Photosensitizer based on 1, 8-naphthalimide and phototherapy application thereof

Abstract

The invention discloses a photosensitizer based on 1, 8-naphthalimide and phototherapy application thereof.The photosensitizer is obtained by taking 1, 8-naphthalimide and acetylpyrrole as raw materials through phosphorus oxychloride condensation and then fluoroborization, the synthesis method is very simple, and separation and purification are convenient. The compound has the characteristic of long wavelength absorption due to the expanded conjugated structure, and the optical characteristic enables the compound to be effectively applied to the field of photodynamic therapy.

Description

Technical Field

[0001] The present invention relates to a photosensitizer based on 1,8-naphthalimide, and more specifically, to a photosensitizer based on 1,8-naphthalimide and its phototherapy application. Background Art

[0002] Photodynamic Therapy (PDT) is a treatment technique based on the generation of Reactive Oxygen Species (ROS) by a photosensitizer under the excitation of light with a specific wavelength to kill tumor cells. This technique has advantages such as high selectivity, minimal trauma, and repeatable treatment, showing broad application prospects in the field of tumor treatment. However, existing photosensitizers still face many challenges in practical applications, such as insufficient light penetration depth, poor tumor tissue targeting, and low singlet oxygen quantum yield, which limit their treatment effects.

[0003] 1,8-Naphthalimide compounds have received extensive attention in the field of photosensitizers due to their excellent photophysical and chemical properties. These compounds have a large conjugated system, and the absorption wavelength can be regulated by molecular structure modification; at the same time, they have a high singlet oxygen quantum yield and good photostability. In recent years, researchers have developed a series of 1,8-naphthalimide derivatives by introducing different functional groups, significantly improving their photodynamic therapy effects. For example, by introducing an electron donor-acceptor structure on the 1,8-naphthalimide skeleton, a red shift of the absorption wavelength and an increase in the ROS generation efficiency have been achieved.

[0004] However, existing 1,8-naphthalimide-based photosensitizers still have some problems that need to be solved urgently: (1) Poor water solubility of some compounds affects their transport and distribution in the body; (2) Their stability in the tumor microenvironment needs to be improved. Therefore, the development of new 1,8-naphthalimide-based photosensitizers with good water solubility and excellent photostability is of great significance for promoting the clinical application of photodynamic therapy. Summary of the Invention

[0005] The main purpose of the present invention is to provide a photosensitizer based on 1,8-naphthalimide and its phototherapy application. The technical solution of the present invention is as follows: A photosensitizer based on 1,8-naphthalimide and its phototherapy application, the chemical structural formula of the compound is:

[0006] The substituent R is selected from any one of chlorine, methoxy, and ethoxy. The chemical structural formula of the compound is: any one of

[0007] The synthesis method of the 1,8-naphthalimide-based photosensitizer described above, the method comprises the following synthesis route:

[0008] The method comprises the following steps: (1) Add 1,8-naphthalimide, acetylpyrrole, and toluene into a reaction flask at room temperature. After ultrasonic dissolution, heat the mixture, and then add phosphorus oxychloride to obtain a reaction solution. (2) After the reaction solution obtained in step (1) is concentrated by rotary evaporation, dissolve it with dichloromethane, and adjust the pH to 8.0 - 9.0 with saturated sodium hydroxide solution. After extraction with a dichloromethane-water system, rotary evaporate to remove the solvent. The obtained residue Np-Cl undergoes a fluoroboration reaction with boron trifluoride etherate complex in the presence of triethylamine. The reaction product is purified by silica gel column chromatography to obtain the target compound Np-BOP. (3) Place the compound Np-BOP prepared in step (2) as a reactant in a reaction flask, use methanol or ethanol as a solvent respectively, and stir the reaction at room temperature to obtain Np-OMe and Np-OEt respectively.

[0009] The feeding sequence of step (1) is compound 1, compound 2, toluene, phosphorus oxychloride; the feeding ratio of compound 1 to phosphorus oxychloride is 1:1 - 3. The feeding sequence and ratio are optimized and designed. First, add the reactants (i.e., compound 1 and compound 2) to ensure sufficient contact of the main reactants. Then, add the solvent to facilitate the control of concentration and temperature. Finally, adding the catalyst can effectively regulate the reaction rate, and the feeding ratio range of 1:1 - 3 can not only ensure the completion of the reaction but also avoid side reactions. If the feeding sequence or ratio is changed, problems such as incomplete reaction, increased by-products, or out-of-control reaction may occur.

[0010] The heating temperature of step (1) is 25 - 120 °C, and the heating time is 3 - 5 hours.

[0011] The feeding sequence of step (2) is compound Np-Cl, triethylamine, boron trifluoride etherate; the feeding ratio of compound Np-Cl to triethylamine and boron trifluoride etherate is 1:5:5.

[0012] The beneficial effects of the present invention are as follows: (1) The compound of the present invention is a photosensitizer based on 1,8-naphthalimide. Through phototoxicity experiments, it is verified that this photosensitizer can efficiently generate reactive oxygen species (ROS) under illumination at a specific wavelength and exhibits significant phototoxicity to Hela tumor cells. This photosensitizer has low toxicity to normal cells under dark conditions, showing good biocompatibility, which is beneficial to improving the safety and selectivity of phototherapy. At the same time, this photosensitizer shows the characteristic of selective accumulation in tumor cells. Combining with the spatio-temporal controllability of illumination, precise tumor-targeted therapy can be achieved. Based on the molecular design of the 1,8-naphthalimide skeleton, this photosensitizer has good photostability and modifiability, providing an important lead compound for the development of new and efficient photodynamic therapy drugs. These characteristics indicate that this photosensitizer has important application value and clinical transformation potential in the field of tumor photodynamic therapy.

[0013] (2) The synthesis reaction conditions of the 1,8-naphthalimide-based photosensitizer described in the present invention are easy to control, and the product purification operation is simple, with general applicability. Description of the Drawings

[0014] Figure 1 is the 1H NMR spectrum of the compound Np-BOP obtained in Example 6.

[0015] Figure 2 is the 1H NMR spectrum of the compound Np-OMe obtained in Example 7.

[0016] Figure 3 is the 1H NMR spectrum of the compound Np-OEt obtained in Example 8.

[0017] Figure 4 are the UV absorption (a) and fluorescence spectra (b) of the compound Np-BOP obtained in Example 6.

[0018] Figure 5 are the UV absorption (a) and fluorescence spectra (b) of the compound Np-OMe obtained in Example 7.

[0019] Figure 6 are the UV absorption (a) and fluorescence spectra (b) of the compound Np-OEt obtained in Example 8.

[0020] Figure 7 is the single crystal diagram of the compound Np-BOP obtained in Example 6.

[0021] Figure 8 are the live / dead cell staining images of the compound Np-BOP obtained in Example 6.

[0022] Figure 9 are the live / dead cell staining images of the compound Np-OMe obtained in Example 7. Detailed Description of the Invention

[0023] The present invention will be further described below in conjunction with embodiments, but the scope of protection required by the present invention is not limited to the scope described in the embodiments.

[0024] Example 1 Weigh 1,8-naphthalimide (Compound 1, 507.55 mg, 3.0 mmol) and acetylpyrrole (Compound 2, 327.39 mg, 3.0 mmol), and dissolve them in 25 mL of toluene. Place the reaction system at room temperature, and slowly add phosphorus oxychloride (275 μL, 3.0 mmol) dropwise under magnetic stirring. Keep the reaction at room temperature for 5 hours, and monitor the reaction progress by thin-layer chromatography (TLC). The reaction does not occur.

[0025] Example 2 Weigh 1,8-naphthalimide (Compound 1, 507.55 mg, 3.0 mmol) and acetylpyrrole (Compound 2, 327.39 mg, 3.0 mmol), and dissolve them in 25 mL of toluene. Place the reaction system in an oil bath and heat it to 120 °C, and slowly add phosphorus oxychloride (275 μL, 3.0 mmol) dropwise under magnetic stirring. Monitor the reaction progress by thin-layer chromatography (TLC, eluent: PE / DCM = 3:1, v / v). After 3 hours, Compound 2 has completely reacted, and there is still a small amount of Compound 1 remaining. After the reaction solution is concentrated by rotary evaporation, it is dissolved in dichloromethane, and the pH is adjusted to 8.0 with saturated sodium hydroxide solution. After extraction with a dichloromethane-water system three times, the solvent is removed by rotary evaporation to obtain the intermediate Np-Cl (125.13 mg, 0.45 mmol). This intermediate undergoes a fluoroboration reaction with boron trifluoride diethyl ether complex (319 μL, 2.25 mmol) in the presence of triethylamine (313 μL, 2.25 mmol). The reaction product is purified by silica gel column chromatography (eluent: PE / DCM = 5:1, v / v) to obtain the target compound Np-BOP, a black solid with a green metallic luster, with a yield of 15%.

[0026]

[0027] Example 3 Weigh 1,8-naphthalimide (Compound 1, 507.55 mg, 3.0 mmol) and acetylpyrrole (Compound 2, 491.10 mg, 4.5 mmol), and dissolve them in 25 mL of toluene. Place the reaction system in an oil bath and heat it to 120 °C. Slowly add phosphorus oxychloride (275 μL, 3.0 mmol) dropwise under magnetic stirring. Monitor the reaction progress by thin-layer chromatography (TLC, eluent: PE / DCM = 3:1, v / v). After 3 hours, Compound 2 has completely reacted, and Compound 1 has basically reacted completely. After the reaction solution is concentrated by rotary evaporation, it is dissolved in dichloromethane, and the pH is adjusted to 8.0 with saturated sodium hydroxide solution. After extraction with a dichloromethane-water system three times, the solvent is removed by rotary evaporation to obtain the intermediate Np-Cl (208.55 mg, 0.75 mmol). This intermediate undergoes a fluoroboration reaction with boron trifluoride diethyl ether complex (531 μL, 3.75 mmol) in the presence of triethylamine (521 μL, 3.75 mmol). The reaction product is purified by silica gel column chromatography (eluent: PE / DCM = 5:1, v / v) to obtain the target compound Np-BOP, a black solid with a green metallic luster, with a yield of 25%. When the amount of Compound 1 is increased by 1.5 mmol relative to Example 2, the yield increases by 10%.

[0028]

[0029] Example 4 Weigh 1,8-naphthalimide (Compound 1, 507.55 mg, 3.0 mmol) and acetylpyrrole (Compound 2, 654.78 mg, 6 mmol), and dissolve them in 25 mL of toluene. Place the reaction system in an oil bath and heat it to 120 °C. Slowly add phosphorus oxychloride (275 μL, 3.0 mmol) dropwise under magnetic stirring. Monitor the reaction progress by thin-layer chromatography (TLC, eluent: PE / DCM = 3:1, v / v). After 3 hours, Compound 2 has completely reacted, and Compound 1 has basically reacted completely. After the reaction solution is concentrated by rotary evaporation, it is dissolved in dichloromethane, and the pH is adjusted to 8.0 with saturated sodium hydroxide solution. After extraction with a dichloromethane-water system three times, the solvent is removed by rotary evaporation to obtain the intermediate Np-Cl (216.89 mg, 0.78 mmol). This intermediate undergoes a fluoroboration reaction with boron trifluoride diethyl ether complex (553 μL, 3.90 mmol) in the presence of triethylamine (543 μL, 3.90 mmol). The reaction product is purified by silica gel column chromatography (eluent: PE / DCM = 5:1, v / v) to obtain the target compound Np-BOP, a black solid with a green metallic luster, with a yield of 26%. When the amount of Compound 1 is increased by 1.5 mmol relative to Example 3, the yield shows no obvious change.

[0030]

[0031] Example 5 Weigh 1,8-naphthalimide (Compound 1, 507.55 mg, 3.0 mmol) and acetylpyrrole (Compound 2, 491.10 mg, 4.5 mmol), and dissolve them in 25 mL of toluene. Place the reaction system in an oil bath and heat it to 120 °C. Slowly add phosphorus oxychloride (550 μL, 6.0 mmol) dropwise under magnetic stirring. The reaction progress was monitored by thin-layer chromatography (TLC, eluent: PE / DCM = 3:1, v / v). After 3 hours, Compound 2 completely reacted, and Compound 1 was basically completely reacted. After the reaction solution was concentrated by rotary evaporation, it was dissolved in dichloromethane, and the pH was adjusted to 8.0 with saturated sodium hydroxide solution. After extraction with dichloromethane-water system for 3 times, the solvent was removed by rotary evaporation to obtain the intermediate Np-Cl (258.60 mg, 0.93 mmol). This intermediate was subjected to a fluoroboration reaction with boron trifluoride ether complex (659 μL, 4.65 mmol) in the presence of triethylamine (647 μL, 4.65 mmol). The reaction product was purified by silica gel column chromatography (eluent: PE / DCM = 5:1, v / v) to obtain the target compound Np-BOP as a black solid with a green metallic luster, and the yield was 31%. When the amount of phosphorus oxychloride was increased by 3 mmol relative to Example 3, the yield increased by 6%.

[0032]

[0033] Example 6 Weigh 1,8-naphthalimide (Compound 1, 507.55 mg, 3.0 mmol) and acetylpyrrole (Compound 2, 491.10 mg, 4.5 mmol), and dissolve them in 25 mL of toluene. Place the reaction system in an oil bath and heat it to 120 °C. Slowly add phosphorus oxychloride (825 μL, 9.0 mmol) dropwise under magnetic stirring. The reaction progress was monitored by thin-layer chromatography (TLC, eluent: PE / DCM = 3:1, v / v). After 3 hours, Compound 2 completely reacted, and Compound 1 was basically completely reacted. After the reaction solution was concentrated by rotary evaporation, it was dissolved in dichloromethane, and the pH was adjusted to 8.0 with saturated sodium hydroxide solution. After extraction with the dichloromethane-water system three times, the solvent was removed by rotary evaporation to obtain the intermediate Np-Cl (300.30 mg, 1.08 mmol). This intermediate was subjected to a fluoroboration reaction with boron trifluoride ether complex (765 μL, 5.40 mmol) in the presence of triethylamine (751 μL, 5.40 mmol). The reaction product was purified by silica gel column chromatography (eluent: PE / DCM = 5:1, v / v) to obtain the target compound Np-BOP, a black solid with a green metallic luster, with a yield of 36%. When the amount of phosphorus oxychloride was increased by 3 mmol relative to Example 5, the yield increased by 5%.

[0034] Take the black solid Np-BOP (3.26 mg, 0.01 mmol) with a green metallic luster and dissolve it in 1 mL of dichloromethane to prepare a stock solution. Take 6 μL of the stock solution and add it to 3 mL of toluene, dichloromethane, ethyl acetate, acetone, and methanol respectively to measure the absorption wavelength of this compound ( Figure 4 ). Use the obtained maximum absorption wavelength to excite this compound to obtain its emission wavelength ( Figure 4 ). This compound has a relatively long absorption wavelength and has double absorption peaks between 400 - 700 nm. Irradiating it with a laser lamp in this wavelength range will cause it to generate reactive oxygen species (ROS), thereby killing tumor cells.

[0035]

[0036] Example 7 Weigh Compound Np-BOP (326.06 mg, 1.0 mmol) and dissolve it in a mixed solvent of 20 mL of methanol and 5 mL of dichloromethane. Place the reaction system under stirring at room temperature, and monitor the reaction progress by thin-layer chromatography (TLC, eluent: PE / DCM = 2:1, v / v). After 5 hours, TLC showed that Compound Np-BOP completely reacted. After the reaction solution was concentrated by rotary evaporation, it was purified by silica gel column chromatography (eluent: PE / DCM = 4:1, v / v) to obtain the rose-red solid product Np-OMe (167.50 mg), with a yield of 52%.

[0037] The magenta solid Np-OMe (3.26 mg, 0.01 mmol) was dissolved in 1 mL of dichloromethane to prepare a mother liquor. 6 μL of the mother liquor was taken and added to 3 mL of toluene, dichloromethane, ethyl acetate, acetone, methanol, and dimethyl sulfoxide respectively to measure the absorption wavelength of the compound ( Figure 5 ). The obtained maximum absorption wavelength was used to excite the compound to obtain its emission wavelength ( Figure 5 ). The compound has a relatively long absorption wavelength and has double absorption peaks between 400 - 700 nm. Irradiating with a laser lamp in this wavelength band will cause it to produce reactive oxygen species (ROS), thereby killing tumor cells.

[0038]

[0039] Example 8 Weigh the compound Np-BOP (322.06 mg, 1.0 mmol) and dissolve it in 25 mL of ethanol. The reaction system was placed under stirring at room temperature, and the reaction progress was monitored by thin-layer chromatography (TLC, developing agent: PE / DCM = 2:1, v / v). After 4 hours, TLC showed that the compound Np-BOP had completely reacted. After the reaction solution was concentrated by rotary evaporation, it was purified by silica gel column chromatography (eluent: PE / DCM = 4:1, v / v) to obtain an orange-red solid product Np-OEt (218.50 mg), with a yield of 65%.

[0040] Take the orange-red solid product Np-OEt (3.36 mg, 0.01 mmol) and dissolve it in 1 mL of dichloromethane to prepare a mother liquor. 6 μL of the mother liquor was taken and added to 3 mL of toluene, dichloromethane, ethyl acetate, acetone, methanol, and dimethyl sulfoxide respectively to measure the absorption wavelength of the compound ( Figure 6 ). The obtained maximum absorption wavelength was used to excite the compound to obtain its emission wavelength ( Figure 6 ). The compound has a relatively long absorption wavelength and has double absorption peaks between 400 - 700 nm. Irradiating with a laser lamp in this wavelength band will cause it to produce reactive oxygen species (ROS), thereby killing tumor cells.

[0041]

[0042] Example 9 Cell Viability and Cytotoxicity Staining Experiment of Compound Np-BOP The cell viability detection experiment was carried out using the Hela cell line. Cells in good growth state were seeded in a six-well plate at a density of 1.0×10 5 cells per well and incubated overnight in an incubator at 37 °C and 5% CO2. Four groups of treatments were set in the experiment: (1) control group (only fresh medium); (2) simple light irradiation group (635 nm, 1.0 W / cm 2); (3) the simple Np-BOP treatment group (medium containing Np-BOP); (4) the Np-BOP combined with light treatment group. After changing the corresponding medium, continue to incubate for 4 hours. Subsequently, the cells in each group were stained with 10 μL of Calein-AM (1.0 mM, live cell staining) and 10 μL of PI (1.0 mM, dead cell staining), and the cells in the second and fourth groups were irradiated with 635 nm laser (1.0 W / cm 2 ) after staining. After all groups were incubated for another 30 minutes, they were observed and photographed using a fluorescence microscope (Olympus, BX51). The experimental results showed that the cell survival rate was the highest in the control group; a small amount of cell death occurred in the simple light irradiation group; partial cell death occurred in the simple Np-BOP treatment group; while in the Np-BOP combined with light treatment group, almost all cancer cells were killed, indicating that Np-BOP has a significant photodynamic therapy effect under the condition of 635 nm laser irradiation.

[0043] Example 10 Cell Viability and Death Staining Experiment of Compound Np-OMe The cell viability detection experiment was carried out using the Hela cell line. Cells in good growth state were seeded in a six-well plate at a density of 1.0×10 5 cells per well and incubated overnight in an incubator at 37 °C and 5% CO2. Four groups of treatments were set in the experiment: (1) the control group (only fresh medium); (2) the simple light irradiation group (635 nm, 1.0 W / cm 2 ); (3) the simple Np-OMe treatment group (medium containing Np-OMe); (4) the Np-OMe combined with light treatment group. After changing the corresponding medium, continue to incubate for 4 hours. Subsequently, the cells in each group were stained with 10 μL of Calein-AM (1.0 mM, live cell staining) and 10 μL of PI (1.0 mM, dead cell staining), and the cells in the second and fourth groups were irradiated with 635 nm laser (1.0 W / cm 2 ) after staining. After all groups were incubated for another 30 minutes, they were observed and photographed using a fluorescence microscope (Olympus, BX51). The experimental results showed that the cell survival rate was the highest in the control group; a small amount of cell death occurred in the simple light irradiation group; partial cell death occurred in the simple Np-OMe treatment group; while in the Np-OMe combined with light treatment group, almost all cancer cells were killed, indicating that Np-OMe has a significant photodynamic therapy effect under the condition of 635 nm laser irradiation.

[0044] Example 11 Solubility Determination Experiment of Compounds Np-BOP, Np-OMe, and Np-OEt Weigh out 1.0 g of the excessive compounds Np-BOP, Np-OMe, and Np-OEt respectively and place them in 9 groups of containers. Each group corresponds to the combination of the temperature (20°C, 40°C, 60°C) and the volume of water (25 mL, 50 mL, 100 mL) in the orthogonal experiment design. After adding the specified volume of deionized water, keep it oscillating at a constant temperature for 24 hours (200 rpm) until the dissolution equilibrium at the corresponding temperature, quickly filter the saturated solution, and use HPLC to measure the concentration of each compound in the filtrate, and calculate the solubility per unit volume (mg / mL) as shown in Table 1: Table 1 Solubility of Np-BOP, Np-OMe, and Np-OEt

[0045] The above embodiments are only the preferred technical solutions of the present invention and should not be regarded as limitations on the present invention. The embodiments in this application and the features in the embodiments can be combined arbitrarily with each other without conflict. The protection scope of the present invention should be the technical solutions recorded in the claims, including the equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, the equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. A photosensitizer based on 1,8-naphthalimide, characterized in that, The chemical structural formula of the compound is as follows: Among them, the substituent R is selected from any one of chlorine, methoxy, and ethoxy.

2. The synthesis method of the photosensitizer based on 1,8-naphthalimide according to claim 1, characterized in that, The method includes the following synthetic route: Among them, the substituent R is selected from any one of methoxy and ethoxy (1) Add 1,8-naphthalimide and acetylpyrrole to a reaction flask at room temperature. After ultrasonic dissolution under solvent conditions, heat the mixture, and add phosphorus oxychloride to obtain the compound Np-Cl; (2) After the reaction solution obtained in step (1) is concentrated by rotary evaporation, dissolve it in dichloromethane, adjust the pH to 8.0 - 9.0 with sodium hydroxide solution, extract it with a dichloromethane-water system, then remove the solvent by rotary evaporation. The obtained residue Np-Cl undergoes a fluoroboration reaction with boron trifluoride diethyl ether complex in the presence of triethylamine. The reaction product is purified by silica gel column chromatography to obtain the target compound Np-BOP; (3) Using the compound Np-BOP prepared in step (2) as a reactant, use methanol or ethanol as a solvent respectively, and stir the reaction at room temperature to obtain Np-OMe and Np-OEt respectively.

3. The method according to claim 2, characterized in that In the step (1), the molar ratio of the feed of 1,8-naphthalimide, acetylpyrrole, and phosphorus oxychloride is 1:1~2:1~3.

4. The method according to claim 2, characterized in that, The heating temperature in the step (1) is 25~120 °C, and the heating time is 3~5 hours.

5. The method according to claim 2, wherein In the step (2), the feed ratio of the compound Np-Cl, triethylamine, and boron trifluoride diethyl ether is 1:5:

5.

6. The method according to claim 2, characterized in that, In the step (1), the feeding order is compound 1, compound 2, toluene, phosphorus oxychloride; in the step (2), the feeding order is compound Np-Cl, triethylamine, boron trifluoride diethyl ether.

7. Use of a photosensitizer based on 1,8-naphthalimide described in claim 1 or a photosensitizer based on 1,8-naphthalimide prepared by the method described in any one of claims 2 - 6 in a drug for photodynamic therapy of cancer tumor cells.

8. The use according to claim 7, characterized in that, The cancer tumor cells include human cervical cancer cells, and the human cervical cancer cells include Hela cells.

9. The use according to claim 7, wherein, The photosensitizer based on 1,8-naphthalimide can generate a large amount of reactive oxygen species (ROS) under the irradiation of a laser within the absorption wavelength range of the photosensitizer.

10. The use according to claim 9, characterized in that, The absorption wavelength range of the photosensitizer is 400 - 700 nm.

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