Use of a triazole compound in the preparation of a drug for preventing and treating ischemic stroke

Abstract

The application provides application of a triazole compound in preparation of a medicine for preventing and treating ischemic stroke, and belongs to the field of biological medicines; the triazole compound is shown as structural formula (I), the medicine further comprises pharmaceutically acceptable adjuvants; and the application of chiral compounds, enantiomers, diastereoisomers, geometric isomers, free forms and pharmaceutically acceptable salts, hydrates, solvates or esters of the triazole compound in preparation of the medicine for preventing and treating ischemic stroke is further provided. Compared with the prior art, the triazole compound has better pharmacokinetic and pharmacodynamic characteristics in the application in preparation of the medicine for preventing and treating ischemic stroke, and has higher clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a triazole compound in the preparation of drugs for the prevention and treatment of ischemic stroke. Background Technology

[0002] Cerebral stroke, also known as apoplexy, is an acute cerebrovascular disease caused by blood vessel blockage preventing blood flow to the brain or by sudden rupture of blood vessels in the brain, resulting in brain tissue damage. It is characterized by high incidence, high disability rate, high mortality rate, and high recurrence rate. However, current treatment options for stroke are very limited. For patients with hyperacute ischemic stroke, intravenous thrombolysis and endovascular therapy are required within the treatment window. However, due to the limited treatment window and the increased risk of bleeding associated with treatment, only about 5% of patients actually benefit from recombinant tissue plasminogen activator (rtPA) therapy. Therefore, finding and discovering drugs with long-acting neuroprotective effects is a key research focus and goal in the prevention and treatment of ischemic stroke.

[0003] Butylphthalide (NBP) is a drug primarily indicated for the treatment of ischemic stroke. NBP acts on multiple pathological aspects of cerebral ischemia. Pharmacodynamic studies have shown that it has a strong anti-cerebral ischemia effect, significantly improving microcirculation and blood flow in the ischemic area, increasing the number of capillaries in the ischemic area, improving cerebral energy metabolism, and reducing neuronal apoptosis. Furthermore, multiple clinical studies have shown that NBP not only improves the symptoms of ischemic stroke patients but also contributes to long-term recovery. However, due to its extremely poor water solubility, hepatotoxicity, and drug resistance, its use as a monotherapy is not entirely ideal.

[0004] In their paper "Design, Synthesis and Antiplatelet Aggregation Activity Study of Ligustrazine-Butylphthalide Derivatives", Zhu Tao et al. synthesized three ligustrazine-butylphthalide derivatives using phthalic anhydride and ligustrazine as raw materials through reactions including free radical substitution, nucleophilic addition of n-butyllithium, catalytic dehydration with p-toluenesulfonic acid, Pd / C hydrogenation reduction, hydrolysis, and esterification. Their structures were confirmed by 1H NMR, 13C NMR, and HR-MS. Among them, (3,5,6-trimethylpyrazine-2-yl)methyl-2-pentanoylbenzoate showed an IC50 inhibition rate of 0.26 mmol / L against adenosine diphosphate (ADP)-induced platelet aggregation, which was superior to that of the parent compounds ligustrazine and butylphthalide. However, as a compound with anti-cerebral ischemia activity, current research on butylphthalide mainly focuses on its anticoagulant effect, and its superior anti-cerebral ischemia activity has not been fully studied and developed.

[0005] In their paper, "Efficacy of Danshen Chuanxiongqin Combined with Butylphthalide Injection in the Treatment of Patients with Acute Ischemic Stroke and Its Effects on Brain Natriuretic Peptide and Chemerin," Chen Zongsheng et al. studied the anti-cerebral ischemia efficacy of butylphthalide combined with Danshen Chuanxiongqin, exploring the efficacy of Danshen Chuanxiongqin combined with butylphthalide injection in the treatment of patients with acute ischemic stroke and its effects on brain natriuretic peptide and serum adipokines (Chemerin). The method used in this paper was as follows: 92 patients with a confirmed diagnosis of acute ischemic stroke were randomly divided into a control group (treated with Danshen Chuanxiongqin) and a combined treatment group (treated with Danshen Chuanxiongqin + butylphthalide injection), with 46 patients in each group. After 2 weeks of continuous treatment, patients' neurological function was assessed using the National Institutes of Health Stroke Scale (NIHSS). Plasma levels of brain natriuretic peptide (BNP), angiotensin II (Ang)-8, interleukin (IL)-6, tumor necrosis factor (TNF)-α, malondialdehyde (MDA), superoxide dismutase (SOD), and chemerin were measured using enzyme-linked immunosorbent assay (ELISA). Hemorheological parameters were analyzed using a whole blood rheometer. Results showed that after treatment, both groups exhibited significantly lower NIHSS scores, low / high shear viscosity, plasma viscosity, erythrocyte aggregation index, and plasma levels of BNP, Ang II, IL-8, TNF-α, MDA, and chemerin compared to pre-treatment levels (P<0.05), with the combined treatment group showing significantly lower levels than the control group (P<0.05). Serum SOD levels significantly increased after treatment in both groups compared to pre-treatment levels (P<0.05), with the combined treatment group showing significantly higher levels than the control group (P<0.05). The prognosis of the combined treatment group was significantly better than that of the control group (P<0.05). In conclusion, the combination of tanshinone and ligustrazine injection can significantly improve neurological function in patients with acute ischemic stroke, resulting in a better prognosis. The authors believe this may be related to reducing plasma BNP, MDA, and chemerin levels, alleviating inflammatory response, enhancing the body's antioxidant capacity, and accelerating blood flow.

[0006] In implementing the embodiments given in the background art, the inventors discovered that the background art has at least the following defects: butylphthalide has extremely poor water solubility and is hepatotoxic and induced drug resistance; thus, its application as a single drug is not ideal.

[0007] Therefore, how to provide an application of butylphthalide-modified compounds in the preparation of drugs with the effect of preventing and treating ischemic stroke, in order to overcome the shortcomings of butylphthalide monotherapy, is a technical problem that those skilled in the art hope to solve. Summary of the Invention

[0008] This invention addresses the shortcomings of existing technologies, such as poor efficacy of single-drug formulations and the lack of applications of compounds with modified butylphthalide structures in the preparation of drugs for the prevention and treatment of ischemic stroke. It provides an application of triazole compounds based on modified butylphthalide structures in the preparation of drugs for the prevention and treatment of ischemic stroke, which can more effectively improve the affected area of ​​the brain in ischemic stroke, enhance cerebral blood circulation, and reduce ischemia-reperfusion injury.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] In this invention, compound B4 or B4 described in the text or images refers to a triazole compound with the molecular formula C. 29 H 39 N7O4, chemical structural formula as shown in formula (Ⅰ):

[0011]

[0012] In a first aspect, the present invention provides the application of triazole compounds in the preparation of drugs for the prevention and treatment of ischemic stroke.

[0013] Preferably, the triazole compound has the molecular formula C2. 29 H 39 N7O4, its general formula is shown in structural formula (Ⅰ):

[0014]

[0015] Preferably, the method for preparing the triazole compound includes the following steps:

[0016] (1) Butylphthalide was hydrolyzed with alkaline solution, pH was adjusted, the solution was concentrated, and chloroacetyl chloride, 4-dimethylaminopyridine and triethylamine were added to react and intermediate 1 was obtained.

[0017] (2) Dissolve intermediate 1 obtained in step (1) with propargylamine, add O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid and N,N-diisopropylethylamine to react and obtain intermediate 2;

[0018] (3) Ligustrazine was brominated with N-bromosuccinic anhydride using benzoyl peroxide as an initiator to obtain intermediate 3;

[0019] (4) Dissolve intermediate 3 obtained in step (3), and then add sodium azide to react and obtain intermediate 4;

[0020] (5) Dissolve intermediate 2 obtained in step (2) and intermediate 4 obtained in step (4), add catalyst to react and obtain intermediate 5;

[0021] (6) The intermediate 5 obtained in step (5) undergoes a substitution reaction with the substituted compound under the action of a catalyst to obtain the triazole compound.

[0022] More preferably, the alkali used in step (1) is selected from at least one of sodium hydroxide, potassium hydroxide and calcium hydroxide.

[0023] More preferably, the hydrolysis conditions in step (1) are: heat preservation at 55-65℃ for 1.5-3 hours, or microwave at 55-65℃ for 30-45 minutes.

[0024] More preferably, the target pH for adjusting the pH in step (1) is 3-4, and the reagent used is selected from one or more of dilute hydrochloric acid, dilute sulfuric acid and dilute nitric acid.

[0025] More preferably, the concentration method in step (1) is extraction by adding an extractant; the extractant is selected from one or both of ethyl acetate and diethyl ether.

[0026] More preferably, the molar ratio of chloroacetyl chloride, 4-dimethylaminopyridine and triethylamine to butylphthalide in step (1) is 1.4-1.6:0.08-0.12:1.4-1.6:1.

[0027] Most preferably, the molar ratio of chloroacetyl chloride, 4-dimethylaminopyridine and triethylamine to butylphthalide in step (1) is 1.5:0.1:1.5:1.

[0028] More preferably, the solvent used for dissolution in step (2) includes carbon dichloride.

[0029] More preferably, the molar ratio of propargylamine to intermediate 1 in step (2) is 1.1-1.3:1.

[0030] Most preferably, the molar ratio of propargylamine to intermediate 1 in step (2) is 1.2:1.

[0031] More preferably, the molar ratio of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid, N,N-diisopropylethylamine and intermediate 1 in step (2) is 1.1-1.3:1.8-2.2:1.

[0032] Most preferably, the molar ratio of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid, N,N-diisopropylethylamine and intermediate 1 in step (2) is 1.2:2:1.

[0033] More preferably, the reaction in step (2) is carried out at a temperature of 20-30°C for 5.5-7 hours.

[0034] Most preferably, the reaction in step (2) is carried out at a temperature of 25°C for 6 hours.

[0035] More preferably, the molar ratio of benzoyl peroxide to tetramethylpyrazine in step (3) is 0.08-0.12:1.

[0036] Most preferably, the molar ratio of benzoyl peroxide to tetramethylpyrazine in step (3) is 0.1:1.

[0037] More preferably, the molar ratio of N-bromosuccinic anhydride to tetramethylpyrazine in step (3) is 0.25-0.35:1.

[0038] Most preferably, the molar ratio of N-bromosuccinic anhydride to tetramethylpyrazine in step (3) is 0.3:1.

[0039] More preferably, the bromination reaction in step (3) is carried out by reflux.

[0040] More preferably, the reflux temperature is 75-85°C and the time is 3.5-5 hours.

[0041] More preferably, the solvent used for dissolution in step (4) includes acetonitrile.

[0042] More preferably, the molar ratio of sodium azide to intermediate 3 in step (4) is 1.15-1.25:1.

[0043] Most preferably, the molar ratio of sodium azide to intermediate 3 in step (4) is 1.2:1.

[0044] More preferably, the reaction conditions in step (4) are microwaved at 55-65°C for 55-70 minutes.

[0045] Most preferably, the reaction conditions in step (4) are microwave at 60°C for 60 min.

[0046] More preferably, the molar ratio of intermediate 2 to intermediate 4 in step (5) is 1:0.9-1.1.

[0047] Most preferably, the molar ratio of intermediate 2 to intermediate 4 in step (5) is 1:1.

[0048] More preferably, the solvent used for dissolution in step (5) includes dichloromethane.

[0049] More preferably, the catalyst in step (5) comprises cuprous thiophene-2-carboxylate (I).

[0050] More preferably, the temperature of the reaction in step (5) is 20-30°C.

[0051] More preferably, the catalyst in step (6) includes cesium carbonate; the molar ratio of cesium carbonate to intermediate 5 is 1.4-1.6:1.

[0052] More preferably, the molar ratio of the substituted compound to intermediate 5 in step (6) is 1.4-1.6:1.

[0053] More preferably, the substituted compound in step (6) is selected from morpholine.

[0054] More preferably, the substitution reaction in step (6) is carried out at 60-70°C for 100-140 min; or at 60-70°C under microwave for 50-65 min.

[0055] Most preferably, the substitution reaction in step (6) is carried out at 65°C for 120 min; or at 65°C under microwave for 60 min.

[0056] Preferably, the minimum dose unit of the drug contains 2-200 mg of B4.

[0057] The smallest unit of dosage for the drug refers to a tablet, a capsule, a sachet of granules, or an injection, etc.

[0058] Preferably, the drugs for preventing and treating ischemic stroke include drugs for preventing ischemic stroke and drugs for treating ischemic stroke.

[0059] Preferably, the drug is in the form of a solid, liquid, or gas.

[0060] More preferably, the solid form is a powder, tablet, granule, pill, hard capsule, soft capsule, cream, ointment, plaster, gel, paste, powder, or patch; the liquid form is a solution, suspension, injection, syrup, liniment, emulsion, tincture, or elixir; and the gas form is an aerosol or spray.

[0061] Most preferably, the drug is in the form of tablets or injections.

[0062] Preferably, the drug is administered via at least one of the following routes: oral, sublingual, oral mucosal, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, nasal, or rectal.

[0063] In a second aspect, the present invention provides the use of chiral compounds, enantiomers, diastereomers, geometric isomers, free forms, and pharmaceutically acceptable salts, hydrates, solvates, or esters of triazole compounds of formula (I) in the preparation of drugs for the prevention and treatment of ischemic stroke.

[0064] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0065] More preferably, the excipients include at least one of the following: binders, fillers, diluents, disintegrants, suspending agents, suspending aids, sustained-release agents, controlled-release agents, lyophilization protectants, coating agents, enteric coatings, lubricants, flow aids, anti-adhesion agents, sweeteners, flavoring agents, plasticizers, opacifiers, solubilizers, humectants, solvents, osmotic pressure regulators, colorants, pigments, surfactants, emulsifiers, water-soluble matrices, fat-soluble matrices, oleic matrices, pore-forming agents, gelling agents, preservatives, buffers, chelating agents, and antioxidants.

[0066] Preferably, the minimum dose unit of the drug contains 2-200 mg of at least one of the enantiomers, diastereomers, geometric isomers, free forms, and pharmaceutically acceptable salts, hydrates, solvates, and esters of a triazole compound of formula (I).

[0067] The smallest unit of dosage for the drug refers to a tablet, a capsule, a sachet of granules, or an injection, etc.

[0068] Preferably, the drugs for preventing and treating ischemic stroke include drugs for preventing ischemic stroke and drugs for treating ischemic stroke.

[0069] Preferably, the drug is in the form of a solid, liquid, or gas.

[0070] More preferably, the solid form is a powder, tablet, granule, pill, hard capsule, soft capsule, cream, ointment, plaster, gel, paste, powder, or patch; the liquid form is a solution, suspension, injection, syrup, liniment, emulsion, tincture, or elixir; and the gas form is an aerosol or spray.

[0071] Preferably, the drug is administered via at least one of the following routes: oral, sublingual, oral mucosal, intravenous, intramuscular, intraperitoneal, subcutaneous, percutaneous, nasal, or rectal.

[0072] Thirdly, the present invention also provides the use of triazole compounds or their enantiomers, diastereomers, geometric isomers, free forms, and pharmaceutically acceptable salts, hydrates, solvates, or esters in the preparation of medicaments for the prevention and treatment of ischemia-reperfusion injury and diseases with similar mechanisms and injury-mediated effects to ischemia-reperfusion injury.

[0073] Preferably, the triazole compound has the molecular formula C2. 29 H 39 N7O4, chemical structural formula as shown in formula (Ⅰ):

[0074]

[0075] Preferably, the disease is selected from coronary heart disease, hypertensive heart disease, pulmonary heart disease, transient ischemic attack, vertebrobasilar insufficiency, vascular dementia, intracranial aneurysm, intracranial vascular malformation, and intracranial artery and sinus thrombosis.

[0076] Fourthly, the present invention also provides the use of a pharmaceutical composition containing an active ingredient in the preparation of a drug for the prevention and treatment of ischemic stroke; the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0077] Preferably, the active ingredient comprises at least one of triazole compounds and chiral compounds, enantiomers, diastereomers, geometric isomers, free forms, and pharmaceutically acceptable salts, hydrates, solvates, and esters of triazole compounds; the molecular formula of the triazole compound is C2. 29 H 39 N7O4, chemical structural formula as shown in formula (Ⅰ):

[0078]

[0079] More preferably, the excipients are selected from at least one of the following groups: adhesives, fillers, diluents, disintegrants, suspending agents, suspending aids, sustained-release agents, controlled-release agents, lyophilization protectants, coating agents, enteric coating materials, lubricants, flow aids, anti-adhesion agents, sweeteners, flavoring agents, plasticizers, opacifiers, solubilizers, humectants, solvents, osmotic pressure regulators, colorants, pigments, surfactants, emulsifiers, water-soluble matrices, fat-soluble matrices, oleic matrices, pore-forming agents, gelling agents, preservatives, buffers, chelating agents, and antioxidants.

[0080] Preferably, the pharmaceutical composition further comprises other active components that have the effect of increasing cerebral blood flow or reducing ischemia-reperfusion injury.

[0081] Compared with the prior art, the present invention has the following beneficial effects:

[0082] This invention provides a novel application of triazole compounds, specifically their use in the preparation of drugs for the prevention and treatment of ischemic stroke. These triazole compounds can effectively reduce the area affected by stroke, increase cerebral blood flow, and improve cerebral ischemia-reperfusion injury. Compared to existing technologies, triazole compounds exhibit superior pharmacokinetic and pharmacodynamic characteristics in the preparation of drugs for the prevention and treatment of ischemic stroke, demonstrating higher clinical application value. Attached Figure Description

[0083] Figure 1 This is a comparative graph showing the protective effect of compound B4 in Example 1 of the present invention on a neuronal cell model induced by hypoxia-reoxygenation in vitro.

[0084] Figure 1 Figures A and B show the effects of compound B4 on the cytotoxicity of SH-SY5Y cells and the proliferation of OGD / R cell models; Figure C shows the effects of compound B4 on the release of LDH from SH-SY5Y cells in the OGD / R cell model.

[0085] Figure 2 This is a comparative graph showing the effects of compounds B4 and A5 in Example 1 of the present invention on the cytotoxicity of SH-SY5Y cells and the proliferation activity of OGD / R cell models.

[0086] Figure 3 This is a graph showing the effect of compound B4 in Example 1 of the present invention on apoptosis and reactive oxygen species levels in the SH-SY5Y cell OGD / R model;

[0087] Figure 3 A and B represent fluorescence results of apoptosis and necrosis; C and D represent fluorescence results of reactive oxygen species.

[0088] Figure 4 The results show the effects of compound B4 in Example 1 of this invention on SOD and MDA levels and Nrf2 nuclear translocation in the SH-SY5Y cell OGD / R model.

[0089] Figure 4 In the table, A and B represent the results of intracellular SOD and MDA content; C represents the Nrf2 nuclear translocation fluorescence results.

[0090] Figure 1-4 In comparison with the control group, ### P < 0.001; compared with the model group (OGD / R) * P < 0.05, *** P < 0.001; compared with the NBP group, & P < 0.05, &&& P < 0.001);

[0091] Figure 5 This is the result of the protective effect of compound B4 in the rat ischemic stroke model in Example 2 of the present invention;

[0092] Figure 5 In the table, A represents the changes in body weight of each group within 7 days of drug administration; B represents the changes in behavioral scores of each group within 7 days of drug administration; C and D represent the cerebral infarction area of ​​each group of rats after 7 days of drug administration; and E and F represent the cerebral blood flow of each group of rats after 7 days of drug administration.

[0093] Figure 5 In comparison with the Sham group, ### P < 0.001; compared with the model group (MCAO / R) ** P < 0.01, *** P < 0.001; compared with the NBP group, & P < 0.05. Detailed Implementation

[0094] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods, instruments, and raw materials used in the following embodiments are all conventional operating methods.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0096] Synthesis of compound B4 (Example)

[0097] Preparation of Intermediate 1

[0098] The structural formula of intermediate 1 is as follows:

[0099]

[0100] Compound NBP (10.0 g, 52.6 mmol) was added to 60 mL of an ethanol-water mixture (2:1 v / v), followed by potassium hydroxide (4.4 g, 78.4 mmol). The mixture was microwaved at 60 °C for 40 min. After the reaction, the solvent was evaporated to dryness. The concentrate was diluted with water, and the pH was adjusted to 3-4 with dilute hydrochloric acid, resulting in the precipitation of a white solid. The solid was extracted three times with ethyl acetate, and the organic layers were combined and dried over anhydrous magnesium sulfate. Chloroacetyl chloride (6.3 mL, 78.9 mmol) was added to the ethyl acetate solution of intermediate 1, followed by the addition of DMAP (642.3 mg, 5.26 mmol) and then triethylamine (11 mL, 78.9 mmol). The mixture was stirred and reacted. After the reaction, the solid was extracted with water, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography with a V (petroleum ether):V (ethyl acetate) ratio of 10:1 to obtain 7.6 g of a pale yellow solid, with a yield of 51%. This crude product was used in the next reaction.

[0101] Preparation of intermediate 2

[0102] The structural formula of intermediate 2 is as follows:

[0103]

[0104] Intermediate 1 (5 g, 17.6 mmol) was dissolved in 30 mL of dichloromethane, followed by the addition of TBTU (6.67 g, 29.6 mmol) and DIPEA (5.8 mL, 44.2 mmol). After the reaction was complete, the mixture was extracted three times with brine, and the organic layer was collected. After removing water, the solvent was evaporated to dryness, yielding the crude product. The crude product was purified by silica gel column chromatography with a ratio of V (petroleum ether):V (ethyl acetate) of 10:1 to give 3.53 g of a pale yellow solid, with a yield of 62.3%. This was used for the next reaction.

[0105] Intermediate 2 1 H NMR, 13 The C NMR and HRMS data are shown below:

[0106] 1 H-NMR (600MHz, CDCl3) δ: 8.09 (dd, J=8.0Hz, 1.1Hz, 1H, H-3), 7.61-7.58 (td, J=8.1Hz, 1.4Hz, 1H, H-5), 7.58-7.56 (dd, J=8 .1Hz,1.5Hz,1H,H-6),7.39(td,1H,J=7.4Hz,1.5Hz,H-4),6.77(m,1H,C(CH)O),4.10(d,2H,CO(CH2)Cl),0.91(t,3H,CH3); 13 C-NMR (150)

[0107] MHz, CDCl3)δ:172.0,166.7,143.7,133.5,131.5,127.7,126.9,126.1,75..0,41.1,36.5,27.9,22.3,14.0; HRMS:Calcd.for C 14 H 17 ClO4(M+Na):307.0708.Found:307.0710

[0108] Preparation of intermediate 3

[0109] The structural formula of intermediate 3 is as follows:

[0110]

[0111] Compound TMP (5 g, 36.7 mmol) was dissolved in carbon tetrachloride (50 mL), followed by the addition of NBS (2.179 g, 12.2 mmol) and BPO (890 mg, 3.6 mmol). The mixture was then microwaved at 65 °C for 1 h. After the reaction was complete, the mixture was filtered, the solvent was evaporated to dryness, and the solution was purified by silica gel column chromatography at a ratio of V (petroleum ether):V (ethyl acetate) of 20:1 to give 1.83 g of a pale white solid, with a yield of 69%. This solid was used in the next reaction.

[0112] Preparation of intermediate 4

[0113] The structural formula of intermediate 4 is as follows:

[0114]

[0115] Dissolve intermediate 4 (500 mg, 2.32 mmol) in acetonitrile (2 ml), then add NaN3, and microwave at 60 °C for 1 h. After the reaction is complete, filter under vacuum, evaporate the solvent to dryness, and use it directly in the next reaction without further processing.

[0116] Preparation of intermediate 5

[0117] The structural formula of intermediate 5 is as follows:

[0118]

[0119] Accurately weigh 2 g (7 mmol) of intermediate 2, dissolve it in 5 mL of dichloromethane, and add intermediate 4 (1.16 g, 7 mmol) and Cutc (260 mg, 7 mmol) sequentially at room temperature. Stir for 2 h at room temperature. After the reaction is complete as detected by TLC, filter and concentrate to obtain the crude product. Purify the crude product by silica gel column chromatography with a V(dichloromethane):V(methanol) ratio of 15:1 to obtain 1.8 g of a pale yellow solid, with a yield of 56%. Used for the next reaction.

[0120] Intermediate 51 H NMR, 13 The C NMR and HRMS data are shown below.

[0121] 1 H-NMR(600MHz, CDCl3)δ:7.72(s,1H,CCHN),7.45-7.42(m,1H,H-3),7.42-7.41(m,1H,H-5),7.41-7.40(m,1H,H-6),7.33-7.29( m,1H,H-4),7.20(m,1H,CONH),6.05(m,1H,C(CH)O),5.61(m,2H,NCH2C),4.74-4.66(m,2H,COCH2Cl),4.04(d,J=14.8Hz,2H,NHC H 2),2.54(s,3H,CH3),2.51(s,3H,CH3),2.50(s,3H,CH3),0.86(t,J=7.2Hz,3H,CH3); 13 C-NMR (150MHz, CDCl3) δ:169.0,167.3,152.1,149.6,148.9,144.8,143.6,138.4,135.3,130.6 ,128.2,127.3,126.2,122.5,75.9,53.2,41.0,36.4,35.6,27.6,22.4,21.7,21.5,20.7,13.9.

[0122] HRMS:Calcd.for C 25 H 31 ClN6O3(M+Na):521.2038.Found:521.2038.

[0123] Preparation of compound B4

[0124]

[0125] Intermediate 5 was prepared as described above; 1 g (1.6 mmol) of intermediate 5 was accurately weighed and placed in a microwave tube, dissolved in 3 ml of tetrahydrofuran, and then cesium carbonate (0.9 g, 3.8 mmol) and morpholine (0.24 ml, 3.8 mmol) were added sequentially. The mixture was microwaved at 65 °C for 1 h. After the reaction was complete as detected by TLC, the mixture was filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (V:V:ethyl acetate = 1:4) to obtain 0.43 g of a yellow viscous liquid, with a yield of 39%. Compound B4... 1 H NMR, 13 The C NMR and HRMS data are shown below.

[0126] 1 H-NMR(600MHz, CDCl3)δ:7.65(s,1H,CCHN),7.54(m,1H,CONH),7.35-7.33(m,1H,H-3),7.33-7.31(m,1H,H-5),7. 31-7.29(m,1H,H-6),7.25-7.21(m,1H,H-4),5.87(m,1H,C(CH)O),5.53(d,J=14.6Hz,2H,NCH2C),4.62(m,2H,NHC H 2),3.62(t,J=4.6Hz,4H,CH2OCH2),3.12(d,J=16.6Hz,2H,COCH2N),2.47(s,3H,CH3),2.47-2. 44,2.42-2.37(m,4H,CH2NCH2),2.44(s,3H,CH3),2.43(s,3H,CH3),0.77(t,J=7.2Hz,3H,CH3); 13 C-NMR (150MHz, CDCl3) δ:169.5,168.2,151.0,148.5,147.9,143.9,142.5,137.6,134.4,129.3,127.0,126.6,125 .0,121.5,73.5,65.7(2C),58.6,52.2(2C),52.1,35.4,34.5,26.5,21.3,20.7,20.5,19.6,12.9.; HRMS:Calcd.for C 29 H 39 N7O4(M+H):550.3135.Found:550.3135.

[0127] Example 1: Protective effect of B4 on an in vitro hypoxia-reoxygenation induced neuronal cell model

[0128] 1. Drugs and reagents

[0129] Compounds B4 and A5 used in this experiment were provided by Researcher Tian Yu of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, according to the above-mentioned synthesis method. The positive control drug butylphthalide (NBP) was purchased from MedChemExpress; the cck-8 kit was purchased from Wuhan Sanying Biotechnology Co., Ltd. Lactate dehydrogenase (LDH) kit, protein quantification (BCA) kit, total superoxide dismutase (T-SOD) assay kit, and malondialdehyde (MDA) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. Reactive oxygen species (ROS) detection kits and apoptosis / necrosis detection kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd. NRF2Rabbit mAb and Cy3 Goat Anti-Rabbit IgG (H+L) were purchased from Wuhan Aibote Biotechnology Co., Ltd.

[0130] 2. Cell culture and establishment of a hypoxia-reoxygenation induced neuronal cell model

[0131] The human neuroblastoma cell line (SH-SY5Y) was obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). SH-SY5Y cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, Gibco, USA), 2 mmol / L glutamine, penicillin (100 U / ml), and streptomycin (100 μg / ml) under humid conditions of 37°C and 5% CO2. Hypoxia-reoxygenation (OGD / R) was performed on SH-SY5Y cells to simulate in vitro brain injury. When the confluence of SH-SY5Y cells exceeded 80%, the original medium was aspirated, and FBS-free, sugar-free medium was added. The cells were then cultured in an anaerobic incubator containing 95% N2 and 5% H2 at 37°C for 3.5 hours (OGD). Afterward, the cells were transferred from the anaerobic incubator to a normal environment and reperfused with normal medium instead of the FBS-free medium (R), and then cultured in a normal incubator for another 12 hours. The control group was treated as follows: when the SH-SY5Y cell confluence was greater than 80%, the original culture medium was aspirated, FBS-free culture medium was added, and then the cells were placed in a normal incubator for culture.

[0132] 3. Cell proliferation activity and lactate dehydrogenase (LDH) detection

[0133] (1) Cell proliferation activity assay (CCK-8)

[0134] Cells in the logarithmic growth phase were harvested at a concentration of 1.2 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 5% CO2 and 37°C until 80% confluence. Then, FBS-free medium containing compounds B4, A5, NBP, and TMP (all at 6.25 μM) was added and incubated for 4 h, followed by OGD / R treatment. After treatment, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 1 h. The plates were then shaken for 5 s, and the absorbance (OD) at 450 nm was measured using a microplate reader. The OD value of each test well was subtracted from the background OD value (serum-free medium with CCK-8, cell-free), and the average ± SD was calculated based on the OD values ​​of each well. Cell viability % = (OD value of treated cells - background OD value) / (OD value of control cells - background OD value) × 100%.

[0135] In this test, compound A5, which also contains a butylphthalide structure, was added as a control; the structural formula of A5 is as follows:

[0136]

[0137] (2) Lactate dehydrogenase (LDH) detection

[0138] After the OGD / R treatment of cells, aspirate the cell culture supernatant from each well, mix thoroughly according to the LDH kit instructions, incubate at room temperature for 5 minutes, and measure the absorbance at a wavelength of 450 nm using a microplate reader.

[0139] 4. Detection of apoptosis and necrosis

[0140] After OGD / R treatment of cells, the original culture medium was aspirated, and cell staining buffer, Hoechst staining solution, and PI staining solution were added at a ratio of 1:1:200. The mixture was thoroughly mixed and incubated at 4°C for 30 minutes. The cells were washed once with PBS and observed and photographed under a fluorescence microscope.

[0141] 5. Detection of reactive oxygen species in cells

[0142] After the OGD / R treatment of cells was completed, the original culture medium was aspirated, and culture medium containing 10 uM DCFH-DA was added to each well. The cells were incubated at 37°C for 60 mins, washed three times with PBS, and photographed under a fluorescence microscope.

[0143] 6. Detection of SOD and MDA in cells

[0144] After OGD / R cell treatment, the original culture medium was aspirated, and 0.25% trypsin was added for digestion at room temperature for 2 minutes. Culture medium was added to stop the digestion. The cells were gently pipetted to remove all liquid and transferred to an EP tube. The tube was then centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected. 1 mL of PBS was added, and the mixture was thoroughly mixed. The cells were centrifuged again at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was used for further processing. PBS was added to the cell pellet, and the cells were sonicated at 300W in an ice-water bath, repeating every 3-5 seconds for 4 cycles. Protein concentration was detected using a protein quantification (BCA) kit. Reagents and samples were added sequentially according to the kit instructions, and the results were analyzed using a microplate reader.

[0145] 7. Detection of Nrf2 nuclear translocation in cells

[0146] After OGD / R cell treatment, the original culture medium was aspirated, and 4% paraformaldehyde was added to each well for fixation for 15 min. The cells were then aspirated and washed three times with PBS. 0.3% Triton X-100 was added for cell permeabilization for 10 min, followed by aspiration and washing three times with PBS. 10% goat serum was added and the cells were incubated at 37°C for 30 min. The cells were then aspirated, and Nrf2 antibody (1:200 dilution) was added to each well, and the cells were incubated overnight at 4°C. The cells were then aspirated and washed three times with PBS. Cy3 Goat Anti-Rabbit IgG (H+L) (1:500 dilution) was added, and the cells were incubated at room temperature for 1 h. The cells were then aspirated, washed three times with PBS, and an anti-fluorescence quencher (containing DAPI) was added. The cells were then photographed under a fluorescence microscope.

[0147] 8. Data Processing

[0148] Data processing was performed using SPSS 16.0 statistical software. Experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was conducted using one-way ANOVA. Pairwise comparisons between experimental groups were performed using t-tests, and P < 0.05 was considered statistically significant.

[0149] 9. Experimental Results

[0150] CCK8 test results are as follows Figure 1 As shown in Figures A and B, both B4 and NBP have significant protective effects in the SH-SY5Y cell OGD / R model. Cellular LDH release is as follows... Figure 1 As shown in Figure C, both B4 and NBP significantly reduced LDH release in the SH-SY5Y cell OGD / R model.

[0151] The comparison results of CCK8 measurement are as follows: Figure 2As shown, B4, A5, NBP, and TMP all exhibit significant protective effects in the SH-SY5Y cell OGD / R model. Compared to A5, NBP, and TMP, B4 demonstrates superior cell-protective efficacy.

[0152] Cellular apoptosis and necrosis levels and cellular reactive oxygen species levels, such as Figure 3 As shown, compared with the Control group, the OGD / R group had significantly higher levels of apoptosis and reactive oxygen species, which decreased after administration of B4 and NBP.

[0153] Cellular SOD and MDA levels, such as Figure 4 As shown in Figures A and B, compared with the Control group, the OGD / R group showed significantly lower SOD levels and significantly higher MDA levels, and achieved remission after treatment with B4 and NBP.

[0154] Cellular Nrf2 nuclear translocation status as follows Figure 4 As shown in C and D, compared with the Control group, the OGD / R group showed increased Nrf2 nuclear translocation, and the administration of B4 and NBP further increased Nrf2 nuclear translocation.

[0155] This indicates that compound B4 can protect neuronal cell models induced by hypoxia-reoxygenation.

[0156] Conclusion: Compound B4 has a significant protective effect on hypoxia-reoxygenation induced neuronal cell models.

[0157] Example 2: Protective effect of B4 on a rat model of ischemic stroke

[0158] 1. Laboratory animals and drug preparation

[0159] SD rats were purchased from Beijing Vital River Laboratory Technology Co., Ltd. and housed in the animal barrier system of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. The environment was SPF grade, with constant temperature (24±2℃), constant humidity (40%-60%), 12 / 12h day and night lighting, and sufficient water and food, as well as a dry environment.

[0160] Preparation of B4: Weigh a certain amount of B4 oily liquid, prepare it with sodium carboxymethyl cellulose (CMC) solution, and sonicate until fully mixed; Preparation of NBP: Weigh a certain amount of butylphthalide oily liquid, prepare it with sodium carboxymethyl cellulose (CMC) solution, and sonicate until fully mixed.

[0161] 2. Establishment of a rat model of ischemic stroke (MCAO / R)

[0162] A rat model of focal cerebral ischemia / reperfusion injury was established using middle cerebral artery embolization. Before the experiment, the nylon suture was cleaned with 75% alcohol, marked 18.5 mm from the bulbar end, and placed in 0.9% sterile saline for later use. The rats were anesthetized with ketamine (80 mg / kg) and toluidine (10 mg / kg) and placed supine on the operating table. A midline incision was made in the neck, and the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (a branch of the ECA) were sequentially dissected. The distal end of the ECA was ligated to the centripetal end of the common carotid artery (CCA). The CCA and ICA were clamped with arterial clamps. A 2 / 3 diameter incision was made in the ECA, and the prepared suture was inserted into the ICA until the blood flow to the ICA was blocked. The suture was then gently ligated. The arterial clamps on the ICA were released, and the nylon suture was inserted into the ICA to a depth of 18.5 ± 0.5 mm to the origin of the anterior cerebral artery (ACA), blocking the blood supply to the middle cerebral artery (MCA). Two hours after MCA occlusion, the nylon suture was removed from the ACA and withdrawn into the ECA to restore normal function, followed by reperfusion for 24 hours.

[0163] 3. Grouping and administration of experimental animals

[0164] Healthy, qualified SPF-grade SD rats weighing 280-330g were selected and randomly divided into 5 groups (n=6 per group): sham-operated group, MCAO / R model group, low-dose B5 administration group (5mg / kg) + MCAO / R, medium-dose B5 administration group (10mg / kg) + MCAO / R, high-dose B5 administration group (20mg / kg) + MCAO / R, and NBP control group (60mg / kg) + MCAO / R. All groups were administered the drugs by gavage for 7 consecutive days. The sham-operated group and the model group were given an equal volume of CMC solution by gavage daily.

[0165] 4. Weight and behavioral scores

[0166] Neurobehavioral assessments and body weight were performed on animals undergoing MCAO / R surgery at 1, 3, 5, and 7 days post-surgery (n = 6 / group / time point). In this experiment, the modified neurological severity score (mNSS) was used to assess the neurological function of rats at different time points according to the internationally recognized MCAO / R postoperative rat neurological deficit scoring system. The total score was 18 points, with higher scores indicating more severe neurological damage.

[0167] 5. Laser Doppler ultrasound for cerebral blood flow detection

[0168] The anesthetized rat was placed flat on a tray. The hair and skin on the top of the rat's head were disinfected with alcohol. The skin on the top of the rat's head was cut open with surgical scissors. The remaining hair at the cut site was cleaned with a cotton ball soaked in physiological saline. The top of the rat's head was then photographed under the laser Doppler blood flow camera.

[0169] 6. TTC staining

[0170] The brain is rapidly harvested within 20 minutes of anesthesia, maintaining its integrity. It is then flash-frozen at -20°C for approximately 10 minutes for easy slicing. The brain is then placed in a specialized brain trough (to accurately calculate the infarct area), typically sliced ​​into 5-6 sections, spaced 2 mm apart. The sections are placed in 2% TTC solution, covered with aluminum foil, and incubated at 37°C for 15 minutes, occasionally turning to ensure even contact with the staining solution. After staining, normal tissue appears rose-red, while infarcted tissue remains white. The brain slices are then fixed in 4% paraformaldehyde for 24 hours, protected from light. The fixed brain slices are photographed on a light-colored operating table using a digital camera. The ischemic area volume ratio is calculated using the formula: (sum of white ischemic area areas in each slice) / (sum of brain slice areas in each slice) × 100%.

[0171] 7. Data Processing

[0172] Data processing was performed using SPSS 16.0 statistical software. Experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was conducted using one-way ANOVA. Pairwise comparisons between experimental groups were performed using t-tests, and P < 0.05 was considered statistically significant.

[0173] 8. Experimental Results

[0174] Changes in rat body weight and behavior, such as Figure 5 As shown in Figures A and B, compared with the MCAO / R group, the high-dose group B4 (20 mg / kg) showed a significant increase in body weight and a decrease in behavioral scores.

[0175] The area of ​​cerebral infarction in rats after 7 days of drug administration is as follows Figure 5 As shown in C and D, compared with the sham surgery group, the MCAO / R group had a significantly increased cerebral infarction area, while the cerebral infarction area of ​​rats was significantly reduced after treatment with B4 and NBP.

[0176] Cerebral blood flow in rats 7 days after administration was as follows Figure 5 As shown in Figures E and F, compared with the sham group, the MCAO / R group had significantly reduced cerebral blood flow, while the rats' cerebral blood flow increased significantly after treatment with B4 and NBP.

[0177] Conclusion: Compound B4 can improve the infarct area and increase cerebral blood flow in rats, and improve cerebral ischemia-reperfusion injury in rats.

[0178] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a triazole compound in the preparation of drugs for the prevention and treatment of ischemic stroke, characterized in that: The molecular formula of the triazole compound is C 29 H 39 N7O4, chemical structural formula as shown in formula (Ⅰ): 。 2. The application according to claim 1, characterized in that: The preparation method of the triazole compound includes the following steps: (1) Butylphthalide was hydrolyzed with alkali, pH was adjusted, the mixture was concentrated, and chloroacetyl chloride, 4-dimethylaminopyridine and triethylamine were added to react and intermediate 1 was obtained. (2) Dissolve intermediate 1 obtained in step (1) with propargylamine, add O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid and N,N-diisopropylethylamine to react and obtain intermediate 2; (3) Ligustrazine was brominated with N-bromosuccinic anhydride using benzoyl peroxide as an initiator to obtain intermediate 3; (4) Dissolve intermediate 3 obtained in step (3), and then add sodium azide to react and obtain intermediate 4; (5) Dissolve intermediate 2 obtained in step (2) and intermediate 4 obtained in step (4), add catalyst to react and obtain intermediate 5; (6) The intermediate 5 obtained in step (5) undergoes a substitution reaction with morpholine under the action of a catalyst to obtain the triazole compound.

3. The application according to claim 2, characterized in that: The alkali used in step (1) is selected from at least one of sodium hydroxide, potassium hydroxide, and calcium hydroxide; the hydrolysis conditions in step (1) are: incubation at 55-65℃ for 1.5-3 hours, or microwave treatment at 55-65℃ for 30-45 minutes; the concentration method in step (1) is extraction with an extractant; the extractant is selected from one or both of ethyl acetate and diethyl ether; the chloroacetyl chloride, 4-dimethylaminopyridine, and triethylamine in step (1) are combined with butanediol... The molar ratio of phthalide is 1.4-1.6:0.08-0.12:1.4-1.6:1; the solvent used for dissolution in step (2) includes carbon dichloride; the molar ratio of propargylamine to intermediate 1 in step (2) is 1.1-1.3:1; the molar ratios of O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid, N,N-diisopropylethylamine to intermediate 1 in step (2) are as follows: 1.1-1.3:1.8-2.2:1; The reaction temperature in step (2) is 20-30℃ and the time is 5.5-7h.

4. The application according to claim 2, characterized in that: The molar ratio of benzoyl peroxide to tetramethylpyrazine in step (3) is 0.08-0.12:1; the molar ratio of N-bromosuccinic anhydride to tetramethylpyrazine in step (3) is 0.25-0.35:1; the bromination reaction in step (3) is carried out by reflux; the solvent used for dissolution in step (4) includes acetonitrile; the molar ratio of sodium azide to intermediate 3 in step (4) is 1.15-1.25:1; the reaction conditions in step (4) are microwaved at 55-65℃ for 55-70 min.

5. The application according to claim 2, characterized in that: The molar ratio of intermediate 2 to intermediate 4 in step (5) is 1:0.9-1.1; the solvent used for dissolution in step (5) includes dichloromethane; the catalyst in step (5) includes cuprous thiophene-2-carboxylate (I); the reaction temperature in step (5) is 20-30℃; the catalyst in step (6) includes cesium carbonate; the molar ratio of cesium carbonate to intermediate 5 is 1.4-1.6:1; the molar ratio of morpholine to intermediate 5 in step (6) is 1.4-1.6:1; the substitution reaction conditions in step (6) are: reaction at 60-70℃ for 100-140 min; or microwave at 60-70℃ for 50-65 min.

6. The use of a triazole compound in the preparation of a drug for the prevention and treatment of cerebral ischemia-reperfusion injury; The molecular formula of the triazole compound is C 29 H 39 N7O4, chemical structural formula as shown in formula (Ⅰ): 。 7. The use of a pharmaceutical composition containing an active ingredient in the preparation of a drug for the prevention and treatment of ischemic stroke, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients; The active ingredient includes triazole compounds; the molecular formula of the triazole compounds is C2. 29 H 39 N7O4, chemical structural formula as shown in formula (Ⅰ): 。 8. The application according to claim 7, characterized in that, The excipients are selected from at least one of the following groups: binders, fillers, disintegrants, suspending agents, suspending aids, sustained-release agents, controlled-release agents, lyophilization protectants, coating agents, enteric coatings, lubricants, flow aids, anti-adhesion agents, sweeteners, flavoring agents, plasticizers, opacifiers, solubilizers, humectants, solvents, osmotic pressure regulators, colorants, surfactants, emulsifiers, water-soluble matrices, fat-soluble matrices, oleic matrices, pore-forming agents, gelling agents, preservatives, buffers, chelating agents, and antioxidants.

9. The application according to claim 7, wherein the pharmaceutical composition further comprises other active components that have the effect of increasing cerebral blood flow or reducing cerebral ischemia-reperfusion injury.

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