Axial substitution of silicon phthalocyanine with a retinamide piperazine and methods of making and using the same

Abstract

This invention discloses a retinamide piperazine axially substituted silica phthalocyanine, its preparation method, and its application, specifically a lysosome-lipid droplet-targeted retinamide piperazine axially substituted silica phthalocyanine, its preparation method, and its application. It involves preparing di-(4-(retinamide piperazine)-1-ethoxy)axially substituted silica (IV) phthalocyanine from 2-((4-retinamide)piperazinyl)ethanol and dichlorosilane(IV) phthalocyanine. This improves the solubility of silica phthalocyanine, enhances its tumor cell targeting ability, and discloses its application as a novel photosensitizer in lysosome-lipid droplet-targeting labeling and fluorescence imaging-guided photodynamic therapy.

Description

Technical Field

[0001] This invention belongs to the field of coordination compounds, and more particularly to axially substituted phthalocyanine retinoic acid piperazine and its preparation method and application. This coordination compound, as an anticancer photosensitizer, can be applied to lysosomal-liposome targeted labeling and fluorescence imaging-guided photodynamic therapy. Background Technology

[0002] Photodynamic therapy (PDT) is a non-invasive and promising treatment for malignant tumors and wet age-related macular degeneration. Photosensitizers are a crucial component of PDT. Phthalocyanines, due to their strong absorption of near-infrared light and high singlet oxygen yield, have become a promising second-generation PDT photosensitizer. However, the low solubility and self-aggregation behavior of phthalocyanines in solvents and physiological environments, as well as their weak targeting ability to tumor cells, affect their singlet oxygen quantum yield and bioavailability, thus impacting their PDT efficacy. Therefore, designing and synthesizing high-performance photosensitizers with organelle-targeting capabilities is our research objective.

[0003] Retinoic acid is a metabolite of vitamin A after oxidation in the human body. It has a skeleton structure composed of a non-aromatic carbon ring structure and a polypentadiene side chain, and plays an important role in living organisms: (1) participating in the regulation of epidermal cell proliferation, differentiation, and apoptosis; (2) playing an immunomodulatory role such as assisting antibody production and inhibiting lymphocyte proliferation; (3) having anti-inflammatory, anti-proliferative, and anti-tumor effects. In addition, topical application of retinoic acid can penetrate the skin quickly and enter cells through the cell membrane and cytoplasm, significantly accelerating the renewal of epithelial cells. It has been proven to be a chemotherapy drug for the treatment of acute promyelocytic leukemia (APL) and has also shown good therapeutic effects on neurocytoma, improving the survival rate of neurocytoma patients. Clinical data show that 9-cis-retinoic acid can prevent prostate cancer and breast cancer. Although retinoic acid has a good therapeutic effect on tumor cells, its high toxicity, poor water solubility, and poor drug targeting have limited its clinical application. By modifying the carboxyl group of retinoic acid, its anti-tumor ability and water solubility can be further improved. Amides or esters formed by retinoic acid with amines or alcohols, or complexes formed with other drug molecules, may have enhanced antitumor effects. Furthermore, the piperazine group can mediate the rapid and efficient uptake of piperazine-substituted phthalocyanines by cancer cells.

[0004] To address the issues of low solubility, easy aggregation, and weak cell-targeting ability of phthalocyanine molecules, this paper considers introducing a sterically hindered retinoic acid (TA)-piperazine (TP-PIP) group with organelle-targeting ability into the axial position of phthalocyanine. This approach aims to impart greater steric hindrance to phthalocyanine, preventing aggregate formation and improving its solubility. Furthermore, the introduction of the TP-PIP group facilitates uptake by cancer cells and enhances their organelle-targeting ability, while also providing both biofluorescence imaging and photodynamic therapy functions. Finally, there are currently no reports, either domestically or internationally, on the replacement of silicon phthalocyanine with retinoic acid-piperazine. Summary of the Invention

[0005] The purpose of this invention is to provide a vitamin A amide piperazine axially substituted silicon phthalocyanine and its preparation method.

[0006] Another object of this invention is to propose the application of a retinamide piperazine axially substituted silica phthalocyanine as a photosensitizer for lysosomal-liposome targeting labeling and fluorescence imaging-guided photodynamic therapy. This object is achieved by providing a retinamide piperazine axially substituted silica phthalocyanine complex with the following structure:

[0007]

[0008] The axially substituted phthalocyanine complex of retinamide piperazine described in this invention involves introducing a sterically hindered retinoamide piperazine (TP-PIP) group with organelle-targeting capability into the axial position of a phthalocyanine, synthesizing a novel organelle-targeting phthalocyanine complex. On one hand, the steric hindrance of retinoamide piperazine inhibits the self-aggregation behavior of phthalocyanines and improves their solubility to a certain extent; on the other hand, retinoamide piperazine can be rapidly taken up by cancer cells and possesses organelle-targeting capability, which can improve the tumor cell targeting ability and bioavailability of phthalocyanines, making the axially substituted retinoamide piperazine phthalocyanine complex a promising fluorescent imaging agent and photosensitizer for photodynamic therapy.

[0009] The method for preparing a retinamide piperazine axially substituted silicophthalocyanine complex according to the present invention includes the following steps:

[0010] (1) 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-methylmorpholine, and retinoic acid were stirred in a dichloromethane solution at 0°C for 0.5 h, and then stirred with 1-(2-hydroxyethyl)piperazine at room temperature to prepare the precursor 2-((4-amido)piperazinyl)ethanol (2-((4-amido)piperazinyl)ethanol is referred to as TP-PIP in this invention); (2) The precursor 2-((4-amido)piperazinyl)ethanol and dichlorosilyl(IV) phthalocyanine were refluxed in a toluene solution in the presence of pyridine to prepare di-(4-(retinoic acid piperazinyl)-1-ethoxy) axially substituted silicon(IV) phthalocyanine (2-(4-(retinoic acid piperazinyl)-1-ethoxy) axially substituted silicon(IV) phthalocyanine is referred to as TP-PIP-SiPc in this invention).

[0011] In this invention, the 2-((4-amido)piperazinyl)ethanol (hereinafter referred to as TP-PIP) is preferably prepared by stirring 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-methylmorpholine, and retinoic acid in a dichloromethane solution at 0°C for 0.5 h, followed by the addition of 1-(2-hydroxyethyl)piperazine, and the reaction is stirred at room temperature for at least 1 h. The reaction is monitored by TLC, and the crude product is purified by silica gel column chromatography using diethyl ether and ethyl acetate as eluents to obtain a brown viscous oil.

[0012] The present invention discloses a retinamide piperazine-substituted silica phthalocyanine complex (the present invention refers to retinamide piperazine axially substituted silica phthalocyanine as TP-PIP-SiPc), preferably 2-((4-amido)piperazinyl)ethanol (TP-PIP) reacted with dichlorosilane(IV) phthalocyanine and pyridine in toluene under reflux at 120°C. The crude product was separated by silica gel column chromatography using dichloromethane and acetone as eluents, and vacuum dried to obtain a blue solid.

[0013] The present invention relates to the application of a retinoic acid piperazine axially substituted phthalocyanine as a phthalocyanine photosensitizer for lysosomal-lipid targeting labeling and fluorescence imaging-guided photodynamic therapy.

[0014] The beneficial effects of this invention are as follows: This invention synthesizes a novel bis-(4-(vitamin A-piperazinyl)-1-ethoxy)axially substituted silicon (IV) phthalocyanine with lysosomal-liposome targeting labeling and fluorescence imaging-guided photodynamic therapy. This novel vitamin A-piperazinyl axially substituted silicon phthalocyanine not only utilizes the steric hindrance of the vitamin A-piperazinyl structure to a certain extent to inhibit the self-aggregation behavior of phthalocyanine, but also improves the solubility of silicon phthalocyanine and enhances its tumor cell targeting ability by introducing vitamin A-piperazinyl into the axial position of silicon phthalocyanine. Simultaneously, it achieves fluorescence imaging and photodynamic therapy, making vitamin A-piperazinyl axially substituted silicon phthalocyanine complexes a class of dual organelle-targeting photosensitizers with application potential. Attached Figure Description

[0015] Figure 1 Fluorescence imaging and lysosomal-liposome localization map of 2-(4-(Vitamin A piperazine)-1-ethoxy) axially substituted silicon (IV) phthalocyanine (TP-PIP-SiPc) in breast cancer cells.

[0016] Figure 2 shows the in vitro photodynamic activity of bis-(4-(Vitamin A piperazine)-1-ethoxy) axially substituted silicon(IV) phthalocyanine (TP-PIP-SiPc) against breast cancer cells. Detailed Implementation

[0017] The present invention will be described in detail below with reference to embodiments: Example 1:

[0018] 1) Synthesis of 2-((4-Vitaminoyl)piperazinyl)ethanol (hereinafter referred to as TP-PIP)

[0019] 2-Chloro-4,6-dimethoxy-1,3,5-triazine (0.176 g, 1 mmol) was dissolved in dichloromethane (20 mL), and N-methylmorpholine (0.33 mL, 15 mmol) was added at 0°C. After 30-40 min, a white suspension was formed, and retinoic acid (0.206 g, 0.5 mmol) was added. After magnetic stirring for 1 h, a clear solution was formed. After returning to room temperature, 1-(2-hydroxyethyl)piperazine (0.123 mL, 1 mmol) was added. The process was detected by TLC. After the reaction was complete, the product was extracted twice with saturated sodium bicarbonate solution (10 mL) and water (10 mL), respectively. The organic phase was recovered, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by alkaline alumina column chromatography using diethyl ether:ethyl acetate = 5:1 (v / v) as the eluent to give 0.353 g of a brown, viscous oily substance, 2-((4-amido)piperazinyl)ethanol (TP-PIP), with a yield of 85.7%. Synthetic characterization: IR (KBr / cm) -1 ): 3391, 2928, 1618, 1445,1280, 972; 1 H NMR (400 MHz, CDCl3, δ / ppm): 1 H NMR (400 MHz, CDCl3, δ / ppm):6.96~5.97 (m, 6H, H 6 ), 3.74~3.71 (t, 6H, H 8 ), 2.65~2.62 (t, 6H, H 9 ), 2.07~1.95(m, 9H, H 4 H 7), 1.73~1.72 (t, 2H, H 3 ), 1.68~1.65 (m, 2H, H 2 ), 1.52~1.48 (t,2H, H 1 ), 1.06 (s, 6H, H 5 ); MALDI-TOF-MS: m / z calc. for [M] + 412.31, found 412.71.

[0020] 2) Synthesis of bis-(4-(vitamin-piperazinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine (hereinafter referred to as TP-PIP-SiPc)

[0021] The TP-PIP (0.206 g, 0.5 mmol) and dichlorosilane(IV) phthalocyanine (SiPcCl2) (0.12 g, 0.2 mmol) obtained in step 1) were dissolved in toluene (30 mL), and pyridine (0.6 mL) was added. The mixture was refluxed at 120 °C with magnetic stirring for 48 h. The solvent was removed by rotary evaporation under reduced pressure. The resulting solid was dissolved in dichloromethane, filtered, and the filtrate was evaporated under reduced pressure to give a blue solid. The solid was purified by alkaline alumina column chromatography using dichloromethane:acetone = 20 / 1 (v / v) as the eluent to give 0.167 g of the blue solid di-(4-(vitaminopyramidinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine, with a yield of 61.25%. Synthetic characterization: IR (KBr / cm) -1 ): 3424, 2924, 2855, 1626, 1429, 1335, 1290, 1123, 1080, 967~914, 759,742; 1 H NMR (400 MHz, CDCl3, δ / ppm): 9.67~9.65 (q, 8H, H 1 ), 8.39~8.37 (q, 8H,H 2 ), 6.82~6.17 (m, 12H, H 7 ), 2.71~2.40 (s, 8H, H 6 ), 2.10~1.93 (m, 12H, H 13 H 14 ), 1.86~1.80 (t, 6H, H 12 ), 1.77~1.71 (t, 4H, H 11 ), 1.67~1.62 (m, 4H, H 10), 1.52~1.49 (m, 4H, H 9 ), 1.09~0.96 (m, 12H, H 8 ), -0.56 (t, 4H, H 3 ), -1.87 (t,4H, H 4 ); MALDI-TOF-MS: m / z calc. for [M] + 1363.84, found 1363.169. Example 2:

[0022] The specific steps are the same as in Example 1: In process 2), pyridine (0.6 mL) was replaced with K2CO3 (0.06 g, 0.4 mmol). Other reaction conditions were the same, yielding 0.096 g of blue powder bis-(4-(vitamin-piperazinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine, with a yield of 35.13%.

[0023] The specific steps are the same as in Example 1: In process 2), TP-PIP is replaced with (0.165 g, 0.4 mmol), and the reaction temperature is changed to 140 °C. o C, the reaction time was changed to 96 h. 0.119 g of blue powder, bis-(4-(vitaminoperipazinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine, was obtained, with a yield of 43.8%. Example 3:

[0024] The specific steps are the same as in Example 1: In step 2), pyridine (2.5 mL) was replaced with NaH (0.048 g, 2 mmol), and other reaction conditions were the same. 0.138 g of 0.138 g of blue powder, bis-(4-(vitamin-piperazinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine, was obtained, with a yield of 50.48%.

[0025] In process 2), SiPcCl2 was replaced with (0.24 g, 0.4 mmol) and TP-PIP was replaced with (0.412 g, 1.0 mmol), while other reaction conditions remained the same. 0.045 g of axially substituted silicon(IV) phthalocyanine (4-(Vitamin A piperazine)-1-ethoxy) powder was obtained, with a yield of 16.5%. Example 4:

[0026] Fluorescence imaging and lysosomal / lipid localization of lysosome-lipid-droplet targeted bis-(4-(vitamin-piperazinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine (TP-PIP-SiPc) in breast cancer cells.

[0027] MCF-7 cells were incubated in 20 mm confocal incubation dishes at 37°C and 5% CO2 for 12 h. After removing the old culture medium, TP-PIP-SiPc was diluted to 4 mM with fresh culture medium and co-incubated with the cells for 10 h. The cells were then washed three times with PBS buffer. Afterward, the cells were stained with LysoTracker® Red DND-99 (50 nM) or BODIPY (8 µM) culture medium solutions at 37°C in the dark for 30 min, and then observed under a confocal microscope. TP-PIP-SiPc: two-photon excitation, excitation wavelength 860 nm, collection wavelength 650–750 nm. LysoTracker® Red DND-99: excitation wavelength 552 nm, collection wavelength 600–650 nm. BODIPY: single-photon excitation, excitation wavelength 488 nm, collection wavelength 500–540 nm.

[0028] Evaluation of the in vitro photodynamic activity of lysosome-liposome-targeted bis-(4-(vitamin-piperazinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine (TP-PIP-SiPc) against breast cancer cells

[0029] To investigate the in vitro photodynamic effects of bis-(4-(vitaminopyramidinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine (TP-PIP-SiPc) prepared in Examples 1-3, cytotoxicity experiments were conducted using the Cell Counting Kit-8 (CCK-8) cell viability assay kit. Cells with a density of 8 × 10⁸ cells were... 3 MCF-7 cells per well were incubated in 96-well plates for 24 h, followed by incubation in DMEM medium of TP-PIP-SiPc at different concentrations (0 µM, 0.2 µM, 0.4 µM, 0.6 µM, 0.8 µM, 1.0 µM) for another 24 h. Then, the cells were treated with a laser (671 nm, 5 mW / cm²). 2 Irradiate cells for 5 min and incubate for 0.5 h. Finally, add 10 mL of CCK-8 reagent to each well and continue incubation for 2 h. Detect the OD value at 450 nm using a multi-mode microplate reader. Cell viability = [OD(drug-treated) - OD(blank)] / [OD(0-drug-treated) - OD(blank)] × 100%.

[0030] After 5 min of laser irradiation, cell viability decreased to 7.54%. The IC50 of bis-(4-(vitaminopyramidinyl)-1-ethoxy)axially substituted silicon(IV) phthalocyanine was 0.4 µM.

Claims

1. A retinamide piperazine axially substituted silyl phthalocyanine, characterized in that: Compounds of the following chemical structure: 。 2. Use of the retinamide piperazine axially substituted silicon phthalocyanine according to claim 1 in the preparation of a drug for the lysosome-lipid droplet targeted labeling and fluorescence imaging guided photodynamic therapy phthalocyanine photosensitizer.

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