Novel small molecule negative allosteric modulators of the μ-opioid receptor

Abstract

Disclosed herein are compounds of Formula (I), and pharmaceutically acceptable salts thereof, that are negative allosteric modulators of the p-opioid receptor (MOR). Also disclosed are methods of inhibiting MOR as well as treating opioid overdose.

Description

S23-339 CZB-293S-PC T-020961 33167 / 59763 NOVEL SMALL MOLECULE NEGATIVE ALLOSTERIC MODULATORS OF THE µ-OPIOID RECEPTOR STATEMENT OF GOVERNMENT INTEREST

[0001] This invention was made with government support under grant number DA057790 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD

[0002] The present disclosure relates generally to novel compounds that negatively modulate the µ-OR and methods for treating opioid overdose. In particular, the disclosure relates to methods for treating fentanyl overdose. BACKGROUND

[0003] Traditional opioid compounds extracted from Papaver somniferum and related semi-synthetic derivates, including morphine, heroin, codeine, and oxycodone, have been used both recreationally and as potent pain relief molecules. These compounds represent best-in-class treatments for acute pain management yet have also been extensively prescribed as long-term analgesic treatments, where their high potential for addiction and abuse have fueled the current opioid overdose epidemic. While ever more patients were becoming addicted to over-prescribed long-term pain management opioids (e.g., oxycodone), fully synthetic, much more potent opioids such as fentanyl have exploded as cheap additives to recreationally-used opioid mixtures. Naloxone (Narcan) is the most common, effective treatment for opioid overdoses but requires larger, repeated doses in response to the more potent fentanyl. All these molecules share a similar mode of action as agonists of the µ-opioid receptor (MOR), albeit with varying affinity and efficacy.

[0004] Agonists and antagonists of the MOR bind at an overlapping orthosteric site in the extracellular vestibule of the receptor and share a set of key interactions with endogenous opioid signaling peptides, namely the enkephalins, endorphins and endomorphins. Given the limitations of the current slate of orthosteric opioid molecules, as well as the severity of the current illegal opioid overdose epidemic, new classes of MOR- modulating compounds with distinct mechanisms of action are highly desirable and have emerged as priorities of the National Institute of Drug Abuse (NIDA).

[0005] While small molecules, including cannabinoids and the selective κOR agonist salvinorin A, have been identified as NAMs of the MOR, their potencies for their selective targets (CB1 and κOR) are much higher than those observed against the MOR (high µM), suggesting they are highly unlikely to display selective MOR-NAM activity in vivo.

[0006] Thus, a need exists for negative allosteric modulators of the MOR and their use in treating opioid overdose.S23-339 CZB-293S-PC T-020961 33167 / 59763 SUMMARY

[0007] Provided herein are compounds having a structure of Formula (I):heterocycle, or 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl each comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and Cyc is substituted with 0 to 3 R1A, each R1Ais independently selected from halo, C1-3alkyl, C1-3alkoxy, CN, NO2, and N(RN)2, R2is C1-6alkyl, C1-6alkoxy, C1-6hydroxyalkyl, –C0-3alkylene–C6-10aryl, or –C0-3alkylene–5-10 membered heteroaryl, wherein the heteroaryl comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and the aryl and heteroaryl ring is substituted with 0 to 3 R2A, each R2Ais independently selected from halo, C1-6alkyl, C1-3haloalkyl, C3-6cycloalkyl, C1-3alkoxy, CN, and N(RN)2, R3is C1- 6alkyl, C1-6alkoxy, N(RN)2, or 4-7-memebred heterocycle having 1-3 ring heteroatoms independently selected from N, O, and S, R4and R5are independently H or RA; or R4and R5join to form a 6-membered fused aromatic ring with 0 to 2 ring N atoms, and is substituted with 0, 1 or 2 RA; each RAis independently C1-6alkyl or C1-6alkoxy, and each RNis independently H or C1-6alkyl, or two RNtogether with the nitrogen to which they are attached form a 4-10 membered heterocycle having 0-2 additional ring heteroatoms independently selected from N, O, and S, with the provisos that when R1is meta-OCH2Ph, R2is CH2Ph with Ph substituted with Me and OMe, R3is NHMe, and R4and R5form a fused unsubstituted phenyl ring, then X is O or NRN.

[0008] Further provided herein are pharmaceutical compositions comprising the compounds as disclosed herein. Also provided herein are methods of inhibiting µ opioid receptor (MOR), comprising contacting MOR with a compound as disclosed herein.

[0009] Also provided herein are methods for treating opioid overdose in a subject suffering therefrom comprising administering to the subject a therapeutically effective amount of a compound as disclosed herein. In many cases, the methods for treating opioid overdose in a subject further comprises administration of a therapeutically effective amount of naloxone either before, after, or concomitantly with administration of the compound.

[0010] Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. While the compounds and methods disclosed herein are susceptible of cases in various forms, the description hereafter includes specific cases with the understanding that the disclosure is illustrative and is not intended to limit the invention to the specific cases described herein.S23-339 CZB-293S-PC T-020961 33167 / 59763 BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows a radioligand binding assay with compounds A1, A2, and A3 compared with S-368, a comparator compound having a structure

[0012] Figure 2 shows a GTP turnover assay for compounds A1 and A2 compared with S-368.

[0013] Figure 3 shows a GTP turnover assay for compounds A3 and A6 compared with S-368.

[0014] Figure 4 shows a radioligand binding assay with compounds A3 and A6 compared with S-368.

[0015] Figure 5 shows (a) a radioligand binding assay and (b) a GTP turnover assay for compounds A4 and A5 compared with S-368.

[0016] Figure 6 shows a radioligand binding assay with compound A7 compared with S-368.

[0017] Figure 7 shows a radioligand binding assay with compounds A8, A9, and A10 compared with S-368. DETAILED DESCRIPTION

[0018] Traditional opioids share a similar mode of action as agonists of the µ-opioid receptor (MOR). Agonists and antagonists of the MOR bind at an overlapping orthosteric site in the extracellular vestibule of the receptor and share interactions with endogenous opioid signaling peptides. Modulation of receptor activity via binding of molecules at alternate sites on the receptor (allosteric sites), rather than agonists / antagonists which bind at traditional orthosteric sites, potentially provides a series of advantages. Specifically, molecules that bind at sites distinct from the MOR orthosteric site and positively or negatively modulate its activity (PAM and NAM, respectively) have the potential to signal more specifically through the µ-opioid receptor (MOR) (rather than its κ- and δ-opioid receptor counterparts). Compounds of the Disclosure

[0019] Disclosed herein are compounds having a structure of Formula (I):pharmaceutically acceptable salts thereof wherein: X is S, NRN, or O;S23-339 CZB-293S-PC T-020961 33167 / 59763 R1is H or L1-Cyc; L1is C0-3alkylene or O-C0-3alkylene; Cyc is C3-6cycloalkyl, C6-10aryl, 4-8 membered heterocycle, or 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl each comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and Cyc is substituted with 0 to 3 R1A; each R1Ais independently selected from halo, C1-3alkyl, C1-3alkoxy, CN, NO2, and N(RN)2; R2is C1-6alkyl, C1-6alkoxy, C1-6hydroxyalkyl, –C0-3alkylene–C6-10aryl, or –C0-3alkylene–5-10 membered heteroaryl, wherein the heteroaryl comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and the aryl and heteroaryl ring is substituted with 0 to 3 R2A; each R2Ais independently selected from halo, C1-6alkyl, C3-6cycloalkyl, C1-3haloalkyl, C1-3alkoxy, CN, and N(RN)2; R3is C1-6alkyl, C1-6alkoxy, N(RN)2, or 4-7-memebred heterocycle having 1-3 ring heteroatoms independently selected from N, O, and S; R4and R5are each independently H or RA; or R4and R5join to form a 6-membered fused aromatic ring with 0 to 2 ring N atoms, and is substituted with 0, 1 or 2 RA; each RAis independently C1-6alkyl or C1-6alkoxy; and each RNis independently H or C1-6alkyl, or two RNtogether with the nitrogen to which they are attached form a 4- 10 membered heterocycle having 0-2 additional ring heteroatoms independently selected from N, O, and S, with the proviso that when R1is meta-OCH2Ph, R2is CH2Ph with Ph substituted with Me and OMe, R3is NHMe, and R4and R5form a fused unsubstituted phenyl ring, then X is O or NRN.

[0020] In various cases, the compound has a structure of Formula (Ia):

[0021] As disclosed herein, R1can be H or L1Cyc. As disclosed herein, L1is C0-3alkylene or O-C0-3alkylene. As disclosed herein, Cyc is C3-6cycloalkyl, C6-10aryl, 4-8 membered heterocycle, or5-10 membered heteroaryl, wherein the heterocycle and heteroaryl each comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and Cyc is substituted with 0 to 3 R1A. In various cases, R1is L1-Cyc. In some cases, L1is C0-3alkylene. In various cases, L1is O–C0-3alkylene. In some cases, Cyc C6-10aryl or 5-10 membered heteroaryl. In some cases, Cyc is phenyl, substituted with 0 to 3 R1A. In some cases, Cyc is pyridyl pyrazolyl, imidazolyl, furanyl, pyrimidinyl, or pyrazinyl, substituted with 0 to 3 R1A. In some cases, Cyc is pyridyl substituted with 0 to 3 R1A. In some cases, Cyc is substituted with 1 or 2 R1A.S23-339 CZB-293S-PC T-020961 33167 / 59763

[0022] As disclosed herein, each R1Acan independently be halo, C1-3alkyl, C1-3alkoxy, CN, NO2, or N(RN)2. In various cases, each R1Ais independently halo, C1-3alkyl, NO2, or N(RN)2.

[0024] In various cases, the compound has a structure of Formula (Ib):

[0025] In various cases, the compound has a structure of Formula (Ic):S23-339 CZB-293S-PC T-020961 33167 / 59763

[0026] As disclosed herein, R2can be C1-6alkyl, C1-6alkoxy, C1-6hydroxyalkyl, –C0-3alkylene–C6-10aryl, or –C0-3alkylene–5-10 membered heteroaryl, wherein the heteroaryl comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and the aryl and heteroaryl ring is substituted with 0 to 3 R2A. In various cases, R2is C1-6alkyl, C1-6hydroxyalkyl, –C0-3alkylene–C6-10aryl, or –C0-3alkylene–5-10 membered heteroaryl, and the aryl and heteroaryl ring is substituted with 0 to 3 R2A.

[0027] As disclosed herein, each R2Acan independently be halo, C1-6alkyl, C3-6cycloalkyl, C1-3haloalkyl, C1- 3alkoxy, CN, or N(RN)2. In various cases, each R2Ais independently halo, C1-6alkyl, C3-6cycloalkyl, C1-3haloalkyl, or C1-3alkoxy.

[0029] As disclosed herein, R3can be C1-6alkyl, C1-6alkoxy, N(RN)2, or 4-7-memebred heterocycle having 1-3 ring heteroatoms independently selected from N, O, and S. In various cases, R3is C1-6alkoxy or N(RN)2. In some cases, R3is N(RN)2and each RNtogether with the nitrogen to which they are attached form a 4-10 membered heterocycle. In some cases, R3is 4-7-membered heterocycle. In some cases, R3is OMe, NHMe, NH2, N(Me)2,

[0030] As disclosed herein, X can be S, O, or NRN. In various cases, X is S. In various cases, X is O. In some cases, X is NRN.

[0031] As disclosed herein, R4can be H or RA. In various cases, R4is H. In some cases, R4is RA.

[0032] As disclosed herein, R5can be H or RA. In various cases, R5is RA. In some cases, R5is H.S23-339 CZB-293S-PC T-020961 33167 / 59763

[0033] As disclosed herein, R4and R5can form a fused 6-membered aromatic ring with 0 to2 ring nitrogen atoms, substituted with 0, 1, or 2 RA.

[0034] As disclosed herein, each RAcan independently be C1-6alkyl, or C1-6alkoxy. In some cases, at least one RAis C1-6alkyl. In some cases, at least one RAis C1-6alkoxy.

[0035] As disclosed herein, each RNcan independently be H or C1-6alkyl, or two RNtogether with the nitrogen to which they are attached form a 4-10 membered heterocycle having 0-2 additional ring heteroatoms independently selected from N, O, and S. In various cases, at least one RNis H. In some cases, two RNtogether with the nitrogen to which they are attached form a 4-10 membered heterocycle having 0-2 additional ring heteroatoms independently selected from N, O, and S.

[0036] Compounds as disclosed herein include those as shown in Table A, or a pharmaceutically acceptable salt thereof. Table AS23-339 CZB-293S-PC T-020961 33167 / 59763

[0037] Unless otherwise indicated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, cis-trans, conformational, and rotational) forms of the structure. For example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in this disclosure, unless only one of the isomers is specifically indicated. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, cis / trans, conformational, and rotational mixtures of the present compounds are within the scope of the disclosure. In some cases, the compounds disclosed herein are stereoisomers. "Stereoisomers" refer to compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds disclosed herein can exist as a single stereoisomer, or as a mixture of stereoisomers. Stereochemistry of the compounds shown herein indicate a relative stereochemistry, not absolute, unless discussed otherwise. As indicated herein, a single stereoisomer, diastereomer, or enantiomer refers to a compound that is at least more than 50% of the indicated stereoisomer, diastereomer, or enantiomer, and in some cases, at least 90% or 95% of the indicated stereoisomer, diastereomer, or enantiomer.

[0038] The compounds disclosed herein that have a double bond can exhibit E or Z (not shown) stereochemistry. In some cases, the compounds of Formula (I) exhibit E stereochemistry. In various cases, the compounds of Formula (I) exhibit Z stereochemistry at the double bond. The compounds of Formula (I) can have any stereochemical configuration at any sp3carbon atoms. In some cases, the compounds of the disclosure are optically pure. As used herein, “optically pure” refers to the predominant presence of one enantiomer of aS23-339 CZB-293S-PC T-020961 33167 / 59763 compound if multiple stereochemical configurations can exist (e.g., at least 99% enantiomeric excess). Unless otherwise indicated, all tautomeric forms of the compounds of the disclosure are within the scope of the disclosure.

[0039] The compounds of the disclosure are defined herein by their chemical structures and / or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.

[0040] As used herein, the term “alkyl” refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty carbon atoms, or one to ten carbon atoms. The term Cn means the alkyl group has “n” carbon atoms. For example, C6alkyl refers to an alkyl group that has 6 carbon atoms. C1-7alkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 6 carbon atoms), as well as all subgroups (e.g., 1-5, 2-5, 3-6, 1, 2, 3, 4, 5, and 6 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2- methylpropyl), and t-butyl (1,1-dimethylethyl). Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0041] As used herein, the term “alkylene” refers to a bivalent saturated aliphatic radical. The term Cnmeans the alkylene group has "n" carbon atoms, e.g., a C1alkylene is CH2. For example, C1-6alkylene refers to an alkylene group having a number of carbon atoms encompassing the entire range, as well as all subgroups, as previously described for "alkyl" groups.

[0042] As used herein, the term “cycloalkyl” specifically refers to a non-aromatic carbocycle. The term Cn means the cycloalkyl group has “n” carbon atoms. For example, C5 cycloalkyl refers to a cycloalkyl group that has 5 carbon atoms in the ring. C5-8 cycloalkyl refers to cycloalkyl groups having a number of carbon atoms encompassing the entire range (i.e., 5 to 10 carbon atoms), as well as all subgroups (e.g., 5-10, 5-9, 5-8, 5-6, 6- 8, 7-8, 5-7, 5, 6, 7, 8, 9 and 10 carbon atoms). Nonlimiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise indicated, a cycloalkyl group can be an unsubstituted cycloalkyl group or a substituted cycloalkyl group.

[0043] As used herein, the term “aryl” refers to an aromatic carbocycle, and can be monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) carbocyclic aromatic ring systems. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, phenanthrenyl, biphenylenyl, indanyl, indenyl, anthracenyl, fluorenyl, tetralinyl. Unless otherwise indicated, an aryl group can be an unsubstituted aryl group or a substituted aryl group.

[0044] As used herein, the term "heterocycle" refers to a non-aromatic ring which contains one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur. Additionally, heterocycles of the disclosure can be monocyclic, bicyclic, bridged, fused or spirocyclic. For example, a heterocycle can be a monocyclic, bicyclic, bridged, fused, or spirocyclic 4-8 membered ring having 1 or 2 or 3 heteroatoms selected from N, O, and S. As another example, a heterocycle can be a 8-10 membered bicyclic, bridged, fused, orS23-339 CZB-293S-PC T-020961 33167 / 59763 spirocyclic group having 1 or 2 or 3 ring heteroatoms selected from N, O, and S in the bicyclic ring. Nonlimiting examples of heterocycle groups include piperidine, piperazine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, and oxazepane.

[0045] As used herein, the term "heteroaryl" refers to a cyclic aromatic ring having heteroatoms in the ring (e.g., a monocyclic aromatic ring with 5-6 total ring atoms, or a fused bicyclic ring with 9-10 total ring atoms), and containing one to three heteroatoms selected from nitrogen, oxygen, and sulfur atom in the aromatic ring. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted. Heteroaryl groups can be isolated (e.g., pyridyl) or fused to another heteroaryl group (e.g., purinyl), a cycloalkyl group (e.g., tetrahydroquinolinyl), a heterocycle group (e.g., dihydronaphthyridinyl), and / or an aryl group (e.g., benzothiazolyl, quinolyl, isoquinolinyl, or quinazolinyl).

[0046] As used herein, the term “alkoxy” refers to a “—O-alkyl” group.

[0047] As used herein, the term “halo” refers to refers to a fluoro (F), chloro (Cl), bromo (Br), or iodo (I) group. Accordingly, a “haloalkyl” refers to an alkyl group substituted with one or more halo atoms.

[0048] As used herein, the term “hydroxyalkyl” refers to refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a hydroxyl group (OH). Such groups include but are not limited to, hydroxymethyl, hydroxyethyl, and the like.

[0049] As used herein, a “substituted” functional group is a functional, group having at least one hydrogen radical that is substituted with a non-hydrogen radical (i.e., a substituent). Examples of non-hydrogen radicals (or substituents) include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkyl, alkynyl, ether, aryl, heteroaryl, heterocycle, hydroxyl, oxy (or oxo), alkoxyl, ester, thioester, acyl, carboxyl, cyano, nitro, amino, sulfhydryl, and halo. When a substituted alkyl group includes more than one non-hydrogen radical, the substituents can be bound to the same carbon or different carbon atoms. Pharmaceutically Acceptable Salts

[0050] As used herein, the term "pharmaceutically acceptable salt" refers to salts of a compound which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue side effects, such as, toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio.

[0051] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds.

[0052] Where the compound described herein contains a basic group, or a sufficiently basic bioisostere, acid addition salts can be prepared by 1) reacting the purified compound in its free-base form with a suitable organicS23-339 CZB-293S-PC T-020961 33167 / 59763 or inorganic acid and 2) isolating the salt thus formed. In practice, acid addition salts might be a more convenient form for use and use of the salt amounts to use of the free basic form.

[0053] Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0054] Where the compound described herein contains a carboxyl group or a sufficiently acidic bioisostere, base addition salts can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base and 2) isolating the salt thus formed. In practice, use of the base addition salt might be more convenient and use of the salt form inherently amounts to use of the free acid form. Salts derived from appropriate bases include alkali metal (e.g., sodium, lithium, and potassium), alkaline earth metal (e.g., magnesium and calcium), ammonium and N+(C1-4alkyl)4 salts. This disclosure also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0055] Basic addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum. The sodium and potassium salts are usually preferred. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. 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 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, dietanolamine, 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, dicyclohexylamine and the like.S23-339 CZB-293S-PC T-020961 33167 / 59763

[0056] Other acids and bases, although not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts.

[0057] It should be understood that a compound disclosed herein can be present as a mixture / combination of different pharmaceutically acceptable salts. Also contemplated are mixtures / combinations of compounds in free form and pharmaceutically acceptable salts. Pharmaceutical Formulations

[0058] Also provided herein are pharmaceutical formulations that include an effective amount of compounds of the disclosure and one or more pharmaceutically acceptable excipients. As used herein, the term “formulation” is used interchangeable with “composition.”

[0059] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and / or ameliorating a disease or condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and / or substantially lessening the chances of a disease or condition in a subject. As used herein, the terms “patient” and “subject” may be used interchangeably and mean animals, such as dogs, cats, cows, horses, and sheep (i.e., non-human animals) and humans. Particular patients or subjects are mammals (e.g., humans). The terms “patient” and “subject” include males and females.

[0060] As used herein, the term “excipient” means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API), suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.

[0061] The compounds of the disclosure can be administered alone or as part of a pharmaceutically acceptable composition or formulation. In addition, the compounds can be administered all at once, as for example, by a bolus injection, multiple times, e.g. by a series of tablets, or delivered substantially uniformly over a period of time, as for example, using transdermal delivery. It is also noted that the dose of the compound can be varied over time.

[0062] The compounds disclosed herein and other pharmaceutically active compounds, if desired, can be administered to a subject or patient by any suitable route, e.g. orally, topically, rectally, parenterally, (for example, subcutaneous injections, intravenous, intramuscular, intrasternal, and intrathecal injection or infusion techniques), or as a buccal, inhalation, or nasal spray. The administration can be to provide a systemic effect (e.g. eneteral or parenteral). All methods that can be used by those skilled in the art to administer a pharmaceutically active agent are contemplated. In some cases, the disclosed formulations can be administered orally or topically.

[0063] Suitable oral compositions or formulations in accordance with the disclosure include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or softS23-339 CZB-293S-PC T-020961 33167 / 59763 capsules, syrups or elixirs. Compositions or formulations suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions.

[0064] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0065] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0066] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0067] The active compounds can also be in microencapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at leastS23-339 CZB-293S-PC T-020961 33167 / 59763 one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0068] The pharmaceutical compositions and formulations described herein may also be administered topically or transdermally, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract, e.g., can be effected in a rectal suppository formulation or in a suitable enema formulation. Dosage forms for topical or transdermal administration of a compound described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, suppositories, or patches.

[0069] For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment, cream, lotion, or gel, containing the active component suspended or dissolved in one or more carriers, and any needed preservatives or buffers as may be required. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

[0070] Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this disclosure. Additionally, the present disclosure contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0071] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For thisS23-339 CZB-293S-PC T-020961 33167 / 59763 purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0072] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0073] In order to prolong the effect of a compound described herein, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0074] Compositions for rectal or vaginal administration are specifically suppositories which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0075] Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.S23-339 CZB-293S-PC T-020961 33167 / 59763

[0076] The pharmaceutical compositions may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0077] The compounds for use in the methods of the disclosure can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0078] The compounds of the disclosure can be administered to a subject or patient at dosage levels in the range of about 0.1 to about 3,000 mg per day. For a normal adult human having a body weight of about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kilogram body weight is typically sufficient. The specific dosage and dosage range that will be used can potentially depend on a number of factors, including the requirements of the subject or patient, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. The determination of dosage ranges and optimal dosages for a particular subject or patient is within the ordinary skill in the art. Methods of Treatment

[0079] The compounds disclosed herein can be used in methods for negatively allosterically modulating the µ opioid receptor (MOR NAM). Allosteric modulators may be able to selectively enhance the activity of certain orthosteric molecules. G-protein coupled receptors (GPCRs) are known to signal through multiple intracellular effectors, including G-proteins, G-protein coupled receptor kinases (GRKs), and the β-arrestins. Orthosteric ligands, including those for the opioid receptors and cannabinoid receptors, are able to activate these different signaling pathways to varying extents, a phenomenon known as bias. So-called biased allosteric modulators (BAMs) are able to combine the advantages of allosteric modulators above while also selectively activating or inhibiting certain desired intracellular pathways. Recently, it has been proposed that MOR NAMs can enhance the anti-overdose properties of naloxone.

[0080] Thus, the disclosure provides a method of negatively modulating MOR comprising contacting the MOR with a therapeutically effective amount of a compound or salt disclosed herein or a formulation thereof, in an amount effective to inhibit MOR activity. In some cases, the contacting occurs in vitro. In some cases, the contacting occurs in vivo. In some cases, the contacting comprises administering to a subject in need thereof. As used herein, the terms “patient” and “subject” may be used interchangeably and mean animals, such as dogs, cats, cows, horses, and sheep (i.e., non-human animals) and humans. In some cases, the patient is a mammal (e.g., human).S23-339 CZB-293S-PC T-020961 33167 / 59763

[0081] Another aspect of the disclosure provides a method of treating opioid overdose in a subject, comprising administering to the subject a therapeutically effective amount of a compound or salt disclosed herein or a formulation thereof. In some cases, the terms “treating”, “treat” or “treatment” and the like can include preventative (e.g., prophylactic) and palliative treatment. In various cases, naloxone can optionally be administered either before, after, or concomitantly with administration of the MOR NAM compound.

[0082] Also contemplated is the use of a compound disclosed herein, or salt thereof, or a formulation comprising a compound or salt disclosed herein for the manufacture of a medicament in the treatment of opioid overdose.

[0083] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities. Embodiments of the Disclosure 1. A compound, or pharmaceutically acceptable salt thereof, having a structure of Formula (I):wherein: X is S, NRN, or O; R1is H or L1-Cyc; L1is C0-3alkylene or O-C0-3alkylene; Cyc is C3-6cycloalkyl, C6-10aryl, 4-8 membered heterocycle, or 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl each comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and Cyc is substituted with 0 to 3 R1A; each R1Ais independently selected from halo, C1-3alkyl, C1-3alkoxy, CN, NO2, and N(RN)2; R2is C1-6alkyl, C1-6alkoxy, C1-6hydroxyalkyl, –C0-3alkylene–C6-10aryl, or –C0-3alkylene–5-10 membered heteroaryl, wherein the heteroaryl comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and the aryl and heteroaryl ring is substituted with 0 to 3 R2A; each R2Ais independently selected from halo, C1-6alkyl, C3-6cycloalkyl, C1-3haloalkyl, C1-3alkoxy, CN, andS23-339 CZB-293S-PC T-020961 33167 / 59763 N(RN)2; R3is C1-6alkyl, C1-6alkoxy, N(RN)2, or 4-7-memebred heterocycle having 1-3 ring heteroatoms independently selected from N, O, and S; R4and R5are each independently H or RA; or R4and R5join to form a 6-membered fused aromatic ring with 0 to 2 ring N atoms, and is substituted with 0, 1 or 2 RA; each RAis independently C1-6alkyl or C1-6alkoxy; and each RNis independently H or C1-6alkyl, or two RNtogether with the nitrogen to which they are attached form a 4-10 membered heterocycle having 0-2 additional ring heteroatoms independently selected from N, O, and S, with the proviso that when R1is meta-OCH2Ph, R2is CH2Ph with Ph substituted with Me and OMe, R3is NHMe, and R4and R5form a fused unsubstituted phenyl ring, then X is O or NRN. 2. The compound or salt of embodiment 1, wherein R1is L1-Cyc. 3. The compound or salt of embodiment 2, wherein L1is C0-3alkylene. 4. The compound or salt of embodiment 2, wherein L1is O-C0-3alkylene. 5. The compound or salt of any one of embodiments 2 to 4, wherein Cyc is phenyl. 6. The compound or salt of any one of embodiments 2 to 4, wherein Cyc is pyridyl, pyrazolyl, imidazolyl, furanyl, pyrimidinyl, or pyrazinyl. 7. The compound or salt of any one of embodiments 2 to 6, wherein Cyc is substituted with 1 or 2 R1A. 8. The compound or salt of any one of embodiments 2 to 7, wherein each R1Ais selected from halo, C1-3alkyl, NO2, and N(RN)2.S23-339 CZB-293S-PC T-020961 33167 / 5976310. The compound or salt of any one of embodiments 1 to 9, having a structure of Formula (Ib):11. The compound or salt of any one of embodiments 1 to 9, having a structure of Formula (Ic):12. The compound of salt of any one of embodiments 1 to 11, wherein R2is C1-6alkyl, C1- 6hydroxyalkyl, –C0-3alkylene–C6-10aryl, or –C0-3alkylene–5-10 membered heteroaryl, the aryl and heteroaryl ring is substituted with 0 to 3 R2A. 13. The compound or salt of any one of embodiments 1 to 12, wherein each R2Ais independently selected from halo, C1-6alkyl, C3-6cycloalkyl, C1-3haloalkyl, and C1-3alkoxy.S23-339 CZB-293S-PC T-020961 33167 / 59763 15. The compound of salt of any one of embodiments 1 to 14, wherein R3is C1-6alkoxy or N(RN)2. 16. The compound of salt of embodiment 15, wherein R3is N(RN)2and each RNtogether with the nitrogen to which they are attached form a 4-10 membered heterocycle. 17. The compound or salt of any one of embodiments 1 to 14, wherein R3is 4-7-membered heterocycle. 18. The compound or salt of any one of embodiments 1 to 14, wherein R3is OMe, NHMe, NH2,19. The compound of salt of any one of embodiments 1 to 18, wherein X is S. 20. The compound of salt of any one of embodiments 1 to 18, wherein X is NRN. 21. The compound of salt of any one of embodiments 1 to 18, wherein X is O. 22. The compound of salt of any one of embodiments 1 to 21, wherein R4is H. 23. The compound or salt of any one of embodiments 1 to 21, wherein R4is RA. 24. The compound or salt of embodiment 23, wherein R4is C1-6alkyl or C1-6alkoxy. 25. The compound of salt of any one of embodiments 1 to 21, wherein R5is H. 26. The compound or salt of any one of embodiments 1 to 21, wherein R5is RA. 27. The compound or salt of embodiment 26, wherein R5is C1-6alkyl or C1-6alkoxy. 28. The compound of salt of any one of embodiments 1 to 21, wherein R4and R5join to form a 6- membered fused aromatic ring with 0 to 2 ring N atoms, and is substituted with 0, 1 or 2 RA. 29. The compound of salt of embodiment 28, wherein R4and R5join to form a 6-membered fused phenyl ring substituted with 0, 1 or 2 RA. 30. A compound, or pharmaceutically acceptable salt thereof, having a structure selected fromS23-339 CZB-293S-PC T-020961 33167 / 59763S23-339 CZB-293S-PC T-020961 33167 / 5976331. A method of inhibiting µ opioid receptor (MOR), comprising contacting MOR with the compound or salt of any one of embodiments 1 to 30 in an amount sufficient to inhibit MOR. 32. A method for treating opioid overdose in a subject suffering therefrom comprising administering to the subject a therapeutically effective amount of the compound or salt of any one of embodiments 1 to 30. 33. The method of embodiment 32, further comprising administration of a therapeutically effective amount of naloxone either before, after, or concomitantly with administration of the compound or salt thereof. 34. The method of embodiment 32 or 33, wherein the opioid is fentanyl. EXAMPLES Experimental Procedures Chemical Materials

[0084] Reagents were purchased from Sigma-Aldrich Chemicals, Ambeed, Chemscene, Chemimpex and used as-is. All reactions were performed under argon atmosphere unless otherwise specified. While performing synthesis, reaction mixtures were purified by silica gel flash chromatography on E. Merck 230–400 mesh silica gel 60 using a Teledyne ISCO CombiFlash Rf instrument with UV detection at 280 and 254 nm. RediSep Rf silica gel normal phase columns were used with a gradient range of 0–80% EtOAc in Hexane. Final clean (purity ≥95%, LC-MS Agilent 1100 Series LC / MSD) compounds were used for the study. NMR spectra were collected using Varian 500 MHz NMR instrument and collected via the Bruker Topspin Software (Bruker Topspin 3.5 pI 6). Chemical shifts are reported in parts per million (ppm) relative to residual solvent peaks at the nearest 0.01 for proton and 0.1 for carbon (CDCl31H: 7.26,13C: 77.1). Peak multiplicity is reported as the NMR spectra were processed with MestreNova software14.2.0, namely s – singlet, d – doublet, t – triplet, q – quartet, m – multiplet for examples. Coupling constant (J) values are expressed in Hz. Mass spectra were obtained using an AgilentS23-339 CZB-293S-PC T-020961 33167 / 59763 1100 Series LC / MSD by electrospray (ESI) ionization with a gradient elution program (Ascentis Express Peptide C18 column, acetonitrile / water 5 / 95 / 95 / 5, 5 minutes, 0.05% formic acid) and UV detection (214 nM / 254 nM). High resolution mass spectra were obtained using a Bruker 10 T APEX -Qe FTICR-MS and the accurate masses are reported for the molecular ion [M+H]+. Purification of µ-opioid receptor and variants

[0085] The mouse µ-opioid receptor was grown and purified as previously described1. Mouse µ-opioid receptor (MOR) with an N-terminal FLAG and C-terminal hexa-histidine tag was expressed as previously described1using the baculovirus method in Spodoptera frugiperda (Sf9) cells. Naloxone was added to 10 µM final concentration upon infection and cells were collected 48 hours post-infection and stored at -80°C for later purification. MOR was extracted from membranes with 0.8% n-dodecyl-ß-D-malopyranoside (DDM; Anatrace), 0.08% cholesterol hemisuccinate (CHS), and 0.3% 3-((3-cholamidopropyl) dimethylammonio)-1- propanesulfonate (CHAPS; Anatrace) in 20 mM HEPES pH 7.5, 500 mM sodium chloride (NaCl), 30% glycerol, 5 mM imidazole, 10 µM naloxone, and the protease inhibitors benzamidine and leupeptin, along with benzonase (Sigma-Aldrich) to degrade cellular DNA. Cells were dounced 30 times on ice and the membranes were allowed to solubilize in the detergent for 2 hours with stirring at 4 °C, followed by centrifugation for 40 minutes at 14k rpm to pellet cell debris. The supernatant was applied to nickel-chelating sepharose resin and bound to resin with end-over-end shaking for 2 hours at 4°C. The resin was then batch washed 4 times followed by washing with 10 column volumes on-column with nickel wash buffer composed of 20 mM HEPES pH 7.4, 500 mM NaCl, 0.1% DDM, 0.01% CHS, 0.03% CHAPS, 5 mM imidazole, 10 µM naloxone, and protease inhibitors leupeptin and benzamidine. The nickel-pure MOR was then eluted in the same buffer with 250 mM imidazole. The nickel elution was initially exchanged to lauryl maltose neopentyl glycol (L-MNG; Anatrace) detergent by incubating with 0.5% L-MNG, 0.17% glycol-diosgenin (GDN; Anatrace) and 0.067% CHS overnight at 4°C.2 mM calcium chloride (CaCl2) was then added and subsequently applied to M1 anti-FLAG immunoaffinity resin. The M1-bound receptor was first washed with 20 mM HEPES pH 7.4, 500 mM NaCl, 0.1% MNG, 0.033% GDN, 0.0133% CHS, 2 mM CaCl2, and 10 µM naloxone. The protein was then washed with 10 column volumes of 20 mM HEPES pH 7.4, 100 mM NaCl, 0.005% MNG, 0.0017% GDN, 0.00067% CHS and 2 mM CaCl2 followed by elution with 20 mM HEPES pH 7.4, 100 mM NaCl, 0.003% MNG, 0.001% GDN, 0.0004% CHS, 5 mM ethylenediaminetetraacetic acid (EDTA) and FLAG peptide. Multimers and dimers of the receptor were removed with size exclusion chromatography on an S20010 / 300 Increase gel filtration column (GE Healthcare) equilibrated with 20 mM HEPES pH 7.4, 100 mM NaCl, 0.003% MNG, 0.001% GDN, 0.0004% CHS. Pure, monomeric apo MOR was spin concentrated to ~150 µM, flash frozen in liquid nitrogen and stored at -80°C until further use.

[0086] The full-length human µ-opioid receptor gene was engineered into an expression vector for use in Expi293F cells (Thermo Fisher), featuring an N-terminal HA signal peptide and FLAG tags, along with a C- terminal hexahistidine tag. This vector was transfected into Expi293F cells that continuously express the tetracycline repressor (Thermo Fisher) using the Expifectamine transfection kit (Thermo Fisher), adhering to theS23-339 CZB-293S-PC T-020961 33167 / 59763 manufacturer's instructions. Expression of the receptor was induced 48 hours post-transfection using doxycycline (4 µg / mL) and sodium butyrate (5 mM) in the presence of naloxone (10 µM). Approximately 30 hours post- induction, cell pellets were harvested and stored at -80°C for subsequent purification. The cells were lysed and the membranes were solubilized using a buffer composed of 20 mM HEPES (pH 7.5), 100 mM NaCl, 20% glycerol, 1% lauryl maltose neopentyl glycol (L-MNG), 0.1% cholesterol hemisuccinate (CHS), 10 µM naloxone, protease inhibitors leupeptin and benzamidine, and benzonase. The receptors were then isolated via anti-FLAG immunoaffinity resin following the same protocol as above. Aggregates and dimers were separated using size exclusion chromatography on an S20010 / 300 Increase gel filtration column (GE Healthcare) equilibrated in a buffer of 20 mM HEPES (pH 7.4), 100 mM NaCl, 0.01% MNG, and 0.001% CHS. The resulting monomeric apo µOR was concentrated to approximately 150 µM, quickly frozen in liquid nitrogen, and maintained at -80°C for future applications. Expression & purification of heterotrimeric G-proteins

[0087] Heterotrimeric Giwas expressed in Trichoplusia ni (T. ni) with the BestBac method (Expression Systems) and purified as previously described.1,2Briefly, T. ni cells were infected with one virus encoding the wild-type human Gαi subunit and another encoding the wild-type human β1γ2 subunits with a histidine tag inserted at the N-terminus of the β subunit. Cells were harvested 48 hours post infection and lysed with hypotonic buffer. Heterotrimeric Gαiβ1γ2proteins were extracted in a buffer containing 1% sodium cholate and 0.05% DDM. The heterotrimer was purified using nickel-chelating sepharose chromatography while removing cholate. Human rhinovirus 3C protease (3C protease) was added to cleave off the histidine tag overnight at 4°C on-column. The flow through was collected and dephosphorylated with lambda protein phosphatase (NEB), calf intestinal phosphatase (NEB) and Antarctic phosphatase (NEB) in the presence of 1 mM manganese chloride (MnCl2). The heterotrimer was further purified by ion exchange chromatography on a MonoQ 10 / 100 GL column (GE Healthcare) in 20 mM HEPES pH 7.5, 1 mM MgCl2, 0.05% DDM, 100 µM TCEP, and 20 µM GDP and eluted with a linear NaCl gradient from 50 to 500 mM. The purified heterotrimer was collected and dialyzed into 20 mM HEPES pH 7.5, 100 mM NaCl, 0.02% DDM, 100 µM TCEP and 20 µM GDP overnight at 4°C, followed by concentration to <250 µM, addition of 20% glycerol and flash-freezing in liquid nitrogen and storage at -80°C until further use. Preparation of µ-opioid receptor membranes

[0088] Cell membranes containing the mouse MOR described above were generated by infecting Sf9 cells in an identical manner to that described above for protein purification. Sf9 cells expressing MOR were resuspended in cold lysis buffer composed of 10 mM HEPES pH 7.4, 10 mM MgCl2, and 20 mM potassium chloride (KCl) with the protease inhibitors leupeptin, benzamidine, and cOmpleteTMEDTA-free protease inhibitor cocktail tablets (Sigma-Aldrich). The lysed cells were spun at 45k rpm for 45 minutes to pellet membranes. The supernatant was removed and membrane pellets were resuspended in cold lysis buffer followed by douncing ~30 times on ice. The dounced membranes were spun again at 45k rpm for 45 minutes. The pellets were resuspended in cold lysisS23-339 CZB-293S-PC T-020961 33167 / 59763 buffer again with the addition of benzonase and dounced a further ~30 times, followed by a further spin at 45k rpm for 45 minutes. Membranes were resuspended in the same lysis buffer above in the presence of 1 M NaCl and dounced a further ~30 times and pelleted. Finally, membranes were washed with the original lysis buffer, dounced, and pelleted. Final washed membranes were resuspended to 2g original pellet mass per 1 mL in lysis buffer; the resulting MOR-containing membranes were flash frozen for later radioligand binding experiments. Membrane binding experiments

[0089] MOR-containing membranes prepared above were diluted 1:1000 in 20 mM HEPES pH 7.4, 100 mM NaCl, and 0.05% bovine serum albumin (BSA). For “competition” binding experiments, membranes were incubated with 2 nM3H-naloxone and serially diluted allosteric modulators for 1.5 hours at room temperature with shaking. Following incubation, membranes were bound to double thick 90 x 120 mm glass fibre Printed Filtermat B filters (Perkin Elmer) and washed with cold binding buffer (20 mM HEPES pH 7.4, 100 mM NaCl) using a MicroBeta Filtermat-96 cell harvester (Perkin Elmer).3H-naloxone bound membranes on Filtermats were measured with a MicroBeta counter (Perkin Elmer) after addition of MultiLex B / HS melt-on scintillator sheets (Perkin Elmer) and data values were plotted as total counts per minute normalized to the highest and lowest average points. GTP turnover assay

[0090] The GTP turnover assay was conducted using a customized GTPase-Glo™ assay protocol (Promega) as described previously2,3. Purified MOR was diluted to 1 µM in 20 mM HEPES pH 7.4, 100 mM NaCl, 0.01% L- MNG, 0.001% CHS, and 20 µM guanosine-5’-triphosphate (GTP) in the presence of various orthosteric (20 µM met-enkephalin, 20 µM naloxone, 20 µM MP, 20 µM H-Tyr-D-Ala-Gly-N(Me)Phe-Gly-OH [DAMGO], 20 µM BU72) and allosteric (serially diluted at 20 µM for 368) ligands and incubated for 1.5 hours at room temperature. Concurrently, Gi purified in DDM was exchanged by incubating with 1% L-MNG and 0.1% CHS for 1 hour on ice. The exchanged Gi was then diluted to 1 µM in 20 mM HEPES pH 7.4, 100 mM NaCl, 0.01% L-MNG, 0.001% CHS, 20 µM guanosine-5’-diphosphate (GDP), 200 µM TCEP, and 20 mM MgCl2. Equal volumes of receptor solutions and Gi solution were mixed and incubated at room temperature for 60 minutes (agonist-bound receptor experiments) or 90 minutes (apo receptor experiments) with gentle shaking. Controls include mixing equal volumes of both buffers (total initial GTP) and equal volumes of 1 µM Gi solution and receptor buffer (intrinsic G protein turnover). Equal volume of GTPase-Glo reagent supplemented with 10 µM adenosine 5’-diphosphate (ADP) in 20 mM HEPES pH 7.4, 100 mM NaCl, 0.01% MNG and 0.001% CHS was added and incubated with gentle shaking for 30 minutes, followed by addition of further equal volume of detection reagent. After brief (10 minute) incubation, luminescence was measured with a MicroBeta counter. Results Synthesis of ComparatorsS23-339 CZB-293S-PC T-020961 33167 / 59763

[0091] Compound (S)-368 (11) was synthesized according the scheme above.

[0092] Step 1. 4-iodo-2-methoxy-1-methylbenzene (2): To a stirred solution of Compound 1 (5 g, 36.49 mmol) in (50 mL) THF was added 15 mL HCl in (25 mL )H2O at 00C. NaNO2 (3.02 g, 43.79 mmol) solution in (10 mL) was added dropwise and reaction mixture was stirred at the same temperature for 20 minutes. A solution of KI in (50 mL) of water was added slowly to the reaction mixture at 00C and stirred at room temperature for another 18 hours. The reaction mixture was diluted with water and EtOAC (100 mL), the organic layer was separated and aqueous layer was extracted with EtOAc (100 mL×2). The combined organic layer was washed with saturated 10% NaOH (50 mL), and saturated Na2S2O3 and water. The organic layer was dried over anhydrous Na2SO4 andS23-339 CZB-293S-PC T-020961 33167 / 59763 evaporated off and the crude reaction mixture was purified by flash chromatography using 1-5% EtOAc:Hexane yielding the iodo compound 2 (4.5 g).1H NMR (500 MHz, CDCl3) δ 7.16 (d, J = 7.7 Hz, 1H), 7.07 (s, 1H), 6.82 (d, J = 7.7 Hz, 1H), 3.78 (s, 3H), 2.13 (s, 3H).13C NMR (126 MHz, CDCl3) δ 158.3, 132.0, 129.4, 126.5, 119.2, 90.4, 55.5, 15.9.

[0093] Step 2. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-methoxy-4-methylphenyl)propanoate (4): Zinc (0.193 g, 0.80 mmol) was dissolved in DMF (2 mL) to it I2 (0.023 g, 0.090 mmol) was added under N2. Compound 3 (0.2 g, 0.60 mmol) and iodine (0.023 g, 0.090 mmol) was added. The reaction was stirred at room temperature for 5 minutes. To this Pd(dba)3 (13.8 mg, 0.015 mmol), S-Phos (12 mg, 0.03 mmol) and 4-iodo-2-methoxy-1- methylbenzene (0.193 g, 0.80 mmol) were added sequentially, and reaction mixture was stirred at room temperature overnight. After, the reaction mixture was diluted with DCM (10 mL) and cold water (10 mL), the organic layer was separated, and the aqueous layer was extracted with DCM (10 mL× 2). The organic layers were combined washed with water, brine and dried over anhydrous Na2SO4. The volatile solvents were evaporated off to furnish crude residue which was purified by flash column chromatography (using 5-10%, EtOAc:hexane) to produce compound 4 (0.130 g).1H NMR (500 MHz, CDCl3) δ 7.04 (d, J = 7.3 Hz, 1H), 6.69 – 6.55 (m, 2H), 4.97 (s, 1H), 4.63–4.51 (m, 1H), 3.81 (s, 3H), 3.73 (s, 3H), 3.17–2.94 (m, 2H), 2.18 (s, 3H), 1.43 (s, 9H).13C NMR (126 MHz, CDCl3) δ 172.4, 156.2, 134.6, 130.6, 121.1, 110.9, 81.9, 55.2, 54.5, 52.4, 38.2, 28.3, 15.9.

[0094] Step 3. methyl (S)-2-amino-3-(3-methoxy-4-methylphenyl)propanoate (5): Compound 4 was dissolved in CH2Cl2, followed by TFA. The solution was worked up according to any method commonly used in the art to provide compound 5 which used in next step without purification.

[0095] Step 4. Ethyl 2-(naphthalen-2-ylthio)acetate (7): Naphthalene-2-thiol (6) (1 g, 6.20 mmol) was dissolved in CH3CN (20 mL) at room temperature to it K2CO3 (1.72 g, 12.4 mmol) and ethyl bromoacetate (55 mL, 5 mmol) was added and reaction was stirred at room temperature overnight. To this 0.5 N NaOH (20 mL) was added and diluted with EtOAc (20 mL). The organic layer was separated, and aqueous layer was extracted with EtOAc (20 mL × 2). The organic layer was washed with brine and dried over anhydrous Na2SO4. The volatile solvents were evaporated off to furnish crude residue which was purified by flash column chromatography (using 1-5%, EtOAc:hexane) to provide compound 7 (0.8 g).1H NMR (500 MHz, CDCl3) δ 7.98 – 7.64 (m, 4H), 7.49 (ddd, J = 13.2, 7.0, 4.1 Hz, 3H), 4.18 (q, J = 7.2 Hz, 2H), 3.75 (s, 2H), 1.22 (t, J = 7.2 Hz, 3H).13C NMR (126 MHz, CDCl3) δ 169.6, 133.7, 132.4, 132.1, 128.6, 128.2, 127.7, 127.6, 127.3, 126.6, 126.1, 61.6, 36.7, 14.1.

[0096] Step 5. Ethyl 2-((1-bromonaphthalen-2-yl)thio)acetate (8): Compound 7 (0.1, 0.4 mmol) was dissolved in CH3CN (5 mL) and cooled to 00C, NBS (0.086 g, 0.48 mmol) was added, and the reaction stirred at same temperature for 45 minutes. After completion of reaction, it was diluted with EtOAc (20 mL), and sat. NaHCO3,and water. The organic layer was separated, and aqueous layer was extracted with EtOAc (10 mL× 2). The combined organic layer was washed with brine and dried over anhydrous Na2SO4. The volatile solvents were evaporated off to furnish crude residue which was purified by flash column chromatography (using 1-5%,S23-339 CZB-293S-PC T-020961 33167 / 59763 EtOAc:hexane) to provide compound 8 (0.088 g).1H NMR (500 MHz, CDCl3) δ 8.24 (d, J = 8.5 Hz, 1H), 7.81 – 7.72 (m, 2H), 7.58 (t, J = 7.2 Hz, 1H), 7.53 – 7.44 (m, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.79 (s, 2H), 1.23 (t, J = 7.1 Hz, 3H).13C NMR (126 MHz, CDCl3) δ 169.1, 134.6, 132.62,132.6128.1, 127.9, 126.9, 126.3, 125.6, 123.3, 61.7, 35.8, 14.0.

[0097] Step 6. Ethyl 2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)thio)acetate (9): Compound 8 (0.02 g, 0.06 mol) was dissolved in 1,4-dioxane:water (2.5 mL, 4:1) and argon was bubbled in for 10 minutes. K2CO3 (0.025 g, 0.18 mmol) and ([1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)) (0.005 g, 0.001 mmol) was added and reaction mixture was stirred under argon at 800C for 4 hours. After, the reaction mixture was dried over anhydrous Na2SO4 and filtered through celite pad, the celite pad washed with EtOAc (20 mL). The volatile solvents were evaporated off and the residue obtained purified by flash column chromatography to provide compound 9 (0.018 g, 69% yield).1H NMR (500 MHz, CDCl3) δ 8.52 (d, J = 2.3 Hz, 1H), 8.21 (s, 1H), 7.87 (dd, J = 12.7, 8.8 Hz, 2H), 7.65 (d, J = 8.7 Hz, 1H), 7.59 – 7.17 (m, 9H), 5.15 (s, 2H), 4.14 (q, J = 7.2 Hz, 2H), 3.55 (s, 2H) 1.20 (t, J = 7.1 Hz, 3H).13C NMR (126 MHz, CDCl3) δ 169.2, 154.5, 143.5, 137.7, 136.0, 135.7, 134.6, 132.8, 132.2, 132.1, 129.1, 128.6, 128.2, 128.0(2C), 127.6, 127.0(2C), 126.5, 126.0, 125.6, 123.6, 70.3, 61.5, 36.2, 14.0.

[0098] Step 7. Methyl(S)-2-(2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4- methylphenyl)propanoate (10): Compound 9 (0.05 g, 0.11 mol) was dissolved in THF:H2O (3 mL, 2:1), to this LiOH (1N, 0.6 mL, 0.58 mmol) was added and reaction stirred overnight. After completion of reaction, it was acidified using 1N HCl and extracted with EtOAc (20 mL× 2), the organic layer was washed with brine and dried over anhydrous Na2SO4. The volatile solvents were evaporated off to give the crude acid. Compound 4 (0.049 g, 0.15 mmol) was dissolved in CH2Cl2 (2 mL) to it TFA (0.5 mL) was added, and reaction stirred for 3 hours. After completion of reaction, the solvent was evaporated off and residue 5 dissolved in CH2Cl2:DMF (3 mL, 2:1) and to it the acid obtained above was added using DMF (1 mL) to this Et3N (0.05 mL, 0.35 mmol), HATU (0.067 g, 0.17 mmol) was added sequentially, and reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with cold water (10 mL) and extracted with EtOAc (20 mL× 2). The combined organic layer was washed with brine and dried over anhydrous Na2SO4.The volatile solvents were evaporated off to furnish crude residue which was purified by flash column chromatography using (1-50%, EtOAc:hexane) to provide compound 10 (0.06 g).1H NMR (500 MHz, CDCl3) δ 8.58 – 8.44 (m, 1H), 8.12 (d, J = 49.7 Hz, 1H), 7.91 – 7.78 (m, 2H), 7.54 – 7.25 (m, 12H), 7.04 (s, 1H), 6.39 (d, J = 6.9 Hz, 1H), 5.15 (s, 1H), 5.06 – 4.94 (m, 1H), 4.85 – 4.67 (m, 1H), 3.72 – 3.60 (m, 5H), 3.57 (s, 3H), 3.09 – 2.96 (m, 2H), 1.99 (m, 3H).13C NMR (100 MHz, CDCl3) δ 171.5, 167.6, 157.7, 143.1, 135.8, 134.4, 133.9, 131.7, 130.5, 129.5, 128.7, 128.7, 128.4, 128.3, 128.2, 127.8, 127.7, 127.3, 126.0, 125.3, 125.2, 120.9, 120.6, 110.3, 70.4, 55.1, 53.4, 52.4, 37.5, 36.6, 15.7.

[0099] Step 8. (S)-2-(2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4- methylphenyl)-N-methylpropanamide (11): Compound 10 was dissolved in MeOH and to it MeNH2 (40% in H2O, 0.25 mL) was added and reaction stirred at room temperature overnight. After completion of reaction, volatile solvents were evaporated off and residue purified by flash chromatography using (80% EtOAc:hexane) to furnishS23-339 CZB-293S-PC T-020961 33167 / 59763 the product compound 11 (7 mg).1H NMR (500 MHz, CDCl3) δ 8.54 (s, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.92 – 7.75 (m, 2H), 7.53 – 7.26 (m, 10H), 7.12 (s, 1H), 6.79 (dd, J = 30.1, 7.3 Hz, 1H), 6.64 – 6.41 (m, 2H), 5.48 (d, J = 24.3 Hz, 1H), 5.18 (ddt, J = 24.7, 18.0, 9.3 Hz, 2H), 4.58 – 4.40 (m, 1H), 3.70 (s, 3H), 3.62 – 3.48 (m, 2H), 3.06 – 2.84 (m, 2H), 2.54 (s, 3H), 2.06 (s, 3H).13C NMR (126 MHz, CDCl3) δ 170.70 (d, J = 4.0 Hz), 167.8, 157.9, 135.9, 135.0, 134.5, 131.9, 130.6, 129.6, 128.7, 128.3, 128.1, 127.71,127.7,127.4, 126.1, 125.4, 124.1, 124.0, 120.7, 110.6, 70.5, 55.2, 38.0, 37.2, 37.0, 36.6, 15.8.HRMS calcd for C36H35N3O4S Na+; 628.2240: HRMS found 628.2245.

[0100] Compound (R)-368 (15) was synthesized according the scheme above, following a similar protocol to the (S) isomer.

[0101] Step 1. Methyl (R)-2-((tert-butoxycarbonyl)amino)-3-(3-methoxy-4-methylphenyl)propanoate (12): The synthetic procedure for compound 4 was followed with Methyl (S)-2-((tert-butoxycarbonyl)amino)-3- iodopropanoate (0.2 g, 0.60 mmol) to provide compound 12 (0.110 g, 56% yield).1H NMR (500 MHz, CDCl3) δ 7.04 (d, J = 7.3 Hz, 1H), 6.66 – 6.55 (m, 2H), 4.99 (s, 1H), 4.57 (s, 1H), 3.81 (s, 3H), 3.72 (s, 3H), 3.12 – 2.94 (m, 2H), 2.18 (s, 3H), 1.43 (s, 9H).

[0102] Step 2. methyl (R)-2-amino-3-(3-methoxy-4-methylphenyl)propanoate (13): Compound 12 was dissolved in CH2Cl2, followed by TFA. The solution was worked up according to any method commonly used in the art to provide compound 13 which used in next step without purification.S23-339 CZB-293S-PC T-020961 33167 / 59763

[0103] Step 3. Methyl(R)-2-(2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4- methylphenyl)propanoate (14): The synthetic procedure for compound 10 was followed, using compound 9 (0.05 g, 0.11 mmol) to provide compound 14 (0.055 g, 78% yield).1H NMR (500 MHz, CDCl3) δ 8.43 (dd, J = 5.6, 2.8 Hz, 1H), 7.84 – 7.68 (m, 2H), 7.53 – 7.15 (m, 10H), 7.09 – 6.88 (m, 2H), 6.58 – 6.34 (m, 1H), 6.34 – 6.22 (m, 2H), 5.10–01 (m, 1H), 4.93 (q, J = 11.6 Hz, 1H), 4.75–4.61 (m, 1H), 3.57 (s, 3H), 3.54–3.51 (m, 2H), 3.49 (s, 3H), 307–2.83 (m, 2H), 1.90 (s, 3H).13C NMR (126 MHz, CDCl3) δ 171.5167.6, 167.5, 157.6, 143.3, 138.1, 138.0, 136.0, 134.3, 134.0, 133.0, 132.8, 131.9, 131.8, 130.5, 129.5, 128.7, 128.0,127.8, 127.7, 127.3, 126.0 , 125.5, 123.7, 123.5, 123.0, 120.9, 120.7, 110.4, 70.4, 55.1, 53.5, 37.5, 36.9, 36.7, 15.7.

[0104] Step 4. (R)-2-(2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4- methylphenyl)-N-methylpropanamide (15): The synthetic procedure for 11 was performed with compound 14 to provide compound 15.1H NMR (500 MHz, CDCl3) δ 8.53 (s, 1H), 8.16 (s, 1H), 7.94 – 7.72 (m, 2H), 7.50 – 7.28 (m, 10H), 7.13 (s, 1H), 6.86 – 6.72 (m, 1H), 6.63 – 6.39 (m, 2H), 5.46 (d, J = 21.9 Hz, 1H), 5.29 – 5.07 (m, 2H), 4.59 – 4.43 (m, 1H), 3.70 (s, 3H), 3.64 (s, 2H), 3.06 – 2.83 (m, 2H), 2.54 (s, 3H), 2.06 (s, 3H).13C NMR (126 MHz, CDCl3) δ 170.7, 167.8, 157.8, 154.8, 143.5, 143.3, 138.15, 138.1, 136.0, 134.9, 134.6, 134.3, 132.9, 131.9, 131.7, 130.6, 129.5, 128.7, 128.3, 128.1, 127.7, 127.3, 126.1, 125.4, 124.0, 123.9, 123.5, 120.7, 110.6, 70.5, 55.2, 38.0, 37.19, 37.0, 26.1, 15.8. HRMS calcd for C36H35N3O4S Na+; 628.2240: HRMS found 628.2243

[0105] Compound (±)-368 (20) was synthesized according the scheme above.S23-339 CZB-293S-PC T-020961 33167 / 59763

[0106] Step 1. Methyl 2-((tert-butoxycarbonyl)amino)-3-(3-methoxy-4-methylphenyl)propanoate (16): The synthetic procedure for compound 4 was performed, but using methyl 2-((tert-butoxycarbonyl)amino)-3- iodopropanoate (0.2 g, 0.60 mmol) provided compound 16 (0.12 g, 61% yield).1H NMR (500 MHz, CDCl3) δ 7.13 – 6.95 (m, 1H), 6.67 – 6.48 (m, 2H), 5.14 – 4.92 (m, 1H), 4.57 (d, J = 7.8 Hz, 1H), 3.80 (s, 3H), 3.72 (s, 3H), 3.05 (dd, J = 12.9, 6.0 Hz, 2H), 2.18 (s, 3H), 1.42 (s, 9H).13C NMR (126 MHz, CDCl3) δ 172.4, 157.7, 155.1, 134.6, 130.6, 125.3, 121.0, 110.9, 79.8, 55.2, 54.5, 52.1, 38.2, 28.3(3C), 15.8.

[0107] Step 2. methyl-2-amino-3-(3-methoxy-4-methylphenyl)propanoate (17): Compound 16 was dissolved in CH2Cl2, followed by TFA. The solution was worked up according to any method commonly used in the art to provide compound 17 which used in next step without purification.

[0108] Step 3. Methyl 2-(2-((1-bromonaphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4-methylphenyl) propanoate (18): The synthetic procedure for compound 10 was used with ethyl 2-((1-bromonaphthalen-2- yl)thio)acetate (8) (0.22 g, 0.68 mmol) provided compound 18 (0.26 g, 76% yield).1H NMR (500 MHz, CDCl3) δ 8.23 (dd, J = 8.6, 1.0 Hz, 1H), 7.82 – 7.74 (m, 1H), 7.71 – 7.65 (m, 1H), 7.60 (ddd, J = 8.4, 6.8, 1.3 Hz, 1H), 7.50 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.18 (dd, J = 15.3, 8.4 Hz, 2H), 6.77 – 6.69 (m, 1H), 6.45 (d, J = 1.7 Hz, 1H), 6.35 (dd, J = 7.5, 1.7 Hz, 1H), 4.81 (ddd, J = 7.8, 6.9, 5.4 Hz, 1H), 3.74 (s, 2H), 3.66 (s, 3H), 3.64 (s, 3H), 3.00 (qd, J = 14.0, 6.2 Hz, 2H), 2.00 (s, 3H).13C NMR (126 MHz, CDCl3) δ 171.4, 167.3, 157.7, 134.0, 133.8, 132.5, 132.4, 130.5, 128.4, 128.2, 128.1, 126.6, 126.3, 125.3, 123.7, 121.9, 120.6, 110.3, 55.1, 53.4, 52.2, 37.5, 36.8, 15.7.

[0109] Step 4.2-(2-((1-bromonaphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4-methylphenyl)-N- methylpropanamide (19): Methyl 2-(2-((1-bromonaphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4- methylphenyl)propanoate (0.250 g, 0.49 mmol) was dissolved in MeOH:CH2Cl2 (10:1, 11 mL) to it NH2Me (40% in H2O, 5 mL) was added and reaction was stirred at room temperature for overnight. After, the volatile solvents were evaporated off to complete dryness and residue used in next step without purification.

[0110] Step 5.2-(2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4- methylphenyl)-N-methylpropanamide (20): The synthetic procedure for compound 11 was followed, using compound 19 to provide compound 20.1H NMR (399 MHz, CDCl3) δ 8.51 (t, J = 2.7 Hz, 1H), 8.15 (s, 1H), 7.94 – 7.72 (m, 2H), 7.52 – 7.17 (m, 11H), 6.81 – 6.70 (m, 1H), 6.54 (m, 1H), 6.46 – 6.40 (m, 1H), 5.89 – 5.66 (m, 1H), 5.25 – 5.06 (m, 2H), 4.54 – 4.47 (m, 1H), 3.66 (s, 3H), 3.58 – 3.47 (m, 2H), 3.01 – 2.86 (m, 2H), 2.54 (s, 3H), 2.04 (s, 3H).13C NMR (100 MHz, CDCl3) δ 170.8, 168.0, 157.8, 154.7, 143.4, 143.2, 138.1,138.0, 136.0, 135.0 134.5, 134.2, 132.9, 131.8, 131.7, 130.5, 129.4, 128.7, 128.63, 128.1,127.7,127.65, 127.3, 126.0, 125.3, 124.01, 123.3, 120.7, 110.6, 70.4, 55.1, 54.9, 38.0, 37.1, 26.0, 15.7. HRMS calcd for C36H35N3O4S Na+; 628.2240: HRMS found 628.224 Synthesis of Compound A1

[0111] Step 1. Ethyl 2-((1-(pyridin-3-yl)naphthalen-2-yl)thio)acetate (21): Following the similar protocol for compound (9), Ethyl 2-((1-bromonaphthalen-2-yl)thio)acetate 8 (0.09 g, 0.27 mol), 3-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyridine (0.085 g, 0.417 mmol) K2CO3 (0.112 g, 0.81 mmol) and ([1,1′-S23-339 CZB-293S-PC T-020961 33167 / 59763 Bis(diphenylphosphino)ferrocene]dichloropalladium(II)) (0.02 g, 0.0271 mmol) yielded product compound 20 (0.06 g).1H NMR (399 MHz, CDCl3) δ 8.62 (d, J = 4.5 Hz, 1H), 8.48 (s, 1H), 7.85 – 7.72 (m, 2H), 7.57 (dd, J = 20.7, 8.2 Hz, 2H), 7.42 – 7.33 (m, 2H), 7.28 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 4.01 (q, J = 7.1 Hz, 2H), 3.46 (s, 2H), 1.08 (t, J = 7.1 Hz, 3H).

[0112] Step 2. Compound A1 (22) (S)-3-(3-methoxy-4-methylphenyl)-N-methyl-2-(2-((1-(pyridin-3- yl)naphthalen-2-yl)thio)acetamido)propenamide (22): Following the similar protocol for compound (10 and 11) Compound 21 (0.04 g, 0.12 mmol) yielded product compound 22 (15 mg).1H NMR (500 MHz, CDCl3) δ 8.82 – 8.72 (m, 1H), 8.58 (d, J = 20.2 Hz, 1H), 7.86 (dd, J = 13.3, 8.5 Hz, 2H), 7.70 (dd, J = 13.8, 7.8 Hz, 1H), 7.60 – 7.47 (m, 2H), 7.43 (t, J = 7.1 Hz, 1H), 7.35 (dd, J = 8.8, 3.1 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.12 – 7.02 (m, 1H), 6.91 – 6.79 (m, 1H), 6.58 (s, 1H), 6.55 – 6.49 (m, 1H), 5.49 (d, J = 33.6 Hz, 1H), 4.51 (p, J = 7.3 Hz, 1H), 3.73 (s, 3H), 3.64 – 3.49 (m, 2H), 3.02 (dt, J = 12.9, 6.2 Hz, 1H), 2.94 – 2.83 (m, 1H), 2.54 (dd, J = 28.4, 4.8 Hz, 3H), 2.08 (s, 3H). Synthesis of Compound A2

[0113] Compound A2 (23) (S)-3-(3-methoxy-4-methylphenyl)-N-methyl-2-(2-(naphthalen-2- ylthio)acetamido)propenamide: Following the similar protocol for compound (10 and 11) Compound 7 (0.045 g, 0.10 mmol) yielded compound 23 (30 mg)1H NMR (500 MHz, CDCl3) δ 8.82 – 8.72 (m, 1H), 8.58 (d, J = 20.2 Hz, 1H), 7.86 (dd, J = 13.3, 8.5 Hz, 2H), 7.70 (dd, J = 13.8, 7.8 Hz, 1H), 7.60 – 7.47 (m, 2H), 7.43 (t, J = 7.1 Hz, 1H), 7.35 (dd, J = 8.8, 3.1 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.12 – 7.02 (m, 1H), 6.91 – 6.79 (m, 1H), 6.58 (s, 1H), 6.55 – 6.49 (m, 1H), 5.49 (d, J = 33.6 Hz, 1H), 4.51 (p, J = 7.3 Hz, 1H), 3.73 (s, 3H), 3.64 – 3.49 (m, 2H), 3.02 (dt, J = 12.9, 6.2 Hz, 1H), 2.94 – 2.83 (m, 1H), 2.54 (dd, J = 28.4, 4.8 Hz, 3H), 2.08 (s, 3H). Synthesis of Compound A3

[0114] Step 1.1-Bromonaphthalen-2-ol (25): The synthetic procedure for 4 was used in this case. Naphthalen-2-ol (24) (0.2 g, 1.38 mmol) yielded product compound 25 (0.21 g).1H NMR (500 MHz, CDCl3) δ 8.07 (d, J = 8.5 Hz, 1H), 7.77 (dd, J = 24.1, 8.3 Hz, 2H), 7.65 – 7.58 (m, 1H), 7.42 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.30 (d, J = 8.8 Hz, 1H), 6.00 (s, 1H).

[0115] Step 2. Ethyl 2-((1-bromonaphthalen-2-yl)oxy)acetate (26): The synthetic procedure for 7 was used in this case. 1-bromonaphthalen-2-ol (25) (0.21 g, 0.95 mmol) yielded product compound 26 (0.18 g).1H NMR (500 MHz, CDCl3) δ 8.26 (d, J = 8.4 Hz, 1H), 7.83 – 7.67 (m, 2H), 7.57 (t, J = 6.9 Hz, 1H), 7.42 (d, J = 7.0 Hz, 1H), 7.16 (d, J = 8.8 Hz, 1H), 4.82 (s, 2H), 4.28 (d, J = 7.0 Hz, 2H), 1.30 (t, J = 6.6 Hz, 3H).

[0116] Step 3. Ethyl 2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)oxy)acetate (27): The synthetic procedure for 9 was used in this case. Ethyl 2-((1-bromonaphthalen-2-yl)oxy)acetate (26) .0.127 g, 0.411 mmol) yielded product compound 27 (0.16 g).1H NMR (500 MHz, CDCl3) δ 8.49 (s, 1H), 8.35 (s, 1H), 7.87 (dd, J = 23.3, 6.5 Hz, 2H), 7.53 (s, 1H), 7.46 (d, J = 6.9 Hz, 3H), 7.37 (dd, J = 18.4, 6.0 Hz, 5H), 7.25 (d, J = 8.7 Hz, 1H), 5.16 (q, J = 11.2 Hz, 2H), 4.74 – 4.54 (m, 2H), 4.22 (d, J = 6.6 Hz, 2H), 1.26 (s, 3H).S23-339 CZB-293S-PC T-020961 33167 / 59763

[0117] Step 4. Compound A3 (28) (S)-2-(2-((1-(5-(Benzyloxy)pyridin-3-yl)naphthalen-2-yl)oxy)acetamido)-3-(3- methoxy-4-methylphenyl)-N-methylpropanamide: Following the similar protocol for compounds 10 and 11 Ethyl 2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)oxy)acetate (0.06 g, 0.14 mmol) yielded product compound 28, as atropisomers (10 mg).1H NMR (399 MHz, CDCl3) δ 8.51 (dd, J = 13.2, 2.8 Hz, 1H), 8.26 (dd, J = 13.2, 1.7 Hz, 1H), 7.95 – 7.83 (m, 2H), 7.55 – 7.28 (m, 9H), 7.17 (dd, J = 9.1, 1.0 Hz, 1H), 6.88 (dd, J = 27.0, 7.5 Hz, 1H), 6.72 (t, J = 8.7 Hz, 1H), 6.58 (dd, J = 13.5, 1.6 Hz, 1H), 6.48 (ddd, J = 34.8, 7.4, 1.6 Hz, 1H), 5.62 (dd, J = 25.1, 4.8 Hz, 1H), 5.29 – 5.09 (m, 2H), 4.58 – 4.40 (m, 3H), 3.69 (d, J = 14.9 Hz, 3H), 2.92 – 2.74 (m, 2H), 2.67 (dd, J = 12.9, 4.8 Hz, 3H), 2.11 (d, J = 4.1 Hz, 3H). Synthesis of Compound A4

[0118] Step 1. Methyl (2-((1-bromonaphthalen-2-yl)thio)acetyl)-L-phenylalaninate (29): The synthetic procedure for compound 10 was used in this case. Ethyl 2-((1-bromonaphthalen-2-yl)thio)acetate 8 (0.22 g, 0.68 mmol) yielded the coupling product compound 29 (0.053 g).1H NMR (399 MHz, CDCl3) δ 8.24 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.66 – 7.58 (m, 1H), 7.50 (ddd, J = 8.1, 6.9, 1.3 Hz, 1H), 7.18 (dd, J = 18.0, 8.3 Hz, 2H), 7.02 – 6.92 (m, 3H), 6.83 (dd, J = 6.8, 2.9 Hz, 2H), 4.82 (q, J = 6.6 Hz, 1H), 3.81 – 3.69 (m, 2H), 3.63 (s, 3H), 3.00 (dd, J = 6.0, 2.9 Hz, 2H).

[0119] Step 2. Compound A4 (30) (S)-2-(2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)-N- methyl-3-phenylpropanamide (30): The synthetic procedure for compound 20 was used in this case. Methyl (2- ((1-bromonaphthalen-2-yl)thio)acetyl)-L-phenylalaninate (29) (0.06 g, 0.13 mmol), yielded the coupling product compound 30 (0.053 g).1H NMR (399 MHz, CDCl3) δ 8.51 (d, J = 2.8 Hz, 1H), 8.13 (dd, J = 4.9, 1.6 Hz, 1H), 7.88 – 7.76 (m, 2H), 7.55 – 7.19 (m, 12H), 7.10 – 6.94 (m, 5H), 5.79 (t, J = 5.1 Hz, 1H), 5.22 – 5.06 (m, 2H), 4.62 – 4.48 (m, 1H), 3.61 – 3.46 (m, 2H), 3.02 – 2.82 (m, 2H), 2.53 (t, J = 5.1 Hz, 3H). Synthesis of Compound A6

[0120] Step 1. Tert-butyl naphthalen-2-ylcarbamate (31): Naphthalen-2-amine (30) (0.5 g, 3.49 mmol) was dissolved in THF (15 mL) to this (Boc)2O (0.762 g, 3.49 mmol) was added and reaction was heated at 55 °C for 16 h. Reaction mixture diluted with H2O (20 mL) and extracted with EtOAc (20 mL× 2), solvents evaporated off and obtained residue purified by flash column using 2-5% EtOAc:hexane to provide compound 31 (0.45 g).1H NMR (500 MHz, CDCl3) δ 8.10 (s, 1H), 7.83 – 7.75 (m, 3H), 7.50 – 7.40 (m, 3H), 7.14 (s, 1H), 1.66 (s, 9H).

[0121] Step 2. Tert-butyl (1-bromonaphthalen-2-yl) carbamate (32): The synthetic procedure for 8 was used in this case. Tert-butyl naphthalen-2-ylcarbamate (31) (0.4 g, 1.64 mmol) yielded product compound 32 (0.48 g).1H NMR (500 MHz, CDCl3) δ 8.39 (d, J = 9.0 Hz, 1H), 8.15 (d, J = 8.5 Hz, 1H), 7.82 – 7.76 (m, 2H), 7.58 – 7.54 (m, 1H), 7.46 – 7.41 (m, 1H), 7.35 (s, 1H), 1.60 (s, 9H).

[0122] Step 3. Ethyl N-(1-bromonaphthalen-2-yl)-N-(tert-butoxycarbonyl)glycinate (33): The synthetic procedure for compound 7 was used in this case. Tert-butyl (1-bromonaphthalen-2-yl) carbamate (32) (0.25 g, 0.77 mmol) yielded product compound 33 (0.208 g).1H NMR (399 MHz, CDcl3) δ 8.31 (d, J = 8.2 Hz, 1H), 7.86 –S23-339 CZB-293S-PC T-020961 33167 / 59763 7.79 (m, 2H), 7.70 (dd, J = 17.9, 8.7 Hz, 1H), 7.57 (ddddd, J = 22.2, 9.6, 8.1, 6.9, 1.3 Hz, 3H), 4.88 (d, J = 17.8 Hz, 1H), 4.20 (dd, J = 7.2, 3.2 Hz, 2H), 3.84 – 3.75 (m, 1H), 1.35 (s, 9H), 1.26 (d, J = 7.1 Hz, 3H).

[0123] Step 4. Methyl (S)-2-(2-((1-bromonaphthalen-2-yl)(tert-butoxycarbonyl)amino)acetamido)-3-(3- methoxy-4-methylphenyl)propanoate (34): The synthetic procedure for compound 10 was used in this case. Ethyl N-(1-bromonaphthalen-2-yl)-N-(tert-butoxycarbonyl)glycinate (33) (0.2 g, 0.49 mmol) yielded product compound 33 (0.198 g, 69%).

[0124] Step 5. Compound A6 (35) (S)-2-(2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)amino)acetamido)-3- (3-methoxy-4-methylphenyl)-N-methylpropanamide: The synthetic procedure for 20 was used in this case. Compound 34 (0.190 g, 0.32 mmol) was subjected to TFA to provide product compound 35 (0.125 g).1H NMR (500 MHz, CDCl3) δ 8.44 (s, 1H), 8.15 (d, J = 7.5 Hz, 1H), 7.84 – 7.69 (m, 2H), 7.49 – 7.25 (m, 10H), 7.14 (t, J = 7.3 Hz, 1H), 6.91 – 6.68 (m, 1H), 6.65 – 6.32 (m, 4H), 5.17 – 4.99 (m, 2H), 4.76 – 4.61 (m, 1H), 3.89 – 3.73 (m, 2H), 3.56 (d, J = 19.8 Hz, 3H), 3.05 (ddd, J = 11.7, 6.9, 4.5 Hz, 1H), 3.00 – 2.85 (m, 1H), 2.64 (d, J = 4.7 Hz, 3H), 2.04 (s, 3H). Synthesis of Compound A5

[0125] Step 1. Methyl (2-((1-bromonaphthalen-2-yl)thio)acetyl)-L-alaninate (36): The synthetic procedure for compound 10 was used in this case. Ethyl 2-((1-bromonaphthalen-2-yl)thio)acetate 8 (0.07 g, 0.21 mmol) yielded the coupling product compound 36 (0.055 g, 68%).1H NMR (500 MHz, CDCl3) δ 8.24 (d, J = 9.5 Hz, 1H), 7.81 – 7.76 (m, 2H), 7.66 – 7.57 (m, 1H), 7.52 – 7.46 (m, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.30 – 7.22 (m, 1H), 4.55 (t, J = 7.3 Hz, 1H), 3.79 (s, 2H), 3.62 (s, 3H), 1.31 (d, J = 7.2 Hz, 3H).

[0126] Step 2. Compound A5 (37) (S)-2-(2-((1-(5-(benzyloxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)-N- methylpropanamide (37): The synthetic procedure for compound 20 was used in this case. Methyl (2-((1- bromonaphthalen-2-yl)thio)acetyl)-L-alaninate (55) (0.055 g, 0.14 mmol) yielded the product compound 37 (0.055 g).1H NMR (399 MHz, CDCl3) δ 8.52 (d, J = 2.8 Hz, 1H), 8.18 (s, 1H), 7.85 (td, J = 10.2, 9.5, 3.0 Hz, 2H), 7.53 – 7.26 (m, 10H), 7.09 (dd, J = 17.2, 7.7 Hz, 1H), 6.04 (s, 1H), 5.19 (qd, J = 11.6, 5.2 Hz, 2H), 4.36 (dq, J = 14.1, 7.0 Hz, 1H), 3.69 – 3.49 (m, 2H), 2.56 (dd, J = 15.0, 4.8 Hz, 3H), 1.30 – 1.19 (m, 3H). Synthesis of Compound A7

[0127] Step 1.1-Bromo-6-methoxynaphthalen-2-ol (38): The synthetic procedure for compound 8 was used in this case.6-methoxynaphthalen-2-ol (37) (0.150 g, 0.86 mmol) yielded product compound 38 (0.125 g).1H NMR (500 MHz, CDCl3) δ 7.94 (d, J = 9.2 Hz, 1H), 7.62 (d, J = 8.9 Hz, 1H), 7.26 – 7.21 (m, 2H), 7.10 (d, J = 2.6 Hz, 1H), 5.79 (s, 1H), 3.91 (s, 4H).

[0128] Step 2. Ethyl 2-((1-bromo-6-methoxynaphthalen-2-yl)oxy)acetate (39): The synthetic procedure for compound 7 was used in this case.1-bromo-6-methoxynaphthalen-2-ol (38) (0.125 g, 0.49 mmol) yielded product compound 39 (0.100 g).S23-339 CZB-293S-PC T-020961 33167 / 59763

[0129] Step 3. Methyl (S)-2-(2-((1-bromo-6-methoxynaphthalen-2-yl)oxy)acetamido)-3-(3-methoxy-4- methylphenyl)propanoate (40): The synthetic procedure for compound 10 was used in this case. Ethyl 2-((1- bromo-6-methoxynaphthalen-2-yl)oxy)acetate (39) (0.125 g, 0.49 mmol) yielded product compound 40 (0.090 g).1H NMR (500 MHz, CDCl3) δ 8.10 (d, J = 9.3 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.29 – 7.23 (m, 1H), 7.11 – 7.05 (m, 2H), 6.97 (dd, J = 7.4, 0.9 Hz, 1H), 6.67 – 6.60 (m, 2H), 5.00 (ddd, J = 8.1, 6.5, 5.6 Hz, 1H), 4.62 (d, J = 6.1 Hz, 2H), 3.92 (s, 4H), 3.76 (s, 3H), 3.73 (s, 3H), 3.22 – 3.05 (m, 2H), 2.17 – 2.14 (m, 3H).

[0130] Step 4. Compound A7 (41) (S)-2-(2-((1-(5-(Benzyloxy)pyridin-3-yl)-6-methoxynaphthalen-2- yl)oxy)acetamido)-3-(3-methoxy-4-methylphenyl)-N-methylpropanamide: The synthetic procedure for compound 20 was used in this case. Compound 40 (0.085 g, 0.16 mmol) yielded product compound 41 (19 mg, 19%).1H NMR (500 MHz, CDCl3) δ 8.50 (dd, J = 16.8, 2.8 Hz, 1H), 8.25 (dd, J = 15.4, 1.7 Hz, 1H), 7.82 – 7.74 (m, 1H), 7.52 – 7.47 (m, 1H), 7.46 – 7.42 (m, 1H), 7.42 – 7.35 (m, 5H), 7.29 (dd, J = 2.8, 1.7 Hz, 1H), 7.17 (dd, J = 2.7, 1.3 Hz, 1H), 7.14 (d, J = 9.0 Hz, 1H), 7.09 (ddd, J = 9.3, 2.6, 0.7 Hz, 1H), 6.90 (ddd, J = 27.8, 7.5, 0.9 Hz, 1H), 6.74 (dd, J = 11.7, 8.0 Hz, 1H), 6.59 (dd, J = 15.1, 1.6 Hz, 1H), 6.50 (ddd, J = 37.2, 7.5, 1.6 Hz, 1H), 5.65 (dd, J = 31.1, 5.0 Hz, 1H), 5.34 – 5.05 (m, 2H), 4.58 – 4.47 (m, 1H), 4.45 – 4.37 (m, 2H), 3.93 (s, 3H), 3.71 (d, J = 16.7 Hz, 4H), 3.02 – 2.78 (m, 2H), 2.69 (dd, J = 15.3, 4.9 Hz, 3H), 2.13 (d, J = 4.5 Hz, 3H). Synthesis of Compound A8

[0131] Step 1. Methyl (S)-2-(2-((2-bromophenyl)thio)acetamido)-3-(3-methoxy-4-methylphenyl)propanoate (42): The synthetic procedure for compounds 7 and 10 were used in this case.2-bromobenzenethiol (1.0 g, 5.34 mmol) yielded product compound 42 (0.310 g).1H NMR (500 MHz, CDCl3) δ 7.56 – 7.52 (m, 1H), 7.21 (d, J = 1.4 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.05 (td, J = 7.6, 1.2 Hz, 2H), 6.92 (dd, J = 7.4, 0.9 Hz, 1H), 6.49 (d, J = 1.6 Hz, 1H), 6.41 (dd, J = 7.5, 1.6 Hz, 1H), 4.80 (ddd, J = 7.9, 6.8, 5.5 Hz, 1H), 3.74 (s, 3H), 3.69 (s, 3H), 3.64 (d, J = 3.2 Hz, 2H), 3.06 – 2.98 (m, 2H), 2.14 (s, 3H).

[0132] Step 2. Compound A8 (43) (S)-2-(2-((2-(5-(benzyloxy)pyridin-3-yl)phenyl)thio)acetamido)-3-(3- methoxy-4-methylphenyl)-N-methylpropanamide: The synthetic procedure for compound 20 was used in this case. Compound 42 (0.2 g, 0.44 mmol) yielded product compound 43 (0.189 g).1H NMR (399 MHz, CDCl3) δ 8.33 (d, J = 66.5 Hz, 2H), 7.47 – 7.12 (m, 12H), 6.92 (d, J = 7.4 Hz, 1H), 6.59 (s, 1H), 6.53 (d, J = 7.0 Hz, 1H), 5.72 (d, J = 5.2 Hz, 1H), 5.16 (s, 2H), 4.50 (q, J = 7.2 Hz, 1H), 3.72 (s, 3H), 3.47 – 3.34 (m, 2H), 2.97 (dd, J = 13.8, 6.6 Hz, 1H), 2.88 (dd, J = 13.7, 7.4 Hz, 1H), 2.64 (d, J = 4.6 Hz, 3H), 2.12 (s, 3H). Synthesis of Compound A9

[0133] Step 1. Methyl (S)-2-(2-((1-(5-hydroxypyridin-3-yl)naphthalen-2-yl)thio)acetamido)-3-(3-methoxy-4- methylphenyl)propanoate (44): The synthetic procedure for compound 20 was used in this case. Compound 19 (0.350 g, 0.69 mmol) subjected to Suzuki coupling using 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3- ol (0.231 g, 1.04 mmol), KOAc (0.202 g, 2.07 mmol), and ([1,1′-S23-339 CZB-293S-PC T-020961 33167 / 59763 Bis(diphenylphosphino)ferrocene]dichloropalladium(II)) (0.028 g, 0.034 mmol) to provide product compound 44 (0.180 g).

[0134] Step 2. Methyl (S)-3-(3-methoxy-4-methylphenyl)-2-(2-((1-(5-((3-nitrobenzyl)oxy)pyridin-3- yl)naphthalen-2-yl)thio)acetamido)propanoate (45): Compound 44 (0.05 , 0.096 mmol) was alkylated using 3- nitrobenzyl bromide (0.031 mg, 0.145 mmol), Cs2CO3(0.062 g, 0.19 mmol), TBAI (4 mg, 0.0096 mmol) in THF gave product compound 45 (38 mg).1H NMR (500 MHz, CDcl3) δ 8.54 (dd, J = 6.8, 2.8 Hz, 1H), 8.33 (dt, J = 7.6, 2.0 Hz, 1H), 8.24 – 8.19 (m, 1H), 7.93 – 7.76 (m, 3H), 7.60 (q, J = 8.0 Hz, 1H), 7.54 – 7.38 (m, 3H), 7.36 – 7.25 (m, 3H), 7.19 – 7.02 (m, 1H), 6.43 (dd, J = 32.8, 1.6 Hz, 1H), 6.36 – 6.26 (m, 1H), 5.23 (s, 1H), 5.15 – 5.01 (m, 1H), 4.89 – 4.74 (m, 1H), 3.69 – 3.61 (m, 6H), 3.58 (s, 2H), 3.12 – 2.96 (m, 2H), 1.98 (d, J = 8.5 Hz, 3H).

[0135] Step 3. Compound A9 (46) (S)-3-(3-methoxy-4-methylphenyl)-N-methyl-2-(2-((1-(5-((3- nitrobenzyl)oxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)propenamide (46): Compound 45 (30 mg, 0.046 mol) was subjected to reaction with methyl amine ( 0.5 mL) in MeOH:DCM (2 mL:0.5 mL) yielded product compound 46 (12 mg)1H NMR (500 MHz, CDCl3) δ 8.55 (dd, J = 5.8, 2.8 Hz, 1H), 8.36 (t, J = 1.9 Hz, 1H), 8.25 – 8.17 (m, 2H), 7.90 – 7.79 (m, 3H), 7.61 (td, J = 7.9, 3.8 Hz, 1H), 7.52 – 7.17 (m, 6H), 6.87 – 6.33 (m, 2H), 5.57 (dd, J = 19.7, 4.9 Hz, 1H), 5.37 – 5.27 (m, 2H), 4.56 – 4.41 (m, 1H), 3.70 (d, J = 20.0 Hz, 3H), 3.66 – 3.61 (m, 1H), 3.54 (dd, J = 16.8, 1.8 Hz, 1H), 3.50 (d, J = 0.5 Hz, 1H), 3.02 – 2.73 (m, 2H), 2.56 (dd, J = 11.7, 4.9 Hz, 3H), 2.06 (d, J = 2.3 Hz, 3H). Synthesis of Compound A10

[0136] Compound A10 (47) (S)-2-(2-((1-(5-((3-aminobenzyl)oxy)pyridin-3-yl)naphthalen-2-yl)thio)acetamido)- 3-(3-methoxy-4-methylphenyl)-N-methylpropanamide was synthesized as follows. Compound 46 (12 mg, 0.018 mmol) was dissolved in MeOH to it SnCl2·2H2O (23 mg, 0.092 mmol) was added and reaction was stirred at 52 °C for 6 h. After completion of reaction crude reaction mixture was subjected to purification using 5-10% MeOH: DCM to provide compound 47 (3.5 mg).1H NMR (500 MHz, CD3OD) δ 7.98 – 7.85 (m, 2H), 7.67 (d, J = 17.6 Hz, 1H), 7.59 – 7.41 (m, 8H), 7.35 – 7.29 (m, 1H), 7.25 (t, J = 8.2 Hz, 1H), 6.72 – 6.68 (m, 1H), 6.67 – 6.62 (m, 1H), 5.42 – 5.25 (m, 2H), 4.54 – 4.49 (m, 1H), 3.70 (d, J = 8.0 Hz, 6H), 3.63 – 3.56 (m, 1H), 3.01 (ddd, J = 19.8, 13.9, 5.7 Hz, 1H), 2.83 – 2.75 (m, 1H), 2.61 (d, J = 13.3 Hz, 3H), 2.01 (d, J = 3.4 Hz, 3H). MOR NAM activity of Compounds A1-A10

[0137] The chemical space around the 368 scaffold was investigated to improve the both the affinity and efficacy for naloxone binding and the pharmacokinetic behavior, specifically its half-life. To that end, various compounds were prepared and evaluated to determine correlations between compound structures and binding and activity. The first series of modifications started with R1and R1A. Specifically, the effects of “terminal” benzene and adjoining ether either in the presence or absence of the pyridine group were investigated with compounds A1 and A2, respectively. Removal of either or both of these groups decrease the affinity of the compounds to3H-naloxone (Fig.1), as seen in the comparator S-368 compound. Further, even at high concentrations (20 µM), neither A1 nor A2 can reverse fentanyl activation of the µOR (Fig.2, Table 1).S23-339 CZB-293S-PC T-020961 33167 / 59763

[0138] Next, the effect of various heteroatoms in place of the sulfur in the thioester in the center of S-368 was investigated. Replacement of the sulfur with -NH was observed to completely abrogate the ability to enhance3H- naloxone affinity (Fig.3) for compound A6. While replacement of the sulfur with an oxygen atom results in a dampened ability to enhance3H-naloxone affinity, compound A3 retains the ability to do so at higher concentrations (Fig.3). Further, while compound A6 was observed to have little ability to inhibit fentanyl activity in GTPase assays even at the highest concentrations (Fig.4, Table 1), compound A3 acts as an efficacious inhibitor of fentanyl-induced µOR activity (Fig.4, Table 1).

[0139] Modifications on the other end of the S-368 scaffold were also evaluated for both activity against signaling and for binding to3H-naloxone by modification of the R2and R2Agroups. Removal of both the methoxy and methyl groups from the benzene ring was investigated and compound A4 was observed to have a greatly dampened ability for enhancing3H-naloxone potency (Fig.5a), indicating the importance of the R2and R2Agroups for interacting with the µOR extracellular vestibule. Accordingly, removal of the entire methoxybenzene moiety results in compound A5, which was unable to enhance3H-naloxone affinity (Fig.5a) and may actually decrease binding at high concentrations. Moreover, compound A4 at high concentrations can somewhat dampen fentanyl-induced signaling through the µOR (Fig.5b, Table 1), while A5 does not (and may instead enhance turnover of the receptor) (Fig.5b, Table 1).

[0140] Further exploration of chemical space commenced with the naphthalene ring of S-368 with either addition of a slightly polar methoxy substituent to the ring or truncation of the naphthalene to a benzene group, with compounds A7 and A8, respectively. Both alterations to the naphthalene result in either a complete loss of 3H-naloxone affinity enhancement for compound A7 (Fig.6) or an actual decrease in3H-naloxone binding for compound A8 (Fig.7). Finally, polar substituents on the terminal benzene group on S-368 were investigated. Specifically, nitroxide or an amine were added to compounds A10 and A9, respectively. Addition of a nitroxide group results in both a decrease in affinity and efficacy in increasing3H-naloxone binding for compound A10 (Fig. 7, Table 1), while amine addition retains some ability to enhance3H-naloxone binding for compound A9 (Fig.7, Table 1). These observations suggest that the 368 compound scaffold can be further explored to increase polarity and potentially result in improved affinity for the µ-OR receptor.

[0141] Table 1.3H-naloxone binding fitted values and corresponding 95% confidence intervals for compounds A1-A10.S23-339 CZB-293S-PC T-020961 33167 / 59763N.T. = not tested.

[0142] All curves were normalized to 100% binding values observed in the absence of allosteric compounds followed by fitting to the binding site. REFERENCES 1. Kumar, K. K. et al. Structure of a Signaling Cannabinoid Receptor 1-G Article Structure. Cell 1–11 (2019) doi:10.1016 / j.cell.2018.11.040. 2. Gregorio, G. G. et al. Single-molecule analysis of ligand efficacy in β2AR-G-protein activation. Nature 547, 68–73 (2017). 3. Hilger, D. et al. Structural insights into differences in G protein activation by family A and family B GPCRs. Science 369, eaba3373 (2020).

Claims

S23-339 CZB-293S-PC T-020961 33167 / 59763 What is claimed is:

1. A compound, or pharmaceutically acceptable salt thereof, having a structure of Formula (I):wherein: X is S, NRN, or O; R1is H or L1-Cyc; L1is C0-3alkylene or O-C0-3alkylene; Cyc is C3-6cycloalkyl, C6-10aryl, 4-8 membered heterocycle, or 5-10 membered heteroaryl, wherein the heterocycle and heteroaryl each comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and Cyc is substituted with 0 to 3 R1A; each R1Ais independently selected from halo, C1-3alkyl, C1-3alkoxy, CN, NO2, and N(RN)2; R2is C1-6alkyl, C1-6alkoxy, C1-6hydroxyalkyl, –C0-3alkylene–C6-10aryl, or –C0-3alkylene–5-10 membered heteroaryl, wherein the heteroaryl comprises 1, 2, or 3 ring heteroatoms selected from N, O, and S, and the aryl and heteroaryl ring is substituted with 0 to 3 R2A; each R2Ais independently selected from halo, C1-6alkyl, C3-6cycloalkyl, C1-3haloalkyl, C1-3alkoxy, CN, and N(RN)2; R3is C1-6alkyl, C1-6alkoxy, N(RN)2, or 4-7-memebred heterocycle having 1-3 ring heteroatoms independently selected from N, O, and S; R4and R5are each independently H or RA; or R4and R5join to form a 6-membered fused aromatic ring with 0 to 2 ring N atoms, and is substituted with 0, 1 or 2 RA; each RAis independently C1-6alkyl or C1-6alkoxy; and each RNis independently H or C1-6alkyl, or two RNtogether with the nitrogen to which they are attached form a 4-10 membered heterocycle having 0-2 additional ring heteroatoms independently selected from N, O, and S, with the proviso that when R1is meta-OCH2Ph, R2is CH2Ph with Ph substituted with Me and OMe, R3is NHMe, and R4and R5form a fused unsubstituted phenyl ring, then X is O or NRN.

2. The compound or salt of claim 1, wherein R1is L1-Cyc.

3. The compound or salt of claim 2, wherein L1is C0-3alkylene.

4. The compound or salt of claim 2, wherein L1is O-C0-3alkylene.S23-339 CZB-293S-PC T-020961 33167 / 59763 5. The compound or salt of any one of claims 2 to 4, wherein Cyc is phenyl.

6. The compound or salt of any one of claims 2 to 4, wherein Cyc is pyridyl, pyrazolyl, imidazolyl, furanyl, pyrimidinyl, or pyrazinyl.

7. The compound or salt of any one of claims 2 to 6, wherein Cyc is substituted with 1 or 2 R1A.

8. The compound or salt of any one of claims 2 to 7, wherein each R1Ais selected from halo, C1- 3alkyl, NO2, and N(RN)2.

10. The compound or salt of any one of claims 1 to 9, having a structure of Formula (Ib):

11. The compound or salt of any one of claims 1 to 9, having a structure of Formula (Ic):S23-339 CZB-293S-PC T-020961 33167 / 59763 12. The compound of salt of any one of claims 1 to 11, wherein R2is C1-6alkyl, C1-6hydroxyalkyl, – C0-3alkylene–C6-10aryl, or –C0-3alkylene–5-10 membered heteroaryl, and the aryl and heteroaryl ring is substituted with 0 to 3 R2A.

13. The compound or salt of any one of claims 1 to 12, wherein each R2Ais independently selected from halo, C1-6alkyl, C3-6cycloalkyl, C1-3haloalkyl, and C1-3alkoxy.

14. The compound of salt of claim 12 or 13 wherein15. The compound of salt of any one of claims 1 to 14, wherein R3is C1-6alkoxy or N(RN)2.

16. The compound of salt of claim 15, wherein R3is N(RN)2 and each RNtogether with the nitrogen to which they are attached form a 4-10 membered heterocycle.

17. The compound or salt of any one of claims 1 to 14, wherein R3is 4-7-membered heterocycle.

18. The compound or salt of any one of claims 1 to 14, wherein R3is OMe, NHMe, NH2, N(Me)2,19. The compound of salt of any one of claims 1 to 18, wherein X is S.

20. The compound of salt of any one of claims 1 to 18, wherein X is NRN.

21. The compound of salt of any one of claims 1 to 18, wherein X is O.

22. The compound of salt of any one of claims 1 to 21, wherein R4is H.

23. The compound or salt of any one of claims 1 to 21, wherein R4is RA.

24. The compound or salt of claim 23, wherein R4is C1-6alkyl or C1-6alkoxy.

25. The compound of salt of any one of claims 1 to 21, wherein R5is H.

26. The compound or salt of any one of claims 1 to 21, wherein R5is RA.S23-339 CZB-293S-PC T-020961 33167 / 59763 27. The compound or salt of claim 26, wherein R5is C1-6alkyl or C1-6alkoxy.

28. The compound of salt of any one of claims 1 to 21, wherein R4and R5join to form a 6- membered fused aromatic ring with 0 to 2 ring N atoms, and is substituted with 0, 1 or 2 RA.

29. The compound of salt of claim 28, wherein R4and R5join to form a 6-membered fused phenyl ring substituted with 0, 1 or 2 RA.

30. A compound, or pharmaceutically acceptable salt thereof, having a structure selected fromS23-339 CZB-293S-PC T-020961 33167 / 5976331. A method of inhibiting µ opioid receptor (MOR), comprising contacting MOR with the compound or salt of any one of claims 1 to 30 in an amount sufficient to inhibit MOR.

32. A method for treating opioid overdose in a subject suffering therefrom comprising administering to the subject a therapeutically effective amount of the compound or salt of any one of claims 1 to 30.

33. The method of claim 32, further comprising administration of a therapeutically effective amount of naloxone either before, after, or concomitantly with administration of the compound or salt thereof.

34. The method of claim 32 or 33, wherein the opioid is fentanyl.

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