A vh032 modified phthalocyanine and preparation method and application thereof

Abstract

This invention discloses a VH032-modified phthalocyanine, its preparation method, and its applications. A chemical synthesis method involving amino and carboxyl coupling is employed. The photosensitizer ZnPc and the Von Hippel-Lindau (VHL) ligand VH032 are linked using either no linker or an alkyl or PEG chain as a linker to construct the VH032-modified phthalocyanine. This drug can degrade VHL after light irradiation and, combined with photodynamic therapy (PDT), efficiently kill tumor cells, achieving a phototoxicity index (PI) of 36397. Utilizing the more active regulation of Cyclin Dependent Kinase 2 / 4 (CDK2 / 4) by VHL in bladder cancer tumor tissue, after light irradiation, this compound exhibits a selectivity index (SI) of 749 for killing bladder cancer cells compared to normal cells, achieving selective killing of tumor cells under light conditions and effectively avoiding the risk of damage to adjacent normal tissues during treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a VH032-modified phthalocyanine, its preparation method, and its application. Background Technology

[0002] Enhancing anticancer efficacy while avoiding toxicity to normal tissues is one of the key scientific challenges in the field of antitumor therapy. Developing drugs with the ability to selectively kill tumor cells is an important strategy for addressing this challenge. Photodegradation-targeting chimeras offer a solution, consisting of a target protein ligand, a linker, and a photosensitizer. After the target ligand binds to the target protein, illumination of the lesion site induces the photosensitizer to generate reactive oxygen species (ROS) that degrade the target protein. By controlling the illumination range, the degradation of the target protein is confined to the lesion site, thus theoretically avoiding off-target toxicity. However, in practical applications, due to the difficulty in defining tumor boundaries and the limitations of existing illumination technologies in precisely defining the illumination area, it is difficult to limit the illumination range to within the tumor boundary. This may lead to damage to adjacent normal tissues while killing cancer cells.

[0003] Von Hippel-Lindau protein (VHL) can exert a pro-cancer effect by regulating the downstream p53 pathway and promoting the activation of Cyclin Dependent Kinase 2 / 4 (CDK2 / 4). Currently, there are no reported photodegradation-targeting chimeras or proteolytic-targeting chimeras that degrade VHL. Summary of the Invention

[0004] Purpose of the invention: To address the risk of photodegradation-targeting chimeric drugs damaging adjacent normal tissues while killing cancer cells, this invention designs VH032-modified phthalocyanine. The compound prepared by this invention generates reactive oxygen species through photoactivation to directly degrade the target protein VHL. Taking advantage of the more active regulation of CDK2 / 4 by VHL in bladder cancer cells, this invention achieves selective killing of tumor cells under light irradiation, avoiding the risk of damage to adjacent tissues caused by photodegradation-targeting chimeric drugs.

[0005] The present invention also provides a method for preparing and applying the VH032-modified phthalocyanine.

[0006] Technical solution: To achieve the above objectives, the present invention provides a VH032-modified phthalocyanine drug, the structural formula of which is shown in Formula I, Formula II, or Formula III:

[0007]

[0008] Formula Ι ZnPc-VH032

[0009]

[0010] Formula II ZnPc-PEG2-VH032

[0011]

[0012] Formula III ZnPc-butly-VH032

[0013] The preparation method of VH032-modified phthalocyanine according to the present invention includes the following steps:

[0014] (1) Synthesis of compound 1 (ZnPc-VH032) of formula I: ZnPc-COOH, (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (VH032-NH2), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and 4-dimethylaminopyridine (DMAP) were dissolved in an organic solvent and reacted at room temperature to obtain compound 1 (ZnPc-VH032) of formula I.

[0015] (2) Synthesis of compound 2 (ZnPc-PEG2-VH032) shown in Formula II: ZnPc-COOH, tert-butyl 9-amino-4,7-dioxanonate, HATU and DMAP were dissolved in an organic solvent and reacted at room temperature to obtain intermediate compound 2a; 2a was dissolved in a mixture of organic solvent and trifluoroacetic acid and reacted at room temperature to obtain intermediate compound 2b; intermediate 2b, VH032-NH2, HATU and DMAP were dissolved in an organic solvent and reacted at room temperature to obtain compound 2 (ZnPc-PEG2-VH032) shown in Formula II.

[0016] (3) Synthesis of compound 3 (ZnPc-butly-VH032) shown in Formula III: ZnPc-COOH, tert-butyl 5-aminovalerate, HATU and DMAP were dissolved in an organic solvent and reacted at room temperature to obtain compound 3a; 3a was dissolved in a mixture of organic solvent and trifluoroacetic acid and reacted at room temperature to obtain compound 3b; compound 3b, VH032-NH2, HATU and DMAP were dissolved in an organic solvent and reacted at room temperature to obtain compound 3 (ZnPc-butly-VH032) shown in Formula III.

[0017] The reaction formula is shown below:

[0018] .

[0019] In step (1), the molar ratio of ZnPc-COOH, VH032-NH2, HATU, DIPEA and DMAP is 1:0.8~1.5:1.8~3:11~15:3~5, the solvent volume is 15~25 mL, the reaction temperature is room temperature, and the stirring reaction time is 4~6 h.

[0020] Preferably, in step (1), the molar ratio of ZnPc-COOH, VH032-NH2, HATU, DIPEA and DMAP is 1:1.1:2:13.7:4, the solvent volume is 20 mL, the reaction temperature is room temperature, and the stirring reaction time is 5 h.

[0021] In step (2), when synthesizing compound 2a, the molar ratio of ZnPc-COOH, tert-butyl 9-amino-4,7-dioxanonate, HATU, DIPEA, and DMAP is 1:0.8~2:1.8~3:6~8:3~5, the reaction temperature is room temperature, and the stirring reaction time is 4~6 h; when synthesizing compound 2b, the reaction temperature is room temperature, and the stirring reaction time is 7~9 h; when synthesizing compound 2, the molar ratio of compound 2b, VH032-NH2, HATU, DIPEA, and DMAP is 1:0.8~1.5:1.8~3:11~15:3~5, the reaction temperature is room temperature, and the stirring reaction time is 4~6 h.

[0022] Preferably, in step (2), when synthesizing compound 2a, the molar ratio of ZnPc-COOH, tert-butyl 9-amino-4,7-dioxanonate, HATU, DIPEA, and DMAP is 1:1:2:6.83:4, the reaction temperature is room temperature, and the stirring reaction time is 5 h; when synthesizing compound 2b, the reaction temperature is room temperature, and the stirring reaction time is 8 h; when synthesizing compound 2, the molar ratio of compound 2b, VH032-NH2, HATU, DIPEA, and DMAP is 1:1.1:2:13.7:4, the reaction temperature is room temperature, and the stirring reaction time is 5 h.

[0023] In step (3), when synthesizing compound 3a, the molar ratio of ZnPc-COOH, tert-butyl 5-aminovalerate, HATU, DIPEA, and DMAP is 1:2~3.5:1.8~3:6~8:3~5, the reaction temperature is room temperature, and the stirring reaction time is 4~6 h; when synthesizing compound 3b, the reaction temperature is room temperature, and the stirring reaction time is 7~9 h; when synthesizing compound 3, the molar ratio of compound 3b, VH032-NH2, HATU, DIPEA, and DMAP is 1:0.8~1.5:1.8~3:11~15:3~5, the reaction temperature is room temperature, and the stirring reaction time is 4~6 h.

[0024] Preferably, in step (3), when synthesizing compound 3a, the molar ratio of ZnPc-COOH, tert-butyl 5-aminovalerate, HATU, DIPEA, and DMAP is 1:2.5:2:6.83:4, the reaction temperature is room temperature, and the stirring reaction time is 5 h; when synthesizing compound 3b, the reaction temperature is room temperature, and the stirring reaction time is 8 h; when synthesizing compound 3, the molar ratio of compound 3b, VH032-NH2, HATU, DIPEA, and DMAP is 1:1.1:2:13.7:4, the reaction temperature is room temperature, and the stirring reaction time is 5 h.

[0025] The application of the VH032-modified phthalocyanine drug described in this invention in the preparation of anticancer drugs.

[0026] The application of the VH032-modified phthalocyanine drug as a photosensitizer in the preparation of anticancer drugs.

[0027] Furthermore, the VH032-modified phthalocyanine compound (ZnPc-PEG2-VH032) described in this invention not only has excellent photosensitizing activity, but can also selectively kill tumor cells under light conditions.

[0028] This invention utilizes a chemical synthesis method involving the coupling of amino and carboxyl groups to modify phthalocyanines using VH032. The photosensitizer ZnPc and the VHL ligand VH032 are linked using either no linker or alkyl or PEG chains as linkers to construct VH032-modified phthalocyanines. The optimal photosensitizer, ZnPc-PEG2-VH032, was selected. VHL can promote cancer development by regulating the downstream p53 pathway and activating CDK2 / 4. In bladder cancer cells (T24), ZnPc-PEG2-VH032 effectively generates ROS after light exposure, degrading VHL. The PI of ZnPc-PEG2-VH032 is 36397 (PI = IC50). 50-黑暗条件-肿瘤细胞 / IC 50-光照条件-肿瘤细胞 The PI was 50 times higher than that of the clinically used phthalocyanine photosensitizer Photosens® (PI = 725). Further studies revealed that ZnPc-PEG2-VH032, after photoexcitation, had a significantly higher ability to kill T24 cells (bladder cancer cells) than SV-HUC-1 cells (normal cells); the SI was as high as 749 (SI = IC50). 50-光照条件-正常细胞 / IC 50-光照条件-肿瘤细胞ZnPc-PEG2-VH032 is currently the photodegradation-targeting chimeric drug with the highest SI (Intensity Sequence) among photosensitizers, a level extremely rare even for photosensitizers. Mechanistic studies revealed no significant difference in VHL expression levels between T24 and SV-HUC-1 cells, but CDK2 / 4 activation levels were significantly higher in T24 cells than in normal cells, suggesting more active VHL regulation of CDK2 / 4 in T24 cells. Simultaneously, T24 cells showed stronger uptake of ZnPc-PEG2-VH032 than SV-HUC-1 cells. Furthermore, inhibition of CDK2 / 4 activation increased the sensitivity of tumor cells to reactive oxygen species damage, further facilitating PDT activity. These factors contribute to the selective killing of tumor cells by ZnPc-PEG2-VH032. In an in vivo animal model of non-muscle-invasive bladder cancer (NMIBC), the therapeutic effect of ZnPc-PEG2-VH032 reached the level of mitomycin C (MMC), a chemotherapy drug recommended in clinical bladder cancer guidelines.

[0029] This invention reveals that although VHL expression levels show no significant difference between bladder cancer cells and normal cells, the activation levels of cyclin-dependent kinase 2 (CDK2 / 4) in bladder cancer cells are significantly higher than in normal cells, suggesting that VHL's regulation of CDK2 / 4 is more active in bladder cancer cells. Therefore, a photodegradable VHL-targeting chimera designed based on this finding holds promise for selectively killing tumor cells, avoiding the risk of damaging adjacent normal tissues while killing cancer cells.

[0030] This invention combines monosubstituted zinc phthalocyanine with VHL target protein ligand VH032 to synthesize and screen the first PDTAC with both high PI and high SI values—ZnPc-PEG2-VH032. Simultaneously, it utilizes the more active regulation of CDK2 / 4 by VHL in T24 cells compared to SV-HUC-1 cells, and the stronger ZnPc-PEG2-VH032 uptake capacity in T24 cells, to achieve the goal of selectively killing tumor cells. This reveals the potential of VHL as a target for precision tumor therapy. Compared with other photosensitizers, the SI value of ZnPc-PEG2-VH032 in this study reached 749 for T24 vs SV-HUC-1 cells, demonstrating a significant advantage in selective tumor cell killing.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0032] (1) The drug prepared by this invention, such as ZnPc-PEG2-VH032, has a PI value of 36397, which is much higher than the approved phthalocyanine photosensitizing drug Photosens® (PI = 725), and has excellent photosensitizing and tumor cell killing activity and high PI value characteristics.

[0033] (2) The drugs prepared in this invention, such as ZnPc-PEG2-VH032, have a much higher ability to kill T24 cells (bladder cancer cells) after photoexcitation than SV-HUC-1 cells (normal cells); the SI is as high as 749 (SI = IC50). 50-光照条件-正常细胞 / IC 50-光照条件-肿瘤细胞 It is currently the photodegradation-targeting chimeric drug with the highest SI, and such a high SI is very rare among photosensitizers.

[0034] (3) The drug prepared by the present invention has significant therapeutic effect on tumors in vivo, which is comparable to MMC, a chemotherapy drug recommended by clinical guidelines for bladder cancer.

[0035] (4) The preparation method of the present invention is simple and efficient, the raw materials are readily available, and it has good industrial production value. Attached Figure Description

[0036] Figure 1 The UV-Vis absorption spectra of the three VH032-modified phthalocyanine compounds of the present invention in aqueous solution are shown below.

[0037] Figure 2 (A) Blotting (A1) and quantitative analysis of VHL at the protein level in T24 cells and SV-HUC-1 cells (A2); (B) Proliferation curves of T24 cells and SV-HUC-1 cells; Blotting (C1) and quantitative analysis of p-CDK2 / 4 at the protein level in T24 cells and SV-HUC-1 cells (C2).

[0038] Figure 3 To determine the effects of different drug treatments on VHL protein in T24 cells, we performed blotting (A1) and quantitative analysis (A2); and blotting (B1) and quantitative analysis (B2) of the thermal stability of VHL protein with ZnPc-PEG2-VH032 and DMSO.

[0039] Figure 4 To compare the cell cycle distribution of T24 cells with p-CDK2 / 4 / 6 protein blotting (A1) and the expression level of p-CDK2 / 4 / 6 protein (A2) under different drug treatment conditions; (B) The effect of CDK2 / 4 / 6 inhibitors on the ROS damage sensitivity of T24 cells;

[0040] Figure 5 Flow cytometry (A) and comparison of uptake capacity (B) of ZnPc-PEG2-VH032 in T24 and SV-HUC-1 cells.

[0041] Figure 6H&E staining sections of bladder tissue from the PBS, ZnPc-PEG2-VH032, MMC and ZnPc-PEG2-VH032+Light treatment groups.

[0042] In each figure, ns indicates no significant difference. . Detailed Implementation

[0043] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0044] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0045] (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (VH032-NH2), CAS No. 1448189-80-7.

[0046] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), CAS No. 125700-67-6.

[0047] 4-Dimethylaminopyridine (DAMP), CAS No. 1122-58-3.

[0048] N,N-Diisopropylethylamine (DIPEA), CAS No. 7087-68-5.

[0049] 9-Amino-4,7-dioxanonanoic acid tert-butyl ester, CAS No. 756525-95-8.

[0050] Dichloromethane (DCM), CAS No. 75-09-2.

[0051] Trifluoroacetic acid (TFA), CAS No. 76-05-1.

[0052] 5-Aminovalerate tert-butyl ester, CAS No. 63984-03-2.

[0053] Commercial phthalocyanine photosensitizer Photosens ® CAS number 122170-90-5.

[0054] ZnPc-COOH synthesis reference: Kebin Ye, Xinlu Bao, Zhikang Song, MingzhuZhang, Shaohua Wei, Jishuang Zhang, Lin Zhou, A mono-substituted aluminumphthalocyanine photosensitizer with high phototoxicity index for effectivebladder cancer treatment, Bioorganic Chemistry, 163 (20125) 108730. https: / / doi.org / 10.1016 / j.bioorg.2025.108730.

[0055] Its structure is as follows:

[0056]

[0057] ZnPc-SFeCO synthesis reference: Lulu Kong, Kaikai Xu, Xinlu Bao, Kebin Ye, DiFan, Ning Wang, Yongming Deng, Wei Wang, Shaohua Wei, and Lin Zhou, mTORC1Selective Nano-Inhibitor by Disrupting the Lysosomal Arginine-SLC38A9-mTORC1-CDKs Axis for Precision Bladder Cancer Therapy, Adv. Mater. 2025,2504798. https: / / doi.org / 10.1002 / adma.202504798 Alternatively, the preparation method described in Example 1 of the patent: A ZnPc-SFeCO compound and its preparation method and application (2025106500767).

[0058] Its structure is as follows: .

[0059] Example 1

[0060] ZnPc-COOH (60 mg, 0.084 mmol), VH032-NH2 (43.15 mg, 0.0924 mmol), HATU (63.84 mg, 0.168 mmol), and DMAP (40.99 mg, 0.336 mmol) were weighed into a round-bottom flask. 200 μL of LDIPEA was added and dissolved in 20 mL of DMF. The mixture was reacted at room temperature for 5 h. The solution was washed with a mixture of methanol and hydrochloric acid (3.5 mL methanol + 0.5 mL hydrochloric acid), centrifuged, and air-dried to obtain the product ZnPc-VH032. MALDI-TOP (m / z) calculated for C 61 H 48 N 12 O5SZn:1126.57, found[M+H] + : 1126.29. 1H N5MR (400 MHz, DMSO) δ 9.19 – 8.85 (m, 9H),8.65 (t, J = 6.0 Hz, 1H), 8.46 (d, J = 1.6 Hz, 1H), 8.14 (dddd, J = 34.3,22.9, 13.2, 7.8 Hz, 9H), 7.77 (dd, J = 8.2, 2.0 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.43 (dd, J = 17.8, 8.4 Hz, 4H), 5.25 (d, J = 3.5 Hz, 1H), 4.90 (d, J =9.1 Hz, 1H), 4.60 – 4.40 (m, 3H), 4.27 (dd, J = 15.9, 5.4 Hz, 1H), 3.89 –3.76 (m, 2H), 2.43 (s, 3H), 2.19 – 2.05 (m, 1H), 1.98 (ddd, J = 12.8, 8.6,4.5 Hz, 1H), 1.11 (d, J = 9.5 Hz, 9H).

[0061]

[0062] Example 2

[0063] ZnPc-COOH (120 mg, 0.168 mmol), tert-butyl 9-amino-4,7-dioxanonate (40 μL), HATU (127.68 mg, 0.336 mmol), and DMAP (81.98 mg, 0.672 mmol) were placed in a round-bottom flask. 200 μL of LIPEA was added, and the mixture was dissolved in 30 mL of DMF. The mixture was reacted at room temperature for 5 h to obtain compound 2a. Compound 2a was dissolved in DCM (10 mL) and TFA (2 mL) and stirred for 8 h to remove BOC. The mixture was washed with water and methanol, centrifuged, and the lower solid layer was dried in a vacuum drying oven to obtain compound 2b. Compound 2b (73 mg, 0.084 mmol), VH032-NH2 (43.15 mg, 0.0924 mmol), HATU (63.84 mg, 0.168 mmol), and DMAP (40.99 mg, 0.336 mmol) were weighed into a round-bottom flask. 200 μL of DIPEA was added, dissolved in 20 mL of DMF, and reacted at room temperature for 5 h. The mixture was washed with a mixture of methanol and hydrochloric acid (3.5 mL methanol + 0.5 mL hydrochloric acid), centrifuged, and air-dried to obtain the product ZnPc-PEG2-VH032. MALDI-TOP(m / z) calculated, for C 68 H 61 N 13 O8SZn: 1185.76, found[M+H] + : 1185.37. 1 H NMR (400 MHz, DMSO) δ 9.25 – 8.89 (m, 9H), 8.76 –8.46 (m, 4H), 8.30 – 8.03 (m, 8H), 7.96 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 6.7Hz, 1H), 7.56 (d, J = 7.2 Hz, 2H), 7.38 (d, J = 9.1 Hz, 4H), 5.15 (s, 1H), 4.57 (d, J = 8.6 Hz, 2H), 4.39 (d, J = 34.7 Hz, 4H), 4.21 (d, J = 11.9 Hz,2H), 3.77 – 3.47 (m, 13H), 2.41 (s, 4H), 1.97 (d, J = 54.1 Hz, 3H), 0.95 (s, 9H).

[0064]

[0065] Example 3

[0066] ZnPc-COOH (120 mg, 0.168 mmol), tert-butyl 5-aminovalerate (75 μL), HATU (127.68 mg, 0.336 mmol), and DMAP (81.98 mg, 0.672 mmol) were placed in a round-bottom flask, and 200 μL of DIPEA was added. The mixture was then dissolved in 30 mL of DMF and reacted at room temperature for 5 h to obtain compound 3a. Compound 3a was dissolved in DCM (10 mL) and TFA (2 mL) and stirred for 8 h to remove BOC, yielding compound 3b. Compound 3b (68 mg, 0.084 mmol), VH032-NH2 (43.15 mg, 0.0924 mmol), HATU (63.84 mg, 0.168 mmol), and DMAP (40.99 mg, 0.336 mmol) were weighed into a round-bottom flask. 200 μL of DIPEA was added, dissolved in 20 mL of DMF, and reacted at room temperature for 5 h. The mixture was washed with a mixture of methanol and hydrochloric acid (3.5 mL methanol + 0.5 mL hydrochloric acid), centrifuged, and air-dried to obtain the product ZnPc-butly-VH032. MALDI-TOP (m / z) calculated for C 66 H 57 N 13 O6SZn: 1225.71, found[M+H] + : 1225.35. 1H NMR (400 MHz, DMSO) δ 9.19 – 8.85 (m, 9H), 8.65 (t, J = 6.0 Hz, 1H), 8.46 (d, J = 1.6 Hz, 1H), 8.14 (dddd, J = 34.3, 22.9, 13.2, 7.8 Hz, 9H), 7.77 (dd, J = 8.2, 2.0Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.43 (dd, J = 17.8, 8.4 Hz, 4H), 5.25 (d,J = 3.5 Hz, 1H), 4.90 (d, J = 9.1 Hz, 1H), 4.60 – 4.40 (m, 3H), 4.27 (dd, J =15.9, 5.4 Hz, 1H), 3.89 – 3.76 (m, 2H), 2.43 (s, 3H), 2.19 – 2.05 (m, 1H),1.98 (ddd, J = 12.8, 8.6, 4.5 Hz, 1H), 1.11 (d, J = 9.5 Hz, 9H).

[0067]

[0068] Example 4

[0069] UV-Vis absorption curve

[0070] The UV absorption spectra of the three VH032-modified phthalocyanine compounds in Examples 1-3 in water were determined using a UV-Vis spectrophotometer.

[0071] like Figure 1 As shown, in the aqueous phase, the absorption spectrum peak shapes and peak intensities of the three molecules ZnPc-VH032, ZnPc-PEG2-VH032, and ZnPc-butyl-VH032 are basically consistent.

[0072] Example 5

[0073] In vitro cell phototoxicity detection

[0074] In vitro cytotoxicity was evaluated using the CCK-8 assay. Due to the significant difference in proliferation rates between the two cell types, 1.25 × 10⁻⁶ cells were used. 4 3.75 × 10 T24 cells 4SV-HUC-1 cells were seeded into 96-well plates. After 24 h, both cell types covered 70% of the bottom area of ​​the culture wells. The culture medium was then replaced with serum-free medium containing ZnPc-VH032, ZnPc-PEG2-VH032, and ZnPc-butyl-VH032 (10, 50, 100, and 200 μM) (SV-HUC-1 cells were incubated with ZnPc-PEG2-VH032 only). After incubation for 24 h, cell viability was measured by the CCK8 assay to evaluate the dark toxicity of the three VH032-modified phthalocyanines at different concentrations in the two cell types.

[0075] 1.25×10 4 T24 cells were seeded into 96-well plates. After 24 h, the culture medium was replaced with serum-free medium containing ZnPc-VH032 (0.5, 1, 5, 10 μM), ZnPc-PEG2-VH032 (1, 5, 10, 15 nM), and ZnPc-butyl-VH032 (1, 5, 10, 15 nM); 3.75 × 10⁻⁶ cells / well. 4 SV-HUC-1 cells were seeded in 96-well plates. After 24 h, the culture medium was replaced with serum-free ZnPc-PEG2-VH032 (0.1, 0.5, 1, 10 μM). After incubation for 15 h, the cells were exposed to a 680 nm LED light source (24.18 mW / cm²). 2 Illuminate the culture plate for 10 min (14.51 J / cm²) 2 After culturing for another 9 h, cell viability was measured using the CCK8 assay to assess the phototoxicity of three VH032-modified phthalocyanines at different concentrations in the two cell types.

[0076] The PI values ​​of ZnPc-VH032, ZnPc-PEG2-VH032, and ZnPc-butyl-VH032 against T24 cells were 166, 3.6 × 10⁻⁶, and 3.6 × 10⁻⁶, respectively. 4 and 2.1×10 4 Compared with ZnPc-VH032 and ZnPc-butyl-VH032, ZnPc-PEG2-VH032 showed a PI value increase of 219 and 1.70 times, respectively. In the clinical application of phototherapy (PDT), due to the difficulty in defining tumor boundaries and the inability of existing light therapy techniques to precisely define the illumination area, it is difficult to confine the illumination range within the tumor boundary. This may lead to damage to adjacent normal tissue while killing cancer cells. Therefore, the killing ability of ZnPc-PEG2-VH032 on SV-HUC-1 (normal cells) was also investigated in this study. Similar to T24 cells, its dark toxicity in SV-HUC-1 cells was also very low (IC50). 50= 190.224 μM), but compared with T24 cells, ZnPc-PEG2-VH032 showed significantly reduced phototoxic activity against SV-HUC-1 cells, with a phototoxic IC50 of 190.224 μM. 50 The PI value was 749 times that of T24 cells, while the PI value was only 45, a significant decrease of 805 times compared to T24 cells (Table 1). Therefore, under the same light conditions, ZnPc-PEG2-VH032's ability to kill T24 cells was far greater than its ability to kill SV-HUC-1 cells, with an PI value as high as 749; achieving the goal of selectively killing tumor cells without controlling the light range. Since there are few reports on SI values ​​in existing studies on photodegradation-targeting chimeras, this invention systematically surveys the data of relevant PDT drugs for reference: Tridib K. Goswami reported that the visible light-activated Ni(II)-curcumin complex for A549 vs HPL1D cells had an SI of 22.1 [Eur. J. Med. Chem., 204 (2020) 112632]; Benzhong Tang's aggregation-induced emission photosensitizer (AIE-PS) for HEPG2 vs L02 cells had an SI of 18.6 [Nat. Commun., 15 (2024) 9999]; Fude Feng designed a mitochondrial-targeting zinc porphyrin mitoZnPor for 4T1 vs L02 cells with an SI of 498 [Chem. Sci., 15 (2024) 20292-20302]. In comparison, the ZnPc-PEG2-VH032 prepared in Example 2 of this invention achieved an SI value of 749 against T24 vs SV-HUC-1 cells, which is the highest SI value among drugs of this class to date, demonstrating a significant advantage in selective killing of tumor cells.

[0077] Table 1

[0078]

[0079] Table 1 shows the phototoxicity IC50 of three VH032-modified phthalocyanine compounds in T24 cells. 50 Dark Poison IC 50 PI value; phototoxic IC50 of three VH032-modified phthalocyanine compounds in SV-HUC-1 cells. 50 Dark Poison IC 50 PI value; phototoxic IC50 of ZnPc-PEG2-VH032 in SV-HUC-1 and T24 cells. 50 Comparison (SI).

[0080] Example 6

[0081] Protein immunoblotting detection

[0082] 1.5×10 6 1 T24 cells and 1.5 × 10 6 SV-HUC-1 cells were washed once with PBS, and 500 μL of IP lysis buffer containing 1% PMSF was added. For p-CDK2 / 4 / 6 protein detection, a phosphatase inhibitor was added at this step. After cell lysis, cells were collected and centrifuged at 12000 rpm at 4 °C for 15 min. The centrifuged cells were quantified using a BCA protein assay kit, and protein loading buffer was added. The cells were then boiled for 10 min to denature the protein. The resulting samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane, which was then blocked with 5% skim milk powder for 1 h. Subsequently, the PVDF membrane was washed three times with Tris-HCl-Tween (TBST) buffer for 8 min each time; incubated with primary antibody (VHL, Gene Tex, catalog number GTX101087; p-CDK2, Proteintech, catalog number 10122-1-AP; p-CDK4, Proteintech, catalog number 11026-1-AP) at 4 °C for 24 h; after washing, incubated with internal control (Vinculin, Proteintech, catalog number 26520-1-AP) for 24 h; after incubation with secondary antibody at room temperature for 2 h, the membrane was placed in a protein chemiluminescence imaging system for luminescence imaging, and the band grayscale was calculated using ImageJ to assess the corresponding protein expression level.

[0083] Although the expression levels of VHL in normal cells and tumor cells are similar ( Figure 2 A1 and Figure 2 A2), but the proliferation rate of T24 cells was significantly higher than that of SV-HUC-1 cells (A2), Figure 2 B); and the CDK2 / 4 activation level in T24 cells was significantly higher than that in normal cells (B); Figure 2 C1 and Figure 2 (C2) Therefore, it is speculated that VHL's regulation of CDK2 / 4 is more active in T24 cells, meaning that VHL's regulatory role in CDK2 / 4 is more critical and sensitive. These factors lead to the more severe impact of VHL degradation on cell survival in T24 cells compared to SV-HUC-1 cells. This example demonstrates that one reason for the drug's photoselectivity on T24 cells is the more active regulation of CDK2 / 4 by VHL in T24 cells, indicating that VHL's regulatory role in CDK2 / 4 is more critical and sensitive.

[0084] Example 7

[0085] ZnPc-PEG2-VH032 binds to VHL and photo-damaged VHL

[0086] Thermal stability test: Wash with PBS 1.5 × 10 6T24 cells were lysed twice with 500 μL of IP buffer containing 1% PMSF for 2 h, and centrifuged at 16500 rpm at low temperature (4 ℃) for 15 min to obtain the supernatant protein. The supernatant was then added to ZnPc-PEG2-VH032 (0.85 μM, DMSO stock solution) or DMSO and incubated in a centrifuge for 2 h. Seven temperature gradients were set for each sample, and the cells were heated in a water bath at 42 ℃, 47 ℃, 52 ℃, 57 ℃, 62 ℃, 67 ℃, and 72 ℃ for 3 min. After cooling, the cells were centrifuged at 16500 rpm at low temperature (4 ℃) for 20 min, and the supernatant was collected. Protein loading buffer was added, and the cells were boiled for 10 min for denaturation. Western blotting was used to detect VHL protein levels under each condition. Photosensitive damage to VHL: 5 × 10⁶ cells were added to the supernatant protein. 5 T24 cells were seeded in 6-well plates and cultured for 24 h. The cells were then divided into two groups. One group was incubated for 24 h with serum-free medium containing VH032-NH2 (10 μM), ZnPc-PEG2-VH032 (1 nM), and VH032-NH2 (10 μM, pre-incubated for 45 min) + ZnPc-PEG2-VH032 (1 nM). The other group was incubated for 24 h with serum-free medium containing ZnPc-PEG2 (1 nM), ZnPc-PEG2-VH032 (1 nM), and VH032-NH2 (10 μM, pre-incubated for 45 min) + ZnPc-PEG2-VH032 (1 nM). After incubation for 15 h, the cells were exposed to a 680 nm LED light source (24.18 mW / cm²). 2 Illuminate the culture plate for 10 min (14.51 J / cm²) 2 After incubation for another 9 h, the drug-treated cells were washed once with PBS, and 200 μL of IP lysis buffer containing 1% PMSF was added to each well. After cell lysis, the cells were collected and centrifuged at 12,000 rpm at 4 °C for 15 min. The centrifuged cells were quantified using a BCA protein assay kit, and protein loading buffer was added. The cells were then boiled for 10 min to denature them. The resulting samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane, which was then blocked with 5% skim milk powder for 1 h. Subsequently, the PVDF membrane was washed three times with TBST buffer for 8 min each time; incubated with primary antibody (VHL, Gene Tex, catalog number GTX101087) at 4 °C for 24 h; after washing, it was incubated with internal control (Vinculin, Proteintech, catalog number 26520-1-AP) for 24 h; after incubation with secondary antibody at room temperature for 2 h, the membrane was placed in a protein chemiluminescence imaging system for luminescence imaging, and the band grayscale was calculated using ImageJ to assess the corresponding protein expression level.

[0087] Western blot analysis was used to detect the expression level of intracellular VHL under different treatments. Compared with the blank control group, there were no differences in VHL expression levels in the VH032 group, the light-only group (to verify the effect of light on cells), the ZnPc-PEG2-VH032 group, the VH032+ZnPc-PEG2-VH032 group, and the ZnPc-PEG2+Light group, while the VHL level in the ZnPc-PEG2-VH032+Light treatment group was significantly downregulated; after cells were pre-incubated with VH032-NH2 to compete for the ZnPc-PEG2-VH032 binding site, the VHL level in the ZnPc-PEG2-VH032+Light group was upregulated. Figure 3 A1 and Figure 3 (A2). The above experimental results demonstrate that the target protein of ZnPc-PEG2-VH032 in killing T24 cells is VHL, and the degradation of VHL by ZnPc-PEG2-VH032 is not due to indiscriminate protein damage from ROS, but rather to the destruction of PDTAC after the VHL ligand binds to the VHL protein. In this embodiment, VH032-NH2 is a ligand of the VHL protein. Direct drug administration first competes for the target site, resulting in less binding of ZnPc-PEG2-VH032, thus reducing the degradation efficiency of the VHL protein, further proving the binding of the drug to the VHL protein.

[0088] Meanwhile, the effect of ZnPc-PEG2-VH032 on the thermostability of VHL protein was investigated using cellular thermal shift assay (CETSA) [J. Am. Chem. Soc., 144 (2022) 10407-10416; Chem. Sci., 10 (2019) 7193-7199; Chem. Sci., 8 (2017) 4626-4633]. After interaction with ZnPc-PEG2-VH032, the thermostability of VHL protein decreased, demonstrating that ZnPc-PEG2-VH032 can effectively bind to VHL (…). Figure 3 B1 and Figure 3 B2).

[0089] Example 8

[0090] Effects on cell cycle and p-CDK2 / 4 / 6

[0091] Cell cycle arrest study: 5×10 5T24 cells were seeded in 6-well plates and cultured for 24 h. The cells were then divided into two groups. One group was incubated for 24 h with serum-free medium containing VH032-NH2 (10 μM), ZnPc-PEG2-VH032 (1 nM), and VH032-NH2 (10 μM, pre-incubated for 30 min) + ZnPc-PEG2-VH032 (1 nM). The other group was incubated for 24 h with serum-free medium containing ZnPc-PEG2 (1 nM, compound 2b), ZnPc-PEG2-VH032 (1 nM), and VH032-NH2 (10 μM, pre-incubated for 30 min) + ZnPc-PEG2-VH032 (1 nM). After incubation for 15 h, the cells were exposed to a 680 nm LED light source (24.18 mW / cm²). 2 Illuminate the culture plate for 10 min (14.51 J / cm²) 2 The cells were returned to the incubator and incubated for another 9 hours. Both groups of cells were treated according to the instructions of the cell cycle assay kit (Beyotime, product number C1052), and the effects of different conditions on the cell cycle were detected by flow cytometry. p-CDK2 / 4 / 6 protein assay: 5 × 10⁶ cells were cultured in the incubator. 5 T24 cells were seeded in 6-well plates and cultured for 24 h. The cells were then divided into two groups. One group was incubated for 24 h with serum-free medium containing VH032-NH2 (10 μM), ZnPc-PEG2-VH032 (1 nM), and VH032-NH2 (10 μM, pre-incubated for 30 min) + ZnPc-PEG2-VH032 (1 nM). The other group was incubated for 24 h with serum-free medium containing ZnPc-PEG2 (1 nM, compound 2b), ZnPc-PEG2-VH032 (1 nM), and VH032-NH2 (10 μM, pre-incubated for 30 min) + ZnPc-PEG2-VH032 (1 nM). After incubation for 15 h, the cells were exposed to a 680 nm LED light source (24.18 mW / cm²). 2 Illuminate the culture plate for 10 min (14.51 J / cm²) 2The cells were returned to the incubator and incubated for another 9 h. After treatment, the cells were washed once with PBS, and 200 μL of IP lysis buffer containing 1% PMSF and a phosphatase inhibitor (Beyotime, product number P1045) were added to each well. After cell lysis, the cells were collected and centrifuged at 12,000 rpm at low temperature (4 ℃) for 15 min. The centrifuged cells were quantified using a BCA protein concentration assay kit, and protein loading buffer was added. The cells were then boiled for 10 min to denature them. The obtained samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane, which was blocked with 5% skim milk powder for 1 h. Subsequently, the PVDF membrane was washed three times with TBST buffer for 8 min each time; incubated with primary antibodies (p-CDK2, Proteintech, catalog number 10122-1-AP; p-CDK4, Proteintech, catalog number 11026-1-AP; p-CDK6, Proteintech, catalog number 14052-1-AP) at 4 °C for 24 h; after washing, it was incubated with internal control (Vinculin, Proteintech, catalog number 26520-1-AP) for 24 h; after incubation with secondary antibody at room temperature for 2 h, the membrane was placed in a protein chemiluminescence imaging system for luminescence imaging, and the band grayscale was calculated using ImageJ to assess the corresponding protein expression level.

[0092] Effects of cell cycle arrest on cellular oxidative damage sensitivity: 1.25 × 10 4 T24 cells were seeded in 96-well plates and cultured for 24 h. The culture medium was then replaced with serum-free medium containing ZnPc-SFeCO (100 nM, a CDK2 / 4 / 6 inhibitor) or serum-free medium without ZnPc-SFeCO. After 24 h of incubation, the medium was replaced with serum-free medium containing H2O2 (500 or 1000 μM). Cell viability was then measured using the CCK8 assay. ZnPc-SFeCO is a CDK2 / 4 / 6 co-inhibitor. The experimental results showing that the addition of this drug resulted in more severe oxidative damage to cells indicate that when the cell cycle is inhibited, the activity of the antioxidant enzyme system decreases, thereby increasing the risk of oxidative damage. Furthermore, the drug of this invention, as demonstrated by previous cyclin and cell cycle assays, affects the cell cycle, further proving that the drug of this invention can also increase oxidative damage sensitivity and enhance the PDT effect.

[0093] Compared with the blank control group, no significant cell cycle arrest was observed in the VH032 group, light group, ZnPc-PEG2-VH032 group, VHL + ZnPc-PEG2-VH032 group, and ZnPc-PEG2 + Light group. The number of G1 phase cells increased in the ZnPc-PEG2-VH032 + Light group. After cells were pre-incubated with VH032-NH2 to compete for the ZnPc-PEG2-VH032 binding site, the number of G1 phase cells decreased in the ZnPc-PEG2-VH032 + Light group. These results indicate that ZnPc-PEG2-VH032 can effectively degrade VHL protein through the PDTAC mechanism, inducing G1 phase cell cycle arrest. Subsequently, the phosphorylation levels of CDK2 / 4 / 6 were detected. The results showed that the level of p-CDK6 was not significantly affected in the ZnPc-PEG2-VH032 + Light group, but the levels of p-CDK2 and p-CDK4 were significantly reduced. Figure 4 A1 and Figure 4 (A2) After cells were pre-incubated with VH032-NH2 to compete for the ZnPc-PEG2-VH032 binding site, the levels of p-CDK2 and p-CDK4 increased in the ZnPc-PEG2-VH032 + Light group. CDK2 and CDK4 are key kinases in the cell cycle, promoting cell transition from G1 to S phase by phosphorylating Rb protein. When VHL is degraded, it inhibits the activity of CDK2 / 4, thereby preventing Rb protein phosphorylation and arresting cells in G1 phase.

[0094] H2O2 is a commonly used oxidative stress inducer that can mimic the physiological environment of cells under oxidative stress conditions. This mimicry is similar to the killing effect of ROS generated by photosensitizers on tumor cells. In the presence of 500 and 1000 μM H2O2 concentrations, the CDK2 / 4 / 6 inhibitor (ZnPc-SFeCO) significantly increased the sensitivity of T24 cells to oxidative damage, indicating that CDK2 and CDK4 inhibition combined with photodynamic therapy has a synergistic sensitizing effect. ZnPc-PEG2-VH032, as a PDTAC drug, can effectively target the VHL protein while exerting photosensitizing effects. Through the specific degradation of VHL by the generated ROS, it causes cell cycle arrest, increases the sensitivity of cells to PDT oxidative damage, and achieves better anti-tumor therapeutic effects. Figure 4 B).

[0095] Example 9

[0096] Differences in ZnPc-PEG2-VH032 uptake between T24 cells and SV-HUC-1 cells

[0097] 6.25×10 41.875 × 10 T24 cells 5 SV-HUC-1 cells were seeded into 24-well plates. After 24 h, both cell types covered 70% of the bottom area of ​​the culture wells. The original culture medium was removed and replaced with serum-free culture medium containing ZnPc-PEG2-VH032 (10 μM). After 15 h of drug incubation, the fluorescence intensity of the drug in the cells was detected by flow cytometry.

[0098] Both SV-HUC-1 cells and T24 cells can effectively take up ZnPc-PEG2-VH032; under the same experimental conditions, the uptake rate of ZnPc-PEG2-VH032 by SV-HUC-1 cells is lower than that by T24 cells. Figure 5 A), the fluorescence intensity of ZnPc-PEG2-VH032 in T24 cells was approximately 2.6 times that in SV-HUC-1 cells. Figure 5 B). The second reason for the photoselectivity of the drug to T24 is the difference in drug uptake.

[0099] Example 10

[0100] ZnPc-PEG2-VH032 in vivo therapy for bladder cancer

[0101] C57BL / 6 mice (female, weighing 16-20 g) were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd. An NMIBC model was then constructed using mCherry gene-transfected mouse bladder cancer cells (mcherry-MB-49) in homologous C57BL / 6 mice. (Methods followed Advanced Materials, 2025, 37, 2504798). NMIBC mice were randomly divided into four groups: control group, MMC positive control group, ZnPc-PEG2-VH032 control group, and ZnPc-PEG2-VH032+Light treatment group. Mouse weight was measured daily. On days 1, 3, 5, 8, and 10, the control group was administered PBS (50 μL), and the MMC positive control group was administered MMC (0.625 mg / kg) via bladder instillation, respectively. −1 ZnPc-PEG2-VH032 (50 μL), ZnPc-PEG2-VH032 control group (0.625 mg kg) −1 The treatment group received ZnPc-PEG2-VH032 (0.625 mg / kg) and the ZnPc-PEG2-VH032+Light treatment group received ZnPc-PEG2-VH032 (0.625 mg / kg). −150 μL), of which the bladder of mice in the ZnPc-PEG2-VH032+Light treatment group was irradiated with LED light for 10 min (12 J / cm²) 2 h after drug administration. 2 After a 10-day treatment period, the mice were euthanized, and the bladders were removed for fluorescence imaging, fixed with paraformaldehyde, embedded in paraffin, and sectioned. The bladders were then stained with hematoxylin and eosin (H&E) and imaged under an optical microscope for pathological analysis.

[0102] Pathological section results showed that the bladders of control mice were filled with a large number of high-density tumor structures; the control group treated with ZnPc-PEG2-VH032 but without light did not show any shrinkage effect, while the ZnPc-PEG2-VH032+Light treatment group showed a similar therapeutic effect to the clinical NMIBC treatment drug MMC, with the bladder tumors basically disappearing and the bladder tissue structure returning to normal after treatment. Figure 6 This study demonstrated that ZnPc-PEG2-VH032 exhibited good antitumor activity after phototherapy, and its therapeutic effect reached the level of MMC, a chemotherapy drug for bladder cancer recommended by clinical guidelines.

Claims

1. A VH032 modified phthalocyanine, characterized in that, The VH032 modified phthalocyanine has the following formula I, formula II or formula III: ； Formula I ； Formula II ； Formula III.

2. A method of preparing a VH032 modified phthalocyanine drug of claim 1, wherein, The method comprises the following steps: (1) Synthesis of compound 1 (ZnPc-VH032) shown in formula I: ZnPc-COOH, (2S, 4R)-1-((S)-2-amino-3, 3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide hydrochloride (VH032-NH2), O-(7-azabenzotriazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU) and 4-dimethylaminopyridine (DMAP) are dissolved in an organic solvent, and the reaction is carried out at room temperature to obtain compound 1 (ZnPc-VH032) shown in formula I; (2) Synthesis of compound 2 (ZnPc-PEG2-VH032) shown in formula II: ZnPc-COOH, 9-amino-4, 7-dioxanonoate tert-butyl ester, HATU and DMAP are dissolved in an organic solvent, and the reaction is carried out at room temperature to obtain intermediate compound 2a; 2a is dissolved in a mixture of an organic solvent and trifluoroacetic acid, and the reaction is carried out at room temperature to obtain intermediate compound 2b; intermediate 2b, VH032-NH2, HATU and DMAP are dissolved in an organic solvent, and the reaction is carried out at room temperature to obtain compound 2 (ZnPc-PEG2-VH032) shown in formula II; (3) Synthesis of compound 3 (ZnPc-butly-VH032) shown in formula III: ZnPc-COOH, 5-aminovaleric acid tert-butyl ester, HATU and DMAP are dissolved in an organic solvent, and the reaction is carried out at room temperature to obtain compound 3a; 3a is dissolved in a mixture of an organic solvent and trifluoroacetic acid, and the reaction is carried out at room temperature to obtain compound 3b; compound 3b, VH032-NH2, HATU and DMAP are dissolved in an organic solvent, and the reaction is carried out at room temperature to obtain compound 3 (ZnPc-butly-VH032) shown in formula III; The reaction formula is as follows: 。 3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of ZnPc-COOH, VH032-NH2, HATU, DIPEA and DMAP is preferably 1:0.8-1.5:1.8-3:11-15:3-5.

4. The production method according to claim 2, characterized by, In step (1), the volume of the solvent is 15-25 mL, the reaction temperature is room temperature, and the stirring reaction time is 4-6 h.

5. The preparation method according to claim 2, characterized in that, In step (2), when synthesizing compound 2a, the molar ratio of ZnPc-COOH, 9-amino-4, 7-dioxanonoate tert-butyl ester, HATU, DIPEA and DMAP is 1:0.8-2:1.8-3:6-8:3-5; the reaction temperature is room temperature, and the stirring reaction time is 4-6 h; when synthesizing compound 2b, the reaction temperature is room temperature, and the stirring reaction time is 7-9 h.

6. The preparation method according to claim 2, characterized in that, In the synthesis of compound 2 in step (2), the molar ratio of compound 2b, VH032-NH2, HATU, DIPEA and DMAP is 1:0.8-1.5:1.8-3:11-15:3-5, the reaction temperature is room temperature, and the stirring reaction time is 4-6 h.

7. The preparation method according to claim 2, characterized in that, In the synthesis of compound 3a in step (3), the molar ratio of ZnPc-COOH, 5-aminovaleric acid tert-butyl ester, HATU, DIPEA and DMAP is 1:2-3.5:1.8-3:6-8:3-5, the reaction temperature is room temperature, and the stirring reaction time is 4-6 h.

8. The preparation method according to claim 2, characterized in that, In the synthesis of compound 3b in step (3), the reaction temperature is room temperature, and the stirring reaction time is 7-9 h; in the synthesis of compound 3, the molar ratio of compound 3b, VH032-NH2, HATU, DIPEA and DMAP is 1:0.8-1.5:1.8-3:11-15:3-5, the reaction temperature is room temperature, and the stirring reaction time is 4-6 h.

9. The use of a VH032 modified phthalocyanine according to claim 1 in the preparation of an anticancer drug.

10. Use according to claim 9, characterized in that, The use of the VH032 modified phthalocyanine as a photosensitizer in the preparation of an anticancer drug.

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