOR-CHO, mMOR-CHO, and hDOR- CHO, in turn) were incubated with GDP (15 pM), [ ³⁵ S]-GTPyS (80 pM), varying concentrations of designed compound, 100 mM NaCl, and TME assay buffer (50 mM Tris- HC1, 3 mM MgCh, 0.2 mM EGTA, pH 7.4) to 500 pL for 1.5 h at 30° C. 10 pM of unlabeled GTPyS was used to determine non-specific binding. To define the maximum effect for each receptor, 5 pM of U-50488, DAMGO, and SCN80 were used as maximally effective concentrations for the KOR, MOR, and DOR, respectively. To separate the bound radioligand from free radioligand after incubation, filtration through a GF/B glass fiber filter paper was performed and the filtrate was rinsed three times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.2) using a Brandel harvester. A scintillation counter was then used to determine results. Dose response curves developed from [ ³⁵ S]-GTPyS data conveyed EC50 and Emax values. Emax values were related to their respective full agonists by (net- stimulated binding by ligand/net-stimulated by maximally effective concentration of full agonist) x 100%. All assays were conducted in duplicate and repeated at least three times. Results were reported as mean ± SEM.

In Vivo Model Methods

Subjects and Approvals

All animals were acquired from Envigo (Frederick, MD, USA). Rats were individually housed, while mice were group housed and both were maintained in temperature- and humidity-controlled vivaria programmed on 12-hr. light/dark cycles (lights on 6:00 AM to 6:00 PM). Food (Teklad Rat Diet, Envigo) and water were provided ad libitum in the home cage. Animal research protocols, maintenance and enrichment were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee and in accordance with the “Guide for the Care and Use of Laboratory Animals”, 8th Ed. (2011). Mice were 6- 8-week-old male Swiss Webster mice weighing approximately 25 - 35 g (n = 6/group). Rats were approximately 11 - 17-week-old male (n=9) and female (n=9) Sprague-Dawley rats with mean weights (± SEM) of X g (± X) and X g (± X) for males and females, respectively. Drugs

Studies in Mice: (-)-Morphine sulfate pentahydrate was purchased from Mallinckrodt (St. Louis, MO) and provided by the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD). (-)-Naltrexone HC1 and naloxone HC1 were purchased from Sigma-Aldrich (St. Louis, MO), norbinaltorphimine di-HCl and naltrindole HC1 were purchased from MedChemExpress (Monmouth Junction, NJ), P-funaltrexamine HC1 was purchased from AABlocks (San Diego, CA). All test compounds synthesized herein were formulated as hydrochloride salts. All known and test compounds were dissolved in double-distilled water and used directly. All drug doses were expressed based on the salt forms listed above and delivered based on individual weights as collected immediately prior to each administration.

Studies in Rats: Fentanyl HC1 was provided by the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD) and dissolved in sterile saline. Nalfurafine HC1, compound 21 and NCF (synthesized herein) were dissolved in sterile saline. All solutions were passed through a 0.22 pm sterile filter (Millex GV, Millipore Sigma, Burlington, MA) before intravenous (IV) administration. All drug doses were expressed based on the salt forms listed above and delivered based on individual weights as collected weekly.

Warm-Water Tail Withdrawal Assay

General Procedure: Warm-water tail withdrawal assays were conducted in Swiss Webster mice. A warm water bath was set to 56 + 0.1 °C. Baseline tail withdrawal latencies (prior to compound administration) were measured and a 2 - 4 s window for this baseline latency was used as exclusion criteria. To prevent tissue damage, a 10 s cutoff was established as the maximum amount of time a mouse’s tail may remain in the warm - water. In studies of agonism, test compounds were administered subcutaneously (SC) and withdrawal latencies were measured 20 min post-injection. In studies of antagonism, test compounds were administered SC 5 min before SC morphine administration and withdrawal latencies were measured 20 min post morphine injection.

Data Analysis: The primary dependent measure was the antinociceptive response calculated as the percentage of the maximum possible effect (%MPE), wherein the maximum possible effect is a tail remaining in the warm - water bath for 10 s and %MPE = [(test - baseline latency )/( 10 - baseline latency)] x 100. %MPE was calculated for each mouse. Data are shown as mean ± SEM. ED50 values were calculated using a least-squares linear regression analysis, followed by the calculation of 95% confidence intervals by the Bliss method. Data were compared using one-way ANOVA as appropriate and a significant ANOVA was followed by a Dunnet’s post hoc test as appropriate. Statistical significance was defined as p<0.05.

Receptor Selectivity Study: Baseline tail withdrawal latencies were measured according to the “general” procedure prior to any compound administration. Mice in nor-BNI groups received 10 mg/kg nor-BNI SC 24 h prior to 0.1 mg/kg test compound SC injection. Mice in [3-FNA groups received 10 mg/kg [3-FNA SC 24 h prior to 0.1 mg/kg test compound SC injection. Mice in NTI groups received 15 mg/kg NTI SC 30 min prior to 0.1 mg/kg test compound SC injection. Mice in the nor-BNI + NTI groups received both nor-BNI and NTI according to the same dosing schedule as mice in receiving a single compound antagonist. Withdrawal latencies for mice in all groups were acquired 20 min post- test-compound administration.

Time-Course Study: Baseline tail withdrawal latencies were measured according to the “general” procedure one hour prior to SC administration of test compound. Immediately following injection, withdrawal latencies were remeasured (time point 0). Withdrawal latencies were acquired after the first 30 min and after every h until test compound effects wore off with a maximum of 10 h post injection.

Tolerance/Cross-Tolerance Study: Baseline tail withdrawal latencies were measured according to the “general” procedure prior to any compound administration. To observe development of tolerance, mice were administered positive control (morphine 10 mg/kg), negative control (vehicle) or test compound SC 2x/day for 4 days with injections spaced 12 h apart. The weight of each mouse was measured prior to every administration. Withdrawal latencies for mice in all groups were acquired 20 min after the second injection of the day. On day 5, to analyze cross-tolerance, separate groups of mice that received morphine injections for 4 days were administered positive control (morphine 10 mg/kg), negative control (vehicle) or test compound SC. Withdrawal latencies for mice in all groups were acquired 20 min post injection.

Locomotor Activity

Apparatus: Six open field activity chambers (Med Associates, St. Albans, VT) were used in the locomotor activity study. Each chamber is located inside a sound-attenuating cubicle (Med Associates) and is equipped with a ventilation system. The interior of the chamber consists of a 27 x 27 cm plexiglass enclosure wired with photo-beam cells and connected to a computer console that monitors the activity of the animal.

Methods: The day before the experiment, mice were acclimated to the activity chambers for a period of 30 min. On the day of the experiment, mice were administered vehicle or test compound SC, placed into the activity chambers immediately and activity was monitored and recorded for a period of 30 min.

Data Analysis: The primary dependent measures are total 1) distance travelled (cm), 2) ambulatory counts, 3) vertical counts, and 4) average speed (cm/s) within the 30 min session. Data are shown as mean ± SEM. Data were compared using one-way or two-way ANOVA as appropriate and a significant ANOVA was followed by a Dunnet’s post hoc test as appropriate. Statistical significance was defined as p<0.05. GPower 3.1.9.7 was used for a post hoc computation of achieved power.

Abuse Liability via a Self-Administration Model

Apparatus and Catheter Maintenance: Twelve modular operant chambers located within sound-attenuating cubicles were assembled as previously reported.107 Following each behavioral session, intravenous catheters were flushed with 0.1 mL of gentamicin (4 mg/mL) and 0.1 mL of heparinized saline (30 units/mL). Catheter patency was verified at least every two weeks and at the conclusion of the study via instantaneous muscle tone loss precipitated by IV methohexital (0.5 mg) administration.

Training: Post surgical implantation of vascular access ports, five days were provided as a recovery period to ensure that the surgical site was properly healed to withstand the tension of the tether. After the recovery period, rats were trained to self-administer fentanyl using the following steps. First, daily 2-hr. behavioral sessions occurring Mon-Sun (approx. 9:30 - 11:30 AM) were used to train rats to respond for IV drug infusions (inf) under a fixed- ratio (FR) 1 / 20-s time out schedule of reinforcement. The “drug” was a 3.2 pg/kg/inf unit dose of fentanyl. This schedule of reinforcement was in effect until the number of earned infusions in a single session was approximately 20. Then, the FR requirement was increased to FR5 and in effect until the number of earned infusions was within 20% of the running mean with no upward or downward trends for three consecutive days.

Once drug self-administration training was complete, the drug syringe attached to the syringe pump and providing within- session drug infusions was intermittently swapped for a saline syringe. This occurred on a double alternation schedule (i.e., DDSSDDSS, wherein D = drug and S = saline) until the number of infusions earned on the first saline day after a drug day was >75% below the number of infusions earned on the drug day preceding it. Upon meeting this criterion, rats were switched to a single alternation schedule (i.e., DSDS) until the number of saline infusions earned was >75% below the number of infusions earned on the drug day preceding it for two consecutive alternations. The same program was used to run saline sessions and saline infusion duration was equivalent to a “drug” (3.2 pg/kg/inf) day.

Methods: Once all training criteria were met, test sessions were inserted into the sequence (i.e., DSTSDTDST, wherein T = test). As a positive control, the fentanyl dose-effect curve (0.32, 1, 3.2, 10 pg/kg/inf) was first established using this schedule. Nalfurafine (0.1, 0.32, 1, 3.2, 10 pg/kg/inf), compound 23 (NCF; 0.32, 1, 3.2, 10 pg/kg/inf [100 pg/kg/inf, n = 1], and compound 21 (0.32, 1, 3.2, 10 pg/kg/inf) were then assessed using the same procedure. Each dose of fentanyl (other than the 3.2 pg/kg/inf test dose), nalfurafine, compound 23 (NCF), and compound 21 was given once in each rat.

Data Analysis: The primary dependent measure was the number of infusions earned per session. Data were compared using one-way repeated measures ANOVA and the Geisser- Greenhouse correction was applied as appropriate (Prism 9, GraphPad, La Jolla, CA, USA). A significant ANOVA was followed by a Dunnet’s post hoc test as appropriate. Statistical significance was defined as p<0.05.

Antagonist-Induced Withdrawal Study

Swiss Webster mice were administered control (morphine 10 mg/kg) or test compound SC 2x/day for 4 days with injections spaced 12 h apart. On day 5, naloxone (1 mg/kg) was administered SC to precipitate withdrawal and mice were individually placed into an open - topped, square, clear plexiglass observation chamber (26 x 26 x 26 cm3) with lines partitioning the bottom into quadrants. Withdrawal signs were monitored for a period of 20 min beginning 3 min post naltrexone injection. Data Analysis: The primary dependent measures were the withdrawal signs including 1) number of escape jumps, 2) number of paw tremors, 3) number of wet dog shakes and 4) presence or absence of diarrhea. Data are shown as mean ± SEM. Data were compared using one-way ANOVA as appropriate and a significant ANOVA was followed by a Dunnet’s post hoc test as appropriate. Statistical significance was defined as p<0.05.

Drug-Distribution Study

Test compound was administered SC and a group of Swiss Webster mice was euthanized by decapitation at each time point (5, 10, 30, 60 min post injection) allowing brain and blood samples to be harvested. Blood samples were centrifuged for 10 min (15,000 g; 4 °C) to collect plasma. Brain and plasma samples were stored at -80 °C for further analysis.

LC/MS Analysis: Identification and quantification of test compound in mouse brain and plasma was performed using a modification of a previously described method with an internal standard of naloxone-d5.119 Compounds were extracted from both blood and brain by a liquid/liquid extraction. Briefly, brain tissue samples were homogenized 1-part tissue to 3-parts water. Seven-point calibration curves (10-1000 ng/mL or ng/g) in plasma, drug free control, a negative control without internal standard in plasma and brain, and quality control specimens in plasma and brain (30, 300 and 750 ng/mL or ng/g) were prepared and analyzed with each batch of samples. Naltrexone-d5, was added at 10 ng/mL concentration to aliquots of either 100 pL for blood or 400 pL for brain homogenate to each calibrator, control or specimen except the negative control. To these samples 0.5 mL of saturated carbonate/ bicarbonate buffer (1:1, pH 9.5) and 2.0 mL of chloroform:2-propanol (8:2) were added. The samples were then mixed and centrifuged. The top, aqueous, layer was aspirated, and the organic layer was transferred to a clean test tube and evaporated to dryness under nitrogen. The samples were then reconstituted in 80:20 water water and transferred to autosampler vials for analysis. The chromatographic separation of test compounds and naloxone-d5 was accomplished using a Shimadzu Nexera X2 liquid chromatography system and a Zorbax XDB-C18 4.6 x 75 mm, 3.5 pm column (Agilent Technologies, Santa Clara, CA). The mobile phases consisted of A) water with 1 g/L ammonium formate and 0.1% formic acid, and B) methanol. The flow rate was set to 1 mL/min. The mobile phase started with 20% B and was increased to 80% B at 1.0 min and held constant for 1.5 min before returning to 20% B. The system detector was a Sciex 6500 QTRAP system with an lonDrive Turbo V source for TurboIonSpray® (Sciex, Ontario, Canada) that had the curtain gas flow rate set at 30 mL/min and the ion source gases 1 and 2 at 60 mL/min. The source temperature was set at 650°C with an ionspray voltage was 5500 V. The declustering potential was 58 eV.

Data Analysis: The primary dependent measures are 1) the plasma concentration of the test compound (pg/mL), 2) the brain concentration of the test compound (pg/g), and 3) the brain-to-plasma concentration ratio calculated as [brain]/[plasma]. Concentrations were determined by a linear regression plot based on peak ratios of the calibrators. Data are shown as mean ± SEM. Data were compared using one-way ANOVA as appropriate and a significant ANOVA was followed by a Dunnet’s post hoc test as appropriate. Statistical significance was defined as p<0.05.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.