Naphthalimide photosensitive dye without heavy atoms as well as preparation method and application of naphthalimide photosensitive dye

Abstract

The invention discloses a heavy-atom-free naphthalimide photosensitive dye as well as a preparation method and application thereof. According to the method, naphthalimide is used as a parent, and a carbazole group is connected with the parent through a single bond to prepare the heavy-atom-free naphthalimide photosensitive dye. Superoxide anion free radicals are generated through a spin-orbit charge transfer mechanism caused by an orthogonal configuration between an electron-donating group carbazole plane and an electron-withdrawing naphthalimide plane, so that tumor cells are killed. The heavy-atom-free naphthalimide photosensitive dye in the method has good hydrophilicity, the morpholine structure contained in the heavy-atom-free naphthalimide photosensitive dye can enable the photosensitive dye to locate lysosome, superoxide anion free radicals are generated in 4T1 cells under the illumination condition to promote apoptosis of tumor cells, and the heavy-atom-free naphthalimide photosensitive dye can be further used as a photosensitive drug for potential cancer photodynamic therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of the synthesis of photosensitizing dyes and their application in the preparation of drugs for photodynamic therapy of cancer, and particularly relates to a naphthalimide photosensitizing dye (NACA) without heavy atoms, a preparation method thereof, and an application thereof. Background Art

[0002] As an emerging technology for treating cancer, photodynamic therapy (PDT) has been favored by people due to its small trauma, high selectivity, low toxicity, and few side effects. During the photodynamic therapy process, under the illumination condition of light with a suitable wavelength, the molecules of the photosensitizing dye are excited from the ground state to the excited singlet state, and then reach the first excited triplet state through intersystem crossing, and undergo a photochemical reaction with oxygen molecules or other substances in the environment to generate highly toxic reactive oxygen species such as singlet oxygen and superoxide anion radicals, which can further promote the apoptosis of tumor cells to achieve the effect of disease treatment.

[0003] As a core component in PDT treatment, the photosensitizing activity of the photosensitizing dye has always been a key factor in improving the efficacy of cancer treatment. At present, there are many design strategies for improving the photosensitizing activity of photosensitizing dyes. Among them, introducing heavy atoms such as halogen atoms with large atomic numbers and transition metal elements into photosensitizing dyes is a widely used method. However, the high biological toxicity of heavy atoms limits their further application in the biological field. Therefore, the development of photosensitizing dyes without heavy atoms and with excellent performance has also become a research hotspot in current cancer photodynamic therapy. Summary of the Invention

[0004] The technical problem solved by the present invention is to provide a naphthalimide photosensitizing dye without heavy atoms and a preparation method thereof. The method uses naphthalimide as the parent body and forms a naphthalimide photosensitizing dye with an orthogonal molecular structure by connecting with carbazole through a single bond. The naphthalimide photosensitizing dye can generate superoxide anion radicals under illumination conditions. The naphthalimide photosensitizing dye is ingeniously designed and simply synthesized. Due to the absence of heavy atoms and having good biocompatibility, the morpholine group in the molecular structure can localize the photosensitizing dye in lysosomes, realizing the efficient generation of superoxide anion radicals in 4T1 cells to kill tumor cells, and can be used as a photosensitizing drug for photodynamic therapy of cancer.

[0005] The present invention adopts the following technical solution to solve the above technical problem. A naphthalimide photosensitizing dye without heavy atoms, characterized in that the structural formula of the naphthalimide photosensitizing dye is as shown in Formula I:

[0006]

[0007] The preparation method of the naphthalimide photosensitizing dye without heavy atoms described in the present invention is characterized in that the specific preparation steps are as follows:

[0008] Step S1: Put morpholine, 2-chloroethylamine and ethanol into a reaction vessel and reflux at 75 - 85 °C. After the reaction is complete, add 4-bromo-1,8-naphthalic anhydride and continue refluxing at 75 - 85 °C to obtain the compound shown in Formula II. The structural formula of the compound shown in Formula II is:

[0009]

[0010] Step S2: Add 3-bromocarbazole and bis(pinacolato)diboron to a reaction vessel, then add a catalyst, a basic substance and dioxane, and reflux at 85 - 95 °C in a non-oxygen gas atmosphere to obtain the compound shown in Formula III. The catalyst is [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (DDPF), and the basic substance is one or more of potassium carbonate, sodium carbonate, potassium acetate and sodium bicarbonate. The structural formula of the compound shown in Formula III is:

[0011]

[0012] Step S3: Add the compound shown in Formula II obtained in Step S1 and the compound shown in Formula III obtained in Step S2 to a reaction vessel, then add a catalyst, a basic substance, dioxane and water, and reflux at 85 - 95 °C in a non-oxygen gas atmosphere to obtain the compound shown in Formula I, that is, a naphthalimide photosensitizing dye without heavy atoms. The catalyst is Pd(PPh 3 ) 4 , and the basic substance is one or more of potassium carbonate, sodium carbonate, potassium acetate and sodium bicarbonate.

[0013] Furthermore, in Step S1, the molar ratio of the fed morpholine, 2-chloroethylamine and 4-bromo-1,8-naphthalic anhydride is 1.5 - 2.5:1:1.

[0014] Furthermore, in Step S2, the molar ratio of the fed 3-bromocarbazole and bis(pinacolato)diboron is 1:1 - 2.

[0015] Furthermore, in Step S3, the molar ratio of the compound shown in Formula III to the compound shown in Formula II is 1 - 2:1, and the volume ratio of dioxane to water is 10:1.

[0016] Furthermore, in Steps S2 and S3, the non-oxygen gas atmosphere is both a nitrogen atmosphere or an argon atmosphere.

[0017] The application of the naphthalimide photosensitizing dye without heavy atoms described in the present invention in the preparation of a photosensitive drug for photodynamic therapy of cancer, wherein the naphthalimide photosensitizing dye without heavy atoms can generate a large amount of reactive oxygen superoxide anion radicals under light irradiation to kill tumor cells.

[0018] The naphthalimide photosensitizing dye without heavy atoms described in the present invention can generate superoxide anion radicals in both solution and 4T1 cells under light irradiation, realizing the photodynamic therapy of tumors.

[0019] The naphthalimide photosensitizing dye without heavy atoms described in the present invention has good hydrophilicity. The morpholine structure it contains can localize the photosensitizing dye in lysosomes, realizing the generation of superoxide anion radicals in 4T1 cells under light irradiation to promote the apoptosis of tumor cells, and can further be used as a photosensitizing drug for potential cancer photodynamic therapy.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: The raw materials used for preparing the naphthalimide photosensitizing dye without heavy atoms shown in Formula I of the present invention are economical and the synthesis is simple. The prepared naphthalimide photosensitizing dye has good biocompatibility and can enter cells. Since it has a morpholine group, it can be localized on lysosomes. After being activated by light, the generated superoxide anion radicals can efficiently and concentratedly damage lysosomes, and then kill tumor cells, and can be used for preparing photosensitizing drugs for photodynamic therapy. Description of the Drawings

[0021] Figure 1 is the 1H NMR spectrum of the compound shown in Formula II in Example 1.

[0022] Figure 2 is the 1H NMR spectrum of the compound shown in Formula III in Example 1.

[0023] Figure 3 is the 1H NMR spectrum of the compound shown in Formula I in Example 1.

[0024] Figure 4 is the water solubility curve of the compound shown in Formula I in Example 1.

[0025] Figure 5 is the fluorescence spectrum of the compound shown in Formula I in Example 1 in different solvents.

[0026] Figure 6 is the spectrogram for detecting the generation of superoxide anion radicals of the compound shown in Formula I in Example 1 in solution.

[0027] Figure 7 is the electron paramagnetic resonance spectrogram for detecting the generation of superoxide anion radicals of the compound shown in Formula I in Example 1 in solution.

[0028] Figure 8 is the dark toxicity and phototoxicity curves of the compound shown in Formula I in Example 1 in 4T1 cells. Detailed Embodiments

[0029] The above content of the present invention will be further described in detail through the following examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Any technology implemented based on the above content of the present invention belongs to the scope of the present invention.

[0030] Example 1

[0031] The specific synthesis steps of the naphthalimide photosensitive dye (NACA) without heavy atoms are as follows:

[0032] (1) Synthesis of the compound shown in Formula II

[0033]

[0034] Weigh 0.1 g (1.2574 mmol) of 2-chloroethylamine and place it in a 50 mL round-bottom flask. Add a certain amount of absolute ethanol as the reaction solvent, and then add 0.2190 mL (2.5138 mmol) of morpholine. After mixing evenly, place the round-bottom flask in an oil bath and heat it up to 80 °C for 4 h. After the reaction is complete, add 0.3481 g of 4-bromo-1,8-naphthalic anhydride to the round-bottom flask, and continue to heat and reflux at 80 °C until the reaction is complete. After the reaction is over, cool it to room temperature, rotary evaporate to remove ethanol, add a small amount of water and filter to obtain a yellow solid, which is the compound shown in Formula II. The nuclear magnetic resonance hydrogen spectrum of the obtained product is as Figure 1 shown, and its nuclear magnetic spectrum is 1 H NMR(600MHz,CDCl 3 )δ8.64(dd,J=7.2,0.9Hz,1H),8.56(dd,J=8.5,0.9Hz,1H),8.39(d,J=7.8Hz,1H),8.03(d,J=7.8Hz,1H),7.87–7.81(m,1H),4.33(t,J=6.9Hz,2H),3.67(s,4H),2.70(t,J=6.4Hz,2H),2.59(s,4H).

[0035] (2) Synthesis of the compound shown in Formula III

[0036]

[0037] Weigh 0.04853 g (0.195 mmol) of 3-bromocarbazole, 0.056 g (0.2925 mmol) of bis(pinacolato)diboron, 4.25 mg (0.01 mmol) of dichlorobis[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) (DDPF), and 0.0575 g (0.585 mmol) of potassium acetate into a 50 mL round-bottom flask. Add a certain amount of dioxane as the reaction solvent and mix well in an ultrasonic bath. Place the round-bottom flask in an oil bath and heat to 90 °C under nitrogen protection for reflux reaction for 15 h. After the reaction is completed, purify by column chromatography to obtain a white solid, namely the compound shown in Formula III. The 1H NMR spectrum of the obtained product is as Figure 2 shown, 1 1H NMR (600 MHz, DMSO-d 6 ) δ 11.41 (s, 1H), 8.46 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.48 (dd, J = 13.8, 8.1 Hz, 2H), 7.39 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 1.33 (s, 12H).

[0038] (3) Synthesis of the compound shown in Formula I

[0039]

[0040] Weigh 100 mg (0.2577 mmol) of the compound shown in Formula III, 0.88 mg (0.4123 mmol) of the compound shown in Formula II, 89.34 mg (0.01289 mmol) of the catalyst Pd(PPh 3 ) 4 and 106.85 mg (0.07731 mmol) of potassium carbonate into a 50 mL round-bottom flask. Then add a certain amount of a mixed reagent of dioxane and water as the reaction solvent and mix well in an ultrasonic bath. Place the round-bottom flask in an oil bath and heat to 90 °C for reflux reaction for 4 h. After the reaction is completed, purify by column chromatography to obtain the compound shown in Formula I, namely a naphthalimide photosensitizing dye without heavy atoms. The 1H NMR spectrum of the obtained product is as Figure 3 shown, 1 1H NMR (600 MHz, CDCl 3)δ8.93(s,1H),8.53(d,J=7.5Hz,1H),8.49(d,J=7.1Hz,1H),8.31(d,J=8.4Hz,1H),8.07(s,1H),7.97(d,J=7.7Hz,1H),7.67(d,J=7.5Hz,1H),7.56(t,J=7.8Hz,1H),7.51(d,J=8.2Hz,1H),7.48(d,J=8.2Hz,1H),7.45(t,J=5.9Hz,2H),7.22(dd,J=9.6,4.0Hz,1H),4.42(t,J=6.7Hz,2H),3.74(t,J=4.4Hz,4H),2.83(t,J=6.7Hz,2H),2.70(s,4H).

[0041] Water solubility detection test of naphthalimide photosensitizing dye without heavy atoms:

[0042] Take the naphthalimide photosensitizing dye without heavy atoms synthesized in Example 1 above and prepare a DMSO stock solution with a concentration of 6.0 mM. Pipette 1 μL of the above stock solution and dilute it to 3 mL of water. Add it in gradient concentrations each time and stir well to ensure that the final added volume of DMSO is less than one-thousandth of the total volume. Measure its absorbance value, and the test results are as Figure 4 shown. The test results show that the naphthalimide photosensitizing dye without heavy atoms has good water solubility, which provides a basic condition for subsequent biological tests.

[0043] Emission spectrum detection test of naphthalimide photosensitizing dye without heavy atoms:

[0044] Pipette 1 μL of the stock solution of the naphthalimide photosensitizing dye without heavy atoms synthesized in Example 1 above into a fluorescence cuvette containing 3 mL of different solvents (ethyl acetate, dichloromethane, chloroform, acetone, petroleum ether, water, methanol, DMF), and measure its emission spectrum. The excitation wavelength is the maximum absorption wavelength in the corresponding solvent. The results are as Figure 5 shown. The results show that the naphthalimide photosensitizing dye without heavy atoms has good fluorescence properties, which is beneficial for subsequent light therapy to kill tumor cells.

[0045] Fluorescence spectrum detection test of the generation of superoxide anions by naphthalimide photosensitizing dye without heavy atoms in solution:

[0046] Add 3 mL of methanol into a quartz cuvette, and then add a certain volume of naphthalimide photosensitizing dye without heavy atoms (final concentration: 10 μM) and DHR-123 (final concentration: 40 μM). Subsequently, irradiate with a xenon lamp at 405 nm for 1 min each time. Then, using 488 nm as the excitation wavelength, measure the corresponding fluorescence intensity on a fluorescence spectrometer. Compare the fluorescence intensity (F) at the maximum absorption and emission wavelength of DHR-123 after each irradiation with the fluorescence intensity (F 0 ) before irradiation. Plot a graph with the irradiation time as the abscissa and the fluorescence intensity ratio as the ordinate. The results are as shown in Figure 6 . The results show that the naphthalimide photosensitizing dye without heavy atoms can generate superoxide anion radicals in solution.

[0047] Fluorescence spectral detection experiment on the generation of superoxide anions by naphthalimide photosensitizing dye without heavy atoms in solution:

[0048] Perform an electron paramagnetic resonance (EPR) spin trapping experiment using 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Add a certain amount of naphthalimide photosensitizing dye without heavy atoms and the DMPO trapping agent into a methanol solution. Test under dark and light (white light, 210 s) conditions respectively to obtain the EPR spectrum of the naphthalimide photosensitizing dye. The results are as shown in Figure 7 . The results show that the naphthalimide photosensitizing dye without heavy atoms can generate a large amount of superoxide anion radicals under light irradiation.

[0049] Test on the cytotoxicity and phototoxicity of naphthalimide photosensitizing dye without heavy atoms to cells:

[0050] Select 4T1 cells as the research object, and characterize the cytotoxic effect of the naphthalimide photosensitizing dye without heavy atoms on 4T1 cells by cell viability. Inoculate at a cell density of 5×10 4 cells / mL into a 96-well plate, with a volume of 100 μL in each well, and culture at 37 °C and 5% (v / v) CO 2 for 24 hours. Then add the naphthalimide photosensitizing dye without heavy atoms to the culture medium at gradient concentrations. Set 5 replicates for each gradient concentration and set a blank control. After culture, detect the cell viability. At the same time, under the same conditions, irradiate with nm light for 20 min, continue to culture in the culture medium for 2 h, and detect the cell viability after irradiation. When detecting, first add 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution into each well, and culture at 37 °C and 5% (v / v) CO 2 for 4 hours. Then remove the original culture medium and add DMSO (150 μL / well), and then measure the OD value with an enzyme-linked immunosorbent assay (ELISA) reader. Repeat three times. The results are as shown in Figure 8As shown. The results show that: the survival rate of 4T1 cells of naphthalimide photosensitive dyes without heavy atoms reaches more than 90% without light irradiation, but naphthalimide photosensitive dyes have good killing ability against 4T1 under 405nm light irradiation, and the survival rate of cells is greatly reduced, providing a theoretical basis and technical support for the elimination of cancer cells in vivo.

[0051] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A naphthylimide photosensitizing dye containing no heavy atoms, characterized in that The structural formula of the naphthalene imide photosensitizer is shown in Formula I:

2. A method for preparing the naphthylimide photosensitizing dye containing no heavy atoms as claimed in claim 1, characterized in that The specific preparation steps are: Step S1, placing morpholine, 2-chloroethylamine and ethanol in a reaction vessel and refluxing at 75-85° C., adding 4-bromo-1,8-naphthalene dicarboxylic anhydride after the reaction is complete and continuing to reflux at 75-85° C. to obtain a compound of formula II, wherein the compound of formula II has the structural formula: Step S2, adding 3-bromocarbazole and diboronic acid pinacol ester to a reaction container, then adding a catalyst, an alkaline substance and dioxane, and reflux reaction at 85-95° C. in a non-oxygen gas atmosphere to obtain a compound shown in formula III, wherein the catalyst is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, and the alkaline substance is one or more of potassium carbonate, sodium carbonate, potassium acetate and sodium bicarbonate. The structural formula of the compound shown in formula III is: Step S3, adding the compound of formula II obtained in step S1 and the compound of formula III obtained in step S2 into a reaction container, then adding a catalyst, an alkaline substance, dioxane and water, and reflux reacting at 85-95° C. in a non-oxygen gas atmosphere to obtain a compound of formula I, i.e., a naphthylimide photosensitizer dye free of heavy atoms, wherein the catalyst is Pd(PPh3)4, and the alkaline substance is one or more of potassium carbonate, sodium carbonate, potassium acetate and sodium bicarbonate.

3. The method for preparing the naphthalene imide photosensitizing dye containing no heavy atoms according to claim 2, characterized in that: The molar ratio of morpholine, 2-chloroethylamine and 4-bromo-1,8-naphthalene dicarboxylic anhydride in step S1 is 1.5-2.5:1:

1.

4. The method for preparing the naphthalimide photosensitizing dye containing no heavy atoms according to claim 2, characterized in that: The molar ratio of 3-bromocarbazole to biboric acid pinacol ester in step S2 is 1:1-2.

5. The method for preparing the naphthalene imide photosensitizing dye containing no heavy atoms according to claim 2, characterized in that: In step S3, the molar ratio of the compound represented by formula III to the compound represented by formula II is 1-2:1, and the volume ratio of dioxane to water is 10:

1.

6. The method for preparing the naphthalimide photosensitizing dye containing no heavy atoms according to claim 2, characterized in that: The non-oxygen gas atmosphere in step S2 and step S3 is a nitrogen atmosphere or an argon atmosphere.

7. Use of the naphthylimide photosensitizer dye containing no heavy atoms as claimed in claim 1 in the preparation of photosensitizer drugs for photodynamic therapy of cancer, wherein the naphthylimide photosensitizer dye containing no heavy atoms can generate a large amount of active oxygen superoxide anion free radicals under light conditions to kill tumor cells.

8. The use according to claim 7, characterized in that: The naphthylimide photosensitizing dye containing no heavy atoms can generate superoxide anion free radicals in both solution and 4T1 cells under light conditions, thereby achieving photodynamic therapy for tumors.

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