Fluorescent hydrogen sulfide donor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a fluorescent hydrogen sulfide donor as well as a preparation method and application thereof. The fluorescent hydrogen sulfide donor has two activation modes: one mode is to activate a phenylboronic acid ester structure to release hydrogen sulfide under the action of active oxygen; and secondly, hydrogen sulfide is released by decomposing a thioacetal structure by means of illumination. The two activation modes enable the release rate of hydrogen sulfide to be accurately controlled. It is worth mentioning that the donor emits blue fluorescence when not releasing the hydrogen sulfide and is converted into green fluorescence after releasing the hydrogen sulfide, so that the release amount of the hydrogen sulfide can be calculated according to the change of the fluorescence ratio, and real-time imaging monitoring is realized. In addition, the donor is simple and convenient in synthesis process and stable in property, and shows huge potential in the field of hydrogen sulfide drug development.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a fluorescent hydrogen sulfide donor, a preparation method thereof, and an application thereof. Background Art

[0002] Hydrogen sulfide (H 2 S) is an important endogenous signaling molecule and has great potential in the treatment of cancer, inflammation, and myocardial ischemia-reperfusion injury. In addition, there are also reports on the treatment of intestinal diseases, reproductive dysfunction, Parkinson's disease, neurodegenerative diseases, and brain injury with H 2 S. These findings highlight the positive role of H 2 S in human health, as well as the potential of synthesizing various H 2 S donors for the treatment of different injuries and diseases. To further explore the role of exogenous H 2 S in organisms, researchers have selected inorganic salts such as sodium sulfide (Na 2 S) and sodium hydrosulfide (NaHS) as H 2 S donors. Although the above-mentioned inorganic salts can release H 2 S when dissolved in water, they cannot control the release time and rate, which may lead to too high H 2 S concentration and thus produce negative effects. In addition, such inorganic salts as H 2 S donors do not have the ability of fluorescence imaging, which greatly limits their practical applications. Therefore, the design and synthesis of controllable H 2 S donors with fluorescence imaging ability are of great significance for the study of H 2 S in biology.

[0003] So far, controllable H 2 S donors with fluorescence imaging ability have been reported, such as pH-triggered, ROS-triggered, enzyme-triggered, and biothiol-triggered modes. Although these donors have made some progress in studying the biological roles of H 2 S, it is important to consider that the organism is a complex environment containing substances such as ROS, enzymes, and biothiols, which may affect the triggering of the above-mentioned H 2 S donors.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention provides a fluorescent hydrogen sulfide donor (named YL-DB), and the structural formula of the fluorescent hydrogen sulfide donor is shown in Formula I:

[0006]

[0007] The YL-DB fluorescent donor designed by the present invention has a core structure composed of the fluorescent parent nucleus 6-(dimethylamino)-2-naphthaldehyde. This parent nucleus is ingeniously connected to the phenylboronic acid ester group capable of specifically recognizing reactive oxygen species through a thioacetal bond. After reacting with reactive oxygen species (ROS), the YL-DB donor will initiate a self-dissolution process, releasing 4-methylenecyclohexyl-2,5-dien-1-one. Subsequently, the formed unstable intermediate thioacetal continues to decompose, ultimately releasing hydrogen sulfide and the fluorescent parent nucleus 6-(dimethylamino)-2-naphthaldehyde. It is worth mentioning that the thioacetal structure of the fluorescent hydrogen sulfide donor can also decompose under light conditions, also releasing hydrogen sulfide and the fluorescent parent nucleus, achieving a unique effect of dual-mode excitation. Due to the change in the substituent at the 1-position of the fluorescent parent nucleus 6-(dimethylamino)-2-naphthaldehyde, the degree of intramolecular charge transfer (ICT) is affected, thus triggering the characteristic of ratio-type fluorescence change.

[0008] Based on the same technical concept, the present invention further provides a preparation method of a fluorescent hydrogen sulfide donor, and the preparation method includes the following steps:

[0009] (I) Preparation of intermediate:

[0010] (I-1) Dissolve (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol in dichloromethane and stir evenly, then add phosphorus tribromide under an inert atmosphere and react to obtain a first mixed solution;

[0011] (I-2) Add saturated sodium bicarbonate solution to the first mixed solution to quench the reaction, extract the aqueous layer with dichloromethane, combine the organic layers, dry with sodium sulfate, filter and concentrate to obtain a residue;

[0012] (I-3) Dissolve the residue in tetrahydrofuran, add thiourea salt and react, then remove the solvent under reduced pressure, dissolve the obtained salt in water, then add dichloromethane, and blow the reaction under an inert gas to obtain a heterogeneous solution;

[0013] (I-4) Add sodium metabisulfite to the heterogeneous solution, reflux the obtained mixture under an inert gas atmosphere, then wash and combine the organic layers, and finally dry, concentrate and purify to obtain a white crystalline solid, which is the intermediate, named (4-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl))phenyl)methanethiol;

[0014] (II) Preparation of fluorescent hydrogen sulfide donor:

[0015] (II-1) Dissolve the intermediate and compound 6-(dimethylamino)-2-naphthaldehyde in anhydrous dichloromethane, and then add boron trifluoride diethyl ether for reaction;

[0016] (II-2) Quench the excessive boron trifluoride diethyl ether with saturated sodium bicarbonate solution, extract with anhydrous dichloromethane, then remove the solvent under reduced pressure and recrystallize with ethanol to obtain the product, which is a fluorescent hydrogen sulfide donor, named 6-[(bis(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)thio)methyl]-N,N-dimethylnaphthalen-2-amine.

[0017] Preferably, in step (I-1), the molar ratio of (4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)phenyl)methanol to phosphorus tribromide is 1:1;

[0018] And / or, in step (I-1), the reaction temperature is 0 °C and the reaction time is 1 h.

[0019] Preferably, in step (I-2), the conditions for the quenching reaction are: add methanol at 0 °C and stir for 15 - 20 min.

[0020] Preferably, in step (I-3), after adding the thiourea salt, react at 24 - 26 °C for 12 - 14 h;

[0021] And / or, in step (I-3), the inert gas is nitrogen and the purging time is 5 - 8 min.

[0022] Preferably, in step (I-4), the inert gas is nitrogen and the reflux time is 4 - 5 h.

[0023] Preferably, in step (II-1), the reaction is: first stir at 0 °C for 2 - 3 h, then raise the temperature to 24 - 26 °C and continue to stir for 1 - 1.5 h.

[0024] Based on the same technical concept, another aspect of the present invention is to provide an application of a fluorescent hydrogen sulfide donor as a fluorescent probe, and an application of the fluorescent hydrogen sulfide donor in the preparation of a visual hydrogen sulfide drug.

[0025] The present invention has many beneficial effects, specifically manifested as:

[0026] 1. The fluorescent hydrogen sulfide donor (YL-DB) of the present invention releases hydrogen sulfide by activating two recognition groups, namely thioacetal and phenylboronic acid ester, which makes up for the deficiency of the current hydrogen sulfide donors that release hydrogen sulfide through a single activation method. Moreover, the hydrogen sulfide release rates of the two are different, making it applicable to a variety of biological conditions and having practical application value. That is, YL-DB can release hydrogen sulfide through the specific interaction between reactive oxygen species and phenylboronic acid ester groups. At the same time, under light illumination conditions, the thioacetal unit in the structure can also be activated to release hydrogen sulfide. The effective combination of this dual-activation mode greatly expands the application scenarios of hydrogen sulfide donors in practical use, enhancing their functionality and operational flexibility.

[0027] 2. When in the state of not releasing hydrogen sulfide, YL-DB can emit blue fluorescence, and its characteristic emission wavelength is located at 420 nm. Once hydrogen sulfide is released, it turns into 6-(dimethylamino)-2-naphthaldehyde that can emit green fluorescence, with an emission wavelength of 540 nm. By monitoring the ratio change of fluorescence, the release amount of hydrogen sulfide can be accurately calculated. This method has significantly improved the measurement accuracy compared with the existing open-type and enhanced-type fluorescent hydrogen sulfide donors, effectively reducing the measurement error, as shown in Formula II.

[0028]

[0029] 3. The rate of hydrogen sulfide release from YL-DB by the activation of reactive oxygen species is 1.5×10 -4 / s, and the reaction rate of hydrogen sulfide release by light illumination activation is 1.05×10 -2 / s. The rate gap between the two methods is large, which can truly achieve the controllable release of hydrogen sulfide.

[0030] 4. YL-DB can maintain the structural stability in a variety of pH environments, and its two activation modes are not affected by the change of pH value. Therefore, it can be widely applied to different cell organelles (such as lysosomes, mitochondria, Golgi apparatus, etc.) of living cells to achieve the precise release of hydrogen sulfide.

[0031] 5. YL-DB can effectively release hydrogen sulfide in a physiological reactive oxygen species environment or under light illumination conditions, and can generate fluorescence signals before and after the release process. Based on this characteristic, YL-DB becomes an ideal choice for preparing visual hydrogen sulfide drugs.

[0032] 6. YL-DB can be used to explore the physiological and pathological effects of hydrogen sulfide, and simultaneously achieve delivery, quantification, and imaging.

[0033] 7. The synthesis conditions of YL-DB are mild. It is obtained by the addition of thiol and aldehyde to form thioacetal. The reaction method is simple, stable, and has a high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0035] Figure 1 It is the synthetic route diagram of the fluorescent hydrogen sulfide donor YL-DB.

[0036] Figure 2 It is the fluorescence change spectrum of the fluorescent hydrogen sulfide donor YL-DB when excited by ultraviolet light.

[0037] Figure 3 It is the fluorescence change spectrum of the fluorescent hydrogen sulfide donor YL-DB when excited by the reactive oxygen species hydrogen peroxide.

[0038] Figure 4 It is the nuclear magnetic resonance spectrum (proton spectrum) of the fluorescent hydrogen sulfide donor YL-DB.

[0039] Figure 5 It is the nuclear magnetic resonance spectrum (carbon spectrum) of the fluorescent hydrogen sulfide donor YL-DB. Detailed implementation manners

[0040] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will describe the technical solutions of the present invention in detail. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts fall within the scope protected by the present invention.

[0041] Embodiment

[0042] This embodiment provides a preparation method of a fluorescent hydrogen sulfide donor, and its synthesis process is as Figure 1 shown, and the preparation method includes the following steps:

[0043] (I) Preparation of intermediate:

[0044] In dry dichloromethane (10 mL), (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (1.2 g, 5.13 mmol) was dissolved and stirred evenly, and then phosphorus tribromide (481 μL, 5.13 mmol) was slowly added under a nitrogen atmosphere. The reaction was stirred at 0 °C for 1 hour. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, 15 mL of saturated sodium bicarbonate solution was added to quench the reaction (10 mL of methanol was added and stirred for 15 min at 0 °C). The aqueous layer was extracted with dichloromethane (3 × 10 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The resulting residue was slightly brown. The residue was further dissolved in dry tetrahydrofuran (15 mL), and thiourea salt (1 g, 13 mmol) was added at room temperature. The reaction was stirred overnight at room temperature. After the reaction was completed (monitored by TLC), the solvent was removed under reduced pressure, and the resulting salt was dissolved in water (20 mL), and then dichloromethane (30 mL) was added. The reaction was purged with nitrogen for 5 minutes. 4 equivalents of sodium metabisulfite (Na 2 S 2 O 5 ) was added to this heterogeneous solution. The resulting mixture was refluxed for 4 hours under a nitrogen atmosphere. Then the solution was cooled to room temperature and washed twice with DCM (20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The crude product obtained was purified by silica gel column chromatography (1% EtOAc / hexane). The resulting compound was a white crystalline solid (1.1 g, 82%), which was the intermediate, named (4-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl))phenyl)methanethiol.

[0045] Intermediate characterization data are as follows: 1 H (400 MHz, CDCl 3 ): δ 7.70 (d, J = 8.0 Hz, 2H), δ 7.26 (d, J = 8.0 Hz, 2H), δ 3.67 (d, J = 7.6 Hz, 2H), δ 1.68 (t, J = 7.6 Hz, 1H), 1.26 (s, 12H).

[0046] (II) Preparation of fluorescent hydrogen sulfide donor:

[0047] Compound (4-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl))phenyl)methanethiol (313.67 mg, 1.25 mmol) and compound 6-(dimethylamino)-2-naphthaldehyde (100 mg, 0.5 mmol) were dissolved in anhydrous dichloromethane (15 mL), and boron trifluoride diethyl etherate (BF 3 ·Et 2O) (200 μL, 0.75 mmol) was stirred at the same temperature for 2 hours, then heated to room temperature (24 - 26 °C) and stirred for an additional 1 hour. The reaction process was monitored by thin-layer chromatography. The excess BF 3 ·Et 2 O was quenched with saturated sodium bicarbonate solution and extracted three times with anhydrous dichloromethane (15 mL × 3). The solvent was removed under reduced pressure, and the product was obtained by recrystallization from ethanol (86.1 mg, 83%).

[0048] The characterization data of the fluorescent hydrogen sulfide donor are as follows: 1 H (400 MHz, DMSO-d 6 ): 7.73 (d, J = 9.1 Hz, 1H), 7.65 (s, 1H), 7.59 (d, J = 7.8 Hz, 4H), 7.56 (d, J = 1.8 Hz, 1H), 7.24 (ddd, J = 18.3, 8.8, 2.2 Hz, 2H), 7.09 (d, J = 8.0 Hz, 4H), 6.91 (d, J = 2.5 Hz, 1H), 4.62 (s, 1H), 3.76 (d, J = 13.4 Hz, 2H), 3.59 (d, J = 13.5 Hz, 2H), 3.00 (s, 6H), 1.31 (s, 24H); 1 H (101 MHz, DMSO-d 6 ): δ 149.3, 142.2, 135.1, 134.8, 132.5, 129.1, 128.8, 127.2, 126.3, 126.1, 125.9, 117.1, 106.0, 84.1, 51.5, 36.4, 25.2, 25.1.

[0049] Detection Example

[0050] Figure 2 is the fluorescence change spectrum of YL-DB upon ultraviolet light excitation. In the present invention, fluorescence spectrophotometer technology was used to monitor the fluorescence spectral changes of the fluorescent donor at different irradiation times. The measurement system was a phosphate buffer solution - dimethyl sulfoxide system (10 mM PBS, pH = 7.4, DMSO = 5%, V / V). The fluorescent donor YL-DB (10 μM) was prepared in a 5 mL cuvette. The sample was subjected to continuous light irradiation for different times from 0 to 15 minutes, and the dynamic changes of the fluorescence spectrum during this process were recorded in detail (specific results are shown in Figure 2 ). According to the ultraviolet absorption spectrum of the donor, its maximum absorption is at 380 nm. Therefore, the fluorescence emission spectrum was measured using an excitation wavelength of 380 nm, and the emission wavelength range was set to 400 - 650 nm (λex = 380 nm, λem = 400 - 650 nm, slit width = 3 nm).

[0051] Figure 3 It is the fluorescence change spectrum of YL-DB when excited by the reactive oxygen species hydrogen peroxide. In this study, fluorescence spectrophotometer technology was used to monitor the fluorescence spectrum changes of the fluorescent donor under different reactive oxygen species stimulations. The measurement system was a phosphate buffer solution-dimethyl sulfoxide system (10 mM PBS, pH = 7.4, DMSO = 5%, V / V). The fluorescent donor YL-DB (10 μM) was configured in a 5 mL cuvette. Hydrogen peroxide (100 μM) was added, and the reaction was carried out at 37 °C for 0 to 12 hours, and the fluorescence emission spectrum under the same conditions was continuously measured. According to the ultraviolet absorption spectrum of the donor, its maximum absorption was at 380 nm. Therefore, the fluorescence emission spectrum was measured using an excitation wavelength of 380 nm, and the emission wavelength range was set to 400 - 650 nm (λex = 380 nm, λem = 400 - 650 nm, slit width = 3 nm).

[0052] Figure 4 It is the measurement result (400 MHz) of the hydrogen spectrum of YL-DB in DMSO-d 6 deuterated solution.

[0053] Figure 5 It is the measurement result (101 MHz) of the carbon spectrum of YL-DB in DMSO-d 6 deuterated solution.

[0054] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. A fluorescent hydrogen sulfide donor, characterized in that: The structural formula of the fluorescent hydrogen sulfide donor is shown in Formula I:

2. The method for preparing the fluorescent hydrogen sulfide donor according to claim 1, characterized in that: The preparation method comprises the following steps: (I) Preparation of intermediates: (I-1) dissolving (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)phenyl)methanol in dichloromethane and stirring the mixture evenly, then adding phosphorus tribromide under an inert atmosphere and reacting the mixture to obtain a first mixed solution; (I-2) adding a saturated sodium bicarbonate solution to the first mixed solution to quench the reaction, extracting the aqueous layer with dichloromethane, and drying the combined organic layers with sodium sulfate, filtering and concentrating to obtain a residue; (I-3) dissolving the residue in tetrahydrofuran, adding thiourea salt to react, then removing the solvent under reduced pressure, dissolving the obtained salt in water, then adding dichloromethane, and reacting under inert gas purge to obtain a heterogeneous solution; (I-4) adding sodium metabisulfite salt to the heterogeneous solution, and reflux the obtained mixture under an inert gas atmosphere, then washing and combining the organic layers, and finally drying, concentrating and purifying to obtain a white crystalline solid, which is an intermediate named (4-(4,4,5-trimethyl-1,3,2-dioxaborolane-2-yl))phenyl)methyl mercaptan; (II) Preparation of fluorescent hydrogen sulfide donor: (II-1) dissolving the intermediate and the compound 6-(dimethylamino)-2-naphthaldehyde in anhydrous dichloromethane, and then adding boron trifluoride etherate to react; (II-2) The excess boron trifluoride etherate is quenched with a saturated sodium bicarbonate solution and extracted with anhydrous dichloromethane. The solvent is then removed under reduced pressure and recrystallized from ethanol to obtain a fluorescent hydrogen sulfide donor named 6-[(bis(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)thio)methyl]-N,N-dimethylnaphthalene-2-amine.

3. The method for preparing the fluorescent hydrogen sulfide donor according to claim 2, characterized in that: In step (I-1), the molar ratio of (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)phenyl)methanol to phosphorus tribromide is 1:1; And / or, in step (I-1), the reaction temperature is 0°C and the reaction time is 1 h.

4. The method for preparing the fluorescent hydrogen sulfide donor according to claim 2, characterized in that: In step (I-2), the quenching reaction is performed by adding methanol at 0°C and stirring for 15 to 20 minutes.

5. The method for preparing the fluorescent hydrogen sulfide donor according to claim 2, characterized in that: In step (I-3), after adding thiourea salt, react at 24 to 26° C. for 12 to 14 hours; And / or, in step (I-3), the inert gas is nitrogen, and the purge time is 5 to 8 minutes.

6. The method for preparing the fluorescent hydrogen sulfide donor according to claim 2, characterized in that: In step (I-4), the inert gas is nitrogen and the reflux time is 4 to 5 hours.

7. The method for preparing the fluorescent hydrogen sulfide donor according to claim 2, characterized in that: In step (II-1), the reaction is: first stirring at 0°C for 2 to 3 hours, then heating to 24 to 26°C and continuing stirring for 1 to 1.5 hours.

8. Use of the fluorescent hydrogen sulfide donor according to claim 1 or the fluorescent hydrogen sulfide donor obtained by the preparation method according to any one of claims 2 to 7 as a fluorescent probe.

9. Use of the fluorescent hydrogen sulfide donor according to claim 1 or the fluorescent hydrogen sulfide donor obtained by the preparation method according to any one of claims 2 to 7 in the preparation of visualized hydrogen sulfide drugs.

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