Photosensitizer for endogenous hydrogen sulfide activation and visible light excitation as well as preparation method and application of photosensitizer

Abstract

The invention discloses a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation as well as a preparation method and application thereof, and relates to the technical field of medical photosensitizers. According to the photosensitizer provided by the invention, BA and NIS react to obtain a BODIPY compound I2BA substituted by double atoms I, so that the intersystem crossing capability is enhanced, and the yield of active oxygen is increased; a target photosensitizer is obtained by introducing a tetraazobenzene ring Cyclone into the para-position of a benzene ring and combining copper ions Cu < 2 + >. The prepared photosensitizer has good light stability and water solubility, can be activated by endogenous hydrogen sulfide in cancer cells, can generate singlet oxygen (1O2) under the irradiation of green light (520 nm), shows excellent photodynamic activity to tumor cells and low dark toxicity to normal cells, has a high photodynamic activity index (PI value), and can be used as a photosensitizer for cancer cells. The compound is successfully used for photodynamic inhibition and elimination of tumors on HCT116 tumor-bearing mice.

Description

Technical Field

[0001] The invention relates to the technical field of medical photosensitizers, and in particular to a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation, and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is an emerging method for treating malignant tumors. Compared with traditional methods such as surgery, chemotherapy and radiotherapy, it has better time selectivity (i.e., it is effective when illuminated and ineffective when not illuminated) and spatial selectivity (i.e., the tumor irradiation area is effective, and the surrounding normal tissues are not irradiated with no toxic effects or very little toxic side effects). Therefore, photodynamic therapy is often considered to be a non-invasive therapy. Photosensitizer, oxygen and light are the three elements of photodynamic therapy, among which photosensitizer is the core. The current research hotspot focuses on the development of new photosensitizers with good biocompatibility, which can efficiently produce reactive oxygen and target cancer cells or cancer tissues, and ultimately play an effective anti-tumor role with small toxic side effects.

[0003] BODIPY dyes have the advantages of large molar absorption coefficient, good photostability and easy structural modification, and are widely used in the design and preparation of new fluorescent probes. When heavy atoms such as I are symmetrically introduced at the 3 and 5 positions of BODIPY dyes, the intersystem crossing (ISC) time is greatly enhanced, thereby transferring more oxygen ( 3 O 2 ) becomes singlet oxygen ( 1 O 2 ), increasing the ROS production rate; then attacking DNA or certain proteins, causing cancer cell apoptosis, necrosis or ferroptosis, etc. At present, there are many iodine-containing BODIPY-type photosensitizers developed based on the above principle, but they often show strong dark toxicity due to the lack of targeting to cancer cells (tissues).

[0004] In order to enhance the specific recognition of cancer cells or tissues and reduce the toxic side effects on normal tissues, masking-activated photosensitizers have great potential. 2 As a gas signaling molecule, H is overexpressed in some cancer cells, especially colorectal cancer and brain glioma. 2 S-specifically activated masked photosensitizers have broad application prospects for improving the targeting of photosensitizers and reducing toxic side effects. Summary of the invention

[0005] The present invention provides a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation, and a preparation method and application thereof. The photosensitizer has good light stability and water solubility, can be activated by endogenous hydrogen sulfide in cancer cells, and can generate singlet oxygen ( 1O 2 ), showing excellent photodynamic activity to tumor cells and low dark toxicity to normal cells, with a high photodynamic activity index (PI value). Based on the above effects, this photosensitizer can be used for photodynamic inhibition and elimination of tumors, to achieve the purpose of improving tumor treatment effects, reducing toxic side effects, and overcoming cancer cell resistance. This is achieved specifically through the following technologies.

[0006] In a first aspect of the present invention, a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation is provided, and its structural formula is shown in Formula I;

[0007]

[0008] Formula I

[0009] Among them, the group R 1 It is the acid ion group of water-soluble copper salt.

[0010] Furthermore, the water-soluble copper salt is one or more of copper perchlorate, copper nitrate, copper sulfate, copper chloride, and copper acetate.

[0011] The second aspect of the present invention also provides a method for preparing a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation, the preparation method being carried out under a nitrogen or inert gas atmosphere and comprising the following steps:

[0012] Take 8-[4-(chloromethyl)benzene]-4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indenene (referred to as "BA") and N-iodosuccinimide (NIS) and add them to the first organic solvent until the reaction is complete, wash, collect and purify to obtain compound I 2 BA, the compound I 2 BA is dissolved in a second organic solvent to obtain a first mixed system;

[0013] Dissolve 1,4,7,10-tetraazacyclododecane in the third organic solvent and add K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 At least one of the above is added dropwise to the first mixed system until the reaction is complete, and then collected, washed and purified to obtain a skeleton intermediate compound I. 2 BA-Cyclen;

[0014] The skeleton intermediate compound I 2BA-Cyclen and the water-soluble copper salt are dissolved in a fourth organic solvent until the reaction is complete, and then collected and purified to obtain the photosensitizer for endogenous hydrogen sulfide activation and visible light excitation.

[0015] Optionally, the first organic solvent, the second organic solvent, the third organic solvent and the fourth organic solvent are respectively one or more of dichloromethane, chloroform, methanol, ethanol and acetonitrile.

[0016] Furthermore, the amount of the substance of 8-[4-(chloromethyl)benzene]-4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indenene and N-iodosuccinimide is 1:(3-8).

[0017] Before the subsequent step reaction, the compound I 2 The BA solid is dissolved in the second organic solvent to obtain a first mixed system.

[0018] Furthermore, the compound I 2 The molar ratio of BA, 1,4,7,10-tetraazacyclododecane and potassium carbonate is 1:(2-6):(3-6).

[0019] Furthermore, the skeleton intermediate compound I 2 BA-Cyclen and water-soluble copper salts 2+ The molar ratio is 1:(0.9-1.2).

[0020] The photosensitizer provided by the present invention is firstly reacted with 8-[4-(chloromethyl)benzene]-4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indenyl (BA) and N-iodosuccinimide (NIS) to obtain a BODIPY compound (I 2 BA), enhancing the L-intersystem crossing (ISC) ability and increasing the L-reactive oxygen species (ROS) yield; introducing a tetranitrogen ring Cyclen at the para position of the benzene ring, and finally combining Cu 2+ , making it have good photostability and water solubility, and can be activated by endogenous hydrogen sulfide in cancer cells to produce singlet oxygen ( 1 O 2 ), showing excellent photodynamic activity against tumor cells and low dark toxicity against normal cells, with a high photodynamic activity index (PI value), and was successfully used for photodynamic inhibition and elimination of tumors in HCT116 tumor-bearing mice.

[0021] The third aspect of the present invention further provides a skeleton intermediate compound, which is used to prepare the photosensitizer for endogenous hydrogen sulfide activation and visible light excitation according to claim 1; the chemical structural formula of the skeleton intermediate compound is shown in Formula II;

[0022]

[0023] Formula II.

[0024] The skeleton intermediate compound shown in the above formula II provided by the present invention contains a heavy atom I modification and has Cu 2+ The intermediate of coordination site Cyclen. This intermediate can be used to bind Cu 2+ The corresponding complex photosensitizer is obtained, which has good endogenous hydrogen sulfide activation and visible light excitation effects.

[0025] In a fourth aspect, the present invention further provides a use of any one of the above-mentioned photosensitizers, or a photosensitizer prepared by any one of the above-mentioned preparation methods, for preparing anticancer drugs; the photosensitizer for endogenous hydrogen sulfide activation and visible light excitation consumes the endogenous hydrogen sulfide of cancer cells and is activated to produce singlet oxygen under green light.

[0026] Compared with the prior art, the present invention is beneficial in that:

[0027] 1. The photosensitizer provided by the present invention has good stability and biocompatibility; it has extremely strong cytotoxic activity under light, and 2 IC of BA-Cyclen-Cu(Ⅱ) against HCT116 at 2 mg / kg 50 As low as 14.8 nM, and almost no toxicity under dark conditions, IC 50 Greater than 240 μM, photodynamic index (PI) greater than 15000.

[0028] 2. When animal experiments were conducted on tumor-bearing mice, the photosensitizer provided by the present invention could consume endogenous hydrogen sulfide in colorectal cancer HCT116 cells, and could strongly inhibit the growth of HCT116 colorectal cancer tumors in tumor-bearing mice, without major organ toxicity and chronic toxicity. After testing, the inhibition rate of small tumors reached 95%, and the inhibition rate of malignant large tumors also reached 35%. The photosensitizer can also effectively inhibit the growth of oxaliplatin-resistant colorectal cancer tumors in tumor-bearing mice, with an inhibition rate of 95% for small tumors and 33% for malignant large tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Preparation of photosensitizer I for Example 2Flowchart of BA-Cyclen-Cu(Ⅱ).

[0030] Figure 2 For the skeleton intermediate compound I 2 BA-Cyclen H NMR spectrum (CDCl 3 , 600 MHz).

[0031] Figure 3 For the skeleton intermediate compound I 2 BA-Cyclen carbon NMR spectrum (CDCl 3 , 100 MHz).

[0032] Figure 4 For the skeleton intermediate compound I 2 High-resolution mass spectrum of BA-Cyclen.

[0033] Figure 5 Target photosensitizer I 2 High-resolution mass spectrum of BA-Cyclen-Cu(Ⅱ).

[0034] Figure 6 For the skeleton intermediate compound I 2 BA-Cyclen and photosensitizer I 2 UV-visible absorption spectrum and fluorescence emission spectrum of BA-Cyclen-Cu(Ⅱ). Figure 6 A represents I 2 BA-Cyclen, I 2 BA-Cyclen-Cu(Ⅱ) and I 2 BA-Cyclen-Cu(Ⅱ)+Na 2 UV-visible absorption spectrum of S; Figure 6 B represents I 2 BA-Cyclen, I 2 BA-Cyclen-Cu(Ⅱ) and I 2 BA-Cyclen-Cu(Ⅱ)+Na 2 Fluorescence emission spectra after S.

[0035] Figure 7 Compound I 2 BA-Cyclen and photosensitizer I 2 The cytotoxic activity of BA-Cyclen-Cu(Ⅱ) on HCT116 colorectal cancer cells (CCK-8). Figure 7 A is the culture result under dark conditions, Figure 7 B is the culture result under light conditions.

[0036] Figure 8For the skeleton intermediate compound I 2 BA-Cyclen and photosensitizer I 2 The results of the BA-Cyclen-Cu(Ⅱ) tolerance experiment on HCT116 tumor-bearing mice (female and male).

[0037] Fig. 9 For the skeleton intermediate compound I 2 BA-Cyclen and photosensitizer I 2 The experimental results of BA-Cyclen-Cu(Ⅱ) inhibiting the proliferation of tumors (tumors) in HCT116 tumor-bearing mice.

[0038] Fig.10 For the skeleton intermediate compound I 2 BA-Cyclen and photosensitizer I 2 The experimental results of BA-Cyclen-Cu(Ⅱ) inhibiting the proliferation of tumors (large tumors, highly malignant) in HCT116 tumor-bearing mice.

[0039] Fig.11 For the skeleton intermediate compound I 2 BA-Cyclen and photosensitizer I 2 Results of the chronic toxicity study of BA-Cyclen-Cu(Ⅱ) on HCT116 tumor-bearing mice (large tumor, high malignancy) (without light).

[0040] Fig.12 Photosensitizer I 2 The experimental results of BA-Cyclen-Cu(Ⅱ) inhibiting the proliferation of tumors (tumors) in oxaliplatin-resistant HCT116 tumor-bearing mice.

[0041] Fig.13 Photosensitizer I 2 The experimental results of BA-Cyclen-Cu(Ⅱ) inhibiting the proliferation of tumors (large tumors, high malignancy) in oxaliplatin-resistant HCT116 tumor-bearing mice. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In some embodiments of the present invention, the photosensitizer provided has a structural formula as shown in Formula I;

[0044]

[0045] Formula I

[0046] Among them, the group R 1 It is the acid ion group of water-soluble copper salt.

[0047] In some embodiments of the present invention, the water-soluble metal salt refers to a water-soluble metal salt, including but not limited to one or more of perchlorates, nitrates, sulfates, hydrochlorides, and acetates.

[0048] In some embodiments of the present invention, the preparation method of the photosensitizer is carried out under nitrogen or inert gas atmosphere, and the main steps are: taking 8-[4-(chloromethyl)benzene]-4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indenene (referred to as BA) and N-iodosuccinimide (NIS) and adding them to a first organic solvent until the reaction is complete, washing, collecting and purifying to obtain compound I 2 BA, the compound I 2 BA is dissolved in a second organic solvent to obtain a first mixed system;

[0049] Dissolve 1,4,7,10-tetraazacyclododecane in the third organic solvent and add K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 At least one of the above is added dropwise to the first mixed system until the reaction is complete, and then collected, washed and purified to obtain a skeleton intermediate compound I. 2 BA-Cyclen;

[0050] The skeleton intermediate compound I 2 BA-Cyclen and the water-soluble copper salt are dissolved in a fourth organic solvent until the reaction is complete, and then collected and purified to obtain the photosensitizer for endogenous hydrogen sulfide activation and visible light excitation.

[0051] In the preparation method of the photosensitizer provided in some embodiments of the present invention, compound I 2 The preparation method of BA is as follows: 8-[4-(chloromethyl)benzene]-4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indenene (BA) and N-iodosuccinimide (NIS) are added to a first organic solvent in a proportion, and stirred to react at room temperature under nitrogen protection (the reaction progress is monitored by TCL) until the reaction is complete; the reaction system is washed with distilled water for several times to remove excess NIS; the organic phase is collected, anhydrous sodium sulfate is added and dried overnight; after suction filtration, the crude product is evaporated and concentrated to obtain a crude product (red viscous solid); and finally, the pure compound I is purified by silica gel column chromatography. 2BA (purple powder).

[0052] In the preparation method of the photosensitizer provided in some embodiments of the present invention, the amount of BA and N-iodosuccinimide is 1:(3-8).

[0053] In the preparation method of the photosensitizer provided in some embodiments of the present invention, the skeleton intermediate compound I 2 The preparation method of BA-Cyclen is as follows: 2 The first mixed system of BA and 1,4,7,10-tetraazacyclododecane (Cyclen); Cyclen is placed in a three-necked flask and dissolved in a third organic solvent, followed by the addition of K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 At least one of the above is stirred at 30°C under nitrogen protection, and slowly dripped into the first mixed system, and continued to stir the reaction; cooled to room temperature, filtered to remove K 2 CO 3 , evaporate all solvent components in the reaction system, dissolve the residue in a fifth organic solvent, and wash with saturated brine; collect the organic layer and dry it over anhydrous sodium sulfate overnight; filter, evaporate and concentrate, and purify by neutral alumina column chromatography (using dichloromethane / methanol = 29:1) to obtain compound I 2 BA-Cyclen.

[0054] In the preparation method of the photosensitizer provided in some embodiments of the present invention, the compound I 2 The molar ratio of BA, 1,4,7,10-tetraazacyclododecane and potassium carbonate is 1:(2-6):(3-6).

[0055] In the preparation method of the photosensitizer provided in some embodiments of the present invention, the skeleton intermediate compound I is used 2 Preparation of photosensitizer I by BA-Cyclen 2 The BA-Cyclen-Cu(Ⅱ) method is as follows: the skeleton intermediate compound I 2 BA-Cyclen and copper nitrate trihydrate were placed in a single-necked flask in proportion, and a fourth organic solvent was added to dissolve the mixture, and the mixture was stirred and reacted at 60°C for 4 h under nitrogen protection. The photosensitizer was obtained after vacuum rotary evaporation.

[0056] In the preparation method of the photosensitizer provided in some embodiments of the present invention, the preparation method of 8-[4-(chloromethyl)benzene]-4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indenyl and N-iodosuccinimide (abbreviated as "BA") can refer to the preparation method of compound "B8" provided in the supplementary materials section of the literature "A high-resolution method to assess cell multinucleation with cytoplasm-localized fluorescent probes" (Hui Wen, Qinghua Cui, Hui Meng, Fangfang Lai, Shufang Wang, Xiang Zhang, Xiaoguang Chen, Huaqing Cui and Dali Yin, Analyst, 2016, 141, 4010-4013): "B8 (351 mg, 17.8%) was obtained from 4-(chloromethyl)benzoyl chloride (1.0 g, 5.32 mmol) and 2,4-dimethyl-pyrrole (1.01 g, 10.6 mmol) as red powder”.

[0057] In the preparation method of the photosensitizer provided in some embodiments of the present invention, the skeleton intermediate compound I 2 BA-Cyclen and water-soluble copper salts 2+ The molar ratio is 1:(0.9-1.2).

[0058] In the preparation method of the photosensitizer provided in some embodiments of the present invention, the first organic solvent, the second organic solvent, the third organic solvent and the fourth organic solvent can be one or more of dichloromethane, chloroform, methanol, ethanol and acetonitrile, respectively.

[0059] In some embodiments of the present invention, in the following embodiments, 2,4-dimethylpyrrole, boron trifluoride ether solution, dichloromethane, N-iodosuccinimide (hereinafter referred to as NIS), 1,4,7,10-tetraazacyclododecane (hereinafter referred to as Cyclen), copper nitrate trihydrate and other reagents were purchased from Shanghai Titan Technology Co., Ltd. Potassium carbonate and methanol were purchased from Sinopharm Group. Nude mice were purchased from Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing.

[0060] Example 1

[0061] The preparation method of the photosensitizer provided in this embodiment is as follows: Figure 1 As shown, compound I is first synthesized 2 BA, and then synthesize the skeleton intermediate compound I 2 BA-Cyclen, finally synthesized photosensitizer I 2 BA-Cyclen-Cu(Ⅱ) finished product; the specific steps include:

[0062] S1. Compound I 2 Synthesis of BA

[0063] BA (256 mg, 0.69 mmol) was dissolved in 30 mL of dichloromethane (the first organic solvent) and added to a 100 mL three-necked flask under N 2 Stir at room temperature under protection.

[0064] Weigh NIS (929 mg, 4.13 mmol) and add it into the three-necked flask. Continue stirring to react. During the reaction, monitor the reaction progress by TLC (Thin Layer Chromatography) until the BA reaction is complete.

[0065] The reaction system was washed with distilled water three times to remove excess NIS;

[0066] The organic phase was collected and dried overnight over anhydrous sodium sulfate;

[0067] Filtration and evaporation to obtain a red viscous solid (crude product);

[0068] The crude product was purified by silica gel column chromatography with ethyl acetate / methanol = 20:1 as eluent to obtain a purple-red powder I 2 BA 348 mg, yield 81.1%. Mass spectrometry analysis results are as follows:

[0069] 1 H NMR (400 MHz, CDCl 3 ) δ 7.56 (d, J= 8.2 Hz, 2H), 7.26 (d, J= 8.2 Hz, 2H), 4.67(s, 2H), 2.65(s, 6H), 1.39(s, 6H). 13 C NMR (100 MHz, CDCl 3 ) δ 157.05,145.23, 140.59, 139.32, 134.83, 131.23,129.50, 128.33, 85.75, 45.37, 17.07,16.02. HRMS(ESI),C 20 H 18 BClF 2I 2 N 2 , m / z:623.9302, [I 2 BA+H] + (Theoretical value: 623.9309).

[0070] S2. Framework intermediate compound I 2 Synthesis of BA-Cyclen.

[0071] The compound I 2 BA (300 mg, 0.48 mmol) was dissolved in 10 mL of the second organic solvent (chloroform) to obtain the first mixed system;

[0072] In N 2 Cyclen (580 mg, 3.37 mmol) was dissolved in 30 mL of chloroform (the third organic solvent) under atmosphere, and K 2 CO 3 (591 mg, 4.28 mmol);

[0073] Slowly add the first mixed system to the above system, and stir and react at 30° C. for 16 h after the addition is complete;

[0074] Filter out K 2 CO 3 , evaporate the solvent, dissolve the residue in 50 mL of dichloromethane, and wash with saturated brine (3×30 mL); collect the organic layer, dry it over anhydrous sodium sulfate overnight; filter, remove the solvent by rotary evaporation, and purify it by column chromatography with neutral alumina in a ratio of dichloromethane / methanol = 29:1 to obtain a dark red powder skeleton intermediate compound I 2 BA-Cyclen 184 mg, yield 48%.

[0075] like Figure 2-4 The skeleton intermediate compound I shown 2 The nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum of BA-Cyclen show the following analysis results: 1 H NMR (600 MHz, CDCl 3 ) δ 7.51 (d, J= 8.1 Hz, 2H), 7.24 (d, J=8.2 Hz, 2H), 3.86 (s, 2H), 2.92 (t, 4H), 2.79 (s, 6H), 2.74 (t, 6H), 2.64 (s, 6H), 1.39 (s, 6H). 13 C NMR (100 MHz, CDCl 3) δ 156.90, 145.18, 141.15, 140.41,133.85, 131.30,130.27, 128.07, 85.87, 60.37, 51.83, 48.46, 46.80, 46.03,17.20, 16.14. HRMS(ESI), C28H37BF2I 2 N 6 , m / z:761.1290, [I 2 BA-Cyclen+H] + (theoretical value: 761.1230); m / z: 381.0688, [I 2 BA-Cyclen+2H] 2+ (Theoretical value: 381.0688).

[0076] S3, Photosensitizer I 2 Synthesis of BA-Cyclen-Cu(Ⅱ).

[0077] The skeleton intermediate compound I 2 BA-Cyclen (130 mg, 0.16 mmol) and copper nitrate trihydrate (Cu(NO) 3 , 61 mg, 0.16 mmol) was dissolved in methanol (the fourth organic solvent, 30 mL) and heated under N 2 The reaction was stirred at 60°C for 4 h under protection. After vacuum rotary evaporation, a reddish brown solid I was obtained. 2 BA-Cyclen-Cu(Ⅱ) 131 mg, i.e. finished photosensitizer, with a yield of 93%.

[0078] The final prepared photosensitizer I 2 The chemical structure of BA-Cyclen-Cu(Ⅱ) is shown in the following formula Ⅲ.

[0079]

[0080] Formula III

[0081] like Figure 5 Photosensitizer I 2 High resolution mass spectrum of BA-Cyclen-Cu(Ⅱ). HRMS(ESI) m / z:411.5271, [I 2 BA-Cyclen+Cu] 2+ (theoretical value: 411.5258); m / z: 868.0515, [I 2 BA-Cyclen+Cu+HCOO] + (Theoretical value: 868.0497).

[0082] Test Example 1: UV-Vis Absorption Spectrum and Fluorescence Spectrum

[0083] UV-visible absorption spectra and fluorescence spectra were measured in PBS buffer (10 mM, 5% DMSO, pH 7.4). 2 BA-Cyclen and I 2 The concentration of BA-Cyclen-Cu(Ⅱ) was 40 μM.

[0084] like Figure 6 As shown, Figure 6 A represents I 2 BA-Cyclen, I 2 BA-Cyclen-Cu(Ⅱ) and I 2 BA-Cyclen-Cu(Ⅱ)+ Na 2 UV-visible absorption spectrum after S; Figure 6 B represents I 2 BA-Cyclen, I 2 BA-Cyclen-Cu(Ⅱ) and I 2 BA-Cyclen-Cu(Ⅱ)+Na 2 Fluorescence emission spectra after S.

[0085] from Figure 6 As can be seen in A, compound I 2 BA-Cyclen and I 2 The maximum absorption peak of BA-Cyclen-Cu(Ⅱ) is at 525 n. Figure 6 As can be seen in B, I 2 BA-Cyclen and I 2 The maximum emission peak of BA-Cyclen-Cu(Ⅱ) is located at 578nm, and the ligand I 2 BA-Cyclen complex I 2 The fluorescence intensity of BA-Cyclen-Cu(Ⅱ) is strong. 2 BA-Cyclen-Cu(Ⅱ) with Na 2 After S, the fluorescence intensity obviously recovered to a certain extent, indicating that S 2- The addition of I 2 BA-Cyclen-Cu(Ⅱ) reaction, taking away Cu 2+ , occurs 2 BA-Cyclen-Cu(Ⅱ) to I 2 Transformation of BA-Cyclen.

[0086] Test Example 2: I 2BA-Cyclen and I 2 CCK-8 in colorectal cancer HCT116 cells under light / dark treatment with BA-Cyclen-Cu(Ⅱ)

[0087] Colorectal cancer HCT116 cells were cultured and incubated in a 96-well plate at 37°C in the dark or under light conditions. The dark conditions were 3 h and 48 h, respectively. The light conditions were 2.5 h + 0.5 h light and 47.5 h + 0.5 h light, respectively.

[0088] The results are as follows Figure 7 As shown in Table 1, Figure 7 A is the culture result under dark conditions, Figure 7 B is the culture result under light conditions. Figure 7 From the experimental results in Table 1, we can see that I 2 BA-Cyclen-Cu(Ⅱ) exhibited excellent PDT effect under light conditions and had weak toxic activity under dark conditions, with its PI reaching 15723 at 48 h; 2 BA-Cyclen compared to I 2 BA-Cyclen-Cu(Ⅱ) is weaker, with a PI value of 1689.

[0089] Table 1 I 2 BA-Cyclen and I 2 IC of BA-Cyclen-Cu(Ⅱ) 50 and PI value

[0090]

[0091] Test Example 3: I 2 BA-Cyclen and I 2 The inhibitory effect of BA-Cyclen-Cu(Ⅱ) on tumors (tumors) in HCT116 tumor-bearing mice.

[0092] Mice were given tumors 7 days in advance and when the tumor volume reached about 80 mm 3 The administration method is as follows: (1) In order to verify I 2 BA-Cyclen and I 2 The toxicity and side effects of BA-Cyclen-Cu(Ⅱ) on HCT116 tumor-bearing mice were all 60 mg / kg; (2) In order to verify the normal dosage, I 2 BA-Cyclen and I 2 The toxicity and side effects of BA-Cyclen-Cu(Ⅱ) on HCT116 tumor-bearing mice, as well as its inhibitory effect on tumors (tumors), I 2 BA-Cyclen and I2 The dosage of BA-Cyclen-Cu(Ⅱ) was 2 mg / kg.

[0093] After the drug administration, the nude mice were returned to the cage. If there were no obvious abnormalities in the nude mice, photodynamic therapy was performed 1 hour later. During the irradiation treatment, the laser output power was 50 mW and the irradiation time was 20 min. The treatment was performed once every 2 days, for a total of 7 times. The therapeutic effect of the drug was evaluated by the tumor inhibition rate, where the tumor inhibition rate = [(average tumor volume of the blank group - average tumor volume of each treatment group) / average tumor volume of the blank group] × 100%. The blank group was the control group without drug administration. The experimental results are as follows Figure 8-9 shown.

[0094] from Figure 8 (The left picture is a female mouse, and the right picture is a male mouse) It can be seen that when the dosage is 60 mg / kg, I 2 BA-Cyclen caused a significant decrease in the body weight of mice and showed toxic side effects, while I 2 BA-Cyclen-Cu(Ⅱ) had almost no effect on the weight of mice, indicating that the I 2 BA-Cyclen-Cu(Ⅱ) has almost no toxic side effects; under the same light conditions, I 2 BA-Cyclen-Cu(Ⅱ) showed a better 2 BA-Cyclen has better tumor inhibition effect.

[0095] from Fig. 9 It can be seen that at the normal dosage of 2 mg / kg, I 2 BA-Cyclen and I 2 BA-Cyclen-Cu(Ⅱ) had no effect on the weight of mice and had significant inhibitory effects on tumors (small tumors). 2 The inhibitory effect of BA-Cyclen-Cu(Ⅱ) was more significant.

[0096] Test Example 4: I 2 The inhibitory effect of BA-Cyclen-Cu(Ⅱ) on tumors (large tumors) in HCT116 tumor-bearing mice.

[0097] Mice were given tumors 20 days in advance and the tumor volume reached about 200 mm. 3 When the drug was injected intratumorally. 2The dosage of BA-Cyclen-Cu(Ⅱ) was 2 mg / kg. After administration, the nude mice were returned to the cage. If there were no obvious abnormalities in the nude mice, photodynamic therapy was performed 1 hour later. During irradiation therapy, the laser output power was 50 mW and the irradiation time was 20 min. Treatment was performed once every 3 days for a total of 8 times. The therapeutic effect of the drug was evaluated by the tumor inhibition rate.

[0098] like Fig.10 As shown, it can be seen that the drug is administered by intratumoral injection. 2 BA-Cyclen-Cu(Ⅱ) also has a certain degree of inhibitory effect on large tumors with high malignancy at a dose of 2 mg / kg and under light conditions. At the same time, H&E staining analysis shows that it is almost non-toxic to major organs such as the heart, liver, spleen, lungs, and kidneys.

[0099] Test Example 5: I 2 BA-Cyclen and I 2 Toxicity of BA-Cyclen-Cu(Ⅱ) to HCT116 tumor-bearing mice (large tumors).

[0100] The experimental conditions were basically the same as those of Experimental Example 4, except that the drug administration method was changed to intraperitoneal injection, and no light was applied.

[0101] like Fig.11 As shown, it can be seen that the drug is administered by intraperitoneal injection. 2 BA-Cyclen and I 2 BA-Cyclen-Cu(Ⅱ) had a weak inhibitory effect on highly malignant tumors at a dose of 2 mg / kg and in the dark, indicating that proper illumination is one of the key factors for photosensitizers to produce efficient anticancer activity. H&E staining also further confirmed that under dark conditions, I 2 BA-Cyclen and I 2 BA-Cyclen-Cu(Ⅱ) is non-toxic to the heart, liver, spleen, lungs and kidneys.

[0102] Test Example 6: I 2 The inhibitory effect of BA-Cyclen-Cu(Ⅱ) on tumors (tumors) in oxaliplatin-resistant HCT116 tumor-bearing mice.

[0103] Mice were given tumors 7 days in advance and then when the tumor volume reached about 80 mm 3 When the drug is injected into the tumor, 2The dosage of BA-Cyclen-Cu(Ⅱ) was 2 mg / kg. After administration, the nude mice were returned to the cage. If there was no obvious abnormality in the nude mice, photodynamic therapy was performed 1 hour later. The laser output power was 50 mW and the irradiation time was 20 minutes. The treatment was performed once every 3 days for a total of 8 times. The therapeutic effect of the drug was evaluated by the tumor inhibition rate.

[0104] The results are as follows Fig.12 As shown, I 2 BA-Cyclen-Cu(Ⅱ) has a significant inhibitory effect on tumors (tumors) in oxaliplatin-resistant HCT116 tumor-bearing mice without affecting the body weight of the mice.

[0105] Test Example 7: I 2 The inhibitory effect of BA-Cyclen-Cu(Ⅱ) on tumors (large tumors) in oxaliplatin-resistant HCT116 tumor-bearing mice.

[0106] Mice were given tumors 20 days in advance and then the tumors reached a volume of approximately 200 mm. 3 When the drug is injected into the tumor, 2 The dosage of BA-Cyclen-Cu(Ⅱ) was 2 mg / kg. After administration, the nude mice were returned to the cage. If there was no obvious abnormality in the nude mice, photodynamic therapy was performed 1 hour later. The laser output power was 50 mW and the irradiation time was 20 minutes. The treatment was performed once every 3 days for a total of 9 times. The therapeutic effect of the drug was evaluated by the tumor inhibition rate.

[0107] The results are as follows Fig.13 As shown, I 2 BA-Cyclen-Cu(Ⅱ) still has a significant inhibitory effect on tumors (large tumors) in oxaliplatin-resistant HCT116 tumor-bearing mice, and does not affect the weight of mice, indicating that it has basically no toxic side effects.

[0108] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A photosensitizer for endogenous hydrogen sulfide activation and visible light excitation, characterized in that: Its structural formula is shown in Formula Ⅰ; Formula I Wherein, the group R1 is an acid ion group of a water-soluble copper salt.

2. The photosensitizer for endogenous hydrogen sulfide activation and visible light excitation according to claim 1, characterized in that: The water-soluble copper salt is one or more of copper perchlorate, copper nitrate, copper sulfate, copper chloride and copper acetate.

3. A method for preparing a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation, characterized in that: The preparation method is carried out under nitrogen or inert gas atmosphere and comprises the following steps: Add 8-[4-(chloromethyl)benzene]-4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indenene and N-iodosuccinimide to a first organic solvent until the reaction is complete, wash, collect and purify to obtain a compound I2BA, and dissolve the compound I2BA in a second organic solvent to obtain a first mixed system; Dissolve 1,4,7,10-tetraazacyclododecane in a third organic solvent and add at least one of K2CO3, Na2CO3, and Cs2CO3, add the first mixed system dropwise until the reaction is complete, collect, wash and purify to obtain a skeleton intermediate compound I2BA-Cyclen; The skeleton intermediate compound I2BA-Cyclen and the water-soluble copper salt are dissolved in a fourth organic solvent until the reaction is complete, and then collected and purified to obtain the photosensitizer for endogenous hydrogen sulfide activation and visible light excitation.

4. The method for preparing a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation according to claim 3, characterized in that: The amount of substance of 8-[4-(chloromethyl)benzene]-4,4-difluoro-1,3,5,7-tetramethyl-4-boron-3a,4a-diaza-s-indenene and N-iodosuccinimide is 1:(3-8).

5. The method for preparing the photosensitizer for endogenous hydrogen sulfide activation and visible light excitation according to claim 3, characterized in that: The molar ratio of the compound I2BA, 1,4,7,10-tetraazacyclododecane and potassium carbonate is 1:(2-6):(3-6).

6. The method for preparing a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation according to claim 3, characterized in that: The skeleton intermediate compound I2BA-Cyclen and the water-soluble copper salt Cu 2+ The molar ratio is 1:(0.9-1.2).

7. A skeleton intermediate compound I2BA-Cyclen, characterized in that: The skeleton intermediate compound I2BA-Cyclen is used to prepare the photosensitizer for endogenous hydrogen sulfide activation and visible light excitation as described in claim 1; the chemical structural formula of the skeleton intermediate compound I2BA-Cyclen is shown in Formula II; Formula II.

8. A photosensitizer for endogenous hydrogen sulfide activation and visible light excitation according to claim 1 or 2, or a photosensitizer for endogenous hydrogen sulfide activation and visible light excitation prepared by the preparation method of any one of claims 3 to 6, characterized in that: Used for preparing anticancer drugs; the photosensitizer used for endogenous hydrogen sulfide activation and visible light excitation consumes the endogenous hydrogen sulfide of cancer cells and is activated to generate singlet oxygen under green light.

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