A precursor compound based on edaravone, preparation method and application

Abstract

The present invention belongs to the technical field of pharmaceutical synthesis, and specifically relates to a precursor compound based on edaravone, a preparation method and an application. The precursor compound based on edaravone of the present invention modifies the exposed hydroxyl group at the 3-position of edaravone through an esterification reaction to form an ester prodrug with a certain stability. After oral administration, the drug can be rapidly metabolized into the original drug in vivo to exert the drug efficacy, which is significantly higher than the effect of the original drug edaravone, can change the administration mode of edaravone, and is suitable for oral administration. Moreover, the prodrug of the present invention can effectively increase the blood drug concentration of the drug after the action of edaravone, achieve the purpose of maintaining the treatment time, and has obvious clinical advantages.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug synthesis, and particularly relates to a precursor compound based on edaravone, a preparation method and an application thereof. Background Art

[0002] Edaravone (Edaravone, MCI-186, PMP), whose chemical name is 3-methyl-1-phenyl-2-pyrazolin-5-one, and the trade name is Bicun, is developed and produced by Mitsubishi in Japan. The chemical structural formula is as follows:

[0003]

[0004] Edaravone is mainly used in clinical treatment of ischemic stroke (IS), amyotrophic lateral sclerosis (ALS), subarachnoid hemorrhage and other diseases. It exerts a protective effect by scavenging free radicals, inhibiting lipid peroxidation, and inhibiting oxidative damage of brain cells, vascular endothelial cells and nerve cells. At present, edaravone has become the first choice drug for brain protection after stroke. However, edaravone has poor gastrointestinal absorption and a fast metabolism rate, making it difficult to reach an effective blood drug concentration, resulting in poor therapeutic effects and ultimately limiting its clinical application.

[0005] At the present stage, the main clinical administration method for treating ischemic stroke with edaravone is intravenous injection, usually lasting for 10-14 days of continuous intravenous injection to consolidate the therapeutic effect on stroke. However, long-term intravenous injection poses great challenges to the economic foundation and physical and mental pressure of patients. At the same time, since edaravone is extremely difficult to dissolve or almost insoluble in water and is prone to oxidation and deterioration, drug excipients such as solubilizer propylene glycol and antioxidant sodium metabisulfite need to be added during the preparation of the preparation, reducing the drug safety.

[0006] Compared with the intravenous injection method, oral administration is more convenient, safe, economical and applicable, and has good compliance, which is the preferred administration method for patients. However, edaravone has a low oral bioavailability and a fast metabolism rate, making it difficult to maintain an effective blood drug concentration and having a poor therapeutic effect on stroke, greatly hindering its clinical use. Existing studies have found that when rats are directly orally gavaged with 30 mg / kg edaravone, it is equivalent to the efficacy of intravenous injection of 1.5 mg / kg, suggesting that the oral bioavailability of edaravone is extremely low and it is not suitable for direct oral administration. Therefore, many pharmaceutical researchers have tried to change the drug dosage form, synthesize prodrugs, etc. to improve the oral bioavailability of edaravone so as to achieve the purpose of changing the administration method of edaravone.

[0007] Patent application CN104257603A discloses an edaravone / cyclodextrin inclusion complex, aiming to achieve oral drug delivery by changing the formulation type. Compared with the edaravone CMC-Na suspension, the relative bioavailability of the obtained inclusion complex after inclusion is 9.8 - 11 times that of the CMC-Na suspension, which can significantly improve the oral bioavailability of edaravone, but it cannot maintain the blood drug concentration. In addition, medicinal chemists have tried to improve the gastrointestinal stability of edaravone by modifying its structure to achieve oral drug delivery. Patent application CN102190622A discloses a carbamate edaravone precursor compound that can be administered orally. The results of its pharmacodynamics in rats show that there is no significant difference in the volume of cerebral infarction and the water content of brain tissue in rats between orally gavaging the edaravone derivative and intravenously injecting an equal amount of edaravone, indicating that the oral bioavailability of this prodrug is not significantly increased compared with directly administering edaravone. The edaravone precursor compound disclosed in patent application CN112714760A can adopt the oral drug delivery method. Its pharmacokinetic results show that its bioavailability is as high as 88.20%. Compared with directly orally administering edaravone (bioavailability is 5.23%), the oral bioavailability is significantly improved, but its maximum blood drug concentration is relatively low, only 1.70 - 1.80 μg / mL.

[0008] Based on the above research status, it can be seen that how to further modify the structure of edaravone to obtain a new compound that can not only effectively increase the oral bioavailability of edaravone, but also increase the blood drug concentration of edaravone, significantly improve its efficacy, and is suitable for oral administration for clinical selection has become an urgent technical problem to be solved. Summary of the Invention

[0009] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a precursor compound based on edaravone. This prodrug can not only significantly improve the oral bioavailability of the original drug edaravone, but also significantly increase the blood drug concentration and improve the efficacy.

[0010] The second object of the present invention is to provide a preparation method of a precursor compound based on edaravone, which has a simple process, mild conditions, and is easy to realize the preparation of the prodrug.

[0011] The third object of the present invention is to provide an application of a precursor compound based on edaravone.

[0012] One of the objects of the present invention is achieved by adopting the following technical solution:

[0013] A precursor compound based on edaravone is a compound shown in Formula I or Formula II:

[0014]

[0015] Among them, in formula I, R1 is an alkyl group having 1 to 10 carbon atoms, an aryl group or a pyridyl group;

[0016] In formula II, R2 is an alkyl group having 1 to 10 carbon atoms or an aryl group.

[0017] To improve the oral bioavailability and blood drug concentration of the drug, preferably, in formula I, R1 is ethyl, isopropyl, tert-butyl, phenyl or pyridyl; in formula II, R2 is propyl, isopropyl, tert-butyl or phenyl.

[0018] More preferably, the edaravone-based prodrug compound is selected from the compounds having the following structures:

[0019]

[0020] The second object of the present invention is achieved by the following technical solution:

[0021] In the preparation method of the above-mentioned edaravone-based prodrug compound, the preparation method of the edaravone-based prodrug compound shown in formula I includes the following steps: reacting edaravone, an acid substance and a condensing agent in a solvent, and then purifying to obtain the edaravone-based prodrug compound shown in formula I;

[0022] The preparation method of the edaravone-based prodrug compound shown in formula II includes the following steps: reacting edaravone, triethylamine and a carboxyl-containing compound in a solvent, and then purifying to obtain the edaravone-based prodrug compound shown in formula II.

[0023] The preparation method of the edaravone-based prodrug compound provided by the present invention modifies the hydroxyl group exposed at the 3-position of edaravone through an esterification reaction to form an ester prodrug with certain stability. After oral administration, the drug can be rapidly metabolized into the original drug in the body to exert its effect, which is significantly higher than the effect of the original drug edaravone, can change the administration method of edaravone, and has obvious clinical advantages. Moreover, the preparation method of the present invention has mild conditions and is easy to operate, and is suitable for industrial preparation of compounds.

[0024] Preferably, the acid substance is one of propionic acid, isobutyric acid, pivalic acid, nicotinic acid; the condensing agent is N,N'-diisopropylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the carboxyl-containing compound is one of n-propyl chloroformate, isopropyl chloroformate, di-tert-butyl dicarbonate.

[0025] More preferably, when preparing the compounds shown in formula I and formula II, the solvent is selected from one of dichloromethane, acetonitrile, acetone, tetrahydrofuran, chloroform.

[0026] Preferably, when preparing the compounds shown in Formula I and Formula II, the reaction is carried out at room temperature for 0.5 to 12 h.

[0027] More preferably, the molar ratio of edaravone, acid substance, and condensing agent during the reaction is 2.9∶(5.74 - 11.5)∶(4.2 - 11.2); the molar ratio of edaravone, triethylamine, and carboxyl group-containing compound during the reaction is (1.7 - 2.9)∶(5.16 - 14.4)∶(5.16 - 14.4).

[0028] The third object of the present invention is achieved by the following technical solution:

[0029] The application of the precursor compound based on edaravone is specifically the application in the preparation of therapeutic drugs for ischemic stroke, Alzheimer's disease, or amyotrophic lateral sclerosis.

[0030] Furthermore, the therapeutic drug is an oral drug; the dosage form of the oral drug is one or more of tablets, soft capsules, hard capsules, solutions, and suspensions.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] The precursor compound based on edaravone provided by the present invention can be rapidly metabolized into the original drug in vivo after oral administration to exert the drug efficacy through structural modification, and the effect is significantly higher than that of the original drug edaravone, which can change the administration mode of edaravone and is suitable for oral administration. Moreover, the prodrug of the present invention can effectively improve the oral bioavailability of edaravone and increase the drug blood concentration to achieve the purpose of maintaining the treatment time, with obvious clinical advantages.

[0033] Therefore, the prodrug provided by the present invention can be used as an active ingredient in the preparation of therapeutic drugs for ischemic stroke, Alzheimer's disease, and amyotrophic lateral sclerosis, and can effectively exert the therapeutic efficacy, with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the TTC staining results and the cerebral infarction area test results of the prodrugs based on the structure of edaravone, namely Compound 2 and Compound 4, in Example 2 and Example 4 of the present invention; wherein Figure A is the TTC staining result, and Figure B is the cerebral infarction area result;

[0035] Figure 2 It is the test results of the prodrugs based on the structure of edaravone, namely Compound 2 and Compound 4, in Example 2 and Example 4 of the present invention on the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA); wherein Figure A is the influence diagram of SOD activity; Figure B is the influence diagram of GSH-Px activity; Figure C is the influence diagram of MDA activity;

[0036] Figure 3 These are the HE staining result diagrams of the edaravone-structure-based prodrugs, namely Compound 2 and Compound 4, of Embodiments 2 and 4 of the present invention.

[0037] Figure 4 These are the fluorescence quantitative result diagrams of ROS tests of the edaravone-structure-based prodrugs, namely Compound 2 and Compound 4, of Embodiments 2 and 4 of the present invention. Specific Embodiments

[0038] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only further clarifications of the present invention and not limitations thereof. Unless otherwise specified, the reagents used in the following embodiments can be obtained from commercial channels.

[0039] Embodiment 1

[0040] The edaravone-structure-based prodrug of this embodiment has the following structural formula, denoted as Compound 1:

[0041]

[0042] The preparation method of Compound 1 includes the following steps: Dissolve edaravone (0.51 g, 2.90 mmol) and propionic acid (0.85 g, 11.20 mmol) in 10 mL of dichloromethane (DCM), then add N,N'-diisopropylcarbodiimide (DIC, 1.45 g, 11.20 mmol), and stir the reaction at room temperature overnight, monitoring the reaction by thin-layer chromatography. Then evaporate DCM under reduced pressure, and purify the product by column chromatography with petroleum ether∶ethyl acetate = 10∶1. Spin-dry the product, redissolve it in n-hexane, and spin-dry it at 50 °C to obtain Compound 1, with a yield of 67.52%.

[0043] The structural characterization results of Compound 1 are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.57–7.49 (m, 2H), 7.47–

[0044] 7.38 (m, 2H), 7.36–7.27 (m, 1H), 6.09 (s, 1H), 2.54 (q, J = 7.5 Hz, 2H), 2.32 (s, 3H), 1.19 (t, J = 7.5 Hz, 3H). 1313C NMR (101 MHz, CDCl3) δ 169.74, 148.95, 144.45, 138.10, 129.04, 128.85, 127.11, 125.07, 123.12, 118.90, 95.80, 77.37, 77.05, 76.73, 43.13, 27.54, 17.05, 14.50, 8.76, 0.01. Among them, 77.37, 77.05, 76.73 are the chemical shifts of C in CDCl3.

[0045] Example 2

[0046] The prodrug based on the structure of edaravone in this example has the following structural formula, denoted as Compound 2:

[0047]

[0048] The preparation method of Compound 2 includes the following steps: Dissolve edaravone (0.51 g, 2.90 mmol) and isobutyric acid (1.01 g, 11.50 mmol) in 25 mL of DCM, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.83 g, 4.20 mmol) and triethylamine (0.29 g, 2.80 mmol), stir at room temperature overnight, and monitor the reaction by thin-layer chromatography. Evaporate DCM under reduced pressure, purify by column chromatography with petroleum ether∶ethyl acetate = 10∶1 to obtain the product. Spin-dry the product, purify it by column chromatography with DCM∶n-hexane = 10∶1 for the second time, and spin-dry at 50 °C to obtain Compound 2 with a yield of 89.16%.

[0049] The structural characterization results of Compound 2 are as follows: 1 1H NMR (400 MHz, CDCl3) δ 7.56–7.48 (m, 2H), 7.47–

[0050] 7.38 (m, 2H), 7.36–7.27 (m, 1H), 6.08 (s, 1H), 2.79–2.62 (m, 1H), 2.33 (s, 3H), 1.20 (dd, J =

[0051] 10.7, 7.0 Hz, 8H). 13 13C NMR (101 MHz, CDCl3) δ 172.35, 148.94, 144.60, 138.04, 128.97, 127.20, 123.39, 95.72, 77.38, 77.06, 76.74, 34.07, 18.64, 14.51. Among them, 77.38, 77.06, 76.74 are the chemical shifts of C in CDCl3.

[0052] Example 3

[0053] The prodrug based on the structure of edaravone in this example has the following structural formula, denoted as Compound 3:

[0054]

[0055] The preparation method of Compound 3 includes the following steps: Dissolve edaravone (0.51 g, 2.90 mmol) and pivalic acid (1.17 g, 11.20 mmol) in 10 mL of DCM, add N,N'-diisopropylcarbodiimide (DIC, 1.45 g, 11.20 mmol), stir and react at room temperature overnight, and monitor the reaction by thin layer chromatography. Evaporate DCM under reduced pressure, purify the product by column chromatography with petroleum ether∶ethyl acetate = 15∶1, spin-dry the product, purify it by column chromatography for the second time with DCM∶n-hexane = 20∶1, and spin-dry at 50 °C to obtain Compound 3 with a yield of 87.47%.

[0056] The structural characterization results of Compound 3 are as follows: 1 H NMR(400MHz,CDCl3)δ7.55–7.47(m,2H),7.47–

[0057] 7.38(m,2H),7.36–7.27(m,1H),6.08(s,1H),2.32(s,3H),1.25(s,9H). 13 C NMR(101MHz,CDCl3)δ173.83,148.94,144.81,137.97,128.91,128.85,127.28,125.06,123.61,118.90,95.59,77.37,77.05,76.74,43.13,39.21,26.92,17.05,14.51. Among them, 77.37, 77.05, and 76.74 are the chemical shifts of C in CDCl3.

[0058] Example 4

[0059] The prodrug based on the structure of edaravone in this example has the following structural formula, denoted as Compound 4:

[0060]

[0061] The preparation method of Compound 4 includes the following steps: Dissolve edaravone (0.51 g, 2.90 mmol) and nicotinic acid

[0062] (0.71 g, 5.74 mmol) was dissolved in 40 mL of acetone. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.10 g, 5.74 mmol) and triethylamine (0.44 g, 2.80 mmol) were added. The reaction was stirred at room temperature overnight, and the reaction was monitored by thin layer chromatography. Acetone was removed by evaporation under reduced pressure, redissolved in DCM, an equal volume of n-hexane was added, and it was placed in a refrigerator at 4 °C overnight to precipitate dicyclohexylurea (DCU). It was filtered, and purified by column chromatography with DCM:acetone = 80:1 to obtain compound 4 with a yield of 44.31%.

[0063] The structural characterization results of compound 4 were as follows: 1 H NMR (400 MHz, CDCl3) δ 9.27 (dd, J = 2.3, 0.8 Hz, 1H), 8.85 (dd, J = 4.9, 1.7 Hz, 1H), 8.33 (dt, J = 8.0, 2.0 Hz, 1H), 7.62–7.54 (m, 2H), 7.49–7.40 (m, 3H), 7.40–7.29 (m, 1H), 6.31 (s, 1H), 2.37 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 160.62, 154.57, 151.38, 149.19, 143.93, 137.95, 137.81, 129.24, 128.85, 127.50, 125.06, 124.27, 123.72, 123.39, 118.90, 95.84, 77.38, 77.06, 76.74, 43.13, 29.71, 17.05, 14.55, 0.01. Among them, 77.38, 77.06, 76.74 are the chemical shifts of C in CDCl3.

[0064] Example 5

[0065] The prodrug based on the structure of edaravone in this example has the following structural formula, denoted as compound 5:

[0066]

[0067] The preparation method of compound 5 includes the following steps: Edaravone (0.31 g, 1.70 mmol) and triethylamine (0.52 g, 5.16 mmol) were dissolved in 25 mL of DCM. n-Propyl chloroformate (0.63 g, 5.16 mmol) was added, and the reaction was stirred at room temperature overnight, and the reaction was monitored by thin layer chromatography. DCM was removed by evaporation under reduced pressure and purified by column chromatography with petroleum ether:ethyl acetate = 20:1 to obtain compound 5 with a yield of 89.23%.

[0068] The structural characterization results of compound 5 were as follows: 11H NMR (400 MHz, CDCl3) δ 7.60–7.52 (m, 2H), 7.43 (t, J = 7.9 Hz, 2H), 7.35–7.27 (m, 1H), 6.09 (s, 1H), 4.18 (t, J = 6.7 Hz, 2H), 2.32 (s, 3H), 1.71 (p, J = 7.2 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H). 13 13C NMR (101 MHz, CDCl3) (101 MHz, CDCl3) δ 151.04, 148.90, 144.72, 137.94, 129.13, 127.14, 122.89, 95.42, 77.37, 77.05, 76.73, 71.38, 21.81, 14.53, 10.01. Among them, 77.37, 77.05, and 76.73 are the chemical shifts of C in CDCl3.

[0069] Example 6

[0070] The prodrug based on the structure of edaravone in this example has the following structural formula, denoted as Compound 6:

[0071]

[0072] The preparation method of Compound 6 includes the following steps: Dissolve edaravone (0.51 g, 2.90 mmol) and triethylamine (1.45 g, 14.4 mmol) in 25 mL of DCM, add isopropyl chloroformate (1.76 g, 14.4 mmol), stir and react at room temperature for 0.5 h, and monitor the reaction by thin layer chromatography. Evaporate DCM under reduced pressure and purify by column chromatography with petroleum ether∶ethyl acetate = 15∶1 to obtain Compound 6.

[0073] The structural characterization results of Compound 6 are as follows: 1 1H NMR (400 MHz, CDCl3) δ 7.60–7.52 (m, 2H), 7.48–

[0074] 7.38 (m, 2H), 7.37–7.26 (m, 1H), 6.08 (s, 1H), 4.91 (hept, J = 6.3 Hz, 1H), 2.32 (s, 3H), 1.29 (d, J = 6.3 Hz, 6H). 1313C NMR (101 MHz, CDCl3) δ 150.38, 148.87, 144.80, 138.00, 129.12, 127.11, 122.89, 95.42, 77.38, 77.06, 76.74, 74.54, 21.50, 14.53, 0.01. Among them, 77.38, 77.06, 76.74 are the chemical shifts of C in CDCl3, and the yield is 67.25%.

[0075] Example 7

[0076] The prodrug based on the edaravone structure in this example has the following structural formula, denoted as Compound 7:

[0077]

[0078] The preparation method of Compound 7 includes the following steps: Dissolve edaravone (0.51 g, 2.90 mmol) and triethylamine (0.85 g, 8.41 mmol) in 25 mL of DCM, add di-tert-butyl dicarbonate (1.83 g, 8.41 mmol), stir and react at room temperature for 0.5 h, and monitor the reaction by thin-layer chromatography. Evaporate DCM under reduced pressure, and purify by column chromatography with petroleum ether∶ethyl acetate = 15∶1 to obtain Compound 7, with a yield of 74.48%.

[0079] The structural characterization results of Compound 7 are as follows: 1 1H NMR (400 MHz, CDCl3) δ 7.60–7.51 (m, 2H), 7.49–7.38 (m, 2H), 7.35–7.26 (m, 1H), 6.05 (s, 1H), 2.32 (s, 3H), 1.45 (s, 9H). 13 13C NMR (101 MHz, CDCl3) δ 148.95, 148.84, 144.93, 138.09, 129.24, 129.08, 127.04, 122.91, 95.52, 85.24, 77.36, 77.05, 76.73, 28.41, 27.42, 14.52, 0.01. Among them, 77.36, 77.05, 76.73 are the chemical shifts of C in CDCl3.

[0080] Test Example 1

[0081] This test example is the pharmacokinetic study in vivo of the prodrugs (Compounds 1–7) based on the edaravone structure prepared in Examples 1–7.

[0082] The experimental method was as follows: Male SD rats of SPF grade, weighing 250±5 g, were fasted for 12 h before the experiment and allowed free access to water. The experiment was randomly divided into 8 groups, namely the edaravone (PMP) intravenous injection group (i.v.), the PMP oral group (p.o.), the compound 1 oral group, the compound 2 oral group, the compound 3 oral group, the compound 4 oral group, the compound 5 oral group, the compound 6 oral group, and the compound 7 oral group. The prodrugs were administered at an equimolar dose with PMP (16 mg / kg). Approximately 0.5 mL of blood was collected from the orbital cavity of the rats at 0.033, 0.083, 0.167, 0.333, 0.5, 0.667, 1, 2, 4, and 8 h after administration. 10 μL of 50% phosphoric acid aqueous solution was added to acidify the blood containing the drug, and the mixture was centrifuged at 4000 rpm for 10 min at 4°C. 100 μL of plasma was taken to precipitate proteins. The drug concentration in plasma was determined by HPLC to obtain the drug-time curve, and then the pharmacokinetic parameters were obtained according to the curve. The experimental results are shown in Table 1. Among them, i.v. represents the intravenous injection group, p.o. represents the oral group, and compounds 1-7 are represented by Compound 1-7. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences between the experimental groups and the PMP (p.o.) group.

[0083] Table 1 Pharmacokinetic parameters of PMP intravenous injection, oral groups, and oral groups of compounds 1-7 of the present invention

[0084]

[0085] As can be seen from Table 1, the areas under the drug-time curves (AUC 0-t ) of PMP (p.o.), compounds 1 (p.o.) - 7 (p.o.) were 3.40±0.76, 5.60±1.49, 6.82±2.81, 7.71±4.54, 7.02±2.90, 9.12±4.56, 3.13±1.37, and 3.56±2.9 mg / L*h, respectively. Compared with the PMP (p.o.) group, the AUC 0-t of compounds 1-7 increased by 1.65, 2.00, 2.27, 2.06, 2.68, 0.92, and 1.05 times, respectively. The maximum drug concentrations in vivo (C max ) of PMP (p.o.), compounds 1 (p.o.) - 7 (p.o.) were 2.43±1.17, 7.19±4.46, 9.89±4.42, 3.19±1.07, 8.30±2.23, 6.88±2.81, 5.14±3.99, and 2.49±1.52 mg / L, respectively. Compounds 1-7 were 2.96, 4.07, 1.31, 3.41, 2.83, 2.12, and 1.02 times higher than the directly orally administered PMP group. And the absolute oral bioavailability (Fa ) They were increased by 1.65, 2.00, 2.27, 2.06, 2.68, 0.92, and 1.05 times respectively. The above results indicate that compared with the original drug of edaravone, the blood drug concentrations and oral bioavailability of Compounds 1-7 of the present invention have been improved to varying degrees.

[0086] Test Example 2

[0087] Combined with the AUC of Comprehensive Test Example 1 0-t , C max and F a results, Compounds 2 and 4 were mainly selected for pharmacodynamic studies here. This test example is an anti-cerebral ischemia study on oral preparations of prodrug Compounds 2 and 4 based on the structure of edaravone.

[0088] The test method is as follows: A rat model of focal cerebral ischemia-reperfusion was established with reference to the modified Zea-Longa suture method. Sodium pentobarbital at 1%, 45 mg / kg, was intraperitoneally injected for anesthesia, and the dosage was adjusted according to the condition of the animals after anesthesia. The rats were fixed supine in the center of the operating table. The area 0.5 cm below the mandible in the anterior cervical region of the rats was disinfected, and a longitudinal incision was made on the right side of the midline of the neck. The muscle and fascia were separated with micro scissors to expose the common carotid artery (CCA) in the field of vision, and the vagus nerve under the CCA was separated. The external carotid artery (ECA) and internal carotid artery (ICA) were found along the CCA. The CCA and ECA were ligated with auxiliary lines, and the ICA was clamped with a micro artery clip. An incision was made about 10 mm below the ligation part of the CCA, and a suture was inserted. The auxiliary line was slightly tightened, the micro artery clip was removed, and the suture was slowly inserted into the ICA until there was slight resistance. The auxiliary line was tightened, the suture was marked, and the incision was sutured. After 1 h,

[0089] reperfusion was performed. The suture was withdrawn into the CCA, and blood supply to the middle cerebral artery was restored. Among them, the sham operation group did not insert the suture.

[0090] The corresponding drugs were administered by oral gavage. PMP (16 mg / kg) was given to the group with the same neck surgery and vascular treatment as the model group (MCAO group), Compound 2 (22.42 mg / kg, equimolar with PMP) was given to the Compound 2 group, Compound 4 (25.64 mg / kg, equimolar with PMP) was given to the Compound 4 group, and normal saline (equal volume to PMP) was given to the sham operation group. After 24 h of drug treatment, the rats were sacrificed and the brains were taken. The residual blood was rinsed with normal saline, and subsequent experiments were carried out. The whole process was carried out at room temperature (24-25 °C).

[0091] The signs of a successful model were Horner's sign and hemiplegia mainly in the contralateral forelimb after the model animals were anesthetized and awake. Various pathological indexes were measured 24 h after reperfusion in the rats. The TTC method was used to detect the cerebral infarction area, and Image J was used to measure the integral of the cerebral infarction focus in the brain tissue. The results are asFigure 1 As shown

[0092] As can be seen from Figure 1 the cerebral infarction volume in the test groups of compound 2 and compound 4 was 15.31±4.92% and 11.23±2.77% (p<0.01) compared with the sham operation MCAO group and the edaravone PMP oral (p.o.) group, respectively, both of which could significantly reduce the cerebral infarction volume, indicating that both compound 2 and compound 4 have better anti-ischemic effects.

[0093] Test Example 3

[0094] This test example investigated the antioxidant capacity and the curative effect on stroke of the prodrugs compound 2 and compound 4 based on the structure of edaravone.

[0095] Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) are markers of oxidative stress and are often used to evaluate the level of cellular oxidative stress. SOD is an important endogenous antioxidant enzyme that plays an antioxidant role by catalyzing the conversion of superoxide radicals generated by cell mitochondria into hydrogen peroxide and oxygen, reducing the damage of reactive oxygen species to cells. As one of the secondary products of lipid oxidation, MDA can exacerbate tissue oxidative damage, so it is used as an important indicator of lipid peroxidation to evaluate the level of tissue oxidative stress. GSH-Px is an antioxidant enzyme present in mitochondria that inhibits lipid peroxidation, thereby protecting cells from oxidative stress damage.

[0096] During the experiment, after 1 h of reperfusion, the rats were respectively given normal saline (equal volume to PMP), PMP, compound 2, and compound 4 (equal molar amount to PMP) according to the grouping. After 24 h of drug treatment, the rats were sacrificed and the brains were taken. The residual blood was rinsed with normal saline, dried and weighed, and the antioxidant factors were detected according to the corresponding instructions. The results are as Figure 2 shown

[0097] As can be seen from Figure 2 the SOD (Figure A) and GSH-Px (Figure B) activities in the compound 2 (p.o.) group and the compound 4 (p.o.) group were significantly increased, while the MDA (Figure C) activity was significantly decreased compared with the MCAO group, indicating that compound 2 and compound 4 have good antioxidant capacity.

[0098] Test Example 4

[0099] Hematoxylin and eosin (HE) staining and immunofluorescence staining of reactive oxygen species (ROS) in brain tissue can directly reflect the degree of neuronal cell damage and the level of oxidative stress in brain tissue. The results of HE staining and the quantitative analysis of immunofluorescence of ROS in brain tissue are shown respectively as Figure 3 , Figure 4 shown below.

[0100] As can be seen from Figure 3 , in the control group (Control group), brain neurons were clearly visible, with deep cytoplasmic staining, round purple-blue nuclei, normal morphology and close arrangement, and no pathological changes. In the model group (MCAO group), the number of cells decreased significantly, the nuclear staining became lighter, the interstitial space became larger, a large number of neurons died, and the integrity of neurons was relatively low. After treatment with compound 2 or compound 4, the number of pyknosis nuclei in the model rats decreased significantly, the number of normal cells increased, and the interstitial space was basically the same as that in the control group. This indicates that compound 2 and compound 4 can significantly reduce brain tissue damage and have a good therapeutic effect on stroke.

[0101] After staining with 4',6-diamidino-2-phenylindole (DAPI) and dihydroethidium (DHE) (dye names), most of the cell nuclei in the brain tissue of the Control group showed fluorescence, and only a small amount of ROS fluorescence was present in the brain tissue, indicating that the amount of ROS in normal brain tissue was relatively small. Further quantitative study on ROS showed that, as can be seen from Figure 4 , compared with the Control group, a large amount of ROS fluorescence was present in the MCAO group, indicating that severe oxidative damage occurred during ischemia-reperfusion in the MCAO group; the ROS fluorescence level in the PMP group was relatively low, and the ROS fluorescence in the compound 2 and compound 4 groups decreased significantly (p<0.001), indicating that the compounds 2 and 4 provided by the present invention can reduce the ROS generated by ischemia-reperfusion in the brain and improve the damage caused by oxidative stress response in the brain.

[0102] In summary, the edaravone-based prodrug provided by the present invention can be rapidly metabolized into the original drug in vivo after oral administration through structural modification to exert its drug efficacy, and the effect is significantly higher than that of the original drug edaravone. It can change the administration mode of edaravone and is suitable for oral administration. Moreover, the prodrug of the present invention can effectively improve the oral bioavailability of edaravone, increase the blood drug concentration of the drug, so as to achieve the purpose of maintaining the treatment time, and has obvious clinical advantages. Therefore, the prodrug provided by the present invention can be used as an active ingredient for the preparation of therapeutic drugs for ischemic stroke, Alzheimer's disease and amyotrophic lateral sclerosis, and can effectively exert its therapeutic effect, having good application prospects.

Claims

1. Use of a precursor compound based on edaravone, characterized in that, The application is the application of a precursor compound of edaravone in the preparation of a therapeutic drug for ischemic stroke; the therapeutic drug is an oral drug; the structure of the precursor compound based on edaravone is:

2. The application of the edaravone-based precursor compound according to claim 1, wherein, The preparation method of the precursor compound based on edaravone comprises the following steps: reacting edaravone, an acid substance, and a condensing agent in a solvent, and then purifying to obtain the precursor compound based on edaravone.

3. Use according to claim 2 of the precursor compound based on edaravone, characterized in that, The acid substance is one of isobutyric acid and nicotinic acid; the condensing agent is N,N'-diisopropylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

4. The application of the edaravone-based precursor compound according to claim 2, wherein The solvent is selected from one of dichloromethane, acetonitrile, acetone, tetrahydrofuran, and chloroform.

5. The use according to claim 2 of the edaravone-based precursor compound, characterized in that, The reaction is carried out at room temperature for 0.5 to 12 h.

6. The use of the edaravone-based precursor compound according to claim 2, wherein The molar ratio of edaravone, the acid substance, and the condensing agent during the reaction is 2.9∶(5.74~11.5)∶(4.2~11.2).

7. Use according to claim 1 of the prodrug compound based on edaravone, characterized in that, The dosage form of the oral drug is one or more of tablets, soft capsules, hard capsules, solutions, and suspensions.

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