A near-infrared fluorescent hydrogen sulfide donor and a preparation method and application thereof

Abstract

The present application relates to a kind of near-infrared fluorescent hydrogen sulfide donor and its preparation method and application, belong to the field of biochemistry technology.The structure formula of the near-infrared fluorescent hydrogen sulfide donor is as follows.The present application combines p-tolyl isothiocyanate with dicyanofluorescent isophorone group, and synthesizes a kind of near-infrared fluorescent hydrogen sulfide donor.The near-infrared fluorescent hydrogen sulfide donor provided by the present application can realize the visualization of hydrogen sulfide release by near-infrared fluorescent response, in addition, can effectively reduce NO2 ‑ Level in inflammatory cells, has great potential in treating inflammation.

Description

Technical Field

[0001] This invention relates to a near-infrared fluorescent hydrogen sulfide donor, its preparation method, and its application, belonging to the field of biochemical technology. Background Technology

[0002] Hydrogen sulfide (H2S) is a key gaseous signaling molecule in living systems, significantly influencing various pathological and physiological states. Its functions include protecting the central nervous system, regulating oxidative stress in the cardiovascular system, and maintaining blood pressure homeostasis. Notably, H2S is considered an important endogenous neuroprotective agent, contributing to antioxidant, anti-inflammatory, and anti-apoptotic effects. Therefore, developing H2S donors that are specifically reactive to substrates and marked by simultaneous fluorescence changes holds considerable promise compared to traditional H2S donors. Such compounds could not only exhibit fluorescence responses to specific conditions or substrates but also serve as effective H2S donors in the treatment of related diseases.

[0003] Existing hydrogen sulfide donors suffer from drawbacks such as low hydrogen sulfide release efficiency, numerous byproducts, and limitations in hydrogen sulfide release detection methods. This invention selects dicyanoisophorone as the fluorophore and p-toluene isothiocyanate as the reaction recognition unit to synthesize a near-infrared fluorescent hydrogen sulfide donor. The structure of the near-infrared fluorescent hydrogen sulfide donor provided by this invention ensures the formation of a single fluorescent product, thereby eliminating potential interference from two fluorophores (primary amine and dihydrothiazolyl carboxylic acid). It performs well in fluorescence response analysis, is suitable for visualizing hydrogen sulfide release in living cells, and can also function as a sustained-release H2S donor, effectively reducing NO in inflammatory cells. 2- level. Summary of the Invention

[0004] To overcome the aforementioned deficiencies in the existing technology, this invention provides a near-infrared fluorescent hydrogen sulfide donor, its preparation method, and its application.

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

[0006] A near-infrared fluorescent hydrogen sulfide donor, the structural formula of which is shown below:

[0007]

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned near-infrared fluorescent hydrogen sulfide donor.

[0009] A method for preparing a near-infrared fluorescent hydrogen sulfide donor includes the following steps:

[0010]

[0011] S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) malononitrile and compound II p-hydroxybenzaldehyde were dissolved in a solvent, and an alkaline catalyst was added dropwise. The mixture was stirred under an inert gas atmosphere to obtain compound III.

[0012] S2: Compound IV p-toluene isothiocyanate and N-bromosuccinic acid imine were dissolved in a solvent, an initiator was added, and the mixture was stirred to obtain compound V;

[0013] S3: Dissolve compound III obtained in S1 and compound V obtained in S2 in a solvent and stir under alkaline and inert gas conditions to obtain compound VI, which is the near-infrared fluorescent hydrogen sulfide donor.

[0014] In the above technical solution, in S1, the molar ratio of (3,5,5-trimethyl-2-cyclohexene-1-diene)malonitrile and p-hydroxybenzaldehyde is 1:1 to 3.

[0015] In the above technical solution, the molar ratio of (3,5,5-trimethyl-2-cyclohexene-1-diene)malononitrile to solvent is 1:30-40.

[0016] In the above technical solution, the volume ratio of the alkaline catalyst to the solvent is 1:30 to 40;

[0017] In the above technical solution, in step S2, the molar ratio of p-toluene isothiocyanate and N-bromosuccinic acid imine is 1:1 to 3.

[0018] In the above technical solution, the molar ratio of N-bromosuccinic acid imine to solvent is 1:10-15.

[0019] In the above technical solution, the molar ratio of p-toluene isothiocyanate to initiator is 10-15:1.

[0020] In the above technical solution, in step S3, the molar ratio of compound III obtained in step S1 and compound V obtained in step S2 is 1:1 to 3.

[0021] In the above technical solution, in step S3, the molar ratio of compound III obtained in step S1 to solvent is 1:80 to 120.

[0022] In the above technical solution, in step S1, the solvent is one of methanol, ethanol, or n-butanol.

[0023] In the above technical solution, in S1, the alkaline catalyst is one of triethylamine, piperidine, or N,N-diisopropylethylamine.

[0024] In the above technical solution, in step S2, the solvent is one of dichloromethane, chloroform, or carbon tetrachloride.

[0025] In the above technical solution, in step S2, the initiator is benzoyl peroxide.

[0026] In the above technical solution, in step S3, the solvent is anhydrous acetonitrile.

[0027] In the above technical solution, the alkaline condition in step S3 is the addition of potassium carbonate and potassium iodide.

[0028] Furthermore, the molar ratio of compound III obtained in S1 to potassium carbonate is 20:1 to 3.

[0029] Furthermore, the molar ratio of compound III obtained in S1 to potassium iodide is 50:1 to 3.

[0030] In the above technical solution, in S1 and S3, the inert gas is one of nitrogen, argon or helium.

[0031] In the above technical solution, in step S1, the stirring reaction conditions are: stirring at 250-350 rpm for 12-14 hours at 80-90°C.

[0032] In the above technical solution, in step S2, the stirring reaction conditions are: stirring at 250-350 rpm for 10-12 hours at 70-80°C.

[0033] In the above technical solution, in step S3, the stirring reaction conditions are: stirring at 250-350 rpm for 1-2 hours at 60-70°C.

[0034] Preferably, the preparation method of the near-infrared fluorescent hydrogen sulfide donor includes the following steps:

[0035]

[0036] S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) malononitrile and compound II p-hydroxybenzaldehyde were dissolved in ethanol, piperidine was added dropwise, and the reaction was carried out under nitrogen and at 80-90℃ with stirring at 250-350 rpm for 12-14 h. After the reaction was completed, the mixture was evaporated under reduced pressure and purified to obtain compound III.

[0037] S2: Compound IV p-toluene isothiocyanate and N-bromosuccinic acid imine were dissolved in carbon tetrachloride, and benzoyl peroxide was added as a catalyst. The mixture was stirred at 250-350 rpm at 70-80°C for 10-12 hours. After the reaction was completed, the mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain compound V.

[0038] S3: Dissolve compound III obtained in S1 and compound V obtained in S2 in anhydrous acetonitrile, add potassium carbonate and potassium iodide, and stir at 250-350 rpm for 1-2 hours under nitrogen and 60-70℃ conditions. After the reaction is completed, cool, filter, concentrate the filtrate under reduced pressure, and purify to obtain compound VI, which is the near-infrared fluorescent hydrogen sulfide donor.

[0039] Another object of the present invention is to provide the application of the above-mentioned near-infrared fluorescent hydrogen sulfide donor in the visualization of hydrogen sulfide release.

[0040] Another object of the present invention is to provide the application of the above-mentioned near-infrared fluorescent hydrogen sulfide donor in the preparation of anti-inflammatory drugs.

[0041] Furthermore, the aforementioned near-infrared fluorescent hydrogen sulfide donor enables visualization of hydrogen sulfide release through near-infrared fluorescence response.

[0042] Furthermore, hydrogen sulfide release can effectively reduce NO in inflammatory cells. 2- level.

[0043] The beneficial effects of this invention are as follows: The near-infrared fluorescent hydrogen sulfide donor prepared by this invention has a structure that facilitates the release of hydrogen sulfide while simultaneously generating a single fluorophore for characterization. This design overcomes the limitation of traditional isothiocyanate structures generating two fluorophores upon response. This invention can be used to release H2S in living cells and visualize its presence through near-infrared fluorescence response; it can also effectively reduce NO in inflammatory cells. 2- Levels of [something] have great potential in treating inflammation. Attached Figure Description

[0044] Figure 1 This is a graph showing the toxicity test of the near-infrared fluorescent hydrogen sulfide donor obtained in Example 1 of the present invention on HeLa cells and RAW 264.7 cells.

[0045] Figure 2 This is an image showing the visualization of hydrogen sulfide release in living cells using the near-infrared fluorescent hydrogen sulfide donor obtained in Example 1 of the present invention.

[0046] Figure 3 The near-infrared fluorescent hydrogen sulfide donor obtained in Example 1 of this invention is effective against NO in inflammatory cells. 2- Horizontal test chart.

[0047] Figure 4 The near-infrared fluorescent hydrogen sulfide donor obtained in Example 1 of this invention visualizes hydrogen sulfide release in cells and reduces NO. 2- A horizontal diagram. Detailed Implementation

[0048] 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.

[0049] 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.

[0050] The near-infrared fluorescent hydrogen sulfide donor of this invention is synthesized according to the following technical route:

[0051]

[0052] Example 1

[0053] A method for preparing a near-infrared fluorescent hydrogen sulfide donor includes the following steps:

[0054] S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) malononitrile (466 mg, 2.5 mmol) and compound II p-hydroxybenzaldehyde (370 mg, 3 mmol) were dissolved in 6 mL of ethanol. After complete dissolution, 150 μL of piperidine was added. The mixture was heated to 80 °C under nitrogen and stirred at 300 rpm for 12 h. The reaction was confirmed to be complete by thin-layer chromatography. The solvent was evaporated under reduced pressure and purified by silica gel column chromatography to obtain compound III (472 mg, yield 65%) as an orange powder.

[0055] S2: N-bromosuccinic acid imine (1.79 g, 10 mmol) and compound IV p-toluene isothiocyanate (1.49 g, 10 mmol) were dissolved in 20 mL of carbon tetrachloride, and then a catalytic amount of benzoyl peroxide (24.2 mg, 0.1 mmol) was added. The mixture was heated to 70 °C and stirred at 300 rpm for 10 h. The reaction was confirmed to be complete by thin-layer chromatography. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure to give white crystalline compound V (1.6 g, yield 55%).

[0056] S3: Compound III (290 mg, 1 mmol) and compound V (342 mg, 1.5 mmol) were dissolved in 5 mL of anhydrous acetonitrile, and K2CO3 (17 mg, 0.1 mmol) and KI (3 mg, 0.02 mmol) were added. The mixture was then heated to 60 °C under nitrogen and stirred at 300 rpm for 2 h. The reaction was monitored by thin-layer chromatography until the reaction was complete. The mixture was cooled, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether to dichloromethane volume ratio of 2:1) to obtain orange solid compound VI, which is the near-infrared fluorescent hydrogen sulfide donor (197 mg, yield 45%). 1H NMR (600MHz, DMSO-d6) δ7.67(d,J=8.8Hz,2H),7.53(d,J=8.5Hz,2H),7.47(d,J=8.5Hz,2H),7.28(d,J=6 .7Hz,2H),7.06(d,J=8.8Hz,2H),6.84(s,1H),5.19(s,2H),2.61(s,2H),2.55–2.52(m,2H),1.02(s,6H). 13 C NMR (150MHz, DMSO-d6) δ170.82,159.90,156.87,138.02,137.24,134.01,130.09,129.96,129.56,129.51,129. 49,127.96,126.55,122.39,115.78,114.51,113.69,75.87,68.98,42.78,40.53,38.65,32.15,27.92.HRMS:m / z calcd for C 27 H 23 ON3SNa[M+Na] + :460.1460,found:460.1460.

[0057] Study on the pharmacological activity of the near-infrared fluorescent hydrogen sulfide donor obtained in Example 1:

[0058] 1. Experimental instruments and reagents

[0059] Experimental instruments: Clean bench (Shanghai Lichen Instrument Technology Co., Ltd.); Incubator (Thermoelectron Corporation); Microplate reader (Thermo Fisher Scientific); Inverted fluorescence microscope (OLYMPUS).

[0060] Experimental reagents: DMEM cell culture medium (high glucose, GIBCO); fetal bovine serum (Hangzhou Sijiqing Co., Ltd.); LPS (TLR4 activator, Beyotime Biotechnology Co., Ltd.); Griess reagent kit (Beyotime Biotechnology Co., Ltd.); CCK-8 (Biosharp); DMSO (Sigma); N-ethylmaleimide (Adamas Reagent Co., Ltd.); cysteine ​​(Adamas Reagent Co., Ltd.); dexamethasone (Shanghai Mairui Biochemical Technology Co., Ltd.).

[0061] Cell lines: human cervical cancer cells HeLa and mouse mononuclear macrophage leukemia cells RAW264.7.

[0062] 2. Experimental Methods

[0063] (1) Cytotoxicity assay: HeLa cells or RAW 264.7 cells were cultured in DMEM containing 10% fetal bovine serum at 37℃ and 5% CO2. Cytotoxicity was studied using the standard CCK-8 assay. HeLa cells or RAW 264.7 cells were seeded into 96-well plates and cultured in a cell culture incubator for 12 hours. The culture medium was then removed, and DMEM containing different drug concentrations was added for another 24 hours. Subsequently, the drug solution was removed, and DMEM containing CCK-8 (10%) was added. After 40 minutes of culture, the OD value of the wells at 450 nm was measured using a microplate reader. Three replicate wells were set for each drug concentration, and the experiment was repeated three times in parallel. Cell viability was calculated using the following formula:

[0064]

[0065] Among them, A s This is the absorbance value of the experimental group, A. b This is the absorbance value of the blank group, A. c This is the absorbance value of the control group.

[0066] (2) Cell imaging experimental method: HeLa cells were sampled at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. The cells were then treated with the near-infrared fluorescent hydrogen sulfide donor prepared in Example 1 of this invention, the H2S probe AzMC, and the biothiol scavenger NEM (N-ethylmaleimide). As a control, one group of cells was pretreated with NEM for 30 minutes to eliminate endogenous biothiols. After administration, the cells were incubated for 45 minutes and then washed three times with PBS buffer. Finally, the treated cells were imaged under the red channel of an inverted fluorescence microscope.

[0067] (3) Anti-inflammatory assay method: RAW264.7 cells were cultured at a density of 4 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 mcg / mL in 96-well plates and cultured at 37°C and 5% CO2. After cell adhesion, the original culture medium was removed, and the cells were divided into 7 groups. The control group received no treatment, while the other 6 experimental groups were treated with LPS, LPS + compound VI (5 μM), LPS + compound VI (10 μM), LPS + compound VI (15 μM), LPS + DEX (dexamethasone), and compound III, respectively, for 24 hours. NO in cells was quantified using a Griess kit. 2- The absorbance was measured at 540 nm and compared with the standard curve provided in the Griess kit: y = 0.00535x + 0.05457 (R0). 2 =0.999) Calculate NO 2-Levels. Three replicate wells were set up for each drug concentration, and the experiment was repeated three times.

[0068] 3. Experimental Results

[0069] The cytotoxicity results of the near-infrared fluorescent hydrogen sulfide donor prepared in Example 1 of this invention against HeLa cells or RAW 264.7 cells are shown in the figure below. Figure 1 It can be seen that when the near-infrared fluorescent hydrogen sulfide donor is cultured at a concentration of 10 μM, at least 90% of HeLa cells maintain good activity within 24 hours, indicating that compound VI has minimal cytotoxicity at concentrations below this threshold, making it suitable for bioimaging applications. For Raw264.7 cells, the cell activity of compounds III and VI remains above 90% in the concentration range of 0–15 μM. Therefore, concentrations of 5, 10, and 15 μM were selected for anti-inflammatory studies.

[0070] The imaging results of HeLa cells using the near-infrared fluorescent hydrogen sulfide donor prepared in Example 1 of this invention are shown in the figure below. Figure 2 As can be seen, cells treated with compound VI alone exhibited obvious red fluorescence, indicating that compound VI was successfully activated and produced a fluorescent response. In contrast, cells pretreated with the biothiol scavenger NEM showed significantly reduced fluorescence in both channels. However, after adding exogenous cysteine ​​(Cys) to NEM-pretreated cells, compound VI and the hydrogen sulfide probe AzMC showed red and blue fluorescence in their respective channels, respectively, indicating that the near-infrared fluorescent hydrogen sulfide donor prepared in this invention can visualize hydrogen sulfide release through near-infrared fluorescence response.

[0071] The near-infrared fluorescent hydrogen sulfide donor prepared in Example 1 of this invention has an effect on NO in inflammatory cells. 2- The results of the horizontal influence are shown in Figure 3 It can be seen that cells incubated with LPS exhibit NO 2- Levels were significantly elevated; in the positive control group, treatment with the potent anti-inflammatory drug dexamethasone (DEX) resulted in NO 2- Accumulation was significantly reduced; NO was observed when cells incubated with LPS were treated with compound VI. 2- The accumulation decreased in a concentration-dependent manner, indicating that compound VI possesses anti-inflammatory properties; byproduct compound III showed an effect on NO in LPS-induced inflammatory cells. 2- The accumulation did not show any inhibitory effect, indicating that the H2S released by compound VI plays a crucial role in anti-inflammation.

[0072] The near-infrared fluorescent hydrogen sulfide donor prepared by this invention has good visualization capabilities for hydrogen sulfide release and good safety, and has the potential to serve as a tool for exploring the anti-inflammatory properties of H2S in biological systems.

Claims

1. A near-infrared fluorescent hydrogen sulfide donor, characterized in that: The structural formula of the near-infrared fluorescent hydrogen sulfide donor is shown below: 。 2. A method for preparing the near-infrared fluorescent hydrogen sulfide donor as described in claim 1, characterized in that: Includes the following steps: S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) malononitrile and compound II p-hydroxybenzaldehyde were dissolved in a solvent, and an alkaline catalyst was added dropwise. The mixture was stirred under an inert gas atmosphere to obtain compound III. S2: Compound IV p-toluene isothiocyanate and N-bromosuccinic acid imine were dissolved in a solvent, an initiator was added, and the mixture was stirred to obtain compound V; S3: Dissolve compound III obtained in S1 and compound V obtained in S2 in a solvent, and stir and react under alkaline and inert gas conditions to obtain compound VI, which is the near-infrared fluorescent hydrogen sulfide donor.

3. The preparation method according to claim 2, characterized in that: In S1, the molar ratio of (3,5,5-trimethyl-2-cyclohexene-1-diene)malonitrile to p-hydroxybenzaldehyde is 1:1~3; the molar ratio of (3,5,5-trimethyl-2-cyclohexene-1-diene)malonitrile to solvent is 1:30~40; and the volume ratio of alkaline catalyst to solvent is 1:30~40. In step S2, the molar ratio of p-toluene isothiocyanate to N-bromosuccinic acid imine is 1:1~3; the molar ratio of N-bromosuccinic acid imine to solvent is 1:10~15; and the molar ratio of p-toluene isothiocyanate to initiator is 10~15:

1. In step S3, the molar ratio of compound III obtained in step S1 to compound V obtained in step S2 is 1:1~3; the molar ratio of compound III obtained in step S1 to the solvent is 1:80~120.

4. The preparation method according to claim 2, characterized in that: In step S1, the solvent is one of methanol, ethanol, or n-butanol; the alkaline catalyst is one of triethylamine, piperidine, or N,N-diisopropylethylamine. In step S2, the solvent is one of dichloromethane, chloroform, or carbon tetrachloride; the initiator is benzoyl peroxide. In step S3, the solvent is anhydrous acetonitrile; the alkaline condition is achieved by adding potassium carbonate and potassium iodide. In S1 and S3, the inert gas is one of nitrogen, argon, or helium.

5. The preparation method according to claim 2 or 4, characterized in that: The molar ratio of compound III obtained in S1 to potassium carbonate is 20:1~3; the molar ratio of compound III obtained in S1 to potassium iodide is 50:1~3.

6. The preparation method according to claim 2, characterized in that: In S1, the stirring reaction conditions are: stirring at 250-350 rpm for 12-14 h at 80-90°C; In S2, the stirring reaction conditions are: stirring at 250-350 rpm for 10-12 h at 70-80°C; In step S3, the stirring reaction conditions are: stirring at 250-350 rpm for 1-2 hours at 60-70°C.

7. The preparation method according to claim 2, characterized in that: Includes the following steps: S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) malononitrile and compound II p-hydroxybenzaldehyde were dissolved in ethanol, piperidine was added dropwise, and the reaction was carried out under nitrogen and at 80-90℃ with stirring at 250-350 rpm for 12-14 h. After the reaction was completed, the mixture was evaporated under reduced pressure and purified to obtain compound III. S2: Compound IV p-toluene isothiocyanate and N-bromosuccinic acid imine were dissolved in carbon tetrachloride, and benzoyl peroxide was added as a catalyst. The mixture was stirred at 250-350 rpm at 70-80℃ for 10-12 h. After the reaction was completed, the mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain compound V. S3: Dissolve compound III obtained in S1 and compound V obtained in S2 in anhydrous acetonitrile, add potassium carbonate and potassium iodide, and stir at 250-350 rpm for 1-2 h under nitrogen and 60-70℃ conditions. After the reaction is completed, cool, filter, concentrate the filtrate under reduced pressure, and purify to obtain compound VI, which is the near-infrared fluorescent hydrogen sulfide donor.

8. The application of the near-infrared fluorescent hydrogen sulfide donor according to claim 1 in the visualization of hydrogen sulfide release.

9. The use of the near-infrared fluorescent hydrogen sulfide donor according to claim 1 in the preparation of anti-inflammatory drugs.

Images