A type-I bioactive photosensitizer targeting histone deacetylase, preparation method and application

Abstract

The present invention provides a type-I type bioactive photosensitizer targeting HDAC, and its preparation method and application in living tumor treatment. The photosensitizer molecular structure is as follows: the core skeleton is quinoxalinone, which is obtained by condensation cyclization, nucleophilic substitution, aldol condensation, ester hydrolysis and amidation, Suzuki coupling reaction by o-phenylenediamine and its derivatives. Only one column chromatography is required during the whole synthesis process, and the preparation is simple. The molecule of the present invention has strong fluorescence emission under blue light radiation, and can produce a large amount of reactive oxygen free radicals such as hydroxyl radicals and superoxide radicals at the same time. The compound of the present invention can also be used for photodynamic therapy of tumor cells, and can also be used to prepare nanoparticles using polyethylene glycol and then be injected intravenously to exert molecular targeted therapy and photodynamic therapy in vivo. In addition, the material of the present invention can induce cell ferroptosis by inhibiting histone deacetylase expression, alleviate hypoxic microenvironment, improve photodynamic therapy effect, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of novel drug development, and in particular to a type-I bioactive photosensitizer targeting histone deacetylase, a preparation method and an application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a treatment that uses photosensitive molecules and laser activation to produce a "photodynamic" reaction to exert an anti-tumor effect. During the PDT process, photosensitizing drugs transfer energy to the surrounding oxygen, generating highly active singlet oxygen. Singlet oxygen can undergo oxidative reactions with nearby biomacromolecules, producing cytotoxicity and thus killing tumor cells. Compared with traditional therapies, the advantage of PDT is that it can use the high temporal and spatial resolution of light for precise and effective treatment, so the side effects are relatively small. Histone deacetylase (HDAC) is a class of proteases that play an important role in the structural modification of chromosomes and the regulation of gene expression. Under normal circumstances, histone acetylation facilitates the dissociation of DNA from the histone octamer, relaxes the nucleosome structure, and enables various transcription factors and co-transcription factors to specifically bind to DNA and activate gene transcription. Within the cell nucleus, histone acetylation and histone deacetylation are in a dynamic equilibrium, co-regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). In cancer cells, overexpression of HDACs leads to enhanced deacetylation, restoring the positive charge of histones and thereby strengthening the interaction between DNA and histones, causing relaxed nucleosomes to become very tight, which is not conducive to the expression of specific genes such as tumor suppressor genes. Histone deacetylase inhibitors (HDACi) can regulate the expression of apoptosis- and differentiation-related proteins by increasing histone acetylation levels in specific chromatin regions, thereby inducing cell apoptosis and differentiation.

[0003] The in-depth research on PDT therapy and the development of HDAC inhibitors have greatly promoted the treatment of malignant tumors, but its application still faces many challenges. First of all, for photosensitizers, since the concentration of reduced glutathione in tumor cells is very high, it can quickly quench the reactive oxygen free radicals generated by photosensitive molecules under light. In addition, the production of reactive oxygen free radicals by most photosensitive molecules depends on the concentration of molecular oxygen (O2), and 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 highly expressed 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.

[0004] Chinese patent CN113501816A, published on October 15, 2021, discloses a quinoxalinone active photosensitizer based on histone deacetylase as a target, its preparation method and application. The active photosensitizer molecule is obtained by cyclization, nucleophilic substitution, aldol condensation, hydroxylamine decomposition and other reactions of o-phenylenediamine and its derivatives. The compound of the present invention can significantly inhibit the activity of HDAC1, 6, and 8, with IC50 of 89±4.6, 34±2.7, and 852±6.3nM respectively. At the same time, the compound produces a large amount of reactive oxygen free radicals under 450nm laser irradiation, which can be used as a potential active photosensitizer. The compound of the invention can be directly added to the culture medium and then used for photodynamic combined with molecular targeted therapy of breast cancer cells (MCF-7). It can also be prepared into nanoparticles and then injected intravenously, followed by laser irradiation, to simultaneously exert molecular targeted therapy and photodynamic therapy in tumor-bearing mice. However, in the aforementioned invention, the active photosensitizer, in addition to reversing the hypoxic microenvironment by inhibiting HDAC, primarily produces singlet oxygen, a reactive oxygen species, to kill cells, resulting in a certain degree of oxygen dependence. Therefore, it is of great significance to develop an oxygen-independent bioactive photosensitizer that can inhibit HDAC to enhance the efficacy of photodynamic therapy.

[0005] However, there are no reports on the type-I bioactive photosensitizer targeting histone deacetylase, its preparation method and application according to the present invention. Summary of the Invention

[0006] The purpose of the present invention is to target the defects of traditional photosensitizers, such as poor effect and high oxygen dependence, and the limitations of small molecule targeted drugs. By searching for key protein targets closely related to hypoxia and oxidative resistance in tumor cells, a bioactive photosensitizer that can induce ferroptosis in tumor cells to reverse the hypoxic microenvironment and is not highly dependent on oxygen is designed and synthesized, thereby enhancing the photodynamic therapy effect. Traditional photosensitizers convert the oxygen around the 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 the photosensitizer. Type-I photosensitizers have a low degree of oxygen dependence and can solve the problem of oxygen dependence of traditional photosensitizers to a certain extent. On the other hand, HDAC inhibitors can drive cells to undergo ferroptosis, which 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.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a type-I bioactive photosensitizer targeting HDAC, wherein the bioactive photosensitizer is a quinoxalinone derivative, and the structural formula of the quinoxalinone derivative is as follows:

[0009]

[0010] The substituent R1 on the left side of the quinoxalinone skeleton is one of methoxy, halogen and methyl, and the substituent is located at any one of positions 5, 6, 7 and 8 of the quinoxalinone ring; n=1, 2, 3, 4, 5.

[0011] In a second aspect, the present invention provides a method for preparing the bioactive photosensitizer as described above, and the reaction scheme is as follows:

[0012]

[0013] The preparation method comprises the following steps:

[0014] S1: Add ethyl pyruvate to a dispersion solvent containing o-phenylenediamine and its derivatives, stir at room temperature, filter after the reaction, and wash the solid with ethanol to obtain quinoxalinone skeleton compound 1a;

[0015] S2: Dispersing the quinoxalinone skeleton compound 1a in a solvent, adding 5-bromothiophene-2-carboxaldehyde and pyridine, filtering after the reaction, and washing the solid with acetic acid to obtain the condensation product 1b;

[0016] S3: Disperse the condensation product 1b in a solvent, add ethyl bromoacetate and potassium carbonate, and purify by column chromatography after the reaction to obtain a bromoquinoxalinone thiophene derivative 1c;

[0017] S4: Disperse the intermediate substituted compound 1c in a solvent, add lithium hydroxide, and after the reaction, acidify to pH 7 by adding a dioxane solution of hydrochloric acid. Remove the solvent using a rotary evaporator, and recrystallize from dichloromethane to obtain the carboxylic acid derivative 1d.

[0018] S5: Disperse the intermediate substituted product 1d in a solvent, add primary amine hydrochloride, a condensing agent 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, and triethylamine, remove the solvent by rotary evaporation after the reaction, and recrystallize from dichloromethane to obtain the carboxylic acid derivative 1e;

[0019] S6: Dispersing the intermediate substituted product 1e in a solvent, adding lithium hydroxide, and after the reaction, acidifying to pH 7 by adding a dioxane solution of hydrochloric acid, removing the solvent by rotary evaporation, and recrystallizing from dichloromethane to obtain the carboxylic acid derivative 1f;

[0020] S7: Disperse the intermediate substituted product 1f in a solvent, add O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, a condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a catalyst 4-dimethylaminopyridine. After the reaction, remove the solvent under reduced pressure, and recrystallize from dichloromethane to obtain 1g of the carboxylic acid derivative.

[0021] S8: 1 g of the intermediate substituent, borate-substituted anthraquinone, catalyst tetrakis-(triphenylphosphine) palladium, and potassium carbonate were added to a Shrek tube and reacted in a mixed solvent of dioxane and water under nitrogen for 20 h. After the reaction, a red solid was obtained by filtration, which was then washed with deionized water, ether, and dichloromethane, respectively, to obtain 1 h of a quinoxalinone anthraquinone derivative;

[0022] S9: The intermediate substituent 1h was dispersed in a solvent, trifluoroacetic acid was added, and the reaction was carried out at 0°C for 3 hours. After the reaction was completed, a red solid was obtained by filtration, and then washed with sodium bicarbonate aqueous solution, deionized water, ether and dichloromethane respectively to obtain a bioactive photosensitizer.

[0023] As a preferred example, the molar ratio of the above-mentioned o-phenylenediamine and its derivatives to ethyl pyruvate is 1:1.3; the solvent is anhydrous ethanol, the reaction temperature is room temperature, and the reaction time is 24 hours.

[0024] More preferably, the molar ratio of the quinoxalinone 1a, 5-bromothiophene-2-carboxaldehyde and pyridine is 1:1.5:5; the solvent is acetic anhydride; the reaction temperature is 120° C., the reaction time is 10 hours, and then the reaction is carried out at room temperature for 12 hours.

[0025] More preferably, the molar ratio of the compound 1b to ethyl bromoacetate is 1:1.5, the solvent is acetone, the reaction temperature is 70° C., and the reaction time is 8 hours.

[0026] More preferably, the molar ratio of the compound 1c to lithium hydroxide is 1:3, the solvent is tetrahydrofuran and water (volume ratio is 3:1), and the reaction is carried out at room temperature for 8 hours.

[0027] More preferably, the molar ratio of the compound 1d, primary amine hydrochloride, condensing agent 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate and triethylamine is 1:1.2:1.2:4, the solvent is dimethylformamide, and the reaction is carried out at room temperature for 8 hours.

[0028] More preferably, the molar ratio of the compound 1e to lithium hydroxide is 1:5, the solvent is tetrahydrofuran and water (volume ratio is 3:1), and the reaction is carried out at room temperature for 8 hours.

[0029] More preferably, the molar ratio of the above-mentioned compound 1f, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the catalyst 4-dimethylaminopyridine is 1:1.5:1.2:0.2, the solvent is dimethylformamide, and the reaction is carried out at room temperature for 6 hours.

[0030] More preferably, the molar ratio of the intermediate 1g, borate-substituted anthraquinone, tetrakis-(triphenylphosphine)palladium and potassium carbonate is 1:1.2:0.05:3.0, the solvent is dioxane and water (volume ratio 3:1); the reaction temperature is 90°C, and the reaction time is 20 hours.

[0031] More preferably, the molar ratio of the intermediate 1h to trifluoroacetic acid is 1:130, the solvent is dichloromethane, the reaction temperature is 0° C., and the reaction time is 3 hours.

[0032] In a third aspect, the present invention provides an application of a Type I bioactive photosensitizer targeting HDAC in image-guided photodynamic therapy of tumors. The bioactive photosensitizer of the present invention has good fluorescence efficiency and strong reactive oxygen generation efficiency, and can be well used in image-guided photodynamic therapy.

[0033] The advantages of the present invention are:

[0034] 1. The present invention provides an active photosensitizer that can target HDAC, which can induce ferroptosis of tumor cells by inhibiting the expression of histone deacetylase, thereby reversing the tumor hypoxic microenvironment and improving the effect of photodynamic therapy.

[0035] 2. The present invention provides a type-I photosensitizer that is not highly dependent on oxygen concentration and can produce reactive oxygen species such as hydroxyl radicals and superoxide radicals in an oxygen-deficient environment.

[0036] 3. The photosensitizer of the present invention can have a high killing effect on cells in vitro and has extremely low dark toxicity.

[0037] 4. The photosensitizer of the present invention can achieve in vivo imaging at the animal level. After constructing tumor-bearing mice, it can be used for image-guided photodynamic therapy of tumor-bearing mice. The above photosensitizer can achieve excellent temporal and spatial resolution in biological imaging experiments.

[0038] The present invention also provides a method for preparing the above-mentioned bioactive photosensitizer, which has cheap raw materials, a simple synthesis route, and is easy to separate, and has good industrial application prospects in the fields of fluorescence imaging and biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Mass spectrometric characterization of QCNHOH.

[0040] Figure 2 This is the H NMR characterization of QCNHOH.

[0041] Figure 3 1 is the absorption and fluorescence spectra of the quinoxalinone derivative (QCNHOH) of the present invention.

[0042] Figure 4 This is a characterization diagram of the quinoxalinone derivative (QCNHOH) of the present invention generating ROS under blue light excitation.

[0043] Figure 5 This study is to study the cellular uptake of QCNHOH.

[0044] Figure 6 For cytotoxicity studies.

[0045] Figure 7 The distribution of QCNHOH in tumor-bearing mice was studied by in vivo imaging. DETAILED DESCRIPTION

[0046] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention record, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the application's appended claims.

[0047] The following Examples 1 to 8 specifically illustrate the synthesis method of the above-mentioned bioactive photosensitizer compound:

[0048] Example 1: Synthesis of intermediate 1a.

[0049] The specific steps include:

[0050] O-phenylenediamine (60 mmol, 6.48 g) was dispersed in anhydrous ethanol (200 mL), and ethyl pyruvate (78 mmol, 9.05 g) was added dropwise under ice bath. The mixture was stirred at room temperature for 24 h. The reaction solution was filtered, and the filter cake was washed with anhydrous ethanol and dried to obtain a white powder, namely compound 1a (8.16 g, yield 85%).

[0051] Example 2: Synthesis of Intermediate 1b

[0052] The specific steps include:

[0053] To a 100 mL dry round-bottom flask equipped with a stirrer, 1a (15 mmol, 2.4 g) and acetic anhydride (24 mL) were added. 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 sand core funnel and then washed with ethanol and acetic acid to obtain a brown-black powder, compound 1b (3.2 g, 65% yield).

[0054] Example 3: Synthesis of Intermediate 1c

[0055] The specific steps include:

[0056] To a 100 mL dry round-bottom flask equipped with a stirrer, 1b (3.3 mmol, 1.1 g), potassium carbonate (5.0 mmol, 690 mg), and 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 moved to 70°C and heated under reflux 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 1c (0.99 g, 72% yield) as a yellow solid. 1 H 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,1 H),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) δ167.2,154.6,151.8,144.0,133.7,132.1,131.1,130.8,1 30.3,130.3,130.1,130.0,114.9,113.2,62.3,43.8,14.3.HRMS(ESI)m / z(M+H) + calculated for C 18 H 16 BrN2O3S:419.0065,observed:419.0058.

[0057] Example 4: Synthesis of Intermediate 1d

[0058] The specific steps include:

[0059] Take a 50mL dry round-bottom flask equipped with a stirrer, add 1c (1.75mmol, 0.75g) and lithium hydroxide (5.25mmol, 123mg), add tetrahydrofuran (25mL) and deionized water (5mL) while stirring at room temperature, and then stir at room temperature for 8h. After the reaction is completed, a dioxane solution of hydrochloric acid (2.0mL, 4mol / L) is added to the reaction solution. Then dichloromethane (100mL) and saturated sodium chloride solution (50mL) are added to the reaction solution, and the organic phase is extracted and separated. The aqueous phase is extracted once with dichloromethane (100mL), 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), which is compound 1d (648mg, 95% yield).

[0060] Example 5: Synthesis of Intermediate 1e

[0061] The specific steps include:

[0062] To a 50 mL dry round-bottom flask equipped with a stirrer, 1d (1.67 mmol, 0.65 g), primary amine hydrochloride (2.0 mmol, 362 mg), and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (2.0 mmol, 1.04 g) were added. Dimethylformamide (20 mL) and triethylamine (6.68 mmol, 0.67 g) were added while stirring at room temperature, and then stirred at room temperature for 8 h. After the reaction was completed, saturated sodium chloride solution (50 mL) was added to the reaction solution, and then extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude product. Finally, a yellow solid was obtained by recrystallization (dichloromethane and petroleum ether), namely compound 1e (827 mg, 96% yield).1 HNMR(600MHz,Chloroform-d)δ8.15(d,J=15.8Hz,1H),7.84(d,J=8.0Hz,1H),7 .53(t,J=7.9Hz,1H),7.48(d,J=8.5Hz,1H),7.41–7.31(m,2H),7.06–6.98(m,2 H),6.45–6.33(m,1H),4.89(s,2H),3.63(s,3H),3.26–3.18(m,2H),2.24(t,J= 7.4Hz, 2H), 1.56 (p, J = 7.5Hz, 2H), 1.47 (p, J = 7.4Hz, 2H), 1.25 (p, J = 7.9Hz, 2H). 13 C NMR (151MHz, CDCl3) δ174.1,166.6,155.1,151.6,143.8,133.8,132.1,131.2,130.8,130.6,130.5 ,130.2,130.0,124.9,121.6,115.0,51.6,47.4,39.5,33.9,29.1,26.3,24.4.HRMS(ESI)m / z(M+H) + calculated for C 23 H 25 BrN3O4S:518.0749,observed:518.0741.

[0063] Example 6: Synthesis of Intermediate 1f

[0064] The specific steps include:

[0065] A 50 mL dry round-bottom flask equipped with a stirrer was added with 1d (1.8 mmol, 1.0 g) and lithium hydroxide (9.0 mmol, 216 mg). Tetrahydrofuran (25 mL) and deionized water (5 mL) were added while stirring at room temperature, and then stirred at room temperature for 8 h. After the reaction was completed, a solution of hydrochloric acid in dioxane (3.0 mL, 4 mol / L) was added to the reaction solution. Dichloromethane (100 mL) and saturated sodium chloride solution (50 mL) were then added to the reaction solution, and the organic phase was extracted and separated. The aqueous phase was extracted once more with dichloromethane (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 obtain the crude product 1e. In a separate 50 mL dry round-bottom flask equipped with a stirrer, 1e, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (2.7 mmol, 316 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.0 mmol, 380 mg), and 4-dimethylaminopyridine (0.36 mmol, 44 mg) were added. Dimethylformamide (20 mL) was added while stirring at room temperature, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, saturated sodium chloride solution (50 mL) was added to the reaction solution, and then extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude product. Finally, a yellow solid was obtained by recrystallization (ethyl acetate and petroleum ether), namely compound 1f (960 mg, total yield of two steps 89%). 1 H NMR (600MHz, DMSO-d6) δ11.00(s,1H),8.34(t,J=5.6Hz,1H),8.14(d,J=15.9Hz,1H),7.8 1(d,J=8.0Hz,1H),7.57(t,J=7.9Hz,1H),7.41–7.36(m,2H),7.34–7.24(m,3H),4.91(s, 2H),4.80(s,1H),3.99–3.83(m,1H),3.55–3.46(m,1H),3.12–2.94(m,2H),1.96(t,J=7. 4Hz,2H),1.67–1.61(m,2H),1.53–1.44(m,6H),1.38(p,J=7.2Hz,2H),1.26–1.16(m,2H). 13C NMR (151MHz, CDCl3) δ165.6,154.1,151.0,143.2,132.8,132.6,132.0,131.5,130.2,129.5,129.2,123.9,121 .6,114.6,113.8,100.8,61.3,44.8,38.6,32.1,28.7,28.7,27.8,25.9,24.7,24.7,18.3.HRMS(ESI)m / z(M+H) + calculated for C 27 H 32 BrN4O5S:603.1277,observed:603.1263.

[0066] Example 7: Synthesis of Intermediate 1g

[0067] The specific steps include:

[0068] To a 100 mL dry Shrek tube equipped with a stirrer, 1f (0.83 mmol, 500 mg), boronate-substituted anthraquinone (333 mg, 1.0 mmol), tetrakis-(triphenylphosphine) palladium (48 mg, 0.042 mmol), and potassium carbonate (344 mg, 2.5 mmol) were added under nitrogen. 1,4-dioxane (30 mL) and deionized water (10 mL) were added while stirring at room temperature. The mixture was then frozen and thawed using liquid nitrogen to remove trace oxygen. Finally, the reaction tube was moved to a 90°C oil bath and heated with stirring for 20 h. After the reaction was complete, the reaction solution was filtered using a sand core funnel to obtain a crude product, which was then washed with deionized water, dichloromethane, and ether, respectively, and finally dried to obtain a red solid, compound 1g (580 mg, 96% yield).

[0069] Example 8: Synthesis of target product

[0070] The specific steps include:

[0071] Take a 100mL dry Shrek tube equipped with a stirrer, add 1g (0.41mmol, 300mg) under nitrogen protection, and add dichloromethane (15mL) while stirring. After cooling the reaction solution to 0°C, trifluoroacetic acid (4.2mL) is added, and finally the reaction tube is kept at 0°C for 3h. After the reaction is completed, the organic solvent is removed using a rotary evaporator to obtain a crude product. The crude product is then transferred to a sand core funnel, washed with chloroform, and finally dried to obtain a red solid, which is the target compound (190mg, yield 72%, mass spectrometry and nuclear magnetic resonance analysis of the molecule are shown in Figure 1 and Figure 2). 1HNMR(600MHz,DMSO-d6)δ8.41(d,J=1.9Hz,1H),8.31–8.19(m,8H),7.96–7.91(m,3H),7.82(d, J=7.9Hz,1H),7.65(d,J=3.9Hz,1H),7.56(t,J=8.0Hz,1H),7.46(d,J=16.0Hz,1H),7.38(t,J=7. 6Hz,1H),7.30(d,J=8.5Hz,1H),4.91(s,2H),3.09–2.99(m,2H),1.90(t,J=7.4Hz,2H),1.48–1.4 1(m,2H),1.37(p,J=7.8,7.0Hz,2H),1.21(t,J=6.9Hz,2H).HRMS(ESI)m / z(M+H)+calculatedfor C36H31N4O6S:647.1964,observed:647.2095.

[0072] Example 9: Characterization of Absorption Spectra, Fluorescence Spectra and Reactive Oxygen Generation Capacity of Bioactive Photosensitizers

[0073] A 0.5 mg / mL DMSO stock solution of QCNHOH was prepared, and then QCNHOH was diluted to a concentration of 10 μg / mL using a mixed solvent of DMSO / CHCl3 at different ratios. The absorption spectrum of QCNHOH was measured by a Thermo Electron-EV300 UV-visible spectrophotometer. The maximum absorption wavelength of QCNHOH was located at 450 nm. The fluorescence spectrum of QCNHOH was then measured by a steady-state time-resolved fluorescence spectrophotometer. The results showed that the maximum emission wavelength of QCNHOH was 700 nm ( Figure 3 ). The efficiency of Type I reactive oxygen species (hydroxyl radicals and superoxide radicals) was then measured using HPF probe and DHR123 probe under light. The experimental results showed that QCNHOH had a high efficiency of reactive oxygen species generation ( Figure 4 ).

[0074] Example 10: Nanoparticle preparation, cellular uptake and killing of cancer cells

[0075] Nanoparticle preparation: Prepare 1 mg / mL QCNHOH in DMSO stock solution A and 10 mg / mL DSPE-PEG 2000 DMSO stock solution B was stored at room temperature and protected from light. 0.5 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). Ultrasonication was performed at 37°C for 30 min to obtain QCNHOH NPs. The organic solvent was removed by ultrafiltration and the concentration was quantified to 100 μg / mL for later use.

[0076] Cell uptake: Mouse breast cancer 4T1 cells (purchased from Starfish Bio) were seeded in 6-well plates at a density of 10 6 / mL, added after adherence. QCNHOH NPs (20 μM) were added at different time points (2h, 4h, 6h, 8h), and then the culture medium was removed for trypsin digestion. The cells were collected and the uptake of QCNHOH NPs was investigated using a flow cytometer ( Figure 5 ).

[0077] Cell killing: 4T1 cells were seeded in a culture dish at a density of 10 5 / mL, after it adheres to the wall, different concentrations of QCNHOH or vorinostat (SAHA) (250nM, 500nM, 1μM, 2μM, 4μM, 8μM, 16μM) are added and cultured for 4h, then irradiated with 450nm laser for 5min (the control group is not irradiated, and the hypoxic group is placed in a hypoxic box after irradiation). Culture is continued for 44h, the culture medium is removed, 100μL of fresh culture medium is added, and then 10μL of CCK-8 is added. After incubation in a 37℃ carbon dioxide incubator for 1h, the absorbance value at 450nm of each group is measured by a microplate reader. The results show that QCNHOH has a good killing effect on cells under normal oxygen content and hypoxic conditions. Moreover, compared with vorinostat, QCNHOH has a stronger killing effect on cells under dark conditions ( Figure 6 ).

[0078] Example 11: In vivo imaging in mice

[0079] The DSPE-PEG in the above nanoparticle preparation 2000 After replacing DSPE-PEG2000-NH2 with photosensitizer, NHS-Cy5.5 was added and stirred at room temperature overnight. Then, the solution was injected into tumor-bearing mice through the tail vein. The fluorescence distribution was observed on an in vivo imaging device (AniView100) at 2h, 4h, 6h, 8h, 12h, and 24h. The mice were killed suddenly at 24h, and the internal organs (heart, liver, spleen, lung, kidney, and tumor) of the mice were taken to observe the in vivo distribution. The results showed that the photosensitizer fluorescence signal could be observed at the tumor site 4h after injection. The signal intensity reached a peak at 6h and then began to decay. It can be found from the fluorescence images of the dissected organs that the photosensitizer mainly accumulated at the tumor tissue site ( Figure 7 ).

[0080] Similarly, it should be understood that in order to streamline the present disclosure and aid in understanding one or more of the various inventive aspects, in the above description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together into a single embodiment, figure, or description thereof. However, this disclosed method should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as reflected in the claims below, inventive aspects lie in less than all the features of the individual embodiments disclosed above. Accordingly, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of the invention.

[0081] It should be noted that the above embodiments illustrate rather than limit the invention, and that those skilled in the art may devise alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between brackets should not be construed as limiting the claims. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The present invention may be implemented by means of hardware comprising several different elements and by means of appropriately programmed computers. In a unit claim enumerating several means, several of these means may be embodied by the same item of hardware. The use of the words first, second, and third etc. does not indicate any order. These words may be interpreted as names.

Claims

1. A type-I bioactive photosensitizer targeting histone deacetylase, characterized in that: The bioactive photosensitizer is a quinoxalinone derivative, and the structural formula of the quinoxalinone derivative is as follows: Among them, the substituent R on the left side of the quinoxalinone skeleton 1 It is one of methoxy, halogen and methyl, and the substituent is located at any of the 5, 6, 7 and 8 positions of the quinoxalinone ring; n=1, 2, 3, 4, 5.

2. The method for preparing a type-I bioactive photosensitizer targeting histone deacetylase according to claim 1, wherein: The reaction route is as follows: R 1 , n as described in claim 1.

3. The preparation method according to claim 2, characterized in that The preparation method comprises the following steps: S1: Disperse the o-phenylenediamine derivative in anhydrous ethanol, add ethyl pyruvate, and stir at room temperature. After the reaction is complete, filter and wash the solid with ethanol to obtain quinoxalinone skeleton compound 1a; S2: Disperse the quinoxalinone skeleton compound 1a in acetic anhydride, add 5-bromothiophene-2-carboxaldehyde and pyridine, filter after the reaction, and wash the solid with acetic acid to obtain the condensation product 1b; S3: Disperse the condensation product 1b in acetone, add ethyl bromoacetate and potassium carbonate, and purify by column chromatography after the reaction to obtain a bromoquinoxalinone thiophene derivative 1c; S4: Derivative 1c is dispersed in a mixed solvent of tetrahydrofuran and water, and lithium hydroxide is added. After the reaction, a solution of hydrochloric acid in dioxane is added to acidify to pH 7. The solvent is removed by rotary evaporation, and the carboxylic acid derivative 1d is obtained by recrystallization from dichloromethane. S5: Derivative 1d was dispersed in dimethylformamide, and primary amine hydrochloride, a condensing agent 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, and triethylamine were added. After the reaction, the solvent was removed using a rotary evaporator, and the carboxylic acid derivative 1e was obtained by recrystallization from dichloromethane. S6: Dispersing the derivative 1e in a mixed solvent of tetrahydrofuran and water, adding lithium hydroxide, and after the reaction, acidifying to pH 7 by adding a dioxane solution of hydrochloric acid, removing the solvent by rotary evaporation, and recrystallizing from dichloromethane to obtain the carboxylic acid derivative 1f; S7: The derivative 1f was dispersed in dimethylformamide, and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, a condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a catalyst 4-dimethylaminopyridine were added. After the reaction, the solvent was removed under reduced pressure, and the carboxylic acid derivative 1g was obtained by recrystallization from dichloromethane. S8: Add 1g of the derivative, borate-substituted anthraquinone, catalyst tetrakis-(triphenylphosphine)palladium, and potassium carbonate into a Shrek tube, and react in a mixed solvent of dioxane and water under nitrogen for 24 hours. After the reaction, filter to obtain a red solid, which is then washed with deionized water, ether, and dichloromethane, respectively, to obtain 1h of the quinoxalinone anthraquinone derivative; S9: The derivative 1h was dispersed in dichloromethane, trifluoroacetic acid was added, and the reaction was carried out at 0°C for 8 hours. After the reaction was completed, a red solid was obtained by filtration, and then washed with sodium bicarbonate aqueous solution, deionized water, ether and dichloromethane respectively to obtain a bioactive photosensitizer.

4. The preparation method according to claim 3, characterized in that In step S1, the molar ratio of the o-phenylenediamine derivative to ethyl pyruvate is 1:1.3; the reaction temperature is room temperature, and the reaction time is 24 hours.

5. The preparation method according to claim 3, characterized in that In step S2, the molar ratio of the quinoxalinone skeleton compound 1a, 5-bromothiophene-2-carboxaldehyde and pyridine is 1:1.5:5; the reaction temperature is 120°C, the reaction time is 10 hours, and then the reaction is carried out at room temperature for 12 hours.

6. The preparation method according to claim 3, characterized in that In step S3, the molar ratio of the condensation product 1b to ethyl bromoacetate is 1:1.5, the reaction temperature is 70° C., and the reaction time is 8 hours.

7. The preparation method according to claim 3, characterized in that In step S4, the molar ratio of the derivative 1c to lithium hydroxide is 1:3, the volume ratio of tetrahydrofuran to water in the mixed solvent is 3:1, and the reaction is carried out at room temperature for 8 hours.

8. The preparation method according to claim 3, characterized in that In step S5, the derivative 1d, primary amine hydrochloride, condensing agent 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate and triethylamine are reacted at a molar ratio of 1:1.2:1.2:4 at room temperature for 8 hours.

9. The preparation method according to claim 3, characterized in that In step S6, the molar ratio of the derivative 1e to lithium hydroxide is 1:5, the volume ratio of tetrahydrofuran to water in the mixed solvent is 3:1, and the reaction is carried out at room temperature for 8 hours.

10. The preparation method according to claim 3, characterized in that In step S7, the molar ratio of the derivative 1f, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the catalyst 4-dimethylaminopyridine is 1:1.5:1.2:0.2, and the reaction is carried out at room temperature for 6 hours.

11. The method for preparing quinoxalinone anthraquinone according to claim 3, characterized in that: In step S8, the molar ratio of 1 g of the derivative, borate-substituted anthraquinone, tetrakis-(triphenylphosphine)palladium and potassium carbonate is 1:1.2:0.05:3.0, the volume ratio of dioxane and water in the mixed solvent is 3:1; the reaction temperature is 90° C., and the reaction time is 20 hours.

12. The preparation method according to claim 3, characterized in that In step S9, the molar ratio of the derivative 1h to trifluoroacetic acid is 1:130, the reaction temperature is 0° C., and the reaction time is 3 hours.

13. Use of the type-I bioactive photosensitizer targeting histone deacetylase according to claim 1 in the preparation of a photodynamic therapy agent.

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