WO2021022718A1 - 一种吡唑并嘧啶类化合物在制备预防/治疗肿瘤药物上的用途 - Google Patents

一种吡唑并嘧啶类化合物在制备预防/治疗肿瘤药物上的用途 Download PDF

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WO2021022718A1
WO2021022718A1 PCT/CN2019/119758 CN2019119758W WO2021022718A1 WO 2021022718 A1 WO2021022718 A1 WO 2021022718A1 CN 2019119758 W CN2019119758 W CN 2019119758W WO 2021022718 A1 WO2021022718 A1 WO 2021022718A1
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compound
cells
drug
crystal
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French (fr)
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何杨
李为民
马蓓蓓
黄日东
陈海
吴琼
周兴龙
柴莹莹
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四川大学华西医院
成都华西精准医学产业技术研究院有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to drug synthesis, and specifically relates to the use of a pyrazolopyrimidine compound in the preparation of a tumor prevention/treatment drug.
  • Lung cancer is the malignant tumor with the highest morbidity and mortality in the world, posing a great threat to human health. According to the biological characteristics, clinical treatment and prognosis of tumors, lung cancer is subdivided into two types: small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). NSCLC is one of the most common types of lung cancer. It is related to the increased production of epithelial cells, accounting for approximately 85% to 90% of lung cancer cases. Non-small cell lung cancer is divided into several subtypes, namely: lung adenocarcinoma, squamous cell carcinoma (SCC) and large cell lung cancer (LCLC).
  • SCC squamous cell carcinoma
  • LCLC large cell lung cancer
  • Adenocarcinoma or lung adenocarcinoma has obvious histological characteristics, with changes in its tissue cells, subatomic structure, and composition, accompanied by changes in organs, bronchi, and mucus.
  • Lung adenocarcinoma accounts for approximately 40% of all primary lung cancers. The growth and spread of malignant cells in lung adenocarcinoma is much slower than other subtypes of lung cancer, so it is easier to detect than other types of lung cancer.
  • SCC usually occurs in one of the left bronchus or the right bronchus. Smoking is the main cause of this type of lung cancer.
  • the clinical manifestations of SCC are generally dyspnea, chest pain and blood in sputum. SCC accounts for approximately 25-30% of primary lung cancers.
  • LCLC is a heterogeneous aggregate of undifferentiated threatening tumors. It does not have the morphological characteristics of small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, and it does not produce mucus. LCLC often originates from the central epithelial cells of the lung and spreads to distant organs. Many studies have shown that there is a close relationship between LCLC and smoking, accounting for about 5-10% of all lung cancers.
  • the drug is the same as other substances.
  • the intramolecular or intermolecular bonding changes, making the molecules or atoms in the crystal.
  • the arrangement in the lattice space changes to form different crystal structures, that is, the same substance has two or more spatial arrangements and unit cell parameters.
  • This phenomenon of different crystal structures is called polymorphism ( polymorphism).
  • different crystal forms will show different melting points and solubility, which will affect the bioavailability of the drug and the subsequent preparation process.
  • the dissolution rate of crystals of different shapes will be different, and the molecular groups on the exposed surface of different crystals will be different, resulting in different drug effects. Therefore, the crystals of the drug will affect the bioavailability, stability, dosage form selection, and efficacy of the drug.
  • Solvate refers to a crystalline substance formed by compound molecules and one or more solvent molecules in a certain combination. It is a ubiquitous form of compound. Solvate is a kind of polymorph, which plays an important role in the fields of medicine, polymer and energy. Especially in the field of medicine and its importance, when the drug is combined with a solvent to form a solvate, the properties exhibited are quite different from that of an unsolvate. For example: the volume, density, refractive index, hygroscopicity and solubility of molecules will be different. When the solubility of different solvates or unsolvates of drugs differs greatly, their bioavailability may differ greatly. For this kind of drugs, if the crystals in the preparation or storage process are not well controlled, the drug may not achieve the therapeutic effect due to the decrease in bioavailability, or the drug may be poisoned due to excessive dosage, which may cause medical accidents. .
  • the purpose of the present invention is to provide a use of pyrazolo[3,4-d]pyrimidine derivatives in the preparation of drugs for preventing and/or treating tumors, mesoporous materials, drug delivery systems, drug carriers, and artificial channels.
  • the present invention provides the use of Compound 1, or its solvate, or its crystal, or its salt in the preparation of drugs for preventing and/or treating tumors;
  • the tumor is lung cancer.
  • the tumor is non-small cell lung cancer.
  • the tumor is lung adenocarcinoma.
  • the drug can inhibit the proliferation, growth and migration of tumor cells, and induce tumor cell apoptosis.
  • the drug can regulate the level of Cyclin E1 protein in tumor cells, regulate the expression of Bcl-2 and Bax, regulate the activity of caspase, and regulate the expression of AMPK-mTOR pathway protein.
  • the drug can reduce the level of Cyclin E1 protein in tumor cells, increase the ratio of Bax/Bcl-2 expression, and increase the activities of Cleaved-Caspase-9 and Cleaved-Caspase-3.
  • the drug can promote the production of reactive oxygen species in tumor cells.
  • the drug can induce autophagic death of tumor cells.
  • the present invention also provides the use of compound 1, or its solvate, or its crystal, or its salt in the preparation of mesoporous materials, drug delivery systems, drug carriers, and artificial channels;
  • solvate is a hydrate of compound 1.
  • the method for preparing the crystals is: taking compound 1, adding it to a mixed solution of methanol and water, dissolving, filtering, taking the liquid, and crystallization to obtain crystals;
  • the volume ratio of methanol to water is 10:1; the mass-volume ratio of the compound 1, the mixed solution of methanol and water is 25mg:8mL; and the dissolution method is Dissolve by heating at 60° C. until it is clear and transparent; the filtering is filtering while it is hot; the crystallization method is static crystallization at room temperature, and the crystallization time is 10-30 days, preferably 10 days or 30 days.
  • CH... ⁇ interaction refers to the non-bonding weak interaction between the CH bond and the ⁇ system
  • the non-bonding weak interaction refers to the general term for various bonds other than covalent bonds, ionic bonds, and metal bonds
  • ⁇ system refers to the system that can form conjugated ⁇ bonds.
  • N-H...N hydrogen bonding refers to the hydrogen bonding between N-H bonds and N atoms.
  • O-H...N hydrogen bonding refers to the hydrogen bonding between O-H bonds and N atoms.
  • Non-porous three-dimensional cross structure refers to a highly cross-linked space structure with non-porous gaps formed by chemical bonding of compound crystal molecules.
  • porous three-dimensional network structure refers to a highly cross-linked spatial structure with pore-like gaps formed by chemical bonding of compound crystal molecules.
  • Enantiomers refer to stereoisomers that are real and mirror images of each other and are not superimposable.
  • Porous material refers to a type of porous material with a pore diameter of 2-50 nm.
  • DDS Drug Delivery Systems
  • Drug Delivery Systems refers to the different administration forms or dosage forms of various therapeutic drugs used in the prevention and treatment of diseases, including injections, tablets, capsules, patches, and aerosols Drugs, suppositories, osmotic pumps, transdermal patches, medicinal strips, implants, mucosal adhesives, etc.
  • Drug carrier refers to a system that can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ.
  • “Artificial channel” refers to a synthetic channel with a function similar to that of natural aquaporin.
  • Solvate refers to a crystalline substance formed by compound molecules and one or more solvent molecules in a certain combination. It is a ubiquitous form of compound. In the process of drug production, there are many processes that must involve solvents. In these processes, the compound will come into close contact with the solvent, and under certain conditions, corresponding solvates will be formed.
  • Hydrophilate is a kind of solvate, which refers to a crystalline substance formed by a certain combination of compound molecules and water molecules.
  • the compound 1 prepared by the present invention has a highly active anti-lung cancer effect, can significantly inhibit the growth of A549 cells, and the inhibitory effect is even better than the positive control drug, making it very useful in the preparation of drugs for preventing and/or treating tumors. Good prospects.
  • the compound 1 of the present invention also has good thermal stability, an obvious layered structure and a relatively high porosity. This structure makes it very useful in the preparation of mesoporous materials, drug-carrying materials, and artificial channel materials. Application potential.
  • Figure 1 Compound 1 inhibits the proliferation of A549 cells.
  • Figure 2 Visual morphology of A549 cells after treatment with different concentrations of Compound 1.
  • Figure 3 The effect of compound 1 on the cell cycle of A549-flow chart.
  • FIG. 4 Western blot analysis of G1 protein in A549 cells (72h).
  • FIG. 5 Transwell experiment detects the effect of compound 1 on the migration of A549 cells.
  • Figure 6 Compound 1 at different concentrations inhibits the migration of A549 cells *P ⁇ 0.05.
  • Figure 7 The effect of compound 1 on A549 cell apoptosis-flow chart.
  • Figure 8 Effect of compound 1 on ROS production in A549 cells.
  • Figure 9 a) the effect of compound 1 on the expression of Bcl-2 and Bax in A549 cells, b) the ratio of Bax/Bcl-2 expression after compound 1 treatment of A549 cells, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 10 The effect of compound 1 on Caspase family proteins in A549 cells.
  • Figure 11 Effect of compound 1 on AMPK-mTOR signaling pathway protein in A549 cells.
  • Figure 12 a) The effect of compound 1 on the autophagy pathway proteins LC3 I and LC3 II of A549 cells; b) the ratio of compound 1 on the autophagy pathway proteins LC3 II/LC3 I of A549 cells, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 13 Molecular conformation of crystal 1, crystal atom numbering and overlap diagram
  • (b) 1-A1 and 1-A2 overlap diagram Front view (left) and top view (right), where different colors represent different atoms: carbon atoms, gray; oxygen atoms, red; nitrogen atoms, blue; hydrogen atoms, white.
  • Figure 14 Molecular packing diagram and hydrogen bond network diagram of crystal 1 (hydrogen bonds are indicated by dashed lines, for clarity, the H atoms that do not form hydrogen bonds are deleted); a) molecular packing diagram along the a axis; b) along a Analytical diagram of the crystal hydrogen bond network in the direction; c) Analytical diagram of the hydrogen bond network along the b direction; d) Analytical diagram of the hydrogen bond network along the c direction.
  • Figure 15 a) View of a four-membered ring and hydrogen bond chain formed by water and molecules of compound 1; b) Intermolecular interaction of two adjacent four-membered rings with a one-dimensional water chain in the center (host molecule It is represented by sticks mode, and the guest molecule water is represented by ball and stick mode); c) a one-dimensional water chain four-membered ring space fiiling schematic diagram at the center; d) a perspective view of a four-membered ring with a water molecule array.
  • Figure 16 The layer-to-layer stacking mode in crystal 1 (the molecules are represented by space filling mode).
  • Figure 17 Hirshfeld surface analysis of crystal 1 a) dnorm surface view front view; b) dnorm surface view back view (white represents the force equivalent to the distance between van der Waals force between atoms; red represents a strong effect shorter than the distance of van der Waals force Force; blue indicates weaker force than van der Waals force), and c) 2D fingerprint pattern; d) dnorm surface map of specific intermolecular interaction force.
  • Figure 18 Molecular conformation, crystal atom numbering and overlapping diagram of crystal 2
  • Figure 19 a) Four-membered ring and hydrogen bond chain formed by compound 1; b) Four-membered ring structure; c) Interaction between adjacent four-membered ring structure and four-membered ring structure; d) Space filling mode of Figure c (The red is a four-membered ring, and the green is a four-membered ring).
  • Figure 20 Crystal 2 a) 2 cross-layer views along the crystallographic b-axis; b) space filling mode of Figure a); c) CH... ⁇ enlarged view of the four-membered ring and the four-membered ring between the crossed layers ; D) An enlarged view of CH... ⁇ between the four-membered rings of the cross layer.
  • Figure 21 The relationship between a) two cross-layer views along the c-axis in crystal 2; b) the space filling mode of Figure a).
  • Figure 22 Hirshfeld surface analysis and 2D fingerprint of crystal 2.
  • Figure 23 The ratio of the contact between atoms in the compound 1 crystal to the surface of Hirshfeld.
  • Figure 24 Thermal analysis results of compound 1: a) DSC curve of compound 1; b) TGA curve of compound 1.
  • Figure 25 The supramolecular morphology of Compound 1 in the solution state (upper left 1 mg/mL normal visual field image, lower left 1 mg/mL enlarged image; upper right 2 mg/mL normal visual field image, lower right 2 mg/mL enlarged image).
  • the raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.
  • N-bromosuccinimide (1.37g, 7.85mmol) was added to the 4-amino-1 tert-butyl-1H-pyrazolo[3,4-d]pyrimidine (1.00g, 5.23mmol) mmol) in 100 mL acetonitrile solution.
  • the reaction mixture was then stirred at 80°C for 4 hours.
  • the reaction mixture was cooled to room temperature, it was extracted with CH 2 Cl 2 (3 ⁇ 60 mL) and H 2 O (30 mL), the organic extracts were combined and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
  • Example 1 (1) Cultivate compound single crystals according to standard recrystallization operation methods. Specifically, the compound 1 (25 mg) prepared in Example 1 was dissolved in 8 ml of a mixed solvent of methanol: water (10:1). After stirring, the mixture was heated to saturation at 60°C until it was clear and transparent. The hot solution is filtered in time with a syringe filter. The solvent was then slowly evaporated at room temperature. On the 30th day, yellow granular crystals 1 were obtained.
  • step (2) Using the same method and the same solvent as in step (1), 30 days were changed to 10 days to obtain yellow granular crystals, ie, crystal 2 of compound 1.
  • Example 2 Weigh 28 mg of compound 1 powder prepared in Example 1, and dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 100 ⁇ M drug solution, filter it with a 0.22 ⁇ M sterile microporous membrane, and place it in- Store in a refrigerator at 20°C away from light. Dilute with RPMI-1640 culture medium to the required concentration before the experiment.
  • DMSO dimethyl sulfoxide
  • TTT tetramethylazazole blue
  • A549 cells are digested with trypsin-EDTA solution, they are collected in a 15ml centrifuge tube, and then centrifuged at 1000rpm for 5min;
  • Matrigel was operated on ice, diluted 1:8, and coated on the upper chamber surface of the bottom membrane of the Transwell chamber, and placed at 37°C for 30 minutes to polymerize Matrigel into a gel. Hydrate the basement membrane before use;
  • Apoptosis was detected by Annexin V-FITC/PI double staining method, the steps are as follows:
  • the compounding concentration is 5% concentrated glue (see Table 6)
  • ECL chemiluminescence luminescent liquid A and B are mixed in a ratio of 1:1, after mixing, put it at room temperature for 1 min until use;
  • the specific operation method is the same as that in 2.11, to detect the expression of AMPK-mTOR pathway protein.
  • the specific operation method is the same as that in 2.11, to detect the expression of autophagy pathway proteins.
  • MTT method detects the proliferation inhibitory effect of each compound 1 on A549 cells
  • Example 7 Take compound 1 synthesized in Example 1, select different concentrations (0, 3.125, 6.25, 12.5, 25, 50 ⁇ M) to test the proliferation inhibition of cells by MTT method, use PP1 as a positive control to evaluate cell viability, and select human lung Adenocarcinoma cells A549 cells were tested, and the experimental results after 72 hours of treatment are shown in Table 7.
  • compound 1 has a more obvious inhibitory effect on A549 cells, and the inhibitory effect is significantly enhanced with the increase of drug concentration (see Figure 1), indicating that compound 1 is anti-A549 cell growth and proliferation It is concentration dependent.
  • the IC 50 value of compound 1 was 2.12 ⁇ M after 72 hours of action on A549 cells, so the present invention selects 2.5 and 5 ⁇ M for morphological influence experiments. Different concentrations of compound 1 (0, 2.5 and 5 ⁇ M) were treated on A549 cells for 72h and observed under a 20X inverted microscope.
  • the PI staining method was used to detect the cell cycle to determine the drug's blocking effect on A549 cells, and further prove the drug's inhibitory effect on cell proliferation.
  • the results of 24h, 48h, and 72h (Table 8, Figure 3) all show that compared with the control group, the ratio of A549 cells treated with compound 1 in the G0/G1 phase increased significantly, and with the increase of the drug concentration, G0/G1 The proportion of cells in phase is also increasing, while the proportion of cells in the corresponding S phase and G2/M phase decreases, which indicates that the drug significantly blocks the cell cycle of A549 cells in the G0/G1 phase, and this blocking effect In a dose-dependent manner, all these changes resulted in cell proliferation inhibition. Further analysis of the related protein Cyclin E1 involved in the regulation of G1 phase, it was found that the level of Cyclin E1 protein in A549 cells decreased significantly with the increase of drug concentration (Figure 4).
  • the AnnexinV-FITC/PI double staining method was used to detect the apoptosis activity of compound 1 inducing A549 cells.
  • the effects of 24h and 48h drugs on cell apoptosis were measured respectively.
  • Figure 7 and Table 9 after 24 hours of treatment of A549 cells with different concentrations of Compound 1, as the drug concentration increases, the number of apoptosis also increases, and the proportion increases.
  • the apoptotic rate increased from 2.77% to 6.41%, and was dose-dependent.
  • the cells showed different degrees of apoptosis.
  • the present invention uses the Western Blot method to detect the expression of Caspase protein.
  • cleaved Caspase-9 (Cleaved-Caspase-9)
  • the expression of cleaved Caspase-3 (Cleaved-Caspase-3) increased significantly, and as the concentration of the compound increased, the activities of Cleaved-Caspase-9 and Cleaved-Caspase-3 also increased.
  • This result shows that compound 1 can increase the activities of Cleaved-Caspase-9 and Cleaved-Caspase-3 in A549 cells through a dose-dependent way, thereby inducing cell apoptosis.
  • This experiment evaluates the level of autophagy by detecting the expression ratio of the two key proteins LC3II/LC3I.
  • the present invention uses thermogravimetric analysis (Ther-mogravimetric Analysis, TGA) and differential scanning calorimetry (Differential s-canning calorimetry, DSC) to characterize the thermal properties of compound 1 prepared in Example 1, and the test process is carried out in air , Heating from room temperature to 400°C, heating rate is 10°C. min -1 .
  • TGA Ther-mogravimetric Analysis
  • DSC differential scanning calorimetry
  • the thermal behavior of the compound mainly includes two processes, endothermic and exothermic processes.
  • the exothermic phase is 132.31°C-144.03°C
  • the peak temperature is 137.35°C
  • the exothermic heat is 11.70J/g.
  • the exotherm at this stage may be a weak exothermic peak formed during the recrystallization of the sample.
  • the two endothermic stages are 186.31°C-199.29°C and 198.29°C-206.78°C, the peak temperature and endothermic heat are respectively 192.43°C, 22.11J/g and 202.05°C, 68.74J/g, these two endothermic stages are the melting of the compound At this stage, the two melting peaks appear because the crystal is an ⁇ crystal.
  • the last endothermic stage is 365.26°C-371.62°C, the peak temperature is 368.33°C, and the endothermic heat is 269.30J/g.
  • This stage is the compound decomposition stage.
  • the compound TGA chart shows a weight loss of 87.97%, and the compound decomposes at a relatively high temperature, indicating that the structure of the compound is relatively stable.
  • the compound 1 prepared in Example 1 was prepared into a 1 mg/mL solution and a 2 mg/mL solution with a methanol:water ratio of 10:1, and the supramolecular morphology of the sample was observed by SEM (see Figure 25).
  • the SEM image of the sample solution at 1 mg/mL shows that at this concentration, compound 1 exists in a regular micro-disc shape and is well dispersed. And the high-resolution SEM image shows that the surface of the microdisk is relatively rough and has an obvious layered structure, suggesting that the compound may be a mesoporous material and can be used to prepare drug-carrying materials.
  • the SEM image of 2 mg/mL showed that at this concentration compound 1 appeared obvious agglomeration, the microdisks were connected together, and the layered structure disappeared.
  • the crystal 1 of compound 1 is a monoclinic crystal system with a P 1 21/n space group crystal.
  • Compound crystal 1 only has a pair of mirror image enantiomers, 1-A1 and 1-A2 (as shown in Figure 13a). It is found through the molecular conformation overlap map, as shown in Figure 13b, that the two phenol groups are relative to the nitrogen-containing heterocyclic ring.
  • the parent core pyrazolo[3,4-d]pyrimidine is in a para-cross conformation.
  • the accumulation of crystals depends on the solvent contained in the crystal---the O atom in the guest molecule H 2 O as the H donor and phenol O12 to form an intermolecular Hydrogen bond; the amino N6 outside the pyrimidine ring acts as a hydrogen bond donor to form a hydrogen bond with the O atom in H 2 O; and the C11 atom on the phenol ring acts as a donor to form a hydrogen bond with the O atom in H 2 O.
  • These three types The hydrogen bonds of the adjacent host molecules are connected into a one-dimensional supramolecular chain extending along the crystallographic a-axis as shown in Figure 14b.
  • the interaction of molecules along the b-axis direction of crystallography mainly relies on phenol group O12 as a hydrogen bond donor to form a hydrogen bond with the N3 atom on the pyrimidine ring of the adjacent molecule.
  • the stacking of molecules in the c-axis direction of crystallography relies on two adjacent molecules with the exo-pyrimidine ring amino N6 as the hydrogen bond donor and the N3 atom in the pyrimidine ring to form intermolecular hydrogen bonds and the C17 on the tert-butyl group as the H donor It forms CH- ⁇ interaction with phenol ring (see Figure 14d).
  • the two conformational enantiomers 1-A1 and 1-A2 of the compound are connected by two types of hydrogen bonds to form a closed four-membered ring structure containing two solvent water molecules in the cavity ( Figure 15a), and there is a strong hydrogen bond between the host molecule and the guest water molecule.
  • Figure 15b For two adjacent four-membered rings, as shown in Figure 15b, there is no direct interaction between them. Instead, they form an intermolecular hydrogen bond with the host molecule through the guest water molecule in the nanocavity as a bridge.
  • the overall arrangement of crystal 1 is analyzed.
  • the four-membered ring formed by the four molecules 1-A11-A11-A21-A2 connected by hydrogen bonding forms along the crystallographic b-axis direction similar to Type II molecular ladder structure (Leong WL, Vittal JJ. One-dimensional coordination polymers: complexity and diversity in structures, properties, and applications. Chemical reviews. 2010; 111: 688-764).
  • the molecular ladder layer1 and the adjacent molecular ladder layer2 are arranged in a parallel stacking manner.
  • adjacent molecular steps layer1 and layer1' interact with weak intermolecular bonds C-H... ⁇ bonds. Therefore, the stack of countless molecular steps forms a beautiful three-dimensional network structure.
  • the Hirshfeld surface analysis and 2D fingerprint of crystal 1 are shown in Figure 17.
  • the dark red spots are attributed to the interaction of NH...N and OH...N between molecules.
  • Other visible spots on the surface are related to the interaction between guest water molecules and host molecules in the crystal.
  • the 2D fingerprint image Figure 17c
  • there are two pairs of sharp peaks pointing to the lower left of the image which are typical N...H and O...H hydrogen bonding interactions, which account for 14.5% and 8.8% of the entire Hirsfeld surface interaction, respectively.
  • a characteristic symmetrical "wing" shape appears in the upper left and lower right corners of the fingerprint.
  • the present invention found that although the crystal 2 cultivated after 10 days and the crystal 1 cultivated after 30 have the same crystal system and space group, they exhibit completely different molecular conformations and crystal arrangements. As shown in Figure 18d, when the host molecule of crystal 1 and the molecule of crystal 2 are overlapped, it is found that the framework of the host molecule of crystal 1 is completely different from that of crystal 2. And crystal 1 has only one pair of enantiomers, while crystal 2 has two different conformations 2-A1 and 2-B2, two pairs of enantiomers (see Figure 18a, b).
  • the first structural unit is a four-membered ring structure (as shown in Figure 19a), which interacts with hydrogen bonds between the same molecules (N6-H6...N3) through intermolecular hydrogen bonds between enantiomers (O12-H12...N3) ) Is formed in the same way as the four-membered ring of crystal 1.
  • the difference from crystal 1 is that crystal 2 also has a structural unit similar to a four-membered ring (as shown in Figure 19b), which has only one hydrogen bond connection mode, relying on the 3-position N on the pyridine ring and the O atom on the phenol group.
  • the formed hydrogen bonds extend wirelessly in the b-axis direction.
  • the structural unit differs from crystal 1 in that the four-membered ring cavity of crystal 1 is occupied by solvent water molecules, and adjacent four-membered rings are stacked in parallel, while the adjacent four-membered rings in crystal 2 are similar to four-membered rings.
  • the ring members are interlaced with each other (see Figure 19c, d), and the cavities are occupied by each other. This is due to the fact that the torsion angle of the molecules is not used in the two crystals.
  • FIG 20a,b The relationship between the two layers of the crystal is shown in Figure 20a,b. Although there is no direct interaction between the four-membered ring and the four-membered ring of the same cross layer, they rely on the four-membered ring of the lower layer and the four-membered ring of the previous cross layer. Two kinds of CH... ⁇ bonds interact. One type of CH... ⁇ bond is shown in Figure 20c. The tert-butyl carbon on the four-membered ring and the pyridine ring in the four-membered ring structure interact through the parallel array of CH... ⁇ The distance is stacked.
  • CH... ⁇ bond is a four-membered phenol ring between the upper and lower cross layers as the hydrogen donor and the pyridine ring in the other four-membered ring passes through weak CH... ⁇ to The distance interaction (see Figure 20d). These two CH... ⁇ effects make the crystal molecules extend infinitely along the c-axis.
  • crystal 2 is formed by repeatedly stacking intersecting layers, forming a shape similar to IX-type molecular ladder (Leong WL, Vittal JJ. One-dimensional coordination polymers: complexity and diversity in structures, properties, and applications. Chemical reviews. 2010; 111:688-764).
  • the surface analysis of crystal 2Hirshfeld and the 2D fingerprint spectrum are shown in Figure 22.
  • the dark red spots in the dnorm surface maps (22a,b) are the interaction between N on the pyrimidine ring and the exocyclic amino N and the N on the pyrimidine ring and the hydroxyl group of phenol O interacts.
  • the 2D fingerprint ( Figure 22c) shows that the contribution value of the force between N...H is 17.5%, the contribution value of the force between O...H is 3.9%, and the contribution value of the force between C...H is 16.2%, H ...The maximum contribution value of interaction between H is 61.0%. It shows that crystal 2 also maintains the stability of the crystal through the interaction between H...H.
  • Figure 23 shows the contribution of various types of intermolecular contacts in the two crystals of compound 1 to the Hirshfeld surface.
  • the interaction between the two crystals is not much different, and both contain H...H, N...H and With the existence of hydrogen bonds such as O...H, the stability of the crystal is mainly maintained by the H...H interaction, and there is almost no ⁇ - ⁇ interaction in the crystal, but there are some differences.
  • the proportion of O...H in crystal 1 has increased significantly. This is because crystal 1 is a hydrate, and the presence of water provides an excess proportion of O...H.
  • the compound 1 prepared by the present invention has a highly active anti-lung cancer effect, can significantly inhibit the growth of A549 cells, and the inhibitory effect is even better than the positive control drug, making it very useful in preparing drugs for preventing and/or treating tumors. Good prospects.
  • the compound 1 of the present invention also has good thermal stability, an obvious layered structure and a relatively high porosity. This structure makes it very useful in the preparation of mesoporous materials, drug-carrying materials, and artificial channel materials. Application potential.

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Abstract

化合物1、或其溶剂化物、或其晶体、或其盐在制备预防和/或治疗肿瘤的药物上的用途；还提供了化合物1、或其溶剂化物、或其晶体、或其盐在制备介孔材料、药物传递系统、药物载体、人工通道上的用途。实验证明，化合物1具有高活性的抗肺癌效果，可显著抑制A549细胞的生长，抑制效果甚至优于阳性对照药物，使其在制备预防和/或治疗肿瘤的药物上具有很好的前景。此外，化合物1还具有良好的热稳定性，具有明显的分层结构和颇高的孔隙率，这种结构使其在制备介孔材料、载药材料、人工通道材料领域具有很好的应用潜力。

Description

一种吡唑并嘧啶类化合物在制备预防/治疗肿瘤药物上的用途 技术领域
本发明属于药物合成，具体涉及一种吡唑并嘧啶类化合物在制备预防/治疗肿瘤药物上的用途。
背景技术
肺癌是全球范围内发病率和死亡率最高的恶性肿瘤，对人类健康造成了极大威胁。根据肿瘤的生物学特性、临床治疗及预后，肺癌被细分为两种类型：小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(Non-small-cell lung carcinoma，NSCLC)。NSCLC是一种最常见的肺癌，它与上皮细胞的产生增加有关，约占肺癌病例的85％～90％。非小细胞肺癌又被分为几种亚型，分别为：肺腺癌，肺鳞癌(squamous cell carcinoma,SCC)和肺大细胞癌(large cell lung cancer,LCLC)。腺癌或者肺腺癌具有明显的组织学特征，其组织细胞、亚原子结构以及组分发生了变化，并伴随有器官、支气管和黏液的变化。肺腺癌在所有原发性肺癌中大约占40％。肺腺癌中恶性细胞的生长和扩散速度比其他亚型肺癌要慢得多，因此比其他类型的肺癌更易检测。SCC通常发生在左支气管或者右支气管其中之一，吸烟是导致这种类型肺癌发生的主要原因。SCC的临床表现一般为呼吸困难，胸痛和痰中带血。SCC大约占原发性肺癌的25-30％。LCLC是未分化的威胁性肿瘤的异质聚集体，它不具有小细胞肺癌，肺腺癌和肺鳞癌的细胞形态学特征也不会产生黏液。LCLC常起源于肺的中央上皮细胞并扩散到远处器官。许多研究表明LCLC与吸烟之间存在密切关联，占所有肺癌的约5-10％。
近十年来，随着精准医学的快速发展，靶向治疗被用于肺癌的临床治疗，并取得显著效果，但靶向治疗耐药性突变以及部分突变基因没有对应的靶向药物是目前临床治疗面临的一个难以解决的问题。
此外，药物作为一种具有药理活性的物质，与其他物质一样，在结晶的过程中，由于受到不同物理化学条件的影响，分子内或者分子间的键合方式发生改变，使得分子或者原子在晶格空间内的排列方式发生变化，形成不同的晶体结构，即同一种物质具有两种或者两种以上的空间排列和晶胞参数，这种不同的晶体结构存在的现象称为多晶型现象(polymorphism)。对同一药物而言，不同晶体的形态会表现出不同的熔点和溶解度从而影响药物的生物利用度，以及后续的制剂工艺。不同形 状的晶体的溶解速率会不同，不同的晶体显露面的分子基团不同，导致药效会有所不同。所以，药物的晶体对药物生物利用度、稳定性、剂型选择，以及疗效等方面都会产生影响。
溶剂化物是指化合物分子与一种或多种溶剂分子以一定的结合形式共同形成的晶体物质，它是化合物的一种普遍存在形式。溶剂化物是属于多晶型的一种，它在医药、高分子及能源等领域都扮演重要角色。特别是在医药领域及其重要，当药物与溶剂一起结合形成溶剂化物后，与非溶剂化物相比所表现出的性质有很大的区别。例如：分子的体积、密度、折射率、吸湿性和溶解度等都会不同。当药物不同溶剂化物或非溶剂化物溶解度差异较大时，其生物利用度可能存在较大的差别。对于这类药物，如果不能很好的控制其在制备或者储存过程中的晶体，可能会因为生物利用度的降低而使药物达不到治疗效果，或因用量过多引起药物中毒，造成医疗事故。
因此，开发具有新型结构的有效低毒的肺癌治疗药物，并研究和掌握药物的不同晶体及其性质，是当前临床用药的迫切需求。
发明内容
本发明的目的在于提供一种吡唑并[3,4-d]嘧啶衍生物在制备预防和/或治疗肿瘤药物、介孔材料、药物传递系统、药物载体、人工通道上的用途。
本发明提供了化合物1、或其溶剂化物、或其晶体、或其盐在制备预防和/或治疗肿瘤的药物上的用途；
化合物1的结构为
Figure PCTCN2019119758-appb-000001
进一步地，所述肿瘤为肺癌。
进一步地，所述肿瘤为非小细胞肺癌。
进一步地，所述肿瘤为肺腺癌。
进一步地，所述药物能够抑制肿瘤细胞的增殖、生长、迁移，诱导肿瘤细胞的凋亡。
进一步地，所述药物能够调控肿瘤细胞中Cyclin E1蛋白水平，调控Bcl-2和Bax的表达，调控胱天蛋白酶的活性，调控AMPK-mTOR通路蛋白的表达。
优选地，所述药物能够降低肿瘤细胞中Cyclin E1蛋白水平，升高Bax/Bcl-2表达量的比值，提高Cleaved-Caspase-9和Cleaved-Caspase-3的活性。
进一步地，所述药物能够促进肿瘤细胞内活性氧的产生。
进一步地，所述药物够诱导肿瘤细胞的自噬性死亡。
本发明还提供了化合物1、或其溶剂化物、或其晶体、或其盐在制备介孔材料、药物传递系统、药物载体、人工通道上的用途；
化合物1的结构为
Figure PCTCN2019119758-appb-000002
进一步地，所述溶剂化物为化合物1的水合物。
进一步地，所述晶体的制备方法为：取化合物1，加入甲醇与水的混合溶液中，溶解，过滤，取液体，析晶，即得晶体；
优选地，所述甲醇与水的混合溶液中，甲醇与水的体积比为10：1；所述化合物1、甲醇与水的混合溶液的质量体积比为25mg：8mL；所述溶解的方式为在60℃下加热溶解至澄清透明；所述过滤为趁热过滤；所述析晶的方式为室温下静置析晶，析晶的时间为10～30天，优选为10天或30天。
本发明中，“C-H…π作用”是指C-H键与π体系之间的非键弱相互作用，非键弱相互作用是指除了共价键、离子键、金属键以外的各种键的总称，π体系指能形成共轭π键的体系。
“N-H…N氢键作用”是指N-H键与N原子之间的氢键作用。
“O-H…N氢键作用”是指O-H键与N原子之间的氢键作用。
“无孔三维交叉结构”是指化合物晶体分子通过化学键相键接形成的无孔状间隙的高度交联的空间结构。
“多孔三维网状结构”是指化合物晶体分子通过化学键相键接形成的具有孔状间隙的高度交联的空间结构。
“对映异构体”是指互为实物与镜像而不可重叠的立体异构体。
“介孔材料”是指孔径介于2-50nm的一类多孔材料。
“药物传递系统”即Drug Delivery Systems(DDS)，是指在防治疾病的过程中所采用的各种治疗药物的不同给药形式或剂型，包括注射剂、片剂、胶囊剂、贴片、气雾剂、栓剂、渗透泵、透皮贴片、药条、植入 剂、粘膜粘附剂等。
“药物载体”是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。
“人工通道”是指具有与天然水通道蛋白相似功能的合成通道。
“溶剂化物”是指化合物分子与一种或多种溶剂分子以一定的结合形式共同形成的晶体物质，它是化合物的一种普遍存在形式。在药物生产过程中，有着许多必须要溶剂参与的过程，在这些过程中，化合物会与溶剂发生紧密接触，在一定的条件下，会形成相应的溶剂化物。
“水合物”属于溶剂化物的一种，是指化合物分子与水分子以一定的结合形式共同形成的晶体物质。
实验证明，本发明制得的化合物1具有高活性的抗肺癌效果，可显著抑制A549细胞的生长，抑制效果甚至优于阳性对照药物，使其在制备预防和/或治疗肿瘤的药物上具有很好的前景。此外，本发明化合物1还具有良好的热稳定性，具有明显的分层结构和颇高的孔隙率，这种结构使其在制备介孔材料、载药材料、人工通道材料领域具有很好的应用潜力。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1：化合物1对A549细胞增殖抑制影响。
图2：不同浓度化合物1处理后A549细胞视觉形态。
图3：化合物1对A549细胞周期影响—流式图。
图4：A549细胞G1蛋白的免疫印迹分析(72h)。
图5：Transwell实验检测化合物1对A549细胞迁移的影响。
图6：不同浓度化合物1抑制A549细胞迁移情况*P<0.05。
图7：化合物1对A549细胞凋亡影响—流式图。
图8：化合物1对A549细胞中ROS产生的影响。
图9:a)化合物1对A549细胞Bcl-2和Bax表达的影响，b)化合物1处理A549细胞后Bax/Bcl-2表达比例，*P<0.05，**P<0.01，***P<0.001。
图10：化合物1对A549细胞Caspase家族蛋白的影响。
图11：化合物1对A549细胞AMPK-mTOR信号通路蛋白的影响。
图12：a)化合物1对A549细胞自噬通路蛋白LC3 I和LC3 II的影响；b)化合物1作用于A549细胞自噬通路蛋白LC3 II/LC3 I的比值，*P<0.05，**P<0.01，***P<0.001。
图13：晶体1的分子构象，晶体原子编号及重叠图(a)构象1-A1及原子编号(左)和对映异构体1-A2；(b)1-A1和1-A2重叠图正视图(左)和俯视图(右)，其中不同颜色代表不同的原子：碳原子，灰色；氧原子，红色；氮原子，蓝色；氢原子，白色。
图14：晶体1分子堆积图和氢键网络图(氢键用虚线表示，为了清晰起见，删除未形成氢键的H原子)；a)沿着a轴的分子堆积图；b)沿着a方向的晶体氢键网络解析图；c)沿着b方向的氢键网络解析图；d)沿着c方向的氢键网络解析图。
图15：a)由水和化合物1的分子形成的一个四元环和氢键链的视图；b)在中心具有一维水链两个相邻层四元环的分子间相互作用(主体分子用sticks模式表示，客体分子水用ball and stick模式表示)；c)在中心有一维水链四元环space fiiling示意图；d)具有水分子阵列的四元环的透视图。
图16：晶体1中层与层之间的堆积方式(分子用space filling模式表示)。
图17：晶体1的Hirshfeld表面分析的a)dnorm表面图正视图；b)dnorm表面图后视图(白色表示等价于范德华力原子间距离的作用力；红色表示短于范德华力距离的强作用力；蓝色表示长于范德华力弱作用力)，和c)2D指纹图谱；d)特定分子间相互作用力的dnorm表面图。
图18：晶体2的分子构象，晶体原子编号及重叠图(a)构象2-A1及原子编号(左)和对映异构体2-A2；(b)构象2-B1原子编号及对映异构体2-B2；c)2-A1和2-A2重叠图；d)1-A1,2-A1和2-B1的重叠图(绿色表示1-A1,黄色表示2-B1，品红表示2-B1)；e)2-B1和2-B2的重叠图。
图19：a)化合物1形成的四元环及氢键链；b)类四元环结构；c)相邻四元环结构与类四元环结构相互作用；d)图c的space filling模式(其中红色为四元环，绿色为类四元环)。
图20：晶体2中a)沿着晶体学b轴方向2个交叉层视图；b)图a)的space filling模式；c)交叉层间四元环与类四元环的C-H…π放大图；d)交叉层类四元环间C-H…π放大图。
图21：晶体2中a)沿c轴的两个交叉层视图的关系.b)图a)的space  filling模式。
图22：晶体2的Hirshfeld表面分析及2D指纹图。
图23：化合物1晶体中各原子间接触占Hirshfeld表面比例。
图24：化合物1热分析结果：a)化合物1的DSC曲线；b)化合物1的TGA曲线。
图25：溶液状态下化合物1的超分子形态(左上1mg/mL正常视野图，左下1mg/mL放大图；右上2mg/mL正常视野图，右下2mg/mL放大图)。
具体实施方式
本发明所用原料与设备均为已知产品，通过购买市售产品所得。
1、实验试剂及设备
下述实施例部分的实验试剂来源如表1所示：
表1：实验试剂
Figure PCTCN2019119758-appb-000003
Figure PCTCN2019119758-appb-000004
下述实施例部分所使用的主要实验设备和分析测试仪器如表2：
表2：化学实验仪器设备
Figure PCTCN2019119758-appb-000005
2、合成方法
实施例1、本发明化合物1的合成
(1)合成5-氨基-4-氰基-1-叔丁基-1H-吡唑(12)：
Figure PCTCN2019119758-appb-000006
在室温下，将叔丁基肼11(0.72g，8.19mmol)和三乙胺(1.70mL，12.29mmol)加入含有20mL无水乙醇的50mL圆底烧瓶中，然后向其中缓慢滴入乙氧基亚甲基丙二腈10(1.00g，8.19mmol)。将反应混合物在78℃下加热3小时。然后将反应溶液冷却至室温并旋干，得到粘稠的橙色固体。随后向其中加入水(30mL)并用CH 2Cl 2(3×60mL)萃取反应。合并有机相用无水硫酸钠干燥，然后减压蒸发溶剂,进行浓缩。获得快速固化的橙黄色胶。将残余物用10％EtOAc的己烷溶液(60mL)分层，并将混合物超声处理。过滤所得结晶固体，用大量10％EtOAc的己烷溶液洗涤并干燥，最终得到浅橙色晶体5-氨基-4-氰基-1-叔丁基-1H-吡唑1.29g,产率为96.3％。 1H NMR(600MHz,DMSO-d 6)δ7.45(s,1H),6.22(s,2H),1.50(s,9H)。 13C NMR(150M,DMSO-d 6)δ150.71,138.01,115.24,74.55,57.76,28.23。
(2)合成4-氨基-1-叔丁基-1H-吡唑并[3,4-d]嘧啶(13)：
Figure PCTCN2019119758-appb-000007
在氮气中，将5-氨基-4-氰基-1-叔丁基-1H-吡唑12(1.00g，6.09mmol)和甲酰胺(15ml)混合物在190℃下加热6小时。用CH 2Cl 2(3×60ml)和H 2O(30ml)萃取混合物。然后合并有机层用无水硫酸钠干燥并真空蒸发。通过硅胶柱色谱法(洗脱：0％～50％CH 2Cl 2/CH 3OH)纯化反应混合物，得到产物13，为白色固体1.16g，产率为55.8％。 1H NMR(600MHz,DMSO-d 6)δ8.14(s,1H),8.03(s,1H),7.58(s,2H),1.69(s,9H)。 13C NMR(150M,DMSO-d 6)δ158.16,154.72,152.72,130.04,101.43,59.23,28.78。
(3)合成4-氨基-3-溴-1-叔丁基-1H-吡唑并[3,4-d]嘧啶(14)：
Figure PCTCN2019119758-appb-000008
在室温下，将N-溴代琥珀酰亚胺(1.37g，7.85mmol)加入到含有4-氨基-1叔丁基-1H-吡唑并[3,4-d]嘧啶(1.00g，5.23mmol)的100mL乙腈溶液中。然后将反应混合物在80℃下搅拌4小时。待反应混合液冷却到室温，用CH 2Cl 2(3×60mL)和H 2O(30mL)萃取，合并有机萃取相后用无水硫酸钠干燥，减压蒸发除去溶剂。随后通过硅胶色谱法梯度洗脱(洗脱剂：CH 2Cl 2)纯化残余物，合并所需的级分，并真空蒸发，得到所需的黄色固体产物0.91g，产率为64.8％。 1H NMR(600M,DMSO-d 6)δ8.20(s,1H),1.67(s,9H)。 13C NMR(150M,DMSO-d 6)δ157.51,155.64,153.52,115.29,100.46,60.61,28.61。
(4)合成3-(4-氨基-1-(叔丁基)-1H-吡唑并[3,4-d]嘧啶-3-基)苯酚(1)：
Figure PCTCN2019119758-appb-000009
将3-羟基苯基硼酸频哪醇酯(0.40g，1.79mmol)和前面反应得到的4-氨基-3-溴-1-叔丁基-1H-吡唑并[3,4-d]嘧啶13(0.40g，1.49mmol)溶于装有1,4-dioxane/H 2O(4：1,25mL)溶剂的100mL圆底烧瓶中。在室温下依次加入K 2CO 3(0.41g，2.98mmol)和PdCl 2dppf(0.11g，0.15mmol)，反应混合物在100℃下搅拌8h。然后将反应液冷却并真空蒸发，使用二氯甲烷作为溶剂将残余物质粘附到硅胶上。进行硅胶柱色谱纯化(洗脱：0％～1％CH 2Cl 2:CH 3OH)并真空蒸发所需级分，得到所需的灰色固体终产物(化合物1)0.30g，产率71.1％。 1H NMR(600M,DMSO-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)。 13C NMR(150M DMSO-d 6)δ158.12,154.59,153.77,141,69,134.48,130.22,118.93,115.62,115.03,98.55,59.62,28.75。HRMS-ESI(m/z)calcd for[M+H] +,284.1433；found,284.1505。
实施例2、本发明化合物1的晶体1和晶体2的制备
(1)按标准重结晶操作方法培养化合物单晶。具体为：将实施例1制得的化合物1(25mg)溶解于8ml甲醇：水(10:1)的混合溶剂中。搅拌后混合物在60℃加热至饱和，直至澄清透明。热溶液及时用注射器过滤器过滤。随后溶剂在室温下缓慢蒸发。于第30天获得了黄色的颗粒状晶体1。
(2)采用与步骤(1)相同的方法和相同的溶剂，将30天改变为10天，获得了黄色颗粒状晶体，即化合物1的晶体2。
以下通过实验例证明本发明化合物的有益效果。
实验例1、本发明化合物抗肿瘤活性评价
一、实验方法
1、实验试剂与仪器
下述实验例中所使用的主要试剂如表3：
表3：实验试剂
Figure PCTCN2019119758-appb-000010
Figure PCTCN2019119758-appb-000011
下述实验例中所使用的主要实验设备如表4：
表4：实验仪器设备
Figure PCTCN2019119758-appb-000012
2、实验方法
2..1溶液的配制
(1)化合物1溶液的配制
称取实施例1制得的化合物1粉末28mg,溶于1mL的二甲亚砜(DMSO)中，配制成100μM的药物溶液，用0.22μM无菌微孔滤膜过滤，分装后置于-20℃冰箱避光保存。实验前用RPMI-1640培养液稀释至所需浓度。
(2)RPMI-1640完全培养配制
配制RPMI-1640+10％FBS+1％PS培养基：5ml FBS(血清)+0.5ml PS(双抗)+44.5ml RPMI-1640，储存于4℃冰箱。
(3)MTT溶液的配制
称取0.5g的四甲基偶氮唑蓝(MTT)粉末，使之在PBS溶液中充分溶解，调节其终浓度为5mg·mL -1，经过0.22μM的无菌微孔滤膜过滤后，放于4℃避光保存。
(4)电泳缓冲液的配制
称取Glycine 93.85g、Tris 15.15g以及SDS 5g使充分溶解在700mL双蒸水中，随后加双蒸水定容至1000mL,使用时用双蒸水稀释5倍。
(5)10×TBS溶液的配制方法
称取Tris碱24.2g,NaCl 80g,用稀盐酸调PH至7.6，双蒸水定容至1L。
(6)1×TBST溶液的配制方法
将0.5mL Tween-20、100mL 10×TBS和900mL双蒸水混合均匀,室温保存
(7)封闭液5％脱脂奶粉的配制
称取5g脱脂奶粉溶于100mL TBST中搅拌至充分溶解，即为5％的脱脂奶粉(W/V)的TBST溶液。
(8)BCA溶液的配制
称取5g BSA在100mL TBST中充分溶解，即为5％BSA(W/V)的TBST溶液。
2..2细胞复苏
(1)将恒温水浴箱加热至37℃，75％酒精擦拭超净工作台的台面，并开启紫外线灯照射30min；
(2)配置RPMI-1640完全培养基15ml，加入到15ml离心管中；
(3)将A549细胞冻存管从液氮中取出，立即放于37℃水浴中，快速并轻轻摇晃，将细胞解冻；
(4)轻轻吹打混匀，用吸管将悬液缓慢滴入含完全培养基的15ml离心管内；
(5)用离心机在20℃，1000rpm转速下离心3min，弃去上清液；
(6)向离心管中加入1ml配置好的RPMI-1640完全培养基中重悬细胞，转移悬液到加入4ml培养基的25cm 2细胞培养瓶内，十字法摇晃混匀；
(7)将培养瓶放置在5％CO 2，37℃的培养箱中培养。
2.3细胞传代
(1)入无菌室之前用肥皂洗手，再用75％的酒精擦拭消毒双手；
(2)倒置显微镜下观察细胞形态，确定A549细胞是否传代及细胞需要稀释的倍数，将培养基、胰酶等37℃下预热；(3)75％酒精擦拭超净工作台的台面；
(4)打开超净工作台的紫外灯照射台面20min左右，关闭紫外灯，打开风机清洁空气除去臭氧；
(5)用移液枪吸去培养瓶中旧培养基，酌情可用2-3ml Hanks液洗去残留的就培养基，或用少量胰酶刷洗一下；
(6)培养瓶中加入胰蛋白酶-EDTA溶液1mL，摇匀后平铺，使其充分覆盖瓶底，置恒温培养箱消化1min；
(7)在倒置显微镜下观察，当细胞收回突起变圆时立即翻转培养瓶，使细胞脱离胰酶，然后将胰酶倒掉；
(8)加入少量的含血清的新鲜培养基终止消化，反复吹打消化好的细胞使其脱壁并分散，1000rpm离心5min,弃掉上清液；
(9)再根据分传瓶数加一定量的含血清的新鲜培养基重悬细胞制成细胞悬液，然后分装到新培养瓶中；
(10)盖好瓶盖，适度拧紧后稍回转，以利于CO 2的进入，将培养瓶放回5％CO 2，37℃的培养箱；
(11)取生长良好的A549细胞做实验。
2.4细胞冻存
(1)冻存前一天更换细胞完全培养基，收集对数生长期的A549细胞；
(2)取一离心管，加入RPMI-1640培养基、胎牛血清和10％二甲基亚砜DMSO，配置细胞冻存液，使他们的比例为7:2:1，置于室温待用；
(3)A549细胞加入胰蛋白酶-EDTA溶液消化后，收集至15ml离心管中，随后以1000rpm离心5min；
(4)弃去上清液，加入配置好的细胞冻存液，轻轻吹至细胞重悬；
(5)将细胞悬液分装至细胞冻存管中，每管1mL～1.5mL,旋紧管口，贴上封口膜，做好冻存记录；
(6)将细胞冻存管置于4℃10min→-20℃30min→-80℃16～18h(或过夜)→转至液氮罐中长期保存。
2.5细胞增殖抑制实验(MTT法)
(1)取对数生长期的A549细胞，用0.25％的胰蛋白酶消化后，收集细胞，调整细胞浓度至1×10 5cell.mL -1，将细胞接种于96孔板中，每孔100μL，同时设置空白对照孔，置于37℃，5％CO 2的饱和湿度的培养箱中培养24h；
(2)更换含有不同浓度化合物1(0、3.125、6.25、12.5、25、50μM)的完全培养基，每个浓度设置3个复孔，继续培养72h；
(3)每孔加入10μL的MTT溶液，继续于培养箱培养4h；
(4)吸弃上清液后，每孔加入100μL的DMSO溶液，于摇床上低速震荡10min，使结晶产物完全溶解；
(5)在酶标仪检测490nm波长下吸光度值，计算细胞活力；
(6)实验重复3次，取平均值。
2.6细胞生长状态实验
(1)取对数生长期的A549细胞，经0.25％胰蛋白酶消化后，收集细胞，调节细胞浓度至2x10 5cell·mL -1，加入6孔板中，每孔加2mL；
(2)于培养箱中培养24h，加入不同浓度化合物1(0、2.5、5μM)；
(3)继续放置在培养箱中培养72h，于倒置显微镜下观察经各浓度化合物1作用后细胞生长及形态变化。
2.7细胞周期实验
(1)取对数生长期的A549细胞，每孔接种1×10 5cell·mL -1接种于6孔板中，每孔加2mL完全培养基,每组设置三个副孔,置于37℃，5％CO 2的饱和湿度的培养箱中培养24h；
(2)更换含有不同浓度化合物1(0、2.5、5μM)的完全培养基，继续培养；
(3)分别培养24h、48h和72h后，0.25％的胰蛋白酶溶液消化细胞，加预冷的磷酸盐缓冲液PBS，以1000rpm离心3min，洗涤细胞两遍；
(4)逐滴加入70％乙醇溶液，4℃避光过夜固定；
(5)细胞固定后，细胞在2000rpm，4℃下离心5min，弃掉上清液，再用PBS洗涤两次；
(6)加入500uL PBS含50ug/mL溴化乙锭(PI)，100ug/mL RNase A，0.2％Triton X-100，，37℃避光孵育30分钟；
(7)用流式细胞仪检测，每组样品检测2万个细胞，利用FlowJo软件获取数据并分析细胞周期分布。
2.8 Transwell细胞迁移实验
(1)将BD公司的Matrigel 4℃过夜冻融，100μL枪头冷藏备用；
(2)实验开始后Matrigel保持在冰上操作，1：8稀释，包被Transwell小室底部膜的上室面，置37℃30min使Matrigel聚合成凝胶。使用前进行基底膜水化；
(3)用0.25％的胰蛋白酶消化A549细胞后，倒掉培养液，PBS溶液清洗2次，用含BSA的无血清培养基重悬，调整细胞密度至5x10 5/mL；
(4)取细胞悬液100μL加入Transwell小室，加入不同浓度化合物1(0、2.5、5、10μM)的处理A549细胞，同时设置空白对照组，每个浓度设置三个复孔；
(5)在24孔板下室加入600μL含20％FBS的培养基，放入37℃，5％CO 2培养箱中培养；
(6)培养24h后，取出Transwell小室，弃去孔中的培养液，用无菌PBS洗两遍，甲醇固定30min,将小室适当风干；
(7)Giemsa染色15min,用棉签轻轻擦掉上层未迁移细胞，用PBS洗三遍；
(8)放在20X的倒置显微镜下随机选择五个视野观察细胞，拍照。
2.9细胞凋亡实验
细胞凋亡采用Annexin V-FITC/PI双染法检测，步骤如下：
(1)取对数生长的A549细胞，计数，按5×10 5cell·mL -1种于6孔板中培养；
(2)更换含有不同浓度化合物1(0、2.5、5μM)的完全培养基，继续培养24h和48h；
(3)把细胞培养液分别吸出至15ml离心管内，用胰蛋白酶消化细胞后，用预冷的PBS洗涤3次，调节细胞浓度至1×10 6cell·mL -1，2000rmp离心5min，吸弃PBS；
(4)每组加入400μL 1X Annexin V结合液重悬细胞，再加入5μL Annexin V-FITC染色液，轻轻混匀后于室温避光条件下孵育5min；
(5)再加入10μL PI染色液上机检测
(6)用流式细胞仪检测，每组样品检测2万个细胞，利用Cell Quest软件获取数据并分析细胞凋亡情况。
2.10 DCFH-DA探针检测活性氧实验
(1)取对数生长期的A549细胞，经0.25％胰蛋白酶消化后，收集细胞，调节细胞浓度至1×10 5cell·mL -1，加入6孔板中，每孔2mL，培养24h；
(2)加入浓度分别为0、2.5、5和10μM的化合物1，作用24h；
(3)按1:1000的比例用无血清培养液将DCFH-DA浓度稀释为10μmol·L -1待用；
(4)胰酶消化收集细胞后，用预冷的PBS洗两次，用1mL的DCFH-DA重悬细胞；
(5)放置在培养箱中避光反应20min，每隔5min混匀一次，使探针和细胞充分接触；
(6)用无血清培养液洗涤细胞三次后，再用1mL重悬细胞；
(7)用流式细胞仪检测平均荧光强度。
2.11 Western Blot法分析化合物1对凋亡相关蛋白表达影响实验
2.11.1细胞总蛋白的提取
(1)取对数生长期的A549细胞接种于6孔板，调节细胞浓度为1x10 5cell/mL -1,放置培养箱中培养24h；
(2)加入浓度分别为0、5、10和20μM的化合物1，作用72h；
(3)胰酶消化后1000rmp离心5min收集细胞，用预冷的PBS洗两次，弃去上清液；
(4)每组细胞加入100μL细胞裂解液，于冰上裂解30min，然后12000rpm 4℃离心10min，小心吸取上清液，-20℃保存。
2.11.2 BCA法测定蛋白质浓度
(1)依次向96孔培养板中加入BSA标准品(0.5mg/mL)0，1，2，4，8，12，16，20uL，然后用预冷的PBS补足总体积至20uL；
(2)将待测样品稀释20倍，每孔20μL加入96孔板中；
(3)加入BCA工作液200uL/孔置于恒温箱中培养30min，冷却至室温；
(4)用酶标仪于562nm处测定各孔吸光度，用水较零；
(5)绘制蛋白标准曲线，并计算待测样品的蛋白浓度。
2.11.3 SDS-PAGE蛋白电泳
(1)配制浓度为12％的分离胶(见表5)
(2)向两块玻璃板中间注入分离胶，避免产生气泡，灌注分离胶至梳子下边缘1cm，轻柔加入双蒸水进行水封；
(3)配制浓度为5％浓缩胶(见表6)
(4)灌注好分离胶后，室温放置30min，待分离胶聚合完全，缓缓倒出上层的双蒸水，用滤纸条吸净残留的双蒸水；
(5)将浓缩胶迅速注入至玻璃板顶，插入梳子以防产生气泡，室温静置30min后待用；
(6)取分装好的细胞总蛋白或浆蛋白，加入5×上样缓冲液5uL，100℃金属浴10min使蛋白变性，离心上样；
(7)于蛋白样品两侧孔中加入4μL预染蛋白marker；
(8)开启电泳仪，电泳分离出所需条带，可终止电泳。
表5：12％分离胶配制(15mL)
Figure PCTCN2019119758-appb-000013
表6：5％的浓缩胶配制(4mL)
Figure PCTCN2019119758-appb-000014
2.11.4转膜
(1)将适当大小的PVDF膜浸泡于甲醇中约30s，然后再放入蒸馏水中浸泡2min，之后转移至电转液；
(2)制作海绵垫-滤纸-分离胶-PVDF膜滤纸-海绵垫“三明治，”放入转膜槽中；
(3)倒入转移缓冲液，放入冷却装置；
(4)于恒压60V的条件下转移100min。转膜结束后，将PVDF膜取出，标记正反面和标准分子量参照蛋白的位置。
2.11.5封闭、一抗孵育、二抗孵育
(1)转膜成功的膜放入配置好的5％脱脂奶粉中，置于封闭液中室温封闭1h左右；封闭后用TBST；
(2)一抗Cleaved-caspase-9、Cleaved-caspase-3、Bax和Bcl-2用5％的脱脂奶粉稀释，4℃孵育过夜，β-actin抗体作为内参；
(3)1×TBST洗膜3次，每次5min，5％的脱脂奶粉稀释二抗，室温孵育1h,最后用1×TBST洗膜3次，每次15min。
2.11.6 ECL显影
(1)ECL化学荧光发光液A和B按1:1的比例混合，混匀后，室温放置1min待用；
(2)将混合好的ECL试剂加到PVDF膜上(1mL/10cm 2)，室温反应1min，化学发光得到条带；
(3)凝胶成像系统拍照。
2.12 Western Blot法分析化合物1对AMPK-mTOR通路蛋白表达影响实验
具体操作方法同2.11，检测AMPK-mTOR通路蛋白的表达。
2.13 Western Blot法分析化合物1对自噬通路蛋白表达影响实验
具体操作方法同2.11，检测自噬通路蛋白的表达。
2.14统计分析
采用Graphpad Prism进行统计分析，组间差异用T检验吗，结果用
Figure PCTCN2019119758-appb-000015
表示，检验结果P<0.05为差异具有统计学意义。
二、实验结果
1、MTT法检测各化合物1对A549细胞的增值抑制作用
取实施例1合成的化合物1，选择不同浓度(0、3.125、6.25、12.5、25、50μM)用MTT法测试细胞的增殖抑制，以PP1作为阳性对照物，对细胞活性进行评价，选择人肺腺癌细胞A549细胞进行实验，作用72h后的实验结果如表7所示。
表7：吡唑并[3,4-d]嘧啶衍生物对A549细胞抑制活性
Figure PCTCN2019119758-appb-000016
从实验结果可以看出，与阳性对照相比，化合物1对A549细胞有更加明显的抑制作用，且随着药物浓度的增高抑制作用明显增强(见图1)，说明化合物1抗A549细胞生长增殖呈浓度依赖性。
2、化合物1对A549细胞生长状态的影响
根据上述MTT实验结果，化合物1作用于A549细胞72h后IC 50值为2.12μM，因此本发明选取2.5、5μM这两个浓度进行形态学影响实验。不同浓度的化合物1(0、2.5和5μM)作用A549细胞72h后，置于20X倒置显微镜下观察。
结果如图2所示，对照组的细胞连接紧密且大小均匀，呈现正常A549细胞的多角形形态。随着化合物1浓度的增加，细胞相互分散，皱缩成圆形，呈现出细胞凋亡相关的表型，再次证明化合物1能够有效抑制A549细胞生长。
3、化合物1对A549细胞周期的影响
采用PI染法检测细胞周期，确定药物对A549细胞的阻滞作用，进一步证明药物对细胞增殖的抑制作用。24h、48h、72h的结果(表8，图3)都显示与对照组相比，化合物1处理后的A549细胞在G0/G1期的比例显著增高，且随着药物浓度的增加，G0/G1期的细胞所占的比例也在增加，而相应的S期和G2/M期的细胞比例下降，这表明药物将A549细胞的细胞周期明显阻滞在G0/G1期，且这种阻滞作用呈药物剂量依赖性，所有这些变化都导致了细胞增殖抑制。进一步分析参与G1期调控的相关蛋白Cyclin E1,发现随着药物浓度的增加A549细胞中Cyclin E1蛋白水平明显下降(图4)。
表8:化合物1对A549细胞周期影响结果
Figure PCTCN2019119758-appb-000017
4、化合物1对A549细胞迁移的影响
通过Transwell TM细胞迁移实验结果(图5、图6)显示，加入化合物1处理A549细胞24小时后，穿过小室膜的细胞数明显少于对照组，且随着药物浓度的增加，细胞数呈现剂量依赖性的减少，在10μM的时候表现出了非常明显的抑制效果(P<0.05)，表明化合物1可以明显抑制A549细胞的迁移能力。
本实验通过AnnexinV-FITC/PI双染法检测化合物1诱导A549细胞凋亡活性。分别测定了24h和48h药物对细胞凋亡的影响。如图7和表9可见，用不同浓度的化合物1处理A549细胞24h后，随着药物浓度的增加，细胞凋亡数也在增加，所占比值增大。与对照组相比凋亡率从2.77％上升至6.41％，且呈剂量依赖关系。当用化合物1处理A549细胞48h后，细胞出现不同程度的凋亡。当药物浓度为2.5μM和5μM时，A549细胞凋亡率分别为14.86％和21.42％，与对照组的凋亡率4.85％相比，比例显著增高。并且也是随着药物浓度增加，细胞凋亡率也逐渐升高。因此，可知化合物1明显诱导A549细胞的凋亡。
表9：化合物1对A549细胞凋亡影响结果
Figure PCTCN2019119758-appb-000018
6、化合物1对A549细胞ROS活性的影响
为了探讨化合物1对A549细胞的作用机制，利用荧光探针DCFH-DA进行活性氧的的检测，采用流式细胞仪检测DCF荧光强度，结果如图8所示，不同浓度的化合物1作用于A549细胞24h后，与阴性对照组NC相比，加入药物后的荧光强度呈升高趋势，因此，此结果显示化合物1可以促进细胞内活性氧的产生。
7、化合物1对A549细胞凋亡相关蛋白Bcl-2家族蛋白的影响
本实验通过Western Blot实验对细胞凋亡通路调控蛋白Bax和Bcl-2进行检测。从图9a可以看出，当A549细胞用化合物1处理72小时后，Bax蛋白的表达量随着化合物浓度的增加明显升高，虽然，Bcl-2蛋白的表达也随着药物浓度增加在上升，但是从图9b可以看出，与对照组相比，加入药物处理后，A549细胞中Bax/Bcl-2表达量的比值显著增高，且随着药物浓度的增加，蛋白表达量比值呈浓度依赖性的增加。当药物浓度达到20μM时，Bax/Bcl-2的表达量与对照组相比增加了6倍多。此结果 说明化合物1能够通过调控Bcl-2和Bax的表达，发挥诱导细胞凋亡的作用。
8、化合物1对A549细胞Caspase家族蛋白表达影响
本发明采用Western Blot方法检测了Caspase蛋白的表达，如图10所示，经过化合物1处理A549细胞72小时后，与对照组相比，被剪切的Caspase-9(Cleaved-Caspase-9)和被剪切的Caspase-3(Cleaved-Caspase-3)的的表达量明显升高，并且随着化合物浓度的加大，Cleaved-Caspase-9和Cleaved-Caspase-3的活性也越高。此结果显示化合物1可以通过剂量依赖途径升高A549细胞内的Cleaved-Caspase-9和Cleaved-Caspase-3的活性，从而诱导细胞凋亡。
9、化合物1对A549细胞AMPK-mTOR通路蛋白的影响
本实验采用Western Blot方法检测AM-PK-mTOR通路蛋白的表达量，如图11a所示，当用化合物1处理A549细胞72h后，与对照组相比，p-AMPK(Thr172)活性明显升高，AMPK正向调控的蛋白p-Raptor的活性显著下降，mTOR下游蛋白P-p70-S6K、p-S6(235/236)以及p-S6(240/244)的活性也明显下降(见图11b)，这些结果都表明化合物1通过调控AMPK-mTOR通路来抑制肿瘤细胞的生长。
10、化合物1对自噬通路蛋白的影响
本实验通过检测LC3II/LC3I这2个关键蛋白的表达比例来评价自噬水平高低。
如图12a所示，当用化合物处理A549细胞72h后，LC3I蛋白的表达水平与对照组相比是在下降的，而LC3II的表达水平是在上升的。见图12b，LC3II/LC3I的比值与对照组相比显著增加，且随着药物浓度的增大，LC3II/LC3I的表达呈现出剂量依赖性的上升。由此可见，化合物1诱导了A549细胞的自噬性死亡，从而使细胞凋亡。
实验例2、本发明化合物1的热学性能测试
本发明采用热重分析(Ther-mogravimetric Analysis,TGA)和差示扫描量热分析(Differential s-canning calorimetry,DSC)表征了实施例1制得的化合物1的热学性质,测试过程在空气中进行，从室温加热到400℃，加热速度为10℃﹒min -1。
从DSC图(见图24a)可以看出化合物的热行为主要包括2个过程， 吸热和放热过程。放热阶段132.31℃-144.03℃，峰温137.35℃，放热量为11.70J/g,此阶段放热可能为样品再结晶过程形成的一个微弱的放热峰。而两个吸热阶段186.31℃-199.29℃和198.29℃-206.78℃，峰温和吸热量分别为192.43℃、22.11J/g和202.05℃、68.74J/g,这两个吸热阶段为化合物熔化阶段，出现这2个熔融峰是因为晶体是一个α晶型的结晶。最后一个吸热阶段365.26℃-371.62℃，峰温为368.33℃，吸热量为269.30J/g,此阶段为化合物分解阶段。化合物TGA图(见图24b)显示失重87.97％，化合物在一个比较高的温度分解，表明此化合物结构较稳定。
实验例3、本发明化合物1的扫描电镜(SEM)
将实施例1制得的化合物1用比例为10:1的甲醇：水分别制备成1mg/mL的溶液和2mg/mL的溶液并通过SEM观察样品的超分子形态(见图25)。
在1mg/mL的样品溶液的SEM图像表明，在此浓度时化合物1以规则的微型圆盘形存在，分散良好。且高分辨率的SEM图显示，微盘表面较粗糙，具有明显的分层结构，提示化合物可能是介孔材料，可用于制备载药材料。而2mg/mL的SEM图像显示，在此浓度化合物1出现明显的团聚现象，微盘之间连通在一起，分层结构消失。
实验例4、本发明化合物和晶体的超分子结构测试
利用X射线单晶衍射法，对实施例2制得的化合物1的晶体1、晶体2进行测试，结果如下：
晶体1的参数:C 15H 17N 5O,Mr＝584.69g.mol -1；monoclinic,space group P 1 21/n 1；
Figure PCTCN2019119758-appb-000019
α＝90°,β＝90.024(8)°,γ＝90°；
Figure PCTCN2019119758-appb-000020
Z＝2；calc density＝1.292g·cm -3；F(000)＝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 mi n＝0.541,T max＝0.745；11668reflections collected；2952independent reflections；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；Largest diff.peak/hole/e:0.31
Figure PCTCN2019119758-appb-000021
and
Figure PCTCN2019119758-appb-000022
晶体1的结构分析：化合物1的晶体1为单斜晶系，P 1 21/n空间群晶体。化合物的晶体1只存在一对镜像对映体，1-A1和1-A2(如图13a)，通过分子构象重叠图发现，如图13b所示，2个苯酚基团相对于含氮杂环母核吡唑并[3,4-d]嘧啶呈对位交叉构象，1-A1和1-A2为互不重 叠的镜像分子，相应的1-A1的二面角(C5-C7-C10-C15)τ 1＝69.050(305)°，1-A2的τ 2＝-69.050(305)°。
由于化合物1中含有大量N原子和O原子，易形成氢键(见表10)，因此该化合物晶体稳定性主要靠氢键来维系。这些氢键使化合物构成稳定的空间三维网状结构(如图14a)。为了全面了解晶体，对晶体1的复杂氢键网络进行解析。首先从晶体学a轴方向开始分析,相邻分子间没有直接相互作用，晶体的堆积靠晶体中包含的溶剂---客体分子H 2O中的O原子作为供H体与苯酚O12形成分子间氢键；嘧啶环外氨基N6作为氢键供体与H 2O中O原子形成氢键；以及苯酚环上C11原子作为供体H体与H 2O中O原子形成氢键，这三种类型的氢键，使得相邻的主体分子被连接成如图14b所示的沿着晶体学a轴方向伸展的一维超分子链。接下来，如图14c所示，沿着晶体学b轴方向分子的相互作用主要靠苯酚基O12作为氢键供体与相邻分子嘧啶环上N3原子形成氢键。最后，在晶体学c轴方向上分子的堆积依靠相邻两个分子嘧啶环外氨基N6作为氢键供体与嘧啶环中N3原子形成分子间氢键以及叔丁基上的C17作为供H体与苯酚环形成C-H-π相互作用(见图14d)。
表10：晶体1的氢键参数
Figure PCTCN2019119758-appb-000023
Figure PCTCN2019119758-appb-000024
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
在晶体1中，化合物的两种构象对映体1-A1和1-A2，以两种类型的氢键连接方式形成了空腔中含有2个溶剂水分子的一个封闭的四元环结构(如图15a)，且主体分子与客体水分子之间有较强的氢键作用。而对于两个相邻的四元环，如图15b所示它们之间没有直接的相互作用，而是通过与纳米空腔中的客体水分子作为桥梁通过其与之主体分子形成 分子间氢键(O2-H2B…O12,O1-H1A…O12,N6-H6A…O2,N6-H6B…O12,C11-H11…O1,C11-H12…O2)相互作用,因此沿着a轴方向形成了一个管状堆积。
在认识了分子局部的连接方式后，对晶体1的整体排列方式进行分析。如图16所示，在同一层以layer1为例，由1-A11-A11-A21-A2四个分子通过氢键键合方式连接成的四元环沿着晶体学b轴方向形成了类似于II型分子阶梯状结构(Leong WL,Vittal JJ.One-dimensional coordination polymers:complexity and diversity in structures,properties,and applications.Chemical reviews.2010；111:688-764)。而分子阶梯layer1与相邻的分子阶梯layer2之间呈平行堆叠的方式排列。沿着晶体学c轴方向，相邻分子阶梯layer1和layer1’以弱分子间键C-H…π键产生相互作用。因此这样无数个分子阶梯的堆叠就形成了一个精美的三维网状结构。
晶体1的Hirshfeld表面分析和2D指纹图谱如图17所示，在晶体1的dnorm表面图中(图17a,b)，深红色斑点归因于分子间N-H…N和O-H…N的相互作用，表面上的其他可见斑点与晶体中的客体水分子和主体分子间相互作用有关。在2D指纹图中(图17c)，有两对尖峰指向图的左下方，是典型的N…H和O…H氢键作用,它们分别占整个Hirsfeld表面相互作用的14.5％和8.8％。在指纹图的左上角和右下角，出现了特征性的对称“翅膀”形状，这是C-H...π相互作用，它占Hirshfeld总表面积的16.3％。图17c中沿着对角线弥散点形成的尖峰表示H…H相互作用，这种相互作用在整个Hirshfeld表面积中占比高达55.5％。表明该晶体主要是通过H…H作用来使其稳定。与之相反的是，C…C作用占仅占Hirshfeld总表面积的0.7％，表明在晶体1中几乎没有π-π堆叠作用。
晶体2参数：C 15H 17N 5O,Mr＝283.33g.mol -1；monoclinic；space group P1 21/n 1；
Figure PCTCN2019119758-appb-000025
α＝90°,β＝113.35(3)°,γ＝90°；
Figure PCTCN2019119758-appb-000026
Z＝8；calc density＝1.284g·cm -3；F(000)＝1200.0；T＝173K；R int＝0.0890；μ＝0.086mm -1；miller indexranges,-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:
Figure PCTCN2019119758-appb-000027
and
Figure PCTCN2019119758-appb-000028
本发明发现经过10天培养出的晶体2与经过30培养出的晶体1虽然拥有相同的晶系与空间群，但是却展现出完全不同的分子构象及晶体排列方式。如图18d，将晶体1的主体分子与晶体2的分子进行重叠发现，晶体1的主体分子框架与晶体2完全不同。且晶体1中只有一对对映异构体，而晶体2中有两个不同的构象2-A1和2-B2，2对对映异构体(见图18a,b)。如图18c，e所示，2-A1,2-A2和2-B1,2-B2分别为互不重叠的镜像分子，相应的2-A1的二面角(C5-C7-C10-C15)τ 1＝45.620(434)°，2-A2的τ 2＝-45.620(434)°，2-B1的τ 3＝45.734(445)°及2-B2的τ 4＝-45.734(445)°。
在晶体2中，有3种氢键连接方式，形成2种结构单元。第一种结构单元是一个四元环结构(如图19a)，通过对映体之间的分子间氢键(O12-H12…N3)与相同分子间的氢键相互作用(N6-H6…N3)形成，这与晶体1四元环作用方式相同。与晶体1不同的是晶体2中还有一种类似与四元环的结构单元(如图19b),它只有一种氢键连接方式，靠吡啶环上的3位N于苯酚基上的O原子形成的氢键(O12’-H12’…N3’)在b轴方向上无线延伸。在结构单元上与晶体1不同的是，晶体1的四元环空腔被溶剂水分子占据，且相邻四元环是平行堆叠的关系，而晶体2中相邻的四元环与类四元环之间是互相交错(见图19c,d)，空腔被彼此占据，这是由于在两种晶体中分子扭转角度不用造成的。
表11：晶体2的氢键参数
Figure PCTCN2019119758-appb-000029
Symmetry codes:(i)x,1+y,z(ii)-x,-y,1-z(iii)x,-1+y,z
晶体2层间关系见图20a,b,虽然同一个交叉层的四元环与类四元环间没有直接的相互作用关系，它们依靠下层四元环与上一个交叉层的类四元环间两种的C-H…π键产生相互作用，一种C-H…π键如图20c,类四元环上的叔丁基碳与四元环结构中吡啶环通过平行阵列的C-H…π作用以
Figure PCTCN2019119758-appb-000030
的距离堆叠。另一种C-H…π键是上下交叉层之间一个类四元环的苯酚环的C14作为供氢体与另一个类四元环中吡啶环通过弱的C-H…π以
Figure PCTCN2019119758-appb-000031
的距离相互作用(见图20d)。这两种C-H…π作用使晶体分子沿c轴方向无限延伸。
从晶体2沿着晶体c轴的视图21a,b可以看出，晶体2由交叉层重 复堆叠而成，形成了类似于IX型分子梯的形状(Leong WL,Vittal JJ.One-dimensional coordination polymers:complexity and diversity in structures,properties,and applications.Chemical reviews.2010；111:688-764)。
对于同一种化合物1的两种晶体，相比与晶体1只存在一个非对映异构体(Z’＝1)的情况,晶体2中存在两种非对映异构体(Z’＝2)，说明晶体1是热力学控制的晶体产物而晶体2是动力学控制的晶体产物。
晶体2Hirshfeld表面分析以及2D指纹图谱如图22所示，dnorm表面图(22a,b)中的深红色斑点，是嘧啶环上N与环外氨基N的相互作用以及嘧啶环上N与苯酚的羟基O相互作用。2D指纹图谱(图22c)显示,N…H之间的作用力贡献值为17.5％，O…H之间作用力贡献值为3.9％，C…H之间作用力贡献值为16.2％，H…H之间相互作用贡献值最高为61.0％。说明晶体2也是通过H…H之间的相互作用来维持晶体的稳定性。
图23显示了化合物1的两种晶体中各种类型分子间接触对Hirshfeld表面的贡献，通过这个图对比发现两种晶体之间的相互作用差别不大，都含有H…H，N…H和O…H等氢键的存在，晶体的稳定性都是主要靠H…H相互作用来维持，并且在晶体中几乎都不存在π-π作用，但也存在一些区别。相较于晶体2，晶体1中O…H占比显著上升，这是由于晶体1是个水合物，水的存在提供了多余的O…H占比。
综上，本发明制得的化合物1具有高活性的抗肺癌效果，可显著抑制A549细胞的生长，抑制效果甚至优于阳性对照药物，使其在制备预防和/或治疗肿瘤的药物上具有很好的前景。此外，本发明化合物1还具有良好的热稳定性，具有明显的分层结构和颇高的孔隙率，这种结构使其在制备介孔材料、载药材料、人工通道材料领域具有很好的应用潜力。

Claims (11)

  1. 化合物1、或其溶剂化物、或其晶体、或其盐在制备预防和/或治疗肿瘤的药物上的用途；
    化合物1的结构为
    Figure PCTCN2019119758-appb-100001
  2. 根据权利要求1所述的用途，其特征在于：所述肿瘤为肺癌。
  3. 根据权利要求2所述的用途，其特征在于：所述肿瘤为非小细胞肺癌。
  4. 根据权利要求3所述的用途，其特征在于：所述肿瘤为肺腺癌。
  5. 根据权利要求1所述的用途，其特征在于：所述药物能够抑制肿瘤细胞的增殖、生长、迁移，诱导肿瘤细胞的凋亡。
  6. 根据权利要求1所述的用途，其特征在于：所述药物能够调控肿瘤细胞中Cyclin E1蛋白水平，调控Bcl-2和Bax的表达，调控胱天蛋白酶的活性，调控AMPK-mTOR通路蛋白的表达。
    优选地，所述药物能够降低肿瘤细胞中Cyclin E1蛋白水平，升高Bax/Bcl-2表达量的比值，提高Cleaved-Caspase-9和Cleaved-Caspase-3的活性。
  7. 根据权利要求1所述的用途，其特征在于：所述药物能够促进肿瘤细胞内活性氧的产生。
  8. 根据权利要求1所述的用途，其特征在于：所述药物够诱导肿瘤细胞的自噬性死亡。
  9. 化合物1、或其溶剂化物、或其晶体、或其盐在制备介孔材料、药物传递系统、药物载体、人工通道上的用途；
    化合物1的结构为
    Figure PCTCN2019119758-appb-100002
  10. 根据权利要求1-9任一项所述的用途，其特征在于：所述溶剂化物为化合物1的水合物。
  11. 根据权利要求1-9任一项所述的用途，其特征在于：所述晶体的制备方法为：取化合物1，加入甲醇与水的混合溶液中，溶解，过滤，取液体，析晶，即得晶体；
    优选地，所述甲醇与水的混合溶液中，甲醇与水的体积比为10：1；所述化合物1、甲醇与水的混合溶液的质量体积比为25mg：8mL；所述溶解的方式为在60℃下加热溶解至澄清透明；所述过滤为趁热过滤；所述析晶的方式为室温下静置析晶，析晶的时间为10～30天，优选为10天或30天。
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