CN111560033A - Synthesis method of light controlled release compound and application of light controlled release compound in tumor treatment - Google Patents

Abstract

A method for synthesizing a light-controlled release compound, the lead compound is composed of BODIPY parent nucleus and chlorambucil, and integrates light-controlled release drug, photodynamic therapy and chemotherapy in the same molecular structure. The invention also discloses the application in tumor treatment. The lead compound in the present invention has simple structure, small molecular weight, has a definite chemical structure, is easy to prepare, purify and further modify, and has the remarkable characteristics of low toxicity to mice, which meets the basic requirements of clinical medicine. In vitro and in vivo experiments proved that the lead compound has precise controlled release of the chemotherapeutic drug chlorambucil under visible light radiation, and the light radiation time is positively correlated with the drug release amount, realizing the precise release of the chemotherapeutic drugs and single-line drugs by single molecules through light stimulation state oxygen, and realized the synergistic treatment of chemotherapy/photodynamic therapy of living tumors. Therefore, the leading compound of the photo-controlled release chemotherapy and photodynamic therapy involved in the present invention has a good application prospect in tumor treatment.

Description

Synthesis method of a light-controlled release compound and its application in tumor therapy

technical field

The invention relates to a method for synthesizing a light-controlled release compound and its application in tumor treatment, and belongs to the field of pharmaceutical chemical synthesis.

Background technique

At present, cancer has become one of the major diseases that threaten human health. Therefore, the effective prevention and treatment of cancer has always been the focus of medical research. Currently, clinical treatments for tumors include chemotherapy, surgery, photodynamic therapy, and radiotherapy [Zhou J, Yu G, Huang F. Chemical Society Reviews, 2017, 46:7021-7053]. Among these treatment options, chemotherapy is currently the mainstay of clinical tumor treatment. However, due to low bioavailability, large toxic and side effects, and multidrug resistance, a single chemotherapy drug cannot meet the clinical treatment requirements, especially in the late stage of the tumor or when metastasis occurs. Therefore, the combination of drugs is often used in clinical practice. The combined use of chemotherapeutic drugs can produce increased or synergistic effects and improve the therapeutic effect; and due to the different mechanisms of action of chemotherapeutic drugs, the occurrence of multidrug resistance can be prevented to a certain extent. However, chemotherapy combination therapy cannot solve the inherent shortcomings of traditional chemotherapy drugs, such as poor specificity, undesired pharmacokinetics and biodistribution, etc., which make chemotherapy drugs kill tumor cells and cause serious damage to normal tissues; The therapeutic effect of therapy on cancer is not necessarily synergistic, but may also be antagonistic, resulting in increased toxic and side effects. Many patients have to terminate therapy due to intolerance, which greatly limits the clinical application of combined therapy [Parhi P, Mohanty C , Sahoo S K. Drug Discovery Today, 2012, 17: 1044-1052; Zhang Bo, Zhang Na. Chinese Journal of New Drugs, 2014, 23: 2514-2520]. Therefore, there is an urgent need to improve the "smart" controlled release of anticancer drugs in combination with other therapeutic regimens.

Stimuli-responsive drug release can realize the timed and localized release of drugs. It is called a non-invasive and efficient new way of personalized "smart" drug delivery, and is expected to achieve "tailor-made" precise drug release. It is the use of certain environmental or conditional factors to cause corresponding changes in some physical or chemical properties of the substance itself, and then play the role of releasing drug molecules. Some people vividly call it "smart matter" or "environmentally sensitive matter". Intelligent stimuli-responsive prodrugs can achieve the advantages of maintaining stable blood drug concentration, reducing the number of administrations, and reducing toxic and side effects, and are especially suitable for prodrug design of anti-tumor drugs with large toxic and side effects. Therefore, the research and development of stimuli-responsive anti-tumor prodrugs is one of the hot areas of research on anti-tumor drugs in recent years. The design of stimuli-responsive anticancer prodrugs is mainly based on the changes in the internal environment of cancer cells and some chemical substances affected by a certain external environment. There are two strategies: endogenous stimulation and exogenous stimulation. Endogenous stimulus response strategies include pH response, enzyme response, redox response of reduced glutathione (GSH), glucose response type, etc. , Zhang W J, et al. Materials Science and Engineering: C, 2017, 71: 1267-1280]. The exogenous stimulus response strategy mainly releases drugs by applying external conditions such as photoactivation to break chemical bonds, light radiation to generate heat to change the physicochemical properties of prodrugs to release drugs, or strategies such as temperature-responsive and field-responsive strategies [Zhang Liuwei, Qian Ming] , Jingyun Wang. Chinese Journal of Chemistry, 2017, 75: 770-782].

Compared with endogenous stimuli-response, exogenous stimuli-response strategies have advantages such as being artificially controllable from the outside world and do not require changes in the chemical environment inside the tissue. Among them, light is a clean, non-invasive and effective stimulus, which has been recognized by humans and can be controlled "at will (intelligence)". This is because light, as an electromagnetic wave, has the characteristics of external artificial control such as wavelength, intensity, light radiation time and radiation location. Accurate "intelligent" personalized and precise drug release can be achieved through the control of light radiation time and light source intensity).

In view of the significant advantages of light regulation, it is of great application significance and clinical value to study chemotherapeutic drugs based on light regulation to achieve "intelligent" precise and controllable drug release on targets.

SUMMARY OF THE INVENTION

What the present invention aims to solve is targeted drugs and multi-therapeutic combination drugs according to the current clinical demand for future personalized medicine, so as to overcome the single imaging and treatment mode, poor diagnosis and treatment effect, and monotherapy's effect on tumors in the current field of malignant tumor diagnosis and treatment. The technical problems of photo-controlled release of in situ chemotherapeutic drugs for malignant tumors and synergistic treatment of photodynamic therapy and chemotherapy are realized.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions:

A method for synthesizing a light-controlled release compound, comprising the following steps: (i) dissolving 4-benzoyl chloride (2 mmol) into a round-bottomed flask containing 100 mL of dichloromethane, and feeding argon gas for 15 min to the round-bottomed flask 4 mmol of 2,4-dimethyl-3-ethyl-1H-pyrrole was added, and then a small amount of trifluoroacetic acid was added dropwise. The mixture was stirred at room temperature for about 12 hours, and 2,3-dichloro-5,6-dicyano was added. 4 mmol of base-1,4-benzoquinone, continued stirring for 4 h, then added 10 mmol of triethylamine and continued to stir for 15 min, slowly added 15 mmol of boron trifluoride ether dropwise under ice bath, the dropping was completed, and the mixture was continued at room temperature Stir overnight; the reaction solution was extracted with dichloromethane (15 mL×3), dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography, eluted with dichloromethane/petroleum ether v:v=1:7, to obtain the lead compound Orange-red solid powder of intermediate BOD-Cl or BOD-OH; (ii) Weigh 0.25 mmol of lead compound intermediate BOD-Cl and 0.25 mmol of chlorambucil and dissolve in a round-bottomed flask containing 10 mL of anhydrous DMF, Potassium carbonate was then added, and the reaction was carried out at 60 °C for 6 h. After monitoring by TLC, the reaction was completed, extracted with ethyl acetate 15 mL × 3, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography with ethyl acetate/petroleum ether v :v=1:7 elution to obtain red powder lead compound BOD-BLB, yield 45%; see the following reaction scheme:

The light-controlled release compound is an organic small molecule, and has multiple functions of light-controlled drug release, photodynamic therapy and chemotherapy in the same molecular structure, and its molecular structure is formed by coupling a BODIPY parent nucleus and chlorambucil; The BODIPY core in the molecular structure is a photodynamic group; chlorambucil is a chemotherapeutic group; an ester bond is a light-responsive group, and its functional structural formula is shown in formula I,

The light-controlled release compound is used for tumor therapy.

The beneficial effects of adopting the above technical scheme are:

The leading compound of photodynamic therapy combined with photodynamic therapy according to the present invention can control the release of chemotherapeutic drugs by light, and generate a large amount of ROS in the photodynamic of tumor lesions, achieve synergistic therapy of photodynamic therapy and chemotherapy, and effectively improve the curative effect.

Description of drawings

Fig. 1 is the ¹ H NMR of the lead compound of the single-molecule multifunctional diagnostic combination according to the present invention.

Fig. 2 is the ¹³ C NMR of the lead compound of the single-molecule multifunctional diagnostic combination according to the present invention.

FIG. 3 is a high-resolution mass spectrogram of the single-molecule multifunctional diagnostic combination lead compound according to the present invention.

FIG. 4 is an infrared spectrum of the lead compound for the single-molecule multifunctional diagnosis and treatment combination according to the present invention.

Fig. 5 is the UV-Vis absorption spectrum (red line) of the single-molecule multifunctional diagnostic combination lead compound of the present invention.

Figure 6 is the fluorescence spectrum (red line) of the single-molecule multifunctional diagnosis and treatment combined lead compound of the present invention.

Fig. 7 is the singlet oxygen (black line) generated by light irradiation of the single-molecule multifunctional diagnostic combination lead compound of the present invention.

FIG. 8 shows the release of chlorambucil from the single-molecule multifunctional diagnosis and treatment combined lead compound of the present invention under different light irradiation times.

Fig. 9 is the change of the peak area of the single-molecule multifunctional diagnosis and treatment combination lead compound of the present invention releasing chlorambucil under light stimulation.

Figure 10 is the fluorescence imaging (10×) of the uptake of the single-molecule multifunctional diagnostic and therapeutic lead compound of the present invention under different incubation times of 4T1 cells.

Figure 11 shows the dark toxicity (a all black) and phototoxicity (b all black) of the single-molecule multifunctional therapeutic combination lead compound of the present invention on 4T1 cells.

Figure 12 shows the therapeutic effect of the single-molecule multifunctional diagnosis and treatment combined lead compound of the present invention on tumor in tumor-bearing mice under different illumination times; (a) changes in tumor volume of tumor-bearing mice in each group during treatment; (b) ) Changes in the body weight of tumor-bearing mice in each group during treatment; (c) tumor size in vitro in each treatment group on day 15; (d) changes in tumor weight in each treatment group at different light times on day 15 (n=4) .

Fig. 13 is the H&E staining images of the tumor tissue in the dark room and under different light irradiation of the single-molecule multifunctional diagnosis and treatment combined lead compound according to the present invention (×40).

Figure 14 is an H&E staining image (×40) of the main organ tissue sections of tumor-bearing mice using the single-molecule multifunctional diagnosis and treatment combined with the lead compound according to the present invention.

Figure 15 shows the hematological parameters of the single-molecule multifunctional therapeutic combination lead compound groups A, B, C, and D under light irradiation for 15 minutes and in a dark room (n=4).

Figure 16 is the hematological parameters of the single-molecule multifunctional therapeutic combination lead compound group E in the light irradiation for 15 minutes and the dark room (n=4).

Figure 17 is a photo of the hemolysis experiment of the single-molecule multifunctional diagnosis and treatment combined lead compound of the present invention.

Detailed ways

The content of the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following descriptions are illustrative rather than limiting of the present invention.

1. The synthetic method of the light-controlled release compound of the present invention;

Including the following steps: (i) dissolving 4-benzoyl chloride (2 mmol) into a round-bottomed flask containing 100 mL of dichloromethane, and passing argon for 15 min, adding 2,4-dimethyl- 4 mmol of 3-ethyl-1H-pyrrole, then a small amount of trifluoroacetic acid was added dropwise, the mixture was stirred at room temperature for about 12 h, 4 mmol of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone was added, After stirring for 4 h, 10 mmol of triethylamine was added and the stirring was continued for 15 min, and 15 mmol of boron trifluoride ether was slowly added dropwise in an ice bath. After the dropping was completed, the mixture was stirred at room temperature overnight; ×3) Extraction, drying with anhydrous sodium sulfate, silica gel column chromatography, eluting with dichloromethane/petroleum ether v:v=1:7, to obtain the lead compound intermediate BOD-Cl or BOD-OH orange-red Solid powder; (ii) Weigh 0.25 mmol of the lead compound intermediate BOD-Cl, and 0.25 mmol of chlorambucil and dissolve it in a round-bottomed flask containing 10 mL of anhydrous DMF, then add potassium carbonate, and react at 60 °C for 6 h. , after the completion of the reaction monitored by TLC, extracted with ethyl acetate 15mL×3, dried with anhydrous sodium sulfate, subjected to silica gel column chromatography, eluted with ethyl acetate/petroleum ether v:v=1:7 to obtain red powder Lead compound BOD-BLB, yield 45%; see the following reaction scheme:

2. The light-controlled release compound of the present invention has an absorption wavelength of 524 nm and a fluorescence emission wavelength of 542 nm, which is in the visible light absorption region.

The light-controlled release compound of the present invention is composed of BODIPY and chlorambucil, and realizes that the same molecule has multiple functions of light-controlled drug release, photodynamic therapy and chemotherapy.

After dissolving the light-controlled release compound of the present invention with methanol, measure its absorbance value on an ultraviolet-visible spectrophotometer, and measure its fluorescence excitation spectrum and fluorescence emission spectrum on a Vary Eclipse fluorescence spectrophotometer. The results are shown in Figures 5 and 5. 6. Its excitation wavelength is 524nm, and its emission wavelength is 542nm, which conforms to the optical characteristics of visible light fluorescent dyes.

3. The light-controlled release compound of the present invention generates singlet oxygen by light irradiation.

The photo-controlled release compound of the present invention generates singlet oxygen by light radiation, which is tested by 1,3-diphenylisobenzofuran (DPBF) bleaching method. DPBF can react quantitatively with singlet oxygen, and its absorbance at 410 nm decreases after the reaction. Take the lead compound intermediate with a concentration of 1 × 10 ^-3 mol/L into 50 μL graduated colorimetric tubes with a stopper, and then add 50 μL of DPBF solution with a concentration of 1 × 10 ^-3 mol/L into it. . Using a diode laser at 524 nm with a power density of 500 mW/cm ² irradiated for different times, the absorption spectrum was measured and the absorbance value at 410 nm was recorded.

4. In vitro drug release properties of the optically controlled release compounds of the present invention.

Take a small amount of the lead compound stock solution and dilute it with methanol water (8:2, v:v) to a concentration of 20 μmol/L and place it in the injection vial, and irradiate the injection vial with a laser light for 0 min, 5 min, 10 min, and 20 min, respectively. , 30min, 40min, 50min, 60min (λ=524 nm, 500mW/cm ² ), then 10 μL was injected into the high performance liquid chromatography system, and the liquid phase conditions were: methanol/water (9:1) as the mobile phase, The flow rate was 0.8 mL/min, and the detection wavelength was 254 nm. In addition, in order to confirm that the lead compound releases chlorambucil only by light stimulation, the lead compound was placed in a light and dark room periodically for 10 minutes, and then the sample was injected into the high performance liquid chromatography system, and the peak area of the lead compound was recorded. Variety.

5. Cell imaging of the light-controlled release compound of the present invention.

After the 4T1 cells were recovered, they were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% double antibody in a carbon dioxide incubator at 37°C, saturated humidity and 5% CO ₂ . When cells have grown to log phase, cell assays can be performed.

Experimental operation: Carefully wash the cultured 4T1 cells with PBS for 2-3 times, add 1 mL of trypsinization treatment, when the cell shape is observed to be translucent and round, add 3 mL of fresh RPMI 1640 medium, and disinfect it with one-time disinfection. Pipette carefully to disperse the cells evenly, then transfer the cell fluid to a 15mL centrifuge tube, centrifuge it with a low-speed centrifuge for 3 min (1000 r/min), pour off the supernatant, add fresh medium, pipette and mix well, use a pipette Pipette 10 μL of cell suspension on a cell counting plate with a liquid gun, count with a cell counter, and then add culture medium to dilute the cells to a concentration of 5000 cells/mL. The cell suspension of this concentration was inoculated into a 24-well plate at 500 μL/well, and incubated in a carbon dioxide incubator for 24 hours. After the cells were observed to adhere to the wall under the microscope, the compound lead compound could be added.

The two compounds to be investigated were dissolved with cell-grade DMSO, diluted with medium, added to the well plate to make the final concentration of the compounds 1 μmol/L, and then continued to be incubated in a carbon dioxide incubator for 0.5h, 1h, 3h, 6h And 12h, when the incubation time is up, carefully remove the medium containing the compound in the well plate, and gently wash with PBS twice, then add a small amount of medium, and place the well plate under a fluorescence microscope to observe and take pictures. The excitation slice wavelength of the blue channel of the fluorescence microscope is 420nm-485nm, and the emission slice wavelength is 515nm, which excites green fluorescence; the green channel excitation slice wavelength is 460nm-550nm, and the emission slice wavelength is 590nm. Excited red fluorescence.

6. Cell phototoxicity test of the light-controlled release compound of the present invention.

After the cultured cells were digested and counted, 100 μL/well of cell suspension was taken and placed in a 96-well plate, and the zero-adjustment well, blank group and drug-added group were set. After placing the 96-well plate in a carbon dioxide incubator for 24 hours, the compound was added, and the incubation was continued for 6 hours. The 96-well plate was irradiated with a 524 nm diode laser lamp (500 mW/cm ² ) for 10 min/well. After irradiation, the cells were incubated for 18 hours, the cell culture medium containing the compound was carefully aspirated, washed twice with PBS, then the medium containing MTT was added, incubated for 4 hours, the culture medium was carefully aspirated, and DMSO was added to each well. 100μL, and the absorbance value at 490nm was detected with a microplate reader.

7. The cell light-dependent experiment of the light-controlled release compound of the present invention.

Change the light time to 0min, 5min, 10min, 15min and 20min, and the rest of the steps are the same as above.

8. In vivo antitumor activity test of the light-controlled release compound of the present invention.

Forty tumor mice were selected and randomly divided into 10 groups: ① and ② blank group: PBS light group and dark room group; ③ and ④ chemotherapy control group: chlorambucil light group and dark room group; ⑤ and ⑥ positive control Group: commercial photosensitizer Ce 6 light group and dark room group; ⑦ and ⑧ control group: BOD-OH administration light group and dark room group; ⑨ and ⑩ stimulated drug release group: BOD-CLB light group and dark room group. Then weigh the body weight of each mouse in each group and measure the length and width of the tumor with a vernier caliper, and then inject 100 μL of PBS, chlorambucil, Ce 6, BOD-OH, BOD-CLB into the tumor of each group of mice , the injected compound concentration was 2 μmol/L. After the injection, each illumination group was irradiated with a diode laser lamp (524 nm, 500 mW/cm ² ) for 10 minutes, and the compound was injected every 4 days and illuminated in a dark room. The mice in the group were only injected with compounds without illumination. The weight of each group of mice was weighed every day and the length and width of the tumor were measured with a vernier caliper. The total treatment time was 15 days.

After 15 days, the mice in each group were sacrificed, and blood samples from the fundus artery were taken and placed in a 1.5 mL EP tube. At the same time, the tumors were stripped and weighed and photographed. Sections stained with H&E were observed and photographed using an Olympus CX41 microscope.

9. In vivo light-dependent experiments of the light-controlled release compounds of the present invention.

The tumor mice were selected, and 100 μL of the lead compound at a concentration of 2 μmol/L was injected into the tumor, and the mice were illuminated for 5 min and 15 min, respectively. The rest of the experimental operations are the same as those in Section 8.

10. Study on the liver and kidney toxicity of the light-controlled release compounds of the present invention.

After the above-mentioned mouse fundus arterial blood was allowed to stand for 30min, the EP tube was placed in a low-temperature high-speed centrifuge, centrifuged at a speed of 12000r/min for 15min, the centrifuge tube was taken out, and the supernatant (serum) was drawn with a pipette gun. In a 25 μL EP tube, the samples were sent to the laboratory for testing of liver function markers and renal function markers.

11. Biocompatibility of the light-controlled release compound of the present invention.

The mouse blood was collected in a 1.5mL heparin tube, then placed in a low-temperature high-speed centrifuge, centrifuged at a speed of 2000r/min for 10min, the supernatant was removed with a pipette, the blood cells in the lower layer were retained, and newly configured PBS was added. , gently mix with a pipette, continue centrifugation, and repeat the above steps until the supernatant does not appear yellow. Dilute the lower blood cells to 10 mL with newly prepared PBS, and then take 0.2 mL of it and add it to the following 1 mL solution prepared in advance, with concentrations of 2 μmol/L, 8 μmol/L, 32 μmol/L, and 64 μmol/L. After adding, shake gently, stand for 2 hours at room temperature and take pictures.

Although some embodiments of the present invention have been presented herein, those skilled in the art should understand that changes may be made to the embodiments herein without departing from the spirit of the present invention. The above-mentioned embodiments are only exemplary, and the embodiments herein should not be construed as limiting the scope of the rights of the present invention.

Claims (3)

1. a synthetic method of a light-controlled release compound, comprising the following steps: (i) 4-benzoyl chloride (2mmol) is dissolved in the round-bottomed flask containing 100mL of methylene chloride, and argon gas is passed into it for 15min. 4 mmol of 2,4-dimethyl-3-ethyl-1H-pyrrole was added to the bottom flask, then a small amount of trifluoroacetic acid was added dropwise, the mixture was stirred at room temperature for about 12 h, and 2,3-dichloro-5,6- Dicyano-1,4-benzoquinone 4 mmol, continue stirring for 4 h, then add 10 mmol of triethylamine and continue stirring for 15 min, slowly add 15 mmol of boron trifluoride diethyl ether dropwise under an ice bath, after the drop is complete, the mixture is cooled to room temperature Continue stirring overnight; the reaction solution was extracted with dichloromethane (15 mL×3), dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography, eluted with dichloromethane/petroleum ether v:v=1:7, to obtain the lead compound Orange-red solid powder of intermediate BOD-Cl or BOD-OH; (ii) Weigh 0.25 mmol of lead compound intermediate BOD-Cl and 0.25 mmol of chlorambucil and dissolve in a round-bottomed flask containing 10 mL of anhydrous DMF, Potassium carbonate was then added, and the reaction was carried out at 60 °C for 6 h. After monitoring by TLC, the reaction was completed, extracted with ethyl acetate 15 mL × 3, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography with ethyl acetate/petroleum ether v :v=1:7 elution to obtain red powder lead compound BOD-BLB, yield 45%; see the following reaction scheme:

2. The application of the light-controlled release compound according to claim 1, wherein the light-controlled release compound is an organic small molecule, and has multiple functions of light-controlled drug release, photodynamic therapy and chemotherapy in the same molecular structure , its molecular structure is formed by coupling BODIPY core and chlorambucil; BODIPY core in its molecular structure is a photodynamic group; chlorambucil is a chemotherapeutic group; ester bond is a light-responsive group group, its functional structural formula is shown in formula I,

3 . The application of the light-controlled release compound according to claim 1 , wherein the light-controlled release compound is used for tumor treatment. 4 .

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