Anti-breast cancer toosendanin derivative as well as preparation method and application thereof
By performing C-12 selective deacetylation of ketolitin, a ketolitin derivative of structural formula I was prepared, which solved the hepatotoxicity problem of ketolitin and improved the anti-breast cancer activity, especially for HER2-positive breast cancer, which is suitable for clinical applications.
Patent Information
- Application Number
- CN202510529613.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-25
- Publication Date
- 2025-08-01
AI Technical Summary
The existing citrus citrus has hepatotoxicity problems in anti-breast cancer treatment, which limits its clinical application, and the existing drugs are not effective in the treatment of HER2-positive breast cancer.
By performing C-12 selective deacetylation of ketolitin, a ketolitin derivative of structural formula I was prepared, and LiHMDS was used as a strong base to react in a tetrahydrofuran solvent to reduce hepatotoxicity and improve anti-breast cancer activity.
The prepared citrus derivatives show better anti-breast cancer activity in the body, especially for HER2-positive breast cancer, and their hepatotoxicity is significantly reduced, which is suitable for clinical promotion.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a toosendanin derivative for anti-breast cancer, a preparation method thereof, and an application thereof. Background Art
[0002] The statements in this section only provide background information related to the disclosure of the present application and may not constitute prior art.
[0003] To date, malignant tumors still seriously threaten the health of the people, with the incidence and mortality rates ranking first in the world. The research and development of anti-tumor drugs in China has a long way to go. Among them, breast cancer has become the number one cancer in the world and is also the main cause of cancer death in Chinese women. Among refractory / recurrent breast cancers, except triple-negative breast cancer (TNBC), human epidermal growth factor receptor 2 positive (HER2+) breast cancer accounts for about 20-25%. Due to its strong invasiveness, high recurrence rate, poor prognosis, easy occurrence of drug resistance, and high brain metastasis rate in the advanced stage (about 50%), it is considered the second most difficult breast cancer to treat.
[0004] Toosendanin (TSN) is an active monomer compound from traditional Chinese medicine Melia toosendan Sieb. et Zucc., which has good anti-tumor activity but causes liver toxicity, limiting its wide clinical use. As is well known, drug-induced liver toxicity is one of the main reasons for the failure of new drug research and development, and about 1 / 3 of drugs are withdrawn from the market due to liver toxicity. Therefore, how to obtain toosendanin derivatives with high efficiency and low toxicity is crucial for the development of toosendanin into a new type of anti-tumor drug.
[0005] In drug research and development, the C-5 selectively hydroxylated product LDDT of triptolide has been proven to be able to reduce nephrotoxicity and enhance immunosuppressive activity. Currently, this compound is in the clinical phase II research stage. The development of toosendanin derivatives has great value for the utilization of limited drug resources. Summary of the Invention
[0006] The purpose of the present invention is to provide a toosendanin derivative with anti-breast cancer activity, a preparation method thereof, and an application thereof, aiming at the current liver toxicity problem of toosendanin. By selectively deacetylating the C-12 of toosendanin, a toosendanin derivative shown in structural formula I is discovered, which has better anti-breast cancer activity than toosendanin and lower in vivo liver toxicity.
[0007] The technical solution of the present invention is as follows:
[0008] On the one hand, the present invention provides a toosendanin derivative, the structural formula of which is shown in the following formula (Ⅰ):
[0009]
[0010] On the other hand, the present invention provides a method for preparing the toosendanin derivative as described above, comprising the following steps:
[0011] The toosendanin and a strong base are reacted in tetrahydrofuran as a solvent at -20°C to room temperature to obtain the product.
[0012] The preparation route is as follows:
[0013]
[0014] Preferably, the strong base is lithium bis(trimethylsilyl)amide (LiHMDS).
[0015] Preferably, the molar ratio of the strong base LiHMDS to toosendanin is 1 - 5:1, preferably 2:1.
[0016] Preferably, the reaction temperature is preferably 0°C to room temperature.
[0017] The present invention also provides the use of the toosendanin derivative as described above in the preparation of a drug for treating or adjuvantly treating breast cancer.
[0018] Preferably, the breast cancer is triple-negative breast cancer or HER2-positive breast cancer.
[0019] A pharmaceutical composition for treating breast cancer comprises the toosendanin derivative as described above and one or more pharmaceutically acceptable carriers or excipients.
[0020] Preferably, in the pharmaceutical composition, the mass ratio of the toosendanin-like derivative to the pharmaceutically acceptable carrier and / or excipient is 1:1 - 1:10.
[0021] Preferably, the pharmaceutical composition can be prepared into tablets, capsules or injections.
[0022] The present invention has no special limitation on the pharmaceutical carrier or the excipient, and the well-known pharmaceutical carriers or excipients in the art can be used, specifically one or more of solid, semi-solid or liquid diluents, fillers or pharmaceutical product adjuvants. In the present invention, the preparation types of the toosendanin derivative pharmaceutical composition preferably include liquid preparations, solid preparations, sprays or aerosols; the liquid preparations preferably include injections, suspensions, emulsions, solutions or syrups; the solid preparations preferably include tablets, capsules, granules or infusion powders. The present invention has no special limitation on the preparation method of the toosendanin derivative pharmaceutical composition, and the well-known preparation methods in the art can be used.
[0023] In the present invention, the administration route of the toosendanin derivative pharmaceutical composition is preferably injection, oral administration or sublingual administration; the injection preferably includes intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection or subcutaneous injection.
[0024] In the present invention, the toosendanin derivative pharmaceutical composition is preferably used in the form of a dosage per unit body weight. In the present invention, the toosendanin derivative pharmaceutical composition is preferably used in the form of a dosage per unit body weight. In the present invention, the dosage per unit body weight is preferably 0.5-20 mg / kg.
[0025] Compared with the existing technology, the beneficial effects of the present invention are as follows:
[0026] 1. The toosendanin derivative (Compound 2) shown in Structural Formula I was discovered, which has better anti-breast cancer activity than toosendanin and lower in vivo liver toxicity; it can replace toosendanin to exert the cancer treatment effect, and at the same time has weak liver toxicity, being suitable for market promotion and use;
[0027] 2. A new preparation method of the toosendanin derivative (Compound 2) shown in Structural Formula I was provided. This method is simple, has the advantages of high yield compared with other preparation methods, and is easy to produce on a large scale. Description of the Drawings
[0028] Figure 1 IC of toosendanin (1) and toosendanin derivative (2) against the in vitro anti-proliferative activities of different breast cancer cells and normal cells 50 value;
[0029] Figure 2 Effect of toosendanin derivative (2) on the colony formation of HER2-positive breast cancer cells (***P < 0.001, ****P < 0.0001);
[0030] Figure 3 In vivo anti-HER2-positive breast cancer activities of toosendanin (1) and toosendanin derivative (2) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 6);
[0031] Figure 4 Liver and kidney tissue sections of the experimental mice after 24 days of administration treatment with toosendanin (1) and toosendanin derivative (2);
[0032] Figure 5 CREA-S, UREA, ALT, and AST indexes in the serum of the experimental mice after 24 days of administration treatment with toosendanin (1) and toosendanin derivative (2) (*P < 0.05, ****P < 0.0001, n = 6). Detailed Embodiments
[0033] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not indicating the manufacturer are conventional products that can be purchased commercially. To better illustrate the present invention, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.
[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.
[0035] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.
[0036] Example 1
[0037] 1. Preparation of Toosendanin Derivative (2)
[0038] To a solution of TSN (1) (200 mg, 0.35 mmol) in THF (5 mL) at 0 °C was added dropwise LiHMDS (1 M solution in THF, 0.70 mL, 0.70 mmol), and then the reaction mixture was allowed to warm to room temperature and stirred for 24 h. The reaction was quenched by the addition of saturated aqueous NaHCO3 (10 mL), and the mixture was extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give the crude product. The crude product was recrystallized twice from petroleum ether / acetone (10:1) to give toosendanin derivative (2) (176 mg, 0.33 mmol, 95% yield) as colorless needle crystals (melting point: 242.5 - 243.8 °C, partially decomposed).
[0039] Characterization of Toosendanin Derivative (2) 1 1H NMR and 13 13C NMR data are shown in Table 1.
[0040] Table 1 Toosendanin Derivative (2) 1 1H and 1313C NMR (CD3CN) data
[0041]
[0042]
[0043] 2. Preparation of Toosendanin Derivative (2)
[0044] At 0 °C, LiHMDS (1 M THF solution, 3.5 mL, 3.5 mmol) was added dropwise to a solution of TSN (1) (1 g, 1.75 mmol) in THF (25 mL). Then the reaction mixture was allowed to warm to room temperature and stirred for 24 h. The reaction was quenched by adding saturated aqueous NaHCO3 (100 mL), and the mixture was extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a crude product. The crude product was recrystallized twice from petroleum ether / acetone (10:1) to give toosendanin derivative (2) (930 mg, 1.75 mmol, yield >99%) as colorless needle crystals.
[0045] 3. Preparation of Toosendanin Derivative (2)
[0046] At 0 °C, LiHMDS (1 M THF solution, 17.5 mL, 17.5 mmol) was added dropwise to a solution of TSN (1) (5 g, 8.75 mmol) in THF (125 mL). Then the reaction mixture was allowed to warm to room temperature and stirred for 24 h. The reaction was quenched by adding saturated aqueous NaHCO3 (100 mL), and the mixture was extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a crude product. The crude product was recrystallized twice from petroleum ether / acetone (10:1) to give toosendanin derivative (2) (4.14 g, 7.78 mmol, yield 89%) as colorless needle crystals.
[0047] Example 2 In Vitro Activity Experiment
[0048] Compounds 1 and 2 of the present invention have significant anti-proliferative activity against tumor cell lines including human triple-negative breast cancer cell lines (HCC1806, MDA-MB-468, MDA-MB-231), HER2-positive breast cancer cell lines (JIMT-1, SKBr3, MCF-7), and human liver cancer cell line HEPG2. And the toxicity of compound 2 to normal cells MCF-10A and 293T is lower than that of compound 1. The experimental methods and results are as follows:
[0049] I. Experimental Methods
[0050] 1. Dissolve the to-be-tested toosendanin (1) and toosendanin derivative (2) samples in DMSO respectively to prepare working solutions with a concentration of 20 μM, and store them for later use.
[0051] 2. Start with a working solution concentration of 20 μM and dilute it twofold into 5 concentration gradients. Take the triple-negative breast cancer cell lines HCC1806 (3500 cells / well), MDA-MB-468 (3650 cells / well), MDA-MB-231 (4500 cells / well), HER2-positive breast cancer cell lines JIMT-1 (4300 cells / well), JIMT-1 (5300 cells / well), MCF-7 (3600 cells / well), MCF-10A (4300 cells / well), 293T (4500 cells / well), and human liver cancer cell line HEPG2 (4000 cells / well) in the logarithmic growth phase, inoculate them in 96-well plates and culture overnight for 24 h, add the gradient-diluted compound working solutions for treatment, set three replicates for each concentration, and set a control group and a blank group at the same time. Observe the cell morphological changes at 24 h, 48 h, and 72 h, and detect the cell viability by MTT.
[0052] Cell viability = (OD of drug treatment group - OD of blank) / (OD of DMSO group - OD of blank).
[0053] 3. IC 50 Determination
[0054] Culture the above cell lines in the logarithmic growth phase in 96-well plates. After 24 h, add 10 μL of the compound passed in the primary screening to each well for re-screening (starting from a concentration of 20 μM and diluting it twofold in 5 concentration gradients) and treat for 72 h. Set three replicates for each concentration. Stain with 5 mg / mL MTT at 37 °C for 2.5 h, remove the culture medium, add 100 μL of DMSO, detect the absorbance at 490 nm, and perform nonlinear regression analysis using GraphPad Prism 10 software to determine the IC 50 Value.
[0055] II. Experimental Results
[0056] See Figure 1In this invention, the anti-proliferative activities of toosendanin (1) and toosendanin derivatives (2) against breast cancer were evaluated in vitro. The results showed that toosendanin (1) and toosendanin derivatives (2) had significant anti-proliferative activities against human triple-negative breast cancer cell lines (HCC1806, MDA-MB-468, MDA-MB-231), HER2-positive breast cancer cell lines (JIMT-1, SKBr3, MCF-7), and human liver cancer cell line HEPG2. Among them, toosendanin derivatives (2) had better anti-HER2-positive breast cancer activity than toosendanin (1), and the toxicity of toosendanin derivatives (2) to normal cells was lower than that of toosendanin (1).
[0057] Example 3 In vitro activity experiment
[0058] According to the in vitro experimental data, it was found that toosendanin derivatives (2) had the best activity against JIMT-1 cells. Therefore, we selected JIMT-1 cells to further observe whether their colony formation could be affected by toosendanin derivatives (2). JIMT-1 cells were seeded into 6-well plates and treated with toosendanin derivatives (2) (0, 0.25, 0.5, 1.0 μM) for 4 hours. The medium containing the reagent was replaced with fresh medium, and the cells were allowed to grow for another 12 days. Then the cells were fixed with methanol and stained with crystal violet.
[0059] The results are shown in Figure 2 , toosendanin derivatives (2) inhibited the colony formation of JIMT-1 cells in a concentration-dependent manner and had good in vitro anti-breast cancer activity.
[0060] Example 4 In vivo activity experiment
[0061] According to the in vitro experimental data, it was found that toosendanin derivatives (2) had the best activity against JIMT-1 cells. Therefore, we selected JIMT-1 cells to establish a nude mouse xenograft tumor model to investigate the in vivo anti-tumor activity of toosendanin derivatives (2). The experimental steps were as follows: After the appropriate JIMT-1 cell line was passaged and cultured, well-grown cells were collected to prepare a tumor cell suspension. 1×10 6 cells were inoculated subcutaneously into mice. When the tumors grew to about 100 mm 3 , they were randomly divided into a normal saline group, a toosendanin derivatives (2) treatment group (1 mg / kg), and a toosendanin (1) control group (1 mg / kg). They were administered by intraperitoneal injection once every 2 days for 24 consecutive days, and the changes in tumor volume over time were observed. Four weeks after drug treatment, blood was collected from the tail vein and the mice were sacrificed. The harvested tumor tissues were quickly frozen in liquid nitrogen. The mouse kidney and liver tissues preserved in 4% paraformaldehyde were dehydrated and paraffin-embedded. The embedded and cooled tissue sections were 5 μm thick. The sections were stained with hematoxylin-eosin, sealed, and observed under an optical microscope.
[0062] The results are shown inFigure 3 , the in vivo anti-breast cancer activity of toosendanin derivative (2) is stronger than that of toosendanin (1), and the tumor inhibition rate is 62%. In addition, from the liver and kidney tissue sections ( Figure 4 ), it can be seen that the in vivo toxicity of toosendanin derivative (2) is lower than that of toosendanin (1). The results of serum biochemical analysis ( Figure 5 ) show that serum toosendanin derivative (2) cannot cause changes in the levels of CREA-S, UREA, AST, and ALT, but toosendanin (1) can significantly increase the levels of AST and ALT, further indicating that the in vivo liver toxicity of toosendanin derivative (2) is lower than that of toosendanin (1).
[0063] Example 5 A pharmaceutical composition for treating breast cancer
[0064] A pharmaceutical composition for treating breast cancer, comprising the toosendanin derivative (2) of the present invention, wherein the toosendanin derivative (2) and the excipient are added in a weight ratio of 1:1, granulated and tabletted. The excipient is specifically a common excipient such as hydroxypropyl cellulose or gelatin.
[0065] Example 6 A pharmaceutical composition for treating breast cancer
[0066] A pharmaceutical composition for treating breast cancer, comprising the toosendanin derivative (2) of the present invention, wherein the toosendanin derivative (2) is made into a capsule preparation.
[0067] Example 7 A pharmaceutical composition for treating breast cancer
[0068] A pharmaceutical composition for treating breast cancer, comprising the toosendanin derivative (2) of the present invention and starch, corn steep liquor, and magnesium stearate, combined to make tablets.
[0069] The content of the natural-like diarylpropane dimer compound in each tablet is 10 mg.
[0070] The preparation method is to mix the toosendanin derivative (2) or its pharmaceutical composition with an auxiliary agent, granulate and tabletten to obtain tablets.
[0071] Example 8 A pharmaceutical composition for treating breast cancer
[0072] A pharmaceutical composition for treating breast cancer, comprising the toosendanin derivative (2) of the present invention, and also comprising auxiliary agents starch and magnesium stearate. It is prepared into a capsule.
[0073] The preparation method is: mix the toosendanin derivative (2) with auxiliary agents starch and magnesium stearate, sieve, uniformly mix in a suitable container, and fill the obtained mixture into hard gelatin capsules.
[0074] In each capsule, the content of the toosendanin derivative (2) is 10 mg.
[0075] Example 9 A pharmaceutical composition for treating breast cancer
[0076] A pharmaceutical composition for treating breast cancer, comprising 2 mg of the toosendanin derivative (2) of the present invention and 10 mg of sodium chloride, and is prepared into an ampoule.
[0077] Preparation method: Dissolve the toosendanin derivative (2) and sodium chloride in an appropriate amount of water for injection, filter the obtained solution, and fill it into an ampoule bottle under sterile conditions to prepare an ampoule.
[0078] The above-described embodiments only represent the specific embodiments of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can be made, and these all belong to the protection scope of the present application.
[0079] Example 10 Optimization of the preparation conditions of the toosendanin derivative (2)
[0080] According to the preparation method of Example 1, adjust the types of strong base and solvent, as well as the reaction temperature and reaction time, and detect the yield of the product. The conditions and results are shown in Table 1 below:
[0081] Table 1: Adjusted reaction conditions and yields a
[0082]
[0083]
[0084] In the table, the a annotation indicates that except for special instructions, the reaction substrate toosendanin is 0.035 mmol and the reaction is carried out in freshly distilled and degassed THF; the b annotation indicates that the raw material has not reacted completely and the raw material is recovered; the c annotation indicates that the reaction substrate toosendanin is 1.75 mmol and the reaction is carried out in freshly distilled and degassed THF; the d annotation indicates that the reaction substrate toosendanin is 8.75 mmol and the reaction is carried out in freshly distilled and degassed THF.
[0085] As can be seen from Table 1, when the strong base LiHMDS is combined with the solvents toluene and 1,4-dioxane, the yield is extremely low, less than 30%; and when combined with Et2O, the yield is also less than 60%. When the solvent THF is combined with the strong bases LDA and NaOH, the yield is also relatively low, less than 34%; when combined with K2CO3, it is even less than 5%. When the solvent THF is combined with the strong base LiHMDS, the yield is generally higher than 80%; when the reaction substrate is 1.75 mmol, the reaction yield is even as high as 99%.
[0086] The above-described embodiments merely represent the specific implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application.
Claims
1. A toosendanin derivative, characterized in that, The structural formula is as shown in the following formula (I):
2. The preparation method of the toosendanin derivative according to claim 1, characterized in that it comprises the following steps: It is obtained by reacting toosendanin and a strong base in tetrahydrofuran as a solvent at -20°C to room temperature.
3. The preparation method of the toosendanin derivative according to claim 2, characterized in that The strong base is lithium bis(trimethylsilyl)amide.
4. The preparation method of the toosendanin derivative according to claim 2, wherein, The molar ratio of the strong base lithium bis(trimethylsilyl)amide to toosendanin is: 1-5:
1.
5. The preparation method of the toosendanin derivative according to claim 2, characterized in that, The molar ratio of the strong base lithium bis(trimethylsilyl)amide to toosendanin is 2:
1.
6. The preparation method of the toosendanin derivative according to claim 2, characterized in that, The reaction temperature is 0°C to room temperature.
7. A pharmaceutical composition for treating breast cancer, characterized in that, It comprises the toosendanin derivative according to claim 1 and one or more pharmaceutically acceptable carriers or excipients, and the mass ratio of the toosendanin derivative to the pharmaceutically acceptable carrier and / or excipient is 1:1 to 1:
10.
8. A pharmaceutical composition for treating breast cancer according to claim 7, wherein, The dosage of the pharmaceutical composition is: 0.5-20 mg / kg calculated based on the content of the active ingredient toosendanin derivative.
9. Use of a toosendanin derivative in the preparation of a drug for treating or adjuvantly treating breast cancer, characterized in that, The toosendanin derivative is the compound according to claim 1.
10. The application according to claim 9, wherein The breast cancer is triple-negative breast cancer or HER2-positive breast cancer.