Near-infrared xanthene dye with high stability and application of near-infrared xanthene dye in field of photo-thermal therapy

Abstract

The invention relates to a near-infrared xanthene dye with high stability and application of the near-infrared xanthene dye in the field of photo-thermal therapy. According to the xanthene dye, keto carbonyl is introduced into a xanthene structure to replace an oxygen bridge of a xanthene compound, the wavelength of the xanthene dye is successfully extended to a near-infrared region, and the emission wavelength of molecules is 850-1050 nm, so that the xanthene dye has better biological penetrability. The introduction of sulfonate radical realizes effective resistance to thiol nucleophilic attack, so that the compound has good chemical stability and light stability. After the nanoparticles are wrapped by DSPE-PEG2000, the photobleaching phenomenon does not occur in five photo-thermal cycle experiments, efficient photo-thermal conversion capability is achieved, and the photo-thermal conversion efficiency is as high as 59%. Meanwhile, an excellent photoacoustic signal is achieved, and a strong photoacoustic signal can be generated under optical excitation and is used for photoacoustic imaging. The photo-thermal reagent prepared from the xanthene dye shows obvious cell killing ability under light excitation, and has relatively low dark toxicity at the same time.

Description

Technical Field

[0001] The present invention relates to a class of near-infrared xanthene dyes with excellent stability and their nanoparticles for use as photothermal agents in photothermal therapy, belonging to the technical field of organic functional materials. Background Art

[0002] Photothermal therapy (PTT), a new precision treatment technology, uses photothermal agents (PTAs) to convert excitation light energy of a specific wavelength into thermal energy, raising the temperature in the targeted area above the thermal tolerance threshold of tumor cells (above 42°C), thereby achieving selective ablation of tumor cells. This technology offers advantages such as non-invasiveness, spatiotemporal controllability, and rapid treatment. By precisely controlling excitation light source parameters (such as wavelength and power density) and PTA concentration, it can effectively kill tumor tissue while significantly reducing toxic side effects on surrounding normal tissues, providing an innovative solution for precision medicine.

[0003] Among existing organic near-infrared photothermal material systems, xanthene dyes have attracted widespread attention due to their tunable optical properties. The absorption / emission wavelengths of these dyes are mainly red-shifted by regulating the extension of the intramolecular conjugated skeleton, so that their absorption peaks can be extended to the near-infrared region, thereby having better biological tissue penetration and lower risk of background light damage. However, studies have revealed that their long conjugated systems have inherent defects - they are prone to nucleophilic attack in high-concentration thiol biological environments, leading to problems such as molecular structure degradation and fluorescence quenching. This lack of photo / chemical stability seriously restricts their application potential in complex physiological environments.

[0004] Based on the above technical bottlenecks, the development of new xanthene-based photothermal reagents with both long-wavelength absorption characteristics and excellent stability has become a key breakthrough in current research. Summary of the Invention

[0005] To address the low stability of long-wavelength xanthene dyes, the present invention aims to provide a method for preparing a near-infrared xanthene dye with excellent stability. It also provides a xanthene dye nanoparticle photothermal agent with excellent photothermal stability and biocompatibility, along with high photothermal conversion efficiency.

[0006] This design provides a near-infrared xanthene dye compound with excellent stability. The series of compounds have the following general structural formula:

[0007] In the general formula, R1 is independently 、 、 、 、 、 、 、 、 、 、 or .

[0008] R2= 、 、 、 、 、 、 、 、 、 、 or .

[0009] R5 is 、 、 、 、 、 、 、 、 、 、 or .

[0010] n is an integer from 1 to 10.

[0011] X - is an anion, the total negative charge of the anion is equal to the total positive charge of the nitrogen-containing groups in the R2 or R5 structure; R3-R4 are each independently selected from H or CH3.

[0012] Y1-Y4 are each independently selected from H, halogen, cyano, amino, alkoxy having 1 to 5 carbon atoms, alkyl having 1 to 5 carbon atoms, nitro, hydroxy, carboxyl and sulfonate.

[0013] In some embodiments, X - Each is independent of BF4 - 、Cl - Br - , I - 、NO3 - 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - or CF3SO3 - .

[0014] In some specific embodiments, in the compound, R3-R4 are each independently selected from H or CH3.

[0015] Y1-Y4 are each independently selected from H, halogen, methyl, and methoxy.

[0016] n is an integer from 1 to 5.

[0017] In some specific embodiments, in the compound, R3-R4 are each independently selected from H or CH3; Y1-Y4 are all H. n is an integer of 1-3.

[0018] In some specific embodiments, in the compound, R1 is independently 、 、 、 、 、 、 、 、 、 、 、 、 or ; R2 is 、 、 、 、 、 、 、 、 、 、 、 、 or .

[0019] R5 is 、 、 、 、 、 、 、 、 、 、 、 、 or .

[0020] In some specific embodiments, in the compound, R1 is independently 、 、 、 、 、 、 .

[0021] R2 is 、 、 、 、 、 、 .

[0022] R5 is 、 、 、 、 、 、 .

[0023] A nanoparticle comprising at least one of the above-mentioned compounds.

[0024] In some specific embodiments, the nanoparticles and the compound are loaded on DSPE-PEG2000 to form nanoparticles.

[0025] In some specific embodiments, the nanoparticles have a diameter of 100-200 nm.

[0026] A photothermal agent comprising at least one of the compounds described above.

[0027] In some specific embodiments, the method for preparing the photothermal reagent comprises dropwise adding a solution of at least one of the compounds described in any one of claims 1 to 5 to an aqueous solution of DSPE-PEG2000 under ultrasound, sonicating for 10 to 90 minutes, and dialyzing using a dialysis bag to obtain the photothermal reagent.

[0028] In some specific embodiments, in the preparation method, the mass ratio of the compound to DSPE-PEG2000 is 0.05-0.2:1.

[0029] Preferably, the mass ratio of the compound to DSPE-PEG2000 is 0.07-0.12:1.

[0030] Use of the compound, the nanoparticles according to claim 6 or the photothermal agent according to claim 9 in the preparation of anti-tumor drugs.

[0031] The method for preparing the near-infrared xanthene dye has the following reaction formula and reaction steps: i) Under argon conditions, compound A, Pd(OAc)2, BINAP, Cs2CO3 and HR1 are heated in an organic solvent to react to obtain compound B; ii) removing water using a water separator, and heating the mixture of compound B, TsOH, and 1,3-propylene glycol to react to obtain compound C; iii) Under argon, a mixture of compound C, TsOH, CuBr2, and NH4Cl2 was dissolved in an organic solvent (1:1 toluene:1,3-ethylene glycol) and heated under reflux for 4-12 hours. The reaction mixture was concentrated, and methanol and DDQ were added to the concentrate, stirred at room temperature for 2 hours. The solvent was removed and purified to obtain compound D.

[0032] The definitions of R1, R2, R3, R4, Y1, Y2, Y3 and Y4 are the same as those in the general structural formula.

[0033] The organic solvent in step i is one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, tetrahydrofuran, dioxane, nitrogen-methylpyrrolidone, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, hexamethylphosphoramide, sulfolane, acetonitrile and benzonitrile, and the reaction temperature is 90-180° C.; the reaction temperature in step ii is 130-170° C., and the reaction temperature in step iii is 100-170° C.

[0034] A specific method for preparing a photothermal reagent is as follows: a specific compound D is dissolved in an organic solvent, and the solution is added dropwise to an aqueous solution containing DSPE-PEG2000 under ultrasound. The solution is ultrasonicated for 30 minutes, and dialyzed using a dialysis bag to obtain an aqueous solution containing nanoparticles.

[0035] The present invention provides a near-infrared xanthene dye with excellent stability. This dye introduces a ketone carbonyl group into the structure of xanthene compounds, replacing the oxygen bridge of the xanthene compound. This successfully extends the wavelength of the xanthene dye into the near-infrared region, with the molecule's emission wavelength between 850 nm and 1050 nm, resulting in improved biopenetration. The introduction of the sulfonate group effectively resists nucleophilic attack by thiols, resulting in excellent chemical and photostability. After being encapsulated into nanoparticles using DSPE-PEG2000, no photobleaching occurred during five photothermal cycle experiments, demonstrating efficient photothermal conversion with a photothermal conversion efficiency of up to 59%. This series of dyes also exhibits excellent photoacoustic signals, generating strong photoacoustic signals under light excitation, making them useful in applications such as photoacoustic imaging. The photothermal reagent prepared from this xanthene dye exhibits significant cell-killing ability under light excitation while also exhibiting low dark toxicity. The near-infrared xanthene dye provided by the present invention has a simple structure, a simple synthesis method, and potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are the normalized absorption and emission spectra of D1 in different solvents.

[0037] Figure 2This is a comparison of the chemical stability of D1 and ortho-methyl molecules.

[0038] Figure 3 This is a comparison of the photostability of D1 and the commercial dye IR780.

[0039] Figure 4 is the particle size distribution diagram of nanoparticles prepared by D1.

[0040] Figure 5 Nanoparticles prepared with different concentrations of D1 were irradiated by 808 nm laser (1.0 W / cm 2 ) in vitro photothermal heating curve under irradiation.

[0041] Figure 6 The nanoparticles prepared by D1 were irradiated by 808nm laser (1.0W / cm 2 ) photothermal cycle experiment under irradiation.

[0042] Figure 7 This is the result of the cell phototoxicity experiment of nanoparticles prepared by D1.

[0043] Figure 8 is the photoacoustic signal intensity diagram of compound D1 at different wavelengths. DETAILED DESCRIPTION

[0044] To make the technical solutions of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention. Example 1

[0045]

[0046] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 minutes. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:25) to obtain a light yellow solid, B1.

[0047]

[0048] A mixture of B1 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C1 as a light yellow solid.

[0049]

[0050] C1 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and sodium benzaldehyde o-sulfonate (2.70 mmol) were dissolved in 2 mL of an equal volume of toluene / ethylene glycol mixed solvent and heated under argon reflux for 5 hours. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and stirring at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to yield D1 as a brown solid, the structure of which was subsequently identified. Example 2

[0051]

[0052] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and dimethylamine hydrochloride (1.2 g) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 minutes. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:20) to obtain B2 as a light yellow solid.

[0053]

[0054] A mixture of B2 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product C2 was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford a light yellow solid.

[0055]

[0056] C2 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and sodium benzaldehyde o-sulfonate (2.70 mmol) were dissolved in 2 mL of an equal volume of toluene / ethylene glycol mixed solvent and heated under argon reflux for 5 hours. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and stirring at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to yield D2, a brown solid. The product was structurally identified. Example 3

[0057]

[0058] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and piperidine (1.2 mL) was dissolved in toluene bubbled with argon (to deoxygenate) for 20 minutes. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:20) to afford B3 as a light yellow solid.

[0059]

[0060] A mixture of B3 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product C3 was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford a light yellow solid.

[0061]

[0062] C3 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and 2-formyl-1,4-benzenedisulfonic acid (2.70 mmol) were dissolved in 2 mL of an equal volume of a toluene / ethylene glycol mixture and heated under reflux for 5 hours under argon. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 5:1) to yield D3 as a brown solid, the structure of which was subsequently identified. Example 4

[0063]

[0064] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 minutes. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:20) to obtain B4 as a light yellow solid.

[0065]

[0066] A mixture of B2 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C4 as a light yellow solid.

[0067]

[0068] C4 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and sodium benzaldehyde o-sulfonate (2.70 mmol) were dissolved in 2 mL of an equal volume of toluene / ethylene glycol mixed solvent and heated under argon reflux for 5 hours. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and stirring at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to yield D4 as a brown solid, the structure of which was subsequently identified. Example 5

[0069]

[0070] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 minutes. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:20) to obtain B5 as a light yellow solid.

[0071]

[0072] A mixture of B5 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product C5 was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford a light yellow solid.

[0073]

[0074] C5 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and 2-formyl-5-methoxybenzenesulfonic acid (2.70 mmol) were dissolved in 2 mL of an equal volume of a toluene / ethylene glycol mixture and heated under reflux for 5 hours under argon. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and stirring at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to yield D5 as a brown solid, the structure of which was subsequently identified. Example 6

[0075]

[0076] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 minutes. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:20) to obtain B6 as a light yellow solid.

[0077]

[0078] A mixture of B6 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product C6 was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford a light yellow solid.

[0079]

[0080] C6 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and 2-formyl-4-nitrobenzenesulfonic acid (2.70 mmol) were dissolved in 2 mL of an equal volume of a toluene / ethylene glycol mixture and heated under reflux for 5 hours under argon. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to yield D6 as a brown solid, the structure of which was subsequently identified. Example 7

[0081]

[0082] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 min. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:20) to afford B7 as a light yellow solid.

[0083]

[0084] A mixture of B7 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product C7 was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford a light yellow solid.

[0085]

[0086] C7 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and 3,4-difluoro-2-formylbenzenesulfonic acid (2.70 mmol) were dissolved in 2 mL of an equal volume of a toluene / ethylene glycol mixture and heated under argon for 5 hours at reflux. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to yield D7 as a brown solid, the structure of which was subsequently identified. Example 8

[0087] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 min. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:20) to afford B8 as a light yellow solid.

[0088]

[0089] A mixture of B8 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C8 as a light yellow solid.

[0090]

[0091] C8 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and 2-(1,1-dimethylethyl)-6-formylbenzenesulfonic acid (2.70 mmol) were dissolved in 2 mL of an equal volume of a toluene / ethylene glycol mixture and heated under reflux for 5 hours under argon. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and stirring at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to yield D8 as a brown solid, the structure of which was subsequently identified. Example 9

[0092]

[0093] Under argon, a mixture of compound A1 (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 minutes. The reaction was heated at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:20) to afford B9 as a light yellow solid.

[0094]

[0095] A mixture of B9 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product C9 was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford a light yellow solid.

[0096]

[0097] C9 (0.54 mmol), p-toluenesulfonic acid (1.08 mmol), cuprous bromide (0.24 mmol), and sodium benzaldehyde o-sulfonate (2.70 mmol) were dissolved in 2 mL of an equal volume of toluene / ethylene glycol mixed solvent and heated under argon reflux for 5 hours. After cooling, 5 mL of methanol was added, followed by the slow addition of DDQ (0.54 mmol), and stirring at room temperature for 2 hours. The reaction mixture was concentrated, acetone was added under an ice bath, and the precipitated solid was filtered. The resulting filter cake was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to yield D9 as a brown solid, the structure of which was subsequently identified. Example 10

[0098] Test the absorption and fluorescence emission spectra of D1 in different solvents: After vacuum drying D1, weigh the exact mass on a 1 / 10,000 balance and put it into a centrifuge tube, prepare a 2 mmol / L mother solution with dimethyl sulfoxide; take an appropriate amount of mother solution and add it to 3 mL of solvent, mix well, and measure its absorption and emission spectra by UV spectrophotometer and fluorescence spectrophotometer. The selected solvents are: dichloromethane, methanol and water. Its absorption wavelength is higher than 800 nm, and the wavelength of the emission peak is greatly affected by the polarity of the solvent. The emission wavelength in water can reach 1006 nm. It is a long-wavelength near-infrared xanthene dye. Its normalized absorption and emission spectra are as follows Figure 1 shown Example 11

[0099] Chemical stability test of D1: First, prepare a D1 aqueous solution (containing 0.5% Tween 80) with an absorbance of about 1. Then add a certain amount of cysteine ​​(Cys) or cysteine ​​to the aqueous solution. After stirring evenly, incubate at 37°C for different time periods. Then record the UV-visible absorption spectrum of the dye at different times. According to the change in the absorption spectrum, select the absorbance at the maximum absorption point and plot it against time ( Figure 2 The concentration of Cys was 100 μM. It was found that after 7 hours of incubation in a thiol environment, the absorbance retention rate of D1 was >95%, while the control molecule (the sulfonate group of D1 was replaced by a methyl group) Only 60%, indicating that the introduction of sulfonate groups gives the molecule excellent ability to resist thiol attack and good chemical stability. Example 12

[0100] Light stability test of D1: D1 and commercial IR780 (CAS: 207399-07-3) were prepared into methanol solutions with an absorption value of about 1. 2 Under 808 nm laser irradiation, the absorption was tested every 3 minutes for a total of 30 minutes to determine the degradation amount. The results are as follows Figure 3 As shown, from Figure 3 As can be seen, the absorption value of D1 only decreased by 8% within 30 minutes, while that of the commercial IR780 decreased by 86%, indicating that the molecule has good photostability and good resistance to photobleaching in fluorescence imaging. Example 13

[0101] Preparation of nanophotothermal reagent: 1 mg of compound D1 was weighed and dissolved in DMF (1 mL). The solution was then added dropwise to 10 mL of Wahaha purified water containing 10 mg of DSPE-PEG2000 under ultrasound. The solution was sonicated for half an hour and dialyzed for 24 hours using a 3500 molecular weight dialysis bag. The aqueous solution was replaced every six hours to obtain a nanoparticle aqueous solution (nanophotothermal reagent). The DSPE-PEG2000 used in this example was purchased from Anaiji. The prepared nanoparticle aqueous solution was tested for particle size using a particle size analyzer, and the particle size distribution diagram is shown in FIG. Figure 4 As shown, it shows that we have successfully encapsulated compound D1 into nanoparticles. Example 14

[0102] In vitro photothermal heating experiment of nanophotothermal reagent: 808 nm laser was used at 1.0 W / cm 2 The light power density was used to irradiate aqueous solutions of D1 nanoparticles with different concentrations (10, 20, 50 μM) and the time was recorded. The temperature rise at different times was recorded and the temperature rise curve was drawn. Figure 5 As shown in the figure, the temperature of 50 μM nanoparticles increased by more than 40 degrees Celsius under light irradiation, indicating that they have good photothermal conversion effect. Example 15

[0103] In vitro photothermal stability experiment of nanoreagents: 50 μM aqueous solution of D1 nanoparticles was prepared and the photothermal stability was tested by 808 nm laser at 1.0 W / cm 2 The solution was irradiated with a light power density of 100 nm and the time was set. Five heating-cooling cycles were repeated and a cycle diagram was drawn as shown in FIG. Figure 6 As shown, the temperature change in each cycle remains basically unchanged, indicating that it has good photothermal stability and photothermal performance. Example 16

[0104] Nano-agent in vitro photothermal therapy experiment: Hela cells were selected for cytotoxicity experiment. The specific operation steps are as follows: Hela cells were seeded into 96-well plates, 100 μL of DMEM medium was added to each well and incubated. When the cell density was about 80%, DMEM medium was used as solvent to prepare nano-photothermal agent solutions of different concentrations (0 μM, 5 μM, 10 μM). The experiment was divided into 3 groups, each with 4 wells. 100 μL of nano-solution of corresponding concentration was added to each well. After incubation with Hela cells for 12 hours, the illumination group was illuminated with 808 nm laser (with an optical power of 800 mW / cm 2 ) Each well was irradiated for 5 min. Figure 7It can be seen that 5 μM of the light group has killed 50% of the cells, and the cell survival rate of 10 μM concentration has dropped to below 30%, while the corresponding dark toxicity cell survival rate is above 85%. The above results show that the photothermal reagent has excellent cell phototoxicity and low dark toxicity, showing excellent cell killing ability, and is a photothermal reagent with great potential. Example 17

[0105] Photoacoustic Performance Exploration: The photoacoustic imaging capability of D1 was tested using the VevoLazr-X system. First, a DMSO solution with a D1 concentration of 100 μM was prepared. A certain amount was pipetted into a capillary tube and placed in a photoacoustic imaging coupling agent. The photoacoustic signal at wavelengths of 750-950 nm was measured under a photoacoustic probe, and a photoacoustic spectrum was obtained. The photoacoustic signal intensity of D1 at different wavelengths was tested, and it was found that under light excitation conditions, compound D1 was able to respond with a strong photoacoustic signal in the near-infrared region (700-1000 nm), demonstrating excellent photoacoustic signal generation capabilities. This characteristic not only gives it the application potential of photoacoustic imaging, but also provides a new adjuvant treatment imaging medium for photothermal therapy. Example 18

[0106] The emission spectra of compounds D4 and D9 in aqueous solution were tested according to the method of Example 10. The emission wavelengths of compounds D4 and D9 were 930 nm and 955 nm, respectively.

Claims

1. Compounds of Formula I-III: In the general formula, R1 is independently R5 is or n is an integer from 1 to 10; X - is an anion, the total negative charge of the anion is equal to the total positive charge of the nitrogen-containing groups in the R2 or R5 structure; R3-R4 are each independently selected from H or CH3; Y1-Y4 are each independently selected from H, halogen, cyano, amino, alkoxy having 1 to 5 carbon atoms, alkyl having 1 to 5 carbon atoms, nitro, hydroxy, carboxyl and sulfonate.

2. The compound according to claim 1, characterized in that R3-R4 are each independently selected from H or CH3; Y1-Y4 are each independently selected from H, halogen, methyl, methoxy; n is an integer from 1 to 5; X - Each is independent of BF4 - 、Cl - Br - , I - 、NO3 - 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - or CF3SO3 - .

3. The compound according to claim 1, characterized in that R3-R4 are each independently selected from H or CH3; Y1-Y4 are all H; n is an integer from 1 to 3.

4. The compound according to any one of claims 1 to 3, characterized in that R1 is independent of each other R2 is R5 is or 5. The compound according to any one of claims 1 to 3, characterized in that R1 is independent of each other R2 is R5 is 6. A nanoparticle, characterized in that Comprising at least one of the compounds according to any one of claims 1 to 5.

7. The nanoparticles according to claim 6, characterized in that The compound is loaded on DSPE-PEG2000 to form nanoparticles.

8. The nanoparticles according to claim 6, characterized in that The diameter of the nanoparticles is 100-200 nm.

9. A photothermal reagent, characterized in that Comprising at least one of the compounds according to any one of claims 1 to 5.

10. The method for preparing the photothermal reagent according to claim 9, characterized in that: A solution of at least one of the compounds according to any one of claims 1 to 5 is added dropwise to a DSPE-PEG2000 aqueous solution under ultrasound, ultrasound is performed for 10 to 90 minutes, and dialyzed using a dialysis bag to obtain the photothermal reagent.

11. The preparation method according to claim 10, characterized in that: The mass ratio of the compound to DSPE-PEG2000 is 0.05-0.2:1; Preferably, the mass ratio of the compound to DSPE-PEG2000 is 0.07-0.12:

1.

12. Use of the compound according to claim 1 in the preparation of anti-tumor drugs.

13. Use of the nanoparticles according to claim 6 in the preparation of anti-tumor drugs.

14. Use of the photothermal agent according to claim 9 in the preparation of anti-tumor drugs.

15. Use of the compound according to claim 1 in the preparation of photoacoustic imaging materials, photothermal reagents, and photothermal materials.

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