A pyrazole amide derivative, its preparation method and use as an anti-liver cancer drug

Abstract

The present invention discloses a pyrazoleamide derivative, its preparation method, and its use as an anti-liver cancer drug. The pyrazoleamide derivative has the following molecular structure: Its molecular design is based on a five-membered heterocycle of pyrazole, bridged by an amide bond to a pyridine heterocycle. Long-term testing has confirmed that it exhibits significant proliferation inhibition against liver cancer cells, making it a promising new anti-liver cancer drug.

Description

Technical Field

[0001] The present invention relates to a pyrazole amide derivative, in particular to a pyrazole amide derivative and a preparation method thereof and use thereof as an anti-liver cancer drug. Background Art

[0002] Liver cancer ranks fifth in incidence and second in mortality in Asia. Currently, surgery and systemic anti-tumor therapy are widely used in the treatment of liver cancer. However, among patients first diagnosed with liver cancer, less than 30% are suitable for radical surgery; therefore, systemic therapy plays a vital role in the management of patients with advanced liver cancer, usually including first-line and second-line anti-tumor treatment options. Although chemotherapy is an important part of multidisciplinary treatment, it kills cancer cells while also causing significant damage to normal cells, leading to a series of adverse reactions, which affect the quality of life of patients. As treatment progresses, the development of drug resistance and the risk of recurrence cannot be ignored. Therefore, there is an urgent need to develop highly selective alternative anti-liver cancer drugs.

[0003] Pyrazole compounds and their derivatives have widespread applications in medicine and pesticides due to their structural variability and high-efficiency, broad-spectrum biological activity. Many currently used anticancer drugs contain pyrazole structures, suggesting that pyrazoles are an important template for the discovery of new anticancer chemotherapeutic agents. Molecular design and development based on the five-membered heterocyclic ring structure of pyrazoles is a key approach to developing drugs with enhanced anticancer efficacy. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a pyrazole amide derivative, a preparation method thereof, and use thereof as an anti-liver cancer drug.

[0005] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0006] According to the first aspect of the present invention, a pyrazole amide derivative is first provided, which has the following molecular structure expression:

[0007]

[0008] The molecular design of this derivative is based on the five-membered heterocyclic ring of pyrazole, and is bridged by an amide bond to the pyridine heterocyclic ring. Long-term experiments have confirmed that it exhibits a significant proliferation inhibitory effect on liver cancer cells and is expected to become a new anti-liver cancer drug.

[0009] According to the second aspect of the present invention, there is also provided a method for preparing the pyrazole amide derivatives as described above, comprising the following steps:

[0010] a. Dissolve 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid (CAS: 500011-86-9) in an organic solvent, add thionyl chloride dropwise under ice bath, and react at room temperature for 2-12 hours. Purify after reaction to obtain an intermediate;

[0011] b. Dissolve the obtained intermediate in a solvent, and add triethylamine dropwise to a solution containing 4-amino-3,5-dichloro-2,6-difluoropyridine (CAS: 2840-00-8) under ice bath, then mix the two and react, stir at room temperature for 10-24 hours, and purify to obtain the pyrazole amide derivative.

[0012] In some preferred examples, in step a, the amount of thionyl chloride added is 3-5 times the molar amount of 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid, for example, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, etc.

[0013] In some preferred examples, in step a, the organic solvent is one or more of dichloromethane, chloroform, acetonitrile, ethyl acetate, ethanol, methanol, and tetrahydrofuran.

[0014] In some preferred examples, after the reaction in step a is completed, vacuum distillation, extraction, and purification are performed to obtain an intermediate.

[0015] In some preferred examples, in step b, the amount of 4-amino-3,5-dichloro-2,6-difluoropyridine added is 1-1.2 times the molar amount of 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid in step a, for example, 1 time, 1.05 times, 1.1 times, 1.15 times, 1.2 times, etc.

[0016] In some preferred examples, in step b, the amount of triethylamine added is 1.1-1.5 times the molar amount of 4-amino-3,5-dichloro-2,6-difluoropyridine, for example, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, etc.

[0017] In some preferred examples, the reaction solvent in step b is one or more of dichloromethane, chloroform, acetonitrile, ethyl acetate, ethanol, methanol, and tetrahydrofuran.

[0018] In some preferred examples, after the reaction in step b is completed, vacuum distillation, extraction, and purification are performed to obtain the pyrazole amide derivatives.

[0019] In the above reaction process of the present invention, it is speculated that the possible reaction process is as follows:

[0020]

[0021] According to the third aspect of the present invention, there is also provided a use of the pyrazoleamide derivatives as described above or the pyrazoleamide derivatives prepared by the method described above as anti-liver cancer drugs.

[0022] The present invention provides a novel derivative having a pyrazole skeleton, an amide bond, and a pyridine hybrid. Studies have found that the novel derivative has a significant effect on inhibiting the proliferation of liver cancer cells. The novel derivative has a short process flow, simple post-processing, and good reproducibility, which is conducive to industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the H NMR spectrum of the T1 compound prepared in Example 1.

[0024] Figure 2 The test results are for evaluating the anticancer activity of drugs in vitro.

[0025] Figure 3 These are the results of liver cancer cell staining experiments after drug treatment.

[0026] Figure 4 The results of Transwell cell invasion assay.

[0027] Figure 5 The results of tumor size comparison in the anticancer activity test of the drug in nude mice.

[0028] Figure 6 The results are the comparison of tumor weights in the anticancer activity test of the drug in nude mice. DETAILED DESCRIPTION

[0029] The present invention will be further described below through specific examples. The examples of the present invention are only for illustration of the present invention and do not limit the scope of the present invention.

[0030] Unless otherwise specified, the raw materials and reagents in the following examples of the present invention can be generally obtained through commercial channels.

[0031] [Example 1]

[0032] a. First, 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid (2 mmol) was dissolved in 10 ml of dichloromethane. Then, thionyl chloride (6 mmol) was slowly added dropwise at 0°C with stirring. After a few minutes, the mixture was transferred to room temperature and stirred for 4 hours. The mixture was distilled under reduced pressure and extracted twice with ethyl acetate and sodium chloride solution. After drying over anhydrous sodium sulfate, it was distilled under reduced pressure to obtain the intermediate.

[0033] b. The intermediate obtained in the above step was dissolved in dichloromethane (10 ml); 4-amino-3,5-dichloro-2,6-difluoropyridine (2 mmol) was then dissolved in dichloromethane (10 ml), and triethylamine (2.4 mmol) was added dropwise at 0°C with stirring. After stirring for several minutes, the dichloromethane solution of the intermediate was slowly added dropwise to the solution. The mixture was transferred to room temperature and stirred overnight. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted twice with ethyl acetate and sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, collected, and subjected to column chromatography using ethyl acetate: petroleum ether = 1:1 to obtain the target product, a pyrazole amide derivative, which was labeled as T1.

[0034] The nuclear magnetic hydrogen spectrum of T1 is as follows Figure 1 As shown, the parsed data is as follows:

[0035] 1H NMR (600MHz, chloroform-d) δ8.46(dd,J=4.7,1.6Hz,1H),7.91(dd,J=8.1,1.6Hz,1H),7.42(dd,J=8.0,4.6Hz,1H),7.07(s,1H).

[0036] [Example 2]

[0037] a. First, 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid (2 mmol) was dissolved in 10 ml of chloroform. Then, thionyl chloride (10 mmol) was slowly added dropwise at 0°C with stirring. After a few minutes, the mixture was transferred to room temperature and stirred for 8 hours. The mixture was distilled under reduced pressure, extracted twice with ethyl acetate and sodium chloride solution, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain the intermediate.

[0038] b. The intermediate obtained in the above step was dissolved in chloroform (10 ml) solution; then 4-amino-3,5-dichloro-2,6-difluoropyridine (2.4 mmol) was dissolved in chloroform (10 ml), and triethylamine (2.2 mmol) was added dropwise under stirring at 0°C. After stirring for several minutes, the chloroform solution of the intermediate was slowly added dropwise to the solution, and the mixture was transferred to room temperature and stirred overnight. The mixture was monitored by TLC. After the reaction was completed, the mixture was extracted twice with ethyl acetate and sodium chloride solution, dried over anhydrous sodium sulfate, and the organic phase was collected and subjected to column chromatography using ethyl acetate: petroleum ether = 1:1 to obtain the target product, a pyrazole amide derivative.

[0039] [Example 3]

[0040] a. First, 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid (2 mmol) was dissolved in 10 ml of acetonitrile. Then, thionyl chloride (8 mmol) was slowly added dropwise at 0°C with stirring. After a few minutes, the mixture was transferred to room temperature and stirred for 10 h. The mixture was distilled under reduced pressure, extracted twice with ethyl acetate and sodium chloride solution, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain the intermediate.

[0041] b. The intermediate obtained in the above step was dissolved in acetonitrile (10 ml) solution; then, 4-amino-3,5-dichloro-2,6-difluoropyridine (2.2 mmol) was dissolved in acetonitrile (10 ml), and triethylamine (3 mmol) was added dropwise under stirring at 0°C. After stirring for several minutes, the acetonitrile solution of the intermediate was slowly added dropwise to the solution. The mixture was transferred to room temperature and stirred overnight. TLC monitoring was performed. After completion of the reaction, the mixture was extracted twice with ethyl acetate and sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, collected, and subjected to column chromatography using ethyl acetate: petroleum ether = 1:1 to obtain the target product, a pyrazole amide derivative.

[0042]

In vitro anticancer activity evaluation

[0043] Test drugs: pyrazole amide derivatives (T1) prepared in Example 1, 5-fluorouracil (5-Fluorouracil)

[0044] Test cancer cells: human liver cancer cell line HepG2

[0045] Cell culture: RPMI Medium 1640 culture medium, 10% calf serum, and 0.01% L-glutamine were used as the culture medium. Cell lines were routinely cultured and passaged at 37°C in a humidified atmosphere of 5% CO2. All experiments used cells in the logarithmic growth phase.

[0046] Evaluation of anticancer activity by CCK-8 method: Digest the cells with 0.25% trypsin and prepare a single cell suspension. Inoculate 6000-7000 cells per well in a 96-well plate and incubate overnight at 37°C and 5% CO2. Add different concentrations of experimental samples. Use blank culture medium and cell suspension without drug action as blank control and negative control, respectively. Use different concentrations of anticancer drug 5-fluorouracil as positive control. Set up 6 replicates for each group and continue to culture for 24 hours. Add 10 μl of CCK-8 solution to each well and continue to culture for 1 hour. Discard the supernatant and detect the absorbance value (A) at 450 nm wavelength with a microplate reader. 450 The average cell inhibition rate was calculated according to the following formula:

[0047] Inhibition rate = (A 阴性对照 –A 实验样品 ) / (A 阴性对照 –A 空白对照 )×100%

[0048] Among them, the in vitro activity inhibition rate of the positive control 5-fluorouracil and the pyrazole amide derivative (T1) experimental sample prepared in Example 1 on the human liver cancer cell line HepG2 at different sample concentrations is as follows: Figure 2 The results showed that the pyrazole amide derivatives prepared by the present invention exhibited significant inhibitory effects on test cancer cells and exhibited superior in vitro anticancer activity compared to the positive control drug 5-fluorouracil. Therefore, the pyrazole amide derivatives of the present invention can be widely used in the field of anticancer drugs and have significant research value and application prospects.

[0049]

Liver cancer cell staining experiment after drug treatment

[0050] The liver cancer cell samples treated with drugs were stained with DAPI and EDU, and the Marge staining effect was superimposed to detect the effect of drugs on the proliferation of liver cancer cells.

[0051] (1) EdU labeling, fixation, washing, and permeabilization of cultured cells

[0052] a. Cell culture methods were the same as above. Human hepatocellular carcinoma cell line HepG2 cells in the logarithmic growth phase were added to a 6-well plate. 90 μg / ml of the pyrazole amide derivative (T1) and 5-fluorouracil (5-Fluorouracil) prepared in Example 1 were added, respectively. A cell suspension without drug treatment served as a blank control.

[0053] b. Prepare 2X EdU working solution: dilute EdU (10 mmol / L) with cell culture medium at a volume ratio of 1:500 to obtain 2X EdU working solution (20 μmol / L).

[0054] c. Add an equal volume of 2X EdU working solution (20 μmol / L) preheated at 37°C to the 6-well plate containing the samples to make the final EdU concentration 1X.

[0055] d. Continue to incubate the cells for 2 hours for cell labeling.

[0056] e. After the EdU labeling of cells was completed, the culture medium was removed and 1 ml of fixative (4% paraformaldehyde P0099) was added and fixed at room temperature for 15 minutes.

[0057] f. Remove the fixative and wash the cells three times with 1 ml of washing solution per well, each time for 3-5 minutes.

[0058] g. Remove the washing solution and add 1 ml of permeabilization solution (PBS containing 0.3% Triton X-100) to each well and incubate at room temperature for 10-15 minutes.

[0059] h. Remove the permeabilization solution and wash the cells 1-2 times with 1 ml of washing solution per well, each time for 3-5 minutes.

[0060] (2) EdU detection

[0061] a. Prepare the Click reaction solution according to the number of samples in the 6-well plate (adjustable in proportion) according to Table 1.

[0062] Table 1

[0063]

[0064] b. Remove the washing solution from the previous step in the 6-well plate.

[0065] c. Add 0.5 ml of Click reaction solution to each well and gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample.

[0066] d. Incubate at room temperature in the dark for 30 minutes.

[0067] e. Aspirate the Click reaction solution and wash three times with washing solution, each time for 3-5 minutes.

[0068] (3) Nuclear staining

[0069] a. Preparation of DAPI solution: dilute DAPI with PBS at a ratio of 1:1000.

[0070] b. Continue with step 2e above. After removing the wash solution, add 1 ml of DAPI solution to each well and incubate at room temperature in the dark for 10 minutes.

[0071] c. Aspirate and remove the DAPI solution.

[0072] d. Wash with detergent three times, 3-5 minutes each time.

[0073] e. Then perform fluorescence detection.

[0074] Specific test results such as Figure 3 As shown, it can be seen that the cell proliferation trend of the blank control group has almost no obvious change, while the cell proliferation of the T1 drug group in the present invention is significantly weakened, and the inhibitory effect on cell proliferation is even significantly better than that of the 5-fluorouracil control group. It can be seen that the pyrazole amide derivatives (T1) provided by the present invention can significantly inhibit the proliferation of HepG-2 cells.

[0075]

Transwell cell invasion assay

[0076] The specific experimental process is as follows:

[0077] A. Prepare Matrigel

[0078] 1. Thaw Matrigel at 4°C overnight and transfer to an ice box before the experiment.

[0079] 2. Place the ice prepared in advance in an ice box. All operations before gelation should be performed on ice. Place the pipette tip, centrifuge tube, and 24-well plate with Transwell chamber in an ice box for pre-cooling. Use the pre-cooled pipette tip to mix the Matrigengel.

[0080] B. Matrigel plating (for invasion assay, skip this step for migration)

[0081] 1. Dilute Matrigel: First, add 8 μL of Matrigel to a pre-cooled 1.5 ml EP tube, then add 64 μL of pre-cooled serum-free medium and mix thoroughly with a pipette tip. (Matrigenge and serum-free medium are diluted in a ratio of 1:8)

[0082] 2. Pipette 60 μL of diluted Matrigel, add it vertically into the upper chamber of Transwewll, and spread it evenly on the bottom.

[0083] 3. Place in an incubator (37°C, 5% CO2) and incubate for 3 hours to allow the Matrigel to polymerize into a thin film. (Note: Avoid creating bubbles during the gel-spreading process and spread the gel evenly.)

[0084] 4. After incubation, remove excess liquid from the upper chamber, add 100 μL of serum-free culture medium to each well, and place in the incubator for 30 minutes to hydrate the basement membrane.

[0085] 5. Aspirate the liquid in the upper chamber and check whether there is liquid passing through the chamber into the lower chamber. If not, the cells can be inoculated.

[0086] C. Preparation of cell suspension

[0087] 1. Cell culture methods were the same as above. Human hepatocellular carcinoma cell line HepG2 cells in the logarithmic growth phase were added to a 6-well plate. 90 μg / ml of the pyrazole amide derivative (T1) and 5-fluorouracil (5-Fluorouracil) prepared in Example 1 were added, respectively. A cell suspension without drug treatment was used as a blank control. The cells were cultured for 24 hours to obtain a cell suspension.

[0088] 2. Before making cell suspension, starve the cells of serum for 12-24 hours to further remove the effects of serum.

[0089] 3. Take cells with a confluence of 70-80%, digest them, centrifuge and discard the waste liquid, then resuspend the cells in serum-free medium and count and adjust the cell density to 2.5×10 5 / mL.

[0090] D. Inoculation of cells

[0091] 1. Add 500 μL of 10% FBS culture medium to the lower chamber of the 24-well plate. Then use tweezers to place the Transwell chamber into the 24-well plate.

[0092] 2. Add 200 μL of cell suspension to each well into the upper chamber of the Transwell.

[0093] 3. Fixation and staining can be performed after 24 hours of incubation in the incubator.

[0094] E. Cell fixation

[0095] 1. Remove the Transwell chamber, aspirate the culture medium, and gently wipe the Matrigengel and cells in the upper chamber with a cotton swab.

[0096] 2. Add 600 μL of 4% paraformaldehyde to a clean well of a 24-well plate, place the chamber in it, and fix it for 20-30 minutes.

[0097] F. Cell staining and counting

[0098] 1. Discard the fixative solution and rinse the chamber once in a 6 cm dish filled with PBS.

[0099] 2. Stain with 0.1% crystal violet for 5-10 minutes, rinse with PBS three times to remove the crystal violet that is not bound to the cells, and gently wipe the upper side of the chamber with a cotton swab to wipe off the dye that is non-specifically bound to the upper surface of the chamber for subsequent microscopic examination.

[0100] 3. After proper air drying, observe and count the cells under a microscope.

[0101] Specific test results such as Figure 4 As shown, it can be seen that the migration and invasion trends of cells in the blank control group showed almost no significant changes, indicating that this light condition did not affect the migration and invasion of HepG-2 cells, while the migration and invasion of cells in the T1 group were significantly weakened, indicating that this drug would significantly inhibit the migration and invasion of HepG-2 cells.

[0102]

Anticancer activity test in nude mice

[0103] Test animals: BALB / C male nude mice, weighing approximately 18-22 g.

[0104] Tumor cell line: human hepatocellular carcinoma cell line (HepG-2).

[0105] Experimental method: HepG-2 cells cultured to the logarithmic growth phase were digested, counted, and diluted to 5×10 6 / mL. Under sterile conditions, 0.2mL / mouse was inoculated subcutaneously on the flank of nude mice. Tumor size was observed and measured daily. 14 days after injection, visible tumors formed under the skin of nude mice. When the tumor diameter was ≥6mm, the volume was calculated using the following formula: Volume = Length × Width 2 / 2, tumor volume reaches 100mm 3 The nude mice were randomly divided into groups and a blank control group (PBS) and a 5-fluorouracil (5-Fluorouracil) control group (20 mg / kg) were set up at the same time. The mice were intraperitoneally injected once a day for 7 consecutive days. After that, the nude mice were euthanized and the tumors were dissected. The tumor size of each group was observed and recorded. The results are shown in the figure below. Figure 5 In addition, the average tumor weight of each group was statistically analyzed, and the results were as follows: Figure 6 As shown in Figure 3, T1 significantly inhibited the growth of transplanted tumor volume, and the body weight of mice remained relatively stable during the treatment process.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be considered within the scope of protection of the present invention.

Claims

1. A pyrazole amide derivative, characterized in that: It has the following molecular structure expression: 。 2. A method for preparing a pyrazole amide derivative according to claim 1, characterized in that: The following processes are included: a. Dissolve 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid in an organic solvent, add thionyl chloride dropwise under ice bath, and react at room temperature for 2-12 hours. Purify after reaction to obtain an intermediate; b. Dissolve the obtained intermediate in a solvent, and add triethylamine dropwise to the solution containing 4-amino-3,5-dichloro-2,6-difluoropyridine under ice bath, then mix the two and react, stir at room temperature for 10-24 hours, and purify to obtain the pyrazole amide derivative.

3. The method for preparing the pyrazole amide derivatives according to claim 2, wherein: In step a, the amount of thionyl chloride added is 3-5 times the molar amount of 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid.

4. The method for preparing the pyrazole amide derivatives according to claim 2 or 3, characterized in that: In step a, the organic solvent is one or more of dichloromethane, chloroform, acetonitrile, ethyl acetate, ethanol, methanol, and tetrahydrofuran.

5. The method for preparing the pyrazole amide derivatives according to claim 2 or 3, characterized in that: After the reaction in step a is completed, the intermediate is obtained by distillation under reduced pressure, extraction, and purification.

6. The method for preparing the pyrazole amide derivatives according to claim 2 or 3, characterized in that: In step b, the amount of 4-amino-3,5-dichloro-2,6-difluoropyridine added is 1-1.2 times the molar amount of 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid in step a.

7. The method for preparing a pyrazole amide derivative according to claim 2 or 3, characterized in that: In step b, the amount of triethylamine added is 1.1-1.5 times the molar amount of 4-amino-3,5-dichloro-2,6-difluoropyridine.

8. The method for preparing the pyrazole amide derivatives according to claim 2 or 3, characterized in that: The reaction solvent in step b is one or more of dichloromethane, chloroform, acetonitrile, ethyl acetate, ethanol, methanol, and tetrahydrofuran.

9. The method for preparing a pyrazole amide derivative according to claim 2 or 3, characterized in that: After the reaction in step b is completed, the product is subjected to reduced pressure distillation, extraction, and purification to obtain the pyrazole amide derivative.

10. Use of the pyrazoleamide derivative according to claim 1 or the pyrazoleamide derivative prepared by the method according to any one of claims 2 to 9 in preparing an anti-liver cancer drug.

Images