A benzoselenadiazole derivative and its preparation method and application

Abstract

The present application belongs to the technical field of benzoselenadiazoles, and in particular relates to a benzoselenadiazole derivative, a preparation method thereof, and an application thereof. The benzoselenadiazole derivative provided by the present application has an electron-donating group at the 4-position and a double-bonded conjugated electron-withdrawing group at the 7-position in its chemical structure, wherein the maximum visible absorption wavelength is in the near-infrared region above 650 nm, the triplet state conversion rate is high, and reactive oxygen species can be generated with high efficiency under light conditions, thereby having a good killing effect on cancer cells and cancer tissues and having no side effects on normal cell tissues. This solves the technical problem in the prior art that the maximum light absorption band of the photosensitizer is outside the near-infrared band, the absorption efficiency of near-infrared rays is low, and the efficiency of generating reactive oxygen species under light is low.

Description

Technical Field

[0001] The present invention relates to the technical field of benzoselenadiazoles, and in particular to a benzoselenadiazole derivative and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy is minimally invasive and features high spatiotemporal precision, making it an important medical technology for treating malignant tumors such as primary hepatocellular carcinoma. Photodynamic therapy involves injecting a photosensitizer into the body via intravenous injection (for malignant tumors on the surface of the skin, the photosensitizer can also be applied to the surface of the skin). After a certain period of time, the tumor tissue is irradiated with light of a specific wavelength. The photosensitizer accumulated in the tumor tissue, under the excitation of light, produces a series of photophysical and chemical reactions, generating cytotoxic reactive oxygen species, thereby killing cancer cells and destroying the tumor tissue.

[0003] However, the maximum light absorption band of the photosensitizers currently used in photodynamic therapy to treat malignant tumors is outside the near-infrared band, the absorption efficiency of near-infrared rays is not high, and the efficiency of generating reactive oxygen species under light is low. Summary of the Invention

[0004] In view of this, the present application provides a benzoselenadiazole derivative and its preparation method and application, which are used to solve the technical problems in the prior art that the maximum light absorption band of the photosensitizer is outside the near-infrared band, the absorption efficiency of near-infrared rays is not high, and the efficiency of generating active oxygen under light is low.

[0005] The first aspect of the present application provides a benzoselenadiazole derivative, the chemical structure of which is shown in Formula I

[0006]

[0007] Wherein, the R1 substituent is selected from any one of the following electron-donating groups:

[0008] ;

[0009] The R2 substituent is selected from any one of the following electron-withdrawing groups:

[0010]

[0011] The R3 substituent is selected from a H atom or a cyano group.

[0012] Preferably, R1 is selected from dimethylamino substitution, R2 is selected from benz[c]indole-2(1H)-one derivatives, and R3 is selected from hydrogen atom.

[0013] Preferably, the chemical formula of the benzoselenadiazole derivative is as shown in Formula A

[0014] Formula A.

[0015] The second aspect of the present application provides a method for preparing a benzoselenadiazole derivative, comprising the following steps:

[0016] Step 1: Dissolve 7-(dimethylamino)benzoselenadiazole-4-aldehyde and 1,2-dimethylbenzindole in a solvent and heat under reflux overnight to obtain a benzoselenadiazole derivative.

[0017] Preferably, in step 1, the preparation method of 7-(dimethylamino)benzoselenadiazole-4-aldehyde comprises the steps of:

[0018] Step 11, in an inert atmosphere, dissolving 4-fluorobenzoselenadiazole in anhydrous ethanol, adding triethylamine and dimethylamine to react to obtain N,N-dimethylbenzoselenadiazole-4-amine;

[0019] Step 12: At room temperature, add N,N-dimethylbenzoselenadiazole-4-amine solution dropwise to phosphorus oxychloride solution, mix, and react to obtain 7-(dimethylamino)benzoselenadiazole-4-aldehyde.

[0020] It should be noted that the yield of N,N-dimethylbenzoselenadiazole-4-amine prepared by the preparation method provided in this application is relatively high, about 90%, while the yield of 7-(dimethylamino)benzoselenadiazole-4-aldehyde is about 44%.

[0021] Preferably, after obtaining the benzoselenadiazole derivative in step 1, after obtaining N,N-dimethylbenzoselenadiazole-4-amine in step 11, and after obtaining 7-(dimethylamino)benzoselenadiazole-4-aldehyde in step 12, the method further includes the steps of separation and purification.

[0022] Preferably, the separation is carried out by vacuum distillation.

[0023] Preferably, the purification is performed by silica gel chromatography.

[0024] It should be noted that the yield of the benzoselenadiazole derivative prepared by the preparation method provided in this application is relatively high, about 62.2%.

[0025] Preferably, in step 11, the inert atmosphere is argon, the reaction temperature is 120-180 degrees Celsius, and the reaction time is 18-30 hours;

[0026] In step 12, the reaction time is 1 to 3 hours.

[0027] The third aspect of the present application provides a use of a benzoselenadiazole derivative in the treatment of malignant tumors.

[0028] It should be noted that the benzoselenadiazole derivatives provided in this application can generate reactive oxygen species with high efficiency under light conditions, have a good killing effect on cancer cells and cancer tissues, and have little toxic side effects on normal cell tissues, so they can be used as photosensitizers for photodynamic therapy of malignant tumors.

[0029] Preferably, the malignant tumor is liver cancer.

[0030] Preferably, the malignant tumor is breast cancer.

[0031] It should be noted that the maximum light absorption band of the benzoselenadiazole derivative provided in this application is located in the near-infrared band, which is suitable for the treatment of malignant tumors in deep tissue locations such as liver cancer. After experiments, it was found that it has extremely high photodynamic activity against triple-negative breast cancer cells, can produce a large number of reactive oxygen species to kill triple-negative breast cancer cells, and maintains extremely low dark toxicity, making it an excellent photosensitizing drug.

[0032] In summary, the present application provides a benzoselenadiazole derivative, a preparation method, and an application thereof. In the present application, a series of benzoselenadiazole derivatives are obtained by expanding the conjugated double bond at the 7-position of benzoselenadiazole. These series of benzoselenadiazole derivatives have high ultraviolet-visible light absorption. The electron-donating group at the 4-position and the conjugated electron-withdrawing group of the double bond at the 7-position of the benzoselenadiazole derivative are further regulated. The maximum visible absorption wavelength of the obtained benzoselenadiazole derivative is in the near-infrared region above 650 nm, so that the benzoselenadiazole derivative provided by the present application has a high triplet state conversion rate, can generate reactive oxygen species with high efficiency under light conditions, has a good killing effect on cancer cells and cancer tissues, and achieves photodynamic therapy for malignant tumors while almost no toxic side effects are added to normal tissues, thereby solving the technical problems in the prior art that the maximum light absorption band of the photosensitizer is outside the near-infrared band, the absorption efficiency of near-infrared rays is not high, and the efficiency of generating reactive oxygen species under light is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0034] Figure 1 This is the hydrogen spectrum of N,N-dimethylbenzoselenadiazole-4-amine in Example 2 of the present application;

[0035] Figure 2This is the hydrogen spectrum of 7-(dimethylamino)benzoselenadiazole-4-aldehyde in Example 2 of the present application;

[0036] Figure 3 This is the hydrogen spectrum of the benzoselenadiazole derivative in Example 2 of the present application;

[0037] Figure 4 This is the mass spectrum of the benzoselenadiazole derivative in Example 2 of the present application;

[0038] Figure 5 This is a spectrum of the benzoselenadiazole derivative in Example 3 of the present application;

[0039] Figure 6 This is the active oxygen staining diagram of the benzoselenadiazole derivative in Example 3 of the present application;

[0040] Figure 7 This is an in vitro cell live-dead staining image of the benzoselenadiazole derivative in Example 3 of the present application;

[0041] Figure 8 This is a graph showing the killing effect of the benzoselenadiazole derivatives in Example 3 of the present application on MDA-MB-231 cells, MCF-7 cells, B16F10 cells, U87 cells, 4T1 cells, and HeLa cells;

[0042] Figure 9 This is a graph showing the inhibition of MDA-MB-231 cell migration by benzoselenadiazole derivatives in Example 3 of the present application. DETAILED DESCRIPTION

[0043] The present application provides a benzoselenadiazole derivative and its preparation method and application, which are used to solve the technical problems in the prior art that the maximum light absorption band of the photosensitizer is outside the near-infrared band, the absorption efficiency of near-infrared rays is low, and the efficiency of generating active oxygen under light is low.

[0044] The following will clearly and completely describe the technical solution of this application in conjunction with the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of this application.

[0045] Example 1

[0046] The present application provides a benzoselenadiazole derivative, the chemical structure of which is shown in Formula I

[0047]

[0048] In Formula I, the R1 substituent is selected from:

[0049]

[0050] The R2 substituent is selected from:

[0051]

[0052] The R3 substituent is selected from a H atom or a cyano group.

[0053] The benzoselenadiazole derivatives shown in Formula I provided in the present application are obtained by expanding the conjugated double bond at the 7-position of benzoselenadiazole. In the chemical formula of the expanded benzoselenadiazole derivatives, the 4-position R1 group is an electron-donating substituent, and the 7-position R2 group is an electron-withdrawing substituent. By regulating the 4-position electron-donating group R1 and the 7-position double bond conjugated electron-withdrawing group R2, the benzoselenadiazole derivatives shown in Formula I with a maximum visible absorption wavelength in the near-infrared region of more than 650 nm can be obtained. The benzoselenadiazole derivatives shown in Formula I contain heavy elements of the oxygen family and have a maximum visible absorption wavelength in the near-infrared region, so that the triplet state conversion rate of the benzoselenadiazole derivatives shown in Formula I is high, and reactive oxygen species can be generated with high efficiency under light conditions. Reactive oxygen species are highly toxic substances that can be introduced by The reactive oxygen species can cause the death of diseased tissues by causing cell apoptosis or necrosis, destroying blood vessels, stimulating immune responses, etc.; in addition, the smaller action radius and shorter lifespan of the reactive oxygen species help to damage only cancer cells or microorganisms exposed to specific light, thereby minimizing side effects on normal cell tissues and greatly improving treatment accuracy; at the same time, since the benzoselenadiazole derivatives shown in Formula I provided by the present application have water solubility, cell membrane permeability and biological tissue penetrability, they can be well accumulated in deep malignant tumors such as liver tumors. The above properties make the benzoselenadiazole derivatives shown in Formula I provided by the present application have a good killing effect on cancer cells and cancer tissues, and almost no toxic side effects on normal tissues while achieving photodynamic therapy for malignant tumors.

[0054] Regarding the selection of substituents in the benzoselenadiazole derivatives shown in Formula I, the 4-substituted R1 group is a dimethylamino group, the 7-substituted R2 group is a benzo[c]indole-2(1H)-one derivative, and R3 is a hydrogen atom. Regarding the selection of benzoselenadiazole derivatives, the present application preferably uses the following three benzoselenadiazole derivatives:

[0055]

[0056] More preferably, the present application preferably uses the benzoselenadiazole derivative represented by the following formula A:

[0057] Formula A, By performing spectral analysis on the benzoselenadiazole derivative represented by Formula A, it was determined that the maximum visible light absorption wavelength of the benzoselenadiazole derivative represented by Formula A was 718 nm, and the benzoselenadiazole derivative represented by Formula A could be detected to produce reactive oxygen species under light conditions. Reactive oxygen species caused cell apoptosis or necrosis, thereby producing a significant killing effect on cells, especially on triple-negative breast cancer cells, thereby inhibiting the invasion of triple-negative breast cancer cells on normal human cells.

[0058] Example 2

[0059] Example 2 of the present application provides a method for preparing a benzoselenadiazole derivative represented by formula A, which includes preparing N,N-dimethylbenzoselenadiazole-4-amine, preparing 7-(dimethylamino)benzoselenadiazole-4-aldehyde, and preparing a benzoselenadiazole derivative.

[0060] The preparation of N,N-dimethylbenzoselenadiazole-4-amine comprises the following steps:

[0061] Under argon, 4-fluorobenzoselenadiazole (800 mg, 3.9 mmol) was dissolved in anhydrous ethanol. Triethylamine (1.65 mL, 19.5 mmol, 5 eqv) and dimethylamine (2 M in THF, 10 eqv) were added. The reaction was carried out at 150°C under argon for 24 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. The product was purified by silica gel chromatography (PE:EA = 10:1) to obtain 813 mg of a red solid A. The yield was 90%. The red solid was N,N-dimethylbenzoselenadiazole-4-amine, and its hydrogen spectrum was shown in Figure 1. Figure 1 shown.

[0062] The reaction formula is

[0063]

[0064] The preparation of 7-(dimethylamino)benzoselenadiazole-4-aldehyde includes the following steps: phosphorus oxychloride (2 eqv) is added dropwise to ultra-dry DMF at 0°C, stirred for 15 minutes, N,N-dimethylbenzoselenadiazole-4-amine (400 mg, 1.7 mmol) is dissolved in DMF (2 mL) and added dropwise to the solution, which is then warmed to room temperature and allowed to react for 2 hours. After the reaction is complete, the solution is poured into ice water and adjusted to neutrality with sodium hydroxide solution. The organic phase is then extracted with ethyl acetate and dried over anhydrous sodium sulfate. The solvent is removed by distillation under reduced pressure, and the product is purified by silica gel chromatography (PE:EA = 5:3) to obtain 198 mg of a red solid B. The yield is 44%. The red solid is 7-(dimethylamino)benzoselenadiazole-4-aldehyde, and its hydrogen spectrum is shown below. Figure 2 shown.

[0065] The reaction formula is

[0066]

[0067] The preparation of benzoselenadiazole derivatives comprises the steps of:

[0068] 7-(Dimethylamino)benzoselenadiazole-4-aldehyde (127 mg, 0.5 mmol) and 1,2-dimethylbenzindole (109 mg, 0.6 mmol) were dissolved in ultra-dry ethanol and heated under reflux overnight. After the reaction, vacuum distillation was performed to obtain a dark blue solid. The product was purified by silica gel chromatography using dichloromethane / methanol (20:1) as the eluent to obtain the final product A: 130 mg, with a yield of 62.2%. Its hydrogen spectrum and mass spectrum are shown in FIG. Figure 3 、 4 shown.

[0069] The reaction formula is

[0070]

[0071] Example 3

[0072] Example 3 of the present application is a performance test of the benzoselenadiazole derivative shown in Formula A to determine its effect on cell photodynamic therapy. The performance test includes spectral testing, intracellular reactive oxygen species imaging test, cell live-dead staining imaging test, cell photodynamic therapy test and triple-negative breast cancer cell migration test.

[0073] The spectrophotometric test is to dissolve the benzoselenadiazole derivative shown in formula A in DMSO solvent and then perform UV-visible spectrophotometry test. The results are as follows: Figure 5 As shown, from Figure 5 It can be seen that the maximum visible light absorption wavelength of the benzoselenadiazole derivative represented by Formula A provided in the present application is 718 nm.

[0074] The intracellular reactive oxygen species imaging test was specifically performed by incubating MDA-MB-231 cells seeded in CLSM-specific cell culture dishes with 1 μM benzoselenadiazole derivatives shown in formula A for 2 hours, and then staining the cells with 10 μM 2,7-dichlorofluorescein diacetate for 20 minutes as a control group. MDA-MB-231 cells seeded in CLSM-specific cell culture dishes were incubated with 1 μM benzoselenadiazole derivatives shown in formula A for 2 hours, and then stained with 690 nm (30 mW / cm 2 The cells were irradiated with LED light for 8 minutes, and then stained with 10 μM 2,7-dichlorofluorescein diacetate for 20 minutes as the experimental group. A blank control group was also set up. The results are shown in Figure 2. Figure 6As shown, the experimental group detected the green signal of the fluorescent product DCF, indicating that the benzoselenadiazole derivative shown in Formula A of the present application can generate active oxygen under light.

[0075] The live-death staining imaging test specifically involved inoculating MDA-MB-231 cells in confocal laser scanning microscopy (CLSM)-specific cell culture dishes for 24 hours. The cell culture dishes included a blank control group, a group containing the benzoselenadiazole derivative of Formula A, and a group containing the benzoselenadiazole derivative of Formula A + light exposure. The blank control group received no treatment. In the experimental group, the cells were incubated with 1 μM of a photosensitizer, the benzoselenadiazole derivative of Formula A, at 37 degrees Celsius for 120 minutes, then washed three times with PBS and continued to incubate in an incubator. The group containing the benzoselenadiazole derivative of Formula A + light exposure was treated with a 690 nm LED light at 30 mW / cm 2 The cells were irradiated with a power density of 100 nm for 3 minutes and then incubated in a cell culture incubator for another 120 minutes. Finally, Calcein AM and Propidium Iodide (PI) were co-stained. The confocal fluorescence of Calcein-AM / PI was monitored. The results were as follows: Figure 7 As shown, from Figure 7 The cell apoptosis signal induced by reactive oxygen species generated in the cells can be observed, with an excitation wavelength of 488 nm, an emission wavelength of the green channel of 490 to 550 nm, and an emission wavelength of the red channel of 610 to 700 nm. No cell apoptosis signal was detected in the control group and the non-illumination experimental group, while the benzoselenadiazole derivative shown in Formula A detected red fluorescence staining induced by cell apoptosis under illumination conditions, which indicates that the reactive oxygen species generated by the benzoselenadiazole derivative shown in Formula A provided in the present application under illumination can cause cell apoptosis or necrosis.

[0076] The cell photodynamic therapy test specifically includes first planting MDA-MB-231 cells, MCF-7 cells, B16F10 cells, U87 cells, 4T1 cells and HeLa cells in a 96-well plate at a cell density of 5000 cells / well and culturing them in a cell culture incubator for 24 hours (37°C, 5% CO2). Then, the benzoselenadiazole derivative shown in formula A is added to DMEM containing 10% fetal bovine serum to prepare solutions of different concentrations, which are added to each well and incubated for 2 hours. The liquid in the well plate is discarded with a pipette and replaced with fresh cell culture medium. Among them, the well plate of the illumination group is illuminated with red light of 690 nm wavelength (30 mW / cm 2) for 8 minutes. After irradiation, the well plate was placed in a cell culture incubator for further incubation for 22 hours. For the dark group well plate, no light treatment was performed. Subsequently, 120 μL of culture medium containing 5 mg / mL MTT was added to each well and the plate was moved to a cell culture incubator for incubation for 4 hours. The culture medium in the plate wells was removed and 150 μL of DMSO was added to each well to fully dissolve the MTT oxidation product. The absorbance at 490 nm in each well was measured with a microplate reader to calculate the cell survival rate. The results are shown as follows: Figure 8 As shown, from Figure 8 It can be seen that, compared with dark conditions, under light conditions, the benzoselenadiazole derivative shown in Formula A can produce a significant killing effect on cells.

[0077] The triple-negative breast cancer cell migration test specifically involves seeding MDA-MB-231 cells at a density of 400,000 per well in a 35 mm well plate and culturing them in a 37°C, 5% CO2 incubator until a monolayer evenly covers the well plate. Three parallel lines are vertically scratched with a 200 μL pipette tip and washed twice with PBS to remove cell debris generated by the scratches. Serum-free culture medium containing the benzoselenadiazole derivative of formula A is then added. After incubation for 2 hours, fresh serum-free culture medium is replaced and the cells are then irradiated with light: 690 nm (30 mW / cm 2 The cells were irradiated with LED light for 8 minutes. At 0 hours, 24 hours, and 48 hours later, the scratched area of ​​the cells was photographed under an inverted fluorescence microscope and the cell migration was recorded. Figure 9 As shown, compared with the blank control group, the benzoselenadiazole derivatives represented by Formula A inhibited the self-repair and migration of MDA-MB-231 cells under both normoxia and hypoxia. This indicates that the benzoselenadiazole derivatives represented by Formula A can prevent the invasion of triple-negative breast cancer cells under light conditions, both normoxia and hypoxia.

[0078] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 application.

Claims

1. A benzoselenadiazole derivative, characterized in that The benzoselenadiazole derivative is shown in Formula A: Formula A.

2. The method for preparing a selenadiazole derivative according to claim 1, characterized in that: The following steps are involved: Step 1: Dissolve 7-(dimethylamino)benzoselenadiazole-4-aldehyde and 1,2-dimethylbenzindole in a solvent and heat under reflux overnight to obtain a benzoselenadiazole derivative.

3. The method for preparing a selenadiazole derivative according to claim 2, wherein: In step 1, the preparation method of 7-(dimethylamino)benzoselenadiazole-4-aldehyde comprises the following steps: Step 11, in an inert atmosphere, dissolving 4-fluorobenzoselenadiazole in anhydrous ethanol, adding triethylamine and dimethylamine to react to obtain N,N-dimethylbenzoselenadiazole-4-amine; Step 12: At room temperature, add N,N-dimethylbenzoselenadiazole-4-amine solution dropwise to phosphorus oxychloride solution, mix, and react to obtain 7-(dimethylamino)benzoselenadiazole-4-aldehyde.

4. The method for preparing a selenadiazole derivative according to claim 2, wherein: After obtaining a benzoselenadiazole derivative in step 1, obtaining N,N-dimethylbenzoselenadiazole-4-amine in step 11, and obtaining 7-(dimethylamino)benzoselenadiazole-4-aldehyde in step 12, the method further includes the steps of separation and purification.

5. The method for preparing a selenadiazole derivative according to claim 3, wherein: In step 11, the inert atmosphere is argon, the reaction temperature is 120-180 degrees Celsius, and the time is 18-30 hours; In step 12, the reaction time is 1 to 3 hours.

6. Use of the benzoselenadiazole derivative according to claim 1 in the preparation of a photosensitizer for photodynamic therapy of malignant tumors, characterized in that: The malignant tumor is selected from liver cancer or breast cancer.

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