Unit dose compositions of AKT inhibitors

Abstract

The present application provides a unit dose composition of an AKT inhibitor, and in particular relates to a pharmaceutical composition in a unit dose form comprising a compound of formula I or a pharmaceutically acceptable salt thereof wherein the compound I or the pharmaceutically acceptable salt thereof has a mass of 5 mg to 400 mg in terms of free alkali. # imgabs0 #

Description

[0001] This application claims the priority of the Chinese patent application filed with the Chinese Patent Office on July 22, 2020, with application number 202010709847.2 and invention name “Unit dose composition of AKT inhibitor”, the entire contents of which are incorporated by reference into this application.

[0002] This application is a divisional application of the Chinese invention patent application with application date of July 22, 2021, application number 202180049206.1, and name “Unit dose composition of AKT inhibitor”. Technical Field

[0003] The present application belongs to the field of pharmaceutical chemistry, and specifically relates to a unit dose composition of an AKT inhibitor, a preparation method thereof, and medical uses thereof. Background Art

[0004] The PI3K / AKT / mTOR pathway, which consists of phosphatidylinositol 3-kinase (PI3K) and its downstream protein AKT (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR), is a very important signal transduction pathway in cells, playing an extremely important biological function in cell growth, survival, proliferation, apoptosis, angiogenesis, autophagy, etc. Abnormal activation of this pathway can cause a series of diseases, including cancer, neuropathy, autoimmune diseases and blood lymphatic system diseases.

[0005] AKT is a serine / threonine kinase that affects cell survival, growth, metabolism, proliferation, migration and differentiation through numerous downstream effectors. More than 50% of human tumors have overactivation of AKT, especially prostate cancer, pancreatic cancer, bladder cancer, ovarian cancer and breast cancer. Overactivation of AKT can lead to tumorigenesis, metastasis and drug resistance.

[0006] AKT has three subtypes: AKT1, AKT2 and AKT3. As a typical protein kinase, each subtype consists of an amino-terminal PH domain (Pleckstrinhomology domain), a central ATP-binding kinase domain and a carboxyl-terminal regulatory domain. The three subtypes have approximately 80% homology in amino acid sequence, with only a large difference in the PH domain and kinase domain junction region.

[0007] At present, targeted drugs for the PI3K / AKT / mTOR signaling pathway are mainly PI3K inhibitors and mTOR inhibitors, and AKT is at the core of this signal transduction pathway. Inhibiting AKT activity can avoid the serious side effects caused by inhibiting upstream PI3K, and can also avoid the negative feedback mechanism caused by inhibiting downstream mTOR that affects the efficacy. Therefore, finding effective and selective AKT inhibitors is an important direction for the current research and development of tumor targeted drugs. CN101631778A discloses a class of cyclopentadienyl [D] pyrimidine derivatives, CN101578273A discloses a class of hydroxylated and methoxylated cyclopentadienyl [D] pyrimidine derivatives, CN101511842A discloses a class of dihydrofuranyl pyrimidine derivatives, and CN101970415A discloses a class of 5H-cyclopentadienyl [d] pyrimidine derivatives. These compounds have an IC of less than 10 μM for inhibiting AKT1. 50 .

[0008] Therefore, there is still a need to develop new AKT inhibitors and use them to treat diseases. Summary of the invention

[0009] The first aspect of the present application provides a pharmaceutical composition in a unit dosage form comprising compound I-0 or a pharmaceutically acceptable salt thereof, wherein the mass of compound I-0 or a pharmaceutically acceptable salt thereof is 5 mg to 400 mg as a free base, and compound I-0 has the following structure:

[0010]

[0011] Wherein, R is selected from C1-C4 alkyl or C3-C6 cycloalkyl;

[0012] X is selected from CH 2 or O.

[0013] In some embodiments, R is selected from methyl, ethyl, n-propyl, isopropyl, tert-butyl, cyclopropyl, cyclopentyl, or cyclohexyl.

[0014] In some typical embodiments, R is selected from methyl, ethyl, isopropyl or cyclopropyl.

[0015] In some more typical embodiments, R is selected from isopropyl or cyclopropyl.

[0016] In some embodiments, X is selected from CH 2 .

[0017] In some embodiments, X is selected from O.

[0018] In some embodiments, compound I-0 is selected from the following compounds:

[0019]

[0020]

[0021] In some embodiments, in the pharmaceutical composition in the unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 10 mg to 400 mg as a free base.

[0022] In some embodiments, in the pharmaceutical composition in the unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 10 mg to 200 mg based on the free base.

[0023] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 10 mg to 150 mg based on the free base.

[0024] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 25 mg to 150 mg as a free base.

[0025] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 25 mg to 100 mg as a free base.

[0026] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 10 mg as a free base.

[0027] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 25 mg as a free base.

[0028] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 50 mg as a free base.

[0029] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 75 mg as a free base.

[0030] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 100 mg as a free base.

[0031] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 150 mg as a free base.

[0032] In some embodiments, in the pharmaceutical composition in the unit dose form, the mass of Compound 1-0 or a pharmaceutically acceptable salt thereof is 200 mg as a free base.

[0033] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of compound 1-0 or a pharmaceutically acceptable salt thereof is 400 mg as a free base.

[0034] In some embodiments, the mass of compound I-0 or a pharmaceutically acceptable salt thereof, calculated as a free base, accounts for 0.1%-99.9% of the total mass of the pharmaceutical composition; preferably 5%-90%; more preferably 25%-65%.

[0035] Another aspect of the present application provides a pharmaceutical composition in a unit dosage form comprising Compound I or a pharmaceutically acceptable salt thereof, wherein the mass of Compound I or a pharmaceutically acceptable salt thereof is 5 mg to 400 mg as a free base, and Compound I has the following structure:

[0036]

[0037] Wherein, R is selected from C1-C4 alkyl or C3-C6 cycloalkyl;

[0038] X is selected from CH 2 or O.

[0039] In some embodiments, R is selected from methyl, ethyl, n-propyl, isopropyl, tert-butyl, cyclopropyl, cyclopentyl, or cyclohexyl.

[0040] In some typical embodiments, R is selected from methyl, ethyl, isopropyl or cyclopropyl.

[0041] In some more typical embodiments, R is selected from isopropyl or cyclopropyl.

[0042] In some embodiments, X is selected from CH 2 .

[0043] In some embodiments, X is selected from O.

[0044] In some embodiments, Compound I is selected from the following compounds:

[0045]

[0046] In some embodiments, Compound 1 is the following compound:

[0047]

[0048] In some embodiments, Compound 1 is the following compound:

[0049]

[0050] In some embodiments, Compound 1 is the following compound:

[0051]

[0052] In some embodiments, the pharmaceutical composition in unit dose form, wherein the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 10 mg to 400 mg based on the free base.

[0053] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 10 mg to 200 mg as a free base.

[0054] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 10 mg to 150 mg as a free base.

[0055] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 25 mg to 150 mg as a free base.

[0056] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 25 mg to 100 mg as a free base.

[0057] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 10 mg as a free base.

[0058] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 25 mg as a free base.

[0059] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 50 mg as a free base.

[0060] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 75 mg as a free base.

[0061] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 100 mg as a free base.

[0062] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 150 mg as a free base.

[0063] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 200 mg as a free base.

[0064] In some embodiments, in the pharmaceutical composition in unit dose form, the mass of Compound 1 or a pharmaceutically acceptable salt thereof is 400 mg as a free base.

[0065] In some embodiments, the mass of Compound I or a pharmaceutically acceptable salt thereof, calculated as a free base, accounts for 0.1%-99.9% of the total mass of the pharmaceutical composition; preferably 5%-90%; more preferably 25%-65%.

[0066] In some embodiments, the pharmaceutical composition in unit dose form further comprises one or more pharmaceutically acceptable carriers, such as excipients, disintegrants, lubricants, etc., and the mass percentage of the pharmaceutically acceptable carrier is 0.1%-99.9%, preferably 10%-95%, and more preferably 35%-75%.

[0067] In some embodiments, the pharmaceutical composition in unit dosage form is a pharmaceutical formulation suitable for oral administration.

[0068] In some embodiments, the pharmaceutical composition in unit dosage form is a liquid preparation or a solid preparation.

[0069] In some typical embodiments, the pharmaceutical composition in unit dosage form is a solid preparation suitable for oral administration, preferably a tablet or capsule.

[0070] For capsules, the pharmaceutically acceptable carrier is preferably selected from fillers and lubricants. Examples of fillers are calcium hydrogen phosphate dihydrate, and examples of lubricants are glyceryl behenate.

[0071] On the other hand, the present application also provides a pharmaceutical composition in unit dosage form comprising compound I-0 or a pharmaceutically acceptable salt thereof for use as a medicament.

[0072] On the other hand, the present application also provides a pharmaceutical composition in unit dosage form comprising Compound I or a pharmaceutically acceptable salt thereof for use as a medicament.

[0073] On the other hand, the present application also provides the use of a pharmaceutical composition in unit dosage form comprising compound I-0 or a pharmaceutically acceptable salt thereof, or compound I or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for preventing and / or treating diseases or disease states mediated by AKT protein kinase.

[0074] On the other hand, the present application also provides the use of a pharmaceutical composition in unit dosage form comprising compound I-0 or a pharmaceutically acceptable salt thereof, or compound I or a pharmaceutically acceptable salt thereof, for preventing and / or treating diseases or disease states mediated by AKT protein kinase.

[0075] On the other hand, the present application also provides a method for preventing and / or treating diseases or disease states mediated by AKT protein kinase, which comprises administering to an individual in need thereof a pharmaceutical composition in unit dosage form comprising compound I-0 or a pharmaceutically acceptable salt thereof, or compound I or a pharmaceutically acceptable salt thereof of the present application.

[0076] On the other hand, the present application also provides a pharmaceutical composition in unit dosage form comprising compound I-0 or a pharmaceutically acceptable salt thereof, or compound I or a pharmaceutically acceptable salt thereof, for preventing and / or treating diseases or disease states mediated by AKT protein kinase.

[0077] In some embodiments, the AKT protein kinase-mediated disease or condition is cancer.

[0078] In some typical embodiments, the cancer is breast cancer, prostate cancer, or ovarian cancer.

[0079] In some typical embodiments, the cancer is prostate cancer.

[0080] Related definitions

[0081] Unless otherwise specified, the following terms used in the specification and claims have the following meanings:

[0082] The present invention compound I-0 includes its tautomeric form. The tautomeric form is derived from the exchange of a single bond with an adjacent double bond accompanied by the transfer of a proton. For example, under certain conditions it can be transformed into Compound For compound accordingly, the tautomers of other compounds in compound I-0 are also within the protection scope of the present application.

[0083] The compounds of the present application or their pharmaceutically acceptable salts include their hydrates, specifically, including hydrates of the compounds and hydrates of pharmaceutically acceptable salts of the compounds.

[0084] Numeric ranges in the present disclosure refer to each integer in a given range. For example, "C1-C4" means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms; "C3-C6" means that the group may have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0085] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including a linear or branched saturated hydrocarbon group having the indicated number of carbon atoms. For example, the term "C1-C4 alkyl" includes C1 alkyl, C2 alkyl, C3 alkyl or C4 alkyl, examples of which include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.

[0086] The term "cycloalkyl" refers to a monocyclic saturated hydrocarbon system, without heteroatoms and without double bonds. Examples of the term "C3-C6 cycloalkyl" include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0087] The term "unit dosage form" refers to a specific form of a product that exists individually and relatively independently in a medicine box or drug packaging, which contains a certain amount of active ingredients. For example, the "unit dosage form" of the present application can be understood as a tablet or a pill of a solid preparation for oral administration. According to commonly used oral solid preparations, the weight of the tablet or pill of the medicine can be any weight applicable in the art, such as 100 mg to 1500 mg, etc.; in other examples, the "unit dosage form" of the present application can be understood as a liquid preparation for oral administration, such as a bottle of oral liquid or a capsule containing liquid. According to commonly used oral liquids or capsules, the volume of the liquid in the bottle or the pill can be any volume applicable in the art, such as 20 μL to 10 ml, etc.

[0088] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness of the free acids and bases of a particular compound without adverse biological effects, such as acid (including organic acids and inorganic acids) addition salts or base addition salts (including organic bases and inorganic bases).

[0089] The pharmaceutically acceptable salts of the present application can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

[0090] The term "pharmaceutically acceptable carrier" refers to carriers that have no significant irritation to the body and do not impair the biological activity and performance of the active compound. It includes but is not limited to any diluent, disintegrant, binder, glidant, wetting agent approved by the State Food and Drug Administration for use in humans or animals.

[0091] Unless otherwise specified, the abbreviations of this application have the following meanings:

[0092] M: mol / L

[0093] mM:mmol / L

[0094] nM: nmol / L

[0095] Boc: tert-Butyloxycarbonyl

[0096] DCM: dichloromethane

[0097] DEA: Diethylamine

[0098] DIEA: N,N-diisopropylethylamine

[0099] HATU: 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0100] RT: retention time

[0101] SFC: Supercritical Fluid Chromatography

[0102] h: hours

[0103] min: minutes

[0104] TK: tyrosine kinase

[0105] SEB: Fluorescence signal enhancer

[0106] HTRF: Homogeneous Time-Resolved Fluorescence

[0107] DTT: dithiothreitol

[0108] QD: Once daily

[0109] po: oral

[0110] TV: tumor volume

[0111] PG: 1,2-propylene glycol

[0112] T / C: relative tumor proliferation rate

[0113] TGI: tumor growth inhibition rate BRIEF DESCRIPTION OF THE DRAWINGS

[0114] In order to more clearly illustrate the technical solutions of the embodiments of the present application and the prior art, the following is a description of the technical solutions required in the embodiments and the prior art.

[0115] The drawings used are briefly introduced. Obviously, the drawings described below are only some embodiments of the present application.

[0116] For those skilled in the art, other drawings can be obtained based on these drawings.

[0117] Figure 1 This is a single molecule schematic diagram of isomer 2 of Example 1;

[0118] Figure 2 This is a schematic diagram of the asymmetric structural unit of the oxalate single crystal of Isomer 2 of Example 1;

[0119] Figure 3 This is a single molecule schematic diagram of isomer 1 of Example 3;

[0120] Figure 4 This is a schematic diagram of the asymmetric structural unit of the oxalate single crystal of isomer 1 in Example 3;

[0121] Figure 5 This is a single molecule schematic diagram of isomer 3 of Example 3;

[0122] Figure 6 Schematic diagram of the asymmetric structural unit of the single crystal of isomer 3 oxalate of Example 3. DETAILED DESCRIPTION

[0123] The pharmaceutical composition of the present application, its preparation method and its application, including the preparation method of the compound, are described in more detail below. However, these specific descriptions do not constitute any limitation to the present application.

[0124] Preparation Example

[0125] Preparation Example 1 Preparation of (R)-4-chloro-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one

[0126]

[0127] a) 2-Methylpropane-1,1,3-tricarboxylic acid trimethyl ester

[0128] Under nitrogen protection, sodium methoxide methanol solution (30wt%, 50.32g) was added to methanol (900mL) at 20°C, then the temperature was raised to 70°C, dimethyl malonate (461.12g) and ethyl crotonate (349.46g) were mixed evenly, and added dropwise to the above sodium methoxide methanol solution, and reacted at 70°C for 3 hours. After the reaction was complete, the solvent was evaporated under reduced pressure, ethyl acetate (1L) was added, and the pH was adjusted to 7-8 with 4M hydrochloric acid, followed by adding 500mL of water, separation, and the organic phase was evaporated under reduced pressure to obtain 777.68g of yellow liquid. 1 HNMR (400 MHz, DMSO-d 6) δ (ppm) 3.67 (s, 3H), 3.65 (s, 3H), 3.59 (s, 3H), 3.56 (d, J = 6.8Hz, 1H), 2.45-2.58 (m, 2H), 2.23-2.29 (m, 1H), 0.93 (d, J = 6.8Hz, 3H).

[0129] b) (R)-2-Methylpropane-1,1,3-tricarboxylic acid trimethyl ester

[0130] At 25°C, disodium hydrogen phosphate (4.5 g) was dissolved in 1.5 L deionized water, and the pH was adjusted to 7.05 with 2N hydrochloric acid. 2-methylpropane-1,1,3-tricarboxylic acid trimethyl ester (150.46 g) and lipase (Candida rugosa, 40 g added in 6 days) were added, and the pH was adjusted to between 7.0 and 7.6 with 2N sodium hydroxide solution. The reaction was carried out at 35°C for 6 days, and the chiral detection ee%>98%, and the chiral detection conditions were (Chiralpak IC, 4.6×250 mm, 5 μm, n-hexane: ethanol=9:1, volume ratio). The reaction solution was cooled to 10°C, and the pH was adjusted to 3-4 with 3M hydrochloric acid. 500 mL of ethyl acetate was added, and the filter cake was washed with ethyl acetate (600 mL), separated, and washed with saturated sodium bicarbonate aqueous solution (100 mL), separated, and the organic phase was concentrated to obtain 26.89 g of a light yellow liquid. 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 3.74 (s, 6H), 3.68 (s, 3H), 3.46 (d, J = 7.2Hz, 1H), 2.71-2.79 (m, 1H), 2. 54 (dd, J=15.6, 4.8Hz, 1H), 2.32 (dd, J=16.0, 8.4Hz, 1H), 1.06 (d, J=6.8Hz, 3H).

[0131] c) (R)-3-(4,6-dihydroxypyrimidin-5-yl)butanoic acid methyl ester

[0132] Under nitrogen protection, formamidine acetate (11.33 g) was dissolved in methanol (200 mL) at 20°C, cooled to 0°C, sodium methoxide methanol solution (30 wt%, 55.62 g) was added dropwise, reacted at 0°C for 60 min, (R)-2-methylpropane-1,1,3-tricarboxylic acid trimethyl ester (24.07 g) in methanol (60 mL) was added dropwise, the temperature was naturally raised to 20°C, and the reaction was carried out for 10 hours. After the reaction was complete, the reaction solution was cooled to 0°C, 3N hydrochloric acid was added to adjust the pH to 5-6, the solvent was evaporated under reduced pressure, and then cooled to 0°C, 3N hydrochloric acid was added to adjust the pH to 3, solid precipitated, solid was collected by suction filtration, the filter cake was washed with ice water (100 mL), and the filter cake was vacuum dried to obtain 18.79 g of white solid, which was directly used in the next step.

[0133] d) (R)-3-(4,6-dichloropyrimidin-5-yl)butyric acid methyl ester

[0134] Under nitrogen protection, (R)-3-(4,6-dihydroxypyrimidin-5-yl)butyric acid methyl ester (14.63g) was dispersed in acetonitrile (70mL) at 22°C, phosphorus oxychloride (26.42g) and diisopropylethylamine (12.51g) were added dropwise, the system released heat significantly, then the temperature was raised to 60°C, the solid gradually dissolved, and the reaction was continued for 18 hours. After the reaction was complete, the reaction solution was cooled to 0°C, 100mL of ethyl acetate was added, the pH was adjusted to 7-8 with saturated sodium bicarbonate solution, extracted with ethyl acetate (50mL×3), and the organic phase was evaporated under reduced pressure to obtain 13.89g of yellow solid, which was directly used in the next step.

[0135] e)(R)-4-Chloro-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one

[0136] At 20°C, (R)-3-(4,6-dichloropyrimidin-5-yl)butyric acid methyl ester (13.89 g) and ammonia water (25-28 wt%, 70 mL) were added to a 100 mL autoclave, heated to 50°C, and reacted for 18 hours. After the reaction was complete, the reaction solution was cooled to 0°C, filtered, and the filter cake was slurried with 30 mL (petroleum ether: ethyl acetate = 10:1, volume ratio) to obtain 7.32 g of a light yellow solid. LC-MS (ESI) m / z: 198 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ (ppm) 1.30 (d, J=7.2Hz, 3H), 2.65-2.69 (m, 1H), 2.86-2.92 (m, 1H), 3.47-3.54 (m, 1H), 8.64 (s, 1H), 10.10 (s, 1H).

[0137] Example 1 Preparation of (R)-4-((1S,6R)-5-((S)-2-(4-chlorophenyl)-3-(isopropylamino)propanoyl)-2,5-diazabicyclo[4.1.0]heptane-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one

[0138]

[0139] Reaction conditions: a) tert-butyl 2,5-diazabicyclo[4.1.0]heptane-2-carboxylate, N-methylpyrrolidone, 4-dimethylaminopyridine; b) hydrogen chloride / 1,4-dioxane (4.0M), dichloromethane; c) (S)-3-((tert-butoxycarbonyl)(isopropyl)amino)-2-(4-chlorophenyl)-propionic acid, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4-dimethylaminopyridine, N,N-dimethylformamide; d) trifluoroacetic acid, dichloromethane.

[0140] a) tert-butyl 5-((R)-5-methyl-7-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)-2,5-diazabicyclo[4.1.0]heptane-2-carboxylate

[0141] Under nitrogen protection, (R)-4-chloro-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one (0.21 g), tert-butyl 2,5-diazabicyclo[4.1.0]heptane-2-carboxylate (0.31 g) and 4-dimethylaminopyridine (0.39 g) were dissolved in N-methylpyrrolidone (5 mL) at 22°C, then heated to 140°C and reacted for 3 hours. After the reaction was complete, the reaction solution was cooled to 20°C, poured into 20 mL of ice water, extracted with ethyl acetate (20 mL×2), washed with saturated brine (10 mL×3), and the solvent was evaporated under reduced pressure. The product was separated by silica gel column chromatography (petroleum ether:ethyl acetate=3:1~1:1) to obtain 0.28 g of a light yellow liquid. LC-MS (ESI) m / z: 360 (M+H).

[0142] b) (5R)-4-(2,5-diazabicyclo[4.1.0]heptane-2-yl)-5-methyl-5,8-dihydropyridin[2,3-d]pyrimidin-7(6H)-one hydrochloride

[0143] 5-((R)-5-methyl-7-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)-2,5-diazabicyclo[4.1.0]heptane-2-carboxylic acid tert-butyl ester (0.28 g) was dissolved in dichloromethane (5 mL) at 20°C, and hydrogen chloride / 1,4-dioxane (4.0 mL) was added to react for 1 hour. After the reaction was complete, the reaction solution was evaporated under reduced pressure to remove the solvent to obtain 0.23 g of a yellow solid, which was directly used in the next step.

[0144] c) tert-Butyl (2S)-2-(4-chlorophenyl)-3-(5-((R)-5-methyl-7-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)-2,5-diazabicyclo[4.1.0]heptane-2-yl)-3-oxopropyl)(isopropyl)carbamate

[0145] Under nitrogen protection, (5R)-4-(2,5-diazabicyclo[4.1.0]heptane-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one hydrochloride (0.20 g) and (S)-3-((tert-butoxycarbonyl)(isopropyl)amino)-2-(4-chlorophenyl)-propionic acid (0.22 g) were dissolved in N,N-dimethylformamide (5 mL) at 20°C, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.59 g) and 4-dimethylaminopyridine (0.48 g) were added, and the mixture was reacted at 25°C for 4 hours. After the reaction was complete, 20 ml of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated brine (10 mL × 2), and the organic phase was evaporated under reduced pressure and separated by column chromatography (dichloromethane: methanol = 50:1) to obtain 0.18 g of a yellow solid. LC-MS (ESI) m / z: 583 (M+H).

[0146] d) (R)-4-((1S,6R)-5-((S)-2-(4-chlorophenyl)-3-(isopropylamino)propanoyl)-2,5-diazabicyclo[4.1.0]heptane-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one

[0147] Tert-butyl (2S)-2-(4-chlorophenyl)-3-(5-((R)-5-methyl-7-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)-2,5-diazabicyclo[4.1.0]heptane-2-yl)-3-oxopropyl)(isopropyl)carbamate (0.18 g) was dissolved in dichloromethane (2 mL) at 20°C, and trifluoroacetic acid (0.86 mL) was added to react for 3 hours. After the reaction was completed, dichloromethane (10 mL) was added to the reaction solution, and 2M sodium hydroxide solution was added dropwise at 0°C to adjust the pH to 12. The organic phase was separated, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain 0.10 g of a yellow solid. After separation by high performance preparative liquid chromatography, isomer 1 (3 mg) and isomer 2 (12 mg) were obtained. High performance preparative liquid chromatography conditions: Chromatographic column: Aglient 5 μm prep-C18 50×21.2 mm, mobile phase A: water (containing 0.1 vol% ammonia (25-28 wt%)); mobile phase B: methanol. Gradient: time 0-10 min, phase B 60-70% (volume ratio).

[0148] Isomer 1: RT 1 =5.3min, LC-MS (ESI) m / z: 483 (M+H).

[0149] Isomer 2: RT = 5.9 min; LC-MS (ESI) m / z: 483 (M+H); 1 HNMR (400 MHz, CDCl 3 )δ(ppm)8.27(d, J=7.6Hz, 1H), 7.92(s, 1H), 7.27-7.30(m, 4H), 4.23-4.29(m, 1H), 3.90-3.95(m, 1H), 3.81-3.85(m, 1H), 3.69-3.72(m, 1H), 3.44-3.59(m, 1 H), 3.20-3.38(m, 3H), 3.01-3.05(m, 1H), 2.70-2.85(m, 3H), 2.47-2.57(m, 1H ), 2.21-2.25(m, 1H), 1.25-1.28(m, 3H), 1.03-1.11(m, 6H), 0.82-0.90(m, 2H).

[0150] According to the configuration determined by single crystal diffraction, isomer 2 was determined to be the title compound of this example:

[0151] Single crystal preparation: Add 30.0 mg of isomer 2 and 2.0 mL of isopropanol to a 5 mL screw-mouth glass bottle, stir for 5 minutes, and the solid will dissolve. Weigh 3.9 mg of oxalic acid dihydrate and add it to the above glass bottle. White solid will gradually precipitate in the glass bottle. Stir at room temperature for 3 hours, and a large amount of white solid will precipitate in the glass bottle. Add 1.0 mL of methanol to the glass bottle, the white solid will gradually disappear, the solution will become clear, and continue to stir for 1 hour. Filter the solution through a 0.22 μm microporous filter membrane into a 3 mL screw-mouth glass bottle, and cover the mouth of the glass bottle with plastic wrap. Use a needle to poke 8 small holes at the mouth of the bottle, and leave it at room temperature for 7 days to obtain a single crystal of the oxalate salt of isomer 2.

[0152] Single crystal diffraction experiment:

[0153] Single crystal X-ray diffractometer: BRUKER D8 VENTURE PHOTON II

[0154] Wavelength: Ga Kα

[0155] Test temperature: 190K

[0156] Computer program used for structure elucidation: SHELXL-2018

[0157] Single crystal data: Molecular formula: C 55 H 72 Cl 2 N 12 O 9 ; Molecular weight: 1116.14; Crystal system: hexagonal; Space group: P 61; Unit cell parameters: α=90°,β=90°,γ=120°;Unit cell volume: The number of molecular formulas contained in the unit cell: Z = 12; calculated density: D calc =0.838g / cm 3 ; R(F o ): 0.0730; R W (F o 2 ): 0.2069; goodness of fit (S): 1.034; Flack parameter: 0.066(9).

[0158] Structural description: Single crystal X-ray diffraction and structural analysis show that the single crystal obtained is the oxalate isopropanol complex of isomer 2. The asymmetric structural unit of the crystal contains four isomer 2 molecules, two oxalic acid molecules and two isopropanol molecules, among which isomer 2 and oxalic acid form oxalate. The schematic diagram of the single molecule of isomer 2 is shown in the figure below. Figure 1 As shown, the asymmetric structural unit of the oxalate single crystal is as follows Figure 2 The structural formula is shown as follows:

[0159]

[0160] Example 2 Preparation of (R)-4-((1S,6R)-5-((S)-2-(4-chlorophenyl)-3(cyclopropylamino)propanoyl)-2,5-diazabicyclo[4.1.0]hept-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one

[0161]

[0162] Reaction conditions: a) triethylamine, di-tert-butyl dicarbonate, dichloromethane; b) sodium bis(trimethylsilyl)amide (2.0 mol / L tetrahydrofuran solution), bromomethyl methyl ether, 2-methyltetrahydrofuran; c) (R)-4-benzyloxazolidin-2-one, diisopropylethylamine, trimethylacetyl chloride, toluene; d) titanium tetrachloride (1 mol / L toluene solution), diisopropylethylamine, dichloromethane; e) hydrogen peroxide solution (30 wt % ), lithium hydroxide monohydrate, tetrahydrofuran, water; f) tert-butyl 2,5-diazabicyclo[4.1.0]heptane-2-carboxylate, 4-dimethylaminopyridine, N-methylpyrrolidone; g) hydrogen chloride dioxane (4.0M); h) 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate, diisopropylethylamine, N,N-dimethylformamide; i) hydrogen chloride dioxane (4.0M).

[0163] a) tert-Butyl cyclopropylcarbamate

[0164] Under nitrogen protection, cyclopropylamine (9.3 g) and triethylamine (19.7 g) were dissolved in dichloromethane (100 mL) at 20°C, di-tert-butyl dicarbonate (35.48 g) was added dropwise at 0°C, and the mixture was reacted at 20°C for 16 h. After the reaction was completed, the solvent was removed to obtain 24.3 g of a colorless liquid. 1 HNMR (400 MHz, CDCl 3 )δ (ppm) 0.47-0.50 (m, 2H), 0.66-0.72 (m, 2H), 1.44 (s, 9H), 2.53 (m, 1H), 4.79 (s, 1H).

[0165] b) tert-Butyl cyclopropyl (methoxymethyl) carbamate

[0166] Under nitrogen protection, tert-butyl cyclopropylcarbamate (24.3 g) was dissolved in 2-methyltetrahydrofuran (100 mL), and sodium bis(trimethylsilyl)amide (120 mL) was added dropwise at 0°C. The reaction solution was stirred at 0°C for 1 hour, and bromomethyl methyl ether (35.7 g) was added dropwise at 0°C. The reaction solution was stirred at 0°C for 6 hours. The reaction solution was poured into 50 g of ice water, separated, and extracted with ethyl acetate (100 mL*2). The reaction solution was directly concentrated to obtain 29.1 g of the product as a colorless liquid, which was directly used in the next step without purification.

[0167] c) (R)-4-Benzyl-3-(2-(4-(chlorophenyl)acetyl)oxazolidin-2-one

[0168] Under nitrogen protection, 2-(4-chlorophenyl)acetic acid (50g), (R)-4-benzyloxazolidin-2-one (45.5g) and diisopropylethylamine (127.3g) were dissolved in toluene (600mL), and trimethylacetyl chloride (38.4g) was added dropwise at 15°C. The reaction solution was refluxed and stirred for 16 hours, and then the reaction solution was poured into 200mL of water, separated, and washed with 120mL of saturated brine. The organic phase was dried and concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=5:1) to obtain 32g of a white solid. 1 HNMR (400 MHz, DMSO-d 6 )δ (ppm) 2.88-3.02 (m, 2H), 4.12-4.37 (m, 4H), 4.64-4.70 (m, 1H), 7.13-7.16 (m, 2H), 7.23-7.32 (m, 5H), 7.39-7.42 (m, 2H).

[0169] d) tert-Butyl ((S)-3-((R)-4-benzyl-2-oxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl)(cyclopropyl)carbamate

[0170] Under nitrogen protection, (R)-4-benzyl-3-(2-(4-(chlorophenyl)acetyl)oxazolidin-2-one (3.48 g) was dissolved in dichloromethane (60 mL), and titanium tetrachloride toluene solution (13 mL) was added dropwise at 0°C. The reaction solution was stirred at 0°C for 2 h, DIPEA (1.49 g) was added dropwise, and the reaction solution was stirred at 0°C for 1.5 h. Cyclopropyl(methoxymethyl)carbamic acid tert-butyl ester (2.77 g) was added dropwise, and the reaction solution was stirred at 0°C for 6 h. The reaction was completed. The reaction solution was then poured into 30 mL of saturated ammonium chloride solution, separated, and washed with 120 mL of saturated brine. The organic phase was dried and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (PE:EA=10:1) to obtain 2.50 g of a colorless oil.

[0171] e) (S)-3-((tert-Butyloxycarbonyl)(cyclopropyl)amino)-2-(4-chlorophenyl)propanoic acid

[0172] Lithium hydroxide monohydrate (0.63 g) was dissolved in water (18 mL), tetrahydrofuran (20 mL) was added, hydrogen peroxide (1.6 mL) was added dropwise at 0°C, ((S)-3-((R)-4-benzyl-2-oxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl)(cyclopropyl)carbamic acid tert-butyl ester (2.50 g) was added at 0°C, the reaction solution was stirred at 0°C for 3 h, a saturated sodium sulfite solution (15 mL) was added to the reaction solution, the reaction was continued for 1.5 h, the pH was adjusted to 3-4 with a saturated potassium hydrogen sulfate solution, the solution was extracted with ethyl acetate (30 mL*2), the organic phase was dried, and the crude product was obtained by concentration. The crude product was separated and purified by column chromatography (PE:EA=1:1) to obtain 1.26 g of a colorless solid. 1 HNMR (400 MHz, DMSO-d 6 )δ(ppm):0.45-0.48(m,2H),0.60-0.64(m,2H),1.30(s,9H),2.19(s,1H),3.61(d , J=7.6Hz, 1H), 3.95 (t, J=8.0Hz, 1H), 7.37 (dd, J=26.8, 8.8Hz, 4H), 12.7 (s, 1H).

[0173] f) 5-((R)-5-methyl-7-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)-2,5-diazabicyclo[4.1.0]heptane-2-carboxylate

[0174] Under nitrogen protection, (R)-4-chloro-5-methyl-5,8-dihydropyridin[2,3-d]pyrimidin-7(6H)-one (300 mg), tert-butyl 2,5-diazabicyclo[4.1.0]heptane-2-carboxylate (455 mg), and 4-dimethylaminopyridine (600 mg) were dissolved in N-methylpyrrolidone (5 mL), stirred at 120°C for 12 h, and then the reaction solution was poured into 50 mL of water, extracted with ethyl acetate (20 mL*2), and washed with 15 mL of saturated brine, the organic phase was dried, and the solvent was removed to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (PE:EA=1:1~1:2) to obtain 400 mg of a yellow solid.

[0175] g) (5R)-4-(2,5-diazabicyclo[4.1.0]hept-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one

[0176] 5-((R)-5-methyl-7-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)-2,5-diazabicyclo[4.1.0]heptane-2-carboxylate (400 mg) was dissolved in dioxane (5 mL), and a solution of hydrogen chloride in dioxane (5 mL) was added dropwise. The mixture was stirred at 25° C. for 2 hours. After the reaction was completed, the reaction solution was directly concentrated to obtain a crude yellow solid, which was directly used in the next step.

[0177] h) tert-Butyl ((S)-2-(4-chlorophenyl)-3-((1R,6S)-5-((R)-5-methyl-7-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)-2,5-diazabicyclo[4.1.0]hept-2-yl)-3-oxopropyl)(cyclopropyl)carbamate

[0178] Under nitrogen protection, (5R)-4-(2,5-diazabicyclo[4.1.0]hept-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one (270 mg), the product of step e) (389 mg), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (474 ​​mg) and diisopropylethylamine (671 mg) were dissolved in N,N-dimethylformamide (10 mL), and the reaction solution was stirred at 25°C for 3 hours to complete the reaction. The reaction solution was then poured into 50 mL of water, extracted with ethyl acetate (20 mL*2), and washed with saturated brine (10 mL*3), the organic phase was dried and concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:2) to obtain 320 mg of a yellow solid.

[0179] i) (R)-4-((1S,6R)-5-((S)-2-(4-chlorophenyl)-3(cyclopropylamino)propanoyl)-2,5-diazabicyclo[4.1.0]hept-2-yl)-5-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-7(6H)-one

[0180] Tert-butyl ((S)-2-(4-chlorophenyl)-3-((1R, 6S)-5-((R)-5-methyl-7-oxo-5,6,7,8-tetrahydropyridinyl[2,3-d]pyrimidin-4-yl)-2,5-diazabicyclo[4.1.0]hept-2-yl)-3-oxopropyl)(cyclopropyl)carbamate (320 mg) was dissolved in dioxane (2.5 mL) and hydrogen chloride in dioxane (2.7 mL) was added dropwise. The reaction solution was stirred at 25° C. for 14 h. After the reaction was completed, the reaction solution was concentrated to obtain a crude product. The pH was adjusted to 13-14 with a saturated potassium carbonate solution, extracted with DCM (10 mL*2), washed with water (10 mL), and desolvated. The product was separated by supercritical fluid chromatography to obtain isomer 1 (61.2 mg) and isomer 2 (31.2 mg). According to the configuration determined by single crystal diffraction, isomer 2 was determined to be the title compound of this example. Separation instrument and conditions: waters SFC200; chromatographic column: Daicel ChiralcelAS, 250×30mm ID, 5μm; mobile phase: A is CO 2 , B is isopropanol (0.1 vol% ammonia water (25-28 wt%)), A:B=70:30 (volume ratio); flow rate 60 mL / min, column temperature 38°C.

[0181] Ultra-high performance convergence chromatography conditions: Column: Daicel Chiralcel AD, 2.1×150 mm ID, 3 μm, Mobile phase A: CO 2 ; Mobile phase B: isopropanol (0.1 vol% DEA), gradient: time 0-8 min, phase B 5-40% (volume ratio); flow rate: 1 mL / min; column temperature: 40°C. Isomer 1: RT = 3.7 min; Isomer 2: RT = 4.6 min.

[0182] Isomer 1: LC-MS (ESI) m / z: 481 (M+H). 1 H NMR (400 MHz, DMSO-d 6)δ (ppm): 0.03-0.12 (m, 2H), 0.25-0.30 (m, 2H), 0.66-0.70 (m, 1H), 0.96-1. 05 (m, 3H), 1.35-1.40 (m, 1H), 1.93-2.11 (m, 2H), 2.29-2.35 (m, 1H), 2.67-2. 77 (m, 2H), 2.80-2.86 (m, 1H), 3.03-3.25 (m, 4H), 3.39-3.48 (m, 1H), 3.69-3. 79 (m, 1H), 4.24-4.34 (m, 2H), 7.34-7.41 (m, 4H), 8.17 (s, 1H), 10.52 (s, 1H).

[0183] Isomer 2: LC-MS (ESI) m / z: 481 (M+H). 1 HNMR (400MHz, DMSO-d6) δ (ppm): 0.14-0.21 (m, 2H), 0.30-0.37 (m, 2H), 0.93-1.07 (m, 4H), 2.03-2.34 (m, 3H), 2.66-2.86 (m, 2H) ), 3.10-3.25(m, 4H), 3.36-3.94(m, 4H), 4.07-4.15(m, 1H), 4.41-4.45(m, 1H), 7.32-7.42(m, 4H), 8.19(s, 1H), 10.48(s, 1H).

[0184] Example 3 Preparation of (S)-5-((1S,6R)-5-((S)-2-(4-chlorophenyl)-3-(isopropylamino)propanoyl)-2,5-diazabicyclo[4.1.0]hept-2-yl)-4-methyl-1,4-dihydro-2H-pyrimidinyl[4,5-d][1,3]oxazin-2-one

[0185] a) 1-(4-amino-6-chloropyrimidin-5-yl)ethanone (Compound 34-1)

[0186] 1-(4,6-Dichloropyrimidin-5-yl)ethanone (2.5 g) was dissolved in tetrahydrofuran (15 mL) at 20°C, and then aqueous ammonia (25-28 wt%, 9 g) was added. The reaction solution was stirred at 20°C for 5 hours, then concentrated and diluted with a small amount of water, and filtered to obtain a white solid. After vacuum drying, 2 g of a white solid was obtained, which was directly used in the next step.

[0187] b) 1-(4-amino-6-chloropyrimidin-5-yl)ethan-1-ol (Compound 34-2)

[0188] At 20°C, 1-(4-amino-6-chloropyrimidin-5-yl)ethanone (1.5 g) was dissolved in methanol (15 mL) and cooled to -10°C. Sodium borohydride (1 g) was added in batches. After completion, the reaction solution was slowly heated to 20°C and stirred for 3 hours. After the reaction was complete, the reaction solution was quenched with saturated aqueous ammonium chloride solution. The reaction solution was then concentrated and slurried with ethyl acetate (20 mL*2). The mother liquor was concentrated to obtain an oily crude product. The crude product was separated by chromatography to obtain 400 mg of a white oily product. LC-MS (ESI) m / z: 174 (M+H).

[0189] c) 5-Chloro-4-methyl-1,4-dihydro-2H-pyrimidin[4,5-d][1,3]oxazin-2-one (Compound 34-3)

[0190] At 20°C, 1-(4-amino-6-chloropyrimidin-5-yl)ethane-1-ol (300 mg) and N,N-diisopropylethylamine (282 mg) were dissolved in tetrahydrofuran (3 mL), then cooled to -5°C and bis(trichloromethyl)carbonate (300 mg) was slowly added, and stirred at -5°C for 0.5 hours. Then the temperature was slowly raised to 18°C ​​and stirred for 1.5 hours. After the reaction was complete, the reaction was quenched with aqueous sodium bicarbonate solution, extracted with ethyl acetate (10 mL*3), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain an oily crude product. The crude product was separated and purified by column chromatography to obtain 108 mg of a white solid. LC / MS(ESI)m / z:200(M+H).

[0191] d) (S)-5-chloro-4-methyl-1,4-dihydro-2H-pyrimidin[4,5-d][1,3]oxazin-2-one (Compound 34-4a) and (R)-5-chloro-4-methyl-1,4-dihydro-2H-pyrimidin[4,5-d][1,3]oxazin-2-one (Compound 34-4b)

[0192] Compound 34-3 was separated by SFC chiral column to obtain the desired target products, compound 34-4a and compound 34-4b.

[0193] SFC chiral separation conditions are as follows: instrument: waters SFC200; separation column: Daicel Chiralcel AD, 250×50 mm ID, 10 μm; mobile phase: A: CO 2 , B: methanol (0.1 vol% ammonia water (25-28 wt%)), A: B = 65:35 (volume ratio); flow rate: 150 mL / min; pressure: 100 bar; column temperature: 38 ° C; detection wavelength: 220 nm; cycle time: 14 min; sample pretreatment: 10 g dissolved in 300 ml MeOH; injection volume: 16 ml.

[0194] Post-treatment: The sample was concentrated at 40°C and then freeze-dried to obtain compound 34-4a and compound 34-4b, respectively.

[0195] Route 1: Preparation of Isomer 1 and Isomer 4

[0196] e) 5-((S)4-methyl-2-oxo-1,4-dihydro-2H-pyrimidinyl[4,5-d][1,3]oxazin-5-yl)-2,5-diazabicyclo[4.1.0]heptane-2-carboxylic acid tert-butyl ester (Compound 34-5a)

[0197] Compound 34-4a (2 g) and tert-butyl 2,5-diazabicyclo[4.1.0]heptane-2-carboxylate (3.58 g) were dissolved in anhydrous MeCN (20 mL), and DIEA (3.89 g) was added. After the reaction solution was purged with nitrogen, the tube was sealed and stirred at 95° C. for 6 hours. After the reaction was completed, the crude product was concentrated to obtain the target product. The crude product was dissolved in DCM, washed with water, and concentrated to obtain the crude product, which was separated and purified by column chromatography (EA:PE=1:1) to obtain 3.2 g of a light brown solid.

[0198] f) (4S)-5-(2,5-diazabicyclo[4.1.0]heptane-2-yl)-4-methyl-1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-one hydrochloride (Compound 34-6a)

[0199] The product of step e) (3.2 g) was dissolved in HCl / i-PrOH (10 mL) and stirred at room temperature for 2 h. After the reaction was completed, the crude product was concentrated and used directly in the next step without purification.

[0200] g) Compound 34-7a

[0201] The product of step f) (3.3 g), (S)-3-((tert-butyloxycarbonyl)(isopropyl)amino)-2-(4-chlorophenyl)propionic acid (4.9 g), HATU (6.32 g), and DIPEA (4.3 g) were dissolved in anhydrous DMF (50 mL), and the reaction solution was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was poured into 100 mL of ethyl acetate, and washed with water (20 mL*3) and 10 mL of saturated brine, and the organic phase was dried and concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:1) to obtain 7.2 g of a brown solid. MS (ESI) m / z: 585 (M+H).

[0202] h) Compound 34-8a

[0203] The product of step g) (7.2 g) was dissolved in MeOH (25 mL), and then HCl / dioxane (70 mL) was added. The reaction solution was stirred at room temperature for 2 h, and then the reaction solution was concentrated to obtain a red oily crude product, which was then dissolved in MeOH (20 mL) and treated with Na 2 CO 3 After liberation, the residue was concentrated to obtain 6 g of crude product.

[0204] i) Isomer 1 and Isomer 4

[0205] Compound 34-8a was separated by SFC chiral column to give isomer 1 and isomer 4.

[0206] SFC separation conditions: Instrument: waters SFC200; Separation column: Daicel Chiralcel AD, 250×50 mm I.D., 10 μm; Mobile phase: A: CO 2 , B: MeOH (0.1 vol% ammonia water (25-28 wt%)), A: B = 75:25; flow rate: 70 mL / min; pressure: 100 bar; column temperature: 38 ° C; detection wavelength: 254 nm; cycle time: 5 min; sample pretreatment: 10 g dissolved in 200 ml MeOH; injection volume: 16 ml.

[0207] Post-treatment: The sample was concentrated at 40°C and then freeze-dried to obtain isomer 1 and isomer 4 respectively.

[0208] Route 2: Preparation of Isomers 2 and 3

[0209] Using compound 34-4b as the starting material, isomer 2 and isomer 3 were prepared according to the method described in route 1.

[0210] Isomer 1: LC-MS m / z: 485 (M+H). 1 HNMR (400 MHz, DMSO-d 6 )δ10.70 (s, 1H), 8.23 ​​(s, 1H), 7.46 (d, J=8.5Hz, 2H), 7.39 (d, J=8.6Hz, 2H), 6.13 (q, J=6.6Hz, 1H), 4.51 (s, 1H), 4.42-4.30 (m, 1H), 3.53-3.45 (m, 1H), 3.28-3.06 (m, 5H), 3.01-2.59 (m, 3H), 1.52-1.34 (m, 4H), 1.08-0.97 (m, 6H), 0.93-0.84 (m, 1H).

[0211] Isomer 4: LC-MS m / z: 485 (M+H). 1 HNMR (400 MHz, DMSO-d6 )δ10.73(s, 1H), 8.23(s, 1H), 7.41–7.32(m, 4H), 6.14(q, J=8.0Hz, 1H), 4. 40–4.36(m, 1H), 4.19–4.11(m, 1H), 3.62–3.51(m, 2H), 3.49–3.35(m, 1H), 3.24–3.05(m, 4H), 2.73–2.63(m, 2H), 1.45(d, J=8.0Hz, 1H), 1.33(d, J=8. 0Hz, 2H), 1.12 (q, J=4.0Hz, 1H), 0.95–0.88 (m, 6H), 0.26 (q, J=4.0Hz, 1H).

[0212] Isomer 2: LC-MS m / z: 485 (M+H). 1 H NMR (400 MHz, DMSO-d 6 )δ10.86(s, 1H), 8.27(s, 1H), 7.54–7.27(m, 4H), 6.32–6.18(m, 1H), 4.69–4.52(m, 1H), 4.27–3.97(m, 2H), 3.6 6–3.43(m, 2H), 3.29–2.92(m, 6H), 2.61–2.55(m, 1H), 1.63–1.58(m, 1H), 1.53–1.28(m, 3H), 1.28–1.12(m, 6H).

[0213] Isomer 3: LC-MS m / z: 485 (M+H). 1 H NMR (400 MHz, DMSO-d 6 )δ10.71 (s, 1H), 8.18 (s, 1H), 7.46–7.39 (m, 1H), 7.36–7.28 (m, 3H), 6.00 (q, J=6.4Hz, 1H), 4.53 (s, 1H), 4.46–4.33 (m, 1 H), 3.56–3.44(m, 2H), 3.26–3.09(m, 5H), 3.00–2.71(m, 2H), 1.43-1.38(m, 3H), 1.10–0.93(m, 7H), -0.07–-0.11(m, 1H).

[0214] Configuration determined by single crystal diffraction:

[0215] (1) Determination of the configuration of isomer 1

[0216] Preparation of single crystal: Weigh compound isomer 1 (50.0 mg) and 3.0 ml of isopropanol into a 5 ml screw-mouth glass bottle, stir for 5 minutes until the solid dissolves. Weigh 13.0 mg of oxalic acid dihydrate into the above glass bottle, and a white solid gradually precipitates out of the glass bottle. Stir at room temperature for 3 hours, and a large amount of white solid precipitates out of the glass bottle. After adding 1.5 ml of methanol and 0.2 ml of purified water to the glass bottle, the white solid gradually disappears, the solution becomes clear, and stirring is continued for 1 hour. The solution is filtered through a 0.22 μm microporous filter membrane into a 20 ml screw-mouth glass bottle, and the mouth of the glass bottle is covered with plastic wrap. Use a needle to poke 8 small holes at the mouth of the bottle, and leave it at room temperature for 10 days to obtain a single crystal of the oxalate salt of isomer 1 compound.

[0217] Single crystal diffraction experiment:

[0218] Single crystal X-ray diffractometer: BRUKER KAPPA APEX-IICCD

[0219] Wavelength: Cu Kα

[0220] Test temperature: 296K

[0221] Computer program used for structure elucidation: SHELXL-2018

[0222] Single crystal data: Molecular formula: C 50 H 60 Cl 2 N 12 O 10 ; Molecular weight: 1060.00; Crystal system: orthorhombic; Space group: C222; Unit cell parameters: α=90°,β=90°,γ=90°;Unit cell volume: The number of molecular formulas contained in the unit cell: Z = 8; calculated density: D calc =1.064g / cm 3 ; R(F o ): 0.0612; R W (F o 2 ): 0.1856; goodness of fit (S): 1.023; Flack parameter: 0.040 (11).

[0223] Structural description: Single crystal X-ray diffraction and structural analysis show that the single crystal obtained is the oxalate of isomer 1. The asymmetric structural unit of the crystal contains two molecules of isomers and one molecule of oxalic acid. The single molecule schematic diagram of compound isomer 1 is shown in Figure 3 As shown, oxalate single crystals Figure 4 The structural formula is shown as follows:

[0224]

[0225] (2) Determination of the configuration of isomer 3

[0226] Single crystal preparation: Single crystals of the oxalate salt of isomer 3 were prepared according to the method described for the preparation of single crystals of isomer 1 above.

[0227] Single crystal diffraction experiment:

[0228] Single crystal X-ray diffractometer: BRUKER D8 VENTURE PHOTON II

[0229] Wavelength: Ga Kα

[0230] Test temperature: 173K

[0231] Computer program used for structure elucidation: SHELXL-2018

[0232] Single crystal data: Molecular formula: C 52 H 64 Cl 2 N 12 O 15 ; Molecular weight: 1168.05; Crystal system: monoclinic; Space group: P2 1 / c; Unit cell parameters: α=90°,β=98.259(3)°,γ=90°;Unit cell volume: The number of molecular formulas contained in the unit cell: Z = 4; calculated density: D calc =1.257g / cm 3 ; R(F o ): 0.0634; R W (F o 2 ): 0.2016; goodness of fit (S): 1.053.

[0233] Structural description: Single crystal X-ray diffraction and structural analysis show that the single crystal obtained is the oxalate hydrate of isomer 3. The asymmetric structural unit of the crystal contains two molecules of isomer 3, two molecules of oxalic acid and one molecule of water, in which isomer 3 and oxalic acid form oxalate. The single molecule schematic diagram of compound isomer 3 is shown in Figure 5 As shown, the asymmetric structural unit of the oxalate single crystal is as follows Figure 6 The structural formula is shown as follows:

[0234]

[0235] Pharmacological activity test

[0236] Example 4 AKT kinase activity test

[0237] 1. Materials and Reagents

[0238] Envision plate reader (Molecular Devices)

[0239] White 384-well plate (Cat.#264706, Thermo)

[0240] Main reagents included in the HTRF kinEASE TK kit (Cat. #62TKOPEC, Cisbio)

[0241] TK-Biotin Substrate

[0242] Streptavidin-XL665

[0243] Europium-labeled tyrosine kinase substrate antibodies

[0244] 5× Enzyme Reaction Buffer

[0245] SEB

[0246] HTRF Assay Buffer

[0247] AKT1 (Cat. No. 01-101, Carna)

[0248] AKT2 (Cat. No. 01-102, Carna)

[0249] AKT3 (Cat.# PV3185, Invitrogen)

[0250] ATP 10mM (Catalog #PV3227, Invitrogen)

[0251] DTT 1M (Catalog #D5545, Sigma)

[0252] MgCl 2 1M (Cat.#M8266, Sigma)

[0253] Compounds of Examples 1-3 of the present application

[0254] Positive control: GDC-0068

[0255] 2. Experimental Procedure

[0256] 2.1 Reagent preparation

[0257] Table 1 Components and concentrations of kinase reaction system

[0258]

[0259] 1× Kinase Reaction Buffer

[0260] 1 mL of 1× kinase reaction buffer for AKT1, 2, 3 contains 200 μL of 5× kinase reaction buffer, 5 μL of

[0261] 1M MgCl 2 , 1 μL 1M DTT, 794 μL ultrapure water.

[0262] 5×TK-biotin substrate and ATP working solution

[0263] The specific concentrations of TK-biotin substrate and ATP are shown in Table 1.

[0264] Dilute substrate and ATP to 5 times the reaction concentration in 1× kinase reaction buffer.

[0265] 5× Kinase Working Solution

[0266] The concentrations used in enzyme screening are shown in Table 1. Prepare 5× enzyme working solution with 1× kinase reaction buffer.

[0267] 4× Streptavidin-XL665 working solution

[0268] The concentration of streptavidin-XL665 in the reaction is shown in Table 1. Prepare 4× streptavidin-XL665 working solution in assay buffer.

[0269] 4× Europium-labeled tyrosine kinase substrate antibody working solution

[0270] The europium-labeled tyrosine kinase substrate antibody was diluted 100-fold with the detection reaction buffer as the working solution.

[0271] 2.2 Experimental Procedure

[0272] After all reagents are prepared as described above, except for the enzyme, and equilibrated to room temperature, start adding samples.

[0273] a) First, dilute the compound stock solution (10mM DMSO solution) with DMSO to 100μM compound solution, and then dilute it with 1× kinase reaction buffer to 2.5μM compound working solution (containing 2.5% DMSO). Use 1× kinase reaction buffer to prepare 2.5% DMSO solution, and then dilute 2.5μM compound working solution with 2.5% DMSO solution, 4-fold gradient dilution 7 times, a total of 8 concentrations (2500nM, 625nM, 156nM, 39nM, 9.8nM, 2.4nM, 0.6nM, 0.15nM) of compound working solution. Add 4μL of the diluted compound working solution to all reaction wells except the control wells, and add 4μL of the previously prepared 2.5% DMSO / kinase buffer solution to the control wells.

[0274] b) Add 2 μL of the previously prepared TK-biotin substrate solution to all reaction wells (see Table 1 for substrate concentrations during enzyme screening).

[0275] c) Add 2 μL of the previously prepared enzyme solution (enzyme concentrations are shown in Table 1) to all reaction wells except the negative wells. For the negative wells, add 2 μL of enzyme corresponding to 1× kinase reaction buffer to make up the volume. Seal the plate with a sealing film, mix well, and incubate at room temperature for 10 minutes to allow the compound and enzyme to fully react and bind.

[0276] d) Add 2 μL of ATP solution to all reaction wells to start the kinase reaction (see Table 1 for ATP concentration and reaction time for enzyme screening).

[0277] e) Prepare the detection solution 5 minutes before the end of the kinase reaction. Use the detection buffer in the kit to prepare the detection solution of streptavidin-XL665 and europium-labeled tyrosine kinase substrate antibody (1:100) (see Table 1 for the detection reagent concentrations during enzyme screening).

[0278] f) After the kinase reaction is completed, add 5 μL of diluted streptavidin-XL665 to all reaction wells, mix well, and immediately add diluted europium-labeled tyrosine kinase substrate antibody detection solution.

[0279] g) Seal the plate and mix well. After reacting at room temperature for 1 hour, use ENVISION (PerkinElmer) instrument to detect fluorescence signals (320nm stimulation, 665nm, 615nm emission). Calculate the inhibition rate of each well through the full activity wells and background signal wells, take the average value of duplicate wells, and use professional drawing analysis software PRISM 6.0 to fit the half inhibition activity (IC50) of each test compound.

[0280] Table 2: Experimental sample addition flow chart

[0281]

[0282]

[0283] 2.3 Data Analysis

[0284] ER = 665nm fluorescence value / 615nm fluorescence value

[0285] Inhibition rate = (ER 阳性对照 -ER 样品 ) / (ER 阳性对照 -ER 阴性对照 )×100%

[0286] 3. Experimental results

[0287] The experimental results are shown in Table 3:

[0288] Table 3: AKT inhibitory activity

[0289]

[0290]

[0291]

[0292] Example 5 Pharmacodynamic evaluation of the compounds of the present application in the LnCap human prostate cancer subcutaneous transplantation model

[0293] 1. Experimental Materials

[0294] 1.1 Experimental animals

[0295] NCG mice, male, 8-10 weeks old (age of mice at the time of tumor cell inoculation), purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Breeding environment: SPF grade.

[0296] 1.2 Test and reference substances

[0297] Test sample: Isomer 2 of Example 1 (hereinafter referred to as Compound 62)

[0298] Example 2 Isomer 2 (hereinafter referred to as Compound 98)

[0299] Example 3 Isomer 4 (hereinafter referred to as Compound 102)

[0300] Positive control: GDC-0068

[0301] 1.3 Reagents

[0302] Androgen tablets, supplier: Aladdin, item number: A1910098.

[0303] FBS: Supplier: Gibco, SKU: 10099141C.

[0304] RPMI1640 culture medium: Supplier: Gibco, catalog number: C22400500BT.

[0305] Matrigel: Supplier: Corning, catalog number: 354234.

[0306] PBS: Supplier: Crownbio, Batch No.: 20190828.

[0307] 2. Experimental methods and procedures

[0308] 2.1 Cells

[0309] LnCap cells were cultured in RPMI1640 medium containing 10 vol% fetal bovine serum (FBS). LnCap cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0310] 2.2 Animal modeling and grouping

[0311] All animals were implanted with androgen tablets subcutaneously one day before cell inoculation. The entire process was performed aseptically. 7 LnCap cells were resuspended in 1:1 PBS and Matrigel (0.2 ml / cell). Tumor growth was observed regularly. When the tumor grew to an average volume of 177.70 mm 3 At the same time, the mice were randomly divided into groups and given drugs according to the tumor size and weight (see Table 4). The tumor volume was calculated as follows: long diameter × short diameter 2 / 2.

[0312] Table 4 LnCap animal model grouping

[0313]

[0314] 2.3 Drug preparation

[0315] The compound of the present application and the positive control were dissolved in a solvent to obtain solutions with concentrations of 1.25 mg / ml, 2.5 mg / ml and 5 mg / ml for compound 62, 2.5 mg / ml for the positive control, 5 mg / ml for compound 98 and 5 mg / ml for compound 102.

[0316] Solvent: The solvent is a mixed solvent of PG, PEG400 and water, PG:PEG400:water=20-40:20-30:30-50 (v:v:v), and any ratio within this ratio range may be used.

[0317] 2.4 Dosage regimen

[0318] The specific dosage regimen is shown in Table 5:

[0319] Table 5 Administration routes, doses and regimens in LnCap animal models

[0320]

[0321]

[0322] 2.5 Data Analysis

[0323] All experimental results were expressed as mean tumor volume ± SEM (mean standard error). Independent sample T test was used to compare whether there was a significant difference in tumor volume between the treatment group and the control group. All data were analyzed using SPSS18.0. p<0.05 was considered to be significantly different.

[0324] T / C (%) = average tumor volume of treatment group / average tumor volume of control group × 100%;

[0325] TGI (%) = (1-T / C) x 100%.

[0326] 2.6 Experimental Results

[0327] The tumor growth of each treatment group and control group is shown in Table 6:

[0328] Table 6 Analysis of drug efficacy of each treatment group in LnCap human prostate cancer tumor model

[0329]

[0330] *Each treatment group vs. vehicle control group

[0331] The inventors have found that the compound I-0 of the present application has the effect of inhibiting the activity of AKT kinase, especially the compound I of the present application (ie, isomer 2 of Example 1, isomer 2 of Example 2, and isomer 4 of Example 3), which exhibits a significant tumor inhibitory effect in in vivo experiments, so the compounds of the present application and the pharmaceutical compositions containing them can be used to prevent and / or treat diseases or disease states mediated by AKT protein kinase. Further, the inventors have found that when the dosage of the pharmaceutical composition is administered to an individual in need, the dosage of the pharmaceutical composition is 0.001-100 mg / kg body weight / day, preferably 0.01-50 mg / kg body weight / day, and more preferably 10-50 mg / kg body weight / day, based on compound I-0 or compound I, which can significantly inhibit the growth of tumors.

[0332] Formulation Examples

[0333] The following are exemplary preparations of the present application. It will be appreciated that the following preparations can be prepared using conventional preparation methods in the art that are well known to those skilled in the art.

[0334] Formulation 1: Capsules containing 25 mg of compound 62

[0335] Element Quality Ratio Unit dose (mg) Compound 62 25.00％ 25.00 Anhydrous calcium hydrogen phosphate 74.00％ 74.00 Glyceryl Behenate 1.00％ 1.00 Total weight 100.00％ 100.00

[0336] a) adding compound 62, anhydrous calcium hydrogen phosphate and glyceryl behenate into a mixer hopper and mixing until uniform;

[0337] b) Filling the mixture into capsules.

[0338] Formulation 2: Capsules containing 50 mg of compound 62

[0339] Element Quality Ratio Unit dose (mg) Compound 62 33.33％ 50.00 Anhydrous calcium hydrogen phosphate 65.67％ 98.50 Glyceryl Behenate 1.00％ 1.50 Total weight 100.00％ 150.00

[0340] Prepare according to the method of Preparation 1.

[0341] Formulation 3: Capsules containing 100 mg of compound 62

[0342] Element Quality Ratio Unit dose (mg) Compound 62 41.67％ 100.00 Calcium Hydrogen Phosphate Dihydrate 57.33％ 137.60 Glyceryl Behenate 1.00％ 2.40 Total weight 100.00％ 240.00

[0343] Prepare according to the method of Preparation 1.

[0344] Formulation 4: Capsules containing 150 mg of compound 62

[0345] Element Quality Ratio Unit dose (mg) Compound 62 62.50％ 150.00 Calcium Hydrogen Phosphate Dihydrate 36.50％ 87.60 Glyceryl Behenate 1.00％ 2.40 Total weight 100.00％ 240.00

[0346] Prepare according to the method of Preparation 1.

[0347] Formulation 5: Capsules containing 200 mg of compound 62

[0348] Element Quality Ratio Unit dose (mg) Compound 62 62.50％ 200.00 Calcium Hydrogen Phosphate Dihydrate 36.50％ 116.8 Glyceryl Behenate 1.00％ 3.2 Total weight 100.00％ 320.00

[0349] Prepare according to the method of Preparation 1.

[0350] Preparation 6: Dry suspension containing 400 mg of compound 62

[0351] Element Quality Ratio Unit dose (mg) Compound 62 62.50％ 400.00 Calcium Hydrogen Phosphate Dihydrate 36.50％ 233.6 Glyceryl Behenate 1.00％ 6.4 Total weight 100.00％ 640.00

[0352] a) adding compound 62, calcium hydrogen phosphate dihydrate and glyceryl behenate into a mixer hopper and mixing until uniform;

[0353] b) Fill the mixture into aluminum bags to form a dry suspension.

[0354] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A pharmaceutical composition in unit dosage form of Compound I, wherein: The mass of compound I or a pharmaceutically acceptable salt thereof is 5 mg to 400 mg as a free base, and compound I has the following structure:

2. The pharmaceutical composition according to claim 1, wherein the mass of Compound I or a pharmaceutically acceptable salt thereof is 10 mg to 400 mg as a free base; preferably 10 mg to 200 mg; more preferably 10 mg to 150 mg; more preferably 25 mg to 150 mg; more preferably 25 mg to 100 mg.

3. The pharmaceutical composition according to claim 1, wherein the mass of Compound I or a pharmaceutically acceptable salt thereof is 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg or 400 mg as a free base.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The mass of compound I or its pharmaceutically acceptable salt calculated as free base accounts for 0.1%-99.9% of the total mass of the pharmaceutical composition.

5. The pharmaceutical composition according to claim 4, wherein The mass of compound I or its pharmaceutically acceptable salt calculated as free base accounts for 5%-90% of the total mass of the pharmaceutical composition.

6. The pharmaceutical composition according to claim 4, wherein The mass of Compound I or its pharmaceutically acceptable salt calculated as free base accounts for 25%-65% of the total mass of the pharmaceutical composition.

7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising one or more pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition in unit dosage form is a pharmaceutical preparation suitable for oral administration.

9. The pharmaceutical composition according to claim 7 for use as a medicament.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a medicament for treating a disease or disease state mediated by AKT protein kinase, wherein: The disease or condition mediated by AKT protein kinase is prostate cancer.

Images