Diglycosylated benzophenoxazine photosensitizer and preparation method and use thereof

Abstract

Disclosed are a diglycosylated benzophenoxazine photosensitizer, and a preparation method and use thereof. The present invention greatly improves the enriched concentration of the photosensitizer in tumor cells by taking full advantage of the enhanced uptake and enhanced glycolysis of carbohydrates by tumor cells and the glycosylation of a selenium-containing benzophenoxazine compound, thereby improving the targeting of a diglycosylated benzophenoxazine photosensitizer involved in the present invention in the treatment of cutaneous tumors and also significantly decreasing the toxic and side effects of the photodynamic therapy. The present invention can efficiently and rapidly inhibit the proliferation of cells of cutaneous squamous cell carcinoma and essentially cause no damage to normal cells.

Claims

1. A diglycosylated benzophenoxazine photosensitizer having a structural formula as follows: ##STR00016## wherein, Et represents ethyl.

2. A method for preparing the diglycosylated benzophenoxazine photosensitizer according to claim 1, comprising the following steps: (1) reacting galactose with a selenium-containing compound (compound 1) in a solvent of ethanol at 65° C.-68° C. to obtain a glycosylated selenium-containing compound (compound 2); (2) reacting with the glycosylated selenium-containing compound obtained in step (1) in a solvent of acetone at a temperature of 160° C.-165° C. to obtain an intermediate product A; reacting 1-naphthylamine with the glycosylated selenium-containing compound obtained in step (1) in a solvent of ethanol by heating to reflux, so as to obtain an intermediate product B; (3) reacting the intermediate product A and the intermediate product B obtained in step (2) at a temperature of 85° C.-90° C. in a solvent of toluene, so as to obtain the diglycosylated benzophenoxazine photosensitizer; wherein the structural formulas of Compound 1, Compound 2, Compound 3, Intermediate Product A, and Intermediate Product B are: Intermediate Product B.

3. The method according to claim 2, wherein in step (1), a molar ratio of galactose to the glycosylated selenium-containing compound is 1-1.5:3-34; and a reaction time is 5-8 minutes.

4. The method according to claim 2, wherein in the reaction of preparing the intermediate product A step (2), a reaction time is 25-35 minutes; and a molar ratio of ##STR00017## to the glycosylated selenium-containing compound is 1:2.5-3.6.

5. The method according to claim 2, wherein in the reaction of preparing the intermediate product B in step (2), a temperature of the solvent of ethanol is 65° C.-70° C.

6. The method according to claim 2, wherein in the reaction of preparing the intermediate product B in step (2), a molar ratio of the 1-naphthylamine to the glycosylated selenium-containing compound is 2-2.5:1-1.5.

7. The method according to claim 2, wherein in step (3), a reaction time is 5-6 minutes; and a molar ratio of the intermediate product A to the intermediate product B is 1:1.

8. An anti-tumor drug comprising the diglycosylated benzophenoxazine photosensitizer according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the structural formula of a diglycosylated benzophenoxazine photosensitizer.

(2) FIG. 2 shows the hydrogen spectrogram of the diglycosylated benzophenoxazine photosensitizer.

(3) FIG. 3 shows the carbon spectrogram of the diglycosylated benzophenoxazine photosensitizer.

(4) FIG. 4 is a diagram showing the pathological sections of a tumor tissue after the external treatment with the photosensitizer.

(5) FIG. 5 is a graph showing Western Blot results of cell proliferation signaling proteins after the treatment with the photosensitizer.

(6) FIG. 6 is a graph showing Western Blot results of cell proliferation-related proteins after the treatment with the photosensitizer.

DETAILED DESCRIPTION OF EMBODIMENTS

(7) The present invention will be further described below with reference to examples.

Example 1: Synthesis of the Diglycosylated Benzophenoxazine Photosensitizer

(8) (1) galactose was reacted with a selenium-containing compound

(9) ##STR00009##
in a solvent of ethanol at 65° C.-68° C. for 5-8 minutes at a dosage ratio of galactose to the selenium-containing compounds of 1-1.5:3-34, wherein the reaction process was as follows:

(10) ##STR00010##

(11) (2) The selenium-containing glycosylated compound obtained in the step (1) was reacted with

(12) ##STR00011##
in a solvent of acetone under the conditions of 160° C.-165° C. for 30 minutes, wherein the reaction process was:

(13) ##STR00012##

(14) (3) 1-naphthylamine was reacted with the glycosylated selenium-containing compound

(15) ##STR00013##
obtained in the step (1) at a dosage ratio of the 1-naphthylamine to the glycosylated selenium-containing compound of 2-2.5:1-1.5 in a solvent of ethanol at 85° C.-87° C. for 15-20 minutes, wherein the reaction process was:

(16) ##STR00014##

(17) (4) The product obtained in the step (3) was reacted with the product obtained in the step (2) under the conditions of 160° C.-170° C. in acetone or toluene as the solvent for 5-6 minutes, wherein the ratio of the two reactants was 1:1, and the reaction process was:

(18) ##STR00015##

(19) The hydrogen spectrogram and carbon spectrogram of the aforementioned diglycosylated benzophenoxazine photosensitizer were shown in FIGS. 2 and 3, respectively. It could be confirmed that the present invention successfully synthesized the aforementioned target product.

Example 2 Inhibition of Proliferation of Cells of Cutaneous Squamous Cell Carcinoma by a Diglycosylated Benzophenoxazine Photosensitizer

(20) The MTT method is a method for detecting cell survival and growth. The detection principle of it is that a succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTTs to water-insoluble blue-violet crystalline formazan and deposit the formazan in the cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and the light absorbance value of it is measured at 490 nm wavelength with an enzyme-linked immunometric meter, which can indirectly reflect the number of living cells.

(21) (1) Experimental Method

(22) The cell line A-431 of cutaneous squamous cell carcinoma was cultured in a DMEM medium that contained 10% calf serum, added with 100 IU/L of penicillin and 100 mg/L of streptomycin, and placed in a constant-temperature incubator at 37° C. and containing 5% CO.sub.2 for conventional culture. After the cells grew to confluence on the bottom of the flask, the medium was discarded, the cells were rinsed twice with a PBS solution, and then digested with 0.25% trypsin and 0.02% EDTA for 5 minutes, and then subcultured into flasks once every 2 to 3 days. The cells at the logarithmic growth phase were taken for experiment.

(23) A-431 cells at the logarithmic growth phase were digested with trypsin, and inoculated into a six-well culture plate, and cultured in a 5% CO.sub.2 incubator at 37° C. When grew to the confluence of 80%-90%, the cells were respectively added with distilled water and 100 nM, 200 nM, 400 nM, 600 nM and 800 nM of the diglycosylated benzophenoxazine photosensitizer referred in the present invention. After incubation for 1 h, the cells were irradiated with 20 J/cm.sup.2 of red light for 15 min. 16 h later, the survival and growth status of the cells were detected by the MTT method.

(24) (2) Experimental Result

(25) The MTT experimental results show that, the diglycosylated benzophenoxazine photosensitizer referred in the present invention can significantly inhibit the proliferation of the A-431 cells, and as the dose of the photosensitizer increases, the inhibition effect on cell proliferation increases. The diglycosylated benzophenoxazine photosensitizer referred in the present invention can achieve a proliferation inhibition effect of 80% at 800 nM. At the same time, the pathological detection results show that the diglycosylated benzophenoxazine photosensitizer referred in the present invention can be specifically enriched in the cells of cutaneous squamous cell carcinoma, and is more concentrated on the membrane structures of important organelles such as the nuclear envelope and endoplasmic reticulum membrane (FIG. 4).

Example 3: Inhibition of Proliferation of A-431 Cells by the Diglycosylated Benzophenoxazine Photosensitizer by Inhibiting Wnt Signaling

(26) The Wnt signaling pathway mainly refers to a classic Wnt signaling pathway mediated by β-Catenin. In the absence of the Wnt signaling, GSK3β can add a phosphate group to the serine/threonine residue at the N-terminus of β-Catenin. The phosphorylated β-Catenin is covalently modified by β-TRCP ubiquitination and then degraded by a proteasome. PP2A dephosphorylation of GSK3β in the Wnt pathway can significantly promote the Wnt signaling. Therefore, by measuring the phosphorylation statuses of GSK3β, β-Catenin and PP2A, we can directly understand whether the Wnt signaling is activated. C-myc and Cyclin D1 as protooncogenes can promote division and translation of the cells of cutaneous squamous cell carcinoma, and ultimately promote cell proliferation. By observing C-myc and Cyclin D1, we can clearly understand the inhibition effect of the photosensitizer of the present invention on proliferation of the A-431 cells.

(27) (1) Experimental Method

(28) The cell line A-431 of cutaneous squamous cell carcinoma was cultured in a DMEM medium that contained 10% calf serum, added with 100 IU/L of penicillin and 100 mg/L of streptomycin, and placed in a constant-temperature incubator at 37° C. and containing 5% CO.sub.2 for conventional culture. After the cells grew to confluence on the bottom of the flask, the medium was discarded, the cells were rinsed twice with a PBS solution, and then digested with 0.25% trypsin and 0.02% EDTA for 5 minutes, and then subcultured into flasks once every 2 to 3 days. The cells at the logarithmic growth phase were taken for experiment.

(29) A-431 cells at the logarithmic growth phase were digested with trypsin, and inoculated into a six-well culture plate, and cultured in a 5% CO.sub.2 incubator at 37° C. When grew to the confluence of 80%-90%, the cells in each well were added with the diglycosylated benzophenoxazine photosensitizer referred in the present invention until the final concentrations were respectively 0 nM, 100 nM, 200 nM, 400 nM, and 800 nM. After incubation for 1 h, the cells were irradiated with 20 J/cm.sup.2 of red light for 15 min. Respectively after 0.5 h and 16 h, the cells were collected to extract total protein, and the p-GSK3β, p-PP2A, p-β-catenin, C-myc, Cyclin D1 and β-Catenin in the cells were detected by the Western Blot method.

(30) (2) Experimental Result

(31) By analysis of Western Blot results of GSK3β, PP2A, β-catenin phosphorylation of the A-431 cells treated with gradient doses of the diglycosylated benzophenoxazine photosensitizer, it is found that the photosensitizer doses of 200 to 800 nM can significantly inhibit the phosphorylation of PP2A and GSK3β. The 100 nM and 400 to 800 nM of the diglycosylated benzophenoxazine photosensitizer referred in the present invention can significantly promote the phosphorylation of β-catenin (FIG. 5). By analysis of the Western Blot results of the expression quantities of β-catenin, Cyclin D1 and C-myc in A-431 cells treated with the gradient doses of the diglycosylated benzophenoxazine photosensitizer, it is found that the expression quantities of β-Catenin, Cyclin D1 and C-myc are increased slightly after treatment with the diglycosylated benzophenoxazine photosensitizer at doses of 100 to 400 nM, but significantly decreased after treatment with the diglycosylated benzophenoxazine photosensitizer at doses of 600 and 800 nM. The results indicate that 600 nM and 800 nM of the diglycosylated benzophenoxazine photosensitizer can significantly inhibit the proliferation of A-431 cells (FIG. 6).