Methylene blue-fingolimod prodrugs activated by hypochlorous acid and methods of making and using the same

Abstract

This invention belongs to the field of biomedical technology and discloses a methylene blue-fingolimod prodrug based on hypochlorous acid activation, its preparation method, and its application. Fingolimod and methylene blue, which has hypochlorous acid scavenging and neuroprotective functions, are bonded together via a hypochlorous acid-sensitive thioether bridge. This design cleverly utilizes the abnormally high concentration of hypochlorous acid within the lesion as "molecular scissors," achieving precise activation of the prodrug at the target site and synergistic release of both drugs. This not only endows fingolimod with targeted noise reduction and enhanced efficacy but also simultaneously implements a combined attack of "detoxification" and "anti-inflammation," thereby more thoroughly breaking the damage cascade and providing a novel synergistic solution for stroke treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a methylene blue-fingolimod prodrug activated by hypochlorous acid, its preparation method and application. Background Technology

[0002] Following ischemic stroke, the secondary injury cascade is central to the deterioration of neurological function. Within this complex pathological network, a vicious cycle of oxidative stress and neuroinflammation, driven by infiltrating immune cells and overactivated microglia, is formed. Hypochlorous acid, a unique reactive oxygen species catalyzed by the myeloperoxidase system, acts as a key hub and amplifier in this vicious cycle, and has become a highly promising therapeutic target.

[0003] Hypochlorous acid possesses extremely strong oxidative activity and protein chlorination capacity, with a destructive power far exceeding that of other reactive oxygen species. In ischemic brain regions, it not only directly attacks neuronal membrane lipids, disrupts mitochondrial function, and induces programmed necrosis, but also abnormally activates pathways such as the NLRP3 inflammasome by modifying specific signaling proteins, thereby dramatically amplifying local oxidative damage signals into a severe inflammatory storm. This characteristic of tightly coupling oxidative damage with inflammatory responses makes it a key "catalyst" driving the transformation of the penumbra into the infarct core.

[0004] To effectively block this destructive process triggered by hypochlorous acid, an ideal drug needs a two-pronged approach: it must be able to eliminate hypochlorous acid and resolve the oxidative crisis, while also precisely regulating the subsequent immune inflammatory response. Based on this, the anti-inflammatory drug fingolimod has come into focus. In vivo studies have demonstrated remarkable anti-inflammatory functions by modulating S1P receptors, effectively inhibiting central lymphocyte infiltration, stabilizing the blood-brain barrier, and regulating glial cell polarization. However, its singular anti-inflammatory mechanism and inability to intervene in oxidative stress limit its therapeutic potential in complex injury environments.

[0005] Therefore, researching novel therapeutic drugs that can synergistically intervene in key aspects of post-ischemic oxidative stress and neuroinflammation is of great significance. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies in the prior art and to provide a methylene blue-fingolimod prodrug based on hypochlorous acid activation, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a methylene blue-fingolimod prodrug activated by hypochlorous acid, the chemical structure of which is shown in the following formula:

[0008]

[0009] In a second aspect, the present invention provides a methylene blue-fingolimod prodrug based on hypochlorous acid activation, the chemical structure of which is shown in the following formula:

[0010]

[0011] In a third aspect, the present invention provides a methylene blue-fingolimod prodrug based on hypochlorous acid activation, the chemical structure of which is shown in the following formula:

[0012]

[0013] In a fourth aspect, the present invention provides a formula I a A method for preparing the methylene blue-fingolimod prodrug with the structure shown is characterized in that the synthetic route of the preparation method is shown in the following formula:

[0014] ,

[0015] The preparation method is as follows: Compound 1 and fingolimod 2 are reacted overnight at 0-20 degrees Celsius under organic base conditions, allowing Compound 1 to selectively react with the amino group of fingolimod. After the reaction is complete, Compound I is obtained by column chromatography. a .

[0016] In a fifth aspect, the present invention provides a formula I b The method for preparing the methylene blue-fingolimod prodrug with the structure shown is illustrated by the following synthetic route:

[0017] ,

[0018] The preparation method includes the following steps:

[0019] S1. Gamma-aminobutyric acid (3) reacts with compound 1 at room temperature overnight under triethylamine (TEA) conditions until the reaction is complete to give compound 4;

[0020] S2. Compound 4 and fingolimod 2 were subjected to an amidation reaction at 0–20°C under the conditions of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA), selectively amide-reacting the amino group of fingolimod to give compound I. b .

[0021] In a sixth aspect, the present invention provides Formula I c The method for preparing the methylene blue-fingolimod prodrug with the structure shown is illustrated by the following synthetic route:

[0022]

[0023] The preparation method includes the following steps:

[0024] S1. Compound 1 was reacted with propanolamine 5 at room temperature overnight under TEA conditions to give intermediate 6;

[0025] S2. Intermediate 6 and phenyl p-nitrochloroformate were reacted overnight at room temperature under TEA conditions to give active intermediate 7;

[0026] S3. Intermediate 7 reacted with fingolimod 2 at room temperature under TEA and 4-dimethylaminopyridine (DMAP) conditions, and the mixture was purified by column chromatography to give compound I. c .

[0027] In a seventh aspect, the present invention provides the use of the above-mentioned methylene blue-fingolimod prodrug in the preparation of a reagent for diagnosing ischemic stroke and / or cerebral ischemia-reperfusion injury.

[0028] In an eighth aspect, the present invention provides the use of the above-described methylene blue-fingolimod prodrug in the preparation of a medicament for treating ischemic stroke and / or cerebral ischemia-reperfusion injury.

[0029] In a ninth aspect, the present invention provides the application of the above-mentioned methylene blue-fingolimod prodrug in the preparation of a fluorescent imaging reagent targeting ischemic brain tissue and cells.

[0030] In a tenth aspect, the present invention provides a pharmaceutical composition comprising the above-described methylene blue-fingolimod prodrug and a pharmaceutically acceptable carrier or excipient.

[0031] The pharmaceutical composition can be formulated into various dosage forms to suit different routes of administration. In specific embodiments, the dosage forms include, but are not limited to, oral dosage forms (such as tablets, capsules, emulsions, suspensions, dispersants, and solutions) and sterile parenteral dosage forms (such as injectable solutions or suspensions). Injectable compositions can be formulated using techniques known in the art, employing suitable dispersants, wetting agents, and suspending agents.

[0032] Commonly used pharmaceutically acceptable carriers or excipients include diluents, fillers, binders, lubricants, surfactants, emulsifiers, stabilizers, antioxidants, colorants, and suitable solvents (such as water, physiological saline solutions, mannitol solutions, etc.). The composition can be administered alone or in combination with other therapeutic agents.

[0033] In practical applications, the dosage of the active ingredient (i.e., the compound of this invention) needs to be determined on a case-by-case basis. The selected dosage level should ensure safety and efficacy for the patient and depends on a variety of factors, including but not limited to: the activity of the specific compound used, the route and timing of administration, the duration of treatment, the in vivo metabolic and excretion characteristics of the composition, possible combination therapies, and the patient's individual condition (e.g., age, sex, weight, general health status, and medical history). Those skilled in the art can determine the appropriate dosage through routine experiments based on these well-known factors.

[0034] Compared to existing technologies, this invention proposes an innovative prodrug strategy: fingolimod is bonded to methylene blue, which has hypochlorous acid clearance and neuroprotective functions, via a hypochlorous acid-sensitive thioether linker. This design cleverly utilizes the abnormally high concentration of hypochlorous acid within the lesion as "molecular scissors," achieving precise activation of the prodrug at the target site and synergistic release of both drugs. This not only endows fingolimod with targeted noise reduction and enhanced efficacy but also simultaneously implements a combined attack of "detoxification" and "anti-inflammation," thereby more thoroughly breaking the damage cascade and providing a novel synergistic solution for stroke treatment. Experiments show that, in vitro, the compound provided by this invention can significantly improve the survival rate of BV2 cells treated with the compound in the OGD / R injury cellular inflammation model, and its protective effect is superior to that of the positive control drug fingolimod. In vivo, in MCAO model mice, the compound can significantly reduce the volume of cerebral infarction and effectively inhibit the expansion of infarct foci, confirming its precise neuroprotective and damage control capabilities. In addition, based on its "hypochlorous acid activation" design principle and fluorescence properties, the compound also has the potential visual diagnostic function for ischemic tissues and cells, thus demonstrating a unique "diagnosis and treatment integration" application advantage in the field of ischemic stroke. Attached Figure Description

[0035] Figure 1 For the detection of compound I of the present invention in Example 4 a -I c Fluorescence spectra obtained from the time and concentration responses to HClO, with wavelength on the x-axis and fluorescence intensity on the y-axis;

[0036] Figure 2 Compound I of the present invention was tested in Example 5. a -I c Statistical analysis of cell viability obtained from cellular-level neuroprotective effects;

[0037] Figure 3 To evaluate compound I of the present invention in Example 6 a -I c Confocal images obtained by selective imaging capability in the OGD / R model and their statistical analysis graphs;

[0038] Figure 4 Compound I of the present invention was tested in Example 7. a -I c TTC-stained images of coronal sections of mouse brains and statistical analysis of infarct volume percentage obtained from in vivo treatment of cerebral ischemia. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0042] This invention discloses a method for preparing a methylene blue-fingolimod prodrug based on hypochlorous acid activation and its application. Specific embodiments are as follows:

[0043] Example 1: 3,7-bis(dimethylamino)-N-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)-10H-phenthiazine-10-carboxamide (I a )

[0044] Under nitrogen protection and at room temperature, 3,7-bis(dimethylamino)-10H-phenthiazine-10-carbonyl chloride (1) (201 mg, 0.58 mmol), fingolimod 2 (196 mg, 0.64 mmol), and triethylamine (290 mg) were dissolved in anhydrous dichloromethane (3 mL), and the mixture was stirred for 48 hours in the dark. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction solution was extracted with dichloromethane and water, the organic phases were combined and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Finally, the crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (3:1 v / v) as the eluent, with a yield of 68%. 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 8.8 Hz, 2H, ArH), 7.09– 6.93 (m, 4H, ArH), 6.72 (d, J = 2.8 Hz, 2H, ArH), 6.64 (dd, J = 8.8, 2.8Hz, 2H, ArH), 5.46 (s, 1H, NH), 3.88 (d, J = 11.6 Hz, 2H, CH), 3.59 (d, J =11.5 Hz, 2H, CH), 2.95 (s, 12H, CH), 2.60 – 2.43 (m, 4H, CH), 1.85 – 1.78 (m,2H, CH), 1.57 (t, J = 7.5 Hz, 2H, CH), 1.31 – 1.22 (m, 10H, CH), 0.89 – 0.84 (m, 3H, CH).

[0045] Example 2: 3,7-bis(dimethylamino)-N-(4-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butane-2-yl)amino)-4-oxobutyl)-10H-phenthiazine-10-carboxamide (I b )

[0046] Preparation of 4-(3,7-bis(dimethylamino)-10H-phenthiazine-10-carbamate)butyric acid (compound 4)

[0047] 3,7-bis(dimethylamino)-10H-phenthiazine-10-carbonyl chloride (1) (348 mg, 1 mmol) and 4-aminobutyric acid (137 mg, 1.1 mmol) were dissolved in dichloromethane, and triethylamine (300 mg) was added. The mixture was reacted at room temperature under N2 protection for 4-5 hours. After the reaction was complete, the mixture was evaporated to dryness, dissolved and extracted with dichloromethane, and the organic layer was concentrated to give a blue solid with a yield of 80%.

[0048] 3,7-Bis(dimethylamino)-N-(4-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butane-2-yl)amino)-4-oxobutyl)-10H-phenthiazine-10-carboxamide (I b Preparation of )

[0049] Under nitrogen protection, compound 4 (282 mg, 0.68 mmol), HATU (306 mg, 0.81 mmol), and DIPEA (103 mg, 0.81 mmol) were dissolved in anhydrous dichloromethane and activated by stirring at room temperature for 30 min. Then, fingolimod 2 (228 mg, 0.75 mmol) was added to the reaction system, and the reaction was continued with stirring for 3 h. After the reaction was complete (monitored by TLC), the mixture was extracted with water and dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, v / v 3:1) to give the target amide product in 60% yield. 1 H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 8.8 Hz, 2H, ArH), 7.06 (m, 4H,ArH), 6.87 (s, 1H, NH), 6.70 (d, J = 2.7 Hz, 2H, ArH), 6.60 (dd, J = 8.9, 2.8Hz, 2H, ArH), 5.11 (s, 1H, NH), 4.65 (d, J = 6.6 Hz, 2H, CH), 3.81 (dd, J =11.9, 4.9 Hz, 2H, CH), 3.60 (dd, J = 11.6, 3.8 Hz, 2H, CH), 3.32 (q, J = 6.1Hz, 2H, OH), 2.92 (s, 12H, CH), 2.53 (q, J = 6.2 Hz, 4H, CH), 2.23 – 2.15 (m,2H, CH), 1.93 – 1.85 (m, 2H, CH), 1.70 (t, J = 6.4 Hz, 2H, CH), 1.60 – 1.53(m, 2H, CH), 1.28 (dd, J = 12.6, 8.6 Hz, 10H, CH), 0.87 (t, J = 6.6 Hz, 3H, CH).

[0050] Example 3: 3-(2,3-dihydroxy-1-(4-hexylphenyl)propyl)carbamoyloxypropyl-1-(2-(10-(dimethylamino)-3,7-dimethylphenthiazin-2-yl)acetyl)carbamate (I) c )

[0051] Preparation of 3,7-bis(dimethylamino)-N-(3-hydroxypropyl)-10H-phenthiazine-10-carboxamide (compound 6)

[0052] 3,7-bis(dimethylamino)-10H-phenthiazine-10-carbonyl chloride (1,348 mg, 1 mmol) and 3-amino-1-propanol (5,150 mg, 2 mmol) were dissolved in dichloromethane, and triethylamine (300 mg) was added. The reaction was carried out at room temperature for 1 h. After the reaction was completed, rapid column chromatography was used to purify the solid to a blue color, with a yield of 86%. The chromatographic data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ7.25 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 2.8 Hz, 2H), 6.66 (dd, J = 8.9, 2.8Hz, 2H), 6.08 (m, 1H), 4.43 (t, J = 5.2 Hz, 1H), 3.43 – 3.35 (m, 9H), 3.10(q, J = 6.3 Hz, 2H), 2.89 (s, 12H), 1.53 (m, 2H).

[0053] Preparation of 3-(3,7-bis(dimethylamino)-10H-phenthiazine-10-carboxamide)propyl(4-nitrophenyl) carbonate (compound 7)

[0054] Compound 6 (193 mg, 0.5 mmol) and phenyl p-nitrochloroformate (121 mg, 0.6 mmol) were dissolved in dichloromethane, and triethylamine (150 mg) was added. The reaction was carried out at room temperature for 1-2 h. After the reaction was completed, the product was purified by rapid column chromatography to obtain a blue-gray solid with a yield of 87%.

[0055] 3-(2,3-dihydroxy-1-(4-hexylphenyl)propyl)carbamoyloxypropyl-1-(2-(10-(dimethylamino)-3,7-dimethylphenthiazin-2-yl)acetyl)carbamate (I) c Preparation of )

[0056] Compound 7 (198 mg, 0.36 mmol) and fingomodide 2 (122 mg, 0.40 mmol) were dissolved in dichloromethane, and DIPEA (183 mg, 1.44 mmol), DMAP (4.40 mg, 0.036 mmol), and HOBt (48.64 mg, 0.36 mmol) were added. The mixture was stirred at room temperature for 24 h. After the reaction was complete (monitored by TLC), the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, v / v 3:1) to obtain the target carbamate product I. c Yield: 28%. 1H NMR(400 MHz, DMSO-d6) δ 7.28 (s, 1H), 7.02 – 6.97 (m, 3H), 6.61 (d, J = 2.8 Hz,3H), 6.57 (d, J = 2.9 Hz, 2H), 6.54 (m, 2H), 5.62 (t, J = 6.4 Hz, 1H), 3.97(t, J = 6.1 Hz, 3H), 3.01 (dd, J = 7.5, 5.0 Hz, 3H), 2.85 (s, 12H), 2.51 –2.38 (m, 5H), 1.72 (dd, J = 6.2, 1.8 Hz, 2H), 1.54 (q, J = 7.2 Hz, 6H), 1.24– 1.13 (m, 10H), 0.83 – 0.76 (m, 3H).

[0057] Example 4: Compound I of the present invention a -I c Spectral properties

[0058] Compound I of the present invention a -I c The solution was dissolved in an aqueous solution of 0.2% DMSO to prepare a detection solution with a concentration of 20 μM.

[0059] The compound I of this invention was detected by fluorescence spectroscopy. a -I c The time- and concentration-responsive properties to HClO were studied. Specifically, the method involved detecting compound I of this invention using fluorescence spectroscopy. a -I c The changes in fluorescence spectra before and after the addition of HClO solution were studied. 660 nm was selected as the excitation wavelength to investigate its fluorescence performance, while a fluorescence spectrometer was responsible for collecting fluorescence emission spectrum data in the 675-900 nm range.

[0060] Figure 1 To detect compound I of the present invention a -I c Fluorescence spectra of time- and concentration-responsive properties to HClO. Figure 1 Figures a-c in the figure represent compound I of the present invention. a -I c Fluorescence spectra of 20 μM DMSO aqueous solution as a function of HClO concentration; Figure 1 Figures d-f in the figure represent compound I of this invention. a -I cQuantitative fluorescence intensity at 683 nm after incubation for different times in 0.2% DMSO aqueous solution with HClO solution (concentration 400 μM) at different concentrations (20 μM).

[0061] Figure 1 Display: Compound I of the present invention a -I b Add HClO solutions of different concentrations (0, 200, 300, 400, 500 μM) to a 0.2% DMSO H2O solution. c Adding different concentrations (0, 10, 50, 100, 400 μM) of HClO solution to a 0.2% DMSO aqueous solution resulted in an increase in fluorescence intensity with increasing concentration. Figure 1 (Figures a, b, and c in the original text), and compound I of the present invention a and I c fluorescence intensity ( Figure 1 (Figures a and c in the diagram) are much higher than I. b fluorescence intensity ( Figure 1 (Figure b in the image). b and compound I of the present invention a I c When HClO solution is added to a 0.2% DMSO aqueous solution, the fluorescence intensity increases with time. Figure 1 (df ​​diagram), and compound I of the present invention a and I c fluorescence intensity ( Figure 1 The d and f graphs in the figure are much higher than I. b fluorescence intensity ( Figure 1 (See diagram e in the original text). This indicates that compound I of the present invention... a and I c Compare I b It exhibits higher sensitivity to HClO.

[0062] Example 5: Test of the neuroprotective effect of the compound of the present invention

[0063] The experiment used an oxygen deprivation (OGD / R) model to induce an inflammatory response in brain microglia (BV2 cells), and fingolimod was selected as a positive control drug.

[0064] BV2 cells in the logarithmic growth phase were selected and subjected to a 1×10⁻⁶ m² / h²⁻¹ m²⁻¹. 4 100 μL of seed cells per well was inoculated into 96-well plates and incubated for 24 h. The cells were then randomly divided into the following six groups:

[0065] Normal control group: cultured under normal conditions (northeaster, sugar-containing DMEM complete medium) throughout the entire process.

[0066] OGD / R model group: The culture medium was replaced with sugar-free DMEM complete medium and placed in an anoxic incubator (with 95% N2 + 5% CO2 mixed gas) for 6 h. After 6 h, the cells were reoxygenated and normal DMEM complete medium was added. The cells were incubated for another 18 h to simulate the reperfusion process.

[0067] Ia-Ic drug administration group: The culture medium was replaced with sugar-free DMEM complete medium and placed in an anaerobic incubator (with 95% N2 + 5% CO2 mixed gas) for 6 h. After 6 h, the cells were reoxygenated and at the same time, complete medium containing 2.5, 5, and 10 µM Ia-Ic was added and incubated for another 18 h to simulate the reperfusion process.

[0068] Fingo positive control group: The culture medium was replaced with sugar-free DMEM complete medium and placed in an anaerobic incubator (with 95% N2 + 5% CO2 mixed gas) for 6 h. After 6 h, the cells were reoxygenated and a complete medium containing 2.5, 5, and 10 µM Fingo was added. The cells were then incubated for another 18 h to simulate the reperfusion process.

[0069] Each group was given CCK-8 reagent, and after 2 hours of reaction, the optical density (OD) value at a wavelength of 450 nm was measured and the cell viability was calculated.

[0070] Cell survival rate formula:

[0071] Survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of normal control group - OD value of blank control group) × 100%;

[0072] The OD value refers to the optical density value. The OD value of the experimental group is the optical density value of the cells after treatment, the OD value of the control group is the optical density value of the untreated cells, and the OD value of the blank control group is the optical density value of the culture medium.

[0073] The statistical analysis of cell survival rates in each group is shown in the figure below. Figure 2 As shown.

[0074] like Figure 2 As shown, BV2 cells exhibited significantly reduced viability after OGD / R treatment, and the viability was further reduced after treatment with up to 10 µM of compound I of this invention. a -I c Cell viability was greater than 85% after treatment. Compound I of this invention. a -I c The compound I of this invention significantly enhanced BV2 cell viability in the 2.5-10 µM dose range compared to the OGD / R treatment group, and exhibited superior activity at most concentrations compared to the positive control drug Fingo, demonstrating its superiority over the OGD / R treatment group. a-I c It can effectively improve cell viability after OGD / R injury, thus proving that the compound of the present invention has a significant neuroprotective effect on ischemic stroke simulated by OGD / R.

[0075] Example 6: Selective Imaging Test of the Compounds of the Present Invention using the OGD / R Model

[0076] Compound I of the present invention was evaluated using a confocal fluorescence imaging system. a -I c Selective imaging capability in a glucose-oxygen deprivation model. The experiment was divided into the following six groups: Compound I of the present invention. a -I c (5 μM) group, OGD / R + 5 μM compound I of the present invention a -I c Group. An oxygen deprivation (OGD / R) model was used to induce an inflammatory response in BV2 cells, and the selective imaging effect of the compounds of this invention in the OGD / R model was investigated. Logarithmically grown BV2 cells were used in a 5×10⁻⁶ cell line. 4 Cells were seeded at a density of 1000 μL per well in confocal dishes and incubated routinely for 24 h. The culture medium was then replaced with sugar-free DMEM complete medium, and the cells were transferred to an anoxic incubator (95% N2 + 5% CO2 mixed gas) for another 6 h of incubation. After 6 h, the cells were reoxygenated, and the culture medium was then replaced with medium containing compound I of this invention. a -I c Cells were treated with (5 μM) DMEM complete medium and incubated for 2 h to initiate and maintain reperfusion. Confocal imaging was performed, and statistical analysis was conducted. The results are as follows: Figure 3 As shown.

[0077] in, Figure 3 Figure a shows images taken by confocal imaging for each experimental group in this embodiment, with a scale bar of 25 μm. Figure 3 Figure b in the middle is a statistical analysis result of the fluorescence intensity of the 640 nm pathway in Figure a.

[0078] The results show ( Figure 3 Compound I of the present invention a -I c While not fluorescent on its own, the compounds of this invention significantly illuminated the model group cells after treatment with an oxygen deprivation / glucose deprivation (OGD / R) model, with a fluorescence signal enhancement of tens of times. Compound I of this invention... a and I c The fluorescence produced at the same concentration is stronger than that of I. b The above results demonstrate that the compound of the present invention has selective fluorescence imaging capability for OGD / R cells.

[0079] Example 7: In vivo therapeutic effect of the compound of the present invention on cerebral ischemia

[0080] To evaluate the therapeutic effect of the compounds of this invention in vivo, male C57BL / 6 mice (age: 8-10 weeks; weight: 22-25 g) were randomly divided into four groups (n=10-12 mice / group):

[0081] Sham surgery group: Only surgical exposure is performed, without inserting suture plugs.

[0082] Model group: Mice underwent MCAO surgery, and the suture embolus was removed 1 hour after ischemia, and reperfusion was restored.

[0083] Fingolimod positive control group: mice underwent MCAO surgery, the suture embolus was removed 1 hour after ischemia, and Fingolimod (2.9 μmol / kg) was injected during reperfusion.

[0084] Ia-Ic treatment group: mice underwent MCAO surgery, the suture embolus was removed 1 hour after ischemia, and compound Ia-Ic (2.9 μmol / kg) was injected during reperfusion.

[0085] Twenty-four hours after reperfusion, the Longa 5-point scale was used to assess neurological deficits in mice. Subsequently, mice were euthanized under deep anesthesia, brain tissue was extracted, and TTC staining was performed. Infarct volume was calculated using image analysis software, and the therapeutic effect of the compound was evaluated as the percentage of infarct volume to the contralateral cerebral hemisphere volume. Results are as follows: Figure 4 As shown.

[0086] Figure 4 Figure a shows TTC staining images of coronal sections of the brains of mice in each group. Normal tissue is stained red, and infarcted areas appear pale white. Figure b is a statistical graph showing the percentage of cerebral infarction volume to the volume of the contralateral cerebral hemisphere in each group of mice (mean ± standard deviation, n = 3).

[0087] Experimental results show that ( Figure 4 Compared to the sham surgery group, the middle cerebral artery occlusion (MCAO) model group showed significantly higher neurological function scores and large-area infarct foci in the brain tissue. Figure 4 a, b). The results of TTC staining confirmed that compound I of this invention... a -I c Both can significantly reduce the volume of cerebral infarction, and their protective effect is superior to that of the MCAO model group, among which I a I c The volume of cerebral infarction was significantly better than that of the positive control Fingolimod group. Figure 4 a, b). In summary, compound I of the present invention a -I cIt has a clear neuroprotective effect and can reduce the area of ​​cerebral infarction and alleviate the pathological damage of brain tissue in mice with cerebral ischemia, providing new potential candidate drugs and strategies for the treatment of cerebral ischemic diseases.

[0088] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A methylene blue-fingolimod prodrug based on hypochlorous acid activation, characterized in that, The chemical structure of the methylene blue-fingolimod prodrug is shown in the following formula:

2. A methylene blue-fingolimod prodrug based on hypochlorous acid activation, characterized in that, The chemical structure of the methylene blue-fingolimod prodrug is shown in the following formula:

3. A methylene blue-fingolimod prodrug based on hypochlorous acid activation, characterized in that, The chemical structure of the methylene blue-fingolimod prodrug is shown in the following formula:

4. A method for preparing the methylene blue-fingolimod prodrug as described in claim 1, characterized in that, The synthetic route of the preparation method is shown in the following formula: ， The preparation method is as follows: Compound 1 and fingolimod 2 are reacted overnight at 0-20 degrees Celsius under organic base conditions, allowing Compound 1 to selectively react with the amino group of fingolimod 2. After the reaction is complete, Compound I is obtained by column chromatography. a .

5. A method for preparing the methylene blue-fingolimod prodrug as described in claim 2, characterized in that, The synthetic route of the preparation method is shown in the following formula: ， The preparation method includes the following steps: S1. Compound 1 reacts with aminobutyric acid 3 at room temperature overnight under triethylamine conditions until the reaction is complete to give compound 4; S2. Compound 4 and fingolimod 2 were subjected to an amidation reaction at 0–20°C under the conditions of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine to give compound I. b .

6. A method for preparing the methylene blue-fingolimod prodrug as described in claim 3, characterized in that, The synthetic route of the preparation method is shown in the following formula: The preparation method includes the following steps: S1. Compound 1 reacts with propanolamine 5 at room temperature under TEA conditions to give intermediate 6; S2. Intermediate 6 reacts with phenyl p-nitrochloroformate at room temperature under TEA conditions to give active intermediate 7; S3. Intermediate 7 reacted with fingolimod 2 at room temperature under TEA and 4-dimethylaminopyridine conditions. After the reaction was complete, compound I was purified by column chromatography. c .

7. The use of a methylene blue-fingolimod prodrug as described in any one of claims 1-3 in the preparation of a reagent for diagnosing ischemic stroke and / or cerebral ischemia-reperfusion injury.

8. The use of a methylene blue-fingolimod prodrug as described in any one of claims 1-3 in the preparation of a medicament for treating ischemic stroke and / or cerebral ischemia-reperfusion injury.

9. The use of a methylene blue-fingolimod prodrug as described in any one of claims 1-3 in the preparation of a fluorescent imaging reagent targeting ischemic brain tissue and cells.

10. A pharmaceutical composition, characterized in that, Includes the methylene blue-fingolimod prodrug as described in any one of claims 1-3, and a pharmaceutically acceptable carrier or excipient.

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