AER270 diagnosis and treatment prodrug with hypochlorous acid response characteristic as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and discloses an AER270 diagnosis and treatment prodrug with hypochlorous acid response characteristic as well as a preparation method and application thereof, the AER270 diagnosis and treatment prodrug has a structure as shown in a general formula I. Based on a core structure of methylene blue (MB), gamma-aminobutyric acid (GABA), gabapentin or aminopropanol is used as a connecting chain, and is coupled with an AQP4 inhibitor AER270 through an ester bond or an ether bond, so that the prodrug is prepared. According to the present invention, the HOCl specific activation rupture release active molecules AER270 and GABA or gabapentin is achieved, the penetrability on the blood brain barrier and the selective treatment on the cerebral apoplexy focus are enhanced, the reduced methylene blue molecule is released, the cerebral apoplexy focus can be specifically lightened, and the diagnosis and treatment integration is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to AER270 protheco-pharmaceutical with hypochlorous acid responsive properties, its preparation method and application. Background Technology

[0002] Neuroinflammation is a common pathological basis for many acute and chronic central nervous system diseases, including stroke, traumatic brain injury, Alzheimer's disease, and multiple sclerosis. Following a stroke (ischemic or hemorrhagic), cytotoxic and vasogenic edema often occur in brain tissue, leading to increased intracranial pressure, further reduced cerebral blood flow, and exacerbated neurological damage. During neuroinflammation, activated astrocytes and microglia produce reactive oxygen species (ROS). Among these, hypochlorous acid, catalyzed by myeloperoxidase, is a key and highly oxidizing effector molecule in the inflammatory microenvironment. Excessive hypochlorous acid not only damages neurons and oligodendrocytes but also amplifies the inflammatory cascade, worsening blood-brain barrier disruption and cerebral edema. In ischemic brain regions, the high concentration of hypochlorous acid (HClO) mediated by myeloperoxidase constitutes a specific pathological microenvironment. Utilizing this characteristic, designing prodrugs specifically activated by HClO has become an advanced targeting strategy.

[0003] Aquaporin 4 (AQP4) is the most abundant aquaporin in the central nervous system, mainly distributed at the terminal feet of astrocytes (sites in contact with blood vessels), and plays a crucial role in the formation and resolution of cerebral edema. In neuroinflammation and brain injury, the expression and function of AQP4 are significantly disrupted, and its mediated cytotoxic cerebral edema is a key factor leading to deterioration of neurological function and poor prognosis. Therefore, inhibiting AQP4 function has become an important strategy for alleviating cerebral edema and treating the aforementioned neurological diseases. The small molecule AQP4 inhibitor N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide (AER-270) has shown low or unstable BBB penetration in animal models, excessively rapid metabolism in vivo, and a short half-life. Post-stroke cerebral edema is a dynamic process lasting several days, requiring drugs to maintain stable and effective intracerebral concentrations within the therapeutic window. Meanwhile, AQP4 plays an important role in other organs throughout the body (such as the kidneys, eyes, and gastrointestinal tract), and inhibiting the expression of AQP4 (or structurally similar members of the AQP family) in other organs may produce serious side effects.

[0004] Therefore, in order to reduce systemic toxicity and improve the treatment effect of stroke, it is necessary to develop a highly central nervous system-targeting and selective protherapeutic drug. Summary of the Invention

[0005] In view of this, the present invention provides an AER270 prodrug with hypochlorous acid responsive properties, its preparation method and application. The AER270 prodrug can rapidly cleave and release the AQP4 inhibitor AER270 in the presence of HClO, while retaining the fluorescence properties of MB. This strategy ensures high local concentrations of the two active ingredients at the lesion site, while reducing systemic exposure, thereby potentially significantly improving the therapeutic effect and reducing side effects.

[0006] The specific technical solution of this invention is as follows:

[0007] In a first aspect, the present invention provides an AER270 prothecochemical drug with hypochlorous acid responsive properties, wherein the AER270 prothecochemical drug has the structure shown in general formula I:

[0008]

[0009] when At that time, the chemical structure of the AER270 prodrug is shown in the following formula:

[0010]

[0011] when At that time, the chemical structure of the AER270 prodrug is shown in the following formula:

[0012]

[0013] when At that time, the chemical structure of the AER270 prodrug is shown in the following formula:

[0014]

[0015] Secondly, the present invention provides a method for preparing the above-mentioned AER270 prothecochemical drug, the synthetic route of which is shown in the following formula:

[0016]

[0017] The preparation method includes the following steps:

[0018] S1. 3,7-bis(dimethylamino)-10H-phenthiazine-10-carbonyl chloride (compound 1) was reacted with methyl γ-aminobutyrate at room temperature overnight under organic base conditions until the reaction was complete. The mixture was purified by column chromatography to obtain compound 2, wherein the organic base was triethylamine or N,N-diisopropylethylamine.

[0019] S2. Hydrolyze compound 2 in an aqueous methanol solution containing NaOH to give 4-(3,7-bis(dimethylamino)-10H-phenthiazin-10-carbamate)butyric acid (compound 3);

[0020] S3,4-(3,7-bis(dimethylamino)-10H-phenthiazine-10-carbamoyl)butyric acid (compound 3) was esterified with the aquaporin inhibitor AER270 under the conditions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP). After the reaction was complete, compound I1 was obtained by column chromatography.

[0021] Thirdly, the present invention provides a method for preparing the above-mentioned AER270 prothecochemical drug, the synthetic route of which is shown in the following formula:

[0022]

[0023] The preparation method includes the following steps:

[0024] S1. Compound 1 reacts with aminopropanol at room temperature overnight under organic base conditions until the reaction is complete, and is purified by column chromatography to obtain compound 4, wherein the organic base is triethylamine or N,N-diisopropylethylamine;

[0025] S2. Compound 4 was etherified with the aquaporin inhibitor AER270 under the conditions of diisopropyl azodicarboxylate (DIAD) and PPh3 to give compound I2.

[0026] Fourthly, the present invention provides a method for preparing the above-mentioned AER270 prothecochemical drug, the synthetic route of which is shown in the following formula:

[0027]

[0028] The preparation method includes the following steps:

[0029] S1. Compound 1 and gabapentin (compound 5) were reacted overnight at room temperature under organic base conditions until the reaction was complete, and the mixture was purified by column chromatography to obtain compound 6, wherein the organic base was triethylamine or N,N-diisopropylethylamine;

[0030] S2. Compound 6 was esterified with the aquaporin inhibitor AER270 under the conditions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-dimethylaminopyridine to give compound I3.

[0031] Fifthly, the present invention provides the application of the above-mentioned AER270 prodrug in the preparation of fluorescence imaging reagents targeting ischemic brain tissues and cells.

[0032] In a sixth aspect, the present invention provides the use of the above-mentioned AER270 pre-diagnostic drug in the preparation of reagents for imaging diagnosis of ischemic stroke and / or cerebral ischemia-reperfusion injury.

[0033] In a seventh aspect, the present invention provides the application of the above-mentioned AER270 prodrug in the preparation of AQP4 inhibitors targeting ischemic brain tissue.

[0034] Eighthly, the present invention provides the use of the above-mentioned AER270 prothecochemical drug in the preparation of a medicament for treating ischemic stroke and / or cerebral ischemia-reperfusion injury.

[0035] Compared with existing technologies, this invention is based on the core structure of methylene blue and uses γ-aminobutyric acid (GABA), gabapentin, or aminopropanol as a linker chain. It is coupled with the AQP4 inhibitor AER270 through ester or ether bonds to achieve HOCl-specific activation and release of active molecules such as AER270 and GABA, enhancing the penetration of the blood-brain barrier and selective treatment of stroke lesions. At the same time, it releases reduced methylene blue (MB) molecules, which emit fluorescence and can specifically illuminate stroke lesions, realizing integrated diagnosis and treatment. It has broad application prospects in the treatment of neuroinflammation and cerebral edema. Attached Figure Description

[0036] Figure 1 The fluorescence spectra of the compounds of the present invention after incubation with different concentrations of HClO in 0.2% DMSO H2O solution are shown. The horizontal axis represents wavelength and the vertical axis represents fluorescence intensity.

[0037] Figure 2 The fluorescence intensity of the compound of the present invention at 683 nm over time after incubation with HClO (500 μM) in 0.2% DMSO H2O solution is shown.

[0038] Figure 3 The results of the test on the specific response of the compound of the present invention to HClO are shown. The horizontal axis represents various substances in the physiological environment (including various anions, cations, oxidizing substances, and reducing substances) and HClO, and the vertical axis represents the fluorescence intensity.

[0039] Figure 4 The results of neuroprotective effects of the compounds of the present invention at the cellular level are shown.

[0040] Figure 5 The results of selective imaging tests using the OGD / R model for the compounds of this invention are shown.

[0041] Figure 6 The results of HClO-responsive imaging tests of the compounds of the present invention are shown.

[0042] Figure 7 The results of fluorescence imaging tests on a mouse model of cerebral ischemia-reperfusion of the compounds of the present invention are shown.

[0043] Figure 8The results of in vivo therapeutic efficacy tests of the compounds of the present invention for cerebral ischemia are shown. Detailed Implementation

[0044] To further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention. They should not be construed as limiting the present invention.

[0045] Example 1: Preparation of methyl 4-(3,7-bis(dimethylamino)-10H-phenthiazine-10-carbamate)butyrate (2)

[0046] 3,7-bis(dimethylamino)-10H-phenthiazine-10-carbonyl chloride (1) (348 mg, 1 mmol) and methyl 4-aminobutyrate (152 mg, 1.1 mmol) were dissolved in dichloromethane, and triethylamine (303 mg, 3 mmol) was added. The reaction was carried out under N2 protection at room temperature for 4-5 hours. After the reaction was complete, the solution was evaporated to dryness to give a blue solid with a yield of 82%. The spectral data are as follows: 1 H NMR (400MHz, CDCl3) δ7.33(d,J=8.8Hz,2H),6.69(d,J=2.8Hz,2H),6.62(dd,J=8.8,2.8Hz,2H),5.05(t, J=5.9Hz,1H),3.65(s,3H),3.25(q,J=6.6Hz,2H),2.93(s,12H),2.33(t,J=7.4Hz,2H),1.84–1.78(m,2H)

[0047] Example 2: Preparation of 2-chloro-5-[(3,5-bis(dimethylamino)-10H-phenthiazine-10-carbamoyl)butyric acid ester (I1)

[0048] Methyl 4-(3,7-bis(dimethylamino)-10H-phenthiazin-10-carbamate)butyrate (2) (200 mg, 1 mmol) and sodium hydroxide (160 mg, 4 mmol) were added to a flask. Ethanol:water (2:1, v / v) was added, and the mixture was reacted at room temperature for 30 min. After the reaction was complete, the ethanol was evaporated, and the mixture was extracted with dichloromethane. The organic layer was concentrated and dissolved in anhydrous dichloromethane with EDCI (240 mg, 1.25 mmol). The mixture was reacted at room temperature for 30 min, and then N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide (320 mg, 0.84 mmol) and 4-dimethylaminopyridine (15 mg, 0.085 mmol) were added. The mixture was reacted at room temperature for 24 h, and then extracted with water. The solution was purified by column chromatography to give a pale blue solid I1 in 44% yield. The spectral data are as follows:

[0049] 1 H NMR (400MHz, CDCl3) δ10.02(s,1H),8.48(s,2H),7.73–7.37(m,4H),7.02(d,J=8.8Hz,2H),6.51(d,J=2.7Hz,2H),6.4 1(dd,J=8.8,2.8Hz,2H),5.25(t,J=6.1Hz,1H),3.35(d,J=6.1Hz,2H),2.91(s,12H),2.74–2.66(m,2H),1.89(s,2H).

[0050] Example 3: Preparation of 3,7-bis(dimethylamino)-N-(3-hydroxypropyl)-10H-phenthiazine-10-carboxamide (5)

[0051] 3,7-bis(dimethylamino)-10H-phenthiazine-10-carbonyl chloride (348 mg, 1 mmol) and 3-amino-1-propanol (150 mg, 2 mmol) were dissolved in dichloromethane, and triethylamine (303 mg, 3 mmol) 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 white solid in 86% yield. The chromatographic data are as follows:

[0052] 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.25 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 2.8 Hz, 2H), 6.66 (dd, J = 8.9, 2.8 Hz, 2H), 6.08 (t, J = 5.6 Hz, 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 (p, J = 6.4 Hz, 2H). Example 4: Preparation of N-(3-(2-((3,5-bis(trifluoromethyl)phenyl)acyl)-4-chloro-phenyl)propyl)-3,7-bis(dimethylamino)-10H-phenthiazine-10-carboxamide (I₂)

[0053] 3,7-Bis(dimethylamino)-N-(3-hydroxypropyl)-10H-phenthiazine-10-carboxamide (371 mg, 1 mmol), N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide (400 mg, 1.1 mmol), and triphenylphosphine (393 mg, 1.5 mmol) were dissolved in anhydrous tetrahydrofuran and stirred at 0 °C for 15 minutes. Then, diisopropyl azodicarboxylate (304 mg, 1.5 mmol) was slowly added dropwise, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was purified by column chromatography in 76% yield. The chromatographic data are as follows:

[0054] 1 H NMR (400MHz, CDCl3) δ10.29(s,1H),8.27(s,2H),8.00(d,J=2.8Hz,1H),7.60(s,1H),7.39(dd,J=8.9,2.8Hz,1H),7.22(s,2H),6.93(d,J=8.8 Hz,1H),6.66(d,J=2.8Hz,2H),6.55(dd,J=8.8,2.9Hz,2H),4.17(t,J=6.0Hz,2H),3.48(q,J=6.1Hz,2H),2.92(s,13H),2.02(p,J=6.1Hz,2H).

[0055] Example 5: Preparation of 2-{1-[(3,7-bis(dimethylamino)-10H-phenthiazine-10-carbamate)methyl]cyclohexyl}acetic acid (6)

[0056] 3,7-bis(dimethylamino)-10H-phenthiazine-10-carbonyl chloride (1) (348 mg, 1 mmol) and gabapentin (5) (245 mg, 1.5 mmol) were dissolved in dichloromethane. Triethylamine (303 mg, 3 mmol) was added, and the mixture was reacted at room temperature for 4 hours. After the reaction was complete, the mixture was extracted and evaporated to dryness to give a pale blue solid 6. Yield: 88%. Spectral data are as follows:

[0057] 1 H NMR (400MHz, CDCl3) δ7.35–7.29(m,2H),7.27(d,J=1.0Hz,1H),6.72(d,J=2.7Hz,2H),6.63(dd,J=8.8,2. 8Hz,2H),5.41(t,J=6.8Hz,1H),3.19(d,J=6.8Hz,2H),2.95(d,J=1.1Hz,13H),2.34(s,2H),1.52(s,11H).

[0058] Example 10: Preparation of 2-(1-((3,7-bis(dimethylamino)-10H-phenthiazine-10-carbamate)methyl)cyclohexyl)acetic acid 4-chloro-2-((3,5-bis(trifluoromethyl)phenyl)carbamoyl)phenyl ester (I3)

[0059] 2-{1-[(3,7-bis(dimethylamino)-10H-phenthiazin-10-carbamate)methyl]cyclohexyl}acetic acid (205 mg, 1 mmol) and EDCI (148 mg, 1.5 mmol) were dissolved in anhydrous dichloromethane and reacted at room temperature for 30 min. Then, N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide (142 mg, 1.5 mol) and 4-dimethylaminopyridine (3 mg, 0.05 mmol) were added, and the reaction was continued at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography to give a pale blue solid I3 in 55% yield. The spectral data are as follows:

[0060] 1 H NMR (400MHz, DMSO-d6) δ11.10(s,1H),8.38(s,2H),7.90–7.83(m,2H),7.68(dd,J=8.7,2.6Hz,1H),7.23(dd,J=8.7,4.6Hz,3H),6.68(d, J=2.8Hz,2H),6.59(dd,J=8.9,2.8Hz,2H),5.76(t,J=6.4Hz,1H),3.16(d,J=6.3Hz,2H),2.85(s,12H),2.40(s,2H),1.37–1.20(m,11H).

[0061] Example 11: Response of the compounds of the present invention to HClO

[0062] The responsiveness of compounds I1, I2, and I3 of this invention to different concentrations of HClO was detected using fluorescence spectroscopy. Specifically, compounds I1, I2, and I3 were mixed with different concentrations (0, 200, 300, 400, and 500 μM) of HClO, and the changes in fluorescence intensity with varying concentrations were measured. The results are as follows: Figure 1 As shown. Among them, Figure 1 AC represent the fluorescence spectra of compounds I1, I2, and I3 (20 μM) of this invention after incubation with different concentrations of HClO in 0.2% DMSO H2O solution. Figure 1 The results showed that when different concentrations (0-500 μM) of HClO were added to compounds I1, I2, and I3 of the present invention, the fluorescence increased with the increase of concentration, proving that the compounds of the present invention have good responsiveness to HClO, and compound I1 of the present invention had the highest fluorescence intensity and the strongest responsiveness.

[0063] The time-response properties of compounds I1, I2, and I3 of this invention to HClO (500 μM) were detected using fluorescence spectroscopy. Specifically, compounds I1, I2, and I3 were mixed with HClO, and the changes in fluorescence intensity over time were measured. The results are as follows: Figure 2As shown. Among them, Figure 2 AC represent the quantitative values ​​of fluorescence intensity at 683 nm over time for compounds I1, I2, and I3 (20 μM) of this invention after incubation in 0.2% DMSO H2O solution with HClO (500 μM). The excitation wavelength was 660 nm, and the emission wavelength was 675-900 nm. Figure 2 As shown, when 500 μM HClO is added, the fluorescence increases with time, reaching its strongest intensity at 20-30 min, and then remains stable, demonstrating that the fluorophore released by this compound has good fluorescence stability.

[0064] Example 12: Test of the specific response of the compound of the present invention to HClO

[0065] The specific HClO response of compound I1 of this invention was detected by fluorescence spectroscopy. To analyze the selectivity of various substances in the physiological environment (including various anions, cations, oxidizing substances, and reducing substances) and HClO– to the compound of this invention, the compound was added to corresponding bioanalytes (such as Na2SO4) at room temperature. + K + Cu 2 + Fe 2+ Zn 2+ Mn 2+ Ca 2+ Mg 2+ The sample was incubated with I-, glucose (Glu), sodium ascorbate (VcNa) and HClO, followed by fluorescence spectroscopy measurements. The results are as follows: Figure 3 As shown. Figure 3 The results show that compound I1 (20 μM) of the present invention was reacted with 1 mM of the corresponding bioanalyte (such as Na) in 0.2% DMSO H2O solution. + K + Cu 2+ Fe 2+ Zn 2+ Mn 2+ Ca 2+ Mg 2+ Fluorescence intensity (mean ± standard deviation, n = 3) after incubation with I-, glucose (Glu), sodium ascorbate (VcNa) and 500 μM HClO.

[0066] The results show ( Figure 3 When different interfering ions were added to the solution of the compound of the present invention, the fluorescence intensity did not change significantly; however, when HClO was added, the solution showed strong fluorescence, proving that the compound of the present invention has good response specificity to HClO.

[0067] Example 13: Test on the protective effect of the compound of the present invention against glucosamine deprivation injury in brain cells

[0068] The oxygen deprivation / glucose deprivation (OGD / R) model was used to induce hypoxia in BV2 cells (mouse microglia differentiation cell line), PC12 cells (adrenal medullary pheochromocytoma cell line of adult rats), SH-SY5Y cells (neuroblastoma subclonal cell line from human bone marrow), and Bend.3 cells (mouse brain microvascular endothelial cell line). AER270 was selected as a positive control drug to study the therapeutic effect of the compounds of this invention. Logarithmically grown cells were used in a 1×10⁻⁶... 4 Cells were seeded at a density of 100 μL / well in 96-well plates and incubated for 24 h. The medium was then replaced with sugar-free DMEM complete medium and incubated for another 6 h in an anoxic incubator (95% N2 + 5% CO2 mixed gas). After 6 h, the cells were reoxygenated, and the medium was replaced with DMEM complete medium again. Cells were then treated with complete medium containing different concentrations (0.01–10 μM) of compounds I1, I2, I3, and AER270 of this invention and incubated for 18 h to initiate and maintain reperfusion. After two hours of CCK-8 reaction, the absorbance at 450 nm was measured and cell viability was calculated.

[0069] Cell survival rate formula:

[0070] Survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%

[0071] The OD value refers to the optical density value. The OD value of the experimental group is the absorbance value of the treated cells, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0072] The test results of the protective effect of the compound of the present invention on brain cells under OGD / R are as follows: Figure 4 As shown.

[0073] Figure 4 A represents the cell viability of BV2 cells treated with OGD / R after incubation and culture for 24 hours with different concentrations (0, 2.5, 5, 10 μM) of compounds I1, I2, I3, and AER270 of this invention. The results showed that cell viability significantly decreased after OGD / R treatment. After incubation with compounds I1, I2, I3, and AER270, compounds I1 and I3 showed better effects on improving BV2 cell viability than the same doses (2.5-10 μM) of I2 and AER270. Figure 4 A).

[0074] Figure 4BD represents PC12 cells treated with OGD / R ( Figure 4 B), SH-SY5Y cells ( Figure 4 C) and Bend3 cells ( Figure 4 D) Cell viability after incubation and culture for 24 hours with different concentrations (0, 0.01, 0.1, 0.5, 1 μM) of compounds I1, I3, and AER270 of the present invention. The results showed that cell viability significantly decreased after OGD / R treatment. After incubation with compounds I1, I3, and AER270 of the present invention, compound I1 showed a better effect on improving cell viability than the same doses (0.01–1 μM) of I3 and AER270. Figure 4 (BD). This study shows that compound I1 of the present invention can effectively improve cell viability after OGD / R injury, demonstrating that compound I1 of the present invention exhibits a significant protective effect against glucose-oxygen deprivation injury in an OGD / R-induced cell inflammation model, suggesting its significant therapeutic potential for ischemic stroke.

[0075] Example 14: Selective imaging testing of the compounds of the present invention using the oxygen deprivation / glucose deprivation (OGD / R) model.

[0076] Confocal fluorescence imaging was used to evaluate the selective imaging ability of the compounds of this invention in a glucose-oxygen deprivation model. Experimental groups were divided into: PBS group, compound groups I1, I2, and I3 (5 μM), and OGD / R + 5 μM compound groups I1, I2, and I3. An inflammatory response in BV2 cells was induced using a glucose-oxygen deprivation model (OGD / R), and the selective imaging effect of the compounds of this invention in the OGD / R model was studied. Logarithmically grown BV2 cells were used in a 5 × 10⁻⁶ cell line. 4 Cells were seeded at a density of 1000 μL / well in confocal dishes and incubated for 24 h. The medium was then replaced with sugar-free DMEM complete medium and incubated for another 6 h in an anoxic incubator (95% N2 + 5% CO2 mixed gas). After 6 h, the cells were reoxygenated, and the medium was replaced again with DMEM complete medium. Cells were then treated with complete medium containing compounds I1, I2, and I3 (5 μM) of this invention and incubated for 2 h to initiate and maintain reperfusion. The PBS group without OGD / R treatment and the compound group of this invention served as controls. Cells were washed three times with PBS, stained with Hoechst 33342 nuclei, and then photographed using confocal microscopy. Statistical analysis was performed, and the results are as follows: Figure 5 As shown.

[0077] in, Figure 5 Image A is a confocal image taken by each experimental group in this embodiment, with a scale bar of 50 μm. Figure 5 B is the result of statistical analysis of fluorescence intensity in Figure A.

[0078] The results show ( Figure 5The compounds of this invention do not produce fluorescence themselves, but after OGD / R treatment, their fluorescence signals are significantly enhanced by tens of times. Among them, I1 produces stronger fluorescence than I2 and I3 at the same concentration. Figure 5 (A, B). This demonstrates that the compounds of the present invention possess selective fluorescence imaging capabilities for OGD / R-damaged cells.

[0079] Example 15: HClO-responsive fluorescence imaging test of the compound of the present invention

[0080] The HClO-responsive imaging ability of compound I1 was evaluated using confocal fluorescence imaging. Experimental groups were divided into three groups: compound I1 (5 μM), compound I1 with HClO + 5 μM, and compound I1 with LPS + 5 μM. BV2 cells were incubated with HClO to study the exogenous HClO-responsive imaging effect of the compound, and an endogenous HClO-responsive imaging effect was studied by inducing an inflammatory response in BV2 cells with LPS. Logarithmically grown BV2 cells were incubated at 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 1000 μL per well in confocal dishes and incubated for 24 h. They were then incubated for 1 h in DMEM complete medium containing HClO (10–100 μM) to provide exogenous HClO, followed by incubation for 1 h with complete medium containing compound I1 (5 μM) of the present invention. Alternatively, they were incubated for 1 h in DMEM complete medium containing LPS (1 μg / ml) to induce endogenous HClO production, followed by incubation for 1 h with complete medium containing compound I1 (5 μM) of the present invention. The untreated group (with compound I1) served as a control. Confocal imaging was performed, and statistical analysis was conducted. The results are as follows: Figure 6 As shown.

[0081] in, Figure 6 Image A is a confocal image taken by each experimental group in this embodiment, with a scale bar of 25 μm. Figure 6 B is the statistical analysis result of the fluorescence intensity of the 640nm pathway in Figure A.

[0082] The results show ( Figure 6 The compound of the present invention does not produce fluorescence on its own, but its fluorescence signal is significantly enhanced after activation by endogenous and exogenous HClO, which proves that the compound of the present invention has HClO-responsive cell imaging ability.

[0083] Example 16: Fluorescence imaging test of the cerebral ischemia-reperfusion model mouse of the compound of the present invention.

[0084] Establishment of a middle cerebral artery occlusion (MCAO) model: Mice were fasted for 12 hours prior to surgery, and the anesthetic was 2.5% tribromoethanol. After anesthesia, the mice were placed supine on the operating table, with their four legs immobilized to ensure unobstructed breathing. The hair on the mouse's neck was removed with scissors, and the common carotid artery, internal carotid artery, and external carotid artery were separated. A suture was inserted into the middle cerebral artery and secured. After 1 hour of ischemia, the suture was removed, and immediately, physiological saline / the compound of this invention (13 μmol / kg) was injected via the tail vein.

[0085] Mice were randomly divided into two groups: 1) the Sham group (sham-operated group): mice underwent vascular dissection without inserting a suture embolism, and were injected intravenously with compound I1 of the present invention at a dose of 13 μmol / kg; 2) the MCAO + compound I1 group: model mice were injected intravenously with compound I1 of the present invention at a dose of 13 μmol / kg. Changes in fluorescence signals in the mouse brain were observed using small animal imaging to evaluate the fluorescence imaging capability of the compound and its targeting of ischemic brain tissue.

[0086] Figure 7 This is a fluorescence imaging test result of the compound of the present invention in a mouse model of cerebral ischemia-reperfusion; wherein, Figure 7 A shows fluorescence imaging of mice in the Sham group, a mouse model of cerebral ischemia-reperfusion injury injected with the compound of the present invention. Figure 7 B represents the average fluorescence intensity (mean ± standard deviation, n = 3) in the mouse brain shown in Figure A.

[0087] The results showed that the compound of the present invention could produce a clear fluorescent signal in the brain. Figure 7 A), and significantly higher than the Sham group, indicating that the compound of the present invention can effectively penetrate the blood-brain barrier and be activated by HClO in ischemic brain tissue, releasing MB fluorescence. This demonstrates that the compound has selective fluorescence imaging capability for ischemic stroke disease models.

[0088] Example 17: In vivo therapeutic effect of the compound of the present invention on cerebral ischemia

[0089] To evaluate the therapeutic effect of the compound of the present invention in vivo, experimental mice were divided into five groups: sham surgery group, middle cerebral artery occlusion (MCAO) group, AER270 group, and compound I1 group of the present invention. After 1 hour of ischemia, the suture embolus was removed, reperfusion was restored, and AER270 (13 μmol / kg) and the compound of the present invention (13 μmol / kg) were immediately injected via the tail vein. 24 hours later, the Longa score was performed to assess neurological deficits. Subsequently, the mice were anesthetized, and brain tissue was harvested for TTC staining to determine infarct volume and evaluate the therapeutic effect of the compound of the present invention.

[0090] Figure 8 The results of in vivo therapeutic efficacy tests of the compounds of the present invention for cerebral ischemia are shown.

[0091] in, Figure 8 Image A shows TTC-stained coronal sections of the brains of mice in each group. Figure 7 B is a statistical graph of the cerebral infarction volume of mice in each group (mean ± standard deviation, n = 3).

[0092] Experimental results show that ( Figure 8 Compared to the sham surgery group, the middle cerebral artery occlusion (MCAO) model group showed large-area infarct lesions in the brain tissue of mice. Figure 8 A, B). TTC staining results confirmed that no infarction was observed in the brain of the Sham group mice, while the right brain tissue of the MCAO group mice showed a large area of ​​paleness, with an infarct volume of 33.4% ± 4.32%, significantly higher than that of the Sham group. However, compared with the MCAO group, the infarct area was significantly reduced in the AER270 group (29.6% ± 1.71%) and the compound I1 group of this invention (11.6% ± 3.08%) (P < 0.0001), indicating that compound I1 of this invention can significantly reduce the infarct volume in mice with middle cerebral artery occlusion, improve the therapeutic effect of the drug, and protect brain tissue. Compound I1 of this invention can significantly reduce the infarct volume, and its protective effect is superior to that of the AER270 group ( Figure 8 (A, B). In summary, the compounds of this invention have a clear neuroprotective effect, and can reduce the area of ​​cerebral infarction and improve 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.

Claims

1. A pre-diagnostic drug for AER270 with hypochlorous acid responsive properties, characterized in that, The chemical structure of the AER270 prodrug is shown in the following formula:

2. A pre-diagnostic drug for AER270 with hypochlorous acid responsive properties, characterized in that, The chemical structure of the AER270 prodrug is shown in the following formula:

3. A pre-diagnostic drug for AER270 with hypochlorous acid responsive properties, characterized in that, The chemical structure of the AER270 prodrug is shown in the following formula:

4. A method for preparing the AER270 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 includes the following steps: S1. 3,7-bis(dimethylamino)-10H-phenthiazine-10-carbonyl chloride and methyl γ-aminobutyrate were reacted overnight at room temperature under organic base conditions until the reaction was complete. The reaction was purified by column chromatography to obtain compound 2, wherein the organic base was triethylamine or N,N-diisopropylethylamine. S2. Hydrolyze compound 2 in an aqueous methanol solution containing NaOH to give 4-(3,7-bis(dimethylamino)-10H-phenthiazin-10-carbamate)butyric acid; S3,4-(3,7-bis(dimethylamino)-10H-phenthiazine-10-carbamoyl)butyric acid was esterified with the aquaporin inhibitor AER270 under the conditions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-dimethylaminopyridine. After the reaction was complete, compound I1 was obtained by column chromatography.

5. A method for preparing the AER270 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 aminopropanol at room temperature overnight under organic base conditions until the reaction is complete, and is purified by column chromatography to obtain compound 4, wherein the organic base is triethylamine or N,N-diisopropylethylamine; S2. Compound 4 was etherified with the aquaporin inhibitor AER270 under the conditions of diisopropyl azodicarboxylate and PPh3 to give compound I2.

6. A method for preparing the AER270 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 and gabapentin were reacted overnight at room temperature under organic base conditions until the reaction was complete, and the reaction was purified by column chromatography to obtain compound 6, wherein the organic base was triethylamine or N,N-diisopropylethylamine; S2. Compound 6 was esterified with the aquaporin inhibitor AER270 under the conditions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-dimethylaminopyridine to give compound I3.

7. The use of the AER270 prodrug as described in any one of claims 1-3 in the preparation of a fluorescence imaging reagent targeting ischemic brain tissue and cells.

8. The use of the AER270 prodrug as described in any one of claims 1-3 in the preparation of reagents for imaging diagnosis of ischemic stroke and / or cerebral ischemia-reperfusion injury.

9. The use of the AER270 prodrug as described in any one of claims 1-3 in the preparation of an AQP4 inhibitor targeting ischemic brain tissue.

10. The use of the AER270 prothecochemical drug as described in any one of claims 1-3 in the preparation of a medicament for the treatment of ischemic stroke and / or cerebral ischemia-reperfusion injury.

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