Near-infrared fluorescent hydrogen sulfide donor as well as preparation method and application thereof

Abstract

The invention relates to a near-infrared fluorescent hydrogen sulfide donor as well as a preparation method and application thereof, and belongs to the technical field of biochemistry. The near-infrared fluorescent hydrogen sulfide donor has the following structural formula. According to the invention, p-tolyl isothiocyanate is combined with dicyanoisophorone fluorophore, such that a near-infrared fluorescence hydrogen sulfide donor is synthesized. The near-infrared fluorescence hydrogen sulfide donor provided by the invention can realize visualization of hydrogen sulfide release through near-infrared fluorescence response, in addition, can also effectively reduce the level of NO2 <-> in inflammatory cells, and has huge potential in the aspect of treating inflammation. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a near-infrared fluorescent hydrogen sulfide donor and a preparation method and application thereof, belonging to the technical field of biochemistry. Background Art

[0002] Hydrogen sulfide (H2S) is a key gaseous signaling molecule in living systems and has a significant impact on various pathological and physiological states. Its roles include protecting the central nervous system, regulating oxidative stress in the cardiovascular system, and maintaining blood pressure homeostasis. Notably, H2S is considered to be an important endogenous neuroprotectant that contributes to antioxidant, anti-inflammatory, and anti-apoptotic effects. Therefore, compared with traditional H2S donors, the development of H2S donors marked by specific reaction substrates and simultaneous fluorescence changes has considerable prospects. Such compounds can not only produce fluorescent responses to specific conditions or substrates, but also effectively serve as H2S donors in the treatment of related diseases.

[0003] Existing hydrogen sulfide donors have defects such as low hydrogen sulfide release efficiency, many byproducts, and limited hydrogen sulfide release detection methods. The present invention selects dicyanoisophorone as a fluorophore and p-toluene isothiocyanate as a reaction recognition unit to synthesize a near-infrared fluorescent hydrogen sulfide donor. The structure of the near-infrared fluorescent hydrogen sulfide donor provided by the present invention ensures the formation of a single fluorescent product, thereby eliminating potential interference from two fluorophores (primary amines and dihydrothiazole carboxylic acids), performs well in fluorescence response analysis, is suitable for visualizing the release of hydrogen sulfide from donors in living cells, and can act as a slow-release H2S donor to effectively reduce NO in inflammatory cells. 2- level. Summary of the invention

[0004] In order to overcome the above-mentioned defects in the prior art, the present invention provides a near-infrared fluorescent hydrogen sulfide donor and a preparation method and application thereof.

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

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

[0007]

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

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

[0010]

[0011] S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) propanedinitrile and compound II p-hydroxybenzaldehyde are dissolved in a solvent, a basic catalyst is added dropwise, and the mixture is stirred under an inert gas to react to obtain compound III;

[0012] S2: dissolving compound IV p-toluene isothiocyanate and N-bromosuccinimide in a solvent, adding a catalytic amount of an initiator, and stirring the reaction to obtain compound V;

[0013] S3: Dissolve the compound III obtained in S1 and the compound V obtained in S2 in a solvent, and react with stirring under alkaline and inert gas conditions to obtain compound VI, which is a 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)malononitrile to p-hydroxybenzaldehyde is 1:1-3.

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

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

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

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

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

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

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

[0022] In the above technical solution, in 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 S2, the solvent is one of dichloromethane, chloroform or carbon tetrachloride.

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

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

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

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

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

[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 S1, the stirring reaction condition is stirring the reaction at 80-90°C and 250-350 rpm for 12-14 hours.

[0032] In the above technical solution, in S2, the stirring reaction condition is stirring the reaction at 70-80°C and 250-350 rpm for 10-12 hours.

[0033] In the above technical solution, in S3, the stirring reaction condition is stirring the reaction at 60-70°C and 250-350 rpm for 1-2h.

[0034] Preferably, the method for preparing the near-infrared fluorescent hydrogen sulfide donor comprises the following steps:

[0035]

[0036] S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) propanonitrile and compound II p-hydroxybenzaldehyde are dissolved in ethanol, piperidine is added dropwise, and the mixture is stirred at 250-350 rpm under nitrogen and 80-90°C for 12-14 hours. After the mixture is reacted, it is evaporated under reduced pressure and purified to obtain compound III;

[0037] S2: dissolving compound IV p-toluene isothiocyanate and N-bromosuccinimide in carbon tetrachloride, adding a catalytic amount of benzoyl peroxide, stirring at 70-80°C and 250-350 rpm for 10-12 hours, filtering after completion, and evaporating the filtrate under reduced pressure to obtain compound V;

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

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

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

[0041] Furthermore, the near-infrared fluorescent hydrogen sulfide donor can realize 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] Beneficial effects of the present invention: The near-infrared fluorescent hydrogen sulfide donor prepared by the present invention has a structure that helps to release hydrogen sulfide while also generating a single fluorophore for characterization. This design overcomes the limitation of the traditional isothiocyanate structure that produces two fluorophores after response. The present invention can be used to release H2S in living cells and visualize its presence through near-infrared fluorescence response; it can effectively reduce NO in inflammatory cells 2- levels, and has great potential in treating inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[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 imaging diagram of the near-infrared fluorescent hydrogen sulfide donor obtained in Example 1 of the present invention visualizing hydrogen sulfide release in living cells.

[0046] Figure 3 The near-infrared fluorescent hydrogen sulfide donor obtained in Example 1 of the present invention is used to treat NO in inflammatory cells. 2- Level test chart.

[0047] Figure 4 The near-infrared fluorescent hydrogen sulfide donor obtained in Example 1 of the present invention visualizes the release of hydrogen sulfide in cells and reduces NO 2- Schematic diagram of the level. DETAILED DESCRIPTION

[0048] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0049] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0050] The near-infrared fluorescent hydrogen sulfide donor of the present 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 comprises the following steps:

[0054] S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) propanonitrile (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. Under nitrogen conditions, the temperature was raised to 80°C and stirred at 300 rpm for 12 h. After the completion of the reaction was confirmed by thin layer chromatography, the solvent was evaporated under reduced pressure and purified by silica gel column chromatography to obtain orange powder compound III (472 mg, yield 65%);

[0055] S2: N-bromosuccinimide (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 temperature was raised to 70°C, and the reaction was stirred at 300 rpm for 10 h. After the completion of the reaction was confirmed by thin layer chromatography, the reaction mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain 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, K2CO3 (17 mg, 0.1 mmol) and KI (3 mg, 0.02 mmol) were added, and then the temperature was raised to 60°C under nitrogen, and the reaction was stirred at 300 rpm for 2 h. The reaction was monitored by thin layer chromatography until the reaction was completed, cooled, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (the volume ratio of petroleum ether to dichloromethane was 2:1) to obtain an orange solid compound VI, which was a 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.); constant temperature incubator (Thermoelectron Corporation); microplate reader (Thermo Fisher Scientific); inverted fluorescence microscope (OLYMPUS).

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

[0061] Cell lines: human cervical cancer cell line HeLa, mouse mononuclear macrophage leukemia cell line RAW264.7.

[0062] 2. Experimental Methods

[0063] (1) Cytotoxicity experimental method: HeLa cells or RAW 264.7 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2. Cytotoxicity was studied using a standard CCK-8 experiment. HeLa cells or RAW264.7 cells were inoculated into a 96-well plate and cultured in a cell culture incubator for 12 hours. The culture medium was then removed, and DMEM containing different drug concentrations was added and cultured for another 24 hours. The drug solution was then removed, and a DMEM solution containing CCK-8 (10%) was added. After 40 minutes of culture, the OD value of the well plate at 450nm was measured using an enzyme reader. Three replicate wells were set for each drug concentration, and the experiment was repeated three times in parallel. The cell viability was calculated according to the following formula:

[0064]

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

[0066] (2) Cell imaging experimental method: HeLa cells were plated at 5×10 3 The cells were seeded in a 96-well plate at a density of 10 cells. The cells were then treated with the near-infrared fluorescent hydrogen sulfide donor prepared in Example 1 of the present invention, the H2S probe AzMC, and the biothiol scavenger NEM (N-ethylmaleimide). As a control, a group of cells were pretreated with NEM for 30 minutes to eliminate endogenous biothiols. After administration, they were incubated for 45 minutes and then washed three times with PBS buffer solution. Finally, the treated cells were imaged under the red channel of an inverted fluorescence microscope.

[0067] (3) Anti-inflammatory experimental method: RAW264.7 cells were cultured at 4×10 4 The cells were seeded in a 96-well plate at a density of 100 cells and cultured at 37°C and 5% CO2. After the cells adhered, the original culture medium was removed and the cells were divided into 7 groups. The control group did not receive treatment, and the remaining 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 for 24 hours. The Griess kit was used to quantify NO in the cells. 2- The absorbance was measured at 540 nm and the standard curve provided in the Griess kit was: y = 0.00535x + 0.05457 (R 2 =0.999) to calculate NO 2-Three replicate wells were set up for each drug concentration, and the experiment was repeated three times.

[0068] 3. Experimental results

[0069] The cytotoxicity of the near-infrared fluorescent hydrogen sulfide donor prepared in Example 1 of the present invention to HeLa cells or RAW 264.7 cells is shown in 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 the HeLa cells maintain good activity within 24 hours, indicating that compound VI has minimal cytotoxicity when the concentration is below this threshold, making it suitable for bioimaging applications; for Raw264.7 cells, the cell activity of compounds III and compound VI remains above 90% in the concentration range of 0 to 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 the present invention are shown in Figure 2 It can be seen that: cells treated only with compound VI showed 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 weakened fluorescence under both channels; however, after adding exogenous cysteine ​​(Cys) to the NEM-pretreated cells, compound VI and the hydrogen sulfide probe AzMC showed red and blue fluorescence under their corresponding channels, respectively, indicating that the near-infrared fluorescent hydrogen sulfide donor prepared by the present invention can realize the visualization of hydrogen sulfide release through near-infrared fluorescence response.

[0071] The near-infrared fluorescent hydrogen sulfide donor prepared in Example 1 of the present invention has an effect on NO in inflammatory cells 2- The horizontal impact results are shown in Figure 3 , it can be seen that cells incubated with LPS showed NO 2- In the positive control group, treatment with the potent anti-inflammatory drug dexamethasone (DEX) led to a significant increase in NO 2- When LPS-incubated cells were treated with compound VI, NO accumulation was significantly reduced. 2- The accumulation decreased in a concentration-dependent manner, indicating that compound VI has anti-inflammatory properties; the byproduct compound III has an anti-inflammatory 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 the present invention has good visualization function of hydrogen sulfide release and good safety, and has the potential to be used 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 as follows:

2. A method for preparing a near-infrared fluorescent hydrogen sulfide donor, characterized in that: The following steps are involved: S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) propanedinitrile and compound II p-hydroxybenzaldehyde are dissolved in a solvent, a basic catalyst is added dropwise, and the mixture is stirred under an inert gas to react to obtain compound III; S2: dissolving compound IV p-toluene isothiocyanate and N-bromosuccinimide in a solvent, adding a catalytic amount of an initiator, and stirring the reaction to obtain compound V; S3: Dissolve the compound III obtained in S1 and the compound V obtained in S2 in a solvent, and react with stirring under alkaline and inert gas conditions to obtain compound VI, which is a 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)malononitrile to p-hydroxybenzaldehyde is 1:1-3; the molar ratio of (3,5,5-trimethyl-2-cyclohexene-1-diene)malononitrile to solvent is 1:30-40; the volume ratio of the alkaline catalyst to solvent is 1:30-40; In S2, the molar ratio of p-toluene isothiocyanate to N-bromosuccinimide is 1:1-3; the molar ratio of N-bromosuccinimide to solvent is 1:10-15; the molar ratio of p-toluene isothiocyanate to initiator is 10-15:1; In S3, the molar ratio of the compound III obtained in S1 to the compound V obtained in S2 is 1:1-3; the molar ratio of the compound III obtained in S1 to the solvent is 1:80-120.

4. The preparation method according to claim 2, characterized in that: In S1, the solvent is one of methanol, ethanol or n-butanol; the alkaline catalyst is one of triethylamine, piperidine or N,N-diisopropylethylamine; In S2, the solvent is one of dichloromethane, chloroform or carbon tetrachloride; the initiator is benzoyl peroxide; In S3, the solvent is anhydrous acetonitrile; the alkaline condition is the addition of 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 the compound III obtained in S1 to potassium carbonate is 20:1-3; the molar ratio of the 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 condition is to stir the reaction at 80-90°C and 250-350 rpm for 12-14 hours; In S2, the stirring reaction condition is stirring the reaction at 70-80°C and 250-350 rpm for 10-12 hours; In S3, the stirring reaction condition is to stir the reaction at 60-70° C. and 250-350 rpm for 1-2 hours.

7. The preparation method according to claim 2, characterized in that: The following steps are involved: S1: Compound I (3,5,5-trimethyl-2-cyclohexene-1-diene) propanonitrile and compound II p-hydroxybenzaldehyde are dissolved in ethanol, piperidine is added dropwise, and the mixture is stirred at 250-350 rpm under nitrogen and 80-90°C for 12-14 hours. After the mixture is reacted, it is evaporated under reduced pressure and purified to obtain compound III; S2: dissolving compound IV p-toluene isothiocyanate and N-bromosuccinimide in carbon tetrachloride, adding a catalytic amount of benzoyl peroxide, stirring at 70-80°C and 250-350 rpm for 10-12 hours, filtering after completion, and evaporating the filtrate under reduced pressure to obtain compound V; S3: Dissolve the compound III obtained in S1 and the compound V obtained in S2 in anhydrous acetonitrile, add potassium carbonate and potassium iodide, and stir the reaction at 250-350 rpm under nitrogen and 60-70°C for 1-2 hours. After the reaction, cool and filter, concentrate the filtrate under reduced pressure, and purify to obtain compound VI, which is a near-infrared fluorescent hydrogen sulfide donor.

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

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

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