A hydrogen sulfide donor based on isothiocyanate structure, and a preparation method and application thereof

Abstract

The present application relates to a kind of hydrogen sulfide donors based on isothiocyanate structure and its preparation method and application, belong to biochemical technical field.The chemical structure formula of the hydrogen sulfide donor is as shown below.The hydrogen sulfide donor described in the present application is with tricyano furan as fluorescent mother nucleus, is constructed by converting its amino into strong electron-withdrawing isothiocyanate group, it can specifically recognize cysteine and release hydrogen sulfide, while accompanied by significant fluorescence enhancement, realize the visual monitoring of release process, solve the existing hydrogen sulfide donor release uncontrollable, single function and the technical problem that release process is difficult to real-time monitoring, it has important significance in the visual tracing of hydrogen sulfide release and the diagnosis and treatment research of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology and relates to a hydrogen sulfide donor based on an isothiocyanate structure, its preparation method and application. Background Technology

[0002] Hydrogen sulfide (H2S), as the third key endogenous gaseous signaling molecule in the human body, plays an irreplaceable role in maintaining health. It is widely involved in important physiological processes such as cardiovascular tension regulation, neuroprotection, anti-inflammatory and anti-apoptotic effects, and maintaining redox homeostasis through protein S-hydrogen sulfide modification. Clinical studies have confirmed that abnormal fluctuations in H2S levels in vivo are closely related to the development of myocardial ischemia, hypertension, neurodegenerative diseases, and various inflammatory conditions. Therefore, developing chemical biological tools that can precisely intervene in and regulate H2S levels in vivo is of paramount importance for exploring its pathophysiological mechanisms and developing related targeted drugs.

[0003] Currently, exogenous H2S donors have become a core research tool in this field, but existing technologies still have significant limitations. Traditional inorganic donors release H2S too rapidly and are extremely difficult to control, easily leading to excessively high local concentrations and cytotoxicity. While existing organic donors have achieved sustained release, most are single-function, belonging to the "silent" donor category. Due to the exceptionally complex in vivo environment, the subcellular distribution, release dynamics, and precise concentration of these donors after entering the body are difficult to monitor in real time. This "black box effect" severely restricts the accurate assessment of their pharmacological effects. Therefore, there is an urgent need to construct a "visible" multifunctional donor that can be intelligently triggered by a specific endogenous substrate (such as cysteine) and accompanied by significant changes in fluorescence signal during H2S release. This would not only enable controlled release and precise in-situ monitoring of H2S but also greatly promote its application in the field of integrated diagnosis and treatment. Summary of the Invention

[0004] To address the technical problems of uncontrollable release, limited functionality, and difficulty in real-time monitoring of the release process in existing hydrogen sulfide donors, this invention provides a hydrogen sulfide donor based on an isothiocyanate structure (TCF-NCS), its preparation method, and its applications. The hydrogen sulfide donor of this invention uses tricyanofuran (TCF) as the fluorescent core, constructed by converting its amino group into a strongly electron-withdrawing isothiocyanate group (-N=C=S). It can release H2S specifically triggered by endogenous cysteine, accompanied by significant fluorescence enhancement, enabling visualized in-situ monitoring of the H2S release process.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A hydrogen sulfide donor based on an isothiocyanate structure, the chemical structural formula of which is shown below: .

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned hydrogen sulfide donor based on the isothiocyanate structure, comprising the following steps: S1: 3-Hydroxy-3-methyl-2-butanone and malononitrile were dissolved in anhydrous ethanol. Using sodium ethoxide as a base, the mixture was first stirred at room temperature, followed by reflux. After the reaction was completed, the mixture was cooled and recrystallized at low temperature. The crystals were then filtered, washed, and dried to obtain a white solid intermediate 1. ; S2: p-Aminobenzaldehyde and intermediate 1 were dissolved in a mixed solvent of tetrahydrofuran (THF) and ethanol (EtOH), and ammonium acetate was added. The reaction was carried out at room temperature under an inert gas atmosphere. After the reaction was complete, the intermediate was purified by vacuum distillation, extraction, drying, and grinding to obtain a purple solid intermediate 2. ; S3: Intermediate 2 was dissolved in anhydrous tetrahydrofuran, and triethylamine (TEA) was added. After stirring in an ice bath, an anhydrous tetrahydrofuran solution of a thiocarbonylating reagent was slowly added dropwise. The reaction was then carried out at room temperature under an inert gas atmosphere. After the reaction was complete, the product was extracted, dried, and purified by column chromatography to obtain an orange-red solid target product. .

[0008] In the above technical solution, in step S1, the molar ratio of 3-hydroxy-3-methyl-2-butanone, malononitrile, and sodium ethoxide is 1:(2.0~2.1):(0.14~0.15).

[0009] In the above technical solution, in step S1, 3-hydroxy-3-methyl-2-butanone and malononitrile are dissolved in anhydrous ethanol, and sodium ethoxide is used as a base. The mixture is first stirred at room temperature for 1-2 h, and then heated under reflux at 80-90 °C for 1-2 h. After the reaction is completed, the mixture is cooled and recrystallized at -20-0 °C. The mixture is then filtered, washed, and dried to obtain a white solid intermediate 1. The solvents for recrystallization and washing are both ethanol.

[0010] In the above technical solution, in step S2, the molar ratio of p-aminobenzaldehyde, intermediate 1 and ammonium acetate is 1:(1.1~1.2):(1.1~1.2).

[0011] In the above technical solution, in step S2, the volume ratio of tetrahydrofuran to ethanol in the mixed solvent is 4:1.

[0012] In the above technical solution, in step S2, p-aminobenzaldehyde and intermediate 1 are dissolved in a mixed solvent of tetrahydrofuran and ethanol, ammonium acetate is added, and the reaction is carried out at room temperature for 16-24 h under nitrogen protection. After the reaction is complete, the crude product is obtained by vacuum distillation, extraction with ethyl acetate, and drying. Subsequently, the crude product is dissolved in anhydrous methanol, ground with the aid of an ultrasonic cleaner, filtered and dried to obtain purple solid intermediate 2.

[0013] In the above technical solution, in step S3, the molar ratio of intermediate 2, triethylamine and thiocarbonylating agent is 1:(5~6):(2~3).

[0014] In the above technical solution, in step S3, intermediate 2 is dissolved in anhydrous tetrahydrofuran, triethylamine is added, and the mixture is stirred for 5-10 min under ice bath conditions. Then, an anhydrous tetrahydrofuran solution of 1,1'-thiocarbonyldiimidazole (TCDI) is added dropwise at a uniform rate over 5 min. The reaction is then carried out at room temperature for 16-24 h under nitrogen protection. After the reaction is complete, the product is extracted with dichloromethane, dried, and purified by column chromatography to obtain an orange-red solid target product.

[0015] Another object of the present invention is to provide the application of the above-described hydrogen sulfide donor based on the isothiocyanate structure in the visualization of hydrogen sulfide release.

[0016] Another object of the present invention is to provide the application of the above-described hydrogen sulfide donor based on the isothiocyanate structure in the preparation of products for the qualitative detection of endogenous cysteine ​​in cells.

[0017] This invention relates to the application of a fluorescent hydrogen sulfide donor with an isothiocyanate structure for the qualitative detection of endogenous cysteine ​​in cells and the visualization of hydrogen sulfide release levels in vitro or in vivo. This invention is suitable for in vitro fluorescence imaging assays and also for in vivo pharmacological mechanism studies. It features a simple preparation method, significant spectral changes, good specificity, and low cytotoxicity. It can be used simultaneously for fluorescence imaging of cysteine ​​and as a tool for long-acting sustained-release hydrogen sulfide.

[0018] The beneficial effects of this invention are as follows: The fluorescent hydrogen sulfide donor obtained by this invention has excellent optical properties, exhibiting a large Stokes shift of 90 nm and red emission of 610 nm, effectively avoiding interference from background fluorescence in biological tissues. Furthermore, it shows significant spectral changes, good specificity, low cytotoxicity, and can stably release hydrogen sulfide for up to 8 hours, making it suitable for both visual tracing of hydrogen sulfide release and research on the diagnosis and treatment of related diseases. Moreover, its preparation method is convenient, the raw materials are readily available, and it is easy to produce. Attached Figure Description

[0019] Figure 1 The fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1 of this invention 1H NMR spectrum; Figure 2 The fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1 of this invention 13 C NMR spectrum; Figure 3 The image shows the HRMS pattern of the fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1 of this invention. Figure 4 The UV absorption and fluorescence emission spectra of the fluorescent hydrogen sulfide donor TCF-NCS before and after the reaction with cysteine ​​in Example 2 of the present invention are shown. Figure 5 (a) is the fluorescence titration curve of cysteine ​​by the fluorescent hydrogen sulfide donor TCF-NCS in Example 3 of the present invention. Figure 5 (b) is a fluorescence titration graph and linear fitting graph of the fluorescent hydrogen sulfide donor TCF-NCS in Example 3 of the present invention; Figure 6 (ab) is a fluorescence response kinetics test diagram of the fluorescent hydrogen sulfide donor TCF-NCS in Example 4 of the present invention. Figure 6 (c) is a photostogram of the fluorescent hydrogen sulfide donor TCF-NCS in Example 4 of the present invention. Figure 6 (de) is a bio-media selectivity diagram of the fluorescent hydrogen sulfide donor TCF-NCS in Example 5 of the present invention, and 6(f) is a pH stability test diagram of the fluorescent hydrogen sulfide donor TCF-NCS in Example 5 of the present invention. Figure 7 (a) is a graph showing the results of H2S release measured by the methylene blue method using the fluorescent hydrogen sulfide donor TCF-NCS in Example 6 of the present invention. Figure 7 (b) is a diagram showing the H2S release results verified using the AzMC probe in Example 6 of this invention; Figure 8 (a) is a graph showing the cytotoxicity test results of the fluorescent hydrogen sulfide donor TCF-NCS on HaCat cells in Example 7 of the present invention. Figure 8 (b) is a fluorescence image of dual-channel visualization imaging of live cells in Example 7 of the present invention. Detailed Implementation

[0020] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0021] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0022] The synthetic route of the fluorescent hydrogen sulfide donor TCF-NCS described in the following examples is as follows: .

[0023] Example 1 A method for preparing a fluorescent hydrogen sulfide donor based on an isothiocyanate structure includes the following steps: (1) Preparation of compound TCF (intermediate 1) 3-Hydroxy-3-methyl-2-butanone (900 mg, 8.8 mmol) and malononitrile (1.2 g, 18.1 mmol) were dissolved in 8 mL of anhydrous ethanol, and sodium ethoxide (90 mg, 1.3 mmol) was added. The mixture was stirred at room temperature for 1 h. The reaction mixture was then refluxed at 80 ºC for 1 h and cooled to room temperature. The reaction mixture was then placed at -20 ºC. After a large amount of solid precipitated, the precipitate was collected by filtration and washed with small amounts of cold ethanol several times to obtain a white solid TCF (950 mg, 45%). 1 H NMR (600 MHz, DMSO-) d 6) δ : 2.37 (s, 3H), 1.60 (s, 6H). (2) Preparation of compound TCF-NH2 (intermediate 2) p-Aminobenzaldehyde (121 mg, 1 mmol) was dissolved in 3.75 mL of a THF / EtOH mixture (4:1 v / v), followed by the addition of TCF (219 mg, 1.1 mmol) and ammonium acetate (85 mg, 1.1 mmol). The reaction mixture was then incubated overnight at room temperature under nitrogen protection. After the reaction was monitored by silica gel thin-layer chromatography to ensure complete reaction, the solvent was evaporated under reduced pressure, and the mixture was redissolved in ethyl acetate. The reaction mixture was washed 2-3 times with saturated brine, and the ethyl acetate layer was collected and dried over anhydrous sodium sulfate. The filtrate was filtered and evaporated under reduced pressure. The crude product was dissolved in as little anhydrous methanol as possible and thoroughly ground using an ultrasonic cleaner. After filtration and drying, a purple solid, TCF-NH2 (170 mg, 56%), was obtained. 1 H NMR (600 MHz, DMSO- d 6) δ 7.88 (d, J = 15.8 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 6.85 (s, 2H), 6.82 (d, J =15.8 Hz, 1H), 6.66 (d, J = 8.6 Hz, 2H), 1.74 (s, 6H). 13 C NMR (151 MHz, DMSO-d 6) δ : 177.90, 176.14, 155.80, 150.39, 134.11, 122.49, 114.68, 114.03, 113.19, 112.57, 108.16, 98.59, 92.00, 51.18, 26.13. (3) Preparation of compound TCF-NCS (target product) TCF-NH2 (60 mg, 0.2 mmol) was dissolved in 3 mL of anhydrous THF, then TEA (165 μL, 1.2 mmol) was added and stirred for 5 min in an ice bath. Next, 2 mL of TCDI (70.8 mg, 0.4 mmol) in anhydrous THF was added slowly using a dropping funnel under nitrogen protection. The reaction was allowed to proceed at room temperature for 24 h, and the reaction was monitored for completeness by silica gel thin-layer chromatography. The solvent was evaporated under reduced pressure, and the mixture was extracted three times with dichloromethane. The dichloromethane layers were combined and dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated. The crude product was further purified by silica gel column chromatography (eluent:PE:EA = 6:1-2:1) to obtain an orange-red solid TCF-NCS (18 mg, 26%). 1 H NMR (600 MHz, DMSO- d 6) δ 8.01 (d, J = 8.6 Hz, 2H), 7.90(d, J = 16.5 Hz, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 16.5 Hz, 1H), 1.79 (s, 6H). 13 C NMR (151 MHz, DMSO- d 6) δ : 177.49, 175.16, 145.80, 134.17, 133.30,131.32, 130.12, 127.29, 116.93, 113.08, 112.25, 111.19, 100.70, 99.97, 55.24,25.52. HRMS (ESI): m / z calcd for C 19 H 12 N4NaOS + [M + Na] + 367.0624. Found 367.0631. Example 2 Evaluation of the photochemical properties of the fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1: In this embodiment, an appropriate amount of donor TCF-NCS was weighed and prepared into a 10 mM stock solution using DMSO. An appropriate amount of cysteine ​​(Cys) was weighed and prepared into a 50 mM stock solution using deionized water. Unless otherwise specified, this concentration of stock solution will be used in subsequent embodiments. An appropriate amount of donor stock solution was diluted to 10 μM with PBS buffer solution (pH = 7.4, 10 mM, 20% DMSO) to prepare two aliquots, one as a blank solution and the other with Cys stock solution added to bring the final concentration to 100 μM. The solutions were then incubated at 37 ºC in the dark for 1 h, and the UV absorption and fluorescence emission spectra of the two solutions were measured.

[0024] Test results are as follows Figure 4 As shown, TCF-NCS exhibits weak autofluorescence; after the addition of cysteine ​​(Cys), the system shows a significant ultraviolet absorption peak at 520 nm and a significant red fluorescence emission signal at 610 nm, with a Stokes shift of 90 nm. Simultaneously, the test solution can be observed to change from pale yellow to red with the naked eye.

[0025] Example 3 Optical response study of the fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1: In this embodiment, a PBS buffer solution (pH = 7.4, 10 mM, 20% DMSO) with a final concentration of 10 μM of donor TCF-NCS was prepared, followed by the addition of Cys stock solution to achieve concentrations of 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 μM. All test systems at different concentrations were incubated at 37 ºC in the dark for 1 h, and then fluorescence emission spectra were measured. Each experimental concentration was measured in triplicate. Detection limit calculation: Line graphs were plotted showing the fluorescence intensity of TCF-NCS at 576 nm and 610 nm as a function of Cys concentration. The linear concentration range of Cys and fluorescence intensity was statistically analyzed, and the linear regression equation was calculated. The detection limit was then calculated using the formula LOD = 3σ / k, where k is the slope of the linear regression equation, and σ is the standard deviation calculated after testing the fluorescence intensity of the blank solution 11 times.

[0026] Cysteine ​​(Cys) at different concentrations (0–500 μM) were added to a 10 μM TCF-NCS test solution. After reacting for 1 h, the fluorescence intensity was measured. The test results are as follows: Figure 5As shown in the figure. The results indicate that when the Cys concentration reaches 300 μM, the fluorescence intensity of the system tends to saturate, increasing by approximately 13 times compared to before the reaction. Within the Cys concentration range of 0–200 μM, the fluorescence intensity of the system exhibits a good linear relationship with the Cys concentration, and the linear regression equation is y = 5.644 × [Cys] + 180.332 (R²). 2 = 0.992). The calculated detection limit (LOD = 3σ / k) of this donor for Cys is 0.922 μM, exhibiting extremely high response sensitivity.

[0027] Example 4 Evaluation of fluorescence kinetics and photostability of the fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1: In this embodiment, a PBS buffer solution (pH = 7.4, 10 mM, 20% DMSO) with a final concentration of 10 μM for the donor TCF-NCS was prepared, and the Cys concentration was fixed at 100 μM. Fluorescence emission spectra were measured for different incubation periods. Each incubation period was measured three times in parallel.

[0028] Photostability experiment: In the fluorescence kinetics experiment of Bcy-NCS and TCF-NCS donors, after the fluorescence intensity reached the plateau period, the fluorescence stability within 1 h was tested using the time-scan function of a fluorescence spectrophotometer.

[0029] Test results are as follows Figure 6 As shown in (a~c), it can be seen that after adding 10 equivalents of cysteine ​​(Cys), the fluorescence intensity of the system increased rapidly within 0-60 min, then the fluorescence enhancement trend gradually slowed down, and reached a stable plateau after 5 h of reaction. Meanwhile, the fluorescence intensity of the blank control group without Cys showed no significant change, indicating that TCF-NCS has good stability in the test solution.

[0030] Example 5 Biomedia selectivity and pH stability of the fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1: In this embodiment, 17 common biological mediators were selected, including: cations (Mg) 2+ Fe 2+ and Fe 3+ ), anion (S2O3) 2- SO3 2- SO4 2- and NO 2-The biological media were prepared as follows: amino acids (Trp, Tyr, Phe, Gly, Lys, and Ser), biothiols (Hcy and GSH), and Na2S·9H2O and H2O2. All biological media were prepared into 50 mM stock solutions for later use. Sixteen aliquots of 10 μM TCF-NCS in PBS buffer (pH = 7.4, 10 mM, 20% DMSO) were prepared, and different biological media stock solutions were added to achieve a concentration of 300 μM. Fluorescence emission spectra were measured after 1 h of incubation. Measurements were repeated three times for each biological media, and corresponding curves and bar charts were plotted.

[0031] In this embodiment, a PBS buffer solution (pH = 7.4, 10 mM, 20% DMSO) with a final concentration of 10 μM TCF-NCS was prepared and divided into 5 equal portions. The pH of four of these portions was adjusted to 5.5, 6.5, 8.5, and 9.5 using a pH meter. Cys stock solution was added to each TCF-NCS test solution to achieve a concentration of 300 μM. After incubation for 1 h, the fluorescence intensity at 610 nm was measured. Measurements were repeated three times under different pH conditions, and the corresponding scatter plots were plotted.

[0032] Test results are as follows Figure 6 As shown in (d~f), regarding the selectivity of biological media, only Cys can induce a significant enhancement of red fluorescence in TCF-NCS, while other sulfur-containing media such as GSH and Hcy, as well as common ions, cannot excite fluorescence signals, indicating that this donor has a very high specific recognition ability for Cys. Regarding pH stability, without the addition of Cys, the fluorescence intensity under all pH conditions is at a very low level, indicating that the donor remains stable within the test range. After the addition of Cys, TCF-NCS can produce significant fluorescence response signals within the physiological pH range of 6.5~8.5, indicating that it is well-suited for complex physiological environments.

[0033] Example 6 H2S release study of the fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1: In this embodiment, a standard curve for H2S concentration was first established and fitted by measuring the absorbance at 670 nm after reacting different concentrations of Na2S solution with MB detection solution at room temperature in the dark for 30 min. Subsequently, TCF-NCS (10 μM) and cysteine ​​(100 μM) were placed in PBS buffer (pH = 7.4, 10 mM, 20% DMSO) and incubated at 37 ºC. Samples were taken at 0.5, 1, 2, 4, 6, and 8 h, and MB detection solution was added, reacting at room temperature in the dark for 30 min. The absorbance at 670 nm was then measured. The absorbance at each time point was substituted into the standard curve to calculate the H2S release concentration of TCF-NCS at different incubation times.

[0034] The ability of TCF-NCS to release H2S was then further verified using the specific fluorescent probe AzMC. The donor (10 μM) and cysteine ​​(Cys, 100 μM) were pre-incubated in PBS buffer (pH 7.4, 10 mM, containing 20% ​​DMSO) at 37 ºC for 2 h, followed by the addition of the AzMC probe (10 μM), and the reaction was carried out in the dark for 30 min. Three parallel control groups were also set up: AzMC only, AzMC + Cys, and AzMC + donor, and were treated in the same dark-protected manner. After the reaction, the fluorescence emission spectra of each system in the range of 400–500 nm were measured using 350 nm as the excitation wavelength.

[0035] Test results are as follows Figure 7 As shown, the concentration of H2S in the system rapidly reaches its peak within 2 hours of reaction and remains stable over the following 6 hours. This result confirms that TCF-NCS possesses excellent long-term sustained-release performance for H2S. Fluorescence spectroscopy results show that the fluorescence intensity of the probe AzMC only significantly increases in reaction systems where TCF-NCS and Cys coexist. This result further corroborates that TCF-NCS has the function of specifically responding to Cys and effectively releasing H2S.

[0036] Example 7 Cytotoxicity and live-cell imaging study of the fluorescent hydrogen sulfide donor TCF-NCS obtained in Example 1: In this embodiment, HaCat cells in the logarithmic growth phase are used at 10 4Cells were seeded at a density of 1 cell / well in 96-well plates and cultured overnight at 37 ºC, 5% CO2 (using DMEM medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin) until adherence. The original medium was discarded, and DMEM medium containing different concentrations of donor TCF-NCS was added, followed by incubation for another 12 h. After treatment, the old medium was discarded, and the cells were washed with PBS buffer. 100 μL of CCK-8 assay solution diluted 10-fold with DMEM was added to each well, and the cells were incubated at 37 ºC, 5% CO2 for another 2–4 h. Finally, the absorbance at 450 nm was read using a microplate reader. Each concentration group was configured with four replicates, and the experiment was repeated three times in parallel to calculate cell viability.

[0037] HaCat cells in the logarithmic growth phase were subjected to 10 4 Cells were seeded at a density of cells / well in 96-well plates and cultured overnight at 37 ºC and 5% CO2 until adherence. The old culture medium was discarded, and the cells were divided into two groups for control treatment: (1) Endogenous response group: DMEM medium containing only donor TCF-NCS (10 μM), only fluorescent probe AzMC (10 μM), and both TCF-NCS and AzMC were added to the cells. (2) Inhibition and exogenous verification group: The cells were pretreated with DMEM containing the inhibitor N-ethylmaleimide (NEM, 500 μM) for 1 h, discarded, and washed with PBS buffer. Then, DMEM medium containing TCF-NCS+AzMC and TCF-NCS+AzMC+Cys (300 μM) were added to the cells. In all the above treatments, the incubation time for the donor was 1 h, Cys was incubated for 30 min, and AzMC was incubated for 30 min. After incubation, each group was washed 2-3 times with PBS buffer. Finally, imaging was performed using an inverted fluorescence microscope: bright-field images of the cells were recorded, and red channel fluorescence images were collected using a green excitation block, while blue channel fluorescence images were collected using a UV excitation block.

[0038] Test results are as follows Figure 8As shown, the donor TCF-NCS exhibits virtually no cytotoxicity within the 10 μM concentration range and maintains extremely high cell viability even at a 20 μM concentration. This result confirms the excellent biocompatibility of TCF-NCS, fully meeting the requirements for in situ fluorescence imaging in live cell systems. Imaging results show that when TCF-NCS is co-incubated with AzMC, significant red (representing maternal recovery) and blue (representing H2S release) dual-channel fluorescence is simultaneously activated within the cells. If endogenous Cys is consumed beforehand using the thiol inhibitor NEM, both channels of fluorescence are suppressed; after supplementing with exogenous Cys, the red and blue fluorescence are significantly restored. This result confirms that the donor TCF-NCS can specifically respond to endogenous Cys in live cells and achieve dual-channel visualization monitoring of in situ H2S release.

[0039] In summary, the fluorescent hydrogen sulfide donor of this invention has a large Stokes shift of 90 nm and red fluorescence emission of 610 nm, which can effectively eliminate the background interference of autofluorescence in biological tissues and is suitable for deep in vivo imaging. At the same time, the donor has high specificity, only responding specifically to cysteine ​​(Cys) and is not affected by cross-interference from other endogenous biological thiols such as GSH and Hcy. In addition, the donor has both long-acting sustained release and visualization functions, and can achieve continuous and stable release of hydrogen sulfide (H2S) in living cells for up to 8 hours. Moreover, the release process is synchronized with the activation of red fluorescence, successfully realizing the visualization monitoring of in-situ sustained drug release, which has great application potential in the field of integrated diagnosis and treatment.

Claims

1. A hydrogen sulfide donor based on an isothiocyanate structure, characterized in that: The chemical structural formula of the hydrogen sulfide donor is shown below: 。 2. The method for preparing the hydrogen sulfide donor based on the isothiocyanate structure according to claim 1, characterized in that: Includes the following steps: S1: 3-Hydroxy-3-methyl-2-butanone and malononitrile were dissolved in anhydrous ethanol. Using sodium ethoxide as a base, the mixture was first stirred at room temperature, followed by reflux. After the reaction was completed, the mixture was cooled and recrystallized at low temperature. The crystals were then filtered, washed, and dried to obtain a white solid intermediate 1. ; S2: p-Aminobenzaldehyde and intermediate 1 were dissolved in a mixed solvent of tetrahydrofuran and ethanol, and ammonium acetate was added. The reaction was carried out at room temperature under an inert gas atmosphere. After the reaction was complete, the intermediate was purified by vacuum distillation, extraction, drying, and grinding to obtain a purple solid intermediate 2. ; S3: Intermediate 2 was dissolved in anhydrous tetrahydrofuran, triethylamine was added, and the mixture was stirred in an ice bath. Then, an anhydrous tetrahydrofuran solution of a thiocarbonylating reagent was slowly added dropwise. The reaction was then carried out at room temperature under an inert gas atmosphere. After the reaction was complete, the product was extracted, dried, and purified by column chromatography to obtain an orange-red solid target product. .

3. The preparation method according to claim 2, characterized in that: In S1, the molar ratio of 3-hydroxy-3-methyl-2-butanone, malononitrile, and sodium ethoxide is 1:(2.0~2.1):(0.14~0.15).

4. The preparation method according to claim 2, characterized in that: In step S1, 3-hydroxy-3-methyl-2-butanone and malononitrile are dissolved in anhydrous ethanol, and sodium ethoxide is used as a base. The mixture is first stirred at room temperature for 1-2 h, and then heated under reflux at 80-90 °C for 1-2 h. After the reaction is completed, the mixture is cooled and recrystallized at -20-0 °C. The product is then filtered, washed, and dried to obtain a white solid intermediate 1. The solvents used for recrystallization and washing are both ethanol.

5. The preparation method according to claim 2, characterized in that: In S2, the molar ratio of p-aminobenzaldehyde, intermediate 1 and ammonium acetate is 1:(1.1~1.2):(1.1~1.2); the volume ratio of tetrahydrofuran to ethanol in the mixed solvent is 4:

1.

6. The preparation method according to claim 2, characterized in that: In step S2, p-aminobenzaldehyde and intermediate 1 are dissolved in a mixed solvent of tetrahydrofuran and ethanol, ammonium acetate is added, and the reaction is carried out at room temperature for 16-24 h under nitrogen protection. After the reaction is complete, the crude product is obtained by vacuum distillation, extraction with ethyl acetate, and drying. The crude product is then dissolved in anhydrous methanol, ground with the aid of an ultrasonic cleaner, filtered and dried to obtain purple solid intermediate 2.

7. The preparation method according to claim 2, characterized in that: In S3, the molar ratio of intermediate 2, triethylamine and thiocarbonylating agent is 1:(5~6):(2~3).

8. The preparation method according to claim 2, characterized in that: In step S3, intermediate 2 is dissolved in anhydrous tetrahydrofuran, triethylamine is added, and the mixture is stirred in an ice bath for 5-10 min. Then, an anhydrous tetrahydrofuran solution of 1,1'-thiocarbonyldiimidazole is added dropwise at a uniform rate over 5 min. The reaction is then carried out at room temperature for 16-24 h under nitrogen protection. After the reaction is complete, the product is extracted with dichloromethane, dried, and purified by column chromatography to obtain an orange-red solid target product.

9. The application of the hydrogen sulfide donor based on the isothiocyanate structure as described in claim 1 in the visualization of hydrogen sulfide release.

10. The use of the hydrogen sulfide donor based on the isothiocyanate structure of claim 1 in the preparation of products for the qualitative detection of endogenous cysteine ​​in cells.

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