Application of IKK kinase inhibitor in preparation of medicine for treating cerebral apoplexy

Abstract

The invention discloses application of an IKK kinase inhibitor in preparation of a medicine for treating cerebral apoplexy. According to the invention, small molecule compounds capable of improving cerebral injury caused by cerebral ischemia / reperfusion are screened, and research results show that the small molecule compounds can specifically inhibit IKK activation and play a role in treating cerebral apoplexy. The invention also utilizes a mouse arterial embolism / reperfusion model to verify that the small molecule compound can significantly reduce the cerebral injury caused by cerebral ischemia / reperfusion. In a word, the invention provides a new thought and a targeted drug for treatment of cerebral apoplexy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of IKK kinase inhibitors in the preparation of drugs for treating stroke. Background Art

[0002] Stroke, also known as apoplexy, is an acute cerebrovascular disease caused by the sudden rupture or blockage of blood vessels in the brain, leading to the interruption of blood supply and subsequent damage to brain tissue. According to the pathological mechanism, stroke can be divided into ischemic stroke (accounting for more than 85%) and hemorrhagic stroke. The former is caused by thrombosis or embolism resulting in vascular occlusion, while the latter is caused by the rupture of cerebral blood vessels. Stroke may also leave permanent disabilities, including motor dysfunction, cognitive impairment, and language disorders, etc., bringing heavy medical and economic burdens to the patient's family and society.

[0003] Currently, the strategies for clinical treatment of stroke are mainly divided into acute-phase intervention and long-term rehabilitation management. The core of acute-phase treatment is to restore blood flow: for ischemic stroke, vascular recanalization can be achieved through intravenous thrombolytic drugs (such as recombinant tissue plasminogen activator tPA) or endovascular thrombectomy, but both have significant limitations. The treatment time window of tPA is narrow, accompanied by the risk of intracranial hemorrhage, and at the same time, the recanalization rate of large vessel occlusion is low; although thrombectomy can effectively open large blood vessels, it depends on advanced imaging evaluation and professional intervention teams, and it is difficult to popularize in primary medical institutions. Hemorrhagic stroke is mainly treated by surgical hemostasis and antihypertensive treatment, lacking specific neuroprotective means. Although neuroprotective drugs (such as free radical scavengers, glutamate receptor antagonists) have shown potential in animal models, they have repeatedly failed in clinical trials, mainly due to problems such as low blood-brain barrier penetration efficiency, insufficient efficacy of single-target drugs caused by the complexity of pathological mechanisms, and poor timeliness of drug delivery.

[0004] The limitations of existing therapies highlight the urgent need in the field of stroke treatment: on the one hand, more than half of the patients cannot receive effective treatment due to missing the thrombolysis / thrombectomy time window or having contraindications; on the other hand, the lack of neuroprotection means that even after vascular recanalization, reperfusion injury, oxidative stress, and inflammatory cascade reactions still continue to exacerbate brain damage. Therefore, the development of new therapeutic drugs, especially small molecule compounds with good blood-brain barrier penetration and a wide treatment time window, has become the key direction to break through the current bottleneck. Small molecule drugs have shown unique value in rapidly intervening in the pathological cascade reaction after stroke, synergistically protecting neurons and vascular units due to their strong chemical structure plasticity, mature synthesis process, easy oral administration, and controllable cost. By high-throughput screening and artificial intelligence-assisted design, mining candidate small molecules with neurovascular protection functions from known compound libraries, or expanding the indications of approved drugs (Drug Repurposing), is expected to accelerate the clinical translation process and provide more universal and safe treatment options for stroke patients. Summary of the Invention

[0005] To solve at least some of the technical problems in the above-mentioned prior art, the present invention discloses the use of IKK kinase inhibitors in the preparation of drugs for treating stroke. The IKK kinase inhibitor of the present invention can specifically inhibit IKK activation, thereby inhibiting NF-κB activation and downstream inflammatory gene expression, and thus playing a role in treating stroke. Specifically, the present invention includes the following content.

[0006] In one aspect of the present invention, there is provided the use of an IKK kinase inhibitor in the preparation of a drug for treating or improving stroke, wherein the IKK kinase inhibitor has the structure shown in Formula I:

[0007]

[0008] Wherein, X1 and X2 each independently selected from heteroatoms of N, S or O, R1 is a heterocyclic group or a substituted heterocyclic group, R2 is a naphthyl group, a heteroaryl group, a phenyl group or a biphenyl group, or a substituted naphthyl group, a heteroaryl group, a phenyl group or a biphenyl group.

[0009] In certain embodiments, the use of the IKK kinase inhibitor according to the present invention in the preparation of a drug for treating or improving stroke, wherein, R1 is selected from furyl, thienyl, pyrrolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, piperidinyl, morpholinyl, piperazinyl or triazinyl.

[0010] In certain embodiments, the use of the IKK kinase inhibitor according to the present invention in the preparation of a drug for treating or improving stroke, wherein, R2 is selected from a phenyl group or a substituted phenyl group, preferably a substituted phenyl group, and more preferably the substituents are selected from halogen, hydroxyl, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, cyano or aryl.

[0011] In certain embodiments, the use of the IKK kinase inhibitor according to the present invention in the preparation of a drug for treating or improving stroke, wherein, the treatment or improvement of the stroke includes at least one of the following:

[0012] (1) Reducing the level of inflammatory factors related to stroke;

[0013] (2) Improving or alleviating brain injury;

[0014] (3) Reducing or decreasing the area of cerebral infarction;

[0015] (4) Alleviating or reducing the complications related to stroke;

[0016] (5) Reducing the mortality rate of stroke.

[0017] In certain embodiments, the IKK kinase inhibitor according to the present invention is used in the preparation of a medicament for treating or improving stroke, wherein the medicament further comprises a pharmaceutically acceptable carrier.

[0018] In certain embodiments, the IKK kinase inhibitor according to the present invention is used in the preparation of a medicament for treating or improving stroke, wherein the pharmaceutically acceptable carrier comprises at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a sweetening agent, a preservative, and an antioxidant.

[0019] In certain embodiments, the IKK kinase inhibitor according to the present invention is used in the preparation of a medicament for treating or improving stroke, wherein the dosage of the IKK kinase inhibitor is 0.01 - 500 mg / Kg.

[0020] In certain embodiments, the IKK kinase inhibitor according to the present invention is used in the preparation of a medicament for treating or improving stroke, wherein the stroke includes ischemic stroke or hemorrhagic stroke.

[0021] In a second aspect of the present invention, there is provided the use of an IKK kinase inhibitor in the preparation of a medicament for the combined treatment of stroke with other medicaments.

[0022] In certain embodiments, according to the use of the present invention, the other medicaments include at least one of a thrombolytic medicament, an antiplatelet medicament, an anticoagulant medicament, a statin, a neuroprotective agent, an antihypertensive medicament, an antiepileptic medicament, and an acid suppressant.

[0023] The present invention screened small molecule compounds capable of improving brain injury caused by cerebral ischemia / reperfusion. The research results show that they can specifically inhibit IKK activation, thereby inhibiting NF-κB activation and downstream inflammatory gene expression, and play a role in treating stroke. The present invention also used a mouse arterial embolization / reperfusion model to verify that the small molecule compound can significantly reduce brain injury caused by cerebral ischemia / reperfusion. In summary, the present invention provides new ideas and targeted medicaments for the treatment of stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shows the effects of the IKK kinase inhibitor on the phosphorylation of IKKα / β, the phosphorylation of IκB, and the total protein level.

[0025] Figure 2 Shows the effects of the IKK kinase inhibitor on the levels of inflammatory factors.

[0026] Figure 3 Shows that the IKK kinase inhibitor can significantly improve brain injury caused by cerebral ischemia / reperfusion. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value therebetween are specifically disclosed. Intermediate values within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Application

[0031] In one aspect of the present invention, there is provided the use of an IKK kinase inhibitor in the preparation of a medicament for treating or improving stroke, wherein the IKK kinase inhibitor has the structure shown in Formula I:

[0032]

[0033] Wherein, X1 and X2 each independently represent a heteroatom selected from N, S or O, R1 is a heterocyclic group or a substituted heterocyclic group, and R2 is a naphthyl group, a heteroaryl group, a phenyl group or a biphenyl group, or a substituted naphthyl group, heteroaryl group, phenyl group or biphenyl group.

[0034] In a preferred embodiment, the IKK kinase inhibitor of the present invention has the structure shown in Formula I, where X1 and X2 are both S, R1 is a heterocyclic group, and R2 is a phenyl group or a substituted phenyl group. In another preferred embodiment, the IKK kinase inhibitor of the present invention has the structure shown in Formula I, where X1 and X2 are both S, R1 is selected from furyl, thienyl, pyrrolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, piperidinyl, morpholinyl, piperazinyl or triazinyl, and R2 is a substituted phenyl group. In a more preferred embodiment, the IKK kinase inhibitor of the present invention has the structure shown in Formula I, where X1 and X2 are both S, R1 is morpholinyl, R2 is a substituted phenyl group, and the substituents are selected from halogen, hydroxyl, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, cyano or aryl. In the most preferred embodiment, the IKK kinase inhibitor of the present invention has the structure shown in Formula I, where X1 and X2 are both S, R1 is morpholinyl, R2 is a substituted phenyl group, and the substituent is C1-C6 alkyl.

[0035] In a specific embodiment, the IKK kinase inhibitor of the present invention, N-(2-morpholinophenyl)-2-[(2-phenylethyl-5,6,7,8-tetrahydrobenzothieno[2,3-d]-4-pyrimidinyl)thio]acetamide, has the structure of Formula II (molecular weight 544.73, molecular formula C 30 H 32 N4O2S2):

[0036]

[0037] The compounds used in the present invention can all be obtained from commercially available products or synthesized by known methods, and are not particularly limited in this regard.

[0038] In the present invention, the treatment or improvement includes at least one of the following:

[0039] (1) Reducing the levels of inflammatory factors related to stroke;

[0040] (2) Improving or alleviating brain injury;

[0041] (3) Reducing or decreasing the cerebral infarction area;

[0042] (4) Alleviating or reducing the complications related to stroke;

[0043] (5) Reducing the mortality rate of stroke;

[0044] (6) Reducing stroke-related nerve damage.

[0045] In the present invention, the term "treating or ameliorating" refers to therapeutic treatment or prophylactic measures, the purpose of which is to prevent or slow down (reduce) an undesired physiological change or disorder, such as the progression of a stroke. Beneficial or desired clinical outcomes include, but are not limited to, the following whether detectable or undetectable outcomes, including relief of symptoms, reduction in the degree of disease, stabilization of the disease state (i.e., not getting worse), delay or slowdown in the progression of the disease, improvement or mitigation of the disease state, and alleviation (whether partial or total). Those in need of treatment include those who already have a stroke or those who need to treat or ameliorate a stroke. In certain embodiments, the treating or ameliorating effect is selected from any one or a combination of relieving symptoms, improving prognosis, reducing the degree of neurological deficit, and enhancing the patient's ability for daily activities.

[0046] A therapeutically effective amount of the IKK kinase inhibitor of the present invention can be administered to a subject in need thereof, such as a mammal (e.g., a human). The mode of administration is not particularly limited, and representative modes of administration include, but are not limited to: oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. Accordingly, the compound of formula I or the compound of formula II of the present invention can be formulated into various clinically acceptable dosage forms, including oral dosage forms, injection dosage forms, topical dosage forms or external use dosage forms, etc.

[0047] The therapeutically effective amount described in the present invention refers to the pharmaceutically recognized effective dosage, that is, the amount of the active compound (i.e., the compound of formula I or the compound of formula II) is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of the compound of formula I or the compound of formula II is usually 0.01 - 500 mg / Kg, preferably 0.5 - 200 mg / Kg or 0.5 - 180 mg / Kg, or 0.5 - 150 mg / Kg, or 0.5 - 120 mg / Kg, or 0.5 - 100 mg / Kg, or 0.5 - 50 mg / Kg, or 0.5 - 40 mg / Kg, or 0.5 - 30 mg / Kg, and most preferably 0.5 - 25 mg / Kg. Exemplary effective dosages are for example 0.5 mg / Kg, 0.75 mg / Kg, 0.95 mg / Kg, 1 mg / Kg, 1.25 mg / Kg, 1.5 mg / Kg, 1.75 mg / Kg, 2 mg / Kg, 2.5 mg / Kg, 2.75 mg / Kg, 3 mg / Kg, 3.25 mg / Kg, 3.5 mg / Kg, 3.75 mg / Kg, 4 mg / Kg, 4.25 mg / Kg, 4.5 mg / Kg, 4.75 mg / Kg, 5 mg / Kg, 5.25 mg / Kg, 5.5 mg / Kg, 5.75 mg / Kg, 6 mg / Kg, 6.25 mg / Kg, 6.5 mg / Kg, 6.75 mg / Kg, 7 mg / Kg, 7.25 mg / Kg, 7.5 mg / Kg, 7.75 mg / Kg, 8 mg / Kg, 8.25 mg / Kg, 8.5 mg / Kg, 8.75 mg / Kg, 9 mg / Kg, 9.25 mg / Kg, 9.5 mg / Kg, 9.75 mg / Kg, 10 mg / Kg, 11 mg / Kg, 12 mg / Kg, 13 mg / Kg, 14 mg / Kg, 15 mg / Kg, 16 mg / Kg, 17 mg / Kg, 18 mg / Kg, 19 mg / Kg, 20 mg / Kg, 21 mg / Kg, 22 mg / Kg, 23 mg / Kg, 24 mg / Kg, 25 mg / Kg. It can be administered as a single dose once a day, can be administered in multiple doses per day, or can be used at intervals.

[0048] In the present invention, the drug containing the IKK kinase inhibitor may further contain a pharmaceutically acceptable carrier. In the present invention, the pharmaceutically acceptable carrier participates in transporting or delivering the medicament from one organ or part of the body to another organ or another part of the body. Each carrier is "acceptable", that is, it is compatible with other components of the formulation (such as the compound of formula I or the compound of formula II) and does not harm the patient.

[0049] In the present invention, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or medium, which includes at least one of diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives and antioxidants. Among them, examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, dispersion media, etc.; fillers include, but are not limited to, starch, lactose, mannitol, microcrystalline cellulose, etc.; absorbents include, but are not limited to, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents include, but are not limited to, water, ethanol, etc.; binders include, but are not limited to, hypromellose, povidone, microcrystalline cellulose, etc.; disintegrants include, but are not limited to, croscarmellose sodium, crospovidone, surfactants, low-substituted hydroxypropyl cellulose, etc.; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, colloidal silicon dioxide, talc, etc.; sweeteners include, but are not limited to, sucralose, acesulfame, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, aspartame, etc.; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, etc.; antioxidants include, but are not limited to, ascorbic acid, methionine, etc.

[0050] In the present invention, stroke includes ischemic stroke or hemorrhagic stroke.

[0051] In the present invention, ischemic stroke includes any one or a combination of mild ischemic stroke, moderate ischemic stroke, moderately severe ischemic stroke, and severe ischemic stroke. In certain embodiments, the ischemic stroke is acute ischemic stroke. In other embodiments, the ischemic stroke is selected from any one or a combination of the ultra-early stage, early stage, acute late stage, recovery stage, and sequela stage of stroke.

[0052] Combined application

[0053] In one aspect of the present invention, there is provided the use of an IKK kinase inhibitor in the preparation of a medicament for the combined treatment of stroke with other drugs.

[0054] In the present invention, the other drugs include but are not limited to at least one of thrombolytic drugs, antiplatelet drugs, anticoagulant drugs, statins, neuroprotective agents, antihypertensive drugs, antiepileptic drugs, and acid-suppressing drugs. The therapeutic drugs for ischemic stroke include but are not limited to: thrombolytic drugs (used within the critical time window), such as alteplase (rt-PA), tenecteplase; antiplatelet drugs, such as aspirin, clopidogrel, ticagrelor; anticoagulant drugs, such as heparin / low molecular weight heparin, warfarin; oral anticoagulants (NOACs), such as dabigatran, rivaroxaban, apixaban; statins, such as atorvastatin, rosuvastatin; neuroprotective agents, such as edaravone, butylphthalide. Other adjuvant antihypertensive drugs, such as labetalol. Volume expansion therapy, such as hydroxyethyl starch. The therapeutic drugs for hemorrhagic stroke include but are not limited to: acute-phase antihypertensive drugs, such as labetalol, nicardipine, urapidil; hemostatic drugs, such as tranexamic acid; drugs for reducing intracranial pressure, such as mannitol; hypertonic saline. In addition, drugs for preventing and treating complications can also be used, such as antiepileptic drugs, such as levetiracetam; acid-suppressing drugs, such as proton pump inhibitors.

[0055] Example

[0056] Inhibitor of kappa B kinase (IKK) is composed of two subunits, α and β, abbreviated as IKKα / β, and is involved in multiple intracellular signal transduction pathways, including apoptosis induced by cytokines and inflammatory responses induced by pathogen-associated pattern molecules. It can phosphorylate the IκBα protein that binds to NF-κB (p65-p50 heterodimer protein) in the cytoplasmic matrix, causing IκBα to be ubiquitinated and degraded, thereby promoting the translocation of NF-κB to the nucleus and mediating the transcription of downstream inflammatory genes. The activation of the NF-κB pathway can upregulate the expression of inflammasome-related genes such as NLRP3 and IL-1β, and further promote the activation of the NLRP3 inflammasome. Whether it is the activation of the NF-κB pathway or the NLRP3 inflammasome, they are both involved in the occurrence and development of various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, ischemic stroke, and multiple sclerosis. Therefore, it is necessary to develop new IKK inhibitors to inhibit the activation of IKK, and then inhibit the activation of the downstream NF-κB pathway and NLRP3 inflammasome, so as to improve nervous system diseases including Alzheimer's disease, Parkinson's disease, ischemic stroke, and multiple sclerosis.

[0057] In this embodiment, through a large number of screenings, a compound with the chemical name N-(2-morpholinophenyl)-2-[(2-phenethyl-5,6,7,8-tetrahydrobenzothieno[2,3-d]-4-pyrimidinyl)thio]acetamide (Compound of Formula II) was finally screened out. It can significantly inhibit the phosphorylation of IKKα / β. For the first time, the present invention reports that it can regulate the activation of IKKα / β. In addition, the present invention also clarifies the role of this compound and IKK in the occurrence of ischemic stroke. Therefore, the present invention provides candidate compounds for targeting IKKα / β and treating ischemic stroke, as follows.

[0058] 1. The Compound of Formula II inhibits the phosphorylation of IKKα / β kinase

[0059] Experimental procedure: Macrophages were seeded into 12-well cell culture plates and divided into 6 groups (Group 1, Group 2, Group 3, Group 4, Group 5, Group 6). Group 1 and Group 2 were negative control groups, and DMSO (equal volume to the drug) and the Compound of Formula II with a final concentration of 12 μM were added respectively. Group 3 was a positive control group, and DMSO with an equal volume to the drug was added. Groups 4 to 6 were added with the Compound of Formula II with final concentrations of 3, 6, and 12 μM respectively. After the cells were cultured for another 0.5 h, lipopolysaccharide (LPS, Sigma-Aldrich) was added. After treatment for 0.5 h, the cells were collected to extract proteins, and the phosphorylation of IKKα / β and IκB kinase was detected by Western blot assay.

[0060] Then, the Western blot assay was performed:

[0061] (1) Place the prepared gel into the electrophoresis tank (with the short plate facing inward). For freshly prepared samples, they can be directly centrifuged at room temperature, mixed well by oscillation and then loaded. Samples stored at -20 °C need to be centrifuged and then heated at 95 °C for 3 - 5 min, mixed well by oscillation and then loaded.

[0062] (2) Electrophoresis: For 30 min, electrophoresis was carried out at 80 V until the marker bands began to separate, and for 45 min, electrophoresis was carried out at 120 V until the individual bands in the marker were separated.

[0063] (3) Transfer: The NC membrane and filter paper need to be soaked in pre-cooled 1× transfer buffer first, and then transferred at a constant current of 250 mA for 1.5 h.

[0064] (4) Blocking: Weigh 2.5 g of skim milk powder, dissolve it in 50 mL of TBST, put the membrane into it, place it on a shaker at room temperature, block for 1 h, and wash the membrane 4 times with 1× TBST, 5 min for each wash.

[0065] (5) Add the antibody diluted with the primary antibody dilution solution, incubate overnight on a shaker at 4 °C, and wash the membrane 4 times with 1× TBST, 5 min for each wash.

[0066] (6) Add the HRP-labeled secondary antibody corresponding to the species of the primary antibody (which can be prepared with the milk powder solution used for blocking), place it on a shaker at room temperature, incubate for 1 h, wash the membrane 4 times with 1×TBST, 5 min for each wash;

[0067] (7) Development: Transfer the membrane to a developing cassette, then add ECL developing solution to the membrane, and then perform X-ray film exposure for development;

[0068] (8) The exposed film is developed and fixed with an automatic film processor, and the imaging result is scanned and digitized with a scanner.

[0069] Table 1 Antibody List

[0070]

[0071]

[0072] The detection results are as Figure 1 shown. Adding the compound of formula II can significantly inhibit the phosphorylation of IKKα / β and IκBα, and further inhibit the degradation of IκBα, and shows a significant dose effect.

[0073] 2. The compound of formula II inhibits the transcription of inflammatory factors

[0074] Experimental procedure: Macrophages are seeded into a 12-well cell culture plate and divided into 5 groups (Group 1, Group 2, Group 3, Group 4, Group 5). Group 1 and Group 2 are negative control groups, and DMSO with the same volume as the drug and the compound of formula II with a final concentration of 12 μM are added respectively. Group 3 is a positive control group, and DMSO with the same volume as the drug is added. Group 4 and Group 5 are added with the compound of formula II with final concentrations of 6 μM and 12 μM respectively. After the cells are cultured for another 0.5 h, lipopolysaccharide (LPS, Sigma-Aldrich) is added. After treatment for 0.5 h, the cells are collected to extract RNA, and the changes in the mRNA levels of inflammatory factors IL-1β, TNF-α and IL-6 are detected by fluorescence quantitative PCR experiment.

[0075] Experimental procedure for fluorescence quantitative PCR: The experiment is carried out according to the SYBR Green fluorescence dye method. Total reaction system for fluorescence quantitative PCR: 10 μl of 2×RealStar Green Fast Mixture with ROX II, 7.5 μL of RNase-free water, 2.5 μl of cDNA, 0.05 μL of Forward primer (100 μM), 0.05 μl of Reverse primer (100 μM).

[0076] PCR procedure: Pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing / extension at 60°C for 45 s, for a total of 40 cycles. Record the fluorescence value during each extension segment. The instrument default melting curve detection program is used, and the 2-ΔΔCt method is adopted to calculate the relative mRNA level.

[0077] Table 2 Fluorescent quantitative PCR primers

[0078]

[0079]

[0080] The detection results are as Figure 2 shown. Adding LPS promoted the increase in the levels of IL-1β, TNF-α, and IL-6. Then adding the compound of formula II could significantly reduce the levels of these inflammatory factors, and showed a significant dose-effect.

[0081] 3. The compound of formula II improves the brain injury caused by cerebral ischemia / reperfusion

[0082] The middle cerebral artery occlusion / reperfusion (MCAO / R) model is a classic animal model for simulating ischemic stroke. In this invention, C57 / BL6J wild-type mice (weight 22 - 25 g, 10 weeks old, male mice) were used to evaluate the improvement effect of the compound of formula II on ischemic stroke through the MCAO / R model.

[0083] The steps for animal modeling were as follows: After anesthetizing the animals with sodium pentobarbital, cut open the neck skin, separate the right common carotid artery and ligate it. Then separate the external carotid artery and the internal carotid artery, and ligate the distal end of the external carotid artery. Next, clamp the internal carotid artery with an artery clip, cut open the external carotid artery, insert a thread embolism into the M2 segment of the middle cerebral artery. During this period, monitor the cerebral blood flow with a laser Doppler cerebral blood flow meter until it drops below 20% of the normal level. After embolization for 60 min, remove the thread embolism, ligate the proximal end of the external carotid artery and then restore the blood supply of the common carotid artery.

[0084] Administration method: The administration group was given the compound of formula II (20 mg / kg / time) once before modeling and 6 h after modeling respectively. The control group was given the same dose of solvent control (1% DMSO + 99% sunflower seed oil). At the same time, in this example, another HCK inhibitor - A419259 was used as a control, and the administration method and dose were the same as those of the compound of formula II. The mice were sacrificed 24 h after modeling, and the mouse brains were taken out and placed in a brain mold for sectioning. Then, the infarct area was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and the size of the infarct focus was statistically analyzed with ImageJ software.

[0085] The results are as Figure 3As shown by the TTC staining results, significant cerebral infarction areas appeared in the MCAO / R model mice, with an average cerebral infarction volume of approximately 30%. In the group given Compound II, the cerebral infarction volume decreased to around 10%. This result indicates that Compound II can significantly reduce the brain damage caused by cerebral ischemia / reperfusion. However, all the mice (100%) given A419259 died within 24 hours. After dissection, it was found that A419259 increased the risk of intracranial hemorrhage after reperfusion in mice with ischemic stroke, which further led to secondary hemorrhagic stroke after ischemic stroke in mice, resulting in the death of the mice. In healthy mice, even when using a dosing dose of 30 mg / kg, no abnormal reactions or deaths were found in the mice. This indicates that the toxicity of A419259 is not due to an excessive dose. In summary, Compound II can play a role in improving acute brain injury in ischemic stroke model mice by targeting the IKK kinase.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an IKK kinase inhibitor in the preparation of a medicament for treating or improving stroke, characterized in that, The IKK kinase inhibitor has the structure shown in Formula I: Wherein, X1 and X2 each independently selected from the heteroatoms N, S or O, R1 is a heterocyclic group or a substituted heterocyclic group, R2 is a naphthyl group, a heteroaryl group, a phenyl group or a biphenyl group, or a substituted naphthyl group, a heteroaryl group, a phenyl group or a biphenyl group.

2. Use of the IKK kinase inhibitor according to claim 1 in the preparation of a medicament for treating or improving stroke, characterized in that, R1 is selected from furyl, thienyl, pyrrolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, piperidyl, morpholinyl, piperazinyl or triazinyl.

3. Use of the IKK kinase inhibitor according to claim 1 in the preparation of a medicament for treating or improving stroke, characterized in that, R2 is selected from a phenyl group or a substituted phenyl group, preferably a substituted phenyl group, and the substituents are preferably selected from halogen, hydroxyl, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, cyano or aryl.

4. Use of the IKK kinase inhibitor according to claim 1 in the preparation of a medicament for treating or improving stroke, characterized in that, The treatment or improvement of the stroke includes at least one of the following: (1) Reducing the level of inflammatory factors related to stroke; (2) Improving or alleviating brain injury; (3) Reducing or decreasing the cerebral infarction area; (4) Alleviating or reducing the complications related to stroke; (5) Reducing the mortality rate of stroke.

5. Use of the IKK kinase inhibitor according to claim 4 in the preparation of a medicament for treating or improving stroke, characterized in that, The drug further comprises a pharmaceutically acceptable carrier.

6. Use of the IKK kinase inhibitor according to claim 5 in the preparation of a medicament for treating or improving stroke, characterized in that, The pharmaceutically acceptable carrier includes at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a sweetening agent, a preservative and an antioxidant.

7. Use of the IKK kinase inhibitor according to claim 6 in the preparation of a medicament for treating or improving stroke, characterized in that, The dosage of the IKK kinase inhibitor is 0.01-500 mg / Kg.

8. Use of the IKK kinase inhibitor according to claim 1 in the preparation of a medicament for treating or improving stroke, characterized in that, The stroke includes ischemic stroke or hemorrhagic stroke.

9. Use of the IKK kinase inhibitor in the preparation of a drug for the combined treatment of stroke with other drugs.

10. The application according to claim 9, wherein The other drugs include at least one of thrombolytic drugs, antiplatelet drugs, anticoagulant drugs, statins, neuroprotective agents, antihypertensive drugs, antiepileptic drugs and acid-suppressing drugs.

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