Dendrimer-modified phthalocyanine photosensitive compound and application thereof

Abstract

The invention provides a dendrimer-modified phthalocyanine photosensitive compound and application thereof, the dendrimer-modified phthalocyanine photosensitive compound has any one structure as shown in formulas I-IV, and the photosensitive compound has the advantages of simple and controllable synthesis, clear structure, strong light killing ability and the like. The water solubility, tumor targeting and biocompatibility of the zinc phthalocyanine photosensitizer can be improved, the defects of a traditional nano photosensitizer delivery system can be overcome, and the zinc phthalocyanine photosensitizer has a good application prospect in the aspect of photodynamic therapy of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of photodynamic therapy drugs, and particularly relates to a dendritic molecule modified phthalocyanine photosensitizer and application thereof. BACKGROUND

[0002] Photodynamic therapy (PDT) is a new type of local targeted therapy method, and its principle is that after a photosensitizer is aggregated to a lesion site, a specific wavelength of light is used for local irradiation of the target lesion site. This process triggers photosensitization reaction at the target site, producing reactive oxygen species (ROS) with cytotoxicity, thereby inducing target cell apoptosis. As a non-invasive treatment method, PDT has good prospects in the treatment of various cancers, especially in the treatment of superficial but extensive lesions such as tumor peritoneal metastasis, and PDT shows unique advantages.

[0003] The photosensitizer is a key component that determines the success of PDT, and current research on PDT also focuses on the exploration of photosensitizers. The physicochemical properties of the photosensitizer and its structural characterization under physiological conditions are crucial to the efficiency of photosensitization process. The second-generation photosensitizer phthalocyanine is a large ring conjugated system composed of four pyrrole structures and four nitrogen atoms, and has a series of advantages, including high quantum yield, good stability, wavelength absorption of 650-800 nm, low dark toxicity and fast in vivo metabolism, etc. The introduction of a metal ion Zn + in the center can improve the quantum yield and prolong the lifetime of the triplet state, thereby further improving the photosensitization performance and antitumor effect of phthalocyanine. However, zinc phthalocyanine has a hydrophobic defect and is easy to aggregate under physiological conditions, resulting in fluorescence quenching. Therefore, it is of great significance to optimize the existing zinc phthalocyanine and develop new photosensitizers with better water solubility, better efficacy and lower toxicity. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a dendritic molecule modified phthalocyanine photosensitizer and a preparation method and application thereof. The photosensitizer of the present application has the advantages of simple and controllable synthesis, clear structure and strong light killing ability, and not only can improve the water solubility, tumor targeting and biocompatibility of zinc phthalocyanine photosensitizer, but also can avoid the deficiencies of traditional nano photosensitizer delivery systems, and has good application prospects in the photodynamic therapy of tumors.

[0005] To achieve the purpose of the present application, the following technical solutions are adopted:

[0006] In a first aspect, the present application provides a dendritic molecule-modified phthalocyanine photosensitizing compound, said dendritic molecule-modified phthalocyanine photosensitizing compound having any one of the following structures of Formula I to IV:

[0007]

[0008]

[0009] wherein R1is selected from or -(CH2) x , wherein m, n are each independently selected from an integer from 0 to 5 (e.g., can be 0, 1, 2, 3, 4, or 5), and x is an integer selected from 2 to 8 (e.g., can be 2, 3, 4, 5, 6, 7, or 8);

[0010] R2, R3, R4, R5are selected from wherein p, q are each independently selected from an integer from 1 to 7 (e.g., can be 1, 2, 3, 4, 5, 6, or 7);

[0011] R' is selected from wherein R a is selected from any one of H, substituted or unsubstituted C1-C10 (e.g., can be C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10) straight chain acyl, substituted or unsubstituted benzoyl, biotin group, amino acid residue, C2-C10 straight chain alkenyl containing 1 to 3 carbon-carbon double bonds, or C2-C10 straight chain alkynyl containing 1 to 3 carbon-carbon triple bonds; R b is selected from any one of hydroxyl or C1-C10 (e.g., can be C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy, amino acid residue, C2-C10 straight chain alkenyl containing 1 to 3 carbon-carbon double bonds, or C2-C10 straight chain alkynyl containing 1 to 3 carbon-carbon triple bonds; any one or more methylene groups in the above-mentioned groups are optionally substituted with a heteroatom, and any one or more hydrogen atoms are optionally substituted with a methyl group or a halogen atom;

[0012] represents the attachment site of the group.

[0013] In the present application, the substituents of the C1-C10 acyl group and the substituents of the benzoyl group are each independently selected from halogen or C1-C10 (e.g., can be C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy.

[0014] Preferably, the R aany one selected from the group consisting of H, acetyl, propionyl, n-butyryl, isobutyryl, t-butoxyacetyl, trifluoroacetyl, pentafluoropropionyl, heptafluorobutyryl, biotin group, trichloroacetyl, benzoyl, 4-fluorobenzoyl, 2,3,4,5,6-pentafluorobenzoyl, 4-trifluoromethylbenzoyl, 3,4-dichlorobenzoyl, or 4-trichloromethylbenzoyl.

[0015] Preferably, the R b any one selected from the group consisting of H, acetyl, propionyl, n-butyryl, isobutyryl, t-butoxyacetyl, trifluoroacetyl, pentafluoropropionyl, heptafluorobutyryl, biotin group, trichloroacetyl, benzoyl, 4-fluorobenzoyl, 2,3,4,5,6-pentafluorobenzoyl, 4-trifluoromethylbenzoyl, 3,4-dichlorobenzoyl, or 4-trichloromethylbenzoyl.

[0016] In the present application, preferably, the dendrimer-modified phthalocyanine photosensitizing compound has any one of the following structures shown in Formulae V-VIII:

[0017]

[0018]

[0019] m, n are each independently an integer selected from -5 (for example, can be 1, 2, 3, 4, 5, etc.);

[0020] R2, R3, R4, R5, R' are the same as defined above.

[0021] Preferably, the dendrimer-modified phthalocyanine photosensitizing compound has any one of the following structures shown in Formulae IX-XII:

[0022]

[0023]

[0024] wherein q is an integer selected from 1-7 (for example, can be 1, 2, 3, 4, 5, 6, or 7);

[0025] R' is the same as defined above.

[0026] Preferably, the dendrimer-modified phthalocyanine photosensitizing compound has any one of the following structures shown in Formulae XIII-XVI:

[0027]

[0028]

[0029]

[0030] wherein, Ra is selected from any one of H, acetyl, propionyl, n-butyryl, isobutyryl, t-butoxy acyl, biotin group, trifluoroacetyl, pentafluoropropionyl, heptafluorobutyryl, trichloroacetyl, benzoyl, 4-fluorobenzoyl, 2,3,4,5,6-pentafluorobenzoyl, 4-trifluoromethylbenzoyl, 3,4-dichlorobenzoyl or 4-trichloromethylbenzoyl; preferably any one of H, t-butoxy acyl, biotin group, trifluoroacetyl, pentafluoropropionyl or heptafluorobutyryl.

[0031] Preferably, the dendritic molecule modified phthalocyanine photosensitizing compound is selected from any one of the following structures of formulae XVII-XX:

[0032]

[0033]

[0034]

[0035] wherein, R b is selected from any one of hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy or n-butoxy, preferably methoxy.

[0036] In the present application, the dendritic molecule modified phthalocyanine photosensitizing compound is selected from any one of the following compounds A1-A13:

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] In a second aspect, the present application provides a tumor-targeting photodynamic therapy drug, which comprises the dendritic molecule modified phthalocyanine photosensitizing compound as described in the first aspect.

[0043] Compared with the prior art, the present application has the following beneficial effects and outstanding advantages:

[0044] (1) The dendritic molecule modified phthalocyanine photosensitizing compound of the present application not only can produce effective photodynamic effect, but also has strong tumor-targeting enrichment effect, and thus can be used as a photosensitizing drug for tumor-targeting photodynamic therapy. Meanwhile, it has good fluorescence imaging characteristics and can be used as a tumor-targeting fluorescence probe to realize the integration of photodynamic therapy and imaging of tumors.

[0045] (2) The preferred dendritic molecule modified phthalocyanine photosensitizing compound of the present application has good tumor targeting and photodynamic therapy effect, has high anticancer activity in various cancer types such as gastric cancer, intestinal cancer, lung cancer, breast cancer and pancreatic cancer, shows high photodynamic therapy effect, and has high safety in in vivo experiments, thus having significant application prospect in the treatment of solid tumors.

[0046] (3) The dendritic molecule modified phthalocyanine photosensitizing compound of the present application can self-assemble in water, and the formation of the self-assembled body can reduce the aggregation of phthalocyanine and reduce the fluorescence quenching phenomenon.

[0047] (4) The preparation process of the dendritic molecule modified phthalocyanine photosensitizing compound of the present application is simple, stable in nature, easy to store, and conducive to large-scale preparation in industrial production, thus having good industrialization prospect. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1A UV-Vis absorption spectrum of unchemically modified phthalocyanine and dendritic molecule modified phthalocyanine photosensitizing molecule.

[0049] Figure 1B Fluorescence spectrum of unchemically modified phthalocyanine and dendritic molecule modified phthalocyanine photosensitizing molecule.

[0050] Figure 2 Fluorescence imaging characteristics of dendritic photosensitizing molecule.

[0051] Figure 3 Uptake ability evaluation of dendritic photosensitizing molecule in gastric normal epithelial cells (GES-1) and gastric cancer cells (SNU638 and SNU668).

[0052] Figure 4 Photodynamic killing effect of dendritic photosensitizing molecule in different solid tumor cells, wherein a-b are gastric cancer cells (AGS and SNU638, respectively), c is an intestinal cancer cell (HCT116), d is a breast cancer cell (T47D), e is a lung cancer cell (A549), and f is a pancreatic cancer cell (PANC-1).

[0053] Figure 5 Penetration effect of dendritic photosensitizing molecule on gastric cancer tumor spheroids.

[0054] Figure 6A Bright field photos of tumor spheroids of MFC treated with dendritic photosensitizing molecule at different time points.

[0055] Figure 6B Bright field photos of tumor spheroids of SNU668 treated with dendritic photosensitizing molecule at different time points.

[0056] Figure 6C Growth of tumor spheroids treated with dendritic photosensitizer.

[0057] Figure 6D Growth of tumor spheroids treated with dendritic photosensitizer.

[0058] Figure 7 In vivo distribution of dendritic photosensitizer. a, Bioluminescence images of peritoneal metastasis and major organs (heart, liver, spleen, lung and kidney); b, Fluorescence images of A9; c, Average fluorescence intensity of A9 in peritoneal metastasis and major organs (heart, liver, spleen, lung and kidney).

[0059] Figure 8 Dynamic monitoring of dendritic photosensitizer for photodynamic therapy of peritoneal metastasis. a, Bioluminescence images of peritoneal metastasis in mice; b, Bioluminescence intensity of peritoneal metastasis in mice.

[0060] Figure 9 Evaluation of the efficacy of dendritic photosensitizer for photodynamic therapy in a mouse model of peritoneal metastasis of gastric cancer. a, Gross pictures of peritoneal metastasis in mice; b, Comparison of tumor weight of peritoneal metastasis in mice; c, Comparison of the number of peritoneal metastasis in mice.

[0061] Figure 10 Evaluation of the safety of dendritic photosensitizer for photodynamic therapy in vivo. a, Changes in body weight of mice during the A9 photodynamic therapy cycle; b, Skin condition of the laser irradiation site (abdomen) during the photodynamic therapy cycle; c, HE staining results of major organs (heart, liver, spleen, lung and kidney) after A9 photodynamic therapy; d-i, Analysis results of liver and kidney function indicators (Albumin (ALB), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Blood Urea Nitrogen (BUN) and Serum creatinine (CREA)) in blood after A9 photodynamic therapy.

[0062] Figure 11 Evaluation of the photodynamic killing effect of dendritic photosensitizer on human gastric cancer organoids. a, Bright field photos of gastric cancer organoids at different time points; b, Changes in the diameter of gastric cancer organoids. DETAILED DESCRIPTION

[0063] The technical solutions of the present application are further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations on the present application.

[0064] The sources of the products in the following examples and application examples are as follows:

[0065]

[0066] Example 1

[0067] The present embodiment provides a dendritic molecule modified phthalocyanine photosensitizing compound (referred to as dendritic photosensitizing molecule) A1, and a preparation method of the dendritic photosensitizing molecule A1 includes the following steps:

[0068]

[0069] (1) Synthesis of intermediate B1

[0070] A 250 mL two-necked round-bottom flask was dried and reserved, and compound 4-iodo phthalonitrile (5.00 g, 19.69 mmol, 1 equiv.), 4-tert-butyl phthalonitrile (14.51 g, 78.74 mmol, 4 equiv.), and zinc chloride (4.02 g, 29.52 mmol, 1.5 equiv.) were sequentially weighed into the two-necked flask, a magnetic stirrer was placed in the reaction system, and the reaction system was protected by argon. N,N-dimethyl ethanolamine (60 mL) was added to the above reaction system, protected from light, and heated to 140°C and refluxed and stirred for 15 hours. TLC detection showed that the reaction was complete, and the concentrated solution was dissolved in dichloromethane, washed with deionized water twice, separated by a separatory funnel, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated solution was purified by column chromatography (petroleum ether: 1,4-dioxane = 18:1) to obtain blue powder solid B1 with a yield of 30%.

[0071] 1 H NMR (500 MHz, DMSO-d6) δ 9.43-8.01 (m, 12H), 1.89 (m, 27H).

[0072] LCMS m / z: calculated value C 44 H 39 IN8Zn, [M] + 870.1, test value [(M+2H) / 2] + 437.2.

[0073] (2) Synthesis of intermediate B2

[0074] A 250 mL two-necked round-bottom flask was oven-dried and charged with compound B1 (5.15 g, 5.91 mmol, 1 equiv.) and tetrakis(triphenylphosphine)palladium (0.68 g, 0.59 mmol, 0.1 equiv.), fitted with a magnetic stir bar, and protected with an argon atmosphere. To the reaction was added toluene (120 mL) and ethynyltri-n-butyltin (5.58 g, 17.72 mmol, 3 equiv.). The reaction was protected from light, warmed to 60 °C, and stirred for 4 h. TLC analysis indicated the reaction was complete. The reaction was concentrated under reduced pressure, the residue was dissolved in dichloromethane, washed with deionized water twice, separated with a separatory funnel, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether: 1,4-dioxane = 19:1). The product obtained after concentration was washed with a small amount of hot methanol twice to give blue powder solid B2 in 30% yield.

[0075] 1 H NMR (500 MHz, DMSO-d6) δ 9.07 (m, 8H), 8.41 - 8.09 (m, 4H), 4.72 - 4.64 (m, 1H), 2.04 - 1.69 (m, 27H).

[0076] LCMS m / z: calculated C 46 H 40 N8Zn, [M] + 768.2, found [M+H] + 769.3.

[0077] (3) Synthesis of intermediate B3

[0078] A 100 mL two-necked round-bottom flask was oven-dried and charged with compound 3-azidopropylamine (4.75 g, 47.50 mmol, 1 equiv.), fitted with a magnetic stir bar, and protected with an argon atmosphere. To the reaction was added super-dry acetonitrile (40 mL) and 1,8-diazabicycloundec-7-ene (3.61 g, 23.75 mmol, 0.5 equiv.). The reaction was cooled to 0 °C with an ice-water bath, and methyl acrylate (10.21 g, 118.75 mmol, 2.5 equiv.) was added dropwise. The reaction was allowed to warm to room temperature and stirred for 2 days. TLC analysis indicated the reaction was complete. The reaction was concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to give white oil B3 in 93% yield.

[0079] 1H NMR (500 MHz, Chloroform-d) δ 3.67 (s, 6 H), 3.31 (t, J = 6.6 Hz, 2 H), 2.74 (t, J = 6.9 Hz, 4 H), 2.47 (t, J = 6.6 Hz, 2 H), 2.43 (t, J = 6.9 Hz, 4 H), 1.69 (m, J = 6.6 Hz, 2 H).

[0080] LCMS m / z: Calculated for C 11 H 20 N4O4, [M] + 272.1, found [M+H] + 273.0.

[0081] (4) Synthesis of Intermediate B4

[0082] Take a 250 mL two-necked round-bottom flask and dry it for use. Compound B3 (12.02 g, 44.19 mmol, 1 equiv.) was weighed into the flask. A magnetic stirrer was added and the reaction system was protected by argon. Anhydrous methanol (120 mL) was added to the reaction system, which was cooled to 0 °C in an ice water bath. Ethylenediamine (53.03 g, 883.80 mmol, 20 equiv.) was slowly added dropwise, and the temperature was allowed to rise to room temperature and stirred for 3 days. TLC detection showed that the reaction was complete. The reaction was concentrated under reduced pressure and then treated. The excess ethylenediamine was treated by adding a mixture of toluene:methanol = 9:1 (V:V) and removing the ethylenediamine by azeotropic distillation under reduced pressure. First, the excess methanol was removed by reduced pressure, then 30 mL of a mixture of toluene / methanol (9:1) was added, and the mixture was concentrated under reduced pressure at 50 °C for 0.5 h using a circulating water vacuum pump, then the oil pump was replaced and the mixture was concentrated under reduced pressure at 50 °C for 0.5 h. Finally, 30 mL of methanol was added and the mixture was concentrated under reduced pressure, and the toluene was removed by azeotropic distillation. The above process was repeated three times. Finally, yellow viscous liquid B4 was obtained in 98% yield.

[0083] 1 H NMR (500 MHz, Chloroform-d) δ 7.14 (t, J = 5.7 Hz, 2 H), 3.27 (m, 6 H), 2.80 (t, J = 5.9 Hz, 4 H), 2.68 (t, J = 6.0 Hz, 4 H), 2.45 (t, J = 6.7 Hz, 2 H), 2.33 (t, J = 6.1 Hz, 4 H), 1.69 (m, J = 6.5 Hz, 2 H).

[0084] LCMS m / z: Calculated for C 13 H 28 N8O2, [M] + 328.2, found [M+H] + 329.6.

[0085] (5) Synthesis of intermediate B5

[0086] Take a 10 mL two-mouth round-bottom flask and dry it for use. Weigh compound B4 (60.0 mg, 0.18 mmol, 1 equiv.) into the two-mouth flask. Put a magnetic stirrer into the flask, and protect the reaction system with argon. Add super-dry acetonitrile (1 mL) into the two-mouth flask. Cool the reaction system to 0 °C, and then slowly add trifluoroacetic anhydride (384 mg, 1.83 mmol, 10 equiv.) dropwise. Stir the reaction system at 0 °C for 1 day. TLC detection shows that the reaction is completed. Concentrate under reduced pressure, dissolve the concentrate in dichloromethane, slowly add saturated aqueous sodium bicarbonate solution, and then extract twice with dichloromethane after no gas is generated. Separate the organic phase by a separating funnel, dry it with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the concentrate by column chromatography (dichloromethane:methanol = 8:1), and finally obtain yellowish liquid B5 with a yield of 26%.

[0087] 1 H NMR (500 MHz, Chloroform-d) δ 8.27 (s, 2H), 7.48 (t, J = 5.6 Hz, 2H), 3.47 - 3.40 (m, 8H), 3.28 (t, J = 6.3 Hz, 2H), 2.68 (t, J = 6.0 Hz, 4H), 2.49 (t, J = 7.0 Hz, 2H), 2.36 (t, J = 6.0 Hz, 4H), 1.73 - 1.64 (m, 2H).

[0088] LCMS m / z: calculated C 17 H 26 F6N8O4, [M] + 520.2, test value [M+H] + 521.2.

[0089] (6) Synthesis of compound A1

[0090] A 10 mL two-necked round-bottom flask was dried and reserved. Compound B2 (44.0 mg, 0.058 mmol, 1.2 equiv.), B5 (25.0 mg, 0.048 mmol, 1 equiv.), copper sulfate pentahydrate (12.0 mg, 0.048 mmol, 1 equiv.), and sodium ascorbate (19.0 mg, 0.096 mmol, 2 equiv.) were sequentially added into the flask. A magnetic stirrer was added, and the reaction system was protected by argon. 5 mL of mixed solvent [tetrahydrofuran: water = 5: 1 (V / V)] was added into the reaction system, which was protected from light, heated to 40°C and stirred for 12 h. TLC detection showed that the reaction was completed. The reaction system was concentrated under reduced pressure, and the concentrate was placed in a freeze dryer to remove water. Finally, blue solid A1 was obtained by basic column chromatography (dichloromethane:methanol = 10:1), with a yield of 58%.

[0091] 1 H NMR (500 MHz, Methanol-d4) δ 8.98 - 6.66 (m, 13H), 3.46 (m, 10H), 2.94 - 2.71 (m, 4H), 2.53 (m, 2H), 2.47 - 2.35 (m, 4H), 2.18 (m, 1H), 2.01 (m, 1H), 1.77 - 1.29 (m, 27H).

[0092] LCMS m / z: calculated C 63 H 66 F6N 16 O4Zn, [M] + 1288.5, test value [M+2H] 2+ 1290.7, [(M+2H) / 2] + 645.4.

[0093] Example 2

[0094] The present embodiment provides a dendritic photosensitive molecule A2, and a preparation method of the dendritic photosensitive molecule A2, which comprises the following steps:

[0095]

[0096] (1) Synthesis of intermediate B6

[0097] Referring to the method of step (5) of Reference Example 1, B4 was used as the starting material to synthesize white solid B6, with a yield of 57%.

[0098] 1H NMR (500 MHz, Methanol-d4) δ 3.37 (t, J = 6.1 Hz, 4H), 3.30 - 3.27 (m, 4H), 3.26 (m, 2H), 2.69 (t, J = 6.8 Hz, 4H), 2.48 (t, J = 7.0 Hz, 2H), 2.30 (t, J = 6.8 Hz, 4H), 1.67 (m, 2H).

[0099] LCMS m / z: calculated for C 19 H 26 F 10 N8O4,[M] + 620.2, found [M+H] + 621.2.

[0100] (2) Synthesis of compound A2

[0101] A2 was synthesized as a blue solid in 57% yield by the method of Reference Example 1, step (6) using B2 and B6 as starting materials.

[0102] 1 H NMR (500 MHz, Methanol-d4) δ 8.99 - 6.81 (m, 13H), 3.59 - 3.38 (m, 10H), 2.90 - 2.66 (m, 4H), 2.52 (m, 2H), 2.44 - 2.30 (m, 4H), 2.18 (m, 1H), 2.01 (m, 1H), 1.28 (m, 27H).

[0103] LCMS m / z: calculated for C 65 H 66 F 10 N 16 O4Zn,[M] + 1388.5, found [M+H] + 1389.7, [(M+2H) / 2] + 695.5.

[0104] Example 3

[0105] The present embodiment provides a dendritic photosensitive molecule A3, and a preparation method of the dendritic photosensitive molecule A3 comprises the following steps:

[0106]

[0107] (1) Synthesis of intermediate B7

[0108] B7 was synthesized as a white solid in 33% yield by the method of Reference Example 1, step (5) using B4 as starting material.

[0109] 1 H NMR (500 MHz, Methanol-d4) δ 3.38 (t, J = 6.2 Hz, 4H), 3.30 - 3.27 (m, 4H), 3.26 (m, 2H), 2.69 (t, J = 6.8 Hz, 4H), 2.48 (t, J = 7.0 Hz, 2H), 2.30 (t, J = 6.8 Hz, 4H), 1.67 (m, 2H).

[0110] LCMS m / z: Calculated for C 21 H 26 F 14 N8O4, [M] + 720.2, found [M+H] + 721.2.

[0111] (2) Synthesis of compound A3

[0112] Referring to the method of step (6) of Reference Example 1, A3 was synthesized from B2 and B7 as starting materials, and was obtained as a blue solid in a yield of 45%.

[0113] 1 H NMR (500 MHz, Methanol-d4) δ 8.49 - 7.02 (m, 13H), 3.47 (m, 10H), 2.89 - 2.63 (m, 4H), 2.56 - 2.23 (m, 7H), 2.18 (m, 1H), 2.01 (m, 1H), 1.79 - 1.27 (m, 27H).

[0114] LCMS m / z: Calculated for C67H66F14N16O4Zn, [M]+1488.5, found [M+H]+1489.9, [(M+2H) / 2]+745.5

[0115] Example 4

[0116] The present embodiment provides a dendritic photosensitive molecule A4, and a preparation method of the dendritic photosensitive molecule A4 includes the following steps:

[0117]

[0118] (1) Synthesis of intermediate B8

[0119] Take a 25 mL two-necked round-bottom flask and dry it for use, and a 10 mL two-necked round-bottom flask and dry it for use, weigh dimethyl itaconate (1.08 g, 6.71 mmol, 10 equiv.) in the 25 mL two-necked flask, put in a magnetic stirrer, and protect the reaction system with argon. After adding super-dry methanol (5 mL), start stirring, and cool the reaction system to 0°C. Dissolve B4 (0.22 g, 0.67 mmol, 1 equiv.) in 2 mL of super-dry methanol in the 10 mL two-necked flask, and protect it with argon. Slowly add the dimethyl itaconate methanol solution to the reaction system in the 25 mL flask, naturally warm to room temperature, and stir for 3 days. TLC detection shows that the reaction is complete, concentrate under reduced pressure, and purify the concentrate by column chromatography (dichloromethane:methanol = 13:1) to obtain white solid B8, with a yield of 63%.

[0120] 1 H NMR (500 MHz, Chloroform-d) δ 7.51 (d, J = 25.0 Hz, 2H), 3.80 (s, 1H), 3.73 (d, J = 3.0 Hz, 6H), 3.70 - 3.63 (m, 3H), 3.54 - 3.42 (m, 4H), 3.38 - 3.20 (m, 6H), 2.75 - 2.56 (m, 7H), 2.48 (m, 2H), 2.40 - 2.25 (m, 4H), 2.07 - 1.75 (m, 4H), 1.72 (m, 1H).

[0121] LCMS m / z: calculated C 25 H 40 F 28 N8O8, [M] + 580.3, test value [M+H] + 581.3.

[0122] (2) Synthesis of compound A4

[0123] Referring to the method of step (6) of Reference Example 1, blue solid A4 is synthesized with B2 and B8 as starting materials, with a yield of 80%.

[0124] 1 H NMR (500 MHz, Methanol-d4) δ 9.15 - 6.66 (m, 13H), 4.69 - 4.05 (m, 2H), 3.80 - 3.60 (m, 6H), 3.40 (m, 3H), 3.04 - 1.40 (m, 27H), 1.33 - 1.21 (m, 27H).

[0125] LCMS m / z: calculated C 71 H 80 N 16O8Zn, [M] + 1348.6, found [M+H] + 1349.8, [(M+2H) / 2] + 675.4.

[0126] Example 5

[0127] The present embodiment provides a dendritic-like photosensitive molecule A5, and a preparation method of the dendritic-like photosensitive molecule A5 includes the following steps:

[0128]

[0129] (1) Synthesis of intermediate B9

[0130] Take a 100 mL two-port round-bottom flask and dry it for standby. Compound B4 (8.00 g, 24.39 mmol, 1 equiv.) is weighed in the two-port flask. A magnetic stirrer is placed in the reaction system, which is protected by argon. Add ultradry methanol (40 mL) to the two-port flask, and cool it to 0°C in an ice water bath. Slowly add methyl acrylate (20.98 g, 243.90 mmol, 10 equiv.) to the reaction system, and naturally warm it to room temperature and stir for 1 day. TLC detection shows that the reaction is complete. Concentrate under reduced pressure, and purify the concentrate by column chromatography (dichloromethane:methanol = 60:1). Finally, white oily B9 is obtained in a yield of 70%.

[0131] 1 H NMR (500 MHz, Chloroform-d) δ 3.69 (s, 12H), 3.44 (t, J = 6.3 Hz, 2H), 3.34 (q, J = 5.6 Hz, 4H), 3.30-3.23 (m, 3H), 2.98 (s, 2H), 2.79 (t, J = 6.6 Hz, 8H), 2.73 (d, J = 6.8 Hz, 5H), 2.60 (t, J = 5.8 Hz, 4H), 2.47 (t, J = 6.6 Hz, 8H), 2.06-1.98 (m, 2H).

[0132] LCMS m / z: calculated C 29 H 52 N8O 10 ,[M] + 672.3, found [M+H] + 673.4.

[0133] (2) Synthesis of intermediate B10

[0134] A 10 mL two-necked round-bottom flask was dried and reserved, and compound B9 (58.0 mg, 0.086 mmol, 1 equiv.) was placed in a magnetic stirrer, and the reaction system was protected by argon. After adding anhydrous methanol (2 mL) to the two-necked flask, stirring was started, and 2-hydroxyethylamine (52.71 mg, 0.86 mmol, 10 equiv.) was slowly added dropwise to the reaction system, which was then warmed to 40°C and stirred for 2 days. TLC detection showed that the reaction was complete, and the reaction was concentrated under reduced pressure. The concentrate was purified by column chromatography (dichloromethane:methanol = 2:1), and a colorless liquid B10 was finally obtained in a yield of 72%.

[0135] 1 H NMR (500 MHz, Methanol-d4) δ 6.34-6.16 (m, 2H), 5.65 (d, J = 9.8 Hz, 1H), 3.61 (q, J = 5.5 Hz, 12H), 3.38-3.32 (m, 4H), 3.27 (t, J = 6.6 Hz, 4H), 2.77 (m, 12H), 2.65-2.44 (m, 8H), 2.37 (t, J = 6.7 Hz, 12H), 1.72 (t, J = 6.8 Hz, 2H).

[0136] LCMS m / z: calculated C 33 H 64 N 12 O 10 ,[M] + 788.5, test value [M+H] + 789.5, [(M+2H) / 2] + 395.3.

[0137] (3) Synthesis of compound A5

[0138] A 10 mL two-necked round-bottom flask was dried and reserved, and compound B2 (32.7 mg, 0.043 mmol, 1.2 equiv.), B10 (28.0 mg, 0.036 mmol, 1 equiv.), copper sulfate pentahydrate (4.4 mg, 0.018 mmol, 0.5 equiv.), and sodium ascorbate (7.0 mg, 0.036 mmol, 1 equiv.) were sequentially placed in the two-necked flask. A magnetic stirrer was placed in the reaction system, which was protected by argon. 3 mL of a mixed solvent [tetrahydrofuran:water = 4:1 (V / V)] was added to the reaction system, which was protected from light, warmed to 45°C, and stirred for 12 h. TLC detection showed that the reaction was complete, and the reaction was concentrated under reduced pressure. The concentrate was placed in a freeze dryer to remove water, and then purified by basic column chromatography (dichloromethane:methanol = 5:1) to obtain a blue solid 2-30 in a yield of 43%.

[0139] 1 H NMR (500 MHz, Methanol-d4) δ 9.37 - 7.51 (m, 13H), 3.57 (m, 8H), 3.26 (m, 8H), 2.94 (m, 3H), 2.74 (m, 10H), 2.61 (m, 4H), 2.50 (m, 4H), 2.41 - 2.02 (m, 13H), 1.77 (m, 27H), 1.41 - 1.38 (m, 2H).

[0140] LCMS m / z: calculated C 79 H 104 N 20 O 10 Zn, [M] + 1556.8, found [(M + 2H) / 2] + 780.0, [(M + 3H) / 3] + 520.6.

[0141] Example 6

[0142] The present embodiment provides a dendritic photosensitive molecule A6, and a preparation method of the dendritic photosensitive molecule A6 comprises the following steps:

[0143]

[0144] (1) Synthesis of intermediate B11

[0145] Take a 250 mL two-port round-bottom flask and dry it for standby. At room temperature, weigh compound B9 (10.00 g, 14.88 mmol, 1 equiv.) into the two-port flask. Put in a magnetic stirrer, and protect the reaction system with argon. After adding ultra-dry methanol (100 mL) into the two-port flask, start stirring. Cool to 0°C in an ice water bath, and then slowly drop ethylenediamine (53.57 g, 892.80 mmol, 60 equiv.) into the reaction system. Naturally warm to room temperature and stir for 3 days. TLC detection shows that the reaction is completed. Concentrate under reduced pressure, and then treat. For the treatment of excess ethylenediamine, add a mixed solution of toluene:methanol=9:1 (V:V), and remove ethylenediamine by azeotropic distillation under reduced pressure. Repeat three times. Refer to the treatment in Example 1, step (4) for details. Finally, yellow viscous liquid B11 is obtained in a yield of 95%.

[0146] 1 H NMR (500 MHz, Methanol-d4) δ 3.37 - 3.33 (m, 4H), 3.26 (t, J = 6.4 Hz, 10H), 2.83 - 2.70 (m, 20H), 2.56 (m, 6H), 2.37 (t, J = 6.9 Hz, 12H), 1.73 (m, 2H).

[0147] LCMS m / z: calculated for C 33 H 68 N 16 O6,[M] + 784.6, found [M+H] + 785.7.

[0148] (2) Synthesis of intermediate B12

[0149] Take a 10 mL two-mouth round-bottom flask and dry it for use. Compound B11 (50.0 mg, 0.064 mmol, 1 equiv.) was weighed into the two-mouth flask. A magnetic stirrer was placed in the flask, and the reaction system was protected by argon. After adding super-dry acetonitrile (1 mL) to the two-mouth flask, stirring was started. The reaction system was cooled to 0°C in an ice-water bath, and then trifluoroacetic anhydride (133.9 mg, 0.64 mmol, 10 equiv.) was slowly added dropwise. After natural warming to room temperature, stirring was continued for 2 days. TLC detection showed that the reaction was completed. The concentrate was dissolved in dichloromethane, and saturated sodium bicarbonate aqueous solution was slowly added. After no gas was generated, the mixture was extracted twice with ethyl acetate, and the organic phase was separated by a separating funnel, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography (dichloromethane:methanol = 7:1), and finally a yellowish liquid B12 was obtained in a yield of 37%.

[0150] 1 H NMR (500 MHz, Methanol-d4) δ 3.47-3.33 (m, 18H), 3.28 (d, J = 6.9 Hz, 4H), 2.84 (m, 12H), 2.64 (m, 6H), 2.41 (m, 12H), 1.77 (q, J = 6.8 Hz, 2H).

[0151] LCMS m / z: calculated for C 41 H 64 F 12 N 16 O 10 ,[M] + 1168.5, found [M+H] + 1169.8, [(M+2H) / 2] + 585.6, [(M+3H) / 3] + 390.7.

[0152] (3) Synthesis of compound A6

[0153] Referring to the method of step (3) of Reference Example 5, B2 and B12 were used as starting materials to synthesize blue solid A6 in a yield of 28%.

[0154] 1H NMR (500 MHz, Methanol-d4) δ 9.33 - 7.18 (m, 13H), 3.18 - 2.98 (m, 6H), 2.83 (m, 16H), 2.68 (m, 6H), 2.55 (m, 6H), 2.36 (m, 18H), 1.85 (m, 27H), 1.60 (m, 2H).

[0155] LCMS m / z: Calculated for C 87 H 104 F 12 N 24 O 10 Zn, [M] + 1936.7, Test value [(M+2H) / 2] + 969.6, [(M+3H) / 3] + 647.5.

[0156] Example 7

[0157] The present embodiment provides a dendritic photosensitive molecule A7, and a preparation method of the dendritic photosensitive molecule A7 comprises the following steps:

[0158]

[0159] (1) Synthesis of intermediate B13

[0160] Referring to the method of Example 6, step (2), B13 was synthesized from B11 as a starting material to obtain a white solid with a yield of 37%.

[0161] 1 H NMR (500 MHz, Methanol-d4) δ 3.39 (t, J = 6.3 Hz, 8H), 3.33 (s, 4H), 3.25 (m, 8H), 2.77 (m, 12H), 2.57 (d, J = 6.5 Hz, 6H), 2.35 (m, 12H), 1.71 (m, 2H), 1.31 (m, 2H).

[0162] LCMS m / z: Calculated for C 45 H 64 F 20 N 16 O 10 , [M] + 1368.5, Test value [M+H] + 1369.8, [(M+2H) / 2] + 685.6.

[0163] (2) Synthesis of compound A7

[0164] Reference Example 5, step (3) using B2 and B13 as starting materials to give A7 as a blue solid in 68% yield.

[0165] 1 H NMR (500 MHz, Methanol-d4) δ 8.51 - 7.31 (m, 13H), 2.95 (m, 4H), 2.88 - 2.67 (m, 16H), 2.60 (m, 6H), 2.55 - 2.44 (m, 6H), 2.43 - 2.06 (m, 18H), 1.80 (m, 27H), 1.58 - 1.49 (m, 2H), 1.49 - 1.44 (m, 2H).

[0166] LCMS m / z: Calculated for C 91 H 104 F 20 N 24 O 10 Zn, [M] + 2136.7, found [(M+2H) / 2] + 1070.3, [(M+3H) / 3] + 714.3.

[0167] Example 8

[0168] The present embodiment provides a dendritic photosensitive molecule A8, a preparation method of the dendritic photosensitive molecule A8 comprising the following steps:

[0169]

[0170] (1) Synthesis of intermediate B14

[0171] Reference Example 6, step (2) using B11 as starting material to give B14 as a white solid in 34% yield.

[0172] 1 H NMR (500 MHz, Chloroform-d) δ 8.42 (s, 3H), 7.61 (m, 5H), 5.46 - 5.28 (m, 1H), 3.52 - 3.46 (m, 8H), 3.43 (q, J = 5.9 Hz, 8H), 3.29 (t, J = 6.6 Hz, 2H), 3.21 (d, J = 6.2 Hz, 2H), 2.70 (m, 12H), 2.51 (q, J = 7.0 Hz, 6H), 2.34 (m, 12H), 1.71 - 1.66 (m, 2H), 1.46 - 1.31 (m, 2H).

[0173] LCMS m / z: Calculated for C 49 H 64 F28 N 16 O 10 ,[M] + 1568.5, found [M+H] + 1369.9, [(M+2H) / 2] + 785.7.

[0174] (2) Synthesis of compound A8

[0175] Referring to the method of Reference Example 4, step (1), B2 and B14 were used as starting materials to synthesize A8 as a blue solid in a yield of 72%.

[0176] 1 H NMR (500 MHz, Methanol-d4) δ 8.87 - 7.86 (m, 13H), 3.39 (m, 10H), 2.94 (m, 4H), 2.74 (m, 15H), 2.58 (m, 5H), 2.54 - 2.43 (m, 6H), 2.42 - 2.03 (m, 18H), 1.81 (m, 27H), 1.55 - 1.48 (m, 2H), 1.43 - 1.38 (m, 4H).

[0177] LCMS m / z: calculated C 95 H 104 F 28 N 24 O 10 Zn, [M] + 2336.7, found [(M+2H) / 2] + 1170.2, [(M+3H) / 3] + 781.0.

[0178] Example 9

[0179] The present embodiment provides a dendritic photosensitive molecule A9, and a preparation method of the dendritic photosensitive molecule A9 includes the following steps:

[0180]

[0181] (1) Synthesis of intermediate B15

[0182] Referring to the method of Reference Example 4, step (1), B2 and B14 were used as starting materials to synthesize A8 as a blue solid in a yield of 72%.

[0183] 1H NMR (500 MHz, Methanol-d4) δ 3.75 (d, J = 15.8 Hz, 16H), 3.68 (m, 4H), 3.42 - 3.34 (m, 21H), 3.28 (d, J = 6.3 Hz, 3H), 2.81 (m, 12H), 2.70 - 2.55 (m, 16H), 2.41 (t, J = 6.9 Hz, 4H), 2.34 (t, J = 6.8 Hz, 8H), 1.76 (m, 2H).

[0184] LCMS m / z: Calculated for C 57 H 92 N 16 O 18 ,[M] + 1288.7, Found [M+H] + 1289.8, [M+Na] + 1311.8, [(M+2H) / 2] + 645.6, [(M+3H) / 3] + 430.8.

[0185] (2) Synthesis of compound A9

[0186] Following the procedure of Reference Example 5, step (3), A9 was synthesized in 38% yield as a blue solid, using B2 and B15 as starting materials.

[0187] 1 H NMR (500 MHz, Methanol-d4) δ 9.39 - 7.23 (m, 12H), 4.68 (m, 2H), 4.10 (m, 1H), 3.73 (m, 4H), 3.63 (m, 12H), 3.60 - 3.55 (m, 4H), 3.52 (m, 4H), 3.24 - 3.15 (m, 15H), 3.03 - 2.84 (m, 4H), 2.83 - 2.42 (m, 30H), 2.41 - 2.27 (m, 4H), 2.20 (m, 8H), 2.02 (m, 2H), 1.83 (m, 27H).

[0188] LCMS m / z: Calculated for C 103 H 132 N 24 O 18 Zn,[M] + 2056.9, Found [M+3H] 3+ 2060.3, [(M+2H) / 2] + 1030.1, [(M+3H) / 3] + 687.5.

[0189] Example 10

[0190] The present embodiment provides a dendritic photosensitive molecule A10, and a preparation method of the dendritic photosensitive molecule A10 includes the following steps:

[0191]

[0192] (1) Synthesis of intermediate B16

[0193] Take a 25 mL two-port round-bottom flask and dry it for standby. Compound B11 (51.0 mg, 0.065 mmol, 1 equiv.) is weighed in the two-port flask. A magnetic stirrer is placed in the flask, and the reaction system is protected by argon. After adding the solvent tetrahydrofuran (4 mL) to the two-port flask, the stirring is started. The reaction system is cooled to 0°C in an ice water bath, and then di-tert-butyl dicarbonate (141.8 mg, 0.65 mmol, 10 equiv.) is slowly added dropwise to the reaction system. After 1 h of reaction at 0°C, the temperature is increased to 45°C, and the stirring is continued for 2 days. TLC detection shows that the reaction is complete. The reaction system is concentrated under reduced pressure, and the concentrate is purified by basic column chromatography (dichloromethane:methanol = 21:1). Finally, B16 is obtained in a yield of 59%.

[0194] 1 H NMR (500 MHz, Chloroform-d) δ 7.59 (m, 5H), 5.66 (s, 4H), 3.28 (m, 21H), 2.75 (s, 12H), 2.56 (s, 7H), 2.45-2.30 (m, 12H), 1.74 (d, J = 8.6 Hz, 2H), 1.42 (s, 36H).

[0195] LCMS m / z: calculated C 53 H 100 N 16 O 14 ,[M] + 1184.8, test value [M+H] + 1185.7, [(M+2H) / 2] + 593.3.

[0196] (2) Synthesis of compound A10

[0197] Referring to the method of step (3) of Reference Example 5, B2 and B16 are used as starting materials to synthesize blue solid A10 in a yield of 35%.

[0198] 1H NMR (500 MHz, Methanol-d4) δ 8.71 - 7.76 (m, 14H), 6.60 (m, 1H), 6.22 (m, 1H), 4.72 (m, 1H), 3.19 (m, 10H), 3.11 (m, 12H), 2.82 (m, 12H), 2.70 (m, 4H), 2.57 (m, 4H), 2.36 (m, 12H), 1.82 (m, 27H), 1.38 (m, 36H).

[0199] LCMS m / z: calculated C 99 H 140 N 24 O 14 Zn, [M] + 1953.0, test value [M+3H] 3+ 1956.7, [(M+2H) / 2] + 978.0, [(M+3H) / 3] + 652.5.

[0200] Example 11

[0201] The present embodiment provides a dendritic photosensitive molecule A11, and a preparation method of the dendritic photosensitive molecule A1112, which comprises the following steps:

[0202]

[0203] (1) Synthesis of intermediate B17

[0204] Take a 25 mL two-necked round-bottom flask and a 10 mL two-necked round-bottom flask, dry them and reserve for use. Weigh B11 (53.0 mg, 0.068 mmol, 1 equiv.) into the 25 mL two-necked flask, put a magnetic stirrer into the reaction system, and protect the reaction system with argon. After adding ultra-dry methanol (2 mL), start stirring. Add (+) biotin-N-succinimidyl ester (232.1 mg, 0.68 mmol, 10 equiv.) into the 10 mL two-necked flask, and protect it with argon, then add ultra-dry dimethyl sulfoxide (0.9 mL). Slowly add the dimethyl sulfoxide solution of biotin into the 25 mL flask, and stir at room temperature for 3 days. White solid is generated in the reaction, and TLC detection shows that the reaction is complete. Concentrate under reduced pressure, wash the concentrate with 5 mL of methanol, collect the precipitate, dissolve the precipitate in 10 mL of deionized water, and sequentially wash it with diethyl ether, dimethyl ether and ethyl acetate. Collect the aqueous phase, and put it into a freeze dryer to remove water. Finally, white solid B17 is obtained, with a yield of 73%.

[0205] 1H NMR (500 MHz, Deuterium Oxide) δ 4.62 (m, 4H), 4.44 (m, 4H), 3.45 (t, J = 6.5 Hz, 2H), 3.40 - 3.28 (m, 26H), 3.01 (m, 4H), 2.90 (t, J = 7.3 Hz, 8H), 2.79 (d, J = 13.0 Hz, 4H), 2.73 (q, J = 6.8, 4.9 Hz, 4H), 2.60 (d, J = 7.2 Hz, 4H), 2.47 (t, J = 7.2 Hz, 8H), 2.27 (t, J = 7.4 Hz, 8H), 1.92 - 1.85 (m, 2H), 1.67 (m, 16H), 1.42 (m, 9H).

[0206] LCMS m / z: calculated for C 73 H 124 N 24 O 14 S4, [M] + 1688.9, found [(M + 2H) / 2] + 845.9, [(M + H + Na) / 2] + 857.0, [(M + 2Na) / 2] + 867.0.

[0207] (2) Synthesis of compound A11

[0208] Following the procedure of Reference Example 5, step (3), A11 was synthesized in 19% yield as a blue solid, using B2 and B17 as starting materials.

[0209] 1 H NMR (500 MHz, DMSO-d6) δ 9.85 (m, 1H), 9.52 - 9.18 (m, 8H), 8.76 - 8.68 (m, 1H), 8.47 - 7.55 (m, 14H), 6.92 - 6.56 (m, 1H), 6.39 (m, 7H), 5.28 (m, 1H), 4.61 (m, 2H), 4.32 - 4.20 (m, 4H), 4.12 (m, 1H), 4.05 (m, 3H), 3.12 - 2.99 (m, 20H), 2.80 - 2.65 (m, 15H), 2.52 (m, 20H), 2.31 (m, 4H), 2.20 (m, 10H), 2.02 (m, 8H), 1.92 (m, 2H), 1.78 (m, 27H), 1.48 (m, 18H).

[0210] LCMS m / z: calculated for C 119 H 164 N 32 O 14Zn, [M] + 2457.1, test value [(M+2H) / 2] + 1230.3, [(M+3H) / 3] + 821.1.

[0211] Example 12

[0212] The particle size and surface charge of A5-A11 prepared in Examples 5-11 were detected:

[0213] Each of 10 mg of A5-A11 prepared in Examples 5-11 was configured into a solution with a concentration of 10 mg / mL, and filtered using a 0.22 μM filter membrane to obtain a mother liquor of seven dendritic-like phthalocyanine photosensitizer molecules. According to the sample concentration requirements, the dendritic-like photosensitizer solution was uniformly diluted to 0.2 mg / mL (for particle size measurement) or 1 mg / mL (for surface charge measurement). The particle size and surface charge of the dendritic-like photosensitizer molecules were detected using a Malvern particle potential instrument (Zetasizer Nano ZS). The experimental results are shown in Table 1.

[0214] Table 1

[0215]

[0216] Table 1 shows the self-assembly effect of the synthesized dendritic-like photosensitizer molecules under physiological conditions. In this embodiment, the particle size and surface potential of these compounds in PBS were measured using dynamic light scattering method. The results show that the dendritic-like photosensitizer molecules modified with different groups have their own unique particle size and charge characteristics: A9 has a smaller size (239.3 nm), a relatively uniform size distribution (PDI is 0.300), and a weak negative charge (-3.26 mV); A5 has a larger size (401.1 nm), a less uniform size distribution (PDI is 0.427), and a strong negative charge (-16.2 mV); A6 to A8 have relatively small sizes, relatively uniform size distributions, and different degrees of positive charges; A10 has a small size (132.5 nm), a relatively uniform size distribution (PDI is 0.081), and a positive charge (27.8 mV); and A11 has a large size (612.9 nm), a less uniform size distribution (PDI is 0.571), and a weak positive charge (4.53 mV).

[0217] Example 13

[0218] The UV-visible absorption spectrum and fluorescence spectrum of A5-A11 prepared in Examples 5-11 were detected by a multifunctional enzyme marker (Thermo Scientific Varioskan LUX):

[0219] For UV-Vis absorption spectrum detection, A5-A11 were diluted to 10 μM in ethanol, and the spectrum was detected in the range of 300-800 nm after shaking with a step of 1 nm, and blank control was used for normalization. For fluorescence spectrum detection, including excitation spectrum and emission spectrum, A5-A11 were diluted to 10 μM in PBS, the excitation spectrum was detected in the range of 300-670 nm with 690 nm as the detection wavelength, and the emission spectrum was detected in the range of 370-800 nm with 350 nm as the excitation wavelength, and blank control was used for normalization.

[0220] Figure 1A UV-Vis absorption spectrum of dendritic photosensitive molecules is shown, Figure 1B Fluorescence spectrum of dendritic photosensitive molecules is shown. UV-Vis spectrum shows that phthalocyanine monomer and dendritic photosensitive molecules have significant Q-band absorption in the range of 650-730 nm, and B-band absorption at about 305-390 nm. The Q-band absorption intensity of the modified dendritic photosensitive molecules is higher than that of the phthalocyanine monomer. Fluorescence spectrum shows that the emission peak of phthalocyanine monomer and dendritic photosensitive molecules is at 690 nm, and the fluorescence intensity is the largest under 610 nm excitation. The excitation and emission intensity of the dendritic photosensitive molecules under physiological conditions is higher than that of the phthalocyanine monomer, indicating that the chemical modification enhances the fluorescence performance, among which A11, A9, A5 perform best.

[0221] Example 14

[0222] The fluorescence imaging characterization of A5-A11 prepared in Examples 5-11 is carried out:

[0223] Each dendritic photosensitive molecule is diluted in ethanol to a concentration of 10 μM, and fluorescence imaging is carried out immediately after shaking uniformly. Fluorescence imaging is detected by a live imaging instrument (PerkinElmer IVIS Spectrum), and the excitation wavelength is 640 nm and the emission wavelength is 700 nm during the detection process.

[0224] Figure 2 The fluorescence imaging image and average fluorescence intensity of phthalocyanine molecules are shown. By comparing the fluorescence imaging of each dendritic photosensitive molecule under 640 nm light excitation, it is found that the seven synthesized dendritic photosensitive molecules all show bright red fluorescence under 700 nm light excitation, which indicates that these dendritic photosensitive molecules have the potential to be used as fluorescence probes, as shown in Figure 2 Under the same excitation conditions, the fluorescence intensity emitted from strong to weak is A9, A7, A8, A10, A5, A11, and A6.

[0225] Example 15

[0226] Tumor cell targeting detection of A5-A11 prepared in Examples 5-11 is carried out:

[0227] The uptake of A5–A11 molecules by normal gastric epithelial cells GES-1 and gastric cancer cells SNU638 and SNU668 was assessed by flow cytometry. Cells were seeded in 12-well plates and incubated with 1 μM dendritic photosensitive molecules for 24 hours after adhesion. Cells were then washed, digested, centrifuged, and resuspended in staining buffer. Flow cytometry was performed using the APC channel, and relative fluorescence intensity was calculated using the formula: Relative fluorescence intensity = (Fluorescence intensity of the drug-treated group - Fluorescence intensity of the control group) / Fluorescence intensity of the control group.

[0228] like Figure 3 As shown, each cell type exhibited a strong uptake capacity for A9, A5, A10, and A11. Furthermore, the uptake of A9, A5, and A10 by each gastric cancer cell was significantly higher than that by normal gastric epithelial cells GES-1. This confirms that dendritic photosensitizers have a high targeting enrichment effect on tumor cells.

[0229] Example 16

[0230] Based on the combined fluorescence spectra, fluorescence imaging, and cell targeting screening results of various dendritic photosensitive molecules, A9 prepared in Example 9 was preferred for phototoxicity and dark toxicity testing of various tumor cells:

[0231] This study used the CCK-8 assay to evaluate the cytotoxicity of the dendritic photosensitizer A9, including dark toxicity and photodynamic toxicity. Cells were seeded in 96-well plates during the logarithmic growth phase, and after 24 hours, different concentrations of A9 solution were added, followed by another 24 hours of culture. In the dark toxicity assay, cell viability was calculated by measuring OD450 after direct addition of CCK-8 solution. In the photodynamic toxicity assay, cells were irradiated with 700 nm light for 10 minutes after drug addition, followed by the addition of CCK-8 solution, and OD450 was measured to calculate viability. The concentrations of A9 were 0.9375, 1.825, 3.75, 7.5, and 15 μg / mL.

[0232] like Figure 4 The photodynamic killing effect of the preferred A9 in different solid tumor cells was demonstrated. By comparing the dark toxicity and photodynamic killing toxicity of the preferred A9 in human gastric cancer cell lines (AGS and SNU638), human colorectal cancer cell line (HCT116), human breast cancer cell line (T47D), human lung cancer cell line (A549), and human pancreatic cancer cell line (PANC-1), the results showed that A9 had low dark toxicity (cell viability above 70%) and high phototoxicity (phototoxic IC50 below 10 μg / ml) in all cancer types, exhibiting a unique photodynamic killing effect.

[0233] Example 17

[0234] Tumor spheroid distribution experiment was performed on the selected A9:

[0235] To improve the efficacy of photodynamic therapy for solid tumors, the synthetic dendritic photosensitizer A9 needs to have good tumor penetration. The spheroid model of gastric cancer cells (SNU668 and MFC) was established by scaffold-free liquid encapsulation method to evaluate the penetration and distribution of A9. The fluorescence distribution of A9 (i.e. Cy5 channel fluorescence) in the spheroids was monitored and photographed using a fluorescence microscope (Leica DMi8) at different incubation times (3 hours, 6 hours, 9 hours, 12 hours, 24 hours and 48 hours). The fluorescence imaging characteristics of A9 were analyzed by three-dimensional imaging using LAS X 2.7.5 software and the fluorescence intensity changes within the maximum diameter range of the spheroids were calculated. As shown in Figure 5 Fig. 1, after incubation of SNU668 and MFC spheroids with A9 for 6 to 24 hours, a significant red fluorescence signal appeared in the center of the spheroids, and the signal increased with time, confirming that A9 can effectively penetrate gastric cancer spheroids.

[0236] Example 18

[0237] The preferred A9 was subjected to photodynamic killing experiments in the spheroid model:

[0238] Subsequently, this example continued to verify the photodynamic killing effect of A9 in the spheroid model of gastric cancer cells. The spheroid model of gastric cancer cells (SNU668 and MFC) was established by scaffold-free liquid encapsulation method, and then randomly divided into four groups, namely: control group (i.e. Ctrl group), drug addition without light group (i.e. A9 group), no drug addition light group (i.e. Ctrl+L group) and drug addition light group (i.e. A9+L group). The A9 group and A9+L group were added with A9-containing medium on the 3rd day of spheroid culture, and the control group and L group were added with blank medium. After 24 hours of continuous culture under the same conditions, the L group and A9+L group were irradiated for 10 minutes on the 4th day using a high-throughput photodynamic screening instrument (PTHS-690-96, wavelength 700 nm, light intensity 6.5 mW / cm2), and then the medium was changed for continuous culture. The spheroids were continuously observed and photographed using a microscope (Leica DMi8), and the spheroid volume was calculated using Image J software. Figure 6A and Figure 6B Fig. 2 shows bright field photos of MFC and SNU668 spheroids at different time points (2nd, 4th, 5th, 6th, 7th days), Figure 6C and Figure 6D Fig. 3 shows the changes in spheroid volume, reflecting the effect of A9 on the growth of gastric cancer spheroids. The growth rates of MFC and SNU668 spheroids under drug addition without light and light only conditions were similar to those of the control group, but the spheroids in the drug addition light group (A9+L) stopped growing or even collapsed after drug administration and 700 nm light irradiation, indicating that A9 has a significant photodynamic killing effect in the spheroid model of gastric cancer.

[0239] Example 19

[0240] In vivo distribution experiment of preferred A9 was performed:

[0241] To evaluate the in vivo tumor targeting property of A9, a mouse model of peritoneal metastasis of gastric cancer was constructed, and the mice were injected intraperitoneally with A948 hours later, the peritoneal metastasis lesions and important organs (heart, liver, spleen, lung and kidney) were removed, washed with normal saline, and observed and imaged using a small animal living imaging instrument (PerkinElmer IVIS Spectrum). The luciferase luminescence signal of the tumor was detected by the bioluminescence detection module of the living imaging instrument, and the fluorescence distribution of A9 was detected by the fluorescence detection module, using an excitation wavelength of 640 nm and an emission wavelength of 700 nm. The average fluorescence intensity of A9 in the peritoneal metastasis lesions and each important organ was calculated using Living Image 4.4 software. As shown in Figure 7 Fig. 1, 48 hours later, the bioluminescence images of peritoneal metastasis lesions (as shown in Figs. 1a and 1b) and the fluorescence images of A9 (as shown in Fig. 1c) were highly coincident, and A9 was mainly enriched in peritoneal metastasis lesions, with an average fluorescence intensity significantly higher than that of other organs, about 4 times that of other organs, confirming the targeting enrichment ability of A9 in the peritoneal metastasis lesions of gastric cancer in vivo, which can be used as a fluorescent probe for tumor targeting.

[0242] Example 20

[0243] Mouse photodynamic therapy experiment of preferred A9 was performed:

[0244] Based on the previously constructed luciferase-labeled gastric cancer cells (SNU638-luc), a mouse model of peritoneal metastasis of gastric cancer was established to evaluate the in vivo anti-tumor effect of A9. The bioluminescence images of luciferase-labeled peritoneal metastasis lesions were dynamically monitored by a small animal living imaging instrument (PerkinElmer IVIS Spectrum), and the average bioluminescence intensity of the tumor was quantitatively analyzed. The results confirmed that the mice in the drug administration and light irradiation group (i.e., A9+L group) showed the best anti-tumor effect, including the slowest tumor growth Figure 8 ), the most limited peritoneal metastasis Figure 9 ), the lightest tumor weight collected Figure 9 ), and the least number of tumor nodules Figure 9 ). The drug administration alone without light irradiation group (i.e., A9 group) showed comparable tumor growth rate, peritoneal metastasis condition, tumor nodule number and tumor weight to the control group (i.e., PBS group). The above results confirmed that A9 also had good photodynamic killing effect in vivo.

[0245] Example 21

[0246] In vivo safety experiment of preferred A9 was performed:

[0247] To evaluate the in vivo safety of A9 photodynamic therapy, after the end of treatment, the hearts, livers, spleens, lungs and kidneys of the mice were fixed, paraffin-embedded and HE-stained to observe histopathological changes. At the same time, the serum liver and kidney function indicators such as albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and serum creatinine (CREA) were detected by blood biochemical analysis to evaluate the toxicity and biocompatibility. In addition, the skin of the irradiated area (the abdomen of the mice) was also examined to evaluate the potential effects of photodynamic therapy and laser irradiation on the skin, including observing whether skin necrosis or photosensitivity reactions occurred.

[0248] The body weight changes of the mice during the A9 photodynamic therapy cycle were monitored (Fig. 2a). Figure 10 a) The monitoring showed that there was no significant difference in the body weight of the mice in each group. The skin condition of the laser irradiation site (abdomen) during the photodynamic therapy cycle was also evaluated, as shown in Fig. 2b. Figure 10 b) The abdominal skin of the mice in the drug and light group was not significantly different from the control group and the simple drug group, and no obvious skin reactions such as skin necrosis (black spots) and photosensitivity reactions (red spots) were observed. In addition, the important organs (heart, liver, spleen, lung and kidney) after A9 photodynamic therapy were collected for HE staining detection, and the results showed that there was no obvious histopathological damage to the heart, liver, spleen, lung and kidney in each group, and there was no obvious organ toxicity (Fig. 2c). Figure 10 c) To evaluate the systemic toxicity after A9 photodynamic therapy, the liver and kidney function indicators such as ALB, ALT, AST, ALP, BUN and CREA in the blood after the treatment cycle were analyzed. Compared with the control group, there was no statistically significant difference in ALB, ALT, AST, ALP, BUN and CREA in the simple drug group and the drug and light group, indicating that the systemic toxicity of A9 photodynamic therapy for gastric cancer peritoneal metastasis was small (Figs. 2d-i). Figure 10 d-i).

[0249] Example 22

[0250] Verification of the preferred photodynamic killing effect of A9 in a human organoid model:

[0251] Finally, a human gastric cancer organoid model was constructed, and the photodynamic effect of A9 in the human gastric cancer organoid was evaluated, Figure 11The bright field photos (as shown in Figure a) and the organoid diameter changes (as shown in Figure b) of the gastric cancer organoids at different time points (2nd, 3rd, 4th, 5th, 6th day) were shown to reflect the effect of A9 on the growth of gastric cancer organoids. The results showed that the growth rate of the organoids in the drug-added non-irradiation group (i.e. A9 group) and the irradiation group (i.e. L group) was comparable to the control group, while the gastric cancer organoids in the drug-added irradiation group (i.e. A9+L group) stopped growing after being administered and irradiated with 700 nm light on the 1st and 2nd day, respectively, and the diameter of the organoids on the 6th day was significantly smaller than that of the control group, indicating that A9 also showed excellent photodynamic killing toxicity in the gastric cancer organoid model.

[0252] The applicant states that the dendritic molecule modified phthalocyanine photosensitizing compound and its application of the present application are illustrated by the above-mentioned examples, but the present application is not limited to the above-mentioned examples, i.e. it does not mean that the present application must rely on the above-mentioned examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of the selected materials of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A dendrimer-modified phthalocyanine photosensitizing compound, characterized in that, The dendritic molecule modified phthalocyanine photosensitizing compound has any one of the structures shown in the following formula I to formula IV: wherein R1is selected from or -(CH2) x - wherein m, n are each independently selected from an integer from 0 to 5, x is an integer selected from 2 to 8; R2, R3, R4, R5are selected from wherein p, q are each independently selected from an integer from 1 to 7; R' is selected from wherein R a and R b is selected from any one of H, OH, substituted or unsubstituted benzoyl, a biotin group, an amino acid residue, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 straight chain acyl, C2-C10 straight chain alkenyl containing 1-3 carbon-carbon double bonds, or C2-C10 straight chain alkynyl containing 1-3 carbon-carbon triple bonds. indicates the point of attachment of the group.

2. The phthalocyanine photosensitizer compound modified with a dendrimer according to claim 1, characterized by, Any one or more of the methylene groups in the group is optionally substituted with a heteroatom, and any one or more of the hydrogen atoms in the group is optionally substituted with a methyl group or a halogen atom.

3. The phthalocyanine photosensitizing compound modified with a dendrimer according to claim 1 or 2, characterized by, said R a is selected from any one of H, acetyl, propionyl, n-butyryl, isobutyryl, t-butoxyacetyl, trifluoroacetyl, pentafluoropropionyl, heptafluorobutyryl, a biotin group, trichloroacetyl, benzoyl, 4-fluorobenzoyl, 2,3,4,5,6-pentafluorobenzoyl, 4-trifluoromethylbenzoyl, 3,4-dichlorobenzoyl, or 4-trichloromethylbenzoyl.

4. The phthalocyanine photosensitizer compound modified with a dendrimer according to any one of claims 1 to 3, characterized in that, The R b is selected from any one of hydroxy, methoxy, ethoxy, n-propoxy, i-propoxy or n-butoxy.

5. The phthalocyanine photosensitizer compound modified with a dendrimer according to any one of claims 1 to 4, characterized in that, The dendritic molecule modified phthalocyanine photosensitizing compound has any one of the structures shown in the following formula V to VIII: m, n are each independently selected from an integer of -5; R2, R3, R4, R5, R' are the same as defined in claim 1.

6. The phthalocyanine photosensitizer compound modified with a dendrimer according to any one of claims 1 to 5, wherein The dendritic molecule modified phthalocyanine photosensitizing compound has any one of the structures shown in the following formula IX to XII: q is selected from an integer of 1-7; R' is the same as defined in claim 1.

7. The phthalocyanine photosensitizer compound modified with a dendrimer according to any one of claims 1 to 6, wherein The dendritic molecule modified phthalocyanine photosensitizing compound has any one of the structures shown in the following formula XIII to XVI: Ra is selected from any one of H, acetyl, propionyl, n-butyryl, isobutyryl, t-butoxyacetyl, biotin group, trifluoroacetyl, pentafluoropropionyl, heptafluorobutyryl, trichloroacetyl, benzoyl, 4-fluorobenzoyl, 2,3,4,5,6-pentafluorobenzoyl, 4-trifluoromethylbenzoyl, 3,4-dichlorobenzoyl or 4-trichloromethylbenzoyl; preferably any one of H, t-butoxyacetyl, biotin group, trifluoroacetyl, pentafluoropropionyl or heptafluorobutyryl.

8. The phthalocyanine photosensitizer compound modified with a dendrimer according to any one of claims 1 to 7, wherein The dendritic molecule modified phthalocyanine photosensitizing compound is selected from any one of the structures shown in the following formula XVII to XX: wherein R b is selected from any one of hydroxy, methoxy, ethoxy, n-propoxy, i-propoxy or n-butoxy, preferably methoxy.

9. The phthalocyanine photosensitizer compound modified with a dendrimer according to any one of claims 1 to 8, characterized in that, The dendritic molecule modified phthalocyanine photosensitizing compound is selected from any one of the following compounds A1 to A13:

10. A tumor-targeting photodynamic therapeutic agent, characterized by comprising: a tumor-targeting moiety; a photosensitizer; and a linker connecting the tumor-targeting moiety and the photosensitizer. The tumor-targeting photodynamic therapy drug comprises the dendritic molecule modified phthalocyanine photosensitizing compound as claimed in any one of claims 1 to 9.

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