A highly stable fluorescent probe for photodynamic therapy of tumors and its preparation method and use

Abstract

The invention discloses a preparation method and use of a fluorescent probe for photodynamic therapy of tumors. The fluorescent probe is prepared in one step by using 2-[3-cyanofuran-2(5H)-ylidene]malononitrile as a reaction raw material and reacting with 6-(dimethylamino)naphthalene-2-formaldehyde, 4-(N-(2-hydroxyethyl)-N-methylamino)benzaldehyde and 4-(N-(2-N,N,N-trimethylethyl)-N-methylamino)benzaldehyde bromide through a Knoven condensation reaction to prepare an organic small molecule probe having a high molar absorption coefficient, a high singlet oxygen generation efficiency and adjustable water solubility in the white light region. The organic small molecule probe of the invention can overcome the disadvantage of poor photostability of organic molecules and can perform photodynamic therapy in mouse tumor tissue for a long time.

Description

Technical Field

[0001] The invention relates to a highly stable fluorescent probe for tumor photodynamic therapy and a preparation method and application thereof, belonging to the field of medical therapy. Background Art

[0002] Photodynamic therapy is an effective medical technology that has emerged in recent years. It treats specific malignant tumor diseases (such as melanoma, esophageal cancer, oropharyngeal cancer and bronchial cancer) and common epidermal diseases (such as age-related macular degeneration, psoriasis and lupus) by combining photosensitizers, light and oxygen. In this process, the photosensitizer absorbs light and the electrons jump to the excited singlet state, and then relax to the triplet state through intersystem crossing. The photosensitizer in the triplet state tends to transfer energy to the surrounding molecules and generate reactive oxygen species with strong oxidative ability. The newly generated reactive oxygen species react with the surrounding biomolecules such as lipids, proteins and nucleic acids, inactivating them and eventually leading to cell death. In short, oxidative damage only occurs at the target site irradiated by light. Because it is a point-to-point treatment, photodynamic therapy has lower biological toxicity, side effects, tissue damage and drug resistance than traditional treatments (surgery, radiotherapy and chemotherapy).

[0003] As exogenous factors, the choice of irradiation light and photosensitizer is a key point in the process of photodynamic therapy. Compared with lasers with a clear spectrum, white light (such as sunlight) has particularly noteworthy advantages, including less damage to tissues, higher safety, wider irradiation area, and easier access. Photosensitizers can be divided into nanomaterials, metal complexes, and small organic molecules. Among the many photosensitizers, small organic molecules have irreplaceable characteristics, such as clear molecular structure, large-scale synthesis and purification, measurable metabolic mechanism, low biological toxicity, high metabolic rate, and strong targeting. Currently, a series of organic molecules, such as porphyrin derivatives, have been approved for use in clinical photodynamic therapy, but their inherent characteristics such as poor water solubility, low extinction coefficient, and small Stokes shift still need to be overcome. Summary of the invention

[0004] The present invention aims to provide a method for preparing and using a highly stable fluorescent probe for photodynamic therapy of tumors. The present invention achieves high molar absorption coefficient and singlet oxygen production efficiency by rationally constructing molecular probes 3CN-Np, 3CN-OH and 3CN-QA, thereby being able to inhibit tumor growth through photodynamic therapy. An organic small molecule probe with high molar absorption coefficient, high singlet oxygen production efficiency and adjustable water solubility in the white light region. The organic small molecule probe of the present invention can overcome the disadvantage of poor photostability of organic molecules and can perform long-term photodynamic therapy in mouse tumor tissues.

[0005] In order to solve the technical problem of the present invention, the proposed technical solution is: a highly stable fluorescent probe, the molecular structure of the probe is any one of the following:

[0006]

[0007] In order to solve the technical problem of the present invention, another technical solution is proposed: a method for preparing the highly stable fluorescent probe, wherein the fluorescent probe is based on 2-[3-cyanofuran-2(5H)-ylidene]malononitrile, and a D-π-A type fluorescent probe is constructed with an aromatic aldehyde through a Knoven condensation reaction, wherein the aromatic aldehyde is 6-(dimethylamino)naphthalene-2-carboxaldehyde, 4-(N-(2-hydroxyethyl)-N-methylamino)benzaldehyde or 4-(N-(2-N,N,N-trimethylethyl)-N-methylamino)benzaldehyde bromide.

[0008] Preferably, the fluorescent probe is synthesized by reacting 2-[3-cyanofuran-2(5H)-ylidene]malononitrile with 6-(dimethylamino)naphthalene-2-carboxaldehyde, 4-(N-(2-hydroxyethyl)-N-methylamino)benzaldehyde and 4-(N-(2-N,N,N-trimethylethyl)-N-methylamino)benzaldehyde bromide respectively; the reaction equation is any one of the following:

[0009]

[0010] Preferably, the molar ratio of 2-[3-cyanofuran-2(5H)-ylidene]malononitrile to the aromatic aldehyde is 1:1-1:1.5.

[0011] Preferably, the reaction temperature is 50-80°C.

[0012] Preferably, the reaction solvent is ethanol.

[0013] Preferably, piperidine is used as a catalyst.

[0014] Preferably, the synthesis of 3CN-OH is as follows:

[0015]

[0016] In a 250 mL round-bottom flask, 1.99 g (10 mmol) of 2-[3-cyanofuran-2(5H)-ylidene]malononitrile, 1.79 g (10 mmol) of 4-(N-(2-hydroxyethyl)-N-methylamino)benzaldehyde and 100 mL of ethanol were added in sequence. After stirring evenly, 2-5 drops of piperidine were added as a catalyst. The reaction solution was heated to 75°C and reacted for 12 hours. After the reaction was completed, it was cooled to room temperature. Solid precipitated and the target product 3CN-OH was obtained by suction filtration with a yield of 70%.

[0017] In order to solve the technical problem of the present invention, another technical solution is proposed: application of the highly stable fluorescent probes, wherein the fluorescent probes 3CN-Np, 3CN-OH and 3CN-QA are highly stable fluorescent probes for tumor photodynamic therapy.

[0018] Preferably, the fluorescent probe has high photostability and can be used to prepare tumor therapeutic drugs.

[0019] Beneficial effects:

[0020] The working principle of the highly stable fluorescent probes 3CN-Np, 3CN-OH and 3CN-QA for tumor photodynamic therapy of the present invention is as follows: The three organic molecular fluorescent probes of the present invention have strong absorption in the 400-700nm white light range, indicating that white light can be used as the excitation light source of the probe. After enhancing the charge transfer ability within the molecule, the molecule has a strong singlet oxygen generation efficiency and can be applied to photodynamic therapy. At the same time, because the molecule has high photostability, it can be used as a photosensitizer for a long time.

[0021] In the molecular structure design, we obtain a large Stokes shift and a high extinction coefficient by enhancing the molecular charge transfer. At the same time, the strong intramolecular charge transfer can reduce the energy level difference between the excited singlet state and the excited triplet state, produce more molecules in the excited triplet state, and transfer the molecular energy to the surrounding environment to obtain more reactive oxygen, thereby enhancing the photodynamic therapy effect of the molecule.

[0022] Compared with existing anti-tumor fluorescent probes, the present invention has the following advantages:

[0023] 1. The fluorescent probe described in the present invention is simple to synthesize and purify.

[0024] 2. The fluorescent probe of the present invention has a high molar absorption coefficient (>10000Lmol -1 cm -1 ).

[0025] 3. The fluorescent probe described in the present invention can stain the membrane structure of cells.

[0026] 4. The fluorescent probe described in the present invention has high photostability.

[0027] 5. The fluorescent probe described in the present invention has strong absorption in the white light region.

[0028] 6. The fluorescent probe described in the present invention has low dark toxicity and high phototoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0030] Figure 1 is (A) absorption spectrum and (B) fluorescence emission spectrum of the molecule of the present invention;

[0031] Figure 2 The molecules of the present invention (A) Active oxygen generation efficiency (Ce6 as reference) (B) Light stability (25mW / cm 2 );

[0032] Figure 3 The staining conditions of the molecules of the present invention in cells are as follows: (A) 3CN-Np stains in the endoplasmic reticulum; (B) 3CN-OH stains in the endoplasmic reticulum; (C) 3CN-QA stains in the cell membrane;

[0033] Figure 4 is the efficiency of active oxygen generation in the molecular cells of the present invention;

[0034] Figure 5 Comparison of dead cells and live cells after 20 minutes of illumination under the confocal system;

[0035] Figure 6 After mice were treated with 3CN-OH, (A) the weight change of mice, (B) the change of tumor volume, (C) the final comprehensive screening of tumor size;

[0036] Figure 7 This is the molecular structure of the fluorescent probe of the present invention. DETAILED DESCRIPTION

[0037] Example 1

[0038] The method for preparing a highly stable fluorescent probe for tumor photodynamic therapy of the present invention comprises the following steps:

[0039] Synthesis of 3CN-Np

[0040]

[0041] In a 250mL round-bottom flask, 1.99g (10mmol) 2-[3-cyanofuran-2(5H)-ylidene]malononitrile, 1.99g (10mmol) 6-(dimethylamino)naphthalene-2-carboxaldehyde and 100mL ethanol were added in sequence, stirred evenly, and 2-5 drops of piperidine were added as a catalyst. The reaction solution was heated to 75°C and reacted for 12 hours. After the reaction was completed, it was cooled to room temperature, and solids precipitated. The target product 3CN-Np was obtained by suction filtration with a yield of 75%. 1H NMR (400MHz, DMSO-d6) δ8.23(s,1H),8.08(d,J=16.1Hz,1H),7.88(dd,J=16.3,9.0Hz,2H),7.71(d,J =8.8Hz,1H),7.35–7.23(m,1H),7.18(d,J=16.1Hz,1H),7.06–6.92(m,1H),3.11(s,6H),1.81(s,6H). 13 C NMR(101MHz,DMSO-d6)δ172.17,152.50,150.20,142.50,133.23,129.54,128.23,127.55,1 24.41,123.10,116.70,112.77,108.12,60.92,53.18,45.34,38.65,36.28.HRMS-ESI:m / z,C 24 H 20 N4O calculated:380.1637,[M+H] + found:381.1702.

[0042] Example 2

[0043] Synthesis of 3CN-OH

[0044]

[0045] In a 250mL round-bottom flask, 1.99g (10mmol) 2-[3-cyanofuran-2(5H)-ylidene]malononitrile, 1.79g (10mmol) 4-(N-(2-hydroxyethyl)-N-methylamino)benzaldehyde and 100mL ethanol were added in sequence, stirred evenly, and 2-5 drops of piperidine were added as a catalyst. The reaction solution was heated to 75°C and reacted for 12 hours. After the reaction was completed, it was cooled to room temperature, and solids precipitated. The target product 3CN-OH was obtained by suction filtration with a yield of 70%. 1 H NMR(400MHz,DMSO-d6)δ7.92(d,J=15.8Hz,1H),7.76(d,J=8.7Hz,2H),7.04 –6.74(m,3H),4.82(s,1H),3.59(d,J=3.5Hz,4H),3.12(s,3H),1.75(s,6H). 13C NMR (101MHz, DMSO) δ177.30,175.33,153.43,149.36,132.92,121.79,113.52,112.69,112.32,112.04,108.10,98 .08,91.68,58.28,54.00,50.75,40.11,39.90,39.69,39.48,39.28,39.07,39.04,38.86,25.65.HRMS-ESI:m / z,C 21 H 20 N4O2for calculated:360.1586,found:[M+H] + 361.1659.

[0046] Example 3

[0047] Synthesis of 3CN-QA

[0048]

[0049] In a 250mL round-bottom flask, 1.99g (10mmol) 2-[3-cyanofuran-2(5H)-ylidene]malononitrile, 3.00g (10mmol) 4-(N-(2-N,N,N-trimethylethyl)-N-methylamino)benzaldehyde bromide and 100mL ethanol were added in sequence, stirred evenly, and 2-5 drops of piperidine were added as a catalyst. The reaction solution was heated to 75°C and reacted for 12 hours. After the reaction was completed, it was cooled to room temperature, and solids precipitated. The target product 3CN-QA was obtained by suction filtration with a yield of 60%. 1 H NMR(400MHz, DMSO-d6)δ7.93(d,J=16.0Hz,1H),7.82(d,J=8.6Hz,2H),7.00–6.85(m,3H),3. 97(t,J=7.9Hz,2H),3.51(t,J=7.9Hz,2H),3.16(s,9H),3.10(d,J=6.2Hz,8H),1.77(s,6H). 13 C NMR (101MHz, DMSO) δ177.30,175.67,152.16,148.84,132.58,122.92,113.25,112.48,111.68, 109.55,98.50,93.90,54.39,54.35,54.31,52.58,51.83,44.79,38.16,25.51.HRMS-ESI:m / z,C 24 H 28N5O for calculated:402.2288,found:[M-Br - ] + 402.2268.

[0050] Example 4

[0051] The oil-water separation coefficients of 3CN-Np, 3CN-OH and 3CN-QA are 3.21, 2.32 and 0.58, respectively.

[0052] Figure 1 is (A) absorption spectrum and (B) fluorescence emission spectrum of the molecule of the present invention;

[0053] The probe concentration was prepared to 5×10 -6 mol / L DMSO solution, and the absorption curve of the samples was tested by UV-visible absorption spectrophotometer. It can be calculated that the molar absorption coefficients of the three samples 3CN-Np, 3CN-OH, and 3CN-QA are 1.72, 3.01, and 1.76×10 4 L mol -1 cm -1 The absorption peaks are at 575nm, 596nm and 559nm, covering the entire visible light region, indicating that all three molecules can use white light as an excitation light source. In the fluorescence spectrum, the fluorescence emission peaks of the three molecules are at 811nm, 673nm and 663nm, respectively, with a Stoke shift of nearly 100nm.

[0054] Figure 2 The molecules of the present invention (A) Active oxygen generation efficiency (Ce6 as reference) (B) Light stability (25mW / cm 2 );

[0055] The probe concentration was prepared to 5×10 -6 mol / L PBS solution, into which 15 times the molar equivalent of DCFH was added as the control group, and the Ce6 concentration was prepared to be 5×10 -6 mol / L PBS solution, add 15 times the molar equivalent of DCFH. Use a white light lamp (25mW / cm 2 ) was used to measure the fluorescence emission spectra of the test solution after irradiation for 10 seconds. A total of 14 tests were performed. As shown in Figure (A), the three probes all showed strong ROS generation capabilities, which were 2.4, 3.4, and 2.7 times that of Ce6, respectively. This indicates that the three probes have better ROS generation efficiency than Ce6 in vitro. 2 The three probes were illuminated with white light and the absorption spectra were tested every 5 minutes for a total of 30 minutes. Figure 2It can be found in B that 3CN-OH and 3CN-QA exhibit very high photostability, and the absorption peak intensity hardly decreases with time, while 3CN-Np shows a certain downward trend.

[0056] Figure 3 The staining conditions of the molecules of the present invention in cells are as follows: (A) 3CN-Np stains in the endoplasmic reticulum; (B) 3CN-OH stains in the endoplasmic reticulum; (C) 3CN-QA stains in the cell membrane;

[0057] The cells were co-cultured with a medium containing 2 μM probe and 1 μM commercial dye for 30 minutes, washed three times with PBS after the culture, and imaged using a laser confocal imager. Through imaging, it was found that 3CN-Np and 3CN-OH could stain the endoplasmic reticulum and had a high overlap with the endoplasmic reticulum commercial dye, while 3CN-QA had a high overlap with the cell membrane commercial dye.

[0058] Figure 4 is the efficiency of active oxygen generation in the molecular cells of the present invention;

[0059] 4T1 cells were selected for the test of intracellular reactive oxygen production efficiency. The cells were cultured for 15 minutes using a culture medium containing 1 μM probe and 15 μM DCFH. The cells were then irradiated with the probe's optimal excitation wavelength, and imaging data of DCFH oxidation to produce DCF was collected every 5 minutes. The test showed that with the increase in illumination time, the fluorescence intensity of DCFH in cells treated with the three probes 3CN-Np, 3CN-OH, and 3CN-QA showed a significant increase, while as a control group, the fluorescence intensity of cells treated with only DCFH did not change significantly. Among them, 3CN-Np increased by 4.8 times, 3CN-OH increased by 5.3 times, and 3CN-QA increased by 4.5 times.

[0060] Figure 5 Comparison of dead cells and live cells after 20 minutes of illumination under the confocal system;

[0061] 4T1 cells were used for the dead cell and live cell experiment after 20 minutes of illumination. The cells were cultured in a medium containing 1 μM probe for 15 minutes, washed three times with the medium, and irradiated with white light for 20 minutes. The cells were then stained with a live / dead cell kit, and the imaging results of the calcein (live cells) and PI (dead cells) channels were tested using a laser confocal imager. The results showed that the dead / live cell ratios of the three probes were 3.02, 3.5, and 3.7, respectively, indicating that the three probes have a strong killing effect on tumor cells.

[0062] Figure 6After mice were treated with 3CN-OH, (A) the weight change of mice, (B) the change of tumor volume, (C) the final comprehensive screening of tumor size;

[0063] When the mice weighed about 17 g, 5×10 5 Tumor cells, when the tumor grows to 100mm 3 Around 1:10 pm, 100 μL PBS, 3CN-OH and Ce6 (100 μmol / L PBS solution) were injected through the tail vein. One hour after the injection, the tumor was irradiated with white light, once a day for 30 minutes each time, and the same dose of sample was added on the seventh day. The changes in the weight of the mice (A), the tumor volume of the mice (B) and the tumor morphology after the mice were killed 14 days later (C) were recorded throughout the process. The results showed that 3CN-OH had almost no toxic side effects, while the tumor volume was significantly reduced, about 0.3 times that of the PBS group and 0.5 times that of the Ce6 group. This shows that 3CN-OH has better tumor inhibition efficiency than the commercial photosensitizer Ce6.

Claims

1. Fluorescent probes 3CN-Np, 3CN-OH and 3CN-QA are used in the preparation of highly stable fluorescent probes for photodynamic therapy of tumors. The molecular structure of the probes is: , , .

2. A highly stable fluorescent probe, characterized in that: The molecular structure of the probe is: or .

3. The method for preparing the highly stable fluorescent probe according to claim 2, characterized in that: The fluorescent probe is based on 2-[3-cyanofuran-2(5H)-ylidene]malononitrile, and is constructed into a DpA type fluorescent probe with aromatic aldehyde through Knoven condensation reaction. The aromatic aldehyde is 6-(dimethylamino)naphthalene-2-carboxaldehyde or 4-(N-(2-N,N,N-trimethylethyl)-N-methylamino)benzaldehyde bromide.

4. The method for preparing a highly stable fluorescent probe according to claim 3, characterized in that: The fluorescent probe is synthesized by reacting 2-[3-cyanofuran-2(5H)-ylidene]malononitrile with 6-(dimethylamino)naphthalene-2-carboxaldehyde and 4-(N-(2-N,N,N-trimethylethyl)-N-methylamino)benzaldehyde bromide respectively; the reaction equation is any one of the following: ； 。 5. The method for preparing a highly stable fluorescent probe according to claim 3, characterized in that: The molar ratio of 2-[3-cyanofuran-2(5H)-ylidene]malononitrile to aromatic aldehyde is 1:1-1:1.

5.

6. The method for preparing a highly stable fluorescent probe according to claim 3, characterized in that: The reaction temperature is 50-80°C.

7. The method for preparing a highly stable fluorescent probe according to claim 3, characterized in that: The reaction solvent is ethanol.

8. The method for preparing a highly stable fluorescent probe according to claim 3, characterized in that: Piperidine was used as catalyst.

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