Taraxasterol nitrobenzoate derivatives and their anti-tumor applications

Abstract

This invention relates to a taraxasterol nitrobenzoate derivative in the field of medicinal chemistry, with the following structural formula: Compound B is obtained by reacting taraxasterol 1 with 4-nitrophthalic anhydride in a suitable alkaline catalyst and organic solvent. This synthetic route offers high yield and is easy to implement. Activity tests demonstrate that the taraxasterol nitrobenzoate derivative shown as B, designed and synthesized in this invention, is a suitable antitumor drug candidate, particularly as a candidate drug for lung cancer.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a taraxasterol nitrobenzoate 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 nitrobenzoate derivative B 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 nitrobenzoate derivative B 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 nitrobenzoate derivative, the structural formula of which is shown in Formula B. .

[0008] The second objective of this invention is to provide a method for preparing taraxasterol nitrobenzoate derivatives. Taraxasterol 1 is reacted with nitrophthalic anhydride in a suitable alkaline catalyst and reaction solvent to obtain compound B and taraxasterol nitrobenzoate derivative isomer byproduct C. 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 nitrobenzoate derivatives in the preparation of antitumor drugs.

[0014] Specifically, the tumor is lung cancer.

[0015] Activity tests have demonstrated that the taraxasterol nitrobenzoate 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 72.64%, 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-position hydroxyl group on the parent structure of taraxasterol, a novel taraxasterol nitrobenzoate derivative B was obtained. Activity tests show that this derivative possesses good antitumor activity, especially against tumors such as lung cancer. Furthermore, the synthetic method of taraxasterol nitrobenzoate derivative B 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 nitrobenzoate derivative B 1 Full HNMR image; Figure 2 For taraxasterol nitrobenzoate derivative B 1 A magnified view of a portion of the HNMR spectrum; Figure 3 Byproduct C, an isomer of taraxasterol nitrobenzoate derivative. 1 Full HNMR image; Figure 4 Byproduct C, an isomer of taraxasterol nitrobenzoate derivative. 1 A magnified view of a portion of the HNMR spectrum; Figure 5 This image shows the morphological changes of cells after treatment with taraxasterol nitrobenzoate derivative B. Figure 6 The graph shows the inhibitory effect of the drug taraxasterol nitrobenzoate derivative B on lung cancer cells at different time points. Detailed Implementation

[0019] The following detailed description illustrates the specific implementation method: Example 1: Preparation of taraxasterol nitrobenzoate derivative B: 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, 0.45 mmol (5.0 equiv) of 4-nitrophthalic anhydride and 0.09 mmol (1.0 equiv) of DMAP were weighed and added to the reactor. The mixture was stirred at 120 °C for 16 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, followed by elution with petroleum ether:ethyl acetate = 1:1 and then with dichloromethane:methanol = 10:1 to obtain the target product, taraxasterol nitrobenzoate derivative B (yield 58.0%), as an off-white solid, and the isomer of taraxasterol nitrobenzoate derivative, byproduct C (yield 17.0%), as an off-white solid.

[0020] Derivative B: 1 H NMR (400 MHz, DMSO) δ 8.47 (s, 1H), 8.38 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 4.70 – 4.55 (m, 3H), 2.39 (s, 1H), 2.19 – 2.09 (m,2H), 2.07 (s, 1H), 1.67 (s, 2H), 1.51 (t, J = 12.6 Hz, 5H), 1.36 (s, 5H), 1.23– 1.05 (m, 9H), 1.00 (s, 6H), 0.93 (s, 6H), 0.84 (s, 6H), 0.80 (s, 3H). Derivative C: 1 H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 8.6 Hz, 1H), 8.16 (s, 1H), 7.87 (d, J = 8.5 Hz, 1H), 4.52 – 4.67 (m, 3H), 2.36 – 2.45 (m, 1H), 2.03 – 2.20(m, 2H), 1.44 – 1.73 (m, 9H), 1.05 – 1.43 (m, 8H), 1.02 – 0.96 (m, 5H), 0.99 (s, 6H), 0.93 (s, 6H), 0.84 (s, 3H), 0.82 (s, 6H). Example 2: Activity test of novel taraxasterol nitrobenzoate derivative B Experimental preparation: Cell line: Human lung cancer cell line A549.

[0021] Culture medium: RPMI-1640 medium; 10% high-quality fetal bovine serum; 1% penicillin and antibiotics.

[0022] Culture conditions: Gas phase: air, 95%; carbon dioxide, 5%. Temperature: 37℃, incubator humidity: 100%. Drug solvent: Dimethyl sulfoxide (DMSO).

[0023] Reagent: Cell Counting Kit-8 (CCK-8) 1. Drug and cell treatment (1) Dissolve taraxasterol (molecular weight: 426.72) and its derivative powder in an appropriate volume of DMSO solution under sterile conditions; (2) When the confluence of human A549 cells reaches 80%-90% after 3 passages, seed the corresponding number of cells into 6-well or 96-well plates according to the experimental type, and culture conditions as described above. (3) On the day of drug treatment, the cell fusion rate in the well should reach about 40%-50%; (4) Discard the old culture medium, wash the cells once with PBS, and add fresh complete culture medium according to the culture system. (5) Set up a control group and a drug treatment group respectively, with three replicates for each group; (6) After the drug is added, place the culture plate into the cell culture incubator to continue culturing; (7) No medium needs to be changed during drug treatment. Cells that have been successfully treated with drugs can be used for subsequent related experiments.

[0024] 2. CCK-8 assay for cell viability (1) Select human A549 cells in the logarithmic growth phase, digest them with trypsin, centrifuge them, and count the cells under a microscope; (2) With 1x10 per hole 4 Cells were seeded in 96-well plates with a culture volume of 100 μL. Blank group, control group and experimental group were set up respectively, and three replicates were set up for each group. The cells were cultured in an incubator. (3) The next day, replace the medium with fresh RPMI-1640 complete medium and add different doses of taraxasterol (Tara) and its derivatives (Tara). der (The drug was administered at concentrations of 0, 5, 10, 20, 40, and 80 μg / ml, respectively), with action times of 24 h, 48 h, and 72 h. (4) Add 10 μL of CCK-8 reagent to each well under light-protected conditions and continue to incubate for 1-2 h. (5) Under light-protected conditions, the absorbance (OD) of each well was measured at a wavelength of 450 nm using an ELISA reader. 450 The absorbance values ​​were determined, and statistical analysis software was used to plot the absorbance curves and calculate the inhibition rate of the drug on cells.

[0025] 3. Observe cell morphological changes under a microscope. (1) Select human A549 cells in the logarithmic growth phase, digest with trypsin, centrifuge, and count the cells under a microscope; (2) With 3x10 per hole 5 One cell was seeded in a 6-well plate with a culture volume of 2 mL. Control and experimental groups were set up and cultured in an incubator. (3) On the second day, replace the medium with fresh RPMI-1640 complete medium and add 40 μg / ml of taraxasterol and its derivatives, and let it react for 48 h. (4) Discard the old culture medium, wash once with PBS, and observe and photograph the morphological changes of cells after drug treatment using a regular optical microscope.

[0026] The results are as follows Figure 5 Table 1 and Figure 6 As shown. 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 nitrobenzoate derivative B.

[0027] 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.

[0028] exist Figure 6 In 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; Dandelion sterol nitrobenzoate derivative B:IC 50 =25.86μg / mL; 48h: taraxasterol: IC 50 =41.70 μg / mL; Dandelion sterol nitrobenzoate derivative B:IC 50 =16.90 μg / mL; 72h: taraxasterol: IC 50 =33.01μg / mL; Dandelion sterol nitrobenzoate derivative B:IC 50 =15.74 μg / mL.

[0029] Table 1. Tumor inhibition rate (%) of taraxasterol nitrobenzoate derivative B

[0030] In Table 1, Tara and Tara der The drug was administered at a concentration of 40 μg / ml for 72 hours. The experimental results in Table 1 show that the taraxasterol nitrobenzoate derivative B of this invention has excellent in vitro antitumor activity. At a drug concentration of 40 μg / ml, it inhibited human lung cancer cells A549 cells by up to 72.64% in vitro, which is stronger than the inhibitory activity of the positive control drug taraxasterol. It can be used as a novel taraxasterol candidate drug for clinical antitumor applications.

[0031] 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 nitrobenzoate derivative, characterized in that, Its structural formula is shown in formula B. 。 2. The method for preparing the taraxasterol nitrobenzoate derivative according to claim 1, characterized in that: Compound B was obtained by reacting taraxasterol 1 with 4-nitrophthalic 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 nitrobenzoate derivative B 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.

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