CN118126038A - Pyrazolopyridine derivative, and preparation method and application thereof - Google Patents

Abstract

The invention discloses pyrazolopyridine derivatives, a preparation method and application thereof, and belongs to the technical field of pharmaceutical chemistry. The pyrazolopyridine derivative has the following structure: Or (b) . The derivatives have good inhibition effects on human pancreatic cancer cells PANC-1, gastric cancer cells AGS, triple negative breast cancer cells HCC1806 and HCC1937, and the IC ₅₀ value of the optimal molecule is in the range of 0.6-2.5 mu M, so that the derivatives can be used as antitumor lead molecules.

Description

Pyrazolopyridine derivatives and preparation methods and applications thereof

Technical Field

The present invention relates to a class of compounds and preparation methods and applications thereof, in particular to pyrazolopyridine derivatives and preparation methods thereof and applications thereof in preparing drugs for treating pancreatic cancer, gastric cancer or breast cancer, and belongs to the technical field of pharmaceutical chemistry.

Background technique

Cancer is a serious disease that threatens human health and is also a major global public health issue. According to the statistics of the World Health Organization (WHO) in 2023, the number of new cancer cases in the world exceeds 20 million, and the number of deaths is nearly 10 million. The global cancer burden is increasing. Based on multiple factors such as indications, treatment costs, and safety, chemotherapy is the primary strategy for tumor treatment at present, and the development of chemical small molecules with anti-tumor activity is the main strategy for the discovery of new anti-tumor drugs. Nitrogen-containing heterocycles and aromatic skeletons are one of the important structures that make up drugs. Among the 164 chemical small molecules approved by the U.S. Food and Drug Administration (FDA) in the past five years, chemical molecules with nitrogen-containing heterocycles and aromatic skeletons accounted for 88% and 87% respectively. Pyrazolopyridine is a common and important nitrogen-containing heterocyclic derivative, which contains steric hindrance and electronic configuration similar to those of indole, azaindole, etc., and has multiple pharmacological activities such as anti-inflammatory, antibacterial, and anti-tumor. The present invention carries out the preparation of molecules with anti-tumor activity based on the pyrazolopyridine parent core and the application evaluation of its anti-cancer activity.

Summary of the invention

The purpose of the present invention is to provide pyrazolopyridine derivatives and a preparation method thereof and use thereof in preparing a drug for treating pancreatic cancer, gastric cancer or breast cancer.

In order to achieve the above object, the present invention adopts the following technical solution:

Pyrazolopyridine derivatives, the structure is shown below:

or/> ;

Wherein, R ₁ is selected from any one of the following structures:

;

_R2 is selected from any one of the following structures:

.

The preparation method of the aforementioned pyrazolopyridine derivatives comprises the following steps:

Step 1, cyclization: dissolving 5-bromo-2-chloronicotinonitrile in anhydrous ethanol, heating to 85° C. and adding hydrazine hydrate dropwise, maintaining the temperature of the reaction system and stirring, and after the reaction is completed, performing low-temperature recrystallization, vacuum filtration, and drying in sequence to obtain compound 2; preferably, the amount ratio of 5-bromo-2-chloronicotinonitrile to hydrazine hydrate is 23 mmol:115 mmol;

Step 2, carbon-carbon coupling: dissolving compound 2, compound A and potassium phosphate in a dioxane aqueous solution, wherein compound A is compound 3, compound 9, compound 12 or compound 15, blowing with nitrogen, adding ditri-tert-butylphosphine palladium to the reaction system, reacting at 80-110° C., and after the reaction, sequentially performing diatomaceous earth vacuum filtration, ethyl acetate and water extraction, and drying to obtain compound B;

Step 3, amidation: dissolving compound B in N, N-dimethylformamide, adding compound C, wherein compound C is bromoacetyl chloride, chloroacetyl chloride, 2,2,2-trichloroethyl chloroformate or isobutyryl chloride, reacting at 20° C., and after the reaction, sequentially performing quenching, extraction with ethyl acetate and water, vacuum rotary evaporation, column column, and drying to obtain the pyrazolopyridine derivative according to claim 1;

Among them, the structures of compound 2, compound 3, compound 9, compound 12 and compound 15 are shown below respectively:

、/> 、/> 、/> , .

Preferably, in step 2, the usage ratio of compound 2, compound A, potassium phosphate and ditri-tert-butylphosphine palladium is 14 mmol: 16.8 mmol: 28 mmol: 0.7 mmol.

Preferably, in step 3, the usage ratio of compound B to compound C is 0.3 mmol:0.6 mmol.

More preferably, it also includes a methylation step, specifically:

After step 2 is completed, compound B is dissolved in N, N-dimethylformamide, sodium hydride is added under an ice bath, and potassium iodide is then added dropwise to react. After the reaction is completed, quenching, extraction with ethyl acetate and water, vacuum rotary evaporation, column chromatography, and drying are performed in sequence to obtain compound D. The methylation is completed, and compound D subsequently replaces compound B for amidation reaction.

Preferably, in the methylation step, the usage ratio of compound B, sodium hydride and potassium iodide is 0.3 mmol:0.45 mmol:0.45 mmol.

The aforementioned pyrazolopyridine derivatives are used in the preparation of anticancer drugs, wherein the anticancer drugs are anti-pancreatic cancer drugs, anti-gastric cancer drugs or anti-breast cancer drugs.

The present invention is beneficial in that:

The present invention has developed a class of novel pyrazolopyridine derivatives, which exhibit good inhibitory effects on human pancreatic cancer cells PANC-1, gastric cancer cells AGS, and triple-negative breast cancer cells HCC1806 and HCC1937. The IC ₅₀ value of the optimal molecule is in the range of 0.6 to 2.5 μM. The optimal molecule can be used as an anti-tumor lead molecule.

(2) The present invention uses a commercial product (5-bromo-2-chloronicotinonitrile) as a substrate and can obtain the above-mentioned novel pyrazolopyridine derivatives through a three-step chemical reaction (cyclization, carbon-carbon coupling, amidation) or a four-step chemical reaction (cyclization, carbon-carbon coupling, methylation, amidation). The preparation method has the advantages of low cost, simple conditions, and easy separation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 and 2 are the hydrogen spectrum and carbon spectrum of compound 5, respectively;

Figures 3 and 4 are the hydrogen spectrum and carbon spectrum of compound 6, respectively;

Figures 5 and 6 are the hydrogen spectrum and carbon spectrum of compound 7, respectively;

Figures 7 and 8 are the hydrogen spectrum and carbon spectrum of compound 8, respectively;

Figures 9 and 10 are the hydrogen spectrum and carbon spectrum of compound 11, respectively;

Figures 11 and 12 are the hydrogen spectrum and carbon spectrum of compound 14, respectively;

Figures 13 and 14 are the hydrogen spectrum and carbon spectrum of compound 17, respectively;

Figures 15 and 16 are the hydrogen spectrum and carbon spectrum of compound 19, respectively;

FIG17 is a graph showing the effect of compound 6 at different concentrations on apoptosis of human triple-negative breast cancer cells HCC1937.

Detailed ways

The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

1. Structure of pyrazolopyridine derivatives

The structure of the pyrazolopyridine derivatives provided by the present invention is as follows:

or/> ;

Wherein, R ₁ is selected from any one of the following structures:

;

_R2 is selected from any one of the following structures:

.

2. Preparation method of pyrazolopyridine derivatives

Example 1

23mmol 5-bromo-2-chloronicotinonitrile (Compound 1) was dissolved in 150mL anhydrous ethanol (EtOH), the reaction system was heated to 85°C and 115mmol hydrazine hydrate ( _H2N - _NH2 ) was added dropwise, the temperature of the reaction system was maintained and the reaction was stirred for 2h. After the reaction was completed, the reaction system was placed in an ice-water bath for low-temperature recrystallization, vacuum filtered, and dried to obtain Compound 2 (yellow crystals) with a crude yield of 83%.

14 mmol of compound 2, 16.8 mmol of compound 3 and 28 mmol of potassium phosphate were dissolved in 80 mL of dioxane aqueous solution (dioxane: water = 2:1, v/v), nitrogen was blown for 10 min, 0.7 mmol of ditri-tert-butylphosphine palladium was added to the reaction system, and the reaction was carried out at 110°C for 72 h. After the reaction was completed, the reaction system was vacuum filtered with diatomaceous earth, extracted with ethyl acetate and water, and dried to obtain compound 4 with a crude yield of 68%.

0.3 mmol of compound 4 was dissolved in 3 mL of N, N-dimethylformamide (DMF), and 0.6 mmol of bromoacetyl chloride was added, and the mixture was reacted at 20°C for 3 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporation was performed with V _{dichloromethane} /V _methanol = 50: 1 column, and compound 5 (white solid) was obtained after drying, with a yield of 47%.

The hydrogen spectrum and carbon spectrum of compound 5 are shown in Figures 1 and 2 respectively. The specific information of each spectrum is as follows:

¹ H NMR ( d ₆ -DMSO, 400 MHz) δ 13.54 (s, 1H), 11.19 (s, 1H), 8.92 (d, J = 2 Hz, 1H), 8.72 (d, J = 2. Hz, 1H), 8.02-7.95 (m, 4H), 4.41 (s, 2H), 3.49-3.42 (m, 1H), 1.20 (s, 3H), 1.18 (s, 3H);

¹³ C NMR ( d ₆ -DMSO, 100 MHz) δ 165.5, 152.2, 149.4, 143.7, 140.2, 136.0, 131.7, 130.0 (2C), 128.2 (2C), 127.5, 108.1, 54.8, 43.4, 15.8 (2C).

According to high resolution mass spectrometry (HR-MS) detection, the hydrogenation calculated value of the molecular weight of compound 5 (2-bromo-N-(5-(4-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide) is 437.1929, and the hydrogenation theoretical value is 437.0283.

Example 2

Compound 4 was prepared by the same method as Example 1.

0.3 mmol of compound 4 was dissolved in 3 mL of N, N-dimethylformamide (DMF), and 0.6 mmol of chloroacetyl chloride was added, and the mixture was reacted at 20°C for 3 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporation was performed with V _{dichloromethane} /V _methanol = 50: 1 column, and compound 6 (white solid) was obtained after drying, with a yield of 48%.

The hydrogen spectrum and carbon spectrum of compound 6 are shown in Figures 3 and 4 respectively. The specific information of each spectrum is as follows:

¹ H NMR ( d ₆ -DMSO, 400 MHz) δ 13.53 (s, 1H), 11.18 (s, 1H), 8.91 (d, J = 2 Hz, 1H), 8.72 (d, J = 2 Hz, 1H), 8.01-7.95 (m, 4H), 4.41 (s, 2H), 3.49-3.42 (m, 1H), 1.20 (s, 3H), 1.18 (s, 3H);

¹³ C NMR ( d ₆ -DMSO, 100 MHz) δ 165.5, 152.2, 149.4, 143.7, 140.2, 136.0, 131.7, 130.0 (2C), 128.2, 127.5, 108.1, 54.8, 15.8 2.

According to high resolution mass spectrometry (HR-MS) detection, the hydrogenation calculated value of the molecular weight of compound 6 (2-chloro-N-(5-(4-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide) is 393.0775, and the hydrogenation theoretical value is 393.0783.

Example 3

Compound 4 was prepared by the same method as Example 1.

0.3 mmol of compound 4 was dissolved in 3 mL of N, N-dimethylformamide (DMF), and 0.6 mmol of 2,2,2-trichloroethyl chloroformate was added, and the mixture was reacted at 20°C for 3 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporation was performed with V _{dichloromethane} /V _methanol = 50: 1 column, and compound 7 (white solid) was obtained after drying with a yield of 49%.

The hydrogen spectrum and carbon spectrum of compound 7 are shown in Figures 5 and 6 respectively. The specific information of each spectrum is as follows:

¹ H NMR ( d ₆ -DMSO, 400 MHz) δ 13.47 (s, 1H), 10.84 (s, 1H), 8.92 (d, J = 2 Hz, 1H), 8.64 (d, J = 2 Hz, 1H), 8.03-7.95 (m, 4H), 5.00 (s, 2H), 3.50-3.43 (m, 1H), 1.20 (s, 3H), 1.19 (s, 3H);

¹³ C NMR ( d ₆ -DMSO, 100 MHz) δ 153.2, 152.1, 149.4, 143.7, 139.7, 136.1, 130.0 (2C), 128.3 (2C), 127.5, 108.4, 96.3, 74.4, 54.7, 15.8 (2C).

According to high resolution mass spectrometry (HR-MS) detection, the hydrogenation calculated value of the molecular weight of compound 7 (2,2,2-trichloroethyl (5-(4-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)carbamate) is 491.0108, and the hydrogenation theoretical value is 491.0109.

Example 4

Compound 4 was prepared by the same method as Example 1.

0.3 mmol of compound 4 was dissolved in 3 mL of N, N-dimethylformamide (DMF), and 0.6 mmol of isobutyryl chloride was added, and the mixture was reacted at 20°C for 3 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporated and filtered with V _{dichloromethane} /V _methanol = 50: 1. After drying, compound 8 (white solid) was obtained with a yield of 37%.

The hydrogen spectrum and carbon spectrum of compound 8 are shown in Figures 7 and 8 respectively. The specific information of each spectrum is as follows:

¹ H NMR ( d ₆ -DMSO, 400 MHz) δ 13.38 (s, 1H), 10.72 (s, 1H), 8.88 (d, J = 2 Hz, 1H), 8.73 (d, J = 2 Hz, 1H), 8.00-7.95 (m, 4H), 3.49-3.42 (m, 1H), 2.81-2.74 (m, 1H), 1.20 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H), 1.15 (s, 3H);

¹³ C NMR ( d ₆ -DMSO, 100 MHz) δ 175.9, 152.2, 149.2, 143.9, 141.1, 135.9, 132.2, 130.0 (2C), 128.3 (2C), 127.2, 108.2, 54.8, 34.5, 20.0 (2C), 15.8 (2C).

According to high resolution mass spectrometry (HR-MS) detection, the calculated value of the molecular weight of compound 8 (N-(5-(4-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)isobutyramide) with sodium addition was 409.0903, and the theoretical value with sodium addition was 409.1305.

Example 5

Compound 2 was prepared by the same method as Example 1.

14 mmol of compound 2, 16.8 mmol of compound 9 and 28 mmol of potassium phosphate were dissolved in 80 mL of dioxane aqueous solution (dioxane: water = 2:1, v/v), nitrogen was blown for 10 min, 0.7 mmol of ditri-tert-butylphosphine palladium was added to the reaction system, and the reaction was carried out at 80°C for 72 h. After the reaction was completed, the reaction system was vacuum filtered with diatomaceous earth, extracted with ethyl acetate and water, and dried to obtain compound 10 with a crude yield of 62%.

0.3 mmol of compound 10 was dissolved in 3 mL of N, N-dimethylformamide (DMF), and 0.6 mmol of chloroacetyl chloride was added, and the mixture was reacted at 20°C for 3 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporation was performed with V _{dichloromethane} /V _methanol = 50: 1 column, and compound 11 (white solid) was obtained after drying with a yield of 62%.

The hydrogen spectrum and carbon spectrum of compound 11 are shown in Figures 9 and 10 respectively. The specific information of each spectrum is as follows:

¹ H NMR ( d ₆ -DMSO, 400 MHz) δ 13.37 (s, 1H), 11.09 (s, 1H), 8.78 (d, J = 2 Hz, 1H), 8.50 (d, J = 2 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 4.40 (s, 2H), 3.80 (s, 3H);

¹³ C NMR ( d ₆ -DMSO, 100 MHz) δ 165.4, 159.5, 151.7, 149.1, 139.6, 130.8, 129.7, 129.1, 128.7 (2C), 115.2 (2C), 108.2, 55.7, 43.4.

According to high resolution mass spectrometry (HR-MS) detection, the calculated value of the molecular weight of compound 11 (2-chloro-N-(5-(4-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide) was 339.0617 and the theoretical value was 339.0619.

Example 6

Compound 2 was prepared by the same method as Example 1.

14 mmol of compound 2, 16.8 mmol of compound 12 and 28 mmol of potassium phosphate were dissolved in 80 mL of dioxane aqueous solution (dioxane: water = 2:1, v/v), nitrogen was blown for 10 min, 0.7 mmol of ditri-tert-butylphosphine palladium was added to the reaction system, and the reaction was carried out at 80°C for 72 h. After the reaction was completed, the reaction system was vacuum filtered with diatomaceous earth, extracted with ethyl acetate and water, and dried to obtain compound 13 with a crude yield of 83%.

0.3 mmol of compound 13 was dissolved in 3 mL of N, N-dimethylformamide (DMF), and 0.6 mmol of chloroacetyl chloride was added, and the mixture was reacted at 20°C for 3 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporation was performed with V _{dichloromethane} /V _methanol = 50: 1 column, and compound 14 (white solid) was obtained after drying, with a yield of 54%.

The hydrogen spectrum and carbon spectrum of compound 14 are shown in Figures 11 and 12 respectively. The specific information of each spectrum is as follows:

¹ H NMR ( d ₆ -DMSO, 400 MHz) δ 13.37 (s, 1H), 11.09 (s, 1H), 8.78 (d, J = 2 Hz, 1H), 8.50 (d, J = 2 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 4.68-4.62 (m, 1H), 4.40 (s, 2H), 1.29 (s, 3H), 1.28 (s, 3H);

¹³ C NMR ( d ₆ -DMSO, 100 MHz) δ 165.4, 157.7, 151.6, 149.1, 139.6, 130.5, 129.7, 129.1, 128.7 (2C), 116.8 (2C), 108.2, 69.8, 43.4, 22.4 (2C).

According to high resolution mass spectrometry (HR-MS) detection, the hydrogenation calculated value of the molecular weight of compound 14 (2-chloro-N-(5-(4-isopropoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide) was 34.1104, and the hydrogenation theoretical value was 345.113.

Example 7

Compound 2 was prepared by the same method as Example 1.

14 mmol of compound 2, 16.8 mmol of compound 15 and 28 mmol of potassium phosphate were dissolved in 80 mL of dioxane aqueous solution (dioxane: water = 2:1, v/v), nitrogen was blown for 10 min, 0.7 mmol of ditri-tert-butylphosphine palladium was added to the reaction system, and the reaction was carried out at 80°C for 72 h. After the reaction was completed, the reaction system was vacuum filtered with diatomaceous earth, extracted with ethyl acetate and water, and dried to obtain compound 16 with a crude yield of 85%.

0.3 mmol of compound 16 was dissolved in 3 mL of N, N-dimethylformamide (DMF), and 0.6 mmol of chloroacetyl chloride was added, and the mixture was reacted at 20°C for 3 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporation was performed with V _{dichloromethane} /V _methanol = 50: 1 column, and compound 17 (white solid) was obtained after drying, with a yield of 50%.

The hydrogen spectrum and carbon spectrum of compound 17 are shown in Figures 13 and 14 respectively. The specific information of each spectrum is as follows:

¹ H NMR ( d ₆ -DMSO, 400 MHz) δ 13.50 (s, 1H), 11.16 (s, 1H), 8.89 (d, J = 2 Hz, 1H), 8.68 (d, J = 2 Hz, 1H), 7.96-7.89 (m, 4H), 7.60 (s, 1H), 4.41 (s, 2H), 3.33 (s, 1H), 1.13 (s, 9H);

¹³ C NMR ( d ₆ -DMSO, 100 MHz) δ 165.5, 152.1, 149.4, 143.7, 141.8, 140.1, 131.3, 127.9 (2C), 127.8, 127.7 (2C), 108.1, 53.9, 43.4, 30.3 (3C).

According to high resolution mass spectrometry (HR-MS) detection, the hydrogenation calculated value of the molecular weight of compound 17 (N-(5-(4-(N-(tert-butyl)aminosulfonyl)cyclohexa-2,4-dien-1-yl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-2-chloroacetamide) was 424.1011, and the hydrogenation theoretical value was 424.1205.

Example 8

Compound 4 was prepared by the same method as Example 1.

0.3 mmol of compound 4 was dissolved in 3 mL of N, N-dimethylformamide (DMF), 0.45 mmol of sodium hydride was added under ice bath, and then 0.45 mmol of potassium iodide was added dropwise to react for 2 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporation was followed by column chromatography with V _{dichloromethane} /V _methanol = 50: 1. After drying, compound 18 (yellow solid) was obtained with a yield of 48%.

0.3 mmol of compound 18 was dissolved in 3 mL of N, N-dimethylformamide (DMF), and 0.6 mmol of chloroacetyl chloride was added, and the mixture was reacted at 20°C for 3 h. After the reaction, the reaction system was quenched with water, extracted with ethyl acetate and water, and vacuum rotary evaporated and filtered with V _{dichloromethane} /V _methanol = 50: 1. After drying, compound 19 (white solid) was obtained with a yield of 42%.

The hydrogen spectrum and carbon spectrum of compound 19 are shown in Figures 15 and 16 respectively. The specific information of each spectrum is as follows:

¹ H NMR ( d ₆ -DMSO, 400 MHz) δ 11.24 (s, 1H), 8.95 (d, J = 2 Hz, 1H), 8.73 (d, J = 2 Hz, 1H), 8.01-7.96 (m, 4H), 4.41 (s, 2H), 3.01 (s, 3H), 3.50-3.43 (m, 1H), 1.20 (s, 3H), 1.19 (s, 3H);

¹³ C NMR ( d ₆ -DMSO, 100 MHz) δ 165.4, 150.3, 149.4, 143.6, 138.9, 136.1, 132.1, 130.0 (2C), 128.3 (2C), 127.4, 108.4, 54.8, 43.4, 34.0, 15.8 (2C).

According to high resolution mass spectrometry (HR-MS) detection, the hydrogenation calculated value of the molecular weight of compound 19 (2-chloro-N-(5-(4-(isopropylsulfonyl)phenyl)-1-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide) was 407.0941, and the hydrogenation theoretical value was 407.0939.

III. Application and evaluation of the anticancer activity of pyrazolopyridine derivatives

1. Cell proliferation inhibition experiment

The MTT colorimetric method was used in vitro to detect the cell proliferation inhibitory ability of the above-mentioned pyrazolopyridine derivatives (Compound 5, Compound 6, Compound 7, Compound 8, Compound 11, Compound 14, Compound 17, Compound 19) on human pancreatic cancer cell line PANC-1, human gastric cancer cell line AGS, and triple-negative breast cancer cells HCC1806 and HCC1937.

Experimental principle: MTT can be reduced to insoluble blue-purple formazan crystals by amber dehydrogenase in the mitochondria of living cells. The crystals can be dissolved by dimethyl sulfoxide (DMSO) and detected by a microplate reader at a wavelength of 570nm. The absorbance reflects the cell survival rate.

The half inhibitory concentration (IC ₅₀ ) refers to the drug concentration required for 50% cell death when tumor cells are exposed to the drug for a certain period of time. By measuring the MTT absorbance of different drug concentrations, the dose response curve can be fitted to calculate the IC ₅₀ value.

The determination method is as follows:

(1) Tumor cells were seeded into 96-well plates at a density of 4000 cells/well and cultured in a _CO2 incubator for 24 h;

(2) The test compound was diluted 1/2 times from 25 μM and incubated with tumor cells for 48 h, with three sets of parallel wells set for each concentration;

(3) After drug treatment, 20 μL of MTT solution (2.5 mg/mL) was added to each well of the plate and incubated in a 37°C incubator for 2 h;

(4) Discard the supernatant, add 150 μL DMSO to each well, and shake thoroughly until the formazan is completely dissolved;

(5) Use an ELISA reader to detect the absorbance of the sample at 570 nm, fit the dose-response curve, and calculate the _IC50 value of the test compound.

After calculation, the toxicity test results of the above compounds on human pancreatic cancer cell line PANC-1, human gastric cancer cell line AGS, triple-negative breast cancer cells HCC1806 and HCC1937 after administration for 48 hours are shown in Table 1.

Table 1 Toxicity test results of each compound on three tumor cells (IC ₅₀ value, μM)

Cell proliferation inhibition experiments show that the compounds developed by the present invention exhibit good in vitro tumor inhibition effects on pancreatic cancer, gastric cancer and triple-negative breast cancer.

(1) Compound 6 has the best inhibitory effect on triple-negative breast cancer cells HCC1806, with an IC ₅₀ value of 2.4 μM;

(2) Compounds 6, 11, 14, 17 and 19 showed the best inhibitory effects on triple-negative breast cancer cells HCC1937, with IC ₅₀ values of 0.6 μM, 1.9 μM, 1.1 μM, 0.7 μM and 1.3 μM, respectively;

(3) Compounds 5, 6, 7, 17 and 19 had the best inhibitory effects on human pancreatic cancer cell PANC-1, with IC ₅₀ values of 2.5 μM, 2.5 μM, 2.6 μM, 1.3 μM and 0.7 μM, respectively;

(4) Compounds 6 and 14 had the best inhibitory effects on the human gastric cancer cell line AGS, with IC ₅₀ values of 1.2 μM and 1.1 μM, respectively;

(5) Compound 6 has good inhibitory effects on triple-negative breast cancer cells HCC1806 and HCC1937, human pancreatic cancer cells PANC-1 and human gastric cancer cell line AGS, and is the preferred compound.

2. Cell apoptosis experiment

Flow cytometry was used in vitro to detect the effect of the preferred compound (compound 6) on the apoptosis of triple-negative breast cancer cells HCC1937.

Experimental principle: Phosphatidylserine (PS) of normal cells is located on the inner side of the cell membrane. When early cell apoptosis occurs, the PS on the inner side of the membrane will be turned outward to the surface of the cell membrane and bind to the fluorescein-labeled membrane annexin V. When cells undergo late apoptosis or necrosis, the permeability of the cell membrane increases, allowing the nucleic acid dye (PI) that cannot enter normal cells to enter the cell and stain the DNA in the cell. The Annexin V and PI double staining method can be used to identify and quantify early apoptotic, late apoptotic and necrotic cells.

The determination method is as follows:

(1) Tumor cells were inoculated into 5× ⁵ cm2 culture dishes at a density of 300,000 cells/dish and cultured in a _CO2 incubator for 24 h;

(2) When the cells to be tested entered the logarithmic growth phase, compound 6 was added at different concentrations (0 μM, 0.5 μM, 1.0 μM, 3.0 μM) for administration;

(3) 24 h after drug treatment, transfer the cells to be tested to a 15 mL centrifuge tube, centrifuge at 1000 g for 5 min, discard the supernatant and collect the cells, and wash twice with PBS;

(4) Add 195 μL of diluted 1× Annexin V Binding Buffer to the cells and resuspend the cells;

(5) Add 5 μL Annexin V-FITC and 5 μL PI dye to the resuspended cells and mix gently;

(6) Incubate at 20°C in the dark for 15 min. Mix by pipetting during incubation. After incubation, perform the test on the microarray machine.

The effects of compound 6 at different concentrations on apoptosis of triple-negative breast cancer cells HCC1937 are shown in Figure 17.

As shown in Figure 17, compound 6 can significantly induce apoptosis of triple-negative breast cancer cells HCC1937. After administration of 3.0 μM for 24 hours, it induces early apoptosis of about 36.2% of triple-negative breast cancer cells HCC1937 and late apoptosis of 1.12% of triple-negative breast cancer cells HCC1937.

It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled 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 embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims (8)

1. Pyrazolopyridine derivative is characterized in that the pyrazolopyridine derivative has the following structure:

Or/> ；

Wherein, R ₁ is selected from any one of the following structures:

；

r ₂ is selected from any one of the following structures:

。

2. the method for producing pyrazolopyridine derivative according to claim 1, comprising the steps of:

step 1, cyclizing: dissolving 5-bromo-2-chloronicotinonitrile in absolute ethyl alcohol, heating to 85 ℃, dropwise adding hydrazine hydrate, maintaining the temperature of a reaction system, stirring, and after the reaction is finished, sequentially carrying out low-temperature recrystallization, vacuum filtration and drying to obtain a compound 2;

Step 2, carbon-carbon coupling: dissolving a compound 2, a compound A and potassium phosphate in dioxane aqueous solution, wherein the compound A is a compound 3, a compound 9, a compound 12 or a compound 15, blowing nitrogen, adding ditri-butyl phosphine palladium into a reaction system, reacting at 80-110 ℃, and sequentially carrying out diatomite vacuum filtration, ethyl acetate and water extraction and drying after the reaction is finished to obtain a compound B;

Step 3, amidation: dissolving a compound B in N, N-dimethylformamide, adding a compound C, wherein the compound C is bromoacetyl chloride, chloroacetyl chloride, 2-trichloroethyl chloroformate or isobutyryl chloride, reacting at 20 ℃, and sequentially quenching, extracting with ethyl acetate and water, performing vacuum rotary evaporation, passing through a column and drying after the reaction is finished to obtain the pyrazolopyridine derivative of claim 1;

Wherein the structures of compound 2, compound 3, compound 9, compound 12 and compound 15 are shown below, respectively:

、/>、/>、/>、。

3. The method for producing pyrazolopyridine derivative according to claim 2, wherein in step 1, the ratio of 5-bromo-2-chloronicotinonitrile to hydrazine hydrate is 23mmol:115mmol.

4. The method for producing pyrazolopyridine derivative according to claim 2, wherein in step 2, the ratio of the amounts of compound 2, compound a, potassium phosphate and di-tri-t-butylphosphine palladium is 14mmol:16.8mmol:28mmol:0.7mmol.

5. The method for producing pyrazolopyridine derivative according to claim 2, wherein in step 3, the ratio of compound B to compound C is 0.3mmol:0.6mmol.

6. The method for producing pyrazolopyridine derivative according to claim 2, further comprising a methylation step, specifically:

After the step 2 is finished, the compound B is dissolved in N, N-dimethylformamide, sodium hydride is added under ice bath, then potassium iodide is added dropwise for reaction, quenching, ethyl acetate and water extraction, vacuum rotary evaporation, column passing and drying are sequentially carried out after the reaction is finished, the compound D is obtained, methylation is finished, and then the compound D is substituted for the compound B for amidation reaction.

7. The method for producing pyrazolopyridine derivative according to claim 6, wherein in the methylation step, the amount ratio of compound B, sodium hydride and potassium iodide is 0.3mmol:0.45mmol:0.45mmol.

8. The use of pyrazolopyridine derivative according to claim 1 in the preparation of an anticancer drug, wherein the anticancer drug is an anti-pancreatic cancer drug, an anti-gastric cancer drug or an anti-breast cancer drug.