A photosensitizer, a photosensitizer prodrug, and a preparation method and application thereof

Abstract

The present invention discloses a photosensitizer, a photosensitizer prodrug, and a preparation method and application thereof, and relates to the field of photodynamic therapy. The compound can target the polyamine transport system in tumor cells, has no dark toxicity and phototoxicity in the blood circulation system and normal tissue cells at pH 7.4, and can produce reactive oxygen under light in the acidic extracellular fluid of the tumor (pH 6.5-6.8) and the lysosome of cancer cells (pH 4.5-5.0), and has phototoxicity. The compound can be used as a photosensitizer prodrug in photodynamic therapy, and effectively inhibits the growth of tumor volume in experimental mice. The photosensitizer prodrug has the following structure:

Description

Technical Field

[0001] The invention relates to the field of photodynamic therapy and is a photosensitizer prodrug material capable of targeting tumor tissue and being activated in the acidic environment of tumor tissue and the lysosome of cancer cells and a preparation method thereof. Background Art

[0002] Photodynamic therapy (PDT) is a safe and minimally invasive treatment that is gaining recognition as an effective alternative for cancer treatment [see: M. Triesscheijn, P. Baas, JHM Schellens, FA Stewart, Oncologist 2006, 11, 1034-1044]. Photodynamic therapy mainly uses light of a specific wavelength to irradiate photosensitizers to produce reactive oxygen species (ROS), especially singlet oxygen, to kill cancer cells. Therefore, photosensitizers are the most critical component of photodynamic therapy. Since the reactive oxygen species produced are lethal to all cells, tumor targeting of photosensitizers is particularly important to avoid side effects during treatment. Generally, photosensitizers obtain initial selectivity by moderately accumulating in tumor tissues, and selective illumination at the tumor site provides further selectivity [see: N.Solban, I. Rizvi, T. Hasan, Laser Surg Med 2006, 38, 522-531]. Improving the tumor targeting of photosensitizers can selectively reduce background accumulation in non-target tissues, thereby inhibiting side effects including edema and urticaria. Ideal photosensitizers should be inactive in non-tumor tissues, but present an active form that can produce reactive oxygen species in tumor tissues. These can be achieved by modifying classic targeting groups and prodrug strategies [see: F. Borgia, R.Giuffrida, E. Caradonna, M. Vaccaro, F. Guarneri, SP Cannavo, Biomedicines2018, 6.]. Generally, these strategies take advantage of the physiological / morphological heterogeneity between tumor tissues and normal tissues. Classic targeting strategies include active targeting and passive targeting. Passive targeting strategies mainly use the Enhanced Permeability and Retention effect (EPR) commonly used in nano-carriers and micellar preparations, but they have limitations (early tumors have no EPR effect) [see: D. Peer, JM Karp, S. Hong, OC FaroKHzad, R. Margalit, R. Langer, Nat Nanotechnol 2007, 2, 751-760.].For active targeting, it is generally achieved by combining a targeting ligand with a photosensitizer or carrier. These photosensitizers or carriers modified with targeting ligands can be overexpressed in tumor tissues to achieve targeting [see: N. Shirasu, SO Nam, M.Kuroki, Anticancer Res 2013, 33, 2823-2831.]. Recently, the use of activatable PDT agents as prodrugs of photosensitizers has attracted attention. Prodrugs can selectively activate in tumor tissues by utilizing the physiological / morphological specificity between tumor tissues and normal tissues and transform into photosensitizers with photodynamic therapy capabilities. This strategy can not only achieve targeted treatment of tumor tissues, but also reduce the side effects during treatment [see: CJ Wang, JG Delcros, L. Cannon, F. Konate, H. Carias, J. Biggerstaff, RA Gardner, O.Phanstiel, J Med Chem 2003, 46, 5129-5138; RA Gardner, JG Delcros, F.Konate, F. Breitbeil, B. Martin, M. Sigman, M. Huang, O. Phanstiel, J MedChem 2004, 47, 6055-6069; YG Wang, KJ Zhou, G. Huang, C. Hensley, XN Huang, XP Ma, T. Zhao, BD Sumer, RJ DeBerardinis, JM Gao, Nat Mater 2014, 13, 204-212; R. Perera, S. Stoykova, BN Nicolay, KN Ross, J. Fitamant, M. Boukhali, J. Lengrand, V. Deshpande, MK Selig, CRFerrone, J. Settleman, G. Stephanopoulos, NJ Dyson, R. Zoncu, S.Ramaswamy, W. Haas, N. Bardeesy, Nature 2015, 524, 361-U251.].However, most current strategies are still based on targeting endogenous receptors or overexpressed enzymes, which may be hampered by tumor heterogeneity and poor expression of tumor-associated stimuli on healthy cells, so it is still challenging to design a universal strategy suitable for multiple tumors. An alternative strategy is to target ubiquitous features of tumors, such as acidic extracellular fluid, rather than endogenous biomarkers. However, it is challenging to design a photosensitizer prodrug that is photodynamically inactive in the weakly alkaline (pH 7.4) blood circulation system and can be activated in the acidic extracellular fluid of tumors. Summary of the invention

[0003] The present invention designs and provides a photosensitizer prodrug material that can target tumor tissues and be activated in the acidic environment of tumor tissues and lysosomes of cancer cells and a preparation method thereof.

[0004] In the present invention, we cleverly designed a class of non-conjugated system compounds containing spermine, fluorine boron and iodine atoms, which have almost no photodynamic activity in the blood circulation system at pH 7.4. The spermine functional group enables the molecule to target the polyamine transport system elevated in tumor tissue, so that the compound is enriched in tumor tissue. In tumor tissue, the compound is activated in the acidic extracellular fluid (pH 6.5-6.8) in the tumor microenvironment and in the lysosomes (pH 4.5-5.0) of cancer cells, and has photodynamic activity, and can produce singlet oxygen under light, thereby selectively killing cancer tissues and cancer cells and reducing damage to normal tissues and cells.

[0005] In order to solve the technical problem of the present invention, the proposed technical solution is: a photosensitizer prodrug having the following structure:

[0006] .

[0007] In order to solve the technical problem of the present invention, another technical solution is proposed: a photosensitizer having the following structure:

[0008] .

[0009] In order to solve the technical problem of the present invention, another technical solution is proposed: the preparation method of the photosensitizer is as follows: diethyl malonate, 1H-pyrrole-2-carboxaldehyde, methylamine hydrochloride and sodium acetate are added to methanol in equal molar amounts, and reacted for 2-4 hours to obtain a yellow oily compound; the yellow oily compound and benzaldehyde are dissolved in dry dichloromethane under nitrogen protection at a molar ratio of 1:2, covered with aluminum foil, and trifluoroacetic acid (TFA) is added as a catalyst at the same time, and the amount of the catalyst is 1-5 times the molar amount of benzaldehyde, stirred at 60°C-100°C for 48-96 hours, and then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in an equal molar amount to benzaldehyde is added and stirred at room temperature for 30 -60 minutes for oxidation; immediately add excess triethylamine and boron trifluoride etherate (, reflux for 12-48 hours, purify and recrystallize with dichloromethane / n-hexane to obtain a solid with a blue metallic luster; dissolve the above solid with a blue metallic luster in ultra-dry acetonitrile, add trifluoroacetic acid as a catalyst under stirring at room temperature, the catalyst amount is 5-10 times the molar amount of the blue metallic luster solid and excess N-iodosuccinimide; heat reflux at 80°C-120°C for 1.5-5 hours and then return to room temperature; purify and recrystallize with dichloromethane / n-hexane to obtain the target compound photosensitizer 1 with a dark green metallic luster; the reaction route is as follows:

[0010]

[0011] In order to solve the technical problem of the present invention, another technical solution is proposed: the preparation method of the photosensitizer prodrug, the photosensitizer 1 and spermine react in equal proportions in a solution to obtain the photosensitizer prodrug; the reaction route is as follows:

[0012]

[0013] Preferably, the method comprises the following steps: adding equimolar amounts of diethyl malonate, 1H-pyrrole-2-carboxaldehyde, methylamine hydrochloride and sodium acetate to methanol at the same time to react at room temperature for 2-4 hours, extracting with dichloromethane after the reaction, drying with anhydrous sodium sulfate after the extraction, and purifying with a silica gel column using dichloromethane as a solvent to obtain a yellow oily compound;

[0014] The yellow oily compound and benzaldehyde are dissolved in dry dichloromethane at a molar ratio of 1:2 under nitrogen protection, covered with aluminum foil, and trifluoroacetic acid (TFA) is added as a catalyst, the amount of the catalyst is 1-5 times the molar amount of benzaldehyde, stirred at 60°C-100°C for 48-96 hours, and then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in an amount equal to that of benzaldehyde is added and stirred at room temperature for 30-60 minutes for oxidation; then an excess of triethylamine and boron trifluoride ether are immediately added, refluxed for 12-48 hours, and the obtained blue solution (with strong pink fluorescence) is washed with water and saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, and purified by silica gel chromatography using dichloromethane containing 8% ethyl acetate as solvent; after purification, recrystallized with dichloromethane / n-hexane to obtain a solid with a blue metallic luster;

[0015] The solid with blue metallic luster is dissolved in ultra-dry acetonitrile, and trifluoroacetic acid is added as a catalyst under stirring at room temperature, the amount of the catalyst is 5-10 times the molar amount of the blue metallic luster solid and an excess of N-iodosuccinimide; heat and reflux at 80℃-120℃ for 1.5-5 hours and then return to room temperature. Wash with saturated sodium thiosulfate solution 3 times to wash away excess N-iodosuccinimide, then dry with anhydrous sodium sulfate, concentrate under reduced pressure, purify with a silica gel column using a developing agent of dichloromethane; recrystallize with dichloromethane / n-hexane to obtain a dark green photosensitizer 1 with metallic luster;

[0016] The dark green metallic photosensitizer 1 is dissolved in dimethyl sulfoxide to obtain a blue-purple solution. An equimolar aqueous solution of spermine is added thereto and mixed evenly. The reaction is stopped when the solution becomes nearly colorless. The solution is lyophilized to obtain the target product, the photosensitizer prodrug 1-spm.

[0017] In order to solve the technical problem of the present invention, another technical solution is proposed: a pharmaceutical composition comprising the photosensitizer prodrug compound and a pharmaceutically acceptable carrier.

[0018] In order to solve the technical problem of the present invention, another technical solution is proposed: a pharmaceutical composition, which is composed of the active ingredient being the photosensitizer prodrug and pharmaceutically acceptable pharmaceutical excipients to form a clinically applicable injection or lyophilized powder injection.

[0019] In order to solve the technical problem of the present invention, another technical solution is proposed: use of the photosensitizer prodrug compound in the preparation of drugs for photodynamic diagnosis and therapy.

[0020] A photosensitizer prodrug compound that can target tumor tissue and be activated in the acidic environment of tumor tissue and the lysosome of cancer cells, named 1-spm, has the following structure:

[0021]

[0022] 1-spm.

[0023] A method for preparing the above-mentioned photosensitizer prodrug compound 1-spm that can target tumor tissue and be activated in the acidic environment of tumor tissue and the lysosome of cancer cells, comprising the following steps:

[0024] Diethyl malonate (1.59 mL), 1H-pyrrole-2-carboxaldehyde (1.05 g), methylamine hydrochloride (0.36 g) and sodium acetate (0.89 g) were added to 50 mL of methanol and reacted at room temperature for 2-4 h. After the reaction, it was extracted with dichloromethane. After the extraction, it was dried with anhydrous sodium sulfate and purified by silica gel chromatography using dichloromethane as solvent to obtain a yellow oily compound. This yellow oily compound (480 mg) and benzaldehyde (200 µL) were dissolved in dry dichloromethane (50 mL) under nitrogen protection, covered with aluminum foil, and 0.5 mL of trifluoroacetic acid (TFA) was added as a catalyst. It was stirred at 60 ° C for 48 h, and then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (460 mg) was added and stirred at room temperature for 30 min for oxidation. Add triethylamine (3 mL) and boron trifluoride etherate (10 mL), reflux for 12 h, wash the obtained blue solution (with strong pink fluorescence) with water and saturated sodium bicarbonate solution, dry over anhydrous magnesium sulfate, and purify it by silica gel column chromatography using dichloromethane containing 8% ethyl acetate as solvent. After purification, recrystallize it with dichloromethane / n-hexane to obtain crystals with blue metallic luster. Dissolve the crystals with blue metallic luster (1.2 g) in ultra-dry acetonitrile, add trifluoroacetic acid (0.5 mL) and N-iodosuccinimide (2.2 g) under stirring at room temperature. Heat and reflux at 80°C for 1.5 h and return to room temperature. Wash it with saturated sodium thiosulfate solution three times to wash off excess NIS, then dry it over anhydrous sodium sulfate, concentrate it under reduced pressure, and purify it with silica gel column chromatography using dichloromethane as the developing solvent. Recrystallize it with dichloromethane / n-hexane to obtain dark green crystals with metallic luster. Take 12 mg of the dark green metallic crystals and dissolve them in 10 mL of dimethyl sulfoxide. The solution is blue-purple in color. Add 10 µL of spermine aqueous solution (1 M) and mix well. Stop the reaction when the solution becomes almost colorless. Freeze-dry the solution to obtain the target product.

[0025] Beneficial Effects of the Invention

[0026] Compared with the prior art, the present invention has the significant advantage of inventing a completely new photosensitizer prodrug material that can target tumor tissues and be activated in the acidic environment of tumor tissues and the lysosomes of cancer cells.

[0027] Activatable photosensitizer prodrugs can be selectively activated in tumor tissues by utilizing the physiological / morphological specificity between tumor tissues and normal tissues and transformed into photosensitizers with photodynamic therapy capabilities. This strategy can not only achieve targeted treatment of tumor tissues, but also reduce side effects during treatment. For example, since the activation of the mTORC1 complex promotes cell growth, enriching mTORC1 on the surface of lysosomes is crucial for its activation. Therefore, the abundance of lysosomes is found to increase in some rapidly proliferating tumor cells. For example, the abundance of lysosomes in pancreatic ductal adenocarcinoma is much higher than that in normal pancreatic cells (see: R. Perera, S. Stoykova, BN Nicolay, KNRoss, J. Fitamant, M. Boukhali, J. Lengrand, V. Deshpande, MK Selig, CRFerrone, J. Settleman, G. Stephanopoulos, NJ Dyson, R. Zoncu, S.Ramaswamy, W. Haas, N. Bardeesy. Nature. 2015, 524, 361-U251; Y. Rabanal-Ruiz, VI Korolchuk. Int. J. Mol. Sci. 2018, 19.). The development of photosensitizers targeting lysosomes can effectively inhibit tumor growth by inhibiting the activation of mTORC1. On the other hand, since the content of lysosomes in normal cells is less than that in tumor cells, it will cause less damage to normal cells. On the other hand, the polyamine transport system overexpressed on the surface of tumor cells is another target. Polyamines (spermine, spermidine, etc.) are key cell growth factors that play an important role in cell proliferation, differentiation, and chromatin conformation maintenance.Under physiological conditions, intracellular polyamine levels are precisely regulated by the polyamine transport system on the cell membrane to maintain the normal operation of the cell cycle (see: CJ Wang, JG Delcros, L. Cannon, F. Konate, H. Carias, J. Biggerstaff, RA Gardner, O.Phanstiel. J. Med. Chem. 2003, 46, 5129-5138; RA Gardner, JG Delcros,F. Konate, F. Breitbeil, B. Martin, M. Sigman, M. Huang, O. Phanstiel; J.Med. Chem. 2004, 47, 6055-6069; C. Moinard, L. Cynober, JP de Bandt.; Clin.Nutr. 2005, 24, 184-197.). The occurrence and development of tumors are often accompanied by abnormal polyamine metabolism. Because the proliferation of tumor cells requires high levels of polyamines in the cells to promote DNA replication, protein synthesis and the formation of blood vessels in tumor tissue, it leads to high expression of the polyamine transport system on the surface of tumor cell membranes, making it easier for them to take up polyamines from the outside into the cells for proliferation activities.

[0028] In the present invention, the difference between tumor tissue and normal tissue is utilized to modify the spermine group on the photosensitizer prodrug molecule to target the polyamine transport system overexpressed on the surface of tumor tissue. In addition, in view of the weak acidity of the tumor tissue microenvironment and the lysosomes with increased abundance in the cells of tumor tissue, the photosensitizer prodrug of the present invention can be converted into a photosensitizer under acidic conditions, so that the photosensitizer prodrug with pH sensitivity will be quickly converted into a photosensitizer with the ability to produce singlet oxygen after entering the tumor cell due to the low pH in the lysosome, and preferentially destroy the lysosome, thereby killing the entire cancer cell.

[0029] The photosensitizer prodrug of the present invention can be prepared into an injection or a lyophilized agent for use, and its photodynamic therapy effect has been verified in tumor cells and tumor-bearing mice.

[0030] In summary, the photosensitizer prodrug in the present invention can provide dual targeting functions at both the tissue and cell levels, and the material has almost no photodynamic activity in the blood circulation system and is only selectively activated in tumor tissues and cancer cells, further reducing damage to normal tissues and reducing side effects on the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The photosensitizer 1 of the present invention1 H NMR spectrum;

[0032] Figure 2 is a high-resolution mass spectrum of the photosensitizer 1 of the present invention;

[0033] Figure 3 Compound 1-spm, which is a photosensitizer prodrug of the present invention 1 H NMR spectrum;

[0034] Figure 4 It is a high-resolution mass spectrum of compound 1-spm, a photosensitizer prodrug of the present invention;

[0035] Figure 5 Schematic diagram of the reversible reaction between the photosensitizer prodrug 1-spm and the photosensitizer 1 of the present invention;

[0036] Figure 6 is the ultraviolet absorption spectrum of the reversible reaction between the photosensitizer prodrug 1-spm and the photosensitizer 1 of the present invention;

[0037] Figure 7 The ultraviolet absorption spectrum of the photosensitizer prodrug 1-spm of the present invention changes with pH;

[0038] Figure 8 The photosensitizer prodrug 1-spm of the present invention cannot produce the ultraviolet absorption spectrum of singlet oxygen;

[0039] Fig. 9 is the ultraviolet absorption spectrum of the photosensitizer 1 of the present invention for generating singlet oxygen;

[0040] Fig.10 The photostability of the photosensitizer prodrug 1-spm of the present invention;

[0041] Fig.11 is the photostability of the photosensitizer 1 of the present invention;

[0042] Fig.12 is the water solubility of the photosensitizer prodrug 1-spm of the present invention;

[0043] Fig.13 The phototoxicity and dark toxicity of the photosensitizer prodrug 1-spm of the present invention in 4T1 cells;

[0044] Fig.14 The photosensitizer prodrug 1-spm of the present invention generates singlet oxygen in cells;

[0045] Fig.15 The photosensitizer prodrug 1-spm of the present invention kills cancer cells during photodynamic therapy;

[0046] Fig.16The photosensitizer prodrug 1-spm of the present invention has tumor targeting in tumor-bearing mice;

[0047] Fig.17 The present invention shows the photodynamic therapy effect of the photosensitizer prodrug 1-spm in tumor-bearing mice. DETAILED DESCRIPTION

[0048] Example 1: A method for preparing the above-mentioned photosensitizer 1 and photosensitizer prodrug 1-spm that can target tumor tissue and be activated in the acidic environment of tumor tissue and lysosomes of cancer cells:

[0049] Diethyl malonate (1.59 mL), 1H-pyrrole-2-carboxaldehyde (1.05 g), methylamine hydrochloride (0.36 g) and sodium acetate (0.89 g) were added to 50 mL of methanol and reacted at room temperature for 2-4 h. After the reaction, it was extracted with dichloromethane. After the extraction, it was dried with anhydrous sodium sulfate and purified by silica gel chromatography using dichloromethane as solvent to obtain a yellow oily compound. This yellow oily compound (480 mg) and benzaldehyde (200 µL) were dissolved in dry dichloromethane (50 mL) under nitrogen protection, covered with aluminum foil, and 0.5 mL of trifluoroacetic acid (TFA) was added as a catalyst. It was stirred at 60 ° C for 48 h, and then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (460 mg) was added and stirred at room temperature for 30 min for oxidation. Triethylamine (3 mL) and boron trifluoride etherate (10 mL) were added and refluxed for 12 h. The obtained blue solution (with strong pink fluorescence) was washed with water and saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, and purified by silica gel chromatography using dichloromethane containing 8% ethyl acetate as solvent. After purification, it was recrystallized from dichloromethane / n-hexane to obtain crystals with blue metallic luster. The crystals with blue metallic luster (1.2 g) were dissolved in ultra-dry acetonitrile, and trifluoroacetic acid (0.5 mL) and N-iodosuccinimide (2.2 g) were added under stirring at room temperature. The mixture was heated to reflux at 80 °C for 1.5 h and then returned to room temperature. The solution was washed three times with saturated sodium thiosulfate solution to wash away excess NIS, then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified using a silica gel chromatography column with dichloromethane as the developing solvent. The photosensitizer 1 with dark green metallic luster was obtained by recrystallization from dichloromethane / n-hexane. High-resolution mass spectrum of photosensitizer 1 ( Figure 1 )and 1 H NMR spectrum ( Figure 2): Calculated m / z [M]- 1111.8002, found 1111.8006. 1H NMR (500 MHz, CDCl2) δ 7.79 (s, 2H), 7.70 – 7.65 (m, 3H), 7.38 – 7.26 (m,2H), 4.41 – 4.36 (m, 4H), 4.23 – 4.18 (m, 4H), 1.40 (t, J = 7.1 Hz, 6H), 1.19(t, J = 7.1 Hz, 6H)

[0050] 12 mg of solid photosensitizer 1 was dissolved in 10 mL of dimethyl sulfoxide. The solution was blue-purple. 10 µL of spermine aqueous solution (1 M) was added and mixed evenly. The reaction was stopped when the solution became almost colorless. The solution was lyophilized to obtain the target product, photosensitizer prodrug 1-spm. The product was a light yellow powder. High-resolution mass spectrometry ( Figure 3 )and 1 H NMR spectrum ( Figure 4 ): Calculated m / z [M]- 1313.0081, found 1313.0084. 1H NMR (500 MHz, CDCl2) δ 8.12 (s, 1H), 7.52 (s, 2H), 7.19 (d, J = 19.6 Hz, 3H), 4.23 – 4.19 (m, 4H), 3.36 (s, 4H), 2.83 – 2.58 (m, 10H), 1.31 (s, 12H).

[0051] Example 2: The above-mentioned photosensitizer prodrug compound 1-spm that can target tumor tissue and be activated in the acidic environment of tumor tissue and the lysosome of cancer cells can be mutually converted with the photosensitizer 1 that has the ability to generate singlet oxygen ( Figure 5 ).

[0052] When a small amount of spermine is added to a dichloromethane solution of photosensitizer 1 that has the ability to generate singlet oxygen, 1 will react quickly to generate the pro-photosensitizer 1-spm that does not have the ability to generate singlet oxygen. When a small amount of trifluoroacetic acid is added to a solution of 1-spm, 1-spm will be converted into 1. This reaction is reversible ( Figure 6 Since spermine reacts directly at the central carbon position of photosensitizer 1 ( Figure 5 ), resulting in the breakage of the conjugated system and the inability to generate singlet oxygen under laser irradiation.

[0053] Example 3: Electronic absorption spectra of the above-mentioned photosensitizer prodrug compound 1-spm that can target tumor tissues and be activated in the acidic environment of tumor tissues and lysosomes of cancer cells as a function of pH ( Figure 7 ).

[0054] In a mixed solution of tetrahydrofuran and water (v / v=1:1), at pH 8.0, the absorption peak of the electronic absorption spectrum of 1-spm is 375 nm. As the pH decreases, the 375 nm absorption peak gradually decreases, and a new absorption peak appears at 583 nm. At pH 2, the absorbance at 583 nm reaches a maximum. This example illustrates that 1-spm can be transformed from a non-conjugated state to a conjugated state under acidified conditions.

[0055] When the photosensitizer prodrug 1-spm is enriched in the tumor site, and because the tumor tissue fluid is acidic (pH = 6.5-6.8), part of the 1-spm will be converted into photosensitizer 1 at low pH to achieve the effect of killing tumor cells under light.

[0056] Example 4: Singlet oxygen generation capability of the above-mentioned photosensitizer prodrug compound 1-spm and photosensitizer 1 that can target tumor tissue and be activated in the acidic environment of tumor tissue and lysosome of cancer cells ( Figure 8 , Fig. 9 ).

[0057] The singlet oxygen generation ability of compound 1-spm was tested in dichloromethane. 1,3-diphenylisobenzofuran (DPBF) was used as a singlet oxygen scavenger. When singlet oxygen is generated in an organic solution, the ultraviolet absorption of DPBF as a singlet oxygen scavenger at 415 nm decreases. Figure 8 As shown in the figure, after being irradiated with a laser (590 nm, 100 mW) for different time periods, the UV absorption data was collected once. After irradiation, the UV absorption of DPBF at 415 nm was almost unchanged, which indicates that 1-spm cannot produce singlet oxygen under illumination.

[0058] The same method was used to test the singlet oxygen generation ability of the conjugated photosensitizer 1 in dichloromethane. Fig. 9 As shown, after irradiation with a laser (590 nm, 100 mW) for different time periods, the ultraviolet absorption of DPBF at 415 nm continued to decrease and reached the bottom after 100 seconds, indicating that photosensitizer 1 can produce singlet oxygen under light.

[0059] Example 5: Photostability and water solubility of the above-mentioned photosensitizer prodrug compound 1-spm that can target tumor tissues and be activated in the acidic environment of tumor tissues and lysosomes of cancer cells, and photostability of photosensitizer 1 ( Fig.10 , Fig.11 ).

[0060] Because long-term illumination is often required during photodynamic therapy, the photostability of the photosensitizer is very important. The pro-photosensitizer 1-spm and photosensitizer 1 in dichloromethane were continuously irradiated with a laser light source (590 nm, 500 mW). Within 3 hours, the UV absorption spectra of the pro-photosensitizer 1-spm and photosensitizer 1 did not change significantly, indicating that the two compounds have good photostability.

[0061] Example 6: Water solubility of the above-mentioned photosensitizer prodrug 1-spm that can target tumor tissues and be activated in the acidic environment of tumor tissues and lysosomes of cancer cells ( Fig.12 ).

[0062] In order to explore the possibility of making the pro-photosensitizer 1-spm into an injection, we conducted the following test on the water solubility of the pro-photosensitizer 1-spm: adding the same concentration of 1-spm to tetrahydrofuran / water solutions with different water contents and testing the UV absorption spectrum respectively. In tetrahydrofuran solutions with different water contents, the UV absorption spectrum of 1-spm did not change significantly, indicating that it has good water solubility.

[0063] Example 7: Phototoxicity and dark toxicity of the above-mentioned photosensitizer prodrug compound 1-spm that can target tumor tissues and be activated in the acidic environment of tumor tissues and lysosomes of cancer cells ( Fig.13 ).

[0064] Photosensitizer prodrug 1-spm can be converted into photosensitizer 1 in acidic organelles such as lysosomes in cells, and can kill cancer cells under light. Different concentrations of 1-spm were added to a 96-well plate inoculated with 4T1 cells that had grown to the logarithmic phase. After incubation for 30 minutes, each well was irradiated with a laser (590 nm, 500 mW) for 10 minutes, and then placed in a cell culture incubator for 12 hours. The cell viability in the well plate was then tested using the MTT method. In addition, the dark toxicity of 1-spm was tested in a 96-well plate under the same conditions without light as a control.

[0065] After illumination, the cell viability was affected to varying degrees according to the different 1-spm concentrations. The cell median lethal concentration (IC 50 ) was 2.11 micromolar. In cells without light exposure, different concentrations of 1-spm showed obvious cytotoxicity, indicating that 1-spm has low dark toxicity and good biocompatibility.

[0066] Example 8: The above-mentioned photosensitizer prodrug compound 1-spm that is capable of targeting tumor tissue and being activated in the acidic environment of tumor tissue and the lysosome of cancer cells generates reactive oxygen species ( Fig.14 ).

[0067] The classic intracellular reactive oxygen fluorescent probe DCFH-DA was used as the detection reagent. DCFH-DA itself has no fluorescence and can pass through the cell membrane into the cell. After entering the cell, it can be hydrolyzed by intracellular esterase into DCFH, which also has no fluorescence. Because DCFH cannot pass through the cell membrane, it can label the reactive oxygen in the cell. The reactive oxygen in the cell can oxidize the non-fluorescent DCFH into DCF with green fluorescence emission. After incubating 1-spm and 10 μM DCFH-DA in the cells for 30 minutes, the cells were placed under a laser confocal microscope for observation. The results are as follows: Fig.14 As shown in the figure, after 1 min of irradiation with a 590 nm (500 mW) laser, green fluorescence appeared in the cells, indicating that singlet oxygen can be produced in the cells after 1 min of laser irradiation. After 10 minutes of irradiation with a 590 nm (500 mW) laser, the green fluorescence intensity in the cells increased, indicating that the production of singlet oxygen in the cells increased with the increase of laser irradiation time. Example 8 shows that 1-spm can produce reactive oxygen in cells under light conditions.

[0068] Example 9: The above-mentioned photosensitizer prodrug compound 1-spm that is capable of targeting tumor tissue and being activated in the acidic environment of tumor tissue and the lysosome of cancer cells kills cancer cells during photodynamic therapy ( Fig.15 ).

[0069] During photodynamic therapy with 1-spm, we used Calcein AM / Propidium Iodide (PI) to perform live / dead cell staining on different groups of cells. Dead cells emit red fluorescence, while live cells emit green fluorescence. Fig.15As shown, the control group without 1-spm and the drug-added group without laser irradiation were observed to have strong green fluorescence emission and no red fluorescence, indicating that the cell activity was good. In the group with 1-spm added after 590 nm (500mW) laser irradiation for 10 min, the green fluorescence intensity was greatly reduced, and a large amount of red fluorescence in the cell nucleus could be clearly observed, which indicated that a large number of cells died, and PI could enter the cell nucleus through the cell membrane that was no longer intact in the dead cells, so that the cell nucleus emitted red fluorescence. In living cells, because PI cannot pass through the intact cell membrane, the cell nucleus in living cells will not be dyed red by PI. On the other hand, in the light-treated drug-added group treated with vitamin C (which can remove singlet oxygen), no red fluorescence was observed in the cell nucleus, which indicates that the photosensitizer causes cell death by producing singlet oxygen. Vitamin C with reducing properties can remove reactive oxygen in cells to avoid cell death. Example 9 proves that 1-spm can produce singlet oxygen and kill cancer cells during photodynamic therapy.

[0070] Example 10: Preparation of injection, the prescription is as follows (based on 1000 vials):

[0071] Take 100 mg of photosensitizer 1 in Example 1, dissolve it in 100 mL of dimethyl sulfoxide, then add 20 mg of spermine to prepare photosensitizer prodrug 1-spm, and add water for injection to 1000 mL. Mix well, filter, and dispense the resulting solution into ampoules under sterile conditions to prepare an injection solution of 1 mL / bottle, with an active ingredient content of 0.12 mg / bottle

[0072] Example 11: Targeting of the above-mentioned photosensitizer prodrug compound 1-spm in tumor-bearing mice ( Fig.16 ).

[0073] BALB / c mice were implanted with 4T1 breast cancer tumors in the axilla and the tumors were grown to 100 mm. 3 Then, the injection solution of Example 10 was injected into the tail vein of the mouse, and then the mouse was placed in a small animal imaging device for photographing and observation at different time points. Fig.16 As shown in Figure 2, 1-spm was enriched in the tumor site 4 h after injection ( Fig.16 ). Example 11 illustrates that 1-spm has tumor targeting properties in experimental animals.

[0074] Example 12: Photodynamic therapy of the above-mentioned photosensitizer prodrug compound 1-spm that can target tumor tissue and be activated in the acidic environment of tumor tissue and lysosomes of cancer cells in tumor-bearing mice ( Fig.17 ).

[0075] The 4T1 breast cancer tumors were transplanted and the tumor size reached 100 mm. 3 BALB / c mice at the time of treatment were used as research subjects and divided into three groups according to phosphate buffer solution, drug-added group and drug-added light group. The mice in the drug-added group and drug-added light group were injected with the injection solution in Example 7 once every two days, and the phosphate buffer solution group was injected with the same dose of phosphate buffer solution. Fig.17 As shown in a, there was no significant difference in the weight of mice in different groups during the whole photodynamic therapy process. This shows that 1-spm has no obvious toxic side effects on the body. After transplantation of 4T1 breast tumors, the tumor size of mice in the control group injected with phosphate buffer solution increased with the time of transplantation, while after the treatment group was irradiated with light (590 nm, 500mW, 30 min), the tumor size of mice in the treatment group did not increase significantly. The growth trend of the tumor size of mice in the drug-treated group without light treatment was similar to that of the control group ( Fig.17 b, c). After 16 days of treatment, the tumors of each group of tumor-bearing mice were removed, and the size of the tumor after photodynamic therapy can be more clearly seen ( Fig.17 d). The tumor volumes of the control group and the drug-treated group increased from the initial 0.108 cm 3 and 0.104 cm 3 Grows to 1.565 cm 3 and 1.239 cm 3 The initial tumor volume and the tumor volume after treatment in the treatment group were 0.103 cm 3 and 0.218 cm 3 The tumor inhibition rate was 86.04%. Example 12 illustrates that 1-spm can effectively inhibit tumor growth in experimental animals.

[0076] Example 13: Injections other than Example 10 can also be made into lyophilized powders:

[0077] The photosensitizer prodrug 1-spm is synthesized according to the method of Example 1. After the reaction is completed and purified, it is freeze-dried and stored. When used, the freeze-dried powder of the required mass can be prepared.

[0078] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A photosensitizer prodrug, characterized in that: It has the following structure: 。 2. A photosensitizer, characterized in that: It has the following structure: 。 3. The method for preparing a photosensitizer according to claim 2, characterized in that: Equimolar amounts of diethyl malonate, 1H-pyrrole-2-carboxaldehyde, methylamine hydrochloride and sodium acetate are added to methanol at the same time, and the reaction is carried out for 2-4 hours to obtain a yellow oily compound; the yellow oily compound and benzaldehyde are dissolved in dry dichloromethane at a molar ratio of 1:2 under nitrogen protection, covered with aluminum foil, and trifluoroacetic acid (TFA) is added as a catalyst at the same time, and the amount of the catalyst is 1-5 times the molar amount of benzaldehyde. Stir at a temperature of 60°C-100°C for 48-96 hours, and then add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in an amount equal to that of benzaldehyde and stir at room temperature for 30-60 minutes. Oxidation; immediately add excess triethylamine and boron trifluoride etherate, reflux for 12-48 hours, purify and recrystallize with dichloromethane / n-hexane to obtain a solid with blue metallic luster; dissolve the above solid with blue metallic luster in ultra-dry acetonitrile, add trifluoroacetic acid as a catalyst under stirring at room temperature, the catalyst amount is 5-10 times the molar amount of the blue metallic luster solid and excess N-iodosuccinimide; heat reflux at 80°C-120°C for 1.5-5 hours and then return to room temperature; purify and recrystallize with dichloromethane / n-hexane to obtain the target compound photosensitizer 1 with dark green metallic luster; the reaction route is as follows: 。 4. The method for preparing a photosensitizer prodrug according to claim 1, characterized in that: Photosensitizer 1 and spermine react in equal proportions in a solution to obtain a photosensitizer prodrug; the reaction route is as follows: 。 5. The method for preparing a photosensitizer prodrug according to claim 4, characterized in that: The method comprises the following steps: adding equimolar amounts of diethyl malonate, 1H-pyrrole-2-carboxaldehyde, methylamine hydrochloride and sodium acetate to methanol at the same time, reacting at room temperature for 2-4 hours, extracting with dichloromethane after the reaction is completed, drying with anhydrous sodium sulfate after the extraction is completed, and purifying with a silica gel column using dichloromethane as a solvent to obtain a yellow oily compound; The yellow oily compound and benzaldehyde are dissolved in dry dichloromethane at a molar ratio of 1:2 under nitrogen protection, covered with aluminum foil, and trifluoroacetic acid (TFA) is added as a catalyst, the amount of the catalyst is 1-5 times the molar amount of benzaldehyde, stirred at 60°C-100°C for 48-96 hours, and then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in an amount equal to that of benzaldehyde is added and stirred at room temperature for 30-60 minutes for oxidation; then an excess of triethylamine and boron trifluoride ether are immediately added, refluxed for 12-48 hours, and a blue solution with strong pink fluorescence is obtained, washed with water and saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, and purified by silica gel chromatography using dichloromethane containing 8% ethyl acetate as solvent; after purification, recrystallized with dichloromethane / n-hexane to obtain a solid with a blue metallic luster; The solid with blue metallic luster is dissolved in ultra-dry acetonitrile, and trifluoroacetic acid is added as a catalyst under stirring at room temperature, the amount of the catalyst is 5-10 times the molar amount of the blue metallic luster solid and an excess of N-iodosuccinimide; heating under reflux at 80°C-120°C for 1.5-5 hours, then returning to room temperature, washing with saturated sodium thiosulfate solution for 3 times to wash away excess N-iodosuccinimide, then drying over anhydrous sodium sulfate, concentrating under reduced pressure, and purifying with a silica gel column using a developing agent of dichloromethane; and recrystallizing with dichloromethane / n-hexane to obtain a dark green photosensitizer 1 with metallic luster; The dark green metallic photosensitizer 1 is dissolved in dimethyl sulfoxide to obtain a blue-purple solution. An equimolar aqueous solution of spermine is added thereto and mixed evenly. The reaction is stopped when the solution becomes nearly colorless. The solution is lyophilized to obtain the target product, the photosensitizer prodrug 1-spm.

6. A pharmaceutical composition, characterized in that: It comprises the photosensitizer prodrug compound according to claim 1 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that: The active ingredient is the photosensitizer prodrug, and pharmaceutically acceptable pharmaceutical excipients are combined to prepare clinically applicable injections and freeze-dried powder injections.

8. Use of the photosensitizer prodrug compound according to claim 1 in the preparation of drugs for photodynamic diagnosis and therapy.

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