Precursor compound based on edaravone structure, and preparation method therefor and use thereof

Abstract

Disclosed in the present application are a precursor compound based on edaravone structure, or a pharmaceutically acceptable salt thereof, a preparation method therefor and the use thereof. The compound or the pharmaceutically acceptable salt thereof of the present application has relatively good water solubility, and can release the free drug edaravone in the brain to cause the concentration of the free drug edaravone accumulated in the brain being significantly higher than that of bulk drug edaravone, thereby prolonging the residence time of the drug in the brain, and thus improving the action effect of the drug.

Description

A precursor compound based on edaravone structure and its preparation method and application Technical Field

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

[0002] Free radicals, also known in chemistry as "free radicals," are groups of atoms containing an unpaired electron. Since electrons must be paired in chemical bonds when atoms form molecules, free radicals must steal an electron from other substances to form stable substances. In chemistry, this phenomenon is called "oxidation." In biological systems, oxygen free radicals, such as superoxide anion radicals, hydroxyl radicals, and lipid oxygen radicals, are primarily involved. Reactive oxygen free radicals in the body have certain functions, such as immune and signaling processes, and the body's built-in antioxidant system plays a crucial role in preventing damage caused by free radicals. However, an imbalance in the antioxidant system's defense mechanisms, excessive free radical production, or introduction of free radicals from the environment can lead to neuronal damage and degeneration. In addition to several environmental or genetic factors, oxidative stress triggers free radicals to attack nerve cells, and the resulting damage and degeneration, particularly in the central nervous system, can be devastating, as seen in stroke, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Parkinson's disease (PD).

[0003] Cerebral stroke, also known as "stroke," is an acute cerebrovascular disease caused by a sudden rupture of a cerebral blood vessel or a blockage that prevents blood flow to the brain, leading to brain tissue damage. This disease not only has a high mortality rate but is also one of the leading causes of disability among adults in my country. The pathological mechanism leading to stroke sequelae and post-treatment disability is the generation of large amounts of reactive oxygen species and free radicals around the brain lesion during reperfusion after cerebral ischemia. These free radicals can irreversibly damage nerve cells and brain cells through cell membrane damage, protein damage, nucleic acid / DNA damage, and the induction of various inflammatory mediators, ultimately leading to nerve or brain cell death. Therefore, clinical treatment for stroke using only intravenous or thrombolytic therapy is unlikely to completely address and prevent the sequelae, disability, and potential risks of free radical neurological damage.

[0004] Similar to stroke, amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a disease characterized by the damage, atrophy, and death of nerve cells. While its pathogenesis remains uncertain, biological evidence suggests a link between the disease and superoxide dismutase (SOD). Mutations in SOD are one of the causes of ALS. This leads to the inability of SOD to break down harmful substances such as free radicals within mitochondria through dismutation reactions, resulting in extensive nerve damage.

[0005] Alzheimer's disease (AD), also known as senile dementia, is the most common neurodegenerative disease. Its primary clinical manifestations include progressive cognitive impairment and memory loss. While the underlying cause of AD remains uncertain, a growing body of research indicates that oxidative stress is a hallmark of AD. Free radicals in oxidative stress can promote the phosphorylation of Aβ and tau proteins, leading to the abnormal accumulation of Aβ and other proteins. They can also cause mutations in neuronal DNA and RNA, contributing to the overall progression of AD.

[0006] Parkinson's disease (PD) is a chronic neurodegenerative disorder that occurs frequently in middle-aged and elderly individuals. Its characteristic pathological changes are degeneration and necrosis of dopaminergic neurons in the substantia nigra pars compacta, which in turn leads to decreased dopamine (DA) levels in the striatum. While its etiology remains unclear, factors such as mitochondrial dysfunction, oxidative stress, unregulated iron metabolism, aging, and excitotoxic neurotoxicity ultimately lead to excessive free radical formation, which in turn causes neuronal death. Scavenging free radicals has become a promising treatment option for PD.

[0007] Therefore, free radical damage is an important factor causing many diseases such as central nervous system damage and degeneration. Free radical scavenging and antioxidant therapy are important means of treating many of the above central nervous system diseases.

[0008] Summary of the Invention

[0009] To overcome the shortcomings of the prior art, the present application aims to provide a precursor compound based on the structure of edaravone, or a pharmaceutically acceptable salt thereof, as well as a preparation method and application thereof. The compound or pharmaceutically acceptable salt provided herein has good water solubility, which increases the drug's concentration and retention time in the brain, thereby enhancing the drug's efficacy.

[0010] The first aspect of the present application provides a precursor compound based on the structure of edaravone or a pharmaceutically acceptable salt thereof, having the general structural formula I

[0011] wherein R1, R2, R3, and R4 are each independently selected from hydrogen, halogen, hydroxyl, thiol, nitro, amino, acetamido, cyano, acetoxy, acetate, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, and trifluoromethoxy;

[0012] X is selected from Nitrogen, CH2, oxygen or sulfur; m is a natural number selected from 0 to 4; A is R6 and R7 are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a C3-C7 cycloalkyl group or a C1-C5 alkyloxy C1-C5 alkyl group; or A is a C3-C10 heterocycle or substituted heterocycle containing 1 N atom.

[0013] In some embodiments, R1, R2, R3, and R4 are all hydrogen; X is arbitrarily selected from CH2 or oxygen; m is 0 or 2; A is selected from Or A is selected from

[0014] In some embodiments, the compound is

[0015] In some embodiments, the salt is prepared by reacting the precursor compound based on the edaravone structure with an acid; the acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, oxalic acid, nicotinic acid, camphoric acid, gluconic acid, glucuronic acid, methanesulfonic acid, ethanesulfonic acid, aminosulfonic acid or p-toluenesulfonic acid.

[0016] The second aspect of the present application provides a method for preparing a precursor compound based on the edaravone structure, comprising:

[0017] When X is CH2, and Or a C3-C10 heterocycle or substituted heterocycle containing 1 N atom is prepared by amination reaction under alkaline conditions Hydrolysis to remove the R5 group yields The precursor compound based on the edaravone structure described in the first aspect of the present application is prepared by acylation reaction with the edaravone;

[0018] Or when X is oxygen, and Prepared by etherification under alkaline conditions Hydrolysis removes the R5 group to obtain The precursor compound based on the structure of edaravone according to the first aspect of the present application is prepared by acylation reaction with edaravone; wherein R1, R2, R3, and R4 are each independently selected from hydrogen, halogen, hydroxyl, thiol, nitro, amino, acetylamino, cyano, acetoxy, acetate, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, and trifluoromethoxy; m is a natural number selected from 0 to 4; and Y is selected from halogen Cl, Br, or I;

[0019] A is R6 and R7 are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a C3-C7 cycloalkyl group, a C1-C5 alkyloxy group, or a C1-C5 alkyl group;

[0020] Alternatively, A is a 3-10 membered heterocyclic ring or a substituted heterocyclic ring containing one nitrogen atom.

[0021] In some embodiments, m is selected from 0 or 2; Y is selected from Cl; R1, R2, R3 and R4 are hydrogen atoms; A is selected from Or A is selected from

[0022] The third aspect of the present application provides an application of a precursor compound based on the structure of edaravone or a pharmaceutically acceptable salt thereof, wherein the precursor compound based on the structure of edaravone is used to prepare a drug for preventing or treating central nervous system damage and central nervous system degeneration diseases.

[0023] In some embodiments, the central nervous system injury and central nervous system degenerative diseases include stroke, amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease.

[0024] In some embodiments, the dosage form of the drug includes tablets, suppositories, soft capsules, hard capsules, solutions, suspensions, aerosols, injections, lyophilized powder injections, sustained-release preparations or various microparticle delivery systems, and is administered orally, intranasally, rectally, percutaneously or by injection.

[0025] In some embodiments, the dosage form of the drug is an injection.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The present application provides a precursor compound based on the structure of edaravone or a pharmaceutically acceptable salt thereof, a preparation method and application thereof. The compound or pharmaceutically acceptable salt thereof of the present application has good water solubility and can release the free drug edaravone in the brain, making it significantly higher than the brain accumulation concentration of the original drug edaravone, prolonging the retention time of the drug in the brain, thereby improving the effect of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a graph comparing the concentrations of a precursor compound based on the structure of edaravone and edaravone in brain tissue according to an embodiment of the present application;

[0029] FIG2 is a graph showing the concentration distribution of commercially available edaravone in different tissues at different times;

[0030] FIG3 is a graph showing the concentration distribution of compound 1 of the present application in different tissues at different times;

[0031] FIG4 is a graph showing the concentration distribution of compound 2 of the present application in different tissues at different times;

[0032] FIG5 is a comparison of brain infarct foci after treatment with compounds 1 to 2 of the present application and the control group;

[0033] Figure 6 is a comparative graph showing the evaluation of intracellular oxidative stress and inflammation levels in rats after treatment with compounds 1 to 2 of the present application and a control group; Figure 6A is a comparative graph showing MDA obtained by different treatments, Figure 6B is a comparative graph showing SOD obtained by different treatments, Figure 6C is a comparative graph showing GSH-Px obtained by different treatments, Figure 6D is a comparative graph showing CAT obtained by different treatments, Figure 6E is a comparative graph showing TNF-α obtained by different treatments, and Figure 6F is a comparative graph showing TGF-β obtained by different treatments;

[0034] FIG7 is a comparison of the results of H&E staining and Nissl staining after treatment with compounds 1 to 2 of the present application and the control group;

[0035] FIG8 is a comparison of the ROS staining results after treatment with compounds 1 to 2 of the present application and the control group. DETAILED DESCRIPTION

[0036] The present application is further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as those generally understood by those skilled in the art to which this application belongs.

[0037] Free radical damage is a significant contributing factor to numerous diseases, including central nervous system damage and degeneration. Free radical scavenging and antioxidant therapy are important treatments for these central nervous system diseases. 3-Methyl-1-phenyl-2-pyrazolin-5-one, also known as edaravone, is a novel brain-protective agent that protects the brain by scavenging free radicals and inhibiting lipid peroxidation, thereby suppressing oxidative damage to brain cells, vascular endothelial cells, and neurons.

[0038] Currently, the main clinical dosage form of edaravone is injection. The pKa of edaravone is 6.9±0.1. Therefore, under physiological pH conditions, about 50% of edaravone is ionized and exists in the anionic form. This form is also the more reactive form and easily reacts with ROS in the body, donating one electron to free radicals to achieve the purpose of scavenging free radicals and producing an antioxidant effect. However, there are two major problems with the clinical application of edaravone: ① It has poor water solubility and is easily oxidized in aqueous solution. The existing technology mainly increases its content in the currently prepared formulations by adding antioxidants and cosolvents during the preparation process, such as sodium bisulfite and cysteine ​​hydrochloride. However, this can cause allergic reactions when applied intravenously, which may bring unnecessary side effects or potential health hazards to patients. ② Due to the existence of the blood-brain barrier, the anionic form of edaravone has difficulty crossing the blood-brain barrier, resulting in a low effective dose reaching the central nervous system, which limits its application in the treatment of brain and central nervous system diseases. Therefore, it is necessary to modify edaravone to obtain edaravone derivatives with certain water solubility and the ability to efficiently pass through the blood-brain barrier, and then release the original drug edaravone in the central nervous system to exert its free radical scavenging and antioxidant effects.

[0039] In response to the deficiencies in the above-mentioned prior art, the present application provides a precursor compound based on the structure of edaravone, and its preparation method and application, which are described in detail below with reference to the accompanying drawings.

[0040] Example 1

[0041] A precursor compound based on the structure of edaravone, Denoted as compound 1, its preparation process is as follows:

[0042] (1) Dissolve (2.021 g, 8.740 mmol) of 3-bromomethyl-benzoic acid methyl ester in 40 mL of acetonitrile, then add 2 eq. of morpholine and react at room temperature for 3 h. After separation by column chromatography, dissolve in 20 mL of methanol, add 4 eq. of sodium hydroxide (NaOH), and react at 55°C for 2 h. Remove the methanol by rotary evaporation, add 20 mL of methanol to dissolve it, and slowly add 200 μL of anhydrous ethanol-hydrogen chloride solution. Filter, rotary evaporation, add 5 mL of methanol to dissolve it, and then add ethyl acetate to adjust the solubility of the solution until a large amount of white precipitate appears. Place it in a refrigerator at 4°C and filter to obtain (5.6 g, 21.78 mmol) of the intermediate.

[0043] (2) Weigh 0.501 g, 2.87 mmol) of edaravone and 1 eq. of the intermediate and dissolve them in 20 mL of dichloromethane (DCM). Add 2 eq. of triethylamine and 3 eq. of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) thereto, react at room temperature for 6 h, separate and purify by column chromatography, spin dry, add 20 mL of ethyl acetate to redissolve, slowly add 200 μL of anhydrous ethanol-hydrogen chloride solution, react in a -10°C cold trap for 2 h, and filter to obtain compound 1 as a white solid powder with a yield of approximately 55.73%.

[0044] The structural characterization results of compound 1 are:

[0045] 1 H NMR (400MHz, Methanol-d4) δ8.29(t,J=1.8Hz,1H),8.19(dt,J=7.9,1.4Hz,1H),7.92(dt,J=7.8,1.5Hz,1H),7.69(t,J=7.8Hz,1H), 7.61–7.53(m,2H),7.48(dd,J=8.7,7.0Hz,2H),7.43–7.34(m,1H),6.30(s,1H),4.46(s,2H),4.02(s,3H),3.78(s,3H),2.34(s,3H).

[0046] Example 2

[0047] A precursor compound based on the structure of edaravone, Denoted as compound 2, its preparation process is as follows:

[0048] Edaravone (0.310 g, 1.781 mmol) and 1.2 eq. of 4-morpholinomethylbenzoic acid were dissolved in 25 mL of DCM until completely dissolved. 2 eq. of N,N'-dicyclohexylcarboximide (DCC) was added and stirred at room temperature for 6 h. The reaction was monitored by plate-spotting, filtered, and the DCM removed by vacuum distillation. 5 mL of n-hexane and 5 mL of DCM were added and the mixture was refrigerated at 4°C for 2 h. The mixture was filtered and the DCM and n-hexane removed by vacuum distillation to yield a mixture containing the intermediate. The mixture was separated by column chromatography, filtered, and the DCM and acetone removed by vacuum distillation. The mixture was reconstituted in 20 mL of ethyl acetate, and 200 μL of anhydrous ethanol-hydrogen chloride solution was slowly added dropwise. The mixture was refrigerated at -10°C for 2 h and filtered to yield compound 2 as a white solid powder in an approximately 60.24% yield.

[0049] The structural characterization results of compound 2 are:

[0050] 1H NMR(400MHz, Methanol-d4)δ8.19–8.09(m,3H),7.81–7.74(m,2H),7.74–7.67(m,0H),7. 65–7.56(m,3H),7.56–7.48(m,2H),7.48–7.40(m,1H),6.43(s,1H),4.47(d,J=9.4Hz,3H ),4.04(dd,J=13.4,3.5Hz,3H),3.80(dddd,J=13.3,11.5,5.9,2.2Hz,3H),3.38(d,J=12 .4Hz,3H),3.28(d,J=3.7Hz,1H),3.23(dd,J=12.2,3.7Hz,2H),2.45(s,1H),2.38(s,3H).

[0051] Example 3

[0052] A precursor compound based on the structure of edaravone, Denoted as compound 3, its preparation process is as follows:

[0053] (1) Dissolve (2.005 g, 13.20 mmol) of methyl p-hydroxybenzoate and 2 eq. of N-chloroethylpyrrolidine hydrochloride in 40 mL of acetonitrile, add 4 eq. of sodium hydroxide, and react at 50°C for 3 h. After separation by column chromatography, dissolve in 20 mL of methanol, add 6 eq. of sodium hydroxide, and react at 55°C for 3 h. Remove the methanol by rotary evaporation, add 20 mL of methanol to dissolve it, and slowly add 200 μL of anhydrous ethanol-hydrogen chloride solution. Filter, rotary evaporation, add 5 mL of methanol to dissolve it, and then add ethyl acetate to adjust the solubility of the solution until a large amount of white precipitate appears. Place it in a refrigerator at 4°C and filter to obtain the intermediate.

[0054] (2) Weigh 0.501 g, 2.87 mmol) of edaravone and 2 eq. of the intermediate, dissolve in 20 mL of DCM, add 4 eq. of triethylamine and 3 eq. of EDC, react at room temperature for 6 h, separate and purify by column chromatography, spin dry, add 20 mL of ethyl acetate to redissolve, slowly add 200 μL of anhydrous ethanol-hydrogen chloride solution dropwise, react at -10°C for 2 h, and filter to obtain compound 3 as a white solid powder with a yield of 58.43%.

[0055] The structural characterization results of compound 3 are:

[0056] 1H NMR (400MHz, Methanol-d4) δ8.09–8.01(m,2H),7.58–7.51(m,2H),7.47(dd,J=8.7,7.0Hz,2H),7.42–7.33(m,1H) ,7.20–7.09(m,2H),6.27(s,1H),4.48–4.41(m,2H),3.70(dd,J=5.6,4.2Hz,2H),2.33(s,3H),2.16–2.08(m,4H).

[0057] Example 4

[0058] A precursor compound based on the structure of edaravone, Denoted as compound 4, its preparation process is as follows:

[0059] (1) Take (0.606g, 3.96mmol) methyl 3-hydroxybenzoate and 2eq. 1-(2-chloroethyl)piperidine hydrochloride, dissolve them in acetonitrile, add 4eq. NaOH, and react at 45°C. Use column chromatography to separate and purify to obtain the first intermediate. Dissolve (1.10g, 4.18mmol) of the first intermediate in methanol, add 2eq. NaOH, and react at 55°C. Monitor the reaction by thin layer chromatography and spin dry. Add methanol, then dropwise add anhydrous ethanol-hydrogen chloride, filter, and spin evaporate. Add 5mL of methanol to redissolve, and then add ethyl acetate to adjust the solubility of the solution until a large amount of white precipitate appears to obtain the second intermediate.

[0060] (2) 0.245 g, 1.40 mmol) of edaravone and 2 eq. of the second intermediate were dissolved in DCM. 4 eq. of triethylamine was added, followed by 3 eq. of EDC. The reaction was allowed to proceed at room temperature with a plate monitor. Column chromatography was performed using a mobile phase of DCM:methanol = 60:1 to obtain compound 4 as a yellow oily liquid with a yield of 46.87%.

[0061] The structural characterization results of compound 4 are as follows:

[0062] 1H NMR (400MHz, Methanol-d4) δ7.76–7.66(m,5H),7.66–7.59(m,13H),7.59–7.45(m,14H),7.44(d,J=3.5Hz,1H),7.43–7.34(m,5H),7.32–7.2 3(m,2H),6.31(s,2H),4.10(q,J=7.1Hz,2H),3.90(s,1H),2.42(s,7H),2.34(s,6H),1.57(s,2H),1.54(d,J=4.9Hz,2H),1.31–1.13(m,5H).

[0063] Example 5

[0064] A precursor compound based on the structure of edaravone, Denoted as compound 5, its preparation process is as follows:

[0065] (1) Take (0.606g, 3.96mmol) of methyl p-hydroxybenzoate and 2eq. of 1-(2-chloroethyl)piperidine hydrochloride, dissolve them in acetonitrile, add 4eq. of NaOH, and react at 45°C. Use column chromatography to separate and purify to obtain the first intermediate. Dissolve (1.10g, 4.18mmol) of the first intermediate in methanol, add 2eq. of NaOH, and react at 55°C. Monitor the reaction with a plate, spin dry, and add methanol. Then add anhydrous ethanol-hydrogen chloride dropwise, filter, and evaporate. After redissolving in 5mL of methanol, add ethyl acetate to adjust the solubility of the solution until a large amount of white precipitate appears to obtain (1.29g, 3.06mmol) of the second intermediate.

[0066] (2) 0.266 g, 1.53 mmol) of edaravone and 2 eq. of the second intermediate were dissolved in DCM. 4 eq. of triethylamine was added, followed by 3 eq. of EDC. The reaction was allowed to proceed at room temperature with a plate monitor. Column chromatography was performed using a mobile phase of DCM:methanol = 80:1 to obtain compound 5 as a yellow oily liquid with a yield of 53.51%.

[0067] The structural characterization results of compound 5 are:

[0068] 1H NMR(400MHz, Methanol-d4)δ8.09–8.01(m,6H),7.58–7.50(m,6H),7.47(dd,J=8.7,7.0Hz,6H),7.42–7.33(m,3H),7.19–7.10 (m,6H),6.27(s,3H),4.52–4.45(m,6H),3.64–3.57(m,9H),3.10(s,2H),2.33(s,9H),1.94(s,4H),1.86(s,4H),1.57(s,1H).

[0069] Experimental Example 1

[0070] Study on brain-targeted distribution of precursor compounds based on the structure of edaravone

[0071] This experiment investigated the in vivo brain targeting of compounds 1 to 5 prepared in this application by a distribution experiment in mice. The specific experimental process is as follows: 100 Kunming mice that met the experimental criteria were randomly divided into 6 groups, including commercially available edaravone (PMP) group, compound 1 group, compound 2 group, compound 3 group, compound 4 group, and compound 5 group. Before the experiment, the tested Kunming mice were fasted for 12 hours and allowed to drink water freely. PMP was administered intravenously at 50 mg / kg, and compounds 1 to 5 were administered in equimolar doses with PMP. After the injection, death occurred in compound 3 group, and the remaining 5 groups were killed by cervical dislocation at 1 minute, and tissue samples such as brain, heart, liver, spleen, lung, and kidney were taken out respectively. Subsequently, each drug was treated according to the in vivo sample treatment method, and 5 Kunming mice were used at each time point.

[0072] Figure 1 shows the levels of edaravone, Compound 1, Compound 2, Compound 4, and Compound 5 in brain tissue at 1 minute. As shown in Figure 1, the compounds of the present application exhibited higher concentrations in brain tissue than commercially available edaravone at the same time point. Compounds 1-5 are denoted as Compound 1-5. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences between the experimental group and the PMP group.

[0073] The Kunming mice were fasted for 12 hours and allowed free access to water. Edaravone was administered intravenously at 50 mg / kg, and Compound 1 and Compound 2 were administered at equimolar doses with PMP. After the injections, the mice were sacrificed by cervical dislocation at 1, 3, 5, 10, and 15 minutes. Tissue samples from the brain, heart, liver, spleen, lung, and kidney were removed. Each drug was then treated as an in vivo sample. Five Kunming mice were used at each time point. The levels of edaravone, Compound 1, and Compound 2 in brain tissue at different time points are shown in Figures 2 to 4.

[0074] As shown in Figures 2 to 4, compared with the PMP group, the amount of edaravone distributed in the brain tissue of the Compound 1 and Compound 2 groups at each time point was significantly increased, indicating that Compound 1 and Compound 2 have significant brain targeting.

[0075] Furthermore, the present application calculated the brain targeting evaluation index after intravenous injection of drugs in mice. The various pharmacokinetic parameters were calculated using DAS (Data Analysis System) software, among which the targeting evaluation index was the relative uptake rate R e and peak concentration ratio C e , these two indicators are used to evaluate the brain targeting of drugs. A value greater than 1 indicates that the drug has brain targeting, and the larger the value, the better the brain targeting effect of the drug. The calculation formula is shown below, and the results of the brain targeting evaluation index after intravenous injection of drugs in mice are statistically shown in Table 1. 脑 =(AUC) 化合物 / (AUC) 依达拉奉 Ce 脑 =(C max ) 化合物 / (C max ) 依达拉奉

[0076] Table 1 Results of brain targeting evaluation indexes of drugs after intravenous injection in mice

[0077] As shown in Table 1, compared with edaravone, the relative uptake rates of the edaravone-based precursor compounds provided by the present application, including Compound 1 and Compound 2, were as high as 5.11-11.76, and the peak concentration ratios were as high as 6.49-12.19. This indicates that compared to commercially available edaravone, the edaravone-based precursor compounds provided by the present application can effectively increase the accumulation of edaravone in the brain and exhibit significant in vivo brain targeting.

[0078] Experimental Example 2

[0079] Study on the Equilibrium Solubility of Edaravone and Its Precursor Compounds

[0080] Using edaravone as a control, the equilibrium solubility of edaravone was compared with that of Compound 1 and Compound 2, which showed good results in Experimental Example 1. During the experiment, excess amounts of edaravone, Compound 1, and Compound 2 were added to a test tube containing 1 mL of ultrapure water. After sonication, supersaturated solutions of each drug were obtained. The experiment was repeated three times in parallel. Each sample was shaken overnight in a room temperature oscillator, centrifuged, and the supernatant was diluted with methanol to determine the equilibrium solubility of each drug. The results are shown in Table 2.

[0081] Table 2 Equilibrium solubility test results of edaravone and its precursor compounds

[0082] As shown in Table 2, the solubility of Compound 1 and Compound 2 provided by the present invention in pure water is 17.22 and 22.61 times that of Edaravone, respectively. This indicates that the present invention can significantly improve the solubility of the drug by structural modification of Edaravone.

[0083] Experimental Example 3

[0084] Study on the intravenous anti-cerebral ischemia effect of a precursor compound based on the structure of edaravone

[0085] The experimental process is as follows: SPF-level male SD rats, weighing 250±5g, were fasted for 12h before the experiment and had free access to water. The rats were randomly divided into 8 groups, namely, edaravone (PMP) group, compound 1 group, compound 2 group, sham operation group, neck surgery and vascular treatment model group (MCAO group), etc., a total of 5 groups.

[0086] A rat model of focal cerebral ischemia-reperfusion was established using a modified Zea-Longa suture method. Rats were anesthetized with 1% sodium pentobarbital at 45 mg / kg via intraperitoneal injection. The dosage was adjusted based on the animal's post-anesthesia condition. The rats were fixed in the supine position in the center of the operating table. The rat's anterior neck was disinfected 0.5 cm below the mandible. A longitudinal incision was made in the mid-right neck. Muscles and fascia were separated using microscissors to expose the common carotid artery (CCA). The vagus nerve beneath the CCA was isolated. The external carotid artery (ECA) and internal carotid artery (ICA) were located along the CCA. Auxiliary sutures were used to ligate the CCA and ECA, and a micro-arteriolar clamp was used to clamp the ICA. An incision was made approximately 10 mm below the CCA ligature. The suture was inserted and lightly tightened. The micro-arteriolar clamp was removed, and the suture was slowly advanced into the ICA until slight resistance was felt. The auxiliary suture was tightened, the suture was marked, and the suture was sutured. One hour later, reperfusion was performed and the suture plug was withdrawn into the CCA to restore blood supply to the middle cerebral artery. No suture plug was inserted in the sham group (Sham).

[0087] The corresponding drugs were administered via intravenous injection. Neck surgery and vascular treatment were similar to those in the model group (MCAO group): PMP (3 mg / kg) was administered, the compound 1 group was administered compound 1 (7.08 mg / kg, equimolar to PMP), the compound 2 group was administered compound 2 (7.08 mg / kg, equimolar to PMP), and the sham-operated group was administered saline (equal volume to PMP). Twenty-four hours after drug treatment, the animals were sacrificed and their brains removed. Residual blood was flushed with saline, and subsequent experiments were performed. The entire procedure was performed at room temperature (24-25°C).

[0088] The success of the model is marked by the development of Horner's sign and hemiplegia, primarily in the contralateral forelimb, after the animal awakens from anesthesia. Pathological parameters were measured 24 hours after reperfusion. The TTC method was used to measure cerebral infarction area, as shown in Figure 5.

[0089] As shown in Figure 5, compared with the edaravone group and the cervical surgery and vascular treatment model group (MCAO group), the brain infarct volume of the compound 1 and compound 2 test groups was reduced, indicating that both compound 1 and compound 2 have better anti-ischemic effects.

[0090] Experimental Example 4

[0091] Experimental study on the antioxidant capacity, anti-inflammatory ability and therapeutic effect of edaravone-based precursor compounds on stroke

[0092] Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and catalase (CAT) are markers of oxidative stress and are commonly used to assess cellular oxidative stress levels. SOD is an important endogenous antioxidant enzyme that catalyzes the conversion of superoxide radicals produced by mitochondria into hydrogen peroxide and oxygen, thereby reducing the cellular damage caused by reactive oxygen species (ROS). MDA, a secondary product of lipid oxidation, can aggravate tissue oxidative damage and is therefore an important indicator of lipid peroxidation and is used to assess tissue oxidative stress levels. GSH-Px is an antioxidant enzyme present in mitochondria that inhibits lipid peroxidation, thereby protecting cells from oxidative stress. CAT, present in cellular peroxisomes, generally has protective and detoxifying effects in the human body, scavenging reactive oxygen species (ROS) produced within the body and is therefore used to assess intracellular oxidative stress levels.

[0093] Tumor necrosis factor-alpha (TNF-α) and transforming growth factor-β (TGF-β) are both cytokines and are often used to assess intracellular inflammation levels. TNF-α, a member of the tumor necrosis factor family, increases in number when the body produces an inflammatory response. It plays an important role in the body's inflammatory and immune responses and is therefore used to assess inflammatory lesions, disease progression, and tissue damage. TGF-β, a member of the transforming growth factor family, is a polypeptide cytokine with multiple biological functions. It is produced by lymphocytes, NK cells, and macrophages and has anti-infective and antiviral effects, making it commonly used to assess inflammation levels.

[0094] In the experiment, rats were administered physiological saline (equal volume to PMP) or PMP, Compound 1, and Compound 2 (equimolar amounts to PMP) according to their respective groups 1 hour after reperfusion. Twenty-four hours after drug treatment, rats were sacrificed and their brains removed. Residual blood was flushed with physiological saline, dried, and weighed. Antioxidant and inflammatory factors were assayed according to the corresponding instructions. The results are shown in Figure 3.

[0095] As shown in Figure 6, compared with the MCAO group, the activities of SOD (Figure 6B), GSH-Px (Figure 6C), and CAT (Figure 6D) in the compound 1 group and compound 2 were significantly increased (p < 0.001), and the activity of MDA (Figure 6A) was significantly decreased (p < 0.001), indicating that compounds 1 and compound 2 have good antioxidant capacity. At the same time, compared with the MCAO group, the activity of TNF-α (Figure 6E) in the compound 1 group and compound 2 was significantly decreased (p < 0.001), and TGF-β (Figure 6F) was significantly increased, indicating that compounds 1 and compound 2 have the ability to inhibit inflammation.

[0096] Hematoxylin and eosin (H&E) staining, Nissl staining (NISSL), and reactive oxygen species (ROS) immunofluorescence staining (RIS) in brain tissue can directly reflect the degree of neuronal damage and oxidative stress in brain tissue. The results of H&E staining, NISSL staining, and quantitative analysis of ROS in brain tissue are shown in Figures 7 and 8, respectively.

[0097] As shown in Figure 7, the brain neurons of the control group (Sham group) are clearly visible, the cytoplasm is darkly stained, the nuclei are purple-blue and round, the morphology is normal and closely arranged, and there are no pathological changes. The number of cells in the model group (MCAO group) is significantly reduced, the nucleus staining becomes lighter, the interstitial space becomes larger, a large number of neurons die, and the integrity is low. After treatment with compound 1 or compound 2, the number of pyknosis in the model rats is significantly reduced, the number of normal cells increases, and the interstitial space is basically the same as that in the control group, indicating that compound 1 and compound 2 can significantly reduce brain tissue damage and have a good therapeutic effect on stroke.

[0098] As shown in Figure 8, after staining with 4',6-diamidino-2-phenylindole (DAPI) and dihydroethidium (DHE), most of the cell nuclei in the brain tissue of the sham group showed fluorescence, and only a small amount of reactive oxygen species fluorescence was present in the brain tissue, indicating that the number of ROS in normal brain tissue is relatively low. Compared with the sham group, the MCAO group had a large amount of reactive oxygen species fluorescence, indicating that the MCAO group had more severe oxidative damage caused by ischemia-reperfusion. The PMP group had a lower level of reactive oxygen species fluorescence, and the compound 1 and compound 2 groups had a significant decrease in reactive oxygen species fluorescence (p < 0.001), indicating that compounds 1 and 2 can reduce the reactive oxygen species generated in the brain due to ischemia-reperfusion and improve the brain's oxidative stress damage.

[0099] The above-mentioned embodiments are only preferred embodiments of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and replacements made by technicians in this field based on the present application shall fall within the scope of protection required by the present application.

Claims

1. A precursor compound based on the structure of edaravone or a pharmaceutically acceptable salt thereof, characterized in that: With the general structural formula Ⅰ wherein R1, R2, R3, and R4 are each independently selected from hydrogen, halogen, hydroxyl, thiol, nitro, amino, acetylamino, cyano, acetoxy, acetate, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, and trifluoromethoxy; X is selected from nitrogen, CH2, oxygen or sulfur; m is a natural number selected from 0 to 4; A is R6 and R7 are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a C3-C7 cycloalkyl group or a C1-C5 alkyloxy group C1-C5 alkyl group; Alternatively, A is a C3-C10 heterocycle or substituted heterocycle containing 1 N atom.

2. The precursor compound based on the edaravone structure or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R1, R2, R3, and R4 are all hydrogen; X is selected from CH2 or oxygen, m is 0 or 2; A is selected from Or A is selected from 3. The precursor compound based on the edaravone structure or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound is 4. The precursor compound based on the edaravone structure or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The salt is prepared by reacting the precursor compound based on the edaravone structure with an acid; The acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, oxalic acid, nicotinic acid, camphoric acid, gluconic acid, glucuronic acid, methanesulfonic acid, ethanesulfonic acid, aminosulfonic acid or p-toluenesulfonic acid.

5. A method for preparing a precursor compound based on the structure of edaravone, characterized in that: include: When X is CH2, and Or a C3-C10 heterocycle or substituted heterocycle containing 1 N atom is prepared by amination reaction under alkaline conditions Hydrolysis to remove the R5 group gives The precursor compound based on the structure of edaravone is prepared by acylation reaction with edaravone as claimed in any one of claims 1 to 3; Or when X is oxygen, and Prepared by etherification under alkaline conditions Hydrolysis to remove the R5 group gives The precursor compound based on the structure of edaravone is prepared by acylation reaction with edaravone as claimed in any one of claims 1 to 3; wherein R1, R2, R3, and R4 are each independently selected from hydrogen, halogen, hydroxyl, thiol, nitro, amino, acetylamino, cyano, acetoxy, acetate, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, and trifluoromethoxy; m is a natural number selected from 0 to 4; Y is selected from halogen Cl, Br or I; A is R6 and R7 are each independently selected from a hydrogen atom, a C1-C6 alkyl group, a C3-C7 cycloalkyl group, a C1-C5 alkyloxy group, and a C1-C5 alkyl group; Alternatively, A is a 3-10 membered heterocyclic ring or a substituted heterocyclic ring containing 1 N atom.

6. The method according to claim 5, characterized in that The m is selected from 0 or 2; Y is selected from Cl; R1, R2, R3 and R4 are hydrogen atoms; A is selected from Or A is selected from 7. A use of a precursor compound based on the structure of edaravone or a pharmaceutically acceptable salt thereof, characterized in that: The precursor compound based on the edaravone structure is used for preparing drugs for preventing or treating central nervous system damage and central nervous system degeneration diseases.

8. The use according to claim 7, characterized in that The central nervous system damage and central nervous system degeneration diseases include stroke, amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease.

9. The use according to claim 7, characterized in that The dosage forms of the drug include tablets, suppositories, soft capsules, hard capsules, solutions, suspensions, aerosols, injections, freeze-dried powder injections, sustained-release preparations or various microparticle delivery systems, and are administered orally, intranasally, rectally, percutaneously or by injection.

10. The use according to claim 9, characterized in that The dosage form of the drug is injection.

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