A highly active wnt pathway inhibitor compound

Abstract

The present application provides a kind of compound for inhibiting Wnt pathway activity and preparation method thereof, and pharmaceutical composition comprising the compound.The present application also provides the use of the compound in preventing and / or treating cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.

Description

[0001] This application is a divisional application of Chinese invention patent application number "202180066465.5". The parent application was filed on October 27, 2021 (PCT international application date), with Chinese national application number "202180066465.5" (PCT international application number is PCT / CN2021 / 126539), and the invention title is "A Highly Active Wnt Pathway Inhibitor Compound".

[0002] The parent application and this divisional application claim priority to Chinese Patent Application No. 202011175894.X, entitled “A Highly Active Wnt Pathway Inhibitor Compound,” filed with the Chinese Patent Office on October 28, 2020, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to a heterocyclic compound, and more particularly to a highly active Wnt pathway inhibitor and its uses. Background Technology

[0004] The Wnt / β-catenin signaling pathway is a conserved pathway throughout biological evolution. In normal somatic cells, β-catenin functions as a cytoskeletal protein, forming a complex with E-cadherin at the cell membrane to maintain homologous cell adhesion and prevent cell migration. When the Wnt signaling pathway is not activated, intracellular β-catenin is phosphorylated and forms a β-catenin degradation complex with APC, Axin, and GSK3β, initiating the ubiquitin system to degrade β-catenin via the proteasome pathway, maintaining intracellular β-catenin levels at a low level. When cells are stimulated by Wnt signaling, Wnt protein binds to the specific receptor Frizzled protein on the cell membrane. The activated Frizzled receptor recruits intracellular Disheveled protein, inhibiting the degradation activity of the β-catenin degradation complex formed by proteins such as GSK3β, thus stabilizing free β-catenin protein in the cytoplasm. β-Catenin, stably accumulated in the cytoplasm, enters the nucleus and binds to the LEF / TCF transcription factor family, initiating the transcription of downstream target genes (such as c-myc, c-jun, Cyclin D1, etc.). Overactivation of the Wnt / β-catenin signaling pathway is closely related to the development of various cancers (including colon cancer, gastric cancer, and breast cancer). For example, abnormal activation of the classical Wnt signaling pathway and intranuclear accumulation of β-catenin protein are widespread in colorectal cancer, and inhibiting Wnt signaling pathway activity can suppress colon cancer proliferation. Mutations in APC are present in more than 85% of colorectal cancers; mutated APC blocks β-catenin phosphorylation and degradation, inducing colorectal cancer development. Furthermore, mutations in Axin and β-catenin itself can also cause intracellular accumulation of β-catenin, activating the Wnt / β-catenin pathway. Summary of the Invention

[0005] In one aspect, the present invention provides compounds having the following structures, as well as pharmaceutically acceptable salts, isotope derivatives, and stereoisomers:

[0006]

[0007] In one aspect, the present invention provides a method for preparing the compound as described above, comprising using compound 1e as an intermediate:

[0008]

[0009] In one embodiment, the preparation of compound 19 includes the following steps:

[0010]

[0011] Step 1: Compounds 19a and 19b were dissolved in N,N-dimethylformamide, and Cs₂CO₃ was added. The reaction solution was heated to 100°C and reacted for 2 hours until the reaction was complete. The reaction solution was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to obtain a white solid, 19c. Step 2: Compound 19c was dissolved in dioxane, and concentrated hydrochloric acid was added. The reaction solution was stirred at room temperature for 1 hour until the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain compound 19d. Step 3: The compound... Compound 19d was dissolved in THF, and NaH was added under ice bath conditions. After stirring for 5 minutes, iodomethane was added, and stirring was continued for another 30 minutes until the reaction was complete. The reaction solution was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to obtain compound 19e. Fourth step: Compounds 19e and 1e were dissolved in n-butanol, and trifluoroacetic acid was added. The reaction solution was heated to 150°C in a microwave oven and reacted for 4 hours until the reaction was complete. The reaction solution was concentrated, and the residue was purified by preparative HPLC to obtain compound 19.

[0012] In one embodiment, the preparation of compound 24 includes the following steps:

[0013]

[0014] Step 1: Under nitrogen protection, SOCl2 was dissolved in anhydrous dichloromethane. After the reaction solution was cooled to -70°C, an anhydrous dichloromethane solution of imidazole and triethylamine was added dropwise. The internal temperature of the reaction solution was controlled below -40°C. After the addition was complete, the reaction solution was cooled back to -70°C and stirred for 10 minutes. Anhydrous dichloromethane solution of compound 24a was then added dropwise. The reaction mixture was slowly heated to room temperature and stirred overnight. The reaction solution was diluted with dichloromethane, and 0.5M citric acid aqueous solution was added to separate the organic phase. The obtained organic phase was washed successively with saturated sodium bicarbonate solution and saturated brine solution until anhydrous... The reaction mixture was dried over sodium sulfate, filtered, and concentrated to obtain compound 24b. The second step involved dissolving compound 24b in a mixture of acetonitrile and water, and then adding ruthenium trichloride and sodium periodate sequentially under ice bath conditions. The reaction mixture was stirred at room temperature for 3 hours to terminate the reaction. The reaction solution was diluted with ethyl acetate, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 24c. The third step involved dissolving compounds 19a, 22a, and 18-crown-6 in dioxane (20 mL), adding potassium carbonate, and heating the reaction system to 60°C for 16 hours until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 24d. Step 4: Compound 24d was dissolved in dioxane, concentrated hydrochloric acid was added, and the reaction solution was stirred at room temperature for 3 hours until the reaction was complete. The reaction solution was concentrated to obtain the hydrochloride salt of compound 24e. Step 5: The hydrochloride salt of compound 24e was dissolved in a mixture of dioxane and N,N-dimethylformamide, N,N-diisopropylethylamine was added, and the reaction solution was heated to 100°C and reacted for 16 hours until the reaction was complete. The reaction solution was concentrated, and the residue was diluted with ethyl acetate and washed successively with saturated ammonium chloride solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to obtain compound 24f; Step 6: Compound 24f was dissolved in anhydrous N,N-dimethylformamide, NaH was added under ice bath, the reaction mixture was stirred for 5 minutes and then iodomethane was added; the reaction solution was heated to room temperature and stirred for 3 hours until the reaction was complete; the reaction solution was diluted with ethyl acetate, washed with saturated brine, the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated; the residue was purified by silica gel column chromatography to obtain compound 24g; Step 7: Compound 24g and 1e were dissolved in n-butanol, trifluoroacetic acid was added, the reaction solution was heated to 150℃ in microwave and reacted for 3 hours until the reaction was complete; the reaction solution was concentrated, and the residue was purified by preparative HPLC to obtain compound 24.

[0015] In one embodiment, the preparation of compound 25 includes the following steps:

[0016]

[0017] Step 1: Under nitrogen protection, SOCl2 was dissolved in anhydrous dichloromethane. After the reaction solution was cooled to -70°C, an anhydrous dichloromethane solution of imidazole and triethylamine was added dropwise. The internal temperature of the reaction solution was controlled below -40°C. After the addition was complete, the reaction solution was cooled back to -70°C and stirred for 10 minutes. An anhydrous dichloromethane solution of compound 25a was then added dropwise. The reaction mixture was slowly heated to room temperature and stirred overnight. The reaction solution was diluted with dichloromethane, and 0.5M citric acid aqueous solution was added to separate the organic phase. The obtained organic phase was washed successively with saturated sodium bicarbonate solution and saturated brine. First, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 25b. Second, compound 25b obtained in the previous step was dissolved in acetonitrile, ruthenium trichloride was added, and then sodium periodate aqueous solution was added dropwise under ice bath conditions. After the addition was complete, the reaction mixture was heated to room temperature and stirred for 4 hours. The reaction solution was diluted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 25c. Third, compounds 19a, 25c, and 18-crown-6 were dissolved in dioxane, potassium carbonate was added, and the reaction system was heated to 80°C and reacted for 16 hours until the reaction was complete. The reaction solution was diluted with ethyl acetate, washed with water and saturated brine respectively, and the organic phase was dried with anhydrous sodium sulfate. After filtration and concentration, compound 25d was obtained. Fourth step: Compound 25d obtained in the previous step was dissolved in dichloromethane, and dioxane hydrochloride solution was added. The reaction solution was stirred at room temperature for 16 hours until the reaction was complete. The reaction solution was concentrated to obtain the hydrochloride salt of compound 25e. Fifth step: The hydrochloride salt of compound 25e was dissolved in a mixture of dioxane and N,N-dimethylformamide, and N,N-diisopropylethylamine was added. The reaction solution was heated to 90°C and reacted for 16 hours until the reaction was complete. The reaction solution was concentrated, and the residue was diluted with ethyl acetate, washed successively with saturated ammonium chloride solution and saturated brine. The organic phase was dried with anhydrous sodium sulfate, and after filtration and concentration, the residue was obtained.The residue was purified by silica gel column chromatography to obtain compound 25f; Step 6: Compound 25f was dissolved in anhydrous N,N-dimethylformamide, NaH was added under ice bath, the reaction mixture was stirred for 30 minutes and then iodomethane was added; the reaction solution was brought to room temperature and stirred for 3 hours until the reaction was complete; the reaction solution was diluted with ethyl acetate, washed with saturated brine, the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated; the residue was purified by silica gel column chromatography to obtain compound 25g; Step 7: Under nitrogen protection, compound 25g was dissolved in anhydrous tetrahydrofuran, the reaction solution was cooled to -70℃, n-butyllithium solution was added dropwise, and after stirring for 30 minutes, iodomethane was added; The reaction mixture was stirred at -70°C for 1 hour and then slowly brought to room temperature. The reaction solution was quenched with saturated ammonium chloride aqueous solution, diluted with ethyl acetate, and washed successively with water and saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated to obtain compound 25h. Step 8: Compound 25h, compound 1e, Pd2(dba)3, t-BuONa) and X-Phos were dispersed in toluene. The reaction system was replaced with nitrogen and heated to 100°C for 16 hours until the reaction was complete. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to obtain crude product, which was then purified by Prep-HPLC to obtain compound 25.

[0018] In one embodiment, the preparation of compound 26 includes the following steps:

[0019]

[0020] Step 1: Compound 19a and triphenylphosphine were dissolved in anhydrous tetrahydrofuran at room temperature. Under nitrogen protection, N,N-diisopropylethylamine and diisopropyl azodicarbonate were added sequentially. After stirring for 30 minutes, an anhydrous tetrahydrofuran solution of compound 26a was added dropwise. The reaction mixture was heated to 70°C and reacted for 16 hours until the reaction was complete. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to obtain compound 26b. Step 2: Compound 26b was dissolved in dichloromethane, and dioxane hydrochloride solution was added. The reaction mixture was stirred at room temperature for 16 hours until the reaction was complete. The reaction mixture was filtered, the filter cake was collected, and dried under reduced pressure to obtain the hydrochloride salt of compound 26c. Step 3: The hydrochloride salt of compound 26c was dissolved in a mixture of dioxane and N,N-dimethylformamide, and N,N-diisopropylethylamine was added. The reaction mixture was heated to 100°C and reacted for 4 hours. The reaction was carried out until complete; the reaction solution was concentrated, and the residue was diluted with ethyl acetate and washed successively with saturated ammonium chloride solution and saturated brine; the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated; the residue was purified by silica gel column chromatography to obtain a white solid 26d; Step 4: Compound 26d was dissolved in anhydrous N,N-dimethylformamide, and NaH was added in portions under ice bath conditions. The reaction mixture was stirred for 15 minutes and then iodomethane was added; the reaction solution was brought to room temperature and stirred for 4 hours until complete; the reaction solution was diluted with ethyl acetate, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 26e; Step 5: Compounds 26e and 1e were dissolved in n-butanol, and trifluoroacetic acid was added. The reaction solution was heated to 150°C in a microwave oven and reacted for 6 hours until complete; the reaction solution was concentrated, and the residue was purified by preparative HPLC to obtain compound 26.

[0021] In one embodiment, (R)-1-(BOC-amino)-2-propanol methanesulfonate 19b in the first step of Example 19 is replaced with (S)-1-(BOC-amino)-2-propanol methanesulfonate, and compound 27 is obtained by similar method and reaction steps.

[0022] In one embodiment, the preparation of compound 28 includes the following steps:

[0023]

[0024] Step 1: Compound 19a and triphenylphosphine were dissolved in anhydrous tetrahydrofuran at room temperature. Under nitrogen protection, N,N-diisopropylethylamine and diisopropyl azodicarbonate were added sequentially. After stirring for 30 minutes, anhydrous tetrahydrofuran solution of compound 28a was added dropwise. The reaction mixture was heated to 70°C and reacted for 16 hours to terminate the reaction. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to obtain compound 28b. Step 2: Compound 28b was dissolved in dichloromethane, and dioxane hydrochloride solution was added. The reaction solution was stirred at room temperature for 16 hours until the reaction was complete. The reaction solution was concentrated to obtain compound 28c. Step 3: Compound 28c and 1e were dissolved in n-butanol, and trifluoroacetic acid was added. The reaction solution was heated to 150°C by microwave and reacted for 5 hours until the reaction was complete. The reaction solution was concentrated, and the residue was purified by preparative HPLC to obtain compound 28.

[0025] In one aspect, the present invention also provides a method for preparing the compound of formula 1e as described above, comprising the following steps:

[0026]

[0027] Step 1: Dissolve 6-trifluoromethyl-3-pyridinemethanol 1a in dichloromethane, add thionyl chloride dropwise under ice bath conditions, and after the addition is complete, raise the reaction temperature to room temperature and stir overnight at 55°C; concentrate the reaction solution to obtain crude yellow oily substance 1b; Step 2: Dissolve compounds 1b and 1c in N,N-dimethylformamide, add potassium carbonate, and stir overnight at room temperature; dilute the reaction solution with ethyl acetate, wash successively with water and saturated brine, dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain yellow solid 1d; Step 3: Dissolve compound 1d and hydroxylamine hydrochloride in ethanol, stir overnight at room temperature; add zinc powder and acetic acid to the reaction solution and heat to 70°C and react overnight; after the reaction is complete, evaporate most of the solvent under reduced pressure, adjust the pH of the residue to 11-12 with 2N sodium hydroxide, filter; extract the filtrate three times with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, filter and concentrate to obtain yellow oily substance 1e.

[0028] In one aspect, the present invention provides a pharmaceutical composition comprising compounds 19, 24-28 as described above and a pharmaceutically acceptable salt.

[0029] In one aspect, the present invention provides the use of compounds 19, 24-28 and pharmaceutically acceptable salts, as well as pharmaceutical compositions thereof, in medicaments for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases, or immune-mediated diseases.

[0030] It is particularly noteworthy that, in this article, when referring to a "compound" having a specific structural formula, it generally also includes its stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotopic derivatives.

[0031] As is known to those skilled in the art, the salts, solvates, and hydrates of a compound are alternative forms of the compound, and they can all be converted into the compound under certain conditions. Therefore, it is particularly noteworthy that when a compound is mentioned herein, its pharmaceutically usable salts are generally also included, and consequently its solvates and hydrates are also included.

[0032] Similarly, when referring to a compound in this article, its prodrug, metabolites, and nitrogen oxides are generally also included.

[0033] The pharmaceutically acceptable salts described in this invention can be formed using, for example, inorganic or organic acids: “Pharmaceutically acceptable salt” means a salt that, within a reasonable medical judgment, is suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and has a reasonable benefit / risk ratio. The salts can be prepared in situ during the final separation and purification of the compounds of this invention, or solely by reacting a free base or free acid with a suitable reagent, as outlined below. For example, the free base function can react with a suitable acid. Furthermore, when the compounds of this invention contain an acidic moiety, suitable pharmaceutically acceptable salts may include metal salts, such as alkali metal salts (e.g., sodium or potassium salts); and alkaline earth metal salts (e.g., calcium or magnesium salts). Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts formed by amino groups with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed using other methods in the art, such as ion exchange.

[0034] The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example by dissolving the compounds of the present invention in a water-miscible organic solvent (e.g., acetone, methanol, ethanol, and acetonitrile), adding an excess of an aqueous solution of an organic or inorganic acid to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid, and then separating the precipitated salt.

[0035] The precursors or metabolites described in this invention can be precursors or metabolites known in the art, as long as they are metabolized and transformed in vivo to form compounds. For example, "prodrug" refers to those prodrugs of the compounds of this invention that, within a reasonable medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio and are effective for their intended use. The term "prodrug" refers to a compound that is rapidly transformed in vivo to produce the parent compound of the above formula, for example, through in vivo metabolism, or through N-demethylation of the compounds of this invention.

[0036] The term "solvate" as used in this invention refers to the physical association of the compound of this invention with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric solvent molecules. "Solvate" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0037] The "stereoisomerism" described in this invention is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism and optical isomerism. Conformational isomerism refers to the phenomenon where organic molecules with a certain configuration undergo different spatial arrangements of atoms or groups of atoms due to the rotation or twisting of carbon or carbon single bonds. Common examples include the structures of alkanes and cycloalkanes, such as the chair conformation and boat conformation in the cyclohexane structure. "Stereoisomers" refer to compounds of this invention containing one or more asymmetric centers, thus allowing them to exist as racemic mixtures and racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers. The compounds of this invention have asymmetric centers, each of which produces two optical isomers. The scope of this invention includes all possible optical isomers and diastereomer mixtures, as well as pure or partially pure compounds. The compounds of this invention can exist as tautomers, which have different hydrogen bonding sites through one or more double bond shifts. For example, ketones and their enol forms are ketone-enol tautomers. All tautomers and mixtures thereof are included in the compounds of this invention. All enantiomers, diastereomers, racemates, mesomates, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof of all compounds of formula (I) are included within the scope of this invention.

[0038] The "isotope derivative" of this invention refers to a molecule in which the compound is isotopically labeled. Commonly used isotopes for isotopic labeling are hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 11 C, 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes:15 O, 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially deuterium. 3 H and carbon 13 C, because they are easy to label and convenient to detect, are more widely used. Some heavy isotopes, such as deuterium (… 2 Substitution with H can enhance metabolic stability and prolong the half-life, thereby reducing the dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques, just like non-isotope-labeled compounds.

[0039] The present invention also provides the use of the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases or immune-mediated diseases.

[0040] Furthermore, the present invention provides pharmaceutical compositions for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases, or immune-mediated diseases, comprising compounds of the present invention as active ingredients.

[0041] Furthermore, the present invention provides a method for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases, or immune-mediated diseases, comprising administering the compound of the present invention to a mammal in need of such treatment.

[0042] When the compounds of the present invention or their pharmaceutically acceptable salts are administered in combination with other anticancer agents or immune checkpoint inhibitors used to treat cancer or tumors, the compounds of the present invention or their pharmaceutically acceptable salts may provide enhanced anticancer effects.

[0043] When the compounds of the present invention or their pharmaceutically acceptable salts are administered in combination with other therapeutic agents for treating inflammatory diseases, autoimmune diseases and immune-mediated diseases, the compounds of the present invention or their pharmaceutically acceptable salts may provide enhanced therapeutic effects.

[0044] In addition, the present invention provides a method for preventing and / or treating tumors, cancer, viral infections, organ transplant rejection, neurodegenerative diseases, attention-related diseases, or autoimmune diseases, comprising administering the compounds of the present invention or the pharmaceutical compositions of the present invention to mammals in need of such treatment.

[0045] The pharmaceutical compositions of the present invention can be formulated into dosage forms for oral or parenteral administration (including intramuscular, intravenous, and subcutaneous routes, and intratumoral injection) according to any of the conventional methods, such as tablets, granules, powders, capsules, syrups, emulsions, microemulsions, solutions, or suspensions.

[0046] Other features of the invention will become apparent as the exemplary embodiments are described. The embodiments are given to illustrate the invention and are not intended to be limiting. The following examples use the methods disclosed in the invention to prepare, separate, and characterize.

[0047] The compounds of the present invention can be prepared in a variety of ways known to those skilled in the art of organic synthesis. They can be synthesized using the methods described below, as well as synthetic methods known in the field of organic synthetic chemistry, or by variations thereof understood by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reaction is carried out in a solvent or solvent mixture suitable for the kit materials used and suitable for the transformation achieved. Those skilled in the art of organic synthesis will understand that the functionalities present on the molecule are consistent with the proposed transformation. This sometimes necessitates determining whether to change the order of synthetic steps or the starting materials to obtain the desired compound of the present invention. Detailed Implementation

[0048] the term

[0049] Unless otherwise specified, the terms used in this application, including the specification and claims, are defined as follows. It must be noted that in the specification and appended claims, unless otherwise clearly indicated, the singular form "a" includes the plural meaning. Unless otherwise specified, conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used. In this application, unless otherwise specified, "or" or "and" refers to "and / or".

[0050] In the specification and claims, the given chemical formula or name shall cover all stereo and optical isomers and racemic products containing such isomers. Unless otherwise specified, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of this invention. Many geometric isomers of C=C double bonds, C=N double bonds, ring systems, etc., may also be present in the compounds, and all such stable isomers are covered within this invention. This invention describes cis- and trans- (or E- and Z-) geometric isomers of the compounds of this invention, which can be separated into mixtures of isomers or separate isomeric forms. The compounds of this invention can be separated in optically active or racemic forms. All methods used to prepare the compounds of this invention and the intermediates prepared therein are considered part of this invention. In the preparation of enantiomers or diastereomers, they can be separated by conventional methods (e.g., by chromatography or fractional crystallization). Depending on the method conditions, the end products of this invention are obtained in free (neutral) or salt form. Both the free form and salts of these end products are within the scope of this invention. If necessary, one form of the compound can be converted into another. A free base or acid can be converted into a salt; a salt can be converted into a free compound or another salt; a mixture of isomers of the present invention can be separated into individual isomers. The compounds of the present invention, their free forms, and salts can exist in a variety of tautomer forms, wherein hydrogen atoms are transposed to other parts of the molecule and thereby the chemical bonds between the atoms of the molecule are rearranged. It should be understood that all possible tautomer forms are included within the scope of this invention.

[0051] Unless otherwise defined, the definitions of substituents in this invention are independent of each other and not related to each other. When any variable appears more than once in any composition or formula of a compound, its definition for each occurrence is independent of its definition for each other occurrence.

[0052] As used herein, the term "patient" refers to an organism treated by the method of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., rodents, apes / monkeys, horses, cattle, pigs, dogs, cats, etc.), and most preferably, humans.

[0053] As used herein, the term "effective amount" means the amount of a drug or agent (i.e., the compound of the present invention) that will elicit a biological or medical response in a tissue, system, animal, or human, as sought by, for example, a researcher or clinician. Furthermore, the term "therapeutic effective amount" means an amount that, compared to a corresponding subject who has not received the aforementioned amount, results in improved treatment, cure, prevention, or reduction of a disease, symptom, or side effect, or a slower rate of progression of a disease or symptom. Effective amounts may be administered, applied, or dosed in one or more ways and are not intended to be limited to a particular formulation or route of administration. The term also includes effective amounts within its scope that enhance normal physiological function.

[0054] The term “treatment” as used in this article includes any effect that results in improvement of a condition, disease, disorder, etc., such as reducing, decreasing, regulating, improving or eliminating, or improving its symptoms.

[0055] The term "pharmaceutical" as used herein refers to compounds, substances, compositions, and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, and / or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0056] As used herein, the phrase "pharmaceutical carrier" refers to a pharmaceutical substance, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulation substance, which relates to carrying or delivering a subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient.

[0057] The term "pharmaceutical composition" means a composition comprising the compounds of the present invention and at least one other pharmaceutical carrier. "Pharmaceutical carrier" refers to a medium commonly accepted in the art for delivering a bioactive agent to an animal (specifically a mammal), including (i) adjuvants, excipients, or mediators such as diluents, preservatives, fillers, flow modifiers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, antibacterial agents, antifungal agents, lubricants, and dispersants, depending on the mode of administration and the nature of the dosage form.

[0058] All features described in this specification (including any claims or abstracts) and / or all steps involved in any method or process may exist in any combination unless certain features or steps are mutually exclusive in the same combination.

[0059] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0060] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0061] The units used in weight-volume percentages in this invention are well known to those skilled in the art, for example, referring to the weight of the solute in 100 ml of solution. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0062] Example

[0063] General process

[0064] When the preparation method is not specified, all raw materials and reagents used in this invention are known products that can be synthesized according to methods known in the art, or can be obtained by purchasing commercially available products. None of the commercially available reagents used require further purification.

[0065] Room temperature refers to 20-30℃.

[0066] Unless otherwise specified in the reaction examples, all reactions were carried out under a nitrogen atmosphere. A nitrogen atmosphere refers to a reaction flask connected to a nitrogen balloon of approximately 1L.

[0067] Hydrogenation reactions are typically carried out under vacuum, filled with hydrogen gas, and repeated three times. A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon of approximately 1L.

[0068] Microwave reaction use Initiator + Microwave Reactor.

[0069] The structure of the compounds of this invention was determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR shifts (δ) were expressed in terms of 10⁻¹⁰. -6 The measurements are given in units of (ppm). NMR determinations are performed using (Bruker Ascend) TM A Model 500 NMR spectrometer was used. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). The following abbreviations are used for NMR signal multiplicity: s = singlet, brs = broad peak, d = doublet, t = triplet, m = multiplet. Coupling constants are listed in J values ​​and measured in Hz.

[0070] LC-MS was performed using a Thermo UltiMate 3000+MSQ PLUS system. HPLC was performed using a Thermo UltiMate 3000 high-performance liquid chromatograph. Reversed-phase preparative chromatography was performed using a Thermo UltiMate 3000 reversed-phase preparative chromatograph. Rapid column chromatography was performed using an Agilent FS-9200T automated column press, and pre-packed silica gel columns were obtained from Sante. Pre-packed column. Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The thickness used for thin-layer chromatography separation and purification of products is 0.4mm to 0.5mm.

[0071] Example 1

[0072] (S)-4,5-dimethyl-2-(((1-((6-(trifluoromethyl)pyridin-3-yl)methyl)-1H-pyrazol-4-yl)methyl)amino)-4,5,9,10-tetrahydro-6H,8H-pyrido[3,2,1-de]pterodin-6-one

[0073]

[0074] Compound 1 was prepared by the following steps:

[0075]

[0076] Step 1: 6-Trifluoromethyl-3-pyridinylmethanol 1a (4.0 g, 22.58 mmol) was dissolved in dichloromethane (20 mL). Thionyl chloride (26.87 g, 225.83 mmol, 16.38 mL) was added dropwise under ice bath conditions. After the addition was complete, the reaction mixture was brought to room temperature and stirred overnight at 55 °C. The reaction solution was concentrated to give a crude, yellow, oily product 1b (4.4 g, 99% yield). ESI-MS (m / z): 196.5 [M+H] + .

[0077] Step 2: Compound 1b (4.4 g, 22.48 mmol) and compound 1c (1.8 g, 18.73 mmol) were dissolved in N,N-dimethylformamide (10 mL), and potassium carbonate (6.47 g, 46.83 mmol) was added. The mixture was stirred overnight at room temperature. The reaction solution was diluted with ethyl acetate, washed successively with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solution was filtered and concentrated to give a yellow solid 1d (4.5 g, 94% yield). ESI-MS (m / z): 256.4 [M+H] + .

[0078] Step 3: Compound 1d (4.5 g, 17.63 mmol) and hydroxylamine hydrochloride (1.73 g, 26.45 mmol) were dissolved in ethanol (20 mL) and stirred overnight at room temperature. Zinc powder (4.58 g, 70.53 mmol) and acetic acid (50 mL) were added to the reaction solution, and the mixture was heated to 70 °C and reacted overnight. After the reaction was complete, most of the solvent was evaporated under reduced pressure. The residue was adjusted to pH 11-12 with 2N sodium hydroxide and filtered. The filtrate was extracted three times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow oily substance 1e (3.5 g, 77% yield). ESI-MS (m / z): 257.6 [M+H] + .

[0079] Step 4: 1f of 2,4-dichloropyrido[3,2-d]pyrimidine (1.7g, 8.50mmol) and 1g of (S)-2-(methylamino)propionate hydrochloride (1.70g, 11.05mmol) were dissolved in tetrahydrofuran (40mL), and triethylamine (2.58g, 25.50mmol, 3.53mL) was added. The mixture was stirred overnight at room temperature. The reaction was monitored by LCMS until completion. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to give a yellow oily substance (1h, 1.1g, yield 46%). ESI-MS (m / z): 281 [M+H] + .

[0080] Step 5: Compound 1h (1.1 g, 3.92 mmol) was dissolved in tetrahydrofuran (20 mL), and hydrochloric acid aqueous solution (6 N, 0.65 mL) and platinum dioxide (88 mg, 0.39 mmol) were added. The reaction system was purged with hydrogen gas using a hydrogen balloon, and stirred at room temperature under hydrogen balloon pressure for 48 hours. The reaction was monitored by LCMS until completion. The reaction solution was diluted with methanol, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give a white solid 1i (900 mg, 90% yield). ESI-MS (m / z): 253 [M+H] + .

[0081] Step 6: Compound 1i (300 mg, 1.19 mmol), compound 1e (395 mg, 1.54 mmol), Pd2(dba)3 (217 mg, 0.23 mmol), t-BuONa (342 mg, 3.56 mmol), and X-Phos (113 mg, 0.23 mmol) were dispersed in toluene (10 mL). The reaction system was purged with nitrogen and heated to 100 °C for 16 hours. The reaction was monitored by LCMS until completion. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography. The crude product was further purified by preparative HPLC to obtain white solid 1 (143 mg, yield 25%). ESI-MS (m / z): 473.2 [M+H]+ ; 1 HNMR(500MHz,DMSO-d6)δ8.61(s,1H),8.13(s,1H),7.86(d,J=8.0Hz,1H),7.82(dd,J=8.0,1.5Hz,1H),7.74(s,1H),7.41(s,1H),6.67(t,J=5.5Hz 1H),5.43(s,2H),4.28-4.16(m,2H),4.10(q,J=7.0Hz,1H),4.04-3.96(m,1H),3.30-3.20(m,1H ), 2.93 (s, 3H), 2.54-2.50 (m, 2H), 1.95-1.85 (m, 1H), 1.84-1.70 (m, 1H), 1.21 (d, J = 7.0Hz, 3H).

[0082] Example 19

[0083] (S)-4,6-dimethyl-N-((1-((6-(trifluoromethyl)pyridin-3-yl)methyl)-1H-pyrazol-4-yl)methyl)-5,6-dihydro-4H-pyrrolo[3,2,1-de]pterodin-2-amine

[0084]

[0085] Compound 19 was prepared by the following steps:

[0086]

[0087] Step 1: Compounds 19a (200 mg, 1.06 mmol) and 19b (808 mg, 3.19 mmol) were dissolved in N,N-dimethylformamide (5 mL), and Cs₂CO₃ (1.39 g, 4.26 mmol) was added. The reaction mixture was heated to 100 °C and reacted for 2 hours. The reaction was monitored by LC-MS until complete. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to give a white solid, 19c (200 mg, 60% yield). ESI-MS (m / z): 253.5 [M-55] + .

[0088] Step 2: Compound 19c (200 mg, 0.64 mmol) was dissolved in dioxane (5 mL), and concentrated hydrochloric acid (118 mg, 0.098 mL) was added. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was monitored by LCMS until completion. The reaction mixture was concentrated under reduced pressure to give compound 19d (135 mg, 99% yield). ESI-MS (m / z): 209.7 [M+1] + .

[0089] Step 3: Compound 19d (135 mg, 0.64 mmol) was dissolved in THF (3 mL), and NaH (77 mg, 1.92 mmol) was added under ice bath conditions. After stirring for 5 minutes, iodomethane (137 mg, 0.97 mmol) was added, and stirring was continued for another 30 minutes. The reaction was monitored by LCMS to ensure complete reaction. The reaction solution was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to give compound 19e (60 mg, 41% yield). ESI-MS (m / z): 223.6 [M+H] + .

[0090] Step 4: Compounds 19e (60 mg, 0.26 mmol) and 1e (103 mg, 0.40 mmol) were dissolved in n-butanol (5 mL), and trifluoroacetic acid (153 mg, 1.35 mmol) was added. The reaction mixture was heated to 150 °C in a microwave oven for 4 hours, and the reaction was monitored by LCMS to ensure completion. The reaction mixture was concentrated, and the residue was purified by preparative HPLC to obtain compound 19 (20 mg, yield 16%). ESI-MS (m / z): 443.6 [M+H] + ; 1 HNMR(500MHz,DMSO-d6)δ8.64(s,1H),8.18(s,1H),7.92-7.81(m,3H),7.49(s,1H),7.28(s,1H),6.09(s,1H),5.47(s,2H),4.3 6(d,J=5.4Hz,2H), 4.18(dd,J=12.3,3.9Hz,1H), 4.07(dd,J=12.4,2.9Hz,1H), 3.99(s,1H), 3.11(s,3H), 1.24(d,J=6.5Hz,3H).

[0091] Example 24

[0092] (R)-5-(methoxymethyl)-4-methyl-N-((1-((6-(trifluoromethyl)pyridin-3-yl)methyl)-1H-pyrazol-4-yl)methyl)-5,6-dihydro-4H-pyrrolo[3,2,1-de]pterodin-6,6-d2-2-amine

[0093]

[0094] Compound 24 was prepared by the following steps:

[0095]

[0096] Step 1: Under nitrogen protection, SOCl2 (1.72 g, 14.47 mmol, 1.05 mL) was dissolved in anhydrous dichloromethane (100 mL). After cooling the reaction solution to -70 °C, an anhydrous dichloromethane solution of imidazole (2.63 g, 38.60 mmol) and triethylamine (2.93 g, 28.95 mmol, 4.01 mL) was added dropwise (10 mL). The reaction was exothermic and the internal temperature of the reaction solution was controlled below -40 °C. After the addition was complete, the reaction solution was recooled to -70 °C and stirred for 10 minutes. An anhydrous dichloromethane solution of compound 24a (2.0 g, 9.65 mmol) (10 mL) was then added dropwise. The reaction mixture was slowly heated to room temperature and stirred overnight. The reaction solution was diluted with dichloromethane, and 0.5 M citric acid aqueous solution was added to separate the organic phase. The obtained organic phase was washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 24b (2.43 g, crude product), which was directly used in the next reaction.

[0097] Step 2: Compound 24b (2.43 g) obtained in the previous step was dissolved in a mixed solution of acetonitrile (55 mL) and water (25 mL). Ruthenium trichloride (99 mg, 0.47 mmol) and sodium periodate (4.10 g, 19.19 mmol) were added sequentially under ice bath conditions. The reaction mixture was stirred at room temperature for 3 hours to terminate the reaction. The reaction solution was diluted with ethyl acetate, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solution was filtered and concentrated to obtain compound 24c (2.2 g, crude product), which was directly used in the next step of the reaction.

[0098] Step 3: Compounds 19a (1.3 g, 6.91 mmol), 22a (2.23 g), and 18-crown-6 (91 mg, 0.34 mmol) were dissolved in dioxane (20 mL), and potassium carbonate (2.87 g, 20.74 mmol) was added. The reaction system was heated to 60 °C and reacted for 16 hours. The reaction was monitored by LCMS until completion. The reaction solution was diluted with ethyl acetate, washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. After filtration and concentration, compound 24d (2.6 g, crude product) was obtained and used directly in the next step. ESI-MS (m / z): 377.5 [M+H] + .

[0099] Step 4: Compound 24d (1.65 g, crude) was dissolved in dioxane (10 mL), and concentrated hydrochloric acid (1.33 mL) was added. The reaction mixture was stirred at room temperature for 3 hours, and the reaction was monitored by LCMS to ensure completion. The reaction mixture was concentrated to obtain the hydrochloride salt of compound 24e (1.2 g, crude), which was directly used in the next step of the reaction. ESI-MS (m / z): 277.4 [M+H] + .

[0100] Step 5: The hydrochloride salt of compound 24e (1.3 g, crude product) was dissolved in a mixture of dioxane (20 mL) and N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (2.68 g, 20.73 mmol, 3.43 mL) was added. The reaction mixture was heated to 100 °C and reacted for 16 hours. The reaction was monitored by LCMS until complete. The reaction solution was concentrated, and the residue was diluted with ethyl acetate and washed successively with saturated ammonium chloride solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to give compound 24f (0.56 g). ESI-MS (m / z): 241.5 [M+H] + .

[0101] Step 6: Compound 24f (0.1 g, 0.41 mmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL). NaH (49 mg, 60% purity, 1.22 mmol) was added under ice bath conditions. After stirring the reaction mixture for 5 minutes, iodomethane (70 mg, 0.49 mmol) was added. The reaction mixture was brought to room temperature and stirred for 3 hours. LC-MS monitoring showed the reaction was complete. The reaction mixture was diluted with ethyl acetate, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated by filtration. The residue was purified by silica gel column chromatography to give compound 24f (50 mg, 47% yield). ESI-MS (m / z): 255.5 [M+H] + .

[0102] Step 7: Compound 24 g (100 mg, 0.39 mmol) and 1e (100 mg, 0.39 mmol) were dissolved in n-butanol (3 mL), and trifluoroacetic acid (134 mg, 1.18 mmol) was added. The reaction solution was heated to 150 °C in a microwave oven for 3 hours, and the reaction was monitored by LCMS to ensure completion. The reaction solution was concentrated, and the residue was purified by preparative HPLC to obtain compound 24 (43 mg, yield 23%). ESI-MS (m / z): 475.4 [M+H] + ; 1 HNMR(500MHz,DMSO-d6)δ8.65(s,1H),8.23(s,1H),7.90-7.84(m,3H),7.52(s,1H),7.37(s,1H),6.14(s,1H),5.48(s,2 H), 4.47-4.36 (m, 2H), 4.17-4.14 (m, 1H), 3.57 (dd, J=10.0, 5.6Hz, 1H), 3.46 (dd, J=9.8, 7.3Hz, 1H), 3.30-3.26 (m, 6H).

[0103] Example 25

[0104] (R)-6-(methoxymethyl)-4,8-dimethyl-N-((1-((6-(trifluoromethyl)pyridin-3-yl)methyl)-1H-pyrazol-4-yl)methyl)-5,6-dihydro-4H-pyrrolo[3,2,1-de]pterodin-2-amine

[0105]

[0106] Compound 25 was prepared by the following steps:

[0107]

[0108] Step 1: Under nitrogen protection, SOCl2 (2.26 g, 19.00 mmol, 1.38 mL) was dissolved in anhydrous dichloromethane (50 mL). After cooling the reaction solution to -70°C, an anhydrous dichloromethane solution (10 mL) of imidazole (3.98 g, 58.47 mmol) and triethylamine (2.96 g, 29.23 mmol, 4.05 mL) was added dropwise. The reaction was exothermic and the internal temperature of the reaction solution was controlled below -40°C. After the addition was complete, the reaction solution was recooled to -70°C and stirred for 10 minutes. An anhydrous dichloromethane solution (10 mL) of compound 25a (3.0 g, 14.62 mmol) was then added dropwise. The reaction mixture was slowly heated to room temperature and stirred overnight. The reaction solution was diluted with dichloromethane, and 0.5 M citric acid aqueous solution was added to separate the organic phase. The obtained organic phase was washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 25b (3.6 g), which was directly used in the next reaction.

[0109] Step 2: Compound 25b (3.6 g) obtained in the previous step was dissolved in acetonitrile (50 mL), and ruthenium trichloride (99 mg, 0.47 mmol) was added. Then, sodium periodate aqueous solution (4.10 g, 19.19 mmol, dissolved in 50 mL of water) was added dropwise under ice bath conditions. After the addition was complete, the reaction mixture was brought to room temperature and stirred for 4 hours. The reaction solution was diluted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 25c (3.5 g, 46% yield of the two-step reaction). 1 HNMR(500MHz,DMSO-d6)δ5.14-5.06(m,1H),4.13-4.06(m,1H),3.85-3.78(m,1H),3.73-3.65(m,2H),3.31(s,3H),1.47(s,9H).

[0110] Step 3: Compounds 19a (2.0 g, 10.64 mmol), 25c (3.70 g, 13.83 mmol), and 18-crown-6 (281 mg, 1.06 mmol) were dissolved in dioxane (50 mL), and potassium carbonate (4.41 g, 31.91 mmol) was added. The reaction mixture was heated to 80 °C and reacted for 16 hours. The reaction was monitored by LCMS until completion. The reaction solution was diluted with ethyl acetate, washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. After filtration and concentration, compound 25d (3.8 g, crude product) was obtained and used directly in the next step. ESI-MS (m / z): 375.4 [M+H] + .

[0111] Step 4: Dissolve compound 25d (3.8 g) obtained in the previous step in dichloromethane (40 mL), add dioxane hydrochloride solution (4 N, 12.66 mL), and stir the reaction solution at room temperature for 16 hours. The reaction was monitored by LCMS to ensure completion. The reaction solution was concentrated to obtain the hydrochloride salt of compound 25e (3.0 g, crude product), which was directly used in the next step of the reaction. ESI-MS (m / z): 275.5 [M+H] + .

[0112] Step 5: The hydrochloride salt of compound 25e (3.6 g, crude product) was dissolved in a mixture of dioxane (50 mL) and N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (7.47 g, 57.77 mmol, 9.57 mL) was added. The reaction mixture was heated to 90 °C and reacted for 16 hours. The reaction was monitored by LCMS to ensure complete reaction. The reaction solution was concentrated, and the residue was diluted with ethyl acetate and washed successively with saturated ammonium chloride solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to give compound 25f (0.84 g). ESI-MS (m / z): 239.6 [M+H] + .

[0113] Step 6: Compound 25f (150 mg, 0.62 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL). NaH (75 mg, 60% purity, 1.89 mmol) was added under ice bath conditions. The reaction mixture was stirred for 30 minutes, followed by the addition of iodomethane (133 mg, 0.94 mmol). The reaction mixture was brought to room temperature and stirred for 3 hours until complete, as monitored by LC-MS. The reaction mixture was diluted with ethyl acetate, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated by filtration. The residue was purified by silica gel column chromatography to give compound 25 g (130 mg, 81% yield). ESI-MS (m / z): 253.5 [M+H] + .

[0114] Step 7: Under nitrogen protection, 25 g (130 mg, 0.51 mmol) of compound was dissolved in anhydrous tetrahydrofuran (3 mL). The reaction solution was cooled to -70 °C, and n-butyllithium solution (2.5 M, 0.82 mL) was added dropwise. After stirring for 30 minutes, iodomethane (292 mg, 2.06 mmol) was added. The reaction mixture was stirred at -70 °C for another hour and then slowly brought to room temperature. The reaction solution was quenched with saturated ammonium chloride aqueous solution, diluted with ethyl acetate, and washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 25 h (100 mg, crude product). ESI-MS (m / z): 267.4 [M+H] + .

[0115] Step 8: Compound 25h (100 mg, crude), compound 1e (124 mg, 0.48 mmol), Pd2(dba)3 (68 mg, 0.074 mmol), t-BuONa (108 mg, 1.12 mmol), and X-Phos (35 mg, 0.074 mmol) were dispersed in toluene (5 mL). The reaction system was purged with nitrogen and heated to 100 °C for 16 hours. The reaction was monitored by LCMS until completion. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to obtain the crude product, which was then purified by Prep-HPLC to obtain compound 25 (13 mg). ESI-MS (m / z): 487.4 [M+H] + ; 1 HNMR(500MHz,DMSO-d6)δ8.65(s,1H),8.29(br s,1H),7.88(d,J=8.0Hz,1H),7.84(d,J=8.0Hz,1H),7.78(s,1H),7.45(s,1H),6.33(br s,1H),5.80(s,1H),5.45(s,2H),4.63(s,1H),4.29(d,J=6.0Hz,2H),3.57(s,2H),3. 43(dd,J=9.5,7.0Hz,1H),3.34-3.30(m,1H),3.24(s,3H),3.01(s,3H),2.33(s,3H).

[0116] Example 26

[0117] 8,8-Difluoro-6-methyl-N-((1-((6-(trifluoromethyl)pyridin-3-yl)methyl)-1H-pyrazol-4-yl)methyl)-6,7,8,9-tetrahydro-3,5,6,9a-tetraazabenzo[cd]azine-4-amine

[0118]

[0119] Compound 26 was prepared by the following steps:

[0120]

[0121] Step 1: Compound 19a (400 mg, 2.13 mmol) and triphenylphosphine (1.67 g, 6.38 mmol) were dissolved in anhydrous tetrahydrofuran (15 mL) at room temperature. Under nitrogen protection, N,N-diisopropylethylamine (1.37 g, 10.64 mmol) and diisopropyl azodicarbonate (1.29 g, 6.38 mmol, 1.25 mL) were added dropwise. After stirring for 30 minutes, anhydrous tetrahydrofuran solution (5 mL) of compound 26a (898 mg, 4.26 mmol) was added dropwise. The reaction mixture was heated to 70 °C and reacted for 16 hours to terminate the reaction. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to give compound 26b (500 mg, 61% yield). ESI-MS (m / z): 325.4 [M-55] + .

[0122] Step 2: Compound 26b (500 mg, 1.31 mmol) was dissolved in dichloromethane (5 mL), and dioxane hydrochloride solution (4 N, 1.64 mL) was added. The reaction mixture was stirred at room temperature for 16 hours, and the reaction was monitored by LCMS to ensure complete reaction. The reaction mixture was filtered, the filter cake was collected, and dried under reduced pressure to obtain the hydrochloride salt of compound 26c (360 mg, yield 86%).

[0123] Step 3: The hydrochloride salt of compound 26c (360 mg, 1.13 mmol) was dissolved in a mixture of dioxane (5 mL) and N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (732 mg, 5.67 mmol) was added. The reaction mixture was heated to 100 °C and reacted for 4 hours. The reaction was monitored by LC-MS until complete. The reaction solution was concentrated, and the residue was diluted with ethyl acetate and washed successively with saturated ammonium chloride solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to give a white solid 26d (250 mg, 90% yield). ESI-MS (m / z): 245.5 [M+H] + .

[0124] Step 4: Compound 26d (300 mg, 1.23 mmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL). NaH (122 mg, 60% purity, 3.07 mmol) was added in portions under ice bath conditions. After stirring the reaction mixture for 15 minutes, iodomethane (261 mg, 1.84 mmol, 0.17 mL) was added. The reaction mixture was brought to room temperature and stirred for 4 hours. LC-MS monitoring showed the reaction was complete. The reaction mixture was diluted with ethyl acetate, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration yielded compound 26e (290 mg, 91% yield). ESI-MS (m / z): 259.5 [M+H] + .

[0125] Step 5: Compounds 26e (100 mg, 0.38 mmol) and 1e (148 mg, 0.57 mmol) were dissolved in n-butanol (4 mL), and trifluoroacetic acid (44 mg, 0.38 mmol) was added. The reaction solution was heated to 150 °C in a microwave oven for 6 hours, and the reaction was monitored by LCMS to ensure completion. The reaction solution was concentrated, and the residue was purified by preparative HPLC to obtain compound 26 (17 mg, yield 9%). ESI-MS (m / z): 479.5 [M+H] + ; 1 HNMR(500MHz,DMSO-d6)δ8.64(s,1H),7.90-7.80(m,3H),7.52-7.40(m,2H),6.26(d,J=2.5Hz,1H) ,5.46(s,2H),4.73(t,J=12.0Hz,2H),4.37(d,J=6.0Hz,2H),4.17(t,J=12.0Hz,2H),3.26(s,3H).

[0126] Example 27

[0127] (R)-4,6-dimethyl-N-((1-((6-(trifluoromethyl)pyridin-3-yl)methyl)-1H-pyrazol-4-yl)methyl)-5,6-dihydro-4H-pyrrolo[3,2,1-de]pterodin-2-amine

[0128]

[0129] By replacing (R)-1-(BOC-amino)-2-propanol methanesulfonate 19b in the first step of Example 19 with (S)-1-(BOC-amino)-2-propanol methanesulfonate, compound 27 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 443.6 [M+H] + ; 1HNMR(500MHz,DMSO-d6)δ12.19(br s,1H),8.64(s,1H),8.34(br s,1H),7.92-7.83(m,3H),7.53(s,1H),7.44(d,J=2.5Hz,1H),6.20(s,1H),5. 49(s,2H),4.44(s,2H),4.29-4.06(m,3H),3.22(s,3H),1.29(d,J=6.5Hz,3H).

[0130] Example 28

[0131] (R)-N-((1-((6-(trifluoromethyl)pyridin-3-yl)methyl)-1H-pyrazol-4-yl)methyl)-7a,8,10,11-tetrahydro-7H-[1,4]oxazin[3,4-h]pyrrolo[3,2,1-de]pterodin-2-amine

[0132]

[0133] Compound 28 was prepared by the following steps:

[0134]

[0135] Step 1: Compound 19a (300 mg, 1.60 mmol) and triphenylphosphine (1.26 g, 4.79 mmol) were dissolved in anhydrous tetrahydrofuran (10 mL) at room temperature. Under nitrogen protection, N,N-diisopropylethylamine (1.03 g, 7.98 mmol) and diisopropyl azodicarbonate (967 mg, 4.79 mmol, 0.94 mL) were added dropwise. After stirring for 30 minutes, anhydrous tetrahydrofuran solution (5 mL) of compound 28a (866 mg, 3.99 mmol) was added dropwise. The reaction mixture was heated to 70 °C and reacted for 16 hours to terminate the reaction. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to give compound 28b (200 mg, 32% yield). ESI-MS (m / z): 331.4 [M-55] + .

[0136] Step 2: Compound 28b (200 mg, 0.51 mmol) was dissolved in dichloromethane (2 mL), and dioxane hydrochloride solution (4 N, 1.29 mL) was added. The reaction mixture was stirred at room temperature for 16 hours, and the reaction was monitored for completeness by LCMS. The reaction mixture was concentrated to obtain compound 28c (100 mg, crude product), which was directly used in the next step of the reaction. ESI-MS (m / z): 251.5 [M+H] + .

[0137] Step 3: Compound 28c (100 mg) and 1e (122 mg, 0.47 mmol) were dissolved in n-butanol (4 mL), and trifluoroacetic acid (45 mg, 0.39 mmol) was added. The reaction solution was heated to 150 °C in a microwave oven for 5 hours, and the reaction was monitored by LCMS to ensure completion. The reaction solution was concentrated, and the residue was purified by preparative HPLC to obtain compound 28 (13 mg). ESI-MS (m / z): 471.5 [M+H] + ; 1 HNMR(500MHz,DMSO-d6)δ12.19(s,1H),8.63(s,1H),8.36(br s,1H),8.01-7.79(m,3H),7.65-7.44(m,2H),6.23(s,1H),5.48(s,2H),4.56-4.40(m,3H),4. 29-4.25(m,1H),4.13-4.02(m,3H),3.93-3.82(m,1H),3.62-3.52(m,2H),3.24-3.17(m,1H).

[0138] Biological screening and results of Wnt pathway inhibitors

[0139] Experiment 1: Construction of Colo205-LUC-TCF / LEF-M1 reporter cell line

[0140] The Colo205 cell line (Chinese Academy of Sciences Cell Bank, Cat#TCHu102) was purchased from the Chinese Academy of Sciences Cell Bank. After expansion and passage, during the exponential growth phase, the cells were transfected with a luciferase reporter plasmid (Promega) driven by the TCF / LEF transcription factor during the lipo3000 transfection method. This plasmid carries an antibiotic resistance gene, allowing for antibiotic selection. Transfection was performed in 10cm culture dishes using standard complete medium without antibiotics. After 2 days, the medium was replaced with antibiotic-containing medium, and culture continued. Thereafter, the antibiotic-containing medium was replaced every 2 days, and the suspended cells were discarded. The original medium was centrifuged to remove cells and debris and retained as adaptation medium. When the cells reached confluence, they were digested, counted, and passaged into 96-well plates, ensuring an average of 1.5 cells / well. Adaptation medium was used for passage. The remaining cells were cryopreserved. After passage, the cells were cultured for 4 hours to allow them to adhere, and then the cell count in each well was observed under a microscope. Each well containing only one cell was labeled as a monoclonal well. The cells were then cultured normally, with the medium changed every two days and observations performed. Wells showing continued monoclonal cell growth were labeled twice and then replaced with normal antibiotic-containing medium. When a monoclonal well filled the wells of a 96-well plate, it was digested and passaged into a 24-well plate. After the 24-well plate reached confluence, it was passaged into one 96-well plate and one 6-well plate. At least six wells in the 96-well plate were treated, with three wells treated with a known Wnt inhibitor and the other three untreated. After 24 hours, fluorescence detection reagent was added to the 96-well plate, and fluorescence intensity was measured. Cell lines showing fluorescence expression without treatment and exhibiting reduced fluorescence after inhibition were selected for further culture. The Colo205-LUC-TCF / LEF-M1 cell line is one of the cell lines selected above. Its growth curve, cell morphology, and cell growth status are similar to those of the original Colo205 cells. Moreover, the ratio of fluorescence signal with and without inhibitor treatment is relatively large among all cell lines. The ratio can reach 4-5 times when inhibited at 4h, which is fully suitable for the subsequent screening of Wnt inhibitors.

[0141] Example 2: Detection of the inhibitory effect of the compound on the Colo205-LUC-TCF / LEF M1 reporter cell line. The Colo205-LUC-TCF / LEF M1 cell line is a reporter cell line stably transfected with the pGL4.49-LUC2-TCF / LEF vector. Its β-catenin Wnt pathway is continuously activated. After the addition of the inhibitor, the Wnt pathway is inhibited, and the expression level of firefly luciferase regulated by the TCF / LEF cis-elements on the vector decreases. Subsequently, after the addition of the detection substrate, the detected light signal decreases accordingly, thus detecting the inhibitory effect of the compound.

[0142] 100 μL of a compound with a maximum concentration of 20 μM was added to each well of a 96-well cell culture plate, with the compound concentration serially diluted 3-fold. Then, 10,000 stable colo205 cells transfected with the reporter gene and 100 μL of culture medium were seeded into each well, along with corresponding positive and negative control wells. The cells were placed in a 5% CO2 cell culture incubator and incubated at 37°C for 4 h. After 4 hours, the culture medium was removed, and 100 μL of reagent containing the corresponding firefly luciferase substrate (Promega) was added to each well. The activity of the luciferase reporter gene was measured. The luminescence intensity was read using SpectraMax in full wavelength mode. The light signal intensity of cells treated only with DMSO served as a positive control, and the light signal intensity of wells without cells served as a negative control. The IC50 concentration of each compound was calculated. The colo 205 reporter gene detection data are summarized in Table 1.

[0143] Table 1

[0144] Example 3: Inhibition of proliferation of the compound on Wnt mutant cell lines (Colo205 and DU4475) and non-Wnt mutant cell lines (HeLa and RKO). The cell lines used in the experiment were Colo205 and DU4475 cell lines with sustained activation of the Wnt pathway and Wnt pathway-dependent proliferation; while HELA and RKO cell lines, which are normally not activated and whose proliferation is not dependent on the Wnt pathway, were used as control cell lines to determine that the inhibitory effect of the compound of the present invention on Wnt-dependent proliferation was not due to other non-specific toxicity.

[0145] Colo205, Du4475, HELA, and RKO cell lines cultured in their respective media were treated during the logarithmic growth phase. Cells were collected and prepared into homogeneous cell suspensions of known concentrations. These suspensions were then added to 96-well cell culture plates, ensuring each well contained 1000 cells. The plates were incubated in a 5% CO2 incubator at 37°C for 20–24 hours. The next day, completely dissolved, three-fold serially diluted compounds were added to each well to achieve a final maximum concentration of 20 μM. The plates were then cultured for another 96 hours. Cell viability was detected using a Promega cell viability assay; higher cell proliferation resulted in a stronger signal. The assay was performed using a SpectraMax in full-wavelength mode. Wells containing only DMSO served as positive control wells, while wells without cell inoculation served as negative control wells. The IC50 values ​​of each compound were calculated for inhibition of proliferation in cells with sustained Wnt activation or proliferation dependence, as well as for inhibition of proliferation in cells without Wnt activation or proliferation independence. The inhibitory effects of the compounds on the Wnt pathway and their toxicity to normal cells were evaluated (Table 2).

[0146] Table 2

[0147]

Claims

1. A method for preparing compounds having the structure of Formula 19, characterized in that, The preparation method of compound 19 includes the following steps: Formula 19 Step 1: Dissolve compounds 19a and 19b in N,N-dimethylformamide, add Cs2CO3, heat the reaction solution to 100℃ and react for 2 hours until the reaction is complete; dilute the reaction solution with water and extract with ethyl acetate; wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate; purify the residue by silica gel column chromatography to obtain white solid 19c; Step 2: Dissolve compound 19c in dioxane, add concentrated hydrochloric acid, and stir the reaction solution at room temperature for 1 hour until the reaction is complete; concentrate the reaction solution under reduced pressure to obtain compound 19d; Step 3: Dissolve compound 19d in THF, add NaH under ice bath, stir for 5 minutes, add iodomethane, continue stirring for half an hour and monitor the reaction to be complete; dilute the reaction solution with water and extract with ethyl acetate; wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate; purify the residue by silica gel column chromatography to obtain compound 19e. Step 4: Dissolve compounds 19e and 1e in n-butanol, add trifluoroacetic acid, and heat the reaction solution in a microwave oven to 150°C for 4 hours until the reaction is complete; concentrate the reaction solution, and purify the residue by preparative HPLC to obtain compound 19.

2. A method for preparing compounds having the structure of formula 24, characterized in that, The preparation method of compound 24 includes the following steps: Formula 24 Step 1: Under nitrogen protection, SOCl2 was dissolved in anhydrous dichloromethane. After the reaction solution was cooled to -70°C, an anhydrous dichloromethane solution of imidazole and triethylamine was added dropwise. The internal temperature of the reaction solution was controlled below -40°C. After the addition was completed, the reaction solution was cooled back to -70°C and stirred for 10 minutes. An anhydrous dichloromethane solution of compound 24a was then added dropwise. The reaction mixture was slowly heated to room temperature and stirred overnight. The reaction solution was diluted with dichloromethane, and 0.5M citric acid aqueous solution was added to separate the organic phase. The obtained organic phase was washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 24b. Step 2: Dissolve compound 24b obtained in the previous step in a mixed solution of acetonitrile and water, and add ruthenium trichloride and sodium periodate sequentially under ice bath conditions; stop the reaction by stirring the reaction mixture at room temperature for 3 hours; dilute the reaction solution with ethyl acetate, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain compound 24c; Step 3: Dissolve compounds 19a, 22a and 18-crown-6 in dioxane, add potassium carbonate, heat the reaction system to 60°C and react for 16 hours until the reaction is complete; dilute the reaction solution with ethyl acetate, wash with water and saturated brine respectively, dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain compound 24d; Step 4: Dissolve compound 24d in dioxane, add concentrated hydrochloric acid, and stir the reaction solution at room temperature for 3 hours until the reaction is complete; concentrate the reaction solution to obtain the hydrochloride salt of compound 24e; Step 5: Dissolve the hydrochloride salt of compound 24e in a mixture of dioxane and N,N-dimethylformamide, add N,N-diisopropylethylamine, and heat the reaction solution to 100°C for 16 hours until the reaction is complete; concentrate the reaction solution, dilute the residue with ethyl acetate, and wash successively with saturated ammonium chloride solution and saturated brine; dry the organic phase with anhydrous sodium sulfate, filter and concentrate; purify the residue by silica gel column chromatography to obtain compound 24f; Step 6: Dissolve compound 24f in anhydrous N,N-dimethylformamide, add NaH under ice bath, stir the reaction mixture for 5 minutes, and then add iodomethane; raise the reaction solution to room temperature and stir for 3 hours until the reaction is complete; dilute the reaction solution with ethyl acetate, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter and concentrate; purify the residue by silica gel column chromatography to obtain compound 24g; Step 7: Dissolve compound 24g and 1e in n-butanol, add trifluoroacetic acid, and heat the reaction solution in a microwave oven to 150°C for 3 hours until the reaction is complete; concentrate the reaction solution, and purify the residue by preparative HPLC to obtain compound 24.

3. A method for preparing compounds having the structure of formula 25, characterized in that, The preparation method of compound 25 includes the following steps: Formula 25 Step 1: Under nitrogen protection, SOCl2 was dissolved in anhydrous dichloromethane. After the reaction solution was cooled to -70°C, an anhydrous dichloromethane solution of imidazole and triethylamine was added dropwise. The internal temperature of the reaction solution was controlled below -40°C. After the addition was completed, the reaction solution was cooled back to -70°C and stirred for 10 minutes. An anhydrous dichloromethane solution of compound 25a was then added dropwise. The reaction mixture was slowly heated to room temperature and stirred overnight. The reaction solution was diluted with dichloromethane, and 0.5M citric acid aqueous solution was added to separate the organic phase. The obtained organic phase was washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 25b. Step 2: Dissolve compound 25b obtained in the previous step in acetonitrile, add ruthenium trichloride, and then add sodium periodate aqueous solution dropwise under ice bath; after the addition is complete, the reaction mixture is heated to room temperature and stirred for 4 hours; the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is dried with anhydrous sodium sulfate, filtered and concentrated to obtain compound 25c; Step 3: Dissolve compounds 19a, 25c and 18-crown-6 in dioxane, add potassium carbonate, heat the reaction system to 80°C and react for 16 hours until the reaction is complete; dilute the reaction solution with ethyl acetate, wash with water and saturated brine respectively, dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain compound 25d. Step 4: Dissolve compound 25d obtained in the previous step in dichloromethane, add dioxane hydrochloride solution, and stir the reaction solution at room temperature for 16 hours until the reaction is complete; concentrate the reaction solution to obtain the hydrochloride salt of compound 25e; Step 5: Dissolve the hydrochloride salt of compound 25e in a mixture of dioxane and N,N-dimethylformamide, add N,N-diisopropylethylamine, and heat the reaction solution to 90°C for 16 hours until the reaction is complete; concentrate the reaction solution, dilute the residue with ethyl acetate, and wash successively with saturated ammonium chloride solution and saturated brine; dry the organic phase with anhydrous sodium sulfate, filter and concentrate; purify the residue by silica gel column chromatography to obtain compound 25f; Step 6: Dissolve compound 25f in anhydrous N,N-dimethylformamide, add NaH under ice bath, stir the reaction mixture for 30 minutes, and then add iodomethane; raise the reaction solution to room temperature and stir for 3 hours until the reaction is complete; dilute the reaction solution with ethyl acetate, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter and concentrate; purify the residue by silica gel column chromatography to obtain compound 25g; Step 7: Under nitrogen protection, 25 g of compound was dissolved in anhydrous tetrahydrofuran. The reaction solution was cooled to -70°C, and n-butyllithium solution was added dropwise. After stirring for 30 minutes, iodomethane was added. The reaction mixture was stirred at -70°C for another hour and then slowly raised to room temperature. The reaction solution was quenched with saturated ammonium chloride aqueous solution, diluted with ethyl acetate, and washed successively with water and saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain compound 25 h. Step 8: Disperse compound 25h, compound 1e, Pd2(dba)3, t-BuONa and X-Phos in toluene. After purging the reaction system with nitrogen, heat to 100℃ and react for 16 hours until the reaction is complete. Concentrate the reaction solution and purify the residue by silica gel column chromatography to obtain the crude product, and then purify it by Prep-HPLC to obtain compound 25.

4. A method for preparing compounds having the structure of Formula 26, characterized in that, The preparation method of compound 26 includes the following steps: Formula 26 Step 1: Compound 19a and triphenylphosphine were dissolved in anhydrous tetrahydrofuran at room temperature. Under nitrogen protection, N,N-diisopropylethylamine and diisopropyl azodicarbonate were added sequentially. After stirring for 30 minutes, anhydrous tetrahydrofuran solution of compound 26a was added dropwise. The reaction mixture was heated to 70°C and reacted for 16 hours until the reaction was complete. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to obtain compound 26b. Step 2: Dissolve compound 26b in dichloromethane, add dioxane hydrochloride solution, stir the reaction solution at room temperature for 16 hours until the reaction is complete; filter the reaction solution, collect the filter cake, and dry it under reduced pressure to obtain the hydrochloride salt of compound 26c. Step 3: The hydrochloride salt of compound 26c was dissolved in a mixture of dioxane and N,N-dimethylformamide, and N,N-diisopropylethylamine was added. The reaction solution was heated to 100°C and reacted for 4 hours until the reaction was complete. The reaction solution was concentrated, and the residue was diluted with ethyl acetate and washed successively with saturated ammonium chloride solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to obtain a white solid 26d. Step 4: Dissolve compound 26d in anhydrous N,N-dimethylformamide, add NaH in portions under ice bath, stir the reaction mixture for 15 minutes and then add iodomethane; raise the reaction solution to room temperature and stir for 4 hours until the reaction is complete; dilute the reaction solution with ethyl acetate, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain compound 26e; Step 5: Dissolve compounds 26e and 1e in n-butanol, add trifluoroacetic acid, and heat the reaction solution in a microwave oven to 150°C for 6 hours until the reaction is complete; concentrate the reaction solution, and purify the residue by preparative HPLC to obtain compound 26.

5. A method for preparing compounds having the structure of formula 28, characterized in that, The preparation method of compound 28 includes the following steps: Formula 28 Step 1: Compound 19a and triphenylphosphine were dissolved in anhydrous tetrahydrofuran at room temperature. Under nitrogen protection, N,N-diisopropylethylamine and diisopropyl azodicarbonate were added sequentially. After stirring for 30 minutes, anhydrous tetrahydrofuran solution of compound 28a was added dropwise. The reaction mixture was heated to 70 °C and reacted for 16 hours to terminate the reaction. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to obtain compound 28b. Step 2: Dissolve compound 28b in dichloromethane, add dioxane hydrochloride solution, and stir the reaction solution at room temperature for 16 hours until the reaction is complete; concentrate the reaction solution to obtain compound 28c. Step 3: Dissolve compounds 28c and 1e in n-butanol, add trifluoroacetic acid, and heat the reaction solution in a microwave oven to 150°C for 5 hours until the reaction is complete; concentrate the reaction solution, and purify the residue by preparative HPLC to obtain compound 28.

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