Novel photosensitizer as well as preparation method and application thereof

[0044] Example 1

[0045]

[0046] (1) Synthesis of Compound I

[0047]D-biotin (300 mg, 1.23 mmol) of 10 ml of methanol suspension was added to sulfoxide (0.3 mL, 4.0 mmol), stirred at room temperature overnight until the reaction liquid clarified and transparent. The white solid was obtained under reduced pressure. It was dispersed into 10 mL of methanol and slowly dripped the hydrated sac (0.48 mL, 10 mmol) while stirring, heated to 70 ° C, and the reaction was 12 h. Cooled to room temperature, distilled under reduced pressure, poured into a large amount of ice-cold methanol, filtered, washed three times with methanol to give a white powder-shaped compound I.

[0048] (2) Compound IIIA synthesis

[0049] In a nitrogen atmosphere, Compound IIA (DCF-TFM, 20.4 mg, 0.025 mmol) was dissolved in 5 mL anhydrous DMF, 0 ° C, add 1- (3-dimethylaminopropyl) -3-ethyl carbon two Asia Amine hydrochloride (EDCI) (24 mg, 0.125 mmol), 1-hydroxybenzene triazole (HOBT) (17 mg, 0.125 mmol) and N, N-diisopropylidelamine (DIEA) (12.4 μL, 0.075 mmol After 2 h, Compound I (13 mg, 0.05 mmol) was added, and stirred at room temperature for 24 h. Decompression distillation was purified by column chromatography (a system = 1/8 of methanol / dichloromethane). The high distortion of the compound IIIA and the nuclear magnetic resonance spectrum pattern are shown in respectively. figure 1 and figure 2 Indicated.

[0050] Example 2

[0051]

[0052] (1) Synthesis of Compound I as in Example 1.

[0053] (2) Synthesis of Compound IIIB

[0054] In a nitrogen atmosphere, compound IIB (Fl, 50 mg, 0.065 mmol) was dissolved in 5 ml of anhydrous DMF, and 1- (3-dimethylaminopropyl) -3-ethyl carbide hydrochloric acid was added at 0 ° C. Salt (EDCI) (62.8 mg, 0.33 mmol), 1-hydroxybenzene triazole (HOBT) (44 mg, 0.33 mmol) and N, N-diisopropylidelamine (DIEA) (32 μL, 0.2 mmol) reaction 2H After the addition, Compound I (33.5 mg, 0.13 mmol) was added, and stirred at room temperature for 24 h. Decompression distillation was purified by column chromatography (methanol / dichloromethane = 1/8) to obtain a photosensitizer compound IIIb. The high distortion of the compound IIIb and the nuclear magnetic resonance spectrometry table show respectively image 3 and Figure 4 Indicated.

[0055] Example 3

[0056]

[0057] (1) Synthesis of Compound I as in Example 1.

[0058] (2) Synthesis of Compound IIIC

[0059] In a nitrogen atmosphere, Compound IIC (PPIX, 30 mg, 0.053 mmol) was dissolved in 3 ml anhydrous DMF, and 1- (3-dimethylaminopropyl) -3-ethyl carbide hydrochloric acid was added at 0 ° C. Salt (EDCI) (10.56 mg, 0.053 mmol), 1-hydroxybenzene triazole (HOBT) (7.43 mg, 0.053 mmol) and N, N-diisopropylamine (DIEA) (8.75 μL, 0.053 mmol) After 2 h, Compound I (15.3 mg, 0.05 mmol) was added, and stirred at room temperature for 24 h. Decapression was purified, and the photosensitizer compound was obtained after purification using column chromatography (methanol / dichloromethane = 1/8). The high distortion mass spectrometry of Compound IIIC and the nuclear magnetic resonance spectrum pattern are shown. Figure 5 and Image 6 Indicated.

[0060] Figure 7 For the absorption of photosensitizer compound IIIA and compound IIA in ethanol, Figure 8 For the absorption of photosensitizer compound IIIb and compound IIB in ethanol, Figure 9 The absorption and emission spectrum of the photosensitizer compound III and the compound IIC in ethanol. Such as Figure 7-9 As shown, the absorption and emission spectrum of the photosensitizer compound II and the compound II are almost uniform, and the introduction of biotin does not affect the photophysical properties of the photosensitizer. In particular, compound IIIA has more widely absorbed in the range of 400-700 nm, making it an ideal photosensitizer to capture white light. The maximum emission of compound IIIa can reach 750 nm. At the same time, the introduction of biotin imparts the ability of the compound IIIA to target tumor cells. It can be seen that the compound IIIA has application prospects in the treatment of malignant tumors in near infrared biomatography and image guidance.

[0061] Figure 10 For different photosensitizers in ethanol 1 O 2 Generate rate graph. choose 1 O 2 Temperature 1,3-diphenylisine benofuran (DPBF) 1 O 2 Detection. Record 20MW / cm 2 White (400-800 nm) was irradiated under irradiation, the absorbance at 412 nm decreased over time. As can be seen from the figure, DPBF is almost unchanged under the respective action of compound III and compound II. Note The introduction of biotin does not affect the ability of the photosensitizer single-line oxygen.

[0062] Figure 11 For the photosensitizer compound IIIA, the compound IIa, and the mixture of compound IIA and compound I in the water O 2 -· Produce capacity graph, Figure 12 For the photosensitizer compound IIIb, the compound IIb, and the compound IIb and the mixture of Compound I in the water O 2 -· Produce capacity graph, Figure 13 For the photosensitizer compound IIIC, compound IIC, and compound IIC and compound I of Compound I in the water O 2 -· Generate capacity graph. Select O 2 -· Capture Chloride Nitroquic Tetrazolia (NBT) Perform O 2 -· Detection. Record 20MW / cm 2 Light (400-800 nm) was irradiated under irradiation, the absorbance at 260 nm decreases over time. Compound II in light O 2 -· The production is significantly less than compound III. The results after mixing with the compound II and the compound I is mixed with the individual compound II. 2 -· The ability has little variation, indicating that the active agent covalently modified by biotin is O 2 -· Production has a positive impact. In particular, Compound IIC is a well-known typical type PS compared to compound IIa, compound IIB, and does not have obvious O. 2 -·. Therefore, the compound IIIC's O 2 -· The generation capability should be attributed to the introduction of biotin. This unexpected biotinylation strategy shows that biotinylated photosensitors can have O 2 -· and 1 O 2 The ability to produce, alleviate the dependence of conventional PDT on oxygen.

[0063] Figure 14 and Figure 15 A common focal fluorescence imaging of two cells having different cells of photosensitizer compound IIIA and compound IIa on biotin expression levels, respectively. The results showed that compound III (10 μm) was incubated with biotin receptor negative (COS-7, African green monkey renal source cells) and biotin receptor positive (MCF-7, breast cancer cells) cells, respectively, in the former There is no fluorescence, and the latter has obvious fluorescence in the cell. Compound IIA (10 μm) was incubated with these two cells for 4 h, and fluorescence occurred in both cells. It can be seen that the introduction of biotin imparts the photosensitizer IIIA tumor target.

[0064] Figure 16 For the photosensitizer compound IIIA, in the case of normal oxygen (21%) and lack of oxygen (1%), the dark toxicity map of MCF-7 cells, Figure 17 For the photosensitizer compound IIIA in the case of normal oxygen (21%) and lack of oxygen (1%), the light toxicity of MCF-7 cells. 12W white light (400-800 nm) LED is used in the photodynamic experiment, the optical density is 20mW / cm 2 The illumination time is 10 min. The results showed that under dark conditions, compound IIIA had almost no toxicity to MCF-7, which showed a good biocompatibility of compound IIIA. In light conditions, MCF-7 cells have a significant inhibitory effect. In addition, the inhibition of Tumor cells can still reach more than 50% in the inhibition of tumor cells. This indicates that the compound IIIA's photocyteticity is mainly attributed to other Ros produced under low oxygen conditions. according to Figure 10 The result, other ROS should be O 2 -·. It can be seen that the compound IIIA can effectively relieve the high-degree dependence of photodynamic treatment on oxygen.

[0065] Figure 18 For normal oxygen (21%) and bare oxygen (1%), there is a light-free photo-sensitizer compound IIIA (A), compound IIC (B), compound IIIC (C) to induce an active oxygen in MCF-7 intracellular . Select 2 ', 7'-dichlorocetate (DCFH-DA) as an active oxygen indicator, can be oxidized by ROS to DCF, and green fluorescence is emitted. Under normal oxygen conditions, the fluorescence of DCF was observed, which means that the compound IIIA, compound IIC and compound III in MCF-7 cells can increase the inner ROS level. Even in an oxygen-deficient environment, the obvious fluorescence of DCF is still detected in MCF-7 cells, which highlights the ability of oxygen to produce active oxygen to compound IIIA and compound IIB, and there is no serious effect. In contrast, there is no oxygen substance to produce any active oxygen substance. This means that biochemical photosensitizers provide other ROS rather than just 1 O 2 To kill tumor cells.

[0066] Figure 19 Under normal oxygen (21%) and bare oxygen (1%), there is a light-sensitive photo-sensitive agent compound IIIA, compound IIC, compound IIIC induced MCF-7 intracellular O 2 -· Production situation. Choose dihydrogen ingot (DHE) as O 2 -· Detection reagents, its oxidation product can be inserted into DNA and red fluorescence. Whether it is normal oxygen or lack of oxygen, it can induce MCF-7 intracellular O, ever-borne oxygen. 2 -· The resulting, and typical secondary photosensitizer compound IIC is not available. Indication of biotin's covalent introduction can indeed induce O 2 -· Produce. And O 2 -· It can also be catalyzed by ultravalase (SOD) within the cells, and converted to other high cytotoxic free radicals (such as OH ·) through Haber-WeissS reactions and FENTON reactions. 2 Reuse. This involves O 2 -·The generated one mechanism will provide a more satisfactory efficacy for PDT.

[0067] Figure 20 Explore the mechanism of biotinylation effects, circulating voltammetry as an external standard with ferrocene (Fc). Such as Figure 20 As shown, all three of these three biotinylated photosensitizers exhibit lower reduction potentials than their correspondingly biotinylated photosensitizers. The anode movement of the photosensitizer reduction potential is advantageous to accept electrons, which makes them more likely to produce more O 2 -·.

[0068] The one-type PDT mechanism is as follows:

[0069] 3 Photosensitizer ＊ + Matrix → matrix -· + Photosensitizer -·

[0070] Photosensitizer -· + Oxygen → photosensitizer + O 2 -·

[0071] Such as Figure 21 The configuration shown, the triple state PS is transmitted to oxygen by electron with an electron with an electron with electrons to form O by electrons that receive adjacent substrates and transmitting external electrons to form O 2 -·. According to the results of the theoretical calculations, the folding conformation of Compound IIIA supports a more efficient electron transfer between biotin moieties and PS portions. Therefore, biotin acts as an electron-rich substrate in the manner in the molecule, which facilitates tilting the one-type PDT mechanism.