CN112480085B

CN112480085B - A compound or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof

Info

Publication number: CN112480085B
Application number: CN202011502210.2A
Authority: CN
Inventors: 刘国强; 王延东
Original assignee: Shanghai Innofucheng Biotechnology Co ltd
Current assignee: Shanghai Innofucheng Biotechnology Co ltd
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2022-10-25
Anticipated expiration: 2040-12-17
Also published as: WO2022127050A1; CN112480085A

Abstract

The invention relates to the field of pharmacy, in particular to a compound or pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof. The invention provides a compound or pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, wherein the chemical structural formula of the compound is shown as a formula I. Compared with similar compounds in the prior art, the compound provided by the invention has the advantages of higher performance, better antioxidation, nerve cell protection and platelet aggregation inhibition activities, is easy to pass through a blood brain barrier, has good oral bioavailability, can greatly improve the compliance and clinical convenience of patients, can be developed into medicines for treating and preventing oxidative stress related diseases such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases and the like, can also be used for treating thromboembolic diseases, and has good industrial prospects.

Description

A compound or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof

Technical Field

The invention relates to the field of pharmacy, in particular to a compound or pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof.

Background

A free radical is an atom, molecule, ion, or group of atoms that has one or more unpaired electrons and is highly reactive. Free radicals are mainly formed during electron transport in the body and are products of aerobic metabolism in normal cells. Aerobic organisms continuously generate free radicals containing oxygen and/or nitrogen with metabolism, mainly including superoxide anion free radical (. O2), hydroxyl free radical (. OH), carboxyl free radical (ROO.), lipoxy free radical, nitric oxide free radical (NO.), nitro free radical (. ONOO-), and the like. Oxygen free radicals are among the most important types, and account for more than 95% of the total free radicals in the human body. Oxygen radicals and H convertible to free radicals ₂ O ₂ Singlet oxygen (1O) ₂ ) And molecules such as ozone are also collectively referred to as Reactive Oxygen Species (ROS).

Free radicals are highly reactive in chemistry and can react with biomolecules (proteins, lipids, sugars, DNA) to alter their structure and function. Under normal conditions, a set of complete antioxidant system is arranged in an organism, and the metabolic balance of free radicals can be maintained. However, in pathological conditions, excessive production or elimination of free radicals can lead to lipid peroxidation damage of biological membranes and macromolecular substances, and trigger pathological oxidative stress in the body.

Research has shown that damage to cellular components such as protein, lipid and DNA caused by oxidative stress is a causative factor in a variety of chronic diseases, such as cardiovascular and cerebrovascular diseases including stroke, atherosclerosis, myocardial infarction, etc., neurodegenerative diseases, diabetes, kidney diseases, retinal diseases, cancer, inflammatory diseases, immune diseases, etc. (Yun-Zhong F, et al. Free radials, antioxidants, and nutrition. Nutrition.2002;18 872-879.. The relationship between antioxidant status of impaired organisms, indicators of oxidative damage and disease is well established.

The brain is one of the most oxygen-loaded and metabolically active organs in the body, and compared with other organs, brain tissues are more susceptible to free radicals and lipid peroxides and oxidative stress. Attack of nerve cells by free radicals may lead to degenerative changes, causing neurodegenerative diseases such as Parkinson's Disease (PD), alzheimer's Disease (AD), multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS). According to research, oxidative stress, mitochondrial dysfunction, excitotoxicity, immune inflammation, apoptosis and the like are main pathogenesis of neurodegenerative diseases. Redox transition metal-catalyzed oxidative stress and free radical generation are believed to play a critical role therein (Mark pm. Metal-catalyzed dispersion of membrane protein and lipid signalling in the pathogenesis of neurogenetic disorders, ann. N. Y. Acad. Sci.2004; 1012. The antioxidant medicine can scavenge or reduce free radicals, protect nerve cells from being damaged by the free radicals, delay and prevent neurodegenerative diseases and play a therapeutic role.

Oxidative stress is also an obvious and important pathological phenomenon in ischemic cerebrovascular diseases, and plays a role in various stages to different degrees. The stimulation of various risk factors such as hypertension, hyperglycemia, hyperlipidemia, infection, overwork and the like can cause the damage of the vascular endothelial function, generate oxidative stress and inflammatory reaction, induce the secretion of various inflammatory factors and start the pathological process of atherosclerosis. Oxidative stress and inflammatory reactions promote each other, which in turn further exacerbates the impairment of endothelial function. Vascular endothelial function is impaired, leading to the entry of Low Density Lipoprotein (LDL) into the endothelium and modification by oxygen radical attack, resulting in the formation of oxidized low density lipoprotein (ox-LDL), which may further induce atherosclerotic plaque formation. While rupture of unstable plaque and thrombus lead to ischemic cerebrovascular disease. Oxidative stress plays an important role in plaque formation, and is a key factor in inducing plaque rupture, which is a trigger for acute cerebrovascular events. Cerebral thrombosis and embolism, which causes the local blood flow reduction or blood supply interruption of brain, cerebral ischemia and anoxia, and cerebral ischemia injury cascade reaction,

ischemia and reperfusion can also cause local free radical burst, lipid peroxidation, and vascular endothelial cell injury, edema of endothelial cell, permeability increase, and brain tissue injury. Oxidative stress can also promote nerve cell necrosis through lipid peroxidation, protein denaturation, and/or DNA modification, and can also initiate nerve cell apoptosis through mitochondrial, endoplasmic reticulum, or death receptor. Therefore, oxidative stress plays a key role in the cascade of cerebral ischemia and reperfusion injury. In conclusion, oxidative stress is involved in the whole process of ischemic cerebrovascular disease initiation from pathology until prognosis recovery. Inhibition of oxidative stress and scavenging of free radicals are important therapeutic strategies throughout the course of ischemic cerebrovascular disease. The antioxidant is used in each stage of ischemic cerebrovascular disease, can bring different benefits to patients, and plays an important role in preventing and treating the occurrence and development of cerebrovascular disease. ( Wang cham, oxidative stress and ischemic cerebrovascular disease, china stroke journal 2008;3 (3): 163-165 )

Oxidative stress is also considered to be closely related to cardiovascular disease, since atherosclerosis is the most prominent pathological basis for cardiovascular disease, and oxidative stress persists throughout atherosclerosis. Oxidative stress plays an important role in the pathological process of cardiovascular diseases such as atherosclerosis, myocardial ischemia and reperfusion injury, myocardial infarction, coronary heart disease, heart failure and the like.

For example, oxygen radicals are formed in the myocardium after ischemia, and can accelerate the formation of oxygen radicals, which can damage the ischemic injury site. Oxidative stress can also damage cell membranes, forming one of the mechanisms of intracellular calcium overload, and in damaged myocardium there can be cardiac contractile dysfunction.

Oxidative stress also plays an important role in the aging process, free radicals leading to aging and various geriatric diseases. Free radicals attack replicating genes, causing mutations in the genes, which induce carcinogenesis. In addition, free radicals are also related to diabetes mellitus and complications of the diabetic vascular system, inflammatory bowel disease, macular degeneration and cataract, rheumatoid arthritis and the like, and free radical damage caused by oxidative stress is involved in the pathological process of the disease.

In conclusion, oxidative stress injury caused by accumulation of free radicals is closely related to the occurrence and development of various diseases. The antioxidant can play a role in preventing or treating related diseases by scavenging free radicals.

Edaravone (Edaravone) is the first free radical scavenger on the market, which can scavenge free radicals and inhibit lipid peroxidation, thereby inhibiting oxidative damage of brain cells, vascular endothelial cells and nerve cells. (K.Toyoda, et al. Free radial coder, edaravone, in tension with internal cardiac array classification. J Neurol Sci,2004 (1-2): 11-17) was approved in Japan as a cerebroprotective agent for the improvement of neurological symptoms, activities of daily life and dysfunctions due to acute cerebral infarction. FDA approval in 2017 was for the treatment of Amyotrophic Lateral Sclerosis (ALS). In addition, edaravone has the potential to be used for preventing and treating various oxidative damages of external organs of the brain and chronic diseases related to free radical damages.

Edaravone belongs to pyrazolone compounds, and can form tautomeric forms including amine forms, ketone forms and enol forms by keto-enol tautomerism under physiological conditions, and is the basis of the antioxidant activity of edaravone in vivo (Kazutoshi Watanabe, et al. How is edaravone effective against oxidation of acid residue biochemical strain and amyotropic latex sensitive J Clin Biochem Nutr.2018;62 (1): 20-38). Preclinical and clinical studies show that edaravone has good free radical scavenging and antioxidant activities, and can protect brain cells, nerve cells and vascular endothelial cells from oxygen radical damage. Although the treatment effect is positive, the method also has obvious defects. Mainly characterized by poor water solubility, low oral bioavailability, clinical administration only by intravenous injection and no prescriptionIt is convenient for clinical use. Furthermore, edaravone, although able to enter the brain to some extent, has limited permeability of the blood-brain barrier. Rat intravenous injection ¹⁴ Only 6% of the plasma radioactivity level (https:// www.accessdata. Fda. Gov /) was found in cerebrospinal fluid (CSF) 5 minutes after C edaravone, affecting its pharmacodynamic performance. The development of an oral effective free radical scavenger which can penetrate the blood brain barrier more easily is clinically unsatisfied with urgent requirements, is expected to improve the curative effect, and is more suitable for long-term use of chronic diseases caused by oxidative stress.

Recently, disclosed in fanglin et al (patent application CN 201810292836.1) is a novel pyrazolol compound which has both effects of resisting platelet aggregation and protecting nerve cells. The compound 1 (Y1502) has the most similar structure to edaravone, and the anti-platelet aggregation activity thereof is similar to edaravone, but the neurocyte protective activity thereof is weaker than that of edaravone. Furthermore, no oral bioavailability data is provided, and no other comparative advantages with edaravone are seen.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a compound or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, which solves the problems of the prior art.

To achieve the above and other related objects, the present invention provides, in one aspect, a compound having a chemical structural formula as shown in formula I:

in another aspect, the present invention provides a process for preparing the above compound, comprising: hydrolyzing a compound of formula III to provide a compound of formula I, the reaction equation is as follows:

in another aspect, the present invention provides the use of a compound as described above, or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, for the manufacture of a medicament.

In another aspect, the present invention provides a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof.

Drawings

FIG. 1 is a graph showing the mean plasma concentration-time curve of male SD rats after gavage or intravenous administration of X1901 in example 7 of the present invention.

Fig. 2 is a graph showing the mean plasma concentration-time curve of male SD rats in example 7 of the present invention after gavage or intravenous administration of Y1502.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

The inventors of the present invention have made extensive practical studies and have unexpectedly found a more advantageous pyrazolone compound which has stronger radical scavenging and antioxidant activities, and has better neuronal protection activity and anti-platelet aggregation activity, and good oral absorption properties, and thus can be used for the preparation of a medicament, and have completed the present invention on this basis.

In a first aspect, the present invention provides a compound, or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, wherein the chemical structural formula of the compound is as shown in formula I:

the compounds of formula I provided above may generally form tautomers and specifically may form a group of enol-keto tautomers. Enol-Keto tautomers (Keto-Enol Tautomerism) generally refer to tautomers that are formed by the formation of a Keto form and an Enol form by the chemical equilibrium between a ketone or an aldehyde and an Enol, i.e., a carbonyl compound such as a ketone or an aldehyde having an acidic α -proton and undergoing proton transfer at different pH values. For example, isomers may include compounds having the chemical structures shown in formula Ia and/or formula II. Specifically, the compounds of formula I provided above may form a group of tautomers, in keto, enol, and amine forms, respectively, with the compounds of formula Ia and/or formula II under certain conditions (e.g., pH = 6-8, or in neutral aqueous solution), as follows:

in the present invention, the term "salt" is to be understood as any form of the active compound used by the present invention, wherein said compound may be in ionic form or charged or coupled to a counter ion (cation or anion) or in solution. This definition may also include quaternary ammonium salts and complexes of the active molecule with other molecules and ions, particularly complexes through ionic interactions. This definition includes in particular physiologically acceptable salts, which term is to be understood as being equivalent to "pharmacologically acceptable salts".

In the present invention, the term "pharmaceutically acceptable salt" generally refers to any salt (generally, this means that it is non-toxic, in particular as a result of counterions) that is physiologically tolerable when used in a suitable manner in therapy, in particular when applied or used in humans and/or mammals. These physiologically acceptable salts may be formed with cations or bases and in the context of the present invention, especially when administered in humans and/or mammals, they are to be understood as being formed by at least one compound provided according to the inventionThe substance, usually an acid (deprotonated), is for example a salt of an anion and at least one physiologically tolerated cation, preferably an inorganic cation. In the context of the present invention, salts with alkali metals and alkaline earth metals, and ammonium cations (NH) may be included in particular ₄ ⁺ ) The salt formed may specifically include, but is not limited to, salts with (mono) or (di) sodium, (mono) or (di) potassium, magnesium or calcium. These physiologically acceptable salts may also be formed with anions or acids, and in the context of the present invention, in particular when administered in humans and/or mammals, they are to be understood as being salts formed by at least one compound provided according to the invention, usually protonated (e.g. on nitrogen), such as a cation and at least one physiologically tolerable anion. In the context of the present invention, salts formed with physiologically tolerable acids, i.e. salts of the particular active compounds with physiologically tolerable organic or inorganic acids, may be included in particular, but not exclusively, with hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid or citric acid.

The term "prodrug" is used in the broadest sense in the present invention and includes those derivatives that can be converted in vivo to the compounds of the invention. Methods for preparing prodrugs of the named functional compounds will be known to those skilled in the art, and may be found, for example, in connection with the disclosure of Krogsgaard-Larsen et al, "Textbook of Drug design and Discovery" (Textbook of Drug design and Discovery) Taylor Francis Press (2002, 4 months).

In the context of the present invention, the term "solvate" refers generally to any form of substance obtained by non-covalent bonding of an active compound according to the invention to another molecule, generally a polar solvent, and may include in particular, but not exclusively, hydrates and alcoholates, such as methanolate.

In a second aspect, the present invention provides a process for the preparation of a compound provided in the first aspect of the present invention, comprising: hydrolyzing the compound of formula III (X1901-6) to provide a compound of formula I, the reaction equation is as follows:

in the preparation method provided by the invention, the hydrolysis reaction can be generally carried out in the presence of alkali. The person skilled in the art may select a suitable kind and amount of base for the above hydrolysis reaction, for example, the base may be an alkali metal hydroxide or the like, more specifically, lithium hydroxide or the like, and further, for example, the amount of the base is usually substantially equal to or in excess with respect to the compound of formula III, and specifically, the molar ratio of the compound of formula III to the base may be 1: 1-5, 1: 1-1.2, 1: 1.2-1.5, 1: 1.5-2, 1: 2-2.5, 1: 2.5-3, 1:3 to 4, or 1:4 to 5.

In the production method provided by the present invention, the reaction can be usually carried out at room temperature to the boiling point of the reaction solvent, and preferably at room temperature. The reaction time of the hydrolysis reaction can be appropriately adjusted by those skilled in the art according to the progress of the reaction, and the method for monitoring the progress of the reaction is known to those skilled in the art, and may be, for example, an analytical method such as chromatography, nuclear magnetic resonance method, etc., and the specific reaction time may be 1 to 24 hours, 1 to 2 hours, 2 to 4 hours, 4 to 8 hours, 8 to 12 hours, or 12 to 24 hours.

In the preparation method provided by the invention, the reaction is usually carried out in the presence of a solvent, wherein the solvent can be a good solvent of the reaction raw materials and needs to include water, so that the reaction raw materials can be dispersed fully and the smooth progress of the reaction can be ensured. The kind and amount of suitable reaction solvent should be known to those skilled in the art, for example, the reaction solvent may include water, and may further include alcohol solvent, and the like, and specifically may be methanol and the like.

In the preparation method provided by the invention, a person skilled in the art can select a suitable method to carry out post-treatment on the reaction product. For example, the post-treatment of the hydrolysis reaction may include: and (4) removing the solvent. After the reaction is finished, the solvent can be removed from the reaction system to provide the compound of formula I and the compound of formula II. The resulting product after removal of the solvent may be further purified (e.g., column chromatography, etc.) to provide compounds of formula I and formula II in greater purity.

In a third aspect, the present invention provides the use of a compound provided in the first aspect of the present invention, or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, in the manufacture of a medicament. The compound provided by the invention has stronger free radical scavenging and antioxidant activity. In addition, the compound has a protective effect on damage (such as glutamic acid-induced cell damage) of nerve cells (such as PC12 cells) in a cell experiment, the survival rate of the nerve cells is remarkably increased under the condition of the existence of the tested compound, and the generation of ROS (reactive oxygen species) of the nerve cells is remarkably inhibited, and the compound has certain dose dependence. Further in animal experiments, the compound is proved to have obvious inhibition effect on platelet aggregation (for example, platelet aggregation induced by ADP), can effectively reduce the cerebral infarction range (for example, in a rat focal cerebral ischemia-reperfusion model), and has certain dose dependence. In the aspect of pharmacokinetics, the compound also has good pharmacokinetic performance and drug distribution tendency, has longer elimination half-life, and can permeate blood brain barrier to realize higher exposure level in brain. It is clear that the above compounds or pharmaceutically acceptable salts, isomers, prodrugs, polymorphs or solvates thereof may be used for the manufacture of a medicament.

In the use provided by the invention, the medicine can be generally used for treating diseases caused by free radical-induced oxidative stress (such as acute oxidative stress, chronic oxidative stress and the like) and/or thrombus. These diseases may be cardiovascular and cerebrovascular diseases (e.g., arteriosclerosis, heart failure, heart disease, cerebral stroke, myocardial ischemia and ischemia reperfusion injury, myocardial infarction, coronary heart disease, heart failure, or the like), neurodegenerative diseases (e.g., senile dementia (AD), parkinson's Disease (PD), multiple Sclerosis (MS), amyotrophic Lateral Sclerosis (ALS), or the like), senile/aging diseases (e.g., arthritis, diabetes and complications (e.g., diabetic cardiomyopathy, diabetic nephropathy, diabetic cerebrovascular disease, diabetic retinopathy, or the like due to diabetic high glucose oxidative stress), osteoarthritis, cataract, macular degeneration, prostatosis, or the like), cancer, liver disease, lung disease, digestive tract disease, kidney disease, infectious disease, and immune disease, or the like.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising a compound provided in the first aspect of the present invention or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

In the present invention, the composition may include one or more pharmaceutically acceptable carriers, which generally refers to carriers used for the administration of therapeutic agents, which do not themselves induce the production of antibodies harmful to the individual receiving the composition, and which are not unduly toxic after administration. Such carriers are well known to those skilled in the art, and relevant information regarding pharmaceutically acceptable carriers is disclosed, for example, in Remington's Pharmaceutical Sciences (Mack pub. Co., n.j.1991). In particular, the carrier may be a combination including, but not limited to, one or more of saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and the like.

In the pharmaceutical composition provided by the invention, the compound or the pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof can be a single effective component, and can also be combined with other active ingredients to form a combined preparation. The other active ingredient may be other various drugs that may be used for the treatment of diseases caused by oxidative stress and/or thrombosis due to free radicals. The amount of active ingredient in the composition will generally be a safe and effective amount, which should be adjusted by the person skilled in the art. For example, the administration amount of the above-mentioned compound or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof as an active ingredient, and the active ingredient of a pharmaceutical composition is generally dependent on the body weight of a patient, the type of application, the condition and severity of the disease. For example, the above-mentioned compound or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof as an active ingredient may be administered in an amount of usually 0.1 to 1000mg/kg/day, 0.1 to 0.5mg/kg/day, 0.5 to 1mg/kg/day, 1 to 2mg/kg/day, 2 to 3mg/kg/day, 3 to 4mg/kg/day, 4 to 5mg/kg/day, 5 to 6mg/kg/day, 6 to 8mg/kg/day, 8 to 10mg/kg/day, 10 to 20mg/kg/day, 20 to 30mg/kg/day, 30 to 40mg/kg/day, 40 to 60mg/kg/day, 60 to 80mg/kg/day, 80 to 100mg/kg/day, 100 to 150mg/kg/day, 150 to 200mg/kg/day, 200 to 300mg/kg/day, or 400 to 300mg/kg/day, more preferably, 400 to 600mg/kg/day, or 800 mg/kg/day.

The compounds provided herein may be adapted for any form of administration, and may be administered orally or parenterally, for example, by pulmonary, nasal, rectal and/or intravenous injection, and more particularly intradermal, subcutaneous, intramuscular, intraarticular, intraperitoneal, pulmonary, buccal, sublingual, nasal, transdermal, vaginal, oral or parenteral administration. One skilled in the art can select a suitable formulation according to the mode of administration, for example, a formulation suitable for oral administration may include, but is not limited to, pills, tablets, chewables, capsules, granules, drops or syrups, and the like, for example, a formulation suitable for parenteral administration may include, but is not limited to, solutions, suspensions, reconstitutable dry preparations or sprays, and the like, for example, a suppository suitable for rectal administration may generally be used.

In a fifth aspect, the invention provides a method of treatment comprising: administering to the subject a therapeutically effective amount of a compound provided by the first aspect of the invention or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, or a pharmaceutical composition provided by the fourth aspect of the invention.

In the present invention, a "subject" generally includes human, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., which would benefit from treatment with the formulation, kit or combined formulation.

In the present invention, a "therapeutically effective amount" generally refers to an amount which, after an appropriate period of administration, is capable of achieving the effect of treating the diseases as listed above.

Compared with similar compounds in the prior art, the compound provided by the invention has the advantages of higher performance, better antioxidation, nerve cell protection and platelet aggregation inhibition activity, easy blood brain barrier passing, good oral bioavailability, greatly improved patient compliance and clinical convenience, can be developed into medicines for treating and preventing oxidative stress related diseases such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases and the like, can also be used for treating thromboembolic diseases, and has good industrial prospect.

The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Example 1

Synthesis of compound X1901:

1. synthesis of X1901-1

Dissolving X1901-SM (100g, 0.819mol) by dichloromethane, cooling to 0 ℃, adding m-CPBA (155g, 0.898mol) of m-chloroperoxybenzoic acid into the solution in batches, and heating to 45 ℃ for reflux reaction for 4 hours after the addition is finished. And monitoring by TLC (thin layer chromatography) until the reaction is finished, cooling the reaction solution to room temperature, filtering, wherein a filter cake is m-chlorobenzoic acid serving as a reaction by-product, and leaching the filter cake with dichloromethane until the filter cake is colorless. The filtrate was quenched m-CPBA with aqueous Na2SO3, separated, the aqueous phase was adjusted to pH9-10 with Na2CO3 solids, extracted 10 times with DCM, all organic phases were combined, washed 1 time with saturated aqueous NaCl solution, and the organic phase was spin dried under reduced pressure to give 106g of X1901-1 as an oily liquid in 94% yield.

2. Synthesis of X1901-2

Dissolving X1901-1 (106g, 0.767mol) in toluene, cooling to 0 deg.C, and dropwise adding POCl ₃ (400 ml), after dropping, the temperature was raised to 90 ℃ to react for 9 hours. TLC monitoring till the reaction is finished, cooling the reaction solution to room temperature, and concentrating under reduced pressure to remove POCl completely ₃ An oil was obtained, which was dissolved with dichloromethane and NaHCO ₃ Adjusting pH of the aqueous solution to 8-9, separating, extracting the aqueous phase with DCM for 4 times, mixing all organic phases, sequentially adding saturated saline water and anhydrous MgSO ₄ Drying and spin-drying under reduced pressure gave 60g of X1901-2 as an oily liquid in 45% yield.

3. Synthesis of X1901-3

60g of X1901-2 (0.383 mol) were dissolved in DCM and m-CPBA (73g, 0.423mol) was added dropwise at 0 ℃ and, after completion, the reaction was stirred at room temperature overnight. And monitoring by TLC (thin layer chromatography) until the reaction is finished, cooling the reaction solution to 10 ℃, filtering, wherein a filter cake is m-chlorobenzoic acid as a reaction by-product, and leaching the filter cake with dichloromethane until the filter cake is colorless. Quenching m-CPBA by using an aqueous Na2SO3 solution, separating liquid, washing an organic phase with a saturated aqueous NaHCO3 solution for 2 times, separating liquid, combining aqueous phases, extracting for 4 times by using DCM, combining the organic phases, washing for 1 time by using a saturated aqueous NaCl solution, evaporating to obtain an oily liquid X1901-3, and performing separation by using n-heptane: EA (1.

4. Synthesis of X1901-4

21g of X1901-4 (0.122 mol) was dispersed in hydrazine hydrate, and the reaction was carried out at 120 ℃ for 4 hours. TLC monitoring till the reaction is finished, cooling the reaction solution to 10 ℃, filtering, and evaporating the filter cake to dryness to obtain 15g of white solid X1901-4 with the yield of 73%.

5. Synthesis of X1901-5

15g of X1901-4 (0.089 mol) was dissolved in 300ml of water, ethyl acetoacetate (29g, 0.22mol) was added dropwise with stirring, and the mixture was heated to 50 ℃ to react for 1 hour. TLC was monitored until the starting material was completely converted to the intermediate, 2eq sodium carbonate (18.9g, 0.178mol) was added, the temperature was raised to 100 deg.C, and TLC was monitored until the reaction was complete. Adding toluene, performing reduced pressure spin-drying on the aqueous solution, dispersing a filter cake with methanol, performing suction filtration to remove insoluble inorganic salts, backwashing an organic phase with water for 3 times, separating liquid, discarding the organic phase, collecting a product mainly in a water phase, performing reduced pressure spin-drying on the water phase to obtain a crude product of X1901-5, and performing column chromatography purification by using DCM-methanol to obtain 6g of X1901-5 with the yield of 29%.

6. Synthesis of X1901-6

6g of X1901-5 (0.026 mol) was dissolved in 60ml of acetic anhydride, and the mixture was heated to 140 ℃ with stirring to react for 10 hours. TLC monitors the reaction until the reaction of the starting material is completed, the reaction solution is cooled to room temperature, concentrated under reduced pressure, the residue is dissolved with 100mL of DCM, the pH is adjusted to 7.0 by saturated sodium bicarbonate, and concentrated under reduced pressure to obtain the crude product. Purification by DCM-MeOH column chromatography gave 4.2g of X1901-6 in 59% yield.

7. Synthesis of X1901

42ml of methanol was dissolved with 4.2g of X1901-6, and 10ml of water was dissolved with lithium hydroxide monohydrate, and the alkali solution was dropwise added to the methanol solution with stirring, followed by stirring at room temperature for 3 hours. TLC monitoring reaction until the raw material reaction is finished, decompression concentration to obtain crude product. Purification by DCM-methanol column chromatography gave 2g of X1901 as a foamy solid in 56% yield. X1901 molecular formula C ₁₁ H ₁₄ N ₄ O ₂ Molecular weight: 234.25,1H-NMR (400MHz, CDCl3): 2.76-2.73 (M, 2H), 4.12 (t, 1H), 3.44 (s, 2H), 2.55-2.45 (M, 6H), 2.24 (s, 3H), LC-MS: M/z =235 (M + 1)

Example 2

Determination of antioxidant activity by ORAC method:

the oxidation resistance index (ORAC) is a standard method for evaluating the oxidation resistance activity, and an azo compound AAPH is used as a peroxyl radical source, fluorescein sodium is used as a fluorescence indicator, a vitamin E water-soluble analogue Trolox is used as a quantitative standard, and a fluorescence microplate analyzer is used for testing the reduction of the fluorescence intensity of free radicals and the protection effect of an antioxidant. In the experiment, trolox or a test sample, fluorescein sodium and potassium phosphate buffer solution are taken to be arranged in the micropores of a 96-pore plate, the temperature is preset for 5min at 37 ℃, AAPH is rapidly added to start reaction, and the fluorescence attenuation curve is tested. And fluorescence decay curves of sodium fluorescein-AAPH and sodium fluorescein + AAPH were measured as controls. ORAC is calculated from the area under the fluorescence decay curve for the antioxidant and the area under the fluorescence decay curve for the free radical in the absence of antioxidant.

ORAC value = [ (AUC) _sample -AUC _+AAPH )/(AUC _Trolox -AUC _+AAPH )]* (Trolox molarity/molarity of sample)

The results show that: x1901 has significant antioxidant activity, ORAC is 20.3. Mu. Mol Trolox, and the antioxidant capacity is stronger than edaravone and Y1502 (4.17 and 11.5. Mu. Mol Trolox).

Example 3

Protective effect on glutamate-induced PC12 cell damage:

PCI2 cells are rat adrenal chromaffin cells, originating from the ectoderm that produces the nervous system, and share many similar characteristics with dopaminergic neurons. The experiment which is often used for replacing dopaminergic neuron to research partial nerve cell function and biological characteristics is one of the most extensive cell lines for researching in vitro nerve cell damage and protection. ( Shafer WJ, atchison WD.Transmitter, ion channel and receptor properties of phytochromycotoma (PC 12) cells: a model for neurooxidative students. Neurooxidative 1991.12 (3): 473-492 )

Glutamate is one of the major neurotransmitters of excitatory synapses in the brain and plays an important role in maintaining normal physiological functions. However, glutamate is a potentially neurotoxic substance that can produce "excitotoxicity" in the nerve cells of the central nervous system, and oxidative stress injury is a key pathological mechanism among them (Sztakowski M, atwell D.triggering and execution of neural death in brain ischemia: two phases of glutamate release by two differential mechanisms. Trends Neurosci,1994, 17 (9): 359-365). Glutamate-induced PC12 cell Reactive Oxygen Species (ROS) levels were detected using flow cytometric fluorescence sorting (FACS) using the fluorescent probe H2 DCFDA.

After the PC12 cells were recovered, they were resuspended in DMEM complete medium and cultured in a constant temperature cell culture chamber at 37 ℃ and 5% CO2. PC12 cells in logarithmic growth phase were seeded in 96-well plates at 5X 10 ³ Cells/well were cultured overnight (37 ℃) in DMEM medium. After 1h incubation with different concentrations of test compound, sodium glutamate (10 mM) was damaged for 24h. Each concentration was set with 3 multiple wells, and the cell viability was observed after culturing for 48h using normal controls and model controls (no sodium glutamate and test compound were added to normal controls, sodium glutamate was added to model controls, but no test compound was added (blank vehicle was used instead), controls and experimental wells were run in parallel).

The survival rate of the cells is detected by an MTT colorimetric method. Cells were treated with MTT (a tetramethylazozolium salt) solution at 37 ℃ for 4h. Finally formazan crystals were dissolved in 120. Mu.L of DMSO, and OD values were measured at 570 nm. Cell viability was a percentage of the OD value of the normal control group.

The experimental results are detailed in table 1. As shown in Table 1, the oxidative stress injury of PC12 cells was induced by sodium glutamate, and the average cell survival rate was reduced to 58.4% as compared with the 100% cell survival rate of the normal control group. X1901 has significant protective effect on glutamic acid induced injured PC12 cells, the cell survival rate is significantly increased at a concentration of more than 1 μ M, and the protective effect is obviously better than that of Y1502 under the same concentration (10 μ M).

TABLE 1 Effect on survival of glutamate induced PC12 cells

Note: # # #: p <0.01; as compared to model controls,.: p <0.01,.: p <0.001; compared with Y1502, the ratio of &: p <0.05, & & &: p <0.001

Example 4

Inhibition of glutamate-induced ROS production by PC12 cells:

after PC12 cells were recovered, they were resuspended in DMEM complete medium and cultured in a constant temperature cell incubator at 37 ℃ and 5% CO2. After the cells are cultured to a logarithmic phase, the cells are digested by 0.25% pancreatin to prepare a single cell suspension, and the cell density is adjusted to 1.5 multiplied by 10 ⁵ Perml, seeded in six well plates, 2mL per well. 5% CO2, incubation in 37 ℃ incubator for 24h. After 24h, the supernatant was aspirated off, the medium containing 10mM sodium glutamate was added, the test compound (as the administration group) or its blank vehicle (0.1% DMSO, as the model control group) was added, and in addition, the normal control group was set, 2mL of DMEM complete medium (containing 0.1% DMSO) was added instead of the sodium glutamate solution. Each of the above groups was prepared with three duplicate wells at 37 ℃ and 5% CO ₂ And culturing in an incubator for 24 hours. After 24h, cells were harvested. The cells were resuspended in 0.1mL of 10. Mu.M H2DCFDA solution and incubated in a 37 ℃ water bath for 30min in the dark. After incubation, centrifugation at 1500r/min for 5min, cells were resuspended in 500 μ L serum-free medium and the Mean Fluorescence Intensity (MFI) was measured using flow cytometry to assess the production of Reactive Oxygen Species (ROS). Two experiments were performed according to the above method.

Experiment one: the in vitro efficacy of test compound X1901 in the glutamate-induced oxidative stress injury model of PC12 was examined and compared with edaravone in parallel at concentrations of 100 μ M, and the results are shown in table 2. As can be seen from Table 2, the addition of sodium glutamate can induce a significant increase in ROS levels in PC12 cells as compared with the normal control group, and the MFI value is increased from 2144 + -202 of the normal control group to 3537 + -32.3 of the model control group; both edaravone and X1901 can obviously reduce MFI value, ROS generation is close to normal value, and X1901 activity is better. The test shows that X1901 can inhibit oxidative stress injury of PC12 cells caused by glutamic acid, and play a role in protecting nerve cells.

TABLE 2 inhibition of ROS production in glutamate-induced PC12 cells (mean. + -. Standard error, n = 3)

Note: MFI: mean fluorescence intensity (mean fluorescence intensity); Δ Δ Δ: p <0.001; as compared to model control group, { v. }: p <0.001,.: p <0.0001.

Experiment two: the dose relationship of the effect of X1901 in inhibiting ROS production was further investigated and compared in parallel with Y1502. X1901 represents the results of three doses of concentration, low, medium and high, as shown in Table 3. As can be seen from table 3, the MFI of the model control group was significantly increased compared to the normal control group (P < 0.001), indicating the presence of significant oxidative stress damage. Each dose group of X1901 significantly reduced MFI (P <0.01 or P < 0.001) compared to the model control group, and was dose-dependent. The efficacy of X1901 was significantly better than Y1502 (P < 0.05) at the same dose (10 μ M) (table 3).

TABLE 3 inhibition of ROS production in PC12 cells induced by glutamate (mean. + -. Standard deviation, n = 3)

Note: compared with the normal control group, ###: p <0.001, compared to model group,.: p <0.01, x: p <0.001. Compared to Y1502, &: p <0.05.

Example 5

Inhibition of ADP-induced platelet aggregation

10 male SD rats adopt a citric acid anticoagulation negative pressure tube to collect rat arterial blood, immediately reverse and mix uniformly, centrifuge for 10min at 800r/min to prepare Platelet Rich Plasma (PRP), absorb the required PRP, concentrate and mix uniformly for later use, centrifuge the residual blood for 20min at 3000r/min to prepare Platelet Poor Plasma (PPP). 490 mu l of PRP is added into a reaction cup containing a magnetic bar, 10 mu l of test samples with different concentrations are added, 10 mu l of normal saline is added into a blank control group, each concentration is repeated for 3 times, incubation is carried out for 5min at 37 ℃, 500 mu l of PPP is added into a reaction cup without the magnetic bar, the temperature is uniformly pre-warmed for 5min, 5 mu l of Adenosine Diphosphate (ADP) with 1mol/ml is added into the PRP for induction stimulation, and a platelet aggregation rate curve is recorded.

Specific results are shown in table 4, and it can be seen from table 4 that each dose group of X1901 significantly inhibited ADP-induced platelet aggregation in vitro (P <0.05, P-woven 0.01 or P < 0.001), and was dose-dependent. The inhibition effect is better than that of Y1502 (P < 0.001) under the same dosage.

TABLE 4 inhibition of ADP-induced platelet aggregation in vitro

Note: comparison with model control group,: p is a radical of<0.05，**：p<0.01，***：p<0.001. In comparison with the case of Y1502, ^&&& ：p<0.001。

example 6

Influence on the range of cerebral infarction of focal cerebral ischemia-reperfusion rats:

male SD rats (purchased from Beijing Wintolite laboratory animal technology, inc., license number: SCXK (Jing) 2016-0006) 70, weighing 250-280g, were divided into 6 groups: a sham operation group and a model group, Y1502 of 12.5mg/kg, X19016.7mg/kg, X1901.4 mg/kg and X1901.8 mg/kg. After the experimental quarantine period is finished, the cells are randomly grouped, and focal cerebral ischemia-reperfusion (MCAO) models are prepared in batches and in parallel by adopting a line-embolism method. (E Z Longa, P R Weinstein, S Carlson, R Cummins, reversible middle core area encapsulation with out creativity in rates.Stroke.1989; 20 (1): 84-91) a single gavage was given 30min prior to molding, and a blank vehicle was given to the model group.

MCAO model: after anesthetizing the rats with chloral hydrate, a pre-treated special embolus was inserted into the right Common Carotid Artery (CCA) and into the Internal Carotid Artery (ICA) until it blocked the beginning of the Middle Cerebral Artery (MCA). After 2h of occlusion of the artery, the tissue was blood reperfused for 24h by carefully removing the filaments to restore blood perfusion. The sham group exposed the carotid artery but did not insert a plug wire.

Evaluation of cerebral infarction scope: the experimental animal is subjected to cerebral ischemia reperfusion for 24h, then brain is dissected and taken out, frozen in a refrigerator for 15min, and then the brain is taken out and subjected to coronal dissection by 4 knives into 5 pieces. The brain slices were quickly placed in 1.2% TTC stain (0.02M K) ₂ HPO ₄ Preparation), incubating at 37 ℃ in the dark for 20min, taking out and placing in tissue fixing solution in the dark for preservation. Normal tissue was stained rose-red and infarcted tissue was white. Each brain plane was placed in sequence on filter paper, white tissue was carefully dug out and weighed, and the infarct size (%) was determined as the percentage of the infarct tissue weight to the total brain weight and the operated side half brain weight. Calculating an inhibition rate: (infarct size in model control group-infarct size in administered group)/infarct size in model control group = infarct suppression rate.

Specific results are shown in table 5, and it can be seen from table 5 that X1901 significantly reduced the infarct size (P <0.05, P < -0.01 or P < 0.001) in the rat operated side and whole brain of MCAO model, and showed dose-dependency. X1901 was superior to Y1502 (table 5).

Table 5 effect on focal cerebral ischemia-reperfusion rat brain tissue infarction scope (n.gtoreq.8,

)

note: p <0.05, p <0.01, p <0.001, compared to the model control group. Compared with Y1502, p <0.05, p <0.01.

Example 7

Rat pharmacokinetics:

12 male SD rats were divided into 4 groups and 3 groups, and the 1 st to 2 nd groups were administered Y1502 by oral gavage and tail vein injection at doses of 10mg/kg and 5mg/kg, respectively, and the 3 rd to 4 th groups were administered X1901 by oral gavage and tail vein injection at doses of 13.4mg/kg and 5mg/kg, respectively. The oral administration group for intragastric administration takes 0.2mL of blood after 5min, 10min, 15min, 30min, 45min, 1.0h, 1.5h, 2.0h, 3.0h, 5.0h, 7.0h and 24.0h after administration; in the tail vein administration group, 0.2mL of blood (anticoagulant is heparin sodium solution containing 16.7mg/mL of sodium metabisulfite) is taken 2min, 5min, 10min, 15min, 30min, 45min, 1.0h, 1.5h, 2.0h, 5.0h, 7.0h and 24.0h after administration. Plasma was centrifuged, drug concentrations were analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated by DAS 3.2.7 software.

As a result: the time course of the drug is shown in FIGS. 1-2, and the pharmacokinetic parameters are shown in tables 6-7. From tables 6 and 7, it can be seen that:

1) Oral gavage, X1901 is rapidly absorbed in rats with a Tmax of 0.25h, a mean t1/2 of 10.12h, and an oral absolute bioavailability of 88% which is much higher than edaravone (5.23%) (Rong, w.t., et al.hydroxypyrophyll fobuytl-beta-cyclodextrins of oral bioavailability of edaravone by modulating drug efflux pump of entries. Journal of Pharmaceutical Sciences,2014;103 (2):730-742). Compared with Y1502, the compound has longer half-life and higher oral bioavailability.

2) Tail vein injection, mean t of X1901 in rats _1/2 It was 20.28h, the mean CLz was 0.34L/h/kg, and the mean apparent distribution volume (Vz) was 9.81L/kg. Compared with Y1502, the composition has longer elimination half-life and higher tissue distribution characteristics. It is beneficial to maintain higher and more durable drug exposure levels in the effector organ or tissue.

TABLE 6 pharmacokinetic parameters of intragastric administration in rats

	Unit of	Y1502	X1901
				Dosage form	mg/kg	10	13.4
AUC _0-24h	h*ng/mL	7791±488	30338±6727
				AUC _0-∞	h*ng/mL	7883±367	31730±7339
C _max	ng/mL	7168±2335	13136±3199
				t _1/2	h	3.14±2.49	10.12±2.98
T _max	h	0.14±0.048	0.25±0
				F	％	64	88

TABLE 7 pharmacokinetic parameters for intravenous administration of rats

	Unit	Y1502	X1901
				Dosage form	mg/kg	5	5
AUC _0-24h	h*ng/mL	6164±5	12911±977
				AUC _0-∞	h*ng/mL	6165±4	14835±1459
t _1/2	h	1.82±0.57	20.28±5.22
				MRT _0-∞	h	2.70±0.47	11.74±3.16
Vz	L/kg	2.13±0.66	9.81±2.08
				CLz	L/h/kg	0.81±0.001	0.34±0.034

Example 8

Distribution of oral administration in brain of rat

6 male SD rats were divided into 2 groups, and administered with equimolar dose of X1901 or edaravone (57.4. Mu. Mol/kg) by gavage, and sacrificed after abdominal bleeding 10min and 1.0h after administration, and brain tissue was collected. Brain tissue sample treatment: the left rat brain is put into a tube with grinding beads, and 5mg/mL sodium metabisulfite normal saline solution is added according to the mass volume ratio of 1. In the mill, 30s of centrifugation at 6000rpm was used for 1 cycle, 4 cycles of milling, and 20s of each cycle was waited for. The homogenate is placed in a refrigerator at-20 ℃ to be frozen for treatment. A50. Mu.L sample of the plasma/brain homogenate was placed in a 1.5mL centrifuge tube, 200. Mu.L of an internal standard working solution (100 ng/mL edaravone in methanol) was added thereto, vortexed for 5min, and then centrifuged at 12000rpm for 10min in a high-speed centrifuge. Taking the supernatant, and analyzing the drug concentration of the supernatant of the tissue homogenate by using an UPLC/MS/MS method.

Specific results are shown in table 8. As shown in Table 8, after the rat orally takes X1901, the drug can be rapidly distributed in the brain tissue, the drug exposure level in the brain is obviously higher than that of edaravone, and the duration is longer.

TABLE 8 concentration of prototype drug (ng/g) in brain tissue after a single gavage administration in rats

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A compound or a pharmaceutically acceptable salt, isomer thereof, wherein the chemical structural formula of the compound is shown as formula I:

the isomers are tautomers formed by keto-enol tautomerism; the isomers include compounds having one of the following chemical structural formulas:

2. a process for preparing a compound of claim 1, comprising: hydrolyzing a compound of formula III to provide a compound of formula I, the reaction equation is as follows:

3. use of a compound according to claim 1 or a pharmaceutically acceptable salt, isomer thereof for the manufacture of a medicament.

4. Use according to claim 3, wherein the medicament is for the treatment of diseases caused by oxidative stress and/or thrombosis induced by free radicals.

5. The use according to claim 4, wherein the diseases caused by oxidative stress and/or thrombosis due to free radicals comprise cardiovascular and cerebrovascular diseases, neurodegenerative diseases, cancer, infectious diseases, arthritis, diabetes and complications, cataracts or macular degeneration.

6. The use of claim 5, wherein the cardiovascular and cerebrovascular diseases comprise atherosclerosis, heart failure, heart disease, cerebral stroke, myocardial ischemia and ischemia reperfusion injury, myocardial infarction or coronary heart disease; and/or, the neurodegenerative disease includes senile dementia, parkinson's disease, multiple sclerosis or amyotrophic lateral sclerosis.

7. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt, isomer thereof.