Synthesis method and application of asymmetric cyanine dye containing dioxaborinine

Abstract

The invention relates to the technical field of fluorescent dyes, in particular to a synthesis method and application of an asymmetric cyanine dye containing dioxaborinine. The synthesis method comprises the following steps: (1) preparing a compound, the asymmetric cyanine dye disclosed by the invention has remarkable advantages in the aspects of synthesis convenience, active oxygen generation efficiency, treatment mode diversity and detection simplicity, and provides a more efficient and more practical novel photosensitizer choice for tumor photodynamic therapy.

Description

Technical Field

[0001] This invention relates to the field of fluorescent dye technology, and more specifically, to a method for synthesizing asymmetric cyanine dyes containing dioxaboroline and their applications. Background Technology

[0002] Photodynamic therapy (PDT) for tumors, as an emerging precision treatment method, has become a research hotspot in the field of tumor treatment due to its advantages such as minimal invasiveness, strong targeting, and synergistic therapeutic effects. The core mechanism of PDT relies on the generation of reactive oxygen species (such as singlet oxygen) by a photosensitizer (usually a fluorescent probe) under light excitation at a specific wavelength. 1 O2 can precisely kill tumor tissue by oxidatively damaging tumor cell structure, disrupting the vascular system, and inducing an immune response. However, the fluorescent probes currently used in clinical practice and research still have many key limitations: (1) low efficiency in generating reactive oxygen species, resulting in slow tumor killing speed and insufficient effect; (2) poor photostability, which is prone to photobleaching or degradation under continuous light irradiation, affecting the durability of treatment; (3) insufficient tumor targeting, with some probes potentially damaging normal tissues due to non-specific distribution, or reducing bioavailability and safety due to poor water solubility and metabolic kinetics. In addition, although traditional photosensitizers such as clinically approved porphyrin derivatives have certain therapeutic effects, they are still limited by problems such as poor water solubility and low extinction coefficient, which restricts the widespread application and improvement of efficacy of PDT.

[0003] To address the aforementioned challenges, developing novel fluorescent probes that combine efficient reactive oxygen species generation, excellent photostability, and precise tumor targeting, and optimizing their synthesis process to achieve stable and scalable preparation, is of great significance for promoting the clinical translation of phototherapy techniques (PDT) and improving the efficacy of tumor treatment. This is also the core technical problem that this invention aims to solve. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a method for synthesizing asymmetric cyanine dyes containing dioxaboroline, the steps of which are as follows: in, Preferably, the dioxaboroline-containing asymmetric cyanine dye comprises: .

[0005] Another objective of this invention is to provide a method for synthesizing asymmetric cyanine dyes containing dioxaboroline, the steps of which are as follows: S1. Synthesis of dioxaboroline derivatives: Aromatic compounds, acetic anhydride, and boron trifluoride diethyl ether ester were added to a round-bottom flask. The reaction mixture was heated and stirred. The reaction was terminated by adding the reaction mixture dropwise to cold water. The precipitate was washed three times with water and dried under vacuum. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as the eluent to obtain dioxaboroline derivatives. S2. Synthesis of N-methylonium salt intermediate: N-methylonium salt and diphenylamino Schiff base compound were added to a round-bottom flask, and acetic anhydride was added dropwise. The reaction mixture was heated and stirred under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and precipitated with isopropyl ether to obtain a black solid. The precipitate was washed three times with water and dried under vacuum. The crude product was purified by silica gel column chromatography using dichloromethane / methanol as eluent to obtain the black solid N-methylonium salt intermediate. S3. Synthesis of asymmetric cyanine dyes containing dioxaboroline with different conjugated chain lengths: N-methylhexane salt intermediate and dioxaboroline derivative were added to a round-bottom flask, and triethylamine was added dropwise. The reaction mixture was heated and stirred under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and dried under vacuum. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as eluent to obtain the product, asymmetric cyanine dye containing dioxaboroline.

[0006] Preferably, in S1, the molar ratio of aromatic compound, acetic anhydride, and boron trifluoride diethyl ether ester is 1:5 to 10:1 to 3, the aromatic compound is pyrene, 9,9-dimethylfluorene, 9,9'-spirodifluorene, triphenylamine, phenylcarbazole, carbazole, or 9-silazine, the heating temperature is 50 to 80°C, the stirring time is 0.5 to 3 hours, and the volume ratio of petroleum ether to dichloromethane is 1:1.

[0007] Preferably, in S2, the molar ratio of N-methylonium salt to diphenylamino Schiff base compound is 1:1~2, and the amount of acetic anhydride added is 30 mL of acetic anhydride per 1 g of N-methylonium salt. The N-methylonium salt is 1,1,2-trimethyl-3-substituted benzoindole iodide, 1,1,2-trimethyl-3-substituted naphthoindole iodide, 1-methyl-2-substituted fluorene iodide, or 1-methyl-2-substituted... - benzoquinoline iodomonium salt, wherein the N-methylonium salt substituent is methyl, ethyl, n-butyric acid, propylsulfonate ion, propyltrimethylammonium ion or propyltriphenylphosphonium ion; wherein the diphenylamino Schiff base compound is N,N-diphenylmethylammonium, malondialdehyde diphenylamine hydrochloride or pentadienal diphenylamine hydrochloride, the heating temperature is 120~140℃, the stirring time is 1~3h, and the volume ratio of petroleum ether to dichloromethane is 1:1.

[0008] Preferably, in S3, the molar ratio of dioxaboroline derivative, N-methylhexane salt intermediate, and triethylamine is 1:1:1.2, and the volume ratio of petroleum ether to dichloromethane is 1:1.

[0009] Another aspect of the present invention aims to provide a method for synthesizing asymmetric cyanine dyes containing dioxaboroline, the steps of which are as follows: S1. Synthesis of deuterated dioxaboroline derivatives: Aromatic compounds were added to toluene and Pd / C catalyst to a reaction vessel, and D2O was introduced. The reaction was stirred under an inert gas atmosphere to obtain deuterated aromatic compounds. Acetic anhydride and boron trifluoride diethyl ether ester were then added. The reaction mixture was heated and stirred. The reaction was terminated by adding the reaction mixture dropwise to cold water. The precipitate was washed three times with water and dried under vacuum. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as eluent to obtain the solid compound deuterated dioxaboroline derivative. S2. Synthesis of N-methylonium salt intermediate: N-methylonium salt and diphenylamino Schiff base compound were added to a round-bottom flask, and acetic anhydride was added dropwise. The reaction mixture was heated and stirred under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and precipitated with isopropyl ether to obtain a black solid. The precipitate was washed three times with water and dried under vacuum. The crude product was purified by silica gel column chromatography using dichloromethane / methanol as eluent to obtain the black solid N-methylonium salt intermediate. S3. Synthesis of asymmetric cyanine dyes containing dioxaboroline with different conjugated chain lengths: N-methylhexane salt intermediate and deuterated dioxaboroline derivative were added to a round-bottom flask, and triethylamine was added dropwise. The reaction mixture was heated and stirred under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and dried under vacuum. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as eluent to obtain the product, asymmetric cyanine dyes containing dioxaboroline.

[0010] Preferably, in S1, the molar ratio of aromatic compound to Pd / C catalyst is 1:1~2, 100 mL L2O is added for every 1 g of aromatic compound, and the reaction is carried out at 220~240°C under an inert gas atmosphere with stirring for 12~24 h. The molar ratio of deuterated aromatic compound, acetic anhydride and boron trifluoride diethyl ether ester is 1:5~10:1~3, and the volume ratio of petroleum ether to dichloromethane is 1:1.

[0011] Preferably, in S2, the molar ratio of N-methylonium salt to diphenylamino Schiff base compound is 1:1~2, and the amount of acetic anhydride added is 30mL of acetic anhydride per 1g of N-methylonium salt. The N-methylonium salt is 1,1,2-trimethyl-3-substituted benzoindoline iodide, 1,1,2-trimethyl-3-substituted naphthoindoline iodide, 1-methyl-2-substituted fluorene iodide, or 1-methyl-2-substituted benzoquinoline iodide. The salt, wherein the N-methylonium salt substituent is methyl, ethyl, n-butyric acid, propylsulfonate ion, propyltrimethylammonium ion, or propyltriphenylphosphonium ion; the diphenylamino Schiff base compound is N,N-diphenylmethylammonium, malondialdehyde diphenylamine hydrochloride, or pentadienaldehyde diphenylamine hydrochloride; the molar ratio of dioxaboroline derivative, N-methylonium salt intermediate, and triethylamine in S3 is 1:1:1.2, and the volume ratio of petroleum ether to dichloromethane is 1:1.

[0012] Another aspect of the present invention is to provide an application of a dioxaboroline-containing asymmetric cyanine dye in early type I / II photodynamic therapy.

[0013] The beneficial effects of this invention are as follows: The synthesis process is simple and efficient: the synthesis route of this dye is simple, easy to operate, and easy to prepare on a large scale, which reduces production costs and process complexity.

[0014] Highly efficient reactive oxygen species generation capacity: It has a high singlet oxygen generation rate, which can significantly improve the efficiency of photodynamic killing and rapidly destroy tumor tissue; it has a strong superoxide anion generation capacity, which further enhances the oxidative stress effect and improves the killing effect on tumor cells.

[0015] Synergistic photodynamic therapy mechanism: It can simultaneously achieve type I and type II photodynamic therapy, exert a synergistic effect, overcome the limitations of a single mechanism, and improve the anti-tumor efficacy.

[0016] Convenient testing: Performance characterization can be performed using only a UV-Vis spectrophotometer and a fluorescence spectrophotometer, simplifying the experimental process and facilitating quality control and practical applications.

[0017] In summary, the asymmetric cyanine dyes of this invention exhibit significant advantages in terms of ease of synthesis, reactive oxygen species generation efficiency, diversity of treatment modalities, and ease of detection, providing a more efficient and practical new photosensitizer option for tumor photodynamic therapy.

[0018] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. Attached Figure Description

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is the 1H NMR spectrum of the probe CyTY-1 prepared in this embodiment of the invention; Figure 2 This is the 1H NMR spectrum of the probe CyTY-2 prepared in this embodiment of the invention; Figure 3 This is the 1H NMR spectrum of the probe CyTY-3 prepared in this embodiment of the invention; Figure 4 This is the absorption diagram of the probe CyTY-1﹣3+DPBF prepared in the embodiments of the present invention under different light sources; Figure 5 This is the absorption diagram of the blank DPBF of this invention under an 808nm 50mW / cm² light source; Figure 6 The fluorescence emission patterns of the probe CyTY-1﹣3+DHR123 prepared in the embodiments of the present invention under different light sources are shown. Figure 7 This is the fluorescence spectrum of the blank DHR123 of this invention under an 808nm 50mW / cm² light source; Figure 8 These are fluorescence emission patterns of the probe CyTY-1﹣3+HPF prepared in the embodiments of the present invention under different light sources; Figure 9 This is the fluorescence spectrum of the blank HPF of this invention under an 808nm 50mW / cm² light source; Figure 10 This is a graph showing the MTT cell survival rate in a cytotoxicity assay using the probe CyTY-1﹣3 according to an embodiment of the present invention. Figure 11 This is a graph showing the survival rate of MTT cells in a cytotoxicity experiment of probe CyTY-1﹣3 under different light source excitation in an embodiment of the present invention. Detailed Implementation

[0020] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0021] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0022] Example 1: Synthesis of Intermediate 1 (a dioxaboroline derivative with a triphenylamine donor) Set up a magnetic stirrer, oil bath, thermometer, condenser, and nitrogen protection device in a 100mL round-bottom flask. Add 245mg (1.0mmol, purity ≥98%) of triphenylamine to the flask, cool to 0℃ in an ice bath, and slowly add 3.6mL of analytical grade acetic anhydride dropwise, stirring for 10min until homogeneous. Then add 1.1mL (purity ≥98%) of boron trifluoride diethyl ether dropwise at a rate of 1-2 drops / second, avoiding violent exothermic reactions. Remove the ice bath, raise the temperature to 60℃ (oil bath temperature 65-70℃), and react with magnetic stirring at 400-500rpm for 1 hour. Separately, use petroleum ether / dichloromethane (1... :1, (Rf≈0.4)) TLC monitoring; after the reaction was completed, the hot solution was slowly poured into 200mL of ice water and stirred vigorously at 800-1000rpm. An orange precipitate was precipitated. After filtration, the filter cake was washed with 3×50mL of cold water and dried under vacuum at 40℃ for 12 hours. It was purified by silica gel column chromatography with 200-300 mesh silica gel (column length 20cm, inner diameter 3cm), using petroleum ether / dichloromethane (1:1, (v / v)) as eluent, at a flow rate of 1-2mL / min. The target component was collected, concentrated under reduced pressure, and dried under vacuum to obtain 250mg of orange solid 2 (yield 72%).

[0023] Example 2: Synthesis of intermediates 3a-3c (benzoindole derivatives) General operating procedures (taking 3a as an example): 177 mg (0.5 mmol, purity ≥98%) of 1,1,2,3-tetramethyl-1H-benzo[e]indoleium salt and 168 mg (0.75 mmol, purity ≥97%) of N,N-diphenylformamidinium were added to a round-bottom flask, and the mixture was purged with nitrogen three times. 3 mL of analytical grade acetic anhydride was slowly added dropwise under ice bath conditions. After the addition was complete, the temperature was raised to 120 °C (oil bath temperature 125-130 °C), and the mixture was stirred at 400-500 rpm for 1 hour under nitrogen protection. The reaction was monitored by TLC using dichloromethane / methanol (20:1, R_f≈0.3). The reaction progress was monitored; after cooling to room temperature (25°C), 30 mL of isopropyl ether was slowly added and stirred for 30 min, resulting in the precipitation of a black precipitate. The precipitate was filtered, washed with 3 × 10 mL of isopropyl ether, and then dried under vacuum at 40°C for 12 hours. Purification was then performed by silica gel column chromatography (20 cm column length, 3 cm inner diameter) using 200-300 mesh silica gel, with dichloromethane / methanol (20:1 → 15:1, (v / v)) as the eluent and a flow rate controlled at 1-2 mL / min. The target fraction was collected, concentrated under reduced pressure, and dried under vacuum to obtain 135 mg of black solid 3a (yield 56%). The synthesis of 3b and 3c used the corresponding substituted benzoindole salts, with the remaining procedures the same as for 3a, yielding yields of 52% and 48%, respectively.

[0024] Example 3: Synthesis of target products CyTY-1, CyTY-2, and CyTY-3 Add 100 mg (0.27 mmol) of intermediate 3a and 78 mg (0.21 mmol) of compound 1 to a round-bottom flask, purging with nitrogen three times; add 500 μL (purity ≥99%) of triethylamine dropwise under ice bath, and after the addition is complete, raise the temperature to 50 °C (oil bath temperature 55-60 °C), and stir the mixture at 400-500 rpm for 1 hour under nitrogen protection. The reaction mixture is then passed through petroleum ether / dichloromethane (1:1, R_f≈0. TLC monitoring of .2)); after cooling to room temperature of 25℃, triethylamine was removed by vacuum concentration, and then purified by silica gel column chromatography (20cm column length, 3cm inner diameter) using petroleum ether / dichloromethane (1:1, (v / v)) as eluent at a flow rate of 1-2mL / min. The target component was collected, concentrated under reduced pressure, and dried under vacuum to obtain 85mg of dark green solid CyTY-1 (yield 67%).

[0025] CyTY-1¹H NMR (400 MHz, CDCl3) δ 8.04 (t, J = 8.0 Hz, 1H), 7.80-7.70 (m, 3H), 7.60-7.50 (m, 5H), 7.40-7.30 (m, 7H), 7.08 (d, J = 16.0 Hz, 1H), 7.04 (t, J = 16.0 Hz, 1H), 6.89 (d, J = 16.0 Hz, 2H), 6.68 (s, 1H), 6.35 (t, J = 16.0 Hz, 1H), 6.02 (d, J = 8.0 Hz, 1H), 5.88 (d, J = 8.0 Hz, 1H), 3.37 (s, 3H), 1.59 (s, 6H). HRMS** (ESI⁺): calcd for C 39 H 34 BF2N2O2⁺ [M⁺] 610.2603, found 610.2609.

[0026] CyTY-2: Add 100 mg (0.25 mmol) of intermediate 3b and 78 mg (0.21 mmol) of compound 1 to a round-bottom flask, purging with nitrogen three times; add 500 μL (purity ≥99%) of triethylamine dropwise under ice bath, and after the addition is complete, raise the temperature to 50℃ (oil bath temperature 55-60℃), and stir the reaction at 400-500 rpm for 1 hour under nitrogen protection. Then, pass the mixture through petroleum ether / dichloromethane (1:1, ... TLC monitoring was performed on the sample with f ≈ 0.2. After cooling to room temperature of 25°C, triethylamine was removed by vacuum concentration. The sample was then purified by silica gel column chromatography (20 cm column length, 3 cm inner diameter) using petroleum ether / dichloromethane (1:1, (v / v)) as eluent at a flow rate of 1-2 mL / min. The target component was collected, concentrated under reduced pressure, and dried under vacuum to obtain 78 mg of reddish-brown solid CyTY-2 (yield 58%).

[0027] CyTY-2 ¹H NMR (400 MHz, CDCl3) δ 8.25 (t, J = 8.0 Hz, 1H), 7.88 (d, J= 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 1H), 7.60-7.50 (m, 4H), 7.40-7.30 (m, 2H), 7.08 (m, 9H), 7.04 (d, J = 16.0 Hz, 1H), 7.04 (d, J = 16.0 Hz, 1H), 6.89 (d, J = 16.0 Hz, 2H), 6.68 (s, 1H), 6.89 (d, J = 16.0 Hz, 1H), 6.01 (s, 1H), 5.98-6.06 (m, 2H), 3.47 (s, 3H), 1.60 (s, 6H). HRMS** (ESI⁺): calcd forC 41 H 36 BF2N2O2⁺ [M⁺] 637.5498, found 637.5487.

[0028] CyTY-3: Add 100 mg (0.24 mmol) of intermediate 3b and 78 mg (0.21 mmol) of compound 1 to a round-bottom flask, purging with nitrogen three times; add 500 μL (purity ≥99%) of triethylamine dropwise under ice bath, and after the addition is complete, raise the temperature to 70°C (oil bath temperature 55-60°C), and stir the mixture at 400-500 rpm for 1 hour under nitrogen protection. The reaction is then carried out using petroleum ether / dichloromethane (1:1, ...). TLC monitoring was performed on the sample with f ≈ 0.2. After cooling to room temperature of 25°C, triethylamine was removed by vacuum concentration. The sample was then purified by silica gel column chromatography (20 cm column length, 3 cm inner diameter) using petroleum ether / dichloromethane (1:1, (v / v)) as eluent at a flow rate of 1-2 mL / min. The target component was collected, concentrated under reduced pressure, and dried under vacuum to obtain 72 mg of reddish-brown solid CyTY-3 (yield 52%).

[0029] CyTY-3 ¹H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 8.0 Hz, 1H), 7.96 (t, J= 8.0 Hz, 2H), 7.51 (m, 4H), 7.60-7.50 (m, 2H), 7.40-7.30 (m, 6H), 7.08 (m, 8H), 6.98 (d, J = 8.0 Hz, 2H), 6.25 (t, J = 16.0 Hz, 2H), 5.96 (d, J = 8.0Hz, 1H), 5.59 (t, J = 16.0 Hz, 1H), 3.37 (s, 3H), 1.93 (s, 6H). HRMS** (ESI⁺): calcd for C 43 H 38 BF2N2O2⁺ [M⁺] 663.5878, found 663.5871.

[0030] like Figures 1 to 3 The image shows the 1H NMR spectra of the prepared probes CyTY-1, CyTY-2, and CyTY-3.

[0031] Example 4: Type II photodynamic therapy activity (singlet oxygen detection) When preparing the solution, weigh 5.92 mg (0.01 mmol) of CyTY-1 and dilute to 10 mL with DMSO to obtain a 1 mM CyTY-1-3 stock solution, which should be stored at 4 °C protected from light; separately weigh 3.98 mg (0.01 mmol) of DPBF and dilute to 10 mL with DMSO to obtain a 1 mg / mL DPBF stock solution, which should be stored at -20 °C protected from light. During the test, 20 μL of LcyTY-1 stock solution was added to 2 mL of deionized water and vortexed for 10 seconds to prepare a 10 μM working solution. Then, 10 μL of DPBF stock solution was added, vortexed for 10 seconds, and transferred to a 1 cm quartz cuvette. The initial absorbance (λ=410 nm) was measured using a UV-Vis spectrophotometer and recorded as A0. Subsequently, the cuvette was placed under an LED light source with a wavelength of 600 nm, a power of 50 mW / cm², and a spot diameter of 1 cm, at a distance of 2 cm from the light source. The absorbance At was measured immediately after 0, 10, 20, 30, 40, 50, 60, 120, 180, 240, and 300 seconds of illumination. The absorbance decrease rate ΔA% was calculated as [(A0-At) / A0]×100%. The blank control group only added DPBF singlet oxygen probe and no photosensitizer. During data processing, ΔA% - time curves were plotted to compare the singlet oxygen generation efficiency of CyTY-1, CyTY-2, and CyTY-3 (e.g., ...). Figure 4 As shown), and plot the blank group ΔA% - time curve (as shown). Figure 5 (As shown).

[0032] Singlet oxygen generation efficiency was assessed by monitoring the rate of decrease in absorbance (ΔA%) at 410 nm using DPBF. For example... Figure 4 As shown, under 600nm illumination, CyTY-1, CyTY-2, and CyTY-3 all exhibited significantly higher ΔA% values ​​within 300s compared to the control commercially available photosensitizer ICG (e.g., CyTY-1 reached 85% ΔA% at 300s, while ICG was only 50%), and the rate of absorbance decrease increased linearly with time. The blank group (DPBF only) showed almost no change (e.g., ...). Figure 5 (As shown).

[0033] These asymmetric cyanine dyes can efficiently induce type II photodynamic reactions, and their singlet oxygen generation efficiency is significantly better than that of traditional photosensitizers. This is attributed to their D-π-AD type conjugated skeleton promoting intersystem crossing (ISC), shortening the singlet triplet energy level difference, and improving energy conversion efficiency.

[0034] Example 5: Type I photodynamic therapy activity (superoxide anion detection) When preparing the solution, the CyTY-1-3 stock solution was prepared in the same way as the Type II detection method. Weigh 3.58 mg (0.01 mmol) of DHR123 and dilute to 1 mL with DMSO to obtain a 10 mM stock solution. After aliquoting, store at -20 °C protected from light. For detection, 20 μL of LcyTY-1 stock solution was added to 2 mL of deionized water and vortexed for 10 seconds to prepare a 10 μM working solution. Then, 2 μL of LDH123 stock solution (final concentration 10 μM) was added, vortexed, and transferred to a 1 cm quartz fluorescence cuvette. The initial fluorescence F0 was measured using a fluorescence spectrophotometer at Ex=488 nm, Em=525 nm, slit width 5 nm / 5 nm, and PMT voltage 700 V. The cuvette was placed under an LED light source with wavelengths of 600 nm / 680 nm / 808 nm, power of 50 mW / cm², and spot diameter of 1 cm (distance 2 cm). After irradiation with light at time gradients of 0, 1, 2, 3, 4, and 5 minutes, the fluorescence intensity Ft was measured immediately. The control group consisted of a blank control containing only a DPBF singlet oxygen probe (this description may differ from the actual detection system; the actual probe type should be used as the standard). During data processing, F / F0-time curves were plotted, and fluorescence enhancement rates were calculated to compare the superoxide anion generation efficiencies of CyTY-1, 2, and 3 (e.g., ...). Figure 6 As shown), and plot the curves corresponding to the blank groups (such as...). Figure 7 (As shown).

[0035] Using DHR123 as a probe, CyTY-1-3 showed a significant increase in fluorescence intensity (F / F0) of DHR123 within 5 minutes under illumination at 600 / 680 / 808 nm (e.g., Figure 6As shown in the figure, the fluorescence of the blank group (without photosensitizer) remained almost unchanged. Among them, CyTY-2 showed the fastest fluorescence enhancement rate under 680nm illumination.

[0036] These dyes can efficiently generate reactive oxygen species (•O2⁻) through the type I mechanism, and the type I and type II mechanisms work synergistically to form a multi-pathway oxidative damage network, thereby enhancing the destructive ability of tumor cells.

[0037] Example 6: Type I photodynamic therapy activity (hydroxyl radical detection) For solution preparation, the CyTY-1-3 stock solution was prepared using the same method as for Type II detection. 4.65 mg (0.01 mmol) of HPF was weighed and diluted to 1 mL with DMSO to obtain a 10 mM stock solution. This solution was aliquoted and stored at -20°C protected from light. For detection, 20 μL of the CyTY-1 stock solution was added to 2 mL of deionized water and vortexed for 10 seconds to prepare a 10 μM working solution. 2 μL of HPF stock solution (final concentration 10 μM) was added, vortexed, and transferred to a 1 cm quartz fluorescence cuvette. The initial fluorescence F0 was measured using a fluorescence spectrophotometer at Ex=488 nm, Em=515 nm, slit width 5 nm / 5 nm, and PMT voltage 700 V. The cuvette was placed under an LED light source with wavelengths of 600 nm / 680 nm / 808 nm, power of 50 mW / cm², and a spot diameter of 1 cm (distance 2 cm). The fluorescence intensity Ft was measured immediately after irradiation at time gradients of 0, 1, 2, 3, 4, and 5 minutes. The control experiment included a blank group (HPF probe only, no photosensitizer) and a dark control group (photosensitizer and HPF probe added, but protected from light). During data processing, F / F0-time curves were plotted to calculate the fluorescence enhancement rate, and the hydroxyl radical generation efficiency of CyTY-1, 2, and 3 was compared (e.g., ...). Figure 8 As shown), and plotted the curves corresponding to the blank group and the dark control group to verify the light-dependent generation of reactive oxygen species (e.g. Figure 9 (As shown).

[0038] When using the HPF probe, hydroxyphenylfluorescein showed enhanced fluorescence in the presence of different photosensors (e.g., Figure 8 As shown in the figure, the dark control group (protected from light) showed no significant changes, confirming that hydroxyl radicals (・OH) are generated after irradiation with CyTY-1-3 photoactivator.

[0039] These dyes can efficiently generate reactive oxygen species (·OH) through the type I mechanism, and the type I and type II mechanisms work synergistically to form a multi-pathway oxidative damage network, thereby enhancing the destructive ability of tumor cells.

[0040] Example 7: Cytotoxicity test of CyTY-1-3 under light-free and light-free conditions 4T1 cells (1×10⁴ cells per well) 4Cells were seeded in 96-well plates. After 12 hours of culture, cells were treated with increasing concentrations of CyTY-1-3 nanoparticles (0, 1, 2, 5, 10, 20, 50 μM) for 24 hours. Subsequently, 10 μL of MTT solution (0.5 mg / mL) was added to each well. - ¹, Prepare with 1×PBS. After incubating for 4 hours, replace with dimethyl sulfoxide (200 μL per well). Measure the absorbance at 490 nm using a microplate reader (e.g., ...). Figure 10 (As shown).

[0041] In vitro phototoxicity assay: 4T1 cells (1 × 10^4 cells per well) were first cultured in 96-well plates for 12 hours. After removing the culture medium, each well was incubated with a gradient of CyTY-1-3 nanoparticles (0, 1, 2, 5, 10, 20, 50 μM) at increasingly higher concentrations (100 μL per well). After 6 hours of incubation, the wells were irradiated for 5 minutes with an 808 nm laser at a power density of 0.5 W cm−2. Finally, cell viability was determined using the standard MTT assay (e.g., ...). Figure 11 (As shown).

[0042] Under dark conditions, the cell viability of CyTY-1, CyTY-2, and CyTY-3 remained above 80% (close to 100%) with increasing dye concentration (0-50 μM). This indicates that the three nanoparticles have extremely low dark toxicity and do not significantly damage 4T1 cells in the absence of light, avoiding the side effects on normal tissues caused by the dark toxicity of traditional photosensitizers, thus laying the foundation for safe clinical application. The three dyes showed no significant difference in dark toxicity (the survival rate curves almost overlapped), indicating that the differences in dye structure did not introduce additional dark toxicity, and the core framework (D-π-AD type asymmetric cyanine) possesses good biocompatibility.

[0043] Comparing the "Dark" and "Light" groups, cell survival rate decreased significantly with increasing dye concentration after light exposure (e.g., at 50 μM, the survival rate of the light-exposed group decreased from approximately 90% in the dark group to approximately 20%). This demonstrates that all three nanoparticles can generate reactive oxygen species (ROS) triggered by an 808 nm laser (0.5 W / cm², 5 min), inducing 4T1 cell death, indicating a clear photodynamic therapy (PDT) effect. With increasing dye concentration (0-50 μM), the percentage of live cells in the light-exposed group continuously decreased, while the percentage of dead cells (purple + blue portion) continuously increased, exhibiting concentration-dependent killing. For example, at 5 μM, the survival rate of the light-exposed group with CyTY-2 had decreased to approximately 60%; at 20 μM, it decreased to approximately 30%, indicating that the treatment intensity can be controlled clinically by adjusting the drug concentration.

[0044] Conclusion: This invention achieves a synergistic improvement in reactive oxygen species generation efficiency, photostability, and tumor targeting in photodynamic therapy through structural innovation and synthesis process optimization of asymmetric cyanine dyes. Its dual-type photodynamic synergistic mechanism and convenient detection system provide a new strategy for precision tumor treatment and have significant clinical translational potential.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for synthesizing asymmetric cyanine dye containing dioxaboroline, characterized in that: The synthesis method steps are as follows: in, 。 2. The method for synthesizing a dioxaboroline-containing asymmetric cyanine dye according to claim 1, characterized in that: The dioxaboroline-containing asymmetric cyanine dyes include: 。 3. The method for synthesizing a dioxaboroline-containing asymmetric cyanine dye according to claim 1, characterized in that: The synthesis method steps are as follows: S1. Synthesis of dioxaboroline derivatives: Aromatic compounds, acetic anhydride, and boron trifluoride diethyl ether ester were added to a round-bottom flask. The reaction mixture was heated and stirred. The reaction was terminated by adding the reaction mixture dropwise to cold water. The precipitate was washed three times with water and dried under vacuum. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as the eluent to obtain dioxaboroline derivatives. S2. Synthesis of N-methylonium salt intermediate: N-methylonium salt and diphenylamino Schiff base compound were added to a round-bottom flask, and acetic anhydride was added dropwise. The reaction mixture was heated and stirred under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and precipitated with isopropyl ether to obtain a black solid. The precipitate was washed three times with water and dried under vacuum. The crude product was purified by silica gel column chromatography using dichloromethane / methanol as eluent to obtain the black solid N-methylonium salt intermediate. S3. Synthesis of asymmetric cyanine dyes containing dioxaboroline with different conjugated chain lengths: N-methylhexane salt intermediate and dioxaboroline derivative were added to a round-bottom flask, and triethylamine was added dropwise. The reaction mixture was heated and stirred under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and dried under vacuum. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as eluent to obtain the product, asymmetric cyanine dye containing dioxaboroline.

4. The method for synthesizing a dioxaboroline-containing asymmetric cyanine dye according to claim 3, characterized in that: In S1, the molar ratio of aromatic compound, acetic anhydride, and boron trifluoride diethyl ether ester is 1:5 to 10:1 to 3. The aromatic compound is pyrene, 9,9-dimethylfluorene, 9,9'-spirodifluorene, triphenylamine, phenylcarbazole, carbazole, or 9-silazine. The heating temperature is 50 to 80°C, the stirring time is 0.5 to 3 hours, and the volume ratio of petroleum ether to dichloromethane is 1:

1.

5. The method for synthesizing a dioxaboroline-containing asymmetric cyanine dye according to claim 3, characterized in that: In S2, the molar ratio of N-methylonium salt to diphenylamino Schiff base compound is 1:1~2. The amount of acetic anhydride added is 30 mL per 1 g of N-methylonium salt. The N-methylonium salt is 1,1,2-trimethyl-3-substituted benzoindole iodide, 1,1,2-trimethyl-3-substituted naphthoindole iodide, 1-methyl-2-substituted fluorene iodide, or 1-methyl-2-substituted benzoindole iodide. The compound is a quinoline iodine salt, wherein the N-methylonium salt substituent is methyl, ethyl, n-butyric acid, propylsulfonate ion, propyltrimethylammonium ion, or propyltriphenylphosphonium ion; the diphenylamino Schiff base compound is N,N-diphenylmethylammonium, malondialdehyde diphenylamine hydrochloride, or pentadienal diphenylamine hydrochloride; the heating temperature is 120~140℃, the stirring time is 1~3h, and the volume ratio of petroleum ether to dichloromethane is 1:

1.

6. The method for synthesizing a dioxaboroline-containing asymmetric cyanine dye according to claim 3, characterized in that: In S3, the molar ratio of dioxaboroline derivative, N-methylhexane salt intermediate, and triethylamine is 1:1:1.2, and the volume ratio of petroleum ether to dichloromethane is 1:

1.

7. The method for synthesizing a dioxaboroline-containing asymmetric cyanine dye according to claim 1, characterized in that: The synthesis method steps are as follows: S1. Synthesis of deuterated dioxaboroline derivatives: Aromatic compounds were added to toluene and Pd / C catalyst to a reaction vessel, and D2O was introduced. The reaction was stirred under an inert gas atmosphere to obtain deuterated aromatic compounds. Acetic anhydride and boron trifluoride diethyl ether ester were then added. The reaction mixture was heated and stirred. The reaction was terminated by adding the reaction mixture dropwise to cold water. The precipitate was washed three times with water and dried under vacuum. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as eluent to obtain the solid compound deuterated dioxaboroline derivative. S2. Synthesis of N-methylonium salt intermediate: N-methylonium salt and diphenylamino Schiff base compound were added to a round-bottom flask, and acetic anhydride was added dropwise. The reaction mixture was heated and stirred under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and precipitated with isopropyl ether to obtain a black solid. The precipitate was washed three times with water and dried under vacuum. The crude product was purified by silica gel column chromatography using dichloromethane / methanol as eluent to obtain the black solid N-methylonium salt intermediate. S3. Synthesis of asymmetric cyanine dyes containing dioxaboroline with different conjugated chain lengths: N-methylhexane salt intermediate and deuterated dioxaboroline derivative were added to a round-bottom flask, and triethylamine was added dropwise. The reaction mixture was heated and stirred under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and dried under vacuum. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as eluent to obtain the product, asymmetric cyanine dyes containing dioxaboroline.

8. The method for synthesizing a dioxaboroline-containing asymmetric cyanine dye according to claim 7, characterized in that: The molar ratio of aromatic compound to Pd / C catalyst in S1 is 1:1~2. 100 mL of L2O is added for every 1 g of aromatic compound. The reaction is carried out at 220~240 °C under an inert gas atmosphere with stirring for 12~24 h. The molar ratio of deuterated aromatic compound, acetic anhydride, and boron trifluoride diethyl ether ester is 1:5~10:1~3. The volume ratio of petroleum ether to dichloromethane is 1:

1.

9. The method for synthesizing a dioxaboroline-containing asymmetric cyanine dye according to claim 7, characterized in that: In S2, the molar ratio of N-methylonium salt to diphenylamino Schiff base compound is 1:1~2. The amount of acetic anhydride added is 30 mL per 1 g of N-methylonium salt. The N-methylonium salt is 1,1,2-trimethyl-3-substituted benzoindoline iodide, 1,1,2-trimethyl-3-substituted naphthoindoline iodide, 1-methyl-2-substituted fluorene iodide, or 1-methyl-2-substituted benzoquinoline iodide. The N-methylonium salt substituent is methyl, ethyl, n-butyric acid, propylsulfonate ion, propyltrimethylammonium ion, or propyltriphenylphosphonium ion; the diphenylamino Schiff base compound is N,N-diphenylmethylammonium, malondialdehyde diphenylamine hydrochloride, or pentadienaldehyde diphenylamine hydrochloride; the molar ratio of dioxaboroline derivative, N-methylonium salt intermediate, and triethylamine in S3 is 1:1:1.2, and the volume ratio of petroleum ether to dichloromethane is 1:

1.

10. The application of a dioxaboroline-containing asymmetric cyanine dye according to claim 1, characterized in that: The application of the aforementioned type I / II photodynamic therapy containing dioxaboroline asymmetric cyanine dye.

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