A pyrazolopyridine compound and its preparation method and application

Abstract

The present invention discloses a pyrazolopyridine compound, its preparation method, and its application, belonging to the field of pharmaceutical technology. The present invention organically combines a pyrazolopyridine analog with a sulfonamide compound using a chemical design method based on skeleton transition and splicing principles. Compared with existing drugs (such as sorafenib), the compound of the present invention has a better anti-cancer effect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a novel pyrazolopyridine analogue and a pharmaceutically acceptable salt or carrier thereof, as well as the use of the compound in treating liver cancer. Background Art

[0002] The latest World Cancer Report, released by the World Health Organization's International Agency for Research on Cancer (IARC), shows that global cancer cases reached 19.29 million, with 9.96 million deaths. Liver cancer ranks third globally in deaths and is one of the top three causes of cancer-related death. Despite significant progress in the diagnosis and treatment of liver cancer, the prognosis is poor, and the disease is prone to metastasis and drug resistance, resulting in a high mortality rate. Therefore, the development of safe, effective, and innovative targeted drugs for liver cancer is of great significance. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention provides a pyrazolopyridine compound, its preparation method, and its application. The preparation method of the present invention organically combines a pyrazolopyridine analog with a sulfonamide compound using a chemical design method based on skeleton hopping and splicing principles to obtain a novel anti-liver cancer bioactive compound with higher activity and improved bioavailability.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] A method for preparing a pyrazolopyridine compound comprises the following steps:

[0006] Step 1: Compound 3, anhydrous potassium phosphate, N,N-dimethylformamide and distilled water, as well as Compound 1 or Compound 2, are heated under gas protection, and tetrakis-triphenylphosphine palladium is added to carry out a mixing reaction;

[0007] Step 2: After cooling, add ammonium chloride solution, extract, dry, filter and concentrate, and then elute and purify by silica gel column chromatography to obtain compound 4 or compound 5; when compound 4 is obtained, go to step 3; when compound 5 is obtained, go to step 4;

[0008] Step 3: Under ice bath, a mixture of compound 4 and sodium hydroxide in dichloromethane was added with three dichloromethane solutions of benzenesulfonyl chlorides, mixed and reacted, extracted, dried, filtered and concentrated, and then eluted and purified by silica gel column chromatography to obtain compound A;

[0009] Step 4: Compound 5, pyridine, and 4-dimethylaminopyridine in dichloromethane were mixed under ice bath, and three dichloromethane solutions of benzenesulfonyl chloride were added, mixed and reacted, extracted, dried, filtered, concentrated, and eluted, and purified by silica gel column chromatography to obtain compound B;

[0010] Wherein, the structural formula of compound A is:

[0011]

[0012] The structural formula of the compound B is:

[0013]

[0014] Wherein, R is H, 4-F or 4-CF3;

[0015] The structural formula of compound 1 is:

[0016] The structural formula of compound 2 is:

[0017] The structural formula of compound 3 is:

[0018] The structural formula of compound 4 is:

[0019] The structural formula of compound 5 is:

[0020] Optionally, after step 1 is completed, the reaction is detected by thin layer chromatography.

[0021] Optionally, ethyl acetate is used as the extraction agent in the extraction process in step 2.

[0022] Optionally, ethyl acetate and petroleum ether are used as eluents in the elution process in step 2, step 3 and step 4.

[0023] Optionally, dichloromethane is used as the extraction agent in the extraction process in step 3 and step 4.

[0024] The second aspect of the present invention relates to a pyrazolopyridine compound having the structural formula:

[0025] or

[0026] R is H, 4-F or 4-CF3.

[0027] The third aspect of the present invention relates to the use of the above-mentioned compound in the preparation of anti-liver cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below with reference to the accompanying drawings.

[0029] Figure 1 A synthetic route for the compounds of this application;

[0030] Figure 2 is the hydrogen spectrum of compound E5 of the present application;

[0031] Figure 3 This is the carbon spectrum of compound E5 of the present application;

[0032] Figure 4 The experimental results of the anti-cancer cell proliferation effect of the compounds of the present application;

[0033] Figure 5 The experimental results of the anti-cancer cell migration and invasion effects of the compounds of the present application;

[0034] Figure 6 These are the experimental results of the compounds of the present application on promoting apoptosis of cancer cells. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1:

[0037] This embodiment discloses a compound A, whose structural formula is:

[0038]

[0039] Wherein, R is H, 4-F or 4-CF3.

[0040] Specifically, the preparation method of compound A is disclosed in this embodiment, such as Figure 1 As shown, the following steps may be specifically included:

[0041] Compound 1 (1 g, 5 mmol), compound 3 (1.25 g, 6 mmol), anhydrous potassium phosphate (K3PO4, 1.59 g, 7.5 mmol), N,N-dimethylformamide (DMF, 60 mL), and distilled water (40 mL) were added to a 250 mL round-bottom flask and reacted under nitrogen for 30 minutes. Tetrakis-triphenylphosphine palladium (Pd(PPh3)4, 289 mg, 0.25 mmol) was then added and the mixture was allowed to react at 100°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. After cooling, saturated ammonium chloride solution was added, and the mixture was extracted three times with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate and petroleum ether (1:9 by volume) as eluents to obtain a light yellow powder in approximately 60% yield. The mass spectrum, melting point, and NMR data are as follows:

[0042] ESI-MS m / z:199.23 (M+H) + .MP:170-172℃. 1 H NMR (400MHz, DMSO-d6) δ11.60(s,1H),8.47(d,J=2.1Hz,1H),8.12(s,1H),8.08(d,J= 2.0Hz,1H),7.89(s,1H),7.47–7.43(m,1H),6.42(dd,J=3.4,1.8Hz,1H),3.87(s,3H). 13 C NMR (101MHz, DMSO-d6) δ147.40,140.39,135.85,127.24,126.60,124.04,120.70,120.41,119.69,99.69,38.65.

[0043] Preparation of Compounds E1-E3

[0044] Under ice, a mixture of compound 4 (198 mg, 1 mmol) and solid sodium hydroxide (100 mg, 2.5 mmol) in dichloromethane (DCM, 5 mL) was placed in a 100 mL round-bottom flask. Three dichloromethane solutions of benzenesulfonyl chloride (4-H, 4-F, or 4-CF3, 2 mmol) were slowly added. The reaction was continued at this temperature for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The pH was adjusted to a weakly acidic state with 2 mol / L hydrochloric acid solution, and the mixture was extracted three times with dichloromethane. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate and petroleum ether (1:9 by volume) as eluents to obtain a light yellow powder in an approximately 70-75% yield.

[0045] The mass spectrum, melting point and NMR data are as follows:

[0046] Compound E1: ESI-MS m / z: 339.39 (M+H) + .MP:220-222℃. 1 H NMR (600MHz, CDCl3) δ8.54(d,J=2.1Hz,1H),8.19(dd,J=8.5,1.1Hz,2H),7.87(d,J=2.1Hz,1H),7.73(s,1H),7.7 1(d,J=4.0Hz,1H),7.61(s,1H),7.57(t,J=7.5Hz,1H),7.48(t,J=7.9Hz,2H),6.58(d,J=4.0Hz,1H),3.96(s,3H). 13C NMR(151MHz,Chloroform-d)δ146.18,143.00,138.54,136.90,134.16,129.18,1 28.08,127.17,127.13,126.30,126.23,124.59,123.16,120.30,105.58,39.32.

[0047] Compound E2: ESI-MS m / z: 357.38 (M+H) + .MP:190-192℃. 1 H NMR (600MHz, CDCl3) δ8.54(d,J=2.1Hz,1H),8.24(dd,J=9.0,5.0Hz,2H),7.88(d,J=2.1Hz,1H),7.7 4(s,1H),7.69(d,J=4.0Hz,1H),7.61(s,2H),7.17–7.13(m,1H),6.59(d,J=4.0Hz,1H),3.96(s,3H). 13 C NMR(151MHz,Chloroform-d)δ166.91,165.20,146.04,143.00,136.91,134.43,131. 17,127.13,126.97,126.31,124.75,123.17,120.22,116.59,116.44,105.71,39.33.

[0048] Compound E3: ESI-MS m / z: 407.38 (M+H) + .MP:186-188℃. 1 H NMR (600MHz, CDCl3) δ8.55(d,J=2.0Hz,1H),8.35(d,J=8.3Hz,2H),7.89(d,J=2.1Hz,1H), 7.78–7.73(m,2H),7.70(d,J=4.0Hz,1H),7.63(s,1H),6.62(d,J=4.0Hz,1H),3.98(s,3H). 13 C NMR(151MHz,Chloroform-d)δ146.06,143.11,141.79,136.74,135.84,135.62,128. 77,127.34,126.92,126.53,126.39,126.36,124.88,123.27,120.18,106.24,39.34.

[0049] Example 2

[0050] This embodiment discloses another compound B, the structural formula of which is:

[0051]

[0052] Wherein, R is H, 4-F or 4-CF3;

[0053] Specifically, the preparation method of compound B is also disclosed in this embodiment, such as Figure 1 As shown, the following steps may be specifically included:

[0054] Preparation of intermediate compound 5

[0055] Compound 2 (1 g, 5 mmol), compound 3 (1.25 g, 6 mmol), anhydrous potassium phosphate (K3PO4, 1.59 g, 7.5 mmol), N,N-dimethylformamide (DMF, 60 mL), and distilled water (40 mL) were added to a 250 mL round-bottom flask and reacted under nitrogen for 30 minutes. Tetrakis-triphenylphosphine palladium (Pd(PPh3)4, 289 mg, 0.25 mmol) was then added and the mixture was allowed to react at 100°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. After cooling, saturated ammonium chloride solution was added, and the mixture was extracted three times with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate and petroleum ether (1:9 by volume) as eluents to obtain a light yellow powder in approximately 65% ​​yield. The mass spectrum, melting point, and NMR data are as follows:

[0056] ESI-MS m / z:175.21(M+H) + .MP:217-219℃. 1 H NMR(600MHz,DMSO-d6)δ8.13(d,J=1.9Hz,1H),7.94(s,1H),7.70(d,J=0.7Hz,1H ),7.53(dd,J=8.5,2.4Hz,1H),6.45(d,J=8.5Hz,1H),5.83(s,2H),3.82(s,3H). 13 C NMR (151MHz, DMSO-d6) δ158.20,144.20,135.08,134.22,126.31,119.74,116.99,107.97,38.55.

[0057] Preparation of compounds E4-E6

[0058] Under ice, a mixture of compound 5 (174 mg, 1 mmol), pyridine (200 μL), and 4-dimethylaminopyridine (DMAP, 0.1 mmol) in dichloromethane (DCM, 5 mL) was placed in a 100 mL round-bottom flask. Three dichloromethane solutions of benzenesulfonyl chloride (4-H, 4-F, or 4-CF3, 2 mmol) were slowly added, and the reaction was continued at this temperature for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The pH was adjusted to a weakly acidic state with 2 mol / L hydrochloric acid solution, and the mixture was extracted three times with dichloromethane. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography using ethyl acetate and petroleum ether (1:9 by volume) as eluents to obtain a light yellow powder in approximately 75-80% yield. The mass spectrum, melting point, and NMR data are as follows:

[0059] Compound E4: ESI-MS m / z: 315.36 (M+H) + .MP:170-172℃. 1 H NMR(600MHz,DMSO-d6)δ8.32(s,1H),8.09(s,1H),7.89(dd,J=7.9,4.8Hz,3H),7.83(s ,1H),7.59(t,J=7.3Hz,1H),7.55(t,J=7.4Hz,2H),7.15(d,J=8.8Hz,1H),3.83(s,3H). 13 C NMR (151MHz, DMSO-d6) δ150.35,141.36,135.87,132.48,129.41,129.04,128.24,127.70,126.67,117.77,113.32,38.69.

[0060] Compound E5 (such as Figure 2 and Figure 3 ): ESI-MS m / z: 333.35 (M+H) + .MP:222-224℃. 1 H NMR (600MHz, DMSO-d6) δ8.31(s,1H),8.10(s,1H),7.95(dd,J=8.9,5.2Hz,2H),7.91(dd,J=8.8 ,2.4Hz,1H),7.83(d,J=0.8Hz,1H),7.38(t,J=8.8Hz,2H),7.15(d,J=8.7Hz,1H),3.83(s,3H). 13C NMR (151MHz, DMSO-d6) δ164.89,163.23,137.93,135.88,129.72,129.66,127.73,117.68,116.22,116.07,113.54,38.69.

[0061] Compound E6: ESI-MS m / z: 383.36 (M+H) + .MP:210-212℃. 1 H NMR(600MHz,DMSO-d6)δ8.27(s,1H),8.09(d,J=8.3Hz,3H),7.96(dd,J=8.9,2.4Hz,1 H),7.92(d,J=8.4Hz,2H),7.84(d,J=0.8Hz,1H),7.22(d,J=8.9Hz,1H),3.83(s,3H). 13 CNMR(151MHz,DMSO-d6)δ151.11,146.16,135.92,131.78,127.81,127.47,126.23,124.46,122.65,117.38,114.36,38.71.

[0062] Example 3

[0063] In this embodiment, a method for experimentally verifying the anti-liver cancer effect of the compounds prepared in the above-mentioned embodiment 1 and embodiment 2 is disclosed, which may specifically include the following steps:

[0064] (1) Cell proliferation ability: HepG2 cells in the logarithmic growth phase were seeded in 96-well plates at 5000 cells / well and cultured in DMEM medium containing 10% FBS. After the cells adhered, the cells were treated with compounds E1-E6 (10 μM) for 48 h, 20 μL MTT (4 mg / mL) was added to each well and cultured for another 4 h at 37°C. 200 μL DMSO was added to each well and shaken at room temperature for 10 min. The optical density was read at 490 nm on a microplate reader. The results are shown as follows: Figure 4 As shown in Figure A, according to the experimental results, compound E5 was screened out with the highest inhibition rate.

[0065] like Figure 4 As shown in Figure 2B, the cell viability of compound E5 at different concentrations (0.78, 1.56, 3.13, 6.25, 12.5, and 25 μM) was determined using the MTT assay described above. The inhibition rate (%) was calculated using the formula: 1-[(OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%. IC was obtained using nonlinear fitting using GraphPad Prism 8 software. 50The experimental results were obtained by at least three experiments. The results of the cell proliferation ability experiment showed that the IC 50 The values ​​are IC 50 =10.06 μM and 9.41 μM.

[0066] (2) Clone-forming ability, such as Figure 4 Figure C: HepG2 cells in the logarithmic growth phase were seeded at 1000 cells / well in a 6-well plate. After overnight culture, the cells were treated with different concentrations of compound E5 (0, 0.5, 1, and 2 μM) for 48 hours. Fresh medium was replaced, and the cells were cultured at 37°C until visible cell colonies formed. The cells were fixed with 4% paraformaldehyde and stained with crystal violet for 15 minutes. The difference in the antiproliferative effect of the drug was evaluated based on the number and size of colonies. The results of the colony formation assay showed that compound E5 had significant antiproliferative activity and was concentration-dependent.

[0067] (3) Cell migration ability, such as Figure 5 As shown in Figure A: Cells in logarithmic growth phase were seeded in a 24-well plate and cultured overnight until 100% confluence was achieved. A scratch was then made perpendicular to the bottom of the plate. The plates were washed three times with 1× PBS and replaced with FBS-free medium. The cells were then treated with various concentrations of compound E5 (0, 2.5, 5, and 10 μM). The cells were incubated at 37°C in a 5% CO2 incubator, and photographed at 0, 24, and 48 hours. The results of the cell scratch healing assay demonstrated that compound E5 exhibited significant anti-migratory activity in a time- and concentration-dependent manner.

[0068] (4) Cell invasion ability, such as Figure 5 B: 5×10 4 200 μL serum-free medium was added to each well, and 700 μL medium containing 10% FBS was added to the bottom. Different concentrations of compound E5 (0.5 μM) were added to the two chambers. The cells were allowed to migrate for 48 hours, and the non-migrating cells on the upper surface of the membrane were gently scraped off with a cotton swab. The migrated cells were fixed in 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 15 minutes. The migrated cells in three randomly selected areas were counted under an inverted microscope. The results of the cell transwell experiment showed that compound E5 had significant anti-invasive ability.

[0069] (5) Mitochondrial membrane potential Figure 6As shown: Cells in logarithmically growing phase were seeded into 6-well plates and cultured overnight. After cells adhered, they were grouped and treated. The culture medium was aspirated and washed three times with PBS for 2 minutes each. 500 μL of 10 μg / mL JC-1 culture medium was added to each well, brought to room temperature, and gently shaken to mix. The cells were incubated at 37°C for 15-20 minutes. After the incubation period, the supernatant was aspirated and the cells were washed twice with PBS. 500 μL of PBS was added and the red fluorescence intensity of JC-1 aggregates and the green fluorescence intensity of JC-1 monomers were observed using a fluorescence microscope. Mitochondrial membrane potential assays showed that treatment of cells with compound E5 resulted in green fluorescence, indicating a decrease in mitochondrial membrane potential. With increasing compound E5 concentration, the green fluorescence intensity gradually increased while the red fluorescence intensity gradually decreased, demonstrating that compound E5 has a significant pro-apoptotic activity in a concentration-dependent manner. Whole-cell lysates were prepared from groups treated with compound E5 at different concentrations (0, 2.5, 5, and 10 μM) and protein was quantified and denatured. The corrected protein lysate was electrophoresed on SDS-PAGE and transferred to a PVDF membrane. After blocking with skim milk for 1 hour, the membrane was incubated with the primary antibody against the target protein at 4°C overnight and the corresponding secondary antibody at room temperature for 1 hour. Chemiluminescent staining was used to detect the protein levels and phosphorylation of key pro-apoptotic proteins such as Caspase 3 and Bcl-2. Western blotting results showed that after treatment of cells with compound E5, the protein levels and phosphorylation levels of Caspase 3 and Bcl-2 gradually increased, further demonstrating the significant pro-apoptotic potential of compound E5.

[0070] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0071] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. Use of a compound in the preparation of an anti-liver cancer drug, wherein the compound has the structural formula: or R is H, 4-F or 4-CF3.

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