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WO2025113038A1 - 一种基于依达拉奉结构的前体化合物及其制备方法和应用 - Google Patents

一种基于依达拉奉结构的前体化合物及其制备方法和应用 Download PDF

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WO2025113038A1
WO2025113038A1 PCT/CN2024/128013 CN2024128013W WO2025113038A1 WO 2025113038 A1 WO2025113038 A1 WO 2025113038A1 CN 2024128013 W CN2024128013 W CN 2024128013W WO 2025113038 A1 WO2025113038 A1 WO 2025113038A1
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acid
edaravone
precursor compound
compound based
compound
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French (fr)
Inventor
李建波
张金洁
塞田叶尔太
张硕
张捷柯
郭军
黄静雯
张梦壹
王辰旭
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Zhengzhou University
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Zhengzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/18—One oxygen or sulfur atom
    • C07D231/20—One oxygen atom attached in position 3 or 5
    • C07D231/22—One oxygen atom attached in position 3 or 5 with aryl radicals attached to ring nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • 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.
  • Free radicals also known as "free radicals" in chemistry, are groups of atoms containing an unpaired electron. Since electrons must appear in pairs in chemical bonds when atoms form molecules, free radicals need to seize an electron from other substances to form stable substances. In chemistry, this phenomenon is called “oxidation”. Oxygen free radicals are mainly involved in biological systems, such as superoxide anion free radicals, hydroxyl free radicals, and lipid oxygen free radicals. Reactive oxygen free radicals in the body have certain functions, such as immunity and signal transduction processes, and the body's built-in antioxidant system plays a decisive role in preventing damage caused by free radicals.
  • oxidative stress causes free radicals to attack nerve cells, especially the damage and degeneration of the central nervous system is catastrophic, such as stroke, amyotrophic lateral sclerosis (ALS), Alzheimer's (AD), Parkinson's disease (PD), etc.
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer's
  • PD Parkinson's disease
  • Cerebral stroke is also known as "stroke”. It is an acute cerebrovascular disease. It is a group of diseases caused by brain tissue damage due to sudden rupture of brain blood vessels or blood vessel blockage that prevents blood from flowing into the brain. 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 that leads to sequelae of cerebral stroke and disability after treatment is that a large amount of reactive oxygen and free radicals are produced around the brain lesions during blood reperfusion after cerebral ischemia. These free radicals will cause irreversible damage to nerve cells and brain cells through cell membrane damage, protein damage, nucleic acid/DNA damage, and induction of various inflammatory mediators, and ultimately lead to the death of nerve cells or brain cells. Therefore, if only intravenous or thrombolytic therapy is used in the clinical treatment of stroke, it is difficult to completely solve and prevent the sequelae and disability problems and hidden dangers caused by free radical nerve damage to patients.
  • ALS amyotrophic lateral sclerosis
  • Lou Gehrig's disease is also a disease characterized by the damage, atrophy and death of nerve cells. Its pathogenesis has not yet been determined, but biology has proven that the disease is associated with superoxide dismutase (SOD). Mutation of SOD superoxide dismutase is one of the causes of ALS, which prevents SOD from decomposing harmful substances such as free radicals in mitochondria through dismutation reactions, thereby causing extensive damage to nerves.
  • SOD superoxide dismutase
  • AD Alzheimer's disease
  • oxidative stress is one of the hallmarks of Alzheimer's disease.
  • the free radicals in the oxidative stress response can promote the production of A ⁇ and tau protein phosphorylation, the abnormal accumulation of A ⁇ and other proteins, and can cause mutations in neuronal DNA and RNA, promoting the overall progression of AD.
  • Parkinson's disease is a chronic neurodegenerative disease that occurs frequently in middle-aged and elderly people. Its characteristic pathological changes are degeneration and necrosis of dopaminergic neurons in the substantia nigra, which in turn leads to a decrease in the content of dopamine (DA) in the striatum. The etiology is still unclear. Mitochondrial dysfunction, oxidative stress, uncontrolled iron metabolism, aging, and excitotoxic neurotoxicity all ultimately lead to excessive formation of free radicals, which in turn lead to neuronal death. Scavenging free radicals has become a promising treatment for PD.
  • Free radical damage is an important influencing 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.
  • the purpose of the present application is to provide a precursor compound based on the structure of edaravone or a pharmaceutically acceptable salt thereof, a preparation method and application.
  • the compound or a pharmaceutically acceptable salt thereof provided in the present application has good water solubility, improves the drug's concentration in the brain, the brain retention time, and thus improves the drug's effect.
  • the first aspect of the present application provides a precursor compound based on the structure of edaravone or a pharmaceutically acceptable salt thereof, having a general structural formula I
  • 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 C1-C5 alkyl group; or A is a C3-C10 heterocycle or substituted heterocycle containing 1 N atom.
  • R1, R2, R3, and R4 are all hydrogen; X is arbitrarily selected from CH2 or oxygen, and m is 0 or 2; A Selected from Or A is selected from
  • the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-phenyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • 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, glucose Uronic acid, methanesulfonic acid, ethanesulfonic acid, aminosulfonic acid or p-toluenesulfonic acid.
  • the second aspect of the present application provides a method for preparing a precursor compound based on the edaravone structure, comprising:
  • 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 alkyl, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, trifluoromethoxy; m is a natural number selected from 0 to 4; Y is a halogen selected from Cl, Br or I;
  • 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;
  • A is a 3-10 membered heterocyclic ring or a substituted heterocyclic ring containing 1 N atom.
  • 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
  • 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.
  • the central nervous system injury and central nervous system degenerative diseases include stroke, amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease.
  • 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.
  • the drug is in the form of an injection.
  • 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 a 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 aggregation concentration of the original drug edaravone, prolonging the retention time of the drug in the brain, thereby improving the effect of the drug.
  • FIG1 is a graph comparing the concentrations of a precursor compound based on the structure of edaravone and edaravone in brain tissue in an embodiment of the present application;
  • FIG2 is a graph showing the concentration distribution of commercially available edaravone in different tissues at different times
  • FIG3 is a concentration distribution diagram of compound 1 of the present application in different tissues at different times;
  • FIG4 is a concentration distribution diagram of compound 2 of the present application in different tissues at different times;
  • FIG5 is a comparison of brain infarction foci after treatment with compounds 1 to 2 of the present application and the control group;
  • Figure 6 is a comparative diagram of the evaluation of intracellular oxidative stress levels and inflammatory levels in rats treated with compounds 1 to 2 of the present application and the control group; wherein Figure 6A is a comparative diagram of MDA obtained by different treatments, Figure 6B is a comparative diagram of SOD obtained by different treatments, Figure 6C is a comparative diagram of GSH-Px obtained by different treatments, Figure 6D is a comparative diagram of CAT obtained by different treatments, Figure 6E is a comparative diagram of TNF- ⁇ obtained by different treatments, and Figure 6F is a comparative diagram of TGF- ⁇ obtained by different treatments;
  • 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;
  • FIG8 is a comparison chart of the ROS staining results after treatment with compounds 1 to 2 of the present application and the control group.
  • 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.
  • 3-Methyl-1-phenyl-2-pyrazol-5-one also known as edaravone, is a new type of brain protective agent. It scavenges free radicals and inhibits lipid peroxidation, thereby inhibiting oxidative damage to brain cells, vascular endothelial cells, and nerve cells, and plays a brain protective role.
  • the main dosage form of edaravone used in clinical practice 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 anionic form. This form is also a more reactive form. It is easy to react with ROS in the body and can provide 1 electron to free radicals to achieve the purpose of scavenging free radicals and produce antioxidant effects.
  • ROS reactive oxygen species
  • the existing technology mainly increases its content in the currently prepared preparations by adding antioxidants and cosolvents during the preparation process, such as adding sodium bisulfite, cysteine hydrochloride, etc., which will cause allergic reactions when used 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 is difficult to pass through the blood-brain barrier, resulting in a small effective dose reaching the central nervous system, which is limited in the treatment of brain central nervous system diseases.
  • the present application provides a precursor compound based on the structure of edaravone and a preparation method and application thereof, which are described in detail below in conjunction with the accompanying drawings.
  • This experiment investigated the brain targeting of compounds 1 to 5 prepared in this application in vivo by a mouse in vivo distribution experiment.
  • the specific experimental process is as follows: 100 Kunming mice that meet the experimental criteria are 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. Among them, PMP was administered intravenously at 50 mg/kg, and compounds 1 to 5 were administered at equimolar doses with PMP.
  • PMP edaravone
  • FIG1 shows the contents of edaravone, compound 1, compound 2, compound 4 and compound 5 in brain tissue at 1 minute.
  • the compounds of the present application have a higher concentration in brain tissue at the same time point than commercially available edaravone.
  • compounds 1 to 5 are represented by Compound 1 to 5. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 indicate that the experimental group is significantly different from the PMP group.
  • the Kunming mice were fasted for 12 hours and allowed to drink water freely.
  • Edaravone was intravenously administered at 50 mg/kg, and compound 1 and compound 2 were administered at equimolar doses with PMP. After the injection, the mice were killed by dislocation of the neck at 1, 3, 5, 10, and 15 minutes, and tissue samples such as brain, heart, liver, spleen, lung, and kidney were taken out respectively. Then, each drug was treated according to the in vivo sample treatment method, and 5 Kunming mice were used at each time point. The contents of Edaravone, compound 1, and compound 2 in brain tissue at different time points are shown in Figures 2 to 4.
  • the present application calculates the brain targeting evaluation index after intravenous injection of drugs in mice.
  • the various pharmacokinetic parameters are calculated using DAS (Data Analysis System) software, where the targeting evaluation indexes are the relative uptake rate Re and the peak concentration ratio Ce . These two indexes are used to evaluate the brain targeting of drugs. A value greater than 1 indicates that the drug has brain targeting, and a value greater than 1 indicates that the drug has brain targeting. 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 the drug in mice are statistically shown in Table 1.
  • the relative uptake rate of the precursor compound based on edaravone provided by the present application is as high as 5.11-11.76, and the peak concentration ratio is as high as 6.49-12.19. This shows that compared with edaravone sold on the market, the precursor compound based on edaravone in the present application can effectively increase the accumulation of edaravone in the brain and has significant in vivo brain targeting.
  • the specific 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.
  • PMP edaravone
  • MCAO group vascular treatment model group
  • the rat focal cerebral ischemia-reperfusion model was established with reference to the modified Zea-Longa suture method.
  • the rats were anesthetized by intraperitoneal injection of sodium pentobarbital 1%, 45 mg/kg, and the dosage was increased or decreased according to the animal's anesthesia condition.
  • the rats were fixed in the center of the operating table in a supine position.
  • the rats were disinfected 0.5 cm below the mandible in the anterior neck area, and a longitudinal incision was made in the middle right of the neck.
  • the muscles and fascia were separated with microscissors to expose the common carotid artery (CCA) to the visual field, and the vagus nerve under the CCA was separated.
  • CCA common carotid artery
  • the external carotid artery (ECA) and the internal carotid artery (ICA) were found along the CCA.
  • the CCA and ECA were ligated with auxiliary lines, and the ICA was clamped with micro-artery clamps.
  • a wound was cut about 10 mm below the ligature of the CCA, and the suture was inserted.
  • the auxiliary line was slightly tightened, the micro-artery clamp was removed, and the suture was slowly sent into the ICA until there was slight resistance, the auxiliary line was tightened, the suture was marked, and the suture was sutured. After 1 hour, reperfusion was performed and the suture was withdrawn into the CCA to restore blood supply to the middle cerebral artery. No suture was inserted in the sham operation group (Sham).
  • the corresponding drugs were administered by intravenous injection. Neck surgery and vascular treatment were the same as the model group (MCAO group), PMP (3 mg/kg) was given, compound 1 group was given compound 1 (7.08 mg/kg, equimolar to PMP), compound 2 group was given compound 2 (7.08 mg/kg, equimolar to PMP), and the sham operation group was given normal saline (same volume as PMP). After 24 hours of drug treatment, the animals were killed and the brains were removed. The residual blood was rinsed with normal saline, and subsequent experiments were performed. The whole process was carried out at room temperature (24-25°C).
  • the sign of the success of the model is the appearance of Horner's sign and hemiplegia mainly in the contralateral forelimb after the model animal wakes up from anesthesia.
  • the various pathological indicators of the rats were measured 24 hours after reperfusion.
  • the TTC method was used to detect the area of cerebral infarction, and the results are shown in Figure 5.
  • SOD Superoxide dismutase
  • GSH-Px glutathione peroxidase
  • MDA malondialdehyde
  • CAT catalase
  • SOD is an important endogenous antioxidant enzyme that catalyzes the conversion of superoxide free radicals produced by cell mitochondria into hydrogen peroxide and oxygen to exert antioxidant effects and reduce the damage of reactive oxygen species to cells.
  • MDA as one of the secondary products of lipid oxidation, can aggravate tissue oxidative damage, so it is a lipid peroxidase.
  • An important indicator of oxidation used 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 and thus protecting cells from oxidative stress damage.
  • CAT exists in the peroxisome of cells and generally has a protective and detoxifying effect on the human body. It can remove reactive oxygen species (ROS) produced in the body, so it is used to evaluate the level of intracellular oxidative stress.
  • ROS reactive oxygen species
  • Tumor necrosis factor-alpha TNF- ⁇
  • TGF- ⁇ transforming growth factor- ⁇
  • TNF- ⁇ TNF- ⁇
  • TGF- ⁇ TGF- ⁇
  • TNF- ⁇ TNF- ⁇
  • TGF- ⁇ TGF- ⁇
  • TGF- ⁇ TGF- ⁇
  • TGF- ⁇ TGF- ⁇
  • TGF- ⁇ TGF- ⁇
  • TGF- ⁇ TGF- ⁇
  • TGF- ⁇ TGF- ⁇
  • TGF- ⁇ is a member of the transforming growth factor family. It is a polypeptide cytokine with multiple biological functions. It is produced by lymphocytes, NK cells and macrophages, and has anti-infection and antiviral effects. It is often used to evaluate the level of inflammation.
  • rats were given physiological saline (equal volume with PMP), PMP, compound 1 and compound 2 with equal molar amount with PMP) according to the grouping 1 hour after reperfusion.
  • the rats were killed and the brains were removed 24 hours after drug treatment.
  • the residual blood was washed with physiological saline, and the weight was weighed after absorbing the water, and the antioxidant factors and inflammatory factors were detected according to the corresponding instructions.
  • the results are shown in Figure 3.
  • NNSSL Nissl staining
  • ROS brain tissue reactive oxygen species immunofluorescence staining
  • the brain neurons of the control group (Sham group) are clearly visible, the cytoplasm is dark, 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.
  • the number of nuclear condensation in the model rats is significantly reduced, the number of normal cells increases, and the interstitial space is basically the same as the control group, indicating that compound 1 and compound 2 can significantly reduce brain tissue damage and have a good therapeutic effect on stroke.

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Abstract

本申请公开了一种基于依达拉奉结构的前体化合物或其药学上可接受的盐、其制备方法和应用，采用本申请的化合物或其药学上可接受的盐，具有较好的水溶性，在脑中可释放出游离药物依达拉奉，使其显著高于原药依达拉奉的脑部聚集浓度，延长了药物在脑部的滞留时间，进而提高了药物的作用效果。

Description

一种基于依达拉奉结构的前体化合物及其制备方法和应用 技术领域
本申请属于有机化学合成技术领域，涉及一种基于依达拉奉结构的前体化合物及其制备方法和应用。
背景技术
自由基，化学上也称为“游离基”，是含有一个不成对电子的原子团。由于原子形成分子时，化学键中电子必须成对出现，因此自由基需要夺取其他物质的一个电子，使自己形成稳定的物质。在化学中，这种现象称为“氧化”。生物体系中主要涉及氧自由基，例如超氧阴离子自由基、羟自由基、脂氧自由基等。体内活性氧自由基具有一定的功能，如免疫和信号传导过程，并且人体的内置抗氧化系统在预防因自由基引起的损伤方面起到了决定性的作用。然而，抗氧化系统的防御机制失衡、自由基的过度产生或从环境引入体内将会导致神经损伤、变性。除若干环境或遗传因素外，氧化性应激引起自由基攻击神经细胞，尤其是对中枢神经损伤、变性的影响是灾难性的，如脑卒中、肌萎缩性侧索硬化症(ALS)、阿兹兹海默(AD)、帕金森病(PD)等。
“脑卒中”(cerebral stroke)又称“中风”，是一种急性脑血管疾病，是由脑部血管突然破裂或因血管阻塞导致血液不能流入大脑而引起脑组织损伤的一组疾病。该病不但死亡率高，也是我国成年人残疾的首要原因之一。其中导致脑中风后遗症和治疗后残疾的病理学机制是脑缺血后血液再灌注时在脑部病灶周围产生大量的活性氧和自由基，这些自由基会通过细胞膜损伤、蛋白质损伤、核酸/DNA损害、以及诱导各种炎症介质的产生等形式，对神经细胞、脑细胞形成非可逆伤害，最终导致神经细胞或脑细胞死亡。因此，针对脑卒中的临床治疗如果只采用静脉或者溶栓治疗，很难彻底解决和防止自由基神经损伤给患者带来的后遗症以及残疾的问题和隐患。
与脑卒中类似，肌萎缩侧索硬化症(ALS)，俗称渐冻人，也是一种以神经细胞受损萎缩和死亡为病理特征的疾病。其发病机制尚无定论，但生物学证明该疾病与超氧化物歧化酶(SOD)有关联，SOD超氧化物歧化酶的突变是肌萎缩侧索硬化症形成的原因之一，导致SOD无法通过歧化反应将线粒体内的自由基等有害物质分解，从而造成对神经的广泛损伤。
阿尔兹海默症(Alzheimer's disease，AD)是一种最常见的神经退行性疾病，又称早老性痴呆症，主要临床表现为进行性的认知功能障碍和记忆力损伤等。虽然目前AD致病因素还不 确定，但越来越多的研究表示，氧化应激为阿尔兹海默的标志之一，氧化应激反应中的自由基可以促进Aβ、tau蛋白磷酸化的产生，Aβ等蛋白的异常累积，并且能使神经元DNA、RNA发生突变，促进AD疾病的整体进展。
帕金森病(Parkinson's disease，PD)是一种多发于中老年人的慢性神经系统退行性疾病，其特征性病理改变为黑质致密部多巴胺能神经元变性、坏死，继而引起纹状体多巴胺(dopamine，DA)含量降低。其病因至今未明确，线粒体功能缺陷、氧化应激、铁代谢失控、衰老与兴奋性神经毒等促发因素最终都引起自由基形成过多、从而引起神经元死亡，清除自由基已成为是充满希望的治疗PD的方案。
因此，自由基损伤是造成中枢神经系统性损伤、变性等众多疾病的重要影响因素，清除自由基及抗氧化治疗是治疗以上众多中枢神经系统疾病的重要手段。
发明内容
为了克服现有技术的不足，本申请的目的在于提供一种基于依达拉奉结构的前体化合物或其药学上可接受的盐、制备方法和应用。本申请提供的化合物或其药学上可接受的盐具有较好的水溶性，提高了药物在脑部的聚集浓度、脑部滞留时间，进而提高了药物的作用效果。
本申请第一方面提供了一种基于依达拉奉结构的前体化合物或其药学上可接受的盐，具有结构通式Ⅰ
其中，R1、R2、R3、R4各自独立地选自氢、卤素、羟基、巯基、硝基、氨基、乙酰氨基、氰基、乙酰氧基、乙酸酯基、C1-C4烷基、C1-C4烷氧基、三氟甲基、三氟甲氧基；
X选自氮、CH2、氧或硫；m选自0至4的自然数；A为R6和R7各自独立地选自氢原子、C1-C6烷基、C3-C7环烷基或C1-C5烷基氧基C1-C5烷基；或者A为含有1个N原子的C3-C10杂环或取代杂环。
在一些实施方式中，R1、R2、R3、R4均为氢；X任意选自CH2或氧，m为0或2；A 选自或者A选自
在一些实施方式中，所述化合物为
在一些实施方式中，所述盐通过所述基于依达拉奉结构的前体化合物与酸制备得到；所述酸包括盐酸、氢溴酸、硫酸、磷酸、乙酸、三氟乙酸、乳酸、丙酮酸、丙二酸、琥珀酸、戊二酸、富马酸、酒石酸、马来酸、柠檬酸、抗坏血酸、草酸、烟酸、樟脑酸、葡萄糖酸、葡萄 糖醛酸、甲磺酸、乙磺酸、氨基磺酸或对甲苯磺酸。
本申请第二方面提供了一种基于依达拉奉结构的前体化合物的制备方法，包括：
当X为CH2时，将与或含有1个N原子的C3-C10杂环或取代杂环在碱性条件下进行胺化反应制得水解脱去R5基团得到将其与依达拉奉通过酰化反应制备得到本申请第一方面所述的基于依达拉奉结构的前体化合物；
或当X为氧时，将与在碱性条件下醚化制备得到水解脱去R5基团，得到将其与依达拉奉通过酰化反应制备得到本申请第一方面所述的基于依达拉奉结构的前体化合物；其中，R1、R2、R3、R4各自独立地选自氢、卤素、羟 基、巯基、硝基、氨基、乙酰氨基、氰基、乙酰氧基、乙酸酯基、C1-C4烷基、C1-C4烷氧基、三氟甲基、三氟甲氧基；m选自0至4的自然数；Y选自卤素Cl、Br或I；
A为R6和R7各自独立地选自氢原子、C1-C6烷基、C3-C7环烷基、C1-C5烷基氧基C1-C5烷基；
或者A为含有1个N原子的3-10元杂环或取代杂环。
在一些实施方式中，所述m选自0或2；Y选自Cl；R1、R2、R3和R4为氢原子；A选自或者A选自
本申请第三方面提供了一种基于依达拉奉结构的前体化合物或其药学上可接受的盐的应用，所述基于依达拉奉结构的前体化合物用于制备预防或治疗中枢神经损伤、中枢神经变性类疾病的药物。
在一些实施方式中，所述中枢神经损伤、中枢神经变性类疾病包括脑卒中、肌萎缩性侧索硬化症、阿兹兹海默或帕金森。
在一些实施方式中，所述药物的剂型包括片剂、栓剂、软胶囊剂、硬胶囊剂、溶液剂、混悬剂、气雾剂、注射剂、冻干粉针剂、缓控释制剂或各种微粒给药系统，通过口、鼻内、直肠、透辟或注射方式进行给药。
在一些实施方式中，所述药物的剂型为注射剂。
相比现有技术，本申请的有益效果在于：
本申请提供了一种基于依达拉奉结构的前体化合物或其药学上可接受的盐、其制备方法和应用，采用本申请的化合物或其药学上可接受的盐，具有较好的水溶性，在脑中可释放出游离药物依达拉奉，使其显著高于原药依达拉奉的脑部聚集浓度，延长了药物在脑部的滞留时间，进而提高了药物的作用效果。
附图说明
图1为本申请实施例中基于依达拉奉结构的前体化合物与依达拉奉在脑组织中浓度对比图；
图2为市售依达拉奉在不同时间、不同组织中的浓度分布图；
图3为本申请化合物1在不同时间、不同组织中的浓度分布图；
图4为本申请化合物2在不同时间、不同组织中的浓度分布图；
图5为本申请化合物1至2与对比组处理后的脑部梗死灶对比图；
图6为本申请化合物1至2与对比组处理大鼠后的细胞内氧化应激水平和炎症水平评价对比图；其中图6A为不同处理方式得到的MDA对比图，图6B为不同处理方式得到的SOD对比图，图6C为不同处理方式得到的GSH-Px对比图，图6D为不同处理方式得到的CAT对比图，图6E为不同处理方式得到的TNF-α对比图，图6F为不同处理方式得到的TGF-β对比图；
图7为本申请化合物1至2与对比组处理后进行H&E染色及尼式染色的结果对比图；
图8为本申请化合物1至2与对比组处理后进行ROS染色的结果对比图。
具体实施方式
下面，结合附图以及具体实施方式，对本申请做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。除有定义外，以下实施例中所用的技术术语具有与本申请所属领域技术人员普遍理解的相同含义。
自由基损伤是造成中枢神经系统性损伤、变性等众多疾病的重要影响因素，清除自由基及抗氧化治疗是治疗以上众多中枢神经系统疾病的重要手段。3-甲基-1-苯基-2-吡唑啉-5-酮，又名依达拉奉，是一种新型脑保护剂，通过清除自由基、抑制脂质过氧化，从而抑制脑细胞、血管内皮细胞、神经细胞的氧化损伤，发挥脑保护作用。
目前依达拉奉临床主要应用的剂型为注射液，依达拉奉的pKa为6.9±0.1，因此在生理pH条件下，有约50％的依达拉奉是离子化的，以阴离子形式存在，该形式也是更具反应性的形式，在体内很容易与ROS反应，可将1个电子提供给自由基而达到清除自由基的目的，产生抗氧化作用。然而，依达拉奉的临床应用存在两个主要问题：①其水溶性差，且在水溶液中极易被氧化，现有技术主要通过在制剂制备过程中加入抗氧化物和助溶剂，增加其在目前制备的制剂中，如加入亚硫酸氢钠、半胱氨酸盐酸盐等，而这在静脉应用时会造成过敏反应，可能会对患者带来不必要的副作用或潜在的健康危害；②由于血脑屏障的存在，阴离子形态的依达拉奉很难透过血脑屏障，造成到达中枢神经系统的有效剂量偏少，在治疗脑部中枢神经系统性疾病方面受限。因此很有必要对依达拉奉进行改造，获得具有一定水溶性、并可高效透过血脑屏障的依达拉奉衍生物，然后再在中枢神经系统释放出原药依达拉奉，发挥其清除自由基、抗氧化的作用。
针对上述现有技术中存在的不足，本申请提供了一种基于依达拉奉结构的前体化合物及其制备方法和应用，下面结合附图进行详细阐述。
实施例1
一种基于依达拉奉结构的前体化合物，记为化合物1，其制备过程如下：
(1)将(2.021g，8.740mmol)3-溴甲基-苯甲酸甲酯溶于40mL乙腈，接着加入2eq.吗啉，置室温下反应3h。使用柱层析分离后溶解于20mL甲醇，再加入4eq.氢氧化钠(NaOH)，置55℃条件下反应2h。旋蒸除去甲醇，加入20mL甲醇复溶，缓慢滴加200μL无水乙醇-氯化氢溶液。过滤、旋蒸，加入5mL甲醇复溶后，再加入乙酸乙酯调节溶液溶解度，直至出现大量白色沉淀，置4℃冰箱静置，过滤即可得到(5.6g，21.78mmol)中间体。
(2)称取(0.501g，2.87mmol)依达拉奉和1eq.中间体溶解于20mL二氯甲烷(DCM)向其中加入2eq.三乙胺和3eq.1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)，室温反应6h，使用柱层析分离提纯后，旋干，加入20mL乙酸乙酯复溶，缓慢滴加200μL无水乙醇-氯化氢溶液，-10℃冷阱反应2h，过滤得到化合物1，白色固体粉末，产率约为55.73％。
化合物1的结构表征结果为：
1H 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).
实施例2
一种基于依达拉奉结构的前体化合物，记为化合物2，其制备过程如下：
将(0.310g，1.781mmol)依达拉奉和1.2eq.4-吗啉甲基苯甲酸溶解于25mLDCM中，完全溶解，加入2eq.N,N’-二环己基碳酰亚胺(DCC)，室温搅拌反应6h。点板监测反应，过滤，减压选蒸除去DCM，加入5mL正己烷和5mL DCM放4℃冰箱静置2h，过滤，减压选蒸除去DCM和正己烷，得到含有中间体的混合产物。将混合物通过柱层析法分离，过滤，减 压选蒸除去DCM和丙酮。加入20mL乙酸乙酯复溶，缓慢滴加200μL无水乙醇-氯化氢溶液，-10℃冷阱反应2h，过滤得到化合物2，白色固体粉末，产率约为60.24％。
化合物2的结构表征结果为：
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).
实施例3
一种基于依达拉奉结构的前体化合物，记为化合物3，其制备过程如下：
(1)将(2.005g，13.20mmol)对羟基苯甲酸甲酯和2eq.N-氯乙基吡咯烷盐酸盐溶于40mL乙腈，加入4eq.氢氧化钠，置50℃条件反应3h。使用柱层析分离后溶解于20mL甲醇，再加入6eq.氢氧化钠，置55℃条件反应3h，旋蒸除去甲醇，加入20mL甲醇复溶，缓慢滴加200μL无水乙醇-氯化氢溶液。过滤、旋蒸，加入5mL甲醇复溶后，再加入乙酸乙酯调节溶液溶解度，直至出现大量白色沉淀。置4℃冰箱静置，过滤即可得到中间体。
(2)称取(0.501g，2.87mmol)依达拉奉和2eq.中间体，溶解于20mL DCM，再加入4eq.三乙胺和3eq.EDC，室温反应6h，使用柱层析分离提纯后，旋干，加入20mL乙酸乙酯复溶，缓慢滴加200μL无水乙醇-氯化氢溶液，-10℃冷井反应2h，过滤得到化合物3，白色固体粉末，产率为58.43％。
化合物3的结构表征结果为：
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).
实施例4
一种基于依达拉奉结构的前体化合物，记为化合物4，其制备过程如下：
(1)取(0.606g，3.96mmol)3-羟基苯甲酸甲酯和2eq.1-(2-氯乙基)哌啶盐酸盐，溶于乙腈，再加入4eq.NaOH，置45℃反应。使用柱层析分离提纯得到第一中间体。将(1.10g，4.18mmol)第一中间体溶于甲醇，加入2eq.NaOH后，置55℃反应，薄层色谱法监测反应、旋干。加入甲醇，再滴加无水乙醇-氯化氢，过滤、旋蒸，加入5mL甲醇复溶后，再加入乙酸乙酯调节溶液溶解度，直至出现大量白色沉淀即可得到第二中间体。
(2)将(0.245g，1.40mmol)依达拉奉和2eq.第二中间体溶于DCM中，加入4eq.三乙胺后，在加入3eq.EDC，置室温下反应，点板监测。使用流动相DCM：甲醇＝60：1进行柱层析分离提纯，得到化合物4，黄色油状液体，产率为46.87％。
化合物4的结构表征结果为：
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),7.32–7.23(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).
实施例5
一种基于依达拉奉结构的前体化合物，记为化合物5，其制备过程如下：
(1)取(0.606g，3.96mmol)对羟基苯甲酸甲酯和2eq.1-(2-氯乙基)哌啶盐酸盐，溶于乙腈，再加入4eq.NaOH，置45℃反应。使用柱层析分离提纯得到第一中间体。将(1.10g，4.18mmol)第一中间体溶于甲醇，加入2eq.NaOH后，置55℃反应，点板监测反应，旋干，加入甲醇。再滴加无水无水乙醇-氯化氢，过滤、旋蒸，加入5mL甲醇复溶后，再加入乙酸乙酯调节溶液溶解度，直至出现大量白色沉淀即可得到(1.29g，3.06mmol)第二中间 体。
(2)将(0.266g，1.53mmol)依达拉奉和2eq.第二中间体溶于DCM中，加入4eq.三乙胺后，再加入3eq.EDC，置室温下反应，点板监测。使用流动相DCM：甲醇＝80：1进行柱层析分离提纯，最后得到化合物5，黄色油状液体，产率为53.51％。
化合物5的结构表征结果为：
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).
实验例1
基于依达拉奉结构的前体化合物的脑靶向分布研究
本实验通过小鼠体内分布实验，考察本申请所制备的化合物1至化合物5的体内脑靶向性。具体实验过程如下：将100只符合实验标准的昆明小鼠随机分为市售依达拉奉(PMP)组、化合物1组、化合物2组、化合物3组、化合物4组、化合物5组等共6组，实验前将受试昆明小鼠禁食12h，并使其自由饮水。其中PMP以50mg/kg静脉给药，化合物1～5与PMP等摩尔剂量给药。注射完毕后，化合物3组出现死亡情况，将其余5组于1min时间点对小鼠脱颈处死，分别取出脑、心，肝，脾，肺，肾等组织样品，随后将各药物按体内样品的处理方法进行，每个时间点采用5只昆明小鼠。
图1示出了1min时间点脑组织中依达拉奉、化合物1、化合物2、化合物4和化合物5的含量，如图1所示，本申请的化合物相较于市售依达拉奉，同样的时间点在脑组织中浓度更大。其中，化合物1～5采用Compound 1～5表示。*p<0.05,**p<0.01,***p<0.001表示实验组相较于PMP组有显著差异。
对受试昆明小鼠禁食12h，并使其自由饮水。其中依达拉奉以50mg/kg静脉给药，化合物1和化合物2与PMP等摩尔剂量给药。注射完毕后，于1、3、5、10、15min时间点对小鼠脱颈处死，分别取出脑、心、肝、脾、肺、肾等组织样品，随后将各药物按体内样品的处理方法进行，每个时间点采用5只昆明小鼠。不同时间点脑组织中依达拉奉、化合物1和化合物2的含量如图2至图4所示。
由图2至图4可知，相比于PMP组，化合物1和化合物2组各时间点在脑组织中分布依达拉奉的量显著提高，表明化合物1和化合物2具有显著的脑靶向性。
进一步地，本申请对小鼠静脉注射药物后的脑靶向性评价指标进行计算。各项药代动力学参数用DAS(Data Analysis System)软件计算，其中靶向性评价指标为相对摄取率Re和峰浓度比Ce，这两项指标用来评价药物的脑部靶向性，大于1表示药物具有脑部靶向性，值越 大表明药物的脑部靶向性效果越好。计算公式如下所示，并将小鼠静脉注射药物后的脑靶向性评价指标结果统计于表1中。
Re脑＝(AUC)化合物/(AUC)依达拉奉
Ce脑＝(Cmax)化合物/(Cmax)依达拉奉
表1小鼠静脉注射后药物的脑靶向评价指标结果
由表1可得本申请化合物1与化合物2与依达拉奉相比，本申请提供的基于依达拉奉的前体化合物的相对摄取率高达5.11-11.76，峰浓度比高达6.49-12.19。表明相对于市面销售依达拉奉，本申请基于依达拉奉的前体化合物能够有效提高依达拉奉在脑部的蓄积量，具有显著的体内脑靶向性。
实验例2
依达拉奉与前体化合物的平衡溶解度研究
以依达拉奉作为对照，比较依达拉奉与实验例1中效果良好的化合物1和化合物2的平衡溶解度。实验时：分别取过量的依达拉奉及化合物1和化合物2，加入到含有1mL超纯水的试管中，超声溶解后得到各药物的过饱和溶液，实验平行重复三次。将各样品置于常温振荡器中过夜振摇，离心并取上清用甲醇稀释，测定各药物的平衡溶解度。结果如表2所示。
表2依达拉奉及其前体化合物的平衡溶解度测试结果
由表2的药物平衡溶解度测定结果可知，本发明提供的化合物1和化合物2在纯水中的溶解度分别是依达拉奉的17.22和22.61倍，由此表明本发明通过对依达拉奉进行结构修饰， 能够使药物的溶解度显著提升。
实验例3
一种基于依达拉奉结构的前体化合物的静脉注射抗脑缺血研究
试验过程具体如下：取SPF级别的雄性SD大鼠，体重250±5g，实验前禁食12h，自由饮水，实验随机分为8组，分别为依达拉奉(PMP)组、化合物1组、化合物2组、假手术组、颈部手术及血管处理同模型组(MCAO组)组等共5组。
参照改良Zea-Longa线栓法建立大鼠局灶性脑缺血再灌注模型。其中使用戊巴比妥钠1％、45mg/kg，向大鼠给予腹腔注射麻醉，并依据动物麻醉后状况酌情增减给药剂量。大鼠呈仰卧位固定于操作台中央。于大鼠颈前区下颌骨下0.5cm处消毒，颈部正中偏右纵行切口，显微剪分离肌肉及筋膜，使颈总动脉(CCA)暴露于视野之中，分离CCA下侧的迷走神经。沿CCA找到颈外动脉(ECA)和颈内动脉(ICA)。使用辅助线结扎CCA及ECA，微动脉夹夹闭ICA。于CCA结扎部下方约10mm处剪出创口，插入线栓，辅助线轻微扎紧，取下微动脉夹，将线栓缓慢送入ICA，直至有轻微阻力为止，扎紧辅助线，标记线栓、缝合。1h后进行再灌输，将线栓抽至CCA内，大脑中动脉恢复血供。其中假手术组(Sham)不插入线栓。
采用静脉注射给予相应药物。颈部手术及血管处理同模型组(MCAO组)给予PMP(3mg/kg)、化合物1组给予化合物1(7.08mg/kg，与PMP等摩尔)、化合物2组给予化合物2(7.08mg/kg，与PMP等摩尔)、假手术组给予生理盐水(与PMP等体积)。给予药物治疗24h后处死并取脑。生理盐水冲洗残血，并进行后续实验。整个过程均在室温(24-25℃)下进行。
模型成功的标志是模型动物麻醉清醒后出现Horner's征及对侧前肢为主的偏瘫。大鼠再灌注24h进行各项病理指标测定。使用TTC法检测脑梗死面积，结果如图5所示。
由图5可知，与依达拉奉组、颈部手术及血管处理同模型组(MCAO组)组相比，化合物1与化合物2试验组的脑部梗死灶体积均降低，表明化合物1及化合物2均具有更好的抗缺血作用。
实验例4
基于依达拉奉结构的前体化合物的抗氧化能力、抑制炎症能力以及脑卒中疗效实验
超氧化物歧化酶(Superoxide dismutase，SOD)、谷胱甘肽过氧化物酶(Glutathione peroxidase，GSH-Px)、丙二醛(Malondialdehyde,MDA)、过氧化氢酶(Catalase，CAT)为氧化应激标志物，常用来评价细胞氧化应激水平。SOD是一种重要的内源性抗氧化酶，通过催化细胞线粒体产生的超氧自由基转化为过氧化氢和氧气来发挥抗氧化作用，降低活性氧对细胞的损伤作用。MDA作为脂质氧化的次级产物之一可加重组织氧化损伤，因此作为脂质过 氧化的重要指标，用来评价组织氧化应激水平。GSH-Px是一种存在于线粒体内的抗氧化酶抑制脂质过氧化，从而保护细胞免受氧化应激，从而保护细胞免受氧化应激损伤。CAT存在于细胞的过氧化物酶体内，一般对于人体具有保护作用和解毒的作用，可以清除体内产生的活性氧物质(Reactive oxygen species，ROS)，因此用来评价细胞内氧化应激水平。
肿瘤坏死因子-α(Tumor necrosis factor-alpha，TNF-α)、转化生长因子-β(transforming growth factor-β，TGF-β)同为细胞因子，常用来评价细胞内炎症水平。TNF-α属于肿瘤坏死因子家族的成员，机体产生炎症反应时数量增多，它在机体的炎症和免疫应答中发挥重要的作用，因此用来评价炎症病变、疾病发展和组织损伤情况。TGF-β属于转化生长因子家族的成员，是多种生物学功能的多肽类细胞因子，由淋巴细胞、NK细胞及巨噬细胞产生，并具有抗感染、抗病毒等作用，常用作评价炎症水平。
试验时，大鼠于再灌注1h后，根据分组分别给予生理盐水(与PMP等体积)，PMP、化合物1及化合物2与PMP等摩尔量)。给予药物治疗24h后处死并取脑。用生理盐水冲洗残血，吸干水分后称重，并按照相应说明书对抗氧化因子及炎症因子进行检测。结果如图3所示。
由图6可知，相较于MCAO组，化合物1组及化合物2中SOD(图6B)、GSH-Px(图6C)、CAT(图6D)活力显著升高(p<0.001)，MDA(图6A)活力显著降低(p<0.001)，表明化合物1和化合物2有良好的抗氧化能力。同时相较于MCAO组，化合物1组及化合物2中的TNF-α(图6E)活力显著下降(p<0.001)，TGF-β(图6F)显著上升，表明化合物1和化合物2有抑制炎症的能力。
H&E染色、尼式染色(NISSL)和脑组织活性氧免疫荧光染色(ROS)能够直接反映出脑组织神经细胞损伤程度与氧化应激水平。HE染色结果、尼式染色结果以及脑组织活性氧免疫荧光(ROS)定量分析结果分别如图7、图8所示。
由图7可知，对照组(Sham组)脑部神经元细胞清晰可见、胞质着色深，胞核呈紫蓝色圆形、形态正常且排列紧密且无病理改变。模型组(MCAO组)细胞数量数显著减少、细胞核染色变浅、间质空隙变大、大量神经元死亡且完整度较低。经化合物1或化合物2治疗后，模型大鼠细胞核固缩数量明显减少，正常细胞数量增加，间质空隙与对照组基本相同，由此表明化合物1与化合物2可显著降低脑组织损伤，对脑卒中有很好的治疗作用。
由图8可知，经4',6-二脒基-2-苯基吲哚(DAPI)与二氢乙锭(DHE)(染料名称)染色后，Sham组脑组织细胞核大部分呈现荧光，仅有少量活性氧荧光存在于脑组织中，提示正常脑组织中ROS存在数量较少。相较于Sham组，MCAO组中活性氧荧光大量存在，表明MCAO组缺血再灌注产生较严重的氧化损伤；PMP组活性氧荧光水平较低，化合物1及化合物2组 活性氧荧光显著降低(p<0.001)，说明化合物1和2能减少脑部因缺血再灌注而产生的活性氧，改善脑部氧化应激反应损伤。
上述实施方式仅为本申请的优选实施方式，不能以此来限定本申请保护的范围，本领域的技术人员在本申请的基础上所做的任何非实质性的变化及替换均属于本申请所要求保护的范围。

Claims (10)

  1. 一种基于依达拉奉结构的前体化合物或其药学上可接受的盐，其特征在于，具有结构通式Ⅰ
    其中，R1、R2、R3、R4各自独立地选自氢、卤素、羟基、巯基、硝基、氨基、乙酰氨基、氰基、乙酰氧基、乙酸酯基、C1-C4烷基、C1-C4烷氧基、三氟甲基、三氟甲氧基；
    X选自氮、CH2、氧或硫；
    m选自0至4的自然数；
    A为R6和R7各自独立地选自氢原子、C1-C6烷基、C3-C7环烷基或C1-C5烷基氧基C1-C5烷基；
    或者A为含有1个N原子的C3-C10杂环或取代杂环。
  2. 如权利要求1所述的基于依达拉奉结构的前体化合物或其药学上可接受的盐，其特征在于，R1、R2、R3、R4均为氢；
    X选自CH2或氧，m为0或2；
    A选自
    或者A选自
  3. 如权利要求1所述的基于依达拉奉结构的前体化合物或其药学上可接受的盐，其特征在于，所述化合物为
  4. 如权利要求1至3任一项所述的基于依达拉奉结构的前体化合物或其药学上可接受的盐，其特征在于，所述盐通过所述基于依达拉奉结构的前体化合物与酸制备得到；
    所述酸包括盐酸、氢溴酸、硫酸、磷酸、乙酸、三氟乙酸、乳酸、丙酮酸、丙二酸、琥珀酸、戊二酸、富马酸、酒石酸、马来酸、柠檬酸、抗坏血酸、草酸、烟酸、樟脑酸、葡萄糖酸、葡萄糖醛酸、甲磺酸、乙磺酸、氨基磺酸或对甲苯磺酸。
  5. 一种基于依达拉奉结构的前体化合物的制备方法，其特征在于，包括：
    当X为CH2时，将与或含有1个N原子的C3-C10杂环或取代杂环在碱性条件下进行胺化反应制得水解脱去R5基团得到将其与依达拉奉通过酰化反应制备得到如权利要求1至3任一项所述的基于依达拉奉结构的前体化合物；
    或当X为氧时，将与在碱性条件下醚化制备得到水解脱去R5基团，得到将其与依达拉奉通过酰化反应制备得到如权利要求1至3任一项所述的基于依达拉奉结构的前体化合物；
    其中，R1、R2、R3、R4各自独立地选自氢、卤素、羟基、巯基、硝基、氨基、乙酰氨基、氰基、乙酰氧基、乙酸酯基、C1-C4烷基、C1-C4烷氧基、三氟甲基、三氟甲氧基；
    m选自0至4的自然数；
    Y选自卤素Cl、Br或I；
    A为R6和R7各自独立地选自氢原子、C1-C6烷基、C3-C7环烷基、C1-C5烷基氧基C1-C5烷基；
    或者A为含有1个N原子的3-10元杂环或取代杂环。
  6. 如权利要求5所述的方法，其特征在于，所述m选自0或2；
    Y选自Cl；
    R1、R2、R3和R4为氢原子；
    A选自
    或者A选自
  7. 一种基于依达拉奉结构的前体化合物或其药学上可接受的盐的应用，其特征在于，所述基于依达拉奉结构的前体化合物用于制备预防或治疗中枢神经损伤、中枢神经变性类疾病的药物。
  8. 如权利要求7所述的应用，其特征在于，所述中枢神经损伤、中枢神经变性类疾病包括脑卒中、肌萎缩性侧索硬化症、阿兹兹海默或帕金森。
  9. 如权利要求7所述的应用，其特征在于，所述药物的剂型包括片剂、栓剂、软胶囊剂、硬胶囊剂、溶液剂、混悬剂、气雾剂、注射剂、冻干粉针剂、缓控释制剂或各种微粒给药系统，通过口、鼻内、直肠、透辟或注射方式进行给药。
  10. 如权利要求9所述的应用，其特征在于，所述药物的剂型为注射剂。
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