A formononetin derivative, a preparation method and application thereof

Abstract

The present application relates to the chemical medicine field, specifically, a kind of formononetin derivative, preparation method and its application, the compound has significant proliferation inhibitory effect to EGFR high expression tumor cell, and activity is significantly better than formononetin;To EGFR low expression tumor cell and normal cell, toxicity is lower, has good target selectivity and biocompatibility;To A431 and HCC827 cell EGFR phosphorylation has good inhibitory effect, and it is dose-dependent;The compound mainly kills tumor cell by inducing apoptosis, with the gradual increase of compound concentration, early apoptotic cell and late apoptotic cell number also gradually increase, it is explained that compound mainly kills tumor cell by inducing apoptosis, provides potential candidate for developing new EGFR targeted antitumor drug, has important research value and application prospect.

Description

Technical Field

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

[0002] Cancer is a major disease that seriously threatens human health, with its incidence and mortality rates continuing to rise, making it a pressing public health challenge worldwide. Currently, clinical treatments for cancer encompass traditional methods such as surgical resection, radiotherapy, and chemotherapy, as well as novel strategies like immunotherapy and targeted therapy. Among these, chemotherapy remains crucial in comprehensive cancer treatment due to its ability to kill tumor cells systemically. However, traditional chemotherapy drugs often suffer from insufficient targeting, significant toxic side effects, and while killing tumor cells, they can easily damage normal tissues. Furthermore, long-term use can lead to drug resistance, limiting their clinical efficacy. Therefore, identifying novel, highly active, and safer anti-tumor candidate compounds from natural products has become a key direction in the development of new anti-tumor drugs.

[0003] Formononetin is a natural isoflavone compound widely found in legumes such as Astragalus membranaceus and Spatholobus suberectus, and is also a core active ingredient in many traditional Chinese medicines. Modern pharmacological studies have confirmed that formononetin possesses multiple biological activities, including antioxidant, anti-inflammatory, and immunomodulatory effects. Its anti-tumor potential has also attracted considerable attention, exhibiting certain inhibitory effects on the proliferation of various tumor cells, including non-small cell lung cancer, bladder cancer, breast cancer, and ovarian cancer. However, natural formononetin itself has significant pharmacokinetic defects, such as poor water solubility, low oral bioavailability, and insufficient in vivo stability, which makes it difficult to fully exert its anti-tumor activity and greatly limits its clinical translation and application.

[0004] Epidermal growth factor receptor (EGFR), a key receptor tyrosine kinase, is highly expressed or abnormally activated in various solid tumors such as lung cancer, breast cancer, and colorectal cancer. Its overactivation can continuously regulate the proliferation, migration, invasion, and apoptosis resistance of tumor cells by mediating downstream signaling pathways such as PI3K / AKT and Ras / ERK. It is one of the driving factors of tumor development and also an important target for the current research and development of anti-tumor targeted drugs.

[0005] Based on this, using natural genistein as a lead compound, chemical structure modification was carried out on the EGFR target to optimize its molecular conformation and target binding activity, in order to obtain novel genistein derivatives with stronger activity, higher selectivity and better pharmacokinetic performance, providing potential candidates for the development of novel EGFR-targeted antitumor drugs, which has important research value and application prospects. Summary of the Invention

[0006] 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):

[0007]

[0008] (I).

[0009] A second objective of this invention is to provide the use of the genistein derivatives in the preparation of drugs for inhibiting EGFR phosphorylation, wherein the genistein derivatives are capable of inhibiting phosphorylation at the EGFRTyr1086 site in A431 cells and HCC827 cells.

[0010] A third objective of this invention is to provide the application of the aforementioned strychnosine derivatives in the preparation of drugs that inhibit the proliferation of EGFR-overexpressing tumor cells.

[0011] A fourth objective of this invention is to provide the application of the aforementioned strychnosine derivatives in the preparation of antitumor drugs.

[0012] Preferably, the tumor includes one or more of squamous cell carcinoma and non-small cell lung cancer.

[0013] The beneficial effects of this invention are as follows: This invention provides a genistein derivative, the compound of which has a significant inhibitory effect on the proliferation of EGFR-overexpressing tumor cells, and its activity is significantly better than that of genistein; it has low toxicity to EGFR-underexpressing tumor cells and normal cells, and has good targeting selectivity and biocompatibility; it has a good inhibitory effect on EGFR phosphorylation in A431 and HCC827 cells in a dose-dependent manner; the compound mainly kills tumor cells by inducing apoptosis, and as the concentration of the compound gradually increases, the number of early apoptotic cells and late apoptotic cells also gradually increases, indicating that the compound mainly kills tumor cells by inducing apoptosis. Attached Figure Description

[0014] Figure 1 This is a schematic diagram showing the results of compound FMN-39 inhibiting the expression of p-EGFR (Tyr1086) protein in A431 and HCC827 cells.

[0015] Note: A: A431 cells, B: HCC827 cells Detailed Implementation

[0016] 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.

[0017] 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.

[0018] This invention provides a styracifoliin derivative, the structure of which is shown in the following formula (Ⅰ):

[0019]

[0020] (I).

[0021] In the following embodiments, formononetin refers to styrax pedunculin.

[0022] Example 1: Preparation method of strychnosine derivatives

[0023] The synthetic route for the strychnosine derivatives described in this invention is as follows:

[0024]

[0025] The specific preparation method of the strychnosine derivatives of the present invention is as follows:

[0026] (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 hours. 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 hour. After the reaction was completed, the solution was cooled to room temperature and filtered to obtain compound 1 (200.66 mg, 74.8%).

[0027] (2) 3,5-Dimethyl-2-pyrrolecarboxaldehyde (123.16 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 at 90 °C overnight. 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 (113.16 mg, 68.5%).

[0028] (3) Compound 2 (165.19 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 minutes. 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-39 (196.50 mg, 47.3%). Its structure was identified as belonging to the stigmophytin derivative class.

[0029] The structural assessment data is as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 8.52 (s,1H), 8.20 (d, J = 8.7 Hz, 1H), 7.94 (s, 1H), 7.71 (d, J = 2.2 Hz, 1H), 7.55(d, J = 8.7 Hz, 3H), 7.42 (dd, J = 8.7, 2.2 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 6.17 (s, 1H), 3.79 (s, 3H), 2.37 (s, 3H), 2.24 (s, 3H).

[0030] 13 C NMR (151 MHz, DMSO-d6) δ 175.24, 163.27, 159.60, 156.57, 154.86,154.72, 146.20, 139.31, 130.60, 127.45, 124.75, 124.23, 124.15, 122.20,120.65, 118.16, 115.13, 114.16, 112.12, 83.31, 55.64, 40.50, 14.44, 14.22.

[0031] Example 2: Inhibitory effect of genistein derivatives FMN-39 and genistein on the proliferation of EGFR-high / low-expressing tumor cells.

[0032] 1. Experimental Methods

[0033] EGFR-high expressing tumor cells (squamous cell carcinoma A431 cells, non-small cell lung cancer HCC827 cells), EGFR-low expressing tumor cells (breast cancer MCF-7 cells), and normal human lung epithelial cells (BEAS-2B) were selected as experimental cell lines (all purchased from Guangzhou Chuangrong Biotechnology Co., Ltd.). After cell resuscitation, cells were cultured and passaged. 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 with 5% CO2 to allow cell adhesion. The next day, the old culture medium was discarded, and fresh culture medium containing different concentration gradients of the compounds FMN-39, gentianin, and gefitinib was added (negative control wells and zeroing wells were also included). Cells were cultured for another 48 h. After incubation, 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 wells were shaken slowly for 10 min to fully dissolve the purple formazan crystals. Finally, the optical density (OD) value of each well was measured at 490 nm using a microplate reader. Cell viability or inhibition rate was calculated based on the OD value to evaluate the inhibitory effect of FMN-39 on the proliferation of different tumor cells.

[0034] 2. Experimental Results

[0035] The results are shown in Table 1. The results indicate that compound FMN-39 significantly inhibited the proliferation of EGFR-high expressing tumor cells (A431, HCC827), and its activity was significantly superior to that of gentiopicrin (gentiopicrin showed no significant toxicity at the concentration gradients tested in this experiment, IC50...). 50 >16 μM); FMN-39 exhibits low toxicity to EGFR-low expressing tumor cells and normal cells, and demonstrates good targeting selectivity and biocompatibility.

[0036] Table 1. IC50 of FMN-39, strychnine, and gefitinib on various cell lines. 50 Values ​​(μM, n=3, x±s)

[0037] Example 3: Effects of compound FMN-39 on p-EGFR (Tyr1086) protein expression in A431 and HCC827 cells

[0038] 1. Experimental Methods

[0039] Logarithmically growing A431 and HCC827 cells were seeded in culture dishes and cultured overnight. Afterward, different concentrations of FMN-39 and gefitinib were added for 1 h, followed by stimulation with 100 ng / mL EGF for 10 min. After stimulation, cells were washed with pre-cooled PBS, lysed on ice for 30 min with RIPA lysis buffer containing phosphatase inhibitors and PMSF, and collected by centrifugation at 12000 rpm for 5 min at 4 °C. Protein concentration was determined by BCA and adjusted to a uniform level. Protein samples were denatured in a boiling water bath for 5 min and then subjected to SDS-PAGE electrophoresis (80 V stacking gel, 120 V separating gel). Transfer to a PVDF membrane at a constant current of 200 mA for 90 min was performed. After blocking with 5% skim milk at room temperature for 1 h, p-EGFR (Tyr1086) primary antibody was added and incubated overnight at 4 °C. After washing with TBST, HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. Imaging was performed using ECL chemiluminescence. Using GAPDH as an internal reference, the relative expression level of p-EGFR protein was calculated by analyzing the gray values ​​of the bands using ImageJ software.

[0040] 2. Experimental Results

[0041] The results are as follows Figure 1 As shown, compound FMN-39 exhibits good inhibitory effects on p-EGFR (Tyr1086) phosphorylation in A431 and HCC827 cells in a dose-dependent manner.

[0042] Example 4: Effect of compound FMN-39 on apoptosis in A431 and HCC827 cells

[0043] 1. Experimental Methods

[0044] A431 and HCC827 cells in logarithmic growth phase were seeded in 6-well plates and cultured overnight. Then, different concentrations of the compound FMN-39 were added for 48 h. The suspended cells in the culture supernatant were collected, and the 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. The fluorescence signal was then detected by flow cytometry, and the proportion of cells in each quadrant was analyzed using software to evaluate the pro-apoptotic effect of FMN-39.

[0045] 2. Experimental Results

[0046] The results are shown in Tables 2 and 3. Compound FMN-39 mainly kills tumor cells by inducing apoptosis. As the concentration of compound FMN-39 gradually increases, the number of early apoptotic cells and late apoptotic cells also gradually increases, indicating that compound FMN-39 mainly kills tumor cells by inducing apoptosis.

[0047] Table 2. Effects of compound FMN-39 on apoptosis in A431 cells (48 h, x±s, n=3)

[0048] Note: Compared with the control group, *P<0.05, **P<0.01

[0049] Table 3 Effect of compound FMN-39 on apoptosis in HCC827 cells (48 h, x±s, n=3)

[0050] Note: Compared with the control group, *P<0.05, **P<0.01

[0051] In summary, this invention provides a genistein derivative, which exhibits significant inhibitory activity against EGFR-overexpressing tumor cells, with activity significantly superior to genistein; it also shows low toxicity against EGFR-underexpressing tumor cells and normal cells, demonstrating good targeting selectivity and biocompatibility; it effectively inhibits EGFR phosphorylation in A431 and HCC827 cells in a dose-dependent manner; and the compound primarily kills tumor cells by inducing apoptosis. As the compound concentration gradually increases, the number of early and late apoptotic cells also gradually increases, indicating that the compound primarily kills tumor cells by inducing apoptosis.

[0052] 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 use of the genistein derivative as described in claim 1 in the preparation of a drug for inhibiting EGFR phosphorylation, characterized in that, The styracin derivatives can inhibit phosphorylation at the EGFRTyr1086 site in A431 and HCC827 cells.

3. The use of the strychnosine derivatives as described in claim 1 in the preparation of drugs that inhibit the proliferation of EGFR-overexpressing tumor cells.

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 types of squamous cell carcinoma and non-small cell lung cancer.

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