Chlorin photosensitizer as well as pharmaceutically acceptable salt, preparation method and application thereof

Abstract

The invention discloses a dihydroporphin photosensitizer, pharmaceutically acceptable salts thereof, a preparation method and application, and belongs to the technical field of medicines. The structure of the photosensitizer is shown as a formula I, and the photosensitizer is prepared from chlorin E6 through esterification, selective hydrolysis and decarboxylation reaction and has the advantages of being single in chemical component, high in stability and high in photodynamic anti-tumor activity. In-vitro and in-vivo experiments show that the compound has a remarkable killing effect on various tumor cells, IC50 is lower than 10 mu g / mL, the tumor inhibition rate is up to 100%, and the compound is suitable for preparing anti-tumor drugs, has no obvious toxic or side effect and has clinical application potential. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a chlorin photosensitizer, a pharmaceutically acceptable salt thereof, a preparation method and an application thereof. Background Art

[0002] Photodynamic Therapy (hereinafter referred to as PDT) is a rapidly developing treatment technology in recent years. Its basic principle is to use light of a specific wavelength to activate a photosensitizer, enabling it to generate reactive oxygen species (such as singlet oxygen) in the body, thereby selectively killing diseased cells, such as tumor cells or pathogens in infected tissues. Due to its advantages such as minimally invasive nature, high selectivity, and fewer systemic side effects, PDT has shown broad application prospects in tumor treatment, dermatological treatment, and the antibacterial field. As the core component of PDT, the performance of the photosensitizer directly determines the quality of the treatment effect. An ideal photosensitizer should have a high singlet oxygen yield, good photochemical stability, appropriate solubility in the body, and the ability to target diseased tissues.

[0003] Among numerous photosensitizers, chlorin compounds have attracted much attention due to their unique molecular structures and excellent photophysical and photochemical properties. Chlorin is a reduced derivative of porphyrin compounds. Its molecule contains a partially saturated pyrrole ring, which causes its absorption spectrum to redshift to the near-infrared region (usually between 650 - 700 nm). This wavelength range has strong tissue penetration ability and is suitable for deep internal treatment in the body. In addition, chlorin photosensitizers generally have a high singlet oxygen quantum yield and good photostability, making them a hot spot in PDT research and application. In recent years, a variety of chlorin photosensitizers have been developed and applied in preclinical or clinical studies, such as Chlorin E6, mTHPC, and other derivatives. These photosensitizers have shown certain curative effects in the treatment of diseases such as solid tumors, oral cancer, and skin cancer, and some products have been approved for clinical use.

[0004] Although chlorin photosensitizers show significant advantages in PDT, there are still some problems and deficiencies in the prior art that need to be urgently solved, such as complex composition, insufficient solubility, poor targeting, and poor stability, which are difficult to store, restricting their further development and wide application.

[0005] In response to the above problems, researchers have attempted to improve the performance of chlorin-based photosensitizers through various technical means in recent years. For example, hydrophilic groups (such as carboxyl, hydroxyl, or polyethylene glycol chains) are introduced through chemical modification to improve the water solubility of the photosensitizer; tumor-specific targeting molecules (such as antibodies or peptides) are grafted to enhance its targeting to diseased tissues; nanotechnology (such as nanoparticle coating) is used to improve the stability and in vivo delivery efficiency of the photosensitizer. In addition, optimizing the synthesis route and developing green chemical methods have also been proposed to simplify the preparation process and increase the yield. For example, it has been reported in the literature that chlorin derivatives are synthesized by a one-pot reaction method, which significantly reduces the reaction steps, but the generality of this method and the performance of the products still need to be further verified.

[0006] However, these improvement measures still have certain limitations. For example, chemical modification may introduce new toxicity or reduce the photochemical activity of the photosensitizer; the preparation of nanocarriers increases the process complexity and cost; the optimized synthesis route may only be applicable to photosensitizers with specific structures and lacks universality. Therefore, there is still much room for improvement in the performance enhancement and preparation process optimization of chlorin-based photosensitizers in the existing technology.

[0007] In summary, as an important research direction of PDT, chlorin-based photosensitizers have made certain progress in the existing technology, but still face challenges in terms of solubility, stability, preparation efficiency, etc. The present invention is an innovative solution proposed under this technical background, aiming to promote the performance improvement and application expansion of chlorin-based photosensitizers through structural innovation and process optimization, and provide support for the technological progress and industrial development of related fields. Summary of the Invention

[0008] In order to overcome the above-mentioned disadvantages and deficiencies of the existing technology and give full play to the advantages of the chlorin-based structure, the present invention provides a chlorin-based photosensitizer, its pharmaceutically acceptable salts, preparation methods and applications. The structure of the photosensitizer is shown in Formula I and is prepared by esterification, selective hydrolysis and decarboxylation reactions of chlorin E6. It has the advantages of single chemical composition, high stability and strong photodynamic anti-tumor activity. In vitro and in vivo experiments show that it has a significant killing effect on a variety of tumor cells, with an IC50 lower than 10 μg / mL and a maximum tumor inhibition rate of 100%. It is suitable for the preparation of anti-tumor drugs, shows no obvious toxic side effects, and has clinical application potential.

[0009] A chlorin-based photodynamic photosensitizer and its pharmaceutically acceptable salts, the structural formula of which is shown in Formula I;

[0010]

[0011] Wherein, R is an unsubstituted C1-6 straight-chain or branched-chain alkyl group, or a C1-6 straight-chain or branched-chain alkyl group substituted by halogen, hydroxyl, amino or alkoxy group.

[0012] A preparation method of a chlorin-based photodynamic photosensitizer, comprising the following steps:

[0013]

[0014] (1) Dissolve chlorin E6 in an alcohol solvent, react under the protection of argon, acidic and heating conditions. After the reaction, concentrate, extract, wash, purify and dry the reaction solution in sequence to obtain Compound 1;

[0015] (2) Add Compound 1 and an aqueous sodium hydroxide solution to ethanol, react under heating under the protection of argon. After the reaction, slowly add a dilute hydrochloric acid solution dropwise to the reaction solution to adjust the pH until a solid precipitates. Filter to obtain a filter cake, wash it with deionized water and dry it to obtain Compound 2, which is directly used in the next step without purification;

[0016] (3) Carry out a decarboxylation reaction on Compound 2 under the conditions of a catalyst and heating. After the reaction and cooling, a crude product is obtained; the crude product is purified by silica gel column chromatography to obtain a chlorin-based photodynamic photosensitizer, i.e., Compound I.

[0017] Further, the alcohol solvent in step (1) is a substituted or unsubstituted C1-6 straight-chain or branched-chain alcohol, including but not limited to monohydric alcohols such as methanol, ethanol, n-butanol, isobutanol and dihydric alcohols such as ethylene glycol, propylene glycol, etc.;

[0018] Further, the heating reaction temperature in step (2) is 40-80 °C, preferably 55-65 °C;

[0019] Further, the pH adjusted by the dilute hydrochloric acid solution in step (2) is 1.0-5.0 °C, preferably 3.0-4.0 °C;

[0020] Further, the catalyst in step (3) is one of anhydrous sodium acetate, soda lime, sodium hydroxide, potassium hydroxide, silver powder;

[0021] Further, the heating temperature in step (3) is 80-300 °C, preferably 150 °C. The heating time is 10-60 min, preferably 30 min. The eluent for silica gel column chromatography separation is dichloromethane / methanol, with a volume ratio of 100:1.

[0022] The present invention also provides the application of the above-mentioned chlorin-based photodynamic photosensitizer and its pharmaceutically acceptable salts in the preparation of photodynamic therapy drugs or their lead compounds.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] The chlorin-based photodynamic photosensitizer involved in the present invention exhibits excellent photodynamic performance, characterized by significant absorption ability in the near-infrared spectral region and excellent photostability. Under red light irradiation, this photosensitizer can efficiently generate reactive oxygen species (ROS), so it is very suitable for use as a photosensitizer.

[0025] In addition, the chlorin-based photodynamic photosensitizer shows strong photoactivity and a relatively high phototherapeutic index, especially showing significant photodynamic effects in anti-tumor treatment. More importantly, no obvious toxic and side effects were found in the experiments, indicating its potential to become a new generation of clinical photosensitizers and can be used for the development of phototherapeutic drugs or as a lead compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly introduced below. It should be noted that the following drawings only show some embodiments of the present invention, so they should not be regarded as limiting the scope of the present invention. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0027] Figure 1 : Chemical structural formula of Compound I-1;

[0028] Figure 2 : UV-vis spectrum of Compound I-1;

[0029] Figure 3 : HR-ESI-MS spectrum of Compound I-1;

[0030] Figure 4 : 1H-NMR spectrum of Compound I-1;

[0031] Figure 5 : 13C-NMR spectrum of Compound I-1;

[0032] Figure 6 : Killing and inhibition curve of Compound I-1 PDT against BGC-823 cells;

[0033] Figure 7 : Killing and inhibition curve of Compound I-1 PDT against BEL-7402 cells;

[0034] Figure 8 : Killing and inhibition curve of Compound I-1 PDT against A549 cells;

[0035] Figure 9 : Killing and inhibition curve of Compound I-1 PDT against A431 cells;

[0036] Figure 10:Inhibitory curve of compound I-1 PDT on KB cells;

[0037] Figure 11 :Comparison of tumor inhibition rates in each group;

[0038] Figure 12 :Tumor growth curves of each group;

[0039] Figure 13 :Comparison of tumor RTV values in each group;

[0040] Figure 14 :Comparison of tumor T / C values in each group;

[0041] Figure 15 :Effect of light dose on singlet oxygen production in the reaction system;

[0042] Figure 16 :Change of singlet oxygen production at different photosensitizer concentrations. Specific implementation mode

[0043] The present invention will be further described in detail below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto. The materials involved in the following embodiments can be obtained from commercial channels without special instructions. The methods involved are all conventional methods without special instructions.

[0044] A chlorin-based photodynamic photosensitizer and its pharmaceutically acceptable salt, the structural formula of which is as Figure 1 shown; wherein R is a substituted or unsubstituted straight-chain or branched-chain alkyl group of C1-6.

[0045] As a preferred technical solution of the present invention, the chlorin-based photodynamic photosensitizer and its pharmaceutically acceptable salt have one of the following structures:

[0046]

[0047] Example 1: Synthesis of chlorin e6 diethyl ester (compound 1-1)

[0048]

[0049] Chlorin e6 (20 mmol) was added to anhydrous ethanol (300 ml), and 98% concentrated sulfuric acid (10 ml) was added dropwise with stirring at room temperature. The reaction was carried out at 55 °C for 2 h under argon protection. 90% of the ethanol was removed from the reaction solution by rotary evaporation, and the residue was poured into deionized water (1500 ml). It was extracted twice with dichloromethane (about 800 ml), the organic phases were combined, and washed successively with saturated brine (400 ml) and deionized water (200 ml). After drying, 8.0 g of brown product was obtained by silica gel column chromatography to obtain compound 1-1.

[0050] Example 2: Synthesis of Chlorophyllide E6 Monoethyl Ester (Compound 2-1)

[0051]

[0052] Compound 1-1 (10 mmol) and aqueous sodium hydroxide solution (16 ml, 0.1 mol / ml) were added to ethanol (1600 ml), and the reaction was carried out at 50 °C for 1 h under argon protection. 1000 ml of deionized water was added for dilution. Under stirring conditions, 10% dilute hydrochloric acid was slowly added dropwise to adjust the pH to 4. The filter cake was obtained by filtration, washed with 300 ml of deionized water, and dried in vacuo at 25 °C to obtain Compound 2-1, which was directly used for the next reaction without purification.

[0053] Example 3: Synthesis of Chlorophyllide-based Photodynamic Photosensitizer (Compound I-1)

[0054]

[0055] The above Compound 2-1 and Ag powder (1 mmol) were mixed evenly and heated to 150 °C for decarboxylation reaction. The reaction time was 30 min. After the reaction was completed and cooled, the crude product was obtained; the crude product was purified by silica gel column chromatography, and the eluent was dichloromethane / methanol with a volume ratio of 100:1 to obtain the chlorophyllide-based photodynamic photosensitizer (Compound I-1). HR-ESI-MS: [M+H]+ = 581.31. The 1H NMR data are shown in Table 1, and the 13C NMR data are shown in Table 2.

[0056] Table 1 1H NMR Analysis Data Table of Compound I-1

[0057]

[0058] Table 2 13C NMR Analysis Data Table of Compound I-1

[0059]

[0060] Example 4: In vitro PDT Antitumor Activity Test of Chlorophyllide-based Photodynamic Photosensitizer

[0061] 1. Materials and Methods

[0062] ① Main Experimental Instruments

[0063] 660 nm semiconductor laser (BWT-660-800), 630 nm semiconductor laser (DIOMED-630 PDT), laser wavelength meter (THORLABS), laser power meter (PM310D), special fiber with Microlens, microplate reader (BIO-RAD Model680), inverted microscope (MOTIC AE31); purification workbench (BOXUN SW-CJ-2FD); electronic balance (METTLER TOLEDO).

[0064] ② Preparation of test samples

[0065] Dissolve compound I-1 in DMSO to prepare a stock solution with a concentration of 30 mM, and then dilute it to the required concentration with the culture medium. The control drug, Xipofen (specification: 5 mg / mL), is a commercially available product of Huading Modern Biological Pharmaceutical Co., Ltd. Dilute it to different concentrations with the culture medium before administration.

[0066] ③ Cell lines

[0067] BGC-823 (human gastric cancer cells), BEL-7402 (human liver cancer cells), A549 (human lung cancer cells), A431 (human epithelial cancer cells), KB (human nasopharyngeal cancer cells) are all derived from the Anti-Cancer Research Center of Xiamen University.

[0068] ④ Experimental procedures

[0069] a. Select adherent tumor cells in the logarithmic growth phase. After digestion with trypsin, prepare a cell suspension with a concentration of 50,000 cells / mL using DMEM culture medium containing 10% fetal bovine serum. Inoculate 100 μL into each well of a 96-well culture plate and incubate overnight in a 5% CO2, 37 °C cell culture incubator. To avoid light interference between different light dose groups, fill the wells between groups with black ink (300 μL) for isolation, as shown below.

[0070] Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Ink Ink Ink Ink Ink Ink Ink Ink Ink Ink Ink Cell Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Ink Ink Ink Ink Ink Ink Ink Ink Ink Ink Ink Ink Cell Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Cell Cell Cell Ink Cell Cell Ink Cell Cell Ink Cell Cell Cell

[0071] b. After overnight incubation of the cells, add drugs for treatment. At the same time, set up control groups such as simple light irradiation, simple compound I-1, positive drug Xipofen (XPF), and drug-free culture medium. Each group has 4 replicate wells.

[0072] c. After 12 h of adding drugs, remove the supernatant culture medium, wash twice with PBS, add 200 μL of fresh culture medium, and then perform laser irradiation. The laser outputs 600 mW through a special fiber with a Microlens at the end, vertically covering 4 wells in the same light dose group. The spot diameter is 2.5 cm, and the surface light power density is 120 mW / cm 2 , with uniform distribution. The experimental group is irradiated with 660 nm laser for 168 s, and the light energy density is 20 J / cm 2; The XPF control group was irradiated with 630 nm laser for 202 s, and the light energy density was 24 J / cm 2 . The negative control group, blank control group, and zero adjustment group were not irradiated with light.

[0073] d. After 24 h of cell light treatment, the supernatant was discarded, and 200 μL of medium containing 0.5 mg / mL MTT was added to each well and cultured at 37 °C for another 4 h. Then, the supernatant was carefully removed, 200 μL of DMSO was added to each well, and shaken at low speed for 15 min to fully dissolve the crystals. Using an enzyme-linked immunosorbent assay (ELISA) reader, the optical density value was measured with 570 nm as the test wavelength and 630 nm as the reference wavelength. A zero adjustment well (cells, culture medium, DMSO) and a control well (cells, culture medium, MTT, DMSO) were set up.

[0074] e. Calculate the survival rate of tumor cells in the drug treatment group according to the following formula:

[0075] Survival rate of tumor cells (%) = (ODexperiment / ODcontrol) × 100%

[0076] Calculate the inhibition rate of the drug on the growth of tumor cells according to the following formula:

[0077] Inhibition rate of tumor cell growth (%) = (1 – ODexperiment / ODcontrol) × 100%

[0078] Plot the dose-effect curve with the inhibition rate of tumor cells at different concentrations of the same sample, and then calculate the median lethal concentration (IC50 value or EC50 value) using the Logit method. When the IC50 of the synthetic compound or pure plant extract is < 10 μg / mL or the IC50 of the crude plant extract is < 20 μg / mL, it is determined that the sample has a killing effect on tumor cells in vitro.

[0079] 2. Experimental results

[0080] The killing and inhibition data of compound I-1 PDT on BGC-823, BEL-7402, A549, A431, and KB cells are shown in the appendix Figures 6 - 10 . The killing IC50 (MTT method) values of compound I-1 PDT on the above 5 kinds of cells are shown in the following table.

[0081] Table 3 Killing IC50 (MTT method) of compound I-1 PDT on 5 kinds of cells

[0082]

[0083] The chlorophyllide-based photodynamic photosensitizer compound I-1 PDT shows significant killing effects on human tumor cells from 5 sources including gastric cancer, esophageal cancer, liver cancer, lung cancer, and nasopharyngeal cancer. The IC50 values are all less than 10 μg / mL, and the killing effect is positively correlated with the drug concentration. However, different cells have different sensitivities to the killing effect of compound I-1 PDT, and their sensitivities are in the order of KB > A431 > A459 > BGC-823 > BEL-7402. Therefore, compound I-1 shows good PDT anti-tumor activity in vitro.

[0084] Example 5: In Vivo PDT Anti-Tumor Activity Test of Chlorophyllide-Based Photodynamic Photosensitizers

[0085] 1. Materials and Methods

[0086] ① Test Animals

[0087] nu / nu nude mice, 18 - 22 g, male

[0088] ② Preparation of Test Samples

[0089] Compound I-1 was dissolved in DMSO to prepare a solution with a concentration of 30 mM as the stock solution, and then diluted to the required dose with normal saline. The control drug, HemoPhot (specification: 5 mg / mL), is a commercially available product from Huading Modern Biotechnology Co., Ltd. Dilute the required dose with normal saline before administration.

[0090] ③ Tumor Strains

[0091] Human lung cancer nude mouse xenograft A549, passaged and preserved in the laboratory of the Anti-Cancer Research Center of Xiamen University

[0092] ④ Preparation of Tumor Models

[0093] Select tumor-bearing animals with good tumor growth and good general condition, and sacrifice them by cervical dislocation. Under sterile conditions, remove the tumor mass, cut it into tumor pieces with a diameter of 2 - 3 mm with a scalpel, and inoculate them subcutaneously at the right thigh root of nude mice with an ascites puncture needle. Seven days later, when the tumor in the right thigh root of the nude mouse grows to a diameter of about 5 - 8 mm, it can be used.

[0094] ⑤ Animal Grouping

[0095] Select 80 qualified tumor-bearing nude mice, measure the length and width of the tumors with a vernier caliper, and stratify and group them according to the tumor volume size, with 8 mice in each group (2 of which are used for histological observation and sampling), for a total of 10 groups. Specifically

[0096] Negative control group, intravenous injection of normal saline through the tail vein;

[0097] Simple light irradiation group, intravenous injection of normal saline through the tail vein and then irradiate the tumor with 660 nm light at 100 J / cm 2 ;

[0098] Simple administration group: The test drug was administered via the tail vein at a dose of 2 mg / kg;

[0099] Experimental group 1: The test drug was administered via the tail vein at a dose of 2 mg / kg, and the tumor was irradiated with 660-nm light at 100 J / cm 2 ;

[0100] Experimental group 2: The test drug was administered via the tail vein at a dose of 2 mg / kg, and the tumor was irradiated with 660-nm light at 50 J / cm 2 ;

[0101] Experimental group 3: The test drug was administered via the tail vein at a dose of 1 mg / kg, and the tumor was irradiated with 660-nm light at 100 J / cm 2 ;

[0102] Experimental group 4: The test drug was administered via the tail vein at a dose of 1 mg / kg, and the tumor was irradiated with 660-nm light at 50 J / cm 2 ;

[0103] Experimental group 5: The test drug was administered via the tail vein at a dose of 1 mg / kg, and the tumor was irradiated with 660-nm light at 25 J / cm 2 ;

[0104] Experimental group 6: The test drug was administered via the tail vein at a dose of 0.5 mg / kg, and the tumor was irradiated with 660-nm light at 50 J / cm 2 ;

[0105] Positive control group: Hypericin was administered via the tail vein at a dose of 6 mg / kg, and the tumor was irradiated with 630-nm light at 100 J / cm 2 .

[0106] The injection solutions for each group were prepared 1 h before the experiment and stored in the dark and refrigerated before use.

[0107] Except for the tumor irradiation step, the animals were strictly kept in the dark for 3 d after drug administration and then raised under dim light.

[0108] ⑥ Irradiation implementation

[0109] When implementing tumor irradiation, first adjust the 660- and 630-nm semiconductor lasers used to lock the output power at the fiber end at 226 mW. Then, align the fiber with a microlens at the end directly towards the target site, let the light beam be perpendicular to the irradiation and completely cover the tumor, and keep the spot diameter at 12 mm. At this time, the surface light power density at the target site is 200 mW / cm 2 , and continuous irradiation for 500 s can obtain a light dose (light energy density) of 100 J / cm 2 .

[0110] ⑦ Observation, recording, and data processing

[0111] After laser irradiation, the length and width of the tumors carried by the experimental animals in each group were measured and recorded every other day. The growth of the animal tumors and the general conditions were observed. The tumor volume (TV) and relative tumor volume (RTV) were calculated according to the methods provided in relevant literature, and a graph of the growth change of the tumor volume was drawn. At the end of the experiment, the animals were sacrificed to dissect the tumors, the tumor weights were measured, and the tumor growth inhibition rate of the drug was calculated.

[0112] The calculation formula for the tumor volume (TV) is: TV = tumor length × tumor width 2 / 2

[0113] The calculation formula for the relative tumor volume (RTV) is: RTV = Vt / V0, where V0 is the TV measured at the time of drug administration, and Vt is the TV obtained from each subsequent observation.

[0114] The evaluation index for anti-tumor activity is the relative tumor proliferation rate T / C (%), and the calculation formula is:

[0115] T / C (%) = [RTV of the treatment group (T) / RTV of the negative control group (C)] × 100%

[0116] 2. Experimental results

[0117] The test results are shown in the appendix Figures 11 - 14 。

[0118] The results of the tumor inhibition rate measurement showed that the tumor inhibition rates of the 0.5 mg / kg × 50 J / cm 2 group, 1 mg / kg × 50 J / cm 2 group and 2 mg / kg × 50 J / cm 2 group were 59.97%, 66.24% and 83.24% respectively. That is, when the light dose remained unchanged, the tumor inhibition rate increased with the increase of the drug dose. The tumor inhibition rates of the 1 mg / kg × 25 J / cm 2 group, 1 mg / kg × 50 J / cm 2 group and 1 mg / kg × 100 J / cm 2 group were 68.87%, 66.24% and 74.21% respectively. That is, when the drug dose remained unchanged, the tumor inhibition rate increased with the increase of the light dose. The 2 mg / kg × 100 J / cm 2 group had the best tumor inhibition effect, and the tumor inhibition rate reached 100%, which was greater than 87.40% of the tumor inhibition effect of the positive control group (Hippoifen 6 mg / kg × 100 J / cm 2 ).

[0119] The results of the relative tumor proliferation rate showed that the 0.5 mg / kg × 50 J / cm 2 group, 1 mg / kg × 50 J / cm 2 group and 2 mg / kg × 50 J / cm 2The relative tumor proliferation rates of the groups were 35.94, 30.96, and 14.38 respectively. That is, when the light dose remained unchanged, the relative tumor proliferation rate decreased with the increase of the administration dose. 1 mg / kg × 25 J / cm 2 group, 1 mg / kg × 50 J / cm 2 group, and 1 mg / kg × 100 J / cm 2 The relative tumor proliferation rates of the groups were 29.92, 30.96, and 25.49 respectively. That is, when the dose remained unchanged, the relative tumor proliferation rate decreased with the increase of the light dose. The tumor suppression effect of the 2 mg / kg × 100 J / cm 2 group was the best, and the relative tumor proliferation rate was 0, which was less than the relative tumor proliferation rate of 11.42 in the positive control group.

[0120] In summary, the chlorin-based photodynamic photosensitizer of the present invention shows great potential for photodynamic tumor treatment.

[0121] Example 6: Determination of the singlet oxygen yield of the chlorin-based photodynamic photosensitizer

[0122] 1. Materials and methods

[0123] ① Main experimental instruments

[0124] The full-wavelength scanning multi-functional reader is a product of Thermo Company, the electronic balance is a product of the Swiss METTLER TOLEDO Group, and the pure water system is a product of Millipore Company. The 660 nm semiconductor laser (BWT-660-800) is equipped with a Microlens optical fiber, a laser wavelength meter (THORLABS), and a laser power meter (PM310D).

[0125] ② Test method

[0126] The detection of the singlet oxygen yield in the aqueous solution adopts the SOSG oxidation method. SOSG is a water-soluble fluorescent probe, which can undergo an internal oxidation reaction with singlet oxygen in the aqueous solution to produce SOSG-EP. Under the excitation of a 488 nm wavelength, SOSG-EP can emit strong green fluorescence with a wavelength of 525 nm. Detecting the green fluorescence intensity in the aqueous solution can reflect the generation of singlet oxygen in the reaction system.

[0127] ③ Experimental steps

[0128]

[0129] 2. Experimental results

[0130] The experiment observed the effects of different light doses (20, 15, 10, 5 J / cm 2 ) on the singlet oxygen yield of Compound I-1, and the results are shown in the appendix Figure 15As shown, in the high-dose group of Compound I-1 (18 μg / ml), within the range of 5 - 20 J / cm 2 , the singlet oxygen yields of each light-dose group were close, being 318.0, 316.0, 324.1, and 310.1 in sequence. At 5 J / cm 2 , the yield of singlet oxygen had reached >90% of the maximum value of singlet oxygen. In the low-dose group of Compound I-1 (3 μg / ml), within the range of 5 - 15 J / cm 2 , the singlet oxygen yield increased with the increase of light irradiation dose, being 79.8, 123.5, 170.8, and 152.8 respectively, and reached the maximum value at 15 J / cm 2 . Further increasing the light dose could not further increase the singlet oxygen yield. The above results indicate that the singlet oxygen yield has reached the maximum value at a light irradiation dose of 15 J / cm 2 and above.

[0131] The experiment observed the production of singlet oxygen at different doses of Compound I-1 (0.5 - 18 μg / ml). The results are as shown in the appendix Figure 16 . When the detection concentration of SOSG was 2 μM and the light dose was 5 J / cm 2 in the detection mode, the singlet oxygen yield of Compound I-1 was proportional to the concentration of Compound I-1. The singlet oxygen yields of the Compound I-1 groups with 18, 9, 6, 3, 1, and 0.5 were 329.4, 256.5, 171.3, 79.8, 34.0, and 14.2 respectively under light stimulation. The above results indicate that Compound I-1 has a strong ability to produce singlet oxygen at a relatively low light irradiation dose, and its yield is proportional to the concentration of Compound I-1.

Claims

1. A chlorin-based photodynamic photosensitizer and its pharmaceutically acceptable salts, characterized in that The structural formula is as shown in Formula I: Wherein, R is an unsubstituted C1-6 straight-chain or branched-chain alkyl group, or a C1-6 straight-chain or branched-chain alkyl group substituted by halogen, hydroxyl, amino or alkoxy group, and R is methyl, ethyl or hydroxyethyl.

2. The chlorophyllide-based photodynamic photosensitizer according to claim 1 and its pharmaceutically acceptable salt, characterized in that: The pharmaceutically acceptable salts are sodium salts, potassium salts or lithium salts.

3. A preparation method of the chlorin-based photodynamic photosensitizer according to claim 1, characterized in that, It includes the following steps: (1) Dissolve chlorin e6 in an alcohol solvent, add concentrated sulfuric acid under argon protection, and heat and react at 50-60 °C. After the reaction is completed, the reaction solution is concentrated, extracted, washed, purified and dried to obtain Compound 1; (2) Add Compound 1 and an aqueous sodium hydroxide solution to ethanol, heat and react at 40-80 °C under argon protection. After the reaction is completed, slowly add a dilute hydrochloric acid solution to the reaction solution to adjust the pH to 1.0-5.0, filter to obtain a filter cake, wash it with deionized water and dry it to obtain Compound 2, which is directly used in the next step without purification; (3) Compound 2 undergoes a decarboxylation reaction under the conditions of a catalyst and heating. After the reaction is completed and cooled, a crude product is obtained; the crude product is purified by silica gel column chromatography to obtain a chlorin-based photodynamic photosensitizer. The structural formula of the chlorin e6 is: The structural formula of the Compound 1 is: The structural formula of the Compound 2 is:

4. The preparation method of the chlorin-based photodynamic photosensitizer according to claim 3, wherein, The alcohol solvent in the step (1) is a substituted or unsubstituted C1-6 straight-chain or branched-chain alcohol, including monohydric alcohols such as methanol, ethanol, n-butanol, isobutanol and dihydric alcohols such as ethylene glycol, propylene glycol.

5. The preparation method of the chlorin-based photodynamic photosensitizer according to claim 3, characterized in that, The heating reaction temperature in the step (2) is 40-80 °C.

6. The preparation method of the chlorin-based photodynamic photosensitizer according to claim 3, characterized in that, The dilute hydrochloric acid solution in the step (2) adjusts the pH to 1.0-5.

0.

7. The preparation method of the chlorin-based photodynamic photosensitizer according to claim 3, wherein, The catalyst in the step (3) is silver powder.

8. The preparation method of the chlorin-based photodynamic photosensitizer according to claim 3, characterized in that, The heating temperature in the step (3) is 100-200 °C; the heating time is 10-60 min; the eluent for silica gel column chromatography separation is dichloromethane / methanol with a volume ratio of 100:

1.

9. Use of the chlorin-based photodynamic photosensitizer according to any one of claims 1-3 and its pharmaceutically acceptable salts in the preparation of an antitumor drug for photodynamic therapy, wherein the antitumor drug is used for the treatment of gastric cancer, liver cancer, lung cancer, epithelial cancer or nasopharyngeal cancer.

10. Use of the chlorophyllide-based photodynamic photosensitizer according to any one of claims 1-3 and its pharmaceutically acceptable salts in the preparation of a medicament for treating benign vascular diseases or a medicament for treating condyloma acuminata, wherein, The benign vascular diseases include age-related macular degeneration and port-wine stain.

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