Preparation and application of tetracoumarin-based beta-beta coupled double-BODIPY near-infrared photothermal agent

Abstract

The invention relates to preparation and application of tetracoumarin-based beta-beta coupled double-BODIPY near-infrared photothermal dye, which are realized by the following method: carrying out C-C coupling reaction on a BODIPY monomer derivative (I) and anhydrous ferric trichloride to obtain a beta-beta coupled double-BODIPY derivative (II), and carrying out reaction on the beta-beta coupled double-BODIPY derivative (II) and 7-(N, N-dimethyl formamide)-1, 2, 3, 4-tetracoumarin-based beta-beta coupled double-BODIPY derivative (II) to obtain the tetracoumarin-based beta-beta coupled double-BODIPY near-infrared photothermal dye. The preparation method comprises the following steps: carrying out Knoevenagel condensation reaction on N, N '-diethylamino) coumarin-3-formaldehyde to obtain the near-infrared photothermal agent tetracoumarin-based beta-beta coupled double BODIPY derivative (III). The preparation method has the advantages of simple reaction steps, mild reaction conditions, good selectivity and wide application range. The photothermal agent has relatively strong near-infrared absorption, excellent photothermal conversion efficiency and cell apoptosis ability, can be used as a novel photothermal therapy anti-tumor and antibacterial drug, and has important scientific significance and application value.

Description

Technical Field

[0001] The invention belongs to the technical fields of organic compound synthesis, functional fluorescent dyes and fine chemical industry, and specifically relates to the preparation and application of a tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent. Background Art

[0002] Photothermal therapy (PTT) is an emerging clinical tumor treatment method. Compared with traditional cancer treatments such as surgery, radiotherapy and chemotherapy, it has shown great application prospects due to its advantages such as low toxicity, low drug resistance, high specificity and low invasiveness. Usually, photosensitizers are excited to transition to the singlet state (S 1 ), and produce a series of photophysical phenomena, including fluorescence, phosphorescence, singlet oxygen and photothermal conversion, etc. PTT uses light-activated functional biomedical and bioactive nanophotothermal agents in the near-infrared region (NIR) to ablate tumor cells through the heat generated by non-radiative transitions during irradiation. Although PTT has many advantages, the shortcomings of photothermal conversion efficiency, biocompatibility, biodegradability, long-term toxicity and the threat of these photothermal agents to the environment remain unresolved.

[0003] Organic molecules can be modified into multifunctional biomaterials due to their easy modification and good biocompatibility. They can be used as photothermal agents in PTT and have attracted extensive attention. However, improving the light penetration, photothermal conversion efficiency and photostability of organic molecules under repeated laser irradiation are still issues worth addressing. In addition to organic dyes such as cyanine dyes and porphyrins, BODIPY has become a new category of PTT applications. Compared with traditional inorganic materials (such as TiO 2 , ZnO, etc.), BODIPY exhibits excellent biocompatibility and photophysical properties. BODIPY monomers release energy mainly through radiative transitions, and the geometric relaxation of the ground state and excited state is small, which is not conducive to its further photothermal application. The dimerization of BODIPY units can be used as a new and effective strategy. BODIPY can achieve spectral range expansion for deep tissue penetration and strong near-infrared emission. At the same time, according to the energy gap law, BODIPY can effectively enhance the non-radiative decay rate through orthogonal coupling at the β-β position. The CF at the meso position of the BODIPY molecule mother nucleus 3The group is a rotation unit with excellent performance. At the same time, the methyl groups at the 3rd and 5th positions of the BODIPY molecular nucleus also have certain chemical activity and can undergo Knoevenagel condensation reaction with aromatic aldehydes. Therefore, the introduction of conjugated groups at the methyl groups at the four positions of the coupled di-BODIPY can form a larger conjugated system with the BODIPY main body, which can not only maintain the rigidity of the BODIPY structure, but also expand the π conjugated system and increase the planarity of the molecule, resulting in the absorption and emission spectra shifting to longer wavelengths. The increase of the vinyl rotation unit can also effectively increase the photothermal conversion efficiency, providing a theoretical basis for the synthesis of BODIPY-based near-infrared photothermal agents.

[0004] Coumarin dyes are a class of natural products with cinnamic acid lactone condensed ring structures with hindered double bond rotation. They are widely valued for their excellent photophysical properties and physiological activities. However, coumarin-based derivatives have been widely studied as fluorescent probes in the fields of materials and medicine, but there are few systematic explorations of their photothermal properties. The appearance of strong fluorescence of the coumarin parent indicates that the energy absorbed in the excited state mainly undergoes relaxation rather than conversion from light energy to heat energy. Therefore, the focus should be on modifying and modifying the structure of coumarin-based photosensitizers.

[0005] Based on the easy modification of the bi-BODIPY fluorescent dye matrix and the excellent photophysical properties of coumarin derivatives, in order to obtain ideal NIR photophysical properties, integrating two fluorophores into one skeleton through combinatorial chemistry is a good regulatory strategy. 3 A tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent was prepared by catalyzing CC coupling reaction and Knoevenagel condensation reaction, which avoids the problems of many synthetic steps, immature reaction, and low selectivity of most near-infrared photothermal dyes. In addition, given that the photothermal dye has excellent photothermal conversion efficiency and cell apoptosis ability, this new near-infrared photothermal agent has important scientific significance and application value. Summary of the invention

[0006] Purpose of the invention: In view of the shortcomings of the prior art, the purpose of the present invention is to provide a tetracoumarin-based β-β-coupled bis-CF 3 -Preparation and application of BODIPY near-infrared photothermal agent.

[0007] Technical solution: In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0008] The preparation and application of a tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent of the present invention are characterized in that the near-infrared photothermal agent has a structural formula shown in the following formula (III):

[0009]

[0010] The preparation and application of a tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent are as follows:

[0011] Step 1: Under argon protection, add BODIPY monomer derivative (I) to dry dichloromethane, then add anhydrous ferric chloride and nitromethane, stir, react for 15 minutes, stop the reaction, add methanol, stir, react for 15 minutes, extract, dry, and separate and purify by silica gel column chromatography to obtain a β-β coupled bis-BODIPY derivative (II);

[0012] Step 2: Under anhydrous conditions, add the β-β coupled bis BODIPY derivative (II) and 7-(N,N′-diethylamino) coumarin-3-carboxaldehyde in a molar ratio of 1:4.0 to dry toluene, then add p-toluenesulfonic acid and piperidine, stir and heat, control the temperature at 80°C, react for 1 hour, stop the reaction, extract, dry, and separate and purify by silica gel column chromatography to obtain a tetracoumarin-based β-β coupled bis BODIPY near-infrared photothermal agent (III).

[0013] The specific chemical reaction formula is as follows:

[0014]

[0015] In the above step 1, the molar ratio of BODIPY monomer derivative (I) and anhydrous ferric chloride is 1:3; the volume ratio of dichloromethane, methanol and nitromethane to the amount of BODIPY monomer derivative (I) is 25 mL: 10 mL: 5 mL: 0.05 mmol.

[0016] In the above step 1, the eluent for silica gel column chromatography separation is 100% dichloromethane.

[0017] In the above step 2, the molar ratio of the β-β coupling bis BODIPY derivative (II), p-toluenesulfonic acid and 7-(N,N′-diethylamino)coumarin-3-carboxaldehyde is 1:0.5:4.0, and the ratio of the volume of toluene and piperidine to the amount of the β-β coupling bis BODIPY derivative (II) is 50 mL:1 mL:1 mmol.

[0018] In the above step 2, the silica gel column chromatography separation eluent is dichloromethane and methanol.

[0019] Beneficial effects of the present invention

[0020] Compared with the prior art, the tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) of the present invention has the following advantages: (1) the preparation method is simple and easy, the synthesis method is mature, and the selectivity is high; (2) the molar extinction coefficient of the photothermal agent is high (>6×10-6 L·mol -1 cm -1 ), the maximum absorption in dimethyl sulfoxide solvent reaches 912nm, which can effectively avoid tissue damage and bulk interference and improve imaging sensitivity; (3) It has a high photothermal conversion efficiency (88%), which is conducive to the realization of photothermal therapy in living experiments; (4) It has low dark toxicity and strong biocompatibility at low power (0.1W / cm 2 )808nm laser excitation can significantly kill cancer cells and induce cancer cell apoptosis through photothermal effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the UV-visible absorption spectra of tetracoumarin-based β-β-coupled biBODIPY near-infrared photothermal agent (III) in different solvents. The concentration of photothermal agent (III) is 1×10 -5 mol / L;

[0022] Figure 2 The photothermal performance changes of tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) in dimethyl sulfoxide under different laser power densities of 808 nm laser irradiation;

[0023] Figure 3 This is a graph of the survival rate of HCT-116 and CT-26 cancer cells treated with different concentrations of tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III). The left side of the bar graph is the dark condition treatment, and the right side is the 808nm laser irradiation condition treatment;

[0024] Figure 4 It is 808nm laser (0.1W / cm 2 ) Fluorescence microscopy imaging of cancer cell apoptosis induced by poly-tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) under irradiation;

[0025] Figure 5 Flow cytometry was used to analyze apoptosis and necrosis in HCT-116 cells treated with tetracoumarin-based β-β-coupled biBODIPY near-infrared photothermal agent (III). DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the specific drawings.

[0027] The structures of β-β-coupled bis-BODIPY dye (II) and tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) were characterized and confirmed by NMR and MALDI-TOF. The instruments used for the detection were: Bruker ARX600 NMR (deuterated chloroform as solvent), Shimadzu UV-3100 UV-visible spectrophotometer (scanning range 300-900nm, optical path slit 2nm), Bruker Dalton Autoflex II matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF MASS), American APExBIO Annexin V-FITC / PI apoptosis detection kit, CytoFLE flow cytometer (BECKMAN COULTER), China Mingmei Optoelectronics Yunnan MF53-N fluorescence microscope.

[0028] Example 1 Preparation of β-β coupled bis-BODIPY derivative (II)

[0029] Under anhydrous conditions, a solution of anhydrous ferric chloride (243 mg, 1.5 mmol) in nitromethane (5 mL) was added to a solution of BODIPY monomer derivative (I) (158 mg, 0.5 mmol) in anhydrous dichloromethane (25 mL), and the mixture was reacted at room temperature for 15 minutes. The reaction solution quickly turned from orange to dark blue purple. Methanol (10 mL) was added and stirred for 15 minutes to quench the reaction, and the mixture was cooled to room temperature, extracted with dichloromethane, washed with water, and the organic layers were combined. The organic solvent was removed by distillation under reduced pressure, and the residue was separated and purified by silica gel column chromatography, with the eluent being 100% dichloromethane, to obtain a dark blue solid product, β-β coupled bis-BODIPY derivative (II) (47 mg, 30%). 1 H NMR (600 MHz, CDCl 3 , ppm): δ=6.21 (s, 2H), 2.58 (s, 6H), 2.37 (s, 6H), 2.34 (d, J=3.0Hz, 6H), 2.08 (d, J=3.0Hz, 6H). 13 C NMR (100 MHz, CDCl 3 , ppm)δ=160.567, 157.185, 144.176, 140.410, 132.399, 130.949, 126.861, 125.037, 123.673, 120.937, 15.838, 15.223, 14.280, 13.884. 19 F NMR (376 MHz, CDCl 3 , ppm): δ=-52.552, -146.097, -146.178, -146.264, -146.347.MALDI-TOF calcd.forC28 H 26 B 2 F 1o N 4 =630.218, found: 630.342.

[0030] Example 2 Preparation of polycoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III)

[0031] Under anhydrous conditions, β-β coupling bis BODIPY (II) agent (630 mg, 1 mmol), 7-(N, N'-diethylamino) coumarin-3-carboxaldehyde (980 mg, 4 mmol) and p-toluenesulfonic acid (86 mg, 0.5 mmol) were dissolved in 50 mL toluene and 1 mL piperidine, heated to 80°C, and reacted for 1 hour. Cooled to room temperature, extracted with dichloromethane, washed with water, the organic layers were combined, the organic solvent was evaporated under reduced pressure, and the residue was separated and purified by silica gel column chromatography, the eluents were dichloromethane and methanol, and a gray-brown solid product, polycoumarin-based β-β coupling bis BODIPY near-infrared photothermal agent (III) (383 mg, 20%) was obtained. 1 H NMR (600 MHz, CDCl 3 , ppm): δ=8.29 (d, J=16.2Hz, 2H), 8.17 (s, 2H), 7.77 (d, J=16.2Hz, 2H), 7.68 (d, J=16.2Hz, 2H), 7.45 (d, J =9.0Hz, 2H), 7.43 (s, 2H), 7.22 (d, J = 9.0Hz, 2H), 7.03 (d, J = 16.2Hz, 2H), 6.96 (s, 2H), 6.61 (dd, J = 9.0, 2. 4Hz, 2H), 6.51 (d, J=2.4Hz, 1H), 6.50 (d, J=2.4Hz, 3H), 6.41 (d, J=1.8Hz, 2H), 3.48-3.44 (m, 8H), 3.41-3. 37 (m, 8H), 2.40 (s, 6H), 2.13 (d, J = 1.2Hz, 6H), 1.25 (t, J = 7.2Hz, 12H), 1.18 (t, J = 7.2Hz, 12H); MALDI-TOF calcd forC 84 H 78 B 2 F 10 N 8 O 8 K=1577.561, found: 1577.000.

[0032] Example 3 UV-visible absorption spectra of tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) in different solvents

[0033] The tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III) prepared in Example 2 was dissolved in DMSO solution to prepare a 3 mmol / L stock solution. The stock solution (3 mM) was added to toluene, tetrahydrofuran, dichloromethane, methanol and dimethyl sulfoxide to a concentration of 1×10 -5 mol / L solution and measure its UV-visible absorption spectrum. Figure 1 This is the UV-visible absorption spectrum of β-β coupled bi-BODIPY near-infrared photothermal agent (III) in different solvents.

[0034] Example 4 Photothermal performance of tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III) in dimethyl sulfoxide

[0035] Take the 3 mmol / L tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III) stock solution prepared in Example 3 and add it to dimethyl sulfoxide to prepare a concentration of 2×10 -5 mol / L solution was used to test its photothermal performance. The tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) showed excellent photothermal conversion efficiency (PCE), with a PCE value of 88%. Figure 2 To characterize the photothermal properties of β-β coupled bis-BODIPY near-infrared photothermal agent (III) in dimethyl sulfoxide under irradiation of different laser power densities of 808 nm laser.

[0036] Example 5 Phototoxicity and dark toxicity determination of tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III)

[0037] Colon cancer cells (HCT-116 cells) and mouse colon cancer cells (CT-26 cells) were cultured in a medium containing 10% fetal bovine serum (FBS), penicillin (80 μg / mL) and streptomycin (80 μg / mL). The culture medium was placed in a constant temperature cell culture incubator at 37°C containing 5% carbon dioxide. The cell density was about 80-90% for cell passaging or other experiments. Phototoxicity and dark toxicity tests were performed using the MTT assay. HCT-116 cells and CT-24 cells (10,000 cells / well) were inoculated into 96-well plates containing DMEM medium (100 μL / well) and cultured in an incubator overnight. Then, 100 μL of near-infrared photothermal agent (III) containing different concentrations (0, 10, 20, 30, 40, 50 μM) was added and cultured for 2 h. The phototoxic group was treated with an 808 nm laser (0.1 W / cm 2) for 5 minutes, and there was no laser irradiation in the dark toxicity group. After incubation for 4 hours, the cells were washed 3 times with PBS and treated with 100μL MTT solution (0.5mg / mL) for another 4 hours. The MTT solution was removed in a light-proof environment, and 150μL DMSO solution was added to each well. After oscillation for 3 minutes, the optical density (OD) value was recorded at 490nm using a microplate reader. The results showed that under dark conditions (no laser), the cell viability rate treated with different concentrations of photothermal agent (III) was almost always higher than 70%. However, when irradiated with 808nm laser, the cell survival rate decreased significantly. This shows that the probe has good biocompatibility and can significantly kill cancer cells under laser excitation. Figure 3 This is a graph of the survival rate of HCT-116 and CT-26 cancer cells under the treatment of different concentrations of polycoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III). The left side of the bar graph is the dark condition treatment, and the right side is the 808nm laser irradiation condition treatment.

[0038] Example 6 Photothermal-induced cell apoptosis assay of tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) Photothermal-induced cell apoptosis was studied by propidium iodide (PI) and calcein (AM) assay. HCT-116 cells (2×10 4 / well) were inoculated into 24-well plates and then divided into four groups: ① control group, cells were incubated without any treatment; ② laser group, cells were irradiated with 808 nm laser (0.1 W / cm 2 ) irradiated the cells for 5 minutes; ③ Sample group, the cells were incubated with 10 μM polycoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) at 37°C for 2 hours; ④ Sample + laser group, the cells were incubated with 10 μM tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) at 37°C for 2 hours, and then irradiated with 808 nm laser (0.1 W / cm 2 ) for 5 minutes. After 2 hours, all four groups were washed three times with PBS aqueous solution and AM (5 μM) and PI (10 μM) were added, incubated at 37°C for 20 minutes, and then imaged using a fluorescence microscope. AM was excited at 494 nm and collected at 514 nm. PI was excited at 540 nm and collected at 605 nm or 655 nm. After 5 minutes of laser irradiation, the sample + laser group showed obvious PI fluorescence in the cell nucleus, while only green fluorescence was observed in the sample group without laser irradiation, and no obvious fluorescence signal was shown. Similarly, in the control group with the same laser exposure duration, no obvious PI fluorescence was observed. These results indicate that the tetracoumarin-based β-β-coupled bisBODIPY near-infrared photothermal agent (III) has excellent photothermal properties, and the near-infrared absorption wavelength also makes it have the potential for in vivo diagnosis and treatment of cancer. Figure 4It is 808nm laser (0.1W / cm 2 ) Fluorescence microscopy imaging of cancer cell apoptosis induced by tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) under irradiation;

[0039] Example 7 Flow cytometry experiment of tetracoumarin-based β-β-coupled biBODIPY near-infrared photothermal dye (III)

[0040] HCT-116 cells were seeded on 6-well plates for 24 h, and then treated with the following conditions: ① Sample group, cells were incubated with 10 μM tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) at 37 °C for 2 h; ② Sample + laser group, cells were incubated with 10 μM tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) at 37 °C for 2 h, and then irradiated with 808 nm laser (0.1 W / cm 2 ) for 5 minutes. After incubation for 2 hours, the cells were stained using a cell apoptosis detection kit and detected by flow cytometry analyzer. After 5 minutes of irradiation, more than 80% of the tumor cells in the sample group survived, while less than 42% of the cells in the sample + laser group survived, further confirming the effective necrosis-inducing ability of the polycoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) on cancer cells. These results indicate that photothermal therapy with the tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) can be performed at low power intensities (0.1 W / cm 2 ) laser irradiation effectively induces tumor cell death. Figure 5 This is a flow cytometry analysis of apoptosis and necrosis in HCT-116 cells treated with tetracoumarin-based β-β-coupled biBODIPY near-infrared photothermal agent (III).

Claims

1. Preparation and application of a polycoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent, characterized in that: Its structural formula is shown in formula (III):

2. The method for preparing a polycoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III) according to claim 1, characterized in that: The preparation method is as follows: firstly, a BODIPY monomer derivative (I) is subjected to a CC coupling reaction with anhydrous ferric chloride to obtain a β-β coupled bis-BODIPY derivative (II), and then the β-β coupled bis-BODIPY derivative (II) is subjected to a Knoevenagel condensation reaction with 7-(N, N′-diethylamino) coumarin-3-carboxaldehyde to obtain a near-infrared photothermal dye tetracoumarin-based β-β coupled bis-BODIPY derivative (III). The reaction formula of the synthesis process is as follows:

3. The method for preparing a tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III) according to claim 2, characterized in that The following steps are involved: Step 1: Under argon protection, add BODIPY monomer derivative (I) to dry dichloromethane, then add anhydrous ferric chloride and nitromethane, stir, react for 15 minutes, terminate the reaction, add methanol, stir, react for 15 minutes, extract, dry, and separate and purify by silica gel column chromatography to obtain a β-β coupled bis-BODIPY derivative (II); Step 2; Under anhydrous conditions, a β-β coupled bis-BODIPY derivative (II) and 7-(N,N′-diethylamino)coumarin-3-carboxaldehyde are added to dry toluene in a molar ratio of 1:4.0, and then p-toluenesulfonic acid and piperidine are added. The mixture is stirred and heated. The temperature is controlled at 80°C. The reaction time is 1 hour. The reaction is terminated, and the mixture is separated and purified by extraction, drying, and silica gel column chromatography to obtain a tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III).

4. The method for preparing a polycoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III) according to claim 3, characterized in that In the step 1, the molar ratio of the BODIPY monomer derivative (I) to anhydrous ferric chloride is 1:3; the volume ratio of dichloromethane, methanol, and nitromethane to the amount of the BODIPY monomer derivative (I) is 25 mL: 10 mL: 5 mL: 0.05 mmol.

5. The method for preparing a tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III) according to claim 3, characterized in that In the step 2, the molar ratio of the β-β coupled bis BODIPY dye (II), p-toluenesulfonic acid and 7-(N,N′diethylamino)coumarin-3-carboxaldehyde is 1:0.5:4.0, and the volume ratio of toluene and piperidine to the amount of the β-β coupled bis BODIPY derivative (II) is 50 mL:1 mL:1 mmol.

6. The use of a tetracoumarin-based β-β coupled bis-BODIPY near-infrared photothermal agent (III) according to claim 1, characterized in that: The photothermal agent is used in photothermal therapy against human and mouse colon cancer cells.

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