Compound, preparation method therefor and use thereof

Abstract

Provided in the present invention are a compound, a preparation method therefor and the use thereof. The compound has a structure shown as formula (A).wherein X represents an acid, and n is a number from 0.1 to 3. The compound has good biological activity and solubility, and has outstanding potential to be manufactured as a drug.

Description

A compound, its preparation method and application

[0001] This application claims priority to an earlier application filed on December 26, 2024, with China National Intellectual Property Administration, patent application number 202411942222.5, entitled "A compound and its preparation method and application"; the entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of compounds, specifically relating to a compound, its preparation method, and its application. Background Technology

[0003] Studies have found that the TP53 gene is the most frequently mutated gene in human cancers, with p53 mutations present in nearly 50% of malignant tumors. TP53 mutations result in the loss of tumor-suppressive transcriptional activity of wild-type p53 through homozygous deletion; and through heterozygous deletion, they manifest as dominant negative effects and gain-of-function effects, such as increased binding to p63 and p73, thereby promoting tumorigenesis and development. Therefore, mutated p53 proteins can be considered a group of heterogeneous proteins, exhibiting varying degrees of loss of normal tumor-suppressive function and gain of oncogenic properties (GOF).

[0004] Although different p53 mutants exhibit varying activities, TP53 missense mutants can be considered proto-oncogenes, promoting tumor migration and contributing to drug resistance and poor prognosis, thus becoming therapeutic targets for drug development. Among them, the oncogenic p53 Y220C mutant exhibits a particularly suitable structure for small molecule stabilizer development. However, while several existing small molecules, such as PK083, PK7088, PK5196, and PC14586, can bind to the hydrophobic cleft of the Y220C mutant, they still exhibit relatively high effective concentrations, thus raising the possibility of off-target effects. Therefore, the development of small molecule stabilizers with better physicochemical properties and biological activity is a clinical necessity. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention provides a compound having the structure shown in Formula A:

[0006] Where X represents acid, and n is a number from 0.1 to 3.

[0007] According to an embodiment of the present invention, the acid is an inorganic acid or an organic acid. For example, the inorganic acid is selected from one or more of hydrochloric acid, phosphoric acid, and sulfuric acid, and the organic acid is selected from one or more of methanesulfonic acid, lactic acid, maleic acid, malic acid, tartaric acid, ethylsulfonic acid, malonic acid, and oxalic acid.

[0008] According to an embodiment of the present invention, n is a number from 0.3 to 2.5, for example, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1.0, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5.

[0009] According to embodiments of the present invention, the organic acid may be its racemic, D-configuration, or L-configuration. For example, the lactic acid may be DL-lactic acid, D-lactic acid, or L-lactic acid; the malic acid may be DL-malic acid, D-malic acid, or L-malic acid; and the tartaric acid may be DL-tartaric acid, D-tartaric acid, or L-tartaric acid.

[0010] In some implementations, when X represents methanesulfonic acid, n is a number from 0.7 to 1.3, such as 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, or 1.3.

[0011] In some implementations, when X represents lactic acid (e.g., DL-lactic acid), n is a number from 0.5 to 1.3, such as 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1.0, 1.05, 1.1, 1.2, or 1.3.

[0012] In some implementations, when X represents maleic acid, n is a number from 0.5 to 1.3, such as 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1.0, 1.05, 1.1, 1.2, or 1.3.

[0013] In some implementations, when X represents malic acid (e.g., L-malic acid), n is a number from 0.9 to 2.2, such as 0.9, 1.0, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, or 2.2.

[0014] In some implementations, when X represents tartaric acid (e.g., L-tartaric acid or D-tartaric acid), n is a number from 0.9 to 2.2, such as 0.9, 1.0, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, or 2.2.

[0015] In some embodiments, when X represents ethylsulfonic acid, n is a number from 0.9 to 1.3, such as 0.9, 1.0, 1.05, 1.1, 1.2 or 1.3.

[0016] In some implementations, when X represents malonic acid, n is a number from 0.5 to 1.3, such as 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 1.0, 1.05, 1.1, 1.2, or 1.3.

[0017] The present invention also provides a method for preparing the above-mentioned compound, comprising: reacting the compound of formula (I) (i.e., 5-amino-1-(tert-butyl)-N-(3-(7-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)pyrazolo[1,5-a]pyridin-2-yl)prop-2-ynyl)-1H-pyrazol-4-carboxamide) with acid X to obtain the compound of formula A;

[0018] The n and X are subject to the limitations described above.

[0019] According to an embodiment of the present invention, the reaction is carried out in a solvent, for example, the solvent is selected from one or more of n-propanol, n-heptane, acetonitrile, water, acetone, tetrahydrofuran and ethyl acetate.

[0020] In some implementations, when X is methanesulfonic acid, malic acid, tartaric acid, or ethylsulfonic acid, the solvent is n-propanol.

[0021] In some embodiments, when X is lactic acid (e.g., DL-lactic acid), the solvent is n-propanol and / or acetonitrile.

[0022] In some embodiments, when X is maleic acid, the solvent is one or more of n-propanol, acetone, and n-heptane, for example, n-propanol, or a mixture of acetone and n-heptane.

[0023] In some embodiments, when X is malonic acid, the solvent is one or more of n-propanol, ethyl acetate, n-heptane, and acetone, for example, a mixed solvent of n-propanol, ethyl acetate, and n-heptane, or a mixed solvent of acetone and n-heptane.

[0024] In some embodiments, when X is oxalic acid, the solvent is n-propanol and / or acetonitrile.

[0025] According to an embodiment of the present invention, the reaction temperature is 40–60°C, for example 45°C, 50°C, or 55°C.

[0026] The present invention also provides a pharmaceutical composition comprising the above-described compounds.

[0027] According to embodiments of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0028] According to embodiments of the present invention, the pharmaceutical composition may further contain one or more therapeutic agents, used in combination with the above-mentioned compounds.

[0029] The present invention also provides the use of the above-described compounds or pharmaceutical compositions in the preparation of anti-tumor drugs containing p53-Y220C mutants, such as in the preparation of p53-Y220C reactivator drugs.

[0030] The present invention also provides a method for treating tumors, comprising administering to a patient a preventive or therapeutically effective amount of the above-mentioned compound and / or pharmaceutical composition; preferably, the tumor is a tumor containing the p53-Y220C mutant.

[0031] According to embodiments of the present invention, the tumors containing the p53-Y220C mutant include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive cell tumor, neuroectodermal tumors, visual pathway and hypothalamic gliomas, breast cancer, bronchial adenoma, Burkitt lymphoma, unknown primary cancers, central nervous system lymphomas, and cerebellar astrocytomas. Cytokine tumors, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, fibroblastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer such as non-small cell lung cancer and small cell lung cancer. Lung cancer, lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma with occult primary carcinoma, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell carcinoma, paranasal sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pine Astrocytoma of the pelvis, pineal germ cell tumor, pituitary adenoma, pleural pulmonary blastoma, plasmacytoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, cutaneous Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy), cancer of unknown primary location, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and nephroblastoma. Beneficial effects

[0032] The compound shown in Formula A has good biological activity and solubility, and has the potential to be developed into a drug.

[0033] Terminology Definitions and Explanations

[0034] The term "pharmaceuticalally acceptable excipient" refers to an excipient that does not cause significant irritation to the organism and does not impair the biological activity and properties of the active compound.

[0035] The term "two or more" refers to two, three, four, five or more kinds.

[0036] The term "patient" refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, with humans being the most preferred.

[0037] The term “therapeutic effective amount” refers to the amount of an active compound or drug that researchers, veterinarians, physicians, or other clinicians are searching for in tissues, systems, animals, individuals, or humans to elicit a biological or medical response. It includes one or more of the following: (1) prevention of disease: e.g., prevention of disease, disorder, or condition in individuals susceptible to disease, disorder, or symptom but not yet experiencing or exhibiting the pathology or symptoms of the disease; (2) suppression of disease: e.g., suppression of disease, disorder, or symptom in individuals experiencing or exhibiting the pathology or symptoms of the disease, disorder, or symptom (i.e., prevention of further development of the pathology and / or symptoms); (3) relief of disease: e.g., relief of disease, disorder, or symptom in individuals experiencing or exhibiting the pathology or symptom of the disease, disorder, or symptom (i.e., reversal of the pathology and / or symptom).

[0038] The term "post-processing" refers to operations commonly used in the art to separate products, such as filtration, centrifugation, and / or drying. Attached Figure Description

[0039] Figure 1 shows the amorphous XRPD spectrum of the compound represented by formula (I);

[0040] Figure 2 shows the XRPD spectrum of free base Type C. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0042] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0043] Nuclear magnetic resonance analysis (NMR) 1 H NMR)

[0044] Several milligrams of solid sample were dissolved in dimethyl sulfoxide-d6 or deuterated methanol solvent and analyzed by nuclear magnetic resonance on a Bruker AVANCE NEO 400 (Bruker, GER).

[0045] X-ray powder diffraction (XRPD)

[0046] The obtained solid samples were analyzed using an ARL Equinox 100 X-ray powder diffractometer. The 2θ scanning angle ranged from 0° to 35°. The testing method was Cu target Kα radiation, voltage 40 kV, current 0.9 mA, and the sample disk was a zero-background sample disk.

[0047] Example 1: Compound of Formula (I)

[0048] Step 1: Synthesis of Compounds 1-2

[0049] At room temperature, water (100 mL) was added to the reaction flask, and sodium hydroxide (11.36 g, 0.284 mol, 3.0 eq) was added with stirring. Then, ethyl 5-amino-1-tert-butyl-1H-pyrazole-4-carboxylate (compound 1-1, 20 g, 94.67 mmol, 1 equiv) was added. The mixture was heated to 60 °C and stirred overnight. After the reaction was complete, the mixture was cooled to room temperature, and the pH was adjusted to 2–2.5 with 4 mol / L hydrochloric acid. The mixture was extracted with ethyl acetate (3 × 150 mL), and the organic phases were combined. The mixture was backwashed with saturated sodium chloride (1 × 50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 1-2 (15.6 mg, 90.1%).

[0050] LCMS:(ESI,m / z):183.95[M+H] + .

[0051] Step 2: Synthesis of 5-amino-1-(tert-butyl)-N-[3-(7-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-3-(2,2,2-trifluoroethylpyrazolo[1,5-a]pyridin-2-yl)prop-2-yn-1-yl]-1H-pyrazol-4-carboxamide (the compound shown in formula (1)).

[0052] At room temperature, 5-amino-1-tert-butyl-1H-pyrazole-4-carboxylic acid (compounds 1-2, 20 mg, 0.109 mmol, 1 eq) and 2-(3-aminoprop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3-(2,2,2-trifluoroethyl)pyrazolo[1,5-a]pyridine-7-amine dihydrochloride (compound 001-12, 41.85 mg, 0.109 mmol, 1.0 eq) A solution of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (45.66 mg, 0.120 mmol, 1.1 eq) and N,N-diisopropylethylamine (70.25 mg, 0.545 mmol, 5 eq) in N,N-dimethylformamide (2 mL) was stirred and reacted for 1 hour. After the reaction was complete, the residue was concentrated under reduced pressure, and the crude product was purified by high-performance liquid chromatography under the following conditions (Column:Xselect). CSHTM Prep C18 5μm 30*150mm OBD; Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL / min; elution gradient: 15% B to 31% B in 7 min; detection wavelength: 254 nm / 220 nm; retention time (min): 6.43), yielded compound 5-amino-1-(tert-butyl)-N-[3-(7-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-3-(2,2,2-trifluoroethyl)pyrazolo[1,5-a]pyridin-2-yl)prop-2-yn-1-yl]-1H-pyrazol-4-carboxamide (9.3 mg, 15.42%), i.e., the compound shown in formula (I).

[0053] LCMS:(ESI,m / z):549.15[M+H] + .

[0054] 1H NMR (400MHz, DMSO-d6) δ8.31(t,J=5.6Hz,1H),7.65(s,1H),7.28(t,J=8.3Hz,1H),7.02(d,J=8.7Hz,1 H),6.31(d,J=7.6Hz,1H),6.25(d,J=9.0Hz,1H),6.18(s,2H),4.87(d,J=49.5Hz,1H),4.28(d,J=5.6H z,2H),3.94–3.81(m,1H),3.75(q,J=11.2Hz,2H),3.06(t,J=10.6Hz,1H),2.79(d,J=10.6Hz,1H),2.3 8–2.22(m,1H),2.21(s,3H),2.14(t,J=10.7Hz,1H),1.98–2.85(m,1H),1.87–1.79(m,1H),1.52(s,9H)

[0055] The synthesis of compound 001-12 is as follows:

[0056] Step 1: Synthesis of Compound 001-2

[0057] Acetonitrile (4.72 g, 114.872 mmol, 3.4 eq) and tetrahydrofuran (150 mL) solution were added to a reaction flask. Butyllithium (44 mL) was added dropwise at -78 °C, and the mixture was stirred for 1 hour. Then, a tetrahydrofuran solution (50 mL) containing 2,6-dichloropyridine (compound 001-1, 5 g, 33.786 mmol, 1 eq) was added dropwise. The reaction was continued at -78 °C for 2 hours, then the temperature was raised to room temperature for 30 minutes. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate (3 × 300 mL), and the organic phases were combined. The mixture was backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give compound 001-2 (4.66 g, 89.49%).

[0058] 1 H NMR (400MHz, DMSO-d6) δ7.92(t,J=7.8Hz,1H),7.52(dd,J=8.0,0.7Hz,1H),7.45(dd,J=7.6,0.7Hz,1H),4.26(s,2H).

[0059] Step 2: Synthesis of Compound 001-3

[0060] 2-(6-chloropyridin-2-yl)acetonitrile (compound 001-2, 5 g, 32.770 mmol, 1 eq) was added to a reaction flask and dissolved in dichloromethane (100 mL). A dichloromethane solution (70 mL) containing 2-[(aminooxy)sulfonyl]-1,3,5-trimethylbenzene (14.11 g, 65.546 mmol, 2.00 eq) was added at 0 °C, and the mixture was stirred at 0 °C for 2 hours. After the reaction was complete, diethyl ether was added, and the solvent was evaporated to dryness to obtain the crude product 001-3 (7 g, 40.65%), which was directly added to the next step.

[0061] Step 3: Synthesis of compound 001-4

[0062] 1-Amino-2-chloro-6-(cyanomethyl)pyridine-1-onium-2,4,6-trimethylbenzenesulfonate (compound 001-3, 8 g, 21.748 mmol, 1 eq) was added to a reaction flask and dissolved in methanol (80.0 mL). Potassium carbonate (6.01 g, 43.496 mmol, 2.00 eq) was added under ice bath conditions, and the mixture was allowed to react overnight at room temperature. After the reaction was complete, the solvent was evaporated, water was added, and the mixture was extracted with dichloromethane (3 × 100 mL). The organic phases were combined, backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50:1) to give compound 001-4 (1.7 g, 39.64%).

[0063] 1 H NMR (400MHz, DMSO-d6) δ7.28(dd,J=8.8,1.2Hz,1H),7.01(dd,J=8.8,7.3Hz,1H),6.75(dd,J=7.3,1.2Hz,1H),5.80(s,1H),5.57(s,2H).

[0064] Step 4: Synthesis of Compound 001-5

[0065] 7-chloropyrazole[1,5-a]pyridine-2-amine (compound 001-4, 1.4 g, 8.353 mmol, 1 eq), 4-dimethylaminopyridine (102.05 mg, 0.835 mmol, 0.1 eq), and di-tert-butyl dicarbonate (2.01 g, 9.188 mmol, 1.1 eq) were added sequentially to a reaction flask and dissolved in 1,4-dioxane (20 mL). The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the mixture was diluted with water and extracted with dichloromethane (3 × 20 mL). The organic phases were combined, backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give compound 001-5 (634 mg, 22.68%).

[0066] LCMS:(ESI,m / z):267.85[M+H] + .

[0067] Step 5: Synthesis of Compound 001-6

[0068] N,N-dimethylformamide (344.03 mg, 4.706 mmol, 2 eq) and tetrahydrofuran (13 mL) were added to a reaction flask. Phosphorus oxychloride (1.08 g, 7.059 mmol, 3 eq) was added at 0 °C, and the reaction was allowed to proceed for 30 minutes. Then, tert-butyl 7-chloropyrido[1,5-a]pyridin-2-yl)carbamate (compound 001-5, 630 mg, 2.353 mmol, 1 eq) was added, and the reaction was allowed to proceed to 40 °C for 30 minutes. After the reaction was complete, saturated sodium carbonate solution was added to quench the reaction. The mixture was extracted with dichloromethane (3 × 20 mL), and the organic phases were combined. The mixture was backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to directly obtain compound 001-6 (791 mg, 90.93%).

[0069] LCMS:(ESI,m / z):296.00[M+H] + .

[0070] Step 6: Synthesis of Compound 001-7

[0071] (7-chloro-3-formylpyridino[1,5-a]pyridin-2-yl)tert-butyl carbamate (compound 001-6, 740 mg, 2.502 mmol, 1 eq) was added to a reaction flask and dissolved in dichloromethane (5 mL) and trifluoroacetic acid (5 mL). The reaction was carried out at room temperature for 30 minutes. After the reaction was complete, the solvent was directly evaporated to obtain crude product 001-7 (800 mg, 138.92%), which was directly added to the next step.

[0072] LCMS:(ESI,m / z):196.00[M+H] + .

[0073] Step 7: Synthesis of Compound 001-8

[0074] In one reaction flask, 2-amino-7-chloropyrazole[1,5-a]pyridine-3-carboxaldehyde (compound 001-7, 490 mg, 2.505 mmol, 1 eq) and p-toluenesulfonic acid hydrate (1.43 g, 7.515 mmol, 3 eq) were added and dissolved in acetonitrile (8 mL). In another reaction flask, sodium nitrite (345.66 mg, 5.010 mmol, 2 eq) and potassium iodide (1.04 g, 6.262 mmol, 2.5 eq) were added and dissolved in water (2 mL). This mixed aqueous solution was then added dropwise to the acetonitrile solution and reacted overnight at room temperature. After the reaction was complete, the mixture was quenched with a saturated sodium thiosulfate aqueous solution, extracted with ethyl acetate (3 × 10 mL), the organic phases were combined, backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give compound 001-8 (180 mg, 23.21%).

[0075] 1 H NMR (400MHz, DMSO-d6) δ9.76(s,1H),8.22(dd,J=8.7,1.2Hz,1H),7.73(dd,J=8.7,7.5Hz,1H),7.54(dd,J=7.6,1.2Hz,1H).

[0076] Step 8: Synthesis of Compound 001-9

[0077] 7-Chloro-2-iodopyrazolo[1,5-a]pyridine-3-carboxaldehyde (compound 001-8, 160 mg, 0.522 mmol, 1 eq) and (triphenylphosphonium)difluoroacetic acid inner salt (372.01 mg, 1.044 mmol, 2 eq) were added to a reaction flask and dissolved in N,N-dimethylformamide (8.0 mL). The reaction was carried out at 60 °C for 2 hours, followed by the addition of tetrabutylammonium fluoride solution (1 M tetrahydrofuran solution, 1.566 mL, 1.566 mmol, 3.00 eq), and the reaction was continued for 1 hour. After the reaction was complete, ethyl acetate was added for dilution, followed by the addition of water. The mixture was extracted with ethyl acetate (3 × 10 mL), and the organic phases were combined. The mixture was backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give compound 001-9 (57 mg, 29.98%).

[0078] 1 H NMR (400MHz, DMSO-d6) δ7.85–7.82(m,1H),7.38(dd,J=8.9,7.3Hz,1H),7.25(dd,J=7.3,1.1Hz,1H),3.78(q,J=11.0Hz,2H).

[0079] Step 9: Synthesis of Compound 001-10

[0080] Under nitrogen protection at 50°C, 7-chloro-2-iodo-3-(2,2,2-trifluoroethyl)pyrazole[1,5-a]pyridine (compound 001-9, 400 mg, 1.110 mmol, 1 eq), N-Boc-aminopropyne (189.42 mg, 1.221 mmol, 1.1 eq), cuprous iodide (21.13 mg, 0.111 mmol, 0.1 eq), tetrakis(triphenylphosphine)palladium (128.22 mg, 0.111 mmol, 0.1 eq) and diisocyanate were released. Propylamine (1.12 g, 11.100 mmol, 10 eq) was dissolved in tetrahydrofuran (6 mL), and the mixture was stirred for 1 hour. After the reaction was complete, the reaction mixture was quenched with water at room temperature and extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated sodium chloride (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:9) to give compound 001-10 (400 mg, 92.96%).

[0081] LCMS:(ESI,m / z):387.95[M+H] + .

[0082] Step 10: Synthesis of Compound 001-11

[0083] Under nitrogen protection, the following compounds were released: {3-[7-chloro-3-(2,2,2-trifluoroethyl)pyrazolo[1,5-a]pyridin-2-yl]prop-2-yn-1-yl} tert-butyl carbamate (compound 001-10, 380 mg, 0.980 mmol, 1 eq), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine dihydrochloride (240.92 mg, 1.176 mmol, 1.2 eq), and (SP-4-1)-[1,3-bis[2,6-bis(1-ethylpropyl)phenyl]-4,5-dichloro-1,3-dihydro-2H-imidazol-2-ylidene]dichloro(2-methylpyridinium) Palladium (82.43 mg, 0.098 mmol, 0.1 eq) and cesium carbonate (1.277 g, 3.920 mmol, 4 eq) were dissolved in 1,4-dioxane (4 mL). The mixture was stirred at 120 °C for 4 hours. After the reaction was complete, the mixture was cooled to room temperature, diluted with water, and the reaction mixture was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, backwashed with saturated sodium chloride solution (1 × 50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10:1) to give compound 001-11 (380 mg, 80.20%).

[0084] LCMS:(ESI,m / z):483.90[M+H] + .

[0085] Step 11: Synthesis of Compound 001-12

[0086] At room temperature, N-[3-(7-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-3-(2,2,2-trifluoroethyl)pyrazol[1,5-a]pyridin-2-yl)prop-2-yn-1-yl] tert-butyl carbamate (compound 001-11, 100 mg, 0.207 mmol, 1 eq) was added to a reaction flask and dissolved in methanol (2 mL). 4 M HCl / 1,4-dioxane (2 mL) was added dropwise at 0 °C, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the residue was concentrated under vacuum to obtain compound 001-12.

[0087] LCMS:(ESI,m / z):383.85[M+H] + .

[0088] Salt formation screening was performed on the compounds shown in Formula I. Solvents for salt formation reactions were selected based on the solubility of the compounds in various commonly used solvents. For 15 acidic compounds, salt formation screening was conducted using methods such as solution suspension, dissolution, and cooling. Ultimately, 12 salt forms were identified: hydrochloride, phosphate, sulfate, methanesulfonate, lactate, maleate, malate, L-tartrate, D-tartrate, ethylsulfonate, malonate, and oxalate. Hydrobromic acid, citric acid, and p-toluenesulfonic acid failed to form salts. The specific process is as follows.

[0089] Example 2

[0090] 40 mg of the compound shown in formula (I) was added to 87.5 μl of n-heptane and 87.5 μl of 1M hydrochloric acid 1,4-dioxane solution. 100 μl of 1,4-dioxane / n-heptane (1v / 1v) was added. After stirring overnight at 50 °C, 400 μl of 1,4-dioxane / n-heptane (1v / 1v) was added. After cooling to room temperature and stirring, the compound of formula A-1 was obtained after post-treatment.

[0091] 1H NMR(400MHz,DMSO-d6)δ9.88(s,1H),8.34(t,J=5.7Hz,1H),7.65(s,1H),7.31(dd,J=8.8,7.6Hz, 1H),7.07(d,J=8.7Hz,1H),6.78(d,J=8.4Hz,1H),6.37–6.29(m,1H),6.19(s,2H),5.21(d,J=47.2 Hz,1H),4.28(d,J=5.6Hz,2H),4.08(d,J=30.1Hz,1H),3.91–3.70(m,3H),3.54–3.45(m,2H),3.17 (d,J=11.8Hz,1H),2.82(d,J=3.8Hz,3H),2.41–2.25(m,1H),2.08(d,J=14.2Hz,1H),1.52(s,9H).

[0092] It was also found that the compound shown in formula (I) could not yield the target compound in hydrochloric acid in n-propanol, acetonitrile, acetone / water (2v / 1v), and tetrahydrofuran / water (2v / 1v). Furthermore, the compound shown in formula (I) also could not yield the target compound in a 1,4-dioxane solution (1M) containing 2.2 eq of hydrochloric acid.

[0093] Example 3

[0094] 40 mg of the compound shown in formula (I) was added to 0.2 ml of n-propanol, followed by 87.5 μl of 1 M n-propanol phosphate solution. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. 0.6 ml of n-heptane was added, and the mixture was heated to 50 °C and stirred for 2-3 hours. The mixture was then cooled to room temperature and stirred for another day. After post-treatment, compound A-2 was obtained.

[0095] 1 H NMR (400MHz, DMSO-d6) δ8.32(t,J=5.7Hz,1H),7.65(s,1H),7.28(t,J=8.2Hz,1H),7.03(d,J=8.7Hz,1H),6.40–6.10(m,4H),4.91(d, J=49.1Hz,2H),4.28(d,J=5.6Hz,3H),3.16(s,2H),2.87(s,2H),2.28(s,4H),2.06–1.92(m,1H),1.86(d,J=12.6Hz,1H),1.51(s,9H).

[0096] It was also found that the compound shown in formula (I) could not yield the target compound in acetonitrile, acetone / water (2v / 1v), and tetrahydrofuran / water (2v / 1v).

[0097] Example 4

[0098] 40 mg of the compound shown in formula (I) was added to 0.6 ml of n-propanol, followed by 87.5 μl of 1 M n-propanol sulfuric acid solution. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature. After stirring at room temperature for a certain period of time, the mixture was post-treated to obtain compound A-3.

[0099] 1 H NMR(400MHz, DMSO-d6)δ9.72(s,1H),8.33(t,J=5.7Hz,1H),7.65(s,1H),7.31(dd,J=8.8,7.6 Hz,1H),7.07(d,J=8.7Hz,1H),6.78(d,J=8.5Hz,1H),6.45–5.96(m,3H),5.21(d,J=47.2Hz,1 H),4.28(d,J=5.6Hz,2H),4.19–4.02(m,2H),3.49(td,J=28.8,26.0,11.7Hz,3H),3.29–3.11 (m,1H),2.83(d,J=3.7Hz,3H),2.30(p,J=11.5,10.4Hz,1H),2.15–2.02(m,1H),1.52(s,9H).

[0100] It was also found that the compound shown in formula (I) could not yield the target compound in acetonitrile and tetrahydrofuran / water (2v / 1v) with sulfuric acid.

[0101] Example 5

[0102] 40 mg of the compound shown in formula (I) was added to 0.6 ml of n-propanol, followed by 87.5 μl of 1 M methanesulfonic acid n-propanol solution. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. After post-treatment, compound A-4 was obtained.

[0103] 1H NMR (400MHz, DMSO-d6) δ9.73 (s, 1H), 8.33 (t, J = 5.7Hz, 1H), 7.65 (s, 1H), 7.31 (dd, J = 8.8, 7. 6Hz,1H),7.07(d,J=8.7Hz,1H),6.78(d,J=8.5Hz,1H),6.35–6.11(m,3H),5.33–5.12(m,1H) ,4.28(d,J=5.7Hz,2H),4.08(d,J=29.8Hz,1H),3.84(dd,J=21.1,9.5Hz,1H),3.50(d,J=12. 3Hz, 2H), 3.19 (s, 1H), 2.83 (s, 3H), 2.31 (s, 4H), 2.10 (dd, J = 17.6, 13.4Hz, 1H), 1.52 (s, 9H).

[0104] A peak of methanesulfonic acid was visible at 2.83 ppm. Based on the NMR integral results, n = 0.9 in compound A-4.

[0105] It was also found that the compound shown in formula (I) could not yield the target compound in acetonitrile, acetone / water (2v / 1v), and tetrahydrofuran / water (2v / 1v). Furthermore, the compound shown in formula (I) also failed to yield the target compound in a 2.2 eq solution of methanesulfonic acid in n-propanol (1M).

[0106] Example 6

[0107] 40 mg of the compound shown in formula (I) was added to 0.4 ml of n-propanol, followed by 87.5 μl of 1 M DL-lactic acid n-propanol solution. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. After post-treatment, compound A-5 was obtained.

[0108] 1H NMR (400MHz, DMSO-d6) δ8.33(t,J=5.7Hz,1H),7.65(s,1H),7.28(dd,J=8.8,7.6Hz,1H),7.03 (d,J=8.7Hz,1H),6.39–6.14(m,4H),4.88(d,J=49.5Hz,1H),4.28(d,J=5.6Hz,2H),4.02(q,J= 6.9Hz,1H),3.87(s,1H),3.75(q,J=11.2Hz,2H),3.06(t,J=11.6Hz,1H),2.79(d,J=11.3Hz,1H ),2.34(d,J=12.7Hz,1H),2.21(s,4H),2.02–1.77(m,2H),1.52(s,9H),1.22(d,J=6.9Hz,2H).

[0109] Lactic acid peaks are visible at 4.02 ppm and 1.22 ppm. Based on the NMR integral results, n = 0.5 in compound A-5.

[0110] Example 7

[0111] 40 mg of the compound shown in formula (I) was added to 0.4 ml of acetonitrile, followed by 87.5 μl of 1 M DL-lactic acid acetonitrile solution. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. After post-treatment, compound A-6 was obtained.

[0112] 1 H NMR (400MHz, DMSO-d6) δ8.33(t,J=5.7Hz,1H),7.66(s,1H),7.28(dd,J=8.8,7.6Hz,1H),7.03(d,J= 8.6Hz,1H),6.36–6.16(m,4H),4.88(d,J=49.4Hz,1H),4.29(d,J=5.6Hz,2H),4.03(q,J=6.9Hz,1H), 3.75(q,J=11.2Hz,3H),3.07(t,J=11.3Hz,1H),2.79(d,J=11.3Hz,1H),2.35(d,J=13.8Hz,1H),2.2 9–2.12(m,4H),1.94(qd,J=11.7,3.5Hz,1H),1.88–1.80(m,1H),1.52(s,9H),1.23(d,J=6.9Hz,3H).

[0113] Lactic acid peaks are visible at 4.03 ppm and 1.23 ppm. Based on the NMR integral results, n = 1 in compound A-6.

[0114] It was also found that the compound shown in formula (I) could not yield the target compound in acetone / water (2v / 1v) and tetrahydrofuran / water (2v / 1v) with DL-lactic acid.

[0115] Example 8

[0116] 40 mg of the compound shown in formula (I) was added to 0.2 ml of n-propanol and 10.2 mg of maleic acid. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. The mixture was then cooled to 5 °C and stirred overnight. After post-treatment, compound A-7 was obtained.

[0117] 1 H NMR(400MHz, DMSO-d6)δ9.73(s,1H),8.33(t,J=5.7Hz,1H),7.65(s,1H),7.31(dd,J=8.7,7.6 Hz,1H),7.07(d,J=8.6Hz,1H),6.75(d,J=8.4Hz,1H),6.30(d,J=7.6Hz,1H),6.19(s,2H),6.0 4(s,2H),5.19(d,J=47.3Hz,1H),4.28(d,J=5.7Hz,2H),4.05(d,J=29.9Hz,1H),3.76(q,J=11 .0Hz,3H),3.10(s,2H),2.78(s,3H),2.39–2.20(m,1H),2.06(d,J=13.6Hz,1H),1.52(s,9H).

[0118] A signal peak of maleic acid was visible at 6.04 ppm, calculated from the NMR integral results, where n = 1.0 in compound A-7.

[0119] Example 9

[0120] 40 mg of the compound shown in formula (I) was added to 0.4 ml of acetone / n-heptane (1v / 1v), and 10.2 mg of maleic acid was added. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. The temperature was then raised to 50 °C and stirred overnight. After post-treatment, compound A-8 was obtained.

[0121] 1H NMR(400MHz,DMSO-d6)δ8.33(t,J=5.7Hz,1H),7.65(s,1H),7.37–7.27(m,1H ),7.05(d,J=8.7Hz,1H),6.57(s,1H),6.31(d,J=7.6Hz,1H),6.19(s,2H),6.0 2(s,1H),5.07(d,J=48.1Hz,1H),4.28(d,J=5.6Hz,2H),3.94(s,1H),3.76(q, J=11.0Hz,2H),2.67(t,J=2.0Hz,4H),2.15(s,1H),1.99(s,2H),1.52(s,9H).

[0122] A signal peak of maleic acid was visible at 6.02 ppm, calculated from the NMR integral results, where n = 0.5 in compound A-8.

[0123] It was also found that the compound shown in formula (I) could not yield the target compound in acetonitrile and acetone / n-heptane (1v / 1v) with maleic acid.

[0124] Example 10

[0125] 40 mg of the compound shown in formula (I) was added to 0.8 ml of n-propanol and 10.7 mg of L-malic acid. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. After post-treatment, compound A-9 was obtained.

[0126] 1 H NMR(400MHz, DMSO-d6)δ8.33(t,J=5.7Hz,1H),7.65(s,1H),7.37–7.24(m,1H),7.04(d,J=8.7Hz, 1H),6.47–6.11(m,4H),4.95(d,J=48.9Hz,1H),4.28(d,J=5.6Hz,2H),4.18(t,J=6.5Hz,1H),3.89 (d,J=29.3Hz,1H),3.76(q,J=11.1Hz,2H),2.95(d,J=11.3Hz,1H),2.64–2.56(m,2H),2.41(dd,J= 15.6,7.1Hz,2H),2.35(s,3H),2.01(dd,J=13.1,9.6Hz,1H),1.89(d,J=12.9Hz,1H),1.52(s,9H).

[0127] Peaks of malic acid were observed at 4.28 ppm, 2.41 ppm, and 2.57 ppm. Based on the NMR integral results, n = 1.5 to 2 was calculated in compound A-9.

[0128] It was also found that the compound shown in formula (I) could not yield the target compound in acetonitrile, acetone / n-heptane (1v / 1v), and ethyl acetate / n-heptane (1v / 1v).

[0129] Example 11

[0130] 40 mg of the compound shown in formula (I) was added to 0.2 ml of n-propanol and 13.1 mg of L-tartaric acid. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. 0.2 ml of n-heptane was added and the mixture was stirred overnight at room temperature. After post-treatment, compound A-10 was obtained.

[0131] 1 H NMR(400MHz,DMSO-d6)δ8.32(t,J=5.7Hz,1H),7.65(s,1H),7.28(dd,J=8.8,7.6Hz, 1H),7.03(d,J=8.7Hz,1H),6.32(t,J=8.3Hz,2H),6.19(s,2H),4.92(d,J=49.1Hz,1H ),4.28(d,J=5.7Hz,2H),4.23(s,2H),3.87(d,J=28.8Hz,2H),3.21(s,3H),2.89(d, J=11.6Hz,2H),2.30(s,4H),2.08–1.94(m,1H),1.87(d,J=11.7Hz,1H),1.52(s,9H).

[0132] A signal peak of L-tartaric acid was visible at 4.28 ppm. Based on the NMR integral results, n = 1 to 1.5 was calculated in compound A-10.

[0133] It was also found that the compound shown in formula (I) could not yield the target compound in acetonitrile, acetone / n-heptane (1v / 1v), and ethyl acetate / n-heptane (1v / 1v).

[0134] Example 12

[0135] 40 mg of the compound shown in formula (I) was added to 0.2 ml of n-propanol and 13.1 mg of D-tartaric acid. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. 0.2 ml of n-heptane was added and the mixture was stirred overnight at room temperature. After post-treatment, compound A-11 was obtained.

[0136] 1 H NMR (400MHz, DMSO-d6) δ8.32(t,J=5.7Hz,1H),7.65(s,1H),7.28(dd,J=8.8,7.6Hz,1H),7.0 3(d,J=8.7Hz,1H),6.33(dd,J=12.2,8.3Hz,2H),6.19(s,2H),4.93(d,J=49.2Hz,2H),4.28( d,J=5.6Hz,2H),4.23(s,3H),3.87(d,J=28.7Hz,2H),3.21(d,J=12.5Hz,2H),2.91(d,J=11. 4Hz,1H),2.31(d,J=2.5Hz,4H),2.00(q,J=12.0Hz,1H),1.87(d,J=12.7Hz,1H),1.51(s,9H).

[0137] A peak of D-tartaric acid was observed at 4.28 ppm, indicating that the compound was successfully prepared. Based on the NMR integral results, n = 1 to 1.5 was calculated in compound A-11.

[0138] It was also found that the compound shown in formula (I) could not yield the target compound in acetonitrile, acetone / n-heptane (1v / 1v), and ethyl acetate / n-heptane (1v / 1v).

[0139] Example 13

[0140] 40 mg of the compound shown in formula (I) was added to 0.8 ml of n-propanol, followed by 87.5 μl of 1 M ethylsulfonic acid n-propanol solution. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. After post-treatment, compound A-12 was obtained.

[0141] 1H NMR(400MHz,DMSO-d6)δ9.73(s,1H),8.33(t,J=5.7Hz,1H),7.65(s,1H),7.32(dd,J=8.7,7.6Hz,1H),7.07 (d,J=8.7Hz,1H),6.78(d,J=8.5Hz,1H),6.31(d,J=7.6Hz,1H),6.20(s,2H),5.21(d,J=47.3Hz,1H),4.28(d ,J=5.7Hz,2H),4.08(d,J=30.3Hz,1H),3.78(p,J=12.0,11.2Hz,3H),3.49(d,J=11.2Hz,2H),3.18(s,1H), 2.82(s,3H),2.36(qd,J=7.5,2.4Hz,3H),2.08(d,J=14.0Hz,1H),1.52(s,9H),1.05(td,J=7.5,0.9Hz,3H).

[0142] The presence of ethylsulfonic acid peaks at 1.05 ppm and 3.78 ppm indicates that the compound was successfully prepared. Based on the NMR integral results, n = 1 was calculated in compound A-12.

[0143] It was also found that the compound shown in formula (I) could not yield the target compound in acetonitrile, acetone / water (2v / 1v), and tetrahydrofuran / water (2v / 1v).

[0144] Example 14

[0145] 40 mg of the compound shown in formula (I) was added to 0.2 ml of n-propanol and 9.1 mg of malonic acid. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. 0.1 ml of n-heptane was added and the mixture was stirred overnight at room temperature. After post-treatment, compound A-13 was obtained.

[0146] 1H NMR (400MHz, DMSO-d6) δ8.33(t,J=5.8Hz,1H),7.65(s,1H),7.29(dd,J=8.7,7.6Hz,1H),7.04( d,J=8.7Hz,1H),6.48(d,J=8.8Hz,1H),6.31(d,J=7.3Hz,1H),6.19(s,2H),5.02(d,J=48.6Hz,1 H),4.28(d,J=5.7Hz,2H),3.94(d,J=28.9Hz,2H),3.76(q,J=11.1Hz,3H),3.08(d,J=11.5Hz,1H ),3.00(s,2H),2.47(s,3H),2.10(q,J=11.8,11.4Hz,1H),1.94(d,J=12.9Hz,1H),1.52(s,9H).

[0147] A peak of malonic acid was observed at 3.00 ppm. Based on the NMR integral results, n = 1.05 was calculated in compound A-13.

[0148] Example 15

[0149] 40 mg of the compound shown in formula (I) was added to 0.6 ml of ethyl acetate / n-heptane (1 v / 1 v), and 9.1 mg of malonic acid was added. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. The temperature was then raised to 50 °C and stirred overnight. After post-treatment, compound A-14 was obtained.

[0150] 1 H NMR(400MHz,DMSO-d6)δ8.33(t,J=5.6Hz,1H),7.65(s,1H),7.35–7.26(m,1H),7.04 (d,J=8.7Hz,1H),6.41(d,J=8.8Hz,1H),6.32(d,J=7.6Hz,1H),6.20(s,2H),4.98(d, J=48.8Hz,1H),4.28(d,J=5.6Hz,2H),3.91(d,J=28.8Hz,2H),3.76(q,J=11.1Hz,3H ),2.95(s,3H),2.39(s,3H),2.14–1.98(m,1H),1.91(d,J=13.4Hz,1H),1.52(s,9H).

[0151] A peak of malonic acid was observed at 2.95 ppm. Based on the NMR integral results, n = 0.65 was calculated in compound A-14.

[0152] Example 16

[0153] 40 mg of the compound shown in formula (I) was added to 0.2 ml of n-propanol and 7.9 mg of oxalic acid. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. After post-treatment, compound A-15 was obtained.

[0154] 1 H NMR (400MHz, DMSO-d6) δ8.34(t,J=5.7Hz,1H),7.65(s,1H),7.30(t,J=8.2Hz,1H),7.06(d,J=8.7Hz,1H ),6.60(d,J=8.6Hz,1H),6.32(d,J=7.6Hz,1H),6.20(s,2H),5.10(d,J=48.0Hz,2H),4.28(d,J=5.6Hz,2 H), 4.01 (d, J = 29.0Hz, 2H), 3.76 (q, J = 11.1Hz, 2H), 3.58 (s, 1H), 3.26 (d, J = 11.9Hz, 1H), 3.08 (dd, J = 39. 2,13.8Hz,1H),2.91–2.78(m,1H),2.62(s,3H),2.19(q,J=12.2Hz,1H),2.07–1.96(m,1H),1.52(s,9H).

[0155] Example 17

[0156] 40 mg of the compound shown in formula (I) was added to 0.6 ml of acetonitrile and 7.9 mg of oxalic acid. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. After post-treatment, compound A-16 was obtained.

[0157] 1H NMR (400MHz, DMSO-d6) δ8.34(t,J=5.7Hz,1H),7.66(s,1H),7.31(dd,J=8.7,7.6Hz,1H),7.06(d,J=8.7Hz,1H ),6.62(d,J=8.6Hz,1H),6.32(d,J=7.6Hz,1H),6.20(s,2H),5.12(d,J=48.0Hz,2H),4.28(d,J=5.7Hz,2H),4 .03(d,J=28.7Hz,2H),3.76(q,J=11.1Hz,2H),3.62(t,J=11.9Hz,1H),3.29(s,1H),3.15(dd,J=39.2,13.7Hz ,1H),2.90(t,J=12.5Hz,1H),2.66(s,3H),2.22(q,J=12.4,11.9Hz,1H),2.01(d,J=13.7Hz,1H),1.52(s,9H).

[0158] It was also found that the compound shown in formula (I) could not yield the target compound when reacted with oxalic acid in ethyl acetate / n-heptane (1v / 1v).

[0159] Example 18

[0160] 40 mg of the compound shown in formula (I) was added to 0.6 ml of acetone / n-heptane (1 v / 1 v), and 9.1 mg of malonic acid was added. The mixture was stirred at 50 °C for 2-3 hours, then cooled to room temperature and stirred overnight. After post-treatment, compound A-17 was obtained.

[0161] 1 H NMR (400MHz, DMSO-d6) δ8.32(t,J=5.7Hz,1H),7.65(s,1H),7.29(dd,J=8.7,7.6Hz,1 H),7.04(d,J=8.6Hz,1H),6.43(d,J=8.5Hz,1H),6.31(d,J=7.6Hz,1H),6.19(s,2H), 4.99(d,J=48.7Hz,1H), 4.28(d,J=5.6Hz,2H), 3.91(d,J=28.4Hz,2H), 3.76(q,J=11. 0Hz, 3H), 2.97 (s, 3H), 2.41 (s, 3H), 2.08 (s, 3H), 1.92 (d, J = 10.1Hz, 1H), 1.52 (s, 9H).

[0162] A peak of malonic acid was observed at 2.97 ppm. Based on the NMR integral results, the n = 0.5 to 1 was calculated in compound A-17.

[0163] Example 19

[0164] The compound shown in formula (I) was rotary evaporated to dryness with a mixed solvent of ethyl acetate and n-heptane to obtain the amorphous form of the compound shown in formula (I) (Figure 1). 50 mg of the amorphous form of the compound shown in formula (I) was weighed as a raw material, 0.2 mL of n-propanol was added to form a suspension, and the mixture was stirred at 50 °C for one week. After centrifuging the suspension, a solid was obtained. This solid was characterized by XRPD (Figure 2, Table 1) and was found to be a crystalline form, which was designated as the free base Type C.

[0165] Table 1

[0166] Test Example 1 Solubility Test

[0167] The following compounds were weighed separately and added to an appropriate amount of water, stirred at 37 °C for 24 h, sampled at 2 h, and the sampled solution was filtered through a 0.22 μm aqueous filter membrane. Samples with relatively high concentrations were appropriately diluted with a diluent, and the signal peak area of the solution was measured by HPLC. Finally, the concentration of the compound in the solution was calculated based on the peak area, the HPLC standard curve of the raw material, and the dilution factor. The results are shown in Table 2.

[0168] Table 2 Solubility Results

[0169] Note: LOQ is the limit of quantitation, and <LOQ means less than 0.0005 mg / mL.

[0170] Among them, the HPLC test conditions in this test example are shown in Table 3:

[0171] Table 3

[0172] The structures of the control compounds in Test Examples 2 - 4 are as follows:

[0173] Test Example 2 In Vitro DNA Binding Activity Experiment

[0174] Time-resolved fluorescence resonance energy transfer (TR-FREt) was used to detect the activity of compounds regulating the binding of the p53-Y220C mutant protein to DNA. First, recombinant Y220C p53DBD protein with a his-tag and a biotin-tagged DNA sequence were prepared. The donor, Eu(Europium)-SA (Streptavidin), binds to the DNA sequence via biotin. The acceptor is a his antibody linked to allophycocyanin (APC), which binds to the his-tag Y220C p53DBD protein. When excited by 340 nm light, the donor Eu(Europium)-SA (Streptavidin) emits fluorescence at 615 nm. If the donor and acceptor are close together, the donor transfers some energy to the acceptor, producing fluorescence at 665 nm. In other words, if the p53 mutant protein is reactivated by a compound and binds close to DNA, the donor energy is transferred to the acceptor, resulting in fluorescence at 615 and 665 nm. If there is no interaction, only 615 nm fluorescence will be observed.

[0175] 1) Experimental Procedure

[0176] Add 4 μL of diluted compound solution to each well of the a.384 microplate.

[0177] Add 4 μL of 4X P53 working solution to each well of the b.384 microplate.

[0178] c. Seal the 384 microplate and equilibrate at room temperature for 60 minutes.

[0179] d. Add 4 μl of biotinylated DNA solution to a 384 microplate.

[0180] e. Add 4 μl of 4X detection solution (MAb Anti-6His-Eu and streptavidin-d2) to each well of the 384 microplate.

[0181] f. Incubate overnight, away from light.

[0182] g. Read the data at wavelengths of 665nm and 615nm on the BMG multi-functional microplate reader.

[0183] 2) Data Analysis:

[0184] a. Calculate the ratio per well (Ratio665nm / 615nm - Ratiobackground)

[0185] b. Percentage of activity (%) activity The calculation is as follows: C = (Ave) _Ac -Ave _Ba ) / (Ave _Dc -Ave _Bd R data=(A-Ave) _Ba -CD) / (D-Ave _Bd )*(Ave _Dc -Ave _Bd ) %Activity=R data / Ave_VC*100

[0186] A: Fluorescence intensity of the sample at 665 nm; D: Fluorescence intensity of the sample at 615 nm;

[0187] Ba: Background fluorescence intensity of the culture plate at 665 nm; Bd: Background fluorescence intensity of the culture plate at 615 nm;

[0188] Dc: Background fluorescence intensity of HIS-E at 615 nm; Ac: Background fluorescence intensity of streptavidin-d2 at 665 nm.

[0189] c. Calculate SC 150 With drawing dose-response curves

[0190] The percentage of activation of p53 mutant protein binding to DNA in the presence of the compound of the present invention, compared with the absence of the compound, is expressed as SC. 150 The value indicates the concentration of the compound required to increase the DNA-binding activity of the p53 mutant protein by 50%. (Using Prism) TM Calculate SC 150 value.

[0191] The reactivation activity of the compounds of this invention against the p53-Y220C mutant protein was determined by the above experiments, and the measured SC... 150 The values ​​are shown in Table 4.

[0192] Table 4. SC of the activity of compounds against p53-Y220C mutant protein 150 value

[0193] Note: SC 150 This indicates the concentration of the compound required to increase protein-DNA binding activity by 50%.

[0194] Experimental conclusion: The above representative compounds can effectively restore the activity of p53-Y220C mutant protein.

[0195] Test Example 3: NUGC-3 Cell Proliferation Experiment

[0196] Experimental process

[0197] 1. Cell Culture

[0198] The cells were cultured strictly in accordance with ATCC requirements.

[0199] Cell culture medium: 1640 medium, 10% serum, 1% penicillin + streptomycin antibiotics.

[0200] Culture conditions: 37℃, 95% air, 5% carbon dioxide.

[0201] 2. Compound treatment and cell suspension preparation

[0202] The compound (20 mM) was serially diluted using DMSO: 10 gradients, 3-fold dilution, double-dopants.

[0203] Prepare positive and negative controls (100% dimethyl sulfoxide).

[0204] The DMSO serially diluted compound and the positive and negative controls (steps 1 and 2) were transferred to 75 nL into 384 cell culture plates using an Echo550.

[0205] After washing the cells twice with PBS, the cells were digested with trypsin and centrifuged to count the cells.

[0206] Experiments can only be conducted when the cell viability is above 90%.

[0207] Seed the cells into 384 cell culture plates, 200 (NUGC-3) cells per well, 30 μL of culture medium.

[0208] The 384 microplates were placed in a cell culture incubator and cultured for 7 days.

[0209] 3 tests

[0210] Add 30 μL of CTG detection reagent to a 384 microplate.

[0211] The 384 microplate was incubated at 37°C with 5% carbon dioxide for 30 minutes in the dark.

[0212] Readings were taken using the Envision multi-functional microplate reader.

[0213] 4. Data Analysis

[0214] %Inhibition is calculated as follows: %Inhibition = 100 - (Signal) cmpd -Signal Ave_BL ) / (Signal Ave_VC-Signal Ave_BL )*100

[0215] Signal cmpd The average signal of compounds in the cell plate

[0216] Signal ave_vc Mean signal of negative control in cell plate

[0217] Signal ave_BL Average signal in blank wells of cell plate

[0218] Computing IC 50 And plot the effect-dose curve.

[0219] Using Graphpad 8.0, the %Inhibition and the logarithm of the compound concentration were fitted to a nonlinear regression (dose response-variable slope) to calculate the IC. 50 .

[0220] The IC50 of the compound of this invention inhibits the proliferation of NUGC-3 cells. 50 The values ​​are shown in Table 5.

[0221] Table 5. IC50 values ​​of selected compounds in NUGC-3 cell proliferation assay 50

[0222] Experimental conclusion: The compound shown in formula (I) exhibits significantly better activity than the control compound in inhibiting the proliferation of NUGC-3 cells containing the p53-Y220C mutation.

[0223] Test Example 4: BXPC-3 3D Cell Proliferation Experiment

[0224] Experimental process

[0225] 1) Preparation of 1% methylcellulose solution

[0226] Weigh 1g of methylcellulose into a glass dish and autoclave. After cooling, add 100mL of the corresponding culture medium, mix vigorously, and stir with a magnetic stirrer at 4℃. After 48h, it will be completely dissolved and stored at 4℃.

[0227] 2) Cell Culture

[0228] Resuscitate cells with appropriate culture medium, maintain cell growth to the logarithmic growth phase, collect cell suspension, centrifuge at 1000 rpm for 5 minutes, remove supernatant after centrifugation, and resuspend cells in an appropriate amount of culture medium.

[0229] 3) 3D culture method to determine the inhibitory effect of compounds on tumor cells

[0230] Day 1: Cell Plating

[0231] Count the cells from the above cell suspension, and freeze a portion of the cells for later use. Adjust the cell suspension concentration by diluting it with culture medium according to a density of 8000 cells per well. Mix 3.5 mL of cell suspension with 6.5 mL of 1% methylcellulose thoroughly, avoiding air bubbles as much as possible. Plate the cells according to the required number of cells for testing. Add 90 μL of cell suspension to each well of a 96-well plate, set up a T0 plate, and culture the cells at 37°C, 5% CO2, and 95% aerosol.

[0232] Day 2: T0 board reading

[0233] Add 10 μL of culture medium containing solvent to each well and perform CTG plate reading. Melt the CTG reagent and equilibrate the cell plate to room temperature for 30 min; add an equal volume of CTG solution to each well (e.g., add 100 μL CTG / 100 μL cell culture medium to a 96-well plate); mix thoroughly for 5 min to lyse the cells. Place the cell plate at room temperature for 10 min to stabilize the luminescence signal, and read the luminescence value using EnVision.

[0234] Day 2: Add the drug to be tested

[0235] Dilute the test compound with culture medium to prepare a 10× positive control solution. Add 10 μL of the drug solution to each well (three replicates per cell concentration). The highest concentration of the test compound was 50 μM, with 9 concentrations and 3.5-fold dilutions. The highest concentration of the positive control was 100 μM, with 9 concentrations and 3-fold dilutions.

[0236] Cell culture was performed at 37°C and 5% CO2.

[0237] Day 9: Plate readings (7 days of drug treatment)

[0238] Melt the CTG reagent and equilibrate the cell culture plate to room temperature for 30 min. Add an equal volume of CTG solution to each well (e.g., add 100 μL CTG / 100 μL cell culture medium to a 96-well plate); mix thoroughly for 5 min to lyse the cells. Place the cell culture plate at room temperature for 10 min to stabilize the luminescence signal, and read the luminescence value using EnVision.

[0239] 4) Data Analysis

[0240] Cell viability is expressed by the formula: (V sample -V Blank ) / (V vehicle control -V Blank Calculate by multiplying V by 100%. sample For the drug treatment group, V vehicle control V represents the average value of the solvent control group. BlankThis represents the average value of the culture medium wells (blank wells). Using GraphPad Prism software, a nonlinear regression model was used to plot an S-shaped dose-survival curve and calculate the IC50. 50 value.

[0241] IC50 of the compound on the inhibition of BXPC-3 3D cell proliferation 50 The values ​​are shown in Table 6.

[0242] Table 6 IC50 values ​​of selected compounds in the BXPC-3 3D cell proliferation assay 50

[0243] Experimental conclusion: The compound shown in formula (I) can significantly inhibit the proliferation of BXPC-3 cells containing the p53-Y220C mutation, and its activity is superior to that of the control compound.

[0244] Test Example 5: Pharmacokinetic Experiment

[0245] I. Free bases Type C and compound A-8 target females and males. Pharmacokinetic study of cynomolgus monkeys after a single oral dose of 100 mpk

[0246] Crab-eating macaques source: Animals transferred from the experimental facility's animal reserve (999M-014).

[0247] Sample collection and processing: Blood samples were collected via the femoral vein or other suitable vein, approximately 1 mL per sample, anticoagulated with K2-EDTA. After collection, blood samples were placed on ice and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 2200g, 10 minutes, 2-8℃). Plasma samples were stored at -80℃ before analysis, and any remaining plasma samples after analysis were also stored at -80℃.

[0248] Experimental procedure:

[0249] Solvent: 5% TPGS + 95% (0.5% MC solution); Administration: Weigh the patient before administration and calculate the dosage based on body weight. Administer orally via gavage once on the day of administration; Blood collection time points: PO: 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h after administration; Sample collection and processing: Collect approximately 1mL of blood via the femoral vein or other suitable vein, using K2-EDTA as anticoagulant. Place blood samples on ice after collection and centrifuge to separate plasma within 1 hour (centrifugation conditions: 2200g, 10 minutes, 2-8℃). Store plasma samples at -80℃ before analysis, and continue to store remaining plasma samples at -80℃ after analysis.

[0250] II. PK experiments were conducted on free base Type C and compound A-8 in cynomolgus monkeys, and the data are as follows:

[0251] Free bases Type C and compound A-8 in female cynomolgus monkeys at 100 mpk max (ng / mL) were 2575.26 and 5568.97, respectively; AUC (0-t) (h*ng / mL) were 36034.51 and 84814.2, respectively; C in males max (ng / mL) were 3189.15 and 5317.7, respectively; AUC (0-t) The (h*ng / mL) values ​​were 49512.51 and 89755.96, respectively; the exposure of compound A-8 in cynomolgus monkeys was 2 to 3 times higher than that of the free base Type C.

[0252] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound having the structure shown in Formula A: in, X represents acid, and n is a number from 0.1 to 3.

2. The compound according to claim 1, wherein, The acid is an inorganic acid or an organic acid; For example, the inorganic acid is selected from one or more of hydrochloric acid, phosphoric acid, and sulfuric acid; For example, the organic acid is selected from one or more of methanesulfonic acid, lactic acid, maleic acid, malic acid, tartaric acid, ethylsulfonic acid, malonic acid, and oxalic acid.

3. The compound according to claim 1, wherein, The n is a number between 0.3 and 2.

5.

4. The compound according to claim 2, wherein, The organic acid is its racemic form, D configuration, or L configuration.

5. The compound according to claim 1, wherein, When X represents methanesulfonic acid, n is a number between 0.7 and 1.3; When X represents lactic acid, n is a number between 0.5 and 1.3; When X represents maleic acid, n is a number between 0.5 and 1.3; When X represents malic acid, n is a number ranging from 0.9 to 2.2; When X represents tartaric acid, n is a number ranging from 0.9 to 2.2; When X represents ethylsulfonic acid, n is a number ranging from 0.9 to 1.3; When X represents malonic acid, n is a number ranging from 0.5 to 1.

3.

6. A method for preparing the compound according to any one of claims 1-5, comprising: The compound shown in formula (I) is reacted with acid X to give a compound having the structure shown in formula A; 7. The preparation method according to claim 6, wherein, The reaction is carried out in a solvent, for example, the solvent is selected from one or more of n-propanol, n-heptane, acetonitrile, water, acetone, tetrahydrofuran and ethyl acetate; And / or, the temperature of the reaction is 40–60°C.

8. The preparation method according to claim 7, wherein, When X is methanesulfonic acid, malic acid, tartaric acid, or ethylsulfonic acid, the solvent is n-propanol; When X is lactic acid, the solvent is n-propanol and / or acetonitrile; When X is maleic acid, the solvent is one or more of n-propanol, acetone, and n-heptane; When X is malonic acid, the solvent is one or more of n-propanol, ethyl acetate, n-heptane, and acetone; When X is oxalic acid, the solvent is n-propanol and / or acetonitrile.

9. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the compound according to any one of claims 1-5; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition further contains one or more therapeutic agents.

10. Use of the compound of any one of claims 1-5 or the pharmaceutical composition of claim 9 in the preparation of a tumor drug against p53-Y220C mutant; Preferably, the tumors containing the p53-Y220C mutant include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive cell tumor, neuroectodermal tumors, visual pathway and hypothalamic gliomas, breast cancer, bronchial adenoma, Burkitt lymphoma, unknown primary cancers, central nervous system lymphomas, and cerebellar astrocytoma. Cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, fibroblastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer such as non-small cell lung cancer and small cell lung cancer. Lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma with occult primary carcinoma, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal carcinoma, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell carcinoma, paranasal sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pineal gland. Astrocytoma, pineal germ cell tumor, pituitary adenoma, pleural pulmonary blastoma, plasmacytoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, cutaneous Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy), cancer of unknown primary location, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and nephroblastoma.

11. A method of treating a tumor, comprising administering to a patient a therapeutically effective amount of the compound of any one of claims 1-5 or the pharmaceutical composition of claim 9; wherein the tumor is a tumor containing a p53-Y220C mutant; Preferably, the tumors containing the p53-Y220C mutant include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive cell tumor, neuroectodermal tumors, visual pathway and hypothalamic gliomas, breast cancer, bronchial adenoma, Burkitt lymphoma, unknown primary cancers, central nervous system lymphomas, and cerebellar astrocytoma. Cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, fibroblastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer such as non-small cell lung cancer and small cell lung cancer. Lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma with occult primary carcinoma, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal carcinoma, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell carcinoma, paranasal sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pineal gland. Astrocytoma, pineal germ cell tumor, pituitary adenoma, pleural pulmonary blastoma, plasmacytoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, cutaneous Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy), cancer of unknown primary location, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and nephroblastoma.

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