A tumor-resistant formononetin derivative, a preparation method and application thereof

Abstract

The present application relates to the chemical medicine field, specifically, it relates to a kind of anti-tumor calophyllol derivative, preparation method and application, the calophyllol derivative has good inhibitory effect on thioredoxin reductase activity, and it is found that the molecule inhibits the activity of thioredoxin reductase by inhibiting the selenium cysteine of thioredoxin reductase carbon end, and then kills tumor cell, and then obtain the anti-tumor candidate drug with higher activity and better pharmacokinetic characteristics.

Description

Technical Field

[0001] This invention relates to the field of chemical medicine, and more specifically, to a strychnosine derivative with anti-tumor properties, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease that seriously threatens human health, ranking first in both incidence and mortality rates among the leading causes of death worldwide, and remains a primary unsolved problem globally. Currently, the main clinical treatments for cancer include traditional methods such as surgical resection, radiotherapy, and chemotherapy, as well as emerging strategies such as immunotherapy and targeted therapy. Chemotherapy, due to its systemic therapeutic advantages, still plays a crucial role in comprehensive cancer treatment. However, traditional chemotherapy drugs generally suffer from poor selectivity and significant toxic side effects. Therefore, identifying highly effective and low-toxicity anti-tumor drug candidates from natural products has become an important direction in new drug development.

[0003] Formononetin is a natural isoflavone compound widely found in various legumes such as Astragalus membranaceus and Spatholobus suberectus, and is one of the main active ingredients in these traditional Chinese medicines. Modern pharmacological studies have shown that formononetin possesses various biological activities, including antioxidant, anti-inflammatory, immunomodulatory, cardiovascular protective, and neuroprotective effects. In recent years, the antitumor activity of formononetin has attracted widespread attention. Studies have confirmed that formononetin exhibits significant inhibitory effects on various tumor cells, including non-small cell lung cancer, bladder cancer, osteosarcoma, cervical cancer, breast cancer, and ovarian cancer. However, formononetin suffers from pharmacokinetic defects such as poor water solubility and low oral bioavailability, which severely limits its potential application in clinical treatment.

[0004] Thioredoxin reductase (TrxR) is an intracellular selenium-containing homodimeric flavin enzyme highly expressed in various tumor tissues. It promotes tumor cell survival and proliferation by maintaining intracellular redox balance. Studies have shown that inhibiting TrxR activity can disrupt the redox homeostasis of tumor cells and selectively induce tumor cell apoptosis; therefore, TrxR has become one of the important targets for targeted cancer therapy. Given the promising antitumor activity of gentianin, using it as a lead compound for structural modification and optimization targeting TrxR holds promise for obtaining antitumor drug candidates with higher activity and better pharmacokinetic properties. Summary of the Invention

[0005] Based on the current state of existing technology research, the primary objective of this invention is to provide a strychnophorin derivative, the structural formula of which is shown in formula (Ⅰ):

[0006]

[0007] (I).

[0008] A second objective of this invention is to provide the application of the aforementioned strychnosine derivatives in inhibiting thioredoxin reductase.

[0009] A third objective of this invention is to provide the application of the aforementioned strychnosine derivatives in the preparation of thioredoxin reductase inhibitors.

[0010] A fourth objective of this invention is to provide the application of the aforementioned strychnosine derivatives in the preparation of antitumor drugs.

[0011] Preferably, the tumor includes one or more of breast cancer, non-small cell lung cancer, liver cancer, and ovarian cancer.

[0012] The beneficial effects of the present invention are as follows: The present invention provides a genistein derivative, which has a good inhibitory effect on thioredoxin reductase activity, and it has been found that this type of molecule inhibits the activity of thioredoxin reductase by inhibiting the selenocysteine ​​at the C-terminus of thioredoxin reductase, thereby killing tumor cells. Attached Figure Description

[0013] Figure 1 This is a schematic diagram showing the cytotoxic activity of the gentianin derivatives FMN-25 and gentianin synthesized in Experiment Example 1 against tumor cells.

[0014] Figure 2 This is a schematic diagram showing the inhibitory effects of compound FMN-25 on thioredoxin reductase and related enzyme activities in vitro.

[0015] Note: A: MDA-MB-231 cells, B: A549 cells, C: HepG2 cells, D: A2780 cells

[0016] Figure 3 This is a schematic diagram showing the apoptosis results of tumor cells treated with compound FMN-25 for 48 hours.

[0017] Note: A: MDA-MB-231 cells, B: A549 cells, C: HepG2 cells, D: A2780 cells Detailed Implementation

[0018] The scope of protection of the present invention will be described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0019] It should be noted that, unless otherwise specified, the reagents and consumables of the present invention in the following embodiments are all from commercial sources, and the methods described can all be obtained from the literature.

[0020] This invention provides a styracifoliin derivative, the structure of which is shown in the following formula (Ⅰ):

[0021]

[0022] (I).

[0023] In the following embodiments, formononetin refers to styrax pedunculin.

[0024] Example 1: Preparation method of strychnosine derivatives

[0025] The synthetic route for the strychnosine derivatives described in this invention is as follows:

[0026]

[0027] The specific preparation method of the strychnosine derivatives of the present invention is as follows:

[0028] (1) Dissolve p-methoxyphenylacetic acid (166.17 mg, 1 mmol) and resorcinol (110.11 mg, 1 mmol) in THF (20 mL), then add boron trifluoride (203.41 mg, 3 mmol), and stir at 50°C for 5 h. After the reaction is complete, transfer to room temperature, add water (10 mL), and continue stirring overnight. After the reaction is complete, filter to obtain the intermediate. Dissolve the intermediate and boron trifluoride (135.61 mg, 2 mmol) in DMF (10 mL), and then slowly add a DMF (5 mL) solution of phosphorus oxychloride (306.66 mg, 2 mmol). The reaction was carried out at room temperature for 3 h. After the reaction was completed, the resulting reaction solution was added dropwise to 10 mL of 37% hydrochloric acid solution at 85°C. After the addition was completed, the solution was refluxed for 1 h. After the reaction was completed, the solution was cooled to room temperature and filtered to obtain compound 1 (200.66 mg, 74.8%).

[0029] (2) 3-Aldehydebenzofuran (146.14 mg, 1 mmol) and malonic acid (124.87 mg, 1.2 mmol) were dissolved in ethanol (20 mL), and piperidine (100 μL) was added. The mixture was refluxed overnight at 90 °C. After the reaction was complete, dilute hydrochloric acid was added to adjust the pH of the solution to weakly acidic. A solid precipitated out. The solid was filtered and washed with water and petroleum ether to obtain compound 2 (136.62 mg, 72.6%).

[0030] (3) Compound 2 (188.18 mg, 1 mmol), HATU (570.36 mg, 1.5 mmol), and DMAP (366.51 mg, 3 mmol) were dissolved in DCM (20 mL), and the mixture was stirred in an ice bath for 30 min. Then, the mixture was transferred to room temperature, and compound 1 (321.92 mg, 1.2 mmol) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted and washed with saturated brine and DCM. After extraction, the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by vacuum rotation, and the mixture was purified by column chromatography (PE: DCM = 2: 1) to obtain compound FMN-25 (231.06 mg, 52.7%). Its structure was identified as belonging to the stigmophyticin derivative class.

[0031] The structural assessment data is as follows: 1 H NMR (600 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.59 (s,1H), 8.32 – 8.28 (m, 2H), 8.27 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.78 (d, J= 2.1 Hz, 1H), 7.63 (d, J = 8.7 Hz, 2H), 7.60 (d, J = 7.0 Hz, 1H), 7.56 (t, J= 7.4 Hz, 1H), 7.51 (dd, J = 8.7, 2.2 Hz, 1H), 7.09 – 7.05 (m, 3H), 3.86 (s,3H).

[0032] 13 C NMR (151 MHz, DMSO-d6) δ 167.40, 156.60, 140.46, 139.82, 139.50,137.77, 133.26, 131.56, 130.61, 130.55, 129.84, 127.90, 127.78, 127.53,126.88, 125.57, 125.48, 125.09, 123.34, 123.19, 120.65, 120.35, 117.76,114.17, 112.07, 55.65.

[0033] Example 2: Cytotoxicity experiment of strychnosine derivatives FMN-25 and strychnosine on tumor cells.

[0034] 1. Experimental Methods

[0035] Breast cancer MDA-MB-231 cells, non-small cell lung cancer A549 cells, liver cancer HepG2 cells, and ovarian cancer A2780 cells (all purchased from Guangzhou Chuangrong Biotechnology Co., Ltd.) were revived and cultured separately. Cells in the logarithmic growth phase were digested, counted, and seeded at a density of 5000 cells per well in 96-well plates. Cells were incubated overnight at 37°C in a 5% CO2 incubator to allow cell adhesion. The next day, the old culture medium was discarded, and fresh culture medium containing different concentrations of the compound FMN-25 (with negative control wells and zeroing wells) was added to each well. Cells were cultured for another 48 h. After the culture, 20 μL of MTT solution (5 mg / mL, dissolved in PBS) was added to each well, and the cells were incubated in the dark for 4 h. The supernatant was then carefully aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plates were then shaken slowly on a shaker for 10 minutes. The purple formazan crystals were fully dissolved using a microplate reader. Finally, the optical density (OD) value of each well was measured at a wavelength of 490 nm. The cell viability or inhibition rate was calculated based on the OD value to evaluate the inhibitory effect of FMN-25 on the proliferation of different tumor cells.

[0036] 2. Experimental Results

[0037] The results are as follows Figure 1 As shown, the results indicate that compound FMN-25 exhibits strong cytotoxicity against MDA-MB-231, A549, HepG2, and A2780 cells.

[0038] Example 3: Inhibitory effect of compound FMN-25 on thioredoxin reductase and related enzyme activities in vitro.

[0039] 1. Experimental Methods

[0040] TrxR (Soluble Biotech, BC1155) and U498C TrxR (U498C TrxR is a mutant enzyme with selenocysteine ​​at position 498 of the active site of TrxR mutated to cysteine, expressed and purified in our laboratory) were dissolved in 50 mM Tris-HCl buffer containing 1 mM EDTA. 80 nM of enzyme was added to each well of a 96-well plate. The plate was pre-incubated with 100 μM NADPH (Soluble Biotech, IN0040) at room temperature for 10 min to fully reduce the enzyme's active site. Then, different concentrations of compound FMN-25 (prepared with DMSO) were added, and the plate was incubated at room temperature for 30 min to inhibit the reaction. A negative control group containing an equal volume of DMSO and a blank control group without enzyme were also included. After incubation, 2 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) (Soluble Biotech, D8350) and 200 mM EDTA were added to each well, respectively. The detection reaction was initiated with a mixture of μM NADPH substrates, and the kinetic changes in absorbance were continuously monitored at a wavelength of 412 nm over 20 min using an enzyme-linked immunosorbent assay (ELISA) reader. The degree of inhibition of TrxR or U498CTrxR activity by FMN-25 was assessed by calculating the enzyme-catalyzed reaction rate.

[0041] 2. Experimental Results

[0042] The results are as follows Figure 2 As shown, compound FMN-25 inhibits the activity of thioredoxin reductase by selectively acting on the selenocysteine ​​active site at the C-terminus of TrxR.

[0043] Example 4: Apoptosis of tumor cells after 48 hours of treatment with compound FMN-25

[0044] 1. Experimental Methods

[0045] MDA-MB-231, A549, HepG2, and A2780 cells in logarithmic growth phase were seeded in 6-well plates and cultured overnight. Different concentrations of the compound FMN-25 were then added for 48 h. Suspended cells in the culture supernatant were collected, and adherent cells were digested with trypsin without EDTA. After centrifugation and washing, the cells were washed with pre-cooled PBS and resuspended in 1× Binding Buffer. Annexin V-FITC and PI were added sequentially for staining at room temperature in the dark for 15-20 min. Fluorescence signals were then detected by flow cytometry, and the proportion of cells in each quadrant was analyzed using software to calculate the total apoptosis rate to assess the pro-apoptotic effect of FMN-25.

[0046] 2. Experimental Results

[0047] The results are as follows Figure 3As shown, compound FMN-25 primarily kills tumor cells by inducing apoptosis. With increasing concentrations of FMN-25, the number of apoptotic cells also gradually increases, indicating that FMN-25 mainly kills tumor cells by inducing apoptosis.

[0048] In summary, this invention provides a genistein derivative that exhibits excellent inhibitory effects on thioredoxin reductase activity. Furthermore, it has been found that this type of molecule inhibits thioredoxin reductase activity by inhibiting the selenocysteine ​​residue at the C-terminus of thioredoxin reductase, thereby killing tumor cells.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strychnosine derivative, characterized in that, The structural formula of the styracifoliin derivatives is shown in formula (Ⅰ): （Ⅰ）。 2. The application of the strychnosine derivatives as described in claim 1 in inhibiting thioredoxin reductase.

3. The use of the strychnosine derivatives as described in claim 1 in the preparation of thioredoxin reductase inhibitors.

4. The use of the strychnosine derivatives as described in claim 1 in the preparation of antitumor drugs.

5. The application as described in claim 4, characterized in that, The tumors mentioned include one or more of breast cancer, non-small cell lung cancer, liver cancer, and ovarian cancer.

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