Opioid receptor agonists and methods of making and using the same

Abstract

The application provides a kind of oxygen heterospirocyclic small molecule compound, and the pharmaceutical composition containing the compound and its purposes as therapeutic agent, especially as MOR receptor agonist and in the preparation of treating and / or preventing pain and other related diseases.The MOR receptor agonist provided by the application has novel structure, shows high activity, has higher selectivity to MOR, and its maximum efficiency Emax also has obvious improvement.

Description

[0001] This application claims priority to Chinese Patent Application No. 202210057938.1, filed on January 19, 2022, entitled "Opioid Receptor Agonist and its Preparation Method and Use", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of medicinal chemistry, specifically relating to a class of oxaspirocyclic small molecule compounds, their preparation methods, pharmaceutical compositions containing the compounds, and their use as therapeutic agents, particularly as MOR receptor agonists, and in the preparation of remedies for treating and preventing pain and related diseases. Background Technology

[0003] Opioid receptors are G protein-coupled receptors (GPCRs) and are targets for the binding of endogenous opioid peptides and opioid drugs. Multiple opioid receptors exist in the human body, mainly including muopioid receptors (MOR), delta opioid receptors (DOR), and kappaopoid receptors (KOR), which are widely distributed in peripheral tissues such as the central nervous system, heart, digestive tract, blood vessels, and kidneys (Nature, 2016, 537(7619):185). MOR has the strongest binding affinity for morphine peptides and is the primary receptor protein site for the action of analgesics such as morphine and fentanyl. Zadina et al. found that the binding affinity of the MOR receptor to morphine 1 (360 pM) was 4,000 times and 15,000 times greater than that of the DOR receptor and KOR receptor to morphine 1, respectively (Science 2001 Vol.293 No: 311-315; Biochem Biophys Res Commun 235:567-570; Life Sci 61:409-415).

[0004] Studies have found that GPCRs mediate and regulate physiological functions mainly through the activation of G protein pathways and β-arrestin pathways. G protein signaling pathways mainly include second messenger systems such as calcium ions, adenylate cyclase, and mitogen-activated protein kinases. The β-arrestin pathway has three main aspects: (1) acting as a negative regulator and interacting with GPCR kinases to induce receptor desensitization of GPCRs, thereby terminating G protein signal transduction; (2) acting as a scaffold protein to recruit endocytic proteins and induce GPCR endocytosis; (3) acting as an adaptor protein to form a complex with downstream signaling molecules of GPCRs, thereby activating signal transduction molecules in a G protein-independent manner. Early studies showed that endogenous enkephalins and the opioid drug etorphine can stimulate G proteins and induce receptor endocytosis, while morphine does not induce receptor endocytosis. This is because morphine exerts its physiological functions through the G protein signaling pathway rather than the β-arrestin pathway (Zhang et al., Proc Natl Acad Sci USA, 1998, 95(12):7157-7162). Studies have found that injecting morphine into β-arrestin2 gene knockout mice resulted in stronger and longer-lasting analgesic effects mediated by G protein signaling (Bohn et al., Science, 1999). This suggests that the difference in ligand-stimulated G protein and / or β-arrestin signaling determines the ligand-specific cellular biological effects of GPCRs. If such ligands exhibit a stronger negative β-arrestin preference, they may even escape β-arrestin-mediated receptor desensitization, leading to prolonged G protein signaling transmission and a stronger analgesic effect. Recent studies have also found that the β-arrestin pathway is associated with several side effects of MOR agonists, such as constipation, respiratory depression, and analgesic tolerance (Science 1999 Vol. 286:2495-2498; J. Pharmacol. Exp. Ther. 2005, 314:1195-1201). Therefore, developing a "biased" MOR agonist drug that can selectively activate the G protein signaling pathway, namely a drug designed with a negative β-arrestin-biased ligand for MOR, would reduce β-arrestin-mediated side effects and have significant clinical value and social significance in the field of analgesia.

[0005] In August 2020, the FDA approved Trevena Inc.'s drug Olinvyk (WO2012129495) for marketing. Currently, there are patents reported on the research and development of G protein-biased MOR agonists, including WO2017063509A1, WO2019205983A1, CN10920641A, WO2019072235A1, CN111662284A, and WO2019052557A1. Although these patents have disclosed a series of G protein-biased MOR agonists, their molecular structures are quite different from the structure provided in this invention, and their efficacy and safety have not yet been confirmed. Clinically, it is still necessary to develop new molecular structures to obtain MOR agonists with better efficacy, selectivity, drug safety, and drug metabolism results. Summary of the Invention

[0006] To address the needs of existing technologies, this invention provides a novel compound that can act as a MOR receptor agonist. This type of compound exhibits high activity, E max It also shows significant improvement and high selectivity for MOR.

[0007] The first aspect of the present invention provides compounds, solvates thereof, stereoisomers thereof, deuterated compounds thereof, or pharmaceutically acceptable salts thereof, as shown in formula (IV) or (V) or (VI).

[0008]

[0009] Among them, ring B and ring C are each independently selected from substituted or unsubstituted aryl groups and substituted or unsubstituted heteroaryl groups;

[0010] Ring D is selected from cycloalkyl and heterocycloalkyl groups;

[0011] R 4 R 5 Each group is independently selected from H, deuterium, alkyl, oxo, alkoxy, hydroxyl, halogen, cyano, alkynyl, alkenyl, and -(CH2). g -O-3 to 12-membered heterocyclic groups, -(CH2) g -O-3 to 12-membered cycloalkyl groups, -(CH2) g -3 to 12-membered cycloalkyl groups, -(CH2) g -3 to 12-membered heterocyclic groups, 5 to 10-membered heteroaryl groups, 5 to 10-membered aryl groups, -S (=O) f -C 1-6 Alkyl, -OC 2-6 alkynyl group, -OC 2-6 Alkenyl; wherein the heterocyclic, heteroaryl, aryl, alkyl, alkynyl, alkenyl, or alkoxy group may optionally be further surrounded by 1 to 3 R groups. 6Replaced;

[0012] Among them, R 6 Each element is independently selected from deuterium, halogen, -OH, and -C. 1-6 Alkyl, -C 1-6 Alkyl-OC 1-6 Alkyl, -OC 1-6 Alkyl, 3- to 6-membered cycloalkyl, -OC 2-6 alkynyl group, -OC 2-6 alkenyl, -C 2-6 alkynyl group, -C 2-6 Alkenyl, amino, carboxylic ester, nitro, cyano, hydroxyalkyl, heterocyclic, aryl, and heteroaryl;

[0013] The value of g is selected from 0, 1, 2, 3, 4, 5, and 6.

[0014] f is selected from 0, 1, and 2;

[0015] p, q, and L are each independently 0, 1, 2, 3, or 4;

[0016] The heteroaryl group, the heterocyclic alkyl group, or the heteroatom on the heterocyclic group are each independently selected from O, S, or N.

[0017] In some embodiments provided by the present invention, the compound is selected from formula (VII), (VIII), (IX), (X), (XI), or (XII):

[0018]

[0019]

[0020] In some embodiments provided by this invention, the compound is selected from the following structures:

[0021]

[0022] In some embodiments provided by this invention, the compound is selected from the following structures:

[0023]

[0024] In some embodiments provided by this invention, the compound is selected from the following structures:

[0025]

[0026] Preferably, the compound, its solvates, stereoisomers, deuterated compounds, or pharmaceutically acceptable salts thereof are selected from the following formula:

[0027]

[0028] Among them, R 4 Independently selected from deuterium, alkyl, oxo, alkoxy, hydroxyl, halogen, cyano, alkynyl, alkenyl, and -(CH2). g -O-3 to 12-membered heterocyclic groups, -(CH2) g -O-3 to 12-membered cycloalkyl groups, -(CH2) g -3 to 12-membered cycloalkyl groups, -(CH2) g -3 to 12-membered heterocyclic groups, 5 to 10-membered heteroaryl groups, 5 to 10-membered aryl groups, -S (=O) f -C 1-6 Alkyl, -OC 2-6 alkynyl group, -OC 2-6 alkenyl;

[0029] q can be selected from 1, 2, 3 or 4.

[0030] In some preferred embodiments provided by this invention, the compounds are selected from:

[0031]

[0032] A second aspect of the invention relates to a pharmaceutical composition comprising the compound shown in the first aspect of the invention, its solvates, stereoisomers, deuterated compounds or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

[0033] The third aspect of the invention provides the use of the compounds, solvates, stereoisomers, deuterated compounds or pharmaceutically acceptable salts thereof shown in the first aspect of the invention, or the pharmaceutical compositions described in the second aspect of the invention, in the preparation of medicaments for the prevention and / or treatment of MOR receptor agonist-mediated diseases.

[0034] In some preferred embodiments of the present invention, the diseases mediated by MOR receptor agonists are selected from pain, immune dysfunction, inflammation, esophageal reflux, neurological and psychiatric diseases, urinary and reproductive diseases, cardiovascular diseases and respiratory diseases; preferably, the pain is selected from postoperative pain, cancer-related pain, neuropathic pain, traumatic pain and inflammation-related pain.

[0035] Terminology Explanation

[0036] The term "C" as used in this invention 2-6 "Alynyl" refers to a straight-chain or branched alynyl group derived from the removal of one hydrogen atom from a 2-6 carbon atom moiety containing a carbon-carbon triple bond, such as ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, etc.

[0037] The term "cycloalkyl" as used in this invention encompasses all possible monocyclic and fused ring systems (including those fused in fused, spiro, or bridged forms); for example, "3-12 membered cycloalkyl" can be a monocyclic, bicyclic, or polycyclic cycloalkyl system (also known as a fused ring system). Unless otherwise specified, a monocyclic system is a cycloalkyl group containing 3-8 carbon atoms, examples including but not limited to: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, etc.

[0038] Fused cycloalkyl groups include fused cycloalkyl groups, bridged cycloalkyl groups, and spirocycloalkyl groups.

[0039] The cycloalkyl group can be 6-11 membered cycloalkyl group or 7-10 membered cycloalkyl group, and representative examples include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane and bicyclo[4.2.1]nonane.

[0040] Spirocycloalkyl groups can be 7-12 membered spirocycloalkyl groups or 7-11 membered spirocycloalkyl groups, examples of which include, but are not limited to: . group.

[0041] The aforementioned bridged cycloalkyl group can be a 6-11 member bridged cycloalkyl group or a 7-10 member bridged cycloalkyl group, examples of which include, but are not limited to: . group.

[0042] As used in this article, the term "heterocyclic alkyl" (or "heterocyclic ring") refers to a monovalent, non-aromatic ring system whose ring atoms consist of carbon atoms and heteroatoms selected from nitrogen, oxygen, sulfur, and phosphorus, and are connected to the parent nucleus or other groups by a single bond; common heterocyclic alkyl groups include (but are not limited to) ethylene oxide, oxetane-3-yl, azirane-3-yl, tetrahydrofuran-2-yl, pyrrolidine-1-yl, pyrrolidine-2-yl, tetrahydro-2-hydropyran-2-yl, tetrahydro-2-hydropyran-4-yl, piperidin-2-yl, piperidin-4-yl, etc.

[0043] As used herein, the term "heterocyclic group" refers to a non-aromatic cyclic group consisting of 3-12 members in which at least one carbon atom of a ring is replaced by a heteroatom selected from O, S, and N, preferably 1-3 heteroatoms, and where the carbon, nitrogen, and sulfur atoms can be oxidized. "3-12 membered heterocyclic group" refers to monocyclic, bicyclic, or polycyclic heterocyclic systems (also known as fused-ring systems), including saturated and partially saturated heterocyclic groups, but excluding aromatic rings. Unless otherwise specified, it includes all possible monocyclic, fused (including fused in fused, spiro, or bridged forms), saturated, and partially saturated cases.

[0044] The monoheterocyclic group can be a 3-8 member monoheterocyclic group, a 3-6 member monoheterocyclic group, a 4-7 member monoheterocyclic group, a 5-7 member monoheterocyclic group, a 5-6 member monoheterocyclic group, a 5-6 member oxygen-containing monoheterocyclic group, a 3-8 member nitrogen-containing monoheterocyclic group, a 5-6 member nitrogen-containing monoheterocyclic group, a 5-6 member saturated monoheterocyclic group, etc., and examples include, but are not limited to: . group.

[0045] Fused heterocycles include fused heterocyclic groups, spirocyclic groups, and bridged heterocyclic groups. They can be saturated, partially saturated, or unsaturated, but are not aromatic.

[0046] The heterocyclic group can be a 6-12 member heterocyclic group, a 7-10 member heterocyclic group, a 6-10 member heterocyclic group, or a 6-12 member saturated heterocyclic group. Representative examples include, but are not limited to: . group.

[0047] The spiroheterocyclic group can be a 6-12 member spiroheterocyclic group, a 7-11 member spiroheterocyclic group, or a 6-12 member saturated spirocyclic group, examples of which include, but are not limited to: Groups;

[0048] The aforementioned bridged heterocyclic group can be a 6-12 member bridged heterocyclic group, a 7-11 member bridged heterocyclic group, or a 6-12 member saturated bridged heterocyclic group, and examples include, but are not limited to: . group.

[0049] As used herein, the term "aryl" (or "aromatic ring") refers to a monovalent monocyclic or polycyclic aromatic ring system (including fused forms) consisting only of carbon and hydrogen atoms. Common aryl groups include (but are not limited to) phenyl, naphthyl, anthraceneyl, phenanthryl, acenaphthene, azulel, fluorenyl, indene, pyrene, etc. The aforementioned aryl groups also include heterocyclic benzoaryl and cycloalkyl benzoaryl groups.

[0050] As used herein, the term "heteroaryl" (or "heteroary ring") refers to a monovalent monocyclic or polycyclic aromatic ring system (including fused forms) whose ring atoms consist of carbon atoms and heteroatoms selected from nitrogen, oxygen, sulfur, and phosphorus. Common heteroaryl groups include (but are not limited to) benzopyrrole, benzofuran, benzothiophene, benzoimidazolyl, benzooxazolyl, benzothiazolyl, azacyclic butyl, carbazole, pyrrole, furanyl, thiaphene, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, indazole, inazinyl, indolyl, quinolinyl, isoquinolinyl, phenazinyl, phenoxazinyl, phenthiazinyl, pteridinyl, purine, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, tetrazolyl, etc. The aforementioned heteroaryl groups also include heterocyclic heteroaryl and cycloalkyl heteroaryl groups.

[0051] The term "stereoisomer" in the present invention refers to the enantiomer produced when the compound contains asymmetric carbon atoms; the cis-trans isomer produced when the compound contains carbon-carbon double bonds or a cyclic structure; and the tautomer produced when the compound contains ketones or oximes. All enantiomers, diastereomers, racemic isomers, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof are included within the scope of this invention. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments are provided to further illustrate the invention in detail. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention are within the scope of protection of this invention.

[0053] As used in this article, room temperature refers to approximately 20-30°C; "overnight" refers to approximately 10-16 hours; 1M, 1N are 1 mmol / L, 1μM is 1 μmol / L, 1mM is 1 mmol / L, 1nM is 1 nmol / L; eq: equivalent.

[0054] Yield or yield = actual mass of synthesized product / theoretical mass of synthesized product × 100%.

[0055] Example 1

[0056] Synthesis of (1R,4R)-4-ethoxy-N-{2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]dec-2-en-9-yl]ethyl}-1,2,3,4-tetrahydronaphthyl-1-amine (compound 141)

[0057]

[0058] Step 1: Synthesis of 2-9-(pyridin-2-yl)-6-oxaspiro[4.5]dec-2-en-9-yl)acetic acid (141-2)

[0059] Compound 141-1 (4.66 g, 18.32 mmol, 1.0 eq) was dissolved in 1 M HCl aqueous solution (23 mL) at room temperature. The solution was heated to 100 °C and stirred under N2 protection for 16 hours. After the reaction was complete, the solution was allowed to cool naturally to room temperature. The aqueous phase was washed with ethyl acetate (100 mL × 3), then the pH of the aqueous phase was adjusted to ~13 with saturated NaOH aqueous solution, washed with ethyl acetate (100 mL × 3), and the pH of the aqueous phase was adjusted to 4–6 with 1 M HCl aqueous solution. The solution was then extracted with ethyl acetate (100 mL × 5). The combined organic phases were washed with brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to give a pale yellow solid, compound 141-2 (4.52 g, yield 90.22%, [M+H)). + :274.17.

[0060] Step 2: Synthesis of (R)-2-9-(pyridin-2-yl)-6-oxaspiro[4.5]dec-2-en-9-yl)acetic acid and (S)phenethylamine salt (141-3)

[0061] At room temperature, ethanol (EtOH, 30 mL) and compound 141-2 (4.52 g, 16.46 mmol) were added to a 100 mL single-necked flask. The mixture was heated to 50 °C and stirred at 50 °C for 0.5 hours. A solution of S-phenylethylamine and ethanol (15 mL) was then slowly added dropwise at 50 °C. After the addition was complete, the mixture was heated to 80 °C and stirred for 1 hour. The mixture was then slowly cooled to room temperature and stirred at room temperature for 12 hours. A white solid precipitated, was filtered, and the solid was washed twice with ethanol (10 mL × 2). The collected white solid was added to 30 mL of ethanol and heated to 80 °C until dissolved. The mixture was stirred at 80 °C for 0.5 hours and gradually cooled to room temperature. After the white solid precipitated, it was filtered, and the filter cake was washed twice with ethanol (10 mL × 2). The solid was collected to obtain white solid 141-3 (1.2 g, ee%: 99.6%).

[0062] Step 3: Synthesis of (R)-2-9-(pyridin-2-yl)-6-oxaspiro[4.5]dec-2-en-9-yl)acetic acid (141-4)

[0063] Compound 141-3 (1.2 g) was dissolved in 20 mL of water, and the pH was adjusted to >9 with 1 M sodium hydroxide solution. The mixture was washed with dichloromethane (50 mL × 3). The aqueous phase was adjusted to pH 4–6 with 1 N hydrochloric acid solution and then extracted with dichloromethane (50 mL × 5). The extracted organic phases were combined and washed with saturated brine (50 mL × 1). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain a pale yellow oily compound 141-4 (600 mg).

[0064] Step 4: Synthesis of N-methyl-N-methoxy-(R)-2-(9-(pyridin-2-yl)-6-oxaspiro[4.5]decene-9-yl)acetamide (141-5)

[0065] Compound 141-4 (600 mg, 2.2 mmol, 1.0 eq) was dissolved in dichloromethane (30 mL) at room temperature. Then, methoxy(methyl)amine hydrochloride (256.93 mg, 2.63 mmol, 1.2 eq), 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (631.22 mg, 3.29 mmol, 1.5 eq), and 4-dimethylaminopyridine (DMAP) (26.82 mg, 219.51 μmol, 0.1 eq) were added sequentially. After stirring for 0.5 h under N2 protection, N,N-diisopropylethylamine (DIPEA) (851.17 mg, 6.59 mmol, 3.0 eq) was added and the mixture was stirred overnight. After the reaction was complete, a saturated NH4Cl solution (100 mL) was added to the reaction solution to quench the reaction, followed by extraction with dichloromethane (100 mL × 5). The organic phases were combined and washed with saturated brine (100 mL × 2), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow viscous substance 141-5 (592 mg, yield: 85.24%), [M+H]. + : 317.17.

[0066] Step 5: Synthesis of compound N-methyl-N-methoxy-(R)-2-(9-(pyridin-2-yl)-6-oxaspiro[4.5]decene-9-yl)acetaldehyde (141-6)

[0067] Compound 141-5 (438.6 mg, 1.39 mmol, 1.0 eq) was dissolved in toluene (Tol, 7 mL) at room temperature. Red-Al (657.9 mg, 2.28 mmol, 1.05 eq) was slowly added dropwise under N2 protection at -40 °C. The mixture was stirred for 4 h after the addition was complete. After the reaction was complete, a 10% citric acid solution (20 mL) was added to the reaction solution to quench the reaction, and then the solution was brought to room temperature. A 10% citric acid aqueous solution (10 mL) was added to the reaction solution, and the solution was brought to room temperature again. The pH was adjusted to 2–3 with 1 mol / L HCl solution, and the solution was extracted with ethyl acetate (30 mL × 1). The aqueous phase was then adjusted to pH 11–13 with 5N NaOH solution, and extracted with dichloromethane (50 mL × 3). The organic phases were combined and washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain an orange-red viscous substance 141-6 (287.2 mg), yield: 80.5%, [M+H]. + : 258.16.

[0068] Step 6: Synthesis of (1R,4R)-4-ethoxy-N-{2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]dec-2-en-9-e]ethyl}-1,2,3,4-tetrahydronaphthyl-1-amine (141)

[0069] Compounds 141-6 (50 mg, 0.19 mmol) and 141-7 (37.2 mg, 0.19 mmol) were dissolved in dichloromethane (DCM) (3 mL) at room temperature. MgSO4 (116.9 mg, 0.97 mmol) was then added, and the mixture was stirred at room temperature for 12 h under nitrogen protection. Sodium borohydride (22.1 mg, 0.58 mmol) was then added, and the mixture was stirred for 2 h. Methanol (1 mL) was then added, and the mixture was stirred for 0.5 h. The mixture was then filtered through diatomaceous earth, and the filtrate was concentrated at room temperature. Large plate (V) plates were then formed. 石油醚(PE) V 乙酸乙酯(EA) =0:1) Purification yielded a yellow viscous substance 141 (35 mg, yield 41.7%), [M+H] + : 433.40.

[0070] 1 H NMR (400MHz, CDCl3) δ8.60-8.56(m, 1H), 7.70-7.64(m, 1H), 7.36-7.32(m, 2H), 7.26-7.19(m, 3H ), 7.18-7.13(m, 1H), 5.66-5.61(m, 1H), 5.49-5.45(m, 1H), 4.39-4.35(m, 1H), 3.95-3.88(m, 1H) , 3.87-3.82(m, 1H), 3.71-3.64(m, 2H), 3.56-3.51(m, 1H), 2.59-2.49(m, 3H), 2.47-2.38(m, 2H) , 2.29-2.22(m, 1H), 2.12-1.98(m, 5H), 1.96-1.90(m, 1H), 1.87-1.77(m, 3H), 1.30-1.20(m, 5H).

[0071] Example 2

[0072] Synthesis of (1R,4R)-4-ethoxy-N-{2-[4'-(pyridin-2-yl)spiro[bicyclo[3.1.0]hexane-3,2'-oxoalkyl]-4'-yl]ethyl}-1,2,3,4-tetrahydronaphthyl-1-amine (compound 143)

[0073]

[0074] Step 1: Synthesis of N-methyl-N-methoxy-(R)-2-(4'-(pyridin-2-yl)tetrahydroxazaspiro[bicyclo[3.1.0]hexane-3,2'-pyran]-4'-ylacetamide (143-1)

[0075] Compound 141-5 (590 mg, 1.86 mmol) was dissolved in DCM (15 mL) under an ice-water bath. Under nitrogen protection, 2 mol / L dimethyl zinc (3.7 mL, 7.46 mmol) and diiodomethane (5.0 g, 18.6 mmol) were added. After the addition was complete, the mixture was moved to room temperature and stirred for 16 h. After the reaction was complete, ethyl acetate (50 mL) was added to the reaction solution, followed by washing with saturated NaHCO3 solution (20 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness and subjected to column chromatography (V). PE V EA =2:1) ​​Purification yielded a yellow viscous substance 143-1 (223 mg, yield 36.26%), [M+H + :331.19.

[0076] Step 2: Synthesis of (R)-2-(4'-(pyridin-2-yl)tetrahydroxazaspiro[bicyclo[3.1.0]hexane-3,2'-pyran]-4'-ylacetaldehyde (143-2)

[0077] Compound 143-1 (223 mg, 0.68 mmol) was dissolved in tetrahydrofuran (THF) (10 mL) at room temperature. Under nitrogen protection at -40 °C, aluminum oxide (335 mg, 70%, 1.16 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred for 4 h. After the reaction was complete, a 10% citric acid aqueous solution (10 mL) was added to the reaction solution. The mixture was then cooled to room temperature, and the pH was adjusted to 2–3 with 1 mol / L HCl solution. The solution was extracted with ethyl acetate (30 mL × 1). The aqueous phase was then adjusted to pH 11–13 with 10% NaOH solution and extracted with dichloromethane (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a brown oily substance 143-2 (160 mg, yield 87.37%), [M+H]. + : 272.23.

[0078] Step 3: Synthesis of (1R,4R)-4-ethoxy-N-{2-[4'-(pyridin-2-yl)spiro[bicyclo[3.1.0]hexane-3,2'-oxoalkyl]-4'-yl]ethyl}-1,2,3,4-tetrahydronaphthyl-1-amine (143)

[0079] At room temperature, compounds 143-2 (50 mg, 0.18 mmol) and 141-7 (35 mg, 0.18 mmol) were dissolved in DCM (3 mL), followed by the addition of MgSO4 (111 mg, 0.92 mmol). The mixture was stirred at room temperature for 12 h under nitrogen protection. Then, NaBH4 (21 mg, 0.55 mmol) was added and stirred for 1 h. Finally, methanol (1 mL) was added and stirred for 0.5 h. The mixture was then filtered through diatomaceous earth, and the filtrate was concentrated at room temperature. Large plate (V) plates were then formed. PE V EA =0:1) Purification yielded a yellow viscous substance 143 (32 mg, yield 39.0%), [M+H] + : 447.43.

[0080] 1 H NMR (400MHz, CDCl3) δ8.56-8.49(m, 1H), 7.69-7.63(m, 1H), 7.39-7.34(m, 1H), 7.33-7.30( m, 1H), 7.26-7.18 (m, 3H), 7.17-7.12 (m, 1H), 4.41-4.36 (m, 1H), 3.84-3.62 (m, 5H), 3.59-3. 48(m, 2H), 2.39-2.28(m, 2H), 2.26-2.21(m, 1H), 2.17-2.04(m, 3H), 1.89-1.80(m, 3H), 1.3 7-1.22(m, 7H), 1.19-1.10(m, 2H), 1.09-0.99(m, 2H), 0.59-0.54(m, 1H), 0.30-0.24(m, 1H).

[0081] Example 3

[0082] Synthesis of (1R,4R)-4-ethoxy-nitro-{2-[9-(pyridin-2-yl)-2,6-dioxaspiro[4,5]decane-9]ethyl}-1,2,3,4-tetrahydronaphthyl-1-amine (compound 156)

[0083]

[0084] Compound 156-1 (50 mg, 0.19 mmol) and compound 141-7 (37.2 mg, 0.19 mmol) were dissolved in DCM (3 mL) at room temperature. MgSO4 (116.9 mg, 0.97 mmol) was then added, and the mixture was stirred at room temperature for 12 h under nitrogen protection. Sodium borohydride (22.1 mg, 0.58 mmol) was then added, and the mixture was stirred for 2 h. Methanol (1 mL) was then added, and the mixture was stirred for 0.5 h. The mixture was then filtered through diatomaceous earth, and the filtrate was concentrated at room temperature. Large plate (V) plates were then formed. PE VEA =0:1) Purification yielded 156 (35 mg, yield 41.7%) of a yellow viscous substance, [M+H] + : 437.27.

[0085] 1 H NMR (400MHz, CDCl3) δ8.60-8.56(m, 1H), 7.70-7.64(m, 1H), 7.36-7.32(m, 2H), 7.26-7.19( m, 3H), 7.18-7.13 (m, 1H), 4.39-4.35 (m, 1H), 3.95-3.88 (m, 1H), 3.87-3.82 (m, 1H), 3.71-3 .64(m, 2H), 3.56-3.51(m, 1H), 2.59-2.49(m, 3H), 2.47-2.38(m, 2H), 2.29-2.22(m, 1H), 2. 12-1.98 (m, 5H), 1.96-1.90 (m, 1H), 1.87-1.77 (m, 3H), 1.45-1.39 (m, 1H) 1.30-1.20 (m, 5H).

[0086] Example 4

[0087] Synthesis of nitrogen-(2-(9-(pyridin-2-yl)-6-oxaspirocyclic[4.5]decane-2-en-9-yl)ethyl)-2,3-dihydro-1-hydro-indene-1-amine (compound 128)

[0088]

[0089] Under nitrogen protection, compound A (100 mg, 0.39 mmol), compound 9 (76 mg, 0.58 mmol), and dichloroethane (DCE, 6 mL) were sequentially added to a single-necked flask. Tetraisopropyl titanate (TIPT, 1.5 mL) was added at room temperature, and the mixture was stirred at 60 °C for 16 h. Sodium borohydride (44 mg, 1.2 mmol) was then added, and the reaction was stirred at 60 °C for 2 h. After the reaction was complete, 1.5 mL of water was added to quench the reaction, followed by the addition of 10 mL of dichloromethane. The mixture was filtered, concentrated at room temperature, and column-secured (V). 二氯甲烷 V 甲醇 =10:1), yielding a yellow viscous compound 128 (20 mg, yield 14%), [M+H] + 375.3.

[0090] 1H NMR (400MHz, CDCl3) δ8.58 (d, J=8.0Hz, 1H), 7.675 (t, J=8.0Hz, 1H), 7.32 (d, J=8.0 Hz, 1H), 7.20-7.10 (m, 5H), 5.61 (brs, 1H), 5.45 (brs, 1H), 4.08-4.04 (m, 1H), 3.94- 3.89(m, 1H), 3.83-3.80(m, 1H), 2.95-2.88(m, 1H), 2.77-2.69(m, 1H), 2.63-2.38( m, 5H), 2.26-2.14 (m, 2H), 2.04-1.94 (m, 3H), 1.81-1.76 (m, 2H), 1.70-1.56 (m, 2H).

[0091] Example 5

[0092] Synthesis of nitrogen-(2-(4'-(pyridin-2-yl)tetrahydroxaspiro[bicyclo[3.1.0]hexane-3,2'-pyran]-4'-yl)ethyl)-2,3-dihydro-1-hydro-indene-1-amine (compound 1)

[0093]

[0094] The preparation method is the same as in Example 4, yielding a yellow viscous compound 1 (7% yield), [M+H]. + 389.3.

[0095] 1 H NMR (400MHz, CDCl3) δ8.59-8.50(m, 1H), 7.67-7.62(m, 1H), 7.38-7.27(m, 2H), 7.20- 7.19(m, 2H), 7.15-7.10(m, 2H), 4.25(brs, 1H), 3.80-3.68(m, 2H), 3.04-2.98(m, 1H), 2.81-2.79(m, 1H), 2.77-2.66(m, 1H), 2.32-2.10(m, 5H), 1.90-1.84(m, 5H), 1.66-1.6 2(m, 1H), 1.30-1.25(m, 2H), 1.11-1.01(m, 3H), 0.55-0.52(m, 1H), 0.24-0.22(m, 1H).

[0096] Comparative Example 1

[0097] Synthesis of N-((3-methoxythiophene-2-yl)methyl)-2-(4'-(pyridin-2-yl)tetrahydroxazolo[bicyclo[3.1.0]hexane-3,2'-pyran]-4'-yl)ethylamine (compound 91)

[0098]

[0099] Under nitrogen protection, intermediate B (100 mg, 0.37 mmol), intermediate 1 (63 mg, 0.44 mmol), magnesium sulfate (882 mg, 7.4 mmol), and dichloromethane (DCM, 6 mL) were sequentially added to a single-necked flask and stirred at room temperature for 16 h. Sodium borohydride (42 mg, 1.1 mmol) was added, and the mixture was stirred for 10 min. Methanol (MeOH, 0.5 mL) was then added, and the reaction was stirred for 2 h. After the reaction was complete, dichloromethane (10 mL) was added, the mixture was filtered, concentrated at room temperature, and the crude product was purified by column chromatography (V... 二氯甲烷 V 甲醇 =10:1), yielding a yellow viscous compound 91 (12.43 mg, yield 8.5%), [M+H] + 399.3.

[0100] 1 ¹H NMR (400 MHz, deuterated chloroform (CDCl₃)) δ 8.53–8.52 (m, ¹H), 7.62 (dt, J₁ = 2.0 Hz, J₂ = 7.6 Hz, ¹H), 7.27 (d, J = 8.0 Hz, ¹H), 7.13–7.07 (m, 2H), 6.77 (d, J = 5.2 Hz) 1H), 3.81-3.66(m, 7H), 2.60-2.53(m, 1H), 2.32-2.28(m, 1H), 2.23-2.19(m, 1H), 2.05-1.96(m, 2H), 1.89-1.80(m, 4H) ), 1.69-1.60(m, 2H), 1.31-1.26(m, 1H), 1.10-1.09(m, 1H), 1.04-1.01(m, 1H), 0.55-0.52(m, 1H), 0.25-0.22(m, 1H).

[0101] Comparative Example 2

[0102] Synthesis of N-((3-methoxythiophene-2-yl)methyl)-2-(9-(pyridin-2-yl)-6-oxaspirocyclic[4.5]decane-2-en-9-yl)ethylamine (compound 83)

[0103]

[0104] Under nitrogen protection, intermediate A (80 mg, 0.31 mmol), intermediate 1 (53 mg, 0.37 mmol), magnesium sulfate (744 mg, 6.2 mmol), and dichloromethane (6 mL) were sequentially added to a single-necked flask and stirred at room temperature for 16 h. Sodium borohydride (35 mg, 0.93 mmol) was added, and the mixture was stirred for 10 min. Methanol (0.5 mL) was then added, and the reaction was stirred for 2 h. After the reaction was complete, dichloromethane (10 mL) was added, the mixture was filtered, concentrated at room temperature, and the crude product was purified by column chromatography (V... 二氯甲烷 V 甲醇 =10:1), yielding a yellow viscous compound 83 (35 mg, yield 29%), [M+H] + 385.3.

[0105] 1 H NMR (400MHz, CDCl3) δ8.57-8.55 (m, 1H), 7.62 (dt, J1=2.0Hz, J2=8.0Hz, 1H), 7.29 (d, J=8.0Hz 1H), 7.13-7.09 (m, 1H), 7.04 (d, J=5.6, Hz 1H), 6.76 (d, J=5.6Hz 1H), 5.61 (brs, 1H), 5.44 (brs, 1H), 3.89-3.86 (m, 1H), 3.83-3.79 (m, 4H), 3.74-3.66 (m, 2H), 2.54-2.37 (m, 5H), 2.14-1.91 (m, 4H), 1.81-1.72 (m, 2H), 1.62-1.56 (m, 1H).

[0106] Comparative Example 3

[0107] Synthesis of nitrogen-((3-methoxythiophene-2-yl)methyl)-2-(9-(pyridin-2-yl)-2,6-dioxaspiro[4.5]decane-9-yl)ethylamine (compound 31)

[0108]

[0109] At room temperature, intermediate C (50 mg, 0.162 mmol) was dissolved in dichloromethane (3 mL), followed by the sequential addition of sodium sulfate (136 mg, 0.96 mmol) and intermediate 1 (41 mg, 0.288 mmol). The reaction was carried out overnight at room temperature under nitrogen protection. After 16 h of reaction, sodium borohydride was added and stirring was continued for 30 min. After the reaction was complete, the mixture was quenched with 15 mL of water, extracted with ethyl acetate (15 × 2 mL), washed with saturated sodium carbonate solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the crude product was purified by column chromatography (V). 石油醚 V 乙酸乙酯=1:1) to obtain 31 (20 mg, yield 22%) of a pale yellow oily liquid, [M+H) + :389.2.

[0110] 1 H NMR (400MHz, chloroform-d (Chloroform-d)) δ8.59 (d, J=8.5, 3.0Hz, 1H), 7.65 (d, J=7.7, 6.0, 4.1, 1.9Hz, 1H), 7.32 (d, J=8.1Hz, 1H), 7.17–7. 12 (m, 1H), 7.05 (d, J = 5.5Hz, 1H), 6.79 (d, J = 5.4Hz, 1H), 4.87 (s, 1H), 3.88–3.82 (m, 3H), 3.79 (s, 3H), 3.75 (d, J = 3.3Hz, 1H), 3.69 (d, J=2.0Hz, 2H), 3.55 (d, J=9.3Hz, 1H), 3.51 (s, 2H), 3.18 (d, J=10.0Hz, 1H), 2.86 (d, J=10.0Hz, 1H), 2.47 (dd, J=11.0, 5.9Hz, 2H), 2.14 (dd, J=10.3, 4.8Hz, 1H), 2.03 (d, J=13.7Hz, 1H), 1.92 (d, J=9.0Hz, 1H), 1.77 (d, J=4.8Hz, 1H), 1.45–1.39 (m, 1H), 1.20–1.12 (m, 1H).

[0111] Biological evaluation

[0112] Op-Mu agonist cAMP assay

[0113] The compounds of this invention can activate the μ-opioid receptor (MOR). Activated MOR regulates intracellular cAMP levels, which then enter the cell nucleus and bind to the cAMP response element (CRE) region of the reporter gene luciferase, initiating reporter gene expression. Luciferase reacts with its substrate to emit fluorescence, and the agonistic activity of the compound can be reflected by measuring the fluorescence signal.

[0114] Experimental methods

[0115] The activity of the compound in inducing MOR and affecting downstream cAMP levels was tested using the following methods.

[0116] 1. Materials and Reagents

[0117]

[0118] 2. Experimental Procedure

[0119] Detection buffer: 1×stimulation buffer, 500μM 1-methyl-3-isobutylxanthine (IBMX), ddH2O.

[0120] Compound preparation: The compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a final concentration of 10 mM. This stock solution was then diluted to a working concentration of 0.08 mM. The compound was serially diluted 4-fold using an Echo pipette, with an initial concentration of 0.08 mM and 10 concentration gradients. 50 nL of each gradient was added to 384 cell culture plates in duplicate, with a final concentration of 0.4 μM. The cell culture plates were then centrifuged at 1000 rpm for 1 min. 50 nL of forskolin (final concentration 1 μM) was then transferred to a 384 cell culture plate using an Echo pipette.

[0121] Cell plating: Thaw the frozen cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash the cells twice with HBSS buffer, resuspend the cells in assay buffer, and adjust the cell density to 5.0 × 10⁶ cells / min. 5 Add 10 μL of the desired cell count / mL to each well of a 384-well plate (5000 cells per well). Shake for 20 seconds, centrifuge at 1000 rpm for 1 minute, and incubate the plate at 23°C for 60 minutes.

[0122] Preparation of standard curve: The standard adenosine-3',5'-cyclic phosphate (cAMP) was serially diluted 4-fold with detection buffer to a total of 8 concentration points, with the highest concentration being 800 nM. 10 μL was added to each well according to the microplate layout diagram.

[0123] Preparation of detection reagents: Dilute Anti cAMP-Cryptate and AMP-d2 to 1× with lysis buffer. Add 10 μL of detection reagent to each well according to the microplate layout diagram, shake for 20 s, centrifuge at 1000 rpm for 1 min, and incubate the cell plate in a 23℃ incubator for 60 min. Read the plate on an Envision microplate reader.

[0124] 3. Results Analysis

[0125] The percentage of activity was calculated using Microsoft Excel. For agonists, the formula %Effect = 100 × (Sample Raw Value - Low Control Average) / (High Control Average - Low Control Average) was used. GraphPad Prism 5 data analysis software was then used, with the Dose-response-Stimulation-log[agonist] vs. response-Variable slope mode selected for fitting analysis to obtain the EC5 values ​​for each tested sample. 50 value.

[0126] The effects of the compounds of this invention on downstream cAMP levels by activating MOR were determined through the above experiments. The experimental results show that this series of compounds exhibits a strong Op-Mu activating effect, among which the EC50 of the typical representative compounds was measured. 50 The values ​​are shown in Table 1. Six control groups were set up: compound 83, compound 91, compound 31, TRV130, TRV130 (racemic), and SHR8554. TRV130, TRV130 (racemic), and SHR8554 have the following structural formulas. The preparation method of TRV130 refers to patent CN103702561A; the preparation method of SHR8554 refers to patent CN107001347B; E max This represents the maximum efficacy of the compound in inducing changes in cAMP levels.

[0127]

[0128] Table 1: Effects of test compounds on cAMP levels of MOR receptors (EC5) 50 and E max

[0129] Compound numbering <![CDATA[EC 50 (nM)]]> <![CDATA[E max ]]> 83 32.61 84.4％ 91 26.42 97.2％ 31 >400 54.0 141 0.7787 71.0％ 143 0.1500 105.4％ 156 5.152 65.6％ SHR8554 2.54 79.9％ TRV130 2.79 75.3％ TRV130 (Rash) 5.749 81.8％

[0130] Preferred compounds of this invention exhibit significant agonistic effects on Mu opioid receptors, and some compounds, such as EC, show similar effects. 50 Value and E max It is far superior to the control group.

[0131] Op-Kappa agonist cAMP assay

[0132] ForsKolin stimulates the release of cAMP in human opioid receptor-overexpressing OPRK1 cells (DiscoveRx), while opioid receptor agonists inhibit forsKolin-stimulated cAMP release. By detecting the inhibitory effect of the test compound on forsKolin-stimulated cAMP release, the agonistic activity of the compound on human opioid receptors can be determined. First, human opioid receptor-overexpressing cell lines were co-incubated with specific concentrations of forsKolin and different concentrations of the test compound. The cAMP levels in the stimulated OPRK1 cells were determined using a time-resolved fluorescence resonance energy transfer (TR-FRET) cAMP immunoassay (LANCEPerKinElmer). The specific method is as follows:

[0133] Detection buffer: 1×stimulation buffer, 500μM IBMX, ddH2O. Compound preparation: The compound was dissolved in DMSO to prepare a stock solution with a final concentration of 10mM. This stock solution was then diluted to a working concentration of 2mM. The compound was serially diluted 4-fold using an Echo, with an initial concentration of 2mM and 10 concentration gradients. 50nL of each gradient was added to a 384 cell plate in duplicate, with a final concentration of 10μM. The cell plates were then centrifuged at 1000rpm for 1min. 50nL of ForsKolin (final concentration of 3μM) was transferred to the 384 cell plate using an Echo.

[0134] Cell plating: Thaw the frozen cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash the cells twice with HBSS buffer, resuspend the cells in assay buffer, and adjust the cell density to 3.0 × 10⁶ cells / mL. 5 10 μL of cAMP was added to each well of a 384-well plate, 3000 cells per well. The plates were vortexed for 20 seconds, centrifuged at 1000 rpm for 1 min, and then incubated at 23°C for 60 min. For the standard curve preparation: cAMP standard was serially diluted 4-fold with assay buffer to obtain 8 concentration points, with the highest concentration being 800 nM. 10 μL of each standard was added to each well according to the microplate layout. For the assay reagent preparation: Anti cAMP-Cryptate and AMP-d2 were diluted to 1× with lysis buffer. 10 μL of the assay reagent was added to each well, vortexed for 20 seconds, centrifuged at 1000 rpm for 1 min, and then incubated at 23°C for 60 min. The plates were then read on an Envision microplate.

[0135] The percentage of activity was calculated using Microsoft Excel software. For agonists, the formula %Effect = 100 × (Sample Raw Value - Low Control Average) / (High Control Average - Low Control Average). GraphPad Prism 5 data analysis software was used, and for agonists, the Dose-response-Stimulation—log[agonist] vs. response—Variable slope mode was selected for fitting analysis to obtain the EC5 of each tested sample. 50 Values. Experimental data are shown in Table 2.

[0136] Table 2: Effects of the tested compounds on ketogenic receptors

[0137] Compound numbering <![CDATA[EC 50 (nM)]]> <![CDATA[E max ]]> TRV130 (Rash) 189.6 56.2％ SHR8554 130 43.2％ 83 10287 47.0％ 91 287.13 38.1％ 141 679.1 31.9％ 143 592.4 39.2％ 156 801.6 37.2％

[0138] The compounds in the embodiments of the present invention showed significantly weaker activity against kinase receptors compared to the control group; this demonstrates that the compounds of the present invention have high selectivity for MOR receptors, suggesting that the compounds in the embodiments of the present invention have fewer side effects.

[0139] Assay on the activity of the β-arrestin signaling pathway of the Mμ opioid receptor

[0140] This study aims to utilize the EC of CHO-K1 / Arrestin / hMOR. 50 The recruitment efficiency of β-Arrestin, an agonist targeting the μ-opioid receptor MOR, was evaluated using EMAX assays. The CHO-K1 / Arrestin / hMOR cell line expressed hMOR fused with a β-galactosidase donor fragment and β-Arrestin fused with a β-galactosidase receptor fragment. When β-arrestin interacts with hMOR, these fragments form an active β-galactosidase. 384-well plates were prepared, with 60 nL / well of serially diluted compound added to each well. 20 μL of CHO-K1 / Arrestin / hMOR cell suspension was injected into the assay plate, resulting in a cell density of 7.5 kC cells / well. The assay plates were incubated at 37°C with 0.5% CO2 (volume fraction, remainder air) for 120 min. 10 μL / well of assay reagent was added to the assay plate using the dragonfly method, and the plates were incubated at room temperature for 60 min. Chemiluminescence signals were detected using Envision, and data were analyzed using XLfit. Experimental data are shown in Table 3.

[0141] Table 3: Effects of the tested compounds on the β-arrestin signaling pathway

[0142] Compound numbering <![CDATA[EC 50 (μM)]]> <![CDATA[E max ]]> TRV130 0.75 7.63％ SHR8554 0.55 3.46％ 141 3.21 4.09％ 143 4.10 6.20％

[0143] The compounds in the embodiments of the present invention have almost no activating effect on the β-arrestin signaling pathway, and the compounds of the present invention have a better bias (cAMP and β-arrestin signaling pathway) compared with the control group, suggesting that the compounds of the present invention have fewer side effects than the control group.

[0144] Testing the blocking effect of the compounds of this invention on hERG potassium current.

[0145] Test system

[0146] Cells: Chinese hamster ovary (CHO) cell line, CHO-hERG cells were used in this experiment.

[0147] Cell culture medium and conditions: The complete culture medium was F12 medium, supplemented with 10% fetal bovine serum and 1% [unclear - possibly a specific ingredient or preparation]. Selective antibiotic (G418), 89 μg / mL hygromycin B (HB). The resuscitation medium was F12 medium supplemented with 10 vol% fetal bovine serum. CHO-hERG cells were grown in a high-humidity incubator at 37°C (±2°C) and 5% CO2 (4% to 8%). Cells were resuscitated with resuscitation medium, passaged in complete medium, and cells used for patch-clamp experiments were replaced with resuscitation medium at the final passage.

[0148] Components of extracellular and intracellular fluids:

[0149] reagents External fluid (mM) Internal fluid (mM) <![CDATA[CaCl2]]> 2 5.37 <![CDATA[MgCl2]]> 1 1.75 KCl 4 120 NaCl 145 - Glucose 10 - HEPES 10 10 EGTA - 5 <![CDATA[Na2ATP]]> - 4 pH 7.3-7.4 7.2-7.3

[0150] Test methods

[0151] (1) Collect CHO-hERG cells in the exponential growth phase and resuspend them in ECS for later use.

[0152] (2) Manual Patch Clamp Test

[0153] hERG currents were recorded using whole-cell patch-clamp technology at room temperature. The patch-clamp amplifier output signal was converted from digital to analog and then filtered using a 2.9 kHz low-pass filter. Data was acquired using Patchmaster Pro software.

[0154] Cells were seeded in cell recording wells and placed on the stage of an inverted microscope. One cell from the recording well was randomly selected for the experiment. The perfusion system was fixed to the stage of the inverted microscope and continuously perfused the cells using an ECS.

[0155] Manual patch-clamp experimental recording microelectrodes were prepared using capillary glass tubes filled with intracellular fluid. On the day of the patch-clamp experiment, electrodes were prepared using borosilicate glass tubes (BF150-117-10, SUTTER INSTRUMENT USA). The resistance of the electrodes after filling with intracellular fluid (ICS) was between 2 and 5 MΩ.

[0156] The clamping voltage was -80mV. The first step involved depolarizing to +60mV and maintaining it for 850ms to open the hERG channel. Then, the voltage was set to -50mV and maintained for 1275ms, generating a bounce current, or tail current. The peak value of this tail current was measured and used for analysis. Finally, the voltage was restored to the clamping voltage (-80mV). During the experiment, this command voltage procedure was repeated every 15s.

[0157] At the beginning of the recording of perfusion with the solvent control working solution, monitor the tail current peak until at least three stable scan curves are obtained, then perfuse the test sample / positive control working solution until the inhibitory effect of the test sample / positive control working solution on the hERG current peak reaches a stable state. Generally, the standard for judging whether a stable state has been reached is the near overlap of the peak values ​​of the three most recent consecutive current curves. After reaching a stable state, continue perfusing the next concentration of test sample. One or more test samples / positive controls, or multiple concentrations of the same drug, can be tested on a single cell. Different test samples / positive controls need to be rinsed with solvent control working solution until the hERG current returns to more than 80% of its value before drug addition. The standard deviation of the inhibition rate of each recorded cell at the same concentration should not exceed 15%.

[0158] The positive control, cisapride, was tested at a concentration of 0.1 μM, and two cells were measured repeatedly. According to scientific literature, 0.1 μM cisapride inhibits hERG currents by more than 50% (Milnes, JT, et al.).

[0159] (3) Manual Patch Clamp Data Acceptance Standards

[0160] Sealing criteria: After whole-cell pattern formation, applying a clamping voltage (-80mV) allows recording of cell membrane-related parameters (Cm, Rm, and Ra). A good whole-cell record should meet the following conditions: path resistance (Rs) less than 10MΩ; membrane resistance (Rm) greater than 500MΩ; and membrane capacitance (Cm) less than 100pF.

[0161] Current magnitude: The peak current amplitude before treatment with the test sample / positive control should be between 400 pA and 5000 pA. Otherwise, discard the cell.

[0162] Leakage current: At a clamping voltage of -80mV, the absolute value of the leakage current should be less than 200pA. The current amplitude will be corrected using the leakage current at -80mV. Scan curves with an absolute leakage current greater than 200pA cannot be used for analysis.

[0163] Data Analysis

[0164] For each cell, the percentage of inhibition for each concentration of the test sample and the positive control was calculated from the recorded current response using the following formula: (1 – test sample / tail peak current recorded after perfusion of the positive control / tail peak current (initial current) recorded after perfusion of the solvent control) × 100%.

[0165] For each concentration, the average of all recorded percentages of cell inhibition was taken, and the IC50 was calculated. 50 The value (half-inhibitory concentration) was obtained from the concentration-effect curve using Hill's fitting method.

[0166] Test results

[0167] The results of some of the compounds in this invention on the suppression of hERG current are shown in Table 4 below;

[0168] Table 4: Suppression Results of Test Compounds on hERG Current

[0169] Compound numbering <![CDATA[IC 50 ]]> 141 ++ 143 ++ 156 ++ SHR8554 + TRV130 +

[0170] Note: 20μM > IC 50 >10μM is ++, 10μM >IC 50 >1μM is positive.

[0171] The compounds in the embodiments of the present invention have higher hERG IC50 compared to the control group. 50 The values ​​showed significant differences, indicating a weaker inhibitory effect on hERG, suggesting that the compounds of this invention have a lower risk of cardiotoxicity.

[0172] Pharmacokinetic experiments

[0173] The compounds under investigation were administered orally or intravenously (solvent 5 vol% DMSO + 10 vol% Solutol (HS-15) + 85 vol% saline) to animals (e.g., mice, rats, dogs, or monkeys), with blood samples collected at fixed time points. Immediately after blood collection, the test tubes were gently inverted at least five times to ensure thorough mixing before being placed on ice. Blood was anticoagulated with heparin and then centrifuged at 8000 rpm for 5 minutes to separate serum from erythrocytes. Serum was pipetted into 2 mL polypropylene tubes, labeled with the compound name and time point, and stored at -40°C for LC-MS analysis. High-concentration samples were diluted with blank plasma for analysis. After sample processing, the substances in the plasma were quantitatively analyzed using LC-MS / MS. Pharmacokinetic parameters were calculated using plasma concentration / time curves obtained in this manner using a validated pharmacokinetic computer program. Experiments showed that the compounds of this invention all exhibited good pharmacokinetic properties.

[0174] Male SD rats were administered the drugs intravenously at the doses listed in Table 5 (each group was administered at an equimolar dose, with the solvent being 5 vol% DMSO + 10 vol% Solutol (HS-15) + 85 vol% saline, 3 rats per group). Blood samples were collected at fixed time points for analysis. The pharmacokinetic parameters of the prototype compounds of some of the compounds of this invention in rat plasma are shown in Table 5 below; in Table 5, IV represents intravenous administration, AUC is the area under the plasma concentration-time curve, and C... max For maximum blood drug concentration, T 1 / 2 To eliminate half-life, Variable is the variable, Mean is the mean, and SD is the standard deviation.

[0175] Table 5: Pharmacokinetic parameters of the test compounds

[0176]

[0177]

[0178] The compounds of the present invention exhibited favorable pharmacokinetic properties in rats; compared with the control group, the AUC (h*ng / mL) of the free base in plasma of the compounds of the present invention was significantly increased.

[0179] Hot plate test for analgesic efficacy in rats

[0180] Female SD rats were used, and the start date of training was designated D0. On D0, the analgesic device was set to 52℃ (52.0±0.5℃). The rats were placed on the hot plate, and the time (s) taken for the rat to lick its hind paw or jump was recorded. If a rat did not show a heat-induced pain response for more than 30 seconds on the hot plate, it was immediately removed, and the pain threshold was recorded as 30 seconds. Rats with sensitive or sluggish responses were culled. On D1, pre-screened rats were collected, and the analgesic device was set to 52℃ (52.0±0.5℃). The rats were placed on the hot plate, and the time (s) taken for the rat to lick its hind paw or jump was recorded. This was repeated three times. The average of the three measurements was used as the baseline pain threshold for the rats. On D1, the rats were randomly divided into groups of eight based on the baseline pain threshold. On day 2, the solvent or compound was administered via tail vein injection according to the dosage groups in the table (equal molar doses for each group, solvent being 5 vol% DMSO + 10 vol% Solutol (HS-15) + 85 vol% saline). On day 2, at 0.5 min, 0.5 h, 1 h, and 3 h after administration, the analgesia meter temperature was set to 52℃ (52.0 ± 0.5℃), and the rats were placed on a hot plate while timing was recorded. The time (s) taken for the rat to lick its hind paw or jump to reach the pain threshold was recorded, with one measurement at each time point. If the rat did not show a thermal pain response on the hot plate for more than 30 seconds, it was immediately removed, and the pain threshold was recorded as 30 seconds. The percentage of pain threshold (MPE) was calculated for each group at each time point to evaluate the in vivo analgesic efficacy of each test sample. Experimental data are expressed as Mean ± SEM. Data between groups were analyzed using GraphPad Prism ANOVA (Two-Way ANOVA or One-Way ANOVA), with P < 0.05 considered statistically significant. The maximum analgesic effect percentage (i.e., pain threshold % MPE) = (post-drug pain threshold - baseline pain threshold) / (30 - baseline pain threshold) × 100%. The experimental results are shown in Table 6 below; in Table 6, Variable represents the variable, Average represents the mean, and SEM represents the standard error.

[0181] Table 6: In vivo analgesic efficacy of the tested compounds

[0182]

[0183]

[0184] Note: Compared with the control group (solvent, IV), ***p<0.001

[0185] The compounds in the embodiments of the present invention showed better analgesic effects compared with the control group, and demonstrated higher pain threshold and longer duration of analgesia in the hot plate analgesic efficacy study in rats.

[0186] Acute toxicity test

[0187] The compounds were administered intravenously once (5 vol% DMSO + 10 vol% Solutol (HS-15) + 85 vol% saline) to SD rats (4-6 dose groups for each compound, 10 rats per group, half female and half male). Clinical observation was conducted after administration. Clinical observation was performed twice on the first day, and once daily from the second day onwards for 14 consecutive days. Behavioral observations included spontaneous activity, neurological behavior, and mortality. The maximum tolerated dose (MTD) and median lethal dose (LD50) of the compounds were obtained. 50 (Value). Experimental results show the MTD and LD50 values ​​of the compound of the present invention after a single intravenous administration in rats. 50 The value was significantly higher than that of the control group, indicating that the compound of the present invention has good safety.

[0188] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from: 、 。 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from: 、 。 3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. Use of the compound of any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3, in the preparation of a medicament for the prevention and / or treatment of MOR receptor agonist-mediated diseases.

5. The use according to claim 4, characterized in that, The diseases mediated by the MOR receptor agonists mentioned are selected from pain, immune dysfunction, inflammation, esophageal reflux, neurological and psychiatric diseases, urinary and reproductive diseases, cardiovascular diseases, and respiratory diseases.

6. The use according to claim 5, characterized in that, The pain is selected from postoperative pain, cancer-related pain, neuropathic pain, traumatic pain, and pain caused by inflammation.

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