Preparation method and application of selenium-containing photosensitizer based on quinoline skeleton

Abstract

The invention discloses a preparation method and application of a selenium-containing photosensitizer based on a quinoline skeleton, the structure of the organic photosensitizer is shown in the specification, the invention develops a novel photosensitizer PDSe series based on selenium atoms, and the photosensitizer can be used for preparing the selenium-containing photosensitizer through unique D-pi-A structural design and the heavy atom effect of the selenium atoms. And an efficient photodynamic therapy effect is achieved. Active oxygen generated by the photosensitizer after illumination activation can directly kill tumor cells and also can significantly enhance the anti-tumor effect through a concentration-dependent dual action mechanism (low-concentration apoptosis induction and high-concentration pyroptosis induction), and the dynamic regulation mechanism not only improves the treatment effect, but also reduces the drug resistance risk of the tumor cells. Particularly, by introducing the PDSE-8 derivative designed by a targeting ligand, mitochondria can be accurately targeted, an overexpressed receptor on the surface of a tumor cell can be recognized, and the tumor selectivity and the drug delivery efficiency are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a method for preparing a class of selenium-containing photosensitizers based on a quinoline skeleton and their applications. Background Technology

[0002] Photodynamic therapy (PDT) has become an important treatment for tumors and infectious diseases due to its advantages such as minimal invasiveness, low systemic toxicity, and repeatability. Photosensitizers are the core component of PDT; their mechanism of action involves absorbing light energy under specific wavelengths of light, converting oxygen molecules in tissues into cytotoxic reactive oxygen species (ROS), thereby selectively killing tumor cells or pathogenic microorganisms. However, existing photosensitizers still have significant limitations in practical applications: ROS generation efficiency is severely constrained by the hypoxic tumor microenvironment; most photosensitizers have insufficient singlet oxygen quantum yield and excessive oxygen dependence; poor tumor targeting leads to insufficient accumulation of photosensitizers at the lesion site, while simultaneously causing photosensitized damage to normal tissues; and the regulation of cell death pathways is too singular, making it difficult to overcome the heterogeneity and drug resistance of tumor cells. These problems severely limit the efficacy of PDT for deep and drug-resistant tumors.

[0003] In recent years, selenium-containing organic photosensitizers have become a research hotspot due to their unique photophysical properties and efficient ROS generation capabilities. The heavy atom effect of selenium atoms can significantly improve the interstitial crossing efficiency while reducing the singlet-triplet energy difference (ΔE). st This enhances the photodynamic activity of photosensitizers. Furthermore, through rational molecular design (such as constructing D-π-A structures), the light absorption, fluorescence emission, and charge transfer properties of photosensitizers can be further optimized, enabling them to play a dual role in tumor imaging and treatment. Therefore, developing photosensitizers with novel structures and their derivatives to overcome the problems of hypoxia and drug resistance in the treatment of solid tumors is of great significance. Summary of the Invention

[0004] Objective of the invention: To address the limitations of existing photosensitizers in terms of reactive oxygen species (ROS) generation efficiency, tumor selectivity, and cell death mechanism regulation, this invention provides an organic photosensitizer and its derivatives based on a quinoline-selenium structure. This organic photosensitizer is a series of near-infrared activated selenium-containing photosensitizers. This series of photosensitizers is designed using molecular engineering optimization, based on a quinoline core and verbena extended conjugated system. It can efficiently generate ROS under near-infrared light irradiation and exhibits good cytotoxicity to tumor cells under light irradiation.

[0005] Furthermore, among the derivatives of the present invention, PDSe-8 exhibits the best phototoxicity and maintains high activity in both oxygen-rich and hypoxic environments through a type I photodynamic mechanism. It can not only significantly inhibit tumor cell growth but also induce both apoptosis and pyroptosis through a concentration-dependent mechanism. Furthermore, PDSe-8 significantly improves tumor selectivity and reduces systemic toxicity by introducing enzyme-responsive groups that specifically bind to tumor-highly expressed proteins such as fibroblast activator protein (FAP), histone deacetylase (HDAC), lysosomal protease (CTSL), nitroreductase (NTR), and quinone oxidoreductase (QR). Triphenylphosphine (TPP)-modified PDSe-8 can specifically target the mitochondria of tumor cells. Mitochondria are the main sites of ROS production in cells. Through mitochondrial enrichment, this modified photosensitizer can efficiently generate reactive oxygen species at the tumor site, effectively overcoming key problems such as hypoxic microenvironment, tumor drug resistance, and insufficient treatment specificity faced by traditional photodynamic therapy. Our in vitro and in vivo experiments demonstrated that PDSe-8 can efficiently kill various tumor cells and significantly inhibit tumor growth in animal models while maintaining low toxicity. This invention provides an important tool for developing novel photodynamic therapy drugs and opens up new avenues for overcoming the problems of hypoxia and drug resistance in the treatment of solid tumors.

[0006] The present invention also provides a method for preparing the photosensitizer and its derivatives and their applications.

[0007] Technical solution: To achieve the above objectives, the present invention provides an organic photosensitizer based on a quinoline-selenium structure, the structure of which is shown below:

[0008]

[0009] The R is selected from any of the following:

[0010]

[0011] The preparation method of the organic photosensitizer based on the quinoline-selenium structure of the present invention includes the following steps:

[0012] Under an inert gas atmosphere, 6-bromo-2-methylquinoline and selenium dioxide were dissolved in an organic solvent. The reaction mixture was heated and stirred, cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. The product was dissolved, extracted, dried, filtered, and concentrated under vacuum to obtain a light yellow crude product S-1.

[0013] Compound S-1 and verbena were dissolved in an organic solvent and then potassium hydroxide was added. The mixture was heated and stirred under an inert gas atmosphere. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed and dried under vacuum to obtain yellow solid S-2.

[0014] Compound S-2 and selenium dioxide were dissolved in an organic solvent and stirred to react. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed under vacuum. The residue was washed, extracted, and the crude product was purified by column chromatography to obtain white solid S-3.

[0015] Compound S-3 and malononitrile were heated and stirred in an organic solvent. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was washed and extracted. The crude product was purified by silica gel column chromatography to give a yellow solid, S-4.

[0016] Compound S-4, palladium acetate, 2,2'-bis(diphenylphosphino)-1,1'-binaphthylene and cesium carbonate were dissolved in an organic solvent and reacted under inert gas protection. The reaction mixture was heated and stirred, and then different imines were added. The reaction mixture was refluxed and reacted. After the reaction was completed, the mixture was cooled to room temperature, extracted and concentrated. The residue was purified by silica gel column chromatography to obtain the target product PDSe-n.

[0017] The imine includes any one of aziridine, pyrrolidine, piperidine, morpholine, N-methylpiperazine, N-ethylpiperazine, 1-(tert-butoxycarbonyl)piperazine, piperazine, thiomorpholine, 4,4-difluoropiperidine, cyclohexylpiperazine, and tert-butyl carbamate.

[0018] The organic photosensitizer derivatives based on the quinoline-selenium structure described in this invention have structures shown in any of the following:

[0019]

[0020]

[0021] The method for preparing the derivative of the present invention includes the following steps:

[0022] Using 1-(tert-butyloxycarbonyl)piperazine as the imine, compound PDSe-7 was obtained. Compound PDSe-7 was dissolved in an organic solvent, and trifluoroacetic acid was added to carry out the reaction. After the reaction was completed, the mixture was extracted and concentrated under reduced pressure. The residue was purified by column chromatography to obtain the target product PDSe-8. Compound PDSe-8, Boc-glycine-L-proline, HATU, and DIPEA were dissolved in an organic solvent and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-8a.

[0023] Compound PDSe-8 and p-nitrobenzyl chloroformate were dissolved in an organic solvent, triethylamine was added, and the mixture was stirred overnight. After the reaction was complete, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-8b.

[0024] Compound PDSe-8, 3-(2,3,5-trimethyl-1,4-benzoquinone)-3-methylbutyric acid, HATU, and DIPEA were dissolved in an organic solvent and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-8c.

[0025] Compound PDSe-8, N-tert-butoxycarbonyl-N'-(acetyl)-L-lysine, HATU, and DIPEA were dissolved in an organic solvent and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-8d.

[0026] Compound PDSe-8, 2-carboxyethyltriphenylphosphine bromide, HOBT, and EDCI were dissolved in an organic solvent, triethylamine was added, and the mixture was stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-8e.

[0027] Palladium acetate, 4,5-bis(diphenylphosphino)-9,9-dimethyloxanthracene, and cesium carbonate were dissolved in an organic solvent and reacted under inert gas protection. Compound S-4 and tert-butyl carbamate were added to the system, and the reaction was continued under reflux. After completion, the mixture was cooled to room temperature, extracted, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain PDSe-12. PDSe-12 was dissolved in an organic solvent, and trifluoroacetic acid was added. The reaction was carried out, extracted, and concentrated under reduced pressure. The residue was purified by column chromatography. The target product PDSe-NH2 was obtained. PDSe-NH2, Boc-L-leucine, HATU, and DIPEA were dissolved in an organic solvent and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-LAP(Boc). Then, PDSe-LAP(Boc) was dissolved in an organic solvent, trifluoroacetic acid was added dropwise, and the mixture was stirred overnight. After the reaction was completed, the solvent was removed under vacuum, and the crude product was purified by column chromatography to obtain PDSe-LAP.

[0028] Compound PDSe-NH2, N-Boc-D-glutamic acid-1-tert-butyl ester, HATU, and DIPEA were dissolved in an organic solvent and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-GGT(Boc). Then, compound PDSe-GGT(Boc) was dissolved in anhydrous dichloromethane, trifluoroacetic acid was added dropwise and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, and the crude product was purified by column chromatography to obtain PDSe-GGT.

[0029] Compound PDSe-NH2, (S)-2-((tert-butoxycarbonyl)amino)propionic acid, HATU, and DIPEA were dissolved in an organic solvent and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-APN(Boc). Then, compound PDSe-APN(Boc) was dissolved in anhydrous dichloromethane, trifluoroacetic acid was added dropwise and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, and the crude product was purified by column chromatography to obtain PDSe-APN.

[0030] The compound PDSe-NH2, N-tert-butoxycarbonyl-N'-(acetyl)-L-lysine, HATU, and DIPEA were dissolved in an organic solvent and stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-HC.

[0031] Compound PDSe-LAP and tert-butoxycarbonyl-L-alanine N-succinimide ester were dissolved in an organic solvent, triethylamine was added, and the mixture was stirred overnight. After the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-AL(Boc). Then, compound PDSe-AL(Boc) was dissolved in anhydrous dichloromethane, trifluoroacetic acid was added dropwise, and the mixture was stirred overnight. After the reaction was completed, the solvent was removed under vacuum, and the crude product was purified by column chromatography to obtain PDSe-AL.

[0032] The present invention relates to the application of the quinoline-selenium-based organic photosensitizer or its derivative as a photosensitizer in the preparation of reagents or drugs that generate ROS.

[0033] The generation of ROS includes both in vitro generation and intracellular generation.

[0034] The application of the quinoline-selenium-based organic photosensitizer or its derivatives in the preparation of reagents or drugs for cancer treatment, as described in this invention.

[0035] The organic photosensitizer based on the quinoline-selenium structure or its derivatives have an inhibitory effect on cancer cells under normoxic and hypoxic conditions.

[0036] Furthermore, the photosensitizers PDSe-n and PDSe-8n both exhibit significant cytotoxicity against cancer cells under light irradiation; the photosensitizers PDSe-8 and PDSe-8n both exhibit significant toxicity against cancer cells under both normoxic and hypoxic conditions.

[0037] The application of the derivatives described in this invention in the preparation of reagents or drugs that target lysosomes and mitochondria.

[0038] Furthermore, the photosensitizer PDSe-8 exhibits a concentration-dependent cell death mechanism: low concentrations induce apoptosis, while high concentrations induce pyroptosis.

[0039] The present invention relates to the application of a series of novel organic photosensitizers based on a quinoline-selenium structure, wherein the application includes the ability of photosensitizers PDSe-n and PDSe-8e to generate singlet oxygen in vitro; the application includes the ability of photosensitizers PDSe-n and PDSe-8e to generate superoxide anions in vitro; the application includes the ability of photosensitizer PDSe-8 to generate ROS in cells under normoxic and hypoxic conditions; the application includes the toxicity of photosensitizers PDSe-n and PDSe-8e to cancer cells under light irradiation; the application includes the cytotoxicity of photosensitizers PDSe-8 and PDSe-8n under normoxic and hypoxic conditions; the application includes the lysosomal targeting of photosensitizer PDSe-8; the application includes the mitochondrial targeting of photosensitizer PDSe-8e; and the application includes the concentration-dependent shift in cell death induced by photosensitizer PDSe-8.

[0040] This invention relates to a photosensitizer based on molecular engineering optimization of a quinoline core and verbena extended conjugated system. The introduction of selenium atoms significantly enhances its photophysical properties. This photosensitizer exhibits strong absorption in the near-infrared region and efficiently generates reactive oxygen species in oxygen-rich / hypoxic environments through a type I photodynamic mechanism, with a significantly higher quantum yield compared to traditional photosensitizers. Furthermore, it possesses unique tumor microenvironment response characteristics, enabling precise targeting through enzyme activation strategies (including but not limited to tumor-overexpressed enzymes such as FAP, HDAC, CTSL, NTR, and QR) and the introduction of organelle targeting groups.

[0041] This invention designs a selenium-containing photosensitizer, providing a multifunctional research tool for studying the dynamic generation of reactive oxygen species and the regulation of cell death mechanisms in the treatment of solid tumors. Combined with near-infrared light excitation with deep tissue penetration and precise targeting technology, it shows great potential for treating deep tumors and overcoming resistance to traditional PDT in clinical translation.

[0042] This invention has revealed that quinoline possesses advantages such as simple and easily modifiable structure and low cytotoxicity. Introducing a quinoline skeleton into the molecular structure can endow it with excellent photophysical and photochemical properties. Therefore, this invention uses quinoline as the core skeleton and combines it with the natural product verbena to extend the conjugated system, designing and synthesizing a series of novel selenium-containing photosensitizers by introducing selenium atoms.

[0043] This invention is based on the design principle of a series of quinoline-selenium photosensitizers: The photosensitizers of this invention use quinoline as the core framework, combined with the natural product verbena to extend the conjugated system, constructing a stable D-π-A (donor-π-acceptor) structure. This design achieves highly efficient intramolecular charge transfer (ICT), significantly improving the photophysical properties of the photosensitizer. Through theoretical calculations and experimental verification, we found that this structure can effectively reduce the singlet-triplet energy difference (ΔE). st This process promotes intersystem crossing (ISC) and thus improves ROS generation efficiency. A selenium atom is innovatively introduced into the molecular structure, utilizing its heavy atom effect to further enhance ISC efficiency. The introduction of selenium not only increases ROS yield but also endows the molecule with unique environmental responsiveness. Experiments show that selenium-containing photosensitizers maintain high ROS generation capacity under both normoxic and hypoxic conditions, providing a new approach to solving the hypoxia problem in solid tumors. Based on PDSe-8 and PDSe-NH2, ten derivatives with targeting functions were further developed. These derivatives can precisely target organelles and recognize receptors overexpressed on the surface of tumor cells, significantly improving tumor selectivity and drug delivery efficiency.

[0044] This invention develops a series of photosensitizers with quinoline-selenium structures, which can effectively achieve photodynamic therapy of tumors.

[0045] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0046] This invention develops a novel PDSe series of photosensitizers based on selenium atoms. These photosensitizers achieve highly efficient photodynamic therapy through a unique D-π-A structural design and the heavy atom effect of selenium atoms. The reactive oxygen species generated after photoactivation not only directly kill tumor cells but also significantly enhance the anti-tumor effect through a concentration-dependent dual-action mechanism (low concentration induces apoptosis, high concentration induces pyroptosis). This dynamic regulatory mechanism not only improves therapeutic efficacy but also reduces the risk of drug resistance in tumor cells. Specifically, by introducing targeted ligand-designed PDSe-8 and PDSe-NH2 derivatives, precise targeting of mitochondria and recognition of overexpressed receptors on the surface of tumor cells can be achieved, significantly improving tumor selectivity and drug delivery efficiency. Attached Figure Description

[0047] Figure 1This is a detection graph showing the in vitro generation of singlet oxygen by the photosensitizer PDSe-n listed in this invention;

[0048] Figure 2 This is a detection graph showing the in vitro generation of singlet oxygen by the photosensitizer PDSe-8e listed in this invention;

[0049] Figure 3 This is a detection graph showing the in vitro generation of superoxide anions by the photosensitizer PDSe-n listed in this invention.

[0050] Figure 4 This is a detection graph showing the in vitro generation of superoxide anions by the photosensitizer PDSe-8e listed in this invention;

[0051] Figure 5 To use the DCFH fluorescent probe to detect intracellular reactive oxygen species generation in U87MG cells treated with PDSe-8 under normoxic (21% O2) and hypoxic (2% O2) conditions, confocal imaging analysis and quantification of cell fluorescence intensity were conducted.

[0052] Figure 6 To detect O2 in PDSe-8-treated U87MG cells under normoxic (21% O2) and hypoxic (2% O2) conditions using a DHE fluorescent probe. -· The generated confocal imaging analysis and quantification of cell fluorescence intensity;

[0053] Figure 7 The MTT plot shows the cytotoxicity of the photosensitizer PDSe-n listed in this invention to various cancer cells (including neurogenic tumor cells SH-SY5Y, U251MG, and U87MG) under light and dark conditions.

[0054] Figure 8 For illumination (530nm, 20mW / cm) 2 The live / dead cell count of U87MG cells treated with the photosensitizer PDSe-8 listed in this invention was determined at 10 min.

[0055] Figure 9 A and B are MTT plots showing the cytotoxicity of PDSe-8a-d to U87MG cells under normoxic (21% O2) and hypoxic (2% O2) conditions, as listed in this invention; C and D are MTT plots showing the cytotoxicity of PDSe-8a-d to bEnd.3 cells under normoxic (21% O2) and hypoxic (2% O2) conditions, as listed in this invention.

[0056] Figure 10 The MTT assay for the 48-hour dark toxicity of PDSe-8a-d to bEnd.3 cells under dark conditions, as described in this invention.

[0057] Figure 11The MTT plot shows the cytotoxicity of PDSe-8e to breast cancer cells (MCF-7) under light and dark conditions in hypoxic (2% O2) conditions, as listed in this invention.

[0058] Figure 12 MTT plot of cytotoxicity of PDSe-8e to normal human umbilical vein endothelial cells (HUVEC) under light and dark conditions as listed in this invention;

[0059] Figure 13 This is a detection diagram of lysosome targeting by the photosensitizer PDSe-8 listed in this invention;

[0060] Figure 14 This is a diagram showing the organelle targeting detection of the photosensitizer PDSe-8e listed in this invention;

[0061] Figure 15 The morphological changes of U87MG cells after different light exposure times after treatment with 0.2 and 0.5 μM PDSe-8. Detailed Implementation

[0062] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0063] Example 1

[0064] A series of novel organic photosensitizers based on the quinoline-selenium structure are prepared as follows:

[0065]

[0066]

[0067] Under an argon (Ar) atmosphere, 6-bromo-2-methylquinoline (2.0 g, 9.0 mmol) and selenium dioxide (SeO2, 2.0 g, 18.0 mmol) were dissolved in anhydrous 1,4-dioxane (10 mL). The reaction mixture was stirred at 60 °C for 5 h. After cooling to room temperature, the solvent was removed by rotary evaporation under reduced pressure. The resulting solid was dissolved in ethyl acetate (100 mL), followed by the addition of 50 mL of water. The organic phase was extracted three times, dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated under vacuum to give a pale yellow crude product S-1 (1.5 g, 72% yield).

[0068] Compound S-1 (2.0 g, 8.5 mmol) and verbenaenolide (1.1 g, 7.0 mmol) were dissolved in methanol (MeOH, 10 mL). Potassium hydroxide (KOH, 779.6 mg, 13.89 mmol) was then added, and the mixture was stirred at 60 °C for 6 h under an argon (Ar) atmosphere. After the reaction was complete, the mixture was cooled to room temperature, filtered, washed, and dried under vacuum to give a yellow solid S-2 (1.2 g, 45% yield).

[0069] Compound S-2 (200 mg, 0.54 mmol) and selenium dioxide (SeO2, 910.2 mg, 8.2 mmol) were dissolved in 6 mL of a mixed solution of 1,4-dioxane and pyridine (5:1) and stirred vigorously. After the reaction was complete, the mixture was cooled to room temperature and the solvent was removed under vacuum. The residue was washed with aqueous copper sulfate solution and extracted with ethyl acetate. The crude product was purified by column chromatography (petroleum ether / ethyl acetate, 8:1) to give a white solid S-3 (89.0 mg, 37% yield).

[0070] Compound S-3 (200 mg, 0.45 mmol) and malononitrile (445.9 mg, 6.75 mmol) were stirred in pyridine (5 mL) at 90 °C for 24 h. After the reaction was complete, the mixture was cooled to room temperature and the solvent was removed under reduced pressure. The resulting residue was washed with aqueous copper sulfate solution and extracted with ethyl acetate. The crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (8:1, v / v) as eluent to give yellow solid S-4 (215.3 mg, 97% yield).

[0071] Compound S-4 (200.0 mg, 0.041 mmol), palladium acetate (Pd(OAc)2, 3.6 mg, 0.016 mmol), 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP, 15.6 mg, 0.025 mmol) and cesium carbonate (Cs2CO3, 202 mg, 0.62 mmol) were dissolved in toluene (5 mL) and reacted under an argon atmosphere. The reaction mixture was activated by stirring at 110 °C for 20 min. Then, 2.05 mmol of amine compounds with different structures were added to the activated system, specifically: PDSe-1: azacyclobutane, PDSe-2: pyrrolidine, PDSe-3: piperidine, PDSe-4: morpholine, PDSe-5: N-methylpiperazine, PDSe-6: N-ethylpiperazine, PDSe-7: 1-(tert-butoxycarbonyl)piperazine, PDSe-9: thiomorpholine, PDSe-10: 4,4-difluoropiperidine, and PDSe-11: cyclohexylpiperazine. The reaction mixture was refluxed at 110 °C for 15 h. After the reaction was complete, the mixture was cooled to room temperature, extracted with ethyl acetate, and concentrated under reduced pressure. The residues were purified by silica gel column chromatography. The eluent systems for each target product PDSe-n were as follows: PDSe-1: petroleum ether / ethyl acetate (v / v: 30:1), PDSe-2: petroleum ether / ethyl acetate (v / v: 50:1), PDSe-3: petroleum ether / ethyl acetate (v / v: 30:1), PDSe-4: petroleum ether / ethyl acetate (v / v: 100:1), PDSe-5: petroleum ether / ethyl acetate (v / v: 30:1), PDSe-6: petroleum ether / ethyl acetate (v / v: 40:1), PDSe-7: petroleum ether / ethyl acetate (v / v: 50:1), PDSe-9: petroleum ether / ethyl acetate (v / v: 40:1), PDSe-10: petroleum ether / ethyl acetate (v / v: 20:1), PDSe-11: petroleum ether / ethyl acetate (v / v: 10:1).

[0072] Compound PDSe-7 (50 mg, 0.08 mmol) was added to a flask and dissolved in 4 mL of anhydrous dichloromethane. At room temperature, 1 mL of trifluoroacetic acid was slowly added dropwise to the reaction solution to achieve a final volume fraction of 20%. The reaction was allowed to proceed for 12 h, followed by dichloromethane extraction and concentration under reduced pressure. The resulting residue was purified by silica gel column chromatography using dichloromethane / methanol as the eluent (v / v: 200:1) to yield the target product PDSe-8.

[0073] Palladium acetate (Pd(OAc)₂, 10.37 mg, 0.046 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (Xantphos, 46.17 mg, 0.080 mmol), and cesium carbonate (Cs₂CO₃, 469.83 mg, 1.44 mmol) were dissolved in toluene (15 mL) and reacted under an argon atmosphere. The reaction mixture was activated by stirring at 100 °C. After the solution turned red, compound S-4 (280.0 mg, 0.57 mmol) and tert-butyl carbamate (80.36 mg, 0.69 mmol) were added to the activated system, and the mixture was refluxed at 100 °C for 12 h. After the reaction was complete, the mixture was cooled to room temperature, extracted with dichloromethane, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (4:1, v / v) as eluent to give a yellow solid PDSe-12 (200 mg, yield 66.5%).

[0074] Compound PDSe-12 (200 mg, 0.38 mmol) was added to a flask and dissolved in 8 mL of anhydrous dichloromethane. At room temperature, 2 mL of trifluoroacetic acid was slowly added dropwise to the reaction solution to achieve a final volume fraction of 20%. The reaction was allowed to proceed for 12 h, followed by dichloromethane extraction and concentration under reduced pressure. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (1:1, v / v) as the eluent to give a red solid PDSe-NH2 (188.2 mg, yield 72.1%). Compound PDSe-8 (50 mg, 0.1 mmol) and Boc-glycine-L-proline (68.07 mg, 0.25 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU 45.63 mg, 0.12 mmol), and N,N-diisopropylethylamine (DIPEA 19.39 mg, 0.15 mmol) were dissolved in 5 mL of dichloromethane and stirred overnight. After the reaction was complete, the solvent was removed under vacuum. The residue was extracted with ethyl acetate. The crude product was purified by column chromatography (dichloromethane / methanol, v / v, 50:1) to give PDSe-8a (42.2 mg, 56% yield).

[0075] Compound PDSe-8 (50 mg, 0.1 mmol) and p-nitrobenzyl chloroformate (21.56 mg, 0.1 mmol) were dissolved in 5 mL of dichloromethane, and 0.1 mmol of triethylamine was added. The mixture was stirred overnight. After the reaction was complete, the solvent was removed under vacuum. The residue was extracted with ethyl acetate. The crude product was purified by column chromatography (petroleum ether / ethyl acetate, v / v, 4:1) to give PDSe-8b (42.7 mg, 63% yield).

[0076] Compound PDSe-8 (50 mg, 0.1 mmol) and 3-(2,3,5-trimethyl-1,4-benzoquinone)-3-methylbutyric acid (62.57 mg, 0.25 mmol), HATU (45.63 mg, 0.12 mmol), and DIPEA (19.39 mg, 0.15 mmol) were dissolved in 5 mL of dichloromethane and stirred overnight. After the reaction was complete, the solvent was removed under vacuum. The residue was extracted with ethyl acetate. The crude product was purified by column chromatography (petroleum ether / ethyl acetate, v / v, 4:1) to give PDSe-8c (53.4 mg, 73% yield).

[0077] Compound PDSe-8 (50 mg, 0.1 mmol) and N-tert-butoxycarbonyl-N'-(acetyl)-L-lysine (70.1 mg, 0.25 mmol), HATU (45.63 mg, 0.12 mmol), and DIPEA (19.39 mg, 0.15 mmol) were dissolved in 5 mL of dichloromethane and stirred overnight. After the reaction was complete, the solvent was removed under vacuum. The residue was extracted with ethyl acetate. The crude product was purified by column chromatography (ethyl acetate / methanol, v / v, 100:1) to give PDSe-8d (53.8 mg, 70% yield).

[0078] Compound PDSe-8 (50 mg, 0.1 mmol) and 2-carboxyethyltriphenylphosphine bromide (62.3 mg, 0.15 mmol), HOBT (18.9 mg, 0.14 mmol), and EDCI (26.8 mg, 0.14 mmol) were dissolved in 5 mL of N,N-dimethylformamide (DMF), and 0.15 mmol of triethylamine was added. The mixture was stirred overnight. After the reaction was complete, the solvent was removed under vacuum. The residue was extracted with ethyl acetate. The crude product was purified by column chromatography (dichloromethane / methanol, v / v, 200:1) to give PDSe-8e (45.8 mg, 77% yield).

[0079]

[0080]

[0081] Boc-L-leucine (161.35 mg, 0.70 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU 265.26 mg, 0.70 mmol), and N,N-diisopropylethylamine (DIPEA 120.23 mg, 0.90 mmol) were dissolved in 4 mL of N,N-dimethylformamide (DMF). After reacting at room temperature for 5 min, compound PDSe-NH2 (200 mg, 0.47 mmol) was added to the activated system, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (4:1, v / v) as eluent to give a yellow solid PDSe-LAP(Boc) (162.1 mg, yield 53.7%).

[0082] The compound PDSe-LAP (Boc) (162.1 mg, 0.25 mmol) was added to a flask and dissolved in 8 mL of anhydrous dichloromethane. At room temperature, 2 mL of trifluoroacetic acid was slowly added dropwise to the reaction solution until the final volume fraction was 20%. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography using (dichloromethane / methanol, v / v, 40:1) as the eluent to give a yellow solid PDSe-LAP (125.7 mg, yield 92.0%).

[0083] N-Boc-D-glutamic acid-1-tert-butyl ester (209.30 mg, 0.70 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU 265.26 mg, 0.70 mmol), and N,N-diisopropylethylamine (DIPEA 120.23 mg, 0.90 mmol) were dissolved in 4 mL of N,N-dimethylformamide (DMF). After reacting at room temperature for 5 min, compound PDSe-NH2 (200 mg, 0.47 mmol) was added to the activated system, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (4:1, v / v) as eluent to give a yellow solid PDSe-GGT(Boc) (152 mg, yield 45.3%).

[0084] The compound PDSe-GGT(Boc) (162.1 mg, 0.25 mmol) was added to a flask and dissolved in 7 mL of anhydrous dichloromethane. At room temperature, 3 mL of trifluoroacetic acid was slowly added dropwise to the reaction solution until the final volume fraction was 30%. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography using (dichloromethane / methanol, v / v, 10:1) as the eluent to give a yellow solid PDSe-GGT (82.3 mg, yield 69.2%).

[0085] (S)-2-((tert-butoxycarbonyl)amino)propionic acid (132.0 mg, 0.70 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU 265.26 mg, 0.70 mmol), and N,N-diisopropylethylamine (DIPEA 120.23 mg, 0.90 mmol) were dissolved in 4 mL of N,N-dimethylformamide (DMF). After reacting at room temperature for 5 min, compound PDSe-NH2 (200 mg, 0.47 mmol) was added to the activated system, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (4:1, v / v) as eluent to give a yellow solid PDSe-APN(Boc) (179.5 mg, yield 63.6%).

[0086] The compound PDSe-APN(Boc) (179.5 mg, 0.30 mmol) was added to a flask and dissolved in 8 mL of anhydrous dichloromethane. At room temperature, 2 mL of trifluoroacetic acid was slowly added dropwise to the reaction solution until the final volume fraction was 20%. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography using (dichloromethane / methanol, v / v, 20:1) as the eluent to give a yellow solid PDSe-APN (103.9 mg, yield 69.7%).

[0087] N-tert-butoxycarbonyl-N'-(acetyl)-L-lysine (100.6 mg, 0.35 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU 132.63 mg, 0.35 mmol), and N,N-diisopropylethylamine (DIPEA 60.12 mg, 0.47 mmol) were dissolved in 4 mL of N,N-dimethylformamide (DMF). After reacting at room temperature for 5 min, compound PDSe-NH2 (100 mg, 0.23 mmol) was added to the activated system, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using (dichloromethane / methanol, v / v, 50:1) as the eluent to give a yellow solid PDSe-HC (35.1 mg, yield 50.2%).

[0088] The compound PDSe-LAP (108.5 mg, 0.20 mmol) was added to a flask and dissolved in 3 mL of N,N-dimethylformamide (DMF). Then, 50 μL of triethylamine was added to the reaction solution, followed by the addition of tert-butoxycarbonyl-L-alanine N-succinimide ester (114.5 mg, 0.40 mmol). The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (4:1, v / v) as the eluent to give a yellow solid PDSe-AL(Boc) (85.7 mg, yield 60.0%).

[0089] The compound PDSe-AL(Boc) (85.7 mg, 0.12 mmol) was added to a flask and dissolved in 8 mL of anhydrous dichloromethane. At room temperature, 2 mL of trifluoroacetic acid was slowly added dropwise to the reaction solution until the final volume fraction was 20%. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography using (dichloromethane / methanol, v / v, 40:1) as the eluent to give a yellow solid PDSe-AL (63.7 mg, yield 86.5%).

[0090] PDSe-1: Red solid (145 mg, yield 76%) 11H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.8 Hz, 2H), 7.71–7.60 (m, 2H), 7.01 (dd, J = 9.1, 2.6 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H), 4.06 (t, J = 7.3 Hz, 4H), 3.49 (t, J = 5.7 Hz, 1H), 3.19 (t, J = 5.5 Hz, 1H), 3.04 (dt, J = 10.4, 5.5 Hz, 1H), 2.47 (p, J = 7.2 Hz, 2H), 2.17 (d, J = 9.6 Hz, 1H), 1.64 (s, 3H), 0.85 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 171.5, 164.3, 150.2, 147.6, 142.7, 134.0, 131.3, 130.6, 129.7, 124.6, 118.5, 117.2, 115.1, 114.2, 103.0, 70.8, 56.1, 52.1, 51.7, 47.4, 42.2, 29.7, 26.6, 22.5, 16.7. MS(ESI): C 26 H 22 N4Se [M + H] + , m / z calcd. 471.1083, found 471.1072.

[0091] PDSe-2, red solid (127.6 mg, yield 65%), 1 1H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H), 7.25 (d, J = 9.0 Hz, 1H), 6.65–6.60 (m, 1H), 3.47 (q, J = 6.2 Hz, 5H), 3.21 (s, 1H), 3.06 (dd, J = 9.7, 5.2 Hz, 1H), 2.18 (d, J = 9.6 Hz, 5H), 1.65 (s, 3H), 0.85 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 171.5, 164.2, 164.0, 150.5, 148.6, 143.4, 134.8, 131.5, 130.3, 129.6, 124.8, 123.4, ll7.0, 115.1, 114.1, 108.0, 71.0, 56.1, 51.7, 49.9, 47.5, 42.2, 29.7, 26.6, 25.7, 24.3, 22.5. MS(ESI): C 27 H 24 N4Se [M + H] +,m / z calcd.485.1239, found 485.1231.

[0092] PDSe-3, red solid (117.1 mg, yield 58%) 1 H NMR (400MHz, CDCl3) δ7.95(dd,J=13.8,8.9Hz,2H),7.76–7.59(m,2H),7.52(d,J=9.5Hz,1H),6.98(s,1H),3.49(t,J=5.8Hz,1H),3.36(t ,J=5.3Hz,4H),3.19(d,J=5.7Hz,1H),3.05(dt,J=10.8,5.4Hz,1H),2.17(d,J=9.6Hz,1H),1.84–1.66(m,6H),1.64(s,3H),0.85(s,3H). 13 C NMR (101MHz, CDCl3) δ171.5,164.2,164.0,150.5,148.6,143.4,134.8,131.5,130.3,129.6,124.8,123.4 ,117.0,115.1,114.1,108.0,71.0,56.1,51.7,49.9,47.5,42.2,29.7,26.6,25.7,24.3,22.5.MS(ESI):C 28 H 26 N4Se[M+H] + ,m / z calcd.499.1396, found 499.1381.

[0093] PDSe-4, red solid (146 mg, yield 72%) 1 H NMR (400MHz, CDCl3) δ7.98 (dd, J=12.4, 9.0Hz, 2H), 7.72 (d, J=8.6Hz, 1H), 7.65 (d,J=0.9Hz,1H),7.49(dd,J=9.3,2.7Hz,1H),6.98(d,J=2.7Hz,1H),3.92(t,J =4.8Hz,4H),3.48(t,J=5.7Hz,1H),3.32(t,J=4.9Hz,4H),3.18(t,J=5.6Hz,1H ), 3.03(dt,J=9.9,5.5Hz,1H),2.15(d,J=9.6Hz,1H),1.62(s,3H),0.82(s,3H). 13C NMR (101MHz, CDCl3) δ171.6,164.2,163.6,149.8,149.2,143.8,135.2,131.8,130.5,129.3,125.2,12 2.4,117.2,115.0,114.0,108.7,71.3,66.7,56.1,51.7,48.9,47.4,42.2,29.7,26.6,22.5.MS(ESI):C 27 H 24 N4Se[M+H] + ,m / z calcd.501.1189, found 501.1211.

[0094] PDSe-5, red solid (131.1 mg, yield 63%) 1 H NMR (400MHz, CDCl3) δ7.98 (dd, J=12.4, 9.0Hz, 2H), 7.73 (d, J=8.6Hz, 1H), 7.67 ( s,1H),7.52(dd,J=9.3,2.7Hz,1H),7.01(d,J=2.7Hz,1H),5.32(s,1H),3.55–3. 49(m,2H),3.45(t,J=5.0Hz,3H),3.20(t,J=5.5Hz,1H),3.06(dt,J=9.7,5.5Hz, 1H), 2.73 (s, 4H), 2.47 (s, 2H), 2.18 (d, J = 9.6Hz, 1H), 1.65 (s, 3H), 0.85 (s, 3H). 13 C NMR (101MHz, CDCl3) δ171.6,164.2,163.8,149.7,149.0,143.7,135.1,131.7,130.4,129.4,125.1,122.9 ,117.1,115.1,114.1,108.3,71.2,56.1,54.9,51.7,48.5,47.4,46.1,42.3,26.6,22.7,22.5.MS(ESI):C 28 H 27 N5Se[M+H] + ,m / zcalcd.514.1505,found 514.1484.

[0095] PDSe-6, red solid (147.5 mg, yield 69%) 11H NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 17.8, 9.0 Hz, 2H), 7.72 (d, J = 8.7 Hz, 1H), 7.66 (s, 1H), 7.53 (d, J = 9.5 Hz, 1H), 7.00 (s, 1H), 3.49 (t, J = 5.6 Hz, 1H), 3.41 (t, J = 5.0 Hz, 4H), 3.19 (t, J = 5.5 Hz, 1H), 3.04 (dt, J = 10.5, 5.5 Hz, 1H), 2.72–2.65 (m, 4H), 2.53 (q, J = 7.2 Hz, 2H), 2.17 (d, J = 9.6 Hz, 1H), 1.64 (s, 3H), 1.18 (t, J = 7.2 Hz, 3H), 0.84 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 171.6, 164.2, 163.8, 149.8, 148.9, 143.7, 135.1, 131.7, 130.4, 129.4, 125.0, 122.9, 117.1, 115.1, 114.1, 108.2, 71.1, 56.1, 52.7, 52.4, 51.7, 48.6, 47.4, 42.3, 29.8, 26.6, 22.5, 12.1. MS (ESI): C 29 H 29 N5Se [M+H] + , m / z calcd. 528.1661, found 528.1653.

[0096] PDSe-7, red solid (191.9 mg, yield 79%), 1 1H NMR (400 MHz, CDCl3) δ 8.00 (t, J = 8.5 Hz, 2H), 7.74 (d, J = 8.7 Hz, 1H), 7.69 (s, 1H), 7.52 (dd, J = 9.4, 2.7 Hz, 1H), 7.01 (d, J = 2.7 Hz, 1H), 3.66 (t, J = 5.1 Hz, 4H), 3.51 (t, J = 2.9 Hz, 1H), 3.34 (dd, J = 6.6, 3.9 Hz, 4H), 3.21 (t, J = 5.5 Hz, 1H), 3.06 (dt, J = 9.5, 5.5 Hz, 1H), 2.18 (d, J = 9.6 Hz, 1H), 1.65 (s, 3H), 0.85 (s, 3H). 13C NMR (101MHz, CDCl3) δ171.6,164.1,163.6,154.7,149.7,149.7,143.8,135.2,131.9,130.5,129.3,125.2,123.3,117 .2,115.0,114.0,108.9,80.2,71.3,56.1,51.7,48.8,47.4,42.3,31.5,31.5,30.1,29.7,28.5,26.6,22.5.MS(ESI):C 32 H 33 N5O2Se[M+H] + ,m / z calcd.600.1873, found 600.1872.

[0097] PDSe-8, red solid (141.7 mg, 70% yield), 1 H NMR (400MHz, CDCl3) δ8.07–7.98 (m, 2H), 7.77 (d, J = 8.6Hz, 1H), 7.70 (s, 1H), 7.52 (dd,J=9.3,2.6Hz,1H),7.04(d,J=2.6Hz,1H),3.90(dt,J=29.7,5.0Hz,4H),3.51( t,J=5.7Hz,1H),3.44(t,J=5.1Hz,4H),3.21(t,J=5.6Hz,1H),3.07(dt,J=10.6,5 .5Hz,1H),2.18(d,J=9.6Hz,1H),1.66(s,3H),1.28(d,J=3.9Hz,1H),0.86(s,3H). 13 C NMR(101MHz,DMSO-d6)δ171.6,164.2,163.7,150.1,149.1,143.8,135.1,131.8,130.4,129.4,125.1 ,123.0,117.1,115.1,114.1,108.4,71.2,56.1,51.7,49.6,47.4,45.8,42.3,26.6,22.5.MS(ESI):C 27 H 25 N5Se[M+H] + ,m / z calcd.500.1348, found 500.1342.

[0098] PDSe-9, red solid (146.5 mg, yield 61%) 11H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.48 (dd, J = 9.4, 2.7 Hz, 1H), 7.00 (s, 1H), 3.80–3.76 (m, 4H), 3.51 (t, J = 5.6 Hz, 1H), 3.21 (t, J = 5.6 Hz, 1H), 3.06 (dt, J = 10.4, 5.4 Hz, 1H), 2.83 (t, J = 4.9 Hz, 4H), 2.18 (d, J = 9.6 Hz, 1H), 1.65 (s, 3H), 0.85 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 171.8, 164.2, 163.7, 149.3, 149.1, 143.6, 134.9, 131.8, 130.7, 129.5, 125.1, 123.4, 117.2, 115.1, 114.0, 109.0, 71.3, 56.1, 51.7, 51.5, 47.4, 42.3, 26.6, 26.6, 22.5. MS(ESI): C 27 H 24 N4SSe [M+H] + , m / z calcd. 517.0960, found 517.0955.

[0099] PDSe-10, red solid (160.3 mg, yield 74%), 1 1H NMR (400 MHz, CD3CN) δ 8.15 (d, J = 8.7 Hz, 1H), 7.98–7.87 (m, 3H), 7.64 (dd, J = 9.3, 2.8 Hz, 1H), 7.25 (d, J = 2.8 Hz, 1H), 3.57 (dd, J = 6.7, 4.9 Hz, 4H), 3.45 (t, J = 5.7 Hz, 1H), 3.27 (t, J = 5.6 Hz, 1H), 3.11 (dt, J = 9.7, 5.5 Hz, 1H), 2.15 (s, 4H), 1.65 (s, 3H), 0.83 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 171.6, 164.1, 163.5, 149.5, 148.6, 143.8, 135.1, 131.9, 130.7, 129.3, 125.2, 123.4, 117.2, 115.0, 114.0, 109.2, 71.4, 56.1, 51.7, 47.4, 46.3, 42.2, 33.8, 33.6, 33.3, 26.6, 22.5. MS(ESI): C 28 H24 F2N4Se[M+H] + ,m / z calcd.535.1208,found535.1202.

[0100] PDSe-11, red solid (124.9 mg, yield 53%) 1 H NMR (400MHz, CDCl3) δ7.97 (dd, J=14.9, 9.0Hz, 2H), 7.72 (d, J=8.6Hz, 1H), 7.66 (s, 1H), 7 .53(dd,J=9.3,2.7Hz,1H),6.99(d,J=2.6Hz,1H),3.50(t,J=5.6Hz,1H),3.39(t,J=4.9Hz ,4H),3.20(t,J=5.5Hz,1H),3.05(dt,J=10.5,5.5Hz,1H),2.81(t,J=5.0Hz,4H),2.35(d, J=10.0Hz,1H),2.17(d,J=9.6Hz,1H),1.99–1.78(m,5H),1.34–1.20(m,5H),0.85(s,3H). 13 C NMR (101MHz, CDCl3) δ171.5,164.2,163.9,149.9,148.9,143.6,135.0,131.7,130.3,129.5,125.0,122.8,117. 1,115.1,114.1,108.1,71.2,63.5,56.1,51.7,49.1,48.9,47.4,42.2,29.0,26.6,26.3,25.9,22.5.MS(ESI):C 33 H 35 N5Se[M+H] + ,m / zcalcd.582.2131, found 582.2145.

[0101] PDSe-8a red solid (42.2 mg, yield 56%): 11H NMR (400 MHz, CDCl3) δ 8.00 (dd, J = 13.8, 8.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 1H), 7.68 (s, 1H), 7.51 (dd, J = 9.4, 2.6 Hz, 1H), 7.01 (d, J = 2.6 Hz, 1H), 5.41 (t, J = 4.6 Hz, 1H), 4.95 (dd, J = 8.0, 3.7 Hz, 1H), 4.07 (dd, J = 17.4, 5.5 Hz, 1H), 3.95 (td, J = 13.8, 12.2, 7.1 Hz, 3H), 3.78 - 3.66 (m, 3H), 3.51 (dt, J = 10.8, 4.4 Hz, 3H), 3.37 (t, J = 6.6 Hz, 3H), 3.21 (t, J = 5.6 Hz, 1H), 3.06 (dt, J = 10.5, 5.6 Hz, 1H), 2.23 - 2.15 (m, 2H), 2.02 (ddq, J = 31.0, 11.4, 6.4, 5.8 Hz, 3H), 1.64 (s, 3H) 1.44 (s, 9H), 0.85 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 171.7, 170.1, 167.3, 164.2, 163.5, 155.9, 149.4, 149.3, 143.9, 135.3, 13¹.9, 130.5, 129.2, 125.3, 123.1, 117.2, 115.1, 114.0, 109.1, 79.6, 71.3, 56.5, 56.1, 55.4, 51.7, 49.00, 48.7, 47.4, 46.1, 45.4, 43.0, 42.3, 41.9, 31.6, 29.0, 28.4, 26.6, 24.7, 22.5. MS (ESI): C 39 H 43 N7O4Se [M + H] + , m / z calcd. 754.2615, found 754.2615.

[0102] PDSe - 8b red solid (42.7 mg, yield 63%): 11H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 8.4 Hz, 2H), 8.00 (dd, J = 13.8, 8.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 1H), 7.68 (s, 1H), 7.60 - 7.48 (m, 3H), 7.02 (d, J = 2.6 Hz, 1H), 5.30 (s, 2H), 3.82 - 3.74 (m, 4H), 3.50 (t, J = 5.8 Hz, 1H), 3.37 (t, J = 5.0 Hz, 4H), 3.20 (t, J = 5.6 Hz, 1H), 3.05 (dt, J = 10.4, 5.6 Hz, 1H), 2.17 (d, J = 9.6 Hz, 1H), 1.65 (s, 3H), 0.85 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 171.6, 164.1, 163.4, 154.7, 149.5, 149.4, 147.7, 144.0, 143.9, 135.3, 131.9, 130.6, 129.2, 128.2, 125.3, 123.9, 123.3, 117.2, 115.0, 114.0, 109.2, 71.4, 65.9, 56.1, 51.7, 47.4, 42.3, 31.6, 26.6, 22.7, 22.5, 14.2. MS (ESI): C 35 1H 30 6N6O4Se [M + H] + , m / z calcd. 679.1567, found 679.1580.

[0103] PDSe - 8c red solid (53.4 mg, yield 73%): 1 1H NMR (400 MHz, CDCl3 δ 8.01 (t, J = 8.6 Hz, 2H), 7.75 (d, J = 8.6 Hz, 1H), 7.69 (s, 1H), 7.52 (dd, J = 9.4, 2.7 Hz, 1H), 7.01 (d, J = 2.8 Hz, 1H), 3.75 (p, J = 5.0 Hz, 4H), 3.51 (t, J = 5.8 Hz, 1H), 3.41 (d, J = 5.2 Hz, 2H), 3.30 (t, J = 5.2 Hz, 2H), 3.21 (t, J = 5.6 Hz, 1H), 3.07 (d, J = 16.0 Hz, 3H), 2.17 (d, J = 2.4 Hz, 4H), 1.95 (dd, J = 13.6, 1.4 Hz, 6H), 1.65 (s, 3H), 1.62 (s, 3H), 1.49 (s, 3H), 0.85 (s, 3H). 13C NMR (101MHz, CDCl3) δ191.4,187.7,171.6,170.6,164.1,163.5,154.3,149 .3,144.0,143.1,138.1,136.6,135.2,132.0,130.6,129.2,125.2,123.1,1 17.2,115.0,114.0,109.0,71.4,56.1,51.7,49.1,48.9,47.4,46.9,45.3, 42.2,41.1,37.8,31.6,28.9,26.6,22.7,22.5,14.2,12.7,12.2.MS(ESI):C 41 H 41 N5O3Se[M+H] + ,m / zcalcd.732.2447,found 732.2454.

[0104] PDSe-8d red solid (53.8 mg, 70% yield): 1 H NMR (400MHz, CDCl3) δ8.01(dd,J=13.6,8.9Hz,2H),7.75(d,J=8.6Hz,1H),7.68(s,1H),7.51(dd,J=9.4,2.6Hz,1H),7 .02(d,J=2.6Hz,1H),5.80(s,1H),5.52(d,J=8.6Hz,1H),4.66(td,J=8.6,4.3Hz,1H),3.97-3.88(m,1H),3.77(ddd,J= 28.4,13.0,6.8Hz,3H),3.50(t,J=5.6Hz,1H),3.45-3.30(m,5H),3.22(dt,J=10.6,5.9Hz,2H),3.05(dt,J=10.6,5.4H z,1H),2.17(d,J=9.6Hz,1H),1.99(s,3H),1.81-1.71(m,3H),1.59(dd,J=14.4,7.2Hz,6H),1.47(s,9H),0.85(s,3H). 13C NMR (101MHz, CDCl3) δ171.6,170.7,170.2,164.1,163.4,155.8,149.6,1 49.2,144.0,135.3,132.0,130.7,129.2,125.3,123.2,117.2,115.0,114 .0,109.3,79.9,71.4,56.1,51.7,49.7,49.2,48.8,47.4,45.3,42.3,41 .9,39.3,33.3,31.6,29.0,28.4,26.6,23.4,22.7,22.5,14.2.MS(ESI):C 40 H 47 N7O4Se[M+H] + ,m / z calcd.770.2928,found 770.2927.

[0105] PDSe-8e red solid (45.8 mg, yield 77%): 1 H NMR(400MHz, CDCl3)δ8.02(d,J=8.6Hz,1H),7.94(d,J=9.2Hz,1H),7.91–7.78(m, 10H),7.76–7.69(m,7H),7.45(d,J=9.4Hz,1H),7.01(s,1H),3.89(d,J=28.2Hz,4 H),3.62(d,J=5.2Hz,2H),3.49(q,J=5.6Hz,1H),3.36(s,4H),3.21(q,J=5.9Hz,3 H),3.05(dt,J=10.8,5.5Hz,1H),2.17(d,J=9.6Hz,1H),1.88(s,3H),0.84(s,3H). 13 C NMR (101MHz, CDCl3) δ171.7,168.2,168.1,164.3,163.7,149.2,149.2,143. 8,135.4,135.1,135.0,133.9,133.9,131.7,130.5,130.4,129.3,125.3,12 2.9,118.9,118.1,117.2,115.1,114.1,109.0,71.1,56.2,51.7,50.6,48.7 ,48.6,47.4,45.2,42.3,41.9,29.7,27.1,26.6,22.5,19.5,18.9.MS(ESI):C 48 H 43 N5OPSe + [M]+ ,m / zcalcd.816.2365,found 816.2365.

[0106] PDSe-12, yellow solid (200 mg, yield 66.5%) 1 H NMR (400MHz, CDCl3) δ8.12(d,J=8.6Hz,1H),8.02(d,J=9.0Hz,1H),7.78(d,J=8.7Hz,1H),7.71(s,1H),7.49–7.44(m,1H),6.77(s, 1H),3.52(t,J=5.7Hz,1H),3.22(t,J=5.5Hz,1H),3.08–3.03(m,1H),2.18(d,J=9.6Hz,1H),1.66(s,3H),1.59(s,9H),0.86(s,3H). 13 C NMR (101MHz, CDCl3) δ171.7,164.0,163.1,152.6,150.5,145.0,137.1,136.3,132.4,130.5,128.8,125.7 ,123.3,117.2,114.9,113.9,113.4,81.3,77.3,71.7,56.1,51.8,47.4,42.2,28.3,26.6,22.5.MS(ESI):C 28 H 26 N4O2Se[M+H] + ,m / z calcd.530.1221,found531.1296.

[0107] PDSe-NH2, red solid (188.2 mg, yield 72.1%) 1 H NMR (400MHz, DMSO) δ8.09–8.02(m,2H),7.96(d,J=8.7Hz,1H),7.74(d,J=9.0Hz,1H),7.20(dd,J=9.0,2.4Hz,1H),6.81(d,J=2.5Hz, 1H),5.97(s,2H),3.24(t,J=5.6Hz,1H),3.10(dt,J=10.5,5.5Hz,1H),2.12(d,J=9.6Hz,1H),1.61(s,3H),1.24(s,1H),0.77(s,3H). 13CNMR(101MHz,DMSO)δ172.0,166.2,164.0,148.9,146.8,142.0,134.3,130.7,130.3,130.2,1 26.3,123.2,117.7,115.7,114.4,105.0,69.8,56.5,51.9,47.3,42.5,26.6,22.6.MS(ESI):C 23 H 18 N4Se[M+H] + ,m / zcalcd.430.0697,found 431.0765.

[0108] PDSe-LAP (Boc), yellow solid (162.1 mg, yield 53.7%). 1 H NMR (400MHz, CDCl3) δ9.14(d,J=23.3Hz,1H),8.25(d,J=21.3Hz,1H),7.90(d,J=8.8Hz,2H ),7.62(d,J=21.7Hz,2H),7.51–7.40(m,1H),5.12(d,J=7.4Hz,1H),4.41(s,1H),3.53(d, J=5.6Hz,1H),3.21–3.16(m,1H),3.10–3.05(m,1H),2.18(dd,J=9.6,2.6Hz,1H),1.84–1. 78(m,2H),1.67(s,3H),1.53(d,J=2.4Hz,9H),1.04–1.00(m,6H),0.86(d,J=15.5Hz,3H). 13 C NMR (101MHz, CDCl3) δ171.8,171.7,171.6,171.5,164.0,136.6,136.4,132.4,130.0,125.7,123.9,123.7,116.8,115.4,11 4.9,113.9,81.0,71.8,56.1,56.0,51.8,47.4,42.2,28.5,28.4,26.6,24.9,23.1,23.1,22.6,22.5,21.7,21.6.MS(ESI):C 34 H 37 N5O3Se[M+H] + ,m / zcalcd.643.2062, found 644.2148.

[0109] PDSe-LAP, yellow solid (125.7 mg, yield 92.0%) 1H NMR(400MHz,MeOD)δ8.37(d,J=2.3Hz,1H),8.23(d,J=8.7Hz,1H),8.02–7.93(m,3H),7.83(dd,J=9.1,2.4Hz,1H),3.66(dd,J=8.4,5.8Hz,1H),3.46(t,J=5.7Hz,1H),3.27(t,J=5.5Hz,1H),3.17–3.11(m,1H),2.17(d,J=9.7Hz,1H),1.88–1.78(m,1H),1.77–1.70(m,1H),1.67(s,3H),1.60(ddd,J=13.9,8.4,6.0Hz,1H),1.04(t,J=6.2Hz,6H),0.85(s,3H). 13 C NMR(101MHz,DMSO)δ175.1,172.2,165.9,162.4,150.8,144.8,138.2,137.4,131.7,129.9,128.9,128.1,125.0,118.1,115.5,115.3,114.2,71.1,56.5,54.33,52.0,47.2,44.0,42.5,26.6,24.7,23.6,22.6,22.4.MS(ESI):C 29 H 29 N5OSe[M+H] + ,m / z calcd.543.1537,found 544.1589.

[0110] PDSe-GGT (Boc), yellow solid (152 mg, yield 45.3%), 1H NMR (400 MHz, CDCl3) δ 9.55 (s, 1H), 8.55 (s, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.05 (d, J = 9.0 Hz, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.75–7.66 (m, 2H), 5.46 (d, J = 8.0 Hz, 1H), 4.28 (d, J = 8.8 Hz, 1H), 3.52 (t, J = 5.7 Hz, 1H) ,3.22(t,J=5.5Hz,1H),3.11–3.05(m,1H),2.55(dd,J=7.6,4.5Hz,2H),2.34(dd,J=8.1,4.5Hz,1H),2 .19(d,J=9.6Hz,1H),1.92(d,J=12.6Hz,1H),1.66(s,3H),1.54(s,9H),1.49(s,9H),0.86(s,3H).13C NMR (101MHz, CDCl3) δ171.7,171.1,164.0,163.1,157.0,150.8,145.5,137.2,136.62,132.4,130.3,128.7,125.8,124.2,117.1 ,115.4,114.9,113.9,83.0,80.9,71.8,56.1,53.1,51.8,47.4,42.2,34.4,31.6,31.2,28.4,28.3,28.0,26.6,22.5.MS(ESI):C 37 H 41 N5O5Se[M+H] + ,m / z calcd.715.2273, found 716.2346.

[0111] PDSe-GGT, yellow solid (82.3 mg, yield 69.2%), ¹H NMR (400 MHz, DMSO) δ 10.89 (s, ¹H), 8.36 (d, J = 2.2 Hz, ¹H), 8.31 (d, J = 8.7 Hz, ¹H), 8.15 (s, ¹H), 8.06 (d, J = 8.7 Hz, ¹H), 7.91–7.84 (m, 2H), 3.52 (s, ¹H), 3.34 (s, ¹H), 3.24 (d, J = 5.4 Hz, ¹H), 3.10 (dd, J = 9.9, 5.2 Hz, ¹H), 2.64 (q, J = 8.6 Hz, 2H), 2.13–2.06 (m, 3H), 1.61 (s, 3H), 0.77 (s, 3H). ¹³C NMR (101MHz, DMSO) δ171.6,170.9,165.9,162.5,150.6,144.7,138.5,137.3,131.6,129.8,128.9,128.0, 124.8,118.0,115.5,115.0,114.2,71.0,56.5,53.3,52.0,47.2,42.5,33.0,27.0,26.6,22.6.MS(ESI):C 28 H 25 N5O3Se[M+H] + ,m / z calcd.559.1123,found 560.1189.

[0112] PDSe-APN(Boc), yellow solid (179.5 mg, yield 63.6%) 1 H NMR(400MHz, CDCl3)δ9.37(d,J=20.0Hz,1H),8.14(d,J=38.4Hz,1H),7.83–7.75(m, 2H),7.57(d,J=25.0Hz,1H),7.50–7.35(m,2H),5.40–5.37(m,1H),4.52(s,1H),3.5 2–3.49(m,1H),3.18–3.14(m,1H),3.09–3.05(m,1H),2.18–2.15(m,1H),1.65(d,J= 5.1Hz, 3H), 1.53 (d, J = 2.4Hz, 9H), 0.88 (d, J = 7.0Hz, 3H), 0.82 (s, 1H), 0.09 (s, 2H). 13CNMR(101MHz, CDCl3)δ172.0,171.6,164.0,136.7,136.4,132.3,130.0,128.2,125.8,123.9,116.8,115.4,114.9,1 13.9,81.0,71.7,56.1,56.0,51.8,47.4,42.3,31.6,28.4,26.6,26.6,22.7,22.6,22.5,17.8,14.1,1.0.MS(ESI):C 31 H 31 N5O3Se[M+H] + ,m / zcalcd.601.1592,found 602.1666.

[0113] PDSe-APN, yellow solid (103.9 mg, yield 69.7%) 1 H NMR (400MHz, DMSO) δ8.46(d,J=8.7Hz,1H),8.39(dd,J=4.4,2.3Hz,1H),8.26(s,1H),8.19(d,J=8.7Hz,1H),8.06(d,J=9.1Hz,1H),7.92–7.88(m,1H), 4.06(t,J=7.0Hz,1H),3.35(s,1H),3.26(s,1H),3.12(dd,J=9.9,5.2Hz,1 H),2.12(d,J=9.6Hz,1H),1.61(s,3H),1.48(d,J=7.0Hz,3H),0.77(s,3H). 13 C NMR (101MHz, CDCl3) δ177.0,175.0,170.6,166.9,156.0,149.8,142.4,142.3,136.6,134.9,133.6,133 .1,129.5,123.0,120.5,120.2,118.9,75.9,61.2,56.7,54.7,52.0,47.3,31.3,27.4,23.0.MS(ESI):C 26 H 23 N5OSe[M+H] + ,m / z calcd.601.1592,found602.1666.

[0114] PDSe-HC, yellow solid (35.1 mg, yield 50.2%) 1H NMR(400MHz, CDCl3)δ9.28(s,1H),8.36(d,J=8.4Hz,1H),8.06–8.02(m,1H),7.97(d,J=9.2Hz,1H),7.73–7.67 (m,2H),7.64–7.58(m,1H),5.84(t,J=5.7Hz,1H),5.42(d,J=7.8Hz,1H),4.34(s,1H),3.52(t,J=5.7Hz,1H),3 .31(dp,J=24.1,6.8Hz,3H),3.20(d,J=5.3Hz,1H),3.08(dd,J=10.0,5.2Hz,1H),2.18(d,J=9.6Hz,1H),2.02( s,5H),1.66(d,J=1.5Hz,3H),1.61(d,J=6.9Hz,2H),1.51(s,9H),0.90(t,J=6.6Hz,1H),0.85(d,J=6.0Hz,3H). 13 C NMR (101MHz, CDCl3) δ171.7,171.5,170.73,164.1,164.0,163.1,163.1,136.7,136.5,132.4,130.1,128.4,125.8,124.2,124.1,117. 0,115.6,114.9,113.9,80.6,71.7,56.1,56.1,55.1,51.8,47.4,42.3,38.7,31.6,28.9,28.4,26.6,23.4,22.6,22.5,22.5.MS(ESI):C 36 H 40 N6O4Se[M+H] + ,m / z calcd.700.2276,found 701.2351.

[0115] PDSe-AL (Boc), yellow solid (85.7 mg, yield 60.0%) 11H NMR (400 MHz, CDCl3) δ 9.10 (d, J = 9.1 Hz, 1H), 8.45 (s, 1H), 8.11–8.01 (m, 2H), 7.72 (dd, J = 8.6, 5.5 Hz, 3H), 6.69 (dd, J = 7.9, 3.6 Hz, 1H), 4.97 (d, J = 4.8 Hz, 1H), 4.67 (t, J = 8.8 Hz, 1H), 4.24–4.19 (m, 1H), 3.53 (d, J = 5.7 Hz, 1H), 3.22 (q, J = 5.2 Hz, 1H), 3.08 (dd, J = 10.0, 5.2 Hz, 1H), 2.18 (d, J = 9.6 Hz, 1H), 1.97 (d, J = 13.7 Hz, 1H), 1.74 (t, J = 6.1 Hz, 1H), 1.48 (s, 9H), 1.00 (dd, J = 14.1, 6.3 Hz, 6H), 0.85 (d, J = 4.0 Hz, 3H), 0.09 (s, 6H). 13 13C NMR (151 MHz, CDCl3) δ 170.6, 163.0, 135.8, 128.9, 127.5, 116.0, 114.7, 113.9, 112.8, 80.3, 70.9, 55.0, 51.7, 50.8, 46.4, 41.2, 39.0, 30.9, 30.6, 28.7, 27.3, 25.6, 25.6, 24.0, 22.1, 21.6, 21.5, 21.5, 20.7, 16.5, 13.1. MS (ESI): C 37 H 42 N6O4Se [M+H] + , m / z calcd. 714.2433, found 715.2508.

[0116] PDSe-AL, yellow solid (63.7 mg, yield 86.5%), 1 1H NMR (400 MHz, DMSO) δ 10.55 (s, 1H), 8.41 (d, J = 2.4 Hz, 2H), 8.24 (s, 1H), 8.17 (d, J = 8.7 Hz, 1H), 8.01 (s, 1H), 7.90–7.87 (m, 1H), 4.56 (s, 1H), 3.36 (d, J = 5.7 Hz, 3H), 3.27 (d, J = 5.4 Hz, 1H), 3.12 (dd, J = 9.8, 5.3 Hz, 1H), 2.14 (s, 1H), 1.71–1.66 (m, 1H), 1.62 (s, 3H), 1.22 (s, 1H), 1.18 (d, J = 6.9 Hz, 3H), 0.96–0.92 (m, 6H), 0.77 (s, 3H). 13C NMR(101MHz,MeOD)δ171.8,171.7,165.0,162.5,151.1,145.2,137.0,136.4,132.0,129.4,128.5,126.5,124.1,117.0, 115.9,114.6,113.3,71.8,70.9,55.9,55.8,52.9,51.9,48.6,41.8,40.6,25.4,24.6,22.1,21.4,20.6,20.5.MS(ESI):C 32 H 34 N6O2Se[M+H] + ,m / z calcd.614.1908, found 615.1979.

[0117] Example 2

[0118] Synthesis and Preliminary Evaluation of Photosensitizers

[0119] The final structure of the photosensitizer prepared in Example 1 was determined by... 1 H and 13 The results were fully confirmed by C10 NMR spectroscopy and mass spectrometry. Then, the effects of PDSe-n and PDSe-8e in phosphate buffer under 530 nm LED light (20 mW / cm²) were further investigated. 2 The ability of ROS generation under irradiation was investigated. The generation of superoxide anions was detected using a dihydroethidium (DHE) assay, and the results were... Figure 1 , 2 The results showed that all photosensitizers could generate superoxide anions, with PDSe-8 exhibiting the highest ROS generation capacity (11-fold increase in fluorescence intensity). Furthermore, the generation of singlet oxygen was further evaluated by 1,3-diphenylisobenzofuran (DPBF) experiments, which were performed by observing the attenuation of the DPBF absorption band at 417 nm. Results ( Figure 3 , 4 The results showed that among these compounds, PDSe-10 exhibited the highest singlet oxygen quantum yield (0.44), while PDSe-8 had a relatively low yield of 0.11. These results indicate that the series of photosensitizers possess characteristics of both type I and type II photosensitizers. Specifically, PDSe-8 exhibited the most significant type I activity, while PDSe-10 exhibited the highest type II activity.

[0120] Example 3

[0121] Detection of intracellular reactive oxygen species using photosensitizer PDSe-8

[0122] To assess the generation of ROS in the whole cell, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a universal ROS probe to detect ROS generation in U87MG cells treated with 0.1 μM PDSe-8 under normoxic (21% O2) and hypoxic (2% O2) conditions. U87MG cells were seeded at a density of 50,000 cells per well in confocal culture dishes. The normoxic group was cultured in an incubator (21% O2, 5% CO2, 37℃) for 24 hours, while the hypoxic group was cultured in a hypoxic incubator (2% O2, 5% CO2, 37℃) for 24 hours. After incubation, the cell culture medium was replaced with medium containing 0.1 μM PDSe-8, and the cells were incubated for 1 hour. Then, the cells were washed three times with PBS, and serum-free medium containing 10 μM DCFH-DA was added. The cells were incubated with the probe for 20 minutes, and then excess probe was removed by washing with PBS. Cells were then irradiated with a 530nm LED lamp for 10 minutes. Finally, ROS production was assessed by imaging the cells using laser scanning confocal microscopy with an excitation wavelength of 488nm and an emission wavelength range of 500-550nm. Results ( Figure 5 The results showed that no significant fluorescence signal was observed in the dark treatment group under any oxygen condition. In contrast, significant fluorescence signals were detected under light conditions, confirming that PDSe-8 can effectively induce intracellular ROS generation under both normoxic and hypoxic conditions.

[0123] Example 4

[0124] Detection of intracellular superoxide anion by photosensitizer PDSe-8 under normoxic and hypoxic conditions

[0125] To further clarify the specific ROS types generated by PDSe-8 under anoxic conditions, dihydroethidine was selected as the O2 source. -· Specific probes. U87MG cells were seeded at a density of 50,000 cells per well in confocal culture dishes. The normoxic group was cultured in an incubator (21% O2, 5% CO2, 37℃) for 24 hours, while the hypoxic group was cultured in a hypoxic incubator (2% O2, 5% CO2, 37℃) for 24 hours. After incubation, the cell culture medium was replaced with medium containing 0.1 μM PDSe-8, and the cells were incubated for 1 hour. Then, the cells were washed three times with PBS. After treatment, serum-free medium containing 10 μM DHE was added to the cells and incubated for 30 minutes. After incubation, excess probes were removed by washing with PBS. Subsequently, the cells were irradiated with a 530 nm LED lamp for 10 minutes. Finally, intracellular superoxide anion concentrations (λ) were detected using laser scanning confocal microscopy. ex =561nm, λ em =570-620nm). For example Figure 6As shown, strong fluorescence signals were observed in U87MG cells treated with 0.1 μM PDSe-8 under both normoxic and hypoxic conditions. In summary, these results strongly demonstrate that PDSe-8 primarily functions as a type I photosensitizer in photodynamic therapy, efficiently generating superoxide anions in both oxygen-rich and hypoxic environments.

[0126] Example 5

[0127] Detection of cytotoxicity of photosensitizer PDSe-n against various cancer cells under light and dark conditions.

[0128] The cytotoxicity of PDSe-n against various cancer cell lines (including neurogenic tumor cells SH-SY5Y, U251MG, and U87MG) was evaluated using the MTT assay. Cells were seeded at a density of 3000 to 5000 cells per well in 96-well plates and cultured overnight at 37°C with 5% CO2. Subsequently, the cells were divided into two groups: a light-exposed group and a dark-exposed group. For the light-exposed group, 0.1 μM PDSe-n (containing 1% DMSO) was added, and the cells were incubated for 1 hour. Then, the cells were exposed to a 530 nm LED lamp (20 mW / cm²). 2 Irradiate cells for 10 minutes, then incubate in a cell culture incubator for 48 hours. For the dark group, add 10 μL of MPDSe-n, then incubate cells in a cell culture incubator for 48 hours in the dark. After incubation, add 0.5% MTT solution to each well and incubate cells for another 4 hours. Then, discard the culture medium and add 150 μL of DMSO to each well. Shake the plate on a shaker for 10 minutes to dissolve the formazan crystals. Measure the absorbance at 490 nm using a microplate reader and calculate the IC50 based on the absorbance data. 50 Value. Result ( Figure 7 This indicates that all probe compounds exhibited extremely low toxicity under dark conditions. However, at 530 nm and 20 mW / cm², [the toxicity was not fully developed]. 2 Significant cytotoxicity was observed under illumination, with U87MG cells being the most sensitive to treatment. Notably, PDSe-8 exhibited the most significant phototoxicity among all the compounds tested, highlighting its potential as an effective photosensitizer for cancer therapy.

[0129] Example 6

[0130] Cytotoxicity of photosensitizer PDSe-8 on U87MG cells under normoxic (21% O2) and hypoxic (2% O2) conditions.

[0131] To evaluate cellular responses under different photodynamic therapy conditions, a live / dead cell staining method was used to assess cell viability. U87MG cells were seeded at a density of 50,000 cells per well in confocal microscope culture dishes and cultured for 24 hours under normoxic conditions (21% O2, 5% CO2, 37°C). The hypoxic group was cultured for 24 hours in a hypoxic incubator (2% O2, 5% CO2, 37°C). Cells were then treated with PDSe-8 medium and incubated for 1 hour. Cells were then washed three times with PBS, and a detection buffer containing 5 μM calcein AM and 5 μM propidium iodide (PI) was added. Cells were co-incubated with the detection buffer for 30 minutes. After incubation, cells were irradiated with a 530 nm LED lamp for 10 minutes and then cultured for another hour in a cell culture incubator. Subsequently, live and dead cells were imaged using a laser scanning confocal microscope. (Green Channel: λ) ex =488nm, λ em =500-550nm; Red channel: λ ex =561nm, λ em =570-620nm). Result ( Figure 8 The results showed that a significant increase in red fluorescence (indicating dead cells) was observed under light conditions, in both normoxic and hypoxic environments, thus confirming type I PDT-induced cell death. Given the remarkable antitumor activity of PDSe-8, further investigation into its mechanism of action was conducted.

[0132] Example 7

[0133] Cytotoxicity of photosensitizer PDSe-8a-d on U87MG cells under normoxic (21% O2) and hypoxic (2% O2) conditions.

[0134] PDSe-8c's poor water solubility limits its further biological applications; therefore, the cytotoxicity of PDSe-8a, PDSe-8b, and PDSe-8d under normoxic and hypoxic conditions was tested. U87MG cell lines were seeded into 96-well plates at 5000 cells per well and cultured overnight in a cell culture incubator (21% O2, 5% CO2, 37℃); the hypoxic group was cultured overnight in a hypoxic incubator (2% O2, 5% CO2, 37℃). The old culture medium was then discarded. Fresh culture medium containing the photosensitizer was added and incubated for 48 h. The light-treated group was incubated with a 530nm LED (20mW / cm²) 2 h after the addition of the drug-containing medium. 2 After irradiation for 10 minutes, the cells were placed in an appropriate incubator and cultured for 48 hours. Cell viability was then detected using the MTT assay. Results ( Figure 9The results showed that after treatment with 0.1 μM PDSe-8a, PDSe-8b, and PDSe-8d in the light-treated group, PDSe-8a and PDSe-8d exhibited cytotoxicity similar to PDSe-8 against U87MG cells, indicating that PDSe-8a and PDSe-8d were activated by the corresponding overexpressed enzymes in U87MG cells to release PDSe-8. PDSe-8b, with the introduction of a nitroreductase responsive group, showed poorer antitumor activity, possibly due to its lower recognition efficiency. To verify whether the photosensitizers modified with enzyme-controlled groups had less toxicity to normal cells, the toxicity of PDSe-8, PDSe-8a, PDSe-8b, and PDSe-8d to bEnd.3 cells was subsequently examined. The results showed that after modification with enzyme-controlled groups, the survival rate of bEnd.3 cells treated with 0.1 μM light (530 nm LED, 10 min) for 48 h was significantly increased compared to PDSe-8.

[0135] Example 8

[0136] PDSe-8a-d cytotoxicity to normal cells (bEnd.3) under dark conditions

[0137] Given the potentially high enzyme activation potential of PDSe-8a and PDSe-8d, their dark toxicity to normal cells was further investigated. bEnd.3 cells were seeded in cell culture dishes at a density of 50,000–100,000 cells per dish and treated with different concentrations of PDSe-8a and PDSe-8d for 48 hours in the dark. Results ( Figure 10 This indicates that PDSe-8a and PDSe-8d significantly reduced the cytotoxicity of bEnd.3, and the IC50 values ​​of the two photosensitizers were significantly lower. 50 The value (greater than 80 μM) is much higher than that of PDSe-8 for bEnd.3 IC. 50 Value (8.22 μM).

[0138] Example 9

[0139] Cytotoxicity of photosensitizer PDSe-8e against breast cancer cells (MCF-7) under light and dark conditions in hypoxic (2% O2) conditions.

[0140] The cytotoxic effects of the photosensitizer PDSe-8e under different conditions were systematically evaluated using the MTT assay. MCF-7 cells were seeded at a density of 3000 to 5000 cells per well in 96-well plates and cultured overnight in a cell culture incubator (2% O2, 5% CO2, 37°C). Cells were then divided into two groups: a light group and a dark control group. For the light group, PDSe-8e was added, and the cells were incubated for 1 hour. Subsequently, the cells were incubated under a 530 nm LED lamp (20 mW / cm²). 2Cells were irradiated for 10 minutes and then incubated in a cell culture incubator for 48 hours. For the dark control group, PDSe-8e was added, and cells were incubated in a cell culture incubator for 48 hours in the dark. After incubation, 0.5% MTT solution was added to each well, and cells were incubated for another 4 hours. Then, the culture medium was discarded, and 150 μL of DMSO was added to each well. The plate was shaken on a shaker for 10 minutes to dissolve the formazan crystals. The absorbance at 490 nm was measured using a microplate reader, and the IC50 was calculated based on the absorbance data. 50 Value. Experimental results show ( Figure 11 Under hypoxic conditions (2% O2), PDSe-8e exhibited significant photodependent cytotoxicity against MCF-7 breast cancer cells. The IC50 value was measured under dark conditions. 50 The value was 3.56 μM, while after irradiation with a 530 nm laser, the IC... 50 The value decreased significantly to 0.22 μM, indicating that it has excellent light control selectivity.

[0141] Example 10

[0142] Cytotoxicity of photosensitizer PDSe-8e against normal human umbilical vein endothelial cells (HUVECs) under light and dark conditions

[0143] In toxicity assessments of normal human umbilical vein endothelial cells (HUVECs), PDSe-8e also demonstrated good biocompatibility and selectivity. Cells were seeded at a density of 3000 to 5000 cells per well in 96-well plates and cultured overnight in a humidified incubator at 37°C and 5% CO2. Subsequently, the cells were divided into two groups: a light-exposed group and a dark-exposed group. For the light-exposed group, PDSe-8e was added, and the cells were incubated for 1 hour. Following this, they were exposed to a 530nm LED lamp (20mW / cm²). 2 Irradiate cells for 10 minutes, then incubate in a cell culture incubator for 48 hours. For the dark group, add PDSe-8e, then incubate cells in a cell culture incubator for 48 hours in the dark. After incubation, add 0.5% MTT solution to each well and incubate cells for another 4 hours. Then, discard the culture medium and add 150 μL DMSO to each well. Shake the plate on a shaker for 10 minutes to dissolve the formazan crystals. Measure the absorbance at 490 nm using a microplate reader and calculate the IC50 based on the absorbance data. 50 Value. Result ( Figure 12 The results showed that the IC50 of HUVEC cells under dark conditions... 50 >20μM, IC under illumination 50>0.8μM confirmed that PDSe-8e has a specific killing effect on tumor cells, while maintaining a low level of toxicity to normal vascular endothelial cells, providing important safety evidence for its clinical application.

[0144] Example 11

[0145] PDSe-8 photosensitizer lysosomal targeting detection

[0146] The subcellular targeting of compound PDSe-8 was verified using confocal microscopy: 0.1 μM PDSe-8 was co-incubated with Lyso-Tracker Green in U87MG cells (50,000 cells per well) for co-localization observation. Figure 13 The excitation wavelength of Lyso-Tracker Green was 488 nm and the emission wavelength was 500-550 nm, while the excitation wavelength of PDSe-8 was 488 nm and the emission wavelength was 663-737 nm. The results showed that PDSe-8 could accurately target lysosomes, with a Pearson correlation coefficient of 0.95, which confirmed the high-efficiency targeting ability of this compound to lysosomes.

[0147] Example 12

[0148] Detection of mitochondrial targeting by photosensitizer PDSe-8e

[0149] TPP, as a classic mitochondrial targeting group, possesses a positive charge and can specifically accumulate in mitochondria with negative membrane potentials through electrostatic interactions. 0.5 μM PDSe-8e was applied to MCF-7 breast cancer cells seeded at a density of 50,000 cells per well, and co-localization was observed with the commercial mitochondrial dye MitoTracker. Confocal laser scanning microscopy revealed… Figure 14 The colocalization coefficient (Pearson correlation coefficient) between PDSe-8e and MitoTracker reached 0.90, confirming the compound's highly efficient targeting ability to mitochondria.

[0150] Example 13

[0151] Morphological changes of U87MG cells treated with 0.2 and 0.5 μM PDSe-8 after different light exposure times.

[0152] U87MG cells were placed in confocal culture dishes and cultured until confluence reached 70%-90%. The cells were then incubated for 1 hour with medium containing 0.2 μM or 0.5 μM PDSe-8, followed by three washes with PBS. Subsequently, complete medium containing 1 μg / mL Hoechst 33258 was added, and the cells were incubated for 15 minutes, followed by three washes with PBS. Finally, the cells were subjected to a 530 nm laser (20 mW / cm²). 2 Cells were irradiated for 10 minutes. Then, a binding solution containing annexin V-FITC was added, and the cells were incubated at room temperature for 20 minutes before confocal imaging. (Blue channel: λ) ex =405nm, λ em =425-475nm; Green channel: λ ex =488nm, λ em =500-550nm; Red channel λ ex =561nm, λ em =570-620nm). Cells treated with different concentrations of PDSe-8 and then exposed to light exhibited two distinctly different morphological changes. Figure 15 In the cell group treated with 0.2 μM PDSe-8, cell shrinkage and nuclear condensation, typical features of apoptosis, were observed. Conversely, cells treated with 0.5 μM PDSe-8 showed cell membrane "vacuolization" without significant nuclear morphological changes, consistent with pyroptosis, and demonstrating that photosensitizers can induce concentration-induced changes in cell death patterns.

Claims

1. A class of selenium-containing photosensitizers based on quinoline skeleton, characterized in that, The structure of the organic photosensitizer is shown below: wherein R is selected from any one of the following:

2. A method for preparing the organic photosensitizer based on quinoline-selenium structure according to claim 1, characterized in that, comprising the steps of: Dissolving 6-bromo-2-methylquinoline and selenium dioxide in an organic solvent, stirring the reaction mixture under an inert gas atmosphere, removing the solvent under reduced pressure using a rotary evaporator after cooling to room temperature, dissolving, extracting, drying, filtering and concentrating under vacuum to obtain a light yellow crude product S-1; Dissolving compound S-1 and verbenone in an organic solvent and then adding potassium hydroxide, stirring the mixture under an inert gas atmosphere, cooling the mixture to room temperature after the reaction is completed, filtering, washing and drying under vacuum to obtain a yellow solid S-2; Dissolving compound S-2 and selenium dioxide in an organic solvent and stirring the reaction, cooling the mixture to room temperature after the reaction is completed, and removing the solvent under vacuum, washing the residue, extracting, and purifying the crude product by column chromatography to obtain a white solid S-3; Stirring compound S-3 and malononitrile in an organic solvent, cooling the mixture to room temperature after the reaction is completed, and removing the solvent under reduced pressure, washing and extracting the obtained residue, and purifying the crude product by silica gel column chromatography to obtain a yellow solid S-4; Dissolving compound S-4, palladium acetate, 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl or 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and cesium carbonate in an organic solvent and reacting under an inert gas atmosphere, stirring the reaction mixture, then adding different imines, refluxing the reaction mixture, cooling the mixture to room temperature after the reaction is completed, extracting and concentrating, and purifying the obtained residue by silica gel column chromatography to obtain the target product PDSe-n.

3. The method of claim 2, wherein, The imine includes any one of azetidine, pyrrolidine, piperidine, morpholine, N-methylpiperazine, N-ethylpiperazine, 1-(tert-butoxycarbonyl)piperazine, piperazine, thiomorpholine, 4,4-difluoropiperidine, cyclohexylpiperazine, tert-butyl carbamate.

4. A derivative of the organic photosensitizer based on quinoline-selenium structure according to claim 1, characterized by, The structure of the derivative is preferably any one of the following:

5. A process for the preparation of a derivative according to claim 4, characterized in that, comprising the steps of: According to the method of claim 2, using imine 1-(tert-butoxycarbonyl)piperazine to obtain compound PDSe-7, dissolving compound PDSe-7 in an organic solvent, adding trifluoroacetic acid, reacting, extracting after the reaction is completed, concentrating under reduced pressure, purifying the obtained residue by column chromatography to obtain the target product PDSe-8, dissolving compound PDSe-8 and Boc-glycine-L-proline, HATU, DIPEA in an organic solvent and stirring overnight, removing the solvent under vacuum after the reaction is completed, extracting the residue, and purifying the crude product by column chromatography to obtain PDSe-8a; Dissolving compound PDSe-8 and p-nitrobenzyl chloroformate in an organic solvent, adding triethylamine and stirring overnight, removing the solvent under vacuum after the reaction is completed, extracting the residue, and purifying the crude product by column chromatography to obtain PDSe-8b; Compound PDSe-8 and 3-(2,3,5-trimethyl-1,4-benzoquinoyl)-3-methylbutanoic acid, HATU, DIPEA were dissolved in an organic solvent and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-8c; Compound PDSe-8 and N-tert-butoxycarbonyl-N'-(acetyl)-L-lysine, HATU, DIPEA were dissolved in an organic solvent and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-8d; Compound PDSe-8 and 2-carboxyethyl triphenylphosphonium bromide, HOBT, EDCI were dissolved in an organic solvent, triethylamine was added, and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-8e; According to the method in claim 2, palladium acetate, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and cesium carbonate were dissolved in an organic solvent and reacted under inert gas protection, compound S-4, tert-butyl carbamate was added to the above system, and the reaction was continued to reflux, after completion, the mixture was cooled to room temperature, extracted, concentrated under reduced pressure, and the obtained crude product was purified by column chromatography to obtain PDSe-12; compound PDSe-12 was dissolved in an organic solvent, trifluoroacetic acid was added, and the reaction was carried out, after the reaction was completed, extraction was carried out, concentrated under reduced pressure, and the obtained residue was purified by column chromatography to obtain the target product PDSe-NH2, compound PDSe-NH2 and Boc-L-leucine, HATU, DIPEA were dissolved in an organic solvent and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-LAP(Boc), then compound PDSe-LAP(Boc) was dissolved in an organic solvent, trifluoroacetic acid was added dropwise and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, and the crude product was purified by column chromatography to obtain PDSe-LAP; Compound PDSe-NH2 and N-Boc-D-glutamic acid-1-tert-butyl ester, HATU, DIPEA were dissolved in an organic solvent and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the residue was extracted, and the crude product was purified by column chromatography to obtain PDSe-GGT(Boc), then compound PDSe-GGT(Boc) was dissolved in an organic solvent, trifluoroacetic acid was added dropwise and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, and the crude product was purified by column chromatography to obtain PDSe-GGT; Compound PDSe-NH2 and (S)-2-((tert-butoxycarbonyl)amino)propanoic acid, HATU, DIPEA were dissolved in an organic solvent and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the residue was extracted, the crude product was purified by column chromatography to obtain PDSe-APN(Boc), then compound PDSe-APN(Boc) was dissolved in an organic solvent, trifluoroacetic acid was added dropwise and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the crude product was purified by column chromatography to obtain PDSe-APN; Compound PDSe-NH2 and N-tert-butoxycarbonyl-N'-(acetyl)-L-lysine, HATU, DIPEA were dissolved in an organic solvent and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the residue was extracted, the crude product was purified by column chromatography to obtain PDSe-HC; Compound PDSe-LAP and tert-butoxycarbonyl-L-alanine N-maleimide ester were dissolved in an organic solvent, triethylamine was added and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the residue was extracted, the crude product was purified by column chromatography to obtain PDSe-AL(Boc), then compound PDSe-AL(Boc) was dissolved in an organic solvent, trifluoroacetic acid was added dropwise and stirred overnight, after the reaction was completed, the solvent was removed under vacuum, the crude product was purified by column chromatography to obtain PDSe-AL.

6. Use of the quinoline-selenium structure-based organic photosensitizer of claim 1 or the derivative of claim 4 as a photosensitizer in the preparation of a ROS-generating reagent or a drug.

7. Use according to claim 6, characterized in that, The ROS generation includes in vitro ROS generation and intracellular ROS generation.

8. Use of the quinoline-selenium structure-based organic photosensitizer of claim 1 or the derivative of claim 4 in the preparation of a reagent or a drug for cancer treatment.

9. Use according to claim 8, characterized in that, The quinoline-selenium structure-based organic photosensitizer or the derivative has an inhibitory effect on cancer cells under normoxic and hypoxic conditions.

10. Use of the derivative of claim 4 in the preparation of a lysosome-targeting and mitochondrion-targeting reagent or a drug.

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