Phenyl-substituted pyrazolopyridine compounds, their preparation methods and uses

Abstract

The present invention discloses phenyl-substituted pyrazolopyridine compounds, their preparation methods and applications, belonging to the technical field of medicinal chemistry. The structures of these compounds are shown as follows: #imgabs0# Among them, R1 is any one of the following structures: #imgabs1#; R2 is any one of the following structures: #imgabs2#. The advantages of the present invention are as follows: These compounds have a proliferation inhibitory effect on a variety of tumor cells and can selectively inhibit the proliferation of triple-negative breast cancer cells MDA-MB-468. Among them, the half-maximal inhibitory concentration IC 50 500 μM, and the selectivity index SI = 8.428, which can be used as a lead compound for anti-triple-negative breast cancer for further development.

Description

Technical Field

[0001] The present invention relates to compounds and their preparation methods and applications, specifically to phenyl-substituted pyrazolopyridine compounds and their preparation methods and applications, belonging to the technical field of medicinal chemistry. Background Art

[0002] Triple-negative breast cancer is a subtype of breast cancer with high malignancy and strong invasiveness, accounting for 15%-20% of all breast cancers. The molecular characteristics of such tumors are the lack of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 and weak immunogenicity, and they are not sensitive to hormone and immunotherapy.

[0003] The pyrazolopyridine structure is the active nucleus of many marketed drugs and has potential antitumor activity. Phenyl substitution based on the nucleus can optimize the physicochemical properties of the molecule and improve its biological activity. This strategy has been successfully applied in the drug development of PARP inhibitors, which are the only approved targeted drugs for the treatment of triple-negative breast cancer.

[0004] Currently, existing drugs cannot meet the clinical treatment needs of triple-negative breast cancer. Exploring new active molecules is the key to current drug research and development for such tumors. Summary of the Invention

[0005] The purpose of the present invention is to: perform phenyl substitution on the active nucleus pyrazolopyridine, prepare and screen phenyl-substituted pyrazolopyridine compounds with anti-triple-negative breast cancer activity.

[0006] In order to achieve the above goal, the present invention adopts the following technical scheme:

[0007] Phenyl-substituted pyrazolopyridine compounds, the structure of which is shown as follows:

[0008]

[0009] Wherein, R1 is any one of the following structures:

[0010] ;

[0011] R2 is any one of the following structures:

[0012] .

[0013] The preparation method of the aforementioned phenyl-substituted pyrazolopyridine compounds includes the following steps:

[0014] (1) Dissolve 5-bromo-2-chloronicotinonitrile in absolute ethanol, and dropwise add hydrazine hydrate at 70°C. After the reaction is completed, perform low-temperature recrystallization, filtration, and drying to obtain compound 2;

[0015] (2) Dissolve compound 2, compound 3 or compound 13, and potassium phosphate in a dioxane / water mixture. Under nitrogen protection, add bis(tri-tert-butylphosphine)palladium(II), and carry out the reaction at 110 °C. After the reaction is completed, filter with diatomaceous earth, extract with ethyl acetate and water, dry the organic phase, precipitate the solid, filter and dry to obtain compound 4 or compound 14;

[0016] (3) Dissolve compound 4 or compound 14 in DMF to obtain an intermediate product solution. Dissolve the carboxylic acid, EDCI and HOBt in DMF to obtain an activated ester mixture. Dropwise add the activated ester mixture to the intermediate product solution, and carry out the reaction at room temperature. After the reaction is completed, quench, precipitate the solid, filter, slurry and dry to obtain the aforementioned phenyl-substituted pyrazolopyridine compounds;

[0017] Among them, the carboxylic acid is 3-phenylpropionic acid, 4-tert-butylphenylacetic acid, 2-(6-bromopyridin-3-yl)acetic acid, 3,4-dimethoxyphenylacetic acid, 2-(benzo[d][1,3]dioxol-5-yl)acetic acid, 3-(p-tolyl)propionic acid, 2-(4-ethoxyphenyl)acetic acid, 4-phenylbutyric acid or 3-(4-methoxyphenyl)propionic acid;

[0018] The structures of compound 2, compound 3, compound 4, compound 13 and compound 14 are shown as follows:

[0019] , , , , .

[0020] Preferably, in step (1), the molar ratio of 5-bromo-2-chloronicotinonitrile to hydrazine hydrate is 1:5.

[0021] Preferably, in step (2), the molar ratio of compound 2 to compound 3 or compound 13 is 5:6.

[0022] Preferably, in step (3), the molar ratio of compound 4 or compound 14 to the carboxylic acid is 3:5.

[0023] Use of the aforementioned phenyl-substituted pyrazolopyridine compounds in the preparation of anti-tumor drugs, wherein the tumor is breast cancer, pancreatic cancer, gastric cancer or lung cancer.

[0024] Preferably, the tumor is triple-negative breast cancer, and the phenyl-substituted pyrazolopyridine compounds are:

[0025] , , , or .

[0026] Preferably, the tumor is pancreatic cancer, and the phenyl-substituted pyrazolopyridine compound is:

[0027] or .

[0028] Preferably, the tumor is gastric cancer, and the phenyl-substituted pyrazolopyridine compound is:

[0029] .

[0030] Preferably, the tumor is lung cancer, and the phenyl-substituted pyrazolopyridine compound is:

[0031] .

[0032] The advantages of the present invention are as follows:

[0033] (1) The present invention selects commercially available and inexpensive pyridine as the substrate, and only needs three chemical reactions: a ring-closure reaction, a Suzuki-Miyaura coupling reaction, and an N-acylation reaction to prepare the target product. The reaction conditions are mild, the cost is low, the product is easy to separate, and the synthesis efficiency is high, which is suitable for large-scale industrial production;

[0034] (2) The phenyl-substituted pyrazolopyridine compounds prepared by the present invention have a proliferation inhibitory effect on a variety of tumor cells (triple-negative breast cancer cells MDA-MB-468, pancreatic cancer cells PANC-1, gastric cancer cells MKN-45, lung cancer cells NCI-H1299), and can selectively inhibit the proliferation of triple-negative breast cancer cells MDA-MB-468. Among them, the half-maximal inhibitory concentration IC 50 = 2.500 µM of compound 8, and the selectivity index SI = 8.428, which can be further developed as a lead compound for anti-triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the 1H NMR spectrum of compound 5;

[0036] Figure 2 is the 13C NMR spectrum of compound 5;

[0037] Figure 3 is the 1H NMR spectrum of compound 6;

[0038] Figure 4 is the 13C NMR spectrum of compound 6;

[0039] Figure 5 is the 1H NMR spectrum of compound 7;

[0040] Figure 6 It is the carbon-13 NMR spectrum of Compound 7;

[0041] Figure 7 It is the proton NMR spectrum of Compound 8;

[0042] Figure 8 It is the carbon-13 NMR spectrum of Compound 8;

[0043] Figure 9 It is the proton NMR spectrum of Compound 9;

[0044] Figure 10 It is the carbon-13 NMR spectrum of Compound 9;

[0045] Figure 11 It is the proton NMR spectrum of Compound 10;

[0046] Figure 12 It is the carbon-13 NMR spectrum of Compound 10;

[0047] Figure 13 It is the proton NMR spectrum of Compound 11;

[0048] Figure 14 It is the carbon-13 NMR spectrum of Compound 11;

[0049] Figure 15 It is the proton NMR spectrum of Compound 12;

[0050] Figure 16 It is the carbon-13 NMR spectrum of Compound 12;

[0051] Figure 17 It is the proton NMR spectrum of Compound 15;

[0052] Figure 18 It is the carbon-13 NMR spectrum of Compound 15;

[0053] Figure 19 It is the proton NMR spectrum of Compound 16;

[0054] Figure 20 It is the carbon-13 NMR spectrum of Compound 16;

[0055] Figure 21 It is the result graph of detecting intracellular DNA content by flow cytometry;

[0056] Figure 22 It is the gel imaging graph of DNA damage and cell cycle-related proteins;

[0057] Figure 23 It is the quantification result graph of the expression levels of DNA damage and cell cycle-related proteins. Detailed implementation manners

[0058] The present invention will be specifically introduced below in conjunction with specific embodiments.

[0059] I. Structure of Phenyl-Substituted Pyrazolopyridine Compounds

[0060] The structure of the phenyl-substituted pyrazolopyridine compounds provided by the present invention is shown as follows:

[0061]

[0062] Among them, R1 is any one of the following structures:

[0063] ;

[0064] R2 is any one of the following structures:

[0065] .

[0066] II. Preparation of the Above Phenyl-Substituted Pyrazolopyridine Compounds

[0067] Example 1

[0068]

[0069] Step 1: Commercially available 5-bromo-2-chloronicotinonitrile (Compound 1, 23 mmol) was added to a reaction vessel containing anhydrous ethanol (75 mL). The reaction system was heated to 70 °C. After the temperature was stabilized, hydrazine hydrate (115 mmol) was added dropwise. After the addition was completed, the reaction system was maintained at 70 °C for a ring-closure reaction (2 h). The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, heating was stopped, and it was allowed to cool naturally. Then, low-temperature recrystallization, vacuum filtration, and drying were carried out in sequence to obtain 5-bromo-1H-indazol-3-amine (Compound 2, yellow crystals) with a yield of 86%.

[0070] Step 2: 5-bromo-1H-indazol-3-amine (Compound 2, 14 mmol), 4-morpholinophenylboronic acid (Compound 3, 16.8 mmol), potassium phosphate (28 mmol), and a dioxane / water mixture (80 mL, volume ratio 2:1) were added to the reaction vessel, stirred and dissolved, purged with nitrogen for 10 min, and bis(tri-tert-butylphosphine)palladium (0.7 mmol) was added. The reaction system was heated to 110 °C for a Suzuki-Miyaura coupling reaction (72 h). The reaction progress was monitored by TLC. After the reaction was completed, it was filtered through diatomaceous earth while it was hot, then extracted with ethyl acetate and water, the organic phase was collected and dried over anhydrous sodium sulfate. Finally, the solid was precipitated by cooling and filtered and dried to obtain 5-(4-morpholinoyl phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, yellow solid) with a crude yield of 65%.

[0071] Step 3: Dissolve 3-phenylpropionic acid (0.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 0.8 mmol) and hydroxybenzotriazole (HOBt, 0.8 mmol) in N,N-dimethylformamide (DMF, 3 mL) to obtain an activated ester mixture. Dissolve 5-(4-morpholinylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, 0.3 mmol) in DMF (3 mL) to obtain an intermediate product solution. Dropwise add the activated ester mixture to the intermediate product solution, and carry out the N-acylation reaction (4 h) at room temperature. Monitor the reaction progress by TLC. After the reaction is completed, quench with water (20 mL), precipitate the solid and filter by suction. Pulp with dichloromethane (10 mL), and dry to obtain the final product (Compound 5, yellow solid) with a yield of 58%.

[0072] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 5) are shown in Figure 1 and Figure 2 , respectively. The spectral information of 1H NMR, 13C NMR and high-resolution mass spectrometry is as follows:

[0073] 1 1H NMR (400 MHz, DMSO-D6): δ 8.87 (s, 1H), 8.54 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.30 (s, 4H), 7.19 (s, 1H), 7.08 (d, J = 8.3 Hz, 2H), 6.56 (s, 2H), 3.76 (s, 4H), 3.45 (s, 2H), 3.18 (s, 4H), 3.01 (t, J = 7.7 Hz, 2H);

[0074] 13 13C-NMR (101 MHz, DMSO-D6): δ 150.93, 150.79, 150.42, 148.09, 141.16, 131.04, 128.40 (3C), 128.34 (2C), 127.38 (2C), 127.16, 126.83, 125.97, 115.42 (2C), 112.54, 66.03 (2C), 48.03 (2C), 37.47, 30.04;

[0075] HR-MS (ESI): m / z [M + H] + C 25 H 25 Calculated value of molecular weight of C24H25N5O2: 428.2082, theoretical value: 428.2081.

[0076] Example 2

[0077]

[0078] First, 5-(4-morpholinoyl-phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4) was prepared according to the methods described in Steps 1 and 2 of Example 1.

[0079] Then, using 4-tert-butylphenylacetic acid as the raw material, 4-tert-butylphenylacetic acid (0.5 mmol), EDCI (0.8 mmol) and HOBt (0.8 mmol) were dissolved in DMF (3 mL) to obtain an activated ester mixture. 5-(4-morpholinoyl-phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, 0.3 mmol) was dissolved in DMF (3 mL) to obtain an intermediate product solution. The activated ester mixture was added dropwise to the intermediate product solution, and the reaction system was subjected to N-acylation reaction at room temperature (4 h). The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction, and a solid was precipitated and filtered by suction. The solid was slurried with ether and dried to obtain the final product (Compound 6, white solid), with a yield of 48%.

[0080] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 6) are shown in Figure 3 and Figure 4 respectively. The spectral information of 1H NMR, 13C NMR and high-resolution mass spectrometry is as follows:

[0081] 1 H NMR (400 MHz, DMSO-D6) δ 8.91 (s, 1H), 8.56 (s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 6.62 (s, 2H), 4.45 (s, 2H), 3.76 (t, J = 4.8 Hz, 4H), 3.18 (t, J = 4.8 Hz, 4H), 1.26 (s, 9H);

[0082] 13 C NMR (101 MHz, DMSO-D6) δ 151.18, 151.00, 150.73, 149.09 (2C), 148.33, 132.25, 131.40, 129.50 (2C), 127.57 (2C), 127.31, 127.04, 125.23 (2C), 115.60 (2C), 112.96, 66.22 (2C), 48.21 (2C), 41.47, 34.33, 31.35 (3C);

[0083] HR-MS (ESI): m / z [M+H] + C 28 H 31 Calculated value of molecular weight of N5O2: 470.2548, theoretical value: 470.2551.

[0084] Example 3

[0085]

[0086] First, 5-(4-morpholinoyl phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4) was prepared according to the methods described in Step 1 and Step 2 of Example 1.

[0087] Then, using 2-(6-bromopyridin-3-yl)acetic acid as the raw material, 2-(6-bromopyridin-3-yl)acetic acid (0.5 mmol), EDCI (0.8 mmol) and HOBt (0.8 mmol) were dissolved in DMF (4 mL) to obtain an activated ester mixture solution. 5-(4-morpholinoyl phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, 0.3 mmol) was dissolved in DMF (3 mL) to obtain an intermediate product solution. The activated ester mixture solution was added dropwise to the intermediate product solution, and the reaction system was subjected to N-acylation reaction at room temperature (4 h). The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction, and the solid was precipitated and filtered. It was slurried with dichloromethane and dried to obtain the final product (Compound 7, yellow solid), with a yield of 74%.

[0088] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 7) are shown in Figure 5 and Figure 6 respectively. The spectral information of 1H NMR, 13C NMR and high-resolution mass spectrometry is as follows:

[0089] 1 H NMR (500 MHz, DMSO-D6) δ 8.90 (s, 1H), 8.83 (s, 1H), 8.59 (s, 1H), 8.18 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 6.73 (s, 2H), 3.76 (t, J = 4.8 Hz, 4H), 3.19 (d, J = 4.8 Hz, 4H);

[0090] 1313C NMR (126 MHz, DMSO-D6) δ 161.99, 151.59, 151.15 (2C), 150.89, 148.37, 143.58, 140.41, 131.96, 130.67, 127.43 (2C), 127.39, 126.96, 126.80, 115.40 (2C), 113.08, 66.02 (2C), 47.98 (2C);

[0091] HR-MS (ESI): m / z [M+H] + C 22 H 19 Calculated for C15H12BrN6O2: 479.0828, found: 479.0826.

[0092] Example 4

[0093]

[0094] First, 5-(4-morpholinocarbonylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4) was prepared according to the methods described in Steps 1 and 2 of Example 1.

[0095] Then, using 3,4-dimethoxyphenylacetic acid as the raw material, 3,4-dimethoxyphenylacetic acid (0.5 mmol), EDCI (0.8 mmol), and HOBt (0.8 mmol) were dissolved in DMF to obtain an activated ester mixture. 5-(4-morpholinocarbonylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, 0.3 mmol) was dissolved in DMF (3.0 mL) to obtain an intermediate product solution. The activated ester mixture was added dropwise to the intermediate product solution, and the reaction system was subjected to N-acylation reaction at room temperature (4 h). The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate and water, and the organic phase was collected. The solid was precipitated by recrystallization and filtered by suction. The solid was slurried with ether (10 mL) and dried to obtain the final product (Compound 8, white solid) with a yield of 39%.

[0096] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 8) are shown in Figure 7 and Figure 8 , respectively. The spectral information of 1H NMR, 13C NMR, and high-resolution mass spectrometry is as follows:

[0097] 11H NMR (400 MHz, DMSO-D6) δ 8.90 (d, J = 2.2 Hz, 1H), 8.56 (d, J = 2.3 Hz, 1H), 7.61 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 7.00 (s, 1H), 6.87 (s, 2H), 6.64 (s, 2H), 4.42 (s, 2H), 3.73 (dd, J = 10.8, 4.2 Hz, 10H), 3.20 - 3.10 (m, 4H);

[0098] 13 13C NMR (101 MHz, DMSO-D6) δ 166.95, 150.75, 150.59, 150.34, 148.29, 147.89, 147.46, 130.98, 127.18 (3C), 126.92, 126.64, 121.49, 115.20 (2C), 113.48, 112.52, 111.56, 65.82 (2C), 55.30 (2C), 47.81 (2C), 41.15;

[0099] HR-MS (ESI): m / z [M+H] + C 26 H 27 Calculated value for C19H21N6O3 molecular weight: 474.2135, theoretical value: 474.2136.

[0100] Example 5

[0101]

[0102] First, 5-(4-morpholinoyl phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4) was prepared according to the methods described in Step 1 and Step 2 of Example 1.

[0103] Then, using 2-(benzo[d][1,3]dioxol-5-yl)acetic acid as the raw material, 2-(benzo[d][1,3]dioxol-5-yl)acetic acid (0.5 mmol), EDCI (0.8 mmol), and HOBt (0.8 mmol) were dissolved in DMF (3 mL) to obtain an activated ester mixture solution. 5-(4-Morpholinocarbonylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, 0.3 mmol) was dissolved in DMF (3 mL) to obtain an intermediate product solution. The activated ester mixture solution was added dropwise to the intermediate product solution, and the reaction system was subjected to an N-acylation reaction (4 h) at room temperature. The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction, and a solid was precipitated and filtered by suction. The solid was slurried with dichloromethane (10 mL) and dried to obtain the final product (Compound 9, yellow solid) with a yield of 63%.

[0104] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 9) are shown in Figure 9 and Figure 10 respectively. The spectral information of the 1H NMR, 13C NMR, and high-resolution mass spectrometry is as follows:

[0105] 1 H NMR (400 MHz, DMSO-D6) δ 8.90 (d, J = 2.3 Hz, 1H), 8.56 (d, J = 2.2 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.7 Hz, 2H), 6.93 (s, 1H), 6.83 (q, J = 7.9 Hz, 2H), 6.62 (s, 2H), 5.98 (s, 2H), 4.40 (s, 2H), 3.80 - 3.73 (m, 4H), 3.18 (s, 4H);

[0106] 13 C NMR (101 MHz, DMSO-D6) δ 151.18, 151.01, 150.74, 148.34, 147.29, 146.16, 131.40, 128.85, 127.59 (2C), 127.32, 127.06, 122.93 (2C), 115.62 (2C), 112.93, 110.41, 108.24, 100.99, 66.23 (2C), 48.22 (2C), 41.61;

[0107] HR-MS (ESI): m / z [M + H] + C 25 H 23 Calculated value for the molecular weight of C25H22N5O4: 458.1832, theoretical value: 458.1823.

[0108] Example 6

[0109]

[0110] First, 5-(4-morpholinylcarbonylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4) was prepared according to the methods described in Step 1 and Step 2 of Example 1.

[0111] Then, using 3-(p-tolyl)propionic acid as the raw material, 3-(p-tolyl)propionic acid (0.5 mmol), EDCI (0.8 mmol), and HOBt (0.8 mmol) were dissolved in DMF (3 mL) to obtain an activated ester mixture. 5-(4-morpholinylcarbonylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, 0.3 mmol) was dissolved in DMF (3 mL) to obtain an intermediate product solution. The activated ester mixture was added dropwise to the intermediate product solution, and the reaction system was subjected to N-acylation reaction at room temperature (4 h). The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction, and the solid was precipitated and filtered by suction. It was slurried with ether and dried to obtain the final product (Compound 10, yellow solid), with a yield of 49%.

[0112] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 10) are shown in Figure 11 and Figure 12 respectively. The spectral information of 1H NMR, 13C NMR, and high-resolution mass spectrometry is as follows:

[0113] 1 H NMR (400 MHz, DMSO-D6) δ 8.87 (d, J = 2.2 Hz, 1H), 8.54 (d, J = 2.3 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 7.8 Hz, 2H), 7.09 (d, J = 8.2 Hz, 4H), 6.55 (s, 2H), 3.76 (t, J = 4.8 Hz, 4H), 3.42 (s, 2H), 3.18 (t, J = 4.8 Hz, 4H), 2.96 (t, J = 7.8 Hz, 2H), 2.26 (s, 3H);

[0114] 13 C NMR (101 MHz, DMSO-D6) δ 150.77, 150.40, 148.06, 138.01, 134.83, 131.01, 128.88 (3C), 128.24 (2C), 128.04, 127.36 (2C), 127.16, 126.81 (2C), 115.41, 112.50, 66.02 (2C), 48.02 (2C), 37.51, 29.62, 20.61;

[0115] HR-MS (ESI): m / z [M+H] + C 26 H 27 Calculated value of molecular weight of N5O2: 442.2240, theoretical value: 442.2238.

[0116] Example 7

[0117]

[0118] First, 5-(4-morpholinoyl phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4) was prepared according to the methods described in Step 1 and Step 2 of Example 1.

[0119] Then, using 2-(4-ethoxyphenyl)acetic acid as the raw material, 2-(4-ethoxyphenyl)acetic acid (0.5 mmol), EDCI (0.8 mmol) and HOBt (0.8 mmol) were dissolved in DMF (3 mL) to obtain an activated ester mixture. 5-(4-morpholinoyl phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, 0.3 mmol) was dissolved in DMF (3 mL) to obtain an intermediate product solution. The activated ester mixture was added dropwise to the intermediate product solution, and the reaction system was subjected to N-acylation reaction (4 h) at room temperature. The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction, and the solid was precipitated and filtered. It was slurried with ether and dried to obtain the final product (Compound 11, pink solid), with a yield of 30%.

[0120] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 11) are shown in Figure 13 and Figure 14 respectively. The spectral information of 1H NMR, 13C NMR and high-resolution mass spectrometry is as follows:[[]]

[0121] 1 H NMR (400 MHz, DMSO-D6) δ 8.90 (d, J = 2.2 Hz, 1H), 8.56 (d, J = 2.3 Hz, 1H), 7.63 (d, J = 8.9 Hz, 2H), 7.25 (d, J = 8.7 Hz, 2H), 7.09 (d, J = 9.0 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.61 (s, 2H), 4.46 - 4.34 (m, 2H), 3.99 (q, J = 7.0 Hz, 2H), 3.80 - 3.73 (m, 4H), 3.22 - 3.15 (m, 4H), 1.30 (t, J = 7.0 Hz, 3H);

[0122] 1313C NMR (101 MHz, DMSO-D6) δ 157.31, 150.94, 150.80, 150.53, 148.11, 131.17, 130.66 (3C), 127.38 (2C), 127.12, 126.82, 115.41 (2C), 114.18 (3C), 112.71, 66.02 (2C), 62.90, 48.01 (2C), 40.88, 14.67;

[0123] HR-MS (ESI): m / z [M+H] + C 26 H 27 Calculated value for C15H14N5O3 molecular weight: 458.2188, theoretical value: 458.2187.

[0124] Example 8

[0125]

[0126] First, 5-(4-morpholinylcarbonylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4) was prepared according to the methods described in Steps 1 and 2 of Example 1.

[0127] Then, using 4-phenylbutyric acid as the raw material, 4-phenylbutyric acid (0.5 mmol), EDCI (0.8 mmol), and HOBt (0.8 mmol) were dissolved in DMF (3 mL) to obtain an activated ester mixture. 5-(4-morpholinylcarbonylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, 0.3 mmol) was dissolved in DMF (3.0 mL) to obtain an intermediate product solution. The activated ester mixture was added dropwise to the intermediate product solution, and the reaction system was subjected to N-acylation reaction at room temperature (4 h). The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate and water, and the organic phase was collected. The solid was precipitated by recrystallization and filtered by suction. The solid was slurried with dichloromethane and dried to obtain the final product (Compound 12, yellow solid), with a yield of 16%.

[0128] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 12) are shown in Figure 15 and Figure 16 , respectively. The spectral information of 1H NMR, 13C NMR, and high-resolution mass spectrometry is as follows:

[0129] 11H NMR (400 MHz, DMSO-D6) δ 8.03 (d, J = 2.3 Hz, 1H), 7.70 (d, J = 2.3 Hz, 1H), 6.82 - 6.74 (m, 2H), 6.50 - 6.30 (m, 5H), 6.29 - 6.21 (m, 2H), 5.72 (s, 2H), 2.93 (s, 4H), 2.34 (dd, J = 6.0, 3.7 Hz, 4H), 2.28 (d, J = 7.5 Hz, 2H), 1.86 (t, J = 7.7 Hz, 2H), 1.17 (p, J = 7.5 Hz, 2H);

[0130] 13 13C NMR (101 MHz, DMSO-D6) δ 168.73, 150.76, 150.40, 148.09, 141.69, 131.00, 128.31 (4C), 127.36 (2C), 127.19, 126.75, 125.79 (2C), 115.40 (2C), 112.48, 66.01 (2C), 48.02 (2C), 34.99, 34.52, 25.91;

[0131] HR-MS (ESI): m / z [M+H] + C 26 H 27 Calculated for C21H21N5O2: 442.2239, found: 442.2238.

[0132] Example 9

[0133]

[0134] First, 5-bromo-1H-indazol-3-amine (Compound 2) was prepared according to the method described in Step 1 of Example 1.

[0135] Then, using 4-(4-methylpiperazin-1-yl)phenylboronic acid pinacol ester (Compound 13) as the raw material, 5-bromo-1H-indazole-3-amine (Compound 2, 14 mmol), 4-(4-methylpiperazin-1-yl)phenylboronic acid pinacol ester (Compound 13, 16.8 mmol), potassium phosphate (28 mmol) and dioxane / water mixture (80 mL, volume ratio 2:1) were added to the reaction vessel, stirred and dissolved, purged with nitrogen for 10 min, and bis(tri-tert-butylphosphine)palladium(0) (0.7 mmol) was added. The reaction system was heated to 110 °C for Suzuki-Miyaura coupling reaction (72 h). The reaction progress was monitored by TLC. After the reaction was completed, it was filtered through diatomaceous earth while hot, then extracted with ethyl acetate and water. The organic phase was collected and dried over anhydrous sodium sulfate. Finally, the solid was precipitated by cooling and filtered by suction. After drying, 5-(4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 14, yellow solid) was obtained with a crude yield of 58%.

[0136] After that, using 3,4-dimethoxyphenylacetic acid as the raw material, 3,4-dimethoxyphenylacetic acid (0.5 mmol), EDCI (0.8 mmol), and HOBt (0.8 mmol) were dissolved in DMF (3 mL) to obtain an activated ester mixture solution. 5-(4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 14, 0.3 mmol) was dissolved in DMF (3 mL) to obtain an intermediate product solution. The activated ester mixture solution was added dropwise to the intermediate product solution, and the reaction system was subjected to N-acylation reaction at room temperature (4 h). The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction, and it was extracted with ethyl acetate and water. The organic phase was collected, the solid was precipitated by recrystallization and filtered by suction, and slurried with ether. After drying, the final product (Compound 15, white solid) was obtained with a yield of 36%.

[0137] The ¹H NMR and ¹³C NMR spectra of the obtained final product (Compound 15) are shown in Figure 17 and Figure 18 respectively. The spectral information of ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry is as follows:

[0138] 11H NMR (400 MHz, DMSO-D6) δ 8.89 (d, J = 2.2 Hz, 1H), 8.55 (d, J = 2.3 Hz, 1H), 7.61 (d, J = 8.9 Hz, 2H), 7.08 (d, J = 8.9 Hz, 2H), 6.99 (d, J = 1.7 Hz, 1H), 6.88 - 6.86 (m, 2H), 6.62 (s, 2H), 3.73 - 3.71 (m, 8H), 3.24 - 3.17 (m, 4H), 2.48 - 2.42 (m, 4H), 2.23 (s, 3H);

[0139] 13 13C NMR (126 MHz, DMSO-D6) δ 150.96, 150.73, 150.52, 148.49, 147.66, 131.25, 127.41, 127.37 (2C), 126.77, 126.66, 121.70, 115.62 (2C), 113.67, 112.73, 111.75, 55.52, 55.43, 54.51 (2C), 47.65 (2C), 45.80 (2C), 41.35, 40.11;

[0140] HR-MS (ESI): m / z [M + H] + C 27 H 30 Calculated value for the molecular weight of C19H24N6O3: 487.2454, theoretical value: 487.2452.

[0141] Example 10

[0142]

[0143] First, 5-(4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 14) was prepared according to the method described in Example 9.

[0144] Then, using 3-(4-methoxyphenyl)propanoic acid as the raw material, 3-(4-methoxyphenyl)propanoic acid (0.5 mmol), EDCI (0.8 mmol), and HOBt (0.8 mmol) were dissolved in DMF (3 mL) to obtain an activated ester mixture solution. 5-(4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 14, 0.3 mmol) was dissolved in DMF (3 mL) to obtain an intermediate product solution. The activated ester mixture solution was added dropwise to the intermediate product solution, and the reaction system was subjected to an N-acylation reaction at room temperature (4 h). The reaction progress was monitored by TLC. After the reaction was completed, water (20 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate and water, and the organic phase was collected. The solid was precipitated by recrystallization and filtered by suction. The solid was slurried with ether and dried to obtain the final product (Compound 16, white solid), with a yield of 28%.

[0145] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 16) are shown in Figure 19 and Figure 20 , respectively. The spectral information of the 1H NMR, 13C NMR, and high-resolution mass spectrometry are as follows:

[0146] 1 H NMR (400 MHz, DMSO-D6) δ 8.86 (d, J = 2.2 Hz, 1H), 8.53 (d, J = 2.2 Hz, 1H), 7.60 (d, J = 8.9 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 9.0 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.55 (s, 2H), 3.71 (s, 3H), 3.40 (s, 2H), 3.24 - 3.17 (m, 4H), 2.94 (t, J = 7.7 Hz, 2H), 2.49 - 2.44 (m, 4H), 2.23 (s, 3H);

[0147] 13 C NMR (101 MHz, DMSO-D6) δ 157.54, 150.88, 150.67, 150.36, 148.01, 132.97 (3C), 131.05 (2C), 129.32, 127.31 (2C), 126.70 (3C), 115.59, 113.74 (2C), 112.50 (2C), 54.97 (2C), 54.49 (2C), 47.63 (2C), 45.76, 37.74, 29.19;

[0148] HR-MS (ESI): m / z [M + H] + C 27 H 30Calculated molecular weight of N6O2: 471.2503, theoretical value: 471.2503.

[0149] III. Cell proliferation inhibition assay

[0150] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method was used to detect the proliferation inhibition ability of the above-mentioned phenyl-substituted pyrazolopyridine compounds against normal cells and different tumor cells.

[0151] 1. Cell lines

[0152] Triple-negative breast cancer cells MDA-MB-468, pancreatic cancer cells PANC-1, gastric cancer cells MKN-45, lung cancer cells NCI-H1299, human umbilical vein endothelial cells HUVEC (normal cells).

[0153] 2. Detection principle

[0154] Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-violet crystalline formazan, which deposits in the cells. Dead cells do not have this function. Within a certain range, the amount of formazan crystals formed is proportional to the number of living cells. DMSO was used to dissolve the formazan in the cells, and the optical absorbance was measured at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance of the non-drug-treated group and the drug-treated group was compared to calculate the cell survival rate.

[0155] Half maximal inhibitory concentration (IC 50 ) refers to the drug concentration at which the tumor cell survival rate drops to 50% within a specific time period. By measuring the absorbance of cells incubated with MTT at different drug concentrations and using Graphpad to plot the dose-response curve, the IC 50 was calculated through the Hill function.

[0156] 3. Detection method

[0157] The cells to be detected were cultured to the logarithmic growth phase, and the cell morphology and density were observed under a microscope.

[0158] The cells to be tested were seeded into 96-well plates at a density of 4000 cells / well and cultured in a 37 °C incubator containing 5% CO2 for 24 h.

[0159] The test compounds were serially diluted by half from 50 µM and added to the 96-well plates seeded with tumor cells. Three parallel wells were set for each concentration, and the cells were incubated with the drugs for 48 h.

[0160] After drug treatment, 20 µL of MTT solution (5.0 mg / mL) was added to each well to make the final concentration 0.5 mg / mL, and the cells were incubated in a 37 °C incubator for 2 h.

[0161] Discard the supernatant, add 150 µL of DMSO to each well, and shake at 300 rpm on an oscillator for 3 min to fully dissolve the formazan.

[0162] Use a microplate reader to detect the absorbance of the sample at 570 nm, fit the dose-response curve, and calculate the half-maximal inhibitory concentration IC 50 and selectivity index SI of the compound to be tested.

[0163] 4. Detection results

[0164] The half-maximal inhibitory concentration IC of the compound to be tested 50 The calculation results are shown in Table 1, and the calculation results of the selectivity index SI are shown in Table 2.

[0165] Table 1 Half-maximal inhibitory concentration IC of the compound to be tested against normal cells and tumor cells 50 (µM)

[0166]

[0167] Table 2 Selectivity index SI of the compound to be tested against tumor cells

[0168]

[0169] Note: N.D. means that it cannot be calculated based on the results of this study.

[0170] As can be seen from Table 1 and Table 2:

[0171] (1) The above-mentioned phenyl-substituted pyrazolopyridine compounds prepared by the present invention have a proliferation inhibitory effect on a variety of tumor cells. Among them, the proliferation inhibitory effect on triple-negative breast cancer cells MDA-MB-468 is the best;

[0172] (2) Compound 8 has strong anti-tumor activity, its sensitive cell line is triple-negative breast cancer cells MDA-MB-468, and the selectivity index SI is the highest (8.428).

[0173] IV. Cell cycle detection experiment

[0174] Detect the effect of compound 8 on the cell cycle of triple-negative breast cancer cells MDA-MB-468 in vitro by flow cytometry.

[0175] 1. Detection principle

[0176] Propidium iodide (PI) is a nucleic acid fluorescent dye. After the intracellular DNA is stained with PI, the intracellular DNA content can be detected by flow cytometry. The cell cycle refers to the entire process experienced by continuously dividing cells from the end of one mitosis to the end of the next mitosis. The DNA content of normal diploid cells (2n) is 2C in the G0 / G1 phase, gradually increases from 2C to 4C in the S phase, and reaches 4C after entering the G2 / M phase. Since PI cannot penetrate the intact cell membrane, ethanol or other membrane-permeabilizing reagents are needed to enhance the cell membrane permeability during sample preparation to achieve accurate detection of the DNA content.

[0177] 2. Detection method

[0178] Inoculate triple-negative breast cancer cells MDA-MB-468 in the logarithmic growth phase at a density of 500,000 cells per dish into a 5×5 cm 2 culture dish and culture in an incubator at 37°C with 5% CO2 for 24 h.

[0179] After the cells to be tested enter the logarithmic growth phase, add compound 8 at different concentrations (0 µM, 0.5 µM, 2.5 µM) for drug treatment.

[0180] After 48 h of drug treatment, transfer the cells to be tested to a 1.5 mL EP tube, centrifuge at 1500 rpm for 5 min, discard the supernatant and collect the cells, and wash the cells 1-2 times with PBS.

[0181] Resuspend the cells with 150 µL of PBS, then gradually add 350 µL of ethanol at 4°C to suspend the cells, and fix them at -20°C for 12 h.

[0182] Centrifuge the fixed cells at 5000 rpm for 4 min at 4°C, discard the supernatant, and then wash the cells 1-2 times with PBS at 4°C.

[0183] Add 200 µL of dye (PI:RNase = 9:1, volume ratio) to the cell pellet to resuspend the cells, incubate at -20°C in the dark for 30 min, pipette and mix well, and then perform on-machine detection.

[0184] 3. Detection results

[0185] The results of detecting the intracellular DNA content by flow cytometry are shown in Figure 21 .

[0186] It can be seen from Figure 21 that after 48 h of treatment with 2.5 µM of compound 8, the proportion of triple-negative breast cancer cells MDA-MB-468 in the G1 phase decreased from 58.4% to 44.8%, the proportion in the S phase changed little, and the proportion of cells in the G2 / M phase increased from 12.5% to 19.8%.

[0187] This indicates that compound 8 can induce G2 / M phase arrest in triple-negative breast cancer cell line MDA-MB-468.

[0188] V. Experiment for detecting the expression levels of DNA damage- and cell cycle-related proteins

[0189] In vitro, Western Blot was used to detect the effects of compound 8 on the expression levels of DNA damage- and cell cycle-related proteins in triple-negative breast cancer cell line MDA-MB-468.

[0190] 1. Detection principle

[0191] SDS-polyacrylamide gel electrophoresis was used to separate intracellular proteins according to their molecular weights, forming bands at different positions in the gel for different molecular weight proteins, and then transferring them to a solid-phase carrier PVDF membrane. Specific primary antibodies were incubated with HRP-labeled secondary antibodies, and the target proteins were detected by substrate color development.

[0192] γ-H2AX is a phosphorylated modification of histone H2AX, which usually appears during DNA double-strand breaks and is an important DNA damage marker. The binding of cyclin-dependent kinase CDK1 and cyclin CyclinB to form a complex is the main factor initiating mitosis. The CDK1 / CyclinB complex is regulated by various feedback mechanisms. In particular, the phosphorylation of CDK1, p-CDK1, is the key to the G2 / M phase transition. Cdc25C is a phosphatase that can dephosphorylate and activate CDK1, driving cells from the G2 phase into the M phase.

[0193] 2. Detection method

[0194] Triple-negative breast cancer cell line MDA-MB-468 in the logarithmic growth phase was seeded in 5×5 cm culture dishes at a density of 500,000 cells per dish and cultured in a 37°C incubator with 5% CO2 for 24 h. 2 After the cells to be tested entered the logarithmic growth phase, different concentrations (0 µM, 0.5 µM, 2.5 µM) of compound 8 were added for drug treatment for 48 h, and then the cells were washed 1-2 times with 4°C PBS on ice.

[0195] After adding 150 - 200 µL of cell lysate and phosphatase inhibitor (RIPA:PMSF = 10:1, volume ratio) and incubating on ice for 1 min, the cells were collected, and then incubated on ice for another 30 min, vortexing every 7 min to ensure that the lysis working solution and phosphatase inhibitor acted fully, and then centrifuged at 12,000 rpm for 15 min at 4°C in a centrifuge.

[0196]

[0197] ​The total protein concentration of cells was determined by the BCA method. Specifically, standard proteins with a concentration of 0.5 mg / mL were added to a 96-well plate at 0 µL, 1 µL, 2 µL, 4 µL, 8 µL, 12 µL, 16 µL, and 20 µL, and filled up to 20 µL with PBS. For the sample proteins, 1 µL of the protein solution was added to each well and filled up to 20 µL with PBS, with 3 parallel replicates. Then, 200 µL of BCA working solution (solution A: solution B = 50:1, volume ratio) was added to each well, incubated at 37 °C for 30 min, and then the OD value at 562 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The total protein concentration of the cells was calculated according to the standard curve.

[0198] 400 µg of protein solution was taken from each sample, 40 µL of 5× protein loading buffer was added, and filled up to 200 µL with PBS. The final concentration of the protein solution was 2 µg / µL, and the protein was denatured by heating in a metal bath at 95 °C for 5 min.

[0199] Prepare a polyacrylamide gel (12% separating gel, 8% stacking gel), and then load the samples at 30 µg per well for a total of 15 µL. Electrophoresis was carried out at 80 V for 30 min and then switched to 120 V until the bromophenol blue band was close to the bottom of the polyacrylamide gel. The protein to be detected was transferred to a methanol-activated PVDF membrane using a wet electrotransfer membrane apparatus, and transfer buffer was added. Transfer was carried out at 106 V in an ice bath for 75 min.

[0200] Incubate with a protein-free rapid blocking solution on a shaker at room temperature for 15 min, then discard the blocking solution. Then wash the PVDF membrane three times with 1×TBST buffer on a shaker, 10 min each time. Then dilute the primary antibody to an appropriate concentration with 1×TBST buffer and add 3 - 5 mL to the corresponding PVDF membrane, incubate overnight at 4 °C.

[0201] Wash the PVDF membrane incubated with the primary antibody three times with 1×TBST buffer, 10 min each time. Then dilute the secondary antibody to an appropriate concentration with 1×TBST buffer and add 3 - 5 mL to the corresponding PVDF membrane, incubate slowly on a shaker at room temperature for 40 min, and then wash the PVDF membrane three times with 1×TBST buffer on a shaker, 10 min each time.

[0202] Mix the ECL developing solution (protected from light) at a ratio of solution A: solution B = 1:1 (volume ratio), evenly drip it onto the membrane (about 200 µL), and place it in a gel imager for development.

[0203] 3. Detection Results

[0204] Graphpad was used for plotting. The relative protein levels of cells in each experimental group and the untreated group were analyzed for gray values using Image J and then normalized. The data are expressed as the mean ± SEM of 3 independent experiments.

[0205] The gel imaging results of DNA damage- and cell cycle-related proteins are shown in Figure 22 , and the quantification results of the expression levels of each protein are shown in Figure 23 .

[0206] It can be seen from Figure 22 and Figure 23 that: The relative expression levels of the DNA double-strand break marker γ-H2AX increased to 308.20% ± 32.36% (p < 0.01) and 521.28% ± 34.41% (p < 0.001) after treatment with 0.5 µM and 2.5 µM of compound 8 for 48 h, respectively; the relative expression level of p-CDK1, the core protein regulating the G2 / M phase transition of the cell cycle, decreased to 72.49% ± 3.65% after treatment with 2.5 µM of compound 8 for 48 h, and the relative expression levels of the G2 / M phase arrest-related proteins Cdc25C and Cyclin B did not change significantly after the drug treatment.

[0207] This indicates that: Compound 8 significantly upregulated the relative expression level of the DNA double-strand break marker γ-H2AX, significantly induced DNA double-strand breaks in cells, slightly downregulated the relative expression level of the marker protein p-CDK1 regulating G2 / M phase arrest, and could induce G2 / M phase cell cycle arrest.

[0208] In summary, compound 5, compound 6, compound 7, compound 8, and compound 15 can be used as drug lead compounds for the preparation of subsequent anti-tumor (triple-negative breast cancer) drugs; the remaining compounds can play a role in terms of the number of compounds for future new drug research and development.

[0209] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention and are not limitations on the implementation modes of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation modes here. Any obvious changes or modifications derived from the technical solution of the present invention still fall within the protection scope of the present invention.

Claims

1. A phenyl-substituted pyrazolopyridine compound, characterized in that, The structure of the phenyl-substituted pyrazolopyridine compounds is as follows: ； Among them, the structure of R2 is: ； R1 is any one of the following structures: ； Alternatively, the structure of R2 is: ； The structure of R1 is: 。 2. The preparation method of the phenyl-substituted pyrazolopyridine compound according to claim 1, characterized in that, It includes the following steps: (1) Dissolve 5-bromo-2-chloronicotinonitrile in absolute ethanol, and dropwise add hydrazine hydrate at 70 °C. After the reaction is completed, perform low-temperature recrystallization, filtration by suction, and drying to obtain Compound 2; (2) Dissolve Compound 2, Compound 3 or Compound 13, and potassium phosphate in a dioxane / water mixture. Under nitrogen protection, add bis(tri-tert-butylphosphine)palladium, and react at 110 °C. After the reaction is completed, filter by suction through diatomaceous earth, extract with ethyl acetate and water, dry the organic phase, precipitate a solid, filter by suction, and dry to obtain Compound 4 or Compound 14; (3) Dissolve Compound 4 or Compound 14 in DMF to obtain an intermediate product solution. Dissolve the carboxylic acid, EDCI, and HOBt in DMF to obtain an activated ester mixture solution. Dropwise add the activated ester mixture solution to the intermediate product solution, and carry out the reaction at room temperature. After the reaction is completed, quench, precipitate a solid, filter by suction, slurry, and dry to obtain the phenyl-substituted pyrazolopyridine compound described in Claim 1; Among them, the carboxylic acid is 3-phenylpropionic acid, 4-tert-butylphenylacetic acid, 2-(6-bromopyridin-3-yl)acetic acid, or 3,4-dimethoxyphenylacetic acid; The structures of Compound 2, Compound 3, Compound 4, Compound 13, and Compound 14 are as follows: 、 、 、 、 。 3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of 5-bromo-2-chloronicotinonitrile to hydrazine hydrate is 1:

5.

4. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of Compound 2 to Compound 3 or Compound 13 is 5:

6.

5. The preparation method according to claim 2, wherein In step (3), the molar ratio of Compound 4 or Compound 14 to the carboxylic acid is 3:

5.

6. Use of the phenyl-substituted pyrazolopyridine compound described in Claim 1 in the preparation of an anti-tumor drug, wherein the tumor is triple-negative breast cancer, pancreatic cancer, or gastric cancer.

7. The application according to claim 6, wherein The tumor is triple-negative breast cancer, and the phenyl-substituted pyrazolopyridine compound is: , , , or .

8. The application according to claim 6, wherein The tumor is pancreatic cancer, and the phenyl-substituted pyrazolopyridine compound is: or 。 9. The application according to claim 6, wherein The tumor is gastric cancer, and the phenyl-substituted pyrazolopyridine compound is: 。

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