A water-soluble thionaphthalimide photosensitizer, a preparation method thereof and an application thereof

Abstract

The present invention discloses a water-soluble thionaphthalimide photosensitizer, a preparation method thereof and an application thereof, belonging to the field of organic optofunctional materials. The photosensitizer of the present invention uses thionaphthalimide containing an electron-donating effect as a parent body, and at the same time introduces a water-soluble polyethylene glycol (PEG) group at its amide position. In an aqueous solution, the photosensitizer can self-assemble into nanoparticles with a particle size of 147 nm without other nanocarriers, and has good absorption at 570 nm. The method of the present invention has the advantages of easily available raw materials, simple synthesis and easy separation. The prepared photosensitizer has good reactive oxygen generation ability under white light irradiation, good water solubility, and good photo, thermal and chemical stability. The ultraviolet-visible absorption tail peak can reach the near-infrared region. The nanoparticles have good photodynamic killing effect on cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of organic optofunctional materials, and mainly relates to the preparation and application of water-soluble thionaphthalimide photosensitizers. Technical Background

[0002] Cancer causes 9 million deaths worldwide every year, becoming a major threat to human health. Traditional treatments include surgical treatment, radiotherapy, and chemotherapy with chemical drugs, but it is difficult to achieve good treatment effects fundamentally. For example, surgical treatment is limited to directly removing visible solid tumors to achieve the treatment purpose, and it is impossible to ensure that all tumor cells are completely removed, resulting in a high risk of tumor recurrence; radiotherapy inevitably damages or kills surrounding tissues and cells due to certain uncontrollability; chemotherapy has great side effects.

[0003] Photodynamic therapy (PDT) is expected to become an important supplement to traditional treatment methods to a certain extent due to its advantages such as high controllability, no drug resistance, and minimally invasive nature. The principle of photodynamic therapy is that photosensitizers can effectively generate reactive oxygen species (ROS) under the conditions of light irradiation and the presence of oxygen, and then damage the structures of biological macromolecules such as nucleic acids and proteins, ultimately leading to apoptosis or necrosis of tumor cells. Photosensitizers play a crucial role in the process of photodynamic therapy, and their quality directly determines the photodynamic therapy effect. Currently used organic small molecule photosensitizers such as porphyrin, methylene blue, BODIPY, etc. all have the defect of low efficiency of reactive oxygen generation and cannot achieve good treatment effects. At the same time, disadvantages such as poor water solubility and unstable structure will also affect their treatment effects in PDT. Summary of the Invention

[0004] In view of this, the synthesis of new photosensitizers with good biocompatibility, high reactive oxygen output, good water solubility, etc. is of great significance for photodynamic therapy of tumors. Compared with traditional photosensitizers, the combination of sulfur substitution and the introduction of strong electron-donating groups highlights the potential of obtaining photosensitizers with high-efficient reactive oxygen (ROS) generation ability in the near-infrared phototherapy window.

[0005] The first aspect of the present invention provides a water-soluble thionaphthalimide photosensitizer, and its structure is as follows:

[0006]

[0007] Wherein, R1 is

[0008] R2 is n = 3

[0009] The present invention also provides a preparation method of the above-mentioned water-soluble thionaphthalimide photosensitizer, and the reaction formula in this method process is as follows:

[0010]

[0011] The above preparation method specifically includes the following steps:

[0012] (1) Synthesis of intermediate compound 2

[0013] Under a N2 atmosphere, 6 mmol of compound 1, 6 - 18 mmol of active amine hydrogen compound or amine hydrogen compound modified with borate ester, 6 - 24 mmol of K2CO3, and 0.02 - 0.06 mmol of Pd(PPh3)4 are dissolved in toluene, heated to 110 °C and refluxed for 24 h, and the reaction is monitored by thin layer chromatography. After the reaction is completed, the reaction solution cooled to room temperature is poured into cold water and extracted with dichloromethane. The organic phase crude product is rotary evaporated, dried, separated by silica gel column chromatography, and eluted with dichloromethane / petroleum ether with a volume ratio of 3:1 to obtain a pale yellow solid product.

[0014] (2) Synthesis of intermediate compound TNP

[0015] Under a N2 atmosphere, 4 mmol of compound 2, 4 - 20 mmol of PEG 208 -OTS, 4 - 16 mmol of K2CO3 are dissolved in 50 ml of acetone, then evacuated three times, heated to 120 °C and refluxed for 12 h, and the reaction is monitored by thin layer chromatography. After the reaction is completed, the reaction solution is rotary evaporated under reduced pressure to remove the solvent, dried, separated by silica gel column chromatography, and eluted with dichloromethane / petroleum ether with a volume ratio of 8:1 to obtain a bright yellow solid product.

[0016] (3) Synthesis of thionaphthalimide compound TNPS

[0017] Under a N2 atmosphere, 3 mmol of compound TNP and 3 - 45 mmol of Lawesson's reagent are dissolved in 15 ml of toluene, heated to 110 °C and refluxed for 36 h, and the reaction is monitored by thin layer chromatography. After the reaction is completed, the reaction solution is rotary evaporated under reduced pressure to remove the solvent, dried, separated by silica gel column chromatography, and eluted with dichloromethane / petroleum ether with a volume ratio of 5:1 to obtain a dark purple solid product.

[0018] Further, the active hydrogen amine compound in step (1) is one of N,N - dimethyl and triphenylamine; the water - soluble group is a PEG chain.

[0019] The preparation of the nanoparticles of the water - soluble thionaphthalimide photosensitizer according to the present invention is characterized in that the compound will spontaneously assemble to form nanoparticles with a particle size of 147 nm in water solubility.

[0020] Further, the application of the nanoparticles is used as a photodynamic reagent.

[0021] Furthermore, the application of the nanoparticles is used as a photosensitizing reagent for killing cancer cells.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The photosensitizing reagent designed and synthesized in the present invention has the advantages of easily available raw materials, simple synthesis, and easy separation.

[0024] (2) The photosensitizing reagent designed and synthesized in the present invention has good water solubility, and has good photo, thermal, and chemical stabilities. The ultraviolet-visible light absorption tail peak can reach the near-infrared region, with high reactive oxygen species efficiency, and has excellent photodynamic killing effects on tumor cells, and can achieve efficient photodynamic therapy for cancer cells.

[0025] (3) The photosensitizing reagent designed and synthesized in the present invention is obtained by thioation with Lawesson's reagent. While the absorption is redshifted, it can further promote intersystem crossing (ISC) and improve the yield of reactive oxygen species.

[0026] (4) The photosensitizer designed and synthesized in the present invention self-assembles into spherical nanoparticles with a size of about 147 nm in aqueous solution, and the size distribution is uniform and stable. After functional modification, it has specific recognition ability and has broad prospects in the field of biological applications. Description of the Drawings

[0027] Figure 1 1H NMR spectrum (CDCl3) of Compound 2;

[0028] Figure 2 1H NMR spectrum (CDCl3) of Compound TNP;

[0029] Figure 3 1H NMR spectrum (CDCl3) of Compound TNPS;

[0030] Figure 4 Visible light-ultraviolet absorption spectrum in Example 4;

[0031] Figure 5 Fluorescence emission in Example 4;

[0032] Figure 6 Test chart of in vitro reactive oxygen species generation ability in Example 5;

[0033] Figure 7 Test chart of reactive oxygen species generation ability measured in cancer cells in Example 6. Detailed Description of the Invention

[0034] Example 1

[0035] Preparation method of a water-soluble thionaphthalimide photosensitizer, comprising the following steps:

[0036] (1) Synthesis of Compound 2

[0037] Under a nitrogen atmosphere, compound 1 (0.3 g, 0.82 mmol) and dimethylamine (0.11 g, 2.46 mmol) were added to a two-necked flask. Subsequently, 30 ml of anhydrous 2-methoxyethanol was added and stirred well to dissolve. The mixture was heated to 120 °C and refluxed for 24 h, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the reaction solution was cooled to room temperature to obtain yellow needle-like crystals, which were then filtered and washed with n-hexane to obtain a bright yellow solid product, compound 2 (0.12 g, yield 43%). 1 H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.6 Hz, 1H), 8.68–8.66 (m, 1H), 8.27 (dd, J = 8.5, 1.2 Hz, 1H), 7.35 (dd, J = 8.9, 7.3 Hz, 2H), 7.53–7.49 (m, 2H), 7.33 (dd, J = 8.5, 7.2 Hz, 2H), 3.16–3.12 (m, 6H).

[0038] (2) Synthesis of Compound 3

[0039] Under a nitrogen atmosphere, compound 2 (0.1 g, 0.3 mmol), PEG 208 -OTS (0.16 g, 0.45 mmol), and K2CO3 (0.17 g, 1.2 mmol) were successively added to a two-necked flask. Subsequently, 15 ml of acetone was added and stirred well to dissolve. The mixture was heated to 60 °C and refluxed for 24 h, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the reaction solution cooled to room temperature was poured into cold water and extracted with dichloromethane. The combined organic layers were dried over anhydrous MgSO4 and concentrated using a rotary evaporator to obtain a crude product. After drying, it was separated by silica gel column chromatography and eluted with dichloromethane / methanol in a volume ratio of 8:1 to obtain a bright yellow solid product 3 (0.10 g, yield 62%).

[0040] (3) Synthesis of Compound 4

[0041] Under a nitrogen atmosphere, compound 3 (0.1 g, 0.2 mmol) and Lawesson's reagent (0.24 g, 0.6 mmol) were added to a two-necked flask. 15 ml of toluene was added and stirred well to dissolve. The mixture was heated to 120 °C and refluxed for 36 h, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the reaction solution cooled to room temperature was poured into cold water and extracted with dichloromethane. The combined organic layers were dried over anhydrous MgSO4 and concentrated using a rotary evaporator to obtain a crude product. After drying, it was separated by silica gel column chromatography and eluted with dichloromethane / petroleum ether in a volume ratio of 5:1 to obtain a black-purple solid product 4 (0.027 g, yield 24%).

[0042]

[0043] Example 2

[0044] A preparation method of a water-soluble thionaphthalimide photosensitizer, comprising the following steps:

[0045] (1) Synthesis of Compound 2

[0046] Under a N2 atmosphere, compound 1 (0.56 g, 1.53 mmol), 4-(diphenylaminophenyl)boronic acid pinacol ester (0.53 g, 1.84 mmol), K2CO3 (0.84 g, 6.12 mmol), and Pd(pp3)4 (0.012 g, 0.091 mmol) were successively added to a three-necked flask, and then 30 ml of anhydrous THF was added and stirred well to dissolve. The mixture was heated to 110 °C and refluxed for 24 h, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the reaction solution cooled to room temperature was poured into cold water and extracted with dichloromethane. The combined organic layers were dried over anhydrous MgSO4 and concentrated using a rotary evaporator to obtain a crude product. After drying, it was separated by silica gel column chromatography and eluted with dichloromethane / petroleum ether with a volume ratio of 6:1 to obtain a pale yellow solid product - Compound 2 (0.66 g, yield 81%).

[0047] The 1H NMR spectrum of Compound 2 is as Figure 1 shown 1 H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.6 Hz, 1H), 8.68–8.66 (m, 1H), 8.47 (dd, J = 8.5, 1.2 Hz, 1H), 7.76 (dd, J = 8.9, 7.3 Hz, 2H), 7.42–7.37 (m, 2H), 7.33 (dd, J = 8.5, 7.2 Hz, 4H), 7.24–7.19 (m, 6H), 7.18–7.07 (m, 5H), 6.93–6.89 (m, 2H).

[0048] (2) Synthesis of Compound TNP

[0049] Under a N2 atmosphere, compound 2 (0.3 g, 0.56 mmol), PEG 208-OTS (0.2 g, 0.56 mmol) and K2CO3 (0.39 g, 2.8 mmol) were successively added to a three-necked flask, and then 15 ml of acetone was added and stirred well to dissolve. The mixture was heated to 60 °C and refluxed for 24 h, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the reaction solution cooled to room temperature was poured into cold water and extracted with dichloromethane. The combined organic layers were dried over anhydrous MgSO4 and concentrated using a rotary evaporator to obtain a crude product. After drying, it was separated by silica gel column chromatography and eluted with dichloromethane / petroleum ether with a volume ratio of 6:1 to obtain a bright yellow solid product TNP (0.32 g, yield 79%).

[0050] The 1H NMR spectrum of TNP is as Figure 2 shown as 1 H NMR (400 MHz, CDCl3) δ 8.68–8.63 (m, 2H), 8.45 (dd, J = 8.3, 1.2 Hz, 1H), 7.81–7.71 (m, 3H), 7.42–7.36 (m, 2H), 7.35–7.29 (m, 4H), 7.24–7.18 (m, 7H), 7.12–7.04 (m, 4H), 4.20 (t, J = 4.9 Hz, 2H), 3.92–3.86 (m, 2H), 3.72–3.59 (m, 12H), 3.38–3.36 (m, 3H).

[0051] (3) Synthesis of compound TNPS

[0052] Under a N2 atmosphere, compound 3 (0.3 g, 0.42 mmol) and Lawesson's reagent (0.85 g, 2.1 mmol) were added to a three-necked flask, and 15 ml of toluene was added and stirred well to dissolve. The mixture was heated to 120 °C and refluxed for 36 h, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the reaction solution cooled to room temperature was poured into cold water and extracted with dichloromethane. The combined organic layers were dried over anhydrous MgSO4 and concentrated using a rotary evaporator to obtain a crude product. After drying, it was separated by silica gel column chromatography and eluted with dichloromethane / petroleum ether with a volume ratio of 6:1 to obtain a dark purple solid product TNPS (0.11 g, yield 36%).

[0053] The 1H NMR spectrum of TNPS is as Figure 3 shown as

[0054] The reaction formula of this example is as follows:

[0055]

[0056] Example 3

[0057] 1. Tests of ultraviolet-visible absorption spectrum and fluorescence emission spectrum

[0058] First, the photophysical properties of compounds TNP and TNPS prepared in Example 1 before and after thionation were investigated by ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy. Compounds TNP and TNPS were prepared into a stock solution of 2×10 -3 mol / L using THF as the solvent for later use. During the test, 10 μL of the stock solution was pipetted into a cuvette, and then 1990 μL of toluene, acetonitrile, or dimethyl sulfoxide was added. After mixing evenly, the ultraviolet-visible absorption spectrum and fluorescence emission spectrum were measured simultaneously.

[0059] It can be seen from Figure 4 that the maximum absorption peak of compound TNP in toluene is at 415 nm, and the maximum absorption peak of compound TNPS after carbonyl thionation in toluene is at 535 nm. With the increase in solvent polarity, the absorption peaks of both show a slight red shift. The thionation of the carbonyl in TNPS results in a 120-nm red shift in the maximum absorption.

[0060] It can be seen from Figure 5 that compared with the strong fluorescence of compound TNP at 580 nm, compound TNPS after carbonyl thionation has almost no fluorescence. Fluorescence quenching indicates that carbonyl thionation enhances the intersystem crossing (ISC) process from the singlet excited state to the triplet excited state, which is beneficial to the generation of reactive oxygen species.

[0061] 2. Test of the ability to generate reactive oxygen species in vitro

[0062] Under white light irradiation, the ability of TPNS to generate reactive oxygen species in vitro was evaluated and compared with the classical commercial photosensitizer methylene blue (MB). The reactive oxygen species indicator 2`,7`-dichlorodihydrofluorescein (DCFH) was used to detect the generation of reactive oxygen species by photosensitizers TNPS and methylene blue (MB) under white light. The test sample prepared into 1×10 -5 mol / L was placed in a container with a magnetic stir bar, and 1 equivalent of DCFH was added to the above mixed solution. The fluorescence emission of the solution was immediately measured. DCFH can be oxidized by reactive oxygen species to 2`,7`-dichlorofluorescein with strong green fluorescence emission (Ex / Em = 504 / 529 nm). Irradiation was carried out with white light of a specific power at 10-s intervals, and the fluorescence emission intensity of the solution was immediately measured after each irradiation until the fluorescence emission intensity remained unchanged.

[0063] It can be seen from Figure 6 that under the irradiation of white light with a specific power, the fluorescence emission intensity of photosensitizer TNPS increased significantly within 120 s and was 117 times the initial fluorescence emission intensity. Under the same conditions, the ability of photosensitizer ROS to generate was 3.2 times that of the classical commercial photosensitizer methylene blue (MB). This indicates that photosensitizer TNPS has excellent ROS generation ability and can be a potential alternative.

[0064] 3. Measurement of the ability of cancer cells to produce reactive oxygen species

[0065] In the present invention, the ability of HEPG-2 cancer cells to produce reactive oxygen species is measured. First, the cancer cells are inoculated in a 96-well plate with a cell density of 5000 cells and incubated for 24 h. After incubation with 10 μM of the thionaphthalimide photosensitizer prepared in Example 1 for 1.5 h, the cells are washed three times with PBS. After adding 10 μM of DCFH and continuing to incubate for 30 minutes, the cells are carefully washed two to three times again with PBS buffer, and 100 μL of PBS buffer is added. After irradiating the cells with white light (25 mW / cm 2 ) for 10 min, fluorescence confocal imaging is performed. For the fluorescence channel excitation wavelength of the indicator DCFH: 502 nm, and the receiving emission band: 515 - 540 nm.

[0066] The HEPG-2 cells are co-incubated with the photosensitizer TNPS for 1.5 hours. After white light irradiation at different times, the cells are washed three times, and the cells are co-stained with calcein and propidium iodide (PI) and incubated for 20 min. Among them, calcein stains live cells (green fluorescence), and propidium iodide (PI) stains dead cells (red fluorescence). As Figure 7 shown, green fluorescence is presented without light irradiation, indicating that singlet oxygen is not generated. Under light irradiation conditions, as the light irradiation time prolongs, the number of dead cells (red fluorescence) increases. When the light irradiation time reaches 10 min, almost all the cells are killed. This result shows that the photosensitizer TNPS indeed has a high PDT efficacy.

Claims

1. Application of a water-soluble thionaphthalimide compound, characterized in that: The water-soluble thionaphthalimide compound is used for preparing a drug with PDT efficacy, and the structure of the water-soluble thionaphthalimide compound is as follows: 。 2. Use of the water-soluble thionaphthalimide compound according to claim 1, characterized in that: The thionaphthalimide compound will spontaneously assemble into nanoparticles with a particle size of 147 nm in water solubility.

3. Use of the water-soluble thionaphthalimide compound according to claim 2, characterized in that: The nanoparticles are used for preparing a photodynamic reagent.

4. Use of the water-soluble thionaphthalimide compound according to claim 2, characterized in that: The nanoparticles are used for preparing a photosensitive reagent for killing cancer cells.

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