Taraxasterol 3-ester derivatives and their anti-tumor applications

Abstract

The present application relates to the field of pharmaceutical chemistry taraxasterol 3-ester derivative, its structural formula is shown as A, preparation is specifically taraxasterol 1 with phthalic anhydride in suitable base catalyst, organic solvent is reacted to obtain compound A, the yield of synthesis route is high, easy to operate implementation.Activity test proves that taraxasterol 3-ester derivative is a suitable antitumor candidate drug, especially as the candidate drug of anti-lung cancer.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a taraxasterol 3-ester derivative and its antitumor applications. Background Technology

[0002] Cancer is a major disease that seriously threatens human health. Statistics show that in 2023, there were 18.5 million new cases of cancer worldwide, and 10.4 million cancer deaths during the same period. The risk of developing cancer increases significantly with age, and multiple factors, including environmental pollution, smoking, excessive alcohol consumption, obesity, and genetics, contribute to the development and progression of cancer. Currently, anti-cancer drugs generally suffer from significant side effects, easy development of drug resistance, and insufficient targeting, severely impacting treatment efficacy and patients' quality of life. Therefore, developing novel, highly effective, low-toxicity, and highly specific anti-cancer drugs is of significant clinical importance.

[0003] Dandelion, a perennial herb of the Asteraceae family, is a traditional Chinese medicine. Its dried whole plant possesses properties of clearing heat and detoxifying, reducing swelling and dissipating nodules, and promoting diuresis. Modern pharmaceutical research shows that dandelion is rich in flavonoids, polysaccharides, and triterpenoids, and its extracts exhibit significant anti-inflammatory, hepatoprotective, choleretic, immunomodulatory, antioxidant, and antitumor effects. Our research group focuses on the antitumor activity of dandelion extracts. Dandelion sterol 1 is a class of natural steroidal compounds isolated from dandelion.

[0004]

[0005] Researchers focused on the antitumor activity of dandelion extract, discovering that taraxasterol 1 possesses certain antitumor potential, but there is still room for improvement in its activity. Based on this, this study modified the structure of taraxasterol to obtain a derivative with stronger antitumor activity. To find candidates for anticancer drugs with better efficacy and stronger activity, this study designed and synthesized the following drug molecule for the first time. Esterification of the 3-hydroxyl group of taraxasterol yielded a novel taraxasterol 3-ester derivative A with a unique structure, which was found to have significant antitumor activity, providing an important foundation for the development of novel antitumor drugs. Summary of the Invention

[0006] The present invention aims to provide a novel taraxasterol 3-ester derivative A to overcome the shortcomings of existing antitumor drugs, such as large toxic side effects, easy drug resistance, and insufficient targeting, as well as the problem that the antitumor activity of natural taraxasterol needs to be improved.

[0007] One objective of this invention is to provide a taraxasterol 3-ester derivative, the structural formula of which is shown in Formula A. .

[0008] The second objective of this invention is to provide a method for preparing taraxasterol 3-ester derivatives, wherein taraxasterol 1 is reacted with phthalic anhydride in a suitable base catalyst and reaction solvent to obtain compound A. The synthetic route is as follows: .

[0009] Preferably, as an improvement, the base catalyst is selected from one or more of triethylamine (Et3N), N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0010] Preferably, as an improvement, the base catalyst is 4-dimethylaminopyridine.

[0011] Preferably, as an improvement, the reaction solvent is selected from one or more of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), chloroform, acetonitrile, tetrahydrofuran, pyridine, or tetrahydrofuran.

[0012] Preferably, as an improvement, the reaction solvent is pyridine.

[0013] A third objective of this invention is to provide the application of taraxasterol 3-ester derivatives in the preparation of antitumor drugs.

[0014] Specifically, the tumor is lung cancer.

[0015] Activity tests demonstrated that the taraxasterol 3-ester derivative designed and synthesized in this invention has excellent anti-tumor effects, especially against lung cancer; the inhibition rate at an in vitro concentration of 40 μg / ml is as high as 73.95%, which is stronger than that of the positive control drug taraxasterol; it can be used as a novel taraxasterol candidate drug for clinical anti-tumor applications.

[0016] The advantages of this invention are: by esterifying the 3-hydroxyl group at the taraxasterol parent structure, a novel taraxasterol 3-ester derivative A was obtained. Activity tests show that this derivative possesses good antitumor activity, especially against tumors such as lung cancer. Furthermore, the synthetic method for taraxasterol 3-ester derivative A of this invention uses readily available raw materials, has a high yield, and is very easy to operate and implement.

[0017] In the above preparation method, the organic solvent can also be selected from other reaction solvents by those skilled in the art, depending on the reaction's requirements for temperature and solvent polarity. The reaction temperature can be appropriately selected according to the reaction type. The reaction time can be monitored and determined by methods such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), or LC-MS. Attached Figure Description

[0018] Figure 1 For taraxasterol 3-ester derivative A 1 Full HNMR image; Figure 2 For taraxasterol 3-ester derivative A 1 A magnified view of a portion of the HNMR spectrum; Figure 3 For taraxasterol 3-ester derivative A 13 Full image of CNMR; Figure 4 For taraxasterol 3-ester derivative A 13 A magnified view of a section of CNMR; Figure 5 Image showing the morphological changes of cells after treatment with taraxasterol 3-ester derivative A; Figure 6 This is a graph showing the inhibitory effect of drug taraxasterol 3-ester derivative A on human lung cancer A549 cells at different time points. Detailed Implementation

[0019] The following detailed description illustrates the specific implementation method: The present invention will be further illustrated by specific embodiments below, but these are not intended to limit the scope of protection of the present invention. Without departing from the concept of the present invention, those skilled in the art can make improvements or combinations to the parameters or conditions of the claims, and these improvements or combinations should also be considered within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. In this invention, taraxasterol was purchased from Hubei Danding Pharmaceutical Technology Co., Ltd., other raw materials and reagents were purchased from Sinopharm Group, and solvents were purchased from Zunyi Shuangju Chemical Co., Ltd. Unless otherwise specified, all reagents used are chemically pure.

[0020] Example 1: Preparation of taraxasterol 3-ester derivative A:

[0021] 0.09 mmol (1.0 equiv) of taraxasterol was weighed and placed in a dry sealed tube. Pyridine (2.0 mL) was added to dissolve it. Then, 0.28 mmol (3.0 equiv) of phthalic anhydride and 0.05 mmol (0.5 equiv) of DMAP were weighed and added to the reactor. The mixture was stirred at 120 °C for 23 hours. Heating and the reaction were stopped. The resulting reaction mixture was transferred to a round-bottom flask and the pyridine was removed by vacuum distillation at 50 °C. The residue was then post-processed by rapid column chromatography. The column chromatography post-processing was performed by dry loading. Unreacted taraxasterol was recovered by elution with petroleum ether:ethyl acetate = 1:1 (recovery rate 55.1%). Then, the target product, taraxasterol 3-ester derivative A (yield 40.0%), was obtained by elution with dichloromethane:methanol = 10:1 as a white solid.

[0022] 1 H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 6.8 Hz, 1H), 7.76 – 7.69 (m, 1H), 7.62 – 7.51 (m, 2H), 4.76 (dd, J = 11.7, 4.5 Hz, 1H), 4.61 (d, J = 6.7 Hz, 2H),2.35 – 2.50 (m, 1H),2.14 – 2.27 (m, 1H), 2.09 (p, J = 6.4 Hz, 1H),1.83 – 1.93(m, 1H), 1.50 – 1.81 (m, 9H), 1.00 – 1.44 (m, 12H), 1.02 (s, 6H), 0.95 (s,6H), 0.89 (s, 3H), 0.88 (s, 3H), 0.86 (s, 3H). 13C NMR(101 MHz, CDCl3) δ 179.68, 168.11, 154.91, 131.93, 131.11,130.05, 129.18, 107.37, 83.35, 55.83, 50.64, 48.88, 42.29, 41.16, 39.63,39.40, 39.09, 38.72, 38.53, 38.30, 37.31, 34.77, 34.23, 28.27, 26.89, 26.39,25.86, 25.75, 23.36, 21.73, 19.73, 18.41, 16.88, 16.54, 16.14, 15.01. Example 2: Activity test of novel taraxasterol 3-ester derivative A Cell lines and solvents Cell line: Human lung cancer cell line A549.

[0023] Culture medium: RPMI-1640 medium; 10% high-quality fetal bovine serum; 1% penicillin and antibiotics.

[0024] Culture conditions: Gas phase: air, 95%; carbon dioxide, 5%. Temperature: 37℃, humidity in incubator: 100%.

[0025] Drug solvent: Dimethyl sulfoxide (DMSO).

[0026] Reagent: Cell Counting Kit-8 (CCK-8).

[0027] Drugs and cell treatment Dissolve taraxasterol and its derivative powder in an appropriate volume of DMSO solution under aseptic conditions. When the confluence of human A549 cells reaches 80%-90% after three passages, seed the corresponding number of cells into 6-well or 96-well plates according to the experimental type, and culture under the conditions described above. On the day of drug treatment, the cell confluence in the wells should reach approximately 40%-50%. Discard the old culture medium, wash the cells once with PBS, and add fresh complete culture medium according to the culture system. Set up a control group and a drug treatment group, with three replicates for each group. After drug addition, place the culture plate in a cell culture incubator for continued culture. No medium change is required during drug treatment; successfully treated cells can be used for subsequent related experiments.

[0028] CCK-8 staining method for detecting antitumor cell activity: implementation plan Cells with a viable cell percentage of over 90% were selected for the experiment. The cell proliferation inhibition assay used the CCK-8 cell viability assay kit. Human A549 cell lines in the logarithmic growth phase were selected, and after trypsin digestion and centrifugation, cell counting was performed under a microscope; cells were counted at 1 x 10⁶ cells per well. 4 Cells were seeded into 96-well plates with a culture volume of 100 μL. Three replicates were set up for each group: a blank control group, a control group, and an experimental group. Cells were incubated in an incubator. The next day, the medium was replaced with fresh RPMI-1640 complete medium, and different doses of taraxasterol (0, 5, 10, 20, 40, and 80 μg / ml) were added for 24 h, 48 h, and 72 h, respectively. Under light-protected conditions, 10 μL of CCK-8 reagent was added to each well, and the incubator was continued for 1-2 h. Under light-protected conditions, the absorbance (OD) of each well was measured at 450 nm using a microplate reader. 450 The absorbance values ​​were analyzed, and statistical analysis software was used to plot absorbance curves and calculate the inhibitory rate of the drug on cells (e.g., ) Figure 6 (and Table 1).

[0029] Microscopic observation of cell morphology changes Human lung cancer A549 cells in the logarithmic growth phase were selected, digested with trypsin, centrifuged, and counted under a microscope; cells were counted at 3 x 10⁻⁶ cells per well. 5 Cells were seeded into 6-well plates with a culture volume of 2 mL. Control and experimental groups were set up and cultured in an incubator. On the second day, the medium was replaced with fresh RPMI-1640 complete medium, and taraxasterol (Tara) and its derivatives (Tara) were added at gradient concentrations (0, 5, 10, 20, 40, 80 μg / mL) of 40 μg / mL. der The cells were treated for a period of time (24h, 48h, 72h); the old culture medium was discarded, and the cells were washed once with PBS. The morphological changes of the cells after drug treatment were observed and photographed using a regular optical microscope, and the cell inhibition rate was tested. The experimental results are as follows: Figure 5 Table 1 and Figure 6 As shown.

[0030] Figure 5 and Figure 6 In this context, Con refers to the blank control, i.e., the solvent DMSO; Tara refers to the positive control, i.e., taraxasterol; Tara der This refers to the test drug, namely taraxasterol 3-ester derivative A.

[0031] exist Figure 5 In the middle, Tara and Tara der The dosage was 40 μg / mL; the duration of action was 24 h and 48 h.

[0032] exist Figure 6In this study, gradient concentration dosing was used for both the positive control and the test drug, specifically administering 0, 5, 10, 20, 40, and 80 μg / ml of taraxasterol (Tara) and its derivatives (Tara). der The growth inhibition rate and IC50 of A549 cells were tested at 24h, 48h, and 72h after drug administration. 50 The values ​​are as follows: Of which 24h: taraxasterol: IC 50 =82.27μg / mL; taraxasterol 3-ester derivative A: IC 50 =35.51 μg / mL; 48h: taraxasterol: IC 50 =41.70 μg / mL; taraxasterol 3-ester derivative A: IC 50 =20.67 μg / mL; 72h: taraxasterol: IC 50 =33.01μg / mL; taraxasterol 3-ester derivative A:IC 50 =16.73μg / mL.

[0033] Table 1. Tumor inhibition rate (%) of taraxasterol 3-ester derivative A

[0034] In Table 1, Tara and Tara der The drug was administered at a concentration of 40 μg / mL for 72 h. The experimental results in Table 1 show that the taraxasterol 3-ester derivative A of this invention has excellent in vitro antitumor activity. At a concentration of 40 μg / mL, it inhibited human lung cancer cells A549 cells by 73.95% in vitro. Compared with the control drug taraxasterol, it has stronger antitumor inhibitory activity. It can be used as a novel taraxasterol candidate drug for clinical antitumor application.

[0035] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A taraxasterol 3-ester derivative, characterized in that, Its structural formula is shown in formula A. 。 2. The method for preparing the taraxasterol 3-ester derivative according to claim 1, characterized in that: Compound A was obtained by reacting taraxasterol 1 with phthalic anhydride in a suitable alkaline catalyst and reaction solvent. The synthetic route is shown in the following formula: 。 3. The preparation method according to claim 2, characterized in that: The base catalyst is selected from one or more of triethylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine or 1,8-diazabicyclo[5.4.0]undec-7-ene.

4. The preparation method according to claim 2, characterized in that: The alkaline catalyst is 4-dimethylaminopyridine.

5. The preparation method according to claim 1, characterized in that: The reaction solvent is selected from one or more of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, pyridine, or tetrahydrofuran.

6. The preparation method according to claim 5, characterized in that: The reaction solvent is pyridine.

7. The use of the taraxasterol 3-ester derivative according to claim 1 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that: The tumor is lung cancer.

Images