Application of oxidized indole compounds targeting the MAGI2 GK domain in the treatment of ischemic stroke

Abstract

This invention relates to the application of a class of oxidized indole compounds targeting the MAGI2GK domain in the preparation of drugs for treating stroke. The compounds of this invention can improve ischemic injury; improve organic and neurobehavioral damage caused by ischemia-reperfusion; reduce ischemia-induced neuronal apoptosis; and reduce oxygen-glucose deficiency-induced cell damage. Experiments conducted by the applicant have shown that the compounds can alleviate neuronal ischemia-reperfusion injury in both OGD cell models of neuronal ischemia and in vivo MCAO models, exhibiting significant neuroprotective effects. These compounds hold promise for further development into novel drugs for treating ischemic stroke and can be formulated into dosage forms suitable for various routes of administration.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of an oxidized indole compound targeting the MAGI2 GK domain in the preparation of a drug for treating ischemic stroke. Background Art

[0002] Ischemic stroke is caused by local cerebral ischemia and permanent cerebral infarction, which leads to ischemic and hypoxic necrosis of brain tissue and corresponding neurological deficits. The disease can cause about 16 million people to become disabled or die each year worldwide. It is estimated that from 2005 to 2050, the economic loss caused by ischemic stroke is about 25,000 US dollars, which has become a heavy burden on today's society and families. Although ischemic stroke is characterized by high morbidity, high mortality and high disability rate, effective treatment measures for ischemic stroke are still very limited. The only stroke drug currently approved by the FDA is a thrombolytic drug, tissue plasminogen activator (rtPA), which needs to be used within 3 hours after the onset of stroke, limiting its scope of application.

[0003] Therefore, there is an urgent need to develop compounds with neuroprotective effects in clinical practice.

[0004] PSD-95 (also known as Discs large 4, DLG4 or synapse-associated protein 90, SAP90) belongs to the membrane-associated guanylate kinase family (Membrane-associated guanylate kinases, MAGUKs) and is located in the postsynaptic density (PSD) of excitatory synapses. PSD is a densely packed multiprotein structure after the synapse, which is rich in neurotransmitter receptors, scaffold proteins, adhesion molecules, signaling enzymes and cytoskeletal components, and plays a role in coordinating the formation and function of synapses. As an important scaffold protein in PSD, PSD-95 mainly contains three PDZ domains, one SH3 domain and one GK domain. It connects membrane surface receptors and intracellular signaling molecules through various domains and plays an important physiological role. Given that PSD-95 plays an important role in cerebral ischemia-reperfusion, many PSD-95-related neuroprotectants have been developed and gradually entered the clinical trial stage, such as NA-1, AVLX-144, SCR-4026, etc.

[0005] MAGI2 (Membrane-associated guanylate kinase inverted protein 2) and PSD95 protein are both members of the MAGIUK family protein. MAGI2 is composed of 6 PDZ domains, 2 WW domains and 1 GK domain. Unlike other members of the MAGUK family, the GK domain of MAGI protein is at the nitrogen end.

[0006] So far, there are no reports on the structures and neuroprotective activities of active small molecules targeting the MAGI2 GK domain. Summary of the invention

[0007] The purpose of the present invention is to provide an application of an oxidized indole compound targeting the MAGI2 GK domain in the preparation of a drug for treating ischemic stroke. The compound is used for the neuroprotective effect of ischemic stroke, laying the foundation for the development of a new drug for treating ischemic stroke.

[0008] The technical solution of the present invention is:

[0009] Application of oxidized indole compounds targeting the MAGI2 GK domain in the preparation of drugs for treating stroke.

[0010] The stroke is ischemic stroke.

[0011] The drug counteracts brain damage caused by transient focal ischemia.

[0012] The drug protects neurons by alleviating neural ischemia-reperfusion injury.

[0013] The dosage of the drug is 0.1-50 mg / Kg.

[0014] The dosage of the drug is 0.5-50 μM / Kg.

[0015] The structure of the aza-oxindole compound targeting the MAGI2 GK domain of the present invention is as follows:

[0016]

[0017] The protein target is the amino acid sequence of the GK domain of the MAGI2 protein:

[0018] MSKSLKKKSHWTSKVHESVIGRNPEGQLGFELKGGAENGQFPYLGEVKPGKVAYESGSKLVSEELLLEVNETPVAGLTIRDVLAVIKHCKDPLRLKCVKQGGIVDKDLRHYLNLRFQKGSVDHELQQIRDNLYLRTVPCTTRPHKEGEVPGVDYIFITVEEFMELEKSGALLESGTYEDNYYGTPKPPAEPAPLLNVTDQILPGATPSAEGKRKRNKSVTNMEKASIEPPEEEEEERPVVNGNGVVITPESSEHDKSAGASGETPSQPYPAPVYSQPEELKDQMDDTKPTKPEENEDS。

[0019] The drugs described in the present invention can be administered orally, parenterally, by inhalation spray, by dropwise administration, or by implantable administration.

[0020] The compounds described in the present invention can improve ischemic injury; improve organic injury and neurobehavioral injury caused by ischemia-reperfusion; reduce ischemia-induced neuronal apoptosis; and reduce oxygen-glucose deprivation-induced cell injury. Experiments by the applicant have shown that the representative compounds can reduce the cerebral ischemic injury area in both the OGD cell model of neuronal ischemia and the in-vivo MCAO model of animals, and have a significant neuroprotective effect. Such compounds are expected to be further developed into new drugs for the treatment of ischemic stroke and can be formulated into dosage forms suitable for various administration routes. Description of the Drawings

[0021] Figure 1 1H NMR spectra of Compound 3 and 3-76-1;

[0022] Figure 2 1 shows the reduction of the cerebral ischemic injury area in rats with middle cerebral artery occlusion (MCAO) by Compound 3-76-1 (intraperitoneal injection); where, Figure 2 1A shows the effect diagram of mouse brain slices stained with TTV;

[0023] Figure 2 1B shows the statistical chart of the damaged area of mouse brain slices, with the displayed value being the mean + SEM. ****p < 0.001, compared with the model group; n = 10;

[0024] Figure 3 2 shows the improvement of the neurobehavioral function in mice with middle cerebral artery occlusion (MCAO) by Compound 3-76-1 (intraperitoneal injection). The neurobehavioral scores of the mice are shown as the mean + SEM, *p < 0.05, compared with the model group; n = 8;

[0025] Figure 4 The average waiting time in the rotarod test of the compound 3-76-1 (intraperitoneal injection). The average waiting time of mice, the displayed value is the mean + SEM, *p < 0.05, compared with the model group; n = 8;

[0026] Figure 5 The compound 3-76-1 has a neuroprotective activity in the oxygen-glucose deprivation (OGD) model of primary neuronal cells in mice. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Reagents and instruments used in the examples

[0029] 1. Reagents

[0030] TTC was purchased from Biosharp (catalog number BS095-25g)

[0031] 4% paraformaldehyde was purchased from Abisson (Cat. No. abs9179)

[0032] Poly-L-Lysine was purchased from Sigma (Cat. No. P4832)

[0033] DMEM high glucose medium was purchased from Gibco (Cat. No. C11995500bt)

[0034] Trypsin-EDTA was purchased from Gibco (Cat. No. 25200056)

[0035] Fetal bovine serum was purchased from Gibco (Cat. No. 10099-141C)

[0036] Penicillin-streptomycin was purchased from Gibco (Cat. No. 15140122)

[0037] TM Neurobasal culture medium was purchased from Gibco (Cat. No. 21103049)

[0038] B-27 TM Supplement (50X) was purchased from Gibco (Cat. No. 17504044)

[0039] GlutaMAX was purchased from Gibco (Cat. No. 35050-061)

[0040] MTS was purchased from Promega (Cat. No. G3580)

[0041] 2. Instruments

[0042] The absorbance value was measured by a multifunctional microplate reader from Molecular Devices.

[0043] Cell culture using Thermo CO2 incubator

[0044] Example 1 Synthesis of Compound 3-76-1

[0045] 1.1 Synthesis of Compound 3-76-1

[0046] Using 1-methyl-1H-pyrrolo[2,3-b]pyridine-2,3-dione and benzyl-substituted maleimide, a Morita-Baylis-Hillman reaction is carried out under the catalysis of a tertiary amine catalyst to generate a target compound in which R1 is substituted with a benzyl group. Adding a base and dimethyl sulfate, a target compound with hydroxymethyl protection at the R2 position is obtained.

[0047]

[0048] Synthesis method of substrate 1:

[0049] Under nitrogen protection, 7-azaindole (2g, 16.95mmol) was dissolved in DMF (10mL), cooled to 0°C, NaH (1.2 equivalents) was added, stirred for 1h, iodomethane (1.1 equivalents) was added, and stirring was continued for 1h. The reaction was quenched with cold water (100mL), extracted with ethyl acetate (3×100mL), dried over anhydrous sodium sulfate, and the solvent was removed to obtain N-methyl-7-azaindole; PCC (5.37g, 25mmol) and silica gel (5.37g) were mixed and ground, 40ml DCE and DCE solution of N-methyl-7-azaindole were added, stirred evenly, and AlCl3 (1.3mol%) was added, and the temperature was raised to 80°C for reaction for 3h; the solvent was removed by rotary evaporation, and the solid was treated with 50ml n-hexane / ethyl acetate (4:1), filtered with a funnel and silica gel, and the filtrate was rotary evaporated to remove the solvent to obtain product 1.

[0050]

[0051] Synthesis method of substrate 2:

[0052] Maleic anhydride (10.6 mmol) was dissolved in 10 ml of ether, aromatic amine was slowly added dropwise, and the mixture was stirred at room temperature for 1 h. The precipitate was filtered, and the filter cake was washed with ether for 2-3 times and dried in air. The dried filter cake was then mixed with 5 ml of acetic anhydride and sodium acetate (30 mol%) and stirred at 70°C for 2 h. The reaction solution was poured into cold water and stirred, filtered, and the solid was washed with water and recrystallized with ethanol to obtain substrate 2.

[0053]

[0054] Synthesis method of compound 3:

[0055] 1-Methyl-1H-pyrrolo[2,3-b]pyridine-2,3-dione 1 (0.1 mmol), maleimide derivative 2k (0.6 mmol) and α-ICD (β-ICD) (20 mol%) were dissolved in 2 ml of toluene, stirred at 50 °C for 24 h, cooled to room temperature, and the reaction solution was purified by silica gel chromatography to obtain chiral compound 3.

[0056]

[0057] Synthesis method of compound 3-76-1:

[0058] Compound 3 (0.1 mmol) was dissolved in 1 ml of DCM, and tetrabutylammonium iodide (1 mol%), NaOH (aq) (0.3 mmol) and dimethyl sulfate (0.2 mmol) were added, and the mixture was stirred at room temperature for 18 h.

[0059]

[0060] 1.2 Confirmation of compound structure

[0061] The structure of the product was characterized using an Agilent 600MHz NMR spectrometer. The 1H NMR data are as follows:

[0062] Compound 3: Yield: 94%; yellow solid, 1 H NMR(600MHz, CDCl3) δ8.29(d,J=5.1Hz,1H),7.61(d,J=7.3Hz,1H),7.32-7.26( m,5H),7.03-7.00(m,1H),6.69(s,1H),4.59(s,2H),3.93(s,1H),3.34(s,3H).

[0063] Compound 3-76-1: Yield 85%; yellow solid, 1H NMR (600MHz, CDCl3) δ8.34-8.28(m,1H),7.54-7.49(m,1H),7.25(dt,J=16.2,6.5Hz, 5H),7.02(dd,J=7.0,5.5Hz,1H),6.90(s,1H),4.53(s,2H),3.38(s,3H),3.18(s,3H).

[0064] The H NMR spectra of compounds 3 and 3-76-1 are shown in Figure 1 .

[0065] Example 2 Testing of the activity of compound 3-76-1 in intervention of cerebral ischemic injury by intraperitoneal injection

[0066] 2.1 Experimental methods

[0067] Middle cerebral artery occlusion (MCAO) in C57 mice:

[0068] C57 mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were anesthetized with 1% sodium pentobarbital, and the hair on the neck was shaved. The left CCA, ICA, and ECA were exposed and separated. The CCA was ligated with a slipknot near the tail end, and then the ECA was ligated and two lines were prepared at the proximal ends of the two ligatures. A cut was made at the proximal end of the ECA ligature and a thread plug of appropriate size was slowly inserted from this mouth. The thread plug was adjusted to enter the ICA about 10mm (with the black dot on the thread plug as a mark), and stopped moving forward after encountering resistance, so that the occlusion of the middle cerebral artery of the mouse can be achieved. Next, the previously prepared thread was tied to fix the thread plug. After completion, the slipknot at the CCA was untied and the timer was counted. After 90 minutes of vascular occlusion, the thread plug was slowly removed and the ECA stump was ligated to restore cerebral blood perfusion. The skin was then sutured and disinfected. A heating pad was used to keep warm during the experiment, and the body temperature of the mice was controlled at 37°C.

[0069] TTC staining method:

[0070] TTC staining is a method that uses TTC to react with succinate dehydrogenase in the mitochondria of living cells to generate red formazan to reflect cell viability, and is often used to evaluate cerebral ischemic injury. The specific operation is: the mice that have completed the MCAO model and injected with 3-76-1 or control solvent are evaluated for behavior at 72 hours of reperfusion, and then the brain is removed by dislocation of the neck. Freeze at -80℃ for 5 minutes, and after the brain tissue is hard enough, coronal continuous sections are made, each with a thickness of 2mm, and 7 sections can be obtained for each mouse brain. After that, the sections are immersed in 2% TTC solution and incubated at 37℃ in the dark for 15 minutes. During this period, the sections are turned over every 5 minutes to ensure uniform staining. After the incubation, the TTC solution is discarded, and 4% paraformaldehyde is added for fixation for 24 hours and then photographed. The red part of the section shows the area with blood perfusion, while the white part indicates the infarct area.

[0071] Rotarod test method:

[0072] After the speed is set and the power is turned on, the wheel can rotate automatically. The rotating rod gradually increases the rotation speed from 2rpm to 50rpm within 5 minutes. The mice are first adapted to the training once, and then three consecutive trials are carried out, each experiment lasting 5 minutes. The rest time between each trial is 30 minutes. The average waiting time for the mouse to fall off the rotating rod in the last three trials is used for analysis.

[0073] Test group: Mice were intraperitoneally injected with the compound at 10 mg / kg, qd, and cerebral infarction area was measured and rotarod test was performed three days after administration.

[0074] Model group: Mice were subjected to MCAO and simultaneously injected with solvent control via tail vein, and other treatments were the same as those of the test group.

[0075] Compounds' protective activity against brain damage:

[0076] The present invention adopts behavioral scoring, rotarod test and TTC staining respectively to evaluate the neurobehavioral damage caused by cerebral ischemia and the infarction condition of the cerebral ischemic area, and determine the protective effect of the compound on cerebral ischemia.

[0077] Results evaluation:

[0078] The smaller the infarct area in the drug-treated group, the lower the behavioral score, and the longer the waiting time for falling in the rotating rod test, indicating that the sample has a stronger protective effect on brain damage.

[0079] 2.2 Experimental Results

[0080] The compound 3-76-1 of the present invention reduces the cerebral infarction area of ​​mice induced by MCAO (see Figure 2 ), the results showed that compared with the model group, the brain damage area in the drug-treated group was significantly lower than that in the model group, indicating that the compound 3-76-1 can alleviate brain damage caused by focal transient ischemia.

[0081] The results of the improvement of 3-76-1 on MCAO-induced neurobehavioral damage in rats (see Figure 3 and Figure 4 ):

[0082] In the neurobehavioral experiment, the model group scored about 2 points, and the 10 mg / kg administration group scored about 1 point. The difference between the model group and the administration group was statistically significant (p<0.05), indicating that the compound 3-76-1 of the present invention can improve the neurobehavioral function of MCAO mice. In the rotating rod experiment, the waiting time for the model group mice to fall was about 20s, and the waiting time for the 10 mg / kg administration group was about 30s. The difference between the model group and the administration group was statistically significant (p<0.05), also indicating that the compound 3-76-1 of the present invention can improve the neurobehavioral function of MCAO mice.

[0083] 2.3 Conclusion

[0084] The compound 3-76-1 of the present invention has the effect of improving the cerebral infarction area and neurobehavior of MCAO mice. Therefore, the present invention is expected to be further developed into a new anti-ischemic stroke therapeutic drug.

[0085] Example 3 Test of Anti-Oxygen Glucose Deprivation (OGD)-Induced Neurological Damage Activity

[0086] 3.1 Experimental samples and experimental methods

[0087] The source of the test samples is the same as that in Example 1.

[0088] Packing:

[0089] Coat the 96-well plate with poly-L-lysine (PLL) to promote subsequent primary neuron attachment: add poly-L-lysine solution to the 96-well plate, 100 μL per well. Then place the 96-well plate in a CO2 incubator overnight to allow poly-L-lysine to bind to the surface of the plate. Afterwards, recover the PLL and rinse the coated 96-well plate 3 times with sterile ultrapure water to remove excess PLL. Finally, place the coated 96-well plate in a CO2 incubator to dry for later use.

[0090] Extraction of primary mouse neuronal cells:

[0091] Pregnant C57 mice at E15–17 were killed by cervical dislocation and disinfected with 75% alcohol. Starting from the lower abdomen of the pregnant mouse, the skin and muscle layers were cut open in turn, the uterus was removed and temporarily immersed in ice-cold sterile DMEM high-glucose medium, and then the fetal mice were removed in turn and the fetal brains were extracted in batches. The meninges were peeled off under a stereomicroscope, and the cortical tissues of the fetal mice were separated and collected. The above process was all carried out on ice, and the immersion solution was DMEM high-glucose medium. Care was taken to avoid contamination during the operation. The isolated mouse cerebral cortex tissue was digested with 0.25% trypsin at 37°C to dissociate the cells. After 5 minutes, the digestion was terminated with seeding solution and the cells were blown away. The cells were filtered through a 70μM cell strainer and counted. After dilution, the cells were seeded in the well plate according to the target concentration. The seeding density of the cells was 10 for 96-well plates. 4 pcs / well; 24-well plate 10 5 After the plate is inoculated, transfer the plate to a CO2 incubator and culture it at 37°C and 5% CO2 for 4-6 hours before replacing it with maintenance medium. After 4-5 days of culture, perform a half-change of medium and continue culturing for 7-9 days before conducting the experiment.

[0092] OGD-induced neuronal cell damage method:

[0093] After 7-9 days of culture of primary cortical neurons of mice, OGD modeling was performed. First, the maintenance medium was replaced with DMEM sugar-free medium, and the cells were cultured in an anaerobic environment (5% CO2, 95% N2) for 2 hours, then replaced with maintenance medium, and the cells were transferred to a normal culture environment and continued to be cultured for 24 hours. Finally, cell viability was detected.

[0094] Test group: After OGD, maintenance culture medium containing different concentrations of test compound solution or positive compound edaravone solution was added by medium replacement, and culture was continued at 37°C and 5% CO2 for 24 hours before detecting cell viability.

[0095] Model group: except that solvent control intervention was used instead of 3-76-1 intervention, other treatments were the same as those of the test group.

[0096] Cell viability test method:

[0097] The present invention uses MTS to determine the vitality of primary cortical neuron cells. After the primary cortical neurons of mice are treated, MTS is added to a 96-well plate, 20 μL per well, and then cultured for 2 hours at 37°C and 5% CO2. During this process, MTS can be reduced to a colored formazan product by a variety of dehydrogenases in the mitochondria of living cells, and the color depth of the system is highly correlated with the number of living cells therein. After incubation for 2 hours, the absorbance of each well at 490 nm is measured using a multifunctional microplate reader to obtain cell vitality data.

[0098] Results Evaluation

[0099] Taking the cell viability of the OGD model group as 100%, the cell viability of the drug-treated group was compared with that of the model group. The higher the cell viability value, the stronger the protective effect of the sample on the cells.

[0100] 3.2 Experimental Results

[0101] The neuron cell protective activity of the compound 3-76-1 of the present invention (see Figure 5 ): The compound has a significant protective effect at concentrations of 5, 10, and 20 μM.

Claims

1. Application of an oxidized indole compound targeting the MAGI2 GK domain in the preparation of a drug for treating stroke, wherein the structure of the oxidized indole compound is: , the stroke is ischemic stroke.

2. The use according to claim 1, characterized in that: The drug counteracts brain damage caused by transient focal ischemia.

3. The use according to claim 1, characterized in that: The drug protects neurons by alleviating neural ischemia-reperfusion injury.

4. The use according to claim 1, characterized in that: The dosage of the drug is 0.1-50 mg / Kg.

5. The use according to claim 4, characterized in that: The dosage of the drug is 0.5-50 μM / Kg.

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