WO2016112874A1 - 二联苯酚在制备防治缺血性脑卒中的药物中的应用 - Google Patents

二联苯酚在制备防治缺血性脑卒中的药物中的应用 Download PDF

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WO2016112874A1
WO2016112874A1 PCT/CN2016/076112 CN2016076112W WO2016112874A1 WO 2016112874 A1 WO2016112874 A1 WO 2016112874A1 CN 2016076112 W CN2016076112 W CN 2016076112W WO 2016112874 A1 WO2016112874 A1 WO 2016112874A1
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Prior art keywords
diphenol
tetraisopropyl
pharmaceutically acceptable
cerebral
formula
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PCT/CN2016/076112
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English (en)
French (fr)
Inventor
王汝涛
陈涛
安龙
赵熠
王惟娇
郭树攀
肖飒
庞箐华
胡慧静
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西安力邦制药有限公司
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Application filed by 西安力邦制药有限公司 filed Critical 西安力邦制药有限公司
Priority to JP2017536945A priority Critical patent/JP2018508479A/ja
Priority to EP16737099.8A priority patent/EP3246022A4/en
Priority to AU2016207117A priority patent/AU2016207117B2/en
Priority to US15/543,473 priority patent/US20180185299A1/en
Publication of WO2016112874A1 publication Critical patent/WO2016112874A1/zh
Priority to US16/789,646 priority patent/US20200179302A1/en

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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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/222—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having aromatic groups, e.g. dipivefrine, ibopamine
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/01—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by replacing functional groups bound to a six-membered aromatic ring by hydroxy groups, e.g. by hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/11—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by reactions increasing the number of carbon atoms
    • C07C37/16—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by reactions increasing the number of carbon atoms by condensation involving hydroxy groups of phenols or alcohols or the ether or mineral ester group derived therefrom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/12—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings
    • C07C39/15—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings with all hydroxy groups on non-condensed rings, e.g. phenylphenol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds

Definitions

  • the invention belongs to the technical field of medicine and relates to a new use of a medicine. Specifically, it relates to a new application of a diphenol in the preparation of a medicament for treating and/or preventing ischemic stroke.
  • stroke has become a common disease that seriously threatens the health of humans, especially middle-aged and older people over the age of 50. It has "high incidence, high disability rate, high mortality, high recurrence rate, and many complications".
  • Ischemic stroke accounts for about 80% of all strokes. It refers to the softening and necrosis of local brain tissue due to blood circulation disorder, ischemia and hypoxia. Mainly due to atherosclerosis and thrombosis in the arteries supplying blood in the brain, narrowing or even occlusion of the lumen, leading to focal acute brain cerebral insufficiency; also due to abnormal objects (solid, liquid, gas) entering the blood circulation.
  • the cerebral arteries or the cervical arteries that supply cerebral blood circulation cause blood flow blockage or sudden decrease in blood flow to produce softened necrosis of brain tissue in the corresponding dominant area.
  • Ischemic brain injury mainly comes from two aspects: (1) insufficient capacity after ischemia, inhibition of ATP-dependent enzyme activity and physiological activity, chloride ion, sodium ion and water influx to cause cell edema, synaptic gap excitatory amino acid (mainly glutamate) aggregation, excessive agonism of glutamate receptors, mediated by NMDA and other receptors, causing increased calcium influx, potassium ion extravasation-induced cell depolarization, also opening voltage-sensitive calcium channels, intracellular Calcium overload, multiple enzymes that overactivate phospholipase, nitric oxide synthase (NOS), produce a series of active metabolites and free radicals, causing cell damage; (2) ischemic tissue in patients with stroke after treatment Obtaining blood perfusion or spontaneous reperfusion, although regaining nutrients, at the same time inevitably caused cerebral ischemia-reperfusion injury, that is, after cerebral ischemia resumed blood supply for a certain period of time, its function not only failed to recover, but appeared More serious
  • EAA excitatory amino acid
  • the excitatory amino acids mainly refer to glutamate (Glu) and aspartame. Acid (asparate, Asp).
  • Esp Excessive excitatory EAA of postsynaptic neurons activates intracellular signal transduction pathways, amplifying the second messenger effect caused by some receptors under normal physiological stimulation, triggering the expression of proinflammatory genes after ischemia.
  • Excitatory amino acids such as Glu and Asp play a key role in ischemic neuronal injury.
  • EAA Glu and Asp in the interstitial
  • the effect is consistency in concentration dependence.
  • the toxic effects of excitatory amino acids on nerve cells are multifaceted: excessive EAA activates its receptors, causing sustained depolarization of excitatory neurons, causing intracellular Ca 2+ overload, resulting in cell necrosis; triggering free radicals (such as Nitric oxide production increases, cytotoxicity is produced by free radicals; participation in various metabolic processes in the brain, hindering the circulation of the tricarboxylic acid, reducing ATP production, and aggravating the toxic effects of EAA on cells.
  • Ischemic brain injury is a complex pathophysiological process involving multiple factors. It is generally thought to be related to irreversible damage caused by oxygen peroxide-induced tissue lipid peroxidation and intracellular calcium overload. Its harmful effects can be summarized as: acting on polyvalent unsaturated fatty acids, causing lipid peroxidation; inducing cross-linking of macromolecules such as DNA, RNA, polysaccharides and amino acids, and cross-linking macromolecules lose their original activity or function.
  • Ca 2+ overload in ischemic brain injury is the result of a combination of various factors, and is also a common pathway for various factors in the process of cerebral ischemic injury.
  • the main roles of Ca 2+ in ischemic brain injury are:
  • Ca 2+ can inhibit ATP synthesis, resulting in energy production barriers.
  • Ca 2+ activates phospholipase on mitochondria, causing damage to mitochondrial membranes.
  • mitochondria play an important role in cellular redox reactions, osmotic pressure, pH, and maintenance of intracytoplasmic signals. Mitochondria are important targets for cell damage.
  • Ca 2+ activates Ca 2+ -dependent phospholipase (mainly phospholipase C and phospholipase A2) to promote the decomposition of membrane phospholipids; free fatty acids, prostaglandins, leukotrienes produced during the decomposition of membrane phospholipids, Lysophospholipids are all toxic to cells; Ca 2+ also activates calcium-dependent proteases to convert intracellular harmless xanthine dehydrogenase into xanthine oxidase, which produces a large number of oxygen free radicals; Ca 2+ activates NOS.
  • Ca 2+ activates NOS.
  • GABA receptor agonist GABA can counteract the excitotoxicity of excitatory amino acids and exert its inhibitory protective effect, representing the drug clomethiazine;
  • NMDA receptor antagonist antagonizes NMDA receptor, thereby inhibiting its mediated calcium influx, representing the drug MK801;
  • Calcium antagonists prevent intracellular calcium overload, prevent vasospasm, increase blood flow, and represent the drug nimodipine;
  • Anti-free radical drugs scavenge free radicals, inhibit lipid peroxidation, thereby inhibiting oxidative damage of brain cells, vascular endothelial cells and nerve cells. Representing the drug edaravone;
  • propofol an anesthetic
  • ischemic stroke In recent years, many studies at home and abroad have found that propofol, an anesthetic, may play a very positive role in ischemic stroke. In animal and ex vivo experiments, and even in some clinical studies, propofol has been shown to have significant protective and therapeutic effects on neurological impairment. Experiments have shown that propofol can not only block sodium ion flux or reduce potassium release-activated Glu release by activating GABA receptors, but also block the inhibition of Glu transport by glial cells after oxide treatment.
  • propofol inhibits extracellular calcium influx through voltage-dependent calcium channels, which may increase the current loss of L-type voltage-dependent calcium channels to some extent Liver rate, thereby reducing calcium influx; propofol binds to specific sites of GABAa receptor, not only increases the frequency of GABA open chloride channels, but also enhances low-affinity GABA binding sites and GABA by positive allosteric regulation Combination; propofol can inhibit the production of proinflammatory cytokines such as TNF, IL-1, IL-6 in the blood of patients with sepsis, and has a strong inhibitory effect at low concentrations; propofol can inhibit the brain The expression of the proapoptotic gene caspase-3 mRNA and the expression of the anti-apoptotic gene Bcl-2 mRNA; propofol can competitively bind to membrane phospholipids, and can form a stable phenoxy group with peroxide, which is actually generated.
  • One of the objects of the present invention is to provide 3,3',5,5'-tetraisopropyl-4,4'-diphenol and pharmaceutically acceptable salts, esterates or solutions thereof. New use of the agent.
  • the present invention provides 3,3',5,5'-tetraisopropyl-4,4'-diphenol and pharmaceutically acceptable salts, esters or solvates thereof for the treatment and/or prevention of ischemic Application in drugs for stroke;
  • the structure of 3,3',5,5'-tetraisopropyl-4,4'-diphenol is as shown in formula (I):
  • the 3,3',5,5'-tetraisopropyl-4,4'-diphenol of the present invention further includes a pharmaceutically acceptable salt, ester or solvate of the compound.
  • diphenol has a strong affinity for GABA receptors and has GABA agonism. It can also antagonize NMDA receptors, regulate calcium channels, limit calcium influx in cells, and have propofol. Stronger antioxidant and free radical scavenging effects. Dihydroxyphenol may fight ischemic stroke damage under a variety of mechanisms. The most important thing is that diphenol does not cause loss of consciousness, so it has important clinical application value in the treatment of various ischemic stroke symptoms.
  • the esterified product of the present invention is a monoester or diester of 3,3',5,5'-tetraisopropyl-4,4'-diphenol; preferably, the esterified product is a a monoethyl ester represented by (II) or a diethyl ester represented by the formula (III):
  • the "monoester or diester of 3,3',5,5'-tetraisopropyl-4,4'-diphenol" as used in the present invention means in the 3, 3', 5, a monoester or diester formed on the 4' and/or 4' phenolic hydroxyl groups of 5'-tetraisopropyl-4,4'-diphenol.
  • Treatment and/or prevention of ischemic stroke as used herein generally refers to the treatment and/or prevention of damage caused by ischemic stroke.
  • Control as used herein means "treatment and/or prevention.”
  • the pharmaceutically acceptable salt of the present invention is a salt of 3,3',5,5'-tetraisopropyl-4,4'-diphenol and an organic acid, an inorganic acid or an alkali metal; Salts are, for example, sulfates, phosphates, hydrochlorides, hydrobromides, acetates, oxalates, citrates, succinates, gluconates, tartrates, p-toluenesulfonates, benzenesulfonic acids a salt, a methanesulfonate, a benzoate, a lactate, a maleate, a lithium salt, a sodium salt, a potassium salt or a calcium salt.
  • Salts are, for example, sulfates, phosphates, hydrochlorides, hydrobromides, acetates, oxalates, citrates, succinates, gluconates, tartrates, p-toluenesulf
  • the 3,3',5,5'-tetraisopropyl-4,4'-diphenol and pharmaceutically acceptable salts and esterified products thereof Or a solvate can be formulated into a pharmaceutical composition containing the pharmaceutical composition 3,3',5,5'-tetraisopropyl-4,4'-diphenol or a pharmaceutically acceptable salt, ester or solvate thereof, and a pharmaceutically acceptable adjuvant.
  • the solvate is formulated into a tablet, capsule, injection, emulsion, liposome, lyophilized powder or microsphere preparation containing the same; the capsule is, for example, a soft capsule.
  • the treatment and/or prevention of ischemic stroke is by improving cerebral ischemia and/or reperfusion of neurological impairment behavior; reducing cerebral ischemia and/or Or reperfusion cerebral infarction volume; reduce the consumption of SOD of endogenous oxygen free radical scavenger in brain tissue, reduce lipid peroxidation damage, reduce serum MDA content; down-regulate Fas expression in brain tissue cells; inhibit brain cell apoptosis; and / Or down-regulation of brain tissue cells IL-1 ⁇ and TNF- ⁇ expression to achieve.
  • the ischemic stroke comprises an injury caused by one or more of the following conditions: cerebral thrombosis, transient ischemic attack, basal ganglia Cavity, atherosclerotic cerebral infarction, lacunar infarction, cerebral embolism and cerebrovascular dementia.
  • the present invention provides a method of treating and/or preventing an ischemic stroke in an animal or a human, the method comprising administering to the test animal or human an effective dose of 3,3', 5, 5'-tetraisopropyl- 4,4'-diphenol and pharmaceutically acceptable salts, esters or solvates thereof, the 3,3',5,5'-tetraisopropyl-4,4'-diphenol structure I):
  • the esterified compound in the method of treating and/or preventing an ischemic stroke in an animal or human according to the present invention, is 3,3', 5, 5'-tetraisopropyl. a monoester or diester of -4,4'-diphenol; preferably, the esterified product is a monoethyl ester represented by formula (II) or a diethyl ester represented by formula (III) :
  • the pharmaceutically acceptable salt is 3, 3', 5, 5'-tetraiso a salt of propyl-4,4'-diphenol with an organic acid, an inorganic acid or an alkali metal; for example, the pharmaceutically acceptable salt is a sulfate, a phosphate, a hydrochloride, a hydrobromide, or an acetic acid.
  • Salt oxalate, citrate, succinate, gluconate, tartrate, p-toluenesulfonate, besylate, methanesulfonate, benzoate, lactate, maleate , lithium salt, sodium salt, potassium salt or calcium salt.
  • the 3,3',5,5'-tetraisopropyl-4 4'-Dihydric phenol and its pharmaceutically acceptable salts, esters or solvates are formulated into pharmaceutical compositions containing 3,3',5,5'-tetraisopropyl-4,4'- Dihydric phenol or a pharmaceutically acceptable salt, ester or solvate thereof, and a pharmaceutically acceptable adjuvant.
  • the 3,3',5,5'-tetraisopropyl-4, 4'-diphenol and pharmaceutically acceptable salts, esters or solvates thereof are formulated into tablets, capsules, injections, emulsions, liposomes, lyophilized powders or microsphere preparations containing the same; For soft capsules.
  • the treating and/or preventing ischemic stroke is by improving cerebral ischemia and / or reperfusion of neurological injury behavior; reduce cerebral ischemia and / or reperfusion cerebral infarction volume; reduce brain tissue endogenous oxygen free radical scavenger SOD consumption, reduce lipid peroxidation damage, while reducing serum MDA content; Down-regulation of Fas expression in brain tissue cells; inhibition of brain cell apoptosis; and / or down-regulation of brain tissue cells IL-1 ⁇ and TNF- ⁇ expression.
  • the ischemic stroke comprises causing one or more of the following conditions: Injury: cerebral thrombosis, transient ischemic attack, basal ganglia, atherothrombotic cerebral infarction, lacunar infarction, cerebral embolism, and cerebrovascular dementia.
  • the 3,3',5,5'-tetraisopropyl-4,4'-diphenol of the present invention is commercially available or can be produced by the method of the present invention.
  • Another object of the present invention is to provide a process for the preparation of 3,3',5,5'-tetraisopropyl-4,4'-diphenol.
  • the present invention provides a process for the preparation of the 3,3',5,5'-tetraisopropyl-4,4'-diphenol, which comprises preparing a compound represented by the formula (IV) The step of 3,3',5,5'-tetraisopropyl-4,4'-diphenol,
  • the 3,3',5,5'-tetraisopropyl-4,4'-dibiphenol is a compound of the formula (IV)
  • the compound shown is dissolved in an organic solvent, preferably the organic solvent is ethyl acetate; and then mixed with an aqueous solution containing a powdered powder; preferably, the aqueous solution containing the powder is formed of a powder of insurance powder and sodium hydroxide.
  • Aqueous solution is a compound of the formula (IV)
  • the compound of formula (IV) is The propofol is dissolved in an organic solvent, preferably, the organic solvent is ethyl acetate; and then an inorganic salt is added to obtain a reaction; preferably, the inorganic salt is silver carbonate and anhydrous magnesium sulfate.
  • the method for producing the 3,3',5,5'-tetraisopropyl-4,4'-diphenol includes the following steps:
  • the organic solvent is ethyl acetate, further preferably, the mass to volume ratio of the propofol to the acetic acid acetic acid is 1 g: 4 to 6 mL; a salt, stirred at room temperature; preferably, the inorganic salt comprises silver carbonate and anhydrous magnesium sulfate; further preferably, the molar ratio of the propofol to the silver carbonate is 1.1 to 1.4:1, the propofol The molar ratio to the anhydrous magnesium sulfate is 1.2 to 1.4:1;
  • the red solid is dissolved in ethyl acetate and mixed with the powder solution.
  • the powder solution is an aqueous solution of the powder and NaOH, optionally stirred, and then the ethyl acetate phase is separated, and the aqueous phase is extracted.
  • the mixture is extracted several times (preferably with ethyl acetate, extracted twice), dried (preferably dried over anhydrous sodium sulfate), filtered and evaporated to dryness (p.
  • a white solid 3,3',5,5'-tetraisopropyl-4,4'-diphenol preferably washed with petroleum ether
  • the method for producing the 3,3',5,5'-tetraisopropyl-4,4'-diphenol includes the following steps:
  • the red solid is dissolved in ethyl acetate and mixed with a powdered solution (preferably an aqueous solution of the powder and NaOH), optionally stirred, then the ethyl acetate phase is separated and the aqueous phase is extracted (preferably with ethyl acetate). Multiple times, preferably twice, drying (preferably anhydrous sodium sulphate), filtration, evaporation of the filtrate (preferably under reduced pressure) to dryness afforded a pale yellow solid, washed (p.
  • a powdered solution preferably an aqueous solution of the powder and NaOH
  • Another object of the present invention is to provide a process for the preparation of 3,3',5,5'-tetraisopropyl-4,4'-diphenol ester.
  • the present invention provides a process for the preparation of a monoethyl ester of the formula (II), which comprises the steps of:
  • 4'-Benzyloxy-3,3',5,5'-tetraisopropylbiphenyl-4-acetate is dissolved in an organic solvent at room temperature, preferably, the organic solvent is methanol, and then a catalyst is added.
  • the catalyst is palladium carbon, evacuated, and hydrogen is introduced; The reaction is warmed, filtered, and the filtrate is evaporated to dryness. The filtrate is evaporated to dryness to give a white solid, i.e., 4'-hydroxy-3,3',5,5'-tetraisopropylbiphenyl-4-acetate.
  • the invention also provides a preparation method of the diethyl ester represented by the formula (III), which comprises the following steps:
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the 3,3',5,5'-tetraisopropyl-4,4'-diphenol and a pharmaceutically acceptable salt, ester or solvate thereof, the pharmaceutical composition Containing at least one 3,3',5,5'-tetraisopropyl-4,4'-diphenol or a pharmaceutically acceptable salt, ester or solvate thereof.
  • composition of the present invention may also be added with one or more pharmaceutically acceptable carriers or excipients as needed.
  • the pharmaceutical composition of the present invention may have a weight percentage of 3,3',5,5'-tetraisopropyl-4,4'-diphenol or a pharmaceutically acceptable salt, ester or solvate thereof of 0.1 to 99.9. %, the remainder being a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be prepared into any pharmaceutically acceptable dosage form, including: tablets, sugar-coated tablets, film-coated tablets, enteric coated tablets, capsules, hard capsules, soft capsules, orally. Liquid, buccal, granules, granules, pills, powders, ointments, dans, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches .
  • the preparation of the present invention is preferably a tablet, a capsule, an injection, an emulsion, a liposome, a lyophilized powder or a microsphere preparation; the capsule is, for example, a soft capsule.
  • the pharmaceutical composition of the present invention may contain conventional excipients such as a binder, a filler, a diluent, a tablet, a lubricant, a disintegrant, a coloring agent, a flavoring agent, and a moisturizing agent.
  • a binder such as a polyethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, polypropylene glycol dimethacrylate, a steaglycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, glycerin, a talct, talct, talct, talct, talct, talct copolymer, graft copolymer, graft copolymer, graft copolymer, graft copolymer, graft copolymer, graft copolymer,
  • Suitable fillers include cellulose, mannitol, lactose and other similar fillers.
  • Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate.
  • Solid oral compositions can be prepared by conventional methods such as mixing, filling, tableting, and the like. Repeated mixing allows the active material to be distributed throughout those compositions that use large amounts of filler.
  • the oral liquid preparation may be in the form of, for example, an aqueous or oily suspension, solution, emulsion, syrup or elixir, or may be a dry product which may be formulated with water or other suitable carrier before use.
  • a liquid preparation may contain a conventional additive such as a suspending agent, an emulsifier, a nonaqueous carrier, a preservative, etc., and if necessary, may contain a conventional flavoring agent or coloring agent.
  • the suspending agent is, for example, sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and/or hydrogenated edible fat;
  • the emulsifier is for example an egg Phospholipids, sorbitan monooleate and/or gum arabic;
  • said non-aqueous carriers (which may include edible oils) such as almond oil, fractionated coconut An oily, oily ester of an ester such as glycerol, propylene glycol and/or ethanol; said preservative being, for example, p-hydroxybenzyl or propylparaben and/or sorbic acid.
  • the prepared liquid unit dosage form contains the active substance of the present invention (ie, 3,3',5,5'-tetraisopropyl-4,4'-diphenol of the present invention, and pharmaceutically acceptable salts and esters thereof And solvates) and sterile carriers.
  • This compound can be suspended or dissolved depending on the carrier and concentration.
  • the solution is usually prepared by dissolving the active substance in a carrier, sterilizing it by filtration before filling it into a suitable vial or ampoule, and then sealing. Excipients such as a local anesthetic, preservative and buffer may also be dissolved in such a carrier.
  • the composition can be frozen after filling the vial and the water removed under vacuum.
  • the pharmaceutical composition of the present invention may optionally be added to a suitable pharmaceutically acceptable carrier when prepared as a medicament, the pharmaceutically acceptable carrier being selected from the group consisting of: mannitol, sorbitol, sodium metabisulfite, sodium hydrogen sulfite, sulfur Sodium sulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid , phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and their derivatives , alginate, gelatin, polyvinylpyrrolidone, glycerin,
  • the pharmaceutical excipient of the present invention may comprise polyethylene glycol, phospholipid, vegetable oil, vitamin E and/or glycerin;
  • the phospholipid may be selected from one or more of soybean phospholipids, egg yolk lecithin, and hydrogenated phospholipids;
  • the vegetable oil may be selected from one or more of soybean oil, olive oil, and safflower oil.
  • the ischemic stroke injury of the present invention involves the following conditions: cerebral thrombosis, transient ischemic attack, basal ganglia, atherothrombotic cerebral infarction, lacunar infarction, cerebral embolism and brain Vascular dementia.
  • cerebral thrombosis cerebral thrombosis
  • transient ischemic attack basal ganglia
  • atherothrombotic cerebral infarction spinal ganglia
  • lacunar infarction cerebral embolism
  • cerebral embolism cerebral embolism and brain Vascular dementia.
  • the above-mentioned conditions usually cause headaches, dizziness, tinnitus, hemiplegia, difficulty swallowing, unclear speech, nausea, vomiting, coma and the like.
  • the application of the present invention is achieved by improving the behavior of neurological injury induced by ischemia and reperfusion.
  • the application of the present invention is achieved by reducing the volume of cerebral infarction following ischemia-reperfusion.
  • the application of the invention is achieved by reducing the consumption of SOD of the endogenous oxygen radical scavenger, reducing lipid peroxidation damage, and reducing serum MDA content.
  • the application of the present invention is achieved by effectively down-regulating the expression of Fas in brain tissue cells.
  • the application of the present invention is achieved by effectively inhibiting brain cell apoptosis.
  • the application of the present invention is achieved by effectively down-regulating the expression of IL-1 ⁇ and TNF- ⁇ in brain tissue cells.
  • the inventor adopts the suture method to establish a middle cerebral artery occlusion animal model (MCAO) with It has the advantages of not craniotomy, small trauma, and accurate control of ischemia and reperfusion time. It is the most classic model of focal cerebral ischemia-reperfusion. It is used in cerebral ischemia test and cerebral ischemia-reperfusion injury at home and abroad. Widely used in drug evaluation.
  • MCAO middle cerebral artery occlusion animal model
  • SOD is the main enzymatic defense system against oxygen free radicals in cells. It removes superoxide anion radicals by disproportionation; MDA acts as a metabolite of lipid peroxidation of oxygen free radicals and biofilm unsaturated fatty acids. Changes indirectly reflect the extent of oxygen free radicals and cellular damage in tissues. Therefore, the activity of SOD and the content of MDA in ischemia-reperfusion can reflect the degree of free radical-induced lipid peroxidation in the body.
  • Cerebral ischemia-reperfusion injury is mainly related to oxidative stress, inflammatory reaction, calcium overload, brain edema and apoptosis.
  • cerebral ischemia-reperfusion due to energy metabolism and various endogenous active substances, the Ca 2+ release in the reservoir is stimulated, and the intracellular Ca 2+ concentration is increased.
  • cerebral ischemia can cause excessive release of EAA from neuronal or glial cell or metabolic pools.
  • EAA can cause intracellular Ca 2+ overload, resulting in increased free radical production.
  • the increase of free radicals and EAA can induce the expression of apoptotic factors such as Fas during cerebral ischemia-reperfusion and promote apoptosis.
  • apoptosis can also reflect the degree of brain cell damage;
  • IL-1 ⁇ and TNF- ⁇ are the main pro-inflammatory factors after brain injury, and participate in the inflammatory response in the ischemic and reperfusion areas.
  • endothelial cells, neurons, astrocytes, and perivascular inflammatory cells in the injured area are activated, triggering an inflammatory response by releasing IL-1 ⁇ and TNF- ⁇ , resulting in neuronal damage.
  • the detection of IL-1 ⁇ and TNF- ⁇ as the initial factors of inflammatory response is important for measuring brain damage after ischemia-reperfusion.
  • the present invention also evaluates the middle cerebral artery occlusion animal model (MCAO) established by the suture method.
  • MCAO middle cerebral artery occlusion animal model
  • the invention proves by experiments that 3,3',5,5'-tetraisopropyl-4,4'-diphenol and pharmaceutically acceptable salts, esters or solvates thereof can effectively reduce cerebral ischemia-reperfusion injury
  • the resulting consumption of SOD activity of endogenous oxygen free radical scavengers reduces lipid peroxidation damage, reduces serum MDA content, effectively down-regulates Fas expression, reduces apoptotic cells, and reduces pro-inflammatory factors IL-1 ⁇ and TNF-
  • ⁇ which has protective effects on neurons in cerebral ischemia-reperfusion rats, and also has a good protective effect on permanent cerebral ischemic injury.
  • PEG400 aqueous solution of compound 1-3 at a concentration of 10 mg/mL the concentration of PEG400 in the PEG400 aqueous solution is 400 mg/ml; GL-22M cryogenic centrifuge (Hubei Site Xiangyi); BI2000 image analyzer (Chengdu Taimeng Co., Ltd.) ); SOD, MDA test kit (Nanjing Institute of Bioengineering); Fas, TUNEL kit (Wuhan Dr. Bioengineering Co., Ltd.); IL-1 ⁇ and TNF- ⁇ kit (Shanghai Hengyuan Biotechnology Co., Ltd.)
  • the reagents are all domestically analytically pure.
  • Rats were intraperitoneally injected with 10% chloral hydrate at 350 mg/kg. After anesthesia with Zea Longa, the neck was cut open, the right common carotid artery was isolated, and the branches of the right external carotid artery were ligated. Cut a small opening into the pre-prepared suture, through the common carotid artery and external carotid artery bifurcation into the internal carotid artery, to the front of the middle cerebral artery, the immersion depth of the tying line 18 ⁇ 19mm; properly fix the suture line, suture the wound layer by layer .
  • the standard of successful preparation of the cerebral ischemia model Horner syndrome appeared on the right side of the rat after waking and left hemiplegia.
  • the sham operation group, the ischemia-reperfusion group and the example group prepared the cerebral ischemia-reperfusion model in strict accordance with the requirements.
  • the sham operation group only entered the external carotid artery. Animals are free to eat and drink after waking up. After 2 hours, the suture was pulled out to achieve reperfusion.
  • the sham operation group, the ischemia-reperfusion group, and the example group were intravenously administered 40 mg/kg of the compound 1-3 of PEG400 solution 30 minutes before reperfusion and 12 hours after reperfusion (the 40 mg/kg means that the rats were given 40 mg per kilogram of body weight). Test compound).
  • the sham operation group and the ischemia-reperfusion group were injected with the same amount of blank PEG400 aqueous solution at the same time point.
  • Each group of rats was scored for neurological impairment after 24 hours of reperfusion. According to Longa's 5-level scale: 0: no neurological damage; grade 1: unable to extend the contralateral forepaw; grade 2: to the contralateral circle; grade 3: to the contralateral side; grade 4: no voluntary activity with consciousness Lost.
  • the perfusion needle was inserted into the ascending aorta through the apex, and the solution was quickly perfused with the physiological saline at 4 ° C until the effluent became clear, followed by perfusion with 4% polyformaldehyde phosphate buffer, and the brain was craniotomy, and 2 mm before and after the crossover.
  • the infarct size of each slice was quantified by imageJ image software, and the ratio of cerebral infarction volume to total brain volume was calculated.
  • the image is analyzed by the camera and input into the image analysis system.
  • Each slice is randomly selected from 5 non-overlapping fields. Five fields are selected for each field to measure the gray value, and the average gray value is calculated. The average gray value is calculated. It is inversely proportional to the positive rate of expression.
  • the brain tissue homogenate was centrifuged at 3000 rpm for 15 min, and the supernatant was taken and the IL-1 ⁇ and TNF- ⁇ were detected in strict accordance with the instructions of the kit.
  • Compounds 1-3 can significantly improve the neurological damage behavior of rats with ischemia-reperfusion injury; can significantly reduce the volume of cerebral infarction in rats with ischemia-reperfusion; can significantly reduce the consumption of SOD in endogenous oxygen free radical scavengers in rats, and reduce Lipid peroxidation damage, while reducing serum MDA content; effectively down-regulating Fas expression in brain tissue cells; effectively inhibiting brain cell apoptosis; effectively down-regulating the expression of IL-1 ⁇ and TNF- ⁇ in rat brain tissue cells.
  • Table 1 The results are shown in Table 1 below.
  • PEG400 aqueous solution of compound 1-3 at a concentration of 10 mg/mL the concentration of PEG400 in the PEG400 aqueous solution is 400 mg/mL; GL-22M cryogenic centrifuge (Hubei Site Xiangyi); BI2000 image analyzer (Chengdu Taimeng Co., Ltd.) ); SOD, MDA test kit (Nanjing Institute of Bioengineering); Fas, TUNEL kit (Wuhan Dr. Bioengineering Co., Ltd.); IL-1 ⁇ and TNF- ⁇ kit (Shanghai Hengyuan Biotechnology Co., Ltd.)
  • the reagents are all domestically analytically pure.
  • Rats were intraperitoneally injected with 10% chloral hydrate at 350 mg/kg. After anesthesia with Zea Longa, the neck was cut open, the right common carotid artery was isolated, and the branches of the right external carotid artery were ligated. Cut a small opening into the pre-prepared suture, through the common carotid artery and external carotid artery bifurcation into the internal carotid artery, to the front of the middle cerebral artery, the immersion depth of the tying line 18 ⁇ 19mm; properly fix the suture line, suture the wound layer by layer .
  • the sham operation group, the permanent cerebral ischemia model group, and the example group were prepared in strict accordance with the requirements.
  • the sham operation group only entered the external carotid artery. Animals are free to eat and drink after waking up.
  • the sham operation group, the permanent cerebral ischemia model group, and the experimental group were intravenously administered 40 mg/kg of the compound 1-3 of the PEG 400 solution 30 min before the insertion of the suture and 12 h after the embolization (the 40 mg/kg indicates the rat per kg body weight). 40 mg of test compound was administered).
  • the sham operation group and the permanent cerebral ischemia model group were injected with the same amount of blank PEG400 aqueous solution at the same time point.
  • Each group of rats was scored for neurological impairment after embolization for 24 h. According to Longa's 5-level scale: 0: no neurological damage; grade 1: unable to extend the contralateral forepaw; grade 2: to the contralateral circle; grade 3: to the contralateral side; grade 4: no voluntary activity with consciousness Lost.
  • the perfusion needle was inserted into the ascending aorta through the apex, and the solution was quickly perfused with the physiological saline at 4 ° C until the effluent became clear, followed by perfusion with 4% polyformaldehyde phosphate buffer, and the brain was craniotomy, and 2 mm before and after the crossover.
  • the infarct size of each slice was quantified by imageJ image software, and the ratio of cerebral infarction volume to total brain volume was calculated.
  • the image is analyzed by the camera and input into the image analysis system.
  • Each slice is randomly selected from 5 non-overlapping fields. Five fields are selected for each field to measure the gray value, and the average gray value is calculated. The average gray value is calculated. It is inversely proportional to the positive rate of expression.
  • the brain tissue homogenate was centrifuged at 3000 rpm for 15 min, and the supernatant was taken and the IL-1 ⁇ and TNF- ⁇ were detected in strict accordance with the instructions of the kit.
  • Compounds 1-3 can significantly improve neurological impairment behavior in rats with permanent cerebral ischemia; significantly reduce cerebral infarction volume in permanent ischemic rats; can significantly reduce endogenous oxygen in rats with permanent cerebral ischemia Free radical scavenger SOD consumption, reduce lipid peroxidation damage, and reduce serum MDA content; effectively down-regulate Fas expression in brain tissue of rats with permanent cerebral ischemia; effectively inhibit brain cell apoptosis; effectively down-regulate permanent brain deficiency Expression of IL-1 ⁇ and TNF- ⁇ in brain tissue of blood model rats.
  • Table 2 The results are shown in Table 2 below.
  • Table 4 show that the therapeutic effect of the diphenyl derivative on the model group is better than that of the positive control drugs propofol and edaravone, and most of the example groups have a significant pharmacological advantage over the positive drug (p ⁇ 0.05).
  • Table 4 only lists the comparison of the pharmacodynamic test data of the Example 1 group with the positive drug as a reference. At the same time, it was found that after the administration of propofol, the righting reflex of the rats disappeared and entered anesthesia, while the rats in other administration groups had no obvious behavioral changes.
  • the formulation of the oily preparation of the dibiphenyl derivative of the present invention can be as shown in Table 5:
  • the formulation of the tablet of the diphenyl derivative of the present invention can be as shown in Table 6:
  • the formulation of the capsule of the biphenyl derivative of the present invention can be as shown in Table 7:
  • the formulation of the emulsion of the dibiphenyl derivative of the present invention can be as shown in Table 8:

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Abstract

本发明公开了3，3'，5，5'-四异丙基-4，4'-二联苯酚及其药用盐、酯化物或溶剂化物在制备预防和治疗缺血性脑卒中的药物中的应用。

Description

二联苯酚在制备防治缺血性脑卒中的药物中的应用 技术领域
本发明属于医药技术领域，涉及一种药物新用途。具体涉及一种二联苯酚在制备治疗和/或预防缺血性脑卒中的药物中的新应用。
背景技术
脑卒中近年来已经成为一种严重威胁人类，特别是50岁以上中老年人健康的常见病，具有“发病率高、致残率高、死亡率高、复发率高、并发症多”即“四高一多”的特点，脑卒中的病人，因各种诱发因素引起脑内动脉狭窄，闭塞或破裂，而造成急性脑血液循环障碍，临床上表现为一过性或永久性脑功能障碍的症状和体征。
根据统计全世界每年有4000万人死于脑卒中，仅中国每年发病人数达200万。现幸存病人700万，其中450万病人不同程度丧失劳动力和生活不能自理。致残率高达75％。中国每年脑卒中患者死亡120万。已得过脑卒中的患者，还易再复发，每复发一次，加重一次。所以，更需要采取有效措施预防复发。
缺血性脑卒中大约占所有脑卒中的80％，其是指局部脑组织因血液循环障碍，缺血、缺氧而发生的软化坏死。主要是由于供应脑部血液的动脉出现粥样硬化和血栓形成，使管腔狭窄甚至闭塞，导致局灶性急性脑供血不足而发病；也有因异常物体(固体、液体、气体)沿血液循环进入脑动脉或供应脑血液循环的颈部动脉，造成血流阻断或血流量骤减而产生相应支配区域脑组织软化坏死。
缺血性脑损伤主要来自两个方面：(1)缺血后产能不足，依赖ATP的酶活性及生理活动受抑制，氯离子、钠离子和水分内流使细胞水肿，突触间隙兴奋性氨基酸(主要为谷氨酸)聚集，过度激动谷氨酸受体，通过NMDA等受体介导引起钙离子内流增加，钾离子外流致细胞去极化，也使电压敏感钙通道开放，细胞内钙超载，过度激活磷脂酶、一氧化氮合酶(NOS)在内的多种酶，产生一系列活性代谢产物和自由基，造成细胞损伤；(2)脑卒中患者在经过治疗后缺血组织获得血液灌注或自发性再灌注，虽然重新获得了养分，但同时不可避免地造成了脑缺血再灌注损伤，即脑缺血一定时间恢复血液供应后，其功能不但未能恢复，却出现了更加严重的脑机能障碍的现象。
缺血性脑损伤涉及非常复杂的病理生理过程，其中各个环节、各种因素间的相互作用尚未完全阐明。但目前已有以下的机制被认为在其中起到关键作用：
(1)兴奋性氨基酸毒性与缺血性脑损伤
大量研究显示缺血期间升高的兴奋性氨基酸(excitatoryaminoacid,EAA)的兴奋毒性在缺血性神经细胞损伤中起重要作用，兴奋性氨基酸主要是指谷氨酸(glutamate,Glu)和天冬氨酸(asparate,Asp)。突触后神经元过度兴奋EAA可活化胞内信号转导通路，使一些受体在正常生理刺激下引起的第二信使效应得到放大，触发缺血后致炎基因表达。Glu与Asp等兴奋性氨基酸在缺血性神经细胞损伤中起关键作用，缺血时间愈长，脑间质Glu与Asp的峰值浓度愈高，神经病理学和神经学损伤愈严重；这与EAA毒性作用为浓度依赖性呈一致性。兴奋性氨基酸对神经细胞的毒性作用是多方面的：过量的EAA激活其受体，引起兴奋性神经元持续去极化，造成细胞内Ca2+超载，结果引起细胞坏死；引发自由基(如一氧化氮)生成增多，通过自由基产生细胞毒性作用；参与脑内多种代谢过程，使三羧酸循环受阻，ATP生成减少，加重EAA对细胞的毒性作用。
(2)自由基及脂质过氧化与缺血性脑损伤
缺血性脑损伤是多因素参与的复杂的病理生理过程。一般认为与氧自由基引起的组织脂质过氧化和细胞内钙超负荷导致的不可逆损伤有关。其有害作用可概括为：作用于多价不饱和脂肪酸，发生脂质过氧化；诱导DNA、RNA、多糖和氨基酸等大分子物质交联，交联后的大分子则失去原来的活性或功能降低；促使多糖分子聚合和降解，自由基可广泛攻击富含不饱和脂肪酸的神经膜与血管，引发脂质过氧化瀑布效应，产生蛋白质变性、多核苷酸链断裂、碱基重新修饰，造成细胞结构的完整性破坏、膜的通透性、离子转运、膜屏障功能均受到严重影响，从而导致细胞死亡。自由基还能导致EAA释放增加，促使脑缺血后再灌注损伤发生。
(3)Ca2+超载与脑缺血性脑损伤
缺血性脑损伤中Ca2+超载是各种因素综合作用的结果，也是造成脑缺血损伤过程中各种因素作用的共同通路。Ca2+在缺血性脑损伤中的作用主要有：
①线粒体功能障碍细胞内外钙平衡紊乱，细胞外Ca2+内流入细胞，主要聚集在线粒体内，Ca2+可抑制ATP合成，使能量生成障碍。Ca2+活化线粒体上的磷脂酶，引起线粒体膜损伤。除ATP合成外，线粒体对细胞氧化还原反应、渗透压、pH值、胞质内信号的维持都有重要作用，线粒体是细胞受损的重要靶目标。
②酶的活化Ca2+活化Ca2+依赖性磷脂酶(主要是磷脂酶C和磷脂酶A2)，促进膜磷脂分解；在膜磷脂分解过程中产生的游离脂肪酸、前列腺素、白三烯、溶血磷脂等均对细胞产生毒害；Ca2+还活化钙依赖蛋白酶，使胞内无害的黄嘌呤脱氢酶转变为黄嘌呤氧化酶，生成大量氧自由基；Ca2+可活化NOS。
实验证实，上述病理生理变化存在着一定的药物可干预性。服用可靠药物长期防治缺 血性脑卒中与停药患者相比，复发率要降低80％以上，死亡率降低90％以上，长期用药超过三年以上患者80％以上无复发危险，极少数轻复发。这为药物性对抗缺血性脑损伤奠定了理论基础。目前用于脑血管病的常见药物主要包括以下几类：
GABA受体激动剂：GABA能够对抗兴奋性氨基酸的兴奋毒性，发挥抑制保护的作用，代表药物氯美噻嗪；
NMDA受体拮抗剂：拮抗NMDA受体，从而抑制其介导的钙离子内流，代表药物MK801；
钙离子拮抗剂：阻止细胞内钙超载、防止血管痉挛、增加血流量，代表药物尼莫地平；
抗自由基药物：清除自由基，抑制脂质过氧化，从而抑制脑细胞、血管内皮细胞和神经细胞的氧化损伤。代表药物依达拉奉；
但目前缺血性脑卒中的具体损伤机制尚未研究清楚，被认为是多种因素相互作用下的非常复杂的病理生理过程，而以上药物的作用机制单一，临床治疗效果仍不确切或有较严重的副作用，从而限制了其在缺血性脑卒中治疗中的应用。
近年来国内外多例研究发现，麻醉药丙泊酚可能对缺血性脑卒中起到非常积极的作用。在动物和离体实验中，甚至部分临床研究中，丙泊酚被证明对神经功能损伤有明显的保护和治疗作用。实验证实，丙泊酚不仅能阻断钠离子流或通过激活GABA受体减少钾离子激活的Glu释放，而且还可阻断氧化物处理后胶质细胞对Glu转运的抑制，两者最终都能降低细胞外Glu浓度，推迟或防止兴奋性神经元死亡；丙泊酚可抑制细胞外经电压依赖性钙通道流入的钙，其可在一定程度上增加L-型电压依赖性钙通道的电流失活率，从而减少钙内流；丙泊酚能与GABAa受体特定位点结合，不仅增加GABA开放氯离子通道的频率，而且通过正性变构调节作用增强低亲和力GABA结合位点与GABA的结合；丙泊酚能抑制败血症患者血中的致炎性细胞因子如TNF、IL-1、IL-6等的产生，且在低浓度时便有较强的抑制作用；丙泊酚可以抑制脑组织的促凋亡基因caspase-3mRNA的表达并提高抗凋亡基因Bcl-2mRNA的表达；丙泊酚能竞争性结合膜磷脂，还能与过氧化物形成稳定的苯氧基，实际上是生成了低活性的自由基取代高活性的自由基，从而减轻后者引发的脂质过氧化级联反应。以上结果提示发明人丙泊酚对抗缺血性脑卒中损伤的机制可能包括抗自由基，抑制脂质过氧化；抑制细胞内钙超载；抑制细胞凋亡等多种。但丙泊酚自身的全身麻醉作用限制了其在缺血性脑卒中治疗中的临床应用。
发明内容
本发明的目的之一在于提供3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶 剂化物的新用途。
为此，本发明提供3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物在制备治疗和/或预防缺血性脑卒中的药物中的应用；所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚的结构如式(I)所示：
Figure PCTCN2016076112-appb-000001
本发明所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚还包括该化合物的药用盐、酯化物或溶剂化物。
发明人经过研究发现：二联苯酚对GABA受体有着非常强的亲和力，并具有GABA激动作用，还能拮抗NMDA受体，调节钙离子通道，限制细胞的钙离子内流，具有较丙泊酚更强的抗氧化性和自由基清除作用。二联苯酚可能会在多种机制下对抗缺血性脑卒中损伤。最为重要的是二联苯酚并不会导致意识丧失，因此其在多种缺血性脑卒中症状治疗中具有重要的临床应用价值。
本发明所述的酯化物为3,3’,5,5’-四异丙基-4,4’-二联苯酚的单酯化物或二酯化物；优选地，所述的酯化物为式(II)所示的单乙酯化物或式(III)所示的二乙酯化物：
Figure PCTCN2016076112-appb-000002
本发明所述的“3,3’,5,5’-四异丙基-4,4’-二联苯酚的单酯化物或二酯化物”是指在所述3,3’,5,5’-四异丙基-4,4’-二联苯酚的4位和/或4’位酚羟基上形成的单酯化物或二酯化物。
本发明所述“治疗和/或预防缺血性脑卒中”通常是指治疗和/或预防由缺血性脑卒中所带来的损伤。本发明所述“防治”是指“治疗和/或预防”。
本发明所述的药用盐为3,3’,5,5’-四异丙基-4,4’-二联苯酚与有机酸、无机酸或碱金属所成的盐；所述药用盐例如为硫酸盐、磷酸盐、盐酸盐、氢溴酸盐、醋酸盐、草酸盐、柠檬酸盐、琥珀酸盐、葡萄糖酸盐、酒石酸盐、对甲苯磺酸盐、苯磺酸盐、甲磺酸盐、苯甲酸盐、乳酸盐、马来酸盐、锂盐、钠盐、钾盐或钙盐。
根据本发明的具体实施方案，在本发明所述应用中，所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物可被配制成药物组合物，该药物组合物含有 3,3’,5,5’-四异丙基-4,4’-二联苯酚或其药用盐、酯化物或溶剂化物，和药用辅料。
根据本发明的具体实施方案，在本发明所述应用中，所述3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物被配制成含其的片剂、胶囊剂、注射剂、乳剂、脂质体、冻干粉或微球制剂；所述胶囊剂例如为软胶囊。
根据本发明的具体实施方案，在本发明所述应用中，所述治疗和/或预防缺血性脑卒中是通过改善脑缺血和/或再灌注神经功能损伤行为；减少脑缺血和/或再灌注脑梗死体积；降低脑组织内源性氧自由基清除剂SOD的消耗，减轻脂质过氧化损伤，同时降低血清MDA含量；下调脑组织细胞Fas表达；抑制脑细胞凋亡；和/或下调脑组织细胞IL-1β和TNF-α表达来实现的。
根据本发明的具体实施方案，在本发明所述应用中，所述缺血性脑卒中包括由以下病症中的一种或几种引起的损伤：脑血栓、短暂性脑缺血发作、基底节腔梗、动脉粥样硬化性血栓性脑梗塞、腔隙性脑梗塞、脑栓塞和脑血管性痴呆。
本发明提供一种治疗和/或预防动物或人类缺血性脑卒中的方法，所述方法包括对受试动物或者人类给予有效剂量的3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物，所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚结构如式(I)所示：
Figure PCTCN2016076112-appb-000003
根据本发明的具体实施方式，在本发明所述的治疗和/或预防动物或人类缺血性脑卒中的方法中，所述酯化物为3,3’,5,5’-四异丙基-4,4’-二联苯酚的单酯化物或二酯化物；优选地，所述的酯化物为式(II)所示的单乙酯化物或式(III)所示的二乙酯化物：
Figure PCTCN2016076112-appb-000004
根据本发明的具体实施方式，在本发明所述的治疗和/或预防动物或人类缺血性脑卒中的方法中，所述的药用盐为3,3’,5,5’-四异丙基-4,4’-二联苯酚与有机酸、无机酸或碱金属所成的盐；例如，所述药用盐为硫酸盐、磷酸盐、盐酸盐、氢溴酸盐、醋酸盐、草酸盐、柠檬酸盐、琥珀酸盐、葡萄糖酸盐、酒石酸盐、对甲苯磺酸盐、苯磺酸盐、甲磺酸盐、苯甲酸盐、乳酸盐、马来酸盐、锂盐、钠盐、钾盐或钙盐。
根据本发明的具体实施方式，在本发明所述的治疗和/或预防动物或人类缺血性脑卒中的方法中，所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物被配制成药物组合物，该药物组合物含有3,3’,5,5’-四异丙基-4,4’-二联苯酚或其药用盐、酯化物或溶剂化物，和药用辅料。
根据本发明的具体实施方式，在本发明所述的治疗和/或预防动物或人类缺血性脑卒中的方法中，所述3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物被配制成含其的片剂、胶囊剂、注射剂、乳剂、脂质体、冻干粉或微球制剂；所述胶囊剂例如为软胶囊。
根据本发明的具体实施方式，在本发明所述的治疗和/或预防动物或人类缺血性脑卒中的方法中，所述治疗和/或预防缺血性脑卒中是通过改善脑缺血和/或再灌注神经功能损伤行为；减少脑缺血和/或再灌注脑梗死体积；降低脑组织内源性氧自由基清除剂SOD的消耗，减轻脂质过氧化损伤，同时降低血清MDA含量；下调脑组织细胞Fas表达；抑制脑细胞凋亡；和/或下调脑组织细胞IL-1β和TNF-α表达来实现的。
根据本发明的具体实施方式，在本发明所述的治疗和/或预防动物或人类缺血性脑卒中的方法中，所述缺血性脑卒中包括由以下病症中的一种或几种引起的损伤：脑血栓、短暂性脑缺血发作、基底节腔梗、动脉粥样硬化性血栓性脑梗塞、腔隙性脑梗塞、脑栓塞和脑血管性痴呆。
本发明所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚，可以在市场上购买到，也可以通过本发明提供的方法制备得到。
本发明的另一个目的在于提供3,3’,5,5’-四异丙基-4,4’-二联苯酚的制备方法。
为此，本发明提供所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚的制备方法，所述方法包括由式(IV)所示的化合物制备所述3,3’,5,5’-四异丙基-4,4’-二联苯酚的步骤，
Figure PCTCN2016076112-appb-000005
根据本发明的具体实施方案，在本发明所述的制备方法中，所述3,3’,5,5’-四异丙基-4,4’-二联苯酚是将式(IV)所示的化合物溶解在有机溶剂中，优选该有机溶剂为乙酸乙酯；然后与含保险粉的水溶液混合反应得到的；优选地，所述含保险粉的水溶液是由保险粉与氢氧化钠形成的水溶液。
根据本发明的具体实施方案，在本发明所述的制备方法中，式(IV)所示的化合物是 将丙泊酚溶于有机溶剂，优选地，该有机溶剂为乙酸乙酯；然后加入无机盐反应得到的；优选地，所述无机盐为碳酸银和无水硫酸镁。
根据本发明的具体实施方案，在本发明所述的制备方法中，所述3,3’,5,5’-四异丙基-4,4’-二联苯酚的制备方法包括如下步骤：
将丙泊酚溶于有机溶剂中，优选地，该有机溶剂为乙酸乙酯中，进一步优选地，所述丙泊酚与所述乙酸乙酸的质量体积比为1g:4～6mL；再加入无机盐，室温搅拌；优选地，所述无机盐包括碳酸银和无水硫酸镁；进一步优选地，所述丙泊酚与所述碳酸银的摩尔比为1.1～1.4:1，所述丙泊酚与所述无水硫酸镁的摩尔比为1.2～1.4:1；
反应完毕后，向反应液内加水，将固体过滤，洗涤(优选采用乙酸乙酯洗涤)，水相去除，干燥乙酸乙酯相(优选采用无水硫酸钠干燥)，过滤，滤液蒸发至干(优选减压蒸发至干)，任选洗涤(优选采用无水甲醇洗涤)，得到枚红色结晶；
将上述枚红色固体溶于乙酸乙酯，并与保险粉溶液混合，优选地，该保险粉溶液为保险粉与NaOH形成的水溶液，任选搅拌，然后将乙酸乙酯相分离出，水相萃取多次(优选采用乙酸乙酯萃取该水相，萃取两次)，干燥(优选采用无水硫酸钠干燥)，过滤，滤液蒸发至干(优选减压蒸发至干)，得到淡黄色固体，洗涤得到白色固体3,3’,5,5’-四异丙基-4,4’-二联苯酚(优选采用石油醚洗涤)。
根据本发明的具体实施方案，在本发明所述的制备方法中，所述3,3’,5,5’-四异丙基-4,4’-二联苯酚的制备方法包括以下步骤：
将丙泊酚溶于有机溶剂(优选乙酸乙酯)中，再加入无机盐(优选碳酸银和无水硫酸镁)，室温搅拌；
反应完毕后，向反应液内加水，将固体过滤，洗涤(优选用乙酸乙酯)，水相去除，干燥乙酯相(优选用无水硫酸钠)，过滤，滤液蒸发(优选减压)至干，任选洗涤(优选加无水甲醇)，得到枚红色结晶；
将上述枚红色固体溶于乙酸乙酯，并与保险粉溶液(优选保险粉与NaOH形成的水溶液)混合，任选搅拌，然后将乙酸乙酯相分离出，水相萃取(优选用乙酸乙酯)多次，优选两次，干燥(优选无水硫酸钠)，过滤，滤液蒸发(优选减压)至干，得到淡黄色固体，洗涤(优选用石油醚)得到白色固体。
本发明另一个目的在于提供3,3’,5,5’-四异丙基-4,4’-二联苯酚酯化物的制备方法。
为此，本发明提供式(II)所示的单乙酯的制备方法，该方法包括以下步骤：
室温下将4’-苄氧基-3,3’,5,5’-四异丙基联苯-4-乙酸酯溶于有机溶剂中，优选地，该有机溶剂为甲醇，再加入催化剂，优选地，该催化剂为钯碳，抽真空，通入氢气；密封后室 温反应，过滤，滤液蒸发除去，优选滤液减压蒸发除去，得到白色固体，即4’-羟基-3,3’,5,5’-四异丙基联苯-4-乙酸酯。
本发明亦提供式(III)所示的二乙酯的制备方法，该方法包括以下步骤：
将4,4’-二羟基-3,3’,5,5’-四异丙基联苯加入到乙酸酐中，氮气保护下，回流反应，将反应液冷却至室温，除去乙酸酐，优选减压除去乙酸酐，向残余物中加水，出现白色固体，洗涤(优选采用乙醇与水洗涤)，干燥，得到3,3’,5,5’-四异丙基联苯-4’4-二乙酸酯。
本发明还提供含有所述3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物的药物组合物，该药物组合物含有至少一种3,3’,5,5’-四异丙基-4,4’-二联苯酚或其药用盐、酯化物或溶剂化物。
根据需要，本发明的药物组合物还可以加入一种或多种药学上可接受的载体或赋形剂。
本发明的药物组合物，3,3’,5,5’-四异丙基-4,4’-二联苯酚或其药用盐、酯化物或溶剂化物所占重量百分比可以是0.1～99.9％，其余为药物可接受的载体。
本发明的药物组合物可以制备成任何可药用的剂型，这些剂型包括：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、滴剂、贴剂。本发明的制剂，优选的是片剂、胶囊剂、注射剂、乳剂、脂质体、冻干粉或微球制剂；所述胶囊剂例如为软胶囊。
本发明的药物组合物，其口服给药的制剂可含有常用的赋形剂，诸如粘合剂、填充剂、稀释剂、压片剂、润滑剂、崩解剂、着色剂、调味剂和湿润剂，必要时可对片剂进行包衣。
适用的填充剂包括纤维素、甘露糖醇、乳糖和其它类似的填充剂。适宜的崩解剂包括淀粉、聚乙烯吡咯烷酮和淀粉衍生物，例如羟基乙酸淀粉钠。适宜的润滑剂包括，例如硬脂酸镁。适宜的药物可接受的湿润剂包括十二烷基硫酸钠。
可通过混合，填充，压片等常用的方法制备固体口服组合物。进行反复混合可使活性物质分布在整个使用大量填充剂的那些组合物中。
口服液体制剂的形式例如可以是水性或油性悬浮液、溶液、乳剂、糖浆剂或酏剂，或者可以是一种在使用前可用水或其它适宜的载体复配的干燥产品。这种液体制剂可含有常规的添加剂，诸如悬浮剂、乳化剂、非水性载体、防腐剂等，如果需要，可含有常规的香味剂或着色剂。所述悬浮剂例如为山梨醇、糖浆、甲基纤维素、明胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶和/或氢化食用脂肪；所述乳化剂例如为卵磷脂、脱水山梨醇一油酸酯和/或阿拉伯胶；所述非水性载体(它们可以包括食用油)例如为杏仁油、分馏椰子 油、诸如甘油的酯的油性酯、丙二醇和/或乙醇；所述防腐剂例如谓对羟基苯甲酯或对羟基苯甲酸丙酯和/或山梨酸。
对于注射剂，制备的液体单位剂型含有本发明的活性物质(即本发明所述3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物)和无菌载体。根据载体和浓度，可以将此化合物悬浮或者溶解。溶液的制备通常是通过将活性物质溶解在一种载体中，在将其装入一种适宜的小瓶或安瓿前过滤消毒，然后密封。辅料例如一种局部麻醉剂、防腐剂和缓冲剂也可以溶解在这种载体中。为了提高其稳定性，可在装入小瓶以后将这种组合物冰冻，并在真空下将水除去。
本发明的药物组合物，在制备成药剂时可选择性的加入适合的药物可接受的载体，所述药物可接受的载体选自：甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素C、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、土温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β－环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙和/或硬脂酸镁等。
优选的，本发明所述药用辅料可以包括聚乙二醇、磷脂、植物油、维生素E和/或甘油；
所述磷脂可以选自大豆磷脂、蛋黄卵磷脂和氢化磷脂中的一种或几种；
所述植物油可以选自大豆油、橄榄油和红花油中的一种或几种。
本发明所述的缺血性脑卒中损伤涉及以下病症：脑血栓、短暂性脑缺血发作、基底节腔梗、动脉粥样硬化性血栓性脑梗塞、腔隙性脑梗塞、脑栓塞和脑血管性痴呆。上述病症通常引起的头痛、眩晕、耳鸣、半身不遂，吞咽困难，说话不清，恶心、呕吐、昏迷等多种情况。
本发明所述的应用，是通过改善缺血再灌注神经功能损伤行为实现的。
本发明所述的应用，是通过减少缺血再灌注脑梗死体积实现的。
本发明所述的应用，是通过降低内源性氧自由基清除剂SOD的消耗，减轻脂质过氧化损伤，同时降低血清MDA含量实现的。
本发明所述的应用，是通过有效下调脑组织细胞Fas表达实现的。
本发明所述的应用，是通过有效抑制脑细胞凋亡实现的。
本发明所述的应用，是通过有效下调脑组织细胞IL-1β和TNF-α表达实现的。
本发明的实施例中，发明人采用线栓法建立的大脑中动脉阻塞动物模型(MCAO)具 有不开颅，创伤小，能准确控制缺血和再灌注时间的优点，是目前最经典的局灶性脑缺血再灌注模型，在国内外脑缺血实验和脑缺血再灌注损伤治疗药物评价中广泛应用。
脑缺血再灌注时，细胞内氧自由基大量增加，极易攻击含不饱和双键的生物膜结构，发生脂质过氧化反应，使膜结构遭到破坏，影响膜的通透性，使离子转运、生物能的产生和细胞器的功能发生一系列病理生理改变，导致神经细胞、胶质细胞和血管内皮细胞损伤。SOD是细胞内主要的对抗氧自由基的酶促防御系统，它通过歧化方式清除超氧阴离子自由基；MDA作为氧自由基与生物膜不饱和脂肪酸发生脂质过氧化反应的代谢产物，其含量变化间接反映组织中氧自由基含量及细胞损伤的程度。因此在缺血再灌注中测定SOD的活性及MDA的含量可反映机体内自由基引发的脂质过氧化反应的程度。
脑缺血再灌注损伤主要与氧化应激反应、炎性反应、钙超载、脑水肿和细胞凋亡等有关。脑缺血再灌注时，由于能量代谢及各种内源活性物质的作用，刺激贮存池的Ca2+释放，细胞内Ca2+浓度升高。同时，脑缺血可引起EAA从神经元或胶质细胞递质池或代谢池中过度释放。EAA可致细胞内Ca2+超载，造成自由基生成增多。而自由基、EAA的增多均可诱导脑缺血再灌注时凋亡因子例如Fas的表达，促进细胞凋亡发生。因此细胞凋亡情况也可反映脑细胞的损伤程度；IL-1β和TNF-α为脑损伤后的主要促炎性因子，参与缺血区和再灌注区的炎性反应。脑缺血-再灌注后，损伤区内皮细胞、神经元、星形细胞和血管周围的炎性细胞被激活，通过释放IL-1β和TNF-α而触发炎症反应，导致神经元损伤。IL-1β、TNF-α作为炎症反应的起始因子检测其含量对衡量缺血-再灌注后的脑损伤有重要意义。
同时，由于脑卒中患者再灌注的发生往往较为滞后，脑组织缺血性“饥饿”伤也不能被忽视，因此本发明同样也使用线栓法建立的大脑中动脉阻塞动物模型(MCAO)评价了二联苯酚在永久性缺血损伤中的保护作用。
本发明通过实验证明：3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物能有效地减少脑缺血再灌注损伤导致的内源性氧自由基清除剂SOD活力的消耗，减轻脂质过氧化损伤，降低血清MDA含量，有效地下调Fas表达，减少凋亡细胞，同时降低了促炎症因子IL-1β和TNF-α的表达，从而对脑缺血再灌注大鼠神经元产生保护作用；同时对永久性脑缺血损伤也有较好的保护作用。
具体实施方式
下面结合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。
实施例1化合物1：3,3’,5,5’-四异丙基-4,4’-联苯二酚
Figure PCTCN2016076112-appb-000006
称取20g丙泊酚用100mL乙酸乙酯溶解，再加入24.75g碳酸银和10g无水硫酸镁，室温搅拌2h，检测反应完毕。向反应液内加水，直至无气泡产生即可，将固体过滤，用乙酸乙酯洗涤，水相去除，乙酸乙酯相用无水硫酸钠干燥1h，过滤，滤液减压蒸发至干，加适量无水甲醇洗涤，得到12.30g枚红色结晶。称取上述枚红色固体7g用100mL乙酸乙酯溶解，此外，27.66g保险粉用1mol/L NaOH溶解后，加入上述所得枚红色固体的乙酸乙酯溶液中，室温搅拌1.5h，检测反应完毕。将乙酸乙酯相分离出，水相用乙酸乙酯萃取两次，无水硫酸钠干燥，过滤，滤液减压蒸发至干，得到5g淡黄色固体，用石油醚洗涤得到4.5g白色固体。1H NMR(300MHz,CDCl3):δ7.22(s,4H),4.81(s,2H),3.27-3.20(m,4H),1.37-1.35(d,24H)。
实施例2化合物2：4’-羟基-3,3’,5,5’-四异丙基联苯-4-乙酸酯
Figure PCTCN2016076112-appb-000007
室温下将4’-苄氧基-3,3’,5,5’-四异丙基联苯-4-乙酸酯(5g，10.27mmol)用200mL甲醇溶解，再加入10％钯碳(570mg)，抽真空，通氢气，反复三次，密封后室温反应10h，将反应液中钯碳过滤，滤液减压蒸发除去，得到4’-羟基-3,3’,5,5’-四异丙基联苯-4-乙酸酯(3.9g，95.73％)，白色固体。1H NMR(300MHz,CDCl3)δ7.19(s,4H),4.86(s,1H),3.37-3.32(m,4H),3.16(s,3H),1.20(d,24H)。
实施例3化合物3：3,3’,5,5’-四异丙基联苯-4’4-二乙酸酯
Figure PCTCN2016076112-appb-000008
将4,4’-二羟基-3,3’,5,5’-四异丙基联苯(5g，14.10mmol)加入到30mL的乙酸酐中，氮气保护下，回流3h后，将反应液冷却至室温，减压除去乙酸酐，向残余物中加水(200mL)，出现白色固体，用10％冷乙醇(100mL)与水(200mL)洗涤，干燥，得到3,3’,5,5’-四异丙基联苯-4’4-二乙酸酯(6g，95.06％)。白色固体。
白色固体，1H NMR(300MHz,CDCl3)δ7.19(s,4H),2.91-2.89(m,4H),2.32(s,6H), 1.19(d,24H)。
实施例4化合物1～3对脑缺血再灌注损伤大鼠神经功能损伤评分、脑梗死体积、脑组织Fas、IL-1β、TNF-α和细胞凋亡的影响
(1)材料：
浓度为10mg/mL的化合物1～3的PEG400水溶液，该PEG400水溶液中PEG400的浓度为400mg/ml；GL-22M低温离心机(湖北赛特湘仪)；BI2000图象分析仪(成都泰盟公司)；SOD、MDA测试盒(南京建成生物工程研究所)；Fas、TUNEL试剂盒(武汉博士德生物工程有限公司)；IL-1β和TNF-α试剂盒(上海恒远生物科技有限公司)其余试剂均为国产分析纯。
(2)方法：
1.实验动物及分组
健康雄性SD大鼠120只，由第四军医大学实验动物中心提供，体重250～300g，随机平均分为5组：假手术组、缺血再灌注组、实施例1组(采用化合物1为供试样品)、实施例2组(采用化合物2为供试样品)和实施例3组(采用化合物3为供试样品)。
2.动物模型制备及处理
大鼠腹腔注射10％水合氯醛350mg/kg麻醉后，参照Zea Longa改良法，取颈部正中切开，分离右侧颈总动脉、结扎右侧颈外动脉各分支，由颈外动脉远端剪一小口置入预先备好的栓线，经颈总动脉与颈外动脉分叉浸入颈内动脉，至大脑中动脉前端，栓线浸入深度18～19mm；妥当固定栓线，逐层缝合伤口。术毕放置于情节保温箱苏醒，脑缺血模型制备成功的标准：大鼠苏醒后右侧出现Horner综合症并左侧偏瘫。假手术组、缺血再灌注组、实施例组严格按照要求制备脑缺血再灌注模型，假手术组栓线只进入颈外动脉。动物苏醒后自由进食进饮。2h后拔出栓线实现再灌注。假手术组、缺血再灌注组、实施例组于再灌注前30min和再灌注12h后分别静脉给药化合物1～3的PEG400水溶液40mg/kg(该40mg/kg表示大鼠每公斤体重给予40mg的测试化合物)。假手术组、缺血再灌注组则于相同时间点注射等量空白PEG400水溶液。
3.大鼠神经功能损伤程度评分
再灌注24h后对每组大鼠进行神经功能损伤程度评分。依据Longa的5级评分法：0级：无神经损伤症状；1级：不能伸展对侧前爪；2级：向对侧转圈；3级：向对侧倾倒；4级：无自主活动伴意识丧失。
4.标本采集及制备
再灌注24h后，每组取8只大鼠处死断头取脑，PBS(pH7.4)涮洗后-20℃放置20min， 均匀切片(厚度2mm)，2％TTC溶液37℃避光染色30min，10％福尔马林固定24h，拍摄照片后分析梗死体积；每组另取8只大鼠麻醉状态下取股动静脉混合血，置于4℃低温离心机，3500r/min离心20min，取上清液于-20℃冰箱保存备检测SOD、MDA。取血后经心尖插入灌注针至升主动脉，以4℃生理盐水快速灌注至流出液变澄亮，继以4％多聚甲醛磷酸缓冲液灌注固定，开颅取脑，取视交叉前后2mm脑组织固定；脱水，透明，浸蜡，包埋；连续切取脑部连续冠状组织病理切片待用；每组余下8只大鼠处死取脑，于冰盘上迅速取缺血侧大脑半球，制备10％脑组织匀浆备用检测IL-1β和TNF-α。
5.相对梗死体积检测
用imageJ图像软件分析量化每片脑片的梗死面积，最后计算求得脑梗死体积占脑总体积的比例。
6.SOD、MDA检测
严格按照试剂盒说明书要求检测SOD和MDA。
7.Fas检测
采用免疫组化法检测。脑组织石蜡切片脱蜡至水，3％H2O2消除内源性过氧化物酶的活性，蒸馏水冲洗3次。枸橼酸钠缓冲液热修复抗原，小牛血清室温下密闭15min；滴加兔抗鼠Fas抗体，4℃过夜，滴加生物素化山羊抗兔IgG，37℃水浴20min，PBS冲洗5min连续4次，DAB显色，充分冲洗，不复染，梯度酒精脱水，二甲苯透明，封片固定。高倍光学显微镜下通过摄像头采集并输入图像分析系统进行图像分析，每张切片随机选取5个不重叠视野，每个视野选取5个区域测量灰度值，计算平均灰度值，其平均灰度值与表达的阳性率成反比。
8.细胞凋亡检测
采用TUNEL法检测。脑组织石蜡切片脱蜡至水，3％H2O2消除内源性过氧化物酶的活性，蒸馏水洗涤2min，连续冲洗3次，加标记液37℃标记2h，加封闭液室温封闭30min，加生物化抗地高辛抗体，37℃反应30min，加SABC37℃反应30min，TBS冲洗5min，连续4次，DAB显色；充分冲洗，苏木素轻度复染，梯度酒精脱水，二甲醛透明，封皮固定。每张切片随机选取半暗带5个不重叠视野，输入图象分析系统，分别计数凋亡细胞个数，取平均值记为凋亡细胞数。
9.IL-1β和TNF-α检测
将脑组织匀浆液以3000rpm低温离心15min后取上清严格按照试剂盒说明书要求检测IL-1β和TNF-α。
(3)结果：
化合物1～3能明显改善缺血再灌注大鼠神经功能损伤行为学；能明显减少缺血再灌注大鼠脑梗死体积；能显著降低大鼠内源性氧自由基清除剂SOD的消耗，减轻脂质过氧化损伤，同时降低血清MDA含量；有效下调脑组织细胞Fas表达；有效抑制脑细胞凋亡；有效下调大鼠脑组织细胞IL-1β和TNF-α表达。结果见下表1所示。
表1
Figure PCTCN2016076112-appb-000009
与缺血再灌注组比较*：p<0.05
实施例5化合物1～3对永久性脑缺血大鼠神经功能损伤评分、脑梗死体积、脑组织Fas、IL-1β、TNF-α和细胞凋亡的影响
(1)材料：
浓度为10mg/mL的化合物1～3的PEG400水溶液，该PEG400水溶液中PEG400的浓度为400mg/mL；GL-22M低温离心机(湖北赛特湘仪)；BI2000图象分析仪(成都泰盟公司)；SOD、MDA测试盒(南京建成生物工程研究所)；Fas、TUNEL试剂盒(武汉博士德生物工程有限公司)；IL-1β和TNF-α试剂盒(上海恒远生物科技有限公司)其余试剂均为国产分析纯。
(2)方法：
1.实验动物及分组
健康雄性SD大鼠120只，由第四军医大学实验动物中心提供，体重250～300g，随机平均分为5组：假手术组、永久性脑缺血模型组、实施例1组(采用化合物1为供试样品)、实施例2组(采用化合物2为供试样品)和实施例3组(采用化合物3为供试样品)。
2.动物模型制备及处理
大鼠腹腔注射10％水合氯醛350mg/kg麻醉后，参照Zea Longa改良法，取颈部正中切开，分离右侧颈总动脉、结扎右侧颈外动脉各分支，由颈外动脉远端剪一小口置入预先备好的栓线，经颈总动脉与颈外动脉分叉浸入颈内动脉，至大脑中动脉前端，栓线浸入深度18～19mm；妥当固定栓线，逐层缝合伤口。术毕放置于情节保温箱苏醒，脑缺血模型制备成功的标准：大鼠苏醒后右侧出现Horner综合症并左侧偏瘫。假手术组、永久性脑缺血模型组、实施例组严格按照要求制备，假手术组栓线只进入颈外动脉。动物苏醒后自由进食进饮。假手术组、永久性脑缺血模型组、实施例组于插入栓线前30min和栓塞后12h分别静脉给药化合物1～3的PEG400水溶液40mg/kg(该40mg/kg表示大鼠每公斤体重给予40mg的测试化合物)。假手术组、永久性脑缺血模型组则于相同时间点注射等量空白PEG400水溶液。
3.大鼠神经功能损伤程度评分
栓塞24h后对每组大鼠进行神经功能损伤程度评分。依据Longa的5级评分法：0级：无神经损伤症状；1级：不能伸展对侧前爪；2级：向对侧转圈；3级：向对侧倾倒；4级：无自主活动伴意识丧失。
4.标本采集及制备
栓塞24h后，每组取8只大鼠处死断头取脑，PBS(pH7.4)涮洗后-20℃放置20min，均匀切片(厚度2mm)，2％TTC溶液37℃避光染色30min，10％福尔马林固定24h，拍摄照片后分析梗死体积；每组另取8只大鼠麻醉状态下取股动静脉混合血，置于4℃低温离心机，3500r/min离心20min，取上清液于-20℃冰箱保存备检测SOD、MDA。取血后经心尖插入灌注针至升主动脉，以4℃生理盐水快速灌注至流出液变澄亮，继以4％多聚甲醛磷酸缓冲液灌注固定，开颅取脑，取视交叉前后2mm脑组织固定；脱水，透明，浸蜡，包埋；连续切取脑部连续冠状组织病理切片待用；每组余下8只大鼠处死取脑，于冰盘上迅速取缺血侧大脑半球，制备10％脑组织匀浆备用检测IL-1β和TNF-α。
5.相对梗死体积检测
用imageJ图像软件分析量化每片脑片的梗死面积，最后计算求得脑梗死体积占脑总体积的比例。
6.SOD、MDA检测
严格按照试剂盒说明书要求检测SOD和MDA。
7.Fas检测
采用免疫组化法检测。脑组织石蜡切片脱蜡至水，3％H2O2消除内源性过氧化物酶的活性，蒸馏水冲洗3次。枸橼酸钠缓冲液热修复抗原，小牛血清室温下密闭15min；滴加 兔抗鼠Fas抗体，4℃过夜，滴加生物素化山羊抗兔IgG，37℃水浴20min，PBS冲洗5min连续4次，DAB显色，充分冲洗，不复染，梯度酒精脱水，二甲苯透明，封片固定。高倍光学显微镜下通过摄像头采集并输入图像分析系统进行图像分析，每张切片随机选取5个不重叠视野，每个视野选取5个区域测量灰度值，计算平均灰度值，其平均灰度值与表达的阳性率成反比。
8.细胞凋亡检测
采用TUNEL法检测。脑组织石蜡切片脱蜡至水，3％H2O2消除内源性过氧化物酶的活性，蒸馏水洗涤2min，连续冲洗3次，加标记液37℃标记2h，加封闭液室温封闭30min，加生物化抗地高辛抗体，37℃反应30min，加SABC37℃反应30min，TBS冲洗5min，连续4次，DAB显色；充分冲洗，苏木素轻度复染，梯度酒精脱水，二甲醛透明，封皮固定。每张切片随机选取半暗带5个不重叠视野，输入图象分析系统，分别计数凋亡细胞个数，取平均值记为凋亡细胞数。
9.IL-1β和TNF-α检测
将脑组织匀浆液以3000rpm低温离心15min后取上清严格按照试剂盒说明书要求检测IL-1β和TNF-α。
(3)结果：
化合物1～3能明显改善永久性脑缺血模型大鼠神经功能损伤行为学；能明显减少永久性缺血模型大鼠脑梗死体积；能显著降低永久性脑缺血模型大鼠内源性氧自由基清除剂SOD的消耗，减轻脂质过氧化损伤，同时降低血清MDA含量；有效下调永久性脑缺血模型大鼠脑组织细胞Fas表达；有效抑制脑细胞凋亡；有效下调永久性脑缺血模型模型大鼠脑组织细胞IL-1β和TNF-α表达。结果见下表2所示。
表2
Figure PCTCN2016076112-appb-000010
Figure PCTCN2016076112-appb-000011
与永久性脑缺血模型组比较*：p<0.05
实施例6本发明与现有阳性药品药效的比较
依照上述实施例4、5所述方法评价丙泊酚和依达拉奉对大鼠缺血再灌注模型和永久脑缺血模型的治疗作用(丙泊酚15mg/kg；依达拉奉3mg/kg，此处15mg/kg表示大鼠每公斤体重给予15mg的丙泊酚，此处3mg/kg表示大鼠每公斤体重给予3mg依达拉奉)，同时观察大鼠给药后行为学变化，实验结果分别如下表3、表4所示。
表3阳性药在大鼠缺血再灌注模型中的药效
Figure PCTCN2016076112-appb-000012
*：相对于实施例1组，p<0.05。
表3结果显示，二联苯衍生物对模型组的治疗效果优于阳性对照药丙泊酚和依达拉奉，且大多数实施例相对于阳性药药效优势明显(p<0.05)，表3仅列出实施例1组药效实验数据与阳性药比较作为参考。同时发现，丙泊酚给药后大鼠翻正反射消失，进入麻醉状态，而其他给药组大鼠无明显行为学变化。
表4阳性药在大鼠永久脑缺血模型中的药效
Figure PCTCN2016076112-appb-000013
*：相对于实施例1，p<0.05。
表4结果显示，二联苯衍生物对模型组的治疗效果优于阳性对照药丙泊酚和依达拉奉，且大多数实施例组相对于阳性药药效优势明显(p<0.05)，表4仅列出实施例1组药效实验数据与阳性药比较作为参考。同时发现，丙泊酚给药后大鼠翻正反射消失，进入麻醉状态，而其他给药组大鼠无明显行为学变化。
实施例7油性制剂
本发明二联苯衍生物的油性制剂的配方可如表5所示：
表5
组分 药量配比
3,3’,5,5’-四异丙基-4,4’-联苯二酚 200mg
四氢呋喃聚乙二醇醚 0.80ml
维生素E醋酸酯 5mg
苯甲醇 50μl
蓖麻油 加至1ml
实施例8片剂
本发明二联苯衍生物的片剂的配方可如表6所示：
表6
组分 药量配比
3,3’,5,5’-四异丙基联苯-4’4-二乙酸酯 200mg
乳糖 140mg
微晶纤维素 100mg
淀粉浆 50mg
羧甲基淀粉钠 10mg
硬脂酸镁 1mg
实施例9胶囊
本发明二联苯衍生物的胶囊的配方可如表7所示：
表7
组分 药量配比
3,3’,5,5’-四异丙基-4,4’-联苯二酚 1g
橄榄油 10g
蛋黄卵磷脂 1.2g
维生素E 0.2g
实施例10乳剂
本发明二联苯衍生物的乳剂的配方可如表8所示：
表8
组分 药量配比
4’-羟基-3,3’,5,5’-四异丙基联苯-4-乙酸酯 1g
大豆油 10g
蛋黄卵磷脂 1.2g
维生素E 0.1g
甘油 2.25g
氢氧化钠 适量
注射用水 加至100ml

Claims (20)

  1. 3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物在制备治疗和/或预防缺血性脑卒中的药物中的应用，所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚结构如式(I)所示：
    Figure PCTCN2016076112-appb-100001
  2. 根据权利要求1所述的应用，其中，所述酯化物为3,3’,5,5’-四异丙基-4,4’-二联苯酚的单酯化物或二酯化物；优选地，所述的酯化物为式(II)所示的单乙酯化物或式(III)所示的二乙酯化物：
    Figure PCTCN2016076112-appb-100002
  3. 根据权利要求1或2所述的应用，其中，所述的药用盐为3,3’,5,5’-四异丙基-4,4’-二联苯酚与有机酸、无机酸或碱金属所成的盐；所述药用盐例如为硫酸盐、磷酸盐、盐酸盐、氢溴酸盐、醋酸盐、草酸盐、柠檬酸盐、琥珀酸盐、葡萄糖酸盐、酒石酸盐、对甲苯磺酸盐、苯磺酸盐、甲磺酸盐、苯甲酸盐、乳酸盐、马来酸盐、锂盐、钠盐、钾盐或钙盐。
  4. 根据权利要求1或2所述的应用，其中，所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物被配制成药物组合物，该药物组合物含有3,3’,5,5’-四异丙基-4,4’-二联苯酚或其药用盐、酯化物或溶剂化物，和药用辅料。
  5. 根据权利要求1或2所述的应用，其中，所述3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物被配制成含其的片剂、胶囊剂、注射剂、乳剂、脂质体、冻干粉或微球制剂；所述胶囊剂例如为软胶囊。
  6. 根据权利要求1或2所述的应用，其中，所述治疗和/或预防缺血性脑卒中是通过改善脑缺血和/或再灌注神经功能损伤行为；减少脑缺血和/或再灌注脑梗死体积；降低脑组织内源性氧自由基清除剂SOD的消耗，减轻脂质过氧化损伤，同时降低血清MDA含量；下调脑组织细胞Fas表达；抑制脑细胞凋亡；和/或下调脑组织细胞IL-1β和TNF-α表达来实现的。
  7. 根据权利要求1或2所述的应用，其中，所述缺血性脑卒中包括由以下病症中的一种或几种引起的损伤：脑血栓、短暂性脑缺血发作、基底节腔梗、动脉粥样硬化性血栓性脑梗塞、腔隙性脑梗塞、脑栓塞和脑血管性痴呆。
  8. 一种治疗和/或预防动物或人类缺血性脑卒中的方法，所述方法包括对受试动物或者人类给予有效剂量的3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物，所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚结构如式(I)所示：
    Figure PCTCN2016076112-appb-100003
  9. 根据权利要求8所述的方法，其中，所述酯化物为3,3’,5,5’-四异丙基-4,4’-二联苯酚的单酯化物或二酯化物；优选地，所述的酯化物为式(II)所示的单乙酯化物或式(III)所示的二乙酯化物：
    Figure PCTCN2016076112-appb-100004
  10. 根据权利要求8或9所述的方法，其中，所述的药用盐为3,3’,5,5’-四异丙基-4,4’-二联苯酚与有机酸、无机酸或碱金属所成的盐；例如，所述药用盐为硫酸盐、磷酸盐、盐酸盐、氢溴酸盐、醋酸盐、草酸盐、柠檬酸盐、琥珀酸盐、葡萄糖酸盐、酒石酸盐、对甲苯磺酸盐、苯磺酸盐、甲磺酸盐、苯甲酸盐、乳酸盐、马来酸盐、锂盐、钠盐、钾盐或钙盐。
  11. 根据权利要求8或9所述的方法，其中，所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物被配制成药物组合物，该药物组合物含有3,3’,5,5’-四异丙基-4,4’-二联苯酚或其药用盐、酯化物或溶剂化物，和药用辅料。
  12. 根据权利要求8或9所述的方法，其中，所述3,3’,5,5’-四异丙基-4,4’-二联苯酚及其药用盐、酯化物或溶剂化物被配制成含其的片剂、胶囊剂、注射剂、乳剂、脂质体、冻干粉或微球制剂；所述胶囊剂例如为软胶囊。
  13. 根据权利要求8或9所述的方法，其中，所述治疗和/或预防缺血性脑卒中是通过改善脑缺血和/或再灌注神经功能损伤行为；减少脑缺血和/或再灌注脑梗死体积；降低脑组织内源性氧自由基清除剂SOD的消耗，减轻脂质过氧化损伤，同时降低血清MDA含量； 下调脑组织细胞Fas表达；抑制脑细胞凋亡；和/或下调脑组织细胞IL-1β和TNF-α表达来实现的。
  14. 根据权利要求8或9所述的方法，其中，所述缺血性脑卒中包括由以下病症中的一种或几种引起的损伤：脑血栓、短暂性脑缺血发作、基底节腔梗、动脉粥样硬化性血栓性脑梗塞、腔隙性脑梗塞、脑栓塞和脑血管性痴呆。
  15. 权利要求1～7中任一项所述的应用中或权利要求8～14中任一项所述的方法中所述的3,3’,5,5’-四异丙基-4,4’-二联苯酚的制备方法，所述方法包括由式(IV)所示的化合物制备所述3,3’,5,5’-四异丙基-4,4’-二联苯酚的步骤：
    Figure PCTCN2016076112-appb-100005
  16. 根据权利要求15所述的方法，其中，所述3,3’,5,5’-四异丙基-4,4’-二联苯酚是将式(IV)所示的化合物溶解在有机溶剂中，优选该有机溶剂为乙酸乙酯；然后与含保险粉的水溶液混合反应得到的；优选地，所述含保险粉的水溶液是由保险粉与氢氧化钠形成的水溶液。
  17. 根据权利要求15或16所述的方法，其中，式(IV)所示的化合物是将丙泊酚溶于有机溶剂，优选地，该有机溶剂为乙酸乙酯；然后加入无机盐反应得到的；优选地，所述无机盐为碳酸银和无水硫酸镁。
  18. 根据权利要求15所述的方法，所述方法包括如下步骤：将丙泊酚溶于有机溶剂中，优选地，该有机溶剂为乙酸乙酯中，再加入无机盐，优选地，所述无机盐包括碳酸银和无水硫酸镁，室温搅拌；
    反应完毕后，向反应液内加水，将固体过滤，洗涤，水相去除，干燥乙酸乙酯相，过滤，滤液蒸发至干，任选洗涤，得到枚红色结晶；
    将上述枚红色固体溶于乙酸乙酯，并与保险粉溶液混合，优选地，该保险粉溶液为保险粉与NaOH形成的水溶液，任选搅拌，然后将乙酸乙酯相分离出，水相采用乙酸乙酯萃取合并，干燥，过滤，滤液蒸发至干，得到淡黄色固体，洗涤得到白色固体3,3’,5,5’-四异丙基-4,4’-二联苯酚。
  19. 权利要求2～7中任一项所述的应用中或权利要求9～14中任一项所述的方法中式(II)所示的单乙酯化物的制备方法，该方法包括如下步骤：
    室温下将4’-苄氧基-3,3’,5,5’-四异丙基联苯-4-乙酸酯溶于有机溶剂中，优选地，该有机溶剂为甲醇，再加入催化剂，优选地，该催化剂为钯碳，抽真空，通入氢气；密封后室温反应，过滤，滤液蒸发至干，得到白色固体，即4’-羟基-3,3’,5,5’-四异丙基联苯-4-乙酸酯。
  20. 权利要求2～7中任一项所述的应用中或权利要求9～14中任一项所述的方法中式(III)所示的二乙酯化物的制备方法，该方法包括如下步骤：
    将4,4’-二羟基-3,3’,5,5’-四异丙基联苯加入到乙酸酐中，氮气保护下，回流反应，将反应液冷却至室温，除去乙酸酐，向残余物中加水，出现白色固体，洗涤，干燥，得到3,3’,5,5’-四异丙基联苯-4’4-二乙酸酯。
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