Silicon phthalocyanine conjugate axially and asymmetrically disubstituted by temozolomide and Boc-1-methyl-D-tryptophan as well as preparation method and application of silicon phthalocyanine conjugate

Abstract

The invention discloses a temozolomide and Boc-1-methyl-D-tryptophan axial asymmetric disubstituted silicon phthalocyanine conjugate as well as a preparation method and application of the temozolomide and Boc-1-methyl-D-tryptophan axial asymmetric disubstituted silicon phthalocyanine conjugate. 1, 3-diimino isoindoline is used as an original raw material, dichlorosilicon phthalocyanine is generated through a ring formation reaction, silicon phthalocyanine with double chains being tetraethylene glycol is obtained through a nucleophilic reaction, and finally, a target product DTPC is generated through an esterification reaction. The target product DTPC can enhance targeting and killing effects on cancer cells, can resist angiogenesis and effectively and synergistically promote anti-tumor immunity, and provides a new thought for improving photodynamic therapy.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a silicon phthalocyanine conjugate with axially asymmetric double substitution of temozolomide and Boc-1-methyl-D-tryptophan, and a preparation method and application thereof. Background Art

[0002] Cancer is a major disease endangering human health. Currently, the treatment methods include radiotherapy, chemotherapy, photodynamic therapy, surgical resection, etc. Among them, photodynamic therapy has great potential because of its selectivity, minimally invasive nature, and relatively low toxicity. It can not only destroy in-situ tumor cells and tissues with reactive oxygen species, but also inhibit the angiogenesis around tumors. At the same time, it can stimulate anti-tumor immunity through immunogenic cell death, and is expected to combat the recurrence and metastasis of malignant tumors with greater treatment difficulty. However, the effects of these actions need to be further improved. The photosensitizers used in photodynamic therapy are mainly porphyrins and phthalocyanines. Compared with zinc phthalocyanine, copper phthalocyanine, etc., two different functional drugs can be simultaneously connected to the axial position of silicon phthalocyanine, which can not only inhibit the aggregation behavior between phthalocyanine molecules, but also is expected to improve the photodynamic therapy effect.

[0003] As a negative feedback enzyme, IDO is highly expressed in tumor cells and can catalyze the conversion of tryptophan into kynurenine, causing local tryptophan depletion, and then affecting the activity and function of CD8 + T cells. The increase of kynurenine will promote the overexpression of regulatory T cells, and ultimately induce tumor immune escape. IDO enzyme inhibitors such as 1-methyl-D-tryptophan can activate CD8 + T cells, effectively inhibiting the recurrence and metastasis of tumors; the chemotherapy drug temozolomide not only has cytotoxicity, is expected to synergistically induce immunogenic cell death caused by photodynamic therapy, but also has the effect of inhibiting angiogenesis and the ability to inhibit tumor metastasis.

[0004] The present invention proposes to connect temozolomide and the active domain Boc-1-methyl-D-tryptophan of IDO enzyme inhibitor with dichlorosilicon phthalocyanine through a tetraethylene glycol chain, so as to improve the targeting, killing ability, angiogenesis inhibition and anti-tumor immune activity effects of photosensitive drugs on malignant tumors, and combat malignant tumors efficiently and precisely from multiple angles. Summary of the Invention

[0005] Aiming at the problem that photosensitive drugs have limited effects on the recurrence and metastasis of malignant tumors with great treatment difficulty, the present invention provides a silicon phthalocyanine conjugate with axially asymmetric double substitution of temozolomide and Boc-1-methyl-D-tryptophan, and a preparation method and application thereof, which not only improve its tumor targeting, but also enhance the killing activity of photosensitizers on tumor cells, the angiogenesis inhibition effect and the anti-tumor immune effect from multiple dimensions.

[0006] To achieve the above invention purpose, the present invention adopts the following technical solutions: A temozolomide and Boc-1-methyl-D-tryptophan axially asymmetrically disubstituted silicon phthalocyanine conjugate, with the chemical structural formula as

[0007] The preparation method of the above axially asymmetrically disubstituted phthalocyanine complex includes the following steps: 1) Add THF dissolved with 1-methyl-tryptophan and NaHCO3 into water, stir at room temperature for 30 - 60 min, add di-tert-butyl dicarbonate, react at room temperature for 24 - 48 h, adjust the pH value to strong acid condition with HCl aqueous solution, extract with EtOAc for several times, collect the organic layer, wash the organic layer with saturated brine, and dry with anhydrous Na2SO4 to obtain compound Boc-1-methyl-D-tryptophan, whose chemical structural formula is: 2) Dissolve 1,3-diaminoisoindoline and silicon tetrachloride in quinoline, heat and stir at 220 °C for 2 - 10 h under N2 protection. After the reaction, filter while it is hot, wash the filter cake with a mixed solution of quinoline, toluene, methanol and acetone for several times, and dry in vacuum to obtain compound SiPC, whose chemical structural formula is: 3) Dissolve SiPC, tetraethylene glycol and pyridine in toluene, heat and react at 110 - 130 °C for 24 - 48 h under N2 protection. First, extract the obtained reaction solution with HCl aqueous solution, then wash the separated organic phase with water, saturated sodium bicarbonate aqueous solution and saturated brine respectively, dry with anhydrous Na2SO4, concentrate under reduced pressure, and subject the obtained crude product to column chromatography on neutral silica gel using a mixed solution of DCM and CH3OH as the eluent to obtain compound 2TEG-PC, whose chemical structural formula is: 4) Dissolve 2TEG-PC, Boc-1-methyl-D-tryptophan and DMAP in DCM, add EDCI under ice bath condition, react at room temperature for 12 - 24 h. First, extract the obtained reaction solution with HCl aqueous solution, then wash the separated organic phase with water, saturated sodium bicarbonate aqueous solution and saturated brine respectively, dry with anhydrous Na2SO4, concentrate under reduced pressure, and subject the obtained crude product to column chromatography on neutral silica gel using a mixed solution of DCM and CH3OH as the eluent to obtain compound D-PC, whose chemical structural formula is: 5) Dissolve compound D-PC, temozolomide acid, and DMAP in DCM. Add EDCI under ice bath conditions and react at room temperature for 24 - 48 h. First, extract the resulting reaction solution with aqueous HCl solution. Wash the separated organic phase with water, saturated sodium bicarbonate aqueous solution, and saturated brine successively, dry it with anhydrous Na2SO4, and concentrate it under reduced pressure. The obtained crude product is passed through a neutral silica gel column using a mixed solution of DCM and CH3OH as the eluent. Collect the product and repeat passing through the neutral silica gel column several times under the same elution and purification conditions, and then purify it through a gel column to obtain the target product DTPC, whose chemical structural formula is: In the above step 1), the molar ratio of 1-methyl-D-tryptophan, NaHCO3, and di-tert-butyl dicarbonate is 1:1.5:1.5 - 1:6:6.

[0008] In the above step 2), the molar ratio of 1,3-diaminoisoindoline and silicon tetrachloride is 1:1 - 1:8.

[0009] In the above step 3), the molar ratio of SiPC, tetraethylene glycol, and pyridine is 1:4:5 - 1:50:10.

[0010] In the above step 4), the molar ratio of 2TEG-PC, Boc-1-methyl-D-tryptophan, DMAP, and EDCI is 1:1:5:5 - 4:1:20:20.

[0011] In the above step 5), the molar ratio of D-PC, temozolomide acid, DMAP, and EDCI is 1:2:5:5 - 1:4:20:20.

[0012] The application of the above-mentioned axially asymmetric double-substituted silicon phthalocyanine conjugate of temozolomide and Boc-1-methyl-D-tryptophan in the preparation of a photodynamic targeted anti-tumor drug.

[0013] The beneficial effects of the present invention are as follows: 1) The silicon phthalocyanine conjugate synthesized in the present invention has a stable structure, no isomers, is easy to synthesize, and is conducive to industrial production; 2) In the silicon phthalocyanine conjugate of the present invention, the active domains of Boc-1-methyl-D-tryptophan and temozolomide can inhibit angiogenesis, stimulate the immune response of tumors, and inhibit the recurrence and metastasis of tumors. Description of the Drawings

[0014] Figure 1 It is a process route diagram for synthesizing Boc-1-methyl-D-tryptophan, temozolomide, and phthalocyanine complex in the present invention.

[0015] Figure 2UV-Vis absorption spectra of DTPC, 2TPC and 2DPC in DMF.

[0016] Figure 3 Dark toxicity (left) and phototoxicity (right) of DTPC, 2TPC and 2DPC in 4T1 tumor cells.

[0017] Figure 4 Inhibitory graphs of migration and invasion of DTPC, 2TPC and 2DPC against 4T1 cells.

[0018] Figure 5 Inhibitory graph of migration of DTPC, 2TPC and 2DPC against Eahy 926 cells. Detailed implementation manners

[0019] To make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific implementation manners, but the present invention is not limited thereto.

[0020] Example: Preparation of axially asymmetric disubstituted silicon phthalocyanine conjugate of temozolomide and Boc-1-methyl-D-tryptophan. Using 1,3-diaminoisoindoline as the raw material, dichlorosilicon phthalocyanine is generated through a cyclization reaction, and silicon phthalocyanine with a double chain of tetraethylene glycol is obtained through a nucleophilic reaction. Finally, the target product DTPC is generated through an esterification reaction. The specific steps are as follows: 1) Add 22 mL of THF containing 763.88 mg (3.5 mmol) of 1-methyl-D-tryptophan and 882.105 mg (10.5 mmol) of NaHCO3 to 22 mL of water, stir at room temperature for 30 min, add 1.1458 g (5.25 mmol) of di-tert-butyl dicarbonate, react at room temperature for 24 h, adjust the pH value of the obtained reaction solution to 1 with 1 M HCl aqueous solution, extract three times with EtOAc, collect the organic layer, wash the organic layer with saturated brine, and dry with anhydrous Na2SO4 to obtain 1.05 g of compound Boc-1-methyl-D-tryptophan, with a yield of 94.3%. Its chemical structural formula is: 1 H NMR (600 MHz, DMSO-d6) δ 7.53 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.13 (dd, J = 15.2, 7.4 Hz, 2H), 7.02 (t, J = 7.4 Hz, 1H), 6.98 (d, J = 6.8 Hz, 1H), 4.13 (dd, J = 17.3, 4.8 Hz, 1H), 3.72 (s, 3H), 3.14–2.94 (m, 2H), 1.33 (s, 9H). HRMS (ESI): Calcd C 25 H 38N2O8[M+H] + : 495.2701, found: 495.2744。

[0021] 2) Dissolve 3.73 g (0.0257 mmol) of 1,3-diaminoisobenzofuran and 4 mL of silicon tetrachloride in 42 mL of quinoline. Heat and stir the reaction at 220 °C for 2.5 h under N2 protection. After the reaction, filter while it is hot. Wash the filter cake three times with a mixed solution of quinoline:toluene:methanol:acetone = 1:3:5:3 (volume ratio), and dry it in vacuum at room temperature. 3.56 g of a purple-red, powdery compound SiPC is collected, with a yield of 65.69%. Its chemical structural formula is: SiPC and its preparation method have been disclosed in the reference [Juan-Juan, Chen, Yi-Zhen, et al. Silicon Phthalocyanines Axially Disubstituted with Erlotinib towards Small Molecular Target-Based Photodynamic Therapy. [J]. Chemmedchem, 2017. DOI: 10.1002 / cmdc.201700384.].

[0022] 3) Dissolve 244 mg (0.4 mmol) of SiPC, 691.75 μL (4 mmol) of tetraethylene glycol and 26 μL of pyridine in 13 mL of toluene. Heat the reaction at 120 °C for 24 h under N2 protection. First, extract the obtained reaction solution with 0.5 M HCl aqueous solution. Wash the separated organic phase successively with water, saturated sodium bicarbonate aqueous solution and saturated brine, dry it with anhydrous Na2SO4, and concentrate it under reduced pressure. The obtained crude product is passed through a neutral silica gel column using a mixed solution of DCM:CH3OH = 20:1 (volume ratio) as the eluent. 246 mg of a blue solid compound 2TEG-PC is collected, with a yield of 66.4%. Its chemical structural formula is: 1 H NMR (500 MHz, DMSO-d6) δ 9.67 (dd, J = 5.6, 3.0 Hz, 8H), 8.51 (dd, J = 5.7, 2.9 Hz, 8H), 4.46 (t, J = 5.6 Hz, 2H), 3.35 (d, J = 5.3 Hz, 4H), 3.19 (t, J = 5.3 Hz, 4H), 3.12–3.08 (m, 4H), 2.86–2.82 (m, 4H), 2.32–2.28 (m, 4H), 1.59–1.55 (m, 4H), 0.34–0.24 (m, 4H), -2.04 (d, J = 5.2 Hz, 4H); HRMS (ESI): Calcd C48 H 50 N8O 10 Si[M+Na] + : 949.3311 found: 949.3355。

[0023] 4) Dissolve 35 mg (0.0378 mmol) of 2TEG-PC, 8.023 mg (0.0252 mmol) of Boc-1-methyl-D-tryptophan, and 48.3084 mg (0.252 mmol) of 4-dimethylaminopyridine (DMAP) in 2.835 mL of DCM. Add 30.79 mg (0.252 mmol) of EDCI under ice bath conditions and react at room temperature for 8 h. First, extract the resulting reaction solution with 0.5 M aqueous HCl solution. Then, wash the separated organic phase successively with water, saturated aqueous sodium bicarbonate solution, and saturated brine, dry it with anhydrous Na2SO4, and concentrate it under reduced pressure. The obtained crude product is passed through a neutral silica gel column using a mixed solution of DCM:CH3OH = 40:1 (volume ratio) as the eluent to collect 18.3 mg of the blue compound D-PC with a yield of 60.8%. Its chemical structural formula is: 1 H NMR (600 MHz, DMSO-d6) δ 9.66 (dd, J = 5.5, 3.0 Hz, 8H), 8.49 (dd, J = 5.7, 2.8 Hz, 8H), 7.40 (d, J = 8.7 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 7.02 (d, J = 5.3 Hz, 1H), 6.91–6.88 (m, 1H), 4.26 (t, J = 7.1 Hz, 1H), 3.52–3.46 (m, 3H), 3.36–3.35 (m, 4H), 3.20–3.13 (m, 8H), 3.08–3.05 (m, 2H), 2.82 (dt, J = 20.5, 4.9 Hz, 4H), 2.32–2.27 (m, 4H), 1.56 (dt, J = 15.8, 5.0 Hz, 4H), 1.31–1.24 (m, 9H), 0.33–0.29 (m, 2H), 0.29–0.24 (m, 2H), -2.07 (dt, J = 15.5, 5.2 Hz, 4H). HRMS (ESI): Calcd C 65 H 70 N 10 O 13 Si[M+Na] + : 1249.4785, found: 1249.479。

[0024] 5) Dissolve 50 mg (0.054 mmol) of D-PC, 63.23 mg (0.324 mmol) of temozolomide acid, and 103.52 mg (0.54 mmol) of DMAP in 7.2 mL of DCM. Add 65.97 mg (0.54 mmol) of EDCI under ice bath conditions and react at room temperature for 24 h. Extract the resulting reaction solution with 0.5 M aqueous HCl solution first. Wash the separated organic phase successively with water, saturated aqueous sodium bicarbonate solution, and saturated brine, dry it with anhydrous Na2SO4, and concentrate it under reduced pressure. Purify the obtained crude product by passing it through a neutral silica gel column with a mixed solution of DCM:CH3OH = 100:1 (v / v) as the eluent, collect the product, repeat passing through the neutral silica gel column once under the same elution and purification conditions, and then purify it by passing through a Sephadex LH-20 gel column with THF as the mobile phase to obtain 22 mg of dark blue pure target product DTPC with a yield of 29%. Its chemical structural formula is: 1 H NMR (600 MHz, DMSO-d6) δ 9.66 (dd, J = 5.6, 2.9 Hz, 8H), 8.81 (s, 1H), 8.49 (dd, J = 5.7, 2.7 Hz, 8H), 7.40 (d, J = 7.2 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.12 (d, J = 7.1 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 7.02 (d, J = 5.5 Hz, 1H), 6.91–6.88 (m, 1H), 4.27–4.23 (m, 1H), 3.83 (s, 3H), 3.60 (s, 3H), 3.35 (d, J = 2.3 Hz, 4H), 3.20–3.13 (m, 8H), 3.08–3.04 (m, 2H), 2.85–2.82 (m, 2H), 2.82–2.79 (m, 2H), 2.32–2.27 (m, 4H), 1.56 (dt, J = 15.4, 4.9 Hz, 4H), 1.31–1.24 (m, 9H), 0.33–0.29 (m, 2H), 0.29–0.24 (m, 2H), -2.07 (dt, J = 15.5, 5.2 Hz, 4H). HRMS (ESI): Calcd C 71 H 73 N 15 O 15 Si[M+Na] + : 1426.5072, found: 1426.5047.

[0025] Comparative Example: Synthesis of 2DPC Dissolve 15 mg (0.0162 mmol) of 2TEG-PC, 20.62 mg (0.065 mmol) of Boc-1-methyl-D-tryptophan, and 31.1 mg (0.162 mmol) of DMAP in 2.1 mL of DCM. Add 19.79 mg (0.162 mmol) of EDCI under ice bath conditions and react at room temperature for 24 h. First, extract the resulting reaction solution with 0.5 M aqueous HCl solution. Then, wash the separated organic phase successively with water, saturated aqueous sodium bicarbonate solution, and saturated brine, dry it with anhydrous Na2SO4, and concentrate it under reduced pressure. The obtained crude product is passed through a neutral silica gel column using a mixed solution of DCM:CH3OH = 40:1 (v / v) as the eluent, collect the product, and repeat passing through the neutral silica gel column 3 times under the same elution and purification conditions to obtain 8.73 mg of the blue compound 2DPC with a yield of 35.3%. 1 H NMR (600 MHz, DMSO-d6) δ 9.65 (dd, J = 5.5, 3.0 Hz, 8H), 8.48 (dd, J = 5.8, 2.8 Hz, 8H), 7.40 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.12 (d, J = 7.8 Hz, 2H), 7.06–7.00 (m, 4H), 6.92–6.88 (m, 2H), 4.16–4.09 (m, 2H), 3.60 (s, 6H), 3.33 (d, J = 1.0 Hz, 4H), 3.30–3.20 (m, 4H), 3.06 (t, J = 4.8 Hz, 4H), 3.03–2.88 (m, 4H), 2.82–2.78 (m, 4H), 2.28 (t, J = 5.1 Hz, 4H), 1.55 (dd, J = 5.8, 4.4 Hz, 4H), 1.29 (s, 18H), 0.26 (t, J = 5.2 Hz, 4H), -2.08 (t, J = 5.2 Hz, 4H). HRMS (ESI): Calcd C 82 H 90 N 12 O 16 Si[M+Na] + : 1565.5999, found: 1565.5968. The chemical structural formula of 2DPC is as follows:

[0026] Comparative Example: Synthesis of 2TPC Dissolve 50 mg (0.054 mmol) of 2TEG-PC, 63.23 mg (0.324 mmol) of TMZ, and 103.52 mg (0.54 mmol) of DMAP in 7.29 mL of DCM. First, extract the resulting reaction solution with 0.5 M aqueous HCl solution. Wash the separated organic phase successively with water, saturated aqueous sodium bicarbonate solution, and saturated brine, dry it over anhydrous Na2SO4, and concentrate it under reduced pressure. The obtained crude product is purified by passing through a neutral silica gel column using a mixed solution of DCM:CH3OH = 40:1 (v / v) as the eluent. Collect the product and repeat passing through the neutral silica gel column 3 times under the same elution and purification conditions to obtain 20 mg of the blue compound 2TPC with a yield of 28.9%. 1 H NMR (600 MHz, DMSO-d6) δ 9.66 (dd, J = 5.5, 3.0 Hz, 8H), 8.81 (s, 2H), 8.50 (dd, J = 5.5, 2.9 Hz, 8H), 3.83 (s, 6H), 3.52–3.48 (m, 4H), 3.32 (d, J = 1.1 Hz, 4H), 3.20–3.16 (m, 4H), 2.87–2.81 (m, 4H), 2.33–2.28 (m, 4H), 1.60–1.54 (m, 4H), 0.31 (d, J = 5.7 Hz, 4H), -2.05 (t, J = 5.2 Hz, 4H). HRMS (ESI): Calcd C 60 H 56 N 18 O 14 Si [M+Na] + : 1303.3885, found: 13030.388. The chemical structural formula of 2TPC is as follows:

[0027] Ultraviolet-visible absorption spectrum Dissolve 2DPC, 2TPC, and DTPC in DMF and measure the absorbance of 2DPC, 2TPC, and DTPC in DMF at different concentrations. The results are shown in Figure 2 .

[0028] Cytotoxicity experiment The MTT method, also known as the colorimetric method, is a method for detecting the survival status of cells. Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble formazan, which deposits in the cells, while dead cells cannot reduce MTT to formazan. Dimethyl sulfoxide can dissolve the formazan in the cells. By measuring the wavelength value at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader, the number of living cells can be indirectly reflected. Within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of cells.

[0029] Select 4T1 cells with good growth status under a microscope. When the cell density reaches about 90%, digest with 1 mL of trypsin (containing EDTA) for 4 minutes, then add 2 mL of medium to terminate digestion, pipette evenly, and then count the cells using a cell counting plate. Dilute the cell solution to 60,000 cells / mL with DMEM medium. Add 100 μL of the above cell culture solution to each well of a 96-well plate. Set 5 replicates for each concentration of DTPC, 2TPC, and 2DPC drugs, and set up a cell blank group and a solvent blank group (5 replicates for each group). Place the 96-well plate in an incubator at 37°C for 24 h.

[0030] Drug addition: Dilute DTPC, 2TPC, and 2DPC to 7 different concentrations with DMSO containing 5% castor oil respectively, and then dilute the above 7 concentrations of drugs 100-fold with DMEM medium. Use a multi-channel pipette to aspirate the old medium in the 96-well plate, add DMSO containing 5% CEL diluted 100-fold to the cell control group, add DMEM medium to the solvent control group, and add the above-prepared solutions to the remaining drug control groups respectively. Continue to place the 96-well plate in the incubator for 18 h.

[0031] Light irradiation: Take out the 96-well plate, carefully aspirate the drug-containing medium in the wells with a multi-channel pipette, then wash each well 3 times with physiological saline to remove the unabsorbed drugs, and finally add 100 μL of medium to each well. Then irradiate with an LED lamp at 670 nm for 2 min, and finally place the irradiated 96-well plate back into the incubator for further incubation.

[0032] Measure the OD value: Add 1 μL of 5 mg / mL MTT solution to each well of the 96-well plate, then place the 96-well plate in an incubator at 37°C for 2 h. Then aspirate the solution in each well with a multi-channel pipette, add 100 μL of DMSO solution to each well again, shake on a shaker for 30 min, and measure the absorbance at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0033] Calculation: Calculate the cell survival rate using the measured absorbance. The calculation formula is as follows: Cell survival rate (%) = (absorbance of the sample group - absorbance of the solvent blank group) / (absorbance of the cell blank group - absorbance of the solvent blank group) × 100.

[0034] Figure 3 It is a comparison chart of the toxicity of DTPC, 2TPC, and 2DPC to 4T1 cells under light and dark conditions. As can be seen from the figure, the simultaneous conjugation of the two drugs can enhance the toxic effect of silicon phthalocyanine photosensitizer on tumor cells.

[0035] Cell migration experiment The scratch assay is a commonly used method to simulate and analyze the migration ability of tumor cells in vitro. The principle of this method is that when the cells reach confluence, a blank area is artificially created on the monolayer of cells, called a "scratch". The edge area of the scratch will gradually grow and migrate into the blank area, thus healing it. To a certain extent, it mimics the process of tumor cell migration in vivo and is very suitable for analyzing cell-cell interactions. By comparing the sizes of the areas before and after the scratch, the migration ability of the cells can be judged.

[0036] Select 4T1 cells and Eahy 926 cells with good growth status under the microscope. When the cell density reaches about 90%, digest with 1 mL of trypsin (containing EDTA) for 4 minutes, then add 2 mL of medium to terminate digestion, pipette evenly, then count with a cell counting plate, and dilute the cell solution with DMEM medium to 300,000 cells / mL. Add 2 mL of the above cell culture medium solution to each well of a 6-well plate (the well plate has been marked), and place the 6-well plate in a 37 °C, 5% carbon dioxide incubator overnight.

[0037] Scratch: The next day, use a 100 μL sterile pipette tip perpendicular to the cell plane to make a scratch on the cell layer at a right angle to the previous mark (try to use the same pipette tip between different wells).

[0038] Washing: After the scratch is made, wash three times with sterile normal saline to wash away non-adherent cells and cells floating at the scratch, so that the gap after scratching is clearly visible.

[0039] Drug addition: Prepare a medium containing 1% fetal bovine serum with a drug concentration of 250 nM, and add 2 mL of each drug to the 6-well plate.

[0040] Photographing: Put the cells into a 37 °C, 5% carbon dioxide incubator, take out the 6-well plate at appropriate times (0, 12, and 24 h), observe the width of the scratch under the microscope and take pictures.

[0041] Calculation: Use Image J software to open and calculate the area of the scratch, and use GraphPad Prism 6.0 statistical analysis software to process the data. The experimental results are expressed as Means±SD.

[0042] Figure 4 A is the migration inhibition graph of Control, 2DPC, 2TPC, and DTPC in 4T1 cells; Figure 5 is the migration inhibition graph of Control, 2DPC, 2TPC, and DTPC in Eahy 926 cells. It can be seen from the figure that the simultaneous conjugation of the two drugs can enhance the migration inhibitory ability of silicon phthalocyanine photosensitizer on cells.

[0043] Cell invasion assay The invasion experiment is mainly used to detect the in vitro invasion ability of tumor cells. Its principle is to separate a high-nutrient culture medium and a low-nutrient culture medium with a polycarbonate membrane. Tumor cells are inoculated into the low-nutrient culture medium, and the pores of the membrane are covered with Matrigel to mimic the extracellular matrix. Tumor cells can enter the lower chamber only by secreting hydrolases and passing through the filter membrane coated with Matrigel through amoeboid movement. By counting the cells that enter the lower chamber, the invasion ability of tumor cells can be evaluated.

[0044] Experiment preparation: One day before the experiment, the aliquoted Matrigel (16 μL) was taken out from -20 °C and placed in a 4 °C refrigerator overnight. The colloid changed from solid to liquid state. The chamber was sterilized under ultraviolet light overnight. 784 μL of serum-free medium was added to every 16 μL of Matrigel, and Matrigel (100 μL per well) was spread on the chamber and placed in an incubator at 37 °C and 5% carbon dioxide for 5 h.

[0045] Seeding: Under the microscope, 4T1 cells with good growth status were selected. When the cell density reached about 90%, they were digested with 1 mL of trypsin (containing EDTA) for 4 minutes, then 2 mL of medium was added to terminate the digestion, and the cells were pipetted evenly. Then, they were counted using a cell counting plate, and the cell solution was diluted to 2 million cells / mL with serum-free DMEM medium and pipetted evenly. 100 μL of the counted medium and 100 μL of the prepared drug (250 nM) were mixed evenly. 100 μL of the cell suspension was pipetted into the upper chamber of the chamber, and 500 μL of medium containing 10% fetal bovine serum (FBS) was added to the 24-well plate (note that no bubbles should be generated between the lower medium and the chamber), and then placed in the incubator for 24 h.

[0046] Fixation: The next day, the 24-well plate was taken out, and the chamber was washed twice with precooled normal saline at 4 °C, and then fixed with methanol for 20 minutes. The chamber was taken out and dried (placed in a fume hood for 10 minutes).

[0047] Staining: 500 μL of 0.1% crystal violet was added, and the chamber was stained at room temperature for 20 minutes. Then, the cells on the upper layer of the microporous membrane of the chamber were carefully wiped off with a cotton swab, and the chamber was washed twice with normal saline.

[0048] Photographing and calculation: Photographs were taken under an inverted microscope. Subsequently, an aqueous solution containing 33% acetic acid was added to the 24-well plate to dissolve the crystal violet in the lower chamber of the chamber. After mixing evenly, the solution was aspirated into a 96-well plate, with 5 replicates in each group and 100 μL in each well. The OD value at 570 nm was measured, and the data were processed using GraphPad Prism 6.0 statistical analysis software. The experimental results were expressed as Means ± SD.

[0049] Figure 4 B and Figure 4C shows the invasion inhibition and quantification graphs of Control, 2DPC, 2TPC, and DTPC in 4T1 cells. From the graph analysis, the simultaneous conjugation of the two drugs can enhance the invasion inhibitory ability of silicon phthalocyanine photosensitizer on cells.

[0050] The above are only the preferred embodiments of the present invention. Any equivalent structural or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. An axially asymmetrically disubstituted silicon phthalocyanine conjugate of temozolomide and Boc-1-methyl-D-tryptophan, characterized in that: The chemical structural formula is 2. A method for preparing an axially asymmetrically disubstituted phthalocyanine complex as described in claim 1, characterized in that: It includes the following steps: 1) Add THF dissolved with 1-methyl-tryptophan and NaHCO3 into water, stir at room temperature for 30 - 60 min, add di-tert-butyl dicarbonate, react at room temperature for 24 - 48 h, adjust the pH value to strong acid condition with HCl aqueous solution, extract with EtOAc for several times, collect the organic layer, wash the organic layer with saturated brine, and dry with anhydrous Na2SO4 to obtain compound Boc-1-methyl-D-tryptophan, whose chemical structural formula is: 2) Dissolve 1,3-diaminoisoindoline and silicon tetrachloride in quinoline, heat and stir the reaction at 220 °C for 2 - 10 h under N2 protection. After the reaction, filter while it is hot, wash the filter cake with a mixed solution of quinoline, toluene, methanol and acetone for several times, and dry under vacuum to obtain compound SiPC, whose chemical structural formula is: 3) Dissolve SiPC, tetraethylene glycol and pyridine in toluene, heat and react at 110 - 130 °C for 24 - 48 h under N2 protection. First, extract the obtained reaction solution with HCl aqueous solution, and then wash the separated organic phase with water, saturated sodium bicarbonate aqueous solution and saturated brine respectively, dry with anhydrous Na2SO4, concentrate under reduced pressure. The obtained crude product is passed through a neutral silica gel column using a mixed solution of DCM and CH3OH as the eluent to obtain compound 2TEG-PC, whose chemical structural formula is: 4) Dissolve 2TEG-PC, Boc-1-methyl-D-tryptophan and DMAP in DCM, add EDCI under ice bath condition, react at room temperature for 12 - 24 h. First, extract the obtained reaction solution with HCl aqueous solution, and then wash the separated organic phase with water, saturated sodium bicarbonate aqueous solution and saturated brine respectively, dry with anhydrous Na2SO4, concentrate under reduced pressure. The obtained crude product is passed through a neutral silica gel column using a mixed solution of DCM and CH3OH as the eluent to obtain compound D-PC, whose chemical structural formula is: 5) Dissolve compound D-PC, temozolomide acid and DMAP in DCM, add EDCI under ice bath condition, react at room temperature for 24 - 48 h. First, extract the obtained reaction solution with HCl aqueous solution, and then wash the separated organic phase with water, saturated sodium bicarbonate aqueous solution and saturated brine respectively, dry with anhydrous Na2SO4, concentrate under reduced pressure. The obtained crude product is passed through a neutral silica gel column using a mixed solution of DCM and CH3OH as the eluent, collect the product, repeat passing through the neutral silica gel column several times under the same elution and purification conditions, and then purify through a gel column to obtain the target product DTPC, whose chemical structural formula is:

3. The preparation method of the axially asymmetrically disubstituted phthalocyanine complex according to claim 2, characterized in that: In step 1), the molar ratio of 1-methyl-D-tryptophan, NaHCO3 and di-tert-butyl dicarbonate is 1:1.5:1.5 - 1:6:

6.

4. The preparation method of the axially asymmetrically disubstituted phthalocyanine complex according to claim 2, characterized in that: In step 2), the molar ratio of 1,3-diaminoisoindoline and silicon tetrachloride is 1:1 - 1:

8.

5. The method for preparing an axially asymmetrically disubstituted phthalocyanine complex according to claim 2, characterized in that: In step 3), the molar ratio of SiPC, tetraethylene glycol and pyridine is 1:4:5 - 1:50:

10.

6. The preparation method of the multi-axially asymmetric double-substituted phthalocyanine complex according to claim 2, characterized in that: In step 4), the molar ratio of 2TEG-PC, Boc-1-methyl-D-tryptophan, DMAP and EDCI is 1:1:5:5 - 4:1:20:

20.

7. The preparation method of the axially asymmetrically disubstituted phthalocyanine complex according to claim 2, characterized in that: In step 5), the molar ratio of D-PC, temozolomide acid, DMAP, and EDCI is 1:2:5:5 to 1:4:20:

20.

8. Use of a silicon phthalocyanine conjugate with axial asymmetric double substitution of temozolomide and Boc-1-methyl-D-tryptophan as claimed in claim 1 in the preparation of a photodynamic targeted anti-tumor drug.

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