A class of N-sulfonylamide-containing nitrogen heterocyclic compounds targeting p53 mutations and their applications

Abstract

The present invention discloses a class of N-sulfonamide-containing nitrogen heterocyclic compounds targeting p53 mutations and their applications. The N-sulfonamide-containing nitrogen heterocyclic compounds of the present invention are compounds represented by Formula I or pharmaceutically acceptable salts thereof. These compounds can enhance the ability of p53 Y220C to bind to DNA and have excellent inhibitory activity against cancer cell proliferation.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals, and further belongs to the field of drugs of p53 Y220C pocket binders with drug-likeness, and particularly relates to a class of N-sulfonamide-containing nitrogen heterocyclic compounds targeting p53 mutations and their applications. Background Art

[0002] The protein product of the TP53 gene (p53) is one of the most important tumor suppressors identified to date. Unlike RB1, CDKN2A, and PTEN, which are lost through homozygous deletion in tumors, TP53 is frequently found to have somatic missense mutations (Alexandrova E M 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 homologs p63 and p73, causing them to lose their normal regulatory functions on the 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 lymphoma and sarcoma growth through apoptosis (Christophorou M A et al. Temporal dissection of p53 function in vitro and in vivo[J]. Nat Genet, 2005, 37(7):718-726; Martins C.P., Brown-Swigart L., Evan G.I. Modeling the Therapeutic Efficacy of p53 Restoration in Tumors. Cell. 2006;127:1323–1334), providing a theoretical basis for tumor treatment by restoring the normal function of p53.In addition to enhancing wild-type p53 activity in tumors with a complete Tp53 genotype, restoring the wild-type protein function of mutant p53 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).

[0003] The core domain of wild-type p53 is unstable, with low thermodynamic and kinetic stability, allowing for rapid cycling between folded and unfolded states. Mutations in these core region residues in mutant p53 lead to enhanced thermodynamic and kinetic instability, resulting in the loss of DNA-binding activity in the core DNA-binding domain. These effects can be used to design ligands that selectively bind to the native state of the 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 specific mutation Y220C mediates the formation of a surface pocket far from the DNA-binding core domain (Joerger A C 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 this p53 mutant and reactivate its transcriptional activity, but with limited potency (Boeckler F M, 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 K Z 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 cancer patients with the p53 Y220C mutation, there is an urgent need for p53 Y220C pocket binders with stronger affinity and better druggability. Summary of the Invention

[0004] The technical problem to be solved by the present invention is that the existing p53 Y220C pocket binders have poor drug-likeness. Therefore, the present invention provides a class of N-sulfonamide nitrogen-containing heterocyclic compounds targeting p53 mutations and their applications. This class of compounds can enhance the ability of p53 Y220C to bind to DNA and has good inhibitory activity against the proliferation of cancer cells represented by gastric cancer cells.

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

[0006]

[0007] Wherein,

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

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

[0010] R 6 is -(CH 2 ) n R 7 ;

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

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

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

[0014]

[0015] Wherein,

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

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

[0018] R 6 is -(CH 2 ) n R 7 ;

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

[0020] R 7 is 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 is N; X 2 is CH or N; X 3 is CH.

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

[0023] As a preferred technical solution, R 6 is -(CH 2 ) n R 7 ; n is 0 or 1; R 7 is 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 is -(CH 2 ) n R 7 ; n is 0 or 1; R 7 is selected from -CH 3 , -CD 3 , -CF 3 , 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 are deuterium; R 4 and R 5 are hydrogen.

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

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

[0029]

[0030] In formula Ib and Ic, is

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

[0032] R 6 is -(CH 2 ) n R 7 ;

[0033] n is 0 or 1;

[0034] R 7 is selected from C1-C6 alkyl, halo C1-C6 alkyl and C3-C6 cycloalkyl.

[0035] Preferably, is selected from -OCH 3 and -OCD 3 ;

[0036] R 4 and R 5 are selected from hydrogen;

[0037] R 6 is -(CH 2 ) n R 7 ; n is 0 or 1; R 7 is selected from -CH 3 , -CD 3 , -CF 3 or cyclopropyl.

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

[0039]

[0040]

[0041]

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

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

[0044] Method 1:

[0045]

[0046] The first step: The compound a-1 undergoes a ring-closing aromatization reaction with ethyl bromopyruvate to obtain the compound a-2; wherein, X is selected from Cl, Br, I;

[0047] The second step: The compound a-2 undergoes a Vilsmeier-Haack reaction to obtain the compound a-3;

[0048] The third step: The compound a-3 reacts with a fluorination reagent to obtain the compound a-4; the fluorination reagent can be methyl fluorosulfonyldifluoroacetate, (triphenylphosphonium) difluoroacetate inner salt, tetrabutylammonium fluoride;

[0049] The fourth step: The compound a-4 undergoes an ester reduction reaction under the action of a reducing agent to obtain the compound a-5; the reducing agent can be sodium borohydride, lithium borohydride, lithium aluminum hydride or diisobutylaluminum hydride;

[0050] The fifth step: The compound a-5 is oxidized under the action of an oxidizing agent to obtain the compound a-6, and the oxidizing agent can be Dess-Martin reagent, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide, sulfur trioxide / pyridine;

[0051] The sixth step: The compound a-6 reacts with the Gilbert reagent to generate the compound a-7;

[0052] The seventh step: The compound a-7 reacts with formaldehyde under the catalysis of a metal reagent to obtain the compound a-8; the metal reagent can be a Cu-containing reagent, such as copper(I) tetraethylacetonitrile hexafluorophosphate;

[0053] The eighth step: The compound a-8 is oxidized to the aldehyde a-9 under the action of an oxidizing agent, and the oxidizing agent can be Dess-Martin reagent, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide, sulfur trioxide / pyridine;

[0054] The ninth step: The compound a-9 undergoes a reductive amination reaction with the compound a-10 to generate the compound a-11; R 1 、R 2 、R 3 、R6 、X 1 、X 2 、X 3 are defined as described above;

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

[0056] Method 2:

[0057]

[0058] Step 1: Compound a-11 and amine a-13 undergo a metal reagent-catalyzed coupling reaction 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 are defined as described above; the metal reagent can be a Pd- or Cu-containing reagent, such as Brettphos Pd G3, Brettphos Pd G4, cuprous iodide;

[0059] Step 2: Compound a-14 removes the Boc protecting agent under acidic conditions to obtain compound a-15; the acidic conditions can be carried out in the presence of an inorganic acid (such as HCl, HBr, H 2 SO 4 ), an organic acid (such as trifluoroacetic acid), a Lewis acid (such as zinc bromide) or other reagents (such as trimethylsilyl iodide);

[0060] Step 3: Compound a-15 and formaldehyde undergo a reductive amination reaction to form compound (I-a).

[0061] Method 3:

[0062]

[0063] Step 1: Compound a-9 and compound a-16 undergo a reductive amination reaction to form compound a-17; wherein, X is selected from Cl, Br, I; Y is selected from C 1 -C 4 alkyl; R 1 、R 2 、R 3 、X 1 、X 2 、X 3 are defined as described above;

[0064] 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 Pd- or Cu-containing reagent, such as Brettphos Pd G3, Brettphos Pd G4, cuprous iodide;

[0065] Step 3: Compound a-18 undergoes a hydrolysis reaction under acidic or basic conditions to form a-19; the acidic conditions can be inorganic acids (such as HCl, HBr, H 2 SO 4 ) or organic acids (such as trifluoroacetic acid); the basic conditions can be inorganic bases (such as LiOH, NaOH, KOH);

[0066] Step 4: Compound a-19 undergoes a condensation reaction with sulfonamide H 2 NS(O) 2 R 6 to form compound (I-a); wherein, the definition of R 6 is as defined above.

[0067] Method 4:

[0068]

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

[0070] 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 Pd- or Cu-containing reagent, such as Brettphos Pd G3, Brettphos Pd G4, cuprous iodide;

[0071] Step 3: Compound a-21 removes the protecting group under acidic or basic conditions to form a-22; the acidic conditions can be inorganic acids (such as HF, HCl, HBr, H 2 SO 4 ) or organic acids (such as trifluoroacetic acid); the basic conditions can be inorganic bases (such as LiOH, NaOH, KOH) or organic base (tetra-n-butylammonium fluoride);

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

[0073] Step 5: Compound a-23 undergoes a reductive amination reaction with sulfonamide a-10 to generate compound (I-a); wherein, R 1 , R 2 , R 3 , R 6 , X 1 , X 2 , X 3 are as defined above;

[0074] Method Five:

[0075]

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

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

[0078] Step 3: Compound a-25 undergoes a reduction reaction to obtain compound a-26; The reducing agent can be sodium borohydride (lithium) or lithium aluminum hydride reagent, etc., as well as their deuterated reagents; R 4 , R 5 are as defined above;

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

[0080] Step 5: Compound a-27 and a-28 undergo a nucleophilic substitution reaction to obtain compound a-29; wherein, R 1 , R 2 , R 3 , X 1 , X 2 , X 3 are as defined above; G is a protecting group, such as p-toluenesulfonyl group, tert-butoxycarbonyl group; Y is selected from C 1 -C 4 alkyl;

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

[0082] Step 7: The 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 Pd- or Cu-containing reagent, such as Brettphos Pd G3, Brettphos Pd G4, cuprous iodide;

[0083] Step 8: Compound a-31 undergoes a hydrolysis reaction under acidic or basic conditions to form a-32; the acidic conditions can be inorganic acids (such as HCl, HBr, H 2 SO 4 ) or organic acids (such as trifluoroacetic acid); the basic conditions can be inorganic bases (such as LiOH, NaOH, KOH);

[0084] Step 9: Compound a-32 undergoes a condensation reaction with sulfonamide H 2 NS(O) 2 R 6 to form compound (I); wherein, the definition of R 6 is as described above.

[0085] The present invention also provides a pharmaceutical composition, which comprises a compound of formula I or a pharmaceutically acceptable salt thereof (such as a therapeutically effective amount) and pharmaceutical excipients.

[0086] The present invention also provides an application of a compound of formula I, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of a p53 mutant pocket binder or a drug for treating and / or preventing diseases related to p53 mutants.

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

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

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

[0090] (3) The diseases related to the p53 mutant are cancers, such as breast cancer, gastric cancer, lung cancer or ovarian cancer.

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

[0092] The cancers such as breast cancer, gastric cancer, lung cancer or ovarian cancer.

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

[0094]

[0095] Among them, X is a halogen (e.g., Br); R 8 is an amino protecting group (e.g., -Boc);

[0096] X 1 、X 2 and X 3 、R 1 、R 2 、R 3 、R 4 、R 5 、R 6 are defined as described above.

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

[0098]

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

[0100] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, the 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 a compound contains a relatively basic functional group, the 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).

[0101] The "-" at the end of the group means that the group is connected to the rest of the molecule through this site. For example, CH 3 -C(=O)- means an acetyl group.

[0102] When a wavy line is attached to the valence bond of the group as in, for example, the wavy line indicates the connection point of the group to the rest of the molecule

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

[0104] The term "alkyl" refers to having a specified number of carbon atoms (e.g., C1 -C 6 ), a linear or branched, monovalent hydrocarbon group of u. Alkyl groups include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0105] The term "alkoxy" refers to the group R X -O-, where R X is defined in the same way as the term "alkyl". Alkoxy groups include but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0106] The term "therapeutically effective amount" refers to the 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 according to the circumstances. The term "patient" refers to any animal in need of treatment or prevention of a disease, usually a mammal, such as a human. Mammals include but are not limited to: cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

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

[0108] The term "treatment" refers to eliminating the cause or relieving the symptoms.

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

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

[0111] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

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

[0113] The positive and progressive effects of the present invention are as follows: The compounds of the present invention can enhance the ability of p53 Y220C to bind to DNA, and have good inhibitory activity against the proliferation of cancer cells represented by gastric cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0116] Figure 3 For 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;

[0117] Figure 4 For the relative body weight change curves of the rats in the control group and each experimental group in the pharmacodynamic evaluation experiment of the rat NUGC-3 human gastric cancer xenograft tumor model in Test Example 10. Detailed implementation manners

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

[0119] In each example, 1 HNMR was recorded by a BRUKER AVANCE NEO 400MHz, JNM-ECZ400s type nuclear magnetic resonance spectrometer, and the chemical shift was expressed in δ (ppm): liquid chromatography-mass spectrometry (LCMS) was recorded by Shimadzu LC-20AD, Agilent 1260 type and Agilent 1200 type mass spectrometers: preparative HPLC separation was carried out using a WATERS Autop, Shimadzu LC20AR type liquid chromatograph

[0120] Abbreviations

[0121]

[0122]

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

[0124]

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

[0126] A mixture of 3-bromo-2-methyl-pyridine Int-1-1 (500 mg, 2.91 mmol), ethyl 3-bromopyruvate (850 mg, 4.36 mmol) and sodium bicarbonate (561 mg, 6.69 mmol) in methyl ethyl ketone (5 mL) was stirred at 85 °C for 16 h, then concentrated to dryness and purified by SGC (0 - 10% EtOAc in PE) to give ethyl 8-nitroindolizine-2-carboxylate Int-1-2 as a grey solid (190 mg, yield 24.4%). LCMS calculated for C 11 H 11 BrNO 2 [M+H] + : m / z = 268.0 / 270.0; found: 267.9 / 269.9; 1 H NMR (400 MHz, CDCl 3 ) δ 7.88 (d, J = 1.6 Hz, 1H), 7.87 - 7.83 (m, 1H), 7.03 - 6.99 (m, 1H), 6.95 (d, J = 6.8 Hz, 1H), 6.43 (t, J = 7.2 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 6.8 Hz, 3H).

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

[0128] A solution of ethyl 8-bromoindolizine-2-carboxylate 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) at 0 °C. The mixture was stirred at 20 °C for 1 h, then quenched slowly 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 ethyl 8-bromo-3-formylindolizine-2-carboxylate Int-1-3 as a yellow gum (10.6 g, crude). LCMS calculated for C 12 H 11 BrNO 3 [M+H] + : m / z = 296.0 / 298.0; found: 295.9 / 297.9.

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

[0130] 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-(triphenylphosphoniumyl)acetate (11.2 g, 31.5 mmol) in DMF (120 mL) was stirred at 60 °C for 2 h. TBAF (47.3 mL, 47.3 mmol, 1 M solution in THF) was added to the mixture. The reaction mixture was stirred at 60 °C for 2 h, then diluted with water (100 mL) and extracted with MTBE (100 mL × 3). The organic layers were combined, dried over sodium sulfate, filtered and concentrated in vacuo to dryness. The residue was purified by SGC (PE with 0 - 5% EtOAc) to give ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate Int-1-4 as a white solid (2.40 g, yield 43.4%). LCMS calculated value C 13 H 12 BrF 3 NO 2 [M+H] + : m / z = 350.0 / 352.0; found: 349.9 / 351.9; 1 H NMR (400 MHz, CDCl 3 ) δ 7.89 (d, J = 7.2 Hz, 1H), 7.12 (s, 1H), 7.05 (d, J = 6.8 Hz, 1H), 6.56 (t, J = 7.2 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.25 (q, J = 10.0 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H).

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

[0132] To a mixture of ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate 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 h to give a yellow solution. The reaction mixture was poured into saturated aqueous ammonium chloride (200 mL), then water (200 mL) was added and the mixture was extracted with EtOAc (150 mL × 3). The organic layers were combined, dried over sodium sulfate, filtered and concentrated in vacuo to dryness. The residue was purified by SGC (PE with 0 - 5% EtOAc) to give [8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]methanol Int-1-5 as a white solid (3.55 g, yield 98.4%). LCMS calculated value C 11 H10 BrF 3 NO[M+H] + : m / z = 308.0 / 310.0; Detected value: 307.9 / 309.9; 1 H NMR (400 MHz, CDCl 3 ) δ 7.85 (d, J = 7.2 Hz, 1H), 7.00 (d, J = 6.8 Hz, 1H), 6.70 (br s, 1H), 6.50 (t, J = 7.2 Hz, 1H), 4.84 (s, 2H), 3.83 (q, J = 10.4 Hz, 2H).

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

[0134] At 0 °C, DMP (7.33 g, 17.2 mmol) was added 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). The reaction system was warmed to room temperature and stirred for 2 h. The reaction solution was poured into saturated aqueous sodium bicarbonate (20 mL), then water (200 mL) was added, and the mixture was extracted with DCM (100 mL × 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to dryness. The residue was purified by SGC (0 - 7% EtOAc in PE) to give 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 as a white solid (2.70 g, yield 76.5%). LCMS calculated value C 11 H 8 BrF 3 NO[M+H] + : m / z = 306.0 / 308.0; Detected value: 305.9 / 307.9.

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

[0136] At 0 °C, 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 (500 mg, 1.63 mmol) was added to a mixed system of dimethyl (1-diazo-2-oxopropyl)phosphonate (470 mg, 2.45 mmol) and potassium carbonate (451 mg, 3.27 mmol) in methanol (15 mL). The reaction system was warmed to room temperature and stirred for 16 h. The reaction solution was diluted with water (50 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to dryness. The residue was purified by SGC (0 - 3% EtOAc in PE) to give the white solid 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (380 mg, yield 77.0%). LCMS calculated value C 12 H 8 BrF 3 N[M+H] + : m / z = 302.0 / 304.0; found: 301.9 / 303.9; 1 H NMR (400 MHz, CDCl 3 ) δ 7.81 (d, J = 7.2 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.79 (s, 1H), 6.52 (t, J = 7.2 Hz, 1H), 3.83 (q, J = 10.0 Hz, 2H), 3.25 (s, 1H).

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

[0138] Under nitrogen protection, copper(I) tetraethylacetonitrile hexafluorophosphate (123 mg, 0.33 mmol) and tributylphosphine (268 mg, 1.32 mmol) were dissolved in toluene (15 mL), and the reaction mixture was stirred at 70 °C for 30 min. Then 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (1.00 g, 3.31 mmol) and aqueous formaldehyde solution (0.18 mL, 6.62 mmol, 36% - 38% content) were added. The mixture was stirred at 70 °C overnight. After completion of the reaction detected by TLC, the mixture was concentrated in vacuo, and the residue was purified by SGC (0 - 15% EtOAc in PE) to give the white solid 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol Int-1-7 (843 mg, yield 76.7%). LCMS calculated value C 13 H 10 BrF 3 NO[M+H] +: m / z = 332.0 / 334.0; Detected value: 332.3 / 334.3; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.38 (d, J = 7.1 Hz, 1H), 7.19 (d, J = 7.1 Hz, 1H), 6.68 (t, J = 7.1 Hz, 1H), 6.61 (s, 1H), 5.34 (t, J = 6.0 Hz, 1H), 4.34 (d, J = 6.0 Hz, 2H), 4.20 - 4.11 (m, 2H).

[0139] Cpd-C: 4 - ((3-(8 - (((3S,4R)-3 - fluoro - 1 - methylpiperidin - 4 - yl)amino)-3-(2,2,2 - trifluoroethyl)indolizin - 2 - yl)prop - 2 - ynyl - 1 - yl)amino)-3 - methoxy - N - methylbenzamide

[0140]

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

[0142] At 0 °C, HATU (18.8 g, 49.5 mmol) was added to a solution of 4 - amino - 3 - methoxybenzoic acid Cpd - C - 1 (7.52 g, 45.0 mmol), methylamine solution (6.71 g, 54.0 mmol, 25% content), and triethylamine (18.8 mL, 135 mmol) in THF (45 mL). 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 obtain the white solid 4 - amino - 3 - methoxy - N - methylbenzamide Cpd - C - 2 (6.08 g, yield 75.0%). LCMS calculated value C 9 H 13 N 2 O 2 [M + H] + : m / z = 181.1; Detected value: 181.2.

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

[0144] To a mixture of 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (600 mg, 1.99 mmol) and 4-amino-3-methoxy-N-methylbenzamide Cpd-C-2 (716 mg, 3.97 mmol) in 1,4-dioxane (12 mL) was added aqueous formaldehyde solution (497 mg, 5.96 mmol, 36%-38% content) and copper(I) bromide (567 mg, 3.97 mmol). The reaction was stirred under microwave irradiation at 100 °C for 1 hour. The residue was purified by SGC (0-30% EtOAc in DCM) to give the yellow solid 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). LCMS calculated for C 22 H 20 BrF 3 N 3 O 2 [M+H] + : m / z = 494.1 / 496.1; found: 494.2 / 496.2.

[0145] Step 3: (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylic acid tert-butyl ester

[0146] Under argon protection, a mixture of 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (1.35 g, 2.73 mmol), (3S,4R)-tert-butyl 4-amino-3-fluoropiperidine-1-carboxylate (1.19 g, 5.46 mmol), BrettPhos Pd G4 (0.50 g, 0.55 mmol), Ruphos (0.25 g, 0.55 mmol) and cesium carbonate (1.78 g, 5.46 mmol) in 1,4-dioxane (50 mL) was stirred at 100 °C for 16 h. The reaction mixture was diluted with EtOAc (200 mL) and washed with water (100 mL), saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by SGC (0 - 50% EtOAc in DCM) to give the yellow solid (3S,4R)-tert-butyl 3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate Cpd-C-4 (1.00 g, yield 58.0%). LCMS calculated value C 32 H 38 F 4 N 5 O 4 [M+H] + : m / z = 632.3; found: 632.5.

[0147] Step 4: 4-((3-(8-(((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

[0148] At 0 °C, hydrochloric acid (7.92 mL, 31.7 mmol, 4N 1,4-dioxane solution) was added 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). 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 the 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 directly used in the next step. LCMS calculated value C 27 H 30 F 4 N 5 O 2 [M+H] + : m / z = 532.2; found: 532.4.

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

[0150] At room temperature, sodium cyanoborohydride (160 mg, 2.54 mmol) was added to a methanol (20 mL) 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 aqueous formaldehyde solution (212 mg, 2.54 mmol, content 36%-38%). The reactants were stirred at room temperature for 1 hour. Saturated sodium bicarbonate solution was added dropwise to the reaction mixture to adjust the pH to 7-8. The aqueous phase was extracted with EtOAc (100 mL×3), 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 preparation to obtain the white solid 4-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C (119 mg, yield 25.2%). LCMS calculated value C 28 H 32 F 4 N 5 O 2 [M+H] + : m / z = 546.2; detected value: 546.2; 1 H NMR (400 MHz, DMSO-d 6)δ8.08 (d, J = 4.6 Hz, 1H), 7.57 (d, J = 6.9 Hz, 1H), 7.41 (dd, J = 8.2, 1.7 Hz, 1H), 7.34 (d, J = 1.7 Hz, 1H), 6.91 (s, 1H), 6.74 (d, J = 8.3 Hz, 1H), 6.51 (t, J = 7.2 Hz, 1H), 5.90 (t, J = 6.2 Hz, 1H), 5.85 (d, J = 7.5 Hz, 1H), 5.54 (d, J = 8.4 Hz, 1H), 4.81 (d, J = 49.8 Hz, 1H), 4.23 (d, J = 6.2 Hz, 2H), 3.92 (q, J = 10.7 Hz, 2H), 3.83 (s, 3H), 3.54 (d, J = 27.9 Hz, 1H), 3.02 (t, J = 10.5 Hz, 1H), 2.80 (d, J = 11.3 Hz, 1H), 2.75 (d, J = 4.5 Hz, 3H), 2.28 - 2.14 (m, 4H), 2.07 (t, J = 11.2 Hz, 1H), 1.97 (ddd, J = 24.0, 12.0, 3.2 Hz, 1H), 1.67 (dd, J = 12.7, 2.7 Hz, 1H).

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

[0152]

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

[0154] 6 - Chloro - 5 - nitropyridine - 2 - carboxylic acid Cpd - 1 - 1 (4.00 g, 19.7 mmol) and cesium carbonate (12.8 g, 39.4 mmol) were stirred in a mixture of DMSO / MeOH (40 mL, 1:1) at 60 °C for 12 h. LCMS detection showed complete consumption of the starting material and the detection of the desired compound. The reaction mixture was poured into ice - water (50 mL) and the pH was adjusted to 5 with 1N hydrochloric acid, and then extracted with EtOAc (30 mL × 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 give 6 - methoxy - 5 - nitropyridine - 2 - carboxylic acid Cpd - 1 - 2 (2.80 g, yield 71.7%, yellow solid). LCMS calculated for C 7 H 7 N 2 O5 [M+H] + : m / z = 199.0; Detected value: 199.1.

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

[0156] A mixture of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-2 (3.20 g, 16.2 mmol), methanesulfonamide (1.54 g, 16.2 mmol), EDCI (4.65 g, 24.3 mmol) and 4-dimethylaminopyridine (3.94 g, 32.4 mmol) in DMF (30 mL) was stirred at room temperature for 8 hours. LCMS detection showed that the starting materials were completely consumed and the desired compound was detected. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to dryness. The residue was purified by SGC (0 - 5% MeOH in DCM) to give 6-methoxy-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-1-3 (2.20 g, yield 49.5%, yellow solid). LCMS calculated value C 8 H 10 N 3 O 6 S[M+H] + : m / z = 276.0; Detected value: 276.1.

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

[0158] Under a hydrogen atmosphere, a solution of palladium on carbon (1.60 g, 5% content) and 6-methoxy-N-(methylsulfonyl)-5-nitropyridine amide Cpd-1-3 (800 mg, 2.91 mmol) in EtOAc (15 mL) was stirred at room temperature for 2 hours. LCMS detection showed that the starting materials were completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure to dryness. 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, yield 70.1%, yellow solid). LCMS calculated value C 8 H 12 N 3 O 4 S[M+H] + : m / z = 246.1; Detected value: 246.0.

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

[0160] At 0 °C, tert-butyldimethylchlorosilane (68.0 mg, 451 μmol) was added portionwise 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). The reaction mixture was stirred at 0 °C for an additional 0.5 h, and then the solid was filtered off. The filtrate was concentrated under reduced pressure to give 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, yield 99.7%, yellow solid). LCMS calculated for C 19 H 24 BrF 3 NOSi[M+H] + : m / z = 446.1 / 448.1; found: 446.0 / 448.0.

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

[0162] Under argon protection, a mixture of 8-bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-1-5 (134 mg, 301 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (79.3 mg, 601 μmol), BrettPhos Pd G3 (54.4 mg, 60.0 μmol) and cesium carbonate (195 mg, 598 μmol) in THF (10 mL) was stirred at 100 °C for 2 h. The reaction mixture was cooled to room temperature and filtered, and then 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, yield 54.9%, white solid). LCMS calculated for C 25 H 36 F 4 N3 OSi[M+H] + : m / z = 498.3; Detected value: 498.0.

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

[0164] At 0 °C, TBAF (0.20 mL, 0.20 mmol, 1 M 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). The reaction mixture was stirred at 0 °C for an additional 1 hour. 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 (50 mL × 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, yield 90.2%, white solid). LCMS calculated value C 19 H 22 F 4 N 3 O[M+H] + : m / z = 384.2; Detected value: 384.1.

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

[0166] At 0 °C, a solution of pyridine sulfur trioxide (780 mg, 4.90 mmol) in DMSO (1 mL) was added dropwise to a solution of 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Cpd-1-7 (625 mg, 1.63 mmol), DMSO (1.20 mL, 16.3 mmol) and diisopropylethylamine (1.50 mL, 8.15 mmol) in DCM (20 mL). The reaction mixture was stirred at 0 °C for an additional 0.5 h. LCMS analysis indicated complete consumption of the starting material and detection of the desired compound. The reaction was quenched with saturated aqueous sodium bicarbonate (100 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to dryness. The residue was purified by SGC (0 - 5% MeOH in DCM) to afford 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Cpd-1-8 (550 mg, yield 88.6%, yellow solid). LCMS calculated value C 19 H 20 F 4 N 3 O[M+H] + : m / z = 382.2; found: 382.1.

[0167] Step 8: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide

[0168] 3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indazol-2-yl)propiolaldehyde 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 titanium(IV) isopropoxide (112 mg, 393 μmol) in a mixture of THF (1 mL) was stirred at 100 °C for 1 h, then the mixed solution was cooled to 0 °C and MeOH (0.5 mL) and sodium cyanoborohydride (12.4 mg, 197 μmol) were added slowly. The reaction mixture was warmed to room temperature and stirred for an additional 10 min. LCMS analysis showed complete consumption of the starting materials and the desired compound was detected. The reaction mixture was poured into ice water (5 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with saturated aqueous sodium bicarbonate (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by SGC (0 - 3% MeOH in DCM) to give the crude product as a yellow solid, which was further purified by preparative HPLC to give 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indazol-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-1 (14.8 mg, yield 18.5%, yellow solid). LCMS calcd for C 27 H 31 F 4 N 6 O 4 S[M+H] + : m / z = 611.2; found: 611.9; 1 H NMR (400 MHz, CDCl 3)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),2.94(d,J=11.0Hz,1H),2.37 - 2.21(m,4H),2.16(t,J=11.1Hz,1H),2.02(d,J=10.0Hz,1H),1.93(td,J=12.2,3.5Hz,1H).

[0169] Example 2: 5 - ((3 - (8 - (((3S,4R) - 3 - fluoro - 1 - methylpiperidin - 4 - yl)amino) - 3 - (2,2,2 - trifluoroethyl)indolizin - 2 - yl)prop - 2 - yn - 1 - yl)amino) - 6 - (methoxy - d 3 ) - N - (methylsulfonyl)pyridine - 2 - carboxamide (Cpd - 2, i.e., Compound 2)

[0170]

[0171]

[0172] Step 1: 6 - (methoxy - d 3 ) - 5 - nitropyridine - 2 - carboxylic acid

[0173] Under nitrogen protection, a mixture of 6 - chloro - 5 - nitropyridine - 2 - carboxylic acid Cpd - 1 - 1 (20.0 g, 98.7 mmol), cesium carbonate (96.5 g, 296 mmol), and deuterated methanol (4.27 g, 118 mmol) in DMSO (200 mL) was stirred at 60 °C for 16 hours. After the reaction was completed as detected by LCMS, the mixture was diluted with water (100 mL) and the pH was adjusted to 3 with 1N hydrochloric acid. The mixture was filtered to collect the precipitate, and the filter cake was dried under vacuum to obtain 6 - (methoxy - d 3 ) - 5 - nitropyridine - 2 - carboxylic acid Cpd - 2 - 1 (12.0 g, crude, yellow solid). The crude product was directly used in the next step. LCMS calculated value C 7 H 4 D 3 N2 O 5 [M+H] + : m / z = 202.1; Detected value: 201.9.

[0174] Step 2: Methyl 6-(methoxy-d 3 )-5-nitropyridine-2-carboxylate

[0175] Under nitrogen protection, 6-(methoxy-d 3 )-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) were stirred at 0 °C for 0.5 h. Then methyl iodide (9.32 g, 179 mmol) was added to the reaction mixture and stirred at 0 °C for 2 h. After completion of the reaction detected by LCMS, the reaction mixture was poured into ice-ammonium chloride aqueous solution (1000 mL), and extracted with EtOAc (1000 mL × 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The residue was purified by SGC (0 - 20% EtOAc in PE) method to obtain methyl 6-(methoxy-d 3 )-5-nitropyridine-2-carboxylate Cpd-2-2 (10.0 g, yield 70.1%, yellow solid). LCMS calculated value C 8 H 6 D 3 N 2 O 5 [M+H]+: m / z = 216.1; Detected value: 215.9.

[0176] Step 3: Methyl 5-amino-6-(methoxy-d 3 )pyridine-2-carboxylate

[0177] Methyl 6-(methoxy-d 3 )-5-nitropyridine-2-carboxylate Cpd-2-2 (10.0 g, 46.5 mmol) was dissolved in EtOAc (100 mL), and palladium on carbon (9.90 g, 5% content) was added. The resulting mixture was stirred in a hydrogen atmosphere at room temperature for 2 h. LCMS detection showed that the raw materials were completely consumed and the desired compound was detected. It was filtered and concentrated to obtain methyl 5-amino-6-(methoxy-d 3 )pyridine-2-carboxylate Cpd-2-3 (8.20 g, yield 90.5%, yellow solid). LCMS calculated value C 8 H 8 D 3 N 2 O 3 [M+H] +: m / z = 186.1; Detected value: 186.2; 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.53 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 5.88 (s, 2H), 3.75 (s, 3H).

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

[0179] To a solution of 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-yn-1-ol Int-1-7 (6.80 g, 20.5 mmol) in DCM (120 mL) under ice bath was slowly added portionwise DMP (17.4 g, 41.0 mmol). The mixture was stirred at room temperature for 2 h. The reaction was quenched with saturated aqueous sodium bicarbonate (50 mL) and extracted with DCM (30 mL × 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-ynal Int-2 (5.70 g, yield 84.3%, gray solid). LCMS calculated value C 13 H 8 BrF 3 NO[M + H] + : m / z = 330.0 / 332.0; Detected value: 330.2 / 332.2; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.47 (s, 1H), 8.49 (d, J = 7.1 Hz, 1H), 7.28 (d, J = 7.1 Hz, 1H), 6.90 (s, 1H), 6.78 (t, J = 7.1 Hz, 1H), 4.31 (q, J = 10.7 Hz, 2H).

[0180] Step 5: 5-((3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )isopropyl pyridine-2-carboxylate

[0181] Under nitrogen protection, 3-[8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl]prop-2-ynal Int-2 (200 mg, 606 μmol), 5-amino-6-(methoxy-d 3) A solution of methyl pyridine-2-carboxylate Cpd-2-3 (135 mg, 730 μmol) and titanium(IV) isopropoxide (517 mg, 1.82 mmol) in THF (2 mL) was stirred at 100 °C for 1 h. The reaction mixture 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 min. LCMS analysis showed complete consumption of the starting material and the desired compound was detected. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by SGC (0 - 25% EtOAc in PE) to give 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 ) isopropyl pyridine-2-carboxylate Cpd-2-4 (190 mg, yield 59.4%, yellow solid). LCMS calculated for C 23 H 19 D 3 BrF 3 N 3 O 3 [M+H] + : m / z = 527.1 / 529.1; found: 526.9 / 529.0.

[0182] Step 6: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 ) isopropyl pyridine-2-carboxylate

[0183] Under nitrogen protection, 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3) A solution of isopropyl picolinate 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 in a sealed tube at 100 °C for 2 h. LCMS detection showed complete consumption of the starting materials and the desired compound was detected. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative 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-d 3 ) isopropyl picolinate Cpd-2-5 (90.0 mg, yield 48.1%, yellow solid). LCMS calculated value C 29 H 31 D 3 F 4 N 5 O 3 [M+H] + : m / z = 579.3; found: 579.0.

[0184] Step 7: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 ) picolinic acid

[0185] 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-d 3)Isopropyl picolinate Cpd-2-5 (40.0 mg, 69.2 μmol), sodium hydroxide (8.28 mg, 207 μmol) in a mixture of THF (0.4 mL), MeOH (0.2 mL) and water (0.1 mL) was stirred at room temperature for 16 h. LCMS analysis showed complete consumption of the starting material and detection of the desired compound. The reaction mixture was concentrated under reduced pressure, diluted with water, adjusted to pH 5 with dilute hydrochloric acid, filtered, and the residue was purified by SGC (0 - 3% MeOH in DCM) to obtain the crude product, which was further 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-d 3 )Picolinic acid Cpd-2-6 (5.50 mg, yield 15.0%, white solid). LCMS calculated value C 26 H 25 D 3 F 4 N 5 O 3 [M+H] + : m / z = 537.2; found: 537.0; 1 1H NMR (400 MHz, CD 3 OD) δ 7.64 (d, J = 7.3 Hz, 1H), 7.36 (d, J = 6.9 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.56 (s, 1H), 6.42 (t, J = 7.2 Hz, 1H), 5.79 (d, J = 7.3 Hz, 1H), 4.90 (s, 1H), 4.19 (s, 2H), 3.69 (q, J = 10.5 Hz, 2H), 3.62 - 3.54 (m, 1H), 3.32 - 3.23 (m, 1H), 3.01 (d, J = 12.2 Hz, 1H), 2.62 - 2.48 (m, 1H), 2.46 - 2.39 (m, 1H), 2.37 (s, 3H), 2.00 - 1.87 (m, 2H).

[0186] Step 8: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )-N-(methylsulfonyl)picolinamide

[0187] 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-d 3 ) A solution of Cpd-2-6 (210 mg, 392 μmol) and CDI (127 mg, 784 μmol) in THF (8 mL) was stirred at 70 °C for 1 hour under argon protection and then cooled to room temperature. Methanesulfonamide (74.5 mg, 784 μmol) was added to the reaction solution, and the mixture was stirred for 10 minutes. Then DBU (120 μL, 789 μmol) was added, and the 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 × 3). The combined organic phases were washed with brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The residue was purified by preparative TLC to obtain 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-d 3 )-N-(Methylsulfonyl)pyridine-2-carboxamide Cpd-2 (50.0 mg, yield 20.8%, white solid). LCMS calculated value C 27 H 28 D 3 F 4 N 6 O 4 S[M+H] + : m / z = 614.2; found: 614.4; 1 H NMR (400 MHz, DMSO-d 6)δ 10.85 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 6.9 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.91 (s, 1H), 6.74 (s, 1H), 6.51 (t, J = 7.2 Hz, 1H), 5.86 (d, J = 7.5 Hz, 1H), 5.56 (d, J = 8.4 Hz, 1H), 4.83 (d, J = 49.6 Hz, 1H), 4.28 (d, J = 6.0 Hz, 2H), 4.05 (s, 1H), 3.95 (q, J = 10.7 Hz, 2H), 3.65 - 3.46 (m, 1H), 3.06 (t, J = 11.0 Hz, 1H), 2.84 (d, J = 11.5 Hz, 1H), 2.37 - 2.23 (m, 1H), 2.22 (s, 3H), 2.13 (t, J = 11.3 Hz, 1H), 2.04 - 1.92 (m, 1H), 1.75 - 1.64 (m, 1H).

[0188] Example 3: 5 - ((3 - (8 - (((3S,4R) - 3 - fluoro - 1 - methylpiperidin - 4 - yl)amino) - 3 - (2,2,2 - trifluoroethyl)indolizin - 2 - yl)prop - 2 - yn - 1 - yl)amino) - 4 - methoxy - N - (methylsulfonyl)pyrimidine - 2 - carboxamide (Cpd - 3, i.e., Compound 3)

[0189]

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

[0191] 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 raw materials were 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 layers were 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) method to obtain tert - butyl (2 - chloro - 4 - methoxypyrimidin - 5 - yl)carbamate Cpd - 3 - 2 (4.80 g, yield 42.5%, white solid). LCMS calculated value C 15 H 23 ClN 3 O5 [M+H] + : m / z = 360.1; Detected value: 360.2.

[0192] Step 2: Methyl 5-((tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate

[0193] Under a carbon monoxide gas atmosphere, a mixture of tert-butyl (2-chloro-4-methoxypyrimidin-5-yl)carbamate Cpd-3-2 (4.30 g, 11.9 mmol), Pd(dppf)Cl 2 (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 h. LCMS detection indicated complete consumption of the starting materials and detection of the desired compound. The reaction mixture was filtered, concentrated under reduced pressure, the residue was diluted with water (100 mL), and extracted with EtOAc (100 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 - 20% EtOAc in PE) to give methyl 5-((tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-3 (2.00 g, yield 59.0%, white solid). LCMS calculated value C 12 H 18 N 3 O 5 [M+H] + : m / z = 284.1; Detected value: 284.1.

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

[0195] At room temperature, 3-(8-bromo-3-(2,2,2-trifluoroethyl)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, then carbon tetrabromide (3.00 g, 9.03 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. LCMS detection showed that the raw materials were completely consumed and the desired compound was detected. 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, yield 53.0%, white solid). LCMS calculated value C 13 H 9 Br 2 F 3 N[M+H] + : m / z = 393.9 / 395.9 / 397.9; detected value: 394.0 / 396.0 / 398.0.

[0196] 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

[0197] Under nitrogen protection, a mixture of 8-bromo-2-(3-bromoprop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-4 (1.37 g, 3.46 mmol), 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 detection showed that the raw materials were completely consumed and the desired compound was detected. The reaction mixture was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by SGC (0 - 25% EtOAc in PE) to give methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-5 (1.60 g, yield 65.1%, white solid). LCMS calculated value C 25 H 25 BrF 3 N 4 O 5[M+H] + : m / z = 597.1 / 599.1; Detected value: 597.2 / 599.2.

[0198] 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

[0199] Under nitrogen protection, 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 in a sealed tube at 95 °C for 2 hours. LCMS detection showed that the raw materials were completely consumed and the desired compound was detected. The reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by SGC (0 - 5% MeOH in DCM) to obtain 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 product, yellow solid). LCMS calculated value C 31 H 37 F 4 N 6 O 5 [M+H] + : m / z = 649.3; Detected value: 649.2.

[0200] 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

[0201] At room temperature, 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, 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 obtain 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 F 4 N 6 O 5 [M+H] + : m / z = 635.3; found: 635.5.

[0202] Step 7: (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)aminocarbonyl)pyrimidin-5-yl)carbamic acid tert-butyl ester

[0203] A solution of 5-((tert-butoxycarbonyl)(3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxypyrimidine-2-carboxylic acid Cpd-3-7 (260 mg, 410 μmol) and CDI (133 mg, 820 μmol) in DMF (4 mL) was stirred at 90 °C for 2 h. The reaction mixture was cooled to room temperature, and then methylsulfonamide (78.0 mg, 820 μmol) and DBU (206 mg, 820 μmol) were added. The reaction mixture was further stirred at 90 °C for 2 h. LCMS analysis showed complete consumption of the starting material and detection of the desired compound. The reaction mixture 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)aminocarbonyl)pyrimidin-5-yl)carbamate Cpd-3-8 (100 mg, crude, yellow solid). LCMS calculated value C 31 H 38 F 4 N 7 O 6 S[M+H] + : m / z = 711.3; found: 711.6.

[0204] Step 8: 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxy-N-(methylsulfonyl)pyrimidine-2-carboxamide

[0205] At 0 °C, trifluoroacetic acid (1 mL) was added to a solution of tert-butyl (3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(4-methoxy-2-((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate Cpd-3-8 (100 mg, 141 μmol) in DCM (3 mL). The reaction mixture was slowly warmed to room temperature and stirred for 2 h. LCMS analysis indicated complete consumption of the starting material and detection of the desired compound. The reaction mixture 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 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 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxy-N-(methylsulfonyl)pyrimidine-2-carboxamide Cpd-3 (8.05 mg, yield 8.8%, yellow solid). LCMS calculated value C 26 H 30 F 4 N 7 O 4 S[M+H] + : m / z = 612.2; found: 612.2; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.05 (s, 1H), 7.60 (d, J = 6.8 Hz, 1H), 6.93 (s, 1H), 6.80 - 6.70 (m, 1H), 6.52 (t, J = 7.2 Hz, 1H), 5.86 (s, 1H), 5.64 (d, J = 8.3 Hz, 1H), 4.87 (d, J = 52.8 Hz, 1H), 4.32 (d, J = 6.1 Hz, 2H), 4.05 (s, 3H), 4.02 - 3.90 (m, 2H), 3.60 (d, J = 10.3 Hz, 1H), 3.23 (s, 3H), 3.18 - 3.08 (m, 1H), 2.91 (d, J = 10.9 Hz, 1H), 2.28 (s, 3H), 2.05 - 1.94 (m, 1H), 1.78 - 1.67 (m, 1H), 1.23 (s, 2H).

[0206] Example 4: N-(Cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )pyridine-2-carboxamide (Cpd-4, i.e., Compound 4)

[0207]

[0208] Step 1: N-(Cyclopropylsulfonyl)-6-(methoxy-d 3 )-5-nitropyridine-2-carboxamide

[0209] At room temperature, to a solution of 6-(methoxy-d 3 )-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) were successively added cyclopropylsulfonamide (1.33 g, 10.9 mmol) and triethylamine (3.32 g, 32.8 mmol). The reaction mixture was stirred at 40 °C for 3 hours. LCMS detection showed complete consumption of the starting materials and detection of the desired compound. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (100 mL) and the pH was adjusted to 2 with 1N aqueous hydrochloric acid. The precipitated solid was filtered, washed with water (100 mL) and dried over anhydrous sodium sulfate to give N-(cyclopropylsulfonyl)-6-(methoxy-d 3 )-5-nitropyridine-2-carboxamide Cpd-4-1 (1.00 g, crude, yellow solid). The crude product was used directly in the next step. LCMS calculated value C 10 H 7 D 3 N 3 O 6 S[M-H] - : m / z = 303.1; found: 303.2.

[0210] Step 2: 5-Amino-N-(cyclopropylsulfonyl)-6-(methoxy-d 3 )pyridine-2-carboxamide

[0211] At room temperature, N-(cyclopropylsulfonyl)-6-(methoxy-d 3)-5-Nitro-pyridine-2-carboxamide Cpd-4-1 (1.00 g, 3.29 mmol, crude) and palladium on carbon (350 mg, 5% content) were stirred in a mixture of THF (15 mL) and MeOH (15 mL) under a hydrogen atmosphere for 16 hours. LCMS analysis showed complete consumption of the starting material and detection of the desired compound. The reaction mixture was filtered and concentrated under reduced pressure to give 5-amino-N-(cyclopropylsulfonyl)-6-(methoxy-d 3 )pyridine-2-carboxamide Cpd-4-2 (730 mg, crude, yellow solid). The crude product was used directly in the next step. LCMS calculated value for C 10 H 9 D 3 N 3 O 4 S[M-H] - : m / z = 273.1; Found: 273.3.

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

[0213] 3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-al Int-2 (500 mg, 1.51 mmol), N-(cyclopropylsulfonyl)-6-(methoxy-d 3 )pyridine-2-carboxamide Cpd-4-2 (395 mg, 1.44 mmol) and acetic acid (91.0 mg, 1.51 mmol) were dissolved in a mixed solvent of MeOH (6 mL) and DCM (4 mL), and the system was stirred at room temperature for 16 hours. Then NaBH 3 CN (286 mg, 4.54 mmol) was added, and the system was stirred for an additional 0.5 hour. LCMS analysis showed complete consumption of the starting material and detection of the desired compound. The reaction mixture was diluted with water (40 mL), and the precipitate was collected by filtration. The precipitate was purified by trituration 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-d 3 )pyridine-2-carboxamide Cpd-4-3 (560 mg, yield 62.8%, yellow solid). LCMS calculated value for C 23 H 16 BrD 3 F 3 N 4 O 4 S[M-H]- : m / z = 586.1 / 588.1; Detected value: 586.2 / 588.2.

[0214] Step 4: N-(Cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )pyridine-2-carboxamide

[0215] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d 3 )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 in a sealed tube at 100 °C for 5 h. LCMS detection showed that the raw materials were 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 combined organic layers were washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na 2 SO 4 dried, 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-d 3 )pyridine-2-carboxamide Cpd-4 (42.4 mg, yield 6.80%, yellow solid). LCMS calculated value C 29 H 30 D 3 F 4 N 6 O 4 S[M + H] + : m / z = 640.2; Detected value: 640.1; 1 H NMR (400 MHz, DMSO-d 6) δ 10.88 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.76 (t, J = 6.0 Hz, 1H), 6.51 (t, J = 7.2 Hz, 1H), 5.86 (d, J = 7.2 Hz, 1H), 5.55 (d, J = 8.0 Hz, 1H), 4.83 (d, J = 49.2 Hz, 1H), 4.28 (d, J = 6.0 Hz, 2H), 3.99 - 3.91 (m, 2H), 3.62 - 3.49 (m, 1H), 3.13 - 3.01 (m, 2H), 2.84 - 2.82 (m, 1H), 2.33 - 2.21 (m, 1H), 2.23 (s, 3H), 2.15 - 2.10 (m, 1H), 2.03 - 1.93 (m, 1H), 1.71 - 1.67 (m, 1H), 1.19 - 1.15 (m, 2H), 1.09 - 1.04 (m, 2H).

[0216] Example 5: N-(Cyclopropylmethanesulfonyl)-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-d 3 )pyridine-2-carboxamide (Cpd-5, i.e., Compound 5)

[0217]

[0218]

[0219] Step 1: N-((Cyclopropylmethyl)sulfonyl)-6-(methoxy-d 3 )-5-nitromethylpyridinecarboxamide

[0220] At room temperature, to a mixed system of 6-(methoxy-d 3 )-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) were successively added (cyclopropylmethyl)sulfonamide (1.69 g, 12.5 mmol) and triethylamine (3.62 g, 35.8 mmol). The reaction mixture was stirred at 40 °C for 3 hours. LCMS detection showed that the raw materials were completely consumed and the desired compound was detected. 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 × 3). The combined organic layers were washed with water (50 mL) and saturated brine (50 mL), and anhydrous Na2 SO 4 Dry, filter and concentrate under reduced pressure. The residue was purified by SGC (0 - 5% MeOH in DCM) to give N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d 3 )-5-nitropyridine-2-carboxamide Cpd-5-1 (1.10 g, yield 29.0%, white solid). LCMS calculated value C 11 H 9 D 3 N 3 O 6 S[M-H] - : m / z = 317.1; found: 316.9.

[0221] Step 2: 5-Amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d 3 )pyridine-2-carboxamide

[0222] At room temperature, a mixture of N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d 3 )-5-nitromethylpyridinecarboxamide Cpd-5-1 (1.10 g, 3.46 mmol) and palladium on carbon (550 mg, 10% content) in THF (20 mL) and MeOH (30 mL) was stirred under a hydrogen atmosphere for 16 h. LCMS analysis showed complete consumption of the starting material and detection of the desired compound. The reaction mixture was filtered and concentrated under reduced pressure to give 5-amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d 3 )pyridine-2-carboxamide Cpd-5-2 (970 mg, crude, yellowish-white solid). The crude product was used directly in the next step. LCMS calculated value C 11 H 11 D 3 N 3 O 4 S[M-H] - : m / z = 287.1; found: 287.1.

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

[0224] 3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-al Int-2 (500 mg, 1.51 mmol), N-(cyclopropylsulfonyl)-6-(methoxy-d 3)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 a mixed system of MeOH (20 mL) and 1,2-dichloroethane (20 mL) were stirred at room temperature for 16 h. Then NaBH 3 CN (181 mg, 2.89 mmol) was added, and the system was stirred at room temperature for an additional 0.5 h. LCMS detection showed complete consumption of the starting materials and detection of the desired compound. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL×3). The combined organic layers were washed with water (50 mL) and saturated brine (50 mL), and dried over anhydrous Na 2 SO 4 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-d 3 )pyridine-2-carboxamide Cpd-5-3 (390 mg, yield 44.9%, yellow solid). LCMS calculated value for C 24 H 20 BrD 3 F 3 N 4 O 4 S[M + H] + : m / z = 602.1 / 604.1; found: 602.3 / 604.3.

[0225] Step 4: N-(Cyclopropylmethylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )pyridine-2-carboxamide

[0226] Under argon protection, 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d 3)A mixture of 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 h. LCMS analysis showed complete consumption of the starting materials and the formation of the desired compound. The reaction mixture was diluted with water (50 mL), adjusted to pH 8 with 1 N aqueous hydrochloric acid and extracted with EtOAc / THF (2:1, 30 mL × 3). The combined organic layers were washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by Prep-HPLC to give the product N-(cyclopropylmethanesulfonyl)-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-d 3 )pyridine-2-carboxamide Cpd-5 (81.2 mg, yield 19.1%, white solid). LCMS calcd for C 30 H 32 D 3 F 4 N 6 O 4 S[M+H] + : m / z = 654.3; found: 654.7; 1 H NMR (400 MHz, DMSO-d 6)δ 10.86 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.77 (t, J = 6.0 Hz, 1H), 6.51 (t, J = 7.2 Hz, 1H), 5.86 (d, J = 7.2 Hz, 1H), 5.55 (d, J = 8.0 Hz, 1H), 4.83 (d, J = 49.2 Hz, 1H), 4.28 (d, J = 6.0 Hz, 2H), 3.98 - 3.90 (m, 2H), 3.62 - 3.48 (m, 1H), 3.43 (d, J = 7.2 Hz, 2H), 3.08 - 3.03 (m, 1H), 2.86 - 2.82 (m, 1H), 2.33 - 2.21 (m, 1H), 2.23 (s, 3H), 2.15 - 2.09 (m, 1H), 2.03 - 1.93 (m, 1H), 1.72 - 1.66 (m, 1H), 1.09 - 1.00 (m, 1H), 0.59 - 0.54 (m, 2H), 0.34 - 0.30 (m, 2H).

[0227] Example 6: N - ((2,2,2 - trifluoroethyl)sulfonyl)-5 - ((3-(8 - (((3S,4R)-3 - fluoro - 1 - methylpiperidin - 4 - yl)amino)-3-(2,2,2 - trifluoroethyl)indolizin - 2 - yl)prop - 2 - yn - 1 - yl)amino)-6-(methoxy - d 3 )pyridine - 2 - carboxamide (Cpd - 6, i.e., Compound 6)

[0228]

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

[0230] At room temperature, 2,2,2 - trifluoroethane - 1 - sulfonyl chloride Cpd - 6 - 1 (750 mg, 4.11 mmol) was added dropwise to a solution of 2,4 - dimethoxybenzylamine (1.37 g, 8.22 mmol) in THF (20 mL). The system was stirred for 1 hour. LCMS detection showed that the raw materials were completely consumed and the desired compound was detected. The reaction solution was concentrated under reduced pressure, and the residue obtained was purified by SGC (0 - 1% MeOH in DCM) to obtain N-(2,4 - dimethoxybenzyl)-2,2,2 - trifluoroethane - 1 - sulfonamide Cpd - 6 - 2 (1.20 g, yield 93.2%, white solid). LCMS calculated value C 11 H 14 F 3 NNaO 4 S[M + Na] +: m / z = 336.1; Detected value: 336.1.

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

[0232] Dissolve N-(2,4-dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide Cpd-6-2 (1.45 g, 4.63 mmol) in DCM (15 mL), slowly add trifluoroacetic acid (15 mL), and stir the system at room temperature for 1 hour. LCMS detection shows that the raw materials are completely consumed, and the desired compound is detected. The reaction solution is concentrated under reduced pressure to obtain 2,2,2-trifluoroethane-1-sulfonamide Cpd-6-3 (851 mg, yield 99.1%, purple solid). The crude product is directly used for the next reaction. 1 1H NMR (400 MHz, DMSO-d 6 ) δ 7.50 (s, 2H), 4.26 (q, J = 10.0 Hz, 2H).

[0233] Step 3: 5-((3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 ) methyl pyridine-2-carboxylate

[0234] Mix 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-al Int-2 (700 mg, 2.33 mmol), 5-amino-6-(methoxy-d 3 ) methyl pyridine-2-carboxylate Cpd-2-3 (432 mg, 2.33 mmol) and acetic acid (280 mg, 4.67 mmol) in MeOH (20 mL), and stir the mixture at room temperature for 16 hours. Then add NaBH 3 CN (440 mg, 7.00 mmol), and continue to stir the system at room temperature for 2 hours. LCMS detection shows that the raw materials are completely consumed, and the desired compound is detected. Dilute the reaction mixture with water (40 mL) and extract with DCM (100 mL × 3). Combine the organic layers, wash with water (50 mL) and saturated brine (50 mL), dry over anhydrous Na 2 SO 4 , filter and concentrate under reduced pressure. The residue is purified by SGC (0 - 70% EtOAc in PE) to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 ) methyl pyridine-2-carboxylate Cpd-6-4 (625 mg, yield 53.7%, yellow solid). LCMS calculated value C 21 1H15 D 3 BrF 3 N 3 O 3 [M+H] + : m / z = 499.1 / 501.1; Detected value: 499.0 / 501.0.

[0235] Step 4: 5 - ((3 - (8 - Bromo - 3 - (2,2,2 - trifluoroethyl) indolizin - 2 - yl) prop - 2 - ynyl) amino) - 6 - (methoxy - d 3 ) pyridine - 2 - carboxylic acid

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

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

[0238] At 0 °C, add triethylamine (0.21 mL, 1.48 mmol) to 5 - ((3 - (8 - bromo - 3 - (2,2,2 - trifluoroethyl) indolizin - 2 - yl) prop - 2 - ynyl) amino) - 6 - (methoxy - d 3) In a mixed system of pyridine-2-carboxylic acid Cpd-6-5 (240 mg, 490 μmol), 2,2,2-trifluoroethane-1-sulfonamide Cpd-6-3 (242 mg, 1.48 mmol) and 2-chloro-1-methylpyridinium iodide (316 mg, 1.24 mmol) in DCM (40 mL). The reaction mixture was stirred at 0 °C for 30 minutes. LCMS detection showed complete consumption of the starting materials and the desired compound was detected. The reaction mixture was diluted with DCM (100 mL) and washed successively with 0.5 N aqueous hydrochloric acid (50 mL), saturated aqueous sodium bicarbonate (50 mL) and saturated brine (50 mL), and anhydrous Na 2 SO 4 dried, 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-d 3 )-N-((2,2,2-trifluoroethyl)sulfonyl)pyridine-2-carboxamide Cpd-6-6 (160 mg, yield 51.3%, white solid). LCMS calculated value C 22 H 15 D 3 BrF 6 N 4 O 4 S[M+H] + : m / z = 630.0 / 632.0; found: 629.9 / 631.9.

[0239] Step 6: N-((2,2,2-trifluoroethyl)sulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )pyridine-2-carboxamide

[0240] Under argon protection, 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3)-N-((2,2,2-Trifluoroethyl)sulfonyl)picolylamide 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 a mixture of THF (13 mL) and NMP (1.3 mL) were stirred in a sealed tube at 100 °C for 4 h. LCMS analysis showed complete consumption of the starting materials and the detection of the desired compound. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with water (40 mL) and brine (40 mL), dried over anhydrous Na 2 SO 4 and 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-d 3 )picolylamide Cpd-6 (14.0 mg, yield 8.09%, white solid). LCMS calculated for C 28 H 27 D 3 F 7 N 6 O 4 S[M + H] + : m / z = 682.2; found: 682.5; 1 HNMR (400 MHz, DMSO-d 6 ) δ 10.53 (s, 1H), 7.64 (dd, J = 16.0, 6.9 Hz, 2H), 7.01 (d, J = 7.5 Hz, 1H), 6.93 (s, 1H), 6.53 (t, J = 6.4 Hz, 1H), 6.50 - 6.33 (m, 1H), 5.90 (d, J = 7.1 Hz, 1H), 5.81 (d, J = 6.4 Hz, 1H), 5.09 (d, J = 47.1 Hz, 1H), 4.70 - 4.33 (m, 2H), 4.26 (d, J = 4.2 Hz, 2H), 3.97 (d, J = 10.4 Hz, 2H), 3.89 - 3.76 (m, 1H), 2.73 (s, 3H), 2.49 - 2.45 (m, 2H), 2.26 - 2.16 (m, 1H), 1.97 - 1.88 (m, 1H).

[0241] Example 7: N-(Cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxypyridine-2-carboxamide (Cpd-7, i.e., Compound 7)

[0242]

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

[0244] At room temperature, 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) were successively added cyclopropylsulfonamide (3.59 g, 29.6 mmol) and triethylamine (7.49 g, 74.1 mmol). The reaction mixture was stirred at 40 °C for 3 hours. LCMS detection showed complete consumption of the starting materials 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 the pH was adjusted to 2 with 1N aqueous hydrochloric acid. The precipitated solid was filtered, washed with water and dried to obtain 6-chloro-N-(cyclopropylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-7-1 (8.90 g, crude, white solid). The crude product was directly used in the next step. LCMS calculated value C 9 H 7 ClN 3 O 5 S[M-H] - : m / z = 304.0 / 306.0; detected value: 303.9 / 305.9.

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

[0246] At room temperature, a solution of sodium methoxide in methanol (16.2 mL, 87.3 mmol, 5.4 mol / L) was added dropwise 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). The reaction solution was stirred at room temperature for 30 minutes. LCMS detection showed complete consumption of the starting materials and the detection of the desired compound. 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 obtain N-(cyclopropylsulfonyl)-6-methoxy-5-nitropyridine-2-carboxamide Cpd-7-2 (6.70 g, crude, white solid). The crude product was directly used in the next step. LCMS calculated value C10 H 10 N 3 O 6 S[M-H] - : m / z = 300.0; Detected value: 300.0.

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

[0248] At room temperature, N-(cyclopropylsulfonyl)-6-methoxy-5-nitropyridine-2-carboxamide Cpd-7-2 (6.70 g, 22.2 mmol) and palladium on carbon (3.30 g, 5% content) were stirred in a mixed system of THF (200 mL) and MeOH (130 mL) under a hydrogen atmosphere at room temperature for 16 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain 5-amino-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide Cpd-7-3 (6.20 g, crude product, white solid). The crude product was directly used in the next reaction. LCMS calculated value C 10 H 12 N 3 O 4 S[M-H] - : m / z = 270.1; Detected value: 270.1.

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

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

[0251] Step 5: N-(Cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxypyridine-2-carboxamide

[0252] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypyridine-2-carboxamide Cpd-7-4 (500 mg, 850 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (226 mg, 1.71 mmol), BrettPhos Pd G3 (77.4 mg, 10.0 μmol) and cesium carbonate (557 mg, 1.71 mmol) in THF (15 mL) and NMP (1.5 mL) was stirred in a sealed tube at 100 °C for 5 h. 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 combined organic layers were washed with water (40 mL) and saturated brine (40 mL), and anhydrous Na 2 SO 4 Dry, filter and concentrate under reduced pressure. The residue was purified by Prep-HPLC to obtain the product N-(cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxypyridine-2-carboxamide Cpd-7 (46.1 mg, yield 8.3%, yellow solid). LCMS calculated value C 29 H 33 F 4 N 6 O 4 S[M+H]+ : m / z = 637.2; Detected value: 637.1; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.83 (s, 1H), 7.68 (d, J = 6.8 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.92 (s, 1H), 6.76 (t, J = 6.0 Hz, 1H), 6.51 (t, J = 7.2 Hz, 1H), 5.86 (d, J = 7.6 Hz, 1H), 5.55 (d, J = 8.4 Hz, 1H), 4.83 (d, J = 48.6 Hz, 1H), 4.28 (d, J = 6.4 Hz, 2H), 4.06 (s, 3H), 4.00 - 3.91 (m, 2H), 3.62 - 3.49 (m, 1H), 3.11 - 3.03 (m, 2H), 2.84 - 2.82 (m, 1H), 2.33 - 2.21 (m, 1H), 2.23 (s, 3H), 2.15 - 2.09 (m, 1H), 2.03 - 1.93 (m, 1H), 1.71 - 1.67 (m, 1H), 1.19 - 1.15 (m, 2H), 1.10 - 1.04 (m, 2H).

[0253] Example 8: N-(Methylsulfonyl)-5-((3-(8-(((3R,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )pyridine-2-carboxamide (Cpd-8, i.e., Compound 8)

[0254]

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

[0256] Under stirring, 2-chloro-1-methylpyridinium iodide (95.0 g, 372 mmol) and methanesulfonamide (35.0 g, 368 mmol) were added to a suspension of 6-chloro-5-nitropyridine-2-carboxylic acid Cpd-1-1 (50.0 g, 247 mmol) and dichloromethane (500 mL). After the system was stirred for 10 minutes, triethylamine (100 g, 988 mmol) was added dropwise. After the addition was complete, the temperature was raised to reflux, and the reaction was continued for about 5 hours. After cooling and concentration, the residue was diluted with water (250 mL) and adjusted to pH = 2 with concentrated hydrochloric acid, and a large amount of yellow solid precipitated. The mixture was stirred at room temperature overnight, filtered, and the filter cake was washed with water (250 mL). The filter cake was dried under vacuum at 50 °C to obtain 6-chloro-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-1 (68.0 g, yield 98.5%, yellow solid). LCMS calculated value C 7 H 5 ClN 3 O 5 S[M-H] - : m / z = 278.0; detected value: 278.1.

[0257] Step 2: 6-(methoxy-d 3 )-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide

[0258] Under argon protection, 6-chloro-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-1 (45.0 g, 161 mmol) and deuterated methanol (17.4 g, 482 mmol) were dissolved in anhydrous N-methylpyrrolidone (450 mL). The temperature of the system was lowered to -15 - 0 °C, and a 1 M solution of potassium tert-butoxide in tetrahydrofuran (338 mL, 338 mmol) was added dropwise. After the addition was complete, the temperature of the system was controlled at -10 - 0 °C and the reaction was carried out for 40 minutes, and then 4 N 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, and a large amount of yellow solid was produced. The mixture was filtered, and the filter cake was washed with water (500 mL) and methyl tert-butyl ether (500 mL). The filter cake was recrystallized from acetonitrile-water to obtain the product 6-(methoxy-d 3 )-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-2 (30.0 g, yield 67.0%, yellow solid). LCMS calculated value C 8 H 7 D 3 N 3 O 6 S[M+H] + : m / z = 279.0; detected value: 279.2.

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

[0260] Mix 6-(methoxy-d 3 )-N-(methylsulfonyl)-5-nitropyridine-2-carboxamide Cpd-8-2 (30.0 g, 108 mmol) with tetrahydrofuran (600 mL) and acetic acid (3.24 g, 53.9 mmol), stir to form a solution, then add methanol (600 mL). After purging with argon, add palladium on carbon (3.00 g, 10% content), purge with argon and then replace with hydrogen, and react at room temperature for 12 hours. Filter through diatomaceous earth, wash the filter cake with tetrahydrofuran, combine the filtrates, and concentrate. Concentrate and dry the residue to obtain the product 5-amino-6-(methoxy-d 3 )-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-8-3 (26.0 g, yield 97.1%, off-white solid), and proceed to the next step without further treatment. LCMS calculated value C 8 H 9 D 3 N 3 O 4 S[M+H] + : m / z = 249.1; detected value: 249.2.

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

[0262] Add 5-amino-6-(methoxy-d 3 )-N-(methylsulfonyl)pyridine-2-carboxamide Cpd-8-3 (7.20 g, 29.0 mmol) and acetic acid (3.14 g, 58.0 mmol) to a mixed solvent of methanol (100 mL) and dichloromethane (100 mL). After dissolving clearly, add Int-2 (10.1 g, 30.5 mmol), stir the system at 10 - 15 °C for 16 hours, and a large amount of turbidity appears in the system. Then add sodium cyanoborohydride (6.00 g, 87.1 mmol), and continue to stir the system for 2 hours. Concentrate the system, add methanol (100 mL), dropwise add water (100 mL) at room temperature, then stir at room temperature for 2 hours, filter, wash the filter cake with water (50 mL) and methyl tert-butyl ether (100 mL), and dry 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-d 3 )pyridine-2-carboxamide Cpd-8-4 (12.9 g, yield 79.0%, off-white solid). LCMS calculated value C21 H 16 D 3 BrF 3 N 4 O 4 S[M+H] + : m / z = 562.0 / 564.0; Detected value: 562.2 / 564.2.

[0263] Step 5: N-(Methylsulfonyl)-5-((3-(8-(((3R,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )pyridine-2-carboxamide

[0264] Under argon protection, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d 3 )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 a mixture of tetrahydrofuran (20 mL) and N,N-dimethylformamide (10 mL) was stirred at 70 °C for 3 hours. The reaction mixture was cooled and concentrated, and the residue was purified by silica gel column chromatography (MeOH:DCM = 1:24) to obtain 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-d 3 )pyridine-2-carboxamide Cpd-8 (830 mg, yield 42.3%, off-white solid). LCMS calculated value C 27 H 28 D 3 F 4 N 6 O 4 S[M+H] + : m / z = 614.2; Detected value: 614.4; 1 H NMR (400 MHz, DMSO-d 6)δ 10.83 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 6.9 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.92 (s, 1H), 6.75 (t, J = 6.1 Hz, 1H), 6.52 (t, J = 7.1 Hz, 1H), 5.86 (d, J = 7.5 Hz, 1H), 5.56 (d, J = 8.4 Hz, 1H), 4.84 (d, J = 49.3 Hz, 1H), 4.28 (d, J = 6.1 Hz, 2H), 3.95 (q, J = 10.8 Hz, 2H), 3.56 (d, J = 28.9 Hz, 1H), 3.07 (t, J = 11.2 Hz, 1H), 2.85 (d, J = 11.1 Hz, 1H), 2.40 - 2.09 (m, 5H), 2.05 - 1.91 (m, 1H), 1.69 (dd, J = 13.2, 4.0 Hz, 1H).

[0265] Example 9: N-(Methylsulfonyl)-5-((3-(8-(((3S,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )pyridine-2-carboxamide (Cpd-9, i.e., Compound 9)

[0266]

[0267] Step 1: N-(Methylsulfonyl)-5-((3-(8-(((3S,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )pyridine-2-carboxamide

[0268] Under argon protection, 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d 3) A mixture of 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 a mixture of tetrahydrofuran (20 mL) and N,N-dimethylformamide (10 mL) was stirred at 70 °C for 3 h. The reaction mixture 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-d 3 ) pyridine-2-carboxamide Cpd-9 (500 mg, yield 26.9%, brown solid). LCMS calculated value C 27 H 28 D 3 F 4 N 6 O 4 S[M+H] + : m / z = 614.2; found: 614.4; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.85 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 6.9 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.78 (s, 1H), 6.74 (d, J = 7.8 Hz, 1H), 6.51 (t, J = 7.2 Hz, 1H), 5.86 (d, J = 7.5 Hz, 1H), 5.74 (d, J = 8.3 Hz, 1H), 4.57 (dtd, J = 49.6, 9.3, 4.7 Hz, 1H), 4.28 (d, J = 6.2 Hz, 2H), 3.95 (q, J = 10.7 Hz, 2H), 3.49 (d, J = 4.2 Hz, 1H), 3.10 (p, J = 5.4 Hz, 1H), 2.77 - 2.62 (m, 1H), 2.24 (s, 3H), 2.19 - 1.90 (m, 3H), 1.61 - 1.39 (m, 1H).

[0269] Example 10: N-(methylsulfonyl)-5-((3-(8-(((3R,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3)Pyridine-2-carboxamide (Cpd-10, i.e., Compound 10)

[0270]

[0271] Step 1: N-(Methylsulfonyl)-5-((3-(8-(((3R,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d 3 )Pyridine-2-carboxamide

[0272] Under argon protection, 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d 3 )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 a mixed system of tetrahydrofuran (20 mL) and N,N-dimethylformamide (10 mL) were 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 obtain 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-d 3 )Pyridine-2-carboxamide Cpd-10 (1.12 g, yield 57.0%, brown solid). LCMS calculated value C 27 H 28 D 3 F 4 N 6 O 4 S[M+H] + : m / z = 614.2; detected value: 614.4; 1 H NMR (400 MHz, DMSO-d 6)δ 10.89 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 6.9 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.81 - 6.78 (m, 2H), 6.51 (t, J = 7.1 Hz, 1H), 5.86 (d, J = 7.4 Hz, 1H), 5.74 (d, J = 8.3 Hz, 1H), 4.57 (dtd, J = 49.6, 9.2, 4.8 Hz, 1H), 4.28 (d, J = 6.1 Hz, 2H), 3.95 (q, J = 10.7 Hz, 2H), 3.59 - 3.41 (m, 1H), 3.10 (dt, J = 10.9, 5.7 Hz, 1H), 2.77 - 2.59 (m, 1H), 2.25 (s, 3H), 2.17 - 1.83 (m, 3H), 1.62 - 1.38 (m, 1H).

[0273] Biological evaluation

[0274] I. Biochemical Activity Experiment: Activity Test of the Compound in Promoting the Binding of p53 Y220C Mutant to DNA

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

[0276] Measure the binding of recombinant His-tagged p53 Y220C protein and biotinylated DNA using fluorescence resonance energy transfer (FRET). For the FRET experiment, measure the binding between the p53 mutant and the DNA sequence by detecting the fluorescence of the interaction between the Eu-conjugated anti-His antibody (Cisbio, 61HI2KLA) and the d2-conjugated streptavidin (Cisbo, 610SADLF) bound to the biotinylated DNA molecule. The compound was prepared as a 2 mM stock solution and serially diluted 10-fold at 1:3 in DMSO. Each 200-fold working concentration of the compound was serially diluted to a 4-fold working concentration. Use Echo to transfer 4 μL of the compound solution row by row to a 384-well assay plate, with 2 replicates per column. Add 4 μL of the p53 solution to the assay plate. Add 4 μL of the biotinylated DNA solution to the assay plate, and then add 4 μL of the detection solution (His-Eu antibody and streptavidin-d2) to each well of the assay plate. Incubate the assay plate overnight in the dark. Read the fluorescence on a BMG (BMG LRBTECH).

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

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

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

[0280] Percentage of activity = R_data / Ave_VC * 100.

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

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

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

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

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

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

[0287] VC: DMSO-treated group.

[0288] The activity percentage and the logarithm of the compound concentration were fitted to a non-linear regression (dose-response - variable slope) using Graphpad 8.0, and the SC was calculated. 150 。

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

[0290]

[0291]

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

[0293]

[0294] The above data show that the compounds of the embodiments of the present invention have good activity in promoting the binding of p53 Y220C mutant to DNA, and are significantly superior to the reference compounds.

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

[0296] According to the activity of dehydrogenases in cells, the anti-proliferation effect of the compounds on the human gastric adenocarcinoma cell line was evaluated, and this activity has a good correlation with the number of live cells. The cell line NUGC-3 (JCRB, JCRB0822) was cultured in RPMI 1640 (Invitrogen, 11875-085) medium supplemented with 10% (v / v) fetal bovine serum (BI, 04-002-1A). The cell line was cultured according to the standard instructions of the American Type Culture Collection. The cell line was identified by short tandem repeat profiling.

[0297] In the proliferation experiment, 1000 NUGC-3 cells were seeded in a 96-well flat-bottom transparent TC-treated plate (Corning, 3903) with 200 μL of medium per well and cultured overnight in the medium to recover. The compounds were added at 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 live cells was measured by adding Cell Counting Kit-8 reagent (MCE, HY-K0301). The absorbance was measured on an Enspire (Perkin elme). The relative survival rate of each group was expressed as the percentage change relative to the positive control group, and then fitted to a four-parameter logit non-linear curve using the program XLFit (IDBS).

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

[0299]

[0300]

[0301] The above data show that the compound of the embodiment of the present invention has good inhibitory activity against the proliferation of NUGC-3 cells, and is significantly superior to the reference compound.

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

[0303] (1) Inhibitory activity test of CYP enzymes at a single concentration

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

[0305] 2. Experimental instruments and reagents:

[0306] Liver microsomes:

[0307] Species Gender Supplier Article number Batch Storage Human Mix Corning 452117 38298 -80℃

[0308] Reagents:

[0309] Compound Supplier Article number Storage Terfenadine Sigma-Aldrich T9652 4℃ Tolbutamide Sigma-Aldrich T0891 4℃ Dipotassium hydrogen phosphate Sinopharm Group 20032117 Room temperature Reduced nicotinamide adenine dinucleotide phosphate Shanghai Aoyi Biotechnology Co., Ltd. N99640-100MG -20℃ Phenacetin Sigma-Aldrich 101690303 Room temperature Diclofenac Sigma-Aldrich D6899-10G Room temperature Mephenytoin Glpbio GC14486 -20℃ Dextromethorphan Sigma-Aldrich D9684-5G Room temperature Midazolam National Institutes for Food and Drug Control PVJT-0H9Z Room temperature Testosterone Adamas Reagents 171265 Room temperature Bupropion Cayman Chemical 10488 -20℃ Amodiaquine Abmole M5412 -20℃ β-Naphthoflavone Sigma-Aldrich N5757-1G 4℃ Sulfaphenazole Sigma-Aldrich S0758-1G 4℃ Benzylnirvanol Shanghai Yuanye Biotechnology Co., Ltd. Y43177-5mg Room temperature Quinidine Internation-Laboratory-USA 1311468-5G Room temperature Ketoconazole Sigma-Aldrich K1003-100MG 4℃

[0310] 3. Experimental method:

[0311] Dissolve 8.71 g of K 2 HPO 4 in 950 mL of water. Adjust the pH to 7.4 with HCl solution. Adjust the volume to 1000 mL with water. Filter the buffer through a 0.45 μm filter. Store in a 4 °C refrigerator for future use. Prepare a stock solution of terfenadine / tolbutamide (1 mg / mL each) with DMSO. Dilute the above stock solution with acetonitrile to make a quenching solution containing 5 / 10 ng / mL (terfenadine / tolbutamide).

[0312] Positive control working solution:

[0313]

[0314] Substrate working solution:

[0315]

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

[0317] Add 238.5 μL of liver microsome working solution to a 1.1 mL tube. Add 1.5 μL of test compound / control working solution / DMSO, and mix well by pipetting several times. Incubate in a shaking water bath at 37 °C for 5 minutes. After pre-incubation, add 60 μL of NADPH working solution, and mix well 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.

[0318] 4. Data processing method and results:

[0319] Calculate the percentage of inhibition (% inhibition) by comparing the metabolite formation of the test compound or control compound with the matrix control.

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

[0321]

[0322]

[0323] * Final concentration of compound is 10 μM; ND: Not detected; NI: No inhibition

[0324] The above data show that the compounds in the examples of the present invention have weak inhibitory activities against CYP series enzymes, and the risk of drug-drug interaction is significantly lower than that of the reference compound.

[0325] (2) Determination of multi-concentration inhibition curve of CYP3A4 / 5 enzyme

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

[0327] 2. Experimental instruments and materials:

[0328] Liver microsomes:

[0329] Species Gender Supplier Article number Batch Storage Human Mix Corning 452117 38298 -80℃

[0330] Reagents:

[0331]

[0332] 3. Experimental method:

[0333] Add 8.71 g of K 2 HPO 4Dissolve in 950 mL of water. Adjust the pH to 7.4 with HCl solution. Adjust the volume to 1000 mL with water. Filter the buffer through a 0.45 μm filter. Store in a 4 °C refrigerator for future use. Prepare a stock solution of terfenadine / tolbutamide (1 mg / mL each) with DMSO. Dilute the above stock solution with acetonitrile to prepare a quenching solution containing 5 / 10 ng / mL (terfenadine / tolbutamide).

[0334] Positive control working solution:

[0335]

[0336]

[0337] Substrate working solution:

[0338]

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

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

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

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

[0343] 4. Data processing method and results:

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

[0345] Table 4: Inhibitory activity of compounds on CYP3A4 / 5 enzyme

[0346]

[0347] ND: Not detected

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

[0349] IV. In vitro metabolic stability evaluation: Liver microsome metabolic stability study:

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

[0351] 2. Experimental reagents and materials:

[0352] Reagents:

[0353]

[0354]

[0355] Materials:

[0356] Name of experimental material Gender Manufacturer Article number Storage conditions Human liver microsomes Mixed male and female Corning 452117 -80℃ Rat liver microsomes Male BioreclamationIVT M00001 -80℃ Mouse liver microsomes Male BioreclamationIVT M00501 -80℃

[0357] 3. Experimental method:

[0358] The test compound and the positive control compound (dextromethorphan as the control compound) were separately dissolved in DMSO to prepare stock solutions with a concentration of 10 mM, and the above stock solutions were diluted with DMSO to 200 μM working solutions. 8.709 g of dipotassium hydrogen phosphate (K 2 HPO 4 ) was dissolved in 950 mL of water, the pH value of the solution was adjusted to 7.4 with hydrochloric acid, and then the volume was made up to 1000 mL with water. After filtration through a 0.22 μm filter membrane, it was stored in a refrigerator at 4 °C for standby. After thawing the liver microsomes of various species (protein concentration 20 mg / mL) in a water bath at 37 °C, the liver microsome working solutions with a protein concentration of 0.629 mg / mL were respectively diluted with phosphate buffer solution. A 5 mM NADPH solution was prepared with the above phosphate buffer solution for standby. A stock solution of 1 mg / mL terfenadine / tolbutamide was prepared with DMSO, and then diluted with a mixed solution of 50% methanol / 50% acetonitrile to prepare a reaction termination solution containing 5 / 10 ng / mL (terfenadine / tolbutamide) internal standard.

[0359] Add 238.5 μL of liver microsome working solutions of different species into 1.1 mL microtubes, and then add 1.5 μL of the test compound working solution or the positive control compound (dextromethorphan) working solution (200 μM). After mixing, pre-incubate in a 37 °C water bath for 5 min. Add 60 μL of NADPH solution to initiate the reaction. After thorough mixing, at the 0, 5, 15, 30, and 60-minute time points after the reaction, transfer 30 μL of the reaction solution to 300 μL of the reaction termination solution respectively. Vortex all the samples vigorously for 1 minute and then centrifuge at 4000 rpm at 4 °C for 15 minutes. Take 100 μL of the supernatant, add 100 μL of pure water, mix evenly, and then perform LC-MS / MS analysis.

[0360] 4. Data processing method:

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

[0362] The calculation of the remaining rate of the compound is as follows:

[0363]

[0364] The intrinsic clearance rate CL int (μL / min / mg protein) = 0.693 * 1000 / T 1 / 2 / protein concentration (0.5 mg protein / mL)

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

[0366]

[0367]

[0368] The above data show that the compound in the examples of the present invention has high metabolic stability in liver microsomes of multiple species and is significantly better than the reference compound.

[0369] V. In vitro metabolic stability evaluation: Hepatocyte metabolic stability test

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

[0371] 2. Experimental materials:

[0372] Species Strain Gender Supplier Human N / A Mix BioIVT Rat Sprague Dawley Male BioIVT Mouse ICR / CD-1 Male BioIVT

[0373] 3 Test design

[0374] 3.1 Preparation of compound working solution

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

[0376] 3.2 Preparation of hepatocytes

[0377] 1) Mix 49.5 mL of Williams’ Medium E and 0.5 mL of GlutaMAX as the incubation solution. The hepatocyte resuscitation solution and the incubation solution were preheated in a 37 °C water bath for at least 15 minutes before use.

[0378] 2) Take a tube of cryopreserved hepatocytes and ensure that the hepatocytes are still in a cryofrozen state before resuscitation. The hepatocytes were quickly placed in a 37 °C water bath and gently shaken until all ice crystals were completely dispersed, then sprayed with 70% ethanol and transferred to a biosafety cabinet.

[0379] 3) Pour the contents of the hepatocyte tube into a centrifuge tube containing 50 mL of resuscitation medium, and centrifuge it at 100 g for 10 minutes. After centrifugation, aspirate the resuscitation medium and add sufficient incubation medium to obtain a cell suspension with a cell density of approximately 1.5×10

[0380] 4) Use Cellometer Vision to count the hepatocytes and determine the viable cell density. The viability of the hepatocytes must be greater than 75%. Dilute the hepatocyte suspension with the incubation medium to a viable cell density of 0.5×10 6 cells / mL.

[0381] 5) Boil a part of the hepatocyte suspension with a density of 0.5×10 6 cells / mL in boiling water for 5 minutes as a negative control. After cell inactivation, it is convenient to examine the substrate conversion mediated by non-cellular enzyme systems.

[0382] 5) Transfer 198 μL of the suspension of viable or inactivated cells to a 96-well deep-well plate, and place the deep-well plate on a vortex and preheat it in an incubator for 10 minutes. Viable cells were incubated in duplicate, and inactivated cells were incubated in single. 6 6) 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 vortex.

[0383] 3.3 Test method

[0384] 1) Transfer 198 μL of the suspension of viable or inactivated cells to a 96-well deep-well plate, and place the deep-well plate on a vortex and preheat it in an incubator for 10 minutes. Viable cells were incubated in duplicate, and inactivated cells were incubated in single.

[0385] 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 vortex.

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

[0387] 4 Data analysis

[0388] All data calculations were performed using Microsoft Excel software. The peak area was detected by extracting the ion chromatogram. By linearly fitting the natural logarithm of the percentage of the parent drug eliminated against time, the in vitro half-life (t 1 / 2 ) of the parent drug was detected.

[0389] The in vitro half-life (t 1 / 2 ) was calculated from the slope:

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

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

[0392] in vitro CLint = kV / N

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

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

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

[0396]

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

[0398] VI. Cardiac safety evaluation: Inhibition activity test of hERG potassium channels

[0399] 1. Experimental purpose:

[0400] Use the manual patch clamp technique to evaluate whether the test compound has a potential inhibitory effect on the voltage-gated potassium channel hERG. By detecting the effects of 5 concentrations of the compound on the hERG channel current, the dose-effect curve of the compound was obtained and the IC50 。

[0401] 2. Experimental instruments and reagents:

[0402]

[0403] 3. Experimental methods:

[0404] Cell line and cell culture: The HEK293 cell line stably expressing the hERG ion channel (product number: K1236) was purchased from Invitrogen. This cell line was 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 increased to 40% - 80% of the bottom area of the culture dish, it was digested with trypsin and passaged three times a week. Before the experiment, the cells were cultured in 3.5 cm culture dishes at a density of 5×10 5 , induced with 1 μg / mL doxycycline for 48 hours, and then the cells were digested and seeded on slides for subsequent manual patch clamp experiments.

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

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

[0407] Experimental procedure: Place the coverslip with HEK293 cells in the Petri dish into the perfusion chamber of the micromanipulation stage. Under an Olympus IX71 or IX73 inverted microscope, position the appropriate cells in the center of the field of view. Use a 10× objective lens to find the tip of the glass electrode and place 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. When approaching the cell, switch to a 40× objective lens for observation. Use the fine adjustment of the micromanipulator to gradually approach the cell surface. Apply negative pressure to form a seal with a resistance higher than 1 GΩ between the electrode tip and the cell membrane. Compensate for the instantaneous capacitive current Cfast in the voltage-clamp mode. Then repeat applying short negative pressures to rupture the membrane and finally form the whole-cell recording mode. Under the condition that the membrane potential is clamped at -60 mV, compensate for the slow capacitive current Cslow, the cell membrane capacitance (Cm), and the input membrane resistance (Ra) respectively. After the cell is stable, change the clamping voltage to -90 mV, set the sampling frequency to 20 kHz, and the filtering frequency to 10 kHz. The detection condition for the leakage current is that the clamping voltage is switched to -80 mV for a duration of 500 ms. The hERG current test method is as follows: Apply a 4.8-second depolarizing command voltage to depolarize the membrane potential from -80 mV to +30 mV, and then instantaneously apply a 5.2-second repolarizing voltage to lower the membrane potential to -50 mV to remove channel inactivation, so as to observe the hERG tail current. The peak value of the tail current is the magnitude of the hERG current. The hERG current used to detect the test compound is continuously recorded for 120 seconds before drug administration to evaluate 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 detection. Test for the inhibitory effect of the test compound on the hERG current: First, use the hERG current measured in the extracellular solution containing 0.1% DMSO as the detection baseline. After the hERG current remains stable for at least 5 minutes, sequentially perfuse the solutions containing the test compound around the cell from low concentration to high concentration. Wait about 5 minutes after each perfusion to allow the compound to fully act on the cell and synchronously record the hERG current. After the recorded current tends to be stable, record the last 5 hERG current values and take their average as the final current value at a specific concentration. After testing the compound, add 450 nM dofetilide to the same cell to completely inhibit its current as the positive control for this cell. At the same time, the positive compound dofetilide is synchronously detected using the same patch-clamp system before and after the test of the test drug to ensure the reliability and sensitivity of the entire detection system. The above test steps will be repeated on two separate test cells (n = 2).

[0408] 4. Data processing method and results:

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

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

[0411] Note: The data is output by the PatchMaster software.

[0412] After perfusing the blank solvent or the compound gradient solution, the average value of 5 consecutive current values obtained stably is taken as the "tail current magnitude 空白 " and the "tail current magnitude 化合物 ".

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

[0414]

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

[0416] Table 7: Inhibitory activity of the compound on hERG

[0417] Compound number <![CDATA[hERG IC 50 (μM) <!-- 45 -->]]> Cpd-A 19 Cpd-B 5.5 Cpd-C 1.4 Cpd-1 >30 Cpd-2 >30

[0418] The above data show that the compounds in the embodiments of the present invention have a relatively low risk of potential cardiotoxicity caused by hERG inhibition, and the safety is significantly better than that of the reference compound.

[0419] VII. Pharmacokinetics study in mice

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

[0421] 2. Experimental instruments and materials:

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

[0423] Instruments:

[0424]

[0425] Reagents:

[0426]

[0427]

[0428] Solvent:

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

[0430] 3. Experimental method:

[0431] Six male mice. Fast the mice in the evening one day before drug administration, and allow free access to water. During the experiment, the mice were allowed free access to food and water. The drugs were administered by intravenous injection or gavage. After drug administration, the animal status was observed and abnormal manifestations were recorded. Blood samples were collected from the orbital sinus at 0.0833, 0.25, 1, 2, 4, 8, and 24 hours after intravenous injection; blood samples were collected from the orbital sinus at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after gavage. Take 20 μL of plasma sample, add 250 μL of acetonitrile (containing dexamethasone as internal standard) to precipitate proteins, centrifuge at 4000 rpm for 20 minutes at 4 °C, take 180 μL of the supernatant and mix it with 180 μL of aqueous solution containing 0.1% formic acid in a 96-well plate, and then take 10 μL of the sample for LC-MS / MS detection.

[0432] 4. Data processing method and results:

[0433] The standard curve was established by the internal standard method, with the theoretical standard curve concentration as the abscissa and the peak area ratio as the ordinate (test compound peak area / internal standard peak area). Using the linear regression method (weighting factor 1 / X2), R 2 > 0.9900. The unknown samples were calculated through the standard curve. The pharmacokinetic parameters were calculated by the non-compartmental analysis model of WinNonlin 8.2 software and presented in the report. The parameters include Clint, C max and AUC, etc.

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

[0435]

[0436] The above data show that the compound in the embodiment of the present invention has a lower clearance rate and a higher oral exposure in mice, and is superior to the reference compound.

[0437] VIII. Pharmacokinetic study in rats

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

[0439] 2. Experimental instruments and materials:

[0440] Animals: SD rats (male)

[0441] Instrument:

[0442]

[0443] Reagent:

[0444]

[0445] Solvent:

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

[0447] 3. Experimental method:

[0448] Six male rats. Fast the rats in the evening one day before drug administration, and allow free access to water. During the experiment, the rats were allowed free access to food and water. Intravenous injection or gavage was used for drug administration, and the animal status was observed and abnormal manifestations were recorded after drug administration. Blood samples were collected from the jugular vein at 0.0833, 0.25, 1, 2, 4, 8, and 24 hours after intravenous injection; blood samples were collected from the jugular vein at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after gavage. Take 50 μL of plasma sample, add 500 μL of acetonitrile (containing dexamethasone as internal standard) to precipitate proteins, centrifuge at 4°C and 4000 rpm for 20 minutes, take 300 μL of the supernatant and mix it with 300 μL of Watson's aqueous solution in a 96-well plate, and then take 5 μL of the sample for LC-MS / MS detection.

[0449] 4. Data processing method and results:

[0450] The standard curve was established by the internal standard method, with the theoretical standard curve concentration as the abscissa and the peak area ratio as the ordinate (test compound peak area / internal standard peak area). Using the linear regression method (weighting factor 1 / X2), R 2 > 0.9900. Calculate the unknown samples through the standard curve. Calculate the pharmacokinetic parameters by the non-compartmental analysis model of WinNonlin 8.2 software and present them in the report. The parameters include Clint, C max and AUC, etc.

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

[0452]

[0453] The above data show that the compound in the embodiment of the present invention has a low clearance rate and a high oral exposure in rats, and is significantly better than the reference compound.

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

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

[0456] 2. Experimental materials:

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

[0458] Reagents:

[0459]

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

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

[0462] 3. Experimental protocol:

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

[0464] Model establishment and grouping: NUGC-3 cells in the exponential growth phase (5×10 6 ) were resuspended in 0.2 mL PBS and Matrigel (1:1) and used for subcutaneous inoculation on the right side of NUNU mice to establish a subcutaneous xenograft model. The health status of the mice and the tumor growth were observed daily after cell inoculation. On the 5th day after inoculation, 36 animals were selected and grouped. The tumor volume of the animals included in the group 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).

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

[0466] Table 10. In Vivo Pharmacodynamic Experiment Design of NUGC-3 Xenograft Tumor Mouse Model

[0467]

[0468]

[0469] 5. Experimental Observation and Result Judgment:

[0470] Experimental Observation: Monitor the health status and death of animals every day. Routine examinations include observing tumor growth and the effects of drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake, body weight changes, appearance signs, or other abnormal conditions. Record the number of animal deaths and side effects within each group based on the number of animals in each group. All clinical symptoms observed during the experiment are recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 ). During the research process, the entire drug administration process, as well as tumor and body weight measurements, are carried out in a laminar flow hood.

[0471] Data Processing: Tumor volume inhibition rate TGI (%): TGI% = (1 - △T / △C) × 100%; where, △C is the tumor volume C of the control group t -C 0 , C 0 is the average tumor volume of the control group at the time of grouping, C t is the average tumor volume of the control group after treatment; △T is the tumor volume T of the treatment group t -T 0 , T0 is the average tumor volume of the treatment group at the time of grouping, T t is the average tumor volume of the treatment group after treatment.

[0472] Statistical Analysis: The results of tumor volume and animal body weight are expressed as Mean (mean value) ± SEM (standard error of the mean). Statistical comparison analysis is performed on the tumor volumes between different groups. All statistical analyses are completed in GraphPad Prism 8.0. The one-way ANOVA method is used to compare whether there are significant differences in tumor volume or tumor weight between groups. Among them, p ≥ 0.05 indicates no significant difference, p < 0.05 indicates a significant difference, and p < 0.001 indicates a highly significant difference.

[0473] 6. Experimental Results:

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

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

[0476] Table 11. In vivo pharmacodynamic experimental results of the NUGC-3 xenograft mouse model

[0477]

[0478]

[0479] Remarks:

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

[0481] b. p-values from the comparative analysis of the mean tumor volume of each dosing group and the mean tumor volume of Vehicle in Group G1

[0482] X. In Vivo Pharmacodynamic Experiments on a Rat Model of NUGC-3 Human Gastric Cancer Xenograft Tumors

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

[0484] 2. Experimental Materials:

[0485] Animals: Female B-Rag2 / IL2rg KO SD rats, 7-9 weeks old;

[0486] Reagents:

[0487]

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

[0489] 3. Experimental Protocol:

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

[0491] Model Establishment and Grouping: NUGC-3 cells in the exponential growth phase (1×10 7 ) were resuspended in 0.2 mL PBS and Matrigel (1:1) and used for subcutaneous inoculation on the right side of B-Rag2 / IL2rg KO SD rats to establish a subcutaneous xenograft tumor model. The health status of the rats and the tumor growth were observed daily after cell inoculation. On the 10th day after inoculation, 24 animals were selected and grouped. The tumor volume of the animals included in the group was approximately 199.0 mm 3 . The experiment was divided into 4 groups, with 6 rats in each group, and dosing began (see Table 12).

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

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

[0494]

[0495] 5. Experimental observation and result judgment:

[0496] Experimental observation: Monitor the health status and death of animals every day. Routine examinations include observing tumor growth and the effects of drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake, body weight changes, appearance signs, or other abnormal conditions. Record the number of animal deaths and side effects within each group based on the number of animals in each group. All clinical symptoms observed during the experiment are recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 ). During the research process, the entire drug administration process, as well as tumor and body weight measurements, are carried out in a laminar flow hood.

[0497] Data processing: Tumor volume inhibition rate TGI TV (%) : TGI TV % = (1 - △T / △C) × 100%; where △C is the tumor volume C t -C 0 of the control group, C 0 is the average tumor volume of the control group at the time of grouping, C t is the average tumor volume of the control group after treatment; △T is the tumor volume T t -T 0 of the treatment group, T0 is the average tumor volume of the treatment group at the time of grouping, and T t is the average tumor volume of the treatment group after treatment.

[0498] Statistical analysis: The results of tumor volume and animal body weight are expressed as Mean (mean) ± SEM (standard error of the mean). Statistical comparison and analysis of tumor volumes between different groups are carried out. All statistical analyses are completed at https: / / d2k.bio / Efficacy / Invivo, and the wilcox.test nominal method is used to compare whether there are significant differences in tumor volume or tumor weight between groups. Among them, p ≥ 0.05 indicates no significant difference, p < 0.05 indicates a significant difference, and p < 0.01 indicates a highly significant difference.

[0499] 6. Experimental results:

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

[0501] On the 28th day after grouping, the average tumor volume of the G1 (Vehicle) group was 2946.2 mm 3 , and 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 , 119.2 mm 3 , and 70.1 mm 3 , respectively. The corresponding tumor volume inhibition rates TGI were 92.9%, 102.9%, and 104.7%, respectively. Statistical analysis showed that there were extremely significant differences in the average tumor volume between the G2, G3, and G4 groups and the Vehicle group (p values were all < 0.01). The above results indicate that Cpd-2 showed good activity in inhibiting the proliferation of NUGC-3 xenografts in rats at each tested dose, and there was a dose-dependence.

[0502] Table 13. In vivo pharmacodynamic experimental results of the NUGC-3 xenograft tumor rat model

[0503]

[0504]

[0505] Remarks:

[0506] p values of the comparative analysis of the average tumor volume of each drug administration group and the average tumor volume of Vehicle in the G1 group.

Claims

1. A nitrogen-containing compound as shown in Formula Ia or a pharmaceutically acceptable salt thereof; 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.

2. The nitrogen-containing compound of formula Ia or a pharmaceutically acceptable salt thereof according to claim 1, 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.

3. The nitrogen-containing compound of formula Ia or a pharmaceutically acceptable salt thereof according to claim 1, 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.

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

5. The nitrogen-containing compound of formula Ia or a pharmaceutically acceptable salt thereof according to claim 1, 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.

6. The nitrogen-containing compound of formula Ia or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: 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.

7. The nitrogen-containing compound of formula Ia or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: 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. The nitrogen-containing compound of formula Ia or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The nitrogen-containing compound as shown in Formula Ia is selected from any of the following compounds:

9. A pharmaceutical composition comprising a nitrogen-containing compound as represented by formula Ia or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 8 and a pharmaceutical excipient.

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

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

12. The use according to claim 11, characterized in that It meets one or both of the following conditions: (1) The p53 mutant has a p53 Y220C mutation at amino acid 220; And the disease associated with p53 mutant described in (2) is breast cancer, gastric cancer, lung cancer or ovarian cancer.

13. Use of the nitrogen-containing compound of formula Ia according to any one of claims 1 to 8, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating and / or preventing cancer; The cancer is breast cancer, gastric cancer, lung cancer or ovarian cancer.

14. A compound represented by formula a-11 or a-33; in, X is halogen; R 8 is an amino protecting group; 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.

15. The compound represented by formula a-11 or a-33 according to claim 14, characterized in that: The compound represented by formula a-11 or a-33 is any of the following compounds;

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