Application of compound WAY-226570 in preparation of anti-hepatoma drugs

Abstract

The invention provides an application of a compound WAY-226570 in preparation of an anti-liver cancer drug. The invention firstly finds that WAY-226570 (the molecular formula is C14H10F3N3) can inhibit the proliferation effect and migration and invasion activity of liver cancer cells, and further, the lenvatinib and WAY-226570 are combined to generate a synergistic inhibition effect on the proliferation of the liver cancer cells, so that the lenvatinib can be used as a combined medicine composition to be applied to the preparation of the medicine for preventing and / or treating the liver cancer, and has a clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and particularly to the application of compound WAY-226570 and a combined drug composition in the preparation of anti-hepatocellular carcinoma drugs. Background Art

[0002] Primary liver cancer (PLC) is one of the most commonly diagnosed cancers globally and the third leading cause of cancer-related deaths. Hepatocellular carcinoma (HCC) accounts for approximately 75 - 85% of all primary liver cancer cases. Its pathogenesis is extremely complex and can be caused by multiple factors, including chronic hepatitis B virus or hepatitis C virus infection, exposure to aflatoxin B1, alcoholic and non-alcoholic fatty diseases, etc. Although recent studies have identified many genes and pathways that affect the development of liver cancer, the treatment and patient prognosis are still not satisfactory. The efficacy of existing therapeutic drugs such as lenvatinib is limited, and drug resistance is prone to occur, which highlights the need for more treatment methods to improve the clinical benefits of HCC treatment.

[0003] Therefore, it is necessary to develop a new anti-hepatocellular carcinoma drug. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of a combined drug composition in the preparation of anti-hepatocellular carcinoma drugs. It is first discovered that compound WAY-226570 (the structural formula is shown as formula I) has significant anti-proliferative activity against hepatocellular carcinoma cells, and when combined with lenvatinib, it can achieve the efficacy of synergistically preventing and / or treating hepatocellular carcinoma.

[0005] The present invention is achieved as follows:

[0006] In the first aspect of the present invention, there is provided the application of WAY-226570 in the preparation of anti-hepatocellular carcinoma drugs, and the structural formula of the WAY-226570 is shown as formula (I):

[0007]

[0008] In the second aspect of the present invention, there is provided the application of a combined drug composition in the preparation of anti-hepatocellular carcinoma drugs, and the combined drug composition contains lenvatinib and WAY-226570 shown as formula (I).

[0009] Furthermore, the anti-hepatocellular carcinoma drug inhibits the proliferation, invasion and metastasis of hepatocellular carcinoma cells.

[0010] Furthermore, the mass ratio of lenvatinib to WAY-226570 is 1:1 - 5. Preferably, the mass ratio of lenvatinib to WAY-226570 is 1:5.

[0011] Furthermore, the application concentration of lenvatinib is 1 μM - 100 μM, and the application concentration of WAY-226570 is 1 μM - 10 μM.

[0012] In the second aspect of the present invention, an anti-hepatocellular carcinoma drug is provided. The anti-hepatocellular carcinoma drug contains lenvatinib and WAY-226570, and the structural formula of WAY-226570 is shown in formula (I).

[0013] Furthermore, the anti-hepatocellular carcinoma drug contains an effective amount of lenvatinib and an effective amount of WAY-226570.

[0014] Furthermore, the anti-hepatocellular carcinoma drug contains a pharmaceutically acceptable carrier and / or excipient. The excipient is selected from one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners or colorants.

[0015] Furthermore, the dosage form of the anti-hepatocellular carcinoma drug is selected from one of granules, tablets, pills, capsules, injections and dispersants.

[0016] In the third aspect of the present invention, the application of WAY-226570 in the preparation of an anti-hepatocellular carcinoma drug is provided.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0018] The present invention first discovers that the compound WAY-226570 (structural formula shown in formula I) has significant anti-proliferation activity against hepatocellular carcinoma cells, and shows a synergistic effect when combined with lenvatinib, significantly enhancing the inhibitory effect on the clone formation and apoptosis of hepatocellular carcinoma cells. Therefore, it can be used as a combined drug composition in the preparation of anti-hepatocellular carcinoma drugs. Description of the Drawings

[0019] Figure 1 : IC50 curves of single-agent WAY-226570 and single-agent lenvatinib against MHCC-97H and PLC / PRF / 5.

[0020] Figure 2 : IC50 curve of the combination of WAY-226570 and lenvatinib.

[0021] Figure 3 : Crystal violet staining results of the cell clone formation experiment. Each row is divided into 5 columns: DMSO control group, treatment groups of 2 μM, 5 μM, 10 μM WAY-226570 and lenvatinib.

[0022] Figure 4 : Crystal violet staining results of the cell clone formation experiment of the combination of WAY-226570 and lenvatinib.

[0023] Figure 5 : Flow cytometry detection of cell apoptosis distribution map.

[0024] Figure 6 : Of the synthesized compound WAY-226570 1 1H NMR spectrum.

[0025] Figure 7 : 19F nuclear magnetic resonance spectrum of the compound WAY-226570. Detailed implementation manners

[0026] The present invention will be specifically described below in conjunction with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are for illustrating the present invention rather than limiting the present invention.

[0027] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0028] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0029] To solve the technical problems of the present invention, the general idea of the present invention is as follows:

[0030] WAY-226570, the structural formula of WAY-226570 is:

[0031]

[0032] The molecular formula of WAY-226570 is C 14 H 10 F3N3, with a molecular weight of 277.2 and a CAS number: 299919-03-2.

[0033] WAY-226570 is commercially available. Optionally, the preparation method is as follows:

[0034] (1) Construction and coupling reaction of trifluoromethyl aromatic ring

[0035] Starting material: Using 4-chloro-2-nitrobenzene trifluoromethyl derivative (or similar aromatic precursor containing trifluoromethyl) as the starting material, the core skeleton is constructed through nitro reduction and substitution reaction of halogenated aromatic ring.

[0036] (2) Nitro reduction: Reduce the nitro group (-NO2) to an amino group (-NH2), for example, using hydrogen / Pd-C catalytic hydrogenation or Fe / HCl reduction system. Reaction conditions: H2 (1 atm), Pd / C (10%), ethanol, room temperature, 2 hours.

[0037] (3) Buchwald-Hartwig amination reaction: Introduce the second nitrogen-containing group by palladium-catalyzed cross-coupling of the amino group with a nitrogen-containing heterocycle (such as pyridine or pyrimidine derivatives).

[0038] Reagents: Pd(OAc)2 (catalyst), Xantphos (ligand), Cs2CO3 (base), toluene, 110 °C, 12 hours.

[0039] (4) Introduction of trifluoromethyl group: Perform electrophilic substitution on the aromatic ring using a trifluoromethylating reagent (such as TMSCF3 or Umemoto reagent).

[0040] Reaction conditions: Catalyzed by CuI, DMSO solvent, 80 °C, 6 hours.

[0041] (5) Purification and crystallization: Purify the crude product by column chromatography (silica gel, gradient elution with ethyl acetate / petroleum ether), and finally crystallize to obtain WAY-226570.

[0042] This application found through experiments that WAY-226570 can inhibit the proliferation, invasion, and metastasis of liver cancer cells.

[0043] Furthermore, when lenvatinib and WAY-226570 are used in combination, it is found that they can produce a synergistic inhibitory effect on the proliferation of liver cancer cells. WAY-226570 can improve the in vitro response of lenvatinib.

[0044] These results indicate that WAY-226570 may be a potential treatment method for HCC treatment.

[0045] The following will specifically describe the application of the combined drug composition of this application in the preparation of anti-liver cancer drugs in combination with examples and experimental data.

[0046] As used herein, "combination" or "combination treatment group" etc. mean that two or more active substances can be administered to a subject simultaneously as individual preparations or in any order as individual preparations successively.

[0047] As used herein, "effective amount" refers to an amount that includes enough to prevent or treat the symptoms or diseases of a medical condition. After being used for a specific patient or medical subject, the following changes can occur: the medical condition to be treated, the overall health of the patient / subject is improved. The effective amount can also be a dosage regimen below the maximum dose to avoid significant side effects or toxic effects.

[0048] WAY-226570 in the embodiments of the present invention was synthesized by WuXi AppTec. The 1 1H NMR spectrum of the synthesized compound WAY-226570 is as Figure 6 shown, and the 19F nuclear magnetic resonance spectrum is as Figure 7 shown.

[0049] Example 1: Verification of the anti-hepatocellular carcinoma activity of WAY-226570 alone

[0050] 1. The anti-proliferation effect of WAY-226570 on two hepatocellular carcinoma cells (MHCC-97H, PLC / PRF / 5) was detected by CCK-8 assay. The specific process is as follows:

[0051] Hepatocellular carcinoma cells (MHCC-97H, PLC / PRF / 5) were seeded in 96-well plates at a density of 5000 cells / well and cultured overnight at 37 °C to adhere.

[0052] Media containing WAY-226570 (concentration gradient: 0 - 100 μM) or lenvatinib (control) were added and cultured for 48 hours.

[0053] Tumor cells were seeded in 96-well plates at a density of 5000 / well and placed in a 37 °C constant temperature incubator overnight to adhere. The next day, 100 μL of the corresponding drug final concentrations (100, 50, 25, 10, 5, 2, 1, 0 μM) diluted with fresh complete medium were changed into each well. Lenvatinib was used as the control drug and cultured in a 37 °C constant temperature incubator. After 48 h, 100 μL of CCK-8 working solution was added per well in a light-proof environment, incubated in the incubator for 1 h, placed in a microplate reader, the detection wavelength was selected at 450 nm, the absorbance (OD) of each well was measured, and the IC50 value was calculated.

[0054] 2. The results are as Figure 1 shown. The half maximal inhibitory concentration (IC50) of WAY-226570 in MHCC-97H and PLC / PRF / 5 cells was 4.610 μM and 6.532 μM respectively; the half maximal inhibitory concentration of lenvatinib in MHCC-97H and PLC / PRF / 5 cells was 53.65 μM and 47.51 μM respectively.

[0055] The above data indicate that the anti-hepatocellular carcinoma activity of WAY-226570 alone (4.610 μM and 6.532 μM) against MHCC-97H and PLC / PRF / 5 is significantly better than that of lenvatinib alone (53.65 μM and 47.51 μM).

[0056] Example 2: Evaluation of the combined effect of WAY-226570 and lenvatinib

[0057] 1. Combined administration regimen:

[0058] The anti-proliferative effect of the combination of WAY-226570 and Lenvatinib on two hepatocellular carcinoma cell lines (MHCC-97H, PLC / PRF / 5) was detected by CCK-8 assay. The specific procedure is as follows:

[0059] Tumor cells were seeded into 96-well plates at a density of 5000 cells / well and incubated overnight at 37 °C in a constant temperature incubator to allow them to adhere. The next day, 100 μL of the corresponding drug final concentrations diluted with fresh complete medium were added to each well. The final concentrations of Lenvatinib were 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0 μM, and the double-drug mixture with 2 μM of WAY-226570 was used. The control group was a mixture of Lenvatinib gradient concentrations and the corresponding volume of DMSO. The plates were incubated at 37 °C in a constant temperature incubator. After 48 h, 100 μL of CCK-8 working solution was added to each well in the dark environment and incubated in the incubator for 1 h. Then, the plates were placed in a microplate reader, and the absorbance (OD) of each well was measured at a detection wavelength of 450 nm.

[0060] 2. Detection results:

[0061] The results are as Figure 2 shown. The half-maximal inhibitory concentration (IC50) of the combination of WAY-226570 and Lenvatinib in MHCC-97H and PLC / PRF / 5 cell lines was 37.95 μM and 15.60 μM, respectively; the IC50 of Lenvatinib alone in the control group in MHCC-97H and PLC / PRF / 5 cell lines was 51.38 μM and 35.60 μM, respectively.

[0062] It was shown that the IC50 values of the combination group for MHCC-97H and PLC / PRF / 5 (37.95 μM and 15.60 μM) were significantly lower than those of Lenvatinib alone (51.38 μM and 35.60 μM).

[0063] Example 3: Cell colony formation inhibition assay

[0064] 1. Experimental method:

[0065] Cell cloning experiments were conducted to investigate the effects of WAY-226570 and Lenvatinib on the colony formation ability of MHCC-97H and PLC / PRF / 5 tumor cells, respectively. The independent survival ability of cells was represented by the colony formation rate and the size of the colonies formed. 1000 cells per well were seeded in 6-well plates and incubated overnight at 37 °C in a constant temperature incubator to allow them to adhere to the wells. The next day, the two drugs were separately diluted with fresh complete medium to a final concentration of (0, 2, 5, 10 μM) in a single-drug mixture. After discarding the supernatant, 2 mL of the corresponding concentration of the single-drug mixture was added to each well. The control group was added with DMSO, and then the plates were placed back into the incubator for further culture. After that, the complete medium containing the above concentrations of the drugs was changed every 2 days. After 2 weeks, the supernatant was discarded, and the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 15 minutes, then the paraformaldehyde was discarded, and the cells were washed 3 times with PBS again. Crystal violet staining solution was added for 20 minutes of staining, and finally, the plates were washed with PBS until the bottom of the wells was transparent and colorless. The plates were left to stand for several days to dry naturally and then photographed.

[0066] The results of colony formation after treatment with different concentrations of drugs showed that WAY-226570 could inhibit the colony formation ability of hepatoma cell lines in a concentration-dependent manner.

[0067] 2. Results:

[0068] The results were as Figure 3 , and the results of colony formation after treatment with different concentrations of WAY-226570 as a single drug showed that WAY-226570 could inhibit the colony formation ability of hepatoma cell lines in a concentration-dependent manner;

[0069] It was known from Figure 4 that the combined treatment group (2 μM WAY-226570 + 10 μM lenvatinib) significantly reduced the number and size of colonies compared with the single-drug groups. WAY-226570 could enhance the ability of lenvatinib to inhibit the colony formation of hepatoma cell lines, and the combination of WAY-226570 and lenvatinib could significantly inhibit the colony formation of hepatoma cell lines.

[0070] Example 4: Cell apoptosis induction experiment

[0071] 1. Experimental design:

[0072] The apoptosis experiment was conducted to investigate the effects of WAY-226570 and Lenvatinib alone and in combination on the apoptosis of MHCC-97H tumor cells. 2×105 cells were seeded in each well of a 6-well plate and incubated overnight at 37°C in a constant temperature incubator to allow them to adhere. The next day, WAY-226570 was diluted to a final concentration of 2 μM and Lenvatinib was diluted to a final concentration of 10 μM with fresh complete medium. Additionally, two mixtures of the drugs used in combination and a control group with only DMSO were set up. Then, the supernatant was discarded, and 2 mL of the corresponding concentration of the drug mixture was added to each well. The cells were then incubated in the incubator for another 48 hours. Subsequently, the supernatant and cells were collected into a flow cytometry tube, and the cells were double-labeled with Annexin V-FITC and PI, and then detected for apoptosis using a flow cytometer.

[0073] 2. Results:

[0074] The results are as Figure 5 shown. The apoptosis rate of the combination group was significantly higher than that of the single-drug group and the control group (DMSO). The combination of WAY-226570 and Lenvatinib could significantly promote the apoptosis of liver cancer cells, confirming the synergistic pro-apoptotic effect.

[0075] Finally, it should also be noted that the term "comprise", "include" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0076] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0077] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of WAY-226570 in the preparation of anti-hepatocellular carcinoma drugs, characterized in that, The structural formula of WAY-226570 is shown as formula (I):

2. Use of the combined pharmaceutical composition in the preparation of an anti-hepatocellular carcinoma drug, characterized in that, The combined pharmaceutical composition contains WAY-226570 as claimed in claim 1 and lenvatinib.

3. The application according to claim 2, characterized in that, The anti-hepatocellular carcinoma drug inhibits the proliferation, invasion and metastasis of hepatocellular carcinoma cells.

4. The application according to claim 2, characterized in that The mass ratio of lenvatinib to WAY-226570 is 1:1 - 5.

5. The application according to claim 2, characterized in that The application concentration of lenvatinib is 1 μM - 100 μM, and the application concentration of WAY-226570 is 1 μM - 10 μM.

6. An anti-hepatocellular carcinoma drug, characterized in that, The anti-hepatocellular carcinoma drug contains lenvatinib and WAY-226570, and the structural formula of WAY-226570 is as shown in formula (I) shown.

7. The anti-hepatocellular carcinoma drug according to claim 6, wherein The anti-hepatocellular carcinoma drug contains an effective amount of lenvatinib and an effective amount of WAY-226570.

8. The anti-hepatocarcinoma drug according to claim 6, characterized in that, The anti-hepatocellular carcinoma drug contains a pharmaceutically acceptable carrier and / or excipient.

9. The application according to claim 6, wherein The dosage form of the anti-hepatocellular carcinoma drug is selected from one of granules, tablets, pills, capsules, injections and dispersants.

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