RU2800295C1 - Optically pure oxaspiro-substituted pyrrolopyrazole derivative, a method of its production and its pharmaceutical use - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a compound represented in formula (I) or a pharmaceutically acceptable salt thereof, wherein R_a and R_b are each independently either hydrogen or C_1-3alkyl; R_c is either hydrogen or C_1-3alkyl; and * indicates either R or S configuration. The invention also relates to the intermediates represented in formulas (I-1) and (II-1).

EFFECT: new compounds have been obtained that can be used in medicine as therapeutic agents for the treatment and prevention of pain and diseases associated with pain.

26 cl, 2 dwg, 2 tbl, 8 ex

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a class of optically pure oxaspiro-substituted pyrrolopyrazole derivatives, processes for their preparation, pharmaceutical compositions containing the derivatives, their use as therapeutic agents, in particular as MOR receptor agonists, and their use in the preparation of drugs for the treatment and prevention of pain and diseases associated with pain.

BACKGROUND OF THE INVENTION

[0002] Opioid receptors are an important class of G protein-coupled receptors (GPCRs) that bind endogenous opioid peptides and opioid drugs. Endogenous opioid peptides are opioid-like active substances that are naturally produced in mammals, and known endogenous opioid peptides are broadly classified into enkephalins, endorphins, dynorphins, and neoendorphins. The corresponding opioid receptors, namely μ (MOR) receptor, δ (DOR) receptor, κ (KOR) receptor, and the like, are present in the central nervous system. Studies have established that the analgesic effect of endogenous opioid peptides mainly depends on the level of expression of opioid receptors, targets that are affected by the analgesic effects of opioid drugs, as well as endogenous opioid peptides.

[0003] Current research suggests that GPCRs mediate and regulate physiological functions primarily through two signaling pathways: the G-protein signaling pathway and the β-arrestin signaling pathway. After a conventional GPCR agonist binds to the receptor, the G-protein signaling pathway is activated, which includes second messenger systems such as calcium ions, adenyl cyclase (AC), and mitogen-activated protein kinases (MAPKs), while the arrestin-associated β-ligand mainly activates the β-arrestin signaling pathway. By mediating GPCR responses, β-arrestin mainly acts as follows: 1) as a negative regulator, it acts together with the G protein-coupled kinase (GRK) receptor to desensitize the GPCR and stop G protein signaling; 2) as a scaffold protein, it recruits endocytic proteins and induces GPCR endocytosis; and 3) as an adapter protein, it forms a complex with downstream GPCR signaling molecules to activate signal transduction molecules such as MAPK, protein tyrosine kinase Src and Akt in a G-protein independent manner. Differences in ligand-stimulated G-protein signaling and/or β-arrestin signaling ultimately determine the ligand-specific cellular biological effects of GPCR.

[0004] MOR is the target of endogenous enkephalins and opioid analgesics such as morphine. Early studies have shown that endogenous enkephalins and opioid drugs such as etorphine can activate G proteins and trigger receptor endocytosis, but morphine does not trigger receptor endocytosis at all because morphine is too weak to combat MOR phosphorylation and can only capture trace amounts of β-arrestin on the cell membrane (Zhang et al., Proc Natl Acad Sci USA, 19 98, 95(12): 7157-7162). Such ligands perform their physiological functions exclusively through the G-protein signaling pathway, and not through the β-arrestin signaling pathway. Studies have shown that the analgesic effect mediated by G-protein signaling is stronger and lasts longer after morphine injection in β-arrestin-2 knockout mice (Bohn et al., Science, 1999). It would be expected that if such ligands are highly negatively associated with β-arrestin and can even avoid β-arrestin-mediated receptor desensitization, the ligands could lead to long-term G-protein signaling and stronger analgesic effects.

[0005] Currently, MOR agonists are disclosed in patent applications WO 2017106547, WO 2017063509, WO 2012129495, WO 2017106306, etc.

[0006] Prolonged use of opioid drugs causes tolerance as well as side effects such as respiratory depression and constipation, which have been shown to be closely related to the functions of β-arrestin. In order to reduce the side effects of opioid drugs, drugs can be designed based on β-arrestin-based negative MOR ligands to reduce β-arrestin-mediated side effects and enhance therapeutic effects.

SUMMARY OF THE INVENTION

[0007] The purpose of the present disclosure is to provide a compound with a novel structure that can be used as an agonist of the MOR receptor.

[0008] In a first aspect, the present disclosure provides a compound represented in formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof:

[0009] where

[0010] each of R _a and R _b is independently hydrogen, halogen (preferably F or Cl), C _1-6 alkyl (preferably C _1-3 alkyl), halogenated C _1-6 alkyl (preferably halogenated C _1-3 alkyl), or halogenated C _1-6 alkoxy (preferably halogenated C _1-3 alkoxy); или R _a и R _b вместе со связанным с ними атомом углерода образуют четырех-шестичленное насыщенное моногетероциклическое кольцо или трех-шестичленное насыщенное моноциклическое кольцо, в котором каждое четырех-шестичленное насыщенное моногетероциклическое кольцо и трех-шестичленное насыщенное моноциклическое кольцо не замещено или замещено одной, двумя или тремя замещающими группами, выбранными из группы, состоящей из циано, гидроксила, гидроксиметила, цианометила, галогена, С _1-3 алкоксила, С _1-3 алкила, -СО(С _1-3 алкила), CONR _a1 R _b1 , NR _a1 R _b1 , -NHCO(C _1-3 алкила), -NHSO ₂ NR _a1 R _b1 , -NHSO ₂ (C _1-3 алкила), -NHSO ₂ NR _a1 R _b1 и -NHSO ₂ (C ₃ - ₆ циклоалкила);

[0011] R_c is hydrogen, halogen (preferably F or Cl), C_1-6 alkyl (preferably C_1-3 alkyl), C_2-6 alkenyl (preferably C_2-4 alkenyl), C_2-6 alkynyl (preferably C_2-4 alkynyl), C_1-6 alkoxy (preferably C_1-3 alkoxy), halogenated C_1-6 alkyl (preferably halogenated C_1-3 alkyl), halogenated C_1-6 alkoxy (preferably halogenated C_1-3 alkoxyl), -CO(C_1-6 alkyl) (preferably -CO(C_1-3 alkyl)), -CONR_a1R_b1, NR_a1R_b1, -NHCO(C_1-6 alkyl) (preferably -NHCO(C_1-3alkyl)), -NHCONR_a1R_1b -NHSO₂(C_1-3 alkyl), -NHSO₂NR_a1R_1b -NHSO₂(C₃-₆cycloalkyl), -SO₂(C_1-3 alkyl), -SO₂NR_a1R_b1, four-six-membered saturated monoheterocyclic ring, С_6-10 aryl or a five- or six-membered monocyclic heteroaryl, in which each four-to-six-membered saturated monoheterocyclic ring, C_6-10 aryl and five- or six-membered monocyclic heteroaryl are unsubstituted or substituted with one, two or three substituent groups selected from the group consisting of cyano, hydroxyl, hydroxymethyl, cyanomethyl, halogen, C_1-3 alkoxy, C_1-3 alkyl, -CO(C_1-3 alkyl), -CONR_alR_1b, NR_alR_1b, -NHCO(C_1-3 alkyl), -NHCONR_alR_1b.], -NHSO₂(C_1-3 alkyl), -NHSO₂NR_alR_bl and -NHSO₂(C₃-₆ cycloalkyl);

[0012] R _a1 and R _b1 are each independently hydrogen, C _1-6 alkyl (preferably C _1-3 alkyl), halogenated C _1-6 alkyl (preferably halogenated C _1-3 alkyl), C _3-6 cycloalkyl, or a four to six membered saturated monoheterocyclic ring; or R _a1 and R _b1 together with their associated nitrogen atom form a four to six membered saturated monoheterocyclic ring in which the four to six membered saturated monoheterocyclic ring is unsubstituted or substituted with one, two or three C _1-3 alkyl groups; And

[0013] * indicates an R or S configuration.

[0014] In another preferred embodiment, the compound represented in formula (I) has the structure represented in formula (I-a) or formula (I-b):

[0015] In another preferred embodiment, R _a and R _b together with their associated carbon atom form a four to six membered saturated monoheterocyclic ring which is selected from the group consisting of azetidine, oxetane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyrrole, piperidine, piperazine, morpholine, thiomorpholine, thiomorpholine-1,1-dioxide and tetrahydropyran.

[0016] In another preferred embodiment, R _a and R _b together with their associated carbon atom form a three to six membered saturated monocyclic ring which is selected from the group consisting of cyclopropyl ring, cyclobutyl ring, cyclopentyl ring and cyclohexyl ring.

[0017] In another preferred embodiment, the five or six membered monocyclic heteroaryl linked to R _c is selected from thiophene, furan, thiazole, imidazole, oxazole, pyrrole, pyrazole, triazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,5-triazole, 1,3,4-triazole, tetrazole, isoxazole, oxadiazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, thiadiazole, pyridine, pyridazine, pyrimidine or pyrazine.

[0018] In another preferred embodiment, the four to six membered saturated monoheterocyclic ring linked to R _c is selected from the group consisting of azetidine, oxetane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyrrole, piperidine, piperazine, morpholine, thiomorpholine, thiomorpholine-1,1-dioxide, and tetrahydropyran.

[0019] In another preferred embodiment, the four to six membered saturated monoheterocyclic ring linked to R _a1 and R _b1 is selected from the group consisting of azetidine, oxetane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyrrole, piperidine, piperazine, morpholine, thiomorpholine, thiomorpholine-1,1-dioxide, and tetrahydropyran.

[0020] In another preferred embodiment, the four to six membered saturated monoheterocyclic ring linked to R _a1 and R _b1 is selected from the group consisting of azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine, thiomorpholine, and thiomorpholine-1,1-dioxide.

[0021] In another preferred embodiment, R _a and R _b are each independently hydrogen, halogen, or C _1-3 alkyl; or R _a and R _b together with their associated carbon atom form a three to six membered saturated monocyclic ring.

[0022] In another preferred embodiment, each of R _a and R _b independently represents hydrogen, fluorine, methyl or ethyl.

[0023] In another preferred embodiment, R _a and R _b together with their associated carbon atom form a cyclopropyl ring, cyclobutyl ring or cyclopentyl ring.

[0024] In another preferred embodiment, R _c is hydrogen, halogen, C _1-3 alkyl, C _1-3 alkoxy, or halogenated C _1-3 alkyl.

[0025] In another preferred embodiment, R _c is hydrogen, fluoro, methyl, ethyl, methoxy, or trifluoromethyl.

[0026] In another preferred embodiment, each of R _a and R _b independently represents hydrogen, fluorine or methyl; and R _c is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0027] In another preferred embodiment, R _a and R _b are each methyl; and R _c is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0028] In another preferred embodiment, R _a and R _b are fluoro; and R _c is hydrogen, fluorine, methyl or ethyl.

[0029] In another preferred embodiment, R _a and R _b are each hydrogen; and R _c is hydrogen, fluorine, methyl, ethyl, or trifluoromethyl.

[0030] In another preferred embodiment, R _a is hydrogen; R _b is methyl; and R _c is methyl or ethyl.

[0031] In another preferred embodiment, R _a and R _b together with their associated carbon atom form a cyclopropyl ring, cyclobutyl ring or cyclopentyl ring; and R _c is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0032] In a second aspect, the present disclosure provides a compound represented by formula (II), or a pharmaceutically acceptable salt, solvate, or prodrug thereof:

[0033] where

[0034] R ₁ and R ₂ are each independently hydrogen, halogen (preferably F or Cl), C _1-6 alkyl (preferably C _1-3 alkyl), halogenated C _1-6 alkyl (preferably halogenated C _1-3 alkyl), or halogenated C _1-6 alkoxy (preferably halogenated C _1-3 alkoxy); или R ₁ и R ₂ вместе со связанным с ними атомом углерода образуют четырех-шестичленное насыщенное моногетероциклическое кольцо или трех-шестичленное насыщенное моноциклическое кольцо, где каждое четырех-шестичленное насыщенное моногетероциклическое кольцо и трех-шестичленное насыщенное моноциклическое кольцо не замещено или замещено одной, двумя или тремя замещающими группами, выбранными из группы, состоящей из циано, гидроксила, гидроксиметила, цианометила, галогена, С _1-3 алкоксила, С _1-3 алкила, -CO(C _1-3 алкила), -CONR ₁₁ R ₁₂ , NR ₁₁ R ₁₂ , -NHCO(C _1-3 алкила), -NHCONR ₁₁ R ₁₂ , -NHSO ₂ (C _1-3 алкила), -NHSO ₂ NR ₁₁ R ₁₂ и -NHSO ₂ (C ₃ - ₆ циклоалкила);

[0035] R₃ is hydrogen, halogen (preferably F or Cl), C_1-6 alkyl (preferably C_1-3 alkyl), C₂-₆ alkenyl (preferably C₂-₄alkenyl), C_2-6 alkynyl (preferably C_2-4 alkynyl), C_1-6 alkoxy (preferably C_1-3 alkoxy), halogenated C_1-6 alkyl (preferably halogenated C_1-3 alkyl), halogenated C_1-6 alkoxy (preferably halogenated C_1-3 alkoxyl), -CO(C_1-6 alkyl) (preferably -CO(C_1-3 alkyl)), -CONR_elevenR₁₂, NR_elevenR₁₂, -NHCO(C_1-6 alkyl) (preferably -NHCO(C_1-3alkyl)), -NHCONR_elevenR₁₂, -NHSO₂(WITH_1-3 alkyl), -NHSO₂NR_elevenR₁₂, -NHSO₂(C₃-₆cycloalkyl), -SO₂(C_1-3 alkyl), -SO₂NR_elevenR₁₂, four-six-membered saturated monoheterocyclic ring, С_6-10 aryl or five or six membered monocyclic heteroaryl and four to six membered saturated monoheterocyclic ring, C_6-10 aryl, where each four to six membered saturated monoheterocyclic ring, C_6-10 aryl and five- or six-membered monocyclic heteroaryl are unsubstituted or substituted with one, two or three substituent groups selected from the group consisting of cyano, hydroxyl, hydroxymethyl, cyanomethyl, halogen, C_1-3 alkoxy, C_1-3 alkyl, -CO(C_1-3 alkyl), -CONR_elevenR₁₂, NR_elevenR₁₂, -NHCO(C_1-3 alkyl), -NHCONR_elevenR₁₂, -NHSO₂(WITH_1-3alkyl), -NHSO₂NR_elevenR₁₂ and -NHSO₂(C_3-6 cycloalkyl);

[0036] R ₁₁ and R ₁₂ are each independently hydrogen, C _1-6 alkyl (preferably C _1-3 alkyl), halogenated C _1-6 alkyl (preferably halogenated C _1-3 alkyl), C _3-6 cycloalkyl, or a four to six membered saturated monoheterocyclic ring; or R ₁₁ and R ₁₂ together with their associated nitrogen atom form a four to six membered saturated monoheterocyclic ring, where the four to six membered saturated monoheterocyclic ring is unsubstituted or substituted with one, two or three C _1-3 alkyl groups; And

[0037] * indicates an R or S configuration.

[0038] In another preferred embodiment, the compound represented in formula (II) has the structure represented in formula (II-a) or formula (II-b):

[0039] In another preferred embodiment, R ₁ and R ₂ together with their associated carbon atom form a four to six membered saturated monoheterocyclic ring which is selected from the group consisting of azetidine, oxetane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyrrole, piperidine, piperazine, morpholine, thiomorpholine, thio orph oline-1,1-dioxide, and tetrahydropyran.

[0040] In another preferred embodiment, R ₁ and R ₂ together with their associated carbon atom form a three to six membered saturated monocyclic ring which is selected from the group consisting of cyclopropyl ring, cyclobutyl ring, cyclopentyl ring and cyclohexyl ring.

[0041] In another preferred embodiment, the five- or six-membered monocyclic heteroaryl linked to R ₃ is selected from the group consisting of thiophene, furan, thiazole, imidazole, oxazole, pyrrole, pyrazole, triazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,5-triazole, 1,3,4- triazole, tetrazole, isoxazole, oxadiazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, thiadiazole, pyridine, pyridazine, pyrimidine and pyrazine.

[0042] In another preferred embodiment, the four to six membered saturated monoheterocyclic ring linked to R ₃ is selected from the group consisting of azetidine, oxetane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyrrole, piperidine, piperazine, morpholine, thiomorpholine, thiomorpholine-1,1-dioxide, and tetrahydropyran.

[0043] In another preferred embodiment, the four to six membered saturated monoheterocyclic ring linked to R ₁₁ and R ₁₂ is selected from the group consisting of azetidine, oxetane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyrrole, piperidine, piperazine, morpholine, thiomorpholine, thiomorpholine-1,1-dioxide, and tetrahydropyran.

[0044] In another preferred embodiment, R ₁₁ and R ₁₂ together with their associated nitrogen atom form a four to six membered saturated monoheterocyclic ring which is selected from the group consisting of azetidine, tetrahydropyrrole, piperidine, piperazine, morpholine, thiomorpholine and thiomorpholine-1,1-dioxide.

[0045] In another preferred embodiment, each of R ₁ and R ₂ independently represents hydrogen, halogen, or C _1-3 alkyl; or R ₁ and R ₂ together with their associated carbon atom form a three to six membered saturated monocyclic ring.

[0046] In another preferred embodiment, each of R ₁ and R ₂ independently represents hydrogen, fluorine, methyl or ethyl.

[0047] In another preferred embodiment, R ₁ and R ₂ together with their associated carbon atom form a cyclopropyl ring, cyclobutyl ring or cyclopentyl ring.

[0048] In another preferred embodiment, R ₃ is hydrogen, halogen, C _1-3 alkyl, C _1-3 alkoxy, or halogenated C _1-3 alkyl.

[0049] In another preferred embodiment, R ₃ is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0050] In another preferred embodiment, each of R ₁ and R ₂ independently represents hydrogen, fluorine or methyl; and R ₃ is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0051] In another preferred embodiment, R ₁ and R ₂ are each methyl; and R ₃ is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0052] In another preferred embodiment, R ₁ and R ₂ are each fluoro; and R ₃ is hydrogen, fluorine, methyl or ethyl.

[0053] In another preferred embodiment, R ₁ and R ₂ are each hydrogen; and R ₃ is hydrogen, fluorine, methyl, ethyl or trifluoromethyl.

[0054] In another preferred embodiment, R ₁ is hydrogen; R ₂ is methyl; and R ₃ is methyl or ethyl.

[0055] In another preferred embodiment, R ₁ and R ₂ together with their associated carbon atom form a cyclopropyl ring, cyclobutyl ring or cyclopentyl ring; and R ₃ is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0056] In another preferred embodiment, the four to six membered saturated monoheterocyclic ring is selected from the following structures:

and hydrogen atoms in

four to six membered saturated monoheterocyclic ring is optionally substituted with one, two or three substituent groups each independently selected from the group consisting of cyano, hydroxyl, hydroxymethyl, cyanomethyl, halogen, C _1-3 alkoxy, C _1-3 alkyl, -CO(C _1-3 alkyl), -CONR _a1 R _b1 , NR _a1 R _b1 , -NHCO(C _{1 -3 alkyl), -NHCONR a1 R b1 , -NHSO 2 (C 1-3 alkyl ) , -NHSO 2} NR _a1 R _b1 and -NHSO ₂ (C _3-6 cycloalkyl).

[0057] In another preferred embodiment, the five- or six-membered monocyclic heteroaryl linked to R _c or R ₃ is selected from the following structures:

and five or six membered monocyclic heteroaryl, optionally substituted with one, two or three substituent groups each independently selected from the group consisting of cyano, hydroxyl, hydroxymethyl, cyanomethyl, halogen, C _1-3 alkoxy, C _1-3 alkyl, -CO(C _1-3 alkyl), -CONR _a1 R _1b, NR _a1 R _1b , -NHCO(C _1-3 alkyl), -NHCONR _a1 R _b1, -NHSO ₂ (C _1-3 alkyl), -NHSO ₂ NR _a1 R _b1 and -NHSO ₂ (C _3-6 cycloalkyl).

[0058] In another preferred embodiment, the compound is selected from the group consisting of:

[0059] In a third aspect, this disclosure provides a pharmaceutical composition that includes a compound, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, according to the first or second aspect of this disclosure, and a pharmaceutically acceptable carrier.

[0060] In a fourth aspect, this disclosure provides the use of a compound or a pharmaceutically acceptable salt, solvate, or prodrug thereof according to the first or second aspect of this disclosure, or a pharmaceutical composition according to the third aspect of this disclosure, in the manufacture of a medicament for the prevention and/or treatment of a disease mediated by an MOR receptor agonist.

[0061] In a fifth aspect, this disclosure provides the use of a compound or a pharmaceutically acceptable salt, solvate, or prodrug thereof according to the first or second aspect of this disclosure, or a pharmaceutical composition according to the third aspect of this disclosure, in the manufacture of a medicament to activate or antagonize MOR receptors.

[0062] In a sixth aspect, this disclosure provides the use of a compound or a pharmaceutically acceptable salt, solvate, or prodrug thereof according to the first or second aspect of this disclosure, or a pharmaceutical composition according to the third aspect of this disclosure, in the manufacture of a medicament for the prevention and/or treatment of pain and a disease associated with pain.

[0063] In another preferred embodiment, the MOR receptor agonist mediated disease is selected from the group consisting of pain, immune dysfunction, inflammation, esophageal reflux, neurological and psychiatric diseases, urological and reproductive diseases, cardiovascular diseases, and respiratory diseases, preferably pain.

[0064] In another preferred embodiment, the pain is selected from the group consisting of post-operative pain, pain caused by cancer, neuropathic pain, traumatic pain, and pain caused by inflammation.

[0065] In another preferred embodiment, the cancer is selected from the group consisting of breast cancer, endometrial cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, fallopian tube tumor, ovarian tumor, hemophilia, and leukemia.

[0066] In a seventh aspect, the present disclosure provides a method for preventing and/or treating a disease mediated by an MOR receptor agonist, which comprises: administering to a patient in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate, or prodrug thereof according to the first or second aspect of the present disclosure, or a pharmaceutical composition according to the third aspect of the present disclosure.

[0067] In an eighth aspect, the present disclosure provides a method for preventing and/or treating pain and a disease associated with pain, which includes: administering to a patient in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate or prodrug thereof in accordance with the first or second aspect of the present disclosure, or a pharmaceutical composition in accordance with the third aspect of the present disclosure.

[0068] In another preferred embodiment, the MOR receptor agonist mediated disease is selected from the group consisting of pain, immune dysfunction, inflammation, esophageal reflux, neurological and psychiatric diseases, urological and reproductive diseases, and cardiovascular diseases, and respiratory diseases, preferably pain.

[0069] In another preferred embodiment, the pain is selected from the group consisting of post-operative pain, pain caused by cancer, neuropathic pain, traumatic pain, and pain caused by inflammation.

[0070] In another preferred embodiment, the cancer is selected from the group consisting of breast cancer, endometrial cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, fallopian tube tumor, ovarian tumor, hemophilia, and leukemia.

[0071] In a ninth aspect, the present disclosure provides a process for preparing a compound represented in formula (I), which includes the following steps:

[0072] carrying out reductive amination of compound 16 and the compound represented in formula (I-1) to obtain the compound represented in formula (I),

[0073] where the groups in each formula are as defined in the description.

[0074] In other preferred embodiments, the compound represented in formula (I-1) has the structure represented in formula (I-1a) or (I-1b):

[0075] In another preferred embodiment, each of R _a and R _b independently represents hydrogen, fluorine or methyl; or R _a and R _b together with their associated carbon atom form a cyclopropyl ring, cyclobutyl ring or cyclopentyl ring; and R _c is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0076] In another preferred embodiment, the reductive amination is carried out in a reductive system containing an inert solvent and a reductant.

[0077] In another preferred embodiment, the inert solvent is C _1-4 alkyl alcohol, toluene, xylene, methyl tert-butyl ether, tetrahydrofuran, 1,4-dioxane, diethyl ether, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, or a combination thereof.

[007S] In another preferred embodiment, the reducing agent is selected from the group consisting of tetrabutylammonium borohydride, sodium malonyloxyborohydride, sodium triacetoxyborohydride, sodium cyanoborohydride, sodium borohydride, lithium borohydride, potassium borohydride and borane.

[0079] In a tenth aspect, the present disclosure provides a process for preparing a compound represented by formula (II), which includes the following steps:

[0080] carrying out reductive amination of compound 16 and the compound represented in formula (II-1) to obtain the compound represented in formula (II),

[0081] where the groups in each formula are as defined in the description.

[0082] In another preferred embodiment, the compound represented in formula (II-1) has the structure represented in formula (II-1a) or (P-1b):

[0083] In another preferred embodiment, each of R ₁ and R ₂ independently represents hydrogen, fluorine or methyl; or R ₁ and R ₂ together with their associated carbon atom form a cyclopropyl ring, cyclobutyl ring or cyclopentyl ring; and R ₃ is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0084] In another preferred embodiment, the reductive amination is carried out in a reaction system containing an inert solvent and a reducing agent.

[0085] In another preferred embodiment, the inert solvent is C _1-4 alkyl alcohol, toluene, xylene, methyl tert-butyl ether, tetrahydrofuran, 1,4-diocene, diethyl ether, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, or combinations thereof.

[0086] In another preferred embodiment, the reducing agent is selected from the group consisting of tetrabutylammonium borohydride, sodium malonyloxyborohydride, sodium triacetoxyborohydride, sodium cyanoborohydride, sodium borohydride, lithium borohydride, potassium borohydride, and borane.

[0087] In an eleventh aspect, the present disclosure provides a compound represented by formula (III), or a salt, enantiomer, diastereomer, racemate, or mixture of diastereomers thereof:

[0088] where

[0089] R _la and R _1b are each independently hydrogen, halogen (preferably F or Cl), C _1-6 alkyl (preferably C _1-3 alkyl), halogenated C _1-6 alkyl (preferably halogenated C _1-3 alkyl), or halogenated C _1-6 alkoxy (preferably halogenated C _1-3 alkoxy); или R _a и R _b вместе со связанным с ними атомом углерода образуют четырех-шестичленное насыщенное моногетероциклическое кольцо или трех-шестичленное насыщенное моноциклическое кольцо, где каждое четырех-шестичленное насыщенное моногетероциклическое кольцо и трех-шестичленное насыщенное моноциклическое кольцо не замещено или замещено одной, двумя или тремя замещающими группами, выбранными из группы, состоящей из циано, гидроксила, гидроксиметила, цианометила, галогена, С _1-3 алкоксила, С _1-3 алкила, -СО(С _1-3 алкила), -CONR _a1 R _b1 , NR _a1 R _b1 , -NHCO(C _1-3 алкила), -NHCONR _a1 R _b1 , -NHSO ₂ (С _1-3 алкила), -NHSO ₂ NR _a1 R _b1 и -NHSO ₂ (C ₃ . ₆ циклоалкила);

[0090] R _1b представляет собой водород, галоген (предпочтительно F или Cl), C _1-6 алкил (предпочтительно С _1-3 алкил), С _2-6 алкенил (предпочтительно С ₂ - ₄ алкенил), С _2-6 алкинил (предпочтительно С ₂ - ₄ алкинил), С _1-6 алкоксил (предпочтительно С _1-3 алкоксил), галогенированный C _1-6 алкил (предпочтительно галогенированный С _1-3 алкил), галогенированный С _1-6 алкоксил (предпочтительно галогенированный С _1-3 алкоксил), -CO(C _1-6 алкил) (предпочтительно -CO(C _1-3 алкил)), -CONR _a1 R _b1 , NR _a1 R _b1 , -NHCO(C _1-6 алкил) (предпочтительно -NHCO(C _1-3 алкил)), -NHCONR _a1 R _b1 , -NHSO ₂ (С _1-3 алкил), -NHSO ₂ NR _a1 R _b1, -NHSO ₂ (C ₃ - ₆ циклоалкил), -SO ₂ (C _1-3 алкил), -SO ₂ NR _a1 R _b1 , четырех-шестичленное насыщенное моногетероциклическое кольцо, С _6-10 арил или пяти- или шестичленный моноциклический гетероарил, где каждое четырех-шестичленное насыщенное моногетероциклическое кольцо, С _6-10 арил и пяти- или шестичленный моноциклический гетероарил не замещены или замещены одной, двумя или тремя замещающими группами, выбранными из группы, состоящей из циано, гидроксила, гидроксиметила, цианометила, галогена, С _1-3 алкоксила, С _1-3 алкила, -CO(C _1-3 алкила), -CONR _a1 R _b1 , NR _a1 R _b1 , -NHCO(С _1-3 алкила), -NHCONR _a1 R _b1 , -NHSO ₂ (С _1-3 алкила), -NHSO ₂ NR _a1 R _b1 и -NHSO ₂ (C _3-6 циклоалкила); And

[0091] R _a1 and R _b1 are each independently hydrogen, C _1-6 alkyl (preferably C _1-3 alkyl), halogenated C _1-6 alkyl (preferably halogenated C _1-3 alkyl), C _3-6 cycloalkyl, or a four to six membered saturated monoheterocyclic ring; or R _a1 and R _b1 together with their associated nitrogen atom form a four to six membered saturated monoheterocyclic ring, where the four to six membered saturated monoheterocyclic ring is unsubstituted or substituted with one, two or three C _1-3 alkyl groups.

[0092] In some embodiments, R _1a and R _1b are each independently hydrogen, halogen, or C _1-3 alkyl; or R _1a and R _1b together with their associated carbon atom form a three to six membered saturated monocyclic ring.

[0093] In some embodiments, each of R _1a and R _1b independently represents hydrogen, fluorine, methyl or ethyl.

[0094] In some embodiments, R _1a and R _1b , together with their associated carbon atom, form a cyclopropyl ring, a cyclobutyl ring, or a cyclopentyl ring.

[0095] In some embodiments, R _1c is hydrogen, halogen, C _1-3 alkyl, C _1-3 alkoxy, or halogenated C _1-3 alkyl.

[0096] In some embodiments, R _1c is hydrogen, fluoro, methyl, ethyl, methoxy, or trifluoromethyl.

[0097] In some embodiments, R _1a and R _1b are each independently hydrogen, fluorine, or methyl; and R _1c is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0098] In some embodiments, R _1a and R _1b are each methyl; and R _1c is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0099] In some embodiments, R _1a and R _1b are each fluorine; and R _1c is hydrogen, fluorine, methyl or ethyl.

[0100] In some embodiments, R _1a and R _1b are each hydrogen; and R _1c is hydrogen, fluorine, methyl, ethyl, or trifluoromethyl.

[0101] In some embodiments, R _1a is hydrogen; R _1b is methyl; and R _1c is methyl or ethyl.

[0102] In some embodiments, R _1a and R _1b , together with their associated carbon atom, form a cyclopropyl ring, a cyclobutyl ring, or a cyclopentyl ring; and R _1c is hydrogen, fluorine, methyl, ethyl, methoxy, or trifluoromethyl.

[0103] In some embodiments, the compound represented in formula (III) has the structure represented in formula (I-1) or formula (II-1):

[0104] where R _a , R _b and R _c in formula (1-1) are those defined in the compound represented in formula (I); R ₁ , R ₂ and R ₃ in formula (II-1) are those defined in the compound represented in formula (II); and * is a chiral carbon atom in the R or S configuration.

[0105] In some embodiments, the compound represented in formula (I-1) has the structure represented in formula (I-1a) or formula (I-1b); and the compound shown in formula (II-1) has the structure shown in formula (II-1a) or formula (II-1b):

[0106] In some embodiments, the compound represented in formula (I-1a) is selected from the following structures:

[0107] In some embodiments, the compound represented in formula (I-1b) is selected from the following structures:

[0108] In some embodiments, the compound represented in formula (II-1a) is selected from the following structures:

[0109] In some embodiments, the compound represented in formula (II-1b) is selected from the following structures:

[0110] The present disclosure also relates to methods for preparing the compounds represented in formula (I-a), formula (I-b), formula (II-a) and formula (II-b), or their salts, and thus synthesize essentially optically pure oxaspiro-substituted pyrrolopyrazole derivatives, and the derivative practically does not contain other stereoisomers. There is a need to provide such compounds in substantially pure stereoisomer form with an isomeric purity of at least 98% ee, most preferably at least 99% ee. Isomeric excess value is defined as a quantitative measurement of the percentage of a major isomer that is greater than the percentage of minor isomers that co-exist with the major isomer, and can be readily measured by appropriate methods established and known in the art, such as chiral high performance liquid chromatography (HPLC), chiral gas chromatography (GC), and nuclear magnetic resonance (NMR) using chiral shift reagents.

[0111] It should be understood that within the scope of this disclosure, the above technical features of the present disclosure and the technical features specifically described below (eg, embodiments) may be combined with each other to create new or preferred technical solutions. Due to space limitations, they will not be repeated in this document.

BRIEF DESCRIPTION OF GRAPHICS

[0112] FIG. 1 is a block diagram of single crystals of compound V5.

[0113] FIG. 2 is a block diagram of single crystals of compound V6.

DESCRIPTION OF EMBODIMENTS

[0114] Through extensive and in-depth research, the inventors unexpectedly discovered such optically pure oxaspiro-substituted pyrrolopyrazole derivatives, which have not only an excellent analgesic effect, but also a good measurement error. In addition, the compounds of the present disclosure have excellent pharmacokinetic properties. Thus, these compounds are expected to be developed as drugs for the treatment and prevention of pain and diseases associated with pain. On this basis, the inventors have completed the present disclosure.

[0115] Definition of terms

[0116] As used herein, the term "alkyl" refers to a linear and branched saturated aliphatic hydrocarbyl group. C _1-10 alkyl refers to an alkyl group having 1 to 10 carbon atoms, C _1-6 alkyl is preferred, and C _1-3 alkyl is more preferred, which have similar definitions. Examples of alkyl more preferably include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1 ,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2.2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl , 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3.3-diethylhexyl, 2,2-diethylhexyl, their various branched isomers, etc.

[0117] As used herein, the term "alkenyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group having carbon-carbon double bonds (C=C), which preferably contains 2 to 10 carbon atoms (C _2-10 ), more preferably contains 2 to 6 carbon atoms (C _2-6 ), and even more preferably contains 2 to 4 carbon atoms (C _2-4 ). Examples of alkenyl include, but are not limited to, vinyl, propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, hexenyl, and the like.

[0118] As used herein, "alkynyl" refers to a linear and branched unsaturated aliphatic hydrocarbon group having carbon-carbon triple bonds that preferably contains 2 to 10 carbon atoms (C _2-10 ), more preferably contains 2 to 6 carbon atoms (C _2-6 ), and even more preferably contains 2 to 4 carbon atoms (C ₂ - ₄ ). Examples of alkynyl include, but are not limited to, ethynyl, propynyl, n-butynyl, isobutynyl, pentynyl, hexynyl, and the like.

[0119] As used herein, the terms "cycloalkyl" and "cycloalkyl ring" may be used interchangeably, and both refer to saturated or partially unsaturated monocyclic or polycyclic hydrocarbon groups. "C ₃ - ₈ cycloalkyl" refers to a cyclic hydrocarbyl group having 3 to 8 carbon atoms, and preferred is C ₃ - ₆ cycloalkyl, which has a similar definition. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc., and cyclopropyl, cyclopentyl, and cyclohexenyl are preferred.

[0120] As used herein, the term "spiro ring" refers to a polycyclic group in which the monocyclic rings share one carbon atom (called a spiro atom), these monocyclic rings may contain one or more double bonds, but none of the rings has a fully conjugated π electron system. According to the number of rings, spiro rings are divided into spiro bicyclic rings and spiro polycyclic rings, with spiro bicyclic rings being preferred. A four-membered/five-membered, five-membered/five-membered or five-membered/six-membered spirobicyclic ring is more preferred. For example:

[0121] As used herein, the term "spiro heterocyclic ring" refers to a polycyclic hydrocarbon in which the monocyclic rings share one atom (referred to as a spiro atom), one or two ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _n (where n is an integer from 0 to 2), and the other atoms in the ring are carbon atoms. These rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. According to the number of rings, spiro-heterocyclic rings are divided into bicyclic spiro-heterocyclic rings and polycyclic spiro-heterocyclic rings, with bicyclic spiro-heterocyclic rings being preferred. A four-membered/five-membered, five-membered/five-membered or five-membered/six-membered bicyclic spiro-heterocyclic ring is more preferred. For example:

[0122] As used herein, the term "bridged ring" refers to a polycyclic group in which two or more common carbons are shared, where the common carbons are referred to as bridging carbons, and the two bridging carbons may be linked by a carbon chain or bond referred to as a bridge. This bridging ring may contain one or more double bonds, but none of the rings has a fully conjugated π electron system. Bicyclic or tricyclic bridged rings are preferred. For example:

[0123] As used herein, the term "bridged heterocyclic ring" refers to a polycyclic group in which two or more common atoms are shared, one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _n (where n is an integer from 0 to 2), and the other ring atoms are carbon atoms. A bridged heterocyclic ring may contain one or more double bonds, but none of the rings has a fully conjugated π electron system. Bridged heterobicyclic or heterotricyclic rings are preferred. For example:

[0124] As used herein, the term "eight to ten membered bicyclic ring" refers to a bridged ring containing two rings with 8 to 10 ring atoms, and the bicyclic ring can be a saturated all-carbon bicyclic ring or a partially unsaturated all-carbon bicyclic ring. Examples of an eight to ten membered bicyclic ring include, but are not limited to,

[0125] As used herein, the term "eight to ten membered heterobicyclic ring" refers to a bridged heterocyclic ring containing two rings with 8 to 10 ring atoms, in which one, two, three, four, or five carbon atoms in the ring are replaced by heteroatoms selected from nitrogen, oxygen, or sulfur. Examples of the eight to ten membered heterobicyclic ring include, but are not limited to, tetrahydroquinoline ring, tetrahydroisoquinoline ring, decahydroquinoline ring, etc.

[0126] As used herein, the term "C _1-8 alkoxy" refers to -O-(C _1-8 alkyl), where alkyl is as defined above. Preferred is C _1-6 alkoxy, preferred is C _1-4 alkoxy, and more preferred is C _1-3 alkoxy. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, isobutoxy, pentyloxy, and the like.

[0127] As used herein, the term "C _3-8 cycloalkoxy" refers to -O-(C _3-8 cycloalkyl), where cycloalkyl is as defined above. Preferred is C ₃ - ₆ cycloalkoxy. Examples of cycloalkoxy include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.

[0128] As used herein, the terms "C _6-10 aryl" and "C _6-10 aromatic ring" can be used interchangeably, both referring to an all-carbon monocyclic or fused polycyclic (i.e., the rings have common adjacent pairs of carbon atoms) group having a conjugated π electron system and refer to an aryl group having 6 to 10 carbon atoms. Phenyl and naphthyl are preferred, and phenyl is more preferred.

[0129] As used herein, the term "one bond" refers to a covalent bond through which two groups are linked.

[0130] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0131] As used herein, the term "halogenated" refers to one or more (eg, one, two, three, four, or five) hydrogen atoms in the group being replaced by a halogen.

[0132] For example, "halogenated C _1-10 alkyl" means that the alkyl group is substituted with one or more (eg, one, two, three, four or five) halogen atoms, in which alkyl is as defined above. Halogenated C _1-8 alkyl is preferred, more preferred is halogenated C _1-6 alkyl, more preferred is halogenated C _1-4 alkyl, and even more preferred is halogenated C _1-3 alkyl. Examples of halogenated C _1-6 alkyl include, but are not limited to, monochloromethyl, dichloromethyl, trichloromethyl, monochloroethyl, 1,2-dichloroethyl, trichloroethyl, monobromoethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, etc.

[0133] In another example, "halogenated Cio alkoxy" means that the alkoxy group is substituted with one or more (eg, one, two, three, four, or five) halogen atoms, wherein the alkoxy is as defined above. Halogenated C _1-8 alkoxy is preferred, more preferred is halogenated C _1-6 alkoxy, more preferred is halogenated C _1-4 alkoxy, and even more preferred is halogenated C _1-3 alkoxy. Halogenated alkoxy groups include, but are not limited to, trifluoromethoxy, trifluoroethoxy, monofluoromethoxy, monofluoroethoxy, difluoromethoxy, difluoroethoxy, and the like.

[0134] In another example, "halogenated C ₃ - ₈ cycloalkyl" means that the cycloalkyl group is substituted with one or more (eg, one, two, three, four, or five) halogen atoms, wherein the cycloalkyl is as defined above. Preferred is C _3-6 cycloalkyl. Examples include, but are not limited to, trifluorocyclopropyl, monofluorocyclopropyl, monofluorocyclohexyl, difluorocyclopropyl, difluorocyclohexyl, etc.

[0135] As used herein, the term "deuterated C _1-8 alkyl" means that the alkyl group is substituted with one or more (eg, one, two, three, four, or five) deuterium atoms, in which alkyl is as defined above. Preferred is deuterated C _1-6 alkyl, and more preferred is deuterated C _1-3 alkyl. Examples of the deuterated alkyl group include, but are not limited to, mono-deuteromethyl, mono-deuteroethyl, di-deuteromethyl, di-deuteroethyl, tri-deuteromethyl, tri-deuteroethyl, etc.

[0136] As used herein, "amino" refers to NH ₂ , "cyano" refers to CN, "nitro" refers to NO ₂ , "benzyl" refers to -CH ₂ -phenyl, "oxo" refers to ═O, "carboxyl" refers to -C(O)OH, "acetyl" refers to -C(O)CH ₃ , "hydroxymethyl" refers to -CH ₂ OH, "hydroxyethyl" refers to -CH ₂ CH ₂ OH or -CHOHCH ₃ , "hydroxyl" refers to -OH, "thiol" refers to SH and "cyclopropylidene" has the following structure:

[0137] As used herein, the terms "heteroaryl ring" and "heteroaryl" may be used interchangeably, both refer to a group having 5 to 10 ring atoms, 6, 10, or 14 shared π electrons in an array of rings, and 1 to 5 heteroatoms in addition to carbon atoms. "Heteroatom" refers to nitrogen, oxygen or sulfur. Five or six membered monocyclic heteroaryl or eight to ten membered bicyclic heteroaryl is preferred.

[0138] As used herein, the term "three-seven-membered (four-seven-membered) saturated monocyclic ring" refers to a saturated all-carbon monocyclic ring containing 3 to 7 ring atoms, and a three- to six-membered saturated monocyclic ring is preferred. Examples of saturated monocyclic rings include, but are not limited to, cyclopropyl ring, cyclobutyl ring, cyclopentyl ring, cyclohexyl ring, cycloheptyl ring, cyclooctyl ring, etc.

[0139] As used herein, the term "three-seven-membered (four-seven-membered) saturated monoheterocyclic ring" means that one, two or three carbon atoms in a three-seven-membered saturated monocyclic ring are replaced by heteroatoms selected from nitrogen, oxygen, or S(O) _t (where t is an integer from 0 to 2), the ring fragment -O-O-, -OS- or -SS- is not included, and the other atoms in the ring are carbon atoms. Preferred is a four to six membered saturated monoheterocyclic ring, and more preferred is a five or six membered saturated monoheterocyclic ring. An example of a saturated monoheterocyclic ring includes, but is not limited to, propylene oxide, azetidine, oxetane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyrrole, piperidine, pyrroline, oxazolidine, piperazine, dioxolane, dioxane, morpholine, thiomorpholine, thiomorpholine-1,1-dioxide, tetrahydropyran, etc.

[0140] As used herein, the terms "five- or six-membered monocyclic heteroaryl ring" and "five- or six-membered monocyclic heteroaryl ring" may be used interchangeably, and both refer to a monocyclic heteroaryl ring having 5 or 6 ring atoms, which includes, but is not limited to, thiophene ring, furan ring, thiazole ring, imidase ring, oxazole ring, pyrrole new ring, pyrazole ring, triazole ring, 1.2.3-triazole ring, 1,2,4-triazole ring, 1,2,5-triazole ring, 1.3.4-triazole ring, tetrazole ring, isoxazole ring, oxadiazole ring, 1,2,3-oxadiazole ring, 1,2,4-oxadiazole ring, 1.2.5-oxadiazole ash ring, 1,3,4-oxadiazole ring, thiadiazole ring, pyridine ring, pyridazine ring, pyrimidine ring, pyrazine ring, etc.

[0141] As used herein, the terms "eight to ten membered bicyclic heteroaryl ring" and "eight to ten membered bicyclic heteroaryl ring" can be used interchangeably, and both refer to a bicyclic heteroaryl ring containing 8 to 10 ring atoms, including, but not limited to, benzofuran, benzothiophene, indole, isoindole, quinoline, isoquinoline, indazole, benzothiazole, benzimidazole, quinazoline, quinoxaline, cinnoline, phthalazine, pyrido[3,2-d]pyrimidine, pyrido[2,3-d]pyrimidine, pyrido[3,4-d]pyrimidine, pyrido[4,3-d]pyrimidine, 1,8-naphthyridine, 1,7-naphthyridine, 1,6-naph thyridine and 1,5-naphthyridine.

[0142] As used herein, the term "substituted" means that each of one or more hydrogen atoms, preferably one to five hydrogen atoms, and more preferably one to three hydrogen atoms in a group, is independently substituted with an appropriate number of substituent groups, i.e. the substituent groups are only in their possible chemical positions, and those skilled in the art can determine (experimentally or theoretically) possible or impossible substitutions without any effort. For example, the bonding of an amino or hydroxyl group having free hydrogen atoms to carbon atoms having unsaturated (eg, olefinic) bonds may be unstable.

[0143] Unless otherwise indicated, the phrase "each substituent group is independently selected from..." as used herein means that when more than one hydrogen atom in a group is substituted with substituent groups, the substituent groups may be of the same type or different types, and the selected substituent groups are independent in terms of their types.

[0144] Unless otherwise indicated, the phrase "...are the same or different, and each independently..." as used herein means that when there is more than one identical substituent group in a general formula, the groups may be the same or different and independent in their types. For example, provided that L is (CR ₀₁ R ₀₂ )s when s is 2, that is, L is (CR ₀₁ R ₀₂ )-(CR ₀₁ R ₀₂ ), where the two groups R ₀₁ or R ₀₂ may be the same or different and independent in their types. For example, L may be C(CH ₃ )(CN)-C(CH ₂ CH ₃ )(OH), C(CH ₃ )(CN)-C(CH ₃ )(OH), or C(CN)(CH ₂ CH ₃ )-C(OH)(CH ₂ CH ₃ ).

[0145] Unless otherwise indicated, any of the groups described herein may be substituted or unsubstituted. Если указанная выше группа является замещенной, при этом замещающие группы предпочтительно представляют собой от одной до пяти групп, каждая из которых независимо выбрана из CN, галогена, С _1-10 алкила (предпочтительно С _1-6 алкила и более предпочтительно C _1-3 алкила), С _1-10 алкоксила (предпочтительно C _1-6 алкоксила и более предпочтительно С _1-3 алкоксила), галогенированного С _1-8 алкила (предпочтительно галогенированного С _1-6 алкила и более предпочтительно галогенированного С _1-3 алкила), С _3-8 циклоалкила (предпочтительно С _3-6 циклоалкила), галогенированного С _1-8 алкоксила (предпочтительно галогенированного С _1-6 алкоксила и более предпочтительно галогенированного С _1-3 алкоксила), C _1-8 алкилзамещенного амидо, амидо, галогенированного С _1-8 С _1-8 алкилзамещенного амидо, ацетила, гидроксила, гидроксиметила, гидроксиэтила, карбоксила, нитро, С _6-10 арила (предпочтительно фенила), С _3-8 циклоалкоксила (предпочтительно С _3-6 циклоалкоксила), С _2-10 алкенила (предпочтительно С _2-6 алкенила и более предпочтительно С _2-4 алкенила), С ₂ - ₁₀ алкинила (предпочтительно С _2-6 алкинила и более предпочтительно С _2-4 алкинила), -CONR _a0 R _b0 , -C(O)O(C _1-10 алкила) (предпочтительно -С(O)O(С _1-6 алкила) и более предпочтительно -C(O)O(C _1-3 алкила)), -СНО, -OC(O)(C _1-10 алкила) (предпочтительно -ОС(O)(С _1-6 алкила) и более предпочтительно -OC(O)(C _1-3 алкила)), -SO ₂ (C _1-10 алкила) (предпочтительно -SO ₂ (C _1-6 алкила) и более предпочтительно -SO ₂ (C _1-3 алкила)), -SO ₂ (C _6-10 арила) (предпочтительно -SO ₂ (С ₆ арила), а именно -SO ₂ -фенила), -СО(С _6-10 арила) (предпочтительно -CO(C ₆ арила), а именно -СО-фенила), четырех-шестичленного насыщенного или ненасыщенного моногетероциклического кольца, четырех-шестичленного насыщенного или ненасыщенного моноциклического кольца, пяти- или шестичленного моноциклического гетероарильного кольца, восьми-десятичленного бициклического гетероарильного кольца, спирокольца, спирогетероциклического кольца, мостикового кольца и мостикового гетероциклического кольца, в которой каждый из R _a0 и R _b0 независимо представляет собой водород или С _1-3 алкил.

[0146] Each substituent group described above may itself also be substituted by the groups described herein.

[0147] When the four-to-six-membered (six-membered) saturated monoheterocyclic ring described herein is substituted, the substituent groups may be in their possible chemical positions. Representative substitutions for typical monoheterocyclic rings are shown below:

substituent, where “substituent” refers to the various substituent groups described herein, and “refers to bonding to other atoms.

[0148] Unless otherwise indicated, when the four to six membered saturated monoheterocyclic ring described herein is a substituent group, it may also be substituted or substituted with one, two or three substituent groups selected from the group consisting of halo, hydroxyl, C_1-3 alkyl, O=, NR_a0Rb_0, hydroxymethyl, hydroxyethyl, carboxyl, -C(O)O(C_1-3 alkyl), acetyl, halogenated C_1-3 alkyl, C₁-₃alkoxy, C₃-₆ cycloalkyl, azetidine, oxetane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyrrole, piperidine, oxazolidine, piperazine, dioxolane, dioxane, morpholine, thiomorpholine, thiomorpholine-1,1-dioxide, tetrahydropyran, thiophene ring, N-alkylpyrrole ring, furan ring, thiazole ring, imidazole ring, oxazole ring, pyrrole ring, pyrazole ring, triazole ring, tetrazole ring, isoxazole ring, oxadiazole ring, thiadiazole ring, pyridine ring, pyridazine ring, pyrimidine ring and pyrazine ring, in which each of R_a0 and R_b0 is independently hydrogen or C_1-3 alkyl.

[0149] A "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0150] "Pharmaceutical acid addition salts" refers to salts formed with inorganic or organic acids that can retain the biological effectiveness of free alkali without causing other side effects.

[0151] "Pharmaceutical base addition salts" include, but are not limited to, salts such as sodium salts, potassium salts, calcium salts, and magnesium salts, inorganic alkalis, and salts such as ammonium salts, triethylamine salts, lysine salts, and organic alkali arginine salts.

[0152] "Solvate" as described herein refers to a complex formed by a compound of the present disclosure and a solvent. Compounds either react in solvents or precipitate or crystallize from solvents. For example, a complex formed by a compound and water is called a "hydrate". Solvates of the compound represented in formula (I) are within the scope of this disclosure.

[0153] The present disclosure further relates to prodrugs of the above compounds. Prodrugs include known amino protecting groups and carboxy protecting groups which are hydrolyzed under physiological conditions or released via enzymatic reactions to give the parent compounds. For specific prodrug preparation methods, see Saulnier, M.G.; Frennesson, D. B.; Deshpande, M.S.; Hansel, S.B. and Vysa, D.M. Bioorg. Med. Chem Lett. 1994, 4, 1985-1990; and Greenwald. R.B.; Choe, Y. H.; Conover, CD.; Shum, K.; Wu, D.; Royzen, M.J. Med. Chem. 2000, 43, 475.

[0154] Typically, a compound or a pharmaceutically acceptable salt, solvate or prodrug thereof of the present disclosure and one or more pharmaceutical carriers may be formulated into suitable formulations for administration. These formulations are suitable for oral administration, rectal administration, topical administration, intraoral administration and other parenteral (eg subcutaneous, intramuscular and intravenous) administration. For example, formulations suitable for oral administration include capsules, tablets, granules, syrups, and the like. The compound of the present disclosure contained in these formulations may be a solid powder or particles, a solution or suspension in an aqueous or non-aqueous liquid, or a water-in-oil or oil-in-water emulsion. The above formulations may be prepared from the active compound and one or more carriers or excipients by general pharmaceutical methods. The carriers mentioned above must be compatible with the active compound or other excipients. For solid formulations, non-toxic carriers commonly used include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like. For liquid formulations, carriers commonly used include water, saline, aqueous glucose, ethylene glycol, polyethylene glycol, and the like. The active compound may form a solution or suspension with the above carrier.

[0155] Compositions of the present disclosure are prepared, dosed, and administered in accordance with medical practice. The "therapeutically effective amount" of the compound to be administered is determined by factors such as the particular disease being treated, the individual being treated, the cause of the disorder, the purpose of the drug, the route of administration, and so on.

[0156] As used herein, the term "therapeutically effective amount" refers to an amount of a compound of the present disclosure that will elicit a biological or medical response in an individual, such as reducing or inhibiting enzyme or protein activity or relieving symptoms, alleviating a disorder, slowing or delaying disease progression, and preventing disease.

[0157] In general, although the effective dose of a compound of the present disclosure used for treatment may vary greatly in the application of the present disclosure depending on the particular application, disease or pathological condition, the dose can be easily determined by those skilled in the art. For each of the respective compositions described herein, and in order to achieve a therapeutic effect in each of the diseases described herein, a suitable therapeutically effective dose of a compound represented in formula (I) or formula (II), or a therapeutically acceptable salt, solvate or prodrug of the present disclosure, which is contained in a pharmaceutical composition of the present disclosure, is usually from 0.1 μg to 5 g per kilogram of patient body weight per day and preferably from 1 to 1000 mg per kilogram body weight per day.

[0158] The required dose is preferably administered as two, three, four, five, six or more dose portions at appropriate intervals throughout the day. These portions of the dose may be administered as a unit dose. For example, each unit dose contains 10 µg to 1000 mg, preferably 50 µg to 500 mg, more preferably 50 µg to 250 mg, and most preferably 50 µg to 10 mg of the active ingredient. Or the dose can be administered by continuous infusion, depending on the need of the patient's disease.

[0159] As used herein, the term "pharmaceutically acceptable carrier" refers to non-toxic, inert and solid or semi-solid substances or liquid excipients, diluents, encapsulating materials, or adjuvants, or excipients of any type, which are compatible with patients, preferably mammals, and more preferably with humans, and are suitable for delivering the active reagent to the target without termination of the reagent.

[0160] As used herein, the term "patient" refers to an animal, preferably a mammal, and more preferably a human. The term "mammal" refers to warm-blooded vertebrate mammals, which include cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice or rats, pigs, and humans.

[0161] As used herein, the term "treatment" refers to alleviating, slowing the progression, attenuating, preventing, or maintaining an existing disease or disorder (eg, cancer). Treatment also includes recovery from one or more symptoms of the disease or disorder, prevention of development, or relief to a certain extent.

[0162] Methods of obtaining

[0163] The experimental method described in the following examples without imposing specific conditions generally follows conventional conditions, such as those described in Sambrook, et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturers.

[0164] Unless otherwise indicated, terms used herein have the same meanings as terms known to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applicable to the present disclosure.

[0165] Methods for preparing compounds of the present disclosure:

[0166] The compounds represented in formula (I) and formula (II) can be obtained using existing methods, such as the following methods, methods equivalent to the following methods, and the methods described in the examples. In the following preparations, the raw starting compound may be in the form of a salt, and the salt may be any pharmaceutically acceptable salt as shown for the compounds represented in formula (I) and formula (II) of the present disclosure.

[0167] Reaction scheme (a)

[0168] In each formula of the above reaction scheme, * represents a chiral carbon atom in the R- or S-configuration (same hereinafter), and all groups are as defined in the description.

[0169] In particular, the compound represented in formula (I) can be obtained by the following method: reductive amination is carried out on the compound represented in formula (I-1) and compound 1b to obtain the compound represented in formula (I). Reductive amination is carried out in an inert solvent, wherein the compound represented in formula (I-1), compound 1b and the reducing agent are reacted at a certain temperature (for example, from -20°C to 80°C, preferably from 0°C to 60°C, and more preferably from 20°C to 60°C) for a period of time (for example, from 0.5 hours to 48 hours, preferably from 0.5 hours to 5 hours. ) to obtain the compound represented in formula (I). An inert solvent and reducing agent known in the art, wherein the reducing agent may be selected from tetrabutylammonium borohydride, sodium malonyloxyborohydride, sodium triacetoxyborohydride, sodium cyanoborohydride, sodium borohydride, lithium borohydride, potassium borohydride, borane, etc. The inert solvent may be selected from C _1-4 alkyl alcohol, toluene, xylene, methyl tert-butyl ether, tetrahydrofuran, 1,4-dioxane, diethyl ether, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, or combinations thereof.

[0170] The compound shown in (I-1) can be produced using the reaction scheme (a-1) or the reaction scheme (a-2).

[0171] Reaction Scheme (a-1)

[0172] In each formula of the above scheme, all groups are as defined in the description.

[0173] In particular, the compound represented in the formula (i-2) can be obtained by condensing the compound represented in the formula (i-1) and (S)-2-methylpropane-2-sulfinamide (in the presence of a catalyst such as titanium ethoxide and titanium isopropoxide); wherein the compound represented in formula (ii-2) is reduced with a reducing agent (e.g. sodium borohydride, lithium trisec-butylborohydride, sodium cyanoborohydride and diisobutylaluminum hydride) and separated by preparative high performance liquid chromatography to give two separate isomers, i.e. the compounds represented in formula (i-3a) and in formula (i-3b); and wherein the compounds represented in formula (i-3a) and formula (i-3b) are subjected to deprotection of the amino group using an acid (for example, chlorine hydride) to obtain compounds represented in formula (I-1a) and formula (I-1b), respectively.

[0174] Reaction Scheme (a-2)

[0175] In each formula of the above scheme, all groups are as defined above.

[0176] In particular, the compound represented in the formula (i-2) can be obtained by condensation of the compound represented in the formula (i-1) and (3)-2-methylpropane-2-sulfinamide (in the presence of a catalyst such as titanium ethoxide and titanium isopropoxide); chiral resolution is carried out on the compound represented in formula (i-2) to give two separate isomers, i.e. compounds represented in formula (i-2a) and formula (i-26); while the compounds represented in formula (i-2a) and formula (i-26), respectively, are reduced with reducing agents (for example, sodium borohydride, lithium tri-sec-butylborohydride, sodium cyanoborohydride and diisobutylaluminum hydride) to obtain compounds represented in formula (i-3a) and in formula (i-3b); while the compounds represented in formula (i-3a) and in formula (i-3b) are subjected to deprotection of the amino group using an acid (for example, chlorine hydride) to obtain compounds represented in formula (I-1a) and formula (I-1b), respectively.

[0177] A chiral excipient, i. e. (S)-2-methylpropane-2-sulfinamide, in reaction schemes (a-1) and (a-2), can be replaced by other chiral excipients having the same chiral inductive function, such as (S)-methylbenzylamine.

[0178] Reaction scheme (b)

[0179] In each formula of the above scheme, * indicates the R or S configuration, and all groups are as defined in the description.

[0180] In particular, the compound represented in formula (II) can be obtained by the following method: reductive amination is carried out on the compound represented in formula (II-1) and compound 1b to obtain the compound represented in formula (II). Reductive amination is carried out in an inert solvent, and wherein the compound represented in formula (II-1), compound 1b and the reducing agent react at a certain temperature (for example, from -20°C to 80°C, preferably from 0°C to 60°C, and more preferably from 20°C to 60°C) for a period of time (for example, from 0.5 hours to 48 hours, preferably from 0.5 hours to 5 hours) to obtain the compound represented in formula (I). An inert solvent and reducing agent known in the art, wherein the reducing agent may be selected from tetrabutylammonium borohydride, sodium malonyloxyborohydride, sodium triacetoxyborohydride, sodium cyanoborohydride, sodium borohydride, lithium borohydride, potassium borohydride, borane, etc. The inert solvent may be selected from C _1-4 alkyl alcohol, toluene, xylene, methyl tert-butyl ether, tetrahydrofuran, 1,4-dioxane, diethyl ether, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, or combinations thereof.

[0181] The compound represented in the formula (II-1) can be obtained using the reaction scheme (b-1) or the reaction scheme (b-2).

[0182] Reaction Scheme (b-1)

[0183] In each formula of the above scheme, all groups are as defined in the description.

[0184] In particular, the compound represented in formula (ii-2) can be obtained by condensing the compound represented in formula (ii-1) and (S)-2-methylpropane-2-sulfinamide (in the presence of a catalyst such as titanium ethoxide and titanium isopropoxide); wherein the compound represented in formula (ii-2) is reduced with a reducing agent (for example, sodium borohydride, lithium trisec-butylborohydride, sodium cyanoborohydride, and diisobutylaluminum hydride) and separated by preparative high performance liquid chromatography to obtain compounds represented in formula (ii-3a) and formula (ii-3b); and wherein the compounds represented in formula (ii-3a) and formula (i-3b) are subjected to amino protection using an acid (for example, chlorine hydride) to obtain compounds represented in formula (II-1a) and formula (II-1b), respectively.

[0185] Reaction Scheme (b-2)

[0186] In each formula of the above scheme, all groups are as defined in the description.

[0187] In particular, the compound represented in the formula (ii-2) can be obtained by condensation of the compound represented in the formula (ii-1) and (R)-2-methylpropane-2-sulfinamide (in the presence of a catalyst such as titanium ethoxide and titanium isopropoxide); chiral resolution is carried out on the compound represented in formula (ii-2) to give two separate isomers, i.e. the compounds represented in formula (ii-2a) and in formula (ii-2b); while the compounds presented in formula (ii-2a) and in formula (ii-2b), respectively, are reduced with reducing agents (for example, sodium borohydride, lithium tri-sec-butylborohydride, sodium cyanoborohydride, diisobutylaluminum hydride) to obtain compounds presented in formula (ii-3a) and in formula (ii-3b); while the compounds represented in the formula (ii-3a) and in the formula (ii-3b) are subjected to deprotection of the amino group using an acid (for example, chlorine hydride) to obtain the compounds represented in the formula (P-1a) and in the formula (II-16), respectively.

[0188] A chiral excipient, i. e. (R)-2-methylpropane-2-sulfinamide, in reaction schemes (b-1) and (b-2), can be replaced by other chiral excipients having the same chiral inductive function, such as (R)-methylbenzylamine.

[0189] Compounds containing an amino group, a carboxyl group, or a hydroxyl group, which are used in the present disclosure, can be obtained from compounds that have been protected by protecting groups commonly used for groups if necessary, and after the reaction process of the above reaction scheme, a known deprotection can be performed.

[0190] Compared with the prior art, the present disclosure mainly has the following advantages.

[0191] The present disclosure provides a range of optically pure oxaspiro-substituted pyrrolopyrazole derivatives with novel structures that exhibit high inhibitory activity (EC ₅₀ is 1 nM to 50 nM, more preferably 1 nM to 30 nM, and most preferably 1 nM to 10 nM) against cAMP with high Emax values (greater than 50%, preferably greater than 8 0%) and have excellent analgesic effects. In addition, the compounds of the present disclosure have small Emax values with respect to β-arrestin (less than 50%, more preferably less than 20%, and most preferably less than 10%) and have a good measurement error. Thus, the compounds of the present disclosure can be formulated into drugs for the treatment and prevention of pain and diseases associated with pain.

[0192] The present disclosure will be further described below with reference to specific examples. It should be understood that these examples are only for the purpose of describing the present disclosure and not limiting the scope of the present disclosure. The experimental method described in the following examples without the introduction of specific conditions, usually corresponds to the usual conditions or conditions recommended by the manufacturers. Unless otherwise stated, percentages and parts are calculated based on weight. Unless otherwise indicated, terms used herein have the same meanings as terms known to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applicable to the present disclosure.

[0193] Reagents and instruments

[0194] ¹ HNMR: Bruker AVANCE-400 NMR spectrometer, internal standard: tetramethylsilane (TMS);

[0195] LC-MS: Agilent 1290 HPLC System/6130/6150 MS LC/MS spectrometer (manufacturer: Agilent), column: Waters BEH/CHS, 50×2.1 mm, 1.7 µm.

[0196] Detection of single crystals was performed using a single crystal X-ray diffractometer Bruker D8 VENTURE.

[0197] Preparative High Performance Liquid Chromatography (pre-HPLC): GX-281 (manufacturer: Gilson).

[0198] An ISCO CombiFlash Rf75 or Rf200 automated chromatograph was used with Agela 4 g, 12 g, 20 g, 40 g, 80 g, and 120 g disposable silica gel columns.

[0199] Known starting materials can be synthesized using methods existing in the art or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Chembee Chemicals, etc.

[0200] In the examples, the reaction process can be monitored using thin layer chromatography (TLC) and compounds can be purified using column chromatography. The developing solvent system used for column chromatography or TLC can be selected from dichloromethane-methanol system, n-hexane-ethyl acetate system, petroleum ether-ethyl acetate system, acetone system, etc., and the volume ratio of solvents is adjusted according to the polarity of the compound.

[0201] As used herein, DCE refers to 1,2-dichloroethane, THF refers to tetrahydrofuran, EA refers to ethyl acetate, PE refers to petroleum ether, DCM refers to dichloromethane, n-BuLi refers to n-butylium, HATU refers to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate y, DMF refers to dimethylformamide, DMSO refers to dimethyl sulfoxide, DIEA or DIPEA refers to N,N-diisopropylethylamine, DBU refers to 1,8-diazabicyclo[5.4.0]undec-7-ene, and DIBAL-H refers to diisobutylaluminum hydride.

[0202] As used herein, room temperature refers to about 20°C to 25°C.

[0203] Intermediate 1b

[0204] (R)-2-(9-(pyridin-2-yl)-6-oxaspiro[4,5]decan-9-yl)acetonitrile (11 g, 43 mmol, purchased from Shanghai Yucheng Pharmaceutical Technology Co., Ltd., CAS) No. 1401031-38-6) was dissolved in DCM solution (100 mL), diisobutylaluminum hydride solution ( 1.0 M, 86 ml) at -78°C and the reaction solution was stirred at -78°C for 1 h. Sodium sulfate decahydrate (30 g) was added, the reaction solution was stirred at room temperature for 0.5 h and filtered, the resulting filtrate was concentrated under reduced pressure to obtain a yellow oily intermediate. The intermediate was dissolved in an ethanol solution (100 ml), a hydrochloric acid solution (6 and 100 ml) was added, and the reaction solution was stirred at 100° C. for 36 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative liquid chromatography to obtain (R)-2-(9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9 -yl) acetaldehyde (intermediate 1b, 6.5 g) in 59% yield. MS m/s (ESI): 260.2 [M+1].

[0205] Intermediate VI

[0206] In step 1: neovaleraldehyde (17.2 g, 0.2 mol) was dissolved in acetonitrile (400 ml), triethylphosphonoacetate (48 g, 0.21 mol) and lithium chloride (8.8 g, 0.21 mol) were added, the reaction solution was cooled to 2-5°C, DBU (30.4 g, 0.2 mol) was added slowly and the reaction solution was kept at room temperature for 18 hours. The reaction solution was concentrated, the resulting residue was diluted with EA (300 ml), washed with water (100 ml × 2), washed with saturated brine (80 ml × 1), dried with anhydrous sodium sulfate and filtered, and the resulting filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (PE/EA=10/1) to give compound V1-1 (25.6 g, yellow oily liquid) in 82% yield. MS m/h (ESI): 157.2 [M+1].

[0207] In step 2: a solution of acetonitrile (80 ml) and DBU (11.4 g, 75 mmol) was added to a sealed tube containing compound VI-1 (7.8 g, 50 mmol) and pyrazole (5.1 g, 75 mmol) was placed, the reaction solution was stirred at 100 ° C for 48 hours, cooled to room temperature, concentrated, the resulting residue times EA (150 ml) was added, washed with water (60 ml x 1) _; washed with brine (30 ml x 1), dried over anhydrous sodium sulfate, filtered, and the resulting filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (PE/EA=6/1) to give compound V1-2 (8.96 g, yellow oil) in 80% yield. MS m/s (ESI): 225.1 [M+1].

[0208] In step 3: Compound V1-2 (8.96 g, 40 mmol) was dissolved in a mixture of methanol (100 ml) and water (20 ml), sodium hydroxide (3.2 g, 80 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour to complete the reaction. The reaction solution was concentrated, the resulting residue was dissolved in water (70 ml), 2 M hydrochloric acid was added to adjust the pH of the mixture to 3-4, the mixture was extracted with DCM (90 ml × 3), the combined organic phase was dried over anhydrous sodium sulfate and filtered, the resulting filtrate was concentrated under reduced pressure to obtain compound V1-3 (7 g, yellow oily liquid) with a yield of 90%. MS m/s (ESI): 197.2 [M+1].

[0209] In step 4: Compound V1-3 (7 g, 35.7 mmol) was dissolved in dry THF (120 ml), the reaction solution was cooled to -70° C. under nitrogen gas protection, and a 2.5 M solution of butyllithium in THF (35.7 ml, 89.2 mmol) was slowly added dropwise. After completion of the dropwise addition, the reaction solution was slowly warmed to -40° C. and stirred for 45 minutes. The reaction solution was quenched with water (3 ml), filtered and the resulting filtrate was concentrated. The resulting residue was purified by silica gel column chromatography (PE/EA=6/1) to give compound VI (4.76 g, yellow oil) in 15% yield. MS m/s (ESI): 179.2 [M+1].

[0210] Intermediate V2

[0211] In step 1: To DMF (80 ml), HATU (45.6 g, 0.12 mol) and DIEA (50 ml, 0.30 ml) were added 1-methylcyclopropanecarboxylic acid (10 mg, 0.10 mol) and N,O-dimethylhydroxylamine hydrochloride (14.6 g, 0.15 mol) were added successively and the reaction solution was kept at room temperature during the night. EA (500 ml) was added. The resulting organic phase was washed with 1 N. hydrochloric acid (100 ml × 2) and saturated sodium chloride solution (50 ml × 2), dried over anhydrous sodium sulfate, filtered, the resulting filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using an eluent system (PE/EA=1/0 to 1/1) to obtain compound V2-1 (11.3 g, pale yellow oily substance) with a yield of 79%. MS m/s (ESI): 144.1 [M+1].

[0212] In step 2: Compound V2-1 (14.5 g, 101 mmol) was dissolved in diethyl ether (90 ml), the reaction solution was cooled to -78°C, and 1 M diisobutylaluminum hydride (152 ml, 152 mmol) was slowly added dropwise. The reaction solution was kept at -78° C. for 2 hours, 1 M HCl (16 ml) was added to quench the reaction, and after the reaction solution was heated to 0° C., 1 M HCl (16 ml) was added again. The resulting solution was dried over anhydrous sodium sulfate (70 g) and filtered to give compound V2-2.

[0213] In step 3: lithium chloride (6.5 g, 153 mmol) was added to acetonitrile (60 ml), triethylphosphonoacetate (24.5 ml, 122 mmol) and DBU (18.1 ml, 121 mmol) were added successively after the reaction solution was cooled to 0°C, compound V2-2 (8.5 g) was added dropwise and the reaction solution was left at room temperature overnight. Water (150 ml) was added, the mixture was extracted with EA (150 ml × 3), the resulting organic phases were combined, the combined organic phase was dried over anhydrous sodium sulfate and filtered, the resulting filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using eluents (PE/EA=1/0-10/1) to give compound V2-3 (10.5 g, colorless oily liquid) with a yield of 68%. MS m/s (ESI): 155.0 [M+1].

[00214] Step 4: Compound V2-3 (10.5 g, 0.068 mol), pyrazole (9.3 g, 0.137 mol) and potassium carbonate (18.8 g, 0.136 mol) were added to DMF (50 ml) and the reaction solution was placed at 65° C. for 45 hours. The reaction solution was cooled to room temperature, EA (300 ml) was added, the resulting organic phase was washed with water (100 ml) and saturated sodium chloride solution (100 ml×2), dried over anhydrous sodium sulfate and filtered, the resulting filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using eluents (PE/EA=1/0-5/1) to give compound V2-4 (12.3 g, colorless oily liquid) with yield 82%. MS m/s (ESI): 223.1 [M+1].

[0215] In step 5: Compound V2-4 (12.3 g, 55.3 mmol) was dissolved in a mixture of THF (50 ml) and methanol (20 ml), lithium hydroxide monohydrate (3.6 g, 85.7 mmol) and water (30 ml) was added, and the reaction solution was stirred at room temperature for 2 hours. 6 M hydrochloric acid was added to adjust the pH of the reaction solution to 3, organic the solvent was removed by centrifugation under reduced pressure, the resulting aqueous phase was extracted with DCM (100 ml×3), the resulting organic phases were combined, the combined organic phase was dried over anhydrous sodium sulfate and filtered, the resulting filtrate was concentrated under reduced pressure, and the resulting residue was purified by preparative liquid chromatography to give compound V2-5 (9.2 g, colorless oily liquid) in 86 %. MS m/s (ESI): 195.0 [M+1].

[0216] In step 6: compound V2-5 (3.05 g, 15.7 mmol) was dissolved in THF (80 ml), the reaction solution was cooled to -78°C, 2.5 M n-butyllithium (15.7 ml, 39.3 mmol) was slowly added dropwise, and the reaction solution was reacted at -78°C for 0.5 h. and reacted at -45°C for Saturated ammonium chloride solution (30 ml) was added to quench the reaction, the reaction solution was extracted with EA (50 ml × 4), the resulting organic phases were combined, the combined organic phase was dried over anhydrous sodium sulfate and filtered, the resulting filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using eluents (PE/EA=1/0 to 3/1) to give compound V2 (460 mg, colorless oily liquid) in 16.7% yield. MS m/h (ESI): 177.0 [M+1].

[0217] Intermediate V3

[0218] In Step 1: Triethylphosphonoacetate (476 ml, 2.4 mol), DBU (365 g, 2.4 mol), lithium chloride (127 g, 3 mol) and acetonitrile (1.2 L) were added to a 3 L single-necked flask, and the reaction solution was stirred at room temperature for 20 minutes. protected by argon gas. The reaction solution was cooled to 0° C. (internal temperature), and isobutyraldehyde (144 g, 2 mol) was slowly added dropwise. The reaction solution was stirred at room temperature for 12 hours. After LC-MS monitored the completion of the reaction, the reaction solution was filtered, and the resulting filter cake was washed with EA (100 ml×2). Water (1 L) was added, then extracted with EA (1.5 L×2), washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and dried by centrifugation to give compound V3-1 (175 g, colorless liquid).

[0219] In Step 2: DMF (1 L) was added to a 3 L single-necked flask, into which compound V3-1 (300 g, 2.1 mol), potassium carbonate (579 g, 4.2 mol) was placed, and pyrazole (287 g, 4.2 mol) was added with stirring, the reaction solution was stirred at 65°C for 18 hours, after the completion of the reaction process was monitored by LC-MS, the reaction solution was dried directly by centrifugation, the resulting solid was mixed with acetonitrile (150 ml) to form a suspension, and the suspension was filtered to obtain a white solid. The white solid was filtered off and the resulting filter cake was washed with EA (500 ml x 2), then washed with brine (300 ml x 3) _, dried over anhydrous sodium sulfate, and dried by centrifugation, and purified by column chromatography (mobile phase - PE containing 5% EA) to give compound V3-2 (258 g, colorless liquid) with a yield of 5 8%. MS m/s (ESI): 211.1 [M+1].

[0220] Step 3: Water (200 ml) was added to potassium hydroxide (133 g, 3.32 mol) to dissolve and the aqueous solution was pre-cooled to 5°C. Compound V3-2 (465 g, 2.21 mol), methanol (0.5 L) and THF (0.5 L) were added to a 3 L flask, pre-cooled aqueous potassium hydroxide solution was added, the reaction solution was stirred for 2 h, concentrated hydrochloric acid was added to adjust the pH of the reaction solution to about 3, the reaction solution was extracted with DCM (800 ml × 2), all obtained organic the phases were combined, the combined organic phase was washed with brine, dried and concentrated to give V3-3 (410.5 g, white solid) in 100% yield. MS m/s (ESI): 183.1 [M+1].

[0221] In step 4: under the protection of nitrogen gas, the compound V3-3 (10.2 g, 0.055 mol) and THF (200 ml) were added to a three-necked flask (500 ml) and the reaction solution was cooled to -75°C. A 2.5 M solution of n-butyllithium in THF (55 ml, 0.137 mol) was slowly added dropwise, and after completion of the dropwise addition, the reaction solution was slowly heated to -10°C and stirred again for 3 h. The organic phase was washed with brine, dried and concentrated to give a brown liquid. The brown liquid was purified by column chromatography (mobile phase was PE containing 30% EA) to give compound V3 (4 g, white solid) in 22.2% yield. MS m/s (ESI): 165.1 [M+1].

[0222] Intermediate V4

[0223] In step 1: 1-(trifluoromethyl)cyclopropane-1-carboxylic acid (6.8g, 44mmol) was dissolved in DMF solution (100ml), dimethylhydroxylamine hydrochloride solution (5.14g, 53mmol), HATU (25g, 66mmol) triethylamine (13.3g, 132mmol) was added and the reaction solution was stirred at room temperature during the night. EA (400 ml) was added, then washed with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate, the resulting organic phase was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using eluents (PE: EA=1: 1) to obtain compound V4-1 (8 g) with a yield of 92%. MS m/s (ESI): 198.1 [M+1].

[0224] In step 2: Compound V4-1 (8 g, 4.06 mmol) was dissolved in THF solution (100 ml), DIBAL-H (1.0 M, 60 ml, 6.1 mmol) was added at -78°C and the reaction mixture of the solution was stirred at -78°C for 1 h. Sodium sulfate decahydrate was added, the resulting solution was stirred for 30 min. and filtered, the resulting filtrate was concentrated under reduced pressure to obtain compound V4-2.

[0225] In step 3: Compound V4-2 (5.6 g, 40.6 mmol) was dissolved in a solution of acetonitrile (100 ml), ethyl diethoxyphosphinyl formate (13.64 g, 60.9 mmol), DBU (12.34 g, 81.2 mmol) and lithium chloride (3.4 g, 81.2 mmol) were added and the reaction solution was stirred at room temperature. temperature during the night. EA (300 ml) was added, then washed with brine sodium bicarbonate and dried over anhydrous sodium sulfate, the resulting organic phase was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using eluents (PE: EA=2: 1) to obtain compound V4-3 (4 g) with a yield of 47%. MS m/s (ESI): 209.1 [M+1].

[0226] In step 4: pyrazole (2.61 g, 38.5 mmol) was dissolved in a DMF solution (100 ml), compound V4-3 (4 g, 19.2 mmol) and potassium carbonate (7.95 g, 57.6 mmol) were added and the reaction solution was stirred at 60° C. overnight. EA (300 ml) was added, then washed with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate, the resulting organic phase was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using eluents (PE: EA=1:1) to obtain compound V4-4 (3.3 g) with a yield of 62%. MS m/s (ESI): 277.1 [M+1].

[0227] In step 5: Compound V4-4 (3.3 g, 12 mmol) was dissolved in a solution of methanol (20 ml) and water (5 ml), lithium hydroxide (0.55 g, 24 mmol) was added, and the reaction solution was stirred at room temperature for 2 hours. Hydrochloric acid solution (3 N) was added to adjust the pH of the reaction solution to 2, the reaction solution was extracted with a mixture of DCM and methane ol (10/1), the resulting organic phase was distilled under reduced pressure, and the resulting residue was purified by preparative liquid chromatography to give compound V4-5 (1.8 g) in 61% yield. MS m/s (ESI): 249.1 [M+1].

[0228] In step 6: Compound V4-5 (1.8 g, 7.26 mmol) was dissolved in THF solution (30 ml), n-butyllithium solution (2.5 M, 7.3 ml, 18.1 mmol) was added at -78°C and the reaction solution was stirred at -20°C for 30 min. An aqueous sodium chloride solution was added, the solution was extracted with EA, the resulting organic phase was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using eluents (PE:EA=1:2) to give compound V4 (700 mg) in 42% yield. MS m/s (ESI): 231.1 [M+1].

[0229] Connection V5

[0230] Compound 3-3a (0.11 g, 0.67 mmol) was dissolved in dichloromethane (8 ml), triethylamine (0.2 g, 2 mmol) and 4-bromobenzenesulfonyl chloride (0.204 g, 0.804 mmol) were added at 0°C, the reaction solution was stirred at 0°C for 1 h, dichloromethane (10 ml) was added, the resulting solution was washed saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the resulting residue was purified by preparative liquid chromatography to give 4-bromo-N-((4S,6S)-6-isopropyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-yl)benzene sulfonamide (Compound V5, 0.06 g, white solid) with yield of 23.3%. MS m/s (ESI): 384.1 [M+1].

[0231] Obtaining single crystals of the compound V5: a small amount of compound V5 was placed in a small beaker, ethyl acetate was added to dissolve, the beaker was sealed with a thin film, and the film was pierced to form small holes. Petroleum ether was placed in a large grinder, a small grinder was placed in a large grinder, a large grinder was sealed and placed at room temperature for crystallization, and crystals were precipitated from the solution. Single crystal X-ray analysis of the crystals was performed using a Bruker D8 VENTURE single crystal X-ray diffractometer, and it was determined that the absolute configuration of the crystals was (4S, 6S). The structure is shown in FIG. 1. Thus, based on the structure of single crystals of compound V5, it was determined that the absolute configuration of compound 3-3a is the configuration (4S, 6S).

[0232] Device Parameters

[0233] Connection V6

[0234] Compound V6 was prepared according to the method for preparing Compound V5. MS m/s (ESI): 384.1 [M+1].

[0235] Preparation of single crystals of compound V6: a small amount of compound V6 was placed in a small beaker, ethyl acetate was added to dissolve, the beaker was sealed with a thin film, and the film was pierced to form small holes. Petroleum ether was placed in a large grinder, a small grinder was placed in a large grinder, a large grinder was sealed and placed at room temperature for crystallization, and crystals were precipitated from the solution. Single crystal X-ray analysis of the crystals was performed using a Bruker D8 VENTURE single crystal X-ray diffractometer, and it was determined that the absolute configuration of the crystals was (4R, 6R). The structure is shown in FIG. 2.

[0236] Device Parameters

[0237] Example 1.

(4S,6S)-6-tert-butyl-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-1a) and

(4S,6R)-6-tert-butyl-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (Н-1b)

[0238] In Step 1: Compound VI (1 g, 5.6 mmol) and ^)-2-methylpropane-2-sulfinamide (1 g, 8.4 mmol) were dissolved in toluene (70 ml), titanium isopropoxide (3 ml) was added, and the reaction solution was stirred at 120°C for 1 hour. The reaction solution was cooled to room temperature, 10 ml of water was added, stirred for 20 minutes. and filtered, sodium borohydride (425 mg, 11.2 mmol) was added to the obtained filtrate, the mixture was stirred for 0.5 h, water (5 ml) was added to the reaction solution, the solution was stirred for 0.5 min. and filtered, the resulting filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (PE/EA=4/1) to give Compound 1-1 (1.26 g, yellow solid) in 85% yield. MS m/s (ESI): 284.1 [M+1].

[0239] In Step 2: Compound 1-1 (0.8 g) was purified by preparative chromatography (preparation conditions: preparative column: C18 column 21.2×250 mm; system: 10 mM NH ₄ HCO ₃ H ₂ O; wavelengths: 254/214 nm; gradients: 30%-60% changes in acetonitrile) to obtain the product 1-2a single configuration (340 mg, retention time 9.5 minutes) and product 1-26 single configuration (300 mg, retention time 10.5 minutes), respectively.

[0240] In step 3: 0.3 g (1 mmol) of compound 1-2a was dissolved in DCM (15 ml), a 4 M dioxane hydrochloride solution was added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated to give compound 1-3a (179 mg, yellow solid) in 100% yield. MS m/s (ESI): 180.1 [M+1].

[0241] На шаге 4: соединение 1-3а (179 мг, 1 ммоль) и соединение 16 (259 мг, 1 ммоль) растворяли в метаноле (30 мл) и реакционный раствор перемешивали при комнатной температуре в течение 1 ч. Добавляли цианоборогидрид натрия (248 мг, 4 ммоль), реакционный раствор перемешивали в течение 5 ч., добавляли воду (5 мл), смесь перемешивали в течение 0,5 мин., фильтровали, полученный фильтрат концентрировали при пониженном давлении и полученный остаток очищали с помощью препаративной хроматографии (условия получения: препаративная колонка: колонка С18 21,2 × 250 мм; система: 10 мМ NH ₄ HCO ₃ H ₂ O; длины волн: 254/214 нм; градиенты: 30%-60% изменений в ацетонитриле) с получением соединения Н-1а одинарной конфигурации (110 мг, белое твердое вещество) с выходом 31%. MS m/s (ESI): 423.2 [M+1]; ¹ Н ЯМР (400 МГц, ДМСО-d6) δ 8,52-8,50 (м, 1Н), 7,70-7,68 (м, 1Н), 7,45-7,42 (м, 1Н), 7,30 (д, J=1,8 Гц, 1H), 7,17-7,15 (м, 1H), 5,71 (д, J=1,7 Гц, 1H), 3,85-3,81 (м, 2Н), 3,57-3,55 (м, 2Н), 2,57-2,55 (м, 1H), 2,46-2,38 (м, 2Н), 2,32-2,30 (м, 1H), 2,01-1,88 (м, 1Н), 1,91-1,63 (м, 4Н), 1,67-1,21 (м, 9Н), 0,91 (с, 9Н), 0,62-0,59 (м, 1Н).

[0242] In step 5: compound 1-36 was obtained from compound 1-26 in accordance with the preparation method in step 3.

[0243] Step 6: Compound H-16 (110 mg, white solid) was prepared from Compound 1-36 according to the preparation method of Step 4 in 31% yield. MS m/s (ESI): 423.2 [M+1]; ¹ H ЯМР (400 МГц, DMSO-d6) δ 8,51-8,48 (м, 1H), 7,71-7,69 (м, 1Н), 7,45-7,43 (м, 1Н), 7,29 (д, J=1,8 Гц, 1Н), 7,18-7,16 (м, 1H), 5,64 (д, J=1,8 Гц, 1H), 3,83-3,81 (м, 2Н), 3,58 (д, J=3,0 Гц, 2Н), 2,54-2,51 (м, 1Н), 2,45-2,26 (м, 3Н), 1,99-1,97(м, 1Н), 1,89-1,64 (м, 4Н), 1,65-1,23 (м, 9Н), 0,91 (с, 9Н), 0,58-0,56 (м, 1Н).

[0244] Example 2.

and

(4S,6R)-6-(1-methylcyclopropyl)-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (Н-2b)

[0245] In step 1: Toluene (20 ml), titanium ethylate (2 ml, 9.54 mmol) were added compound V2 (460 mg, 2.61 mmol) and (R)-2-methylpropane-2-sulfinamide (475 mg, 3.92 mmol) and the reaction solution was stirred at 120°C for 2 hours, water (5 ml) was added to quench the reaction, the reaction solution was filtered, the resulting filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using eluents (PE/EA=1/0-1/1) to obtain compound 2-1 (612 mg, yellow oily liquid) in 84% yield. MS m/s (ESI): 280.1 [M+1].

[0246] In step 2: compound 2-1 (612 mg, 2.19 mmol) was added to methanol (50 ml). After the mixture was cooled to 0°C, sodium borohydride (166 mg, 4.39 mmol) was added, the reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative liquid chromatography (preparative column: C18 column 21.2×250 mm; system: 10 mM NH ₄ HCO ₃ H ₂ O; wavelengths: 254/214 nm; gradients: 30%-60% change in acetonitrile) to monoisomer 2-2a (254 mg, colorless oily liquid, retention time 9 min.) in 41% yield and monoisomer 2-26 (242 mg, colorless oily liquid, retention time 10 min.) in 39% yield, respectively. MS m/s (ESI): 282.1 [M+1].

[0247] In step 3: compound 2-2a (254 mg, 0.90 mmol) was added to methanol (20 ml). A 1,4-dioxane solution of 4 M hydrochloric acid (4.5 ml, 18 mmol) was added and the reaction solution was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, saturated sodium bicarbonate solution (30 ml) was added, the mixture was extracted with a mixture of DCM and methanol (15:1) (50 ml × 4), the resulting organic phases were combined, the combined organic phases were dried over anhydrous sodium sulfate and filtered, the resulting filtrate was concentrated under reduced pressure to give one isomeric compound 2-3a (151 mg, pale yellow oily liquid) in 94% yield. MS m/s (ESI): 178.1 [M+1].

[0248] Step 4: Compound 2-3a (151 mg, 0.85 mmol), Compound 16 (243 mg, 0.94 mmol) and sodium cyanoborohydride (268 mg, 4.26 mmol) were added to methanol (20 mL). The reaction solution was reacted at 40° C. for 4 hours and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative liquid chromatography (preparative column: C18 column 21.2×250 mm; system: 10 mM NH ₄ HCO ₃ H ₂ O; wavelengths: 254/214 nm; gradients: 30%-60% changes in acetonitrile) to give compound H-2a (186.2 5 mg, white solid) in 52% yield. MS m/s (ESI): 421.1 [M+1]; ¹ Н ЯМР (400 МГц, CD ₃ OD) δ 8,53-8,48 (м, 1H), 7,79-7,72 (м, 1Н), 7,50 (д, J=8,1 Гц, 1H), 7,43 (д, J=1,8 Гц, 1H), 7,24-7,21 (м, 1Н), 5,89 (дд, 3=1,9, 0,7 Гц, 1H), 4,02 (т, J=7,8 Гц, 1Н), 3,77-3,71 (м, 2Н), 3,49 (т, J=7,8 Гц, 1Н), 2,81-2,73 (м, 1Н), 2,70-2,59 (м, 1H), 2,53 (д, J=11,7 Гц, 1Н), 2,41 (дд, J=13,7, 1,9 Гц, 1H), 2,11-2,00 (м, 3Н), 1,91 (д, J=13,8 Гц, 1H), 1,77-1,38 (м, 8Н), 1,13-1,06 (м, 1H), 0,84 (с, 3Н), 0,76-0,69 (м, 2Н), 0,60-0,55 (м, 1H), 0,51-0,42 (м, 1Н), 0,38-0,29 (м, 1H).

[0249] In step 5: compound 2-26 (242 mg, 0.86 mmol) was added to methanol (20 ml). A 1,4-dioxane solution of 4 M hydrochloric acid (4.3 ml, 17.2 mmol) was added and the reaction solution was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, saturated sodium bicarbonate solution (30 ml) was added, the mixture was extracted with a mixture of DCM and methanol (15: 1) (50 ml × 4), the resulting organic phases were combined, the combined organic phase was dried over anhydrous sodium sulfate and filtered, the resulting filtrate was concentrated under reduced pressure to give one isomeric compound 2-36 (151 mg, pale yellow oily liquid) in 99% yield. MS m/s (ESI): 178.1 [M+1].

[0250] Step 6: Compound 2-36 (151 mg, 0.85 mmol), Compound 16 (243 mg, 0.94 mmol) and sodium cyanoborohydride (267 mg, 4.25 mmol) were added to methanol (10 mL). The reaction solution was reacted at 40° C. for 4 hours and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative liquid chromatography (preparative column: C18 column 21.2×250 mm; system: 10 mM NH ₄ HCO ₃ H ₂ O; wavelengths: 254/214 nm; gradients: 30%-60% changes in acetonitrile) to give compound H-26 (215.6 2 mg, white solid) in 60% yield. MS m/s (ESI): 421.1 [M+1]; ЯМР (400 МГц, CD ₃ OD) δ 8,54-8,49 (м, 1H), 7,78-7,74 (м, 1Н), 7,51 (д, J=8,1 Гц, 1H), 7,42 (д, 3=1,9 Гц, 1H), 7,25-7,22 (м, 1H), 5,91-5,80 (м, 1Н), 4,02 (т, J=7,8 Гц, 1Н), 3,82-3,66 (м, 2Н), 3,49 (т, J=7,8 Гц, 1H), 2,79-2,72 (м, 1H), 2,63-2,47 (м, 2Н), 2,41 (дд, J=13,7, 1,9 Гц, 1H), 2,16-1,97 (м, 3Н), 1,91 (д, J=13,8 Гц, 1H), 1,79-1,35 (м, 9Н), 1,12-1,06 (м, 1Н), 0,83 (с, 3Н), 0,74-0,69 (м, 2Н), 0,61-0,53 (м, 1Н), 0,49-0,44 (м, 1Н), 0,38-0,29 (м, 1Н).

[0251] Example 3.

(4S,6S)-6-isopropyl-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-3a) and (4S,6R)-6-isopropyl-N-(2-((R)-9-(pyridine-2- yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (Н-3b)

[0252] In step 1: Compound V3 (17 g, 103.5 mmol) was dissolved in toluene (250 ml), (R)-2-methylpropane-2-sulfinamide (18.8 g, 155 mmol) and titanium isopropoxide (117 g, 414 mmol) and the reaction solution was stirred at 110°C for 16 hours. The reaction solution was cooled to room temperature , water (80 ml) was added, DCM (600 ml) was added, the mixture was filtered, the resulting precipitate was washed with DCM (100 ml) and stratified, the resulting organic phase was washed with brine (100 ml), dried over anhydrous sodium sulfate, concentrated under reduced pressure, the resulting concentrate was purified by column chromatography (mobile phase - PE containing 30% EA) to give compound 3-1 (20.3 g, brown liquid) in 73.5% yield. MS m/s (ESI): 268.1 [M+1].

[0253] In Step 2: Compound 3-1 (0.39 g, 1.45 mmol) and methanol (5 ml) were added to a single-necked flask, the mixture was cooled to 0° C. and sodium borohydride (0.11 g, 2.9 mmol) was added slowly. The reaction solution was stirred at room temperature for 1.5 hours. After the reaction solution was cooled to 0°C, ice water (20 ml) was added and the mixture was extracted with DCM (50 ml×2). Объединенную органическую фазу промывали насыщенным солевым раствором (20 мл), сушили над безводным сульфатом натрия и сушили с помощью центрифугирования, а полученный остаток очищали с помощью препаративной хроматографии (препаративная колонка: колонка С18 21,2 × 250 мм; система: 10 мМ NH ₄ HCO ₃ H ₂ O; длины волн: 254/214 нм; градиенты: 28%-30% изменений в ацетонитриле) с получением одного диастереоизомерного соединения 3-2а (150 мг, белое твердое вещество, время удерживания 9,5 мин) с выходом 38,4%: МС м/з (ИЭР):270.0 [М+1] и одно диастереоизомерное соединение 3-26 (100 мг, белое твердое вещество, время удерживания 10,5 мин.) с выходом 25,6%: МС м/з (ИЭР):270,0 [М+1].

[0254] In step 3: Compound 3-2a (120 mg, 0.45 mmol) and methanol (3 ml) were added to a single-necked flask, the mixture was cooled to 0°C and a solution of hydrogen chloride in methanol (1.2 ml, 4.5 mmol, 4 mol/l) was slowly added. The reaction solution was stirred at room temperature for 1.5 hours. Saturated sodium bicarbonate was added to adjust the pH of the reaction solution to about 8, the mixture was extracted with DCM (50 ml × 2), all the resulting organic phases were combined, the combined organic phase was washed with brine, dried and concentrated, to obtain compound 3-3a (70 mg, yellow liquid) with a yield of 95.12%. MS m/s (ESI): 166.1 [M+1].

[0255] На шаге 4: соединение 3-За (50 мг, 0,3 ммоль) растворяли в метаноле (5 мл), добавляли соединение 16 (78,3 мг, 0,3 ммоль) и цианборгидрид натрия (94 мг, 1,5 ммоль) и реакционный раствор перемешивали при 20°С в течение 3 ч. Добавляли воду (20 мл), смесь экстрагировали с помощью ДХМ (30 мл × 2), объединенную органическую фазу сушили над безводным сульфатом натрия, концентрировали при пониженном давлении и очищали полученный концентрат с помощью препаративной хроматографии (препаративная колонка: колонка С18 21,2 × 250 мм; система: 10 мМ NH ₄ HCO ₃ H ₂ O; длины волн: 254/214 нм; градиенты: 30%-60% изменений в ацетонитриле) с получением соединения Н-3а одинарной конфигурации (10,12 мг, белое твердое вещество) с выходом 8,26%. The absolute configuration of the H-3a junction was determined by inference based on the absolute configuration of the V5 junction. MS m/z (ESI): 409.2 [M+1]; ¹ H ЯМР (400 МГц, CD ₃ OD) δ 8,51 (ддд, 3=4,9, 1,8, 0,8 Гц, 1H), 7,79-7,71 (м, 1H), 7,50 (д, J=8,1 Гц, 1Н), 7,41 (д, J=1,9 Гц, 1H), 7,22 (ддд, J=7,5, 4,9, 1,0 Гц, 1H), 5,87 (дд, 3=1,9, 0,6 Гц, 1H), 4,07 (ддд, J=21,0, 11,7, 6,2 Гц, 2Н), 3,81-3,69 (м, 2Н), 2,73-2,58 (м, 2Н), 2,52 (дд, J=14,0, 2,3 Гц, 1Н), 2,48-2,35 (м, 2Н), 2,12-1,97 (м, 2Н), 1,95-1,82 (м, 2Н), 1,78-1,65 (м, ЗН), 1,64-1,36 (м, 5Н), 1,14-1,04 (м, 1Н), 0,99 (д, J=7,0 Гц, 3Н), 0,79-0,65 (м, 4Н).

[0256] In Step 3: Compound 3-26 (100 mg, 0.37 mmol) and methanol (3 mL) were added to a single-necked flask, the mixture was cooled to 0°C, and a solution of hydrogen chloride in methanol (0.93 mL, 3.7 mmol, 4 mol/L) was slowly added. The reaction solution was stirred at room temperature for 1.5 hours. Saturated sodium bicarbonate was added to adjust the pH of the reaction solution to about 8, the mixture was extracted with DCM (50 ml × 2), all the resulting organic phases were combined, the combined organic phase was washed with brine, dried and concentrated to give compound 3-36 (50 mg, yellow liquid) in 81.2% yield. MS m/s (ESI): 166.1 [M+1].

[0257] На шаге 6: соединение 3-36 (50 мг, 0,3 ммоль) растворяли в метаноле (5 мл), добавляли соединение 16 (78,3 мг, 0,3 ммоль) и цианборгидрид натрия (94 мг, 1,5 ммоль) и реакционный раствор перемешивали при 20°С в течение 3 ч. К реакционному раствору добавляли воду (20 мл), смесь экстрагировали с помощью ДХМ (30 мл × 2), объединенную органическую фазу сушили над безводным сульфатом натрия, концентрировали при пониженном давлении и очищали полученный концентрат с помощью препаративной хроматографии (препаративная колонка: колонка С18 21,2 × 250 мм; система: 10 мМ NH ₄ HCO ₃ H ₂ O; длины волн: 254/214 нм; градиенты: 30%-60% изменений в ацетонитриле) с получением соединения Н-3б одинарной конфигурации (30,01 мг, белое твердое вещество) с выходом 24,5%. MS m/z (ESI): 409.3 [M+1]; ¹ Н ЯМР (400 МГц, CD ₃ OD) δ 8,59-8,52 (м, 1Н), 7,79 (тд, J=8,0, 1,9 Гц, 1H), 7,54 (д, J=8,1 Гц, 1Н), 7,43 (д, J=1,8 Гц, 1H), 7,27 (ддд, J=7,4, 4,9, 0,9 Гц, 1H), 5,86 (дд, J=1,9, 0,7 Гц, 1H), 4,11 (ддд, J=15,3, 10,4, 6,3 Гц, 2Н), 3,86-3,71 (м, 2Н), 2,69 (от, J=13,l, 7,8 Гц, 1H), 2,63-2,51 (м, 2Н), 2,50-2,39 (м, 2Н), 2,15 (тд, J=11,4, 4,7 Гц, 1H), 2,09-1,98 (м, 1Н), 1,91 (ддд, J=15,3, 13,5, 8,6 Гц, 2Н), 1,83-1,38 (м, 8Н), 1,16-1,08 (м, 1H), 1,02 (д, J=7,0 Гц, 3Н), 0,81-0,64 (м, 4Н).

[0258] Example 4.

(4S,6S)-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-6-(1-(trifluoromethyl)cyclopropyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-4-amine (H-4a) and (4S,6R)-N-(2-((R)-9-(pyri din-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-6-(1-(trifluoroethyl)cyclopropyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-4,)

[0259] In step 1: Compound V4 (700 mg, 3.04 mmol) was dissolved in a solution of toluene (10 ml), (R)-2-methylpropane-2-sulfinamide (442 mg, 3.65 mmol) and titanium isopropoxide (0.5 ml), and the reaction solution was stirred at 120°C for 2 hours. The resulting organic phase was concentrated under reduced pressure and the resulting residue purified by thin layer chromatography using eluents (PE:EA=3:7) to give compound 4-1 (600 mg) in 49% yield. MS m/s (ESI): 334.1 [M+1].

[0260] На шаге 2: соединение 4-1 (600 мг, 1,8 ммоль) растворяли в метанольном растворе (10 мл), добавляли боргидрид натрия (205 мг, 5,4 ммоль) при 0°С и реакционный раствор перемешивали при комнатной температуре в течение 2 ч. Добавляли воду, смесь фильтровали, полученный фильтрат концентрировали при пониженном давлении и полученный остаток очищали с помощью препаративной жидкостной хроматографии (условия получения: препаративная колонка: колонка С18 21,2 × 250 мм; система: 10 мМ NH ₄ HCO ₃ H ₂ O; длины волн: 254/214 нм; градиенты: 28%-30% изменений в ацетонитриле), с получением соединения 4-2а одинарной конфигурации (150 мг, время удерживания 8,5 мин.) с выходом 25% и соединения 4-26 одинарной конфигурации (150 мг, время удерживания 9,5 мин.) с выходом 25%. MS m/s (ESI): 336.1 [M+1].

[0261] In step 3: compound 4-2a (150 mg, 0.45 mmol) was dissolved in methanol (10 ml), a solution of hydrogen chloride in THF (4 M, 2 ml) was added, and the reaction solution was stirred at room temperature for 4 hours. DCM (50 ml) was added, then washed with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate, and the resulting organic phase was concentrated at reduced pressure to give compound 4-3a (100 mg).

[0262] In step 4: Compound 16 (112 mg, 0.43 mmol) was dissolved in a methanol solution (10 ml), compound 4-3a (100 mg, 0.43 mmol) and sodium cyanoborohydride (55 mg, 0.86 mmol) were added, and the reaction solution was stirred at 40°C for 4 hours. Water was added, the mixture was filtered, the resulting filtrate was concentrated under reduced pressure, and the resulting residue was purified by preparative liquid chromatography to give compound H-4a (72.70 mg) in 36% yield. MS m/s (ESI): 475.1 [M+1]; ¹ H ЯМР (400 МГц, CD ₃ OD): δ 8,51-8,50 (м, 1Н), 7,70-7,67 (м, 1H), 7,45-7,43 (д, J=8 Гц, 1H), 7,35 (с, 1H), 7,18-7,15 (м, 1H), 5,75 (д, 3=1,2 Гц, 1Н), 4,42-4,38 (т, J=8 Гц, 1Н), 3,84-3,82 (м, 1Н), 3,57-3,52 (м, 2Н), 2,93-2,89 (м, 1H), 2,42-2,30 (м, 3Н), 1,97-1,74 (м, 5Н), 1,57-1,31 (м, 10Н), 1,02-0,80 (м, 2Н), 0,61-0,55 (м, 1Н).

[0263] In step 5: Compound 4-3b was prepared from compound 4-2b according to the preparation method in step 3.

[0264] Step 6: Compound H-4b (90.52 mg) was prepared from Compound 4-3b according to the preparation method of Step 4 in 44% yield. MS m/s (ESI): 475.1 [M+1]; ¹ Н ЯМР (400 МГц, CD ₃ OD): δ 8,51-8,49 (м, 1H), 7,72-7,68 (м, 1H), 7,44-7,42 (д, J=8 Гц, 1Н), 7,34 (с, 1Н), 7,18-7,16 (м, 1Н), 5,70 (д, J=1,2 Гц, 1Н), 4,41-4,37 (т, J=8 Гц, 1H), 3,86-3,84 (м, 1H), 3,58-3,56 (м, 2Н), 2,90-2,87 (м, 1H), 2,32-2,29 (м, 3Н), 2,00-1,74 (м, 5Н), 1,64-1,30 (м, 9Н), 0,97-0,90 (м, 3Н), 0,81-0,78 (м, 1H).

[0265] Example 5.

(4R,6S)-6-tert-butyl-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-5a) and

(4R,6R)-6-tert-butyl-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-5b)

[0266] Two compounds were obtained in accordance with the preparation methods of Example 1. The difference was that the compound (R)-2-methylpropane-2-sulfinamide was replaced by the compound (S)-2-methylpropane-2-sulfinamide, and compound H-5a was obtained with a single configuration and compound H-56 with a single configuration: MS m / s (ESI): 423.2 [M + 1].

[0267] Example 6.

(4R,6S)-6-(1-methylcyclopropyl)-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-4-amine (H-ba) and (4R,6R)-6-(1-methylcyclopropyl)-N-(2-((R)- 9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (Н-6b)

[0268] Two compounds were obtained in accordance with the preparation methods of Example 2. The difference was that the compound (R)-2-methylpropane-2-sulfinamide was replaced by the compound (S)-2-methylpropane-2-sulfinamide, and compound H-6a with a single configuration and compound H-6b with a single configuration were obtained: MS m / s (ESI): 421.1 [M + 1].

[0269] Example 7.

(4R,6S)-6-isopropyl-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-7a) and (4R,6R)-6-isopropyl-N-(2-((R)-9-(pyridine-2 -yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-7b)

[0270] Two compounds were obtained in accordance with the preparation methods of Example 3. The difference was that the compound (R)-2-methylpropane-2-sulfinamide was replaced by the compound (S)-2-methylpropane-2-sulfinamide, and compound H-7a with a single configuration and compound H-76 with a single configuration were obtained: MS m / s (ESI): 409.3 [M + 1].

[0271] Example 8.

(4R,6S)-N-(2-((R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-6-(1-(trifluoromethylcyclopropyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-8a) and (4R,6R)-N-(2-((R)-9-(pyridine -2-yl)-6-oxaspiro[4.5]decan-9-yl)ethyl)-6-(1-(trifluoromethyl)cyclopropyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-4-amine (H-8b)

[0272] Two compounds were obtained in accordance with the preparation methods of Example 4. The difference was that the compound (R)-2-methylpropane-2-sulfinamide was replaced by the compound (S)-2-methylpropane-2-sulfinamide, and compound H-8a with a single configuration and compound H-8b with a single configuration were obtained: MS m / s (ESI): 475.1 [M + 1].

[0273] Bioanalysis

[0274] The cell lines used in the following assays are the PathHunter® CHO-K1 OPRM1 β-arrestin cell line from DiscoverX, Cat #93-0213C2, Lot #13K0402.

[0275] The reagents used, suppliers, catalog number and storage temperature are listed below:

[0276] Assay Complete™ 107 Cell Culture Kit, DiscoverX, 92-3107G, -20°C;

[0277] Assay Complete™ Thaw Reagent, DiscoverX, 92-4002TR, -20°C;

[0278] Assay Complete™ Cell Separation Reagent, DiscoverX, 92-0009, -20°C;

[0279] Assay Complete™ Cell Coating Reagent, DiscoverX, 93-0563R2, -20°C;

[0280] Pathhunter Discovery Kit, DiscoverX, 93-0001, -20°C;

[0281] PBS (lx) 0.0067M (P04), Hyclone, SH30256.01, 4°C;

[0282] DMSO, Sigma, D5879-100ML, room temperature;

[0283] NKH477, Sigma, 1603, -20°C; And

[0284] IBMX, Tocris, 15879, -20°C.

[0285] The tools, models and vendors used are listed below:

[0286] Countsatr BioMed, IM1200, ALIT;

[0287] Microscope, 1X51, OLYMPUS;

[0288] Centrifuge, 5804, Eppendorf;

[0289] Thermostatic water bath, DK-S420, Shanghai Shenxian thermostatic equipment factory;

[0290] Cell Incubator, 3111, Thermo;

[0291] Biological safety cabinet BSC-1300IIA2, AIRTECH;

[0292] White Opaque OptiPlate-384, 6007290, Perkin Elmer;

[0293] Victor X5 multimode tablet reader, PerkinElmer; And

[0294] TC Treated White Opaque Culture Plate-384, 6007680, PerkinElmer.

[0295] Test Example 1 HTRF-cAMP cell experiment

[0296] Experimental Methods and Steps

[0297] I. Cell resuscitation

[0298] 1. The resuscitation solution was removed from the refrigerator at 4°C and preheated over a water bath at 37°C for 15 minutes.

[0299] 2. The P6 cells were removed from the liquid nitrogen container, the frozen cell cryopreservation tube was immediately placed in a water bath at 37° C. and gently shaken for 30 seconds to 1 minute. until the appearance of small ice crystals or until the cells are completely thawed.

[0300] 3. The cryopreservation tube was carefully sterilized with 70% alcohol and wiped dry.

[0301] 4. Cells were centrifuged to remove cryoprotectant and resuspended in pre-warmed fresh resuscitation solution.

[0302] a. 3 ml of the pre-warmed cell resuscitation solution was aspirated and placed in a 15 ml centrifuge tube.

[0303] b. Cells were centrifuged at 1300 rpm. within 3 min.

[0304] c. The cryoprotectant supernatant was removed, and the cells were resuspended with 4 ml of pre-warmed resuscitation solution.

[0305] 5. The cell suspension was transferred to a T25 cell culture flask and cultured with 5% CO ₂ at 37°C for 24 hours.

[0306] 6. After culturing the cells for 24 hours, the resuscitation solution in the cell culture flask was replaced with pre-warmed cell culture medium.

[0307] P. Cell subculture

[0308] 1. After the cell growth density in the T25 culture flask exceeded 70%, the cells were digested with a cell dissociation solution for passage.

[0309] a. The culture flask medium was removed by aspiration, 4 ml of prewarmed PBS was added, the culture flask was gently shaken to wash the cells, and the PBS was removed by aspiration.

[0310] b. 1 ml of the cell dissociation solution was aspirated and added to a T25 culture flask.

[0311] c. The culture flask was shaken repeatedly to completely cover the culture flask with the dissociation solution and placed in a 5% CO ₂ incubator at 37° C. for 5 min.

[0312] The cell culture flask was removed and the cells were observed under a microscope to see if the cells had dissociated.

[0313] e. 3 ml of prewarmed cell culture medium was added to stop digestion.

[0314] e. The cell culture flask was gently washed again with the cell culture medium, the cell suspension was collected and placed in a 15 ml centrifuge tube.

[0315] f. Cells were centrifuged at 1300 rpm. within 3 min. and the supernatant was removed.

[0316] and. The cells were resuspended with 3 ml of cell culture medium.

[0317] 2. Cell subculture was performed at a ratio of 1:3 (i.e., 1 ml of cell resuspension solution and 3 ml of cell culture medium were placed in each T25 cell culture flask).

[0318] III. Seeding cells in a plate

[0319] 1. Step 2.2.1 (a to i) was repeated until P8 generation cells were obtained. Cells were counted and resuspended with 2×/1 mM IBMX stimulation buffer until the cell density was 1.2×10 ⁶ cells/ml.

[0320] 2. A cell solution at a density of 1.2×10 ⁶ cells/ml was inoculated into a 384-well plate using a multichannel pipette at 10 μl/well (ie 12,000 cells/well).

[0321] IV. cAMP analysis

[0322] 1. Received the appropriate reagents, and the compounds were obtained in accordance with the tables for obtaining drug solutions.

[0323] a. 1x Stimulation Buffer: Add 1 ml stock 5x Stimulation Buffer and mix uniformly with 4 ml distilled water.

[0324] b. 2x/1 mM IBMX Stimulation Buffer (5 ml): 10 µl of IBMX stock solution at 500 mM concentration was added to 4990 µl of cell culture medium and mixed evenly by gentle pipetting.

[0325] c. Gradient dilution table for positive morphine drug:

[0326] g. Prior to dilution, the compound was dissolved in DMSO to a storage concentration of 10 mM.

[0327] Dilution preparation table for TRV130 positive drug and various compounds:

[0328] e. 50 μM NK477 (1 ml): 1 μl of 50 mM NKH477 stock solution was added to 999 μl of 1x stimulation buffer and mixed uniformly by shaking.

[0329] Reagents for detection

[0330] A. cAMP cryptate reaction solution (donor, lyophilized): 1 ml of 5x cAMP cryptate stock solution was added to 4 ml of 1x lysis and detection buffer and gently mixed until uniform.

[0331] B. Anti-cAMP-c12 reaction solution (acceptor, lyophilized): 1 ml of 5x anti-cAMP-c12 stock solution was added to 4 ml of 1x lysis and detection buffer and gently mixed until homogeneous.

[0332] 2. Steps of cAMP analysis

[0333] a. 12,000 cells were inoculated into each well containing 10 μl of 2x IBMX Stimulation Buffer.

[0334] b. 8 µl of a diluted compound sample solution was added to the cells in each well.

[0335] c. 2 µl of the prepared 10* NKH477 solution was added to each well.

[0336] d. Cells were incubated at 37°C for 45 minutes.

[0337] e. 10 µl cAMP-c12 and 10 µl anti-cAMP-cryptate reaction solution were added.

[0338] e. Cells were incubated in the dark at room temperature for 60 minutes.

[0339] f. The cell culture plate was read with HTRF.

[0340] 3. RFU detection and plate reading

[0341] After incubating the cells for 60 minutes. the plate with all samples was subjected to detection and read by homogeneous fluorescence with time resolution.

[0342] Data analysis

[0343] The data, which included two signal values at 665 nm and 620 nm, respectively, was exported from the appropriate software to a computer connected to a multi-mode plate reader. The formula for calculating the ratio was: ratio = signal value at 665 nm/signal value at 620 nm × 10,000. Data were analyzed using GraphPad Prism software. The best fitting curve was logarithm (agonist) vs. response. Compound EC50 values were determined by non-linear regression analysis of computerized dose-response curves; PEC50=-logEC50 (the unit of EC50 values was mol); % maximum morphine effect value=(compound sample-blank ratio)/TOP x 100 (Note: TOP represents the upper end of the morphine sample-to-blank ratio curve, which was analyzed and fitted using Graphpad Prism software). The results are presented in table 1:

[0344] Table 1 Compound activity on cAMP

[0345] Test Example 2 β-Arrestin Cell Experiment

[0346] Experimental method and steps

[0347] I. Cell resuscitation

[0348] 1. The resuscitation solution was removed from the refrigerator at 4°C and preheated over a water bath at 37°C for 15 minutes.

[0349] 2. P6 generation cells were removed from the liquid nitrogen container, the frozen cell culture tube was immediately placed in a water bath at 37° C. and gently shaken for 30 seconds to 1 minute. until the appearance of small ice crystals or until the cells are completely thawed.

[0350] 3. The culture tube was thoroughly sterilized with 70% alcohol and wiped dry.

[0351] 4. Cells were centrifuged to remove cryoprotectant and resuspended in pre-warmed fresh resuscitation solution.

[0352] a. 3 ml of the pre-warmed cell resuscitation solution was aspirated and placed in a 15 ml centrifuge tube.

[0353] b. Cells were centrifuged at 1300 rpm. within 3 min.

[0354] c. The supernatant was removed and the cells were resuspended with 4 ml of pre-warmed resuscitation solution.

[0355] 5. The cell suspension was transferred to a T25 cell culture flask and cultured with 5% CO ₂ at 37°C for 24 hours.

[0356] 6. After culturing the cells for 24 hours, the resuscitation solution in the cell culture flask was replaced with pre-warmed cell culture medium.

[0357] P. Cell subculture

[0358] 1. After the cell growth density in the T25 culture flask exceeded 70%, the cells were digested with a cell dissociation solution for passage.

[0359] a. The culture flask medium was removed by aspiration, 4 ml of prewarmed PBS was added, the culture flask was gently shaken to wash the cells, and the PBS was removed by aspiration.

[0360] b. 1 ml of the cell dissociation solution was aspirated and added to a T25 culture flask.

[0361] c. The culture flask was shaken repeatedly to completely cover the culture flask with the dissociation solution and placed in a 5% CO ₂ incubator at 37° C. for 5 min.

[0362] The cell culture flask was removed and the cells were observed under a microscope to see if the cells had dissociated.

[0363] e. 3 ml of prewarmed cell culture medium was added to stop digestion.

[0364] e. The cell culture flask was gently washed again with the cell culture medium, the cell suspension was finally transferred to a 15 ml centrifuge tube.

[0365] f. Cells were centrifuged at 1300 rpm. within 3 min. and the supernatant was removed.

[0366] i. The cells were resuspended with 3 ml of cell culture medium.

[0367] 2. Cell subculture was performed at a ratio of 1:3 (i.e., 1 ml of cell resuspension solution and 3 ml of cell culture medium were placed in each T25 cell culture flask).

[0368] 3. Step 2.2.1 (from a to i) was repeated until P8 generation cells were obtained.

[0369] III. Seeding cells in a plate

[0370] 1. 20 µl of the cell suspension was pipetted and the number of cells was measured using a cell counter.

[0371] 2. Cells were centrifuged at 1300 rpm. within 3 min. for cell sedimentation.

[0372] 3. The supernatant was removed and the appropriate cell culture solution was added until the cell density was 2×10 ⁵ cells/ml.

[0373] 4. Cell solution at a density of 2×10 ⁵ cells/ml was inoculated into a 384-well plate using a multichannel pipette at 20 μl/well (ie 4000 cells/well).

[0374] 5. The cell-seeded 384-well plate was placed in a 5% CO ₂ incubator at 37°C for 24 hours.

[0375] IV. β-arrestin assay

[0376] 1. Compounds were prepared according to the following dilution tables.

[0377] a. Gradient dilution table for positive morphine drug:

[0378] b. Prior to dilution, the compound was dissolved in DMSO to a storage concentration of 10 mM.

[0379] Dilution preparation table for TRV130 positive drug and various compounds:

[0380] 2. To a 384-well plate, 5 µl of diluted compound sample solution was added.

[0381] 3. After adding the samples, the 384-well plate was placed in a 5% CO ₂ incubator at 37° C. for 90 minutes.

[0382] V. RLU Definition

[0383] 1. Before completion of compound incubation, a detection working solution (in the dark) was prepared according to the following ratio. Then, 12.5 µl of detection working solution was added to each well, and the cells were incubated with shaking in the dark at room temperature for 1 hour.

[0384]

[0385] 2. After the end of compound incubation, 12.5 μl of the above working solution was added to each well, and the cells were incubated with shaking in the dark at room temperature and 80 rpm. within 1 hour

[0386] 3. After completion of the incubation, the plate was read using a multi-mode plate reader.

[0387] Data analysis

[0388] The data was exported from the appropriate software to a computer connected to a multi-mode plate reader and analyzed using the GraphPad Prism software. The best fitting curve was logarithm (agonist) vs. response. Compound EC50 values were determined by non-linear regression analysis of computerized dose-response curves; PEC50=-logEC50 (the unit of EC50 values was mol); % maximum morphine effect value = (compound sample RLU value empty well RLU value)/TOP×100 (Note: TOP represents the upper end of the morphine sample RLU versus empty well RLU curve as analyzed and fitted using Graphpad Prism software). The results are presented in table 2:

[0389] Table 2. Results of testing compounds on β-arrestin

[0390] Table 1 and Table 2 show that representative compounds of the present disclosure have high cAMP inhibitory activity and high Emax values. In addition, the compounds of the present disclosure have small Emax values with respect to β-arrestin and have a good measurement error.

[0391] All documents referred to in this disclosure are incorporated herein by reference as if each document were separately incorporated by reference. In addition, it should be understood that after reading the above teaching content of this disclosure, various changes or modifications to this disclosure may be made by those skilled in the art, and these equivalent forms also fall within the scope of the appended formulas of this disclosure.

Claims (57)

1. The compound represented in formula (I) or a pharmaceutically acceptable salt thereof: where each of R _a and R _b independently represents hydrogen or C _1-3 alkyl; R _c is hydrogen or C _1-3 alkyl; And * indicates R or S configuration. 2. A compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented in formula (I) has the structure represented in formula (I-a) or formula (I-b): where R _a and R _b are each independently C _1-3 alkyl; And R _c is hydrogen or C _1-3 alkyl. 3. A compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R _a and R _b are each independently hydrogen, methyl or ethyl. 4. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R _a and R _b are each independently methyl. 5. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R _c is hydrogen, methyl or ethyl. 6. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R _c is hydrogen or methyl. 7. A compound or pharmaceutically acceptable salt thereof according to claim 2, wherein R _a and R _b are each independently methyl and R _c is hydrogen or methyl. 8. A compound or pharmaceutically acceptable salt thereof according to claim 2, wherein R _a and R _b are each methyl and R _c is hydrogen. 9. The compound represented in formula (II), or a pharmaceutically acceptable salt thereof: where each of R ₁ and R ₂ independently represents hydrogen or C _1-3 alkyl; or R ₁ and R ₂ together with their associated carbon atom form a three to six membered saturated monocyclic ring, where the three to six membered saturated monocyclic ring is unsubstituted; R ₃ is hydrogen or C _1-3 alkyl; And * indicates R or S configuration. 10. A compound or a pharmaceutically acceptable salt thereof according to claim 9, wherein the compound represented in formula (II) has the structure represented in formula (II-a) or formula (II-b): where R ₁ and R ₂ are each independently C _1-3 alkyl; or R ₁ and R ₂ together with the carbon atom to which they are attached, form a three to six membered saturated monocyclic ring, while the three to six membered saturated monocyclic ring is unsubstituted; And R ₃ is hydrogen or C _1-3 alkyl. 11. The compound or its pharmaceutically acceptable salt according to claim 9, where each of R ₁ and R ₂ independently represents hydrogen, methyl or ethyl; or R ₁ and R ₂ together with their associated carbon atom form a cyclopropyl ring, a cyclobutyl ring or a cyclopentyl ring. 12. A compound or a pharmaceutically acceptable salt thereof according to claim 9, wherein R ₁ and R ₂ are each independently hydrogen, methyl or ethyl. 13. The compound or pharmaceutically acceptable salt thereof according to claim 9, wherein R ₁ and R ₂ together with their associated carbon atom form a cyclopropyl ring. 14. A compound or a pharmaceutically acceptable salt thereof according to claim 9, wherein R ₃ is hydrogen, methyl or ethyl. 15. A compound or pharmaceutically acceptable salt thereof according to claim 9, wherein R ₃ is hydrogen or methyl. 16. A compound or pharmaceutically acceptable salt thereof according to claim 10, wherein R ₁ and R ₂ are each independently methyl and R ₃ is hydrogen, methyl or ethyl. 17. A compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of: 18. A compound or a pharmaceutically acceptable salt thereof according to claim 9, wherein the compound is selected from the group consisting of: 19. The compound represented in the formula (III) where each of R _1a and R _1b independently represents hydrogen or C _1-3 alkyl; or R _1a and R _1b together with their associated carbon atom form a three to six membered saturated monocyclic ring, where the three to six membered saturated monocyclic ring is unsubstituted; R _1c is hydrogen or C _1-3 alkyl. 20. The compound of claim 19 wherein R _1a and R _1b are each independently hydrogen or methyl and R _1c is hydrogen, methyl or ethyl. 21. A compound according to claim 19 wherein R _1a and R _1b are each methyl and R _1c is hydrogen, methyl or ethyl. 22. The compound of claim 19 wherein R _1a and R _1b are each methyl and R _1c is hydrogen. 23. A compound according to claim 19, wherein R _1a and R _1b together with their associated carbon atom form a cyclopropyl ring and R _1c is methyl or ethyl. 24. An intermediate compound of an oxaspiro-substituted pyrrolopyrazole derivative, wherein the intermediate compound has the structure shown in formula (I-1) or formula (II-1): where R _a , R _b and R _c are as defined in paragraph 1; R ₁ , R ₂ and R ₃ are as defined in paragraph 9; And * represents a chiral carbon atom in the R or S configuration. 25. An intermediate compound according to claim 24, wherein the compound represented in formula (I-1) has the structure represented in formula (I-1a) or formula (I-1b); and the compound shown in formula (II-1) has the structure shown in formula (II-1a) or formula (II-1b): 26. An intermediate compound according to claim 25, wherein the compound represented in formula (I-1a) is selected from the group consisting of: wherein the compound represented in formula (I-1b) is selected from the group consisting of: wherein the compound represented in formula (II-1a) is selected from the group consisting of: wherein the compound represented in formula (II-1b) is selected from the group consisting of: