CN110548032B - Application of pyrazolopyrimidine compound in preparation of medicines for preventing/treating tumors - Google Patents

Abstract

The invention provides an application of a compound 1, or a solvate thereof, or a crystal thereof, or a salt thereof in preparing a medicament for preventing and/or treating tumors; also provided is the use of compound 1, or a solvate, or crystal, or salt thereof, in the preparation of mesoporous materials, drug delivery systems, drug carriers, artificial channels. Experiments prove that the compound 1 prepared by the invention has high-activity lung cancer resistance effect, can obviously inhibit the growth of A549 cells, has inhibition effect even better than that of a positive control medicament, and has good prospect in preparing medicaments for preventing and/or treating tumors. In addition, the compound 1 provided by the invention has good thermal stability, an obvious layered structure and quite high porosity, and the structure has good application potential in the fields of mesoporous materials, drug carrying materials and artificial channel materials.

Description

Application of pyrazolopyrimidine compound in preparation of medicines for preventing/treating tumors

Technical Field

The invention belongs to medicine synthesis, and in particular relates to application of pyrazolopyrimidine compound in preparation of medicines for preventing/treating tumors.

Background

Lung cancer is the malignant tumor with highest morbidity and mortality in the world, and poses a great threat to human health. Lung cancer is subdivided into two types, based on its biological properties, clinical treatment and prognosis: small cell lung cancer (small cell lung cancer, SCLC) and Non-small cell lung cancer (Non-small-cell lung carcinoma, NSCLC). NSCLC is one of the most common lung cancers, which is associated with increased production of epithelial cells, accounting for about 85% to 90% of lung cancer cases. Non-small cell lung cancer is also divided into several subtypes, respectively: lung adenocarcinoma, lung squamous carcinoma (squamous cell carcinoma, SCC) and lung large cell carcinoma (large cell lung cancer, LCLC). Adenocarcinoma or adenocarcinoma of the lung has obvious histological features, and changes in tissue cells, subatomic structures and components are accompanied by changes in organs, bronchi and mucus. Lung adenocarcinoma accounts for approximately 40% of all primary lung cancers. Malignant cells in lung adenocarcinoma grow and spread much slower than other subtypes of lung cancer and are therefore more detectable than other types of lung cancer. SCC usually occurs in one of the left or right bronchi, and smoking is the leading cause of this type of lung cancer. Clinical manifestations of SCC are typically dyspnea, chest pain and bloody sputum. SCC accounts for approximately 25-30% of primary lung cancer. LCLC is a heterogeneous aggregate of undifferentiated threatening tumors that does not have the cellular morphological features of small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma nor produce mucus. Lclclclc often originates in the central epithelial cells of the lung and diffuses to distant organs. Many studies have shown a close association between LCLC and smoking, accounting for about 5-10% of all lung cancers.

In recent decades, with the rapid development of accurate medicine, targeted therapy is used for clinical treatment of lung cancer and achieves remarkable effects, but the fact that targeted therapy drug resistance mutation and partial mutant genes have no corresponding targeted drugs is a difficult problem faced by current clinical treatment.

In addition, as a substance having pharmacological activity, as with other substances, in the course of crystallization, the intramolecular or intermolecular bonding pattern changes due to the influence of different physicochemical conditions, so that the arrangement of molecules or atoms in the lattice space changes, forming different crystal structures, i.e., the same substance has two or more spatial arrangements and unit cell parameters, and the phenomenon in which such different crystal structures exist is called polymorphism (polymorphism). For the same drug, the morphology of different crystals may exhibit different melting points and solubilities, which affect the bioavailability of the drug, and subsequent formulation processes. The dissolution rate of crystals of different shapes will be different and the molecular groups on the exposed surfaces of different crystals will be different, resulting in different drug effects. Therefore, the crystals of the medicine have influence on the aspects of bioavailability, stability, dosage form selection, curative effect and the like.

Solvates refer to crystalline substances of a compound molecule in a certain bound form with one or more solvent molecules, which is a ubiquitous form of compounds. The solvate belongs to a polymorphic form and plays an important role in the fields of medicine, polymers, energy sources and the like. Particularly in the medical field and of importance, when a drug is combined with a solvent to form a solvate, the properties exhibited are greatly different from those exhibited by non-solvates. For example: the volume, density, refractive index, hygroscopicity, solubility, etc. of the molecules will vary. There may be a large difference in bioavailability when the solubility of the drug is large in different solvates or non-solvent compounds. If the crystals of the medicine cannot be well controlled in the preparation or storage process, the medicine can not achieve the treatment effect due to the reduction of bioavailability or can be poisoned due to excessive dosage, so that medical accidents are caused.

Therefore, development of an effective low-toxicity lung cancer therapeutic drug with a novel structure, and research and palm holding of different crystals and properties of the drug are urgent demands of current clinical medication.

Disclosure of Invention

The invention aims to provide application of pyrazolo [3,4-d ] pyrimidine derivative in preparing medicines for preventing and/or treating tumors, mesoporous materials, medicine delivery systems, medicine carriers and artificial passages.

The invention provides an application of a compound 1, or a solvate thereof, or a crystal thereof, or a salt thereof in preparing a medicament for preventing and/or treating tumors;

the structure of the compound 1 is

Further, the tumor is lung cancer.

Further, the tumor is non-small cell lung cancer.

Further, the tumor is lung adenocarcinoma.

Further, the medicine can inhibit proliferation, growth and migration of tumor cells and induce apoptosis of the tumor cells.

Furthermore, the medicine can regulate and control the level of Cyclin E1 protein in tumor cells, regulate and control the expression of Bcl-2 and Bax, regulate and control the activity of caspase and regulate and control the expression of AMPK-mTOR channel protein.

Preferably, the medicine can reduce the level of the Cyclin E1 protein in tumor cells, increase the ratio of the Bax/Bcl-2 expression quantity and improve the activities of clear-Caspase-9 and clear-Caspase-3.

Further, the medicament can promote the generation of active oxygen in tumor cells.

Further, the drug is capable of inducing autophagic death of tumor cells.

The invention also provides application of the compound 1, or a solvate thereof, or a crystal thereof, or a salt thereof in preparing mesoporous materials, drug delivery systems, drug carriers and artificial channels;

the structure of the compound 1 is

Further, the solvate is a hydrate of compound 1.

Further, the preparation method of the crystal comprises the following steps: adding the compound 1 into a mixed solution of methanol and water, dissolving, filtering, taking liquid, and crystallizing to obtain crystals;

preferably, in the mixed solution of methanol and water, the volume ratio of methanol to water is 10:1, a step of; the mass volume ratio of the mixed solution of the compound 1, the methanol and the water is 25mg:8mL; the dissolution mode is that heating and dissolving are carried out at 60 ℃ until the solution is clear and transparent; the filtering is carried out while the filtering is hot; the crystallization mode is standing crystallization at room temperature, and the crystallization time is 10-30 days, preferably 10 or 30 days.

In the present invention, "C-H … pi action" means a non-bond weak interaction between a C-H bond and a pi system, and the non-bond weak interaction means a generic term for various bonds other than covalent bonds, ionic bonds and metallic bonds, and the pi system means a system capable of forming conjugated pi bonds.

"N-H … N hydrogen bonding" refers to hydrogen bonding between an N-H bond and an N atom.

"O-H … N hydrogen bonding" refers to hydrogen bonding between O-H bonds and N atoms.

"nonporous three-dimensional cross-structure" refers to a highly crosslinked spatial structure of nonporous interstices formed by chemical bond phase bonding of compound crystal molecules.

By "porous three-dimensional network" is meant a highly crosslinked spatial structure with pore-like interstices formed by chemical bond phase bonding of the compound crystal molecules.

"enantiomers" refer to stereoisomers that are physical and mirror images of each other and that are non-superimposable.

"mesoporous materials" refers to a class of porous materials having a pore size of between 2 and 50 nm.

"drug delivery system" or Drug Delivery Systems (DDS) refers to various forms or dosage forms of therapeutic drugs used in the course of disease control, including injections, tablets, capsules, patches, aerosols, suppositories, osmotic pumps, transdermal patches, drug strips, implants, mucoadhesives, and the like.

"drug carrier" refers to a system that alters the manner in which a drug enters the body and the distribution within the body, controls the release rate of the drug, and delivers the drug to a targeted organ.

"artificial channel" refers to a synthetic channel having a similar function to a natural aquaporin.

"solvate" refers to a crystalline material of a compound molecule that is co-formed with one or more solvent molecules in a bound form, which is a ubiquitous form of compounds. In the pharmaceutical manufacturing process, there are many processes in which solvents are necessary, and in which the compounds are brought into close contact with the solvents, under certain conditions, the corresponding solvates are formed.

"hydrate" is one of the solvates, and refers to a crystalline substance formed by a compound molecule and water molecules in a certain combination mode.

Experiments prove that the compound 1 prepared by the invention has high activity lung cancer resistance, can obviously inhibit the growth of A549 cells, has inhibition effect even better than that of a positive control medicament, and has good prospect in preparing medicaments for preventing and/or treating tumors. In addition, the compound 1 provided by the invention has good thermal stability, an obvious layered structure and quite high porosity, and the structure has good application potential in the fields of mesoporous materials, drug carrying materials and artificial channel materials.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be further described in detail with reference to the following embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

Fig. 1: compound 1 has an inhibitory effect on a549 cell proliferation.

Fig. 2: visual morphology of a549 cells after treatment with different concentrations of compound 1.

Fig. 3: compound 1 effect on a549 cell cycle-flow chart.

Fig. 4: immunoblot analysis of the G1 protein of a549 cells (72 h).

Fig. 5: transwell experiments examined the effect of Compound 1 on A549 cell migration.

Fig. 6: compound 1 inhibited a549 cell migration at different concentrations P <0.05.

Fig. 7: compound 1 effect on apoptosis of a549 cells-flow chart.

Fig. 8: effect of compound 1 on ROS production in a549 cells.

Figure 9:a) effect of compound 1 on Bcl-2 and Bax expression in a549 cells, b) ratio of Bax/Bcl-2 expression after treatment of a549 cells with compound 1P <0.05, P <0.01, P <0.001.

Fig. 10: effect of compound 1 on a549 cell Caspase family proteins.

Fig. 11: effect of compound 1 on the a549 cell AMPK-mTOR signaling pathway protein.

Fig. 12: a) Effect of compound 1 on a549 cell autophagy pathway proteins LC3I and LC3 II; b) Compound 1 acts on the ratio of a549 cell autophagy pathway protein LC3II/LC3I, < P <0.05, < P <0.01, < P <0.001.

Fig. 13: the molecular conformation of crystal 1, crystal atomic number and overlap map (a) conformations 1-A1 and atomic number (left) and enantiomer 1-A2; (b) 1-A1 and 1-A2 overlap front view (left) and top view (right), wherein different colors represent different atoms: carbon atoms, grey; oxygen atom, red; nitrogen atom, blue; hydrogen atom, white.

Fig. 14: crystal 1 molecular stacking diagram and hydrogen bond network diagram (hydrogen bonds are indicated by broken lines, H atoms not forming hydrogen bonds are deleted for clarity); a) A molecular stacking diagram along the a-axis; b) A crystal hydrogen bond network analysis chart along the a direction; c) A hydrogen bond network resolution graph along the b direction; d) Hydrogen bond network resolution along the c-direction.

Fig. 15: a) A view of one four-membered ring and hydrogen bond chain formed by water and molecules of compound 1; b) Intermolecular interactions with two adjacent layers of four-membered rings of one-dimensional water chains in the center (host molecules are represented by the sticks mode, guest molecule water is represented by the ball and stick mode); c) A one-dimensional water chain four-ring space mapping schematic diagram is arranged in the center; d) Perspective view of a four-membered ring with an array of water molecules.

Fig. 16: the layer-to-layer stacking pattern (molecules are shown in space film mode) in crystal 1.

Fig. 17: a front view of a) dnorm surface plot of Hirshfeld surface analysis of crystal 1; b) The dnorm surface plot back view (white indicates forces equivalent to van der waals interatomic distances; red represents a strong force shorter than the van der waals force distance; blue represents weaker force than van der waals force), and c) 2D fingerprint; d) Dnorm surface plot of specific intermolecular interactions.

Fig. 18: the molecular conformation of crystal 2, crystal atomic number and overlap map (a) conformations 2-A1 and atomic number (left) and enantiomer 2-A2; (B) conformational 2-B1 atomic number and enantiomer 2-B2; c) 2-A1 and 2-A2 overlap maps; d) 1-A1,2-A1 and 2-B1 (green represents 1-A1, yellow represents 2-B1, and magenta represents 2-B1); e) 2-B1 and 2-B2.

Fig. 19: a) A four-membered ring and a hydrogen bond chain formed by the compound 1; b) A quaternary ring-like structure; c) The adjacent four-ring structure interacts with the similar four-ring structure; d) Space filtering mode of graph c (where red is a four-membered ring and green is a four-membered ring like).

Fig. 20: 2 cross-layer views of a) in crystal 2 along crystallographic b-axis direction; b) The space filtering mode of FIG. a); c) C-H … pi enlarged view of crossed interlayer four-membered ring and quasi-four-membered ring; d) Cross-layer class four-membered inter-loop C-H … pi amplification.

Fig. 21: relationship of a) two cross-layer views along the c-axis in crystal 2 b) space filling mode of figure a).

Fig. 22: hirshfeld surface analysis and 2D fingerprint of crystal 2.

Fig. 23: the contact between atoms in the compound 1 crystal accounts for the proportion of Hirshfeld surface.

Fig. 24: compound 1 thermal analysis results: a) DSC curve of compound 1; b) TGA profile of compound 1.

Fig. 25: the supramolecular morphology of compound 1 in solution (top left 1mg/mL normal field of view, bottom left 1mg/mL magnified view; top right 2mg/mL normal field of view, bottom right 2mg/mL magnified view).

Detailed Description

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

1. Experimental reagent and equipment

The experimental reagent sources for the following examples section are shown in table 1:

table 1: experimental reagent

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The main experimental equipment and analytical test equipment used in the examples section below are as in table 2:

table 2: chemical experimental instrument

2. Synthesis method

EXAMPLE 1 Synthesis of Compound 1 of the present invention

(1) Synthesis of 5-amino-4-cyano-1-tert-butyl-1H-pyrazole (12):

tert-butylhydrazine 11 (0.72 g,8.19 mmol) and triethylamine (1.70 mL, 12.29 mmol) were added to a 50mL round-bottom flask containing 20mL of absolute ethanol at room temperature, and ethoxymethylenemalononitrile 10 (1.00 g,8.19 mmol) was slowly added dropwise thereto. The reaction mixture was heated at 78 ℃ for 3 hours. Then the reaction is carried out The solution was cooled to room temperature and dried by spinning to give a viscous orange solid. Water (30 mL) was then added thereto and the mixture was treated with CH ₂ Cl ₂ (3X 60 mL) extraction reaction. The combined organic phases were dried over anhydrous sodium sulfate, and then the solvent was evaporated under reduced pressure and concentrated. A fast setting orange-yellow gum was obtained. The residue was partitioned with 10% etoac in hexanes (60 mL) and the mixture was sonicated. The resulting crystalline solid was filtered, washed with a large amount of 10% EtOAc in hexane and dried to finally give 1.29 g of 5-amino-4-cyano-1-tert-butyl-1H-pyrazole as pale orange crystals in 96.3% yield. ¹ H NMR(600MHz,DMSO-d ₆ )δ7.45(s,1H),6.22 (s,2H),1.50(s,9H)。 ¹³ C NMR(150M,DMSO-d ₆ )δ150.71,138.01, 115.24,74.55,57.76,28.23。

(2) Synthesis of 4-amino-1-tert-butyl-1H-pyrazolo [3,4-d ] pyrimidine (13):

a mixture of 5-amino-4-cyano-1-tert-butyl-1H-pyrazole 12 (1.00 g,6.09 mm. Ol.) and formamide (15 ml) was heated at 190℃for 6 hours under nitrogen. By CH ₂ Cl ₂ (3X 60 ml) and H ₂ O (30 ml) extracts the mixture. The combined organic layers were then dried over anhydrous sodium sulfate and evaporated in vacuo. By silica gel column chromatography (elution: 0% -50% CH) ₂ Cl ₂ /CH ₃ OH) purification of the reaction mixture gave product 13 as a white solid 1.16g in 55.8% yield. ¹ H NMR(6 00MHz,DMSO-d ₆ )δ8.14(s,1H),8.03(s,1H),7.58(s,2H),1.69(s, 9H)。 ¹³ C NMR(150M,DMSO-d ₆ )δ158.16,154.72,152.72,130.04,1 01.43,59.23,28.78。

(3) Synthesis of 4-amino-3-bromo-1-tert-butyl-1H-pyrazolo [3,4-d ] pyrimidine (14):

n-bromosuccinimide (1.37 g,7.85 mmol) was added to a solution containing 4-amino-1 tert-amino at room temperature butyl-1H-pyrazolo [3,4-d ]]Pyrimidine (1.00 g,5.23 mmol) in 100mL of acetonitrile. The reaction mixture was then stirred at 80℃for 4 hours. After the reaction mixture was cooled to room temperature, it was cooled by CH ₂ Cl ₂ (3X 60 mL) and H ₂ O (30 mL) was extracted, the organic extracts were combined, dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure. Followed by gradient elution by chromatography on silica gel (eluent: CH) ₂ Cl ₂ ) The residue was purified, the desired fractions were combined and evaporated in vacuo to give the desired product as a yellow solid, 0.91g, 64.8% yield. ¹ H NMR(600M, DMSO-d ₆ )δ8.20(s,1H),1.67(s,9H)。 ¹³ C NMR(150M,DMSO-d ₆ )δ 157.51,155.64,153.52,115.29,100.46,60.61,28.61。

(4) Synthesis of 3- (4-amino-1- (tert-butyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) phenol (1):

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pinacol 3-hydroxyphenylborate (0.40 g,1.79 mmol) was reacted with 4-amino-3-bromo-1-tert-butyl-1H-pyrazolo [3,4-d ] as obtained in the preceding reaction]Pyrimidine 13 (0.40 g,1.49 mmol) was dissolved in 1,4-dioxane/H ₂ O (4:1, 25 mL) solvent in a 100mL round bottom flask. Sequentially adding K at room temperature ₂ CO ₃ (0.41 g,2.98 mmol) and PdCl ₂ dppf (0.11 g,0.15mm mol), the reaction mixture was stirred at 100℃for 8h. The reaction solution was then cooled and evaporated in vacuo, and the residue was adhered to silica gel using dichloromethane as solvent. Purifying by silica gel column chromatography (eluting: 0% -1% CH) ₂ Cl ₂ :CH ₃ OH) and evaporating the desired fraction in vacuo to give the desired end product (compound 1) as an grey solid in a yield of 71.1% and 0.30 g. ¹ H NMR(600M,DMS O-d ₆ )δ9.69(s,1H),8.23(s,1H),7.35-7.32(t,J＝8.1Hz,1H),7.06-7.05 (d,J＝7.2Hz,2H),6.87-6.86(dd,J＝8.2Hz,1H),1.74(s,9H)。 ¹³ C N MR(150M DMSO-d ₆ )δ158.12,154.59,153.77,141,69,134.48,130.2 2,118.93,115.62,115.03,98.55,59.62,28.75。HRMS-ESI(m/z)calcd for[M+H] ⁺ ,284.1433；found,284.1505。

Example 2 preparation of crystals 1 and 2 of Compound 1 of the present invention

(1) The compound single crystals were grown according to standard recrystallization procedures. The method comprises the following steps: compound 1 (25 mg) obtained in example 1 was dissolved in 8ml of methanol: in a mixed solvent of water (10:1). After stirring the mixture was heated to saturation at 60 ℃ until clear and transparent. The hot solution was filtered in time with a syringe filter. The solvent was then slowly evaporated at room temperature. Yellow granular crystals 1 were obtained on day 30.

(2) Using the same method and the same solvent as in step (1), 30 days was changed to 10 days, and yellow granular crystals, namely, crystals 2 of compound 1 were obtained.

The beneficial effects of the compounds of the invention are demonstrated below by experimental examples.

Experimental example 1 evaluation of antitumor Activity of the Compound of the present invention

1. Experimental method

1. Experimental reagent and instrument

The main reagents used in the following experimental examples are shown in table 3:

table 3: experimental reagent

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The main experimental equipment used in the following experimental examples is shown in table 4:

table 4: experimental instrument

2. Experimental method

2.1 preparation of solutions

(1) Preparation of Compound 1 solution

28mg of the compound 1 powder prepared in example 1 was weighed, dissolved in 1mL of Dimethylsulfoxide (DMSO), prepared into a 100. Mu.M drug solution, filtered through a 0.22. Mu.M sterile microporous filter membrane, and stored in a refrigerator at-20℃in the absence of light after packaging. The culture broth RPMI-1640 was diluted to the desired concentration before the experiment.

(2) RPMI-1640 complete culture preparation

RPMI-1640+10% fbs+1% ps medium was prepared: 5ml FBS (serum) +0.5ml PS (diabody) +44.5ml RPMI-1640 was stored in a refrigerator at 4 ℃.

(3) Preparation of MTT solution

0.5g of tetramethylazoblue (MTT) powder was weighed and dissolved in PBS to adjust the final concentration to 5 mg/mL ^-1 Filtering with 0.22 μm sterile microporous membrane, and storing at 4deg.C in dark place.

(4) Preparation of running buffer

Glycine 93.85g, tris 15.15g and SDS 5g were weighed to be fully dissolved in 700m L double distilled water, then double distilled water was added to fix the volume to 1000mL, and the double distilled water was used for dilution by 5 times.

(5) Preparation method of 10 XTBS solution

24.2g of Tris base and 80g of NaCl are weighed, the PH is regulated to 7.6 by dilute hydrochloric acid, and the volume is fixed to 1L by double distilled water.

(6) Preparation method of 1 XTBST solution

Mixing 0.5mL Tween-20, 100mL 10 XTBS and 900mL double distilled water uniformly, and preserving at room temperature

(7) Preparation of 5% skim milk powder as sealing liquid

5g of skim milk powder was weighed and dissolved in 100mL of TBST and stirred until it was sufficiently dissolved, i.e., a 5% TBST solution of skim milk powder (W/V).

(8) Preparation of BCA solution

5g of BSA was weighed and dissolved well in 100mL TBST, namely a TBST solution of 5% BSA (W/V).

2.2 cell resuscitation

(1) Heating the constant-temperature water bath to 37 ℃, wiping the table surface of the ultra-clean workbench with 75% alcohol, and starting an ultraviolet lamp to irradiate for 30min;

(2) Preparing 15ml of RPMI-1640 complete culture medium, and adding the culture medium into a 15ml centrifuge tube;

(3) Taking the A549 cell cryopreservation tube out of the liquid nitrogen, immediately placing the tube in a water bath at 37 ℃, rapidly and gently shaking the tube, and thawing the cells;

(4) Gently blowing and mixing, and slowly dripping the suspension into a 15ml centrifuge tube containing the complete culture medium by using a suction tube;

(5) Centrifuging at 20deg.C and 1000rpm for 3min, and removing supernatant;

(6) 1ml of the prepared RPMI-1640 complete medium was added to a centrifuge tube to resuspend the cells, and the suspension was transferred to 25cm with 4ml of medium ² In the cell culture flask, shaking and uniformly mixing by a cross method;

(7) The flask was placed in 5% CO ₂ Culturing in an incubator at 37 ℃.

2.3 passage of cells

(1) Washing hands with soap before entering the aseptic chamber, and wiping and sterilizing the hands with 75% alcohol;

(2) Observing cell morphology under an inverted microscope, determining whether the A549 cells are passaged or not, and preheating a culture medium, pancreatin and the like at 37 ℃ by multiple of dilution of the cells; (3) wiping the table top of the ultra-clean workbench with 75% alcohol;

(4) Turning on an ultraviolet lamp of the ultra-clean workbench to irradiate the table top for about 20min, turning off the ultraviolet lamp, and turning on a fan to clean air to remove ozone;

(5) Sucking the old culture medium from the culture flask with a pipette, and optionally washing the residual culture medium with 2-3ml Hanks solution or brushing with a small amount of pancreatin;

(6) Adding 1mL of trypsin-EDTA solution into a culture flask, shaking uniformly, spreading, covering the bottom of the flask sufficiently, and placing into a constant temperature incubator for digestion for 1min;

(7) Observing under an inverted microscope, immediately turning over the culture flask when the cell retraction protuberance is rounded, separating the cells from pancreatin, and then pouring out the pancreatin;

(8) Adding a small amount of fresh culture medium containing serum to stop digestion, repeatedly blowing and beating digested cells to remove walls and disperse the cells, centrifuging at 1000rpm for 5min, and discarding supernatant;

(9) Adding a certain amount of fresh culture medium containing serum according to the number of the branch transfer bottles to re-suspend cells to prepare cell suspension, and then sub-packaging the cell suspension into a new culture bottle;

(10) The bottle cap is covered, and the bottle cap is slightly rotated after being moderately screwed, so as to be beneficial to CO ₂ Is put back into 5% CO ₂ An incubator at 37 ℃;

(11) And taking well-grown A549 cells for experiments.

2.4 cryopreservation of cells

(1) Changing a cell complete culture medium one day before freezing and storing, and collecting A549 cells in a logarithmic growth phase;

(2) Taking a centrifuge tube, adding RPMI-1640 culture medium, fetal calf serum and 10% dimethyl sulfoxide DMSO, preparing cell cryopreservation solution, enabling the ratio of the cell cryopreservation solution to be 7:2:1, and standing at room temperature for standby;

(3) A549 cells were digested with trypsin-EDTA solution, collected into 15ml of a centrifuge tube, and subsequently centrifuged at 1000rpm for 5min;

(4) Discarding the supernatant, adding the prepared cell freezing solution, and lightly blowing to the cell for resuspension;

(5) Subpackaging the cell suspension into cell freezing tubes, wherein each tube is 1-1.5 mL, screwing a tube orifice, attaching a sealing film, and making freezing records;

(6) The cell freezing tube is placed in a liquid nitrogen tank for long-term preservation after being placed in the liquid nitrogen tank for 10min at 4 ℃ to 30min at minus 20 ℃ to 16-18 h (or overnight) at minus 80 ℃.

2.5 cell proliferation inhibition assay (MTT method)

(1) Taking A549 cells in logarithmic growth phase, digesting with 0.25% trypsin, collecting cells, and adjusting cell concentration to 1×10 ⁵ cell.mL ^-1 Cells were seeded in 96-well plates at 100. Mu.L per well with blank wells placed at 37℃in 5% CO ₂ Culturing in an incubator with saturated humidity for 24 hours;

(2) Changing complete culture medium containing different concentrations of compound 1 (0, 3.125, 6.25, 12.5, 25, 50 μm), setting 3 duplicate wells for each concentration, and culturing for 72 hr;

(3) Adding 10 mu L of MTT solution into each hole, and continuously culturing in an incubator for 4 hours;

(4) After the supernatant is sucked and removed, 100 mu L of DMSO solution is added into each hole, and the mixture is vibrated for 10min on a shaking table at a low speed to completely dissolve the crystallization product;

(5) Detecting absorbance value at 490nm wavelength by using an enzyme-labeled instrument, and calculating cell activity;

(6) Experiments were repeated 3 times and the average was taken.

2.6 cell growth State experiment

(1) Taking A549 cells in logarithmic growth phase, digesting with 0.25% trypsin, collecting cells, and regulating cell concentration to 2x10 ⁵ cell·mL ^-1 Adding into a 6-hole plate, and adding 2mL of the solution into each hole;

(2) Culturing in an incubator for 24 hours, and adding compound 1 (0, 2.5 and 5 mu M) with different concentrations;

(3) The cells were cultured in an incubator for 72 hours, and the growth and morphological changes of the cells after the effect of each concentration of Compound 1 were observed under an inverted microscope.

2.7 cell cycle experiments

(1) Taking A549 cells in logarithmic growth phase, inoculating 1×10 cells in each well ⁵ cell·mL ^-1 Inoculating into 6-well plate, adding 2mL of complete culture medium into each well, arranging three auxiliary wells in each group, placing at 37deg.C, and placing 5% CO ₂ Culturing in an incubator with saturated humidity for 24 hours;

(2) Changing the complete culture medium containing different concentrations of compound 1 (0, 2.5, 5 μm) and continuing the culture;

(3) After culturing for 24h, 48h and 72h respectively, the cells were digested with 0.25% trypsin solution, pre-chilled phosphate buffer PBS was added, and the cells were centrifuged at 1000rpm for 3min and washed twice;

(4) Dropwise adding 70% ethanol solution, and fixing at 4deg.C overnight in dark;

(5) After cell fixation, the cells were centrifuged at 2000rpm at 4℃for 5min, the supernatant was discarded and washed twice with PBS;

(6) Adding 500uL PBS containing 50ug/mL ethidium bromide (PI), 100ug/mL RNase A,0.2% Triton X-100, and incubating at 37deg.C for 30min in the absence of light;

(7) Each group of samples was tested for 2 ten thousand cells using flow cytometry, data were acquired using FlowJo software and cell cycle distribution was analyzed.

2.8Transwell cell migration experiments

(1) Freezing and thawing Matrigel of BD company at 4deg.C overnight, and refrigerating 100 μl gun head;

(2) Matrigel was kept on ice for operation after the start of the experiment, 1:8, diluting, coating the upper chamber surface of the bottom membrane of the Transwell chamber, and placing at 37 ℃ for 30min to polymerize Matrigel into gel. Performing basement membrane hydration using an advancing process;

(3) After digesting A549 cells with 0.25% trypsin, the culture solution was decanted, washed 2 times with PBS solution, resuspended in serum-free medium containing BSA, and the cell density was adjusted to 5X10 ⁵ /mL；

(4) Taking 100 mu L of cell suspension, adding into a Transwell chamber, adding treated A549 cells of compound 1 (0, 2.5, 5 and 10 mu M) with different concentrations, setting a blank control group, and setting three compound wells for each concentration;

(5) 600 μl of 20% FBS-containing medium was added to the 24-well plate chamber, and the mixture was placed at 37deg.C in 5% CO ₂ Culturing in an incubator;

(6) After 24h of culture, taking out a Transwell chamber, discarding culture solution in the hole, washing twice with sterile PBS, fixing with methanol for 30min, and properly air-drying the chamber;

(7) Giemsa was stained for 15min, the upper non-migrated cells were gently rubbed off with a cotton swab and washed three times with PBS;

(8) Five fields of view were randomly selected under a 20X inverted microscope to view cells and photographed.

2.9 apoptosis experiments

Apoptosis was detected by Annexin V-FITC/PI double staining, as follows:

(1) Logarithmic growth of A549 cells was taken, counted and measured 5X 10 ⁵ cell·mL ^-1

Culturing in a 6-well plate;

(2) Changing complete culture medium containing different concentrations of compound 1 (0, 2.5, 5 μm), and culturing for 24 hr and 48 hr;

(3) The cell culture solution was aspirated into 15ml centrifuge tubes, and after digesting the cells with trypsin, the cells were washed 3 times with pre-chilled PBS, and the cell concentration was adjusted to 1X 10 ⁶ cell·mL ^-1 Centrifuging at 2000rmp for 5min, and removing PBS;

(4) Adding 400 mu L of 1 Xannexin V binding solution into each group to re-suspend cells, adding 5 mu L of Annexin V-FITC staining solution, gently mixing, and incubating for 5min at room temperature under dark conditions;

(5) Then 10 mu L of PI staining solution is added for on-machine detection

(6) And detecting by using a flow cytometer, detecting 2 ten thousand cells in each group of samples, acquiring data by using Cell Quest software, and analyzing apoptosis conditions.

2.10DCFH-DA Probe detection reactive oxygen species experiment

(1) Taking A549 cells in logarithmic growth phase, digesting with 0.25% trypsin, collecting cells, and regulating cell concentration to 1×10 ⁵ cell·mL ^-1 Adding into 6-hole plates, and culturing for 24h with 2mL of each hole;

(2) Compound 1 was added at concentrations of 0, 2.5, 5 and 10 μm, respectively, for 24h;

(3) DCFH-DA was diluted to 10. Mu. Mol.L in serum-free medium at a ratio of 1:1000 ^-1 For standby application;

(4) After pancreatin digestion to collect cells, washing twice with pre-chilled PBS, resuspending cells with 1mL of DCFH-DA;

(5) Placing in an incubator to react for 20min in a dark place, and uniformly mixing every 5min to ensure that the probe is fully contacted with the cells;

(6) After washing the cells three times with serum-free medium, the cells were resuspended in 1 mL;

(7) The average fluorescence intensity was measured by flow cytometry.

2.11Western Blot analysis of Compound 1 Effect experiment on apoptosis-related protein expression

2.11.1 extraction of Total cellular proteins

(1) Inoculating A549 cells in logarithmic growth phase into 6-well plate, and regulating cell concentration to 1×10 ⁵ cell/mL ^-1 Placing in an incubator for culturing for 24 hours;

(2) Compound 1 was added at concentrations of 0, 5, 10 and 20 μm, respectively, and allowed to act for 72h;

(3) Centrifuging at 1000rmp for 5min after pancreatin digestion to collect cells, washing twice with pre-cooled PBS, and discarding supernatant;

(4) mu.L of cell lysate was added to each cell group, lysed on ice for 30min, and then centrifuged at 12000rpm for 10min at 4℃and the supernatant carefully aspirated and stored at-20 ℃.

Determination of protein concentration by 2.11.2BCA method

(1) BSA standards (0.5 mg/mL) 0,1,2, 4,8, 12, 16, 20uL were added sequentially to 96-well plates, and then the total volume was made up to 20uL with pre-chilled PBS;

(2) Diluting the sample to be tested by 20 times, and adding 20 mu L of each well into a 96-well plate;

(3) Adding 200 uL/hole of BCA working solution, placing in an incubator, culturing for 30min, and cooling to room temperature;

(4) Measuring the absorbance of each hole at 562nm by using an enzyme-labeled instrument, and using water to be zero;

(5) And drawing a protein standard curve, and calculating the protein concentration of the sample to be detected.

2.11.3SDS-PAGE protein electrophoresis

(1) Preparation of a separation gel with a concentration of 12% (see Table 5)

(2) Injecting separating glue between the two glass plates to avoid generating bubbles, injecting the separating glue to the lower edge of the comb by 1cm, and gently adding double distilled water for water sealing;

(3) Concentrated gel with concentration of 5% was prepared (see Table 6)

(4) Pouring the separating gel, standing at room temperature for 30min, slowly pouring out the double distilled water on the upper layer after the separating gel is completely polymerized, and sucking the residual double distilled water with a filter paper strip;

(5) Rapidly injecting concentrated glue onto the top of a glass plate, inserting a comb to prevent bubbles, and standing at room temperature for 30min for later use;

(6) Taking the packaged cell total protein or plasma protein, adding 5 Xloading buffer solution 5uL, carrying out metal bath at 1 ℃ for 10min to denature the protein, and centrifuging and loading;

(7) Adding 4 mu L of pre-dyed protein markers into holes on two sides of a protein sample;

(8) The electrophoresis apparatus is started, the required bands are separated by electrophoresis, and the electrophoresis can be stopped.

Table 5:12% separating gel preparation (15 mL)

Table 6:5% concentrated gum formulation (4 mL)

2.11.4 transfer film

(1) Soaking PVDF film in proper size in methanol for 30s, soaking in distilled water for 2min, and transferring to electrotransfer liquid;

(2) Preparing a foam-rubber cushion-filter paper-separating gel-PVDF membrane filter paper-foam-cushion sandwich, and putting the foam-cushion sandwich into a membrane transferring groove;

(3) Pouring transfer buffer solution and putting into a cooling device;

(4) Transfer at constant pressure 60V for 100min. After the transfer, the PVDF membrane is taken out and the positions of the front and back sides and the standard molecular weight reference protein are marked.

2.11.5 closure, primary antibody incubation, secondary antibody incubation

(1) Placing the film which is successfully transferred into prepared 5% skimmed milk powder, and placing the skimmed milk powder into a sealing liquid for sealing for about 1h at room temperature; TBST is used after sealing;

(2) Diluting the primary antibodies of clear-caspase-9, clear-caspase-3, bax and Bcl-2 with 5% skimmed milk powder, and incubating at 4deg.C overnight, wherein the beta-actin antibody is used as an internal reference;

(3) The membrane was washed 3 times with 1 XTBE for 5min each time, the secondary antibody was diluted with 5% nonfat dry milk, incubated at room temperature for 1h, and finally washed 3 times with 1 XTBE for 15min each time.

2.11.6ECL development

(1) ECL chemiluminescent light-emitting liquid A and ECL chemiluminescent light-emitting liquid B are mixed according to the proportion of 1:1, and are placed at room temperature for 1min for standby;

(2) The mixed ECL reagent was applied to PVDF membrane (1 mL/10 cm) ² ) Reacting for 1min at room temperature, and obtaining a strip by chemiluminescence;

(3) The gel imaging system takes a photograph.

2.12Western Blot analysis of Compound 1 Effect experiment on AMPK-mTOR pathway protein expression

The specific procedure is as described in 2.11, and the expression of the AMPK-mTOR pathway protein is detected.

2.13Western Blot analysis of Compound 1 Effect experiment on autophagy pathway protein expression

The specific procedure is as in 2.11, and the expression of autophagy pathway proteins is detected.

2.14 statistical analysis

Statistical analysis was performed using Graphpad Prism, and differences between groups were tested using T-test, results were used

Indicating the test result P<A difference of 0.05 is statistically significant.

2. Experimental results

1. MTT method for detecting proliferation inhibition effect of each compound 1 on A549 cells

The proliferation inhibition of the cells was tested by MTT method using compound 1 synthesized in example 1 at different concentrations (0, 3.125, 6.25, 12.5, 25, 50. Mu.M), the activity of the cells was evaluated using PP1 as a positive control, and the experiment was performed by selecting human lung adenocarcinoma cells A549 cells, and the experimental results after 72 and h actions are shown in Table 7.

Table 7: pyrazolo [3,4-d ] pyrimidine derivatives having inhibitory activity against a549 cells

From the experimental results, compared with the positive control, the compound 1 has more obvious inhibition effect on the A549 cells, and the inhibition effect is obviously enhanced along with the increase of the drug concentration (see figure 1), which shows that the compound 1 has concentration dependence on the growth and proliferation of the anti-A549 cells.

2. Effect of Compound 1 on the growth State of A549 cells

According to the MTT assay results, compound 1 acted on the A549 cells for 72h and then acted on the IC ₅₀ The value was 2.12. Mu.M, so the invention selects two concentrations of 2.5 and 5. Mu.M for morphological influence experiments. Compound 1 (0, 2.5 and 5. Mu. At various concentrationsM) after 72h of action on A549 cells, they were observed under a 20X inverted microscope.

As a result, as shown in FIG. 2, the cells of the control group were tightly connected and uniformly sized, exhibiting a polygonal morphology of normal A549 cells. With the increase of the concentration of the compound 1, cells are mutually dispersed, wrinkled into circles and present a phenotype related to apoptosis, and the compound 1 is proved to be capable of effectively inhibiting the growth of A549 cells.

3. Effect of Compound 1 on A549 cell cycle

The cell cycle is detected by adopting a PI staining method, the blocking effect of the drug on the A549 cells is determined, and the inhibition effect of the drug on cell proliferation is further proved. The results of 24h, 48h, 72h (table 8, fig. 3) all showed that the ratio of the a549 cells treated with compound 1 was significantly increased in the G0/G1 phase compared to the control group, and the ratio of the cells in the G0/G1 phase was also increased with increasing drug concentration, while the corresponding ratio of the cells in the S phase and the G2/M phase was decreased, indicating that the drug significantly blocked the cell cycle of the a549 cells in the G0/G1 phase, and that the blocking effect was drug dose-dependent, and that all these changes resulted in inhibition of cell proliferation. Further analysis of the related protein Cyclin E1 involved in G1-phase regulation revealed a significant decrease in Cyclin E1 protein levels in a549 cells with increasing drug concentration (fig. 4).

TABLE 8 results of Compound 1 effect on A549 cell cycle

4. Effect of Compound 1 on migration of A549 cells

By Transwell ^TM The results of the cell migration experiments (fig. 5 and 6) showed that the cell number passing through the cell membrane was significantly smaller than that of the control group 24 hours after the a549 cells were treated with the compound 1, and the cell number showed a dose-dependent decrease with the increase of the drug concentration, and a very significant inhibitory effect was exhibited at 10 μm (P<0.05 Indicated that compound 1 can significantly inhibit the migration ability of a549 cells.

The experiment detects the apoptosis activity of the compound 1 induced A549 cells through an annexin V-FITC/PI double-staining method. The effect of 24h and 48h drugs on apoptosis was measured, respectively. As can be seen in fig. 7 and table 9, after treatment of a549 cells with different concentrations of compound 1 for 24h, the number of apoptosis increased with increasing drug concentration, and the ratio was increased. The apoptosis rate increased from 2.77% to 6.41% compared to the control group, and was dose dependent. After 48h of treatment of a549 cells with compound 1, the cells appeared to undergo different degrees of apoptosis. The apoptosis rate of A549 cells was 14.86% and 21.42% respectively at drug concentrations of 2.5. Mu.M and 5. Mu.M, with significantly higher ratios than the apoptosis rate of 4.85% for the control group. And also as the drug concentration increases, the rate of apoptosis increases gradually. Thus, it was found that compound 1 significantly induced apoptosis of a549 cells.

Table 9: compound 1 effect on a549 apoptosis results

6. Effect of Compound 1 on ROS Activity of A549 cells

In order to investigate the mechanism of action of compound 1 on a549 cells, the detection of active oxygen was performed using the fluorescent probe DCFH-DA, and the fluorescence intensity of DCF was detected using a flow cytometer, as shown in fig. 8, after compound 1 at different concentrations acted on a549 cells for 24 hours, the fluorescence intensity after drug addition tended to increase compared to the negative control NC, and therefore, this result showed that compound 1 could promote the production of intracellular active oxygen.

7. Effect of Compound 1 on A549 apoptosis-related protein Bcl-2 family proteins

The apoptosis channel regulatory proteins Bax and Bcl-2 are detected by Western Blot experiments. It can be seen from FIG. 9a that the expression level of Bax protein significantly increased with increasing concentration of compound after the A549 cells were treated with compound 1 for 72 hours, while the expression level of Bcl-2 protein also increased with increasing concentration of drug, it can be seen from FIG. 9b that the ratio of Bax/Bcl-2 expression level in the A549 cells significantly increased after the drug addition treatment, as compared with the control group, and the protein expression level ratio increased with increasing concentration of drug. When the drug concentration reached 20. Mu.M, the Bax/Bcl-2 expression level was increased by more than 6-fold compared to the control group. This result demonstrates that compound 1 can exert an apoptosis-inducing effect by regulating the expression of Bcl-2 and Bax.

8. Effect of Compound 1 on A549 cell Caspase family protein expression

As shown in FIG. 10, after the A549 cells are treated for 72 hours by adopting a Western Blot method, compared with a control group, the expression amounts of sheared Caspase-9 (clean-Caspase-9) and sheared Caspase-3 (clean-Caspase-3) are obviously increased, and the activities of the clean-Cas-9 and the clean-Caspase-3 are also higher along with the increase of the concentration of the compounds. This result shows that compound 1 can increase the activity of clear-Caspase-9 and clear-Caspase-3 in a549 cells via a dose-dependent pathway, thereby inducing apoptosis.

9. Effect of Compound 1 on the A549 cell AMPK-mTOR pathway protein

In the experiment, as shown in FIG. 11a, when the A549 cells are treated for 72h by using the compound 1, the activity of P-AMPK (Thr 172) is obviously increased compared with the control group, the activity of P-Raptor of the forward regulation protein of AMPK is obviously reduced, and the activities of P-P70-S6K, P-S6 (235/236) and P-S6 (240/244) of the downstream protein of mTOR are also obviously reduced (see FIG. 11 b), and the results show that the compound 1 can inhibit the growth of tumor cells by regulating the AMPK-mTOR channel.

10. Effect of Compound 1 on autophagy pathway proteins

The autophagy level was assessed by measuring the expression ratio of the 2 key proteins LC3II/LC 3I.

As shown in fig. 12a, the expression level of LC3I protein was decreased and the expression level of LC3II was increased when the a549 cells were treated with the compound for 72h, compared to the control group. See fig. 12b, the ratio LC3II/LC3I increases significantly compared to the control group, and the LC3II/LC3I expression showed a dose-dependent increase with increasing drug concentration. From this, it can be seen that compound 1 induced autophagic death of a549 cells, thereby apoptosis.

Experimental example 2 thermal Property test of inventive Compound 1

The thermal properties of the compound 1 prepared in example 1 were characterized by thermogravimetric analysis (Ther-mogravimetric Analysis, TGA) and Differential Scanning Calorimetry (DSC), the test procedure being carried out in air, heating from room temperature to 400℃at a rate of 10℃for a period of minutes ^-1 。

From the DSC plot (see fig. 24 b), it can be seen that the thermal behavior of the compounds consists essentially of 2 processes, endothermic and exothermic. The exothermic stage 132.31-144.03 ℃ and the peak temperature 137.35 ℃ have an exothermic amount of 11.70J/g, and the exothermic stage may be a weak exothermic peak formed in the sample recrystallization process. And two endothermic phases 186.31 ℃ to 199.29 ℃ and 198.29 ℃ to 206.78 ℃, peak temperature and endothermic quantity are 192.43 ℃, 22.11J/g and 202.05 ℃, 68.74J/g respectively, which are compound melting phases, the 2 melting peaks occur because the crystal is a crystal of alpha crystal form. The final endothermic stage 365.26-371.62 deg.C, peak temperature 368.33 deg.C and endothermic capacity 269.30J/g is the compound decomposition stage. The TGA profile of the compound (see fig. 24 a) shows a weight loss of 87.9% and the compound breaks down at a relatively high temperature, indicating that the compound is structurally stable.

Experimental example 3 Scanning Electron Microscope (SEM) of Compound 1 of the invention

Compound 1 prepared in example 1 was purified using methanol in a ratio of 10:1: water was prepared as a 1mg/mL solution and a 2mg/mL solution, respectively, and the supramolecular morphology of the samples was observed by SEM (see FIG. 25).

SEM images of the sample solution at 1mg/mL showed that compound 1 was present in a regular mini-disc shape at this concentration, well dispersed. And the SEM image with high resolution shows that the surface of the micro-disc is rough and has an obvious layered structure, which suggests that the compound may be a mesoporous material and can be used for preparing drug-carrying materials. And SEM images of 2mg/mL showed that at this concentration compound 1 showed significant agglomeration, the microdisks were connected together and the layered structure disappeared.

Experimental example 4 supramolecular Structure test of the Compounds and crystals of the invention

Crystals 1 and 2 of the compound 1 obtained in example 2 were tested by an X-ray single crystal diffraction method, and the results were as follows:

parameters of Crystal 1C ₁₅ H ₁₇ N ₅ O,Mr＝584.69g.mol ^-1 ；monoclinic,space g roup P 1 21/n 1；

α＝9 0°,β＝90.024(8)°,γ＝90°；

Z＝2；calc density＝1.292g·cm ^-3 ；F(0 00)＝620.0；T＝100K；R _int ＝0.0781；μ＝0.088mm ^-1 ；miller index ranges ,-7≤h≤7,-11≤k≤13,-29≤l≤29；θ _max ＝52.04°,θ _min ＝3.36°；T _min ＝0.541,T _max ＝0.745；11668reflections collected；2952independent reflec tions；Data/restraints/parameters:2952/0/206；Goodness-of-fit on F ² ＝1.010； R ₁ ＝0.0540；wR ₂ ＝0.1432；R indexes(all data)R ₁ ＝0.0767；wR ₂ ＝0.1561；Lar gest diff.peak/hole/e:/>

and/>

Structural analysis of Crystal 1: crystal 1 of compound 1 is monoclinic, P1/n space group crystal. Crystal 1 of the compound has only one pair of mirror enantiomers, 1-A1 and 1-A2 (as shown in FIG. 13 a), and is found by molecular conformational overlap, as shown in FIG. 13b, 2 phenol groups relative to the nitrogen-containing heterocyclic parent nucleus pyrazolo [3,4-d ] ]Pyrimidine is in para-crossed conformation, 1-A1 and 1-A2 are mirror image molecules which are not overlapped with each other, and the dihedral angle (C5-C7-C10-C15) tau of the corresponding 1-A1 ₁ τ of 1-A2 = 69.050 (305) ° ₂ ＝-69.050(305)°。

Since compound 1 contains a large number of N atoms and O atoms, hydrogen bonds are easily formed (see table 10), the crystal stability of the compound is mainly maintained by the hydrogen bonds. These hydrogen bonds allow the compounds to form a stable spatial three-dimensional network (see fig. 14 a). In order to fully understand the crystal, the complex hydrogen bond network of the crystal 1 is analyzed. Firstly, from the analysis of crystallographic a-axis direction, adjacent molecules have no direct interaction, and crystals are accumulated by solvent-guest molecule H contained in the crystals ₂ O atoms in O are used as H donor to form intermolecular hydrogen bond with phenol O12; pyrimidine exocyclic amino N6 as hydrogen bond donor and H ₂ O atoms in O form hydrogen bonds; c11 atom on phenol ring as donor H and H ₂ The O atoms in O form hydrogen bonds, these three types of hydrogen bonds, so that adjacent host molecules are connected into one-dimensional supramolecular chains extending along the crystallographic a-axis direction as shown in fig. 14 b. Next, as shown in fig. 14c, the molecular interactions along the crystallographic b-axis form hydrogen bonds with the N3 atom on the pyrimidine ring of the adjacent molecule mainly by phenol O12 as hydrogen bond donor. Finally, the stacking of molecules in the crystallographic C-axis direction relies on the adjacent two molecules of pyrimidine exocyclic amino N6 as hydrogen bond donors to form intermolecular hydrogen bonds with N3 atoms in the pyrimidine ring and C17 on tert-butyl groups as H donor to form C-H-pi interactions with phenol ring (see FIG. 14 d).

Table 10: hydrogen bonding parameters of Crystal 1

Symmetry codes:(i)x,-1+y,z(ii)-x,-y,1-z(iii)2-x,1-y,1-z(iv)1-x,-y,1-z

In crystal 1, the two conformational enantiomers 1-A1 and 1-A2 of the compound form a closed four-membered ring structure containing 2 solvent water molecules in the cavity in two types of hydrogen bonding modes (as shown in fig. 15 a), and the host molecule and the guest water molecule have stronger hydrogen bonding effect. Whereas for two adjacent four-membered rings there is no direct interaction between them as shown in fig. 15B, but rather a tubular stack is formed along the a-axis by interaction with guest water molecules in the nanocavity as a bridge through which intermolecular hydrogen bonds are formed with their host molecules (O2-H2B … O12, O1-H1a … O12, N6-H6a … O2, N6-H6B … O1, C11-H11 … O1, C11-H12 … O2).

After the local connection mode of the molecules was recognized, the overall arrangement mode of the crystal 1 was analyzed. As shown in FIG. 16, in the same layer, taking layer1 as an example, four rings of molecules 1-A11-A11-A21-A2 connected by hydrogen bonding form a stepped structure similar to a type II molecule along the crystallographic b axis (Leong WL, visual JJ. One-dimensional coordination polymers: complexity and diversity in structures, properties, and applications. Chemical reviews.2010; 111:688-764). The molecular step layers 1 and the adjacent molecular step layers 2 are arranged in parallel and stacked. Along the crystallographic C-axis direction, adjacent molecular steps layer1 and layer1' interact with weak intermolecular bonds C-H … pi bonds. The stack of such numerous molecular steps thus forms an elegant three-dimensional network.

Hirshfeld surface analysis and 2D fingerprint of Crystal 1 As shown in FIG. 17, in the dnorm surface plot of Crystal 1 (FIGS. 17a, b), dark red spots are attributed to intermolecular interactions of N-H … N and O-H … N, other visible spots on the surface are associated with guest water molecules and host molecular interactions in the crystal. In the 2D fingerprint (fig. 17 c), there are two pairs of spikes pointing to the bottom left of the graph, typical N … H and O … H hydrogen bonding interactions, which account for 14.5% and 8.8% of the overall Hirsfeld surface interactions, respectively. At the upper left and lower right corners of the fingerprint pattern, a characteristic symmetric "wing" shape appears, which is a C-h..pi. interaction that accounts for 16.3% of the total surface area of Hirshfeld. The sharp peak formed along the diagonal dispersion point in fig. 17c represents an H … H interaction which accounts for up to 55.5% of the total Hirshfeld surface area. It is shown that the crystals are stabilized mainly by the action of H … H. In contrast, the C … C effect accounted for only 0.7% of the total Hirshfeld surface area, indicating little pi-pi stacking effect in crystal 1.

Crystal 2 parameters: c (C) ₁₅ H ₁₇ N ₅ O,Mr＝283.33g.mol ^-1 ；monoclinic；space group P 1 21/n 1；

α＝90°,β＝ 113.35(3)°,γ＝90°；

Z＝8；calc density＝1.284g·cm ^-3 ； F(000)＝1200.0；T＝173K；R _int ＝0.0890；μ＝0.086mm ^-1 ；miller index ranges,-19≤h≤19,-13≤k≤13,-23≤l≤22；θ _max ＝52.698°,θ _min ＝2.908°； T _min ＝0.0674,T _max ＝0.745；23885reflections collected；5936independent reflections；Data/restraints/parameters:5936/0/390；Goodness-of-fit on F ² ＝0.987；R ₁ ＝0.0565；wR ₂ ＝0.1383；R indexes(all data)R ₁ ＝0.1249； wR ₂ ＝0.1746；Largest diff.peak/hole/e:/>

and/>

The present invention found that crystal 2 grown over 10 days and crystal 1 grown over 30 had the same crystal system and space group, but exhibited completely different molecular conformations and crystal arrangement. As shown in fig. 18d, the main molecule of crystal 1 and the molecule of crystal 2 are overlapped, and it is found that the main molecule frame of crystal 1 is completely different from crystal 2. And only one pair of enantiomers in crystal 1, and two different conformations 2-A1 and 2-B2,2 enantiomers in crystal 2 (see FIGS. 18a, B). As shown in FIGS. 18C and e, 2-A1,2-A2 and 2-B1,2-B2 are mirror image molecules, respectively, which do not overlap each other, and the dihedral angle (C5-C7-C10-C15) τ of the corresponding 2-A1 ₁ τ of 2-A2 = 45.620 (434) ° ₂ -45.620 (434) °, τ of 2-B1 ₃ τ of = 45.734 (445) ° and 2-B2 ₄ ＝-45. 734(445)°。

In crystal 2, there are 3 hydrogen bonding modes, forming 2 structural units. The first structural unit is a four-membered ring structure (FIG. 19 a), formed by intermolecular hydrogen bonding between enantiomers (O12-H12 … N3) and the same intermolecular hydrogen bonding interactions (N6-H6 … N3), in the same manner as the four-membered ring of crystal 1. Unlike crystal 1, there is also a structural unit similar to a four-membered ring in crystal 2 (see FIG. 19 b), which has only one hydrogen bonding means, and the hydrogen bond (O12 ' -H12' … N3 ') formed by the O atom of the 3-position N on the phenol group on the pyridine ring extends wirelessly in the b-axis direction. Unlike crystal 1 in the structural unit, the quaternary ring cavity of crystal 1 is occupied by solvent water molecules, and adjacent quaternary rings are in parallel stacked relationship, while adjacent quaternary rings and quasi-quaternary rings in crystal 2 are staggered (see fig. 19c, d), and the cavities are occupied by each other, because the molecular twist angle is not used in both crystals.

Table 11: hydrogen bonding parameters of Crystal 2

Symmetry codes:(i)x,1+y,z(ii)-x,-y,1-z(iii)x,-1+y,z

The relationship between the layers of the crystal 2 is shown in FIGS. 20a and b, although the four-membered rings of the same cross layer and the four-membered ring-like rings have no direct interaction, they depend on the interaction between the lower four-membered ring and the two C-H … pi bonds between the four-membered ring-like rings of the previous cross layer, one C-H … pi bond is shown in FIG. 20C, the tertiary butyl carbon on the four-membered ring-like ring and the pyridine ring in the four-membered ring structure act through the parallel array of C-H … pi bonds

Is a distance stack of (c). Another C-H … pi bond is characterized in that C14 of phenol ring with four-membered ring between upper and lower crossing layers is used as hydrogen donor and pyridine ring in the other four-membered ring passes through weak C-H … pi to ∈ ->

Distance interaction of (2)(see FIG. 20 d). These two C-H … pi actions allow infinite extension of the crystal molecules along the C-axis.

From the views 21a, b of crystal 2 along the c-axis of the crystal, it can be seen that crystal 2 is repeatedly stacked from the cross layers, forming a shape resembling a molecular ladder of IX (Leong WL, vittal JJ. One-dimensional coordination polymers: complexity and diversity in structures, properties, and applications. Chemical reviews 2010; 111:688-764).

For both crystals of the same compound 1, the presence of both diastereomers (Z '=2) in crystal 2, compared to the case where only one diastereomer (Z' =1) is present in crystal 1, indicates that crystal 1 is a thermodynamically controlled crystal product and crystal 2 is a kinetically controlled crystal product.

The crystal 2Hirshfeld surface analysis and 2D fingerprint are shown in FIG. 22, and the dark red spots in the dnorm surface (22 a, b) are the interactions of N on the pyrimidine ring with exocyclic amino N and the interactions of N on the pyrimidine ring with the hydroxyl group O of phenol. The 2D fingerprint (fig. 22C) shows that the force contribution between N … H is 17.5%, the force contribution between O … H is 3.9%, the force contribution between C … H is 16.2%, and the interaction contribution between H … H is 61.0% at the highest. Crystal 2 is also illustrated to maintain crystal stability by the interaction between H … H.

FIG. 23 shows the contributions of various types of intermolecular contacts in two crystals of Compound 1 to the Hirshfeld surface, by comparison of which it was found that the interaction between the two crystals was not very different, both contained the presence of hydrogen bonds such as H … H, N … H and O … H, the stability of the crystals was maintained mainly by the H … H interaction, and there was almost no pi-pi effect in the crystals, but some differences. The O … H duty cycle in crystal 1 is significantly increased compared to crystal 2 because crystal 1 is a hydrate and the presence of water provides an excess O … H duty cycle.

In conclusion, the compound 1 prepared by the invention has high-activity lung cancer resistance, can obviously inhibit the growth of A549 cells, has inhibition effect even better than that of a positive control medicament, and has good prospect in preparing medicaments for preventing and/or treating tumors. In addition, the compound 1 of the invention has good thermal stability, obvious layered structure and quite high porosity, and the structure has good application potential in the fields of mesoporous materials, drug carrying materials and artificial channel materials.

Claims (1)

1. A process for producing crystals of compound 1, characterized by:

the structure of the compound 1 is

The parameters of the crystal are as follows: c (C) ₁₅ H ₁₇ N ₅ O,Mr＝283.33g.mol ^-1 ；monoclinic；space group P 1 21/n 1；/>

α＝90°,β＝113.35(3)°,γ＝90°；/>

Z=8; the preparation method of the crystal comprises the following steps: adding the compound 1 into a mixed solution of methanol and water, dissolving, filtering, taking liquid, and crystallizing to obtain crystals; in the mixed solution of the methanol and the water, the volume ratio of the methanol to the water is 10:1, a step of; the mass volume ratio of the mixed solution of the compound 1, the methanol and the water is 25mg:8mL; the dissolution mode is that heating and dissolving are carried out at 60 ℃ until the solution is clear and transparent; the filtering is carried out while the filtering is hot; the crystallization mode is standing crystallization at room temperature, and the crystallization time is 10 days. />

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