CN118027013A

CN118027013A - Bioactive photosensitizer targeting poly ADP-ribose polymerase, preparation method and application

Info

Publication number: CN118027013A
Application number: CN202410067968.XA
Authority: CN
Inventors: 施雷雷; 樊连峰
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2024-01-17
Filing date: 2024-01-17
Publication date: 2024-05-14

Abstract

The invention discloses a bioactive photosensitizer targeting poly ADP-ribose polymerase, a preparation method and application thereof. The bioactive photosensitizer is a quinoxalinone derivative, and the structural formula of the quinoxalinone derivative is as follows: Wherein R ¹ is one of methoxy, methyl and halogen, and the substituent position of R ¹ is at least one of 5,6,7 and 8 positions of the quinoxalinone ring. The active photosensitizer provided by the invention is used as a PARP inhibitor, can induce cell synthesis to death, can promote oxidation state and oxygen concentration in cells, and can improve the hypoxia microenvironment in tumors, so that the photodynamic treatment effect is further enhanced, namely, the PDT therapy and the PARP inhibitor are synergistically enhanced.

Description

Bioactive photosensitizer targeting poly ADP-ribose polymerase, preparation method and application

Technical Field

The invention relates to the field of novel drug development, in particular to a quinoxalinone-derived bioactive photosensitizer of targeting poly ADP-ribose polymerase, a preparation method and an anti-tumor application thereof.

Background

Photodynamic therapy (PDT) is a therapy that uses photoactive molecules to produce a "photodynamic" response under excitation by light of a specific wavelength to act as an anti-tumor agent. During PDT, photosensitizing drugs are capable of generating Reactive Oxygen Species (ROS) by energy or electron transfer. ROS can undergo oxidation reactions with nearby biological macromolecules, producing cytotoxicity and thus killing tumor cells.

PDT has the advantage over conventional therapies that it enables accurate and efficient treatment with high temporal-spatial resolution of light, and thus has fewer side effects. However, due to the higher concentration of the reduced glutathione in the tumor cells, the active oxygen free radicals generated by the photosensitive molecules under the illumination can be rapidly quenched; in addition, most of the photosensitive molecules generate active oxygen radicals which depend on the concentration of molecular oxygen (O ₂), and the anaerobic microenvironment inside the solid tumor can limit the effect of photodynamic therapy to a large extent.

Poly (ADP-ribose) polymerase (PARP), a ribozyme having catalytic activity of transglycosylase, plays an important role in DNA damage repair, transcriptional regulation, chromatin dynamics, hypoxia response, metabolism, cell death, genomic stability, and the like mainly by modification with poly (ADP-ribosylation, PARylation). The PARP family has a total of 18 members, of which PARP1, the most critical member of the PARP family, exerts an intracellular 80-90% PARylation modification. PARP1 mainly acts as a sensor during DNA damage repair. Once DNA breaks occur, the zinc finger structure of PQRP a is able to rapidly bind to the damaged site of DNA and initiate DNA repair by PAR recruiting proteins associated with DNA damage repair. DNA damage stimulation is capable of inducing the catalytic activity of PARP1, synthesizing PAR chains on itself, histones and nonhistones via PARylation. PARP1 is a critical temporal and spatial organiser for the whole DNA repair process and the repair pathway of Base Excision Repair (BER) cannot function when the PARP1 gene is deleted. When the PARP1 gene has defects, DNA damage can accumulate in cells, and finally cell cycle arrest and even cell death are caused, so that the PARP becomes a hot tumor target, and the research and development of the action mechanism of the PARP inhibitor are promoted.

PARP inhibitors, through binding to the catalytic site of PARP1 or PARP2, result in the inability of PARP proteins to shed from the site of DNA damage. PRAP bound to DNA causes DNA replication to stall and DNA replication to fail, resulting in cell cycle arrest and thus cell death. There are 4 PARP inhibitors currently available in the domestic market: olaparib, nilaparib, fluxazopali and pamiopalide are mainly used for the treatment or maintenance treatment of ovarian cancer, breast cancer, prostate cancer and the like.

The intensive research of PDT therapy and the development of PARP inhibitors have greatly promoted malignant tumor treatment, but the application thereof still faces a plurality of challenges. Specifically, for photosensitizers, due to the high concentration of reduced glutathione in tumor cells, active oxygen radicals generated by photosensitizers under illumination can be rapidly quenched, however, the anaerobic microenvironment inside solid tumors can greatly limit the effect of photodynamic therapy. Compared with the traditional chemotherapy drugs, the target-based small molecule inhibitor has relatively low side effects caused by the fact that the target-based small molecule inhibitor can inhibit key proteins in tumor cells, and is better in patient tolerance. But the off-target effect and drug resistance of small molecule inhibitors have been a problem in the field of tumor therapy.

Disclosure of Invention

Aiming at the defects of poor effect of the traditional photosensitizer and limitations of small molecular targeted drugs, the invention designs and synthesizes a bioactive photosensitizer which can induce tumor cells to synthesize and death and can simultaneously efficiently generate ROS by searching key protein targets related to DNA repair in the tumor cells, thereby improving photodynamic treatment effect.

In order to achieve the above purpose, the 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:

wherein R ¹ is one of methoxy, methyl and halogen, and the substituent position of R ¹ is at least one of 5,6,7 and 8 positions of the quinoxalinone ring.

Optionally, the structural formula of the quinoxalinone derivative is:

the invention also provides a preparation method of the bioactive photosensitizer of the targeting poly ADP-ribose polymerase, which comprises the following reaction routes:

the method comprises the following steps:

Step 1, carrying out amidation reaction on the compound (1 d) and the compound (1 h) to obtain a compound (1 i);

step 2, carrying out Suzuki coupling reaction on the compound (1 i) and the borate substituted anthraquinone (1 j) to obtain the compound (1).

Optionally, in step 1, condensing agent 1H-benzotriazole-1-yl oxygen tripyrrolidinylphosphonium hexafluorophosphate and organic base triethylamine are also added, and the compound (1H): compound (1 d): condensing agent: organic base=1 (1-2): 2-5.

Alternatively, the compound (1 d) is prepared by the following method:

Step 1.1, performing amidation condensation reaction on the compound (1 a) and the compound (1 b) to obtain a compound (1 c);

Step 1.2, removing the amino protection of the compound (1 c) to obtain a compound (1 d).

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

Optionally, the condensing agent O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and the catalyst diisopropylethylamine are also added in the step 1.1.

Alternatively, the compound (1 h) is prepared by the following method:

Step S1, performing condensation reaction on the compound (1 e) and 5-bromothiophene-2-formaldehyde to form a compound (1 f);

Step S2, performing nucleophilic substitution reaction on the compound (1 f) and ethyl bromoacetate under alkaline conditions to generate a compound (1 g);

Step S3, hydrolyzing the compound (1 g) to obtain a compound (1 h).

Optionally, in the step S1, a catalyst pyridine is also added, wherein the molar ratio of the compound (1 e), the 5-bromothiophene-2-formaldehyde and the catalyst is 1: (1-2): (1-5).

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

Optionally, in step 2, catalyst tetra- (triphenylphosphine) palladium and inorganic base are also added, compound (1 i): borate substituted anthraquinone (1 j): catalyst: inorganic base=1 (1-2): (0.03-0.1): (2-5), molar ratio.

The invention also provides application of the bioactive photosensitizer targeting poly ADP-ribose polymerase in photodynamic therapy.

Compared with the prior art, the invention has the following beneficial effects:

1) The PARP-targeted active photosensitizer provided by the invention can inhibit DNA repair of tumor cells and induce death of the tumor cells by inhibiting expression of poly ADP ribose polymerase, thereby improving photodynamic treatment effect.

2) The PARP-targeted active photosensitizer provided by the invention has strong capability of generating active oxygen free radicals, and can generate active oxygen species in a hypoxic tumor microenvironment.

3) The PARP-targeted active photosensitizer provided by the invention has a very high killing effect on ovarian cancer cells and has very low dark toxicity on normal cells.

4) The PARP-targeted active photosensitizer provided by the invention can realize living body imaging at animal level and can be used for image-guided photodynamic therapy of tumor-bearing mice. The photosensitizer can realize good time and space resolution in the biological imaging experiment and can be applied to photodynamic therapy guided by fluorescence imaging.

Drawings

FIG. 1 is a mass spectral characterization of QTABI.

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum characterization of QTABI.

FIG. 3 is a graph showing the absorption and fluorescence spectra of QTABI of the present invention.

FIG. 4 is a graph representing the generation of ROS by QTABI of the present invention under excitation of blue light.

FIG. 5 is a graph of intracellular ROS detection by cell pair QTABI.

FIG. 6 is a QTABI study of cytotoxicity against ovarian cancer.

Fig. 7 is a distribution of in vivo small animal imaging studies QTABI in tumor-bearing mice.

Detailed Description

The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

the method comprises the following steps:

step 1, compound 1d and compound 1h are subjected to amidation reaction to obtain compound 1i.

In order to improve the reaction efficiency, in the step 1, a condensing agent 1H-benzotriazole-1-yl oxygen tripyrrolidinylphosphonium hexafluorophosphate and an organic base are also added, and a compound 1H: compound 1d: condensing agent: organic base=1 (1-2): 2-5. The organic base includes: triethylamine, diisopropylethylamine, and the like. For example, a carboxylic acid derivative (compound 1 h) was dispersed in a solvent, a condensing agent PyBOP and triethylamine were added, and after stirring at room temperature for 1 hour, an amine derivative (compound 1 d) was added, and after the completion of the reaction, the solvent was removed by a rotary evaporator, and purification by column chromatography was performed to obtain intermediate 1i. Compound 1h: compound 1d: condensing agent: the molar ratio of the catalyst is 1:1.1:1.1:4, the solvent is DMF, and the reaction is carried out for 8 hours at room temperature.

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

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

In some embodiments, to facilitate the condensation reaction, a condensing agent O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and a catalyst, such as an organic base, e.g., diisopropylethylamine, are also added to step 1.1. For example, the benzoic acid derivative, the o-phenylenediamine derivative and the HATU are added into a dispersion solvent, DIPEA is added, stirring is carried out at room temperature, after the reaction is finished, saturated saline water is used for washing, ethyl acetate extraction is carried out, then liquid separation, drying, filtration and concentration are carried out to obtain a crude product, and column chromatography is used for purifying to obtain an amide intermediate; the intermediate is dissolved in acetic acid, stirred at 120 ℃, and purified by column chromatography after the reaction is finished to obtain benzimidazole intermediate 1c.

Step 1.2, removing the amino protection of the compound 1c to obtain a compound 1d.

In some embodiments, deprotection is by acid. The acid may be hydrochloric acid or trifluoroacetic acid, etc. For example, benzimidazole intermediate 1c is dispersed in methanol, a dioxane solution of hydrochloric acid is added, stirred at room temperature, and after the reaction is completed, intermediate 1d is obtained by purification by column chromatography. The molar ratio of the benzimidazole derivative (compound 1 c) to the hydrochloric acid (dioxane solution) is 1:10; the solvent is anhydrous methanol, the reaction temperature is room temperature, and the reaction time is 8 hours.

The compound 1h was prepared by the following method:

Step S1, performing condensation reaction on a compound 1e and 5-bromothiophene-2-formaldehyde to form a compound 1f; in order to increase the reaction rate, pyridine is also added as a catalyst, wherein the molar ratio of the compound 1e to the 5-bromothiophene-2-formaldehyde to the catalyst is 1: (1-2): (1-5). As an example, quinoxalinone skeleton compound 1e is dispersed in a solvent, 5-bromothiophene-2-carbaldehyde and pyridine are added, and after the reaction is completed, the solid is filtered and washed with acetic acid and diethyl ether, respectively, to obtain a condensation product 1f. The molar ratio of the quinoxalinone (compound 1 e), the 5-bromothiophene-2-formaldehyde and the pyridine is 1:1.5:5; the solvent is acetic anhydride; the reaction temperature was 120℃and the reaction time was 10 hours, followed by reaction at room temperature for 12 hours.

Step S2, performing nucleophilic substitution reaction on the compound 1f and ethyl bromoacetate under alkaline conditions to generate a compound 1g; the molar ratio of the compound 1f to ethyl bromoacetate is 1: (1-2). The alkaline condition is that adding alkali such as potassium carbonate or sodium carbonate into the solution to make its pH value larger than 7 so as to promote nucleophilic substitution reaction. As an example, condensation product 1f was dispersed in a solvent, ethyl bromoacetate and potassium carbonate were added, and after the reaction was completed, column chromatography was performed to obtain a bromoquinoxalinone thiophene derivative (compound 1 g). The molar ratio of the compound 1f to the ethyl bromoacetate is 1:1.5, the solvent is acetone, the reaction temperature is 70 ℃, and the reaction time is 8 hours.

Step S3, hydrolyzing 1g of the compound to obtain a compound 1h. The hydrolysis is carried out under conventional alkaline conditions (lithium hydroxide or sodium hydroxide, etc.). For example, 1g of the intermediate was dispersed in a solvent, lithium hydroxide was added, and after the reaction was completed, a dioxane solution containing hydrochloric acid was acidified to ph=7, the solvent was removed by a rotary evaporator, and the resultant was recrystallized from methylene chloride to obtain a carboxylic acid derivative for 1h. The molar ratio of 1g of the compound to lithium hydroxide is 1:5, the solvent is Tetrahydrofuran (THF) and water (volume ratio is 3:1), and the reaction is carried out for 8 hours at room temperature.

Step 2, carrying out Suzuki coupling reaction on the compound 1i and borate substituted anthraquinone 1j to obtain a compound 1. The Suzuki coupling reaction, also known as Suzuki reaction, is an organic coupling reaction: under the catalysis of the zero-valent palladium complex, aryl or alkenyl boric acid or boric acid ester and chlorine, bromine, iodo-aromatic hydrocarbon or olefin are cross-coupled.

In some examples, the catalyst tetrakis- (triphenylphosphine) palladium and an inorganic base, compound 1i: borate substituted anthraquinone 1j: catalyst: inorganic base=1 (1-2): (0.03-0.1): (2-5), molar ratio. As an example, intermediate compound 1i, borate substituted anthraquinone 1j, catalyst tetra- (triphenylphosphine) palladium and potassium carbonate were added to a schlenk tube, reacted in a mixed solvent of dioxane and water under nitrogen protection for 16 hours, after the reaction was completed, filtered to obtain red solid, and then washed with deionized water, diethyl ether and dichloromethane, respectively, to obtain quinoxalinone anthraquinone derivative QTABI (compound 1). The molar ratio of the intermediate compound 1i to the borate substituted anthraquinone 1j to the tetra- (triphenylphosphine) palladium to the potassium carbonate is 1:1.2:0.05:3.0, and the solvent is dioxane to water (volume ratio is 3:1); the reaction temperature was 90℃and the reaction time was 16 hours.

The following examples 1 to 10 specifically illustrate the synthesis of the above-described bioactive photosensitizer compounds:

the reagents and starting materials used in the examples were all commercially available.

The reagent abbreviations used in the examples represent the following full names:

QTABI: (2- (4- { [ (2- {3- [ (1E) -2- [5- (9, 10-dioxo-9, 10-dihydro-anthracene-2-yl) thiophen-2-yl ] vinyl ] -2-oxoquinoxalin-1-yl } acetyl) amino ] methyl } phenyl) -1H-benzo [ d ] imidazole-4-carboxamide

PyBOP: 1H-benzotriazol-1-yloxy tripyrrolidinylphosphonium hexafluorophosphate

HATU: o- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate

DMF: n, N-dimethylformamide

DIPEA: diisopropylethylamine

PBS: phosphate buffered saline

DAPI 4', 6-diamidino-2-phenylindole

CCK-8: cell Counting Kit-8 cell counting reagent

DMSO: dimethyl sulfoxide.

Example 1

Synthesis of intermediate 1 c. The method comprises the following steps:

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, stirring was performed at normal temperature for 24h, the reaction solution was washed with saturated brine, extracted with ethyl acetate, and the organic phase was separated, dried and concentrated to give a crude product, which was purified by column chromatography to give a white powder. The obtained white powder was dissolved in acetic acid (AcOH), heated at 120 ℃ for reaction for 8 hours, and after the reaction was completed, the reaction mixture was concentrated, and purified by column chromatography to give the title compound 1c (650 mg, yield 59%).

Synthesis of intermediate 1 d. The method comprises the following steps:

25mL of a dry round-bottomed flask equipped with a stirrer was taken, compound 1c (1.78 mmol,650 mg) and methanol (8 mL) were added, followed by dropwise addition of hydrochloric acid (dioxane solution, 4mol/L,4.45 mL) and stirring at room temperature for 8h. After the completion of the reaction, the crude product was concentrated, and then purified by column chromatography to give pale yellow powder, which was compound 1d (350 mg, yield 74%).

Synthesis of intermediate 1 f. The method comprises the following steps:

100mL of a dry round-bottomed flask equipped with a stirrer was taken, compound 1e (15 mmol,2.4 g) and acetic anhydride (24 mL) were added, pyridine (5.9 mL) and 5-bromothiophene-2-carbaldehyde (22.5 mmol,4.25 g) were added with stirring at room temperature, and the reaction solution was then moved to 120℃and heated for 10h. After the reaction solution was cooled, stirring was continued at room temperature for 12 hours. After the reaction was completed, the solid was collected by filtration using a sand core funnel, and then washed with ethanol and acetic acid to obtain a tan powder, which was compound 1f (3.2 g, yield 65%).

Synthesis of intermediate 1 g. The method comprises the following steps:

100mL of a dry round-bottom flask equipped with a stirrer was taken, compound 1f (3.3 mmol,1.1 g), potassium carbonate (5.0 mmol,690 mg) and tetrabutylammonium iodide as catalyst (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, and the reaction solution was then brought to 70℃under reflux with heating for 8h. After the reaction liquid was cooled, the organic solvent was removed by a rotary evaporator to obtain a crude product, which was then purified by column chromatography (petroleum ether/ethyl acetate=8:1 to 5:1) to obtain a yellow solid, namely, 1g (0.99 g, yield 72％).¹H NMR(600MHz,Chloroform-d)δ8.19(d,J＝15.8Hz,1H),7.86(dd,J＝8.0,1.5Hz,1H),7.49(ddd,J＝8.6,7.2,1.5Hz,1H),7.38-7.33(m,1H),7.37(d,J＝15.9Hz,1H),7.07(dd,J＝8.3,1.2Hz,1H),7.04-7.00(m,2H),5.05(s,2H),4.26(q,J＝7.2Hz,2H),1.28(t,J＝7.1Hz,3H).¹³C NMR(151MHz,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 (C ₁₈H₁₆BrN₂O₃ S): 419.0065; detected value: 419.0058) of the compound.

Synthesis of intermediate 1 h. The method comprises the following steps:

50mL of a dry round-bottomed flask equipped with a stirrer was taken, 1g (1.75 mmol,0.75 g) of the compound and lithium hydroxide (5.25 mmol,123 mg) were added, tetrahydrofuran (25 mL) and deionized water (5 mL) were added with stirring at room temperature, and then stirred at room temperature for 8h. After completion of the reaction, dioxane solution (2.0 mL,4 mol/L) of hydrochloric acid was added to the reaction mixture. Subsequently, methylene chloride (100 mL) and a saturated sodium chloride solution (50 mL) were added to the reaction solution, the organic phase was separated by extraction, the aqueous phase was extracted once with methylene chloride (100 mL), and the organic phase was separated and combined with the previous organic phase, and dried over anhydrous sodium sulfate. The organic phase was then filtered and distilled under reduced pressure to give a crude product. Finally, by recrystallization (dichloromethane and petroleum ether), a yellow solid was obtained as compound 1h (648 mg, yield 95%).

Synthesis of intermediate 1 i. The method comprises the following steps:

50mL of a dry round bottom flask equipped with a stirrer was taken, 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 with stirring at room temperature, and then stirred at room temperature for 1h. Then, amine derivative 1d (0.38 mmol,100 mg) was added to the reaction solution, and stirring was continued at room temperature for 8 hours. After the completion of the reaction, DMF was removed by rotary evaporator to give crude product, which was finally subjected to column chromatography to give yellow solid as compound 1i (210 mg, yield) 87％).¹H NMR(600MHz,DMSO-d₆)δ13.53(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).

Synthesis of the target product QTABI. The method comprises the following steps:

35mL of a dry Shi Laike-tube equipped with a stirrer was taken, compound 1i (0.2 mmol,128 mg), borate 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 protection, DMF (6 mL) and deionized water (2 mL) were added under stirring at room temperature, then frozen using liquid nitrogen, thawed to remove trace oxygen, and finally the reaction tube was moved to a 90℃oil bath with heating stirring for 16h. After the reaction, the reaction solution was filtered using a sand core funnel to obtain a crude product, which was washed with deionized water, dichloromethane and diethyl ether, respectively, and finally dried to obtain a red solid, which was compound QTABI (70 mg, yield 46％).HRMS(ESI)m/z(M+H)⁺calculated for C₄₅H₃₁N₆O₅S:767.2077,observed:767.1961, mass spectrum analysis chart is shown in FIG. 1. QTABI nuclear magnetic resonance hydrogen spectrum characterization is shown in FIG. 2) ,¹H NMR(600MHz,DMSO-d₆)δ9.40(s,1H),8.89(t,J＝6.0Hz,1H),8.42(d,J＝1.9Hz,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＝3.9Hz,1H),7.61(t,J＝7.9Hz,1H),7.50(d,J＝16.0Hz,1H),7.47-7.38(m,4H),7.35-7.25(m,1H),5.06(s,2H),4.41(d,J＝6.0Hz,2H).

Absorption spectrum, fluorescence spectrum and active oxygen generating capacity characterization of bioactive photosensitizer

A stock solution of QTABI mg/mL DMSO was prepared, QTABI was diluted to a solution of 10. Mu.g/mL by a mixed solvent of DMSO/CHCl ₃ in different proportions, and the absorption spectrum was measured by a Thermo Electron-EV300 UV-visible spectrophotometer with a maximum absorption wavelength of QTABI at 451nm. The fluorescence spectrum of QTABI was then measured by a steady state time resolved fluorescence spectrophotometer, resulting in a QTABI maximum emission wavelength of 679nm (FIG. 3). The active oxygen production efficiency was then measured by a 2',7' -dichlorofluorescein Diacetate (DCFH) probe, and the experimental result showed that QTABI had a very high active oxygen production efficiency (FIG. 4), the abscissa thereof represents the irradiation time, and the ordinate I/I ₀ represents the ratio of absorbance of the mixed solution of DCFH and QTABI after illumination to the initial absorbance.

Cellular uptake and killing of ovarian cancer cells

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. Mu.g/mL) was added after adherence and incubated for 4 hours, and the medium was removed. After replacement of the new medium, 2',7' -dichlorofluorescein diacetate (DCFH-DA) (5. Mu.M) was added for an additional incubation of 0.5 hours, after which the medium was discarded and washed 3 times with PBS after irradiation with blue light (60 mW/cm ², 450 nm) for 5 minutes. After replacement of the new medium, DAPI was added for an additional 10 minutes of incubation and washing with PBS. Finally, the fluorescent image of DCFH-DA staining on cells was observed using CLSM (DCFH-DA was excited at 495nm and emission spectra were collected at 500-550 nm), as shown in FIG. 5. Wherein, the Merge group is QTABI +ROS+DAPI mix. It can be seen that the ROS group can observe that DCFH is oxidized to generate obvious green fluorescence after illumination, which indicates that QTABI can generate obvious active oxygen under the intracellular illumination condition.

Ovarian cancer cell killing: HEYA8 was placed in a petri dish at a density of 10 ⁵/mL, after which QTABI (0,0.01. Mu.g/mL, 0.1. Mu.g/mL, 1. Mu.g/mL, 10. Mu.g/mL, 100. Mu.g/mL) was added at various concentrations after adherence, and after further incubation for 4 hours, the incubation was continued with a 450nm laser for 5min (control was not irradiated) and continued for 44 hours. The medium was then removed, 100. Mu.L of fresh medium was added, followed by 10. Mu.L of CCK-8, and after incubation for 1h in a 37℃carbon dioxide incubator, the absorbance values at 450nm of each group were determined by means of a microplate reader (FIG. 6). The results indicated that IC ₅₀ had a value of 22.9 μg/ml in the absence of light and IC ₅₀ had a value of 0.041 μg/ml in the presence of light.

In vivo imaging in mice

Nanoparticle preparation: 0.5mg/mL of QTABI DMSO stock solution A and 10mg/mL of DSPE-PEG ₂₀₀₀-NH₂ DMSO stock solution B were prepared and stored at room temperature in a dark place. 1.0mL of stock solution A and 0.25mL of stock solution B are added into 10mL of ultrapure water under the ultrasonic condition (100 w), ultrasonic treatment is carried out for 30min at 37 ℃, and then QTABI NPs ultrafiltration is carried out to remove the organic solvent, and the organic solvent is quantified to 100 mug/mL. NHS-Cy5.5 was added to the nanoparticle obtained above, and stirred overnight at room temperature, and then the reaction solution was diluted and ultrafiltered to give a photosensitizer concentration of 100. Mu.g/mL. The nanoparticle tails were then intravenously injected into tumor-bearing mice, and fluorescence distribution was observed in a biopsy imager (AniView) at 0h,2h,4h,6h,8h,12h, and quenched at 12h, taking the internal viscera (heart, liver, spleen, lung, kidney, tumor) of the mice for the observation period (fig. 7). The results show that 2 hours after administration the drug starts to enrich in tumor tissue, peaks at 6-8 hours and then gradually decreases due to the onset of metabolism.

The bioactive photosensitizer provided by the invention has good fluorescence efficiency and strong active oxygen species generation capacity under the excitation of blue light, and can be well used for image-guided photodynamic therapy.

In summary, the photosensitizer converts oxygen around tumor tissue into singlet oxygen after being excited by light of a specific wavelength, thereby exerting photodynamic therapy effect. However, tumor tissue is often hypoxic, limiting the therapeutic efficacy of photosensitizers. The active photosensitizer provided by the invention is used as a PARP inhibitor, can induce cell synthesis to death, can promote oxidation state and oxygen concentration in cells, and can improve the hypoxia microenvironment in tumors, so that the photodynamic treatment effect is further enhanced, namely, the PDT therapy and the PARP inhibitor are synergistically enhanced.

While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims

1. A bioactive photosensitizer targeting poly ADP-ribose polymerase, characterized in that the bioactive photosensitizer is a quinoxalinone derivative having the structural formula:

2. The bioactive photosensitizer targeting a poly ADP-ribose polymerase of claim 1 wherein the quinoxalinone derivative has the structural formula:

3. a method of preparing a biologically active photosensitizer targeting poly ADP-ribose polymerase according to claim 1, characterized by the following reaction scheme:

the method comprises the following steps:

4. The method for preparing a bioactive photosensitizer targeting poly ADP-ribose polymerase as claimed in claim 3, wherein in step 1, condensing agent 1H-benzotriazole-1-yl oxy tripyrrolidinylphosphonium hexafluorophosphate and organic base triethylamine are added, compound (1H): compound (1 d): condensing agent: organic base=1 (1-2): 2-5.

5. A method of preparing a biologically active photosensitizer targeting poly ADP-ribose polymerase as claimed in claim 3 wherein the compound (1 d) is prepared by:

6. The method for producing a biologically active photosensitizer targeting poly ADP-ribose polymerase as claimed in claim 5 wherein in step 1.1, the molar ratio of compound (1 a) to compound (1 b) is 1-2:1.

7. The method for preparing a bioactive photosensitizer targeting poly ADP-ribose polymerase as claimed in claim 5 wherein the condensing agent O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate and catalyst diisopropylethylamine are also added in step 1.1.

8. A method of preparing a biologically active photosensitizer targeting poly ADP-ribose polymerase as claimed in claim 3 wherein the compound (1 h) is prepared by:

Step S3, hydrolyzing the compound (1 g) to obtain a compound (1 h).

9. The method for preparing the bioactive photosensitizer targeting poly ADP-ribose polymerase as claimed in claim 8, wherein in step S1, pyridine is added as a catalyst, wherein the molar ratio of the compound (1 e), 5-bromothiophene-2-formaldehyde and the catalyst is 1: (1-2): (1-5).

10. The method of claim 8, wherein in step S2, the molar ratio of compound (1 f) to ethyl bromoacetate is 1: (1-2).

11. The method for preparing a bioactive photosensitizer targeting poly ADP-ribose polymerase as claimed in claim 3, wherein in step 2, catalyst tetra- (triphenylphosphine) palladium and inorganic base are added, compound (1 i): borate substituted anthraquinone (1 j): catalyst: inorganic base=1 (1-2):

(0.03-0.1) and (2-5) in terms of mole ratio.

12. Use of a biologically active photosensitizer targeting poly ADP-ribose polymerase according to claim 1 in photodynamic therapy.