Triarylmethane type I photosensitive dye, and synthesis method and application thereof

Abstract

The invention discloses a triarylmethane type I photosensitive dye as well as a synthesis method and application thereof. The structure of the dye is shown as a general formula I; intramolecular charge separation is optimized by regulating substituent groups, and the generation efficiency of superoxide anions and hydroxyl radicals is remarkably improved. According to the synthesis method, 4-diphenylaminobenzaldehyde or 4-vinyl benzaldehyde is used as a raw material, efficient preparation is achieved through the steps of condensation, oxidation and the like, the raw material is easy to obtain, and operation is easy and convenient. The dye has strong absorption at the wavelength of 600-630 nm, is suitable for red light triggered photodynamic therapy (PDT), especially keeps high reactive oxygen yield under the hypoxic condition, and breaks through the oxygen dependence limitation of traditional PDT. Experiments show that the dye can induce ferroptosis of tumor cells, has the functions of fluorescence imaging and treatment, and has wide application prospects in the fields of antitumor drugs, in-vivo fluorescence labeling, photoresponsive functional preparations and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of photosensitizing dyes and relates to a triarylmethane type I photosensitizing dye, a synthesis method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a treatment method based on photosensitizer (PS), molecular oxygen and light source of specific wavelength. The reactive oxygen species (ROS) generated by light excitation can selectively damage biological macromolecules (lipids, proteins and DNA) in cells, thereby inducing cell death. Compared with traditional treatment methods, photodynamic therapy uses the targeting of light sources to selectively eliminate tumors and avoid damage to normal tissues; it can reduce the scope of surgery, be safe, minimally invasive, and have no drug resistance; it can activate immune function and reduce recurrence. In photodynamic therapy, the excellent performance of photosensitizers is the key to eliminating tumors. However, most of the current photosensitizers mainly produce tumors through singlet oxygen ( 1 O2) to eliminate tumors. This oxygen-dependent mechanism is easily restricted by the hypoxic microenvironment of the tumor, resulting in low reactive oxygen production, which seriously limits the clinical application of photodynamic therapy.

[0003] Triarylmethane (TAM) dyes are a typical type I photosensitizer that provides a new approach to solving the above problems. Unlike type II photosensitizers that rely on singlet oxygen (1O2), triarylmethane dyes can directly generate superoxide anions (O2 ·- ) and hydroxyl radicals (·OH), and can achieve oxygen recycling through intracellular Fenton / Fenton-like reactions, significantly alleviating the oxygen-dependent limitation of PDT. However, due to the rapid rotation of the aromatic ring, the excited state energy of traditional triarylmethane dyes is more easily dissipated as heat through non-radiative transitions, resulting in low yields of type I ROS and making it difficult to scale up production.

[0004] Therefore, developing an efficient triarylmethane photosensitizer with a donor-acceptor structure, long-wavelength absorption, and scalable synthesis, improving the efficiency of type I reactive oxygen species production through molecular design, and providing innovative solutions to the PDT bottleneck are still technical problems that need to be solved urgently. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the purpose of the present invention is to provide a synthesis and application of TAM dye (triarylmethane type I photosensitizer dye) with DA structure, which can promote O2 ·- and ·OH, inducing ferroptosis in cells.

[0006] In order to achieve the above objectives, the first aspect of the present application is to protect a triarylmethane dye having a DA structure, the general structural formula of which is as follows.

[0007]

[0008] Among them, R 1 and R 2 are each independently selected from any one of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkylthio or alkylamino having 1-18 carbon atoms; more preferably selected from any one of hydrogen, halogen, alkoxy, alkylthio or alkylamino having 1-10 carbon atoms; further preferably selected from any one of hydrogen, alkoxy, alkylthio or alkylamino having 1-6 carbon atoms; and most preferably selected from any one of hydrogen, alkoxy or alkylamino having 1-2 carbon atoms.

[0009] R 3 、R 4 Each is independently selected from any one of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl or aryl having 1-18 carbon atoms; more preferably selected from any one of hydrogen, alkyl and cycloalkyl having 1-10 carbon atoms; further preferably selected from any one of alkyl and cycloalkyl having 1-5 carbon atoms; most preferably selected from any one of alkyl and cycloalkyl having 1-3 carbon atoms.

[0010] Y - Selected from halogen ion, p-toluenesulfonate ion, ClO4 - 、CH3COO - or CF3COO - Any one of; most preferably any one of halide ion or p-toluenesulfonate ion. Halide ions include Cl - Br - , I - wait.

[0011] The second aspect of the present application is to protect the synthesis method of the above-mentioned triarylmethane dye, comprising the following steps:

[0012] The chemical reaction equation is as follows:

[0013]

[0014] (1) 1 、R 2 Substituted 4-diphenylaminobenzaldehyde and p-toluenesulfonic acid are mixed in a molar ratio of 1:0.5-5, fully dissolved in an organic solvent I, and reacted at 60-150°C (more preferably 80-120°C, most preferably 105-115°C) for a period of time, and then 2-6 eq of R-containing 3 、R 4The substituted aniline is reacted for 5-20 h (more preferably 6-15 h, most preferably 8-12 h) to give intermediate 1.

[0015] (2) Intermediate 1 and chlorobenzoquinone are mixed in a molar ratio of 1:0.5-5, fully dissolved in an organic solvent II, reacted at 10-50°C (more preferably 20-40°C, most preferably 25-30°C) for 0.5-8h (more preferably 1-4h, most preferably 2-3h), and purified to obtain dye I.

[0016] (3) 4-vinylbenzaldehyde, containing R 1 、R 2 Substituted 4-bromotriphenylamine, triphenylphosphine, palladium acetate and an inorganic base are mixed and evacuated. Under nitrogen protection, an organic solvent III is added and heated under reflux at 75-120°C (more preferably 80-120°C, most preferably 100-110°C) for 18-48 hours (more preferably 20-30 hours, most preferably 22-25 hours). 4-vinylbenzaldehyde and R-containing 1 、R 2 The molar ratio of the substituted 4-bromotriphenylamine is 1:0.5-5, and the amount of triphenylphosphine, palladium acetate and inorganic base added is 0.1-1 eq. After the reaction is completed, the intermediate 2 is obtained by extraction, drying and purification.

[0017] (4) The intermediate 2 and p-toluenesulfonic acid are mixed in an organic solvent IV at a molar ratio of 1:0.5-5, and the mixture is fully reacted at 60-150°C (more preferably 80-120°C, most preferably 105-115°C), and 2-6 eq of R-containing 3 、R 4 The substituted aniline is reacted for 5-20 h (more preferably 6-15 h, most preferably 8-12 h) to give intermediate 3.

[0018] (5) Intermediate 3 and chlorobenzoquinone are mixed in a molar ratio of 1:0.5-5 and dissolved in an organic solvent V. The mixture is reacted at 10-50°C (more preferably 20-40°C, most preferably 25-30°C) for 0.5-8h (more preferably 1-4h, most preferably 2-3h), and dye II is purified.

[0019] For the technical solution described above, in the above steps (1) and (4), it is further preferred that the R 1 、R 2 Substituted 4-diphenylaminobenzaldehyde, p-toluenesulfonic acid and R-containing 3 、R 4 Substituted aniline and intermediate 2, p-toluenesulfonic acid and R 3 、R 4The molar ratio of the substituted aniline is 1:(1-3):(2-5); more preferably, the molar ratio is 1:(1-2):(2-4); most preferably, the molar ratio is 1:(1-1.5):(2-3).

[0020] For the technical solution described above, it is further preferred that the organic solvent I or IV is independently selected from at least one of toluene, methanol, ethanol, and benzene; further preferably, it is independently selected from at least one of toluene, methanol, and ethanol; most preferably, it is independently selected from at least one of toluene and methanol.

[0021] For the technical solution described above, in the above steps (1) and (4), it is further preferred that the purification is performed by column chromatography, and the purification solvent is selected from any one of petroleum ether, hexane, dichloromethane, ethyl acetate, and ether, or a mixed solvent of several combinations thereof; further preferred that the purification solvent is selected from any one of petroleum ether, hexane, dichloromethane, and ethyl acetate, or a mixed solvent of several combinations thereof. Most preferred that the purification solvent is selected from a mixed solvent of petroleum ether and dichloromethane.

[0022] For the technical solution described above, in the above steps (2) and (5), it is further preferred that the molar ratios of the intermediate 1 to tetrachlorobenzoquinone and the intermediate 2 to tetrachlorobenzoquinone are both 1:(1-3); further preferably, the molar ratios are both 1:(1-2); most preferably, the molar ratios are both 1:(1-1.5).

[0023] For the technical solution described above, it is further preferred that the organic solvent II or V is independently selected from any one of toluene, benzene, methanol, ethanol, dichloromethane, and ethyl acetate, or a mixed solvent of several combinations thereof; further preferably, it is independently selected from any one of toluene, methanol, ethanol, and dichloromethane, or a mixed solvent of several combinations thereof; most preferably, it is independently selected from any one of toluene and dichloromethane.

[0024] For the technical solution described above, in the above steps (2) and (5), it is further preferred that the purification is column chromatography purification, and the purification solvent is selected from any one or a mixed solvent of dichloromethane, ethyl acetate, methanol, ethanol, ether, acetone, and propanol; further preferably, the purification solvent is selected from any one or a mixed solvent of dichloromethane, ethyl acetate, methanol, ethanol, and propanol. Most preferably, the purification solvent is selected from a mixed solvent of dichloromethane and methanol.

[0025] For the technical solution described above, in the above step (3), it is further preferred that the 4-vinylbenzaldehyde, containing R 1 、R 2The molar ratio of substituted 4-bromotriphenylamine, triphenylphosphine, palladium acetate and inorganic base is 1:(0.5-3):(0.1-0.6):(0.1-0.5):(0.1-0.6); more preferably, the molar ratio is 1:(1-2):(0.1-0.4):(0.1-0.3):(0.1-0.4); most preferably, the molar ratio is 1:(1-1.5):(0.1-0.2):(0.1-0.2):(0.1-0.2).

[0026] For the technical solution described above, it is further preferred that the organic solvent III is selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, and acetonitrile; even more preferably, the organic solvent III is selected from at least one of N,N-dimethylformamide and N,N-dimethylacetamide; most preferably, the organic solvent III is selected from N,N-dimethylacetamide.

[0027] For the technical solution described above, it is further preferred that the extraction solvent in step (3) is water and dichloromethane; it is further preferred that the purification is performed by column chromatography, and the purification solvent is selected from any one or a combination of petroleum ether, hexane, dichloromethane, ethyl acetate, methanol, ethanol, ether, acetone, and propanol; it is further preferred that the purification solvent is selected from any one or a combination of petroleum ether, hexane, dichloromethane, and ethyl acetate. Most preferably, the purification solvent is selected from a mixed solvent of petroleum ether and dichloromethane.

[0028] The third aspect of this application is to protect the application of triarylmethane dyes in the fields of biology and medicine.

[0029] Preferably, the triarylmethane dye is used in the preparation of living fluorescent labeling preparations, anti-tumor drug compositions, and photoresponsive functional preparations;

[0030] Preferably, the preparation is used for in vitro fluorescence detection of biological samples or in vivo distribution tracing;

[0031] Preferably, the composition comprises an effective amount of the dye or a pharmaceutically acceptable carrier;

[0032] Preferably, the preparation is selected from at least one of a reactive oxygen species generator, a fluorescent imaging probe, and a ferroptosis inducer.

[0033] Dye I absorbs at approximately 600 nm, while Dye II absorbs at approximately 630 nm. Both dyes exhibit excellent biosafety and efficient reactive oxygen species generation, and have potential applications in bioimaging of cells, tissues, and in vivo, as well as in tumor therapy.

[0034] Furthermore, Compound 2 can rapidly enter cells and accumulate in subcellular organelles. Upon exposure to light, it can generate fluorescence and reactive oxygen species, enabling its application in photodynamic therapy to achieve integrated diagnosis and treatment. Upon exposure to low-intensity red light, Compound 2 can generate large amounts of highly toxic reactive oxygen species, disrupting cellular homeostasis and inducing ferroptosis at low concentrations, effectively killing cancer cells.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention provides a method for synthesizing two triarylmethane dyes having a DA structure, which has the advantages of readily available raw materials, simple operation and low cost.

[0037] (2) Compared with traditional crystal violet dyes, the two dyes described in the present invention have red-shifted absorption and emission wavelengths, making them more suitable for deep in vivo imaging and tumor treatment.

[0038] (3) The dye of the present invention can aggregate in PBS buffer. Compared with traditional triarylmethane dyes, its ROS generation ability is significantly enhanced ( Figure 3 ).

[0039] (4) Compound 2 of the present invention has good biocompatibility and phototoxicity, and can generate a large amount of O2 under light conditions. ·- and ·OH, leading to the accumulation of LPO in cells and effectively light-induced ferroptosis.

[0040] (5) The dye of the present invention maintains a high ROS production rate under hypoxic conditions (Example 9 shows that the cell survival rate under hypoxia is less than 30%), breaking through the oxygen dependence limitation of traditional PDT and being particularly suitable for the treatment of solid tumors.

[0041] (6) The present invention realizes the integration of diagnosis and treatment through DA structural design. Compound 2 has both fluorescent tracer properties under 630nm light ( Figure 2 ) and ferroptosis induction ( Figure 6 ) dual functions, simplifying the treatment monitoring process. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0043] Figure 1 are the UV-visible absorption spectra of compounds 2 and 4 in different solvents.

[0044] Figure 2 Figure 2 is the fluorescence emission spectra of compounds 2 and 4 in different solvents.

[0045] Figure 3 This is a graph showing the reactive oxygen species generation test of compounds 2 and 4 in water and PBS buffer.

[0046] Figure 4 This is a test diagram of the active oxygen generation of compound 2 in cells.

[0047] Figure 5 This is the MTT assay diagram of compound 2.

[0048] Figure 6 This is a test chart of cellular lipid peroxide accumulation of compound 2. DETAILED DESCRIPTION

[0049] The following will be combined with the attached Figure 1-6 The present invention is further described in detail with reference to Examples 1-10. It is apparent that the described embodiments are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0050] Unless otherwise specified, the terms used herein have the following meanings.

[0051] The term "alkyl group" used in the present invention includes straight-chain alkyl groups and branched-chain alkyl groups.

[0052] The term "MTT" as used in the present invention refers to a method for detecting cell survival and growth.

[0053] The instruments and equipment used in the examples are:

[0054] In the column chromatography process of the present invention, column chromatography silica gel with 200-300 mesh and 100-200 mesh purchased from Qingdao Meigao Group Co., Ltd. and analytical pure quartz sand with 20-40 mesh purchased from Tianda Chemical Reagent Factory were used.

[0055] In the process of detecting compounds, the mass spectrometer used was the Synapt G2-Si HDMS high-resolution mass spectrometer from Waters Corporation of the United States, and the nuclear magnetic resonance instrument used was the 600M fully digital superconducting nuclear magnetic resonance instrument from Bruker Scientific Instruments of Switzerland.

[0056] The absorption and emission spectra of the dyes were measured using an Agilent Cary 60 UV-visible spectrophotometer and a Cary Eclipse fluorescence spectrophotometer.

[0057] The dye intracellular reactive oxygen species test and lipid peroxide accumulation test experiments were measured using an Olympus FV1000 single-photon confocal microscope from Japan.

[0058] The cytotoxicity test was measured using Varioskan LUX Multimode Microplate Reader from Thermofisher, USA.

[0059] Example 1 Preparation of Compound 1.

[0060] The structural formula of compound 1:

[0061]

[0062] Preparation of compound 1

[0063] 4-Dianilinobenzaldehyde (547 mg, 1 eq), N,N-dimethylaniline (535 mg, 2.2 eq), and p-toluenesulfonic acid (380 mg, 1.1 eq) were added to a round-bottom flask, followed by 12 mL of toluene. The mixture was heated to reflux at 110°C and stirred for 12 hours. After the reaction, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation under reduced pressure. The crude product was dissolved in dichloromethane and purified by column chromatography to remove excess p-toluenesulfonic acid. The mixture was then dissolved in dichloromethane, and tetrachlorobenzoquinone (735 mg, 1.5 eq) was added, and the reaction was stirred for 2 hours.

[0064] After the reaction, the solvent was removed by rotary evaporation under reduced pressure, and the residue was purified by silica gel column separation (dichloromethane / methanol=50:1) to obtain compound 1 (black powder, 12 mg, 1.13%).

[0065] The characterization results of compound 1 are as follows: HRMS (ESI + )m / z:M + C 35 H 34 N3 + Calculated value: 496.2747; actual value: 496.2745. 1 H NMR (600MHz, MeOD) δ7.51 (d, J = 7.8 Hz, 1H), 7.48–7.43 (m, 8H), 7.31 (d, J = 7.8 Hz, 6H), 7.03 (dd, J = 9.1, 2.7 Hz, 6H), 6.68 (s, 1H), 3.30 (s, 12H).

[0066] Example 2 Preparation of Compound 2.

[0067] The structural formula of compound 2:

[0068]

[0069] Preparation of compound 2

[0070] 4-Dianilinobenzaldehyde (549 mg, 1 eq), N,N-diethylaniline (660 mg, 2.2 eq), and p-toluenesulfonic acid (383 mg, 1.1 eq) were added to a round-bottom flask, followed by 12 mL of toluene. The mixture was heated under reflux at 110°C and stirred for 12 hours. After the reaction, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation under reduced pressure. The crude product was dissolved in dichloromethane and purified by column chromatography to remove excess p-toluenesulfonic acid. The mixture was then dissolved in dichloromethane, and tetrachlorobenzoquinone (738 mg, 1.5 eq) was added, and the reaction was stirred for 2 hours.

[0071] After the reaction, the solvent was removed by rotary evaporation under reduced pressure and the residue was purified by silica gel column separation (dichloromethane / methanol=50:1) to obtain compound 2 (black powder, 28 mg, 2.83%).

[0072] The characterization results of compound 2 are as follows: HRMS (ESI + )m / z:M + C 39 H 42 N3 + Calculated value 552.3373; actual value 552.3366. 1 H NMR(600MHz,MeOD)δ7.72(d,J=8.3Hz,1H),7.47–7.43(m,6H),7.33–7.21(m,9H), 7.05–7.00(m,5H),6.87(d,J=8.0Hz,1H),3.79–3.53(m,8H),1.35–1.28(m,12H).

[0073] Example 3 Preparation of Compound 3.

[0074] The structural formula of compound 3:

[0075]

[0076] Preparation of compound 3

[0077] 4-Dianilinobenzaldehyde (546 mg, 1 eq), julolidine (763 mg, 2.2 eq), and p-toluenesulfonic acid (380 mg, 1.1 eq) were added to a round-bottom flask, followed by 12 mL of toluene. The mixture was heated to reflux at 110°C and stirred for 8 h. After the reaction, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation under reduced pressure. The crude product was dissolved in dichloromethane and purified by column chromatography to remove excess p-toluenesulfonic acid. The mixture was then dissolved in dichloromethane, and tetrachlorobenzoquinone (718 mg, 1.5 eq) was added, and the reaction was stirred for 2 h.

[0078] After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure and the residue was purified by silica gel column separation (dichloromethane / methanol=50:1) to obtain compound 3 (black powder, 300 mg, 23.59%).

[0079] The characterization results of compound 3 are as follows: HRMS (ESI + )m / z:M + C 43 H 42 N3 + Calculated value: 600.3373; actual value: 600.3370. 1 H NMR(600MHz,MeOD)δ7.44(dd,J=8.8,7.2Hz,4H),7.30–7.25(m,6H),7.11(d,J=9.0Hz,2H),6.95 (d,J=9.0Hz,2H),6.89(s,4H),3.52(t,J=5.8Hz,8H),2.74(t,J=6.3Hz,8H),2.02–1.98(m,8H).

[0080] Example 4 Preparation of Compound 4.

[0081] The structural formula of compound 4:

[0082]

[0083] Example 4.1

[0084]

[0085] 4-Vinylbenzaldehyde (341 mg, 1 eq), 4-bromotriphenylamine (995 mg, 1.2 eq), triphenylphosphine (140 mg, 0.2 eq), palladium acetate (60 mg, 0.1 eq), and potassium carbonate (69 mg, 0.2 eq) were mixed and added to a two-necked round-bottom flask. The mixture was evacuated using a circulating water vacuum pump. Under nitrogen protection, 12 mL of N,N-dimethylacetamide was injected via syringe. The mixture was heated under reflux at 110°C for 24 h.

[0086] After the reaction, the solvent was removed by rotary evaporation under reduced pressure. The reaction mixture was then extracted with water and dichloromethane to remove the catalyst and inorganic salts, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was then dissolved in a small amount of dichloromethane and purified by silica gel column separation (petroleum ether / dichloromethane = 5:1) to obtain intermediate 2 (yellow solid, 570 mg, 60.8%).

[0087] Preparation of compound 4

[0088] Intermediate 2 (188 mg, 1 eq), N,N-diethylaniline (168 mg, 2.2 eq), and p-toluenesulfonic acid (132 mg, 1.5 eq) were added to a round-bottom flask. 12 mL of toluene was then added. The mixture was heated to reflux at 110°C and stirred for 12 hours. After the reaction, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation under reduced pressure. The crude product was dissolved in dichloromethane and purified by column chromatography to remove excess p-toluenesulfonic acid. Subsequently, the mixture was dissolved in dichloromethane, and chloranil (185 mg, 1.5 eq) was added, and the reaction was stirred for 2 hours.

[0089] After the reaction, the solvent was removed by rotary evaporation under reduced pressure. The crude product was then dissolved in a small amount of dichloromethane and purified by silica gel column separation (dichloromethane / methanol = 40:1) to obtain compound 4 (black powder, 5 mg, 1.45%).

[0090] The characterization results of compound 4 are as follows: HRMS (ESI + )m / z:M + C 47 H 48 N3 + Calculated value: 654.3843; actual value: 654.3843. 1 H NMR (600MHz, MeOD) δ7.77 (dd, J=8.4, 3.0Hz, 2H), 7.52 (d, J=8.7Hz, 2H), 7.48–7.18 (m, 12H), 7.16–6. 94(m,10H),6.90(d,J=9.1Hz,1H),6.69(d,J=8.1Hz,1H),3.78–3.54(m,8H),1.33(t,J=7.1Hz,12H).

[0091] Example 5 Preparation of Compound 5.

[0092] The structural formula of compound 5:

[0093]

[0094] Preparation of compound 5

[0095] Intermediate 2 (189 mg, 1 eq), julolidine (198 mg, 2.2 eq), and p-toluenesulfonic acid (135 mg, 1.5 eq) were added to a round-bottom flask, followed by 12 mL of toluene. The mixture was heated to reflux at 110°C and stirred for 10 h. After the reaction, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation under reduced pressure. The crude product was dissolved in dichloromethane and purified by column chromatography to remove excess p-toluenesulfonic acid. The mixture was then dissolved in dichloromethane, and tetrachlorobenzoquinone (180 mg, 1.5 eq) was added, and the reaction was stirred for 2 h.

[0096] After the reaction, the solvent was removed by rotary evaporation under reduced pressure. The crude product was then dissolved in a small amount of dichloromethane and purified by silica gel column separation (dichloromethane / methanol = 40:1) to obtain compound 5 (black powder, 35 mg, 9.47%).

[0097] The characterization results of compound 5 are as follows: HRMS (ESI + )m / z:M + C 51 H 48 N3 + Calculated value: 702.3843; actual value: 702.3842. 1 H NMR(600MHz,MeOD)δ7.68(d,J=8.4Hz,2H),7.49(d,J=8.7Hz,2H),7.42–7.21(m,8H),7.19–7.06(m,6H),7.00 (d, J=8.7Hz, 2H), 6.96 (d, J=8.0Hz, 4H), 3.56 (t, J=5.8Hz, 8H), 2.75 (t, J=6.3Hz, 8H), 2.01 (t, J=5.0Hz, 8H).

[0098] Example 6 Absorption and fluorescence spectra of compound 2 and compound 4

[0099] For UV-Vis absorption and fluorescence spectroscopy, 7.5 μL of the sample test stock solution was pipetted and dissolved in 3 mL of each solvent to obtain a 5 μM test sample solution for absorption and fluorescence emission spectroscopy. All tests were performed at 25°C.

[0100] Depend on Figure 1 The maximum absorption wavelength of compound 2 in organic solvents is around 595 nm, while that of compound 4 is around 630 nm, indicating that the double bond effectively extends the conjugation degree of the TMA dye. Furthermore, compounds 2 and 4 form a broad absorption peak between 540 and 650 nm in PBS buffer, indicating that they are likely to aggregate in an aqueous physiological environment, forming aggregate particles.

[0101] Depend on Figure 2 Compounds 2 and 4 produced almost no fluorescence in either ethanol or PBS buffer. However, after adding BSA to the test solvent PBS buffer or increasing the solvent viscosity (a mixed solvent of ethanol and propylene glycol), a significant increase in fluorescence intensity was observed, with compound 4 exhibiting a fluorescence intensity greater than 2. These results suggest that the dye may produce strong fluorescence in vivo and could be used to monitor therapeutic processes.

[0102] Example 7 Detection of Reactive Oxygen Species Generation by Compounds 2 and 4

[0103] DHR123 was used as superoxide anion (O2 ·- ) detection indicator. When it reacts with superoxide anion radicals in solution, it is oxidized to the cationic rhodamine 123, which emits bright green fluorescence. Therefore, the presence of superoxide anion radicals can be qualitatively evaluated based on changes in the fluorescence spectrum of DHR123. Simultaneously, hydroxyphenyl fluorescein (HPF) was selected as a hydroxyl radical (·OH) detection indicator. In the HPF system, the push-pull electron system of the fluorescein chromophore is destroyed, resulting in fluorescence quenching. However, in the presence of ·OH, the electron-withdrawing group is oxidized and dissociated, restoring the strong push-pull electron system and releasing the strong green fluorescence of the fluorescein. Therefore, the degree of recovery of HPF green fluorescence after illumination can be used to qualitatively evaluate the ability of the photosensitizer to produce ·OH.

[0104] Procedure: A test solution containing 5 μM dye and 1 μM probe was prepared in water and PBS buffer in a 1×1 cm cuvette, and then placed under 630 nm monochromatic light (optical power density: 10 mW / cm 2 ) for different durations, and fluorescence spectra were measured at regular intervals using a fluorescence spectrometer (excitation wavelength: 488 nm; slit width: 5.5 nm). A negative control solution containing no light or the test dye was used, and the level of reactive oxygen species generated by the different compounds was evaluated by comparing changes in fluorescence intensity at 526 nm or 516 nm.

[0105] Depend on Figure 3 It can be seen that both compounds 2 and 4 can generate superoxide anions and hydroxyl radicals to a certain extent. In aqueous solution, the ROS generation capacity of compound 4 is greater than 2. Compound 2 shows obvious aggregation in PBS buffer, which significantly enhances its ROS level in PBS buffer.

[0106] Example 8 Detection of Intracellular Reactive Oxygen Species Generation by Compound 2

[0107] The experiment used the total ROS probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), the hydroxyl radical probe HPF and the superoxide anion radical probe dihydroethidium (DHE) to detect the generation of ROS in MCF-7 cells. DCFH-DA itself has no fluorescence. After entering the cell, it can be hydrolyzed by the esterase in the cell into 2,7-dichlorodihydrofluorescein (DCFH), allowing the probe to be loaded into the cell. The reactive oxygen species in the cell can oxidize DCFH to generate fluorescent 2,7-dichlorofluorescein (DCF), thereby detecting the generation of ROS in the cell. DHE is a cell membrane permeable blue probe that reacts with O2 ·-The reaction generates 2-hydroxyethidium, which, upon intercalation into nucleic acids, produces red fluorescence that can be detected by a fluorescence spectrometer. All three probes can be used to detect ROS production within cells using the following steps.

[0108] Procedure: MCF-7 cells were evenly seeded in a 35 mm confocal culture dish and washed three times with PBS after reaching 50% density. The cells were randomly divided into four treatment groups: control group (incubated with 2 mL PBS buffer for 90 min, then added with 2 μM ROS probe and incubated for 30 min), illumination group (incubated with 2 mL PBS buffer for 90 min, then added with 2 μM ROS probe and incubated for 30 min, then irradiated with 630 nm LED light at 30 mW / cm 2 The cells were exposed to light for 15 min), the drug-added group (1.5 μM compound 2 in PBS was incubated for 90 min, followed by the addition of 2 μM ROS probe and incubation for 30 min), the drug-added light group (1.5 μM compound 2 in PBS was incubated for 90 min, followed by the addition of 2 μM ROS probe and incubation for 30 min, and then the cells were exposed to 630 nm LED light at 30 mW / cm 2 After treatment, all four groups were washed three times with PBS, and 2 mL of PBS buffer was added. Fluorescence images of each channel were recorded using a laser confocal scanning microscope. The excitation wavelength of the three ROS probes was 488 nm, and the emission wavelengths were 490-570 nm (DCFH-DA), 490-560 nm (HPF), and 550-620 nm (DHE), respectively.

[0109] like Figure 4 As shown, after the addition of DCFH-DA, the illumination group exhibited a distinct green fluorescence signal, while the other groups showed almost no fluorescence, indicating that compound 2 significantly generated reactive oxygen species under illumination. Furthermore, significant intracellular generation of hydroxyl radicals and superoxide anion radicals was also detected under illumination. These results demonstrate that compound 2 possesses a robust capacity to generate type I reactive oxygen species within cells.

[0110] Example 9 Cytotoxicity test of compound 2

[0111] The (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the killing ability of photosensitizers on MCF-7 tumor cells under normoxic and hypoxic conditions. The principle is as follows: succinate dehydrogenase in the mitochondria of living cells can reduce MTT to water-insoluble blue-purple crystalline formazan, while dead cells cannot.

[29] DMSO can dissolve formazan, and the absorbance at 490 nm can be measured using an enzyme-linked immunosorbent assay (ELISA) to indirectly reflect the number of living cells.

[0112] Normoxia incubation group: MCF-7 cells were evenly seeded in a 96-well plate and washed three times with PBS after reaching 70% density. 100 μL of compound 2 solution containing 0, 0.2, 0.5, 0.8, 1 and 1.2 μM prepared with complete culture medium was added and incubated for 90 min. The cells were divided into two groups. One group was illuminated with a 630 nm LED light at 30 mW / cm 2 One group was exposed to light for 15 minutes, while the other group was kept in the dark for 15 minutes. After washing with PBS buffer, the cells were incubated together for 12 hours. The cells were washed with PBS buffer, and 100 μL of complete medium containing MTT (0.5 mg / mL) was added to the 96-well plate. The plate was incubated for 4 hours. After removing the solution, 100 μL of DMSO solution was added to each well to dissolve the formazan. The absorbance at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA).

[0113] Hypoxic incubation group: The operation method is basically the same as above, but after adding compound 2, both groups of cells must be incubated in a hypoxic environment. The light group must be treated with light under hypoxic conditions. Then, both groups of cells are incubated under normoxic conditions. Subsequent operations are the same.

[0114] The absorbance at 490 nm obtained from the test was quantitatively converted into cell viability using the following formula. The experimental group consisted of cells cultured with different concentrations of photosensitizing dye, the blank group consisted of cells cultured with only culture medium, and the control group consisted of cells cultured without photosensitizing dye. OD is the absorbance of formazan in DMSO solution at 490 nm.

[0115]

[0116] Depend on Figure 5 It can be seen that compound 2 does not cause significant damage to tumor cells under dark conditions, indicating that it has good biocompatibility. Under light, the inhibitory effect of compound 2 on tumor cells is significantly concentration-dependent. 2 When cells were irradiated for 15 minutes under hypoxic conditions, a single dose of 1.2 μM of compound 2 reduced tumor cell survival to below 10%. Furthermore, compound 2 also exhibited a certain degree of cell-killing ability under hypoxic conditions. These experiments demonstrate that compound 2, as a type I photosensitizer, can effectively generate reactive oxygen species, overcome tumor hypoxia, and induce cell death.

[0117] Example 10 Detection of Intracellular Lipid Peroxide Accumulation of Compound 2

[0118] The accumulation of lipid peroxidation (LPO) is the direct cause of ferroptosis. Glutathione peroxidase 4 (GPX4) can clear LPO and is a key enzyme involved in regulating cell ferroptosis. Downregulation of its activity is conducive to the occurrence of ferroptosis. In order to verify ferroptosis, the experiment used C11-bodipy probe to detect the accumulation of LPO. The specific operation is as follows: MCF-7 cells were evenly seeded in a 35mm confocal culture dish, and after reaching 50% density, they were washed 3 times with PBS. The cells were randomly divided into 4 groups for treatment, namely the control group (2mL complete medium was incubated for 90min, and then 2μM C11-bodipy probe was added and incubated for 30min), the light group (2mL complete medium was incubated for 90min, and then 2μMC11-bodipy probe was added and incubated for 30min, and then 630nm LED light was used at 30mW / cm 2 light for 15 min), drug-treated group (1.5 μM compound 2 incubated for 90 min followed by 2 μM C11-bodipy probe incubated for 30 min), drug-treated light-treated group (1.5 μM compound 2 incubated for 90 min followed by 2 μM C11-bodipy probe incubated for 30 min, then irradiated with 630 nm LED light at 30 mW / cm 2 After treatment, all four groups were washed three times with PBS and supplemented with 2 mL of complete culture medium. Fluorescence images of each channel were recorded using a confocal laser scanning microscope. The excitation wavelength of the C11-bodipy probe was 488 nm, and the emission wavelength was 500-560 nm.

[0119] Depend on Figure 6 It can be seen that after the cells were treated with light or compound 2, only weak green fluorescence was observed; while a stronger green fluorescence signal was observed in the drug-treated light group, indicating that the LPO content in the cells increased after treatment, effectively inducing the occurrence of ferroptosis.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A triarylmethane dye having a DA structure, the structure of which is as shown in general formula I: in, R 1 and R 2 are each independently selected from any one of hydrogen, halogen, alkyl having 1 to 18 carbon atoms, cycloalkyl, alkenyl, alkynyl, alkoxy, alkylthio or alkylamino; R 3 、R 4 are each independently selected from any one of hydrogen, an alkyl group having 1 to 18 carbon atoms, a cycloalkyl group, an alkenyl group, an alkynyl group or an aryl group; Y - Selected from halogen ion, p-toluenesulfonate ion, ClO4 - 、CH3COO - or CF3COO - Any one of .

2. The synthetic method of triarylmethane dye according to claim 1, wherein The following steps are involved: The chemical reaction equation is as follows: (1) 1 、R 2 Substituted 4-diphenylaminobenzaldehyde and p-toluenesulfonic acid are mixed in a molar ratio of 1:0.5-5, fully dissolved in organic solvent I, reacted at 60-150°C for a period of time, and then 2-6eq of R 3 、R 4 The substituted aniline is reacted for 5-20 h to obtain intermediate 1; (2) Intermediate 1 and chloranil are mixed in a molar ratio of 1:0.5-5, fully dissolved in an organic solvent II, reacted at 10-50° C. for 0.5-8 h, and purified to obtain dye I; (3) 4-vinylbenzaldehyde, containing R 1 、R 2 Substituted 4-bromotriphenylamine, triphenylphosphine, palladium acetate and inorganic base are mixed and vacuumed, and under the protection of N2, organic solvent III is added and heated under reflux at 75-120℃ for 18-48h; 4-vinylbenzaldehyde and R-containing 1 、R 2 The molar ratio of the substituted 4-bromotriphenylamine is 1:0.5-5, and the amount of each of triphenylphosphine, palladium acetate and inorganic base added is 0.1-1eq; after the reaction, the intermediate 2 is obtained by extraction, drying and purification; (4) The intermediate 2 and p-toluenesulfonic acid were mixed in an organic solvent IV at a molar ratio of 1:0.5-5, and the mixture was fully reacted at 60-150°C for a period of time, and 2-6 eq of R 3 、R 4 The substituted aniline is reacted for 5-20 h to obtain intermediate 3; (5) Intermediate 3 and chlorobenzoquinone are mixed in a molar ratio of 1:0.5-5, dissolved in an organic solvent V, reacted at 10-50° C. for 0.5-8 h, and purified to obtain dye II.

3. The method according to claim 2, wherein: In the above steps (1) and (4), the R 1 、R 2 Substituted 4-diphenylaminobenzaldehyde, p-toluenesulfonic acid and R-containing 3 、R 4 Substituted aniline and intermediate 2, p-toluenesulfonic acid and R 3 、R 4 The molar ratio of the substituted anilines is 1:(1-3):(2-5).

4. The method according to claim 2, wherein: The organic solvent I or IV is independently selected from at least one of toluene, methanol, ethanol, and benzene; the organic solvent II or V is independently selected from any one of toluene, benzene, methanol, ethanol, dichloromethane, and ethyl acetate, or a mixed solvent of several combinations thereof; the organic solvent III is selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, and acetonitrile.

5. The method according to claim 2, wherein: In the above steps (1) and (4), the purification is performed by column chromatography, and the purification solvent is selected from any one of petroleum ether, hexane, dichloromethane, ethyl acetate, and diethyl ether, or a mixed solvent of several combinations thereof.

6. The method according to claim 2, wherein: In the above steps (2) and (5), the molar ratios of the intermediate 1 to chloranil and the molar ratio of the intermediate 2 to chloranil are both 1:(1-3).

7. The method according to claim 2, wherein: In the above step (3), the 4-vinylbenzaldehyde, containing R 1 、R 2 The molar ratio of substituted 4-bromotriphenylamine, triphenylphosphine, palladium acetate and inorganic base is 1:(0.5-3):(0.1-0.6):(0.1-0.5):(0.1-0.6).

8. The method according to claim 2, wherein: In the step (3), the extraction solvents are water and dichloromethane; the purification is performed by column chromatography, and the purification solvent is selected from any one of petroleum ether, hexane, dichloromethane, ethyl acetate, methanol, ethanol, ether, acetone, and propanol, or a mixed solvent of several combinations thereof; in the steps (2) and (5), the purification is performed by column chromatography, and the purification solvent is selected from any one of dichloromethane, ethyl acetate, methanol, ethanol, ether, acetone, and propanol, or a mixed solvent of several combinations thereof.

9. Use of the triarylmethane dye according to claim 1 in the fields of biology and medicine.

10. The use according to claim 9, characterized in that: The applications include the application of triarylmethane dyes in the preparation of living fluorescent labeling preparations, anti-tumor drug compositions, and light-responsive functional preparations.

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