Hydrogen sulfide donors that release near-infrared light in response to reactive oxygen species and methods of making and using the same

Abstract

The application discloses a hydrogen sulfide donor (HSD-BDP) capable of releasing near-infrared light in response to active oxygen, and a preparation method and application thereof. A structural formula of the HSD-BDP is as follows. The compound can generate a near-infrared fluorescent compound while releasing and delivering H2S, so as to monitor the process of H2S release in vitro and in vivo in real time, monitor H2S release in a complex biological system in situ and in real time, thereby visualizing and quantifying the released H2S, exerting biological activity of the H2S, and reducing toxicity of the H2S.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to hydrogen sulfide donor drugs, particularly to a hydrogen sulfide donor compound that can release near-infrared light upon response to reactive oxygen species, its preparation method, and its application. Background Technology

[0002] Hydrogen sulfide (H2S) is the third endogenous gaseous signaling molecule after nitric oxide (NO) and carbon monoxide (CO). H2S plays a crucial physiological regulatory role in the cardiovascular, nervous, immune, and digestive systems, such as vasodilation, anti-inflammation, anti-oxidation, and anti-apoptosis. However, the physiological functions of H2S exhibit a typical "concentration-dependent" pattern; that is, its abnormal expression (too low or too high) is closely associated with various diseases, such as hypertension, atherosclerosis, neurodegenerative diseases, cancer, and ischemia / reperfusion injury. Therefore, precise regulation and detection of H2S in vivo are of great significance for understanding its physiological and pathological functions and developing related therapeutic drugs.

[0003] To apply H2S to research and treatment, researchers have developed various H2S donor molecules, such as sodium hydrosulfide (NaHS), GYY4137, and organic donors based on structures like allyl sulfides. However, these traditional H2S donors have significant limitations:

[0004] 1. Uncontrollable release: Most of them release H2S spontaneously and slowly under physiological conditions, lacking responsiveness to specific pathological environments, making it impossible to precisely control the release site, timing, and dosage of H2S. This non-specific release may cause off-target effects and limit its therapeutic efficacy while also bringing potential toxic side effects.

[0005] 2. Lack of real-time monitoring methods: Existing donors cannot provide real-time, traceable feedback signals when releasing H2S. Researchers typically need to rely on complex detection techniques (such as electrodes, fluorescent probes, and chromatography) to indirectly determine H2S release. These methods are cumbersome to operate, have low spatiotemporal resolution, and are difficult to apply to in vivo real-time imaging, which greatly hinders in-depth research on the metabolic kinetics and pharmacodynamics of H2S in vivo. Summary of the Invention

[0006] Based on this, the present invention provides a hydrogen sulfide donor that can release near-infrared light upon response to reactive oxygen species. This compound can generate near-infrared fluorescent compounds while releasing and delivering H2S to monitor the process of H2S release in vitro and in vivo in real time. It can monitor H2S release in complex biological systems in situ and in real time, thereby visualizing and quantifying the released H2S to exert its biological activity and reduce toxicity.

[0007] The present invention includes the following technical solutions.

[0008] In one aspect, the present invention provides a compound HSD-BDP, the structural formula of which is as follows:

[0009]

[0010] HSD-BDP.

[0011] In one aspect, the present invention provides a method for preparing the compound HSD-BDP, comprising the following steps:

[0012] (1) Under the action of a base, p-aminoacetophenone and 3,4-dimethoxybenzaldehyde react to give compound 1;

[0013] (2) Under the action of alkali, compound 1 reacts with nitromethane to give compound 2;

[0014] (3) Compound 2 reacts with ammonium acetate to give compound 3;

[0015] (4) Under the action of alkali, compound 3 reacts with boron trifluoride diethyl ether to give HSD-BDP-NH2;

[0016] (5) Under the action of alkali, HSD-BDP-NH2 reacts with compound 4 to obtain compound HSD-BDP;

[0017] The reaction formula is as follows:

[0018] .

[0019] Thirdly, the present invention provides applications of the compound HSD-BDP, including: the application of the compound HSD-BDP in the preparation of a hydrogen sulfide donor drug; the application of the compound HSD-BDP in the preparation of a drug for treating cerebral ischemia-reperfusion; the application of the compound HSD-BDP in the preparation of an anti-apoptotic drug; the application of the compound HSD-BDP in the preparation of an anti-inflammatory drug; and the application of the compound HSD-BDP in the preparation of an antioxidant drug.

[0020] In a fourth aspect, the present invention provides a hydrogen sulfide donor drug prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the compound HSD-BDP;

[0021] The hydrogen sulfide donor drug can be used to treat cerebral ischemia-reperfusion, or to inhibit apoptosis, or to reduce inflammation, or to act as an antioxidant.

[0022] The compound HSD-BDP prepared by this invention has the following advantages and beneficial effects:

[0023] The compound HSD-BDP provided by this invention can release near-infrared light and hydrogen sulfide upon response to reactive oxygen species. This compound can generate a near-infrared fluorescent compound while releasing and delivering H2S, and H2S is highly correlated with fluorescence intensity. The release dose of H2S can be determined by observing the change in fluorescence intensity, thereby enabling real-time monitoring of the H2S release process in vitro and in vivo. It can also monitor H2S release in complex biological systems in situ and in real time, thereby visualizing and quantifying the released H2S to exert its biological activity.

[0024] The compound HSD-BDP of this invention does not release H2S under normal physiological conditions, but can responsively release hydrogen sulfide and near-infrared light at pathological sites of ROS such as H2O2. This allows for visualization and quantification of the released H2S during disease treatment, thereby better leveraging the biological activity of H2S, reducing its toxic side effects on normal tissues, and improving its biosafety.

[0025] The compound HSD-BDP of this invention can smoothly enter the cells at the disease site and release fluorescence and H2S under intracellular ROS stimulation, thereby effectively reducing the level of reactive oxygen species in pathological cells, protecting cells from ROS damage, and exhibiting excellent anti-inflammatory, antioxidant and anti-apoptotic activities. Moreover, its activity is superior to that of the positive control drug edaravone, and it can be used to prepare drugs for the treatment of cerebral ischemia-reperfusion. Attached Figure Description

[0026] Figure 1 This is the hydrogen spectrum of compound 1.

[0027] Figure 2 This is the carbon spectrum of compound 1.

[0028] Figure 3 This is the hydrogen spectrum of compound 2.

[0029] Figure 4 This is the carbon spectrum of compound 2.

[0030] Figure 5 This is the hydrogen spectrum of compound 3.

[0031] Figure 6 This is the carbon spectrum of compound 3.

[0032] Figure 7 This is the proton NMR spectrum of compound HSD-BDP-NH2.

[0033] Figure 8 This is the carbon spectrum of compound HSD-BDP-NH2.

[0034] Figure 9 This is the proton NMR spectrum of compound HSD-BDP.

[0035] Figure 10 This is the carbon spectrum of compound HSD-BDP.

[0036] Figure 11 This is the high-resolution mass spectrum of compound HSD-BDP.

[0037] Figure 12 The UV absorption spectra of 10 μM HSD-BDP reacted with 100 μM H2O2 for 0 hours and 6 hours.

[0038] Figure 13 The fluorescence spectrum is shown after 10 μM HSD-BDP reacts with 0 – 800 μM H2O2 for 2 hours.

[0039] Figure 14 The fluorescence and hydrogen sulfide release diagrams are shown for 100 μM HSD-BDP reacting with 1000 μM H2O2 over 0-48 hours.

[0040] Figure 15 This is a standard curve showing the fit between fluorescence intensity and hydrogen sulfide concentration.

[0041] Figure 16 The HPLC chromatograms show the reactions of HSD-BDP, HSD-BDP-NH2, and HSD-BDP with H2O2.

[0042] Figure 17 This is a schematic diagram illustrating the mechanism by which HSD-BDP generates fluorescence and H2S after being triggered by H2O2.

[0043] Figure 18 The diagram shows the protective effect of HSD-BDP on PC-12 cells in an oxygen-glucose deprivation / reoxygenation model.

[0044] Figure 19 The effect of HSD-BDP on ROS levels in PC-12 cells in an oxygen-glucose deprivation / reoxygenation model.

[0045] Figure 20 The image shows the fluorescence and H2S emission of HSD-BDP in cells.

[0046] Figure 21 The effect of HSD-BDP on hypoxia-induced apoptosis in PC-12 cells. Detailed Implementation

[0047] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0048] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0049] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0050] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0051] Some embodiments of the present invention involve a compound HSD-BDP, the structural formula of which is as follows:

[0052] .

[0053] Some embodiments of the present invention involve a method for preparing the compound HSD-BDP, comprising the following steps:

[0054] (1) Under the action of a base, p-aminoacetophenone and 3,4-dimethoxybenzaldehyde react to give compound 1;

[0055] (2) Under the action of alkali, compound 1 reacts with nitromethane to give compound 2;

[0056] (3) Compound 2 reacts with ammonium acetate to give compound 3;

[0057] (4) Under the action of alkali, compound 3 reacts with boron trifluoride diethyl ether to give HSD-BDP-NH2;

[0058] (5) Under the action of alkali, HSD-BDP-NH2 reacts with compound 4 to obtain compound HSD-BDP;

[0059] The reaction formula is as follows:

[0060] .

[0061] In some embodiments, the molar ratio of p-aminoacetophenone, 3,4-dimethoxybenzaldehyde and base in step (1) is 1:0.8-1.2:1-1.5.

[0062] In some embodiments, the reaction solvent in step (1) is a mixture of ethanol and water, with the volume ratio of ethanol to water preferably being 2-4:1, and the ratio of the amount of ethanol added to p-aminoacetophenone preferably being 1 mL-2 mL:1 mmol.

[0063] In some embodiments, the base in step (1) is sodium hydroxide.

[0064] In some embodiments, the temperature of the reaction in step (1) is 15°C-30°C and the reaction time is 6-10 hours.

[0065] In some embodiments, the molar ratio of compound 1, nitromethane and base in step (2) is 1:8-12:4-6.

[0066] In some embodiments, the reaction solvent in step (2) is methanol and / or ethanol.

[0067] In some embodiments, the base in step (2) is at least one of diethylamine, potassium hydroxide, sodium hydroxide and potassium carbonate.

[0068] In some embodiments, the reaction solvent in step (2) is methanol, and the base is potassium carbonate.

[0069] In some embodiments, the temperature of the reaction in step (2) is 60°C-80°C and the reaction time is 12-18 hours.

[0070] In some embodiments, the molar ratio of compound 2 and ammonium acetate in step (3) is 1:30-40, preferably 1:34-36.

[0071] In some embodiments, the reaction solvent in step (3) is n-butanol and / or ethanol.

[0072] In some embodiments, the temperature of the reaction in step (3) is 70°C-120°C and the reaction time is 12 hours-56 hours.

[0073] In some embodiments, the temperature of the reaction in step (3) is 75°C-85°C and the reaction time is 45-50 hours.

[0074] In some embodiments, the molar ratio of compound 3, boron trifluoride ether and base in step (4) is 1:16-20:8-12.

[0075] In some embodiments, the base in step (4) is N,N-diisopropylethylamine.

[0076] In some embodiments, the reaction solvent in step (4) is dichloromethane.

[0077] In some embodiments, the temperature of the reaction in step (4) is 15°C-30°C and the reaction time is 20-28 hours.

[0078] In some embodiments, the molar ratio of HSD-BDP-NH2, compound 4 and base in step (5) is 1:2.5-3.5:5-6.

[0079] In some embodiments, the base in step (5) is triethylamine and / or sodium carbonate, preferably sodium carbonate.

[0080] In some embodiments, the reaction solvent in step (5) is dichloromethane and / or tetrahydrofuran, preferably dichloromethane and tetrahydrofuran in a volume ratio of 1:0.8-1.2.

[0081] In some embodiments, the temperature of the reaction in step (5) is 15°C-30°C and the reaction time is 20-28 hours.

[0082] In some embodiments, the preparation of compound 4 includes the following steps: reacting 4-(hydroxymethyl)phenylboronic acid pinacol ester with a sulfur source to obtain compound 4.

[0083] In some embodiments, the molar ratio of the 4-(hydroxymethyl)phenylboronic acid pinacol ester to the sulfur source is 1:2-3.

[0084] In some embodiments, the sulfur source is phosgene.

[0085] In some embodiments, the solvent for the reaction of 4-(hydroxymethyl)phenylboronic acid pinacol ester and the sulfur source is 1,4-dioxane.

[0086] In some embodiments, the reaction temperature of 4-(hydroxymethyl)phenylboronic acid pinacol ester and sulfur source is 15°C-30°C, and the reaction time is 20-28 hours.

[0087] This invention significantly improves reaction yield by designing the reaction route and optimizing the reaction conditions at each step.

[0088] Some embodiments of the present invention relate to the use of the compound HSD-BDP in the preparation of hydrogen sulfide donor drugs.

[0089] Some embodiments of the present invention relate to the use of the compound HSD-BDP in the preparation of a medicament for the treatment of cerebral ischemia-reperfusion.

[0090] Some embodiments of the present invention relate to the use of the compound HSD-BDP in the preparation of anti-apoptotic drugs.

[0091] Some embodiments of the present invention relate to the use of the compound HSD-BDP in the preparation of anti-inflammatory drugs.

[0092] Some embodiments of the present invention relate to the use of the compound HSD-BDP in the preparation of antioxidant pharmaceuticals.

[0093] In some embodiments of the present invention, there is a hydrogen sulfide donor drug prepared from an active ingredient and pharmaceutically acceptable excipients, said active ingredient comprising the compound HSD-BDP;

[0094] The hydrogen sulfide donor drug can be used to treat cerebral ischemia-reperfusion, or to inhibit apoptosis, or to reduce inflammation, or to act as an antioxidant.

[0095] The following are specific examples.

[0096] Example 1: Preparation of hydrogen sulfide donor (HSD-BDP)

[0097] This embodiment provides a method for preparing a hydrogen sulfide donor (HSD-BDP), and its synthetic route is as follows:

[0098]

[0099] S1. Dissolve 13.5 g (100 mmol) of p-aminoacetophenone in 140 mL of ethanol, add 16.6 g (100 mmol) of 3,4-dimethoxybenzaldehyde, and stir until completely dissolved. Add 50 mL of 10% sodium hydroxide aqueous solution dropwise to the reaction system and stir at room temperature for 8 hours. After the reaction is complete (monitored by thin-layer chromatography), filter the reaction solution, and wash the precipitate three times with ice water to obtain compound 1 (21.0 g, 74%). 1H NMR (400 MHz, Chloroform-d) δ 7.94 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 15.5 Hz, 1H), 7.42 (d,J = 15.5 Hz, 1H), 7.22 (dd, J = 8.3, 1.9 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.71 (d, J = 8.2 Hz, 2H), 3.95 (s, 3H), 3.92 (s,3H). 13 C NMR (101 MHz, Chloroform-d) δ 188.23, 151.07, 150.86, 149.18, 143.38,131.01, 128.77, 128.27, 122.81, 119.95, 114.17, 111.15, 110.13, 55.99.

[0100] S2. Compound 1 (5 g, 17.7 mmol) was dissolved in 80 mL of methanol, and potassium carbonate (12.2 g, 88.3 mmol) and nitromethane (9.48 mL, 176.6 mmol) were added. The mixture was heated to 70 °C and refluxed for 15 hours. After the reaction was complete (monitored by thin-layer chromatography), the mixture was washed with water and extracted three times with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 2 (4.65 g, 76%). 1 H NMR (400 MHz, Chloroform- d ) δ 7.81 – 7.76 (m, 2H), 6.82 (d, J = 1.1 Hz,2H), 6.79 (d, J = 1.4 Hz, 1H), 6.65 (d, J = 8.4 Hz, 2H), 4.83 (dd, J = 12.4,6.3 Hz, 1H), 4.66 (dd, J = 12.3, 8.4 Hz, 1H), 4.21 – 4.09 (m, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.40 – 3.23 (m, 2H). 13 C NMR (101 MHz, Chloroform- d) δ195.00, 151.51, 149.15, 148.47, 131.95, 130.59, 126.94, 119.22, 113.82,111.51, 111.03, 79.92, 55.92 (d, J = 7.9 Hz), 41.00, 39.36.

[0101] S3. Compound 2 (4.65 g, 13.5 mmol) and ammonium acetate (36.45 g, 472.9 mmol) were dissolved in 70 mL of ethanol. The reaction mixture was heated to 82 °C with stirring and refluxed for 48 hours. After the reaction mixture was cooled to room temperature, it was filtered and the crude product was washed with cold ethanol to give the desired compound 3 (2.23 g, 55%). 1 H NMR (400 MHz, DMSO-d6) δ 7.76 (d, J = 8.7 Hz, 4H), 7.70 (dd, J = 8.3, 2.0 Hz, 2H), 7.58 (d,J = 2.0 Hz, 2H), 7.36 (s, 2H), 7.01 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 8.7 Hz,4H), 5.98 (s, 4H), 3.83 (s, 6H), 3.71 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ173.38, 153.79, 151.87, 149.27, 149.07, 148.95, 140.49, 128.54, 127.38,121.82, 119.32, 114.61, 113.75, 112.69, 112.08, 56.04, 55.90.

[0102] S4. Compound 3 (2.5 g, 4.2 mmol) was dissolved in 300 mL of anhydrous dichloromethane, and N,N-diisopropylethylamine (7.256 mL, 42 mmol) was added. The mixture was heated to 30 °C and stirred for 10 min, then boron trifluoride diethyl ether (9.256 mL, 75.6 mmol) was added, and the mixture was stirred at room temperature for 24 h. After the reaction was complete (monitored by thin-layer chromatography), the solvent was removed under reduced pressure, the mixture was washed with water, and extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to give compound HSD-BDP-NH2 (1.5 g, 56%). 1H NMR (400MHz, DMSO-d6) δ 8.02 (d, J = 8.7 Hz, 4H), 7.78 (d, J = 10.4 Hz, 2H), 7.64 (s,2H), 7.45 (s, 2H), 7.07 (d, J = 8.6 Hz, 2H), 6.70 (d, J = 8.9 Hz, 4H), 6.25 (s, 4H), 3.85 (s, 6H), 3.77 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 155.52,152.62, 150.27, 149.26, 144.19, 140.00, 132.38, 125.90, 122.50, 118.39,118.02, 114.06, 112.74, 112.22, 56.11, 56.04.

[0103] S5. Dissolve 1.17 g (5 mmol) of 4-hydroxymethylphenylboronic acid pinacol ester in 10 mL of 1,4-dioxane, add phosgene (800 μL, 10 mmol), and stir the mixture at room temperature for 24 hours under N2 protection. After the reaction is complete (monitored by thin-layer chromatography), remove the solvent under reduced pressure. The crude product (compound 4) can be used directly in the next reaction without further purification.

[0104] S6. HSD-BDP-NH2 (353 mg, 0.545 mmol) was dissolved in 15 mL of anhydrous tetrahydrofuran under ice bath and N2 protection. Sodium carbonate (321 mg, 3.03 mmol) was added, followed by dropwise addition of compound 4 (510 mg, 1.636 mmol) in 15 mL of anhydrous dichloromethane. The reaction was stirred at room temperature for 24 hours. After the reaction was complete (monitored by thin-layer chromatography), the mixture was filtered, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain HSD-BDP (107.6 mg, 16%). 1 HNMR (400 MHz, Chloroform- d) δ 8.16 – 7.93 (m, 6H), 7.81 (d, J = 7.5 Hz, 4H), 7.69 – 7.50 (m, 6H), 7.41 (d, J = 8.4 Hz, 2H), 6.99 – 6.85 (m, 4H), 6.74 –6.64 (m, 2H), 5.53 (s, 4H), 3.95 (s, 6H), 3.83 – 3.79 (m, 6H), 1.35 (s, 24H). 13 C NMR (101 MHz, Chloroform- d ) δ 190.53, 171.23, 150.63, 149.80, 148.99,143.18, 140.36, 137.23, 135.07 (d, J = 9.2 Hz), 132.80, 130.29, 128.09,127.84, 127.54, 122.81, 122.28, 114.78, 112.40, 112.17, 111.14 (d, J = 6.6Hz), 83.92, 74.76, 56.09 – 55.83 (m), 24.86. ESI-MS: [C 64 H 66 B3F2N5O 10 S2] + ([M + H)) + ) was calculated as 1200.4577, found 1200.4583

[0105] Example 2 Preparation of hydrogen sulfide donor intermediate (compound 2)

[0106] The base and solvent are shown in Table 1 below. Other raw materials and their amounts, as well as the reaction process, are the same as step S2 in Example 1. The yield of compound 2 obtained is shown in Table 1.

[0107] Table 1

[0108]

[0109] Example 3 Preparation of hydrogen sulfide donor intermediate (compound 3)

[0110] The solvent, reaction time, and reaction temperature are shown in Table 2 below. Other raw materials and their amounts, as well as the reaction process, are the same as step S3 in Example 1. The yield of compound 3 obtained is shown in Table 2.

[0111] Table 2

[0112]

[0113] Example 4: Preparation of HSD-BDP

[0114] The base and solvent are shown in Table 3 below. Other raw materials and their amounts, as well as the reaction process, are the same as step S6 in Example 1. The yield of the prepared HSD-BDP is shown in Table 3.

[0115] Table 3

[0116]

[0117] Example 5: Determination of UV absorption spectra before and after the reaction of HSD-BDP with H2O2

[0118] The spectroscopic determination of HSD-BDP was performed in PBS (10 mM, pH 7.4) containing 50% THF. 12 mg of HSD-BDP was accurately weighed into a 2 mL centrifuge tube, and 1 mL of THF was added to prepare a 10 mM HSD-BDP stock solution. A certain amount of PBS solution, HSD-BDP stock solution, and H2O2 solution were added to a 50 mL flask to achieve a final HSD-BDP concentration of 10 μM and a final H2O2 concentration of 100 μM. The flask was then placed in a 37°C water bath for 6 h. After the reaction, the absorbance at 650–850 nm was measured using a UV-Vis spectrophotometer.

[0119] Experimental results are as follows Figure 12 As shown, the maximum absorption peak of HSD-BDP is located at 743 nm. After reacting with 100 μH2O2 at 37 °C for 6 h, its maximum absorption peak red-shifts to 764 nm, indicating that HSD-BDP can react with H2O2 to cause its absorption peak to red-shift.

[0120] Example 6: Fluorescence spectroscopy determination of HSD-BDP after reaction with different concentrations of H2O2

[0121] Accurately pipette 10 μL of HSD-BDP stock solution into a 50 mL round-bottom flask, add a certain amount of H2O2 solution and PBS solution to make the final concentration of HSD-BDP 10 μM. The final concentrations of H2O2 were 0, 10, 20, 40, 80, 100, 200, 400, and 800 μM, respectively. After reacting in a 37℃ water bath for 2 h, the fluorescence intensity at 770–870 nm was measured using a fluorophotometer. (Excitation wavelength: 755 nm; slit width: 9 / 9 nm)

[0122] Experimental results are as follows Figure 13As shown, the fluorescence intensity increases with increasing H2O2 concentration. This is because with increasing H2O2 concentration, more H2O2 molecules in the solution react with HSD-BDP molecules, resulting in a greater amount of HSD-BDP-NH2 near-infrared fluorophores and stronger fluorescence.

[0123] Example 7: Real-time monitoring of fluorescence intensity changes and H2S release changes after the reaction of H2O2 with HSD-BDP.

[0124] Accurately weigh 60 mg HSD-BDP into a 2 mL centrifuge tube and add 1 mL THF to prepare a 50 mM HSD-BDP stock solution. Accurately weigh 5 mg hydrogen sulfide probe (WSP-1) into a 2 mL centrifuge tube and add 338 μL DMSO to prepare a 5 mM WSP-1 stock solution. Accurately weigh 1 mg carbonic anhydrase (CA) into a 2 mL centrifuge tube and add 1 mL PBS to prepare a 1 mg / mL CA stock solution. Accurately weigh 3.9 mg sodium sulfide (Na2S) into a 50 mL centrifuge tube and add 50 mL water to prepare a 1 mM Na2S stock solution.

[0125] Construction of H2S standard curve: Accurately pipette 1, 5, 10, 30, 50, 100, and 200 μL of Na2S stock solution into 5 mL centrifuge tubes. Add a certain amount of WSP-1 stock solution and PBS to make the final concentration of WSP-1 25 μM, thus preparing 1, 5, 10, 30, 50, 100, and 200 μM Na2S standard solutions. After stirring in a 37℃ water bath for 2 h, measure the fluorescence intensity at 515 nm under excitation light of 465 nm using a microplate reader. Fit the standard curve using solution concentration and fluorescence intensity.

[0126] Accurately pipette 6 μL of 50 mM HSD-BDP stock solution into a 10 mL round-bottom flask. Add a certain amount of H2O2 solution, WSP-1 stock solution, and CA stock solution to make the final concentrations of HSD-BDP 100 μM, H2O2 1000 μM, WSP-1 25 μM, and CA 100 μg / mL. Then place the flask in a 37℃ water bath for reaction. Measure the fluorescence intensity at 1, 3, 5, 8, 12, 24, and 48 h (fluorescence channel of H2S: excitation wavelength 465 nm; emission wavelength: 515 nm; fluorescence channel of HSD-BDP-NH2: excitation wavelength: 755 nm; emission wavelength: 856 nm).

[0127] Experimental results are as follows Figure 14 and Figure 15As shown, the fluorescence intensity and the amount of H2S released increase continuously as the reaction proceeds. The release curves show a certain similarity between the two, and linear fitting results indicate a correlation between H2S and fluorescence intensity (R = 0.97869). This result demonstrates that the release dose of H2S can be determined by observing changes in fluorescence intensity, thus achieving visualization of H2S gas release.

[0128] Example 8: Response Mechanism of HSD-BDP

[0129] Accurately weigh 12 mg of HSD-BDP into a 2 mL centrifuge tube and add 1 mL of CH3CN to prepare a 10 mM HSD-BDP stock solution. Accurately weigh 6.46 mg of HSD-BDP-NH2 into a 2 mL centrifuge tube and add 1 mL of CH3CN to prepare a 10 mM HSD-BDP-NH2 stock solution. Take 10 μL of each HSD-BDP and HSD-BDP-NH2 stock solution and add 990 μL of mobile phase to dilute their concentrations to 100 μM. Take 10 μL of the HSD-BDP stock solution and add H2O2 solution and mobile phase to achieve a final HSD-BDP concentration of 100 μM and a final H2O2 concentration of 1000 μM. After reacting at 37℃ for 6 h, analyze the samples using high-performance liquid chromatography (HPLC). A SuperLuC18-AQ5 column (4.6 mm × 250 mm, 5 μm) was used, with acetonitrile and water (7:3, v / v, 1 mL / min, 724 nm) as the mobile phase.

[0130] HPLC analysis results are as follows Figure 16 As shown, the reaction pathway of HSD-BDP responding to ROS and simultaneously releasing fluorescence with H2S is as follows: Figure 17 As shown, firstly, the borate ester group of HSD-BDP hydrolyzes to boric acid, which is then oxidized by H2O2 to form phenol. Under alkaline conditions, phenol oxide anion is formed. The phenol oxide anion undergoes 1,6-elimination through electron-donating conjugation to generate the fluorescent compound HSD-BDP-NH2 and carbonyl sulfide (COS). Finally, COS is catalyzed by CA to generate H2S.

[0131] Example 9: Protective effect of HSD-BDP on PC-12 cells in an oxygen-glucose deprivation / reoxygenation model.

[0132] PC-12 cells were used at a rate of 1 × 10⁻⁶ 4Cells were seeded per well in 96-well plates. After cell attachment, the culture medium was replaced with RPMI-1640 sugar-free medium. The plates were then placed in a sealed culture jar with an anaerobic gas-generating bag for 6 hours of anaerobic culture. After 6 hours, the plates were removed and the culture medium was replaced with RPMI-1640 complete medium containing 1–20 μM HSD-BDP, 5 μM HSD-BDP-NH2, or 5 μM edaravone (EDR). The plates were then returned to the cell culture incubator and cultured for 24 hours. The cells were then washed with PBS and 100 μL of 10% CCK-8 solution was added. After incubation for another hour, the absorbance at 450 nm was measured using a microplate reader.

[0133] The results are as follows Figure 18 As shown, PC-12 neurons treated with HSD-BDP donor (5 μM) after oxygen-glucose deprivation / reoxygenation showed a significant increase in survival rate compared to the positive control EDR. This indicates that HSD-BDP can effectively protect neurons from reactive oxygen species damage during reoxygenation and maintain neurons in a normal state.

[0134] Example 10 Measurement of reactive oxygen species levels in cells

[0135] PC-12 cells were used at a rate of 1 × 10⁻⁶ 5 Cells were seeded per well in 24-well plates. After cell attachment, oxygen-glucose deprivation / reoxygenation was performed on the cells according to the method in Example 9. The culture medium was discarded, and the cells were washed three times with PBS. 500 μL of reactive oxygen species probe (DCFH-DA) was added to each well, and the cells were incubated in a cell culture incubator for 30 minutes. After washing with PBS, 200 μL of Hoechst staining solution was added and stained for 5 minutes. Cell images were then captured under laser confocal microscopy.

[0136] Experimental results are as follows Figure 19 As shown, the ROS level of PC-12 cells increased significantly after oxygen-glucose deprivation / reoxygenation treatment, while the ROS level of PC-12 cells was significantly reduced after HSD-BDP treatment.

[0137] Example 11: Fluorescence generation and H2S production in cells

[0138] PC-12 cells were used at a rate of 1 × 10⁻⁶ 5 Cells were seeded per well in 24-well plates. After cell adhesion, the cells were subjected to oxygen-glucose deprivation / reoxygenation as described in Example 9. During reoxygenation, the culture medium used was RPMI-1640 complete medium containing 5 μM HSD-BDP and 5 μM hydrogen sulfide probe. After 24 hours of culture, the culture medium was discarded, the cells were washed three times with PBS, and stained with 200 μL Hoechst staining solution for 5 minutes. Cell images were then captured using laser confocal microscopy.

[0139] Experimental results are as follows Figure 20 As shown, because neither the control group nor the model group received the HSD-BDP donor, no fluorescence was observed in their self-reported fluorescence channel or H2S fluorescence channel. The HSD-BDP-NH2 group received the fluorophore that responds to HSD-BDP, thus exhibiting self-reported fluorescence in this channel; however, since the molecule does not produce H2S, no fluorescence was observed in the H2S fluorescence channel. After co-incubation with PC-12 cells treated with oxygen-glucose deprivation / reoxygenation, both self-reported fluorescence and H2S release were observed intracellularly, indicating that HSD-BDP molecules can successfully enter cells and release fluorescence and H2S upon intracellular ROS stimulation.

[0140] Example 12 Assay for Apoptosis

[0141] PC-12 cells were used at a rate of 5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates. After cell attachment, the cells were subjected to oxygen-glucose deprivation / reoxygenation as described in Example 9. The culture medium was discarded, the cells were washed with PBS, and 1 mL of EDTA-free trypsin was added for digestion and cell collection. The cells were resuspended in 100 μL of binding buffer and the cell suspension was transferred to flow cytometry tubes. 5 μL of Annexin V-FITC was added and the cells were incubated at room temperature in the dark for 5 minutes. Then, 10 μL of PI was added and the cells were incubated for 5 minutes. The cells were then analyzed by flow cytometry.

[0142] Experimental results are as follows Figure 21 As shown, the proportion of apoptotic cells in PC-12 neurons increased significantly after oxygen-glucose deprivation / reoxygenation treatment, but the proportion of apoptotic cells decreased significantly after treatment with HSD-BDP donor (5 μM), indicating that HSD-BDP has excellent anti-apoptotic activity.

[0143] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The compound HSD-BDP has the following structural formula: 。 2. A method for preparing the compound HSD-BDP according to claim 1, characterized in that, Includes the following steps: (1) Under the action of a base, p-aminoacetophenone and 3,4-dimethoxybenzaldehyde react to give compound 1; (2) Under the action of alkali, compound 1 reacts with nitromethane to give compound 2; (3) Compound 2 reacts with ammonium acetate to give compound 3; (4) Under the action of alkali, compound 3 reacts with boron trifluoride diethyl ether to give HSD-BDP-NH2; (5) Under the action of alkali, HSD-BDP-NH2 reacts with compound 4 to obtain compound HSD-BDP; The reaction formula is as follows: 。 3. The method for preparing compound HSD-BDP according to claim 2, characterized in that, The molar ratio of p-aminoacetophenone, 3,4-dimethoxybenzaldehyde, and the base in step (1) is 1:0.8-1.2:1-1.5; and / or, The reaction solvent in step (1) is a mixture of ethanol and water; and / or, The base mentioned in step (1) is sodium hydroxide; and / or, The reaction temperature in step (1) is 15℃-30℃ and the reaction time is 6 hours-10 hours.

4. The method for preparing compound HSD-BDP according to claim 2, characterized in that, The reaction solvent in step (1) is a mixture of ethanol and water, with a volume ratio of ethanol to water of 2-4:

1.

5. The method for preparing compound HSD-BDP according to claim 2, characterized in that, The reaction solvent in step (1) is a mixture of ethanol and water, and the ratio of the amount of ethanol added to p-aminoacetophenone is 1 mL-2 mL: 1 mmol.

6. The method for preparing compound HSD-BDP according to claim 2, characterized in that, In step (2), the molar ratio of compound 1, nitromethane, and base is 1:8-12:4-6; and / or, The reaction solvent in step (2) is methanol and / or ethanol; and / or, The base mentioned in step (2) is at least one of diethylamine, potassium hydroxide, sodium hydroxide, and potassium carbonate; and / or, The reaction temperature in step (2) is 60℃-80℃, and the reaction time is 12 hours-18 hours.

7. The method for preparing compound HSD-BDP according to claim 2, characterized in that, In step (3), the molar ratio of compound 2 and ammonium acetate is 1:30-40; and / or, The reaction solvent in step (3) is n-butanol and / or ethanol; and / or, The reaction temperature in step (3) is 70℃-120℃, and the reaction time is 12 hours-56 hours.

8. The method for preparing compound HSD-BDP according to claim 7, characterized in that, The molar ratio of compound 2 and ammonium acetate in step (3) is 1:34-36.

9. The method for preparing compound HSD-BDP according to claim 7, characterized in that, The reaction temperature in step (3) is 75℃-85℃, and the reaction time is 45 hours-50 hours.

10. The method for preparing compound HSD-BDP according to claim 2, characterized in that, In step (4), the molar ratio of compound 3, boron trifluoride diethyl ether, and base is 1:16-20:8-12; and / or, The base mentioned in step (4) is N,N-diisopropylethylamine; and / or, The reaction solvent in step (4) is dichloromethane; and / or, The reaction temperature in step (4) is 15℃-30℃, and the reaction time is 20 hours-28 hours.

11. The method for preparing compound HSD-BDP according to claim 2, characterized in that, In step (5), the molar ratio of HSD-BDP-NH2, compound 4, and base is 1:2.5-3.5:5-6; and / or, The base mentioned in step (5) is triethylamine and / or sodium carbonate; and / or, The reaction solvent in step (5) is dichloromethane and / or tetrahydrofuran; and / or, The reaction temperature in step (5) is 15℃-30℃, and the reaction time is 20 hours-28 hours.

12. The method for preparing compound HSD-BDP according to claim 11, characterized in that, The reaction solvent in step (5) is dichloromethane and tetrahydrofuran in a volume ratio of 1:0.8-1.

2.

13. The method for preparing compound HSD-BDP according to any one of claims 2-12, characterized in that, The preparation of compound 4 includes the following steps: reacting 4-(hydroxymethyl)phenylboronic acid pinacol ester with a sulfur source to obtain compound 4.

14. The method for preparing compound HSD-BDP according to claim 13, characterized in that, The molar ratio of the 4-(hydroxymethyl)phenylboronic acid pinacol ester to the sulfur source is 1:2-3.

15. The method for preparing compound HSD-BDP according to claim 13, characterized in that, The sulfur source is phosgene.

16. The method for preparing compound HSD-BDP according to claim 13, characterized in that, The solvent for the reaction of 4-(hydroxymethyl)phenylboronic acid pinacol ester and sulfur source is 1,4-dioxane.

17. The method for preparing compound HSD-BDP according to claim 13, characterized in that, The reaction temperature of 4-(hydroxymethyl)phenylboronic acid pinacol ester with sulfur source is 15℃-30℃, and the reaction time is 20 hours-28 hours.

18. The use of the compound HSD-BDP of claim 1 in the preparation of a hydrogen sulfide donor drug, or in the preparation of a drug for treating cerebral ischemia-reperfusion, or in the preparation of an anti-apoptotic drug, or in the preparation of an anti-inflammatory drug, or in the preparation of an antioxidant drug.

19. A hydrogen sulfide donor drug, characterized in that, It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the compound HSD-BDP of claim 1; The hydrogen sulfide donor drug can be used to treat cerebral ischemia-reperfusion, or to inhibit apoptosis, or to reduce inflammation, or to act as an antioxidant.

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