Phenyl-substituted pyrazolopyridine compound as well as preparation method and application thereof

Abstract

The invention discloses a phenyl-substituted pyrazolopyridine compound as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The structure of the compound is as follows: # imgabs 0 #, wherein R1 is any one of the following structures: # imgabs 1 #; and R2 is any one of the following structures: # imgabs2. The compound has the beneficial effects that the compound has a proliferation inhibition effect on various tumor cells and can selectively inhibit proliferation of triple negative breast cancer cells MDA-MB-468, the half inhibitory concentration IC50 of the compound 8 is 2.500 M, the selection index SI is 8.428, and the compound 8 can be deeply developed as a lead compound for resisting triple negative breast cancer.

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 a variety of marketed drugs and has potential antitumor activity. Phenyl substitution based on the nucleus can optimize the physicochemical properties of the molecule and improve 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: Phenyl-substituted pyrazolopyridine compounds, the structure of which is shown as follows:

[0007] Among them, R1 is any one of the following structures: ; R2 is any one of the following structures: .

[0008] The preparation method of the foregoing phenyl-substituted pyrazolopyridine compounds includes the following steps: (1) Dissolve 5-bromo-2-chloronicotinonitrile in absolute ethanol, dropwise add hydrazine hydrate at 70°C, and after the reaction is completed, perform low-temperature recrystallization, filtration, and drying to obtain compound 2; (2) Compound 2, Compound 3 or Compound 13 and potassium phosphate are dissolved in a dioxane / water mixture, di-tri-tert-butyl phosphine palladium is added under nitrogen protection, and the reaction is carried out at 110° C. After the reaction is completed, diatomaceous earth is filtered, ethyl acetate and water are extracted, the organic phase is dried, and the precipitated solid is filtered and dried to obtain Compound 4 or Compound 14; (3) dissolving compound 4 or compound 14 in DMF to obtain an intermediate product solution, dissolving carboxylic acid, EDCI and HOBt in DMF to obtain an activated ester mixture, adding the activated ester mixture dropwise to the intermediate product solution, and reacting the reaction system at room temperature. After the reaction is completed, the reaction is quenched, the solid is precipitated, filtered, slurried, and dried to obtain the aforementioned phenyl-substituted pyrazolopyridine compound; Wherein, 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; The structures of Compound 2, Compound 3, Compound 4, Compound 13 and Compound 14 are shown below: , , , , .

[0009] Preferably, in step (1), the molar ratio of 5-bromo-2-chloronicotinonitrile to hydrazine hydrate is 1:5.

[0010] Preferably, in step (2), the molar ratio of compound 2 to compound 3 or compound 13 is 5:6.

[0011] Preferably, in step (3), the molar ratio of compound 4 or compound 14 to carboxylic acid is 3:5.

[0012] The use of the above-mentioned phenyl-substituted pyrazolopyridine compound in the preparation of anti-tumor drugs, wherein the tumor is breast cancer, pancreatic cancer, gastric cancer or lung cancer.

[0013] Preferably, the tumor is triple-negative breast cancer, and the phenyl-substituted pyrazolopyridine compound is: , , , or .

[0014] Preferably, the tumor is pancreatic cancer, and the phenyl-substituted pyrazolopyridine compound is: or .

[0015] Preferably, the tumor is gastric cancer, and the phenyl-substituted pyrazolopyridine compound is: .

[0016] Preferably, the tumor is lung cancer, and the phenyl-substituted pyrazolopyridine compound is: .

[0017] The advantages of the present invention are as follows: (1) The present invention uses commercially available and inexpensive pyridine as a substrate, and only requires 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; (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 of compound 8 is 2.500 μM, 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

[0018] Figure 1 is the 1H NMR spectrum of compound 5; Figure 2 is the 13C NMR spectrum of compound 5; Figure 3 is the 1H NMR spectrum of compound 6; Figure 4 is the 13C NMR spectrum of compound 6; Figure 5 is the 1H NMR spectrum of compound 7; Figure 6 is the 13C NMR spectrum of compound 7; Figure 7 is the 1H NMR spectrum of compound 8; Figure 8 is the 13C NMR spectrum of compound 8; Figure 9 is the 1H NMR spectrum of compound 9; Figure 10 is the 13C NMR spectrum of compound 9; Figure 111H NMR spectrum of compound 10; Figure 12 13C NMR spectrum of compound 10; Figure 13 1H NMR spectrum of compound 11; Figure 14 13C NMR spectrum of compound 11; Figure 15 1H NMR spectrum of compound 12; Figure 16 13C NMR spectrum of compound 12; Figure 17 1H NMR spectrum of compound 15; Figure 18 13C NMR spectrum of compound 15; Figure 19 1H NMR spectrum of compound 16; Figure 20 13C NMR spectrum of compound 16; Figure 21 Result graph of detecting intracellular DNA content by flow cytometry; Figure 22 Gel imaging graph of DNA damage and cell cycle related proteins; Figure 23 Quantification result graph of the expression levels of DNA damage and cell cycle related proteins. Detailed implementation manners

[0019] The present invention will be specifically introduced below in combination with specific embodiments.

[0020] I. Structure of phenyl-substituted pyrazolopyridine compounds The structure of the phenyl-substituted pyrazolopyridine compounds provided by the present invention is shown as follows:

[0021] Among them, R1 is any one of the following structures: ; R2 is any one of the following structures: .

[0022] II. Preparation of the above phenyl-substituted pyrazolopyridine compounds

[0023] Example 1

[0024]

[0025] Step 1: Add commercially available 5-bromo-2-chloronicotinonitrile (Compound 1, 23 mmol) into a reaction vessel containing anhydrous ethanol (75 mL). Heat the reaction system to 70 °C. After the temperature stabilizes, gradually add hydrazine hydrate (115 mmol) dropwise. After the addition is complete, maintain the reaction system at 70 °C for a ring-closure reaction (2 h). Monitor the reaction progress by thin-layer chromatography (TLC). After the reaction is completed, stop heating and allow it to cool naturally. Then, perform low-temperature recrystallization, vacuum filtration, and drying successively to obtain 5-bromo-1H-indazol-3-amine (Compound 2, yellow crystals) with a yield of 86%.

[0026] Step 2: Add 5-bromo-1H-indazol-3-amine (Compound 2, 14 mmol), 4-morpholinylphenylboronic acid (Compound 3, 16.8 mmol), potassium phosphate (28 mmol), and a dioxane / water mixture (80 mL, volume ratio 2:1) into the reaction vessel. Stir to dissolve, bubble nitrogen for 10 min, add bis(tri-tert-butylphosphine)palladium (0.7 mmol), and heat the reaction system to 110 °C for a Suzuki-Miyaura coupling reaction (72 h). Monitor the reaction progress by TLC. After the reaction is completed, filter while hot through diatomaceous earth, then extract with ethyl acetate and water. Collect the organic phase and dry it with anhydrous sodium sulfate. Finally, cool to precipitate a solid and filter by suction and dry to obtain 5-(4-morpholinylphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine (Compound 4, yellow solid) with a crude yield of 65%.

[0027] Step 3: Dissolve 3-phenylpropanoic 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 solution. 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. Gradually add the activated ester mixture solution dropwise to the intermediate product solution. The reaction system undergoes an N-acylation reaction at room temperature (4 h). Monitor the reaction progress by TLC. After the reaction is completed, quench with water (20 mL), precipitate a solid and filter by suction, slurry with dichloromethane (10 mL), and dry to obtain the final product (Compound 5, yellow solid) with a yield of 58%.

[0028] The nuclear magnetic resonance hydrogen spectrum and carbon spectrum of the obtained final product (Compound 5) are shown in Figure 1 and Figure 2 respectively. The spectral information of the nuclear magnetic resonance hydrogen spectrum, carbon spectrum, and high-resolution mass spectrum is as follows: 11H 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); 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; HR-MS (ESI): m / z [M+H] + C 25 H 25 Calculated value for C21H20N5O2 molecular weight: 428.2082, theoretical value: 428.2081.

[0029] Example 2

[0030]

[0031] 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.

[0032] 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 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 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%.

[0033] The 1H NMR and 13C NMR spectra of the obtained final product (Compound 6) are shown in Figure 3 and Figure 4, the spectral information of proton nuclear magnetic resonance (¹H NMR), carbon nuclear magnetic resonance (¹³C NMR), and high-resolution mass spectrometry (HR-MS) is as follows: 1 ¹H 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); 13 ¹³C 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); HR-MS (ESI): m / z [M + H] + C 28 H 31 Calculated value of molecular weight of C₁₉H₂₀N₅O₂: 470.2548, theoretical value: 470.2551.

[0034] Example 3

[0035]

[0036] 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.

[0037] 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-morpholinoylphenyl)-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 a solid was precipitated and filtered by suction. The solid was slurried with dichloromethane and dried to obtain the final product (Compound 7, yellow solid), with a yield of 74%.

[0038] 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: 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); 13 C 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); HR-MS (ESI): m / z [M + H] + C 22 H 19 The calculated value of the molecular weight of C22H20BrN6O2 is 479.0828, and the theoretical value is 479.0826.

[0039] Example 4

[0040] 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.

[0041] 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-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. It was slurried with ether (10 mL) and dried to obtain the final product (Compound 8, white solid) with a yield of 39%.

[0042] 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: 1 H NMR (400 MHz, DMSO-D6) δ 8.90 (d, J = 2.2 Hz, 1H), 8.56 (d, J = 2.3 Hz, 1H), 7.61 (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); 13 C 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; HR-MS (ESI): m / z [M + H] + C 26 H 27Calculated molecular weight of N6O3: 474.2135, theoretical value: 474.2136.

[0043] Example 5

[0044]

[0045] 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.

[0046] 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. 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 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%.

[0047] 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 1H NMR, 13C NMR and high-resolution mass spectrometry is as follows: 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); 1313C 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; HR-MS (ESI): m / z [M+H] + C 25 H 23 Calculated value for C15H18N5O4 molecular weight: 458.1832, theoretical value: 458.1823.

[0048] Example 6

[0049]

[0050] 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.

[0051] Then, using 3-(p-tolyl)propanoic acid as the raw material, 3-(p-tolyl)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. 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 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, and a solid was precipitated and filtered. The solid was slurried with ether and dried to obtain the final product (Compound 10, yellow solid), with a yield of 49%.

[0052] 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 the 1H NMR, 13C NMR, and high-resolution mass spectrometry is as follows: 11H 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); 13 13C 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; HR-MS (ESI): m / z [M+H] + C 26 H 27 Calculated for C25H24N5O2: 442.2240, found: 442.2238.

[0053] Example 7

[0054] 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.

[0055] 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-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 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. The solid was slurried with ether and dried to obtain the final product (Compound 11, pink solid) with a yield of 30%.

[0056] The 1H NMR and 13C NMR spectra of the 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: 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); 13 C 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; HR-MS (ESI): m / z [M+H] + C 26 H 27 Calculated value for the molecular weight of C

[0057] Example 8

[0058]

[0059] 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.

[0060] 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 solution. 5-(4-Morpholinoyl phenyl)-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 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. 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%.

[0061] 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: 1 H 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); 13 C 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; HR-MS (ESI): m / z [M + H] + C 26 H 27 The calculated value of the molecular weight of C24H23N5O2: 442.2239, the theoretical value: 442.2238.

[0062] Example 9

[0063]

[0064] First, 5-bromo-1H-indazole-3-amine (Compound 2) was prepared according to the method described in Step 1 of Example 1.

[0065] 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%.

[0066] 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%.

[0067] 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: 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); 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; HR-MS (ESI): m / z [M + H] + C 27 H 30 Calculated value for C25H28N6O3 molecular weight: 487.2454, theoretical value: 487.2452.

[0068] Example 10

[0069] 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.

[0070] 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. 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 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 extraction was carried out with ethyl acetate and water. The organic phase was collected, recrystallized to precipitate a solid, which was then filtered by suction, slurried with ether, and dried to obtain the final product (Compound 16, white solid) with a yield of 28%.

[0071] 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 1H NMR, 13C NMR and high-resolution mass spectrometry is as follows: 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); 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; HR-MS (ESI): m / z [M + H] + C 27 H 30 Calculated value of molecular weight of C25H23N6O2: 471.2503, theoretical value: 471.2503.

[0072] III. Cell proliferation inhibition experiment The MTT colorimetric method was used to detect the proliferation inhibition ability of the above-mentioned phenyl-substituted pyrazolopyridine compounds on normal cells and different tumor cells.

[0073] 1. Cell lines Triple-negative breast cancer cell line MDA-MB-468, pancreatic cancer cell line PANC-1, gastric cancer cell line MKN-45, lung cancer cell line NCI-H1299, human umbilical vein endothelial cell line HUVEC (normal cell).

[0074] 2. Detection principle Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-violet crystalline formazan, which is deposited in the cells. However, 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 is used to dissolve the formazan in the cells, and the absorbance is measured at 570 nm using a microplate reader. By comparing the absorbance values of the untreated group and the treated group, the cell survival rate can be calculated.

[0075] The half-maximal inhibitory concentration (IC 50 50%) is the drug concentration at which the survival rate of tumor cells 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 50% is calculated using the Hill function.

[0076] 3. Detection method The cells to be detected are cultured until the logarithmic growth phase, and the cell morphology and density are observed under a microscope.

[0077] The cells to be tested are seeded in a 96-well plate at a density of 4000 cells / well and cultured in a 37°C incubator containing 5% CO2 for 24 h.

[0078] The test compound is serially diluted by a factor of two starting from 50 µM and added to the 96-well plate seeded with tumor cells. Three parallel wells are set for each concentration, and the cells are incubated with the drug for 48 h.

[0079] After drug treatment, 20 µL of MTT solution (5.0 mg / mL) is added to each well to a final concentration of 0.5 mg / mL, and the cells are incubated in a 37°C incubator for 2 h.

[0080] The supernatant is discarded, 150 µL of DMSO is added to each well, and the plate is shaken on an oscillator at 300 rpm for 3 min to completely dissolve the formazan.

[0081] The absorbance of the samples is measured at 570 nm using a microplate reader, the dose-response curve is fitted, and the half-maximal inhibitory concentration IC 50 50% and selectivity index SI of the test compound are calculated.

[0082] 4. Detection results The calculated results of the half-maximal inhibitory concentration IC 50 50% of the test compound are shown in Table 1, and the calculated results of the selectivity index SI are shown in Table 2.

[0083] Table 1 Half-maximal inhibitory concentration IC 50 50% of the test compound against normal cells and tumor cells (µM)

[0084] Table 2 Selectivity Index SI of the Test Compounds against Tumor Cells

[0085] Note: N.D. means that it cannot be calculated based on the results of this study.

[0086] As can be seen from Table 1 and Table 2: (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; (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).

[0087] IV. Cell Cycle Detection Experiment The effect of compound 8 on the cell cycle of triple-negative breast cancer cells MDA-MB-468 was detected by flow cytometry in vitro.

[0088] 1. Detection Principle 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-breaking reagents need to be used to enhance the cell membrane permeability during sample preparation to achieve accurate detection of the DNA content.

[0089] 2. Detection Method Triple-negative breast cancer cells MDA-MB-468 in the logarithmic growth phase were seeded in 5×5 cm 2 culture dishes at a density of 500,000 cells / dish and cultured in a 37°C incubator with 5% CO2 for 24 h.

[0090] After the test cells entered the logarithmic growth phase, different concentrations (0 µM, 0.5 µM, 2.5 µM) of compound 8 were added for drug treatment.

[0091] After 48 h of drug treatment, the test cells were transferred to 1.5 mL EP tubes, centrifuged at 1500 rpm for 5 min, the supernatant was discarded and the cells were collected, and washed 1-2 times with PBS.

[0092] The cells were resuspended with 150 µL of PBS, and then 350 µL of ethanol at 4°C was added dropwise to suspend the cells, and fixed at -20°C for 12 h.

[0093] Centrifuge the fixed cells at 5000 rpm for 4 min at 4 °C, discard the supernatant, and then wash 1 - 2 times with PBS at 4 °C.

[0094] Add 200 µL of dye (PI:RNase = 9:1, volume ratio) to the cell pellet to resuspend the cells, incubate in the dark at -20 °C for 30 min, pipette to mix well, and then perform on-machine detection.

[0095] 3. Detection Results The results of detecting the intracellular DNA content by flow cytometry are shown in Figure 21 .

[0096] It can be seen from Figure 21 that after treatment with 2.5 µM of compound 8 for 48 h, the proportion of triple-negative breast cancer cells MDA-MB-468 in G1 phase decreased from 58.4% to 44.8%, the proportion in S phase changed little, and the proportion of cells in G2 / M phase increased from 12.5% to 19.8%.

[0097] This indicates that compound 8 can cause G2 / M phase arrest in triple-negative breast cancer cells MDA-MB-468.

[0098] V. Experiment for Detecting the Expression Levels of DNA Damage and Cell Cycle-related Proteins Detect the effects of compound 8 on the expression levels of DNA damage and cell cycle-related proteins in triple-negative breast cancer cells MDA-MB-468 in vitro by Western Blot.

[0099] 1. Detection Principle Separate the intracellular proteins according to their molecular weights by SDS-polyacrylamide gel electrophoresis, so that proteins with different molecular weights form bands at different positions in the gel, and transfer them to the solid-phase carrier PVDF membrane. Incubate with specific primary antibody and HRP-labeled secondary antibody, and detect the target protein by substrate color development.

[0100] 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, especially the phosphorylation of CDK1, p-CDK1, which is the key to the G2 / M phase transition. Cdc25C is a phosphatase that can dephosphorylate and activate CDK1, driving the cell from G2 phase into M phase.

[0101] 2. Detection Method Inoculate triple-negative breast cancer cells MDA-MB-468 in the logarithmic growth phase at a density of 500,000 cells per dish into 5×5 cm 2 culture dishes and place them in an incubator with 5% CO2 at 37℃ for 24 h.

[0102] 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 for 48 h, and then wash them 1-2 times with PBS at 4℃ on ice.

[0103] Add 150-200 µL of cell lysate and phosphatase inhibitor (RIPA:PMSF = 10:1, volume ratio), act on the cells on ice for 1 min, then collect the cells, continue to act on ice for 30 min, vortex every 7 min to make the lysis working solution and phosphatase inhibitor act fully, and then centrifuge at 12,000 rpm for 15 min in a 4℃ centrifuge.

[0104] Determine the total protein concentration of the cells by the BCA method. Specifically, add the standard protein with a concentration of 0.5 mg / mL to the 96-well plate at 0 µL, 1 µL, 2 µL, 4 µL, 8 µL, 12 µL, 16 µL, 20 µL, and make up to 20 µL with PBS. Add 1 µL of the sample protein solution to each well and make up to 20 µL with PBS. Prepare 3 parallel replicates. Then add 200 µL of BCA working solution (A solution:B solution = 50:1, volume ratio) to each well, incubate at 37℃ for 30 min, and then measure the OD value at 562 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the total protein concentration of the cells according to the standard curve.

[0105] Take 400 µg of protein solution from each sample, add 40 µL of 5× protein loading buffer respectively, and make up to 200 µL with PBS. The final concentration of the protein solution is 2 µg / µL. Heat the protein solution at 95℃ in a metal bath for 5 min to denature the protein.

[0106] Prepare a polyacrylamide gel (12% separating gel, 8% stacking gel), and then load the samples at 30 µg per well, a total of 15 µL. Electrophorese at 80 V for 30 min, then change to 120 V until the bromophenol blue band is close to the bottom of the polyacrylamide gel, and then stop electrophoresis. Transfer the protein to be tested to a methanol-activated PVDF membrane with a wet electrotransfer membrane apparatus, add transfer buffer, and transfer at 106 V in an ice bath for 75 min.

[0107] Incubate with protein-free rapid blocking solution on a shaker at room temperature with slow shaking for 15 min, then discard the blocking solution. Then wash the PVDF membrane three times with 1×TBST buffer on a shaker for 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, and incubate overnight at 4℃.

[0108] Wash the PVDF membrane incubated with the primary antibody three times with 1×TBST buffer for 10 minutes each time. Then add 3 - 5 mL of the secondary antibody diluted to an appropriate concentration with 1×TBST buffer to the corresponding PVDF membrane, and slowly incubate it on a shaker at room temperature for 40 minutes. After that, wash the PVDF membrane three times with 1×TBST buffer on the shaker for 10 minutes each time.

[0109] Mix the ECL chromogenic solution (protected from light) in a ratio of solution A:solution B = 1:1 (by volume), and evenly drip it onto the membrane (about 200 µL), then place it in a gel imager for development.

[0110] 3. Detection Results Use Graphpad to plot the graph. After analyzing the gray values of the relative protein levels of each experimental group and the untreated group of cells using Image J and normalizing them, the data are expressed as the mean ± SEM of 3 independent experiments.

[0111] 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 .

[0112] From Figure 22 and Figure 23 it can be seen 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 hours 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 hours, and the relative expression levels of the G2 / M phase arrest-related proteins Cdc25C and CyclinB did not change significantly after the drug treatment.

[0113] This indicates that: compound 8 significantly up-regulates the relative expression level of the DNA double-strand break marker γ-H2AX, significantly causes DNA double-strand breaks in cells, slightly down-regulates the relative expression level of the marker protein p-CDK1 that regulates G2 / M phase arrest, and can cause G2 / M phase cell cycle arrest.

[0114] 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 providing the number of compounds for future new drug research and development.

[0115] It should be noted that the above embodiments are merely examples given for clearly illustrating the present invention, rather than limitations on the implementation manners 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 manners here. Any obvious changes or modifications derived from the technical solutions 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 compound is shown below: ； Wherein, R1 is any one of the following structures: ； R2 is any of the following structures: 。 2. The method for preparing the phenyl-substituted pyrazolopyridine compound according to claim 1, characterized in that: The following steps are involved: (1) Dissolve 5-bromo-2-chloronicotinonitrile in anhydrous ethanol, add hydrazine hydrate dropwise at 70°C, recrystallize at low temperature after the reaction, filter and dry to obtain compound 2; (2) Compound 2, Compound 3 or Compound 13 and potassium phosphate are dissolved in a dioxane / water mixture, di-tri-tert-butyl phosphine palladium is added under nitrogen protection, and the reaction is carried out at 110° C. After the reaction is completed, diatomaceous earth is filtered, ethyl acetate and water are extracted, the organic phase is dried, and the precipitated solid is filtered and dried to obtain Compound 4 or Compound 14; (3) dissolving compound 4 or compound 14 in DMF to obtain an intermediate product solution, dissolving carboxylic acid, EDCI and HOBt in DMF to obtain an activated ester mixture, adding the activated ester mixture dropwise to the intermediate product solution, reacting the reaction system at room temperature, quenching after completion of the reaction, precipitating a solid, filtering, slurrying and drying to obtain the phenyl-substituted pyrazolopyridine compound according to claim 1; Wherein, 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; The structures of Compound 2, Compound 3, Compound 4, Compound 13 and Compound 14 are shown below: 、 、 、 、 。 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.

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