Hypoxia-responsive beta-carboline quinoline azoium and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a beta-carboline quinoline azo oxonium based on hypoxia response and a preparation method and application thereof. The application constructs a brain ischemia activated beta-carboline quinoline azo oxonium shown in the following formula. The beta-carboline quinoline azo oxonium realizes focal specific release of NO through reduction and fracture of an azo bond, and can integrate near-infrared fluorescence imaging function to provide real-time visual monitoring for treatment response.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a hypoxia-responsive β-carbolinequinoline azomonium, its preparation method, and its application. Background Technology

[0002] Ischemic stroke, a leading cause of disability and death worldwide, involves multiple mechanisms in its pathological cascade, including energy metabolism breakdown, oxidative stress outbreaks, amplified neuroinflammatory cascades, and disruption of the blood-brain barrier (BBB). Based on these complex pathological mechanisms, researchers have begun to focus on innovative approaches to improve treatment outcomes.

[0003] The "Double-Edged Sword" Effect of NO and Treatment Bottlenecks Nitric oxide (NO), as a neutral hydrophobic signaling molecule, exhibits a significant "double-edged sword" effect. Under physiological conditions, low concentrations of NO (<10 nmol / L) produced by endothelial nitric oxide synthase (eNOS) exert a protective effect through vasodilation and inhibition of platelet aggregation (DOI: 10.1038 / aps.2012.82).

[0004] Existing treatment strategies have limitations. Although thrombolytic therapy can partially restore blood flow, its narrow time window (<4.5 hours) and bleeding risk limit its applicability to certain populations. Neuroprotective agents have poor clinical efficacy due to low BBB penetration efficiency and insufficient lesion targeting. Although exogenous NO delivery can alleviate ischemic injury, existing NO donors suffer from a lack of targeting, short half-life (1-10 seconds), and non-specific release, making it difficult to achieve precise spatiotemporal control, which severely restricts clinical translation. There is an urgent need to develop innovative strategies that combine precise delivery and controllable release.

[0005] Key findings revealed that the ischemic penumbra exhibits a unique hypoxia-reductase coexistence microenvironment within 6 hours of ischemic stroke onset. Immunofluorescence data confirmed significantly high expression of cytochrome P450 reductase (CPOR) and iNOS in neurons and glial cells, with their activity preserved through subcellular compartmentalization under oxidative stress. The unique hypoxic microenvironment (<0.5% O2) of the ischemic penumbra provides a revolutionary entry point for targeted drug design. Developing highly effective and low-toxicity therapeutic agents using a prodrug strategy, leveraging the hypoxia-reductase expression conditions during cerebral ischemia, is of great significance for the efficient treatment of ischemic stroke. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies in the prior art and to provide a hypoxia-responsive β-carbolinequinoline azomonium, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a hypoxia-responsive β-carbolinequinolineazobium, wherein the β-carbolinequinolineazobium has the structure shown in Formula I:

[0008]

[0009] A second aspect of the present invention provides a method for preparing β-carbamolinequinoline azomonium with the structure shown in Formula I, the method comprising the following steps:

[0010] S1. Compound 1 reacts with 4-nitrosobenzyl alcohol under acetic acid catalysis to form azo compound 3;

[0011] S2. Azo compound 3 undergoes an Appel bromination reaction in the presence of CBr4 and PPh3 to give compound 4;

[0012] S3. Compound 4 reacts with (E)-1-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-onium salt diazoxide-1-oxide at 0–10°C to give β-carbolinequinoline azomonium (I, CPN).

[0013] The synthesis route is shown below:

[0014]

[0015] In some embodiments of the present invention, in step S1, the molar ratio of compound 1 to 4-nitrosobenzyl alcohol is 7.9:31.7, and the reaction temperature is room temperature.

[0016] In some embodiments of the present invention, in step S2, the molar ratio of compound 3, CBr4 and PPh3 is 2:4.1:4.1, and the reaction temperature is room temperature.

[0017] In some embodiments of the present invention, in step S3, the molar ratio of compound 4 to (E)-1-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-onium salt diazoxide-1-oxide is 1.1:1.2.

[0018] A third aspect of the present invention provides a pharmaceutical composition comprising β-carbamolinequinoline azomonium of Formula I and a pharmaceutically acceptable carrier or excipient.

[0019] In a fourth aspect, the invention provides the use of β-carbolinequinoline azomonium with the structure shown in Formula I in the preparation of a medicament for fluorescence imaging diagnosis of ischemic stroke.

[0020] In a fifth aspect, the invention provides the application of β-carbamolinequinoline azomonium with the structure shown in Formula I in the preparation of near-infrared fluorescence imaging reagents targeting the penumbra region of cerebral ischemia.

[0021] In a sixth aspect, the invention provides the use of β-carbolinequinoline azomonium of Formula I in the preparation of a neuroprotective drug for treating ischemic stroke and ischemic brain injury.

[0022] The present invention has the following advantages over the prior art:

[0023] The compounds of this invention significantly improved the survival rate of OGD / R-damaged cells in a stroke-induced oxygen deprivation (OGD / R) induced cellular inflammation model. In a mouse model of cerebral ischemia-reperfusion, the compounds, after tail vein injection, entered the brain and were activated by reductases in a hypoxic environment, producing a significant fluorescent signal, successfully visualizing ischemic stroke lesions. Simultaneously, monitoring the release of NO in the brain further confirmed their excellent targeted diagnostic capability for stroke.

[0024] The efficacy of the compounds of this invention in treating ischemic brain injury has been fully validated. Compared with the MCAO group, the compounds of this invention significantly reduced the infarct volume in mice, effectively alleviated cerebral edema, reduced forelimb asymmetry and right-turning preference, lowered neurological scores, and protected neurological function. The successful development of the compounds of this invention demonstrates significant advantages in the diagnosis and treatment of ischemic stroke. Attached Figure Description

[0025] Figure 1 As a control with a CQ fluorescent probe, the ultraviolet absorption spectrum and fluorescence emission spectrum of the compound of the present invention before and after addition to rat liver microsomes and NADPH are shown.

[0026] Figure 2 This is a fluorescence emission spectrum of the reductase response of the compound of the present invention over time, with the horizontal axis representing wavelength and the vertical axis representing fluorescence intensity.

[0027] Figure 3 This is a graph showing the test results of the specific response ability of the compounds of this invention to reductase;

[0028] Figure 4 This is a graph showing the test results of the cytotoxicity and cellular-level neuroprotective effects of the compounds of this invention;

[0029] Figure 5 This is a fluorescence imaging test result of the compound of the present invention in a mouse model of cerebral ischemia-reperfusion.

[0030] Figure 6 These are TTC-stained images and statistical analysis bar charts of coronal sections of the mouse brains in each group in Example 11. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Example 1: Preparation of 4-((E)-2-(6-((E)-(4-(hydroxymethyl)phenyl)azo)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-onium salt (3)

[0035] Compound 1 (CQ, 0.96 g, 7.9 mmol) and 4-nitrosobenzyl alcohol (2.03 g, 31.7 mmol) were dissolved in ethanol (5 ml) at room temperature, and an equal volume of acetic acid (5 ml) was added. The mixture was stirred for 12 h. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 50:1, v / v) to give a yellow solid 3 in 66% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.37 (d, J = 6.5 Hz, 1H, ArH), 8.93 (m, 2H,2ArH), 8.84 (m, 1H, ArH), 8.59 (dd, J = 9.2 Hz, 1H, ArH), 8.53 (m, 1H, ArH),8.47 (m, 1H, ArH), 8.32 (m, 1H, ArH), 8.27 (m, 1H, ArH), 8.23 ​​(m, 1H, ArH),8.18 (m, 2H, 2ArH), 8.12 (d, J = 7.6 Hz, 1H, ArH), 8.01 (m, 2H, ArH, CH=C),7.95 (m, 1H, CH=C), 4.54 (s, 3H, CH3), 4.27 (s, 3H, CH3), 3.92 (s, 2H, CH2), 3.15 (s, 3H, CH3).

[0036] Example 2: Preparation of 4-((E)-2-(6-((E)-(4-(bromomethyl)phenyl)azo)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-onium salt (4)

[0037] Compound 3 (1.25 g, 2 mmol) was dissolved in anhydrous THF (10 ml) under N2 conditions, and carbon tetrabromide (1.35 g, 4.1 mmol) was added. The mixture was stirred at room temperature for 1 h, followed by the addition of triphenylphosphine (1.14 g, 4.1 mmol) and stirring for another 5 h. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was extracted with DCM (10 ml) and washed with brine (3 × 10 ml). The organic phase was slurried with DCM and petroleum ether to precipitate a yellow solid 4, with a yield of 71%. ESI-MS (m / z): calcd for C 32 H 27 N5Br + : 560.1444, found 560.1450.

[0038] Example 3: Preparation of (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-onth-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazeninyl)benzyl)oxy)-1-(2-(hydroxymethyl)pyrrolidine-1-yl)diazenin-1-oxide (I, CPN)

[0039] (E)-1-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-onium salt diazoxide-1-oxide (0.20 g, 1.2 mmol) was dissolved in THF (3 ml) at 0 °C under nitrogen protection. After stirring at 0 °C for 5 min, a solution of compound 4 (0.38 g, 1.1 mmol) in DMF (1 ml) was added to the reaction mixture at 0 °C. The mixture was stirred at room temperature for 4 hours. After the reaction was completed as monitored by TLC, the mixture was extracted with DCM, concentrated, and purified by column chromatography to obtain compound CPN in 75% yield. 1 H NMR(DMSO-d6, 400 MHz) δ 9.05 (m, 1H, ArH), 8.69 - 8.63 (m, 1H, ArH), 8.57 (m,2H, ArH), 8.42 (m, 1H, ArH), 8.37 - 8.33 (m, 1H, ArH), 8.33 - 8.28 (m, 1H,ArH), 8.21 - 8.11 (m, 2H, ArH), 7.98 - 7.90 (m, 1H, ArH), 7.88 - 7.77 (m, 1H,ArH), 7.72 - 7.65 (m, 1H, CH), 7.42 - 7.33 (m, 1H, ArH), 7.06 (m, 1H, CH),6.84 (m, 2H, ArH), 5.11 (d, J = 6.7 Hz, 2H, CH2), 4.34 - 4.15 (m, 3H, CH3),3.81 (s, 1H, OH), 3.17 (d, J = 1.3 Hz, 3H, CH3), 3.01 (s, 2H, CH2), 2.22 (d, J= 8.5 Hz, 1H, CH), 1.71 - 1.62 (m, 3H, CH3), 1.21 (m, 4H, CH2), 1.06 (m, 2H,CH2); HRMS (ESI) m / z calcd for C 37 H 37 N8O3 + 641.2983; found, 641.2981.

[0040] Example 4: The activation characteristics of the compound under hypoxic conditions were evaluated using ultraviolet-fluorescence spectroscopy.

[0041] The specific experimental steps are as follows: First, CPN solution (20 μM) was mixed with 0.1 M phosphate buffer (pH 7.4), followed by the addition of a suspension of rat liver microsomes (mainly P450 reductase, final protein concentration 0.5 mg / mL) containing 0.5 mg / mL protein. After pre-incubation at 37℃, NADPH was added to a final concentration of 1 mM to initiate the reaction. Under hypoxic conditions, UV absorption spectrophotometer data from 200-600 nm were collected, and fluorescence emission spectrometer was used to detect changes in fluorescence emission spectra at an excitation wavelength of 436 nm. CQ was used as a fluorescent probe control. The obtained UV absorption and fluorescence emission spectra are shown below. Figure 1 As shown.

[0042] Figure 1 Figure a shows the UV absorption spectrum of the compound of the present invention at 20 μM before and after the addition of rat liver microsomes and NADPH (the horizontal axis represents wavelength and the vertical axis represents absorbance); Figure b shows the fluorescence spectrum of the compound of the present invention at 20 μM before and after the addition of rat liver microsomes and NADPH (the horizontal axis represents wavelength and the vertical axis represents fluorescence intensity); Figure c shows the UV absorption spectrum of CQ (20 μM) in DMSO / H2O (v:v=1:99) solution; and Figure d shows the fluorescence spectrum of CQ (20 μM) in DMSO / H2O (v:v=1:99) solution.

[0043] The results showed that the compound of this invention (CPN probe) exhibited a significant ultraviolet absorption peak at 436 nm. Figure 1 a), while the maximum absorption peak of the fluorophore CQ is located at 446 nm (a), Figure 1 c). Under hypoxic activation conditions, the UV absorption peak of CPN underwent a redshift of approximately 10 nm (from 436 nm to 446 nm). Figure 1 a), which completely coincides with the absorption peak of CQ. Fluorescence spectroscopy analysis showed that the background fluorescence emission of CPN was almost negligible, but after microsomal / NADPH hypoxia activation, it produced a fluorescence signal at 656 nm, with an intensity increase of 26 times (a). Figure 1 (b) confirms that the hypoxic microenvironment can specifically trigger probe activation.

[0044] Example 5: The time dependence of the fluorescence intensity of the compound of the present invention after adding rat liver microsomes and NADPH under hypoxic conditions was detected by fluorescence spectroscopy.

[0045] The specific method is as follows:

[0046] First, the compound CPN of this invention was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. For testing, the stock solution was diluted to 20 µM (pH 7.4) with ultrapure water. In the experiment, the reaction system was pre-incubated at 37°C, and then NADPH (final concentration 1 mM) was added to initiate the reaction. Subsequently, the fluorescence spectrum changes at different time points were monitored under hypoxic conditions, and the results are as follows: Figure 2 As shown.

[0047] Figure 2 Figure a shows the fluorescence spectrum of the compound of the present invention after the addition of liver microsomes and NADPH under hypoxic conditions, measured over time. Figure b shows the fluorescence spectrum of the compound of the present invention at 656 nm as a function of incubation time.

[0048] The results showed that the fluorescence intensity of the compound CPN of the present invention increased significantly by about 18 times within 5 minutes after the addition of liver microsomes and NADPH under hypoxic conditions, and continued to increase with the extension of incubation time, with the highest increase reaching about 46 times. This indicates that the compound of the present invention has high sensitivity and time-dependent fluorescence enhancement characteristics under hypoxic conditions.

[0049] Example 6: Evaluation of the compound's specific recognition ability for reductase using fluorescence spectroscopy.

[0050] CPN (40µM) was combined with various physiologically disruptive substances (including Na+). + K + Cu 2+ Fe 2+ Zn 2+ Mn 2+ Ca 2+ Mg 2+ I - GSH, H2O2, Glu, and VcNa (all at 1 mM) were mixed in 0.1 M phosphate buffer (pH 7.4) and incubated at room temperature for 3 hours. The experimental group was additionally supplemented with rat liver microsomes (final protein concentration 0.5 mg / mL) and NADPH (final concentration 1 mM). Fluorescence spectroscopy parameters were set to excitation wavelength 446 nm and emission wavelength range 650-900 nm. Results are shown below. Figure 3 As shown.

[0051] Figure 3 The compound of the present invention (40 μM) was shown to be reacted with the corresponding bioanalyte (Na) in DMSO solution at 1 mM. + K + Cu 2+ Fe 2+ Zn 2+ Mn 2+ Ca 2+Mg 2+ I - Fluorescence intensity (mean ± standard deviation, n = 3) after 3 hours of incubation with Glu, VcNa, liver microsomes and NADPH.

[0052] The results showed that at a concentration of 1 mM, interfering substances (such as Ca) 2+ Cu 2+ Fe 2+ In the presence of metal ions and redox reactive substances such as GSH and H2O2, the fluorescence signal of the system did not fluctuate significantly. However, after introducing rat liver microsomes and NADPH under hypoxic conditions, the fluorescence intensity at 656 nm was significantly enhanced, confirming that the compound of this invention has a highly specific response to reductase activity in a hypoxic microenvironment.

[0053] Example 7: Test of the neuroprotective effect of the compound of the present invention at the cellular level

[0054] The cytotoxicity of the compounds in this invention was first assessed using an in vitro cytotoxicity assay using the CCK-8 assay. A vial of rat adrenal medullary pheochromocytoma differentiation cell line PC12 (Shanghai Institute of Cell Biology, China) in a healthy exponential growth phase was digested and prepared into 1×10⁻⁶ cells. 4 Cell suspension of cells / mL was seeded into 96-well plates and cultured at 37°C in the dark for 24 hours. Then, CPN, CQ and NaNO2 (0, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1 µM) were added, and the cells were cultured for another 24 hours. After incubation with CCK-8 for 2 hours, the absorbance at 450 nm was measured and the cell viability was calculated.

[0055] An oxygen-glucose deprivation / reoxygenation (OGD / R) cell model was used to simulate the brain ischemia-reperfusion injury environment. PC12 neurons were introduced at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured for 24 hours. After OGD treatment in a three-gas incubator (95% N2 + 5% CO2) for 6 hours, the medium was replaced with complete medium and reoxygenated for 24 hours. After reoxygenation, the experimental groups were treated with CPN, CQ, and NaNO2 (0, 0.0625, 0.125, 0.25, 0.5, and 1 µM, respectively). Cell viability was detected by CCK-8 assay.

[0056] Cell survival rate formula:

[0057] 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%

[0058] 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.

[0059] The cytotoxicity and neuroprotective effects of the compounds of this invention were tested as follows: Figure 4 As shown.

[0060] Figure 4 Figure a shows the survival rate of PC12 cells treated with different concentrations (0, 0.031, 0.062, 0.125, 0.25, 0.5, and 1 µM) of the compounds of this invention and CQ. Figure b shows the cell viability of OGD / R-stimulated PC12 cells treated with different concentrations of CPN, CQ, and NaNO2 (0, 0.062, 0.125, 0.25, 0.5, and 1 µM).

[0061] The results showed that CCK-8 cytotoxicity data indicated that CPN and CQ, within a concentration range of 0.125 - 1 µM, could maintain cell viability above 80%. Figure 4 a), which demonstrates the low cytotoxicity of the compounds of this invention. Further observation of glucose-oxygen deprivation stroke cell activity revealed that under OGD / R treatment, cell viability decreased to 50%; however, when treated with 0.5-1 µM CPN, cell viability gradually increased, reaching nearly 90% at a 1 µM concentration, exhibiting a strong neuroprotective effect. This effect was significantly superior to the same concentrations of NaNO2 and CQ groups (a). Figure 4 (b) Notably, CQ also exhibited a certain neuroprotective effect in the concentration range of 0.5–1 µM. These results demonstrate that the compounds of this invention can effectively alleviate OGD / R-induced neuronal damage and possess potential for therapeutic application in ischemic stroke.

[0062] Example 10: Imaging performance of the compound of the present invention in ischemic brain parenchyma using in vivo fluorescence imaging technology.

[0063] The specific method involves establishing a middle cerebral artery occlusion (MCAO) model of cerebral ischemia / reperfusion injury: C57BL / 6J mice weighing 20±2 g were anesthetized with 2.5% tribromoethanol, and their neck vessels were exposed. A suture embolism was inserted into the internal carotid artery to induce ischemia for 1 hour, followed by a tail vein injection of the compound of this invention (5 mg / kg). Using the AniView100 multi-modal animal in vivo imaging system under 650 nm near-infrared illumination, changes in brain signals were dynamically monitored at 1, 2, 4, 8, 12, and 24 hours post-administration. Ex vivo imaging of organs such as the brain, heart, liver, spleen, lungs, and kidneys was performed at the 4-hour peak time point (n=3). In the Sham group, only the common carotid artery was dissected, and the wound was immediately sutured.

[0064] Figure 5 This is a graph showing the results of the targeted imaging capability test of the compound of the present invention in a mouse model of cerebral ischemia-reperfusion.

[0065] Figure 5 Figure a shows fluorescence images of a mouse model of cerebral ischemia-reperfusion, a sham-operated mouse, and a normal mouse injected with the compound of the present invention. Figure b shows the mean fluorescence intensity (mean ± standard deviation, n = 3) of the mouse brain in Figure a. Figure c shows fluorescence images of the isolated brain tissue from the mouse model of cerebral ischemia-reperfusion, a sham-operated mouse, and a normal mouse injected with the compound of the present invention. Figure d shows the mean fluorescence intensity (mean ± standard deviation, n = 3) of the isolated brain tissue in Figure c.

[0066] Figure 5 The results showed that the compound of this invention produced a significant brain fluorescence signal 1 hour after administration (Figure a) during in vivo dynamic monitoring, with the signal intensity increasing over time to a peak at 4 hours (Figure b). The fluorescence intensity of the brain tissue in the administration group was 4-5 times that of the Sham control group and remained there for up to 24 hours. Ex vivo organ imaging (Figures c and d) also showed a significant fluorescence signal in the brain of the CPN group, which was 3 times that of the Sham control group. Furthermore, a certain fluorescence signal was also present in the kidneys, indicating that the compound of this invention can be cleared through renal metabolism. The fluorescence intensity in the sham-operated group and the blank control group remained at background levels, eliminating interference from surgical trauma. This demonstrates that the compound of this invention possesses precise brain ischemia-targeting imaging capabilities and a clear metabolic pathway characteristic.

[0067] Example 11: Detection of the therapeutic effect of the compound CPN of the present invention on ischemic stroke using a middle cerebral artery occlusion (MCAO) model.

[0068] The specific method was as follows: One hour after ischemia, PBS, NaNO2 (0.815 and 1.63 mg / kg), or CPN (2.5 and 5 mg / kg) were administered via tail vein injection (n=6). A sham-operated group and a CPN+PTIO (0.5 mg / kg pretreatment group) were also included. Twenty-four hours post-surgery, neurobehavioral assessments were performed (cylinder test to detect forelimb asymmetry, cornering test to observe turning preference, and Zea Longa scoring system to grade the degree of neurological damage). Mice were then sacrificed to obtain brain tissue. The degree of cerebral edema was calculated using the wet-to-dry weight method: (wet weight - dry weight) / wet weight × 100% (n=6). Simultaneously, brain tissue was subjected to TTC staining (frozen at -20℃ for 20 minutes, cut into 1 mm coronal sections, incubated in 2% TTC solution at 37℃ in the dark for 20 minutes, and fixed with 4% PFA for 24 hours). Infarct volume was quantified using ImageJ (n=3). TTC staining images and statistical analysis of coronal sections of the brains of mice in each group are shown below. Figure 6 As shown.

[0069] Figure 6 Figure a shows TTC-stained images of coronal sections of the brains of mice in each group. Figure b is a statistical graph of cerebral infarction volume in each group of mice (mean ± standard deviation, n = 3). Figure c shows the neurological function scores of mice in each group (mean ± standard deviation, n = 6). Figure d shows cerebral edema (%) (mean ± standard deviation, n = 6). Figure e shows the forelimb asymmetry rate measured by the cylinder test (mean ± standard deviation, n = 6). Figure f shows the right deflection rate in the corner turning test (mean ± standard deviation, n = 6).

[0070] Figure 6 The results showed that the degree of cerebral edema improved in a dose-dependent manner in all CPN intervention groups at different concentrations (Figures b and d). The 5 mg / kg CPN treatment group significantly reduced the infarct volume to 8.5%, which was superior to the 0.815 mg / kg NaNO2 group (42.5%), the 1.63 mg / kg NaNO2 group (40.6%), the 2.5 mg / kg CPN group (15.5%), and the 5 mg / kg CPN+PTIO group (31.3%). Neurological function assessments showed that the CPN treatment group was significantly superior to the MCAO group in terms of Zea Longa score, asymmetry rate of forelimb use in the cylinder test, and right-turn preference in the cornering test (Figures c, e, and f), and also superior to all NaNO2 dose groups and the CPN+PTIO group. These results demonstrate that the compounds of this invention have good neuroprotective effects, can significantly reduce the infarct area, and improve the pathological structure of ischemic mice, providing a new potential strategy for the treatment of ischemic diseases.

[0071] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A hypoxia-responsive β-carbolinequinolineazobium, wherein the β-carbolinequinolineazobium has the structure shown in Formula I:

2. A method for preparing β-carbolinequinoline azomonium as described in claim 1, characterized in that, The preparation method includes the following steps: S1. Compound 1 reacts with 4-nitrosobenzyl alcohol under acetic acid catalysis to generate azo compound 3; S2. Azo compound 3 undergoes an Appel bromination reaction in the presence of CBr4 and PPh3 to give compound 4; S3. Compound 4 was reacted with (E)-1-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-onium salt diazene-1-oxide at 0-10 °C to give β-carbolinequinoline azoonium; The synthesis route is shown below:

3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of compound 1 to p-4-nitrosobenzyl alcohol is 7.9:31.7, and the reaction temperature is room temperature.

4. The preparation method according to claim 2, characterized in that, In step S2, the molar ratio of compound 3, CBr4 and PPh3 is 2:4.1:4.1, and the reaction temperature is room temperature.

5. The preparation method according to claim 2, characterized in that, In step S3, the molar ratio of compound 4 to (E)-1-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-onium salt diazoxide-1-oxide is 1.1:1.

2.

6. A pharmaceutical composition, characterized in that, This includes the β-carbolinequinoline azomonium as described in claim 1, and pharmaceutically acceptable carriers or excipients.

7. The use of β-carbolinequinoline azomonium as described in claim 1 in the preparation of a drug for fluorescence imaging diagnosis of ischemic stroke.

8. The application of β-carbolinequinoline azomonium as described in claim 1 in the preparation of near-infrared fluorescence imaging reagents targeting the penumbra region of cerebral ischemia.

9. The use of β-carbolinequinoline azomonium as described in claim 1 in the preparation of a neuroprotective drug for treating ischemic stroke and ischemic brain injury.

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