Sulfur-containing compounds with active amino groups, and methods of making and using the same

Abstract

The application discloses a sulfur-containing compound with active amino groups and a preparation method and application thereof. The compound has the following structural formula. The compound can be used as a hydrogen sulfide donor, can generate a fluorophore while releasing and delivering H2S, can monitor the process of H2S release in vitro and in vivo in real time, can monitor H2S release in a complex biological system in situ and in real time, can visualize and quantify the released H2S, can exert the biological activity of the H2S, and can reduce the toxicity.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, and in particular relates to a self-reporting hydrogen sulfide donor compound with an active amino group, its preparation method, and its application. Background Technology

[0002] H2S mainly works by opening ATP-sensitive potassium channels (K2S). ATP H2S regulates the functions of the nervous and cardiovascular systems. Furthermore, it exerts biological effects through various intracellular signaling pathways (such as MAPK and NF-κB). Discovered biological effects of H2S include: smooth muscle relaxation, angiogenesis, neurotransmitter transmission, regulation of apoptosis, inhibition of platelet aggregation, and promotion of renal water and sodium excretion. H2S can act alone or synergistically with two other gaseous neurotransmitters (NO or CO) to regulate various physiological or pathological processes in organisms. Studies have shown that the broad biological activity of H2S is dose-dependent; certain concentrations of H2S exhibit anti-inflammatory, antioxidant, neuromodulatory, vascular modulatory, lung-protective, kidney-protective, gastric-protective, and cardioprotective effects.

[0003] Traditionally, H2S is delivered through direct inhalation. Later, sulfur-containing inorganic salts, sulfur-containing natural products, and small organic molecules were developed as commonly used hydrogen sulfide donors. Studies have found that inorganic sulfides such as sodium sulfide (Na2S) and sodium hydrosulfide (NaHS) have therapeutic potential for a wide range of diseases and exhibit anti-inflammatory and pro-inflammatory effects. Natural organic sulfur-containing compounds have been confirmed to exist in various plants, with structural types mainly involving organic polysulfides and isothiocyanates. Small organic molecule hydrogen sulfide donors can slowly release H2S and exert their biological activity in in vitro and in vivo experiments.

[0004] However, existing methods for transporting H2S or hydrogen sulfide donors have the following drawbacks:

[0005] 1. Direct inhalation of H2S makes it difficult to accurately adjust the concentration, and excessive H2S can lead to toxicity.

[0006] 2. The hydrolysis of widely used inorganic salt sulfides rapidly releases H2S, posing a challenge to accurate quantification. In vivo experiments can cause toxic reactions due to the large amount of H2S released in a short period of time.

[0007] 3. The most common natural H2S donors, such as garlic extract diallyl trisulfide (DATS) and diallyl disulfide (DADS), usually produce some byproducts unrelated to H2S release and their release capacity is unstable.

[0008] 4. Typical small organic hydrogen sulfide donors lack specificity or reactivity to in vivo biomarkers and cannot quantify the release kinetics and distribution curves of H2S in vivo. Summary of the Invention

[0009] Based on this, the present invention provides a sulfur-containing compound with an active amino group, which can act as a hydrogen sulfide donor. While releasing and delivering H2S, it can generate a fluorophore 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.

[0010] The structural formula of the sulfur-containing compound with an active amino group in this invention is as follows:

[0011]

[0012] This invention also provides a method for preparing a sulfur-containing compound with an active amino group, comprising the following steps:

[0013] S1: 4-Bromo-1,8-naphthalenedicarboxylic anhydride and sodium azide react in a solvent to give compound 1;

[0014] S2: Under the action of acid, compound 1 and triphenylphosphine react in a solvent. The reaction solution is neutralized with alkali to obtain compound 2.

[0015] S3: Compound 2 and N-tert-butoxycarbonyl-1,4-butanediamine react in a solvent to give compound 3;

[0016] S4: Under the action of alkali, compound 3 and sulfur source react in solvent to obtain compound 4;

[0017] S5: Under the action of a base, compound 4 and 4-(hydroxymethyl)phenylboronic acid pinacol ester react in a solvent to give compound 5;

[0018] S6: Remove the tert-butyloxycarbonyl protecting group from compound 5 to obtain compound HSDF-NH2, which is the sulfur-containing compound with an active amino group;

[0019] The reaction formula is as follows:

[0020]

[0021] In some embodiments, the molar ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride and sodium azide in step S1 is 1:1 to 1.5.

[0022] In some embodiments, the molar ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride and sodium azide in step S1 is 1:1.1 to 1.2.

[0023] In some embodiments, the solvent in step S1 is a mixture of water and N,N-dimethylformamide in a volume ratio of 1:20-25.

[0024] In some embodiments, the temperature of the reaction in step S1 is 90°C-110°C, and the reaction time is 10-20 minutes.

[0025] In some embodiments, the molar ratio of compound 1, triphenylphosphine, and acid in step S2 is 1:0.6-1.0:1-1.5.

[0026] In some embodiments, the molar ratio of compound 1, triphenylphosphine, and acid in step S2 is 1:0.8-0.9:1.2-1.3.

[0027] In some embodiments, the acid in step S2 is hydrochloric acid.

[0028] In some embodiments, the solvent in step S2 is a mixture of water and tetrahydrofuran in a volume ratio of 1:4-6.

[0029] In some embodiments, the concentration of the acid in the water is 0.4 mol / L to 0.6 mol / L.

[0030] In some embodiments, the base in step S2 is sodium hydroxide.

[0031] In some embodiments, the temperature of the reaction in step S2 is 15°C-40°C, and the reaction time is 20-40 minutes.

[0032] In some embodiments, the molar ratio of compound 2 and N-tert-butoxycarbonyl-1,4-butanediamine in step S3 is 1:1-3.

[0033] In some embodiments, the molar ratio of compound 2 and N-tert-butoxycarbonyl-1,4-butanediamine in step S3 is 1:1.5-2.5.

[0034] In some embodiments, the molar ratio of compound 2 and N-tert-butoxycarbonyl-1,4-butanediamine in step S3 is 1:1.8-2.2.

[0035] In some embodiments, the solvent in step S3 is N,N-dimethylformamide.

[0036] In some embodiments, the reaction temperature in step S3 is 90°C-110°C, and the reaction time is 6 hours-24 hours.

[0037] In some embodiments, the reaction temperature in step S3 is 95°C-105°C, and the reaction time is 10-14 hours.

[0038] In some embodiments, the molar ratio of compound 3, sulfur source and base in step S4 is 1:1.5-2.5:1.5-2.5.

[0039] In some embodiments, the molar ratio of compound 3, sulfur source and base in step S4 is 1:1.8-2.2:1.8-2.2.

[0040] In some embodiments, the base in step S4 is sodium bicarbonate.

[0041] In some embodiments, the sulfur source in step S4 is phosgene or 1,1'-thiocarbonyldiimidazole (TCDI).

[0042] In some embodiments, the solvent in step S4 is selected from one or more of dichloromethane, acetone, and tetrahydrofuran.

[0043] In some embodiments, the temperature of the reaction in step S4 is 0°C-35°C, and the reaction time is 10-20 hours.

[0044] In some embodiments, the molar ratio of compound 4, 4-(hydroxymethyl)phenylboronic acid pinacol ester and base in step S5 is 1:1-4:1-4.

[0045] In some embodiments, the molar ratio of compound 4, 4-(hydroxymethyl)phenylboronic acid pinacol ester and base in step S5 is 1:2-3:2-3.

[0046] In some embodiments, the alkali in step S5 is sodium hydride.

[0047] In some embodiments, the solvent in step S5 is tetrahydrofuran.

[0048] In some embodiments, the temperature of the reaction in step S5 is 0°C-35°C, and the reaction time is 12 hours-28 hours.

[0049] In some embodiments, the method for removing the tert-butyloxycarbonyl protecting group from compound 5 in step S6 includes: reacting compound 5 with a deBoc reagent in a solvent, and neutralizing it with a base to obtain the compound; wherein the deBoc reagent is trifluoroacetic acid and / or oxalyl chloride.

[0050] In some embodiments, the base in step S6 is sodium bicarbonate.

[0051] In some embodiments, the solvent in step S6 is dichloromethane and / or methanol.

[0052] In some embodiments, the Boc removal reagent in step S6 is oxaloyl chloride, and the solvent is methanol.

[0053] In some embodiments, the temperature of the reaction in step S6 is 15°C-40°C, and the reaction time is 20-40 minutes.

[0054] The present invention also provides applications of the above-mentioned compound HSDF-NH2, including the following technical solutions.

[0055] Application of compound HSDF-NH2 in the preparation of hydrogen sulfide donor drugs.

[0056] Application of compound HSDF-NH2 in the preparation of anti-inflammatory drugs.

[0057] Application of compound HSDF-NH2 in the preparation of drugs for the treatment of myocardial infarction.

[0058] The compound with an active amino group, its preparation method, and its application, as described in this invention, have the following advantages and beneficial effects:

[0059] This invention constructs a sulfur-containing compound with an active amino group by linking pinacol phenylboronic acid ester with 4-amino-N-(4-aminobutyl)-1,8-naphthalimide via a thiocarbamate. This compound can act as a hydrogen sulfide donor to deliver H2S. While delivering H2S, it can generate a fluorophore and can be used as a fluorescent probe to monitor the process of H2S release in vitro and in vivo in real time. The fluorescence of 4-amino-N-(4-aminobutyl)-1,8-naphthalimide in this compound is quenched by the electron-withdrawing group of the thiocarbamate, resulting in the absence of fluorescence in the compound HSDF-NH2 itself. Under ROS conditions such as H2O2, the borate ester group in HSDF-NH2 is easily oxidized and cleaved, releasing the fluorophore through autoincineration. Under the catalysis of carbonic anhydrase, H2S is generated from COS, thus transforming the non-fluorescent HSDF-NH2 into the fluorescent HSDG-NH2. The fluorescence change allows for in-situ and real-time monitoring of H2S release in complex biological systems, enabling visualization and quantification of the released H2S, thereby better leveraging the biological activity of H2S and improving its biosafety.

[0060] Furthermore, the compound HSDF-NH2 of the present invention contains an active amino group, which enhances the water solubility of the drug, improves its drug-like properties, and facilitates the preparation of novel drug formulations.

[0061] The synthesis method of the compound of the present invention has the advantages of simple and readily available raw materials, short synthesis steps, simple process, and high yield. Attached Figure Description

[0062] Figure 1 The image shows the fluorescence response of HSDF-NH2 to hydrogen peroxide.

[0063] Figure 2 This is an HPLC chromatogram of H2O2-induced HSDF-NH2 conversion.

[0064] Figure 3 A diagram illustrating the mechanism of HSDF-NH2 release and H2S monitoring.

[0065] Figure 4 The fluorescence response diagrams of HSDF-NH2 in the presence of different substances are shown.

[0066] Figure 5 The effect of HSDF-NH2 on the viability of H9c2 cardiomyocytes in a hypoxia / reoxygenation injury model.

[0067] Figure 6 The effect of HSDF-NH2 on the viability of rat hippocampal cells in an oxygen-glucose deprivation / reoxygenation model.

[0068] Figure 7 The effect of HSDF-NH2 on LDH release in rat neurons after modeling an oxygen-glucose deprivation / reoxygenation model. Detailed Implementation

[0069] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the present invention and should not be construed as limiting the invention.

[0070] 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.

[0071] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0072] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0073] The following are specific examples.

[0074] Example 1: Preparation of hydrogen sulfide donor (HSDF-NH2)

[0075] This embodiment provides a method for preparing a hydrogen sulfide donor (HSDF-NH2), and the synthetic route is as follows:

[0076]

[0077] S1. 4-Bromo-1,8-naphthalenedicarboxylic anhydride (6.5 g, 23.45 mmol) was added to 35 mL of N,N-dimethylformamide and stirred for 30 minutes until dissolved. Sodium azide (1.75 g, 26.9 mmol) was dissolved in 1.5 mL of water and added to the reaction system. The mixture was heated to 100 °C and reacted for 15 minutes. After the reaction was complete, it was cooled to room temperature, water was added to precipitate the precipitate, and the precipitate was collected and washed three times with water and ethanol. The precipitate was purified by column chromatography to give compound 1 (4.48 g, 80%). 1 HNMR(400MHz,Chloroform-d)δ8.70–8.52(m,3H),7.86–7.77(m,1H),7.54(d,J=8.0Hz,1H). 13 CNMR(101MHz,Chloroform-d)δ160.47,159.93,145.20,134.37,133.94,131.37,130.33,127.32,124.51,118.81,115.12,114.60.

[0078] S2. Compound 1 (4 g, 23.45 mmol) was suspended in a mixture of 300 mL tetrahydrofuran and 60 mL 0.5 M hydrochloric acid aqueous solution. Triphenylphosphine (5.25 g, 20 mmol) was then slowly added to the suspension with stirring. After stirring at room temperature for 30 minutes, the mixture was neutralized with 20 mL of 2 M sodium hydroxide aqueous solution to remove the solvent. The resulting mixture was diluted with ethyl acetate and filtered to give a crude solid product. Purification by column chromatography yielded compound 2 (3.5 g, 70%). 1H NMR (400MHz, DMSO-d6) δ8.62(d,J=8.4Hz,1H),8.36(d,J=7.2Hz,1H),8.12(d,J=8.5Hz,1H),7.73(s,2H),7.62(t,J=7.9Hz,1H),6.82(d,J=8.5Hz,1H). 13 C NMR (101MHz, DMSO-d6) δ162.42,160.73,154.33,136.25,133.37,132.94,131.09,124.72,119.70,118.58,109.16,102.63.

[0079] S3. Compound 2 (639.57 mg, 3 mmol) was dissolved in 15 mL of N,N-dimethylformamide. Then, N-tert-butoxycarbonyl-1,4-butanediamine (1.148 mL, 6 mmol) was added, and the reaction mixture was stirred at 100 °C for 12 hours. After the reaction was complete, the solution was cooled to room temperature and poured into ice-cold water. The precipitate was filtered, washed with water and diethyl ether, and purified by column chromatography to obtain compound 3 (689.7 mg, 60%). 1 H NMR (400MHz, DMSO-d6) δ8.60(d,J=8.4Hz,1H),8.41(d,J=7.2Hz,1H),8.18(d,J=8.4Hz,1H),7.63(t,J=7.8Hz,1H),7.42(s,2H),6.84(d,J= 8.4Hz,1H),6.77(t,J=5.8Hz,1H),3.99(t,J=7.1Hz,2H),2.93(q,J=6.6Hz,2H),1.58(p,J=7.3Hz,2H),1.41(q,J=7.3Hz,2H),1.35(s,9H). 13 C NMR(101MHz,DMSO-d6)δ164.24,163.37,156.03,153.14,134.38,131.42,130.1 4,129.72,124.40,122.24,119.83,108.62,108.03,77.79,28.71,27.70,25.78.

[0080] S4. Under ice-water bath conditions, a dichloromethane solution (284 μl, 3.725 mmol of phosgene dissolved in 25 mL of dichloromethane) was added dropwise to a solution of compound 3 (714.63 mg, 1.865 mmol) and sodium bicarbonate (315 mg, 3.75 mmol) in dichloromethane (25 mL). The reaction mixture was reacted at 0 °C for 30 minutes, and then stirred at room temperature for 16 hours. After removing the solvent, compound 4 (450 mg, 57%) was obtained by column chromatography. 1 H NMR (400MHz, Chloroform-d) δ8.64(dd,J=7.3,1.1Hz,1H),8.52(d,J=7.9Hz,1H),8.45(dd,J=8.4,1.1Hz,1H),7.85(dd,J=8.4,7.4Hz,1H),7. 63(d,J=7.9Hz,1H),4.67(s,1H),4.18(t,J=7.4Hz,2H),3.20(q,J=6.7Hz,2H),1.77(q,J=9.5,8.6Hz,2H),1.62(p,J=7.0Hz,2H),1.44(s,9H). 13 C NMR(101MHz,Chloroform-d)δ163.63,163.12,155.93,133.96,132.17,131.26,128.8 8(d,J=8.1Hz),127.90,127.48,124.44,123.11,121.02,40.05,28.42,27.59,25.41.

[0081] S5. Sodium hydride (40 mg, 1 mmol) was added to 10 mL of ultra-dry tetrahydrofuran. 4-(hydroxymethyl)phenylboronic acid pinacol ester (234.14 mg, 1 mmol) was added to this solvent at 0 °C. Then, a tetrahydrofuran solution of compound 4 (212.57 mg, 0.5 mmol) was added dropwise (10 mL). The resulting mixture was stirred at 0 °C for 30 minutes, then at room temperature for 24 hours. After the reaction was complete, it was quenched by adding brine, and the reactants were extracted with ethyl acetate. Compound 5 (82.41 mg, 25%) was purified by column chromatography. 1H NMR(400MHz,Chloroform-d)δ9.29(s,1H),8.54(dd,J=20.2,7.6Hz,2H),8.30(d,J=8.5Hz,1H),7.91(s,1H),7.74(dd,J=24.6,7.8Hz,3H),7.38–7.26( m,2H),5.61(s,2H),4.71(s,1H),4.14(t,J=7.4Hz,2H),3.16(q,J=6.8Hz,2 H),1.73(p,J=7.5Hz,2H),1.58(q,J=7.3Hz,2H),1.41(s,9H),1.35(s,12H). 13 C NMR(101MHz,Chloroform-d)δ189.47,163.95,163.45,137.66,135.04,131.58,131.39,128.9 0,128.31,127.47,127.18,122.99,120.65,83.97,73.48,39.91,28.41,27.52,25.39,24.87.

[0082] S6. Compound 5 (65.928 mg, 0.1 mmol) was dissolved in 2 mL of dichloromethane, and then 1 mL of trifluoroacetic acid was added dropwise. The mixture was stirred at room temperature for 30 minutes. After the reaction was complete, the solvent was removed under reduced pressure, and the reaction system was diluted with dichloromethane. The pH was adjusted to 7-8 by adding saturated sodium bicarbonate aqueous solution. After extraction with dichloromethane, the solution was purified by column chromatography to obtain HSDF-NH2 (11 mg, 20%). 1 H NMR (400MHz, DMSO-d6) δ8.51(dd,J=10.0,7.5Hz,2H),8.38(dd,J=8.5,1.1Hz,1H),7.93–7.87(m,2H),7.68(d,J=7.5Hz,2H),7.42(d,J=7.5Hz ,2H),5.58(s,2H),4.14(s,2H),4.08(t,J=6.7Hz,2H),2.82(t,J=7.4Hz,2H),1.71(p,J=6.7Hz,2H),1.61(q,J=8.4,7.6Hz,2H),1.29(s,12H). 13C NMR(151MHz,DMSO-d6)δ163.94,163.47,158.43(q,J=31.4Hz),139.49,134.99,131.56,131.32,130.35,128.76,127 .71,127.49,122.84,120.62,118.63,116.65,114.66,84.19,73.99,71.79,49.05,39.09,25.41,25.13(d,J=3.5Hz).

[0083] Example 2: Preparation of hydrogen sulfide donor intermediate (compound 3)

[0084] The solvent, reaction temperature and time, and starting material molar ratio are shown in Table 1 below. Other starting 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 1.

[0085] Table 1

[0086]

[0087]

[0088] Example 3: Preparation of hydrogen sulfide donor intermediate (compound 4)

[0089] The reactants and solvents are shown in Table 2 below. Other raw materials and their amounts, as well as the reaction process, are the same as step S4 in Example 1. The yield of compound 4 obtained is shown in Table 2.

[0090] Table 2

[0091] 1 Phosgene dichloromethane 57％ 2 Phosgene acetone 45％ 3 Phosgene Tetrahydrofuran 35％ 4 TCDI Tetrahydrofuran 43％

[0092] Example 4: Preparation of hydrogen sulfide donor intermediate (compound 5)

[0093] The raw material composition is shown in Table 3 below. Other raw materials and their amounts, as well as the reaction process, are the same as step S5 in Example 1. The yield of compound 5 obtained is shown in Table 3.

[0094] Table 3

[0095]

[0096] Example 5 Preparation of HSDG-NH2

[0097] The Boc removal reagent and solvent are shown in Table 4 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 HSDG-NH2 is shown in Table 4.

[0098] Table 4

[0099] 1 Trifluoroacetic acid dichloromethane 20％ 2 Oxaloyl chloride methanol 35％

[0100] Example 6 Preparation of HSDG-NH2

[0101]

[0102] Compound 3 (191.56 mg, 0.5 mmol) was dissolved in 2 mL of dichloromethane, and then 1 mL of trifluoroacetic acid was added dropwise. The mixture was stirred at room temperature for 30 minutes. After the reaction was complete, the solvent was removed under reduced pressure, and the reaction system was diluted with dichloromethane. The pH was adjusted to 7–8 by adding saturated sodium bicarbonate aqueous solution. After extraction with dichloromethane, the solution was purified by column chromatography to obtain HSDG-NH2 (70.8 mg, 50%). 1 H NMR (400MHz, DMSO-d6) δ8.64(d,J=8.4Hz,1H),8.42(d,J=7.2Hz,1H),8.18(d,J=8.4Hz,1H),7.64(t,J=7.8Hz,1H),7.51(s,2H),6.86 (d,J=8.4Hz,1H),4.04(t,J=6.7Hz,2H),3.48(s,3H),2.82(t,J=7.4Hz,2H),1.67(dt,J=13.6,6.7Hz,2H),1.58(p,J=7.2,6.1Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ164.36,163.45,159.11,158.80,153.36,134.47,131.51,129.92,124.39,119.07,108.65,49.02,25.33,25.16.

[0103] Example 7: Fluorescence response of HSDF-NH2 to hydrogen peroxide

[0104] Experimental methods and procedures: Prepare 3 mL of HSDF-NH2 (10 μM) solution, then add carbonic anhydrase (CA, 25 μg / mL) and H2O2 (final concentration 100 μM), and then test on a fluorescence spectrometer at 37 °C. Specifically, dissolve HSDF-NH2 in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. Prepare a 1 mg / mL carbonic anhydrase stock solution using PBS 7.4 buffer, and dilute the H2O2 solution tenfold with PBS 7.4 buffer. Form solutions with a final sample concentration of 10 μM, a final H2O2 concentration of 100 μM, and a final carbonic anhydrase concentration of 25 μg / mL in flasks. After each incubation period, excite with a light source at an excitation wavelength of 434 nm and measure the change in fluorescence intensity of HSDF-NH2 over time in the presence of H2O2.

[0105] During the experiment, HPLC was used to confirm the generation of HSDG-NH2. A cds 2.x HPLC system equipped with a C18 column (SuperLu, 5 μm, 250 mm × 4.6 mm) was used. The mobile phase consisted of acetonitrile and ammonium acetate buffer (8:2, v / v, 25 mM, pH 6.0). The flow rate was 1 mL / min, and the eluent was detected at 254 nm.

[0106] Experimental results are as follows Figure 1 As shown: 10 μM HSDF-NH2 showed no significant fluorescence emission in the range of 480–700 nm (λex = 434 nm). However, in the presence of H2O2 and carbonic anhydrase, a new peak fluorescence intensity at 546 nm increased with time, corresponding to the formation of HSDG-NH2 (e.g., Figure 2 (As shown). This result clearly demonstrates that H2O2 triggers the selective cleavage of borate-based thiocarbamates from the HSDF-NH2 molecule to form HSDG-NH2, resulting in the emission spectrum (λem = 546 nm).

[0107] The experimental results show that the HSDF-NH2 hydrogen sulfide donor of this invention can generate a fluorophore while delivering H2S, and can be used as a fluorescent probe to monitor the H2S release process in vitro and in vivo in real time. The mechanism of H2S release and monitoring by this fluorescent probe is shown in the diagram below. Figure 3As shown, the HSDF-NH2 of this invention is constructed by linking pinacol phenylboronic acid ester with 4-amino-N-(4-aminobutyl)-1,8-naphthalimide via a thiocarbamate. The fluorescence of the 4-amino-N-(4-aminobutyl)-1,8-naphthalimide is quenched by the electron-withdrawing group of the thiocarbamate, resulting in HSDF-NH2 itself being non-fluorescent. Under ROS conditions such as H2O2, the borate ester group in HSDF-NH2 is easily oxidized and cleaved, releasing the fluorophore through autoincineration, and generating H2S from COS under the catalysis of carbonic anhydrase. In other words, HSDF-NH2 can consume ROS to release carbonyl sulfide (COS), which is then hydrolyzed into H2S by ubiquitous carbonic anhydrase (CA), thus transforming the non-fluorescent HSDF-NH2 into the fluorescent HSDG-NH2, accompanied by a fluorescence change. This fluorescence change allows for in-situ and real-time monitoring of H2S release in complex biological systems, enabling visualization and quantification of the released H2S.

[0108] Example 8: Fluorescence response of HSDF-NH2 in the presence of different substances

[0109] Experimental methods and procedures: Relevant tests were performed using 10 μM HSDF-NH2 solution in 10 mL DMSO-PBS solution (1:9, v / v, 10 mM pH = 7.40) and appropriate concentrations of various ROS and biological substances. Spectra were recorded after incubation with various ROS at 37 °C.

[0110] The concentrations of each ROS and biological substance are as follows:

[0111] Hydrogen peroxide (100 μM): Commercial hydrogen peroxide solution diluted with deionized water.

[0112] Sodium hypochlorite (100 μM): Commercial sodium hypochlorite standard solution, diluted with deionized water.

[0113] TBHP (100 μM): Commercial tert-butyl hydroperoxide, diluted with deionized water.

[0114] Hydroxyl radical (100 μM): According to the Fenton reaction, ferrous ions and aqueous hydrogen peroxide solution can react to produce hydroxyl radicals. Therefore, a hydroxyl radical solution (50 mM) can be prepared by adding aqueous hydrogen peroxide solution (500 mM) to a ferrous sulfate solution (50 mM), and then diluting with deionized water.

[0115] Singlet oxygen (100 μM): A singlet oxygen solution (50 mM) can be prepared by adding a 100 mM aqueous solution of hydrogen peroxide to a hypochlorous acid aqueous solution (50 mM), and then diluting with deionized water.

[0116] Peroxynitrite (100 μM): Hydrogen peroxide solution (0.7 M, 1.5 ml) and sodium nitrite solution (0.6 M, 3 ml) were acidified with hydrochloric acid (0.6 M, 1.5 ml). NaOH solution (1.5 M, 3 ml) was added within 1–2 seconds to make the solution alkaline. The resulting solution was divided into small aliquots and stored at -80 °C. Before use, the aliquots were thawed immediately. A certain amount of the prepared peroxynitrite stock solution was added to 0.1 M NaOH, and the concentration of peroxynitrite was determined by measuring the absorbance of the solution at 302 nm. The extinction coefficient of the peroxynitrite solution in 0.1 M NaOH at 302 nm was 1670 M. -1 cm -1 .

[0117] Calcium chloride (30μM): Commercial calcium chloride, in deionized water solvent.

[0118] Magnesium chloride (30 μM): Commercial magnesium chloride, in deionized water solvent.

[0119] Ferrous chloride (30 μM): Commercial ferrous chloride, in deionized water solvent.

[0120] Zinc sulfate (30 μM): Commercial zinc sulfate, in deionized water solvent.

[0121] Ferric chloride (30μM): Commercial ferric chloride, using deionized water as the solvent.

[0122] Copper chloride (30μM): Commercial copper chloride, using deionized water as a solvent.

[0123] Glucose (1.5mM): Commercial glucose, in deionized water solvent.

[0124] Arginine (1.5mM): Commercial arginine, in deionized water solvent.

[0125] Serine (1.5mM): Commercial serine, in deionized water solvent.

[0126] Experimental results are as follows Figure 4 As shown, the results indicate that the presence of other substances did not significantly increase fluorescence intensity, suggesting that HSDF-NH2 exhibits high selectivity in complex biological environments. Reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), are important substances affecting the occurrence and development of ischemia-reperfusion injury, playing a crucial role in physiological processes. The compounds of this invention, with their high selectivity for ROS responsiveness, can achieve precise treatment, reduce side effects, and improve drug completeness.

[0127] Example 9: Protective effect of HSDF-NH2 on cells

[0128] (I) Protective effect of HSDF-NH2 on H9c2 cardiomyocytes in a hypoxia / reoxygenation injury model

[0129] A MIRI model of H9c2 cardiomyocytes was established using the following procedure: Ischemia was simulated using non-glucose DMEM medium. After incubation for 6 hours in an anaerobic glove box at 37°C, cells were returned to a conventional incubator and incubated for 4 hours in fresh medium containing high glucose to simulate reperfusion. The effect of HSDF-NH2 on H9c2 cardiomyocyte apoptosis was assessed using an annexin V-FITC apoptosis detection kit. MIRI model cells were seeded in six-well plates (1 × 10⁶ cells per well). 5 Cells (number of cells) were treated with or without HSDF-NH2 or propranolol for 24 hours, and then stained with annexin V-fluorescein isothiocyanate in binding buffer for 15 minutes at room temperature. Subsequently, cells were labeled with PI and apoptotic cells were assessed using flow cytometry.

[0130] Experimental results are as follows Figure 5 As shown, HSDF-NH2 has a protective effect on H9c2 cardiomyocytes in a hypoxia / reoxygenation injury model, and the protective effect is positively correlated with the dose. In the cellular environment, HSDF-NH2 is activated by ROS, releasing H2S to inhibit apoptosis and provide cellular protection against oxidative stress. The protective effect of HSDF-NH2 on H9c2 cardiomyocytes in a hypoxia / reoxygenation injury model at a concentration of 2 μM is comparable to that of the positive control propranolol.

[0131] (II) Protective effect of HSDF-NH2 on neurons

[0132] A rat hippocampal oxygen deprivation / reoxygenation (OGD / R) model was established using the following procedure: Primary cultured hippocampal neurons were cultured normally in culture dishes (37°C, 5% CO2) for 6 days. On the seventh day, the culture medium was replaced with preheated Erl balanced salt solution without glucose. The cells were then cultured at 37°C for 2 hours in an incubator containing 95% N2 and 5% CO2 as the OGD period, and O2 was measured using an O2 analyzer. After 2 hours of culture, the cultures were returned to a normal oxygen incubator with normal medium containing HSDF-NH2 for another 24 hours as the reoxygenation period. Cell viability was then measured using the MTT assay, and neuronal damage was evaluated by detecting lactate dehydrogenase (LDH).

[0133] Experimental results are as follows Figure 6 and Figure 7 As shown: In the rat hippocampal oxygen-glucose deprivation / reoxygenation (OGD / R) model, after adding different doses of HSDF-NH2, both the MTT test and LDH release experiment showed that HSDF-NH2 had a protective effect on neurons, and the protective effect was positively correlated with the dose.

[0134] 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.

[0135] 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. A compound with the following structural formula: 。 2. A method for preparing the compound according to claim 1, characterized in that, Includes the following steps: S1: 4-Bromo-1,8-naphthalenedicarboxylic anhydride and sodium azide react in a solvent to give compound 1; S2: Under the action of acid, compound 1 and triphenylphosphine react in a solvent. The reaction solution is neutralized with alkali to obtain compound 2. S3: Compound 2 and N-tert-butoxycarbonyl-1,4-butanediamine react in a solvent to give compound 3; S4: Under the action of alkali, compound 3 and sulfur source react in solvent to obtain compound 4; S5: Under the action of a base, compound 4 and 4-(hydroxymethyl)phenylboronic acid pinacol ester react in a solvent to give compound 5; S6: Remove the tert-butoxycarbonyl protecting group from compound 5 to obtain compound HSDF-NH2, which is a hydrogen sulfide donor compound; The reaction formula is as follows: 。 3. The method for preparing the compound according to claim 2, characterized in that, The molar ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride and sodium azide in step S1 is 1:1~1.5; and / or, The solvent mentioned in step S1 is a mixed solvent of water and N,N-dimethylformamide with a volume ratio of 1:20-25; and / or, The reaction temperature in step S1 is 90℃-110℃, and the reaction time is 10 minutes-20 minutes.

4. The method for preparing the compound according to claim 2, characterized in that, In step S2, the molar ratio of compound 1, triphenylphosphine, and acid is 1:0.6-1.0:1-1.5; and / or, The acid mentioned in step S2 is hydrochloric acid; and / or, The solvent mentioned in step S2 is a mixed solvent of water and tetrahydrofuran with a volume ratio of 1:4-6; and / or, The base mentioned in step S2 is sodium hydroxide; and / or, The reaction temperature in step S2 is 15℃-40℃, and the reaction time is 20 minutes-40 minutes.

5. The method for preparing the compound according to claim 2, characterized in that, In step S3, the molar ratio of compound 2 and N-tert-butoxycarbonyl-1,4-butanediamine is 1:1-3; and / or, The solvent mentioned in step S3 is N,N-dimethylformamide; and / or, The reaction temperature in step S3 is 90℃-110℃, and the reaction time is 6 hours-24 hours.

6. The method for preparing the compound according to claim 2, characterized in that, In step S4, the molar ratio of compound 3, the sulfur source, and the base is 1:1.5-2.5:1.5-2.5; and / or, The base mentioned in step S4 is sodium bicarbonate; and / or, The sulfur source mentioned in step S4 is phosgene or 1,1'-thiocarbonyldiimidazole; and / or, The solvent mentioned in step S4 is selected from one or more of dichloromethane, acetone, and tetrahydrofuran; and / or, The reaction temperature in step S4 is 0℃-35℃, and the reaction time is 10 hours-20 hours.

7. The method for preparing the compound according to claim 2, characterized in that, In step S5, the molar ratio of compound 4, 4-(hydroxymethyl)phenylboronic acid pinacol ester, and the base is 1:1-4:1-4; and / or, The base mentioned in step S5 is sodium hydride; and / or, The solvent mentioned in step S5 is tetrahydrofuran; and / or, The reaction temperature in step S5 is 0℃-35℃, and the reaction time is 12 hours-28 hours; and / or, The method for removing the tert-butoxycarbonyl protecting group from compound 5 in step S6 includes: reacting compound 5 with a deBoc reagent in a solvent, and neutralizing with a base to obtain the compound; the deBoc reagent is trifluoroacetic acid and / or oxalyl chloride.

8. The method for preparing the compound according to claim 7, characterized in that, In step S5, the molar ratio of compound 4, 4-(hydroxymethyl)phenylboronic acid pinacol ester, and the base is 1:2-3:2-3; and / or, The base mentioned in step S6 is sodium bicarbonate; and / or, The solvent mentioned in step S6 is dichloromethane and / or methanol; and / or, The reaction temperature in step S6 is 15℃-40℃, and the reaction time is 20 minutes-40 minutes.

9. The method for preparing the compound according to claim 8, characterized in that, The Boc removal reagent in step S6 is oxalyl chloride, and the solvent is methanol.

10. The use of the compound of claim 1 in the preparation of a hydrogen sulfide donor drug, an anti-inflammatory drug, or a drug for the treatment of myocardial infarction.

11. An intermediate compound for a hydrogen sulfide donor, characterized in that, Its structural formula is as follows: or .

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