N-sulfonamide nitrogen-containing fused heterocyclic compound, and application thereof

Abstract

Disclosed in the present invention are an N-sulfonamide nitrogen-containing fused heterocyclic compound and an application thereof. The N-sulfonamide nitrogen-containing fused heterocyclic compound is a compound as shown in formula I, an isotope marker thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a prodrug thereof. The compound can improve the DNA-binding ability of p53 Y220C, and has good inhibitory activity on cancer cell proliferation.

Description

A class of N-sulfonylamide nitrogen-containing fused heterocyclic compounds and their applications

[0001] This application claims the benefit of priority to Chinese Patent Application No. 2023113669427, filed on October 20, 2023, the benefit of priority to Chinese Patent Application No. 2023117838674, filed on December 22, 2023, the benefit of priority to Chinese Patent Application No. 2024102622351, filed on March 7, 2024, and the benefit of priority to Chinese Patent Application No. 2024112753956, filed on September 11, 2024. The entire text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of medicine, and further belongs to the field of druggable p53 Y220C pocket binder medicines, and specifically relates to a class of N-sulfonylamide nitrogen-containing fused heterocyclic compounds and applications thereof. Background Art

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

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

[0005] Summary of the Invention

[0006] The present invention addresses the poor drugability of existing p53 Y220C pocket binders. To address this issue, the present invention provides a class of N-sulfonylamide nitrogen-containing fused heterocyclic compounds and their applications. These compounds can enhance the ability of p53 Y220C to bind DNA and exhibit excellent inhibitory activity against the proliferation of cancer cells, particularly gastric cancer cells.

[0007] The present invention provides a nitrogen-containing compound as shown in Formula I, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt solvate or its prodrug;

[0008] in,

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

[0010] R 1 、R 2 、R 3 、R 4 、R 5 are each independently selected from hydrogen and deuterium;

[0011] R 6 -(CH2) n R 7 ;

[0012] n is 0, 1, 2, or 3;

[0013] R 7 Selected from C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl and C3-C6 cycloalkyl.

[0014] As a preferred technical solution, the nitrogen-containing compound as shown in Formula I, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt, or its prodrug is a nitrogen-containing compound as shown in Formula Ia, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt, or its prodrug;

[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 、R5 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 is 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 are each 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 3is 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] In Formulas Ib and Ic, for

[0030] R 1 、R 2 、R 3 、R 4 and R 5 are each independently selected from hydrogen and deuterium;

[0031] R 6 -(CH2) n R 7 ;

[0032] n is 0 or 1;

[0033] R 7 Selected from C1-C6 alkyl, halogenated C1-C6 alkyl and C3-C6 cycloalkyl.

[0034] Preferably, Selected from -OCH3 and -OCD3;

[0035] R 4 and R 5 selected from hydrogen;

[0036] R 6 -(CH2) n R 7 ; n is 0 or 1; R 7 Selected from -CH3, -CD3, -CF3 or cyclopropyl.

[0037] As a preferred technical solution, the compound of formula I is selected from the following compounds:

[0038] Its enantiomers or mixtures of the two in any ratio (e.g. racemates).

[0039] The present invention also provides methods for preparing the compound of Formula I, its isotope-labeled products, enantiomers, diastereomers, solvates, or pharmaceutically acceptable salts thereof, including the following methods:

[0040] Method 1:

[0041] 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;

[0042] Step 2: Compound a-2 undergoes Vilsmeier-Haack reaction to obtain compound a-3;

[0043] Step 3: Compound a-3 reacts with a fluorination agent to obtain compound a-4; the fluorination agent can be methyl fluorosulfonyldifluoroacetate, (triphenylphosphonium) difluoroacetic acid inner salt, or tetrabutylammonium fluoride;

[0044] 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;

[0045] Step 5: Compound a-5 is oxidized to obtain compound a-6 under the action of an oxidant, wherein the oxidant may be Dess-Martin reagent, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide, or sulfur trioxide / pyridine;

[0046] Step 6: Compound a-6 reacts with Gilbert reagent to generate compound a-7;

[0047] Step 7: Compound a-7 reacts with formaldehyde under the catalysis of a metal reagent to obtain compound a-8; the metal reagent can be a Cu-containing reagent, such as cuprous tetraacetonitrile hexafluorophosphate;

[0048] Step 8: Compound a-8 is oxidized to aldehyde a-9 under the action of an oxidant, which may be Dess-Martin reagent, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide, or sulfur trioxide / pyridine;

[0049] Step 9: Compound a-9 undergoes reductive amination reaction with compound a-10 to generate compound a-11; R 1 、R 2 、R 3 、R 6 、X 1 、X 2 、X 3 The definition of is the same as above;

[0050] Step 10: Compound a-11 undergoes a metal reagent-catalyzed coupling reaction with amine a-12 to obtain compound (Ia); the metal reagent can be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, or cuprous iodide.

[0051] Method 2:

[0052] 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;

[0053] Step 2: Compound a-14 is subjected to acidic conditions to remove the Boc protecting agent to obtain compound a-15; the acidic conditions can be carried out in the presence of an inorganic acid (such as HCl, HBr, H2SO4), an organic acid (such as trifluoroacetic acid), a Lewis acid (such as zinc bromide) or other reagents (such as trimethylsilyl iodide);

[0054] Step 3: Compound a-15 undergoes reductive amination reaction with formaldehyde to generate compound (Ia).

[0055] Method 3:

[0056] 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;

[0057] Step 2: Compound a-17 undergoes a metal reagent-catalyzed coupling reaction with amine a-12 to obtain compound a-18; the metal reagent can be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, or cuprous iodide;

[0058] Step 3: Compound a-18 undergoes hydrolysis under acidic or alkaline conditions to produce a-19; the acidic conditions can be inorganic acids (such as HCl, HBr, H2SO4) or organic acids (such as trifluoroacetic acid); the alkaline conditions can be inorganic bases (such as LiOH, NaOH, KOH);

[0059] Step 4: Compound a-19 and sulfonamide H2NS(O)2R 6A condensation reaction occurs to generate compound (Ia); wherein R 6 The definition of is the same as above.

[0060] Method 4:

[0061] Step 1: Compound a-8 is subjected to a hydroxyl protection reaction with a suitable protecting agent to produce compound a-20; wherein X is selected from Cl, Br, and I; M is a protecting group, such as trimethylsilyl, tert-butyldimethylsilyl, methoxymethyl, tert-butoxycarbonyl, or 2-tetrahydropyranyl; the protecting agent may be trimethylchlorosilane, tert-butyldimethylchlorosilane, chloromethyl methyl ether, di-tert-butyl dicarbonate, or 3,4-dihydro-2H-pyran;

[0062] Step 2: Compound a-20 undergoes a metal reagent-catalyzed coupling reaction with amine a-12 to obtain compound a-21; the metal reagent can be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, or cuprous iodide;

[0063] Step 3: Compound a-21 is deprotected under acidic or alkaline conditions to produce a-22; the acidic conditions can be inorganic acids (such as HF, HCl, HBr, H2SO4) or organic acids (such as trifluoroacetic acid); the alkaline conditions can be inorganic bases (such as LiOH, NaOH, KOH) or organic bases (tetra-n-butylammonium fluoride);

[0064] Step 4: Compound a-22 is oxidized to obtain compound a-23 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;

[0065] Step 5: Compound a-23 undergoes reductive amination reaction with sulfonamide a-10 to generate compound (Ia); wherein R 1 、R 2 、R 3 、R 6 、X 1 、X 2 、X 3 The definition of is the same as above;

[0066] Method 5:

[0067] 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;

[0068] Step 2: Compound a-24 is esterified to obtain compound a-25;

[0069] Step 3: Compound a-25 is reduced to obtain compound a-26; the reducing agent may be sodium borohydride (lithium) or lithium aluminum hydride reagent, or their deuterated reagents; R 4 、R 5 The definition of is the same as above;

[0070] Step 4: Compound a-26 is halogenated to generate compound a-27; wherein X is selected from Cl, Br, and I;

[0071] Step 5: Compound a-27 reacts with a-28 to obtain compound a-29 through nucleophilic substitution reaction; 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;

[0072] Step 6: Compound a-29 removes the protecting group G to obtain a-30;

[0073] Step 7: Compound a-30 undergoes a metal reagent-catalyzed coupling reaction with amine a-12 to obtain compound a-31; the metal reagent can be a reagent containing Pd or Cu, such as Brettphos Pd G3, Brettphos Pd G4, or cuprous iodide;

[0074] Step 8: Compound a-31 undergoes hydrolysis reaction under acidic or alkaline conditions to produce a-32; the acidic conditions can be inorganic acids (such as HCl, HBr, H2SO4) or organic acids (such as trifluoroacetic acid); the alkaline conditions can be inorganic bases (such as LiOH, NaOH, KOH);

[0075] Step 9: Compound a-32 and sulfonamide H2NS(O)2R 6 A condensation reaction occurs to generate compound (I); wherein R 6 The definition of is the same as above.

[0076] The present invention also provides a pharmaceutical composition comprising a compound as shown in Formula I, its isotope-labeled substance, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt (eg, an effective therapeutic amount) and pharmaceutical excipients.

[0077] The present invention also provides a compound as shown in Formula I, its isotope-labeled substance, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt, or the use of the pharmaceutical composition in the preparation of a p53 mutant pocket binder or a drug for treating and / or preventing diseases associated with p53 mutants.

[0078] In a preferred technical solution, the application satisfies one or more of the following conditions:

[0079] (1) The p53 mutant has a mutation at amino acid 220, for example, p53 Y220C;

[0080] (2) the pocket binder increases the ability of the p53 mutant to bind to DNA;

[0081] (3) The disease associated with p53 mutants is cancer, such as breast cancer, gastric cancer, lung cancer or ovarian cancer.

[0082] The present invention also provides a compound as shown in Formula I, its isotope-labeled substance, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt, or the use of the pharmaceutical composition in the preparation of a drug for treating and / or preventing cancer.

[0083] The cancer is, for example, breast cancer, gastric cancer, lung cancer or ovarian cancer.

[0084] The present invention also provides a method for treating and / or preventing cancer, characterized in that a therapeutically effective amount of a compound as shown in Formula I, its isotope label, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof is administered to a patient.

[0085] The cancers mentioned can all be the same as those described above.

[0086] 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);

[0087] Wherein, X is a halogen (such as Br); R 8 is an amino protecting agent (e.g. -Boc);

[0088] 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.

[0089] The compound represented by formula a-11 or a-32 is preferably any of the following compounds:

[0090] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0091] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0092] The term "solvate" refers to a substance formed by the combination of a compound and a solvent (including but not limited to water, methanol, ethanol, etc.). Solvates are divided into stoichiometric solvates and non-stoichiometric solvates.

[0093] The term "pharmaceutically acceptable salt solvate" refers to a compound formed by combining with a pharmaceutically acceptable acid or base and a solvent (including but not limited to water, methanol, ethanol, etc.). The amount of solvent may be stoichiometric or non-stoichiometric.

[0094] A "-" at the end of a group means that the group is attached to the rest of the molecule through that site. For example, CH3-C(=O)- means an acetyl group.

[0095] When the group valence bond has a wavy line When, for example, In the example, the wavy line indicates the point of attachment of the group to the rest of the molecule.

[0096] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0097] The term "alkyl" refers to a linear or branched, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, and the like.

[0098] The term "alkoxy" refers to a group R X -O-, R XThe same definition as the term "alkyl". Alkoxy includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like.

[0099] 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.

[0100] The term "pharmaceutical excipients" refers to all substances contained in pharmaceutical preparations other than the active pharmaceutical ingredient (API). These substances are generally classified into two categories: excipients and additives. For details, see the Pharmacopoeia of the People's Republic of China (2020 Edition) and the Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0101] The term "treat" refers to eliminating the cause or alleviating the symptoms of a disease.

[0102] The term "prevent" refers to reducing the risk of developing a disease.

[0103] The term "patient" refers to any animal, typically a mammal, such as a human, that needs to be treated or prevented. Mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like.

[0104] The term "PG" represents a protecting group, such as a Boc protecting group and the like.

[0105] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0106] The reagents and raw materials used in the present invention are commercially available.

[0107] The positive progress of the present invention is that the compound of the present invention can enhance the ability of p53 Y220C to bind to DNA and has good inhibitory activity on the proliferation of cancer cells represented by gastric cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG1 is a graph showing the tumor growth curves of the control group and each experimental group in the pharmacodynamic evaluation experiment of the NUGC-3 human gastric cancer xenograft tumor model in mice of Test Example 9;

[0109] FIG2 is a curve showing the relative body weight changes of mice in the control group and each experimental group in the pharmacodynamic evaluation experiment of the NUGC-3 human gastric cancer xenograft tumor model in Test Example 9;

[0110] FIG3 is a graph showing 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 in Test Example 10;

[0111] FIG4 is a curve showing the relative body weight change 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 of Test Example 10. DETAILED DESCRIPTION

[0112] The present invention is further illustrated below by way of examples, but the present invention is not limited thereto. Unless otherwise indicated, the actual operations disclosed herein will employ conventional techniques of organic synthesis, cell biology, cell culture, and molecular biology within the skill of the art.

[0113] In each embodiment, 1 HNMR was recorded on a BRUKER AVANCE NEO 400 MHz, JNM-ECZ400s NMR instrument, and chemical shifts are expressed in δ (ppm). Liquid chromatography-mass spectrometry (LCMS) was recorded on Shimadzu LC-20AD, Agilent 1260, and Agilent 1200 mass spectrometers. Preparative HPLC separations were performed using a WATERS Autop, Shimadzu LC20AR liquid chromatograph.

[0114] Preparation of intermediates Int-1 and Int-1-7

[0115] Step 1: Ethyl 8-bromoindolizine-2-carboxylate

[0116] A mixture of 3-bromo-2-methyl-pyridine Int-1-1 (500 mg, 2.91 mmol), ethyl 3-bromopyruvate (850 mg, 4.36 mmol) and sodium bicarbonate (561 mg, 6.69 mmol) in butanone (5 mL) was stirred at 85°C for 16 hours, then concentrated to dryness and purified by SGC (0-10% EtOAc in PE) to give ethyl 8-nitroindoleazine-2-carboxylate Int-1-2 as a gray solid (190 mg, 24.4% yield). LCMS calculated value C 11 H 11 BrNO2[M+H] + :m / z=268.0 / 270.0; Detection value: 267.9 / 269.9; 1H NMR(400MHz, CDCl3)δ7.88(d,J=1.6Hz,1H),7.87-7.83(m,1H),7.03-6.99(m,1H),6.9 5(d,J=6.8Hz,1H),6.43(t,J=7.2Hz,1H),4.37(q,J=7.2Hz,2H),1.40(t,J=6.8Hz,3H).

[0117] Step 2: Ethyl 8-bromo-3-formylindolizine-2-carboxylate

[0118] At 0 ° C, a solution of 8-bromoindolizine-2-carboxylic acid ethyl ester Int-1-2 (5.34 g, 19.9 mmol) in DCM (130 mL) was added dropwise to a solution of phosphorus oxychloride (5.19 g, 33.8 mmol) in DMF (130 mL). The mixture was stirred at 20 ° C for 1 hour, then slowly quenched with saturated aqueous sodium bicarbonate (300 mL) and extracted with DCM (200 mL). The separated organic layers were combined, dried over sodium sulfate, filtered and concentrated to dryness. The residue was purified by SGC (0-10% EtOAc in PE) to give 8-bromo-3-formylindolizine-2-carboxylic acid ethyl ester Int-1-3 (10.6 g, crude) as a yellow gum. LCMS calculated value C 12 H 11 BrNO3[M+H] + :m / z=296.0 / 298.0; Detection: 295.9 / 297.9.

[0119] Step 3: Ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate

[0120] A mixture of ethyl 8-bromo-3-formylindolizine-2-carboxylate Int-1-3 (8.50 g, 15.8 mmol) and ethyl 2,2-difluoro-2-(triphenylphosphonium)acetate (11.2 g, 31.5 mmol) in DMF (120 mL) was stirred at 60°C for 2 hours. TBAF (47.3 mL, 47.3 mmol, 1 M solution in tetrahydrofuran) was added to the mixture. The reaction mixture was stirred at 60°C for 2 hours, then diluted with water (100 mL) and extracted with MTBE (100 mL x 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% EtOAc in PE) to afford ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate Int-1-4 (2.40 g, 43.4% yield) as a white solid. LCMS calculated value C 13 H 12 BrF3NO2[M+H]+ :m / z=350.0 / 352.0; Detection value: 349.9 / 351.9; 1 H NMR (400MHz, CDCl3) δ7.89(d,J=7.2Hz,1H),7.12(s,1H),7.05(d,J=6.8Hz,1H),6.56( t,J=7.2Hz,1H),4.40(q,J=7.2Hz,2H),4.25(q,J=10.0Hz,2H),1.42(t,J=7.2Hz,3H).

[0121] Step 4: (8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)methanol

[0122] To a mixture of 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylic acid ethyl ester Int-1-4 (4.10 g, 11.7 mmol) in THF (120 mL) was added DIBAL-H (35.1 mL, 35.1 mmol, 1 M solution in toluene). The reaction was stirred at -10°C for 3 hours to give a yellow solution. The reaction solution was poured into a saturated aqueous ammonium chloride solution (200 mL), followed by the addition of water (200 mL) and the mixture was extracted with EtOAc (150 mL x 3). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% EtOAc in PE) to give [8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl]methanol Int-1-5 (3.55 g, 98.4% yield) as a white solid. LCMS calculated value: C 11 H 10 BrF3NO[M+H] + :m / z=308.0 / 310.0; Detection value: 307.9 / 309.9; 1 H NMR (400MHz, CDCl3) δ7.85 (d, J = 7.2Hz, 1H), 7.00 (d, J = 6.8Hz, 1H), 6.70 (br s, 1H), 6.50 (t, J = 7.2Hz, 1H), 4.84 (s, 2H), 3.83 (q, J = 10.4Hz, 2H).

[0123] Step 5: 8-Bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde

[0124] To a solution of [8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]methanol Int-1-5 (3.55 g, 11.5 mmol) in DCM (400 mL) was added DMP (7.33 g, 17.2 mmol) at 0°C. The reaction system was warmed to room temperature and stirred for 2 hours. The reaction solution was poured into a saturated aqueous sodium bicarbonate solution (20 mL), followed by addition of water (200 mL) and extraction with DCM (100 mL×3). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-7% EtOAc in PE) to give 8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-carbaldehyde Int-1-6 (2.70 g, 76.5% yield) as a white solid. LCMS calculated value: C 11 H8BrF3NO[M+H] + :m / z=306.0 / 308.0; Detected value: 305.9 / 307.9.

[0125] Step 6: 8-Bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine

[0126] To a mixture of dimethyl (1-diazo-2-oxopropyl)phosphonate (470 mg, 2.45 mmol) and potassium carbonate (451 mg, 3.27 mmol) in methanol (15 mL) was added 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 (500 mg, 1.63 mmol) at 0°C. The reaction system was warmed to room temperature and stirred for 16 hours. The reaction solution was diluted with water (50 mL) and extracted with EtOAc (50 mL×3). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-3% EtOAc in PE) to give 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (380 mg, 77.0% yield) as a white solid. LCMS calculated value: C 12 H8BrF3N[M+H] + :m / z=302.0 / 304.0; Detection value: 301.9 / 303.9; 1 H NMR (400MHz, CDCl3) δ7.81 (d, J = 7.2Hz, 1H), 7.03 (d, J = 7.2Hz, 1H), 6.79 (s, 1H), 6.52 (t, J = 7.2Hz, 1H), 3.83 (q, J = 10.0Hz, 2H), 3.25 (s, 1H).

[0127] Step 7: 3-[8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol

[0128] Under nitrogen, cuprous tetraacetonitrile hexafluorophosphate (123 mg, 0.33 mmol) and tributylphosphine (268 mg, 1.32 mmol) were dissolved in toluene (15 mL). The reaction mixture was stirred at 70°C for 30 minutes. 8-Bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (1.00 g, 3.31 mmol) and aqueous formaldehyde (0.18 mL, 6.62 mmol, 36%-38% content) were then added. The mixture was stirred at 70°C overnight. After completion of the reaction by TLC, the mixture was concentrated in vacuo, and the residue was purified by SGC (0-15% EtOAc in PE) to afford 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol Int-1-7 (843 mg, 76.7% yield) as a white solid. LCMS calculated value C 13 H 10 BrF3NO[M+H] + :m / z=332.0 / 334.0; Detection value: 332.3 / 334.3; 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=7.1Hz,1H),7.19(d,J=7.1Hz,1H),6.68(t,J=7.1H z,1H),6.61(s,1H),5.34(t,J=6.0Hz,1H),4.34(d,J=6.0Hz,2H),4.20-4.11(m,2H).

[0129] 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

[0130] Step 1: 4-Amino-3-methoxy-N-methylbenzamide

[0131] 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 was added HATU (18.8 g, 49.5 mmol). The reaction was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by SGC (0-4% MeOH in DCM) to give 4-amino-3-methoxy-N-methylbenzamide Cpd-C-2 (6.08 g, 75.0% yield) as a white solid. LCMS calculated for C9H 13 N2O2[M+H] + :m / z=181.1; Detected value: 181.2.

[0132] Step 2: 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

[0133] To a mixture of 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (600 mg, 1.99 mmol) and 4-amino-3-methoxy-N-methylbenzamide Cpd-C-2 (716 mg, 3.97 mmol) in 1,4-dioxane (12 mL) was added aqueous formaldehyde solution (497 mg, 5.96 mmol, 36%-38% content) and cuprous bromide (567 mg, 3.97 mmol). The reaction was stirred at 100°C under microwave irradiation for 1 hour. The residue was purified by SGC (0-30% EtOAc in DCM) to give 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (480 mg, 48.9% yield) as a yellow solid. LCMS calculated value: C 22 H 20 BrF3N3O2[M+H] + :m / z=494.1 / 496.1; Detected value: 494.2 / 496.2.

[0134] Step 3: tert-Butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate

[0135] Under argon, a mixture of 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (1.35 g, 2.73 mmol), (3S,4R)-tert-butyl 4-amino-3-fluoropiperidine-1-carboxylate (1.19 g, 5.46 mmol), BrettPhos Pd G4 (0.50 g, 0.55 mmol), Ruphos (0.25 g, 0.55 mmol) and cesium carbonate (1.78 g, 5.46 mmol) in 1,4-dioxane (50 mL) was stirred at 100 ° C. for 16 hours. The reaction mixture was diluted with EtOAc (200 mL) and washed with water (100 mL) and 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)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylaminoformyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylic acid tert-butyl ester 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.

[0136] 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

[0137] To a solution of tert-butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate Cpd-C-4 (1.00 g, 1.58 mmol) in DCM (16 mL) at 0°C was added hydrochloric acid (7.92 mL, 31.7 mmol, 4N 1,4-dioxane solution). The mixture was transferred to room temperature and stirred for 1 hour. The mixture was concentrated to dryness, diluted with water (30 mL), and the pH was adjusted to 7-8 with saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM (100 mL × 2), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid 4-((3-(8-(((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-5 (450 mg, crude). The crude product was used directly in the next reaction. LCMS calculated value C 27 H 30 F4N5O2[M+H] + :m / z=532.2; Detected value: 532.4.

[0138] 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

[0139] To a solution of 4-((3-(8-(((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-5 (450 mg, 0.85 mmol), acetic acid (0.02 mL, 0.42 mmol), and formaldehyde (212 mg, 2.54 mmol, 36%-38%) in methanol (20 mL) was added sodium cyanoborohydride (160 mg, 2.54 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. Saturated sodium bicarbonate solution was added dropwise to the reaction mixture to adjust the pH to 7-8. The aqueous phase was extracted with EtOAc (100 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative method to give 4-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C (119 mg, 25.2% yield) as a white solid. LCMS calculated value: C 28 H 32 F4N5O2[M+H] + :m / z=546.2; Detection value: 546.2; 1 H NMR (400MHz, DMSO-d6) δ8.08(d,J=4.6Hz,1H),7.57(d,J=6.9Hz,1H),7.41(dd,J=8.2,1.7Hz,1H),7.34(d,J=1.7Hz,1H),6.91(s,1H), 6.74(d,J=8.3Hz,1H),6.51(t,J=7.2Hz,1H),5.90(t,J=6.2Hz,1H),5.85(d,J=7.5Hz,1H),5.54(d,J=8.4Hz,1H),4.81(d,J=49.8Hz,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).

[0140] Example 1: 5-[3-[8-[[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino]-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-ynylamino]-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide (Cpd-1, i.e., Compound 1)

[0141] Step 1: 6-Methoxy-5-nitropyridine-2-carboxylic acid

[0142] 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 analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was poured into ice water (50 mL) and adjusted to pH 5 with 1N hydrochloric acid. The mixture was then extracted with EtOAc (30 mL x 3). The combined organic layers were 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 afford 6-methoxy-5-nitropyridine-2-carboxylic acid Cpd-1-2 (2.80 g, 71.7% yield, yellow solid). LCMS calculated for C7H7N2O5 [M+H] + :m / z=199.0; Detection value: 199.1.

[0143] Step 2: 6-Methoxy-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide

[0144] 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 analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were 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 afford 6-methoxy-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-1-3 (2.20 g, 49.5% yield, as a yellow solid). LCMS calculated for C8H 10 N3O6S[M+H] + :m / z=276.0; Detection value: 276.1.

[0145] Step 3: 5-amino-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide

[0146] Under a hydrogen atmosphere, a solution of palladium on 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 analysis showed complete consumption of the starting material and the detection of the desired compound. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% MeOH in DCM) to give 5-amino-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-1-4 (500 mg, 70.1% yield, yellow solid). LCMS calculated value: C8H 12 N3O4S[M+H] + :m / z=246.1; Detection value: 246.0.

[0147] Step 4: 8-Bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine

[0148] 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) was added portionwise at 0°C. The reaction mixture was stirred at 0°C for 0.5 h, after which the solid was removed by filtration. The filtrate was concentrated under reduced pressure to a residue, which was purified by SGC (0-30% EtOAc in PE) to afford 8-bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-1-5 (134 mg, 99.7% yield, yellow solid). LCMS calculated value: C 19 H 24 BrF3NOSi[M+H] + :m / z=446.1 / 448.1; Detected value: 446.0 / 448.0.

[0149] 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

[0150] 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.

[0151] 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

[0152] TBAF (0.20 mL, 0.20 mmol, 1 M solution in tetrahydrofuran) 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)indolizin-8-amine Cpd-1-6 (82.1 mg, 165 μmol) in THF (5 mL) at 0°C. The reaction was stirred at 0°C for 1 hour. LCMS analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction was quenched with saturated aqueous sodium bicarbonate (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% 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; Detection value: 384.1.

[0153] Step 7: 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propynal

[0154] 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) at 0°C. The reaction was stirred at 0°C for 0.5 hours. LCMS analysis indicated complete consumption of the starting material and the presence of the desired compound. The reaction was quenched with saturated aqueous sodium bicarbonate (100 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0-5% 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.

[0155] Step 8: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propan-2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide

[0156] A mixture of 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propioaldehyde 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. The mixed solution was then cooled to 0°C, and MeOH (0.5 mL) and sodium cyanoborohydride (12.4 mg, 197 μmol) were slowly added. The reaction solution was allowed to warm to room temperature and stirring continued for 10 minutes. LCMS analysis indicated complete consumption of the starting material, 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 aqueous sodium bicarbonate 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 preparative method 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).

[0157] 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)

[0158] Step 1: 6-(Methoxy-d3)-5-nitropyridine-2-carboxylic acid

[0159] Under nitrogen protection, a mixture of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (20.0 g, 98.7 mmol), cesium carbonate (96.5 g, 296 mmol) and deuterated methanol (4.27 g, 118 mmol) in DMSO (200 mL) was stirred at 60°C for 16 hours. After the reaction was completed, the mixture was diluted with water (100 mL) and the pH was adjusted to 3 with 1N hydrochloric acid. The mixture was filtered to collect the precipitate, and the filter cake was dried under vacuum to obtain 6-(methoxy-d3)-5-nitropyridine-2-carboxylic acid Cpd-2-1 (12.0 g, crude product, yellow solid). The crude product was used directly in the next reaction. LCMS calculated value: C7H4D3N2O5[M+H] + :m / z=202.1; Detected value: 201.9.

[0160] Step 2: 6-(Methoxy-d3)-5-nitropyridine-2-carboxylic acid methyl ester

[0161] Under nitrogen, a mixture of 6-(methoxy-d3)-5-nitropyridine-2-carboxylic acid Cpd-2-1 (12.0 g, 59.7 mmol) and cesium carbonate (58.4 g, 179 mmol) in DMSO (120 mL) was stirred at 0°C for 0.5 hours. Iodomethane (9.32 g, 179 mmol) was then added to the reaction mixture, which was stirred at 0°C for 2 hours. After completion of the reaction, as determined by LCMS, the reaction mixture was poured into an ice-cold aqueous ammonium chloride solution (1000 mL) and extracted with EtOAc (1000 mL x 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to yield the crude product. The residue was purified by SGC (0-20% EtOAc in PE) to yield methyl 6-(methoxy-d3)-5-nitropyridine-2-carboxylate Cpd-2-2 (10.0 g, 70.1% yield, as a yellow solid). LCMS calculated value for C8H6D3N2O5[M+H]+: m / z=216.1; found value: 215.9.

[0162] Step 3: 5-amino-6-(methoxy-d3)pyridine-2-carboxylic acid methyl ester

[0163] Methyl 6-(methoxy-d3)-5-nitropyridine-2-carboxylate Cpd-2-2 (10.0 g, 46.5 mmol) was dissolved in EtOAc (100 mL), palladium on carbon (9.90 g, 5% content) was added, and the resulting mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. LCMS analysis showed complete consumption of the starting material and the detection of the desired compound. Filtration and concentration afforded methyl 5-amino-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-3 (8.20 g, 90.5% yield, 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).

[0164] Step 4: 3-[8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-ynal

[0165] 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-cooling, DMP (17.4 g, 41.0 mmol) was slowly added portionwise. The mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated aqueous sodium bicarbonate (50 mL) and extracted with DCM (30 mL x 3). The combined organic phases were concentrated in vacuo, and the residue was purified by SGC (0-15% EtOAc in PE) to give 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol Int-2 (5.70 g, 84.3% yield, 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).

[0166] Step 5: 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

[0167] Under nitrogen, a solution of 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-ynal Int-2 (200 mg, 606 μmol), methyl 5-amino-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-3 (135 mg, 730 μmol), and tetraisopropyl titanate (517 mg, 1.82 mmol) in THF (2 mL) was stirred at 100°C for 1 hour. The reaction was cooled to 25°C, MeOH (2 mL) and sodium cyanoborohydride (57.1 mg, 0.91 mmol) were added, and stirring was continued for 10 minutes. LCMS analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by SGC (0-25% EtOAc in PE) to give isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-4 (190 mg, 59.4% yield, yellow solid). LCMS calculated value C 23 H 19 D3BrF3N3O3[M+H] + :m / z=527.1 / 529.1; Detected value: 526.9 / 529.0.

[0168] Step 6: Isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate

[0169] Under nitrogen, a solution of isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-4 (170 mg, 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 analysis indicated complete consumption of the starting material and the presence of the desired compound. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative method to give isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-5 (90.0 mg, 48.1% yield, yellow solid). LCMS calculated value C 29 H 31 D3F4N5O3[M+H] + :m / z=579.3; Detection value: 579.0.

[0170] 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

[0171] A mixture of isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate Cpd-2-5 (40.0 mg, 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 analysis indicated complete consumption of the starting material and the presence of the desired compound. The reaction mixture was concentrated under reduced pressure, diluted with water, and adjusted to pH 5 with dilute hydrochloric acid. The product was filtered, and the residue was purified by SGC (0-3% MeOH in DCM) to give a crude 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, 15.0% yield, white solid). LCMS calculated value: C 26 H 25 D3F4N5O3[M+H] + :m / z=537.2;Detection value:537.0; 1 H NMR (400MHz, CD3OD) δ7.64(d,J=7.3Hz,1H),7.36(d,J=6.9Hz,1H),6.94(d,J=7.8H z,1H),6.56(s,1H),6.42(t,J=7.2Hz,1H),5.79(d,J=7.3Hz,1H),4.90(s,1H),4.1 9(s,2H),3.69(q,J=10.5Hz,2H),3.62-3.54(m,1H),3.32-3.23(m,1H),3.01(d,J= 12.2Hz,1H),2.62-2.48(m,1H),2.46-2.39(m,1H),2.37(s,3H),2.00-1.87(m,2H).

[0172] 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

[0173] 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 and then cooled to room temperature. Methanesulfonamide (74.5 mg, 784 μmol) was added to the reaction mixture, stirred for 10 minutes, and then DBU (120 μL, 789 μmol) was added. Stirring was continued 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 x 3). The combined organic phases were washed with brine (50 mL x 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, 20.8% yield, 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).

[0174] 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)

[0175] Step 1: tert-Butyl (tert-Butoxycarbonyl)(2-chloro-4-methoxypyrimidin-5-yl)carbamate

[0176] Under nitrogen protection, a mixture of 2-chloro-4-methoxypyrimidin-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 starting 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, 42.5% yield, white solid). LCMS calculated value C 15 H 23 ClN3O5[M+H] + :m / z=360.1; Detected value: 360.2.

[0177] Step 2: Methyl 5-((tert-Butyloxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate

[0178] 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 analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction solution was filtered and concentrated under reduced pressure. The residue was diluted with water (100 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by SGC (0-20% EtOAc in PE) to give methyl 5-((tert-butyloxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-3 (2.00 g, 59.0% yield, white solid). LCMS calculated value C 12 H 18 N3O5[M+H] + :m / z=284.1; Detected value: 284.1.

[0179] Step 3: 8-Bromo-2-(3-bromoprop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine

[0180] At room temperature, 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-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, followed by the addition of carbon tetrabromide (3.00 g, 9.03 mmol). The reaction mixture was stirred at room temperature for 2 hours. LCMS analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by SGC (0-10% 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, 53.0% yield, white solid). LCMS calculated value C 13 H9Br2F3N[M+H] + :m / z=393.9 / 395.9 / 397.9; Detected values: 394.0 / 396.0 / 398.0.

[0181] Step 4: 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

[0182] Under nitrogen, 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), methyl 5-((tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate 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 analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was cooled to room temperature, filtered, and the filtrate 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, 65.1% yield, white solid). LCMS calculated value C 25 H 25 BrF3N4O5[M+H] + :m / z=597.1 / 599.1; Detection value: 597.2 / 599.2.

[0183] Step 5: 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

[0184] Under nitrogen, a mixture of 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 (1.35 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 at 95°C in a sealed tube for 2 hours. LCMS analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was cooled to room temperature, 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.

[0185] Step 6: 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

[0186] 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, yellow solid). LCMS calculated value: C 30 H 35 F4N6O5[M+H] + :m / z=635.3; Detection value: 635.5.

[0187] 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

[0188] 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 another 2 hours. LCMS analysis 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 layers were 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)indolizin-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.

[0189] 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

[0190] 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, 8.8% yield, 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).

[0191] 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)

[0192] Step 1: N-(cyclopropylsulfonyl)-6-(methoxy-d3)-5-nitropyridine-2-carboxamide

[0193] 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) was added cyclopropylsulfonamide (1.33 g, 10.9 mmol) and triethylamine (3.32 g, 32.8 mmol) in sequence at room temperature. The reaction mixture was stirred at 40°C for 3 hours. LCMS analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (100 mL) and adjusted to pH 2 with 1N aqueous hydrochloric acid. The precipitated solid was filtered, washed with water (100 mL), and dried over anhydrous sodium sulfate to yield N-(cyclopropylsulfonyl)-6-(methoxy-d3)-5-nitropyridine-2-carboxamide Cpd-4-1 (1.00 g, crude, yellow solid). The crude product was used directly in the next reaction. 10 H7D3N3O6S[MH] - :m / z=303.1; Detected value: 303.2.

[0194] Step 2: 5-amino-N-(cyclopropylsulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide

[0195] 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 under a hydrogen atmosphere for 16 hours. LCMS detection showed that the starting 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 used directly in the next reaction. LCMS calculated value C 10 H9D3N3O4S[MH] - :m / z=273.1;Detection value:Found:273.3.

[0196] 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

[0197] 3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-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. NaBH3CN (286 mg, 4.54 mmol) was then added and the system was stirred for another 0.5 hours. LCMS analysis showed that the starting 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 give 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, 62.8% yield, yellow solid). LCMS calculated value C 23 H 16 BrD3F3N4O4S[MH] - :m / z=586.1 / 588.1; Detection: 586.2 / 588.2.

[0198] 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

[0199] 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)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-4 (42.4 mg, 6.80% yield, 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).

[0200] 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)

[0201] Step 1: N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)-5-nitromethylpicolinamide

[0202] 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) was added (cyclopropylmethyl)sulfonamide (1.69 g, 12.5 mmol) and triethylamine (3.62 g, 35.8 mmol) at room temperature. The reaction mixture was stirred at 40°C for 3 hours. LCMS analysis indicated complete consumption of the starting material and the presence of the desired compound. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (100 mL), adjusted to pH 2 with 1N aqueous hydrochloric acid, and extracted with DCM (100 mL x 3). The combined organic layers were 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, 29.0% yield, white solid). LCMS calculated value C 11 H9D3N3O6S[MH] - :m / z=317.1; Detected value: 316.9.

[0203] Step 2: 5-amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)pyridine-2-carboxamide

[0204] 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 under a hydrogen atmosphere for 16 hours. LCMS detection showed that the starting 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 reaction. LCMS calculated value C 11 H 11 D3N3O4S[MH] - :m / z=287.1;Detection value:Found:287.1.

[0205] 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

[0206] 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 stirring continued at room temperature for 0.5 hours. LCMS analysis indicated complete consumption of the starting material, and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL x 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 give 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, 44.9% yield, yellow solid). LCMS calculated value: C 24 H 20 BrD3F3N4O4S[M+H]+ :m / z=602.1 / 604.1; Detected value: 602.3 / 604.3.

[0207] 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

[0208] 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, and extracted with EtOAc / THF (2:1, 30 mL x 3). The combined organic layers were 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 yield the product, 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 (81.2 mg, 19.1% yield, 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).

[0209] 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)

[0210] Step 1: N-(2,4-dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide

[0211] 2,2,2-Trifluoroethane-1-sulfonyl chloride Cpd-6-1 (750 mg, 4.11 mmol) was added dropwise to a solution of 2,4-dimethoxybenzylamine (1.37 g, 8.22 mmol) in THF (20 mL) at room temperature, and the system was stirred for 1 hour. LCMS analysis showed that the starting material was completely consumed and the desired compound was detected. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by SGC (0-1% MeOH in DCM) to give N-(2,4-dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide Cpd-6-2 (1.20 g, 93.2% yield, white solid). LCMS calculated value C 11 H 14 F3NNaO4S[M+Na] + :m / z=336.1; Detected value: 336.1.

[0212] Step 2: 2,2,2-Trifluoroethane-1-sulfonamide

[0213] 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 mixture was stirred at room temperature for 1 hour. LCMS analysis indicated complete consumption of the starting material and the presence of the desired compound. The reaction mixture was concentrated under reduced pressure to afford 2,2,2-trifluoroethane-1-sulfonamide Cpd-6-3 (851 mg, 99.1% yield, purple solid). The crude product was used directly in the next reaction. 1 H NMR (400MHz, DMSO-d6) δ7.50 (s, 2H), 4.26 (q, J = 10.0Hz, 2H).

[0214] Step 3: Methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxylate

[0215] A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-aldehyde Int-2 (700 mg, 2.33 mmol), methyl 5-amino-6-(methoxy-d3)pyridine-2-carboxylate 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. NaBH3CN (440 mg, 7.00 mmol) was then added, and the system was stirred at room temperature for another 2 hours. LCMS analysis indicated complete consumption of the starting material, and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL x 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 give methyl 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-6-4 (625 mg, 53.7% yield, yellow solid). LCMS calculated value: C 21 H 15 D3BrF3N3O3[M+H] + :m / z=499.1 / 501.1; Detection value: 499.0 / 501.0.

[0216] 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

[0217] Methyl 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-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 analysis showed complete consumption of the starting material and the detection of the desired compound. 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, 84.4% yield, white solid). LCMS calculated value C 20 H 13 D3BrF3N3O3[M+H] + :m / z=485.1 / 487.1; Detection value: 485.0 / 487.0.

[0218] 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

[0219] 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) at 0°C. The reaction was stirred at 0°C for 30 minutes. LCMS analysis indicated complete consumption of the starting material and the presence of the desired compound. The reaction mixture was diluted with DCM (100 mL) and washed sequentially with 0.5N aqueous hydrochloric acid (50 mL), saturated aqueous sodium bicarbonate (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, 51.3% yield, white solid). LCMS calculated value: C 22 H 15 D3BrF6N4O4S[M+H] + :m / z=630.0 / 632.0; Detected value: 629.9 / 631.9.

[0220] 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

[0221] 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 complete consumption of the starting material 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)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)pyridine-2-carboxamide Cpd-6 (14.0 mg, 8.09% yield, white solid). LCMS calculated value C 28 H 27 D3F7N6O4S[M+H] + :m / z=682.2;Detection value:682.5; 1 H NMR (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=6.4Hz ,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).

[0222] 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)

[0223] Step 1: 6-Chloro-N-(cyclopropylsulfonyl)-5-nitropyridine-2-carboxamide

[0224] To a mixture of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (5.00 g, 24.7 mmol) and 2-chloro-1-methylpyridinium iodide (7.57 g, 29.6 mmol) in DCM (50 mL) at room temperature were added cyclopropylsulfonamide (3.59 g, 29.6 mmol) and triethylamine (7.49 g, 74.1 mmol). The reaction system was stirred at 40°C for 3 hours. LCMS analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (100 mL) and adjusted to pH 2 with 1N aqueous hydrochloric acid. The precipitated solid was filtered, washed with water, and dried to yield 6-chloro-N-(cyclopropylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-7-1 (8.90 g, crude, white solid). The crude product was used directly in the next reaction. LCMS calculated value C9H7ClN3O5S[MH] - :m / z=304.0 / 306.0; Detected value: 303.9 / 305.9.

[0225] Step 2: N-(cyclopropylsulfonyl)-6-methoxy-5-nitropyridine-2-carboxamide

[0226] To a solution of 6-chloro-N-(cyclopropylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-7-1 (8.90 g, 29.1 mmol) in methanol (90 mL) was added a methanol solution of sodium methoxide (16.2 mL, 87.3 mmol, 5.4 mol / L) dropwise at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. LCMS analysis showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was quenched with 1N aqueous hydrochloric acid and the pH was adjusted to 2. The precipitated solid was filtered, washed with water, and dried to give N-(cyclopropylsulfonyl)-6-methoxy-5-nitropyridine-2-carboxamide Cpd-7-2 (6.70 g, crude, white solid). The crude product was used directly in the next reaction. LCMS calculated value C 10 H 10 N3O6S[MH] - :m / z=300.0; Detection value: 300.0.

[0227] Step 3: 5-amino-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide

[0228] 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 at room temperature under a hydrogen atmosphere for 16 hours. LCMS detection showed that the starting 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 product, white solid). The crude product was used directly in the next reaction. LCMS calculated value C 10 H 12 N3O4S[MH] - :m / z=270.1; Detected value: 270.1.

[0229] 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

[0230] 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. NaBH3CN (570 mg, 9.09 mmol) was then added, and the system was stirred at room temperature for an additional 2 hours. LCMS analysis indicated complete consumption of the starting material and the detection of the desired compound. The reaction mixture was quenched with saturated aqueous sodium bicarbonate (50 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were 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, 28.2% yield, yellow solid). LCMS calculated value C 23 H 21 BrF3N4O4S[M+H] +:m / z=585.0 / 587.0; Detection: 585.3 / 587.3.

[0231] 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

[0232] 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 analysis indicated complete consumption of the starting material and the detection of the desired compound. 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, 8.3% yield, 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).

[0233] 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)

[0234] Step 1: 6-Chloro-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide

[0235] To a stirred suspension of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (50.0 g, 247 mmol) in dichloromethane (500 mL) were added 2-chloro-1-methylpyridinium iodide (95.0 g, 372 mmol) and methanesulfonylamine (35.0 g, 368 mmol). The system was stirred for 10 minutes, followed by the dropwise addition of triethylamine (100 g, 988 mmol). After the addition was complete, the temperature was raised to reflux and the reaction was continued for approximately 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 give 6-chloro-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-1 (68.0 g, 98.5% yield, yellow solid). LCMS calculated value: C7H5ClN3O5S[MH] - :m / z=278.0; Detection value: 278.1.

[0236] Step 2: 6-(Methoxy-d3)-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide

[0237] 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 1 M potassium tert-butoxide tetrahydrofuran solution (338 mL, 338 mmol) was added dropwise. After the addition was complete, 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 product was filtered, and the filter cake was rinsed with water (500 mL) and methyl tert-butyl ether (500 mL). The filter cake was recrystallized from acetonitrile to obtain the product 6-(methoxy-d3)-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-2 (30.0 g, 67.0% yield, yellow solid). LCMS calculated value: C8H7D3N3O6S [M+H] + :m / z=279.0; Detection value: 279.2.

[0238] Step 3: 5-amino-6-(methoxy-d3)-N-(methylsulfonyl)pyridine-2-carboxamide

[0239] 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) and stirred to form a solution. Methanol (600 mL) was then added. After argon replacement, palladium on carbon (3.00 g, 10% content) was added. The argon atmosphere was replaced with hydrogen, and the reaction was allowed to proceed at room temperature for 12 hours. Filter through celite, rinse the filter cake with tetrahydrofuran, and the combined filtrates were concentrated. The residue was concentrated to dryness to afford 5-amino-6-(methoxy-d3)-N-(methylsulfonyl)pyridine-2-carboxamide (Cpd-8-3) (26.0 g, 97.1% yield, off-white solid), which was carried forward to the next step without further processing. LCMS calculated value: C8H9D3N3O4S [M+H] + :m / z=249.1; Detected value: 249.2.

[0240] 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

[0241] 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 dissolution, Int-2 (10.1 g, 30.5 mmol) was added and 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. The mixture was filtered, and the filter cake was rinsed with water (50 mL) and methyl tert-butyl ether (100 mL). It was dried under oil pump vacuum to obtain 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 (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.

[0242] 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

[0243] 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, 42.3% yield, 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).

[0244] 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)

[0245] 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

[0246] 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).

[0247] 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)

[0248] 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

[0249] 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)δ 10.89(s,1H),7.68(d,J=8.0Hz,1H),7.55(d,J=6.9Hz,1H),7.08(d,J=8.1Hz,1H),6.81-6.7 8(m,2H),6.51(t,J=7.1Hz,1H),5.86(d,J=7.4Hz,1H),5.74(d,J=8.3Hz,1H),4.57(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(d t,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).

[0250] Biological evaluation

[0251] 1. Biochemical Activity Assay: Testing the Compound's Activity in Promoting the Binding of the p53 Y220C Mutant to DNA

[0252] Homogeneous time-resolved fluorescence (HTRF) experiments were used to measure the reactivation effects of compounds on the p53 Y220C mutant. Recombinant His-tagged p53 Y220C (94-312) for HTRF experiments was expressed in Escherichia coli and purified to 90% purity using a Ni-NTA column. Biotinylated DNA for HTRF experiments was synthesized by Pharmaron Chemicals; the specific sequence is 5'-ATTAGGCATGTCTAGGCATGTCTAGG-3'.

[0253] Fluorescence resonance energy transfer (FRET) was used to measure the binding of recombinant His-tagged p53 Y220C protein to biotinylated DNA. For the FRET experiment, the binding between the p53 mutant and the DNA sequence was measured by detecting the fluorescence of the interaction between an anti-His antibody conjugated to Eu (Cisbio, 61HI2KLA) and d2-conjugated streptavidin (Cisbio, 610SADLF) bound to the biotinylated DNA molecule. Compounds were prepared as 2 mM stocks and serially diluted 1:3 in DMSO to 10 concentrations. Each 200-fold working concentration of compound was diluted sequentially to a 4-fold working concentration. Using an Echo, 4 μL of compound solution was transferred row by row to a 384 assay plate, with 2 replicates per column. 4 μL of p53 solution was added to the assay plate. 4 μL of biotinylated DNA solution was added to the assay plate, and then 4 μL of detection solution (His-Eu antibody and streptavidin-d2) was added to each well of the assay plate. The assay plates were incubated overnight and protected from light. Fluorescence was read on a BMG (BMG LRBTECH).

[0254] Calculate the ratio of each well (Ratio_665nm / 615nm-Ratio_Background). The activity % is calculated as follows:

[0255] C=(Ave_Ac-Ave_Ba) / (Ave_Dc-Ave_Bd)

[0256] R data=(A-Ave_Ba-C*D) / (D-Ave_Bd)*(Ave_Dc-Ave_Bd)

[0257] Activity% = R data / Ave_VC * 100.

[0258] A: Fluorescence intensity of the sample at 665 nm;

[0259] D: fluorescence intensity of the sample at 615 nm;

[0260] Ba: fluorescence intensity of the plate background at 665 nm;

[0261] Bd: fluorescence intensity of the plate background at 615 nm;

[0262] Dc: fluorescence intensity of HIS-Eu background at 615 nm;

[0263] Ac: fluorescence intensity of streptavidin-d2 background at 665 nm;

[0264] VC: DMSO-treated group.

[0265] 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 .

[0266] Table 1: Activity of compounds in promoting the binding of p53 Y220C mutant to DNA

[0267] Cpd-A is selected from WO2021061643A1; Cpd-B is selected from WO2022213975A1;

[0268] 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.

[0269] 2. Cell activity assay: NUGC-3 cell proliferation inhibition assay

[0270] The antiproliferative effects of the compounds on human gastric adenocarcinoma cell lines were evaluated based on cellular dehydrogenase activity, which correlated well with viable cell counts. The NUGC-3 cell line (JCRB, JCRB0822) was cultured in RPMI 1640 (Invitrogen, 11875-085) supplemented with 10% (v / v) fetal bovine serum (BI, 04-002-1A). The cell lines were cultured according to the standard protocols of the American Type Culture Collection. The cell lines were authenticated by their short tandem repeat profiles.

[0271] In the proliferation experiment, 1000 NUGC-3 cells were seeded in a 96-well flat-bottomed transparent TC-treated plate (Corning, 3903) in 200 μL of culture medium per well and cultured overnight in culture medium to recover. Compounds were added in increasing concentration gradients, and DMSO treatment was used as a positive control. After 5 days of treatment, the plate was equilibrated to room temperature for 30 minutes, and the number of viable cells was measured by adding the cell counting kit-8 reagent (MCE, HY-K0301). The absorbance was measured on Enspire (Perkin Elme). The relative survival rate of each group was expressed as a percentage change relative to the positive control group and then fitted to a four-parameter logit nonlinear curve using the program XLFit (IDBS).

[0272] Table 2: Antiproliferation activity of compounds on NUGC-3 cells

[0273] 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.

[0274] 3. Evaluation of drug-drug interactions related to metabolic enzymes

[0275] (1) CYP enzyme inhibitory activity test at a single concentration

[0276] 1. Experimental purpose: To test the inhibitory effect of compounds on CYP series enzymes at a single concentration.

[0277] 2. Experimental instruments and reagents:

[0278] Liver microsomes:

[0279] Reagents:

[0280] 3. Experimental methods:

[0281] Dissolve 8.71 g of K2HPO4 in 950 mL of water. Adjust the pH to 7.4 with HCl solution. Bring the volume to a final volume of 1000 mL with water. Filter the buffer through a 0.45 μm filter. Store in a refrigerator at 4°C for future use. Prepare a stock solution of terfenadine / tolbutamide (1 mg / mL each) in DMSO. Dilute this stock solution with acetonitrile to prepare a quenching solution containing 5 / 10 ng / mL (terfenadine / tolbutamide).

[0282] Positive control working solution:

[0283] Substrate working solution:

[0284] Thaw liver microsomes in a 37°C water bath before use. Prepare a liver microsome working solution (final concentration 0.5 mg / mL). Prepare a 5 mM NADPH working solution in phosphate buffered saline. Make a 2 mM working solution from the above stock solution in DMSO.

[0285] 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. Pre-incubate in a shaking water bath at 37°C for 5 minutes. After pre-incubation, 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. 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.

[0286] 4. Data processing methods and results:

[0287] Percent inhibition (% Inhibition) is calculated using metabolite formation of the test or control compound compared to vehicle control.

[0288] Table 3: Inhibitory activity of compounds against CYP enzymes*

[0289] *The final concentration of the compound was 10 μM; ND: not detected; NI: no inhibition

[0290] The above data show that the Example compounds of the present invention have weak inhibitory activity against CYP series enzymes and the risk of drug interactions is significantly lower than that of the reference compounds.

[0291] (II) CYP3A4 / 5 enzyme multi-concentration inhibition curve determination

[0292] 1. Experimental purpose: To determine the concentration inhibition curve of the compound on CYP3A4 / 5 enzyme.

[0293] 2. Experimental instruments and materials:

[0294] Liver microsomes:

[0295] Reagents:

[0296] 3. Experimental methods:

[0297] Dissolve 8.71 g of K2HPO4 in 950 mL of water. Adjust the pH to 7.4 with HCl solution. Bring the volume to a final volume of 1000 mL with water. Filter the buffer through a 0.45 μm filter. Store in a refrigerator at 4°C for future use. Prepare a stock solution of terfenadine / tolbutamide (1 mg / mL each) in DMSO. Dilute this stock solution with acetonitrile to prepare a quenching solution containing 5 / 10 ng / mL (terfenadine / tolbutamide).

[0298] Positive control working solution:

[0299] Substrate working solution:

[0300] Thaw liver microsomes in a 37°C water bath before use. Prepare a liver microsome working solution (final concentration 0.5 mg / mL). Prepare a 5 mM NADPH working solution in phosphate buffered saline. Make a 2 mM working solution from the above stock solution in DMSO. Dilute the above stock solution with acetonitrile (4-fold dilution, 6 non-zero concentrations).

[0301] 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. Pre-incubate in a shaking water bath at 37°C for 5 minutes. After pre-incubation, 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. 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.

[0302] 4. Data processing methods and results:

[0303] Graph Pad Prism was used to calculate the half-maximal inhibitory concentration (IC 50 ).

[0304] Table 4: Inhibitory activity of compounds against CYP3A4 / 5 enzymes

[0305] ND: Not Detected

[0306] The above data show that the example compounds of the present invention have no obvious inhibitory activity on CYP3A4 / 5 enzymes, and the risk of drug interactions is significantly lower than that of the reference compounds.

[0307] IV. In vitro metabolic stability evaluation: Liver microsome metabolic stability study:

[0308] 1. Experimental purpose: To evaluate the metabolic stability of the test compound in human / rat / mouse liver microsomes.

[0309] 2. Experimental reagents and materials:

[0310] Reagents:

[0311] Material:

[0312] 3. Experimental methods:

[0313] The test compound and the positive control compound (dextromethorphan as the control compound) were dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with DMSO to a 200 μM working solution. 8.709 g of dipotassium hydrogen phosphate (K2HPO4) was dissolved in 950 mL of water, the pH of the solution was adjusted to 7.4 with hydrochloric acid, and then the volume was made up to 1000 mL with water. After filtering with a 0.22 μm filter, the solution was placed in a refrigerator at 4°C for storage. Various types of liver microsomes (protein concentration 20 mg / mL) were melted in a 37°C water bath and diluted with phosphate buffer to obtain a liver microsome working solution with a protein concentration of 0.629 mg / mL. A 5 mM NADPH solution was prepared using the above phosphate buffer for use. A 1 mg / mL stock solution of terfenadine / tolbutamide was prepared in DMSO, and then diluted with a 50% methanol / 50% acetonitrile mixture to form a reaction stop solution containing 5 / 10 ng / mL (terfenadine / tolbutamide) as an internal standard.

[0314] 238.5 μL of the working solution of liver microsomes from different species was added to a 1.1 mL microcentrifuge tube. 1.5 μL of the working solution of the test compound or the positive control compound (dextromethorphan) (200 μM) was added to each tube. Mix thoroughly and pre-incubate in a 37°C waterbath for 5 minutes. The reaction was initiated by adding 60 μL of NADPH solution. After thorough mixing, 30 μL of the reaction solution was pipetted into 300 μL of the reaction stop solution at 0, 5, 15, 30, and 60 minutes after the reaction. Samples at all time points were vortexed vigorously for 1 minute and centrifuged at 4000 rpm for 15 minutes at 4°C. 100 μL of the supernatant was added to 100 μL of purified water, mixed thoroughly, and then analyzed by LC-MS / MS.

[0315] 4. Data processing methods:

[0316] 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 ):

[0317] The compound residual rate is calculated as follows:

[0318] Intrinsic clearance CL int (μL / min / mg protein)=0.693*1000 / T 1 / 2 / Protein concentration (0.5mg protein / mL)

[0319] Table 5: Metabolic stability of compounds in human / rat / mouse liver microsomes

[0320] 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.

[0321] 5. In vitro metabolic stability evaluation: Hepatocyte metabolic stability test

[0322] 1. Objective: To determine the metabolic stability of the compound in human / rat / mouse hepatocytes.

[0323] 2. Experimental Materials:

[0324] 3 Experimental design

[0325] 3.1 Preparation of compound working solution

[0326] The test substance and control drug verapamil powder were prepared into high concentration stock solutions 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.

[0327] 3.2 Preparation of hepatocytes

[0328] 1) Mix 49.5 mL Williams' Medium E and 0.5 mL GlutaMAX as incubation medium.

[0329] Preheat the incubation solution and the incubation solution in a 37°C water bath for at least 15 minutes before use.

[0330] 2) Take a vial of cryopreserved hepatocytes and ensure they remain frozen 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.

[0331] 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 approximately 1.5 × 10 6 cells / mL of cell suspension.

[0332] 4) Count the hepatocytes and determine the viable cell density using a Cellometer Vision. The viability of the hepatocytes must be greater than 75%. Dilute the hepatocyte suspension with incubation medium to a viable cell density of 0.5 × 10 6 cells / mL.

[0333] 5) A portion with a density of 0.5×10 6 A hepatocyte suspension containing 10 cells / mL was inactivated by boiling in water for 5 minutes as a negative control. This inactivation facilitates the investigation of substrate conversion mediated by non-cellular enzymes.

[0334] 3.3 Test methods

[0335] 1) Transfer 198 μL of live or inactivated cell suspension to a 96-well deep-well plate. Place the plate on a vortex and preheat in an incubator for 10 minutes. Incubate live cells in duplicate and inactivated cells in a single replicate.

[0336] 2) Add 2 μL of 100 μM test substance or verapamil to each well to initiate the reaction, and place the deep-well plate back on the incubator vortexer.

[0337] 3) Incubate the sample. At 0, 15, 30, 60, 90, and 120 minutes, aspirate 25 μL of the suspension and terminate the reaction by adding 150 μL of acetonitrile containing internal standards (200 nM alprazolam, 200 nM labetalol, 2 μM ketoprofen, and 200 nM caffeine). Vortex for 10 minutes and centrifuge at 3220 g and 4°C for 30 minutes. After centrifugation, aspirate 100 μL of the supernatant and mix with 100 μL of ultrapure water for UPLC-MS / MS analysis.

[0338] 4 Data Analysis

[0339] All data calculations were performed using Microsoft Excel. Peak areas were determined by extracting ion spectra. The in vitro half-life (t 1 / 2 ).

[0340] In vitro half-life (t 1 / 2 ) is calculated by slope:

[0341] in vitro 1 / 2 =0.693 / k

[0342] In vitro intrinsic clearance (unit: μL / min / 10 6 cells) were calculated using the following formula:

[0343] in vitro CLint=kV / N

[0344] V = incubation volume per well (0.2 mL);

[0345] N = number of cells per well (0.1 × 10 6 cells)

[0346] Table 6: Metabolic stability of compounds in human / rat / mouse hepatocytes

[0347] 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.

[0348] VI. Cardiac Safety Assessment: hERG Potassium Channel Inhibitory Activity Test

[0349] 1. Experimental purpose:

[0350] Manual patch clamp technique was used to evaluate whether the test compound has a potential inhibitory effect on the voltage-gated potassium channel hERG. This experiment measured the effect of five concentrations of the compound on the hERG channel current, obtained the compound dose-effect curve and calculated the IC 50 .

[0351] 2. Experimental instruments and reagents:

[0352] 3. Experimental methods:

[0353] 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 plated at 5 × 10 5 The cells were cultured at a density of 100 μg / mL 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.

[0354] 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, and 110 potassium fluoride (adjust pH to 7.2 with potassium hydroxide). The osmotic pressure of the solution should be controlled between 280 and 300 mOsmol / kg. Filter the solution before use and store at 4°C.

[0355] Preparation of test compound solutions: Following standard operating procedures, test compounds were dissolved in DMSO to a final concentration of 10 mM stock solution. The stock solution was serially diluted 1:3 with DMSO to yield three intermediate concentrations (mM): 3.33, 1.11, and 0.37. Prior to the experiment, the intermediate solutions were further diluted 1:1000 with extracellular fluid to yield a series of working solutions. A 30 μM working solution was prepared from the 10 mM stock solution at a 1:333.33 ratio, resulting in final concentrations (μM): 30, 10, 3.33, 1.11, and 0.37. The DMSO content in the working solutions ranged from 0.1% to 0.3% (v / v). Five working solutions of 30, 10, 3.33, 1.11, and 0.37 μM were used to determine the potential inhibitory effects of the compounds on hERG channels and to fit dose-response curves and calculate IC50 values.

[0356] Experimental Procedure: Place a small glass slide containing HEK293 cells in a culture dish into the perfusion chamber on the microscope stage. Under an Olympus IX71 or IX73 inverted microscope, center the field of view with an appropriate cell. Using a ×10x objective, locate the tip of the glass electrode and position it in the center of the field of view. Then, use the micromanipulator to lower the electrode while adjusting the coarse focus knob to slowly approach the cell. Once close to the cell, switch to a ×40x objective for observation and use the micromanipulator to fine-tune the electrode, gradually bringing it closer to the cell surface. Apply negative pressure to create a seal with a resistance greater than 1 GΩ between the electrode tip and the cell membrane. Compensate for the transient capacitive current, Cfast, in voltage-clamp mode. Repeated brief negative pressure bursts are then applied to disrupt the membrane, ultimately establishing whole-cell recording mode. With the membrane potential clamped at -60 mV, compensate for the slow capacitive current, Cslow, the cell membrane capacitance (Cm), and the 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 leakage current detection conditions are: the clamping voltage is changed to -80mV, and the time course is 500ms. The hERG current test method is as follows: a 4.8-second depolarization command voltage is applied to depolarize the membrane potential from -80mV to +30mV, and then a 5.2-second repolarization voltage is applied instantaneously to reduce the membrane potential to -50mV to eliminate 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 is recorded continuously for 120 seconds before administration to assess the stability of the hERG current generated by the test cells. Only stable cells within the acceptable range of the evaluation criteria can enter the subsequent compound testing. Test of the inhibitory effect of the test compound on hERG current: First, the hERG current measured in 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 compound to be tested 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 stabilizes, 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 end of 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).

[0357] 4. Data processing methods and results:

[0358] Data quality control standards: initial sealing resistance greater than 1GΩ; series resistance less than 15MΩ and the series resistance voltage error less than 5mV; leakage current under the detection voltage is less than 50% of the current value under this condition; tail current is greater than the platform current of the pre-pulse, and the initial tail current value is greater than 250pA; membrane rupture resistance Ra is less than 15MΩ; the tail current decay rate per minute is less than 2.5%.

[0359] Data analysis: Only data that meet the above criteria can be analyzed according to the following steps:

[0360] Note: Data is output by PatchMaster software.

[0361] After perfusing blank solvent or compound gradient solution, the average value of the 5 consecutive current values ​​obtained was calculated and used as the "tail current size" 空白 ” and “tail current size 化合物 ”.

[0362] The current suppression percentage is calculated using the following formula.

[0363] The dose-effect curve was fitted and the IC was calculated using Graphpad Prism 8.0 software. 50 value.

[0364] Table 7: Inhibitory activity of compounds on hERG

[0365] 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.

[0366] 7. Pharmacokinetic studies in mice

[0367] 1. Experimental purpose: To test the pharmacokinetics of the compound in CD-1 mice.

[0368] 2. Experimental instruments and materials:

[0369] Animals: CD-1 mice (male)

[0370] instrument:

[0371] Reagents:

[0372] Solvent:

[0373] Intravenous injection: 40% HP-β-CD aqueous solution; intragastric administration: 0.2% HPC + 0.5% Tween80 aqueous solution.

[0374] 3. Experimental methods:

[0375] Six male mice were enrolled. They were fasted in the evening before administration and had free access to water. Mice were allowed to eat and drink freely during the experiment. The mice were administered intravenously or orally. After administration, the animals were observed and any abnormalities were recorded. Blood was collected from the orbital cavity at 0.0833, 0.25, 1, 2, 4, 8, and 24 hours after intravenous injection; and at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after oral gavage. A 20 μL plasma sample was collected, and 250 μL of acetonitrile (containing dexamethasone as an internal standard) was added to precipitate the protein. The sample was centrifuged at 4000 rpm for 20 minutes at 4°C. 180 μL of the supernatant was mixed with 180 μL of an aqueous solution containing 0.1% formic acid in a 96-well plate. A 10 μL sample was then collected for LC-MS / MS analysis.

[0376] 4. Data processing methods and results:

[0377] 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 using the standard curve. Pharmacokinetic parameters were calculated using the non-compartmental analysis model of WinNonlin 8.2 software and presented in the report. Parameters included Clint, C max and AUC, etc.

[0378] Table 8: Pharmacokinetic parameters of compounds in CD-1 mice

[0379] The above data show that the example compounds of the present invention have lower clearance rates and higher oral exposures in mice and are superior to the reference compounds.

[0380] 8. Pharmacokinetic studies in rats

[0381] 1. Experimental purpose: To test the pharmacokinetics of the compound in SD rats.

[0382] 2. Experimental instruments and materials:

[0383] Animals: SD rats (male)

[0384] instrument:

[0385] Reagents:

[0386] Solvent:

[0387] Intravenous injection: 40% HP-β-CD aqueous solution; intragastric administration: 0.2% HPC + 0.5% Tween80 aqueous solution.

[0388] 3. Experimental methods:

[0389] Six male rats were enrolled. They were fasted in the evening before dosing and had free access to water. Rats were allowed to eat and drink freely during the experiment. The rats were administered intravenously or orally. After administration, the animals were observed and any abnormalities were recorded. Blood was collected by jugular vein puncture at 0.0833, 0.25, 1, 2, 4, 8, and 24 hours after intravenous injection; and by jugular vein puncture at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after oral gavage. A 50 μL plasma sample was collected, and 500 μL of acetonitrile (containing dexamethasone as an internal standard) was added to precipitate the protein. The sample was centrifuged at 4000 rpm for 20 minutes at 4°C. 300 μL of the supernatant was mixed with 300 μL of Watson's aqueous solution in a 96-well plate. Another 5 μL sample was collected for LC-MS / MS analysis.

[0390] 4. Data processing methods and results:

[0391] 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 using the standard curve. Pharmacokinetic parameters were calculated using the non-compartmental analysis model of WinNonlin 8.2 software and presented in the report. Parameters include Clint, C max and AUC, etc.

[0392] Table 9: Pharmacokinetic parameters of the compounds in SD rats

[0393] The above data show that the example compounds of the present invention have lower clearance rates and higher oral exposures in rats and are significantly better than the reference compounds.

[0394] IX. In vivo efficacy studies in a NUGC-3 humanized gastric cancer xenograft mouse model

[0395] 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 NUNU female mice.

[0396] 2. Experimental Materials:

[0397] Animals: 6-8 week old female NUNU mice;

[0398] Reagents:

[0399] Cpd-A solvent: 0.2% HPC, 0.5% Tween80 in water (w / v / v);

[0400] Cpd-2 solvent: 5% DMSO / 10% Solutol HS15 / 85% pH 4.65±0.10acetate buffer.

[0401] 3. Experimental plan:

[0402] Cell culture: NUGC-3 cells were cultured in RPMI-1640 medium containing 10% FBS.

[0403] Modeling and grouping: NUGC-3 cells (5×10 6 ) were resuspended in 0.2 mL of PBS and Matrigel (1:1) and inoculated subcutaneously on the right side of NUNU mice to establish a subcutaneous xenograft tumor model. The health status of the mice and tumor growth were observed daily after cell inoculation. On the fifth day after inoculation, 36 animals were selected and grouped. The tumor volume of the enrolled animals was approximately 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).

[0404] 4. Experimental design: Table 10 shows 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±0.10 acetate 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.

[0405] Table 10. In vivo efficacy experimental design of NUGC-3 xenograft mouse model

[0406] 5. Experimental observation and result judgment:

[0407] 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.

[0408] 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 when grouped, 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.

[0409] Statistical Analysis: Tumor volume and animal body weight are expressed as mean ± SEM. Tumor volume was statistically compared between groups. All statistical analyses were performed in GraphPad Prism 8.0. One-way ANOVA was used to compare significant differences in tumor volume or weight between groups. A p value of ≥ 0.05 indicated no significant difference, a p value of < 0.05 indicated a significant difference, and a p value of < 0.001 indicated a highly significant difference.

[0410] 6. Experimental results:

[0411] The tumor growth curves for the control and experimental groups in the pharmacodynamic evaluation of the NUGC-3 human gastric cancer xenograft model in mice are shown in Figure 1, and the relative body weight changes of the mice in this model are shown in Figure 2. The mean tumor volume, tumor volume inhibition rate (TGI) (%), and comparative results for each group at the experimental endpoint are shown in Table 11.

[0412] On day 19 after grouping, the average tumor volume in 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 mm3 , 399.1mm 3 , 615.1mm 3 , 202.2mm 3 and 102.5mm 3 , and 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 ​​< 0.05); among them, the average tumor volume of the G5 and G6 groups was significantly different from that of the Vehicle group (p values ​​< 0.001). The above results indicate 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 all tested doses, and the activity 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%) was better than the tumor inhibitory activity of the double compound Cpd-A at a dosage (100 mg / kg, QD) (TGI = 81.3%), indicating that Cpd-2 has better anti-tumor activity than Cpd-A.

[0413] Table 11. In vivo efficacy test results of NUGC-3 xenograft mouse model

[0414] Remark:

[0415] a. TGI (%) = [1-(T 19 -T0) / (V 19 -V0)]×100

[0416] b. P value of the comparison analysis between the average tumor volume of each drug-treated group and the average tumor volume of the G1 group Vehicle

[0417] 10. In vivo efficacy study of NUGC-3 human gastric cancer xenograft rat model

[0418] 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.

[0419] 2. Experimental Materials:

[0420] Animals: 7-9 week old female B-Rag2 / IL2rg KO Sprague-Dawley rats;

[0421] Reagents:

[0422] Solvent: 5% DMSO / 10% Solutol HS15 / 85% pH 4.65±0.10acetate buffer;

[0423] 3. Experimental plan:

[0424] Cell culture: NUGC-3 cells were cultured in RPMI-1640 medium containing 10% FBS.

[0425] Modeling and grouping: NUGC-3 cells (1×10 7 ) were resuspended in 0.2 mL of PBS and Matrigel (1:1) and inoculated subcutaneously on the right side of B-Rag2 / IL2rg KO SD rats to establish a subcutaneous xenograft tumor model. Following cell inoculation, the rats' health and tumor growth were observed daily. On day 10 after inoculation, 24 animals were selected and grouped. The tumor volume of the enrolled animals was approximately 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).

[0426] 4. Experimental Design: Table 12 shows the grouping and dosing information for the efficacy experiment. Rats in group G1 were orally administered with a vehicle control (5% DMSO / 10% Solutol HS 15 / 85% pH 4.65±0.10 acetate buffer) once daily for 28 days. Rats in group G2 were orally administered with Cpd-2 at a dose of 6.25 mg / kg once daily for 28 days. Rats in group G3 were orally administered with Cpd-2 at a dose of 12.5 mg / kg once daily for 28 days. Rats in group G4 were orally administered with Cpd-2 at a dose of 25 mg / kg once daily for 28 days.

[0427] Table 12. In vivo efficacy experimental design of NUGC-3 xenograft rat model

[0428] 5. Experimental observation and result judgment:

[0429] 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 2During the study, the entire dosing process as well as tumor and body weight measurements were performed in a clean bench.

[0430] 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 when grouped, 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.

[0431] Statistical Analysis: Tumor volume and animal body weight are expressed as mean ± SEM. Tumor volumes between groups were statistically compared. All statistical analyses were performed at https: / / d2k.bio / Efficacy / Invivo. Significant differences in tumor volume or weight between groups were compared using the Wilcoxon nominal test, with p ≥ 0.05 indicating no significant difference, p < 0.05 indicating a significant difference, and p < 0.01 indicating a highly significant difference.

[0432] 6. Experimental results:

[0433] The tumor growth curves for the control and experimental groups in the pharmacodynamic evaluation of the NUGC-3 human gastric cancer xenograft model in rats are shown in Figure 3 , and the relative body weight changes of the rats in this model are shown in Figure 4 . The mean tumor volume, tumor volume inhibition (TGI) (%), and comparative results for each group at the endpoint are shown in Table 13 .

[0434] On day 28 after grouping, the average tumor volume in 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 3The corresponding tumor volume inhibition rates (TGIs) were 92.9%, 102.9%, and 104.7%, respectively. Statistical analysis showed that the mean tumor volumes of the G2, G3, and G4 groups were significantly different from those of the Vehicle group (all p values ​​< 0.01). These results demonstrate that Cpd-2 exhibits potent inhibitory activity against NUGC-3 xenograft tumors in rats at all tested doses, with a dose-dependent pattern.

[0435] Table 13. In vivo efficacy test results of NUGC-3 xenograft rat model

[0436] Remark:

[0437] The p value of the comparative analysis of the average tumor volume of each drug-treated group and the average tumor volume of the G1 group Vehicle.

Claims

1. A nitrogen-containing compound as shown in formula I, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt solvate or its prodrug; 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 according to claim 1, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt, or its prodrug, characterized in that: The nitrogen-containing compound, its isotope label, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt, or its prodrug as shown in Formula Ia is a nitrogen-containing compound, its isotope label, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt, or its prodrug; 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 according to claim 1 or 2, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt, or its prodrug, characterized in that: X 1 N; X 2 is 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, its isotope label, its solvate, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt solvate or its prodrug 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 as claimed in claim 1 or 2, the compound as shown, its isotope label, its solvate, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt solvate or its prodrug, 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), an isotope-labeled substance thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a solvate of a pharmaceutically acceptable salt thereof, or a prodrug thereof according to any one of claims 1 to 5, 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 according to claim 1, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt, or its prodrug, characterized in that: 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 of formula (I) as claimed in claim 1, its isotope-labeled substance, its solvate, its pharmaceutically acceptable salt, its solvate of a pharmaceutically acceptable salt or its prodrug, characterized in that: The compound of formula (I) is selected from the following compounds: Its enantiomers or mixtures of both in any ratio.

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

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

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

14. A method for treating and / or preventing cancer, characterized in that: The method comprises administering to the patient a therapeutically effective amount of a compound of formula I as described in any one of claims 1 to 8, an isotope-labeled substance, an enantiomer, a diastereomer, a solvate or a pharmaceutically acceptable salt thereof.

15. 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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