WO2023123468A1 - 心脑血管药物及其应用 - Google Patents

心脑血管药物及其应用 Download PDF

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Publication number
WO2023123468A1
WO2023123468A1 PCT/CN2021/143944 CN2021143944W WO2023123468A1 WO 2023123468 A1 WO2023123468 A1 WO 2023123468A1 CN 2021143944 W CN2021143944 W CN 2021143944W WO 2023123468 A1 WO2023123468 A1 WO 2023123468A1
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compound
group
formula
pharmaceutically acceptable
pharmaceutical composition
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PCT/CN2021/143944
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English (en)
French (fr)
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漆又毛
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杭州奥默医药股份有限公司
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Priority to PCT/CN2021/143944 priority Critical patent/WO2023123468A1/zh
Publication of WO2023123468A1 publication Critical patent/WO2023123468A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/724—Cyclodextrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase

Definitions

  • the invention belongs to the field of medicines, and more specifically relates to cardiovascular and cerebrovascular medicines and applications thereof.
  • Acute pulmonary embolism is the third largest acute cardiovascular syndrome in the world after myocardial infarction and stroke. The annual incidence of pulmonary embolism is (39-115) per 100,000 people, and the incidence has been on the rise in recent years.
  • thrombolytic drugs include urokinase, streptokinase,reteplase, etc.
  • antithrombotic drugs include antiplatelet drugs and anticoagulants.
  • Edaravone is a drug developed and marketed in Japan for the treatment of acute onset of cerebral infarction, improving neurological symptoms, activities of daily living and dysfunction caused by acute cerebral infarction. Although it has been approved for marketing in China, it has not been approved by mainstream countries in Europe and America recognized. Edaravone composition (edaravone camphenol) obtained some positive results in phase III clinical research and has been approved for marketing in China. Approved by the US FDA and recommended by guidelines.
  • butylphthalide is proposed as a class II recommendation for improving cerebral microcirculation, while there is no recommended neuroprotective agent in foreign clinical guidelines. It can be said that the field of neuroprotective agents recognized in the global mainstream medicine is still blank.
  • the inventor conceived to develop a new type of drug for the treatment of neurological damage in stroke, which can not only effectively treat thrombotic diseases with thrombolysis, but also protect the brain nerves. It has great clinical significance and practical value.
  • the object of the present invention is to provide a kind of cardiovascular medicine and application thereof.
  • a compound of formula I or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition or preparation, the pharmaceutical composition or preparation comprising formula I as an active ingredient compound, and the pharmaceutical composition or formulation is used for:
  • R 1 is selected from the group consisting of hydroxyl, C1-C6 alkoxy, halogen, substituted or unsubstituted -O-C1-C6 alkyl, Wherein the substitution is sulfonic acid group or hydroxyl substitution;
  • n is a positive integer of 1-6;
  • the carbon atom is a chiral carbon atom, and the chirality is selected from the group:
  • R 4 is a metal ion selected from the group consisting of Na + , K + , Li + or Cs + ;
  • R 5 is C1-C6 alkyl, C3-C8 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or heteroaryl;
  • R 2 and R 3 are each independently H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C2-C6 hydroxyalkyl or -(C1-C3 alkylene base)-COOH;
  • n is a positive integer of 6-12.
  • the pharmaceutical composition is used for thrombosis prevention and thrombolytic therapy; or for preventing and/or treating cerebral ischemia-reperfusion injury, and inhibiting neuronal ferroptosis caused by cerebral ischemia-reperfusion , or subside vascular inflammation.
  • the thrombus is selected from the group consisting of vascular endothelial injury thrombus, arteriovenous bypass thrombus, cerebral ischemia-reperfusion injury, or a combination thereof.
  • the cerebral ischemia-reperfusion injury includes: cerebral infarction, cerebral edema and/or neuronal ferroptosis.
  • the anti-inflammatory treatment is anti-vascular inflammation treatment.
  • the vascular inflammation includes: vascular inflammation caused by high sugar and high fat.
  • the pharmaceutical composition or preparation is used to prevent and/or treat thrombosis-related diseases.
  • the thrombosis-related diseases are selected from the group consisting of cerebral thrombosis, cerebral infarction (acute ischemic stroke) and the resulting neuron damage to nerve tissue, cerebral edema, myocardial infarction, pulmonary Embolism, or a combination thereof.
  • the thrombosis-related diseases include infarction-related diseases.
  • the pharmaceutical composition or preparation is used for anti-inflammatory treatment.
  • the pharmaceutical composition or preparation is used for the treatment of neuron damage caused by cerebral infarction, traumatic brain injury, cerebral hemorrhage or brain tumor surgery.
  • R 1 is selected from the following group: hydroxyl, methoxy,
  • R 4 is Na + .
  • R 5 is CH 3 .
  • n 6, 7 or 8.
  • R 1 is selected from the following group: hydroxyl, methoxy,
  • R2 and R3 are each independently H, methyl, hydroxypropyl, hydroxyethyl or carboxymethyl.
  • the compound of formula I is selected from the group consisting of methylcyclodextrin, carboxymethylcyclodextrin, hydroxyethyl- ⁇ -ethylcyclodextrin, hydroxypropyl- ⁇ -cyclodextrin Alcohol or sulfobutyl ether- ⁇ -cyclodextrin.
  • the core of the compound of formula I is cyclodextrin.
  • the compound of formula I is selected from the following group:
  • the pharmaceutical composition further contains additional active ingredients.
  • the additional active ingredient is selected from the group consisting of butylphthalide, urokinase, edaravone, or combinations thereof.
  • the pharmaceutical composition or preparation contains the compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.
  • the dosage form of the pharmaceutical composition or preparation is selected from the group consisting of injection, freeze-dried powder injection, sustained release, controlled release, enteric-coated tablet or capsule, and granule.
  • the pharmaceutical composition or preparation contains 0.001-99wt%, preferably 0.1-90wt%, more preferably 1-80wt% of the compound of formula I or its pharmaceutically acceptable Salts are accepted by weight of the total composition.
  • the second aspect of the present invention provides the use of a compound of formula I and urokinase composition for the preparation of medicines for treating thrombosis-related diseases.
  • the third aspect of the present invention provides the use of a compound of formula I and butylphthalide for the preparation of a medicament for treating nerve damage caused by cerebral infarction.
  • a pharmaceutical composition or preparation containing (a) an active ingredient comprising a compound of formula I or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable Carrier, the pharmaceutical composition or preparation is used for:
  • the weight ratio of the two components is 1:20 to 20:1, preferably 1:10 to 10:1, more preferably 1 :5 to 5:1.
  • the fifth aspect of the present invention provides a kit, characterized in that the kit includes:
  • the nth pharmaceutical composition contains the nth compound or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier; wherein , n is any positive integer in 2-8;
  • both the first compound and the nth compound are compounds of formula I or a pharmaceutically acceptable salt thereof, or at least one of the first compound is a compound of formula I or a pharmaceutically acceptable salt thereof and the nth compound is additional activity
  • the compound of formula I is as defined in claim 1;
  • the sixth aspect of the present invention provides a method for treating a disease, said disease as claimed in claim 1, comprising the step of: administering a compound of formula I or a pharmaceutically acceptable salt thereof to a subject in need, wherein, formula I
  • the compound is as defined in claim 1.
  • the subject is a mammal.
  • the subject is human.
  • a class of compounds with the structure shown in formula I can prevent and/or treat thrombosis-related diseases; anti-inflammatory treatment; treatment of cerebral infarction, traumatic brain injury, cerebral hemorrhage or cerebral Neuronal damage caused by tumor surgery has a significant effect.
  • the compound of formula I has a good therapeutic effect on brain nerve damage caused by stroke.
  • the compound of formula I of the present invention can be used for thrombosis prevention, thrombolysis, anti-inflammatory treatment and the like in thrombotic diseases. On this basis, the present invention has been accomplished.
  • halogen refers to F, Cl, Br and I.
  • C1-C6 alkyl refers to a linear or branched alkyl group comprising 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl group, neopentyl, popentyl, or similar groups.
  • C2-C6 alkenyl refers to a straight-chain or branched alkenyl group with 2-6 carbon atoms containing a double bond, including without limitation ethenyl, propenyl, butenyl, isobutenyl, amyl alkenyl and hexenyl etc.
  • C2-C6 alkynyl refers to a straight or branched chain alkynyl having 2 to 6 carbon atoms containing a triple bond, including without limitation ethynyl, propynyl, butynyl, isobutynyl base, pentynyl and hexynyl, etc.
  • C1-C6 hydroxyalkyl refers to a linear or branched chain alkyl group containing one hydroxyl group with 1-6 carbon atoms, including without limitation hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl group, etc., preferably a C1-C3 hydroxyalkyl group.
  • C3-C8 cycloalkyl refers to a cyclic alkyl group having 3-8 carbon atoms in the ring, including without limitation cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl , Cyclooctyl, etc.
  • C1-C6 alkoxy refers to a straight-chain or branched alkoxy group having 1-6 carbon atoms, including without limitation methoxy, ethoxy, propoxy, isopropoxy and Butoxy, etc. Preference is given to C1-C4 alkoxy.
  • aromatic ring or “aryl” has the same meaning, preferably “C6-C10 aryl”.
  • C6-C10 aryl refers to an aromatic ring group having 6-10 carbon atoms without heteroatoms in the ring, such as phenyl, naphthyl and the like.
  • heteroaryl denotes a heteroaromatic system comprising 1 to 4 heteroatoms including nitrogen, oxygen and S(O)r (where r is an integer 0, 1, 2) heteroatoms, for example , 4-8 membered heteroaryl refers to a heteroaromatic system containing 4-8 ring atoms, 4-10 membered heteroaryl refers to a heteroaromatic system containing 4-10 ring atoms, including but not limited to pyrrolyl, Furyl, thienyl, pyrazolyl, thiazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridyl, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, benzimidazolyl, triazole Base etc.
  • the groups described in the present invention may be substituted with substituents selected from the group consisting of halogen, acyloxy, cyano, amino, nitric Group, carboxyl, amido, carboxymethyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, C3-C6 cycloalkyl, C3-C6 halocycloalkyl, hydroxyl C1-C4 alkyl, C5-C7 cycloalkenyl, phenyl, naphthyl, etc.
  • substituents selected from the group consisting of halogen, acyloxy, cyano, amino, nitric Group, carboxyl, amido, carboxymethyl, C1-C6 alkyl, C1-C6
  • pharmaceutically acceptable salts of compounds of formula I are also included.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention with an acid or a base which is suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the polymers of the invention with acids.
  • Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Maleic acid, lactic acid, malic acid,
  • the compound of formula I of the present invention can be prepared by methods well known to those skilled in the art, and there is no special limitation on the reaction parameters of each step. In addition, typical compounds of the present invention are also commercially available.
  • the chiral carbon atom may be in the R configuration or the S configuration, or a mixture of both.
  • the active substance is a compound of formula I,
  • R 1 is selected from the group consisting of hydroxyl, C1-C6 alkoxy, halogen, substituted or unsubstituted -O-C1-C6 alkyl, Wherein the substitution is sulfonic acid group or hydroxyl substitution;
  • n is a positive integer of 1-6;
  • the carbon atom is a chiral carbon atom, and the chirality is selected from the group:
  • R 4 is a metal ion selected from the group consisting of Na + , K + , Li + or Cs + ;
  • R 5 is C1-C6 alkyl, C3-C8 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or heteroaryl;
  • R 2 and R 3 are each independently H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C2-C6 hydroxyalkyl or -(C1-C3 alkylene base)-COOH;
  • n is a positive integer of 6-12.
  • R is selected from the group consisting of hydroxyl, methoxy,
  • R 4 is Na + .
  • R5 is CH3 .
  • n 6, 7 or 8.
  • R is selected from the group consisting of hydroxyl, methoxy,
  • R2 and R3 are each independently H, methyl, hydroxypropyl, hydroxyethyl or carboxymethyl.
  • the compound of formula I is selected from the group consisting of methylcyclodextrin, carboxymethylcyclodextrin, hydroxyethyl- ⁇ -ethylcyclodextrin, hydroxypropyl- ⁇ -cyclodextrin or sulfobutyl ether-beta-cyclodextrin.
  • the core of the compound of formula I is cyclodextrin.
  • the compound of formula I is selected from the group consisting of:
  • compositions and methods of administration are provided.
  • the present invention also provides a pharmaceutical composition, comprising a pharmaceutically acceptable carrier and one or more safe and effective doses of the compounds described in the present invention.
  • the compound of the present invention has excellent antithrombotic activity, the compound of the present invention and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and drugs containing the compound of the present invention as the main active ingredient
  • the composition can be used to treat, prevent and alleviate diseases related to embolism.
  • the pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof within a safe and effective amount range and a pharmaceutically acceptable excipient or carrier.
  • safe and effective dose refers to: the amount of the compound is sufficient to obviously improve the condition without causing severe side effects.
  • the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-1000 mg of the compound of the present invention per dose.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low enough toxicity. "Compatibility” herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound.
  • Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
  • cellulose and derivatives thereof such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as talc
  • solid lubricants such as stearic acid , magnesium stearate
  • calcium sulfate such
  • the pharmaceutical composition is injection, capsule, tablet, pill, powder or granule.
  • the mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration .
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostea, or
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner.
  • coatings and shell materials such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner.
  • Examples of usable embedding components are polymeric substances and waxy substances.
  • the active compounds can also be in microencapsulated form, if desired, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.
  • inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and
  • compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required, if necessary.
  • the compound of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds such as antithrombotic drugs.
  • the treatment method of the present invention can be used alone or in combination with other treatment methods or drugs.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage when administered, for a person with a body weight of 60kg, the daily
  • the dosage is usually 1-2000 mg, preferably 50-1000 mg.
  • factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the compound of formula I of the present invention has an excellent therapeutic effect on cranial nerve damage caused by cerebral apoplexy.
  • the compound of formula I of the present invention can be used for thromboprophylaxis and thrombolytic therapy of thrombotic diseases without causing bleeding.
  • compound 1 has excellent safety performance and less toxic and side effects.
  • Example 1 Evaluation experiment on the effect of samples on the prevention of thrombosis caused by vascular endothelial injury
  • compound (3) 4.00ng/tail dose group p>0.05 its thrombosis prevention effect was 17%, compound (1), compound (2), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10) and compound (11) 4.00ng/tail dose group p ⁇ 0.01, p ⁇ 0.05, p ⁇ 0.001, p ⁇ 0.001, p ⁇ 0.001, p ⁇ 0.001, p ⁇ 0.001, p ⁇ 0.01, p ⁇ 0.001, the thrombosis prevention effect is 66%, 39%, 63%, 64%, 86%, 73%, 73% , 78%, 64% and 79%.
  • compound (6), compound (11), compound (9), compound (7), compound (8) and compound (1) at 4.00ng/tail dose group, for ponatinib-induced vascular
  • the zebrafish with endothelial injury thrombosis has a very significant preventive effect, the thrombosis prevention (%) index is 66%-86%, and the thrombosis prevention index of 30ug/ml acetylsalicylic acid is 101%.
  • Experimental method Randomly select 2dpf wild-type AB strain zebrafish in a 24-well plate, set 6 replicate wells in each group, and treat 10 zebrafish in each well.
  • the samples were administered intravenously at 500ng/tail dose, and the concentration of the positive control "C48/80" was 1.5 ⁇ g/mL.
  • a normal control group was set up, and the volume of each well was 1 mL.
  • compound (2) and compound (1) have no or substantially no risk of sensitization, and “compound (10)” has a low risk of sensitization.
  • the wild-type AB strain zebrafish was treated with 4 ⁇ g/mL ponatinib for 18 hours to establish a model of vascular endothelial injury thrombus in zebrafish. Randomly select 150 5dpf wild-type AB strain zebrafish in a six-well plate, 30 in each well (experimental group). Compound (1) 2.06, 6.18ng/ tail dose was given by injection, and the positive control drug acetylsalicylic acid was given a concentration of 30 ⁇ g/mL in water solution.
  • a normal control group ie, standard dilution water was used to treat zebrafish
  • a model control group were set up, and each well ( Experimental group) with a capacity of 3mL.
  • the rest of the experimental groups were given ponatinib in water solution to induce vascular endothelial injury thrombus model in zebrafish.
  • compound (1) was co-treated with Ponatinib for a period of time, and then stained with o-dianisidine.
  • compound (1) has a significant preventive effect on ponatinib-induced vascular endothelial injury thrombus in zebrafish, and its preventive effect has a certain dose-dependent effect. Superior to the positive control acetylsalicylic acid.
  • Grouping divide into 5 groups, 10 in each group.
  • the model control group was given the solvent, and the test drug compound (1) and the positive control group were given intravenous injection, respectively, for 3 consecutive days, once a day, and the model was established 30 minutes after the last administration.
  • Model preparation 50 SD male rats were anesthetized by ip with pentobarbital sodium 60mg/kg. The supine position was fixed, the skin was incised at the midline of the neck, the fascia and muscle tissue were separated, the left common carotid artery and the right external jugular vein were freed, and arteriovenous (A-V) bypass surgery was performed with a polyethylene tube with built-in silk thread. Clamp the proximal end of the carotid artery, insert one section of the polyethylene tube into the artery, and insert the other section into the vein to complete the bypass operation.
  • A-V arteriovenous
  • the arterial clamp was opened, and the blood flowed from the left common carotid artery to the right external jugular vein through the bypass tube, and the blood flow was opened to form a bypass circulation.
  • the thrombus was collected 15 minutes later.
  • Test indicators Open the blood flow after 15 minutes, take out the silk thread, blot the residual liquid, weigh the silk thread with thrombus attached, and calculate the wet weight of the thrombus; dry it in an oven at 50°C, weigh it, and calculate the dry weight of the thrombus. Thrombosis inhibition rate was calculated. After the experiment, the tail was cut to detect the bleeding time.
  • Example 5 Effect of compound (1) preventive administration on cerebral ischemia-reperfusion injury in rats
  • mice were randomly divided into 6 groups, which were set as sham operation group, model control group, positive control drug ozagrel (6mg/kg) group, test drug compound (1) high (67.5mg/kg), Middle (22.5mg/kg), low (7.5mg/kg) dose groups.
  • the administration group was pre-administered by tail vein injection, and the sham operation group and the model control group were given equal volumes of normal saline once a day for 3 consecutive days.
  • the rat MCAO/R model was established, and reperfusion was performed after 2 hours of ischemia.
  • 24 hours after reperfusion with MCAO neurobehavioral scores were performed on the rats to determine the neurological function of the rats; the rate of cerebral infarction and brain content water volume.
  • Compound (1) preventive administration can reduce the rate of cerebral infarction and brain water content in rats with cerebral ischemia-reperfusion injury, and improve the neuromotor function of rats.
  • Example 6 Effect of compound (1) combined with urokinase (i.v) on ischemic brain injury in rats induced by autologous thrombus and thrombin
  • SD rat cerebral ischemic injury model was prepared by injecting autologous thrombus and thrombin into the internal carotid artery through the external carotid artery to embolize the middle cerebral artery of rats. (only separate the external carotid artery); model control group (both groups are given equal volume of normal saline); urokinase (5000U/kg) group; compound (1) (22.5mg/kg) group, every group of 20 ( 10 were used to detect cerebral infarction rate, 10 were used for pathological detection), 2 hours after modeling, the tail vein was slowly injected (1 ml per minute) once, and the blood flow rate was monitored after thrombin was injected and 120 minutes after administration. decline situation.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • HE hematoxylin Essence-eosin
  • Intravenous administration of compound (1), urokinase and compound (1) + urokinase after autologous thrombus and thrombin-induced cerebral ischemic injury model can significantly reduce the percentage of cerebral blood flow decline and the cerebral infarct size of rats and brain water content, and improve pathological damage and behavioral changes, in which compound (1) + urokinase has the most obvious improvement in cerebral ischemic damage caused by autologous thrombus and thrombin.
  • mice were randomly divided into 11 groups, which were set as sham operation group, model control group, intravenous injection of positive control drug edaravone (6mg/kg, i.v.) group, intravenous injection of positive control butylphthalide injection ( 5mg/kg, i.v.) group, intravenous compound (1) high (24mg/kg, i.v.), medium (12mg/kg, i.v.), low (6mg/kg, i.v.) dose group, oral administration of positive drug butylphthalide Soft capsule (60mg/kg, i.g.) group, high (60mg/kg, i.g.), middle (30mg/kg, i.g.), low (15mg/kg, i.g.) dose group of compound (1) administered orally.
  • positive control drug edaravone 6mg/kg, i.v.
  • positive control butylphthalide injection 5mg/kg, i.v.
  • the cerebral ischemia-reperfusion model of middle cerebral artery occlusion (MCAO) in male rats was established by internal carotid artery suture method, and the ischemia time was 90min and then reperfusion. 1 hour after reperfusion, the tail vein was administered and the oral administration was administered once. After 24 hours, the neurobehavioral score was performed on the rats to determine the neurological function of the rats; 2,3,5-triphenyltetrazolium chloride (TTC) The cerebral infarction rate and brain water content of rats were measured by dyeing method.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • each dose group of compound (1) and each positive drug group can reduce the neurological function score, cerebral infarction rate and brain water content of MCAO/R rats to varying degrees, wherein compound (1) (24mg/kg, i.v.) and compound (1) (60mg/kg, i.g.) group had the most obvious effect.
  • Compound (1) can reduce the rate of cerebral infarction and brain water content in rats with cerebral ischemia-reperfusion injury after therapeutic administration, and improve the neuromotor function of rats.
  • Example 8 Compound 1, Compound 3 and Compound 5 Inhibit Neuronal Ferroptosis During Cerebral Ischemia-Reperfusion
  • the experimental data are expressed as means ⁇ SD, and the statistical differences between groups were tested by one-way ANOVA and Tukey's test, and a p value less than 0.05 was considered to have a significant difference.
  • CCK8 assay was used to detect the cell viability of the cells in each group. Results are presented as Mean ⁇ SD. ***p ⁇ 0.0001 compared to control group. # p ⁇ 0.05, ## p ⁇ 0.01 compared to the model group.
  • the cell viability of the model group was significantly lower than that of the control group (p ⁇ 0.0001); when the concentration of compound 3 was greater than 10 ⁇ M, the cell viability was significantly improved compared with the model group (p ⁇ 0.05); compound 5 and compound 1 When the concentration is greater than 5 ⁇ M, the cell viability is significantly increased compared with the model group (p ⁇ 0.05).
  • Compound 1, Compound 3 and Compound 5 can significantly increase Erastin-induced rat neuron cell viability and GPX4 mRNA expression, and significantly reduce intracellular lipid ROS levels and iron content.
  • the above results show that Compound 1, Both compound 3 and compound 5 can inhibit Erastin-induced ferroptosis in rat neurons.
  • Compound (1), compound (3) and compound (5) were co-treated with high-sugar and high-fat diet for 30 hours, and treated to 9dpf.
  • Ten zebrafish were randomly selected in each experimental group and the tail veins and tail veins of zebrafish were collected under a fluorescent microscope. The fluorescence intensity of macrophages around the vein was used to evaluate the regression effect of compound (1), compound (3) and compound (5) on vascular inflammation in high-glucose and high-fat model zebrafish by statistical analysis results.
  • Prescription amount Compound (1) 1000mg glycerin 10g Sodium chloride 3.5g Sodium citrate dihydrate 150mg 0.1M hydrochloric acid or sodium hydroxide Appropriate amount water for injection 500ml Solution pH range 5.0-6.5
  • Table 22 compound (1) formula table for preparing freeze-dried powder injection

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Abstract

本发明提供了心脑血管药物及其应用。具体地，本发明化合物具有式(I)所示结构，其中，各基团定义如说明书中所述；本发明还公开了所述化合物在治疗血栓性疾病疾病、抗炎和治疗神经损伤方面的用途。

Description

＜心脑血管药物及其应用＞ 技术领域
本发明属于药物领域，更具体地涉及心脑血管药物及其应用。
背景技术
中国心血管病患病率处于持续上升阶段，推算心血管病现患人数3.30亿。脑卒中发病率在我国仍呈现上升趋势，新发人数约占全球的40％，死亡人数约占全球的30％，带病生存的卒中患者高达1300万，我国已成为卒中终生风险最高和疾病负担最重的国家。据估计，未来10年中国冠心病发生率会持续升高，急性心肌梗死的发生率也会急剧攀升，预计到2030年以后，中国大概会有2000多万的急性心肌梗死患者。急性肺栓塞是仅次于心肌梗死和脑卒中的全球第三大急性心血管综合征，肺栓塞的年发病率为(39-115)人/10万人，且近年来发病率呈上升趋势。
发生血栓性疾病时，临床上主要采用手术取栓、溶栓、抗栓治疗。常用的溶栓药物包括尿激酶、链激酶、阿替普酶、瑞替普酶等。常用的抗栓药物包括抗血小板药和抗凝药。溶栓药物和口服抗栓药物虽然有较好的预防和治疗效果，但缺点是均有导致出血的风险，因而在使用期间必须对凝血机制进行严密的监测。
由于在脑卒中发生期间,发生脑部血管闭塞,血供终止,对神经元细胞造成损害,因此临床需要对受损的神经元细胞进行治疗。
依达拉奉是日本开发上市的用于治疗脑梗死急性发作,改善急性脑梗死所致的神经症状、日常生活活动能力和功能障碍的药物，虽已在中国批准上市,但尚未被欧美主流国家认可。依达拉奉组合物(依达拉奉莰醇)III期临床研究获得部分阳性结果,中国已批准上市,但由于其临床方案设计和国外目前神经保护剂公认临床研究指南的要求不一致,因此未得到美国FDA批准及指南推荐。
中国针对缺血性脑卒中诊治指南中仅提出丁苯酞作为改善脑微循环用药的II级推荐意见，而国外临床指南中尚无可推荐的神经保护剂。可以说全球主流医学上公认的神经保护剂领域仍为空白。
为此，本发明人设想,开发一种新型的针对脑卒中神经损害的治疗药物，既能有效地对血栓性疾病进行溶栓治疗，同时又能起到保护脑神经的作用，将填补神经保护剂领域的空白,满足心脑血管疾病领域临床上未能满足的需求，具有重大的临床意义和实用价值。
发明内容
本发明目的在于提供一种心脑血管药物及其应用。
在本发明的第一方面，提供了一种式I化合物或其药学上可接受的盐的用途， 用于制备一药物组合物或制剂，所述药物组合物或制剂包含作为活性成分的式I化合物，并且所述药物组合物或制剂用于：
(a)预防和/或治疗血栓相关性疾病；
(b)抗炎治疗；和/或
(c)治疗脑梗、脑外伤、脑出血或脑肿瘤手术所导致的神经元损伤；
Figure PCTCN2021143944-appb-000001
式中，
R 1选自下组：羟基、C1-C6烷氧基、卤素、取代或未取代的-O-C1-C6烷基、
Figure PCTCN2021143944-appb-000002
其中所述的取代为磺酸基或羟基取代；
其中，m为1-6的正整数；
Figure PCTCN2021143944-appb-000003
中
Figure PCTCN2021143944-appb-000004
的碳原子为手性碳原子，所述的手性选自下组：
Figure PCTCN2021143944-appb-000005
Figure PCTCN2021143944-appb-000006
R 4为选自下组的金属离子：Na +、K +、Li +或Cs +；
R 5为C1-C6烷基、C3-C8环烷基、C2-C6烯基、C2-C6炔基、芳基或杂芳基；
R 2和R 3各自独立地为H、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C2-C6羟烷基或-(C1-C3亚烷基)-COOH；
n为6-12的正整数。
在另一优选例中，所述的药物组合物用于血栓预防、溶栓治疗；或用于预防和/或治疗脑缺血再灌注损伤、抑制脑缺血再灌注所导致的神经细胞铁死亡、或消退血管炎症。
在另一优选例中，所述的血栓选自下组：血管内皮损伤性血栓、动静脉旁路血栓、脑缺血再灌注损伤、或其组合。
在另一优选例中，所述的脑缺血再灌注损伤包括：脑梗死、脑水肿和/或神经细胞铁死亡。
在另一优选例中，所述的抗炎治疗为抗血管炎症治疗。
在另一优选例中，所述的血管炎症包括：高糖高脂导致的血管炎症。
在另一优选例中，所述药物组合物或制剂用于预防和/或治疗血栓相关性疾病。
在另一优选例中，所述的血栓相关性疾病选自下组：脑血栓、脑梗死(急性缺血性脑卒中)及其导致的神经组织的神经元损伤、脑水肿、心肌梗死、肺栓塞，或其组合。
在另一优选例中，所述的血栓相关性疾病包括梗塞相关性疾病。
在另一优选例中，所述药物组合物或制剂用于抗炎治疗。
在另一优选例中，所述药物组合物或制剂用于脑梗、脑外伤、脑出血或脑肿瘤手术所导致的神经元损伤的治疗。
在另一优选例中，R 1选自下组：羟基、甲氧基、
Figure PCTCN2021143944-appb-000007
Figure PCTCN2021143944-appb-000008
在另一优选例中，m＝1。
在另一优选例中，R 4为Na +。
在另一优选例中，R 5为CH 3。
在另一优选例中，n为6、7或8。
在另一优选例中，R 1选自下组：羟基、甲氧基、
Figure PCTCN2021143944-appb-000009
Figure PCTCN2021143944-appb-000010
在另一优选例中，R 2和R 3各自独立地为H、甲基、羟丙基、羟乙基或羧甲基。
在另一优选例中，所述的式I化合物选自下组：甲基环糊精、羧甲基环糊精、羟乙基-β-乙环糊精、羟丙基-β-环糊精或磺丁基醚-β-环糊精。
在另一优选例中，所述的式I化合物母核为环糊精。
在另一优选例中，所述的式I化合物为
Figure PCTCN2021143944-appb-000011
在另一优选例中，所述的式I化合物选自下组：
Figure PCTCN2021143944-appb-000012
在另一优选例中，所述的药物组合物还含有额外的活性成分。
在另一优选例中，所述的额外的活性成分选自下组：丁苯酞、尿激酶、依达拉奉，或其组合。
在另一优选例中，所述药物组合物或制剂含有作为活性成分的式(I)化合物或其药学上可接受的盐和药学上可接受的载体。
在另一优选例中，所述药物组合物或制剂的剂型选自下组：注射液、冻干粉针、缓释、控释、肠溶片剂或胶囊、颗粒剂。
在另一优选例中，所述的药物组合物或制剂中含有0.001-99wt％，较佳地0.1-90wt％，更佳地1-80wt％的作为活性成分的式I化合物或其药学上可接受的盐，按组合物的总重量计。
本发明的第二方面，提供了一种式I化合物与尿激酶组合物的用途，用于制备治疗血栓相关性疾病的药物。
本发明的第三方面，提供了一种式I化合物与丁苯酞组合物的用途，用于制备治疗脑梗死所导致的神经损伤的药物。
本发明的第四方面，提供了一种药物组合物或制剂，含有(a)活性成分，所述活性成分包括式I化合物或其药学上可接受的盐；以及(b)药学上可接受的载体，所述药物组合物或制剂用于：
(1)预防和/或治疗血栓相关性疾病；
(2)抗炎治疗；和/或
(3)治疗脑梗、脑外伤、脑出血或脑肿瘤手术所导致的神经元损伤。
在另一优选例中，当所述活性成分含有两种组分时，两种组分的重量比为1:20至20:1，较佳地1:10至10:1，更佳地1:5至5:1。
本发明的第五方面，提供了一种药盒，其特征在于，所述的药盒包括：
(1)第一容器，以及位于所述容器内的第一药物组合物，所述的第一药物组合物含有第一化合物或其药学上可接受的盐，以及药学上可接受的载体；
(2)第n容器，以及位于所述容器内的第n药物组合物，所述的第n药物组合物含有第n化合物或其药学上可接受的盐；以及药学上可接受的载体；其中，n为2-8中任一正整数；
其中，第一化合物和第n化合物均为式I化合物或其药学上可接受的盐，或第一化合物为式I化合物或其药学上可接受的盐和第n化合物中至少一个为额外的活性物质，其中，式I化合物如权利要求1中所定义；
和/或(3)任选的使用说明书。
本发明的第六方面，提供了一种疾病的治疗方法，所述的疾病如权利要求1所述，包括步骤：给需要对象施用式I化合物或其药学上可接受的盐，其中，式I化合物如权利要求1中所定义。
在另一优选例中，所述的对象为哺乳动物。
在另一优选例中，所述的对象为人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现了一类结构如式I所示化合物对预防和/或治疗血栓相关性疾病；抗炎治疗；治疗脑梗、脑外伤、脑出血或脑肿瘤手术所导致的神经元损伤均有显著的效果。实验表明，所述的式I化合物对脑卒中引起的脑神经损伤具有良好的治疗效果。本发明的式I化合物可用于血栓性疾病的血栓预防及溶栓、抗炎的治疗等。在此基础上，完成了本发明。
术语：
术语“卤素”指F、Cl、Br和I。
术语“C1-C6烷基”是指包括1-6个碳原子的直链或支链的烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、新戊基、特戊基、或类似基团。
术语“C2-C6烯基”是指具有2-6个碳原子的含有一个双键的直链或支链烯基，非限制性地包括乙烯基、丙烯基、丁烯基、异丁烯基、戊烯基和己烯基等。
术语“C2-C6炔基”是指具有2-6个碳原子的含有一个三键的直链或支链炔基，非限制性地包括乙炔基、丙炔基、丁炔基、异丁炔基、戊炔基和己炔基等。
术语“C1-C6羟烷基”是指具有1-6个碳原子的含有一个羟基的直链或支链烷基，非限制性地包括羟甲基、羟乙基、羟丙基、羟丁基等，优选为C1-C3羟烷基。
术语“C3-C8环烷基”是指在环上具有3-8个碳原子的环状烷基，非限制性地包括环丙基、环丁基、环戊基、环己基、环庚基、环辛基等。
术语“C1-C6烷氧基”是指具有1-6个碳原子的直链或支链烷氧基，非限制性地包括甲氧基、乙氧基、丙氧基、异丙氧基和丁氧基等。优选为C1-C4烷氧基。
术语“芳环”或“芳基”具有相同的含义，优选为“C6-C10芳基”。术语“C6-C10芳基”是指在环上不含杂原子的具有6-10个碳原子的芳香族环基，如苯基、萘基等。
术语“杂芳基”表示包含1至4个杂原子的杂芳族体系，所述杂原子包括氮、氧和S(O)r(其中r是整数0、1、2)的杂原子，例如，4-8元杂芳基指含有4-8个环原子的杂芳族体系，4-10元杂芳基指含有4-10个环原子的杂芳族体系，包括但不限于吡咯基、呋喃基、噻吩基、吡唑基、噻唑基、咪唑基、噁唑基、异噁唑基、吡啶基、吡喃基、哒嗪基、嘧啶基、吡嗪基、苯并咪唑基、三唑基等。
本发明所述的基团除非特别说明是“取代或未取代的”，否则本发明的基团均可被选自下组的取代基所取代：卤素、酰氧基、氰基、氨基、硝基、羧基、酰胺基、羧基甲基、C1-C6烷基、C1-C6烷氧基、C1-C6卤代烷基、C2-C6烯基、C2-C6卤代烯基、C2-C6炔基、C2-C6卤代炔基、羟基、C3-C6环烷基、C3-C6卤代环烷基、羟基C1-C4烷基、C5-C7环烯基、苯基、萘基等。
Figure PCTCN2021143944-appb-000013
表示基团的接合位置。
活性物质
如本文所用，术语“本发明化合物”和“本发明活性成分”可互换使用，指式I化合物。
Figure PCTCN2021143944-appb-000014
在本发明中，还包括式I化合物的药学上可接受的盐。术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括 无机盐和有机盐。一类优选的盐是本发明聚合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。
本发明的式I化合物可采用现有技术中本领域技术人员熟知的方法进行制备，对各个步骤的反应参数没有特别限制。此外，本发明的典型化合物也可通过市售方式获得。
如本文所用，在式I化合物中，如果存在手性碳原子，则手性碳原子可以为R构型，也可以为S构型，或二者的混合物。
本发明中，所述的活性物质为式I化合物，
Figure PCTCN2021143944-appb-000015
式中，
R 1选自下组：羟基、C1-C6烷氧基、卤素、取代或未取代的-O-C1-C6烷基、
Figure PCTCN2021143944-appb-000016
其中所述的取代为磺酸基或羟基取代；
其中，m为1-6的正整数；
Figure PCTCN2021143944-appb-000017
中
Figure PCTCN2021143944-appb-000018
的碳原子为手性碳原子，所述的手性选自下组：
Figure PCTCN2021143944-appb-000019
Figure PCTCN2021143944-appb-000020
R 4为选自下组的金属离子：Na +、K +、Li +或Cs +；
R 5为C1-C6烷基、C3-C8环烷基、C2-C6烯基、C2-C6炔基、芳基或杂芳基；
R 2和R 3各自独立地为H、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C2-C6羟烷基或-(C1-C3亚烷基)-COOH；
n为6-12的正整数。
一个实施方式中，R 1选自下组：羟基、甲氧基、
Figure PCTCN2021143944-appb-000021
Figure PCTCN2021143944-appb-000022
另一个实施方式中，m＝1。
另一个实施方式中，R 4为Na +。
另一个实施方式中，R 5为CH 3。
另一个实施方式中，n为6、7或8。
另一个实施方式中，R 1选自下组：羟基、甲氧基、
Figure PCTCN2021143944-appb-000023
Figure PCTCN2021143944-appb-000024
另一个实施方式中，R 2和R 3各自独立地为H、甲基、羟丙基、羟乙基或羧甲基。
另一个实施方式中，所述的式I化合物选自下组：甲基环糊精、羧甲基环糊精、羟乙基-β-乙环糊精、羟丙基-β-环糊精或磺丁基醚-β-环糊精。
另一个实施方式中，所述的式I化合物母核为环糊精。
另一个实施方式中，所述的式I化合物为
Figure PCTCN2021143944-appb-000025
另一个实施方式中，所述的式I化合物选自下组：
Figure PCTCN2021143944-appb-000026
Figure PCTCN2021143944-appb-000027
药物组合物和施用方法
本发明还提供了一种药物组合物，包含药学上可接受的载体和一种或多种安全有效量的本发明所述的化合物。
由于本发明化合物具有优异的抗血栓活性，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解与栓塞相关的疾病。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明化合物/剂，更佳地，含有10-1000mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2021143944-appb-000028
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
所述的药物组合物为注射剂、囊剂、片剂、丸剂、散剂或颗粒剂。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、 葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的其他化合物(如抗血栓药物)联合给药。
本发明治疗方法可以单独施用，或者与其它治疗手段或者治疗药物联用。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选50～1000mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
(a)本发明的式I化合物对脑卒中引起的脑神经损伤具有优良治疗效果。
(b)本发明的式I化合物可用于血栓性疾病的血栓预防及溶栓治疗且不引起出血。
(c)本发明的式I化合物具有抗炎效果。
(d)本发明的式I化合物中化合物1具有优良的安全性能，毒副作用小。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1 样品对血管内皮损伤性血栓预防作用评价实验
实验方法：随机选取420尾5dpf野生型AB品系斑马鱼于六孔板中，每孔(实验组)30尾。注射给予化合物(1)、化合物(2)、化合物(3)、化合物(4)、化合物(5)、化合物(6)、化合物(7)、化合物(8)、化合物(9)、化合物(10)和化合物(11)(结构式见表1)，剂量为4.00ng/尾剂量，水溶给予阳性对照药乙酰水杨酸30.0μg/mL浓度，同时设置正常对照组(即标准稀释水处理斑马鱼)和模型对照组，每孔(实验组)容量为3mL。除正常对照组外，其余实验组均水溶给予普纳替尼以诱发斑马鱼血管内皮损伤性血栓模型。在28℃孵育条件下，样品与普纳替尼共同处理一段时间后，用邻联茴香胺进行染色。
染色后每组随机选取10尾斑马鱼在显微镜下拍照，采用NIS-Elements D 3.20高级图像处理软件对斑马鱼心脏红细胞染色强度进行分析，以心脏红细胞染色强度的统计学结果评价样品对血管内皮损伤性血栓预防作用。
血栓预防作用计算公式如下：
Figure PCTCN2021143944-appb-000029
用SPSS软件进行统计学分析，p<0.05表明具有显著性差异。
实验结果：
表1样品对血管内皮损伤性血栓预防作用(n＝10)
Figure PCTCN2021143944-appb-000030
Figure PCTCN2021143944-appb-000031
Figure PCTCN2021143944-appb-000032
与模型对照组比较，*p<0.05，**p<0.01，***p≤0.001。
实验结果：化合物(1)、化合物(2)、化合物(3)、化合物(4)、化合物(5)、化合物(6)、化合物(7)、化合物(8)、化合物(9)、化合物(10)和化合物(11)的4.00ng/尾剂量组斑马鱼心脏红细胞染色强度分别为4397、3655、3045、4300、4340、4938、4584、4580、4709、4326和4749像素。
与模型对照组比较，化合物(3)4.00ng/尾剂量组p>0.05，其血栓预防作用分别为17％，化合物(1)、化合物(2)、化合物(4)、化合物(5)、化合物(6)、化合物(7)、化合物(8)、化合物(9)、化合物(10)和化合物(11)4.00ng/尾剂量组p<0.01、p<0.05、p<0.001、p<0.001、p<0.001、p<0.001、p<0.001、p<0.001、p<0.01、p<0.001，其血栓预防作用分别为66％、39％、63％、64％、86％、73％、73％、78％、64％和79％。
实验结论：化合物(1)、化合物(2)、化合物(4)、化合物(5)、化合物(6)、化合物(7)、化合物(8)、化合物(9)、化合物(10)和化合物(11)在本实验条件下对普纳替尼诱发的血管内皮损伤性血栓斑马鱼有明显预防作用。
尤其是，化合物(6)、化合物(11)、化合物(9)、化合物(7)、化合物(8)和化合物(1)，在4.00ng/尾剂量组时，对于普纳替尼诱发的血管内皮损伤性血栓斑马鱼有非常显著预防作用，血栓预防作用(％)指标为66％-86％，而30ug/ml的乙酰水杨酸的血栓预防作用指标为101％。
实施例2 样品诱发致敏风险评价
实验方法：随机选取2dpf野生型AB品系斑马鱼于24孔板中，每组设置6个复孔，每孔均处理10尾斑马鱼。分别静脉注射给予样品500ng/尾剂量，阳性对照“C48/80”1.5μg/mL浓度，同时设置正常对照组，每孔容量为1mL。
各实验组均水溶给予BAPNA。在28℃处理1天后，将液体转移至96孔板中，200μL/孔，利用酶标仪检测各实验组类胰蛋白酶(Tryptase)表达水平的相对吸光度值(OD值)，以OD值的统计学分析结果评价样品致敏风险。统计学结果采用mean±SE表示。
Figure PCTCN2021143944-appb-000033
用SPSS软件进行统计学分析，p<0.05表明具有显著性差异；
表2各样品的致敏风险
Figure PCTCN2021143944-appb-000034
实验结果：即使在实施例1的4ng/尾的剂量的基础上提高125倍(即在500ng/ 尾)的剂量条件下，测试的本发明化合物的致敏风险较低。代表性地，如表2所示，测试的本发明化合物(1)、化合物(2)、化合物(10)致敏风险均显著低于阳性对照的致敏风险。
其中，“化合物(2)”和“化合物(1)”没有或基本上没有致敏风险，“化合物(10)”具有低致敏风险。
实验结论：结合实施例1和2的实验结果，即综合考虑安全性及有效性，本发明选择化合物(1)作为优选化合物进行后续实验。
实施例3 化合物1对血管内皮损伤性血栓预防作用评价
实验方法：用4μg/mL普纳替尼处理野生型AB品系斑马鱼18h，建立斑马鱼血管内皮损伤性血栓模型。随机选取150尾5dpf野生型AB品系斑马鱼于六孔板中，每孔(实验组)30尾。注射给予化合物(1)2.06、6.18ng/尾剂量，水溶给予阳性对照药乙酰水杨酸30μg/mL浓度，同时设置正常对照组(即标准稀释水处理斑马鱼)和模型对照组，每孔(实验组)容量为3mL。除正常对照组外，其余实验组均水溶给予普纳替尼以诱发斑马鱼血管内皮损伤性血栓模型。在28℃孵育条件下，化合物(1)与普纳替尼共同处理一段时间后，用邻联茴香胺进行染色，染色后每组随机选取10尾斑马鱼在显微镜下拍照，采用NIS-Elements D 3.10高级图像处理软件对斑马鱼心脏红细胞染色强度进行分析，以心脏红细胞染色强度的统计学结果评价化合物(I)对血管内皮损伤性血栓预防作用。血栓预防作用计算公式如下：
Figure PCTCN2021143944-appb-000035
用方差分析和Dunnett's T-检验进行统计学分析，p<0.05表明具有显著性差异，提供具有代表性的实验图谱。
表3化合物(I)对血管内皮损伤性血栓预防作用(n＝10)
Figure PCTCN2021143944-appb-000036
与模型对照组比较，***p<0.001。
实验结果：化合物(1)2.06ng/尾和6.18ng/尾剂量组斑马鱼心脏红细胞染色强度分别为4601、5044像素，与模型对照组比较p<0.001，其血栓预防作用分别为83％、99％。
实验结论：化合物(1)对普纳替尼诱发的斑马鱼血管内皮损伤性血栓具有显著的预防作用且其预防效果有一定的剂量依赖性，化合物(1)对血管内皮损伤性血栓的预防作用优于阳性对照乙酰水杨酸。
实施例4 化合物(1)对大鼠动静脉旁路血栓的影响
实验方法：
分组：分为5组，每组10只。模型对照组给予溶剂，受试药化合物(1)和阳性对照组分别静脉注射给予，连续给药3d，每天一次，末次给药30min后造模。
模型制备：SD雄性大鼠50只，戊巴比妥钠60mg/kg经ip麻醉。仰卧位固定，颈正中线切开皮肤，分离筋膜、肌肉组织，游离左侧颈总动脉和右侧颈外静脉，以内置丝线的聚乙烯管进行动-静脉(A-V)搭桥手术，动脉夹夹住颈动脉近心端，聚乙烯管一段插入动脉，另一段插入静脉，完成搭桥手术。于末次给药30min后，打开动脉夹，血液由左侧颈总动脉经搭桥管流向右侧颈外静脉，血流开通形成旁路循环，15min后取血栓。
检测指标：15min后开放血流，取出丝线，吸干残液，称取附有血栓的丝线重量，并计算血栓湿重；50℃烘箱烤干，称重并计算血栓干重。计算血栓形成抑制率。试验结束后剪尾检测出血时间。
表4化合物(1)对大鼠动静脉旁路血栓形成的影响(
Figure PCTCN2021143944-appb-000037
n＝10)
Figure PCTCN2021143944-appb-000038
注：与模型组比较， *P<0.05， **P<0.01， ***P<0.001；
实验结果：与模型对照组比较，静脉注射化合物(1)10mg/kg、20mg/kg、40mg/kg后，可不同程度抑制大鼠动静脉旁路血栓形成，血栓湿重、干重抑制率分别为19.4％(P>0.005)、15.9％(P>0.005)；21.9％(P<0.001)、26.8％(P<0.001)； 34.3％(P<0.001)、37.8％(P<0.001)；静脉给予奥扎格雷16mg/kg，血栓湿重、干重抑制率为34％(P<0.001)、34.1％(P<0.01)。
实验结论：化合物(1)抗血栓效果有剂量依赖性，其抗血栓作用与奥扎格雷相当或更优。
表5化合物(1)对大鼠出血时间的影响(
Figure PCTCN2021143944-appb-000039
n＝10)
Figure PCTCN2021143944-appb-000040
注：与模型组比较， ***P<0.001。
实验结果：与模型对照组比较，静脉给予不同剂量的化合物(1)对大鼠出血时间无显著影响；阳性对照静脉给予奥扎格雷显著延长大鼠的出血时间(P<0.01)。
实验结论：化合物(1)的出血实验显示了非剂量依赖性，由此证明化合物(1)无明显出血风险。
实施例5 化合物(1)预防给药对大鼠脑缺血再灌注损伤的影响
实验方法：SD大鼠随机分为6组，设为伪手术组、模型对照组、阳性对照药奥扎格雷(6mg/kg)组、受试药化合物(1)高(67.5mg/kg)、中(22.5mg/kg)、低(7.5mg/kg)剂量组。给药组预先尾静脉注射给药，伪手术组和模型对照组给予等容积的生理盐水，每天给药一次，连续给药3天。末次给药后10分钟，建立大鼠MCAO/R模型，缺血2小时后进行再灌注。MCAO再灌后24小时，对大鼠进行神经行为学评分，测定大鼠神经功能；2,3,5-氯化三苯基四氮唑(TTC)染色法测定大鼠脑梗死率和脑含水量。
表6化合物(1)预防给药对局灶性脑缺血(MCAO)/再灌注大鼠脑梗死率的影响
(数据采用Mean±SD表示，n＝8)
Figure PCTCN2021143944-appb-000041
Figure PCTCN2021143944-appb-000042
**P<0.01，与模型对照组比较；*P<0.05，与模型对照组比较。
表7化合物(1)预防给药对局灶性脑缺血(MCAO)/再灌注大鼠脑含水量的影响
(数据采用Mean±SD表示，n＝8)
Figure PCTCN2021143944-appb-000043
##P<0.01，与伪手术组比较；**P<0.01，与模型对照组比较；*P<0.05，与模型对照组比较。
表8化合物(1)预防给药对局灶性脑缺血(MCAO)/再灌注大鼠神经行为学的影响
(数据采用Mean±SD表示，n＝8)
Figure PCTCN2021143944-appb-000044
**P<0.01，与模型对照组比较；*P<0.05，与模型对照组比较。
实验结果：化合物(1)各剂量组与阳性药奥扎格雷组均能降低MCAO/R大鼠的神经功能评分、脑梗死率和脑含水量。
实验结论：化合物(1)预防给药可以降低大鼠脑缺血再灌注损伤脑梗死率和脑含水量，改善大鼠神经运动功能。
实施例6 化合物(1)与尿激酶联合用药(i.v)对自体血栓和凝血酶所致大鼠缺 血性脑损伤的影响
实验方法：采用经颈外动脉向颈内动脉注入自体血栓和凝血酶栓塞大鼠大脑中动脉的方法，制备SD大鼠脑缺血损伤模型，按行为学评分随机分为5组，假手术组(仅分离颈外动脉)；模型对照组(两组均给以等容积的生理盐水)；尿激酶(5000U/kg)组；化合物(1)(22.5mg/kg)组，每组20只(10只用于检测脑梗死率，10只用于病理检测)，造模后2h尾静脉缓慢注射(每分钟1毫升)给药一次，并监测注射凝血酶后、给药后120min时血流量的下降情况。24h后对大鼠进行神经行为学评分，测定大鼠神经功能；2,3,5-氯化三苯基四氮唑(TTC)染色法测定大鼠脑梗死率和脑含水量，并用苏木精-伊红(HE)染色法观察脑缺血后2h给药的大鼠大脑病理损伤。
实验结果：
表9化合物(1)(i.v)对大鼠自体血栓和凝血酶所致脑缺血损伤后脑血流量的影响
(数据采用Mean±SD表示，n＝10)
Figure PCTCN2021143944-appb-000045
**P<0.01，与模型对照组比较； ##P<0.01，与注射凝血酶后自身组比较。
表10化合物(1)(i.v)对大鼠神经行为学的影响
(数据采用Mean±SD表示，n＝10)
Figure PCTCN2021143944-appb-000046
*P<0.05，与模型对照组比较。
表11化合物(1)(i.v)对自体血栓和凝血酶所致大鼠脑缺血损伤脑梗死率及脑含水量的影响(mean±S.D.，n＝10)
Figure PCTCN2021143944-appb-000047
##P<0.01，与空白对照组比较；*P<0.05，**P<0.01，与模型对照组比较。
表12化合物(1)(i.v)对自体血栓和凝血酶所致脑缺血损伤
对大鼠脑缺血区病理学损伤评分的影响(mean±SD，n＝10)
Figure PCTCN2021143944-appb-000048
*P<0.05，**P<0.01，与模型对照组比较。
自体血栓和凝血酶所致脑缺血损伤模型后静脉给予化合物(1)、尿激酶和化合物(1)+尿激酶能显著降低模型大鼠的脑血流量的下降百分比、大鼠的脑梗死面积和脑含水量，并改善病理学损伤及行为学变化，其中化合物(1)+尿激酶对自体血栓和凝血酶所致脑缺血损伤的改善水平最为明显。
实验结论：化合物(1)(i.v)及其与尿激酶联合给药可以降低自体血栓和凝血酶所致缺血性脑损伤大鼠的的脑梗死率、脑含水量及病理损伤，改善大鼠神经运动功能，表现出较好的抗缺血性脑损伤作用。
实施例7 化合物(1)治疗给药对大鼠脑缺血再灌注损伤的影响
实验方法：SD大鼠随机分为11组，设为伪手术组、模型对照组、静注阳性对照药依达拉奉(6mg/kg，i.v.)组、静注阳性对照丁苯酞注射液(5mg/kg，i.v.)组、静注化合物(1)高(24mg/kg，i.v.)、中(12mg/kg，i.v.)、低(6mg/kg，i.v.)剂量组、灌胃阳性药丁苯酞软胶囊(60mg/kg，i.g.)组、灌胃化合物(1)高(60mg/kg，i.g.)、中(30mg/kg，i.g.)、低(15mg/kg，i.g.)剂量组。采用颈内动脉线栓法制备雄性大鼠大脑中动脉阻塞(MCAO)脑缺血再灌注模型，缺血时间90min然后再灌。于再灌注后1h尾静脉给药及口服给药1次，24h后对大鼠进行神经行为学评分，测定大鼠神经功能；2,3,5-氯化三苯基四氮唑(TTC)染色法测定大鼠脑梗死率和脑含水 量。
表13化合物(1)治疗给药对局灶性脑缺血(MCAO)/再灌注大鼠脑梗死率的影响
(数据采用Mean±SD表示，n＝8)
Figure PCTCN2021143944-appb-000049
**P<0.01，与模型对照组比较；*P<0.05，与模型对照组比较。
表14化合物(1)治疗给药对局灶性脑缺血(MCAO)/再灌注大鼠脑含水量的影响
(数据采用Mean±SD表示，n＝8)
Figure PCTCN2021143944-appb-000050
Figure PCTCN2021143944-appb-000051
##P<0.01，与伪手术组比较；**P<0.01，与模型对照组比较；*P<0.05，与模型对照组比较。
表15化合物(1)预防给药对局灶性脑缺血(MCAO)/再灌注大鼠神经行为学的影响
(数据采用Mean±SD表示，n＝8)
Figure PCTCN2021143944-appb-000052
**P<0.01，与模型对照组比较；*P<0.05，与模型对照组比较。
实验结果：化合物(1)各剂量组与各阳性药组均能不同程度降低MCAO/R大鼠的神经功能评分、脑梗死率和脑含水量，其中化合物(1)(24mg/kg，i.v.)和化合物(1)(60mg/kg，i.g.)组效果最明显。
实验结论：化合物(1)治疗给药可以降低大鼠脑缺血再灌注损伤脑梗死率，脑含水量，并改善大鼠神经运动功能。
实施例8 化合物1、化合物3和化合物5抑制脑缺血再灌注时神经细胞铁死亡的研究
实验方法：原代神经元细胞使用含Erastin(终浓度为50μM)的培养基孵育24h后，换含不同浓度的受试药和Erastin(终浓度为50μM)的培养基继续孵育24h。孵育结束后，Real time PCR检测各组细胞中GPX4的mRNA表达，CCK8实验检测各组细胞的细胞活力，C11-BODIPY探针法检测各组细胞内的脂质ROS水平，PGSK探针法检测细胞内的铁含量。
实验数据以means±SD表示，组间统计学差异采用one-way ANOVA和 Tukey's检验，p值小于0.05认为有显著性差异。
实验结果：
表16不同浓度化合物1、化合物3和化合物5对Erastin诱导的神经细胞细胞活力的影响
Figure PCTCN2021143944-appb-000053
孵育结束后，CCK8实验检测各组细胞的细胞活力。结果以Mean±SD呈现。与对照组相比***p<0.0001。与模型组相比 #p<0.05， ##p<0.01。
由表16可以看出，模型组细胞活力与对照组相比显著降低(p<0.0001)；化合物3浓度大于10μM时细胞活力与模型组相比有明显提高(p<0.05)；化合物5和化合物1浓度大于5μM时细胞活力与模型组相比明显上升(p<0.05)。
表17不同浓度化合物1、化合物3和化合物5对Erastin诱导的神经细胞内的脂质ROS水平的影响
Figure PCTCN2021143944-appb-000054
孵育结束后，C11-BODIPY探针法检测各组细胞内的脂质ROS水平。结果以 Mean±SD呈现。与对照组相比***p<0.0001；与模型组相比 #p<0.05， ##p<0.01。
由表17可以看出，模型组细胞内脂质ROS水平与对照组相比显著上升(p<0.0001)；化合物3浓度大于10μM时细胞内脂质ROS水平与模型组相比明显下降(p<0.05)；化合物5和化合物1浓度大于5μM时细胞内脂质ROS水平与模型组相比有显著降低(p<0.01)。
表18不同浓度化合物1、化合物3和化合物5对Erastin诱导的神经细胞细胞内的铁含量的影响
Figure PCTCN2021143944-appb-000055
孵育结束后，PGSK探针法检测细胞内的铁含量。结果以Mean±SD呈现。与对照组相比***p<0.0001；与模型组相比 #p<0.05， ##p<0.01。
由表18可以看出，模型组细胞内PGSK探针荧光强度与对照组相比明显减弱，铁含量显著升高(p<0.0001)；化合物3浓度大于10μM时细胞内铁含量与模型组相比明显降低(p<0.05)；化合物5和化合物1浓度大于5μM时细胞内铁含量与模型组相比显著下降(p<0.05)。
表19不同浓度化合物1、化合物3和化合物5对Erastin诱导的神经细胞GPX4 mRNA表达的影响
Figure PCTCN2021143944-appb-000056
Figure PCTCN2021143944-appb-000057
孵育结束后，Real time PCR检测各组细胞中GPX4的mRNA表达。结果以Mean±SD呈现。与对照组相比 ***p<0.0001；与模型组相比 #p<0.05， ##p<0.01。
由表19可以看出，模型组细胞GPX4 mRNA表达与对照组相比显著降低(p<0.0001)；化合物3浓度大于5μM时细胞GPX4 mRNA表达水平与模型组相比明显升高(p<0.05)；化合物5和化合物1浓度大于2.5μM时细胞GPX4 mRNA表达水平与模型组相比有显著上升(p<0.01)。
实验结论：化合物1、化合物3和化合物5均能显著性增加Erastin诱导的大鼠神经元细胞活力和GPX4 mRNA表达，显著性降低细胞内脂质ROS水平和铁含量，以上研究结果表明化合物1、化合物3和化合物5均能抑制Erastin诱导的大鼠神经元细胞铁死亡。
实施例9 化合物1、化合物3和化合物5对血管炎症消退作用评价
实验方法：随机选取360尾5dpf巨噬细胞荧光斑马鱼于烧杯中，每个烧杯30尾，静脉注射给予化合物(1)，化合物(3)，化合物(5)，阳性对照组水溶给予阿托伐他汀钙30μg/mL浓度，同时设置正常对照组(养鱼用水处理斑马鱼)和模型对照组，每个烧杯(实验组)容量为25mL。除正常对照组外均水溶给予高糖高脂饲料。化合物(1)，化合物(3)和化合物(5)与高糖高脂饲料共同处理30h，处理至9dpf，每个实验组随机选择10尾斑马鱼在荧光显微镜下采集斑马鱼尾静脉内及尾静脉周围巨噬细胞荧光强度，以统计分析结果评价化合物(1)，化合物(3)，化合物(5)对高糖高脂模型斑马鱼血管炎症的消退作用。
Figure PCTCN2021143944-appb-000058
实验结果：
表.20供试品对高糖高脂斑马鱼血管炎症消退作用(n＝10)
Figure PCTCN2021143944-appb-000059
Figure PCTCN2021143944-appb-000060
与模型对照组比较，***p<0.001。
实验结论：在本实验剂量条件下，化合物(1)，化合物(3)和化合物(5)对高糖高脂模型斑马鱼血管炎症具有显著的消退作用。
实施例10 注射液的制备
表21化合物(1)制备注射液的配方表
处方 处方量
化合物(1) 1000mg
甘油 10g
氯化钠 3.5g
二水枸橼酸钠 150mg
0.1M盐酸或氢氧化钠 适量
注射用水加到 500ml
溶液PH范围 5.0-6.5
取全量80％注射用水，依次加入处方量氯化钠、二水枸橼酸钠、甘油、化合物(1)，搅拌使溶解，以0.1M盐酸或氢氧化钠调节溶液pH值至5.0-6.5。以注射用水定容即得。分装至10ml的玻璃瓶，热压灭菌，得到该化合物注射液。每瓶含20mg的化合物(1)。
实施例11 冻干粉针的制备
表22化合物(1)制备冻干粉针的配方表
处方 处方量 作用
化合物(1) 0.1～0.5g 活性成分
氢氧化钠或盐酸 适量 pH调节剂
注射用水 加至2ml 溶剂
将处方量的化合物(1)投入80％处方量的注射用水中，搅拌溶解；加入注射用水至处方量。往上述药液中投入活性炭(0.1～0.5％)，搅拌数十分钟；活性炭脱色，过滤脱碳；使用氢氧化钠溶液(或盐酸溶液)调节药液的pH值。取样进行半成品检测。经过0.22μm过滤器二次过滤，灌装。按设定冻干参数进行冻干、压塞、轧盖、包装。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种式I化合物或其药学上可接受的盐的用途，其特征在于，用于制备一药物组合物或制剂，所述药物组合物或制剂包含作为活性成分的式I化合物，并且所述药物组合物或制剂用于：
    (a)预防和/或治疗血栓相关性疾病；
    (b)抗炎治疗；和/或
    (c)治疗脑梗、脑外伤、脑出血或脑肿瘤手术所导致的神经元损伤；
    Figure PCTCN2021143944-appb-100001
    式中，
    R 1选自下组：羟基、C1-C6烷氧基、卤素、取代或未取代的-O-C1-C6烷基、
    Figure PCTCN2021143944-appb-100002
    其中所述的取代为磺酸基或羟基取代；
    其中，m为1-6的正整数，
    Figure PCTCN2021143944-appb-100003
    中
    Figure PCTCN2021143944-appb-100004
    的碳原子为手性碳原子，所述的手性选自下组：
    Figure PCTCN2021143944-appb-100005
    Figure PCTCN2021143944-appb-100006
    R 4为选自下组的金属离子：Na +、K +、Li +或Cs +；
    R 5为C1-C6烷基、C3-C8环烷基、C2-C6烯基、C2-C6炔基、芳基或杂芳基；
    R 2和R 3各自独立地为H、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C2-C6羟烷基或-(C1-C3亚烷基)-COOH；
    n为6-12的正整数。
  2. 如权利要求1所述的用途，其特征在于，所述的血栓相关性疾病选自下组：脑血栓、脑梗死(急性缺血性脑卒中)及其导致的神经组织的神经元损伤、脑水肿、心肌梗死、肺栓塞，或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述药物组合物或制剂用于预防和/或治疗血栓相关性疾病。
  4. 如权利要求1所述的用途，其特征在于，所述药物组合物或制剂用于脑梗、脑外伤、脑出血或脑肿瘤手术所导致的神经元损伤的治疗。
  5. 如权利要求1所述的用途，其特征在于，所述药物组合物或制剂的剂型选自下组：注射液、冻干粉针、缓释、控释、肠溶片剂或胶囊、颗粒剂。
  6. 一种式I化合物与尿激酶组合物的用途，其特征在于，用于制备治疗血栓相关性疾病的药物。
  7. 一种式I化合物与丁苯酞组合物的用途，其特征在于，用于制备治疗脑梗死、脑外伤、脑出血或脑肿瘤手术所导致的神经元损伤。
  8. 一种药物组合物或制剂，其特征在于，含有(a)活性成分，所述活性成分包括式I化合物或其药学上可接受的盐；以及(b)药学上可接受的载体，所述药物组合物或制剂用于：
    (1)预防和/或治疗血栓相关性疾病；
    (2)抗炎治疗；和/或
    (3)治疗脑梗、脑外伤、脑出血或脑肿瘤手术所导致的神经元损伤。
  9. 一种药盒，其特征在于，所述的药盒包括：
    (1)第一容器，以及位于所述容器内的第一药物组合物，所述的第一药物组合物含有第一化合物或其药学上可接受的盐，以及药学上可接受的载体；
    (2)第n容器，以及位于所述容器内的第n药物组合物，所述的第n药物组合物含有第n化合物或其药学上可接受的盐；以及药学上可接受的载体；其中，n为2-8中任一正整数；
    其中，第一化合物和第n化合物均为式I化合物或其药学上可接受的盐，或第一化合物为式I化合物或其药学上可接受的盐和第n化合物中至少一个为额外的活性物质，其中，式I化合物如权利要求1中所定义；
    和/或(3)任选的使用说明书。
  10. 一种疾病的治疗方法，所述的疾病如权利要求1所述，其特征在于，包括步骤：给需要对象施用式I化合物或其药学上可接受的盐，其中，式I化合物如权利要求1中所定义。
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