Preparation and application of a tetra-coumarin-based beta-beta coupled double bodipy near-infrared photothermal agent

Abstract

The present application relates to a kind of four coumarin-based β-β coupling double BODIPY near-infrared photothermal dyes and its application, by the following method is realized: BODIPY monomer derivative (I) and anhydrous ferric chloride occur C-C coupling reaction and obtain β-β coupling double BODIPY derivative (II), then use β-β coupling double BODIPY derivative (II) and 7-(N,N' diethyl amido) coumarin-3-formaldehyde occurs Knoevenagel condensation reaction and obtain near-infrared photothermal agent four coumarin-based β-β coupling double BODIPY derivative (III).The preparation method reaction step is simple, reaction condition is mild, selectivity is better, and the scope is wide.This kind of photothermal agent has strong near-infrared absorption, excellent photothermal conversion efficiency and cell apoptosis ability, can be used as new photothermal therapy antitumor and antibacterial drug, this new type of near-infrared photothermal agent has important scientific significance and application value.

Description

Technical Field

[0001] This invention belongs to the fields of organic compound synthesis, functional fluorescent dyes and fine chemical technology, and specifically relates to the preparation and application of a tetracoumarin-based β-β-coupled bisBODIPY near-infrared photothermal agent. Background Technology

[0002] Photothermal therapy (PTT), as an emerging clinical cancer treatment method, shows great promise compared to traditional cancer treatments such as surgery, radiotherapy, and chemotherapy due to its advantages of low toxicity, low drug resistance, high specificity, and low invasiveness. Typically, photosensitizers are excited to a singlet state (S1) under light irradiation, producing a series of photophysical phenomena, including fluorescence, phosphorescence, singlet oxygen, and photothermal conversion. PTT utilizes photoactivated functional biomedical and bioactive nanophotothermal agents in the near-infrared (NIR) region, ablating tumor cells through the heat generated by non-radiative transitions during irradiation. Despite the many advantages of PTT, drawbacks such as photothermal conversion efficiency, biocompatibility, biodegradability, long-term toxicity, and the environmental threats posed by these photothermal agents remain unresolved.

[0003] Organic molecules have attracted widespread attention as photothermal agents in photothermal transducers (PTTs) due to their ease of modification and good biocompatibility, which allow them to be modified into multifunctional biomaterials. However, improving the light transmittance, photothermal conversion efficiency, and photostability under repeated laser irradiation of organic molecules remains a challenge. Besides organic dyes such as cyanine and porphyrin, BODIPY has emerged as a new category for PTT applications. Compared to traditional inorganic materials (such as TiO2 and ZnO), BODIPY exhibits superior biocompatibility and photophysical properties. The BODIPY monomer primarily releases energy through radiative transitions, but its geometric relaxation in both the ground and excited states is relatively small, which is not conducive to its further photothermal applications. Dimerization of the BODIPY unit can serve as a new and effective strategy. BODIPY can achieve spectral range expansion, enabling deep tissue penetration and near-infrared strong emission. Furthermore, according to the bandgap law, BODIPY can effectively enhance the nonradiative decay rate through orthogonal coupling at the β-β positions. The CF3 group at the meso position of the BODIPY molecule's core is a high-performance rotating unit, and the methyl groups at the 3 and 5 positions of the BODIPY molecule's core also possess certain chemical activity, capable of undergoing Knoevenagel condensation reactions with aromatic aldehydes. Therefore, by introducing conjugated groups into the methyl groups at the four positions of the coupling double BODIPY, a larger conjugated system can be formed with the BODIPY host, which can not only maintain the rigidity of the BODIPY structure, but also extend the π conjugated system and increase the planarity of the molecule, resulting in the absorption and emission spectra shifting to longer wavelengths. The increase in vinyl rotation units can also effectively increase the photothermal conversion efficiency, providing a theoretical basis for the synthesis of BODIPY-type near-infrared photothermal agents.

[0004] Coumarin dyes are a class of natural products with a fused-ring structure of cinnamic acid lactones and hindered double bond rotation, and they have attracted much attention due to their excellent photophysical properties and physiological activities. However, while coumarin derivatives have been extensively studied as fluorescent probes in materials science and medicine, their photothermal properties have been less systematically explored. The strong fluorescence of the coumarin parent compound indicates that the energy absorbed in the excited state mainly undergoes relaxation rather than a conversion from light energy to heat energy. Therefore, the modification and alteration of the structure of coumarin-based photosensitizers should be emphasized.

[0005] Based on the ease of modification of the parent bis-BODIPY fluorescent dyes and the excellent photophysical properties of coumarin derivatives, integrating two fluorophores into a single framework via combinatorial chemistry is a promising regulatory strategy for obtaining ideal NIR photophysical properties. A tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent was prepared via FeCl3-catalyzed CC coupling and Knoevenagel condensation reactions, avoiding the problems of numerous synthesis steps, immature reactions, and low selectivity found in most near-infrared photothermal dyes. Furthermore, given the excellent photothermal conversion efficiency and apoptosis-inducing ability of this photothermal dye, this novel near-infrared photothermal agent has significant scientific and application value. Summary of the Invention

[0006] Purpose of the invention: In view of the shortcomings of the existing technology, the purpose of this invention is to provide a preparation and application of a tetracoumarin-based β-β-coupled bisCF3-BODIPY near-infrared photothermal agent.

[0007] Technical solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0008] The present invention relates to the preparation and application of a tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent, characterized in that the near-infrared photothermal agent has the following structural 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, the BODIPY monomer derivative (I) was added to dry dichloromethane, followed by anhydrous ferric chloride and nitromethane. The mixture was stirred and reacted for 15 minutes. The reaction was then stopped, methanol was added, and the mixture was stirred and reacted for 15 minutes. The mixture was then extracted, dried, and purified by silica gel column chromatography to obtain the β-β coupled bisBODIPY derivative (II).

[0012] Step 2: Under anhydrous conditions, β-β-coupled bis-BODIPY derivative (II) and 7-(N,N′-diethylamino)coumarin-3-carboxaldehyde were added to dry toluene at a molar ratio of 1:4.0. Then, p-toluenesulfonic acid and piperidine were added, the mixture was stirred and heated at 80°C for 1 hour. The reaction was then stopped. The mixture was extracted, dried, and purified by silica gel column chromatography to obtain tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III).

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

[0014]

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

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

[0017] In step 2 above, the molar ratio of β-β-coupled bis-BODIPY derivative (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 β-β-coupled bis-BODIPY derivative (II) is 50mL:1mL:1mmol.

[0018] In step 2 above, the eluents for silica gel column chromatography are 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 (2) It has a maximum absorption of 912 nm in dimethyl sulfoxide solvent, which can effectively avoid tissue damage and bulk interference and improve imaging sensitivity; (3) It has a high photothermal conversion efficiency (88%), which is beneficial for realizing photothermal therapy in in vivo experiments; (4) It has low dark toxicity and strong biocompatibility, and can be used at low power (0.1 W / cm²). 2 Excitation by an 808nm laser can significantly kill cancer cells and induce apoptosis of cancer cells through photothermal effect. Attached Figure Description

[0021] Figure 1 This is the UV-Vis absorption spectrum of tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) in different solvents, with a concentration of 1×10⁻⁶. -5 mol / L;

[0022] Figure 2 The photothermal properties of tetracoumarin-based β-β-coupled bisBODIPY near-infrared photothermal agent (III) in dimethyl sulfoxide were observed under different laser power densities from an 808 nm laser.

[0023] Figure 3 This is a survival graph 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 chart represents the dark condition treatment, and the right side represents the 808nm laser irradiation condition treatment.

[0024] Figure 4 It is an 808nm laser (0.1W / cm²). 2 Fluorescence microscopy image of cancer cell apoptosis induced by polytetracosinyl β-β-coupled double BODIPY near-infrared photothermal agent (III) under irradiation.

[0025] Figure 5 The study used flow cytometry to analyze apoptosis and necrosis in HCT-116 cells treated with tetracoumarin-based β-β-coupled double BODIPY near-infrared photothermal agent (III). Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying 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 detection were: a Bruker ARX600 NMR spectrometer (deuterated chloroform as solvent), a Shimadzu UV-3100 UV-Vis spectrophotometer (scanning range 300–900 nm, optical path slit 2 nm), a Brook Dalton Autoflex II matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MASS), an APExBIO Annexin V-FITC / PI apoptosis detection kit (USA), a CytoFLE flow cytometer (BECKMAN COULTER), and a Yunnan MF53-N fluorescence microscope from Mingmei Optoelectronics (China).

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

[0029] Under anhydrous conditions, a solution of 158 mg (0.5 mmol) of BODIPY monomer derivative (I) in 25 mL of anhydrous dichloromethane was added to a solution of 243 mg (1.5 mmol) of anhydrous ferric chloride in 5 mL of nitromethane, and the reaction was allowed to proceed at room temperature for 15 minutes. The orange solution rapidly turned deep blue-purple. The reaction was quenched by adding 10 mL of methanol and stirring for 15 minutes. After cooling to room temperature, the mixture was extracted with dichloromethane, washed with water, and the organic layers were combined. The organic solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography using 100% dichloromethane as the eluent to obtain a deep blue solid product, β-β-coupled bis-BODIPY derivative (II) (47 mg, 30%). 1 H NMR (600MHz, CDCl3, 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 (100MHz, CDCl3, ppm) δ = 160.567, 157.185, 144.176, 140.410, 132.399, 13 0.949, 126.861, 125.037, 123.673, 120.937, 15.838, 15.223, 14.280, 13.884. 19 F NMR (376MHz, CDCl3, ppm): δ=-52.552, -146.097, -146.178, -146.264, -146.347.MALDI-TOF calcd.forC 28 H 26 B2F 1o N4=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-coupled 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 of toluene and 1 mL of piperidine, heated to 80 °C, and reacted for 1 hour. After cooling to room temperature, the mixture was extracted with dichloromethane, washed with water, and the organic layers were combined. The organic solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain a grayish-brown solid product, polycoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) (383 mg, 20%).1 H NMR (600MHz, CDCl3, 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), 3.4 1-3.37 (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 B2F 10 N8O8K=1577.561, found: 1577.000.

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

[0033] A 3 mmol / L stock solution was prepared by dissolving the tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) prepared in Example 2 in DMSO solution. The stock solution (3 mM) was then added to toluene, tetrahydrofuran, dichloromethane, methanol, and dimethyl sulfoxide to prepare a concentration of 1 × 10⁻⁶ m³ / L. -5 The ultraviolet-visible absorption spectrum of the solution was measured. Figure 1 The images show the UV-Vis absorption spectra of β-β coupled bis-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 bisBODIPY near-infrared photothermal agent (III) stock solution prepared in Example 3, add it to dimethyl sulfoxide, and prepare a concentration of 2×10 -5The photothermal performance of the tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) was tested using a mol / L solution. The agent exhibited superior photothermal conversion efficiency (PCE) of 88%. Figure 2 To characterize the photothermal properties of β-β coupled bisBODIPY near-infrared photothermal agent (III) in dimethyl sulfoxide under different laser power densities of an 808 nm laser.

[0036] Example 5: Determination of phototoxicity and dark toxicity 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 medium was placed in a constant temperature cell culture incubator at 37°C with 5% carbon dioxide. The cell density was approximately 80-90% for cell passaging or other experiments. Phototoxicity and dark toxicity assays were performed using the MTT assay. HCT-116 cells and CT-24 cells (10,000 cells / well) were seeded into 96-well plates containing DMEM medium (100 μL / well) and cultured overnight. Then, 100 μL of near-infrared photothermal agent (III) at different concentrations (0, 10, 20, 30, 40, 50 μM) was added and the cells were cultured for 2 h. The phototoxicity group was treated with an 808 nm laser (0.1 W / cm²). 2 Cells were treated for 5 min (dark toxicity group, no laser irradiation). After 4 hours of incubation, cells were washed three times with PBS and treated again for 4 hours with 100 μL MTT solution (0.5 mg / mL). The MTT solution was removed in a dark environment, and 150 μL DMSO solution was added to each well. After shaking for 3 minutes, the optical density (OD) value was recorded at 490 nm using a microplate reader. The results showed that under dark conditions (no laser), the cell viability of cells treated with different concentrations of photothermal agent (III) was almost always higher than 70%. However, when irradiated with an 808 nm laser, the cell viability decreased significantly. This indicates that the probe has good biocompatibility and can significantly kill cancer cells under laser excitation. Figure 3 This is a survival graph of HCT-116 and CT-26 cancer cells treated with different concentrations of polycoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III). The left side of the bar chart represents the dark condition treatment, and the right side represents the 808nm laser irradiation condition treatment.

[0038] Example 6. Assay of Photothermal-Induced Apoptosis by Tetracoumarin-Based β-β-Conjugated Bis-BODIPY Near-Infrared Photothermal Agent (III): Photothermal-induced apoptosis was studied using propidium iodide (PI) and calcein (AM) assays. HCT-116 cells (2 × 10⁻⁶) were used... 4 Cells were seeded 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 an 808nm laser (0.1W / cm²). 2 ① Irradiate cells for 5 minutes; ③ Sample group: incubate cells with 10 μM coumarin-based β-β-coupled double BODIPY near-infrared photothermal agent (III) at 37℃ for 2 hours; ④ Sample + laser group: incubate cells with 10 μM tetracoumarin-based β-β-coupled double BODIPY near-infrared photothermal agent (III) at 37℃ for 2 hours, then irradiate with an 808nm laser (0.1W / cm²). 2 Irradiation lasted 5 minutes. Two hours later, all four groups were washed three times with PBS aqueous solution and incubated with AM (5 μM) and PI (10 μM) at 37 °C for 20 minutes, followed by imaging using fluorescence microscopy. 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 significant PI fluorescence in the cell nucleus, while the sample group without laser irradiation only showed green fluorescence without significant fluorescence signal. Similarly, no significant PI fluorescence was observed in the control group with the same laser exposure duration. These results indicate that the tetracoumarin-based β-β-coupled bisBODIPY near-infrared photothermal agent (III) possesses excellent photothermal properties, and its near-infrared absorption wavelength also makes it a potential candidate for in vivo diagnosis and treatment of cancer. Figure 4 It is an 808nm laser (0.1W / cm²). 2 Fluorescence microscopy image of cancer cell apoptosis induced by tetracoumarin-based β-β-coupled double BODIPY near-infrared photothermal agent (III) under irradiation.

[0039] Example 7: Flow Cytometry Experiment with Tetracoumarin-Based β-β-Conjugated Bis-BODIPY Near-Infrared Photothermal Dye (III)

[0040] HCT-116 cells were seeded into 6-well plates for 24 hours, and then treated with the following conditions: ① Sample group: cells were incubated with 10 μM tetracoumarin-based β-β-conjugated double BODIPY near-infrared photothermal agent (III) at 37°C for 2 hours; ② Sample + laser group: cells were incubated with 10 μM tetracoumarin-based β-β-conjugated double BODIPY near-infrared photothermal agent (III) at 37°C for 2 hours, and then irradiated with an 808 nm laser (0.1 W / cm²). 2After 5 minutes of incubation, the cells were stained using an apoptosis detection kit and analyzed by flow cytometry. After 5 minutes of irradiation, over 80% of tumor cells in the sample group survived, while less than 42% of cells survived in the sample + laser group, further confirming the effective necrosis-inducing ability of polycoumarin-based β-β-coupled double BODIPY near-infrared photothermal agent (III) on cancer cells. These results indicate that photothermal therapy with tetracoumarin-based β-β-coupled double BODIPY near-infrared photothermal agent (III) can be effective even at low power intensities (0.1 W / cm²). 2 Laser irradiation can effectively induce 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 double BODIPY near-infrared photothermal agent (III).

Claims

1. A polycoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent, characterized in that, Its structural formula is shown in equation (III):

2. The preparation method of the 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: First, the BODIPY monomer derivative (I) undergoes a CC coupling reaction with anhydrous ferric chloride to obtain a β-β-coupled bis-BODIPY derivative (II). Then, the β-β-coupled bis-BODIPY derivative (II) undergoes 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 formulas for the synthesis process are as follows:

3. The preparation method of a tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) according to claim 2, characterized in that... Includes the following steps: Step 1: Under argon protection, the BODIPY monomer derivative (I) was added to dry dichloromethane, followed by anhydrous ferric chloride and nitromethane. The mixture was stirred and reacted for 15 minutes. After the reaction was stopped, methanol was added and stirred for another 15 minutes. The mixture was then extracted, dried, and purified by silica gel column chromatography to obtain the β-β-coupled bis-BODIPY derivative (II). Step 2: Under anhydrous conditions, β-β-coupled bis-BODIPY derivative (II) and 7-(N,N′-diethylamino)coumarin-3-carboxaldehyde were added to dry toluene at a molar ratio of 1:4.

0. Then, p-toluenesulfonic acid and piperidine were added, the mixture was stirred and heated at 80°C for 1 hour. The reaction was then stopped. The product was extracted, dried, and purified by silica gel column chromatography to obtain tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III).

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

5. The preparation method of a tetracoumarin-based β-β-coupled bis-BODIPY near-infrared photothermal agent (III) according to claim 3, characterized in that... In step 2, the molar ratio of 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 molar ratio of β-β-coupled bis-BODIPY derivative (II) is 50 mL:1 mL:1 mmol.

6. The application of the 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 the preparation of a photothermal therapy drug for treating human and mouse colon cancer cells.

Images