Photosensitizer for type I photodynamic therapy and preparation method thereof

Abstract

The invention discloses a photosensitizer for type I photodynamic therapy and a preparation method thereof, and belongs to the field of photosensitizers. The photosensitizer is prepared from phenothiazine, n-butyl bromide, phosphorus oxychloride, N-bromosuccinimide, 5-formyl-2-thiopheneboronic acid, 1, 4-dimethyl pyridine-1-onium iodide and potassium hexafluorophosphate through a five-step reaction, and type I active oxygen can be effectively generated in a small molecule state. The photosensitizer disclosed by the invention has a great prospect in the field of type I photodynamic therapy.

Description

Technical Field

[0001] The present invention relates to the field of photosensitizers, and in particular to a photosensitizer for type I photodynamic therapy and a preparation method thereof. Background Art

[0002] Photodynamic therapy (PDT) is a non-invasive cancer treatment method that uses photosensitizers (PS) and light of a specific wavelength to generate reactive oxygen species (ROS), which lead to cell death through apoptosis or necrosis. PDT has been shown to be effective against various types of cancer. Compared with traditional cancer treatments, it has the advantages of high specificity, non-invasiveness, low toxicity and repeatability. With the help of specifically distributed photosensitizers, the tumor is locally illuminated by light of a specific wavelength to activate the photosensitizer (PS). The excited PS transfers its energy to molecular oxygen, thereby generating cytotoxic ROS. After PS absorbs photons, it changes from the ground state to the singlet excited state, and may then return to the ground state or enter the triplet state with slightly lower energy through intersystem crossing. At this time, on the one hand, free radical reactive oxygen species such as O2 can be generated through the electron transfer process. ·- , •OH, etc. We call this type of photodynamics. On the other hand, the energy generated can also be directly transferred to the molecular oxygen in the surrounding environment to form 1 O2, which is called type II photodynamic therapy. However, the efficacy of PDT depends largely on the PS used, which has triggered the research on new PS. In the past few decades, many PS have been developed, striving to have minimal dark toxicity while significantly increasing the yield of reactive oxygen species, and also hoping to have organelle targeting capabilities and properties such as red light or near-infrared (NIR) absorption. However, due to weak photon capture ability and / or short absorption wavelength, many PS are still poor in inhibiting solid tumors or preventing tumor regeneration. In the above two types of photodynamic therapy, compared with the photosensitizers used in type II photodynamic therapy, type I photosensitizers can effectively utilize limited oxygen in hypoxic tumors to produce reactive oxygen species, thereby causing great damage to biological molecules and achieving the purpose of treating tumors. Therefore, the development of type I photosensitizers is very necessary in the treatment of most solid tumors. Summary of the invention

[0003] The purpose of the present invention is to solve the above problems in the prior art and to provide a photosensitizer for type I photodynamic therapy and a preparation method thereof.

[0004] In order to achieve the above object, the present invention adopts the following technical scheme:

[0005] A photosensitizer NF for type I photodynamic therapy, the chemical structure of which is as follows:

[0006]

[0007] The synthetic route of the photosensitizer NF is as follows:

[0008]

[0009] The specific steps include:

[0010] 1) Synthesis of intermediate 1: Phenothiazine and n-butyl bromide were dissolved in dimethyl sulfoxide, potassium hydroxide was added, and after the reaction was completed, the pH was adjusted, and the product was extracted with an organic solvent, and then eluted and purified by column chromatography to obtain intermediate 1;

[0011] 2) Synthesis of intermediate 2: phosphorus oxychloride is slowly added dropwise to N,N-dimethylformamide under an ice bath, intermediate 1 prepared in step 1) is dissolved in dichloromethane and slowly added dropwise to the above solution, heated to react, after the reaction is completed, poured into ice water, the pH is adjusted, extracted with an organic solvent, then eluted, and purified by column chromatography to obtain intermediate 2;

[0012] 3) Synthesis of intermediate 3: intermediate 2 was dissolved in chloroform solution, N-bromosuccinimide was added, and after the reaction was completed, an organic solvent was used for extraction, followed by elution and purification by column chromatography to obtain intermediate 3;

[0013] 4) Synthesis of intermediate 4: intermediate 3 and 5-formyl-2-thiopheneboronic acid were dissolved in tetrahydrofuran solution, [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride and potassium carbonate were added, and the solution was heated under nitrogen protection for reaction. After the reaction was completed and the solution was cooled to room temperature, it was poured into saturated brine, extracted with an organic solvent, and then eluted and purified by column chromatography to obtain intermediate compound 4;

[0014] 5) The intermediate 4 prepared in step 4) and 1,4-dimethylpyridinium-1-ium iodide were dissolved in an ethanol solution, piperidine was added, and the reaction was heated. After the reaction was completed and the solution was cooled to room temperature, a solid was precipitated, which was filtered to obtain a solid, washed with ethanol, and then purified by column chromatography. The solid was then dissolved in a methanol solution, a saturated potassium hexafluorophosphate solution was added, and stirred. After the reaction was completed, the solid was filtered and washed to obtain a compound NF.

[0015] The solution photophysical properties of the photosensitizer NF of the present invention are as follows: in a dimethyl sulfoxide solution, the ultraviolet absorption wavelength of NF is 487 nm, and the fluorescence emission wavelength is 635 nm.

[0016] Evaluation of the performance of the photosensitizer of the present invention in generating active oxygen:

[0017] (1) Producing O2 ·-Ability: After adding the active oxygen detection reagent DHR123 to the PBS solution of NF, the solution was irradiated with a light source for different time periods to detect the fluorescence changes of the solution. The results showed that in the PBS solution containing NF, the fluorescence emission intensity of the active oxygen detection reagent DHR123 continued to increase as the irradiation time continued to increase. This shows that NF can effectively produce type I active oxygen O2 ·- .

[0018] (2) Ability to generate •OH: After adding the active oxygen detection reagent HPF to the PBS solution of NF, the solution was irradiated with a light source for different time periods and the fluorescence changes of the solution were detected. The results showed that in the PBS solution containing NF, the fluorescence emission intensity of the active oxygen detection reagent HPF continued to increase as the irradiation time continued to increase. This shows that NF can effectively generate type I active oxygen •OH.

[0019] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0020] (1) The photosensitizer of the present invention has good ultraviolet absorption and fluorescence emission properties and has good applicability;

[0021] (2) The photosensitizer of the present invention can effectively generate type I reactive oxygen species O2 ·- and •OH, which has great potential for application in type I photodynamic therapy;

[0022] (3) The photosensitizer of the present invention is 2 ) produces O2 ·- It is 1.29 times that of the reference CV, and the •OH produced is 49.08 times that of the reference CV.

[0023] (4) The photosensitizer synthesis route of the present invention is simple and low-cost, which is conducive to practical application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the UV absorption spectrum of NF;

[0025] Figure 2 is the fluorescence emission spectrum of NF;

[0026] Figure 3 The fluorescence intensity changes of DHR123 in the presence of NF under different irradiation time of light source;

[0027] Figure 4 O2 production by NF and CV under different light irradiation time ·- rate;

[0028] Figure 5 The fluorescence intensity changes of HPF under different irradiation time of light source in the presence of NF;

[0029] Figure 6 is the rate at which NF and CV produce •OH under different light source irradiation times. DETAILED DESCRIPTION

[0030] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0031] Example 1

[0032] The present invention discloses a photosensitizer NF for type I photodynamic therapy, wherein the specific synthesis steps of the photosensitizer are as follows:

[0033] Synthesis of intermediate 1: Add 5 g of phenothiazine, 5.15 g of n-butyl bromide, 4.22 g of potassium hydroxide and 30 mL of dimethyl sulfoxide to a 100 mL round-bottom flask and stir for 6 h. Then adjust the pH to neutral with 37% HCl. Extract with dichloromethane and wash with water. Dry the organic phase with anhydrous sodium sulfate. After filtration, the crude product was obtained by vacuum distillation. The crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 80:1). 4.87 g of light green oil was obtained with a yield of 76%. 1 H NMR (500MHz, CDCl3) δ 7.19-7.15 (m, 4H), 6.95-6.89 (m, 4H), 3.87 (s, 2H), 1.84-1.78(m, 2H), 1.51-1.44 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H).

[0034] Synthesis of intermediate 2: 5 mL of phosphorus oxychloride was slowly added dropwise to 7 mL of N,N-dimethylformamide at 0°C. After stirring for 30 min, 4.6 g of intermediate 1 was dissolved in 15 mL of dichloromethane and slowly added dropwise to a round-bottom flask under an ice-water bath. The resulting mixture was heated to reflux for 12 h and then poured into ice water. Sodium bicarbonate was added to adjust the pH value. Extracted with dichloromethane and washed with water. The organic phase was dried over anhydrous sodium sulfate. After filtration, the crude product was obtained by vacuum distillation. The crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 20:1). 4.1 g of yellow solid was obtained with a yield of 80%. 1H NMR (500 MHz, CDCl3) δ 9.79 (s, 1H), 7.64 (dd, J = 8.4, 1.9 Hz, 1H), 7.59 (d, J = 1.9 Hz,1H), 7.19 - 7.10 (m, 2H), 6.97 (t, J = 7.3 Hz, 1H), 6.90 (t, J = 9.1 Hz, 2H), 3.90 (t, J = 7.2 Hz, 2H), 1.84 - 1.77 (m, 2H), 1.46 (m, 2H), 0.95 (t, J = 7.4Hz, 3H).

[0035] Synthesis of intermediate 3: Dissolve 1.5 g of intermediate 2 in 10 mL of chloroform. Add 1.41 g of N-bromosuccinimide to a round-bottom flask. Stir the mixture in the dark at room temperature for 12 h. Extract with dichloromethane and wash with water. Dry the organic phase over anhydrous sodium sulfate. After filtration, obtain a crude product by vacuum distillation. The crude product is separated and purified by column chromatography (petroleum ether: ethyl acetate = 15:1). 1.49 g of light yellow solid, yield 77%. 1 H NMR (500 MHz, CDCl3) δ 9.80 (s, 1H), 7.65 (dd, J = 8.4, 1.9 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.25 - 7.22 (m,2H), 6.91 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 8.6 Hz, 1H), 3.88 - 3.84 (m, 2H), 1.80 -1.74 (m, 2H), 1.50 - 1.42 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H).

[0036] Synthesis of intermediate 4: Add 0.2 g of intermediate 3, 0.13 g of 5-formyl-2-thiopheneboronic acid, 15 mL of tetrahydrofuran, 22 mg of [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride and 0.38 g of potassium carbonate to a 100 mL two-necked flask. Heat to 80 °C under nitrogen protection and react for 10 h. After the reaction, cool to room temperature, pour into saturated brine, extract with dichloromethane, and wash with water. The organic phase is dried over anhydrous sodium sulfate. After filtration, the crude product is obtained by vacuum distillation. The crude product is separated and purified by column chromatography (petroleum ether: ethyl acetate = 8:1). 0.17 mg of orange-red solid is obtained with a yield of 79%. 1H NMR (500MHz, CDCl3) δ 9.87 (s, 1H), 9.81 (s, 1H), 7.71 (d, J = 3.9 Hz, 1H), 7.66 (dd,J = 8.4, 1.8 Hz, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.45 (dd, J = 8.5, 1.9 Hz,1H), 7.39 (d, J = 2.1 Hz, 1H), 7.30 (d, J = 3.9 Hz, 1H), 6.91 (dd, J = 17.7,8.5 Hz, 2H), 3.92 (s, 2H), 1.84 - 1.78 (m, 2H), 1.52 - 1.45 (m, 2H), 0.97 (t,J = 7.4 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 191.10, 184.35, 151.94, 149.61,144.40, 141.92, 139.82, 131.68, 130.77, 128.38, 128.33, 126.55, 125.25,125.03, 124.03, 123.28, 117.40, 116.34, 47.35, 28.61, 19.73, 14.05. HR-MS(m / s): Calculated (2[M]+Na) + : 809.1607, found: 809.1603.

[0037] Synthesis of NF: 100 mg of intermediate 4, 149 mg of 1,4-dimethylpyridin-1-ium iodide, 50 μL of piperidine and 20 mL of ethanol were added to a 50 mL round-bottom flask and heated to 80 °C for 12 h. The solid was filtered and washed with ethanol. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 30:1). The black solid was dissolved in 5 mL of methanol, saturated potassium hexafluorophosphate solution was added, stirred for 4 h, filtered, washed, and dried in vacuo. 149 mg of black solid was obtained with a yield of 68%. 1H NMR(500 MHz, DMSO-d6) δ 8.85 – 8.79 (m, 4H), 8.24 – 8.11 (m, 6H), 7.90 (d, J =16.3 Hz, 1H), 7.62 – 7.53 (m, 5H), 7.51 (d, J = 4.0 Hz, 1H), 7.40 (d, J =16.1 Hz, 1H), 7.22 (d, J = 16.0 Hz, 1H), 7.18 – 7.12 (m, 2H), 4.25 – 4.21 (m,6H), 3.97 (t, J = 7.2 Hz, 2H), 1.71 (m, 2H), 1.44 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 152.62, 146.40, 146.13, 145.46, 145.40,145.34, 144.17, 141.01, 139.89, 139.56, 134.14, 134.00, 133.77, 133.53,129.34, 128.34, 127.29, 126.57, 125.89, 125.25, 124.36, 123.86, 123.72,123.58, 123.49, 123.13, 121.97, 121.87, 117.07, 116.59, 47.38, 47.29, 47.13,28.70, 19.79, 14.10. HR-MS(m / s): Calculated [M] 2+ :286.6131, found: 286.6139.

[0038] Example 2 Photophysical properties of photosensitizers

[0039] Solution UV absorption spectrum test: Use a 1 / 10,000 balance to accurately weigh 4.3 mg NF, dissolve it in 5 mL dimethyl sulfoxide, and make a mother solution with a concentration of 1 mM. Use a pipette to accurately take 40 µL of the mother solution, add 1960 µL dimethyl sulfoxide, and dilute it to a test solution with a concentration of 20 µM. Pour the solution into a cuvette and use a UV spectrophotometer to test the UV absorption spectrum. The UV absorption wavelength is 487 nm, and the scanning range is 300~700 nm.

[0040] Solution fluorescence spectrum test: 4.3 mg NF was accurately weighed with a 10,000 molecular weight balance and dissolved in 5 mL dimethyl sulfoxide to prepare a 1 mM stock solution. 40 µL of the stock solution was accurately taken with a pipette and added to 1960 µL dimethyl sulfoxide to dilute to a test solution with a concentration of 20 µM. The solution was poured into a cuvette and the spectrum was tested using a fluorescence photometer with an excitation wavelength of 487 nm and a scanning range of 500-900 nm.

[0041] like Figure 1 and Figure 2 The ultraviolet absorption wavelength and fluorescence emission wavelength of NF are 487 nm and 635 nm, respectively.

[0042] Example 3 Evaluation of the ability of photosensitizers to generate type I reactive oxygen species

[0043] Photosensitizer produces O2 ·- Test: Use PBS as solvent to prepare a mixed solution of NF and DHR123, where the concentrations of NF and DHR123 are both 10 µM. Pour the test solution into a fluorescent cuvette, use a light source to illuminate the solution in the cuvette from above, and then use a fluorescence photometer to test the spectrum, where the excitation wavelength of DHR123 is 488 nm, the emission wavelength is 520 nm, and the scanning range is 500~700 nm. Figure 3 As shown in Figure 2, with the increase of solution irradiation time, the fluorescence emission intensity of DHR123 increased significantly, indicating that NF can effectively generate O2 ·- .

[0044] Positive reference CV produces O2 ·- Test: Use PBS as solvent to prepare a mixed solution of CV and DHR123, where the concentrations of CV and DHR123 are both 10 µM. Pour the test solution into a fluorescent cuvette, use a light source to illuminate the solution in the cuvette from above, and then use a fluorescence photometer to test the spectrum. Figure 4 As shown, O2 of NF and CV ·- The photosensitizer NF of the present invention is generated at a rate of 1:1 under white light (50 mw / cm 2 ) produces O2 ·- It is 1.29 times the reference CV.

[0045] Test of •OH produced by photosensitizer: Use PBS as solvent to prepare a mixed solution of NF and HPF, where the concentrations of NF and HPF are both 10 µM. Pour the test solution into a fluorescent cuvette, use a light source to illuminate the solution in the cuvette from above, and then use a fluorescence photometer to test the spectrum, where the excitation wavelength of HPF is 490 nm, the emission wavelength is 515 nm, and the scanning range is 500~700 nm. Figure 5 As shown in the figure, with the increasing irradiation time of the solution, the fluorescence emission intensity of HPF increased significantly, indicating that NF can effectively produce •OH.

[0046] Test of •OH produced by positive reference CV: Use PBS as solvent to prepare a mixed solution of CV and HPF, where the concentrations of CV and HPF are both 10 µM. Pour the test solution into a fluorescent cuvette, use a light source to illuminate the solution in the cuvette from above, and then use a fluorescence photometer to test the spectrum. Figure 6 The photosensitizer of the present invention generates 49.08 times more •OH in NF than in CV.

[0047] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A photosensitizer for type I photodynamic therapy, characterized in that: The chemical structural formula of the photosensitizer is as follows: , the photosensitizer is referred to as NF.

2. A photosensitizer for type I photodynamic therapy according to claim 1, characterized in that: The NF has ultraviolet absorbing properties.

3. A photosensitizer for type I photodynamic therapy according to claim 2, characterized in that: The ultraviolet absorption wavelength of the NF is 487 nm.

4. A photosensitizer for type I photodynamic therapy according to claim 1, characterized in that: The NF has the property of fluorescence emission.

5. A photosensitizer for type I photodynamic therapy according to claim 4, characterized in that: The fluorescence emission wavelength of the NF is 635 nm.

6. A photosensitizer for type I photodynamic therapy according to claim 1, characterized in that: The NF can generate type I reactive oxygen species O2 ·- .

7. A photosensitizer for type I photodynamic therapy according to claim 1, characterized in that: The NF can generate type I reactive oxygen species •OH.

8. The method for preparing a photosensitizer for type I photodynamic therapy according to any one of claims 1 to 7, characterized in that: The synthetic route of the NF is as follows:

Images