Synthesis method of 3-piperidine-phenyl urea derivative

Abstract

The invention provides a synthesis method of a 3-piperidine-phenyl urea derivative. Specifically, the invention provides a preparation method of a compound as shown in a formula I. The preparation method comprises the following steps.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical synthesis, and particularly relates to a synthesis method of 3-piperidine-phenyl urea derivative. BACKGROUND

[0002] Glioma is a malignant tumor originating from glial cells, usually occurring in the central nervous system, especially in the brain and spinal cord. This tumor usually grows rapidly, posing a serious threat to patient health. In the fields of molecular biology and drug research and development, the study of glioma is crucial. Researchers explore potential molecular mechanisms and signaling pathways by deeply understanding the biological characteristics of glioma cells, seeking new therapeutic targets and drugs. Among them, the Wnt / β-catenin signaling pathway is of great concern due to its key role in cell proliferation, differentiation, and stem cell self-renewal. This pathway plays an important role in various tumors, including glioma, and thus becomes one of the focuses of research.

[0003] In summary, there is an urgent need in the art to develop a synthesis method of 3-piperidine-phenyl urea derivative. SUMMARY

[0004] The purpose of the present application is to provide a synthesis method of 3-piperidine-phenyl urea derivative.

[0005] In a first aspect of the present application, a method for preparing a compound of formula I (i.e. compound C37-28R) is provided,

[0006]

[0007] The method comprises the following steps:

[0008]

[0009] (a) reacting 3-bromophenol with tert-butyl 2-bromo-2-methyl-propionate in the presence of potassium carbonate to obtain compound 1;

[0010] (b) reacting compound 1 with (R)-tert-butyl piperidine-3-carbamate in the presence of cesium carbonate, Pd2(dba)3 and Ruphos to obtain compound 2;

[0011] (c) deprotecting compound 2 in the presence of trifluoroacetic acid (TFA) to obtain compound 3;

[0012] (d) reacting compound 3 with compound 4 in the presence of DIPEA and CDI in dichloromethane to obtain compound 5; wherein,

[0013] the molar ratio of compound 3 to compound 4 is 1 : 1.0 ~ 1.5, preferably 1 : 1.0 ~ 1.1 ;

[0014] the molar ratio of compound 3, CDI and DIPEA is 1 : 1.0 ~ 1.5 : 3 ~ 6, preferably 1 : 1.0 ~ 1.1 : 4 ~ 4.1 ;

[0015] the reaction temperature of the reaction is 10 ~ 30 °C, preferably 15 ~ 25 °C; and

[0016] the reaction time of the reaction is 5 ~ 24 hours, preferably 10 ~ 15 hours;

[0017] (e) reacting compound 5 with compound 6 in the presence of potassium acetate and Pd(dppf)Cl2in a mixed solvent of an organic solvent and water to obtain compound 7;

[0018] (f) deprotecting compound 7 in the presence of trifluoroacetic acid (TFA) to obtain compound 8;

[0019] (g) reacting compound 8 with glycine methyl ester hydrochloride in the presence of DIPEA and HATU to obtain compound 9;

[0020] (h) hydrolyzing compound 9 in the presence of lithium hydroxide in a mixed solvent of an organic solvent and water to obtain compound 10; and

[0021] (i) reacting compound 10 with (S)-2-Boc-aminomethyl-pyrrolidine in the presence of DIPEA and HATU to obtain the compound of formula I.

[0022] In another preferred example, in step (a), the molar ratio of 3-bromophenol to tert-butyl 2-bromo-2-methyl-propionate is 1 : 2.5 ~ 3.5; preferably 1 : 2.9 ~ 3.

[0023] In another preferred example, in step (a), the molar ratio of 3-bromophenol to potassium carbonate is 1 : 2.5 ~ 3.5; preferably 1 : 2.9 ~ 3.

[0024] In another preferred example, in step (a), the reaction temperature of the reaction is 80 ~ 90 °C, preferably 85 °C.

[0025] In another preferred example, in step (a), the reaction time of the reaction is 5 ~ 24 hours, preferably 8 ~ 12 hours.

[0026] In another preferred example, the reaction of step (a) is carried out in acetonitrile.

[0027] In another preferred embodiment, in step (a), the mass volume ratio of 3-bromophenol to acetonitrile is 1 g: 10-30 ml; preferably 1 g: 15-20 ml.

[0028] In another preferred embodiment, step (a) comprises the steps of providing a solution of 3-bromophenol in acetonitrile, adding tert-butyl 2-bromo-2-methyl-propionate and potassium carbonate, and conducting the reaction at 80-90 °C to obtain compound 1 after work-up.

[0029] In another preferred embodiment, in step (b), the molar ratio of compound 1 to (R)-tert-butyl piperidin-3-aminocarboxylate is 1 : 2-4; preferably 1 : 2.5-3.

[0030] In another preferred embodiment, in step (b), the molar ratio of compound 1, cesium carbonate, Pd2(dba)3 and Ruphos is 1 : 3-5: 0.05-0.15: 0.15-0.25; preferably 1 : 3.5-4: 0.1-0.12: 0.2-0.25.

[0031] In another preferred embodiment, in step (b), the reaction temperature of the reaction is 90-100 °C, preferably 90 °C.

[0032] In another preferred embodiment, in step (b), the reaction time of the reaction is 36-60 hours, preferably 48 hours.

[0033] In another preferred embodiment, the reaction of step (b) is conducted in toluene.

[0034] In another preferred embodiment, in step (b), the mass volume ratio of compound 1 to toluene is 1 g: 30-60 ml; preferably 1 g: 30-40 ml.

[0035] In another preferred embodiment, step (b) comprises the steps of providing a solution of compound 1 in toluene, adding (R)-tert-butyl piperidin-3-aminocarboxylate, cesium carbonate, Pd2(dba)3 and Ruphos, conducting the reaction under nitrogen protection at 90-100 °C with continuous stirring for 36-60 hours, and obtaining compound 2 after work-up.

[0036] In another preferred embodiment, in step (c), the molar volume ratio of compound 2 to trifluoroacetic acid (TFA) is 1 mmol: 0.3-0.6 ml; preferably 1 mmol: 0.4-0.45 ml.

[0037] In another preferred embodiment, in step (c), the reaction temperature of the deprotection reaction is 10-30 °C; preferably 15-25 °C.

[0038] In another preferred embodiment, in step (c), the reaction time of the deprotection reaction is 2-4 hours; preferably 3 hours.

[0039] In another preferred embodiment, the deprotection reaction of step (c) is carried out in dichloromethane (DCM).

[0040] In another preferred embodiment, in step (c), the mass-volume ratio of compound 2 to dichloromethane is 1 g: 10-30 ml; preferably 1 g: 20 ml.

[0041] In another preferred embodiment, step (c) comprises the steps of providing a solution of compound 2 in dichloromethane, adding trifluoroacetic acid, stirring the reaction at room temperature for 2-4 hours, and obtaining compound 3 after work-up.

[0042] In another preferred embodiment, in step (d), the mass-volume ratio of compound 3 to tetrahydrofuran is 1 g: 10-30 ml; preferably 1 g: 20 ml.

[0043] In another preferred embodiment, step (d) comprises the steps of providing a solution of compound 3, compound 4, DIPEA and CDI in dichloromethane, stirring the reaction at room temperature for 5-24 hours (preferably 10-15 hours) under nitrogen protection, and obtaining compound 5 after work-up.

[0044] In another preferred embodiment, in step (e), the molar ratio of compound 5 to compound 6 is 1: 1.0-1.5; preferably 1: 1.2-1.3.

[0045] In another preferred embodiment, in step (e), the molar ratio of compound 5, potassium acetate and Pd(dppf)Cl2 is 1: 3-5: 1: 0.005-0.015; preferably 1: 4-4.1: 0.01-0.011.

[0046] In another preferred embodiment, in step (e), the reaction temperature is 70-90°C, preferably 80°C.

[0047] In another preferred embodiment, in step (e), the reaction time is 24-48 hours, preferably 36 hours.

[0048] In another preferred embodiment, in step (e), the organic solvent is dioxane.

[0049] In another preferred embodiment, in step (e), the volume ratio of organic solvent to water is 5-7: 1, preferably 6: 1.

[0050] In another preferred embodiment, in step (e), the mass-volume ratio of compound 5 to organic solvent is 1 g: (40-60) ml; preferably 1 g: (50-55) ml.

[0051] In another preferred embodiment, step (e) comprises the steps of providing a solution of compound 5 in an organic solvent, adding potassium acetate, compound 6 and Pd(dppf)Cl2, adding water, stirring under nitrogen protection for 24-48 hours (preferably 36 hours), and post-treatment to obtain compound 7.

[0052] In another preferred embodiment, in step (f), the molar ratio of compound 7 to trifluoroacetic acid (TFA) is 1 mmol: 10-15 ml; preferably 1 mmol: 12-13 ml.

[0053] In another preferred embodiment, in step (f), the reaction temperature of the deprotection reaction is 10-30°C; preferably 15-25°C.

[0054] In another preferred embodiment, in step (f), the reaction time of the deprotection reaction is 3-10 h; preferably 6 h.

[0055] In another preferred embodiment, in step (f), the deprotection reaction is carried out in dichloromethane (DCM).

[0056] In another preferred embodiment, in step (f), the mass-volume ratio of compound 7 to organic solvent is 1 g: 30-70 ml; preferably 1 g: 40-45 ml.

[0057] In another preferred embodiment, step (f) comprises the steps of providing a solution of compound 7 in dichloromethane, adding TFA, stirring at room temperature for 3-10 h (preferably 6 h), and post-treatment to obtain compound 8.

[0058] In another preferred embodiment, in step (g), the molar ratio of compound 8 to glycine methyl ester hydrochloride is 1:1.0-1.5; preferably 1:1.2-1.3.

[0059] In another preferred embodiment, in step (g), the molar ratio of compound 8, DIPEA and HATU is 1:3-5:1.0-1.5; preferably 1:4.0-4.1:1.1-1.2.

[0060] In another preferred embodiment, in step (g), the reaction temperature is 10-30°C, preferably 15-25°C.

[0061] In another preferred embodiment, in step (g), the reaction time is 3-10 hours, preferably 6 hours.

[0062] In another preferred embodiment, in step (g), the reaction is carried out in THF.

[0063] In another preferred embodiment, in step (g), the mass to volume ratio of compound 8 to THF is 1 g: 50-100 ml; preferably 1 g: 65-70 ml.

[0064] In another preferred embodiment, step (g) comprises the steps of providing a solution of compound 8 in THF, adding glycine methyl ester hydrochloride, DIPEA and HATU, stirring at room temperature for 3-10 hours (preferably 6 hours), and working up to give compound 9.

[0065] In another preferred embodiment, in step (h), the molar ratio of compound 8 to lithium hydroxide is 1 : (3-5); preferably 1 : 3.9-4.

[0066] In another preferred embodiment, in step (h), the reaction temperature of the hydrolysis reaction is 10-30 °C, preferably 15-25 °C.

[0067] In another preferred embodiment, in step (h), the reaction time of the hydrolysis reaction is 2-6 hours, preferably 4 hours.

[0068] In another preferred embodiment, in step (h), the organic solvent is THF, methanol, or a combination thereof; preferably a mixture of THF and methanol; more preferably, in the mixture of THF and methanol, the volume ratio of THF to methanol is (3-5): 1; most preferably, in the mixture of THF and methanol, the volume ratio of THF to methanol is 4: 1.

[0069] In another preferred embodiment, in step (h), the volume ratio of organic solvent to water is 4-6: 1; preferably 5: 1.

[0070] In another preferred embodiment, in step (h), the mass to volume ratio of compound 8 to organic solvent is 1 g: 60-80 ml; preferably 1 g: 65-70 ml.

[0071] In another preferred embodiment, step (h) comprises the steps of providing a solution of compound 9 in THF, adding a mixture of lithium hydroxide in water and methanol, stirring at room temperature for 2-6 hours (preferably 4 hours), and working up to give compound 10.

[0072] In another preferred embodiment, in step (i), the molar ratio of compound 10 to compound of formula I is 1 : 1.1-1.4; preferably 1 : 1.2-1.3.

[0073] In another preferred embodiment, in step (i), the molar ratio of compound 10, DIPEA and HATU is 1 : 3-5: 1.0-1.5; preferably 1 : 4.0-4.1 : 1.1-1.2.

[0074] In another preferred embodiment, in step (i), the reaction temperature of the reaction is 10-30°C, preferably 15-25°C.

[0075] In another preferred embodiment, in step (i), the reaction time of the reaction is 3-10 hours, preferably 6 hours.

[0076] In another preferred embodiment, the reaction of step (i) is carried out in THF.

[0077] In another preferred embodiment, in step (i), the mass-volume ratio of compound 10 to THF is 1 g: 20-60 ml; preferably 1 g: 30-40 ml.

[0078] In another preferred embodiment, step (i) comprises the steps of providing a solution of compound 10 in THF, adding (S)-2-Boc-aminomethyl-pyrrolidine, DIPEA and HATU, stirring the reaction at 10-30°C (preferably 15-25°C) for 3-10 hours (preferably 6 hours), and working up to obtain the compound of formula I.

[0079] In another preferred embodiment, the compound of formula I is used for inhibiting the Wnt / β-catenin signaling pathway.

[0080] In another preferred embodiment, the compound of formula I is used for treating or preventing a disease related to the Wnt / β-catenin signaling pathway (such as a tumor, preferably a glioma).

[0081] In another preferred embodiment, the compound of formula I is used for treating or preventing a tumor (preferably a glioma).

[0082] In another preferred embodiment, the compound of formula I is used as a quality control product.

[0083] In a second aspect of the present application, there is provided a compound of formula I.

[0084] In a third aspect of the present application, there is provided the use of a compound of formula I in the preparation of (i) a Wnt / β-catenin signaling pathway inhibitor, (ii) a medicament for treating or preventing a disease related to the Wnt / β-catenin signaling pathway and / or treating or preventing a tumor (preferably a glioma).

[0085] It should be understood that, within the scope of the present application, all combinations of the above-described technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be interchanged and combined to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0086] Figure 1Results of the test example 1 QPCR experiment are shown.

[0087] Figure 2 Results of the test example 1 CCK-8 experiment are shown. DETAILED DESCRIPTION

[0088] The inventors have made long and intensive studies. It was unexpectedly found that the coupling reaction using the S-configuration compound 3 and compound 4 as reactants under the reaction conditions of CDI, DIPEA could obtain the desired coupling product of S-configuration with a high yield (86%). Based on this, the inventors completed the present application.

[0089] TERMINOLOGY

[0090] Unless otherwise specified, each term or phrase as used herein has its ordinary meaning as understood by one of ordinary skill in the art.

[0091] As used herein, the terms "comprising", "containing" or "including" mean that various components can be used together in a mixture or composition of the present application. Thus, the terms "consisting essentially of and "consisting of are included in the term "comprising".

[0092] As used herein, "work-up" refers to the process of isolating the target product from the reaction system and optionally subjecting to further purification. Generally, work-up can include one or more of the following operations: filtration, concentration, extraction, washing (such as washing with saturated brine), drying, chromatography, etc.

[0093] 3-Piperidinophenylurea derivatives

[0094] As used herein, the term "compound of the present application" or "3-piperidinophenylurea derivative" can be used interchangeably to refer to the compound as shown below (i.e. compound of Formula I or compound C37-28R). The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound as described in the first aspect of the present application.

[0095]

[0096] As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0097] As used herein, the term "pharmaceutically acceptable salt" refers to a salt form of a drug in the field of pharmacy, i.e., a salt formed by combining a drug with an acid or a base. Such a salt form can improve the solubility, stability, bioavailability, etc. of the drug, thereby enhancing the efficacy of the drug and the applicability of the drug. The pharmaceutically acceptable salt according to the present application can be a hydrochloride, a sulfate, a citrate, a phosphate, an acetate, an oxalate, a succinate, a fumarate, a maleate, a malate, a propionate, or a trifluoroacetate of the compound.

[0098] In addition to salt forms, the present application provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present application. Additionally, prodrugs can be converted by chemical or biochemical methods into the compounds of the present application in an ex vivo environment. For example, a prodrug can slowly convert into the compound of the present application when placed in a transdermal patch reservoir containing a suitable enzyme or chemical reagent.

[0099] Certain compounds of the present application can exist in unsolvated as well as solvated forms, including hydrated forms (i.e., hydrates). Solvated forms are generally equivalent to unsolvated forms and are included in the scope of the present application. Certain compounds of the present application can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent and are intended to be within the scope of the present application.

[0100] Certain compounds of the present application possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers) are all intended to be within the scope of the present application. When the compounds provided herein have an indicated stereochemistry (indicated as R or S, or with dashed or wedged bonds), it is to be understood by those skilled in the art that those compounds are substantially free of other isomers (e.g., at least 80%, 90%, 95%, 98%, 99% and up to 100% free of other isomers).

[0101] The compounds of the present application can also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds can be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or non-radioactive isotopes, such as deuterium ( 2 H) or carbon-13 ( 13C). In addition to the uses described herein, such isotopic variants can provide additional uses. For example, isotopic variations of the compounds of the application can have additional uses, including but not limited to use as diagnostic and / or imaging agents, or as cytotoxic / radiotoxic therapeutic agents. In addition, isotopic variations of the compounds of the application can have altered pharmacokinetic and pharmacodynamic properties, which can be advantageous in a number of situations. All isotopic variations of the compounds of the application, whether radioactive or not, are within the scope of the present application.

[0102] Pharmaceutical compositions and methods of administration

[0103] Since the compounds of the present application have excellent BCL9 / β-catenin interaction inhibitory activity and tumor cell growth inhibitory effect, the compounds of the present application and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present application as an active ingredient can be used for the treatment, prevention, and alleviation of tumors (especially glioma).

[0104] In some embodiments, the present application provides a pharmaceutical composition comprising a compound provided by the present application or a pharmaceutically acceptable salt thereof. In other embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0105] The pharmaceutical composition of the present application comprises a safe and effective amount of the compound of the present application or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. The "safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 0.1-2000 mg of the compound of the present application per dose, preferably 1-1000 mg of the compound of the present application per dose, and more preferably 10-500 mg of the compound of the present application per dose. Preferably, the "dose" is a capsule or a tablet.

[0106] As used herein, the term "therapeutically effective amount" means any amount of a drug that, when used alone or in conjunction with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of progression to a more serious condition or disability as a result of the disease. A "therapeutically effective amount" of a drug of the present application also includes a "prophylactically effective amount," which is any amount of a drug that, when administered alone or in conjunction with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of a disease, inhibits the development or recurrence of the disease.

[0107] "Pharmaceutically acceptable carrier" means one or more compatible solid or liquid filler or gel materials which are suitable for use in humans and which are nontoxic at the dosages employed. "Compatible" means that the composition containing the carrier does not abnormally affect the biological activity or stability of the compound of the present application. Examples of pharmaceutically acceptable carriers are celluloses and their derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween®, Span®, lecithin, etc.), wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0108] The mode of administration of the compounds or pharmaceutical compositions of the present application is not critical. Representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical.

[0109] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or elixirs. Compositions for parenteral injection can comprise physiologically acceptable, sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Dosage forms for topical administration of the compounds of the present application include ointments, powders, patches, sprays, and inhalers.

[0110] The compounds of the present application can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0111] When the pharmaceutical composition is used, a safe and effective amount of the compound of the present application is administered to a mammal (e.g., a human) in need of treatment, wherein the amount is administered in a dosage that is pharmaceutically effective, and the daily dosage is generally 1 to 2000 mg, preferably 20 to 500 mg, for a 60 kg body weight. Of course, the specific dose will also depend on the route of administration, the health condition of the patient, and the like, which are within the skill of the skilled practitioner.

[0112] Preparation method

[0113] The present application also provides a method for preparing the compounds of the present application. The preparation method is shown in the following scheme:

[0114]

[0115] Each step is as described in the first aspect. ​

[0116] In some embodiments, the reagents and conditions for each step are as follows: (a) K2CO3, ACN, 85 °C, overnight, 95% yield; (b) Pd2(dba)3, Ruphos, Cs2CO3, toluene, 90 °C, 24 h, 62% yield; (c) TFA, CH2Cl2, rt, 3 h, 85% yield; (d) CDI, DIPEA, dry DCM, rt, overnight, 86% yield d; (e) AcOK, Pd(dppf)Cl2, 1,4-dioxane / EtOH / H2O (5:2:1), 85 °C, 24 h, 76% yield; (f) TFA, CH2Cl2, rt, 5 h, 100% yield; (g) HATU, DIPEA, THF, rt, overnight, 81% yield; (h) LiOH, H2O, THF, rt, 3 h, 97% yield; (i) HATU, DIPEA, THF, rt, overnight, 76% yield. (j) DIPEA, DCM, 0-rt.

[0117] The main advantages of the present application include:

[0118] High safety: CDI is a relatively safe reagent that does not produce gas during operation, while triphosgene is a toxic gas that needs to be used under specific conditions (such as a well-ventilated operation room).

[0119] Easy operation: CDI preparation of urea reaction conditions are relatively mild, room temperature reaction can be, relatively simple operation, without complex control conditions, while the use of triphosgene needs more strict operation control, including safety and environmental considerations.

[0120] High product purity: CDI reaction products usually have high purity, which can reduce subsequent separation and purification steps, while triphosgene prepared urea may be accompanied by other by-products or impurities, which need more purification steps.

[0121] Environmental protection: CDI preparation of urea does not release toxic waste gas, relatively environmentally friendly; while triphosgene preparation of urea releases harmful substances such as chlorocyanide, which has a negative impact on the environment.

[0122] Wide range of applications: CDI can not only be used for the preparation of urea, but also for the synthesis of other amine compounds, with more extensive application potential and flexibility.

[0123] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, which are usually carried out according to the conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0124] Example 1: Synthesis of tert-butyl 2-(3-bromophenoxy)-2-methylpropionate (1)

[0125]

[0126] In a 100 mL dry reaction flask, 3-bromophenol (1.20 g, 6.94 mmol) was first added and dissolved in 20 mL of acetonitrile. Then, tert-butyl 2-bromo-2-methylpropionate (4.42 g, 20.81 mmol) and potassium carbonate (2.89 g, 20.81 mmol) were added, and the mixture was refluxed at 85 °C overnight. The progress of the reaction was monitored by thin-layer chromatography (petroleum ether: ethyl acetate = 10:1). After cooling the reaction mixture to room temperature, unreacted potassium carbonate was removed by filtration. After concentrating the filtrate, 30 mL of water was added to the residue, and extraction was performed using ethyl acetate (30 mL × 3). The organic phase was collected, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the solution was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 30:1) to obtain a colorless and transparent liquid product 1 (1.78 g, yield 95%).

[0127] Example 2: Synthesis of (S)-2-(3-(tert-butoxycarbonyl)amino)piperidin-1-yl)phenoxy)-2-methylpropionate tert-butyl ester (2)

[0128]

[0129] In a dry 100 mL three-necked flask, intermediate 1 (0.90 g, 2.86 mmol) was dissolved in 30 mL of toluene. Then, (R)-piperidine-3-carbamate tert-butyl ester (1.72 g, 8.57 mmol), cesium carbonate (3.72 g, 11.42 mmol), Pd2(dba)3 (0.25 g, 0.29 mmol), and Ruphos (0.26 g, 0.58 mmol) were added. Under nitrogen protection, the reaction mixture was heated to 90 °C and stirred continuously for 48 hours. The reaction progress was monitored using TLC plates (petroleum ether:ethyl acetate = 5:1). The reaction mixture was then cooled to room temperature, and the filtrate was concentrated after filtering out insoluble matter. The residue was purified by silica gel column chromatography with petroleum ether and ethyl acetate as eluent (V:V = 20:1) to give a pale yellow oily product 2 (0.78 g, yield 62%).

[0130] Example 3: Synthesis of (S)-2-(3-aminopiperidin-1-yl)phenoxy)-2-methylpropionate tert-butyl ester (3)

[0131]

[0132] Weigh intermediate 2 (1.0 g, 2.99 mmol), dissolve it in 10 mL of DCM in a 50 mL dry reaction flask, add 1.3 mL of TFA, stir at room temperature for 3 h, and monitor the reaction progress by HPLC. Once the reaction proceeds to the point where the starting material disappears and the main product is the de-Boc product, stop the reaction by adding 50 mL of saturated Na₂CO₃ aqueous solution, and extract with 30 mL of DCM. Wash the organic phase with saturated brine, dry with anhydrous Na₂SO₄, and concentrate under reduced pressure to obtain a yellow oily liquid product 3 (0.74 g, yield 85%).

[0133] Example 4: Synthesis of (S)-2-(3-(3-(3-(4-bromobenzyl)-3-cyclopropylurea)piperidin-1-yl)phenoxy)-2-methylpropionate tert-butyl ester (5)

[0134]

[0135] Intermediate 3 (1 g, 2.99 mmol), starting material 4 (0.74 g, 3.27 mmol), CDI (N,N′-carbonyldiimidazole, 0.49 g, 3.02 mmol), and DIPEA (1.55 g, 11.99 mmol) were added to a 100 mL dry reaction flask and dissolved in 20 mL of dry dichloromethane. Under nitrogen protection, the mixture was stirred overnight at room temperature, and the reaction was monitored by TLC (petroleum ether: ethyl acetate = 3:1). After the reaction was complete, the solvent was removed by concentration, and 50 mL of water was added to the reaction solution. The mixture was extracted and separated by DCM (30 mL × 3). The organic phase was collected, washed with 50 mL of saturated brine, and dried over anhydrous Na2SO4. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1 as eluent) to obtain a pale yellow viscous oil 5 (1.51 g, yield 86%).

[0136] Example 5: Synthesis of (S)-2-(3-(3-(4-(1H-pyrazol-4-yl)benzyl)-3-cyclopropylureoyl)piperidin-1-yl)phenoxy)-2-methylpropionate tert-butyl ester (7)

[0137]

[0138] Intermediate 5 (0.67 g, 1.14 mmol) was dissolved in 30 mL of dioxane and added to a 100 mL dry reaction flask. Potassium acetate (0.44 g, 4.57 mmol), intermediate 6 (1-Boc-pyrazole-boronic acid pinacol ester, 0.41 g, 1.37 mmol), and Pd(dppf)Cl2 (0.084 g, 0.012 mmol), along with 5 mL of water, were weighed and added to the reaction system. The reaction was stirred at 80 °C for 36 h under nitrogen protection. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 1:1). After the reaction was complete, the reaction solution was cooled to room temperature, and insoluble matter was removed by filtration. The filtrate was concentrated. The residual solution was purified by silica gel column chromatography [petroleum ether: ethyl acetate = 1:1 (V:V)] to finally obtain a viscous solid product 7 (0.46 g, yield 76%).

[0139] Example 6: Synthesis of (S)-2-(3-(3-(4-(1H-pyrazol-4-yl)benzyl)-3-cyclopropylureoyl)piperidin-1-yl)phenoxy)-2-methylpropionic acid (8)

[0140]

[0141] Intermediate 7 (0.45 g, 0.78 mmol) was dissolved in 20 mL of DCM into a 100 mL dry reaction flask, and then 10 mL of TFA was added. The mixture was stirred at room temperature for 6 h, and the reaction was detected by TLC plate. The reaction solution was concentrated, and the residual solution was dissolved with a small amount of DCM. The solution was concentrated again under reduced pressure to remove the dissolution. This operation was repeated 3 times to obtain crude product 8 (0.42 g, yield 100%), which was a pale yellow viscous solid. No purification was required, and it was used directly in the next reaction.

[0142] Example 7: Synthesis of (S)-(2-(3-(3-(4-(1H-pyrazol-4-yl)benzyl)-3-cyclopropylureoyl)piperidin-1-yl)phenoxy)-2-methylpropionyl)glycine methyl ester (9)

[0143]

[0144] Intermediate 8 (0.44 g, 0.85 mmol) was weighed and dissolved in 30 mL of THF into a 100 mL dry reaction flask. Glycine methyl ester hydrochloride (0.13 g, 1.02 mmol), DIPEA (0.44 g, 3.40 mmol), and HATU (0.35 g, 0.94 mmol) were then added, and the mixture was stirred at room temperature for 6 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:1). 30 mL of water was added to the reaction mixture, and the mixture was extracted with DCM (30 mL × 3). The organic phase was collected, washed with 40 mL of saturated brine, and dried over anhydrous Na₂SO₄. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1 as eluent) to obtain a pale yellow viscous solid product 9 (0.41 g, yield 81%).

[0145] Example 8: Synthesis of (S)-(2-(3-(3-(4-(1H-pyrazol-4-yl)benzyl)-3-cyclopropylureoyl)piperidin-1-yl)phenoxy)-2-methylpropionyl)glycine (10)

[0146]

[0147] Compound 9 (0.44 g, 0.76 mmol) was weighed and dissolved in 20 mL of THF in a 50 mL dry reaction flask. Lithium hydroxide (0.14 g, 3.02 mmol) was dissolved in 5 mL of water, then 5 mL of methanol was added and mixed well. Finally, the mixture was added to the reaction system, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC plate (petroleum ether: ethyl acetate = 1:1). The reaction solution was concentrated, and 20 mL of water was added to the residue. The pH was adjusted to about 4-5 with 1 M HCl, and the solution was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with 40 mL of saturated brine, dried over anhydrous Na2SO4, and the organic phase was concentrated to give a pale yellow solid compound 10 (0.38 g, yield 97%).

[0148] Example 9: Synthesis of 2-({3-[(3S)-3-{[({[4-(1H-pyrazol-4-yl)phenyl]methyl}amino)carbonyl]amino}hexahydropyridin-1-yl]phenyl}oxy)-N-{2-[(3R)-3-hydroxytetrahydro-1H-pyrrolo-1-yl]-2-oxoethyleneethyl}-2-methylpropionamide (C37-28R)

[0149]

[0150] Weigh intermediate 10 (0.42 g, 0.74 mmol), dissolve it in 15 mL of THF in a 100 mL dry reaction flask, and then add (S)-2-Boc-aminomethyl-pyrrolidine (0.18 g, 0.90 mmol), DIPEA (0.39 g, 2.99 mmol), and HATU (0.31 g, 0.82 mmol). Stir the mixture at room temperature for 6 hours. The reaction was monitored by TLC plate (ethyl acetate). The reaction solution was concentrated, the residue was dissolved in 40 mL of dichloromethane, extracted with water (30 mL × 3), washed with saturated brine, dried over anhydrous Na2SO4, and the organic phase was concentrated and purified by silica gel column chromatography (ethyl acetate as eluent) to obtain a pale yellow solid product C37-28R. The product was dissolved in 1 mL of acetonitrile, and then 1 mL of pure water was added. The mixture was thoroughly mixed and placed in a -80℃ freezer for 4 h. Then it was freeze-dried for 12 h to obtain a white solid powder, namely C37-28R (0.43 g, yield 76%). 1 H NMR (400MHz, DMSO-d6) δ8.03 (s, 3H), 7.55 (d, J = 8.1Hz, 2H), 7.25-7.16 (m, 3H), 6.79 (s,2H),6.57(d,J=8.1Hz,1H),6.38(s,1H),6.13(s,1H),4.38-4.15(m,3H),3.99-3 .83(m,2H),3.75(s,1H),3.54-3.35(m,4H),3.35-3.20(m,2H),3.04(d,J=10.6Hz,1 H),2.85(d,J=10.8Hz,1H),1.99-1.73(m,4H),1.66(d,J=10.7Hz,1H),1.42(s,6H). 13 C NMR (101MHz, DMSO-d6) δ174.26,167.04(d,J=5.0Hz),158.61,157.91,156.08(d,J=2.5Hz),138.75,131.73,131.13,130.04(d,J=2.9Hz),128.00 ,125.46,121.49,80.98,70.04,68.19,53.67,45.70,44.15,43.11,41.8 7(d,J=16.1Hz),34.38,32.66,30.11,26.14-25.28(m),23.12.ESI-Mass for C 32 H 41 N7O5: m / z(M+H) + 644.72.

[0151] Test Example 1:

[0152] (1) qPCR experimental method:

[0153] U87 (human glioblastoma cell line), U251 (human glioma cell line), and GL261 (mouse glioma cell line) cells were treated with different concentrations (10, 5, 2.5, 1.25, 0.625 μM) of C37-28R for 24 h. After treatment, the supernatant was discarded, and 500 μL of Trizol (Accurate Biology, A3A2199) was added to each well. After mixing by pipetting, the cells were lysed at room temperature for 10 min, and the mixture was transferred to 1.5 mL EP tubes. 100 μL of chloroform was added to each tube, and the mixture was thoroughly mixed and allowed to separate into layers. Then, the cells were centrifuged at 12000 rpm and 4 °C for 10 min. The supernatant was carefully aspirated using a 200 μL pipette tip and transferred to a new 1.5 mL EP tube. An equal volume of isopropanol was added to the new tube, and the mixture was thoroughly mixed and centrifuged at 2000 rpm and 4 °C for 10 min. Carefully remove the supernatant and wash the precipitate with 500 μL of 75% ethanol. Centrifuge at 12000 rpm, 4°C for 10 minutes. Carefully remove the ethanol, allow the precipitate to air dry, add 20 μL of DEPC water to dissolve the RNA precipitate, and measure the RNA concentration using a microplate reader. Isolate total RNA (500 ng) from the cells and reverse transcribe it into cDNA using Evo-mmLv reverse transcription premix. Then use... Green premixed Pro Taq (Accurate Biology, AG11706, AG11701) and the StepOne-Plus real-time PCR system (Applied Biosystems) were used to dilute and quantify cDNA. The 2^(-ΔCT) for each concentration gradient was calculated, and the inhibition rate was calculated as (1 - experimental wells / control wells) × 100%. The IC50 value was determined using GraphPad Prism 8 software. The test results are shown below. Figure 1 (The IC50 values ​​of compound C37-28R against RNA expression in U87, U251, and GL261 cells were 4.892 μM, 6.671 μM, and 6.357 μM, respectively).

[0154] (2) CCK-8 experimental method

[0155] Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) assay. U87, U251, and GL261 cells were seeded into 96-well plates (flat-bottomed clear plates) at a density of 10,000 cells / 100 μL DMEM (10% FBS) per well. The next day, observe the cell status. Once the cells are well adhered, begin adding the drug. Dilute the target product with DMEM (2% FBS) using a serial dilution technique, resulting in concentrations of 20, 10, 5, 2.5, 1.2, 0.625, 0.3125, 0.15625, and 0 μM (equal volume of DMSO). Add 100 μL of the target product at each concentration to each well, performing two replicates for each concentration gradient. Leave three blank wells (containing only DMEM and 2% FBS, without cells). Incubate at 37°C for 24 hours. Add 10 μL of CCK-8 enhancement solution to each well: Since the amount of CCK-8 added to each well is relatively small, there is a possibility of reagent residue on the well walls, which could cause errors. Gently tap the culture plate after adding the reagent to help mix. Incubate for 0.5-4 hours: The amount of Formazan formed varies depending on the cell type; in most cases, 1 hour of incubation is sufficient. If the color development is insufficient, continue culturing to confirm the optimal conditions. Absorbance was measured at 450 nm and 600 nm (excluding interference from reagent background color and the absorbance of the well plate itself); the final absorbance was calculated as OD450nm-OD600nm. All experiments were repeated three times, and the average value was used to calculate the inhibition rate; Inhibition rate = [(Ac-As) / (Ac-Ab)]×100%. As: absorbance of experimental wells (containing cell culture medium, CCK-8, and the test drug); Ac: absorbance of control wells (containing cell culture medium, CCK-8, and no test drug); Ab: absorbance of blank wells (containing no cells, test drug, culture medium, and CCK-8). IC50 was determined using GraphPad Prism 8 software. 50 Value. See test results. Figure 2 (The IC50 values ​​of compound C37-28R for inhibiting the growth of U87, U251, and GL261 cells were 5.868 μM, 7.936 μM, and 5.701 μM, respectively).

[0156] Regulators of the Wnt / β-catenin signaling pathway, such as ICG-001, have been studied and shown potential anti-tumor activity, providing a theoretical basis for the development of novel therapeutic strategies. Therefore, this application experimentally evaluates the effects of the novel compound C37-28R on glioma cell growth and signaling pathways, which not only contributes to understanding its mechanism of action but may also offer new hope and directions for future clinical treatment development.

[0157] Glioma cell lines U87, U251, and GL261 were selected for experiments. The expression of Axin2, a specific target gene of the Wnt / β-catenin signaling pathway, was detected using quantitative real-time PCR (qPCR). The results showed that Axin2 gene expression was significantly inhibited after treatment with C37-28R. Furthermore, CCK-8 cell proliferation assays demonstrated that C37-28R exhibited significant growth-inhibiting activity against U87, U251, and GL261 cell lines. These findings suggest that C37-28R may exert its growth-inhibiting effect by inhibiting the Wnt / β-catenin signaling pathway, and its inhibitory activity is comparable to that of the known Wnt / β-catenin inhibitor ICG-001. This provides important clues for further research into its mechanism of action and potential therapeutic applications.

[0158] These experimental results not only contribute to a deeper understanding of the effects of C37-28R on the biological characteristics of glioma cells, but also suggest that it may be a potential candidate compound for anti-glioma drugs.

[0159] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A process for preparing a compound of Formula I, ###0001### Formula I said process comprising the steps of: (a) reacting 3-bromophenol with tert-butyl 2-bromo-2-methyl-propionate in the presence of potassium carbonate to obtain compound 1; (b) reacting compound 1 with (R)-tert-butyl piperidin-3-ylcarbamate in the presence of cesium carbonate, Pd2(dba)3 and Ruphos to obtain compound 2; (c) deprotecting compound 2 in the presence of trifluoroacetic acid (TFA) to obtain compound 3; (d) reacting compound 3 with compound 4 in the presence of DIPEA and CDI in dichloromethane to obtain compound 5; wherein, the molar ratio of compound 3 to compound 4 is 1 : 1.0 to 1.5, preferably 1 : 1.0 to 1.1; the molar ratio of compound 3, CDI and DIPEA is 1 : 1.0 to 1.5 : 3 to 6, preferably 1 : 1.0 to 1.1 : 4 to 4.1; the reaction temperature is 10 to 30 °C, preferably 15 to 25 °C; and the reaction time is 5 to 24 hours, preferably 10 to 15 hours; (e) reacting compound 5 with compound 6 in the presence of potassium acetate and Pd(dppf)Cl2 in a mixture of organic solvent and water to obtain compound 7; (f) deprotecting compound 7 in the presence of trifluoroacetic acid (TFA) to obtain compound 8; (g) reacting compound 8 with glycine methyl ester hydrochloride in the presence of DIPEA and HATU to obtain compound 9; (h) hydrolyzing compound 9 in the presence of lithium hydroxide in a mixture of organic solvent and water to obtain compound 10; and (i) reacting compound 10 with (S)-2-Boc-aminomethyl-pyrrolidine in the presence of DIPEA and HATU to obtain a compound of Formula I. wherein Step (d) comprises the steps of providing a solution of compound 3, compound 4, DIPEA and CDI in dichloromethane, stirring the reaction under nitrogen at room temperature for 5 to 24 hours (preferably 10 to 15 hours) and work-up to obtain compound 5. The process comprises one or more features selected from the group consisting of: 1) in step (a), the molar ratio of 3-bromophenol to tert-butyl 2-bromo-2-methyl-propionate is 1 : 2.5 to 3.5; preferably 1 : 2.9 to 3; 2) in step (b), the molar ratio of compound 1 to (R)-tert-butyl piperidin-3-ylcarbamate is 1 : 2 to 4; preferably 1 : 2.5 to 3; 3) in step (e), the molar ratio of compound 5 to compound 6 is 1 : 1.0 to 1.5; preferably 1 : 1.2 to 1.3; 4) in step (g), the molar ratio of compound 8 to glycine methyl ester hydrochloride is 1 : 1.0 to 1.5; preferably 1 : 1.2 to 1.3; 5) in step (i), the molar ratio of compound 10 to a compound of Formula I is 1 : 1.1 to 1.4; preferably 1 : 1.2 to 1.

3. ​ ​ ​ ​ ​ ​ ​ 2. The production method according to claim 1, wherein ​ 3. The production method according to any one of claims 1 to 2, wherein ​ ​ ​ ​ ​ ​ 4. The production method according to any one of claims 1 to 2, wherein comprising one or more features selected from the group consisting of: 1) in step (a), the molar ratio of 3-bromophenol to potassium carbonate is 1:2.5-3.5; preferably 1:2.9-3; 2) in step (b), the molar ratio of compound 1, cesium carbonate, Pd2(dba)3 and Ruphos is 1:3-5:0.05-0.15:0.15-0.25; preferably 1:3.5-4:0.1-0.12:0.2-0.25; 3) in step (c), the molar volume ratio of compound 2 to trifluoroacetic acid (TFA) is 1 mmol:0.3-0.6 ml; preferably 1 mmol:0.4-0.45 ml; 4) in step (e), the molar ratio of compound 5, potassium acetate and Pd(dppf)Cl2 is 1:3-5:1:0.005-0.015; preferably 1:4-4.1:0.01-0.011; 5) in step (f), the molar volume ratio of compound 7 to trifluoroacetic acid (TFA) is 1 mmol:10-15 ml; preferably 1 mmol:12-13 ml; 6) in step (g), the molar ratio of compound 8, DIPEA and HATU is 1:3-5:1.0-1.5; preferably 1:4.0-4.1:1.1-1.2; 7) in step (h), the molar ratio of compound 8 to lithium hydroxide is 1:3-5; preferably 1:3.9-4; 8) in step (i), the molar ratio of compound 10, DIPEA and HATU is 1:3-5:1.0-1.5; preferably 1:4.0-4.1:1.1-1.

2. comprising one or more features selected from the group consisting of:

5. The production method according to any one of claims 1 to 2, wherein 1) in step (a), the reaction temperature of the reaction is 80-90 °C, preferably 85 °C; 2) in step (b), the reaction temperature of the reaction is 90-100 °C, preferably 90 °C; 3) in step (c), the reaction temperature of the deprotection reaction is 10-30 °C; preferably 15-25 °C; 4) in step (e), the reaction temperature of the reaction is 70-90 °C, preferably 80 °C; 5) in step (f), the reaction temperature of the deprotection reaction is 10-30 °C; preferably 15-25 °C; 6) in step (g), the reaction temperature of the reaction is 10-30 °C, preferably 15-25 °C; 7) in step (h), the reaction temperature of the hydrolysis reaction is 10-30 °C, preferably 15-25 °C; 8) in step (i), the reaction temperature of the reaction is 10-30 °C, preferably 15-25 °C. comprising one or more features selected from the group consisting of:

6. The production method according to any one of claims 1 to 2, wherein 1) in step (a), the reaction time of the reaction is 5-24 hours, preferably 8-12 hours; 2) in step (b), the reaction time of the reaction is 36-60 hours, preferably 48 hours; 3) in step (c), the reaction time of the deprotection reaction is 2-4 h; preferably 3 h; ​ 4) in step (e), the reaction time of the reaction is 24-48 hours, preferably 36 hours; 5) in step (f), the reaction time of the deprotection reaction is 3-10 hours, preferably 6 hours; 6) in step (g), the reaction time of the reaction is 3-10 hours, preferably 6 hours; 7) in step (h), the reaction time of the hydrolysis reaction is 2-6 hours, preferably 4 hours; and 8) in step (i), the reaction time of the reaction is 3-10 hours, preferably 6 hours.

7. The production method according to any one of claims 1 to 2, wherein comprising one or more features selected from the group consisting of: 1) the reaction of step (a) is carried out in acetonitrile; 2) the reaction of step (b) is carried out in toluene; 3) the deprotection reaction of step (c) is carried out in dichloromethane (DCM); 4) in step (e), the organic solvent is dioxane; 5) the deprotection reaction of step (f) is carried out in dichloromethane (DCM); 6) in step (g), the reaction is carried out in THF; 7) in step (h), the organic solvent is THF, methanol, or a combination thereof; preferably, a mixture of THF and methanol; more preferably, in the mixture of THF and methanol, the volume ratio of THF to methanol is 3-5:1; most preferably, in the mixture of THF and methanol, the volume ratio of THF to methanol is 4:1; and 8) the reaction of step (i) is carried out in THF.

8. The production method according to claim 7, wherein comprising one or more features selected from the group consisting of: 1) in step (a), the mass-to-volume ratio of 3-bromophenol to acetonitrile is 1 g:10-30 ml; preferably, 1 g:15-20 ml; 2) in step (b), the mass-to-volume ratio of compound 1 to toluene is 1 g:30-60 ml; preferably, 1 g:30-40 ml; 3) in step (c), the mass-to-volume ratio of compound 2 to dichloromethane is 1 g:10-30 ml; preferably, 1 g:20 ml; 4) in step (d), the mass-to-volume ratio of compound 3 to tetrahydrofuran is 1 g:10-30 ml; preferably, 1 g:20 ml. 5) in step (e), the volume ratio of the organic solvent to water is 5-7:1, preferably 6:1; and / or, the mass-to-volume ratio of compound 5 to the organic solvent is 1 g:40-60 ml; preferably, 1 g:50-55 ml; 6) in step (f), the mass-to-volume ratio of compound 7 to the organic solvent is 1 g:30-70 ml; preferably, 1 g:40-45 ml; 7) in step (g), the mass-to-volume ratio of compound 8 to THF is 1 g:50-100 ml; preferably, 1 g:65-70 ml; 8) in step (h), the volume ratio of the organic solvent to water is 4-6:1; preferably, 5:1; and / or, the mass-to-volume ratio of compound 8 to the organic solvent is 1 g:60-80 ml; preferably, 1 g:65-70 ml; and 9) in step (i), the mass-to-volume ratio of compound 10 to THF is 1 g:20-60 ml; preferably, 1 g:30-40 ml.

9. The production method according to claim 8, wherein comprising one or more features selected from the group consisting of: 1) Step (a) comprises the steps of providing a solution of 3-bromophenol in acetonitrile, adding tert-butyl 2-bromo-2-methyl-propionate and potassium carbonate, and conducting the reaction at 80-90 °C, followed by work-up to give compound 1; 2) Step (b) comprises the steps of providing a solution of compound 1 in toluene, adding (R)-tert-butyl piperidin-3-ylcarbamate, cesium carbonate, Pd2(dba)3 and Ruphos, stirring the reaction under nitrogen at 90-100 °C for 36-60 hours, followed by work-up to give compound 2; 3) Step (c) comprises the steps of providing a solution of compound 2 in dichloromethane, adding trifluoroacetic acid, stirring the reaction at room temperature for 2-4 hours, followed by work-up to give compound 3; 4) Step (e) comprises the steps of providing a solution of compound 5 in an organic solvent, adding potassium acetate, compound 6 and Pd(dppf)Cl2, adding water, stirring the reaction under nitrogen for 24-48 hours (preferably 36 hours), followed by work-up to give compound 7; 5) Step (f) comprises the steps of providing a solution of compound 7 in dichloromethane, adding TFA, stirring the reaction at room temperature for 3-10 h (preferably 6 h), followed by work-up to give compound 8; 6) Step (g) comprises the steps of providing a solution of compound 8 in THF, adding glycine methyl ester hydrochloride, DIPEA and HATU, stirring the reaction at room temperature for 3-10 hours (preferably 6 hours), followed by work-up to give compound 9; 7) Step (h) comprises the steps of providing a solution of compound 9 in THF, adding lithium hydroxide in a mixture of water and methanol, stirring the reaction at room temperature for 2-6 hours (preferably 4 hours), followed by work-up to give compound 10; and 8) Step (i) comprises the steps of providing a solution of compound 10 in THF, adding (S)-2-Boc-aminomethyl-pyrrolidine, DIPEA and HATU, stirring the reaction at 10-30 °C (preferably 15-25 °C) for 3-10 hours (preferably 6 hours), followed by work-up to give a compound of formula I.

10. A compound of formula I or a pharmaceutically acceptable salt thereof,

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