Formamide pyrazole compound as well as pharmaceutical composition and application thereof

Abstract

The invention discloses a formamide pyrazole compound as well as a pharmaceutical composition and application thereof. The compound has a structure as shown in a formula (I), has relatively strong anti-proliferative activity on a TRK xDFG mutation drug-resistant cell strain Ba / F3-LMNA-NTRK1-G667C, can effectively down-regulate the TRKAG667C protein level in a Ba / F3-LMNA-NTRK1-G667C cell, has an application value as a TRK degradation agent, is used for preparing an anti-tumor drug, and particularly provides a new drug molecule for treating mutation drug-resistant cancers.

Description

Technical Field

[0001] The present invention relates to a formamide pyrazole compound, a pharmaceutical composition and an application thereof, and in particular to a formamide pyrazole compound capable of targeted protein degradation, a pharmaceutical composition and an application thereof. Background Art

[0002] Tropomyosin receptor kinase (TRK) belongs to the transmembrane receptor tyrosine kinase family and includes three subtypes: TRKA, TRKB, and TRKC, encoded by the neurotrophic receptor tyrosine kinase 1 (NTRK1), NTRK2, and NTRK3 genes, respectively. The TRK protein structure consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain with kinase activity.

[0003] TRK receptors specifically bind to four neurotrophic factor (NT) ligands, including nerve growth factor (NGF) that binds to TRKA, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 / 5 (NT-4 / 5) that bind to TRKB, and neurotrophin-3 (NT-3) that binds to three TRK receptors. When specific TRK ligands bind to TRK receptors in the extracellular domain and dimerize, key tyrosine residues in the activation loop of the kinase domain are phosphorylated, which in turn leads to phosphorylation of tyrosine residues on both sides of the tyrosine kinase domain, docking cytoplasmic proteins and enzymes, driving the activation of downstream signaling pathways (Ras / Raf / MAPK pathway, mTOR / PI3K / Akt pathway, PLCγ / PKC pathway), regulating neuronal survival and differentiation, and further regulating cell proliferation, differentiation, metabolism, and apoptosis.

[0004] Abnormal activation of TRKs drives tumor development and progression. The mechanisms primarily include gene point mutations, variant splicing, and gene fusions. The most well-established mechanism is NTRK gene fusion, which arises from interchromosomal or intrachromosomal rearrangements, in which the 3' region of the NTRK gene, containing the kinase domain, fuses with the 5' fusion partner gene to produce a new fusion gene. The NTRK chimeric oncoprotein expressed by the fusion gene can be constitutively activated in a ligand-independent manner, leading to sustained activation of TRK downstream signaling pathways and a high risk of tumorigenesis. NTRK fusions are major oncogenic drivers of some rare tumors, such as infantile fibrosarcoma (IFS), secretory breast cancer (SBC), mammary analogous secretory carcinoma (MASC), and congenital mesoblastic nephroma (CMN).

[0005] Currently, the U.S. Food and Drug Administration (FDA) has approved two first-generation TRK inhibitors—larotrectinib (LOXO-101) and entrectinib (RDX-101)—and one second-generation TRK inhibitor, repotrectinib (TPX-0005), for the treatment of patients with NTRK gene fusions. Furthermore, the second-generation TRK inhibitor selitrectinib (LOXO-195) has entered Phase II clinical trials. However, clinical results have shown that both first- and second-generation TRK inhibitors have developed acquired resistance due to mutations in the drug's kinase domain, and have failed to address the common drawbacks of small molecule kinase inhibitors, such as poor pharmacokinetic properties and potential off-target toxicities.

[0006]

[0007] Targeted protein degradation (TPD) is a new strategy to induce the degradation of pathogenic proteins. It can use the protein homeostasis system to directly hit the target protein under human intervention, achieving highly selective degradation of the target. As a new therapeutic paradigm, this technology is different from the possessive-driven events of traditional inhibitors. It can efficiently complete the degradation of multiple target proteins with catalytic properties. Current studies have shown that targeted protein degradation can not only expand the drugability of more targets, but also play an important role in overcoming drug resistance. The TPD technologies currently under development mainly include proteolysis targeting chimeras (PROTACs), molecular glues (molecular glue) and hydrophobic tags (HyT).

[0008] PROTACs is a special type of protein that induces targeted protein degradation through the ubiquitin-proteasome system. It is a molecular tool that achieves precise regulation of proteins by causing the target protein to be ubiquitin-tagged and sent to the proteasome for degradation. PROTACs consists of a ligand that binds to the protein of interest (POI) and another ligand that can recruit E3 ubiquitin ligase through a linker (linker). The chemically induced proximity between POI and E3 ligase leads to ubiquitination of POI and subsequent degradation of POI by the ubiquitin-proteasome system. PROTACs can still participate in a new round of degradation after the target protein is degraded and can be reused to achieve continuous ubiquitination and degradation of POI.

[0009] Molecular glues are drugs or small molecules used for protein regulation. Their mechanism of action is to regulate protein function and metabolism by enhancing protein interactions. Unlike traditional enzyme inhibitors or receptor agonists, molecular glues primarily function by promoting protein binding, altering their conformation or function, and thereby influencing cell signaling and metabolic processes.

[0010] A hydrophobic tag is a specific bifunctional molecule that consists of a segment that specifically binds to a target protein, a hydrophobic segment that mimics protein misfolding, and a linker between the two segments. Regarding the principles of HyT technology, current research suggests that: (1) the hydrophobic tag in HyT technology causes an imbalance in protein homeostasis on the protein surface, thereby recruiting molecular chaperones to the protein, further inducing protein ubiquitination and degradation; (2) by binding a known target protein ligand to the hydrophobic tag, the unfolded state of the target protein can be simulated, allowing it to be directly recognized by the molecular chaperone.

[0011] Currently, the research on protein hydrolysis targeting chimeras (PROTACs) technology is relatively mature. Although the specific mechanism of action of HyT technology is still unclear, its advantages such as high degradation efficiency, fewer hydrogen bond acceptor donors, and low molecular weight also give it broad research prospects. The discovery of molecular glue degraders is accidental and their development is difficult. Summary of the Invention

[0012] Purpose of the invention: The first purpose of the present invention is to provide a carboxamide pyrazole compound with targeted protein degradation activity, the second purpose is to provide a pharmaceutical composition with the compound as an active ingredient, and the third purpose is to provide a pharmaceutical application of the compound and its pharmaceutical composition.

[0013] Technical solution: The carboxamide pyrazole compound of the present invention has a structure of formula (I), and also includes stereoisomers, deuterated compounds, pharmaceutically acceptable salts or mixtures thereof:

[0014]

[0015] in:

[0016] X is selected from the following structural fragments:

[0017]

[0018] Y chooses white N or CH;

[0019] R 1 A structural fragment selected from the following:

[0020]

[0021] R 2Independently selected from unsubstituted or substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-4 Alkoxy-substituted C 1-4 Alkyl, C 3-10 Cycloalkyl, C 3-10 Aromatic ring group, 3 to 10 membered heterocyclic group containing 0 to 5 N, S, O ring heteroatoms; or C 3-10 Heterocyclic substituted C 1-8 Alkyl, and C 1-8 The alkyl group contains 0 to 5 halogens, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy; wherein the substituent is selected from 1 to 5 -OH, =O, -OMe, -CH3, -CF3, -Cl, -F, -CO2Et, cyclohexyl, piperazinyl;

[0022] R 3 is selected from hydrogen or halogen;

[0023] R 4 independently selected from halogen, -CN, C 1-4 Alkyl, C 3-6 Cycloalkyl, 0 to 3 halogen-substituted C 1-3 Alkyl, -OH, C 1-3 Alkoxy, 0 to 3 halogen-substituted C 1-3 alkoxy;

[0024] m is selected from integers of 1 to 20, and n is selected from 0 or 1.

[0025] The present invention conducts in-depth research on PROTAC technology and Hyt technology and designs a carboxamide pyrazole TRK protein degrader.

[0026] Preferably, in the structure, R 2 Selected from unsubstituted or substituted methyl, ethyl, isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetane, piperidinyl, piperazinyl; wherein the substituent is selected from 1 to 5 -OH, =O, -OMe, -CH3, -CF3, -Cl, -F, -CO2Et, cyclohexyl, piperazinyl.

[0027] Preferably, in the structure, R 3 is selected from hydrogen, fluorine or chlorine.

[0028] Preferably, in the structure, R 4is selected from fluorine, chlorine, methyl, ethyl, isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 0 to 3 fluorine or chlorine substituted C 1-3 Alkyl, methoxy, ethoxy, 0 to 3 fluorine or chlorine substituted C 1-3 Alkoxy.

[0029] Preferably, in the structure, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0030] Most preferably, the carboxamide pyrazole compound is selected from any one of the following compounds:

[0031]

[0032]

[0033]

[0034] Preferably, the pharmaceutically acceptable salt is a salt formed by the compound and any one of the following acids:

[0035] Hydrochloric acid, hydrobromic acid, sulfuric acid, carbonic acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, acidic amino acid or ferulic acid.

[0036] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting the compounds with specified substituents with relatively nontoxic acids or bases. When the compound contains relatively acidic functional groups, base addition salts can be obtained by contacting the free form of the compound with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compound contains relatively basic functional groups, acid addition salts can be obtained by contacting the free form of the compound with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid (forming sulfate or bisulfate), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, Acids such as citric acid, tartaric acid and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (such as arginine, etc.) and glucuronic acid. When certain specific compounds contain basic and acidic functional groups, they can be converted into either base or acid addition salts. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated to regenerate the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubility in polar solvents.

[0037] "Pharmaceutically acceptable salts" can be synthesized from parent compounds containing acid or basic groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0038] Preferably, the stereoisomer is an isomer with chiral C and N introduced. In the chemical structure, the bond " / " indicates an unspecified configuration, that is, if there are chiral isomers in the chemical structure, the bond " / " can be or or include both or Two configurations.

[0039] Preferably, the isotopic compound is one that contains unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, the compound can be labeled with a radioactive isotope, such as tritium ( 3 H), iodine-125 ( 125 I) or C-14(14 C). For example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of this invention.

[0040] Preferably, the tautomers are isomers formed by conjugated interconversion of double bonds in unsaturated heterocyclic rings, including carbon-carbon double bond interconversion, carbon-heteroatom, and heteroatom-heteroatom double bond interconversion, such as tautomers formed by double bond interconversion in imidazole ring systems and pyrazole rings.

[0041] Preferably, the prodrug is an ester or amide prodrug with a carboxyl group, a hydroxyl group or an amino group introduced therein, more preferably a C1-C4 alkyl ester, a C1-C4 carboxylate or a C1-C4 alkyl amide.

[0042] Preferably, the solvate is a small molecule binding state formed by the compound and solvent molecules, more preferably a hydrate or alcoholate; the solvate can further form a salt with a corresponding acid to obtain a salt of the solvate.

[0043] Preferably, the isotope compound is a compound in which hydrogen is replaced by deuterium.

[0044] Preferably, the crystal is a specific crystal structure formed by the compound during the crystallization process, including different crystal forms of the compound itself, as well as different crystal forms of its salts, solvates, and salts of solvates.

[0045] The pharmaceutical composition of the present invention comprises the carboxamide pyrazole compound of the present invention and a pharmaceutically acceptable carrier.

[0046] Preferably, the pharmaceutical composition is in the form of a tablet, capsule, powder, syrup, liquid, suspension, lyophilized powder injection, or injection.

[0047] "Pharmaceutically acceptable carriers" may be excipients widely used in the field of drug production. Excipients are primarily used to provide a safe, stable, and functional pharmaceutical composition. They may also provide methods to dissolve the active ingredient at a desired rate after administration to a subject, or to promote effective absorption of the active ingredient after administration of the composition to a subject. The pharmaceutical excipients may be inert fillers, or may provide a certain function, such as stabilizing the overall pH value of the composition or preventing degradation of the active ingredient of the composition. The pharmaceutical excipients may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0048] The pharmaceutical compositions of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0049] The pharmaceutical compositions of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ophthalmic, rectal, topical, or parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention can also be in a controlled-release or sustained-release dosage form (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0050] The carboxamide pyrazole compound or the pharmaceutical composition thereof of the present invention is used in the preparation of drugs for preventing and / or treating diseases mediated by tropomyosin receptor kinase.

[0051] Preferably, the drug is for preventing and / or treating diseases related to tropomyosin receptor kinase dysfunction caused by tropomyosin receptor kinase gene amplification, overexpression, mutation or fusion.

[0052] Preferably, the drug is a drug for preventing and / or treating pain or cancer.

[0053] More preferably, the drug is a drug for preventing and / or treating lung cancer, hematological malignancies, prostate cancer, breast cancer, ovarian cancer, glioma, pancreatic cancer, hepatobiliary carcinoma, papillary thyroid cancer, colon cancer, head and neck squamous cell carcinoma or melanoma.

[0054] More preferably, the drug is a drug for preventing and / or treating cancer-related neuropathic pain, bone metastasis cancer pain, cancer visceral pain, and cancer breakthrough pain.

[0055] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0056] The compound designed by the present invention and its pharmaceutical composition can effectively degrade TRK protein, DC 50 The value reached the nanomolar concentration level, (DC 50 The optimal value is below 100 nM), and the D max The value reaches more than 50% (D max The optimal value is higher than 90%); it has broad application prospects, better activity than existing protein degraders, and is expected to solve the acquired drug resistance problem of existing TRK kinase small molecule inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The compound is effective for TRKA in Ba / F3-LMNA-NTRK1-G667C cells. G667C Protein degradation activity results. DETAILED DESCRIPTION

[0058] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0059] The experimental procedures in the following examples, unless otherwise specified, generally followed conventional conditions or those recommended by the manufacturers. The various commonly used chemical reagents used in the examples are commercially available. Unless otherwise defined, all technical and scientific terms have the same meanings as those commonly understood by those skilled in the art.

[0060] Example 1: 5-((2-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-2-carbonylethyl)amino)-1-cyclopropyl-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide

[0061]

[0062] (1) Preparation of intermediate T1: 5-amino-4-cyano-1-cyclopropyl-1H-pyrazole-3-carboxylic acid ethyl ester

[0063]

[0064] Reagents and conditions: (a) EtONa, EtOH, rt, 1 h, yield 60%; (b) 1) POCl3, 130°C, 1 h, yield 97%; 2) MeOH, 0°C, 1 h, yield 88%; (c) Et3N, MeOH, Ar, 0°C, 1 h, yield 47%.

[0065] The raw material, malononitrile (50.00 g, 756.85 mmol), was dissolved in anhydrous ethanol (200 mL). Diethyl oxalate (121.67 g, 832.54 mmol) was added, and the mixture was placed in an ice bath. An ethanolic solution of sodium ethoxide (71.80 mL, 20% wt.%, 832.54.00 mmol) was added dropwise under ice bath conditions. The temperature was raised to room temperature and allowed to react for 1 h. After completion of the reaction, the solvent in the mixture was evaporated under reduced pressure to yield crude intermediate 1-2 (85 g, 451.84 mmol).

[0066] Intermediate 1-2 (85 g, 451.84 mmol) was dissolved in phosphorus oxychloride (200 mL, 6.66 mol) and stirred at room temperature for 15 minutes. The mixture was then heated to 130°C for 1 hour. After the reaction, the mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, and the residue was placed in an ice bath. Methanol (300 mL) was slowly added dropwise with stirring. The mixture was stirred in an ice bath for 30 minutes. After concentration under reduced pressure, the product was purified by column chromatography (PE / EA, 2:1, v / v) to obtain pure intermediate 1-3 (69.7 g, 386.87 mmol).

[0067] Cyclopropylhydrazine hydrochloride (3.62 g, 33.30 mmol) was dissolved in anhydrous ethanol (50 mL). The argon atmosphere was replaced and the reaction flask was placed in an ice bath. Anhydrous triethylamine (5.79 mL, 41.63 mmol) was added to the reaction system under ice bath conditions and stirring continued for 20 min. A solution of Intermediate 1-3 (5 g, 27.75 mmol) in anhydrous ethanol (5 mL) was then slowly added to the reaction system under ice bath conditions and stirring continued at 0°C for 2 h. After the reaction, the mixture was concentrated under reduced pressure and purified by column chromatography (DCM) to obtain crude Intermediate T1. The solvent was removed under reduced pressure and the product was slurried with ethyl acetate to obtain pure Intermediate T1 (2.88 g, 13.08 mmol) as a white solid. ESI-MS m / z: 221.1 [M+H]+.

[0068] (2) Preparation of Intermediate T2: N-(5-amino-6-fluoropyridin-3-yl)-2-(4-(trifluoromethoxy)phenyl)acetamide

[0069]

[0070] Reagents and conditions: (a) T4P, Et3N, rt, 10 h, yield 91%; (b) 4N HCl in dioxane, rt, 1 h, yield 96%.

[0071] The starting material, tert-butyl-(5-amino-2-fluoropyridin-3-yl)carbamate (2-1, 1.00 g, 4.40 mmol), was dissolved in anhydrous tetrahydrofuran (20 mL). 2-(4-(trifluoromethoxy)phenyl)acetic acid (2-2, 1.16 g, 5.28 mmol) was added with stirring at room temperature. Anhydrous triethylamine (3.06 mL, 22.00 mmol) and a solution of n-butylphosphonic anhydride in ethyl acetate (6.94 mL, 50% wt.%, 11.00 mmol) were then added sequentially. The mixture was stirred at room temperature for 10 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with water (40 mL x 3), washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE / EA, 2:1, v / v) to obtain pure intermediate 2-3 (1.72 g, 4.01 mmol).

[0072] Intermediate 2-3 (1.72 g, 4.01 mmol) was dissolved in 15 mL of 4N hydrochloric acid in dioxane and stirred at room temperature for 1 hour. After the reaction, the solvent was partially removed under reduced pressure, and 10 mL of 2N sodium hydroxide was added to neutralize the remaining hydrochloric acid and dilute the mixture. The mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with water (20 mL x 3), washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain pure intermediate T2 (1.26 g, 3.83 mmol) as a white solid. ESI-MS m / z: 330.1 [M+H] + .

[0073] (3) Preparation of target compound JBP-1: 5-((2-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-2-carbonylethyl)amino)-1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide.

[0074]

[0075] Reagents and conditions: (a) Cs2CO3, CH3CN, 85℃, 10h, yield 35~78%; (b) TFA, DCM, rt, 2h, yield 80~92%; (c) HATU, DIPEA, DMF, rt, 10h, yield 50~81%; (d) 20% NaOH aqueous, THF, 60℃, 2h, yield 85~95%; (e) HATU, DIPEA, DMF, rt, 10h, yield 47~60%; (f) acetaldoxime, Pd(OAc)2, triphenylphosphine, EtOH, H2O, Ar, 105℃, 12h, yield 66~73%.

[0076] Intermediate T1 (500 mg, 2.27 mmol) was dissolved in anhydrous acetonitrile (10 mL). Cesium carbonate (1.85 g, 5.68 mmol) was added, and the mixture was heated to 85°C and stirred for 10 min. Tert-butyl bromoacetate (3-1a, 487 mg, 2.50 mmol) was then added to the reaction system, and the reaction was continued at 85°C for 10 h. Upon completion of the reaction, the solvent was removed by concentration, and the mixture was diluted with water. The mixture was extracted with ethyl acetate (40 mL × 3) and washed with water (40 mL × 3). The organic phases were combined and washed with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA, 3:1, v / v) to afford Intermediate 3-2a (579 mg, 1.73 mmol).

[0077] Intermediate 3-2a (579 mg, 1.73 mmol) was dissolved in dichloromethane (10 mL), and 5 mL of trifluoroacetic acid was added. The mixture was reacted at room temperature for 1 h. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain intermediate 3-3a (433 mg, 1.56 mmol).

[0078] Intermediate 3-3a (300 mg, 1.08 mmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL). 2-(7-Azabenzotriazole)-N,N-N',N'-tetramethyluronium hexafluorophosphate (615 mg, 1.62 mmol) and N,N-diisopropylethylamine (563 μL, 3.23 mmol) were added. The mixture was stirred at room temperature for 30 min. Bicyclo[2.2.1]hept-5-en-2-ylmethanamine (J1, 133 mg, 1.08 mmol) was added, and the reaction was allowed to proceed at room temperature for 10 h. Upon completion of the reaction, the mixture was diluted with an appropriate amount of water and extracted with ethyl acetate (50 mL x 3). The organic phases were combined and extracted with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (DCM / MeOH, 100:1, v / v) to afford Intermediate 3-4a (311 mg, 811.05 μmol).

[0079] Intermediate 3-4a (311 mg, 811.05 μmol) was dissolved in tetrahydrofuran (5 mL), and 20 mL of 20% aqueous sodium hydroxide solution was added. The mixture was reacted at 60°C for 2 h. After completion of the reaction, the mixture was extracted with ethyl acetate (40 mL × 2). The aqueous phase was retained and adjusted to weak acidity with 2N aqueous hydrochloric acid. The mixture was then extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with water (50 mL × 2), and then with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the crude product of intermediate 3-5a (267 mg, 751.27 μmol) without further purification.

[0080] The crude intermediate 3-5a (300 mg, 844.12 μmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL), and 2-(7-azabenzotriazole)-N,NN′,N′-tetramethyluronium hexafluorophosphate (401 mg, 1.06 mmol) and N,N-diisopropylethylamine (368 μL, 2.11 mmol) were added. The mixture was stirred at room temperature for 30 min, and N-(5-amino-6-fluoropyridin-3-yl)-2-(4-(trifluoromethoxy)phenyl)acetamide (T2, 232 mg, 703.44 μmoL) was added. The reaction was carried out at room temperature for 10 h. After the reaction is completed, add an appropriate amount of water to dilute, extract with ethyl acetate (50 mL × 3), combine the organic phases, and extract with saturated brine. Add anhydrous sodium sulfate to the organic phase and dry it. After concentration, column chromatography (DCM / MeOH, 80:1, v / v) is performed to obtain intermediate 3-6a (211 mg, 316.51 μmoL).

[0081] Intermediate 3-6a (100 mg, 150.01 μmol) was dissolved in 20 mL of a mixture of ethanol and water (v / v = 4:1). Acetaldehyde oxime (35 mg, 600.03 vmol), palladium acetate (6 mg, 26.73 μmol), and triphenylphosphine (6 mg, 22.88 μmol) were added. The reaction was carried out at 105°C under argon for 12 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (DCM / MeOH, 50:1, v / v) to afford the target compound JBP-1 (69 mg, 100.78 μmol) in a 67% yield as a white solid. 1H NMR (300MHz, DMSO-d6) δ10.60 (s, 1H), 10.05 (s, 1H), 9.14 (s, 1H), 8.59 (dd, J=8.8, 2.5Hz, 1H), 8.28 (t, J=2.2Hz, 1H), 8.08 ( t, J=5.7Hz, 1H), 8.00 (t, J=6.4Hz, 1H), 7.46 (d, J=8.7Hz, 2H), 7.33 (d, J=7.9Hz, 3H), 6.18-5.91 (m, 2H), 4.20 (d, J=6.2Hz, 2H ), 3.74 (s, 2H), 3.61 (tt, J=7.3, 3.7Hz, 1H), 2.88 (dt, J=12.7, 6.1Hz, 1H), 2.79-2.67 (m, 3H), 2.22-1.95 (m, 1H), 1.77 (ddd, J =12.4, 9.2, 3.7Hz, 1H), 1.31 (dd, J=8.1, 2.1Hz, 2H), 1.23 (d, J=2.9Hz, 2H), 1.13-1.08 (m, 2H), 0.46 (dt, J=11.2, 4.0Hz, 1H). 13 C NMR (100MHz, DMSO-d6) δ169.7, 169.1, 165.7, 162.4, 154.7, 151.3 (d, J=256.8Hz), 147.6, 138.9, 137.5, 136.9 (d, J=26.0Hz), 135.4, 134.7, 132. 8, 131.6, 125.9, 121.8, 121.4, 120.4 (d, J=28.7Hz), 98.9, 49.4, 47.7, 44.1, 43.2, 42.5, 42.3, 40.8, 38.8, 31.9, 30.2, 8.4. HR-MS (ESI) m / z: calcd for C 32 H 32 F4N8O5[M+H] + 685.2505found685.2520.

[0082] Example 2: 5-((4-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-4-carbonylbutyl)amino)-1-cyclopropyl-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide

[0083]

[0084] The synthesis method of compound JBP-1 was similar to that in Example 1, except that the linker fragment 3-1a was replaced with 3-1b. The product was a white solid with a final step yield of 66%. 1 H NMR (300MHz, DMSO-d6) δ10.63 (s, 1H), 10.05 (s, 1H), 9.20 (s, 1H), 8.58 (d, J=8.1Hz, 1H), 8.35-8.19 (m, 1H), 7.90 (d, J=38. 1Hz, 1H), 7.55 (t, J=6.6Hz, 1H), 7.46 (d, J=8.0Hz, 2H), 7.36 (d, J=8.1Hz, 2H), 7.32 (s, 1H), 6.18-6.03 (m, 1H), 5.98-5.89 (m , 1H), 3.74 (s, 2H), 3.63 (s, 1H), 3.49 (q, J=6.9Hz, 2H), 2.78 (d, J=21.5Hz, 3H), 2.62 (dd, J=13.6, 9.0Hz, 1H), 2.16 (t, J=6.9 Hz, 3H), 1.72 (d, J=9.6Hz, 3H), 1.29 (d, J=7.5Hz, 2H), 1.17 (d, J=8.0Hz, 3H), 1.09 (d, J=7.3Hz, 2H), 0.43 (d, J=11.5Hz, 1H). 13 C NMR (100MHz, DMSO-d6) δ171.6, 169.7, 165.8, 162.4, 155.3, 151.4 (d, J=241.2Hz) , 147.6, 139.0, 137.3, 136.9 (d, J = 15.9Hz), 135.4, 134.7 (d, J = 3.7Hz), 132.9, 13 1.6, 126.0, 121.4, 122.9-118.7 (m), 120.4 (d, J=28.8Hz), 99.0, 49.3, 44.8, 44.1 , 43.1, 42.5, 42.3, 40.8, 38.8, 32.8, 32.0, 30.2, 27.0, 8.1.HR-MS (ESI) m / z: calcd for C 34 H 36 F4N8O5[M+H] + 713.2818found 713.2841.

[0085] Example 3: 5-((6-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-6-carbonylhexyl)amino)-1-cyclopropyl-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide

[0086]

[0087] The synthesis method of compound JBP-1 was similar to that in Example 1, except that the linker fragment 3-1a was replaced with 3-1c. The product was a white solid with a final step yield of 73%. 1 H NMR (400MHz, DMSO-d6) δ10.62 (s, 1H), 10.00 (s, 1H), 9.17 (s, 1H), 8.59 (dd, J=8.8, 2 .5Hz, 1H), 8.28 (t, J=2.1Hz, 1H), 7.76 (t, J=5.6Hz, 1H), 7.56 (t, J=6.4Hz, 1H), 7.51 -7.42 (m, 2H), 7.39-7.27 (m, 3H), 6.12 (dd, J=5.7, 3.0Hz, 1H), 5.93 (dd, J=5.7, 2.9H z, 1H), 3.74 (s, 2H), 3.64 (tt, J=7.2, 3.7Hz, 1H), 3.49 (q, J=6.8Hz, 2H), 2.81 (dd, J=1 3.1, 6.4Hz, 1H), 2.74 (d, J=11.4Hz, 2H), 2.62 (ddd, J=13.1, 9.0, 5.7Hz, 1H), 2.15 (q d, J=8.8, 4.3Hz, 1H), 2.05 (t, J=7.3Hz, 2H), 1.74 (ddd, J=11.4, 9.1, 3.8Hz, 1H), 1.5 3(p, J=6.8, 6.4Hz, 4H), 1.38-1.30(m, 2H), 1.30-1.27(m, 1H), 1.24-1.21(m, 2H), 1. 17 (d, J=8.2Hz, 1H), 1.10 (dt, J=7.1, 3.6Hz, 2H), 0.43 (ddd, J=11.4, 4.4, 2.5Hz, 1H). 13C NMR (100MHz, DMSO-d6) δ172.1, 169.7, 165.8, 162.4, 155.4, 151.3 (d, J=232.5Hz), 14 7.6, 138.9, 137.3, 136.9 (d, J=12.0Hz), 135.4, 134.7 (d, J=3.9Hz), 132.9, 131.6, 125 .9, 121.4, 120.5 (d, J = 256.0Hz), 120.4 (d, J = 29.0Hz), 98.7, 49.3, 45.1, 44.1, 43.0, 4 2.5, 42.3, 40.8, 38.8, 35.7, 32.1, 30.5, 30.2, 26.2, 25.5, 8.2. HR-MS (ESI) m / z: calcd for C 36 H 40 F4N8O5[M+H] + 741.3131found 741.3150.

[0088] Example 4: 5-((8-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-8-carbonyloctyl)amino)-1-cyclopropyl-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide

[0089]

[0090] The synthesis method of compound JBP-1 was similar to that in Example 1, except that the linker fragment 3-1a was replaced by 3-1d. The product was a white solid with a final step yield of 70%. 1H NMR(400MHz,DMSO-d6)δ10.61(s,1H),10.00(s,1H),9.22-9.10(m,1H),8.59(dd,J=8.7,2.6Hz,1H),8.28(t,J=2.1Hz,1H),7.80(dt,J=51.5,5.7Hz,1H),7.56(t,J=6.4Hz,1H),7.46(d,J=8.7Hz,2H),7.33(d,J=8.1Hz,3H),6.22-5.83(m,2H),3.74(s,2H),3.63(tt,J=7.2,3.7Hz,1H),3.48(q,J=6.7Hz,2H),2.80(dt,J=12.8,6.2Hz,1H),2.73(d,J=10.3Hz,2H),2.61(ddd,J=13.1,9.0,5.7Hz,1H),2.20-2.08(m,1H),2.03(t,J=7.2Hz,2H),1.73(ddd,J=11.4,9.1,3.8Hz,1H),1.55-1.45(m,4H),1.32(d,J=7.5Hz,5H),1.22(t,J=3.5Hz,4H),1.16(d,J=8.0Hz,1H),1.09(td,J=7.6,5.2Hz,2H),0.43(ddd,J=11.5,4.5,2.5Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ172.2,169.6,165.9,162.41,155.48,151.33(d,J=232.7Hz),147.67(d,J=1.9Hz),138.9,137.3,136.8(d,J=16.3Hz),135.4,134.7(d,J=3.9Hz),132.9,131.5,125.9,121.3,120.5(d,J=255.8Hz),120.4(d,J=28.9Hz),98.8,49.3,45.2,44.1,43.0,42.5,42.35,40.8,38.9,35.8,32.1,30.6,30.2,29.0,28.8,26.5,25.7,8.2.HR-MS(ESI)m / z:calcd forC 38 H 44 F4N8O5[M+H] + 769.3444found 769.3471.

[0091] Example 5: 5-((1-(bicyclo[2.2.1]hept-5-en-2-yl)-1-carbonyl-5,8,11-trioxa-2-azatridec-13-yl)amino)-1-cyclopropyl-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide

[0092]

[0093] (1) Prepare intermediates T1 and T2. The synthesis method refers to (1) and (2) in Example 1.

[0094] (2) The target compound JBP-5 was prepared by the synthesis method described in Example 1 (3), except that the linker fragment 3-1a was replaced by 4-1, and the hydrophobic fragment bicyclo[2.2.1]hept-5-en-2-ylmethylamine (J1) was replaced by bicyclo[2.2.1]hept-5-en-2-carboxylic acid (J2).

[0095]

[0096] Reagents and conditions: (a) CsCO3, CH3CN, 85℃, 10h, yield 81%; (b) TFA, DCM, rt, 2h, yield 90%; (c) HATU, DIPEA, DMF, rt, 10h, yield 67%; (d) 20% NaOH aqueous, THF, 60℃, 2h, yield 89%; (e) HATU, DIPEA, DMF, rt, 10h, yield 53%; (f) acetaldoxime, Pd(OAc)2, triphenylphosphine, EtOH, H2O, Ar, 105℃, 12h, yield 59%.

[0097] The product JBP-5 is a light yellow oily liquid. 1H NMR (400MHz, DMSO-d6) δ10.61 (s, 1H), 10.02 (s, 1H), 9.15 (s, 1H), 8.60 (dt, J=9.0, 2.7Hz, 1H), 8.28 (t, J=2.1Hz, 1H), 7.89 (t, J=5.6Hz, 1H), 7.72 (t, J=6.2Hz, 1H), 7.60 (t, J=5.7Hz, 1H), 7.46 (d, J=8.7Hz, 2H), 7.32 (d, J=8.2Hz, 2H), 7.31-7.23 (m, 1H), 6.18-5.76 (m, 2H), 3.74 (s, 2H) ), 3.68-3.64 (m, 2H), 3.58 (t, J = 5.0Hz, 2H), 3.52 (d, J = 1.9Hz, 4H), 3.49 (d, J = 4.6Hz, 4H), 3.40 (t, J = 6.1Hz, 1H), 3.24-3.07 (m, 3H), 2.78 (dd d, J=12.8, 8.8, 3.3Hz, 2H), 2.13-1.92 (m, 1H), 1.81-1.58 (m, 2H), 1.29 (ddd, J=11.2, 4.3, 2.5Hz, 1H), 1.24-1.21 (m, 2H), 1.17-1.03 (m, 4H). 13 C NMR (100MHz, Benzene-d6) δ179.8, 178.0, 174.3, 170.4, 167.0, 159.9, 156.0 (d, J=232.5Hz), 152.3 (d, J=1.9Hz), 143.1 (d, J=78.5Hz), 141.4, 140.1, 139.4 (d, J=3.9Hz), 137.3, 136.2, 13 0.5, 126.0, 125.2 (d, J=255.8Hz), 125.1 (d, J=29.0Hz), 103.6, 75.2, 74.9, 74.9, 74.7, 74.2, 74.2, 54.4, 52.0, 50.7, 49.7, 48.4, 47.2, 46.1, 36.7, 34.9, 33.5, 12.9. HR-MS (ESI) m / z: calcd for C 38 H 44 F4N8O8[M+H] + 817.3291found 817.3320.

[0098] Example 6: 5-((1-(bicyclo[2.2.1]hept-5-en-2-yl)-1,14-dicarbonyl-5,8,11-trioxa-2,15-diazaoctadec-18-yl)amino)-1-cyclopropyl-N 3-(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide

[0099]

[0100] (1) Prepare intermediates T1 and T2. The synthesis method refers to (1) and (2) in Example 1.

[0101] (2) Preparation of Intermediate 5-3: 1-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-carbonyl-5,8,11-trioxa-2-azatetradecane-14-oic acid

[0102]

[0103] Reagents and conditions: (a) HATU, DIPEA, DMF, rt, 10 h, yield 82%; (b) TFA, DCM, rt, 1 h, yield 96%.

[0104] Bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (J2, 179 mg, 1.30 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (675 mg, 1.30 mmol), and N,N-diisopropylethylamine (419 mg, 3.24 mmol) were dissolved in anhydrous N,N-dimethylformamide (DMF) and stirred at room temperature for 20 minutes. Tert-butyl 3-[2-(2-(2-aminoethoxy)ethoxy)ethoxy]propanoate (5-1, 300 mg, 1.08 mmol) was then added to the reaction system, and the reaction was continued at room temperature for 10 hours. After the reaction, an appropriate amount of water was added for dilution, and the mixture was extracted with EA (40 mL × 3). The organic phases were combined and extracted with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (DCM / MeOH, 80:1, v / v) to obtain intermediate 5-2 (351 mg, 882.99 μmol).

[0105] Intermediate 5-2 (351 mg, 882.99 μmol) was dissolved in dichloromethane (10 mL), and 5 mL of trifluoroacetic acid was added under stirring. The mixture was reacted at room temperature for 1 h. After the reaction was completed, the mixture was directly concentrated to obtain intermediate 5-3 (289 mg, 846.50 μmol).

[0106] (3) Preparation of target compound JBP-6: 5-((1-(bicyclo[2.2.1]hept-5-en-2-yl)-1,14-dicarbonyl-5,8,11-trioxa-2,15-diazaoctadecane-18-yl)amino)-1-cyclopropyl-N 3-(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide.

[0107]

[0108] Reagents and conditions: (a) Cs2CO3, CH3CN, 85℃, 10h, yield 57%; (b) 20% NaOH aqueous, THF, 60℃, 2h, yield 72%; (c) HATU, DIPEA, DMF, rt, 10h, yield 53%; (d) H2O2, 20% NaOHaqueous, tetrabutylammonium hydrogen sulfate, DCM, rt, 1h, yield 66%; (e) TFA, DCM, rt, 1h, yield 89%; (f) HATU, DIPEA, DMF, rt, 10h, yield 35%.

[0109] Intermediate T1 (500 mg, 2.27 mmol) was dissolved in 20 mL of anhydrous acetonitrile, and Cs2CO3 (1.09 g, 5.68 mmol) was added. The reaction was allowed to react at 85°C for 10 min. N-Boc-3-aminopropyl bromide (5-4, 648.75 mg, 2.72 mmol) was then added to the reaction system, and the reaction was continued at 85°C overnight. After the reaction was completed, the solvent was removed by concentration, and the mixture was extracted with ethyl acetate (40 mL x 3). The organic phases were combined and extracted with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE / EA, 3:1, v / v) to afford Intermediate 5-5 (490 mg, 1.30 mmol).

[0110] Intermediate 5-5 (500 mg, 1.32 mmol) was dissolved in 10 mL of tetrahydrofuran, and 20% aqueous NaOH solution (20 mL) was added. The mixture was reacted at 60°C for 1 h. After completion of the reaction, the mixture was extracted twice with ethyl acetate (30 mL). The aqueous phase was retained and then adjusted to a slightly acidic pH with 2N aqueous HCl. The mixture was then extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford Intermediate 5-6 (333 mg, 953.09 μmol).

[0111] Intermediate 5-6 (300 mg, 858.64 μmol), 2-(7-azabenzotriazole)-NNN',N'-tetramethyluronium hexafluorophosphate (491.52 mg, 944.50 μmol), and N,N-diisopropylethylamine (448.69 μL, 2.58 mmol) were dissolved in anhydrous N,N-dimethylformamide (DMF) and stirred at room temperature for 20 min. After 20 min, Intermediate T2 (339.25 mg, 1.03 mmol) was added to the reaction system, and the reaction was continued at room temperature for 10 h. After completion of the reaction, the mixture was diluted with an appropriate amount of water and extracted with EA (40 mL x 3). The organic phases were combined and extracted with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE / EA, 4:1, v / v) to afford Intermediate 5-7 (302 mg, 457.14 μmol).

[0112] Intermediate 5-7 (300 mg, 454.11 μmol) was dissolved in dichloromethane (6 mL) and placed in an ice bath. 30% aqueous H2O2 (6 mL), tetrabutylammonium hydrogen sulfate (900 mg, 2.65 mmol), and 20% aqueous NaOH (5 mL) were added sequentially. After completion of the reaction, the mixture was diluted with water and extracted with EA (40 mL × 3). The organic phases were combined and extracted with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (DCM / MeOH, 40:1, v / v) to afford Intermediate 5-8 (204 mg, 300.60 μmol).

[0113] Intermediate 5-8 (200 mg, 294.70 μmol) was dissolved in dichloromethane (10 mL), and 5 mL of trifluoroacetic acid was added with stirring. The mixture was reacted at room temperature for 1 h. After the reaction was completed, the mixture was directly concentrated to obtain intermediate 5-9 (152 mg, 262.74 μmol).

[0114] Intermediate 5-3 (100 mg, 292.91 μmol), 2-(7-azabenzotriazole)-N,NN',N'-tetramethyluronium hexafluorophosphate (167 mg, 439.36 μmol), and N,N-diisopropylethylamine (153 μL, 878.72 μmol) were dissolved in anhydrous N,N-dimethylformamide (DMF) and stirred at room temperature for 20 min. Intermediate 5-9 (169 mg, 292.91 μmol) was then added to the reaction system, and the reaction continued at room temperature for 10 h. After completion of the reaction, the mixture was diluted with an appropriate amount of water and extracted with EA (40 mL x 3). The organic phases were combined and extracted with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (DCM / MeOH, 40:1, v / v) to afford the target compound JBP-6 (92 mg, 102.00 μmol) as a white solid. 1H NMR(400MHz,DMSO-d6)δ10.62(s,1H),10.00(s,1H),9.19(s,1H),8.60(dd,J=8.7,2.6Hz,1H),8.29(t,J=2.2Hz,1H),7.92(q,J=7.0,6.3Hz,1H),7.69(ddd,J=14.6,5.9,3.0Hz,1H),7.61(t,J=5.6Hz,1H),7.54(t,J=6.6Hz,1H),7.46(d,J=8.6Hz,2H),7.32(d,J=7.8Hz,2H),5.93(ddd,J=110.5,5.6,2.9Hz,2H),4.20-3.94(m,2H),3.74(s,2H),3.59(t,J=6.4Hz,2H),3.48(d,J=3.0Hz,4H),3.34(d,J=6.0Hz,2H),3.14(dd,J=12.3,6.0Hz,4H),2.76(dt,J=11.7,3.4Hz,2H),2.30(t,J=6.4Hz,2H),1.70(ddd,J=11.2,9.2,3.7Hz,1H),1.66-1.59(m,2H),1.35(tdd,J=10.5,7.1,5.0Hz,2H),1.31-1.27(m,2H),1.26(d,J=4.6Hz,2H),1.24(d,J=2.4Hz,2H),1.22(d,J=4.2Hz,2H),1.11(dt,J=7.0,3.4Hz,2H),0.88(d,J=4.9Hz,1H). 13 CNMR(100MHz,Benzene-d6)δ178.1,175.2,174.3,172.1,170.5,167.0,159.9,157.8-151.5(m),143.6,141.8,140.1,139.4(d,J=3.9Hz),137.2,136.7(d,J=16.3Hz),136.2,133.7,130.6,126.0,125.1(d,J=28.9Hz),103.8,74.8,74.8,74.7,74.2,72.5,71.9,54.4,50.7,48.4,47.2,41.2,36.7,35.6,34.9,33.5,28.3,27.5,19.0,15.9,12.8.HR-MS(ESI)m / z:calcd for C 42 H 51 F4N9O9[M+H] +902.3819found 902.3838.

[0115] Example 7: 1-cyclopropyl-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-5-((5-carbonyl-5-(((1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)amino)pentyl)amino)-1H-pyrazole-3,4-dicarboxamide

[0116]

[0117] (1) Prepare intermediates T1 and T2. The synthesis method refers to (1) and (2) in Example 1.

[0118] (2) The target compound JBP-7 was prepared by the same synthesis method as that described in Example 1 (3), except that the linker fragment 3-1a was replaced by 6-1, and the hydrophobic fragment bicyclo[2.2.1]hept-5-en-2-ylmethylamine (J1) was replaced by (1R)-1,7,7-trimethylbicyclo[2.2.1]heptane-2-amine (J3).

[0119]

[0120] Reagents and conditions: (a) Cs2CO3, CH3CN, 85℃, 10h, yield 67%; (b) TFA, DCM, rt, 2h, yield 91%; (c) HATU, DIPEA, DMF, rt, 10h, yield 77%; (d) 20% NaOH aqueous, THF, 60℃, 2h, yield 90%; (e) HATU, DIPEA, DMF, rt, 10h, yield 55%; (f) acetaldoxime, Pd(OAc)2, triphenylphosphine, EtOH, H2O, Ar, 105℃, 12h, yield 78%.

[0121] The product JBP-7 was a white solid. 1H NMR (300MHz, DMSO-d6) δ10.60 (s, 1H), 10.05 (s, 1H), 9.14 (s, 1H), 8.59 (dd, J=8.8, 2.5Hz, 1H), 8.28 (t, J=2.2Hz, 1H), 8.08 (t , J=5.7Hz, 1H), 8.00 (t, J=6.4Hz, 1H), 7.46 (d, J=8.7Hz, 2H), 7.33 (d, J=7.9Hz, 3H), 6.18-5.91 (m, 2H), 4.20 (d, J=6.2Hz, 2H), 3.74 (s, 2H), 3.61 (tt, J=7.3, 3.7Hz, 1H), 2.88 (dt, J=12.7, 6.1Hz, 1H), 2.79-2.67 (m, 3H), 2.22-1.95 (m, 1H), 1.77 (ddd, J=1 2.4, 9.2, 3.7Hz, 1H), 1.31 (dd, J=8.1, 2.1Hz, 2H), 1.23 (d, J=2.9Hz, 2H), 1.11 (d, J=5.9Hz, 2H), 0.46 (dt, J=11.2, 4.0Hz, 1H). 13 C NMR (100MHz, DMSO-d6) δ172.3, 169.6, 165.8, 162.4, 155.4, 150.2, 147.6, 138.9, 1 35.4, 134.7 (d, J = 3.9Hz), 133.0 (d, J = 14.3Hz), 131.6, 126.0, 121.4, 120.5 (d, J = 2 55.8Hz), 120.4 (d, J=28.8Hz), 99.0, 53.0, 49.7, 48.1, 45.0, 44.8, 42.5, 36.0, 35. 5, 32.1, 30.3, 28.2, 27.9, 23.2, 20.1, 18.9, 14.2, 8.2, 8.1. HR-MS (ESI) m / z: calcd forC 37 H 44 F4N8O5[M+H] + 757.3444found 757.3478.

[0122] Example 8: 5-(((1-(bicyclo[2.2.1]hept-5-ene-2-carbonyl)azetidin-3-yl)methyl)amino)-1-cyclopropyl-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide

[0123]

[0124] The synthesis method of compound 5 in Example 5 was referred to, except that the linker fragment 4-1 was replaced with 3-(bromomethyl)azetidine. The product JBP-8 was a white solid. 1 H NMR (500MHz, DMSO-d6) δ10.60 (s, 1H), 10.03 (s, 1H), 9.18 (s, 1H), 8.59 (dt, J=8.7, 2.0Hz, 1H), 8.29 (t, J=2.1Hz, 1H), 7 .58 (dt, J=27.0, 6.5Hz, 1H), 7.47 (d, J=8.6Hz, 2H), 7.33 (d, J=8.3Hz, 3H), 6.17-5.70 (m, 2H), 4.26 (dt, J=22.0, 8.4Hz, 1H), 4.02-3.89 (m, 1H), 3.83 (t, J=8.9Hz, 1H), 3.75 (s, 2H), 3.60 (ddd, J=85.3, 9.9, 4.3Hz, 4H), 3.04 (d, J=3.5Hz, 1H), 2.86-2.74 (m, 3H), 1.81-1.70 (m, 1H), 1.25 (d, J=7.2Hz, 4H), 1.12 (td, J=6.6, 2.3Hz, 2H), 0.87 (dt, J=12.4, 7.2Hz, 1H). 13 C NMR (150MHz, DMSO-d6) δ174.0 (d, J=7.9Hz), 169.7, 165.7 (d, J=3.1Hz), 162.3, 155.1 (d, J=4.6Hz), 152.2, 150.6, 147.6, 139.0 (d , J=2.7Hz), 136.9 (d, J=16.4Hz), 135.4, 134.7 (d, J=3.7Hz), 133.0 (d, J=24.7Hz), 131.6, 126.1, 121.4 (d, J=511.9Hz), 121.4, 120 .4 (d, J=29.1Hz), 99.5 (d, J=8.9Hz), 53.2 (d, J=10.7Hz), 51.0 (d, J=15.5Hz), 49.7 (d, J=9.5Hz), 48.4 (d, J=22.0Hz), 44.7, 42.5, 4 2.3, 40.8, 32.0 (d, J = 7.2Hz), 29.4 (d, J = 4.8Hz), 29.2 (d, J = 5.2Hz), 8.2 (d, J = 7.1Hz), 8.1 (d, J = 3.1Hz). HR-MS (ESI) m / z: calcdfor C 34 H 34 F4N8O5[M+H] + 711.2661found 711.2671.

[0125] Example 9: 5-((5-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-5-carbonylpentyl)amino)-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide

[0126]

[0127] (1) Prepare intermediate T3. The synthesis method is similar to that of (1) in Example 1, except that cyclopropylhydrazine hydrochloride is replaced by isopropylhydrazine hydrochloride.

[0128]

[0129] Reagents and conditions: (a) EtONa, EtOH, rt, 1 h, yield 60%; (b) 1) POCl3, 130°C, 1 h, yield 97%; 2) MeOH, 0°C, 1 h, yield 88%; (c) Et3N, MeOH, Ar, 0°C, 1 h, yield 52%.

[0130] (2) Prepare intermediate T2. The synthesis method is similar to that of (2) in Example 1.

[0131] (3) The target compound JBP-9 was prepared by the same synthesis method as in Example 1 (3), except that the intermediate T1 was replaced by T3 and the linker fragment 3-1a was replaced by 6-1.

[0132] The product JBP-9 was a white solid. 1H NMR (300MHz, DMSO-d6) δ10.62 (s, 1H), 10.13 (s, 1H), 9.22 (s, 1H), 8.69 (dd, J=8.7, 2.5Hz, 1H), 8.30 (t, J=2.2Hz, 1H), 7.85 (dt, J=38.4, 5.7Hz, 1H ), 7.49 (d, J=8.6Hz, 2H), 7.35 (d, J=8.2Hz, 1H), 7.10 (t, J=6.4Hz, 2H), 6.19-6.04 (m, 1H), 5.94 (dd, J=5.8, 2.8Hz, 1H), 4.65 (p, J=6.6Hz, 1H), 3.7 6 (s, 2H), 3.18 (q, J = 6.8Hz, 2H), 2.88-2.78 (m, 1H), 2.74 (d, J = 3.9Hz, 2H ), 2.70-2.57(m, 1H), 2.10(t, J=6.6Hz, 3H), 1.73(ddd, J=12.5, 9.2, 3.7H z, 1H), 1.57 (dt, J=12.0, 5.8Hz, 3H), 1.48 (s, 3H), 1.46 (s, 3H), 1.30 (d, J=6.7Hz, 1H), 1.20 (t, J=9.7Hz, 2H), 0.44 (ddd, J=11.4, 4.4, 2.5Hz, 1H). 13 C NMR (75MHz, DMSO-d6) δ171.9, 169.7, 165.8, 162.3, 153.6, 149.6, 147.7, 139.8 , 137.3, 136.9 (d, J = 7.4Hz), 135.5, 134.8 (d, J = 3.2Hz), 132.9, 131.6, 125.5, 1 21.4, 120.6 (d, J=29.0Hz), 120.6 (d, J=255.3Hz), 100.8, 50.6, 49.4, 47.1, 44. 1, 43.0, 42.5, 42.3, 38.9, 35.4, 30.2, 30.0, 23.1, 22.4. HR-MS (ESI) m / z: calcd for C 35 H 40 F4N8O5[M+H] + 729.3131found 729.3155.

[0133] Example 10: 5-((5-((((3R,5R,7R)-adamantan-1-yl)methyl)amino)-5-carbonylpentyl)amino)-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide

[0134]

[0135] The synthesis method of compound JBP-9 in Example 9 was used, except that the hydrophobic fragment bicyclo[2.2.1]hept-5-en-2-ylmethanamine (J1) was replaced with ((3R,5R,7R)-adamantan-1-yl)methanamine. The product JBP-10 was a white solid. 1 HNMR (300MHz, DMSO-d6) δ10.62 (s, 1H), 10.13 (s, 1H), 9.22 (s, 1H), 8.69 (dd, J=8.8, 2.5Hz, 1H), 8.30 (t, J=2.2Hz , 1H), 7.63 (t, J=6.3Hz, 1H), 7.49 (d, J=8.4Hz, 2H), 7.40 (s, 1H), 7.35 (d, J=8.2Hz, 2H), 7.11 (t, J=6.4Hz, 1H), 4.6 5 (p, J=6.5Hz, 1H), 3.76 (s, 2H), 3.19 (q, J=6.6Hz, 2H), 2.76 (d, J=6.2Hz, 2H), 2.15 (t, J=6.8Hz, 2H), 1.99-1.83 (m , 4H), 1.66 (s, 1H), 1.62 (s, 3H), 1.57 (s, 3H), 1.54-1.51 (m, 2H), 1.48 (s, 3H), 1.45 (s, 3H), 1.40 (d, J=2.8Hz, 6H). 13 C NMR (75MHz, DMSO-d6) δ172.4, 169.7, 165.8, 162.2, 153.6, 152.7, 147.6, 139.8, 135.5, 134.8, 132.8 (d, J=15.2Hz), 131.6, 125.6, 121.4, 120 .6 (d, J=29.1Hz), 120.6 (d, J=257.0Hz), 100.7, 50.6, 50.5, 47.1, 42.5, 40.3, 37.0, 35.4, 34.0, 30.0, 28.1, 23.1, 22.4. HR-MS (ESI) m / z: calcd for C 38 H 46 F4N8O5[M+H] + 771.3600found 771.3623.

[0136] Example 11: 5-((4-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-4-carbonylbutyl)amino)-1-cyclopentyl-N 3-(2-Fluoro-5-(2-(4-(trifluoromethyl)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide

[0137]

[0138] (1) Prepare intermediate T4. The synthesis method is similar to that of (1) in Example 1, except that cyclopropylhydrazine hydrochloride is replaced by cyclopentylhydrazine hydrochloride.

[0139]

[0140] Reagents and conditions: (a) EtONa, EtOH, rt, 1 h, yield 60%; (b) 1) POCl3, 130°C, 1 h, yield 97%; 2) MeOH, 0°C, 1 h, yield 88%; (c) Et3N, MeOH, Ar, 0°C, 1 h, yield 61%.

[0141] (2) Prepare intermediate T5. The synthesis method refers to (2) in Example 1, except that 2-(4-(trifluoromethoxy)phenyl)acetic acid (2-2) is replaced by 2-(4-(trifluoromethyl)phenyl)acetic acid (7-2).

[0142]

[0143] Reagents and conditions: (a) T4P, Et3N, rt, 10 h, yield 91%; (b) 4N HCl in dioxane, rt, 1 h, yield 96%.

[0144] (3) The target compound JBP-11 was prepared by the same synthesis method as that described in Example 1 (3), except that the linker fragment 3-1a was replaced by 3-1b, the intermediate T1 was replaced by the intermediate T4, and the intermediate T2 was replaced by the intermediate T5.

[0145] The product JBP-11 was a white solid. 1H NMR (500MHz, DMSO-d6) δ10.62 (s, 1H), 10.02 (s, 1H), 9.18 (s, 1H), 8.68 (dd, J=8.7, 2.6Hz, 1H), 8.27 (t, J=2.1Hz, 1H), 7.78 (t, J=5.8Hz, 1H), 7. 70 (d, J=8.1Hz, 2H), 7.57 (d, J=8.1Hz, 2H), 7.42-7.34 (m, 1H), 7.03 (t, J=6.6Hz, 1H), 6.13 (dd, J=5.8, 3.0Hz, 1H), 5.95-5.92 (m, 1H), 4.91-4.6 9(m, 1H), 3.82(s, 2H), 3.17(q, J=7.0Hz, 2H), 2.85-2.78(m, 1H), 2.76- 2.72 (m, 2H), 2.67-2.60 (m, 1H), 2.16 (td, J=7.4, 4.6Hz, 4H), 2.07-1.97 (m, 4H), 1.88 (p, J=6.4Hz, 2H), 1.75-1.71 (m, 2H), 1.66 (tt, J=6.9, 2.5Hz, 2H), 1.29 (dt, J=4.6, 2.9Hz, 2H), 0.44 (ddd, J=11.4, 4.3, 2.6Hz, 1H). 13 C NMR (150MHz, DMSO-d6) δ171.5, 169.3, 165.7, 162.2 (d, J=6.5Hz), 154.1, 152.0 (d, J=50.6Hz) , 145.4, 140.7, 139.6, 137.3, 137.3-136.1(m), 133.8-131.5(m), 132.9, 130.6, 125.6(d, J=3 .7Hz), 126.5-124.6 (m), 123.9, 120.6 (d, J=28.5Hz), 100.9, 59.4, 49.3, 47.0, 44.1, 43.0 (d, J=6.5Hz), 42.3, 40.8, 38.8, 32.8, 30.2, 26.7, 24.8, 24.2, 22.5, 14.4. HR-MS (ESI) m / z: calcd for C 36 H 40 F4N8O4[M+H] + 725.3181found 725.3211.

[0146] Example 12: 5-((5-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-5-carbonylpentyl)amino)-N 3-(3-(2-(4-chlorophenyl)acetylamino)phenyl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide

[0147]

[0148] (1) Prepare intermediate T3. The synthesis method is similar to that of (1) in Example 9.

[0149] (2) Intermediate T6 was prepared by the same synthesis method as that described in Example 1 (2), except that tert-butyl-(5-amino-2-fluoropyridin-3-yl)carbamate (2-1) was replaced by tert-butyl-(3-aminophenyl)carbamate (8-1), and 2-(4-(trifluoromethoxy)phenyl)acetic acid (2-2) was replaced by 2-(4-chlorophenyl)acetic acid (8-2).

[0150]

[0151] Reagents and conditions: (a) T4P, Et3N, rt, 10 h, yield 93%; (b) 4N HCl in dioxane, rt, 1 h, yield 95%.

[0152] (3) The target compound JBP-12 was prepared by referring to (3) in Example 9, except that the intermediate T2 was replaced by the intermediate T6.

[0153] The product JBP-12 was a white solid. 1H NMR (400MHz, DMSO-d6) δ10.26 (s, 1H), 10.08 (s, 1H), 9.34 (s, 1H), 8.08 (t, J=2.0H z, 1H), 7.77 (t, J=5.7Hz, 1H), 7.43 (dt, J=8.2, 1.4Hz, 1H), 7.41-7.36 (m, 4H), 7.34 (d, J=1.8Hz, 1H), 7.33-7.25 (m, 2H), 7.07 (t, J=6.5Hz, 1H), 6.12 (dd, J=5.8, 3.0H z, 1H), 5.93 (dd, J=5.8, 2.9Hz, 1H), 4.60 (p, J=6.5Hz, 1H), 3.65 (s, 2H), 3.15 (q, J= 6.8Hz, 2H), 2.73 (d, J=6.0Hz, 2H), 2.63 (ddd, J=13.0, 8.9, 5.7Hz, 1H), 2.15 (tt, J =7.4, 4.0Hz, 1H), 2.07 (t, J = 7.0Hz, 2H), 1.72 (ddd, J = 11.4, 9.1, 3.8Hz, 1H), 1.63- 1.54 (m, 2H), 1.51 (dd, J=14.1, 6.9Hz, 2H), 1.46 (d, J=6.5Hz, 6H), 1.30-1.27 (m, 1 H), 1.24 (d, J=10.6Hz, 1H), 1.19-1.17 (m, 1H), 0.43 (ddd, J=11.5, 4.5, 2.5Hz, 1H). 13 C NMR (151MHz, DMSO-d6) δ171.97169.25, 166.10, 162.28, 153.70, 140.85, 1 39.84, 138.43, 137.31, 135.44, 132.94, 131.77, 131.48, 129.28, 128.68, 116.74, 115.75, 112.54, 100.57, 50.56, 49.39, 47.24, 44.16, 43.06, 42.9 5, 42.36, 38.92, 35.46, 30.24, 30.04, 23.12, 22.39. HR-MS (ESI) m / z: calcd for C 35 H 42 ClN7O4[M+Na] + 682.2879found 682.2881.

[0154] Example 13: 5-((5-((2-(2,6-dicarbonylpiperidin-3-yl)-1,3-dicarbonylisoindolin-4-yl)amino)pentyl)amino)-N 3-(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide

[0155]

[0156] (1) Prepare intermediate T2. The synthesis method refers to (2) in Example 1.

[0157] (2) Prepare intermediate T3. The synthesis method refers to (1) in Example 9.

[0158] (3) Preparation of target compound JBP-13: 5-((5-((2-(2,6-dicarbonylpiperidin-3-yl)-1,3-dicarbonylisoindole-4-yl)amino)pentyl)amino)-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide.

[0159]

[0160] Reagents and conditions: (a) Cs2CO3, CH3CN, 85℃, 10h, yield 88%; (b) 20% NaOH aqueous, THF, 60℃, 2h, yield 92%; (c) HATU, DIPEA, DMF, rt, 10h, yield 51%; (d) TFA, DCM, rt, 2h, yield 94%; (e) DIPEA, DMSO, 90℃, 2h, yield 80%; (f) acetaldoxime, Pd(OAc)2, triphenylphosphine, EtOH, H2O, Ar, 105℃, 12h, yield 67%.

[0161] Intermediate T3 (500 mg, 2.25 mmol) was dissolved in anhydrous acetonitrile (10 mL), and cesium carbonate (1.47 g, 4.50 mmol) was added. The mixture was heated to 85°C and stirred for 10 min. 5-(tert-Butyloxycarbonylamino)pentyl bromide (9-1, 719 mg, 2.70 mmol) was then added to the reaction system, and the reaction was continued at 85°C for 10 h. Upon completion of the reaction, the solvent was removed by concentration, the mixture was diluted with water, and extracted with ethyl acetate (40 mL × 3). The organic phases were combined and washed with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA, 2:1, v / v) to afford Intermediate 9-2 (836 mg, 1.98 mmol).

[0162] Intermediate 9-2 (800 mg, 1.90 mmol) was dissolved in tetrahydrofuran (10 mL), and 10 mL of 20% aqueous NaOH solution was added. The mixture was heated to 60°C and stirred for 2 h. After the reaction was completed, 40 mL of water was added for dilution, and the mixture was extracted with 40 mL of ethyl acetate. The aqueous phase was collected and adjusted to a weak acidity with 2N hydrochloric acid. The mixture was extracted with ethyl acetate (40 mL × 3) and washed with water (40 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain Intermediate 9-3 (662 mg, 1.74 mmol).

[0163] Intermediate 9-3 (200 mg, 527.06 μmol), 2-(7-azabenzotriazole)-NNN′, N′-tetramethyluronium hexafluorophosphate (251 mg, 658.83 μmol), and N,N-diisopropylethylamine (230 μL, 1.32 mmol) were dissolved in 3 mL of anhydrous N,N-dimethylformamide (DMF). The reaction was stirred at room temperature for 20 min, and N-(5-amino-6-fluoropyridin-3-yl)-2-(4-(trifluoromethoxy)phenyl)acetamide (T2, 145 mg, 439.22 μmol) was added and the reaction was carried out at room temperature for 10 h. After the reaction is completed, an appropriate amount of water is added to dilute, and the mixture is extracted with ethyl acetate (50 mL×3). The organic phases are combined and washed with saturated brine. The organic phase is added with anhydrous sodium sulfate and dried. After concentration, column chromatography (DCM / MeOH, 100:1, v / v) is performed to obtain intermediate 9-4 (155 mg, 224.41 μmol).

[0164] Intermediate 9-4 (200 mg, 289.56 μmol) was dissolved in dichloromethane (8 mL), and 4 mL of trifluoroacetic acid was added. The reaction was allowed to react at room temperature for 2 h. After completion of the reaction, the solvent was removed under reduced pressure, and the mixture was diluted with ethyl acetate. 2N NaOH was added to adjust the system to a weak alkaline state. The mixture was extracted with ethyl acetate (20 mL × 3), washed with water (20 mL × 3), and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain Intermediate 9-5 (161 mg, 272.61 μmol).

[0165] Intermediate 9-5 (150 mg, 253.99 μmol) was dissolved in 4 mL of dimethyl sulfoxide (DMSO), and N,N-diisopropylethylamine (66 μL, 380.98 μmol) was added. The mixture was stirred at room temperature for 10 min, and 2-(2,6-dicarbonylpiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (J4, 70 mg, 253.99 μmol) was added. The temperature was raised to 90°C and the reaction was allowed to react for 2 h. After the reaction, the reaction solution was cooled to room temperature, diluted with an appropriate amount of water, extracted with ethyl acetate (20 mL × 3), washed with water (20 mL × 3), and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM / MeOH, 120:1, v / v) to obtain Intermediate 9-6 (172 mg, 203.12 μmol).

[0166] Intermediate 9-6 (160 mg, 188.95 μmol), acetaldehyde oxime (45 mg, 761.83 μmol), palladium acetate (7 mg, 31.18 μmol), and triphenylphosphine (8 mg, 30.50 μmol) were dissolved in 15 mL of a 4 / 1 ethanol / water (v / v) mixture under argon atmosphere at 105°C for 10 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (DCM / MeOH, 80:1, v / v) to afford the target compound JBP-13 (110 mg, 127.19 μmol) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ11.10 (s, 1H), 10.60 (s, 1H), 10.13 (s, 1H), 9.17 (s, 1H), 8.66 (d, J = 7.7Hz, 1H), 8.27 (t, J = 2.2Hz, 1H), 7.57 (d d, J=8.6, 7.1Hz, 1H), 7.49-7.44 (m, 2H), 7.40 (s, 1H), 7.36-7.31 (m, 2H), 7.11-6.99 (m, 3H), 6.56 (t, J=6.0Hz, 1H), 5.05 (dd, J=12.9, 5.4Hz, 1H), 4.63 (p, J=6.4Hz, 1H), 3.74 (s, 2H), 3.28 (t, J=6.8Hz, 2H), 3.17 (q, J=6.6Hz, 2H), 2.93-2.82 (m, 1H), 2.58 (d, J=17.7Hz, 1 H), 2.03 (dd, J=8.9, 3.9Hz, 1H), 1.58 (p, J=7.4Hz, 4H), 1.44 (d, J=6.5Hz, 6H), 1.37-1.32 (m, 1H), 1.29 (s, 1H), 1.25 (d, J=3.7Hz, 1H).13 C NMR (151MHz, DMSO-d6) δ173.24, 170.54, 169.69, 169.39, 167.77, 165.84, 162.28, 153.72, 147.69, 146.89, 139.86, 136.74, 135.46, 134.76 (d, J=3.5Hz), 132.88, 132.78 (d, J=1.8Hz) , 132.69, 131.59, 125.62, 121.38, 117.65, 110.85, 109.54, 100.81, 50.63, 49.03, 47.27, 42 .53, 42.27, 31.62, 31.45, 30.31, 30.10, 28.80, 23.97, 22.63, 22.43. HR-MS (ESI) m / z: calcd for C 40 H 40 F4N 10 O8[M+H] + 865.3039found865.3062.

[0167] Example 14: 5-((4-(2-(9H-fluoren-9-yl)acetylamino)butyl)amino)-N 3 -(2-Fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide

[0168]

[0169] (1) Prepare intermediate T2. The synthesis method refers to (2) in Example 1.

[0170] (2) Prepare intermediate T3. The synthesis method refers to (1) in Example 9.

[0171] (3) Preparation of target compound JBP-14: 5-((4-(2-(9H-fluoren-9-yl)acetylamino)butyl)amino)-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide.

[0172]

[0173] Reagents and conditions: (a) Cs2CO3, CH3CN, 85℃, 10h, yield 82%; (b) TFA, DCM, rt, 2h, yield 93%; (c) EDCI, HOBt, THF, rt, 12h, yield 60%; (d) 20% NaOH aqueous, THF, 60℃, 2h, yield 91%; (e) HATU, DIPEA, DMF, rt, 10h, yield 55%; (f) acetaldoxime, Pd(OAc)2, triphenylphosphine, EtOH, H2O, Ar, 105℃, 12h, yield 69%.

[0174] Intermediate T3 (500 mg, 2.25 mmol) was dissolved in anhydrous acetonitrile (10 mL), and cesium carbonate (1.47 g, 4.50 mmol) was added. The mixture was heated to 85°C and stirred for 10 min. 4-(tert-Butoxycarbonylamino)butanebromide (10-1, 624 mg, 2.47 mmol) was then added to the reaction system, and the reaction was continued at 85°C for 10 h. Upon completion of the reaction, the solvent was removed by concentration, the mixture was diluted with water, and extracted with ethyl acetate (40 mL × 3). The organic phases were combined and washed with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA, 2:1, v / v) to afford Intermediate 10-2 (728 mg, 1.85 mmol).

[0175] Intermediate 10-2 (700 mg, 1.78 mol) was dissolved in dichloromethane (10 mL), and 5 mL of trifluoroacetic acid was added. The mixture was allowed to react at room temperature for 2 h. After completion of the reaction, the solvent was removed under reduced pressure, and the mixture was diluted with ethyl acetate. 2N NaOH was added to adjust the system to a weak alkaline state. The mixture was extracted with ethyl acetate (40 mL × 3), washed with water (440 mL × 3), and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain Intermediate 10-3 (486 mg, 1.66 mmol).

[0176] Intermediate 10-3 (450 mg, 1.53 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL). Fluoreneacetic acid (J5, 413 mg, 1.84 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (441 mg, 2.30 mmol), and 1-hydroxybenzotriazole (415 mg, 3.07 mmol) were added sequentially. The mixture was allowed to react at room temperature for 12 h. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate (40 mL × 3), and washed with water (40 mL × 3). The organic phases were combined and washed with saturated brine. The organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM / MeOH, 150:1, v / v) to obtain Intermediate 10-4 (460 mg, 920.71 μmol).

[0177] Intermediate 10-4 (450 mg, 900.69 μmol) was dissolved in tetrahydrofuran (10 mL), and 10 mL of 20% aqueous NaOH solution was added. The mixture was heated to 60°C and stirred for 2 h. After completion of the reaction, 40 mL of water was added for dilution, and the mixture was extracted with 40 mL of ethyl acetate. The aqueous phase was collected and adjusted to a weak acidity with 2N hydrochloric acid. The mixture was extracted with ethyl acetate (40 mL × 3) and washed with water (40 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain Intermediate 10-5 (387 mg, 820.68 μmol).

[0178] Intermediate 10-5 (300 mg, 636.18 μmol), 2-(7-azabenzotriazole)-N,NN′,N′-tetramethyluronium hexafluorophosphate (302 mg, 795.23 μmol), and N,N-diisopropylethylamine (277 μL, 1.59 mmol) were dissolved in 3 mL of anhydrous N,N-dimethylformamide (DMF). The reaction was stirred at room temperature for 20 min, and N-(5-amino-6-fluoropyridin-3-yl)-2-(4-(trifluoromethoxy)phenyl)acetamide (T2, 175 mg, 530.15 μmol) was added and the reaction was carried out at room temperature for 10 h. After the reaction is completed, an appropriate amount of water is added to dilute, and the mixture is extracted with ethyl acetate (50 mL×3). The organic phases are combined and washed with saturated brine. The organic phase is added with anhydrous sodium sulfate and dried. After concentration, column chromatography (DCM / MeOH, 120:1, v / v) is performed to obtain intermediate 10-6 (230 mg, 293.82 μmol).

[0179] Intermediate 10-6 (100 mg, 127.75 μmol), acetaldehyde oxime (30 mg, 510.99 μmol), palladium acetate (4 mg, 19.16 μmol), and triphenylphosphine (5 mg, 19.16 μmol) were dissolved in 15 mL of a 4 / 1 ethanol / water (v / v) mixture under argon atmosphere at 105°C for 10 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (DCM / MeOH, 90:1, v / v) to afford the target compound JBP-14 (55 mg, 68.68 μmol) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ10.66 (s, 1H), 10.12 (s, 1H), 9.21 (s, 1H), 8.66 (dd, J=8.9, 2.5Hz, 1H), 8.28 (t, J=2.2Hz, 1H), 7.97 (t, J=5.4Hz, 1H), 7.73 (s, 1H), 7.72 (t, J=1.0Hz, 1H), 7.51 (t, J=0.9Hz, 1H), 7.49 (d, J=3.0Hz, 2H), 7.46 (d, J=1.9Hz, 1H), 7.41 (s, 1H), 7.33 (dd, J=7. 5, 1.3Hz, 4H), 7.27 (ddd, J=7.5, 6.2, 1.2Hz, 2H), 7.06 (t, J=6.3Hz, 1H), 6.42 (d, J=9.4Hz, 1H), 4.63 (p, J=6.5Hz, 1H), 3.75 (s, 2H), 3.13 ( d, J=6.1Hz, 2H), 3.07 (d, J=5.6Hz, 2H), 2.67 (s, 2H), 1.45 (d, J=6.5Hz, 6H), 1.43-1.41 (m, 3H), 1.41-1.35 (m, 2H), 1.23 (d, J=4.2Hz, 2H). 13C NMR (101MHz, DMSO-d6) δ170.47, 169.29, 165.42, 161.87, 153.29, 149.64, 148.87 , 147.23, 147.22, 147.14, 139.39, 138.69, 135.06, 134.35 (d, J=3.7Hz), 131.17, 1 28.61, 127.66, 125.29 (d, J=2.2Hz), 123.97, 120.99, 119.98, 100.21, 79.57, 50.2 5, 46.62, 44.28, 42.08, 37.94, 27.36, 26.37, 26.21, 22.02. HR-MS (ESI) m / z: calcd forC 41 H 40 F4N8O8[M+H] + 801.3131found 801.3076.

[0180] Example 15: Evaluation of the anti-proliferative activity of compounds on Ba / F3-LMNA-NTRK1-G667C cells

[0181] 1. Experimental materials and instruments

[0182] Table 1 Experimental reagents and equipment

[0183]

[0184] 2. Experimental steps

[0185] (1) Prepare different concentrations of compounds: Dilute the compound to 20 times the final test concentration in growth medium.

[0186] (2) Cell inoculation and drug administration: After centrifugation, the cells were resuspended in growth medium, counted using a cell counter, and the cell suspension was prepared to the desired density. 95 μL of the cell suspension was added to each well of a 96-well plate. 5 μL of drug-containing medium was then added to the corresponding well. A positive control group, a negative control group, and a vehicle control group were also established. Finally, the 96-well plate was placed in a 37°C, 5% CO2 incubator and cultured for 72 h.

[0187] (3) Absorbance measurement: Add 10 μL of CCK8 solution to each well of a 96-well plate in the dark. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 1 h. Remove the 96-well plate and measure the absorbance at 450 nm using a microplate reader.

[0188] (4) Calculate the inhibition rate and drug IC of each group 50IC was calculated using SPSS (Staffstical Package for the Social Science) software using the probability unit weighted regression method (Bliss method). 50 .

[0189] Inhibition Rate(Inh%)=100%-[(RLU compound -RLU blank ) / (RLU control -RLU blank )]×100%

[0190] 3. Experimental results

[0191] The inhibitory activity (IC 50 ) are shown in the table below.

[0192] Table 2 Inhibitory activity of compounds on Ba / F3-LMNA-NTRK1-G667C cells

[0193]

[0194]

[0195] As shown in Table 2, the carboxamide pyrazole TRK protein degrader small molecules designed in the present invention have excellent anti-proliferative activity against mutant cells, among which the cell inhibition IC values ​​of compounds JBP-2 to JBP-5, JBP-7, JBP-9 to JBP-11, and JBP-14 are 50 The value was lower than 500 nM.

[0196] Example 16: Preferred compounds for TRKA G667C Evaluation of protein degradation activity

[0197] 1. Experimental materials and instruments

[0198] Table 3 Experimental reagents and equipment

[0199]

[0200] 2. Experimental steps (Western Blot experiment)

[0201] (1) Total cell protein extraction and quantification: Wash the cultured cells twice with pre-chilled PBS, then add pre-chilled cell lysis buffer (RIPA:RMSF = 100:1). Transfer to a centrifuge tube and place on ice for 30 min. Then centrifuge at 14,000 rpm for 30 min at 4°C. Take 2 μL of the supernatant and quantify it using a BCA kit. Determine the loading volume based on the measured concentration. Take an appropriate amount of supernatant and add 5× Loading Buffer, mix well, and heat in a 95°C water bath for 10 min.

[0202] (2) Gel Preparation and Electrophoresis: Prepare the separating gel and stacking gel according to the instructions. Pour them into the glass plate and remove the comb after solidification. Run the sample at a constant voltage of 200V for 40 minutes.

[0203] (3) Transfer and blocking: After electrophoresis, the samples on the gel were transferred to a PVDF membrane under a constant current (300 mA, 80 min). The membrane was then blocked in 5% skim milk TBST buffer for 2 hours at room temperature.

[0204] (4) Antibody incubation: After blocking, the membrane was incubated with the diluted antibody at a ratio of 16 h at 4°C. The PVDF membrane was then washed with TBST on a shaker (5 × 5 min). After washing, the membrane was incubated with the diluted secondary antibody on a constant temperature shaker (37°C) for 1 h, and the PVDF membrane was then washed with TBST on a shaker (5 × 5 min).

[0205] (5) Development and exposure: Add developer solution onto the PVDF membrane and expose it in a chemiluminescence imager. Quantification is performed using ImageJ software.

[0206] 3. Experimental results

[0207] Table 4 Degradation activity of preferred compounds in Ba / F3-LMNA-NTRK1-G667C

[0208]

[0209] a 24 hours of administration, 6 independent test concentrations, TRKA G667C Concentration required for 50% protein reduction;

[0210] b TRKA was achieved at a concentration of 1 μM G667C Maximum protein degradation rate.

[0211] Depend on Figure 1 As shown in Table 4, the preferred compounds can reduce TRKA at a concentration of 0.5 μM. G667CThe DC of compounds JBP-9 and JBP-11 was higher than 50%, showing excellent protein degradation activity. 50 The values ​​were lower than 0.1 μM, and compounds JBP-3, JBP-9, JBP-10 and JBP-11 were able to achieve protein degradation rates of more than 85% at a dosage concentration of 1 μM.

Claims

1. A carboxamide pyrazole compound, characterized in that: Having the structure of formula (I), also including stereoisomers, deuterated compounds, pharmaceutically acceptable salts or mixtures thereof: in: X is selected from the following structural fragments: Y is selected from N or CH; R 1 A structural fragment selected from the following: R 2 Independently selected from unsubstituted or substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-4 Alkoxy-substituted C 1-4 Alkyl, C 3-10 Cycloalkyl, C 3-10 Aromatic ring group, 3 to 10 membered heterocyclic group containing 0 to 5 N, S, O ring heteroatoms; or C 3-10 Heterocyclic substituted C 1-8 Alkyl, and C 1-8 Alkyl is replaced by 0 to 5 halogens, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 14 Alkoxy substituted; wherein the substituent is selected from 1 to 5 -OH, =O, -OMe, -CH3, -CF3, -C1, -F, -CO2Et, cyclohexyl, piperazinyl; R 3 is selected from hydrogen or halogen; R 4 independently selected from halogen, -CN, C 1-4 Alkyl, C 3-6 Cycloalkyl, 0 to 3 halogen-substituted C 1-3 Alkyl, -OH, C 1-3 Alkoxy, 0 to 3 halogen-substituted C 1-3 alkoxy; m is selected from integers of 1 to 20, and n is selected from 0 or 1.

2. The carboxamide pyrazole compound according to claim 1, characterized in that In the structure, R 2 Selected from unsubstituted or substituted methyl, ethyl, isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetane, piperidinyl, piperazinyl; wherein the substituent is selected from 1 to 5 -OH, =O, -OMe, -CH3, -CF3, -C1, -F, -CO2Et, cyclohexyl, piperazinyl.

3. The carboxamide pyrazole compound according to claim 1, characterized in that In the structure, R3 is selected from hydrogen, fluorine or chlorine.

4. The carboxamide pyrazole compound according to claim 1, characterized in that In the structure, R 4 Selected from fluorine, chlorine, methyl, ethyl, isopropyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 0 to 3 fluorine or chlorine substituted C 1-3 Alkyl, methoxy, ethoxy, 0 to 3 fluorine or chlorine substituted C 1-3 Alkoxy.

5. The carboxamide pyrazole compound according to claim 1, characterized in that m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

6. The carboxamide pyrazole compound according to claim 1, characterized in that Any one compound selected from the following: 5-((2-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-2-carbonylethyl)amino)-1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide; 5-((4-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-4-carbonylbutyl)amino)-1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide; 5-((6-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-6-carbonylhexyl)amino)-1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide; 5-((8-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-8-carbonyloctyl)amino)-1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide; 5-((1-(bicyclo[2.2.1]hept-5-en-2-yl)-1-carbonyl-5,8,11-trioxa-2-azatridecan-13-yl)amino)-1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide; 5-((1-(bicyclo[2.2.1]hept-5-en-2-yl)-1,14-dicarbonyl-5,8,11-trioxa-2,15-diazaoctadec-18-yl)amino)-1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide; 1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-5-((5-carbonyl-5-(((1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)amino)pentyl)amino)-1H-pyrazole-3,4-dicarboxamide; 5-(((1-(bicyclo[2.2.1]hept-5-ene-2-carbonyl)azetidin-3-yl)methyl)amino)-1-cyclopropyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide; 5-((5-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-5-carbonylpentyl)amino)-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide; 5-((5-((((3R,5R,7R)-adamantan-1-yl)methyl)amino)-5-carbonylpentyl)amino)-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide; 5-((4-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-4-carbonylbutyl)amino)-1-cyclopentyl-N 3 -(2-fluoro-5-(2-(4-(trifluoromethyl)phenyl)acetylamino)pyridin-3-yl)-1H-pyrazole-3,4-dicarboxamide; 5-((5-((bicyclo[2.2.1]hept-5-en-2-ylmethyl)amino)-5-carbonylpentyl)amino)-N 3 -(3-(2-(4-chlorophenyl)acetylamino)phenyl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide; 5-((5-((2-(2,6-dicarbonylpiperidin-3-yl)-1,3-dicarbonylisoindolin-4-yl)amino)pentyl)amino)-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide; 5-((4-(2-(9H-fluoren-9-yl)acetylamino)butyl)amino)-N 3 -(2-fluoro-5-(2-(4-(trifluoromethoxy)phenyl)acetylamino)pyridin-3-yl)-1-isopropyl-1H-pyrazole-3,4-dicarboxamide.

7. The carboxamide pyrazole compound according to claim 1, characterized in that The pharmaceutically acceptable salt is a salt formed by the compound and any one of the following acids: Hydrochloric acid, hydrobromic acid, sulfuric acid, carbonic acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, acidic amino acid or ferulic acid.

8. A pharmaceutical composition, characterized in that The invention comprises the carboxamide pyrazole compound according to claim 1 and a pharmaceutically acceptable carrier.

9. Use of the carboxamide pyrazole compound according to claim 1 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing and / or treating diseases mediated by tropomyosin receptor kinase.

10. The use according to claim 9, characterized in that The medicine is a medicine for preventing and / or treating pain or cancer.

Images