Bioactive photosensitizer targeting poly(ADP-ribose) polymerase, preparation method and use

Abstract

Disclosed in the present invention are a bioactive photosensitizer targeting poly(ADP-ribose) polymerase (PARP), a preparation method and a use. The bioactive photosensitizer is a quinoxalinone derivative. The structural formula of the quinoxalinone derivative is as shown in formula (I), wherein R1 is one of methoxy, methyl, and halogen, and substituent R1 is located at at least one of positions 5, 6, 7, and 8 on a quinoxalinone ring. The active photosensitizer provided in the present invention, also acting as a PARP inhibitor, can induce synthetic lethality in cells. During the process, the oxidation state and the oxygen concentration in the cells can be improved, and a hypoxic microenvironment in a tumor is improved, thus further enhancing the effect of photodynamic therapy, that is, the PDT therapy and the PARP inhibitor achieve a synergistic effect.

Description

Bioactive photosensitizer targeting poly (ADP-ribose) polymerase, preparation method and application Technical Field

[0001] The present invention relates to the field of novel drug development, and in particular to a quinoxalinone-derived bioactive photosensitizer targeting poly (ADP-ribose) polymerase, a preparation method thereof, and anti-tumor applications thereof. Background Art

[0002] Photodynamic therapy (PDT) is an anti-tumor therapy that utilizes photosensitive molecules to generate a "photodynamic" reaction when stimulated by light of a specific wavelength. During PDT, photosensitizing drugs can generate highly active reactive oxygen species (ROS) through energy or electron transfer. ROS can oxidize nearby biomacromolecules, generating cytotoxicity and killing tumor cells.

[0003] Compared to traditional therapies, PDT has the advantage of being able to utilize the high temporal and spatial resolution of light for precise and effective treatment, resulting in fewer side effects. However, due to the high concentration of reduced glutathione within tumor cells, it can rapidly quench the reactive oxygen free radicals generated by photosensitive molecules under light irradiation. In addition, the generation of reactive oxygen free radicals by most photosensitive molecules depends on the concentration of molecular oxygen (O2), and the hypoxic microenvironment within solid tumors can greatly limit the effectiveness of photodynamic therapy.

[0004] Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme with transglycosidase catalytic activity. It primarily functions through poly(ADP-ribosylation) (PARylation) modifications, playing important roles in DNA damage repair, transcriptional regulation, chromatin dynamics, hypoxia response, metabolism, cell death, and genomic stability. The PARP family comprises 18 members, of which PARP1 is the most critical, responsible for 80-90% of PARylation modifications in cells. During DNA damage repair, PARP1 primarily functions as a sensor. Upon DNA breakage, the zinc finger structure of PARP1 rapidly binds to the DNA damage site and, through PARylation, recruits proteins involved in DNA damage repair to initiate DNA repair. DNA damage stimulates PARP1 catalytic activity, leading to the synthesis of PAR chains on itself, histones, and non-histone proteins through PARylation. PARP1 is a key temporal and spatial organizer of the entire DNA repair process. Genetic deletion of PARP1 impairs the base excision repair (BER) pathway. When the PARP1 gene is defective, DNA damage accumulates in the cell, eventually leading to cell cycle arrest or even cell death. This makes PARP a popular tumor target and prompts the research and development of the mechanism of action of PARP inhibitors.

[0005] PARP inhibitors bind to the catalytic sites of PARP1 or PARP2, preventing the PARP protein from falling off the DNA damage site. When bound to DNA, PARP can cause DNA replication forks to stall and DNA replication to fail, leading to cell cycle arrest and cell death. Currently, there are four PARP inhibitors approved for marketing in China: Olaparib, Niraparib, Fluzoparib, and Pamiparib, which are mainly used for the treatment or maintenance of ovarian cancer, breast cancer, and prostate cancer.

[0006] The in-depth research on PDT therapy and the development of PARP inhibitors have greatly promoted the treatment of malignant tumors, but their application still faces many challenges. Specifically, for photosensitizers, due to the high concentration of reduced glutathione in tumor cells, they can quickly quench the reactive oxygen free radicals produced by photosensitive molecules under light. However, the hypoxic microenvironment inside solid tumors will greatly limit the effect of photodynamic therapy. Compared with traditional chemotherapy drugs, target-based small molecule inhibitors have relatively fewer side effects and better patient tolerance because they can inhibit key proteins in tumor cells. However, the off-target effects and drug resistance of small molecule inhibitors have always been difficult problems in the field of tumor treatment. Summary of the Invention

[0007] The purpose of the present invention is to address the poor effectiveness of traditional photosensitizers and the limitations of small molecule targeted drugs. By identifying key protein targets related to DNA repair in tumor cells, a bioactive photosensitizer that can induce synthetic lethality in tumor cells and efficiently generate ROS is designed and synthesized, thereby improving the efficacy of photodynamic therapy.

[0008] [Corrected 27.02.2025 according to Rule 26] To achieve the above object, the present invention provides a bioactive photosensitizer targeting poly (ADP-ribose) polymerase, wherein the bioactive photosensitizer is a quinoxalinone derivative, and the structural formula of the quinoxalinone derivative is as follows:

[0009] [Corrected 27.02.2025 in accordance with Article 26]

[0010] Among them, R 1 is one of methoxy, methyl, and halogen, R 1 The substituent is located at at least one of the 5, 6, 7, and 8 positions of the quinoxalinone ring.

[0011] [Corrected 27.02.2025 in accordance with Rule 26] Optionally, the structural formula of the quinoxalinone derivative is:

[0012] [Corrected 27.02.2025 in accordance with Article 26]

[0013] [Corrected 27.02.2025 according to Rule 26] The present invention also provides a method for preparing the above-mentioned bioactive photosensitizer targeting poly (ADP-ribose) polymerase, the reaction scheme of which is as follows:

[0014] [Corrected 27.02.2025 in accordance with Article 26]

[0015] The method includes:

[0016] Step 1: Compound (1d) and compound (1h) undergo amidation reaction to obtain compound (1i);

[0017] Step 2: Compound (1i) undergoes Suzuki coupling reaction with boronate-substituted anthraquinone (1j) to obtain compound (1).

[0018] Optionally, in step 1, a condensing agent 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate and an organic base triethylamine are further added, and the ratio of compound (1h): compound (1d): condensing agent: organic base = 1: (1~2): (1~2): (2~5).

[0019] [Corrected 27.02.2025 according to Rule 26] Alternatively, the compound (1d) is prepared by the following method:

[0020] [Corrected 27.02.2025 in accordance with Article 26]

[0021] Step 1.1, compound (1a) and compound (1b) undergo amidation condensation reaction to obtain compound (1c);

[0022] Step 1.2, compound (1c) is deprotected from the amino group to obtain compound (1d).

[0023] Optionally, in step 1.1, the molar ratio of compound (1a) to compound (1b) is (1-2):1.

[0024] Optionally, in step 1.1, a condensing agent O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and a catalyst diisopropylethylamine are further added.

[0025] [Corrected 27.02.2025 according to Rule 26] Alternatively, the compound (1h) is prepared by the following method:

[0026] [Corrected 27.02.2025 in accordance with Article 26]

[0027] Step S1, compound (1e) is subjected to a condensation reaction with 5-bromothiophene-2-carboxaldehyde to form compound (1f);

[0028] Step S2: Compound (1f) reacts with ethyl bromoacetate under alkaline conditions to undergo a nucleophilic substitution reaction to produce compound (1g);

[0029] Step S3: Compound (1g) is hydrolyzed to obtain compound (1h).

[0030] Optionally, in step S1, a catalyst pyridine is further added, and the molar ratio of the compound (1e), 5-bromothiophene-2-carboxaldehyde, and the catalyst is 1:(1-2):(1-5).

[0031] Optionally, in step S2, the molar ratio of the compound (1f) to ethyl bromoacetate is 1:(1-2).

[0032] Optionally, in step 2, a catalyst tetrakis-(triphenylphosphine)palladium and an inorganic base are further added, and the molar ratio of compound (1i): borate-substituted anthraquinone (1j): catalyst: inorganic base is 1: (1-2): (0.03-0.1): (2-5).

[0033] The present invention also provides a use of the above-mentioned bioactive photosensitizer targeting poly (ADP-ribose) polymerase in photodynamic therapy.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1) The active photosensitizer targeting PARP provided by the present invention can inhibit the expression of poly (ADP-ribose) polymerase, hinder the DNA repair of tumor cells, induce tumor cell death, and thus improve the effect of photodynamic therapy.

[0036] 2) The active photosensitizer targeting PARP provided by the present invention has a strong ability to generate reactive oxygen free radicals and can produce reactive oxygen species in the hypoxic tumor microenvironment.

[0037] 3) The active photosensitizer targeting PARP provided by the present invention has a high killing effect on ovarian cancer cells and has very low dark toxicity to normal cells.

[0038] 4) The PARP-targeting active photosensitizer provided by this invention enables in vivo imaging in animals and can be used for image-guided photodynamic therapy in tumor-bearing mice. These photosensitizers achieve excellent temporal and spatial resolution in bioimaging experiments and are suitable for use in fluorescence imaging-guided photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows the mass spectrometry characterization of QTABI.

[0040] Figure 2 shows the H NMR spectrum of QTABI.

[0041] FIG3 is a graph showing the absorption and fluorescence spectra of QTABI of the present invention.

[0042] FIG4 is a characterization diagram of ROS generation by QTABI of the present invention under blue light excitation.

[0043] FIG5 is a graph showing the intracellular ROS detection of cells in response to QTABI.

[0044] Figure 6 shows the toxicity study of QTABI on ovarian cancer cells.

[0045] Figure 7 shows the distribution of QTABI in tumor-bearing mice studied by in vivo imaging of small animals. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0047] [Corrected 27.02.2025 according to Rule 26] The present invention also provides a method for preparing the above-mentioned bioactive photosensitizer targeting poly (ADP-ribose) polymerase, the reaction scheme of which is as follows:

[0048] [Corrected 27.02.2025 in accordance with Article 26]

[0049] The method includes:

[0050] Step 1: Compound 1d is subjected to amidation reaction with compound 1h to obtain compound 1i.

[0051] To improve reaction efficiency, a condensing agent (1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate) and an organic base are added in step 1. The molar ratio of compound 1h:compound 1d:condensing agent:organic base is 1:(1-2):(1-2):(2-5). Organic bases include triethylamine and diisopropylethylamine. For example, a carboxylic acid derivative (compound 1h) is dispersed in a solvent, and the condensing agents PyBOP and triethylamine are added. After stirring at room temperature for 1 hour, an amine derivative (compound 1d) is added. After completion of the reaction, the solvent is removed using a rotary evaporator, and intermediate 1i is purified by column chromatography. The molar ratio of compound 1h:compound 1d:condensing agent:catalyst is 1:1.1:1.1:4. The solvent is DMF, and the reaction is carried out at room temperature for 8 hours.

[0052] The compound 1d can be prepared by the following method:

[0053] In step 1.1, compound 1a and compound 1b undergo an amidation condensation reaction to obtain compound 1c; the molar ratio of compound 1a to compound 1b is (1-2):1. As an example, the molar ratio of the benzoic acid derivative (compound 1a) to the o-phenylenediamine derivative (compound 1b) is 1.1:1; the solvent is anhydrous DMF, the reaction temperature is room temperature, and the reaction time is 24 hours.

[0054] In some embodiments, to promote the condensation reaction, a condensing agent, O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and a catalyst are further added in step 1.1. The catalyst is an organic base, for example, diisopropylethylamine. For example, DIPEA is added to a benzoic acid derivative, an o-phenylenediamine derivative, and a HATU dispersant, and stirred at room temperature. After the reaction, the mixture is washed with saturated brine, extracted with ethyl acetate, and then separated, dried, filtered, and concentrated to obtain a crude product, which is then purified by column chromatography to obtain an amide intermediate. The intermediate is dissolved in acetic acid, stirred at 120°C, and purified by column chromatography to obtain the benzimidazole intermediate 1c.

[0055] Step 1.2, compound 1c is deprotected from the amino group to obtain compound 1d.

[0056] In some embodiments, deprotection is performed by acid. The acid may be hydrochloric acid or trifluoroacetic acid. For example, benzimidazole intermediate 1c is dispersed in methanol, a solution of hydrochloric acid in dioxane is added, and the mixture is stirred at room temperature. After the reaction is complete, intermediate 1d is purified by column chromatography. The molar ratio of the benzimidazole derivative (compound 1c) to hydrochloric acid (in dioxane) is 1:10. The solvent is anhydrous methanol, the reaction temperature is room temperature, and the reaction time is 8 hours.

[0057] The compound 1h was prepared by the following method:

[0058] In step S1, compound 1e undergoes a condensation reaction with 5-bromothiophene-2-carboxaldehyde to form compound 1f. To increase the reaction rate, a catalyst, pyridine, is added. The molar ratio of compound 1e, 5-bromothiophene-2-carboxaldehyde, and catalyst is 1:(1-2):(1-5). As an example, quinoxalinone scaffold compound 1e is dispersed in a solvent, 5-bromothiophene-2-carboxaldehyde and pyridine are added. After the reaction, the mixture is filtered and the solid is washed with acetic acid and diethyl ether, respectively, to obtain the condensation product 1f. The molar ratio of quinoxalinone (compound 1e), 5-bromothiophene-2-carboxaldehyde, and pyridine is 1:1.5:5. The solvent is acetic anhydride. The reaction temperature is 120°C for 10 hours, followed by 12 hours at room temperature.

[0059] In step S2, compound 1f undergoes a nucleophilic substitution reaction with ethyl bromoacetate under alkaline conditions to produce compound 1g. The molar ratio of compound 1f to ethyl bromoacetate is 1:(1-2). Alkaline conditions refer to the addition of a base such as potassium carbonate or sodium carbonate to a solution with a pH greater than 7 to promote the nucleophilic substitution reaction. As an example, the condensation product 1f is dispersed in a solvent, ethyl bromoacetate and potassium carbonate are added, and after completion of the reaction, column chromatography is performed to purify the product to yield a bromoquinoxalinone thiophene derivative (compound 1g). The molar ratio of compound 1f to ethyl bromoacetate is 1:1.5. The solvent is acetone, the reaction temperature is 70°C, and the reaction time is 8 hours.

[0060] In step S3, compound 1g is hydrolyzed to obtain compound 1h. The hydrolysis is carried out under conventional alkaline conditions (such as lithium hydroxide or sodium hydroxide). For example, intermediate 1g is dispersed in a solvent and lithium hydroxide is added. After the reaction is completed, a solution of hydrochloric acid in dioxane is added to acidify the solution to pH 7. The solvent is removed using a rotary evaporator, and the compound is recrystallized from dichloromethane to obtain the carboxylic acid derivative 1h. The molar ratio of compound 1g to lithium hydroxide is 1:5, and the solvent is tetrahydrofuran (THF) and water (3:1 by volume). The reaction is carried out at room temperature for 8 hours.

[0061] In step 2, compound 1i undergoes a Suzuki coupling reaction with boronate-substituted anthraquinone 1j to obtain compound 1. The Suzuki coupling reaction, also known as the Suzuki reaction, is an organic coupling reaction in which an aryl or alkenyl boronic acid or boronate ester cross-couples with a chlorine-, bromine-, or iodine-substituted aromatic hydrocarbon or olefin under the catalysis of a zero-valent palladium complex.

[0062] In some embodiments, a catalyst, tetrakis(triphenylphosphine)palladium, and an inorganic base are also added. The molar ratio of compound 1i: boronate-substituted anthraquinone 1j: catalyst: inorganic base is 1: (1-2): (0.03-0.1): (2-5). As an example, intermediate compound 1i, boronate-substituted anthraquinone 1j, catalyst tetrakis(triphenylphosphine)palladium, and potassium carbonate are added to a Shrek tube and reacted in a dioxane and water mixture under nitrogen for 16 hours. After completion of the reaction, a red solid is filtered and then washed with deionized water, ether, and dichloromethane to obtain the quinoxalinone anthraquinone derivative QTABI (Compound 1). The molar ratio of intermediate compound 1i, boronate-substituted anthraquinone 1j, tetrakis(triphenylphosphine)palladium, and potassium carbonate is 1:1.2:0.05:3.0. The solvent is dioxane and water (3:1 by volume). The reaction temperature is 90°C and the reaction time is 16 hours.

[0063] The following Examples 1 to 10 specifically illustrate the synthesis method of the above-mentioned bioactive photosensitizer compound:

[0064] The reagents and raw materials involved in the examples are all commercially available.

[0065] The full names of the reagent abbreviations used in the examples are as follows:

[0066] QTABI: (2-(4-{[(2-{3-[(1E)-2-[5-(9,10-dioxyylidene-9,10-dihydroanthracen-2-yl)thiophen-2-yl]vinyl]-2-oxyylidenequinoxalin-1-yl}acetyl)amino]methyl}phenyl)-1H-benzo[d]imidazole-4-carboxamide)

[0067] PyBOP: 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate

[0068] HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0069] DMF: N, N-dimethylformamide

[0070] DIPEA: diisopropylethylamine

[0071] PBS: Phosphate buffered saline

[0072] DAPI: 4',6-diamidino-2-phenylindole

[0073] CCK-8: Cell Counting Kit-8 cell counting reagent

[0074] DMSO: dimethyl sulfoxide. Example 1

[0075] The synthesis of intermediate 1c includes the following steps:

[0076] Benzoic acid derivative 1a (3 mmol, 753 mg), o-phenylenediamine derivative 1b (3.3 mmol, 498 mg), and HATU (3.3 mmol, 1.25 g) were dispersed in anhydrous DMF (10 mL). DIPEA (6 mmol, 774 mg) was then added and stirred at room temperature for 24 h. The reaction solution was washed with saturated brine and extracted with ethyl acetate. The organic phase was separated, dried, and concentrated to obtain the crude product, which was purified by column chromatography to obtain a white powder. The resulting white powder was dissolved in acetic acid (AcOH) and heated at 120°C for 8 h. After the reaction, the reaction solution was concentrated and purified by column chromatography to obtain a light yellow solid, compound 1c (650 mg, 59% yield).

[0077] The synthesis of intermediate 1d comprises the following steps:

[0078] To a 25 mL dry round-bottom flask equipped with a stirrer, compound 1c (1.78 mmol, 650 mg) and methanol (8 mL) were added dropwise. Hydrochloric acid (4 mol / L in dioxane, 4.45 mL) was then added dropwise. Stir at room temperature for 8 h. After the reaction, the crude product was concentrated and purified by column chromatography to yield a pale yellow powder, compound 1d (350 mg, 74% yield).

[0079] The synthesis of intermediate 1f comprises the following steps:

[0080] Compound 1e (15 mmol, 2.4 g) and acetic anhydride (24 mL) were added to a 100 mL dry round-bottom flask equipped with a stirrer. Pyridine (5.9 mL) and 5-bromothiophene-2-carboxaldehyde (22.5 mmol, 4.25 g) were added with stirring at room temperature. The reaction mixture was then heated to 120°C for 10 h. After cooling, stirring was continued at room temperature for 12 h. After the reaction was completed, the solid was collected by filtration using a fritted funnel and washed with ethanol and acetic acid to obtain a brown-black powder, compound 1f (3.2 g, 65% yield).

[0081] The synthesis of intermediate 1g includes the following steps:

[0082] To a 100 mL dry round-bottom flask equipped with a stirrer, compound 1f (3.3 mmol, 1.1 g), potassium carbonate (5.0 mmol, 690 mg), and the catalyst tetrabutylammonium iodide (0.66 mmol, 244 mg) were added. Acetone (20 mL) and ethyl bromoacetate (5.0 mmol, 610 mg) were added with stirring at room temperature. The reaction mixture was then heated to 70°C and refluxed for 8 h. After cooling, the organic solvent was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 8:1 to 5:1) to afford compound 1g (0.99 g, 72% yield) as a yellow solid. 1H NMR (600 MHz, Chloroform-d)δ8.19 (d,J= 15.8 Hz, 1H), 7.86 (dd,J= 8.0, 1.5 Hz, 1H), 7.49 (ddd,J= 8.6, 7.2, 1.5 Hz, 1H), 7.38 – 7.33 (m, 1H), 7.37 (d,J= 15.9 Hz, 1H), 7.07 (dd,J= 8.3, 1.2 Hz, 1H), 7.04 – 7.00 (m, 2H), 5.05 (s, 2H), 4.26 (q,J= 7.2 Hz, 2H), 1.28 (t,J= 7.1 Hz, 3H). 13 C NMR (151 MHz, CDCl3)δ167.2, 154.6, 151.8, 144.0, 133.7, 132.1, 131.1, 130.8, 130.3, 130.3, 130.1, 130.0, 114.9, 113.2, 62.3, 43.8, 14.3. HRMS (ESI) m / z (M+H) + Calculated value (C 18 H 16 BrN2O3S): 419.0065; Detected: 419.0058.

[0083] The synthesis of intermediate 1h comprises the following steps:

[0084] Take a 50 mL dry round-bottom flask equipped with a stirrer, add compound 1g (1.75 mmol, 0.75 g) and lithium hydroxide (5.25 mmol, 123 mg), add tetrahydrofuran (25 mL) and deionized water (5 mL) while stirring at room temperature, and then stir at room temperature for 8 hours. After the reaction is completed, a dioxane solution of hydrochloric acid (2.0 mL, 4 mol / L) is added to the reaction solution. Then, dichloromethane (100 mL) and saturated sodium chloride solution (50 mL) are added to the reaction solution, and the organic phase is extracted and separated. The aqueous phase is extracted once with dichloromethane (100 mL), the organic phase is separated, and combined with the previous organic phase and dried over anhydrous sodium sulfate. The organic phase is then filtered and distilled under reduced pressure to obtain a crude product. Finally, a yellow solid is obtained by recrystallization (dichloromethane and petroleum ether), namely compound 1h (648 mg, yield 95%).

[0085] The synthesis of intermediate 1i comprises the following steps:

[0086] To a 50 mL dry round-bottom flask equipped with a stirrer, compound 1h (0.41 mmol, 165 mg) and PyBOP (0.49 mmol, 237 mg) were added. DMF (8 mL) and triethylamine (4 mmol, 154 mg) were added while stirring at room temperature, and the mixture was stirred at room temperature for 1 hour. The amine derivative 1d (0.38 mmol, 100 mg) was then added to the reaction solution, and stirring was continued at room temperature for 8 hours. After completion of the reaction, DMF was removed using a rotary evaporator to obtain the crude product, which was then purified by column chromatography to obtain a yellow solid, compound 1i (210 mg, 87% yield). 1 H NMR (600 MHz, DMSO-d6) δ 13.53 (s, 1H), 9.34 (s, 1H), 8.91 (s, 1H), 8.25 – 8.05 (m, 3H), 7.89 – 7.67 (m, 4H), 7.58 (s, 1H), 7.48 – 7.18 (m, 8H), 5.02 (s, 2H), 4.38 (s, 2H).

[0087] The synthesis of the target product QTABI includes the following steps:

[0088] To a dry 35 mL Shrek tube equipped with a stirrer, compound 1i (0.2 mmol, 128 mg), boronate-substituted anthraquinone (80 mg, 0.24 mmol), tetrakis(triphenylphosphine)palladium (12 mg, 0.01 mmol), and potassium carbonate (83 mg, 0.6 mmol) were added under nitrogen. DMF (6 mL) and deionized water (2 mL) were added with stirring at room temperature. The mixture was then frozen and thawed with liquid nitrogen to remove trace oxygen. The reaction tube was then transferred to a 90°C oil bath and heated with stirring for 16 h. After completion of the reaction, the reaction solution was filtered using a fritted funnel to obtain the crude product, which was then washed with deionized water, dichloromethane, and ether, respectively, and finally dried to obtain a red solid, compound QTABI (70 mg, 46% yield). HRMS (ESI) m / z (M+H) + calculated for C 45 H 31 N6O5S: 767.2077, observed: 767.1961, mass spectrum analysis is shown in Figure 1. The nuclear magnetic resonance hydrogen spectrum characterization of QTABI is shown in Figure 2. 1H NMR (600 MHz, DMSO-d6)δ9.40 (s, 1H), 8.89 (t,J= 6.0 Hz, 1H), 8.42 (d,J= 1.9 Hz, 1H), 8.35 – 8.12 (m, 7H), 7.99 – 7.91 (m, 3H), 7.88 – 7.80 (m, 2H), 7.79 – 7.69 (m, 2H), 7.67 (d,J= 3.9 Hz, 1H), 7.61 (t,J= 7.9 Hz, 1H), 7.50 (d,J= 16.0 Hz, 1H), 7.47 – 7.38 (m, 4H), 7.35 – 7.25 (m, 1H), 5.06 (s, 2H), 4.41 (d,J= 6.0 Hz, 2H).

[0089] Characterization of Absorption Spectra, Fluorescence Spectra and Reactive Oxygen Generation Capacity of Bioactive Photosensitizers

[0090] A 0.5 mg / mL stock solution of QTABI was prepared in DMSO. The solution was then diluted to a concentration of 10 µg / mL using various DMSO / CHCl₃ mixtures. The absorption spectrum was measured using a Thermo Electron-EV300 UV-visible spectrophotometer, revealing a maximum absorption wavelength of 451 nm. The fluorescence spectrum of QTABI was then measured using a steady-state time-resolved fluorescence spectrophotometer, revealing a maximum emission wavelength of 679 nm (Figure 3). The ROS generation efficiency was then measured using a 2',7'-dichlorofluorescein diacetate (DCFH) probe. The results demonstrate a high ROS generation efficiency (Figure 4). The horizontal axis represents irradiation time, and the vertical axis, I / I₀, represents the ratio of the absorbance of the DCFH and QTABI mixture after irradiation to the initial absorbance.

[0091] Cellular uptake and killing of ovarian cancer cells

[0092] Intracellular ROS detection: Human ovarian cancer cells HEYA8 (source: ATCC) were seeded in 6-well plates at a density of 10 6 / mL, QTABI (10 μg / ml) was added after the cells attached to the wall and incubated for 4 hours, then the medium was removed. After replacing the new medium, 2',7'-dichlorofluorescein diacetate (DCFH-DA) (5 μM) was added and incubated for another 0.5 hours, and then blue light (60 mW / cm 2After irradiation with QTABI (QTABI, ROS, and DAPI) for 5 minutes, the culture medium was discarded and the cells were washed three times with PBS. Fresh culture medium was replaced, and DAPI was added for another 10 minutes of incubation before washing with PBS. Finally, fluorescent images of DCFH-DA staining on the cells were observed using CLSM (DCFH-DA was excited at 495 nm and the emission spectrum was collected between 500 and 550 nm), as shown in Figure 5. The Merge group consisted of a mixture of QTABI, ROS, and DAPI. Clearly, in the ROS group, DCFH oxidation produced a distinct green fluorescence upon illumination, indicating that QTABI can generate significant reactive oxygen species (ROS) under intracellular illumination.

[0093] Ovarian cancer cell killing: HEYA8 cells were seeded in a culture dish at a density of 10 5 / mL, and after they adhered to the wall, different concentrations of QTABI (0, 0.01µg / ml, 0.1µg / ml, 1µg / ml, 10µg / ml, 100µg / ml) were added. After further incubation for 4 hours, they were irradiated with a 450 nm laser for 5 minutes (the control group was not irradiated) and incubated for another 44 hours. The culture medium was then removed, and 100 µL of fresh culture medium was added, followed by 10 µL of CCK-8. The cells were incubated in a 37°C CO2 incubator for 1 hour, and the absorbance at 450 nm of each group was measured using a microplate reader (Figure 6). The results showed that under the absence of light, the IC 50 The value was 22.9 µg / ml, and the IC 50 The value was 0.041 µg / ml.

[0094] In vivo imaging in mice

[0095] Nanoparticle preparation: Prepare 0.5 mg / mL QTABI DMSO stock solution A and 10 mg / mL DSPE-PEG 2000NH2DMSO stock solution B was stored at room temperature and protected from light. 1.0 mL of stock solution A and 0.25 mL of stock solution B were added to 10 mL of ultrapure water under ultrasonic conditions (100 W) and sonicated at 37°C for 30 min to obtain QTABI NPs. The organic solvent was removed by ultrafiltration and the concentration was quantified to 100 µg / mL. NHS-Cy5.5 was added to the resulting nanoparticles and stirred at room temperature overnight. The reaction solution was then diluted and ultrafiltered to a photosensitizer concentration of 100 µg / mL. These nanoparticles were then injected into tumor-bearing mice through the tail vein. Fluorescence distribution was observed using an in vivo imaging system (AniView 100) at 0, 2, 4, 6, 8, and 12 hours. Sudden death occurred at 12 hours, and the mice's internal organs (heart, liver, spleen, lungs, kidneys, and tumors) were harvested for observation of their in vivo distribution (Figure 7). Results showed that drug accumulation in tumor tissue began 2 hours after administration, peaked between 6 and 8 hours, and then gradually decreased due to metabolism.

[0096] The bioactive photosensitizer of the present invention has good fluorescence efficiency and strong reactive oxygen species generation ability under blue light excitation, and can be well used in image-guided photodynamic therapy.

[0097] In summary, photosensitizers convert oxygen surrounding tumor tissue into singlet oxygen after being excited by light of a specific wavelength, thereby exerting a photodynamic therapy effect. However, tumor tissue is usually hypoxic, which limits the therapeutic effect of photosensitizers. The active photosensitizer provided by the present invention, which also acts as a PARP inhibitor, can induce cell synthetic lethality. This process can increase the oxidation state and oxygen concentration in the cells, improve the hypoxic microenvironment inside the tumor, and further enhance the photodynamic therapy effect, that is, PDT therapy and PARP inhibitors synergistically enhance the effect.

[0098] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. [Corrected according to Rule 26 on 27.02.2025] A bioactive photosensitizer targeting poly ADP-ribose polymerase, characterized in that, The bioactive photosensitizer is a quinoxalinone derivative, and the structural formula of the quinoxalinone derivative is as follows: Among them, R 1 is one of methoxy, methyl, and halogen, and the substituent position of R 1 is at least at one of the 5, 6, 7, and 8 positions of the quinoxalinone ring.

2. [Corrected according to Rule 26 on 27.02.2025] The bioactive photosensitizer targeting poly (ADP-ribose) polymerase according to claim 1, characterized in that, The structural formula of the quinoxalinone derivative is as follows:

3. [Corrected according to Rule 26 on 27.02.2025] A method for preparing a bioactive photosensitizer targeting poly ADP-ribose polymerase according to claim 1, characterized in that, The reaction route is as follows: The method includes: Step 1, the amidation reaction of compound (1d) and compound (1h) to obtain compound (1i); Step 2, the Suzuki coupling reaction of compound (1i) and borate-substituted anthraquinone (1j) to obtain compound (1).

4. The preparation method of the bioactive photosensitizer targeting poly ADP-ribose polymerase according to claim 3, characterized in that, In Step 1, condensing agent 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate and organic base triethylamine are also added, and the molar ratio of compound (1h): compound (1d): condensing agent: organic base = 1: (1 - 2): (1 - 2): (2 - 5).

5. [Corrected according to Rule 26 on 27.02.2025] The method for preparing a bioactive photosensitizer targeting poly ADP-ribose polymerase according to claim 3, characterized in that, The compound (1d) was prepared by the following method: Step 1.1, the amidation condensation reaction of compound (1a) and compound (1b) to obtain compound (1c); Step 1.2, the removal of the amino protecting group from compound (1c) to obtain compound (1d).

6. The preparation method of the bioactive photosensitizer targeting poly (ADP-ribose) polymerase according to claim 5, characterized in that, In Step 1.1, the molar ratio of compound (1a) to compound (1b) is (1 - 2):

1.

7. The preparation method of the bioactive photosensitizer targeting poly ADP-ribose polymerase according to claim 5, characterized in that, In Step 1.1, condensing agent O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and catalyst diisopropylethylamine are also added.

8. [Corrected according to Rule 26 on 27.02.2025] The method for preparing a bioactive photosensitizer targeting poly ADP-ribose polymerase according to claim 3, characterized in that, The compound (1h) is prepared by the following method: Step S1, the compound (1e) undergoes a condensation reaction with 5-bromothiophene-2-carbaldehyde to form the compound (1f); Step S2, compound (1f) and ethyl bromoacetate undergo a nucleophilic substitution reaction under alkaline conditions to form compound (1g); Step S3, compound (1g) is hydrolyzed to obtain compound (1h).

9. The preparation method of the bioactive photosensitizer targeting poly ADP-ribose polymerase according to claim 8, characterized in that, In Step S1, catalyst pyridine is also added, and the molar ratio of compound (1e), 5-bromothiophene-2-carbaldehyde, and the catalyst is 1: (1 - 2): (1 - 5).

10. The preparation method of the bioactive photosensitizer targeting poly ADP ribose polymerase according to claim 8, characterized in that, In Step S2, the molar ratio of compound (1f) to ethyl bromoacetate is 1: (1 - 2).

11. The preparation method of the bioactive photosensitizer targeting poly ADP-ribose polymerase according to claim 3, characterized in that, In Step 2, catalyst tetrakis(triphenylphosphine)palladium and inorganic base are also added, and the molar ratio of compound (1i): borate-substituted anthraquinone (1j): catalyst: inorganic base = 1: (1 - 2): (0.03 - 0.1): (2 - 5) in terms of molar ratio.

12. Use of a bioactive photosensitizer targeting poly(ADP-ribose) polymerase as described in claim 1 in photodynamic therapy.

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