Oxaspiro derivatives and uses thereof

Abstract

The application relates to an oxaspiro derivative and application thereof, and belongs to the technical field of organic compound synthesis and medical application. The oxaspiro derivative has the structure shown in formula I. The oxaspiro derivative is a G-protein biased dual-site MOR agonist, can effectively inhibit the change of the cAMP concentration in cells, then helps to relieve pain, has high analgesic activity, and cannot cause respiratory depression and gastrointestinal adverse reactions.

Description

Technical Field

[0001] This invention relates to an oxaspirocyclic derivative and its applications, belonging to the field of organic compound synthesis and pharmaceutical application technology. Background Technology

[0002] μ-opioid receptors (MORs) belong to the G protein-coupled receptor (GPCR) superfamily and are widely distributed in the central and peripheral nervous systems. They are the main pharmacological targets for the potent analgesic effects of opioids. However, traditional opioids (such as morphine and fentanyl), as balance agonists, can simultaneously activate both the G protein signaling pathway and the β-arrestin signaling pathway after binding to their receptors. According to the theory of functional selectivity, the G protein signaling pathway mainly mediates analgesic activity; while activation of the β-arrestin signaling pathway is generally considered to be closely related to receptor internalization, desensitization, respiratory depression, and gastrointestinal adverse reactions (such as constipation). Therefore, developing G protein-biased MOR agonists that can preferentially activate the G protein signaling pathway and minimize β-arrestin recruitment has become a core strategy for improving the therapeutic window and safety of opioids.

[0003] Oxylidene (TRV130), as the first approved G protein-biased MOR agonist, has initially validated the feasibility of this research direction. However, its pharmacological properties and clinical safety remain limited, failing to completely eliminate side effects such as respiratory depression, gastrointestinal reactions, and addiction. Furthermore, TRV130 carries dose-dependent safety risks: a cumulative daily dose exceeding 27 mg significantly increases the risk of QTc interval prolongation (suggesting hERG channel blockade); simultaneously, some patients have experienced abnormally elevated alanine aminotransferase (ALT) levels, indicating potential hepatotoxicity. These safety concerns directly limit the dosage flexibility and the achievement of maximum therapeutic efficacy in clinical administration of this drug.

[0004] Optimization studies for these compounds have primarily focused on structural modifications of their orthosteric sites within the receptor. However, relying solely on orthosteric site modifications presents a bottleneck in precisely regulating receptor conformation to achieve better signal bias, making it difficult to further separate therapeutic effects from adverse reactions. Existing research indicates that the sodium allosteric site of the μ-opioid receptor is a key allosteric site regulating receptor conformation and its signaling pathways, and targeting this site helps reduce the side effects of opioid drugs (Pharmacol Rev. 2019 Oct;71(4):571-595). However, current technologies lack a technical solution based on a G protein-biased agonist backbone to design a bitopic ligand that can extend to this sodium allosteric site, which limits further improvements in the safety of these compounds. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an oxaspirocyclic derivative and its applications.

[0006] This invention is achieved through the following technical solution:

[0007] The first aspect of the present invention provides an oxaspirocyclic derivative having the formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof;

[0008]

[0009] Wherein, ring A is selected from phenyl or heteroaryl, and the heteroaryl group is selected from one of pyrroleyl, furanyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, pyridyl, pyrazinyl, pyridazinyl, indolyl, isoydinolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinolinyl, isoquinolinyl, quinazolinyl, or quinoxalinyl.

[0010] R1 is selected from: (1) C substituted with halogens 1-6 Alkyl; (2) C 2-6 (3) A 5- or 6-membered heteroaryl group, wherein the heteroaryl group contains 1 to 3 heteroatoms selected from N, O or S, and the heteroaryl group is optionally substituted by one or more substituents independently selected from: amino, C 1-4 Alkyl, carbamoyl C 1-4 Alkyl group; (4) a 4- to 6-membered saturated heterocyclic group, wherein the saturated heterocyclic group contains 1 to 2 heteroatoms selected from nitrogen or oxygen atoms, and the saturated heterocyclic group is connected to the parent nucleus through its cyclic nitrogen atom; or (5) a group N(R) a (R) b );

[0011] R2 has the structure shown in general formula (a):

[0012]

[0013] Where n is selected from 1, 2 or 3;

[0014] R3 is selected from phenyl, 5-10 heteroaryl, 5-10 heterocyclic or 5-10 bicyclic heterocyclic, wherein each of the phenyl, heteroaryl, heterocyclic or bicyclic is independently optionally substituted by a halogen, C1-C6 alkyl or C1-C6 alkoxy group.

[0015] In R2, the methylene chain -(CH2) n One of the -CH2- units is optionally substituted with a hydroxyl, carbonyl, or amino group.

[0016] According to a preferred embodiment of the present invention, the group N(R) a (R) b In ), R a Selected from hydrogen or C 1-4 Alkyl; Rb is selected from: (1) C 1-6 Alkyl groups, which are selected from one or more hydroxyl groups, C 1-4 Alkoxy, amino, amino groups substituted with amino protecting groups, single C 1-4 Alkylamino, bis(C) 1-4 Alkylamino, carbamoyl, mono-C 1-4 Alkyl carbamoyl or bis-C 1-4 Substituents of alkylcarbamoyl groups; or (2) 4- to 6-membered saturated heterocyclic groups, optionally substituted with one or more groups selected from C 1-4 Alkyl, amino protecting groups, or oxo substituents are used for substitution.

[0017] According to a preferred embodiment of the present invention, in the general formula of the compound represented by Formula I,

[0018] Ring A is pyridinyl;

[0019] R1 is selected from one of the following groups: ethyl, propyl, (trifluoromethyl)vinyl, 2-aminopyrimidin-4-yl, 1-(2-amino-2-acetyl)-1H-pyrazol-4-yl, pyrrolidone-1-yl, (oxetane-3-yl)amino, (azacyclobutane-3-yl)amino, (1-(tert-butoxycarbonyl)azacyclobutane-3-yl)amino, (tetrahydro-2H-pyran-4-yl)amino, (tetrahydro-2H-thiaran-4-yl)amino. (-yl)amino, (piperidin-4-yl)amino, (1-methylpiperidin-4-yl)amino, (1-(tert-butoxycarbonyl)piperidin-4-yl)amino, (2-aminoethyl)amino, (2-(dimethylamino)ethyl)amino, (2-(tert-butoxycarbonyl)amino)ethyl)amino, (2-hydroxyethyl)amino, (2-methoxyethyl)amino, (carbamoylmethyl)amino, (N,N-dimethylcarbamoylmethyl)amino;

[0020] R2 is (2-methoxythiophene-3-yl)methyl.

[0021] The pharmaceutically acceptable salts described in this invention refer to salts that are pharmaceutically considered safe, effective, and suitable for use in pharmaceutical preparations.

[0022] According to a preferred embodiment of the present invention, the pharmaceutically acceptable salt is a salt formed with an inorganic acid or an organic acid, or a salt formed with an inorganic base or an organic base; wherein the inorganic acid is sulfuric acid, hydrochloric acid, phosphoric acid, or nitric acid; the organic acid is acetic acid, oxalic acid, citric acid, fumaric acid, or p-benzenesulfonic acid; the inorganic base is sodium hydroxide, potassium hydroxide, or ammonia; and the organic base is methylamine, ethylamine, or ethanolamine.

[0023] According to a preferred embodiment of the present invention, the oxaspirocyclic derivative represented by Formula I is selected from any of the following structural compounds:

[0024]

[0025]

[0026]

[0027] .

[0028] The preparation method of the above-mentioned oxaspirocyclic derivatives is selected from one of the following methods:

[0029] a. The preparation methods of compounds BH-002-19, BH-002-21, and BH-002-23 are as follows:

[0030] (1) Preparation of intermediate I-1

[0031]

[0032] Reagents and conditions: (i) Cyclopentanone, 75% sulfuric acid solution, 0℃ to room temperature; (ii) Dys-Martin oxidant, dichloromethane, 0℃ to room temperature; (iii) Methyltriphenylphosphine bromide, potassium tert-butoxide, tetrahydrofuran, 0℃ to room temperature;

[0033] (2) Preparation of BH-002-19, BH-002-21 and BH-002-23

[0034]

[0035] Reagents and conditions: (i) Methyl mercaptoacetate, sodium methoxide, dimethylaminoformamide, 70 °C; (ii) Potassium peroxymonosulfonate, methanol, room temperature; (iii) Intermediate I-1, potassium phosphate, (4,4'-di-tert-butyl-2,2'-bipyridine)bis[(2-pyridyl)phenyl]iridium; (iii) hexafluorophosphate, acetonitrile, 440 nm visible light, nitrogen, room temperature; (iv) Sodium hydroxide, methanol:water mixture (volume ratio 1:1), 45 °C. ℃; (v) Trichloroacetyl azide, copper acetate, 4-dimethylaminopyridine, acetonitrile, 80℃; (vi) Tetrabutylammonium fluoride, tetrahydrofuran, room temperature; (vii) 3-methoxy-2-thiophenecarboxaldehyde, acetic acid, sodium borohydride, dichloroethane, room temperature; (viii) Substituted unsaturated borate ester, tetra-triphenylphosphine palladium, sodium carbonate, a mixed solvent of 1,4-dioxane:water in a volume ratio of 10:1, 100 ℃, nitrogen;

[0036] R1 is 2-aminopyrimidin-4-yl, 1-(2-amino-2-acetyl)-1H-pyrazol-4-yl, or 1-(trifluoromethyl)vinyl.

[0037] b. The preparation methods and synthetic routes of compounds BH-002-25, BH-002-29, BH-002-31, BH-002-33, BH-002-35, BH-002-37, BH-002-41, BH-002-43, BH-002-45, BH-002-49, BH-002-51, and BH-002-53 are as follows:

[0038]

[0039] Reagents and conditions: (i) substituted amino groups, sodium tert-butoxide, tris(dibenzylacetone)palladium, 2-(dicyclohexylphosphine)-3,6-dimethoxy-2'-4'-6'-tri-1-propyl-11'-biphenyl, 1,4-dioxane, 100 °C, nitrogen;

[0040] R1 is selected from (oxetane-3-yl)amino, (aza-butane-3-yl)amino, (pyrrolidine-3-yl)amino, (piperidin-4-yl)amino, (1-methylpiperidin-4-yl)amino, (tetrahydro-2H-pyran-4-yl)amino, (tetrahydro-2H-thiaran-4-yl)amino, (2-aminoethyl)amino, (2-(dimethylamino)ethyl)amino, (2-hydroxyethyl)amino, (2-methoxyethyl)amino, (carbamoylmethyl)amino, or (N,N-dimethylcarbamoylmethyl)amino.

[0041] c. The preparation methods and synthetic routes of compounds BH-002-55 and BH-002-57 are as follows:

[0042]

[0043] Reagents and conditions: (i) Alkylboronic acid, potassium phosphate, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex, 1,4-dioxane, 100 °C, nitrogen;

[0044] R1 is ethyl and n-propyl.

[0045] d. A method for preparing compounds BH-002-27, BH-002-39, and BH-002-47, wherein the method comprises: removing the tert-butyloxycarbonyl protecting group from compounds BH-002-25, BH-002-37, and BH-002-45, respectively, to obtain the corresponding target compounds.

[0046] e. The preparation methods and synthetic routes of compounds BH-002-18, BH-002-20, and BH-002-22 are as follows:

[0047]

[0048] Reagents and conditions: (i) Thiourea, 25 wt% hydrochloric acid solution, 140 ℃; (ii) Methyl chloroacetate, potassium carbonate, acetone, room temperature; (iii) Potassium peroxymonosulfonate, methanol, room temperature; (iv) Intermediate I-1, potassium phosphate, (4,4'-di-tert-butyl-2,2'-bipyridine)bis[(2-pyridyl)phenyl]iridium; (III) hexafluorophosphate, acetonitrile, 440 nm visible light, nitrogen, room temperature; (v) Sodium hydroxide, methanol:water mixed solvent = 1:1, 45 ℃; (vi) Trichloroacetyl azide, copper acetate, 4-dimethylaminopyridine, acetonitrile, 80 ℃; (vii) Tetrabutylammonium fluoride, tetrahydrofuran, room temperature; (viii) 3-Methoxy-2-thiophenecarboxaldehyde, acetic acid, sodium borohydride, dichloroethane, room temperature; (ix) Substituted unsaturated borate ester, tetra-triphenylphosphine palladium, sodium carbonate, a mixed solvent of 1,4-dioxane:water in a volume ratio of 10:1, 100 °C, nitrogen;

[0049] R1 is 2-aminopyrimidin-4-yl, 1-(2-amino-2-acetyl)-1H-pyrazol-4-yl, and 1-(trifluoromethyl)vinyl.

[0050] f. The preparation methods and synthetic routes for compounds BH-002-24, BH-002-28, BH-002-30, BH-002-32, BH-002-34, BH-002-36, BH-002-40, BH-002-42, BH-002-44, BH-002-48, BH-002-50, and BH-002-52 are as follows:

[0051]

[0052] Reagents and conditions: (i) substituted amino groups, sodium tert-butoxide, tris(dibenzylacetone)palladium, 2-(dicyclohexylphosphine)-3,6-dimethoxy-2'-4'-6'-tri-1-propyl-11'-biphenyl, 1,4-dioxane, 100 °C, nitrogen;

[0053] R1 is (oxetane-3-yl)amino, (aza-butane-3-yl)amino, (pyrrolidine-3-yl)amino, (piperidin-4-yl)amino, (1-methylpiperidin-4-yl)amino, (tetrahydro-2H-pyran-4-yl)amino, (tetrahydro-2H-thiaran-4-yl)amino, (2-aminoethyl)amino, (2-(dimethylamino)ethyl)amino, (2-hydroxyethyl)amino, (2-methoxyethyl)amino, (carbamoylmethyl)amino, or (N,N-dimethylcarbamoylmethyl)amino.

[0054] The preparation methods and synthetic routes for compounds g, BH-002-54, and BH-002-56 are as follows:

[0055]

[0056] Reagents and conditions: (i) alkylboronic acid, potassium phosphate, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex, 1,4-dioxane, 100 °C, nitrogen;

[0057] R1 is ethyl or n-propyl.

[0058] h, a method for preparing compounds BH-002-26, BH-002-38, and BH-002-46, the method comprising: removing the tert-butyloxycarbonyl protecting group from compounds BH-002-24, BH-002-36, and BH-002-44 respectively to obtain the corresponding target compounds.

[0059] The room temperature described in this invention is 20-30 ℃.

[0060] The isomers of compound BH-002-42 were obtained by chiral analysis and preparation.

[0061] A second aspect of the invention provides a pharmaceutical composition comprising an oxaspirocyclic derivative having the form of Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0062] The third aspect of the invention provides the use of the oxaspirocyclic derivative of Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the second aspect of the invention, in the preparation of a medicament for the prevention and / or treatment of μ-opioid receptor agonist-mediated diseases.

[0063] According to a preferred embodiment of the present invention, the related diseases mediated by the μ-opioid receptor agonist are selected from pain, inflammatory diseases, gastroesophageal reflux disease, neurological and psychiatric disorders, respiratory diseases, or diarrhea.

[0064] According to a preferred embodiment of the present invention, the pain is selected from postoperative pain, cancer-related pain, neuropathic pain, traumatic pain, migraine, and pain caused by inflammation.

[0065] A fourth aspect of the present invention provides a G-protein biased two-site MOR agonist comprising the oxaspirocyclic derivatives described above.

[0066] The beneficial effects of this invention are:

[0067] 1. The oxaspirocyclic derivatives of the present invention, as G-protein biased dual-site MOR agonists, can effectively inhibit changes in intracellular cAMP concentration, thereby helping to relieve pain and can be used as analgesics.

[0068] 2. The oxaspirocyclic derivatives of the present invention are novel G-protein biased agonists.

[0069] 3. The oxaspirocyclic derivatives of the present invention have high analgesic activity and do not cause respiratory depression or gastrointestinal adverse reactions. Attached Figure Description

[0070] Figure 1 This is a graph showing the analgesic effect of the compound in a mouse model of acute heat pain.

[0071] Figure 2 This is a graph showing the analgesic effect of the compound in a mouse model of acute inflammatory pain.

[0072] Figure 3 These are the results of a mouse small intestinal transport inhibition experiment for some of the compounds;

[0073] Figure 4 These are the results of respiratory inhibition experiments on some of the compounds. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0075] The control drugs were oxalidin (TRV130), fentanyl, and morphine, all commercially available products.

[0076] Example 1: Preparation of 9-methylene-6-oxaspiro[4.5]decane (intermediate I-1)

[0077]

[0078] Intermediate I-1

[0079] 3-Buten-1-ol (41.63 mmol) was dissolved in water, followed by the addition of cyclopentanone (62.45 mmol). 9 mL of 75% concentrated sulfuric acid solution was slowly added at 0 °C. After 15 min, the reaction was allowed to return to room temperature. The reaction was monitored by TLC after 10 h. After the reaction was complete, the pH was adjusted to 7 with 2 M NaOH solution, and the mixture was extracted with dichloromethane (45 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 1, a colorless oil in 20% yield. 1HNMR (400 MHz, CDCl3) δ 3.93 – 3.74 (m, 2H), 3.53 (td, J = 12.0, 2.6 Hz, 1H), 1.90 – 1.77 (m, 4H), 1.75 – 1.66 (m, 2H), 1.64 – 1.56 (m, 3H), 1.54 – 1.34(m, 3H).

[0080] Compound 1 (3.57 mmol) was dissolved in 5 mL of dichloromethane, and Dysmart reagent (5.35 mmol) was slowly added at 0 °C. After 15 min, the reaction was brought to room temperature, and the reaction was monitored by TLC after 4 h. After the reaction was complete, the reaction was quenched with saturated sodium bicarbonate solution and saturated sodium thiosulfate solution, extracted with dichloromethane (20 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 2, a colorless oil in 53% yield. 1 HNMR (400 MHz, CDCl3) δ 3.95 (t, J = 6.1 Hz, 2H), 2.47 (s, 2H), 2.43 (t, J =6.1 Hz, 2H), 1.91 – 1.82 (m, 2H), 1.80 – 1.69 (m, 2H), 1.62 (m, 2H), 1.56 –1.48 (m, 2H).

[0081] Triphenylmethylphosphine bromide (4.67 mmol) was dissolved in 10 mL of ultra-dry tetrahydrofuran, and potassium tert-butoxide (4.67 mmol) was added at 0 °C. After 15 min, an ultra-dry tetrahydrofuran solution of compound 2 (3.11 mmol) was added. The reaction was brought back to room temperature after 30 min. The reaction was monitored by TLC after 6 h. After the reaction was completed, the reaction was quenched with water, extracted with dichloromethane (20 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give intermediate I-1, a colorless oil, with a yield of 40%. 1 H NMR (400 MHz, CDCl3) δ 4.74 (s,1H), 4.68 (s, 1H), 3.67 (t, J = 5.7 Hz, 2H), 2.25 – 2.16 (m, 4H), 1.81 – 1.64(m, 4H), 1.61 – 1.46 (m, 4H).

[0082] Example 2: Preparation of 2-(4-(6-(4-(2-((((3-methoxythiophene-2-yl)methyl)amino)ethyl)-1-oxaspiro[5.5]undecane-4-yl)pyridin-2-yl)-1H-pyrazol-1-yl)acetamide (BH-002-19)

[0083]

[0084] BH-002-19

[0085] 10 mmol of 2,6-dibromopyridine was dissolved in 5 mL of N,N-dimethylformamide, followed by the addition of methyl mercaptoacetate (23 mmol) and sodium methoxide (25 mmol) in sequence. The reaction was carried out at 70 °C. The reaction was monitored by TLC after 8 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (30 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 5, a yellow oil, in 14% yield. 1 H NMR (400 MHz, Chloroform-d) δ 7.35 (t, J = 7.8 Hz, 1H), 7.20 – 7.18 (m, 1H), 7.18 – 7.16(m, 1H), 3.94 (s, 2H), 3.78 (s, 3H).

[0086] Compound 5 (1.96 mmol) was dissolved in 10 mL of methanol, followed by the addition of potassium peroxide monosulfonate (5.89 mmol), and the reaction was carried out at room temperature. The reaction was monitored by TLC after 16 h. After the reaction was complete, the solvent was removed by filtration and concentration under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 6 as a colorless oil in 87% yield. 1 H NMR (400 MHz, Chloroform-d) δ 8.07 –8.04 (m, 1H), 7.87 – 7.82 (m, 1H), 7.77 – 7.74 (m, 1H), 4.49 (s, 2H), 3.72(s, 3H).

[0087] Compound 6 (0.4 mmol) was dissolved in 1 mL of acetonitrile. Intermediate I-1 (1.2 mmol) prepared in Example 1, potassium phosphate (1.2 mmol), and (4,4'-di-tert-butyl-2,2'-bipyridine)bis[(2-pyridyl)phenyl]iridium(III) hexafluorophosphate (4 μmol) were added sequentially under nitrogen protection at room temperature and visible light at 440 nm. The reaction was monitored by TLC after 48 h. After the reaction was complete, the solvent was removed under reduced pressure, quenched with water, and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 7, a colorless oil in 52% yield. 1 H NMR (400 MHz, Chloroform-d) δ 7.49 (t, J = 7.7 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 3.70 (ddd, J = 20.9, 10.2, 7.1Hz, 2H), 3.57 (s, 3H), 2.34 (ddd, J = 14.4, 7.6, 2.5 Hz, 2H), 2.16 (t, J =11.0 Hz, 1H), 2.06 – 2.01 (m, 1H), 1.84 (d, J = 6.3 Hz, 2H), 1.77 – 1.71 (m,1H), 1.66 – 1.63 (m, 2H), 1.54 – 1.42 (m, 5H), 1.17 – 1.09 (m, 1H), 0.72 (dt,J = 12.7, 8.5 Hz, 1H).

[0088] Compound 7 (2.67 mmol) was dissolved in 5 mL of methanol solution, followed by an equal volume of water, and then sodium hydroxide (8 mmol). The reaction was carried out at 45 °C. The reaction was monitored by TLC after 5 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. The pH was then adjusted to 3 with 1 M HCl solution, and the mixture was extracted with ethyl acetate (25 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude compound 8.

[0089] The crude compound 8 (approximately 2.66 mmol) was dissolved in 5 mL of acetonitrile, followed by the sequential addition of trichloroacetyl azide (3.19 mmol), copper acetate (27 μmol), and 4-dimethylaminopyridine (27 μmol), and reacted at 80 °C. The reaction was monitored by TLC after 8 h. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by silica gel column chromatography to give compound 9, a colorless oil, in 51% yield. 1 H NMR (400 MHz, Chloroform-d) δ 7.44 (t, J = 7.8 Hz, 1H), 7.26 (d, J = 7.9 Hz, 1H), 7.19 (d, J = 7.0 Hz, 1H), 4.67 (d, J = 6.7 Hz, 1H), 4.57 (s, 2H), 3.65 (dt, J = 22.1, 8.0 Hz, 2H), 3.10 – 2.99 (m, 1H), 2.73 (dt,J = 13.5, 6.7 Hz, 1H), 2.30 (d, J = 13.8 Hz, 2H), 1.96 – 1.88 (m, 1H), 1.81(d, J = 13.7 Hz, 1H), 1.67 (td, J = 9.8, 5.7 Hz, 3H), 1.43 (p, J = 11.0, 9.2Hz, 4H), 1.09 (d, J = 13.1 Hz, 1H), 0.73 – 0.63 (m, 1H).

[0090] Compound 9 (1.36 mmol) was dissolved in 4 mL of tetrahydrofuran, followed by the addition of tetrabutylammonium fluoride (5.42 mmol) under nitrogen atmosphere, and the reaction was carried out at room temperature. The reaction was monitored by TLC after 4 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (15 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude compound 10.

[0091] The crude compound 10 (approximately 1.36 mmol) was dissolved in 3 mL of ultradry dichloroethane, followed by the addition of 2.71 mmol of 3-methoxy-2-thiophenecarboxaldehyde and 1.36 mmol of acetic acid. The reaction was carried out at room temperature. After 8 h, the reaction solution was concentrated under reduced pressure, 4 mL of methanol was added, and sodium borohydride (5.42 mmol) was added at 0 °C. The reaction was then brought back to room temperature. The reaction was monitored by TLC after 2 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (25 mL × 3), and the organic phases were combined. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give intermediate I-2, a pale yellow oil with a yield of 92%. 1 H NMR (400 MHz, Chloroform-d) δ 8.59 (d, J = 2.4 Hz, 1H), 7.72 (dd, J = 8.4, 2.4 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 5.5 Hz, 1H), 6.78 (d, J = 5.5 Hz, 1H), 3.78 (s, 3H), 3.75 – 3.64 (m, 4H), 2.48 (td, J = 11.1, 5.1 Hz, 1H), 2.38 (d,J = 13.8 Hz, 1H), 2.27 (d, J = 13.8 Hz, 1H), 2.08 (td, J = 11.2, 4.8 Hz, 1H), 1.91 (dd, J = 10.5, 5.6 Hz, 2H), 1.73 – 1.64 (m, 9H), 1.48 (p, J = 8.8, 7.5Hz, 3H), 1.14 (d, J = 14.6 Hz, 1H), 0.67 (dd, J = 13.4, 8.6 Hz, 1H).

[0092] Intermediate I-2 (107 μmol) was dissolved in 1 mL of 1,4-dioxane and 100 μL of water, followed by the sequential addition of 1-acetamido-4-boronate pinacol ester (161 μmol), potassium carbonate (215 μmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex (11 μmol). The reaction was purged with nitrogen and carried out at 100 °C. The reaction was monitored by TLC after 8 h. After the reaction was complete, the reaction was quenched with water, extracted with ethyl acetate (10 mL × 3), and the organic phases were combined. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by preparative thin-layer chromatography to obtain the target product BH-002-19, a yellow-brown oily substance with a yield of 60%. 1H NMR(400 MHz, Chloroform-d) δ 8.06 (s, 1H), 8.00 (s, 1H), 7.60 (t, J = 7.9 Hz,1H), 7.28 (s, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 5.4 Hz, 1H), 6.74(d, J = 5.4 Hz, 1H), 6.40 (s, 1H), 5.82 (s, 1H), 4.86 (s, 2H), 3.81 – 3.61(m, 7H), 2.57 (td, J = 11.0, 4.7 Hz, 1H), 2.49 (d, J = 13.5 Hz, 1H), 2.38 (d,J = 13.6 Hz, 1H), 2.24 – 2.13 (m, 2H), 2.06 – 1.93 (m, 1H), 1.90 (d, J = 13.6Hz, 1H), 1.81 – 1.58 (m, 4H), 1.56 – 1.42 (m, 3H), 1.37 – 1.33 (m, 1H), 1.15 – 1.11 (m, 1H), 0.74 – 0.61 (m, 1H). ESI-MS: m / z 510.52. [M+H] + C 27 H 35 N5O3S[509.25].

[0093] Example 3:

[0094] The preparation method is the same as described in Example 2, except that 2-aminopyrimidine-5-boronic acid pinacol ester is used instead of 1-acetamido-4-boronic acid pinacol ester to prepare BH-002-21.

[0095]

[0096] BH-002-21

[0097] Yellow oily substance, yield 87%. 1H NMR (400 MHz, Chloroform-d) δ 8.94 (s, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 7.01 (d, J = 5.2 Hz, 1H), 6.73 (d, J = 5.5 Hz, 1H), 5.43 – 5.36 (m, 2H), 3.79– 3.68 (m, 7H), 2.61 – 2.48 (m, 2H), 2.40 (d, J = 13.6 Hz, 1H), 2.22 – 2.11(m, 2H), 2.03 – 1.89 (m, 2H), 1.77 – 1.67 (m, 3H), 1.66 – 1.59 (m, 1H), 1.47 (s, 3H), 1.38 – 1.32 (m, 1H), 1.15 – 1.08 (m, 1H), 0.66 (s, 1H). ESI-MS: m / z 480.51. [M+H] + C 26 H 33 N5O2S [479.24].

[0098] Example 4:

[0099] The preparation method is the same as that described in Example 2, except that 1-(trifluoromethyl)ethylene borate hexanol ester is used instead of 1-acetamido-4-boronol ester to prepare BH-002-23.

[0100]

[0101] BH-002-23

[0102] Brown oily substance, yield 81%. 1H NMR (400 MHz, Chloroform-d) δ 7.69 – 7.60 (m,1H), 7.32 (t, J = 10.9 Hz, 2H), 7.10 (d, J = 9.5 Hz, 1H), 6.79 – 6.72 (m,1H), 6.52 (s, 2H), 6.08 (s, 1H), 3.87 – 3.80 (m, 3H), 3.76 – 3.71 (m, 4H), 2.63 – 2.57 (m, 1H), 2.47 (d, J = 13.1 Hz, 1H), 2.34 – 2.27 (m, 1H), 2.22 –2.18 (m, 1H), 2.08 – 2.03 (m, 1H), 1.93 – 1.85 (m, 2H), 1.71 – 1.61 (m, 4H), 1.50 – 1.42 (m, 4H), 1.09 – 1.03 (m, 1H), 0.62 – 0.54 (m, 1H). ESI-MS: m / z481.53. [M+H] + C 25 H 31 F3N2O2S [480.21].

[0103] Example 5: Preparation of tert-butyl(2-((6-(9-(2-((((3-methoxythiophene-2-yl)methyl)amino)ethyl)-6-oxaspiro[4.5]decane-9-yl)pyridin-2-yl)amino)ethyl)carbamate (BH-002-25)

[0104]

[0105] BH-002-25

[0106] Intermediate I-2 (107 μmol) obtained in Example 2 was dissolved in 1 mL of 1,4-dioxane, and N-tert-butoxycarbonyl-1,2-ethylenediamine (161 μmol), cesium carbonate (215 μmol), 2-(dicyclohexylphosphine)-3,6-dimethoxy-2'-4'-6'-tris-1-propyl-11'-biphenyl (21 μmol) and tris(dibenzylacetone)dipalladium (11 μmol) were added sequentially. The reaction was purged with nitrogen and carried out at 100 °C. The reaction was monitored by TLC after 8 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (10 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by preparative thin-layer chromatography to obtain the target product BH-002-25, a yellow oily substance, with a yield of 14%. 1H NMR (400 MHz, DMSO-d6) δ 7.32 – 7.25 (m,2H), 6.92 (d, J = 5.4 Hz, 1H), 6.86 (t, J = 5.7 Hz, 1H), 6.48 (d, J = 7.3 Hz,1H), 6.37 (t, J = 5.5 Hz, 1H), 6.23 (d, J = 8.1 Hz, 1H), 3.72 (s, 3H), 3.63 –3.57 (m, 4H), 3.29 – 3.19 (m, 2H), 3.12 – 3.05 (m, 2H), 2.46 – 2.40 (m, 1H),2.38 – 2.24 (m, 2H), 2.09 – 1.95 (m, 1H), 1.85 – 1.75 (m, 1H), 1.65 (d, J =13.5 Hz, 2H), 1.56 – 1.49 (m, 3H), 1.47 – 1.40 (m, 5H), 1.37 (s, 12H), 1.17 –1.10 (m, 1H), 0.86 – 0.79 (m, 1H). ESI-MS: m / z 545.83. [M+H] + C 29 H 44 N4O4S[544.31].

[0107] Example 6:

[0108] The preparation method is the same as that described in Example 5, except that 2-dimethylaminoethylamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-29.

[0109]

[0110] BH-002-29

[0111] Yellow oily substance, yield 35%. 1H NMR (400 MHz, Chloroform-d) δ 7.30 (t, J = 7.8Hz, 1H), 7.12 (d, J = 5.5 Hz, 1H), 6.77 (d, J = 5.5 Hz, 1H), 6.50 (d, J = 7.4Hz, 1H), 6.27 (d, J = 8.2 Hz, 1H), 5.54 (s, 1H), 3.91 – 3.81 (m, 2H), 3.79 (s, 3H), 3.77 – 3.67 (m, 2H), 3.61 – 3.43 (m, 2H), 2.88 – 2.74 (m, 2H), 2.64(td, J = 11.5, 4.9 Hz, 1H), 2.47 (s, 6H), 2.38 – 2.24 (m, 3H), 2.04 (td, J =12.4, 4.4 Hz, 1H), 1.82 – 1.68 (m, 3H), 1.66 – 1.55 (m, 2H), 1.53 – 1.44 (m,4H), 1.42 – 1.36 (m, 1H), 1.33 – 1.26 (m, 1H), 1.24 – 1.16 (m, 1H), 0.81 –0.72 (m, 1H). ESI-MS: m / z 474.06. [M+H] + C 26 H 40 N4O2S [472.29].

[0112] The preparation method is the same as that described in Example 5, except that glycineamide is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-31.

[0113]

[0114] BH-002-31

[0115] Yellow oily substance, yield 35%. 1H NMR (400 MHz, Chloroform-d) δ 7.31 – 7.22 (m,1H), 7.14 (d, J = 5.5 Hz, 1H), 6.68 (d, J = 5.5 Hz, 1H), 6.44 (d, J = 7.3 Hz,1H), 6.26 (d, J = 7.4 Hz, 1H), 5.44 (s, 1H), 4.06 – 3.92 (m, 4H), 3.70 (s,3H), 3.66 (d, J = 9.9 Hz, 2H), 3.03 (s, 3H), 2.95 (s, 3H), 2.81 – 2.72 (m,1H), 2.42 – 2.34 (m, 1H), 2.25 – 2.12 (m, 3H), 1.97 – 1.89 (m, 1H), 1.72 –1.63 (m, 3H), 1.58 – 1.50 (m, 3H), 1.43 – 1.36 (m, 3H), 1.15 – 1.11 (m, 1H),0.69 – 0.63 (m, 1H). ESI-MS: m / z 487.42. [M+H] + C 26 H 38 N4O3S [486.27].

[0116] The preparation method is the same as that described in Example 5, except that ethanolamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-33.

[0117]

[0118] BH-002-33

[0119] Yellow oily substance, yield 67%. 1H NMR (400 MHz, Chloroform-d) δ 7.34 (t, J = 7.8Hz, 1H), 7.07 (d, J = 5.5 Hz, 1H), 6.78 (d, J = 5.5 Hz, 1H), 6.55 (d, J = 7.5Hz, 1H), 6.27 (d, J = 8.2 Hz, 1H), 4.90 (t, J = 6.1 Hz, 1H), 3.85 – 3.68 (m,9H), 3.48 (t, J = 5.2 Hz, 2H), 3.34 (s, 1H), 2.66 – 2.55 (m, 1H), 2.35 – 2.22(m, 3H), 1.98 – 1.87 (m, 1H), 1.80 – 1.71 (m, 3H), 1.68 – 1.60 (m, 2H), 1.54 – 1.39 (m, 5H), 1.32 – 1.28 (m, 1H), 0.84 – 0.80 (m, 1H). ESI-MS: m / z 446.89.[M+H] + C 24 H 35 N3O3S [445.24].

[0120] The preparation method is the same as that described in Example 5, except that 2-methoxyethylamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-35.

[0121]

[0122] BH-002-35

[0123] Yellow oily substance, yield 63%. 1H NMR (400 MHz, Chloroform-d) δ 7.33 (t, J = 7.8Hz, 1H), 7.10 (d, J = 5.5 Hz, 1H), 6.78 (d, J = 5.5 Hz, 1H), 6.54 (d, J = 7.5Hz, 1H), 6.21 (d, J = 8.2 Hz, 1H), 4.73 (t, J = 5.7 Hz, 1H), 3.89 – 3.81 (m,2H), 3.79 (s, 3H), 3.77 – 3.67 (m, 2H), 3.59 (t, J = 5.3 Hz, 2H), 3.49 (t, J= 5.4 Hz, 2H), 3.39 (s, 3H), 2.64 (td, J = 11.2, 4.9 Hz, 1H), 2.37 – 2.25 (m,3H), 1.99 (td, J = 11.9, 11.3, 4.6 Hz, 1H), 1.82 – 1.70 (m, 3H), 1.66 – 1.56(m, 2H), 1.55 – 1.35 (m, 5H), 1.24 – 1.19 (m, 1H), 0.81 – 0.74 (m, 1H). ESI-MS: m / z 460.15. [M+H] + C 25 H 37 N3O3S [459.26].

[0124] The preparation method is the same as that described in Example 5, except that 1-tert-butoxycarbonyl-3-aminocyclobutane is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-37.

[0125]

[0126] BH-002-37

[0127] A brownish-yellow oily substance, yield 49%. 1H NMR (400 MHz, Chloroform-d) δ 7.35 (t, J =7.8 Hz, 1H), 7.12 (d, J = 5.2 Hz, 1H), 6.77 (d, J = 5.3 Hz, 1H), 6.60 (d, J =7.5 Hz, 1H), 6.18 (d, J = 7.9 Hz, 1H), 4.89 – 4.82 (m, 1H), 4.44 (d, J = 6.7Hz, 1H), 4.28 – 4.20 (m, 2H), 3.91 – 3.83 (m, 2H), 3.79 (s, 3H), 3.77 – 3.68(m, 4H), 2.68 – 2.59 (m, 1H), 2.32 – 2.22 (m, 3H), 2.04 – 1.95 (m, 1H), 1.82 – 1.68 (m, 4H), 1.65 – 1.55 (m, 3H), 1.49 – 1.40 (m, 12H), 1.24 – 1.14 (m,1H), 0.80 – 0.68 (m, 1H). ESI-MS: m / z 557.54. [M+H] + C 30 H 44 N4O4S [556.31].

[0128] The preparation method is the same as that described in Example 5, except that 3-oxacyclobutamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-41.

[0129]

[0130] BH-002-41

[0131] A light yellow oily substance, yield 59%. 1H NMR (400 MHz, Chloroform-d) δ 7.35 (t, J =7.9 Hz, 1H), 7.07 (d, J = 5.4 Hz, 1H), 6.77 (d, J = 5.5 Hz, 1H), 6.59 (d, J =7.5 Hz, 1H), 6.18 (d, J = 8.1 Hz, 1H), 4.98 – 4.82 (m, 4H), 4.54 (q, J = 5.7Hz, 2H), 3.79 (s, 3H), 3.76 (d, J = 6.4 Hz, 2H), 3.71 (d, J = 8.1 Hz, 2H), 3.10 (s, 1H), 2.55 (td, J = 11.4, 4.9 Hz, 1H), 2.34 – 2.25 (m, 2H), 2.19 (td,J = 11.3, 4.8 Hz, 1H), 1.92 (td, J = 11.9, 4.4 Hz, 1H), 1.78 (d, J = 13.6 Hz,1H), 1.74 – 1.57 (m, 4H), 1.54 – 1.45 (m, 3H), 1.43 – 1.36 (m, 1H), 1.20 –1.15 (m, 1H), 0.80 – 0.68 (m, 1H). ESI-MS: m / z 458.10. [M+H] + C 25 H 35 N3O3S[457.24].

[0132] The preparation method is the same as that described in Example 5, except that tetrahydropyrrole is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-43.

[0133]

[0134] BH-002-43

[0135] Yellow oily substance, yield 47%. 1H NMR (400 MHz, Chloroform-d) δ 7.34 (t, J = 7.9Hz, 1H), 7.06 (d, J = 5.6 Hz, 1H), 6.76 (d, J = 5.5 Hz, 1H), 6.48 (d, J = 7.4Hz, 1H), 6.12 (d, J = 8.3 Hz, 1H), 3.82 (d, J = 11.7 Hz, 1H), 3.77 (s, 3H), 3.76 – 3.68 (m, 3H), 3.39 (t, J = 6.5 Hz, 4H), 2.96 (s, 1H), 2.61 (td, J =11.1, 5.0 Hz, 1H), 2.39 (t, J = 13.7 Hz, 2H), 2.27 (td, J = 11.0, 5.0 Hz,1H), 1.97 (t, J = 6.2 Hz, 4H), 1.94 – 1.88 (m, 1H), 1.79 (d, J = 13.4 Hz,1H), 1.72 – 1.57 (m, 4H), 1.53 – 1.39 (m, 4H), 1.30 – 1.26 (m, 1H), 0.85 –0.77 (m, 1H). ESI-MS: m / z 456.57. [M+H] + C 26 H 37 N3O2S [455.26].

[0136] The preparation method is the same as that described in Example 5, except that 1-tert-butoxycarbonyl-4-aminopiperidine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-45.

[0137]

[0138] BH-002-45

[0139] Yellow oily substance, yield 37%. 1H NMR (400 MHz, Chloroform-d) δ 7.32 (td, J =8.0, 3.4 Hz, 1H), 7.10 (t, J = 4.7 Hz, 1H), 6.77 (t, J = 4.7 Hz, 1H), 6.55 –6.51 (m, 1H), 6.18 (dd, J 2.31 (t, J = 14.2 Hz, 3H), 2.07 – 1.96 (m, 4H), 1.81 – 1.70 (m, 3H), 1.63 –1.57 (m, 2H), 1.49 – 1.44 (m, 14H), 1.41 – 1.33 (m, 3H), 1.23 – 1.16 (m, 1H), 0.86 – 0.78 (m, 1H). ESI-MS: m / z 585.72. [M+H] + C 32 H 48 N4O4S [584.34].

[0140] The preparation method is the same as that described in Example 5, except that 4-amino-1-methylpiperidine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-49.

[0141]

[0142] BH-002-49

[0143] Yellow oily substance, yield 26%. 1H NMR (400 MHz, Chloroform-d) δ 7.30 (t, J = 6.7Hz, 1H), 7.06 (d, J = 5.5 Hz, 1H), 6.78 (d, J = 4.1 Hz, 1H), 6.52 (d, J = 6.3Hz, 1H), 6.18 (d, J = 7.1 Hz, 1H), 4.39 – 4.28 (m, 1H), 3.84 – 3.64 (m, 9H), 2.93 – 2.83 (m, 2H), 2.59 – 2.53 (m, 1H), 2.37 (s, 3H), 2.32 – 2.19 (m, 4H),2.10 – 2.01 (m, 2H), 1.90 (t, J = 16.9 Hz, 1H), 1.80 – 1.57 (m, 8H), 1.50 –1.41 (m, 4H), 1.13 – 1.03 (m, 1H), 0.81 – 0.69 (m, 1H). ESI-MS: m / z 499.47.[M+H] + C 28 H 42 N4O2S [498.30].

[0144] The preparation method is the same as that described in Example 5, except that 4-aminotetrahydropyran is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-51.

[0145]

[0146] BH-002-51

[0147] Yellowish-brown oily substance, yield 21%. 1H NMR (400 MHz, Chloroform-d) δ 7.32 (t, J =7.8 Hz, 1H), 7.06 (d, J = 5.5 Hz, 1H), 6.77 (d, J = 5.5 Hz, 1H), 6.54 (d, J =7.5 Hz, 1H), 6.18 (d, J = 8.2 Hz, 1H), 4.29 (d, J = 7.7 Hz, 1H), 4.00 (dt, J= 11.7, 3.6 Hz, 2H), 3.89 – 3.81 (m, 1H), 3.78 (s, 3H), 3.77 – 3.68 (m, 4H), 3.52 (td, J = 11.6, 2.3 Hz, 2H), 1.72 – 1.46 (m, 10H), 1.44 – 1.38 (m, 1H), 1.23 – 1.15 (m, 1H), 0.84– 0.76 (m, 1H). ESI-MS: m / z 486.50. [M+H] + C 27 H 39 N3O3S [485.27].

[0148] The preparation method is the same as that described in Example 5, except that tetrahydrothiaran-4-amine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-53.

[0149]

[0150] BH-002-53

[0151] Yellow oily substance, yield 41%. 1H NMR (400 MHz, Chloroform-d) δ 7.33 (dd, J =13.8, 6.1 Hz, 1H), 7.12 (dt, J = 14.8, 8.1 Hz, 1H), 6.79 (dd, J = 11.8, 5.5Hz, 1H), 6.54 (dd, J = 11.9, 7.5 Hz, 1H), 6.17 (dd, J = 12.0, 7.8 Hz, 1H), 4.45 – 4.32 (m, 1H), 3.91 – 3.61 (m, 9H), 2.81 – 2.61 (m, 5H), 2.38 – 2.21(m, 5H), 2.09 – 1.97 (m, 1H), 1.86 – 1.71 (m, 3H), 1.68 – 1.57 (m, 3H), 1.55 – 1.42 (m, 4H), 0.94 – 0.71 (m, 2H). ESI-MS: m / z 502.79. [M+H] + C 27 H 39 N3O2S2[501.25].

[0152] Example 7: Preparation of 2-(9-(6-ethylpyridin-2-yl)-6-oxaspiro[4.5]decane-9-yl)-N-((3-methoxythiophene-2-yl)methyl)ethane-1-amine (BH-002-55)

[0153]

[0154] BH-002-55

[0155] Intermediate I-2 (107 μmol) obtained in Example 2 was dissolved in 1 mL of 1,4-dioxane, followed by the sequential addition of ethylboric acid (161 μmol), potassium phosphate (537 μmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex (11 μmol). The reaction was purged with nitrogen and carried out at 100 °C. The reaction was monitored by TLC after 8 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (10 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by preparative thin-layer chromatography to obtain the target product BH-002-55, a yellow oily substance with a yield of 43%. 1H NMR (400 MHz, Chloroform-d) δ 7.51 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 5.5 Hz, 1H), 7.07 (d,J = 7.9 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 5.5 Hz, 1H), 3.96 –3.85 (m, 2H), 3.79 (s, 3H), 3.72 (dd, J = 6.8, 2.6 Hz, 2H), 2.78 – 2.63 (m,3H), 2.45 – 2.26 (m, 3H), 2.07 (td, J = 12.3, 11.6, 4.7 Hz, 1H), 1.94 – 1.80 (m, 2H), 1.77 – 1.56 (m, 4H), 1.51 – 1.41 (m, 3H), 1.24 (t, J = 7.5 Hz, 4H), 1.09 – 1.02 (m, 1H), 0.68 – 0.55 (m, 1H). ESI-MS: m / z 415.09. [M+H] + C 24 H 34 N2O2S [414.23].

[0156] Example 8

[0157] The preparation method is the same as that described in Example 7, except that n-propylboronic acid is used instead of ethylboronic acid to prepare BH-002-57.

[0158]

[0159] BH-002-57

[0160] A brownish-yellow oily substance, yield 37%. 1H NMR (400 MHz, Chloroform-d) δ 7.50 (t, J =7.8 Hz, 1H), 7.15 (d, J = 5.5 Hz, 1H), 7.08 (d, J = 7.9 Hz, 1H), 6.92 (d, J =7.5 Hz, 1H), 6.77 (d, J = 5.4 Hz, 1H), 3.97 – 3.84 (m, 2H), 3.80 (s, 3H), 3.73 (d, J = 6.5 Hz, 2H), 2.76 – 2.64 (m, 3H), 2.42 (d, J = 13.4 Hz, 1H), 2.38 – 2.25 (m, 2H), 2.09 (td, J = 11.9, 11.2, 4.4 Hz, 1H), 1.94 – 1.81 (m,2H), 1.71 (q, J = 7.7 Hz, 3H), 1.67 – 1.56 (m, 2H), 1.52 – 1.42 (m, 4H), 1.07– 1.02 (m, 1H), 0.93 (t, J = 7.4 Hz, 3H), 0.67 – 0.55 (m, 1H). ESI-MS: m / z429.19. [M+H] + C 25 H 36 N2O2S [428.25].

[0161] Example 9: Preparation of N-(6-(9-(2-(((3-methoxythiophene-2-yl)methyl)amino)ethyl)-6-oxaspiro[4.5]decane-9-yl)pyridin-2-yl)ethane-1,2-diamine (BH-002-27)

[0162]

[0163] BH-002-27

[0164] BH-002-25 (215 μmol) prepared in Example 5 was dissolved in 1,4-dioxane hydrochloride solution (4.0 M, 1 mL) at 0 °C and reacted at room temperature. The reaction was monitored by TLC after 1 h. After the reaction was complete, the pH of the reaction solution was adjusted to 7-8 with saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane (15 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by preparative thin-layer chromatography to obtain the target product BH-002-27, a yellow oily substance, with a yield of 8%. 1H NMR (400 MHz, DMSO-d6) δ 7.55 (d, J = 5.5 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.04 (d, J =5.6 Hz, 1H), 6.75 (t, J = 5.0 Hz, 1H), 6.57 (d, J = 7.4 Hz, 1H), 6.36 (d, J =8.2 Hz, 1H), 4.01 (s, 2H), 3.79 (s, 3H), 3.63 – 3.58 (m, 2H), 3.53 – 3.44 (m,2H), 3.02 – 2.95 (m, 2H), 2.75 – 2.66 (m, 1H), 2.38 – 2.23 (m, 3H), 2.03 –1.93 (m, 1H), 1.76 – 1.64 (m, 3H), 1.61 – 1.31 (m, 9H), 1.19 – 1.12 (m, 1H), 0.88 – 0.76 (m, 1H). ESI-MS: m / z 445.61.[M+H] + C 24 H 36 N4O2S [444.26].

[0165] Example 10

[0166] The preparation method is the same as that described in Example 9, except that BH-002-37 is used instead of BH-002-25 to obtain BH-002-39.

[0167]

[0168] BH-002-39

[0169] Yellow oily substance, yield 25%. 1H NMR (400 MHz, Chloroform-d) δ 7.34 (d, J = 5.5Hz, 1H), 6.84 – 6.77 (m, 2H), 6.52 – 6.45 (m, 2H), 4.87 – 4.77 (m, 1H), 4.45– 4.39 (m, 1H), 4.36 – 4.28 (m, 1H), 4.24 – 4.12 (m, 4H), 3.81 (s, 3H), 3.71 – 3.65 (m, 2H), 2.74 – 2.67 (m, 1H), 2.37 – 2.28 (m, 2H), 2.23 – 2.16 (m,1H), 2.11 – 2.03 (m, 1H), 1.98 – 1.91 (m, 1H), 1.75 – 1.66 (m, 3H), 1.62 –1.55 (m, 3H), 1.48 – 1.42 (m, 3H), 1.37 – 1.34 (m, 1H), 0.71 – 0.64 (m, 1H).ESI-MS: m / z 457.58. [M+H] + C 25 H 36 N4O2S [456.26].

[0170] The preparation method is the same as that described in Example 9, except that BH-002-45 is used instead of BH-002-25 to obtain BH-002-47.

[0171]

[0172] BH-002-47

[0173] Yellow oily substance, yield 53%. 1H NMR (400 MHz, Chloroform-d) δ 7.32 (d, J = 5.5Hz, 1H), 7.29 – 7.26 (m, 1H), 6.78 (d, J = 5.5 Hz, 1H), 6.44 (d, J = 7.5 Hz,1H), 6.40 (d, J = 8.2 Hz, 1H), 5.66 – 5.48 (m, 1H), 4.17 – 4.08 (m, 2H), 3.79 (s, 3H), 3.73 (t, J = 7.4 Hz, 2H), 3.24 – 3.08 (m, 2H), 2.82 – 2.71 (m, 1H),2.42 (t, J = 11.1 Hz, 1H), 2.32 (d, J = 13.2 Hz, 2H), 2.23 (d, J = 12.2 Hz, 2H), 2.12 (d, J = 13.3 Hz, 1H), 2.07 – 1.84 (m, 4H), 1.79 – 1.69 (m, 2H), 1.69 – 1.54 (m, 3H), 1.53 – 1.32 (m, 6H), 1.23 – 1.16 (m, 1H), 0.79 – 0.71(m, 1H). ESI-MS: m / z 485.72. [M+H] + C 27 H 40 N4O2S [484.29].

[0174] Example 11: Preparation of 2-(3-(6-(9-(2-((((3-methoxythiophene-2-yl)methyl)amino)ethyl)-6-oxaspiro[4.5]decane-9-yl)pyridin-3-yl)-1H-pyrazol-1-yl)acetamide (BH-002-18)

[0175]

[0176] BH-002-18

[0177] 2,5-Dibromopyridine (10 mmol) was added to 10 mL of 25 wt% hydrochloric acid solution, followed by thiourea (12 mmol), and the reaction was carried out at 140 °C. The reaction was monitored by TLC after 12 h. After the reaction was completed, 1 M hydrochloric acid solution was added to precipitate the product, which was then washed with n-hexane and dried to give crude compound 12 as a yellow solid.

[0178] The crude compound 12 (approximately 9 mmol) was dissolved in 15 mL of acetone, followed by the addition of potassium carbonate (9.90 mmol) and methyl chloroacetate (13.49 mmol). The reaction was carried out at room temperature. The reaction was monitored by TLC after 4 h. After the reaction was complete, the reaction solution was filtered and concentrated under reduced pressure to obtain the crude compound 13, a yellow oily substance. 1 H NMR (400 MHz, Chloroform-d) δ 8.45 (d, J = 2.4 Hz, 1H), 7.61 (dd, J = 8.5, 2.4 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.96 (s, 2H), 3.74 (s, 3H).

[0179] The crude compound 13 was dissolved in 10 mL of methanol, followed by the addition of potassium peroxymonosulfonate, and the reaction was carried out at room temperature. The reaction was monitored by TLC after 16 h. After the reaction was complete, the solvent was removed by filtration and concentration under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 14, a colorless oil, in 50% yield. 1 H NMR (400 MHz, Chloroform-d) δ 8.83 – 8.78 (m, 1H), 8.13(dd, J = 8.3, 2.2 Hz, 1H), 7.99 (dd, J = 8.3, 0.7 Hz, 1H), 4.47 (s, 2H), 3.71(s, 3H).

[0180] Compound 14 (0.4 mmol) was dissolved in 1 mL of acetonitrile. Intermediate I-1 (1.2 mmol), potassium phosphate (1.2 mmol), and (4,4'-di-tert-butyl-2,2'-bipyridine)bis[(2-pyridyl)phenyl]iridium(III) hexafluorophosphate (4 μmol) were added sequentially under nitrogen protection at room temperature and visible light at 440 nm. The reaction was monitored by TLC after 48 h. After the reaction was complete, the solvent was removed under reduced pressure, the mixture was quenched with water, and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 15 as a colorless oil in 39% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.61 (d, J = 2.5 Hz, 1H), 7.75 (dd, J =8.5, 2.4 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 3.79 – 3.63 (m, 2H), 3.56 (s,3H), 2.40 – 2.26 (m, 2H), 2.19 – 2.08 (m, 1H), 2.08 – 1.97 (m, 1H), 1.90 (d,J = 13.7 Hz, 1H), 1.86 – 1.69 (m, 3H), 1.63 (d, J = 9.2 Hz, 2H), 1.55 – 1.39(m, 4H), 1.12 (dd, J = 12.9, 8.2 Hz, 1H), 0.68 (dt, J = 13.2, 8.6 Hz, 1H).

[0181] Compound 15 (2.67 mmol) was dissolved in 5 mL of methanol solution, followed by an equal volume of water, and then sodium hydroxide (8 mmol). The reaction was carried out at 45 °C. The reaction was monitored by TLC after 5 h. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure, and the pH was adjusted to 3 with 1 M HCl solution. The mixture was extracted with ethyl acetate (25 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude compound 16.

[0182] The crude compound 16 (approximately 2.66 mmol) was dissolved in 5 mL of acetonitrile, followed by the sequential addition of trichloroacetyl azide (3.19 mmol), copper acetate (27 μmol), and 4-dimethylaminopyridine (27 μmol), and the reaction was carried out at 80 °C. The reaction was monitored by TLC after 8 h. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by silica gel column chromatography to give compound 17, a colorless oil, in 51% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.62 (d, J = 2.5 Hz, 1H), 7.77 (dd, J = 8.5, 2.5 Hz, 1H), 7.22 (d, J = 8.5 Hz, 1H), 4.77 – 4.54 (m,3H), 3.79 – 3.64 (m, 2H), 3.08 (td, J = 15.0, 5.6 Hz, 1H), 2.75 (td, J =14.6, 14.0, 5.8 Hz, 1H), 2.45 – 2.26 (m, 2H), 1.98 (ddd, J = 15.3, 9.7, 6.0Hz, 1H), 1.89 (d, J = 13.7 Hz, 1H), 1.75 – 1.63 (m, 4H), 1.53 – 1.40 (m, 4H), 1.12 (t, J = 9.8 Hz, 1H), 0.68 (dt, J = 13.6, 8.8 Hz, 1H).

[0183] Compound 17 (1.36 mmol) was dissolved in 4 mL of tetrahydrofuran, followed by the addition of tetrabutylammonium fluoride (5.42 mmol) under nitrogen atmosphere, and the reaction was carried out at room temperature. The reaction was monitored by TLC after 4 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (15 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude compound 18.

[0184] The crude compound 18 (approximately 1.36 mmol) was dissolved in 3 mL of ultradry dichloroethane, followed by the addition of 2.71 mmol of 3-methoxy-2-thiophenecarboxaldehyde and 1.36 mmol of acetic acid. The reaction was carried out at room temperature. After 8 h, the reaction solution was concentrated under reduced pressure, 4 mL of methanol was added, and sodium borohydride (5.42 mmol) was added at 0 °C. The reaction was then brought back to room temperature. The reaction was monitored by TLC after 2 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (25 mL × 3), and the organic phases were combined. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to give intermediate I-3, a pale yellow oil with a yield of 92%. 1H NMR (400 MHz, DMSO-d6) δ 8.64 (d, J = 2.2 Hz, 1H), 7.96 (ddd, J = 8.5, 3.7, 2.5 Hz, 1H), 7.45 (dd, J = 8.6, 2.0 Hz, 1H), 7.30 (d, J = 5.5 Hz, 1H), 6.92 (d, J = 5.5Hz, 1H), 3.72 (s, 3H), 3.62 – 3.45 (m, 4H), 2.37 – 2.27 (m, 3H), 2.02 – 1.91(m, 1H), 1.81 (d, J = 16.1 Hz, 2H), 1.66 – 1.60 (m, 2H), 1.53 (td, J = 11.4,8.7, 4.9 Hz, 2H), 1.45 – 1.39 (m, 3H), 1.38 – 1.33 (m, 2H), 1.02 – 0.95 (m,1H), 0.69 – 0.58 (m, 1H).

[0185] Intermediate I-3 (107 μmol) was dissolved in 1 mL of 1,4-dioxane and 100 μL of water, followed by the sequential addition of 1-acetamido-4-boronate pinacol ester (161 μmol), potassium carbonate (215 μmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane dichloride complex (11 μmol). The reaction was purged with nitrogen and carried out at 100 °C. The reaction was monitored by TLC after 8 h. After the reaction was complete, the reaction was quenched with water, extracted with ethyl acetate (10 mL × 3), and the organic phases were combined. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by preparative thin-layer chromatography to obtain the target product BH-002-18, a yellow oily substance with a yield of 64%. 1H NMR(400 MHz, Chloroform-d) δ 8.69 (d, J = 2.4 Hz, 1H), 7.89 (s, 1H), 7.79 (s,1H), 7.69 (dd, J = 8.2, 2.4 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.05 (d, J =5.5 Hz, 1H), 6.76 (d, J = 5.5 Hz, 1H), 6.33 (s, 1H), 5.70 (s, 1H), 4.88 (s,2H), 3.83 – 3.69 (m, 7H), 2.63 – 2.53 (m, 1H), 2.43 (d, J = 13.5 Hz, 1H), 2.32 (d, J = 13.6 Hz, 1H), 2.19 (td, J = 11.4, 5.4 Hz, 1H), 2.00 (td, J =11.9, 10.7, 4.8 Hz, 1H), 1.91 (d, J = 13.7 Hz, 1H), 1.85 – 1.57 (m, 5H), 1.57 – 1.46 (m, 3H), 1.40 – 1.34 (m, 1H), 1.20 – 1.11 (m, 1H), 0.77 – 0.65 (m,1H). ESI-MS: m / z 510.21. [M+H] + C 27 H 35 N5O3S [509.25].

[0186] Example 12

[0187] The preparation method is the same as that described in Example 11, except that 2-aminopyrimidine-5-boronic acid pinacol ester is used instead of 1-acetamido-4-boronic acid pinacol ester to prepare BH-002-20.

[0188]

[0189] BH-002-20

[0190] A pale yellow solid, with a yield of 87%. 1H NMR (400 MHz, Chloroform-d) δ 8.66 (d, J = 2.4Hz, 1H), 8.53 (s, 2H), 7.75 – 7.69 (m, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.08(d, J = 5.4 Hz, 1H), 6.75 (d, J = 5.5 Hz, 1H), 5.25 (s, 2H), 3.85 – 3.70 (m,7H), 2.70 – 2.56 (m, 1H), 2.43 (d, J = 13.5 Hz, 1H), 2.32 (d, J = 13.7 Hz,1H), 2.23 (d, J = 4.4 Hz, 1H), 2.11 – 1.99 (m, 1H), 1.97 – 1.86 (m, 2H), 1.76 – 1.59 (m, 4H), 1.57 – 1.46 (m, 3H), 1.45 – 1.36 (m, 1H), 1.19 – 1.10 (m,1H), 0.77 – 0.65 (m, 1H). ESI-MS: m / z 480.16. [M+H] + C 26 H 33 N5O2S [479.24].

[0191] The preparation method is the same as that described in Example 11, except that 1-(trifluoromethyl)ethylene borate hexanol ester is used instead of 1-acetamido-4-boronic acid pinacol ester to prepare BH-002-22.

[0192]

[0193] BH-002-22

[0194] Yellowish-brown oily substance, yield 39%. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 7.85(d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.28 (d, J = 5.6 Hz, 1H), 6.91(d, J = 5.5 Hz, 1H), 6.27 (s, 1H), 6.18 (s, 1H), 3.71 (s, 3H), 3.66 – 3.51(m, 4H), 2.44 – 2.28 (m, 3H), 2.01 – 1.89 (m, 1H), 1.89 – 1.75 (m, 2H), 1.71– 1.64 (m, 2H), 1.60 – 1.33 (m, 7H), 1.05 – 0.95 (m, 1H), 0.70 – 0.61 (m,1H). ESI-MS: m / z 481.56. [M+H] + C 25 H 31 F3N2O2S [480.21].

[0195] Example 13: Preparation of tert-butyl(2-((6-(9-(2-((((3-methoxythiophene-2-yl)methyl)amino)ethyl)-6-oxaspiro[4.5]decane-9-yl)pyridin-3-yl)amino)ethyl)carbamate (BH-002-24)

[0196]

[0197] BH-002-24

[0198] Intermediate I-3 (107 μmol) obtained in Example 11 was dissolved in 1 mL of 1,4-dioxane, and N-tert-butoxycarbonyl-1,2-ethylenediamine (161 μmol), cesium carbonate (215 μmol), 2-(dicyclohexylphosphine)-3,6-dimethoxy-2'-4'-6'-tris-1-propyl-11'-biphenyl (21 μmol) and tris(dibenzylacetone)dipalladium (11 μmol) were added sequentially. The reaction was purged with nitrogen and carried out at 100 °C. The reaction was monitored by TLC after 8 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (10 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by preparative thin-layer chromatography to obtain the target product BH-002-24, a yellow oily substance, with a yield of 38%. 1H NMR (400 MHz, Chloroform-d) δ 7.94 (s,1H), 7.13 (d, J = 5.5 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 6.87 (dd, J = 8.6,2.9 Hz, 1H), 6.80 (d, J = 5.5 Hz, 1H), 5.04 – 4.95 (m, 1H), 4.26 (s, 1H), 3.95 – 3.79 (m, 5H), 3.71 (d, J = 9.5 Hz, 2H), 3.44 – 3.37 (m, 2H), 3.28 –3.22 (m, 2H), 2.81 – 2.56 (m, 4H), 2.39 – 2.27 (m, 2H), 2.19 (d, J = 13.5 Hz,1H), 2.01 – 1.91 (m, 1H), 1.84 – 1.68 (m, 3H), 1.66 – 1.58 (m, 2H), 1.54 –1.40 (m, 14H), 1.20 – 1.14 (m, 1H), 0.75 – 0.63 (m, 1H). ESI-MS: m / z 545.57.[M+H] + C 29 H 44 N4O4S [544.31].

[0199] Example 14:

[0200] The preparation method is the same as that described in Example 13, except that 2-dimethylaminoethylamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-28.

[0201]

[0202] BH-002-28

[0203] Yellow oily substance, yield 30%. 1H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 2.9Hz, 1H), 7.20 (d, J = 5.5 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 6.91 (d, J = 8.6Hz, 1H), 6.80 (d, J = 5.5 Hz, 1H), 4.87 (s, 1H), 4.15 – 3.91 (m, 2H), 3.83 (s, 3H), 3.68 (dd, J = 8.0, 2.9 Hz, 2H), 3.29 – 3.24 (m, 2H), 2.88 – 2.75 (m,2H), 2.51 – 2.38 (m, 7H), 2.27 (d, J = 13.4 Hz, 1H), 2.13 (d, J = 13.5 Hz, 1H), 2.07 – 1.89 (m, 2H), 1.80 – 1.69 (m, 2H), 1.65 – 1.56 (m, 2H), 1.52 –1.31 (m, 6H), 1.16 – 1.07 (m, 1H), 0.71 – 0.58 (m, 1H). ESI-MS: m / z 473.54.[M+H] + C 26 H 40 N4O2S [472.29].

[0204] The preparation method is the same as that described in Example 13, except that glycineamide is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-30.

[0205]

[0206] BH-002-30

[0207] Yellow oily substance, yield 53%. 1H NMR (600 MHz, Chloroform-d) δ 7.97 (d, J = 2.9Hz, 1H), 7.10 – 7.04 (m, 2H), 6.87 (dd, J = 8.6, 2.9 Hz, 1H), 6.77 (d, J =5.4 Hz, 1H), 4.93 (t, J = 4.3 Hz, 1H), 3.87 (d, J = 4.2 Hz, 2H), 3.78 (s,3H), 3.77 – 3.70 (m, 4H), 3.05 (d, J = 2.6 Hz, 6H), 2.59 (td, J = 11.7, 10.4,5.5 Hz, 1H), 2.34 (d, J = 16.5 Hz, 1H), 2.25 – 2.20 (m, 2H), 2.15 (s, 1H), 1.91 (td, J = 12.1, 10.4, 5.2 Hz, 1H), 1.83 (d, J = 13.6 Hz, 1H), 1.76 – 1.69(m, 2H), 1.67 – 1.60 (m, 2H), 1.51 – 1.43 (m, 3H), 1.41 – 1.35 (m, 1H), 1.17 – 1.11 (m, 1H), 0.74 – 0.66 (m, 1H). ESI-MS: m / z 487.48. [M+H] + C 26 H 38 N4O3S[486.27].

[0208] The preparation method is the same as that described in Example 13, except that ethanolamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-32.

[0209]

[0210] BH-002-32

[0211] Yellow oily substance, yield 23%. 1H NMR (600 MHz, Chloroform-d) δ 8.00 (d, J = 2.9Hz, 1H), 7.09 – 7.04 (m, 2H), 6.89 (dd, J = 8.6, 2.9 Hz, 1H), 6.78 (d, J =5.5 Hz, 1H), 4.09 (s, 1H), 3.85 (t, J = 5.2 Hz, 2H), 3.79 (s, 3H), 3.77 –3.69 (m, 4H), 3.29 (t, J = 5.1 Hz, 2H), 2.57 (td, J = 10.9, 5.5 Hz, 1H), 2.45– 2.31 (m, 3H), 2.25 – 2.19 (m, 2H), 1.92 – 1.86 (m, 1H), 1.83 (d, J = 13.7Hz, 1H), 1.75 – 1.67 (m, 2H), 1.65 – 1.59 (m, 2H), 1.52 – 1.45 (m, 3H), 1.41– 1.36 (m, 1H), 1.19 – 1.14 (m, 1H), 0.76 – 0.68 (m, 1H). ESI-MS: m / z 446.53.[M+H] + C 24 H 35 N3O3S [445.24].

[0212] The preparation method is the same as that described in Example 13, except that 2-methoxyethylamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-34.

[0213]

[0214] BH-002-34

[0215] Yellow oily substance, yield 30%. 1H NMR (400 MHz, Chloroform-d) δ 7.98 (d, J = 2.9Hz, 1H), 7.07 (d, J = 6.1 Hz, 2H), 6.87 (dd, J = 8.6, 2.9 Hz, 1H), 6.77 (d, J= 5.5 Hz, 1H), 4.01 (s, 1H), 3.84 – 3.75 (m, 5H), 3.74 – 3.69 (m, 2H), 3.63(t, J = 5.1 Hz, 2H), 3.41 (s, 3H), 3.33 – 3.24 (m, 2H), 2.66 – 2.55 (m, 1H),2.34 (d, J = 12.9 Hz, 1H), 2.29 – 2.19 (m, 2H), 1.97 – 1.87 (m, 1H), 1.82 (d,J = 13.6 Hz, 1H), 1.77 – 1.70 (m, 2H), 1.67 – 1.59 (m, 2H), 1.53 – 1.37 (m,5H), 1.19 – 1.13 (m, 1H), 0.75 – 0.65 (m, 1H). ESI-MS: m / z 460.15. [M+H] + C 25 H 37 N3O3S [459.26].

[0216] The preparation method is the same as that described in Example 13, except that 1-tert-butoxycarbonyl-3-aminocyclobutane is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-36.

[0217]

[0218] BH-002-36

[0219] Yellow oily substance, yield 20%. 1H NMR (600 MHz, Chloroform-d) δ 7.89 (d, J = 2.9Hz, 1H), 7.07 (dd, J = 16.0, 7.0 Hz, 2H), 6.80 – 6.73 (m, 2H), 4.33 – 4.27(m, 2H), 4.21 – 4.13 (m, 1H), 4.04 (d, J = 6.6 Hz, 1H), 3.79 (s, 3H), 3.78 –3.74 (m, 3H), 3.73 – 3.69 (m, 3H), 2.55 (td, J = 10.9, 5.2 Hz, 1H), 2.36 –2.31 (m, 1H), 2.25 – 2.16 (m, 2H), 1.94 – 1.86 (m, 1H), 1.84 (d, J = 13.7 Hz,1H), 1.76 – 1.69 (m, 2H), 1.66 – 1.60 (m, 2H), 1.53 – 1.42 (m, 15H), 1.41 –1.36 (m, 1H), 1.28 – 1.24 (m, 1H), 1.17 – 1.12 (m, 1H), 0.74 – 0.66 (m, 1H).ESI-MS: m / z 557.68. [M+H] + C 30 H 44 N4O4S [556.31].

[0220] The preparation method is the same as that described in Example 13, except that 3-oxacyclobutamine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-40.

[0221]

[0222] BH-002-40

[0223] Yellow oily substance, yield 43%. 1H NMR (600 MHz, Chloroform-d) δ 7.81 (d, J = 2.9Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 5.4 Hz, 1H), 6.71 (d, J = 5.4Hz, 1H), 6.68 (dd, J = 8.6, 2.9 Hz, 1H), 4.94 (t, J = 6.7 Hz, 2H), 4.59 –4.53 (m, 1H), 4.49 (td, J = 6.2, 2.3 Hz, 2H), 4.09 (d, J = 7.0 Hz, 1H), 3.72(s, 3H), 3.69 – 3.63 (m, 4H), 2.49 (td, J = 10.9, 5.3 Hz, 1H), 2.26 (d, J =13.0 Hz, 1H), 2.17 – 2.10 (m, 2H), 1.83 (td, J = 12.1, 10.6, 4.9 Hz, 1H), 1.77 (d, J = 13.7 Hz, 1H), 1.69 – 1.62 (m, 2H), 1.60 – 1.54 (m, 2H), 1.45 –1.36 (m, 4H), 1.34 – 1.29 (m, 1H), 1.12 – 1.05 (m, 1H), 0.68 – 0.60 (m, 1H).ESI-MS: m / z 458.45. [M+H] + C 25 H 35 N3O3S [457.24].

[0224] The preparation method is the same as that described in Example 13, except that tetrahydropyrrole is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-42.

[0225]

[0226] BH-002-42

[0227] Yellow oily substance, yield 24%. 1H NMR (600 MHz, Chloroform-d) δ 7.89 (d, J = 2.9Hz, 1H), 7.08 (t, J = 7.2 Hz, 2H), 6.79 – 6.76 (m, 2H), 3.81 (d, J = 14.0 Hz,1H), 3.79 (s, 3H), 3.77 (d, J = 6.7 Hz, 1H), 3.75 – 3.71 (m, 2H), 3.29 (t, J= 6.5 Hz, 4H), 2.64 (td, J = 10.4, 9.8, 5.6 Hz, 1H), 2.35 (dd, J = 13.6, 2.7Hz, 1H), 2.29 (td, J = 10.6, 3.8 Hz, 1H), 2.23 (d, J = 15.7 Hz, 1H), 2.03 (t,J = 6.6 Hz, 4H), 1.95 – 1.90 (m, 1H), 1.82 (d, J = 13.6 Hz, 1H), 1.78 – 1.71(m, 2H), 1.66 – 1.59 (m, 2H), 1.52 – 1.43 (m, 4H), 1.41 – 1.36 (m, 1H), 1.19 – 1.14 (m, 1H), 0.76 – 0.66 (m, 1H). ESI-MS: m / z 456.21. [M+H] + C 26 H 37 N3O2S[455.26].

[0228] The preparation method is the same as that described in Example 13, except that 1-tert-butoxycarbonyl-4-aminopiperidine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-44.

[0229]

[0230] BH-002-44

[0231] A brownish-yellow oily substance, yield 22%. 1H NMR (600 MHz, Chloroform-d) δ 7.95 (d, J =2.9 Hz, 1H), 7.06 (dd, J = 7.1, 1.6 Hz, 2H), 6.84 (dd, J = 8.6, 2.9 Hz, 1H), 6.77 (d, J = 5.5 Hz, 1H), 4.13 – 4.03 (m, 2H), 3.79 (s, 3H), 3.76 (d, J =16.7 Hz, 2H), 3.73 – 3.70 (m, 2H), 3.53 – 3.49 (m, 1H), 3.42 – 3.37 (m, 1H), 2.97 – 2.89 (m, 2H), 2.58 (td, J = 11.1, 5.6 Hz, 1H), 2.33 (d, J = 13.6 Hz,1H), 2.27 – 2.20 (m, 2H), 2.05 – 2.01 (m, 2H), 1.95 – 1.87 (m, 1H), 1.83 (d,J = 13.7 Hz, 1H), 1.75 – 1.70 (m, 2H), 1.66 – 1.59 (m, 2H), 1.50 – 1.45 (m,14H), 1.41 – 1.33 (m, 4H), 1.19 – 1.15 (m, 1H), 0.73 (dt, J = 13.5, 8.8 Hz,1H). ESI-MS: m / z 585.58. [M+H] + C 32 H 48 N4O4S [584.34].

[0232] The preparation method is the same as that described in Example 13, except that 4-amino-1-methylpiperidine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-48.

[0233]

[0234] BH-002-48

[0235] Yellow oily substance, yield 13%. 1H NMR (400 MHz, Chloroform-d) δ 7.96 (d, J = 2.9Hz, 1H), 7.08 – 7.01 (m, 2H), 6.83 (dd, J = 8.6, 2.9 Hz, 1H), 6.77 (d, J =5.5 Hz, 1H), 3.78 (s, 3H), 3.72 (d, J = 6.5 Hz, 4H), 3.54 – 3.48 (m, 1H), 3.30 – 3.23 (m, 1H), 2.86 (d, J = 9.4 Hz, 2H), 2.55 (td, J = 11.0, 5.2 Hz, 1H), 2.37 – 2.30 (m, 4H), 2.23 – 2.03 (m, 8H), 1.85 (dd, J = 15.5, 12.1 Hz,2H), 1.73 – 1.60 (m, 4H), 1.54 – 1.44 (m, 5H), 1.21 – 1.14 (m, 1H), 0.79 –0.67 (m, 1H). ESI-MS: m / z 499.19. [M+H] + C 28 H 42 N4O2S [498.30].

[0236] The preparation method is the same as that described in Example 13, except that 4-aminotetrahydropyran is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-50.

[0237]

[0238] BH-002-50

[0239] Yellow oily substance, yield 41%. 1H NMR (400 MHz, Chloroform-d) δ 7.98 (d, J = 3.1Hz, 1H), 7.06 (t, J = 7.0 Hz, 2H), 6.86 (dd, J = 8.6, 3.0 Hz, 1H), 6.78 (d, J= 5.5 Hz, 1H), 4.03 (dt, J = 11.9, 3.4 Hz, 2H), 3.79 (s, 3H), 3.72 (d, J =6.6 Hz, 4H), 3.52 (t, J = 11.3 Hz, 4H), 2.55 (td, J = 11.2, 5.5 Hz, 1H), 2.34(d, J = 13.1 Hz, 1H), 2.26 – 2.17 (m, 2H), 2.05 (d, J = 13.2 Hz, 2H), 1.93 –1.82 (m, 2H), 1.75 – 1.61 (m, 5H), 1.54 – 1.40 (m, 7H), 1.20 – 1.14 (m, 1H),0.77 – 0.67 (m, 1H). ESI-MS: m / z 486.34. [M+H] + C 27 H 39 N3O3S [485.27].

[0240] The preparation method is the same as that described in Example 13, except that tetrahydrothiaran-4-amine is used instead of N-tert-butoxycarbonyl-1,2-ethylenediamine to prepare BH-002-52.

[0241]

[0242] BH-002-52

[0243] Yellow oily substance, yield 32%. 1H NMR (400 MHz, DMSO-d6) δ 7.91 (d, J = 2.8 Hz,1H), 7.30 (d, J = 5.5 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.93 (d, J = 5.5 Hz,1H), 6.88 (dd, J = 8.6, 2.9 Hz, 1H), 5.63 (d, J = 8.3 Hz, 1H), 3.73 (s, 3H), 3.64 (s, 2H), 3.55 (d, J = 7.3 Hz, 2H), 3.17 (d, J = 3.1 Hz, 1H), 2.74 – 2.65(m, 4H), 2.43 – 2.36 (m, 1H), 2.30 (d, J = 13.4 Hz, 1H), 2.23 (d, J = 13.4Hz, 1H), 2.15 (d, J = 12.8 Hz, 2H), 2.03 – 1.96 (m, 1H), 1.82 – 1.70 (m, 1H), 1.66 (d, J = 13.7 Hz, 1H), 1.61 (d, J = 5.6 Hz, 1H), 1.54 – 1.33 (m, 10H), 1.07 – 0.99 (m, 1H), 0.71 – 0.60 (m, 1H). ESI-MS: m / z 502.18. [M+H] + C 27 H 39 N3O2S2 [501.25].

[0244] Example 15: Chiral preparation of the BH-002-42 isomer

[0245] Chiral preparation conditions:

[0246] Instrument: High-performance liquid chromatography (HPLC) preparative chromatograph.

[0247] Chromatographic column: Chiral column, inner diameter 2.1 cm, column length 25 cm, particle size 5 μm.

[0248] Column temperature: 40 ℃

[0249] Mobile phase: Methanol / diethylamine = 100 / 0.05 (v / v)

[0250] Detection wavelength: 254 nm

[0251] Flow rate: 15 mL / min

[0252] The corresponding components were collected and concentrated under reduced pressure to obtain BH-002-42-isomer 1 (light brown oily substance) and BH-002-42-isomer 2 (light brown oily substance).

[0253] BH-002-42-Isomer 1 Chiral analysis: Retention time 7.791 min, ee value 98% (Column: Superchiral R-AG (Chiralway), 0.46 ID; Mobile phase: Methanol / Diethanolamine = 100 / 0.05 (v / v)).

[0254] BH-002-42-Isomer 2 Chiral analysis: Retention time 11.981 min, ee value 98% (Column: Superchiral R-AG (Chiralway), 0.46 ID; Mobile phase: Methanol / Diethanolamine = 100 / 0.05 (v / v)).

[0255] Example 16: Preparation of 2-(9-(5-ethylpyridin-2-yl)-6-oxaspiro[4.5]decane-9-yl)-N-((3-methoxythiophene-2-yl)methyl)ethane-1-amine (BH-002-54)

[0256]

[0257] BH-002-54

[0258] Intermediate I-3 (107 μmol) obtained in Example 11 was dissolved in 1 mL of 1,4-dioxane, followed by the sequential addition of ethylboric acid (161 μmol), potassium phosphate (537 μmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane dichloride complex (11 μmol). The reaction was purged with nitrogen and carried out at 100 °C. The reaction was monitored by TLC after 8 h. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (10 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by preparative thin-layer chromatography to obtain the target product BH-002-54, a brownish-yellow oily substance with a yield of 22%. 1H NMR (400 MHz, DMSO-d6) δ 8.37 (d, J = 2.4 Hz, 1H), 7.56 (dd, J = 8.1, 2.4 Hz, 1H), 7.34 (d,J = 8.2 Hz, 1H), 7.27 (d, J = 5.4 Hz, 1H), 6.91 (d, J = 5.4 Hz, 1H), 3.71 (s,3H), 3.62 – 3.54 (m, 4H), 2.60 (q, J = 7.6 Hz, 2H), 2.42 – 2.27 (m, 3H), 1.92(td, J = 11.1, 4.5 Hz, 1H), 1.82 (td, J = 12.0, 4.5 Hz, 1H), 1.75 (d, J =13.5 Hz, 1H), 1.69 – 1.59 (m, 2H), 1.56 – 1.32 (m, 7H), 1.19 (t, J = 7.6 Hz,3H), 1.03 – 0.94 (m, 1H), 0.70 – 0.57 (m, 1H). ESI-MS: m / z 415.76. [M+H] + C 24 H 34 N2O2S [414.23].

[0259] Example 17

[0260] The preparation method is the same as that described in Example 16, except that n-propylboronic acid is used instead of ethylboronic acid to prepare BH-002-56.

[0261]

[0262] BH-002-56

[0263] A brownish-yellow oily substance, yield 35%. 1H NMR (600 MHz, Chloroform-d) δ 8.31 (d, J =2.3 Hz, 1H), 7.43 (dd, J = 8.1, 2.4 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.12(d, J = 5.5 Hz, 1H), 6.77 (d, J = 5.5 Hz, 1H), 3.95 – 3.84 (m, 2H), 3.80 (s,3H), 3.74 – 3.71 (m, 2H), 2.69 (td, J = 11.2, 5.6 Hz, 1H), 2.56 (t, J = 7.7Hz, 2H), 2.37 – 2.29 (m, 2H), 2.24 (d, J = 13.7 Hz, 1H), 2.05 (td, J = 12.2,10.2, 5.2 Hz, 1H), 1.91 – 1.85 (m, 2H), 1.75 (t, J = 9.0 Hz, 1H), 1.68 – 1.62(m, 4H), 1.51 – 1.44 (m, 4H), 1.39 – 1.36 (m, 1H), 1.13 – 1.08 (m, 1H), 0.94(t, J = 7.3 Hz, 3H), 0.70 – 0.64 (m, 1H). ESI-MS: m / z 429.79. [M+H] + C 25 H 36 N2O2S [428.25].

[0264] Example 18: Preparation of N-(6-(9-(2-(((3-methoxythiophene-2-yl)methyl)amino)ethyl)-6-oxaspiro[4.5]decane-9-yl)pyridin-3-yl)ethane-1,2-diamine (BH-002-26)

[0265]

[0266] BH-002-26

[0267] The BH-002-24 (215 μmol) prepared in Example 13 was dissolved in 1,4-dioxane hydrochloride solution (4.0 M, 1 mL) at 0 °C and reacted at room temperature. The reaction was monitored by TLC after 1 h. After the reaction was complete, the pH of the reaction solution was adjusted to 7-8 with saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane (15 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by preparative thin-layer chromatography to obtain the target product BH-002-26, a yellow oily substance, with a yield of 19%. 1 H NMR (500MHz, Chloroform-d) δ 8.08 (s, 1H), 7.34 (d, J = 5.5 Hz, 1H), 7.07 (s, 2H), 6.85 (d, J = 5.5 Hz, 1H), 5.88 (s, 1H), 4.28 (d, J = 14.1 Hz, 1H), 4.16 (d, J= 14.2 Hz, 1H), 3.88 (s, 3H), 3.70 – 3.61 (m, 2H), 3.60 – 3.51 (m, 2H), 3.30 – 3.21 (m, 2H), 2.94 – 2.85 (m, 1H), 2.68 – 2.59 (m, 1H), 2.31 – 1.93 (m,5H), 1.70 (d, J = 12.6 Hz, 2H), 1.63 – 1.49 (m, 3H), 1.46 – 1.32 (m, 5H), 1.15 – 1.11 (m, 1H), 0.70 – 0.60 (m, 1H). ESI-MS: m / z 445.63. [M+H] + C 24 H 36 N4O2S [444.26].

[0268] Example 19

[0269] The preparation method is the same as that described in Example 18, except that BH-002-36 is used instead of BH-002-24 to obtain BH-002-38.

[0270]

[0271] BH-002-38

[0272] Yellow oily substance, yield 33%. 1H NMR (600 MHz, Chloroform-d) δ 7.98 (s, 1H), 7.36 (d, J = 5.4 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 7.00 (d, J = 9.4 Hz, 1H), 6.87 (d, J = 5.5 Hz, 1H), 6.64 (s, 1H), 4.55 – 4.47 (m, 1H), 4.41 – 4.32 (m,2H), 4.28 (d, J = 13.9 Hz, 1H), 4.21 – 4.12 (m, 3H), 3.88 (s, 3H), 3.67 –3.61 (m, 2H), 2.94 – 2.85 (m, 1H), 2.62 – 2.56 (m, 1H), 2.25 – 2.17 (m, 1H), 2.16 – 2.08 (m, 2H), 2.05 – 2.00 (m, 1H), 1.74 – 1.67 (m, 2H), 1.61 – 1.54(m, 2H), 1.48 – 1.41 (m, 3H), 1.38 – 1.32 (m, 3H), 1.13 – 1.10 (m, 1H), 0.66– 0.60 (m, 1H). ESI-MS: m / z 457.72. [M+H] + C 25 H 36 N4O2S [456.26].

[0273] The preparation method is the same as that described in Example 18, except that BH-002-44 is used instead of BH-002-24 to obtain BH-002-46.

[0274]

[0275] BH-002-46

[0276] Yellow oily substance, yield 74%. 1H NMR (400 MHz, DMSO-d6) δ 7.98 (s, 1H), 7.55 (d,J = 5.5 Hz, 1H), 7.42 (s, 1H), 7.29 (s, 1H), 7.17 (s, 1H), 7.03 (d,J = 5.6Hz, 1H), 6.00 – 5.93 (m, 1H), 4.07 – 3.97 (m, 2H), 3.79 (s, 3H), 3.63 – 3.48(m, 3H), 3.28 (d, J = 12.3 Hz, 2H), 3.04 – 2.92 (m, 2H), 2.72 – 2.62 (m, 1H),2.35 – 2.18 (m, 3H), 2.07 – 1.87 (m, 3H), 1.78 – 1.57 (m, 5H), 1.54 – 1.31(m, 7H), 1.09 – 0.99 (m, 1H), 0.74 – 0.64 (m, 1H). ESI-MS: m / z 485.67. [M+H] + C 27 H 40 N4O2S [484.29].

[0277] Experiment 1: For MOR, cAMP accumulation inhibition assay and β-arrestin recruitment assay were performed at the cellular level, with fentanyl and TRV-130 as positive controls.

[0278] 1) The agonistic activity of oxaspirocyclic derivatives on the G protein signaling pathway of MOR

[0279] Experimental Principle

[0280] This assay measures cAMP produced by GPCR-regulated adenylate cyclase activity. The principle is based on europium (Eu) chelate-labeled cAMP tracers and the competitive binding site competition between the generated cAMP and cAMP-specific monoclonal antibodies. When the antibody binds to the Eu-labeled cAMP tracer, a 320 or 340 nm light pulse excites the Eu chelate molecules of the tracer. The energy released by the excited Eu molecules is transferred to the ULight molecules of the antibody via FRET, resulting in light emission at 665 nm. The remaining energy of the Eu molecules emits light at 615 nm. Maximum TR-FRET signal is achieved in the absence of free cAMP. When free cAMP produced by stimulated cells competitively binds to the ULight-mAb with the Eu-cAMP tracer, the TR-FRET signal decreases.

[0281] Experimental methods

[0282] 1. HEK293 cell transfection

[0283] (1) Materials required for preheating include: DMEM cell culture medium, PBS washing solution, trypsin, and 6-well or 12-well plates required for cell plating.

[0284] (2) Take out HEK293 cells, remove the culture medium, wash with PBS, add trypsin, digest for about 1 min until the cells fall off, then add culture medium to stop digestion, mix well, measure the concentration, dilute the cells to 1.5-2.0 ×106 cells / mL, plate them, mix well and place them in a 37 ℃, 5% CO2 incubator overnight for 12-16 h.

[0285] (3) The next day, observe the cell adhesion, then perform transfection, according to the 6-well plate system, beat and mix well, and let stand for 10 min.

[0286] (4) Add the incubated transfected sample along the side wall into the 6-well plate, mix gently, and incubate overnight at 37 ℃ in a 5% CO2 incubator for 24 h.

[0287] 2. cAMP signal detection

[0288] (1) More than 24 hours after transfection, prepare the following items: PBS washing solution, enzyme-free digestion solution, HBSS buffer, IBMX stock solution, 1 M HEPES stock solution, LANCE cAMP kit.

[0289] (2) Remove the supernatant culture medium of HEK293, add PBS buffer to wash the residual culture medium, then add enzyme-free digestion solution. After the cells detach, add cAMP working solution to stop the digestion.

[0290] (3) Transfer all cells to EP tubes, centrifuge at 1000 rpm and 25 ℃ for 5 min, discard the supernatant, resuspend the cells in cAMP working solution, and test the concentration.

[0291] (4) Prepare ligand solutions with gradient concentrations and add fosklin to pre-stimulate adenylate cyclase to increase the intracellular cAMP concentration.

[0292] (5) Add cells to a 384-well plate and briefly centrifuge to remove cells to the bottom of the plate. Then add ligands, briefly centrifuge to remove ligands to the bottom of the plate, and incubate for 30 min. Then add Eu-cAMP tracer solution, briefly centrifuge to remove solution to the bottom of the plate. Then add ULignt-anti-cAMP solution, briefly centrifuge to remove solution to the bottom of the plate, and incubate in the dark for 30 min.

[0293] (6) Data was detected using an Envision multichannel reader with channels of 665 nm and 620 nm wavelengths. The readings were taken using the ratio of 665 nm to 620 nm. Data was processed using GraphPad Prism software.

[0294] The effects of the compounds of this invention on downstream cAMP levels by activating MOR were determined through the above experiments, and the results are shown in Table 1. In Table 1, Span represents the maximum effect of some compounds in the examples on the cAMP signaling pathway by activating MOR (the maximum effect of fentanyl is 100%).

[0295] 2) β-arrestin pathway activating activity of oxaspirocyclic derivatives on MOR

[0296] Experimental Principle

[0297] The receptor's C-terminus is linked to LgBiT, and the N-terminus of arrestin is linked to SmBiT. After co-expression of both, the addition of a ligand will activate the receptor to recruit arrestin, and if this activates the receptor, LgBiT and SmBiT will automatically bind together to form the NanoBiT complex. This complex emits a measurable fluorescent signal, and the strength of the interaction between the two proteins can be assessed by measuring the fluorescence signal intensity of the NanoBiT complex.

[0298] Experimental methods

[0299] 1. HEK293 cell transfection

[0300] (1) Materials required for preheating include: DMEM cell culture medium, PBS washing solution, trypsin, and 6-well or 12-well plates required for cell plating.

[0301] (2) Take out HEK293 cells, remove the culture medium, wash with PBS, add trypsin, digest for about 1 min until the cells fall off, then add culture medium to stop digestion, mix well, measure the concentration, dilute the cells to 1.5-2.0 ×106 cells / mL, plate them, mix well and place them in a 37 ℃, 5% CO2 incubator overnight for 12-16 h.

[0302] (3) The next day, observe the cell adhesion, then perform transfection, according to the 6-well plate system, beat and mix well, and let stand for 10 min.

[0303] (4) Add the incubated transfected sample along the side wall into the 6-well plate, mix gently, and incubate overnight at 37 ℃ in a 5% CO2 incubator for 24 h.

[0304] 2. Recruitment signal detection

[0305] (1) More than 24 hours after transfection, prepare the following items: PBS washing solution, enzyme-free digestion solution, and BSA stock solution.

[0306] (2) Remove the supernatant culture medium of HEK293, add PBS buffer to wash the residual culture medium, add enzyme-free digestion solution, and after the cells detach, add PBS solution containing 0.1% BSA.

[0307] (3) Transfer the cells to an EP tube, centrifuge at 1000 rpm and 25°C for 5 min, discard the supernatant, resuspend the cells in PBS, and test the concentration.

[0308] (4) Prepare ligand solutions with gradient concentrations, add cells to a 384-well plate, add coelentrin, and briefly centrifuge to remove the cells to the bottom of the plate. Then add the ligands, briefly centrifuge to remove the ligands to the bottom of the plate, and incubate in the dark for 10 minutes.

[0309] (5) Use Envision multi-channel plate reader to detect data, use Luminance channel, and use GraphPadPrism software to process data.

[0310] Results: The activation activity of the compounds of this invention in activating the β-arrestin signaling pathway was determined by the above experiments, and the results are shown in Table 1. In Table 1, Span represents the maximum effect of the compounds on the β-arrestin signaling pathway (the maximum effect of fentanyl is 100%). The results show that BH-002-25, BH-002-42, BH-002-43, and BH-002-56 have high agonistic activity on the G protein signaling pathway, while BH-002-25, BH-002-42, and BH-002-56 have extremely low agonistic activity on the β-arrestin signaling pathway.

[0311] Table 1. Results of cAMP accumulation inhibition and β-arrestin recruitment experiments of the compounds.

[0312]

[0313] Experimental Example 2: Taking compound BH-002-42 and its isomers as examples, in vivo analgesic activity was tested using a hot plate test.

[0314] 1) Analgesic activity test of oxaspirocyclic derivatives in mice induced by hot plate stimulation of acute thermal pain

[0315] Experimental Objective

[0316] The analgesic activity of the compounds of this invention in a mouse model of acute heat pain was tested.

[0317] Experimental methods

[0318] Healthy female SPF-grade ICR mice, weighing 18-20 g, were selected before testing. Mice were placed on a hot plate at 52 ± 0.5 ℃ for screening. Mice that licked their hind paws for less than 5 s or more than 30 s, or those that jumped, were discarded. Mice were divided into a blank group (physiological saline), a solvent group (10% DMSO + 10% Tween-80 + 80% physiological saline), a positive control group (TRV130), and high, medium, and low dose groups of the test compound, with 8 mice in each group. The baseline value (T1) for licking the hind paw or jumping was used before drug administration, and the average value for each group was calculated. Different concentration solutions were prepared and administered according to the different doses for each group. The blank group received subcutaneous injection of physiological saline. The test drug, physiological saline, or solvent was injected intraperitoneally at 10 mL / kg 5 min before the test. Five min after drug administration, the mice were placed on a hot plate at 52 ± 0.5 ℃ to observe and record the latency (T2) for licking the hind paw or jumping. Mice that did not respond for more than 60 s were discontinued to avoid burns.

[0319] Experimental data are expressed as mean ± standard error (mean ± SEM). One-way ANOVA was used for inter-group comparisons, and Tukey's test was used for multiple comparisons. A p-value < 0.05 was considered statistically significant. Data analysis was performed using the nonlinear fitting module in GraphPadPrism 10.0 statistical software to calculate the median effective dose (ED). 50 .

[0320] Results: The analgesic activity of the compounds of this invention in mice for acute febrile pain was determined by the above experiments, and the results are shown in Figure 1. BH-002-42 and its isomers exhibit high analgesic activity in mice (EDS of BH-002-42 is shown in Figure 1). 50 The ED of BH-002-42-2 is 4.64 mg / kg. 50 (4.83 mg / kg).

[0321] 2) Analgesic activity test of oxaspirocyclic derivatives in mice for acetic acid-induced acute inflammatory pain

[0322] Experimental Objective

[0323] The analgesic activity of the compounds of this invention in an acute inflammatory pain model in mice was tested.

[0324] Experimental methods

[0325] Healthy male SPF-grade ICR mice, weighing 18-20 g, were divided into a blank group (physiological saline), a solvent group (10% DMSO + 10% Tween-80 + 80% physiological saline), a positive control group (TRV130), and high, medium, and low dose groups of the test compound, with 8 mice in each group. Different concentration solutions were prepared according to different doses for each group and administered. The final administration volume for each test group was 10 mL / kg. The mice were injected subcutaneously and then intraperitoneally with 0.9% acetic acid saline solution 5 min later to establish the model. The mice were placed in a transparent observation box, and the latency of the first wriggling movement after administration and the number of wriggling movements within 20 min were observed and recorded. The average values ​​of each group were calculated.

[0326] Experimental data are expressed as mean ± standard error (mean ± SEM). One-way ANOVA was used for inter-group comparisons, and Tukey's test was used for multiple comparisons. A p-value < 0.05 was considered statistically significant. The writhing inhibition rate (%) was calculated for each drug-treated group. Data analysis was performed using the nonlinear fitting module in GraphPad Prism 10.0 statistical software, and the ED was calculated. 50 value.

[0327] Results: The analgesic activity of the compounds of this invention in mice for acute inflammatory pain was determined by the above experiments, and the results are shown in Figure 2. BH-002-42 and its isomers exhibited high analgesic activity in mice (EDS of BH-002-42 was [missing information]). 50 The ED of BH-002-42-2 is 0.34 mg / kg. 50 (0.36 mg / kg).

[0328] 3) In vivo gastrointestinal transport experiment of oxaspirocyclic derivatives in mice

[0329] Experimental Objective

[0330] The degree of constipation caused by the compound of this invention was tested in mice.

[0331] Experimental methods

[0332] Healthy male SPF-grade ICR mice, weighing 18-20 g, were fasted for 12 hours before the experiment but allowed free access to water. They were randomly divided into four groups: a blank control group (physiological saline), a solvent group (10% DMSO + 10% Tween-80 + 80% physiological saline), a positive control group (TRV130, morphine), and a test compound group (n=8 per group). Different concentrations of solutions were prepared for each group according to different dosages. The final drug volume for each test group was 10 mL / kg. The drugs were administered intraperitoneally, followed by gavage administration of a charcoal meal (5% activated charcoal suspended in 5% gelatin in physiological saline solution) 20 min later. The mice were euthanized by cervical dislocation 30 min later. Dissection was performed to measure the distance the charcoal meal was propelled and the total length of the small intestine (from the stomach to the cecum). The propulsion rate and small intestinal transport inhibition rate were calculated. The charcoal meal propulsion rate for each group was calculated using the following formula: Propulsion rate (%) = Charcoal meal propulsion distance / Total small intestinal length × 100%. Experimental data are expressed as mean ± standard error (mean ± SEM). One-way ANOVA was performed between groups, and Tukey's test was used for multiple comparisons. A p < 0.05 was considered statistically significant.

[0333] Experimental results

[0334] Results: The gastrointestinal transport of the compound of this invention in mice was determined by the above experiments, and the results are shown below. Figure 3 At equivalent analgesic doses, neither BH-002-42 nor BH-002-42-2 caused constipation in mice.

[0335] Experiment Example 3: Whole-body volume recording experiment of oxaspirocyclic derivatives in mice

[0336] Experimental Objective

[0337] Testing the degree of respiratory inhibition of the compound of the present invention in mice

[0338] Experimental methods

[0339] Healthy male SPF-grade ICR mice, weighing 18-20 g, were randomly divided into four groups: a blank control group (saline), a solvent group (10% DMSO + 10% Tween-80 + 80% saline), a positive control group (TRV130, morphine), and a test compound group (n=6 per group). Different concentrations of solutions were prepared and administered to each group according to different dosages. The final drug volume for each test group was 10 mL / kg. Before the experiment, the mice were placed in an observation chamber for 30 min to acclimatize, and baseline respiratory parameters (including respiratory rate, tidal volume, and minute ventilation) were recorded for 20 min before drug administration. Subsequently, appropriate treatments (saline or drug) were administered via intraperitoneal injection, and changes in respiratory parameters were continuously monitored and recorded for 60 min after drug administration. The entire experimental process was conducted in a quiet environment. After the experiment, data were exported at 5-minute intervals to calculate the average respiratory rate, tidal volume, and minute ventilation for each group. Ultimately, the ratio of respiratory parameters (respiratory rate, tidal volume, minute ventilation) after drug administration to the baseline values ​​before drug administration was used as the efficacy evaluation index.

[0340] Results: The mouse whole-body volume plethysmography experiment of the compound of this invention was conducted according to the above experimental results, as shown in the figure below. Figure 4 At equivalent analgesic doses, neither BH-002-42 nor BH-002-42-2 caused respiratory depression in mice.

Claims

1. An oxaspirocyclic derivative of Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof; in, Ring A is selected from phenyl or heteroaryl, wherein the heteroaryl group is selected from one of pyrroleyl, furanyl, thiopheneyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, pyridyl, pyrazinyl, pyridazinyl, indolyl, isindolyl, benzimidazolyl, benzofuranyl, benzothiopheneyl, quinolinyl, isoquinolinyl, quinazolinyl, or quinoxalinyl. R1 is selected from: (1) C substituted with halogens 1-6 Alkyl; (2) C 2-6 (3) A 5- or 6-membered heteroaryl group, wherein the heteroaryl group contains 1 to 3 heteroatoms selected from N, O or S, and the heteroaryl group is optionally substituted by one or more substituents independently selected from: amino, C 1-4 Alkyl, carbamoyl C 1-4 Alkyl group; (4) a 4- to 6-membered saturated heterocyclic group, wherein the saturated heterocyclic group contains 1 to 2 heteroatoms selected from nitrogen or oxygen atoms, and the saturated heterocyclic group is connected to the parent nucleus through its cyclic nitrogen atom; or (5) a group N(R) a (R) b ); R2 has the structure shown in general formula (a): Where n is selected from 1, 2, or 3; R3 is selected from phenyl, 5-10 heteroaryl, 5-10 heterocyclic or 5-10 bicyclic heterocyclic, wherein each of the phenyl, heteroaryl, heterocyclic or bicyclic is independently optionally substituted by a halogen, C1-C6 alkyl or C1-C6 alkoxy group. In R2, the methylene chain -(CH2) n One of the -CH2- units is optionally substituted with a hydroxyl, carbonyl, or amino group.

2. The oxaspirocyclic derivative according to claim 1, or its stereoisomers, tautomers, or pharmaceutically acceptable salts, characterized in that, In Formula I, the group N(R) a (R) b In ), R a Selected from hydrogen or C 1-4 Alkyl; Rb is selected from: (1) C 1-6 Alkyl groups, which are selected from one or more hydroxyl groups, C 1-4 Alkoxy, amino, amino groups substituted with amino protecting groups, single C 1-4 Alkylamino, bis(C) 1-4 Alkylamino, carbamoyl, mono-C 1-4 Alkyl carbamoyl or bis-C 1-4 Substituents of alkylcarbamoyl groups; or (2) 4- to 6-membered saturated heterocyclic groups, optionally substituted with one or more C-groups. 1-4 Alkyl, amino protecting groups, or oxo substituents are used for substitution.

3. The oxaspirocyclic derivative according to claim 1, or its stereoisomers, tautomers, or pharmaceutically acceptable salts, characterized in that, In the general formula of the compound shown in Formula I, Ring A is pyridinyl; R1 is selected from one of the following groups: ethyl, propyl, (trifluoromethyl)vinyl, 2-aminopyrimidin-4-yl, 1-(2-amino-2-acetyl)-1H-pyrazol-4-yl, pyrrolidone-1-yl, (oxetane-3-yl)amino, (azacyclobutane-3-yl)amino, (1-(tert-butoxycarbonyl)azacyclobutane-3-yl)amino, (tetrahydro-2H-pyran-4-yl)amino, (tetrahydro-2H-thiaran-4-yl)amino. (-yl)amino, (piperidin-4-yl)amino, (1-methylpiperidin-4-yl)amino, (1-(tert-butoxycarbonyl)piperidin-4-yl)amino, (2-aminoethyl)amino, (2-(dimethylamino)ethyl)amino, (2-(tert-butoxycarbonyl)amino)ethyl)amino, (2-hydroxyethyl)amino, (2-methoxyethyl)amino, (carbamoylmethyl)amino, (N,N-dimethylcarbamoylmethyl)amino; R2 is (2-methoxythiophene-3-yl)methyl.

4. The oxaspirocyclic derivative according to claim 1, or its stereoisomers, tautomers, or pharmaceutically acceptable salts, characterized in that, The pharmaceutically acceptable salt is a salt formed with an inorganic acid or an organic acid, or a salt formed with an inorganic base or an organic base; wherein the inorganic acid is sulfuric acid, hydrochloric acid, phosphoric acid, or nitric acid; the organic acid is acetic acid, oxalic acid, citric acid, fumaric acid, or p-benzenesulfonic acid; the inorganic base is sodium hydroxide, potassium hydroxide, or ammonia; and the organic base is methylamine, ethylamine, or ethanolamine.

5. The oxaspirocyclic derivative according to claim 1, or its stereoisomers, tautomers, or pharmaceutically acceptable salts, characterized in that, The oxaspirocyclic derivatives represented by Formula I are selected from any of the following structural compounds: 。 6. A pharmaceutical composition comprising an oxaspirocyclic derivative of formula I as claimed in claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. The use of the oxaspirocyclic derivative of Formula I as described in claim 1, or its stereoisomers, tautomers, or pharmaceutically acceptable salts, or the pharmaceutical composition of claim 6, in the preparation of a medicament for the prevention and / or treatment of μ-opioid receptor agonist-mediated diseases.

8. The application according to claim 7, characterized in that, The diseases mediated by μ-opioid receptor agonists are selected from pain, inflammatory diseases, gastroesophageal reflux disease, neurological and psychiatric disorders, respiratory diseases, or diarrhea.

9. The application according to claim 8, characterized in that, The pain is selected from postoperative pain, cancer pain, neuropathic pain, traumatic pain, migraine, and pain caused by inflammation.

10. A G-protein biased two-site MOR agonist, comprising the oxaspirocyclic derivative of claim 1.

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