CN116669734A - Crystal form of pyrimido five-membered nitrogen heterocyclic derivative and preparation method thereof - Google Patents

Abstract

The present disclosure relates to a crystalline form of a pyrimido five-membered nitrogen heterocyclic derivative and a preparation method thereof. In particular, the present disclosure relates to different crystal forms of a compound represented by formula (I) and a preparation method thereof, and the crystal forms of the compound represented by formula (I) provided by the present disclosure have good stability, and can be better used for clinical treatment.

Description

Crystal form of pyrimido five-membered nitrogen heterocyclic derivative and preparation method thereof

The present application claims priority from chinese patent application 202011563824.1 with filing date 2020/12/25. The present application incorporates the entirety of the above-mentioned chinese patent application.

Technical Field

The present disclosure relates to a crystal form of pyrimidine five-membered nitrogen heterocyclic derivative, a preparation method and a medical application thereof, and belongs to the field of pharmacy.

Background

The Src homology 2-containing protein tyrosine phosphatase 2 (Src homology domain 2containing tyrosine phosphatase-2, SHP 2) is an evolutionarily conserved non-receptor Protein Tyrosine Phosphatase (PTP) encoded by a PTPN11 gene, mainly consists of two SH2 domains (N-SH 2 and C-SH 2) and a PTP catalytic domain, is widely expressed in various tissues of human beings, and plays an important role in maintaining tissue development, cell homeostasis and the like. SHP2 is associated with signaling through the Ras-mitogen-activated protein kinase, JAK-STAT, or phosphoinositide 3-kinase AKT pathway. Mutations in the PTPN11 gene and subsequent mutations in SHP2 have been identified in a variety of human diseases, such as Noonan Syndrome (Noonan Syndrome), leopard Syndrome (Leopard Syndrome), juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myelogenous leukemia, and breast, lung and colon cancers (same as Claim 19). Thus, SHP2 represents a target that can be attractive for the development of new therapies for the treatment of various diseases.

Published patent applications for related studies of SHP2 targets are WO2018136264A, WO2015003094A, WO2018160731A, WO2018130928A1, WO2018136265A, WO2018172984A, WO2018081091, WO2016203405, WO2017211303A, WO2018013597A, etc.; currently, both the North SHP2 inhibitor TNO155 and the JAB-3068 SHP2 inhibitor JACOBIO are in phase I clinical stage, and no product of the target point is marketed, so that more efficient new SHP2 inhibitors still need to be developed continuously in order to provide new and effective anticancer drugs for patients.

The patent application of WO2020259679A discloses a compound shown in a formula (I), the crystal forms of the compound are necessary to be researched in order to meet the requirement of medication,

disclosure of Invention

The present disclosure provides a crystalline form a of a compound of formula (I),

the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 4.847, 9.801, 13.778, 14.770, 15.444, 26.077.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern of form a of a compound of formula (I), expressed as diffraction angle 2θ, having characteristic peaks at 4.847, 9.801, 13.138, 13.778, 14.770, 15.444, 18.363, 19.856, 21.092, 23.371, 26.077, 28.130.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern of form a of a compound of formula (I) as represented by the angle of diffraction 2θ, having characteristic peaks at 4.847, 9.801, 13.138, 13.778, 14.770, 15.444, 18.363, 19.856, 21.092, 22.034, 23.371, 24.460, 26.077, 28.130, 28.970, 31.894, 32.920, 33.916, 38.924.

In an alternative embodiment, the present disclosure provides a crystalline form a of a compound of formula (I) having an X-ray powder diffraction pattern as shown in figure 2.

The present disclosure provides a process for the preparation of form a of a compound of formula (I) selected from:

a) Mixing a compound shown in a formula (I) with a solvent I, dissolving, crystallizing and separating out, wherein the solvent I is selected from

At least one of dichloromethane and chloroform; or (b)

b) Mixing a compound shown in a formula (I) with a solvent II, and crystallizing and separating out, wherein the solvent II is at least one selected from tetrahydrofuran, ethyl acetate, toluene, acetone, methanol, ethanol, acetonitrile, methyl tertiary butyl ether, water, isopropyl ether, butanone and n-hexane; or (b)

c) Mixing a compound shown in a formula (I) with a solvent III to obtain a solution, mixing the solution with a solvent IV, and crystallizing and separating out the solution, wherein the solvent III is at least one selected from methanol, N-dimethylformamide and N-methylpyrrolidone; the solvent IV is at least one selected from acetonitrile, ethyl acetate, isopropyl acetate, methyl tertiary butyl ether and isopropyl ether.

The present disclosure provides a form B of a compound of formula (I) having an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, with characteristic peaks at 4.606, 9.110, 11.423, 13.745, 16.006, 22.973.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern of form B of a compound of formula (I), expressed as diffraction angle 2θ, having characteristic peaks at 4.606, 9.110, 11.423, 13.745, 16.006, 18.349, 22.973, 25.285, 27.505, 29.262.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern of form B of a compound of formula (I), expressed in terms of diffraction angle 2θ, having characteristic peaks at 4.606, 9.110, 11.423, 13.745, 16.006, 18.349, 19.767, 22.973, 24.700, 25.285, 27.505, 29.262, 31.451, 32.407, 34.072, 35.983, 37.216, 38.388.

A preparation method of a B crystal form of a compound shown in a formula (I) comprises the steps of dissolving the compound shown in the formula (I) in methanol, filtering, mixing with methyl tertiary butyl ether, and crystallizing.

The present disclosure provides a C-crystal form of a compound represented by formula (I), an X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, having characteristic peaks at 8.905, 12.920, 13.428, 14.074, 18.458, 22.519.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern of a compound of formula (I) in the form C, expressed as diffraction angle 2θ, having characteristic peaks at 8.905, 12.920, 13.428, 14.074, 16.104, 17.996, 18.458, 18.965, 20.580, 22.519, 23.949, 26.395, 28.795, 31.748.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, of form C of a compound of formula (I), having characteristic peaks at 8.905, 12.920, 13.428, 14.074, 16.104, 17.996, 18.458, 18.965, 20.580, 22.519, 23.949, 25.011, 26.395, 27.226, 28.379, 28.795, 30.041, 31.748, 32.487, 35.578, 37.978, 41.393.

A process for preparing the C crystal form of compound shown in formula (I) includes such steps as mixing the compound shown in formula (I) with at least one solvent chosen from dioxane, N-methylpyrrolidone and N, N-dimethylformamide to obtain the solution, mixing the solution with at least one solvent chosen from methyl tert-butyl ether and isopropanol, and crystallizing.

The present disclosure provides a D-form of a compound of formula (I) having an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, with characteristic peaks at 4.766, 9.594, 14.089, 14.471, 18.981, 19.609, 25.987.

In alternative embodiments, the present disclosure provides a D-form of a compound of formula (I) having an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, with characteristic peaks at 4.766, 9.594, 13.461, 14.089, 14.471, 16.602, 18.981, 19.609, 20.238, 22.616, 24.187, 24.770, 25.264, 25.987, 29.123, 30.380, 32.714, 34.688, 39.042, 39.535, 44.382.

A process for the preparation of form D of a compound of formula (I) selected from the following processes:

a) Mixing a compound shown in a formula (I) with methanol to obtain a solution, mixing the solution with isopropyl ether, and crystallizing out;

b) Mixing the compound shown in the formula (I) with ethanol-water, dissolving, volatilizing and crystallizing.

The present disclosure provides an E-form of a compound of formula (I) having an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, with characteristic peaks at 4.603, 9.209, 13.920, 15.097, 19.700, 25.454.

In alternative embodiments, the present disclosure provides an E-form of a compound of formula (I) having an X-ray powder diffraction pattern with characteristic peaks at 4.603, 9.209, 12.888, 13.920, 15.097, 18.641, 19.194, 19.700, 20.529, 22.876, 25.454, 25.664, 27.775, 29.504, expressed in terms of diffraction angle 2θ.

In alternative embodiments, the present disclosure provides an E-form of a compound of formula (I) having an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, with characteristic peaks at 4.603, 9.209, 12.888, 13.920, 15.097, 18.641, 19.194, 19.700, 20.529, 21.863, 22.876, 23.659, 24.349, 25.054, 25.454, 25.664, 27.203, 27.698, 27.775, 28.906, 29.504, 29.965, 30.747, 31.944, 34.430, 38.756, 39.263, 42.577.

A process for the preparation of form E of a compound of formula (I) selected from:

a) Mixing a compound shown in a formula (I) with a solvent V to obtain a solution, mixing the solution with a solvent VI, and crystallizing and separating out the solution, wherein the solvent V is at least one selected from N, N-dimethylformamide, methanol, dimethyl sulfoxide and dichloromethane; the solvent VI is at least one selected from isopropyl ether, methyl tertiary butyl ether, isopropanol, dioxane, acetone, n-hexane, toluene and acetonitrile; or (b)

b) Mixing the compound shown in the formula (I) with a dichloromethane-acetonitrile solvent, dissolving, and crystallizing to separate out.

The present disclosure provides an X-ray powder diffraction pattern of form F of a compound of formula (I), expressed as diffraction angle 2θ, having characteristic peaks at 4.656, 14.068, 15.183, 18.858, 23.235.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern of a compound of formula (I) in the form F, expressed as diffraction angle 2θ, having characteristic peaks at 2θ angles 4.656, 9.341, 12.670, 13.443, 14.068, 14.850, 15.183, 18.858, 19.436, 20.518, 23.235, 25.591, 28.386, 29.543.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, of form F of a compound of formula (I), having characteristic peaks at 4.656, 9.341, 12.670, 13.443, 14.068, 14.850, 15.183, 16.122, 18.032, 18.858, 19.436, 20.518, 22.150, 22.571, 23.235, 24.442, 24.863, 25.591, 26.585, 27.765, 28.386, 28.934, 29.543, 30.245, 31.113, 32.116, 32.491, 36.608, 38.199, 38.620, 40.679, 43.206.

The present disclosure provides a process for preparing form F of a compound of formula (I), by mixing the compound of formula (I) with ethanol-water and crystallizing.

The present disclosure provides a form G of a compound of formula (I) having an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, with characteristic peaks at 4.869, 9.735, 13.290, 14.713, 20.020.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern of a compound of formula (I) in the form G, expressed as diffraction angle 2θ, having characteristic peaks at 4.869, 9.735, 10.863, 13.290, 14.713, 14.968, 17.852, 19.399, 20.020, 20.593, 21.796, 22.793, 24.251, 24.681, 25.510, 25.907, 26.327, 27.617, 30.155.

In alternative embodiments, the present disclosure provides an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, of form G of a compound of formula (I), having characteristic peaks at 4.869, 9.735, 10.863, 13.290, 14.713, 14.968, 17.852, 19.399, 20.020, 20.593, 21.796, 22.793, 24.251, 24.681, 25.510, 25.907, 26.327, 27.617, 28.051, 29.696, 30.155, 31.101, 32.405, 33.402, 35.019, 39.615, 41.021, 45.193, 46.644, 54.898.

A preparation method of a G crystal form of a compound shown in a formula (I) comprises the steps of mixing the compound shown in the formula (I) with ethanol-water, dissolving, mixing with water, crystallizing, and drying at 40 ℃ for 3 days.

The present disclosure provides an H-crystalline form of a compound of formula (I), having an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, with characteristic peaks at 8.608, 12.983, 13.476, 17.716, 20.144, 23.371.

In alternative embodiments, the present disclosure provides an H-crystalline form of a compound of formula (I) having an X-ray powder diffraction pattern, expressed as diffraction angle 2θ, with characteristic peaks at 8.608, 11.238, 12.983, 13.476, 17.716, 18.227, 19.392, 20.144, 21.435, 21.914, 22.935, 23.371, 23.814, 25.416, 30.620.

In alternative embodiments, the present disclosure provides an H-form of a compound of formula (I) having an X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, with characteristic peaks at 8.608, 11.238, 11.718, 12.983, 13.476, 14.485, 17.716, 18.227, 19.392, 20.144, 21.435, 21.914, 22.935, 23.371, 23.814, 25.416, 26.499, 28.195, 28.887, 30.620, 31.767, 32.874, 33.970, 34.839, 35.854, 37.045, 38.478, 39.958, 41.311, 43.925, 44.582.

A preparation method of H crystal form of a compound shown in a formula (I) comprises the steps of mixing the compound shown in the formula (I) with ethanol-water, and crystallizing out.

In certain embodiments, the methods of making the crystalline forms described in the present disclosure further comprise a filtration, washing, or drying step.

The present disclosure provides a form A, a form B, a form C, a form D, a form E, a form F, a form G and a form H of a compound of formula (I) prepared by the above preparation method.

In alternative embodiments, the compounds of formula (I) and their corresponding crystalline forms in the present disclosure have an abundance of at least 20% per deuterium atom (D).

In alternative embodiments, the compounds of formula (I) and their corresponding crystalline forms of the present disclosure have an abundance of at least 50% per deuterium atom (D).

In alternative embodiments, the compounds of formula (I) and their corresponding crystalline forms in the present disclosure have an abundance of at least 90% per deuterium atom (D).

In alternative embodiments, the compounds of formula (I) and their corresponding crystalline forms of the present disclosure have an abundance of at least 98% per deuterium atom (D).

The present disclosure also provides a pharmaceutical composition comprising a crystalline form of a compound of formula (I) as described above, or a crystalline form of a compound of formula (I) as prepared by the process described above, or a mixture thereof, and optionally from a pharmaceutically acceptable carrier, diluent or excipient.

The present disclosure also provides pharmaceutical compositions prepared from the crystalline forms of the compounds of formula (I) described above.

The present disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing a crystalline form of the compound of formula (I) described above, or a crystalline form of the compound of formula (I) prepared by the method described above, or a mixture thereof, with a pharmaceutically acceptable carrier, diluent or excipient.

The present disclosure also provides the use of a crystalline form of a compound of formula (I) as described above, or a crystalline form of a compound of formula (I) as prepared by the process described above, or a mixture thereof, or a composition as described above, or a composition as prepared by the process described above, in the manufacture of a medicament for the treatment of a disease or disorder associated with SHP2 modulation.

The disclosure also provides the use of the crystal form of the compound shown in the formula (I), or the crystal form of the compound shown in the formula (I) prepared by the method, or a mixture thereof, or the composition prepared by the method, in preparing a medicament for preventing and/or treating tumor or cancer.

The disclosure also provides the use of the crystal form of the compound shown in the formula (I), or the crystal form of the compound shown in the formula (I) prepared by the method, or the composition prepared by the method, in preparing a medicament for preventing or treating noonan syndrome and leopard skin syndrome.

The disclosure also provides the use of the crystal form of the compound shown in the formula (I), or the crystal form of the compound shown in the formula (I) prepared by the method, or the composition prepared by the method, in preparing medicines for preventing or treating juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myelogenous leukemia, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, pancreatic cancer, head and neck squamous cell carcinoma, gastric cancer, liver cancer, anaplastic large cell lymphoma and glioblastoma.

The "2θ or 2θ angle" described in the present disclosure refers to a diffraction angle, θ is a bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2 theta is + -0.20, and can be-0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

The term "crystallization" as used in this disclosure includes, but is not limited to, stirring crystallization, cooling crystallization, beating crystallization, and volatilizing crystallization.

The term "differential scanning calorimetric analysis or DSC" in the present disclosure refers to measuring the temperature difference and the heat flow difference between a sample and a reference object during the temperature rising or constant temperature process of the sample, so as to characterize all physical changes and chemical changes related to thermal effects, and obtain phase change information of the sample.

The drying temperature in the present disclosure is generally 25 ℃ to 100 ℃, preferably 40 ℃ to 70 ℃, and can be either normal pressure drying or reduced pressure drying.

Unless otherwise indicated, when a position is specifically designated as deuterium (D), that position is understood to be deuterium (i.e., at least 10% deuterium incorporation) having an abundance that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). The natural abundance of the compound in the examples can be at least 1000 times greater than the abundance of deuterium, at least 2000 times greater than the abundance of deuterium, at least 3000 times greater than the abundance of deuterium, at least 4000 times greater than the abundance of deuterium, at least 5000 times greater than the abundance of deuterium, at least 6000 times greater than the abundance of deuterium, or higher than the abundance of deuterium.

The contents of WO2020259679a are incorporated by reference into the present disclosure.

Drawings

FIG. 1 shows an amorphous XRPD pattern for a compound of formula (I).

Figure 2 XRPD pattern of crystalline form a of compound of formula (I).

FIG. 3 TGA spectrum of compound A crystal form shown in formula (I).

FIG. 4 shows a DSC spectrum of the compound A crystal form shown in the formula (I).

FIG. 5 shows a front-to-back XRPD comparison of compound A crystalline form DVS of formula (I).

FIG. 6 shows an XRPD pattern for form B of compound (I).

FIG. 7 shows an XRPD pattern for form C of compound (I).

FIG. 8 shows an XRPD pattern for form D of compound (I).

FIG. 9 shows an XRPD pattern for form E of compound of formula (I).

FIG. 10 shows an XRPD pattern for form F of compound (I).

FIG. 11 shows an XRPD pattern for form G of compound (I).

FIG. 12 is an XRPD pattern for form H of the compound of formula (I).

Detailed Description

The present invention is further illustrated in detail by the following examples and experimental examples. These examples and experimental examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Test conditions of the instrument used for the experiment:

the structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shift (. Delta.) is given in units of 10-6 (ppm). NMR was performed using a Bruker AVANCE-400 nuclear magnetic resonance apparatus with deuterated dimethyl sulfoxide (DMSO-d 6), deuterated chloroform (CDCl 3), deuterated methanol (CD 3 OD) as the solvent and Tetramethylsilane (TMS) as the internal standard.

MS was measured using Shimadzu 2010 Mass Spectrometer or Agilent 6110A MSD mass spectrometer.

HPLC was performed using Agilent 1260DAD high pressure liquid chromatography (Sunfire C18X14.6 mm column) and Thermo U3000 high pressure liquid chromatography (Gimini C18X14.6 mm column).

HPLC was performed using a Shimadzu LC-20A systems, shimadzu LC-2010HT series or Agilent 1200 LC high pressure liquid chromatograph (Ultimate XB-C18.0 x 150mm column or Xtime C18.1 x 30mm column).

Chiral HPLC analysis was performed using a chiral HPLC analysis of 3 μm, chiral pak AD-3×4.6mm I.D.,3 μm, chiral pak AS-3 100×4.6mm I.D.,3 μm, chiral pak OD-3×4.6mm I.D.,3 μm, chiral Cel OJ-H150×4.6mm I.D.,5 μm, chiral Cel J-3×4.6mm I.D.,3 μm, chiral column;

the thin layer chromatography silica gel plate uses a smoke table yellow sea HSGF254 or Qingdao GF254 silica gel plate, the specification of the silica gel plate used by the Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

Column chromatography generally uses 100-200 mesh, 200-300 mesh or 300-400 mesh of yellow sea silica gel as a carrier.

The chiral preparation column used DAICEL CHIRALPAK IC (250 mm. Times.30 mm,10 μm) or Phenomnex-Amylose-1 (250 mm. Times.30 mm,5 μm).

The CombiFlash flash rapid prep instrument used CombiFlash Rf150 (teldyne ISCO).

Average inhibition rate of kinase and IC ₅₀ The values were measured using a NovoStar microplate reader (BMG, germany).

Known starting materials of the present disclosure may be synthesized using or following methods known in the art, or may be purchased from ABCR GmbH & co.kg, acros Organics, aldrich Chemical Company, shaog chemical technology (Accela ChemBio Inc), dary chemicals, and the like.

The examples are not particularly described, and the reaction can be carried out under an argon atmosphere or a nitrogen atmosphere.

An argon or nitrogen atmosphere means that the reactor flask is connected to a balloon of argon or nitrogen of about 1L volume.

The hydrogen atmosphere is defined as the reaction flask being connected to a balloon of hydrogen gas of about 1L volume.

The pressure hydrogenation reaction uses a Parr 3916 model EKX hydrogenometer and a clear blue QL-500 type hydrogen generator or HC2-SS type hydrogenometer.

The hydrogenation reaction is usually vacuumized, filled with hydrogen and repeatedly operated for 3 times.

The microwave reaction used was a CEM Discover-S908860 type microwave reactor.

The examples are not specifically described, and the solution refers to an aqueous solution.

The reaction temperature is room temperature and is 20-30 deg.c without specific explanation in the examples.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), the developing reagent used for the reaction, the system of eluent for column chromatography employed for purifying the compound and the developing reagent system of thin layer chromatography included: a: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: the volume ratio of petroleum ether/ethyl acetate/methanol is regulated according to the polarity of the compound, and can be regulated by adding a small amount of alkaline or acidic reagents such as triethylamine, acetic acid and the like.

XRPD is X-ray powder diffraction detection: the determination was performed using a brukend type 8X-ray diffractometer, specifically collecting information: cu anode (40 kV,40 mA), cu-K alpha 1 rayKα2 rayKbeta raysScanning mode: θ/2θ, scan range: 3-48 deg.. DSC is differential scanning calorimeter: the measurement is carried out by adopting METTLER TOLEDO DSC < + >, the heating rate is 10 ℃/min, the temperature is 25-350 ℃, and the nitrogen purging speed is 50mL/min.

TGA is thermogravimetric analysis: METTLER TOLEDO TGA2 is adopted for detection, the heating rate is 10 ℃/min, the specific temperature range is referred to the corresponding map, and the nitrogen purging speed is 50mL/min.

DVS is dynamic moisture adsorption: surface Measurement Systems advantage 2, humidity from 50% is adopted, the range of humidity is 0% -95%, the step is 10%, the quality change within 360min is less than 0.002%, and the process is circulated for two circles.

The metabolite 14 structure is as follows:

example 1

(S) -1'- (8- ((2-amino-3-chloropyridin-4-yl) thio) imidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-amine

First step

(3-bromopyridin-2-yl) methanol 1b

Compound 1a (17.2 g,79.6 mmol) was dissolved in methanol (50 mL) and sodium borohydride (15.1 g, 390 mmol) was added at 0deg.C. The reaction system was stirred at room temperature for 12 hours. After completion of the reaction, a saturated aqueous ammonium chloride solution (600 mL) was added, followed by extraction with ethyl acetate (200 mL. Times.3). The organic phases were combined, washed with saturated sodium chloride (200 mL. Times.2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 1b (9.7 g, yield: 64.8%) as a white solid.

MS(ESI)m/z 187.8[M+H] ⁺

¹ H NMR：(400MHz，MeOD-d ₄ )δ＝8.52(d，J＝4.8Hz，1H)，8.01(dd，J＝1.2，8.0Hz，1H)，7.26(dd，J＝4.4，6.4Hz，1H)，4.77(s，2H).

Second step

3-bromo-2- (chloromethyl) pyridine 1c

Compound 1b (9.70 g,51.6 mmol) was dissolved in dichloromethane (20 mL) and thionyl chloride (7.48 mL,103 mmol) was added at room temperature. Stirring was carried out at room temperature for 3 hours. After completion of the reaction, saturated aqueous sodium bicarbonate (300 mL) was added at 0deg.C, and the mixture was extracted with dichloromethane (80 mL. Times.3). The combined organic phases were washed with saturated sodium chloride (100 mL) and dried over anhydrous sodium sulfate. Concentration under reduced pressure gave compound 1c (10.3 g, yield: 96.9%) as a pink oil.

MS(ESI)m/z 207.7[M+H] ⁺

¹ H NMR(400MHz，Methanol-d4)□□＝8.55-8.45(m，1H)，8.12-7.99(m，1H)，7.37-7.21(m，1H)，4.84-4.80(m，2H).

Third step

1- (tert-butyl) 4-ethyl 4- ((3-bromopyridin-2-yl) methyl) piperidine-1, 4-dicarboxylic acid ester 1e

Compound 1c (9.97 g,38.7 mmol) was dissolved in tetrahydrofuran (80 mL) under nitrogen and LDA (13.5 mL,2M in tetrahydrofuran and n-hexane) was added dropwise at-78deg.C. After the completion of the dropwise addition, the mixture was stirred at-78℃for 1 hour. Compound 1d (8.8 g,35.07 mmol) was then added dropwise at-78℃and stirring continued at-78℃for 9 hours. After completion of the reaction, a saturated aqueous ammonium chloride solution (400 mL), extraction with ethyl acetate (100 mL. Times.3) and washing with a saturated sodium chloride solution (100 mL. Times.2) were added, and the mixture was dried over anhydrous sodium sulfate. The crude product was concentrated in vacuo and purified by silica gel chromatography eluting with petroleum ether and ethyl acetate to give compound 1e (14.8 g, yield: 89.4%) as a yellow oil.

MS(ESI)m/z 429.0[M+H] ⁺

Fourth step

4- ((3-bromopyridin-2-yl) methyl) -1- (tert-butoxycarbonyl) piperidine-4-carboxylic acid 1f

Compound 1e (14.8 g,34.6 mmol) was dissolved in methanol (3 mL), and aqueous sodium hydroxide solution (13.8 g, 348 mmol, dissolved in 40mL of water) was added at 0deg.C and stirred at 80deg.C for 12 hours. After completion of the reaction, the reaction mixture was concentrated, to which were added ethyl acetate (300 mL) and water (300 mL). Saturated aqueous sodium hydroxide (10 mL) was added to adjust the pH to 12, the aqueous phase was separated and washed with ethyl acetate (80 ml×2). To the resulting aqueous phase was added 2N hydrochloric acid (25 mL) to adjust the pH to 3 and extracted with ethyl acetate (100 mL. Times.3). The organic phases were combined, washed with saturated sodium chloride solution (150 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 1f (11.4 g, yield: 82.4%)

MS(ESI)m/z 344.0[M-56+H] ⁺

Fifth step

Tert-butyl 5-carbonyl-5, 7-dihydrospiro [ cyclopenta [ b ] pyridine-6, 4 '-piperidine ] -1' -carboxylic acid ester 1g

Sodium hydride (60% kerosene mixture, 1.32g,33.1 mmol) was added to tetrahydrofuran (100 mL) of compound 1f (11.0 g,27.6 mmol) under nitrogen at-15 ℃. Stirring at-15℃for 1 hour. The reaction mixture was cooled to-78℃and a 2.5M n-hexane solution (16.5 mL,41.3 mmol) of n-butyllithium was added dropwise thereto, followed by stirring at-78℃for 1 hour. After completion of the reaction, a saturated aqueous ammonium chloride solution (400 mL) was added thereto at 0℃and extracted with ethyl acetate (100 mL. Times.3). The organic phases were combined, washed with saturated sodium chloride (100 mL. Times.2), and dried over anhydrous sodium sulfate. The crude product was concentrated in vacuo and purified by silica gel chromatography eluting with methylene chloride and methanol to give 1g (4.60 g, yield: 55.2%) of a white solid compound.

MS(ESI)m/z 246.9[M-56+H] ⁺ .

¹ H NMR(400MHz，Methanol-d4)□□＝8.82(dd，J＝1.6，4.8Hz，1H)，8.12(dd，J＝1.6，7.6Hz，1H)，7.50(dd，J＝4.8，7.6Hz，1H)，4.08(td，J＝3.6，13.6Hz，2H)，3.25(s，2H)，3.12(br s，2H)，1.88-1.77(m，2H)，1.51(br s，2H)，1.49(s，9H).

Sixth step

Tert-butyl (S) -5- ((S) -tert-butylsulfinylamino) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridine-6, 4 '-piperidine ] -1' -carboxylic acid ester 1i

Tetraethyltitanate (9.4 mL,44.6 mmol) was added to 1g (4.50 g,14.9 mmol) of anhydrous toluene (80 mL) of the compound under nitrogen atmosphere and stirred at room temperature for 10 minutes. Then, the compound (5.4 g,44.6 mmol) was added to the reaction mixture for 1 hour, and the mixture was reacted at 120℃for 5 hours. After cooling to 0deg.C and adding lithium borohydride (1.58 g,89.2 mmol) for further 30 minutes, the temperature was raised to room temperature and stirred for 1 hour. After completion of the reaction, methanol (20 mL) was added dropwise at 0deg.C. Water (100 mL) and ethyl acetate (100 mL) were added and stirred for 5 minutes. The suspension was filtered off through celite and washed with ethyl acetate (300 mL) and water (300 mL). The combined organic phases were washed with saturated sodium chloride (500 mL) and dried over anhydrous sodium sulfate. Concentrating under reduced pressure to obtain crude product, eluting with petroleum ether and ethyl acetate by silica gel chromatography to obtain yellow solid compound 1i (4.40 g, yield: 72.6%)

MS(ESI)m/z 408.1[M+H] ⁺

Seventh step

(S) -N- ((S) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-yl) -2-methylpropane-2-sulfinamide 1j

Compound 1i (4.40 g,10.8 mmol) was dissolved in dichloromethane (15 mL), trifluoroacetic acid (5 mL) was added at 0deg.C, and the mixture was stirred at 0deg.C for 1 hour. Concentration under reduced pressure gave a crude product, which was added to 4M aqueous sodium hydroxide to ph=11. Chloroform and isopropanol (volume ratio 3:1) were extracted (30 mL. Times.3), and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration under reduced pressure gave product 1j (3.32 g, yield: 100%) as a yellow oil.

MS(ESI)m/z 307.9[M+H] ⁺

Eighth step

(S) -N- ((S) -1'- (8-bromoimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-yl) -2-methylpropan-2-sulfinamide 1l

Compound 1j (3.30 mg,10.7 mmol) and compound 1k (2.50 g,10.7 mmol) were dissolved in dimethyl sulfoxide (40 mL) under nitrogen, diisopropylethylamine (7.7 g,59.8 mmol) was added, and stirred at 90℃for 2 hours. Ethyl acetate (50 mL) and water (100 mL) were added, extracted with ethyl acetate (50 ml×2), the organic phases were combined, washed with saturated sodium chloride solution (50 ml×3), and dried over anhydrous sodium sulfate. Concentrating under reduced pressure to obtain crude product, and purifying with silica gel chromatography eluting with dichloromethane and methanol to obtain compound 1l (2.96 g, yield: 54.6%)

MS(ESI)m/z 503.1[M+H] ⁺

¹ H NMR(400MHz，METHANOL-d4)□＝8.41(d，J＝4.8Hz，1H)，7.97(s，1H)，7.92(d，J＝1.5Hz，1H)，7.81(d，J＝7.5Hz，1H)，7.66(d，J＝1.5Hz，1H)，7.32(dd，J＝5.0，7.5Hz，1H)，4.61(br s，2H)，3.95-3.83(m，2H)，3.30-3.21(m，2H)，2.99(d，J＝16.6Hz，1H)，2.40(dt，J＝4.0，12.7Hz，1H)，2.14(dt，J＝3.6，12.4Hz，1H)，1.82(br d，J＝13.3Hz，1H)，1.54(br d，J＝12.3Hz， 1H)，1.36(s，9H).

Ninth step

(S) -N- ((S) -1'- (8- ((2-amino-3-chloropyridin-4-yl) thio) imidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-yl) -2-methylpropan-2-sulfinamide 1N

1l (70 mg,0.14 mmol) and 1m (33 mg,0.21mmol, prepared as described in patent application WO2015107495A 1) were dissolved in 1, 4-dioxane (1 mL) and diisopropylethylamine (54 mg,0.42 mmol) was added at room temperature under nitrogen. Tri (dibenzylideneacetone) dipalladium (13 mg,0.014 mmol) and 2-dicyclohexylphosphine-2 ',6' -dimethoxybiphenyl (14 mg,0.028 mmol) were added thereto, and the mixture was heated and stirred at 110℃for 12 hours. After completion of the reaction, the reaction mixture was filtered, and the obtained filtrate was concentrated, and the residue was purified by C-18 reverse phase chromatography with water and methanol to give compound 1n (45 mg, yield: 55.1%) as a brown oil.

MS(ESI)m/z 583.1[M+H] ⁺

Tenth step

(S) -1'- (8- ((2-amino-3-chloropyridin-4-yl) thio) imidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-amine 1

Compound 1n (25 mg,0.035 mmol) was dissolved in 1, 4-dioxane, and a1, 4-dioxane solution (0.2 mL, 4N) of hydrogen chloride was added at 0℃to react at 2-7℃for 1 hour. After completion of the reaction, water (30 mL) was added, and extraction was performed with ethyl acetate (15 mL. Times.2). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and the residue was concentrated under reduced pressure and purified by C-18 reverse phase chromatography to give compound 1 (3.9 mg, yield: 19.0%).

MS(ESI)m/z 479.1[M+H] ⁺

¹ H NMR：(400MHz，MeOD-d ₄ )δ＝8.38(d，J＝4.8Hz，1H)，8.06(s，1H)，7.90-7.84(m，2H)，7.57(s，1H)，7.50(d，J＝5.2Hz，1H)，7.30(dd，J＝5.6，7.6Hz，1H)，5.90(d，J＝6.0Hz，1H)，4.16(s，1H)，4.06(br d，J＝13.6Hz，2H)，3.48-3.36(m，2H)，3.30-3.24(m，1H)，3.01(br d，J＝16.4Hz，1H)，2.20-2.01(m，2H)，1.80-1.71(m，1H)，1.61-1.53(m，1H).

Example 2

(S) -1'- (8- ((3-chloro-2- (methylamino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-amine

First step

(S) -N- ((S) -1'- (8-bromo-7-methylimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-yl) -2-methylpropan-2-sulfinamide 2b

Compound 1j (260 mg,0.85 mmol) and compound 2a (271mg, 1.10 mmol) were dissolved in dimethyl sulfoxide (3 mL), diisopropylethylamine (547 mg,4.23 mmol) was added, and the mixture was stirred at 90℃for 1 hour. Water (30 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (30 mL. Times.3). The combined organic phases were washed with saturated sodium chloride solution (50 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give a crude product, which was purified by silica gel chromatography eluting with methanol, methylene chloride to give compound 2b (370 mg, yield: 84.5%)

MS(ESI)m/z 518.8[M+H] ⁺

1H NMR(400MHz，Methanol-d4)δ＝8.39(d，J＝4.8Hz，1H)，7.82(d，J＝1.2Hz，1H)，7.79(d，J＝8.0Hz，1H)，7.55(d，J＝1.6Hz，1H)，7.30(dd，J＝4.8Hz，7.6Hz，1H)，3.90-3.81(m，2H)，3.37-3.32(m，1H)，3.29-3.17(m，3H)，3.00-2.92(m，1H)，2.57(s，3H)，2.37(td，J＝4.4Hz，12.8Hz，1H)，2.13(td，J＝4.4Hz，13.2Hz，1H)，1.83-1.75(m，1H)，1.56-1.49(m，1H)，1.34(s，9H).

Second step

(S) -N- ((S) -1'- (8- ((3-chloro-2- (methylamino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-yl) -2-methylpropan-2-sulfinamide 2d

Compound 2b (50 mg,0.10 mmol), compound 2c (77 mg,0.39 mmol) and potassium phosphate (41 mg,0.19 mmol) were dissolved in 1, 4-dioxane (1 mL) and nitrogen was replaced three times with stirring. Under nitrogen atmosphere, 1, 10-phenanthroline (3.5 mg,0.02 mmol) and cuprous iodide (1.8 mg,0.01 mmol) were rapidly added, replaced three times with nitrogen, and heated and stirred at 130℃for 10 hours. Water (50 mL) was added to the reaction solution and extracted with ethyl acetate (40 mL. Times.3), the organic phases were combined, washed with saturated sodium chloride solution (70 mL. Times.2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product, which was purified by silica gel chromatography using methylene chloride and methanol to give compound 2d (36 mg, yield: 58.5%).

MS(ESI)m/z 611.1[M+H] ⁺

Third step

(S) -1'- (8- ((3-chloro-2- (methylamino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-amine 2

Compound 2d (36 mg,0.059 mmol) was dissolved in dry dioxane (1 mL), and a solution of hydrogen chloride in 1, 4-dioxane (1 mL, 4N) was added dropwise at 10℃and reacted at 10℃for 15 minutes. To the suspended reaction solution was added water (30 mL), and extracted with ethyl acetate (30X 3). The aqueous phase was adjusted to ph=8 with saturated aqueous sodium bicarbonate and extracted with chloroform (40 ml×4). All organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by high performance liquid chromatography and lyophilized to give compound 2 (2.3 mg, yield: 7.7%).

MS(ESI)m/z 507.3[M+H] ⁺

1H NMR(400MHz，Methanol-d4)δ＝8.35(d，J＝4.4Hz，1H)，7.85(d，J＝7.6Hz，1H)，7.76(d，J＝1.6Hz，1H)，7.58(d，J＝5.6Hz，1H)，7.48(d，J＝1.6Hz，1H)，7.29(dd，J＝5.2Hz，7.6Hz，1H)，5.75(d，J＝6.0Hz，1H)，4.12-4.00(m，3H)，3.46-3.34(m，2H)，3.29-3.23(m，1H)，3.01-2.92(m，4H)，2.55(s，3H)，2.17-2.01(m，2H)，1.74(d，J＝13.6Hz，1H)，1.53(d，J＝13.6Hz，1H).

Example 3

(S) -1'- (8- ((3-chloro-2- ((methyl-d 3) amino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-amine

Intermediate 3e

2- ((methyl-d) ₃ ) Amino) -3-chloropyridine-4-thiol sodium salt

First step

3-chloro-4-iodo-N- (methyl)-d ₃ ) Pyridin-2-amine 3b

Compound 3a (3.0 g,12 mmol) and methyl-d ₃ Amine hydrochloride (1.2 g,16 mmol) was dissolved in DMSO (50 mL) and DIEA (5.8 mL,35 mmol) was added. The reaction was carried out at 70℃for 12 hours. After completion of the reaction, an ice-water mixture (50 mL), filtration, and washing with ice water (50 mL. Times.3) were added. The obtained solid was dried under reduced pressure to obtain compound 3b (2.8 g, yield: 83%).

MS(ESI)m/z 272.0[M+H] ⁺

¹ H NMR：(400MHz，CDCl ₃ )δ＝7.67(d，J＝5.2Hz，1H)，7.02(d，J＝5.2Hz，1H)，5.14(br s，1H)。

Second step

3- ((3-chloro-2- ((methyl-d) ₃ ) Amino) pyridin-4-yl) thio) propanoic acid ethyl ester 3d

Compound 3b (2.7 g,10 mmol) was dissolved in dichloromethane (30 mL), ethyl 3-thiopropionate 3c (2.0 g,15 mmol), tris (dibenzylideneacetone) (0.46 g,0.50 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (0.58 g,0.99 mmol) and N, N-diisopropylethylamine (4.9 mL,30 mmol) were added. The reaction was carried out for 3 hours at 100℃under a nitrogen atmosphere. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel chromatography using ethyl acetate and petroleum ether to give compound 3d (2.5 g, yield: 90%).

MS(ESI)m/z 278.1[M+H] ⁺

¹ H NMR(400MHz，CDCl ₃ )δ＝7.96(d，J＝5.6Hz，1H)，6.45(d，J＝5.6Hz，1H)，5.00(brs，1H)，4.19(q，J＝7.2Hz，2H)，3.23(t，J＝7.6Hz，2H)，2.72(t，J＝7.6Hz，2H)，1.28(t，J＝7.2Hz，3H)。

Third step

2- ((methyl-d) ₃ ) Amino) -3-chloropyridine-4-thiol sodium intermediate 3e

Compound 3d (2.4 g,8.6 mmol) was dissolved in tetrahydrofuran (25 mL) and sodium ethoxide ethanol solution (3.5 g,10mmol,20% w/w) was added at 0deg.C and reacted for 1 hour at 0deg.C. After completion of the reaction, the reaction mixture was concentrated, and a mixed solution (20 mL) of 50:1 methyl tert-butyl ether and methylene chloride was added thereto, followed by filtration and washing (10 mL. Times.3) with 50:1 methyl tert-butyl ether and methylene chloride. The resulting solid was dried under vacuum to give intermediate 3e (2.0 g, yield: 99%).

MS(ESI)m/z 178.0[M+H] ⁺

¹ H NMR(400MHz，DMSO_d ₆ )δ＝7.12(d，J＝5.6Hz，1H)，6.43(d，J＝5.2Hz，1H)，5.29(s，1H)。

Fourth step

(S) -N- ((S) -1' - (8- ((3-chloro-2- ((methyl-d) ₃ ) Amino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c]Pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ]]Pyridine-6, 4' -piperidines]-5-yl) -2-methylpropan-2-sulfinamide 3f

Compound 2b (200 mg,0.39 mmol) intermediate 3e (156 mg,0.77 mmol) was dissolved in 1, 4-dioxane (5 mL) under nitrogen, and cuprous iodide (74 mg,0.39 mmol), N, N' -dimethylethylenediamine (34 mg,0.39 mmol) and potassium phosphate (246 mg,1.2 mmol) were added and reacted by heating at 130℃under nitrogen for 15 hours. After completion of the reaction, aqueous ammonia (30 mL) and ethyl acetate (15 mL) were added, the aqueous phase was extracted with ethyl acetate (25 mL. Times.3), all the organic phases were combined, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The obtained residue was purified by silica gel chromatography eluting with methylene chloride and methanol to give compound 3f (130 mg, yield: 55%).

MS(ESI)m/z 614.3[M+H] ⁺

¹ H NMR(400MHz，CDCl ₃ )δ＝8.47(d，J＝4.4Hz，1H)，7.68(d，J＝5.6Hz，1H)，7.64(d，J＝7.2Hz，1H)，7.48(dd，J＝1.2，9.2Hz，2H)，7.17(dd，J＝4.8，7.6Hz，1H)，5.73(d，J＝5.6Hz，1H)，5.28(s，1H)，5.03(s，1H)，4.64(d，J＝10.0Hz，1H)，4.05-3.93(m，2H)，3.73(d，J＝10.0Hz，1H)，3.33-3.16(m，3H)，2.94(d，J＝16.4Hz，1H)，2.54(s，3H)，2.52-2.44(m，1H)，2.13(dt，J＝4.0，12.4Hz，1H)，1.82-1.74(m，1H)，1.52-1.44(m，1H)，1.30(s，9H)。

Fifth step

(S) -1' - (8- ((3-chloro-2- ((methyl-d) ₃ ) Amino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c]Pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ]]Pyridine-6, 4' -piperidines]-5-amine 3

Compound 3f (130 mg,0.21 mmol) was dissolved in dry dichloromethane (4.5 mL), and a solution of hydrogen chloride in 1, 4-dioxane (1.5 mL, 4N) was added dropwise at 0deg.C and reacted at 20deg.C for 1 hour. To the reaction solution was added 0.1M aqueous sodium hydroxide solution (30 mL) to adjust ph=14, followed by extraction with dichloromethane (30 ml×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse phase chromatography eluting with 0.1% aqueous ammonia and acetonitrile to give compound 3 (65 mg, yield: 41%). The solid was amorphous as measured by X-ray powder diffraction and the XRPD pattern is shown in figure 1.

MS(ESI)m/z 510.2[M+H] ⁺

¹ H NMR(400MHz，MeOD_d4)δ＝8.36(d，J＝4.4Hz，1H)，7.86(d，J＝7.6Hz，1H)，7.76(d，J＝1.6Hz，1H)，7.57(d，J＝5.6Hz，1H)，7.47(d，J＝1.6Hz，1H)，7.29(dd，J＝4.8，7.6Hz，1H)，5.75(d，J＝6.0Hz，1H)，4.11(s，1H)，4.05(br d，J＝13.6Hz，2H)，3.45-3.35(m，2H)，3.27(d，J＝16.8Hz，1H)，2.97(d，J＝16.4Hz，1H)，2.55(s，3H)，2.17-2.03(m，2H)，1.74(br d，J＝14.0Hz，1H)， 1.53(br d，J＝13.6Hz，1H)。

Example 4

(S) -1'- (8- ((2- (bis (methyl-d 3) amino) -3-chloropyridin-4-yl) thio) -7-methylimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-amine

Intermediate 4a

2- (di (methyl-d) ₃ ) Amino) -3-chloropyridine-4-thiol sodium salt

The synthesis step of intermediate 4a is seen in intermediate 3e, in which the compound dimethyl-d ₆ -amine hydrochloride substitution of methyl-d ₃ -amine hydrochloride preparation to obtain the aforementioned intermediate 4a.

MS(ESI)m/z 195.1[M+H] ⁺

¹ H NMR(400MHz，DMSO_d ₆ )δ＝7.24(d，J＝5.2Hz，1H)，6.79(d，J＝5.2Hz，1H)。

The synthetic procedure for compound 4 is described in example 3, wherein compound 4 is prepared by substituting intermediate 4a for intermediate 3 e.

MS(ESI)m/z 527.2[M+H] ⁺

¹ H NMR(400MHz，MeOD_d ₄ )δ＝8.36(d，J＝4.4Hz，1H)，7.86(d，J＝7.6Hz，1H)，7.77(d，J＝1.6Hz，1H)，7.72(d，J＝5.2Hz，1H)，7.47(d，J＝1.6Hz，1H)，7.29(dd，J＝5.2，7.6Hz，1H)，6.06(d，J＝5.6Hz，1H)，4.11(s，1H)，4.06(br d，J＝13.6Hz，2H)，3.44-3.37(m，2H)，3.25(s，1H)，2.97(d，J＝16.8Hz，1H)，2.56(s，3H)，2.14-2.03(m，2H)，1.75(br d，J＝13.2Hz，1H)，1.54(br d，J＝13.6Hz，1H)。

Example 5

(S) -1'- (8- ((3-chloro-2- ((methyl-d 2) amino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-amine

Intermediate 5f

First step

N, N-bis (4-methoxybenzyl) methylamine-d ₂ 5b

Compound 5a (4.0 g,16 mmol) was dissolved in methanol (50 mL) and aqueous deuterated formaldehyde (3.7 g,23mmol,20% w/w) and acetic acid (0.93 g,16 mmol) were added at room temperature. Sodium cyanoborohydride (2.9 g,47 mmol) was added thereto and the reaction was continued at room temperature for 15 hours. After the completion of the reaction, the reaction mixture was concentrated, and then 2M sodium hydroxide solution was added to adjust the pH to 9 to 10, followed by extraction with ethyl acetate (30 mL. Times.3). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the obtained residue was purified by silica gel chromatography eluting with petroleum ether and ethyl acetate to give compound 5b (4.0 g, yield: 95%).

MS(ESI)m/z 274.3[M+H] ⁺

¹ H NMR：(400MHz，CDCl ₃ )δ＝7.28-7.25(m，4H)，6.89-6.84(m，4H)，3.88-3.74(m，7H)，3.45(s，4H)。

Second step

N- (4-methoxybenzyl) methane-d ₂ -amine hydrochloride 5c

Compound 5b (1.0 g,3.7 mmol) was dissolved in methanol (20 mL), and 10% palladium on carbon (water content 1%,100 mg), 20% palladium hydroxide (100 mg) and concentrated hydrochloric acid (0.5 mL) were added. The reaction was carried out at 80℃for 12 hours under a hydrogen atmosphere of 50 psi. Filtration, washing with methanol (30 mL. Times.3) and drying of the filtrate under reduced pressure gave compound 5c (0.69 g, yield: 99%).

MS(ESI)m/z 153.8[M+H] ⁺

¹ H NMR(400MHz，DMSO-d ₆ )δ＝9.18(br s，2H)，7.45(d，J＝8.4Hz，2H)，6.97(d，J＝8.4Hz，2H)，4.01(t，J＝5.6Hz，2H)，3.76(s，3H)，2.44(br s，1H)。

Third step

3-chloro-4-iodo-N- (4-methoxybenzyl) -N- (methyl-d ₂ ) Pyridin-2-amine 5d

Compound 5c (428 mg,3.4 mmol) and compound 3a (787 mg,3.1 mmol) were dissolved in DMSO (10 mL) and DIEA (2.0 g,15 mmol) was added. The reaction was carried out at 60℃for 15 hours. After completion of the reaction, an ice-water mixture (100 mL) was added, followed by extraction with ethyl acetate (30 mL. Times.3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography eluting with petroleum ether and ethyl acetate to give compound 5d (590 mg, yield: 49%).

MS(ESI)m/z 391.0[M+H] ⁺

¹ H NMR：(400MHz，CDCl ₃ )δ＝7.76(d，J＝5.2Hz，1H)，7.35(d，J＝5.2Hz，1H)，7.32-7.27(m，2H)，6.95-6.81(m，2H)，4.45(s，2H)，3.82(s，3H)，2.80(s，1H)。

Fourth step

3- ((3-chloro-2- ((4-methoxybenzyl) (methyl-d) ₂ ) Amino) pyridin-4-yl) thio) propanoic acid ethyl ester 5e

Compound 5d (560 mg,1.5 mmol) was dissolved in 1, 4-dioxane (8 mL), ethyl 3-thiopropionate 3c (304 mg,2.3 mmol), tris (dibenzylideneacetone) dipalladium (69 mg,0.076 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (87 mg,0.15 mmol) and N, N-diisopropylethylamine (586 mg,4.5 mmol) were added. The reaction was carried out for 5 hours at 100℃under a nitrogen atmosphere. After completion of the reaction, filtration, concentration of the filtrate under reduced pressure, and purification of the resulting residue by silica gel chromatography eluting with ethyl acetate and petroleum ether gave compound 5e (614 mg, yield: 94%).

MS(ESI)m/z 397.1[M+H] ⁺

¹ H NMR(400MHz，CDCl ₃ )δ＝8.07(d，J＝5.6Hz，1H)，7.34-7.29(m，2H)，6.93-6.83(m，2H)，6.71(d，J＝5.2Hz，1H)，4.43(s，2H)，4.20(q，J＝7.2Hz，2H)，3.81(s，3H)，3.24(t，J＝7.6Hz，2H)，2.80(s，1H)，2.75(t，J＝7.6Hz，2H)，1.29(t，J＝7.2Hz，3H)。

Fifth step

3-chloro-2- ((4-methoxybenzyl) (methyl-d) ₂ ) Amino) pyridine-4-thiol sodium intermediate 5f

Compound 5e (614 mg,1.4 mmol) was dissolved in tetrahydrofuran (8 mL) and sodium ethoxide ethanol solution (552 mg,1.7mmol,20% w/w) was added at 0deg.C and reacted for 1 hour at 0deg.C. After completion of the reaction, the reaction mixture was concentrated, a mixed solution of methyl t-butyl ether and methylene chloride (6 mL, v/v=50/2) was added, and the mixture was filtered and washed with methyl t-butyl ether (10 ml×3). The resulting solid was dried under vacuum to give intermediate 5f (445 mg, yield: 98%).

MS(ESI)m/z 297.1[M+H] ⁺

¹ H NMR(400MHz，Methanol_d ₄ )δ＝7.46(d，J＝5.4Hz，1H)，7.32-7.22(m，2H)，7.17(d，J＝5.6Hz，1H)，6.92-6.76(m，2H)，4.25(s，2H)，3.77(s，3H)，2.61(s，1H)。

Sixth step

(S) -N- ((S) -1' - (8- ((3-chloro-2- ((4-methoxybenzyl) (methyl-d) ₂ ) Amino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c]Pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ]]Pyridine-6, 4' -piperidines]-5-yl) -2-methylpropan-2-sulfinamide 5g

Compound 2b (153 mg,0.30 mmol) intermediate 5f (188 mg,0.59 mmol) was dissolved in 1, 4-dioxane (5 mL) under nitrogen, and cuprous iodide (56 mg,0.30 mmol), N, N' -dimethylethylenediamine (52 mg,0.59 mmol) and potassium phosphate (188 mg,0.89 mmol) were added and reacted by heating at 130℃under nitrogen for 15 hours. After completion of the reaction, aqueous ammonia (20 mL) and ethyl acetate (10 mL) were added, the aqueous phase was extracted with ethyl acetate (20 mL. Times.3), all the organic phases were combined, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The obtained residue was purified by silica gel chromatography eluting with methylene chloride and methanol to give 5g (190 mg, yield: 45%) of the compound.

MS(ESI)m/z 733.3[M+H] ⁺

Seventh step

(S) -1' - (8- ((3-chloro-2- ((methyl-d) ₂ ) Amino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c]Pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ]]Pyridine-6, 4' -piperidines]-5-amine 5

5g (160 mg,0.22 mmol) of the compound was dissolved in TFA (3 mL) and reacted at room temperature for 4 hours. Then, a solution of hydrogen chloride in 1, 4-dioxane (1 mL, 4N) was added dropwise at 0℃and the reaction was carried out at room temperature for 1 hour. To the reaction solution was added 0.1M aqueous sodium hydroxide solution (30 mL) to adjust ph=14, followed by extraction with dichloromethane (30 ml×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel chromatography eluting with methylene chloride, methanol to give compound 5 (51 mg, yield: 46%).

MS(ESI)m/z 509.2[M+H] ⁺

¹ H NMR(400MHz，CDCl ₃ )δ＝8.45(d，J＝4.4Hz，1H)，7.71(d，J＝5.2Hz，1H)，7.67(d，J＝7.2Hz，1H)，7.56(d，J＝1.2Hz，1H)，7.44(d，J＝1.6Hz，1H)，7.18(dd，J＝5.2，7.6Hz，1H)，5.78(d，J＝5.6Hz，1H)，5.03(d，J＝4.4Hz，1H)，4.11(s，1H)，4.04-3.93(m，2H)，3.33(q，J＝11.6Hz，2H)，3.25(d，J＝16.8Hz，1H)，3.00(br s，1H)，2.93(d，J＝16.8Hz，1H)，2.58(s，3H)，2.11(dt， J＝4.0，12.8Hz，1H)，2.02(dt，J＝4.4，12.4Hz，1H)，1.81-1.73(m，1H)，1.52-1.47(m，1H)。

Example 6

(S) -1'- (8- ((3-chloro-2- ((methyl-d) amino) pyridin-4-yl) thio) -7-methylimidazo [1,2-c ] pyrimidin-5-yl) -5, 7-dihydrospiro [ cyclopenta [ b ] pyridin-6, 4' -piperidin ] -5-amine

Intermediate 6f

Intermediate 6f

N, N-bis (4-methoxybenzyl) methylamine-d 6a

Compound 5a (3.0 g,12 mmol) was dissolved in dry methanol (30 mL) and aqueous formaldehyde (2.6 mL,37% w/w) and acetic acid (0.67 mL,12 mmol) were added at room temperature. The reaction was carried out at room temperature for 2 hours, and sodium borodeuteride (0.97 g,23 mmol) was added at 0℃and the reaction was carried out at room temperature for 1.5 hours. After completion of the reaction, the reaction mixture was concentrated, water (50 mL) was added, and extraction was performed with ethyl acetate (30 mL. Times.3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 6a (3.3 g, yield: 94%).

¹ H NMR：(400MHz，CDCl ₃ )δ＝7.30-7.25(m，4H)，6.91-6.84(m，4H)，3.81(s，6H)，3.45(s，4H)，2.13(s，2H)。

Second step

(4-methoxybenzyl) (methyl-d) carbamic acid benzyl ester 6b

Compound 6a (3.3 g,12 mmol) was dissolved in toluene (30 mL), benzyl chloroformate (4.1 mL,29 mmol) was added, and the mixture was reacted at 120℃under nitrogen for 14 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, ethyl acetate (50 mL), water (20 mL), and saturated brine (20 mL. Times.2) were added thereto, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with petroleum ether and ethyl acetate to give compound 6b (3.8 g crude).

¹ H NMR(400MHz，CDCl ₃ )δ＝7.43-7.31(m，5H)，7.21(d，J＝7.6Hz，1H)，7.13(d，J＝7.6Hz，1H)，6.91-6.82(m，2H)，5.20(s，2H)，4.45(s，2H)，3.82(s，3H)，2.86(d，J＝10.8Hz，2H)。

Third step

N- (4-methoxybenzyl) methane-d-amine hydrochloride 6c

Compound 6b (3.8 g,12 mmol) was dissolved in methanol (40 mL) and 10% palladium on carbon (1.0 g) was added. The reaction was carried out at 40℃for 16 hours under a hydrogen atmosphere. Filtration, washing with methanol (80 mL) and drying of the filtrate under reduced pressure gave compound 6c (2 g, yield: 99%).

MS(ESI)m/z 152.9[M+H] ⁺

Fourth step

3-chloro-4-iodo-N- (4-methoxybenzyl) -N- (methyl-d) pyridin-2-amine 6d

Compound 6c (2 g,12 mmol) and compound 3a (2.3 g,8.8 mmol) were dissolved in DMSO (4 mL) and DIEA (4.3 mL,26 mmol) was added. The reaction was carried out at 60℃for 5 hours. After completion of the reaction, water (50 mL) was added thereto, followed by extraction with ethyl acetate (30 mL. Times.3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography eluting with petroleum ether and ethyl acetate to give compound 6d (2.6 g, yield: 76%).

MS(ESI)m/z 390.0[M+H] ⁺

¹ H NMR：(400MHz，CDCl ₃ )δ＝7.76(d，J＝4.8Hz，1H)，7.35(d，J＝5.2Hz，1H)，7.32-7.27(m，2H)，6.92-6.83(m，2H)，4.45(s，2H)，3.81(s，3H)，2.82(s，2H)。

Fifth step

3- ((3-chloro-2- ((4-methoxybenzyl) (methyl-d) amino) pyridin-4-yl) thio) propanoic acid ethyl ester 6e

Compound 6d (2.6 g,6.7 mmol) was dissolved in 1, 4-dioxane (30 mL), ethyl 3-thiopropionate 3c (1.3 g,10 mmol), tris (dibenzylideneacetone) dipalladium (310 mg,0.34 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (390 mg,0.67 mmol) and N, N-diisopropylethylamine (3.3 mL,20 mmol) were added. The reaction was carried out for 6 hours at 100℃under a nitrogen atmosphere. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel chromatography using ethyl acetate and petroleum ether to give compound 6e (2.3 g, yield: 88%).

MS(ESI)m/z 396.1[M+H] ⁺

¹ H NMR(400MHz，CDCl ₃ )δ＝8.07(d，J＝5.2Hz，1H)，7.34-7.28(m，2H)，6.93-6.82(m，2H)，6.72(d，J＝5.2Hz，1H)，4.43(s，2H)，4.20(q，J＝7.2Hz，2H)，3.81(s，3H)，3.24(t，J＝7.6Hz，2H)，2.81(s，2H)，2.75(t，J＝7.6Hz，2H)，1.29(t，J＝6.8Hz，3H)。

Sixth step

3-chloro-2- ((4-methoxybenzyl) (methyl-d) amino) pyridine-4-thiol sodium intermediate 6f

Compound 6e (2.3 g,5.9 mmol) was dissolved in tetrahydrofuran (25 mL) and sodium ethoxide ethanol solution (2.4 g,7.1mmol,20% w/w) was added at 0deg.C and reacted for 1 hour at 0deg.C. After completion of the reaction, the reaction mixture was concentrated, a mixed solution of methyl t-butyl ether and methylene chloride (40 mL, v/v=50/2) was added, and the mixture was filtered and washed with methyl t-butyl ether (15 ml×3). The resulting solid was dried under vacuum to give intermediate 6f (1.7 g, yield: 91%).

¹ H NMR(400MHz，DMSO-d6)δ＝7.29-7.23(m，3H)，6.89-6.79(m，3H)，4.12(s，2H)，3.72(s，3H)，3.34(s，2H)。

The synthetic procedure for compound 6 is described in example 5, wherein the aforementioned compound 6 is prepared by substituting intermediate 6f for intermediate 5 f.

MS(ESI)m/z 508.2[M+H] ⁺

¹ H NMR(400MHz，CDCl ₃ )δ＝8.47(d，J＝4.4Hz，1H)，7.68(d，J＝5.6Hz，1H)，7.64(d，J＝7.2Hz，1H)，7.48(dd，J＝1.2，9.2Hz，2H)，7.17(dd，J＝4.8，7.6Hz，1H)，5.73(d，J＝5.6Hz，1H)，5.28(s，1H)，5.03(s，1H)，4.64(d，J＝10.0Hz，1H)，4.05-3.93(m，2H)，3.73(d，J＝10.0Hz，1H)，3.33-3.16(m，3H)，2.94(d，J＝16.4Hz，1H)，2.54(s，3H)，2.52-2.44(m，1H)，2.13(dt，J＝4.0，12.4Hz，1H)，1.82-1.74(m，1H)，1.52-1.44(m，1H)，1.30(s，9H)。

Biological evaluation

The present disclosure is explained in further detail below in conjunction with test examples, which are not meant to limit the scope of the present disclosure.

Test example 1, detection of SHP2 phosphatase Activity by Compounds of the present disclosure

1. Experimental material and instrument

Instrument name	Equipment manufacturer	Model number
Constant temperature oscillator	IMB	MB-1002A
Microplate reader	MDSpectraMax	M5

2. Experimental procedure

0.2nM of the full-length SHP2 (aa 1-593) recombinantly expressed, 0.5nM of the activated polypeptide IRS1 (sequence: H2N-LN (pY) IDLDLY (dPEG 8) LST (pY) ASINFQK-amide) with double phosphorylation sites and a series of concentrations of test compounds (final concentrations of 1 ≡M,0.3 ≡M,0.1 ≡M,0.03 ≡M,0.01 ≡M,0.003 ≡M,0.001 ≡M,0.0003 ≡M,0.0001 ≡M,0.00003 ≡M) were added to the phosphatase reaction solution (60mM HEPES,PH 7.5 0.005%Brij-35, 75mM NaCl,75mM KCl,1mM EDTA,5mM DTT) and the reaction solution was shaken at room temperature (350 rpm) for 30 minutes. After 30 minutes of reaction at room temperature with 30 ≡M substrate DiFMUP, the phosphatase reaction was terminated with 5 ≡L reaction terminating solution (60mM HEPES,pH 7.5,0.2%SDS). Ex358nm/Em455 fluorescence values were read on a fluorescence plate reader MD SpectraMax.

IC of compound ₅₀ The values were calculated using a four parameter logic method, where x in the following formula represents the logarithmic form of the compound concentration; f (x) represents an effect value (inhibition rate of cell proliferation under the concentration condition): f (x) =((A-D)/(1+ ((x/C)/(B))) +D. A, B, C and D are four parameters. Different concentrations correspond to different inhibition rates of phosphatase activity, an inverse curve is made, and the IC of the inhibitor is calculated from the curve ₅₀ . Calculation of IC for Compounds with Primer premier 6.0 ₅₀ 。

The in vitro activity of the disclosed compound on SHP2 is determined by the above test, and an orally active SHP2 inhibitor SHP099 is selected as a positive drug, the structure of the compound is disclosed in the document J.Med.chem.2016, 59, 7773-7782, and the specific compound is purchased from Shanghai Haoyuan biological medicine science and technology Co., ltd (Medchemxpress.cn).

Measured IC ₅₀ The values are shown in Table 1.

TABLE 1 Compound pair SHP2 phosphatase IC ₅₀

Examples numbering	IC 50 (nM)	Examples numbering	IC 50 (nM)
SHP099	79	1	1.7
2	2.1	3	4.5
5	4.8	6	4.7

Test example 2 in vitro metabolism stability test of rat liver microsomes

The intrinsic clearance of the test compound was calculated by measuring the compound concentration in the reaction system using LC/MS, and the in vitro metabolic stability in rat liver microsomes was evaluated.

mu.L of a mixture of rat liver microsomes (male Wistar Han strain, available from Corning Corp., cat. 452511) at 1.1236mg/mL and 25. Mu.L of 10mM NADPH were added to the incubation plate. Mix with vortex for 10 seconds. Incubate in a 37℃water bath for 8 minutes. 2.5. Mu.L of the test compound or positive control, 100. Mu.M, was added to the incubation plate to initiate the reaction. Incubation was continued in a 37 ℃ water bath after 12 seconds of mixing on vortex. The reaction was stopped by transferring 20. Mu.L of the incubation system to a stop plate containing 100. Mu.L of cold stop solution at 0.5, 5, 10, 15, 20 and 30 minutes, respectively, and vortexing for 2 minutes. The final plate was centrifuged at 4000rpm for 20 minutes, then allowed to stand at 4℃for 30 minutes and centrifuged at 4000rpm for 20 minutes. Transfer 40 μl of each compound supernatant to a 96 well master plate and dilute the sample by adding 160 μl of purified water.

The obtained sample is quantified by an ion chromatogram, and the residual rate is calculated according to the peak area of the compound to be detected or the positive control. Slope k was determined from a linear regression of natural log values of residual rate versus incubation time using Microsoft Excel.

The in vitro half-life (in vitro t 1/2) was calculated from the slope: in vitro t 1/2= - (0.693/k)

The in vitro half-life was converted to intrinsic clearance (in vitro CLint, μl/min/mg protein) using the following equation:

in vitro clint= (0.693/t 1/2) × (incubation volume (μl)/protein amount (mg))

The intrinsic clearance values of the rat liver microsomes are shown in Table 2.

TABLE 2 intrinsic clearance of rat liver microsomes

Test example 3 in vivo pharmacokinetic experiments in rats

The concentration of the drug in plasma at various times after intragastric administration of the compounds of the invention was determined using LC/MS method using rats as test animals. The pharmacokinetic behavior of the compound of the invention in rats was studied and its pharmacokinetic profile was evaluated.

Test animals: 3 healthy 6-8 week male SD rats per group

Pharmaceutical formulation

A certain amount of the medicine is weighed, and 0.5% of hydroxypropyl methylcellulose, 0.1% of Tween 80 and 99.4% of water by volume are added to prepare a white suspension of 1 mg/mL.

Administration of drugs

SD rats were administered by gavage overnight fast with a reference dose of 7.5mg/kg and example 1 at a dose of 5mg/kg.

Operation of

Rats were given the compounds of the invention by gavage, 0.2mL of blood was collected from the jugular vein 0.25, 0.5, 1, 2, 4, 8, 24 hours after administration, and the plasma was isolated by centrifugation at 4000 rpm for 5 minutes at 4℃in a tube containing EDTA-K2 and stored at-75 ℃.

Determination of the content of the test compounds in rat plasma after administration of different concentrations of the drug by gavage: rat plasma was taken at 50. Mu.L at each time after administration, 200. Mu.L of acetonitrile solution of dexamethasone (50 ng/mL) as an internal standard was added, vortex-mixed for 30 seconds, centrifuged at 4℃for 15 minutes at 4700 rpm, and the plasma sample was taken as a supernatant diluted three times with water and taken as 2.0. Mu.L for LC/MS/MS analysis.

Pharmacokinetic parameter results

The rat pharmacokinetic parameters of the compounds of the invention are shown in table 3 below.

TABLE 3 rat pharmacokinetic parameters of Compounds

Test example 4 in vivo pharmacokinetic experiments in cynomolgus monkeys

The concentration of the drug in the plasma at various times after the compound of the invention is administered by intragastric administration of the cynomolgus monkey was determined using LC/MS method with cynomolgus monkey as the subject animal. The pharmacokinetic behavior of the compound of the invention in the cynomolgus monkey body is studied, and the pharmacokinetic characteristics are evaluated.

Test animals: 3 male cynomolgus monkeys of 2-5 years old were healthy in each group;

pharmaceutical formulation

Gastric lavage administration: a certain amount of the medicine is weighed, and 0.5% of hydroxypropyl methylcellulose, 0.1% of Tween 80 and 99.4% of water by volume are added to prepare a white suspension of 1 mg/mL.

Administration of drugs

The cynomolgus monkey is fed by stomach irrigation after being fasted overnight, and the dosage is 5mg/kg.

Operation of

The compound of the invention is administrated by stomach irrigation of a cynomolgus monkey, 0.2mL of the compound is collected from peripheral veins 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration, and the compound is placed in a test tube containing EDTA-K2, centrifuged at 2000 rpm for 10 minutes at 2-8 ℃ to separate plasma, and the plasma is preserved at-75 ℃.

Determining the content of a compound to be detected in the blood plasma of the cynomolgus monkey after the gastric lavage administration of the medicines with different concentrations: at each time after administration, 55. Mu.L of cynomolgus monkey plasma was taken, 200. Mu.L of acetonitrile solution of internal standard verapamil or dexamethasone was added, vortex mixed for 30 seconds, centrifuged at 3900 rpm at 4℃for 15 minutes, and the supernatant was diluted three times with water for 15. Mu.L for LC/MS/MS analysis.

Pharmacokinetic parameter results

The cynomolgus monkey pharmacokinetic parameters of the compounds of the invention are shown in table 4 below.

TABLE 4 cynomolgus monkey pharmacokinetic parameters

Test example 5, caco-2 permeability experiment

Determination of apparent permeability coefficient (P) of analytical drugs by Caco-2 cell model using liquid chromatography tandem mass spectrometry (LC/MS/MS) _app )。

Density at Caco-2 cells (purchased from ATCC) of 7.92X 10 ⁵ cells/cm ² 210. Mu.L of HBSS (25mM HEPES,pH 7.4 containing 50. Mu.M quinidine, 30. Mu.M benzofuranone, 20. Mu.M sulfasalazine) containing 10. Mu.M test compound was added to the top of a well (available from Corning Co.), while immediately taking 10. Mu.L of the sample to a 96-well plate to which 90. Mu.L of HBSS (25mM HEPES,pH 7.4 containing 50. Mu.M quinidine, 30. Mu.M benzofuranone, 20. Mu.M sulfasalazine) had been added as an initial loading end sample, and 800. Mu.L of HBSS (25mM HEPES,pH 7.4 containing 50. Mu.M quinidine, 30. Mu.M benzofuranone, 20. Mu.M sulfasalazine) was added to the base end. Incubate at 37℃for 2 hours. 10. Mu.L of the sample was pipetted from the top into 96-well deep well plates containing 90. Mu.L of HBSS (25mM HEPES,pH 7.4, containing 50. Mu.M quinidine, 30. Mu.M benzabroclone, 20. Mu.M sulfasalazine) at 45 min and 2 hr time points, respectively. 100. Mu.L of sample was pipetted from the substrate end into 96-well deep well plates at 45 min and 2 hr time points, respectively. Then 3 volumes of pre-chilled internal standard were added per well. 1000rmp vortexed for 10 min and centrifuged at 4000rpm for 20 min. 100. Mu.L of samples were taken per well, and 3 samples were mixed together with 100. Mu.L of pure water for LC/MS/MS analysis.

Data were calculated using Microsoft Excel and peak areas were calculated from chromatograms. Apparent permeability coefficient (Papp) is in units of cm/s and is calculated using the following formula:

C _R For the concentration of the compound to be measured at the substrate end (superscript "120" or "45" is the sampling time in minutes), C _D For the top test compound concentration (superscript "1)20 "or" 45 "is the sampling time in units of: minute), area is the membrane surface Area (0.33 em ² ) The time is the total transit time (75X 60 seconds).

The apparent permeability coefficient values of Caco-2 cells are shown in Table 5.

TABLE 5 apparent osmotic coefficients of Caco-2 cells

Examples numbering	Papp (A-B) (10 -6 ，cm/s)
1	2.71
2	12.05
3	17.22

Test example 6 CYP inhibition test

Representative substrate metabolic reactions of the human major 5 CYP subtypes (CYP 1A2, CYP2C9, CYP2C19, CYP2D6, CYP3 A4/5) were assessed using 150 donor mixed human liver microsomes (available from Corning under accession number 452117). The effect of different concentrations of the compound to be tested on the metabolic reaction of phenacetin (CYP 1A 2), diclofenac sodium (CYP 2C 9), S-mefenacet (CYP 2C 19), bufuralol hydrochloride (2D 6) and midazolam (CYP 3A 4/5) is measured by liquid chromatography tandem mass spectrometry (LC/MS/MS).

mu.M of phenacetin, 10. Mu.M of diclofenac sodium, 35. Mu. M S-mefenacet, 5. Mu.M of bufuralol hydrochloride, 3. Mu.M of midazolam, 1mM NADPH, test compound (concentrations of 0.1, 0.3, 1, 3, 10, 30. Mu. Mol/L, respectively) or 200. Mu.L (100 mmol/L phosphate buffer, pH 7.4) of a reaction system of positive compound or a blank with mixed human liver microsomes (0.2 mg/mL), containing 0.3% DMSO, 0.6% acetonitrile, 0.1% methanol, respectively, were incubated at 37℃for 5 minutes. 200. Mu.L of acetonitrile containing 3% formic acid and 40nM of internal standard verapamil was then added and centrifuged at 4000rpm for 50 minutes. Cooling on ice for 20 min, and centrifuging at 4000rpm for 20 min to precipitate protein. 200. Mu.L of the supernatant was subjected to LC/MS/MS analysis.

Peak areas were calculated from chromatograms. The residual activity ratio (%) was calculated using the following formula:

peak area ratio = metabolite peak area/internal standard peak area

Residual activity ratio (%) =peak area ratio of test compound group/peak area ratio of blank group

CYP half-maximal Inhibitory Concentration (IC) ₅₀ ) Calculated by Excel XLfit 5.3.1.3.

Measured CYP half maximal inhibitory concentration (IC ₅₀ ) The values are shown in Table 6.

TABLE 6 half inhibition concentration of CYP by compounds (IC ₅₀ )

EXAMPLE 7 preparation of form A of the Compound of formula (I)

In the fifth step of example 1, column chromatography is performed by using a methanol-dichloromethane solvent system, a solid is obtained by rotary evaporation, and the solid is detected by X-ray powder diffraction, an XRPD spectrum is shown in figure 2, and a characteristic peak position is shown in table 7, so that the solid is defined as a crystal form A; the TGA spectrum (figure 3) shows that the weight loss of the A crystal form is 1.33% in the range of 25-260 ℃, and the DSC spectrum (figure 4) shows that the A crystal form has an endothermic peak, and the peak is 241.49 ℃; the X-ray powder diffraction pattern before and after DVS shows that no conversion of the DVS front and back crystalline forms occurred, see fig. 5.

TABLE 7 characteristic peaks of form A

EXAMPLE 8 preparation of form A of the Compound of formula (I)

The preparation process of the A crystal form comprises the step of solid-liquid separation, and the crystal form is determined through X-ray powder diffraction detection.

TABLE 8 preparation of form A of the compound of formula (I)

EXAMPLE 9 preparation of form B of the Compound of formula (I)

50mg of the compound A of formula (I) is weighed, 1ml of methanol is added for dissolution, filtration, 5ml of methyl tertiary butyl ether is added, stirring is carried out overnight at normal temperature, suction filtration is carried out, the mixture is washed with methyl tertiary butyl ether, the solid is obtained after drying, the solid is detected by X-ray powder diffraction, an XRPD spectrum is shown in figure 6, and the characteristic peak positions are shown in table 9, and the solid is defined as the B crystal form.

Table 9.B characteristic peaks of crystalline forms

EXAMPLE 10 preparation of form C of the Compound of formula (I)

Weighing 10mg of a crystal form A of a compound shown in a formula (I), adding 0.1ml of N-methylpyrrolidone, dissolving, adding 1.0ml of isopropanol, standing at room temperature, stirring overnight, filtering to obtain a solid, detecting by X-ray powder diffraction, and defining an XRPD spectrum as a C crystal form, wherein the characteristic peak positions are shown in a table 10; TGA spectrum shows that the weight loss of the C crystal form is 8.83% between 25 and 220 ℃; DSC spectrum shows that the C crystal form has two endothermic peaks, wherein the peaks are 197.87 ℃ and 241.33 ℃ respectively.

Table 10. Characteristic peaks of c crystalline forms

Preparation of D-form of Compound of example 11, formula (I)

Weighing 4mg of the A crystal form of the compound shown in the formula (I), adding 0.2ml of methanol, dissolving, adding 1.0ml of isopropyl ether, standing at room temperature, stirring overnight, suction filtering, drying to obtain solid, detecting by X-ray powder diffraction, and defining the XRPD spectrum as the D crystal form, wherein the characteristic peak position is shown in table 11; TGA spectrum shows that the weight loss of the D crystal form is 2.63% at 25-215 ℃; DSC spectrogram shows that the D crystal form has two endothermic peaks, wherein the peaks are 195.92 ℃ and 243.03 ℃ respectively.

Table 11. Characteristic peaks of d crystalline forms

Example 12 preparation of E Crystal form of Compound of formula (I)

Weighing 10mg of a crystal form A of a compound shown in a formula (I), adding 0.3ml of dichloromethane, dissolving, adding 1.0ml of acetonitrile, stirring overnight at 50 ℃, carrying out suction filtration, drying at 40 ℃ to obtain a solid, detecting by X-ray powder diffraction, wherein an XRPD (X-ray diffraction) spectrum is shown in figure 9, and defining the characteristic peak position as a crystal form E as shown in table 12; TGA spectrum shows that the E crystal form has weight loss of 0.33% at 25-65 ℃ and 6.95% at 65-220 ℃, and DSC spectrum shows that the E crystal form has two endothermic peaks at 193.32 ℃ and 243.68 ℃ respectively.

Table 12. Characteristic peaks of form e

Preparation of F Crystal form of Compound of example 13, formula (I)

Weighing 100mg of the E crystal form of the compound shown in the formula (I), adding 5ml of 80% water-ethanol, stirring overnight at normal temperature, carrying out suction filtration, washing with water, drying at 40 ℃ for 2 hours to obtain a solid, detecting by X-ray powder diffraction, wherein an XRPD spectrogram is shown in figure 10, and defining the characteristic peak position as the F crystal form as shown in table 13; TGA spectrum shows that the F crystal form loses weight by 3.02% at 25-70 ℃ and loses weight by 0.47% at 70-260 ℃; DSC spectrum shows that the F crystal form has two endothermic peaks, the peak is 80.36 ℃ and 243.71 ℃, and one exothermic peak is 188.21 ℃.

TABLE 13 characteristic peaks of F crystalline forms

Example 14 preparation of the G Crystal form of the Compound of formula (I)

Weighing 500mg of E crystal form of a compound shown in formula (I), adding 12.5ml of 20% water-ethanol for dissolution, adding 37.5ml of water, stirring overnight at normal temperature, carrying out suction filtration, washing with water, drying at 40 ℃ for 3 days to obtain a solid, carrying out X-ray powder diffraction detection, and defining the XRPD spectrum as G crystal form, wherein the characteristic peak position is shown in table 14; TGA spectrum shows that the weight loss of the G crystal form is 0.62% between 25 and 220 ℃; DSC spectrogram shows that the G crystal form has an endothermic peak, and the peak is 243.37 ℃; one exotherm peak, 187.40 ℃.

Table 14. Characteristic peaks of g crystalline form

Example 15 preparation of H Crystal form of Compound of formula (I)

Weighing 100mg of the E crystal form of the compound shown in the formula (I), 5ml of 80% water-ethanol, stirring overnight at normal temperature, carrying out suction filtration, washing with water, drying at 30 ℃ for 2 hours to obtain a solid, detecting by X-ray powder diffraction, wherein an XRPD spectrum is shown in figure 12, and defining the characteristic peak position as an H crystal form as shown in table 15; TGA spectrum shows that the weight loss of the H crystal form is 3.64% between 25 and 60 ℃; DSC spectrum shows that the H crystal form has three endothermic peaks, the peaks are respectively 50.81 ℃, 67.63 ℃ and 243.52 ℃, and one exothermic peak is 190.16 ℃.

Table 15. Characteristic peaks of H crystalline form

EXAMPLE 16 influence factor stability study

The A crystal form, the F crystal form, the G crystal form and the H crystal form are placed in an open and flat way, the stability of the sample under the conditions of illumination (4500 Lux), high temperature (40 ℃ and 60 ℃) and high humidity (RH 75% and RH 92.5%) is respectively inspected, and the sampling inspection period is 30 days.

Table 16. Stability data for form factor influence

Table 17. Stability data for form f influencing factors

Table 18. Stability data for form factor of influence

Table 19. Stability data for h form factor

Conclusion: the influence factor experiment shows that the A crystal form has better physical and chemical stability under the high-temperature and high-humidity conditions; F. the G, H crystal form has slightly poorer chemical and physical stability under the high-temperature and high-humidity condition.

Example 17 long term/accelerated stability study

Placing the A crystal form, the F crystal form, the G crystal form and the H crystal form at 25 ℃,60% RH and 40 ℃,75% RH, 5 ℃ and-20 ℃ respectively, and checking the stability of the B crystal form, the F crystal form, the G crystal form and the H crystal form under the condition of filling nitrogen gas

Table 20. Long term acceleration stability data for form a

Table 21. Long term acceleration stability data for form f

Table 22. Long term acceleration stability data for form g

Table 23. Long term acceleration stability data for form h

Conclusion: the long-term acceleration experiment shows that: the crystal form A and the crystal form F have good physical and chemical property stability under the long-term acceleration condition, and the crystal form G and the crystal form H are subjected to crystal transformation under the acceleration condition.

Example 18 hygroscopicity study of forms A, F, G and H

Surface Measurement Systems advantage 2, at 25deg.C, humidity from 50%, humidity range of 0% -95%, step of 10%, and judging whether each gradient quality change dM/dT is less than 0.002%, TMAX is less than 360min, and circulating for two circles.

TABLE 24 hygroscopicity data

Claims (15)

1. An X-ray powder diffraction pattern of a compound of formula (I) in the form of a crystal form A expressed in terms of diffraction angle 2 theta,

   there are characteristic peaks at 4.847, 9.801, 13.778, 14.770, 15.444, 26.077, preferably X-ray powder diffraction patterns expressed in terms of diffraction angle 2θ, at 4.847, 9.801, 13.138, 13.778, 14.770, 15.444, 18.363, 19.856, 21.092, 23.371, 26.077, 28.130, most preferably X-ray powder diffraction patterns expressed in terms of diffraction angle 2θ, at 4.847, 9.801, 13.138, 13.778, 14.770, 15.444, 18.363, 19.856, 21.092, 22.034, 23.371, 24.460, 26.077, 28.130, 28.970, 31.894, 32.920, 33.916, 38.924, and particularly preferably X-ray powder diffraction patterns thereof are as shown in fig. 2.
2. A B crystal form of a compound shown in a formula (I),

   The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 4.606, 9.110, 11.423, 13.745, 16.006, 22.973.
3. A C crystal form of a compound shown in a formula (I),

   the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 8.905, 12.920, 13.428, 14.074, 18.458, 22.519.
4. A D crystal form of a compound represented by the formula (I),

   the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 4.766, 9.594, 14.089, 14.471, 18.981, 19.609, 25.987.
5. A form E of a compound of formula (I),

   the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 4.603, 9.209, 13.920, 15.097, 19.700, 25.454.
6. A F crystal form of a compound shown in a formula (I),

   the X-ray powder diffraction pattern expressed in terms of diffraction angle 2 theta has characteristic peaks at 4.656, 14.068, 15.183, 18.858, 23.235.
7. A G crystal form of a compound of formula (I),

   the X-ray powder diffraction pattern expressed in terms of diffraction angle 2 theta has characteristic peaks at 4.869, 9.735, 13.290, 14.713, 20.020.
8. A H crystal form of a compound shown in a formula (I),

   the X-ray powder diffraction pattern expressed in terms of diffraction angle 2 theta has characteristic peaks at 8.608, 12.983, 13.476, 17.716, 20.144 and 23.371.
9. The crystalline form of the compound of formula (I) according to any one of claims 1 to 8, wherein the error range of the 2Θ angle is ± 0.20.
10. The crystalline form of the compound of formula (I) according to any one of claims 1 to 9, wherein the compound of formula (I) has an abundance of at least 20%, preferably at least 50%, most preferably at least 90%, particularly preferably at least 98% per deuterium atom (D).
11. A process for the preparation of form a of a compound of formula (I) according to any one of claims 1, 9 to 10, selected from:

    a) Mixing a compound shown in a formula (I) with a solvent I, dissolving, crystallizing and separating out, wherein the solvent I is at least one of dichloromethane and chloroform; or (b)

    b) Mixing a compound shown in a formula (I) with a solvent II, and crystallizing and separating out, wherein the solvent II is at least one selected from tetrahydrofuran, ethyl acetate, toluene, acetone, methanol, ethanol, acetonitrile, methyl tertiary butyl ether, water, isopropyl ether, butanone and n-hexane; or (b)

    c) Mixing a compound shown in a formula (I) with a solvent III to obtain a solution, mixing the solution with a solvent IV, and crystallizing and separating out the solution, wherein the solvent III is at least one selected from methanol, N-dimethylformamide and N-methylpyrrolidone; the solvent IV is at least one selected from acetonitrile, ethyl acetate, isopropyl acetate, methyl tertiary butyl ether and isopropyl ether.
12. A pharmaceutical composition comprising the following ingredients:

    1) The crystalline form of the compound of formula (I) according to any one of claims 1 to 10, or a mixture thereof and

    2) Optionally from a pharmaceutically acceptable carrier, diluent or excipient.
13. A process for the preparation of a pharmaceutical composition comprising contacting a crystalline form of a compound of formula (I) according to any one of claims 1 to 10, or a mixture thereof, with

    2) Optionally a step of mixing from a pharmaceutically acceptable carrier, diluent or excipient.
14. Use of a crystalline form of a compound of formula (I) according to any one of claims 1 to 10, or a mixture thereof, or a composition according to claim 12, for the manufacture of a medicament for the treatment of a disease or condition associated with SHP2 modulation.
15. Use of a crystalline form of a compound of formula (I) according to any one of claims 1 to 10, or a mixture thereof, or a composition according to claim 12, for the manufacture of a medicament for the treatment of tumors or cancers, preferably juvenile myelomonocytic leukemias, neuroblastomas, melanomas, acute myelogenous leukemias, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, pancreatic cancer, squamous cell carcinoma of the head and neck, gastric cancer, liver cancer, anaplastic large cell lymphomas and glioblastomas.