CN110467618B - Porous supermolecular structure crystal and preparation method thereof - Google Patents

Abstract

The present invention provides pyrazolo [3,4-d]Crystals of a pyrimidine-based compound, said crystals being pyrazolo [3,4-d]Supermolecular hydrate formed by pyrimidine compounds and water molecules; wherein the pyrazolo [3,4-d ] is]The pyrimidine compound is compound 9 or compound 7. Experimental results show that the crystals of the compound 7 and the compound 9 form a novel porous supermolecular framework structure containing a plurality of water channels, particularly the crystals of the compound 9 also have good thermal stability, and have good application prospects in the fields of artificial channel materials, drug carriers and porous materials; the compound 7 and the compound 9 can effectively inhibit the release level of the inflammatory factor IL-6, the compound 9 can inhibit the protein levels of phosphorylation NF-kappa B p65, phosphorylation Akt, phosphorylation STAT3 and adhesion molecule ICAM-1, and the crystal prepared by the invention has a very good application prospect in preparing medicines for treating inflammatory diseases.

Description

Porous supermolecular structure crystal and preparation method thereof

Technical Field

The invention belongs to medicine synthesis, and particularly relates to a porous supermolecular structure crystal of a pyrazolo [3,4-d ] pyrimidine compound and a preparation method thereof.

Background

Inflammation is closely related to the occurrence and development of most diseases, including coronary heart disease, atherosclerosis, diabetes, tumors, etc., and has become a hallmark feature of various diseases in human beings. Inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta) and the like play an important role in the process of generating and developing inflammation. With the continuous and deep research on the inflammatory signal transduction pathway, IL-6 has become an effective therapeutic target for acute and chronic inflammation, and plays an important role in the treatment of inflammatory diseases.

Currently, a variety of monoclonal antibodies directed against inhibitors of the IL-6 signaling cascade, including anti-IL-6, interleukin-6 receptor (IL-6R) and soluble glycoprotein 130(sgp130Fc), have been developed, some of which are undergoing clinical trials. The use of monoclonal antibodies is often associated with adverse reactions such as aplastic anemia and congestive heart failure. In contrast, small molecule compound drugs generally have better bioavailability and are advantageous for manufacturing, among other properties, than monoclonal antibody drugs. Therefore, the development of new IL-6 related small molecule inhibitors has a very good prospect.

Pyrazolo [3,4-d ] pyrimidines belong to nitrogen-containing fused ring compounds, are isosteres of purines, and exhibit various pharmacological effects due to their similar structures to purines. Therefore, pyrazolo [3,4-d ] pyrimidines have been the focus of research in the field of pharmacology. Pyrazolo [3,4-d ] pyrimidines have been reported to have a variety of pharmacological activities such as antibacterial, antiviral, anticancer, anti-inflammatory, antihypertensive, antidiabetic, xanthine oxidase inhibition, and gout treatment.

In addition, some nucleoside analogs can form a relatively stable supramolecular structure through the Watson-Crick base pairing principle, hydrogen bond interaction and pi-pi stacking self-assembly, and can play an important role in the fields of pharmacy and materials. Therefore, in the early stage of drug development, it is important to understand the relationship between the physical structure and properties of compound crystals and the self-assembly rule of molecules during the crystallization process of the compound.

In order to face the current situation that the existing pyrazolo [3,4-d ] pyrimidine compounds generally have the problems of low anti-inflammatory efficiency, strong toxic and side effects and the like in anti-inflammatory action, the IL-6 related micromolecule inhibitor with more novel structures is prepared, and high-quality crystals of the micromolecule inhibitor are further prepared, so that the method has very important significance in guiding the design of novel medicinal compounds with ideal physical and mechanical properties.

Disclosure of Invention

The invention aims to provide a novel crystal of a pyrazolo [3,4-d ] pyrimidine compound.

The invention provides a crystal of a pyrazolo [3,4-d ] pyrimidine compound, which is a supermolecular hydrate formed by the pyrazolo [3,4-d ] pyrimidine compound and water molecules;

wherein the pyrazolo [3,4-d ] pyrimidine compound is a compound 9 or a compound 7:

further, the crystal is a porous supramolecular structure.

Further, the pyrazolo [3,4-d ] pyrimidine small molecule derivative is a compound 9, the crystal is a single crystal, the single crystal belongs to a monoclinic system, and the space group is C2/C.

Further, the crystal has unit cell parameters of:

α＝90°，β＝105.786±0.009°，γ＝90°。

further, the crystal comprises the enantiomers shown as 9-A1 and 9-A2:

among them, 9-A1, τ₁41.441; in 9-A2, τ₂＝-41.441；τ₁、τ₂Are dihedral angles of C5-C7-C10-C15 in 9-A1 and 9-A2 respectively.

Furthermore, the porous supermolecular structure of the crystal is a columnar structure, the columnar structure is formed by self-assembling 9-A1 and 9-A2 and water molecules, and the water molecules are positioned in a cavity in the column of the columnar structure; in the columnar structure, the molar ratio of 9-A1 to 9-A2 to water molecules is 1: 1: 2.

further, the differential scanning calorimetry curve of the crystal is shown in FIG. 21.

Further, the thermogravimetric analysis curve of the crystal is shown in FIG. 22.

Further, the variable temperature powder X-ray diffraction curve of the crystal is shown in figure 23.

Further, the pyrazolo [3,4-d ] pyrimidine small molecule derivative is a compound 7, the crystal belongs to a triclinic system, and the space group is P-1.

Further, the crystal has unit cell parameters of:

α＝71.752±0.009°，β＝65.956±0.008°，γ＝74.378±0.010°。

further, the crystals comprise the enantiomers shown in 7-A1 and 7-A2:

wherein, in 7-A1, τ₃56.615; in 7-A2, τ₄＝-56.615；τ₃、τ₄Are dihedral angles of C5-C7-C10-C15 in 7-A1 and 7-A2 respectively.

Further, the porous supermolecular structure of the crystal is a pipeline structure, the pipeline structure is formed by self-assembling 7-A1 and 7-A2 and water molecules, and the water molecules are positioned in the middle of the pipeline; in the pipeline structure, the molar ratio of 7-A1, 7A2 and water molecules is 1: 1: 1.

the invention also provides a method for preparing the crystal, which comprises the following steps: adding pyrazolo [3,4-d ] pyrimidine compounds into a mixed solution of methanol and water, dissolving, filtering, taking liquid, and crystallizing to obtain crystals of the compounds; wherein the pyrazolo [3,4-d ] pyrimidine compound is the compound 9 or the compound 7.

Further, in the mixed solution of methanol and water, the volume ratio of methanol to water is (8-12): 1;

the mass-to-volume ratio of the mixed solution of the pyrazolo [3,4-d ] pyrimidine compound, methanol and water is (3-7) mg: 1 mL;

the crystallization is standing crystallization, the crystallization temperature is room temperature, and the crystallization time is 15-26 days;

further, in the mixed solution of methanol and water, the volume ratio of methanol to water is 10: 1;

the mass-volume ratio of the mixed solution of the pyrazolo [3,4-d ] pyrimidine compound, methanol and water is 5 mg: 1 mL;

the temperature of the crystallization is room temperature, and the time is 15 days or 26 days.

The 'water channel' of the invention means: in the single crystal structure, the water molecules form a linear structure, and the linear structure is formed under the action of hydrogen bonds.

"supramolecules" refers to: two or more molecules are combined together by means of intermolecular interaction to form a complex and organized aggregate, and certain integrity is maintained, so that the complex and organized aggregate has a definite microstructure and macroscopic characteristics.

The "porous supramolecular framework structure" refers to a spatial structure having pore-like gaps formed by two or more molecules bonded together by intermolecular interaction.

"duct structure" means: circular or round-like, hollow structures.

The "columnar structure" means: non-circular, angular, hollow structures.

"Artificial channel" refers to a synthetic channel with similar function as a natural aquaporin.

"solvate" refers to a crystalline substance formed by the combination of a molecule of a compound and one or more molecules of a solvent, which is a ubiquitous form of the compound. In the pharmaceutical manufacturing process, there are many processes in which a solvent is necessary, and in these processes, the compound comes into close contact with the solvent and under certain conditions, the corresponding solvate is formed.

"hydrate" is a kind of solvate, and refers to a crystal substance formed by the compound molecules and water molecules together in a certain combination form.

"drug carrier" refers to a system that alters the way and distribution of drugs into the body, controls the rate of release of drugs, and delivers drugs to targeted organs.

"enantiomers" refers to stereoisomers that are true to mirror images of one another and do not overlap.

Experimental results show that the crystals of the compound 7 and the compound 9 form a novel porous supermolecular framework structure containing a plurality of water channels, particularly the crystals of the compound 9 also have good thermal stability, and have good application prospects in the fields of artificial channel materials, drug carriers and porous materials; the compound 7 and the compound 9 can effectively inhibit the release level of the inflammatory factor IL-6, the compound 9 can inhibit the protein levels of phosphorylation NF-kappa B p65, phosphorylation Akt, phosphorylation STAT3 and adhesion molecule ICAM-1, and the crystal prepared by the invention has a very good application prospect in preparing medicines for treating inflammatory diseases.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1, flow chart of apoptosis results for Compound 7.

Figure 2. inhibition of a549 cell cycle by compound 7-flow diagram.

Figure 3. effect of compound 9 on a549 cell apoptosis-flow diagram.

Figure 4. inhibition of a549 cell cycle by compound 9-flow diagram.

FIG. 5 shows the inhibitory effect of compounds on the level of inflammatory factor IL-6, in which HX08-4 corresponds to Compound 7 of the present invention, HX08-9 corresponds to Compound 9 of the present invention: (I)

n is 3, P < 0.05, P < 0.01, P < 0.001vs Medium group). FIG. 6 Western blotting method for detecting NF-kB, MAPK and JAK-STAT signal channel related protein expression level (

P < 0.05, p < 0.01, p < 0.001vs IL-4 group). a) Effect of compound 9 on the expression levels of NF- κ B p6 and phosphorylated NF- κ Bp65, b) statistical results of compound 9 on the expression levels of phosphorylated NF- κ B p65, c) effect of compound 9 on the expression levels of STAT3Ser727 and phosphorylated STAT3Ser727, d) effect of compound 9 on the expression levels of phosphorylated Akt Ser 473.

FIG. 7 Western blotting method for detecting ICAM-1 protein expression level, (b)

P < 0.05, p < 0.01, p < 0.001vs IL-4 group). a) Effect of compound 9 on ICAM-1 expression levels, b) statistical results of compound 9 affecting ICAM-1 expression levels.

FIG. 8 is the ORTEP diagram of the molecular configuration of different conformations of Compound 1 and its overlay. a) The ORTEP map of the molecular configuration of the conformation of Compound 1A1, B) the ORTEP map of the molecular configuration of the conformation of Compound 1A2, c) the ORTEP map of the molecular configuration of the conformation of Compound 1B1, d) the ORTEP map of the molecular configuration of the conformation of Compound 1B2, e) the overlap maps of the molecular configurations of the different conformations of Compound 1.

FIG. 9 shows hydrogen bonding interactions between the crystal stacking diagram and 4 conformations of Compound 1; a) a crystal stacking diagram; b) hydrogen bonding interactions (N-butyl at N9 was omitted for clarity of presentation of hydrogen bonding interactions).

FIG. 10. the crystal of Compound 1 pi-pi accumulates the distance between two parallel planes.

FIG. 11 ORTEP map of different conformations of the crystal structure of Compound 7 and their conformational overlays. a) ORTEP map of molecular configuration of the 7A1 conformation, b) ORTEP map of molecular configuration of the 7A2 conformation, c) overlap map of molecular configuration of different conformations of compound 7.

FIG. 12 shows the crystal stacking and intermolecular hydrogen bonding interaction of Compound 7. a) A crystal stacking diagram; b) hydrogen bonding interactions.

FIG. 13 shows the interaction between the crystals of compound 7 and water molecules and the weak interaction between molecules.

FIG. 14 ORTEP map of different conformations of the crystal structure of Compound 9 and their conformational overlays. a) ORTEP map of molecular configuration of the compound 9A1 conformation, b) ORTEP map of molecular configuration of the compound 9A2 conformation, c) overlap map of molecular configuration of different conformations of compound 9.

FIG. 15 shows the intermolecular interactions of the interdigitated four-membered rings in the crystal structure of Compound 9. a) Intermolecular interactions in two interdigitated quaternary rings, b) an interdigitated quaternary ring space-filling map, c) intermolecular interactions in quaternary rings, d) intermolecular interactions in two adjacent layers.

FIG. 16 shows the connection between two adjacent four-membered rings in the crystal structure of Compound 9. a) The intermolecular interaction between two adjacent quaternary rings, b) the intermolecular hydrogen bond interaction between the adjacent quaternary rings, and c) the pi-pi stacking action between the adjacent quaternary rings.

FIG. 17 shows a molecular packing diagram of a crystal of Compound 9.

FIG. 18. d of Compound 9_normShape index (shape index) and curves (curves)s) surface map (the surface is set transparent for visualization of functional groups in the molecule). a) d_normSurface map, b) shape index surface map, c) curved surface map.

FIG. 19 shows the interaction and arrangement of Hirshfeld surface of compound 9 crystal with surrounding molecules.

FIG. 20. 2D fingerprint of compound 9 crystal and contribution of each contact point to Hirshfeld surface, respectively.

FIG. 21 Differential Scanning Calorimetry (DSC) curve of Compound 9.

FIG. 22. thermogravimetric analysis (TGA) curve of Compound 9.

FIG. 23 is a temperature swing powder X-ray diffraction (PXRD) pattern for Compound 9.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Wherein, the sources of part of experimental reagents are shown in the table 1:

TABLE 1 part of the test reagents

The main instruments are as follows:

(1) thin layer chromatography plate: 60F245 type, 0.2mm, Merck, Germany

(2) Column chromatography silica gel: silica gel H-GF254, 200-mesh, 300-mesh, Qingdao ocean factory

(3) A mass spectrum analyzer: Q-TOF spectrometer, ESI ionization source, Bruker, Germany

(4) Nuclear magnetic resonance apparatus: AV II-400MHz, AV II-600MHz or AV II-800MHz, TMS as internal standard, Germany Bruker.

The following are the synthetic procedures for specific compounds of the invention:

example 1 preparation of Compounds 1, 7 and 9

1. Preparation of compound 1:

(1) 1-n-butyl-3-bromo-4-chloro-1H-pyrazolo [3,4-d]Pyrimidine (11 a): 3-bromo-4-chloro-1H-pyrazolo [3,4-d ] was added to a 250mL round-bottom flask]Pyrimidine 10(1g,4.3mmol) and 150mL of dry acetonitrile are dissolved with stirring, potassium carbonate (1.19g,8.6mmol) and 3mL of iodo-n-butyl are added in that order (dropwise), stirred at room temperature for 10 minutes, and then the temperature is raised to 80 ℃ and refluxed. The reaction was continued for about 3h, and TLC monitoring indicated that the reaction was complete (developer: CH)₂Cl₂/CH₃OH 95/5). After the reaction solution was cooled, 50mL of water and CH were added₂Cl₂(100 mL. times.3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product is separated by column chromatography on silica gel (eluent: CH)₂Cl₂) To obtain a white solid product, namely 1-n-butyl-3-bromo-4-chloro-1H-pyrazolo [3,4-d]Pyrimidine 1.06g, yield: 85 percent.¹H NMR(600MHz，CDCl₃)：δ0.93(t,J＝7.4,3H),1.31(m,2H),1.90(m,2H),4.44(t,J＝7.2,2H),8.71(s,1H).¹³CNMR(151MHz,CDCl₃)：δ13.69,19.97,31.61,48.11,112.36,119.27,153.87,155.22,155.31.

(2) 1-n-butyl-3-bromo-4-methoxy-1H-pyrazolo [3,4-d]Pyrimidine (12 a): 1-n-butyl-3-bromo-4-chloro-1H-pyrazolo [3,4-d]Pyrimidine 11a (0.5g,1.74mmol) was dissolved in 50mL dry CH₃To the OH solution, 15mL of 2MCH was added₃ONa solution. The mixture was reacted at 65 ℃ for about 3.5 hours, and the completion of the reaction was monitored by TLC (developer: CH)₂Cl₂/CH₃OH 95/5). After the reaction solution was cooled to room temperature, 30mL of water and CH were added₂Cl₂(150 mL. times.3), combining the organic phases, drying with an appropriate amount of anhydrous sodium sulfate, filtering, concentrating, and separating by column chromatography on silica gel (eluent: CH)₂Cl₂) To obtain a white solid product, namely 1-n-butyl-3-bromo-4-methoxy-1H-pyrazolo [3,4-d]Pyrimidine 0.36g, yield: 73 percent. HRMS-ESI (M/z) calcd for [ M + H [ ]]⁺,285.0273；found,285.1153.

(3) 1-n-butyl-3- (3-phenol) -4-methoxy-1H-pyrazolo [3,4-d]Pyrimidine (1): 1-n-butyl-3-bromo was added sequentially to a 100mL round-bottomed flask containing 75mL of a1, 4-dioxane/water (4:1) mixed solution-4-methoxy-1H-pyrazolo [3,4-d]Pyrimidine 12a (0.20g,0.70mmol), 3-hydroxyphenylboronic acid pinacol ester (0.23g,1.05mmol), K₂CO₃(0.19g,1.4mmol)，PdCl₂dppf (0.13g,0.18mmol) was stirred well at room temperature until completely dissolved. The reaction was placed in an oil bath and heated to 100 ℃ under reflux for about 2h, and the end of the reaction was monitored by TLC (developer: CH)₂Cl₂/CH₃OH-95/5), the solution turned from orange-yellow to black. After the reaction solution was cooled, 30mL of water and CH were added₂Cl₂(100 mL. times.3), combining the organic phases, drying with an appropriate amount of anhydrous sodium sulfate, filtering, concentrating, and separating by column chromatography on silica gel (eluent: CH)₂Cl₂/CH₃OH 100/1-100/3) to give the product 1-n-butyl-3- (3-phenolic) -4-methoxy-1H-pyrazolo [3, 4-d) as a pale yellow solid]Pyrimidine 0.16g, yield: 73.9 percent.¹H NMR(600MHz,CDCl₃):δ0.93(t,J＝7.4,3H),1.36(m,2H),1.95(m,2H),4.16(s,3H),4.49(t,J＝7.2,2H),6.91(dd,J＝8.0,2.3,1H),7.33(t,J＝7.9,1H),7.56(s,1H),7.63(d,J＝7.7,1H),8.58(s,1H).¹³C NMR(151MHz,CDCl₃):δ13.82,20.12,31.95,47.59,54.62,100.44,115.71,116.04,121.34,129.85,133.91,144.04,155.02,155.64,156.11,164.42.HRMS-ESI(m/z)calcd for[M+H]⁺,299.1430；found,299.1505.

2. Preparation of compound 7:

(1) 1-isopropyl-3-bromo-4-amino-1H-pyrazolo [3,4-d]Pyrimidine (15): following the procedure for the alkylation synthesis of 11a described above gave 15 in yields: 83 percent. HRMS-ESI (M/z) calcd for [ M + H [ ]]⁺,256.0120；found,256.0197.

(2) 1-isopropyl-3- (3-phenolic) -4-amino-1H-pyrazolo [3,4-d]Pyrimidine (7): the specific operation is shown in a compound 1 synthesis method through a Suzuki coupling reactionMethod, yield: 76 percent.¹H NMR(600MHz,DMSO):δ1.48(d,J＝6.7,6H),5.05(m,1H),6.87(dd,J＝8.2,1.4,1H),7.07(s,1H),7.08(d,J＝2.9,1H),7.34(t,J＝8.0,1H),8.23(s,1H),9.70(s,1H).¹³C NMR(151MHz,DMSO):δ21.78,48.04,97.35,114.92,115.69,118.85,130.28,134.38,143.26,153.21,155.41,157.83,158.01.HRMS-ESI(m/z)calcd for[M+H]⁺,270.1277；found,270.1357.

3. Preparation of compound 9:

(1) 1-tert-butyl-5-amino-1H-pyrazole-4-carbonitrile (17): tert-butylhydrazine hydrochloride (8g,64.20mmol) and triethylamine (6.50g,64.20mmol) were dissolved in 500mL of anhydrous ethanol, and then 2-ethoxymethylenemalononitrile (7.83g,64.20mmol) was slowly added dropwise to the reaction flask with stirring. Heat to 78 ℃ reflux for about 3h, and monitor by TLC the reaction to completion (developer: PE/EtOAc ═ 1: 1). After the reaction mixture was cooled to room temperature, the solvent was removed by concentration under reduced pressure, and 100mL of water and CH were added₂Cl₂(300 mL. times.3) and the combined organic phases were dried over an appropriate amount of anhydrous sodium sulfate and concentrated to give an orange-yellow viscous solid. The crude product obtained is dissolved in 60mL of ethyl acetate-n-hexane (1:9) mixed solution, ultrasonic treatment is carried out for about 5min, standing is carried out, a large amount of crystal form precipitate is observed to be generated, filtration is carried out (the ethyl acetate-n-hexane mixed solution is washed for 3 times), and drying is carried out to obtain 10.11g of orange solid product 1-tert-butyl-5-amino-1H-pyrazole-4-nitrile, wherein the yield is as follows: 96 percent.¹H NMR(600MHz,DMSO):δ1.50(s,9H),6.22(s,2H),7.44(s,1H).¹³C NMR(151MHz,DMSO):δ28.22,58.75,74.54,115.23,138.00,150.71.HRMS-ESI(m/z)calcd for[M+Na]⁺,187.1062；found,187.0968.

(2) 1-tert-butyl-4-amino-1H-pyrazolo [3,4-d]Pyrimidine (18): a100 mL round-bottomed flask was charged with 1-tert-butyl-5-amino-1H-pyrazole-4-carbonitrile (5g,30.47mmol), a formamide solution80mL, the mixture was heated to 180 ℃ in an oil bath and after about 5h, the reaction was monitored by TLC for completion (developer: PE/EtOAc 1: 1). After the reaction solution was cooled, 50mL of water and CH were added₂Cl₂(200 mL. times.3), the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: PE/EtOAc: 4/1-1/1) to give the product 1-tert-butyl-4-amino-1H-pyrazolo [3,4-d ] as a white solid]Pyrimidine 5.82g, yield: 91 percent.¹H NMR(600MHz,DMSO):δ＝1.69(s,9H),8.03(s,1H),8.14(s,1H).¹³C NMR(151MHz,DMSO)：δ28.76,59.22,101.42,130.03,152.71,154.71,158.16.HRMS-ESI(m/z)calcd for[M+H]⁺,192.1171；found,192.1254.

(3) 1-tert-butyl-3-bromo-4-amino-1H-pyrazolo [3,4-d ] pyrimidine (19): weighing 1-tert-butyl-4-amino-1H-

Pyrazolo [3,4-d]183 g (15.70mmol) of pyrimidine are dissolved in 250mL of anhydrous acetonitrile, and N-bromosuccinimide (NBS, 4.19g,23.55mmol) is added with stirring at room temperature until complete dissolution. Heating to 100 deg.C and refluxing for about 2h, and monitoring by TLC for reaction completion (developing solvent: CH)₂Cl₂/CH₃OH ═ 95/5), after the reaction solution was cooled to room temperature, extracted with dichloromethane/water (× 3), the organic phases were combined, dried over an appropriate amount of anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: CH (CH)₂Cl₂) To obtain a white solid product, namely 1-tert-butyl-3-bromo-4-amino-1H-pyrazolo [3,4-d]Pyrimidine 4.22g, yield: 89 percent.¹H NMR(600MHz,DMSO):δ1.67(s,9H),8.20(s,1H).¹³C NMR(151MHz,DMSO):δ28.60,60.60,100.45,115.28,153.51,155.63,157.50.HRMS-ESI(m/z)calcd for[M+H]⁺,270.0276；found,270.0355.

(4) 1-tert-butyl-3- (5-hydroxy-3-pyridyl) -4-amino-1H-pyrazolo [3,4-d]Pyrimidine (9): through Suzuki coupling reaction, the concrete operation details are shown in the synthesis method of the compound 1, 1-tertiary butyl-3-bromo-4-amino-1H-pyrazolo [3,4-d]Pyrimidine (1.45g,5.43mmol) takes part in the reaction to give the product 1-tert-butyl-3- (5-hydroxy-3-pyridyl) -4-amino-1H-pyrazolo [3,4-d as a grey solid]Pyrimidine 0.58g, yield: and 47 percent. Wherein the developing agent: CH (CH)₂Cl₂/CH₃OH 10/1, eluent: CH (CH)₂Cl₂/CH₃OH＝100:1-100:5。¹H NMR(600MHz,DMSO):δ1.75(s,9H),7.38(dd,J＝2.5,2.0,1H),8.20(d,J＝2.7,1H),8.25(s,1H),8.29(d,J＝1.8,1H),10.16(s,1H).¹³C NMR(151MHz,DMSO)δ29.16,60.32,99.29,121.91,130.11,138.39,139.21,140.03,154.11,154.47,155.17,158.73.HRMS-ESI(m/z)calcd for[M+H]+,285.1386；found,285.1457.

Example 2 preparation of single crystals of Compounds 1, 7 and 9

Single crystals of the compound were obtained by slow solvent evaporation according to standard recrystallization procedures. The method comprises the following specific steps:

1) single crystal culture conditions and method for compound 1

Compound 1(50mg) was weighed out and dissolved in 10ml of a methanol/water (10:1) mixed solution, and heated with stirring until the solution became clear and near saturation. Filtering while hot, standing at room temperature to slowly volatilize the solution, and obtaining colorless needle-shaped single crystal 1 suitable for X-ray single crystal diffraction analysis after 10 days.

2) Single crystal growth conditions and methods for Compound 7

Compound 1 was replaced with Compound 7 in the same manner as Compound 1, and colorless small bulk single crystal 7 suitable for X-ray single crystal diffraction analysis was precipitated after 26 days.

3) Single crystal culture conditions and methods for compound 9

Compound 1 was replaced with Compound 9 by the same method as Compound 1, and colorless thin strip-like single crystal 9 suitable for X-ray single crystal diffraction analysis was precipitated after 15 days.

The beneficial effects of the compound of the invention are proved by the following experimental examples, and the experimental materials, instruments and experimental methods are as follows:

1 experimental materials and instruments:

1.1 the materials and reagents required for the experiments are listed in Table 2.

TABLE 2 partial experimental materials and reagents

1.2 biological experiment main instruments:

(1)CO₂an incubator: thermo311 type, Thermo, USA

(2) Fluorescence inverted microscope: model TE2000-U, Nikon, Japan

(3) Flow cytometry: FACS Valibur, BD Co., USA

(4) The full-automatic scanning type multifunctional microplate reader comprises: multiskan Sky, Thermo, usa

(5)4 ℃/-20 ℃ refrigerator: HXC-158, Haier, China

(6) -80 ℃ ultra low temperature refrigerator: forma, Thermo, USA

(7) Agarose gel electrophoresis apparatus: E-Gel PowerSnap, Invitrogen, USA

(8) Ultrasonic cell disruptor: JYD-650, Shanghai letters instruments Ltd, China

(9) Constant temperature shaking table: SPH-211B-GZ, Shanghai Pingyi experiment Co., Ltd, China

(10) Full-automatic gel imaging system: GelDoc, Bio-Rad Inc., USA

(11) Desk type micro freezing centrifuge: 22R, Beckman, USA

(12) High-speed low-temperature centrifuge: d37520, Heraeus, Germany

(13) Cell viability analyzer: Vi-Cell, Beckman, USA

(14) Circulating constant-temperature water bath box: ZSXH-618/625, Shanghai Zhicheng Analyzer manufacturing, Inc

1.3 supermolecular structure and property research characterization instrument:

(1) x-ray single crystal diffraction analysis: german Bruker APEX-II CCD diffractometer

Mono-colored Mo-K alpha rays of graphite.

(2) Thermal analysis (DSC/TGA): simultaneous thermal analyzer of Mettler TOLEDO TGA/DSC 1 in the Netherlands

(3) Temperature-variable polycrystalline powder X-ray diffraction analysis (VT-PXRD): Panalytical-Empyrean type X-ray diffractometer in the Netherlands

(4) Scanning Electron Microscope (SEM): high resolution INSPECTF50 model scanning electron microscope

(5) Ultraviolet-visible absorption spectrum analysis: UV-3600 spectrophotometer

2 cell culture

A549 cells with human alveolar epithelialization were cultured in RPMI1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% streptomycin. Subpackaging into culture bottle, screwing the bottle cap, and rotating slightly after appropriate screwing to facilitate CO₂The cells were incubated at 37 ℃ in 5% CO₂An incubator. Wherein A549 cells were purchased from Chinese academy of sciences (China).

3 cell proliferation inhibition assay (MTT)

Adherent cells were digested with 0.25% trypsin, single cell suspensions were prepared in RPMI1604 medium containing 10% fetal bovine serum, and seeded at a density of 2000 cells per well in 96-well plates in 100ul per well volume. Transfer the plates to CO₂In an incubator at 37 ℃ with 5% CO₂Incubate for 24 hours under conditions. Different concentrations of drug were added to each well, 3 replicate wells were set, and incubation was continued for 72 hours. 20 μ L of thiazolyl blue tetrazolium bromide (MTT) was added to each well according to the manual, incubation was continued for 4 hours, the culture was terminated, and the culture supernatant in the wells was carefully aspirated away. The 490nm wavelength is selected, the absorbance of each well is set on an enzyme linked immunosorbent assay instrument, and the result is recorded. With time as the horizontal axisThe light absorption values are plotted on the vertical axis. And calculating the median Inhibitory Concentration (IC) by Bliss method₅₀). Each experiment was repeated at least 3 times.

4 cell cycle assay (Flow Cytometry)

Cell cycle determination using flow detection method, first PI (propidium iodide) marker staining, then by flow cytometry PI detection cell population cycle distribution. And adding pancreatin containing EDTA into the A549 cells for digestion, adding DMEM containing 10% FBS after digestion to stop digestion, slightly blowing and beating the cells, and collecting the cell suspension into a centrifuge tube. Centrifuge at 1000rpm/min for 10 minutes and discard the supernatant. 5ml of pre-cooled 70-80% ethanol was added dropwise, vortexed to mix the cells, and left overnight at 4 ℃ in the dark. 1000 ℃ and 1500rpm/min, centrifuging for 10 minutes, and discarding the supernatant. The cells were then washed 2 times with PBS, followed by a PI-containing PBS solution to remove all ethanol. Cells were resuspended in 0.5mL PI/RNase stain and incubated for 30 min at room temperature in the dark. And (4) lightly blowing, uniformly mixing, filtering into a flow tube, storing the sample at 4 ℃ in a dark place, and detecting by an up-flow cytometer within 1 hour.

5 apoptosis assay (Flow Cytometry)

A549 cells were cultured in 6-well plates, and different concentrations of compound and DMSO were added as blanks, respectively. After 24 hours, cells were harvested using 0.25% trypsin without EDTA and washed 2 times with PBS solution. Staining was then performed using an apoptosis detection kit according to the instructions. And then detecting and analyzing by using a flow cytometer.

6 enzyme-linked immunosorbent assay (ELISA)

The concentration of the target protein in the sample is determined using the double antibody Sandwich (Sandwich) method. Samples or standards of different concentrations were added to the corresponding wells at 100. mu.L/well, the reaction wells were sealed with a sealing plate (clear), and incubated at room temperature for 120 minutes. The PBST plates were washed 5 times and the last time was patted dry on thick absorbent paper. Biotinylated antibody 100. mu.L/well was added, the reaction wells were sealed with a sealing plate and incubated at room temperature for 60 minutes. The plate was washed 5 times and also patted dry on thick absorbent paper for the last time. 100 μ L of horse radish peroxidase labeled Streptavidin (HRP-Streptavidin) was added per well, the reaction wells were sealed with a sealing membrane (white), and incubated for 20 min at room temperature in the dark. The plate was washed 5 times and patted dry for the last time. Adding 100 mu L/hole of color reagent TMB solution, sealing the reaction hole by a sealing plate membrane (white), and incubating for 20 minutes at room temperature in the dark until the standard substance and the sample have very obvious color change. Add stop solution 50 μ L/well, mix well and measure absorbance value at 450nm immediately. IL-6 was tested using a specific ELISA kit according to the instructions.

7 Western immunoblot analysis (Western Blotting)

1) Preparation of SDS-PAGE gels

Preparation of SDS-PAGE gels A10% Tris-glycine SDS-PAGE was prepared as an example: prepared according to the method of double distilled water 3.18ml, 30% polyacrylamide solution 2.66ml, 1.5M Tris-HCl solution (pH 8.8)2ml, 10% ammonium persulfate 0.08ml, 10% Sodium Dodecyl Sulfate (SDS) solution 0.08ml, Tetramethyldiethylamine (TEMED) solution (added just before use) 0.0032 ml.

2) SDS-PAGE electrophoresis

The prepared gel plate was fixed to the electrophoresis apparatus, and the shutter thereunder was opened. Adding Tris-glycine electrophoresis buffer working solution, removing air bubbles at the bottom of the gel, adding samples according to a preset sequence, and then connecting with a power supply. Power supply setting: after about 2 hours when bromophenol blue reaches the bottom of the separation gel at 100V, 400A, the power is turned off and the glass plate is removed to stop electrophoresis.

3) Rotary film

Sequentially placing a rotating membrane clamp soaked by a porous gasket, two pieces of accurate filter paper, separation glue, a PVDF membrane and two pieces of accurate filter paper; the membrane, filter paper and gel need to be aligned and must not have air bubbles. And putting the clamp into a membrane conversion electrophoresis tank, adding CAPS buffer solution at 4 ℃ into the electrophoresis tank, opening condensed tap water and a stirrer, and connecting a power supply to carry out membrane conversion. Power supply setting: 50V, 400 mA. The film transfer was complete after about 2.5 hours.

4) Immunostaining

The PVDF membrane after membrane conversion is slightly soaked in a pure methanol solution and then is put into TBS buffer solution for washing. Blocking buffer (TBST) was added and the mixture was then blocked for 2 hours on a shaker at room temperature. The membranes were washed with TBST solution (4X 10min), added with primary antibody diluted 1:300 or 1:500 (in blocking buffer), and incubated on a shaker at room temperature for 2 hours. TBST solution (3X 5min), adding 1:1000 diluted secondary antibody (dissolved in blocking buffer), incubating in a gentle shaking table at room temperature for 1.5 h, washing with TBST solution (3X 5min), and staining with ECL chemiluminescence kit for 1 min for X-ray film exposure or scanning.

8 statistical analysis

Statistical analysis of all experimental results was performed using SPSS 19.0 software to

Indicated, t-test compares differences between groups.

Experimental example 1 anti-inflammatory Activity of Compounds of the invention

The inflammatory factor IL-6 is an effective therapeutic target for acute and chronic inflammation, and plays an important role in the treatment process of inflammatory diseases. The present invention refers to the method of Muller et al, with uridine as a positive control, and IL-4-induced IL-6 release from human lung carcinoma cells A549 in a cell inflammation model to evaluate the level of IL-6 downregulation of synthetic compounds. And evaluating the biological activities of the compound, such as cell proliferation inhibition activity, cell apoptosis, cycle and the like.

1.1 MTT assay for determining the Effect of Compounds of the present invention on inhibition of A549 cell proliferation

The MTT method is adopted to respectively test the inhibition rates of the compounds with different concentrations on the cells of the human lung cancer cell A549 after 24 hours, and the concentrations of the compounds are respectively 3.125 MuM, 6.25 MuM, 12.5 MuM, 25 MuM and 50 MuM. Compound IC₅₀The values are given in the following table (Table 3). As can be seen, the compounds 7 and 9 show certain cell proliferation inhibition activity, and the IC is measured after 24 hours of drug action₅₀The values were 41.01. mu.M, 44.81. mu.M, and 69.62. mu.M, respectively.

TABLE 3 IC of Compounds acting on A549 cells₅₀Value of

1.2 apoptosis and cell cycle assay (Flow Cytometry)

Cell cycle assays were performed on compound 7 and compound 9 and apoptosis was detected (figures 1-4, tables 4-5). The experiment selects human lung cancer cell A549 cell as detection system, the control group is blank control (DMSO), the test concentrations of the compounds are 5 muM, 10 muM and 15 muM respectively, and the compounds are detected by flow cytometry after acting on human lung cancer cell A549 cell for 24h, 48h and 72 h. In the apoptosis results plot, cells in quadrant Q1 were necrotic cells, quadrant Q2 were late apoptotic cells, cells in the quadrant Q3 region were early apoptotic cells, and cells in quadrant Q4 represented live cells.

As can be seen from the test results, the effect of compound 9 on the cells is small, and when the concentration of the compound is 15 μ M, the total apoptosis of the cells is only 15.66% after 72 h.

In addition, when the compound acted on a549 cells for 72h, compound 7 had a more significant effect of inducing apoptosis of a549 cells and exhibited dose dependence as compared to the control group (fig. 1). Suggesting that the anti-proliferative activity of compound 7 on a549 cells may be related to its ability to induce apoptosis. These results indicate that compound 7 may have some anti-cancer activity.

TABLE 4 Effect of Compound 7 on apoptosis of A549 cells

TABLE 5 Effect of Compound 9 on A549 apoptosis

1.3 ELISA method for determining the Effect of Compounds on IL-4-induced IL-6 secretion levels from A549 cells

The results of the measurement of the level of the inflammatory factor IL-6 by the compound are shown in FIG. 5, and the concentrations of the compound are 1. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M, respectively.

The experimental result shows that after the compounds 7 and 9 with different concentrations act on a cell inflammation model for 24 hours, the IL-6 water level is reduced to a certain extent, wherein the compound 9 has the strongest inhibition effect. And the compounds all show good dose dependence, and the anti-inflammatory activity of the compounds is gradually enhanced along with the increase of the concentration. After reaching 5 μ M, the compound had better IL-6 downregulation than uridine (uridine), a positive control, and began to exhibit better anti-inflammatory effects.

The apoptosis and cycle experiment results of the combined compound show that the compound can inhibit the release level of IL-6 without being influenced by apoptosis.

1.4 inflammation-related Signal pathway exploration (Western Blotting)

The invention respectively tests the protein level of phosphorylated NF-kappa B p65, phosphorylated STAT3(Ser727) and phosphorylated Akt (Ser473) after the compound acts, and detects the change of key regulatory factors in the channels by using a Western blotting method. Wherein beta-ACTIN is used as an internal reference. The invention also measures the protein expression of the adhesion molecule ICAM-1 after the compound acts.

The results showed that the level of phosphorylated NF- κ B p65, phosphorylated STAT3(Ser727) and phosphorylated Akt (Ser473) protein in the drug group was reduced compared to the control group after the effect of compound 9 (figure 6). The protein level of adhesion molecule ICAM-1 was also reduced in a dose-dependent manner (FIG. 7). Therefore, the compound 9 has obvious anti-inflammatory activity and has very good application prospect as a potential medicament for treating inflammatory diseases.

Experimental example 2 Single Crystal Structure and Property characterization of the Compound of the present invention

The single crystals of 3 compounds ( compounds 1, 7, 9) were subjected to structural analysis and characterization of properties.

2.1 Crystal Structure of Compound 1

The crystal structure of compound 1 was determined by X-ray single crystal diffraction. The results showed that it belongs to the triclinic system with the space group P-1. The single crystal structure data and structure refinement data are shown in table 6. In the single crystal structure, τ (C5-C7-C11-C12) is defined as the twist angle, and the compound has four conformations (A1: τ)₁＝33.591,A2:τ₂＝33.591,B1:τ₃＝34.812,B2:τ₄-34.812), the molecular structure and atomic numbering of the different conformations are shown in figure 8. The molecular stacking diagram of the compound along the c-axis is shown in FIG. 9a, 4 structuresSuch as pi-pi stacking between 1-B1/1-B2 via intramolecular weak hydrogen bonding (C12-h.. O6), intermolecular hydrogen bonding interactions (O14-h.. N3, C12-h.. O14) and 1-B1/1-B2 to form a Zigzag type arrangement, which extends indefinitely to form a 3D network structure (fig. 9, 10).

TABLE 6. unit cell data and refinement parameters for Compound 1

TABLE 7 Hydrogen bonding parameters for Compound 1 crystals

Symmetry codes:¹1-X,1-Y,1-Z；²1-X,2-Y,-Z

2.2 Crystal Structure of Compound 7

The single crystal structure of compound 7 was tested by X-ray single crystal diffraction. The results showed that it belongs to the triclinic system with the space group P-1. Table 8 is crystal structure data and structure refinement data of compound 7 single crystal. Definition of τ (C5-C7-C10-C11) as twist angle, the compounds have a total of 2 conformations (A1: τ)₁＝56.615,A2:τ₂-56.615) (fig. 11). Hydrogen bonds are formed between different conformations by trans Watson-Crick base pairing (N6-h.. N1), and phenolic hydroxyl groups are hydrogen bonded to N3 between the same conformations (O14-h.. N3), thus forming a four-membered ring structure, with 2 identical water molecules (objects) contained in the ring (fig. 12). However, it was found by analysis that 2 water molecules in the ring did not interact molecularly with the four-membered ring of the layer, and the water molecules formed hydrogen bonds with the phenolic hydroxyl groups of the compounds in the two adjacent layers (FIG. 13). Meanwhile, a weak intermolecular interaction (C-H.. pi.) exists between layers, and the distance

(FIG. 13).

TABLE 8. unit cell data and refinement parameters for Compound 7

TABLE 9 Hydrogen bonding parameters for Compound 7 crystals

Symmetry codes:¹+X,1+Y,+Z；²-1+X,+Y,+Z

2.3 Crystal Structure of Compound 9

The single crystal structure of compound 9 was determined by x-ray single crystal diffraction. The results showed that it belongs to the monoclinic system and the space group was C2/C. The unit cell data and structure refinement data for the compounds are summarized in table 10. The guest water molecule is also included in the crystal structure of compound 9, resulting in a more complex 3D structure than compound 7. With τ (C5-C7-C10-C11) as the twist angle, fig. 14 shows two conformations of the crystal, i.e., a1(τ ═ 41.441), a2(τ ═ 41.441). Hydrogen bonding patterns of compounds: the conformations a1, a2 are interconnected by water bridges (O14-h.. Ow, Ow-h.. N12), defining the plane as a Layer (for example: Layer 1). Layer 1 and Layer 3 are arranged in the same way, and C19 on the t-butyl with the same conformation in the two layers is used as a hydrogen bond donor to form a hydrogen bond with N1 on the mother nucleus of the other Layer, so that 4 water molecules are formed as a water bridge to form a four-membered ring structure with 2 pairs of conformations. Layer2 and Layer 4 are hydrogen bonded in the same manner as Layer 1/Layer 3, and the formed 2 four-membered rings (Cycle P: pink, Cycle B: blue) are crossed with each other at an angle of 43.722 DEG and extend longitudinally and infinitely along the c-axis alternately through tert-butyl. The phenol ring between adjacent layers has pi-pi interaction

And a hydrogen bond is formed by N6 and the phenolic hydroxyl group O14 (fig. 15). ② the same Layer of ring-to-ring interaction, taking Layer2/4 as an example: two adjacent four-membered ring cycles B1/B2 passing through the 6-position NH₂As hydrogen bond donors to form a trans Watson-Crick base pairing pattern with N1, by which the pairing pattern extends transversely and infinitely along the b-and a-axes; black oval circle position in the figureThere is also a weak pi-pi interaction

(FIG. 16).

FIG. 17 is a molecular stacking diagram of a compound, in which molecules of different conformations and water molecules are self-assembled by 6 hydrogen bond modes (Table 11) and 2 weak pi-pi interactions to form a columnar structure, and 4 water columns are located in cavities in the column.

TABLE 10 unit cell data and refinement parameters for Compound 9

TABLE 11 Hydrogen bonding parameters for Compound 9 crystals

Symmetry codes:¹+X,1-Y,1/2+Z；²1-X,1-Y,1-Z

The molecular structure of compound 9 was introduced into crystall explorer17.5 software, and the crystal structure was subjected to Hirshfeld surface analysis. D of Compound 9 is set forth in FIG. 18_normShape index (shape index) and curved surface (curvedness) surface maps. d_normThe bright red highlighted area in the surface map indicates strong intermolecular interactions at this atom. FIG. 19 shows visually at d_normThe number and arrangement of the bright red protruding areas in the surface map and the surrounding molecules with hydrogen bonding interactions. The adjacent triangles of blue and red can be clearly seen in the shape index surface map, and the feature indicates that the interaction of pi-pi exists. From the 2D fingerprint (fig. 20) it can be seen that the diffuse points with blue tails on both sides of the diagonal are typical H.The two pairs of spikes pointing to the lower left corner are the points where n. In addition to these interactions, the C.. H interaction contributed 8.9% of the Hirshfeld surface, with the pair of blue "wings" appearing in the upper right corner being typical of C-H.. pi stacking.

Differential Scanning Calorimetry (DSC) test, thermogravimetric analysis (TGA) test and temperature-variable powder X-ray diffraction (PXRD) test are respectively carried out on the compound 9 in a single-crystal powder state, and the supermolecular structural properties of the compound 9 are characterized. Compound 9 has 3 endothermic peaks in the Differential Scanning Calorimetry (DSC) (fig. 21), Peak:204.70 ℃, Δ H-29.17 mJ, respectively, with corresponding enthalpy changes; peak 266.12 deg.C,. DELTA.H-139.36 mJ; peak 363.25 deg.C,. DELTA.H-239.42 mJ. There were 2 steps in the thermogravimetric analysis (TGA) (FIG. 22), the first weight loss step being observed starting at about 40 ℃ and corresponding to the volatilization of water molecules in the crystal structure (weight loss: 15.35%); the second weight loss step lost 69.92% of its weight. From the VT-PXRD plot (fig. 23), it can be seen that the peak shape of the compound substantially matches the calculated value when the temperature is below 65 ℃, and that the peak shape changes when the temperature is increased to 105 ℃, indicating that the compound crystal form has changed.

From the above-described single crystal analysis of the compounds 1, 7 and 9 of the present invention, it was found that the intramolecular or intermolecular linkage pattern and the molecular stacking pattern of each compound showed a significant difference due to the difference in the substituent at the N1, C3 or C4 position. When the substituent at position C4 is amino, i.e. compound 7, compound 9, hydrogen bond mediated base pairing is the common mode of supramolecular structure for both compounds, and hydrogen bonds are formed between different conformations by trans Watson-Crick base pairing. Among them, single crystals of compound 7 and compound 9 belong to supramolecular hydrates. The compound 7 forms hydrogen bonds with N7 through a base hydrogen bond pairing mode, a four-membered ring supramolecular structure is finally formed, molecules are stacked to form a pipeline structure, and solvent water molecules exist in the middle of the pipeline. The different conformations of compound 9 also form a columnar structure through more complex hydrogen bonding interactions and pi-pi interaction self-assembly, with 4 water columns located in the column cavity. Meanwhile, various interactions existing in the compound 9 are systematically analyzed through Hirshfeld surface analysis; the structure and thermodynamic properties of the compound 9 in a powder state are tested by analysis means such as DSC, TGA and VT-PXRD, and the compound 9 is found to have better thermal stability.

In conclusion, the crystals of the compound 7 and the compound 9 form a novel porous supermolecular framework structure containing a plurality of water channels, particularly the crystals of the compound 9 have good thermal stability, and have good application prospects in the fields of artificial channel materials, drug carriers and porous materials; the compound 7 and the compound 9 can effectively inhibit the release level of the inflammatory factor IL-6, the compound 9 can inhibit the protein levels of phosphorylation NF-kappa B p65, phosphorylation Akt, phosphorylation STAT3 and adhesion molecule ICAM-1, and the crystal prepared by the invention has a very good application prospect in preparing medicines for treating inflammatory diseases.

Claims (8)

1. A crystal of a pyrazolo [3,4-d ] pyrimidine compound, characterized in that: the crystal is a supermolecular hydrate formed by pyrazolo [3,4-d ] pyrimidine compounds and water molecules;

wherein the pyrazolo [3,4-d ] pyrimidine compound is a compound 9:

the crystal is a porous supermolecular structure;

the crystal is a single crystal, the single crystal belongs to a monoclinic system, and the space group is C2/C;

the crystal has the unit cell parameters as follows: α is 90 °, β is 105.786 ± 0.009 °, γ is 90 °;

the crystals comprise the enantiomers shown in 9-A1 and 9-A2:

wherein9-A1, tau₁41.441; in 9-A2, τ₂＝-41.441；τ₁、τ₂Are dihedral angles C5-C7-C10-C15 in 9-A1 and 9-A2 respectively;

the porous supermolecular structure of the crystal is a columnar structure, the columnar structure is formed by self-assembling 9-A1, 9-A2 and water molecules, and the water molecules are positioned in a cavity in a column of the columnar structure; in the columnar structure, the molar ratio of 9-A1 to 9-A2 to water molecules is 1: 1: 2.

2. the crystal of claim 1, wherein: the differential scanning calorimetry curve of the crystal is shown in FIG. 21.

3. The crystal of claim 1, wherein: the thermogravimetric analysis curve of the crystal is shown in FIG. 22.

4. The crystal of claim 1, wherein: the variable temperature powder X-ray diffraction curve of the crystal is shown in figure 23.

5. A crystal of a pyrazolo [3,4-d ] pyrimidine compound, characterized in that: the crystal is a supermolecular hydrate formed by pyrazolo [3,4-d ] pyrimidine compounds and water molecules;

wherein the pyrazolo [3,4-d ] pyrimidine compound is a compound 7:

the crystal is a porous supermolecular structure;

the crystal belongs to a triclinic system, and the space group is P-1;

the crystal has the unit cell parameters as follows: 71.752 ± 0.009 ° α ═ 0.009 °, 65.956 ± 0.008 ° β ═ 74.378 ± 0.010 ° γ;

the crystals comprise the enantiomers shown in 7-A1 and 7-A2:

wherein, in 7-A1, τ₃56.615; in 7-A2, τ₄＝-56.615；τ₃、τ₄Are dihedral angles C5-C7-C10-C15 in 7-A1 and 7-A2 respectively;

the porous supermolecular structure of the crystal is a pipeline structure, the pipeline structure is formed by self-assembling 7-A1, 7-A2 and water molecules, and the water molecules are positioned in the middle of the pipeline; in the pipeline structure, the molar ratio of 7-A1 to 7-A2 to water molecules is 1: 1: 1.

6. a method for producing the crystal according to any one of claims 1 to 5, characterized in that: the method comprises the following steps: adding pyrazolo [3,4-d ] pyrimidine compounds into a mixed solution of methanol and water, dissolving, filtering, taking liquid, and crystallizing to obtain crystals of the compounds; wherein the pyrazolo [3,4-d ] pyrimidine compound is compound 9 or compound 7 according to any one of claims 1 to 5.

7. The method of claim 6, wherein: in the mixed solution of the methanol and the water, the volume ratio of the methanol to the water is (8-12): 1;

the mass-to-volume ratio of the mixed solution of the pyrazolo [3,4-d ] pyrimidine compound, methanol and water is (3-7) mg: 1 mL;

and the crystallization is standing crystallization, the crystallization temperature is room temperature, and the crystallization time is 15-26 days.

8. The method of claim 7, wherein: in the mixed solution of methanol and water, the volume ratio of methanol to water is 10: 1;

the mass-volume ratio of the mixed solution of the pyrazolo [3,4-d ] pyrimidine compound, methanol and water is 5 mg: 1 mL;

the temperature of the crystallization is room temperature, and the time is 15 days or 26 days.

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