Deuterated indolizine compounds, methods of making and using the same

Abstract

The application discloses a kind of deuterated indolizine compounds and application.The application provides a kind of compound as shown in formula (I), its isotope marker, its solvate, its pharmaceutically acceptable salt, the solvate of its pharmaceutically acceptable salt or its prodrug.The compound can enhance the ability of p53Y220C binding DNA, and has better inhibitory activity to cancer cell proliferation.

Description

A class of deuterated indoleazine compounds and their preparation method and application

[0001] This application claims the benefit of Chinese patent application No. 2023113176966, filed on October 12, 2023. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the field of medicine, and specifically relates to a class of deuterated indoleazine compounds and a preparation method and application thereof. Background Art

[0003] The protein product of the TP53 gene (p53) is considered to be one of the most important tumor suppressors to date. Unlike RB1, CDKN2A, and PTEN, which are lost in tumors through homozygous deletion, TP53 is often found to have somatic missense mutations (Alexandrova EM et al. p53 loss-of-heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain-of-function in vivo. Cell Death Dis, 2017, 8(3): e2661-e2661). However, to date, targeting p53 mutations has not been validated in clinical treatment (Levine A J. Targeting therapies for the p53 protein in cancer treatments. Annu Rev Cancer Biol, 2019, 3: 21-34). TP53 mutant cells accumulate high levels of mutant p53 protein, which has a dominant-negative effect on wild-type (WT) p53 and its homologous proteins p63 and p73, causing them to lose their normal regulatory functions on cell cycle and apoptosis (Boettcher S et al. A dominant-negative effect drives selection of Tp53 missense mutations in myeloid malignancies. Science, 2019, 365(6453): 599-604). Experimental results of conditional regulation of p53 expression in mouse tissues have shown that in different mouse models, restoration of p53 expression can inhibit the growth of lymphoma and sarcoma through cell apoptosis (Christophorou MA et al. Temporal dissection of p53 function in vitro and in vivo [J]. Nat Genet, 2005, 37 (7): 718-726; Martins CP, Brown-Swigart L., Evan GI Modeling the Therapeutic Efficacy of p53 Restoration in Tumors. Cell. 2006; 127: 1323–1334), providing a theoretical basis for tumor treatment by restoring the normal function of p53.In addition to enhancing wild-type p53 activity in tumors with an intact Tp53 genotype, restoring its wild-type protein function in tumors expressing mutant p53 is also a promising cancer treatment strategy (Yu X et al. Allele-specific p53 mutant reactivation. Cancer cell, 2012, 21(5): 614-625; Chen S et al. Arsenic trioxide rescues structural p53 mutations through a cryptic allosteric site. Cancer cell, 2021, 39(2): 225-239.e8).

[0004] The core domain of wild-type p53 is unstable, with low thermodynamic and kinetic stability, allowing rapid cycling between folded and unfolded states. Mutation of these core region residues in mutant p53 leads to enhanced thermodynamic and kinetic instability, thereby causing the core DNA binding domain to lose DNA binding activity. These effects can be used to design ligands that selectively bind to the native state of p53 protein to reverse the thermodynamic and kinetic denaturation caused by these mutations (Zhang S et al. Advanced Strategies for Therapeutic Targeting of Wild-Type and Mutant p53 in Cancer. Biomolecules. 2022, 12 (4): 548). Notably, the Y220C mutation mediates the formation of a surface pocket away from the DNA-binding core domain (Joerger AC et al. Structural basis for understanding oncogenic p53 mutations and designing rescue drugs. Proc Natl Acad Sci, 2006, 103(41): 15056-15061), making it an ideal target for treating tumors with this mutation. So far, many Y220C pocket binders have been developed to stabilize the p53 mutant and reactivate its transcriptional activity, but their efficacy is limited (Boeckler FM, et al. Targeted rescue of a destabilized mutant of p53 by an in silico screened drug. Proc Natl Acad Sci, 2008, 105(30): 10360-10365; Guiley KZ et al. A small molecule reacts with the p53 somatic mutant Y220C to rescue wild-type thermal stability. Cancer Discov, 2022: CD-22). Therefore, for patients with tumors carrying the p53 Y220C mutation, there is an urgent need for p53 Y220C pocket binders with stronger affinity and greater drugability.

[0005] Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the existing p53 Y220C pocket binders have poor drugability. To this end, the present invention provides a deuterated indoleazine compound and its application. The deuterated indoleazine compound can enhance the ability of p53 Y220C to bind to DNA and has excellent inhibitory activity on the proliferation of cancer cells represented by gastric cancer cells.

[0007] The present invention provides a compound represented by formula (I), an isotope-labeled compound thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a prodrug thereof;

[0008] in,

[0009] X 1 、X 2 and X 3 are each independently selected from CH or N;

[0010] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 are each independently selected from hydrogen or deuterium, and at least one of them is deuterium.

[0011] As a preferred technical solution, X 1 N; X 2 is CH or N; X 3 For CH.

[0012] As a preferred technical solution, X 1 N; X 2 and X 3 For CH.

[0013] As a preferred technical solution, R 1 、R 2 and R 3 is deuterium; R 4 、R 5 、R 6 、R 7 and R 8 For hydrogen.

[0014] As a preferred technical solution, R 1 、R 2 、R 3 、R 4 and R 5 is hydrogen; R 6 、R 7 and R 8For deuterium.

[0015] As a preferred technical solution, R 1 、R 2 、R 3 、R 6 、R 7 and R 8 is hydrogen; R 4 and R 5 For deuterium.

[0016] As a preferred technical solution, the compound of formula (I) is specifically selected from the following compounds:

[0017] The present invention also provides methods for preparing the compound represented by formula (I), its isotope-labeled products, enantiomers, diastereomers, solvates, or pharmaceutically acceptable salts thereof, including the following methods:

[0018] Method 1:

[0019] Step 1: Compound a-1 undergoes a ring-closing aromatization reaction with ethyl bromopyruvate to obtain compound a-2; wherein X is selected from Cl, Br, and I;

[0020] Step 2: Compound a-2 undergoes Vilsmeier-Haack reaction to obtain compound a-3;

[0021] Step 3: Compound a-3 reacts with a fluorination agent to obtain compound a-4; the fluorination agent can be methyl fluorosulfonyldifluoroacetate, (triphenylphosphonium) difluoroacetic acid inner salt, or tetrabutylammonium fluoride;

[0022] Step 4: Compound a-4 undergoes ester reduction reaction under the action of a reducing agent to obtain compound a-5; the reducing agent may be sodium borohydride, lithium borohydride, lithium aluminum tetrahydride or diisobutylaluminum hydride;

[0023] Step 5: Compound a-5 is oxidized to obtain compound a-6 under the action of an oxidant, wherein the oxidant may be Dess-Martin reagent, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide, or sulfur trioxide / pyridine;

[0024] Step 6: Compound a-6 reacts with Gilbert reagent to generate compound a-7;

[0025] Step 7: Compound a-7 reacts with formaldehyde under the catalysis of a metal reagent to obtain compound a-8; the metal reagent can be a Cu-containing reagent, such as cuprous tetraacetonitrile hexafluorophosphate;

[0026] Step 8: Compound a-8 is oxidized to aldehyde a-9 under the action of an oxidant, which may be Dess-Martin reagent, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide, or sulfur trioxide / pyridine;

[0027] Step 9: Compound a-9 undergoes reductive amination reaction with compound a-10 to generate compound a-11; R 1 、R 2 、R 3 、R 6 、R 7 、R 8 、X 1 、X 2 、X 3 The definition of is the same as above;

[0028] Step 10: Compound a-11 undergoes a metal reagent-catalyzed coupling reaction with amine a-12 to obtain compound (Ia); the metal reagent can be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, or cuprous iodide.

[0029] Method 2:

[0030] Step 1: Compound a-11 undergoes a metal reagent-catalyzed coupling reaction with amine a-13 to obtain compound a-14; wherein X is selected from Cl, Br, I; R 1 、R 2 、R 3 、R 6 、R 7 、R 8 、X 1 、X 2 、X 3 The definition is the same as above; the metal reagent can be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, cuprous iodide;

[0031] Step 2: Compound a-14 is subjected to acidic conditions to remove the Boc protecting agent to obtain compound a-15; the acidic conditions can be carried out in the presence of an inorganic acid (such as HCl, HBr, H2SO4), an organic acid (such as trifluoroacetic acid), a Lewis acid (such as zinc bromide) or other reagents (such as trimethylsilyl iodide);

[0032] Step 3: Compound a-15 undergoes reductive amination reaction with formaldehyde to generate compound (Ia).

[0033] Method 3:

[0034] Step 1: Compound a-9 undergoes a reductive amination reaction with compound a-16 to generate compound a-17; wherein Y is selected from a C1-C4 alkyl group; R 1 、R 2 、R 3 、X 1 、X 2 、X 3 The definition of is the same as above;

[0035] Step 2: Compound a-17 undergoes a metal reagent-catalyzed coupling reaction with amine a-12 to obtain compound a-18; the metal reagent can be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, or cuprous iodide;

[0036] Step 3: Compound a-18 undergoes hydrolysis under acidic or alkaline conditions to produce a-19; the acidic conditions can be inorganic acids (such as HCl, HBr, H2SO4) or organic acids (such as trifluoroacetic acid); the alkaline conditions can be inorganic bases (such as LiOH, NaOH, KOH);

[0037] Step 4: Compound a-19 and amine H2NCR 6 R 7 R 8 A condensation reaction occurs to generate compound (Ia); wherein R 6 、R 7 、R 8 The definition of is the same as above.

[0038] Method 4:

[0039] Step 1: Compound a-9 is subjected to Pinnick oxidation reaction to obtain compound a-20;

[0040] Step 2: Compound a-20 is subjected to esterification reaction to obtain compound a-21;

[0041] Step 3: Compound a-21 is reduced to obtain compound a-22; the reducing agent may be sodium borohydride (lithium) or lithium aluminum hydride reagent, or their deuterated reagents; R 4 、R 5 The definition of is the same as above;

[0042] Step 4: Compound a-22 is subjected to Mitsunobu coupling reaction to obtain compound a-24; R 1 、R 2 、R 3 、R 4 、R 5 、X 1 、X2 、X 3 The definition of is the same as above; G is an electron-withdrawing protecting group, such as p-toluenesulfonyl, p-nitrobenzenesulfonyl, m-nitrobenzenesulfonyl, etc.;

[0043] Step 5: Compound a-24 removes the protecting group G to obtain a-25;

[0044] Step 6: Compound a-25 undergoes a metal reagent-catalyzed coupling reaction with amine a-12 to obtain compound a-26; the metal reagent can be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, or cuprous iodide;

[0045] Step 7: Compound a-26 undergoes hydrolysis under acidic or alkaline conditions to produce a-27; the acidic conditions can be inorganic acids (such as HCl, HBr, H2SO4) or organic acids (such as trifluoroacetic acid); the alkaline conditions can be inorganic bases (such as LiOH, NaOH, KOH);

[0046] Step 8: Compound a-27 and amine H2NCR 6 R 7 R 8 A condensation reaction occurs to generate compound (I); wherein R 6 、R 7 、R 8 The definition of is the same as above.

[0047] The present invention also provides a pharmaceutical composition comprising a compound represented by formula (I), its isotope-labeled substance, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

[0048] The present invention also provides a compound as represented by formula (I), its isotope-labeled substance, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt, or the use of said pharmaceutical composition in the preparation of a p53 mutant pocket binder or a drug for treating and / or preventing diseases associated with p53 mutants.

[0049] In a preferred technical solution, the application satisfies one or more of the following conditions:

[0050] (1) The p53 mutant has a mutation at amino acid 220, for example, p53 Y220C;

[0051] (2) the pocket binder increases the ability of the p53 mutant to bind to DNA;

[0052] (3) The disease associated with p53 mutants is cancer, such as breast cancer, gastric cancer, lung cancer or ovarian cancer.

[0053] The present invention also provides the use of the compound represented by formula (I), its isotope-labeled substance, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt, or the pharmaceutical composition in the preparation of drugs for treating and / or preventing cancer.

[0054] The cancer is, for example, breast cancer, gastric cancer, lung cancer or ovarian cancer.

[0055] The present invention also provides a method for treating and / or preventing cancer, characterized in that a therapeutically effective amount of a compound as represented by formula (I), its isotope label, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt is administered to a patient.

[0056] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0057] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0058] The term "solvate" refers to a substance formed by the combination of a compound and a solvent (including but not limited to water, methanol, ethanol, etc.). Solvates are divided into stoichiometric solvates and non-stoichiometric solvates.

[0059] The term "pharmaceutically acceptable salt solvate" refers to a compound formed by combining with a pharmaceutically acceptable acid or base and a solvent (including but not limited to water, methanol, ethanol, etc.). The amount of solvent may be stoichiometric or non-stoichiometric.

[0060] A "-" at the end of a group means that the group is attached to the rest of the molecule through that site. For example, CH3-C(=O)- means an acetyl group.

[0061] When the valence bond of a group is marked with a wavy line " ”, for example, in “ ", the wavy line indicates the point of attachment of the group to the rest of the molecule

[0062] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0063] The term "alkyl" refers to a linear or branched, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, and the like.

[0064] The term "alkoxy" refers to a group R X -O-, R X The same definition as the term "alkyl". Alkoxy includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like.

[0065] The term "therapeutically effective amount" refers to an amount administered to a patient that is sufficient to effectively treat a disease. The therapeutically effective amount will vary depending on the type of compound, the type of disease, the severity of the disease, the age of the patient, etc., but can be adjusted by those skilled in the art as appropriate.

[0066] The term "pharmaceutical excipients" refers to all substances contained in pharmaceutical preparations other than the active pharmaceutical ingredient (API). These substances are generally classified into two categories: excipients and additives. For details, see the Pharmacopoeia of the People's Republic of China (2020 Edition) and the Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0067] The term "treat" refers to eliminating the cause or alleviating the symptoms of a disease.

[0068] The term "prevent" refers to reducing the risk of developing a disease.

[0069] The term "patient" refers to any animal, typically a mammal, such as a human, that needs to be treated or prevented. Mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like.

[0070] The term "PG" represents a protecting group, such as a Boc protecting group and the like.

[0071] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0072] The reagents and raw materials used in the present invention are commercially available.

[0073] The positive progress of the present invention is that the compound of the present invention can enhance the ability of p53 Y220C to bind to DNA and has good inhibitory activity on the proliferation of cancer cells represented by gastric cancer cells. DETAILED DESCRIPTION

[0074] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0075] In each embodiment, 1 HNMR was recorded by a BRUKER AVANCE NEO 400 MHz, JNM-ECZ400s nuclear magnetic resonance instrument, and the chemical shift is expressed in δ (ppm); liquid chromatography-mass spectrometry (LCMS) was recorded by Shimadzu LC-20AD, Agilent 1260 and Agilent 1200 mass spectrometers; preparative HPLC separations were performed using a WATERS Autop, Shimadzu LC20AR liquid chromatograph.

[0076] abbreviation

[0077] Preparation of intermediates Int-1 and Int-2

[0078] Preparation of intermediates Int-1 and Int-2

[0079] Step 1: Ethyl 8-bromoindolizine-2-carboxylate

[0080] A mixture of 3-bromo-2-methyl-pyridine Int-1-1 (500 mg, 2.91 mmol), ethyl 3-bromopyruvate (850 mg, 4.36 mmol) and sodium bicarbonate (561 mg, 6.69 mmol) in butanone (5 mL) was stirred at 85°C for 16 hours, then concentrated to dryness and purified by SGC (0-10% EtOAc in PE) to give ethyl 8-nitroindoleazine-2-carboxylate Int-1-2 as a gray solid (190 mg, 24.4% yield). LCMS calculated value C 11 H 11 BrNO2[M+H] + :m / z=268.0 / 270.0; Detection value: 267.9 / 269.9; 1H NMR(400MHz, CDCl3)δ7.88(d,J=1.6Hz,1H),7.87-7.83(m,1H),7.03-6.99(m,1H),6.9 5(d,J=6.8Hz,1H),6.43(t,J=7.2Hz,1H),4.37(q,J=7.2Hz,2H),1.40(t,J=6.8Hz,3H).

[0081] Step 2: Ethyl 8-bromo-3-formylindolizine-2-carboxylate

[0082] At 0 ° C, a solution of 8-bromoindolizine-2-carboxylic acid ethyl ester Int-1-2 (5.34 g, 19.9 mmol) in DCM (130 mL) was added dropwise to a solution of phosphorus oxychloride (5.19 g, 33.8 mmol) in DMF (130 mL). The mixture was stirred at 20 ° C for 1 hour, then slowly quenched with saturated aqueous sodium bicarbonate (300 mL) and extracted with DCM (200 mL). The separated organic layers were combined, dried over sodium sulfate, filtered and concentrated to dryness. The residue was purified by SGC (0-10% EtOAc in PE) to give 8-bromo-3-formylindolizine-2-carboxylic acid ethyl ester Int-1-3 (10.6 g, crude) as a yellow gum. LCMS calculated value C 12 H 11 BrNO3[M+H] + :m / z=296.0 / 298.0; Detection: 295.9 / 297.9.

[0083] Step 3: Ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate

[0084] A mixture of ethyl 8-bromo-3-formylindolizine-2-carboxylate Int-1-3 (8.50 g, 15.8 mmol) and ethyl 2,2-difluoro-2-(triphenylphosphonium)acetate (11.2 g, 31.5 mmol) in DMF (120 mL) was stirred at 60°C for 2 hours. TBAF (47.3 mL, 47.3 mmol, 1 M solution in tetrahydrofuran) was added to the mixture. The reaction mixture was stirred at 60°C for 2 hours, then diluted with water (100 mL) and extracted with MTBE (100 mL x 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% EtOAc in PE) to afford ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate Int-1-4 (2.40 g, 43.4% yield) as a white solid. LCMS calculated value C 13 H 12 BrF3NO2[M+H]+ :m / z=350.0 / 352.0; Detection value: 349.9 / 351.9; 1 H NMR (400MHz, CDCl3) δ7.89(d,J=7.2Hz,1H),7.12(s,1H),7.05(d,J=6.8Hz,1H),6.56( t,J=7.2Hz,1H),4.40(q,J=7.2Hz,2H),4.25(q,J=10.0Hz,2H),1.42(t,J=7.2Hz,3H).

[0085] Step 4: (8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)methanol

[0086] To a mixture of 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylic acid ethyl ester Int-1-4 (4.10 g, 11.7 mmol) in THF (120 mL) was added DIBAL-H (35.1 mL, 35.1 mmol, 1 M solution in toluene). The reaction was stirred at -10°C for 3 hours to obtain a yellow solution. The reaction solution was poured into a saturated aqueous ammonium chloride solution (200 mL), followed by the addition of water (200 mL) and the mixture was extracted with EtOAc (150 mL x 3). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% EtOAc in PE) to obtain [8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl]methanol Int-1-5 (3.55 g, 98.4% yield) as a white solid. LCMS calculated value: C 11 H 10 BrF3NO[M+H] + :m / z=308.0 / 310.0; Detection value: 307.9 / 309.9; 1 H NMR (400MHz, CDCl3) δ7.85 (d, J = 7.2Hz, 1H), 7.00 (d, J = 6.8Hz, 1H), 6.70 (br s, 1H), 6.50 (t, J = 7.2Hz, 1H), 4.84 (s, 2H), 3.83 (q, J = 10.4Hz, 2H).

[0087] Step 5: 8-Bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde

[0088] To a solution of [8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]methanol Int-1-5 (3.55 g, 11.5 mmol) in DCM (400 mL) was added DMP (7.33 g, 17.2 mmol) at 0°C. The reaction system was warmed to room temperature and stirred for 2 hours. The reaction solution was poured into a saturated aqueous sodium bicarbonate solution (20 mL), followed by addition of water (200 mL) and extraction with DCM (100 mL×3). The organic layers were combined, dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-7% EtOAc in PE) to give 8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-carbaldehyde Int-1-6 (2.70 g, 76.5% yield) as a white solid. LCMS calculated value: C 11 H8BrF3NO[M+H] + :m / z=306.0 / 308.0; Detected value: 305.9 / 307.9.

[0089] Step 6: 8-Bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine

[0090] To a mixture of dimethyl (1-diazo-2-oxopropyl)phosphonate (470 mg, 2.45 mmol) and potassium carbonate (451 mg, 3.27 mmol) in MeOH (15 mL) at 0°C was added 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 (500 mg, 1.63 mmol). The reaction system was warmed to room temperature and stirred for 16 hours. The reaction solution was diluted with water (50 mL) and extracted with EtOAc (50 mL×3). The organic layers were combined, dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-3% EtOAc in PE) to give 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (380 mg, 77.0% yield) as a white solid. LCMS calculated value C 12 H8BrF3N[M+H] + :m / z=302.0 / 304.0; Detection value: 301.9 / 303.9; 1 H NMR (400MHz, CDCl3) δ7.81 (d, J = 7.2Hz, 1H), 7.03 (d, J = 7.2Hz, 1H), 6.79 (s, 1H), 6.52 (t, J = 7.2Hz, 1H), 3.83 (q, J = 10.0Hz, 2H), 3.25 (s, 1H).

[0091] Step 7: 3-[8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol

[0092] Under nitrogen, cuprous tetraacetonitrile hexafluorophosphate (123 mg, 0.33 mmol) and tributylphosphine (268 mg, 1.32 mmol) were dissolved in toluene (15 mL). The reaction mixture was stirred at 70°C for 30 minutes. 8-Bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (1.00 g, 3.31 mmol) and aqueous formaldehyde (0.18 mL, 6.62 mmol, 36%-38% content) were then added. The mixture was stirred at 70°C overnight. After completion of the reaction by TLC, the mixture was concentrated in vacuo, and the residue was purified by SGC (0-15% EtOAc in PE) to afford 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol Int-1-7 (843 mg, 76.7% yield) as a white solid. LCMS calculated value C 13 H 10 BrF3NO[M+H] + :m / z=332.0 / 334.0; Detection value: 332.3 / 334.3; 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=7.1Hz,1H),7.19(d,J=7.1Hz,1H),6.68(t,J=7.1H z,1H),6.61(s,1H),5.34(t,J=6.0Hz,1H),4.34(d,J=6.0Hz,2H),4.20-4.11(m,2H).

[0093] Step 8: 3-[8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-ynal

[0094] To a 0°C solution of 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol Int-1-7 (6.80 g, 20.5 mmol) in DCM (120 mL) was slowly added DMP (17.4 g, 41.0 mmol) in portions. The mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated aqueous sodium bicarbonate (50 mL) and extracted with DCM (30 mL x 3). The combined organic phases were concentrated in vacuo, and the residue was purified by SGC (0-15% EtOAc in PE) to give 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol Int-2 (5.70 g, 84.3% yield) as a gray solid. LCMS calculated value: C 13 H8BrF3NO[M+H] +:m / z=330.0 / 332.0; Detection value: 330.2 / 332.2; 1 H NMR (400MHz, DMSO-d6) δ9.47(s,1H),8.49(d,J=7.1Hz,1H),7.28(d,J=7.1Hz,1H),6.90(s,1H),6.78(t,J=7.1Hz,1H),4.31(q,J=10.7Hz,2H).

[0095] Cpd-C:4-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

[0096] Step 1: 4-Amino-3-methoxy-N-methylbenzamide

[0097] To a solution of 4-amino-3-methoxybenzoic acid Cpd-C-1 (7.52 g, 45.0 mmol), methylamine solution (6.71 g, 54.0 mmol, 25% content), and TEA (18.8 mL, 135 mmol) in THF (45 mL) at 0°C was added HATU (18.8 g, 49.5 mmol). The reaction was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by SGC (0-4% MeOH in DCM) to give 4-amino-3-methoxy-N-methylbenzamide Cpd-C-2 (6.08 g, 75.0% yield) as a white solid. LCMS calculated for C9H 13 N2O2[M+H] + :m / z=181.1; Detected value: 181.2.

[0098] Step 2: 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

[0099] To a mixture of 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (600 mg, 1.99 mmol) and 4-amino-3-methoxy-N-methylbenzamide Cpd-C-2 (716 mg, 3.97 mmol) in 1,4-dioxane (12 mL) was added aqueous formaldehyde solution (497 mg, 5.96 mmol, 36%-38% content) and cuprous bromide (567 mg, 3.97 mmol). The reaction was stirred at 100°C under microwave irradiation for 1 hour. The residue was purified by SGC (0-30% EtOAc in DCM) to give 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (480 mg, 48.9% yield) as a yellow solid. LCMS calculated value: C 22 H 20 BrF3N3O2[M+H] + :m / z=494.1 / 496.1; Detected value: 494.2 / 496.2.

[0100] Step 3: tert-Butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate

[0101] Under argon, a mixture of 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (1.35 g, 2.73 mmol), (3S,4R)-tert-butyl 4-amino-3-fluoropiperidine-1-carboxylate (1.19 g, 5.46 mmol), BrettPhos Pd G4 (0.50 g, 0.55 mmol), Ruphos (0.25 g, 0.55 mmol), and cesium carbonate (1.78 g, 5.46 mmol) in 1,4-dioxane (50 mL) was stirred at 100° C. for 16 hours. The reaction mixture was diluted with EtOAc (200 mL) and washed with water (100 mL), saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0-50% EtOAc in DCM) to give tert-butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate Cpd-C-4 (1.00 g, 58.0% yield) as a yellow solid. LCMS calculated value: C 32 H 38 F4N5O4[M+H] + :m / z=632.3; Detection value: 632.5.

[0102] Step 4: 4-((3-(8-(((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

[0103] To a solution of tert-butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate Cpd-C-4 (1.00 g, 1.58 mmol) in DCM (16 mL) at 0°C was added hydrochloric acid (7.92 mL, 31.7 mmol, 4N 1,4-dioxane solution). The mixture was transferred to room temperature and stirred for 1 hour. The mixture was concentrated to dryness, diluted with water (30 mL), and the pH was adjusted to 7-8 with saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM (100 mL × 2), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid 4-((3-(8-(((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-5 (450 mg, crude). The crude product was used directly in the next reaction. LCMS calculated value C 27 H 30 F4N5O2[M+H] + :m / z=532.2; Detected value: 532.4.

[0104] Step 5: 4-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

[0105] To a solution of 4-((3-(8-(((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-5 (450 mg, 0.85 mmol), acetic acid (0.02 mL, 0.42 mmol), and formaldehyde (212 mg, 2.54 mmol, 36%-38%) in MeOH (20 mL) was added sodium cyanoborohydride (160 mg, 2.54 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. Saturated sodium bicarbonate solution was added dropwise to the reaction mixture to adjust the pH to 7-8. The aqueous phase was extracted with EtOAc (100 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative method to give 4-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C (119 mg, 25.2% yield) as a white solid. LCMS calculated value: C 28 H 32 F4N5O2[M+H] + :m / z=546.2; Detection value: 546.2; 1 H NMR (400MHz, DMSO-d6) δ8.08(d,J=4.6Hz,1H),7.57(d,J=6.9Hz,1H),7.41(dd,J=8.2,1.7Hz,1H),7.34(d,J=1.7Hz,1H),6.91(s,1H),6.74(d,J =8.3Hz,1H),6.51(t,J=7.2Hz,1H),5.90(t,J=6.2Hz,1H),5.85(d,J=7.5Hz,1H),5.54(d,J=8.4Hz ,1H),4.81(d,J=49.8Hz,1H),4.23(d,J=6.2Hz,2H),3.92(q,J=10.7Hz,2H),3.83(s,3H),3.54(d, J=27.9Hz,1H),3.02(t,J=10.5Hz,1H),2.80(d,J=11.3Hz,1H),2.75(d,J=4.5Hz,3H),2.28-2.14( m,4H),2.07(t,J=11.2Hz,1H),1.97(ddd,J=24.0,12.0,3.2Hz,1H),1.67(dd,J=12.7,2.7Hz,1H).

[0106] Example 1: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methyl-d3)pyridine-2-carboxamide (Cpd-1, i.e., Compound 1)

[0107] Step 1: 6-Methoxy-5-nitropyridine-2-carboxylic acid

[0108] To a solution of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (10.3 g, 51.0 mmol) in MeOH (200 mL) was added sodium methoxide (28.3 mL, 153 mmol, 5.4 N MeOH solution) at 0°C under argon. The mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure to remove volatiles. The residue was diluted with water (200 mL), acidified with 3N hydrochloric acid to a pH of 4-5, and then extracted with DCM (300 mL x 3). The combined organic layers were washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and concentrated to afford 6-methoxy-5-nitropyridine-2-carboxylic acid Cpd-1-2 (9.72 g, crude) as a white solid, which was used in the next step without further purification. LCMS calculated for C7H5N2O5 [MH] - :m / z=197.0; Detection value: 197.1.

[0109] Step 2: 6-methoxy-N-(methyl-d3)-5-nitropyridine-2-carboxamide

[0110] To a mixture of 6-methoxy-5-nitropyridine-2-carboxylic acid Cpd-1-2 (1.51 g, 7.61 mmol), trideuteromethylamine (0.31 g, 9.13 mmol), and DIPEA (2.95 g, 22.8 mmol) in DMF (30 mL) was added HATU (3.47 g, 9.13 mmol), and the mixture was stirred at room temperature for 2 hours. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with EtOAc (200 mL x 2). The organic phase was washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by SGC (0-5% MeOH in DCM) to afford 6-methoxy-N-(methyl-d3)-5-nitropyridine-2-carboxamide Cpd-1-3 (0.63 g, 38.7% yield) as a yellow solid. LCMS calculated for C8H7D3N3O4 [M+H] + :m / z=215.1; Detected value: 215.3.

[0111] Step 3: 5-amino-6-methoxy-N-(methyl-d3)pyridine-2-carboxamide

[0112] A mixture of 6-methoxy-N-(methyl-d3)-5-nitropyridine-2-carboxamide Cpd-1-3 (630 mg, 2.94 mmol), iron powder (657 mg, 11.8 mmol), and ammonium chloride (1.26 g, 23.5 mmol) in water (10 mL) and ethanol (20 mL) was stirred at 60°C for 2 hours. The reaction mixture was filtered, the filter cake was washed with ethanol (50 mL), and the filtrate was concentrated. The residue was purified by SGC (0-10% MeOH in DCM) to afford 5-amino-6-methoxy-N-(methyl-d3)pyridine-2-carboxamide Cpd-1-4 (440 mg, 81.2% yield) as a white solid. LCMS calculated for C8H9D3N3O2 [M+H] + :m / z=185.1; Detected value: 185.2.

[0113] Step 4: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methyl-d3)pyridine-2-carboxamide

[0114] A solution of 5-amino-6-methoxy-N-(methyl-d3)pyridine-2-carboxamide Cpd-1-4 (240 mg, 1.30 mmol), 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-ynal Int-2 (473 mg, 1.43 mmol), and acetic acid (0.07 mL, 1.30 mmol) in MeOH (10 mL) was stirred at 60°C under argon for 1 hour. The mixture was cooled to room temperature, and sodium cyanoborohydride (246 mg, 3.91 mmol) was added. The mixture was stirred at room temperature for 2 hours. Aqueous sodium bicarbonate was added to the residue to adjust the pH to 7-8. The aqueous phase was extracted with EtOAc (100 mL x 2). The combined organic phases were washed with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by SGC (10-50% MeOH in PE) to give 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methyl-d3)pyridine-2-carboxamide Cpd-1-5 as a yellow solid (400 mg, 61.6% yield). LCMS calculated value: C 21 H 16 D3BrF3N4O2[M+H] + :m / z=498.1 / 500.1; Detected value:498.1 / 500.1.

[0115] Step 5: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methyl-d3)picolinamide

[0116] A solution of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methyl-d3)pyridine-2-carboxamide Cpd-1-5 (400 mg, 0.80 mmol), (3S,4R)-3-fluoro-1-methyl-4-piperidinamine (265 mg, 2.01 mmol), Cs2CO3 (523 mg, 1.61 mmol), and BrettPhos Pd G3 (145 mg, 0.16 mmol) in THF (15 mL) was stirred at 100°C under nitrogen for 12 hours. The residue was poured into water (50 mL). The aqueous phase was extracted with EtOAc (100 mL x 2). The combined organic phases were washed with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by preparative method to give 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methyl-d3)picolinamide Cpd-1 (74.3 mg, 16.8% yield) as a white solid. LCMS calculated value: C 27 H 28 D3F4N6O2[M+H] + :m / z=550.3; Detection value: 550.2; 1H NMR (400MHz, DMSO-d6) δ8.14(s,1H),7.57(d,J=7.0Hz,1H),7.54(d,J=7.9Hz,1H),7.03(d,J=8.0Hz,1H),6.91(s,1 H),6.51(t,J=7.2Hz,1H),6.32(t,J=6.2Hz,1H),5.85(d,J=7.5Hz,1H),5.54(d,J=8.4Hz,1H),4.81(d,J=49.9Hz,1H ),4.24(d,J=6.1Hz,2H),4.01(s,3H),3.93(q,J=10.5Hz,2H),3.54(d,J=27.7Hz,1H),3.02(t,J=10.8Hz,1H),2.80 (d,J=11.0Hz,1H),2.28-2.15(m,4H),2.07(t,J=11.1Hz,1H),1.97(dt,J=11.8,8.7Hz,1H),1.67(d,J=11.0Hz,1H).

[0117] Example 2: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-methylpyridine-2-carboxamide (Cpd-2, i.e., Compound 2)

[0118] Step 1: 6-(Methoxy-d3)-5-nitropyridine-2-carboxylic acid

[0119] Under nitrogen protection, a mixture of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (20.0 g, 98.7 mmol), cesium carbonate (96.5 g, 296 mmol) and deuterated methanol (4.27 g, 118 mmol) in DMSO (200 mL) was stirred at 60°C for 16 hours. After the reaction was completed, the mixture was diluted with water (100 mL) and the pH was adjusted to 3 with 1N hydrochloric acid. The mixture was filtered to collect the precipitate, and the filter cake was dried under vacuum to obtain a yellow solid 6-(methoxy-d3)-5-nitropyridine-2-carboxylic acid Cpd-2-1 (12.0 g, crude product). The crude product was used directly in the next reaction. LCMS calculated value C7H4D3N2O5[M+H] + :m / z=202.1; Detected value: 201.9.

[0120] Step 2: 6-(Methoxy-d3)-5-nitropyridine-2-carboxylic acid methyl ester

[0121] Under nitrogen, a mixture of 6-(methoxy-d3)-5-nitropyridine-2-carboxylic acid Cpd-2-1 (12.0 g, 59.7 mmol) and cesium carbonate (58.4 g, 179 mmol) in DMSO (120 mL) was stirred at 0°C for 0.5 hours. Iodomethane (9.32 g, 179 mmol) was then added to the reaction mixture, which was stirred at 0°C for 2 hours. After completion of the reaction, as determined by LCMS, the reaction mixture was poured into an ice-cold aqueous ammonium chloride solution (1000 mL) and extracted with EtOAc (1000 mL x 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to yield the crude product. The residue was purified by SGC (0-20% EtOAc in PE) to yield methyl 6-(methoxy-d3)-5-nitropyridine-2-carboxylate Cpd-2-2 (10.0 g, 70.1% yield) as a yellow solid. LCMS calculated for C8H6D3N2O5 [M+H] + :m / z=216.1; Detected value: 215.9.

[0122] Step 3: 5-amino-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester

[0123] 6-(Methoxy-d3)-5-nitropyridine-2-carboxylic acid methyl ester Cpd-2-2 (10.0 g, 46.5 mmol) was dissolved in EtOAc (100 mL), and Pd / C (9.90 g, 5% content) was added. The resulting mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. LCMS analysis showed complete consumption of the starting material and the detection of the desired compound. Filtration and concentration gave 5-amino-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester Cpd-2-3 (8.20 g, 90.5% yield) as a yellow solid. LCMS calculated value: C8H8D3N2O3 [M+H] + :m / z=186.1;Detection value:186.2; 1 H NMR (400MHz, DMSO-d6) δ7.53(d,J=8.0Hz,1H),6.85(d,J=8.0Hz,1H),5.88(s,2H),3.75(s,3H).

[0124] Step 4: Isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate

[0125] Under nitrogen, a solution of 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-ynal Int-2 (200 mg, 0.61 mmol), methyl 5-amino-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-3 (135 mg, 0.73 mmol), and tetraisopropyl titanate (517 mg, 1.82 mmol) in THF (2 mL) was stirred at 100°C for 1 hour. The reaction was cooled to 25°C, MeOH (2 mL) and sodium cyanoborohydride (57.1 mg, 0.91 mmol) were added, and stirring was continued for 10 minutes. LCMS analysis showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by SGC (0-25% EtOAc in PE) to give isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-4 (190 mg, 59.4% yield) as a yellow solid. LCMS calculated value: C 23 H 19 D3BrF3N3O3[M+H] + :m / z=527.1; Detection value: 526.9.

[0126] Step 5: Isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate

[0127] Under nitrogen, a solution of isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-4 (170 mg, 0.322 mmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (85.1 mg, 0.64 mmol), Brettphos Pd G3 (58.3 mg, 0.06 mmol), and cesium carbonate (210 mg, 0.65 mmol) in 1,4-dioxane (2 mL) was stirred at 100°C for 2 hours in a sealed tube. LCMS analysis indicated complete consumption of the starting material and the presence of the desired compound. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative method to give yellow solid 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid isopropyl ester Cpd-2-5 (90.0 mg, yield 48.1%). LCMS calculated value C 29 H 31 D3F4N5O3[M+H] + :m / z=579.3; Detection value: 579.0.

[0128] Step 6: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid

[0129] A mixture of isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-5 (40.0 mg, 0.07 mmol), NaOH (8.28 mg, 0.21 mmol) in THF (0.4 mL), MeOH (0.2 mL) and H2O (0.1 mL) was stirred at room temperature for 16 hours. LCMS analysis indicated complete consumption of the starting material and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, diluted with water, and adjusted to pH 5 with dilute hydrochloric acid. The product was filtered and the residue was purified by SGC (0-3% MeOH in DCM) to give a crude yellow solid. The crude product was then purified by preparative purification to give 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid Cpd-2-6 (5.50 mg, 15.0% yield) as a white solid. LCMS calculated value: C 26 H 25 D3F4N5O3[M+H] + :m / z=537.2;Detection value:537.0; 1 H NMR (400MHz, CD3OD) δ7.64(d,J=7.3Hz,1H),7.36(d,J=6.9Hz,1H),6.94(d,J=7.8H z,1H),6.56(s,1H),6.42(t,J=7.2Hz,1H),5.79(d,J=7.3Hz,1H),4.90(s,1H),4.1 9(s,2H),3.69(q,J=10.5Hz,2H),3.62-3.54(m,1H),3.32-3.23(m,1H),3.01(d,J= 12.2Hz,1H),2.62-2.48(m,1H),2.46-2.39(m,1H),2.37(s,3H),2.00-1.87(m,2H).

[0130] Step 7: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-methylpyridine-2-carboxamide

[0131] HATU (78.0 mg, 0.21 mmol) was added to a solution of 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid Cpd-2-6 (92.0 mg, 0.17 mmol), DIPEA (66.0 mg, 0.51 mmol), and methylamine hydrochloride (6.00 mg, 0.21 mmol) in anhydrous DMF (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. LCMS analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was diluted with EtOAc (50 mL) and washed with saturated aqueous sodium bicarbonate (30 mL x 2). The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% MeOH in DCM) to give 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-methylpyridine-2-carboxamide (46.8 mg, 47.6% yield) as a light yellow solid. LCMS calculated value: C 27 H 28 D3F4N6O2[M+H] + :m / z=550.3; Detection value: 550.2; 1 H NMR (400MHz, DMSO-d6) δ8.16(q,J=4.4Hz,1H),7.57(d,J=6.8Hz,1H),7.53(d,J=8.0Hz,1H),7.03(d,J=8.0H z,1H),6.91(s,1H),6.51(t,J=7.2Hz,1H),6.32(t,J=6.0Hz,1H),5.85(d,J=7.6Hz,1H),5.54(d,J=8.4Hz,1H ),4.82(d,J=50.4Hz,1H),4.24(d,J=6.0Hz,2H),3.93(q,J=10.8Hz,2H),3.58-3.51(m,1H),3.03(t,J=11.2 Hz,1H),2.82-2.79(m,4H),2.30-2.15(m,4H),2.12-2.06(m,1H),2.03-1.90(m,1H),1.68(d,J=10.4Hz,1H).

[0132] Example 3: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)amino)-6-methoxy-N-methylpyridine-2-carboxamide (Cpd-3, i.e., Compound 3)

[0133] Step 1: 3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propanoic acid

[0134] At room temperature, a solution of sodium chlorite (438 mg, 4.85 mmol) and anhydrous potassium dihydrogen phosphate (1.67 g, 12.1 mmol) in water (10 mL) was slowly added dropwise to a mixed solution of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propanal Int-2 (800 mg, 2.42 mmol) and 2-methyl-2-butene (1.70 g, 24.2 mmol) in tert-butanol (6 mL) and dichloromethane (24 mL). After the addition was complete, the system was stirred at room temperature for 12 hours. LCMS analysis indicated complete consumption of the starting material. Ethyl acetate (15 mL) and water (15 mL) were added to the reaction mixture in sequence. The pH was adjusted to weak acidity with hydrochloric acid (1 N). The aqueous phase was extracted with ethyl acetate (10 mL × 2). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propanoic acid Cpd-3-1 (700 mg, crude). The crude product was used directly in the next reaction. LCMS calculated value C 13 H8BrF3NO2[M+H] + :m / z=346.0 / 348.0; Detection: 346.1 / 348.1.

[0135] Step 2: Methyl 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propanoate

[0136] Under argon protection, iodomethane (623 mg, 4.39 mmol) was slowly added dropwise to a mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propanoic acid Cpd-3-1 (760 mg, 2.20 mmol) and potassium carbonate (911 mg, 6.59 mmol) in acetonitrile (10 mL). After the addition was complete, the system was stirred at room temperature for 2 hours. LCMS analysis showed that the starting material was completely consumed. The reaction system was filtered and the filter cake was washed with ethyl acetate (10 mL × 3). The filtrate was concentrated and the residue was purified by SGC (PE with 0-10% EtOAc) to obtain methyl 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propanoate Cpd-3-2 as a white solid (400 mg, 50.6% yield). LCMS calculated value C 14 H 10 BrF3NO2[M+H] + :m / z=360.0 / 362.0; Detected value: 360.1 / 362.1.

[0137] Step 3: 3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1,1-d2-1-ol

[0138] Under argon protection, lithium aluminum tetrahydride deuterate (44.9 mg, 1.47 mmol) was added portionwise to a solution of methyl 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propanoate Cpd-3-2 (350.0 mg, 972 μmol) in tetrahydrofuran (10 mL) at 0°C. The system was slowly warmed to room temperature and stirred for 0.5 hours. LCMS analysis showed complete consumption of the starting material. The reaction mixture was cooled to 0°C and quenched by the addition of water (10 mL) and 15% sodium hydroxide (15 mL) solution. After stirring at room temperature for 0.5 hours, the solid was removed by filtration through a pad of Celite. The filtrate was extracted with ethyl acetate (10 mL x 2). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by SGC (0-20% EtOAc in PE) to obtain 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1,1-d2-1-ol Cpd-3-3 as a white solid (100 mg, 30.8% yield). LCMS calculated value: C 13 H8D2BrF3NO[M+H] + :m / z=334.0 / 336.0; Detected value: 334.2 / 336.2.

[0139] Step 4: 8-Bromo-2-(3-bromopropyl-1-yn-1-yl-3,3-d2)-3-(2,2,2-trifluoroethyl)indolizine

[0140] Carbon tetrabromide (134 mg, 404 μmol) was added to a solution of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1,1-d2-1-ol Cpd-3-3 (90.0 mg, 270 μmol) and triphenylphosphine (54.5 mg, 539 μmol) in dichloromethane (2 mL) at room temperature. The system was stirred at room temperature for 2 hours. LCMS analysis indicated complete consumption of the starting material. The reaction mixture was concentrated, and the residue was purified by SGC (0-10% EtOAc in PE) to give 8-bromo-2-(3-bromopropyl-1-yn-1-yl-3,3-d2)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-4 (50.0 mg, 46.7% yield) as a blue solid. LCMS calculated value: C 13 H7D2Br2F3N[M+H] + :m / z=395.9 / 397.9 / 399.9; Detected value: 395.9 / 397.9 / 399.9.

[0141] Step 5: methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(tert-butyloxycarbonyl)amino)-6-methoxypyridine-2-carboxylate

[0142] Under argon protection, a solution of 8-bromo-2-(3-bromopropyl-1-yn-1-yl-3,3-d2)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-4 (317 mg, 797 μmol), 5-((tert-butoxycarbonyl)amino)-6-methoxypyridine-2-carboxylic acid methyl ester (225 mg, 797 μmol) and cesium carbonate (4347 mg, 3.19 mmol) in acetonitrile (5 mL) was stirred at 40 ° C for 2 hours. LCMS detection showed that the starting material was completely consumed. The solid was filtered and washed with ethyl acetate (10 mL x 3). The filtrate was concentrated and the residue was purified by SGC (0-30% EtOAc in PE) to give 8-bromo-2-(3-bromopropyl-1-yn-1-yl-3,3-d2)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-5 (400 mg, 83.8% yield) as a white solid. LCMS calculated value: C 26 H 24 D2BrF3N3O5[M+H] + :m / z=598.1 / 600.1; Detected value: 598.2 / 600.2.

[0143] Step 6: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(tert-butyloxycarbonyl)amino)-6-methoxypyridine-2-carboxylic acid

[0144] At room temperature, a solution of lithium hydroxide (160 mg, 6.68 mmol) in water (1 mL) was added to a mixture of 8-bromo-2-(3-bromopropyl-1-yn-1-yl-3,3-d2)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-5 (400 mg, 668 μmol) in methanol (4 mL) and tetrahydrofuran (4 mL). The system was stirred for 2 hours. LCMS detection showed that the starting material was completely consumed. Ethyl acetate (10 mL) and water (10 mL) were added to the reaction mixture in sequence, and then the pH was adjusted to weak acidity with hydrochloric acid (1 N). The aqueous phase was extracted with ethyl acetate (10 mL × 2), and the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a white crude product, 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(tert-butyloxycarbonyl)amino)-6-methoxypyridine-2-carboxylic acid Cpd-3-6 (400 mg, crude). The crude product was used directly in the next reaction. LCMS calculated value C 25 H 22 D2BrF3N3O5[M+H] + :m / z=584.1 / 586.1; Detected value: 584.2 / 586.2.

[0145] Step 7: tert-Butyl (3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(2-methoxy-6-(methylcarbamoyl)pyridin-3-yl)carbamate

[0146] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(tert-butoxycarbonyl)amino)-6-methoxypicolinic acid Cpd-3-6 (400 mg, 685 μmol), methylamine hydrochloride (142 mg, 1.37 mmol), HATU (312 mg, 821 μmol) and N,N-diisopropylethylamine (265 mg, 2.05 mmol) in DMF (4 mL) was stirred at room temperature for 2 hours. LCMS detection showed that the starting material was completely consumed. The reaction system was diluted with water (10 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by SGC (0-50% EtOAc in PE) to obtain tert-butyl 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(2-methoxy-6-(methylcarbamoyl)pyridin-3-yl)carbamate Cpd-3-7 (300 mg, yield 73.3%) as a yellow solid. LCMS calculated value C 26 H 25 D2BrF3N4O4[M+H] + :m / z=597.1 / 599.1; Detection value: 597.2 / 599.3.

[0147] Step 8: Tert-butyl (3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(2-methoxy-6-(methylcarbamoyl)pyridin-3-yl)carbamate

[0148] Under argon protection, a solution of tert-butyl 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(2-methoxy-6-(methylcarbamoyl)pyridin-3-yl)carbamate Cpd-3-7 (200 mg, 335 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (66.4 mg, 502 μmol), Brttephos Pd G3 (30.35 mg, 33.5 μmol), Ruphos (31.24 mg, 67.0 μmol) and cesium carbonate (218 mg, 670 μmol) in tetrahydrofuran (4 mL) was stirred at 95 ° C for 2 hours. LCMS detection showed that the starting material was completely consumed. The system was cooled to room temperature and diluted with water (10 mL), extracted with ethyl acetate (10 mL×3), the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by SGC (0-5% MeOH in DCM) to obtain tert-butyl 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(2-methoxy-6-(methylcarbamoyl)pyridin-3-yl)carbamate Cpd-3-8 (100 mg, 46.1% yield) as a yellow solid. LCMS calculated value: C 32 H 37 D2F4N6O4[M+H] + :m / z=649.3; Detection: 649.4.

[0149] Step 9: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)amino)-6-methoxy-N-methylpicolinamide

[0150] At 0°C, trifluoroacetic acid (0.5 mL) was added to a solution of tert-butyl 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)(2-methoxy-6-(methylcarbamoyl)pyridin-3-yl)carbamate Cpd-3-8 (180 mg, 278 μmol) in dichloromethane (5 mL). The system was warmed to room temperature and stirring was continued for 2 hours. LCMS detection showed that the starting material was completely consumed. The reaction was quenched with saturated aqueous sodium bicarbonate (10 mL) and extracted with ethyl acetate (10 mL × 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative method to give 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl-1,1-d2)amino)-6-methoxy-N-methylpyridine-2-carboxamide Cpd-3 (50.4 mg, 50.1% yield) as a white solid. LCMS calculated value: C 27 H 29 D2F4N6O2[M+H] + :m / z=549.3; Detection value: 549.3; 1 H NMR (400MHz, DMSO-d6) δ8.17(d,J=4.9Hz,1H),7.55(dd,J=14.5,7.4Hz,2H),7.03(d,J=7.9Hz,1 H),6.91(s,1H),6.51(t,J=7.2Hz,1H),6.32(s,1H),5.85(d,J=7.5Hz,1H),5.56(d,J=8.4Hz,1H ),4.82(d,J=49.4Hz,1H),4.01(s,3H),3.93(q,J=11.6,11.1Hz,2H),3.62-3.45(m,1H),3.03(t ,J=11.7Hz,1H),2.80(d,J=4.8Hz,4H),2.18(s,4H),2.12-1.90(m,2H),1.67(d,J=14.0Hz,1H).

[0151] Biological evaluation

[0152] 1. Biochemical Activity Assay: Testing the Compound's Activity in Promoting the Binding of the p53 Y220C Mutant to DNA

[0153] Homogeneous time-resolved fluorescence (HTRF) experiments were used to measure the reactivation effects of compounds on the p53 Y220C mutant. Recombinant His-tagged p53 Y220C (94-312) for HTRF experiments was expressed in Escherichia coli and purified to 90% purity using a Ni-NTA column. Biotinylated DNA for HTRF experiments was synthesized by Pharmaron Chemicals; the specific sequence is 5'-ATTAGGCATGTCTAGGCATGTCTAGG-3'.

[0154] Fluorescence resonance energy transfer (FRET) was used to measure the binding of recombinant His-tagged p53 Y220C protein to biotinylated DNA. For the FRET experiment, the binding between the p53 mutant and the DNA sequence was measured by detecting the fluorescence of the interaction between an anti-His antibody conjugated to Eu (Cisbio, 61HI2KLA) and d2-conjugated streptavidin (Cisbio, 610SADLF) bound to the biotinylated DNA molecule. Compounds were prepared as 2 mM stocks and serially diluted 1:3 in DMSO to 10 concentrations. Each 200-fold working concentration of compound was diluted sequentially to a 4-fold working concentration. Using an Echo, 4 μL of compound solution was transferred row by row to a 384 assay plate, with 2 replicates per column. 4 μL of p53 solution was added to the assay plate. 4 μL of biotinylated DNA solution was added to the assay plate, and then 4 μL of detection solution (His-Eu antibody and streptavidin-d2) was added to each well of the assay plate. The assay plates were incubated overnight and protected from light. Fluorescence was read on a BMG (BMG LRBTECH).

[0155] Calculate the ratio of each well (ratio_665nm / 615nm-ratio_background). The activity % is calculated as follows:

[0156] C=(Ave_Ac-Ave_Ba) / (Ave_Dc-Ave_Bd)

[0157] R data=(A-Ave_Ba-C*D) / (D-Ave_Bd)*(Ave_Dc-Ave_Bd)

[0158] Activity% = R data / Ave_VC * 100.

[0159] A: Fluorescence intensity of the sample at 665 nm;

[0160] D: fluorescence intensity of the sample at 615 nm;

[0161] Ba: fluorescence intensity of the plate background at 665 nm;

[0162] Bd: fluorescence intensity of the plate background at 615 nm;

[0163] Dc: fluorescence intensity of HIS-Eu background at 615 nm;

[0164] Ac: fluorescence intensity of streptavidin-d2 background at 665 nm;

[0165] VC: DMSO-treated group.

[0166] The logarithm of the activity % and compound concentration was fitted to a nonlinear regression (dose response-variable slope) using Graphpad 8.0 to calculate the SC 150 .

[0167] Table 1: Activity of compounds in promoting the binding of p53 Y220C mutant to DNA

[0168] Cpd-A: Selected from patent WO2021061643A1.

[0169] Cpd-B: Selected from patent WO2022213975A1.

[0170] The above data show that the example compounds of the present invention have good activity in promoting the binding of p53 Y220C mutant to DNA and are significantly better than the reference compounds.

[0171] 2. Cell activity assay: NUGC-3 cell proliferation inhibition assay

[0172] The antiproliferative effect of the drug on human gastric adenocarcinoma cell lines was assessed based on cellular dehydrogenase activity, which correlated well with viable cell counts. The NUGC-3 cell line (JCRB, JCRB0822) was cultured in RPMI 1640 (Invitrogen, 11875-085) supplemented with 10% (v / v) fetal bovine serum (BI, 04-002-1A). The cell lines were cultured according to the standard protocols of the American Type Culture Collection. The cell lines were authenticated by their short tandem repeat profiles.

[0173] In the proliferation experiment, 1000 NUGC-3 cells were seeded in a 96-well flat-bottomed transparent TC-treated plate (Corning, 3903) in 200 μL of culture medium per well and cultured overnight in culture medium to recover. Compounds were added in increasing concentration gradients, and DMSO treatment was used as a positive control. After 5 days of treatment, the plate was equilibrated to room temperature for 30 minutes, and the number of viable cells was measured by adding the cell counting kit-8 reagent (MCE, HY-K0301). The absorbance was measured on Enspire (Perkin Elme). The relative survival rate of each group was expressed as a percentage change relative to the positive control group and then fitted to a four-parameter logit nonlinear curve using the program XLFit (IDBS).

[0174] Table 2: Antiproliferation activity of compounds on NUGC-3 cells

[0175] Cpd-A: Selected from patent WO2021061643A1.

[0176] Cpd-B: Selected from patent WO2022213975A1.

[0177] The above data show that the example compounds of the present invention have good inhibitory activity on NUGC-3 cell proliferation and are significantly better than the reference compounds.

Claims

1. A compound as represented by formula (I), an isotope-labeled substance thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a prodrug thereof, characterized in that: in, X 1 , X 2 and X 3 are each independently selected from CH or N; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 Each is independently selected from hydrogen or deuterium, and at least one of them is deuterium.

2. A compound of formula (I) as claimed in claim 1, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt or its prodrug, characterized in that: X 1 N; X 2 CH or N; X 3 For CH.

3. A compound of formula (I) as claimed in claim 1, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt or its prodrug, characterized in that: X 1 N; X 2 and X 3 For CH.

4. A compound of formula (I), an isotope-labeled substance thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a prodrug thereof according to any one of claims 1 to 3, characterized in that: R 1 , R 2 and R 3 for deuterium; R 4 , R 5 , R 6 , R 7 and R 8 For hydrogen.

5. A compound of formula (I), an isotope-labeled substance thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a prodrug thereof according to any one of claims 1 to 3, characterized in that: R 1 , R 2 , R 3 , R 4 and R 5 is hydrogen; R 6 , R 7 and R 8 For deuterium.

6. A compound of formula (I), an isotope-labeled substance thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a prodrug thereof according to any one of claims 1 to 3, characterized in that: R 1 , R 2 , R 3 , R 6 , R 7 and R 8 is hydrogen; R 4 and R 5 For deuterium.

7. A compound of formula (I) as claimed in claim 1, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt or its prodrug, characterized in that: The compound of formula (I) is selected from the following compounds:

8. A pharmaceutical composition comprising a compound of formula (I) as described in any one of claims 1 to 7, its isotope-labeled substance, enantiomer, diastereomer, solvate or a pharmaceutically acceptable salt thereof and a pharmaceutical excipient.

9. Use of a compound of formula (I) as described in any one of claims 1 to 7, its isotope-labeled substance, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 8 in the preparation of a p53 mutant pocket binder or a drug for treating and / or preventing a disease associated with a p53 mutant.

10. The use according to claim 9, characterized in that It meets one or more of the following conditions: (1) The p53 mutant has a mutation at amino acid 220, for example, p53 Y220C; (2) the pocket binder increases the ability of the p53 mutant to bind to DNA; (3) The disease associated with p53 mutant is cancer, such as breast cancer, gastric cancer, lung cancer or ovarian cancer.

11. Use of a compound of formula (I) as described in any one of claims 1 to 7, its isotope-labeled substance, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating and / or preventing cancer.

12. The use according to claim 11, wherein the cancer is breast cancer, gastric cancer, lung cancer or ovarian cancer.

13. A method for treating and / or preventing cancer, characterized in that: The method comprises administering to the patient a therapeutically effective amount of a compound of formula (I) as described in any one of claims 1 to 7, an isotope-labeled substance, an enantiomer, a diastereomer, a solvate or a pharmaceutically acceptable salt thereof.

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