Photosensitizer for type Ⅰ photodynamic therapy and preparation method thereof

Abstract

The application relates to a photosensitizer for type I photodynamic therapy and a preparation method thereof, and belongs to the photosensitizer field. The photosensitizer is obtained through five-step reactions of phenothiazine, n-bromobutane, phosphorus oxychloride, N-bromosuccinimide, 5-aldehyde-2-thiophene boronic acid, 1,4-dimethylpyridine-1-iodonium and potassium hexafluorophosphate, and can effectively generate type I active oxygen in a small molecule state. The photosensitizer has great prospects in the field of type I photodynamic therapy.

Description

Technical Field

[0001] This invention relates to the field of photosensitizers, and more particularly to a photosensitizer for type I photodynamic therapy and its preparation method. Background Technology

[0002] Photodynamic therapy (PDT) is a non-invasive cancer treatment method that utilizes photosensitizers (PS) and light of specific wavelengths to generate reactive oxygen species (ROS), leading to cell death through apoptosis or necrosis. PDT has been proven effective against various types of cancer and offers advantages over traditional cancer treatments, including high specificity, non-invasiveness, low toxicity, and the ability to be repeated. Using specifically distributed photosensitizers, localized illumination of the tumor with light of specific wavelengths activates the PS. The excited PS then transfers its energy to molecular oxygen, generating cytotoxic ROS. After absorbing photons, the PS transitions from its ground state to a singlet excited state, and may then return to the ground state or enter a slightly lower-energy triplet state through intersystem crossing. At this point, free radical reactive oxygen species such as O2 can be generated through electron transfer processes. ·- ,•OH, etc., we call this type of photodynamic therapy Type I. On the other hand, the energy generated can also be directly transferred to molecular oxygen in the surrounding environment, forming... 1 O2, this is called type II photodynamic therapy. However, the efficacy of PDT largely depends on the photosensitizer used, which has led to research into novel photosensitizers. Over the past few decades, many photosensitizers have been developed, aiming to significantly increase the yield of reactive oxygen species while minimizing dark toxicity, and also hoping to possess organelle targeting capabilities and red or near-infrared (NIR) absorption properties. However, due to weak photon trapping ability and / or short absorption wavelengths, many photosensitizers remain ineffective in inhibiting solid tumors or preventing tumor regeneration. In the two types of photodynamic therapy mentioned above, compared to the photosensitizers used in type II photodynamic therapy, type I photosensitizers can effectively utilize limited oxygen in hypoxic tumors to generate reactive oxygen species, thereby causing significant damage to biomolecules and achieving the goal of treating tumors. Therefore, the development of type I photosensitizers is highly needed for the treatment of most solid tumors. Summary of the Invention

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

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A photosensitizer NF for type I photodynamic therapy has the following chemical structure:

[0006]

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

[0008]

[0009] Specifically, the following steps are included:

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

[0011] 2) Synthesis of intermediate 2: Phosphorus oxychloride was slowly added dropwise to N,N-dimethylformamide under ice bath conditions. Intermediate 1 prepared in step 1) was dissolved in dichloromethane and slowly added dropwise to the above solution. The reaction was heated. After the reaction was completed, it was poured into ice water, the pH was adjusted, and it was extracted with an organic solvent. Then it was 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, it was extracted with an organic solvent, then eluted, and purified by column chromatography to obtain intermediate 3;

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

[0014] 5) Dissolve intermediate 4 and 1,4-dimethylpyridine-1-onium iodide prepared in step 4) in ethanol solution, add piperidine, heat to react, and after the reaction is complete, allow the solution to cool to room temperature. A solid precipitates out, is filtered to obtain the solid, washed with ethanol, and then purified by column chromatography. It is then dissolved in methanol solution, saturated potassium hexafluorophosphate solution is added and stirred. After the reaction is complete, it is filtered and washed to obtain compound NF.

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

[0016] Evaluation of the reactive oxygen species generation performance of the photosensitizer of this invention:

[0017] (1) O2 is generated ·-Ability: After adding the reactive oxygen species (ROS) detection reagent DHR123 to a PBS solution containing NF, the solution was irradiated with a light source for different times, and the fluorescence changes were detected. The results showed that in the PBS solution containing NF, the fluorescence emission intensity of the ROS detection reagent DHR123 continuously increased with the extension of irradiation time. This indicates that NF can effectively generate type I reactive oxygen species (O2). ·- .

[0018] (2) Ability to generate •OH: After adding the reactive oxygen species (ROS) detection reagent HPF to the NF-containing PBS solution, the solution was irradiated with a light source for different times, and the fluorescence changes of the solution were detected. The results showed that in the NF-containing PBS solution, the fluorescence emission intensity of the HPF detection reagent continuously increased with the extension of irradiation time. This indicates that NF can effectively generate type I reactive oxygen species •OH.

[0019] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

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

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

[0022] (3) The photosensitizer of the present invention is effective under white light (50 mw / cm²). 2 O2 is generated under these conditions. ·- It is 1.29 times that of the reference CV, and the amount of •OH produced is 49.08 times that of the reference CV.

[0023] (4) The photosensitizer of the present invention has a simple synthesis route and low cost, which is conducive to practical application and promotion. Attached Figure Description

[0024] Figure 1 The ultraviolet absorption spectrum of NF;

[0025] Figure 2 The fluorescence emission spectrum of NF;

[0026] Figure 3 The fluorescence intensity of DHR123 under different exposure times in the presence of NF is shown.

[0027] Figure 4 O2 generation for NF and CV under different light source illumination times ·- The rate;

[0028] Figure 5 The change in fluorescence intensity of HPF under different exposure times in the presence of NF;

[0029] Figure 6 The rate at which •OH is generated by NF and CV under different light source irradiation times. Detailed Implementation

[0030] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0031] Example 1

[0032] This invention discloses a photosensitizer NF for type I photodynamic therapy. The specific synthesis steps of this photosensitizer are as follows:

[0033] Synthetic Intermediate 1: 5 g of phenothiazine, 5.15 g of n-butane bromide, 4.22 g of potassium hydroxide, and 30 mL of dimethyl sulfoxide were added to a 100 mL round-bottom flask and stirred for 6 h. The pH was then adjusted to neutral with 37% HCl. Extraction was performed 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 purified by column chromatography (petroleum ether:ethyl acetate = 80:1). 4.87 g of a 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] Synthetic 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 under reflux for 12 h and then poured into ice water. Sodium bicarbonate was added to adjust the pH. Extraction was performed 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 purified by column chromatography (petroleum ether:ethyl acetate = 20:1). 4.1 g of a 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] Synthetic intermediate 3: 1.5 g of intermediate 2 was dissolved in 10 mL of chloroform. 1.41 g of N-bromosuccinimide was added to a round-bottom flask. The mixture was stirred at room temperature in the dark for 12 h. 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 purified by column chromatography (petroleum ether:ethyl acetate = 15:1). 1.49 g of pale yellow solid, 77% yield. 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] Synthetic intermediate 4: 0.2 g of intermediate 3, 0.13 g of 5-aldehyde-2-thiopheneboronic acid, 15 mL of tetrahydrofuran, 22 mg of [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride, and 0.38 g of potassium carbonate were added to a 100 mL two-necked flask. The mixture was heated to 80 °C and reacted for 10 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, poured into saturated brine, 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 purified by column chromatography (petroleum ether:ethyl acetate = 8:1). 0.17 mg of an orange-red solid was 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-onium iodide, 50 μL of piperidine, and 20 mL of ethanol were added to a 50 mL round-bottom flask, and the mixture was heated to 80 °C and reacted for 12 h. The solid was obtained by filtration, washed with ethanol, and the crude product was purified by column chromatography (dichloromethane:methanol = 30:1). A black solid was obtained, which was then dissolved in 5 mL of methanol, and a saturated potassium hexafluorophosphate solution was added and stirred for 4 h. The mixture was filtered, washed, and dried under vacuum. 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 = 7.4Hz, 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] UV absorption spectroscopy of the solution: Accurately weigh 4.3 mg NF using a 0.01 g / mL balance and dissolve it in 5 mL of dimethyl sulfoxide to prepare a 1 mM stock solution. Accurately pipette 40 µL of the stock solution and add 1960 µL of dimethyl sulfoxide to dilute to a 20 µM test solution. Pour the solution into a cuvette and measure the UV absorption spectrum using a UV spectrophotometer. The UV absorption wavelength is 487 nm, and the scanning range is 300–700 nm.

[0040] Solution fluorescence spectroscopy: Accurately weigh 4.3 mg NF using a 1 / 10,000 molecular weight balance, dissolve it in 5 mL of dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. Accurately pipette 40 µL of the stock solution and add it to 1960 µL of DMSO to dilute to a 20 µM test solution. Pour the solution into a cuvette and use a fluorophotometer to measure the spectrum. The excitation wavelength is 487 nm, and the scanning range is 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 generates O2 ·- Test: A mixed solution of NF and DHR123 was prepared using PBS as the solvent, with each NF and DHR123 at a concentration of 10 µM. The test solution was poured into a fluorescent cuvette, and the solution in the cuvette was illuminated from above using a light source. The spectrum was then measured using a fluorophotometer, with the excitation wavelength of DHR123 at 488 nm, the emission wavelength at 520 nm, and the scan range at 500–700 nm. Figure 3 As shown, the fluorescence emission intensity of DHR123 increased significantly with increasing solution irradiation time, indicating that NF can effectively generate O2. ·- .

[0044] Positive reference CV produces O2 ·- The test: A mixed solution of CV and DHR123 was prepared using PBS as the solvent, with each CV and DHR123 concentration being 10 µM. The test solution was poured into a fluorescent cuvette, and the solution in the cuvette was illuminated from above using a light source. The spectrum was then measured using a fluorophotometer. Figure 4 As shown, O2 is the result of NF and CV. ·- Generation rate. The photosensitizer NF of the present invention exhibits high efficiency under white light (50 mw / cm²). 2 O2 is generated under these conditions. ·- It is 1.29 times that of the reference CV.

[0045] Test for •OH generation by photosensitizer: A mixed solution of NF and HPF was prepared using PBS as solvent, with each NF and HPF concentration of 10 µM. The test solution was poured into a fluorescent cuvette, and the solution in the cuvette was illuminated from above using a light source. The spectrum was then measured using a fluorophotometer, with the excitation wavelength of HPF at 490 nm and the emission wavelength at 515 nm, and the scanning range at 500–700 nm. Figure 5 As shown, the fluorescence emission intensity of HPF increases significantly with the continuous increase of solution irradiation time, indicating that NF can effectively generate •OH.

[0046] Test for the generation of •OH by the positive reference CV: Prepare a mixed solution of CV and HPF using PBS as the solvent, with each CV and HPF concentration being 10 µM. Pour the test solution into a fluorescent cuvette, illuminate the solution in the cuvette from above using a light source, and then measure the spectrum using a fluorometer. Figure 6 The figure shows the •OH generation rates of NF and CV. The photosensitizer of the present invention generates •OH in NF 49.08 times that of the reference CV.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent substitutions, and improvements made by those skilled in the art to the above embodiments without departing from the scope of the technical solution of the present invention, based on the technical essence of the present invention, shall still fall 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 abbreviated as NF.

2. The method for preparing a photosensitizer for type I photodynamic therapy according to claim 1, characterized in that, The synthesis route of NF is as follows:

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