CN116801881A - Opioid receptor agonist, preparation method and application thereof - Google Patents

Abstract

The invention provides oxaspiro small molecule compounds, a pharmaceutical composition containing the compounds and application of the compounds as therapeutic agents, particularly as MOR receptor agonists and in preparing medicaments for treating and/or preventing related diseases such as pain. The MOR receptor agonist with novel structure provided by the invention has high activity, higher selectivity to MOR and obviously improved maximum efficiency Emax.

Description

Opioid receptor agonists and preparation methods and uses thereof

This application claims priority to the Chinese patent application filed with the China Patent Office on January 19, 2022, with application number 202210057938.1 and the invention title is "Opioid Receptor Agonist and Preparation Method and Use thereof", the entire content of which is incorporated by reference. in this application.

Technical field

The invention belongs to the field of medicinal chemistry, and specifically relates to a class of oxaspirocyclic small molecule compounds, their preparation methods and pharmaceutical compositions containing the compounds as well as their use as therapeutic agents, especially as MOR receptor agonists and in the preparation of therapeutic and Use in medicines to prevent pain and other related diseases.

Background technique

The opioid receptor is a G Protein-Coupled Receptor (GPCR), which is the binding target of endogenous opioid peptides and opioid drugs. There are many types of opioid receptors in the human body, including three types: mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opoid receptor (KOR). Distributed in the central nervous system, heart, digestive tract, blood vessels, kidneys and other peripheral tissues (Nature, 2016, 537(7619):185). MOR has the strongest binding ability to morphine peptides and is the main receptor protein site for analgesics such as morphine and fentanyl. Zadina et al. found that the binding ability of MOR receptor to morphinin 1 (360pM) is 4000 times and 15000 times that of DOR receptor and KOR receptor and morphinin 1 (Science 2001 Vol. 293 No: 311-315; Biochem Biophys Res Commun 235:567-570; Life Sci 61:409-415).

Studies have found that GPCR mediates and regulates physiological functions mainly through activating the G protein pathway and β-arrestin pathway. The G protein signaling pathway mainly includes second messenger systems such as calcium ions, adenylyl cyclase, mitogen-activated protein kinase, etc. The β-arrestin pathway has three main aspects: (1) As a negative regulatory factor, it interacts with GPCR kinases to cause receptor desensitization of GPCRs, thus terminating G protein signal transduction; (2) As a scaffold protein, it recruits endocytic proteins to induce GPCR internalization. Endocytosis; (3) As an adapter protein, it forms a complex with GPCR downstream signaling molecules and activates signal transduction molecules in a G protein-independent manner. Early studies have shown that endogenous enkephalins and the opioid etorphine can activate G proteins and trigger receptor endocytosis, while morphine does not trigger receptor endocytosis. This is because morphine activates G protein signaling pathways rather than β -arrestin pathway to exert its physiological functions (Zhang et al., Proc Natl Acad Sci USA, 1998, 95(12):7157-7162). Studies have found that after injecting morphine into β-arrestin2 knockout mice, the analgesic effect mediated by G protein signaling is stronger and lasts longer (Bohn et al., Science, 1999). It can be seen that the difference in ligand-stimulated G protein and/or β-arrestin signals determines the ligand-specific cell biological effects of GPCRs. If such ligands have a stronger negative β-arrestin preference, they can even escape Receptor desensitization mediated by β-arrestin prolongs the G protein signal transmission time and increases the analgesic effect. In recent years, studies have found that the β-arrestin pathway is related to multiple 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, it is necessary to develop a "biased" MOR agonist drug that can selectively activate the G protein signaling pathway, that is, a negative β-arrestin-biased ligand design drug for MOR, which can reduce the side effects mediated by β-arrestin. The field of pain has significant clinical value and social significance.

The FDA approved the marketing application of Trevena Inc's drug Olinvyk (WO2012129495) in August 2020. The current patents reported on the research and development of G protein-biased MOR agonists include WO2017063509A1, WO2019205983A1, CN10920641A, WO2019072235A1, CN111662284A, WO 2019052557A1, etc., although these Patents have disclosed a series of G protein-biased MOR agonists, but their molecular structures are quite different from those provided by the present invention, and their efficacy and safety have not yet been confirmed, and new molecular structures still need to be developed clinically. , to obtain MOR agonists with better efficacy, selectivity, medicinal safety, and drug metabolism results.

Contents of the invention

In response to the needs of the existing technology, the present invention provides a compound with a novel structure that can be used as a MOR receptor agonist. This type of compound exhibits high activity, a significant improvement in E _max , and a high selectivity for MOR.

The first aspect of the present invention provides compounds represented by formula (IV) or (V) or (VI), their solvates, stereoisomers, deuterated compounds, or pharmaceutically acceptable salts thereof,

Wherein, Ring B and Ring C are each independently selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl;

Ring D is selected from cycloalkyl and heterocycloalkyl;

R ⁴ and R ⁵ are each independently selected from H, deuterium atom, alkyl group, oxo, alkoxy group, hydroxyl, halogen, cyano group, alkynyl group, alkenyl group, -(CH ₂ ) _g -O-3 to 12 Membered heterocyclyl, -(CH ₂ ) _g -O-3 to 12-membered cycloalkyl, -(CH ₂ ) _g -3 to 12-membered cycloalkyl, -(CH ₂ ) _g -3 to 12-membered heterocycle base, 5- to 10-membered heteroaryl, 5- to 10-membered aryl, -S(=O) _f -C _1-6 alkyl, -OC _2-6 alkynyl, -OC _2-6 alkenyl; wherein, The heterocyclic group, heteroaryl group, aryl group, alkyl group, alkynyl group, alkenyl group, and alkoxy group may be optionally further substituted by 1 to 3 R ⁶ ;

Among them, R ⁶ is each independently selected from deuterium atom, halogen, -OH, -C _1-6 alkyl, -C _1-6 alkyl-OC _1-6 alkyl, -OC _1-6 alkyl, 3 to 6-membered ring alkyl, -OC _2-6 alkynyl, -OC _2-6 alkenyl, -C _2-6 alkynyl, -C _2-6 alkenyl, amino, carboxylate group, nitro, cyano group , hydroxyalkyl, heterocyclyl, aryl and heteroaryl;

The g is selected from 0, 1, 2, 3, 4, 5, 6;

The f is selected from 0, 1, 2;

p, q, L are each independently 0, 1, 2, 3 or 4;

The heteroatom on the heteroaryl group, the heterocycloalkyl group or the heterocyclyl group is each independently selected from O, S or N.

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

In some embodiments provided by the present invention, the compound is selected from the following formula:

In some embodiments provided by the present invention, the compound is selected from the following formula:

In some embodiments provided by the present invention, the compound is selected from the following formula:

Preferably, the compound, its solvate, stereoisomer, deuterated compound, or pharmaceutically acceptable salt thereof, the compound is selected from the following formula:

Among them, R ⁴ is independently selected from deuterium atom, alkyl group, oxo, alkoxy group, hydroxyl group, halogen, cyano group, alkynyl group, alkenyl group, -(CH ₂ ) _g -O-3 to 12-membered heterocyclic group , -(CH ₂ ) _g -O-3 to 12-membered cycloalkyl, -(CH ₂ ) _g -3 to 12-membered cycloalkyl, -(CH ₂ ) _g -3 to 12-membered heterocyclyl, 5 to 10-membered heteroaryl, 5 to 10-membered aryl, -S(=O) _f -C _1-6 alkyl, -OC _2-6 alkynyl, -OC _2-6 alkenyl;

q is selected from 1, 2, 3 or 4.

In some preferred embodiments provided by the present invention, the compound is selected from:

The second aspect of the present invention relates to a pharmaceutical composition, which includes the compound shown in the first aspect of the present invention, its solvate, stereoisomer body, deuterated compound or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The third aspect of the present invention provides the compounds shown in the first aspect of the present invention, their solvates, stereoisomers, deuterated compounds or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described in the second aspect of the present invention. Use in the preparation of drugs for preventing and/or treating MOR receptor agonist-mediated related diseases.

In some preferred embodiments provided by the present invention, the related 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 the group consisting of postoperative pain, cancer-induced pain, neuropathic pain, traumatic pain and inflammation-induced pain.

Terminology explanation

The term "C _2-6 alkynyl" used in the present invention refers to a linear or branched alkynyl group derived from an alkyne moiety of 2 to 6 carbon atoms containing a carbon-carbon triple bond by removing one hydrogen atom, such as ethynyl, propyne base, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, etc.

The above-mentioned "cycloalkyl" in the present invention includes all possible single rings and fused rings (including fused in the form of parallel, spiro, and bridge); for example: "3-12-membered cycloalkyl" can be a single ring. cyclic, bicyclic, or polycyclic cycloalkyl systems (also called fused ring systems). Unless otherwise specified, a monocyclic system is a cyclic hydrocarbyl group containing 3 to 8 carbon atoms. Examples include but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, Cycloheptyl, cyclooctyl, etc.

Fused-ring cycloalkyl groups include pendant cycloalkyl groups, bridged cycloalkyl groups, and spirocycloalkyl groups.

The cyclic cycloalkyl group can be a 6-11 membered cyclic cycloalkyl group or a 7-10 membered cyclic cycloalkyl group. 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.

The spirocycloalkyl group can be a 7-12-membered spirocycloalkyl group or a 7-11-membered spirocycloalkyl group. Examples include but are not limited to: group.

The above-mentioned bridged cycloalkyl group can be a 6-11-membered bridged cycloalkyl group or a 7-10-membered bridged cycloalkyl group. Examples thereof include but are not limited to: group.

The term "heterocycloalkyl" (or "heteroalicyclic") as used herein refers to a monovalent monocyclic non-aromatic ring system in which the ring atoms are composed of carbon atoms and heteroatoms selected from nitrogen, oxygen, sulfur and phosphorus. composed of, and connected to the parent core or other groups through a single bond; common heterocycloalkyl groups include (but are not limited to) oxiranyl, oxetan-3-yl, azetidine- 3-yl, tetrahydrofuran-2-yl, pyrrolidin-1-yl, pyrrolidin-2-yl, tetrahydro-2hydro-pyran-2-yl, tetrahydro-2hydro-pyran-4-yl, Piperidin-2-yl, piperidin-4-yl, etc.

The term "heterocyclyl" as used herein refers to a 3-12 membered non-aromatic cyclic group in which at least one ring carbon atom is replaced by a heteroatom selected from O, S, and N, preferably 1-3 heteroatoms, and at the same time Including carbon atoms, nitrogen atoms and sulfur atoms can be oxygenated. "3-12 membered heterocyclyl" refers to a monocyclic heterocyclyl, a bicyclic heterocyclyl system or a polycyclic heterocyclyl system (also known as a fused ring system), including saturated and partially saturated heterocyclyl, but Excludes aromatic rings. Unless otherwise specified, it includes all single rings, fused rings (including fused in the form of union, spiro, and bridge), saturated, and partially saturated rings that may be formed.

The monoheterocyclyl group can be a 3-8-membered monoheterocyclyl group, a 3-6-membered monoheterocyclyl group, a 4-7-membered monoheterocyclyl group, a 5-7-membered monoheterocyclyl group, or a 5-6-membered monoheterocyclyl group. , 5-6-membered oxygen-containing monoheterocyclyl, 3-8-membered nitrogen-containing monoheterocyclyl, 5-6-membered nitrogen-containing monoheterocyclyl, 5-6-membered saturated monoheterocyclyl, etc. Examples include but are not Limited to: group.

Condensed heterocycles include pendant heterocyclyl, spiroheterocyclyl and bridged heterocyclyl, which can be saturated, partially saturated or unsaturated, but are not aromatic.

The heterocyclyl group can be a 6-12-membered heterocyclyl group, a 7-10-membered heterocyclyl group, a 6-10-membered heterocyclyl group, or a 6-12-membered saturated heterocyclyl group. Representative examples include but are not limited to : group.

The spiroheterocyclyl group can be a 6-12-membered spiroheterocyclyl group, a 7-11-membered spiroheterocyclyl group, or a 6-12-membered saturated spiroheterocyclyl group. Examples thereof include but are not limited to: group;

The above-mentioned bridged heterocyclyl group can be a 6-12-membered bridged heterocyclyl group, a 7-11-membered bridged heterocyclyl group, or a 6-12-membered saturated bridged heterocyclyl group. Examples thereof include but are not limited to: group.

The term "aryl" (or "aryl ring") used herein refers to a monovalent monocyclic or polycyclic (including fused form) aromatic ring system, which is composed only of carbon atoms and hydrogen atoms; common aryl groups Including (but not limited to) phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthyl, azulenyl, fluorenyl, indenyl, pyrenyl, etc. The above-mentioned aryl groups also include heterocyclyl aryl groups and cycloalkyl aryl groups;

The term "heteroaryl" (or "heteroaryl ring") as used herein refers to a monovalent monocyclic or polycyclic (including fused forms) aromatic ring system, the ring atoms of which are carbon atoms and selected from nitrogen, oxygen , composed of heteroatoms of sulfur and phosphorus; common heteroaryl groups include (but are not limited to) benzopyrrolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl, benzothiazolyl , azetidinyl, carbazolyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, indazolyl, indazine base, indolyl, quinolyl, isoquinolinyl, phenazinyl, phenoxazinyl, phenothiazinyl, pteridinyl, purinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyridyl, Triazolyl, tetrazolyl, etc. The above-mentioned heteroaryl group also includes heterocyclylheteroaryl and cycloalkylheteroaryl.

The "stereoisomer" of the compound shown in the present invention means that when asymmetric carbon atoms exist in the compound, enantiomers will be produced; when a carbon-carbon double bond or cyclic structure exists in the compound, cis-trans isomers will occur body; when ketones or oximes are present in a compound, tautomers will be produced, including enantiomers, diastereomers, racemic isomers, cis-trans isomers, and tautomers of all compounds Isomers, geometric isomers, epimers and mixtures thereof are all included in the scope of the present invention.

Detailed ways

In order to make the purpose, technical solutions, and advantages of the present invention more clear, the following examples are given to further describe the present invention in detail. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art fall within the scope of protection of the present invention.

As used herein, room temperature refers to about 20-30°C; "overnight" refers to about 10h to 16h; 1M, 1N is 1mmol/L, 1μM is 1μmol/L, 1mM is 1mmol/L, 1nM is 1nmol/L; eq :equivalent;

Yield or productivity = mass of actual synthesized product/mass of theoretical synthesized product × 100%.

Example 1

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

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

Compound 141-1 (4.66g, 18.32mmol, 1.0eq) was dissolved in 1M HCl aqueous solution (23mL) at room temperature. After the temperature was raised to 100°C, the reaction was stirred for 16 hours under _N2 protection. After the reaction is completed, allow it to cool to room temperature naturally, wash the aqueous phase with ethyl acetate (100 mL The aqueous phase was adjusted to pH 4-6 with 1M HCl aqueous solution, and extracted with ethyl acetate (100 mL×5). The extracted organic phases were combined, washed with brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain light yellow solid compound 141-2 (4.52 g, yield 90.22%, [M+H] ⁺ : 274.17.

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

At room temperature, add ethanol (EtOH, 30 mL) and compound 141-2 (4.52 g, 16.46 mmol) into a 100 mL single-neck bottle, heat to 50°C, stir at 50°C for 0.5 hours, and slowly add S dropwise at 50°C. - A solution of phenylethylamine and ethanol (15 mL). After the addition is completed, the temperature is raised to 80°C and stirred for 1 hour. Cool slowly to room temperature and stir at room temperature for 12 hours. A white solid precipitated, filtered, and the solid was washed twice with ethanol (10 mL × 2). The white solid obtained was collected and added to 30 mL of ethanol, heated to 80°C, and allowed to dissolve. Stir at 80°C for 0.5 hours, gradually cool to room temperature, filter after the white solid precipitates, wash the filter cake twice with ethanol (10mL×2), collect the solid, and obtain white solid 141-3 (1.2g, e.e.%: 99.6%).

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

Dissolve compound 141-3 (1.2g) in 20mL of water, adjust the pH to >9 with 1M sodium hydroxide solution, wash with dichloromethane (50mL×3), and adjust the aqueous phase to pH=4~6 with 1N hydrochloric acid solution. Extract with dichloromethane (50 mL × 5), combine the extracted organic phases, wash with saturated brine (50 mL × 1), dry over anhydrous sodium sulfate, filter, and spin the filtrate to obtain light yellow oily compound 141-4 (600 mg).

Step 4: Nitrogen-methyl-nitrogen-methoxy-(R)-2-(9-(pyridin-2-yl)-6-oxaspiro[4.5]decene-9-yl)acetamide (141 -5) synthesis

At room temperature, compound 141-4 (600 mg, 2.2 mmol, 1.0 eq) was dissolved in dichloromethane (30 mL), and then methoxy (methyl)amine hydrochloride (256.93 mg, 2.63 mmol, 1.2 eq) was added sequentially. , 1-ethyl-3(3-dimethylpropylamine)carbodiimide) (EDCI) (631.22mg, 3.29mmol, 1.5eq) and 4-dimethylaminopyridine (DMAP) (26.82mg, 219.51μmol, 0.1eq), stir for 0.5h under _N2 protection, then add N,N-diisopropylethylamine (DIPEA) (851.17mg, 6.59mmol, 3.0eq) and stir the reaction overnight. After the reaction is completed, NH ₄ Cl saturated solution (100 mL) was added to the reaction solution to quench, and then extracted with dichloromethane (100 mL × 5). The organic phases were combined and washed with saturated brine (100 mL × 2), and anhydrous Na Dried over ₂ SO ₄ and filtered, the filtrate was concentrated under reduced pressure to obtain yellow thick substance 141-5 (592 mg, yield: 85.24%), [M+H] ⁺ : 317.17.

Step 5: Compound nitrogen-methyl-nitrogen-methoxy-(R)-2-(9-(pyridin-2-yl)-6-oxaspiro[4.5]decene-9-yl)acetaldehyde ( 141-6) synthesis

Compound 141-5 (438.6 mg, 1.39 mmol, 1.0 eq) was dissolved in toluene (Tol, 7 mL) at room temperature, and red aluminum (Red-Al) (657.9 mg) was slowly added dropwise at -40°C under N ₂ protection. , 2.28mmol, 1.05eq), stir and react for 4 hours after the addition is completed. After the reaction is completed, add a 10% mass fraction of citric acid solution (20 mL) to the reaction solution to quench it, then move to room temperature, add a mass fraction of 10% citric acid aqueous solution (10 mL) to the reaction solution, and then move to At room temperature, use 1 mol/L HCl solution to adjust pH=2~3, and extract with ethyl acetate (30mL×1); use 5N NaOH solution to adjust the aqueous phase to pH=11~13, and use methylene chloride (50mL×3) Extract, combine the organic phases and wash with saturated brine (50 mL × 1), dry with anhydrous sodium sulfate, filter, and the filtrate is concentrated under reduced pressure to obtain orange-red thick substance 141-6 (287.2 mg), yield: 80.5%. [M+H] ⁺ :258.16.

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

Compound 141-6 (50 mg, 0.19 mmol) and compound 141-7 (37.2 mg, 0.19 mmol) were dissolved in dichloromethane (DCM) (3 mL) at room temperature, and then MgSO ₄ (116.9 mg, 0.97 mmol) was added. Under nitrogen protection, stir at room temperature for 12 h, then add sodium borohydride (22.1 mg, 0.58 mmol), stir for 2 h, then add methanol (1 mL), and stir for 0.5 h. Then filter through diatomaceous earth, and the filtrate is concentrated at room temperature, and purified by large plate (V _{petroleum ether (PE)} : V _{ethyl acetate (EA)} = 0:1) to obtain yellow thick substance 141 (35 mg, yield 41.7%). [M+H] ⁺ :433.40.

¹ H NMR (400MHz, CDCl ₃ ) δ 8.60-8.56 (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).

Example 2

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

Step 1: Nitrogen-methyl-nitrogen-methoxy-(R)-2-(4'-(pyridin-2-yl)tetrahydroxaspiro[bicyclo[3.1.0]hexane-3,2' -Synthesis of -pyran]-4'-acetamide (143-1)

Under an ice-water bath, compound 141-5 (590 mg, 1.86 mmol) was dissolved in DCM (15 mL). Under nitrogen protection, 2 mol/L dimethylzinc (3.7 mL, 7.46 mmol) and diiodomethane (5.0 g, 18.6 mmol), after the addition is completed, move to room temperature and stir for 16 hours. After the reaction, ethyl acetate (50 mL) was added to the reaction solution, and then washed with saturated NaHCO ₃ solution (20 mL × 2). The organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was spin-dried and subjected to column chromatography (V _PE : V _EA = 2:1) and purified to obtain yellow thick substance 143-1 (223 mg, yield 36.26%), [M+H] ⁺ : 331.19.

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

Compound 143-1 (223 mg, 0.68 mmol) was dissolved in tetrahydrofuran (THF) (10 mL) at room temperature, and red aluminum (335 mg, 70%, 1.16 mmol) was slowly added dropwise at -40°C under nitrogen protection. After completion, stir the reaction for 4 hours. After the reaction is completed, add a 10% citric acid aqueous solution (10 mL) to the reaction solution, then move to room temperature, adjust pH = 2 to 3 with 1 mol/L HCl solution, and use ethyl acetate (30 mL × 1) Extraction; the aqueous phase is then adjusted to pH=11~13 with 10% NaOH solution, extracted with dichloromethane (50mL×3), the organic phases are combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a brown oil. Product 143-2 (160 mg, yield 87.37%), [M+H] ⁺ : 272.23.

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

Dissolve compound 143-2 (50 mg, 0.18 mmol) and compound 141-7 (35 mg, 0.18 mmol) in DCM (3 mL) at room temperature, then add MgSO ₄ (111 mg, 0.92 mmol), and stir at room temperature under nitrogen protection. 12h, then add NaBH ₄ (21 mg, 0.55 mmol), and stir for 1 h, then add methanol (1 mL), and stir for 0.5 h. It was then filtered through diatomaceous earth, and the filtrate was concentrated at room temperature and purified using a large plate (V _PE : V _EA = 0:1) to obtain yellow viscous substance 143 (32 mg, yield 39.0%), [M+H] ⁺ : 447.43.

¹ H NMR (400MHz, CDCl ₃ ) δ 8.56-8.49 (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), 1.37-1.22(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).

Example 3

(1R,4R)-4-ethoxy-nitrogen-{2-[9-(pyridin-2-yl)-2,6-dioxaspiro[4.5]decane-9]ethyl}-1, Synthesis of 2,3,4-tetralin-1-amine (compound 156)

Dissolve compound 156-1 (50 mg, 0.19 mmol) and compound 141-7 (37.2 mg, 0.19 mmol) in DCM (3 mL) at room temperature, then add MgSO ₄ (116.9 mg, 0.97 mmol), and under nitrogen protection, Stir at room temperature for 12 h, then add sodium borohydride (22.1 mg, 0.58 mmol), stir for 2 h, then add methanol (1 mL), and stir for 0.5 h. It was then filtered through diatomaceous earth, and the filtrate was concentrated at room temperature and purified by large plate (V _PE : V _EA = 0:1) to obtain yellow thick substance 156 (35 mg, yield 41.7%), [M+H] ⁺ : 437.27.

¹ H NMR (400MHz, CDCl ₃ ) δ 8.60-8.56 (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).

Example 4

Nitrogen-(2-(9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-2-en-9-yl)ethyl)-2,3-dihydro-1hydro- Synthesis of inden-1-amine (compound 128)

Under nitrogen protection, compound A (100 mg, 0.39 mmol), compound 9 (76 mg, 0.58 mmol), and dichloroethane (DCE, 6 mL) were added to a one-neck bottle in sequence, and tetraisopropyl titanate (TIPT, TIPT, 1.5 mL), stirred at 60°C for 16 hours, then added sodium borohydride (44 mg, 1.2 mmol), and stirred at 60°C for 2 hours. After the reaction is completed, add 1.5 mL of water to quench, add 10 mL of dichloromethane, filter, and concentrate at room temperature through the column (V _{dichloromethane} : V _methanol = 10:1) to obtain yellow viscous compound 128 (20 mg, yield 14%) ), [M+H] ⁺ :375.3.

¹ H NMR (400MHz, CDCl ₃ ) δ8.58 (d, J=8.0Hz, 1H), 7.675 (t, J=8.0Hz, 1H), 7.32 (d, J=8.0Hz, 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).

Example 5

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

For the preparation method, refer to Example 4, and obtain yellow viscous compound 1 (yield 7%), [M+H] ⁺ : 389.3.

¹ H NMR (400MHz, CDCl ₃ ) 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), 1.66-1.62(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).

Comparative example 1

Nitrogen-((3-methoxythiophen-2-yl)methyl)-2-(4'-(pyridin-2-yl)tetrahydroxaspiro[bicyclo[3.1.0]hexane-3,2 Synthesis of '-pyran]-4'-yl)ethylamine (compound 91)

Under nitrogen protection, add 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) into a single-neck bottle in sequence, and stir at room temperature. At 16h, add sodium borohydride (42 mg, 1.1 mmol), stir for 10 minutes, add methanol (MeOH, 0.5 mL), and stir for 2 hours. After the reaction was completed, dichloromethane (10 mL) was added, filtered, concentrated at room temperature, and the crude product was purified by column chromatography (V _{dichloromethane} : V _methanol = 10:1) to obtain yellow viscous compound 91 (12.43 mg, yield 8.5%) ), [M+H] ⁺ :399.3.

¹ H NMR (400MHz, deuterated chloroform (CDCl ₃ )) δ8.53-8.52 (m, 1H), 7.62 (dt, J1=2.0Hz, J2=7.6Hz, 1H), 7.27 (d, J=8.0Hz 1H), 7.13-7.07(m, 2H), 6.77(d, J=5.2Hz 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).

Comparative example 2

Nitrogen-((3-methoxythiophen-2-yl)methyl)-2-(9-(pyridin-2-yl)-6-oxaspiro[4.5]decane-2-ene-9- Synthesis of ethylamine (compound 83)

Under nitrogen protection, add 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) into a single-neck bottle in sequence, and stir at room temperature for 16 hours. Add sodium borohydride (35 mg, 0.93 mmol), stir for 10 minutes, add methanol (0.5 mL), and stir for 2 hours. After the reaction was completed, dichloromethane (10 mL) was added, filtered, concentrated at room temperature, and the crude product was purified by column chromatography (V _{dichloromethane} : V _methanol = 10:1) to obtain yellow thick compound 83 (35 mg, yield 29%) , [M+H] ⁺ :385.3.

¹ H NMR (400MHz, CDCl ₃ ) 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).

Comparative example 3

Nitrogen-((3-methoxythiophen-2-yl)methyl)-2-(9-(pyridin-2-yl)-2,6-dioxaspiro[4.5]decan-9-yl) Synthesis of ethylamine (compound 31)

At room temperature, Intermediate C (50 mg, 0.162 mmol) was dissolved in dichloromethane (3 mL), and then sodium sulfate (136 mg, 0.96 mmol) and Intermediate 1 (41 mg, 0.288 mmol) were added sequentially. The reaction was carried out overnight at room temperature under nitrogen protection. After reacting for 16 hours, add sodium borohydride and continue stirring for 30 minutes. After the reaction is complete, quench with 15 mL of water, add ethyl acetate (15 × 2 mL) for extraction, and then wash with saturated sodium carbonate solution (10 mL × 2). The organic phase is dried over anhydrous sodium sulfate, filtered, and the crude product is subjected to column layer After analytical purification (V _{petroleum ether} : V _{ethyl acetate} = 1:1), light yellow oily liquid 31 (20 mg, yield 22%) was obtained, [M+H] ⁺ : 389.2.

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).

biological evaluation

Op-Mu agonist cAMP test experiment

Compounds of the invention can activate μ-opioid receptors (MOR). Activated MOR can regulate the level of cAMP in cells. cAMP enters the nucleus and binds to the cAMP response element (CRE) region of the reporter gene luciferase (Luciferase), initiating the expression of the reporter gene. Luciferase reacts with its substrate to emit fluorescence, and the agonistic activity of the compound is reflected by measuring the fluorescence signal.

experimental method

The activity of the compounds of the Examples in agonizing MOR and affecting changes in downstream cAMP levels was tested by the following method.

1.Materials and reagents

2. Experimental steps

Detection buffer: 1×stimulation buffer, 500 μM 1-methyl-3-isobutylxanthine (IBMX), ddH ₂ O.

Compound preparation: Dissolve the compound in dimethyl sulfoxide (DMSO) and prepare a stock solution with a final concentration of 10mM. Dilute it to a working concentration of 0.08mM. Use an Echo pipette to perform a 4-fold gradient dilution of the compound. The initial concentration is 0.08mM. , 10 concentration gradients, and add 50nL to the 384 cell plate respectively, in double wells, with a final concentration of 0.4μM, and then centrifuge the cell plate at 1000rpm for 1 minute. Use an Echo pipette to transfer 50 nL of Forskolin (final concentration is 1 μM) into the 384 cell plate.

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 with detection buffer, and adjust the cell density to 5.0× ¹⁰⁵ cells/ mL, add to 384-well plate, 10 μL per well, 5000 cells. Shake for 20 s, centrifuge at 1000 rpm for 1 min, and place the cell plate in a 23°C incubator for 60 min.

Preparation of the standard curve: Use the detection buffer to perform a 4-fold gradient dilution of the standard adenosine-3',5'-cyclic phosphate (cAMP), with a total of 8 concentration points. The highest concentration is 800nM. Follow the microplate layout diagram for each step. Add 10 μL to the well.

Preparation of detection reagents: Dilute Anti cAMP-Cryptate and AMP-d2 to 1× with lysis buffer, add 10 μL detection reagent to each well according to the microplate layout, shake for 20 s, centrifuge at 1000 rpm for 1 min, and place the cell plate at 23°C. Incubate in the incubator for 60 minutes; read the plate on an Envision microplate reader.

3. Result analysis

Use Microsoft Excel software to calculate the activity percentage. For agonists, use the formula %Effect=100×(Sample Raw Value-Low Control Average)/(High Control Average-Low Control Average). Use GraphPad Prism 5 data analysis software. For agonists, use Dose-response-Stimulation—log[agonist]vs.response—Variable slope mode was used for fitting analysis to obtain the EC ₅₀ value of each test sample.

The changes in downstream cAMP levels caused by the compounds of the present invention stimulating MOR were measured through the above experiments. The experimental results show that this series of compounds exhibit strong Op-Mu agonistic effects. The measured EC ₅₀ values of typical representative compounds are shown in Table I. Among them, 6 groups of controls were set up, namely compound 83, compound 91, compound 31, TRV130, TRV130 (racemic) and SHR8554, among which TRV130, TRV130 (racemic) and SHR8554 respectively 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 is the maximum potency of the compound to cause changes in cAMP levels.

Table 1: EC ₅₀ and E _max of test compounds affecting cAMP levels at MOR receptors

The preferred compounds in the embodiments of the present invention have obvious agonistic effects on Mu opioid receptors, and the EC ₅₀ values and E _max of some compounds are much better than those of the control group.

Op-Kappa agonist cAMP test experiment

ForsKolin (forskolin) can stimulate the release of cAMP from human K opioid receptor high-expressing cell line-OPRKI cells (DiscoveRx), while K opioid receptor agonists can inhibit the cAMP release stimulated by forsKolin. By detecting the inhibitory effect of the test compound on forsKolin-stimulated cAMP release, the agonistic activity of the compound on the human K-sheet receptor can be determined. First, a certain concentration of forsKolin and different concentrations of test compounds are used to incubate with human high-expression K opioid receptor cell lines. A time-resolved fluorescence resonance energy transfer (TR-FRET)-based cAMP immunoassay (LANCEPerKinElmer) was used to determine cAMP levels in stimulated OPRK1 cells. The specific method is as follows:

Assay buffer: 1×stimulation buffer, 500μM IBMX, ddH ₂ O. Compound preparation: Dissolve the compound in DMSO and prepare a stock solution with a final concentration of 10mM, dilute it to a working concentration of 2mM, use Echo to perform a 4-fold gradient dilution of the compound, with an initial concentration of 2mM, 10 concentration gradients, and add 50nL to 384. In the cell plate, double wells were prepared with a final concentration of 10 μM, and then the cell plate was centrifuged at 1000 rpm for 1 min; use Echo to transfer 50 nL ForsKolin (final concentration is 3 μM) to the 384 cell plate.

Cell plating: Thaw the frozen cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash the cells twice with HBSS buffer, resuspend the cells with detection buffer, and adjust the cell density to 3.0× ¹⁰⁵ cells/ mL, add to 384-well plate, 10 μL per well, 3000 cells. Shake for 20 s, centrifuge at 1000 rpm for 1 min, and place the cell plate in a 23°C incubator for 60 min. Preparation of the standard curve: Use the detection buffer to perform a 4-fold gradient dilution of the standard cAMP, with a total of 8 concentration points. The highest concentration is 800nM. Add 10 μL to each well according to the microwell plate layout. Preparation of detection reagent: Dilute the intermediates Anti cAMP-Cryptate and AMP-d2 to 1× with lysis buffer, add 10uL detection reagent to each well, shake for 20s, centrifuge at 1000rpm for 1min, and place the cell plate in a 23°C incubator for 60min. . Finally read the plate on Envision.

Use Microsoft Excel software to calculate the activity percentage, and for agonists use the formula %Effect=100×(Sample Raw Value-Low Control Average)/(High Control Average-Low Control Average). Using GraphPad Prism 5 data analysis software, the Dose-response-Stimulation—log[agonist]vs.response—Variable slope mode was used for fitting analysis for agonists, and the EC ₅₀ value of each test sample was obtained. The experimental data are shown in Table 2.

Table 2: Effect of test compounds on K opioid receptors

The activity of the compounds of the present invention in stimulating K opioid receptors is significantly weaker than that of the control group; it shows that the compounds of the present invention have high selectivity for MOR receptors, and it is speculated that the compounds of the present invention have lower side effects.

Activity test experiment of β-arrestin signaling pathway of Mμ opioid receptor

This study aimed to evaluate the β-Arrestin recruitment efficiency of agonists targeting the μ-opioid receptor MOR through EC ₅₀ and EMAX assays of CHO-K1/Arrestin/hMOR. The CHO-K1/Arrestin/hMOR cell line expresses hMOR fused to the β-galactosidase donor fragment and β-Arrestin fused to the β-galactosidase receptor fragment. When β-arrestin interacts with hMOR, these fragments form active β-galactosidase. Prepare a 384-well plate, drop 60 nL/well of serially diluted compounds into the 384-well plate, and inject 20 μL of CHO-K1/Arrestin/hMOR cell suspension into the assay plate. The cell density is 7.5k cells/well. Incubate the analysis plate at 37°C with 0.5% CO ₂ (volume fraction, the rest is air) for 120 minutes. Add 10 μL/well detection reagent to the analysis plate using the dragonfly method. The culture dish is incubated at room temperature for 60 minutes. The chemiluminescence signal is detected by Envision. Use XLfit for data analysis. The experimental data are shown in Table 3.

Table 3: Effect of test compounds on β-arrestin signaling pathway

The compounds of the present invention have almost no activating effect on the β-arrestin signaling pathway, and the compounds of the present invention have better bias (cAMP and β-arrestin signaling pathways) compared with the control group. It is speculated that the compounds of the present invention have better effects than the control group. Lower side effects.

Test the blocking effect of the compound of the present invention on hERG potassium current

Test system

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

Cell culture medium and culture conditions: The complete medium is F12 medium, supplemented with 10% fetal bovine serum, 1% Selective antibiotic (G418), 89μg/mL hygromycin B (HB). The recovery medium is F12 medium supplemented with 10vol% fetal bovine serum. CHO-hERG cells were grown in a high-humidity incubator at 37°C (±2°C), 5% CO ₂ (4% to 8%). Cells were revived with recovery medium and passaged in complete medium. Cells used for patch-clamp experiments were replaced with recovery medium at the last passage.

Extracellular and intracellular fluid components:

experiment method

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

(2) Manual patch clamp test

hERG currents were recorded under whole-cell patch-clamp technique, and the recording temperature was room temperature. The output signal of the patch clamp amplifier is digital-to-analog conversion and 2.9KHz low-pass filtered. Data recording was collected using Patchmaster Pro software.

Cells were seeded in a cell recording tank and placed on the stage of an inverted microscope, and a cell in the recording tank was randomly selected for experiment. The perfusion system was fixed on the stage of an inverted microscope and continuously perfused with ECS.

Manual patch-clamp recording microelectrodes were prepared using capillary glass tubes filled with intracellular fluid. On the day of the patch clamp test, use borosilicate Glass tubes (BF150-117-10, SUTTER INSTRUMENT USA) were used to prepare electrodes. After the electrode is filled with ICS, the resistance is between 2-5MΩ.

The clamping voltage was -80mV, and the first step depolarized to +60mV and maintained open hERG channels for 850ms. Then, the voltage is set to -50mV and maintained for 1275ms, resulting in a rebound current or tail current, and the peak value of the tail current is measured and used for analysis. Finally, the voltage returns to the clamping voltage (-80mV). During the test, this command voltage program is repeated every 15 seconds.

At the beginning of recording the perfusion of the vehicle control working solution, monitor the tail current peak until more than 3 scanning curves stabilize, then the test article/positive control working solution to be tested can be perfused until the test article/positive control working solution changes the hERG current The inhibitory effect of the peak reaches a steady state. Generally, the basic overlap of the three most recent consecutive current curve peaks is used as the criterion for judging whether the state is stable. After reaching a stable state, continue to perfuse the next concentration of test sample. One or more test articles/positive controls, or multiple concentrations of the same drug, can be tested on one cell. Different test articles/positive controls need to be rinsed with vehicle control working solution until the hERG current returns to 80% before adding the drug. % or more in size. The standard deviation of the inhibition rate of each recorded cell at the same concentration does not exceed 15%.

The test concentration of positive control cisapride was 0.1 μM, and the assay was repeated for two cells. According to reports in the scientific literature, cisapride at 0.1 μM inhibits hERG currents by more than 50%. (Milnes, J.T., et al.).

(3) Manual patch clamp data acceptance criteria

Sealing standard: After the whole cell mode is formed, a clamping voltage (-80mV) is applied, and the cell membrane related parameters (Cm, Rm and Ra) can be recorded. A good whole-cell recording should meet the following conditions: path resistance (Rs) less than 10MΩ; membrane resistance (Rm) greater than 500MΩ and membrane capacitance (Cm) less than 100pF.

Current size: The peak current amplitude before the test article/positive control substance acts is between 400pA and 5000pA. Otherwise, discard the cell.

Leakage current: Under the 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. Scanning curves with an absolute value of leakage current greater than 200pA cannot be used for analysis.

data analysis

For each cell, the percent inhibition of each concentration of test article and positive control was calculated from the recorded current response using the following formula: (1 – Tail peak current recorded after test article/positive control perfusion/vehicle control perfusion Recorded tail peak current (initial current)) × 100%.

The percent inhibition of all cells recorded for each concentration was averaged, and the IC ₅₀ value (half inhibitory concentration) was derived from the concentration effect curve by Hill fitting.

test results

The specific inhibition results of some compounds of the present invention on hERG current are shown in Table 4 below;

Table 4: Inhibition results of hERG current by test compounds

Note: 20μM>IC ₅₀ >10μM is ++, 10μM>IC ₅₀ >1μM is +.

The compounds of the present invention have a higher hERG IC ₅₀ value than the control group, with a significant difference, and show a weaker inhibitory effect on hERG, indicating that the risk of cardiotoxicity of the compounds of the present invention is low.

Pharmacokinetic experiments

The compound to be studied is administered orally or intravenously in a single dose (vehicle 5vol% DMSO + 10vol% Solutol (HS-15) + 85vol% saline) to animals (such as mice, rats, dogs or monkeys), and is taken at fixed time points. Blood. Immediately after blood sample collection, gently invert the test tube at least 5 times to ensure thorough mixing and place on ice. The blood was anticoagulated with heparin and then centrifuged at 8000 rpm for 5 minutes to separate serum from red blood cells. Use a pipette to aspirate the serum and transfer it to a 2 mL polypropylene tube, mark the name of the compound and time point, and store it in a -40°C refrigerator until LC-MS analysis. When high concentration samples are diluted with blank plasma for measurement. After sample processing, the substances in the plasma were quantitatively analyzed using LCMS/MS. The plasma concentration/time curve obtained in this way was used to calculate pharmacokinetic parameters using a validated pharmacokinetic computer program. Experiments have found that the compounds of the present invention all have good pharmacokinetic properties.

After intravenous administration of doses in the five groups in Table 5 to SD male rats (each group was administered an equimolar dose, the vehicle was 5vol% DMSO + 10vol% Solutol (HS-15) + 85vol% saline, 3 rats in each group). Blood tests were taken at fixed time points. The pharmacokinetic parameters of prototype compounds of some compounds of the present invention in rat plasma are as follows in Table 5; in Table 5, IV refers to intravenous administration, AUC is the area under the plasma concentration-time curve, and C _max is the maximum plasma concentration. , T _1/2 is the elimination half-life, Variable is the variable, Mean is the mean, and SD is the standard deviation.

Table 5: Pharmacokinetic parameters of test compounds

The compounds of the present invention exhibit good pharmacokinetic properties in rats; compared with the control group, the AUC (h*ng/mL) of the free base of the compounds of the present invention in plasma is significantly increased.

Experiment on analgesic efficacy of hot plate method in rats

For female SD rats, the date the rat started training was recorded as D0. At D0, set the temperature of the pain meter to 52°C (52.0±0.5°C), place the rat on the hot plate and time it at the same time, and record the time (s) it takes for the rat to reach the pain threshold when licking its hind paws or jumping. If the rat does not show heat pain response on the hot plate for more than 30 s, the rat is taken out immediately, and the pain threshold is recorded as 30 s. Eliminate sensitive and unresponsive rats. On D1, take pre-screened rats, set the temperature of the pain meter to 52°C (52.0±0.5°C), place the rats on the hot plate and time, and record the pain threshold when the rats lick their hind paws or jump. time (s), measured three times in total. The average value of three times was used as the baseline pain threshold of rats. On D1, rats were randomly divided into groups according to the baseline pain threshold, with 8 animals in each group. On D2, the vehicle or compound was administered via tail vein injection according to each dose group in the table (equal molar doses were administered in each group, The solvent is 5vol% DMSO + 10vol% Solutol (HS-15) + 85vol% saline). On D2, 0.5min, 0.5h, 1h, and 3h after the administration of rats in each group, the temperature of the pain meter was set to 52°C (52.0±0.5°C). The rats were placed on the hot plate and timed, and the appearance of the rats was recorded. The time (s) it takes to reach the pain threshold when licking the hind paw or jumping, measured once at each time point. If the rat does not show heat pain response on the hot plate for more than 30 s, the rat is taken out immediately, and the pain threshold is recorded as 30 s. Calculate the %MPE of the pain threshold at each time point in each group, and evaluate the analgesic efficacy of each test sample in vivo. The experimental data are expressed as Mean ± SEM, and the data between each group were subjected to analysis of variance (ANOVA) test (Two Way ANOVA or One-Way ANOVA) using GraphPad Prism. P<0.05 was considered to be significantly different. Maximum analgesic effect percentage (ie, pain threshold %MPE) = (pain threshold after administration - basic pain threshold) / (30 - basic pain threshold) × 100%. The experimental results are in Table 6 below; in Table 6, Variable is a variable, Average is the mean and SEM is the standard error.

Table 6: In vivo analgesic efficacy of test compounds

Note: ***p<0.001 compared with the control group (vehicle, IV)

The compounds of the embodiments of the present invention exhibit better analgesic effects than the control group. In the hot plate method analgesic efficacy study in rats, the compounds showed higher pain thresholds and longer lasting analgesic effects.

acute toxicity test

A single intravenous administration of the compound (vehicle 5vol% DMSO + 10vol% Solutol (HS-15) + 85vol% saline) was administered to SD rats (4-6 dose groups were set for each compound, 10 rats per dose group, half female) , conduct clinical observation after administration. Clinical observation was conducted twice on the first day and once a day starting from the second day for 14 consecutive days. Including behavioral observations, autonomous activities, nervous system behavior and death conditions, etc., the maximum tolerated dose (MTD value) and the median lethal dose (LD ₅₀ value) of the compound are obtained. The experimental results show that the MTD value and LD ₅₀ value of the compound of the present invention after a single intravenous administration in rats are significantly improved compared with the control group, indicating that the compound of the present invention has good safety.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

1. A compound represented by the formula (IV) or (V) or (VI), a solvate, stereoisomer, deuterated compound, or a pharmaceutically acceptable salt thereof,

   wherein ring B, ring C are each independently selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl;

   ring D is selected from cycloalkyl, heterocycloalkyl;

   R ⁴ 、R ⁵ each independently selected from H, deuterium atom, alkyl, oxo, alkoxy, hydroxy, halogen, cyano, alkynyl, alkenyl, - (CH) ₂ ) _g -O-3 to 12 membered heterocyclyl, - (CH) ₂ ) _g -O-3 to 12 membered cycloalkyl, - (CH) ₂ ) _g -3 to 12 membered cycloalkyl, - (CH) ₂ ) _g -3 to 12 membered heterocyclyl, 5 to 10 membered heteroaryl, 5 to 10 membered aryl, -S (=o) _f -C _1-6 Alkyl, -O-C _2-6 Alkynyl, -O-C _2-6 Alkenyl groups; wherein the heterocyclyl, heteroaryl, aryl, alkyl, alkynyl, alkenyl, alkoxy may optionally be further substituted with 1 to 3R ⁶ Substituted;

   the R is ⁶ Each independently selected from deuterium atoms, halogen, -OH, -C _1-6 Alkyl, -C _1-6 alkyl-O-C _1-6 Alkyl, -O-C _1-6 Alkyl, 3-to 6-membered cycloalkyl, -O-C _2-6 Alkynyl, -O-C _2-6 Alkenyl, -C _2-6 Alkynyl, -C _2-6 Alkenyl, amino, carboxylate, nitro, cyano, hydroxyalkyl, heterocyclyl, aryl, heteroaryl;

   the g is selected from 0, 1, 2, 3, 4, 5 and 6;

   Said f is selected from 0, 1, 2;

   p, q, L are each independently 0, 1, 2, 3 or 4;

   the heteroaryl, the heterocycloalkyl, or the heteroatoms on the heterocyclyl are each independently selected from O, S or N.
2. The compound, solvate, stereoisomer, deuterated compound, or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of formula (vii) or (viii) or (ix) or (x) or (xi) or (xii):
3. the compound, solvate, stereoisomer, deuterated compound, or pharmaceutically acceptable salt thereof according to claim 1 wherein the compound is selected from the group consisting of the following structures:
4. the compound, solvate, stereoisomer, deuterated compound, or pharmaceutically acceptable salt thereof according to claim 1 wherein the compound is selected from the group consisting of the following structures:
5. the compound, solvate, stereoisomer, deuterated compound, or pharmaceutically acceptable salt thereof according to claim 1 wherein the compound is selected from the group consisting of the following structures:

   wherein R is ⁴ Independently selected from deuterium atom, alkyl group, oxo group, alkoxy group, hydroxy group, halogen, cyano group, alkynyl group, alkenyl group, - (CH) ₂ ) _g -O-3 to 12 membered heterocyclyl, - (CH) ₂ ) _g -O-3 to 12 membered cycloalkyl, - (CH) ₂ ) _g -3 to12 membered cycloalkyl, - (CH) ₂ ) _g -3 to 12 membered heterocyclyl, 5 to 10 membered heteroaryl, 5 to 10 membered aryl, -S (=o) _f -C _1-6 Alkyl, -O-C _2-6 Alkynyl, -O-C _2-6 Alkenyl groups;

   q is selected from 1, 2, 3 or 4.
6. The compound, solvate, stereoisomer, deuterated compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of:
7. a pharmaceutical composition comprising a compound of any one of claims 1 to 5, a solvate, stereoisomer, deuterated compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
8. Use of a compound according to any one of claims 1 to 6, a solvate, stereoisomer, deuterated compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 7, for the manufacture of a medicament for the prevention and/or treatment of a disease associated with the modulation of the MOR receptor agonist.
9. The use according to claim 8, wherein said MOR receptor agonist mediated related disorders are selected from pain, immune dysfunction, inflammation, esophageal reflux, neurological and psychiatric disorders, urinary and reproductive disorders, cardiovascular disorders and respiratory disorders.
10. The use according to claim 9, wherein the pain is selected from postoperative pain, pain caused by cancer, neuropathic pain, traumatic pain and pain caused by inflammation.