N-sulfonyl amide nitrogen-containing fused heterocycle compound targeting p53 mutation and application thereof

Abstract

The invention discloses an N-sulfonyl amide nitrogen-containing fused heterocycle compound targeting p53 mutation and an application of the N-sulfonyl amide nitrogen-containing fused heterocycle compound. The N-sulfonyl amide nitrogen-containing fused heterocycle compound disclosed by the invention is a compound as shown in a formula I or a pharmaceutically acceptable salt thereof. The compound can improve the ability of p53 Y220C to bind DNA, and has better inhibitory activity on cancer cell proliferation. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and further belongs to the field of drug-forming p53 Y220C pocket binder medicine, and specifically relates to a class of N-sulfonylamide nitrogen-containing fused heterocyclic compounds targeting p53 mutations and applications thereof. Background Art

[0002] 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, targeting p53 mutations has not been validated in clinical treatment to date (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). The experimental results of conditional regulation of p53 expression in mouse tissues showed that in different mouse models, the restoration of p53 expression can inhibit the growth of lymphoma and sarcoma through cell apoptosis (Christophorou M A et al. Temporal dissection of p53 function in vitro and in vivo [J]. Nat Genet, 2005, 37 (7): 718-726; Martins C. P., Brown-Swigart L., Evan G I 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 acryptic allosteric site. Cancer cell, 2021, 39(2): 225-239.e8).

[0003] The core domain of wild-type p53 is unstable, with low thermodynamic and kinetic stability, allowing rapid cycling between folded and unfolded states. Mutations in these core region residues in mutant p53 lead 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, this particular mutation, Y220C, 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 the 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 with p53 Y220C mutations, there is an urgent need for p53 Y220C pocket binders with stronger affinity and more drugability. Summary of the invention

[0004] 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 class of N-sulfonylamide nitrogen-containing fused heterocyclic compounds targeting p53 mutations and their applications. The compounds can enhance the ability of p53 Y220C to bind to DNA and have better inhibitory activity on the proliferation of cancer cells represented by gastric cancer cells.

[0005] The present invention provides a nitrogen-containing compound as shown in Formula I or a pharmaceutically acceptable salt thereof;

[0006]

[0007] in,

[0008] X 1 , X 2 and X 3 are each independently selected from CH and N;

[0009] R 1 , R 2 , R 3 , R 4 , R 5 are each independently selected from hydrogen and deuterium;

[0010] R 6 -(CH2) n R 7 ;

[0011] n is 0, 1, 2 or 3;

[0012] R 7 Selected from C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl and C3-C6 cycloalkyl.

[0013] As a preferred technical solution, the nitrogen-containing compound or a pharmaceutically acceptable salt thereof as shown in Formula I is a nitrogen-containing compound or a pharmaceutically acceptable salt thereof as shown in Formula Ia;

[0014]

[0015] in,

[0016] X 1 , X 2 and X 3 are each independently selected from CH or N;

[0017] R 1 , R 2 , R 3 , R 4 , R 5 are each independently selected from hydrogen or deuterium;

[0018] R 6 -(CH2) n R 7 ;

[0019] n is 0, 1, 2 or 3;

[0020] R 7 Selected from C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl or C3-C6 cycloalkyl.

[0021] As a preferred technical solution, X 1 N; X 2 CH or N; X 3 For CH.

[0022] As a preferred technical solution, X 1 N; X 2 and X 3 For CH.

[0023] As a preferred technical solution, R 6 -(CH2) n R 7 ; n is 0 or 1; R 7 Selected from C1-C3 alkyl, deuterated C1-C3 alkyl, halogenated C1-C3 alkyl or C3-C6 cycloalkyl.

[0024] As a preferred technical solution, R 6 -(CH2) n R 7 ; n is 0 or 1; R 7 Selected from -CH3, -CD3, -CF3, cyclopropyl.

[0025] As a preferred technical solution, R 1 , R 2 , R 3 , R 4 , R 5 Each is independently selected from hydrogen or deuterium, and at least one of them is deuterium.

[0026] As a preferred technical solution, R 1 , R 2 and R 3 is deuterium; R 4 and R 5 For hydrogen.

[0027] As a preferred technical solution, R 1 , R 2 and R 3 is hydrogen; R 4 and R 5 For deuterium.

[0028] As a preferred technical solution, the general formula I is the general formula Ib or Ic;

[0029]

[0030] In Formulas Ib and Ic, for

[0031] R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from hydrogen and deuterium;

[0032] R 6 -(CH2) n R 7 ;

[0033] n is 0 or 1;

[0034] R 7 Selected from C1-C6 alkyl, halogenated C1-C6 alkyl and C3-C6 cycloalkyl.

[0035] Preferably, Selected from -OCH3 and -OCD3;

[0036] R 4 and R 5 Selected from hydrogen;

[0037] R 6 -(CH2) n R 7 ; n is 0 or 1; R 7 Selected from -CH3, -CD3, -CF3 or cyclopropyl.

[0038] As a preferred technical solution, the compound of formula I is selected from the following compounds:

[0039]

[0040]

[0041]

[0042] Its enantiomers or mixtures of the two in any ratio (e.g. racemates).

[0043] The present invention also provides a method for preparing the compound shown in Formula I or a pharmaceutically acceptable salt thereof. The method includes the following methods:

[0044] Method 1:

[0045]

[0046] 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;

[0047] Step 2: Compound a-2 undergoes Vilsmeier-Haack reaction to obtain compound a-3;

[0048] Step 3: Compound a-3 reacts with a fluorination agent to obtain compound a-4; the fluorination agent may be methyl fluorosulfonyl difluoroacetate, (triphenylphosphonium) difluoroacetic acid inner salt, or tetrabutylammonium fluoride;

[0049] 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;

[0050] Step 5: Compound a-5 is oxidized under the action of an oxidant to obtain compound a-6. The oxidant may be Dess-Martin reagent, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide, or sulfur trioxide / pyridine;

[0051] Step 6: Compound a-6 reacts with Gilbert reagent to generate compound a-7;

[0052] Step 7: Compound a-7 reacts with formaldehyde under the catalysis of a metal reagent to obtain compound a-8; the metal reagent may be a Cu-containing reagent, such as cuprous tetraacetonitrile hexafluorophosphate;

[0053] Step 8: Compound a-8 is oxidized to aldehyde a-9 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;

[0054] Step 9: Compound a-9 undergoes a reductive amination reaction with compound a-10 to generate compound a-11; R 1 , R 2 , R 3 , R 6 , X 1 , X 2 , X 3 The definition of is the same as above;

[0055] Step 10: Compound a-11 and amine a-12 undergo a coupling reaction catalyzed by a metal reagent to obtain compound (Ia); the metal reagent may be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, and cuprous iodide.

[0056] Method 2:

[0057]

[0058] 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 , 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;

[0059] Step 2: Compound a-14 is subjected to acidic conditions to remove the Boc protecting agent to obtain compound a-15; the acidic conditions may 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);

[0060] Step 3: Compound a-15 undergoes a reductive amination reaction with formaldehyde to generate compound (Ia).

[0061] Method 3:

[0062]

[0063] Step 1: Compound a-9 undergoes a reductive amination reaction with compound a-16 to generate compound a-17; wherein X is selected from Cl, Br, I; Y is selected from C1-C4 alkyl; R 1 , R 2 , R 3 , X 1 , X 2 , X 3 The definition of is the same as above;

[0064] Step 2: Compound a-17 and amine a-12 undergo a coupling reaction catalyzed by a metal reagent to obtain compound a-18; the metal reagent may be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, cuprous iodide;

[0065] Step 3: Compound a-18 undergoes hydrolysis reaction under acidic or alkaline conditions to generate a-19; the acidic conditions may be inorganic acids (such as HCl, HBr, H2SO4) or organic acids (such as trifluoroacetic acid); the alkaline conditions may be inorganic bases (such as LiOH, NaOH, KOH);

[0066] Step 4: Compound a-19 and sulfonamide H2NS(O)2R 6 A condensation reaction occurs to generate compound (Ia); wherein R 6 The definition of is the same as above.

[0067] Method 4:

[0068]

[0069] Step 1: Compound a-8 is subjected to a hydroxyl protection reaction with a suitable protecting agent to generate compound a-20; wherein X is selected from Cl, Br, I; M is a protecting group, such as trimethylsilyl, tert-butyldimethylsilyl, methoxymethyl, tert-butoxycarbonyl, 2-tetrahydropyranyl; the protecting agent may be trimethylsilyl chloride, tert-butyldimethylsilyl chloride, chloromethyl methyl ether, di-tert-butyl dicarbonate, 3,4-dihydro-2H-pyran;

[0070] Step 2: Compound a-20 undergoes a coupling reaction with amine a-12 catalyzed by a metal reagent to obtain compound a-21; the metal reagent may be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, cuprous iodide;

[0071] Step 3: Compound a-21 is deprotected under acidic or alkaline conditions to generate a-22; the acidic conditions may be inorganic acids (such as HF, HCl, HBr, H2SO4) or organic acids (such as trifluoroacetic acid); the alkaline conditions may be inorganic bases (such as LiOH, NaOH, KOH) or organic bases (tetra-n-butylammonium fluoride);

[0072] Step 4: Compound a-22 undergoes oxidation reaction under the action of an oxidant to obtain compound a-23, wherein the oxidant may be Dess-Martin reagent, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide, or sulfur trioxide / pyridine;

[0073] Step 5: Compound a-23 undergoes a reductive amination reaction with sulfonamide a-10 to generate compound (Ia); wherein R 1 , R 2 , R 3 , R 6 , X 1 , X 2 , X3 The definition of is the same as above;

[0074] Method 5:

[0075]

[0076] Step 1: Compound a-9 is subjected to Pinnick oxidation reaction to obtain compound a-24; wherein X is selected from Cl, Br, and I;

[0077] Step 2: Compound a-24 is subjected to esterification reaction to obtain compound a-25;

[0078] Step 3: Compound a-25 is subjected to a reduction reaction to obtain compound a-26; the reducing agent may be a reagent containing sodium (lithium) borohydride or lithium aluminum hydride, and their deuterated reagents; R 4 , R 5 The definition of is the same as above;

[0079] Step 4: Compound a-26 is subjected to a halogenation reaction to generate compound a-27, wherein X is selected from Cl, Br, and I;

[0080] Step 5: Compound a-27 and a-28 undergo nucleophilic substitution reaction to obtain compound a-29; wherein R 1 , R 2 , R 3 , X 1 , X 2 , X 3 The definition is the same as above; G is a protecting group, such as p-toluenesulfonyl, tert-butoxycarbonyl; Y is selected from C1-C4 alkyl;

[0081] Step 6: Compound a-29 removes the protecting group G to obtain a-30;

[0082] Step 7: Compound a-30 undergoes a coupling reaction with amine a-12 catalyzed by a metal reagent to obtain compound a-31; the metal reagent may be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, cuprous iodide;

[0083] Step 8: Compound a-31 undergoes hydrolysis reaction under acidic or alkaline conditions to generate a-32; the acidic conditions may be inorganic acids (such as HCl, HBr, H2SO4) or organic acids (such as trifluoroacetic acid); the alkaline conditions may be inorganic bases (such as LiOH, NaOH, KOH);

[0084] Step 9: Compound a-32 and sulfonamide H2NS(O)2R 6 A condensation reaction occurs to generate compound (I); wherein R 6The definition of is the same as above.

[0085] The present invention also provides a pharmaceutical composition, which includes a compound as shown in Formula I or a pharmaceutically acceptable salt thereof (eg, a therapeutically effective amount) and a pharmaceutical excipient.

[0086] The present invention also provides a use of a compound as shown in formula I, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of a p53 mutant pocket binder or a drug for treating and / or preventing a disease associated with a p53 mutant.

[0087] In a preferred technical solution, the application satisfies one or more of the following conditions:

[0088] (1) The p53 mutant has a mutation at amino acid 220, for example, p53 Y220C;

[0089] (2) the pocket binder increases the ability of the p53 mutant to bind to DNA;

[0090] (3) The disease associated with p53 mutant is cancer, such as breast cancer, gastric cancer, lung cancer or ovarian cancer.

[0091] The present invention also provides a use of a compound as shown in Formula I, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of a drug for treating and / or preventing cancer.

[0092] The cancer is, for example, breast cancer, stomach cancer, lung cancer or ovarian cancer.

[0093] The present invention also provides a compound represented by formula a-11 or a-32 (which can be used to prepare the compound represented by formula I above);

[0094]

[0095] Wherein, X is a halogen (such as Br); R 8 is an amino protecting agent (e.g. -Boc);

[0096] X 1 , X 2 and X 3 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 The definitions are the same as above.

[0097] The compound represented by the formula a-11 or a-32 is preferably any of the following compounds;

[0098]

[0099] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0100] 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, please refer to Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0101] 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)- refers to an acetyl group.

[0102] When the group valence bond has a wavy line When, for example, The wavy line indicates the point of attachment of the group to the rest of the molecule.

[0103] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0104] The term "alkyl" refers to a linear or branched, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6). Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, and the like.

[0105] 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.

[0106] 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. The term "patient" refers to any animal, typically a mammal, such as a human, that needs to be treated or prevented from a disease. Mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

[0107] The term "pharmaceutical excipients" refers to all substances contained in pharmaceutical preparations other than active pharmaceutical ingredients, which are generally divided into two categories: excipients and additives. For details, please refer to the "Pharmacopoeia of the People's Republic of China (2020 Edition)" and Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0108] The term "treating" refers to removing the cause or alleviating the symptoms.

[0109] The term "prevention" refers to reducing the risk of developing a disease.

[0110] The term "PG" represents a protecting group, such as a Boc protecting group and the like.

[0111] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0112] The reagents and raw materials used in the present invention are commercially available.

[0113] The positive and progressive effects of the present invention are that the compound of the present invention can enhance the ability of p53 Y220C to bind to DNA and has a better inhibitory activity on the proliferation of cancer cells represented by gastric cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 This is the tumor growth curve of the control group and each experimental group in the pharmacodynamic evaluation experiment of the mouse NUGC-3 human gastric cancer xenograft tumor model in Test Example 9;

[0115] Figure 2 This is the relative weight change curve of mice in the control group and each experimental group in the pharmacodynamic evaluation experiment of the mouse NUGC-3 human gastric cancer xenograft tumor model in Test Example 9;

[0116] Figure 3 This is the tumor growth curve of the control group and each experimental group in the pharmacodynamic evaluation experiment of the rat NUGC-3 human gastric cancer xenograft tumor model in Test Example 10;

[0117] Figure 4 This is the relative body weight change curve of rats in the control group and each experimental group in the pharmacodynamic evaluation experiment of the rat NUGC-3 human gastric cancer xenograft tumor model in Test Example 10. DETAILED DESCRIPTION

[0118] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. Unless otherwise indicated, the actual operation disclosed in this application will adopt conventional techniques of organic synthesis, cell biology, cell culture and molecular biology within the technical scope of the art.

[0119] In each embodiment, 1 HNMR was recorded by BRUKER AVANCE NEO 400MHz, JNM-ECZ400s NMR, chemical shifts are expressed in δ (ppm); LCMS was recorded by Shimadzu LC-20AD, Agilent 1260 and Agilent 1200 mass spectrometers; preparative HPLC separation was performed using WATERS Autop, Shimadzu LC20AR liquid chromatographs

[0120] abbreviation

[0121]

[0122]

[0123] Preparation of intermediates Int-1 and Int-1-7

[0124]

[0125] Step 1: Ethyl 8-bromoindolizine-2-carboxylate

[0126] 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 obtain gray solid 8-nitroindolazine-2-carboxylic acid ethyl ester Int-1-2 (190 mg, yield 24.4%). 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).

[0127] Step 2: Ethyl 8-bromo-3-formylindolizine-2-carboxylate

[0128] 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 value: 295.9 / 297.9.

[0129] Step 3: Ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate

[0130] A mixture of 8-bromo-3-formyl indolizine-2-carboxylic acid ethyl ester 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 tetrahydrofuran solution) 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×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-carboxylic acid ethyl ester Int-1-4 (2.40 g, yield 43.4%) 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).

[0131] Step 4: (8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)methanol

[0132] DIBAL-H (35.1 mL, 35.1 mmol, 1 M toluene solution) was added 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). The reactants were 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), and then water (200 mL) was added and the mixture was extracted with EtOAc (150 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 (0-5% EtOAc in PE) to obtain a white solid [8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl]methanol Int-1-5 (3.55 g, yield 98.4%). 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).

[0133] Step 5: 8-Bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde

[0134] DMP (7.33 g, 17.2 mmol) was added to a solution of [8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl]methanol Int-1-5 (3.55 g, 11.5 mmol) in DCM (400 mL) 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), and then water (200 mL) was added and extracted 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 a white solid 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxaldehyde Int-1-6 (2.70 g, yield 76.5%). LCMS calculated value C 11 H8BrF3NO[M+H] + :m / z=306.0 / 308.0; Detected value: 305.9 / 307.9.

[0135] Step 6: 8-Bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine

[0136] At 0°C, 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 (500 mg, 1.63 mmol) was added to a mixture of (1-diazo-2-oxopropyl)phosphonic acid dimethyl ester (470 mg, 2.45 mmol) and potassium carbonate (451 mg, 3.27 mmol) in methanol (15 mL). 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, yield 77.0%) 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).

[0137] Step 7: 3-[8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol

[0138] Under nitrogen protection, cuprous tetraacetonitrile hexafluorophosphate (123 mg, 0.33 mmol) and tributylphosphine (268 mg, 1.32 mmol) were dissolved in toluene (15 mL), and the reaction mixture was stirred at 70 ° C for 30 minutes. Then 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl) indolizine Int-1 (1.00 g, 3.31 mmol) and formaldehyde aqueous solution (0.18 mL, 6.62 mmol, content 36%-38%) were added. The mixed system was stirred at 70 ° C overnight. After the reaction was completed by TLC detection, the mixed system was concentrated in vacuo, and the residue was purified by SGC (0-15% EtOAc in PE) to obtain a white solid 3-[8-bromo-3-(2,2,2-trifluoroethyl) indolizine-2-yl] prop-2-yn-1-ol Int-1-7 (843 mg, yield 76.7%). 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).

[0139] 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

[0140]

[0141] Step 1: 4-amino-3-methoxy-N-methylbenzamide

[0142] HATU (18.8 g, 49.5 mmol) was added 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 triethylamine (18.8 mL, 135 mmol) in THF (45 mL) at 0°C. 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 value: C9H 13 N2O2[M+H] + :m / z=181.1; Detected value: 181.2.

[0143] Step 2: 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

[0144] 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 for 1 hour under microwave irradiation. 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.

[0145] Step 3: (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-carboxylic acid tert-butyl ester

[0146] Under argon protection, 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 (3S,4R)-tert-butyl 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.

[0147] 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

[0148] 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 adjusted to pH 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)indolizine-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 step. LCMS calculated value C 27 H 30 F4N5O2[M+H] + :m / z=532.2; Detected value: 532.4.

[0149] 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

[0150] At room temperature, sodium cyanoborohydride (160 mg, 2.54 mmol) was added 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 aqueous solution (212 mg, 2.54 mmol, content 36%-38%) in methanol (20 mL), and the reactants were 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), and the organic phases were combined and washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative method to give a white solid 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, yield 25.2%). 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,1 H),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).

[0151] Example 1: 5-[3-[8-[[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino]-3-(2,2,2-trifluoroethyl)indolizine-2-yl]prop-2-ynylamino]-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide (Cpd-1, i.e., Compound 1)

[0152]

[0153] Step 1: 6-Methoxy-5-nitropyridine-2-carboxylic acid

[0154] 6-Chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (4.00 g, 19.7 mmol) and cesium carbonate (12.8 g, 39.4 mmol) were stirred in a mixture of DMSO / MeOH (40 mL, 1:1) at 60 °C for 12 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was poured into ice water (50 mL) and adjusted to pH 5 with 1N hydrochloric acid, and extracted with EtOAc (30 mL×3). The organic layers were combined, 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 obtain 6-methoxy-5-nitropyridine-2-carboxylic acid Cpd-1-2 (2.80 g, yield 71.7%, yellow solid). LCMS calculated value C7H7N2O5[M+H] + :m / z=199.0; Detection value: 199.1.

[0155] Step 2: 6-methoxy-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide

[0156] A mixture of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-2 (3.20 g, 16.2 mmol), methanesulfonamide (1.54 g, 16.2 mmol), EDCI (4.65 g, 24.3 mmol) and 4-dimethylaminopyridine (3.94 g, 32.4 mmol) in DMF (30 mL) was stirred at room temperature for 8 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (30 mL×3). The organic layers were combined, 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 obtain 6-methoxy-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-1-3 (2.20 g, yield 49.5%, yellow solid). LCMS calculated for C8H 10 N3O6S[M+H] +:m / z=276.0; Detection value: 276.1.

[0157] Step 3: 5-amino-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide

[0158] Under hydrogen atmosphere, a solution of palladium carbon (1.60 g, 5% content) and 6-methoxy-N-(methylsulfonyl)-5-nitropyridineamide Cpd-1-3 (800 mg, 2.91 mmol) in EtOAc (15 mL) was stirred at room temperature for 2 hours. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% MeOH in DCM) to obtain 5-amino-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-1-4 (500 mg, yield 70.1%, yellow solid). LCMS calculated value C8H 12 N3O4S[M+H] + :m / z=246.1; Detection value: 246.0.

[0159] Step 4: 8-Bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine

[0160] Tert-butyldimethylsilyl chloride (68.0 mg, 451 μmol) was added in portions to a solution of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Int-1-7 (100 mg, 302 μmol) and imidazole (61.3 mg, 901 μmol) in DCM (5 mL) at 0°C. The reaction mixture was stirred at 0°C for 0.5 h and then filtered to remove the solid. The filtrate was concentrated under reduced pressure to obtain a residue which was purified by SGC (0-30% EtOAc was added to PE) to obtain 8-bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-1-5 (134 mg, yield 99.7%, yellow solid). LCMS calculated value C 19 H 24 BrF3NOSi[M+H] + :m / z=446.1 / 448.1; Detection value:446.0 / 448.0.

[0161] Step 5: 2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-amine

[0162] Under argon protection, a mixture of 8-bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-1-5 (134 mg, 301 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (79.3 mg, 601 μmol), BrettPhos Pd G3 (54.4 mg, 60.0 μmol) and cesium carbonate (195 mg, 598 μmol) in THF (10 mL) was stirred at 100° C. for 2 hours. The reaction mixture was cooled to room temperature, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0-7% methanol in DCM) to give 2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-amine Cpd-1-6 (82.1 mg, 54.9% yield, white solid). LCMS calculated value C 25 H 36 F4N3OSi[M+H] + :m / z=498.3; Detection value: 498.0.

[0163] Step 6: 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol

[0164] At 0°C, TBAF (0.20 mL, 0.20 mmol, 1 M tetrahydrofuran solution) was added dropwise to a solution of 2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3-(2,2,2-trifluoroethyl)indolizine-8-amine Cpd-1-6 (82.1 mg, 165 μmol) in THF (5 mL). The reaction solution was stirred at 0°C for 1 hour. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution (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 (0-5% MeOH in DCM) to give 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Cpd-1-7 (57.0 mg, 90.2% yield, white solid). LCMS calculated value C 19 H 22 F4N3O[M+H] +:m / z=384.2; Detected value: 384.1.

[0165] Step 7: 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propynal

[0166] At 0°C, a solution of pyridine sulfur trioxide (780 mg, 4.90 mmol) in DMSO (1 mL) was added dropwise to a solution of 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Cpd-1-7 (625 mg, 1.63 mmol), DMSO (1.20 mL, 16.3 mmol) and diisopropylethylamine (1.50 mL, 8.15 mmol) in DCM (20 mL). The reaction solution was stirred at 0°C for 0.5 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction solution was quenched with saturated aqueous sodium bicarbonate solution (100 mL) and extracted with EtOAc (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 (0-5% MeOH in DCM) to give 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propynal Cpd-1-8 (550 mg, 88.6% yield, yellow solid). LCMS calculated value C 19 H 20 F4N3O[M+H] + :m / z=382.2; Detection value: 382.1.

[0167] Step 8: 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-(methylsulfonyl)pyridine-2-carboxamide

[0168] A mixture of 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)propyne aldehyde Cpd-1-8 (50.0 mg, 131 μmol), 5-amino-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-1-4 (38.6 mg, 157 μmol) and tetraisopropyl titanate (112 mg, 393 μmol) in THF (1 mL) was stirred at 100 ° C for 1 hour, and then the mixed solution was cooled to 0 ° C, MeOH (0.5 mL) and sodium cyanoborohydride (12.4 mg, 197 μmol) were slowly added, and the reaction solution was warmed to room temperature and continued to stir for 10 minutes. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was poured into ice water (5 mL) and extracted with EtOAc (20 mL×3). The organic layers were combined, washed with saturated sodium bicarbonate aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by SGC (0-3% MeOH in DCM) to give a yellow solid crude product, which was purified by preparation to give 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)propan)-2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-1 (14.8 mg, yield 18.5%, yellow solid). LCMS calculated value C 27 H 31 F4N6O4S[M+H] + :m / z=611.2; Detection value: 611.9; 1H NMR (400MHz, CDCl3) δ9.70 (s, 1H), 7.85 (d, J = 8.0Hz, 1H), 7.32 (d, J = 7.0Hz, 1H), 6.98 (d, J = 8.0Hz, 1H), 6.53 (t, J = 7. 2Hz,1H),6.48(s,1H),5.81(d,J=7.3Hz,1H),5.18(t,J=5.9Hz,1H),4.85(d,J=49.1Hz,1H),4.29(d,J=6.0Hz,2H),4. 16(d,J=9.4Hz,1H),4.03(s,3H),3.68(q,J=10.1Hz,2H),3.59-3.43(m,1H),3.41(s,3H),3.24(t,J=11.0Hz,1H),2.9 4(d,J=11.0Hz,1H),2.37-2.21(m,4H),2.16(t,J=11.1Hz,1H),2.02(d,J=10.0Hz,1H),1.93(td,J=12.2,3.5Hz,1H).

[0169] 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-(methylsulfonyl)pyridine-2-carboxamide (Cpd-2, i.e., compound 2)

[0170]

[0171]

[0172] Step 1: 6-(Methoxy-d3)-5-nitropyridine-2-carboxylic acid

[0173] 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 by LCMS, 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 6-(methoxy-d3)-5-nitropyridine-2-carboxylic acid Cpd-2-1 (12.0 g, crude product, yellow solid). The crude product was directly used in the next step. LCMS calculated value C7H4D3N2O5[M+H] + :m / z=202.1; Detected value: 201.9.

[0174] Step 2: 6-(Methoxy-d3)-5-nitropyridine-2-carboxylic acid methyl ester

[0175] Under nitrogen protection, 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. Then iodomethane (9.32 g, 179 mmol) was added to the reaction mixture and stirred at 0°C for 2 hours. After the reaction was completed by LCMS, the reaction mixture was poured into an ice-ammonium chloride aqueous solution (1000 mL) and extracted with EtOAc (1000 mL×2). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by SGC (0-20% EtOAc in PE) to obtain 6-(methoxy-d3)-5-nitropyridine-2-carboxylic acid methyl ester Cpd-2-2 (10.0 g, yield 70.1%, yellow solid). LCMS calculated value C8H6D3N2O5[M+H]+: m / z=216.1; detected value: 215.9.

[0176] Step 3: 5-amino-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester

[0177] 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), palladium carbon (9.90 g, 5% content) was added, and the resulting mixture was stirred in a hydrogen atmosphere at room temperature for 2 hours. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. Filtration and concentration gave 5-amino-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester Cpd-2-3 (8.20 g, yield 90.5%, 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).

[0178] Step 4: 3-[8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-ynal

[0179] DMP (17.4 g, 41.0 mmol) was slowly added in portions to a 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) under ice bath. The mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution (50 mL) and extracted with DCM (30 mL×3). The organic phases were combined and concentrated in vacuo. 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-ynal Int-2 (5.70 g, yield 84.3%, 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).

[0180] Step 5: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid isopropyl ester

[0181] Under nitrogen protection, a THF (2mL) solution of 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl]prop-2-ynal Int-2 (200 mg, 606 μmol), 5-amino-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester Cpd-2-3 (135 mg, 730 μmol) and tetraisopropyl titanate (517 mg, 1.82 mmol) was stirred at 100 ° C for 1 hour. The reactant was cooled to 25 ° C, MeOH (2 mL) and sodium cyanoborohydride (57.1 mg, 0.91 mmol) were added and stirred for 10 minutes. LCMS detection showed that the raw 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 layer was 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, yellow solid). LCMS calculated value C 23 H 19 D3BrF3N3O3[M+H] + :m / z=527.1 / 529.1; Detection value: 526.9 / 529.0.

[0182] 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 isopropyl ester

[0183] Under nitrogen protection, a solution of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid isopropyl ester Cpd-2-4 (170 mg, 322 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (85.1 mg, 645 μmol), Brettphos Pd G3 (58.3 mg, 64.3 μmol) and cesium carbonate (210 mg, 644 μmol) in 1,4-dioxane (2 mL) was stirred at 100 ° C for 2 hours in a sealed tube. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product 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-d3)pyridine-2-carboxylic acid isopropyl ester Cpd-2-5 (90.0 mg, yield 48.1%, yellow solid). LCMS calculated value C 29 H 31 D3F4N5O3[M+H] + :m / z=579.3; Detection value: 579.0.

[0184] 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)pyridine-2-carboxylic acid

[0185] A mixture 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 isopropyl ester Cpd-2-5 (40.0 mg, 69.2 μmol), sodium hydroxide (8.28 mg, 207 μmol) in THF (0.4 mL), MeOH (0.2 mL) and water (0.1 mL) was stirred at room temperature for 16 hours. LCMS detection showed that the starting material was completely consumed 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, filtered, and the residue was purified by SGC (0-3% MeOH in DCM) to give a crude product, which 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, yield 15.0%, white solid). LCMS calculated value C 26 H 25 D3F4N5O3[M+H] + :m / z=537.2; Detection value: 537.0; 1 HNMR (400MHz, CD3OD) δ7.64(d,J=7.3Hz,1H),7.36(d,J=6.9Hz,1H),6.94(d,J=7.8 Hz,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).

[0186] Step 8: 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-(methylsulfonyl)pyridine-2-carboxamide

[0187] 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 (210 mg, 392 μmol) and CDI (127 mg, 784 μmol) in THF (8 mL) was stirred at 70 ° C for 1 hour under argon protection and then cooled to room temperature. Methanesulfonamide (74.5 mg, 784 μmol) was added to the reaction solution, stirred for 10 minutes, and then DBU (120 μL, 789 μmol) was added and stirred at 70 ° C for 1 hour. The mixture was concentrated, water (30 mL) was added, and the aqueous phase was extracted with EtOAc (50 mL×3). The combined organic phase was washed with brine (50 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. 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)amino)-6-(methoxy-d3)-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-2 (50.0 mg, yield 20.8%, white solid). LCMS calculated value C 27 H 28 D3F4N6O4S[M+H] + :m / z=614.2;Detection value: 614.4; 1 H NMR (400MHz, DMSO-d6) δ10.85(s,1H),7.67(d,J=8.0Hz,1H),7.58(d,J=6.9Hz,1H),7.07(d,J=8.1Hz,1H),6.91 (s,1H),6.74(s,1H),6.51(t,J=7.2Hz,1H),5.86(d,J=7.5Hz,1H),5.56(d,J=8.4Hz,1H),4.83(d,J=49.6Hz,1H ),4.28(d,J=6.0Hz,2H),4.05(s,1H),3.95(q,J=10.7Hz,2H),3.65-3.46(m,1H),3.06(t,J=11.0Hz,1H),2.84( d,J=11.5Hz,1H),2.37-2.23(m,1H),2.22(s,3H),2.13(t,J=11.3Hz,1H),2.04-1.92(m,1H),1.75-1.64(m,1H).

[0188] 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)amino)-4-methoxy-N-(methylsulfonyl)pyrimidine-2-carboxamide (Cpd-3, i.e., Compound 3)

[0189]

[0190] Step 1: tert-Butyl (tert-butyloxycarbonyl)(2-chloro-4-methoxypyrimidin-5-yl)carbamate

[0191] Under nitrogen protection, a mixture of 2-chloro-4-methoxypyrimidine-5-amine Cpd-3-1 (5.00 g, 3.13 mmol), di-tert-butyl dicarbonate (14.4 g, 6.57 mmol), triethylamine (15.8 g, 15.7 mmol) and 4-dimethylaminopyridine (383 mg, 3.13 mmol) in THF (50 mL) was stirred at 40 ° C for 1 hour. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (50 mL×3). The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by SGC (0-5% EtOAc in PE) to give tert-butyl (tert-butyloxycarbonyl)(2-chloro-4-methoxypyrimidin-5-yl)carbamate Cpd-3-2 (4.80 g, yield 42.5%, white solid). LCMS calculated value C 15 H 23 ClN3O5[M+H] + :m / z=360.1; Detected value: 360.2.

[0192] Step 2: 5-((tert-Butyloxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylic acid methyl ester

[0193] Under a carbon monoxide atmosphere, a mixture of tert-butyl (tert-butyloxycarbonyl) (2-chloro-4-methoxypyrimidin-5-yl) carbamate Cpd-3-2 (4.30 g, 11.9 mmol), Pd (dppf) Cl2 (1.72 g, 2.38 mmol) and triethylamine (3.61 g, 35.7 mmol) in MeOH (43 mL) was stirred at 100 ° C for 16 hours. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction solution was filtered, the reaction solution was concentrated under reduced pressure, the residue was diluted with water (100 mL), and extracted with EtOAc (100 mL × 3). The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by SGC (0-20% EtOAc in PE) to give methyl 5-((tert-butyloxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-3 (2.00 g, yield 59.0%, white solid). LCMS calculated value C 12 H 18 N3O5[M+H] + :m / z=284.1; Detected value: 284.1.

[0194] Step 3: 8-Bromo-2-(3-bromoprop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine

[0195] At room temperature, 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-ol Cpd-1-7 (2.00 g, 6.02 mmol) and triphenylphosphine (3.16 g, 12.0 mmol) were dissolved in DCM (30 mL) and stirred for 10 minutes, and then carbon tetrabromide (3.00 g, 9.03 mmol) was added. The reaction solution was stirred at room temperature for 2 hours. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction solution was diluted with water (50 mL) and extracted with EtOAc (50 mL×3). The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by SGC (0-10% EtOAc in PE) to give 8-bromo-2-(3-bromoprop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-4 (1.55 g, yield 53.0%, white solid). LCMS calculated value C 13 H9Br2F3N[M+H] + :m / z=393.9 / 395.9 / 397.9; Detection value: 394.0 / 396.0 / 398.0.

[0196] Step 4: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylic acid methyl ester

[0197] Under nitrogen protection, a mixture of 8-bromo-2-(3-bromoprop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-4 (1.37 g, 3.46 mmol), 5-((tert-butyloxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylic acid methyl ester Cpd-3-3 (980 mg, 3.46 mmol) and cesium carbonate (1.88 g, 13.8 mmol) in acetonitrile (20 mL) was stirred at 40 ° C for 12 hours. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by SGC (0-25% EtOAc in PE) to give methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-5 (1.60 g, yield 65.1%, white solid). LCMS calculated value C 25 H 25 BrF3N4O5[M+H] + :m / z=597.1 / 599.1; Detection value: 597.2 / 599.2.

[0198] Step 5: 5-((tert-Butyloxycarbonyl)(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)-4-methoxypyrimidine-2-carboxylic acid methyl ester

[0199] Under nitrogen protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylic acid methyl ester Cpd-3-5 (1.35 g, 2.26 mmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (448 mg, 3.39 mmol), Brettphos Pd G3 (205 mg, 226 μmol), Ruphos (211 mg, 452 μmol) and cesium carbonate (1.47 g, 4.52 mmol) in THF (20 mL) was stirred in a sealed tube at 95°C for 2 hours. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by SGC (0-5% MeOH in DCM) to give methyl 5-((tert-butoxycarbonyl)(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)-4-methoxypyrimidine-2-carboxylate Cpd-3-6 (1.20 g, crude, yellow solid). LCMS calculated value C 31 H 37 F4N6O5[M+H] + :m / z=649.3; Detection value: 649.2.

[0200] Step 6: 5-((tert-Butyloxycarbonyl)(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)-4-methoxypyrimidine-2-carboxylic acid

[0201] At room temperature, 5-((tert-butoxycarbonyl)(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)-4-methoxypyrimidine-2-carboxylic acid methyl ester Cpd-3-6 (1.20 g, 1.85 mmol) was dissolved in a mixed solvent of THF (8 mL) and MeOH (8 mL), and water (2 mL) and lithium hydroxide (443 mg, 18.5 mmol) were added. The system was stirred at room temperature for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction solution was adjusted to pH 4 with 1.0 N HCl, diluted with water (20 mL) and extracted with DCM (20 mL×3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 5-((tert-butoxycarbonyl)(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)-4-methoxypyrimidine-2-carboxylic acid Cpd-3-7 (400 mg, crude product, yellow solid). LCMS calculated value: C 30 H 35 F4N6O5[M+H] + :m / z=635.3; Detection value: 635.5.

[0202] Step 7: 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)(4-methoxy-2-((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate

[0203] A solution of 5-((tert-butoxycarbonyl)(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)-4-methoxypyrimidine-2-carboxylic acid Cpd-3-7 (260 mg, 410 μmol) and CDI (133 mg, 820 μmol) in DMF (4 mL) was stirred at 90°C for 2 hours. The reaction solution was cooled to room temperature, and then methylsulfonamide (78.0 mg, 820 μmol) and DBU (206 mg, 820 μmol) were added. The reaction solution was stirred at 90°C for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction solution was cooled to room temperature, diluted with water (50 mL), and extracted with DCM (50 mL×3). The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Prep-HPLC to give tert-butyl (3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)(4-methoxy-2-((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate Cpd-3-8 (100 mg, crude, yellow solid). LCMS calculated value C 31 H 38 F4N7O6S[M+H] + :m / z=711.3; Detected value: 711.6.

[0204] Step 8: 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)-4-methoxy-N-(methylsulfonyl)pyrimidine-2-carboxamide

[0205] At 0°C, trifluoroacetic acid (1 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)(4-methoxy-2-((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate Cpd-3-8 (100 mg, 141 μmol) in DCM (3 mL). The reaction solution was slowly warmed to room temperature and stirred for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction solution was added dropwise to a mixture of saturated sodium bicarbonate solution (10 mL) and ice water (15 mL), and extracted with DCM (10 mL×3). The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Prep-HPLC 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)-4-methoxy-N-(methylsulfonyl)pyrimidine-2-carboxamide Cpd-3 (8.05 mg, yield 8.8%, yellow solid). LCMS calculated value C 26 H 30 F4N7O4S[M+H] + :m / z=612.2; Detection value: 612.2; 1 H NMR(400MHz,DMSO-d6)δ8.05(s,1H),7.60(d,J=6.8Hz,1H),6.93(s,1H),6.80-6.70(m,1H),6 .52(t,J=7.2Hz,1H),5.86(s,1H),5.64(d,J=8.3Hz,1H),4.87(d,J=52.8Hz,1H),4.32(d,J=6 .1Hz,2H),4.05(s,3H),4.02-3.90(m,2H),3.60(d,J=10.3Hz,1H),3.23(s,3H),3.18-3.08(m ,1H),2.91(d,J=10.9Hz,1H),2.28(s,3H),2.05-1.94(m,1H),1.78-1.67(m,1H),1.23(s,2H).

[0206] Example 4: N-(cyclopropylsulfonyl)-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-carboxamide (Cpd-4, i.e., compound 4)

[0207]

[0208] Step 1: N-(cyclopropylsulfonyl)-6-(methoxy-d3)-5-nitropyridine-2-carboxamide

[0209] At room temperature, cyclopropylsulfonamide (1.33 g, 10.9 mmol) and triethylamine (3.32 g, 32.8 mmol) were added to a solution of 6-(methoxy-d3)-5-nitropyridine-2-carboxylic acid Cpd-2-1 (2.20 g, 10.9 mmol) and 2-chloro-1-methylpyridinium iodide (3.35 g, 13.1 mmol) in dichloromethane (40 mL) in sequence. The reaction mixture was stirred at 40°C for 3 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, the residue was diluted with water (100 mL), and the pH was adjusted to 2 with 1N hydrochloric acid aqueous solution. The precipitated solid was filtered, washed with water (100 mL) and dried over anhydrous sodium sulfate to obtain N-(cyclopropylsulfonyl)-6-(methoxy-d3)-5-nitropyridine-2-carboxamide Cpd-4-1 (1.00 g, crude product, yellow solid). The crude product was used directly in the next step. LCMS calculated value C 10 H7D3N3O6S[MH] - :m / z=303.1; Detected value: 303.2.

[0210] Step 2: 5-amino-N-(cyclopropylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide

[0211] At room temperature, a mixture of N-(cyclopropylsulfonyl)-6-(methoxy-d3)-5-nitro-pyridine-2-carboxamide Cpd-4-1 (1.00 g, 3.29 mmol, crude) and palladium carbon (350 mg, 5% content) in THF (15 mL) and MeOH (15 mL) was stirred for 16 hours under a hydrogen atmosphere. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure to give 5-amino-N-(cyclopropylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-4-2 (730 mg, crude, yellow solid). The crude product was directly used in the next step. LCMS calculated value C 10 H9D3N3O4S[MH] - :m / z=273.1; Detection value:Found:273.3.

[0212] Step 3: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide

[0213] 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yne-1-aldehyde Int-2 (500 mg, 1.51 mmol), N-(cyclopropylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-4-2 (395 mg, 1.44 mmol) and acetic acid (91.0 mg, 1.51 mmol) were dissolved in a mixed solvent of MeOH (6 mL) and DCM (4 mL), and the system was stirred at room temperature for 16 hours. Then NaBH3CN (286 mg, 4.54 mmol) was added, and the system continued to stir for 0.5 hours. LCMS detection showed that the raw material was completely consumed, and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and the filter cake was collected by filtration. The filter cake was purified by slurrying with methanol to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-4-3 (560 mg, yield 62.8%, yellow solid). LCMS calculated value C 23 H 16 BrD3F3N4O4S[MH] - :m / z=586.1 / 588.1; Detection value: 586.2 / 588.2.

[0214] Step 4: N-(cyclopropylsulfonyl)-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-carboxamide

[0215] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-4-3 (560 mg, 950 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (252 mg, 1.90 mmol), BrettPhos Pd G3 (86.3 mg, 100 μmol) and cesium carbonate (620 mg, 1.90 mmol) in THF (13 mL) and NMP (1.3 mL) was stirred at 100 ° C in a sealed tube for 5 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL×3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by Prep-HPLC to give the product N-(cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-4 (42.4 mg, yield 6.80%, yellow solid). LCMS calculated value C 29 H 30 D3F4N6O4S[M+H] + :m / z=640.2; Detection value: 640.1; 1 H NMR (400MHz, DMSO-d6) δ10.88(s,1H),7.68(d,J=8.0Hz,1H),7.58(d,J=7.2Hz,1H),7.08(d,J=8.0Hz,1H),6.92(s, 1H),6.76(t,J=6.0Hz,1H),6.51(t,J=7.2Hz,1H),5.86(d,J=7.2Hz,1H),5.55(d,J=8.0Hz,1H),4.83(d,J=49.2Hz,1 H),4.28(d,J=6.0Hz,2H),3.99-3.91(m,2H),3.62-3.49(m,1H),3.13-3.01(m,2H),2.84-2.82(m,1H),2.33-2.21( m,1H),2.23(s,3H),2.15-2.10(m,1H),2.03-1.93(m,1H),1.71-1.67(m,1H),1.19-1.15(m,2H),1.09-1.04(m,2H).

[0216] Example 5: N-(cyclopropylmethylsulfonyl)-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-carboxamide (Cpd-5, i.e., compound 5)

[0217]

[0218]

[0219] Step 1: N-((Cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)-5-nitromethylpicolinamide

[0220] At room temperature, (cyclopropylmethyl)sulfonamide (1.69 g, 12.5 mmol) and triethylamine (3.62 g, 35.8 mmol) were added to a mixture of 6-(methoxy-d3)-5-nitropyridine-2-carboxylic acid Cpd-2-1 (2.40 g, 11.9 mmol) and 2-chloro-1-methylpyridinium iodide (3.35 g, 13.1 mmol) in DCM (80 mL) in sequence. The reaction mixture was stirred at 40 °C for 3 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, the residue was diluted with water (100 mL), the pH was adjusted to 2 with 1N hydrochloric acid aqueous solution and extracted with DCM (100 mL×3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by SGC (0-5% MeOH in DCM) to give N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)-5-nitropyridine-2-carboxamide Cpd-5-1 (1.10 g, yield 29.0%, white solid). LCMS calculated value C 11 H9D3N3O6S[MH] - :m / z=317.1; Detected value: 316.9.

[0221] Step 2: 5-amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide

[0222] At room temperature, a mixture of N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)-5-nitromethylpicolinamide Cpd-5-1 (1.10 g, 3.46 mmol) and palladium carbon (550 mg, 10% content) in THF (20 mL) and MeOH (30 mL) was stirred for 16 hours under a hydrogen atmosphere. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure to give 5-amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-5-2 (970 mg, crude, yellow-white solid). The crude product was used directly in the next step. LCMS calculated value C 11 H 11 D3N3O4S[MH] - :m / z=287.1; Detection value:Found:287.1.

[0223] Step 3: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide

[0224] A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-aldehyde Int-2 (500 mg, 1.51 mmol), N-(cyclopropylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-5-2 (416 mg, 1.44 mmol), acetic acid (86.6 mg, 1.44 mmol) and sodium sulfate (1.02 g, 7.21 mmol) in MeOH (20 mL) and 1,2-dichloroethane (20 mL) was stirred at room temperature for 16 hours. NaBH3CN (181 mg, 2.89 mmol) was then added and the system was stirred at room temperature for another 0.5 hours. LCMS detection indicated that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL×3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by SGC (0-70% EtOAc in PE) to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-5-3 (390 mg, yield 44.9%, yellow solid). LCMS calculated value C 24 H 20 BrD3F3N4O4S[M+H] +:m / z=602.1 / 604.1; Detected value: 602.3 / 604.3.

[0225] Step 4: N-(cyclopropylmethylsulfonyl)-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-carboxamide

[0226] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-5-3 (390 mg, 650 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (171 mg, 1.29 mmol), BrettPhos Pd G3 (58.7 mg, 60.0 μmol) and cesium carbonate (422 mg, 1.29 mmol) in DMF (12 mL) was stirred at 100 °C for 5 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL), adjusted to pH 8 with 1N aqueous hydrochloric acid solution and extracted with EtOAc / THF (2:1, 30 mL×3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by Prep-HPLC to give the product N-(cyclopropylmethylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-5 (81.2 mg, yield 19.1%, white solid). LCMS calculated value C 30 H 32 D3F4N6O4S[M+H] + :m / z=654.3; Detection value: 654.7; 1H NMR (400MHz, DMSO-d6) δ10.86(s,1H),7.66(d,J=8.0Hz,1H),7.58(d,J=7.2Hz,1H),7.07(d,J=8.0Hz,1H),6.92(s,1H),6.77( t,J=6.0Hz,1H),6.51(t,J=7.2Hz,1H),5.86(d,J=7.2Hz,1H),5.55(d,J=8.0Hz,1H),4.83(d,J=49.2Hz,1H),4.28(d,J=6.0Hz ,2H),3.98-3.90(m,2H),3.62-3.48(m,1H),3.43(d,J=7.2Hz,2H),3.08-3.03(m,1H),2.86-2.82(m,1H),2.33-2.21(m,1H),2 .23(s,3H),2.15-2.09(m,1H),2.03-1.93(m,1H),1.72-1.66(m,1H),1.09-1.00(m,1H),0.59-0.54(m,2H),0.34-0.30(m,2H).

[0227] Example 6: N-((2,2,2-trifluoroethyl)sulfonyl)-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-carboxamide (Cpd-6, i.e., Compound 6)

[0228]

[0229] Step 1: N-(2,4-dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide

[0230] At room temperature, 2,2,2-trifluoroethane-1-sulfonyl chloride Cpd-6-1 (750 mg, 4.11 mmol) was added dropwise to a THF (20 mL) solution of 2,4-dimethoxybenzylamine (1.37 g, 8.22 mmol), and the system was stirred for 1 hour. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction solution was concentrated under reduced pressure, and the residue was purified by SGC (0-1% MeOH in DCM) to obtain N-(2,4-dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide Cpd-6-2 (1.20 g, yield 93.2%, white solid). LCMS calculated value C 11 H 14 F3NNaO4S[M+Na] +:m / z=336.1; Detected value: 336.1.

[0231] Step 2: 2,2,2-Trifluoroethane-1-sulfonamide

[0232] N-(2,4-dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide Cpd-6-2 (1.45 g, 4.63 mmol) was dissolved in DCM (15 mL), trifluoroacetic acid (15 mL) was slowly added, and the system was stirred at room temperature for 1 hour. LCMS detection showed that the raw material was completely consumed, and the desired compound was detected. The reaction solution was concentrated under reduced pressure to obtain 2,2,2-trifluoroethane-1-sulfonamide Cpd-6-3 (851 mg, yield 99.1%, purple solid). The crude product was directly used for the next step reaction. 1 H NMR (400MHz, DMSO-d6) δ7.50 (s, 2H), 4.26 (q, J = 10.0Hz, 2H).

[0233] Step 3: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester

[0234] A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-aldehyde Int-2 (700 mg, 2.33 mmol), 5-amino-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester Cpd-2-3 (432 mg, 2.33 mmol) and acetic acid (280 mg, 4.67 mmol) in MeOH (20 mL) was stirred at room temperature for 16 hours. Then NaBH3CN (440 mg, 7.00 mmol) was added and the system was stirred at room temperature for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL×3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by SGC (0-70% EtOAc in PE) to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester Cpd-6-4 (625 mg, yield 53.7%, yellow solid). LCMS calculated value C 21 H 15 D3BrF3N3O3[M+H] + :m / z=499.1 / 501.1; Detection value:499.0 / 501.0.

[0235] Step 4: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid

[0236] 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester Cpd-6-4 (300 mg, 600 μmol) and sodium hydroxide (72.1 mg, 1.80 mmol) were dissolved in a mixed solvent of THF (9 mL), MeOH (6 mL) and water (1 mL), and the system was stirred at 50 ° C for 1 hour. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, diluted with water (20 mL) and adjusted to pH 5 with 1N hydrochloric acid. The solid was collected by filtration and dried to give 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid Cpd-6-5 (246 mg, yield 84.4%, white solid). LCMS calculated value C 20 H 13 D3BrF3N3O3[M+H] + :m / z=485.1 / 487.1; Detection value: 485.0 / 487.0.

[0237] Step 5: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-((2,2,2-trifluoroethyl)sulfonyl)pyridine-2-carboxamide

[0238] At 0°C, triethylamine (0.21 mL, 1.48 mmol) was added to a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylic acid Cpd-6-5 (240 mg, 490 μmol), 2,2-trifluoroethane-1-sulfonamide Cpd-6-3 (242 mg, 1.48 mmol) and 2-chloro-1-methylpyridinium iodide (316 mg, 1.24 mmol) in DCM (40 mL). The reaction solution was stirred at 0°C for 30 minutes. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction solution was diluted with DCM (100 mL) and washed with 0.5N aqueous hydrochloric acid solution (50 mL), saturated aqueous sodium bicarbonate solution (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by SGC (0-8% MeOH in DCM) to give 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-((2,2,2-trifluoroethyl)sulfonyl)pyridine-2-carboxamide Cpd-6-6 (160 mg, yield 51.3%, white solid). LCMS calculated value C 22 H 15 D3BrF6N4O4S[M+H] + :m / z=630.0 / 632.0; Detected value: 629.9 / 631.9.

[0239] Step 6: N-((2,2,2-trifluoroethyl)sulfonyl)-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-carboxamide

[0240] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-((2,2,2-trifluoroethyl)sulfonyl)pyridine-2-carboxamide Cpd-6-6 (160 mg, 250 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (67.0 mg, 510 μmol), BrettPhos Pd G3 (23.0 mg, 30.0 μmol) and cesium carbonate (165 mg, 510 μmol) in THF (13 mL) and NMP (1.3 mL) was stirred in a sealed tube at 100° C. for 4 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL×3). The organic layers were combined and washed with water (40 mL) and brine (40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by SGC (0-8% MeOH in DCM) to give the product N-((2,2,2-trifluoroethyl)sulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-6 (14.0 mg, yield 8.09%, white solid). LCMS calculated value C 28 H 27 D3F7N6O4S[M+H] + :m / z=682.2;Detection value:682.5; 1 HNMR(400MHz,DMSO-d6)δ10.53(s,1H),7.64(dd,J=16.0,6.9Hz,2H),7.01(d,J=7.5Hz,1H), 6.93(s,1H),6.53(t,J=6.4Hz,1H),6.50-6.33(m,1H),5.90(d,J=7.1Hz,1H),5.81(d,J=6.4H z,1H),5.09(d,J=47.1Hz,1H),4.70-4.33(m,2H),4.26(d,J=4.2Hz,2H),3.97(d,J=10.4Hz,2 H),3.89-3.76(m,1H),2.73(s,3H),2.49-2.45(m,2H),2.26-2.16(m,1H),1.97-1.88(m,1H).

[0241] Example 7: N-(Cyclopropylsulfonyl)-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-methoxypyridine-2-carboxamide (Cpd-7, i.e., Compound 7)

[0242]

[0243] Step 1: 6-Chloro-N-(cyclopropylsulfonyl)-5-nitropyridine-2-carboxamide

[0244] At room temperature, cyclopropylsulfonamide (3.59 g, 29.6 mmol) and triethylamine (7.49 g, 74.1 mmol) were added to a mixture of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (5.00 g, 24.7 mmol) and 2-chloro-1-methylpyridine iodide (7.57 g, 29.6 mmol) in DCM (50 mL) in sequence. The reaction system was stirred at 40 ° C for 3 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, the residue was diluted with water (100 mL), and the pH was adjusted to 2 with 1N hydrochloric acid aqueous solution. The precipitated solid was filtered, washed with water and dried to obtain 6-chloro-N-(cyclopropylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-7-1 (8.90 g, crude product, white solid). The crude product was directly used in the next step reaction. LCMS calculated value C9H7ClN3O5S [MH] - :m / z=304.0 / 306.0; Detected value: 303.9 / 305.9.

[0245] Step 2: N-(cyclopropylsulfonyl)-6-methoxy-5-nitropyridine-2-carboxamide

[0246] At room temperature, a methanol solution of sodium methoxide (16.2 mL, 87.3 mmol, 5.4 mol / L) was added dropwise to a methanol (90 mL) solution of 6-chloro-N-(cyclopropylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-7-1 (8.90 g, 29.1 mmol). The reaction solution was stirred at room temperature for 30 minutes. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was quenched with 1N aqueous hydrochloric acid solution and the pH was adjusted to 2. The precipitated solid was filtered, washed with water and dried to obtain N-(cyclopropylsulfonyl)-6-methoxy-5-nitropyridine-2-carboxamide Cpd-7-2 (6.70 g, crude product, white solid). The crude product was directly used in the next step. LCMS calculated value C 10 H 10 N3O6S[MH] -:m / z=300.0; Detection value: 300.0.

[0247] Step 3: 5-amino-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide

[0248] At room temperature, a mixture of N-(cyclopropylsulfonyl)-6-methoxy-5-nitropyridine-2-carboxamide Cpd-7-2 (6.70 g, 22.2 mmol) and palladium carbon (3.30 g, 5% content) in THF (200 mL) and MeOH (130 mL) was stirred in a hydrogen atmosphere at room temperature for 16 hours. LCMS detection showed that the raw material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure to give 5-amino-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide Cpd-7-3 (6.20 g, crude, white solid). The crude product was directly used in the next step. LCMS calculated value C 10 H 12 N3O4S[MH] - :m / z=270.1; Detected value: 270.1.

[0249] Step 4: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide

[0250] A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-aldehyde Int-2 (1.00 g, 3.03 mmol), 5-amino-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide Cpd-7-3 (820 mg, 3.03 mmol) and acetic acid (180 mg, 3.03 mmol) in MeOH (15 mL) was stirred at room temperature for 16 hours. Then NaBH3CN (570 mg, 9.09 mmol) was added and the system was stirred at room temperature for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (50 mL) and extracted with EtOAc (20 mL×3), and the organic layers were combined and washed with water (40 mL), saturated brine (40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by slurrying with dichloromethane to give 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide Cpd-7-4 (500 mg, yield 28.2%, yellow solid). LCMS calculated value C 23 H 21BrF3N4O4S[M+H] + :m / z=585.0 / 587.0; Detection value: 585.3 / 587.3.

[0251] Step 5: N-(cyclopropylsulfonyl)-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-methoxypyridine-2-carboxamide

[0252] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide Cpd-7-4 (500 mg, 850 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (226 mg, 1.71 mmol), BrettPhos Pd G3 (77.4 mg, 10.0 μmol) and cesium carbonate (557 mg, 1.71 mmol) in THF (15 mL) and NMP (1.5 mL) was stirred in a sealed tube at 100° C. for 5 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL×3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by Prep-HPLC to give the product N-(cyclopropylsulfonyl)-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-methoxypyridine-2-carboxamide Cpd-7 (46.1 mg, yield 8.3%, yellow solid). LCMS calculated value C 29 H 33 F4N6O4S[M+H] + :m / z=637.2; Detection value: 637.1; 1H NMR (400MHz, DMSO-d6) δ10.83(s,1H),7.68(d,J=6.8Hz,1H),7.58(d,J=6.8Hz,1H),7.08(d,J=8.4Hz,1H),6.92(s,1H) ,6.76(t,J=6.0Hz,1H),6.51(t,J=7.2Hz,1H),5.86(d,J=7.6Hz,1H),5.55(d,J=8.4Hz,1H),4.83(d,J=48.6Hz,1H),4. 28(d,J=6.4Hz,2H),4.06(s,3H),4.00-3.91(m,2H),3.62-3.49(m,1H),3.11-3.03(m,2H),2.84-2.82(m,1H),2.33-2. 21(m,1H),2.23(s,3H),2.15-2.09(m,1H),2.03-1.93(m,1H),1.71-1.67(m,1H),1.19-1.15(m,2H),1.10-1.04(m,2H).

[0253] Example 8: N-(methylsulfonyl)-5-((3-(8-(((3R,4S)-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-carboxamide (Cpd-8, i.e., Compound 8)

[0254]

[0255] Step 1: 6-Chloro-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide

[0256] Under stirring, 2-chloro-1-methylpyridinium iodide (95.0 g, 372 mmol) and methanesulfonylamine (35.0 g, 368 mmol) were added to a suspension of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (50.0 g, 247 mmol) and dichloromethane (500 mL). After stirring the system for 10 minutes, triethylamine (100 g, 988 mmol) was added dropwise. After the addition was complete, the temperature was raised to reflux and the reaction was continued for about 5 hours. The mixture was cooled and concentrated, and the residue was diluted with water (250 mL) and adjusted to pH = 2 with concentrated hydrochloric acid. A large amount of yellow solid precipitated. The mixture was stirred at room temperature overnight, filtered, and the filter cake was rinsed with water (250 mL). The filter cake was dried under vacuum at 50°C to obtain 6-chloro-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-1 (68.0 g, yield 98.5%, yellow solid). LCMS calculated value C7H5ClN3O5S[MH] -:m / z=278.0; Detection value: 278.1.

[0257] Step 2: 6-(Methoxy-d3)-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide

[0258] Under argon protection, 6-chloro-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-1 (45.0 g, 161 mmol) and deuterated methanol (17.4 g, 482 mmol) were dissolved in anhydrous N-methylpyrrolidone (450 mL). The system was cooled to -15-0 ° C, and 1M potassium tert-butoxide tetrahydrofuran solution (338 mL, 338 mmol) was added dropwise. After the addition was completed, the system was temperature controlled at -10 to 0 ° C for 40 minutes, and then 4N hydrochloric acid (135 mL) was added dropwise to terminate the reaction. The system was concentrated under reduced pressure, and water (1.62 L) was added dropwise with stirring. A large amount of yellow solid was produced. The filter cake was filtered, and rinsed with water (500 mL) and methyl tert-butyl ether (500 mL). The filter cake was recrystallized from acetonitrile water to obtain the product 6-(methoxy-d3)-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-2 (30.0 g, yield 67.0%, yellow solid). LCMS calculated value C8H7D3N3O6S[M+H] + :m / z=279.0; Detected value: 279.2.

[0259] Step 3: 5-amino-6-(methoxy-d3)-N-(methylsulfonyl)pyridine-2-carboxamide

[0260] 6-(Methoxy-d3)-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-2 (30.0 g, 108 mmol) was mixed with tetrahydrofuran (600 mL) and acetic acid (3.24 g, 53.9 mmol) to form a solution, and then methanol (600 mL) was added. After argon replacement, palladium carbon (3.00 g, 10% content) was added, and the argon was replaced with hydrogen. The reaction was carried out at room temperature for 12 hours. Filtered with diatomaceous earth, the filter cake was washed with tetrahydrofuran, and the filtrate was combined and concentrated. The residue was concentrated and dried to obtain the product 5-amino-6-(methoxy-d3)-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-8-3 (26.0 g, yield 97.1%, off-white solid), which was directly used for the next step without further treatment. LCMS calculated value C8H9D3N3O4S[M+H] + :m / z=249.1; Detected value: 249.2.

[0261] Step 4: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide

[0262] To 5-amino-6-(methoxy-d3)-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-8-3 (7.20 g, 29.0 mmol) and acetic acid (3.14 g, 58.0 mmol) was added a mixed solvent of methanol (100 mL) and dichloromethane (100 mL). After the solution became clear, Int-2 (10.1 g, 30.5 mmol) was added. The system was stirred at 10-15 °C for 16 hours. A large amount of turbidity appeared in the system. Then sodium cyanoborohydride (6.00 g, 87.1 mmol) was added and the system was stirred for 2 hours. The system was concentrated, methanol (100 mL) was added, water (100 mL) was added dropwise at room temperature, and then stirred at room temperature for 2 hours, filtered, the filter cake was rinsed with water (50 mL) and methyl tert-butyl ether (100 mL), and dried under oil pump vacuum to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-8-4 (12.9 g, yield 79.0%, off-white solid). LCMS calculated value C 21 H 16 D3BrF3N4O4S[M+H] + :m / z=562.0 / 564.0; Detected value: 562.2 / 564.2.

[0263] Step 5: N-(methylsulfonyl)-5-((3-(8-(((3R,4S)-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-carboxamide

[0264] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-8-4 (1.80 g, 3.20 mmol), (3R,4S)-3-fluoro-1-methylpiperidin-4-amine (500 mg, 3.80 mmol), BrettPhos Pd G3 (600 mg, 662 μmol) and cesium carbonate (3.44 g, 10.6 mmol) in tetrahydrofuran (20 mL) and N,N-dimethylformamide (10 mL) was stirred at 70 °C for 3 hours. The system was cooled and concentrated, and the residue was purified by silica gel column chromatography (MeOH:DCM=1:24) to give the product N-(methylsulfonyl)-5-((3-(8-(((3R,4S)-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-carboxamide Cpd-8 (830 mg, yield 42.3%, off-white solid). LCMS calculated value C 27 H 28 D3F4N6O4S[M+H] + :m / z=614.2;Detection value: 614.4; 1 H NMR (400MHz, DMSO-d6) δ10.83(s,1H),7.67(d,J=8.0Hz,1H),7.58(d,J=6.9Hz,1H),7.07(d,J=8.1Hz,1H) ,6.92(s,1H),6.75(t,J=6.1Hz,1H),6.52(t,J=7.1Hz,1H),5.86(d,J=7.5Hz,1H),5.56(d,J=8.4Hz,1H), 4.84(d,J=49.3Hz,1H),4.28(d,J=6.1Hz,2H),3.95(q,J=10.8Hz,2H),3.56(d,J=28.9Hz,1H),3.07(t,J= 11.2Hz,1H),2.85(d,J=11.1Hz,1H),2.40-2.09(m,5H),2.05-1.91(m,1H),1.69(dd,J=13.2,4.0Hz,1H).

[0265] Example 9: N-(methylsulfonyl)-5-((3-(8-(((3S,4S)-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-carboxamide (Cpd-9, i.e., Compound 9)

[0266]

[0267] Step 1: N-(methylsulfonyl)-5-((3-(8-(((3S,4S)-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-carboxamide

[0268] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-8-4 (1.80 g, 3.20 mmol), (3S,4S)-3-fluoro-1-methylpiperidin-4-amine (400 mg, 3.02 mmol), BrettPhos Pd G3 (600 mg, 662 μmol) and cesium carbonate (3.44 g, 10.6 mmol) in tetrahydrofuran (20 mL) and N,N-dimethylformamide (10 mL) was stirred at 70 °C for 3 hours. The system was cooled and concentrated, and the residue was purified by silica gel column chromatography (MeOH:DCM=1:24) to give the product N-(methylsulfonyl)-5-((3-(8-(((3S,4S)-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-carboxamide Cpd-9 (500 mg, yield 26.9%, brown solid). LCMS calculated value C 27 H 28 D3F4N6O4S[M+H] + :m / z=614.2;Detection value: 614.4; 1H NMR (400MHz, DMSO-d6) δ10.85(s,1H),7.68(d,J=8.0Hz,1H),7.55(d,J=6.9Hz,1H),7.07(d,J=8.1Hz, 1H),6.78(s,1H),6.74(d,J=7.8Hz,1H),6.51(t,J=7.2Hz,1H),5.86(d,J=7.5Hz,1H),5.74(d,J=8.3Hz ,1H),4.57(dtd,J=49.6,9.3,4.7Hz,1H),4.28(d,J=6.2Hz,2H),3.95(q,J=10.7Hz,2H),3.49(d,J=4. 2Hz,1H),3.10(p,J=5.4Hz,1H),2.77-2.62(m,1H),2.24(s,3H),2.19-1.90(m,3H),1.61-1.39(m,1H).

[0269] Example 10: N-(methylsulfonyl)-5-((3-(8-(((3R,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-carboxamide (Cpd-10, i.e., Compound 10)

[0270]

[0271] Step 1: N-(methylsulfonyl)-5-((3-(8-(((3R,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-carboxamide

[0272] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-8-4 (1.80 g, 3.20 mmol), (3R,4R)-3-fluoro-1-methylpiperidin-4-amine (500 mg, 3.78 mmol), BrettPhos Pd G3 (600 mg, 662 μmol) and cesium carbonate (3.44 g, 10.6 mmol) in tetrahydrofuran (20 mL) and N,N-dimethylformamide (10 mL) was stirred at 70 °C for 3 hours. The system was cooled and concentrated, and the residue was purified by silica gel column chromatography (MeOH:DCM=1:24) to give the product N-(methylsulfonyl)-5-((3-(8-(((3R,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-carboxamide Cpd-10 (1.12 g, yield 57.0%, brown solid). LCMS calculated value C 27 H 28 D3F4N6O4S[M+H] + :m / z=614.2;Detection value: 614.4; 1 H NMR (400MHz, DMSO-d6) δ10.89(s,1H),7.68(d,J=8.0Hz,1H),7.55(d,J=6.9Hz,1H),7.08(d,J=8.1 Hz,1H),6.81-6.78(m,2H),6.51(t,J=7.1Hz,1H),5.86(d,J=7.4Hz,1H),5.74(d,J=8.3Hz,1H),4.5 7(dtd,J=49.6,9.2,4.8Hz,1H),4.28(d,J=6.1Hz,2H),3.95(q,J=10.7Hz,2H),3.59-3.41(m,1H),3 .10(dt,J=10.9,5.7Hz,1H),2.77-2.59(m,1H),2.25(s,3H),2.17-1.83(m,3H),1.62-1.38(m,1H).

[0273] Biological evaluation

[0274] 1. Biochemical activity experiment: Activity test of compounds promoting the binding of p53 Y220C mutant to DNA

[0275] Homogeneous time-resolved fluorescence (HTRF) experiments were used to measure the reactivation effect of compounds on the p53 Y220C mutant. Recombinant His-tagged p53 Y220C (94-312) for HTRF experiments was expressed in E. coli and purified to 90% purity by Ni-NTA column. The biotin-labeled DNA used for HTRF experiments was synthesized by Kanglong Chemical, and the specific sequence was 5'-ATTAGGCATGTCTAGGCATGTCTAGG-3'.

[0276] The binding of recombinant His-tagged p53 Y220C protein and biotin-labeled DNA was measured using fluorescence resonance energy transfer (FRET). For FRET experiments, the binding between p53 mutants and DNA sequences was measured by detecting the fluorescence of the interaction between an anti-His antibody conjugated to Eu (Cisbio, 61HI2KLA) and d2-conjugated streptavidin (Cisbo, 610SADLF) bound to a biotin-labeled DNA molecule. Compounds were prepared as 2mM stock solutions and serially diluted 10 concentrations in 1:3 in DMSO. Each 200-fold working concentration compound was diluted sequentially to a 4-fold working concentration. 4 μL of compound solution was transferred row by row to a 384 assay plate using Echo, with 2 replicates per column. 4 μL of p53 solution was added to the assay plate. 4 μL of biotin DNA solution was added to the assay plate, and then 4 μL of the assay 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).

[0277] Calculate the ratio for each well (Ratio_665nm / 615nm - Ratio_background). The activity % is calculated as follows:

[0278] C=(Ave_Ac-Ave_Ba) / (Ave_Dc-Ave_Bd)

[0279] R data=(A-Ave_Ba-C*D) / (D-Ave_Bd)*(Ave_Dc-Ave_Bd)

[0280] Activity% = R data / Ave_VC * 100.

[0281] A: Fluorescence intensity of the sample at 665 nm;

[0282] D: Fluorescence intensity of the sample at 615 nm;

[0283] Ba: fluorescence intensity of the plate background at 665 nm;

[0284] Bd: fluorescence intensity of the plate background at 615 nm;

[0285] Dc: fluorescence intensity of HIS-Eu background at 615 nm;

[0286] Ac: fluorescence intensity of streptavidin-d2 background at 665 nm;

[0287] VC: DMSO-treated group.

[0288] 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 .

[0289] Table 1: Activity of compounds in promoting the binding of p53 Y220C mutant to DNA

[0290]

[0291]

[0292] Cpd-A is selected from WO2021061643A1; Cpd-B is selected from WO2022213975A1;

[0293]

[0294] 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.

[0295] 2. Cell activity experiment: Proliferation inhibition activity test of NUGC-3 cells

[0296] The antiproliferative effect of the compounds on human gastric adenocarcinoma cell lines was evaluated based on the activity of dehydrogenases in the cells, which correlated well with the number of viable cells. The cell line NUGC-3 (JCRB, JCRB0822) was cultured in RPMI 1640 (Invitrogen, 11875-085) medium supplemented with 10% (v / v) fetal bovine serum (BI, 04-002-1A). The cell lines were cultured according to the standard instructions of the American Type Culture Collection. The cell lines were identified by short tandem repeat profiles.

[0297] In the proliferation experiment, 1000 NUGC-3 cells were seeded in a 96-well flat-bottom 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).

[0298] Table 2: Proliferation inhibition activity of compounds on NUGC-3 cells

[0299]

[0300]

[0301] 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.

[0302] 3. Evaluation of drug-drug interactions related to metabolic enzymes

[0303] (I) CYP enzyme inhibitory activity test at a single concentration

[0304] 1. Experimental purpose: To test the inhibitory effect of the compound on CYP series enzymes at a single concentration.

[0305] 2. Experimental instruments and reagents:

[0306] Liver microsomes:

[0307] Species gender supplier Part Number batch storage people mix Corning 452117 38298 -80℃

[0308] Reagents:

[0309] Compound supplier Part Number store Terfenadine Sigma-Aldrich T9652 4℃ Tolbutamide Sigma-Aldrich T0891 4℃ Dipotassium hydrogen phosphate Sinopharm Group 20032117 Room temperature Nicotinamide adenine dinucleotide phosphate, reduced Shanghai AngYi Biotechnology Co., Ltd. N99640-100MG -20℃ Phenacetin Sigma-Aldrich 101690303 Room temperature Diclofenac Sigma-Aldrich D6899-10G Room temperature Mephenytoin Glpbio GC14486 -20℃ Dextromethorphan Sigma-Aldrich D9684-5G Room temperature Midazolam National Institute for Food and Drug Control PVJT-0H9Z Room temperature Testosterone Adamas reagent 171265 Room temperature Bupropion Cayman Chemical Company 10488 -20℃ Amodiaquine Abmole M5412 -20℃ β-Naphthoflavone Sigma-Aldrich N5757-1G 4℃ Sulfadiazine Sigma-Aldrich S0758-1G 4℃ Benzylnilanol Shanghai Yuanye Biotechnology Co., Ltd. Y43177-5mg Room temperature Quinidine International-Laboratory-USA 1311468-5G Room temperature Ketoconazole Sigma-Aldrich K1003-100MG 4℃

[0310] 3. Experimental methods:

[0311] Dissolve 8.71 g K2HPO4 in 950 mL water. Adjust pH to 7.4 with HCl solution. Adjust to final volume of 1000 mL with water. Filter buffer through 0.45 μm filter. Store in 4°C refrigerator for future use. Prepare terfenadine / tolbutamide (1 mg / mL each) stock solution in DMSO. Dilute the above stock solution with acetonitrile to prepare a quenching solution containing 5 / 10 ng / mL (terfenadine / tolbutamide).

[0312] Positive control working solution:

[0313]

[0314] Substrate working solution:

[0315]

[0316] Thaw liver microsomes in a 37°C water bath before use. Prepare liver microsome working solution (final concentration of the system: 0.5 mg / mL). Prepare 5 mM NADPH working solution with phosphate buffer. Prepare the above stock solution into 2 mM working solution with DMSO.

[0317] Add 238.5 μL of liver microsome working solution to a 1.1 mL tube. Add 1.5 μL of test compound / control working solution / DMSO and mix by pipetting several times. Preincubate in a shaking water bath at 37°C for 5 minutes. After preincubation, add 60 μL of NADPH working solution and mix by pipetting several times. Incubate in a shaking water bath at 37°C for 10 minutes. After incubation, immediately add 500 μL of quenching solution and vortex for 1 minute. Centrifuge all samples at 4,000 rpm for 15 minutes at 4°C. Aliquot 300 μL of the above supernatant for further LC-MS / MS analysis.

[0318] 4. Data processing methods and results:

[0319] The percent inhibition (% Inhibition) was calculated using metabolite formation of the test or control compound compared to vehicle control.

[0320] Table 3: Inhibitory activity of compounds against CYP enzymes*

[0321]

[0322]

[0323] *The final concentration of the compound was 10 μM; ND: not detected; NI: no inhibition

[0324] The above data show that the Example compounds of the present invention have weak inhibitory activity on CYP series enzymes, and the risk of drug interaction is significantly lower than that of the reference compounds.

[0325] (II) Determination of multi-concentration inhibition curves of CYP3A4 / 5 enzyme

[0326] 1. Experimental purpose: To determine the concentration inhibition curve of the compound on CYP3A4 / 5 enzyme.

[0327] 2. Experimental instruments and materials:

[0328] Liver microsomes:

[0329] Species gender supplier Part Number batch storage people mix Corning 452117 38298 -80℃

[0330] Reagents:

[0331]

[0332] 3. Experimental methods:

[0333] Dissolve 8.71 g K2HPO4 in 950 mL water. Adjust pH to 7.4 with HCl solution. Adjust to final volume of 1000 mL with water. Filter buffer through 0.45 μm filter. Store in 4°C refrigerator for future use. Prepare terfenadine / tolbutamide (1 mg / mL each) stock solution in DMSO. Dilute the above stock solution with acetonitrile to prepare a quenching solution containing 5 / 10 ng / mL (terfenadine / tolbutamide).

[0334] Positive control working solution:

[0335]

[0336]

[0337] Substrate working solution:

[0338]

[0339] Thaw liver microsomes in a 37°C water bath before use. Prepare liver microsome working solution (final concentration of the system 0.5 mg / mL). Prepare 5 mM NADPH working solution with phosphate buffer. Prepare the above stock solution to 2 mM working solution with DMSO. Dilute the above stock solution with acetonitrile (4-fold dilution, 6 non-zero concentrations).

[0340] Add 238.5 μL of liver microsome working solution to a 1.1 mL tube. Add 1.5 μL of test compound / control working solution / DMSO,

[0341] Mix well by pipetting several times. Pre-incubate in a 37℃ shaking water bath for 5 minutes. After pre-incubation, add 60μL NADPH working solution and mix well by pipetting several times.

[0342] Incubate in a shaking water bath at 37°C for 10 minutes. Immediately after incubation, add 500 μL of quenching solution and vortex for 1 minute. Centrifuge all samples at 4,000 rpm for 15 minutes at 4°C. Aliquot 300 μL of the above supernatant for further LC-MS / MS analysis.

[0343] 4. Data processing methods and results:

[0344] Graph Pad Prism was used to calculate the half inhibitory concentration (IC 50).

[0345] Table 4: Inhibitory activity of compounds against CYP3A4 / 5 enzymes

[0346]

[0347] ND: Not Detected

[0348] The above data show that the example compounds of the present invention have no obvious inhibitory activity on CYP3A4 / 5 enzyme, and the risk of drug interaction is significantly lower than that of the reference compounds.

[0349] IV. Evaluation of in vitro metabolic stability: Study on metabolic stability of liver microsomes:

[0350] 1. Experimental purpose: To evaluate the metabolic stability of the test compounds in human / rat / mouse liver microsomes.

[0351] 2. Experimental reagents and materials:

[0352] Reagents:

[0353]

[0354]

[0355] Material:

[0356] Experimental material name gender Manufacturer Part Number Storage conditions Human liver microsomes Mixed Corning 452117 -80℃ Rat liver microsomes male BioreclamationIVT M00001 -80℃ Mouse liver microsomes male BioreclamationIVT M00501 -80℃

[0357] 3. Experimental methods:

[0358] The test compound and the positive control compound (dextromethorphan as the control compound) were dissolved in DMSO to prepare a 10mM stock solution, and the stock solution was diluted with DMSO to a 200μM working solution. 8.709g of potassium dihydrogen phosphate (K2HPO4) was dissolved in 950mL of water, the pH value of the solution was adjusted to 7.4 with hydrochloric acid, and then water was added to make up to 1000mL. After filtering with a 0.22μm filter membrane, it was placed in a 4-degree refrigerator for standby use. After various types of liver microsomes (protein concentration 20mg / mL) were melted in a 37-degree water bath, they were diluted with phosphate buffer solution to obtain a liver microsome working solution with a protein concentration of 0.629mg / mL. A 5mM NADPH solution was prepared with the above-mentioned phosphate buffer for standby use. A 1 mg / mL stock solution of terfenadine / tolbutamide was prepared with DMSO, and then diluted with a mixed solution of 50% methanol / 50% acetonitrile to form a reaction stop solution containing 5 / 10 ng / mL (terfenadine / tolbutamide) as an internal standard.

[0359] Take 238.5μL of different species of liver microsome working solution and add it to a 1.1mL microtube, add 1.5μL of the working solution of the test compound or the working solution of the positive control compound (dextromethorphan) (200μM), mix well and pre-incubate in a 37-degree water bath for 5min. Add 60μL of NADPH solution to start the reaction. After thorough mixing, at 0, 5, 15, 30 and 60 minutes after the reaction, pipette 30μL of the reaction solution and add it to 300μL of the reaction stop solution. Vortex the samples at all time points vigorously for 1 minute and centrifuge them at 4000rpm at 4℃ for 15 minutes. Take 100μL of the supernatant and add 100μL of pure water to mix well before LC-MS / MS analysis.

[0360] 4. Data processing methods:

[0361] The slope (ke) was measured by plotting the natural logarithm of the percentage of the remaining compound against time, and T was calculated according to the first-order kinetic formula. 1 / 2 and intrinsic clearance (CL int ):

[0362] The compound residual rate is calculated as follows:

[0363]

[0364] Intrinsic clearance CL int (μL / min / mg protein)=0.693*1000 / T 1 / 2 / Protein concentration (0.5mgprotein / mL)

[0365] Table 5: Metabolic stability of compounds in human / rat / mouse liver microsomes

[0366]

[0367]

[0368] The above data show that the example compounds of the present invention have high metabolic stability in liver microsomes of multiple species and are significantly better than the reference compounds.

[0369] 5. In vitro metabolic stability evaluation: hepatocyte metabolic stability test

[0370] 1. Experimental purpose: To determine the metabolic stability of compounds in human / rat / mouse hepatocytes.

[0371] 2. Experimental Materials:

[0372] Species strain gender supplier people N / A mix BioIVT Rat Sprague Dawley male BioIVT Mouse ICR / CD-1 male BioIVT

[0373] 3 Experimental design

[0374] 3.1 Preparation of compound working solution

[0375] The test substance and control drug verapamil powder were prepared into high concentration stock solution with DSMO, and diluted to 100 μM working solution with 50% acetonitrile / water before use. The final concentration of the test substance and verapamil was 1 μM.

[0376] 3.2 Preparation of hepatocytes

[0377] 1) Mix 49.5 mL Williams' Medium E and 0.5 mL GlutaMAX as incubation medium.

[0378] Preheat the incubation solution and the culture medium in a 37°C water bath for at least 15 minutes before use.

[0379] 2) Take a tube of cryopreserved hepatocytes and ensure that the hepatocytes are still in a low-temperature frozen state before thawing. Quickly place the hepatocytes in a 37°C water bath and gently shake until all ice crystals are dispersed, spray with 70% ethanol and transfer to a biosafety cabinet.

[0380] 3) Pour the contents of the hepatocyte tubules into a centrifuge tube containing 50 mL of recovery medium and centrifuge at 100 g for 10 minutes. After centrifugation, aspirate the recovery medium and add enough incubation medium to obtain a cell density of about 1.5×10 6 cells / mL of cell suspension.

[0381] 4) Count the hepatocytes and determine the viable cell density using Cellometer Vision. The hepatocyte viability must be greater than 75%. Dilute the hepatocyte suspension with incubation medium to a viable cell density of 0.5×10 6 cells / mL.

[0382] 5) A portion with a density of 0.5×10 6 The hepatocyte suspension with a concentration of 10 cells / mL was inactivated by boiling water for 5 minutes as a negative control. After the cells are inactivated, it is convenient to examine the substrate conversion mediated by the non-cellular enzyme system.

[0383] 3.3 Test methods

[0384] 1) Transfer 198 μL of live cell or inactivated cell suspension to a 96-well deep-well plate, place the deep-well plate on a vortex and preheat in an incubator for 10 minutes. Live cells are incubated in parallel, and inactivated cells are incubated in parallel.

[0385] 2) Add 2 μL of 100 μM test substance or verapamil to each well to start the reaction, and place the deep-well plate back on the incubator vortexer.

[0386] 3) Incubate the sample, take 25 μL of the suspension at 0, 15, 30, 60, 90 and 120 minutes, add 150 μL of acetonitrile containing internal standard (200 nM alprazolam, 200 nM labetalol, 2 μM ketoprofen, 200 nM caffeine) to terminate the reaction. Vortex for 10 minutes, centrifuge at 3220g and 4°C for 30 minutes, take 100 μL of the supernatant and mix with 100 μL of ultrapure water for UPLC-MS / MS analysis.

[0387] 4 Data analysis

[0388] All data calculations were performed using Microsoft Excel. Peak areas were detected by extracting ion spectra. The in vitro half-life (t 1 / 2 ).

[0389] In vitro half-life (t 1 / 2 ) is calculated by the slope:

[0390] in vitro 1 / 2 =0.693 / k

[0391] In vitro intrinsic clearance (unit: μL / min / 10 6 cells) was calculated using the following formula:

[0392] in vitro CLint=kV / N

[0393] V = incubation volume per well (0.2 mL);

[0394] N = number of cells per well (0.1×10 6 cells)

[0395] Table 6: Metabolic stability of compounds in human / rat / mouse hepatocytes

[0396]

[0397] The above data show that the example compounds of the present invention have high metabolic stability in human / rat / mouse hepatocytes and are significantly better than the reference compounds.

[0398] VI. Cardiac safety evaluation: hERG potassium channel inhibitory activity test

[0399] 1. Experimental purpose:

[0400] Manual patch clamp technique was used to evaluate whether the test compound had a potential inhibitory effect on the voltage-gated potassium channel hERG. This experiment detected the effect of 5 concentrations of the compound on the hERG channel current, obtained the dose-effect curve of the compound and calculated the IC50 .

[0401] 2. Experimental instruments and reagents:

[0402]

[0403] 3. Experimental methods:

[0404] Cell lines and cell culture: HEK293 cell lines stably expressing hERG ion channels (Cat. No. K1236) were purchased from Invitrogen. The cell lines were cultured in a medium containing 85% DMEM, 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acid solution, 100 U / mL penicillin-streptomycin solution, 25 mM HEPES, 5 μg / mL blasticidin and 400 μg / mL geneticin. When the cell density grew to 40% to 80% of the bottom area of ​​the culture dish, the cells were digested by trypsin and passaged three times a week. Before the experiment, the cells were cultured at 5×10 5 The cells were cultured at a density of 1.5 cm in a 3.5 cm dish and induced for 48 h by adding 1 μg / mL doxycycline. The cells were then digested and seeded on glass slides for subsequent manual patch clamp experiments.

[0405] Solution preparation: Extracellular solution (in mM): 132 sodium chloride, 4 potassium chloride, 3 calcium chloride, 0.5 magnesium chloride, 11.1 glucose and 10 HEPES (adjust pH to 7.35 with sodium hydroxide). Intracellular solution (in mM): 10 EGTA, 10 HEPES, 10 potassium chloride, 10 sodium chloride, 110 potassium fluoride (adjust pH to 7.2 with potassium hydroxide). The osmotic pressure of the solution is controlled between 280 and 300 mOsmol / kg. The solution needs to be filtered and stored at 4°C before use.

[0406] Preparation of test compound solution: According to standard operating procedures, the test compound was dissolved in DMSO and prepared into a stock solution with a final concentration of 10mM. The stock solution was diluted with DMSO as the solvent at a ratio of 1:3 to form three other intermediate concentration solutions, with concentrations (mM): 3.33, 1.11 and 0.37. Before the experiment, the intermediate solution of the test compound gradient was diluted again with extracellular fluid at a ratio of 1:1000 to form a series of working solutions. The 30μM working solution was prepared into a working solution with a ratio of 1:333.33 times using the 10mM stock solution, and its final concentrations were (μM): 30, 10, 3.33, 1.11 and 0.37. The content of DMSO in the working solution was 0.1-0.3% (volume ratio). Five working solutions with different concentration gradients of 30, 10, 3.33, 1.11, and 0.37μM were used to determine the potential inhibitory effect of the compound on the hERG channel and to fit the dose-effect curve and calculate IC50.

[0407] Experimental procedure: Place a small glass slide containing HEK293 cells in a culture dish in the perfusion tank of the microscope operation table. Under an Olympus IX71 or IX73 inverted microscope, place the appropriate cell in the center of the field of view, use the ×10x objective to find the tip of the glass electrode and place it in the center of the field of view. Then use the micromanipulator to move the electrode down and adjust the coarse focus knob to slowly approach the cell. When it is almost close to the cell, switch to the ×40x objective for observation, and use the micromanipulator to fine-tune the gear to gradually approach the surface of the cell. Apply negative pressure to form a seal with a resistance higher than 1GΩ between the electrode tip and the cell membrane. Compensate for the instantaneous capacitive current Cfast in voltage clamp mode. Then repeatedly apply short negative pressure to break the membrane, and finally form a whole-cell recording mode. Under the condition that the membrane potential is clamped at -60mV, compensate for the slow capacitive current Cslow, cell membrane capacitance (Cm) and input membrane resistance (Ra). After the cells stabilize, the clamping voltage is changed to -90mV, the sampling frequency is set to 20kHz, and the filtering frequency is 10kHz. The detection condition of the leakage current is that the clamping voltage is changed to -80mV and the time course is 500ms. The hERG current test method is as follows: apply a 4.8-second depolarization command voltage to depolarize the membrane potential from -80mV to +30mV, and then instantly apply a 5.2-second repolarization voltage to reduce the membrane potential to -50mV to remove channel inactivation, so that the hERG tail current can be observed. The peak value of the tail current is the magnitude of the hERG current. The hERG current used to detect the test compound was recorded continuously for 120 seconds before administration to evaluate the stability of the hERG current generated by the test cells. Only stable cells within the acceptance range of the evaluation criteria can enter the subsequent compound detection. Test of the inhibitory effect of the test compound on the hERG current: First, the hERG current measured in the extracellular fluid containing 0.1% DMSO is used as the detection baseline. After the hERG current remains stable for at least 5 minutes, the solution containing the test compound is perfused around the cells in sequence from low concentration to high concentration. After each perfusion, wait for about 5 minutes to allow the compound to fully act on the cells and record the hERG current synchronously. After the recorded current tends to stabilize, record the last 5 hERG current values, and take the average value as the final current value at a specific concentration. After testing the compound, add 450nM dofetilide to the same cell to completely inhibit its current as a positive control for the cell. At the same time, the positive compound dofetilide is synchronously detected using the same patch clamp system before and after the test drug experiment to ensure the reliability and sensitivity of the entire detection system. The above test steps will be repeated on two separate test cells (n=2).

[0408] 4. Data processing methods and results:

[0409] Data quality control standards: initial sealing resistance greater than 1GΩ; series resistance less than 15MΩ and series resistance voltage error less than 5mV; leakage current under detection voltage less than 50% of the current value under this condition; tail current greater than the platform current of the pre-pulse, initial tail current value greater than 250pA; membrane rupture resistance Ra less than 15MΩ; tail current decay rate per minute less than 2.5%.

[0410] Data analysis: Only data that meets the above criteria can be analyzed according to the following steps:

[0411] Note: Data is output by PatchMaster software.

[0412] After perfusing the blank solvent or compound gradient solution, the average of the five consecutive current values ​​obtained was calculated and used as the "tail current magnitude" and "tail current size". 空白 ” and “tail current size 化合物 ”.

[0413] The current suppression percentage is calculated by the following formula.

[0414]

[0415] The dose-effect curve was fitted and the IC was calculated using Graphpad Prism 8.0 software. 50 value.

[0416] Table 7: Inhibitory activity of compounds on hERG

[0417] Compound No. <![CDATA[hERG IC 50 (μM) <!-- 45 -->]]> Cpd-A 19 Cpd-B 5.5 Cpd-C 1.4 Cpd-1 >30 Cpd-2 >30

[0418] The above data show that the example compounds of the present invention have a lower risk of potential cardiac toxicity caused by hERG inhibition and are significantly safer than the reference compounds.

[0419] 7. Pharmacokinetic studies in mice

[0420] 1. Experimental purpose: To test the pharmacokinetics of the compound in CD-1 mice.

[0421] 2. Experimental instruments and materials:

[0422] Animals: CD-1 mice (male)

[0423] instrument:

[0424]

[0425] Reagents:

[0426]

[0427]

[0428] Solvent:

[0429] Intravenous injection: 40% HP-β-CD aqueous solution; intragastric administration: 0.2% HPC + 0.5% Tween80 aqueous solution.

[0430] 3. Experimental methods:

[0431] 6 male mice. Fasting treatment was performed in the evening of the day before administration, and water was freely available. During the experimental process, mice were free to eat and drink, and the drugs were administered by intravenous injection or gavage. After administration, the animal status was observed and abnormal manifestations were recorded. Blood was collected from the eye sockets at 0.0833, 0.25, 1, 2, 4, 8 and 24 hours after intravenous injection; blood was collected from the eye sockets at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after gavage. Take 20 μL of plasma sample, add 250 μL of acetonitrile (containing dexamethasone as internal standard) to precipitate protein, centrifuge at 4°C, 4000 rpm for 20 minutes, take 180 μL of supernatant and mix it with 180 μL of aqueous solution containing 0.1% formic acid in a 96-well plate, and then take 10 μL of sample for LC-MS / MS detection.

[0432] 4. Data processing methods and results:

[0433] The internal standard method was used to establish a standard curve, with the theoretical standard curve concentration as the horizontal axis and the peak area ratio as the vertical axis (test compound peak area / internal standard peak area). The linear regression method (weighting factor 1 / X2) was used, R 2 >0.9900. Unknown samples were calculated by standard curve. Pharmacokinetic parameters were calculated by non-compartmental analysis model of WinNonlin 8.2 software and presented in the report, including Clint, C max and AUC, etc.

[0434] Table 8: Pharmacokinetic parameters of compounds in CD-1 mice

[0435]

[0436] The above data show that the example compounds of the present invention have lower clearance rate and higher oral exposure in mice and are superior to the reference compounds.

[0437] 8. Pharmacokinetic study in rats

[0438] 1. Experimental purpose: To test the pharmacokinetics of the compound in SD rats.

[0439] 2. Experimental instruments and materials:

[0440] Animals: SD rats (male)

[0441] instrument:

[0442]

[0443] Reagents:

[0444]

[0445] Solvent:

[0446] Intravenous injection: 40% HP-β-CD aqueous solution; intragastric administration: 0.2% HPC + 0.5% Tween80 aqueous solution.

[0447] 3. Experimental methods:

[0448] 6 male rats. Fasting treatment was performed in the evening of the day before administration, and water was freely available. Rats were free to eat and drink during the experiment. The rats were intravenously injected or gavaged. The animal status was observed after administration and abnormal manifestations were recorded. Blood was collected by jugular vein puncture at 0.0833, 0.25, 1, 2, 4, 8 and 24 hours after intravenous injection; blood was collected by jugular vein puncture at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after gavage. Take 50μL of plasma sample, add 500μL of acetonitrile (containing dexamethasone as internal standard) to precipitate protein, centrifuge at 4℃, 4000rpm for 20 minutes, take 300μL of supernatant and mix with 300μL of Watson's aqueous solution in a 96-well plate, and then take 5μL of sample for LC-MS / MS detection.

[0449] 4. Data processing methods and results:

[0450] The internal standard method was used to establish a standard curve, with the theoretical standard curve concentration as the horizontal axis and the peak area ratio as the vertical axis (test compound peak area / internal standard peak area). The linear regression method (weighting factor 1 / X2) was used, R 2 >0.9900. Unknown samples were calculated by standard curve. Pharmacokinetic parameters were calculated by non-compartmental analysis model of WinNonlin 8.2 software and presented in the report, including Clint, C max and AUC, etc.

[0451] Table 9: Pharmacokinetic parameters of the compounds in SD rats

[0452]

[0453] The above data show that the example compounds of the present invention have lower clearance rate and higher oral exposure in rats, and are significantly better than the reference compounds.

[0454] IX. In vivo efficacy study of NUGC-3 human gastric cancer xenograft mouse model

[0455] 1. Experimental purpose: To test the in vivo pharmacodynamic activity of the compound in the NUNU female mouse subcutaneous xenograft tumor model of human gastric cancer (NUGC-3).

[0456] 2. Experimental Materials:

[0457] Animals: Female NUNU mice, 6-8 weeks old;

[0458] Reagents:

[0459]

[0460] Cpd-A solvent: 0.2% HPC, 0.5% Tween80 in water (w / v / v);

[0461] Cpd-2 solvent: 5% DMSO / 10% Solutol HS15 / 85% pH 4.65±0.10acetate buffer.

[0462] 3. Experimental plan:

[0463] Cell culture: NUGC-3 cells were cultured in RPMI-1640 medium containing 10% FBS.

[0464] Modeling and grouping: NUGC-3 cells (5×10 6 ), resuspended in 0.2 mL PBS and matrix gel (1:1) and inoculated subcutaneously on the right side of NUNU mice to establish a subcutaneous transplant tumor model. The health status of the mice and tumor growth were observed daily after cell inoculation. On the 5th day after inoculation, 36 animals were selected and grouped. The tumor volume of the animals in the group was about 150.0 mm 3 The experiment was divided into 6 groups, with 6 mice in each group, and drug administration was started (see Table 10).

[0465] 4. Experimental design: Table 10 is the grouping and dosing information of the efficacy experiment. The mice in group G1 were orally administered with a vehicle control (5% DMSO / 10% Solutol HS15 / 85% pH 4.65±0.10acetate buffer) once a day for 19 days. The mice in group G2 were orally administered with Cpd-A at a dose of 50 mg / kg once a day for 19 days. The mice in group G3 were orally administered with Cpd-A at a dose of 100 mg / kg once a day for 19 days. The mice in group G4 were orally administered with Cpd-2 at a dose of 25 mg / kg once a day for 19 days. The mice in group G5 were orally administered with Cpd-2 at a dose of 50 mg / kg once a day for 19 days. The mice in group G6 were orally administered with Cpd-2 at a dose of 100 mg / kg once a day for 19 days.

[0466] Table 10. In vivo efficacy experiment design of NUGC-3 xenograft tumor mouse model

[0467]

[0468]

[0469] 5. Experimental observation and result judgment:

[0470] Experimental observation: The health status and mortality of animals were monitored daily. Routine examinations included observing the effects of tumor growth and drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake, weight changes, physical signs or other abnormal conditions. The number of deaths and side effects of animals in each group was recorded based on the number of animals in each group. Clinical symptoms observed during the experiment were recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ). During the study, the entire dosing process as well as tumor and body weight measurements were performed in a clean bench.

[0471] Data processing: Tumor volume inhibition rate TGI (%): TGI% = (1-△T / △C) × 100%; where △C is the tumor volume C of the control group t -C0, C0 is the average tumor volume of the control group at the time of grouping, C t is the average tumor volume of the control group after treatment; △T is the tumor volume T of the treatment group t -T0, T0 is the average tumor volume of the treatment group at the time of grouping, T t is the average tumor volume of the treatment group after treatment.

[0472] Statistical analysis: The tumor volume and animal body weight results are expressed as Mean ± SEM (mean standard error). The tumor volumes of different groups were statistically compared and analyzed. All statistical analyses were completed in GraphPad Prism 8.0. The one-way ANOVA method was used to compare whether there were significant differences in tumor volume or tumor weight among the groups, where p ≥ 0.05 was no significant difference, p < 0.05 was a significant difference, and p < 0.001 was a very significant difference.

[0473] 6. Experimental results:

[0474] The tumor growth curves of the control group and each experimental group in the pharmacodynamic evaluation experiment of the mouse NUGC-3 human gastric cancer xenograft tumor model are shown in Figure 2. Figure 1 The relative weight changes of mice in this model are shown in Figure 2The average tumor volume, tumor volume inhibition rate TGI (%) and comparison results of each group of animals at the end of the experiment are shown in Table 11.

[0475] On the 19th day after grouping, the average tumor volume of the G1 (Vehicle) group was 1482.2 mm 3 The average tumor volumes of the G2 (Cpd-A, 50 mg / kg, QD), G3 (Cpd-A, 100 mg / kg, QD), G4 (Cpd-2, 25 mg / kg, QD), G5 (Cpd-2, 50 mg / kg, QD), and G6 (Cpd-2, 100 mg / kg, QD) groups were 1208.1 mm 3 , 399.1mm 3 , 615.1mm 3 , 202.2mm 3 and 102.5mm 3 , the corresponding tumor volume inhibition rates TGI were 20.6%, 81.3%, 65.1%, 96.1% and 103.6%, respectively. Statistical analysis showed that the average tumor volume of G3, G4, G5 and G6 was significantly smaller than that of the Vehicle group (p values ​​were all <0.05); among them, the average tumor volume of G5 and G6 groups was significantly different from that of the Vehicle group (p values ​​were all <0.001). The above results show that compound Cpd-A exhibited good activity in inhibiting the proliferation of NUGC-3 transplanted tumors in mice at a high dose of 100 mg / kg, and Cpd-2 exhibited good activity in inhibiting the proliferation of NUGC-3 transplanted tumors in mice at each test dose, and was dose-related. At the same time, the tumor inhibitory activity of Cpd-2 at a lower dosage (such as 50 mg / kg, QD) (TGI = 96.1%) is better than the tumor inhibitory activity of Cpd-A at a dose of twice that of the compound Cpd-A (100 mg / kg, QD) (TGI = 81.3%), indicating that Cpd-2 has better anti-tumor activity than Cpd-A.

[0476] Table 11. In vivo efficacy test results of NUGC-3 xenograft tumor mouse model

[0477]

[0478]

[0479] Remark:

[0480] a. TGI (%) = [1-(T 19 -T0) / (V 19 -V0)]×100

[0481] b. p value of the comparison analysis between the average tumor volume of each drug-treated group and the average tumor volume of G1 group Vehicle

[0482] 10. In vivo efficacy study of NUGC-3 human gastric cancer xenograft rat model

[0483] 1. Experimental purpose: To test the in vivo pharmacodynamic activity of the compound in a subcutaneous xenograft tumor model of human gastric cancer (NUGC-3) in B-Rag2 / IL2rg KO SD female rats.

[0484] 2. Experimental Materials:

[0485] Animals: 7-9 week old female B-Rag2 / IL2rg KO SD rats;

[0486] Reagents:

[0487]

[0488] Solvent: 5% DMSO / 10% Solutol HS15 / 85% pH 4.65±0.10acetate buffer;

[0489] 3. Experimental plan:

[0490] Cell culture: NUGC-3 cells were cultured in RPMI-1640 medium containing 10% FBS.

[0491] Modeling and grouping: NUGC-3 cells (1×10 7 ), resuspended in 0.2 mL PBS and matrix gel (1:1) and used for subcutaneous inoculation on the right side of B-Rag2 / IL2rg KO SD rats to establish a subcutaneous transplant tumor model. The health status of the rats and tumor growth were observed daily after cell inoculation. On the 10th day after inoculation, 24 animals were selected and grouped. The tumor volume of the enrolled animals was about 199.0 mm 3 The experiment was divided into 4 groups, with 6 rats in each group, and drug administration was started (see Table 12).

[0492] 4. Experimental design: Table 12 is the grouping and dosing information of the efficacy experiment. Rats in group G1 were orally administered with a vehicle control (5% DMSO / 10% Solutol HS15 / 85% pH 4.65±0.10 acetate buffer) once a day for 28 days. Rats in group G2 were orally administered with Cpd-2 at a dose of 6.25 mg / kg once a day for 28 days. Rats in group G3 were orally administered with Cpd-2 at a dose of 12.5 mg / kg once a day for 28 days. Rats in group G4 were orally administered with Cpd-2 at a dose of 25 mg / kg once a day for 28 days.

[0493] Table 12. In vivo efficacy experiment design of NUGC-3 xenograft tumor rat model

[0494]

[0495] 5. Experimental observation and result judgment:

[0496] Experimental observation: The health status and mortality of animals were monitored daily. Routine examinations included observing the effects of tumor growth and drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake, weight changes, physical signs or other abnormal conditions. The number of deaths and side effects of animals in each group was recorded based on the number of animals in each group. Clinical symptoms observed during the experiment were recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ). During the study, the entire dosing process as well as tumor and body weight measurements were performed in a clean bench.

[0497] Data processing: tumor volume inhibition rate TGI TV (%): TGI TV % = (1-△T / △C) × 100%; where △C is the tumor volume of the control group C t -C0, C0 is the average tumor volume of the control group at the time of grouping, C t is the average tumor volume of the control group after treatment; △T is the tumor volume T of the treatment group t -T0, T0 is the average tumor volume of the treatment group at the time of grouping, T t is the average tumor volume of the treatment group after treatment.

[0498] Statistical analysis: Tumor volume and animal weight results are expressed as Mean ± SEM. Statistical comparison and analysis of tumor volume among different groups were performed. All statistical analyses were completed in https: / / d2k.bio / Efficacy / Invivo. The wilcox.test nominal method was used to compare whether there were significant differences in tumor volume or tumor weight among the groups, where p ≥ 0.05 indicated no significant difference, p < 0.05 indicated significant difference, and p < 0.01 indicated extremely significant difference.

[0499] 6. Experimental results:

[0500] The tumor growth curves of the control group and each experimental group in the pharmacodynamic evaluation experiment of the rat NUGC-3 human gastric cancer xenograft tumor model are shown in Figure 2. Figure 3 The relative weight changes of rats in this model are shown in Figure 4The average tumor volume, tumor volume inhibition rate TGI (%) and comparison results of each group of animals at the end of the experiment are shown in Table 13.

[0501] On the 28th day after grouping, the average tumor volume of the G1 (Vehicle) group was 2946.2 mm 3 The average tumor volumes of the G2 (Cpd-2, 6.25 mg / kg, QD), G3 (Cpd-2, 12.5 mg / kg, QD) and G4 (Cpd-2, 25 mg / kg, QD) groups were 395.0 mm 3 、119.2mm 3 and 70.1mm 3 , the corresponding tumor volume inhibition rates TGI were 92.9%, 102.9% and 104.7% respectively. Statistical analysis showed that the average tumor volume of G2, G3 and G4 groups was significantly different from that of Vehicle group (all p values ​​< 0.01). The above results show that Cpd-2 showed good activity in inhibiting the proliferation of NUGC-3 transplanted tumors in rats at each tested dose, and it was dose-dependent.

[0502] Table 13. In vivo efficacy test results of NUGC-3 xenograft tumor rat model

[0503]

[0504]

[0505] Remark:

[0506] The p value of the comparative analysis of the average tumor volume of each drug-treated group and the average tumor volume of G1 group Vehicle.

Claims

1. A nitrogen-containing compound as shown in Formula I or a pharmaceutically acceptable salt thereof; in, X 1 , X 2 and X 3 are each independently selected from CH and N; R 1 , R 2 , R 3 , R 4 , R 5 are each independently selected from hydrogen and deuterium; R 6 -(CH2) n R 7 ; n is 0, 1, 2 or 3; R 7 Selected from C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl and C3-C6 cycloalkyl.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The nitrogen-containing compound or a pharmaceutically acceptable salt thereof as shown in Formula I is a nitrogen-containing compound or a pharmaceutically acceptable salt thereof as shown in Formula Ia; 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 are each independently selected from hydrogen or deuterium; R 6 -(CH2) n R 7 ; n is 0, 1, 2 or 3; R 7 Selected from C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl or C3-C6 cycloalkyl.

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: X 1 N; X 2 CH or N; X 3 for CH; Or, X 1 N; X 2 and X 3 For CH.

4. The compound of formula I, the compound shown, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: R 6 -(CH2) n R 7 ; n is 0 or 1; R 7 Selected from C1-C3 alkyl, deuterated C1-C3 alkyl, halogenated C1-C3 alkyl or C3-C6 cycloalkyl.

5. The compound of formula I, the compound shown, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: R 6 -(CH2) n R 7 ; n is 0 or 1; R 7 Selected from -CH3, -CD3, -CF3 or cyclopropyl.

6. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: R 1 , R 2 and R 3 is deuterium, R 4 and R 5 is hydrogen; Or, R 1 , R 2 and R 3 is hydrogen, R 4 and R 5 For deuterium.

7. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Formula I is Formula Ib or Ic; In Formulas Ib and Ic, for R 1 , R 2 , R 3 , R 4 , R 5 are each independently selected from hydrogen or deuterium; R 6 -(CH2) n R 7 ; n is 0 or 1; R 7 Selected from C1-C6 alkyl, halogenated C1-C6 alkyl and C3-C6 cycloalkyl; Preferably, in Formula Ib and Ic, Selected from -OCH3 and -OCD3; R 4 and R 5 Selected from hydrogen; R 6 -(CH2) n R 7 ; n is 0 or 1; R 7 Selected from -CH3, -CD3, -CF3 or cyclopropyl.

8. A compound as shown in formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound of formula (I) is selected from any one of the following compounds: Its enantiomers or mixtures of both in any ratio.

9. A pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 8 and a pharmaceutical excipient.

10. Use of a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9, in the preparation of a p53 mutant pocket binder or a drug for treating and / or preventing a disease associated with a p53 mutant.

11. The use according to claim 10, 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; And the disease associated with p53 mutant described in (3) is cancer, such as breast cancer, gastric cancer, lung cancer or ovarian cancer.

12. Use of the compound of formula I according to any one of claims 1 to 8, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating and / or preventing cancer; The cancer is breast cancer, gastric cancer, lung cancer or ovarian cancer.

13. A compound represented by formula a-11 or a-32; in, X is halogen; R 8 It is an amino protective agent; X 1 , X 2 and X 3 , R 1 , R 2 , R 3 , R 4 , R 5 and R 6 The definitions are as described in any one of claims 1 to 8; The compound represented by the formula a-11 or a-32 is preferably any of the following compounds;

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