Di-(4-(4-(2-(1, 1-oxyimidoyl) ethyl) piperazine-1-formyl) phenyl) boric acid axial substituted silicon phthalocyanine and preparation method and application thereof
By synthesizing axially substituted phthalocyanine bis-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid (PPBA-SiPc), the problem of poor therapeutic effect of photodynamic therapy in hypoxic environment was solved, and highly efficient photodynamic therapy was achieved in MCF-7 breast cancer cells.
Patent Information
- Application Number
- CN202511651324.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-12
- Publication Date
- 2026-01-23
AI Technical Summary
Existing photodynamic therapy is not effective in tumor tissues under hypoxic conditions. Traditional photosensitizers are difficult to produce cytotoxic substances effectively under hypoxic conditions, resulting in suboptimal treatment effects.
We designed and synthesized axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid. This compound can be rapidly absorbed by MCF-7 cells in a hypoxic environment and achieves highly efficient photodynamic therapy through endoplasmic reticulum targeting.
PPBA-SiPc exhibits good photodynamic therapy under hypoxic conditions, effectively killing MCF-7 breast cancer cells. Moreover, the preparation method is simple and suitable for industrial production.
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Figure CN121378320A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present invention belongs to the field of complexes, especially bis-(4-(4-(2-(1,1-oxyl) diimidoyl)ethyl)piperazine-1-carbonyl)phenyl)boronic acid axially substituted silicon phthalocyanine and its method of preparation, the complexes are applied as photosensitizers in the treatment of MCF-7 breast cancer cells. BACKGROUND
[0002] Photodynamic therapy (PDT) is an alternative cancer treatment that employs a photosensitizer (PS) which is activated by a specific wavelength of light, resulting in the production of cytotoxic singlet oxygen (1O2) as the main reactive oxygen species (ROS). The photo-generated ROS effectively oxidize vital cellular components and macromolecules, leading to tumor cell death.
[0003] Metallophthalocyanines (MPc) are known photosensitizers for PDT. MPc containing diamagnetic central metals (such as zinc, silicon or aluminum) are efficient generators of cytotoxic singlet oxygen. Central metals such as silicon are particularly important as they allow for axial ligation, which reduces the tendency of Pcs to aggregate. Silicon phthalocyanines (SiPc) are known photosensitizers for PDT, many of which are in various stages of clinical trials. Many of them are in different stages of clinical trials.
[0004] Phenylboronic acid (PBA) was discovered to have antibacterial activity more than a century ago and is nontoxic to the environment, with antibacterial and antitumor activity. PBA is considered one of the most promising cancer cell binding modules due to its ability to form reversible and dynamic boronate covalent bonds, and exploring its chemical versatility is crucial for the development of new anticancer therapies. PBA and sialic acid can form reversible borate salts, which facilitate the uptake of PBA-functionalized biomaterials by tumor tissues.
[0005] Despite the promising outlook of PDT, the pre-existing hypoxia in tumor tissues remains a major challenge and has profound biological and therapeutic implications. Hypoxia is particularly important in PDT as it is heavily dependent on oxygen availability. The continuous oxygen consumption during PDT further exacerbates hypoxia, and suboptimal PDT outcomes are typically achieved under hypoxic conditions. Various strategies have been developed to address hypoxia, including oxygen supplementation during PDT (e.g., using catalase mimetic metal nanoparticles) or oxygen-independent therapies to facilitate better treatment outcomes. While many catalase mimetics show promise in releasing oxygen, the release / leakage of excess metal ions can lead to acute toxicity. Such systems are also typically complex and laborious, requiring the complex integration of oxygen-producing catalysts and inorganic photosensitizer components. Although limited, a few reports have demonstrated some success using type I photochemical reactions, which result in the production of highly toxic free radicals (superoxide (O2-·) and hydroxyl (·OH) radicals with some efficacy under hypoxic conditions.
[0006] Alternatively, due to the inherent oxygen gradient between normal and tumor tissues, hypoxic microenvironments can be used for tumor-targeted therapy. To this end, research attention has been given to the design of hypoxia-activated prodrugs (HAPs). HAPs are selectively activated by reductive metabolism in hypoxic environments, generating cytotoxic substances inside cells, and thus they are potential hypoxia-targeting agents. Despite their promise, HAPs alone have shown only suboptimal anticancer effects. Therefore, the development of new PSs to respond to the oxygen barrier of PDT remains urgent. SUMMARY
[0007] One of the purposes of the present application is to provide a silicon phthalocyanine complex axially substituted with di-(4-(4-(2-(1,1-oxodiamide)ethyl)piperazine-1-formyl)phenyl)boronic acid.
[0008] Another purpose of the present application is to provide a method for preparing a silicon phthalocyanine complex axially substituted with di-(4-(4-(2-(1,1-oxodiamide)ethyl)piperazine-1-formyl)phenyl)boronic acid.
[0009] Another purpose of the present application is to provide a method for preparing a silicon phthalocyanine complex axially substituted with di-(4-(4-(2-(1,1-oxodiamide)ethyl)piperazine-1-formyl)phenyl)boronic acid.
[0010] The purpose of the present application is achieved as follows: The silicon phthalocyanine complex axially substituted with di-(4-(4-(2-(1,1-oxodiamide)ethyl)piperazine-1-formyl)phenyl)boronic acid according to the present application is a piperazine group connected to the axial position of silicon phthalocyanine to form a piperazine silicon phthalocyanine (PIP-SiPc), and then di-(4-(4-(2-(1,1-oxodiamide)ethyl)piperazine-1-formyl)phenyl)boronic acid is further synthesized to form a silicon phthalocyanine complex axially substituted with PPBA-SiPc. The PPBA-SiPc can be rapidly absorbed by MCF-7 cells and exhibit good photodynamic efficacy.
[0011] The silicon phthalocyanine complex axially substituted with di-(4-(4-(2-(1,1-oxodiamide)ethyl)piperazine-1-formyl)phenyl)boronic acid according to the present application is a piperazine group connected to the axial position of silicon phthalocyanine to form a piperazine silicon phthalocyanine (PIP-SiPc), and then di-(4-(4-(2-(1,1-oxodiamide)ethyl)piperazine-1-formyl)phenyl)boronic acid is further synthesized to form a silicon phthalocyanine complex axially substituted with PPBA-SiPc. The PPBA-SiPc can be rapidly absorbed by MCF-7 cells and exhibit good photodynamic efficacy. .
[0012] The method for preparing a silicon phthalocyanine complex axially substituted with di-(4-(4-(2-(1,1-oxodiamide)ethyl)piperazine-1-formyl)phenyl)boronic acid according to the present application comprises the following steps: 1) 4-carboxyphenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 4-dimethylaminopyridine (DMAP) are mixed and completely dissolved in a round-bottom flask containing an organic solvent; 2) Place the flask in a water bath at room temperature and stir. Then, add piperazine-based axially substituted silicon phthalocyanine (PIP-SiPc) and an amine solvent, and sonicate until the PIP-SiPc is completely dissolved. 3) React in a room temperature water bath. After the reaction is complete, remove the organic solvent using a vacuum rotary evaporator and dry the residue in an oven. 4) The final product was obtained by purification by column chromatography.
[0013] The organic solvent in step 1) is one or more of tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide; The organic solvent in step 1) is N,N-dimethylformamide; The stirring time in step 2) is 10-30 minutes; preferably 15 minutes. In step 2), the amine solvent is triethylamine; In step 3), the water bath reaction time is 50-80 hours; the oven temperature is 50-70°C. In step 4), the column chromatography eluent is one or more of ethyl acetate, tetrahydrofuran, DMSO, and DMF; preferably ethyl acetate and tetrahydrofuran (2-4:1).
[0014] A pharmaceutical preparation containing an axially substituted silylphthalocyanine of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid, wherein the pharmaceutical preparation is an injection or an oral preparation; further, the pharmaceutical preparation is an injection.
[0015] Another objective of this invention is to propose the application of axially substituted silyl phthalocyanine of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid in the treatment of MCF-7 breast cancer cells.
[0016] The beneficial effects of the present invention are as follows: Compared with phthalocyanines with carboxyl, fluorinated functional groups or triphenylamine as terminal groups in previous patent literature, the axially substituted phthalocyanine of borate synthesized in the present invention, bis-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid axially substituted phthalocyanine (PPBA-SiPc) with endoplasmic reticulum targeting function, has good photophysical and photochemical properties.
[0017] The boric acid axially substituted silica phthalocyanine preparation method of the present invention is simple to operate and requires no precision instruments, significantly lowering the technical threshold. By optimizing the raw material ratio and reaction temperature, the formation of by-products is reduced, and the yield is significantly improved compared with traditional processes. Its reaction conditions are mild and controllable, with strong repeatability, and can stably produce high-quality products, fully meeting the needs of large-scale industrial production.
[0018] Meanwhile, co-staining CLSM and phototoxicity evaluation showed that the axially substituted phthalocyanine borate of the present invention has good effects as an endoplasmic reticulum targeting marker and a fluorescence imaging-guided photodynamic photosensitizer in the treatment of MCF-7 breast cancer cells. Attached Figure Description
[0019] Figure 1 CLSM diagram of co-staining PPBA-SiPc (2 μM) with ER-Tracker, BODIPY, Lyso-Tracker and Mito-Tracker.
[0020] Figure 2 Cell viability graphs under different concentrations of PPBA-SiPc (0, 6.25, 12.5, 25, 50, 100 nM). Detailed Implementation
[0021] The present invention will be described in detail below with reference to embodiments: Example 1 (1) Synthesis of bis-(1-ethoxypiperazine)silylphthalocyanine (PIP-SiPc) In a 100 mL round-bottom flask, first add 20 mL of anhydrous toluene, then add 0.536 g of dichlorosilylphthalocyanine, and stir until the solid is completely dissolved; then add 0.357 g of 1-(2-hydroxyethyl)piperazine and 0.16 mL of pyridine dropwise, and stir for 10 minutes to homogenize the system.
[0022] Attach a spherical condenser to the round-bottom flask, heat it in an oil bath to 120°C (the oil bath temperature needs to be stable, with an error of ±2°C), and reflux for 8-10 hours with magnetic stirring (300-400 rpm). Observe the color change of the solution during this period (from light blue to dark blue).
[0023] After the reaction was complete, the oil bath was removed and the solution was cooled to room temperature. The solution was then transferred to a 50 mL rotary evaporator and evaporated at 45 °C and -0.09 MPa vacuum until no solvent was distilled off, yielding a dark blue viscous crude product.
[0024] Add 8 mL of a CHCl3 / n-hexane mixed solvent (1:4 volume ratio) to the crude product (i.e., 1.6 mL CHCl3 + 6.4 mL n-hexane), sonicate to dissolve, and then let stand in a refrigerator at 4°C for 12 hours. Collect the blue crystals by filtration, and dry them at 60°C and -0.095 MPa vacuum for 4 hours to obtain the target product with a yield of 62%.
[0025] (2) Synthesis of axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid 1) Mix 4-carboxyphenylboronic acid (0.050 g, 0.300 mmol) with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (0.0300 g, 0.100 mmol) and 4-dimethylaminopyridine (DMAP) (0.0100 g, 0.100 mmol) and dissolve them completely in a round-bottom flask containing 15 mL of N,N-dimethylformamide.
[0026] 2) Place the flask in a water bath at room temperature and stir for 15 min. Then, add 0.0500 g of piperazine-based axially substituted phthalocyanine (PIP-SiPc) and 5 μL of triethylamine, and sonicate until PIP-SiPc is completely dissolved.
[0027] 3) React in a water bath at room temperature for 72 h. After the reaction is complete, remove the organic solvent using a vacuum rotary evaporator and dry the residue in a 60°C oven.
[0028] 4) Purification was performed by column chromatography using ethyl acetate and tetrahydrofuran (3:1) as eluents, with a final yield of 61%.
[0029] FT-IR ν max / cm −1 : 3390 (OH), 2974 (Aromatic, CH), 2925 (Alphatic, CH), 1649 (C=O), 1080 (Si-O). 1 H NMR (400 MHz, DMSO δ / ppm) 9.65 (d, J = 4.8,3.0 Hz, 8H), 8.50-8.47 (d, 8H), 8.23 (s, 4H), 7.80 (m, J = 7.6 Hz, 4H), 6.96(m, J = 7.4 Hz, 4H), 2.13 (t, 8H), 0.33 (t, 8H), -0.69 (t, 4H), -2.02 (t,4H), MALDI-TOF-MS, m / z Calc: 1094.4, Found: 1094.6 [M] + .
[0030] Example 2 (1) Synthesis of bis-(1-ethoxypiperazine)silylphthalocyanine (PIP-SiPc) The synthesis method and steps are the same as in Example 1. (2) Synthesis of axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid The synthesis steps were the same as in Example 1. Specifically, in step 1), the organic solvent was tetrahydrofuran; in step 2), the stirring time was 10 min; in step 3), the water bath reaction time was 50 h; the oven temperature was 50 °C; and in step 4), the column chromatography eluent was ethyl acetate and tetrahydrofuran (2:1), with a final yield of 59%.
[0031] Example 3 (1) Synthesis of bis-(1-ethoxypiperazine)silylphthalocyanine (PIP-SiPc) The synthesis method and steps are the same as in Example 1. (2) Synthesis of axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid The synthesis steps were the same as in Example 1. Specifically, in step 1), the organic solvent was dimethyl sulfoxide; in step 2), the stirring time was 30 min; in step 3), the water bath reaction time was 80 h; the oven temperature was 70 °C; and in step 4), the column chromatography eluent was ethyl acetate and tetrahydrofuran (4:1), with a final yield of 60%.
[0032] Comparative Example 1 (1) Synthesis of bis-(1-ethoxypiperazine)silylphthalocyanine (PIP-SiPc) The synthesis method and steps are the same as in Example 1. (2) Synthesis of axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid Unlike Example 1, the organic solvent in step 1) is ethanol, and the final yield is 32%.
[0033] Comparative Example 2 (1) Synthesis of bis-(1-ethoxypiperazine)silylphthalocyanine (PIP-SiPc) The synthesis method and steps are the same as in Example 1. (2) Synthesis of axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid Unlike Example 1, the stirring time in step 2) was 50 min; the final yield was 52%.
[0034] Comparative Example 3 (1) Synthesis of bis-(1-ethoxypiperazine)silylphthalocyanine (PIP-SiPc) The synthesis method and steps are the same as in Example 1. (2) Synthesis of axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid Unlike Example 1, the water bath reaction time in step 3) is 30 hours, and the final yield is 39%.
[0035] Comparative Example 4 (1) Synthesis of bis-(1-ethoxypiperazine)silylphthalocyanine (PIP-SiPc) The synthesis method and steps are the same as in Example 1. (2) Synthesis of axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid Unlike Example 1, in step 4), the column chromatography eluent was ethyl acetate and tetrahydrofuran (1:1), and the final yield was 54%.
[0036] Comparative Example 5 (1) Synthesis of bis-(1-ethoxypiperazine)silylphthalocyanine (PIP-SiPc) The synthesis method and steps are the same as in Example 1. (2) Synthesis of axially substituted silyl phthalocyanine (PPBA-SiPc) of di-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid 0.050 g (0.300 mmol) of 4-carboxyphenylboronic acid was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (0.0300 g, 0.100 mmol), 4-dimethylaminopyridine (DMAP) (0.0100 g, 0.100 mmol), and 0.0500 g of piperazine-based axially substituted silica phthalocyanine (PIP-SiPc) and completely dissolved in a round-bottom flask containing 15 mL of N,N-dimethylformamide. The reaction was carried out at room temperature for 50 h. After the reaction was completed, the mixture was rotary evaporated, dried, and purified by column chromatography, with a final yield of 41%.
[0037] Verification of Examples 1. Uptake of PPBA-SiPc in MCF-7 cells Different concentrations (2 μM, 4 μM, 6 μM, 8 μM) of PPBA-SiPc were added and incubated with cells for 12 hours. Fluorescence changes were observed under a confocal microscope in confocal dishes (PPBA-SiPc, QPBA-SiPc, and SPBA-SiPc:λ). ex =860nm, λ em =650-750 nm).
[0038] 2. Organelle localization of PPBA-SiPc in MCF-7 cells PPBA-SiPc (2 μM), QPBA-SiPc (4 μM), and SPBA-SiPc (4 μM) were added to cells and incubated together. Cells were washed with PBS, and the four organelle fluorescent probes were added to confocal dishes for staining for 30 min. Imaging was then performed under a confocal microscope. Figure 1 ) (PPBA-SiPc, QPBA-SiPc and SPBA-SiPc: λ ex =860 nm, λ em =650-750 nm; BODIPY: λ ex =488 nm, λ em =490-550 nm; Mito-Tracker: λ ex =488 nm, λ em =490-550 nm; Lyso-Tracker: λ ex =552 nm, λ em =550-600 nm; ER-Tracker: λ ex =488 nm, λ em =490-550 nm).
[0039] from Figure 1 It can be seen that PPBA-SiPc has the best organelle localization effect in MCF-7 cells.
[0040] 3. In vitro photodynamic therapy of PPBA-SiPc on MCF-7 breast cancer cells Evaluation of phototoxicity and cytotoxicity of PPBA-SiPc Different concentrations of PPBA-SiPc solutions (0, 6.25, 12.5, 25, 50, 100 nM) were incubated overnight with cells in a constant temperature incubator. The culture plates were then irradiated with a 671 nm laser at an intensity of 110 mW / cm² for 5 min, followed by a second incubation for 4 h. The phototoxicity and dark toxicity of the cells were then evaluated. Figure 2 ) from Figure 2It can be seen that PPBA-SiPc has a significant inhibitory effect on MCF-7 breast cancer cells.
Claims
1. A di-(4-(4-(2-(1,1-oxodiimideo)ethyl)piperazine-1-formyl)phenyl)boronic acid axially substituted silylphthalocyanine, with the following structural formula: 。 2. A method for preparing axially substituted silyl phthalocyanine of di-(4-(4-(2-(1,1-oxodiimideo)ethyl)piperazine-1-formyl)phenyl)boronic acid, comprising the following steps: 1) Mix 4-carboxyphenylboronic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 4-dimethylaminopyridine (DMAP) and dissolve them completely in a round-bottom flask containing organic solvent; 2) Place the flask in a water bath at room temperature and stir. Then, add piperazine-based axially substituted phthalocyanine (PIP-SiPc) and an amine solvent, and sonicate until PIP-SiPc is completely dissolved. 3) React in a room temperature water bath. After the reaction is complete, remove the organic solvent using a vacuum rotary evaporator and dry the residue in an oven. 4) The final product was obtained by purification by column chromatography.
3. The preparation method according to claim 2, characterized in that, The organic solvent in step 1) is one or more of tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide.
4. The preparation method according to claim 3, characterized in that, The organic solvent in step 1) is N,N-dimethylformamide.
5. The preparation method according to claim 2, characterized in that, The stirring time in step 2) is 10-30 min; preferably 15 min.
6. The preparation method according to claim 2, characterized in that, In step 2), the amine solvent is triethylamine.
7. The preparation method according to claim 2, characterized in that, In step 3), the water bath reaction time is 50-80 hours; the temperature in the oven is 50-70°C.
8. The preparation method according to claim 2, characterized in that, In step 4), the column chromatography eluent is one or more of ethyl acetate, tetrahydrofuran, DMSO, and DMF; preferably, the ratio of ethyl acetate to tetrahydrofuran is 2-4:1; further, in step 4), the column chromatography eluent is ethyl acetate to tetrahydrofuran in a ratio of 3:
1.
9. A pharmaceutical preparation containing the silicophthalocyanine according to any one of claims 1-8, characterized in that, The pharmaceutical preparation is an injectable or oral preparation; further, the pharmaceutical preparation is an injectable preparation.
10. The use of bis-(4-(4-(2-(1,1-oxodiimide)ethyl)piperazine-1-formyl)phenyl)boronic acid axially substituted phthalocyanine as an endoplasmic reticulum targeting label and fluorescence imaging-guided photodynamic photosensitizer in the preparation of a drug for treating MCF-7 breast cancer cells, as described in claim 1.