HK1260817A1 - Pyrrolopyrimidine crystal for preparing jak inhibitor - Google Patents

Description

Crystals of pyrrolopyrimidine compounds as JAK inhibitors

Cross Reference to Related Applications

This application claims priority and benefit to the chinese patent application No. 201610435947.4 filed on 16/06/2016 with the intellectual property office of china, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present application belongs to the field of medicinal chemistry. In particular, the application relates to crystals, crystal compositions, pharmaceutical compositions, and preparation methods and uses of a pyrrolopyrimidine compound (3R) -3- [ 3-amino-4- (7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl ] -3-cyclopentylpropionitrile as a JAK inhibitor.

Background

Janus kinases (JAKs) are a class of non-receptor tyrosine kinases (PTKs) that exist within cells and transmit cytokine stimulatory signals through the JAK-STAT pathway. The JAK-STAT pathway conducts extracellular chemical signals through the cell membrane to gene promoters located on DNA within the nucleus, ultimately affecting changes in DNA transcription and activity levels in the cell. The JAK-STAT pathway consists of three major components: 1) a receptor; 2) janus kinases (JAK) and 3) Signal Transducer and Activator of Transcription (STAT). The receptor may be activated by interferons, interleukins, growth factors or other chemical messengers, activation leading to autophosphorylation of JAKs; the STAT proteins then bind to phosphorylated receptors, allowing STAT to be phosphorylated by JAKs; phosphorylated STAT proteins are then isolated from the receptor, dimerized, and translocated into the nucleus to bind to specific DNA sites and alter transcription (Scott, m.j., c.j. godshall et al (2002). "Jaks, STATs, Cytokines, and septis" ClinDiagn Lab Immunol 9 (6): 1153-9).

The JAK family plays a role in cellular proliferation and functional cytokine-dependent regulation of immune responses. Currently, there are four known mammalian JAK family members: JAK1, JAK2, JAK3 and TYK2(Tyrosine kinase 2). The size range of the JAK protein is 120-140kDa, which contains 7 conserved JAK Homology (JH) domains; one of them is a functional catalytic kinase domain and the other is a pseudokinase (pseudokinase) domain, which effectively exerts a regulatory function and/or functions as a docking site for STATs (Scott, Godshall et al 2002, supra).

At present, there are many reports of inhibitors of janus kinases, of which a number of JAK inhibitors are disclosed in chinese patent application No. 201410784461.2, filed 12/16 2014 (incorporated herein by reference in its entirety), including (3R) -3- [ 3-amino-4- (7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl ] -3-cyclopentylpropanenitrile compounds represented by formula I below:

in addition to therapeutic efficacy, drug developers have attempted to provide suitable forms of active molecules with properties that are drugs. The chemical stability, solid state stability and shelf life of the active ingredient are all very important factors from the viewpoint of obtaining a commercially viable production process or from the viewpoint of producing a pharmaceutical composition containing the active compound. Therefore, providing a form with the desired properties is crucial for drug development.

Summary of The Invention

In one aspect of the present application there is provided crystalline form a of the compound of formula I,

an X-ray diffraction (XRD) pattern of crystal A of the compound represented by formula I has diffraction angles, 2 theta, of 9.35 DEG + -0.2 DEG, 11.93 DEG + -0.2 DEG, 16.32 DEG + -0.2 DEG, 21.23 DEG + -0.2 DEG, 23.13 DEG + -0.2 DEG and 25.58 DEG + -0.2 deg.

In another aspect, the present application provides a process for preparing crystal a of the compound of formula I, the process comprising the steps of:

1) dissolving a compound shown in a formula I in a crystallization solvent, wherein the crystallization solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, ethylene glycol monomethyl ether, diethyl ether, isopropyl ether, methyl tert-butyl ether, dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, 1-butanone, 2-butanone, ethyl acetate, ethyl formate, methyl acetate, isopropyl acetate, dichloromethane, chloroform, water or a mixed solvent of any two or more of the solvents; and

2) and (4) crystallizing.

In another aspect, the present application provides a crystalline composition wherein crystal a of the compound of formula I comprises more than 50%, preferably more than 80%, more preferably more than 90%, and most preferably more than 95% by weight of the crystalline composition.

Another aspect of the present application provides a pharmaceutical composition comprising an effective amount of crystal a of the compound of formula I, or a crystalline composition comprising crystal a of the compound of formula I.

In another aspect of the application, there is provided a use of crystal a of the compound represented by formula I, or the above crystal composition or the above pharmaceutical composition in the preparation of a medicament for treating or preventing a janus kinase-mediated disease.

In yet another aspect, the present application provides a crystalline form B of a compound of formula I:

an X-ray diffraction (XRD) pattern of crystal B of the compound represented by the formula I has diffraction peaks with 2 theta of 8.97 DEG + -0.2 DEG, 9.39 DEG + -0.2 DEG, 12.90 DEG + -0.2 DEG, 17.70 DEG + -0.2 DEG, 20.31 DEG + -0.2 DEG and 23.63 DEG + -0.2 deg.

In another aspect, the present application provides a process for preparing crystal B of the compound of formula I, the process comprising the steps of:

1) dissolving a compound shown as a formula I in acetonitrile; and

2) and (4) crystallizing.

In another aspect, the present application provides a crystalline composition wherein the crystalline form B of the compound of formula I comprises more than 50%, preferably more than 80%, more preferably more than 90%, and most preferably more than 95% by weight of the crystalline composition.

Another aspect of the present application provides a pharmaceutical composition comprising an effective amount of crystal B of the compound of formula I, or a crystalline composition comprising crystal B of the compound of formula I.

In another aspect of the application, there is provided a use of crystal B of the compound represented by formula I, or the above crystal composition or the above pharmaceutical composition in the manufacture of a medicament for treating or preventing a janus kinase-mediated disease.

Drawings

Figure 1 is an XRD pattern of crystal a of the compound of formula I (example 2, method 1).

FIG. 2 is a DSC of crystal A of the compound of formula I (example 2, method 1).

FIG. 3 is an XRD pattern of crystal A of the compound of formula I (example 2, method 2, ethanol-ethyl acetate (4: 1)).

Figure 4 is an XRD pattern of crystal a of the compound of formula I (example 2, method 3).

Figure 5 is an XRD pattern of crystal B (example 3) of the compound of formula I.

FIG. 6 is a DSC of crystal B of the compound represented by formula I (example 3).

Detailed Description

In one aspect, the present application provides a crystalline form a of a compound of formula I:

an X-ray diffraction (XRD) pattern of the crystal A of the compound shown in the formula I has diffraction peaks with 2 theta of 9.35 degrees, 11.93 degrees, 16.32 degrees, 21.23 degrees, 23.13 degrees and 25.58 degrees +/-0.2 degrees; typically have diffraction peaks with 2 θ of 9.35 °, 11.93 °, 16.32 °, 18.82 °, 20.54 °, 21.23 °, 23.13 °, and 25.58 ° ± 0.2 °; more typically, diffraction peaks at 9.35 °, 10.93 °, 11.93 °, 14.46 °, 16.32 °, 18.82 °, 20.54 °, 21.23 °, 21.66 °, 23.13 °, 25.58 °, and 26.34 ° ± 0.2 ° 2 θ; further typically have diffraction peaks at 2 θ of 9.35 °, 10.93 °, 11.93 °, 14.46 °, 16.32 °, 17.28 °, 18.82 °, 19.25 °, 20.54 °, 21.23 °, 21.66 °, 22.15 °, 23.13 °, 24.09 °, 25.58 °, and 26.34 ° ± 0.2 °.

In some embodiments of the present application, in an X-ray diffraction (XRD) pattern of crystal a of the compound of formula I of the present application, a peak having the largest relative intensity appears at a diffraction peak position where 2 θ is 11.93 °, 16.32 °, or 21.23 ° ± 0.2 °; preferably, the peak with the largest relative intensity occurs at the diffraction peak position with 2 θ of 11.93 ° ± 0.2 °.

In some embodiments of the present application, the compound of formula I of the present application has an X-ray diffraction (XRD) pattern in which the peak three positions before relative intensity appears at the diffraction peak position at 9.35 °, 11.93 °, 16.32 °, 21.23 °, 23.13 ° or 25.58 ° ± 0.2 ° 2 θ.

In some embodiments of the present application, the compound of formula I of the present application has the following X-ray diffraction peak of crystal a:

serial number	2θ±0.2(°)	Relative Strength (%)	Serial number	2θ±0.2(°)	Relative Strength (%)
						1	9.35	43.1	11	20.54	41.6
2	10.82	16.0	12	21.23	75.9
						3	10.93	17.0	13	21.66	38.1
4	11.93	100.0	14	22.15	26.9
						5	13.65	15.8	15	23.13	55.5
6	14.46	18.6	16	23.47	14.5
						7	16.32	58.1	17	24.09	23.5
8	17.28	13.9	18	25.58	54.3
						9	18.82	40.9	19	26.34	33.8
10	19.25	22.3	20	30.02	15.8

In some embodiments of the present application, the compound of formula I has an X-ray diffraction pattern of crystal a as shown in figure 1.

In some embodiments of the present application, the DSC profile of crystal a of the compound of formula I is shown in figure 2.

In some embodiments of the present application, the compound of formula I has an X-ray diffraction pattern of crystal a as shown in figure 3.

In some embodiments of the present application, the compound of formula I has an X-ray diffraction pattern of crystal a as shown in figure 4.

In another aspect, the present application provides a process for preparing crystal a of the compound of formula I, the process comprising the steps of:

1) dissolving a compound shown in a formula I in a crystallization solvent, wherein the crystallization solvent is selected from methanol, ethanol, n-propanol isopropanol, n-butanol, isobutanol, tert-butanol, ethylene glycol monomethyl ether, diethyl ether, isopropyl ether, methyl tert-butyl ether, dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, 1-butanone, 2-butanone, ethyl acetate, ethyl formate, methyl acetate, isopropyl acetate, dichloromethane, chloroform, water or a mixed solvent of any two or more of the solvents; and

2) devitrification and optionally filtration, washing and/or drying.

In some embodiments of the present application, the crystallization solvent for preparing the crystals a of the compound represented by formula I is ethanol, isopropyl ether, ethyl acetate, acetone, dichloromethane, water, or a mixed solvent of any two or more of the above solvents; preferably ethanol, ethanol-ethyl acetate mixed solvent, ethanol-water mixed solvent, ethanol-isopropyl ether mixed solvent, acetone, ethyl acetate or dichloromethane.

In some embodiments of the present application, the crystallization solvent for preparing the crystals a of the compound represented by formula I is preferably ethanol or a mixed solvent comprising ethanol; more preferably, the other solvent in the mixed solvent comprising ethanol is selected from methanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, ethylene glycol monomethyl ether, diethyl ether, isopropyl ether, methyl t-butyl ether, dioxane, tetrahydrofuran, 2-methyl tetrahydrofuran, acetone, 1-butanone, 2-butanone, ethyl acetate, ethyl formate, methyl acetate, isopropyl acetate, dichloromethane, chloroform or water.

In some embodiments of the present application, crystals A of the compound of formula I are prepared using a compound of formula I (in g of weight units) in a ratio to the amount of crystallization solvent (in mL of volume units) selected from the group consisting of 1: 5 to 1: 50, preferably 1: 7.5, 1: 10, 1: 12, 1: 15, 1: 18, 1: 20, 1: 25, 1: 30, 1: 35, 1: 40, 1: 45 or 1: 50, more preferably 1: 7.5 to 1: 30.

In some embodiments of the present application, when the crystallization solvent for preparing the crystals a of the compound represented by formula I is selected from a mixed solvent comprising ethanol, the content (by volume) of ethanol is 10% to 90%; preferably 10%, 20%, 25%, 30%, 33%, 40%, 50%, 60%, 66%, 70%, 75%, 80% or 90%.

In some embodiments of the present application, when the crystallization solvent for preparing the crystals a of the compound represented by formula I is selected from mixed solvents comprising ethanol, the ratio (by volume) of ethanol to other solvents is 9: 1 to 1: 9; preferably 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8 or 1: 9.

In some embodiments of the present application, crystallization may be performed by cooling, for example, to 0-5 ℃. In some embodiments of the present application, crystallization may be performed by concentration under reduced pressure.

In another aspect, the present application provides a crystalline composition comprising crystalline form a of the compound of formula I. In some embodiments of the present application, the amount of crystal a of the compound of formula I in the crystalline composition is 50% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, by weight of the crystalline composition.

Another aspect of the present application provides a pharmaceutical composition comprising crystal a of the compound of formula I, comprising an effective amount of crystal a of the compound of formula I, or a crystalline composition comprising crystal a of the compound of formula I. In addition, the pharmaceutical composition may or may not contain pharmaceutically acceptable carriers, excipients and/or vehicles.

In another aspect of the application, there is provided a use of crystal a of the compound represented by formula I, or the above crystal composition or the above pharmaceutical composition in the preparation of a medicament for treating or preventing a janus kinase-mediated disease.

Another aspect of the present application provides a method for treating or preventing a janus kinase-mediated disease, which comprises administering to a mammal in need thereof a therapeutically effective amount of crystal a of the compound represented by formula I above, or the above crystal composition, or the above pharmaceutical composition.

Another aspect of the present application provides crystal a of the compound represented by formula I above, or the above crystal composition, or the above pharmaceutical composition, for use in treating or preventing a janus kinase-mediated disease.

In yet another aspect, the present application provides a crystalline form B of a compound of formula I:

an X-ray diffraction (XRD) pattern of crystal B of the compound represented by the formula I has diffraction peaks with 2 theta of 8.97 degrees, 9.39 degrees, 12.90 degrees, 17.70 degrees, 20.31 degrees and 23.63 degrees +/-0.2 degrees; typically have diffraction peaks with 2 θ of 8.97 °, 9.39 °, 12.90 °, 16.54 °, 17.70 °, 19.20 °, 20.31 °, 22.78 °, and 23.63 ° ± 0.2 °; more typically, diffraction peaks at 2 θ of 8.97 °, 9.39 °, 11.24 °, 12.90 °, 14.56 °, 16.54 °, 17.70 °, 19.20 °, 20.31 °, 22.23 °, 22.78 °, 23.63 °, and 25.55 ° ± 0.2 °.

In some embodiments of the present application, in an X-ray diffraction (XRD) pattern of crystal B of the compound of formula I of the present application, a peak having the largest relative intensity appears at a diffraction peak position at 9.39 °, 17.70 ° or 23.63 ° ± 0.2 ° 2 θ; preferably, the peak with the largest relative intensity occurs at the diffraction peak position with 2 θ of 17.70 ° ± 0.2 °.

In some embodiments of the present application, the X-ray diffraction peak of crystal B of the compound of formula I of the present application has the following characteristics:

serial number	2θ±0.2(°)	Relative strength(％)	Serial number	2θ±0.2(°)	Relative Strength (%)
						1	8.97	40.7	11	20.31	36.1
2	9.39	47.4	12	20.41	24.3
						3	11.24	17.9	13	21.03	21.3
4	12.90	34.4	14	21.96	18.6
						5	12.96	32.8	15	22.23	18.9
6	14.56	14.4	16	22.78	23.1
						7	16.54	22.1	17	23.50	46.9
8	17.15	40.4	18	23.63	65.0
						9	17.70	100.0	19	25.55	17.3
10	19.20	24.4	----	----	----

In some embodiments of the present application, the compound of formula I has crystal B with an X-ray diffraction pattern as shown in figure 5.

In some embodiments of the present application, the DSC profile of crystal B of the compound of formula I is shown in figure 6.

The crystal B of the compound shown in the formula I is an acetonitrile compound of the compound shown in the formula I, wherein the molar weight ratio of the compound shown in the formula I to the acetonitrile is selected from 1: 0.5-1: 2.0, and preferably 1: 0.5, 1: 1, 1: 1.5 or 1: 2.0.

In another aspect, the present application provides a process for preparing crystal B of the compound of formula I, the process comprising the steps of:

1) dissolving a compound shown as a formula I in acetonitrile; and

2) devitrification and optionally filtration, washing and/or drying.

In some embodiments of the present application, crystals B of the compound of formula I are prepared in a ratio of the compound of formula I (in g of weight units) to the amount of acetonitrile used as the crystallization solvent (in mL of volume units) selected from the group consisting of 1: 5 to 1: 50, preferably 1: 7.5, 1: 10, 1: 12, 1: 15, 1: 18, 1: 20, 1: 25, 1: 30, 1: 35, 1: 40, 1: 45 or 1: 50, more preferably 1: 10 to 1: 25.

In some embodiments of the present application, crystallization may be performed by cooling, for example, to 0-5 ℃.

In another aspect, the present application provides a crystalline composition comprising crystalline form B of the compound of formula I. In some embodiments of the present invention, the amount of crystal B of the compound of formula I is 50% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more by weight of the crystalline composition.

Another aspect of the present application provides a pharmaceutical composition comprising crystal B of the compound of formula I, comprising an effective amount of crystal B of the compound of formula I, or a crystalline composition comprising crystal B of the compound of formula I. In addition, the pharmaceutical composition may or may not contain pharmaceutically acceptable carriers, excipients and/or vehicles.

In another aspect of the application, there is provided a use of crystal B of the compound represented by formula I, or the above crystal composition or the above pharmaceutical composition in the manufacture of a medicament for treating or preventing a janus kinase-mediated disease.

Another aspect of the present application provides a method for treating or preventing a janus kinase-mediated disease, which comprises administering to a mammal in need thereof a therapeutically effective amount of crystal B of the compound represented by formula I above, or the above crystal composition, or the above pharmaceutical composition.

Another aspect of the present application provides crystal B of the compound represented by formula I above, or the above crystal composition, or the above pharmaceutical composition, for use in treating or preventing a janus kinase-mediated disease.

In the present application, the X-ray diffraction pattern is determined by the following method: the instrument comprises the following steps: bruker D8 ADVANCE X-ray diffractometer; the method comprises the following steps: target: cu: K-Alpha; wavelength of lightTube pressure Voltage: 40 kV; pipe flow Current: 40 mA; scanning range: 4-40 degrees; scanning speed: 0.1 second per step and 0.02 degree per step.

In the present application, Differential Scanning Calorimetry (DSC) is measured by the following method: the instrument comprises the following steps: a Mettler DSC-1 differential scanning calorimeter; the method comprises the following steps: a sample (-5 mg) was placed in a DSC aluminum pan for testing by the method: 30-300 ℃ and the heating rate is 10 ℃/min.

It is noted that in X-ray diffraction spectroscopy, the diffraction pattern obtained from a crystalline compound is often characteristic for a particular crystalline form, where the relative intensities of the bands (especially at low angles) may vary due to the dominant orientation effects resulting from differences in crystallization conditions, particle size, and other measurement conditions. Therefore, the relative intensities of the diffraction peaks are not characteristic of the crystal form in question, and when judging whether the diffraction peaks are the same as the known crystal form, the relative positions of the peaks rather than their relative intensities should be noted. In addition, there may be slight errors in the position of the peaks for any given crystalline form, which is also well known in the crystallography art. For example, the position of the peak may shift due to a change in temperature when analyzing the sample, movement of the sample, calibration of the instrument, or the like, and the measurement error of the 2 θ value may be about ± 0.2 °. Therefore, this error should be taken into account when determining each type of structure. The peak position is usually expressed in the XRD pattern by 2 θ angle or plane distance d, with a simple conversion relationship between: d ═ λ/2sin θ, where d represents the interplanar spacing, λ denotes the wavelength of the incident X-rays, and θ denotes the diffraction angle. For the same crystal form of the same compound, the peak positions of the XRD spectrum have similarity on the whole, and the error of relative intensity is likely to be larger. It should also be noted that in the identification of mixtures, the loss of a portion of the diffraction lines may be due to, for example, a reduction in the amount of the compound, in which case it is not necessary to rely on all the bands observed in the high purity sample, and even one band may be characteristic of a given crystal.

In addition, DSC measures the transition temperature when a crystal absorbs or releases heat due to a change in its crystal structure or melting of the crystal. For the same crystal form of the same compound, the thermal transition temperature and melting point errors in successive analyses are typically within about 5 ℃, which when we say a compound has a given DSC peak or melting point means that the DSC peak or melting point is ± 5 ℃. DSC provides an auxiliary method to distinguish different crystal forms. Different crystal morphologies can be identified by their different transition temperature characteristics.

The janus kinase-mediated diseases described herein include, but are not limited to, tumors (e.g., lymphoma, leukemia). The lymphomas described herein may include, but are not limited to, Hodgkin's disease or Non-Hodgkin's lymphoma (Non-Hodgkin's lymphoma), including but not limited to B-cell lymphoma (B-cell lymphoma) or T-cell lymphoma (T-cell lymphoma). The leukemia described herein includes, but is not limited to, Acute lymphocytic leukemia (Acute lymphocytic leukemia), Chronic lymphocytic leukemia (Chronic lymphocytic leukemia), Acute myelogenous leukemia (Acute myelogenous leukemia), and Chronic myelogenous leukemia (Chronic myelocytic leukemia).

In the present application, the term "pharmaceutical composition" refers to a formulation of one or more compounds of the present application with carriers, excipients and/or vehicles generally accepted in the art for the delivery of biologically active compounds to organisms (e.g., humans). The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

The term "carrier" is defined as a compound that facilitates the introduction of the compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is commonly used as a carrier because it facilitates the introduction of certain organic compounds into cells or tissues of an organism.

"pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that is approved by the national drug administration for use in humans or livestock.

By "therapeutically effective amount" is meant an amount of a compound of the present application which, when administered to a mammal, preferably a human, is sufficient to effect treatment of a viral infection in the mammal, preferably a human, as defined below. The amount of a compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and this disclosure.

As used herein, "treatment" encompasses treatment of a viral infection in a mammal, preferably a human, suffering from the viral infection and includes:

(i) inhibiting the viral infection, i.e. preventing its development;

(ii) relief of, i.e., cause recovery from, the viral infection; or

(iii) Alleviating symptoms caused by viral infection.

All solvents used herein are commercially available and can be used without further purification. The reaction is generally carried out under inert nitrogen in an anhydrous solvent.

In the present application, proton nmr data are recorded on a BRUKER AVANCE III HD 500M spectrometer with chemical shifts expressed as (ppm) at tetramethylsilane low field; mass spectra were measured at Waters ACQUITY UPLC + XEVO G2 QTof. The mass spectrometer was equipped with an electrospray ion source (ESI) operating in either positive or negative mode.

The crystal A and the crystal B of the compound shown in the formula I have the advantages of high purity, high crystallinity, good stability and the like; meanwhile, the preparation method of the compound crystal A and the crystal B shown in the formula I is simple, the solvent is cheap and easy to obtain, the crystallization condition is mild, and the method is suitable for industrial production.

The following examples further illustrate the present invention in non-limiting detail. They should not be considered as limiting the scope of the invention but merely as being exemplary illustrations and representative of the invention. The solvents, reagents, raw materials and the like used in the present application are all commercially available chemically pure or analytically pure products.

Example 1 (3R) -3- { 3-amino-4- { 7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } -3-cyclopentylpropanenitrile (I)

Step A: 3-Cyclopentylacrylic acid

Cyclopentylformaldehyde (344.4g, 3.51mol, 1.17eq.) was added dropwise to a 5M malonic acid (312g, 3.0mol, 1.0eq.) solution in pyridine at room temperature, followed by stirring for 10 minutes, followed by slow addition of piperidine (6.2g, 0.075mol, 0.025eq.) followed by reaction for 1 hour at room temperature. Heating to 70-80 ℃, stirring and reacting for 8 hours, concentrating under reduced pressure and evaporating to remove the solvent, adjusting the pH of the residue to 3.0 by concentrated hydrochloric acid, extracting by ethyl acetate for three times, combining organic phases, washing by 2.5M sodium hydroxide solution for five times, adjusting the pH of a water layer by concentrated hydrochloric acid to 3.0, extracting by ethyl acetate for three times, combining organic layers, washing by water for three times, washing by saturated salt water, drying by anhydrous sodium sulfate, filtering and concentrating under reduced pressure to obtain 3-cyclopentyl acrylic acid (391.2g, yield: 93%).¹H NMR(500MHz，CDCl₃)：7.08(dd，J＝15.6，8.1Hz，1H)、5.81(dd，J＝15.6，1.1Hz，1H)、11.25(s，1H)、2.64(m，1H)、1.63(m，2H)、1.42(m，2H)、1.86(m，2H)、1.72(m，2H)；HRMS(ESI)calcd.for C₈H₁₂O₂[M-H]^-139.0765；Found：139.0760。

And B: 5-cyclopentyl-pyrazolidin-3-ones

Under stirring at room temperature, adding 80% hydrazine hydrate (253.5g, 4.05mol, 1.5eq.) dropwise into cyclopentyl acrylic acid (378g, 2.7mol, 1.0eq.), heating to 70-80 ℃, stirring for reaction for 6 hours, cooling to 0-10 ℃, stirring for crystallization, filtering, washing a filter cake twice, and drying by blowing at 45 ℃ for 12 hours to obtain 5-cyclopentyl pyrazolidin-3-one (292.5g, 68% yield).

And C: R-5-cyclopentyl-pyrazolidin-3-one-D-tartrate

D-tartaric acid (135g, 0.9mol, 0.5eq.) was added to an acetone solution of 5-cyclopentylpyrazoline-3-one (278g, 1.8mol, 1.0eq.) with stirring at room temperature, the mixture was reacted for 2 hours with stirring, then crystallized, filtered, the cake was slurried with acetone for 5 times, and dried by forced air at 50 ℃ to obtain R-5-cyclopentylpyrazoline-3-one-D-tartrate (241g, 88% yield, 99.5% ee).

Step D: r-5-cyclopentyl-pyrazolidin-3-one

Adding R-5-cyclopentyl pyrazolidin-3-one-D-tartrate (228g, 0.75mol, 1.0eq.) into a 4M sodium hydroxide (52.2g, 2.61mol, 1.74eq.) solution under stirring at room temperature, extracting with dichloromethane, combining organic layers, drying over anhydrous magnesium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain R-5-cyclopentyl pyrazolidin-3-one (100.6g, 85.2% yield, 99.5% ee value))。¹H-NMR(500MHz，CDCl₃)8.93(s，1H)、5.15(s，1H)、1.89(m，1H)、1.67(m，2H)、1.55(m，2H)、1.47(m，2H)、1.26(m，1H)、1.14(m，1H)；HRMS(ESI)calcd.for C₈H₁₄N₂O[M+H]⁺155.1179；Found：155.1183。

Step E: 4-chloro-7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidine

4-Chloropyrrolo [2, 3-d ] was added under ice bath]Pyrimidine (200g, 1.3mol, 1.0eq.) solution in N, N-dimethylformamide was added 60% NaH (62.4g, 1.56mol, 1.2eq.), and after stirring at room temperature for 1 hour, 2- (trimethylsilyl) ethoxymethyl chloride (SEMCl, 260g, 1.56mol, 1.2eq) was slowly added dropwise with cooling in an ice bath. After the addition, stirring and reacting for 1 hour in ice bath, adding water for quenching, extracting by ethyl acetate, combining organic phases, washing by saturated saline solution, drying by anhydrous sodium sulfate, filtering, decompressing and concentrating filtrate to obtain residue, and purifying by silica gel column chromatography to obtain 4-chloro-7- { [2- (trimethylsilyl) ethoxy group]Methyl } -7H-pyrrolo [2, 3-d)]Pyrimidine (312.2g, 91.8% yield).¹H-NMR(500MHz，CDCl₃)：8.64(s，1H)、7.38(d，J＝3.6Hz，1H)、6.65(d，J＝3.6Hz，1H)、5.64(s，2H)、3.52(t，J＝8.2Hz，2H)、0.90(t，J＝8.2Hz，2H)、-0.07(s，9H)；HRMS(ESI)calcd.for C₁₂H₁₈N₃OSi[M+H]⁺284.0980；Found：284.0995。

Step F: 2-cyano-2- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } acetic acid ethyl ester

Potassium carbonate (207g, 1.5mol, 3.0eq.) was added to 4-chloro-7- { [2- (trimethylsilyl) ethoxy ] at room temperature with stirring]Methyl } -7H-pyrrolo [2, 3-d)]Pyrimidine (142g, 0.5mol, 1.0eq.) and ethyl cyanoacetate (85g, 0.75mol, 1.5 e)q) DMF solution, heating to 120 ℃, stirring for reaction for 4 hours, cooling to room temperature, adding water for quenching, stirring for crystallization, filtering, washing filter cake with water, and drying by air blast at 50 ℃ to obtain 2-cyano-2- {7- { [2- (trimethylsilyl) ethoxy group]Methyl } -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl } acetic acid ethyl ester (167g, 92.6% yield).¹H-NMR(500MHz，CDCl₃)：13.46(s，1H)、8.45(s，1H)、7.56(d，J＝3.6Hz，1H)、7.18(d，J＝3.6Hz，1H)、5.56(s，2H)、4.32(q，J＝7.1Hz，2H)、3.52(t，J＝8.2Hz，2H)、1.27(t，J＝7.1Hz，3H)、0.83(t，J＝8.2Hz，2H)、-0.08(s，9H)；HRMS(ESI)calcd.for C₁₇H₂₄N₄O₃Si[M+H]⁺361.1690；Found：361.1699。

Step G: 2- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } acetonitrile

Sodium chloride (263g, 4.5mol, 10eq.) was added to 2-cyano-2- {7- { [2- (trimethylsilyl) ethoxy ] 2-cyano-2 at room temperature with stirring]Methyl } -7H-pyrrolo [2, 3-d)]And (3) heating the mixed solution of pyridine-4-yl ethyl acetate (162.2g, 0.45mol and 1.0eq.) and N-methylpyrrolidone and water to 160-170 ℃, and stirring the mixture to react for 30 hours. Quenching with water, extracting with ethyl acetate, washing organic layer with saturated saline, drying with anhydrous sodium sulfate, filtering, concentrating the residue, and purifying with silica gel column chromatography to obtain 2- (7- { [2- (trimethylsilyl) ethoxy]Methyl } -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) acetonitrile (98.6g, 76% yield).¹H-NMR(500MHz，CDCl₃)：8.18(s，1H)、7.77(d，J＝3.4Hz，1H)、6.83(d，J＝3.4Hz，1H)、5.65(s，2H)、4.56(s，2H)、3.52(t，J＝7.6Hz，2H)、0.82(t，J＝7.6Hz，2H)、-0.10(s，9H)；HRMS(ESI)calcd.forC₁₄H₂₀N₄OSi[M+H]⁺289.1479；Found：289.1498。

Step H: 3- (dimethylamino) -2- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } acrylonitrile

DMF-DMA (119g, 1.0mol, 3.0eq.) was added to a solution of 2- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } acetonitrile (95g, 0.33mol, 1.0eq.) in DMF, the mixture was heated to reflux for 2 hours, cooled to room temperature, stirred with water for crystallization, filtered, the filter cake washed with water, and dried by forced air at 50 ℃ to give 3- (dimethylamino) -2- (7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) acrylonitrile (106.5g, 94% yield).

¹H-NMR(500MHz，CDCl₃)：8.50(s，1H)、8.38(s，1H)、7.26(d，J＝3.7Hz，1H)、7.18(d，J＝3.7Hz，1H)、5.56(s，2H)、3.49(t，J＝8.4Hz，2H)、3.43(s，3H)、3.23(s，3H)、0.87(t，J＝8.4Hz，2H)、-0.10(s，9H)；；HRMS(ESI)calcd.for C₁₇H₂₅N₅OSi[M+H]⁺344.1901；Found：344.1907。

Step I: (R) -3- { 3-amino-4- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } -3-cyclopentylpropionic acid

While stirring at room temperature, potassium acetate (1.5eq) was added to 3- (dimethylamino) -2- {7- { [2- (trimethylsilyl) ethoxy group]Methyl } -7H-pyrrolo [2, 3-d)]Heating pyrimidine-4-yl acrylonitrile (68.7g, 0.2mol, 1.0eq.) and R-5-cyclopentyl pyrazolidin-3-one (37.0g, 0.24mol, 1.2eq.) in N-methyl pyrrolidone solution, and stirring for reaction for 12 hours at 120-130 ℃. Adding water for quenching, extracting by ethyl acetate, washing an organic layer for three times by water, washing by saturated salt water, and drying by anhydrous sodium sulfate. Filtering, concentrating under reduced pressure to obtain residue, purifying by silica gel column chromatography to obtain (R) -3- { 3-amino-4- {7- { [2- (trimethylsilyl) ethoxy]Methyl } -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl } -1H-pyrazol-1-yl } -3-cyclopentylpropionic acid (37.6g, 40.1% yield, ee 99.8%).¹H-NMR(500MHz，CDCl₃)：8.74(s，1H)、7.96(s，1H)、7.32(d，J＝3.4Hz，1H)、6.67(d，J＝3.4Hz，1H)、5.63(m，2H)、4.19(t，J＝8.2Hz，2H)、3.52(m，1H)、3.52(t，J＝8.4Hz，2H)、3.09(dd，J＝16.7，8.2Hz，1H)、2.87(d，J＝16.7Hz，1H)、2.41(m，1H)、1.87(m，1H)、1.69(m，1H)、1.60(m，2H)、1.51(m，2H)、1.15(m，1H)、0.91(t，J＝8.4Hz，2H)、-0.06(s，9H)；HRMS(ESI)calcd.for C₁₇H₂₅N₅OSi[M+H]⁺471.2534；Found：471.2538。

Step J: (R) -3- {3- (2, 5-dioxopyrrol-1-yl) -4- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } -3-cyclopentylpropionic acid

To 0.2M of (R) -3- { 3-amino-4- {7- { [2- (trimethylsilyl) ethoxy group was added under stirring at room temperature]Methyl } -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl]-1H-pyrazol-1-yl } -3-cyclopentylpropionic acid (35.0g, 74.3mmol, 1.0eq.) in toluene solution succinic anhydride (10.4g, 104mmol, 1.4eq.) was added and the reaction was heated to reflux (water cut) for 14H under nitrogen. Concentrating under reduced pressure to remove solvent, dissolving the residue in ethyl acetate, washing with water, washing with saturated sodium bicarbonate solution and saturated sodium chloride solution, adding anhydrous sodium sulfate and active carbon into the ethyl acetate layer, stirring, drying, decolorizing, filtering, and concentrating under reduced pressure to obtain (R) -3- {3- (2, 5-dioxopyrrol-1-yl) -4- {7- { [2- (trimethylsilyl) ethoxy ] ethyl]Methyl } -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl } -1H-pyrazol-1-yl } -3-cyclopentylpropionic acid (39g, 70.6mmol, 95% yield).¹H-NMR(500MHz，CDCl₃)：8.65(s，1H)、8.28(s，1H)、7.28(d，J＝3.7Hz，1H)、6.62(d，J＝3.7Hz，1H)、5.59(d，J＝11.1Hz，1H)、5.53(d，J＝11.1Hz，1H)、4.44(td，J＝9.9，3.2Hz，1H)、3.48(m，2H)、3.02(dd，J＝16.8，10.0Hz，1H)、2.83(m，1H)、2.43(m，1H)、1.78(m，1H)、1.69(m，1H)、1.61(m，1H)、1.52(m，1H)、1.51(m，1H)、1.50(m，2H)、1.14(m，1H)、0.88(m，2H)、-0.07(s，9H)；HRMS(ESI)calcd.for C₂₇H₃₆N₆O₅Si[M+H]⁺553.2589；Found：553.2603。

Step K: (R) -3-cyclopentyl-3- [3- (2, 5-dioxopyrrol-1-yl) -4- (7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl ] propanamide

Oxalyl chloride (20.0g, 158mmol, 2.5eq.) is added dropwise to a 0.18M solution of (R) -3- {3- (2, 5-dioxopyrrol-1-yl) -4- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } -3-cyclopentylpropionic acid (35.0g, 63.3mmol, 1.0eq.) in dichloromethane with stirring under nitrogen atmosphere, DMF (0.1g, 1.3mmol, 0.02eq.) is added dropwise after addition, the reaction is stirred at room temperature for 1 hour, the solvent is evaporated under reduced pressure, sodium threads are added, THF is dried and dissolved by distillation, the solution is added dropwise to a 2M solution of aqueous ammonia (20.0, 0.32mol, 5.0eq.) in THF, and the reaction is stirred for 30 minutes, the reaction mixture was concentrated under reduced pressure to remove THF, cooled in an ice bath to crystallize for 2 hours, filtered, the filter cake was washed with water and dried by forced air at 50 ℃ to give (R) -3-cyclopentyl-3- {3- (2, 5-dioxopyrrol-1-yl) -4- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } propionamide (29.8g, 85.5% yield).

¹H-NMR(500MHz，CDCl₃)：8.65(s，1H)、8.24(s，1H)、7.32(d，J＝3.7Hz，1H)、6.63(d，J＝3.7，1H)、6.12(s，1H)、5.60(d，J＝11.1Hz，1H)、5.56(d，J＝11.1Hz，1H)、5.44(s，1H)、4.40(td，J＝10.6，3.2Hz，1H)、3.47(dd，J＝9.1，7.5Hz，2H)、2.99(dd，J＝14.4，11.0Hz，1H)、2.91(s，4H)、2.67(dd，J＝14.4，3.3Hz，1H)、2.48(m，1H)、1.84(m，1H)、1.66(m，1H)、1.58(m，2H)、1.57(m，1H)、1.50(m，1H)、1.31(m，1H)、1.21(m，1H)、0.88(dd，9.1，7.5，2H)、-0.08(s，9H)；；HRMS(ES)calcd.for C₂₇H₃₇N₇O₄Si[M+H]⁺552.2749；Found：552.2759。

Step L: (R) -3-cyclopentyl-3- [3- (2, 5-dioxopyrrol-1-yl) -4- (7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl ] propionitrile

To a solution of 0.2M (R) -3-cyclopentyl-3- {3- (2, 5-dioxopyrrol-1-yl) -4- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3- ] pyrimidin-4-yl } -1H-pyrazol-1-yl } propionamide (25g, 45.3mmol, 1.0eq) in dichloromethane was added dropwise phosphorus oxychloride (27.8g, 181mmol, 4.0eq.) with stirring in an ice bath, and after completion of the reaction, the mixture was stirred at room temperature for 2 hours, quenched with water, washed with water for the organic layer, added with anhydrous magnesium sulfate and activated carbon, dried with stirring, and decolorized. Filtration and concentration under reduced pressure removed the solvent to give (R) -3-cyclopentyl-3- {3- (2, 5-dioxopyrrol-1-yl) -4- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } propionitrile (22.2g, 41.7mmol, 92% yield).

¹H-NMR(500MHz，CDCl₃)：8.70(s，1H)、8.35(s，1H)、7.35(d，J＝3.7Hz，1H)、6.66(d，J＝3.7Hz，1H)、5.62(d，J＝10.8Hz，1H)、5.58(d，J＝10.8Hz，1H)、4.30(m，1H)、3.50(m，2H)、3.09(dd，J＝16.8，4.3Hz，1H)、3.01(dd，J＝16.8，4.3Hz，1H)、2.94(s，4H)、2.62(m，1H)、1.96(m，1H)、1.69(m，2H)、1.60(m，1H)、1.58(m，2H)、1.27(m，2H)、0.90(t，J＝8.3Hz，2H)、-0.06(s，9H)；HRMS(ESI)calcd.for C₂₇H₃₅N₇O₃Si[M+H]⁺534.2643；Found：534.2657。

Step M: (R) -3-cyclopentyl-3- {3- (2, 5-dioxopyrrol-1-yl) -4- { (7-hydroxymethyl) -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } propionitrile

To a solution of 0.2M (R) -3-cyclopentyl-3- {3- (2, 5-dioxopyrrol-1-yl) -4- {7- { [2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } propionitrile (20g, 37.5mmol, 1.0eq.) in dichloromethane was added dropwise a solution of 47% boron trifluoride in diethyl ether (34g, 112.5mmol, 3.0eq.) with stirring in an ice bath, and the reaction was stirred at room temperature for 4 hours after completion of addition. Adding water for quenching, adjusting the pH value to 6-7 by using a 10% NaOH solution, extracting by using ethyl acetate, washing an organic layer by using water, washing by using saturated salt solution, stirring and drying by using anhydrous magnesium sulfate. Filtering, concentrating under reduced pressure to give (R) -3-cyclopentyl-3- [3- (2, 5-dioxopyrrol-1-yl) -4- (7-hydroxymethyl) -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl ] propionitrile (14.4g, 88.5% yield)

¹H-NMR(500MHz，CDCl₃)：8.54(s，1H)、8.31(s，1H)、7.31(d，J＝3.7Hz，1H)、6.52(d，J＝3.7Hz，1H)、5.68(d，J＝10.9Hz，1H)、5.61(d，J＝10.9Hz，1H)、4.32(m，1H)、3.13(dd，J＝17.2，7.9Hz，1H)、3.03(dd，J＝17.2，4.3Hz，1H)、2.94(s，4H)、2.62(m，1H)、1.98(m，1H)、1.74(m，1H)、1.65(m，1H)、1.64(m，2H)、1.30(m，1H)、1.29(m，2H)；HRMS(ESI)calcd.forC₂₂H₂₃N₇O₃[M+H]⁺434.1935；Found：434.1944。

And step N: (R) -3- [ 3-amino-4- (7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl ] -3-cyclopentylpropanenitrile (I)

To a 0.2M solution of (R) -3- {3- (2, 5-dioxopyrrol-1-yl) -4- { (7-hydroxymethyl) -7H-pyrrolo [2, 3-d ] pyrimidin-4-yl } -1H-pyrazol-1-yl } -3-cyclopentylpropanenitrile (12g, 27.7mmol, 1.0eq.) in methanol was added dropwise, 80% hydrazine hydrate (8.7g, 138mmol, 5.0eq.) under stirring at room temperature, after which the mixture was heated to reflux for 8 hours, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate, washed with water, washed with brine, and dried over anhydrous sodium sulfate overnight. Filtration and concentration under reduced pressure gave (R) -3- [ 3-amino-4- (7H-pyrrolo [2, 3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl ] -3-cyclopentylpropanenitrile (I) (7.7g, yield 87%, ee value 99.8%).

¹H-NMR(500MHz，CDCl₃)：11.73(s，1H)、8.79(s，1H)、8.06(s，1H)、7.32(d，J＝3.5Hz，1H)、6.62(d，J＝3.5Hz，1H)、5.03(s，2H)、4.05(td，J＝9.5，3.5Hz，1H)、3.12(dd，J＝17.1，8.9Hz，1H)、2.91(dd，J＝17.1，3.6Hz，1H)、2.54(m，1H)、1.74(m，1H)、1.63(m，4H)、1.27(m，1H)、1.26(m，2H)；HRMS(ESI)calcd.for C₁₇H₁₉N₇[M+H]⁺322.1775；Found：322.1783。

EXAMPLE 2 Compound of formula I Crystal A

Method 1

2.0g of the compound of the formula I obtained in example 1 is taken, 24mL of absolute ethyl alcohol is added, the mixture is heated to reflux and clear, the temperature is reduced to 0-5 ℃, stirring is carried out for crystallization for 4 hours, the filtration is carried out, a filter cake is rinsed by 2mL of absolute ethyl alcohol, and the product is dried under reduced pressure at 50 ℃ to obtain 1.62g of the product with 81 percent of yield.

Method 2

Taking 4 parts of 2.0g of the compound of the formula I obtained in example 1, respectively adding 20mL of absolute ethyl alcohol-ethyl acetate (4: 1, 2: 1, 1: 1 and 1: 4) mixed solvent, heating to reflux and dissolving, cooling to 0-5 ℃, stirring and crystallizing for 4 hours, filtering, leaching a filter cake with 2mL of ethyl acetate, and drying at 50 ℃ under reduced pressure to obtain 1.34g, 1.06g, 1.00g and 1.60g of products, wherein the yield is 67%, 53%, 50% and 80%.

Method 3

2.0g of the compound of the formula I obtained in example 1 is taken, 20mL of absolute ethyl alcohol-water (4: 1) mixed solution is added, the mixture is heated to reflux and clear, the temperature is reduced to 0-5 ℃, stirring is carried out for crystallization for 4 hours, the mixture is filtered, 2mL of absolute ethyl alcohol is used for leaching a filter cake, and the product is dried under reduced pressure at 50 ℃ to obtain 1.6g of the product with the yield of 80%.

Method 4

Taking 2.0g of the compound of the formula I obtained in example 1, adding 15mL of acetone, heating to reflux and dissolving, cooling to 0-5 ℃, stirring and crystallizing for 4 hours, filtering, leaching a filter cake with 2mL of acetone, and drying at 50 ℃ under reduced pressure to obtain 1.22g of a product with 61% yield.

Method 5

2.0g of the compound of formula I obtained in example 1 was taken, 50mL of ethyl acetate was added, the mixture was heated to reflux and clear, and the solvent was evaporated by concentration under reduced pressure to give 1.98g of the product in 99% yield.

Method 6

2.0g of the compound of formula I obtained in example 1 was taken, 60ml of dichloromethane was added, the mixture was heated to reflux and clear, and the solvent was evaporated by concentration under reduced pressure to obtain 2.0g of a product with 100% yield.

Method 7

2.0g of the compound of the formula I obtained in example 1 is taken, 24mL of absolute ethanol is added, the mixture is heated to reflux and clear, 120mL of isopropyl ether is dropwise added, the temperature is reduced to 0-5 ℃, stirring is carried out for crystallization for 4 hours, the mixture is filtered, 2mL of isopropyl ether is used for leaching a filter cake, and the product is dried under reduced pressure at 50 ℃ to obtain 1.56g of the product with 78% yield.

Typical XRD patterns and DSC patterns of crystalline a of the compound of formula I are shown in figures 1 and 2, respectively (example 2, method 1).

Another typical XRD pattern of crystalline A compound of formula I is shown in figure 3 (example 2, method 2, ethanol-ethyl acetate (4: 1)).

A further typical XRD pattern of crystalline a compound of formula I is shown in figure 4 (example 2, method 3).

EXAMPLE 3 Crystal B of the Compound of formula I

2.0g of the compound of the formula I obtained in example 1 is taken, 25mL of acetonitrile is added, the mixture is heated to reflux and clear, the temperature is reduced to 0-5 ℃, the mixture is stirred and crystallized for 4 hours, the mixture is filtered, 2mL of acetonitrile is used for leaching a filter cake, and the mixture is dried under reduced pressure at 50 ℃ to obtain 1.82g of a product with the yield of 91 percent.

Crystalline B compound of formula I is its acetonitrile compound, and its typical XRD and DSC patterns are shown in fig. 5 and 6, respectively.

Example 4 stability experiment

The crystal A obtained by the method 1 of example 2 and the crystal B obtained by the method 3 were placed in an open clean container, placed at 60 ℃, sampled and tested on days 5 and 10 respectively, and the test results were compared with the initial test results on day 0, and the test results are shown in the following table:

example 5 biological Activity assay

1. Enzymatic Activity of Compounds (IC)₅₀) Detection of

Adopts homogeneous time-resolved fluorescence (HTRF) method to establish a kinase activity detection platform of JAK2 (wild type) for compound activationAnd (4) measuring the sex. Compounds were diluted 3-fold in 100% DMSO starting at 1mM (11 concentrations in total), and 4. mu.L of each concentration was added to 96. mu.L of reaction buffer (50mM HEPES, pH7.4, 10mM MgCl)₂1mM EGTA, 0.01% Tween-20, 0.005% BAS, 2mM DTT, 2.5. mu.L was added to a 384 well plate (OptiPlate-384, available from Perkinelmer), 5. mu.L of JAK2 kinase (available from Cama) was added, the mixture was centrifuged and mixed, and 2.5. mu.L of ATP (final concentration of K is appropriate) was added_mValue) and TK peptide: (KinEASE^TMTK, purchased from Cisbio) mixture the reaction was started (total reaction volume 10. mu.L). The 384-well plate was placed in an incubator at 23 ℃ for 120 minutes, and then 5. mu.L of Eu3+ cryptate-layered anti-phosphotyrosine antibody (purchased from Cisbio), 5. mu.L of Streptavidin-XL-665 (manufactured by Cisbio) were addedKinEASE^TMTK, purchased from Cisbio) stop reaction. After 1 hour incubation in the incubator, fluorescence values (320nm excitation, detecting 665nm and 620nm emission, ratio for enzyme activity) were read on Envision (purchased from PerkinElmer). The activity of the enzyme was determined at 11 concentrations and the data calculated using GraFit6.0 software (Erithocus software) to give the IC of the compound of formula I₅₀The value is obtained. The results show that the IC of the compound of formula I and the control Ruxolitinib₅₀Values were all < 20 nM.

2. Determination of efficacy in a mouse subcutaneous xenograft tumor model

SPF grade Balb/c nude mice, female, 5-6 weeks old, 0.1mL Ba/F3-JAK2V617F cell suspension (containing 1 × 10) suspended in serum-free culture medium⁷cells, 50% MatriGel) was injected subcutaneously into the right side of each mouse. The average tumor volume reaches about 500mm³When the tumor is to be treated, the tumor-bearing mice are killed, the tumor tissues are picked up aseptically, cut into small pieces, and implanted into Balb/c nude mice subcutaneously on the left and right sides until the average tumor volume reaches about 100mm³When the tumor size and body weight of each mouse are measured, the tumor size is randomly measured from small to largeGrouping and adjusting the average body weight of each group of animals to be at the same level. The 5 groups were negative control group, positive control group, low, medium and high dose groups, and 5 mice in each group were administered on the same day, 2 times daily for 14 consecutive days during which tumor volume and body weight were measured 2 times per week. At the end of the experiment, mice were sacrificed and spleens were isolated and weighed.

Measuring the longest diameter (L) of tumor and the maximum transverse diameter (W) in the vertical direction during the test, and calculating the tumor volume (V), V (mm)³)＝L×W²Tumor growth inhibition ratio TGI (%) -100% × (1- (T)_t-T₀)/(V_t-V₀))，T_tMean tumor volume, T, for each measurement in the treatment group₀Mean tumor volume in treatment group, V_tMean tumor volume, V, for each measurement in the control group₀Mean tumor volume when grouped for control group.

The results are shown in the following table:

as can be seen from the data in the table, in a Ba/F3-JAK2V617F tumor-bearing mouse model, the tumor inhibition effect of the hydrochloride of the compound shown in the formula I in an animal body is measured, and the hydrochloride of the compound shows a dose-dependent inhibition effect on the growth of the Ba/F3-JAK2V617F tumor, and the tumor inhibition effect is very obvious. After 14 days of oral twice daily administration of the hydrochloride salt of the compound of formula I (100mg/kg), the inhibition of Tumor Growth (TGI) reached 85.8%, whereas the inhibition of Tumor Growth (TGI) was only 64.5% for the positive control Ruxolitinib (100mg/kg) under equivalent conditions. The 50mg/kg hydrochloride of the compound shown in the formula I has obvious tumor inhibition effect, and the TGI reaches 68.4 percent, which is equivalent to the tumor inhibition effect of 100mg/kg positive control Ruxolitinib.

3. Determination of pharmacokinetics in adult male/female SD rats

Healthy adult female SD rats are from Beijing Wittingle laboratory animal technology Limited, and the rats are divided into 2 groups, 3 rats in each group are respectively orally taken for single intragastric administration to be tested to obtain a sample suspension (30 mg/kg). Animals were fasted overnight prior to the experiment, with the fasting time ranging from 10 hours prior to dosing to 4 hours post-dosing. Blood was collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-dose. After isoflurane anesthesia using a small animal anesthesia machine, 0.4mL of whole blood was collected through the fundus venous plexus, placed in a heparin anticoagulation tube, the sample was centrifuged at 4 ℃ and 4200rpm for 5min, and the plasma was transferred to a centrifuge tube and stored at-80 ℃ until analysis. Samples from plasma were extracted using protein precipitation and the extracts were analyzed by LC/MS/MS.

And (4) supplementary notes: a. the data are from a pharmacological review published by the U.S. food and drug administration, FDA.

PK of rats (30mg/kg PO) showed that the data for the compound of formula I was superior to that of Ruxolitinib.

4. Determination of pharmacokinetics of adult beagle dog

The study used 4 healthy adult beagle dogs, from beijing marts biotechnology limited. The study was divided into two times: for the first time, animals (2 males and females each) were given a single intravenous injection at a dose of 5 mg/kg; the second time, one week later, the same group of animals (2 males and females each) were given a single gavage at a dose of 10 mg/kg. Gavage animals were fasted overnight before the experiment, with the fasting time ranging from 10 hours before dosing to 4 hours after dosing. The animals in the intravenous group were not food restricted. The intravenous administration group collected blood at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours post-administration; the gavage group collected blood at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours post-dose. Animals were lightly anesthetized with isoflurane, approximately 0.4mL of whole blood was collected from the orbital venous plexus using a glass blood collection tube, placed in a heparin anticoagulation tube, the sample centrifuged at 4 ℃, 4200rpm for 5min, and the plasma transferred to a centrifuge tube and stored at-80 ℃ until analysis. Samples from plasma were extracted using protein precipitation and the extracts were analyzed by LC/MS/MS.

And (4) supplementary notes: a. the data are from a pharmacological review published by the U.S. food and drug administration, FDA.

PK in dogs (10mg/kg PO, 5mg/kg IV) showed that the AUC after IV administration of compound of formula I was comparable to the positive control Ruxolitinib, but the bioavailability was better with oral administration (114% vs 57%).

Claims (20)

1. Crystal a of a compound of formula I:

an X-ray diffraction pattern of crystal A of the compound represented by the formula I has diffraction peaks with 2 theta of 9.35 DEG + -0.2 DEG, 11.93 DEG + -0.2 DEG, 16.32 DEG + -0.2 DEG, 18.82 DEG + -0.2 DEG, 20.54 DEG + -0.2 DEG, 21.23 DEG + -0.2 DEG, 23.13 DEG + -0.2 DEG and 25.58 DEG + -0.2 deg.

2. Crystal a of the compound of formula I according to claim 1 having an X-ray diffraction pattern with diffraction peaks, in terms of 2 Θ, of 9.35 ° ± 0.2 °, 10.93 ° ± 0.2 °, 11.93 ° ± 0.2 °, 14.46 ° ± 0.2 °, 16.32 ° ± 0.2 °, 18.82 ° ± 0.2 °, 20.54 ° ± 0.2 °, 21.23 ° ± 0.2 °, 21.66 ° ± 0.2 °, 23.13 ° ± 0.2 °, 25.58 ° ± 0.2 ° and 26.34 ° ± 0.2 °.

3. Crystal a of the compound of formula I according to claim 1 having an X-ray diffraction pattern with diffraction peaks, in terms of 2 Θ, of 9.35 ° ± 0.2 °, 10.93 ° ± 0.2 °, 11.93 ° ± 0.2 °, 14.46 ° ± 0.2 °, 16.32 ° ± 0.2 °, 17.28 ° ± 0.2 °, 18.82 ° ± 0.2 °, 19.25 ° ± 0.2 °, 20.54 ° ± 0.2 °, 21.23 ° ± 0.2 °, 21.66 ° ± 0.2 °, 22.15 ° ± 0.2 °, 23.13 ° ± 0.2 °, 24.09 ° ± 0.2 °, 25.58 ° ± 0.2 ° and 26.34 ° ± 0.2 °.

4. A process for the preparation of a crystal a of the compound of formula I according to claim 1, which process comprises the steps of:

1) dissolving a compound shown in a formula I in a crystallization solvent, wherein the crystallization solvent is selected from ethanol, isopropyl ether, ethyl acetate, acetone, dichloromethane, water or a mixed solvent of any two or more of the solvents; and

2) crystallizing, filtering, washing and drying.

5. The production method according to claim 4, wherein the crystallization solvent is ethanol, an ethanol-ethyl acetate mixed solvent, an ethanol-water mixed solvent, an ethanol-isopropyl ether mixed solvent, acetone, ethyl acetate, or dichloromethane.

6. A crystalline composition, wherein crystal a of the compound of formula I as defined in claim 1 constitutes more than 50% by weight of the crystalline composition.

7. The crystalline composition as defined in claim 6, wherein the crystalline a of the compound of formula I as defined in claim 1 is more than 80% by weight of the crystalline composition.

8. The crystalline composition as defined in claim 6, wherein the crystalline a of the compound of formula I as defined in claim 1 is more than 90% by weight of the crystalline composition.

9. The crystalline composition as defined in claim 6, wherein the crystalline a of the compound of formula I as defined in claim 1 is greater than 95% by weight of the crystalline composition.

10. A pharmaceutical composition comprising an effective amount of crystal a of the compound of formula I as claimed in claim 1 or the crystalline composition as claimed in claim 6.

11. Use of crystal a of a compound of formula I according to claim 1, a crystalline composition according to claim 6, or a pharmaceutical composition according to claim 10 for the manufacture of a medicament for the treatment or prevention of a condition mediated by janus kinases.

12. Crystal B of a compound of formula I:

an X-ray diffraction pattern of crystal B of the compound represented by the formula I has diffraction peaks at 2 theta of 8.97 DEG + -0.2 DEG, 9.39 DEG + -0.2 DEG, 12.90 DEG + -0.2 DEG, 16.54 DEG + -0.2 DEG, 17.70 DEG + -0.2 DEG, 19.20 DEG + -0.2 DEG, 20.31 DEG + -0.2 DEG, 22.78 DEG + -0.2 DEG and 23.63 DEG + -0.2 DEG;

the crystal B is an acetonitrile compound.

13. Crystal B of the compound of formula I according to claim 12 having an X-ray diffraction pattern with diffraction peaks, in terms of 2 Θ, of 8.97 ° ± 0.2 °, 9.39 ° ± 0.2 °, 11.24 ° ± 0.2 °, 12.90 ° ± 0.2 °, 14.56 ° ± 0.2 °, 16.54 ° ± 0.2 °, 17.70 ° ± 0.2 °, 19.20 ° ± 0.2 °, 20.31 ° ± 0.2 °, 22.23 ° ± 0.2 °, 22.78 ° ± 0.2 °, 23.63 ° ± 0.2 ° and 25.55 ° ± 0.2 °.

14. A process for the preparation of crystal B of the compound of formula I according to claim 12, which process comprises the steps of:

1) dissolving a compound shown as a formula I in acetonitrile; and

2) crystallizing, filtering, washing and drying.

15. A crystalline composition, wherein crystal B of the compound of formula I as defined in claim 12 constitutes more than 50% by weight of the crystalline composition.

16. The crystalline composition as defined in claim 15, wherein the crystalline B of the compound of formula I as defined in claim 12 is more than 80% by weight of the crystalline composition.

17. The crystalline composition as defined in claim 15, wherein the crystalline B of the compound of formula I as defined in claim 12 constitutes more than 90% by weight of the crystalline composition.

18. The crystalline composition as defined in claim 15, wherein the crystalline B of the compound of formula I as defined in claim 12 is greater than 95% by weight of the crystalline composition.

19. A pharmaceutical composition comprising an effective amount of crystalline B of the compound of formula I according to claim 12 or the crystalline composition according to claim 15.

20. Use of crystal B of the compound of formula I according to claim 12, the crystalline composition according to claim 15, or the pharmaceutical composition according to claim 19 for the manufacture of a medicament for the treatment or prevention of a janus kinase mediated disease.