Molten heterocyclic compound containing nitrogen (nsulfonamide), and its application

Description

MOLTEN HETEROCYCLIC COMPOUND CONTAINING NITROGEN NSULFONAMIDE AND ITS APPLICATION SPECIFICATION

[0001] This application claims priority rights over Chinese patent application 2023113669427 filed on October 20, 2023, Chinese patent application 2023117838674 filed on December 22, 2023, Chinese patent application 2024102622351 filed on March 7, 2024, and Chinese patent application 2024112753956 filed on September 11, 2024. The content of the above Chinese patent applications is incorporated herein by reference in its entirety. TECHNICAL AREA

[0002] This disclosure pertains to the pharmaceutical field, in addition to the pharmaceutical field of the binding agent in p53 Y220C pockets with medicamentability capability, and is particularly related to a class of nitrogen-containing N-sulfonamide fused heterocyclic compounds and their use. CONTEXT

[0003] The protein product of the TP53 gene (p53) is currently considered one of the most important tumor suppressor factors. Unlike RB1, CDKN2A, and PTEN, which are lost in tumors by homozygous deletion, TP53 frequently exhibits somatic missense mutations (Alexandrova EM et al. Loss of heterozygosity of p53 is a necessary prerequisite for stabilization and gain of function of the p53 mutant in vivo. Cell Death Dis, 2017, 8(3): e2661-e2661). Petition 870260067420, dated 08 / 07 / 2026, page 8 / 113 2 / 106 However, to date, therapy targeting p53 mutations has not been validated in clinical treatment (Levine AJ. p53 protein-targeted therapies in cancer treatments. Annu Rev Cancer Biol, 2019, 3: 21-34). Cells with TP53 mutations accumulate high levels of the mutant p53 protein, which exerts dominant-negative effects on wild-type (WT) p53 and its homologous proteins p63 and p73, causing them to lose their normal regulatory functions in 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 indicate that restoring p53 expression could inhibit the growth of lymphomas and sarcomas through apoptosis in different mouse models (Christophorou M. A et al. Temporal dissection of p53 function in vitro and in vivo [J].Nat Genet, 2005, 37(7): 718-726; Martins CP, Brown-Swigart L., Evan GI Modeling the efficacy and effectiveness of p53 restoration in tumors. Cellular. 2006; 127:1323-1334), providing a theoretical basis for leveraging the restoration of normal p53 function in tumor treatment. In addition to increasing wild-type p53 activity in tumors with intact Tp53 genotypes, restoring wild-type protein function in tumors expressing mutant p53 is also a promising strategy for cancer treatment (Yu X et al. Allele-specific mutant p53 reactivation. Cancer Cell, 2012, 21(5): 614-625; Chen S et al. Arsenic trioxide rescues p53 structural mutations through an enigmatic allosteric site. Cancer Cell, 2021, 39(2): 225-239. e8).

[0004] The central domain of the wild-type p53 is unstable, with low stability. Petition 870260067420, dated 08 / 07 / 2026, page 9 / 113 3 / 106 thermodynamic and kinetic, allowing a rapid transformation between folded and unfolded states. Mutation of residues in the central p53 region leads to increased thermodynamic and kinetic instability, causing the central DNA-binding domain to lose DNA-binding activity. Such 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 treatments for the rapid alteration of wild-type and mutant p53 in cancer. Biomolecules. 2022, 12(4):548). It is worth noting that the special Y220C mutation mediates the formation of a superficial pocket away from the central DNA-binding domain (Joerger AC et al. Structural basis for understanding oncogenic p53 mutations and designing salvage drugs. Proc Natl Acad Sci, 2006, 103(41): 15056-15061), making it an ideal target for treating tumors that possess this mutation.Until now, many Y220C pocket-binding agents have been developed to stabilize this p53 mutant and reactivate its transcriptional activity, but with limited efficacy (Boeckler FM, et al. Targeted rescue of a destabilized p53 mutant by a drug screened in silico. Proc Natl Acad Sci, 2008, 105(30): 10360-10365; Guiley K, Z et al. A small molecule reacts with the somatic p53 Y220C mutant to rescue wild-type thermal stability. Cancer Discov, 2022: CD-22). Therefore, for patients with p53 Y220C mutated tumors, there is an urgent need to develop p53 Y220C pocket-binding agents with higher affinity and better treatment capability. SUMMARY

[0005] The technical issue to be resolved by this disclosure is the poor pharmacology of existing p53 Y220C pocket binding agents. For this reason, Petition 870260067420, dated 08 / 07 / 2026, page 10 / 113 4 / 106 This disclosure provides a class of nitrogen-containing N-sulfonamide fused heterocyclic compounds and their use. This class of compounds can promote the binding of the p53 Y220C protein to DNA and possess improved inhibitory activity against the proliferation of cancerous cells, represented by gastric cancer cells.

[0006] The present disclosure provides a nitrogen-containing compound of formula I, a compound labeled with isotopes, a solvate thereof, an acceptable medicinal salt, a solvate of the acceptable medicinal salt, or a prodrug thereof;

[0007] where

[0008] X1, X2 and X3 are selected independently between CH and N;

[0009] R1, R2, R3, R4 and R5 are selected independently from hydrogen and deuterium;

[0010] R6e -(CH2)nR7;

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

[0012] R7 is selected from C1-C6 alkyl, C1-C6 deuterated alkyl, C1-C6 haloalkyl and C3-C6 cycloalkyl.

[0013] As a preferred technical solution, the nitrogen-containing compound of the formula Petition 870260067420, dated 08 / 07 / 2026, page 11 / 113 5 / 106 I, the isotope-labeled compound, its solvate, the acceptable medicinal salt, the solvate of the acceptable pharmaceutical salt, or the prodrug is a nitrogen-containing compound of formula Ia, an isotope-labeled compound, a solvate thereof, an acceptable medicinal salt, a solvate of the acceptable medicinal salt, or a prodrug of that salt;

[0014] where

[0015] Xi, X2 and X3 are selected independently between CH and N;

[0016] R1, R2, R3, R4 and R5 are selected independently from hydrogen and deuterium;

[0017] R6e -(CH2)nR7;

[0018] n is 0, 1, 2 or 3;

[0019] R7 is selected from C1-C6 alkyl, C1-C6 deuterated alkyl, C1-C6 haloalkyl and C3-C6 cycloalkyl.

[0020] As a preferred technical solution, X1 is N; X2 is CH or N; X3 is CH.

[0021] As a preferred technical solution, X1 is N; X2 and X3 are CH.

[0022] As a preferred technical solution, R6 is -(CH2)nR7; n is 0 or 1; R7 is selected from C1-C3 alkyl, deuterated C1-C3 alkyl, C1-C3 haloalkyl and C3-C6 cycloalkyl.

[0023] As a preferred technical solution, R6 is -(CH2)nR7; n is 0 or 1; R7 is selected from -CH3, -CD3, -CF3 and cyclopropyl. Petition 870260067420, dated 08 / 07 / 2026, page 12 / 113 6 / 106

[0024] As a preferred technical solution, R1, R2, R3, R4 and R5 are selected independently from hydrogen and deuterium, and at least one of them is deuterium.

[0025] As a preferred technical solution, R1, R2 and R3 are deuterium; R4 and R5 are hydrogen.

[0026] As a preferred technical solution, R1, R2 and R3 are hydrogen; R4 and R5 are deuterium.

[0027] As a preferred technical solution, formula I is a formula Ib or Ic; Ib Ic THE A“r3A“r3Ar3

[0028] In formulas Ib and Ic, r1 R2 is R1 R2 or R1 r2

[0029] R1, R2, R3, R4 and R5 are selected independently from hydrogen and deuterium;

[0030] R6 is -(CH2)nR7;

[0031] n is 0 or 1;

[0032] R7 is selected from C1-C6 alkyl, C1-C6 haloalkyl and C3-C6 cycloalkyl. L-R2

[0033] Preferably, r1 is selected between -OCH3 and -OCD3;

[0034] R4e R5s are selected from hydrogen;

[0035] R6 is -(CH2)nR7; n is 0 or 1; R7 is selected from -CH3, -CD3, -CF3 and cyclopropyl. Petition 870260067420, dated 08 / 07 / 2026, page 13 / 113 7 / 106

[0036] As a preferred technical solution, the compound of formula I is selected from among the following compounds:

[0037] Petition 870260067420, dated 08 / 07 / 2026, page 14 / 113 8 / 106 , an enantiomer of this type, or a mixture of the two in any ratio (e.g., racemate).

[0038] This disclosure also provides a method of preparation for the compound of formula I, a compound labeled with an isotope, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof. Comprising the following methods:

[0039] Method 1: Petition 870260067420, dated 08 / 07 / 2026, page 15 / 113 9 / 106

[0040] Step 1: Performing a cycloaromatization reaction between compound a-1 and ethyl bromopyruvate to obtain compound a-2; wherein X is selected from Cl, Bre, and I;

[0041] Step 2: Performing the Vilsmeier-Haack reaction on compound a-2 to obtain compound a-3;

[0042] Step 3: Reacting compound a-3 with a fluorinating reagent to obtain compound a-4; wherein the fluorinating reagent may be methyl fluorosulfonyldifluoroacetate, (triphenylphosphonium) difluoroacetate or tetrabutylammonium fluoride;

[0043] Step 4: C Performing a reduction reaction of the ester group on compound a-4 in the presence of a reducing agent to obtain compound a-5; wherein the reducing agent may be sodium borohydride, lithium borohydride, lithium aluminum hydride or diisobutylaluminum hydride; Petition 870260067420, dated 08 / 07 / 2026, page 16 / 113 10 / 106

[0044] Step 5: Oxidizing compound a-5 in the presence of an oxidizing agent to obtain compound a-6; wherein the oxidizing agent may be Dess-Martin periodinane, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide or sulfur trioxide / pyridine;

[0045] Step 6: Reacting compound a-6 with Gilbert's reagent to generate compound a-7;

[0046] Step 7: Reacting compound a-7 with formaldehyde under the catalysis of a metallic reagent to obtain compound a-8; wherein the metallic reagent may be a Cu-containing reagent, such as tetrakis(acetonitrile)copper(I) hexafluorophosphate;

[0047] Step 8: Oxidizing compound a-8 to aldehyde a-9 in the presence of an oxidizing agent; wherein the oxidizing agent may be Dess-Martin periodinane, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide or sulfur trioxide / pyridine;

[0048] Step 9: Performing a reductive amination reaction between compound a-9 and compound a-10 to generate compound a-11; wherein R1, R2, R3, R6, X1, X2 and X3 are defined as above;

[0049] Step 10: Performing a metal reagent-catalyzed coupling reaction between compound a-11 and amine a-12 to obtain compound (Ia); wherein the metal reagent may be a Pd or Cu-containing reagent, such as BrettPhos Pd G3, BrettPhos Pd G4 or cuprous iodide.

[0050] Method 2: Petition 870260067420, dated 08 / 07 / 2026, page 17 / 113 11 / 106

[0051] Step 1: Performing a metal reagent-catalyzed cross-coupling reaction between compound a-11 and amine a-13 to obtain compound a-14; wherein X is selected from Cl, Br and I; R1, R2, R3, R6, X1, X2 and X3 are defined as above; the metal reagent may be a Pd or Cu-containing reagent, such as BrettPhos Pd G3, BrettPhos Pd G4 or cuprous iodide;

[0052] Step 2: Removing the Boc protecting group from compound a-14 under acidic conditions to obtain compound a-15; wherein acidic conditions can be obtained in the presence of inorganic acids (e.g., HCl, HBr or H2SO4), organic acids (e.g., trifluoroacetic acid), Lewis acids (e.g., zinc bromide) or other reagents (e.g., iodotrimethylsilane);

[0053] Step 3: Performing a reductive amination reaction between compound a15 and formaldehyde to generate compound (la).

[0054] Method 3: Petition 870260067420, dated 08 / 07 / 2026, page 18 / 113 12 / 106

[0055] Step 1: Performing a reductive aminoation reaction between compound a-9 and compound a-16 to generate compound a-17; wherein X is selected from Cl, Br and I; Y is selected from C1-C4 alkyl; R1, R2, R3, X1, X2 and X3 are defined as above;

[0056] Step 2: Performing a metal-catalyzed cross-coupling reaction between compound a-17 and amine a-12 to obtain compound a-18; wherein the metal reagent may be a Pd- or Cu-containing reagent, such as BrettPhos Pd G3, BrettPhos Pd G4 or cuprous iodide;

[0057] Step 3: Performing a hydrolysis reaction of compound a-18 to generate compound a-19 under acidic or basic conditions; wherein acidic conditions can be obtained in the presence of inorganic acids (e.g., HCl, HBr or H2SO4) or organic acids (e.g., trifluoroacetic acid); basic conditions can be obtained in the presence of inorganic bases (e.g., LiOH, NaOH or KOH);

[0058] Step 4: Performing a condensation reaction between compound a-19 and sulfonamide H2NS(O)2R6 to generate compound (la); wherein R6 is defined as above.

[0059] Method 4: Petition 870260067420, dated 08 / 07 / 2026, page 19 / 113 13 / 106

[0060] Step 1: Performing a hydroxyl protection reaction between compound a-8 and a suitable protecting agent to generate compound a-20; wherein X is selected from Cl, Br and I; M is a protecting group, such as trimethylsilyl, tert-butyldimethylsilyl, methoxymethyl, tert-butoxycarbonyl or 2-tetrahydropyranyl; the protecting agent may be trimethylchlorosilane, tert-butyl-dimethylchlorosilane, chloromethyl methyl ether, di-tert-butyl dicarbonate or 3,4-dihydro-2H-pyran;

[0061] Step 2: Performing a metal-catalyzed cross-coupling reaction between compound a-20 and amine a-12 to obtain compound a-21; wherein the metal reagent may be a Pd- or Cu-containing reagent, such as BrettPhos Pd G3, BrettPhos Pd G4 or cuprous iodide;

[0062] Step 3: Removing the protecting group from compound a-21 to generate compound a-22 under acidic or basic conditions; wherein acidic conditions can be obtained in the presence of inorganic acids (e.g., HF, HCl, HBr or H2SO4) or organic acids (e.g., trifluoroacetic acid); basic conditions can be obtained in the presence of inorganic bases (e.g., LiOH, NaOH or KOH) or organic bases (e.g., tetrabutylammonium fluoride);

[0063] Step 4: Carrying out an oxidation reaction of compound a-22 in the presence of an oxidizing agent to obtain compound a-23; wherein the oxidizing agent may Petition 870260067420, dated 08 / 07 / 2026, page 20 / 113 14 / 106 Dess-Martin periodinan, Swern oxidation reagent, pyridinium chlorochromate, manganese dioxide or sulfur trioxide / pyridine;

[0064] Step 5: Performing a reductive amination reaction between compound a23 and sulfonamide a-10 to generate compound (la); wherein R1, R2, R3, R6, X1, X2 and X3 are defined as above;

[0065] Method 5:

[0066] Step 1: Performing a Pinnick oxidation reaction on compound a-9 to obtain compound a-24; wherein X is selected from Cl, Br and I;

[0067] Step 2: Performing an esterification reaction on compound a-24 to obtain compound a-25;

[0068] Step 3: Performing a reduction reaction on compound a-25 to obtain compound a-26; wherein the reducing agent may be a reagent containing borohydride. Petition 870260067420, dated 08 / 07 / 2026, page 21 / 113 15 / 106 of sodium, lithium borohydride or lithium aluminum hydride, as well as their deuterated reagents; R4 and R5 are defined as above;

[0069] Step 4: Performing a halogenation reaction on compound a-26 to obtain compound a-27; wherein X is selected from Cl, Br and I;

[0070] Step 5: Performing a nucleophilic substitution reaction between compounds a-27 and a-28 to obtain compound a-29; wherein R1, R2, R3, X1, X2 and X3 are defined as above; G is a protecting group, such as p-toluenesulfonyl or tert-butoxycarbonyl; Y is selected from C1-C4 alkyl;

[0071] Step 6: Removing the protecting group G from compound a-29 to obtain compound a-30;

[0072] Step 7: Performing a metal-catalyzed cross-coupling reaction between compound a-30 and amine a-12 to obtain compound a-31; wherein the metal reagent may be a Pd- or Cu-containing reagent, such as BrettPhos Pd G3, BrettPhos Pd G4 or cuprous iodide;

[0073] Step 8: Performing a hydrolysis reaction on compound a-31 to generate compound a-32 under acidic or basic conditions; wherein acidic conditions can be obtained in the presence of inorganic acids (e.g., HCl, HBr or H2SO4) or organic acids (e.g., trifluoroacetic acid); basic conditions can be obtained in the presence of inorganic bases (e.g., LiOH, NaOH or KOH);

[0074] Step 9: Performing a condensation reaction between compound a-32 and sulfonamide H2NS(O)2R6 to generate compound (I); wherein R6 is defined as above.

[0075]

[0075] This disclosure also provides a pharmaceutical composition comprising the compound of Formula I, an isotopically labeled compound, Petition 870260067420, dated 08 / 07 / 2026, page 22 / 113 16 / 106 enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof (e.g., in a therapeutically effective amount), and a pharmaceutical excipient.

[0076]

[0076] This disclosure also provides for the use of the compound of Formula I, of an isotopically labeled compound, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof, or of the pharmaceutical composition, in the manufacture of a p53 mutant pocket binding agent or of a medicament for the treatment and / or prevention of a p53 mutant-related disease.

[0077] In a preferred technical solution, the described use satisfies one or more of the following conditions:

[0078] (1) The p53 mutant has a mutation at amino acid 220, such as p53 Y220C;

[0079] (2) The pocket linking agent promotes the binding of p53 mutants to DNA;

[0080] (3) The disease associated with p53 mutants is cancer, such as breast cancer, gastric cancer, lung cancer, or ovarian cancer.

[0081] This disclosure also provides for the use of the compound of Formula I, of an isotopically labeled compound, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof, or of the pharmaceutical composition, in the manufacture of a medicament for the treatment and / or prevention of cancer.

[0082] Cancer is, for example, breast cancer, stomach cancer, lung cancer, or ovarian cancer.

[0083] This disclosure also provides a method for the treatment and / or prevention of cancer, characterized by comprising administering to a patient a therapeutically effective amount of the compound of Formula I, of an isotopically labeled compound, enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof. Petition 870260067420, dated 08 / 07 / 2026, page 23 / 113 17 / 106

[0084] Cancer is defined as above.

[0085] This disclosure also provides a compound of formula a-11 or a-32 (which can be used to prepare the above compound of formula I); a-33

[0086] Where X is a halogen (e.g., Br); R8 is an amino protecting group (e.g., Boc).

[0087] X1, X2, X3, R1, R2, R3, R4, R5 and R6 are defined as above.

[0088] The compound of formula a-11 or a-32 is preferably any of the following compounds;

[0089] The term pharmaceutically acceptable refers to a substance that is relatively non-toxic, safe, and suitable for use in patients.

[0090] 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 relatively acidic functional groups, the corresponding base addition salt can be obtained by contacting a compound with a sufficient amount of pharmaceutically acceptable base in an inert solvent. Petition 870260067420, dated 08 / 07 / 2026, page 24 / 113 18 / 106 appropriate. When a compound contains relatively basic functional groups, the corresponding acid addition salt can be obtained by contacting a compound with a sufficient amount of acceptable pharmaceutical 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).

[0091] The term solvate refers to a substance formed by the combination of a compound with a solvent (including, but not limited to: water, methanol, ethanol, etc.). Solvates are classified into stoichiometric solvates and non-stoichiometric solvates.

[0092] The term pharmaceutically acceptable salt solvate refers to a substance formed by combining a compound with a pharmaceutically acceptable acid or base and a solvent (including, but not limited to: water, methanol, ethanol, etc.). The amount of solvent present here may be stoichiometric or non-stoichiometric.

[0093] The - at the end of a group indicates that the group is connected to the rest of the molecule through that location. For example, CH3-C(=O)- refers to an acetyl group.

[0094] When the valence bond of a group is marked with a wavy line ww', as in 1, the wavy line indicates the point where the group is connected to the rest of the molecule.

[0095] The term halogen refers to fluorine, chlorine, bromine, or iodine.

[0096] The term alkyl refers to a monovalent hydrocarbon group, linear or branched chain, containing a specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, Petition 870260067420, dated 07 / 08 / 2026, p. 25 / 113 19 / 106 n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl.

[0097] The term alkoxy refers to the RX-O- group, where RX is defined as in the term alkyl. Alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0098] The term therapeutically effective amount refers to an amount administered to a patient that is sufficient to treat a disease effectively. The therapeutic amount effectively varies depending on the type of compound, type of disease, severity of the disease, age of the patient, etc., but can be adjusted by experts in the art as needed.

[0099] The term pharmaceutical excipient refers to all substances contained in pharmaceutical preparations, except active pharmaceutical ingredients, and is generally divided into two categories: vehicles and additives. For details, see Pharmacopoeia of the People's Republic of China (2020 Edition) and Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0100] The term treat / treat refers to eliminating the cause of the disease or relieving the symptoms.

[0101] The term prevent / prevent refers to reducing the risk of developing a disease.

[0102] The term patient refers to any animal, usually a mammal, such as a human, that needs treatment or disease prevention. Mammals include, but are not limited to: cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

[0103] The term PG refers to a protecting group, such as the Boc protecting group. Petition 870260067420, dated 07 / 08 / 2026, p. 26 / 113 20 / 106

[0104] According to common knowledge in the art, the various preferred conditions above can be combined arbitrarily to obtain the various preferred embodiments of this disclosure.

[0105] The reagents and raw materials used in this disclosure are commercially available.

[0106] The positive and progressive effect of this disclosure is that the compounds in this disclosure can promote the binding of p53 Y220C to DNA and possess improved inhibitory activity against the proliferation of cancerous cells represented by gastric cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] FIG. 1 shows the tumor growth curves of the control group and each treatment group in the efficacy study using the NUGC-3 human gastric cancer xenograft model, described in Experimental Example IX.

[0108] FIG. 2 shows the curves of relative variation in body weight of mice in the control group and each treatment group in the efficacy study using the NUGC-3 human gastric cancer xenograft model, described in Experimental Example IX.

[0109] FIG. 3 shows the tumor growth curves of the control group and each treatment group in the efficacy study using the NUGC-3 human gastric cancer xenograft model in rats, described in Experimental Example X.

[0110] FIG. 4 shows the curves of the relative variation in rat body weight in the control group and in each treatment group in the efficacy study of the NUGC-3 rat xenograft model for human gastric cancer described in test example X. Petition 870260067420, dated 08 / 07 / 2026, page 27 / 113 21 / 106 DETAILED DESCRIPTION OF PREFERRED CUSTOMIZATION

[0111] The present disclosure is described in more detail by examples below, but is not limited to the scope of the examples provided. Unless otherwise indicated, the actual operations disclosed in this disclosure will employ conventional techniques of organic synthesis, cell biology, cell culture and molecular biology within the technical scope of the art.

[0112] In each example, 1HNMR was recorded by a JNM-ECZ400 nuclear magnetic resonance instrument on BRUKER AVANCE NEO 400 MHz, and the chemical shift was expressed in δ (ppm); Chromatography-based mass spectrometry (LCMS) was recorded by a Shimadzu LC-20AD, Agilent 1260 and Agilent 1200 mass spectrometer; HPLC reparative separation was performed using a WATERS Autop, Shimadzu LC20AR liquid chromatograph.

[0113] Table 1: BrettPhos Pd G3 Methanesulfonate(2- Dicyclohexylphosphino-3,6-dimethoxy2',4',6'-tri-i-propyl-1,1'-biphenia)(2'amino-1,1'-biphenyl-2-yl)paladium(II) Methanesulfonate(2- Dicyclohexylphosphine-3,6dimethoxy-2',4',6'-tri-i-propyl1,1 '-biphenia)(2'-amino-1,1 'biphenyl-2-yl)paladium(II) BrettPhos Pd G4 Metanesulfonate(2Dicyclohexylphosphine-3,6-dimethoxy2',4',6'-tri-i-propyl-1,1 '-biphenyl)(2'methylamino-1,1'-biphenyl-2yl)paladium(II) Methanesulfonate(2-Dicyclohexylphosphino-3,6dimethoxy-2',4',6'-tri-i-propyl1,1 '-biphenyl)(2'-methylamino-1,1 'biphenyl-2-yl)paladium(II) CDI N,N '-Carbonyldiimidazole N,N '-Carbonyldiimidazole DBU 1,8-Diazabicyclo[5,4,0]undec-7ene 1,8-Diazabicyclo[5,4,0]undec7-ene DCM Dichloromethane Dichloromethane Petition 870260067420, 07 / 08 / 2026, pág. 28 / 113 22 / 106 DIBAL-H Diisobutylamine hydride Diisobutylamine hydride DMF N,N-Dimethylformamide N,N-Dimethylformamide DMP Dess-Martin periodinane Dess-Martin periodinane DMSO Dimethyl sulfoxide Dimethyl sulfoxide EtOAc Ethyl acetate Ethyl acetate EDCI 1-Ethyl-(3-dimethylaminopropyl)carbonyldimimide hydrochloride 1-Ethyl-(3-dimethylaminopropyl)carbonyldimimide hydrochloride HATU 2-(7-Azabenzotriazol-1-yl)N,N,N',N'-tetramethyluronium hexafluorophosphate 2-(7-Azabenzotriazol-1-yl)N,N,N',N'-tetramethyluronium hexafluorophosphate MTBE Methyl tert-butyl ether Methyl tert-butyl ether MeOH Methanol Methanol NaBH3CN Sodium cyanoborohydride Sodium cyanoborohydride Na2SO4 Sodium sulfate Sodium sulfate NMP N-Methylpyrrolidone N-Methylpyrrolidone Pd(dppf)Cl2 [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) Physical Education Petroleum ether Petroleum ether Ruphos 2-Dicyclohexylphosphino-2',6'-di-isopropoxy-1,1'-biphenyl 2-Dicyclohexylphosphino-2',6'-diiso-propoxy-1,1'-biphenyl SGC Silica gel column chromatography Silica gel column chromatography Next Tetrabutylammonium fluoride Tetrabutylammonium fluoride CHÁ Triethylamine Triethylamine THF Tetrahydrofuran Tetrahydrofuran

[0114] Preparation of intermediates Int-1 and Int-1-7 Petition 870260067420, dated 08 / 07 / 2026, page 29 / 113 23 / 106 Br lnt-1-1 POCI3, DMF 20°C, 1h Step 2 1. Ph3P*CF2CO2', DMF, 60°C, 2h 2. TBAF, THF, 60°C, 2h Br lnt-1-3 Step 3 K2CO3, MeOH 0-25°C, 16h Step 6 Cu(MeCN)4PF6, n-Bu3P HCHO, Toluene, 70°C, 6 PM Step 7 Brlnt-1-7 λ OH

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

[0116] A mixture of 3-bromo-2-methylpyridine lnt-1-1 (500 mg, 2.91 mmol), ethyl 3-bromopyruvate (850 mg, 4.36 mmol) and sodium bicarbonate (561 mg, 6.69 mmol) in butanone (5 mL) was mixed at 85°C for 16 hours, then concentrated to dryness and purified by the SGC method (0 to 10% EtOAc added to PE) to obtain ethyl 8-bromoindolizine-2-carboxylate lnt-1-2 (190 mg, 24.4% yield) as gray solid. LCMS calculated for Ci1 HnBrNCte [M+H]+: m / z = 268.0 / 270.0; found: 267.9 / 269.9; iH NMR (400 MHz, CDCl) 4.37 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 6.8 Hz, 3H).

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

[0118] A solution of ethyl 8-bromoindolizine-2-carboxylate lnt-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 hour, then slowly tempered with saturated aqueous sodium bicarbonate solution (300 mL) and extracted with DCM (200 mL). The organic layers Petition 870260067420, dated 08 / 07 / 2026, page 30 / 113 24 / 106 separate samples were combined, dried over Na2SO4, filtered, and concentrated to dryness. The residue was purified by SGC (0 to 10% EtOAc added to PE) to obtain ethyl 8-bromo-3-formylindolizine-2-carboxylate Int-1-3 (10.6 g, crude product) as a yellow colloid. LCMS calculated for C12HuBrNO3 [M+H]+: m / z = 296.0 / 298.0; found: 295.9 / 297.9.

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

[0120] A mixture of ethyl 8-bromo-3-formylindolizine-2-carboxylate Int-1-3 (8.50 g, 15.8 mmol) and 2,2-difluoro-2-triphenylphosphanioyl acetate (11.2 g, 31.5 mmol) in DMF (120 mL) was mixed for 2 hours at 60°C. TBAF (47.3 mL, 47.3 mmol, 1M in THF) was added to the mixture. The reaction mixture was stirred at 60°C for 2 hours, then diluted with water (100 mL) and extracted with MTBE (100 mL χ³). The organic layers were combined, dried over Na₂SO₄, filtered, and concentrated to dryness under reduced pressure. The residue was purified by the SGC method (0 to 5% EtOAc added to PE) to obtain ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate Int-1-4 (2.40 g, 43.4% yield) as a white solid. LCMS calculated for C13H12BrF3NO2 [M+H]+: m / z = 350.0 / 352.0; found: 349.9 / 351.9; 1H NMR (400 MHz, CDCl3) 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).

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

[0122] DIBAL-H (35.1 mL, 35.1 mmol, 1M in toluene) was added 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). The reaction mixture was stirred at -10°C for 3 hours to obtain a yellow solution. The reaction mixture was poured into a saturated aqueous solution of Petition 870260067420, dated 08 / 07 / 2026, page 31 / 113 25 / 106 ammonium chloride (200 mL), then water (200 mL) was added, and the mixture was extracted with EtOAc (150 mL χ 3). The organic layers were combined, dried over Na2SO4, filtered, and concentrated until dry under reduced pressure. The residue was purified by the SGC method (0 to 5% EtOAc added to PE) to obtain (8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)methanol Int-1-5 (3.55 g, yield 98.4%) as a white solid. LCMS calculated for CiiHicBrFaNO [M+H]+: m / z = 308.0 / 310.0; found: 307.9 / 309.9; RMN1H (400 MHz, CDCla) 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 = 10.4Hz, 2H).

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

[0124] DMP (7.33 g, 17.2 mmol) was added to a solution of (8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)methanol Int-1-5 (3.55 g, 11.5 mmol) in DCM (400 mL) at 0°C. The reaction system was heated to room temperature and stirred for 2 hours. The reaction mixture was poured into saturated aqueous sodium bicarbonate solution (20 mL), then added with water (200 mL) and extracted with DCM (100 mL χ 3). The organic layers were combined, dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by the SGC method (0 to 7% EtOAc added to PE) to obtain 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 (2.70 g, 76.5% yield) as a white solid. LCMS calculated for CuHsBrFaNO [M+H]+: m / z = 306.0 / 308.0; found: 305.9 / 307.9.

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

[0126] At 0°C, 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 (500 mg, 1.63 mmol) was added to a mixture of dimethyl(1-diazo-2-oxopropyl)phosphonate Petition 870260067420, dated 08 / 07 / 2026, page 32 / 113 26 / 106 (470 mg, 2.45 mmol) and potassium carbonate (451 mg, 3.27 mmol) in methanol (15 mL). The reaction system was heated to room temperature and stirred for 16 hours. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL x 3). The organic layers were combined, dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by the SGC method (0 to 3% EtOAc added to PE) to obtain 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (380 mg, 77.0% yield) as a white solid. LCMS calculated for Ci2H8BrF3N [m+H]+: m / z = 302.0 / 304.0; found: 301.9 / 303.9; RMN1H (400 MHz, CDCl3) 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).

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

[0128] Under a nitrogen atmosphere, tetrakis(acetonitril), copper(I), hexafluorophosphate (123 mg, 0.33 mmol) and tributylphosphine (268 mg, 1.32 mmol) were dissolved in toluene (15 mL), and the reaction mixture was stirred at 70°C for 30 minutes. Then, 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (1.00 g, 3.31 mmol) and aqueous formaldehyde solution (0.18 mL, 6.62 mmol, 36% to 38% content) were added. The mixture was stirred at 70°C overnight. After completion of the reaction detected by TLC, the mixture was concentrated under vacuum. The residue was purified by the SGC method (0 to 15% EtOAc added to PE) to obtain 3-(8-bromo-3(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-ol Int-1-7 (843 mg, 76.7% yield) as a white solid.LCMS calculated for CnHwBrF3NO [M+H]+: m / z = 332.0 / 334.0; found: 332.3 / 334.3; RMN1H (400 MHz, DMSO-d6) 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, J, = 1H), 1H), 4.34 (d, J = 6.0 Hz, 2H), 4.20–4.11 (m, 2H). Petition 870260067420, dated 08 / 07 / 2026, p. 33 / 113 27 / 106

[0129] Cpd-C: 4-((3-(8-((((3S,4R)-3-fluoro-1 -methylpiperidine-4-yl)amino)-3-(2,2,2trifluoroethyl)indolyzin-2-yl)prop-2-in-1 -methoxylbenza)-da-3-methyl

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

[0131] HATU (18.8 g, 49.5 mmol) was added to a solution of 4-amino-3-methoxybenzoic acid Cpd-C-1 (7.52 g, 45.0 mmol), methylamine solution (6.71 g, 54.0 mmol, 25% content) and triethylamine (18.8 mL, 135 mmol) in THF (45 mL) at 0°C. The reaction was stimulated at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by the SGC method (0 to 4% MeOH added to DCM) to obtain 4-amino-3-methoxy-N-methylbenzamide Cpd-C-2 (6.08 g, 75.0% yield) as a white solid. LCMS calculated for C9H13N2O2 [M+H]+: m / z = 181.1; Found: 181.2.

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

[0133] Aqueous solution of formaldehyde (497 mg, 5.96 mmol, 36% to 38% content) and cuprous bromide (567 mg, 3.97 mmol) were added to a mixture of 8 Petition 870260067420, dated 08 / 07 / 2026, p. 34 / 113 28 / 106 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). The reaction was stimulated under microwave irradiation at 100°C for 1 hour. The residue was purified by the SGC method (0 to 30% EtOAc added to DCM) to obtain 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (480 mg, 48.9% yield) as a yellow solid. LCMS calculated for C22H20BrF3N3O2 [M+H]+: m / z = 494.1 / 496.1; found: 494.2 / 496.2.

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

[0135] Under an argon atmosphere, a mixture of 4-((3-(8-bromo-3-(2,2,2trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (1.35 g, 2.73 mmol), tert-butyl (3S,4R)-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 mixed at 100°C for 16 hours. The reaction mixture was diluted with EtOAc (200 mL), washed with water (100 mL) and saturated brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 50% EtOAc added to DCM) to obtain tert-butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate Cpd-C-4 (1.00 g, 58.0% yield) as a yellow solid. LCMS calculated for C32H38F4N5O4 [M+H]+: m / z = 632.3; found: 632.5.

[0136] Step 4: 4-((3-(8-((((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2. Petition 870260067420, dated 08 / 07 / 2026, p. 35 / 113 29 / 106 trifluoroethyl)indolyzin-2-yl)prop-2-in-1 -yl)amino)-3-methoxy-N-methylbenzamide

[0137] HCl (7.92 mL, 31.7 mmol, 4N in 1,4-dioxane) 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) at 0°C. The mixture was heated to room temperature and stirred for 1 hour. The mixture was concentrated to dryness, diluted with water (30 mL), and the pH adjusted to 7 to 8 with saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM (100 mL χ 2), and the combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated to obtain 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 product) as a yellow solid. The crude product was used directly in the reaction of the next step. LCMS calculated for C27H30F4N5O2 [M+H]+: m / z = 532.2; found: 532.4.

[0138] Step 5: 4-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

[0139] Sodium cyanoborohydride (160 mg, 2.54 mmol) was added to a solution of 4-((3-(8-((((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2yl)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, 36% to 38%) in methanol (20 mL) at room temperature, and the reaction mixture was stirred for 1 hour at room temperature. The pH was adjusted to 7 to 8 by adding saturated sodium bicarbonate solution dropwise to the reaction mixture. The aqueous phase was extracted with EtOAc (100 mL χ 3). The organic phases were Petition 870260067420, dated 08 / 07 / 2026, page 36 / 113 30 / 106 combined and washed with saturated brine (50 mL χ 3), dried over anhydrous Na2SO4, filtered and concentrated. The residue was subjected to preparatory purification to obtain 4-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C (119 mg, 25.2% yield) as a white solid.LCMS calculated for C28H32F4N5O2 [M+H]+: m / z = 546.2; found: 546.2; RMN1H (400 MHz, DMSO-d6) δ 8.08 (d, J = 4.6 Hz, 1H), 7.57 (d, J = 6.9 Hz, 1H), 7.41 (dd, J = 8.2, 1.7 Hz, 1H), 7.6 J = 1.9 (d (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), 4, J 4,8 (1H (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), 2.80 (d, J= 11.3 Hz, 1H), 2.75 (d, J = 4.5 Hz, 3H), 2,282.14 (m, 4H), 2.07 (t, J = 11.2 Hz, 1H), 1.97 (ddd, 1, J = 12,0 1.67 (dd, J= 12.7, 2.7 Hz, 1H).

[0140] Example 1: 5-((3-(8-(((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3(2,2,2-trifluoroethyl)indolyzin-2-yl)prop-2-in-1 -(yl)methyl)aminosulfonyl-6-6 (Cpd-1, or soybean, Compound 1) in O2N Cpd-1-1 is, 2h stage 3 Pd / C, H2, EtOAc Petition 870260067420, dated 07 / 08 / 2026, p. 37 / 113 31 / 106 ο >ζΝ Ο„ Η2Ν-(\ / W Ο HN-S0 X Cpd-1-4 reagent 1) Ti( / -PrO)4, THF, 100°C, 1h 2)MeOH, NaBHjCN, 10min, rt step 8

[0141] Step 1: 6-methoxy-5-nitropicolinic acid

[0142] A mixture of 6-methoxy-5-nitropicolinic acid Cpd-1-1 (4.00 g, 19.7 mmol) and cesium carbonate (12.8 g, 39.4 mmol) in DMSO / MeOH (40 mL, 1:1) was mixed at 60°C for 12 hours. LC-MS analysis showed that the starting material was completely consumed and that the desired compound was detected. The reaction mixture was poured into ice-cold water (50 mL), the pH was adjusted to 5 with 1 N HCl, and the aqueous phase was extracted with ethyl acetate (30 mL χ 3). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by the SGC method (0 to 5% MeOH added to DCM) to obtain 6-metaxy-5-nitropicolinic acid Cpd-1-2 (2.80 g, 71.7% yield, yellow solid). LCMS calculated for C7H7N2O5 [M+H]+: m / z = 199.0; found: 199.1.

[0143] Step 2: 6-methoxy-N-(methylsulfonyl)-5-nitropicolinamide

[0144] A mixture of 6-methoxy-5-nitropicolinic acid Cpd-1-2 (3.20 g, 16.2 mmol), Petition 870260067420, dated 08 / 07 / 2026, page 38 / 113 32 / 106 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. LC-MS analysis showed that the starting material was completely consumed and that the desired compound was detected. The reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (30 mL χ³). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous Na₂SO₄, filtered and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0 to 5% MeOH in DCM) to give 6-methoxy-N-(methylsulfonyl)-5-nitropicolinamide Cpd1-3 (2.20 g, 49.5% yield, yellow solid). LC-MS was calculated for C8H10N3O6S [M+H]+: m / z = 276.0; found: 276.1.

[0145] Step 3: 5-amino-6-methoxy-N-(methylsulfonyl)picolinamide

[0146] Under a hydrogen atmosphere, a mixture of palladium on carbon (1.60 g, 5 wt%) and 6-methoxy-N-(methylsulfonyl)-5-nitropicolinamide Cpd-1-3 (800 mg, 2.91 mmol) in ethyl acetate (15 mL) was stirred at room temperature for 2 hours. LC-MS analysis showed that the starting material was completely consumed and that the desired compound was detected. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0 to 5% MeOH in DCM) to give 5amino-6-methoxy-N-(methylsulfonyl)picolinamide Cpd-1-4 (500 mg, 70.1% yield, yellow solid). LC-MS calculated for C8H12N3O4S [M+H]+: m / z = 246.1; found: 246.0.

[0147] Step 4: 8-bromo-2-(3-(((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2:2:2trifluoroethyl)indolizine. Petition 870260067420, dated 08 / 07 / 2026, page 39 / 113 33 / 106

[0148] Tert-butyldimethylchlorosilane (68.0 mg, 451 μmol) was added in portions to a solution of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Int-1-7 (100 mg, 302 μmol) and imidazole (61.3 mg, 901 μmol) in DCM (5 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 hours and then filtered to remove the solids. The filtrate was concentrated under reduced pressure to provide a residue, which was purified by silica gel column chromatography (0 to 30% ethyl acetate in petroleum ether) to provide 8-bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-1-5 (134 mg, 99.7% yield, yellow solid). LC-MS calculated for C19H24BrF3NOSi [M+H]+: m / z = 446.1 / 448.1; found: 446.0 / 448.0.Step 5: 2-(3-((tert-Butyldimetilsilyl)oxi)prop-1-yn-1-yl)-N((3S,4R)-3-fluoro-1-metilpiperidina-4-yl)-3-(2,2,2-trifluoroethyl)indolizine-8-amine.

[0149] Under an argon atmosphere, a mixture of 8-bromo-2-(3-(((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-1-5 (134 mg, 301 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (79.3 mg, 601 μmol), BrettPhos Pd G3 (54.4 mg, 60.0 μmol) and cesium carbonate (195 mg, 598 μmol) in THF (10 mL) was stirred at 100 °C for 2 hours. The reaction mixture was cooled to room temperature, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 7% methanol in DCM) to provide 2(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3(2,2,2-trifluoroethyl)indolizin-8-amine Cpd-1-6 (82.1 mg, 54.9% yield, white solid). LC-MS calculated for C25H36F4N3OSi [M+H]+: m / z = 498.3; found: 498.0.

[0150] Step 6: 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol. Petition 870260067420, dated 07 / 08 / 2026, p. 40 / 113 34 / 106

[0151] TBAF (0.20 mL, 0.20 mmol, 1 M in THF) was added dropwise to a solution of 2-(3-(((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-amine Cpd-1-6 (82.1 mg, 165 μmol) in THF (5 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour. LC-MS analysis showed that the starting material was completely consumed and that the desired compound was detected. The reaction mixture was neutralized with a saturated aqueous solution of sodium bicarbonate (50 mL) and extracted with ethyl acetate (50 mL x 3). The organic phases were combined and dried over Na2SO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0 to 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)prop2-yn-1-ol Cpd-1-7 (57.0 mg, 90.2% yield, white solid).LC-MS calculated for C19H22F4N3O [M+H]+: m / z = 384.2; found: 384.1.

[0152] Step 7: 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizin-2-yl)prop-2-inal.

[0153] A solution of the sulfur trioxide-pyridine complex (780 mg, 4.90 mmol) in DMSO (1 mL) was added dropwise to a solution of 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Cpd-1-7 (625 mg, 1.63 mmol), DMSO (1.20 mL, 16.3 mmol) and diisopropylethylamine (1.50 mL, 8.15 mmol) in DCM (20 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 hours. LC-MS analysis showed that the starting material was completely consumed and that the desired compound was detected. The reaction mixture was neutralized with a saturated aqueous solution of sodium bicarbonate (100 mL) and extracted with ethyl acetate (100 mL x 3). The organic phases were combined, Petition 870260067420, dated 08 / 07 / 2026, page 41 / 113 35 / 106 dried over Na2SO4, filtered and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0 to 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-inal Cpd-1-8 (550 mg, 88.6% yield, yellow solid). LC-MS calculated for C19H20F4N3O [M+H]+: m / z = 382.2; found: 382.1.

[0154] Step 8: 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)picolinamide.

[0155] A mixture of 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propioaldehyde Cpd-1-8 (50.0 mg, 131 μmol), 5-amino-6-methoxy-N-(methylsulfonyl)picolinamide Cpd-1-4 (38.6 mg, 157 μmol) and tetraisopropyl titanate (112 mg, 393 μmol) in THF (1 mL) was mixed at 100°C for 1 hour. The mixed solution was then cooled to 0°C, slowly added with MeOH (0.5 mL) and sodium cyanoborohydride (12.4 mg, 197 μmol), and the reaction mixture was heated to room temperature and stirred for a further 10 minutes. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was poured into gel water (5 mL) and extracted with EtOAc (20 mL χ³). The organic layers were combined, washed with saturated aqueous sodium bicarbonate solution (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure.The residue was purified by the SGC method (0 to 3% MeOH added to DCM) to obtain a crude product as a yellow solid, which was subjected to preparative purification to obtain 5-((3-(8((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)picolinamide Cpd-1 (14.8 mg yield). Petition 870260067420, dated 08 / 07 / 2026, page 42 / 113 36 / 106 of 18.5%, solid yellow). LCMS calculated for C27H31F4N6O4S [M+H]+: m / z = 611.2; found: 611.9; RMN1H (400 MHz, CDCI3) δ 9.70 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 7.0 Hz, 1H), 6.98 (d, J = 8.0 Hz, J, 3 1, 2, 6), 6.48 (s, 1H), 5.81 (d, 4 = 7.3 Hz, 1H), 5.18 (t, J = 5.9 Hz, 1H), 4.85 (d, J = 49.1 Hz, 1H), 4.29 (d, J = 6.9, J , 4, 2H 1H), 4.03 (s, 3H), 3.68 (q, J= 10.1 Hz, 2H), 3,593.43 (m, 1H), 3.41 (s, 3H), 3.24 (t, J = 11.0 Hz, 1H), 2.94 (d, 1, 1H), J 2.372.21 (m, 4H), 2.16 (t, 4=11.1 Hz, 1H), 2.02 (d, 4 = 10.0 Hz, 1H), 1.93 (td, 4 = 12.2, 3.5 Hz, 1H).

[0156] Example 2: 5-((3-(8-(((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3(2,2,2-trifluoroethyl)indolyzin-2-yl)prop-2-in-1 -yl)amino)-6-(methoxy-d3)-N(methylsulfonyl)picolinamide (Cpd-2, or soybean, Compound 2) NO2Νθ2 'i02NH2°^DX _O. n Í|X Cs2CO3, CD3OD í| Μ Ί6 x>N DMSO, 60°C, 16h D COOH stage 1 COOH Cpd-1-1 Cpd-2-1 FJ F \ A DMP, DCM l· UR 0-25°C, 2h L UH Λ \ I stage 4 Br lnt-1-7. D CH3..Cs2CO3> Π T Ap Pd / C.H2><y DMSO, 0°C, 2h J EA, ta, 2h X> N 1 step 2 step 3 L 0 0 O^O Cpd-2-2 Cpd-2-3 FFJ Cpd-2-3 AA _ ________► LV—==—1) Ti( / -PrO)4, THF, 100°C, 1h O-3 B10, MeBH stagePath5 FF z^X>NH2 F ' do ii j / zcd3 ^n JL qn . íí^na °x — f—► I Γ 7—=—\ ZN O BrettPhos Pd G3, γ X / —V HN—C / Cs2CO3, dioxane Br X= / 0-\ 100°C, 2h .Ç [ Cpd-2-4 stage , CD x- NO 6 ' Na \-Ά / . MeSO2NH2 * THF / MeOH / H2o' HN-Ü CDI, DBU, THP rt,16h Aih X= / 0H 70°C,2h stage 7 | stage 8 | —XF Cpd-2-6 zN^· > cd3 \ 0 - H\ W / \= / 0-( Cpd-2-5 PF FX / ,CD3 θ LL / —=—\ An 0 ΗΝΛ / λ^ ^NH ,NH V o=s. Cpd-2 ''° Petition 870260067420, dated 08 / 07 / 2026, p. 43 / 113 37 / 106

[0157] Stage 1: 6-(methoxy-d3)-5-nitropicolinic acid

[0158] Under a nitrogen atmosphere, a mixture of 6-chloro-5-nitropicolinic 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 complete, as verified by LC-MS, the reaction mixture was diluted with water (100 mL) and the pH adjusted to 3 with 1 N HCl. The mixture was filtered to collect the precipitate, and the resulting solid was dried under vacuum to give 6(methoxy-d3)-5-nitropicolinic acid Cpd-2-1 (12.0 g, crude product, yellow solid). The crude product was used directly in the subsequent step. LC-MS calculated for C7H4D3N2O5 [M+H]+: m / z = 202.1; found: 201.9.

[0159] Step 2: Methyl 6-(methoxy-d3)-5-nitropicolinate.

[0160] Under a nitrogen atmosphere, a mixture of 6-(methoxy-d3)-5-nitropicolinic acid Cpd-2-1 (12.0 g, 59.7 mmol) and cesium carbonate (58.4 g, 179 mmol) in DMSO (120 mL) was stirred at 0 °C for 0.5 hours. Then, iodomethane (9.32 g, 179 mmol) was added, and the reaction mixture was stirred at 0 °C for 2 hours. After completion of the reaction, as verified by LC-MS, the reaction mixture was poured into chilled aqueous ammonium chloride solution (1000 mL) and extracted with ethyl acetate (1000 mL χ2). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated to provide a crude product. The residue was purified by silica gel column chromatography (0 to 20% ethyl acetate in petroleum ether) to give methyl 6-(methoxy-d3)-5-nitropicolinate Cpd-2-2 (10.0 g, 70.1% yield, yellow solid). LC-MS calculated for C8H6D3N2O5 [M+H]+: m / z = 216.1; found: 215.9.

[0161] Step 3: Methyl 5-amino-6-(methoxy-d3)picolinate. Petition 870260067420, dated 08 / 07 / 2026, page 44 / 113 38 / 106

[0162] Methyl 6-(methoxy-d3)-5-nitropicolinate Cpd-2-2 (10.0 g, 46.5 mmol) was dissolved in ethyl acetate (100 mL). Then, palladium on carbon (9.90 g, 5% by mass) was added, and the resulting mixture was stirred for 2 hours at room temperature under a hydrogen atmosphere. LC-MS analysis showed that the starting material was completely consumed and that the desired compound was detected. The reaction mixture was filtered and concentrated to give methyl 5-amino-6-(methoxy-d3)picolinate Cpd-2-3 (8.20 g, 90.5% yield, yellow solid). LC-MS calculated for C8H8D3N2O3[M+H]+: m / z = 186.1; found: 186.2. Ή NMR (400 MHz, DMSO-d6) 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).

[0163] Step 4: 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde.

[0164] DMP (17.4 g, 41.0 mmol) was slowly added portion by portion 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) in an ice bath. The mixture was then stirred at room temperature for 2 hours. The reaction was quenched with a saturated aqueous solution of sodium bicarbonate (50 mL) and extracted with DCM (30 mL χ 3). The organic phases were combined and concentrated under vacuum. The residue was purified by the SGC method (0 to 15% EtOAc added to PE) to obtain 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)propylaldehyde Int-2 (5.70 g, 84.3% yield, gray solid). LCMS calculated for CnHsBrFaNO [M+H]+: m / z = 330.0 / 332.0; found: 330.2 / 332.2; NMR1H (400 MHz, DMSO-d6) 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 = 10.7Hz, 2H). Petition 870260067420, dated 07 / 08 / 2026, p. 45 / 113 39 / 106

[0165] Step 5: Isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2yn-1-yl)amino)-6-(methoxy-d3)picolinate

[0166] Under a nitrogen atmosphere, a solution of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propioaldehyde Int-2 (200 mg, 606 μmol), methyl 5-amino-6(methoxy-d3)picolinate Cpd-2-3 (135 mg, 730 μmol) and tetraisopropyl titanate (517 mg, 1.82 mmol) in THF (2 mL) was stirred at 100°C for 1 hour. The reaction mixture was cooled to 25°C, then added with MeOH (2 mL) and sodium cyanoborohydride (57.1 mg, 0.91 mmol), and stirred for a further 10 minutes. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL χ 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated.The residue was purified by the SGC method (0 to 25% EtOAc added to PE) to obtain isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6(methoxy-d3)picolinate Cpd-2-4 (190 mg, 59.4% yield, yellow solid). LCMS calculated for C^HwDaBrFaNaOa [M+H]+: m / z = 527.1 / 529.1; found: 526.9 / 529.0.

[0167] Step 6: Isopropyl 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)-6-(methoxy-d3)picolinate

[0168] Under a nitrogen atmosphere, a solution of isopropyl 5-((3-(8-bromo-3(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)picolinate Cpd-2-4 (170 mg, 322 μmol), (3S,4R)-3-fluoro-1-methylpiperidine-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 mixed at 100°C for 2 hours in a sealed tube. Detection Petition 870260067420, dated 08 / 07 / 2026, page 46 / 113 LCMS 40 / 106 showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was filtered and the filtrate concentrated under reduced pressure. The crude product was subjected to preparative purification to obtain isopropyl 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)picolinate Cpd-2-5 (90.0 mg, 48.1% yield, yellow solid). LCMS calculated for C29H31D3F4N5O3[M+H]+: m / z = 579.3; found: 579.0.

[0169] Step 7: 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)picolinic acid.

[0170] A mixture of isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy3)picolinate Cpd-2-5 (40.0 mg, 69.2 μmol) and sodium hydroxide (8.28 mg, 207 μmol) in THF (0.4 mL), MeOH (0.2 mL), and water (0.1 mL) was mixed at room temperature for 16 hours. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, diluted with water, adjusted to pH = 5 with dilute HCl, and filtered. The residue was purified by the SGC method (0 to 3% MeOH added to DCM) to obtain a crude product, which was then subjected to preparative purification to obtain 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)picolinic acid Cpd-2-6 (5.50 mg, 15.0% yield, white solid).LCMS calculated for C26H25D3F4N5O3 [M+H]+: m / z = 537.2; encontrado: 537.0; RMN1H (400 MHz, CD3OD) 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. Petition 870260067420, 07 / 08 / 2026, pág. 47 / 113 41 / 106 (q, J = 10.5 Hz, 2H), 3.62-3.54 (m, 1H), 3.32-3.23 (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).

[0171] Step 8: 5-((3-(8-((((3S,4R)-3-Fluoro-1-methylpiperidine-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)-6-(methoxi-d3)-N-(methylsulfonyl)picolinamide [0172) A solution of 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidine-4-yl)amino)-3(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)picic acid Cpd-2-6 (210 mg, 392 μmol) and CDI (127 mg, 784 μmol) in THF (8 mL) were mixed at 70°C for 1 hour under an argon atmosphere and then cooled to room temperature. Methanesulfonamide (74.5 mg, 784 μmol) was added to the reaction mixture and stirred for 10 minutes, then DBU (120 μL, 789 μmol) was added and stirred for another 1 hour at 70°C. The mixture was concentrated, then water (30 mL) was added and the aqueous phase was extracted with EtOAc (50 mL χ³). The combined organic phases were washed with brine (50 mL χ³), dried over anhydrous Na₂SO₄, filtered, and the filtrate concentrated under vacuum. The residue was subjected to preparatory purification to obtain 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidine-4-yl)amino)-3(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-(methylsulfonyl)picolinamide Cpd-2 (50.0 mg, 20.8% yield, white solid).LCMS calculated for C27H28D3F4N6O4S [M+H]+: m / z = 614.2; found: 614.4;1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 6.9 Hz, 1H), 7.1,1 = 8,1 (d (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 = 4, =19), 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 = 1.3m, 2, 1.5 Hz), 1H), 2.22 (s, 3H), 2.13 (t, J =. Petition 870260067420, dated 08 / 07 / 2026, p. 48 / 113 42 / 106 11.3 Hz, 1H), 2.04–1.92 (m, 1H), 1.75–1.64 (m, 1H).

[0173] Example 3: 5-((3-(8-(((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3(2,2,2-trifluoroethyl)indolyzin-2-yl)prop-2-in-1 -yl)amino)-4-methoxy-N(methylsulfonyl)pyrimidine-2-carboxamide (Cpd-3, or soybean, Compound 3) Cpd-3-1 Boc2O, tea, dmap, thf 40°C.1 h step 1 Pd(dppf)CI2, TEA, MeOH_ 100°C, 16 h step 2 'BocHN Cpd-3-3 CBr4, PPh3, DCM \ ta, 2h OH step 3 Cpd-3-2 Br Cpd-3-5 RuPhos, Brettphos Pd G3 Cpd-3-3 Cs2CO3, ACN 40°C, 12h stage 4 Cs2CO3, THF, 95°C, 2 h step 5

[0174] Step 1: tert-butyl (tert-butoxycarbonyl)(2-chloro-4-methoxypyrimidine-5yl)carbamate.

[0175] Under a nitrogen atmosphere, a mixture of 2-chloro-4-methoxypyrimidine-5amine 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) to THF Petition 870260067420, dated 08 / 07 / 2026, page 49 / 113 43 / 106 (50 mL) was mixed at 40°C for 1 hour. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (50 mL χ 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by the SGC method (0 to 5% EtOAc added to PE) to obtain tert-butyl (tertbutoxycarbonyl) (2-chloro-4-methoxypyrimidin-5-yl)carbamate Cpd-3-2 (4.80 g, 42.5% yield, white solid). LCMS calculated for C15H23ClN3O5[M+H]+: m / z = 360.1; found: 360.2.

[0176] Step 2: methyl 5-((tert-butoxycarbonyl)amino)-4-methoxypyrimidin-2-carboxylate.

[0177] Under a carbon monoxide atmosphere, a mixture of tert-butyl(tertbutoxycarbonyl)(2-chloro-4-methoxypyrimidin-5-yl)carbamate Cpd-3-2 (4.30 g, 11.9 mmol), Pd(dppf)Cl2 (1.72 g, 2.38 mmol) and triethylamine (3.61 g, 35.7 mmol) in MeOH (43 mL) was stirred at 100°C for 16 hours. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure. The residue was diluted with water (100 mL) and extracted with EtOAc (100 mL χ 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by the SGC method (0 to 20% EtOAc added to PE) to obtain methyl 5-((tert-butoxycarbonyl)amino)-4-methoxypyrimidin-2-carboxylate Cpd-3-3 (2.00 g, 59.0% yield, white solid). LCMS calculated for C12H18N3O5 [M+H]+: m / z = 284.1; found: 284.1.

[0178] Step 3: 8-bromo-2-(3-bromoprop-1-in-1-yl)-3-(2,2,2-trifluoroethyl)indolizine.

[0179] At room temperature, 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolyzin-2-yl)prop-2 Petition 870260067420, dated 08 / 07 / 2026, p. 50 / 113 44 / 106 in-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 initial material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL χ³). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified by the SGC method (0 to 10% EtOAc added to PE) to obtain 8-bromo-2-(3-bromoprop-1-yn-1-yl)-3(2,2,2-trifluoroethyl)indolizine Cpd-3-4 (1.55 g, 53.0% yield, white solid). LCMS calculated for CnHgB^FaN [M+H]+: m / z = 393.9 / 395.9 / 397.9; found: 394.0 / 396.0 / 398.0.

[0180] Step 4: methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidin-2-carboxylate.

[0181] Under a nitrogen atmosphere, 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-methoxypyrimidin-2-carboxylate Cpd-3-3 (980 mg, 3.46 mmol) and cesium carbonate (1.88 g, 13.8 mmol) in acetonitrile (20 mL) was mixed at 40°C for 12 hours. LCMS detection showed that the initial material was 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 the SGC method (0 to 25% EtOAc added to PE) to obtain methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidin-2-carboxylate Cpd-3-5 (1.60 g, Petition 870260067420, dated 08 / 07 / 2026, page 51 / 113 45 / 106 yield of 65.1%, white solid). LCMS calculated for C25H25BrF3N4O5 [M+H]+: m / z = 597.1 / 599.1; found: 597.2 / 599.2.

[0182] Step 5: methyl 5-((tert-butoxycarbonyl)(3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxypyrimidin-2carboxylate.

[0183] Under a nitrogen atmosphere, a mixture of methyl 5-((3-(3-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidin-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 mixed at 95°C for 2 hours in a sealed tube. LCMS detection showed that the initial material was 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 the SGC method (0 to 5% MeOH added to 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-methoxypyrimidin-2-carboxylate Cpd-3-6 (1.20 g, crude product, yellow solid).LCMS calculated for C31H37F4N6O5 [M+H]+: m / z = 649.3; found: 649.2.

[0184] Step 6: 5-((tert-butoxycarbonyl)(3-(8-((((3S,4R)-3-fluoro-1methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4methoxypyrimidin-2-carboxylic acid.

[0185] At room temperature, methyl 5-((tert-butoxycarbonyl)(3-(8-((((3S,4R)-3fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino)-4-methoxypyrimidin-2-carboxylate Cpd-3-6 (1.20 g, 1.85 mmol) was Petition 870260067420, dated 08 / 07 / 2026, page 52 / 113 46 / 106 dissolved in a mixed solvent of THF (8 mL), MeOH (8 mL) and water (2 mL). Lithium hydroxide (443 mg, 18.5 mmol) was added to it. The system was stirred at room temperature for 2 hours. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was adjusted to pH = 4 with 1N HCl, diluted with water (20 mL) and extracted with DCM (20 mL χ 3). The combined organic phases were dried over anhydrous Na2SO4 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)prop2-yn-1-yl)amino)-4-methoxypyrimidin-2-carboxylic acid) Cpd-3-7 (400 mg, crude product, yellow solid). LCMS calculated for C30H35F4N6O5 [M+H]+: m / z = 635.3; found: 635.5.

[0186] Etapa 7: tert-butyl (3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-in-1 -yl)(4-methoxy-2((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate.

[0187] 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-methoxypyrimidin-2-carboxylic acid) Cpd-3-7 (260 mg, 410 μmol) and CDI (133 mg, 820 μmol) in DMF (4 mL) was stirred at 90°C for 2 hours. The reaction mixture was cooled to room temperature, and then methanesulfonamide (78.0 mg, 820 μmol) and DBU (206 mg, 820 μmol) were added. The reaction mixture was stirred at 90°C for a further 2 hours. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was cooled to room temperature, diluted with water (50 mL), and extracted with DCM (50 mL χ³). The combined organic layers were washed with saturated brine (50 mL). Petition 870260067420, dated 08 / 07 / 2026, page 53 / 113 47 / 106 seconds over anhydrous, filtered and concentrated Na2SO4. The residue was purified by Prep-HPLC to obtain tert-butyl (3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-in-1 -yl)(4-methoxy-2((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate) Cpd-3-8 (100 mg, raw product, bitter solid). LCMS calculated for C31H38F4N7O6S [M+H]+: m / z = 711.3; encountered: 711.6.

[0188] Etapa 8: 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizin-2-yl)prop-2-in-1-yl)amino)-4-methoxy-N-(methylsulfonyl)pyrimidin-2carboxamida).

[0189] 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)prop2-yn-1-yl)(4-methoxy-2-((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate) Cpd-3-8 (100 mg, 141 μmol) in DCM (3 mL) at 0°C. The reaction mixture was slowly heated to room temperature and stirred for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction 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 χ 3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated.The residue was purified by Prep-HPLC to obtain 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-in-1-yl)amino)-4-methoxy-N(methylsulfonyl)pyrimidin-2-carboxamida) Cpd-3 (8.05 mg, yield of 8.8%, bitter solid). LCMS calculated for C26H30F4N7O4S [M+H]+: m / z = 612.2; crossed: 612.2; RMN1H (400 MHz, DMSO-d6) δ 8.05 (s, 1H), 7.60 (d, J = 6.8 Hz, 1H), 6.93 (s,. Petition 870260067420, 07 / 08 / 2026, pág. 54 / 113 48 / 106 Η), 6.80-6.70 (m, 1 Η), 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).

[0190] Example 4: N-(cyclopropylsulfonyl)-5-((3-(8-((((3S,4R)-3-fluoro-1methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino)-6-(methoxy-d3)picolinamide (Cpd-4, i.e. Compound 4)

[0191] Step 1: N-(cyclopropylsulfonyl)-6-(methoxy-d3)-5-nitropicolinamide.

[0192] At room temperature, cyclopropylsulfonamide (1.33 g, 10.9 mmol) and triethylamine (3.32 g, 32.8 mmol) were sequentially added to a solution of 6-(methoxy-d3)-5-nitropicolinic 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). The reaction mixture was stirred at 40°C for 3 hours. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture Petition 870260067420, dated 08 / 07 / 2026, page 55 / 113 49 / 106 was concentrated under reduced pressure, and the residue was diluted with water (100 mL). The pH was adjusted to 2 with aqueous 1N HCl solution. The precipitated solid was filtered, washed with water (100 mL), and dried over anhydrous Na2SO4 to obtain N(cyclopropylsulfonyl)-6-(methoxy-d3)-5-nitropicolinamide Cpd-4-1 (1.00 g, crude product, yellow solid). The crude product was used directly in the reaction of the next step. LCMS calculated for C10H7D3N3O6S [MH]-: m / z = 303.1; found: 303.2.

[0193] Step 2: 5-amino-N-(cyclopropylsulfonyl)-6-(methoxy-d3)picolinamide.

[0194] At room temperature, a mixture of N-(cyclopropylsulfonyl)-6-(methoxy-d3)5-nitropicolinamide Cpd-4-1 (1.00 g, 3.29 mmol, crude product) and palladium on carbon (350 mg, 5% content) in THF (15 mL) and MeOH (15 mL) was mixed for 16 hours under a hydrogen atmosphere. LCMS detection showed that the initial 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-(methoxy-d3)picolinamide Cpd-4-2 (730 mg, crude product, yellow solid). The crude product was used directly in the reaction of the next step. LCMS calculated for C10H9D3N3O4S [MH]-: m / z = 273.1; Found: 273.3.

[0195] Step 3: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d3)picolinamide.

[0196] 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propioaldehyde Int-2 (500 mg, 1.51 mmol), 5-amino-N-(cyclopropylsulfonyl)-6-(methoxy-d3)picolinamide Cpd-4-2 (395 mg, 1.44 mmol), and acetic acid (91.0 mg, 1.51 mmol) were dissolved in MeOH (6 mL) and DCM (4 mL), and the system was stirred at room temperature for 16 hours. Then, NaBH3CN (286 mg, 4.54 mmol) was added, and the system was stirred for a further 0.5 hours. LCMS detection showed that the initial material was completely Petition 870260067420, dated 08 / 07 / 2026, page 56 / 113 50 / 106 consumed and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and filtered to collect the filter cake. The filter cake was purified by suspension with methanol to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d3)picolinamide Cpd-4-3 (560 mg, 62.8% yield, yellow solid). LCMS calculated for C23H16BrDaF3N4O4S [MH]-: m / z = 586.1 / 588.1; found: 586.2 / 588.2.

[0197] Step 4: N-(cyclopropylsulfonyl)-5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxyd3)picolinamide.

[0198] Under an argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy3)picolinamide Cpd-4-3 (560 mg, 950 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4amine (252 mg, 1.90 mmol), BrettPhos Pd G3 (86.3 mg, 100 μmol) and cesium carbonate (620 mg, 1.90 mmol) in THF (13 mL) and NMP (1.3 mL) was stirred at 100°C for 5 hours in a sealed tube. LCMS detection showed that the initial 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 χ 3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure.The residue was purified by Prep-HPLC to obtain the product N-(cyclopropylsulfonyl)-5-((3(8-(((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)picolinamide)Cpd-4 (42.4 mg, 6.80% yield, yellow solid). LCMS calculated for C29H30D3F4N6O4S [M+H]+: m / z = 640.2; found: 640.1; 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 7.68 (d, J = 8.0 Hz,. Petition 870260067420, dated 07 / 08 / 2026, p. 57 / 113 51 / 106 Η), 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, = 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).

[0199] Example 5: N-((cyclopropylmethyl)sulfonyl)-5-((3-(8-((((3S,4R)-3-fluoro-1 methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino)-6-(methoxy-d3)picolinamide (Cpd-5, i.e. Compound 5) cd30 CMPI, TEA, DCM 40°C, 3h stage 1 Pd / C, H2 THF / MeOH ta, 4 PM stage 2 Cpd-2-1

[0200] Step 1: N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)-5-nitropicolinamide.

[0201] At room temperature, (cyclopropylmethyl)sulfonamide (1.69 g, 12.5 mmol) and triethylamine (3.62 g, 35.8 mmol) were sequentially added to a mixture of 6-(methoxy-d3)-5-nitropicolinic 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). The reaction mixture was stirred at 40°C for 3 hours. LCMS detection showed that the initial material was Petition 870260067420, dated 08 / 07 / 2026, page 58 / 113 52 / 106 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 aqueous 1N HCl solution and extracted with DCM (100 mL χ 3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by SGC (0 to 5% MeOH added to DCM) to obtain N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)-5-nitropicolinamide Cpd-5-1 (1.10 g, 29.0% yield, white solid). LCMS calculated for C11H9D3N3O6S [MH]-: m / z = 317.1; found: 316.9.

[0202] Step 2: 5-amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)picolinamide.

[0203] At room temperature, a mixture of N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)-5-nitropicolinamide 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 mixed for 16 hours under a hydrogen atmosphere. LCMS detection showed that the initial 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-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)picolinamide Cpd-5-2 (970 mg, crude product, yellow-white solid). The crude product was used directly in the reaction of the next step. LCMS calculated for C11H11D3N3O4S [MH]-: m / z = 287.1; found: 287.1.

[0204] Step 3: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)picolinamide.

[0205] A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Int-2 (500 mg, 1.51 mmol), 5-amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy Petition 870260067420, dated 07 / 08 / 2026, p. 59 / 113 53 / 106 d3)picolinamide Cpd-5-2 (416 mg, 1.44 mmol), acetic acid (86.6 mg, 1.44 mmol), and Na2SO4 (1.02 g, 7.21 mmol) in MeOH (20 mL) and 1,2-dichloroethane (20 mL) were stirred at room temperature for 16 hours. Then, NaBH3CN (181 mg, 2.89 mmol) was added, and the system was stirred for another 0.5 hours at room temperature. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL χ 3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by the SGC method (0 to 70% EtOAc added to PE) to obtain 5-((3(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N((cyclopropylmethyl)sulfonyl)-6-(methoxy-d3)picolinamide Cpd-5-3 (390 mg, yield of 44.9%, yellow solid).LCMS calculated for C24H20BrD3F3N4O4S [M+H]+: m / z = 602.1 / 604.1; found: 602.3 / 604.3.

[0206] Step 4: N-((cyclopropylmethyl)sulfonyl)-5-((3-(8-((((3S,4R)-3-fluoro-1methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6(methoxy-d3)picolinamide.

[0207] Under an argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy3)picolinamide Cpd-5-3 (390 mg, 650 μmol), (3S,4R)-3-fluoro-1-methylpiperidin-4amine (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 mixed at 100°C for 5 hours. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL). Petition 870260067420, dated 08 / 07 / 2026, page 60 / 113 54 / 106 adjusted to pH = 8 with aqueous 1N HCl solution and extracted with EtOAc / THF (2 : 1.30 mL χ 3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by Prep-HPLC to obtain the product N-(((cyclopropylmethyl)sulfonyl)-5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidine-4-yl)amino)3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)picolinamide Cpd-5 (81.2 mg, 19.1% yield, white solid).LCMS calculado para C30H32D3F4N6O4S [M+H]+: m / z = 654,3; encontrado: 654,7; RMN1H (400 MHz, DMSO-d6) 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).

[0208] Exemplo 6: 5-((3-(8-((((3S,4R)-3-fluoro-1-metilpiperidin-4-il)amino)-3(2,2,2-trifluoroetil)indolizin-2-il)prop-2-in-1-il)amino)-6-(metoxi-d3)-N-((2,2,2trifluoroetil)sulfonil)picolinamida (Cpd-6, ou seja, Composto 6)C

[0209] Etapa 1: N-(2,4-dimetoxibenzil)-2,2,2-trifluoroetano-1-sulfonamida.

[0210] 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), and the system was stirred for 1 hour. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by the SGC method (0 to 1%). Petition 870260067420, dated 08 / 07 / 2026, page 61 / 113 55 / 106 of MeOH added to 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 for C11H14F3NNaO4S [M+Na]+: m / z = 336.1; found: 336.1.

[0211] Step 2: 2,2,2-trifluoroethane-1-sulfonamide

[0212] N-(2,4-dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide Cpd-6-2 (1.45 g, A solution of 4.63 mmol) was dissolved in DCM (15 mL), and trifluoroacetic acid (15 mL) was slowly added, and then the system was stirred for 1 hour at room temperature. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure to obtain 2,2,2-trifluoroethane-1-sulfonamide Cpd-6-3 (851 mg, 99.1% yield, purple solid). The crude product was used directly in the reaction of the next step. 1H NMR (400 MHz, DMSO-d6) δ 7.50 (s, 2H), 4.26 (q, J = 10.0 Hz, 2H).

[0213] Step 3: methyl 5-{[(3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino]}-6-(methoxy-d3)picolinate.

[0214] A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-inal Int2 (700 mg, 2.33 mmol), methyl 5-amino-6-(methoxy-d3)picolinate Cpd-2-3 (432 mg, 2.33 mmol), and acetic acid (280 mg, 4.67 mmol) in MeOH (20 mL) was mixed at room temperature for 16 hours. Then, NaBHaCN (440 mg, 7.00 mmol) was added, and the system was stirred for another 2 hours at room temperature. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL χ 3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na2SO4, and filtered. Petition 870260067420, dated 08 / 07 / 2026, page 62 / 113 56 / 106 and concentrated under reduced pressure. The residue was purified by the SGC method (0 to 70% EtOAc added to PE) to obtain methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)picolinate Cpd-6-4 (625 mg, 53.7% yield, yellow solid). LCMS calculated for C21H15D3BrF3N3O3 [M+H]+: m / z = 499.1 / 501.1; found: 499.0 / 501.0.

[0215] Step 4: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino)-6-(methoxy-d3)picolinic acid.

[0216] Methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)6-(methoxy-d3)picolinate Cpd-6-4 (300 mg, 600 μmol) and sodium hydroxide (72.1 mg, 1.80 mmol) were dissolved in a mixed solvent of THF (9 mL), MeOH (6 mL), and water (1 mL), and the system was stirred at 50°C for 1 hour. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, diluted with water (20 mL), and adjusted to pH = 5 with 1N HCl. The solid was collected by filtration and dried to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2yn-1-yl)amino)-6-(methoxy-d3)picolinic acid Cpd-6-5 (246 mg, yield 84.4%, white solid). LCMS calculated for C20H13D3BrF3N3Ü3 [M+H]+: m / z = 485.1 / 487.1; Found: 485.0 / 487.0.

[0217] Step 5: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino)-6-(methoxy-d3)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide.

[0218] Triethylamine (0.21 mL, 1.48 mmol) was added to a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy3)picolinic acid Cpd-6-5 (240 mg, 490 μmol), 2,2,2-trifluoroethane-1-sulfonamide Cpd-63 (242 mg, 1.48 mmol) and 2-chloro-1-methylpyridinium iodide (316 mg, 1.24 mmol) in DCM Petition 870260067420, dated 08 / 07 / 2026, page 63 / 113 57 / 106 (40 mL) at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was diluted with DCM (100 mL) and washed sequentially with 0.5N HCl aqueous solution (50 mL), saturated sodium bicarbonate aqueous solution (50 mL), and saturated brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by the SGC method (0 to 8% MeOH added to DCM) to obtain 5-((3-(8-bromo3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide Cpd-6-6 (160 mg, 51.3% yield, white solid). LCMS calculated for C22H15DaBrF6N4O4S [M+H]+: m / z = 630.0 / 632.0; found: 629.9 / 631.9.

[0219] Step 6: 5-((3-(8-((((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-((2,2,2trifluoroethyl)sulfonyl)picolinamide.

[0220] Under an argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide Cpd-6-6 (160 mg, 250 μmol), (3S,4R)-3-fluoro-1-methylpiperidine-4-amine (67.0 mg, 510 μmol), BrettPhos Pd G3 (23.0 mg, 30.0 μmol) and cesium carbonate (165 mg, 510 μmol) in THF (13 mL) and NMP (1.3 mL) was mixed at 100°C for 4 hours in a sealed tube. LCMS detection showed that the initial 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 χ 3). The organic layers were combined and washed with water (40 mL) and brine (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Petition 870260067420, dated 07 / 08 / 2026, p. 64 / 113 58 / 106 The residue was purified by SGC (0 to 8% MeOH added to DCM) to obtain the product 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidine-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide Cpd-6 (14.0 mg, yield 8.09%, white solid). LCMS calculated for C28H27D3F7N6O4S [M+H]+: m / z = 682.2; found: 682.5; RMN1H (400 MHz, DMSO-d6) 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).

[0221] Exemplo 7: N-(cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1 metilpiperidina-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizina-2-yl)prop-2-in-1 il)amino)-6-metoxipicolinamida (Cpd-7, ou seja, Compound 7)

[0222] Etapa 1: 6-chloro-N-(cyclopropylsulfonyl)-5-nitropicolinamide. Petition 870260067420, 07 / 08 / 2026, pág. 65 / 113 59 / 106

[0223] At room temperature, cyclopropylsulfonamide (3.59 g, 29.6 mmol) and triethylamine (7.49 g, 74.1 mmol) were sequentially added to a mixture of 6-chloro-5-nitropicolinic 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). The reaction system was stirred at 40°C for 3 hours. LCMS detection showed that the initial material was 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). The pH was adjusted to 2 with 1N aqueous HCl solution. The precipitated solid was filtered, washed with water, and dried to obtain 6-chloro-N-(cyclopropylsulfonyl)-5-nitropicolinamide Cpd-7-1 (8.90 g, crude product, white solid). The crude product was used directly in the reaction of the next step. LCMS calculated for CgHyClNaOõS [MH]-: m / z = 304.0 / 306.0; found: 303.9 / 305.9.

[0224] Step 2: N-(cyclopropylsulfonyl)-6-methoxy-5-nitropicolinamide.

[0225] At room temperature, a solution of sodium methoxide (16.2 mL, 87.3 mmol, 5.4 mol / L) in methanol was added dropwise to a solution of 6-chloro-N(cyclopropylsulfonyl)-5-nitropicolinamide Cpd-7-1 (8.90 g, 29.1 mmol) in methanol (90 mL). The reaction mixture was stirred at room temperature for 30 minutes. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was tempered with aqueous 1N HCl solution, and the pH adjusted to 2. The precipitated solid was filtered, washed with water, and dried to obtain N-(cyclopropylsulfonyl)-6-methoxy-5-nitropicolinamide Cpd-7-2 (6.70 g, crude product, white solid). The crude product was used directly in the reaction of the next step. LCMS calculated for C10H10N3O6S [MH]-: m / z = 300.0; found: 300.0. Petition 870260067420, dated 08 / 07 / 2026, page 66 / 113 60 / 106

[0226] Step 3: 5-amino-N-(cyclopropylsulfonyl)-6-methoxypicolinamide.

[0227] At room temperature, a mixture of N-(cyclopropylsulfonyl)-6-methoxy-5-nitropicolinamide Cpd-7-2 (6.70 g, 22.2 mmol) and palladium on carbon (3.30 g, 5% content) in THF (200 mL) and MeOH (130 mL) was stirred for 16 hours at room temperature under a hydrogen atmosphere. LCMS detection showed that the initial 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-methoxypicolinamide Cpd-7-3 (6.20 g, crude product, white solid). The crude product was used directly in the reaction of the next step. LCMS calculated for C10H12N3O4S [MH]-: m / z = 270.1; found: 270.1.

[0228] Step 4: 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypicolinamide.

[0229] A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)propylaldehyde (Int-2) (1.00 g, 3.03 mmol), 5-amino-N-(cyclopropylsulfonyl)-6-methoxypicolinamide Cpd7-3 (820 mg, 3.03 mmol) and acetic acid (180 mg, 3.03 mmol) in MeOH (15 mL) was mixed at room temperature for 16 hours. Then, NaBH3CN (570 mg, 9.09 mmol) was added, and the system was stirred for a further 2 hours at room temperature. LCMS detection showed that the initial material was completely consumed and the desired compound was detected. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution (50 mL) and extracted with EtOAc (20 mL χ 3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by suspension with dichloromethane to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-N Petition 870260067420, dated 08 / 07 / 2026, page 67 / 113 61 / 106 (cyclopropylsulfonyl)-6-methoxypicolinamide Cpd-7-4 (500 mg, 28.2% yield, yellow solid). LCMS calculated for C23H2iBrF3N4O4S [M+H]+: m / z = 585.0 / 587.0; found: 585.3 / 587.3.

[0230] Step 5: N-(cyclopropylsulfonyl)-5-((3-(8-(((3S, 4R)-3-fluoro-1-methylpiperidin-4yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6methoxypicolinamide.

[0231] Under an argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypicolinamide Cpd-7-4 (500 mg, 850 μmol), (3S,4R)-3-fluoro-1-methylpiperidine-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 mixed at 100°C for 5 hours in a sealed tube. LCMS detection showed that the initial 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 χ 3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure.The residue was purified by Prep-HPLC to obtain the product N-(cyclopropylsulfonyl)-5-((3(8-(((3S,4R)-3-fíuoro-1-methylpiperidine-4-yl)amino)-3-(2,2,2-trífíuoroethyl)indolizine-2yl)prop-2-in-1-yl)amino)-6-methoxypicolinamide Cpd-7 (46.1 mg, yield of 8.3%, bitter solid). LCMS calculated for C29H33F4N6O4S [M+H]+: m / z = 637.2; crossed: 637.1; RMN1H (400 MHz, DMSO-d6) 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. Petition 870260067420, 07 / 08 / 2026, pág. 68 / 113 62 / 106 (m, 1 Η), 3.11-3.03 (m, 2H), 2.84-2.82 (m, 1H), 2.33-2.21 (m, 1H), 2.23 (s, 3H), 2.152.09 (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).

[0232] Example 8: 5-((3-(8-(((3R,4S)-3-fluoro-1-methylpiperidine-4-yl)amino)-3 (2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-in-1-yl)amino)-6-(methoxy-d3)-N(methylsulfonyl)picolinamide (Cpd-8, ou seja, Compound 8) or CMPI, DCM stage 1 CD3OD, KO'Bu NMP5etapa 2 Pd / C, Hz, AcOH MeOH / THF etapa 3 Cpd-8-3 Br lnt-2 1)AcOH, MeOH / DCM 10~15°C, 16h 2) NaBH3CN,ta,2h etapa 4 BrettPhos Pd G3, CS2CO3 THF / DMF, 70°C, 3h .xNH2't etapa 5

[0233] Etapa 1: 6-chloro-N-(methylsulfonyl)-5-nitropicolinamide.

[0234] 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-nitropicolinic acid Cpd-1-1 (50.0 g, 247 mmol) in dichloromethane (500 mL) with stirring. After stirring for 10 minutes, triethylamine (100 g, 988 mmol) was added dropwise. The reaction mixture was then heated to reflux and reacted for about 5 hours. After cooling and concentrating, the residue was diluted with water (250 mL) and adjusted to pH = 2 with concentrated HCl. A large amount of yellow solid precipitated. The mixture was stirred at room temperature overnight, subjected to suction filtration, and the filter cake was rinsed with water (250 mL). The filter cake was dried at 50°C under vacuum to obtain 6-chloro-N-(methylsulfonyl)-5-nitropicolinamide Cpd-8-1 (68.0 g, 98.5% yield, yellow solid). LCMS Petition 870260067420, dated 08 / 07 / 2026, page 69 / 113 63 / 106 calculated for C7H5ClN3O5S [MH]-: m / z = 278.0; found: 278.1.

[0235] Step 2: 6-(methoxy-d3)-N-(methylsulfonyl)-5-nitropicolinamide.

[0236] Under an argon atmosphere, 6-chloro-N-(methylsulfonyl)-5-nitropicolinamide Cpd8-1 (45.0 g, 161 mmol) and deuterated methanol (17.4 g, 482 mmol) were dissolved in anhydrous NMP (450 mL), then the system was cooled to -15°C to 0°C, and a 1M solution of potassium tert-butoxide in tetrahydrofuran (338 mL, 338 mmol) was added dropwise. The system temperature was then controlled between -10°C and 0°C, and the reaction was stirred for 40 minutes, after which 4N HCl (135 mL) was added dropwise to quench the reaction. The system was concentrated under reduced pressure, and water (1.62 L) was added dropwise with stirring. A large quantity of yellow solid was produced and filtered. The filter cake was rinsed with water (500 mL) and methyl tert-butyl ether (500 mL), and the filter cake was recrystallized with acetonitrile water to obtain the product 6-(methoxy-d3)-N-(methylsulfonyl)-5-nitropicolinamide Cpd-8-2 (30.0 g, 67.0% yield, yellow solid).LCMS calculated for C8H7D3N3O6S [M+H]+: m / z = 279.0; found: 279.2.

[0237] Step 3: 5-amino-6-(methoxy-d3)-N-(methylsulfonyl)picolinamide.

[0238] 6-(methoxy-d3)-N-(methylsulfonyl)-5-nitropicolinamide Cpd-8-2 (30.0 g, 108 mmol) was mixed with THF (600 mL) and acetic acid (3.24 g, 53.9 mmol) and stirred to form a solution, then methanol (600 mL) was added. After argon substitution, palladium on carbon (3.00 g, 10% content) was added, and the argon was replaced by hydrogen, and then the reaction was stirred at room temperature for 12 hours. The reaction mixture was filtered through diatomaceous earth, and the filter cake was rinsed with THF. The filtrate was combined and Petition 870260067420, dated 08 / 07 / 2026, pp. 70 / 113 64 / 106 concentrate. The residue was dried to obtain the product 5-amino-6-(methoxy-d3)-N(methylsulfonyl)picolinamide Cpd-8-3 (26.0 g, 97.1% yield, dirty white solid), which was used directly in the next step without further treatment. LCMS calculated for C8H9D3N3O4S [M+H]+: m / z = 249.1; found: 249.2.

[0239] Step 4: 8-bromo-2-(3-((2-(methoxy-d3)-6(((methylsulfonyl)carbamoyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-3-(2,2,2trifluoroethyl)indolizine.

[0240] A mixed solvent of methanol (100 mL) and dichloromethane (100 mL) was added to 5-amino-6-(methoxy-d3)-N-(methylsulfonyl)picolinamide Cpd-8-3 (7.20 g, 29.0 mmol) and acetic acid (3.14 g, 58.0 mmol). When the mixture became clear, Int-2 (10.1 g, 30.5 mmol) was added to it. The system was stirred between 10°C and 15°C for 16 hours. A large amount of turbidity was produced in the system, and then sodium cyanoborohydride (6.00 g, 87.1 mmol) was added, and the system was stirred for a further 2 hours. The system was concentrated, methanol (100 mL) was added, water (100 mL) was added dropwise at room temperature, then it was stirred at room temperature for 2 hours and filtered.The filter cake was rinsed with water (50 mL) and methyl tert-butyl ether (100 mL), and vacuum dried with an oil pump to obtain 8-bromo-2-(3-((2-(6-((methylsulfonyl)carbamoyl)-2-(methoxy3)pyridine-3-yl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-8-4 (12.9 g, 79.0% yield, dirty white solid). LCMS calculated for C21H16D3BrF3N4O4S [M+H]+: m / z = 562.0 / 564.0; found: 562.2 / 564.2.

[0241] Step 5: 5-((3-(8-(((3R,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-3Petition 870260067420, of 07 / 08 / 2026, page 71 / 113 65 / 106 (2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N(methylsulfonyl)picolinamide.

[0242] Under an argon atmosphere, a mixture of 8-bromo-2-(3-((2-(methoxyd3)-6-((methylsulfonyl)carbamoyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-3-(2,2,2trifluoroethyl)indolizine Cpd-8-4 (1.80 g, 3.20 mmol), (3R,4S)-3-fluoro-1methylpiperidine-4-amine (500 mg, 3.80 mmol), BrettPhos Pd G3 (600 mg, 662 μmol), and cesium carbonate (3.44 g, 10.6 mmol) in THF (20 mL) and DMF (10 mL) were mixed 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 5-((3-(8-(((3R,4S)-3fluoro-1-methylpiperidine-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2yn-1-yl)amino)-6-(methoxy-d3)-N-(methylsulfonyl)picolinamide Cpd-8 (830 mg, 42.3% yield, dirty white solid).LCMS calculated for C27H28D3F4N6O4S [M+H]+: m / z = 614.2; found: 614.4; RMN1H (400 MHz, DMSO-d6) 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), 6, 6, 1H (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 = 49,6 (1H), 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), 2,0.4 2.05–1.91 (m, 1H), 1.69 (dd, J = 13.2, 4.0 Hz, 1H).

[0243] Example 9: 5-((3-(8-(8-((3S,4S))-3-fluoro-(((3S,4S)-amino)-3(2,2,2-trifluoroethyl)indolyzine-2-yl)prop-2-in-1-yl)sep-amino)-6-(methoxy-d-sulfone,9)da)(Cpicethyl Compound 9) Petition 870260067420, dated 08 / 07 / 2026, p. 72 / 113 66 / 106 BrettPhos Pd G3, Cs2CO3 THF / DMF, 70°C stage 1

[0244] Stage 1: 5-((3-(8-(((3S,4S)-3-fluoro-1-methylpiperidine-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizine-2-yl)prop-2-in-1-yl)amino)-6-(methoxy-d3)-N(methylsulfonyl)picolinamida.

[0245] Under an argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy3)picolinamide Cpd-8-4 (1.80 g, 3.20 mmol), (3S,4S)-3-fluoro-1-methylpiperidine-4amine (400 mg, 3.02 mmol), BrettPhos Pd G3 (600 mg, 662 pmol) and cesium carbonate (3.44 g, 10.6 mmol) in THF (20 mL) and DMF (10 mL) were mixed 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 5-((3-(8-(((3S,4S)-3-fluoro-1-methylpiperidine-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N(methylsulfonyl)picolinamide Cpd-9 (500 mg, 26.9% yield, brown solid).LCMS calculated for C27H28D3F4N6O4S [M+H]+: m / z = 614.2; found: 614.4; RMN1H (400 MHz, DMSO-d6) 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), 7.7 Hz, 14 (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, 2, 18, 79.4, 4 (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, 2H4), 2.19–1.90 (m, 3H), 1.61–1.39 (m, 1H). Petition 870260067420, dated 08 / 07 / 2026, p. 73 / 113 67 / 106

[0246] Example 10: 5-((3-(8-(((3R,4R)-3-fluoro-1-methylpiperidine-4-yl)amino)-3(2,2,2-trifluoroethyl)indolyzine-2-yl)prop-2-in-1 -yl)amino)-6-(methoxy-d3)-N(methylsulfonyl)picolinamide (Cpd-10, or soybean, Compound 10)

[0247] Etapa 1: 5-((3-(8-(((3R,4R)-3-fluoro-1-methylpiperidine-4-yl)amino)-3-(2,2,2trifluoroethyl)indolizine-2-yl)prop-2-in-1-yl)amino)-6-(methoxy-d3)-N(methylsulfonyl)picolinamida.

[0248] Under an argon atmosphere, a mixture of 8-bromo-2-(3-((2-(methoxy-d3)-6(((methylsulfonyl)carbamoyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-3-(2,2,2trifluoroethyl)indolizine) Cpd-8-4 (1.80 g, 3.20 mmol), (3R,4R)-3-fluoro-1methylpiperidine-4-amine (500 mg, 3.78 mmol), BrettPhos Pd G3 (600 mg, 662 pmol) and cesium carbonate (3.44 g, 10.6 mmol) in THF (20 mL) and DMF (10 mL) were mixed 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 5-((3-(8-(((3R,4R)-3-fluoro-1-methylpiperidine-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizine-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d3)-N-(methylsulfonyl)picolinamide Cpd-10 (1.12 g, 57.0% yield, brown solid).LCMS calculated for C27H28D3F4N6O4S [M+H]+: m / z = 614.2; found: 614.4; NMR1H (400 MHz, DMSO-d6) 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), . Petition 870260067420, dated 07 / 08 / 2026, p. 74 / 113 68 / 106 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).

[0249] Biological assessment E

[0250] Biochemical assay: Most compounds promote the binding of the p53 Y220C mutant to DNA.

[0251] The time-resolved homogeneous fluorescence (HTRF) assay was used to measure the reactivation effect of the compounds on the p53 Y220C mutant. The recombinant His-labeled p53 Y220C (94-312) used for HTRF assays was expressed in E. coli and purified to 90% purity using a Ni-NTA column. The biotin-labeled DNA used for HTRF assays was synthesized by Pharmaron Inc., with the specific sequence 5'-ATTAGGGTCTAGGCATGTCTAGG-3'.

[0252] The binding of recombinant Him-labeled p53 Y220C protein and biotin-labeled DNA was measured using fluorescence resonance energy transfer (FRET). For FRET assays, the binding between p53 mutants and DNA sequences was measured by detecting the fluorescence interaction between an anti-His antibody (Cisbio, 61HI2KLA) conjugated with Eu and d2-conjugated streptavidin (Cisbio, 610SADLF) bound to a biotin-labeled DNA molecule. Compounds were prepared in 2 mM standard solutions and serially diluted to 10 concentrations at a 1:3 ratio in DMSO. Each compound at a working concentration of 200 times was sequentially diluted to a working concentration of 4 times. 4 μL of compound solution were transferred row by row to a 384-well assay plate using Echo, containing 2 Petition 870260067420, dated 08 / 07 / 2026, pp. 75 / 113 69 / 106 replicates per column. 4 μL of p53 solution were added to the assay plate. 4 μL of biotin DNA solution were added to the assay plate, and then 4 μL of the assay solution (His-I antibody and streptavidin-d2) were added to each well of the assay plate. The assay plate was incubated overnight and protected from light. Fluorescence was detected on the BMG (BMG LRBTECH).

[0253] The ratio of each well was calculated (ratio_665 nm / 615 nm-ratio_background). The percentage of activity was calculated as follows:

[0254] C = (Ave_Ac-Ave_Ba) / (Ave_Dc-Ave_Bd)

[0255] R data = (A-Ave_Ba-C*D) / (D-Ave_Bd)*(Ave_Dc-Ave_Bd)

[0256] Activity % = R data / Ave_VC*100.

[0257] A: fluorescence intensity of the sample at 665 nm;

[0258] D: fluorescence intensity of the sample at 615 nm;

[0259] Ba: fluorescence intensity of the plate background at 665 nm;

[0260] Bd: fluorescence intensity of the plate background at 615 nm;

[0261] DC: fluorescence intensity of the HIS-EU background at 615 nm;

[0262] Ac: background fluorescence intensity of streptavidin-d2 at 665 nm;

[0263] VC: DMSO treatment group.

[0264] The SC150 was calculated using Graphpad 8.0 to fit the logarithm of percentage activity and compound concentration to a nonlinear regression (slope of the dose-response variable).

[0265] Table 2: Activity of compounds that promote the binding of the p53 mutant Y220C with DNA Composite number SC150 (nM) Cpd-A 10 Petition 870260067420, dated 08 / 07 / 2026, page 76 / 113 70 / 106 Cpd-B 15 Cpd-C 4.6 Cpd-1 1.0 Cpd-2 0.58 Cpd-3 0.74 Cpd-4 0.35 Cpd-5 1.1 Cpd-6 1.7 Cpd-7 0.64

[0266] CPD-A is selected from WO2021061643A1; CPD-B is selected from WO2022213975A1;

[0267] The data above show that the representative compounds of the present disclosure have good activity in promoting the binding of p53 Y220C mutants to DNA, and are clearly superior to the reference compounds.

[0268] II. Cellular assay: It is a proliferation effect of nti in NUGC-3 cells

[0269] The antiproliferative effect was evaluated against the human gastric adenocarcinoma cell line based on dehydrogenase activity in the cells, which showed a good correlation with the number of live cells. The NUGC-3 cell line (JCRB, JCRB0822) was cultured in RPMI 1640 medium (Invitrogen, 11875-085) supplemented with 10% (v / v) fetal bovine serum (BI, 04-002-1A), according to standard instructions from the American Type Culture Collection. The support line was authenticated by short tandem repeat profiling.

[0270] In the proliferation assay, 1000 NUGC-3 cells were seeded in a Petition 870260067420, dated 08 / 07 / 2026, page 77 / 113 71 / 106 flat-bottomed, transparent 96-well plates were treated with TC (Corning, 3903) in 200 μL of medium per well, and allowed to recover ED overnight in culture medium. Compounds were added at increasing concentrations, with DMSO treatment serving as a positive control. After 5 days of treatment, the plate was equilibrated at room temperature for 30 minutes, and the number of live cells was measured by adding the Cell Counting Kit reagent (MCE, HY-K0301). Absorption was measured using an Enspire (Perkin Elme). The relative viability of each group was presented as the percentage change from the positive control group and then fitted to a four-parameter nonlinear logit curve using the XLFit program (IDBS).

[0271] Table 3: Antiproliferation activity of compounds in NUGC-3 cells Composite number NUGC-3 IC50 (pM) Cpd-A 0.78 Cpd-B 0.36 Cpd-C 0.23 Cpd-1 0.10 Cpd-2 0.058 Cpd-4 0.097 Cpd-5 0.081 Cpd-6 0.19 Cpd-7 0.070 Cpd-8 0.34 Cpd-9 0.55 Cpd-10 0.90

[0272] The data above show that the representative compounds of the present disclosure have good inhibitory activity on the proliferation of NUGC-3 cells, and are clearly superior to the reference compounds s. Petition 870260067420, dated 07 / 08 / 2026, p. 78 / 113 72 / 106

[0273] III. Evaluation of drug-drug interactions related to metabolic enzymes

[0274] (i) Test of inhibitory activity on the ISOCYP s enzyme at a single concentration

[0275] 1. Experimental purpose: To test the inhibitory effect of compounds on ISOCYP enzymes at a single concentration.

[0276] 2. Experimental instrument and reagents:

[0277] Liver microsome: Species Genus Supplier Cat. No. Lot Storage Human Mix Corning 452117 38298 -80°C

[0278] Table 4: Reagents: Compound Supplier Cat. No. Storage Terfenadine Sigma-Aldrich T9652 4°C Tolbutamide Sigma-Aldrich T0891 4°C Potassium hydrogen phosphate Sinopharm 20032117 Room temperature Reducing nicotinamide adenine dinucleotide phosphate Shanghai ABCONE Biotechnology Co., Ltd. N99640-100MG -20°C Phenacetin Sigma-Aldrich 101690303 Room temperature Diclofenac Sigma-Aldrich D6899-10G Room temperature Mephenytoin Glpbio GC14486 -20°C Dextromethorphan Sigma-Aldrich D9684-5G Room temperature Midazolam National Institute of Food Control PVJT-0H9Z Room temperature Petition 870260067420, dated 08 / 07 / 2026, pp. 79 / 113 73 / 106 Testosterone and Medications Adamas Reagent 171265 Room temperature Bupropion Cayman Islands Chemical Company 10488 -20°C Amodiaquine Abmole M5412 -20°C β-Naphtholavone Sigma-Aldrich N5757-1G 4°C Sulfaphenazole Sigma-Aldrich S0758-1G 4°C Benzylnirvanol Shanghai Yuanye BioTechnology Co., Ltd. Y43177-5 mg Room temperature Quinidine International Laboratory-USA 1311468- 5G Room temperature Ketoconazole Sigma-Aldrich K1003- 100MG 4°C

[0279] 3. Experimental methods:

[0280] 8.71 g of K2HPO4 were dissolved in 950 mL of water. The pH was adjusted to 7.4 with HCl solution. The mixture was brought to a final volume of 1000 mL with water. The buffer was filtered through a 0.45 μm filter. The buffer was stored in a refrigerator at 4°C for future use. Stock solutions of terfenadine / tolbutamide (1 mg / mL each) were prepared with DMSO. The above standard solution was diluted with acetonitrile to produce a quenching solution containing 5 / 10 ng / mL (terfenadine / tolbutamide).

[0281] Table 5: Operating solutions with positive control: CYP enzyme inhibitor Solution concentration Working solution concentration (mM) Incubation concentration (pM) Stock solution volume (pL) Total volume (pL) Format Petition 870260067420, dated 08 / 07 / 2026, pages 80 / 113 74 / 106 Standard (mM) 1A2 β-Naphthoflavone 10 2 10 10 50 5 in 1 2C9 Sulfaphenazole 10 2 10 10 2C1 9 Bentilnirvanol 5 1 5 10 2D6 Quinidin 10 2 10 10 3A4 / 5 Ketoconazole 10 2 10 10 Ketoconazole 2 2 10 50 50 Single

[0282] Table 6: Working solutions with substrate: CYP Enzyme Substrate Standard solution concentration (mM) Working solution concentration (mM) Incubation concentration (pM) Stock solution volume (pL) Total volume (pL) Format 1A2 Phenacetin 250 50 50 2 10 5 in 1 2C9 Diclofenac 50 10 10 2 2C19 Mephenytoin 225 45 45 2 2D6 Dextromethorphan 25 5 5 2 3A4 / 5 Midazolam 12.5 2.5 2.5 2 Petition 870260067420, dated 08 / 07 / 2026, pages 81 / 113 75 / 106 Testosterone 50 50 50 5 5 Single

[0283] Liver microorganisms were thawed in a water bath at 37°C before use. A working solution with liver microsomes (final concentration of 0.5 mg / mL) was prepared. A 5 mM NADPH working solution was prepared with phosphate-buffered saline. The above standard solution was prepared in a 2 mM functional solution with DMSO.

[0284] 238.5 μL of liver microsome working solution were added to a 1.1 mL tube. 1.5 μL of test compound / control working solution / DMSO were added and mixed well by pipetting several times. The mixture was pre-incubated in a water bath with shaking at 37°C for 5 minutes. After pre-incubation, 60 μL of NADPH working solution were added and mixed well by pipetting several times. The mixture was incubated in a water bath with shaking at 37°C for 10 minutes. Immediately after incubation, 500 μL of quenching solution were added and vortexed for 1 minute. All samples were centrifuged at 4,000 rpm for 15 minutes at 4°C. 300 μL of the above supernatants were aliquoted for further LC-MS / MS analysis.

[0285] 4. Data processing methods and results:

[0286] The inhibition rate (% inhibition) is calculated using the formation of metabolites of the test compound or the control compound compared to the matrix control.

[0287] Table 7: Inhibitory activity of compounds on ISOCYP* enzymes Compound Number Inhibition Rate (%) CYP1A 2 CYP2C 9 CYP2C1 9 CYP2D6 CYP3A4 / 5 (Mida- CYP3A4 / 5 (Testos- Petition 870260067420, dated 07 / 08 / 2026, p. 82 / 113 76 / 106 zolam) terone) Cpd-A 12 38 42 5.5 83 ND Cpd-B 0.56 21 31 3.6 61 75 Cpd-C 18 43 67 9.5 79 ND Cpd-1 NI 6.7 NI 1.6 9.8 3.7 Cpd-2 NI 8.9 8.4 7.2 8.7 NI

[0288] *The final concentration of the compound is 10 μM; ND: not detected; NI: no inhibition

[0289] The data above show that the compounds in this disclosure have relatively weak inhibitory activity on ISO CYP enzymes, and the risk of drug-drug interaction is significantly lower than that of the reference compounds.

[0290] (ii) Determination of the multiconcentration oral inhibitory activity curve of CYP3A4 / 5 enzymes

[0291] 1. Experimental objective: To determine the concentration that inhibits the activity curve of compounds in CYP3A4 / 5 enzymes.

[0292] 2. Experimental instrument and materials:

[0293] Table 8: Liver microsome: Species Genus Supplier Cat. No. Lot Storage Human Mix Corning 452117 38298 -80°C

[0294] Table 9: Reagents: Compound Supplier Cat. No. Storage Terfenadine Sigma-Aldrich T9652 4°C Tolbutamide Sigma-Aldrich T0891 4°C Potassium hydrogen phosphate Sinopharm 20032117 Room temperature Petition 870260067420, dated 08 / 07 / 2026, page 83 / 113 77 / 106 Reduction of nicotinamide adenine dinucleotide phosphate Shanghai ABCONE Biotechnology Co., Ltd. N99640- 100MG -20°C Midazolam National Institute of Food and Drug Control PVJT-0H9Z Room temperature Testosterone Adamas Reagent 171265 Room temperature Ketoconazole Sigma-Aldrich K1003-100MG 4°C

[0295] 3. Experimental methods:

[0296] 8.71 g of K2HPO4 were dissolved in 950 mL of water. The pH was adjusted to 7.4 with HCl solution. The mixture was brought to a final volume of 1000 mL with water. The buffer was filtered through a 0.45 μm filter. The buffer was stored in a refrigerator at 4°C for future use. Stock solutions of terfenadine / tolbutamide (1 mg / mL each) were prepared with DMSO. The above standard solution was diluted with acetonitrile to produce a quenching solution containing 5 / 10 ng / mL (terfenadine / tolbutamide).

[0297] Table 10: Operating solutions with positive control: Enzyme CYP Inhibitor Concentration of original solution (mM) Concentration of working solution (mM) Incubation concentration (pM) Volume of stock solution (pL) Total volume (pL) 3A4 / 5 Ketoconazole 10 2 10 10 50 Ketoconazole 2 2 10 50 50 Petition 870260067420, dated 08 / 07 / 2026, page 84 / 113 78 / 106 ol

[0298] Table 11: Working solutions with substrate: CYP Enzyme Substrate Original solution concentration (mM) Working solution concentration (mM) Incubation concentration (pM) Stock solution volume (pL) Total volume (pL) 3A4 / 5 Midazolam 12.5 2.5 2.5 2 10 Testosterone 50 50 50 5 5

[0299] Liver microorganisms were thawed in a water bath at 37°C prior to use. A working solution with liver microsomes (final system concentration of 0.5 mg / mL) was prepared. A 5 mM NADPH working solution was prepared with phosphate-buffered saline. The above standard solution was prepared in a 2 mM functional solution with DMSO. The above standard solution was diluted with acetonitrile (4-fold dilution, 6 non-zero concentrations).

[0300] 238.5 μL of liver microsome working solution were added to a 1.1 mL tube. 1.5 μL of test compound / control working solution / DMSO were added and mixed well by pipetting several times. The mixture was pre-incubated in a water bath with shaking at 37°C for 5 minutes. After pre-incubation, 60 μL of NADPH working solution were added and mixed well by pipetting several times. The mixture was incubated in a water bath with shaking at 37°C for 10 minutes. Immediately after incubation, 500 μL of quenching solution were added and vortexed for 1 minute. Petition 870260067420, dated 08 / 07 / 2026, page 85 / 113 79 / 106 All samples were centrifuged at 4,000 rpm for 15 minutes at 4°C. 300 μL of the above supernatants were aliquoted for additional LC-MS / MS analysis.

[0301] 4. Data processing methods and results:

[0302] The half-maximum inhibitory concentration (IC50) was calculated using GraphPad Prism.

[0303] Table 12: Inhibitory activity of compounds on CYP3A4 / 5 enzymes Composite number CYP3A4 / 5 IC50 (pM) (Midazolam) CYP3A4 / 5 IC50 (pM) (Testosterone) Cpd-A 2.3 0.89 Cpd-B 5.8 3.2 Cpd-C 3.8 ND Cpd-1 >50 >50 Cpd-2 >50 >50

[0304] ND: Not detected

[0305] The data above show that the representative compounds of this disclosure do not exhibit evident inhibitory activity on CYP3A4 / 5 enzymes, and the risk of drug-drug interaction is significantly lower than that of the reference compounds.

[0306] IV. In vitro assessment of metabolic stability: Study on the metabolic stability of liver microsomes:

[0307] 1. Experimental purpose: To evaluate the metabolic stability of the test compound in human / rat / mouse liver microsomes.

[0308] 2. Experimental reagents and materials:

[0309] Table 13: Reagents: Petition 870260067420, dated 08 / 07 / 2026, page 86 / 113 80 / 106 Reagent Name Manufacturer Cat. No. Storage Conditions Terfenadine Sigma-Aldrich T9652 2 to 8°C Tolbutamide Sigma-Aldrich T0891 2 to 8°C K2HPO4 SCR 20032117 RT Reduction of nicotinamide adenine dinucleotide phosphate (NADPH) ACROS 32874250 0 -20°C

[0310] Table 14: Materials: Genus Name Manufacturer Cat. Microsome Conditions Genus Corning 452117 -80°C Male Microsome BioreclamationIVT M00001 -80°C Male Microsome BioreclamationIVT M00501 -80°C

[0311] 3. Experimental methods:

[0312] The test compound and the positive control compound (dextromethorphan as the control compound) were dissolved in DMSO to prepare a standard solution with a concentration of 10 mM, and the above standard solution was diluted to a 200 μM working solution with DMSO. 8.709 g of potassium hydrogen phosphate (K2HPO4) were dissolved in 950 mL of water, and the pH of the solution was adjusted to 7.4 with hydrochloric acid, and then the volume was adjusted to 1000 mL with water. After filtration using a 0.22 μm filter membrane, the mixture was stored in a refrigerator at 4°C for later use. After liver microsomes of various species (protein concentration of 20 mg / mL) were thawed in a water bath at 37°C, the liver microorganisms were diluted Petition 870260067420, dated 08 / 07 / 2026, page 87 / 113 81 / 106 with a phosphate buffer, respectively, to obtain a functional solution of liver microsomes with a protein concentration of 0.629 mg / mL. A 5 mM NADPH solution was prepared using the above phosphate buffer for later use. A 1 mg / mL terfenadine / tolbutamide stock solution was prepared with DMSO and then diluted with a 50% methanol / 50% acetonitrile mixed solution to a reaction stop solution containing 5 / 10 ng / mL (terfenadine / tolbutamide) internal standard.

[0313] 238.5 μL of working solutions of liver microsomes of various species were added to a 1.1 mL microtube, and 1.5 μL of the working solution of the test compound or the working compound of the positive control compound (dextromethorphan) (200 μM) were added, respectively, well mixed, and pre-incubated in a water bath at 37°C for 5 minutes. 60 μL of NADPH solution were added to initiate the reaction, and after thorough mixing, at 0, 5, 15, 30, and 60 minutes post-reaction, 30 μL of the reaction mixture were pipetted and added to 300 μL of the reaction stop solution. Samples from all time points were vigorously vortexed for 1 minute and centrifuged at 4000 rpm for 15 minutes at 4°C. 100 μL of the supernatant were added to 100 μL of pure water and mixed well for LC-MS / MS analysis.

[0314] 4. Data processing methods:

[0315] The slope (ke) was determined by plotting the natural logarithm of the percentage of compound remaining against time, and T1 / 2 and intrinsic clearance (CLint) were calculated based on the first-order kinetic formula:

[0316] The compound remaining rate is calculated as follows: Petition 870260067420, dated 08 / 07 / 2026, page 88 / 113 82 / 106 % remaining rate ratio of compound peak area to internal standard peak area at each time point ratio of compound peak area to internal standard peak area at initial T0 time point x 100

[0317] Intrinsic clarity CLint ^L / min / mg of protein) = 0.693 * 1000 / T1 / 2 / protein concentration (0.5 mg of protein / mL)

[0318] Table 15: Metabolic stability of compounds in human / rat / mouse liver microsomes Composite number Species In vitro t1 / 2 (min) In vitro Clint ^L / min / mg protein) Cpd-A Human 41 33.6 Rat 30 46.1 Rat 27 50.7 Cpd-C Human 49 28.3 Rat 23 61.5 Rat 17 83.9 Cpd-1 Human 136 7.3 Rat 28 35.0 Rat 72 13.7 Cpd-2 Human >256 <5.4 Rat 119 11.7 Rat 110 12.6

[0319] The above data show that the representative compounds of this disclosure have excellent metabolic stability in liver microorganisms of various species and are clearly superior to the reference compounds.

[0320] V. In vitro assessment of metabolic stability: Stability test Petition 870260067420, dated 08 / 07 / 2026, page 89 / 113 83 / 106 metabolic in hepatocytes

[0321] 1. Experimental purpose: To determine the metabolic stability of compounds S in human / rat / mouse hepatocytes.

[0322] Table 16: Experimental materials: Species Deformity Gender Supplier Human N / A Mix BioIVT Sprague Dawley Rat Male BioIVT ICR / CD-1 Rat Male BioIVT

[0323] 3. Experimental design

[0324] 3.1 Preparation of the composite working solution

[0325] The test compound and the verapamil powder control drug were prepared in a high-concentration stock solution using DMSO, and diluted in a 100 μM working solution with 50% acetonitrile / water before use, with final concentrations of 1 μM for the test compound and verapamil.

[0326] 3.2 Hepatocyte preparation

[0327] 1) 49.5 mL of Williams Medium E and 0.5 mL of GlutaMAX were mixed as an incubation solution. The hepatocyte recovery solution and the incubation solution were pre-warmed in a water bath at 37°C for at least 15 minutes before use.

[0328] 2) A tube of cryopreserved hepatocytes was removed, ensuring that the hepatocytes were still in a cryogenically frozen state before resuscitation. The hepatocytes were quickly placed in a water bath at 37°C and gently shaken until all ice crystals were completely dispersed, sprayed with 70% ethanol, and transferred to a Petition 870260067420, dated 08 / 07 / 2026, pages 90 / 113 84 / 106 biosafety cabinet.

[0329] 3) The contents of the hepatocyte tube were poured into a centrifuge tube containing 50 mL of recovery medium, which was centrifuged at 100 g for 10 minutes. After centrifugation, the recovery medium was aspirated, and sufficient incubation medium was added to obtain a cell suspension with a cell density of approximately 1.5 χ 106 cells / mL.

[0330] 4) Vision in a cellometer was used to count hepatocytes and determine the density of viable cells. The survival rate of hepatocytes should be greater than 75%. The hepatocyte suspension was diluted with incubation medium to a viable cell density of 0.5 χ 106 cells / mL.

[0331] 5) A portion of the hepatocyte suspension with a density of 0.5 χ 106 cells / mL was boiled in water for 5 minutes to inactivate it as a negative control. After cell inactivation, substrate conversion mediated by non-cellular enzymes could be easily investigated.

[0332] 3.3 Experimental methods

[0333] 1) 198 μL of the live or inactivated cell suspension were transferred to a 96-well deep well plate, which was then placed in a vortex and pre-warmed in an incubator for 10 minutes. Live cells were incubated in duplicate, while inactivated cells were incubated in isolation.

[0334] 2) 2 μL of 100 μM test compound or verapamil were added to each well to initiate the reaction, and the deep well plate was placed back into the incubator vortex.

[0335] 3) 25 μL of the suspension were taken from the samples incubated for 0, 15, 30, 60, 90, and 120 minutes, respectively, and added to 150 μL of internal standards. Petition 870260067420, dated 08 / 07 / 2026, pages 91 / 113 85 / 106 containing acetonitrile (200 nM alprazolam, 200 nM labetalol, 2 μM ketoprofen and 200 nM caffeine) to complete the reaction. The mixture was vortexed for 10 minutes and centrifuged at 3220 g and 4°C for 30 minutes. After centrifugation was complete, 100 μL of the supernatant and 100 μL of ultrapure water were thoroughly mixed for UPLC-MS / MS analysis.

[0336] 4. Data analysis

[0337] All data calculations were performed using Microsoft Excel software. Peak areas were detected by extracting the ion chromatogram. The in vitro half-life (t1 / 2) of the original drug was detected by linear fitting of the natural logarithm of the percentage of the original drug eliminated over time.

[0338] The in vitro half-life (t^ / 2) was calculated using the slope:

[0339] In vitro T1 / 2 = 0.693 / K

[0340] Intrinsic in vitro clearance (unit μL / min / 106 cells) was calculated using the following formula:

[0341] in vitro CLint = kV / N

[0342] V = incubation volume per well (0.2 mL);

[0343] N = number of cells per well (0.1 x 106 cells)

[0344] Table 17: Metabolic stability of compounds in human / rat / mouse hepatocytes Composite Number Species In vitro t1 / 2 (min) In vitro Clint (pL / min / 106 cells) Cpd-A Human 61 22.7 Rat 73 19.0 Rat 164 8.5 Cpd-B Human 82 16.8 Petition 870260067420, dated 08 / 07 / 2026, pages 92 / 113 86 / 106 Rat 22 63.8 Rat 7 197.9 Cpd-C Human 75 18.4 Rat 88 15.7 Rat 44 31.8 Cpd-1 Human >511 <2.7 Rat >511 <2.7 Rat >511 <2.7 Cpd-2 Human >511 <2.7 Rat >511 <2.7 Rat >511 <2.7

[0345] The above data show that the representative compounds of this disclosure have excellent metabolic stability in human / rat / mouse hepatocytes and are clearly superior to the reference compounds.

[0346] VI. Cardiac safety assessment: hERG potassium channel inhibitory activity test.

[0347] 1. Experimental purpose:

[0348] The manual contact clamp technique was used to evaluate whether the tested compounds have potential inhibitory effects on the voltage-dependent potassium channel hERG. This experiment was conducted to detect the effect of compounds at five concentrations on the hERG channel current, obtaining a dose-effect curve and calculating the IC50.

[0349] Table 18. Experimental instrument and reagents: Experimental Materials Supplier (Cat. No.) 6 cm Cell Culture Dish 3.5 cm Cell Culture Dish Shanghai Yes Service Biotech, Inc. (150462) Shanghai Yes Service Biotech, Inc. (150460) Petition 870260067420, dated 08 / 07 / 2026, pages 93 / 113 87 / 106 cm Dialyzed fetal bovine serum Shanghai Bohan Biotechnology Co., Ltd. (BS-0005-500) DMSO Beijing Solarbio Science & Technology Co., Ltd. (D8371) DMEM medium Thermo Fisher Scientific (China) Co., Ltd. (10569044) HEPES Thermo Fisher Scientific (China) Co., Ltd. (15630080) Trypsin Thermo Fisher Scientific (China) Co., Ltd. (12604) Phosphate-buffered saline solution Beijing Solarbio Science & Technology Co., Ltd. (without calcium and magnesium ions) (D1040) Streptomycin penicillin solution Thermo Fisher Scientific (China) Co., Ltd. (15140122) MEM non-essential amino acid solution Thermo Fisher Scientific (China) Co., Ltd. (11140050) Geneticin (G418) Thermo Fisher Scientific (China) Co., Ltd. (11811031) Blasticidin Thermo Fisher Scientific (China) Co., Ltd. (R21001) Polylysine Thermo Fisher Scientific (China) Co., Ltd. (P4832) Dofetilide Beijing Express Technology Development Co., Ltd. (D525700) Doxycycline Sigma-Aldrich (Shanghai) Trading Co., Ltd.(D9891) Thermo Fisher Scientific (China) Co., Ltd. carbon dioxide incubator (Thermo371) NARISHIGE Company glass electrode extraction machine from Japan (PC-10 Puller). Petition 870260067420, dated 08 / 07 / 2026, pages 94 / 113 88 / 106 Micromanipulator positioner, Qingdao Sources Optics Co., Ltd. perfusion system (MC1000e), American ALA Company (VM8 drug gravity delivery system), vacuum pump, German company Chemvak (V300), osmometer, Ashern Company (Osmo310).

[0350] 3. Experimental methods:

[0351] Cell line and cell culture: The HEK293 cell line stably expressing the hERG ion channel (Cat. No.: K1236) was acquired from Invitrogen Company. The 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 cell density increased to 40% to 80% of the bottom area of ​​the culture dish, the cells were digested with trypsin and passed three times a week. Prior to the experiment, cells were cultured in a 3.5 cm culture dish with a density of 5 χ 105, induced with 1 μg / mL of doxycycline for 48 hours, and then the cells were digested and seeded onto glass slides for subsequent manual patch clamp experiments.

[0352] Solution preparation: Extracellular fluid (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 fluid (in mM): 10 EGTA, 10 HEPES, 10 potassium chloride, 10 sodium chloride and 110 potassium fluoride (pH adjusted to 7.2 with potassium hydroxide). The osmotic pressure of the solution was controlled between 280 and 300 mOsmol / kg. The solution was filtered and stored at 4°C before use.

[0353] Preparation of the composite test solution: Following the procedure Petition 870260067420, dated 08 / 07 / 2026, pages 95 / 113 Following standard operating procedure 89 / 106, the test compounds were dissolved in DMSO and prepared in a standard solution with a final concentration of 10 mM. The standard solution was gradually diluted with DMSO as solvent in a 1:3 ratio in three other intermediate concentration solutions at concentrations of (mM): 3.33, 1.11, and 0.37, respectively. Before the start of the experiment, the intermediate gradient solution of the test compound was again diluted with extracellular fluid in a 1:1000 ratio in working solutions of a series of concentrations, and the 30 μM working solution was prepared in a 1:333.33 ratio using a 10 mM standard solution, whose final concentrations were (mM): 30, 10, 3.33, 1.11, and 0.37, respectively. The DMSO content in the functional solution was 0.1 to 0.3% (v / v).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 compounds on hERG channels and to fit the dose-effect curve and calculate the IC50.

[0354] Experimental procedure: A coating loaded with HEK293 cells in a culture dish was placed in the perfusion chamber of the microscope stage. A suitable cell was placed in the center of the field of view under an inverted Olympus IX71 or IX73 microscope, and the tip of the glass electrode was found using a *10 objective lens, placed in the center of the field of view. Then, the electrode was moved downwards using a micromanipulator, while the coarse focus screw was adjusted to slowly bring the electrode closer to the cell. At the moment of approaching the cell, the objective lens was changed to a *40 objective lens for observation, and an electrode was gradually brought closer to the cell surface using a micromanipulator for fine adjustment. The pressure Petition 870260067420, dated 08 / 07 / 2026, pages 96 / 113 A negative 90 / 106 voltage was applied to form a seal between the electrode tip and the cell membrane with a resistance greater than 1 GΩ. The transient capacitive current Cfast was compensated in voltage clamp mode. Brief negative pressure was then applied repeatedly to break the membrane, eventually forming a whole-cell recording mode. Under the condition that the membrane potential was fixed at -60 mV, the slow capacitive current Cslow, the cell membrane capacitance (Cm), and the input membrane resistance (Ra) were compensated, respectively. After cell stability, the clamp voltage was changed to -90 mV, the sampling frequency was set to 20 kHz, and the filtering frequency was 10 kHz. The condition for detecting leakage current was that the clamp voltage changed to -80 mV and the duration was 500 ms.The hERG current test method was as follows: a 4.8-second depolarizing command voltage was applied to raise the membrane potential from -80 mV to +30 mV, and then a 5.2-second repolarizing voltage was instantaneously applied to reduce the membrane potential to -50 mV, removing channel inactivation and thus allowing observation of the hERG tail current. The maximum value of the tail current was the magnitude of the hERG current. The hERG current used to detect the test compound was continuously recorded for 120 seconds prior to administration to assess the stability of the hERG current generated by the test cells. Only stable cells within the acceptable range of the evaluation criteria could proceed to subsequent testing of the compound.Testing the inhibitory effect of the test compound on hERG current: First, the hERG current measured in extracellular fluid containing 0.1% DMSO was used as a detection baseline. After the hERG current... Petition 870260067420, dated 08 / 07 / 2026, pages 97 / 113 91 / 106 remained stable for at least 5 minutes. The solution containing the test compound was sequentially perfused around the cells, from low to high concentration. After each perfusion was complete, approximately 5 minutes were allowed for the compounds to fully act on the cells, and hERG currents were recorded synchronously. The last 5 hERG current values ​​were recorded after the currents to be recorded tended to stabilize, and the average value was taken as the final current value at a specific concentration. After testing the compound, 450 nM dofetilide was added to the same cell, and the current was completely inhibited, serving as a positive control for that cell.At the same time, the positive compound dofetilide was tested before and after the end of the experiment with the test drug, using the same patch clamp system for synchronous detection, ensuring the reliability and sensitivity of the entire detection system. The above test steps were repeated in two separate test cells (n=2).

[0355] 4. Data processing methods and results:

[0356] Data quality control standards: Initial sealing resistance was greater than 1 GΩ; Series resistance was less than 15 MΩ and the voltage error of the series resistance was less than 5 mV; Leakage current at the detection voltage was less than 50% of the current value at that condition; Tail current was greater than the pre-pulse plateau current, with an initial tail current value greater than 250 pA; Diaphragm breakdown resistance Ra was less than 15 MΩ; Tail current decay rate per minute was less than 2.5%.

[0357] Data analysis: Only data that meet the above criteria can be analyzed according to the following steps. Petition 870260067420, dated 08 / 07 / 2026, pages 98 / 113 92 / 106

[0358] Note: The data is generated by PatchMaster software.

[0359] After being infused with blank solvent or composite gradient solutions, 5 consecutive stable current values ​​were obtained, and the average value was calculated, respectively, as blank tail current and composite tail current.

[0360] The current suppression percentage is calculated using the following formula: tail current compound - tail current positive drug^ tail current inhibition rate = (1--;-------------------;----------------------) x 100 tail current bank - tail current positive drug

[0361] The dose-effect curve was fitted using Graphpad Prism 8.0 software and the IC50 value was calculated.

[0362] Table 19: Inhibitory activity of compounds on hERG hERG Composite Number IC50 (pM) Cpd-A 19 Cpd-B 5.5 Cpd-C 1.4 Cpd-1 >30 Cpd-2 >30

[0363] The data above show that the representative compounds of this disclosure have a lower risk of possible cardiotoxicity caused by hERG inhibition, and their safety is obviously superior to that of the reference compounds.

[0364] VII. In vivo pharmacokinetic study in mice

[0365] 1. Experimental purpose: To test the pharmacokinetics of compounds in CD-1 mice.

[0366] 2. Experimental instrument and materials:

[0367] Animals: CD-1 mice (male)

[0368] Table 20: Instrument: Petition 870260067420, dated 08 / 07 / 2026, pages 99 / 113 93 / 106 Instrument Name Manufacturer Instrument Number API4000 Qtrap-Shimadzu AB Sciex / Shimadzu 20AT TW-017-0002 Eppendorf refrigerated centrifuge large benchtop Sartorius electronic balance Sptlabtech pipetting workstation Vortex oscillator DRAGON LABORATORY TW-007-0017 TW-001-0013 TW-013-0016 TW-005-0013 High-capacity tissue grinder Ningbo Scientz Biotechnology TW-011-0002 Co., Ltd.

[0369] Table 21: Reagents: Manufacturer Name: Cat. No. Lot Number: Acetonitrile Sigma-Aldrich 34851 Methanol Sigma-Aldrich 75851G DMSO Fisher Scientific D159-4 Formic Acid Sigma-Aldrich F112034 PBS Shanghai Basal Media B310KJ F222M7L201 WXBD8013V 186403 G2122206 H211017

[0370] Vehicle:

[0371] Intravenous injection: 40% HP^-CD in water; intragastric administration: 0.2% HPC + 0.5% Tween80 in water.

[0372] 3. Experimental methods:

[0373] 6 male rats. Fasting treatment was performed the night before administration, with free access to water. During the experiment, the mice were allowed to eat and drink freely, and were administered by intravenous injection or gavage. After administration, animal status was observed and abnormal behaviors were recorded. Blood was collected from the orbit in Petition 870260067420, dated 08 / 07 / 2026, pages 100 / 113 94 / 106 0.0833, 0.25, 1.2, 4, 8, and 24 hours after intravenous injection, and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intragastric administration. 20 μL of plasma sample were collected and added to 250 μL of acetonitrile (containing dexamethasone as an internal standard) to precipitate proteins. The mixture was centrifuged at 4°C and 4000 rpm for 20 minutes, and 180 μL of the supernatant were removed and mixed with 180 μL of an aqueous solution containing 0.1% formic acid in a 96-well plate. Then, 10 μL of the sample were collected for LC-MS / MS detection.

[0374] 4. Data processing methods and results:

[0375] The standard curve was established using the internal standard method, with the theoretical concentration of the standard curve as the X-axis and the peak area ratio as the Y-axis (peak area of ​​the test compound / peak area of ​​the internal standard). The linear regression method (weighting factor 1 / X2) was used, R2>0.9900. Unknown samples were calculated using the standard curve. Pharmacokinetic parameters were calculated using the non-compartmental analysis model of WinNonlin 8.2 software and presented in a report, including Clint, Cmax, AUC, etc.

[0376] Table 22: Pharmacokinetic parameters of compounds in mice CD-1 Compound number IV 1 mg / kg PO 10 mg / kg Clint (mL / min / kg) AUC0-t (hr*pM) Cmax (pM) AUC0-t (hr*pM) Cpd-A 18 1.7 2.2 10.4 Cpd-2 15 1.7 5.9 15.7

[0377] The data above show that the representative compound of this disclosure has lower clearance and higher oral exposure in mice, being Petition 870260067420, dated 08 / 07 / 2026, pages 101 / 113 95 / 106 superior to the reference compound.

[0378] VIII. In vivo pharmacokinetic study in rats

[0379] 1. Experimental purpose: To test the pharmacokinetics of compounds in rats SD.

[0380] 2. Experimental instrument and materials:

[0381] Animal: SD rats (male)

[0382] Table 23: Instrument: Instrument Name Manufacturer Instrument Number API4000 Qtrap-Shimadzu 20AT AB Sciex / Shimadzu TW-017-0002 Large Benchtop Refrigerated Centrifuge Eppendorf TW-007-0017 Pipetting Workstation Sptlabtech TW-013-0016 Vortex Oscillator DRAGON LABORATORY TW-005-0013 High-Capacity Tissue Grinder Ningbo Scientz Biotechnology Co., Ltd. TW-011-0002

[0383] Table 24: Reagents: Name Manufacturer Cat. No. Lot Number Acetonitrile Sigma-Aldrich 34851 WXBD8733V Methanol Sigma-Aldrich 75851G P2478457 DMSO Fisher Scientific D4540 BCCH3267 Formic acid Sigma-Aldrich F112034 B2320164 PBS Shanghai Basal Media B310KJ K211007

[0384] Vehicle:

[0385] Intravenous injection: 40% HP^-CD in water; intragastric administration: 0.2% HPC + 0.5% Tween80 in water. Petition 870260067420, dated 08 / 07 / 2026, pages 102 / 113 96 / 106

[0386] 3. Experimental methods:

[0387] 6 male rats. Fasting treatment was performed the night before administration, with free access to water. During the experiment, rats were allowed to eat and drink freely, being administered by intravenous injection or gavage. After administration, animal status was observed and abnormal behaviors were recorded. Blood was collected by jugular vein puncture at 0.0833, 0.25, 1, 2, 4, 8, and 24 hours after intravenous injection; Blood B was collected by jugular vein puncture at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intragastric administration. 50 μL of the plasma sample were collected and added with 500 μL of acetonitrile (containing dexamethasone as an internal standard) to precipitate proteins. The mixture was centrifuged at 4°C and 4000 rpm for 20 minutes, and 300 μL of the supernatant were removed and mixed with 300 μL of Watsons aqueous solution in a 96-well plate. Then, 5 μL of the sample were collected for LC-MS / MS detection.

[0388] 4. Data processing methods and results:

[0389] The standard curve was established using the internal standard method, with the theoretical concentration of the standard curve as the X-axis and the peak area ratio as the Y-axis (peak area of ​​the test compound / peak area of ​​the internal standard). The linear regression method (weighting factor 1 / X2) was used, R2>0.9900. Unknown samples were calculated using the standard curve. Pharmacokinetic parameters were calculated using the non-compartmental analysis model of WinNonlin 8.2 software and presented in a report, including Clint, Cmax, AUC, etc.

[0390] Table 25: Pharmacokinetic parameters of compounds in SD rats Petition 870260067420, dated 08 / 07 / 2026, pages 103 / 113 97 / 106 Compound Number IV 1 mg / kg PO 10 mg / kg Clint (mL / min / kg) AUC0-t (hr*pM) Cmax (pM) AUC0-t (hr*pM) Cpd-A 7.1 4.2 3.2 35.8 Cpd-2 3.3 8.4 8.9 58.4

[0391] The data above demonstrate that the representative compound of this disclosure exhibits lower clearance and higher oral exposure in rats compared to the reference compound, indicating a superior pharmacokinetic profile.

[0392] IX. In vivo efficacy study in a NUGC-3 mouse model of human gastric cancer xenotransplantation.

[0393] 1. Experimental purpose: To test the in vivo antitumor activity of compounds in the NUGC-3 subcutaneous human gastric cancer xenotransplant model in female NUNU mice.

[0394] 2. Experimental materials:

[0395] Animals: 6-8 week old female NUNU mice;

[0396] Table 26: Reagents: Reagent name: Supplier: Cat. No. Lot number Double antibody penicillin- Gibco 15240-062 2441816 Streptomycin Trypsin-EDTA Gibco 25200-072 2756239 RPMI-1640 Gibco C22400500BT 6123180 DPBS Corning 21-031-CVC 21031095 FBS Dongling FBSV500 A22JV Matrigel Corning 354234 3012001 Glacial acetic acid Alfa Aesar 36289 U02F788 Tween80 Sigma P1754 WXBD4333V Dimethyl sulfoxide (DMSO) Leyan 1083196 Lg0913304311 Solutol Sigma 42966 BCCJ5912 Petition 870260067420, dated 08 / 07 / 2026, pages 104 / 113 98 / 106

[0397] Vehicle Cpd-A: 0.2% HPC, 0.5% Tween80 in water (v / v / v);

[0398] Cpd-2 Vehicle: 5% DMSO / 10% Solutol HS 15 / 85% pH 4.65 ± 0.10 acetate buffer.

[0399] 3. Experimental protocol:

[0400] Cell culture: NUGC-3 cells were cultured in RPMI-1640 medium containing 10% FBS.

[0401] Molding and pooling: NUGC-3 cells in the exponential growth phase (5 χ 106) were harvested, resuspended in 0.2 mL of PBS and Matrigel (1:1), and then inoculated subcutaneously into the right flank of NUNU mice to establish a subcutaneous xenograft model. Mice were observed daily for healthy status and tumor growth after cell inoculation. On the fifth day after inoculation, 36 animals were selected, pooled, and the tumor volume of the animals in the group was approximately 150.0 mm3. The study was divided into 6 groups, with 6 mice in each group, and administration was performed on the same day (see Table 10).

[0402] 4. Experimental design: Table 10 shows the grouping and administration information for the efficacy experiment. Mice in group G1 received an orally administered vehicle control (5% DMSO / 10% Solutol HS 15 / 85% pH 4.65 ± 0.10 acetate buffer) once daily for a total of 19 days. Mice in group G2 received Cpd-A orally once daily at a dose of 50 mg / kg for a total of 19 days. Mice in group G3 received Cpd-A orally once daily at a dose of 100 mg / kg for a total of 19 days. Mice in group G4 received Cpd-2 orally once daily at a dose of 25 mg / kg for a total of 19 days. Mice in group G5 received Cpd-2 for Petition 870260067420, dated 07 / 08 / 2026, pp. 105 / 113 Mice in group G9 / 106 received Cpd-2 orally once daily at a dose of 50 mg / kg for a total of 19 days. Mice in group G6 received Cpd-2 orally once daily at a dose of 100 mg / kg for a total of 19 days.

[0403] Table 27: Table 10. In vivo efficacy study design of the NUGC-3 xenograft mouse model Group N Compound Dose (mg / kg) Administration Volume (mL / kg) Route of Administration Administration Frequency (post-grouping) G1 6 Vehicle — 10 PO QD*19 Days G2 6 Cpd-A 50 10 PO QD*19 Days G3 6 Cpd-A 100 10 PO QD*19 Days G4 6 Cpd-2 25 10 PO QD*19 Days G5 6 Cpd-2 50 10 PO QD*19 Days G6 6 Cpd-2 100 10 PO QD*19 Days

[0404] 5. Experimental observation and analysis of results:

[0405] Experimental observation: The health status and mortality of the animals were monitored daily. Routine checks included observing the effect of tumor growth and drug treatment on normal animal behavior, such as mobility, food and water consumption, body weight gain / loss, eye / fur tangling, and any other abnormal effects. The number of deaths and side effects in the animals in each group was recorded based on the number of animals in each group. Clinical symptoms observed during the study were recorded in the raw data. Tumor volume calculation formula: Tumor volume (mm3) = 0.5 χ (long tumor diameter χ short diameter2). During Petition 870260067420, dated 08 / 07 / 2026, pages 106 / 113 100 / 106 The research process, the entire administration process, as well as tumor and body weight measurements, were performed in a Laminar Flow Cabinet.

[0406] Data processing: Tumor growth inhibition percentage TGI (%): TGI % = (1 - ΔT / Δ^ χ 100%; W, where Δ^ is the tumor volume Ct C0 for the control group, C0 is the mean tumor volume of the control group at the time of pooling, and Ct is the mean tumor volume of the control group after treatment; ΔT is the tumor volume Tt - T0 for the treated group, T0 is the mean tumor volume of the treated group at the time of pooling, and Tt is the mean tumor volume of the treatment group after treatment.

[0407] Statistical analysis: Tumor volumes and body weight of the animals are expressed as mean ± SEM. Tumor volumes between different groups were statistically compared and analyzed. All statistical analyses were performed using GraphPad Prism 8.0. One-way ANOVA was used to compare whether there was a significant difference in tumor volume or weight between the groups, where p > 0.05 was considered as no significant difference, p < 0.05 was considered as having a significant difference, and p < 0.001 was considered as having an extremely significant difference.

[0408] 6. Experimental results:

[0409] The tumor growth curves of the control group and each experimental group in the efficacy study of the NUGC-3 xenograft model in mice are shown in FIG. 1, and the relative variations in body weight of the mice in the model are shown in FIG. 2. The mean tumor volume, the percentage of inhibition of TGI tumor growth (%), and the results of animal comparisons in each group at the end of the experiment are presented. Petition 870260067420, dated 08 / 07 / 2026, pages 107 / 113 101 / 106 in Table 11.

[0410] On day 19 after pooling, the mean tumor volume of group G1 (Vehicle) was 1482.2 mm3, and the mean tumor volumes of groups 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) were 1208.1 mm3, 399.1 mm3, 615.1 mm3, 202.2 mm3 and 102.5 mm3, respectively. The corresponding percentages of tumor growth inhibition (TGI) were 20.6%, 81.3%, 65.1%, 96.1%, and 103.6%, respectively. Statistical analysis showed that the mean tumor volume in groups G3, G4, G5, and G6 was significantly smaller than in the Vehicle group (p < 0.05 for all); however, the difference in mean tumor volume between groups G5 and G6 and the Vehicle group was extremely significant (p < 0.001 for both). The above results demonstrate that the reference compound Cpd-A showed good antitumor efficacy in the subcutaneous mouse model at a high dose of 100 mg / kg.In contrast, the representative compound of this disclosure, Cpd-2, showed good antitumor efficacy at all doses tested in this model, with dose-dependent activity. Additionally, at lower doses (e.g., 50 mg / kg once daily), Cpd-2 (TGI = 96.1%) showed greater inhibitory activity against tumor growth than Cpd-A (TGI = 81.3%), even when administered at twice the dose (100 mg / kg once daily), indicating that Cpd-2 has superior antitumor efficacy compared to Cpd-A.

[0411] Table 28. Results of the in vivo efficacy study of the mouse model with NUGC-3 xenograft Tumor volume group (mm3) GI tract (%)a (at 19 days) p-valueb Petition 870260067420, dated 08 / 07 / 2026, pages 108 / 113 102 / 106 (at 19 days) G1: Vehicle 1,482.2 ± 333.8 — — G2: CPD-A, 50 mg / kg 1,208.1 ± 320.9 20.6 0.8127 G3: CPD-A, 100 mg / kg 399.1 ± 106.3 81.3 0.0037 G4: Cpd-2, 25 mg / kg 615.1 ± 157.6 65.1 0.0243 G5: CPD-2, 50 mg / kg 202.2 ± 57.9 96.1 0.0006 G6: Cpd-2, 100 mg / kg 102.5 ± 42.1 103.6 0.0003

[0412] Note: the. TGI (%) = [1 - (T19 - T0) / (Vi9 - V0)] * 100 b. p-value of the comparative analysis of the mean tumor volume of each administration group and the mean tumor volume of the vehicle in group G1

[0413] X. In vivo efficacy study in human gastric cancer using a NUGC-3 xenograft mouse model

[0414] 1. Experimental objective: To test the in vivo antitumor activity of compounds in the NUGC-3 xenograft model of human subcutaneous gastric cancer in B-Rag2 / IL2rg KO SD rats.

[0415] 2. Experimental materials:

[0416] Animals: 7- to 9-week-old female B-Rag2 / IL2rg KO SD rats;

[0417] Table 29: Reagents: Reagent Name Supplier Cat. No. Lot Number Phosphate-buffered saline (PBS) BasalMedia B310KJ L211110 RPMI-1640 medium BasalMedia L210KJ D211212 Trypsin BasalMedia S310KJ B121201 Fetal bovine serum ExcellBio FSP500 012C-0530A Penicillin-streptomycin with double antibody 100* BasalMedia S110JV H121101 Petition 870260067420, dated 08 / 07 / 2026, pages 109 / 113 103 / 106 Matrigel CORNING 354234 3012001 Solutol HS15 MeiluneBio MB1809 A0413D Acetic acid Taicang Hushi NA 20180322 Sodium acetate Sigma-Aldrich S2889 WXBD3974v Ultrapure water Guangzhou NA NA Watsons DMSO Sigma-Aldrich V900090 WXBF1310V

[0418] Vehicle: 5% DMSO / 10% Solutol HS 15 / 85% pH 4.65 ± 0.10 acetate buffer;

[0419] 3. Experimental protocol:

[0420] Cell culture: NUGC-3 cells were cultured in RPMI-1640 medium containing 10% FBS.

[0421] Molding and pooling: NUGC-3 cells in the exponential growth phase (1 χ 107) were harvested, resuspended in 0.2 mL of PBS and Matrigel (1:1), and then inoculated subcutaneously into the right flank of B-Rag2 / IL2rg KO SD rats to establish a subcutaneous xenograft model. Rats were observed daily for healthy status and tumor growth after cell inoculation. On the tenth day after inoculation, 24 animals were selected, pooled, and the tumor volume of the animals in the group was approximately 199.0 mm3. The study was divided into 4 groups, with 6 rats in each group, and administration was performed on the same day (see Table 12).

[0422] 4. Experimental design: Table 30 is the grouping and administration information for the efficacy study. Rats in group G1 received an orally administered vehicle control (5% DMSO / 10% Solutol HS 15 / 85% pH 4.65 ± 0.10 acetate buffer) once daily for a total of 28 days. Rats in group G2 received Cpd-2 orally once daily at a dose of 6.25 mg / kg for a total of 28 days. Rats in group G3 received Cpd-2 orally once daily at a dose of 12.5 mg / kg, Petition 870260067420, dated 08 / 07 / 2026, pages 110 / 113 104 / 106 totaling 28 days. Rats in group G4 received Cpd-2 orally once a day at a dose of 25 mg / kg, totaling 28 days.

[0423] Table 30. In vivo efficacy study design of the NUGC-3 xenograft mouse model Group N Compound Dose of administration (mg / kg) Volume of administration (mL / kg) Route of administration Frequency of administration G1 6 Vehicle — 10 PO QD χ 28 days G2 6 Cpd-2 6.25 10 PO QD χ 28 days G3 6 Cpd-2 12.5 10 PO QD χ 28 days G4 6 Cpd-2 25 10 PO QD χ 28 days

[0424] 5. Experimental observation and analysis of results:

[0425] Experimental observation: The health status and mortality of the animals were monitored daily. Routine checks included the effect of tumor growth and drug treatment on normal animal behavior, such as mobility, food and water consumption, body weight gain / loss, eye / fur tangling, and any other abnormal effects. The number of deaths and side effects in the animals in the group was recorded based on the number of animals in each group. Clinical symptoms observed during the study were recorded in the raw data. Tumor volume calculation formula: Tumor volume (mm3) = 0.5 χ (long tumor diameter χ short diameter2). During the research process, the entire administration process, as well as tumor and body weight measurements, were performed in a Laminar Flow Cabinet.

[0426] Data processing: Percentage of tumor growth inhibition TGITV (%): TGITV % = (1 - ΔT / Δ^ χ 100%; W, where Δ0 is the tumor volume Petition 870260067420, dated 08 / 07 / 2026, pages 111 / 113 105 / 106 Ct - C0 for the control group, C0 is the mean tumor volume of the control group at the time of pooling, and Ct is the mean tumor volume of the control group after treatment; ΔT is the tumor volume. Tt - T0 for the treated group, T0 is the mean tumor volume of the treated group at the time of pooling, and Tt is the mean tumor volume of the treatment group after treatment.

[0427] Statistical analysis: Tumor volumes and body weight of the animals are expressed as mean ± SEM. Tumor volumes between different groups were statistically compared and analyzed. All statistical analyses were completed at https: / / d2k.bio / Efficacy / Invivo. The nominal wilcox.test method was used to compare whether there was a significant difference in tumor volume or weight between the groups, where p > 0.05 was considered as no significant difference, p < 0.05 was considered as having a significant difference, and p < 0.01 was considered as having an extremely significant difference.

[0428] 6. Experimental results:

[0429] The tumor growth curves of the control group and each experimental group in the efficacy study of the NUGC-3 xenograft model of human gastric cancer in rats are shown in FIG. 3, and the relative variations in body weight of the rats in the model are shown in FIG. 4. The mean tumor volume, the percentage of inhibition of GI tumor growth (%), and the comparative results of the animals in each group at the end of the experiment are presented in Table 13.

[0430] On day 28 after pooling, the mean tumor volume of group G1 (Vehicle) was 2946.2 mm3, and the mean tumor volumes of groups G2 (Cpd-2, 6.25 mg / kg, QD), G3 (Cpd-2, 12.5 mg / kg, QD), and G4 (Cpd-2, 25 mg / kg, QD) were Petition 870260067420, dated 08 / 07 / 2026, pages 112 / 113 106 / 106 The mean tumor volumes were 395.0 mm3, 119.2 mm3, and 70.1 mm3, respectively. The corresponding percentages of tumor growth inhibition (TGI) were 92.9%, 102.9%, and 104.7%, respectively. Statistical analysis showed that the difference in mean tumor volume between groups G2, G3, and G4 and the Vehicle group was extremely significant (p < 0.01 for all). The results above show that the representative compound of this disclosure, Cpd-2, exhibits good antitumor efficacy in the subcutaneous NUGC-3 xenograft model at each test dose, and the activity is dose-dependent.

[0431] Table 31. Results of the in vivo study of the rat xenograft model NUGC-3 Group Tumor volume (mm3) (Mean ± SEM) (at 28 days) TGITV (%) (at 28 days) p-values ​​G1: Vehicle, QD χ² 28 days, postal 2946.2 ± 167.4 / / G2: Cpd-2, 6.25 mg / kg, QD χ² 28 days, postural 395.0 ± 63.1 92.9 0.0022 G3: Cpd-2, 12.5 mg / kg, QD χ² 28 days, post-op 119.2 ± 10.2 102.9 0.0022 G4: Cpd-2, 25 mg / kg, QD χ² 28 days, post-op 70.1 ± 14.9 104.7 0.0022

[0432] Note:

[0433] p value of the comparative analysis of the mean tumor volume of each administration group and the mean tumor volume of the Vehicle in group G1.

Claims

1. A nitrogen-containing compound of formula I, an isotopically labeled compound thereof, a solvate thereof, a pharmacologically acceptable salt thereof, or a solvate of the pharmacologically acceptable salt thereof, characterized in that the structure of formula I is represented by: wherein X1, X2 and X3 * * are each independently selected from CH and N; R1, R2, R3, R4 and R5 are each independently selected from hydrogen and deuterium; R6 is -(CH2)nR7; n is 0, 1, 2 or 3; R7 is selected from C1-C6 alkyl, deuterated C1-C6 alkyl, C1C6 haloalkyl and C3-C6 cycloalkyl.

2. Compound of formula I, the corresponding isotopically labeled compound, the corresponding solvate, the corresponding pharmaceutically acceptable salt, or the solvate of the corresponding pharmaceutically acceptable salt, according to claim 1, characterized by formula Iser represented by formula Ia; Petition 870260039952, dated 04 / 29 / 2026, p. 21 / 239 2 / 8 3. Compound of formula I, the isotopically labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to any one of claims 1 or 2, characterized in that X1 is N; X2 is CH or N; X3 is CH; or, X1 is N; X2 and X3 are CH.

4. Compound of formula I, the isotopically labeled compound, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that R6 is -(CH2)nR7; n is 0 or 1; R7 is selected from C1-C3 alkyl, deuterated C1-C3 alkyl, C1-C3 haloalkyl, and C3-C6 cycloalkyl.

5. Compound of formula I, the corresponding isotopically labeled compound, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that R6 is -(CH2)nR7; n is 0 or 1; R7 is selected from -CH3, -CD3, -CF3, and cyclopropyl. Petition 870260039952, dated 29 / 04 / 2026, p. 22 / 239 3 / 8 6.

7. Compound of formula I, the corresponding isotopically labeled compound, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that R1, R2, and R3 are deuterium; R4 and R5 are hydrogen; or, R1, R2, and R3 are hydrogen; R4 and R5 are deuterium.Compound of formula I, the corresponding isotopically labeled compound, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that formula I is formula Ib or Ic; Ib Ic in formulas Ib and Ic, λ*3 is R1 R or R1, R2, R3, R4 and R5 are each independently selected from hydrogen and deuterium; R6 is -(CH2)nR7; n is 0 or 1; R7 is selected from C1-C6 alkyl, C1-C6 haloalkyl and C3-C6 cycloalkyl.

8. Compound of formula I, the corresponding isotopically labeled compound, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of Petition 870260039952, dated 29 / 04 / 2026, p.23 / 239 4 / 8 pharmaceutically acceptable salt thereof, according to claim 7, characterized in that in formulas Ib and Ic, R' R2 is selected from -OCH3 and -OCD3; R4 and R5 are selected from hydrogen; R6 is -(CH2)nR7; n is 0 or 1; R7 is selected from -CH3, -CD3, -CF3 and cyclopropyl.

9. Compound of formula I, the corresponding isotopically labeled compound, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that formula I is selected from the following compounds: Petition 870260039952, dated 29 / 04 / 2026, p. 24 / 239 5 / 8 Petition 870260039952, dated 29 / 04 / 2026, p. 25 / 239 6 / 8 any proportion. an enantiomer thereof, or a mixture of the two in 10. A pharmaceutical composition comprising the compound of formula I, the corresponding isotopically labeled compound, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that it is a pharmaceutical excipient.

11. An application of the compound of formula I, the corresponding isotopically labeled compound, the corresponding solvate, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that it is used in the manufacture of a mutant p53 Y220C binding site agent, the binding site agent increasing the ability of the mutant p53 to bind to DNA.

12. A use of the pharmaceutical composition according to claim 10, characterized in that in the manufacture of a Y220C p53 site binding agent, Petition 870260039952, dated 04 / 29 / 2026, p. 26 / 239 7 / 8 the site binding agent increases the ability of the mutant p53 to bind to DNA.

13. A use of the compound of formula I, the corresponding isotopically labeled compound, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that it is in the manufacture of a medicament for the treatment of gastric cancer.

14. Compound of formula a-11 or a-32; a-33 a-11 wherein X is halogen; R8 is an amino protecting group; X1, X2 and X3 are each independently selected from CH and N; R1, R2 and R3 are each independently selected from hydrogen and deuterium; R6 is -(CH2)nR7; N is 0, 1, 2, or 3; R7 is selected from C1-C6 alkyl. C1-C6 deuterated alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl.

15. The compound of formula a-11 or a-32, according to claim 14, characterized in that the compound of formula a-11 or a-32 is any one of the following compounds; r.

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