An organic selenium compound and its application in preparing anticancer drugs

Abstract

The present invention provides a novel organic selenium compound and a synthesis method thereof, as well as the use of the compound in preparing a drug for preventing and / or treating cancer. The organic selenium compound provided by the present invention combines a natural active compound with selenium, so that the anti-cancer activities of the two produce a synergistic effect, and increases its safety and anti-cancer effect, and has great potential in preventing and treating cancer.

Description

Technical Field

[0001] The invention belongs to the field of drug synthesis, and specifically relates to a novel organic selenium compound and a synthesis method thereof, as well as application of the compound in preparing drugs for preventing and / or treating cancer. Background Art

[0002] Due to factors such as an aging population, unhealthy lifestyles, and environmental pollution, cancer has become a common and highly prevalent disease worldwide, and the number of cancer patients continues to rise every year. Cancer drug treatments, including chemotherapy and targeted therapy, have made significant progress, but still face many challenges and pain points. Chemotherapy drugs have non-specific toxicity, relatively limited clinical targets, relatively narrow indications for targeted therapy, and are prone to drug resistance, similar to chemotherapy.

[0003] Selenium is one of the essential trace elements in organisms. It has many special functions for human health and is known as the "spark of life" and the "king of anti-cancer". Selenium is the active center of glutathione peroxidase (GSH-Px) and has a strong antioxidant effect. Selenium deficiency in the human body can lead to the occurrence of dozens of diseases. my country has made significant progress in preventing Keshan disease, Kaschin-Beck disease and liver cancer through selenium supplementation. The anti-cancer effect of selenium is mainly achieved through its antioxidant, immunomodulatory, inhibition of cancer cell proliferation and angiogenesis, and promotion of DNA repair. However, most of the selenium used in clinical practice is inorganic selenium, such as sodium selenite and selenium disulfide. However, inorganic selenium has the disadvantages of large toxic side effects and low bioavailability. Currently, a few organic selenium drugs such as ebselen are in the clinical trial stage. There are very few organic selenium drugs that can be used for clinical anti-cancer in the future.

[0004] Active ingredients isolated from Chinese herbal medicines have always played an important role in anti-cancer, such as paclitaxel and camptothecin. my country has rich Chinese herbal medicine resources and thousands of years of experience in traditional Chinese medicine, but its anti-cancer advantages have not been fully utilized. The combination of anti-cancer active structures of Chinese herbal medicines and selenium is still a blank in the field of drug synthesis. Combining natural active compounds with selenium to synthesize new organic selenium drugs can produce synergistic anti-cancer activities of the two, increase their safety and anti-cancer effects, and have great potential in the prevention and treatment of cancer. Summary of the invention

[0005] In order to overcome the deficiencies in the prior art, the present invention synthesized a new organic selenium compound with anti-cancer effect, which has high safety, good anti-cancer effect and low drug resistance.

[0006] In a first aspect, the present invention provides an organic selenium compound having the structure shown below:

[0007] .

[0008] The organic selenium compound provided by the present invention combines selenium element with the traditional Chinese medicine monomer p-hydroxycinnamic acid through chemical bonds to form a new selenium-containing active molecule, so that the two can play a synergistic role.

[0009] In a second aspect, the present invention provides a method for preparing the organic selenium compound.

[0010] The organic selenium compound of the present invention is prepared by the following synthetic route:

[0011] (1) ;

[0012] (2) ;

[0013] (3) .

[0014] As a preferred embodiment of the present invention, the step (1) comprises: taking selenium powder, mixing it with distilled water under nitrogen protection, slowly adding sodium borohydride aqueous solution while stirring, and after the system is clarified, adjusting 2-chloroacetic acid to a pH value of 9-10 with a saturated sodium carbonate solution, adding it to the above reaction solution, and stirring at room temperature; collecting the reaction solution, adjusting the pH value to 2-3 with dilute hydrochloric acid, extracting with ethyl acetate, collecting the organic phase, removing water, concentrating, and recrystallizing to obtain 2,2'-selenodiacetic acid.

[0015] As a preferred embodiment of the present invention, the step (2) comprises: dissolving 2,2'-selenodiacetic acid in anhydrous tetrahydrofuran at -5 to 5°C under nitrogen protection, adding oxalyl chloride dropwise, and then stirring the reaction at 38 to 42°C; evaporating the solvent in the reaction solution to obtain 2,2'-selenodiacetyl chloride.

[0016] As a preferred embodiment of the present invention, the step (3) comprises: under nitrogen protection, preparing an anhydrous tetrahydrofuran solution of p-hydroxycinnamic acid, adding pyridine and then precooling in an ice bath; slowly dropping an anhydrous tetrahydrofuran solution of 2,2'-selenodiacetyl chloride, and then stirring the reaction at 33-37 °C; evaporating the reaction solution to dryness, re-dissolving it with ethyl acetate and then washing it with water, extracting and collecting the organic phase, removing water, and concentrating it to obtain a crude product of 3,3'-(((2,2'-selenobis(acetyl))bis(oxy))bis(4,1-phenylene))diacrylic acid. The obtained crude product can be purified by conventional methods in the art such as semi-preparative liquid chromatography to obtain a pure product of the target compound.

[0017] In a third aspect, the present invention provides use of an organic selenium compound in the preparation of a drug for preventing and / or treating cancer.

[0018] As a preferred embodiment of the present invention, the cancer is selected from one or more of melanoma, liver cancer, lung cancer, gastric cancer, and cervical cancer.

[0019] As a preferred embodiment of the present invention, the drug inhibits the proliferation of cancer cells. Experimental results show that the organic selenium compound provided by the present invention has a good inhibitory effect on the proliferation of melanoma, liver cancer, lung cancer, gastric cancer and cervical cancer cells, and has no killing effect on normal human cells.

[0020] As a preferred embodiment of the present invention, the drug promotes apoptosis of cancer cells. Experimental results show that the organic selenium compound provided by the present invention promotes apoptosis of cancer cells, especially mouse melanoma B16 cells and human gastric cancer HGC-27 cells, which promote apoptosis as the concentration increases.

[0021] As a preferred embodiment of the present invention, the drug blocks cancer cells in the S phase and inhibits DNA synthesis. Experimental results show that the organic selenium compound provided by the present invention has a certain effect on the cell cycle of human gastric cancer HGC-27 cells. As the drug concentration increases, the proportion of cells in the S phase increases, indicating that the DNA replication process is blocked, and it can be inferred that the regulatory mechanism of the cell cycle has changed.

[0022] Compared with the prior art, the present invention combines the natural active compound p-hydroxycinnamic acid with selenium to synthesize a new type of organic selenium drug, filling the gap in the combined use of the anti-cancer active structure of traditional Chinese medicine and selenium, making the anti-cancer activities of the two produce a synergistic effect, and increasing its safety and anti-cancer effect, which has great potential in the prevention and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 , Compound 1 1 H NMR spectrum (600 MHz, D 2 O);

[0024] Figure 2 , ESI-MS spectrum of compound 1;

[0025] Figure 3 , Compound I-1 1 H NMR spectrum (600 MHz, DMSO- d 6 );

[0026] Figure 4 , ESI-MS spectrum of compound I-1;

[0027] Figure 5 , Compound I-2 1 H NMR spectrum (600 MHz, DMSO- d 6 );

[0028] Figure 6 , ESI-MS spectrum of compound I-2;

[0029] Figure 7 , Compound I-3 1 H NMR spectrum (600 MHz, DMSO- d 6 );

[0030] Figure 8 , ESI-MS spectrum of compound I-3;

[0031] Fig. 9 , optical microscope images of B16 cells, Hela cells, HGC-27 cells and L-O2 cells under different culture conditions;

[0032] Fig.10 , B16 cells and HGC-27 cells apoptosis results of flow cytometry under different culture conditions;

[0033] Fig.11 , flow cytometry results of HGC-27 cells under different culture conditions. DETAILED DESCRIPTION

[0034] The following examples are provided to further illustrate various aspects of the present invention. These examples are non-limiting and should not be construed as limiting any aspect of the present invention. The scope of protection of the present invention is limited only by the claims. Without departing from the scope of the claims, those skilled in the art may make various modifications and improvements to various aspects of the present invention, and these modifications and improvements also belong to the scope of protection of the present invention.

[0035] In addition, it should be noted that, unless otherwise specified, the various materials and reagents used in the following examples are commonly used materials and reagents in the art and can be obtained through conventional commercial channels; the methods used are conventional methods known to those skilled in the art.

[0036] Example 1: Synthesis of Compound I-1

[0037] Compound I-1 is 4,4'-((2,2'-selenobis(acetyl))bis(oxy))bis(3-methoxybenzoic acid), and has the following structure:

[0038]

[0039] Compound I-1 was synthesized by the following method:

[0040] (1) Synthesis of 2,2'-selenodiacetic acid (Compound 1): Weigh 1.6 g (0.02 mol) of selenium powder and place it in a 150 ml three-necked flask. Add 10 mL of distilled water and stir under nitrogen. Then slowly drop 1.5 g (0.04 mol) of sodium borohydride dissolved in 10 mL of distilled water. The selenium powder gradually dissolves and the reaction solution becomes clear. Dissolve 3.8 g (0.04 mol) of 2-chloroacetic acid in 20 mL of distilled water, adjust the pH to 9-10 with saturated sodium carbonate solution, then add it to the above reaction solution and stir at room temperature for 12 h. After the reaction is completed, filter the reaction solution, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, concentrate to a viscous state, extract with ethyl acetate, combine the organic layers, dry over anhydrous sodium sulfate, concentrate and crystallize, and then recrystallize with ethyl acetate to obtain 2.58 g of white solid with a yield of 64.9%. 1 H NMR (600 MHz, D 2 O): δ 3.469 (s, 4H) (e.g. Figure 1 ESI-MS( m / z ):196.9 [MH] - (like Figure 2 as shown).

[0041] (2) Synthesis of 2,2'-selenodiacetyl chloride (Compound 2): Under nitrogen protection, 0.394 g (0.002 mol) of compound 1 was dissolved in 20 mL of anhydrous tetrahydrofuran at 0°C. 0.35 ml (0.004 mol) of oxalyl chloride mixture was added dropwise to the above solution and the reaction was continued at 40°C for 2 h. The solvent was removed by rotary evaporation for 48 h to obtain a yellow oily substance, which was 2,2'-selenodiacetyl chloride. The oil was operated in an anhydrous dry environment and used directly in the next step.

[0042] (3) Synthesis of compound I-1: Under nitrogen protection, 0.672 g (0.004 mol) of vanillic acid was dissolved in 10 mL of anhydrous tetrahydrofuran, 0.5 mL of pyridine was added, and the mixture was pre-cooled in an ice bath. Compound 2 obtained in step (2) was dissolved in 5 ml of anhydrous tetrahydrofuran, and the mixture was slowly added dropwise to the pre-cooled solution, and stirred at 35°C for 6 h. The reaction solution was evaporated to dryness under reduced pressure, dissolved in 20 mL of ethyl acetate, washed with water, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was eluted with a semi-preparative liquid phase with a gradient of acetonitrile / water (volume ratio 30:70 to 100:0) to obtain purified compound I-1. Its structural characterization information is as follows: 1 H NMR (600 MHz, DMSO- d 6 ): δ 7.622 (d, J = 1.8 Hz, 2H), 7.574 (dd,J = 8.4, 1.8 Hz, 2H),7.232 (d, J = 8.4 Hz, 2H), 3.849 (s, 4H), 3.842 (s, 6H) Figure 3 ESI-MS( m / z ):497.2 [MH] - (like Figure 4 as shown).

[0043] Example 2: Synthesis of Compound I-2

[0044] Compound I-2 is 3,3'-(((2,2'-selenobis(acetyl))bis(oxy))bis(3-methoxy-4,1-phenylene))diacrylic acid, and has the following structure:

[0045]

[0046] By using the method provided in Example 1, only in step (3), 0.672 g (0.004 mol) of vanillic acid is replaced by 0.776 g (0.004 mol) of ferulic acid to synthesize compound I-2. Its structural characterization information is as follows: 1 H NMR (600MHz, DMSO- d 6 ): δ 7.580 (d, J = 16.2 Hz, 2H), 7.501 (d, J =1.8 Hz, 2H), 7.272 (dd, J = 8.4, 1.8 Hz, 2H), 7.134 (d, J = 8.4 Hz, 2H), 6.593 (d, J = 16.2 Hz, 2H), 3.834(s, 6H), 3.825 (s, 4H) Figure 5 ESI-MS( m / z ): 549.4 [MH] - (like Figure 6 as shown).

[0047] Example 3: Synthesis of Compound I-3

[0048] Compound I-3 is 3,3'-(((2,2'-selenobis(acetyl))bis(oxy))bis(4,1-phenylene))diacrylic acid, and has the following structure:

[0049]

[0050] By using the method provided in Example 1, only in step (3), 0.672 g (0.004 mol) of vanillic acid is replaced by 0.656 g (0.004 mol) of p-hydroxycinnamic acid to synthesize compound I-3. Its structural characterization information is as follows: 1 HNMR (600 MHz, DMSO- d 6 ): δ 7.748 (d, J = 8.4 Hz, 4H), 7.596 (d, J = 16.2 Hz, 2H),7.178 (d, J = 8.4 Hz, 4H), 6.516 (d, J = 16.2 Hz, 2H), 3.816 (s, 4H,) (e.g. Figure 7 ESI-MS( m / z ): 489.0 [MH] - (like Figure 8 as shown).

[0051] Experimental example

[0052] This experiment verifies the antitumor activity of compounds I-1, I-2 and I-3, using Chinese medicine monomers (vanillic acid, ferulic acid, p-hydroxycinnamic acid), resveratrol and 5-fluorouracil for synthesizing the above compounds as controls. The cell lines involved in the experiment include: (1) mouse melanoma B16 cells, purchased from Beijing Solebao Company, incubated in RPMI1640 medium containing 10% fetal bovine serum in 5% CO 2 (2) Human hepatocellular carcinoma HepG2 cells were purchased from Beijing Solebao Company and cultured in MEM medium containing 10% fetal bovine serum at 5% CO 2 , 37°C; (3) Human lung cancer A549 cells were purchased from Beijing Solebao Company and cultured in F12-K medium containing 10% fetal bovine serum at 5% CO 2 (4) Human gastric cancer HGC-27 cells were purchased from Beijing Solebao Company and cultured in RPMI1640 medium containing 10% fetal bovine serum at 5% CO 2 , 37°C; (5) Human cervical cancer Hela cells were purchased from Beijing Solebao Company and cultured in RPMI1640 medium containing 10% fetal bovine serum at 5% CO 2 , 37°C; (6) Normal human liver L-O2 cells were purchased from Shanghai Huiying Company and cultured in RPMI1640 medium containing 10% fetal bovine serum at 5% CO 2, 37°C; (7) Human umbilical vein HUVEC cells were purchased from Shanghai Huiying Company and cultured in F12 medium containing 10% fetal bovine serum at 5% CO 2 , 37°C. According to the actual growth status of the cells, the cells were routinely subcultured according to the cell instructions. After the cells were cultured for more than three generations and the growth status was stable, they were used for experiments.

[0053] 1. Cell inhibition rate detection

[0054] 100 µL was seeded in 96-well plates at a density of 1 × 10 5 cells / mL in 5% CO 2 , cultured at 37°C for 12 h, 100 µL of medium containing different drug concentrations was added to each well, 5-fluorouracil and resveratrol were used as positive controls, and six concentrations of 5, 10, 20, 50, 100, and 200 µM were set, with three parallel groups for each concentration. After drug treatment, 20 µL of 5 mg / mL thiazolyl blue (MTT) was added to each well for 4 h, the supernatant was aspirated, 150 µL of DMSO was added, and the cells were shaken for 5 min. The absorbance was detected at 570 nm on a microplate reader to calculate the cell survival rate (IC 50 ).

[0055] IC 50 The value represents the concentration of compound required to achieve 50% inhibition rate on tumor cells. The test results are shown in Table 1. Cell microscopy imaging after the action of different types and concentrations of drugs is shown in Table 1. Fig. 9 The results showed that compound I-3 had a good inhibitory effect on the proliferation of melanoma, liver cancer, lung cancer, gastric cancer and cervical cancer cells, and had no killing effect on normal human cells.

[0056]

[0057] (II) Cell apoptosis

[0058] The degree of apoptosis was detected by Annexin V-FITC / Propidium Iodide (ANNEXIN V-FITC / PI) apoptosis detection kit. Cells in the logarithmic growth phase were made into cell suspensions and then cultured at a cell density of 1×10 5 1 mL per well of a six-well plate was inoculated at 37°C and 5% CO 2 Culture for 12 h. Add 1 ml of medium containing different concentrations of drugs to each well and continue to culture for 48 h. Digest and collect cells from each well, centrifuge at 1000 rpm for 5 min, wash once with pre-cooled PBS, reselect cells with 100 µL binding buffer, add 5 µL of annexin V-FITC, mix well, incubate at room temperature in the dark for 5 min, add 5 µL of propidium iodide, and add 400 µL of PBS, and immediately pass through a mesh to detect using a flow cytometer.

[0059] The results are as follows Fig.10 The results showed that compound Ⅰ-3 promoted apoptosis of mouse melanoma B16 cells and human gastric cancer HGC-27 cells as the concentration increased.

[0060] 3. Cell cycle analysis

[0061] The cells in the logarithmic growth phase were made into a cell suspension and then cultured at a cell density of 1×10 5 1 mL per well of a six-well plate was inoculated at 37°C and 5% CO 2 Culture for 12 h. Add 1 ml of medium containing different concentrations of drugs to each well and continue to culture for 48 h. Digest and collect cells from each well, centrifuge at 1000 rpm for 5 min, wash once with pre-cooled PBS, add 500 µL of 70% pre-cooled ethanol and fix at 4℃ for 2 h to overnight. Wash the fixative with PBS before staining, add 100 µL of RNase A solution to the cell pellet, resuspend the cells, and bathe at 37℃ for 30 min. Add 400PI staining solution and mix well, incubate at 4℃ in the dark for 30 min. Detect by flow cytometry through the sieve, and record the red fluorescence at 488 nm.

[0062] The results are as follows Fig.11 The results showed that compound Ⅰ-3 had a certain effect on the cell cycle of human gastric cancer HGC-27 cells. As the drug concentration increased, the proportion of cells in the S phase increased, indicating that the DNA replication process was blocked. It can be inferred that the regulatory mechanism of the cell cycle has changed.

[0063] Unless otherwise specified, the scientific and technological terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The present invention may also be implemented using any methods and materials similar to or equivalent to those described herein, and specific implementation methods and preferred methods and materials are described herein without limiting the present invention in any way.

Claims

1. An organic selenide, characterized in that It has the following structure: 。 2. The method for preparing the organic selenide according to claim 1, characterized in that: Prepared according to the following synthetic route: （1） ； （2） ； （3） 。 3. The preparation method according to claim 2, characterized in that: The step (1) comprises: taking selenium powder, mixing it with distilled water under nitrogen protection, slowly adding sodium borohydride aqueous solution while stirring, and after the system is clarified, adjusting 2-chloroacetic acid to a pH value of 9-10 with a saturated sodium carbonate solution, adding the solution to the reaction solution, and stirring at room temperature; collecting the reaction solution, adjusting the pH value to 2-3 with dilute hydrochloric acid, extracting with ethyl acetate, collecting the organic phase, removing water, concentrating, and recrystallizing to obtain 2,2'-selenodiacetic acid.

4. The preparation method according to claim 2 or 3, characterized in that: The step (2) comprises: dissolving 2,2'-selenodiacetic acid in anhydrous tetrahydrofuran at -5 to 5°C under nitrogen protection, adding oxalyl chloride dropwise, and then stirring the reaction at 38 to 42°C; evaporating the solvent in the reaction solution to obtain 2,2'-selenodiacetyl chloride.

5. The preparation method according to claim 2 or 3, characterized in that: The step (3) comprises: preparing an anhydrous tetrahydrofuran solution of p-hydroxycinnamic acid under nitrogen protection, adding pyridine and then precooling in an ice bath; slowly adding a solution of 2,2'-selenodiacetyl chloride in anhydrous tetrahydrofuran dropwise, and then stirring the reaction at 33-37° C.; evaporating the reaction solution to dryness, re-dissolving it with ethyl acetate and then washing it with water, extracting and collecting the organic phase, removing water, and concentrating it to obtain a crude product of 3,3'-(((2,2'-selenobis(acetyl))bis(oxy))bis(4,1-phenylene))diacrylic acid.

6. Use of the compound of claim 1 in the preparation of a drug for preventing and / or treating cancer; the cancer is selected from one or more of melanoma, liver cancer, lung cancer, gastric cancer, and cervical cancer.

7. The use according to claim 6, characterized in that: The drug inhibits the proliferation of cancer cells.

8. The use according to claim 6, characterized in that: The drug promotes apoptosis of cancer cells.

9. The use according to claim 6, characterized in that: The drug blocks cancer cells in the S phase, inhibiting their DNA synthesis.

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