A method for preparing peptide amide compounds and intermediates thereof

Abstract

A method for preparing a peptide amide compound represented by formula (N) and an intermediate thereof. The method has mild reaction conditions, simple operation, high reaction yield, high product purity, convenient post-treatment, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a method for preparing a peptide amide compound and an intermediate thereof. Background Art

[0002] Opioids have been used to treat pain for thousands of years. They mainly exert their physiological effects by binding to the three known classic opioid receptors, μ, δ, and κ. These three receptors are all members of the G protein-coupled receptor family, mainly distributed in the central nervous system, and also exist in many peripheral tissues. Among them, the most classic drug is morphine, which mainly exerts analgesic effects through the action of μ opioid receptors. In addition, commonly used clinical analgesics also include other μ opioid receptor drugs, such as traditional opioid drugs represented by dihydromorphone and fentanyl.

[0003] However, μ-opioid receptor drugs can produce a variety of side effects after long-term use, such as tolerance, dependence, respiratory depression, and effects on gastrointestinal motility, which not only increases the cost of treatment, but also affects the patient's recovery cycle. Some non-opioid injections, such as acetaminophen and NSAIDs (non-steroidal anti-inflammatory drugs), have limited use and dosage due to their poor analgesic effects; in addition, they also have certain side effects, such as acetaminophen increasing liver toxicity and NSAIDs (non-steroidal anti-inflammatory drugs) causing various gastrointestinal diseases.

[0004] With the increasing pressure of life and work in modern society and the arrival of an aging society, and the vital role of opioid receptors in the treatment of different types of pain, finding new opioid drugs with high analgesic activity and low toxic side effects has important scientific and social significance.

[0005] WO2019015644 discloses a κ opioid receptor agonist represented by formula (N), which has a novel structure and good analgesic effect. Therefore, it is of great significance to study its preparation process.

[0006] Summary of the invention

[0007] The object of the present invention is to provide a method for preparing a peptide amide compound represented by formula (N) and an intermediate thereof, wherein the method has low-cost starting materials, mild reaction conditions, simple operation, high yield, high product purity, convenient post-treatment, and is suitable for industrial production.

[0008] Compared with the method for preparing the compound represented by formula (N) disclosed in WO2019015644, the present invention has the following optimizations:

[0009] (1) The present invention optimizes the process of generating the compound of formula (I) from the compound of formula (G). In the prior art, 2,7-diazaspiro[3.5]nonane-7-carboxylic acid benzyl ester is used as the starting material, which needs to be acetylated first and then the N-benzyloxycarbonyl group is removed before the condensation reaction can be carried out. In this step, the present invention directly uses 1-(2,7-diazaspiro[3.5]nonane-2-yl)ethanone hydrochloride as the starting material, omitting the step of removing the N-benzyloxycarbonyl protecting group, avoiding the use of precious metals (palladium carbon) in the process of removing the protecting group, reducing the reaction steps and saving costs.

[0010] (2) The reaction conditions for preparing the compound of formula (I) from the compound of formula (G), the compound of formula (L) from the compound of formula (J), the compound of formula (F) from the compound of formula (D), and the compound of formula (C) from the compound of formula (A) were optimized. 0.3-1.2 equivalents of copper chloride were added to the reaction, which was beneficial to maintaining the configuration of the chiral compound, so that the diastereomers of the generated intermediates were effectively controlled.

[0011] (3) The post-treatment process is optimized, avoiding the use of high performance liquid chromatography preparative analysis and ion exchange resin process to separate products in the prior art. Although column chromatography is still used in the purification method, rapid silica gel column chromatography is used, which greatly reduces the amount of silica gel used and can be purified quickly, making it suitable for industrial production.

[0012] The present invention optimizes the post-treatment of each reaction step and uses conventional simple operation steps, which is more suitable for industrial production, improves the reaction yield, and is easy to control and monitor the operation process and product, and is environmentally friendly.

[0013] In one aspect, the present invention provides a method for preparing a compound represented by formula (N), which method does not require purification by preparative chromatography.

[0014]

[0015] According to some specific embodiments of the present invention, the method comprises the following steps:

[0016] (1) dissolving the compound of formula (M) in water (preferably purified water), adding an organic solvent for extraction, separating the liquids, and collecting the aqueous phase;

[0017] (2) adding an alkaline agent dropwise to the aqueous phase obtained in step (1) to adjust the pH of the aqueous phase to alkaline (preferably pH 9-10);

[0018] (3) extracting and separating the aqueous phase (the aqueous phase adjusted to alkaline pH) obtained in step (2) with an organic solvent, filtering and concentrating the organic phase (until no obvious fraction is left) to obtain compound (N);

[0019]

[0020] Wherein, HA is hydrochloric acid, phosphoric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid or methanesulfonic acid.

[0021] n is selected from 1, 2 or 3, preferably 2.

[0022] According to some specific embodiments of the present invention, the method further comprises the following purification step:

[0023] (4) separating and purifying the compound (N) obtained in step (3) by silica gel column chromatography (rapid separation and purification), collecting the eluted fractions, and concentrating them;

[0024] (5) The concentrated solution obtained in step (4) is dissolved in an organic solvent and / or a mixed solution of purified water (an organic solvent, or a mixed solution of an organic solvent and purified water), filtered, and concentrated to obtain a purified compound (N).

[0025] According to some specific embodiments of the present invention, the organic solvent in steps (1) and (3) is selected from dichloromethane, ethyl acetate, 2-methyltetrahydrofuran, chloroform or isopropyl acetate; the alkaline agent in step (2) is selected from ammonia water, or a solution of sodium bicarbonate, sodium hydroxide or potassium hydroxide (preferably an aqueous solution);

[0026] According to some specific embodiments of the present invention, the eluent in step (4) (the eluent used for silica gel column chromatography separation and purification in step (4)) is selected from a mixed solution of dichloromethane and / or anhydrous methanol (dichloromethane, or a mixed solution of dichloromethane and anhydrous methanol);

[0027] According to some specific embodiments of the present invention, the mesh size of the silica gel in step (4) is 200-300 meshes.

[0028] According to some specific embodiments of the present invention, the organic solvent in step (5) is selected from methanol, ethanol or isopropanol.

[0029] According to some specific embodiments of the present invention, the eluent in step (4) is selected from a mixed solution of dichloromethane and anhydrous methanol (volume ratio = 50:1 to 5:1);

[0030] According to some specific embodiments of the present invention, the organic solvent and / or purified water mixed solution used in step (5) is selected from a mixed solution of methanol and purified water (volume ratio = 1:8 to 1:5).

[0031] According to some specific embodiments of the present invention, the method for preparing the compound represented by formula (N) comprises the following steps:

[0032] (1) dissolving the compound of formula (M) in water at 10±10°C, preferably 10±5°C, adding dichloromethane solution for extraction, separating the liquids, and collecting the aqueous phase;

[0033] (2) adding aqueous ammonia to the aqueous phase obtained in step (1) at 10±10°C, preferably 10±5°C, to adjust the pH of the aqueous phase to 10-11; regularly measuring the pH value of the reaction solution with a pH meter, and if the pH value drops, adding aqueous ammonia to the reaction solution to maintain the pH value of the reaction solution at 9-10;

[0034] (3) Allow to stand to separate the layers, extract the aqueous phase with a dichloromethane solution, separate the layers, filter the organic phase (through diatomaceous earth filtration), and concentrate the filtrate under reduced pressure at 30±5°C to obtain a concentrated solution;

[0035] (4) taking silica gel (preferably 200-300 mesh silica gel) and dichloromethane wet-loading a column, using dichloromethane and anhydrous methanol (volume ratio = 50:1 to 5:1) as eluent, and separating and purifying the concentrated solution obtained in step (3) by column chromatography; collecting the eluted fractions, controlling the temperature to be below 30±5° C. and concentrating the fractions under reduced pressure to obtain a concentrated solution;

[0036] (5) dissolving the concentrated solution obtained in step (4) in a mixed solution of methanol and purified water (volume ratio = 1:8 to 1:5) at 20±10° C., preferably 20±5° C., filtering (filtering with a glass sand funnel), combining the filtrates, and transferring the filtrates to a freeze dryer for freeze drying to obtain the compound (N);

[0037]

[0038] Wherein, HA is hydrochloric acid, phosphoric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid or methanesulfonic acid.

[0039] n is selected from 1, 2 or 3, preferably 2.

[0040] In certain embodiments, the mass ratio of water to compound (M) in step (1) is 9:1 to 12:1, preferably 10:1.

[0041] In certain embodiments, the alkaline reagent added dropwise in step (2) is aqueous ammonia, and the mass ratio of aqueous ammonia to compound (M) is 1.5:1 to 1:1, preferably 1.15:1.

[0042] In certain embodiments, the mass of silica gel in step (4) is 2.5 to 3 times the mass of the concentrated solution obtained in step (3).

[0043] In certain embodiments, the method further comprises the following further purification steps:

[0044] (a) reacting the compound (N) obtained in step (5) with HA in an organic solvent; and recrystallizing to obtain crystals;

[0045] (b) dissolving the crystals obtained in step (a) in water, extracting with an organic solvent, separating the liquids, and collecting the aqueous phase; dropping an alkaline reagent into the aqueous phase to adjust the pH of the aqueous phase to alkaline; extracting the aqueous phase adjusted to alkaline pH with an organic solvent, separating the liquids, filtering the organic phase and concentrating (until no obvious fraction is left) to obtain compound (N).

[0046] In certain embodiments, (a) is to react the concentrated solution obtained in step (4) with HA in an organic solvent; and recrystallize to obtain crystals;

[0047] In certain embodiments, the recrystallization of step (a) comprises precipitating crystals using an anti-solvent (preferably methyl tert-butyl ether).

[0048] In certain embodiments, the organic solvent of step (b) is selected from dichloromethane, ethyl acetate, 2-methyl-tetrahydrofuran, chloroform or isopropyl acetate; and the alkaline agent is selected from aqueous ammonia, sodium bicarbonate, sodium hydroxide or potassium hydroxide.

[0049] In certain embodiments, the method further comprises the following further purification steps:

[0050] (a) dissolving the concentrated solution obtained in step (4) in an organic solvent at 15±5°C, and adding HA dropwise to react; adding methyl tert-butyl ether dropwise to the reaction solution under stirring at 20±5°C to precipitate crystals, filtering under nitrogen protection, and washing the filter cake with methyl tert-butyl ether;

[0051] (b) at 10±5°C, dissolving the filter cake obtained in step (a) in water, adding dichloromethane solution for extraction, separating the liquids, and collecting the aqueous phase; dropping ammonia water into the aqueous phase, adjusting the pH of the aqueous phase to 10-11, so that the pH value of the reaction solution is maintained at 9-10; after the reaction is completed, standing and stratifying, extracting the aqueous phase with dichloromethane solution, separating the liquids, filtering the organic phase through diatomaceous earth, and concentrating the filtrate under reduced pressure at 30±5°C to obtain a concentrated solution; at 20±5°C, dissolving the concentrated solution with a mixed solution of methanol and purified water (volume ratio = 1:8-1:5), filtering with a glass sand funnel, combining the filtrates, and transferring the filtrate to a freeze dryer for freeze drying to obtain the compound (N).

[0052] In certain embodiments, the organic solvent of step (a) is ethanol.

[0053] According to some specific embodiments of the present invention, the method further comprises taking the compound of formula (L) as a raw material and reacting in the presence of an acidic reagent and dichloromethane to prepare a compound of formula (M),

[0054]

[0055] Wherein, P is an amino protecting group; preferably tert-butyloxycarbonyl, N-benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl; more preferably tert-butyloxycarbonyl.

[0056] In certain embodiments, the acidic agent is selected from hydrochloric acid, phosphoric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid or methanesulfonic acid.

[0057] In certain embodiments, the reaction temperature is 15°C ± 10°C.

[0058] In certain embodiments, the acidic reagent is selected from trifluoroacetic acid, and the mass ratio of compound (L) to trifluoroacetic acid is 1.6:1 to 1.8:1, preferably 1.7:1.

[0059] In a second aspect, the present invention provides a method for preparing a compound of formula (L), comprising using a compound of formula (J) and a compound of formula (K) as raw materials, in the presence of a condensing agent and copper chloride (preferably copper chloride dihydrate) to prepare a compound of formula (L):

[0060]

[0061] The condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one (DHOBT) or BOP; preferably any one or a mixture of any one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole;

[0062] The solvent used in the reaction is selected from any one of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile or a mixture of any of them; preferably any one of N,N-dimethylformamide and dichloromethane or a mixture of any of them.

[0063] The reaction temperature is selected from -5 to 30°C; preferably 0 to 20°C;

[0064] P is an amino protecting group; preferably tert-butyloxycarbonyl, N-benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl; more preferably tert-butyloxycarbonyl.

[0065] In certain embodiments, the mass ratio of compound (J) to compound (K) is 3:1 to 4:1; preferably 3.32:1 and 3.69:1.

[0066] In certain embodiments, the condensing agent used in the reaction is selected from 1-hydroxybenzotriazole (HOBT), and the mass ratio of compound (J) to HOBT is 4:1 to 6:1; preferably 5.24:1 and 4.61:1.

[0067] In certain embodiments, the mass ratio of compound (J) to copper chloride (dihydrate) is 2:1 to 3:1; preferably 2.57:1 and 2.59:1.

[0068] In certain embodiments, the condensing agent used in the reaction is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl), and the mass ratio of compound (J) to EDC.HCl is 2.5:1 to 3.5:1; preferably 3.28:1 and 3.34:1.

[0069] In certain embodiments, the compound of formula (J) and the compound of formula (K) are prepared in the presence of a condensing agent, copper chloride (preferably copper chloride dihydrate) and a base to obtain the compound of formula (L).

[0070] In certain embodiments, the base is selected from triethylamine or N,N-diisopropylethylamine.

[0071] In certain embodiments, the mass ratio of compound (J) to base is 3:1 to 5:1; preferably 4.5:1 and 4.95:1.

[0072] In certain embodiments, the solvent used in the reaction is selected from dichloromethane, and the mass ratio of compound (J) to dichloromethane is 20:1 to 25:1; preferably 23.66:1.

[0073] In certain embodiments, the mass volume ratio of compound (J) to dichloromethane is 1:10 to 1:15; preferably 1:13.

[0074] According to some specific embodiments of the present invention, the method further comprises the preparation of the compound of formula (J), comprising: using the compound of formula (I) as a raw material to prepare the compound of formula (J),

[0075]

[0076] The base used in the reaction is selected from an organic base or an inorganic base; preferably KOH, NaOH, lithium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, sodium methoxide, sodium ethoxide; more preferably lithium hydroxide (monohydrate).

[0077] The solvent used in the reaction is selected from any one of ethanol, methanol, tetrahydrofuran, dimethyl sulfide, ethyl acetate, water or a mixture of any of them;

[0078] The reaction temperature is selected from -10 to 10°C; preferably -5 to 5°C;

[0079] P is an amino protecting group; preferably tert-butyloxycarbonyl, N-benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl; more preferably tert-butyloxycarbonyl.

[0080] In certain embodiments, the base used in the reaction is lithium hydroxide (monohydrate), and the mass ratio of compound (I) to lithium hydroxide (monohydrate) is 1:65 to 1:67, preferably 1:67.

[0081] In certain embodiments, the solvent used in the reaction is tetrahydrofuran, and the mass ratio of compound (I) to tetrahydrofuran is 8:1 to 9:1, preferably 8.5:1.

[0082] In certain embodiments, the mass ratio of lithium hydroxide (monohydrate) to water is 20:1 to 25:1, preferably 22.75:1.

[0083] According to some specific embodiments of the present invention, the method further comprises the preparation of the compound of formula (I), comprising: in the presence of a condensing agent and copper chloride (preferably copper chloride dihydrate), using the compound of formula (G) and the compound of formula (H) as raw materials to prepare the compound of formula (I),

[0084]

[0085] The condensing agent used in the reaction is selected from any one or a mixture of any several of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or N,N'-carbonyldiimidazole or BOP; preferably any one or a mixture of any several of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole.

[0086] The solvent used in the reaction is selected from any one of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile or a mixture of any of them; preferably any one of N,N-dimethylformamide and dichloromethane or a mixture of any of them.

[0087] The reaction temperature is selected from -5 to 30°C; preferably 0 to 20°C.

[0088] P is an amino protecting group; preferably tert-butyloxycarbonyl, N-benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl; more preferably tert-butyloxycarbonyl.

[0089] In certain embodiments, the mass ratio of compound (G) to compound (H) is 1.5:1 to 2:1; preferably 1.78:1.

[0090] In certain embodiments, the condensing agent used in the reaction is selected from 1-hydroxybenzotriazole (HOBT), and the mass ratio of compound (G) to HOBT is 3:1 to 4:1; preferably 3.2:1 and 3.85:1.

[0091] In certain embodiments, the mass ratio of compound (G) to cupric chloride (dihydrate) is 2:1 to 3:1; preferably 2.57:1 and 2.59:1.

[0092] In certain embodiments, the condensing agent used in the reaction is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl), and the mass ratio of compound (G) to EDC.HCl is 2:1 to 2.5:1, preferably 2.29:1.

[0093] In certain embodiments, the compound of formula (G) and the compound of formula (H) undergo condensation reaction in the presence of a condensing agent, copper chloride and a base to produce the compound of formula (I).

[0094] In certain embodiments, the base is selected from triethylamine or N,N-diisopropylethylamine.

[0095] In certain embodiments, the mass ratio of compound (G) to N,N-diisopropylethylamine is 2.5:1 to 3.5:1; preferably 2.72:1 and 3.16:1.

[0096] In certain embodiments, the mass ratio of compound (G) to triethylamine is 3:1 to 5:1; preferably 4.5:1 and 4.95:1.

[0097] In certain embodiments, the solvent used in the reaction is selected from dichloromethane, and the mass ratio of compound (G) to dichloromethane is 15:1 to 20:1; preferably 18.6:1.

[0098] In certain embodiments, the mass volume ratio of compound (G) to dichloromethane is 1:15 to 1:25; preferably 1:19.

[0099] According to some specific embodiments of the present invention, the method further comprises the preparation of the compound of formula (G), comprising: using the compound of formula (F) as a raw material to prepare the compound of formula (G),

[0100]

[0101] The base used in the reaction is selected from an organic base or an inorganic base; preferably KOH, NaOH, lithium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, sodium methoxide, sodium ethoxide; more preferably lithium hydroxide (monohydrate).

[0102] The solvent used in the reaction is selected from any one or a mixture of any of ethanol, methanol, tetrahydrofuran, dimethyl sulfide, ethyl acetate, acetonitrile or water;

[0103] The reaction temperature is selected from -10 to 10°C; preferably -5 to 5°C;

[0104] P is an amino protecting group; preferably tert-butyloxycarbonyl, N-benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl; more preferably tert-butyloxycarbonyl.

[0105] In certain embodiments, the base used in the reaction is selected from lithium hydroxide (monohydrate), and the mass ratio of compound (F) to lithium hydroxide (monohydrate) is 90:1 to 95:1; preferably 93.4:1.

[0106] In certain embodiments, the solvent used in the reaction is selected from tetrahydrofuran, and the mass ratio of compound (F) to tetrahydrofuran is 8:1 to 9:1, preferably 8.8:1.

[0107] In some embodiments, the mass ratio of lithium hydroxide (monohydrate) to water is 20:1 to 30:1; preferably 28.4:1

[0108] According to some specific embodiments of the present invention, the method further comprises a method for preparing a compound of formula (F), comprising: preparing a compound of formula (F) using a compound of formula (D) as a raw material in the presence of a condensing agent,

[0109]

[0110] The condensing agent used in the reaction is selected from any one or a mixture of any several of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; preferably any one or a mixture of any several of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole.

[0111] The solvent used in the reaction is selected from any one of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile or a mixture of any of them; preferably any one of N,N-dimethylformamide and dichloromethane or a mixture of any of them.

[0112] The reaction temperature is selected from -5 to 30°C; preferably 0 to 20°C.

[0113] HA is selected from hydrochloric acid, phosphoric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid or methanesulfonic acid; preferably trifluoroacetic acid.

[0114] In certain embodiments, the mass ratio of compound (D) to compound (E) is 1.2:1 to 1.8:1; preferably 1.53:1.

[0115] In certain embodiments, the condensing agent used in the reaction is selected from 1-hydroxybenzotriazole (HOBT), and the mass ratio of compound (D) to HOBT is 2.5:1 to 3:1; preferably 2.87:1.

[0116] In certain embodiments, the condensing agent used in the reaction is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl), and the mass ratio of compound (D) to EDC.HCl is 1.5:1 to 2:1; preferably 1.78:1.

[0117] In certain embodiments, the compound of formula (F) is prepared from the compound of formula (D) as a starting material in the presence of a condensing agent and a base.

[0118] In certain embodiments, the base is selected from triethylamine or N,N-diisopropylethylamine.

[0119] In certain embodiments, the mass ratio of compound (D) to N,N-diisopropylethylamine is 2:1 to 3:1; preferably 2.64:1.

[0120] In certain embodiments, the mass ratio of compound (D) to triethylamine is 3:1 to 5:1; preferably 4.5:1 and 4.95:1.

[0121] In certain embodiments, the solvent used in the reaction is selected from dichloromethane, and the mass ratio of compound (D) to dichloromethane is 1:10 to 1:15; preferably 1:13.26.

[0122] In certain embodiments, the method for preparing the compound of formula (F) comprises: using the compound of formula (D) and the compound of formula (E) as raw materials to prepare the compound of formula (F),

[0123]

[0124] According to some specific embodiments of the present invention, the method further comprises the preparation of the compound of formula (D), comprising: using the compound of formula (C) as a raw material to prepare the compound of formula (D),

[0125]

[0126] The method comprises: preparing a compound of formula (D) from a compound of formula (C) in the presence of dichloromethane and trifluoroacetic acid.

[0127] According to some specific embodiments of the present invention, the method further comprises the preparation of the compound of formula (D), comprising: using the compound of formula (C) as a raw material to prepare the compound of formula (D),

[0128]

[0129] The method comprises: dissolving a compound of formula (C) in dichloromethane, and dropping trifluoroacetic acid to prepare a compound of formula (D).

[0130] In certain embodiments, the method comprises: dissolving the compound of formula (C) in dichloromethane, adding trifluoroacetic acid dropwise at 0±5°C, and maintaining the reaction at 15±5°C to prepare the compound of formula (D).

[0131] In certain embodiments, the mass ratio of compound (C) to trifluoroacetic acid is 1:2 to 1:2.5; preferably 1:2.33.

[0132] In certain embodiments, the mass ratio of compound (C) to dichloromethane is 6:1 to 3:1, preferably 4:1.

[0133] According to some specific embodiments of the present invention, the method further comprises the preparation of a compound represented by formula (C), comprising: in the presence of a condensing agent, using a compound of formula (A) as a raw material to prepare a compound of formula (C)

[0134]

[0135] The condensing agent used in the reaction is selected from any one or a mixture of any several of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; preferably any one or a mixture of any several of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole.

[0136] The solvent used in the reaction is selected from any one of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile or a mixture of any of them; preferably any one of N,N-dimethylformamide and dichloromethane or a mixture of any of them.

[0137] The reaction temperature is selected from -5 to 30°C; preferably 0 to 20°C.

[0138] In certain embodiments, the mass ratio of compound (A) to compound (B) is 1.2:1 to 1.8:1; preferably 1.46:1.

[0139] In certain embodiments, the condensing agent used in the reaction is selected from HOBT (1-hydroxybenzotriazole), and the mass ratio of compound (A) to HOBT is 1:1 to 1.5:1; preferably 1.27:1.

[0140] In certain embodiments, the condensing agent used in the reaction is selected from EDC.HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and the mass ratio of compound (A) to EDC.HCl is 1:1.1 to 1:1.5; preferably 1:1.27.

[0141] In certain embodiments, the compound of formula (C) is prepared from the compound of formula (A) in the presence of a condensing agent, copper chloride (preferably copper chloride dihydrate) and a base.

[0142] In certain embodiments, the base is selected from triethylamine or N,N-diisopropylethylamine.

[0143] In certain embodiments, the mass ratio of compound (A) to base is 1:1 to 1.5:1; preferably 1.17:1.

[0144] In certain embodiments, the solvent used in the reaction is selected from dichloromethane, and the mass ratio of compound (A) to dichloromethane is 1:25 to 1:35, preferably 1:33.

[0145] In certain embodiments, the method for preparing the compound of formula (C) comprises: using the compound of formula (A) and the compound of formula (B) as raw materials to prepare the compound of formula (C),

[0146]

[0147] In a third aspect, the present invention relates to a method for preparing a compound represented by formula (N), wherein the method comprises the following steps:

[0148] In the first step, the compound of formula (A) and the compound of formula (B) undergo condensation reaction to generate a compound of formula (C);

[0149] The second step is to remove the protecting group (amino protecting group) on the nitrogen atom of the compound of formula (C) to generate the compound of formula (D) (the protecting agent is removed in the presence of an acidic reagent);

[0150] In the third step, the compound of formula (D) undergoes a condensation reaction with the compound of formula (E) to generate a compound of formula (F);

[0151] The fourth step is to hydrolyze the compound of formula (F) to obtain the compound of formula (G);

[0152] In the fifth step, the compound of formula (G) undergoes a condensation reaction with the compound of formula (H) to generate a compound of formula (I);

[0153] Step 6, hydrolyzing the compound of formula (I) to obtain the compound of formula (J);

[0154] Step 7, reacting the compound of formula (J) with the compound of formula (K) to generate the compound of formula (L);

[0155] Step 8, removing the protecting group (amino protecting group) on the nitrogen atom of the compound of formula (L) to generate a compound of formula (M) (removing the protecting agent in the presence of an acidic reagent);

[0156] The ninth step is to free the compound of formula (M) by alkali to generate compound (N).

[0157]

[0158] in,

[0159] HA is selected from hydrochloric acid, phosphoric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid or methanesulfonic acid; preferably trifluoroacetic acid;

[0160] n is selected from 1, 2 or 3, preferably 2;

[0161] P is an amino protecting group; preferably selected from tert-butyloxycarbonyl, N-benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl; more preferably tert-butyloxycarbonyl.

[0162] According to some specific embodiments of the present invention, the method comprises the following steps:

[0163] The first step is to react the compound of formula (A) with the compound of formula (B) in the presence of a condensing agent and a base to generate a compound of formula (C); the condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine;

[0164] In the second step, the compound of formula (C) is prepared in the presence of dichloromethane and trifluoroacetic acid to obtain the compound of formula (D);

[0165] The third step is to react the compound of formula (D) with the compound of formula (E) to generate the compound of formula (F) in the presence of a condensing agent and a base; the condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine;

[0166] Step 4, preparing a compound of formula (G) from the compound of formula (F) in the presence of tetrahydrofuran and lithium hydroxide;

[0167] The fifth step is to react the compound of formula (G) with the compound of formula (H) to generate the compound of formula (I) in the presence of a condensing agent, copper chloride and a base; the condensing agent used in the reaction is selected from any one or a mixture of several of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of several of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine;

[0168] Step 6, preparing a compound of formula (J) from the compound of formula (I) in the presence of tetrahydrofuran and lithium hydroxide;

[0169] Step 7: In the presence of a condensing agent, copper chloride and a base, the compound of formula (J) reacts with the compound of formula (K) to generate a compound of formula (L); the condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine;

[0170] Step 8, preparing a compound of formula (M) from the compound of formula (L) in the presence of dichloromethane and trifluoroacetic acid;

[0171] The ninth step is to dissolve the compound of formula (M) in water, add dichloromethane solution for extraction, separate the liquids, and collect the aqueous phase; drop ammonia water into the aqueous phase, adjust the pH of the aqueous phase to 10-11, and maintain the pH value of the reaction solution at 9-10 to obtain compound (N).

[0172] According to some specific embodiments of the present invention, the method comprises the following steps:

[0173] The first step is to react the compound of formula (A) with the compound of formula (B) in the presence of a condensing agent and a base to generate a compound of formula (C); the condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine;

[0174] The second step is to dissolve the compound of formula (C) in dichloromethane and dropwise add trifluoroacetic acid to prepare the compound of formula (D);

[0175] The third step is to react the compound of formula (D) with the compound of formula (E) to generate the compound of formula (F) in the presence of a condensing agent and a base; the condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine;

[0176] The fourth step is to dissolve the compound of formula (F) in tetrahydrofuran, and add a lithium hydroxide aqueous solution to prepare a compound of formula (G);

[0177] The fifth step is to react the compound of formula (G) with the compound of formula (H) to generate the compound of formula (I) in the presence of a condensing agent, copper chloride (preferably copper chloride dihydrate) and a base; the condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine;

[0178] Step 6, dissolving the compound of formula (I) in tetrahydrofuran, adding lithium hydroxide aqueous solution to prepare a compound of formula (J);

[0179] Step 7: In the presence of a condensing agent, copper chloride (preferably copper chloride dihydrate) and a base, the compound of formula (J) reacts with the compound of formula (K) to generate a compound of formula (L); the condensing agent used in the reaction is selected from any one or a mixture of several of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of several of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine;

[0180] Step 8, dissolving the compound of formula (L) in dichloromethane, and adding trifluoroacetic acid dropwise to prepare a compound of formula (M);

[0181] The ninth step is to dissolve the compound of formula (M) in water, add dichloromethane solution for extraction, separate the liquids, and collect the aqueous phase; drop ammonia water into the aqueous phase, adjust the pH of the aqueous phase to 10-11, and maintain the pH value of the reaction solution at 9-10 to obtain compound (N).

[0182] According to some specific embodiments of the present invention, wherein, in the first step, the third step, the fifth step and the seventh step, the condensing agent used in the reaction is selected from any one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole or any mixture of several thereof, the solvent used in the reaction is selected from any one of N,N-dimethylformamide and dichloromethane or any mixture of several thereof; the base used in the reaction is N,N-diisopropylethylamine; the reaction temperature is selected from -5 to 30°C;

[0183] According to some specific embodiments of the present invention, the ninth step further comprises a post-treatment step after the reaction is completed, and the post-treatment step comprises:

[0184] (1) After the reaction in step 9 is completed, the reaction solution is allowed to stand and separate, the aqueous phase is extracted with a dichloromethane solution and separated, the organic phase is filtered through diatomaceous earth, and the filtrate is concentrated under reduced pressure at 30±5° C. to obtain a concentrated solution;

[0185] (2) taking silica gel (preferably 200-300 mesh silica gel) and dichloromethane wet-loading a column, using dichloromethane and anhydrous methanol (volume ratio = 50:1 to 5:1) as eluent, and eluting the concentrated solution obtained in step (1) by column chromatography (gradient elution); collecting the eluted fractions, controlling the temperature to be below 30±5° C. and concentrating the fractions under reduced pressure to obtain a concentrated solution;

[0186] (3) dissolving the concentrated solution obtained in step (2) in ethanol at 15±5°C, and adding HA dropwise to react; adding methyl tert-butyl ether dropwise to the reaction solution at 20±5°C with stirring to precipitate crystals, filtering under nitrogen protection, and washing the filter cake with methyl tert-butyl ether;

[0187] (4) at 10±5°C, the filter cake obtained in step (3) was dissolved in water, and a dichloromethane solution was added for extraction and separation, and the aqueous phase was collected; ammonia water was added dropwise to the aqueous phase to adjust the pH of the aqueous phase to 10-11, so that the pH value of the reaction solution was maintained at 9-10; after the reaction was completed, the aqueous phase was allowed to stand for stratification, the aqueous phase was extracted with a dichloromethane solution and separated, the organic phase was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure at 30±5°C to obtain a concentrated solution; at 20±5°C, the concentrated solution was dissolved with a mixed solution of methanol and purified water (volume ratio = 1:8-1:5), filtered with a glass sand funnel, the filtrates were combined, and the filtrates were transferred to a freeze dryer for freeze drying to obtain the compound (N).

[0188] P is an amino protecting group; preferably selected from tert-butyloxycarbonyl, N-benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl; more preferably tert-butyloxycarbonyl.

[0189] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0190] The method of using extraction in the post-treatment of the reaction in the present invention is a conventional method in the art. The solvent for extraction can be selected according to the solubility of the product and the solubility of the organic solvent in water. Common extraction solvents include but are not limited to one or more mixed solvents of dichloromethane, chloroform, ethyl acetate, methyl acetate, isopropyl acetate, ether, isopropyl ether, methyl tert-butyl ether, methanol and ethanol. The number of extractions can be appropriately increased or decreased according to the amount of product remaining in the aqueous phase. The organic phase after extraction is optionally further subjected to conventional washing and / or drying treatment in the art.

[0191] The elements carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds of the present invention include their isotopes, and the elements carbon, hydrogen, oxygen, sulfur or nitrogen involved in the groups and compounds of the present invention are optionally further replaced by 1 to 5 of their corresponding isotopes, wherein the isotopes of carbon include 12 C. 13 C and 14 C, hydrogen isotopes include protium (H), deuterium (D, also called heavy hydrogen), tritium (T, also called super tritium), oxygen isotopes include 16 O. 17 O and 18 O, sulfur isotopes include 32 S. 33 S. 34 S and 36 S, isotopes of nitrogen include 14 N and 15 N, isotope of fluorine 19 F, isotopes of chlorine include 35 Cl and 37 Isotopes of Cl and bromine include 79 Br and 81 Br.

[0192] "Optionally" or "optionally" means that the subsequently described event or circumstance can but need not occur, including instances where the event or circumstance occurs or does not occur.

[0193] In the present invention, HATU is 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate. COMU is (1-cyano-2-ethoxy-2-oxyethylmethyleneoxy)dimethylaminomorpholinium carbon hexafluorophosphate. BOP is benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate.

[0194] The reaction process of the present invention is tracked by HPLC, HNMR or thin layer chromatography to determine whether the reaction is completed.

[0195] In the present invention, the internal temperature refers to the temperature of the reaction system. DETAILED DESCRIPTION

[0196] The technical solution of the present invention is described in detail below in conjunction with embodiments, but the protection scope of the present invention includes but is not limited to them.

[0197] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR shifts (δ) were measured in 10 -6The unit of (ppm) is given. NMR measurements were performed using a Bruker Avance III 400 and Bruker Avance 300 nuclear magnetic spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), deuterated acetonitrile (CD3CN) as the internal standard and tetramethylsilane (TMS) as the internal standard.

[0198] MS was measured using Agilent 6120B (ESI) and Agilent 6120B (APCI).

[0199] Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C18 100×4.6 mm) was used for HPLC analysis.

[0200] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.15mm-0.20mm, and the specification of the thin layer chromatography separation and purification product adopts a specification of 0.4mm-0.5mm.

[0201] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0202] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as Titan Technology, Anage Chemical, Shanghai Demo, Chengdu Cologne Chemical, Shaoyuan Chemical Technology, and Bailingwei Technology.

[0203] The ratio shown in the silica gel column chromatography of the present invention is a volume ratio.

[0204] The room temperature is 20℃~30℃.

[0205] Boc is tert-butyloxycarbonyl.

[0206] HOBT is 1-hydroxybenzotriazole.

[0207] EDC.HCl is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. DHOBT or DHOBt: 3-hydroxy-1,2,3-benzotriazine-4(3H)-one, CAS number is 28230-32-2.

[0208] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, CAS number 148893-10-1.

[0209] COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholinium-carbonium hexafluorophosphate, CAS No. 1075198-30-9.

[0210] BOP: benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, CAS No. 56602-33-6.

[0211] Example 1

[0212] 7-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)-2-acetyl-2,7-diazaspiro[3.5]nonane(N)

[0213] (R)-N-((R)-1-(2-acetyl-2,7-diazaspiro[3.5]nonan-7-yl)-6-amino-1-oxohexan-2-yl)-2-((R)-2-((R)-2-amino-3-phenylpropanamido)-3-phenylpropanamido)-4-methylpentanamide(N)

[0214]

[0215] Step 1: (R)-methyl 2-((R)-2-((tert-butoxycarbonyl)amino)-3-phenylpropylamino)-4-methylglutaric acid (C)

[0216] (R)-methyl 2-((R)-2-((tert-butoxycarbonyl)amino)-3-phenylpropanamido)-4-methylpentanoate(C)

[0217]

[0218] Add 0.362kg of compound (A), 0.53kg of compound (B), 0.284kg of HOBT and 12.00kg of dichloromethane to the reactor in sequence, stir and cool to 5±5℃. Keep warm to 5±5℃ and add 0.46kg of EDC.HCl. After addition, add 0.31kg of N,N-diisopropylethylamine dropwise. After addition, let it naturally heat up to 15±5℃, and take samples for central control (monitoring the content of compound (B)) after about 3 hours of reaction until the content of compound (B) is ≤0.5%, and stop the reaction. After the reaction is completed, quench the reaction with 5.00kg of 7.0% sodium bicarbonate aqueous solution. Let stand for stratification, combine the organic phases and transfer them to the reactor, and wash with 5.00kg of 1mol / L hydrochloric acid solution and 5.00kg of 7.0% sodium bicarbonate solution in sequence. Add 3.00kg of purified water to the organic phase under stirring, and let stand for separation after stirring. The organic phases were combined and concentrated under reduced pressure at 35±5°C. 0.73 kg of ethyl acetate was added to the concentrate to dissolve, 4.22 kg of n-hexane was added, stirred for crystallization, filtered, and the filter cake was rinsed with 0.66 kg of n-hexane. The filter cake was collected and weighed to obtain 0.665 kg of off-white solid of compound (C), with a yield of 83.4%.

[0219] HPLC: 99.56%.

[0220] 1 H NMR(400MHz,CD3OD)δ7.28–7.20(m,5H),4.53-4.46(m,1H),4.33-4.30(m,1H),3.68(s,3H) ,3.12–3.09(m,1H),3.07–2.80(m,1H),1.68-1.58(m,3H),1.36(s,9H),0.95–0.90(m,6H).

[0221] LCMS m / z=393.2 [M+1].

[0222] Step 2: (R)-methyl 2-((R)-2-amino-3-phenylpropionamido)-4-methylglutaric acid 2,2,2-trifluoroacetic acid (D)

[0223] (R)-methyl 2-((R)-2-amino-3-phenylpropanamido)-4-methylpentanoate 2,2,2-trifluoroacetate(D)

[0224]

[0225] Add 2.62 kg of dichloromethane and 0.662 kg of compound (C) to the reactor, stir and cool to 5±5°C. Keep warm at 5±5°C and add 1.54 kg of trifluoroacetic acid dropwise. After the addition, naturally raise the temperature to 15±5°C and react for about 4 hours, then take a sample for central control (monitor the content of compound (C)) until the content of compound (C) is ≤0.5%, and stop the reaction. Control the temperature of the reaction solution to below 35±5°C and concentrate under reduced pressure. Under stirring, transfer the concentrated residue to a reactor containing 5.30 kg of methyl tert-butyl ether. Stir and crystallize, filter, and wash the filter cake with 0.70 kg of methyl tert-butyl ether. Combine the filter cakes and vacuum dry for about 12 hours. Obtain 0.66 kg of off-white solid compound (D) with a yield of 95%.

[0226] HPLC: 99.70%.

[0227] 1 H NMR(400MHz,CD3OD)δ7.38–7.31(m,5H),4.52-4.50(m,1H),4.14-4.12(m,1H),3.71 (s,3H),3.31–3.30(m,1H),3.05–3.03(m,1H),1.65-1.61(m,3H),0.97–0.92(m,6H).

[0228] LCMS m / z=293.2 [M+1].

[0229] Step 3: (6R,9R,12R)-methyl 6,9-dibenzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxa-3-oxa-5,8,11-triazatridec-13-oic acid (F)

[0230] (6R,9R,12R)-methyl 6,9-dibenzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oate(F)

[0231]

[0232] Add 0.66kg of compound (D), 0.43kg of compound (E), 0.23kg of HOBT and 8.75kg of dichloromethane to the reactor in sequence. After addition, stir and cool to 5±5℃. Keep the internal temperature at 5±5℃ and add 0.25kg of N,N-diisopropylethylamine dropwise. Keep the temperature at 5±5℃ and add 0.37kg of EDC.HCl. After addition, raise the temperature to 15±5℃. After about 3 hours of reaction, take a sample for central control (monitor the content of compound (D)) until the content of compound (D) is ≤0.3%, and stop the reaction. After the reaction is completed, quench the reaction with 5.00kg of 7.0% sodium bicarbonate aqueous solution. Let stand for stratification, combine the organic phases and transfer them to the reactor, and wash with 5.00kg of 1mol / L hydrochloric acid aqueous solution and 5.00kg of 7.0% sodium bicarbonate aqueous solution in sequence. Add 2.50kg of purified water to the organic phase, stir and let stand for separation. The organic phases were combined and concentrated under reduced pressure at 35±5°C. 1.62 kg of ethyl acetate was added to the concentrate, and 5.94 kg of n-hexane was added to stir and crystallize. Filter, wash the filter cake with 0.66 kg of n-hexane, and collect the filter cake. The combined filter cakes were dried under reduced pressure to obtain 0.780 kg of off-white solid of compound (F), with a yield of 89%.

[0233] HPLC: 99.28%.

[0234] 1 H NMR (400MHz, CD3OD) δ7.28–7.16(m,10H),4.67-4.66(m,1H),4.46-4.43(m,1H),4.23-4.22(m,1H),3.68(s,3H ),3.12–3.09(m,1H),2.99–2.98(m,1H),2.97–2.95(m,1H),1.66-1.58(m,3H),1.34(s,9H),0.94–0.89(m,6H).

[0235] LCMS m / z=540.2 [M+1].

[0236] Step 4: (6R,9R,12R)-6,9-dibenzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxa-3-oxa-5,8,11-triazatridecane-13-oic acid (G)

[0237] (6R,9R,12R)-6,9-dibenzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oic acid(G)

[0238]

[0239] Add 6.66kg of tetrahydrofuran and 0.76kg of compound (F) to the reactor, stir and cool to 0±5℃. Keep the temperature at 0±5℃, and add 71.00g of lithium hydroxide (monohydrate) and 2.50kg of purified water. The addition takes about 1 hour. After the addition, continue to keep the temperature at 0±5℃, and take samples for central control (monitor the content of compound (F)) after about 3 hours of reaction until the content of compound (F) is ≤0.2%, and stop the reaction. Keep the temperature at 0±5℃, add 6.31kg of ethyl acetate, and adjust the pH of the reaction solution to about 3-4 with 3.0mol / L hydrochloric acid aqueous solution. Separate the liquids, and wash the organic phase with 10% sodium chloride solution (3.00kg×1). Combine the organic phases and dry with anhydrous sodium sulfate. Filter, wash the filter cake with 0.90kg of ethyl acetate, and combine the filtrate. Control the temperature of the filtrate below 40±5℃ and concentrate under reduced pressure. Refining: Add 3.33 kg of ethyl acetate to the concentrate, add 4.88 kg of n-hexane and stir to crystallize. Filter, wash the filter cake with 0.66 kg of n-hexane, and collect the filter cake. Take samples to detect related substances and isomers. If the isomer is greater than 0.3%, re-refine until the isomer is ≤ 0.3%. Combine the filter cakes and vacuum dry. Obtain 0.716 kg of off-white solid (G) of the compound, with a yield of 95%.

[0240] HPLC: 99.50%.

[0241] 1 H NMR (400MHz, CD3OD) δ7.25–7.16(m,10H),4.68-4.66(m,1H),4.25-4.23(m,1H),4.12-4.10(m,1H),3.1 5-3.12(m1H),3.02–3.01(m,1H),2.98–2.93(m,1H),1.64-1.61(m,3H),1.33(s,9H),0.94–0.88(m,6H).

[0242] LCMS m / z=526.4 [M+1].

[0243] Step 5: (6R, 9R, 12R, 15R)-methyl 6,9-dibenzyl-15-(4-((tert-butyloxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-acid salt (I)

[0244] (6R,9R,12R,15R)-methyl 6,9-dibenzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-oate(I)

[0245]

[0246] Add 0.508kg of compound (G), 0.30kg of compound (H), 0.132kg of HOBT and 9.45kg of dichloromethane to the reactor. Start stirring and cool to 5±5℃. Keep the internal temperature at 5±5℃ and add 0.187kg of N,N-diisopropylethylamine dropwise. After addition, add 0.198kg of copper chloride (dihydrate). Keep the temperature at 5±5℃ and add 0.223kg of EDC.HCl. After addition, continue to keep the temperature and stir for about 10 minutes. Raise the temperature to 15±5℃, react for about 3 hours and take samples for central control (monitor the content of compound (G)) until HPLC detects compound (G) ≤0.3%, then stop the reaction. After the reaction is completed, quench the reaction with dilute ammonia solution (3.50kg×2). Let stand and separate the organic phase. Add 2.50kg of 1mol / L hydrochloric acid aqueous solution to the organic phase under stirring, stir and let stand to separate the liquid. The organic phase was washed with 2.50 kg of 7.0% sodium bicarbonate aqueous solution. The layers were separated by standing, and the combined organic phases were transferred to a reactor. Samples of the organic phase were tested for compound (G). If compound (G)>0.2%, it was washed once with 7.0% sodium bicarbonate (2.0 kg×1). 3.00 kg of purified water was added to the organic phase under stirring, and the mixture was allowed to stand for separation after stirring. The combined organic phases were concentrated under reduced pressure at 30±5°C. Refining: The concentrated solution was heated to 30±5°C with 4.65 kg of dichloromethane to dissolve, and then transferred to a reactor. Cooled to 5±5°C, 5.80 kg of n-hexane was added dropwise, and stirred for crystallization. Filtered, the filter cake was rinsed with 0.66 kg of n-hexane, and the filter cake was collected. During sampling, the compound (G) was controlled to ≤0.1%; otherwise, the compound (I) refining process was continued and refined again until it met the quality standards. The combined filter cakes were dried under controlled temperature and reduced pressure. 0.686 kg of off-white solid of compound (I) was obtained, with a yield of 94%.

[0247] HPLC: 99.54%.

[0248] 1H NMR (400MHz, CD3OD) δ7.25–7.18(m,10H),4.68(m,1H),4.42(m,1H),4.36(m,1H),4.23(m,1H),3.70 (s,3H),3.12–3.01(m,5H),2.73–2.71(m,1H),1.68-1.58(m,5H),1.36(s,22H),0.93–0.92(m,6H).

[0249] LCMS m / z=768.4 [M+1].

[0250] The present invention screened and optimized the conditions for adding copper chloride (dihydrate) in the step of preparing compound (I) from compound (G), as shown in Table 1.

[0251] Add 1.58g of compound (G), 0.89g of compound (H), 0.49g of HOBT and 30ml of dichloromethane to the reactor. Start stirring and cool to 5±5℃. Maintain the internal temperature at 5±5℃ and drop 0.50g of N,N-diisopropylethylamine. After addition, add 0.61g of copper chloride (dihydrate). Keep the temperature at 5±5℃ and add 0.69g of EDC.HCl. After addition, continue to keep the temperature and stir for about 10 minutes. Raise the temperature to 15±5℃, react for about 2 hours, take samples and start central control, and record the changes of related substances of compound (I) within 2, 4, and 6 hours respectively.

[0252] Add 2.63g of compound (G), 1.43g of compound (H), 0.68g of HOBT and 30ml of dichloromethane to the reactor. Start stirring and cool to 5±5℃. Maintain the internal temperature at 5±5℃ and drop 0.78g of N,N-diisopropylethylamine. After addition, add 1.00g of EDC.HCl. After addition, continue to stir for about 10 minutes. Raise the temperature to 15±5℃, react for about 2 hours, take samples and start central control, and record the changes of related substances of compound (I) within 2, 4, and 6 hours respectively.

[0253] Table 1: Screening of the effect of adding copper chloride (dihydrate) to compound I in the synthesis of compound G on isomers

[0254] NO. Copper chloride dosage Reaction time Reaction temperature Compound I (%) Isomers (%) 1 none 2h 5±5℃-15±5℃ 96.81 0.24 2 none 4h 5±5℃-15±5℃ 97.39 0.18 3 none 6h 5±5℃-15±5℃ 97.46 0.18 4 1.2eq 2h 5±5℃-15±5℃ 97.09 0.05 5 1.2eq 4h 5±5℃-15±5℃ 97.12 0.04 6 1.2eq 6h 5±5℃-15±5℃ 97.30 0.04

[0255] The data in the above table show that the addition of 1.0-1.2 equivalents of copper chloride (dihydrate) in the reaction is beneficial to maintaining the configuration of the chiral compound, so that the diastereoisomers of the generated intermediates are effectively controlled. As the peptide chain increases, the difficulty of purifying and removing isomers increases.

[0256] Step 6: (6R,9R,12R,15R)-6,9-dibenzyl-15-(4-((tert-butyloxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-oic acid (J)

[0257] (6R,9R,12R,15R)-6,9-dibenzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobuty l-2,2-dimethyl-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-oic acid(J)

[0258]

[0259] Add 5.80 kg of tetrahydrofuran and 0.68 kg of compound (I) to the reactor. Start stirring and cool to 0±5°C. Add 45.50 g of lithium hydroxide (monohydrate) and 2.00 kg of purified water dropwise, and keep warm at 0±5°C during the addition. After the addition is completed, continue to keep warm at 0±5°C. After about 3 hours of reaction, take samples for central control (monitor the content of compound (I)) until HPLC detects that the content of compound (I) is ≤0.2%, and stop the reaction. Keep warm at 0±5°C, add 6.30 kg of ethyl acetate and drop 1.20 kg of 3.0 mol / L hydrochloric acid aqueous solution, and adjust the pH of the reaction solution to 3-4. Separate the liquids, and extract the aqueous phase once with ethyl acetate (2.70 kg×1). Combine the organic phases and wash with 10% sodium chloride aqueous solution (3.00 kg×1). Combine the organic phases and dry the organic phases with anhydrous sodium sulfate. Filter, wash the filter cake with 1.50 kg of ethyl acetate, and combine the filtrate. Concentrate the filtrate under reduced pressure. After adding 6.10 kg of ethyl acetate to a 20 L concentration bottle and stirring to dissolve, 4.86 kg of n-hexane was added and stirred to crystallize, and the mixture was filtered to collect the filter cake. The filter cakes were combined and dried under reduced pressure to obtain 0.636 kg of off-white solid of compound (J), with a yield of 92%.

[0260] HPLC: 99.26%.

[0261] 1H NMR (400MHz, CD3OD) δ7.25–7.17(m,10H),4.64(m,1H),4.44(m,1H),4.35(m,1H),4.24(m,1H),3.70 (s,3H),3.10–3.01(m,5H),2.99–2.98(m,1H),1.63-1.45(m,5H),1.36(s,22H),0.95–0.91(m,6H).

[0262] LCMS m / z=754.2 [M+1].

[0263] Step 7: (L) tert-butyl ((10R, 13R, 16R, 19R)-10-(2-acetyl-2,7-diazaspiro[3.5]nonane-7-carbonyl)-16-benzyl-13-isobutyl-2,2-dimethyl-4,12,15,18-tetraoxo-20-phenyl-3-oxo-5,11,14,17-tetraaza-19-yl) carbamate compound (L)

[0264] (L)tert-butyl((10R,13R,16R,19R)-10-(2-acetyl-2,7-diazaspiro[3.5]nonane-7-carbonyl)-16-benzyl-13-i sobutyl-2,2-dimethyl-4,12,15,18-tetraoxo-20-phenyl-3-oxa-5,11,14,17-tetraazaicosan-19-yl)carbamate

[0265]

[0266] Add 0.634kg of compound (J), 0.191kg of compound (K), 0.121kg of HOBT and 15.00kg of dichloromethane to the reactor. Start stirring and cool to 5±5℃. Maintain the internal temperature at 5±5℃ and add 0.128kg of N,N-diisopropylethylamine dropwise. After addition, add 0.171kg of copper chloride (dihydrate). Maintain the internal temperature at 5±5℃ and add 0.19kg of EDC.HCl. After addition, continue stirring for about 10 minutes. Raise the temperature to 15±5℃, and take a sample for central control (monitoring the content of compound (J)) after about 3 hours of reaction. Stop the reaction when the results of two sampling tests of the central control reaction liquid do not change by more than 1.0%. After the reaction is completed, quench the reaction with dilute ammonia solution (2.80kg×2). Let stand and separate the organic phase. Add 2.50kg of 1mol / L hydrochloric acid aqueous solution to the organic phase under stirring, let stand for separation, and transfer the organic phase to a reactor. Wash the organic phase with 2.50kg of 7.0% sodium bicarbonate aqueous solution. Let stand for stratification, combine the organic phases and transfer them to a reactor. Add 3.00kg of purified water to the organic phase under stirring, let stand for separation after stirring. Add 80.00g of activated carbon to the organic phase for decolorization. Pad 0.5kg of diatomaceous earth, filter, wash the filter cake with 1.50kg of dichloromethane, and combine the filtrate. Combine the organic phases and concentrate under reduced pressure at 30±5℃. Add 1.23kg of dichloromethane to a 20L concentration bottle, add 4.15kg of methyl tert-butyl ether under stirring, and stir and slurry. Add 3.20kg of n-hexane and stir and slurry. Filter, wash the filter cake with 1.20kg of n-hexane, and collect the filter cake. Combine the filter cakes and dry under reduced pressure. 0.642 kg of compound (L) was obtained as a light yellow solid, with a yield of 92%.

[0267] HPLC: 95.13%.

[0268] 1 H NMR (400MHz, CD3OD) δ7.28–7.16(m,10H),4.62(m,1H),4.41(m,1H),4.21(m,1H),3.60-3.48(m,2H),3.45-3.43(m,4H),3.31 –3.30(m,2H),3.10–3.01(m,5H),2.99–2.98(m,1H),,1.83(m,4H),1.74(m,3H),1.7(m,4H),1.36(s,22H),0.93–0.90(m,6H).

[0269] LCMS m / z=904.5 [M+1].

[0270] Step 8: (M) 7-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)-2-acetyl-2,7-diazaspiro[3.5]nonane ditrifluoroacetate (M)

[0271] (M)(R)-N-((R)-1-(2-acetyl-2,7-diazaspiro[3.5]nonan-7-yl)-6-amino-1-oxohexan-2-yl)-2-((R)-2-((R)-2-amino-3-phenylpropanamido)-3-phenylpropanamido)-4-methylpentanamide Ditrifluoroacetic acid(M)

[0272]

[0273] Add 9.28 kg of dichloromethane to the reactor, and then add 0.70 kg of compound (L). Keep the internal temperature at 15°C ± 5°C, add 5.37 kg of trifluoroacetic acid to the reactor, and complete the addition in about 1 hour. Raise the temperature to 20°C ± 5°C, and take a sample for central control (monitoring the content of compound (L)) after about 4 hours of reaction until HPLC detects compound (L) ≤ 0.5%, then stop the reaction. Concentrate the reaction solution under reduced pressure at 30 ± 5°C, dissolve the concentrate in 5.54 kg of anhydrous methanol, and then add 15.54 kg of methyl tert-butyl ether and stir to crystallize. Filter under nitrogen protection, wash the filter cake with 0.74 kg of methyl tert-butyl ether, and obtain 0.61 kg of compound (M), with a yield of 85%.

[0274] HPLC: 99.05%.

[0275] The present invention screened and optimized the conditions for adding copper chloride (dihydrate) in the steps of preparing compound (L) from compound (J) and then preparing compound (M) from compound (L), as shown in Table 2.

[0276] Compound (J) Synthesis of Compound (L): Add 2.26g of Compound (J), 0.612g of Compound (K), 0.49g of HOBT and 30ml of dichloromethane to the reactor. Start stirring and cool to 5±5℃. Maintain the internal temperature at 5±5℃ and drop 0.50g of N,N-diisopropylethylamine. After addition, add 0.61g of copper chloride (dihydrate). Maintain the internal temperature at 5±5℃ and add 0.69g of EDC.HCl. After addition, continue stirring for about 10 minutes. Raise the temperature to 15±5℃, react for about 3 hours and take a sample for central control. Stop the reaction when the change in the results of two sampling tests of the central control reaction liquid does not exceed 1.0%. After the reaction is completed, quench the reaction with dilute ammonia solution (40ml×2). Let stand and separate the organic phase. Add 25ml of 1mol / L hydrochloric acid aqueous solution to the organic phase under stirring, let stand and separate the liquid, and transfer the organic phase to the reactor. The organic phase was washed with 25 ml of 7.0% sodium bicarbonate aqueous solution. The mixture was allowed to stand for stratification, and the combined organic phases were transferred to a reactor. 30 ml of purified water was added to the organic phase under stirring, and the mixture was allowed to stand for separation after stirring. 5.00 g of diatomaceous earth was applied, and the filter cake was washed with 20 ml of dichloromethane, and the filtrate was combined. The combined organic phases were concentrated under reduced pressure at 30±5°C. 10 ml of dichloromethane was added to the concentration bottle, and 40 ml of methyl tert-butyl ether was added under stirring, and the mixture was stirred and slurried. Then 60 ml of n-hexane was added and the mixture was stirred and slurried. Filter, wash the filter cake with 20 ml of n-hexane, and collect the filter cakes. The combined filter cakes were dried under reduced pressure. 3.60 g of compound (L) was obtained, with a yield of 95%.

[0277] Compound (L) Synthesis of Compound (M): Add 10 ml of dichloromethane to the reactor, and then add 1.50 g of compound (L). Maintain the internal temperature at 15°C ± 5°C, and drop 5.0 ml of trifluoroacetic acid into the reactor. The addition is completed in about 1 hour. Raise the temperature to 20°C ± 5°C, react for about 4 hours, take a sample for central control, and take samples to detect changes in related substances. The data statistics are shown in the table below.

[0278] Table 2: Screening of the effect of adding copper chloride (dihydrate) to compound M in the synthesis of compound I on isomers

[0279] NO. Copper chloride dosage Reaction time Reaction temperature Compound M (%) Isomers (%) 1 none 6h 5±5℃-15±5℃ 91.68 6.04 2 1.2eq 6h 5±5℃-15±5℃ 97.26 0.23 3 0.6eq 6h 5±5℃-15±5℃ 98.88 0.15 4 0.3eq 6h 5±5℃-15±5℃ 98.42 0.21

[0280] The data in the above table show that the addition of 0.3-1.2 equivalents of copper chloride (dihydrate) in the reaction is beneficial to maintaining the configuration of the chiral compound, so that the diastereoisomers of the generated intermediates are effectively controlled. As the peptide chain increases, the difficulty of purifying and removing the isomers increases. The more isomers there are, the lower the refining and purification yield. Considering all factors, 0.3-1.2 equivalents of copper chloride (dihydrate) are added to maintain the isomeric configuration when synthesizing pentapeptides.

[0281] Step 9: 7-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)-2-acetyl-2,7-diazaspiro[3.5]nonane (N)

[0282] (R)-N-((R)-1-(2-acetyl-2,7-diazaspiro[3.5]nonan-7-yl)-6-amino-1-oxohexan-2-yl)-2-((R)-2-((R)-2-amino-3-phenylpropanamido)-3-phenylpropanamido)-4-methylpentanamide(N)

[0283]

[0284] Start stirring, pump 5.20kg of purified water into the reactor, and control the temperature at 10±5℃. Add 0.52kg of the wet product (undried) of the compound (M) in the previous step. Extract the aqueous phase with dichloromethane (2.50kg×2). Keep the temperature at 10±5℃, add 0.60kg of ammonia solution to the reactor, adjust the pH of the aqueous phase to 10-11, and measure the pH value of the reaction solution with a pH meter. If the pH value decreases, add ammonia water in time to maintain the pH value of the reaction solution at 9-10. Let stand and separate, and extract the aqueous phase with dichloromethane (3.00kg×3 times). Combine the organic phases and wash the organic phases once with saturated brine. Dry the organic phase with anhydrous sodium sulfate, filter it with diatomaceous earth, and wash the filter cake with 2.50kg of dichloromethane. Combine the organic phases and concentrate under reduced pressure at 30±5℃. Weigh 200-300 mesh silica gel, which is about 2.5-3.0 times the mass of the concentrate, and use dichloromethane wet method to pack the column. Pour the concentrate into the silica gel column, and use dichloromethane about 6.12kg, dichloromethane and anhydrous methanol (volume ratio 50:1-5:1) mixed solution gradient elution in turn (first use dichloromethane and anhydrous methanol (volume ratio 50:1 about 6.02kg) to elute, then use dichloromethane and anhydrous methanol (volume ratio 20:1 about 3.06kg) to elute, and collect them separately. This is the front component, which is mainly impurities. Then use dichloromethane and anhydrous methanol (volume ratio 20:1 about 3.06 kg), the eluent was changed to dichloromethane and anhydrous methanol (volume ratio of 10:1, about 15.30kg), dichloromethane and anhydrous methanol (volume ratio of 8:1, about 9.18kg), dichloromethane and anhydrous methanol (volume ratio of 7:1, about 9.18kg), dichloromethane and anhydrous methanol (volume ratio of 6:1, about 9.18kg), dichloromethane and anhydrous methanol (volume ratio of 5:1, about 6.15kg). Use a 5L beaker to collect the washing liquid in turn and send it for inspection of related substances. Combine the washing liquids that meet the collection standards, control the temperature below 30±5℃ and concentrate under reduced pressure until no obvious fraction flows out). Collect the eluent and control the temperature below 30±5℃ and concentrate under reduced pressure. The temperature was controlled at 20±5°C, and the concentrate was stirred and dissolved with a mixed solution of methanol and purified water (300ml:2400ml), filtered through a glass sand funnel, and the filter cake was washed with a mixed solution of purified water and methanol (volume ratio = 10:1). The filtrates were combined and transferred to a freeze dryer for freeze drying to obtain 0.262kg of off-white powder compound (N), with a yield of 26.40%.

[0285] HPLC: 99.46%.

[0286] 1H NMR(400MHz,CD3OD)δ7.27-7.20(m,10H),4.86-4.84(m,1H),4.69-4.66(m, 1H),4.45-4.41(m,1H),3.96-3.92(m,2H),3.73-3.63(m,4H),3.54–3.40(m, 3H),3.15–3.11(m,1H),2.96–2.88(m,2H),2.64-2.57(m,3H),1.87(s,3H), 1.83-1.74(m,5H),1.63-1.57(m,4H),1.52-1.36(m,4H),0.95-0.90(m,6H).

[0287] LCMS m / z=352.8,704.5[M+1].

[0288] Example 2

[0289] Step 1: (L) tert-butyl ((10R, 13R, 16R, 19R)-10-(2-acetyl-2,7-diazaspiro[3.5]nonane-7-carbonyl)-16-benzyl-13-isobutyl-2,2-dimethyl-4,12,15,18-tetraoxo-20-phenyl-3-oxo-5,11,14,17-tetraaza-19-yl) carbamate compound (L)

[0290] (L)tert-butyl((10R,13R,16R,19R)-10-(2-acetyl-2,7-diazaspiro[3.5]nonane-7-carbonyl)-16-benzyl-13-i sobutyl-2,2-dimethyl-4,12,15,18-tetraoxo-20-phenyl-3-oxa-5,11,14,17-tetraazaicosan-19-yl)carbamate

[0291]

[0292] Add 0.634g of compound (J), 0.191g of compound (K), 0.165g of DHOBt and 25mL of dichloromethane to the reaction bottle. Turn on magnetic stirring and cool to 0-5℃. Keep the internal temperature at 0-5℃ and add 0.38mL of 2,2,6,6-tetramethylpiperidine dropwise. Keep the internal temperature at 0-5℃ and add 2.0mL of DMF solution of 0.433g of COMU. After the addition, keep the internal temperature at 0-5℃, continue the reaction for 2 hours, take a sample for control (monitor the content of compound (J)), and stop the reaction when the raw material in the reaction liquid is less than 0.5%. After the reaction is completed, add 25mL of 7% sodium bicarbonate solution to the system to quench the reaction. Let stand, separate the liquids, and collect the organic phase. Wash the organic phase with 25mL of 1N hydrochloric acid aqueous solution, let stand, separate the liquids, and collect the organic phase. The organic phase was then washed with 20 mL of saturated brine, allowed to stand, separated, the organic phase was collected, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, 3.0 mL of dichloromethane was added to the concentration bottle, and stirred to dissolve. 12.0 mL of methyl tert-butyl ether was added thereto under stirring, stirred and slurried, and then 12.0 mL of n-hexane was added and stirred and slurried. Filtered by suction, the filter cake was washed with 3.0 mL of n-hexane, the filter cake was collected, and dried under reduced pressure to obtain 0.67 g of compound (L) as a white solid, with a yield of 88%.

[0293] HPLC: 98.85%.

[0294] Step 2: (M) 7-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)-2-acetyl-2,7-diazaspiro[3.5]nonane ditrifluoroacetate (M)

[0295] (M)(R)-N-((R)-1-(2-acetyl-2,7-diazaspiro[3.5]nonan-7-yl)-6-amino-1-oxohexan-2-yl)-2-((R)-2-((R)-2-amino-3-phenylpropanamido)-3-phenylpropanamido)-4-methylpentanamide Ditrifluoroacetic acid

[0296]

[0297] Add 0.20 g of compound (L) to the reaction bottle, then add 10 mL of dichloromethane and start stirring. At room temperature, add 1.0 mL of trifluoroacetic acid to the reaction bottle. After the dropwise addition, sample the reaction for 4 hours to control (monitor the content of compound (L)), and stop the reaction until HPLC detects compound (L) ≤ 0.5%. Remove the solvent under reduced pressure, add 3.0 mL of methanol to the concentration bottle, stir to dissolve, add 9 mL of methyl tert-butyl ether dropwise, and stir to crystallize. Filter by suction, collect the filter cake, wash the filter cake with 1.0 mL of methyl tert-butyl ether, and dry in vacuo to obtain 0.18 g of compound (M), with a yield of 87%.

[0298] HPLC: 99.02%.

[0299] Example 3

[0300] The first step: add 0.36kg of compound (N) obtained in step nine of Example 1 and 2.84kg of anhydrous ethanol to the reactor, and control the temperature to 15±5°C. Turn on the stirring and stir for about 1.0 hour to completely dissolve the solid. Keep warm at 15±5°C and add 0.13kg of trifluoroacetic acid. Continue stirring for about 0.5 hour after the addition. Control the temperature to 20±5°C and add 8.0kg of methyl tert-butyl ether dropwise for about 1.5 hours. Stir and crystallize for about 0.5 hour. Filter under nitrogen protection and wash the filter cake with 0.74kg of methyl tert-butyl ether. Take a sample and send it for testing. If the relevant substances of the crude trifluoroacetate of compound (N) do not meet the quality standards, continue the recrystallization process (dissolve in anhydrous ethanol and add methyl tert-butyl ether to precipitate crystals) and recrystallize again (the mass is recorded as 0.40kg and the yield is 85% of the theoretical dry weight) and send it for inspection. If it does not meet the standards, continue the following operations. Add 0.40 kg of the crude trifluoroacetate of compound (N) obtained above and 3.16 kg of anhydrous ethanol to the reactor, control the temperature to 15 ± 5 ° C, and stir for about 0.5 hours. Cool down to 15 ± 5 ° C, add 8.88 kg of methyl tert-butyl ether dropwise, and add dropwise for about 2 hours. Stir and crystallize for about 0.5 hours. Filter under nitrogen protection, and wash the filter cake with 0.74 kg of methyl tert-butyl ether. Take a sample for testing. If it still does not meet the concentrated quality standard, continue to repeat the refining and purification operation to obtain 3.2 kg of compound (N) trifluoroacetic acid wet product.

[0301] Step 2: Start stirring, pump 3.2 kg of purified water into the reactor, and control the temperature at 10±5°C. Add 0.32 kg of the trifluoroacetic acid concentrate of the compound (N) in the previous step. Stir for about 0.5 hours to completely dissolve the solid. Extract the aqueous phase with dichloromethane (0.96 kg × 2). Keep the temperature at 10±5°C, drop 0.16 kg of ammonia solution into the reactor, adjust the pH of the aqueous phase to 9-10 and stir for about 0.5 hours. Measure the pH value of the reaction solution with a pH meter every 20 minutes. If the pH value decreases, add ammonia water in time to maintain the pH value of the reaction solution at 9-10. Let it stand for stratification, and extract the aqueous phase with dichloromethane (3.2 kg × 3). Combine the organic phases and wash the organic phase once with 1.00 kg of salt water (preparation method: 0.1 kg sodium chloride + 0.90 kg purified water). Dry 0.50 kg of anhydrous sodium sulfate in the organic phase for about 0.5 hours. The filter cake was washed with 1.50 kg of dichloromethane. The washing liquid was combined, the temperature was controlled at below 30 ± 5 ° C, and the concentrated liquid was stirred with about 1.50 kg of methanol and purified water (volume ratio = 1: 8-1: 5) for about 0.5 hours to completely dissolve, filtered through a glass sand funnel, and the filter cake was washed with purified water and methanol (volume ratio = 10: 1). The filtrate was combined and transferred to a freeze dryer for freeze drying. (Divided into 4 2L single-mouth bottles, each bottle is about 400 mL) The freeze dryer parameters are set, and the sampling is controlled to control the moisture content <2%. (The temperature and vacuum degree are recorded about every 4 hours during the freeze drying process; the moisture content is tested for about 120 hours). The samples are sent for testing. If the moisture does not meet the quality standards, continue to freeze dry until it meets the quality standards of fine products. The finished compound (N) (0.16 kg, yield 26%) HPLC: 99.7%.

Claims

1. A method for preparing a compound of formula (L), comprising using a compound of formula (J) and a compound of formula (K) as raw materials, preparing a compound of formula (L) in the presence of a condensing agent and cupric chloride The condensation agents used in the reaction are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; The solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; P is tert-butyloxycarbonyl.

2. The preparation method according to claim 1, wherein The solvent used in the reaction is selected from any one of N,N-dimethylformamide and dichloromethane or any mixture of several thereof; The reaction temperature is selected from -5 to 30°C.

3. The preparation method according to claim 1, wherein The method also includes the preparation of the compound of formula (J), comprising: using the compound of formula (I) as a raw material to prepare the compound of formula (J), The base used in the reaction is selected from KOH, NaOH, lithium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, sodium methoxide or sodium ethoxide; The solvent used in the reaction is selected from any one or a mixture of any of ethanol, methanol, tetrahydrofuran, dimethyl sulfide, ethyl acetate, acetonitrile or water; P is tert-butyloxycarbonyl.

4. The preparation method according to claim 3, wherein The method also includes the preparation of the compound of formula (I), comprising: in the presence of a condensing agent and cupric chloride, using the compound of formula (G) and the compound of formula (H) as raw materials to prepare the compound of formula (I), The condensation agents used in the reaction are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; The solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; P is tert-butyloxycarbonyl.

5. The preparation method according to claim 4, wherein The solvent used in the reaction is selected from any one of N,N-dimethylformamide and dichloromethane or any mixture of several thereof; The reaction temperature is selected from -5 to 30°C.

6. The preparation method according to claim 4, wherein The method also includes the preparation of the compound of formula (G), comprising: using the compound of formula (F) as a raw material to prepare the compound of formula (G), The base used in the reaction is selected from KOH, NaOH, lithium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, sodium methoxide or sodium ethoxide; The solvent used in the reaction is selected from any one or a mixture of any of ethanol, methanol, tetrahydrofuran, dimethyl sulfide, ethyl acetate, acetonitrile or water; P is tert-butyloxycarbonyl.

7. A method for preparing a compound of formula (M), comprising taking a compound of formula (L) as a raw material, reacting in the presence of an acidic reagent and dichloromethane to prepare a compound of formula (M), further comprising taking a compound of formula (J) and a compound of formula (K) as raw materials, reacting in the presence of a condensing agent and cupric chloride to prepare a compound of formula (L), HA is selected from trifluoroacetic acid; n is selected from 1, 2 or 3; The acidic reagent is selected from trifluoroacetic acid; The condensation agents used in the reactions of formula (J) and formula (K) are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; The solvent used in the reaction of the compound of formula (J) and the compound of formula (K) is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; P is tert-butyloxycarbonyl.

8. A method for preparing a compound represented by formula (N), wherein the method comprises the following steps: In the first step, the compound of formula (A) and the compound of formula (B) undergo condensation reaction to generate a compound of formula (C); In the second step, the amino protecting group on the nitrogen atom of the compound of formula (C) is removed to generate the compound of formula (D); In the third step, the compound of formula (D) undergoes a condensation reaction with the compound of formula (E) to generate a compound of formula (F); The fourth step is to hydrolyze the compound of formula (F) to obtain the compound of formula (G); In the fifth step, the compound of formula (G) undergoes a condensation reaction with the compound of formula (H) to generate a compound of formula (I); Step 6, hydrolyzing the compound of formula (I) to obtain the compound of formula (J); Step 7, reacting the compound of formula (J) with the compound of formula (K) to generate the compound of formula (L); Step 8, removing the amino protecting group on the nitrogen atom of the compound of formula (L) to generate a compound of formula (M); The ninth step, the compound of formula (M) is freed by alkali to generate compound (N); in, HA is selected from trifluoroacetic acid; n is selected from 1, 2 or 3; P is tert-butyloxycarbonyl; The first step is to react the compound of formula (A) with the compound of formula (B) in the presence of a condensing agent and a base to generate a compound of formula (C); the condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine; In the second step, the compound of formula (C) is prepared in the presence of dichloromethane and trifluoroacetic acid to obtain the compound of formula (D); The third step is to react the compound of formula (D) with the compound of formula (E) to generate the compound of formula (F) in the presence of a condensing agent and a base; the condensing agent used in the reaction is selected from any one or a mixture of any of HATU, COMU, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2,2,6,6-tetramethylpiperidine, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one or BOP; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine; Step 4, preparing a compound of formula (G) from the compound of formula (F) in the presence of tetrahydrofuran and lithium hydroxide; The fifth step is to react the compound of formula (G) with the compound of formula (H) in the presence of a condensing agent, copper chloride and a base to generate a compound of formula (I); the condensing agent used in the reaction is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine; Step 6, preparing a compound of formula (J) from the compound of formula (I) in the presence of tetrahydrofuran and lithium hydroxide; Step 7: In the presence of a condensing agent, copper chloride and a base, the compound of formula (J) reacts with the compound of formula (K) to generate a compound of formula (L); the condensing agent used in the reaction is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; the solvent used in the reaction is selected from any one or a mixture of any of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, tetrahydrofuran or acetonitrile; the base used in the reaction is triethylamine or N,N-diisopropylethylamine; Step 8, preparing a compound of formula (M) from the compound of formula (L) in the presence of dichloromethane and trifluoroacetic acid; The ninth step is to dissolve the compound of formula (M) in water, add dichloromethane solution for extraction, separate the liquids, and collect the aqueous phase; drop ammonia water into the aqueous phase, adjust the pH of the aqueous phase to 10-11, and maintain the pH value of the reaction solution at 9-10 to obtain compound (N).

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