GLP-1 receptor agonist / GLP-1 secretion-promoting double-target compound

Abstract

The invention relates to design, preparation and application of a double-target compound which has activity on both a human glucagon-like peptide-1 receptor (GLP-1R) and a KCNH6 potassium channel. The compound plays a dual role of a GLP-1 receptor agonist and an endogenous GLP-1 secretagogue by activating GLP-1R and blocking KCNH6, and can be used for treating obesity, type 2 diabetes mellitus and related complications thereof.

Description

Technical Field

[0001] This application relates to, but is not limited to, the field of medicinal chemistry, and particularly to the design, preparation, and application of a class of dual-target compounds that are active against both human glucagon-like peptide-1 receptor (GLP-1R) and KCNH6 potassium channels. Background Technology

[0002] Over the past few decades, the prevalence of diabetes has been steadily rising. People with diabetes have a two to four times higher risk of cardiovascular disease than the general population, and it can also lead to serious consequences such as blindness, amputation, and kidney failure, making it a significant public health issue. Currently, there are two main forms of diabetes: type 1 and type 2. The primary cause of type 1 diabetes is the attack of pancreatic beta cells by the body's immune system, leading to impaired insulin secretion. Type 2 diabetes is the most common form of diabetes, accounting for approximately 90% of all cases, and is mainly associated with relative insulin deficiency and insulin resistance.

[0003] Glucagon-like peptide-1 (GLP-1) is an incretin responsible for glucose homeostasis, secreted by L cells of the intestinal epithelium, and exerts its physiological effects by binding to its receptor. Insufficient GLP-1 secretion is also one of the pathogenic mechanisms of type 2 diabetes. The GLP-1 receptor (GLP-1R) belongs to the G protein-coupled receptor subfamily; when GLP-1 binds to GLP-1R, it triggers a series of biological effects. Currently, peptide drugs based on GLP-1 analogs, such as exenatide, liraglutide, and smegglutide, are widely used in patients with type 2 diabetes and simple obesity, showing significant effects in reducing weight and improving glucose tolerance, while also possessing advantages such as protecting cardiopulmonary and renal function. Because GLP-1 analogs require subcutaneous injection and are often accompanied by gastrointestinal adverse reactions such as nausea and vomiting, oral non-peptide small molecule GLP-1 receptor agonists have become a hot topic in new drug development. Several non-peptide small molecule agonists have entered clinical trials, including Danuglipron developed by Pfizer, Orforglipron developed by Eli Lilly, and HRS-7535 developed by Hengrui Medicine. However, no small molecule GLP-1 receptor agonist is currently available for clinical use. Furthermore, long-term use of GLP-1 receptor agonists can lead to a decrease in basal metabolic rate, resulting in reduced energy expenditure and weight rebound, which remains a significant problem that urgently needs to be addressed in this field.

[0004] Human delayed rectifier potassium channels (KCNH6) are repolarizing potassium channels. Studies have shown that drugs acting on intestinal KCNH6 channels can significantly improve glucose tolerance, promote GLP-1 secretion, and increase insulin sensitivity, without affecting cardiac KCNH6 channel function. Therefore, this invention aims to discover compounds that are active against both GLP-1 receptors and intestinal KCNH6 channels, particularly novel compounds with good biological properties that can be safely applied to humans. The compounds of this invention act as GLP-1 receptor agonists and endogenous GLP-1 secretagogues by activating GLP-1 receptors and blocking KCNH6 potassium channels, and can be used to treat obesity, type 2 diabetes, and related complications (see appendix). Figure 1 ). Summary of the Invention

[0005] This invention provides molecular structures for compounds of general formula (I) that define their functions, including dual-target inhibitory activity against GLP-1R and KCNH6, with these two components exhibiting a synergistic effect. GLP-1R and KCNH6 together constitute a single drug molecule. These compounds are active against both GLP-1R and KCNH6 channels. This invention also relates to pharmaceutical compositions comprising these compounds and their use in medicaments for treating diseases such as diabetes.

[0006] (I)

[0007] This invention aims to protect the drug design concept and realized drug design entities that achieve the synergistic effect of "GLP-1R agonism" and "KCNH6 inhibition" through reasonable chemical bonds and methods.

[0008] This invention relates to a drug design method and concept that combines "GLP-1R agonist" and "KCNH6 inhibitor" to synergistically exert weight loss and blood sugar lowering effects. It includes the use of various methods to construct drugs that synergistically lower blood sugar through dual-target, dual-mechanism GLP-1R and KCNH6.

[0009] The compound of this invention has a molecular structure constructed by combining a "GLP-1R activating" moiety and a "KCNH6 inhibiting" moiety. Its characteristic is that the "GLP-1R activating" moiety can independently activate GLP-1R.

[0010] The compound described in this invention, “GLP-1R activator”, can independently activate GLP-1R. Features include, but are not limited to, the structures of the following general formulas (II) and (III), and the claims also include isomers and deuterated products of general formulas (II) and (III).

[0011] (II) (III)

[0012] In general formula (II), the chemical structures R1 and R2 can be access sites for the Linker, either independently or jointly.

[0013] The X part may contain the following structures:

[0014] The Y part can contain the following structures:

[0015] The Y1 portion contains a benzene ring with any five-membered or six-membered structure, or a substituted benzene ring (containing a combination of any one or more of methyl, ethyl, hydroxyl, amino, mercapto, fluorine, chlorine, and bromine), or a pyridine ring, or an aliphatic five-membered ring, or an aliphatic six-membered ring, or a nitrogen-oxygen five-, six-, or seven-membered aliphatic heterocycles, including but not limited to the following representative structures:

[0016] Part Y2 includes:

[0017] In general formula (II), the chemical structures R1 and R2 can be access sites for the Linker, either independently or jointly.

[0018] The compound of the present invention is characterized in that the Linker portion comprises the following structure: a unit of any type with a length of 1 to 30 atoms.

[0019] Specifically, the Linker includes, but is not limited to, the following structures, or any group of structures composed of the following structures.

[0020] For example, the structural units of ethylene glycol:

[0021] Or units composed of fatty acid chains:

[0022] Units composed of unsaturated chains:

[0023] Or carboxylic acid:

[0024] Or a unit composed of aromatic compounds:

[0025] Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units:

[0026] In the heteroatom-containing unit, R can be independently: alkyl, alkoxy, or hydrogen.

[0027] Preferably, the alkyl group is specifically methyl, ethyl, propyl, isopropyl, n-butyl, or tert-butyl; and the alkoxy group is -OEt or -OMe.

[0028] In particular, a linker can be composed of multiple sugar groups such as monosaccharides and disaccharides, or a polypeptide composed of a single amino acid, a dipeptide, or multiple amino acids.

[0029] The "KCNH6 inhibiting portion" of the compound described in this invention can independently inhibit KCNH6. Features include, but are not limited to, the structures of the following general formulas (IV) and (V): (IV)

[0030] R1, R2, R3, and R4 can be any structure, either independently or collectively, including methoxy, ethoxy, alkyl groups of length 1 to 5, amino groups, carboxylic acids, etc.

[0031] For example, the structural units of ethylene glycol:

[0032] Or units composed of fatty acid chains:

[0033] Units composed of unsaturated chains:

[0034] Or a unit composed of aromatic compounds:

[0035] Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units:

[0036] In the heteroatom-containing unit, R can be independently: alkyl, alkoxy, or hydrogen.

[0037] Preferably, the alkyl group is specifically methyl, ethyl, propyl, isopropyl, n-butyl, or tert-butyl; and the alkoxy group is -OEt or -OMe.

[0038] R1, R2, and R3 and R4 can form a ring structure independently or together, such as: , ,

[0039] The Linker interface can exist on the aromatized entity, or on the R1, R2, R3, and R4 parts.

[0040] In particular, the KCNH6 inhibition part of the structure includes various salt forms of general formula (Ⅳ), such as quaternary ammonium salt forms of various anions such as chloride ion, bromide ion, iodide ion, and sulfate ion.

[0041] The molecular structure as shown in Formula V is claimed: (V)

[0042] In the formula, P, Q, and R can be independent, together, or in combination as the linker's connection sites.

[0043] P can be constructed as a chemical entity linked to the V core structure via amide bonds, including but not limited to the following structures.

[0044] P can be n has a length of 0 to 10. P can be n has a length of 1 to 5 P can be

[0045] The L1 portion can be any structure, either independently or collectively, including methoxy, ethoxy, alkyl groups of length 1 to 5, amino, carboxylic acids, etc.

[0046] For example, the structural units of ethylene glycol:

[0047] Or units composed of fatty acid chains:

[0048] Units composed of unsaturated chains:

[0049] Or a unit composed of aromatic compounds:

[0050] Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units:

[0051] P can be

[0052] The L2 portion can be any structure, either independently or collectively, including methoxy, ethoxy, alkyl groups of length 1 to 5, amino, carboxylic acids, etc.

[0053] For example, the structural units of ethylene glycol:

[0054] Or units composed of fatty acid chains:

[0055] Units composed of unsaturated chains:

[0056] Or a unit composed of aromatic compounds:

[0057] Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units:

[0058] According to claim 4, the compound “KCNH6 moiety” can also be obtained by reducing the quaternary ammonium salt structure in the above general formula (IV) to obtain a tertiary amine structure, as shown in general formula (VI): (VI)

[0059] R1, R2, R3, and R4 can be any structure, either independently or collectively, including methoxy, ethoxy, alkyl groups of length 1 to 5, amino groups, carboxylic acids, etc.

[0060] For example, the structural units of ethylene glycol:

[0061] Or units composed of fatty acid chains:

[0062] Units composed of unsaturated chains:

[0063] Or a unit composed of aromatic compounds:

[0064] Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units:

[0065] In the heteroatom-containing unit, R can be independently: alkyl, alkoxy, or hydrogen.

[0066] Preferably, the alkyl group is specifically methyl, ethyl, propyl, isopropyl, n-butyl, or tert-butyl; and the alkoxy group is -OEt or -OMe.

[0067] R1, R2, and R3 and R4 can form a ring structure independently or together, such as: , ,

[0068] In particular, the KCNH6 inhibition moiety is constructed in various salt forms of general formula (VI), such as hydrochloride, silver nitrate, hydrogen bromide, sulfate and other structures.

[0069] The claims also include the deuterated drug structure formed by one or more substitutions and the mixture of stereoisomers with unresolved chiral structures constructed by modifying the chemical stereoconformation, and the corresponding pharmaceutically acceptable salts.

[0070] Claims include the following chemical structural entities, including the following chemical structures and related deuterated drug structures constituted by single or multiple substitutions and stereoisomers constructed by modifying chemical stereoconformities, chiral unresolved mixtures, and corresponding pharmaceutically acceptable salts. Attached Figure Description Figure 1 This explains the mechanism of action of the small molecule dual-target site in this invention. Figure 2 The results of the intraperitoneal glucose tolerance test (IPGTT) in mice for RK-gk-303, a representative compound of the dual GLP-1 receptor agonist / GLP-1 secretion promoter compounds of this invention. Detailed Implementation

[0071] The following description is intended to enable those skilled in the art to understand and implement this application, and is illustrated in conjunction with specific applications and their requirements. Those skilled in the art will readily understand various modifications to the disclosed embodiments, which can be applied to other embodiments and applications without departing from the spirit and scope of this application. Therefore, the scope of this application is not limited to the illustrated embodiments, but should cover all broad contents conforming to the scope of the appended claims.

[0072] The terminology used in this application is for describing particular embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein should be considered to include the plural forms. Furthermore, the terms “comprising” or “including,” as used in this specification, indicate the presence of the stated features, elements, steps, operations, components, and / or parts, but do not preclude the possibility of the presence or addition of one or more other features, elements, steps, operations, components, parts, and / or combinations thereof.

[0073] According to one aspect of this application, a variety of compounds are provided. These compounds can activate GLP-1R and / or block KCNH6, exerting a dual effect as a GLP-1 receptor agonist and / or an endogenous GLP-1 secretagogue.

[0074] Example 1: Synthesis of compound 4.

[0075] Reaction formula:

[0076]

[0077] Preparation method:

[0078] Step 1: Preparation of compound 2.

[0079] 9,10-dimethoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinoline (2).

[0080] Compound 1 (12.00 g, 32.3 mmol) and anhydrous methanol (120 mL) were added sequentially to a dry 120 mL three-necked flask at room temperature. The system was cooled to 0 °C, and sodium borohydride (4.28 g, 113.2 mmol) was slowly added. The mixture was slowly brought to room temperature overnight. LCMS monitoring showed that the reaction was complete. The reaction solution was diluted with water (200 mL), and a black solid precipitated. The mixture was filtered under reduced pressure, and the filter cake was collected, dried, and compound 2 (8.16 g, black solid) was given, with a yield of 47.46%.

[0081] LCMS(ESI): m / z 340.1 [M+H] + ;RT = 0.911 (2.5 min).

[0082] 1 H NMR (400 MHz, DMSO- d 6 ): δ 6.92-6.84 (m, 3H), 6.67 (s, 1H), 5.94 (s, 2H), 4.06 (d, J = 15.6 Hz, 1H), 3.77 (s, 3H), 3.73 (s, 3H), 3.39 (s, 4H), 3.12-3.07 (m, 1H), 2.93-2.88 (m, 1H), 2.62-2.57 (m, 2H).

[0083] Step 2: Preparation of compound 3.

[0084] 9,10-dimethoxy-7-(2-methoxy-2-oxoethyl)-5,6,7,8,13,13a-hexahydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (3).

[0085] In a dry 250 mL single-necked flask at room temperature, compound 2 (6.33 mg, 18.62 mmol), acetonitrile (120 mL), methyl bromoacetate (14.24 g, 93.09 mmol), potassium carbonate (12.8 g, 93.09 mmol), and potassium iodide (0.31 g, 1.86 mmol) were added sequentially. The mixture was heated to 40 °C and reacted for 4 hours. After the reaction was complete, the reaction solution was filtered, and the filtrate was collected and concentrated under reduced pressure. The residue was slurried with methyl tert-butyl ether, filtered under reduced pressure, and the filter cake was collected and dried to give compound 3 (2.35 g, nearly white solid), yield: 30.59%.

[0086] LCMS(ESI): m / z 412.3 [M+H] + ;RT = 1.452 min (2.50 min).

[0087] 1 H-NMR (400 MHz, DMSO- d 6 ): δ 7.20-7.09 (m, 2H), 7.01-6.90 (m, 2H), 6.09-6.03 (m, 2H), 5.75 (s, 1H), 5.30-4.80 (m, 3H), 4.49-4.40 (m, 1H), 4.15-3.92 (m, 3H), 3.84-3.69 (m, 10H), 3.23-3.11 (m, 2H).

[0088] Step 3: Preparation of compound 4.

[0089] 7-(carboxymethyl)-9,10-dimethoxy-5,6,7,8,13,13a-hexahydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (4).

[0090] Compound 3 (135 mg, 0.27 mmol), tetrahydrofuran (2 mL), water (2 mL), and lithium hydroxide (66 mg, 2.74 mmol) were added sequentially to a dry 25 mL single-necked flask at room temperature. The mixture was stirred at room temperature and reacted for 0.5 hours. After the reaction was complete, the solution was concentrated under reduced pressure. The residue was purified by preparative HPLC (0.05% HCl) to give compound 4 (40 mg, yellow solid), yield: 33.62%.

[0091] LCMS(ESI): m / z 398.2 [M+H] + ;RT = 4.892&5.159 min (13.00 min).

[0092] 1 H-NMR (400 MHz, DMSO- d 6 ): δ 7.19-7.09 (m, 2H), 7.01-6.89 (m, 2H), 6.08-6.03 (m, 2H), 5.40 (d, J = 16.4 Hz, 0.5H), 5.27-5.22 (m, 0.5H), 5.15-5.11 (m,0.5H), 5.08-5.94 (m, 1H), 4.74 (d, J =16.8 Hz, 0.5H), 4.51-4.48 (m, 0.5H), 4.42-4.30 (m, 1H), 4.07-3.98 (m, 0.5H), 3.88-3.72 (m, 8H), 3.22-3.09 (m, 4H).

[0093] Example 2: Synthesis of compound 6.

[0094] Reaction formula:

[0095]

[0096] Preparation method:

[0097] Step 1: Preparation of compound 4.

[0098] 9-hydroxy-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (4) Compound 1 (10 g, 26.9 mmol) was added to a dry 100 mL single-necked flask at room temperature. The mixture was heated to 195 °C under reduced pressure and reacted for 3 h to give crude compound 4 (4.3 g, red solid), yield: 49.65%.

[0099] LCMS(ESI): m / z 322.1 [M+H] + ;RT = 1.290 (2.5 min).

[0100] 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.09 (s, 1H), 8.00 (s, 1H), 7.62 (s, 1H), 7.23 (d, J = 7.6 Hz, 1H), 6.96 (s, 1H), 6.37 (d, J = 7.6 Hz, 1H), 6.10 (s, 2H), 4.49 (t, J = 5.6 Hz, 2H), 3.74 (s, 3H), 3.04 (t, J = 5.6 Hz, 2H).

[0101] Step 2: Preparation of compound 5.

[0102] 9-(2-(tert-butoxy)-2-oxoethoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (5).

[0103] In a dry 100 mL single-necked flask at room temperature, compound 4 (1.00 g, 3.11 mmol), acetonitrile (20 mL), tert-butyl bromoacetate (3.00 g, 15.53 mmol), potassium carbonate (2.10 g, 15.53 mmol), and potassium iodide (0.05 g, 0.31 mmol) were added sequentially. The mixture was heated to 40 °C and reacted for 4 hours. After the reaction was complete, the reaction solution was filtered under reduced pressure, and the filter cake was collected. The filter cake was slurried with methyl tert-butyl ether, filtered under reduced pressure, and the filter cake was collected to give crude product 5 (500 mg, black solid), yield: 36.9%.

[0104] LCMS(ESI): m / z 436.2 [M+H] + ;RT = 1.719 min (2.50 min).

[0105] 1H-NMR (400 MHz, DMSO- d 6 ): δ 9.93 (s, 1H), 8.94 (s, 1H), 8.20 (d, J = 8.8Hz, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.80 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 4.97-4.94 (m, 4H), 4.03 (s, 3H), 3.21 (s, 2H), 1.42 (s, 9H).

[0106] Step 3: Preparation of compound 6.

[0107] 9-(carboxymethoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (6).

[0108] Compound 5 (100 mg, 0.29 mmol), dichloromethane (3 mL), and trifluoroacetic acid (1 mL) were added sequentially to a dry 25 mL single-necked flask at room temperature. The mixture was stirred at room temperature and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified by preparative HPLC (0.05% HCl) to give compound 6 (11.54 mg, yellow solid), yield: 13.16%.

[0109] LCMS(ESI): m / z 380.1 [M+H] + ;RT = 4.159 min (13.00 min).

[0110] 1 H-NMR (400 MHz, DMSO- d 6 ): 9.99 (s, 1H), 8.92 (s, 1H), 8.19 (d, J = 9.0Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.80 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 4.95-4.91 (m, 4H), 4.04 (s, 3H), 3.22-3.19 (m, 2H).

[0111] Example 3: Synthesis of compound 7.

[0112] Reaction formula:

[0113]

[0114] Preparation method:

[0115] Step 1: Preparation of compound 7.

[0116] 2-((10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-yl)oxy)acetic acid (7).

[0117] Compound 6 (30 mg, 0.08 mmol) and anhydrous methanol (3 mL) were added sequentially to a dry 25 mL single-necked flask at room temperature. The mixture was heated to 75 °C until the starting material was completely dissolved, followed by the slow addition of sodium borohydride (12 mg, 0.32 mmol). The mixture was stirred at 75 °C for 3 hours. The reaction was monitored by LCMS until completion, at which point the reaction solution was cooled to room temperature. Compound 7 (4.0 mg, yellow solid) was purified by preparative HPLC (0.05% FA) in 13.23% yield.

[0118] LCMS(ESI): m / z 384.1 [M+H] + ;RT = 0.920 min (2.50 min).

[0119] 1 H-NMR (400 MHz, DMSO- d 6 ): δ 6.91-6.83 (m, 3H), 6.67 (s, 1H), 5.95 (d, J =2.40 Hz, 2H), 4.56 (s, 2H), 4.24 (d, J =16.0 Hz, 1H), 3.76 (s, 3H), 3.43-3.33(m, 2H), 3.07-3.04 (m, 1H), 2.90-2.87 (m, 1H), 2.63-2.57 (m, 3H), 2.50-2.44(m, 1H).

[0120] Example 4: Synthesis of compound 12.

[0121] Reaction formula:

[0122]

[0123] Preparation method:

[0124] Step 1: Preparation of compound 9.

[0125] 5-bromo-4-fluoro-1-methyl-1H-indazole (9) Sodium hydride (1.21 g, 30.23 mmol) and N,N-dimethylformamide (20 mL) were added sequentially to a dry 100 mL three-necked flask at room temperature. The mixture was purged with nitrogen three times, and the temperature was lowered to 0 °C. A solution of compound 8 (5.00 g, 23.25 mmol) in N,N-dimethylformamide (30 mL) was slowly added dropwise. The mixture was stirred at room temperature for 0.5 h. The temperature was then lowered to 0 °C, and iodomethane (3.96 g, 27.90 mmol) was slowly added dropwise. The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was poured into water (500 mL) and extracted with ethyl acetate (100 mL × 2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 3:1) to give compound 9 (3.41 g, yellow solid), yield: 62.02%.

[0126] LCMS(ESI): m / z 230.9 [M+H] + ;RT = 1.168 min (2.50 min).

[0127] 1 H-NMR (400 MHz, CDCl3): δ 8.02 (s, 1H), 7.47-7.43 (m, 1H), 7.07 (d, J =8.8 Hz, 1H), 4.07 (s, 3H).

[0128] Step 2: Preparation of compound 10.

[0129] tert-butyl (S)-3-(3-(4-fluoro-1-methyl-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4-fluoro -3,5-dimethylphenyl)-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylat (10).

[0130] In a dry 50 mL three-necked flask at room temperature, (S)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-3-(2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester (400 mg, 0.91 mmol), N-methylpyrrolidone (12 mL), compound 9 (249 mg, 1.09 mmol), potassium carbonate (301 mg, 2.17 mmol), cuprous iodide (207 mg, 1.09 mmol), and (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (155 mg, 1.09 mmol) were added sequentially. The mixture was purged with nitrogen three times, heated to 105 °C, and reacted for 3 hours. LCMS monitoring was performed until the reaction was complete. The reaction mixture was poured into water (120 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give compound 10 (400 mg, yellow solid), yield: 74.88%.

[0131] LCMS(ESI): m / z 590.2 [M+H] + ;RT = 1.931 min (2.50 min).

[0132] 1 H-NMR (400 MHz, CDCl3): δ 8.11 (s, 1H), 7.46-7.42 (m, 1H), 7.24 (d, J =8.8 Hz, 1H), 7.12 (d, J = 6.4 Hz, 2H), 6.57 (d, J = 0.4 Hz, 1H), 6.30 (d, J = 2.4Hz, 1H), 5.37-5.26 (m, 1H), 4.10 (s, 3H), 3.40-3.36 (m, 1H), 2.80-2,77 (m,2H), 2.40-2.35 (m, 1H), 2.27-2.25 (m, 6H), 1.50 (s, 9H), 1.36 (d, J = 6.8 Hz, 3H).

[0133] Step 3: Preparation of compound 11.

[0134] (S)-1-(4-fluoro-1-methyl-1H-indazol-5-yl)-3-(2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-1,3-dihydro-2H-imidazol-2-one hydrochloride (11).

[0135] Compound 10 (500 mg, 1.76 mmol), dichloromethane (5 mL), and dioxane hydrochloride (4 M, 5 mL) were added sequentially to a dry 50 mL single-necked flask at room temperature. The mixture was stirred at room temperature and reacted for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to give compound 11 (446 mg, yellow solid), yield: 100.00%.

[0136] LCMS(ESI): m / z 490.2 [M-HCl+H] + ;RT = 1.238 min (2.50 min).

[0137] Step 4: Preparation of compound 12.

[0138] 3-((1S,2S)-1-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-2-((S)-3-(3-(4-fluoro-1-methyl-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4-fluoro -3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine-5-carbonyl)-1H-indol-1-yl)-2-methylcyclopropyl)-1,2,4-oxadiazol-5(4H)-one (12).

[0139] In a dry 25 mL single-necked flask at room temperature, 5-((S)-3,3-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carboxylic acid (150 mg, 0.36 mmol), N,N-dimethylformamide (3 mL), compound 11 (192 mg, 0.36 mmol), N,N-diisopropylethylamine (0.24 mL, 1.46 mmol), and HATU (208 mg, 0.54 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 0.5 hours. LCMS monitoring showed that the reaction was complete. The reaction solution was purified by prep-HPLC (0.05% NH4HCO3) to give compound 12 (117 mg, white solid), yield: 36.35%.

[0140] LCMS(ESI): m / z 883.5 [M+H] + ;RT = 5.153 min (13.00 min).

[0141] 1 H-NMR (400 MHz, DMSO- d 6 ): δ 11.254 (s, 1H), 8.19 (s, 1H), 7.54-7.51 (m, 2H), 7.41-7.35 (m, 2H), 7.25-7.22 (m, 1H), 7.16 (d, J =6.4 Hz, 2H), 6.91 (s,1H), 6.84 (s, 1H), 6.78 (s, 1H), 5.54 (s, 1H), 4.44 (s, 1H), 4.08 (s, 3H),3.73-3.71 (m, 2H), 3.55 (s, 1H), 3.05 (s, 1H), 2.88-2.81 (m, 1H), 2.25 (d, J =1.6 Hz, 6H), 1.72-1.46 (m, 11H), 1.28 (s, 3H), 1.19 (s, 6H).

[0142] Example 5: Synthesis of compound 24.

[0143] Reaction formula:

[0144]

[0145] Preparation method:

[0146] Step 1: Preparation of compound 14.

[0147] 2-bromo-6-methyl-4-nitrobenzenediazonium tetrafluoroborate (14) Compound 13 (38.60 g, 167.06 mmol), acetic acid (87 mL), and a 40% aqueous solution of tetrafluoroboric acid were added to a dry 1 L single-necked flask. The system was cooled to 0 °C, and then a solution of tert-butyl nitrite (22.40 g, 217.18 mmol) in acetic acid (57 mL) was slowly added. The mixture was stirred at 0 °C and reacted for 4 hours. The reaction was monitored by LCMS until it was complete. The reaction system was cooled to -40 °C, and 200 mL of diethyl ether was added, resulting in the precipitation of a solid. The mixture was stirred at -40 °C for 0.5 hours, filtered under reduced pressure, and the filter cake was collected to give compound 14 (47.01 g, pale yellow solid), yield: 85.30%.

[0148] LCMS (ESI): m / z 241.9 [M+H] + ;RT = 0.400 (2.50 min).

[0149] Step 2: Preparation of compound 15.

[0150] 1-bromo-2-fluoro-3-methyl-5-nitrobenzene (15) Compound 14 (10.00 g, 30.39 mmol) was added to a dry 500 mL single-necked flask, heated to 155 °C, and stirred for 0.5 hours. The reaction was monitored by LCMS until complete. The crude product was purified by column chromatography (pure petroleum ether) to give compound 15 (3.74 g, white solid), yield: 52.60%.

[0151] 1 H NMR (400 MHz, DMSO-) d 6 ): δ 8.40-8.39 (m, 1H), 8.29-8.27 (m, 1H), 2.40 (d, J = 2.0 Hz, 3H).

[0152] 19 F NMR (376 MHz, DMSO- d 6): δ -100.72.

[0153] Step 3: Preparation of compound 16.

[0154] 3-bromo-4-fluoro-5-methylaniline (16) Compound 15 (21.00 g, 89.73 mmol), ethyl acetate (200 mL), and stannous dichloride monohydrate (80.99 g, 358.94 mmol) were added sequentially to a dry 500 mL single-necked flask at room temperature. The mixture was heated to 80 °C and reacted for 16 hours. The reaction was monitored by LCMS until completion. The reaction mixture was cooled to room temperature, and diatomaceous earth and 200 mL of ethyl acetate were added. The pH was adjusted to 8 with saturated sodium bicarbonate solution. The mixture was filtered under reduced pressure, and the filtrate was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered again, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 20:1) to give compound 16 (11.21 g, yellow solid), yield: 61.01%.

[0155] LCMS (ESI): m / z 206.0 [M+H] + ;RT = 0.998 min (2.50 min).

[0156] 1 H NMR (400 MHz, DMSO-) d 6 ): δ 6.63-6.61 (m, 1H), 6.42-6.39 (m, 1H), 5.10 (s, 2H), 2.13 (d, J = 2.0 Hz, 3H).

[0157] Step 4: Preparation of compound 17.

[0158] (3-bromo-4-fluoro-5-methylphenyl)hydrazine (17).

[0159] Compound 4 (9.02 g, 44.22 mmol) and hydrochloric acid (6 M, 135 mL) were added sequentially to a dry 500 mL single-necked flask at room temperature. The system was cooled to 0 °C, and a 40 mL solution of sodium nitrite (3.67 g, 53.06 mmol) in water was slowly added dropwise. The mixture was stirred at 0 °C, and after 2 hours of reaction, a 60 mL solution of stannous dichloride monohydrate (39.9 g, 176.88 mmol) in concentrated hydrochloric acid (12 M). The mixture was stirred at 0 °C, and after 2 hours of reaction, the reaction mixture was monitored by LCMS. Once the reaction was complete, the reaction solution was diluted to pH 14 with 6 M potassium hydroxide aqueous solution and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude compound 17 (8.58 g, yellow solid), yield: 85.18%.

[0160] LCMS (ESI): m / z 221.0 [M+H] + ;RT = 0.825 min (2.50 min).

[0161] Step 5: Preparation of compound 18.

[0162] tert-butyl (S)-3-amino-2-(3-bromo-4-fluoro-5-methylphenyl)-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (18).

[0163] Compound 17 (10.06 g, 45.94 mmol), ethanol (100 mL), and (2S)-3-cyano-2-methyl-4-oxopiperidin-1-carboxylic acid tert-butyl ester (10.95 g, 45.94 mmol) were added sequentially to a dry 250 mL single-necked flask at room temperature. The mixture was heated to 80 °C and reacted for 16 hours. After the reaction was complete, the reaction mixture was diluted with water and extracted three times with ethyl acetate. The organic phase was collected, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 4:1) to give compound 18 (15.86 g, yellow solid), yield: 78.70%.

[0164] LCMS (ESI): m / z 440.9 [M+H] + ;RT = 1.558 min (2.50 min).

[0165] 1H NMR (400 MHz, DMSO-) d 6 ): δ 7.65-7.63 (m, 1H), 7.50-7.48 (m, 1H), 5.35 (s, 2H), 5.14-5.03 (m, 1H), 4.06-4.00 (m, 1H), 3.06-2.98 (m, 1H), 2.50-2.44 (m, 2H), 2.32 (d, J = 1.6 Hz, 3H), 1.44 (s, 9H), 1.25 (d, J = 4.8 Hz, 3H).

[0166] Step 6: Preparation of compound 19.

[0167] tert-butyl (S)-2-(3-bromo-4-fluoro-5-methylphenyl)-3-(3-(2,2-dimethoxyethyl)ureido)-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (19).

[0168] In a dry 25 mL three-necked flask at room temperature, triphosgene (0.74 g, 2.49 mmol) and tetrahydrofuran (7 mL) were added sequentially. The system was purged with nitrogen three times, cooled to 0 °C, and a tetrahydrofuran (3 mL) solution of compound 18 (1.00 g, 2.27 mmol) and N,N-diisopropylethylamine (1.46 g, 11.34 mmol) was slowly added dropwise. The mixture was stirred at room temperature and reacted for 2 hours. Then, the mixture was cooled to 0 °C, and dimethyl acetal (1.19 g, 11.35 mmol) was added. The reaction was continued at room temperature for 2 hours, and the reaction was monitored by LC-MS until completion. The reaction solution was diluted with water and extracted three times with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 4:1) to give compound 19 (1.13 g, yellow solid), yield: 80.30%.

[0169] LCMS (ESI): m / z 571.9 [M+H] + ;RT = 1.453 (2.50 min).

[0170] Step 7: Preparation of compound 20.

[0171] tert-butyl (S)-2-(3-bromo-4-fluoro-5-methylphenyl)-4-methyl-3-(2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (20).

[0172] Compound 19 (14.74 g, 25.84 mmol) and tetrahydrofuran (150 mL) were added sequentially to a dry 250 mL single-necked flask at room temperature. The mixture was heated to 60 °C and reacted for 2 hours. After the reaction was complete, the reaction solution was diluted with water and extracted three times with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give compound 20 (6.85 g, yellow solid), yield: 52.37%.

[0173] LCMS(ESI): m / z 508.1 [M+H] + ;RT = 1.423 min (2.5 min).

[0174] 1 H NMR (400 MHz, CDCl3): δ 10.17 (s, 1H), 7.45 (d, J = 2.8 Hz, 1H), 7.20(d, J = 3.6 Hz, 1H), 6.40 (s, 1H), 6.16 (s, 1H), 5.30 (s, 1H), 4.13-4.10 (m,1H), 3.10 (s, 1H), 2.78 (s, 2H), 2.27 (s, 3H), 1.49 (s, 9H), 1.27 (d, J = 6.8Hz, 3H).

[0175] Step 8: Preparation of compound 21.

[0176] tert-butyl (S)-2-(4-fluoro-3-(hydroxymethyl)-5-methylphenyl)-4-methyl-3-(2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (21).

[0177] In a dry 250 mL single-necked flask at room temperature, compound 20 (4.82 g, 9.52 mmol), 1,4-dioxane (50 mL), tributyltin methanol (3.67 g, 11.42 mmol), and chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium (0.75 g, 0.95 mmol) were added sequentially. The mixture was purged with nitrogen three times and heated to 80 °C for 16 hours. The reaction was monitored by LCMS. After completion, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give compound 21 (3.96 g, yellow solid), yield: 90.10%.

[0178] LCMS(ESI): m / z 458.2 [M+H] + ;RT = 1.057 min (2.5 min).

[0179] 1 H NMR (400 M Hz, CDCl3): δ 10.18 (s, 1H), 7.21 (d, J = 4.4 Hz, 1H), 7.15(s, 1H), 6.26 (s, 1H), 6.12 (s, 1H), 5.21-5.15 (m, 1H), 4.55 (d, J = 4.8 Hz,2H), 4.13-4.11 (m, 1H), 3.89 (s, 1H), 3.11 (s, 1H), 2.77 (s, 2H), 2.24 (s,3H), 1.49 (s, 9H), 1.27 (d, J = 6.8 Hz, 3H).

[0180] Step 9: Preparation of compound 22.

[0181] tert-butyl (S)-3-(3-(4-fluoro-1-methyl-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4-fluoro-3-(hydr oxymethyl)-5-methylphenyl)-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (22).

[0182] In a dry 250 mL single-necked flask at room temperature, compound 21 (3.96 g, 8.66 mmol), N-methylpyrrolidone (50 mL), 5-bromo-4-fluoro-1-methyl-1H-indazole (2.38 g, 10.39 mmol), cuprous iodide (1.98 g, 10.39 mmol), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (1.48 g, 10.39 mmol), and potassium carbonate (2.87 g, 20.77 mmol) were added sequentially. The mixture was purged with nitrogen three times, heated to 100 °C, and reacted for 16 hours. The reaction was monitored by LC-MS. After completion, the reaction solution was filtered, diluted with water, and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to give compound 22 (4.36 g, yellow solid), yield: 83.14%.

[0183] LCMS(ESI): m / z 606.3 [M+H] + ;RT = 1.361 min (2.5 min).

[0184] 1 H NMR (400 M Hz, CDCl3): δ 8.09 (s, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.35 (d, J = 3.2 Hz, 1H), 7.27-7.21 (m, 2H), 6.57 (d, J =1.6 Hz, 1H), 6.31(s, 1H),5.36-5.21 (m, 1H), 4.69 (s, 2H), 4.51-4.26 (m, 1H), 4.09 (s, 3H), 3.14 (s,1H), 2.79 (d, J= 2.8 Hz, 2H), 2.27 (d, J = 1.8 Hz, 3H), 1.50 (s, 9H), 1.35 (d, J =6.8 Hz, 3H).

[0185] Step 10: Preparation of compound 23.

[0186] (S)-1-(4-fluoro-1-methyl-1H-indazol-5-yl)-3-(2-(4-fluoro-3-(hydroxymethyl)-5-methylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-1,3-dihydro-2H-imidazol-2-one (23).

[0187] Compound 22 (1.80 g, 2.98 mmol), dichloromethane (6 mL), and trifluoroacetic acid (2 mL) were added to a dry 100 mL single-necked flask at room temperature. The mixture was stirred at room temperature and reacted for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to give crude compound 23 (2.31 g, black oil), yield: 100%.

[0188] LCMS(ESI): m / z 506.3 [M+H] + , RT = 1.069 min; m / z 602.3 [M+COCF3] + , RT = 1.360 (2.5 min).

[0189] Step 11: Preparation of compound 24.

[0190] 3-((1S,2S)-1-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-2-((S)-3-(3-(4-fluoro -1-methyl-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4-fluoro-3-(hyd roxymethyl)-5-methylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine-5-carbonyl)-1H-indol-1-yl)-2-methylcyclopropyl)-1,2,4-oxadiazol-5(4H)-one (24).

[0191] Compound 23 (248 mg, 0.41 mmol), N,N-dimethylformamide (5 mL), N,N-diisopropylethylamine (266 mg, 2.07 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (235 mg, 0.62 mmol) were added to a dry 100 mL single-necked flask at room temperature. The mixture was stirred at room temperature and reacted for 16 hours. After the reaction was complete, the reaction solution was diluted with water and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC to give compound 24 (101 mg, white solid), yield: 27.41%.

[0192] LCMS(ESI): m / z 899.5 [M+H] + RT = 8.683 min (13.00 min).

[0193] 1 H-NMR (400 MHz, DMSO- d 6 ): δ 11.62 (s, 1H), 8.21 (s, 1H), 7.53 (s, 2H), 7.44-7.41 (m, 3H), 7.27-7.23 (m, 2H), 6.93 (s, 1H), 6.87 (s, 1H), 6.81 (s, 1H), 5.57 (s, 1H), 5.12 (s, 1H), 4.59 (s, 2H), 4.46 (s, 1H), 4.10 (s, 3H), 3.74 (d,J = 8.4 Hz, 2H), 3.58 (s, 1H), 2.97-2.91 (m, 2H), 2.27 (s, 3H), 1.73-1.49 (m, 11H), 1.30 (s, 3H), 1.21 (s, 6H).

[0194] Example 6: Synthesis of compound 28.

[0195] Reaction formula:

[0196]

[0197] Preparation method:

[0198] Step 1: Preparation of compound 25.

[0199] 5-bromo-4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (25).

[0200] In a dry 100 mL single-necked flask at room temperature, 5-bromo-4-fluoro-1H-indazole (2.0 g, 9.30 mmol), dichloromethane (35 mL), 3,4-dihydro-2H-pyran (978 mg, 11.63 mmol), and p-toluenesulfonic acid monohydrate (80 mg, 0.47 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 20 hours. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate aqueous solution (20 mL) and extracted with ethyl acetate (50 mL × 2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 50:1 to 1:1) to give compound 25 (2.2 g, white solid), yield: 79.07%.

[0201] 1 H NMR (400 MHz, DMSO- d 6 ): δ 8.29 (s, 1H), 7.66-7.59 (m, 2H), 5.91-5.88(m, 1H), 3.89-3.79 (m, 1H), 3.77-3.72 (m, 1H), 2.41-2.32 (m, 1H), 2.06-1.96(m, 2H), 1.79-1.60(m, 1H), 1.59-1.44(m, 2H).

[0202] Step 2: Preparation of compound 26.

[0203] tert-butyl (4S)-3-(3-(4-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2 -(4-fluoro-3,5-dimethylphenyl)-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (26).

[0204] In a dry 50 mL three-necked flask at room temperature, (S)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-3-(2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester (2.0 g, 4.53 mmol), N-methylpyrrolidone (30 mL), compound 25 (2.03 g, 6.79 mmol), potassium carbonate (1.50 g, 10.87 mmol), cuprous iodide (1.04 g, 5.44 mmol), and (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (1.55 g, 10.87 mmol) were added sequentially. The mixture was purged with nitrogen three times and heated to 100 °C for 16 hours. LCMS monitoring was performed until the reaction was complete. The reaction mixture was poured into water (120 mL) and extracted with ethyl acetate (50 mL × 2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase column chromatography (0.1% NH4HCO3) to give compound 26 (1.70 g, yellow solid), yield: 56.88%.

[0205] LCMS (ESI): m / z 660.1 [M+H] + ; RT = 1.894 min (2.50 min).

[0206] Step 3: Preparation of compound 27.

[0207] (S)-1-(4-fluoro-1H-indazol-5-yl)-3-(2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-1,3-dihydro-2H-imidazol-2-one (27).

[0208] Compound 26 (1.45 g, 1.76 mmol), dichloromethane (5 mL), and dioxane hydrochloride (4 M, 5 mL) were added sequentially to a dry 50 mL single-necked flask at room temperature. The mixture was stirred at 40 °C and reacted for 10 hours. The reaction solution was concentrated under reduced pressure, followed by the addition of dichloromethane (5 mL) and trifluoroacetic acid (5 mL). The mixture was stirred at room temperature and reacted for 2 hours. LCMS monitoring showed that the reaction was complete. The reaction solution was then concentrated under reduced pressure to give crude compound 27 (1.30 g, yellow solid), yield: 100.00%.

[0209] LCMS (ESI): m / z 476.1 [M+H] + ; RT = 0.801 min (2.50 min).

[0210] Step 4: Preparation of compound 28.

[0211] 3-((1S,2S)-1-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-2-((S)-3-(3-(4-fluoro-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4-fluoro-3,5 -dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine-5-carbonyl)-1H-indol-1-yl)-2-methylcyclopropyl)-1,2,4-oxadiazol-5(4H)-one (28).

[0212] In a dry 25 mL single-necked flask at room temperature, 5-((S)-3,3-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carboxylic acid (84 mg, 0.18 mmol), N,N-dimethylformamide (3 mL), compound 27 (95 mg, 0.23 mmol), N,N-diisopropylethylamine (114 mg, 0.86 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (101 mg, 0.27 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. The reaction was monitored by LCMS. Once complete, the reaction solution was... prep-Compound 28 (10.79 mg, white solid) was prepared and purified by HPLC (0.05% NH4HCO3) in a yield of 7.03%.

[0213] LCMS(ESI): m / z 869.5 [M+H] + ;RT = 10.882 min (13.00 min).

[0214] 1 H-NMR (400 MHz, DMSO- d 6 ): δ 13.37 (s, 1H), 11.62 (s, 1H), 8.22 (s, 1H), 7.51 (s, 1H), 7.43-7.39 (m, 2H), 7.31 (s, 1H), 7.25-7.23 (m, 1H), 7.16 (d, J =6.0 Hz, 2H), 6.91 (s, 1H), 6.85 (d, J =4.80 Hz, 1H), 6.77 (d, J =4.0 Hz, 1H),5.56 (s, 1H), 4.43 (s, 1H), 3.73-3.71 (m, 2H), 3.54 (s, 1H), 2.85 (s, 2H),2.25 (d, J =2.0 Hz, 6H), 1.72-1.68 (m, 4H), 1.62-1.46 (m, 6H), 1.28 (s, 4H), 1.19 (s, 6H).

[0215] Example 7: Synthesis of compound 33.

[0216] Reaction formula:

[0217]

[0218] Preparation method:

[0219] Step 1: Preparation of compound 29.

[0220] 2-(5-bromo-4-fluoro-1H-indazol-1-yl)ethan-1-ol (29) In a dry 25 mL three-necked flask at room temperature, compound 8 (800 mg, 3.72 mmol), N,N-dimethylformamide (10 mL), 2-bromoethane-1-ol (697 mg, 5.58 mmol), potassium iodide (926 mg, 5.58 mmol), and potassium carbonate (1540 mg, 11.16 mmol) were added sequentially. The mixture was purged with nitrogen three times and heated to 70°C for 16 hours. After the reaction was complete, the mixture was cooled to room temperature, poured into water, and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give compound 29 (810 mg, white solid), yield: 84.03%.

[0221] 1 H NMR (400 MHz, DMSO- d 6 ): δ 8.23 ​​(s, 1H), 7.59-7.52 (m, 2H), 4.88-4.86 (m, 1H), 4.48-4.46 (m, 2H), 3.82-3.78 (m, 2H).

[0222] Step 2: Preparation of compound 30.

[0223] tert-butyl (S)-3-(3-(4-fluoro-1-(2-hydroxyethyl)-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4-fl uoro-3,5-dimethylphenyl)-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (30).

[0224] In a dry 50 mL three-necked flask at room temperature, tert-butyl(S)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-3-(2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (114 mg, 0.26 mmol), N-methylpyrrolidone (10 mL), compound 29 (100 mg, 0.39 mmol), cuprous iodide (59 mg, 0.31 mmol), potassium carbonate (85 mg, 0.62 mmol), and (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (88 mg, 0.62 mmol) were added sequentially. The mixture was purged with nitrogen three times and heated to 100 °C, and the reaction was allowed to proceed overnight. LCMS monitoring was performed until the reaction was complete. The reaction solution was cooled to room temperature, poured into water, and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by reversed-phase column chromatography (0.1% FA) to give compound 30 (100 mg, light gray solid), yield: 84.03%.

[0225] LCMS(ESI): m / z 620.2 [M+H] + ; RT = 1.814 min (2.50 min).

[0226] 1 H NMR (400 MHz, DMSO- d 6 ): δ 8.31 (s, 1H), 7.63 (d, J = 8.8 Hz, 1H),7.41-7.37 (m, 1H), 7.14 (d, J = 6.4 Hz, 2H), 7.04 (s, 1H), 6.93 (s, 1H), 5.15(s, 1H), 4.87-4.86 (m, 1H), 4.05 (s, 2H), 3.81 (d, J = 4.2 Hz, 2H), 3.12 (s, 2H), 2.75-2.63 (m, 2H), 2.24 (s, 6H), 1.44 -1.43 (m, 12H).

[0227] Step 3: Preparation of compound 31.

[0228] (S)-9-(2-(5-(3-(5-(tert-butoxycarbonyl)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihyd ro-1H-imidazol-1-yl)-4-fluoro-1H-indazol-1-yl)ethoxy)-2-oxoethoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (31).

[0229] In a dry 50 mL single-necked flask at room temperature, compound 30 (200 mg, 0.32 mmol), dichloromethane (5 mL), 9-(carboxymethoxy)-10-methoxy-5,6-dihydro-[1,3]dioxo[4,5-g]isoquinolino[3,2-a]isoquinolino-7-onium (123 mg, 0.32 mmol), triethylamine (98 mg, 0.97 mmol), 4-dimethylaminopyridine (40 mg, 0.32 mmol), and 2-chloro-1-methylpyridine iodide (182 mg, 0.71 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (0.1% FA) to give compound 31 (200 mg, yellow solid), yield: 63.10%.

[0230] LCMS(ESI): m / z 981.2 [M+H] + ; RT = 1.325 min (2.50 min).

[0231] Step 4: Preparation of compound 32.

[0232] (S)-9-(2-(2-(4-fluoro-5-(3-(2-(4-fluoro-3,5-dimethylphenyl))-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro- 1H-imidazol-1-yl)-1H-indazol-1-yl)ethoxy)-2-oxoethoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (32).

[0233] Compound 31 (200 mg, 0.203 mmol), dichloromethane (5 mL), and trifluoroacetic acid (1 mL) were added sequentially to a dry 50 mL single-necked flask at room temperature. The mixture was stirred at room temperature and reacted for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to give crude compound 32 (170 mg, gray solid), yield: 94.65%.

[0234] LCMS(ESI): m / z 883.1 [M+H] + ; RT = 0.980 min (2.50 min).

[0235] Step 5: Preparation of compound 33.

[0236] 9-(2-(2-(5-(3-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl))-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihyd ro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6 ,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-fluoro-1H-indazol-1-y l)ethoxy)-2-oxoethoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (33).

[0237] In a dry 25 mL single-necked flask at room temperature, compound 32 (170 mg, 0.25 mmol), N,N-dimethylformamide (4 mL), 5-((S)-3,3-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carboxylic acid (119 mg, 0.29 mol), N,N-diisopropylethylamine (0.17 mL, 1.25 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (143 mg, 0.38 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 5 hours. LCMS monitoring was performed. After the reaction was complete, the mixture was poured into water (40 mL) and extracted with ethyl acetate (15 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was then treated with... prep- Preparative purification by HPLC yielded compound 33 (53.19 mg, yellow solid), yield: 21.64%.

[0238] LCMS(ESI): m / z 1274.3 [M+H] + ; RT = 1.426 min (2.50 min).

[0239] 1 H-NMR (400 MHz, DMSO- d6 ): δ 9.76 (s, 1H), 8.81 (s, 1H), 8.14 (s, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.71 (s, 1H), 7.50 (s, 2H), 7.40-7.38 (m, 2H), 7.23 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 6.4 Hz, 2H), 7.02 (s,1H), 6.89-6.80 (m, 3H), 6.13 (s, 2H), 5.55 (s, 1H), 4.94 (s, 2H), 4.89-4.86(m, 2H), 4.71 (d, J = 4.4 Hz, 2H), 4.62 (d, J = 4.8 Hz, 2H), 4.44 (s, 1H), 3.95 (s, 3H), 3.72 (d, J = 8.4 Hz, 2H), 3.55 (s, 1H), 3.20-3.17 (m, 3H), 2.89-2.85(m, 2H), 2.24(s, 6H), 1.71-1.68 (m, 3H), 1.61-1.43 (m,7H), 1.27 (s, 3H), 1.18(s, 6H).

[0240] Example 8: Synthesis of compound 35.

[0241] Reaction formula:

[0242]

[0243] Preparation method:

[0244] Step 1: Preparation of compound 34.

[0245] 5-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-3-(3 -(4-fluoro-1-methyl-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)-2-fluoro-3-methylbenzyl 2-bromoacetate(34).

[0246] In a dry 100 mL single-necked flask at room temperature, compound 24 (500 mg, 0.56 mmol), dichloromethane (10 mL), bromoacetic acid (93 mg, 0.67 mmol), N,N'-dicyclohexylcarbodiimide (172 mg, 0.83 mmol), and 4-dimethylaminopyridine (14 mg, 0.11 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give compound 34 (454 mg, white solid), yield: 80.03%.

[0247] LCMS(ESI): m / z [M+H] + ;1021.1 [M+H] + ;RT = 1.844 (2.50 min).

[0248] 1 H NMR (400 MHz, DMSO- d 6):δ 11.74 (s, 1H), 8.30 (s, 1H), 7.64 (d, J =8.8 Hz, 1H), 7.54-7.48 (m, 2H), 7.41-7.33 (m, 4H), 7.26 (d, J = 8.4 Hz, 1H),7.10 (s, 1H), 6.99-6.95 (m, 2H), 5.60-5.58 (m, 1H), 5.25 (s, 3H), 4.41-4.38(m, 1H), 4.17 (s, 2H), 4.13 (s, 3H), 3.72-3.63 (m, 3H), 3.05-3.00 (m, 1H),2.92-2.89 (m, 1H), 2.30 (s, 3H), 1.78-1.59 (m, 7H), 1.43 (d, J = 5.9 Hz, 2H),1.32-1.27 (m, 3H), 1.19-1.17 (m, 6H).

[0249] Step 2: Preparation of Compound 35.

[0250] 7-(2-((5-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-3-(3-(4-fluoro-1-methyl-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)-2-fluoro-3-methylbenzyl)oxy)-2-oxoethyl)-9,10-dimethoxy-5,6,7,8,13,13a-hexahydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (35).

[0251] In a dry 100 mL single-necked flask at room temperature, compound 34 (200 mg, 0.196 mmol), acetonitrile (10 mL), 9,10-dimethoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxo[4,5-g]isoquinolino[3,2-a]isoquinoline (200 mg, 0.588 mmol), potassium carbonate (136 mg, 0.980 mmol), and potassium iodide (4 mg, 0.02 mmol) were added sequentially. The mixture was heated to 60 °C and reacted for 16 hours. The reaction was monitored by LCMS. After completion, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was used... prep- Preparative purification by HPLC yielded product 35 (50.89 mg, yellow solid), yield: 20.28%.

[0252] LCMS(ESI): m / z 1278.7 [M+H] + ;RT = 1.452 min (2.50 min).

[0253] 1 H-NMR (400 MHz, DMSO- d 6 ): δ 11.74 (s, 1H), 8.27 (s, 1H), 7.61 (d, J =8.8 Hz, 1H), 7.53 (s, 1H), 7.49-7.46 (m, 2H), 7.42-7.40 (m, 1H), 7.35 (s,1H), 7.27 (d, J = 8.4 Hz, 1H), 7.15-7.08 (m, 2H), 7.06-6.94 (m, 3H), 6.84-6.82 (m, 2H), 6.06-6.00 (m, 2H), 5.58 (s, 1H), 5.37-5.30 (m, 3H), 5.01-4.91(m, 3H), 4.54-4.38 (m, 3H), 4.11-4.07 (m, 4H), 4.13-4.07 (m, 4H), 3.98-3.91(m, 2H), 3.81 (s, 4H), 3.73-3.69 (m, 6H), 3.16-3.01 (m, 6H), 2.91-2.87 (m,1H), 2.27 (d, J = 13.2 Hz, 3H), 1.78-1.56 (m, 5H), 1.43 (s, 2H), 1.28 (s,1H), 1.25 (s, 3H), 1.19 (s, 5H).

[0254] Example 9: Synthesis of compound 36.

[0255] Reaction formula:

[0256]

[0257] Preparation method:

[0258] Step 1: Preparation of compound 36.

[0259] 9-(2-((5-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1, 2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-3-(3-(4-fluoro-1-methyl-1H-indazol-5-yl)-2-oxo-2,3- dihydro-1H-imidazol-1-yl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)-2-fluoro-3-methylbe nzyl)oxy)-2-oxoethoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (36).

[0260] In a dry 100 mL single-necked flask at room temperature, compound 34 (200 mg, 0.196 mmol), tetrahydrofuran (10 mL), 9-hydroxy-10-methoxy-5,6-dihydro-[1,3]dioxo[4,5-g]isoquinolino[3,2-a]isoquinolino-7-onium (190 mg, 0.588 mmol), and potassium carbonate (136 mg, 0.980 mmol) were added sequentially. The mixture was heated to 60 °C and reacted for 16 hours. The reaction was monitored by LCMS. After completion, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was used... prep- Preparative purification by HPLC yielded product 36 (38.59 mg, yellow solid), yield: 15.6%.

[0261] LCMS(ESI): m / z 1260.7 [M+H] + ;RT = 1.433 min (2.50 min).

[0262] 1 H-NMR (400 MHz, DMSO- d 6 ): δ 11.75 (s, 1H), 9.83 (s, 1H), 8.86 (s, 1H), 8.21 (s, 1H), 8.12 (d, J = 9.2 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.75 (s,1H), 7.57-7.51 (m, 2H), 7.4-7.40 (m, 2H), 7.34 (s, 2H), 7.27 (d, J = 8.4 Hz,1H), 7.09 (s, 2H), 6.95 (s, 1H), 6.16 (s, 2H), 5.58 (d, J = 6.4 Hz, 1H), 5.26(s, 2H), 5.06 (s, 2H), 4.87 (s, 2H), 4.39 (d, J = 10.4 Hz, 1H), 4.05 (s, 3H), 3.94 (s, 3H), 3.73-3.71 (m, 2H), 3.17 (s, 3H), 3.04-3.01 (m, 1H), 2.89-2.85(m, 1H), 2.26 (s, 3H), 1.78-1.56 (m, 8H), 1.41 (d, J = 4.2 Hz, 2H), 1.28 (s,5H), 1.19 (s, 6H).

[0263] Example 10: Synthesis of compound 37.

[0264] Reaction formula:

[0265]

[0266] Preparation method:

[0267] Step 1: Preparation of compound 37.

[0268] 5-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-3-(3 -(4-fluoro-1-methyl-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)-2-fluoro-3-methylbenzyl 2-((10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-yl)oxy)acetate (37).

[0269] In a dry 100 mL single-necked flask at room temperature, compound 34 (200 mg, 0.196 mmol), tetrahydrofuran (10 mL), 10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-ol (191 mg, 0.588 mmol), and potassium carbonate (136 mg, 0.980 mmol) were added sequentially. The mixture was heated to 60 °C and reacted for 16 hours. The reaction was monitored by LCMS. After completion, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was used... prep- Preparative purification by HPLC yielded product 37 (60.01 mg, pale yellow solid), yield: 24.2%.

[0270] LCMS(ESI): m / z 1264.3 [M+H] + ;RT = 1.408 min (2.50 min).

[0271] 1 H-NMR (400 MHz, DMSO- d 6): δ 11.74 (s, 1H), 10.38 (s, 1H), 8.28 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.54-7.48 (m, 2H), 7.42-7.40 (m, 3H), 7.28-7.25 (m,1H), 7.12-7.05 (m, 3H), 7.00-6.99 (d, J = 6.4 Hz, 2H), 6.94 (s, 1H), 6.83 (s,1H), 6.03 (s, 2H), 5.59 (d, J = 6.0 Hz, 1H), 5.28 (s, 2H), 4.86-4.80 (m, 3H), 4.64 (s, 1H), 4.44-4.38 (m, 2H), 4.10 (s, 3H), 3.82-3.79 (m, 2H), 3.71 (s,6H), 3.42 (s, 1H), 3.17-3.01 (m, 2H), 2.91-2.87 (m, 2H), 2.30 (s, 3H), 1.78-1.49 (m, 8H), 1.43 (s, 2H), 1.32-1.29 (m, 5H), 1.19 (s, 6H).

[0272] Example 11: Synthesis of compound 38.

[0273] Reaction formula:

[0274]

[0275] Preparation method:

[0276] Step 1: Preparation of compound 38.

[0277] In a dry 100 mL single-necked flask at room temperature, compound 4 (40 mg, 0.1 mmol), compound 28 (87 mg, 0.1 mmol), dichloromethane (10 mL), N,N'-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and 4-dimethylaminopyridine (2 mg, 0.01 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give compound 38 (92 mg, yellow solid).

[0278] LCMS(ESI): m / z [M+H] + 1248.6 [M+H] + ; 1 H NMR (400 MHz, DMSO- d 6 ): δ8.91 (dd, J = 8.4, 5.0 Hz, 1H), 8.36 (dd, J =5.0, 0.6 Hz, 1H), 8.00 (d, J = 7.0 Hz, 1H), 7.75 – 7.60 (m, 4H), 7.46 – 7.38(m, 1H), 7.26 (d, J = 5.0 Hz, 2H), 7.19 (dd, J = 8.4, 1.8 Hz, 1H), 6.72 (d, J = 8.3Hz, 1H), 6.65 – 6.50 (m, 3H), 5.95 (s, 2H), 5.48 (q, J = 6.9 Hz, 1H), 5.11 (d, J = 14.5 Hz, 1H), 4.98 – 4.84 (m, 2H), 4.20 (d, J = 14.5 Hz, 1H), 4.07 (d, J = 14.4Hz, 1H), 4.03 – 3.82 (m, 3H), 3.88 – 3.67 (m, 7H), 3.65 (ddd, J = 12.2, 7.3,5.0 Hz, 1H), 3.43 (ddd, J = 12.0, 7.3, 5.0 Hz, 1H), 3.33 (ddd, J = 17.1, 5.9, 1.0Hz, 1H), 3.19 – 2.83 (m, 6H), 2.53 – 2.38 (m, 2H), 2.25 (dd, J = 5.2, 3.0 Hz,1H), 2.20 (s, 6H), 2.08 – 1.79 (m, 4H), 1.47 (d, J = 7.0 Hz, 3H), 1.21 (d, J =20.1 Hz, 5H), 1.07 (d, J = 5.0 Hz, 3H).

[0279] Example 12: Synthesis of compound 39.

[0280] Reaction formula:

[0281]

[0282] Preparation method:

[0283] Step 1: Preparation of compound 39.

[0284] In a dry 100 mL single-necked flask at room temperature, compound 6 (40 mg, 0.1 mmol), compound 28 (87 mg, 0.1 mmol), dichloromethane (10 mL), N,N'-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and 4-dimethylaminopyridine (2 mg, 0.01 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give compound 39 (53 mg, yellow solid).

[0285] LCMS(ESI): m / z [M+H] + ;1231.1 [M+H] + .

[0286] 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.81 (d, J = 0.7 Hz, 1H), 8.43 (dd, J = 5.0, 0.6 Hz, 1H), 8.29 (dd, J = 2.2, 0.7 Hz, 1H), 8.07 – 7.95 (m, 3H), 7.78 (d, J =8.3 Hz, 1H), 7.75 – 7.64 (m, 3H), 7.49 (d, J = 5.0 Hz, 2H), 7.44 – 7.37 (m,1H), 7.25 – 7.14 (m, 2H), 7.10 – 7.02 (m, 1H), 6.82 (t, J = 1.0 Hz, 1H), 6.16(q, J = 7.0 Hz, 1H), 6.00 (s, 2H), 5.38 (q, J = 13.3 Hz, 2H), 4.86 (dt, J= 12.7, 5.6 Hz, 1H), 4.48 (dt, J = 12.7, 5.6 Hz, 1H), 4.00 – 3.88 (m, 1H), 3.83 (s,3H), 3.79 – 3.59 (m, 2H), 3.43 (ddd, J = 12.0, 7.3, 5.0 Hz, 1H), 3.17 – 2.89(m, 5H), 2.53 – 2.38 (m, 1H), 2.29 – 2.21 (m, 1H), 2.24 (s, 7H), 2.08 – 1.80(m, 5H), 1.35 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 20.1 Hz, 5H), 1.05 (d, J = 5.0 Hz, 3H).

[0287] Example 13: Synthesis of compound 44.

[0288] Reaction formula:

[0289]

[0290] Preparation method:

[0291] Step 1: Preparation of compound 40.

[0292] (2R,3S,4R,5S)-2-(acetoxymethyl)-5-(1,3-dioxoisoindolin-2-yl)-6-(2-hydroxyethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (40).

[0293] The compound with CAS number 90405-42-8 can be obtained through references or commercial customization. 435 mg (1 mmol) of the compound with CAS number 90405-42-8 was added to a dry 100 mL single-necked flask, along with anhydrous dichloromethane and 433 mg (10 mmol) of trichloroacetonitrile. The reaction was stirred in an ice bath at 0°C, and 15 mg (0.1 mmol) of DBU was added. Stirring continued for 2 hours, and the mixture was slowly brought to room temperature. After the reaction was completed by TLC monitoring, the solvent was removed by rotary evaporation, and the product was purified by silica gel to obtain the intermediate.

[0294] The intermediate and 124 mg (2 mmol) of ethylene glycol were dissolved together in anhydrous dichloromethane. 4 mg (0.02 mmol) of TMSOTf was added at -78 °C. After stirring for 2 hours, the mixture was slowly brought to room temperature. After the reaction was completed by TLC monitoring, the solvent was removed by rotary evaporation. The product was purified by silica gel column chromatography to obtain compound 40, a yellow oil liquid, 189 mg, yield 39%.

[0295] LCMS(ESI): m / z [M+H] + ;480.4 [M+H] + . Step 2: Preparation of Compound 41 (2R,3S,4R,5S)-2-(acetoxymethyl)-5-(1,3-dioxoisoindolin-2-yl)-6-(2-(tosyloxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (41) 480 mg (1 mmol) of compound 40 was dissolved in anhydrous dichloromethane and stirred until dissolved. 209 mg (1.1 mmol) of TsCl and 167 mg (1.2 mmol) of triethylamine were added under ice bath conditions. Stirring was continued under ice bath conditions for 2 hours, and the mixture was slowly brought to room temperature. After the reaction was monitored by TLC until complete, the solvent was removed by rotary evaporation. The mixture was washed with ethyl acetate and saturated brine, and the organic phase was separated. Anhydrous sodium sulfate was added, and the sodium sulfate was removed by filtration. Ethyl acetate was removed by rotary evaporation. Purification was performed by silica gel column chromatography to give 41,481 mg of a grayish-white solid, in 76% yield.

[0296] LCMS(ESI): m / z [M+H] + 634.3 [M+H] + . Step 3: Preparation of Compound 42 (2R,3S,4R,5S)-2-(acetoxymethyl)-6-(2-(5-bromo-4-fluoro-1H-indazol-1-yl)ethoxy)-5-(1,3-dioxoisoindolin-2-yl)tetrahydro-2H-pyran-3,4-diyl diacetate (42) In a dry 25 mL three-necked flask at room temperature, compound 8 (800 mg, 3.72 mmol), N,N-dimethylformamide (10 mL), compound 41 (1.90 g, 3 mmol), potassium iodide (926 mg, 5.58 mmol), and potassium carbonate (1540 mg, 11.16 mmol) were added sequentially. The mixture was purged with nitrogen three times and heated to 40°C for 72 hours. After the reaction was complete, the mixture was cooled to room temperature, poured into water, and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give compound 42 (1.03 g, white solid), yield: 51%.

[0297] LCMS(ESI): m / z [M+H] + 677.8 [M+H] + . Step 4: Preparation of compound 43 (2R,3S,4R,5S)-2-(acetoxymethyl)-6-(2-(5-(3-((S)-5-(tert-butoxycarbonyl)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[ 4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-fluoro-1H-indazol-1-yl)ethoxy)-5-(1,3-dioxoisoindolin-2-yl)tetrahydro-2H-pyran-3,4-diyl diacetate(43) In a dry 50 mL three-necked flask at room temperature, tert-butyl(S)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-3-(2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (114 mg, 0.26 mmol), N-methylpyrrolidone (10 mL), compound 42 (264 mg, 0.39 mmol), cuprous iodide (59 mg, 0.31 mmol), potassium carbonate (85 mg, 0.62 mmol), and (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (88 mg, 0.62 mmol) were added sequentially. The mixture was purged with nitrogen three times and heated to 80 °C, and the reaction was allowed to proceed overnight. LCMS monitoring was performed until the reaction was complete. The reaction solution was cooled to room temperature, poured into water, and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by reversed-phase column chromatography (0.1% FA) to give compound 43 (108 mg, light gray solid), yield: 40.06%.

[0298] LCMS(ESI): m / z 1038.1 [M+H] + ; RT = 2.114 min (2.50 min).

[0299] Step 5: Preparation of Compound 44 (2R,3S,4R,5S)-2-(acetoxymethyl)-5-(1,3-dioxoisoindolin-2-yl)-6-(2-(4-fluoro-5-(3-((S)-2-(4-fluoro-3,5-dimethylphenyl))-4-methyl-4,5,6 ,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-1H-indazol-1-yl)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate(44) 50 mg (0.048 mmol) of compound 43 was dissolved in anhydrous dichloromethane in an ice bath, and 0.5 ml of dioxane hydrochloric acid solution was added. After the reaction was completed by LCMS monitoring, the solvent was removed by rotary evaporation to obtain 43 mg of brown oil containing compound 44, with a yield of 95.83%.

[0300] LCMS(ESI): m / z 938.0 [M+H] +. Step 6: Preparation of Compound 45 (2R,3S,4R,5S)-2-(acetoxymethyl)-6-(2-(5-(3-((S)-5-(5-((S)-3,3-dimethyltetrahydro-2H-pyran-4-yl))-1-((1S ,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-2-(4-fluoro- 3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-i midazol-1-yl)-4-fluoro-1H-indazol-1-yl)ethoxy)-5-(1,3-dioxoisoindolin-2-yl)tetrahydro-2H-pyran-3,4-diyl diacetate(45) In a dry 25 mL single-necked flask at room temperature, compound 44 (234 mg, 0.25 mmol), N,N-dimethylformamide (4 mL), 5-((S)-3,3-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carboxylic acid (119 mg, 0.29 mol), N,N-diisopropylethylamine (0.17 mL, 1.25 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (143 mg, 0.38 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 5 hours. LCMS monitoring was performed. After the reaction was complete, the mixture was poured into water (40 mL) and extracted with ethyl acetate (15 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was then treated with... prep- Preparative purification by HPLC yielded compound 45 (54.16 mg, pale yellow solid), yield: 16.28%.

[0301] LCMS(ESI): m / z 1330.9 [M+H] + ; RT = 1.996 min (2.50 min).

[0302] Step 7: Preparation of Compound 46 3-((1S,2S)-1-(2-((4S)-3-(3-(1-(2-(((3S,4R,5S,6R))-3-amino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro -2H-pyran-2-yl)oxy)ethyl)-4-fluoro-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4-fluoro -3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine-5-carbonyl)-5-((S)-2,2- dimethyltetrahydro-2H-pyran-4-yl)-1H-indol-1-yl)-2-methylcyclopropyl)-1,2,4-oxadiazol-5(4H)-one (46) In a dry 25 mL single-necked flask under ice bath conditions, compound 45 (133 mg, 0.1 mmol), ethanol solution, and NaOEt (6.8 mg, 0.1 mmol) were added sequentially. The reaction was allowed to proceed for 30 min. After LCMS monitoring showed complete removal of the acetyl groups from the starting material, hydrazine monohydrate (7.6 mg, 0.15 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After LCMS monitoring showed the starting material had disappeared and converted to product 46, the solvent was removed by rotary evaporation, followed by extraction with ethyl acetate, washing with saturated brine, drying to anhydrous sodium sulfate, filtration, and concentration of the filtrate under reduced pressure. The residue was then... prep- Preparative purification by HPLC yielded compound 46 (54.16 mg, pale yellow solid), yield: 56.17%.

[0303] LCMS(ESI): m / z 1074.5 [M+H] + ; RT = 1.375 min (2.50 min).

[0304] 1 H NMR (400 MHz, DMSO- d 6) δ 8.19 (dd, J = 5.0, 0.6 Hz, 1H), 8.00 (d, J =7.0 Hz, 1H), 7.82 – 7.64 (m, 4H), 7.46 – 7.37 (m, 2H), 7.26 (d, J= 5.0 Hz, 2H), 7.18 (dd, J = 8.3, 1.8 Hz, 1H), 5.37 (q, J = 6.9 Hz, 1H), 4.72 (t, J = 7.2 Hz,1H), 4.69 – 4.49 (m, 2H), 4.51 – 4.41 (m, 1H), 4.39 – 4.23 (m, 2H), 4.14(ddd, J = 12.1, 7.2, 5.7 Hz, 1H), 4.01 – 3.84 (m, 2H), 3.80 – 3.54 (m, 6H), 3.49 – 3.22 (m, 4H), 3.13 (qd, J = 5.8, 5.1 Hz, 1H), 3.09 – 2.93 (m, 3H), 2.53– 2.40 (m, 1H), 2.25 (dd, J = 5.2, 3.0 Hz, 1H), 2.20 (s, 6H), 2.07 – 1.79 (m,4H), 1.51 (d, J = 6.9 Hz, 3H), 1.21 (d, J = 20.1 Hz, 5H), 1.05 (d, J = 5.0 Hz, 3H).

[0305] Example 14: Synthesis of compound 47.

[0306] Reaction formula:

[0307]

[0308] Preparation method:

[0309] Step 1: Preparation of Compound 47 In a dry 100 mL single-necked flask at room temperature, compound 4 (40 mg, 0.1 mmol), compound 46 (107 mg, 0.1 mmol), dichloromethane (10 mL), N,N'-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and 4-dimethylaminopyridine (2 mg, 0.01 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. The reaction was monitored by TLC. After completion, the reaction solution was concentrated under reduced pressure. The residue was used... prep- Compound 47 (25 mg, yellow solid) was obtained by preparative purification by HPLC.

[0310] LCMS(ESI): m / z 1454.8 [M+H] + ; RT = 1.165 min (2.50 min).

[0311] 1 H NMR (400 MHz, DMSO- d 6) δ 8.94 (d, J = 9.2 Hz, 1H), 8.18 (d, J = 4.9 Hz, 1H), 8.00 (d, J = 7.0 Hz, 1H), 7.80 – 7.67 (m, 2H), 7.67 – 7.55 (m, 2H), 7.56 –7.38 (m, 4H), 7.22 – 7.15 (m, 1H), 7.09 (dd, J = 8.4, 1.8 Hz, 1H), 6.83 (d, J =0.9 Hz, 1H), 6.77 – 6.64 (m, 3H), 5.95 (s, 2H), 5.41 – 5.28 (m, 2H), 4.82 –4.70 (m, 3H), 4.72 – 4.62 (m, 2H), 4.54 (t, J = 6.4 Hz, 2H), 4.44 – 4.33 (m,2H), 4.33 – 4.12 (m, 2H), 4.10 – 3.53 (m, 18H), 3.43 (ddd, J = 12.0, 7.3, 5.1Hz, 1H), 3.34 – 3.04 (m, 5H), 2.94 – 2.74 (m, 3H), 2.53 – 2.38 (m, 1H), 2.25(dd, J = 5.2, 3.0 Hz, 1H), 2.22 (s, 6H), 2.07 – 1.81 (m, 4H), 1.55 (d, J = 7.0Hz, 3H), 1.23 (d, J = 20.1 Hz, 5H), 1.03 (d, J = 5.0 Hz, 3H).

[0312] Example 15: Synthesis of compound 48.

[0313] Reaction formula:

[0314]

[0315] Preparation method:

[0316] Step 1: Preparation of compound 48 In a dry 100 mL single-necked flask at room temperature, compound 6 (40 mg, 0.1 mmol), compound 46 (107 mg, 0.1 mmol), dichloromethane (10 mL), N,N'-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and 4-dimethylaminopyridine (2 mg, 0.01 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. The reaction was monitored by TLC. After completion, the reaction solution was concentrated under reduced pressure. The residue was used... prep- Preparative purification by HPLC yielded compound 48 (31 mg, yellow solid).

[0317] LCMS(ESI): m / z 1436.8 [M+H] + ; RT = 1.167 min (2.50 min).

[0318] 1 H NMR (400 MHz, DMSO- d 6) δ 8.62 (dd, J = 2.1, 0.7 Hz, 1H), 8.18 (d, J =4.9 Hz, 1H), 8.04 – 7.87 (m, 4H), 7.80 – 7.67 (m, 2H), 7.66 – 7.55 (m, 2H),7.51 (d, J = 8.5 Hz, 1H), 7.48 – 7.39 (m, 3H), 7.22 – 7.14 (m, 2H), 7.13 – 7.02(m, 2H), 6.00 (s, 2H), 5.34 (q, J = 7.0 Hz, 1H), 4.81 – 4.43 (m, 11H), 4.18(dddd, J = 8.4, 6.9, 5.7, 2.0 Hz, 1H), 4.10 – 3.54 (m, 10H), 3.83 (s, 3H), 3.43(ddd, J = 12.0, 7.3, 5.1 Hz, 1H), 3.36 – 3.25 (m, 1H), 3.12 (td, J= 5.6, 1.0 Hz,2H), 2.93 – 2.74 (m, 3H), 2.53 – 2.38 (m, 1H), 2.25 (dd, J = 5.2, 3.0 Hz, 1H), 2.22 (s, 6H), 2.07 – 1.82 (m, 4H), 1.55 (d, J = 7.0 Hz, 3H), 1.23 (d, J = 20.1Hz, 5H), 1.03 (d, J = 5.0 Hz, 3H).

[0319] Example 16: Synthesis of compound 49.

[0320] Reaction formula:

[0321]

[0322] Preparation method:

[0323] Step 1: Preparation of compound 49.

[0324] In a dry 100 mL single-necked flask at room temperature, compound 7 (40 mg, 0.1 mmol), compound 46 (107 mg, 0.1 mmol), dichloromethane (10 mL), N,N'-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and 4-dimethylaminopyridine (2 mg, 0.01 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. The reaction was monitored by TLC. After completion, the reaction solution was concentrated under reduced pressure. The residue was used... prep- Compound 49 (33 mg, pale yellow solid) was prepared and purified by HPLC.

[0325] LCMS(ESI): m / z 1439.7 [M+H] + ; RT = 1.431 min (2.50 min).

[0326] 1 H NMR (400 MHz, DMSO- d 6) δ 8.18 (d, J = 4.9 Hz, 1H), 8.00 (d, J = 7.0 Hz, 1H), 7.88 (d, J= 9.3 Hz, 1H), 7.80 – 7.67 (m, 2H), 7.66 – 7.55 (m, 2H), 7.55 –7.39 (m, 4H), 7.22 – 7.15 (m, 1H), 7.09 (dd, J = 8.4, 1.8 Hz, 1H), 6.89 (dt, J =8.5, 1.0 Hz, 1H), 6.82 – 6.69 (m, 2H), 6.60 (t, J = 1.0 Hz, 1H), 6.01 – 5.93(m, 2H), 5.34 (q, J = 7.0 Hz, 1H), 4.81 – 4.63 (m, 4H), 4.65 – 4.50 (m, 3H), 4.47 (d, J = 6.9 Hz, 1H), 4.20 (dddd, J = 8.7, 6.9, 5.7, 2.0 Hz, 1H), 4.11 – 4.02(m, 1H), 4.08 – 3.96 (m, 2H), 4.01 – 3.84 (m, 2H), 3.81 (s, 3H), 3.89 – 3.64(m, 3H), 3.68 – 3.52 (m, 5H), 3.43 (ddd, J = 12.0, 7.3, 5.1 Hz, 1H), 3.36 –3.25 (m, 1H), 3.22 (ddd, J = 12.2, 6.0, 4.6 Hz, 1H), 3.12 (ddd, J = 12.2, 6.1,4.5 Hz, 1H), 3.01 – 2.59 (m, 8H), 2.53 – 2.38 (m, 1H), 2.25 (dd, J = 5.2, 3.0Hz, 1H), 2.22 (s, 6H), 2.07 – 1.82 (m, 4H), 1.55 (d, J = 7.0 Hz, 3H), 1.23 (d, J = 20.1 Hz, 5H), 1.03 (d, J = 5.0 Hz, 3H).

[0327] Example 17: Synthesis of compound 50.

[0328] Reaction formula:

[0329]

[0330] Preparation method:

[0331] Step 1: Preparation of Compound 50 tert-butyl (4S)-3-(3-(4-fluoro-1-(2-(2-((10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-yl)oxy)acetoxy)ethyl)-1H-indaz ol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (50) In a dry 50 mL single-necked flask at room temperature, compound 30 (200 mg, 0.32 mmol), dichloromethane (5 mL), compound 7 (123 mg, 0.32 mmol), triethylamine (98 mg, 0.97 mmol), 4-dimethylaminopyridine (40 mg, 0.32 mmol), and 2-chloro-1-methylpyridine iodide (182 mg, 0.71 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (0.1% FA) to give compound 50 (190 mg, yellow solid).

[0332] LCMS(ESI): m / z 985.2 [M+H] + ; RT = 1.839 min (2.50 min).

[0333] Step 2: Preparation of compound 51.

[0334] 2-(4-fluoro-5-(3-((S)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-1H-indazol-1-yl)ethyl 2-((10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-yl)oxy)acetate (51) Compound 50 (200 mg, 0.203 mmol), dichloromethane (5 mL), and trifluoroacetic acid (1 mL) were added sequentially to a dry 50 mL single-necked flask at room temperature. The mixture was stirred at room temperature and reacted for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to give crude compound 51 (170 mg, gray solid).

[0335] LCMS(ESI): m / z 885.5 [M+H] + ; RT = 1.230 min (2.50 min).

[0336] Step 3: Preparation of compound 52.

[0337] 2-(5-(3-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)- 2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-fluoro-1H-indazol-1-yl)ethyl 2-((10-methoxy-5,8,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-yl)oxy)acetate (52).

[0338] In a dry 25 mL single-necked flask at room temperature, compound 51 (170 mg), N,N-dimethylformamide (4 mL), 5-((S)-3,3-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carboxylic acid (119 mg, 0.29 mol), N,N-diisopropylethylamine (0.17 mL, 1.25 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (143 mg, 0.38 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 5 hours. After the reaction was complete, the solution was poured into water (40 mL) and extracted with ethyl acetate (15 mL × 2). Collect the organic phase, dry it with anhydrous sodium sulfate, filter it, and concentrate the filtrate under reduced pressure. The residue is then... prep- Preparative purification by HPLC yielded compound 52 (51.23 mg, yellow solid).

[0339] LCMS(ESI): m / z 1278.8 [M+H] + ; RT = 1.516 min (2.50 min).

[0340] 1 H-NMR (400 MHz, DMSO- d6) δ 8.23 ​​– 8.15 (m, 1H), 8.00 (d, J = 7.0 Hz, 1H), 7.77 (dd, J = 8.5, 5.0 Hz, 1H), 7.75 – 7.64 (m, 3H), 7.45 – 7.37 (m, 2H),7.26 (d, J = 5.0 Hz, 2H), 7.18 (dd, J = 8.5, 1.7 Hz, 1H), 6.89 (dt, J = 8.5, 1.0Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.73 (d, J = 1.0 Hz, 1H), 6.61 (t, J = 1.0 Hz,1H), 6.01 – 5.93 (m, 2H), 5.36 (q, J = 7.0 Hz, 1H), 4.81 – 4.69 (m, 3H), 4.64(dt, J = 10.2, 6.4 Hz, 1H), 4.44 (dt, J = 14.8, 6.4 Hz, 1H), 4.28 (dt, J = 10.2, 6.4 Hz, 1H), 3.95 (dt, J = 12.0, 5.3 Hz, 1H), 3.84 – 3.52 (m, 8H), 3.43 (ddd, J =12.0, 7.3, 5.0 Hz, 1H), 3.27 – 2.89 (m, 7H), 2.77 – 2.59 (m, 3H), 2.53 – 2.38(m, 1H), 2.25 (dd, J = 5.2, 3.0 Hz, 1H), 2.21 (s, 6H), 2.07 – 1.79 (m, 4H), 1.51 (d, J = 6.9 Hz, 3H), 1.21 (d, J = 20.1 Hz, 5H), 1.05 (d, J = 4.9 Hz, 3H).

[0341] Example 18: Synthesis of compound 58.

[0342] Reaction formula:

[0343]

[0344] Preparation method:

[0345] Step 1: Preparation of compound 53.

[0346] 2-(5-bromo-4-fluoro-1H-indazol-1-yl)ethan-1-amine (53).

[0347] In a dry 25 mL three-necked flask at room temperature, compound 8 (800 mg, 3.72 mmol), N,N-dimethylformamide (10 mL), tert-butyl (2-bromoethyl)carbamate (1.25 g, 5.58 mmol), potassium iodide (926 mg, 5.58 mmol), and potassium carbonate (1540 mg, 11.16 mmol) were added sequentially. The mixture was purged with nitrogen three times, heated to 70°C, and reacted for 16 hours. After LCMS monitoring, the reaction mixture was cooled to room temperature, poured into water, and extracted twice with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give a white solid. The solid was dissolved in dichloromethane, and 2 mL of 6M dioxane hydrochloric acid solution was added. The reaction was allowed to proceed for 1 hour. The solvent was removed by vacuum to obtain 903 mg of a white solid.

[0348] LCMS (ESI): m / z 258.1 [M+H] + .

[0349] Step 2: Preparation of compound 54.

[0350] allyl (S)-(1-((2-(5-bromo-4-fluoro-1H-indazol-1-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (54).

[0351] In a dry 25 mL single-necked flask at room temperature, compound 53 (258 mg), N,N-dimethylformamide (4 mL), ((allyloxy)carbonyl)-L-phenylalanine (249 mg), N,N-diisopropylethylamine (200 mg), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (350 mg) were added sequentially. The mixture was stirred at room temperature and reacted for 5 hours. After the reaction was complete, the mixture was poured into water (40 mL) and extracted with ethyl acetate (15 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative silica gel column chromatography to give compound 54 (448 mg, white solid).

[0352] LCMS (ESI): m / z 489.1 [M+H] + .

[0353] Step 3: Preparation of compound 55.

[0354] tert-butyl (S)-3-(3-(1-(2-((R)-2-((allyloxy)carbonyl)amino)-3-phenylpropanamido)ethyl)-4-fluoro-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1 H-imidazol-1-yl)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (55).

[0355] In a dry 50 mL three-necked flask at room temperature, (S)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-3-(2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester (2.0 g, 4.53 mmol), N-methylpyrrolidone (30 mL), compound 54 (6.79 mmol), potassium carbonate (1.50 g, 10.87 mmol), cuprous iodide (1.04 g, 5.44 mmol), and (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (1.55 g, 10.87 mmol) were added sequentially. The mixture was purged with nitrogen three times and heated to 100 °C for 16 hours. LCMS monitoring was performed until the reaction was complete. The reaction solution was poured into water (120 mL) and extracted with ethyl acetate (50 mL × 2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase column chromatography (0.1% NH4HCO3) to give compound 55 (1.90 g, white solid).

[0356] LCMS (ESI): m / z 850.4 [M+H] + .

[0357] Step 4: Preparation of compound 56.

[0358] allyl ((R)-1-((2-(4-fluoro-5-(3-((S)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyrid in-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-1H-indazol-1-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (56).

[0359] Compound 55 (1 g, 1.76 mmol), dichloromethane (5 mL), and dioxane hydrochloride (4 M, 5 mL) were added sequentially to a dry 50 mL single-necked flask at room temperature. The mixture was stirred at 40 °C and reacted for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to give crude compound 56 (0.83 g, white solid).

[0360] Step 5: Preparation of compound 57.

[0361] allyl ((R)-1-((2-(5-(3-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl))-1-((1S,2S)-2-methyl-1-( 5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-2-(4-fluoro-3,5-dim ethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H -imidazol-1-yl)-4-fluoro-1H-indazol-1-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (57).

[0362] In a dry 25 mL single-necked flask at room temperature, 5-((S)-3,3-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carboxylic acid (84 mg, 0.18 mmol), N,N-dimethylformamide (3 mL), compound 56 (172 mg, 0.23 mmol), N,N-diisopropylethylamine (114 mg, 0.86 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (101 mg, 0.27 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. The reaction was monitored by LCMS. Once complete, the reaction solution was... prep- Compound 57 (30.14 mg, white solid) was prepared and purified by HPLC (0.05% NH4HCO3).

[0363] LCMS (ESI): m / z 1143.6 [M+H] + .

[0364] Step 6: Preparation of compound 58.

[0365] (R)-2-amino-N-(2-(5-(3-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl))-1-((1S,2S)-2- methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-2-(4- (58)

[0366] Compound 57 (114 mg, 0.1 mmol), Pd(PPh3)4 (11 mg, 0.01 mmol), and K2CO3 (0.2 mmol) were added to a dry 25 ml single-necked flask and dissolved in alcohol. The mixture was stirred overnight at room temperature, and the solvent was removed by rotary evaporation. The solution was purified by silica gel column chromatography to obtain 952 mg of compound 58 as a white solid.

[0367] LCMS (ESI): m / z 1059.9 [M+H] + .

[0368] 1 H NMR (400 MHz, DMSO- d 6) δ 8.18 (d, J = 4.9 Hz, 1H), 8.00 (d, J = 7.0 Hz,1H), 7.86 – 7.64 (m, 7H), 7.41 (dd, J = 2.1, 0.5 Hz, 1H), 7.30 – 7.14 (m, 8H), 5.71 (dd, J = 7.2, 5.7 Hz, 1H), 5.49 – 5.32 (m, 2H), 4.54 – 4.33 (m, 2H), 3.95(dt, J = 12.0, 5.3 Hz, 1H), 3.80 – 3.34 (m, 7H), 3.13 – 2.93 (m, 4H), 2.85(ddt, J= 14.5, 6.7, 0.9 Hz, 1H), 2.53 – 2.40 (m, 1H), 2.25 (dd, J = 5.2, 3.0 Hz,1H), 2.21 (s, 6H), 2.07 – 1.80 (m, 4H), 1.52 (d, J = 7.0 Hz, 3H), 1.21 (d, J =19.9 Hz, 5H), 1.05 (d, J = 5.0 Hz, 3H).

[0369] Example 19: Synthesis of compound 59.

[0370] Reaction formula:

[0371]

[0372] Preparation method:

[0373] Step 1: Preparation of compound 59.

[0374] 7-(2-(((R)-1-((2-(5-(3-((S))-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2, 4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazo lo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-fluoro-1H-indazol-1-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-y l)amino)-2-oxoethyl)-9,10-dimethoxy-5,6,7,8,13,13a-hexahydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (59).

[0375] In a dry 100 mL single-necked flask at room temperature, compound 4 (40 mg, 0.1 mmol), compound 58 (105 mg, 0.1 mmol), dichloromethane (10 mL), N,N'-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and 4-dimethylaminopyridine (2 mg, 0.01 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. The reaction was monitored by TLC. After completion, the reaction solution was concentrated under reduced pressure. The residue was used... prep- Preparative purification by HPLC yielded compound 59 (31 mg, yellow solid).

[0376] LCMS(ESI): m / z 1439.7 [M+H] + ; RT = 1.197 min (2.50 min).

[0377] 1 H NMR (400 MHz, DMSO- d 6) δ 8.68 (d, J = 9.4 Hz, 1H), 8.18 (dd, J 7.09 (dd, J = 8.4, 1.8 Hz, 1H), 6.82(d, J = 1.1 Hz, 1H), 6.77 – 6.64 (m, 3H), 5.95 (s, 2H), 5.41 – 5.28 (m, 2H), 4.76 (d, J = 14.4 Hz, 1H), 4.72 – 4.58 (m, 2H), 4.52 (dt, J = 14.9, 5.5 Hz, 1H), 4.50 – 4.34 (m, 2H), 4.28 (d, J = 16.4 Hz, 1H), 4.02 (ddd, J= 12.0, 5.8, 4.9 Hz,1H), 3.95 – 3.72 (m, 9H), 3.67 – 3.40 (m, 4H), 3.34 – 3.02 (m, 5H), 2.98 –2.74 (m, 4H), 2.52 – 2.37 (m, 2H), 2.32 (dd, J = 5.0, 3.0 Hz, 1H), 2.22 (s,6H), 2.04 – 1.85 (m, 4H), 1.55 (d, J = 7.0 Hz, 3H), 1.23 (d, J = 20.1 Hz, 5H), 1.03 (d, J = 5.0 Hz, 3H).

[0378] Example 20: Synthesis of compound 60.

[0379] Reaction formula:

[0380]

[0381] Preparation method:

[0382] Step 1: Preparation of compound 60.

[0383] 9-(2-(((R)-1-((2-(5-(3-((S))-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro- 1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2 H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-fluoro-1H-indazol-1-yl)ethyl)amino)-1-oxo-3-phen ylpropan-2-yl)amino)-2-oxoethoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium (60).

[0384] In a dry 100 mL single-necked flask at room temperature, compound 6 (40 mg, 0.1 mmol), compound 46 (105 mg, 0.1 mmol), dichloromethane (10 mL), N,N'-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and 4-dimethylaminopyridine (2 mg, 0.01 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. The reaction was monitored by TLC. After completion, the reaction solution was concentrated under reduced pressure. The residue was used... prep- Preparative purification by HPLC yielded compound 60 (39 mg, yellow solid).

[0385] LCMS(ESI): m / z 1421.9 [M+H] + ; RT = 1.188 min (2.50 min).

[0386] 1 H NMR (400 MHz, DMSO- d 6) δ 8.62 (dd, J = 2.1, 0.7 Hz, 1H), 8.18 (dd, J =5.0, 0.6 Hz, 1H), 8.06 (d, J = 9.4 Hz, 1H), 8.04 – 7.90 (m, 3H), 7.80 – 7.67(m, 3H), 7.66 – 7.55 (m, 2H), 7.53 (d, J = 8.5 Hz, 1H), 7.48 – 7.39 (m, 3H), 7.29 – 7.14 (m, 7H), 7.13 – 7.02 (m, 2H), 6.00 (s, 2H), 5.34 (q, J = 7.0 Hz,1H), 4.79 – 4.37 (m, 8H), 4.02 (ddd, J = 12.0, 5.8, 4.9 Hz, 1H), 3.86 (ddd, J =12.1, 5.7, 4.9 Hz, 1H), 3.83 (s, 3H), 3.66 – 3.41 (m, 5H), 3.17 – 3.03 (m,3H), 2.98 – 2.74 (m, 4H), 2.52 – 2.37 (m, 2H), 2.32 (dd, J= 5.0, 3.0 Hz, 1H), 2.22 (s, 6H), 2.04 – 1.85 (m, 4H), 1.55 (d, J = 7.0 Hz, 3H), 1.23 (d, J = 20.1Hz, 5H), 1.03 (d, J = 5.0 Hz, 3H).

[0387] Example 21: Synthesis of compound 61.

[0388] Reaction formula:

[0389]

[0390] Preparation method:

[0391] Step 1: Preparation of compound 61.

[0392] (2R)-N-(2-(5-(3-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4 -oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)-2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-py razolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-fluoro-1H-indazol-1-yl)ethyl)-2-(2-((10-methoxy-5,8 ,13,13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-9-yl)oxy)acetamido)-3-phenylpropanamide (61).

[0393] In a dry 100 mL single-necked flask at room temperature, compound 7 (40 mg, 0.1 mmol), compound 46 (107 mg, 0.1 mmol), dichloromethane (10 mL), N,N'-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and 4-dimethylaminopyridine (2 mg, 0.01 mmol) were added sequentially. The mixture was stirred at room temperature and reacted for 3 hours. The reaction was monitored by TLC. After completion, the reaction solution was concentrated under reduced pressure. The residue was used... prep- Compound 61 (55 mg, pale yellow solid) was obtained by HPLC preparative purification.

[0394] LCMS(ESI): m / z 1424.8 [M+H] + ; RT = 1.581 min (2.50 min).

[0395] 1 H NMR (400 MHz, DMSO- d 6) δ 8.18 (dd, J = 5.0, 0.6 Hz, 1H), 8.04 – 7.96(m, 2H), 7.82 (t, J = 4.3 Hz, 1H), 7.80 – 7.67 (m, 2H), 7.66 – 7.55 (m, 2H),7.53 (d, J = 8.5 Hz, 1H), 7.48 – 7.39 (m, 3H), 7.29 – 7.15 (m, 6H), 7.09 (dd, J =8.4, 1.8 Hz, 1H), 6.89 (dt, J = 8.5, 1.0 Hz, 1H), 6.82 – 6.69 (m, 2H), 6.60 (t, J = 1.0 Hz, 1H), 6.01 – 5.93 (m, 2H), 5.34 (q, J = 7.0 Hz, 1H), 4.74 – 4.38 (m,5H), 4.02 (ddd, J = 12.0, 5.8, 4.9 Hz, 1H), 3.86 (ddd, J= 12.2, 5.7, 4.9 Hz,1H), 3.81 (s, 3H), 3.71 – 3.41 (m, 7H), 3.27 – 3.03 (m, 3H), 3.01 – 2.59 (m,9H), 2.52 – 2.37 (m, 2H), 2.32 (dd, J = 5.0, 3.0 Hz, 1H), 2.22 (s, 6H), 2.04 –1.85 (m, 4H), 1.55 (d, J = 7.0 Hz, 3H), 1.23 (d, J = 20.1 Hz, 5H), 1.03 (d, J = 5.0Hz, 3H).

[0396] Example 22: Synthesis of compound 65.

[0397] Reaction formula:

[0398]

[0399] Preparation method:

[0400] Step 1: Preparation of compound 63.

[0401] 5-chloro-2-ethoxy-N-((4-(4-fluorobenzyl)morpholin-2-yl)methyl)-4-isocyanatobenzamide (63).

[0402] 421 mg of compound 62 was dissolved in anhydrous DCM. The reaction was placed in an ice bath at 0°C, and 296 mg of triphosgene was added. After stirring for 1 hour, the reaction was slowly brought to room temperature. After stirring for another hour, unreacted triphosgene and solvent were removed by rotary evaporation to obtain a white solid 63. This product did not require purification and could be used directly in the next step.

[0403] Step 2: Preparation of compound 64.

[0404] 3-((1S,2S)-1-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-2-((S)-3-(3-(4-fluoro-1-(3-hydroxypropyl)-1H-indazol-5-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-2-(4- fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridine-5-carbonyl)-1H-indol-1-yl)-2-methylcyclopropyl)-1,2,4-oxadiazol-5(4H)-one (64).

[0405] Compound 64 can be synthesized by referring to the synthesis steps of compound 33.

[0406] Step 3: Preparation of compound 65.

[0407] 3-(5-(3-((S)-5-(5-((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-1-((1S,2S)-2-methyl-1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)cyclopropyl)-1H-indole-2-carbonyl)- 2-(4-fluoro-3,5-dimethylphenyl)-4-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-yl)-2-oxo-2,3-dihydro-1H-imidazol-1-yl)-4-fluoro-1H-indazol-1-yl)propyl (2-chloro-5-ethoxy-4-(((4-(4-fluorobenzyl)morpholin-2-yl)methyl)carbamoyl)phenyl)carbamate (65).

[0408] 1 mmol of compound 63 and 1 mmol of compound 64 were dissolved in anhydrous tetrahydrofuran and stirred overnight at 50 °C. After the reaction was complete as monitored by TLC, the solvent was removed by rotary evaporation, the mixture was dissolved in ethyl acetate, and extracted with saturated brine to separate the organic phase. Ethyl acetate was removed by rotary evaporation, and compound 65 was purified by silica gel column chromatography to obtain 998 mg of white solid.

[0409] LCMS(ESI): m / z 1374.8 [M+H] + ; RT = 2.111 min (2.50 min).

[0410] 1 H NMR (400 MHz, DMSO- d 6) δ 8.88 (s, 1H), 8.22 – 8.14 (m, 1H), 8.08 (s, 1H), 8.04 – 7.93 (m, 2H), 7.92 – 7.80 (m, 2H), 7.71 (d, J = 7.0 Hz, 1H),7.68 – 7.60 (m, 2H), 7.50 – 7.42 (m, 3H), 7.37 – 7.25 (m, 3H), 7.20 – 7.05(m, 3H), 5.69 (q, J = 6.9 Hz, 1H), 4.54 – 4.00 (m, 8H), 3.92 (ddd, J = 12.0, 5.8,4.9 Hz, 1H), 3.85 – 3.50 (m, 7H), 3.49 – 3.31 (m, 2H), 3.25 (ddd, J = 12.9,5.3, 4.6 Hz, 1H), 3.08 – 2.88 (m, 4H), 2.85 – 2.69 (m, 2H), 2.58 (ddd, J =12.7, 6.7, 5.3 Hz, 1H), 2.53 – 2.40 (m, 1H), 2.29 – 2.21 (m, 1H), 2.24 (s,6H), 2.19 – 1.80 (m, 7H), 1.59 (d, J = 6.9 Hz, 3H), 1.37 (t, J = 6.9 Hz, 3H), 1.21 (d, J = 19.9 Hz, 5H), 1.08 (d, J = 4.9 Hz, 3H).

[0411] Example 23: KCNH6 inhibitory effect.

[0412] A stable HEK293 cell line expressing KCNH6 was constructed. Cells were seeded at a density of 1000 cells / well in 384-well black-walled permeable cell culture plates and cultured overnight at 37°C in a 5% CO2 incubator. Loading buffer from the FluxOR II potassium channel assay kit (Thermo, catalog number: F20017) was added to each well, and the plates were incubated at room temperature in the dark for 1 hour. Then, different concentrations of the test compound and the negative control compound (DMSO) were added to the culture plates and incubated at room temperature for 10 minutes. Stimulation buffer was added, and fluorescence intensity (excitation wavelength 480 nm, emission wavelength 540 nm) was continuously measured at 1-second intervals for 2 minutes using a Tecan Spark multi-well microplate reader (Tecan, Switzerland). The inhibition percentage of each compound relative to DMSO was calculated, and the IC50 was obtained by nonlinear regression fitting. 50 (Half-maximum inhibition concentration), the results are shown in Table 1 below.

[0413] Example 24: Activity testing method for dual-target, dual-mechanism hypoglycemic drugs.

[0414] Step 1: In vitro GLP-1 secretion assay.

[0415] This application describes the ability of compounds to promote GLP-1 secretion in the STC-1 cell line. Cells were cultured in DMEM medium (Gibco, catalog number: 10965092) containing 10% FBS (Gibco, catalog number: 10091148). Cells that had grown to 80-90% confluence were then planted at 4 × 10⁻⁶ cells / year. 5 STC-1 cells were seeded at a density of 10 cells / well into sterile 12-well plates and incubated overnight at 37°C with 5% CO2. 20 μL of solutions containing different concentrations of the test compound and a negative control solution were added to each well, with three replicates for each drug. Cells were incubated at 37°C with 5% CO2 for 1 hour. The culture medium was discarded, and the cells were washed with HBSS solution, incubated for 30 minutes with glucose-free Krebs–Ringer buffer (KRBB), and then incubated for another 2 hours in KRBB solution containing 10 mM glucose, 10 μM Forskolin, and 10 μM IBMX. The supernatant was collected. STC-1 cells were lysed using protein lysis buffer, and the GLP-1 content in the supernatant and cell lysate was detected using a GLP-1 ELISA kit (Crystal Chem, catalog number: 81506). GLP-1 secretion was normalized to the GLP-1 content in the cell lysate. The percentage of GLP-1 secretion measured at each compound concentration to the percentage of GLP-1 secretion under negative control compound stimulation was calculated, and a nonlinear regression analysis was performed relative to the concentration of the added compound. An EC was fitted using a four-parameter logarithmic equation. 50(Half-maximum effective concentration), the results are shown in Table 1 below.

[0416] Step 2: In vitro human GLP-1 receptor agonist experiment.

[0417] This application describes the determination of the agonistic activity of compounds on the GLP-1 receptor in the HEK-293 cell line overexpressing human GLP1-R. Cells were cultured in DMEM medium (Gibco, catalog number: 10965092) containing 10% FBS (Gibco, catalog number: 10091148). Cells grown to 80-90% confluence were seeded at a density of 5000 cells / well into sterile, non-bottom-transparent white 384-well plates, leaving 24 blank wells for plotting a standard curve. The plates were incubated overnight at 37°C in a 5% CO2 incubator. Different concentrations of standards from a cAMP assay kit (Cisbio, catalog number: 62AM4PEC) were added to the blank wells at 10 μL per well, followed by 10 μL of stimulation buffer without the compound, with three replicates for each concentration. The cell culture medium was discarded, and 20 μL of stimulation buffer containing different concentrations of the test compound was added to the cell wells, with three replicates for each drug. Cells were cultured at 37°C in a 5% CO2 incubator for 1 hour. cAMP concentration was detected using an instrument with an excitation wavelength of 320 nm and emission wavelengths of 665 nm and 620 nm. The amount of cAMP produced in each well was converted to the maximum percentage of the observed response in the presence of human GLP-1. Nonlinear regression analysis was performed using the maximum percentage of response relative to the concentration of the added compound, and EC was fitted with a four-parameter logarithmic equation. 50 The results of the activity assay are shown in Table 1 below.

[0418] Step 3: Intraperitoneal glucose tolerance test (IPGTT).

[0419] Humanized GLP-1 receptor mice were fasted overnight and then administered the test compounds or solvents via gavage. One hour after gavage, a 20% (w / v) glucose solution was injected intraperitoneally in a volume equal to 10 μL of the mouse's body weight (g). Blood glucose concentrations in the tail vein were measured at 0, 15, 30, 60, and 120 minutes after the glucose load. Blood glucose was measured using a Johnson & Johnson One-Touch blood glucose meter and its accompanying test strips. The area under the curve (AUC) was calculated based on the time-varying parameters after drug administration to evaluate the blood glucose-lowering effects of each compound.

[0420] In this application, mice were administered RK-gk-303 at the same molar concentration (1 μmol / kg) along with the KCNH6 inhibitor Berberin and the GLP-1 receptor agonist Orforglipron, followed by IPGTT assay. The results showed that RK-gk-303 had a superior hypoglycemic effect compared to Berberin and Orforglipron. Figure 2 Humanized GLP-1 receptor mice were administered 1 μmol / kg Berberin, Orforgliprin, RK-gk-303, or a control solution by gavage for 1 hour, followed by IPGTT assay and blood glucose AUC levels. The glucose concentration was 2 g / kg.

[0424] Appendix 1: List of representative patent holders, their alternative names, and activities.

[0425]

[0426]

[0427]

Claims

1. The drug design method and concept shown in general formula (Ⅰ). This involves combining human glucagon-like peptide-1 receptor (GLP-1R) agonism and KCNH6 potassium channel inhibition to achieve a dual-target synergistic effect of GLP-1 receptor agonists and endogenous GLP-1 secretagogues. This includes single chemical structural entities constructed using various methods, and dual-target compounds acting on both GLP-1 receptor agonists and KCNH6. （Ⅰ） 2. The compound according to claim 1, characterized in that, The "GLP-1R activator" moiety can independently activate GLP-1R. The "GLP-1R activator" moiety of the compound includes, but is not limited to, the structures of the following general formulas (II) and (III), and the claims also include isomers and deuterated products of general formulas (II) and (III): （Ⅱ） （Ⅲ） In general formula (II), the chemical structures R1 and R2 can be access sites for the Linker, either independently or jointly. The X part may contain the following structures: ； The Y part can contain the following structures: ； The Y1 portion contains a benzene ring with any five-membered or six-membered structure, or a substituted benzene ring (containing a combination of any one or more of methyl, ethyl, hydroxyl, amino, mercapto, fluorine, chlorine, and bromine), or a pyridine ring, or an aliphatic five-membered ring, or an aliphatic six-membered ring, or a nitrogen-oxygen five-, six-, or seven-membered aliphatic heterocycles, including but not limited to the following representative structures: ； Part Y2 includes: ； In general formula (II), the chemical structures R1 and R2 can be access sites for the Linker, either independently or jointly.

3. The compound according to claim 1, characterized in that, The "Linker" part of the compound includes, but is not limited to, the following structures, or any structural group consisting of the following structures; For example, the structural units of ethylene glycol: ； Or units composed of fatty acid chains: ； Units composed of unsaturated chains: ； Or carboxylic acid: ； Or a unit composed of aromatic compounds: ； Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units: ； In the heteroatom-containing unit, R can be independently: alkyl, alkoxy, or hydrogen; Preferably, the alkyl group is specifically methyl, ethyl, propyl, isopropyl, n-butyl, or tert-butyl; and the alkoxy group is -OEt or -OMe. In particular, a linker can be composed of multiple sugar groups such as monosaccharides and disaccharides, or a polypeptide composed of a single amino acid, a dipeptide, or multiple amino acids.

4. The compound according to claim 1, characterized in that, The "KCNH6 inhibition" part can independently inhibit KCNH6. The compounds include, but are not limited to, the structures of the following general formulas (IV) and (V): （Ⅳ） R1, R2, R3, and R4 can be any structure, either independently or collectively, including methoxy, ethoxy, alkyl with a length of 1 to 5, amino, carboxylic acid, etc. For example, the structural units of ethylene glycol: ； Or units composed of fatty acid chains: ； Units composed of unsaturated chains: ； Or a unit composed of aromatic compounds: ； Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units: ； In the heteroatom-containing unit, R can be independently: alkyl, alkoxy, or hydrogen; Preferably, the alkyl group is specifically methyl, ethyl, propyl, isopropyl, n-butyl, or tert-butyl; and the alkoxy group is -OEt or -OMe. R1, R2, and R3 and R4 can form a ring structure independently or together, such as: 、 、 ； The Linker interface can exist on the aromatized entity, or on the R1, R2, R3, and R4 parts; In particular, the KCNH6 repressor moiety includes various salt forms of general formula (Ⅳ), such as quaternary ammonium salts with anionic structures of chloride, bromide, iodide, sulfate, etc. The molecular structure as shown in Formula V is claimed: （Ⅴ） In the formula, P, Q, and R can be independent, together, or in combination as the linker's connection sites: P can be constructed as a chemical entity linked to the V core structure via amide bonds, including but not limited to the following structures: P can be n has a length of 0 to 10. P can be n has a length of 1 to 5 P can be ; The L1 part can be any structure, either independently or collectively, including methoxy, ethoxy, alkyl, amino, carboxylic acid, etc. For example, the structural units of ethylene glycol: ； Or units composed of fatty acid chains: ； Units composed of unsaturated chains: ； Or a unit composed of aromatic compounds: ； Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units: ； P can be ; The L2 part can be any structure, either independently or collectively, including methoxy, ethoxy, alkyl, amino, carboxylic acid, etc. For example, the structural units of ethylene glycol: ； Or units composed of fatty acid chains: ； Units composed of unsaturated chains: ； Or a unit composed of aromatic compounds: ； Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units:

5. According to claim 4, the compound "KCNH6 moiety" can also be obtained by reducing the quaternary ammonium salt structure in the above general formula (IV) to obtain a tertiary amine structure, as shown in general formula (VI): （Ⅵ） R1, R2, R3, and R4 can be any structure, either independently or collectively, including methoxy, ethoxy, alkyl with a length of 1 to 5, amino, carboxylic acid, etc. For example, the structural units of ethylene glycol: ； Or units composed of fatty acid chains: ； Units composed of unsaturated chains: ； Or a unit composed of aromatic compounds: ； Or units containing heteroatoms, such as sulfur, nitrogen, phosphine, etc., including but not limited to the following fragment units: ； In the heteroatom-containing unit, R can be independently: alkyl, alkoxy, or hydrogen; Preferably, the alkyl group is specifically methyl, ethyl, propyl, isopropyl, n-butyl, or tert-butyl; and the alkoxy group is -OEt or -OMe. R1, R2, and R3 and R4 can form a ring structure independently or together, such as: 、 、 ； The Linker interface can also exist on the aromatized entity, or on the R1, R2, R3, and R4 parts; In particular, the KCNH6 inhibitory moiety can be constructed in various salt forms of the general formula (VI), such as hydrochloride, silver nitrate, hydrogen bromide, sulfate, etc. The claims also include the deuterated drug structure formed by one or more substitutions and the mixture of chiral unresolved stereoisomers constructed by modifying the chemical stereoconformation, and the corresponding pharmaceutically acceptable salts. Claims include the following chemical structural entities, including the following chemical structures and related deuterated drug structures constituted by single or multiple substitutions and stereoisomers constructed by modifying chemical stereoconformities, chiral unresolved mixtures, and corresponding pharmaceutically acceptable salts.

6. A compound selected from Table 1: Table 1.

Images