Preparation method and application of nitroreductase response hydrogen sulfide donor with near-infrared fluorescence report function

Abstract

The invention discloses a preparation method and application of a nitroreductase response hydrogen sulfide donor with a near-infrared fluorescence report function, and belongs to the technical field of medicine preparation. The preparation method comprises the following steps: mixing m-nitrophenol, K2CO3 and IR-780 iodide in a first organic solvent, reacting, extracting, and carrying out rotary evaporation to obtain a finished product 1; dissolving the finished product 1 in a second organic solvent, co-heating and refluxing with tin chloride and concentrated hydrochloric acid, adding an alkaline solution, and carrying out suction filtration, extraction, rotary evaporation, sand making and column chromatography to obtain a finished product 2; slowly dripping thiophosgene into the third organic solvent solution of the finished product 2, fully reacting, extracting, performing rotary evaporation, making sand, and passing through a column to obtain a finished product 3; and dissolving 4-nitrobenzyl alcohol, the finished product 3 and NaH in a fourth organic solvent, reacting under an ice bath condition, and carrying out sand making, column passing and rotary evaporation to obtain the hydrogen sulfide donor. The hydrogen sulfide donor provided by the invention is helpful for promoting the clinical application of hydrogen sulfide therapy in the aspects of clinical application and research value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical preparation, and in particular relates to a preparation method and application of a nitroreductase-responsive hydrogen sulfide donor with a near-infrared fluorescence reporter function. Background Art

[0002] Hydrogen sulfide (H2S) is a gaseous signaling molecule with a wide range of physiological functions. Studies have shown that H2S not only protects the cardiovascular system and plays a role in diseases such as hypertension, atherosclerosis, and myocardial ischemia, but also inhibits the progression of lung and liver fibrosis. However, the current lack of efficient and specific methods and drugs to regulate intracellular H2S levels and to track its distribution in the body has severely hindered the clinical application of H2S therapy and the study of its biological effects.

[0003] Nitroreductases are cytoplasmic enzymes that rely on flavin mononucleotide or flavin adenine dinucleotide. Under the action of DADH or DADPH, they can reduce aromatic nitro compounds, thereby catalyzing a variety of metabolic reactions in the human body. They are widely present in various biological systems and are used to maintain homeostasis. Studies have found that nitroreductases are closely related to hypoxic diseases.

[0004] However, the H2S release rate of existing nitroreductase donors is significantly affected by environmental pH, redox state, and substrate concentration, making precise control difficult. In addition, some donors may generate toxic byproducts (such as nitrite and thiosulfate) during metabolism, which may lead to cumulative toxicity with long-term use. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a preparation method and application of a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function to solve the above problems.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a preparation method and application of a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function, comprising the following steps:

[0008] S1. Dissolve m-nitrophenol and potassium carbonate in a first organic solvent and stir for a first time, add IR-780 iodide, and stir at room temperature for a second time to obtain 2-[(E)-2-[3-[(E)-2-(3,3-dimethyl-1-propylindolin-2-yl)vinyl]-2-(3-nitrophenoxy)cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-1-propyl-3H-indole cation;

[0009] S2, dissolving 2-[(E)-2-[3-[(E)-2-(3,3-dimethyl-1-propylindolin-2-yl)vinyl]-2-(3-nitrophenoxy)cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-1-propyl-3H-indole cation in a second organic solvent, mixing, and then heating and reflux with tin chloride and concentrated hydrochloric acid for a second time; adjusting the pH value to neutral with an alkaline solution, filtering and removing precipitated impurities, taking the filtrate and rotary evaporating to remove the second organic solvent in the filtrate, and then extracting with a third organic solvent, taking the lower organic phase, rotary evaporating, sand making, and column column to prepare (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium (HCA);

[0010] S3, dissolving (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium in a third organic solvent, reacting with thiophosgene in an ice-water bath for a third time, extracting with the third organic solvent, rotary evaporation, sand making, and column chromatography to obtain (E)-2-[2-(6-isothiocyanate-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium;

[0011] S4. Dissolve (E)-2-[2-(6-isothiocyanate-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium, 4-nitrobenzyl alcohol and sodium hydride in a fourth organic solvent, add a third organic solvent dropwise to promote the reaction, and then react for a fourth time under ice bath conditions to obtain a nitroreductase-responsive hydrogen sulfide donor with a near-infrared fluorescence reporter function.

[0012] Furthermore, in step S2, the ratio of tin chloride and concentrated hydrochloric acid in mg:mL is 100:119, and the ratio of 2-[(E)-2-[3-[(E)-2-(3,3-dimethyl-1-propylindolin-2-yl)vinyl]-2-(3-nitrophenoxy)cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-1-propyl-3H-indole cation and the second organic solvent in mg:mL is 300:15.

[0013] Furthermore, in step S3, the ratio of (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium, thiophosgene, and the third organic solvent is 300:109:12 on a mg:mg:mL basis.

[0014] Furthermore, in step S4, the ratio of (E)-2-[2-(6-isothiocyanate-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium, 4-nitrobenzyl alcohol, NaH, the fourth organic solvent, and the third organic solvent is 180:93:24:5:0.5 on a mg:mg:mg:mL:mL basis.

[0015] Furthermore, the first organic solvent is acetonitrile; the second organic solvent is methanol; the alkaline solvent is a 10% concentration K2CO3 solution; the third organic solvent is dichloromethane; and the fourth organic solvent is tetrahydrofuran.

[0016] Furthermore, in step S1, the first time is 15 minutes, and the second time is 12 hours; in step S3, the third time is 3 hours; and in step S4, the fourth time is 4 hours.

[0017] Furthermore, the hydrogen sulfide donor is as follows:

[0018]

[0019] Furthermore, a nitroreductase responsive to a hydrogen sulfide donor with a near-infrared fluorescence reporter function is used to prepare a drug for sustained or controlled release of hydrogen sulfide in vitro or in vivo;

[0020] and / or, the nitroreductase-responsive hydrogen sulfide donor is used for in vitro or in vivo fluorescent visualization of hydrogen sulfide-producing drugs;

[0021] and / or, the nitroreductase-responsive hydrogen sulfide donor is used as a drug for inhibiting oxidative stress and protecting the cardiovascular system in vitro or in vivo;

[0022] And / or, use of the nitroreductase-responsive hydrogen sulfide donor as a nitroreductase fluorescent probe in vitro or in vivo.

[0023] Furthermore, a nitroreductase responsive to a hydrogen sulfide donor with near-infrared fluorescence reporter function is used to treat cardiovascular diseases, specifically to treat ischemia-reperfusion injury.

[0024] The beneficial effects of the present invention are:

[0025] 1. The nitroreductase-responsive hydrogen sulfide donor prepared by the present invention is designed based on the structure of a cyanine dye and 4-nitrobenzyl alcohol. First, regarding the control of hydrogen sulfide release, this hydrogen sulfide donor is nitroreductase-responsive. In the presence of nitroreductase, the hydrogen sulfide donor can responsively consume the nitroreductase and release hydrogen sulfide. This property makes it very suitable for the preparation of drugs for sustained or controlled release of hydrogen sulfide in vitro or in vivo, achieving precise control of hydrogen sulfide release.

[0026] 2. In terms of imaging, the hydrogen sulfide donor has a lipophilic cationic structure and can be delivered to the mitochondria of myocardial cells injured by ischemia-reperfusion. Combined with its responsiveness to nitroreductase, in the area of ​​ischemia-reperfusion injury, the hypoxic cellular environment leads to the specific high expression of nitroreductase. The donor molecule is specifically activated to release H2S, accompanied by the release of fluorescent molecules, thereby quantifying the release of hydrogen sulfide molecules and marking the ischemic risk area, accurately diagnosing the scope of damage. At the same time, this fluorescent molecule has strong absorption characteristics in the near-infrared region, with an emission wavelength of 750-830nm, which can penetrate 2-3cm of tissue depth, effectively overcoming the depth limitation of visible light imaging and achieving clear imaging of deep tissue. In the area of ​​ischemia-reperfusion injury, its fluorescence signal is significantly enhanced compared to normal tissue, and the signal-to-noise ratio far exceeds the clinical threshold, which can clearly show the boundary of the injury and provide strong support for accurate diagnosis.

[0027] 3. In terms of quantitative fluorescence detection, the nitroreductase-responsive hydrogen sulfide donors of the present invention exhibit unique fluorescence properties: under specific conditions, they can emit a fluorescent signal, and the fluorescence intensity is quantitatively correlated with the amount of hydrogen sulfide released, thus providing an effective means for quantitative detection of hydrogen sulfide. Based on this property, the hydrogen sulfide donors of the present invention not only have the potential to be developed into fluorescently visualized drugs for hydrogen sulfide production in vitro or in vivo, allowing for intuitive monitoring of the hydrogen sulfide production process, but also demonstrate their value as fluorescent probes for nitroreductases, enabling specific detection and imaging analysis of nitroreductase activity in biological systems.

[0028] 4. In cardiovascular disease, ischemia-reperfusion injury (IRI) is caused by a massive influx of blood into ischemic tissue, and its severity is proportional to the duration of tissue ischemia. Ischemia-reperfusion can lead to the release of ROS and pro-inflammatory mediators in ischemic tissue, inducing inflammatory cell infiltration and causing numerous complications, such as apoptosis and tissue necrosis, and even organ failure. Hydrogen sulfide can mitigate IRI through mechanisms including scavenging various reactive oxygen species, regulating the anti-inflammatory function of macrophages, reducing cell death, alleviating leukocyte aggregation, improving vascular microcirculatory disorders, and interacting with nitric oxide (NO).

[0029] 5. The hydrogen sulfide donor of the present invention, in terms of clinical application and research value, helps promote the clinical application of hydrogen sulfide therapy, provides a feasible method for preparing drugs that slowly release or control the release of hydrogen sulfide in hypoxic cell microenvironments, and can monitor the hydrogen sulfide release process by means of fluorescent quantitative detection; at the same time, the nitroreductase-responsive hydrogen sulfide donor also has the effects of reducing the production of reactive oxygen species, alleviating ischemia-reperfusion injury, and protecting the cardiovascular system, providing new options for the design of related drugs; most importantly, the nitroreductase-responsive hydrogen sulfide donor of the present invention can further promote the study of the biological effect mechanism of hydrogen sulfide, and promote people's in-depth understanding and exploration of the treatment of hydrogen sulfide-related diseases.

[0030] 6. The method for preparing the nitroreductase-responsive hydrogen sulfide donor of the present invention has the advantages of simple preparation process, strong operability and high product purity, and has promotion and application value in the field of biomedicine technology.

[0031] 7. The hydrogen sulfide donor synthesized by the present invention can specifically recognize nitroreductase. By introducing a nitroreductase-specific recognition site to adjust the fluorescence properties of the hydrogen sulfide donor, the hydrogen sulfide donor has the advantages of high sensitivity, high specificity, and real-time monitoring.

[0032] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to make the purpose, technical solutions and beneficial effects of the invention clearer, the present invention is described with the following drawings:

[0034] Figure 1 This is a schematic diagram of the preparation route of the hydrogen sulfide donor of the present invention;

[0035] Figure 2 This is the fluorescence intensity-time change curve after the hydrogen sulfide donor of the present invention reacts with nitroreductase. DETAILED DESCRIPTION

[0036] A method for preparing a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function.

[0037] Example 1

[0038] S1. First, m-nitrophenol, potassium carbonate, and IR-780 iodide are weighed in a mass ratio of 313:700:500, respectively. Then, m-nitrophenol and potassium carbonate are dissolved in 4 mL of acetonitrile and reacted at room temperature for 15 minutes. Then, IR-780 iodide is added and the reaction is continued at room temperature for 12 hours. After the reaction is complete, the mixture is extracted with dichloromethane, and the lower organic phase is removed and rotary evaporated to obtain 2-[(E)-2-[3-[(E)-2-(3,3-dimethyl-1-propylindolin-2-yl)vinyl]-2-(3-nitrophenoxy)cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-1-propyl-3H-indole cation;

[0039] S2. In a two-necked flask under vacuum, tin chloride was dissolved in concentrated hydrochloric acid at a mass ratio of 100:119, and 2-[(E)-2-[3-[(E)-2-(3,3-dimethyl-1-propylindolin-2-yl)vinyl]-2-(3-nitrophenoxy)cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-1-propyl-3H-indole cation was dissolved in 15 mL of methanol at a mass ratio of 300:15. The mixture was heated in an oil bath at 70°C. The alcohol solution was added to a two-necked flask and heated under reflux for 12 hours. After the reaction, the pH was adjusted to neutral using a potassium carbonate solution. Precipitated impurities were removed by suction filtration, and methanol in the filtrate was removed by rotary evaporation. The product was then extracted with dichloromethane, and then filtered through a column using dichloromethane and methanol. After the product was eluted, the product was rotary evaporated to obtain (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium (HCA).

[0040] S3, weigh (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium, thiophosgene, and dichloromethane in a mass ratio of 300:109:12; (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium; Dissolve 2-(6-isothiocyanate-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium in a dichloromethane solution, then add thiophosgene dropwise in an ice-water bath and react for 3 hours, then remove dichloromethane by rotary evaporation and prepare sand, then pass through a column with dichloromethane and methanol, and after the product is eluted, obtain (E)-2-[2-(6-isothiocyanate-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium;

[0041] S4. Dissolve (E)-2-[2-(6-isothiocyanate-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium, 4-nitrobenzyl alcohol and NaH in a mass ratio of 180:93:24 in 5 mL of tetrahydrofuran, and then add 0.5 mL of dichloromethane to promote the reaction. Then mix evenly in an ice bath at -10°C. After the reaction is completed, perform rotary evaporation and sand making, and pass through a column with dichloromethane and methanol. After the product elution is completed, a nitroreductase hydrogen sulfide donor with near-infrared fluorescence reporting function is obtained.

[0042] During the preparation process of the present invention, the reaction between concentrated hydrochloric acid and stannic chloride provides a reducing environment to reduce the product. The pH is adjusted to neutral because the product is unstable under strong acid conditions. Neutral conditions can enhance product stability. Furthermore, impurities may precipitate during the pH adjustment process, which can be removed by filtration. In step S4, tetrahydrofuran is used because the reaction requires an ethereal environment. Adding a third organic solvent to the fourth organic solvent to aid dissolution can accelerate the reaction.

[0043] In this example, the mass of the nitroreductase hydrogen sulfide donor was 0.332 g, and the yield was 61%. The results of H-NMR spectrum detection of the hydrogen sulfide donor were: 1 H NMR(400MHz,MeOD)δ8.79(dd,J=14.9,5.6Hz,1H),8.29(d,J=8.6Hz,1H),7.91(t,J=2.7Hz,1H),7.72–7.6 6(m,2H),7.56(q,J=8.1Hz,2H),7.47(dt,J=8.5,5.8Hz,2H),7.37(s,1H),7.30–7.20(m,2H),7.01(dd,J=8 .3,2.5Hz,1H),6.64(d,J=8.3Hz,1H),6.55(dd,J=14.9,2.9Hz,1H),5.39(s,1H),4.36(t,J=7.4Hz,2H),2. 77(dt,J=28.7,6.1Hz,4H),1.97(dd,J=10.0,4.8Hz,4H),1.39(s,4H),1.28(s,4H),1.10(d,J=7.4Hz,3H).

[0044] The nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporting function prepared by the invention is used in preparing a medicine for sustained or controlled release of hydrogen sulfide in vitro or in vivo.

[0045] or applied to drugs that produce hydrogen sulfide by fluorescence visualization in vitro or in vivo;

[0046] or used in drugs for treating ischemia-reperfusion injury in vivo;

[0047] Or it can be used as a fluorescent probe for nitroreductase in vitro or in vivo.

[0048] Detection and Analysis

[0049] 1. Nitroreductase responsiveness to hydrogen sulfide donors with near-infrared fluorescence reporter

[0050] P1. Prepare 1 mL of the nitroreductase hydrogen sulfide donor solution prepared in Example 1 at a concentration of 10 μM, and add nitroreductase to a final concentration of 100 μM;

[0051] P2. After incubation at 37°C for different time periods (0, 5, 10, 15, 20, 30, and 45 minutes), the fluorescence intensity was measured using an F7000 fluorescence spectrometer. The fluorescence curve of the change in fluorescence intensity over time of the hydrogen sulfide donor in the presence of nitroreductase was recorded to detect whether the hydrogen sulfide donor could effectively react with the nitroreductase and the reaction rate.

[0052] The fluorescence intensity-time curve of the reaction between P3 and nitroreductase is shown in Figure 2. Figure 2 As shown, from Figure 2 The analysis showed that the nitroreductase hydrogen sulfide donor had no obvious fluorescence emission at 660-720nm, but after interacting with nitroreductase, a new peak appeared at F690nm.

[0053] The nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporting function is designed based on the structure of cyanine dyes and 4-nitrobenzyl alcohol.

[0054] In terms of imaging diagnosis and treatment, hydrogen sulfide donors have a lipophilic cationic structure and can be delivered to the mitochondria of myocardial cells injured by ischemia-reperfusion. Combined with the response characteristics of nitroreductase, in the area of ​​ischemia-reperfusion injury, the hypoxic cellular environment leads to the specific high expression of nitroreductase, and the donor molecules are specifically activated to release H2S, accompanied by the release of fluorescent molecules, thereby quantifying the release of hydrogen sulfide molecules and marking the ischemic risk area, accurately diagnosing the scope of damage. At the same time, this fluorescent molecule has strong absorption characteristics in the near-infrared region, with an emission wavelength of 750-830nm, which can penetrate 2-3cm tissue depth, effectively overcoming the depth limitation of visible light imaging and achieving clear imaging of deep tissue. In the area of ​​ischemia-reperfusion injury, its fluorescence signal is significantly enhanced compared to normal tissue, and the signal-to-noise ratio far exceeds the clinical threshold, which can clearly display the boundary of the injury and provide strong support for accurate diagnosis.

[0055] In terms of controlling hydrogen sulfide release, nitroreductase is highly expressed in hypoxic tissues. The nitroreductase hydrogen sulfide donor is nitroreductase-responsive. In a cell microenvironment with high nitroreductase expression, the hydrogen sulfide donor can responsively respond to the nitroreductase and release hydrogen sulfide. This characteristic makes the hydrogen sulfide donor very suitable for the preparation of drugs for sustained or controlled release of hydrogen sulfide for hypoxic tissues, so as to achieve precise control of hydrogen sulfide release.

[0056] In terms of quantitative fluorescence detection, the nitroreductase described herein exhibits unique fluorescence properties in response to hydrogen sulfide donors: under specific conditions, the donor can emit a fluorescent signal, and the fluorescence intensity is quantitatively correlated with the amount of hydrogen sulfide released, thus providing an effective means for quantitative detection of hydrogen sulfide release. Based on this property, the hydrogen sulfide donor described herein not only has the potential to be developed as a fluorescently visualized drug for hydrogen sulfide production in vitro or in vivo, allowing for intuitive monitoring of hydrogen sulfide production, but also demonstrates its value as a fluorescent probe for nitroreductase, enabling specific detection and imaging analysis of nitroreductase activity in biological systems with high nitroreductase expression.

[0057] In cardiovascular disease, ischemia-reperfusion injury (IRI) is caused by massive reperfusion of blood into ischemic tissue following tissue infarction, and its severity is proportional to the duration of tissue ischemia. Ischemia-reperfusion can lead to the release of various reactive oxygen species and proinflammatory mediators from ischemic tissue, inducing inflammatory cell infiltration and causing numerous complications, such as apoptosis and tissue necrosis, and even organ failure. Hydrogen sulfide can mitigate IRI through mechanisms including scavenging various reactive oxygen species, regulating the anti-inflammatory function of macrophages, reducing cell death, alleviating leukocyte aggregation, improving vascular microcirculatory disorders, and interacting with nitric oxide (NO).

[0058] The hydrogen sulfide donor of the present invention, in terms of clinical application and research value, helps promote the clinical application of hydrogen sulfide therapy, provides a feasible method for preparing drugs for sustained or controlled release of hydrogen sulfide, and can monitor the hydrogen sulfide release process through fluorescent quantitative detection; at the same time, the nitroreductase-responsive hydrogen sulfide donor also has the effects of reducing the production of reactive oxygen species, alleviating ischemia-reperfusion injury, and protecting the cardiovascular system, providing a new option for the design of related drugs; most importantly, the nitroreductase-responsive hydrogen sulfide donor of the present invention can further promote research on the biological effect mechanism of hydrogen sulfide, and promote people's in-depth understanding and exploration of the treatment of hydrogen sulfide-related diseases.

[0059] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function, characterized in that: The following steps are involved: S1. Dissolve m-nitrophenol and potassium carbonate in a first organic solvent and stir for a first time, add IR-780 iodide, and stir at room temperature for a second time to obtain 2-[(E)-2-[3-[(E)-2-(3,3-dimethyl-1-propylindolin-2-yl)vinyl]-2-(3-nitrophenoxy)cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-1-propyl-3H-indole cation; S2, dissolving 2-[(E)-2-[3-[(E)-2-(3,3-dimethyl-1-propylindolin-2-yl)vinyl]-2-(3-nitrophenoxy)cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-1-propyl-3H-indole cation in a second organic solvent, mixing, and then heating and reflux with tin chloride and concentrated hydrochloric acid for a second time; adjusting the pH value to neutral with an alkaline solution, filtering and removing precipitated impurities, taking the filtrate and rotary evaporating to remove the second organic solvent in the filtrate, and then extracting with a third organic solvent, taking the lower organic phase, rotary evaporating, sand making, and column column to prepare (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium (HCA); S3, dissolving (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium in a third organic solvent, reacting with thiophosgene in an ice-water bath for a third time, extracting with the third organic solvent, rotary evaporation, sand making, and column chromatography to obtain (E)-2-[2-(6-isothiocyanate-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium; S4. Dissolve (E)-2-[2-(6-isothiocyanate-2,3-dihydro-1H-xanthene-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium, 4-nitrobenzyl alcohol and sodium hydride in a fourth organic solvent, add a third organic solvent dropwise to promote the reaction, and then react for a fourth time under ice bath conditions to obtain a nitroreductase-responsive hydrogen sulfide donor with a near-infrared fluorescence reporter function.

2. The method for preparing a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function according to claim 1, characterized in that: In step S2, the ratio of tin chloride and concentrated hydrochloric acid is 100:119 in mg:mL, and the ratio of 2-[(E)-2-[3-[(E)-2-(3,3-dimethyl-1-propylindolin-2-yl)vinyl]-2-(3-nitrophenoxy)cyclohex-1-en-1-yl]vinyl]-3,3-dimethyl-1-propyl-3H-indole cation and the second organic solvent is 300:15 in mg:mL.

3. The method for preparing a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function according to claim 2, characterized in that: In step S3, (E)-2-[2-(6-amino-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium, thiophosgene, and the third organic solvent are calculated on a mg:mg:mL basis in a ratio of 300:109:

12.

4. The method for preparing a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function according to claim 4, characterized in that: In step S4, (E)-2-[2-(6-isothiocyanate-2,3-dihydro-1H-xanthen-4-yl)vinyl]-3,3-dimethyl-1-propyl-3H-indol-1-ium, 4-nitrobenzyl alcohol, NaH, the fourth organic solvent, and the third organic solvent are calculated on a mg:mg:mg:mL:mL basis in a ratio of 180:93:24:5:0.

5.

5. The method for preparing a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function according to claim 3, characterized in that: The first organic solvent is acetonitrile; the second organic solvent is methanol; the alkaline solvent is a 10% concentration K2CO3 solution; the third organic solvent is dichloromethane; and the fourth organic solvent is tetrahydrofuran.

6. The method for preparing a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function according to claim 4, characterized in that: In the step S1, the first time is 15 minutes and the second time is 12 hours; in the step S3, the third time is 3 hours; and in the step S4, the fourth time is 4 hours.

7. The method for preparing a nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function according to claim 1, characterized in that: The schematic structural diagram of the hydrogen sulfide donor to which the nitroreductase responds is shown below:

8. A nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function according to any one of claims 1 to 7, for use in preparing a drug for sustained or controlled release of hydrogen sulfide in vitro or in vivo; and / or, the nitroreductase-responsive hydrogen sulfide donor is used for in vitro or in vivo fluorescent visualization of hydrogen sulfide-producing drugs; and / or, the nitroreductase-responsive hydrogen sulfide donor is used as a drug for inhibiting oxidative stress and protecting the cardiovascular system in vitro or in vivo; And / or, use of the nitroreductase-responsive hydrogen sulfide donor as a nitroreductase fluorescent probe in vitro or in vivo.

9. A nitroreductase-responsive hydrogen sulfide donor with near-infrared fluorescence reporter function according to any one of claims 1 to 6, for use in treating cardiovascular diseases, particularly ischemia-reperfusion injury.

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