A benzofuran derivative with anti-tumor activity, preparation method and application thereof

Abstract

The invention discloses a benzofuran derivative, a preparation method and an application thereof. The benzofuran derivative has a structural formula of 3-(3-(3-hydroxyphenyl)thio)benzofuran-2-yl)-1-methylindole-2-one, and the benzofuran derivative is constructed in one step by a Meyer-Schuster rearrangement / nucleophilic addition / intramolecular cyclization tandem reaction under boron trifluoride ether conditions. The benzofuran derivative obtained by the invention has better anti-tumor efficacy, especially has a stronger inhibitory effect on prostate cancer cells, and has a wide range of pharmaceutical uses.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine preparation, and specifically relates to a benzofuran derivative, a preparation method and an application thereof Background Art

[0002] Currently, there are various ways to clinically treat tumors, including chemotherapy, radiotherapy, targeted therapy, etc. Radiotherapy is a more common treatment method, but radiotherapy has side effects such as large toxicity, limited anti-tumor effect, and poor patient tolerance. Therefore, finding drugs with good therapeutic effects, high bioavailability and little harm to the body of tumor patients has become the key in the field of cancer treatment.

[0003] The benzofuran structure is a type of compound skeleton that is widely present in natural products and drug molecules. Natural products containing the benzofuran skeleton exhibit good biological activities, such as anti-tumor, anti-fungal, antioxidant, anti-inflammatory, etc., so the synthesis of this type of compound is of great significance. Although compounds with a simple benzofuran structure have certain anti-tumor effects, the direct use effect is not good, and the compound needs to be further modified. How to obtain a benzofuran derivative with good drug effect, excellent anti-tumor effect and low toxicity and side effects is the key to studying this type of compound.

[0004] Patent CN 108976187A discloses a substituted benzofuran derivative and its preparation and use in the preparation of anti-tumor drugs, which are mainly used to treat breast cancer, mesothelioma and kidney cancer, and have significant therapeutic effects and few toxic side effects. However, the compound is relatively complex, with a long synthesis route and a low synthesis yield. In addition, only the therapeutic effect on the above-mentioned cancers is disclosed, and the therapeutic effect on other tumors is not reflected.

[0005] Patent CN113582978A discloses a 3-thiobenzofuran derivative and a method for synthesizing the same. The compound disclosed in the patent is similar in structure to the benzofuran derivative claimed in the present invention. The patent only discloses the structural formula and preparation method of the compound similar in structure to the present invention, but does not disclose the use of the compound.

[0006] Therefore, according to the information disclosed in the prior art, further developing benzofuran derivatives and obtaining a compound with good therapeutic effect on various types of tumor cells has become the key to studying benzofuran derivative anti-tumor drugs. Summary of the invention

[0007] In order to solve the problems existing in the prior art and provide a safe and effective method for tumor drug treatment, one of the purposes of the present invention is to provide a class of benzofuran derivatives with anti-tumor activity; the second purpose of the present invention is to provide a method for preparing benzofuran derivatives simply and quickly; the third purpose of the present invention is to apply the above-mentioned benzofuran derivatives to prepare clinical anti-tumor drugs; the above-mentioned purposes of the present invention are achieved by the following technical solutions:

[0008] The benzofuran derivative 3a with antitumor activity has the following structural formula:

[0009]

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned benzofuran derivatives, wherein the preparation method is:

[0011]

[0012] Specifically, the solvent used in the preparation of compound 3a of the present invention is one or more of C1-C4 alcohol, acetonitrile, ethyl acetate, chloroform, and dichloroethane;

[0013] Furthermore, in the present invention, the molar ratio of compound 1a to compound 2a for preparing compound 3a is 1 to 3:1, preferably 1.5:1.

[0014] Furthermore, the reaction temperature for preparing compound 3a of the present invention is -5-5°C, preferably 0°C.

[0015] Furthermore, the molar ratio of boron trifluoride etherate to compound 2a for preparing compound 3a of the present invention is 1-3.5:1, preferably 1.6:1.

[0016] Another object of the present invention is to provide the use of compound 3a and its pharmaceutically acceptable salts, solvates and hydrates in the preparation of anti-tumor drugs.

[0017] Furthermore, the pharmaceutically acceptable salt described in the present invention is one of sodium salt, citrate, phosphate, acetate and sulfate.

[0018] Furthermore, the tumor of the present invention is selected from lung cancer, gastric cancer, prostate cancer and breast cancer.

[0019] Furthermore, the present invention provides a pharmaceutical preparation consisting of compound 3a or its pharmaceutically acceptable salt, solvate and hydrate in combination with a pharmaceutically acceptable carrier or diluent, wherein the pharmaceutically acceptable carrier includes an inert solid filler or diluent or a sterile aqueous solution or organic solution.

[0020] Furthermore, the pharmaceutical preparation of the present invention is a tablet, capsule, pill, spray, aerosol or injection.

[0021] Furthermore, the injection described in the present invention is a freeze-dried powder preparation.

[0022] Beneficial effects of the present invention:

[0023] (1) The benzofuran derivatives or pharmaceutically acceptable salts thereof provided by the present invention can effectively inhibit the spread of tumor cells, especially prostate cancer cells, and can effectively reduce and inhibit the growth of PC-3 cells.

[0024] (2) The present invention provides a new benzofuran derivative, which has a simple preparation method, good anti-tumor effect, and is suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the H NMR spectrum of the benzofuran derivative 3a obtained in Example 1;

[0026] Figure 2 This is the C NMR spectrum of the benzofuran derivative 3a obtained in Example 1;

[0027] Figure 3 Results of cell live-death experiments under different concentrations of compound 3a;

[0028] Figure 4 The results of the PC-3 cell responses exposed to compound 3a;

[0029] Figure 5 The results of Western blotting analysis of the effect of compound 3a on apoptotic proteins;

[0030] Figure 6 These are the results of the anti-tumor experiment of compound 3a in animals. DETAILED DESCRIPTION

[0031] The present invention is further described below by way of examples. It should be correctly understood that the examples of the present invention are only used to illustrate the present invention, rather than to limit the present invention, so simple improvements to the present invention based on the method of the present invention are within the scope of protection claimed by the present invention.

[0032] In this example, the NMR data used tetramethylsilane as an internal standard, and the 1H NMR and 13C NMR spectra were measured on a Bruker AV 600 in DMSO-d6, and the HRMS data were measured on a Varian QFT-ESI.

[0033] I: Preparation of compound 3a

[0034]

[0035] The synthesis method of compound 1a can be found in the literature "Org. Biomol. Chem., 2018, 16, 6133. Compound 2a and boron trifluoride etherate were purchased from Anaiji.

[0036] Compound 1a (0.2 mmol) and compound 2a 3-hydroxybenzenethiol (0.4 mmol) were dissolved in a solution of dichloromethane (2 mL), and BF3·Et2O (0.2 mmol) was added dropwise. The mixture was stirred at 25°C for 12 h. The crude product was then distilled under reduced pressure. The product 3a was purified by column chromatography (petroleum ether / EtOAc=8:1) with a yield of 92% and a product purity of more than 97%.

[0037] The obtained product 3a was characterized, and its characterization data are as follows:

[0038] 1H NMR (CDCl3, 400MHz): δ7.46 (d, J = 8.0Hz, 1H), 7.33 (m, 2H), 7.26–7.16 (m, 2H), 7.08–6.98 (m, 3H), 6.91 (d, J = 8.0Hz, 1H), 6.84 (d, J = 7.6Hz, 1H), 6.75 (s, 1H), 6.65 (s, 1H), 6.59 (d, J = 7.6Hz, 1H), 5.31 (s, 1H), 3.26 (s, 3H); see the attached spectrum Figure 1

[0039] 13C NMR (CDCl3, 100MHz): δ173.2, 156.6, 156.2, 154.7, 144.1, 136.7, 130.0, 129.0, 128.7, 126.0, 125.3, 124.5, 123.6, 123.1, 120.3, 119.2, 113.9, 113.4, 111.6, 109.8, 108.7, 45.1, 26.7; see the attached spectrum Figure 2

[0040] HRMS-ESI(m / z):calcd for C23H17NO3S[M+H]+:388.1002; found 388.1006. 2: Cell experiment

[0041] Prostate cancer PC-3 cells, triple-negative breast cancer cells MDA-MB-231, lung cancer cells A549, the culture medium is the complete culture medium of each cell, and the CCK-8 kit is purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Abiraterone acetate is obtained by the preparation method disclosed in CN111349138. In addition, the structural formula of the compound I described in the present invention is:

[0042]

[0043] The structural formula of compound II is:

[0044]

[0045] The preparation methods of the above-mentioned compounds I and II are shown in patent CN113582978A.

[0046] Specific experiments:

[0047] PC-3 cell CCK-8 assay

[0048] Cell viability was detected by CCK-8 method. Cells with a density of 3000-5000 PC-3 cells / well were seeded on 96-well plates. After cell attachment, the cells were treated with different dilutions or solvent control DMSO for 48 hours. Then, CCK-8 reagent was added and cultured at 37°C and 5% CO2 for 1 hour. The absorbance was measured at 450nm using a microplate spectrophotometer (Thermo). The cell survival rate of the control group was 100%. For IC50, cells were treated with different concentrations of compounds (0, 0.78125μM, 1.5625μM, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM) for 48 hours, cell viability was measured, and the inhibition rate was calculated. The CCK-8 assay was performed three times, and each time was repeated at least three times. The IC50 of compound 3a for PC-3 cells was 3.45±1.16μM using prism statistical software.

[0049] CCK-8 assay for MDA-MB-231 cells

[0050] Cell viability was detected by CCK-8 method. Cells with a density of 3000-5000 MDA-MB-231 cells / well were inoculated on 96-well plates. After cell attachment, the cells were treated with different dilutions or solvent control DMSO for 48 hours. Then, CCK-8 reagent was added and cultured at 37°C and 5% CO2 for 1 hour. The absorbance was measured at 450nm using a microplate spectrophotometer (Thermo). The cell survival rate of the control group was 100%. For IC50, cells were treated with different concentrations of compounds (0, 0.78125μM, 1.5625μM, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM) for 48 hours, cell viability was measured, and the inhibition rate was calculated. The CCK-8 assay was performed three times, with at least three replicates each time. The IC50 of compound 3a on MDA-MB-231 cells was studied using prism statistical software, and the obtained IC50 value was 52.18±5.65μM.

[0051] A549 cell CCK-8 assay

[0052] The cell viability was detected by CCK-8 method. Cells with a density of 3000-5000 A549 cells / well were inoculated on 96-well plates. After the cells adhered to the wall, the cells were treated with different dilutions or solvent control DMSO for 48 hours. Then, CCK-8 reagent was added and cultured at 37°C and 5% CO2 for 1 hour. The absorbance was measured at 450nm using a microplate spectrophotometer (Thermo). The cell survival rate of the control group was 100%. For IC50, cells were treated with different concentrations of compounds (0, 0.78125μM, 1.5625μM, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM) for 48 hours, cell viability was measured, and the inhibition rate was calculated. The CCK-8 assay was performed three times, with at least three replicates each time. The IC50 value of compound 3a for A549 cells was studied using prism statistical software. The obtained IC50 was 86.79±7.65 μM.

[0053] In addition, the IC50 values ​​of prostate cancer cells PC-3, triple-negative breast cancer cells MDA-MB-231 and lung cancer cells A549 were tested by the same method as in Example 1 using the same amounts of Compound I, Compound II and Abiraterone acetate as in Compound 3a. The specific test results are as follows:

[0054] Table 1 IC50 values ​​of different compounds at the same dosage against different cancer cells

[0055]

[0056] Note: The above IC50 value is greater than 100 μM, indicating that the compound has a poor inhibitory effect on the cells and has poor drugability against the cancer cells.

[0057] Cell live or dead

[0058] PC-3 cells were seeded on 6-well plates and left overnight to adhere. The cells were divided into 4 groups (0 μM, 3 μM, 6 μM, 12 μM). After different treatments, the cells were stained with calcein AM / PI for 0.5 h according to the manufacturer's instructions and then observed with an inverted fluorescence microscope.

[0059] Apoptosis and necrosis assay

[0060] PC-3 cancer cells were inoculated into 6-well plates, cultured at 37°C for 24 h, and divided into 4 groups according to the above method. Cell apoptosis was detected using the annexinV-PE / 7-AAD apoptosis detection kit.

[0061] Western blotting analysis

[0062] PC-3 cancer cells were inoculated in 6-well plates, cultured at 37°C for 24 hours, and divided into 4 groups according to the above method. After different treatments, the cells were collected by trypsin digestion and the cell pellets were collected by centrifugation. Total protein was extracted from PC-3 cells using RIPA lysis buffer. The protein concentration was determined using the BCA protein detection kit, and western blotting was performed using standard methods. The proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. Then, the PVDF membrane was blocked with 5% milk for 1 hour and incubated with the primary antibody at 4°C for 12 hours. Subsequently, the membrane was washed 3 times with TBST and incubated with the secondary antibody for 2 hours at room temperature. After rinsing 3 times with TBST, the PVDF membrane was observed using an enhanced ECL detection kit.

[0063] In vivo anti-tumor experiment in mice

[0064] PC-3 tumor-bearing mice with a tumor volume of 120 mm3 were randomly divided into 4 groups (n≥5): body weight and tumor size were recorded every other day after treatment. Tumor volume was calculated as follows: Tumor volume = (width 2 × length) / 2. All mice were photographed and euthanized 2 weeks after injection, and the tumors were removed and photographed. The collected tumors were immediately fixed in 4% paraformaldehyde solution, followed by standard dehydration and paraffin embedding. The embedded tissues were then sliced ​​into 4 μm thick sections and subjected to standard TUNEL and H&E histological analysis.

[0065] H&E staining

[0066] The paraffin sections were fixed in xylene and hydrated with gradient ethanol. The sections were stained with hematoxylin and eosin according to the direction and observed under a fluorescence microscope.

[0067] TUNEL staining

[0068] Conventional paraffin sections were dewaxed in xylene, dehydrated and permeabilized in Triton-X100 for 60 min, washed twice in PBST, and then incubated with fluorescein-conjugated dUTP and TdT (TUNEL reagent) for 1 h in the dark at 37 ° C. After TUNEL staining, they were washed twice with PBST at room temperature for 5 min each. Then, the sections were incubated with Hoechst 33342 (100 ng / mL PBS) at room temperature in the dark for 15 min and washed twice with PBST at room temperature for 5 min each. The slides were immediately examined with a fluorescence microscope.

[0069] Biological experiments:

[0070] In the in vitro experiment, the half maximal inhibitory concentration (IC50 value) of compound 3a on PC-3 cells was 3.45±1.16μM, and the anti-tumor activity of the compound was further evaluated by live / dead staining of prostate cancer PC-3 cell line. Figure 3 As shown, with the increase of compound 3a concentration, the number of dead cells increased, which was consistent with the results of CCK-8 assay.

[0071] To verify whether the observed antiproliferative effect was caused by apoptosis, prostate cancer cells were treated with different concentrations (3 μM, 6 μM, and 12 μM) of 3ad for 24 h and then analyzed by flow cytometry. Figure 4 As shown, a concentration-dependent response was observed for PC-3 cells exposed to compound 3a. The apoptosis rates of PC-3 cells caused by compound 3a were 10.27% (3 μM), 38.09% (6 μM) and 41.99% (12 μM), respectively, indicating that compound 3a has the ability to promote cell apoptosis in a concentration-dependent manner within a certain concentration range. At the same time, the effect of compound 3a on apoptotic proteins was further explored. BCL-2 and BAX are closely related to cell apoptosis. Figure 5 As shown, the expression level of Bcl-2 protein in the experimental group was decreased, and the expression level of Bax protein was significantly upregulated. The results showed that compound 3a could induce cell apoptosis.

[0072] Based on the in vitro experimental results of compound 3a, a preliminary efficacy experiment was conducted on a PC-3 mouse transplant tumor model, and PC-3 cells were injected into mice via the lower back. 3The mice were randomly divided into four groups. Cisplatin 25 mg / kg was used as positive control (intraperitoneal injection once a day), and compound 3a 15 mg / kg and 25 mg / kg were intraperitoneally injected once a day. Figure 6 As shown in a, compound 3a significantly inhibited tumor growth at a concentration of 25 mg / kg, and the body weight of mice did not decrease significantly, indicating the effectiveness and safety of compound 3a ( Figure 6 b and 6c). The targeting of tumor tissue was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and hematoxylin-eosin (H&E) staining experiments. The results showed that after treatment with compound 3a, the number of apoptotic tumor cells in mice increased significantly, especially the compound 3ad at a concentration of 25 mg / kg had a significant killing effect on tumor cells, as shown in Figure 6 .

[0073] Although the above describes the specific implementation methods of the present invention, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A benzofuran derivative having antitumor activity, characterized in that: The structural formula is as follows:

2. A method for preparing the benzofuran derivative according to claim 1, characterized in that: The preparation route of the benzofuran derivative is:

3. The preparation method according to claim 2, characterized in that: The solvent used in the preparation method is one or more of C1-C4 alcohol, acetonitrile, ethyl acetate, chloroform and dichloroethane.

4. The preparation method according to claim 2, characterized in that: The molar ratio of the compound 1a to the compound 2a is 1 to 3:

1.

5. The preparation method according to claim 2, characterized in that: The molar ratio of compound 1a to compound 2a is 1.5:

1.

6. The preparation method according to claim 2, characterized in that: The reaction temperature is -5-5°C.

7. The preparation method according to claim 2, characterized in that: The reaction temperature is 0°C.

8. The preparation method according to claim 2, characterized in that: The molar ratio of boron trifluoride ether to compound 2a is 1-3.5:

1.

9. The preparation method according to claim 2, characterized in that: The molar ratio of boron trifluoride ether to compound 2a is 1.6:

1.

10. Use of the compound according to claim 1 and its pharmaceutically acceptable salt in the preparation of drugs for lung cancer, prostate cancer and triple-negative breast cancer.

11. The use according to claim 10, characterized in that: The pharmaceutically acceptable salt is one of sodium salt, citrate, phosphate, acetate and sulfate.

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