Pyrazolo [1, 5-alpha] pyrimidine compound as well as synthesis method and application thereof

Abstract

The invention discloses a pyrazolo [1, 5-alpha] pyrimidine compound as well as a synthesis method and application thereof, the structural general formula of the pyrazolo [1, 5-alpha] pyrimidine compound is shown in the specification, R is 4-NO2-Ph-, 3-NO2-Ph-, 2-NO2-Ph-, 3-F-Ph-or 4-F-Ph-, and the invention also specifically discloses a pyrazolo [1, 5-alpha] pyrimidine compound as well as a preparation method and application of the pyrazolo [1, 5-alpha] pyrimidine compound. The invention relates to a synthesis method of a [1, 2, 5-alpha] pyrimidine compound and application of the compound in preparation of medicines for preventing or / and treating liver cancer. The pyrazol [1, 5, a] pyrimidine compound synthesized by the invention has a good inhibition effect on liver cancer HepG2 cells, the antitumor activity of the pyrazol [1, 5, a] pyrimidine compound in the liver cancer HepG2 cells is superior to that of a positive control drug CX-4945 and a compound I-1, and particularly, compounds I-2 and I-5 have excellent liver cancer HepG2 cell proliferation inhibition performance.

Description

Technical Field

[0002] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a class of pyrazolo[1,5-α]pyrimidine compounds, their synthesis methods, and applications. Background Technology

[0004] Pyrazolo[1,5-a]pyrimidine compounds have been reported in numerous publications, exhibiting various biological activities including antitumor, antiviral, antibacterial, anti-inflammatory, central nervous system drug, and cardiovascular effects (Turowec JP, Duncan JS, French AC, et al. Protein kinase CK2 is a constitutively active enzyme that promotes cellsurvival: strategies to identify CK2 substrates and manipulate its activity in mammalian cells. Methods Enzymol, 2010, 484:471-493). Casein kinase 2 (CK2) is a ubiquitous and highly specific protein serine or threonine kinase, generally found in a tetrameric complex composed of α and β subunits, and possesses biological targeting properties. Inhibition of CK2 is thought to affect multiple pathways simultaneously, participating in the regulation of cell growth, proliferation, and differentiation, playing a crucial role in cell survival and apoptosis, and promoting cell survival by regulating apoptosis signaling pathways (Pack M, Wrublewsky S, Montenarh M, et al. SGC-CK2-1 Is an Efficient Inducer of Insulin Production and Secretion in Pancreatic β-Cells. Pharmaceutics, 2021, 14). Alterations in CK2 function are associated with a variety of human diseases, including cancer, and can be used to treat cancer, infectious diseases, inflammatory conditions, neurodegenerative diseases, pain, and immune disorders (Haddach M, Tran JA, Pierre F, et al. Pyrazolopyrimidines and Related Heterocycles as CK2 Inhibitors. US20110152240(2024-05-29)).For example, SGC-CK2-1 (Carrow Wells, David H, Drewry, Julie E, Pickett, Alison D, et al. SGC-CK2-1: the first selective chemical probe for the pleiotropic kinase CK2. ChemRxiv.2020, 1) is a highly efficient, ATP-competitive CK2 chemical probe that is selective for both CK2 subtypes, CK2α and CK2α' (Trembley JH, Unger GM, Tobolt DK, et al. Systemic administration of antisense oligonucleotides simultaneously targeting CK2α and α' subunits reduce orthotopic xenograft prostate tumors in mice. Molecular & Cellular Biochemistry, 2011, 356(1-2):21-35.), with IC50 values ​​of 36 nM and 16 nM, respectively. The same article also reported on the CK2-1 analog N-{5-[(3-cyano-7-cyclopropylaminopyrazole[1,5,-α]pyrimidine-5-)amino]-2-methylphenyl}-acetamide (compound I-1), with IC50 values ​​of 3.2 nM and 1.8 nM, respectively. However, there are currently no records of pyrazolo[1,5-a]pyrimidine compounds inhibiting the proliferative activity of HepG2 liver cancer cells.

[0005] Summary of the Invention

[0007] The purpose of this invention is to provide a class of pyrazolo[1,5-α]pyrimidine compounds and a method for their synthesis. The pyrazolo[1,5-α]pyrimidine compounds synthesized by this method have good inhibitory activity against the proliferation of HepG2 liver cancer cells and can be further used to prepare drugs for the prevention and / or treatment of liver cancer.

[0008] To achieve the above objectives, this invention employs the following technical solution: a class of pyrazolo[1,5-α]pyrimidine compounds, with the following general structural formula:

[0009]

[0010] Where R is 4-NO2-Ph-, 3-NO2-Ph-, 2-NO2-Ph-, 3-F-Ph-, or 4-F-Ph-.

[0011] The pyrazolo[1,5-α]pyrimidine compounds of this invention specifically include compounds with the following structures:

[0012] , , , , ;

[0013] The IC50 values ​​of the corresponding compounds inhibiting the proliferation of HepG2 liver cancer cells were 40.60 μM, 32.36 μM, 40.24 μM, 38.37 μM, 36.77 μM, and 39.25 μM, respectively.

[0014] The synthetic method of the pyrazolo[1,5-α]pyrimidine compound of the present invention specifically involves the following steps: Compound 4, i.e., 5,7-dichloropyrazolo[1,5-a]pyrimidine-3-carboxynitrile, is reacted with cyclopropylamine to obtain compound 5, i.e., 5-chloro-7-cyclopropylaminopyrazolo[1,5-a]pyrimidine-3-carboxynitrile; then, compound 5 is reacted with a substituted aniline compound to obtain the target compound I, i.e., the pyrazolo[1,5-α]pyrimidine compound, wherein the substituted aniline compound is o-nitroaniline, m-nitroaniline, p-nitroaniline, m-fluoroaniline, or p-fluoroaniline. The corresponding synthetic route is as follows:

[0015] .

[0016] Furthermore, the specific synthesis process of compound 5 is as follows: compound 4 and anhydrous ethanol are added to a reaction vessel and heated and stirred at 40~60℃ to dissolve. Cyclopropylamine is then added to carry out the reaction. The reaction solution changes from pale yellow turbidity to milky white suspension. The reaction process is analyzed by thin-layer chromatography. The developing solvent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:2. After the reaction is completed, heating is stopped and the temperature is lowered to room temperature to obtain compound 5.

[0017] Furthermore, the specific synthesis process of compound I is as follows: compound 5, substituted aniline compounds, cesium carbonate, palladium dichloride, sodium acetate, and 1,1'-binaphthyl-2,2'-bisdiphenylphosphine are added to a sealed tube, followed by the addition of 1,4-dioxane and sealing. The tube is heated to 110-130°C for reaction. After the reaction is completed, heating is stopped, and the tube is allowed to cool to room temperature. Dichloromethane and water are added, and the tube is allowed to separate into layers. The lower organic phase is washed with water, and the organic phase is concentrated and separated by column chromatography to obtain compound I.

[0018] The use of the pyrazolo[1,5-α]pyrimidine compound described in this invention in the preparation of drugs for the prevention and / or treatment of liver cancer.

[0019] The application of the pyrazolo[1,5-α]pyrimidine compound described in this invention in the preparation of a drug for inhibiting the proliferation of HepG2 liver cancer cells.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: The pyrazole[1,5,a]pyrimidine compounds I-2 to I-6 synthesized in the present invention have good inhibitory effects on HepG2 liver cancer cells, and their anti-tumor activity in HepG2 liver cancer cells is superior to that of the positive control drug CX-4945 and compound I-1. In particular, compounds I-2 and I-5 show excellent inhibitory performance on the proliferation of HepG2 liver cancer cells. Attached Figure Description

[0022] Figure 1 The IC50 curve of compound CX-4945 inhibiting HepG2 liver cancer cells.

[0023] Figure 2 The IC50 curve of compound I-1 inhibiting HepG2 liver cancer cells is shown.

[0024] Figure 3 The IC50 curve of compound I-2 inhibiting HepG2 liver cancer cells is shown.

[0025] Figure 4 The IC50 curve of compound I-3 inhibiting HepG2 liver cancer cells is shown.

[0026] Figure 5 The IC50 curve of compound I-4 inhibiting HepG2 liver cancer cells is shown.

[0027] Figure 6 The IC50 curve of compound I-5 inhibiting HepG2 liver cancer cells is shown.

[0028] Figure 7 The IC50 curve of compound I-6 inhibiting HepG2 liver cancer cells is shown. Detailed Implementation

[0030] The technical solution and its effects of the present invention will be described in detail below with reference to specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of the present invention is not limited to the following embodiments.

[0031] The chemical reactions in the embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. To obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0032] The reactions described herein can be monitored using any suitable method known in the art. For example, product formation can be monitored using broad-spectrum methods such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C), infrared spectroscopy, liquid chromatography-mass spectrometry, etc.

[0033] While preferred embodiments of this application are described herein, these embodiments are provided by way of example only. It should be understood that variations of the embodiments described herein can also be used to implement this application. Those skilled in the art will understand that various variations, changes, and substitutions may occur without departing from the scope of this application. It should be understood that the scope of protection of each aspect of this application is determined by the claims, and the methods and structures within the scope of these claims, as well as their equivalents, are all within the scope of these claims.

[0034]

[0035] 1.1 Synthesis of N-(2-methyl-5-nitrophenyl)acetamide (compound 2)

[0036] 20.0 g of compound 1 was placed in a three-necked flask, and 100 mL of a mixture of acetic acid and fuming nitric acid in a volume ratio of 1:1 was slowly added dropwise while stirring at 0 °C. The mixture was heated to 50 °C in an oil bath for 6 h, cooled to room temperature, poured into ice water, filtered under reduced pressure, and dried to obtain 23.98 g of compound 2, with a yield of 95.00% and a purity of 99.80%.

[0037] 1H NMR (600MHz DMSO-d6) δ:

[0038] 2.14(s,3H,-CH3),2.35(s,3H,-CH3),7.48-7.50(d,1H,=CH-),7.90-7.93(t,1H,=CH-),8.49-8.50(d,1H,=CH-),9.59(s,1H,-NH-).

[0039] 1.2 Synthesis of N-(5-amino-2-methylphenyl)acetamide (compound 3)

[0040] 100.0 g of compound 2, 1000 mL of anhydrous ethanol, and 12.0 g of Raney nickel were added to a high-pressure reactor. The reaction was carried out at 0.80 MPa and 75 °C for 7 h. The mixture was then cooled and filtered to remove the Raney nickel. The filtrate was concentrated under reduced pressure to a material ratio of 1:2 (m / v). The mixture was then cooled to 0 °C and stirred for 2 h. After filtration, the filter cake was a white crystalline flocculent solid, namely 82.64 g of compound 3, with a yield of 98.69% and a purity of 99.37%.

[0041] 1H NMR (600MHz DMSO-d6) δ:

[0042] 2.02(s,6H,-CH3),4.83(s,2H,-NH2),6.30-6.32(d,1H,=CH-),6.70(s,1H,=CH-),6.81-6.83(d,1H,=CH-),9.01(s,1H,-NH-).

[0043] 1.3 Synthesis of 5-chloro-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylonitrile (compound 5)

[0044] 10.0 g of compound 4 and 80 mL of anhydrous ethanol were added to a round-bottom flask and heated and stirred at 50 °C to dissolve. Then, 8.0 g of cyclopropylamine was added, and the reaction solution immediately changed from a pale yellow turbidity to a milky white suspension. The reaction was allowed to proceed for 2 h, and the reaction progress was analyzed by thin-layer chromatography using a 5:2 volume ratio mixture of petroleum ether and ethyl acetate as the developing solvent. After the reaction was completed, heating was stopped and the solution was cooled to room temperature to obtain 10.76 g of compound 5, with a yield of 97.80% and a purity of 99.5%.

[0045] 1H NMR (600MHz DMSO-d6) δ:

[0046] 0.74-0.78(m,2H,-CH2),0.86-0.91(m,2H,-CH2-),2.73-2.78(m,1H,-CH-),6.61(s,1H,=CH-),8.68(s,1H,=CH-),9.24(s,1H,-NH-).

[0047] 1.4 Synthesis of N-{5-[(3-cyano-7-cyclopropylaminopyrazole[1,5,-α]pyrimidin-5-)amino]-2-methylphenyl}-acetamide (compound I-1)

[0048]

[0049] Compound 5 (0.50 g), 4-methyl-3-ethylaminoaniline (compound 3) (0.52 g), cesium carbonate (1.0 g), palladium dichloride (0.11 g), sodium acetate (0.10 g), and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) (0.67 g) were added to a 15 mL sealed tube. Then, 10 mL of 1,4-dioxane was added and the tube was sealed. The mixture was heated to 120 °C and reacted for 12 h. Heating was then stopped, and the mixture was allowed to cool to room temperature. 50 mL of dichloromethane and 50 mL of water were added, and the mixture was allowed to separate into layers. The lower organic phase was washed twice with 50 mL of water. The organic phase was concentrated and separated by column chromatography (petroleum ether:ethyl acetate = 1:4, V / V) to obtain compound I-1 (0.18 g), with a yield of 24.00% and a purity of 98.82%.

[0050] 1H NMR (600MHz DMSO-d6) δ:

[0051] 0.69-0.72(q,2H,-CH2-),0.78-0.83(m,2H,-CH2-),1.99(s,3H,-CH3),2.16(s,3H,-CH3),2.59(s,1H,-CH-),6.01(s,1H,-N H-),7.14-7.16(d,1H,=CH-),7.64(s,1H,=CH-),8.20(s,1H,=CH-),8.34(s,1H,=CH-),9.25(s,1H,-NH-),9.60(s,1H,-NH-).

[0052] 13C NMR (600MHz DMSO-d6) δ: 168.68, 157.43, 151.40, 148.75, 145.51, 138.53, 137.07, 125.61, 76.78, 76.58, 60.23, 23.83, 23.79, 17.73, 7.01.

[0054] 1.5 5-(4-fluorophenylamino)-3-cyano-7-(cyclopropylamino)pyrazole[1,5,-α]pyrimidine (compound I-2)

[0055]

[0056] Compound 5 (0.50 g), 4-fluoroaniline (0.36 g), cesium carbonate (1.03 g), palladium dichloride (0.11 g), sodium acetate (0.10 g), and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) (0.65 g) were added to a 15 mL sealed tube. Then, 10 mL of 1,4-dioxane was added and the tube was sealed. The mixture was heated to 120 °C and reacted for 12 h. Heating was stopped, and the mixture was allowed to cool to room temperature. 50 mL of dichloromethane and 50 mL of water were added, and the mixture was allowed to separate into layers. The lower organic phase was washed twice with 50 mL of water. The organic phase was concentrated and separated by column chromatography (petroleum ether:ethyl acetate = 1:4, V / V) to obtain compound I-2 (0.25 g), with a yield of 40.65% and a purity of 99.82%.

[0057] 1H NMR (600MHz DMSO-d6) δ:

[0058] 0.71-0.74(m,2H,-CH2-),0.80-0.83(m,2H,-CH2-),2.59-2.61(m,1H,-CH-),5.94(s,1H,=CH-),7.18 -7.20(m,2H,=CH-),7.75-7.77(m,2H,=CH-),8.24(s,1H,-NH-),8.36(s,1H,-NH-),9.68(s,1H,=CH-).

[0059] 13C NMR(600MHz DMSO-d6) δ: 158.79, 157.29, 157.21, 151.32, 148.78, 145.51, 137.18, 121.62, 121.57, 115.83, 115.69, 115.28, 76.97, 76.71, 23.78, 7.01.

[0061] 1.6 5-(3-fluorophenylamino)-3-cyano-7-(cyclopropylamino)pyrazole[1,5,-α]pyrimidine (compound I-3)

[0062]

[0063] Compound 5 (0.50 g), 3-fluoroaniline (0.36 g), cesium carbonate (1.03 g), palladium dichloride (0.11 g), sodium acetate (0.10 g), and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) (0.67 g) were added to a 15 mL sealed tube. Then, 6 mL of 1,4-dioxane was added and the tube was sealed. The mixture was heated to 120 °C and reacted for 8 h. Heating was then stopped, and the mixture was allowed to cool to room temperature. 50 mL of dichloromethane and 50 mL of water were added, and the mixture was allowed to separate into layers. The lower organic phase was washed twice with 50 mL of water. The organic phase was concentrated and separated by column chromatography (petroleum ether:ethyl acetate = 1:4, V / V) to give compound I-3 (0.22 g), with a yield of 35.77% and a purity of 99.15%.

[0064] 1H NMR (600MHz DMSO-d6) δ:

[0065] 0.74-0.75(m,2H,-CH2-),0.81-0.84(m,2H,-CH2-),2.62(m,1H,-CH-),6.00(s,1H,=CH-),6.79-6.83(t,1H,=C H-),7.32-7.40(m,2H,=CH-),7.95-7.98(d,1H,=CH-),8.34(s,1H,-NH-),8.40(s,1H,-NH-),9.88(s,1H,=CH-).

[0066] 13C NMR(600MHz DMSO-d6) δ: 163.58,161.99,157.04,151.15,148.90,145.59,142.78,142.70,130.66,130 .60,115.23,115.14,108.66,108.52,106.30,106.13,77.38,77.32,23.80,7.02.

[0068] 1.7 5-(4-nitrophenylamino)-3-cyano-7-(cyclopropylamino)pyrazole[1,5,-α]pyrimidine (compound I-4)

[0069]

[0070] Compound 5 (0.50 g), 4-nitroaniline (0.44 g), cesium carbonate (1.03 g), palladium dichloride (0.11 g), sodium acetate (0.10 g), and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) (0.65 g) were added to a 15 mL sealed tube. Then, 6 mL of 1,4-dioxane was added and the tube was sealed. The mixture was heated to 120 °C and reacted for 8 h. Heating was then stopped, and the mixture was allowed to cool to room temperature. 50 mL of dichloromethane and 50 mL of water were added, and the mixture was allowed to separate into layers. The organic phase was washed twice with 50 mL of water. The organic phase was concentrated and separated by column chromatography (petroleum ether:ethyl acetate = 3:5, V / V) to obtain compound I-4 (0.19 g), with a yield of 264% and a purity of 98.7%.

[0071] 1H NMR (600MHz DMSO-d6) δ:

[0072] 0.73-0.77(q,2H,-CH2-),0.83-0.88(q,2H,-CH2-),2.65-2.66(t,1H,-CH-),6.11(s,1H,=CH-),8.02- 8.04(d,2H,=CH-),8.23-8.25(d,2H,=CH-),8.46(s,1H,-NH-),8.52(s,1H,-NH-),10.36(s,1H,=CH-).

[0073] 13C NMR (600MHz DMSO-d6) δ: 156.43, 150.77, 149.22, 147.41, 145.95, 141.15, 125.53, 118.48, 114.94, 78.25, 77.93, 23.86, 7.04.

[0075] 1.8 5-(3-nitrophenylamino)-3-cyano-7-(cyclopropylamino)pyrazole[1,5,-α]pyrimidine (compound I-5)

[0076]

[0077] Compound 5 (0.50 g), 3-nitroaniline (0.44 g), cesium carbonate (1.03 g), palladium dichloride (0.11 g), sodium acetate (0.10 g), and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) (0.65 g) were added to a 15 mL sealed tube. Then, 10 mL of 1,4-dioxane was added and the tube was sealed. The mixture was heated to 120 °C and reacted for 12 h. Heating was stopped, and the mixture was allowed to cool to room temperature. 50 mL of dichloromethane and 50 mL of water were added, and the mixture was allowed to separate into layers. The lower organic phase was washed twice with 50 mL of water. The organic phase was concentrated and separated by column chromatography (petroleum ether:ethyl acetate = 1:4, V / V) to obtain compound I-5 (0.21 g), with a yield of 29.16% and a purity of 99.80%.

[0078] 1H NMR (600MHz DMSO-d6) δ:

[0079] 0.73-0.77(q,2H,-CH2-),0.83-0.88(m,2H,-CH2-),2.66(s,1H,-CH-),6.03(s,1H,=CH-),7.60-7.64(t,1H,=CH-),7.82-7. 85(q,1H,=CH-),8.04-8.07(q,1H,=CH-),8.42(s,1H,-NH-),8.43(s,1H,=CH-),8.97-8.98(t,1H,-NH-),10.15(s,1H,=CH-).

[0080] 13C NMR(600MHz DMSO-d6) δ: 156.83, 150.90, 149.04, 148.59, 145.73, 142.16, 130.39, 125.16, 116.51, 114.87, 113.32, 77.71, 77.46, 23.86, 7.02.

[0082] 1.9 5-(2-nitrophenylamino)-3-cyano-7-(cyclopropylamino)pyrazole[1,5,-α]pyrimidine (compound I-6)

[0083]

[0084] Compound 5 (0.50 g), 2-nitroaniline (0.44 g), cesium carbonate (1.03 g), palladium dichloride (0.11 g), sodium acetate (0.10 g), and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) (0.65 g) were added to a 15 mL sealed tube. 10 mL of 1,4-dioxane was added, and the tube was sealed. The mixture was heated to 120 °C and reacted for 12 h. Heating was then stopped, and the mixture was allowed to cool to room temperature. 50 mL of dichloromethane and 50 mL of water were added, and the mixture was allowed to separate into layers. The lower organic phase was washed twice with 50 mL of water. The organic phase was concentrated and separated by column chromatography (petroleum ether:ethyl acetate = 1:4, V / V) to give compound I-6 (0.25 g), with a yield of 34.72% and a purity of 99.59%.

[0085] 1H NMR (600MHz DMSO-d6) δ:

[0086] 0.74-0.75(d,2H,-CH2-),0.84-0.85(d,2H,-CH2-),2.65-2.67(t,1H,-CH-),6.10(s,1H,=CH-),7.30-7.34(t,1H,=CH-),7.70 -7.74(t,1H,=CH-),7.81-7.83(d,1H,=CH-),8.00-8.02(d,1H,=CH-),8.37(s,1H,-NH-),8.42(s,1H,=CH-),9.96(s,1H,=CH-).

[0087] 13C NMR (600MHz DMSO-d6) δ: 156.53, 150.62, 149.29, 145.88, 142.36, 125.65, 125.62, 124.38, 114.80, 77.49, 77.31, 23.91, 7.03.

[0089] 2. Antiproliferative activity of compound I against HepG2 cells

[0090] The antiproliferative activity of compound I against HepG2 cells was detected using the MTT assay.

[0091] 2.1 Solution Preparation

[0092] 2.1.1 Preparation of cell culture medium:

[0093] Remove the fetal bovine serum stored at -20°C and thaw it slowly at 4°C. Then, place it in a constant temperature water bath at 37°C until completely thawed. Preheat the DMEM culture medium (hereinafter referred to as culture medium) in a constant temperature water bath at 37°C for 20 minutes. After sterilizing the culture medium and serum, place them in a clean bench. After sterilizing the bottle and tube openings, pour out 50 mL of blank culture medium from the culture medium bottle, then pour 50 mL into the remaining culture medium, invert and mix well. Then, mark the date and store at 4°C for later use.

[0094] 2.1.2 Preparation of MTT solution:

[0095] Add 4 tubes of MTT powder to 200 mL of PBS for cell use (concentration of 5 mg / mL), then filter in a clean bench and dispense into 10 mL tubes, about 8 mL per tube. Store at -20°C for later use. Protect from light throughout the preparation process.

[0096] 2.1.3 Preparation of different drug concentrations of Compound I:

[0097] A certain amount of compound I was weighed and added to DMSO (concentration of 1 mM, compound I:DMSO = 1 mol: 1 L) to dissolve it. The final concentrations in 100 μL wells were 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, and 128 μM.

[0098] 2.2 Detection of the antiproliferative activity of compound I against HepG2 cells using the MTT assay

[0099] HepG2 cells were passaged and counted, then seeded into 96-well plates with 3 replicates per group. After seeding, the plates were placed in an incubator for incubation. When the cells grew to cover 50% of the bottom of the wells, the drugs were prepared, the old culture medium was discarded, the drugs were added, and then the plates were incubated for 24 hours.

[0100] Beforehand, the MTT stored at -20℃ was preheated at 37℃ for 15 minutes in a constant temperature water bath to fully melt it. Then, 10 μL of MTT was added to each well and incubated in an incubator for 4 hours.

[0101] After incubation, remove the 96-well plate from the incubator, remove all liquid with a pipette, add 150 μL of DMSO to each well, place it on a shaker to mix thoroughly, and then measure the absorbance at 490 nm using a microplate reader.

[0102] The data was then statistically analyzed using GraphPad Prism to obtain its IC50 value.

[0103] Table 1. HepG2 activity data of different compounds

[0104]

[0105] 2.3 Conclusion

[0106] The IC50 values ​​of compound I (see Table 1) were 73.44 μM, 40.60 μM, 32.36 μM, 40.24 μM, 38.37 μM, 36.77 μM, and 39.25 μM, respectively. This indicates that pyrazole[1,5,a]pyrimidine compound I exhibited good inhibitory activity against HepG2 cells. The antitumor activity of compounds I-2 to I-6 synthesized in this invention in HepG2 liver cancer cells was superior to the positive control drug CX-4945, with compounds I-2 and I-5 showing excellent inhibitory properties against HepG2 cell proliferation.

[0107] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A class of pyrazolo[1,5-α]pyrimidine compounds, characterized in that... The general structural formula is: Where R is 4-NO2-Ph-, 3-NO2-Ph-, 2-NO2-Ph-, 3-F-Ph-, or 4-F-Ph-.

2. The pyrazolo[1,5-α]pyrimidine compound according to claim 1, characterized in that... Specifically, compounds with the following structures are included: 、 、 、 、 ； The IC50 values ​​of the corresponding compounds inhibiting the proliferation of HepG2 liver cancer cells were 40.60 μM, 32.36 μM, 40.24 μM, 38.37 μM, 36.77 μM, and 39.25 μM, respectively.

3. A method for synthesizing the pyrazolo[1,5-α]pyrimidine compound according to claim 1, characterized in that... The specific synthetic process is as follows: Compound 4, namely 5,7-dichloropyrazolo[1,5-a]pyrimidine-3-carboxylonitrile, is reacted with cyclopropylamine to obtain compound 5, namely 5-chloro-7-cyclopropylaminopyrazolo[1,5-a]pyrimidine-3-carboxylonitrile; then compound 5 is reacted with substituted aniline compounds to obtain target compound I, namely pyrazolo[1,5-α]pyrimidine compound, wherein the substituted aniline compounds are o-nitroaniline, m-nitroaniline, p-nitroaniline, m-fluoroaniline, or p-fluoroaniline. The corresponding synthetic route is as follows: 。 4. The method for synthesizing the pyrazolo[1,5-α]pyrimidine compound according to claim 1, characterized in that... The specific synthesis process of compound 5 is as follows: compound 4 and anhydrous ethanol are added to a reaction vessel and heated and stirred at 40~60℃ to dissolve. Cyclopropylamine is then added to carry out the reaction. The reaction solution changes from pale yellow turbidity to milky white suspension. The reaction process is analyzed by thin-layer chromatography. The developing solvent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:

2. After the reaction is completed, heating is stopped and the temperature is lowered to room temperature to obtain compound 5.

5. The method for synthesizing the pyrazolo[1,5-α]pyrimidine compound according to claim 1, characterized in that... The specific synthesis process of compound I is as follows: compound 5, substituted aniline compounds, cesium carbonate, palladium dichloride, sodium acetate, and 1,1'-binaphthyl-2,2'-bisdiphenylphosphine are added to a sealed tube, followed by the addition of 1,4-dioxane and sealing. The tube is heated to 110-130°C for reaction. After the reaction is completed, heating is stopped, and the tube is allowed to cool to room temperature. Dichloromethane and water are added, and the tube is allowed to separate into layers. The lower organic phase is washed with water, and the organic phase is concentrated and separated by column chromatography to obtain compound I.

6. The use of the pyrazolo[1,5-α]pyrimidine compound according to claim 1 or 2 in the preparation of medicaments for the prevention and / or treatment of liver cancer.

7. The use of the pyrazolo[1,5-α]pyrimidine compound according to claim 1 or 2 in the preparation of a drug for inhibiting the proliferation of HepG2 liver cancer cells.

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