CN112812121B

CN112812121B - Pyridone modified zinc phthalocyanine and preparation method and application thereof

Info

Publication number: CN112812121B
Application number: CN202110032421.2A
Authority: CN
Inventors: 魏少华; 周林; 李燕青; 王冲冲
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2021-01-11
Filing date: 2021-01-11
Publication date: 2022-05-03
Anticipated expiration: 2041-01-11
Also published as: CN112812121A

Abstract

The invention discloses pyridone modified zinc phthalocyanine and a preparation method and application thereof, wherein the structural formula of the pyridone modified zinc phthalocyanine is shown as follows, the novel zinc phthalocyanine is simple and convenient to prepare and can be used as a photosensitive medicament for photodynamic therapy, the novel zinc phthalocyanine has multiple anti-tumor mechanisms, after being absorbed by tumor cells, the novel zinc phthalocyanine can reduce the expression level of hypoxia inducible factor-1, can be excited by red and near infrared light to generate active oxygen, can store redundant singlet oxygen and slowly release at physiological temperature, and finally obtains good photodynamic anti-tumor activity.

Description

Pyridone modified zinc phthalocyanine and preparation method and application thereof

Technical Field

The invention belongs to an anti-tumor photosensitizer, and particularly relates to pyridone modified zinc phthalocyanine and a preparation method and application thereof.

Background

Malignant tumors are one of the major diseases threatening human health. Photodynamic therapy (PDT) is an approved clinical method for tumor therapy. The principle of photodynamic therapy is that the photosensitizer enriched in tumor tissue is irradiated by light, and after being excited, the photosensitizer reacts with surrounding oxygen to generate active oxygen, so that tumor cells are killed. Insufficient penetration depth of a light source for exciting the photosensitizer, low utilization rate of singlet oxygen generated by the photosensitizer, and resistance generated by up-regulation of hypoxia inducible factor-1 (HIF-1) to treatment under hypoxic condition are three major bottleneck problems in PDT treatment process.

The excitation wavelength of conventional photosensitizers is mostly in the visible range. The tissue penetration depth of visible light is insufficient, so PDT is mainly used for superficial tumor treatment at present in clinic. Near Infrared (NIR) light has a strong tissue penetration capability and a high transmission efficiency in biological tissues, and is very suitable for PDT treatment of deep tumors. Therefore, the photosensitizer which can be excited by visible light and near infrared light can realize the synchronous treatment of superficial and deep tumors.

Singlet oxygen (singlet oxygen,¹O₂) Is the primary active oxygen species in PDT and has strong capacity of oxidizing and damaging tumor cells. However, itVery short lifetime in physiological environments (<40 μ s). Therefore, their utility in PDT is limited, many¹O₂Loss of quenching may occur immediately prior to oxidative damage to the tumor cells.

Furthermore, hypoxic microenvironments are an important feature of solid tumors. And PDT treatment itself is also an oxygen-consuming process. PDT treatment further exacerbates tumor hypoxia. Under hypoxic conditions, HIF-1 expression is upregulated. HIF-1 upregulation promotes tumor tissue angiogenesis, triggers tumor metastasis and resistance of cells to apoptosis. Furthermore, HIF-1 upregulation can enhance cellular resistance to various cancer treatments, including PDT. Thus, HIF-1 expression levels are positively correlated with patient mortality and treatment tolerance.

Disclosure of Invention

The purpose of the invention is as follows: the novel pyridone modified zinc phthalocyanine can reduce the expression level of hypoxia inducible factor-1 after being taken by tumor cells, can be excited by red and near infrared light to generate active oxygen, can store redundant singlet oxygen and slowly release the singlet oxygen at physiological temperature, and finally obtains good photodynamic anti-tumor activity, and the novel phthalocyanine is suitable for photodynamic therapy photosensitive anti-tumor therapy.

The invention also provides a preparation method and application of the pyridone modified zinc phthalocyanine.

The technical scheme is as follows: in order to achieve the above object, the pyridone modified zinc phthalocyanine has the following structural formula:

the preparation method of the pyridone modified zinc phthalocyanine comprises the following steps:

(1) synthesis of an intermediate 1: in CH₃In CN, 4-nitro benzyl bromide, 2-hydroxypyridine and K₂CO₃Mixing, stirring overnight under inert gas atmosphere, drying under reduced pressure, mixing with water, extracting, drying, and purifyingObtaining an intermediate 1 after the reaction;

(2) synthesis of intermediate 2: in methanol, intermediate 1, HCOONH₄Pd/C, reacting in an inert gas atmosphere, drying the product under reduced pressure, dissolving, extracting and drying, and then filtering and drying to obtain an intermediate 2;

(3) synthesis of pyridone modified zinc phthalocyanine ZnPc-PYR: in DMF, ZnPc, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl) and 1-hydroxybenzotriazole monohydrate (HOBt) are mixed, the mixture is stirred, an intermediate 2 dissolved in the DMF is dripped into the mixed solution, the mixture is continuously stirred for 1, then the solution is poured into acetone, precipitates are collected by centrifugation, the precipitates are dissolved in an acidic aqueous solution, a supernatant is obtained after centrifugation, the pH is adjusted, a solid product is purified by centrifugation again, and the pyridone modified zinc phthalocyanine ZnPc-PYR is obtained after washing and vacuum drying;

the reaction formula is shown as follows:

wherein, the 4-nitrobenzyl bromide, the 2-hydroxypyridine and the K in the step (1)₂CO₃In a molar ratio of 1: 1-2: 1-2; CH (CH)₃The dosage of CN is 10-50 mL; k₂CO₃The dosage of the composition is 1.45-4.35 g; the reaction temperature is 50-100 ℃. The reaction time is 2-24 h.

Preferably, the reaction temperature in step (1) is 80 ℃ and stirring is carried out overnight under a nitrogen atmosphere.

Wherein the dosage of the intermediate 1 in the step (2) is 0.1-2 g, and HCOONH₄The dosage of the Pd/C is 0.1-3 g, and the dosage of the Pd/C is 0.02-0.3 g; the reaction temperature is 0-80 ℃; the reaction time is 0.5-24 h.

Preferably, the step (2) is reacted at room temperature for 6 hours under a nitrogen atmosphere.

Wherein the molar ratio of the intermediate 2, ZnPc, EDC & HCl and HOBt in the step (3) is 8-10: 1: 8-10; wherein the two DMF dosages are 1-10mL for the first time and 1-5mL for the second time. The reaction temperature of the mixture is 0-60 ℃. The reaction time is 0.5 to 24 hours.

Preferably, in the step (2), the mixture is stirred at 25 ℃ for 1 hour, the intermediate 2 dissolved in DMF is dropped into the mixture, and stirring is continued at 0 ℃ for 1 hour.

The synthetic route of ZnPc used in the present invention is as follows:

the specific synthetic process can be referred to documents: photosensizer and Autophagy Promoter compounded ROS reactive Dendrimer-Assembled Carrier for Synergistic Enhancement of Tumor Growth Suppression, Small 2018,14,1802337

The invention relates to application of pyridone modified zinc phthalocyanine in preparation of photodynamic medicaments.

The pyridone modified zinc phthalocyanine is used as a photosensitizer in preparation of photodynamic medicaments, has good photodynamic anti-tumor activity, and is suitable for photodynamic therapy photosensitive anti-tumor treatment.

The bipyridyl modified zinc phthalocyanine has multiple anti-tumor mechanisms, and can reduce the expression level of hypoxia inducible factor-1 and reduce the tolerance of tumor cells to PDT treatment after being taken by the tumor cells; can be excited by red light (665nm) and near infrared light (808nm) to generate active oxygen, so as to realize synchronous treatment of superficial and deep tumors; pyridone structures can store excess¹O₂And slowly released and lifted at physiological temperature (37℃)¹O₂The utilization efficiency of (2). Finally, good photodynamic anti-tumor activity is obtained.

The traditional zinc phthalocyanine photosensitizer is excited by red light, the penetration depth of the red light to tissues is limited, and the traditional zinc phthalocyanine photosensitizer is only suitable for treating superficial tumors. In contrast, the near infrared (700-. The ZnPc-PYR has stronger absorption intensity about 665nm, which indicates that the ZnPc-PYR can be excited by red light to generate ROS for treating superficial tumors. In addition, ZnPc-PYR also has tail absorbance at about 800nm, and can excite to generate ROS in near infrared light for treating deep tumors.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) after the pyridone modified zinc phthalocyanine ZnPc-PYR is taken by tumor cells, the expression level of hypoxia inducible factor-1 can be reduced, and the tolerance of the tumor cells to PDT treatment can be reduced;

(2) the ZnPc-PYR prepared by the invention can be excited by red (665nm) and near infrared light (808nm) to generate active oxygen, so that the synchronous treatment of superficial and deep tumors is realized;

(3) the ZnPc-PYR prepared by the invention can be stored¹O₂And slowly released and lifted at physiological temperature (37℃)¹O₂The utilization efficiency of (2);

(4) the preparation method of ZnPc-PYR is simple and convenient, has wide raw material source, high activity and diversified sensitization mechanism, can be used as a photosensitizer to be applied to the preparation of photodynamic medicaments, and has good photodynamic anti-tumor activity.

Drawings

FIG. 1 is a synthetic flow chart of a pyridone-modified zinc phthalocyanine ZnPc-PYR of the invention;

FIG. 2 is a schematic diagram of ZnPc-PYR decreasing the expression level of HIF-1 α in cells;

FIG. 3 is a graph showing the cell activity of HeLa cells under different light conditions under normoxic (A1) and hypoxic (A2) conditions; cell activity patterns of 4T1 cells under normoxic (B1) and hypoxic (B2) conditions and different light conditions;

FIG. 4 is a schematic diagram of singlet oxygen generated by ZnPc-PYR under the conditions of 665nm and 808nm light excitation;

FIG. 5 is a schematic representation of the in vitro ROS production of ZnPc-PYR under different lighting conditions;

FIG. 6 is a schematic view of the storage and release of singlet oxygen by ZnPc-PYR;

FIG. 7 is a schematic diagram showing the production, storage and release of ZnPc-PYR in the cell of 1O2 after 665nm and 808nm illumination;

FIG. 8 is a graph showing the comparative effect of ZnPc-PYR on the inhibition of tumor growth under the light excitation conditions of 665nm, 808nm and 665nm +808nm in a subcutaneous transplantation tumor model mouse.

Detailed Description

The invention will be better understood from the following examples. It is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.

Example 1

(1) In 20mL of CH₃To CN were added 4-nitrobenzyl bromide (2.3g), 2-hydroxypyridine (1g) and K₂CO₃(2.9g) and stirred overnight at 80 ℃ under a nitrogen atmosphere. After completion of the reaction, the product was dried under reduced pressure, mixed with water, extracted with DCM, anhydrous Na₂SO₄Drying and column chromatography (PE/EA-5/1, DCM/MeOH-100/1, V/V) to afford intermediate 1;

intermediate 1 compound prepared:¹H NMR(400MHz,d₆-DMSO),δ(ppm)8.22(d,J＝8.8Hz,2H),7.86(dd,J₁₂＝2.0Hz,J₂₃＝6.8Hz,1H),7.53-7.44(m,3H),6.45(d,J＝8.8Hz,1H),6.30(td,J₁₂＝1.6Hz,J₂₃＝5.2Hz,1H),5.24(s,2H).¹³C NMR(100MHz,d₆-DMSO):δ(ppm)161.88,147.27,145.54,140.96,139.73,129.06,124.16,123.86,120.45,106.29,51.41.MS-ESI(m/z):calculated for C₁₂H₁₀N₂O₃ 230.07.Found[M-H]^-229.0.

(2) to 20mL of MeOH were added intermediate 1(1g, 4.3mmol), HCOONH₄(1.5g) and Pd/C (150mg, 10% Pd) were mixed and reacted at room temperature for 6 hours under a nitrogen atmosphere. The product was dried under reduced pressure after Pd/C filtration and redissolved in DCM and Na₂CO₃The solution was washed 3 times and the aqueous phase was extracted with DCM. The combined extracts were extracted with anhydrous Na₂SO₄Drying, filtering, and removing the solvent by rotary evaporation to obtain an intermediate 2;

intermediate 2 compounds of preparationAn object:¹H NMR(400MHz,d₆-DMSO),δ(ppm)7.69(dd,J₁₂＝2.4Hz,J₂₃＝6.8Hz,1H),7.41-7.34(m,1H),7.02(d,J＝8.4Hz,2H),6.50(d,J＝8.4Hz,2H),6.38(dd,J₁₂＝0.8Hz,J₂₃＝8.4Hz,1H),6.19(td,J₁₂＝1.2Hz,J₂₃＝5.6Hz,1H),5.22-5.00(m,2H),4.88(s,2H).¹³C NMR(100MHz,d₆-DMSO):δ(ppm)161.83,148.65,140.15,139.23,129.68,124.70,120.18,114.22,105.76,51.09.MS-ESI(m/z):calculated for C₁₂H₁₀N₂O₃200.09.Found[M-H]^-201.2.

3) ZnPc (56mg), EDC & HCl (76.68mg) and HOBt (54.05mg) were mixed in 5mL of DMF and stirred at 25 ℃ for 1 hour. The mixture was placed in an ice-water bath, and intermediate 2(80mg) dissolved in 2ml of dmdmdff was added dropwise to the mixture. After the reaction was stirred at 0 ℃ for 1 hour, the mixture was then poured into acetone (80mL), the precipitate was collected by centrifugation, dissolved in an acidic aqueous solution (pH 4), and after centrifugation, the supernatant was obtained and the pH of the supernatant was adjusted to 10 with 10% (wt%) NaOH, and the solid product was purified again by centrifugation, then washed several times with EA, and dried under vacuum to obtain the pyridone-modified phthalocyanine derivative ZnPc-PYR. The scheme for preparing the pyridone modified phthalocyanine derivative is shown in figure 1.

The prepared ZnPc-PYR:¹H NMR(400MHz,d₆-DMSO):δ(ppm)10.32(dd,J₁₂＝6.4Hz,J₂₃＝25.6Hz,4H),9.04-8.36(m,12H),8.20(d,J＝34.4Hz,8H),7.79(s,16H),7.47-7.20(m,16H),6.44(d,J＝8.8Hz,4H),6.25(s,4H),5.08(s,8H).¹³CNMR(100MHz,d₆-DMSO):δ(ppm)165.14,162.79,161.90,161.75,160.52,160.33,157.35,140.49,139.65,139.47,139.10,133.77,133.02,133.60,128.68,124.34,121.01,120.33,118.92,118.54,106.01,51.19.

example 2

Example 2 was prepared identically to example 1, except that: in the step (1), the molar ratio of 4-nitrobenzyl bromide to 2-hydroxypyridine is 1:2, 2-hydroxypyridine (0.5g), CH₃The dosage of CN is 10 mL; k₂CO₃The dosage of the medicine is 1.45 g; the reaction temperature is 50 ℃, andthe reaction time is 24 hours; the dosage of the intermediate 1 in the step (2) is 0.1g, HCOONH₄The dosage of the Pd/C is 0.1g, and the dosage of the Pd/C is 0.02 g; the reaction temperature is 0 ℃; the reaction time is 24 h; the molar ratio of the intermediate 2, ZnPc, EDC & HCl and HOBt in the step (3) is 10:1:10: 10; wherein the amount of DMF used in the two times is 1mL, and the reaction temperature of the mixture is 0 ℃. The reaction time was 24 h.

Example 3

Example 3 was prepared identically to example 1, except that: in step (1), the molar ratio of 4-nitrobenzyl bromide to 2-hydroxypyridine is 1:1, 2-hydroxypyridine (1.5g), CH₃The dosage of CN is 50 mL; k₂CO₃The dosage of the composition is 4.35 g; the reaction temperature is 100 ℃, and the reaction time is 2 hours; the dosage of the intermediate 1 in the step (2) is 2g, HCOONH₄The dosage of the Pd/C is 3g, and the dosage of the Pd/C is 0.3 g; the reaction temperature is 80 ℃; the reaction time is 0.5 h; the molar ratio of the intermediate 2, ZnPc, EDC & HCl and HOBt in the step (3) is 8:1:10: 10; wherein the amount of DMF used twice is 10mL for the first time and 5mL for the second time, and the reaction temperature of the mixture is 60 ℃. The reaction time was 0.5 h.

Example 4

Detection of reduction of HIF-1 alpha expression level in cells by ZnPc-PYR

ZnPc-PYR has low water solubility. Thus, a stock solution of ZnPc-PYR was prepared in DMSO at a concentration of 10^-3mol/L. ZnPc-PYR tends to aggregate and precipitate after addition to an aqueous solution. Cremophor EL is a nonionic surfactant having excellent solubility and emulsifying activity. And Cremophor EL has extremely low toxicity and is widely used for improving the solubility of drugs. Therefore, in the present invention, 0.001% Cremophor EL was used as a solubilizer for ZnPc-PYR for all physical and chemical properties and activity studies.

Hypoxia inducible factor 1(HIF-1) is composed of constitutively expressed subunits (HIF-1 beta) and O₂Regulatory subunit (HIF-1 alpha). Down-regulation of HIF-1 α can represent expression of HIF-1. HeLa cells were cultured in an anoxic incubator (1% O)₂、5％CO₂Anoxic pretreatment at 37 ℃). After culturing for 24 hours, the cells were cultured in 1mL of serum-free DMEM medium ([ ZnPc-PYR) containing ZnPc-PYR obtained in example 1]6 μ M) for 4 hoursCell lysates were used to lyse cell extract proteins using EDTA (5mM) which acts as an iron chelator to reduce intracellular iron concentration and inhibit the rapid degradation of HIF-1 α. The effect of reducing the expression level of HIF-1 alpha in cells by ZnPc-PYR is detected by SDS-PAGE electrophoresis and ECL imaging technology. Blank, DMSO and Cremophor EL groups were set as controls to demonstrate that HIF-1 α down-regulation in cells is derived from ZnPc-PYR.

As shown in FIG. 2, the DMSO group and the Cremophor EL group did not cause down-regulation of cellular HIF-1. alpha. expression. However, the ZnPc-PYR can obviously reduce the expression level of HIF-1 alpha of tumor cells, the upregulation of the HIF-1 alpha plays a leading role in the tumor progression processes of angiogenesis, metastasis, apoptosis resistance and the like, and the upregulation of the HIF-1 alpha is related to PDT treatment resistance. Therefore, the ZnPc-PYR of the invention can inhibit HIF-1 alpha and is an effective way to improve the treatment efficiency of PDT.

Cells were all cultured in a three-gas incubator (1% O)₂,5％CO₂At 37 ℃, solutions used in the hypoxic experiment are all deoxygenated by nitrogen. The cells were cultured in a 96-well plate for 24 hours, the original culture solution was removed, a fresh medium containing ZnPc-PYR (6. mu.M) was added, and the cells were incubated in an incubator at 665nm (1W cm)^-2) Irradiating with laser for 4min at 808nm (1W cm)^-2) The laser was irradiated for 4min and incubation in the incubator was continued overnight. After adding 100. mu.L of a fresh medium containing 10% MTT to each well, after 4 hours, formazan crystals were dissolved in DMSO, and the OD value of the optical density at 570nm was measured using a microplate reader, and the amount of formazan crystals produced was counted as the number of living cells. As shown in FIG. 3, ZnPc-PYR also has good two-light induced PDT activity on HeLa cells under hypoxic conditions. The same experiment was also performed on 4T1 cells, and similar results were obtained. This property is of great importance for the in vivo treatment of PDT, since both hypoxic and normoxic regions are present in solid tumors. (in FIG. 3, A1 and B1 indicate the toxicity of HeLa and 4T1 cells under normoxic conditions; A2 and B2 indicate the toxicity of HeLa and 4T1 cells under normoxic conditions), which indicates that the HIF-1 down-regulation function of ZnPc-PYR can enhance the sensitivity of tumor cells to PDT treatment under hypoxia conditions.

Example 5

The detection of singlet oxygen generated by ZnPc-PYR under the excitation condition of 665nm and 808nm light

ADPA and¹O₂the reaction generates its endoperoxide, which in turn leads to a decrease in the absorption intensity at the characteristic absorption peak of ADPA (λ max ═ 378 nm). The decrease in absorption intensity caused by oxidation of ADPA in aqueous solution was monitored¹O₂Is generated.

This process was tested using a commercial singlet oxygen probe ADPA (9, 10-diphenylanthracenylpropionic acid, sigma aldrich, inc.) which oxidizes ADPA to nonabsorbent endoperoxides, resulting in a decrease in its absorbance. The ZnPc-PYR prepared in example 1 was dissolved in an aqueous solution of 0.001% CrEL ([ ZnPc-PYR)]10 μ M) and then mixed with ADPA (100 μ M) (volume ratio of ZnPc-PYR solution to ADPA aqueous solution 10: 1). 665nm (0.4W cm^-2) LED and 808nm (0.25W cm)^-2) A laser is used as the excitation light source. The absorption spectrum of the mixed solution after the light exposure time was recorded.

As shown in fig. 4, the ADPA absorption spectrum intensity gradually decreased with increasing light absorption time under the conditions of 665nm and 808nm light irradiation. Shows that the ZnPc-PYR can be excited by 665nm laser and can also be excited by 808nm laser to generate singlet oxygen

The in vitro total ROS generating capacity of ZnPc-PYR is tested by taking DCFH-DA as a total active oxygen probe (Sigma Aldrich, Co., Ltd.). Intracellular ROS can oxidize non-fluorescent DCFH to generate fluorescent DCF, and the green fluorescence intensity is in direct proportion to the intracellular ROS level. The specific process comprises the following steps: HeLa cells were cultured in DMEM containing 10% NBS and incubated at 37 ℃ with 5% CO₂In a humidified incubator. After overnight incubation in confocal laser culture dish, the cells were incubated with ZnPc-PYR (6. mu.M) for 4 hours. Thereafter, the original culture medium was discarded, and the HeLa cells were incubated with DCFH-DA fluorescent probe for one hour at 665nm (0.4W cm)^-2) Light for 3 min, 808nm (0.25W cm)^-2) Light for 15 minutes, 665+808nm (665nm LED for 3 minutes followed by 808nm LED for 15 minutes). After cell collection, the green fluorescence intensity at 488nm excitation was quantified using flow cytometry. The green fluorescence signal of the living cells is observed by laser confocal observation at the maximum excitation wavelength of 488nm and the maximum emission wavelength of 525 nm.

As shown in FIG. 5, ZnPc-PYR efficiently generates ROS in cancer cells under 665 and 808nm light. Furthermore, total ROS levels were significantly higher for cells irradiated at 665+808nm than for cells treated at 665 or 808nm alone. The ZnPc-PYR can generate a large amount of ROS to oxidize and damage tumor cells under the double-wavelength excitation of 665nm and 808nm, and PDT treatment of superficial and deep tumors can be realized.

Example 6

Detection of singlet oxygen released by ZnPc-PYR storage

To verify the feasibility of ZnPc-PYR continuously releasing singlet oxygen under non-illuminated conditions, a commercial singlet oxygen probe ADPA was used to examine the process. Before running the assay, the ZnPc-PYR prepared in example 1 ([ ZnPc-PYR)]10 μ M) was dissolved in a 0.001% CrEL aqueous solution and then mixed with ADPA (100 μ M) (volume ratio of ZnPc-PYR solution to ADPA aqueous solution 10: 1). 665nm (0.4W cm^-2) An LED is used as the excitation light source. The absorption spectrum of ADPA during intermittent light and heating in the light space (37 ℃ C., dark) was recorded (after 30 seconds of light irradiation, the cycle was repeated 4 times while avoiding light for 30 minutes).

As shown in FIG. 6, the illumination process may result in ADPA at 378nm (λ)_maxof ADPA) absorption intensity is reduced, which shows that illumination can trigger the PDT process of ZnPc-PYR to generate singlet oxygen; likewise, physiological temperature may trigger the ADPA to continue to decrease in absorbance at 378nm when stored in the dark. The pyridone structure of ZnPc-PYR can store singlet oxygen generated in the PDT process of ZnPc-PYR and slowly release the stored singlet oxygen at physiological temperature, and the process can improve¹O₂The utilization efficiency of the PDT is improved.

Furthermore, by HeLa intracellularly¹O₂The generation detection further proves that the ZnPc-PYR is irradiated under 665 and 808nm light¹O₂Storage and release functions. Detecting intracellular by using SOSG as probe (Saimeishoer science and technology (China)) and adopting laser confocal and cell flow meter¹O₂And (4) generating. The specific process comprises the following steps: detection of drug presence within cells using flow cytometry and laser confocal¹O₂Storage and release capacity. HeLa cellsCulturing in 6-well plate or laser confocal culture dish overnight, incubating ZnPc-PYR (6 μ M) for 4 hours, discarding the original culture solution, adding serum-free fresh DMEM, immediately irradiating one group, placing in an incubator for continuous incubation, irradiating the other group after 6 hours, simultaneously adding SOSG singlet oxygen probes (5 μ M) into the two groups, incubating the incubator for one hour, collecting cells, detecting fluorescence intensity by using a cell flow instrument or washing the cells for three times by using PBS, and observing the fluorescence intensity by using laser confocal measurement. 665nm LED lamp for 3 minutes and 808nm LED lamp for 15 minutes.

As shown in FIG. 7, ZnPc-PYR produces a large amount of light immediately at 665 or 808nm in cancer cells¹O₂. After two groups of cells after illumination are continuously incubated for 6 hours in a dark incubator at 37 ℃, the fluorescence intensity of the cells is continuously enhanced, which indicates that the ZnPc-PYR cells can be continuously released¹O₂。

Example 7

In vivo detection of anti-cancer Activity

In a mouse subcutaneous transplantation tumor model animal body, the ZnPc-PYR can inhibit the tumor growth under different illumination conditions. The experimental procedure is as follows, ZnPc-PYR is injected into tumor-bearing nude mice through tail vein, after 6h (administration concentration is 5mg/kg mice), the tumor position is illuminated by different illumination conditions (665nm group: 665nm LED power is 0.9W/cm)²The illumination time is 30 minutes; 808nm group: the power of a 808nm laser is 0.3W/cm²The illumination time is 7 minutes; 665nm +808nm group: the power of the 665nm LED is 0.9W/cm²The illumination time is 30 minutes, and the power of a 808nm laser is 0.3W/cm²And the light irradiation time was 7 minutes). The drug was administered on

days

0, 2, 4, 6,8,10, and 12, and light irradiation was performed daily from the first day. The whole treatment process lasts for 14 days, the size of the tumor tissue is measured every day, and the change rule of the tumor volume is recorded.

As shown in fig. 8, the results of the in vivo antitumor activity study showed that the ability to inhibit tumor growth was significantly stronger in the 665nm +808nm group than in the 665nm and 808nm groups. Indicating that ZnPc-PYR can be excited by 665nm and 808nm to generate active oxygen. 808nm light has better penetration capability for deep and mid tissue than 665 nm. Therefore, the ZnPc-PYR can realize the high-efficiency PDT treatment of deep-surface and superficial tumors.

Claims

1. A pyridone-modified zinc phthalocyanine, which has a structural formula shown as follows:

2. a method for preparing a pyridone-modified zinc phthalocyanine according to claim 1, comprising the steps of:

(1) synthesis of an intermediate 1: in CH₃In CN, 4-nitro benzyl bromide, 2-hydroxypyridine and K₂CO₃Mixing, stirring overnight under the atmosphere of inert gas, drying the product under reduced pressure, mixing with water, extracting, drying, and purifying to obtain an intermediate 1;

(2) synthesis of intermediate 2: in methanol, intermediate 1, HCOONH₄Pd/C, reacting in an inert gas atmosphere, drying the product under reduced pressure, dissolving, extracting and drying, and then filtering and evaporating to obtain an intermediate 2;

(3) synthesis of pyridone modified zinc phthalocyanine ZnPc-PYR: in DMF, ZnPc, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl) and 1-hydroxybenzotriazole monohydrate (HOBt) are mixed, the mixture is stirred, an intermediate 2 dissolved in the DMF is dripped into the mixed solution, stirring is continued, then the solution is poured into acetone, precipitates are collected by centrifugation, the precipitates are dissolved in an acidic aqueous solution, a supernatant is obtained after centrifugation, the pH is adjusted, a solid product is purified by centrifugation again, and the pyridone modified zinc phthalocyanine ZnPc-PYR is obtained after washing and vacuum drying;

the reaction formula is shown as follows:

3. root of herbaceous plantThe method according to claim 2, wherein the 4-nitrobenzyl bromide, 2-hydroxypyridine and K in step (1) are reacted with each other₂CO₃In a molar ratio of 1: 1-2: 1-2; CH (CH)₃The dosage of CN is 10-50 mL; k₂CO₃The dosage of the composition is 1.45-4.35 g; the reaction temperature is 50-100 ℃, and the reaction time is 2-24 h.

4. The method according to claim 2, wherein the amount of the intermediate 1 used in the step (2) is 0.1 to 2g, HCOONH₄The dosage of the Pd/C is 0.1-3 g, and the dosage of the Pd/C is 0.02-0.3 g; the reaction temperature is 0-80 ℃; the reaction time is 0.5 to 24 hours.

5. The preparation method according to claim 2, wherein the molar ratio of the intermediate 2, ZnPc, EDC-HCl and HOBt in the step (3) is 8-10: 1: 8-10; the dosage of DMF is 1-10mL for the first time and 1-5mL for the second time; the reaction temperature is 0-60 ℃; the reaction time is 0.5 to 24 hours.

6. Use of the pyridone-modified zinc phthalocyanine of claim 1 in the preparation of a photodynamic medicament.

7. The use according to claim 6, wherein the pyridone-modified zinc phthalocyanine is used as a photosensitizer in the preparation of a photodynamic medicament.