Deuterated derivatives of ATX inhibitors and their applications

Abstract

Disclosed are a compound of formula (I), its stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals, or pharmaceutical compositions containing them, and use thereof as ATX inhibitors in the preparation of drugs for treating related diseases. The groups in formula (I) are as defined in the specification.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a deuterated derivative of an ATX inhibitor, its stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals, and use thereof in preparing medicines for treating related diseases. Background Art

[0002] Autotaxin (ATX) is an extracellular secretory enzyme (also known as ENPP2). Its main physiological function is to hydrolyze lysophosphatidylcholine (LPC) to generate biologically active lysophosphatidic acid (LPA) and choline. ATX hydrolyzes LPC and is the main source of LPA in the blood. LPA acts on LPA receptors (at least 6 types of LPA 1-6 ) and produce a series of physiological activities, including cell proliferation, survival, movement, etc. The ATX-LPA signaling pathway is involved in many pathological processes, including angiogenesis, autoimmune diseases, inflammation, fibrosis, neurodegeneration and pain. Among them, fibrosis and tumors are the most widely studied, especially idiopathic pulmonary fibrosis (IPF). In 2014, the FDA approved two new drugs, Pirfenidone and the triple angiokinase inhibitor Nintedanib, for the treatment of IPF. Their main function is to prevent the further development and deterioration of IPF. They have no obvious effect on the treatment of IPF itself, so people have been committed to finding more effective drugs for the treatment of IPF. The ATX inhibitor drug GLPG-1690 has made relatively advanced clinical progress (clinical phase III) and is used to treat IPF. Its phase II clinical trial has shown good efficacy. Summary of the invention

[0003] The purpose of the present invention is to provide a deuterated ATX inhibitor, which has the advantages of novel structure, good efficacy, high bioavailability, small side effects, rapid onset and long-term effect.

[0004] The present invention relates to a compound represented by formula (I), its stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals.

[0005]

[0006] Among them, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 are each independently selected from hydrogen or deuterium;

[0007] The condition is that R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 At least one group is selected from deuterium and the compound of formula (I) is not the following structure:

[0008]

[0009] In certain embodiments, R 22 Selected from H;

[0010] In certain embodiments, R1 is selected from deuterium;

[0011] In certain embodiments, R2, R3 are selected from deuterium;

[0012] In certain embodiments, R6, R7 are selected from deuterium;

[0013] In certain embodiments, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 Selected from deuterium;

[0014] In certain embodiments, R 16 Selected from deuterium;

[0015] In certain embodiments, R 17 Selected from deuterium;

[0016] In certain embodiments, R 18 , R 19 , R 20 , R 21 Selected from deuterium;

[0017] In certain embodiments, R 23 , R 24 Selected from deuterium;

[0018] In certain embodiments, R 25 , R 26 , R 27 At least one of is selected from deuterium;

[0019] In certain embodiments, R 25 , R 26 , R 27 Selected from deuterium;

[0020] In certain embodiments, R 28 , R 29 , R 30 Selected from deuterium;

[0021] In certain embodiments, R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 Selected from deuterium;

[0022] In certain embodiments, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 25 , R 26 , R 27 Selected from deuterium;

[0023] In certain embodiments, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 23 , R24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 Selected from deuterium;

[0024] In certain embodiments, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 Selected from deuterium;

[0025] In certain embodiments, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 18 , R 19 , R 20 , R 21 Selected from deuterium;

[0026] In certain embodiments, R 23 , R 24 , R 28 , R 29 , R 30 , R 18 , R 19 , R 20 , R 21 Selected from deuterium;

[0027] In certain embodiments, R 25 , R 26 , R 27 , R 18 , R 19 , R 20 , R 21 Selected from deuterium;

[0028] In certain embodiments, R1, R 25 , R 26 , R 27 Selected from deuterium;

[0029] In certain embodiments, R1, R 23 , R 24 , R 28 , R 29 , R 30 Selected from deuterium;

[0030] In certain embodiments, R6, R7, R 25 , R 26 , R 27 Selected from deuterium;

[0031] In certain embodiments, R6, R7, R 23 , R 24 , R 28 , R 29 , R 30 Selected from deuterium;

[0032] In certain embodiments, R2, R3, R 25 , R 26 , R 27 Selected from deuterium;

[0033] In certain embodiments, R2, R3, R 23 , R 24 , R 28 , R 29 , R 30 Selected from deuterium.

[0034] The compound of formula (I) of the present invention, its stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal, wherein the compound is selected from one of the following structures:

[0035]

[0036]

[0037] The present invention further provides a more specific crystalline form I of the compound of formula (A), which has high purity, low hygroscopicity, good solubility, resistance to high temperature, high humidity and strong light, high stability, suitable particle size, easy filtration, good fluidity, and is suitable for the preparation of pharmaceutical dosage forms.

[0038]

[0039] The crystalline form I of the compound of formula (A) has an X-ray powder diffraction pattern using Cu-Kα radiation, with characteristic diffraction peaks at the following 2θ positions: 9.35±0.2°, 10.32±0.2°, 12.08±0.2°, and 15.10±0.2°.

[0040] Furthermore, the X-ray powder diffraction pattern of the crystalline form I of the compound of formula (A) also has characteristic diffraction peaks at the following 2θ positions: 17.29±0.2°, 18.26±0.2°, and 20.58±0.2°.

[0041] Furthermore, the X-ray powder diffraction pattern of the crystalline form I of the compound of formula (A) also has characteristic diffraction peaks at the following 2θ positions: 6.92±0.2°, 7.23±0.2°, 13.46±0.2°, 19.47±0.2°, and 24.85±0.2°.

[0042] Further, the crystalline form I of the compound of formula (A) has an X-ray powder diffraction pattern substantially as follows Figure 1 shown.

[0043] Furthermore, the X-ray powder diffraction peaks of the crystalline form I of the compound of formula (A) are substantially as shown in Table 1.

[0044] Further, the TGA curve of the crystalline form I of the compound of formula (A) is substantially as follows Figure 2 As shown, its DSC diagram is basically as Figure 3 shown.

[0045] Furthermore, the crystal form I of the compound of formula (A) of the present invention has a crystal particle size of less than 100 μm; in some embodiments, the crystal particle size is less than 90 μm; in some embodiments, the crystal particle size is less than 80 μm; in some embodiments, the crystal particle size is less than 70 μm; in some embodiments, the crystal particle size is less than 60 μm; in some embodiments, the crystal particle size is less than 50 μm.

[0046] The present invention also provides a method for preparing the crystalline form I of the compound of formula (A), which comprises: adding the crude compound (A) to an alcohol solvent, heating and stirring, cooling to room temperature after dissolution, crystallizing, filtering, and obtaining the crystalline form I.

[0047] In some embodiments, in the above preparation method, the alcohol solvent is selected from methanol, ethanol, propanol, isopropanol or a combination thereof.

[0048] In some embodiments, in the above preparation method, the ratio of compound (A) to alcohol solvent is 1:5-8 g / mL. In some embodiments, the ratio of compound (A) to alcohol solvent is 1:5-6 g / mL.

[0049] In some embodiments, in the above preparation method, the temperature is raised to 60-90° C. In some embodiments, the temperature is raised to 70-80° C. In some embodiments, the temperature is raised to 75° C.

[0050] The crystalline form I of compound (A) of the present invention is present at about 5% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 10% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 15% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 20% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 25% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 30% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 35% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 40% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 45% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 50% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about 55% to about 100% by weight of the raw material drug; in certain embodiments, it is present at about In some embodiments, the bulk drug is present at about 60% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 65% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 70% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 75% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 80% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 85% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 90% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 95% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 98% to about 100% by weight of the bulk drug; in some embodiments, the bulk drug is present at about 99% to about 100% by weight of the bulk drug; in some embodiments, substantially all of the bulk drug is Form I of Compound (A) of the present invention, i.e., the bulk drug is substantially phase-pure crystals.

[0051] The present invention further provides compounds of formula (II-A), formula (II-B), and formula (II-C) as shown below:

[0052]

[0053] The compounds of formula (II-A), formula (II-B) and formula (II-C) serve as intermediates of compound (A).

[0054] The present invention further provides a method for preparing a compound of formula (A), comprising the following steps:

[0055]

[0056] The compound of formula (II-C) is dissolved in an organic solvent A, and reacted with 2-chloro-1-(3-hydroxyazetidin-1-yl)ethanone and a base at 70-90° C. to obtain a compound of formula (A).

[0057] In some embodiments, in the above preparation method, the compound of formula (II-C) is dissolved in an organic solvent A, and reacted with 2-chloro-1-(3-hydroxyazetidine-1-yl)ethanone and a base at preferably 72 to 88°C for 3 to 10 hours; more preferably at 75 to 85°C for 4 to 8 hours; and more preferably at 80°C for 6 hours.

[0058] In some embodiments, in the above preparation method, the organic solvent A is selected from at least one of acetonitrile, 2-methyltetrahydrofuran, ethylbenzene, methyl ethyl formate, and diethylene glycol tert-butyl ether.

[0059] In some embodiments, in the above preparation method, the organic solvent A is selected from acetonitrile.

[0060] In some embodiments, in the above preparation method, the base is at least one of potassium carbonate and sodium carbonate.

[0061] In some embodiments, in the above preparation method, the base is potassium carbonate.

[0062] Furthermore, the preparation method of the compound of formula (A) further comprises the following steps:

[0063]

[0064] Compound (II-B) is dissolved in an organic solvent C, trifluoroacetic acid is added, and the reaction is carried out to obtain a compound of formula (II-C).

[0065] Furthermore, the preparation method of the compound of formula (A) further comprises the following steps:

[0066]

[0067] Compound (II-A) is dissolved in organic solvent B, cooled to 0°C under nitrogen protection, and a reducing agent is added. After the reaction is completed, iodomethane-d3 is added to obtain compound (II-B).

[0068] Furthermore, the preparation method of the compound of formula (A) further comprises the following steps:

[0069]

[0070] The compound of formula (II-D), 2-chloro-4-(4-fluorophenyl)thiazole-5-cyano, 2,6-lutidine and a non-protonic polar solvent are reacted at 60-80°C to obtain compound (II-A).

[0071] The preparation of the compound of formula (A) of the present invention further comprises the following steps:

[0072]

[0073] i. a compound of formula (II-D), 2-chloro-4-(4-fluorophenyl)thiazole-5-cyano, 2,6-dimethylpyridine and a non-protonic polar solvent, reacting at 60 to 80° C. to obtain a compound (II-A);

[0074] ii. Dissolve the compound (II-A) obtained in step i in an organic solvent B, cool to 0° C. under nitrogen protection, add a reducing agent, and after the reaction is completed, add iodomethane-d3 to obtain a compound (II-B);

[0075] iii. Dissolve the compound (II-B) obtained in step ii in an organic solvent C, add trifluoroacetic acid, and react to obtain a compound of formula (II-C).

[0076] The preparation method of the compound of formula (A) of the present invention comprises the following steps:

[0077]

[0078] Furthermore, in the above preparation method, the aprotic polar solvent is at least one of N,N-dimethylacetamide, dimethylformamide, hexamethylphosphoramide, acetonitrile, acetone, and dimethyl sulfoxide.

[0079] Furthermore, the aprotic polar solvent is N,N-dimethylacetamide.

[0080] Furthermore, in the above preparation method, the organic solvent B is at least one of tetrahydrofuran, N,N-dimethylacetamide, dimethylformamide, acetonitrile and acetone.

[0081] Furthermore, the organic solvent B is tetrahydrofuran.

[0082] Furthermore, in the above preparation method, the reducing agent is sodium hydride.

[0083] Furthermore, in the above preparation method, the organic solvent C is at least one of dichloromethane, methyl acetate, dimethyl carbonate, propylene glycol methyl ether acetate, and nylon acid dimethyl ester.

[0084] Furthermore, the organic solvent C is dichloromethane.

[0085] As another technical solution of the present invention, the present invention provides a pharmaceutical composition, which contains the compound described in any one of the aforementioned technical solutions, its stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals, or Form I of the compound of formula (A), and a pharmaceutically acceptable carrier and / or excipient.

[0086] As another technical solution of the present invention, the present invention provides a compound described in any one of the above technical solutions, a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal thereof, or a use of the above composition in the preparation of a drug for treating an ATX-mediated disease. The ATX-mediated disease is idiopathic pulmonary fibrosis.

[0087] It is understood that thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) are well known in the art, and the melting peak height of the TGA curve and the DSC curve depends on many factors related to sample preparation and instrument geometry, while the peak position is relatively insensitive to experimental details. Therefore, in some embodiments, the crystalline compound of the present invention has a TGA graph and a DSC graph with characteristic peak positions, and has substantially the same properties as the TGA graph and the DSC graph provided in the accompanying drawings of the present invention, and the measurement value error tolerance is within ±5°C, and is generally required to be within ±3°C.

[0088] It is understood that the numerical values ​​described and protected by the present invention are approximate values. The variation in the numerical values ​​may be due to the calibration of the equipment, equipment error, purity of the crystal, crystal size, sample size and other factors.

[0089] It is to be understood that the crystal forms of the present invention are not limited to characteristic spectra that are completely identical to the characteristic spectra described in the accompanying drawings disclosed in the present invention, such as XRD, DSC, TGA, DVS, and isothermal adsorption curves. Any crystal forms having characteristic spectra that are substantially the same or essentially the same as those described in the accompanying drawings fall within the scope of the present invention.

[0090] the term

[0091] A "therapeutically effective amount" refers to that amount of a compound that will elicit the physiological or medical response of a tissue, system or subject that is being sought, and includes an amount of the compound that, when administered to a subject, is sufficient to prevent the occurrence or alleviate to some extent one or more symptoms of the disorder or condition being treated.

[0092] “Room temperature”: 10℃-30℃, >30%RH.

[0093] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention which retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, or the free base is obtained by reacting with a non-toxic inorganic acid or organic acid.

[0094] "Pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or their stereoisomers, solvates, pharmaceutically acceptable salts, cocrystals, deuterated forms, and other components, wherein the other components include physiologically / pharmaceutically acceptable carriers and / or excipients.

[0095] "Carrier" refers to a system that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered compound, and can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug and deliver the drug to the targeted organ. Non-limiting examples include microcapsules and microspheres, nanoparticles, liposomes, etc.

[0096] "Excipient" refers to a substance that is not a therapeutic agent in itself but is used as a diluent, adjuvant, binder and / or vehicle that is added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can serve a variety of functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents and sweeteners. Examples of pharmaceutical excipients include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose and cross-linked carboxymethylcellulose (e.g., cross-linked sodium carboxymethylcellulose); (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other nontoxic compatible substances used in pharmaceutical preparations.

[0097] "Stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and conformational isomers.

[0098] "Solvate" refers to a substance formed by a stoichiometric or non-stoichiometric amount of a solvent that is bound to the compound or salt of the present invention by non-covalent forces between molecules. When the solvent is water, it is a hydrate.

[0099] "Co-crystal" refers to a crystal formed by the active pharmaceutical ingredient (API) and the co-crystal former (CCF) under the action of hydrogen bonds or other non-covalent bonds, in which the pure state of API and CCF are solid at room temperature and there is a fixed stoichiometric ratio between the components. Co-crystal is a multi-component crystal, including binary eutectics formed between two neutral solids and multi-component eutectics formed between neutral solids and salts or solvates. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 is the X-ray powder diffraction pattern of Compound A Form I using Cu-Kα radiation;

[0101] Figure 2 is a TGA curve diagram of Compound A Form I;

[0102] Figure 3 is a DSC curve diagram of Compound A Form I;

[0103] Figure 4 is the PLM image of Compound A Form I;

[0104] Figure 5 This is the DVS diagram of Compound A Form I. DETAILED DESCRIPTION

[0105] The content of the present invention will be described in detail below through examples. If no specific conditions are specified in the examples, the experimental method according to conventional conditions is carried out. The examples are given to better illustrate the content of the present invention, but it should not be understood that the content of the present invention is limited to the examples. Those skilled in the art can make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still belongs to the protection scope of the present invention.

[0106] Unless otherwise specified, raw materials were purchased from Titan Technology, Anage Chemical, Shanghai Demo, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Nanjing Yaoshi, WuXi AppTec and J&K Technology.

[0107] Instrument information and methods

[0108] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or (and) mass spectrometry (MS). NMR shifts (δ) are given in units of 10-6 (ppm). NMR measurements were performed using (Bruker Avance III 400 and Bruker Avance 300) NMR spectrometers, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD) as the solvent, and tetramethylsilane (TMS) as the internal standard;

[0109] MS was measured using (Agilent 6120B (ESI) and Agilent 6120B (APCI));

[0110] HPLC determination was performed using an Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C18 100×4.6 mm, 3.5 μM);

[0111] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) uses a specification of 0.15mm-0.20mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm-0.5mm;

[0112] Column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier.

[0113] X-ray powder diffraction

[0114] The XRPD pattern was collected on an X-ray powder diffraction analyzer produced by PANalytacal, and the scanning parameters are shown in the following table.

[0115]

[0116] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)

[0117] The TGA and DSC images were collected on a TA Q5000 / 5500 thermogravimetric analyzer and a TA 2500 differential scanning calorimeter, respectively. The test parameters are listed in the following table.

[0118]

[0119]

[0120] Dynamic moisture sorption (DVS)

[0121] The dynamic moisture sorption (DVS) curves were collected on the DVS Intrinsic of SMS (Surface Measurement Systems).

[0122] The relative humidity at 25°C was corrected using the deliquescent points of LiCl, Mg(NO3)2 and KCl. The DVS test parameters are listed in the table below.

[0123]

[0124] Polarized Light Microscopy (PLM)

[0125] Polarized light microscopy data were acquired with an Axio Lab.A1 upright microscope at room temperature.

[0126] Brief description:

[0127] NMP: N-methylpyrrolidone;

[0128] THF: tetrahydrofuran;

[0129] HATU: 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate;

[0130] PE: petroleum ether;

[0131] EA: ethyl acetate;

[0132] DCM: dichloromethane;

[0133] MeOH: methanol;

[0134] Acetone: acetone;

[0135] EtOH: ethanol;

[0136] ACN: acetonitrile;

[0137] H2O: water.

[0138] Intermediate 1:

[0139] (But-1-yn-1-yl-d5)trimethylsilane (Intermediate 1)

[0140] (but-1-yn-1-yl-d5)trimethylsilane

[0141]

[0142] Under room temperature, trimethylsilyl acetylene (1a) (10.07 g, 102.5 mmol) was dissolved in dry tetrahydrofuran (45 mL). After the system was cooled to -78 ° C, n-butyl lithium (45 mL, 2M n-hexane solution) was added dropwise. After the addition was complete, the mixture was transferred to an ice-water bath and continued to react for 20 minutes. Then the mixture was cooled to -78 ° C, hexamethylphosphoric acid triamine (18.37 g, 102.5 mmol) was added dropwise, and stirring was continued for 30 minutes under this condition. Then iodoethane-d5 (1b) (15.00 g, 93.2 mmol) was added dropwise. After the addition was complete, the mixture was naturally heated to room temperature and reacted overnight. Water (20 mL) was added to the reaction solution to quench the reaction, and the liquid was separated. The aqueous phase was extracted with ether (20 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled at atmospheric pressure. The fractions from 80 ° C to 100 ° C were collected. The title compound (but-1-yn-1-yl-d5)trimethylsilane (Intermediate 1) was obtained as a colorless transparent liquid (10.0 g, yield 74%).

[0143] Intermediate 2:

[0144] 2-Chloro-1-(3-hydroxyazetidin-1-yl-3-d)ethan-1-one (Intermediate 2)

[0145] 2-chloro-1-(3-hydroxyazetidin-1-yl-3-d)ethan-1-one

[0146]

[0147] Step 1: tert-Butyl 3-hydroxyazetidine-1-carboxylate-3-d (2b)

[0148] tert-butyl 3-hydroxyazetidine-1-carboxylate-3-d

[0149]

[0150] 1-Boc-3-azetidinone (2a) (5.13 g, 30 mmol) was dissolved in tetrahydrofuran (50 mL), stirred evenly, and sodium borohydride-d4 (1.26 g, 30 mmol) was added in batches, and reacted at room temperature for 1 hour. Water (20 mL) was added to quench the reaction, and after stirring for 10 minutes, ethyl acetate (100 mL) and saturated sodium bicarbonate (100 mL) were added, and the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound tert-butyl 3-hydroxyazetidine-1-carboxylate-3-d (2b), a transparent oil (4.6 g, yield 87%).

[0151] 1 H NMR (400MHz, CDCl3) δ4.14 (d, 2H), 3.79 (d, 2H), 1.44 (s, 9H).

[0152] Step 2:

[0153] Azetidine-3-d-3-ol (2c)

[0154] Azetidin-3-d-3-ol

[0155]

[0156] The compound tert-butyl 3-hydroxyazetidine-1-carboxylate-3-d (2b) (4.6 g, 26.3 mmol) was dissolved in dichloromethane (24 mL), and trifluoroacetic acid (8 mL) was added dropwise to the system. After the addition was complete, the reaction was carried out at room temperature for 1 h, and the mixture was concentrated under reduced pressure to obtain the trifluoroacetate salt of azetidine-3-d-3-ol (2c), a brown oil (4.94 g, yield 100%). The crude product was directly used for the next step reaction.

[0157] Step 3:

[0158] 2-Chloro-1-(3-hydroxyazetidin-1-yl-3-d)ethan-1-one (Intermediate 2)

[0159] 2-chloro-1-(3-hydroxyazetidin-1-yl-3-d)ethan-1-one

[0160]

[0161] The trifluoroacetate (4.94 g, 26.3 mmol) of azetidine-3-d-3-ol (2c) was dissolved in water (40 mL), stirred evenly, and potassium carbonate (12.4 g, 60 mmol) was added in batches. After the system stopped bubbling, dichloromethane (40 mL) was added. Chloroacetyl chloride (3.39 g, 30 mmol) was added dropwise under ice bath conditions. After the addition was complete, the mixture was reacted at room temperature for 2 hours. Ethyl acetate (100 mL) and water (100 mL) were added, and the layers were separated. The aqueous phase was extracted with ethyl acetate (100 mL). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain the target compound 2-chloro-1-(3-hydroxyazetidine-1-yl-3-d)ethan-1-one (intermediate 2) as a white solid (0.85 g, yield 21%).

[0162] 1H NMR (400MHz, CDCl3) δ 4.49 (d, 1H), 4.30 (d, 1H), 4.15 (d, 1H), 3.94 (d, 1H), 3.90 (s, 2H), 3.01 (s, 1H).

[0163] Intermediate 3:

[0164] Ethyl 2-bromoacetate-d2 (Intermediate 3)

[0165] ethyl 2-bromoacetate-d2

[0166]

[0167] Dissolve acetic acid-d4 (3a) (1.0 g, 15.6 mmol) in trifluoroacetic anhydride (6 mL), stir evenly, add 4-dimethylaminopyridine (30 mg, 0.25 mmol), heat the system to 60 ° C, slowly add bromine (0.98 mL) under this condition, and add for 1 hour. After the addition is complete, continue to react under this condition for 1 hour, then cool to room temperature, and introduce nitrogen into the system until the volume of the system is reduced to about 1 mL. Add ethanol (20 mL), react at 60 ° C overnight, and then cool to room temperature. Concentrate under reduced pressure, add ether (100 mL), wash with saturated brine (100 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain ethyl 2-bromoacetate-d2 (intermediate 3), a light yellow liquid (1.65 g, yield 62.6%).

[0168] 1 H NMR (400MHz, CDCl3) δ4.24 (q, J=7.1Hz, 1H), 1.30 (t, J=7.1Hz, 2H).

[0169] Intermediate 4:

[0170]

[0171] first step:

[0172] 4-Fluorobenzoic acid methyl ester-2,3,5,6-d4(4b)

[0173] methyl 4-fluorobenzoate-2,3,5,6-d4

[0174]

[0175] 4-Fluorobenzoic acid-2,3,5,6-d4 (4a) (1.0 g, 6.94 mmol) was dissolved in N,N-dimethylformamide (10 mL), cooled to 0°C, potassium carbonate (1.92 g, 13.9 mmol) was added, and the reaction was continued for 5 minutes under this condition, and dimethyl sulfate (1.05 g, 8.33 mmol) was added dropwise to the system. After the addition, the reaction was continued at room temperature for 1 hour. Water (20 mL) was added to quench the reaction, and ethyl acetate was used for extraction (50 mL × 2). The organic phases were combined, washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate (v / v) = 8: 1) to obtain the target compound 4-fluorobenzoic acid methyl ester-2,3,5,6-d4 (4b) as a transparent liquid (0.96 g, yield 87%).

[0176] 1 H NMR (400MHz, CDCl3) δ3.91 (s, 3H).

[0177] Step 2:

[0178] 3-(4-Fluorophenyl-2,3,5,6-d4)-3-oxopropionitrile (4c)

[0179] 3-(4-fluorophenyl-2,3,5,6-d4)-3-oxopropanenitrile

[0180]

[0181] Dissolve methyl 4-fluorobenzoate-2,3,5,6-d4 (4b) (0.96 g, 6.1 mmol) in toluene (8 mL), cool to -10 ° C under nitrogen protection, add acetonitrile (1.46 g, 36.3 mmol), and slowly drop sodium bis(trimethylsilyl)amide (6 mL, 12 mmol, 2 M in THF) into the system. After complete dropwise addition, quench the reaction with hydrochloric acid (20 mL, 1N aqueous solution). Warm to room temperature, extract with ethyl acetate (50 mL × 2), combine the organic phases, wash with saturated brine (50 mL × 1), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the target compound 3-(4-fluorophenyl-2,3,5,6-d4)-3-oxopropionitrile (4c) as a light yellow solid (0.92 g, yield 91%).

[0182] 1 H NMR (400MHz, CDCl3) δ4.05 (s, 2H).

[0183] Step 3:

[0184] 2-Amino-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (4d)

[0185] 2-amino-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile

[0186]

[0187] 3-(4-fluorophenyl-2,3,5,6-d4)-3-oxopropionitrile (4c) (0.92 g, 5.5 mmol) was dissolved in ethanol (12 mL), pyridine (0.44 g, 5.5 mmol) was added, and the temperature was raised to 70°C for reaction for 15 minutes. After cooling to room temperature, thiourea (0.84 g, 11 mmol) and iodine (1.4 g, 5.5 mmol) in ethanol (8 mL) were slowly added dropwise to the system. After complete addition, the reaction was allowed to proceed at room temperature for 1 hour. Sodium thiosulfate (10 mL, 1N aqueous solution) was used to quench the reaction. After filtration, the filter cake was dried to obtain the target compound 2-amino-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (4d) as a white solid (0.82 g, yield 67%).

[0188] Step 4:

[0189] 2-Chloro-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (Intermediate 4)

[0190] 2-chloro-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile

[0191]

[0192] Dissolve cupric chloride (0.60 g, 4.5 mmol) in acetonitrile (8 mL), add tert-butyl nitrite (0.67 g, 5.6 mmol) dropwise to the system, and stir at room temperature for 30 minutes. Add 2-amino-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (4d) (0.82 g, 3.67 mmol) to the system in batches. After the addition is complete, continue to react at room temperature for 1 hour. Quench the reaction with hydrochloric acid (10 mL, 1N aqueous solution). Add ethyl acetate (80 mL), wash with saturated brine (50 mL), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The residue is separated by silica gel column chromatography (dichloromethane) to obtain the target compound 2-chloro-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (intermediate 4) as a light yellow solid (0.68 g, yield 76%).

[0193] Embodiment 1:

[0194] 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 1)

[0195] 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0196]

[0197] first step:

[0198] tert-Butyl 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylate (1C)

[0199] tert-butyl 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylate(1C)

[0200]

[0201] In a 500mL reaction bottle, NMP (220mL) was added, stirring was started, and then 2-chloro-5-fluoro-4-iodopyridine (1A) (44.00g, 170.9mmol), 1-Boc-piperazine (1B) (47.75g, 256.4mmol) and potassium carbonate (47.24g, 341.8mmol) were added in sequence, and the reaction was stopped after heating to 150°C for 3 hours. The reaction solution was cooled to room temperature, and the reactant was slowly added to ice water (500mL), and a white solid was precipitated. The mixture was stirred for 0.5 hours and filtered. The filter cake was washed with water and then pulped with n-hexane (200mL), filtered, and dried to obtain the target compound 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylic acid tert-butyl ester (1C), a white solid (50g, yield 64%).

[0202] LCMS m / z=316.2[M+1] + .

[0203] Step 2:

[0204] 4-(2-(2-(But-1-yn-1-yl-d5)-5-fluoropyridin-4-yl)piperazine-1-carboxylic acid tert-butyl ester (1D)

[0205] tert-butyl 4-(2-(but-1-yn-1-yl-d5)-5-fluoropyridin-4-yl)piperazine-1-carboxylate

[0206]

[0207] 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylic acid tert-butyl ester (1C) (23.18 g, 71.6 mmol), (but-1-yn-1-yl-d5) trimethylsilane (intermediate 1) (9.4 g, 131.3 mmol), bistriphenylphosphine palladium dichloride (5.03 g, 7.2 mmol), 1,3-bis(diphenylphosphino)propane (4.43 g, 10.7 mmol) and cesium fluoride (22.8 g, 143.3 mmol) were added to dimethyl sulfoxide (184 mL) in sequence, the system was replaced with nitrogen three times, and the reaction was carried out at 95° C. for 4 hours. After the reaction was completed, it was cooled to room temperature, water (200 mL) was added, the aqueous phase was extracted with ethyl acetate (200 mL×3), the combined organic phases were washed with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (PE:EA=20:1-5:1) to give tert-butyl 4-(2-(2-(but-1-yn-1-yl-d5)-5-fluoropyridin-4-yl)piperazine-1-carboxylate (1D), a light brown viscous liquid (10.15 g, yield 41.9%).

[0208] LCMS m / z=339.2[M+1] + .

[0209] Step 3:

[0210] tert-Butyl 4-(2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1F)

[0211] tert-butyl 4-(2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0212]

[0213] In a 250 mL reaction bottle, add ethanol (100 mL) and tert-butyl 4-(2-(2-(but-1-yn-1-yl-d5)-5-fluoropyridin-4-yl)piperazine-1-carboxylate (1D) (10.15 g, 30.0 mmol), cool to 0°C, add 2-[(aminooxy)sulfonyl]-1,3,5-trimethylbenzene (1E) (11.00 g, 51.1 mmol) in batches, then add sodium bicarbonate (5.04 g, 60.0 mmol) to the reaction, react at room temperature for 2 hours, and then add potassium carbonate ( 8.29g, 60.0mmol) was added to the reaction, and the reaction was allowed to proceed overnight at room temperature. Water (150mL) was added to the reaction solution, and the aqueous phase was extracted with ethyl acetate (200mL×2), and the combined organic phases were washed with saturated sodium chloride (100mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography to obtain the target compound 4-(2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (1F), as a white solid (7.53g, yield 71%).

[0214] LCMS m / z=354.3[M+1] + .

[0215] Step 4:

[0216] 4-(2-(Ethyl-d5)-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (1G)

[0217] tert-butyl 4-(2-(ethyl-d5)-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0218]

[0219] Dissolve tert-butyl 4-(2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1F) (7.50 g, 21.2 mmol) in methanol (37.5 mL) and acetic acid (7.5 mL), cool to 5°C, and then dissolve sodium nitrite (2.93 g, 42.4 mmol) in water (10 mL) and add dropwise to the reaction solution. After addition, react at room temperature for 16 hours. Add water (30 mL) dropwise to the reaction solution. After addition, filter, wash the filter cake with water (10 mL×2), and dry to obtain the target compound tert-butyl 4-(2-(ethyl-d5)-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1G) as a crude dark green solid (11.5 g).

[0220] LCMS m / z=383.3[M+1] + .

[0221] Step 5:

[0222] tert-Butyl 4-(3-amino-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1H)

[0223] tert-butyl 4-(3-amino-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0224]

[0225] Dissolve 4-(2-(ethyl-d5)-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (1G) (11.5 g, crude product) in ethanol (40 mL) and water (20 mL), then add ammonium chloride (15.78 g, 295 mmol) and iron powder (8.23 g, 147 mmol) to the reaction, react at 65°C for 20 minutes, and filter. The filtrate is extracted with dichloromethane (50 mL×3), the organic phases are combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain the target compound 4-(3-amino-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (1H) as a crude product, a yellow solid (7.85 g).

[0226] LCMS m / z=369.2[M+1] + .

[0227] Step 6:

[0228] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1J)

[0229] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-(ethyl-d5)-6-fiuoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0230]

[0231] 4-(3-amino-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (1H) (7.85 g, 21.3 mmol), 2-chloro-4-(4-fluorophenyl)thiazole-5-cyano (1I) (6.1 g, 25.6 mmol) (reference J. Med. Chem. 2017, 60, 3580-3590, DOI: 10.1021 / acs.jmedchem.7b00032), 2,6-dimethylpyridine (3.42 g, 31.9 mmol) and N,N-dimethylacetamide (40 mL) were heated to 70°C and reacted for 4 hours. 10% sodium chloride solution (100 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (150 mL×3). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1J) as a crude yellow-brown viscous substance (15.50 g).

[0232] LCMS m / z=571.2[M+1] + .

[0233] Step 7:

[0234] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1K)

[0235] tert-butyl4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0236]

[0237] The crude product (15.50 g) of tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1J) was dissolved in tetrahydrofuran (60 mL), cooled to 0°C under nitrogen protection, and sodium hydroxide (1.3 g, 32.6 mmol, 60% wt) was added in batches. After the addition was completed, the reaction was continued for 10 minutes, and then iodomethane (4.63 g, 32.6 mmol) was added dropwise to the system. After the addition was completed, the reaction was continued at room temperature for 30 minutes. Water (100 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (100 mL×2). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1K) as a yellow solid (11.56 g, four-step yield 93%).

[0238] Step 8:

[0239] 2-((2-(ethyl-d5)-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (1L)

[0240] 2-((2-(ethyl-d5)-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile hydrochloride

[0241]

[0242] The compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1K) (11.56 g, 19.8 mmol) was dissolved in dichloromethane (110 mL), trifluoroacetic acid (35 mL) was added dropwise to the system, and the reaction was carried out for about 3 hours, and then concentrated under reduced pressure. Dichloromethane (200 mL) was added to the residue, and the pH was adjusted to 8 with saturated sodium bicarbonate. The layers were separated, and the aqueous phase was extracted with dichloromethane (200 mL×2). The organic phases were combined, washed with saturated brine (300 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound 2-((2-(ethyl-d5)-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (1 L) as a yellow solid (10.7 g).

[0243] Step 9:

[0244] 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 1)

[0245] 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0246]

[0247] 2-((2-(ethyl-d5)-6-fluoro-5-(piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (1L) (10.7 g) was dissolved in acetonitrile (100 mL), and 2-chloro-1-(3-hydroxyazetidine-1-yl)ethanone (1M) (3.63 g, 24.3 mmol, synthesized according to the method of reference J.Med.Chem. 2017, 60, 3580-3590) and potassium carbonate (6.11 g, 44.2 mmol) were added to the system in sequence, and the temperature was raised to 80°C and the reaction was allowed to react overnight. Water (100 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (150 mL×3). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (dichloromethane / methanol=20:1) to give the title compound 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidine-1-yl)-2-oxoethyl)piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 1) (6.2 g, 53% for two steps).

[0248] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 8.18-8.10 (m, 2H), 7.20-7.11 (m, 2H), 6.45 (d, 1H), 4.67 (s, 1H), 4.50-4.40 (m, 1H), 4.3 2-4.23 (m, 1H), 4.15-4.07 (m, 1H), 3.93-3.85 (m, 1H), 3.58 (s, 3H), 3.30-3.20m, 4H), 3.19-3.06 (m, 2H), 2.85-2.67 (m, 5H).

[0249] LCMS m / z=598.1[M+1] + .

[0250] Embodiment 2:

[0251] 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 2)

[0252] 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0253]

[0254] first step:

[0255] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (2A)

[0256] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0257]

[0258] The crude product (9.2 g, 16.1 mmol) of tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (1J) was dissolved in tetrahydrofuran (60 mL), cooled to 0°C under nitrogen protection, and sodium hydrogen sulfide (0.77 g, 19.3 mmol, 60% wt) was added in batches. After the addition was completed, the reaction was carried out under this condition for 10 minutes, and then iodomethane-d3 (2.80 g, 19.3 mmol) was added dropwise to the system. After the addition was completed, the reaction was carried out at room temperature for 30 minutes. Water (100 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (100 mL×2). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (2A) as a yellow solid (8.0 g, yield 84.5%).

[0259] Step 2:

[0260] 2-((2-(ethyl-d5)-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (2B)

[0261] 2-((2-(ethyl-d5)-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0262]

[0263] The compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-(ethyl-d5)-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (2A) (8.0 g, 13.6 mmol) was dissolved in dichloromethane (90 mL), and trifluoroacetic acid (30 mL) was added dropwise to the system. After reacting for about 3 hours, the mixture was concentrated under reduced pressure. Dichloromethane (100 mL) was added to the residue, and the pH was adjusted to 8 with saturated sodium bicarbonate. The liquids were separated, and the aqueous phase was extracted with dichloromethane (100 mL×3). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound 2-((2-(ethyl-d5)-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (2B) as a yellow solid (6.40 g, yield 96.3%).

[0264] Step 3:

[0265] 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 2)

[0266] 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0267]

[0268] 2-((2-(ethyl-d5)-6-fluoro-5-(piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (2B) (6.40 g, 13.1 mmol) was dissolved in acetonitrile (100 mL), and 2-chloro-1-(3-hydroxyazetidine-1-yl)ethanone (1M) (2.16 g, 14.4 mmol) and potassium carbonate (3.62 g, 26.2 mmol) were added to the system in sequence, and the temperature was raised to 80°C and the reaction was carried out for 5 hours. Water (60 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (dichloromethane / methanol=20:1) to give the title compound 2-((2-(ethyl-d5)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 2) (5.1 g, 64.8% for two steps).

[0269] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 8.20-8.11 (m, 2H), 7.20-7.11 (m, 2H), 6.45 (d, 1H), 4.67 (s, 1H), 4.50-4 .40 (m, 1H), 4.32-4.24 (m, 1H), 4.15-4.07 (m, 1H), 3.94-3.86 (m, 1H), 3.31-3.06 (m, 6H), 2.86-2.66 (m, 5H).

[0270] LCMS m / z=601.2[M+1] + .

[0271] Embodiment 3:

[0272] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 3)

[0273] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fiuorophenyl)thiazole-5-carbonitrile

[0274]

[0275]

[0276] first step:

[0277] tert-Butyl 4-(2-(But-1-yn-1-yl)-5-fluoropyridin-4-yl)piperazine-1-carboxylate (3A)

[0278] tert-butyl 4-(2-(but-1-yn-1-yl)-5-fluoropyridin-4-yl)piperazine-1-carboxylate

[0279]

[0280] 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylic acid tert-butyl ester (1C) (20.1 g, 62 mmol), (but-1-yn-1-yl)trimethylsilane (9.3 g, 74 mmol), bistriphenylphosphine palladium dichloride (4.4 g, 6.3 mmol), 1,3-bis(diphenylphosphino)propane (3.84 g, 9.3 mmol) and cesium fluoride (19.0 g, 125 mmol) were added to dimethyl sulfoxide (160 mL) in sequence, the system was replaced with nitrogen three times, and the reaction was carried out at 95° C. for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, water (160 mL) was added, the aqueous phase was extracted with ethyl acetate (200 mL×3), the combined organic phases were washed with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (PE:EA=20:1-5:1) to give tert-butyl 4-(2-(but-1-yn-1-yl)-5-fluoropyridin-4-yl)piperazine-1-carboxylate (3A) as a light brown viscous liquid (11.0 g, yield 53%).

[0281] LCMS m / z=334.2[M+1] + .

[0282] Step 2:

[0283] tert-Butyl 4-(2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3B)

[0284] tert-butyl 4-(2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0285]

[0286] In a 250 mL reaction bottle, add ethanol (100 mL) and tert-butyl 4-(2-(but-1-yn-1-yl)-5-fluoropyridin-4-yl)piperazine-1-carboxylate (3A) (11.0 g, 33 mmol), cool to 0°C, add 2-[(aminooxy)sulfonyl]-1,3,5-trimethylbenzene (1E) (10.7 g, 49.5 mmol) in batches, and then add sodium bicarbonate (5.5 g, 66 mmol) to the reaction. After the addition is completed and react at room temperature for 2 hours, potassium carbonate (9.12 g, 66 mmol) is added to the reaction and the reaction is allowed to react overnight at room temperature. Water (200 mL) was added to the reaction solution, and the aqueous phase was extracted with ethyl acetate (200 mL×2). The combined organic phases were washed with saturated sodium chloride (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain the target compound 4-(2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (3B) as a white solid (7.8 g, yield 68%).

[0287] LCMS m / z=349.2[M+1] + .

[0288] Step 3:

[0289] tert-Butyl 4-(2-ethyl-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3C)

[0290] tert-butyl 4-(2-ethyl-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0291]

[0292] Dissolve tert-butyl 4-(2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3B) (7.40 g, 22.4 mmol) in methanol (40 mL) and acetic acid (8 mL), cool to 5°C, then dissolve sodium nitrite (3.09 g, 44.8 mmol) in water (10 mL), slowly dropwise add to the reaction solution, and react at room temperature for 16 hours. Add water (30 mL) dropwise to the reaction solution, filter after complete addition, wash the filter cake with water (10 mL×2), and dry to obtain the target compound tert-butyl 4-(2-ethyl-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3C) as a crude dark green solid (10.8 g).

[0293] LCMS m / z=378.3[M+1] + .

[0294] Step 4:

[0295] tert-Butyl 4-(3-amino-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3D)

[0296] tert-butyl 4-(3-amino-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0297]

[0298] 4-(2-ethyl-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (3C) (10.8 g, crude product) was dissolved in ethanol (40 mL) and water (20 mL), and then ammonium chloride (14.98 g, 0.28 mol) and iron powder (7.84 g, 0.14 mol) were added to the reaction, and the reaction was carried out at 65° C. for 20 minutes, and filtered. The filtrate was extracted with dichloromethane (60 mL×3), the organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound 4-(3-amino-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (3D) as a crude yellow solid (7.12 g, two-step yield 87.5%).

[0299] LCMS m / z=364.2[M+1] + .

[0300] Step 5:

[0301] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3E)

[0302] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0303]

[0304] 4-(3-Amino-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (3D) (7.12 g, 19.6 mmol), 2-chloro-4-(4-fluorophenyl)thiazole-5-cyano (1I) (5.61 g, 23.5 mmol), 2,6-dimethylpyridine (3.15 g, 29.4 mmol) and N,N-dimethylacetamide (40 mL) were heated to 70°C and reacted for 5 hours. 10% sodium chloride solution (100 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (150 mL×3). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3E) as a yellow-brown solid (10.0 g, yield 90.3%).

[0305] LCMS m / z=566.2[M+1] + .

[0306] Step 6:

[0307] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3F)

[0308] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0309]

[0310] Dissolve tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3E) (10.0 g, 17.7 mmol) in tetrahydrofuran (60 mL), cool to 0°C under nitrogen protection, add sodium hydrogen sulfide (0.85 g, 21.2 mmol, 60% wt) in batches, and after the addition is completed, react for 10 minutes, then add iodomethane-d3 (3.07 g, 21.2 mmol) dropwise to the system. After the addition is completed, react at room temperature for 30 minutes. Water (100 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (100 mL × 2). The organic phases were combined, washed with saturated brine (100 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3F) as a crude yellow solid (11.7 g).

[0311] LCMS m / z=583.2[M+1] + .

[0312] Step 7:

[0313] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (3G)

[0314] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile hydrochloride

[0315]

[0316] The crude product (11.7 g) of the compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3F) was dissolved in dichloromethane (100 mL), and difluoroacetic acid (33 mL) was added dropwise to the system. After reacting for about 3 hours, the mixture was concentrated under reduced pressure. Dichloromethane (200 mL) was added to the residue, and the pH was adjusted to 8 with saturated sodium bicarbonate. The layers were separated, and the aqueous phase was extracted with dichloromethane (200 mL×2). The organic phases were combined, washed with saturated brine (300 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (3G) as a crude product of a yellow solid (10.0 g).

[0317] LCMS m / z=483.2[M+1] + .

[0318] Step 8:

[0319] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 3)

[0320] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fiuorophenyl)thiazole-5-carbonitrile

[0321]

[0322] The crude product (10.0 g) of 2-((2-ethyl-6-fluoro-5-(piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (3G) was dissolved in acetonitrile (100 mL), and 2-chloro-1-(3-hydroxyazetidine-1-yl)ethanone (1M) (3.35 g, 22.4 mmol) and potassium carbonate (5.55 g, 40.1 mmol) were added to the system in sequence, and the temperature was raised to 80°C for reaction for 6 hours. Water (100 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (150 mL×3). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (dichloromethane / methanol=20:1) to give the title compound 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidine-1-yl)-2-oxoethyl)piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 3) (7.6 g, 72.1% for three steps).

[0323] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1II), 8.20-8.10 (m, 2II), 7.20-7.11 (m, 2II), 6.45 (d, 1H), 4.67 (s, 1H), 4.50-4.40 (m, 1H), 4.3 2-4.24(m, 1H), 4.16-4.08(m, 1H), 3.94-3.86(m, 1H), 3.29-3.07(m, 6H), 2.78(s, 4H), 2.72(q, 2H), 2.58(s, 1H), 1.32(t, 3H).

[0324] LCMS m / z=596.3[M+1] + .

[0325] Embodiment 4:

[0326] 2-((2-(ethyl)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl-2,2,3,3,5,5,6,6-d8)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 4)

[0327] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl-2,2,3,3,5,5,6,6-d8)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0328]

[0329]

[0330] first step:

[0331] Tert-butyl 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8(4B)

[0332] ten-butyl 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8

[0333]

[0334] In a 250 mL reaction bottle, NMP (93 mL) was added, followed by 2-chloro-5-fluoro-4-iodopyridine (1A) (9.3 g, 36.2 mmol), tert-butyl piperazine-1-carbonate-2,2,3,3,5,5,6,6-deuterium 8 (4A) (4.5 g, 24.2 mmol) and potassium carbonate (6.7 g, 48.3 mmol), and the temperature was raised to 150°C for 3 hours before stopping the reaction. The reaction solution was cooled to room temperature, and the reactant was slowly added to ice water (200 mL), a white solid precipitated, stirred for 0.5 hours, and filtered. The filter cake was washed with water and slurried with n-hexane (100 mL), filtered, and dried to give the target compound tert-butyl 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8 (4B) as a white solid (10.01 g, yield 85%).

[0335] LCMS m / z=324.3[M+1] + .

[0336] Step 2:

[0337] 4-(2-(tert-1-yn-1-yl)-5-fluoropyridin-4-yl)piperazine-1-carboxylic acid tert-butyl ester-2,2,3,3,5,5,6,6-d8(4C)

[0338] tert-butyl

[0339] 4-(2-(but-1-yn-1-yl)-5-fluoropyridin-4-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8

[0340]

[0341] Tert-butyl 4-(2-chloro-5-fluoropyridin-4-yl)piperazine-1-carboxylate (4B) (2.00 g, 6.2 mmol), (but-1-yn-1-yl)trimethylsilane (0.94 g, 7.4 mmol), bistriphenylphosphine palladium dichloride (0.44 g, 0.6 mmol), 1,3-bis(diphenylphosphino)propane (0.38 g, 0.9 mmol) and cesium fluoride (1.89 g, 12.4 mmol) were added to dimethyl sulfoxide (16 mL) in sequence, the system was replaced with nitrogen three times, and the reaction was carried out at 95° C. for 5 hours. After the reaction was completed, the mixture was cooled to room temperature, water (20 mL) was added, the aqueous phase was extracted with ethyl acetate (20 mL×3), the combined organic phases were washed with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (PE:EA=15:1-5:1) to give 4C (1.20 g, yield 57%).

[0342] LCMS m / z=342.3[M+1] + .

[0343] Step 3:

[0344] tert-Butyl 4-(2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8(4D)

[0345] tert-butyl 4-(2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8

[0346]

[0347] In a 100mL reaction bottle, add ethanol (12mL) and 4C (1.20g, 3.5mmol), cool to 0℃, add 2-[(aminooxy)sulfonyl]-1,3,5-trimethylbenzene (1E) (1.14g, 5.3mmol) in batches, then add sodium bicarbonate (0.59g, 7.0mmol) to the reaction, react at room temperature for 2 hours, then add potassium carbonate (0.97g, 7.0mmol) to the reaction, and react at room temperature overnight. Add water (150mL) to the reaction solution, extract the aqueous phase with ethyl acetate (200mL×2), combine the organic phases, wash with saturated sodium chloride (100mL), dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product of 4D (2.0g).

[0348] LCMS m / z=357.2[M+1] + .

[0349] Step 4:

[0350] tert-Butyl 4-(2-ethyl-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8(4E)

[0351] tert-butyl 4-(2-ethVl-6-fluoro-3-nitrosopyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8

[0352]

[0353] The crude product 4D (2.0 g, about 5.6 mmol) of the previous step was dissolved in methanol (10.0 mL) and acetic acid (2.0 mL), cooled to 5-10°C, and then an aqueous solution (2.0 mL) of sodium nitrite (0.77 g, 11.2 mmol) was added dropwise to the reaction solution, and the reaction was carried out at room temperature for 16 hours. Water (20 mL) was added dropwise to the reaction solution, and the reaction solution was filtered after the addition was completed. The filter cake was washed with water (5 mL×2) and dried to obtain the crude product 4E (2.00 g).

[0354] Step 5:

[0355] 4-(3-amino-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester-2,2,3,3,5,5,6,6-d8(4F)

[0356] tert-butyl 4-(3-amino-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8

[0357]

[0358] The crude product of 4E (2.00 g, about 5.2 mmol) from the previous step was dissolved in ethanol (10 mL) and water (10 mL), and then ammonium chloride (2.78 g, 52.0 mmol) and iron powder (1.46 g, 26.0 mmol) were added to the reaction, and the reaction was carried out at 65°C for 2 h, and filtered. The filtrate was extracted with EA (10 mL×3), the organic phases were combined, washed with saturated brine (10 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (DCM: MeOH=30:1) to obtain 4F (0.92 g, yield 48%).

[0359] LCMS m / z=372.3[M+1] + .

[0360] Step 6:

[0361] tert-Butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8(4G)

[0362] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8

[0363]

[0364] 4F (0.92 g, 2.5 mmol), 2-chloro-4-(4-fluorophenyl)thiazole-5-carbonitrile (1I) (0.72 g, 3.0 mmol), 2,6-lutidine (0.40 g, 3.8 mmol) and N,N-dimethylacetamide (5 mL) were heated to 70°C and reacted overnight. 10% sodium chloride solution (20 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 mL×3). The organic phases were combined, washed with saturated brine (20 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and then subjected to column chromatography (DCM: MeOH (v / v) = 30:1) to obtain 4G (1.00 g, yield 70%).

[0365] LCMS m / z=574.3[M+1] + .

[0366] Step 7:

[0367] tert-Butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8(4H)

[0368] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate-2,2,3,3,5,5,6,6-d8

[0369]

[0370] Dissolve 4G (15.50 g) in tetrahydrofuran (10 mL), cool to 0 ° C under nitrogen protection, add sodium hydride (0.10 g, 2.1 mmol, 60% wt) in batches, react for 10 minutes under this condition, and then add iodomethane (0.16 mL, 2.1 mmol) dropwise to the system. After the addition, warm to room temperature and react for 1 hour. Add 10 mL of methanol to quench the reaction, spin dry the reaction solution to obtain a crude product of 4H (1.20 g), which can be directly reacted in the next step without purification.

[0371] LCMS m / z=588.3[M+1] + .

[0372] Compound 4H can also be synthesized by the following method:

[0373]

[0374] At room temperature, raw material 4J (synthesized with reference to WO2019228403A1, Intermediate 3) (948 mg, 2.0 mmol), N-Boc-piperazine-d8 (580 mg, 3.0 mmol), tridibenzylideneacetone dipalladium (183 mg, 0.2 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (230 mg, 0.4 mmol)) and sodium tert-butoxide (576 mg, 6.0 mmol)) were added to dry 1,4-dioxane (20 mL), replaced with nitrogen 3 times, and then heated to 105 ° C for 2 h. After cooling to room temperature, it was spin-dried and directly column chromatographed (PE: EA = 5: 1 to 2: 1) to obtain a brown foamy solid 4H (530 mg, yield 45%).

[0375] LCMS m / z=588.3[M+1] + .

[0376] Step 8: 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl-2,2,3,3,5,5,6,6-d8)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (4I)

[0377] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl-2,2,3,3,5,5,6,6-d8)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0378]

[0379] Trifluoroacetic acid (5.0 mL) was added dropwise to a dichloromethane (15.0 mL) solution of compound 4H (1.20 g crude product) obtained in the previous step under nitrogen protection at 0°C, and then the temperature was naturally raised to room temperature and stirring was continued for 2 h. After the reaction solution was dried, 10 mL of dichloromethane was added, and then the solution was neutralized with saturated sodium carbonate to pH = 8, extracted with dichloromethane (10 mL × 2), washed with water (10 mL × 1), washed with saturated sodium chloride (10 mL × 1), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product of 4I (0.90 g), which was directly used for the next step without purification.

[0380] LCMS m / z-488.3[M+1] + .

[0381] Step 9:

[0382] 2-((2-(ethyl)-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl-2,2,3,3,5,5,6,6-d8)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 4)

[0383] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl-2,2,3,3,5,5,6,6-d8)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0384]

[0385] The crude product of compound 4I (860 mg, about 1.8 mmol), 2-chloro-1-(3-hydroxyacridin-1-yl)ethan-1-one (296 mg, 2.0 mmol) and potassium carbonate (500 mg, 3.6 mmol) were added to acetonitrile (50 mL) at room temperature, and then the temperature was raised to 80°C for reaction for 5 h. After cooling to room temperature, the mixture was filtered with suction, and the filter cake was washed with dichloromethane (30 mL×2). The filtrate was concentrated and then subjected to column chromatography (DCM: MeOH=30:1) to obtain compound 4 (880 mg, yield 81%).

[0386] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 8.19-8.11 (m, 2H), 7.20-7.12 (m, 2H), 6.44 (d, 1H), 4.67 (s, 1H), 4.50-4.40 (m, 1H), 4.32 -4.24(m, 1H), 4.16-4.09(m, 1H), 3.94-3.86(m, 1H), 3.58(s, 3H), 3.24-3.06(m, 2H), 2.72(q, 2H), 2.59(s, 1H), 1.32(t, 3H).

[0387] LCMS m / z=601.2[M+1] + .

[0388] Embodiment 5:

[0389] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl-3-d)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 5)

[0390] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl-3-d)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0391]

[0392]

[0393] first step:

[0394] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (5A)

[0395] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0396]

[0397] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (3E) (2.8 g, 5.0 mmol)) was dissolved in tetrahydrofuran (30 mL), cooled to 0°C under nitrogen protection, and sodium hydrogen sulfide (0.24 g, 6.0 mmol, 60% wt) was added in batches. After the addition was completed, the reaction was carried out under this condition for 10 minutes, and then iodomethane (1.42 g, 10 mmol) was added dropwise to the system. After the addition was completed, the reaction was carried out at room temperature for 30 minutes. Water (50 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×2). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (5A) as a crude yellow solid (2.8 g).

[0398] LCMS m / z=580.3[M+1] + .

[0399] Step 2:

[0400] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (5B)

[0401] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0402]

[0403] The crude product (2.8 g) of tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (5A) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (15 mL) was added dropwise to the system. After reacting for about 3 hours, the mixture was concentrated under reduced pressure. Dichloromethane (100 mL) was added to the residue, and the pH was adjusted to 8 with saturated sodium bicarbonate. The liquids were separated, and the aqueous phase was extracted with dichloromethane (100 mL×2). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (5B) as a yellow solid (2.0 g, two-step yield 83.3%).

[0404] LCMS m / z=480.1[M+1] + .

[0405] Step 3:

[0406] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl-3-d)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 5)

[0407] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl-3-d)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0408]

[0409] 2-((2-ethyl-6-fluoro-5-(piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (0.31 g, 0.65 mmol) was dissolved in acetonitrile (10 mL), and 2-chloro-1-(3-hydroxyazetidine-1-yl-3-d)ethan-1-one (Intermediate 2) (0.15 g, 1.0 mmol) and potassium carbonate (0.27 g, 2.0 mmol) were added to the system in sequence, and the temperature was raised to 80°C for reaction for 6 hours. Water (50 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (50 mL×3). The organic phases were combined, washed with saturated brine (50 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (dichloromethane / methanol=20:1) to give the title compound 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidine-1-yl-3-d)-2-oxoethyl)piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 5) (90 mg, 23%).

[0410] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 8.18-8.10 (m, 2H), 7.20-7.10 (m, 2H), 6.45 (d, 1H), 4.43 (d, 1H), 4. 25 (d, 1H), 4.11 (dd, 1H), 3.89 (d, 1H), 3.58 (s, 3H), 3.28-3.06 (m, 7H), 2.81-2.66 (m, 6H), 1.31 (t, 3H).

[0411] LCMS m / z=594.2[M+1] + .

[0412] Embodiment 6:

[0413] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl-1,1-d2)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 6)

[0414] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl-1,1-d2)piperazin-1-yl)pyra zolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0415]

[0416] first step:

[0417] Ethyl 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazin-1-yl)acetate-d2(6A)

[0418] ethyl 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazin-1-yl)acetate-d2

[0419]

[0420] To a solution of 2-((2-ethyl-6-fluoro-5-(piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (5B) (0.31 g, 0.65 mmol) in acetonitrile (10 mL) were added potassium carbonate (0.14 g, 1 mmol) and ethyl 2-bromoacetate-d2 (intermediate 3) (0.17 g, 1 mmol) in turn, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was diluted with saturated brine (30 mL) and extracted with ethyl acetate (30 mL×2). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to give ethyl 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazin-1-yl)acetate-d2(6A) as a light yellow solid (0.32 g, 86.7%).

[0421] LC-MS (ESI): m / z -568.2 [M+H] + .

[0422] Step 2:

[0423] 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazin-1-yl)acetic acid-2,2-d2(6B)

[0424] 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazin-1-yl)acetic-2,2-d2 acid

[0425]

[0426] Ethyl 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazin-1-yl)acetate-d2(6A) (0.32 g, 0.56 mmol) was dissolved in tetrahydrofuran (6 mL), methanol (2 mL) and water (2 mL) were added, stirred evenly, lithium hydroxide monohydrate (84 mg, 2 mmol) was added, and the mixture was reacted at room temperature for 1 hour. The pH was adjusted to 5 with hydrochloric acid (1N aqueous solution), extracted with ethyl acetate (50 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazine-1-yl)acetic acid-2,2-d2(6B) as a light yellow solid (0.24 g, 79% yield).

[0427] LCMS m / z=540.2[M+1] + .

[0428] Step 3:

[0429] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl-1,1-d2)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 6)

[0430] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl-1,1-d2)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0431]

[0432] 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazine-1-yl)acetic acid-2,2-d2(6B) (0.24 g, 0.45 mmol) was dissolved in N,N-dimethylformamide (5 mL), and triethylamine (0.15 g, 1.5 mmol) and HATU (0.19 g, 0.5 mmol) were added to the system in sequence. After stirring for 10 minutes, a solution of 4-hydroxyazetidine hydrochloride (0.7 mmol, 76 mg) in N,N-dimethylformamide (5 mL) was added and the reaction was allowed to react at room temperature for 1 hour. Water (30 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (50 mL×3). The organic phases were combined, washed with saturated brine (50 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (dichloromethane / methanol-20:1) to give the title compound 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidine-1-yl)-2-oxoethyl-1,1-d2)piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 6) (0.12 g, 45%).

[0433] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 8.19-8.10 (m, 2H), 7.20-7.12 (m, 2H), 6.45 (d, 1H), 4.67 (s, 1H), 4.49-4.40 (m, 1H), 4.30-4. 24 (m, 1H), 4.16-4.08 (m, 1H), 3.96-3.87 (m, 1H), 3.58 (s, 3H), 3.26 (s, 4H), 2.84 (s, 4H), 2.72 (q, 2H), 2.59 (s, 1H), 1.32 (t, 3H).

[0434] LCMS m / z=595.3[M+1] + .

[0435] Embodiment 7:

[0436] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxy-azetidin-1-yl-3-d)-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 7)

[0437] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl-3-d)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0438]

[0439] The crude product (300 mg) of 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (3G) was dissolved in acetonitrile (5 mL), and intermediate 2 (103 mg, 0.7 mmol) and potassium carbonate (172 mg, 1.3 mmol) were added to the system in sequence, and the temperature was raised to 80°C for reaction for 6 hours. Water (10 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (10 mL×3). The organic phases were combined, washed with saturated brine (10 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (dichloromethane / methanol (v / v)=20:1) to obtain white solid compound 7 (200 mg, 54%).

[0440] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 8.17-8.13 (m, 2H), 7.18-7.14 (m, 2H), 6.45 (d, 1H), 4.46-4.44 (m, 1H), 4.29-4.26 (m, 1H), 4.14-4.10(m, 1H), 3.92-3.89(m, 1H), 3.26-3.15(m, 6H), 2.83(s, 4H), 2.75-2.69(m, 2H), 2.54(s, 1H), 1.32(t, 3H).

[0441] LCMS m / z=597.3[M+1] + .

[0442] Embodiment 8:

[0443] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl-1,1-d2)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 8)

[0444] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl-1,1-d2)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0445]

[0446] first step:

[0447] Ethyl 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazin-1-yl)acetate-d2(8A)

[0448] ethyl 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazin-1-yl)acetate-d2

[0449]

[0450] Potassium carbonate (0.21 g, 1.5 mmol) and ethyl 2-bromoacetate-d2 (intermediate 3) (0.17 g, 1 mmol) were added to a solution of compound 3G (0.35 g, 0.73 mmol) in acetonitrile (10 mL) in sequence, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was diluted with saturated brine (30 mL) and extracted with ethyl acetate (30 mL×2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether (v / v)=1:1) to give a light yellow solid (0.38 g, yield 92%).

[0451] Step 2:

[0452] 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazin-1-yl)acetic acid-2,2-d2(8B)

[0453] 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazin-1-yl)acetic-2,2-d2 acid

[0454]

[0455] Ethyl 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazin-1-yl)acetate-d2(8A) (0.38 g, 0.67 mmol) was dissolved in tetrahydrofuran (9 mL), methanol (3 mL) and water (3 mL) were added, stirred evenly, lithium hydroxide monohydrate (126 mg, 3 mmol) was added, and the mixture was reacted at room temperature. After 1 hour, the pH was adjusted to 5 with hydrochloric acid (1N aqueous solution), extracted with ethyl acetate (50 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazine-1-yl)acetic acid-2,2-d2(8B) as a light yellow solid (0.32 g, yield 88%).

[0456] Step 3:

[0457] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl-1,1-d2)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 8)

[0458] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl-1,1-d2)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile

[0459]

[0460] 2-(4-(3-((5-cyano-4-(4-fluorophenyl)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]piperidin-5-yl)piperazine-1-yl)acetic acid-2,2-d2(8B) (0.32 g, 0.59 mmol) was dissolved in N,N-dimethylformamide (10 mL), and triethylamine (0.20 g, 2.0 mmol) and HATU (0.27 g, 0.7 mmol) were added to the system in sequence. After stirring for 10 minutes, a solution of 4-hydroxyazetidine hydrochloride (1 mmol, 0.11 g) in N,N-dimethylformamide (5 mL) was added and the reaction was allowed to react at room temperature for 1 hour. Water (30 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (50 mL×3). The organic phases were combined, washed with saturated brine (50 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (dichloromethane / methanol=20:1) to give 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidine-1-yl)-2-oxoethyl-1,1-d2)piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound 8) (0.28 g, 79%).

[0461] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 8.19-8.11 (m, 2H), 7.20-7.11 (m, 2H), 6.44 (d, 1H), 4.67 (s, 1H), 4.49-4.41 (m, 1H) , 4.32-4.24(m, 1H), 4.15-4.08(m, 1H), 3.94-3.84(m, 1H), 3.23(s, 4H), 2.79-2.66(m, 6H), 2.62(s, 1H), 1.32(t, 3H).

[0462] LCMS m / z=598.2[M+1] + .

[0463] Embodiment 9:

[0464] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (Compound 9)

[0465] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile

[0466]

[0467] first step:

[0468] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (9A)

[0469] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0470]

[0471] 4-(3-Amino-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylic acid tert-butyl ester (3D) (1.02 g, 2.8 mmol), 2-chloro-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (intermediate 4) (0.68 g, 2.8 mmol) and 2,6-dimethylpyridine (0.60 g, 5.6 mmol) were added successively to N,N-dimethylacetamide (20 mL), heated to 70°C and reacted for 5 hours. 10% sodium chloride solution (60 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (60 mL×3). The organic phases were combined, washed with saturated brine (60 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate (v / v) = 3:1) to give the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (9A) as a yellow-brown solid (1.02 g, yield 64%).

[0472] LCMS m / z=570.2[M+1] + .

[0473] Step 2:

[0474] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (9B)

[0475] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0476]

[0477] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (9A) (0.51 g, 0.89 mmol) was dissolved in N,N-dimethylformamide (10 mL), and potassium carbonate (0.27 g, 2 mmol) was added. Methyl iodide (0.19 g, 1.34 mmol) was added dropwise to the system. After addition, the mixture was reacted at room temperature for 30 minutes. Water (50 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×2). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (9B) as a yellow solid (0.52 g, yield 100%).

[0478] LCMS m / z=584.2[M+1] + .

[0479] Step 3:

[0480] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (9C)

[0481] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile

[0482]

[0483] The compound (3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)(methyl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (9B) (0.52 g, 0.89 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (3 mL) was added dropwise to the system, and after reacting for about 2 hours, the mixture was concentrated under reduced pressure to obtain the trifluoroacetate salt of the target compound 9C (0.58 g, brown viscous substance), and the crude product was directly subjected to the next step reaction.

[0484] LCMS m / z=484.2[M+1] + .

[0485] Step 4:

[0486] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (Compound 9)

[0487] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile

[0488]

[0489] The trifluoroacetate salt of compound 9C (0.58 g) was dissolved in acetonitrile (15 mL), and potassium carbonate (0.41 g, 3 mmol) and 2-chloro-1-(3-hydroxyazetidin-1-yl)ethanone (1 M) (0.22 g, 1.5 mmol) were added to the system in sequence, and the temperature was raised to 80°C for reaction for 3 hours. Water (50 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated brine (50 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (dichloromethane / methanol=20:1) to give the title compound 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (Compound 9) (0.39 g, 73% for two steps).

[0490] 1H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 6.45 (d, 1H), 4.67 (s, 1H), 4.50-4.41 (m, 1H), 4.32-4.24 (m, 1H), 4.15-4.08 (m, 1H) , 3.94-3.86 (m, 1H), 3.58 (s, 3H), 3.22 (s, 4H), 3.17-3.10 (m, 2H), 2.78 (s, 4H), 2.72 (q, 2H), 2.65 (s, 1H), 1.32 (t, 3H).

[0491] LCMS m / z=597.3[M+1] + .

[0492] Embodiment 10:

[0493] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (Compound 10)

[0494] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile

[0495]

[0496] first step:

[0497] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (10A)

[0498] tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate

[0499]

[0500] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (9A) (0.51 g, 0.89 mmol)) was dissolved in N,N-dimethylformamide (10 mL), potassium carbonate (0.27 g, 2 mmol) was added, and iodomethane-d3 (0.19 g, 1.34 mmol) was added dropwise to the system. After addition, the reaction was carried out at room temperature for 30 minutes. Water (50 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×2). The organic phases were combined, washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the target compound tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (10A) as a yellow solid (0.51 g, yield 98%).

[0501] LCMS m / z=587.2[M+1] + .

[0502] Step 2:

[0503] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (10B)

[0504] 2-((2-ethyl-6-fluoro-5-(piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile

[0505]

[0506] Tert-butyl 4-(3-((5-cyano-4-(4-fluorophenyl-2,3,5,6-d4)thiazol-2-yl)(methyl-d3)amino)-2-ethyl-6-fluoropyrazolo[1,5-a]pyridin-5-yl)piperazine-1-carboxylate (10A) (0.51 g, 0.88 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (3 mL) was added dropwise to the system. After reacting for about 2 hours, the mixture was concentrated under reduced pressure to give the trifluoroacetate salt of the target compound 10B (0.62 g, brown viscous substance), and the crude product was directly used for the next step.

[0507] LCMS m / z=487.3[M+1] + .

[0508] Step 3:

[0509] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (Compound 10)

[0510] 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazin-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile

[0511]

[0512] The trifluoroacetate salt of compound 10B (0.62 g) was dissolved in acetonitrile (15 mL), and potassium carbonate (0.46 g, 3.3 mmol) and 2-chloro-1-(3-hydroxyazetidin-1-yl)ethanone (1 M) (0.22 g, 1.5 mmol) were added to the system in sequence, and the temperature was raised to 80°C for reaction for 4 hours. Water (60 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (60 mL×3). The organic phases were combined, washed with saturated brine (60 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (dichloromethane / methanol (v / v) = 20:1) to give the title compound 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidine-1-yl)-2-oxoethyl)piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl-2,3,5,6-d4)thiazole-5-carbonitrile (Compound 10) (0.35 g, 66% for two steps).

[0513] 1 H NMR (400MHz, CDCl3) δ8.27 (d, 1H), 6.44 (d, 1H), 4.66 (s, 1H), 4.50-4.40 (m, 1H), 4.30-4.22 (m, 1H), 4.15-4. 05 (m, 1H), 3.93-3.85 (m, 1H), 3.21 (s, 4H), 3.18-3.04 (m, 2H), 2.90 (s, 1H), 2.77-2.66 (m, 6H), 1.32 (t, 3H).

[0514] LCMS m / z=600.3[M+1] + .

[0515] Example 11: Preparation of Compound A Crystalline Form I

[0516] The free base (9.1 g, 15.3 mmol) of 2-((2-ethyl-6-fluoro-5-(4-(2-(3-hydroxyazetidin-1-yl)-2-oxoethyl)piperazine-1-yl)pyrazolo[1,5-a]pyridin-3-yl)(methyl-d3)amino)-4-(4-fluorophenyl)thiazole-5-carbonitrile (Compound A, i.e., Compound 3) prepared in Example 3 was added to ethanol (50 mL), heated to 75°C, stirred for 2 h to dissolve, and gradually cooled to room temperature under slow stirring to precipitate a large amount of white solid. After suction filtration, the filter cake was washed with ethanol (10 mL) and vacuum dried at 40°C for 24 h to obtain Form I of Compound A, a white solid (6.2 g, yield 68%). Its XRPD pattern is as follows Figure 1 The XRPD diffraction peaks are listed in Table 1. The TGA graph is listed in Figure 2 , the DSC diagram is listed in Figure 3 The results show that the sample loses 1.3% of its weight when heated to 150°C, and has an endothermic peak at 178.7°C (starting temperature). The dynamic moisture sorption instrument (DVS) graph is listed in Figure 4 , at 25℃ / 80%RH the moisture adsorption is 0.20%. The PLM graph is listed in Figure 5 The results showed that the grain size was less than 50 μm.

[0517] Table 1 XRPD diffraction peak list of Compound A Form I

[0518]

[0519]

[0520] Test Case

[0521] 1. Solubility test

[0522] The dynamic solubility of Compound A Form I (Example 11) in water and three biological solvents was evaluated. The dynamic solubility of the sample in four solvent systems of water, SGF, FaSSIF and FeSSIF was measured at 37°C using a rotary mixing method (25 rpm) at a feed concentration of 5-10 mg / mL (1, 2, 4 and 24 hours). The sample at each time point was centrifuged (12000 rpm, 5 min) and filtered (0.45 μm PTFE filter head), and the HPLC concentration and pH value of the filtrate were determined. The solubility test results are summarized in Table 2.

[0523] Table 2 Summary of dynamic solubility test results

[0524]

[0525] S: solubility (mg / mL);

[0526] LOQ: 0.03 μg / mL

[0527] The initial pH of H2O, SGF, FaSSIF and FeSSIF were 6.9, 1.8, 6.5 and 5.0, respectively.

[0528] Preparation of biosolvents

[0529] Preparation of simulated gastric fluid (SGF): weigh 0.1g NaCl and 0.05g Trinaton X-100 into a 50mL volumetric flask, add purified water to dissolve. Add 67.5μL concentrated hydrochloric acid (12M), adjust the pH to 1.8 with 1M hydrochloric acid or 1M NaOH solution. Add purified water to make up to volume.

[0530] Preparation of simulated fasting intestinal fluid (FaSSIF): Weigh 0.17g anhydrous NaH2PO4, 0.021g NaOH and 0.31g NaCl into a 50mL volumetric flask. Add about 48mL purified water to dissolve, and adjust the pH to 6.5 with 1M hydrochloric acid or 1M NaOH solution. Add purified water to make up to volume, and weigh 0.11g SIF powder to dissolve.

[0531] Preparation of simulated fed intestinal fluid (FeSSIF): Take 0.41 mL of glacial acetic acid, 0.20 g of NaOH, and 0.59 g of NaCl into a 50 mL volumetric flask. Add about 48 mL of purified water to dissolve, and adjust the pH to 5.0 with 1 M hydrochloric acid or 1 M NaOH solution. Add purified water to make up to volume, and weigh in 0.56 g of SIF powder to dissolve.

[0532] 2. Stability test

[0533] After the crystal form I (Example 11) was placed under 25°C / 60%RH and 40°C / 75%RH conditions for 12 days, the sample purity was tested and the crystal form transformation was observed. The results are shown in Table 3.

[0534] Table 3 Crystal stability evaluation

[0535]

[0536] Conclusion: The crystalline form I of compound A of the present invention has good stability.

[0537] Biological experiments

[0538] 1. In vitro experiments

[0539] Autotaxin is a plasma phosphodiesterase that converts lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), therefore LPA formation is used to assess the potency of autotaxin inhibitors.The potency of the compounds was assessed in pooled ex vivo human plasma.

[0540] Plasma was collected after centrifugation of heparinized whole blood. 5-μL of serial dilutions of the test compound or DMSO were added to 95-μL plasma and incubated at 37°C for 2 h before adding the stop solution (40 mM sodium hydrogen phosphate buffer containing 30 mM citric acid, pH = 4). LPA in plasma was detected by LC-MSMS before and after incubation. To determine the concentration of LPA 18:2 or 20:4 in the study plasma, calibration standards of LPA 18:2 or 20:4 were prepared by serial dilution in butanol: 20,000, 10,000, 5,000, 2,000, 1,000, 500, 200, 100, 50, 20, and 10 ng / mL. 3 μL of calibration standard solution was added to 27.0 μL of blank plasma in a 1.5 mL microcentrifuge tube to generate a 1X calibration standard. 30.0 μL of standard or study plasma was added to a 1.5 mL microcentrifuge tube. 200 μL of butanol (containing 25.0 ng / mL LPA17:0 for internal control) was added to each 1.5 mL microcentrifuge tube containing the study plasma or calibration standard. After vortexing for 1 minute and centrifuging at 10000 rpm for 10 min, 180 μL of the supernatant was transferred to a 96-well plate and quantified with the standard using LC / MS / MS for the concentration of LPA18:2 in plasma. Briefly, 8 μL of the solution was injected for LC-MS / MS analysis using an ACQUITY UPLC BEH C18 column (2.1 x 50 mm, 1.7 μm), using mobile phase A [20 mM NH4OAC aqueous solution (0.1% FA)] and mobile phase B [5 mM NH4OAC aqueous solution / 0.2% FA in ACN = 5:95]. Mass spectrometer parameter optimization for LPA18:2 was performed by deprotonating the molecular ions at m / z 433.2 ([MH]-) for LPA18:2 and 457.2 for LPA20:4, and abundant product ions at m / z 152.8 were obtained for both LPA18:2 and LPA20:4. Quantitative data were acquired in multiple reaction monitoring (MRM) negative electrospray ionization mode.

[0541] The inhibition rate of LPA production by different concentrations of compounds was determined by comparing the changes in LPA levels in incubated and unincubated plasma, and the IC of the compound was calculated. 50 To calculate the inhibition rate, the relative concentration of each well = the concentration of each well - the average concentration of the baseline.

[0542] Inhibition rate % = (average relative concentration of control wells - average relative concentration of test wells) / average relative concentration of control wells × 100%. The curve was plotted as inhibition rate (y-axis) and compound concentration (x-axis), and was fitted using GraphPad Prism 7.0 using logarithm (inhibitor) and normalized response (variable slope).

[0543] Table 4 IC of test compounds for inhibition of LPA formation 50 (nM)

[0544] Example LPA formation (18∶2) LPA formation (20∶4) 1 46.05 48.51 2 51.96 55.27 3 42.66 44.47 4 51.79 54.81

[0545] 2. Pharmacokinetic testing

[0546] 2.1 Pharmacokinetic test in rats

[0547] Experimental purpose: To administer the test substance to SD rats by single-dose intravenous and oral gavage, determine the concentration of the test substance in rat plasma, and evaluate the pharmacokinetic characteristics and bioavailability of the test substance in rats.

[0548] Experimental subject: the compound of Example 3 of the present invention.

[0549] Experimental animals: Male SD rats, about 220 g, 6 to 8 weeks old, 6 rats / compound, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0550] Experimental method: On the day of the experiment, 6 SD rats were randomly divided into groups according to their body weight. They were fasted but not watered for 12-14 hours one day before administration and fed 4 hours after administration. The administration was performed according to Table 5.

[0551] Table 5 Dosage information

[0552]

[0553] *Dosage is based on free base.

[0554] 2.2 Biological sample collection

[0555] Before and after drug administration, 0.1 mL of blood was collected from the eye socket under isoflurane anesthesia and placed in an EDTAK2 centrifuge tube. The tube was centrifuged at 5000 rpm and 4°C for 10 min to collect plasma.

[0556] Sample collection time points for group G1: 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 24 h.

[0557] Sample collection time points for group G2: 0, 15, 30 min, 1, 2, 4, 6, 8, 24 h.

[0558] All samples were stored at -80°C before analysis.

[0559] 2.3 Sample pretreatment

[0560] Take 30 μL of plasma sample, standard curve and quality control sample, add 200 μL of acetonitrile solution containing internal standard, vortex mix, centrifuge at 4°C, 12000 rpm for 10 min. Take 170 μL of supernatant in 96-well plate for LC-MS / MS analysis, and the injection volume is 2 μL.

[0561] The main pharmacokinetic parameters were analyzed by non-compartmental model using WinNonlin 8.0 software. The experimental results are shown in Table 6.

[0562] Table 6 Pharmacokinetics in rats

[0563]

[0564] Conclusion: The compounds of the present invention, especially compounds 2, 9, and 10, have good pharmacokinetics and high bioavailability in rats, and compounds 5, 6, and 7 have rapid onset of action.

[0565] 2.2 Pharmacokinetic test in mice

[0566] Purpose of the experiment: To evaluate the pharmacokinetic characteristics and bioavailability of the test substance in mice.

[0567] Experimental animals: C57 mice, 25 g, male, 6-8 weeks old, 36 mice, purchased from Chengdu Dashuo Experimental Animal Co., Ltd., production license number: SCXK (Chuan) 2020-030.

[0568] Test subjects: Compound I-1 (Compound 101 of patent WO2019228403A1) and Compound 3.

[0569] Experimental Design:

[0570] Table 7 Dosage information

[0571]

[0572]

[0573] Intravenous administration solvent: 5% DMA + 5% Solutol + 90% Saline; intragastric administration solvent: 0.5% MC

[0574] Before and after administration, 0.06 mL of blood was collected from the eye socket under isoflurane anesthesia, placed in an EDTAK2 centrifuge tube, and centrifuged at 5000 rpm at 4°C for 10 min to collect plasma. Blood collection time points: 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 10, 24 h. All plasma samples were stored at -80°C before analysis and testing. The experimental results are shown in Table 8.

[0575] Table 8 Pharmacokinetics in mice

[0576]

[0577] Conclusion: The compounds of the present invention, especially compound 3, have good pharmacokinetics and high bioavailability in mice.

[0578] 2.3 Pharmacokinetic testing in dogs

[0579] Purpose of the study: To evaluate the pharmacokinetic characteristics and bioavailability of the test substance in dogs.

[0580] Experimental animals: Beagle dogs, 8-10 kg, male, 0.5-1.5 years old, 12 dogs, purchased from Beijing Mas Biotechnology Co., Ltd., production license number: SCXK (Beijing) 2016-001.

[0581] Test subjects: Compound I-1 (Compound 101 of patent WO2019228403A1) and Compound 3.

[0582] Experimental Design:

[0583] Table 9 Dosage information

[0584]

[0585] Intravenous administration solvent: 5% DMSO + 5% Solutol + 90% Saline;

[0586] Intragastric administration solvent: 1% DMSO + 1% Solutol + 98% (0.5% MC);

[0587] Before and after administration, 1.0 mL of blood was collected from the forelimb vein, placed in an EDTAK2 centrifuge tube, and centrifuged at 5000 rpm at 4°C for 10 min to collect plasma. Blood collection time points: 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 10, 12, 24 h. All plasma samples were stored at -80°C before analysis and testing. The experimental results are shown in Table 10.

[0588] Table 10 Pharmacokinetics in Dogs

[0589]

[0590] Conclusion: The compounds of the present invention, especially compound 3, have good pharmacokinetics and high bioavailability in dogs.

[0591] 2.4 Pharmacokinetic studies in monkeys

[0592] Purpose of the study: To evaluate the pharmacokinetic characteristics and bioavailability of the test substance in monkeys.

[0593] Experimental animals: 12 cynomolgus monkeys, 2.4-5.9 kg, male, 3-5.5 years old, purchased from Suzhou Xishan Zhongke Experimental Animal Co., Ltd., production license number: SCXK (Su) 2018-0001.

[0594] Test subjects: Compound I-1 (Compound 101 of patent WO2019228403A1) and Compound 3.

[0595] Experimental Design:

[0596] Table 11 Dosage information

[0597]

[0598] Intravenous administration solvent: 5% DMSO + 5% Solutol + 90% Saline;

[0599] Oral administration solvent: 1% DMSO + 1% Solutol + 98% (0.5% CMC-Na)

[0600] Before and after administration, 1.0 mL of blood was collected from the forelimb vein, placed in an EDTAK2 centrifuge tube, and centrifuged at 5000 rpm and 4°C for 10 min to collect plasma. Blood collection time points: 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 10, 12, 24 h. All plasma samples were stored at -80°C before analysis and testing. The experimental results are shown in Table 12.

[0601] Table 12 Monkey Pharmacokinetics

[0602]

[0603] Conclusion: The compounds of the present invention, especially compound 3, have good pharmacokinetics and high bioavailability in monkeys.

[0604] 2.5 hERG potassium channel action test

[0605] Test compounds: Compound I-1 (Compound 101 of Patent WO2019228403A1) and Example Compound 3.

[0606] The effects of the example compounds on hERG potassium channel (human Ether-a-go-go Related Gene potassium channel) current were tested using the electrophysiological manual patch clamp method.

[0607] Table 13 Experimental materials and instruments

[0608] Experimental Materials Provider (Product Number) 6cm cell culture dish Shanghai Huayasichuang Biotechnology Co., Ltd. (150288) 3.5cm cell culture dish Shanghai Huayasichuang Biotechnology Co., Ltd. (153066) Dialyzed fetal bovine serum Shanghai Bohan Biotechnology Co., Ltd. (BS-0005-500) DMSO Merck KGaA (102952) DMEM medium Thermo Fisher Scientific (China) Co., Ltd. (10569) HEPES Thermo Fisher Scientific (China) Co., Ltd. (15630080) Trypsin Thermo Fisher Scientific (China) Co., Ltd. (12604) Phosphate buffer (without calcium and magnesium ions) Thermo Fisher Scientific (China) Co., Ltd. (14190) Penicillin-Streptomycin Solution Thermo Fisher Scientific (China) Co., Ltd. (15140-122) MEM Non-Essential Amino Acid Solution Thermo Fisher Scientific (China) Co., Ltd. (11140) Geneticin (G418) Thermo Fisher Scientific (China) Co., Ltd. (11811031) Blasticidin Thermo Fisher Scientific (China) Co., Ltd. (R21001) Poly-L-lysine Thermo Fisher Scientific (China) Co., Ltd. (P4832) Dofelder Beijing Ipress Technology Development Co., Ltd. (D525700) Doxycycline Sigma-Aldrich (Shanghai) Trading Co., Ltd. (D9891) Experimental instruments Provider(Model) Carbon dioxide incubator Thermo Fisher Scientific (China) Co., Ltd. (371) Drawing machine Sutter Company, USA (P-97) Micromanipulator Siskiyou, USA (MC1000e) Sutter Corporation (ROE-200; MP285) Amplifier HEKA, Germany (EPC10) Amplifier AXON Corporation (Multiclamp 700B) microscope Olympus China Co., Ltd. (IX51 / 71 / 73) Perfusion system ALA Company of the United States (VM8 gravity dosing system)

[0609] Cell lines and cell culture:

[0610] HEK293 cell lines stably expressing hERG ion channels were purchased from Invitrogen. The cell lines were cultured in a medium containing 85% DMEM, 10% dialyzed fetal bovine serum, 0.1 mM MEM non-essential amino acid solution, 100 U / mL penicillin-streptomycin solution, 25 mM HEPES, 5 μg / mL blasticidin and 400 μg / mL geneticin (G418). When the cell density grew to 40% to 80% of the bottom area of ​​the culture dish, the cells were digested by trypsin and passaged three times a week. Before the experiment, the cells were cultured at 5×10 5 The cells were cultured at a density of 1.54 μg / nL in a 6 cm dish and induced for 48 h by adding 1 μg / nL doxycycline. The cells were then digested and seeded on glass slides for subsequent manual patch clamp experiments.

[0611] Solution preparation:

[0612] Extracellular solution (in mM): 132 NaCl, 4 KCl, 3 CaCl2, 0.5 MgCl2, 11.1 Glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).

[0613] Preparation of test compound solution:

[0614] The test compound was first dissolved in DMSO and prepared into a stock solution with a final concentration of 30 mM. The original stock solution was then diluted with DMSO in a certain ratio to form four other gradient series solutions with concentrations of 10, 3.33, 1.11 and 0.37 mM, respectively. Before the experiment, the gradient series solution of the test compound was diluted again with extracellular fluid at a ratio of 1:1000 to form a series of working solutions with gradient concentrations, with final concentrations of 30, 10, 3.33, 1.11 and 0.37 μM, respectively. The five working solutions with different gradient concentrations were used to determine the potential inhibitory effect of the compound on the hERG potassium channel and to fit the dose-effect curve and calculate the IC 50 .

[0615] Experimental steps:

[0616] 1. Place the small glass slide containing HEK293 cells in the culture dish into the perfusion tank on the microscope operation table.

[0617] 2. Place the appropriate cell in the center of the field of view under an Olympus IX51, IX71 or IX73 inverted microscope, use a ×10 objective lens to find the tip of the glass electrode and place it in the center of the field of view. Then use the micromanipulator to move the electrode down while adjusting the coarse focusing knob to slowly bring the electrode close to the cell.

[0618] 3. When approaching the cell, switch to a ×40 objective lens for observation and use the micromanipulator to fine-tune the gear to gradually bring the electrode closer to the cell surface.

[0619] 4. Apply negative pressure to form a seal with a resistance higher than 1GΩ between the electrode tip and the cell membrane.

[0620] 5. In the voltage clamp mode, the instantaneous capacitive current C fast Then, short negative pressure is repeatedly applied to break the membrane, eventually forming the whole-cell recording mode.

[0621] 6. Under the condition that the membrane potential is clamped at -60mV, the slow capacitive current C slow , cell membrane capacitance (Cm) and input membrane resistance (Ra) were compensated respectively.

[0622] 7. After the cells are stable, change the clamping voltage to -90mV, set the sampling frequency to 20kHz, and the filtering frequency to 10kHz. The detection conditions for leakage current are: the clamping voltage is changed to -80mV, and the time course is 500ms.

[0623] 8. The hERG current test method is as follows: apply a 4.8-second depolarization command voltage to depolarize the membrane potential from -80mV to +30mV, then instantly apply a 5.2-second repolarization voltage to reduce the membrane potential to -50mV to remove channel inactivation, so that the hERG tail current can be observed. The peak value of the tail current is the magnitude of the hERG current.

[0624] 9. The hERG current used to detect the test compound was recorded for 120 seconds before administration to evaluate the stability of the hERG current generated by the test cells. Only stable cells within the acceptable range of the evaluation criteria can enter the subsequent compound testing.

[0625] 10. Test of the inhibitory effect of the test compound on hERG current: First, the hERG current measured in the extracellular fluid containing 0.1% DMSO is used as the detection baseline. After the hERG current remains stable for at least 5 minutes, the solution containing the test compound is perfused around the cells from low concentration to high concentration. After each perfusion, wait for about 5 minutes to allow the compound to fully act on the cells and record the hERG current synchronously. After the recorded current tends to stabilize, record the last 5 hERG current values, and take the average value as the final current value at a specific concentration. After testing the compound, add 150nM dofelide to the same cell to completely inhibit its current as a positive control for the cell. At the same time, the positive compound dofelide is synchronously detected using the same patch clamp system before and after the test drug experiment to ensure the reliability and sensitivity of the entire detection system.

[0626] Data analysis: The current inhibition percentage was calculated using the following formula. The data were output by PatchMaster or Clampex10.2 software. The dose-effect curve was fitted and the IC was calculated using Graphpad Prism 8.0 software. 50 The experimental results are shown in Table 11.

[0627]

[0628] Table 14 Test results

[0629] Serial number Test samples <![CDATA[hERG IC 50 [μM]]]> 1 Compound I-1 9.368 2 Dofetilide 0.016 3 Compound 3 23.113

[0630] The widely recognized and used criteria for evaluating whether a compound has an inhibitory effect on hERG potassium ion channels are as follows: Inhibition is not significant: IC 50 >10μM; Moderate inhibition: 1μM<IC 50 <10μM; significant inhibitory effect: IC 50 <1μM. From the test results, compound 3 has no significant inhibitory effect on hERG potassium ion channel.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from one of the following structures:

2. Form I of the compound of formula (A), It is characterized in that Using Cu—Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 9.35±0.2°, 10.32±0.2°, 12.08±0.2° and 15.10±0.2°.

3. The crystalline form I of the compound of formula (A) according to claim 2, wherein its X-ray powder diffraction pattern also has characteristic diffraction peaks at the following 2θ positions: 17.29±0.2°, 18.26±0.2° and 20.58±0.2°.

4. The crystalline form I of the compound of formula (A) according to claim 3, wherein its X-ray powder diffraction pattern also has characteristic diffraction peaks at the following 2θ positions: 6.92±0.2°, 7.23±0.2°, 13.46±0.2°, 19.47±0.2° and 24.85±0.2°.

5. The crystalline form I of the compound of formula (A) according to claim 4, whose X-ray powder diffraction pattern is substantially as shown in Figure 1.

6. The crystalline form I of the compound of formula (A) according to claim 2, whose TGA curve is substantially as shown in FIG. 2, and whose DSC graph is substantially as shown in FIG.

3.

7. Compound of formula (II-C) shown below 8. A method for preparing a compound of formula (A), characterized in that: Contains the following steps The compound of formula (II-C) is dissolved in an organic solvent A, and reacted with 2-chloro-1-(3-hydroxyazetidin-1-yl)ethanone and a base at 70-90° C. to obtain a compound of formula (A).

9. The preparation method according to claim 8, characterized in that: The organic solvent A is selected from at least one of acetonitrile, 2-methyltetrahydrofuran, ethylbenzene, ethyl methyl formate and diethylene glycol tert-butyl ether; and the base is at least one of potassium carbonate and sodium carbonate.

10. The preparation method according to claim 8, characterized in that: The preparation method of the compound of formula (A) further comprises the following steps: i. a compound of formula (II-D), 2-chloro-4-(4-fluorophenyl)thiazole-5-cyano, 2,6-dimethylpyridine and a non-protonic polar solvent, reacting at 60 to 80° C. to obtain a compound (II-A); ii. Dissolve the compound (II-A) obtained in step i in an organic solvent B, cool to 0° C. under nitrogen protection, add a reducing agent, and after the reaction is completed, add iodomethane-d3 to obtain a compound (II-B); iii. Dissolve the compound (II-B) obtained in step ii in an organic solvent C, add trifluoroacetic acid, and react to obtain a compound of formula (II-C).

11. The preparation method according to claim 10, characterized in that: The aprotic polar solvent is at least one of N,N-dimethylacetamide, dimethylformamide, hexamethylphosphoramide, acetonitrile, acetone and dimethyl sulfoxide; the organic solvent B is at least one of tetrahydrofuran, N,N-dimethylacetamide, dimethylformamide, acetonitrile and acetone; the reducing agent is sodium hydride; the organic solvent C is at least one of dichloromethane, methyl acetate, dimethyl carbonate, propylene glycol methyl ether acetate and nylon acid dimethyl ester.

12. A pharmaceutical composition, characterized in that Containing the compound of claim 1 or a pharmaceutically acceptable salt thereof, or containing the crystalline form I according to any one of claims 2 to 6, and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition, characterized in that Containing the compound of claim 1 or a pharmaceutically acceptable salt thereof, or containing the crystalline form I according to any one of claims 2 to 6, and a pharmaceutically acceptable excipient.

14. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the crystalline form I comprising any one of claims 2 to 6, or the composition of claim 12 or 13 in the preparation of a medicament for treating an ATX-mediated disease, wherein the ATX-mediated disease is idiopathic pulmonary fibrosis.

Images