Deuterated oxadiazinone compounds and uses thereof

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a deuterated molecular glue that can selectively induce the formation of a type 3A phosphodiesterase (PDE3A) and SLFN12 complex and apoptosis of tumor cells, a preparation method thereof, and an application thereof in tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a molecular glue capable of selectively inducing the formation of a type 3A phosphodiesterase (PDE3A) and SLFN12 complex and apoptosis of tumor cells, a preparation method thereof, and an application thereof in tumor treatment. Background Art

[0002] Phosphodiesterase (PDE) is a superfamily enzyme system that can mediate the hydrolysis of intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) (Chen J, Liu N, Huang YP, et al. Nature Comm 2021; 12: 6204), and regulate the concentration of intracellular cAMP and cGMP. Since the second messenger signaling pathways mediated by cAMP and cGMP are involved in regulating various cell functions, PDE can affect intracellular signal cascade reactions, conduction and regulation of different physiological processes by regulating the activity of cAMP and cGMP, such as cell proliferation and apoptosis, inflammatory response, contraction of cardiomyocytes and smooth muscle cells, permeability of endothelial cells, immune response, etc. (Fertig BA and Baillie GS. J Cardiovasc Dev Dis 2018; 5: 8; Nourian YH, Salimian J, Ahmadi A, et al. Biochem Biophys Rep 2023; 34: 101438). According to its hydrolysis specificity for cAMP or cGMP, PDE can be divided into 11 families, among which PDE4, 7, and 8 are cAMP-specific, PDE5, 6, and 9 are cGMP-specific, and PDE1, 2, 3, 10, and 11 can hydrolyze cAMP and cGMP at the same time (Ahmad F, Murata T, Simizu K, et al. Oral Dis 2015; 21: e25-e50; Barone I, Giordano C, Bonofiglio D, et al. Oncotarget 2017; 8: 99179-99202).

[0003] There are two subtypes of PDE3 enzyme, PDE3A and PDE3B (Begum N, Shen WX, Manganiello V. Curr Opin Pharmacol 2011; 11: 725-9), located on chromosomes 11 and 12 respectively. PDE3A is mainly distributed in the heart, vascular smooth muscle, platelets and oocytes, and can effectively regulate myocardial and smooth muscle contraction, platelet aggregation and oocyte maturation. Although multiple PDE3 inhibitors have been used to treat cardiovascular diseases, studies have found that long-term inhibition of PDE3A enzyme activity can cause palpitations and sudden death in heart patients, reduce platelet production and affect oocyte maturation and other toxic side effects (Ai YW, He HB, Chen PH, et al. Nature Comm 2020; 11: 3236). In addition, current PDE3A inhibitors mainly target the enzyme catalytic region of the cAMP / cGMP binding site. Due to the high homology of the catalytic region of the PDE family, the selectivity of PDE3A inhibitors is different, and the toxic side effects are large. For example, the traditional inhibitor anagrelide can inhibit both PDE3A and PDE2, causing headache, palpitations, diarrhea, vomiting and many other adverse reactions (Birgegard G, Brorkholm M, Kutti J, et al. Haematologica 2004; 89: 520-7). Milrinone can cause arrhythmia, angina pectoris, hypotension and other adverse symptoms. Therefore, improving the safety of PDE3A inhibitors is a top priority in the development of PDE3A drugs.

[0004] The latest studies have shown that multiple compounds with completely different chemical structures, such as anagrelide, DMNDP and nailfine, can induce the formation of PDE3A and SLFN12 complexes in the form of molecular glues. The formation of the complex stabilizes the protein level of intracellular SLFN12 (under normal conditions, SLFN12 is degraded through the ubiquitination pathway and the level in the cell is very low). The PDE3A-SLFN12 complex can bind to downstream ribosomal RNA (rRNA) (Li DR, Chen J, Ai YW, et al. Mol Cell 2019; 75: 1103-1116), blocking the binding of SRP (signal recognition particle) to the ribosome. Since the ribosome needs to be transported to the endoplasmic reticulum membrane with the assistance of SRP to further synthesize membrane secretory proteins, the binding of PDE3A-SLFN12 to rRNA can lead to a decrease in the protein levels of anti-apoptotic proteins Bcl-2 and Mcl-1, making it unable to inhibit the activity of Bax and Bak. The oligomerization of Bax and Bak further promotes the increase of mitochondrial membrane permeability, the release of cytochrome and the induction of tumor cell apoptosis (Li DR, Chen J, Ai YW, et al. Mol Cell 2019; 75: 1103-1116). It was also found that nauclefine does not inhibit the hydrolysis activity of PDE3A on cAMP (Ai YW, He HB, Chen PH, et al. Nature Comm 2020; 11: 3236). It mainly induces apoptosis of tumor cells through the PDE3A-SLFN12 complex, thereby avoiding the risk of toxic side effects caused by the inhibition of PDE3A enzyme activity.

[0005] The present invention is based on the PDE3A-SLFN12-dependent apoptosis mechanism, and aims to develop a molecular glue compound that can efficiently mediate the interaction between PDE3A and SLFN12, thereby promoting apoptosis of tumor cells. The molecular glue of the present invention has high pharmacological and pharmacodynamic activity while reducing the risk of inhibiting PDE enzyme activity, and has high therapeutic potential in tumors expressing PDE3A and SLFN12. Summary of the invention

[0006] The present invention provides a deuterated PDE3A-SLFN12 complex, which can balance the safety and effectiveness of the drug, and on the basis of ensuring the in vitro inhibitory activity of the drug, can also improve the pharmacokinetic properties and increase the metabolic stability of the drug in liver microsomes.

[0007] In order to achieve the above object, the present invention provides a compound of formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof or a mixture thereof:

[0008]

[0009] in,

[0010] R1 is selected from hydrogen or deuterium;

[0011] R2 is undeuterated or one or more times deuterated or fully deuterated C1-C4 alkyl;

[0012] R3, R4, R5, R6 are each independently selected from hydrogen or deuterium;

[0013] Ring A is selected from phenyl or 5-6 membered heteroaryl;

[0014] R a is selected from halogen, amino, C1-C4 alkyl or C3-C6 cycloalkyl; the C1-C4 alkyl may be optionally substituted by one or more halogen or deuterium;

[0015] n is selected from 0, 1 or 2; with the additional proviso that at least one of R1 and R2 is deuterated or deuterated.

[0016] In some preferred embodiments, the present invention provides compounds of formula (I):

[0017] R1 is selected from hydrogen or deuterium;

[0018] R2 is undeuterated or one or more times deuterated or fully deuterated C1-C4 alkyl;

[0019] R3, R4, R5, R6 are each independently selected from hydrogen or deuterium;

[0020] Ring A is selected from phenyl, 5-6 membered heteroaryl or 5-9 membered heterocyclyl;

[0021] R a is selected from hydrogen, halogen, amino, undeuterated or one or more times deuterated or fully deuterated C1-C4 alkyl, C1-C4 haloalkyl, -CF2CH2OH, 3-6 membered heterocyclyl, C(=O)NH2, C(=O)NHCH3 or C3-C6 cycloalkyl;

[0022] n is selected from 0, 1 or 2;

[0023] Provided that at least one of R1 and R2 is deuterated or deuterated.

[0024] In some preferred embodiments, in the compound of formula (I) above, R3, R4, R5, and R6 are selected from hydrogen.

[0025] In some preferred embodiments, in the compound of the above formula (I), R1 is selected from hydrogen or deuterium; R2 is selected from CH3, CH2D, CHD2 or CD3.

[0026] In some more preferred embodiments, R1 is selected from hydrogen or deuterium; R2 is selected from CH3 or CD3; for example, R1 is selected from hydrogen and R2 is selected from CD3; or R1 is selected from deuterium and R2 is selected from CH3; or R1 is selected from deuterium and R2 is selected from CD3.

[0027] In some more preferred embodiments, the compound of formula (I) has the structure shown in the following formula (I-1):

[0028]

[0029] Wherein, R1 is selected from hydrogen or deuterium, and ring A, R a , n is as defined in any of the items herein.

[0030] In some embodiments, the compound of formula (I) has the structure shown in formula (II):

[0031]

[0032] in,

[0033] Ring A is selected from phenyl or 5-6 membered heteroaryl;

[0034] R a is selected from hydrogen, halogen, amino, C1-C4 alkyl or C3-C6 cycloalkyl; the C1-C4 alkyl may be optionally substituted by one or more halogen or deuterium; n is selected from 0, 1 or 2.

[0035] In some preferred embodiments, in the compounds of formula (I), formula (I-1) and formula (II), ring A is selected from phenyl,

[0036]

[0037] In some preferred embodiments, in the compounds of formula (I), formula (I-1) and formula (II), R a is selected from hydrogen, halogen, amino, C1-C4 alkyl or C3-C6 cycloalkyl; the C1-C4 alkyl may be optionally substituted by one or more halogen or deuterium; n is selected from 1 or 2.

[0038] In some more preferred embodiments, in the compounds of formula (I), formula (I-1) and formula (II), ring A is selected from phenyl; R a is selected from F, Cl, CF3, CHF2, amino, cyclopropyl, CH3, CH2D, CHD2 or CD3; n is selected from 1 or 2.

[0039] In some more preferred embodiments, in the compounds of formula (I), formula (I-1) and formula (II), ring A is selected from phenyl; R a Selected from F.

[0040] In some preferred embodiments, the compounds described in the above formula (I), formula (I-1) and formula (II), their pharmaceutically acceptable salts, their stereoisomers, tautomers or mixtures thereof are selected from but not limited to the following compounds:

[0041]

[0042]

[0043] Another aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any one of the above-mentioned compounds, pharmaceutically acceptable salts thereof, stereoisomers thereof, tautomers thereof or mixtures thereof.

[0044] Another aspect of the present invention provides the use of any of the above-mentioned compounds, pharmaceutically acceptable salts thereof, stereoisomers, tautomers or mixtures thereof or pharmaceutical compositions thereof for preparing drugs for regulating the interaction between PDE3A and SLFN12.

[0045] In some preferred embodiments, any of the above-mentioned compounds, pharmaceutically acceptable salts thereof, stereoisomers, tautomers thereof or mixtures thereof or pharmaceutical compositions thereof can enhance and / or promote the interaction between PDE3A and SLFN12.

[0046] In some preferred embodiments, the compound described in any one of the above items, its pharmaceutically acceptable salt, its stereoisomer, tautomer or mixture thereof or its pharmaceutical composition can be used to prepare a drug for treating and / or preventing a disease; preferably, wherein the disease is a hyperproliferative disease; preferably, the hyperproliferative disease is a cancer disease, and the cancer disease is brain cancer (specifically glioma, more specifically glioblastoma, astrocytoma), breast cancer (specifically ductal carcinoma and adenocarcinoma), cervical cancer, AML (specifically erythroleukemia), lung cancer (specifically NSCLC adenocarcinoma and SCLC), skin cancer (specifically melanoma), esophageal cancer (specifically squamous cell carcinoma), ovarian cancer, (specifically teratoma, adenocarcinoma), pancreatic cancer, prostate cancer and sarcoma.

[0047] In some more preferred embodiments, any of the above-mentioned compounds, pharmaceutically acceptable salts thereof, stereoisomers, tautomers or mixtures thereof, or pharmaceutical compositions thereof can be used to prepare drugs for melanoma.

[0048] The selection of any substituent in any embodiment of the present invention can be combined with each other, and the technical solution after the combination is still included in the protection scope of the present invention.

[0049] The deuterated compound of the present invention refers to a compound in which the deuterium isotope content of deuterium at the deuterium substitution position is at least greater than the natural deuterium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, and more preferably greater than 99%. Since the deuterium content in the generated deuterated compound is much higher than the 0.015% content in nature, it can be regarded as a new type of compound.

[0050] The present invention studies the deuterated structure of the compound and ultimately achieves the following beneficial effects:

[0051] 1. The deuterated compounds of the present invention have good in vitro cell inhibitory activity and show equivalent or better efficacy than non-deuterated compounds; in terms of in vivo efficacy in mice, the deuterated compounds of the present invention show better tumor inhibition effect.

[0052] 2. The present invention has studied the position and amount of deuteration and found that compounds with different positions and amounts of deuteration show different effects on liver microparticle stability and pharmacokinetics. When the deuteration position and amount are selected at a specific position, they show better liver microparticle stability and pharmacokinetic properties than non-deuterated compounds. However, when the deuteration position and amount are not selected appropriately, they show worse liver microparticle stability.

[0053] 3. The present invention improves the metabolism of the compound in the organism by selecting a suitable deuterated site, increases the drug concentration of the compound in the body, and thus improves the efficacy of the drug.

[0054] Although it is generally believed that since CD bonds are more stable than CH bonds, deuterated drugs are less likely to break CD bonds during chemical reactions, and their half-life will be extended. However, not all CH bonds can improve the bioavailability of drugs after deuteration. Only when the deuterated position is the rate-determining step of drug metabolism and degradation, it is possible to affect the bioavailability of drugs. The metabolic process of biological systems has its complexity. Compared with corresponding non-deuterated drugs, the changes in the pharmacokinetic properties of deuterated drugs show great contingency and unpredictability. Deuterium at certain sites not only cannot extend the half-life, but may shorten it (Scott L. Harbeson, Roger D. Tung. Deuterium in Drug Discovery and Development, P405-406).

[0055] In fact, the success rate of improving the pharmacokinetic properties and enhancing the efficacy of deuterated compounds is not high. Most deuterated compounds (about >80%) have poorer or no changes in the pharmacokinetic properties and in vivo biological activities than their non-deuterated parent compounds. The changes in drug metabolism properties or activities after deuteration are unpredictable (Studies with deuterated drugs", Journal of Pharmaceutical Sciences, 1975, 64(3): 367-391). So far, only a small number of deuterated drugs have been approved, precisely because of the unpredictability of the effect of deuterium modification on the pharmacokinetic properties of drugs. Therefore, the actual preparation and testing of deuterated molecules must be carried out to determine the differences in metabolism between deuterated drugs and non-deuterated drugs. The present application finally obtained compounds with excellent pharmacokinetics by studying deuterated compounds. DETAILED DESCRIPTION

[0056] In order to make the present invention more easily understood, the present invention will be described in detail below in conjunction with embodiments. These embodiments are only for illustration and are not intended to limit the application scope of the present invention.

[0057] Terms and related definitions:

[0058] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, the following terms have the meanings ascribed to them below unless otherwise indicated.

[0059] As used herein, "deuterated" refers to one or more hydrogens in a compound or group being replaced by deuterium. Deuterated can be monosubstituted, disubstituted, polysubstituted or fully substituted. The term "one or more deuterated" and "one or more deuterated" can be used interchangeably.

[0060] Unless otherwise specified, D in the present invention represents deuterium ( 2 H).

[0061] The term "independently" means that when more than one substituent is selected from a number of possible substituents, those substituents may be the same or different; or when a substituent occurs multiple times, each occurrence may be selected from the same or different.

[0062] The term "alkyl" refers to a straight or branched saturated hydrocarbon group consisting only of carbon atoms and hydrogen atoms. "C1-C6 alkyl" includes C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl and C1 alkyl. Specifically, it includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl.

[0063] The "halogen" described in the present invention refers to fluorine, chlorine, bromine and iodine, preferably fluorine and chlorine.

[0064] The "amino group" described in the present invention refers to the NH2 group.

[0065] The "cycloalkyl" of the present invention refers to a saturated monocyclic or polycyclic (such as spirocyclic, fused or bridged) hydrocarbon ring. For example, the term "C3-C6 cycloalkyl" refers to a saturated monocyclic or polycyclic ring having 3 to 6 ring carbon atoms, and non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.

[0066] The "heterocyclic group" described in the present invention refers to a monocyclic, bicyclic or tricyclic group composed of carbon atoms and one or more heteroatoms, including monocyclic, spirocyclic, bridged and condensed ring forms, wherein the cyclic group is saturated or partially unsaturated (non-aromatic), wherein at least one ring has at least one heteroatom (O, S or N), and the heteroatom-containing ring preferably has 1, 2 or 3 heteroatoms selected from O, S and N.

[0067] The "heteroaryl" described in the present invention refers to a monocyclic or condensed polycyclic monovalent group with aromaticity, wherein at least one (e.g., 1, 2, 3 or 4) ring atoms are heteroatoms selected from N, O and S, and the remaining ring atoms are C. Specific non-limiting examples include, but are not limited to, pyridine, pyrimidine, pyrazine, imidazole, pyrazole, and the like.

[0068] The compounds of the present invention contain one or more asymmetric centers (also called chiral centers), and the compounds and intermediates of the present invention may exist in different tautomeric forms, all of which are included in the protection scope of the present invention. The present invention includes all isomers (e.g., enantiomers, diastereomers and geometric isomers (or conformational isomers)) of the structure.

[0069] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a vertical asymmetric plane due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby being able to rotate plane polarized light;

[0070] The term "tautomer" (or "tautomeric form") refers to structural isomers with different energies that can be interconverted through a low energy barrier. For example, proton tautomers (or prototropic tautomers) include (but are not limited to) interconversion via proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-iminol isomerization, etc.

[0071] The words "include", "comprising" and "containing" and their equivalents in the present invention should be understood as an open and non-exclusive meaning, that is, "including but not limited to", meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

[0072] Unless otherwise specified in the present invention, the solution mentioned in the reaction of the present invention is an aqueous solution.

[0073] The term "room temperature" in the present invention refers to a temperature between 10°C and 25°C.

[0074] Unless otherwise stated, the raw materials used in the present invention are commercially available.

[0075] Intermediate 1 4-Bromo-N-methoxy-N-methyl-3-(trifluoromethyl)benzamide

[0076]

[0077] To a single-necked flask, 4-bromo-3-trifluoromethylbenzoic acid (4.00 g, 14.87 mmol, CAS: 161622-14-6) and dichloromethane (60 mL) were added, followed by N,N-diisopropylethylamine (4.80 g, 37.17 mmol, CAS: 7087-68-5), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.42 g, 17.84 mmol, CAS: 25952-53-8) and 1-hydroxybenzotriazole (HOBT) (2.41 g, 17.84 mmol, CAS: 2592-95-2). The mixture was stirred at room temperature for 10 minutes, and then dimethylhydroxylamine hydrochloride (1.60 g, 16.36 mmol, CAS: 6638-79-5) was added, and the reaction solution was stirred at room temperature for 2 hours. Add water (100 mL) to the reaction solution for dilution, extract twice with dichloromethane (100 mL), combine the organic phases, wash twice with water (80 mL), wash twice with saturated brine (100 mL), dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography (petroleum ether / ethyl acetate = 4:1) to obtain the title compound as an off-white solid (4.22 g, yield 91%). LC-MS: m / z[M+H] + =312.

[0078] Referring to the table below, the following intermediates 6, 9, and 20 were prepared by referring to the preparation method of intermediate 1, except that the raw materials described in the "Raw Materials" column were used to replace the corresponding raw materials.

[0079]

[0080] Intermediate 2 4'-Fluoro-N-methoxy-N-methyl-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxamide

[0081]

[0082] 4-Bromo-N-methoxy-N-methyl-3-(trifluoromethyl)benzamide (2.00 g, 6.40 mmol, intermediate 1), 4-fluorophenylboric acid (0.99 g, 7.04 mmol, CAS: 1765-93-1), bistriphenylphosphine palladium dichloride (0.46 g, 0.64 mmol, CAS: 13965-03-2), potassium carbonate (2.65 g, 19.20 mmol) and N, N-dimethylformamide (20 mL) were added to a single-necked flask. After nitrogen replacement three times, the reaction solution was reacted at 80 ° C for 5 hours under a nitrogen atmosphere. The reaction solution was cooled to room temperature, water (100 mL) was added to the reaction solution to dilute it, and ethyl acetate (50 mL) was extracted twice. The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 6:1) to obtain the title compound as a white solid (1.81 g, yield 86%). LC-MS: m / z[M+H] + =328.

[0083] Intermediate 3 (S)-1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-hydroxypropan-1-one-2,3, 3,3-d4

[0084]

[0085] 1-(4'-Fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)propan-1-one-2,2,3,3,3-d5

[0086]

[0087] Add activated magnesium chips (0.84 g, 35.10 mmol), two iodine particles and anhydrous tetrahydrofuran (5 mL) into a three-necked flask, then add a solution of deuterated bromoethane (0.44 g, 3.86 mmol, CAS: 3675-63-6) in anhydrous tetrahydrofuran (4 mL). Heat the reaction solution with a hair dryer until the temperature reaches 60 ° C, add 1,2-dibromoethane (0.1 mL) to initiate the reaction, and at the same time, add the remaining deuterated bromoethane (1.56 g, 13.68 mmol, CAS: 3675-63-6) in anhydrous tetrahydrofuran (14 mL) solution dropwise and keep the reaction initiation state. After the addition is complete, the reaction solution is stirred at room temperature for 40 minutes. Then, a solution of 4'-fluoro-N-methoxy-N-methyl-2-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxamide (1.80 g, 5.50 mmol, intermediate 2) in anhydrous tetrahydrofuran (10 mL) was slowly added dropwise to the above reaction solution. After the addition was complete, the mixture was reacted at room temperature for 2 hours. The reaction solution was cooled to below 0°C, and a saturated aqueous ammonium chloride solution (150 mL) was slowly added to the reaction solution to quench the reaction. The mixture was extracted twice with ethyl acetate (50 mL), the organic phases were combined, and the mixture was washed once with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 15:1) to obtain the title compound as a white solid (1.12 g, yield 67%).

[0088] (E)-tert-butyl((1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)prop-1-en-1-yl-2,3, 3,3-d4)oxy)dimethylsilane

[0089]

[0090] To a single-necked flask, 1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)propan-1-one-2,2,3,3,3-d5 (1.10 g, 3.65 mol), triethylamine (1.11 g, 10.95 mmol) and dichloromethane (50 mL) were added. The temperature of the reaction solution was lowered to below 0°C, and tert-butyldimethylsilyl trifluoromethanesulfonate (1.93 g, 7.30 mmol, CAS: 69739-34-0) was slowly added dropwise. The reaction solution was kept at 0°C for 5 minutes, and then heated to room temperature for 2 hours. The reaction solution was cooled to below 0°C, saturated aqueous ammonium chloride solution (100 mL) was slowly added to the reaction solution to quench the reaction, extracted twice with dichloromethane (50 mL), the organic phases were combined, washed once with saturated brine (50 mL), the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to give the title compound as a white solid (1.22 g, yield 81%).

[0091] (S)-1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-hydroxypropan-1-one-2,3,3,3-d4

[0092]

[0093] Methylsulfonamide (0.27 g, 2.89 mmol) and AD-mix-α (9.60 g, CAS: 153130-59-7) were dissolved in a mixed solvent of tert-butyl alcohol (30 mL) and water (30 mL). The reaction solution was cooled in an ice bath, and (E)-tert-butyl((1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)prop-1-en-1-yl-2,3,3,3-d4)oxy)dimethylsilane (1.20 g, 2.89 mmol) was added. The reaction solution was naturally warmed to room temperature and stirred overnight. Saturated sodium sulfite solution (100 mL) was added to the reaction solution to quench the reaction, and ethyl acetate (50 mL) was used to extract twice. The organic phases were combined and washed once with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 8:1) to obtain the title compound as a white solid (0.99 g, yield 108%). LC-MS: m / z [M+H] + = 317.

[0094] Referring to the table below, the following intermediates 7, 10, 11, 15, 19, and 22 were prepared by referring to the preparation method of intermediate 3, except that the raw materials described in the "Raw Materials" column were used to replace the corresponding raw materials.

[0095]

[0096]

[0097] Intermediate 4N-methoxy-N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(tri (fluoromethyl)benzamide

[0098]

[0099] To a three-necked flask, 4-bromo-N-methoxy-N-methyl-3-(trifluoromethyl)benzamide (1 g, 3.2 mmol, intermediate 1), bipyraclostrobin (2.4 g, 8.6 mmol, CAS: 73183-34-3), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (259.5 mg, 0.32 mmol, CAS: 95464-05-4), potassium acetate (942 mg, 9.6 mmol) and dimethyl sulfoxide (4 mL) were added. The atmosphere was replaced with nitrogen three times, and the reaction mixture was heated and stirred at 100 °C overnight under a nitrogen atmosphere. Water (20 mL) was added to the reaction solution for dilution, and ethyl acetate (20 mL) was used for extraction twice. The organic phases were combined, washed twice with water (30 mL), and washed twice with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 3:1) to obtain the title compound as a white solid (775 mg, yield 67%). LC-MS: m / z [M+H]+ =360.

[0100] Intermediate 5 3-(Trifluoromethyl)-4-(3-(trifluoromethyl)-1H-pyrazol-1-yl)benzoic acid

[0101]

[0102] Methyl 3-(trifluoromethyl)-4-(3-(trifluoromethyl)-1H-pyrazol-1-yl)benzoate

[0103]

[0104] Dissolve 2-fluoro-5-trifluoromethylbenzoic acid methyl ester (4.5g, 20.27mmol, CAS: 556112-92-6) in N, N-dimethylformamide (23mL), then add 3-(trifluoromethyl) pyrazole (2.7g, 19.8mmol, CAS: 20154-03-4) and anhydrous potassium carbonate (4.23g, 30.6mmol), and heat the reaction solution to 100°C and react overnight. The reaction solution was cooled to room temperature, diluted with water (60mL), extracted twice with ethyl acetate (200mL), the organic phases were combined, washed twice with water (100mL), washed twice with saturated brine (50mL), and the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain a colorless liquid title compound (4.36g, yield 65%). LC-MS: m / z[M+H] + =339.

[0105] 3-(Trifluoromethyl)-4-(3-(trifluoromethyl)-1H-pyrazol-1-yl)benzoic acid

[0106]

[0107] Methyl 3-(trifluoromethyl)-4-(3-(trifluoromethyl)-1H-pyrazol-1-yl)benzoate (4.36 g, 13 mmol) was dissolved in a mixed solvent of tetrahydrofuran (44 mL) and water (14 mL), and then lithium hydroxide (0.93 g, 39 mmol) was added. The reaction solution was reacted at room temperature for 2 hours. 1N hydrochloric acid was added dropwise to the reaction solution to adjust the pH to about 6, and ethyl acetate (200 mL) was used for extraction twice. The organic phases were combined, washed twice with water (100 mL), and washed twice with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the yellow liquid title compound (4.1 g, yield 97%). LC-MS: m / z[M+H] + =325.

[0108] Intermediate 8 4-(1-(difluoromethyl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)benzoic acid

[0109]

[0110] Methyl 4-(1-(difluoromethyl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)benzoate

[0111]

[0112] Methyl 4-bromo-3-(trifluoromethyl)benzoate (2.0 g, 7.06 mmol, CAS: 107317-58-8), 1-(difluoromethyl)-4-boronic acid pinacol ester-1H-pyrazole (2.58 g, 10.60 mmol, CAS: 1206640-82-5) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane complex (517 mg, 0.70 mmol, CAS: 95464-05-4) were dissolved in 1,4-dioxane (10 mL), and potassium carbonate (2.92 g, 21.20 mmol) and water (2 mL) were added, and the atmosphere was replaced with nitrogen three times. The reaction mixture was reacted at 90 ° C overnight. The reaction solution was cooled to room temperature, diluted with water (50 ml), extracted twice with ethyl acetate (50 mL), the organic phases were combined, washed twice with water (50 mL), washed twice with saturated brine (50 mL), the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain the title compound as a white solid (2.2 g, yield 97%). LC-MS: m / z [M+H] + =321.

[0113] 4-(1-(Difluoromethyl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)benzoic acid

[0114]

[0115] Dissolve methyl 4-(1-(difluoromethyl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)benzoate (1.0 g, 3.12 mmol) in a mixed solvent of tetrahydrofuran (5 mL) and water (2.5 mL), then add lithium hydroxide (224.4 mg, 9.37 mmol), and react at room temperature for 3 hours. Add 1N hydrochloric acid to the reaction solution to adjust the pH to about 6, extract twice with ethyl acetate (20 mL), combine the organic phases, wash twice with water (30 mL), wash twice with saturated brine (30 mL), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the title compound as a white solid (0.63 g, yield 66%). LC-MS: m / z[MH] - =305.

[0116] Intermediate 12 (S)-5-(4-bromo-3-(trifluoromethyl)phenyl)-6-(methyl-d 3 )-3,6-dihydro-2H-1,3,4- Oxadiazine-2-one-6-d

[0117]

[0118] (S)-1-(4-bromo-3-(trifluoromethyl)phenyl)-2-hydroxypropan-1-one-2,3,3,3-d4 (1.07 g, 3.55 mmol, intermediate 11), methyl carbazate (640 mg, 7.10 mmol, CAS: 6294-89-9) and 4 drops of 0.1 N hydrochloric acid were dissolved in methanol (12 mL) and refluxed at 70 ° C for 5 hours. The reaction solution was concentrated under reduced pressure, and a 1.6 M solution of sodium methoxide in methanol (3.35 mL, 5.36 mmol) was added, and the mixture was reacted at room temperature overnight. Water (50 mL) was slowly added to the reaction solution to quench the reaction, and ethyl acetate (50 mL) was extracted twice. The organic phases were combined and washed once with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane / methanol = 20:1) to obtain the title compound as a white solid (775 mg, yield 64%). LC-MS: m / z[M+H] + =341.

[0119] Referring to the table below, the following intermediate 16 was prepared by referring to the preparation method of intermediate 12, except that the raw materials described in the "Raw Materials" column were used to replace the corresponding raw materials.

[0120]

[0121]

[0122] Intermediate 13 1-(Methyl-d3)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

[0123]

[0124] 4-Bromo-1-(methyl-d3)-1H-pyrazole

[0125]

[0126] Sodium hydride (312.7 mg, 7.48 mmol, 60% stored in mineral oil) was dissolved in tetrahydrofuran (3 mL), replaced with nitrogen three times, and the reaction solution was cooled to 0 ° C, and then 4-bromo-1H-pyrazole (1 g, 6.8 mmol, CAS: 2075-45-8) was added in batches. The reaction solution was stirred at 0 ° C for 10 minutes, and then stirred for 30 minutes after warming to room temperature. After the reaction solution was cooled to 0 ° C, deuterated iodomethane (1.28 g, 8.84 mmol, CAS: 865-50-9) was slowly added dropwise. After the dropwise addition, the reaction solution was stirred at room temperature overnight. The reaction solution was directly concentrated, and the residue was slurried with methyl tert-butyl ether (10 mL), filtered, and the filtrate was concentrated to obtain the title compound (700 mg, yield 63%) as a colorless oil. LC-MS: m / z[M+H] + =164.

[0127] 1-(Methyl-d3)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

[0128]

[0129] Add 4-bromo-1-(methyl-d3)-1H-pyrazole (700mg, 4.26mmol), biboronic acid pinacol ester (2.16g, 8.53mmol, CAS: 73183-34-3), [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (309.69mg, 0.42mmol, CAS: 72287-26-4), potassium acetate (1.25g, 12.80mmol) and 1,4-dioxane (10mL) to a single-mouth bottle, replace the nitrogen three times, and heat the reaction solution at 110°C under nitrogen atmosphere and stir overnight. The reaction solution is filtered through diatomaceous earth, the filter cake is washed twice with ethyl acetate (20mL), and the filtrate is concentrated to obtain the title compound as a yellow solid (1.8g crude product). LC-MS: m / z[M+H] + =212.

[0130] Referring to the table below, the following intermediate 25 was prepared by referring to the preparation method of intermediate 13, except that the raw materials described in the "Raw Materials" column were used instead of the corresponding raw materials.

[0131]

[0132]

[0133] Intermediate 14 1-(4-chloro-3-(trifluoromethyl)phenyl)propan-1-one-3,3,3-d 3

[0134]

[0135] 4-Chloro-3-(trifluoromethyl)acetophenone (8g, 35.9mmol, CAS: 129825-11-2) was dissolved in anhydrous tetrahydrofuran (80mL), replaced with nitrogen three times, cooled to -78°C, added with 1M lithium bistrimethylsilylamide tetrahydrofuran solution (39.4mL, 39.4mmol, CAS: 4039-32-1) and stirred at -78°C for 1 hour, then added with deuterated iodomethane (5.455g, 37.63mmol, CAS: 865-50-9), and the reaction solution was stirred at -78°C for 2 hours. The reaction solution was poured into ice water (200mL), extracted twice with ethyl acetate (100mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 3:1) to obtain the title compound as a white solid (4g, yield 46%).

[0136] Intermediate 17 7-Oxaspiro[3.5]non-1-en-2-yl trifluoromethanesulfonate

[0137]

[0138] 7-Oxaspiro[3.5]nonane-2-one (400 mg, 2.86 mmol, synthesized according to the method provided in patent WO2011130146A1) was added to anhydrous tetrahydrofuran (5 mL), and the solution was stirred at -78 ° C for 10 minutes, and then lithium diisopropylamide (2.15 mL, 4.3 mmol, 2M tetrahydrofuran / n-hexane solution) was slowly added dropwise, and the mixture was stirred at -78 ° C for 30 minutes, and then N-phenylbis(trifluoromethanesulfonyl)imide (1.02 g, 2.86 mmol, CAS: 37595-74-7) was added dropwise in anhydrous tetrahydrofuran (2 mL), and the mixture was stirred at room temperature for 3 hours. Water (30 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted twice with ethyl acetate (30 mL). The organic phases were combined and washed once with saturated brine (10 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to obtain the title compound as a yellow oil (300 mg, yield 39%). LC-MS: m / z [M+H] + =273.

[0139] Intermediate 18 N-methoxy-N-methyl-4-(7-oxaspiro[3.5]non-2-yl)-3-(trifluoromethyl)benzamide

[0140]

[0141] N-Methoxy-N-methyl-4-(7-oxaspiro[3.5]non-1-en-2-yl)-3-(trifluoromethyl)benzamide

[0142]

[0143] N-methoxy-N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)benzamide (633 mg, 1.76 mmol, intermediate 4), 7-oxaspiro[3.5]non-1-en-2-yl trifluoromethanesulfonate (400 mg, 1.47 mmol, intermediate 17), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (259.5 mg, 0.32 mmol, CAS: 72287-26-4) and potassium carbonate (609 mg, 4.41 mmol) were added to 1,4-dioxane (4 mL) and water (0.8 mL), and the atmosphere was replaced with nitrogen three times. The mixture was heated and stirred at 90 °C overnight under nitrogen protection. The reaction solution was cooled to room temperature, water (30 mL) was added to dilute the reaction solution, and ethyl acetate (30 mL) was used to extract twice. The organic phases were combined, washed twice with water (30 mL), and washed twice with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 3:2) to obtain the title compound (290 mg, yield 56%). LC-MS: m / z [M+H] + =356.

[0144] N-Methoxy-N-methyl-4-(7-oxaspiro[3.5]nonan-2-yl)-3-(trifluoromethyl)benzamide

[0145]

[0146] N-methoxy-N-methyl-4-(7-oxaspiro[3.5]non-1-en-2-yl)-3-(trifluoromethyl)benzamide (290 mg, 0.82 mmol) was dissolved in methanol (5 mL), palladium carbon (60 mg) was added, and hydrogen was replaced three times. The mixture was stirred at 25 ° C in a hydrogen atmosphere overnight. The reaction solution was filtered through diatomaceous earth, and water (20 mL) was added to the filtrate for dilution. Ethyl acetate (20 mL) was extracted twice, and the organic phases were combined, washed twice with water (30 mL), and washed twice with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 2:1) to obtain the title compound (246 mg, yield 84%). LC-MS: m / z[M+H] + =358.

[0147] Intermediate 21 1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)propan-1-one-2,2-d 2

[0148]

[0149] 1-(4-Chloro-3-(trifluoromethyl)phenyl)propan-1-one

[0150]

[0151] 4-Chloro-N-methoxy-N-methyl-3-(trifluoromethyl)benzamide (1000 mg, 3.88 mmol, intermediate 20) was dissolved in tetrahydrofuran (10 mL), replaced with nitrogen 3 times, cooled to -78 ° C, and 1M ethylmagnesium bromide tetrahydrofuran solution (9.7 mL, 9.7 mmol) was slowly added, and the mixture was stirred at 25 ° C for 2 hours. The reaction solution was poured into a saturated aqueous ammonium chloride solution (50 mL), extracted twice with ethyl acetate (30 mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain the title compound (800 mg, yield 91%).

[0152] 1-(4'-Fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)propan-1-one

[0153]

[0154] 1-(4-chloro-3-(trifluoromethyl)phenyl)propan-1-one (800 mg, 3.38 mmol), 4-fluorophenylboric acid (473.06 mg, 3.38 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (161.18 mg, 0.34 mmol, CAS: 564483-18-7), chloro(2-dicyclohexylphosphino-2',4' ,6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (301.66mg, 0.34mmol, CAS:1310584-14-5) and cesium carbonate (2203.16mg, 6.76mmol) were added to dioxane (10mL) and water (2mL), replaced with nitrogen three times, and the mixture was stirred at 100°C overnight. The reaction solution was filtered through diatomaceous earth, diluted with water (30mL), extracted twice with ethyl acetate (20mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 3:1) to obtain the title compound (801mg, yield 80%).

[0155] 1-(4'-Fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)propan-1-one-2,2-d2

[0156]

[0157] 1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)propan-1-one (800 mg, 2.72 mmol) was dissolved in deuterated acetone (10 mL), sodium acetate (221.5 mg, 2.72 mmol) and 1-butyl-3-methylimidazole-3-chloride (235.6 mg, 1.36 mmol, CAS: 79917-90-1) were added, and the mixture was stirred at 80°C overnight. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain the title compound (800 mg, yield 90%).

[0158] Intermediate 23 1-Cyclopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

[0159]

[0160] 4-Bromo-1-cyclopropyl-1H-pyrazole (2.0 g, 10.8 mmol, CAS: 1151802-23-1), bipyraclostrobin (5.5 g, 21.6 mmol, CAS: 73183-34-3), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (700 mg, 1.08 mmol, CAS: 72287-26-4) and potassium acetate (2.16 g, 21.6 mmol) were added to 1,4-dioxane (40 mL), and the atmosphere was replaced with nitrogen three times. The reaction solution was heated and stirred at 100 °C overnight under a nitrogen atmosphere. The reaction solution was filtered through celite, water (100 mL) was added to the filtrate for dilution, and the mixture was extracted twice with ethyl acetate (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain the title compound (1.3 g, yield 52%). LC-MS: m / z [M+H] + =235.

[0161] Intermediate 24 2,2-Difluoro-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolidone oxazol-1-yl)ethan-1-ol

[0162]

[0163] Ethyl 2-(4-bromo-1H-pyrazol-1-yl)-2,2-difluoroacetate

[0164]

[0165] Under nitrogen protection and ice bath conditions, sodium hydride (1197.52 mg, 29.94 mmol, 60% stored in mineral oil) was added in batches to a solution of 4-bromo-1H-pyrazole (4000 mg, 27.22 mmol, CAS: 2075-45-8) in N,N-dimethylformamide (40 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction solution was cooled to 0°C, 2-bromo-2,2-difluoroacetic acid methyl ester (3.3 mL, 29.94 mmol, CAS: 683-98-7, d = 1.7125 g / mL) was slowly added dropwise, and the reaction solution was stirred at room temperature overnight after the addition was completed. Saturated ammonium chloride solution (100 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted three times with ethyl acetate (40 mL). The organic phases were combined, washed once with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered and concentrated, and separated and purified by column chromatography (petroleum ether / ethyl acetate = 50:1 to 10:1) to give the title compound (3000 mg, yield 41%) as a colorless oil.

[0166] 2-(4-Bromo-1H-pyrazol-1-yl)-2,2-difluoroethan-1-ol

[0167]

[0168] Under ice bath conditions, sodium borohydride (267 mg, 7.06 mmol) was added in batches to a solution of ethyl 2-(4-bromo-1H-pyrazol-1-yl)-2,2-difluoroacetate (1900 mg, 7.06 mmol) in tetrahydrofuran (20 mL), and the mixture was stirred at room temperature for 2 hours. Saturated ammonium chloride solution (40 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted 3 times with ethyl acetate (20 mL). The organic phases were combined, washed once with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain the title compound as a colorless liquid (1200 mg, yield 75%).

[0169] 2,2-Difluoro-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)ethyl 1-ol

[0170]

[0171] 2-(4-bromo-1H-pyrazol-1-yl)-2,2-difluoroethane-1-ol (900 mg, 3.96 mmol), biboronic acid pinacol ester (1.21 g, 4.76 mmol, CAS: 73183-34-3), [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (290.09 mg, 0.4 mmol, CAS: 72287-26-4) and potassium acetate (778.17 mg, 7.93 mmol) were added to 1,4-dioxane (10 mL), and the nitrogen atmosphere was replaced 3 times. The reaction solution was heated and stirred at 95 ° C under a nitrogen atmosphere overnight. The reaction solution was filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 20:1 to 10:1) to obtain a colorless liquid title compound (600 mg, yield 55%). LC-MS: m / z[M+H] + =275.

[0172] Example 1

[0173] (S)-5-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-6-(methyl-d3)-3,6-dihydro-2H- 1,3,4-oxadiazine-2-one-6-d

[0174]

[0175] (S)-1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-hydroxypropan-1-one-2,3,3,3-d4 (0.98 g, 2.70 mmol, intermediate 3), methyl carbazate (486 mg, 5.40 mmol, CAS: 6294-89-9) and 2 drops of 1.5N hydrochloric acid were dissolved in methanol (20 mL) and refluxed at 70°C for 2 hours. The reaction solution was concentrated under reduced pressure, and a 1.6 M methanol solution of sodium methoxide (8.60 mL, 13.7 mmol) was added, and the mixture was reacted at room temperature overnight. Water (100 mL) was slowly added to the reaction solution to quench the reaction, and the reaction was extracted twice with ethyl acetate (50 mL). The organic phases were combined and washed once with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The product was purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to give the title compound as a white solid (339 mg, yield 35%). 1 H NMR (400MHz, DMSO-d6) δ11.30(s,1H),8.16(d,J=1.6Hz,1H),8.05(dd,J=8.0,1.6Hz ,1H),7.53(d,J=8.0Hz,1H),7.41-7.38(m,2H),7.33-7.29(m,2H); LC-MS:m / z[M+H] + =357.

[0176] Example 2

[0177] (S)-6-(methyl-d3)-5-(3-(trifluoromethyl)-4-(3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-3, 6-Dihydro-2H-1,3,4-oxadiazin-2-one-6-d

[0178]

[0179] The title compound was synthesized in a manner similar to Example 1 by replacing (S)-1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-hydroxypropan-1-one-2,3,3,3-d4 (Intermediate 3) with (S)-2-hydroxy-1-(3-(trifluoromethyl)-4-(3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)propan-1-one-2,3,3,3-d4 (Intermediate 7). 1 H NMR (400MHz, DMSO-d6) δ11.41 (s, 1H), 8.39 (s, 1H), 8.26 (d, J = 2.0Hz, 1H), 8.20 (dd ,J=8.0,1.6Hz,1H),7.86(d,J=8.4Hz,1H),7.06(d,J=2.4Hz,1H); LC-MS:m / z[M+H] + =397.

[0180] Example 3

[0181] (S)-5-(4-(1-(difluoromethyl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)phenyl)-6-(methyl-d3)-3, 6-Dihydro-2H-1,3,4-oxadiazin-2-one-6-d

[0182]

[0183] The title compound was synthesized in a similar manner to Example 1 by replacing (S)-1-(4-(1-(difluoromethyl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)phenyl)-2-hydroxypropan-1-one-2,3,3,3-d4 (Intermediate 10) with (S)-1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-hydroxypropan-1-one-2,3,3,3-d4 (Intermediate 3). 1 H NMR (400MHz, DMSO-d6) δ11.31(s,1H),8.49(s,1H),8.16(d,J=1.6Hz,1H),8.05(d,J=8.0 ,1.6Hz,1H),7.99(s,1H),7.90(t,J=58.8Hz,1H),7.70(d,J=8.0Hz,1H); LC-MS:m / z[M+H] + =379.

[0184] Example 4

[0185] (S)-5-(4-(2-aminopyridin-4-yl)-3-(trifluoromethyl)phenyl)-6-(methyl-d3)-3,6-dihydro-2H- 1,3,4-oxadiazine-2-one-6-d

[0186]

[0187] (S)-5-(4-bromo-3-(trifluoromethyl)phenyl)-6-(methyl-d3)-3,6-dihydro-2H-1,3,4-oxadiazin-2-one-6-d (100 mg, 0.29 mmol, intermediate 12), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (97 mg, 0.44 mmol, CAS: 1195995-72-2), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (21 mg, 0.029 mmol, CAS: 72287-26-4) and potassium carbonate (120 mg, 0.86 mmol) were dissolved in a mixed solvent of 1,4-dioxane (2 mL) and water (0.5 mL), and the atmosphere was replaced with nitrogen three times. The reaction solution was heated at 90° C. and stirred overnight. Water (20 mL) was added to the reaction solution for dilution, and the mixture was extracted twice with ethyl acetate (20 mL). The organic phases were combined, washed twice with water (30 mL) and twice with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The title compound (27 mg, yield 26%) was obtained by reverse phase preparation and purification under neutral conditions. 1H NMR (400MHz, DMSO-d6) δ11.31(s,1H),8.15(d,J=1.8Hz,1H),8.05(dd,J=8.0,1.8Hz,1H),7.96(d,J= 5.2Hz,1H),7.50(d,J=8.0Hz,1H),6.44(d,J=5.2Hz,1H),6.38(s,1H),6.10(s,2H); LC-MS:m / z[M+H] + =355.

[0188] Example 5

[0189] (S)-6-(methyl-d3)-5-(4-(1-(methyl-d3)-1H-pyrazol-4-yl)-3-(trifluoromethyl)phenyl)-3,6- Dihydro-2H-1,3,4-oxadiazin-2-one-6-d

[0190]

[0191] The title compound was synthesized in a similar manner to Example 4 by replacing 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (CAS: 1195995-72-2) with 1-(methyl-d3)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (Intermediate 13). 1 H NMR (400MHz, DMSO-d6) δ11.25 (s, 1H), 8.11 (d, J = 2.0Hz, 1H), 7.99 (dd, J = 8.0, 2.0Hz,1H),7.97(s,1H),7.63(s,2H),7.62(d,J=8.0Hz,1H); LC-MS:m / z[M+H] + =346.

[0192] Example 6

[0193] (S)-5-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-6-(methyl-d3)-3,6-dihydro-2H- 1,3,4-oxadiazine-2-one

[0194]

[0195] (S)-5-(4-chloro-3-(trifluoromethyl)phenyl)-6-(methyl-d3)-3,6-dihydro-2H-1,3,4-oxadiazine-2-one (100 mg, 0.34 mmol, intermediate 16), p-fluorophenylboric acid (52.06 mg, 0.37 mmol, CAS: 1765-93-1), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (16.12 mg, 0.03 mmol, CAS: 564483- 18-7), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (30.18 mg, 0.03 mmol, CAS: 1310584-14-5) and cesium carbonate (220.4 mg, 0.68 mmol) were dissolved in a mixed solvent of 1,4-dioxane (4 mL) and water (1 mL), replaced with nitrogen three times, and the reaction solution was heated and stirred at 100°C overnight. Water (30 mL) was added to the reaction solution for dilution, and ethyl acetate (15 mL) was used for extraction twice. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative liquid chromatography (acetonitrile and water containing 0.1% formic acid) to obtain the title compound as a white solid (18 mg, yield 15%). 1 H NMR(400MHz,DMSO-d6)δ11.32(s,1H),8.16(d,J=1.9Hz,1H),8.05(dd,J=8.1,1.9Hz,1H), 7.52(d,J=8.1Hz,1H),7.42-7.37(m,2H),7.36-7.27(m,2H),5.94(s,1H); LC-MS:m / z[M+H] + =356.

[0196] Example 7

[0197] (S)-5-(4-(7-oxaspiro[3.5]nonan-2-yl)-3-(trifluoromethyl)phenyl)-6-(methyl-d3)-3,6-difluorophenyl Hydrogen-2H-1,3,4-oxadiazine-2-one-6-d

[0198]

[0199] The title compound was synthesized in a manner similar to Example 1 by replacing (S)-1-(4-(7-oxaspiro[3.5]non-2-yl)-3-(trifluoromethyl)phenyl)-2-hydroxypropan-1-one-2,3,3,3-d4 (Intermediate 19) with (S)-1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-hydroxypropan-1-one-2,3,3,3-d4 (Intermediate 3). 1H NMR (400MHz, DMSO-d6) δ11.22(s,1H),8.00(d,J=7.6Hz,1H),7.99(s,1H),7.82(d,J=8.4Hz,1H),3.86-3.75(m,1H),3.64-3 .53(m,2H),3.51-3.42(m,2H),2.31-2.20(m,2H),2.02–1.96(m,2H),1.76-1.65(m,2H),1.60-1.49(m,2H); LC-MS:m / z[M+H] + =387.

[0200] Example 8

[0201] (S)-5-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-6-methyl-3,6-dihydro-2H-1,3,4- Oxadiazine-2-one-6-d

[0202]

[0203] The title compound was synthesized in a similar manner to Example 1 by replacing (S)-1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-hydroxypropan-1-one-2-d (Intermediate 22) with (S)-1-(4'-fluoro-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-hydroxypropan-1-one-2,3,3,3-d4 (Intermediate 3). 1 H NMR(400MHz,DMSO-d6)δ11.30(s,1H),8.17–8.10(m,1H),8.04(dd,J=8.2,1.9Hz,1H),7.52(d ,J=8.1Hz,1H),7.39(dd,J=8.5,5.5Hz,2H),7.35–7.26(m,2H),1.46(s,3H); LC-MS:m / z[M+H] + =354.

[0204] Example 9

[0205] (S)-5-(4-(1-cyclopropyl-1H-pyrazol-4-yl)-3-(trifluoromethyl)phenyl)-6-(methyl-d3)-3,6-dione Hydrogen-2H-1,3,4-oxadiazine-2-one-6-d

[0206]

[0207] The title compound was synthesized in a similar manner to Example 4 by replacing 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (Intermediate 23) with 1-cyclopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (CAS: 1195995-72-2). 1H NMR (400MHz, DMSO-d6) δ11.26(s,1H),8.11(d,J=1.9Hz,1H),8.05(s,1H),7.98(d,J=8.0Hz,1H),7.68–7.59( m,2H),3.82(dq,J=7.5,3.6Hz,1H),1.10(dd,J=5.1,2.7Hz,2H),1.01(dd,J=7.4,4.8Hz,2H); LC-MS:m / z[M+H] + =369.

[0208] Example 10

[0209] (S)-5-(4-(1-(1,1-difluoro-2-hydroxyethyl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)phenyl)-6-(methyl d3)-3,6-dihydro-2H-1,3,4-oxadiazine-2-one-6-d

[0210]

[0211] The title compound was synthesized in a similar manner to Example 4 by replacing 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)ethan-1-ol (Intermediate 24) with 2,2-difluoro-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (CAS: 1195995-72-2). 1 H NMR (400MHz, DMSO-d6) δ11.30(s,1H),8.43(s,1H),8.16(d,J=1.6Hz,1H),8.04(dd,J=8.4,2.0Hz,1H),7 .95(s,1H),7.70(d,J=8.0Hz,1H),6.06(t,J=6.4Hz,1H),4.28(td,J=12.0,6.4Hz,2H); LC-MS:m / z[M+H] + =409.

[0212] Embodiment 11

[0213] (S)-5-(4-(1-(oxetan-3-yl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)phenyl)-6-(methyl- d3)-3,6-dihydro-2H-1,3,4-oxadiazin-2-one-6-d

[0214]

[0215] The title compound was synthesized in a similar manner to Example 4 by replacing 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (CAS: 1195995-72-2) with 1-(oxetan-3-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (Intermediate 25). 1H NMR(400MHz,DMSO-d6)δ11.26(s,1H),8.15(s,1H),8.12(s,1H),8.03–7.97(m,1H),7.80 (s,1H),7.67(d,J=8.2Hz,1H),5.68(p,J=6.9Hz,1H),4.99–4.90(m,4H); LC-MS:m / z[M+H] + =385.

[0216] Biological detection methods:

[0217] Test Example 1. SK-MEL-3 cell proliferation assay

[0218] 1. Experimental purpose: To detect the inhibitory activity of compounds on SK-MEL-3 cell proliferation.

[0219] 2. Experimental materials:

[0220] 2.1 Drugs: (Promega; G7573); McCoy's5A Medium (GIBCO; 16600-082); Fetal bovine serum (ExCell Bio; FND500) and Dimethyl sulfoxide (DMSO; Solarbio; D8371)

[0221] 2.2 Compounds: Compounds prepared in Examples of the present invention and reference compounds

[0222] 2.3 Experimental instruments / materials: 37°C incubator (Thermo; HERA cell vios 160i), clean bench (AIRTECH; VS-1300L-U), inverted microscope (OPTEC; BDS400); cell counter (Count star; IC1000); microplate reader (Molecular Devices; SpectraMax Paradigm); 96-well plate (Biosahrp; BS-MP-96W).

[0223] 3. Experimental Methods

[0224] SK-MEL-3 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. The cells were placed in McCoys-5A (Gibco) medium (containing 10% fetal bovine serum; ExCell bio), and 1% penicillin and streptomycin were added, and cultured in a cell culture incubator under 37°C, 5% CO2 and saturated humidity.

[0225] After the cells are full, the cells are subcultured into 96-well plates (5000 cells / well). After overnight culture, compounds at concentrations of 0.001nM, 0.005nM, 0.026nM, 0.128nM, 0.64nM, 3.2nM, 16nM, 80nM, 400nM and 2000nM are added to the cells. 0.1% DMSO and culture medium are set as solvent and negative control, respectively. Each sample has 2 replicates. After the compounds are mixed, they are cultured in a 37-well, 5% CO2 incubator for 72h. After treatment, the 96-well plate is placed at room temperature for 30min, and 50μL (Promega), and placed on an orbital shaker for 2 minutes, and then placed at room temperature in the dark for 10 minutes. The 96-well plate was placed in SpectraMax Paradigm (Molecular Devices) to test the luminance signal value.

[0226] Cell activity inhibition rate (%) = 100-(RLUcompound-RLUblank) / (RLUcontrol-RLUblank)*100%, Control is the 0.1DMSO treatment group, blank is the medium treatment group, and the compound IC is calculated using the nonlinear regression function "Dose-Response-Inhibition" in Graphpad Prism 7.0 50 And draw the dose-effect curve.

[0227] Table 1: In vitro anti-proliferation IC of several examples in SK-MEL-3 cells 50 value

[0228]

[0229] - indicates that the activity was not determined.

[0230] As can be seen from the above table, the deuterated compounds of the present invention have comparable or better inhibitory activity on cells than the undeuterated compounds. Test Example 2. A2058 Cell Proliferation Assay

[0231] A2058 cells were purchased from Shanghai Fuheng Biotechnology. The cells were resuspended in RPMI160 medium + 10% fetal bovine serum (Giboco) and then cultured in a cell culture incubator at 37°C, 5% CO2 and saturated humidity. When the confluence of A2058 cells reached 80%-90%, the cells were digested and counted. The counted cells were plated in a 96-well plate (2000 cells / well). After the cells were cultured for 24 hours, the test compounds or control compounds at concentrations of 2000nM, 400nM, 80nM, 16nM, 3.2nM, 0.64nM, 0.128nM, 0.0256nM and 0.00512nM were added, and the final concentration of DMSO was 0.2%. Each sample had 2 replicate wells, and 0.2% DMSO and culture medium controls were set. The compounds were mixed and cultured in a cell culture incubator for 96 hours. After compound treatment, 50 μL of (Promega), shake and mix for 2 minutes, and then incubate at room temperature in the dark for 10 minutes. SpectraMax Paradigm (Molecular Devices) was used to test the luminance signal value of the 96-well plate. Cell activity inhibition rate (%) = 100-(RLUcompound-RLUblank) / (RLUcontrol-RLUblank)*100%, where control is the 0.2% DMSO treatment group and blank is the culture medium blank.

[0232] Table 2: In vitro anti-proliferation IC of several examples in A2058 cells 50 value

[0233]

[0234] Test Example 3. Liver microsome metabolic stability

[0235] Phase I metabolic stability test uses an in vitro incubation system of liver microsomes to simulate the physiological conditions of liver microsomes and add redox coenzymes (NADPH) for related reactions. First, 445 μL of mouse or human liver microsome working solution (mouse: Cat no. M1000, Xenotech; human: Cat no. 452117, Corning) with a concentration of 0.56 mg / mL was added to two 96-well culture plates, and they were named T60 incubation plate (with NADPH added) and non co-factor (NCF) 60 incubation plate (without NADPH).

[0236] After the above incubation plate was placed in a 37°C water bath for pre-incubation for about 10 minutes, 5 μL of the test compound or control compound (testosterone, diclofenac and propafenone) at a concentration of 100 μM was added. 50 μL of potassium phosphate buffer was added to each well of the NCF60 incubation plate to start the reaction. A T0 stop 96 plate was prepared, and 180 μL of stop solution (containing 200 ng / mL tolbutamide and 200 ng / mL labetalol in acetonitrile) and 6 μL of NADPH regeneration system working solution were added in advance. 54 μL of sample was taken from the T60 incubation plate to the T0 stop plate (T0 sample generation). 44 μL of NADPH regeneration system working solution was added to each well of the T60 incubation plate to start the reaction. Only 54 μL of microsome working solution, 6 μL of NADPH regeneration system working solution and 180 μL of stop solution were added to the Blank plate. Therefore, in the samples of the test or control compound, the final reaction concentration of the compound, testosterone, diclofenac and propafenone was 1 μM, the concentration of liver microsomes was 0.5 mg / mL, and the final concentrations of DMSO and acetonitrile in the reaction system were 0.01% (v / v) and 0.99% (v / v), respectively.

[0237] After incubation for an appropriate time (e.g., 5, 15, 30, 45, and 60 minutes), 180 μL of stop solution (containing 200 ng / mL tolbutamide and 200 ng / mL labetalol in acetonitrile) was added to each well of the stop plate, and then 60 μL of sample was taken from the T60 incubation plate to terminate the reaction. All sample plates were shaken and centrifuged at 3220 g for 20 minutes, and then 80 μL of supernatant was taken from each well and diluted into 240 μL of pure water for liquid chromatography tandem mass spectrometry analysis.

[0238] Table 3: Metabolic stability of liver microsomes

[0239]

[0240]

[0241] As can be seen from the above table, compared with the non-deuterated compound, the clearance rate of the deuterated compound of the present invention in mouse liver microsomes is reduced and the half-life is significantly prolonged, indicating that the deuterated compound of the present invention is metabolized more slowly than the non-deuterated compound and has a potential longer in vivo action time.

[0242] From the above effects, the following structure-activity relationship can be preliminarily obtained:

[0243] 1) For the compounds of the present invention, in the structure Part, deuteration of specific positions of R1 and R2 is a key position that affects the metabolic stability of the deuterated compound of the present invention, and different deuteration amounts have significantly different effects on liver metabolic stability, such as the deuterated compounds of Examples 1, 6 and 2 (R1 is H or D, R2 is CD3), the liver clearance rate in vivo is reduced compared to the undeuterated compound, showing a significantly prolonged half-life, and the metabolic stability of the molecule is improved, while the deuterated compound of Example 8 (R1 is D, R2 is CH3) has a reduced liver metabolic stability compared to the undeuterated compound molecule. That is, the metabolism and pharmacokinetic effects of the drug after deuteration are difficult to predict, and the specific deuterated site (R1 is H or D, R2 is CD3) selected by the present invention can significantly improve the drug half-life.

[0244] 2) Based on the preferred deuterated site, the present invention has made various designs for the rightmost ring A, such as Examples 3, 4, 5, 7, 9, 10, and 11. The results show that in the human liver microsome stability test, The different structures of some of them also achieved better metabolic stability than the existing technology, indicating that ring A and its substituted R a The moiety is not a major site affecting the metabolism of the compounds of the invention.

[0245] Test Example 4. Pharmacokinetic Evaluation in Mice

[0246] The clear solution of the test compound was injected into ICR mice via the tail vein (overnight fasting) or administered to ICR mice by gavage (fed). The intravenous and oral administration dose distribution was 1 mg / kg and 5 mg / kg. Blood was collected from cheek puncture at 0h (before administration) and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24h after intravenous injection, and 50 μL of blood was placed in an anticoagulant tube with sodium heparin added. The mixture was vortexed and centrifuged at 6000rpm for 3 minutes at 4°C; blood was collected from cheek puncture at 0h (before administration) and 0.083, 0.25, 1, 2, 4, 6, 8, 24h after oral gavage, and placed in an anticoagulant tube with sodium heparin added. The mixture was vortexed and centrifuged at 6000rpm for 3 minutes. The plasma drug concentration was determined by LC-MS / MS, and the relevant pharmacokinetic parameters were calculated by the non-compartmental linear logarithmic trapezoidal method using Phoenix WinNonlin8.2.0 pharmacokinetic software.

[0247] Table 4: Pharmacokinetic parameters in mice

[0248]

[0249] As can be seen from the table above, the half-life of the deuterated compound 1 substituted at a specific site of the present invention in mice is significantly prolonged compared with the undeuterated compound, and the AUC is also better than that of the comparative compound, showing a significant difference and having better pharmacokinetic properties. Test Example 5. PDE3A enzyme activity inhibition experiment

[0250] The present invention uses isotope-labeled 3 H]cAMP was used as a substrate to test the inhibitory activity of compounds on PDE3A (IC 50 ). Dilute the tritiated substrate cAMP stock solution 100 times with pure water as the mother solution, and then dilute the substrate mother solution with assay buffer (50mMTris.HCl, 10mM MgCl2, 0.5mM DTT, pH 8.0) to make the cpm value of the liquid scintillation counter 20000-30000. Add 58μL of the diluted substrate solution to a 1mL EP tube, slowly add 2μL of a series of gradient concentration compound solutions (50μM, 12.5μM, 3.125μM, 781.25nM, 195.31nM, 48.83nM, 12.21nM, 3.05nM, 0.76nM, 0.19nM), mix well, and add 2μL of 0.1% DMSO to the control group. Add 40 μL of PDE3A protein solution diluted to an appropriate concentration with assay buffer, and add 40 μL of assay buffer to the negative control group, and let the reaction stand at room temperature for 15 minutes. Then add 200 μL of 0.2M ZnSO4 and 200 μL of 0.2M BaOH2 solution in sequence to terminate the reaction, shake and mix evenly, and centrifuge for 5 minutes. Take 430 μL of the supernatant, transfer it to a scintillation tube to which 2.5 mL of scintillation fluid has been added, mix evenly, and measure the radioactivity in the supernatant with a liquid scintillation counter to determine the enzyme activity. Enzyme activity inhibition rate (%) = 100-(RLUcompound-RLUblank) / (RLUcontrol-RLUblank)*100%, Control is the DMSO treatment group, blank is the negative control group, and the nonlinear regression function "Dose-Response-Inhibition" in Graphpad Prism 7.0 is used to calculate the compound IC 50 And draw the dose-effect curve.

[0251] Table 5: IC50 values ​​for PDE3A enzyme activity inhibition

[0252]

[0253] Long-term inhibition of PDE3A enzyme activity can cause palpitations and sudden death of heart patients, reduce platelet production, and affect oocyte maturation and other toxic side effects. Therefore, within a certain range, the higher the IC50 value of PDE3A enzyme activity inhibition, the better the safety of the drug. As can be seen from the above table, compounds 1, 3, and 4 of the present invention have better safety than the comparative example compounds.

[0254] Test Example 6. In vivo efficacy experiment in mice

[0255] The Balb / c nude mice used in this experiment were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. or Jiangsu Jicui Yaokang Biotechnology Co., Ltd. They were female, 6-8 weeks old, and weighed 18-22 grams. After the animals arrived, they were isolated and quarantined in the experimental environment for 7 days before the experiment began. SK-MEL-3 cells in the logarithmic growth phase (Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60325, plus matrix gel, volume ratio of 1:1) were subcutaneously inoculated into the right back of the mice, and group dosing began when the average tumor volume reached about 100-150mm3. Oral QD was administered continuously for 21 days, and the tumor was measured and weighed 3 times a week. The tumor volume was measured by vernier caliper, and the formula was TV = 0.5×a×b 2 , where a is the long diameter of the tumor, b is the short diameter of the tumor, and the tumor weight is measured after the experiment. The anti-tumor efficacy of the compound is evaluated by TGI (%). TGI (%) reflects the tumor growth inhibition rate, TGI (%) = [(1-(average tumor volume at the end of administration of a treatment group-average tumor volume at the beginning of administration of the treatment group)) / (average tumor volume at the end of treatment of the solvent control group-average tumor volume at the beginning of treatment of the solvent control group)] × 100%.

[0256] Conclusion: Compared with the non-deuterated compound, the deuterated compound in the example of the present application has a better inhibitory effect on the growth of the mouse SK-MEL-3 subcutaneous transplanted tumor model.

[0257] Those skilled in the art will recognize that the scope of the present application is not limited to the various specific implementations and examples described above, but that various modifications, replacements, or recombinations can be made without departing from the spirit of the present application, which all fall within the scope of protection of the present application.

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof or a mixture thereof: in, R1 is selected from hydrogen or deuterium; R2 is selected from CD3; R3, R4, R5, R6 are selected from hydrogen; Ring A is selected from phenyl, 5-6 membered heteroaryl or 5-9 membered heterocyclyl; R a is selected from hydrogen, halogen, amino, undeuterated or one or more times deuterated or fully deuterated C1-C4 alkyl, C1-C4 haloalkyl, -CF2CH2OH, 3-6 membered heterocyclyl, C(=O)NH2, C(=O)NHCH3 or C3-C6 cycloalkyl; n is selected from 0, 1 or 2.

2. The compound of formula (I) according to claim 1, its pharmaceutically acceptable salt, its stereoisomer, tautomer or mixture thereof, characterized in that: The compound of formula (I) has the structure shown in formula (II): Among them, ring A, R a and n are as defined in claim 1.

3. The compound according to any one of claims 1 to 2, its pharmaceutically acceptable salt, its stereoisomer, tautomer or a mixture thereof, characterized in that: Ring A is selected from phenyl, R a is selected from hydrogen, halogen, amino, undeuterated or one or more times deuterated or perdeuterated C1-C4 alkyl, C1-C4 haloalkyl or C3-C6 cycloalkyl; n is selected from 1 or 2.

4. The compound according to any one of claims 1 to 2, its pharmaceutically acceptable salt, its stereoisomer, tautomer or a mixture thereof, characterized in that: Ring A is selected from phenyl; R a is selected from F, Cl, CF3, CHF2, amino, cyclopropyl, CH3, CH2D, CHD2 or CD3; n is selected from 1 or 2.

5. The compound according to any one of claims 1 to 2, its pharmaceutically acceptable salt, its stereoisomer, tautomer or a mixture thereof, characterized in that: Ring A is selected from phenyl; R a Selected from F.

6. The compound according to claim 1, its pharmaceutically acceptable salt, its stereoisomer, tautomer or a mixture thereof, characterized in that: Selected from the following compounds:

7. A pharmaceutical composition, characterized in that It comprises a pharmaceutically acceptable carrier and the compound according to any one of claims 1 to 6, its pharmaceutically acceptable salt, its stereoisomer, tautomer or a mixture thereof.

8. Use of the compound according to any one of claims 1 to 6, its pharmaceutically acceptable salt, its stereoisomer, tautomer or mixture thereof, or the pharmaceutical composition according to claim 7 in the preparation of a drug for regulating the interaction between PDE3A and SLFN12.

9. The use according to claim 8, characterized in that The drug is capable of enhancing and / or promoting the interaction between PDE3A and SLFN12.

10. Use of the compound according to any one of claims 1 to 6, its pharmaceutically acceptable salt, its stereoisomer, tautomer or mixture thereof, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating and / or preventing cancer diseases, wherein the cancer diseases are brain cancer, breast cancer, cervical cancer, AML, lung cancer, skin cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer and sarcoma.

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