CN111793032B

CN111793032B - Pyrazolone compounds and preparation method and application thereof

Info

Publication number: CN111793032B
Application number: CN201910277582.0A
Authority: CN
Inventors: 陈雏; 吴燕; 李彬; 杜蕾蕾
Original assignee: Sichuan Academy of Chinese Medicine Sciences SACMS
Current assignee: Sichuan Academy of Chinese Medicine Sciences SACMS
Priority date: 2019-04-08
Filing date: 2019-04-08
Publication date: 2021-11-19
Anticipated expiration: 2039-04-08
Also published as: CN111793032A

Abstract

The invention discloses pyrazolone compounds, belonging to the technical field of pharmaceutical chemistry, wherein the pyrazolone compounds have a structure shown in formula (I):

(I) wherein: r₁、R₂Independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, R₃、R₄Independently selected from hydrogen, methyl, methylene; the invention also discloses pharmaceutically acceptable salts and tautomers of the compounds; the compound can reduce cell damage caused by oxidative stress, can penetrate through a blood brain barrier and can remove free radicals, the concentration of the compound in brain tissues is higher than that of edaravone, and the metabolic stability of the compound in the brain tissues is better; and the water solubility is better, the physicochemical property is more stable, and the preparation method is suitable for preparing various solid and liquid preparations, especially injections, and can be used for preparing medicaments for preventing and/or treating oxidative stress injury related diseases.

Description

Pyrazolone compounds and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicinal chemistry, in particular to pyrazolone compounds and a preparation method and application thereof.

Background

Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-ketone, 3-methyl-1-phenyl-2-pyrazolin-5-one) is a common clinical neuroprotective agent, and is mainly used for improving neurological symptoms, daily life activity and dysfunction caused by acute cerebral infarction and delaying the progress of patients with amyotrophic lateral sclerosis.

Edaravone is a potent radical scavenger. Structurally, there are three tautomers of edaravone molecules in which the enol structure can be converted to the anionic form.

Under the condition of physiological pH value, 50% of edaravone exists in an anion form, and the anion form has strong free radical scavenging capacity and can transfer electrons to free radicals and convert the free radicals into radicals without oxidation activity, so that the damage of the free radicals to tissue cells is reduced or eliminated (Mallotus, et al, Chinese clinical pharmacology and therapeutics, 2011,16: 341-.

Oxidative stress and neuroinflammation are common bases for various brain and nervous system diseases. Oxidative stress refers to cell damage caused by an increase in reactive oxygen species such as superoxide anions, hydrogen peroxide, nitric oxide, and hydroxyl radicals. Brain tissue is rich in unsaturated fatty acids and is susceptible to oxidative stress damage. Cerebral infarction and subarachnoid hemorrhage are the most common cerebrovascular diseases, and severe oxidative and inflammatory injury can occur after the occurrence of the cerebrovascular diseases. The edaravone can capture excessive free radicals generated in the ischemic or hemorrhagic state of brain tissues, inhibit lipid peroxidation and relieve oxidative damage to nerve cells, vascular endothelial cells and lipids. Edaravone can inhibit the activity of xanthine and hypoxanthine oxidase, reduce the generation of free radicals, enhance the activity of superoxide dismutase, inhibit the irreversible damage of free radicals to proteins and nucleic acids, and reduce the death of nerve cells in the late stage of cerebral infarction (Zhao Xiaoying, et al. anesthetic safety and quality control, 2018,2: 107-.

Brain tissue consumes high oxygen, and the intrinsic antioxidant substances are relatively lack, and are very sensitive to oxidative stress. Neurons belong to terminal cells which are difficult to renew, and long-life nerve cells are easy to accumulate oxidative damage. In fact, nerve damage and glial activation caused by chronic oxidative stress are considered to be closely related to the pathogenesis and disease progression of neurodegenerative diseases such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, etc. (Zhao Chunyang, et al. pharmaceutical science, 2015,50, 375-. Edaravone can delay the progress of neurodegenerative disease to some extent and has the potential effect of improving the neurobehavioral performance of patients (Shu-Sheng Jiano et al proceedings of the National Academy of Sciences,2015,112, 5225-5230).

Besides the antioxidant activity based on free radical removal, edaravone also has a strong anti-inflammatory effect, and can reduce the content of inflammatory factors of patients with cerebral hemorrhage, reduce the level of serum inflammatory factors of patients with cerebral infarction, relieve inflammatory injury and improve nerve functions. Edaravone can inhibit the decrease of cerebral blood flow around cerebral ischemic tissues, improve the survival rate of nerve cells, alleviate vascular endothelial cell injury, prevent the progression of cerebral edema and cerebral infarction, alleviate the accompanying neurological symptoms, and inhibit delayed neuronal death (Zhang rock, et al, J. China neurotrauma surgery, 2017,3: 44-47).

Because the edaravone has short half-life and high metabolism speed in vivo, the marketed formulation of the edaravone is only injection in order to take effect quickly and achieve higher drug concentration in brain. However, edaravone is poorly water soluble (3mg/mL, 20 ℃), increasing the difficulty of formulation and quality control. When the injection is prepared, a pH regulator is required to be added to enable the solution to be alkalescent so as to improve the solubility, but the edaravone has poor stability under the alkaline condition, and an antioxidant is required to be added so as to reduce the oxidation of the edaravone, so that the quality control difficulty in the production and storage processes of the injection is increased, and the risk of adverse reaction of a patient during use is increased. The edaravone injection can be used only by diluting with normal saline to avoid the precipitation and degradation of components. This poses a disadvantageous limitation for clinical combinations.

Edaravone has been reported to be present in cerebrospinal fluid at concentrations of 50% to 60% of plasma. However, due to the presence of the cerebrospinal fluid-brain barrier in the body, there may be significant differences in drug concentration in cerebrospinal fluid and brain tissue, and the drug concentration in cerebrospinal fluid is not in fact simply equivalent to the drug concentration in brain tissue. The inventor finds that the drug concentration in the brain is only about 5% of that in the plasma after rats or mice are injected with edaravone intravenously. It follows that edaravone has a poor ability to cross the blood-brain barrier into brain tissue. As a neuroprotective drug, this undoubtedly limits the exertion of edaravone's antioxidant activity in the brain.

Disclosure of Invention

In order to overcome the defects of edaravone and obtain a better neuroprotective medicament, the applicant proposes the invention.

The invention aims to provide pyrazolone compounds and pharmaceutically acceptable salts thereof. The pyrazolone compound has a structure shown in a general formula I:

wherein: r₁、R₂Independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, R₃、R₄Independently selected from hydrogen, methyl, methylene.

The pyrazolone compound provided by the invention comprises keto-type, enol-type and amine-type tautomers thereof under applicable conditions, namely tautomers similar to the edaravone.

The pyrazolone compound of the present invention is further preferably selected from the following compounds:

the term "pharmaceutically acceptable salt" as used herein means a salt of a compound of formula I with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid, or with an organic acid such as methanesulfonic acid, ethanesulfonic acid, oxalic acid, succinic acid, malic acid, tartaric acid, fumaric acid, maleic acid, citric acid, malonic acid, acetic acid, lactic acid, benzoic acid, phthalic acid, salicylic acid, p-toluenesulfonic acid, mandelic acid, nicotinic acid, ascorbic acid, glutamic acid, aspartic acid or lysine.

The second objective of the present invention is to provide a preparation method of the pyrazolone compound, which adopts a technical scheme that the pyrazolone compound can be prepared by the following steps:

the method comprises the following specific steps:

(1) preparation of a compound of formula ii:

the compound in the general formula II can be obtained by nucleophilic substitution reaction of 4-chloro-or bromo-substituted ethyl acetoacetate and derivatives thereof with secondary amine. The reaction solvent is selected from methanol, ethanol, isopropanol, n-butanol, tetrahydrofuran, acetonitrile, ethyl acetate, dichloromethane and water, or a mixed solvent of the solvents; the acid-binding agent is selected from sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium acetate, potassium acetate and triethylamine; the reaction temperature varies depending on the reagents or solvents, etc., and is usually from-10 ℃ to 150 ℃, preferably from 20 ℃ to 70 ℃; the reaction time is not limited, and is usually 0.5 to 12 hours.

(2) Preparation of a compound of general formula i:

reacting the compound of the general formula II with phenylhydrazine to obtain the compound of the general formula I. The reaction solvent is selected from methanol, ethanol, isopropanol, n-butanol, tetrahydrofuran, acetonitrile, ethyl acetate, dichloromethane, or mixture thereof; the reaction temperature varies depending on the reagents or solvents, etc., and is usually from-10 ℃ to 150 ℃, preferably from 10 ℃ to 100 ℃; the reaction time is not limited, and is usually 1 hour to 24 hours.

The third object of the present invention is to provide a pharmaceutical composition comprising a safe and effective amount of one or more pyrazolones and pharmaceutically acceptable salts thereof.

The term "composition" as used herein refers to a product formed by mixing more than one substance or component. The composition of the present invention comprises 1 wt% (weight percentage) to 95 wt%, preferably 10 wt% to 80 wt%, of the above pyrazolone compound and its pharmaceutically acceptable salt, based on the total weight of the pharmaceutical composition.

As used herein, a "safe and effective amount" means that the compound of the present invention is administered in an amount sufficient to significantly ameliorate the condition without causing serious side effects. The safe and effective amount of the compound of the present invention may vary depending on the route of administration, age, weight, type of disease and severity of the disease in the patient, etc. For a human weighing 60kg, the daily dose administered is generally from 10mg to 1000mg, preferably from 25mg to 500 mg. Can be administered in one or more divided doses.

As one of the preferable embodiments, the pharmaceutical composition can be further added with one or more pharmaceutically acceptable carriers to prepare pharmaceutically acceptable pharmaceutical preparations.

The term "pharmaceutically acceptable carrier" as used herein means one or more compatible solid or liquid fillers or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of blending with and between the pyrazolones of the present invention without significantly reducing the efficacy of the pyrazolones. Pharmaceutically acceptable carriers include sugars (e.g., sucrose, lactose, glucose, etc.), starches (e.g., potato starch, corn starch, etc.), celluloses and derivatives thereof (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, microcrystalline cellulose, etc.), dextrins and derivatives thereof, aerosil, gelatin, talc, calcium sulfate, solid lubricants (e.g., magnesium stearate, stearic acid, etc.), vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, castor oil, cottonseed oil, corn germ oil, palm oil, etc.), alcoholic solvents (e.g., ethanol, propylene glycol, glycerol, mannitol, sorbitol, polyethylene glycol, etc.), surfactants, isotonicity adjusting agents, pH adjusting agents, emulsifiers, wetting agents, colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water.

The medicine preparation of the present invention includes tablet, capsule, pill, granule, dripping pill, powder, lozenge, injection, freeze dried powder for injection, syrup, oral liquid, plaster, inhalation powder spray and spray, and is administrated through oral taking, intravenous injection, intramuscular injection, subcutaneous injection, sublingual, transdermal, oral cavity spray and nasal cavity spray.

The fourth purpose of the present invention is to provide the use of the above pyrazolone compound and its pharmaceutically acceptable salt, or the above pharmaceutical composition, for preparing a medicament for preventing and/or treating diseases associated with oxidative stress injury.

The "diseases associated with oxidative stress injury" described in the present invention include alzheimer's disease, parkinson's disease, huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral edema, and traumatic brain injury.

In another preferred embodiment, the "use" refers to the preparation of a medicament for improving neurological symptoms, activities of daily life and dysfunction caused by acute cerebral infarction.

In another preferred embodiment, the "use" refers to the preparation of a medicament for delaying the progression of a patient with amyotrophic lateral sclerosis.

Compared with the prior art, the invention has the beneficial effects that: the pyrazolone compound disclosed by the invention can relieve cell damage caused by oxidative stress; the pyrazolone compound disclosed by the invention can penetrate through a blood brain barrier, the concentration of the pyrazolone compound in brain tissues is higher than that of edaravone, and the pyrazolone compound in the brain tissues has better metabolic stability; the pyrazolone compound disclosed by the invention has good water solubility and stable physicochemical properties, and is suitable for preparing various solid and liquid preparations, especially injections; the pyrazolone compound disclosed by the invention is low in toxicity and good in safety.

Drawings

FIG. 1 is H₂O₂A graph of cell viability results for the induced cell injury assay;

FIG. 2 is a graph showing the number of viable neuronal cells in the same part of the brain region of hippocampal CA1 and striatal CPu in the rat focal ischemia test.

Detailed Description

The invention will be further explained with reference to the drawings.

EXAMPLE 1 preparation of Compound E-1

40ml of a tetrahydrofuran solution (2mol/L) of dimethylamine, 15ml of water and 11g of sodium carbonate were sequentially added to a 100ml round-bottomed flask, and the mixture was stirred at room temperature for 5 minutes. The reaction system is placed in an ice bath, 6.5g of 4-chloroacetoacetic acid ethyl ester is slowly dropped, the temperature is slowly raised to the room temperature after the dropping is finished, and then the reaction is carried out for 6 hours at the temperature of 40 ℃. Filtering the reaction solution, concentrating the filtrate, separating with silica gel column, performing gradient elution with dichloromethane-methanol (100: 0-10: 1, v/v), combining the fractions according to TLC, concentrating, evaporating the solvent to obtain ethyl 4- (N, N-dimethylamino) acetoacetate, and obtaining dark brown red liquid 3.1g with a yield of 45%.

In a 25ml round-bottom flask, 2.2g of ethyl 4- (N, N-dimethylamino) acetoacetate, 10ml of absolute ethanol and 1.3g of phenylhydrazine are sequentially added, the mixture is reacted for 12 hours at 20 ℃, filtered, washed by a small amount of ethyl acetate, and the precipitate is dried under reduced pressure to obtain a compound E-1, 1.6g of white powder with the yield of 57 percent.

¹H-NMR(600MHz，D₂O)：δ7.47(2H，d)，δ7.42(2H，t)，δ7.29(1H，t)，δ4.67(2H，s)，δ3.99(2H，s)，δ2.77(6H，s)；¹³C-NMR(150MHz，CD₃OD)：δ162.9，δ141.9，δ138.6，δ129.1，δ126.8，δ123.9，δ87.3，δ55.3，δ42.2；HR-TOF-MS(m/z)：218.1289([M+H]⁺The theoretical value is as follows: 218.1293, [ C ]₁₂H₁₆N₃O]⁺)。

EXAMPLE 2 preparation of the hydrochloride salt of Compound E-1

Adding compound E-1680 mg and methanol 40ml into a 100ml round bottom flask in sequence, stirring and dissolving at 60 ℃, introducing HCl gas, recovering the solvent to about 10ml under reduced pressure, standing and crystallizing at-20 ℃, filtering, washing the precipitate with a small amount of absolute ethyl alcohol, drying at 50 ℃ under reduced pressure for 6 hours to obtain hydrochloride of the compound E-1, wherein the white sand crystal is 540mg, and the yield is 79%. The hydrochloride salt of E-1 is readily soluble in water (110mg/ml, 20 ℃).

¹H-NMR(600MHz，CD₃OD)：δ7.73(2H，d)，δ7.51(2H，t)，δ7.40(1H，t)，δ5.20(2H，s)，δ4.35(2H，s)，δ2.95(6H，s)。¹³C-NMR(150MHz，CD₃OD)：δ154.8，δ140.8，δ136.5，δ128.9，δ127.7，δ122.8，δ90.3，δ53.4，δ42.0。

EXAMPLE 3 preparation of hydrochloride salt of Compound E-4

50ml of water, 50ml of ethyl acetate, 5.5g of potassium carbonate and 11.7g of diethylamine were sequentially added to a 250ml round-bottom flask, and the mixture was stirred at room temperature for 10 minutes. The reaction system is placed in an ice bath, 6.5g of 4-chloroacetoacetic acid ethyl ester is slowly dropped, and the temperature is slowly raised to 60 ℃ after dropping for reaction for 2 hours. The aqueous layer was separated, the ethyl acetate layer was washed with saturated brine and then adjusted to pH 3 with 2mol/L hydrochloric acid, and the resulting acid aqueous layer was separated and adjusted to pH 9 with 2mol/L sodium hydroxide. The basified aqueous layer was extracted with ethyl acetate. The ethyl acetate extract was evaporated under reduced pressure to remove the solvent, to give ethyl 4- (N, N-diethylamino) acetoacetate as a dark brown-red liquid (2.0 g) in a yield of 25%.

A25 ml round bottom flask was charged with ethyl 4- (N, N-diethylamino) acetoacetate 1.0g, methanol 5ml, and phenylhydrazine 0.6g in this order, and reacted at 50 ℃ for 3 hours. Evaporating the reaction solution to dryness, adding ethyl acetate to dissolve the reaction solution, then extracting the reaction solution by using water, concentrating a water layer, separating the water layer by using a reverse phase silica gel chromatographic column, performing gradient elution by using 10-20% ethanol, combining target fractions, adjusting the pH value to be 3 by using 4mol/L hydrochloric acid, and evaporating the target fractions under reduced pressure to dryness to obtain a compound E-4 hydrochloride, wherein the content of a light yellow solid is 0.34g, and the yield is 28%.

¹H-NMR(600MHz，CD₃OD)：δ7.73(2H，d)，δ7.48(2H，t)，δ7.36(1H，t)，δ5.06(2H，s)，δ4.31(2H，s)，δ3.30(4H，m)，δ1.39(6H，t)。¹³C-NMR(150MHz，CD₃OD)：δ154.3，δ140.8，δ137.4，δ128.7，δ127.1，δ122.3，δ89.7，δ48.4，δ8.1。

As the compounds of the invention all contain alkyl-substituted N atoms, namely the compounds belong to alkalescent compounds, and the water solubility can be better after the compounds are salified with acid. For basic drug molecules, the salt form is commonly used in the art, unless the solubility of the basic drug molecule is good enough, for the compounds of the present application, because they are salts of weakly basic compounds, the ionic state (salt) is less when they are dissolved in pure water or enter the body as if they are predominantly molecular (free base); therefore, since neither the free base nor the salt form thereof has a substantial difference in the radical scavenging ability, the in vivo activity, the metabolism, and the like, the following examples mainly use the salt form to conduct the relevant effect experiments.

Example 4ABTS free radical scavenging ability

Taking a proper amount of ABTS, and taking water as a solvent to prepare a solution with the concentration of 7.4 mmol/L. Get K₂S₂O₈Adding water to dissolve the mixture in a proper amount to prepare a solution with the concentration of 2.6 mmol/L. Taking ABTS stock solution and K₂S₂O₈Mixing the stock solutions in equal volume, standing at room temperature in dark place for 12-16 hr, diluting with pH 7.4 phosphate buffer solution 40-50 times (the absorbance value of the solution at 734nm is 0.7 + -0.02), and getting ABTS⁺And (4) working fluid. Taking a proper amount of a test article, taking water as a solvent to prepare a solution with the concentration of about 1mmol/L, and gradually diluting the solution with water into test article solutions with 5 series of concentrations. Precisely sucking 0.2ml of each test solution, and respectively adding ABTS⁺9.8ml of working solution is shaken up, kept stand for 6min and the absorbance value A is measured at 734 nm. 0.2ml of water and ABTS⁺Working solution 9.8ml of mixed solution is used as a control, and the absorbance value A is measured at 734nm₀Water was used as blank reference.

Clearance (%) - (1-A/A)₀)×100％

ABTS with Compound concentration as abscissa⁺Clearance is ordinate, curve is fitted, and ABTS clearance of each compound is calculated⁺IC of free radicals₅₀The value is obtained. The results show that edaravone scavenges ABTS under this test condition⁺IC of free radicals₅₀ABTS scavenging value of 14.7. mu.M, E-1 and its hydrochloride⁺IC of free radicals₅₀The values are respectively 11.1 mu M and 11.2 mu M, and E-4 and the hydrochloride thereof clear ABTS⁺IC of free radicals₅₀The values were 9.4. mu.M and 9.9. mu.M, respectively. The result shows that the pyrazolone compound disclosed by the invention has stronger free radical scavenging capacity.

Example 5 pairs H₂O₂Inducing cell damageProtective effect of

Taking PC12 cells in logarithmic growth phase, adjusting cell density to 1 × 10⁶Inoculating to 96-well plate at 100 μ L/well, and placing in 5% CO₂The culture was carried out in an incubator at 37 ℃ for 24 hours. The culture medium was changed, the administration groups were pretreated for 1 hour by adding different test compounds (final concentration: 10. mu.M) to the groups, and H was added to the groups except for the normal group ("Con" group in FIG. 1)₂O₂(final concentration 200. mu.M) caused free radical damage to the cells, which were cultured for an additional 24 hours. Then, the cell viability of each group was measured according to the MTT method, i.e., 5mg/mL MTT 15. mu.L was added to each well, the culture was continued for 4 hours, the supernatant was discarded, 200. mu.L DMSO was added to each well, the shaking was repeated for 5 minutes until the blue-violet crystals were completely dissolved, and the cell viability was measured and calculated by a microplate reader at 490nm wavelength, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the pyrazolone compounds disclosed by the invention can obviously reduce cell damage caused by oxidative stress (and model group, namely H)₂O₂Group comparison of P<0.05，**P<0.01)。

Example 6 neuroprotective Effect on cerebral ischemic mice

SPF-grade male C57 mice were randomly grouped into 8 mice each, and a cerebral ischemia model with bilateral common carotid artery transient ligation was prepared (Free radial Biology and medicine.2011,50: 1780-1786; neuropharmacology.2014,77: 453-464). At ischemia-reperfusion time, 24 hours and 48 hours, equimolar doses of compound (edaravone 3mg/kg, E-1 hydrochloride 4.38mg/kg, E-4 hydrochloride 4.86mg/kg) or blank vehicle (Sham and Model groups) were injected intravenously, respectively. 72 hours after reperfusion, neuro-behavioral assessment was performed, and animals were sacrificed to take brain Nissl staining and the number of viable neuronal cells at the same site in hippocampal CA1 region and striatal CPu brain region was examined, and the results are shown in FIG. 2.

In contrast to the Sham group (Sham group), transient ligation of bilateral common carotid arteries in mice to cause cerebral ischemia caused neurobehavioral disturbance, neuronal loss, and excessive astrocyte activation in the Model group (Model group) animals. After the mouse is subjected to cerebral ischemia reperfusion, the tested compound is injected into the vein, and E-1 hydrochloride and E-4 hydrochloride both show neuroprotective effect, so that the neurobehavioral disturbance of the mouse in 24 hours, 48 hours and 72 hours after operation under the cerebral low perfusion state can be improved, the neuron cell number of hippocampus and striatum brain area can be increased, and the effect is superior to that of edaravone.

Example 7 blood brain Barrier permeability and Metabolic stability

Male SD rats weighing 200-220 g were randomly divided into 2 groups of 6 rats, fasted for 12 hours, and given an equimolar dose of E-1 hydrochloride (12.7mg/5ml/kg) or edaravone (8.7mg/5ml/kg) intravenously. Each group died 3 animals each 5min and 60min after dosing. Centrifuging at low temperature to prepare blood plasma, adding 2 times of physiological saline into brain, and grinding to prepare brain homogenate. Plasma and brain homogenate samples were assayed by UPLC precipitation of protein. The blood brain barrier permeability of the compound is evaluated by the concentration ratio of the compound in brain tissue to plasma (B/P value), and the larger the B/P value is, the easier the compound is to penetrate the blood brain barrier.

Chromatographic conditions are as follows: waters Acquity UPLC (containing high pressure binary pump, vacuum degasser, autosampler, column oven, DAD detector); chromatographic column Hypersil-Gold C18(2.1 mm. times.50 mm, 3 μm); the mobile phase was ammonium acetate buffer (pH 4.0 adjusted with acetic acid) -methanol (75:25, v/v); the flow rate is 0.4 ml/min; the column temperature is 30 ℃; the detection wavelength was 240 nm.

The results are shown in the following table:

the above results indicate that E-1 hydrochloride is more permeable to the blood brain barrier and has better metabolic stability in the brain than edaravone.

EXAMPLE 8 preparation of tablets

1. Prescription (1000 tablets):

e-4 hydrochloride	100g
		Starch	50g
Microcrystalline cellulose	20g
		Magnesium stearate	2g
Sodium carboxymethylcellulose	5g
		50% ethanol	Proper amount of

2. The preparation method comprises the following steps: pulverizing compound E-4, starch, microcrystalline cellulose and sodium carboxymethylcellulose, mixing, making into soft mass with 50% ethanol as wetting agent, sieving with 20 mesh sieve, granulating, drying at 60-80 deg.C, grading, adding magnesium stearate, mixing, tabletting, and coating.

EXAMPLE 9 preparation of injection

1. Prescription (1000 counts):

compound E-1	10g
		Sodium chloride	18g
Water for injection is added to	2000ml

2. The preparation method comprises the following steps: sequentially adding sodium chloride and compound E-1 into water for injection, stirring to dissolve, adding water for injection to a sufficient amount, stirring, filtering the solution, bottling, and sterilizing.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A pyrazolone compound and a pharmaceutically acceptable salt thereof, characterized in that: the pyrazolone compound is selected from the following formulas:

2. the pyrazolone compound and a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: the pharmaceutically acceptable salt is selected from hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, ethanesulfonate, oxalate, succinate, malate, tartrate, fumarate, maleate, citrate, malonate, acetate, lactate, benzoate, phthalate, salicylate, p-toluenesulfonate, mandelate, nicotinate, ascorbate, glutamate, aspartate, lysine.

3. The process for the preparation of compound E-1 and compound E-4 hydrochloride according to claim 1, wherein,

the preparation method of the compound E-1 comprises the following steps: 40ml of tetrahydrofuran solution of dimethylamine is sequentially added into a 100ml round-bottom flask, the concentration of the tetrahydrofuran solution is 2mol/L, 15ml of water and 11g of sodium carbonate, and the mixture is stirred for 5 minutes at room temperature; slowly dripping 6.5g of 4-chloroacetoacetic acid ethyl ester into the reaction system in an ice bath, slowly heating to room temperature after dripping, and then reacting for 6 hours at 40 ℃; filtering the reaction solution, concentrating the filtrate, separating with silica gel column, performing gradient elution with dichloromethane-methanol at a ratio of 100: 0-10: 1, v/v, combining the fractions according to TLC, concentrating, and evaporating the solvent to obtain 4- (N, N-dimethylamino) ethyl acetoacetate;

adding 2.2g of 4- (N, N-dimethylamino) ethyl acetoacetate, 10ml of absolute ethyl alcohol and 1.3g of phenylhydrazine into a 25ml round-bottom flask in sequence, reacting for 12 hours at 20 ℃, filtering, washing with ethyl acetate, and drying a precipitate under reduced pressure to obtain a compound E-1;

the preparation method of the hydrochloride of the compound E-4 comprises the following steps: 50ml of water, 50ml of ethyl acetate, 5.5g of potassium carbonate and 11.7g of diethylamine are sequentially added into a 250ml round-bottom flask, and stirred for 10 minutes at room temperature; slowly dripping 6.5g of 4-chloroacetoacetic acid ethyl ester into the reaction system in an ice bath, and slowly heating to 60 ℃ after dripping for reacting for 2 hours; separating out a water layer from the reaction liquid, washing an ethyl acetate layer by using saturated saline solution, adjusting the pH value of the ethyl acetate layer to be 3 by using 2mol/L hydrochloric acid, and adjusting the pH value of the separated acid water layer to be 9 by using 2mol/L sodium hydroxide; extracting the alkalized water layer with ethyl acetate; distilling the ethyl acetate extract under reduced pressure to remove the solvent to obtain 4- (N, N-diethylamino) ethyl acetoacetate;

adding 1.0g of 4- (N, N-diethylamino) ethyl acetoacetate, 5ml of methanol and 0.6g of phenylhydrazine into a 25ml round-bottom flask in sequence, and reacting for 3 hours at 50 ℃; evaporating the reaction solution to dryness, adding ethyl acetate to dissolve the reaction solution, extracting the reaction solution by using water, concentrating a water layer, separating the water layer by using a reverse phase silica gel chromatographic column, performing gradient elution by using 10 to 20 percent ethanol, combining target fractions, adjusting the pH value to be 3 by using 4mol/L hydrochloric acid, and evaporating the target fractions to dryness under reduced pressure to obtain a compound E-4 hydrochloride.

4. A pharmaceutical composition containing the pyrazolone compound according to claim 1 and a pharmaceutically acceptable salt thereof, characterized in that: the pharmaceutical composition comprises: (a) a therapeutically effective amount of one or more selected from the group consisting of the pyrazolones and pharmaceutically acceptable salts thereof according to claim 1; and (b) one or more pharmaceutically acceptable carriers.

5. Use of the pyrazolone compound according to claim 1 and a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 4 for the preparation of a medicament for the prevention and/or treatment of diseases associated with oxidative stress injury.

6. Use according to claim 5, characterized in that: the disease is neurodegenerative disease caused by chronic oxidative stress.

7. Use according to claim 6, characterized in that: the neurodegenerative disease caused by chronic oxidative stress is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis and amyotrophic lateral sclerosis.

8. Use according to claim 5, characterized in that: the disease is selected from ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral edema, and traumatic brain injury.