A single-photon and two-photon bimodal molecular probe for high-sensitivity detection of endogenous H2S in cells and tissues

Abstract

The application belongs to the technical field of organic synthesis, and relates to a kind of single two-photon double-mode excitation high-sensitivity detection cell and tissue endogenous H2S molecular probe. The application relies on the skeleton of the fluorine cyanin as near-infrared dye, introduces naphthalene derivative with two-photon absorption characteristics, and aromatic nitroso structure, and synthesizes two two-photon near-infrared fluorescent molecular probes. The molecular probe provided by the application has the ability to combine with H2S, can specifically recognize H2S under physiological and pathological environment, realizes single-molecule double-mode excitation and separate two-pathway fluorescence imaging visualization monitoring of endogenous H2S fluctuation level in organism.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology and relates to a molecular probe excited by single and two photons in two modes and its application. Background Technology

[0002] Hemoglobin (H2S) is an important gaseous signaling molecule that can induce vasodilation, mediate neurotransmission, lower blood pressure, and regulate insulin release. In myocardial ischemia-reperfusion injury, H2S exhibits a potent cardioprotective effect. Furthermore, H2S plays a crucial role in the treatment of Alzheimer's disease, stroke, and various cancers and liver diseases. However, many fundamental questions regarding the physiological concentration and mechanism of action of H2S remain unanswered. Therefore, monitoring changes in H2S levels in biological systems is essential for a deeper understanding of the complex manifestations of H2S under physiological and pathological conditions.

[0003] Traditional techniques for analyzing H2S concentrations, such as colorimetric detection, electrochemical methods, and chromatography, typically require complex sample preparation and cell or tissue destruction, making them unsuitable for detection in living systems. Currently, while numerous fluorescent probes have been reported for in-situ H2S detection and imaging, probes capable of simultaneously detecting endogenous H2S using both single- and two-photon excitation modes have not yet been reported. Therefore, the development of such dual-mode excitation probes remains a crucial challenge. Summary of the Invention

[0004] This invention aims to address the problems and deficiencies existing in the prior art. This invention redesigns the triggering structure of the H2S molecular probe, thereby achieving single-molecule dual-mode excitation and dual-pathway fluorescence imaging for visual monitoring of fluctuations in endogenous H2S levels in biological organisms.

[0005] On the one hand, the present invention relates to compounds having structures as shown in formula (I) and / or as indicated.

[0006]

[0007] On the other hand, the present invention provides a method for preparing the above-mentioned compound, comprising:

[0008]

[0009] Furthermore, the present invention provides methods for preparing compounds of formula (I) and formula (II):

[0010] Specifically, the preparation method of the compound shown in formula (I) includes: reacting p-nitrobenzyl bromide and 2,3,3-trimethylindole to obtain the compound shown in formula (1-1-1); reacting N,N-dimethylformamide, dichloromethane, and phosphorus oxychloride to obtain the compound shown in formula (1-1-2); reacting the compound shown in formula (1-1-1), the compound shown in formula (1-1-2), and methanol to obtain the compound shown in formula (1-1-3); reacting 6-hydroxy-2-naphthal, malononitrile, and toluene to obtain the compound shown in formula (1-2-1); and reacting the compound shown in formula (1-1-3) and the compound shown in formula (1-2-1) to obtain the compound shown in formula (I).

[0011] Specifically, the preparation method of the compound shown in formula (II) includes: reacting p-nitrobenzyl bromide and 2,3,3-trimethylindole to obtain the compound shown in formula (1-1-1); reacting N,N-dimethylformamide, dichloromethane, and phosphorus oxychloride to obtain the compound shown in formula (1-1-2); reacting the compound shown in formula (1-1-1), the compound shown in formula (1-1-2), and methanol to obtain the compound shown in formula (1-1-3); reacting 6-hydroxy-2-naphthal, 4,6-dihydroxy-2-mercaptopyrimidine, and anhydrous ethanol to obtain the compound shown in formula (1-2-2); and reacting the compound shown in formula (1-1-3) and the compound shown in formula (1-2-2) to obtain the compound shown in formula (II).

[0012] Furthermore, the present invention provides methods for preparing compounds of formula (I) and formula (II):

[0013] Specifically, the preparation method of the compound shown in formula (I) includes: nucleophilic substitution of p-nitrobenzyl bromide and 2,3,3-trimethylindole to obtain the compound shown in formula (1-1-1); N,N-dimethylformamide, dichloromethane, and phosphorus oxychloride to obtain the compound shown in formula (1-1-2) via Wilsmeer-Hacker reaction; elimination reaction of the compound shown in formula (1-1-1), the compound shown in formula (1-1-2), and methanol to obtain the compound shown in formula (1-1-3); Knoevenagel reaction of 6-hydroxy-2-naphthal, malononitrile, and toluene to obtain the compound shown in formula (1-2-1); and Williamson ether synthesis reaction of the compound shown in formula (1-1-3) and the compound shown in formula (1-2-1) to obtain the compound shown in formula (I).

[0014] Specifically, the preparation method of the compound shown in formula (II) includes: nucleophilic substitution of p-nitrobenzyl bromide and 2,3,3-trimethylindole to obtain the compound shown in formula (1-1-1); N,N-dimethylformamide, dichloromethane, and phosphorus oxychloride to obtain the compound shown in formula (1-1-2) via the Wilsmeer-Hacker reaction; the compound shown in formula (1-1-1), the compound shown in formula (1-1-2), and methanol to obtain the compound shown in formula (1-1-3) via an elimination reaction; 6-hydroxy-2-naphthal, 4,6-dihydroxy-2-mercaptopyrimidine, and anhydrous ethanol to obtain the compound shown in formula (1-2-2) via the Knoevenagel reaction; and the compound shown in formula (1-1-3) and the compound shown in formula (1-2-2) to obtain the compound shown in formula (II) via the Williamson ether synthesis reaction.

[0015] Furthermore, the present invention provides methods for preparing compounds of formula (I) and formula (II):

[0016] Specifically, the preparation method of the compound shown in formula (1-1-1) includes: dissolving p-nitrobenzyl bromide in toluene and heating it under reflux in a high-temperature oil bath. Weighing 2,3,3-trimethylindole dissolved in toluene and adding it dropwise to the above solution, and continuing the reaction under high-temperature reflux for a period of time. After the reaction is complete, cooling the reaction solution and cooling it in an ice bath, filtering the precipitate through a Buchner funnel, and repeatedly washing with petroleum ether. After drying, the compound shown in formula (1-1-1) is obtained.

[0017] Specifically, the preparation method of the compound shown in formula (1-1-2) includes: adding twice-dried N,N-dimethylformamide to dichloromethane and stirring in an ice bath for a period of time. Then, dissolving phosphorus oxychloride in dried dichloromethane and slowly adding it dropwise to the above reaction system using a constant-pressure dropping funnel, continuing to stir in an ice bath for a period of time. After the solution in the constant-pressure funnel has been completely added, adding cyclohexanone pretreated with molecular sieves, and continuing to stir for a period of time. Cooling, and after the flask has returned to room temperature, refluxing it in a high-temperature oil bath for a period of time. After the reaction is complete, pouring the reaction solution while hot into a large beaker containing ice, and incubating overnight. Filtering the precipitate through a Buchner funnel to obtain the compound shown in formula (1-1-2).

[0018] Specifically, the preparation method of the compound shown in formula (1-1-3) includes: weighing the compounds shown in formula (1-1-1) and formula (1-1-2) and dissolving them in a small amount of methanol; gradually adding methanol dropwise to the desired volume during heating, so that the solids dissolve and react simultaneously to reduce photolysis products. The reaction is carried out in the dark for a period of time. After the reaction is complete, the reaction is stopped, the reaction solution is cooled to room temperature, and evaporated to dryness to obtain the crude product. Further separation and purification using a silica gel column chromatography yields the compound shown in formula (1-1-3).

[0019] Specifically, the preparation method of the compound shown in formula (1-2-1) includes: weighing 6-hydroxy-2-naphthaldehyde and malononitrile into a mixture of anhydrous ethanol and toluene, and stirring under reflux in a high-temperature oil bath for a period of time. After the reaction is complete, the reaction solution is cooled to room temperature, and the solvent is evaporated under reduced pressure to obtain the crude product. The crude product is further separated and purified by silica gel column chromatography to obtain the compound shown in formula (1-2-1).

[0020] Specifically, the preparation method of the compound shown in formula (1-2-2) includes: weighing 6-hydroxy-2-naphthaldehyde and 4,6-dihydroxy-2-mercaptopyrimidine into anhydrous ethanol, refluxing and stirring in a high-temperature oil bath for a period of time until the reaction is complete, and then cooling the reaction solution to room temperature. The resulting liquid is cooled in an ice bath under light-protected conditions and then centrifuged at high speed. The precipitate is placed in an ice bath and acetone is added and stirred thoroughly, all while avoiding light. The precipitate is filtered through a Buchner funnel and dried to obtain the compound shown in formula (1-2-2).

[0021] Specifically, the preparation method of the compound shown in formula (I) includes: weighing the compound shown in formula (1-2-1) and sodium hydride into dry N,N-dimethylformamide. Stirring at room temperature for a period of time under nitrogen protection. Then, weighing the compound shown in formula (1-1-3) into dry N,N-dimethylformamide and dissolving it, injecting it into the above reaction system using a syringe. Stirring at room temperature in the dark for a period of time until the reaction is complete, adding a mixture of dichloromethane and saturated sodium bicarbonate solution to the reaction system. Collecting the organic layer, drying it with anhydrous MgSO4, and then evaporating the solvent to obtain the crude product. Further separation and purification of the crude product using silica gel column chromatography yields the compound shown in formula (I).

[0022] Specifically, the preparation method of the compound shown in formula (II) includes: weighing the compound shown in formula (1-2-2) and sodium hydride into dry N,N-dimethylformamide. Stirring at room temperature for a period of time under nitrogen protection. Then, weighing the compound shown in formula (1-1-3) into dry N,N-dimethylformamide and dissolving it, injecting it into the above reaction system. Stirring at room temperature in the dark for a period of time until the reaction is complete, then stopping the reaction. Adding water to the reaction system produces a precipitate, which is filtered and then thoroughly stirred in a mixture of lyophilized NaCl solution and acetone under ice bath conditions. Filtering the precipitate through a Buchner funnel yields the compound shown in formula (II).

[0023] Furthermore, the present invention provides methods for preparing compounds of formula (I) and formula (II):

[0024] Specifically, the preparation method of the compound shown in formula (1-1-1) includes: placing p-nitrobenzyl bromide in toluene and heating it in an oil bath at 90–150°C until the solution is clear. Then, placing 2,3,3-trimethylindole in toluene and sonicating it to dissolve it. This is added dropwise to the clear solution, and the solution changes from colorless to pale yellow. After continuing the reaction for 1–20 minutes, the solution changes from pale yellow to red. The reaction is continued at 90–150°C under reflux for 9–15 hours. After the reaction is complete, a small amount of purplish-red solid is produced in the flask. The reaction is then stopped, and the reaction solution is cooled to room temperature and further cooled in an ice bath for 1–2 hours to ensure more complete precipitation of the product, resulting in a large amount of purplish-red solid precipitating out. The precipitate is filtered to obtain a purplish-red solid, which is then washed three times with petroleum ether. After drying, a purplish-red powder is obtained.

[0025] Specifically, the preparation method of the compound shown in formula (1-1-2) includes: mixing dried N,N-dimethylformamide and dichloromethane, and stirring in an ice bath at -5 to 0°C for 10 to 50 min. Then, dissolving phosphorus oxychloride in twice-dried dichloromethane and slowly adding it dropwise to the above mixed solution, continuing to stir in the ice bath for 1 to 5 h. After the phosphorus oxychloride solution is completely added, cyclohexanone is added, and stirring continues for 10 to 60 min. Subsequently, the ice bath is removed, and after the reaction returns to room temperature, it is refluxed in an oil bath at 20 to 80°C for 1 to 5 h. After the reaction is complete, the reaction is stopped, and the reaction solution is poured into a large beaker containing ice while still hot, and left overnight. The precipitate is filtered to obtain a bright yellow solid. After drying, a bright yellow powder is obtained.

[0026] Specifically, the preparation method of the compound shown in formula (1-1-3) includes: placing the compounds shown in formula (1-1-1) and formula (1-1-2) in methanol and reacting them in an oil bath at 50–100°C in the dark for 12–36 h. After the reaction is complete, a small amount of green precipitate is produced. The reaction is then stopped, the reaction solution is cooled to room temperature, and the solvent is evaporated using a rotary evaporator to obtain a green solid crude product. The crude product is further separated and purified by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1) to obtain the compound shown in formula (1-1-3).

[0027] Specifically, the preparation method of the compound shown in formula (1-2-1) includes: dissolving 6-hydroxy-2-naphthaldehyde and malononitrile in a mixture of anhydrous ethanol and toluene (v:v = 9:1), and stirring under reflux in an oil bath at 50–100°C for 10–30 h. After the reaction is complete, the reaction is stopped, the reaction solution is cooled to room temperature, and the solvent is evaporated using a rotary evaporator to obtain a yellow solid crude product. The crude product is further separated and purified by silica gel column chromatography (eluent: petroleum ether: dichloromethane = 4:1) to obtain the compound shown in formula (1-2-1).

[0028] Specifically, the preparation method of the compound shown in formula (1-2-2) includes: placing 6-hydroxy-2-naphthaldehyde and 4,6-dihydroxy-2-mercaptopyrimidine in anhydrous ethanol and stirring under reflux in an oil bath at 50–100°C for 1–5 h. After the reaction is complete, a large amount of orange-red solid is produced in the flask. The reaction is then stopped, and the reaction solution is cooled to room temperature. The resulting liquid is cooled in an ice bath under light-protected conditions and then centrifuged at high speed (10000 r / min) for 6–10 minutes to obtain a tightly aggregated product, which is then immediately filtered. The precipitate is placed in an ice bath and acetone is added, and the mixture is stirred thoroughly under light-protected conditions to obtain the compound shown in formula (1-2-2).

[0029] Specifically, the preparation method of the compound shown in formula (I) includes: placing the compound shown in formula (1-2-1) and sodium hydride in dry N,N-dimethylformamide. Stirring at room temperature for 10–60 min under nitrogen protection. Then, placing the compound shown in formula (1-1-3) in dry N,N-dimethylformamide, sonicating to dissolve it, and injecting it into the above reaction system using a syringe. Stirring at room temperature in the dark for 12–36 h. After the reaction is confirmed to be complete, the reaction is stopped. A mixture of dichloromethane and saturated sodium bicarbonate solution (v:v = 4:1) is added to the reaction system to separate and preliminarily purify the product to remove N,N-dimethylformamide. The organic layer is collected, dried over anhydrous MgSO4, and the solvent is evaporated using a rotary evaporator to obtain a green solid crude product. The crude product is further separated and purified by silica gel column chromatography (eluent: dichloromethane:methanol = 30:1) to obtain the green compound shown in formula (I).

[0030] Specifically, the preparation method of the compound shown in formula (II) includes: placing the compound shown in formula (1-2-2) and sodium hydride in dry N,N-dimethylformamide. Stirring at room temperature for 10–50 min under nitrogen protection. Then, placing the compound shown in formula (1-1-3) in dry N,N-dimethylformamide, sonicating to dissolve it, and injecting it into the above reaction system using a syringe. Stirring at room temperature in the dark for 12–36 h. After the reaction is confirmed to be complete, the reaction is stopped. A large amount of water is added to the reaction system and shaken, producing a dark red precipitate. After filtration, a mixture of lyophilized NaCl solution and acetone (v:v = 1:3) is stirred thoroughly in an ice bath to remove easily photolytic impurities. The precipitate is filtered through a Buchner funnel to obtain a dark red solid crude product. The crude product is further purified by silica gel column chromatography (eluent: dichloromethane:methanol = 30:1) to obtain the green compound shown in formula (II).

[0031] On the other hand, the present invention provides a molecular probe, such as the compound shown in formula (I) and / or the compound shown in formula (II).

[0032] Furthermore, the molecular probe provided by this invention has specific recognition of H2S.

[0033] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0034] This invention improves upon the traditional triggering unit by introducing a naphthalene derivative above the cyanine skeleton, exhibiting two-photon absorption characteristics; and introducing an aromatic nitrosyl structure below, using the nitrosyl structure as a key response site, which has the ability to bind to H2S, thereby achieving specific recognition of H2S. This invention provides a molecular framework suitable for single-molecule dual-mode excitation under physiological and pathological conditions, and synthesizes a molecular probe that can specifically recognize H2S, thereby enabling visualized monitoring of exogenous and endogenous H2S fluctuation levels in vivo through separate dual-pathway fluorescence imaging. Attached Figure Description

[0035] Figure 1 These are the steps for synthesizing compounds YCN and YNH.

[0036] Figure 2 The image shows the NMR spectrum of compound 1-1-3.

[0037] Figure 3 The image shows the NMR spectrum of compound 1-1-3.

[0038] Figure 4 This is the nuclear magnetic resonance spectrum of the probe YCN.

[0039] Figure 5 The image shows the nuclear magnetic resonance spectrum of the probe YNH.

[0040] Figure 6 Selectivity test of probe YCN for H2S: 1 is Mg 2+ ;2 is Zn 2+ ;3 is Fe 2+ ;4 is Fe 3+ ;5 is SO4 2- 6 represents NO2 - 7 represents HCO3 - ;8 is OH - ;9 is S 2- ;10 is S2O3 - ;11 is CO3 2- ;12 is CH3COO - ;13 is F - ;14 is SCN - ;15 is Cl -; 16 is GSH; 17 is Hcy; 18 is Cys; (a) is the short-wave region: Ex = 410nm, Em = 510nm, slit width is 2nm; (b) is the long-wave region: Ex = 710nm, Em = 800nm, slit width is 3nm.

[0041] Figure 7 The selectivity test of probe YNH for H2S was performed using Mg. 2+ ;2 is Zn 2+ ;3 is Fe 2+ ;4 is Fe 3+ ;5 is SO4 2- 6 represents NO2 - 7 represents HCO3 - ;8 is OH - ;9 is S 2- ;10 is S2O3 - ;11 is CO3 2 ;12 is CH3COO - ;13 is F - ;14 is SCN - ;15 is Cl - ; 16 is GSH; 17 is Hcy; 18 is Cys; (a) is the short-wave region: Ex = 390nm, Em = 522nm, slit width is 2nm; (b) is the long-wave region: Ex = 710nm, Em = 810nm, slit width is 3nm.

[0042] Figure 8 The dual-channel fluorescence emission spectra of probe YCN in response to different concentrations of H2S are shown: (a) shows the change in fluorescence emission intensity at 510 nm; (b) shows the relationship between the fluorescence intensity of probe YCN at 510 nm and the H2S concentration; (c) shows the change in fluorescence emission intensity at 800 nm; and (d) shows the relationship between the fluorescence intensity of probe YCN at 800 nm and the H2S concentration.

[0043] Figure 9 The dual-channel fluorescence emission spectra of probe YNH in response to different concentrations of H2S are shown: (a) fluorescence emission intensity variation at 522 nm; (b) relationship between fluorescence intensity of probe YNH and H2S concentration at 522 nm; (c) fluorescence emission intensity variation at 810 nm; (d) relationship between fluorescence intensity of probe YNH and H2S concentration at 810 nm.

[0044] Figure 10 The dual-channel fluorescence emission spectra of probe YCN in response to H2S at different time ranges are shown: (a) fluorescence emission intensity variation at 510 nm; (b) fluorescence intensity of probe YCN at 510 nm versus time; (c) fluorescence emission intensity variation at 800 nm; (d) fluorescence intensity of probe YCN at 800 nm versus time.

[0045] Figure 11 The dual-channel fluorescence emission spectra of probe YNH in response to H2S at different time ranges are shown: (a) fluorescence emission intensity variation at 522 nm; (b) fluorescence intensity of probe YNH at 522 nm versus time; (c) fluorescence emission intensity variation at 810 nm; (d) fluorescence intensity of probe YNH at 810 nm versus time.

[0046] Figure 12 The images show the dual-channel fluorescence emission spectra of probe YCN within different pH ranges. (a) shows the fluorescence emission intensity change of probe YCN at 510 nm within the pH range of 2–9; (b) shows the fluorescence emission intensity change of probe YCN at 800 nm within the pH range of 2–9; (c) shows the fluorescence emission intensity change of probe YCN in response to H2S at 510 nm within the pH range of 2–9; (d) shows the fluorescence emission intensity change of probe YCN in response to H2S at 800 nm within the pH range of 2–9; (e) shows the effect of pH on the response of probe YCN to H2S at 510 nm; and (f) shows the effect of pH on the response of probe YCN to H2S at 800 nm. The concentration of probe YCN was 10 μmol / L, and the concentration of H2S was 100 μmol / L.

[0047] Figure 13 The images show the dual-channel fluorescence emission spectra of probe YNH at different pH ranges. (a) shows the fluorescence emission intensity change of probe YNH at 522 nm within the pH range of 2–9; (b) shows the fluorescence emission intensity change of probe YNH at 810 nm within the pH range of 2–9; (c) shows the fluorescence emission intensity change of probe YNH in response to H2S at 522 nm within the pH range of 2–9; (d) shows the fluorescence emission intensity change of probe YNH in response to H2S at 810 nm within the pH range of 2–9; (e) shows the effect of pH on the response of probe YNH to H2S at 522 nm; and (f) shows the effect of pH on the response of probe YNH to H2S at 810 nm. The concentration of probe YCN was 10 μmol / L, and the concentration of H2S was 50 μmol / L.

[0048] Figure 14 The effects of different concentrations of probes YCN and YNH on HeLa cell survival are shown. (a) represents probe YCN; (b) represents probe YNH.

[0049] Figure 15 Confocal imaging of exogenous and endogenous H2S in cells using the YCN probe. (a) Dark field; (b) Bright field; (c) Collection and emission channels (405–640 nm); (d) Overlay image; Excitation wavelength 405 nm, scale bar 40 μm; YCN concentration 10 μmol / L.

[0050] Figure 16 Confocal imaging of exogenous and endogenous H2S in cells using the YNH probe. (a) Dark field; (b) Bright field; (c) Collection and emission channels (405–640 nm); (d) Superimposed image. Excitation wavelength was 405 nm, scale bar was 40 μm; YNH concentration was 10 μmol / L. Detailed Implementation

[0051] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0052] Example 1

[0053] This embodiment provides a method for synthesizing compounds YCN and YNH.

[0054] like Figure 1 As shown, the specific synthesis methods for compounds YCN and YNH are as follows:

[0055] 1. Synthesis of compound 1-1-1

[0056] Weigh 259.24 mg (1.20 mmol) of compound p-nitrobenzyl bromide into 20 mL of toluene and heat in an oil bath at 90–150 °C until the solution is clear. Then, weigh 238.85 mg (1.50 mmol) of compound 2,3,3-trimethylindole into 20 mL of toluene and sonicate until dissolved. Add this solution dropwise to the clear solution; the solution changes from colorless to pale yellow. After continuing the reaction for 1–20 min, the solution changes from pale yellow to red. Continue the reaction at 90–150 °C for 9–15 hours. After the reaction is complete, a small amount of purplish-red solid is produced in the flask. Then stop the reaction, cool the reaction solution to room temperature, and further cool it in an ice bath for 1–2 hours to ensure more complete precipitation, resulting in a large amount of purplish-red solid. Filter the precipitate through a Buchner funnel to obtain the purplish-red solid, and wash the purplish-red solid three times with petroleum ether (20 mL). After drying, a purplish-red powder is obtained, which is the compound shown in formula (1-1-1).

[0057] 2. Synthesis of compound 1-1-2

[0058] First, 20 mL of dry N,N-dimethylformamide and dichloromethane were poured into a single-necked round-bottom flask and stirred in an ice bath at -5 to 0°C for 10 to 50 min. Then, 23.46 g (153.00 mmol) of phosphorus oxychloride was dissolved in 20 mL of twice-dried dichloromethane and slowly added dropwise to the round-bottom flask using a constant-pressure dropping funnel, while stirring in an ice bath for 1 to 5 h. After the solution in the constant-pressure funnel had been completely added, 50.1 g (51.00 mmol) of cyclohexanone (pre-treated with 10 g of 0.45 μm molecular sieve) was added to the round-bottom flask, and stirring was continued for 10 to 60 min. Subsequently, the ice bath was removed, and after the flask returned to room temperature, it was refluxed in an oil bath at 20 to 80°C for 1 to 5 h. After the reaction was complete, the reaction was stopped, and the reaction solution was poured into a large beaker containing ice while still hot and left overnight. The precipitate was filtered through a Buchner funnel to obtain a bright yellow solid. After drying, a bright yellow powder was obtained, which is the compound shown in formula (1-1-2).

[0059] 3. Synthesis of compound 1-1-3

[0060] Weigh 2.95 g (10.00 mmol) of the compound shown in formula (1-1-1) and 1.21 g (7.00 mmol) of the compound shown in formula (1-1-2) into 60 mL of methanol, and react in an oil bath at 50–100 °C in the dark for 12–36 h. After the reaction is complete, a small amount of green precipitate is formed. Then the reaction is stopped, the reaction solution is cooled to room temperature, and the solvent is evaporated using a rotary evaporator to obtain a green solid crude product. The crude product is further purified by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1) to obtain the compound shown in formula (1-1-3) (NMR spectrum shown in [reference needed]). Figure 2 ).

[0061] 4. Synthesis of Compound 1-2-1

[0062] 1.03 g (6.00 mmol) of compound 6-hydroxy-2-naphthal and 792.76 mg (12.00 mmol) of malononitrile were dissolved in 60 mL of a mixture of anhydrous ethanol and toluene (v:v = 9:1). The mixture was refluxed and stirred in an oil bath at 50–100 °C for 10–30 h. After the reaction was complete, the reaction was stopped, and the reaction solution was cooled to room temperature. The solvent was evaporated using a rotary evaporator to obtain a yellow solid crude product. The crude product was further purified by silica gel column chromatography (eluent: petroleum ether: dichloromethane = 4:1) to obtain the compound shown in formula (1-2-1) (NMR spectrum shown in [reference needed]). Figure 3 ).

[0063] 5. Synthesis of Compound 1-2-2

[0064] 1.21 g (7.00 mmol) of 6-hydroxy-2-naphthal and 1.01 g (7.00 mmol) of 4,6-dihydroxy-2-mercaptopyrimidine were weighed into 60 mL of anhydrous ethanol and refluxed and stirred in an oil bath at 50–100 °C for 1–5 h. After the reaction was complete, a large amount of orange-red solid was produced in the flask. The reaction was then stopped, and the reaction solution was cooled to room temperature. The resulting liquid was cooled in a 500 mL ice bath under light-protected conditions and then centrifuged at high speed (10000 r / min) for 6–10 minutes to obtain a tightly aggregated product, which was immediately filtered. The precipitate was placed in an ice bath and allowed to stand, and 70 mL of acetone was added. The mixture was stirred thoroughly under light-protected conditions to obtain the compound shown in formula (1-2-2).

[0065] 6. Synthesis of compound YCN

[0066] First, weigh 220.23 mg (1.00 mmol) of the compound shown in formula (1-2-1) and 24 mg (1.00 mmol) of sodium hydride into 10 mL of dry N,N-dimethylformamide. Stir at room temperature for 10–60 min under nitrogen protection. Then, weigh 1.09 g (1.50 mmol) of the compound shown in formula (1-1-3) into 10 mL of dry N,N-dimethylformamide, sonicate to dissolve, and inject into the above reaction system using a syringe. Stir at room temperature in the dark for 12–36 h. After the reaction is confirmed to be complete, stop the reaction. Add a mixture of dichloromethane and saturated sodium bicarbonate solution (v:v = 4:1) to the reaction system to separate and preliminarily purify the product, removing N,N-dimethylformamide. Collect the organic layer, dry it with anhydrous MgSO4, and evaporate the solvent using a rotary evaporator to obtain a green solid crude product. The crude product was further purified by silica gel column chromatography (eluent: dichloromethane:methanol = 30:1) to obtain the green compound shown in formula (I) (NMR spectrum shown in [reference image]). Figure 4 ), which is represented as probe YCN.

[0067] 7. Synthesis of compound YNH

[0068] First, weigh 298.32 mg (1.00 mmol) of the compound shown in formula (1-2-2) and 24 mg (1.00 mmol) of sodium hydride into 10 mL of dry N,N-dimethylformamide. Stir at room temperature for 10–50 min under nitrogen protection. Then, weigh 1.09 g (1.50 mmol) of the compound shown in formula (1-1-3) into 10 mL of dry N,N-dimethylformamide, sonicate to dissolve, and inject into the above reaction system using a syringe. Stir at room temperature in the dark for 12–36 h. After the reaction is confirmed to be complete, stop the reaction. Add a large amount of water to the reaction system and shake; a dark red precipitate is produced. After filtration, a mixture of lyophilized NaCl solution and acetone (v:v = 1:3) is stirred thoroughly in an ice bath to remove easily photodegradable impurities. Filter the precipitate through a Buchner funnel to obtain a dark red solid crude product. The crude product was further purified by silica gel column chromatography (eluent: dichloromethane:methanol = 30:1) to obtain the green compound shown in formula (II) (NMR spectrum shown in [reference image]). Figure 5 ), which is represented as probe YNH.

[0069] Example 2

[0070] This embodiment provides experimental studies on the selectivity of probes YCN and YNH for H2S.

[0071] The inventor used metal cations (Mg) 2+ Zn 2+ Fe 2+ Fe 3+ ), anion (SO4) 2- NO2 - HCO3 - OH - S2 - S2O3 - CO3 2- CH3COO - F - SCN - Cl - As well as biothiols (GSH, Hcy, Cys) and other interfering substances, the fluorescence intensity of their reactions with probe YCN (10 μmol / L) and probe YNH (10 μmol / L) was measured. Figure 6 As shown, the probe YCN only exhibits strong fluorescence changes in response to H2S. Specifically, the excitation and emission wavelengths of the probe YCN in the short-wavelength region are Ex = 410 nm and Em = 510 nm, respectively, with a slit width of 2 mm; while the excitation and emission wavelengths in the long-wavelength region are Ex = 710 nm and Em = 800 nm, respectively, with a slit width of 3 mm. Figure 7As shown, the probe YNH showed strong fluorescence changes only to H2S. Specifically, the excitation and emission wavelengths of the probe YNH in the short-wavelength region were Ex = 390 nm and Em = 522 nm, respectively, with a slit width of 2 mm. The excitation and emission wavelengths of the probe YNH in the long-wavelength region were Ex = 710 nm and Em = 810 nm, respectively, with a slit width of 3 mm.

[0072] Therefore, probes YCN and YNH exhibit very high fluorescence response selectivity for H2S and have specific recognition of H2S.

[0073] Example 3

[0074] This embodiment provides experimental research on the detection of H2S and the detection limit of probes YCN and YNH.

[0075] The fluorescence emission spectra of the probe YCN after reacting with different concentrations of H2S (0–100 μmol / L) were measured under simulated physiological conditions (PBS 10 mmol / L, pH = 7.40, 37℃). Figure 8 As shown, the probe YCN itself exhibits weak fluorescence emission at 510 nm (excitation wavelength 410 nm) and strong fluorescence emission at 800 nm (excitation wavelength 710 nm). Upon interaction with H₂S, the fluorescence emission at 510 nm gradually increases with increasing H₂S concentration, while the fluorescence emission at 800 nm gradually decreases (until quenched) with increasing H₂S concentration. Furthermore, when the H₂S concentration is in the range of 0–100 μmol / L, the probe YCN shows a good linear relationship with H₂S.

[0076] The fluorescence emission spectra of the probe YNH after reacting with different concentrations of H2S (0–50 μmol / L) were measured under simulated physiological conditions (PBS 10 mmol / L, pH = 7.40, 37℃). Figure 9 As shown, the probe YNH itself exhibits weak fluorescence emission at 522 nm (excitation wavelength 390 nm) and strong fluorescence emission at 810 nm (excitation wavelength 710 nm). When reacting with H2S, the fluorescence emission at 522 nm remains essentially unchanged with increasing H2S concentration, while the fluorescence emission at 810 nm gradually weakens (until quenched) with increasing H2S concentration.

[0077] Example 4

[0078] This embodiment provides a research experiment on the dynamic testing of H2S using probes YCN and YNH.

[0079] Under simulated physiological conditions (PBS 10 mmol / L, pH = 7.40, 37℃), the fluorescence emission spectra of probe YCN (10 μmol / L) in response to H2S (100 μmol / L) were measured at different time periods (0–150 min). Figure 10 As shown in (b), under 410 nm excitation, the fluorescence emission intensity of the probe YCN at 510 nm remained essentially constant over time. Upon addition of H₂S, as the reaction proceeded, the fluorescence emission intensity of the probe YCN at 510 nm gradually increased, accompanied by a slight red shift. After 150 min, the fluorescence emission intensity reached its maximum value and remained essentially constant. Figure 10 As shown in (d), under 710 nm excitation, the fluorescence emission of probe YCN at 800 nm remained essentially constant over time. Upon the addition of H2S, as the reaction proceeded, the fluorescence emission of probe YCN at 800 nm rapidly decreased, accompanied by a slight blue shift. After 150 min, the fluorescence emission intensity reached its minimum and remained essentially constant.

[0080] Under simulated physiological conditions (PBS 10 mmol / L, pH = 7.40, 37℃), the fluorescence emission spectra of probe YNH (10 μmol / L) in response to H2S (50 μmol / L) were measured at different time periods (0–150 min). Figure 11 As shown in (b), under 390 nm excitation, the fluorescence emission of the probe YNH at 522 nm remained essentially constant over time. Upon the addition of H₂S, although the fluorescence emission of the probe YNH at 522 nm increased with the continuation of the reaction, the increase was not significant. Figure 11 As shown in (d), under 710 nm excitation, the fluorescence emission of the probe YNH at 810 nm remained essentially constant over time. Upon the addition of H₂S, the fluorescence emission of the probe YNH at 810 nm rapidly decreased as the reaction proceeded, reaching a minimum intensity after 150 min, and remained essentially constant thereafter.

[0081] Example 5

[0082] This embodiment provides experimental research on the effects of probes YCN and YNH on H2S at different pH levels.

[0083] The fluorescence emission spectra of the probe YCN in response to H2S (100 μmol / L) were measured under different pH conditions. Figure 12 As shown in (a), under 410 nm excitation, the fluorescence emission of the probe YCN at 510 nm remained essentially unchanged with pH (2–9). Figure 12As shown in (c), after adding H2S and reacting for 90 min, the fluorescence emission of the probe YCN at 510 nm remained basically unchanged with the change of pH (2-9). Figure 12 As shown in (b), under 710 nm excitation, the fluorescence emission of the probe YCN at 800 nm remained essentially unchanged with pH (2-9). Figure 12 As shown in (d), when H2S is added and the reaction is carried out for 90 min, the fluorescence emission of the probe YCN at 800 nm remains unchanged with the change of pH (2-9). Figure 12 The effect of pH on the response of probe YCN to H2S at 510 nm is shown in (e). Figure 12 Figure (f) shows the effect of pH on the response of probe YCN to H2S at 800 nm. Therefore, probe YCN can detect H2S under physiological conditions.

[0084] The fluorescence emission spectra of the probe YNH in response to H2S (50 μmol / L) were measured under different pH conditions. Figure 13 As shown in (a), under 390 nm excitation, the fluorescence emission of the probe YNH itself at 522 nm remained basically unchanged with the change of pH (2-9). Figure 13 As shown in (c), after adding H2S and reacting for 90 min, the fluorescence emission of the probe YNH at 522 nm remained basically unchanged with the change of pH (2-9). Figure 13 As shown in (b), under 710 nm excitation, the fluorescence emission of the probe YNH itself at 810 nm remained basically unchanged with the change of pH (2-9). Figure 13 As shown in (d), after adding H2S and reacting for 90 min, the fluorescence emission of the probe YNH at 810 nm remained unchanged with the change of pH (2-9); Figure 13 As shown in (e), the effect of pH on the probe YNH response to H2S at 522 nm; Figure 13 As shown in (f), the effect of pH on the response of probe YNH to H2S at 810 nm is demonstrated. Therefore, probe YNH can detect H2S under physiological conditions.

[0085] Example 6

[0086] This embodiment provides cytotoxicity studies of probes YCN and YNH.

[0087] The cytotoxicity of probes YCN and YNH was evaluated using the MTT assay. Succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. The formazan crystals are then dissolved in the cells using dimethyl sulfoxide (DMSO), and the absorbance is measured at 490 nm using a microplate reader, indirectly reflecting the number of living cells. Figure 14 As shown, when the concentrations of probes YCN and YNH reach 16 μmol / L, the survival rate of HeLa cells remains no less than 70%. Therefore, probes YCN and YNH exhibit good biocompatibility and can be safely applied to analytical studies at the live cell level.

[0088] Example 7

[0089] This embodiment provides a research experiment on co-fluorescence imaging of probes YCN and YNH in living cells.

[0090] HeLa cells were selected as the experimental subject, and the imaging results are as follows: Figure 15 As shown. The H2S content in cells was divided into three groups, and the endogenous and exogenous H2S were detected using the probe YCN: Group 1 HeLa cells were incubated with 10 μmol / L fluorescent probe YCN for 1 h. After incubation, they were washed three times with PBS, and the emitted fluorescence was captured using a confocal microscope (Ex = 405 nm, Em = 640 nm). Group 2 HeLa cells were incubated with 100 μM H2S for 2.5 h, and then washed three times with PBS. Then, 10 μmol / L probe YCN was added to the HeLa cells, and incubation was continued for 1 h. After incubation, they were washed three times with PBS, and confocal imaging was performed. Group 3 HeLa cells were treated with NEM (200 μM) for 30 min, and then washed three times with PBS. Then, 10 μmol / L probe YCN was added to the HeLa cells, and incubation was continued for 1 h. After incubation, they were washed three times with PBS, and confocal imaging was performed.

[0091] Endogenous and exogenous H2S were detected using the probe YNH, and the imaging results are as follows: Figure 16 As shown. The first group of HeLa cells was incubated with 10 μmol / L probe YNH for 1 h. After incubation, the cells were washed three times with PBS, and the emitted fluorescence was captured using a confocal microscope (Ex = 405 nm, Em = 640 nm). The second group of HeLa cells was incubated with (50 μM) H2S for 2.5 h, and then washed three times with PBS. Then, 10 μmol / L probe YNH was added to the HeLa cells, and incubation was continued for 1 h. After incubation, the cells were washed three times with PBS, and confocal imaging was performed. The third group of HeLa cells was treated with NEM (100 μM) for 30 min, and then washed three times with PBS. Then, 10 μmol / L probe YNH was added to the HeLa cells, and incubation was continued for 1 h. After incubation, the cells were washed three times with PBS, and confocal imaging was performed.

[0092] The addition of probe YCN resulted in significant fluorescence in the cells. The addition of H2S further enhanced the fluorescence intensity, while the addition of NEM significantly weakened the fluorescence. Similarly, the addition of probe YNH resulted in significant fluorescence in the cells. Therefore, probes YCN and YNH specifically detect H2S within cells.

[0093] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A compound, characterized in that, It has a structure as shown in equation (I), 。 2. The method for preparing the compound according to claim 1, characterized in that, include: 。 3. A compound, characterized in that, It has a structure as shown in equation (II), 。 4. The method for preparing the compound according to claim 3, characterized in that, include: 。 5. The method for preparing the compound according to claim 2 or claim 4, characterized in that, When preparing the compound shown in formula (1-1-2), the N,N-dimethylformamide used was subjected to two drying processes.

6. The method for preparing the compound according to claim 4, characterized in that, The compound shown in formula (1-2-2) was prepared in complete darkness.

7. The method for preparing the compound according to claim 4, characterized in that, When preparing the compound shown in formula (II), the impurity was removed by lyophilizing a mixture of NaCl solution and acetone under ice bath conditions.

8. A molecular probe, characterized in that, Includes the compound of claim 1 and / or the compound of claim 3.

9. The molecular probe according to claim 8, characterized in that, Specific recognition of H2S.

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