WO2019183793A1 - N-羟基吡啶酮类化合物及其用途 - Google Patents

N-羟基吡啶酮类化合物及其用途 Download PDF

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WO2019183793A1
WO2019183793A1 PCT/CN2018/080648 CN2018080648W WO2019183793A1 WO 2019183793 A1 WO2019183793 A1 WO 2019183793A1 CN 2018080648 W CN2018080648 W CN 2018080648W WO 2019183793 A1 WO2019183793 A1 WO 2019183793A1
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group
compound
bicyclo
alkynyl
alkyl
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PCT/CN2018/080648
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French (fr)
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李剑
章海燕
胡凌昊
冯红玄
王维
朱进
毛斐
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华东理工大学
中国科学院上海药物研究所
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Priority to CN201880024104.2A priority Critical patent/CN110536687B/zh
Priority to PCT/CN2018/080648 priority patent/WO2019183793A1/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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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

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  • the present invention relates to an N-hydroxypyridone compound and use thereof.
  • Cerebral stroke also known as acute cerebrovascular diseases or cerebral vascular accident (CVA) is a brain damage caused by sudden rupture of blood vessels in the brain or blood circulation disorder caused by vascular obstruction.
  • Group disease also known as stroke. Stroke can be divided into ischemic stroke and hemorrhagic stroke, of which ischemic stroke is more common, accounting for about 87%.
  • Ischemic stroke is a nerve injury caused by blood supply disorders.
  • the key to treating stroke is to re-pass the blood vessels to improve the blood supply of the ischemic brain tissue and protect the nerve cells in the ischemic and hypoxic environment, and prevent the nerve cells.
  • Injury and death At present, the main treatment for early ischemic stroke (within 3-6 hours) is thrombolytic therapy.
  • thrombolytic therapy At present, many patients cannot reach the hospital within the optimal time window of thrombolytic therapy (within 3 hours), and the stroke is still CT (computed tomography) or MRI (Magnetic Resonance Imaging) is required to eliminate hemorrhagic stroke, and it takes more than 30 minutes. Therefore, patients with acute ischemic stroke are less likely to receive thrombolytic therapy.
  • Neuroprotective agent is a drug for treating nerve damage after acute ischemia or reperfusion. It can protect brain tissue, improve the tolerance of nerve cells to ischemia and hypoxia, accelerate the recovery of neurological function, and significantly improve the prognosis of patients. Neuroprotective agents play an important role in the mid-to-late and recovery phase of acute ischemic stroke therapy, especially in patients with recovery after thrombolysis and patients who miss the time window of thrombolytic therapy.
  • the object of the present invention is to provide a novel N-hydroxypyridone compound having neuroprotective activity and 1-hydroxy-4-methyl-6-(2-bicyclo[2,2,1]heptane)-2- A new anti-stroke drug for pyridone, these compounds can be used to prepare new anti-stroke drugs.
  • a compound of formula I a stereoisomer, a pharmaceutically acceptable salt thereof, or a mixture thereof, for the manufacture of a medicament for the treatment of stroke,
  • R 1, R 2 are each independently hydrogen, halogen, C 1 -C 8 alkyl, C 3-8 cycloalkyl, C 2-8 alkynyl group, C 2- 8 alkenyl, benzyl or C 6 -10 aryl;
  • n 1, 2, 3 or 4
  • R 4 and R 5 are independently hydrogen, C 1 -C 10 alkyl, C 3 -C 8 monocyclic cycloalkyl, C 2-8 alkenyl, C 2 - 8 alkynyl, C 6-10 aryl or diaziridine substituted C 5-8 alkynyl;
  • R, R 1 and the attached C together form a substituted or unsubstituted C 6-10 aryl group, the substituent on the aryl group is H, halogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 2-8 alkynyl, C 2-8 alkenyl or benzyl.
  • the treatment refers to inhibiting nerve cell damage or inhibiting nerve cell death.
  • the present application provides 1-hydroxy-4-methyl-6-(2-bicyclo[2,2,1]heptane)-2-pyridinone or a stereoisomer thereof Or a mixture of different configurations or a pharmaceutically acceptable salt thereof and a pharmaceutical composition containing the same, for use in the preparation of a medicament for treating stroke or for treating stroke,
  • the stereoisomer is an endo isomer or an exo isomer
  • Mixtures of different configurations include a mixture of an endo isomer and an exo isomer.
  • R 1 and R 2 are each independently hydrogen, halogen, C 1 -C 6 alkyl, C 3-6 cycloalkane, C 2 - 5 alkyne, C 2-5 alkene, benzyl or Phenyl.
  • R 1 together with the attached C form a phenyl group.
  • R 2 is H; and R 3 is a C 3-6 cycloalkane.
  • the C 7-9 bridged cycloalkyl group is selected from the group consisting of: a bicyclo[2,2,1]heptyl group Bicyclo[2,2,2]octyl Bicyclo[4,1,1]octyl Bicyclo[3,2,1]octyl Bicyclo[3,2,2]decyl And bicyclo[3,3,1]decyl
  • R 3 is a C 7-9 bridged cycloalkane
  • the compound of formula I is an internal configurational isomer, an external configuration isomer, or an internal configuration isomer and A mixture of conformational isomers.
  • the above C 7-9 bridged cycloalkyl group is an endo isomer or an exo isomer
  • the pharmaceutically acceptable salt is an alkaline earth metal, an alkali metal, an aluminum, a transition metal salt of the compound of Formula I, or a salt of a compound of Formula I and an organic base.
  • the organic base is selected from the group consisting of ethanolamine, diethanolamine, N-ethylethanolamine, N-methylethanolamine, triethanolamine, diethylaminoethanol, 2-amino-2-methyl-n-propanol, dimethylaminoisopropyl Alcohol, tert-butylamine, triisopropanolamine, ethylenediamine, morpholine, piperidine, piperazine, cyclohexylamine, tributylamine, dodecylamine, dimethyldodecylamine, triethylamine benzylamine, two Benzylamine, N-methylpiperazine, 4-methylcyclohexylamine, N-methylmorpholine, methylamine and ethylamine.
  • the preferred salt is an ethanolamine salt.
  • the preferred salt is
  • the compound is:
  • R 3 is C 3 -C 8 monocyclic cycloalkyl, C 7-9 bridged cycloalkyl, H, halogen, C 1-8 alkyl, C 2-8 alkynyl, C 2-8 alkenyl or Benzyl;
  • R 1 and R 2 are each independently hydrogen, halogen, C 1 -C 8 alkyl, C 3-8 cycloalkyl, C 2-8 alkynyl, C 2-8 alkenyl, benzyl or C 6-10 aryl base;
  • R, R 1 and the attached C together form a substituted or unsubstituted C 6-10 aryl group
  • the substituent on the aryl group is H, halogen, C 1-8 alkyl, C 3-8 cycloalkyl, C 2-8 alkynyl, C 2-8 alkenyl or benzyl.
  • the C7-9 bridged cycloalkyl group is attached to the N-hydroxypyridone in such a manner that the carbon on the pyridone ring is bonded to the bridged carbon or non-bridged carbon of the bridged cycloalkyl group as a carbon-carbon single bond.
  • R 1 and R 2 are each independently hydrogen, halogen, C 1 -C 6 alkyl, C 3-6 cycloalkane, C 2 - 5 alkyne, C 2-5 alkene, benzyl or Phenyl.
  • R 1 together with the attached C form a phenyl group.
  • R 2 is H; and R 3 is a C 3-6 cycloalkane.
  • the C 7-9 bridged cycloalkyl group is selected from the group consisting of: a bicyclo[2,2,1]heptyl group Bicyclo[2,2,2]octyl Bicyclo[4,1,1]octyl Bicyclo[3,2,1]octyl Bicyclo[3,2,2]decyl And bicyclo[3,3,1]decyl
  • R 3 is a C 7-9 bridged cycloalkane
  • the compound of formula I is an internal configurational isomer, an external configuration isomer, or an internal configuration isomer and A mixture of conformational isomers.
  • the above C 7-9 bridged cycloalkyl group is an endo isomer or an exo isomer
  • the pharmaceutically acceptable salt is an alkaline earth metal, an alkali metal, an aluminum, a transition metal salt of the compound of Formula I, or a salt of a compound of Formula I and an organic base.
  • the organic base is selected from the group consisting of ethanolamine, diethanolamine, N-ethylethanolamine, N-methylethanolamine, triethanolamine, diethylaminoethanol, 2-amino-2-methyl-n-propanol, dimethylaminoisopropyl Alcohol, tert-butylamine, triisopropanolamine, ethylenediamine, morpholine, piperidine, piperazine, cyclohexylamine, tributylamine, dodecylamine, dimethyldodecylamine, triethylamine benzylamine, two Benzylamine, N-methylpiperazine, 4-methylcyclohexylamine, N-methylmorpholine, methylamine and ethylamine.
  • the preferred salt is an ethanolamine salt.
  • the preferred salt is
  • the compound is:
  • a pharmaceutical composition characterized in that the pharmaceutical composition comprises:
  • a pharmaceutically acceptable carrier is selected from:
  • “Pharmaceutically acceptable carrier” means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By “compatibility” it is meant herein that the components of the composition are capable of intermingling with the compounds of the invention and with each other without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid).
  • magnesium stearate magnesium stearate
  • calcium sulfate vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.
  • the carrier is selected from the group consisting of diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubrication Agent, or a combination thereof.
  • the pharmaceutical composition is formulated as a solid dosage form or a liquid dosage form, preferably for oral administration.
  • the solid dosage form comprises capsules, tablets, pills, powders, and granules.
  • the liquid dosage form comprises a pharmaceutically acceptable emulsion, solution, suspension, syrup or elixir.
  • a method for preventing and/or treating stroke which comprises administering a compound of the second aspect, a stereoisomer thereof, a pharmaceutically acceptable salt or a mixture thereof to a subject in need thereof .
  • the administration means intravenous administration, intraperitoneal administration, inhalation administration, sublingual administration, rectal administration, intramuscular administration, subcutaneous administration, or oral administration.
  • Figure 1 shows the results of I-1 reduction of cerebral infarction area of MCAO ( Middle cerebral artery occlusion ) and improvement of neurobehavioral function:
  • A TTC staining of brain slices;
  • B Brain lesions Regional statistical map;
  • the dose of edaravone was 10 mg/kg and the dose of CPX was 3 mg/kg.
  • the inventors of the present application have extensively and intensively studied to develop a novel N-hydroxypyridone compound for the first time, and selected an oxygen glucose deprivation (OGD) model constructed by SH-SY5Y cells.
  • OGD oxygen glucose deprivation
  • the neuroprotective activity was tested and the anti-ischemic injury activity was tested by the model of focal cerebral ischemia (MCAO) in rats.
  • MCAO focal cerebral ischemia
  • the activity results showed that these compounds have good neuroprotective activity, and some compounds are expressed in animal models. It has better anti-ischemic injury activity than the existing anti-stroke drugs and has a good prospect for treating stroke.
  • the present invention has been completed.
  • the N-hydroxypyridone compound of the present invention is a compound of the formula II, III, IV, or a stereoisomer thereof, or a pharmaceutically acceptable salt, or a carboxy group which is degradable in vivo.
  • Acid ester prodrug :
  • R 1 , R 2 are independently H, Cl, Br, I, F, C 1-8 alkyl, C 3-8 cycloalkyl, C 2-8 alkynyl, C 2-8 alkenyl Benzyl.
  • R 3 is C 7-9 bridged cycloalkyl, H, Cl, Br, I, F, C 1-8 alkyl, C 3-8 cycloalkyl, C 2-8 alkynyl, C 2-8 alkenyl, benzyl base.
  • R 4 , R 5 are independently C 2-8 alkynyl, aziridine substituted C 5-8 alkynyl, H, Cl, Br, I, F, C 1-8 alkyl, C 3-8 cycloalkyl, C 1-8 alkenyl and benzyl.
  • R 6 and R 7 are independently H, Cl, Br, I, F, C 1-8 alkyl, C 3-8 cycloalkyl, C 2-8 alkynyl, C 2-8 alkenyl, benzyl.
  • R 8 is C 7-9 bridged cycloalkyl, H, Cl, Br, I, F, C 1-8 alkyl, C 3-8 cycloalkyl, C 2-8 alkynyl, C 2-8 alkenyl Benzyl.
  • R 9 is C 7-9 bridged cycloalkyl, C 3-8 cycloalkyl
  • R 10 is H, Cl, Br, I, F, C 1-8 alkyl, C 3-8 naphthenic A group, a C 2-8 alkynyl group, a C 2-8 alkenyl group, a benzyl group.
  • the carbonyl carbon number is 1, the nitrogen atom is 2, clockwise from small to large, and the substitution site of R 10 may be the 5, 6, 7, and 8 positions on the isoquinolinone ring.
  • R 1 , R 2 , R 6 , R 7 , R 10 are each independently selected from the group consisting of: H, Br, I, methyl.
  • R 3 , R 8 , R 9 are each independently selected from the group consisting of cyclohexyl, bicyclo[2,2,1]heptane.
  • R 4 and R 5 are each independently selected from the group consisting of H, methyl, propyl, isopropyl, cyclopentyl, phenyl, n-heptyl, 5-biaziridine-1-g Alkynyl.
  • the N-hydroxypyridone compound of the present invention can be optionally prepared by the following route.
  • R 4 and R 5 are each independently H, methyl, propyl, isopropyl, cyclopentyl, phenyl, n-heptyl, 5-biaziridine-1-heptynyl.
  • the N-hydroxypyridone compound of the present invention has good neuroprotective activity, has a good prospect for treating stroke, and can be used for preparing a medicament for treating stroke.
  • 1-hydroxy-4-methyl-6-(2-bicyclo[2,2,1]heptane)-2-pyridinone was also found.
  • a stereoisomer thereof or a mixture of different configurations or a pharmaceutically acceptable salt thereof and a pharmaceutical composition containing the same exhibit an anti-ischemic injury activity against an existing anti-stroke drug in an animal model, and can inhibit nerves Cell damage and death play a role in the treatment of stroke.
  • 1-hydroxy-4-methyl-6-(2-bicyclo[2,2,1]heptane)-2-pyridone also known as 2(1H)-Pyridinone, 6-bicyclo [2.2.1] Hept-2-yl-1-hydroxy-4-methyl- (CAS search name), or 6-Bicyclo [2.2.1] hept-2-yl-1-hydroxy-4-methyl-2(1H)-pyridinone.
  • This structural formula represents two optical isomers, an endo isomer and an exo isomer, respectively.
  • the internal conformational isomer of the compound and a mixture of internal and external conformations can inhibit the damage and death of nerve cells to treat stroke.
  • Example 2 The petroleum ether filtrate of Example 2 was cooled to minus 20 degrees Celsius, and the insoluble matter was filtered, and the filtrate was concentrated to give II-3.
  • Example 6 The same starting materials, reagents and preparation methods were the same as in Example 6 except that the methanol solution of methylamine was replaced with 3-bisaziridine-6-heptynylamine to obtain III-6.
  • o-iodobenzoic acid is dissolved in dichloromethane, two equivalents of thionyl chloride are added, and two hundred equivalents of N,N-dimethylformamide is used as a catalyst for three hours at room temperature, and distilled under reduced pressure.
  • the solvent was used to prepare crude o-iodobenzoyl chloride.
  • O-iodobenzoyl chloride and an equivalent amount of triethylamine were simultaneously added to methanol at room temperature for 20 minutes to obtain methyl ortho-iodobenzoate.
  • SH-SY5Y is a human neuroblastoma cell that has the characteristics of a nerve cell.
  • Oxygen glucose deprivation (OGD) model deprives the cultured cells of oxygen, glucose and serum deprivation. After a period of time, the normal culture conditions are restored, and the cell viability is detected after 24 hours of culture. The model simulates clinical cerebral ischemic reperfusion injury. The effect of oxygen glucose deprivation on the activity of SH-SY5Y cells was detected by MTT colorimetric assay. The compounds were initially evaluated for neuroprotective effects, especially for ischemic induction. The improvement of damage.
  • test compound was dissolved in DMSO, formulated into a 10 mM mother liquor, and stored at -20 ° C.
  • SH-SY5Y cells were subcultured in MEM/F12 medium containing 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin. The cells were digested with 0.25% trypsin and suspended in MEM/F12 medium containing 10% fetal bovine serum.
  • SH-SY5Y cells were seeded on a 96-well culture plate at a density of 3.5 ⁇ 10 5 cells/mL, inoculated in a volume of 100 ⁇ L/well, and cultured in a 37 ° C incubator containing 5% CO 2 .
  • the oxygen-deficient injury group and the administration group were all washed with the sugar-free EBSS balanced salt solution, and then replaced with DMEM (sugar-free) medium, and the corresponding concentration of the test compound was added to the drug-administered group.
  • the group was added with the corresponding solvent control and placed in an anaerobic apparatus (containing 85% N 2 , 10% H 2 , 5% CO 2 ) for 2 h.
  • the normal control group was changed to a DMEM medium containing sugar and serum, and cultured in a 37 ° C incubator containing 5% CO 2 for the same time.
  • the oxygen plate was taken out from the anaerobic apparatus, and the oxygen-deficient injury group and the administration group were supplemented with serum and glucose, and the final concentration of glucose was 1 g/L.
  • 5 mg/mL MTT 100 ⁇ L/well was added for viable cell staining.
  • DMSO 100 ⁇ L/well was added, and shaken on a shaker to fully dissolve.
  • the OD value of each group was measured at a wavelength of 490 nm.
  • the survival rate of nerve cells induced by oxygen glucose deprivation after administration was calculated according to the following formula:
  • the cell viability of the normal control group was set to 100%, and the data showed the percentage compared to the normal control group, which is the average of three independent experiments.
  • the test compounds were tested in two groups.
  • the cell viability of group 1 (OGD) was 48.56%, and the cell viability of group 2 (OGD) was 54.92%.
  • the cell viability of the normal control group was set to 100%, and the data showed the percentage compared to the normal control group, which is the average of three independent experiments.
  • the cell viability value of the test model group (OGD group) was 54.23%. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 compared to the model group (OGD group).
  • the middle cerebral artery (MCA) is the frequent site of stroke in the population. Therefore, the model of focal cerebral ischemia caused by blocking MCA is a widely used animal model. The neurobehavioral score recorded by this model is used. And TTC (2,3,5-Triphenyte-trazoliumchloride, 2,3,5-triphenyltetrazolium chloride) staining, respectively, can reflect the neurobehavioral damage caused by cerebral ischemia and infarction in cerebral ischemic area By evaluating the first two indicators, the protective effect of the compound on cerebral ischemia can be reflected. Therefore, the in vivo pharmacological evaluation of the ethanolamine salt of the I-1 having a better activity in Example 16 was carried out by this method.
  • the transient middle cerebral artery embolization model is mainly performed according to the method described by Longa, et al; Stroke, 1989.
  • the rats were anesthetized with chloral hydrate (400mg/Kg, ip), and the midline incision was made in the neck.
  • the tissue was separated, and the right internal carotid artery and the external carotid artery were bifurcated.
  • the thin line on the common carotid artery was alive, and the neck was ligated.
  • the novel N-hydroxypyridone compound provided by the invention has a simple molecular structure, a simple preparation process, and shows strong neuroprotective activity in the SH-SY5Y cell oxygen glucose deprivation model experiment, so it is expected to be developed into a drug for treating stroke.
  • the present invention also relates to the novel use of 1-hydroxy-4-methyl-6-(2-bicyclo[2,2,1]heptane)-2-pyridone for the treatment of stroke, transient MCAO in rats
  • 1-hydroxy-4-methyl-6-(2-bicyclo[2,2,1]heptane)-2-pyridinone can significantly reduce the area of cerebral infarction and improve the neurobehavioral function of rats. This indicates that the compound is expected to be developed into a drug for the treatment of stroke.

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Abstract

一种式（I）所示的化合物，其立体异构体，药学上可接受的盐或它们的混合物，所述式I所示的化合物，其立体异构体，药学上可接受的盐或它们的混合物能够抑制神经细胞损伤和神经细胞死亡，能够用于制备治疗脑卒中的药物。

Description

N-羟基吡啶酮类化合物及其用途 技术领域
本发明涉及一种N-羟基吡啶酮类化合物及其用途。
背景技术
脑卒中(cerebral stroke)又称急性脑血管病(acute cerebrovascular diseases)或脑血管意外(cerebral vascular accident，CVA)是由于脑部血管突然破裂或因血管阻塞造成血液循环障碍而引起脑组织损伤的一组疾病，也称之为中风。脑卒中可分为缺血性脑卒中和出血性脑卒中，其中缺血性脑卒中较为常见，约占87％左右。
缺血性脑卒中是由于供血障碍导致的神经损伤，治疗脑卒中的关键在于重通血管改善缺血部位脑组织的血流供应和保护处于缺血缺氧环境下的神经细胞，阻止神经细胞的损伤和死亡。目前缺血性脑卒中发生的早期(3—6小时内)主要治疗手段是溶栓治疗，然而，很多患者不能在溶栓治疗的最佳时间窗(3小时以内)内到达医院，并且中风还需要CT(computed tomography,电子计算机断层扫描)或MRI(Magnetic Resonance Imaging，磁共振成像)来排除出血性中风，由此还需要30分钟以上。因此，急性缺血性中风患者接受溶栓治疗的几率很低。
神经保护剂是治疗急性缺血或再灌注后神经损伤的药物，它可以保护脑组织，提高神经细胞对缺血缺氧的耐受，加快患者神经功能的恢复，明显改善患者预后。神经保护剂在急性缺血性脑卒中治疗的中后期和恢复期具有重要的地位，尤其对于溶栓后恢复期的患者和错过溶栓治疗时间窗的患者而言。
目前国内的神经保护剂类小分子中风治疗药物只有依达拉奉和丁苯酞两种，然而由于个体差异等原因，并非所有患者应用现有药物都能取得很好疗效。
发明内容
本发明的目的在于提供一种具有神经保护活性的新型N-羟基吡啶酮类化合物和1-羟基-4-甲基-6-(2-双环[2,2,1]庚烷)-2-吡啶酮的抗脑卒中新用途，这些化合物可以用于制备新型抗中风药物。
本发明的第一方面，提供式I所示的化合物，其立体异构体，药学上可接受的盐，或它们的混合物的用途，用于制备治疗脑卒中的药物，
Figure PCTCN2018080648-appb-000001
式中，R 1、R 2各自独立为氢、卤素、C 1-C 8烷基、C 3-8环烷基、C 2-8炔基、C 2- 8烯基、苄基或C 6-10芳基；
R 3为C 3-C 8的单环环烷基；R为溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；或者R 3为C 7- 9桥环烷基、H、卤素、C 1-8烷基、C 2-8炔基或C 2-8烯基，R为氢、溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；
其中n为1、2、3或4，R 4、R 5独立为氢、C 1-C 10烷基、C 3-C 8的单环环烷基、C 2-8烯基、C 2-8炔基、C 6-10芳基或双吖丙啶取代的C 5-8炔基；
或者R、R 1与相连的C共同形成取代或未取代的C 6-10芳基，该芳基上的取代基为H、卤素、C 1-8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基或苄基。
在另一优选例中，所述治疗是指抑制神经细胞损伤或抑制神经细胞死亡。
在另一优选例中，本申请提供下式所示1-羟基-4-甲基-6-(2-双环[2,2,1]庚烷)-2-吡啶酮或其立体异构体或不同构型的混合物或其药学上可以接受的盐和含有其的药物组合物的用途，用于制备治疗脑卒中的药物或治疗脑卒中，
Figure PCTCN2018080648-appb-000002
所述的立体异构体为内构型(endo isomer)或外构型(exo isomer)；
Figure PCTCN2018080648-appb-000003
不同构型的混合物包括内构型(endo isomer)，外构型(exo isomer)两种构型的混合物。
在另一优选例中，R 1、R 2各自独立为氢、卤素、C 1-C 6烷基、C 3-6环烷烃、C 2- 5炔烃、C 2-5烯烃、苄基或苯基。
在另一优选例中，R为溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；其中n为1、2、3或4，R 4、R 5独立为氢、C 1-C 8烷基、C 3-C 6的单环环烷基、C 2-5烯基、C 2-5炔基、苯基或双吖丙啶取代的C 5-8炔基；
或者R、R 1与相连的C共同形成苯基。
在另一优选例中，当R、R 1与相连的C共同形成苯基时，R 2为H；R 3为C 3-6环烷烃。
在另一优选例中，C 7-9桥环烷基选自：双环[2,2,1]庚烷基即
Figure PCTCN2018080648-appb-000004
双环[2,2,2]辛烷基即
Figure PCTCN2018080648-appb-000005
双环[4，1，1]辛烷基即
Figure PCTCN2018080648-appb-000006
双环[3,2,1]辛烷基即
Figure PCTCN2018080648-appb-000007
双环[3,2,2]壬烷基即
Figure PCTCN2018080648-appb-000008
和双环[3,3,1]壬烷基即
Figure PCTCN2018080648-appb-000009
在另一优选例中，当R 3为C 7-9桥环烷烃时，式I所示的化合物为内构型异构体、外构型异构体、或内构型异构体和外构型异构体的混合物。上述C 7-9桥环烷基为内构型(endo isomer)或外构型(exo isomer)
在另一优选例中，所述药学上可接受的盐为式I所示的化合物的碱土金属、碱金属、铝、过渡金属盐，或者是式I所示的化合物与有机碱形成的盐，所述有机碱选自：乙醇胺、二乙醇胺、N-乙基乙醇胺，N-甲基乙醇胺、三乙醇胺、二乙氨基乙醇、2-氨基-2-甲基-正丙醇、二甲氨基异丙醇、叔丁胺、三异丙醇胺、乙二胺、、吗啉、哌啶、哌嗪、环已胺、三丁胺、十二胺、二甲基十二胺、三乙胺苄胺、二苄胺、N-甲基哌嗪、4-甲基环己胺、N-甲基吗啉、甲胺和乙胺。
在另一优选例中，优选的盐为乙醇胺盐。
在另一优选例中，优选的盐为
Figure PCTCN2018080648-appb-000010
在另一优选例中，所述化合物为：
Figure PCTCN2018080648-appb-000011
Figure PCTCN2018080648-appb-000012
本发明的第二方面，提供式I所示的化合物、其立体异构体、药学上可接受的盐、或它们的混合物，
Figure PCTCN2018080648-appb-000013
式中，R 3为C 3-C 8的单环环烷基、C 7-9桥环烷基、H、卤素、C 1-8烷基、C2-8炔基、C2-8烯基或苄基；
R 1、R 2各自独立为氢、卤素、C 1-C 8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基、苄基或C 6-10芳基；
R为溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；其中n为1、2、3或4，R 4、R 5独立为氢、C 1-C 10烷基、C 3-C 8的单环环烷基、C 2-8烯基、C 2-8炔基、C 6-10芳基或双吖丙啶取代的C 5-8炔基；
或者R、R 1与相连的C共同形成取代或未取代的C 6-10芳基，该芳基上的取代基为H、卤素、C 1-8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基或苄基。在另一优选例中，C 7-9桥环烷基与N-羟基吡啶酮的连接方式是吡啶酮环上的碳以碳碳单键与桥环烷基的桥头碳或非桥头碳连接。
在另一优选例中，R 1、R 2各自独立为氢、卤素、C 1-C 6烷基、C 3-6环烷烃、C 2- 5炔烃、C 2-5烯烃、苄基或苯基。
在另一优选例中，R为溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；其中n为0、1、2、3或4，R 4、R 5独立为氢、C 1-C 8烷基、C 3-C 6的单环环烷基、C 2-5烯基、C 2-5炔基、苯基或双吖丙啶取代的C 5-8炔基；
或者R、R 1与相连的C共同形成苯基。
在另一优选例中，当R、R 1与相连的C共同形成苯基时，R 2为H；R 3为C 3-6环烷烃。
在另一优选例中，C 7-9桥环烷基选自：双环[2,2,1]庚烷基即
Figure PCTCN2018080648-appb-000014
双环[2,2,2]辛烷基即
Figure PCTCN2018080648-appb-000015
双环[4，1，1]辛烷基即
Figure PCTCN2018080648-appb-000016
双环[3,2,1]辛烷基即
Figure PCTCN2018080648-appb-000017
双环[3,2,2]壬烷基即
Figure PCTCN2018080648-appb-000018
和双环[3,3,1]壬烷基即
Figure PCTCN2018080648-appb-000019
在另一优选例中，当R 3为C 7-9桥环烷烃时，式I所示的化合物为内构型异构体、外构型异构体、或内构型异构体和外构型异构体的混合物。上述C 7-9桥环烷基为内构型(endo isomer)或外构型(exo isomer)
在另一优选例中，所述药学上可接受的盐为式I所示的化合物的碱土金属、碱金属、铝、过渡金属盐，或者是式I所示的化合物与有机碱形成的盐，所述有机碱选自：乙醇胺、二乙醇胺、N-乙基乙醇胺，N-甲基乙醇胺、三乙醇胺、二乙氨基乙醇、2-氨基-2-甲基-正丙醇、二甲氨基异丙醇、叔丁胺、三异丙醇胺、乙二胺、、吗啉、哌啶、哌嗪、环已胺、三丁胺、十二胺、二甲基十二胺、三乙胺苄胺、二苄胺、N-甲基哌嗪、4-甲基环己胺、N-甲基吗啉、甲胺和乙胺。
在另一优选例中，优选的盐为乙醇胺盐。
在另一优选例中，优选的盐为
Figure PCTCN2018080648-appb-000020
在另一优选例中，所述化合物为：
Figure PCTCN2018080648-appb-000021
本发明的第三方面，提供一种药物组合物，其特征在于，所述药物组合物包含：
第二方面所述的化合物、其立体异构体、药学上可接受的盐、羧酸酯前药或者它们的混合物；和
药学上可接受的载体。
“药学上可接受的载体”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
Figure PCTCN2018080648-appb-000022
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。
在另一优选例中，所述的载体选自下组：稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂、或其组合。
在另一优选例中，所述的药物组合物配制为固体剂型或液体剂型，较佳适用于口服给药。
在另一优选例中，固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在另一优选例中，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。
本发明的第四方面，提供一种预防和/或治疗脑卒中的方法，向有需要的对象施用第二方面所述的化合物、其立体异构体、药学上可接受的盐或者它们的混合物。
在另一优选例中，所述施用是指通过静脉给药、腹腔给药、吸入给药、舌下给药、直肠给药、肌内给药、皮下给药、口服给药。
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。说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一赘述。
附图说明
图1为I-1降低大鼠MCAO( Middle cerebral artery occlusion，大脑中动脉栓塞)的脑梗死区域并改善其神经行为学功能结果图：(A)脑片TTC染色图；(B)脑片损伤区域统计图；(C)神经行为学评分；显示值为平均值， ###p<0.001与假手术组(Sham)相比， *p<0.05， **p<0.01与模型组(Vehicle)相比，n＝15～18。依达拉奉剂量为10mg/kg，CPX剂量为3mg/kg。
具体实施方式
本申请的发明人经过广泛而深入地研究，首次研发出一种结构新颖的N-羟基吡啶酮类化合物，选取SH-SY5Y细胞构建的氧糖剥夺(OGD,oxygen glucose deprivation，氧糖剥夺)模型测试神经细胞保护活性，以线栓法大鼠局灶性脑缺血(MCAO)模型测试抗缺血损伤活性，活性结果显示，这类化合物具有良好的神经保护活性，部分化合物在动物模型上表现出优于现有抗中风药物的抗缺血损伤活性，具有良好的治疗脑卒中前景。在此基础上，完成了本发明。
N-羟基吡啶酮类化合物
在另一优选例中，本发明的N-羟基吡啶酮类化合物为式Ⅱ，Ⅲ，Ⅳ所示化合 物，或其立体异构体，或药学上可接受的盐，或可在体内降解的羧酸酯前药：
Figure PCTCN2018080648-appb-000023
式Ⅱ中：R 1，R 2独立地为H、Cl、Br、I、F、C 1-8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基、苄基。R 3为C 7-9桥环烷基、H、Cl、Br、I、F、C 1-8烷基、C 3-8环烷基、C2-8炔基、C2-8烯基、苄基。
式Ⅲ中：R 4，R 5独立地为C 2-8炔基、双吖丙啶取代的C 5-8炔基、H、Cl、Br、I、F、C 1-8烷基、C 3-8环烷基、C 1-8烯基及、苄基。R 6，R 7独立地为H、Cl、Br、I、F、C 1-8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基、苄基。R 8为C 7-9桥环烷基、H、Cl、Br、I、F、C 1-8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基、苄基。
式Ⅳ中：R 9为C 7-9桥环烷基、C 3-8环烷基；R 10为H、Cl、Br、I、F、C 1-8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基、苄基。以羰基碳编号为1，氮原子为2，顺时针从小到大编号，R 10的取代位点可以是异喹啉酮环上的5,6,7,8位。
在另一优选例中，R 1，R 2，R 6，R 7，R 10各自独立地选自：H、Br、I、甲基。
在另一优选例中，R 3,R 8,R 9各自独立地选自：环己基，双环[2,2,1]庚烷。
在另一优选例中，R 4、R 5各自独立地选自H、甲基、丙基、异丙基、环戊基、苯基、正庚基、5-双吖丙啶-1-庚炔基。
制备方法
本发明的N-羟基吡啶酮类化合物，可选用以下路线制备。
路线1：
Figure PCTCN2018080648-appb-000024
路线2：
Figure PCTCN2018080648-appb-000025
R 4,R 5各自独立地为H、甲基、丙基、异丙基、环戊基、苯基、正庚基、5-双吖丙啶-1-庚炔基。
路线3：
Figure PCTCN2018080648-appb-000026
用途
本发明的N-羟基吡啶酮类化合物具有良好的神经保护活性，具有良好的治疗脑卒中前景，能够用于制备治疗脑卒中的药物。
此外还发现1-羟基-4-甲基-6-(2-双环[2,2,1]庚烷)-2-吡啶酮
Figure PCTCN2018080648-appb-000027
或其立体异构体或不同构型的混合物或其药学上可以接受的盐和含有其的药物组合物在动物模型上表现出优于现有抗中风药物的抗缺血损伤活性，可以抑制神经细 胞的损伤和死亡起到治疗脑卒中的作用。
1-羟基-4-甲基-6-(2-双环[2,2,1]庚烷)-2-吡啶酮，也可以称做2(1H)-Pyridinone,6-bicyclo[2.2.1]hept-2-yl-1-hydroxy-4-methyl-(CAS检索名)，或者6-Bicyclo[2.2.1]hept-2-yl-1-hydroxy-4-methyl-2(1H)-pyridinone。
该结构式代表两种光学异构体，分别为Ⅰ内构型(endo isomer)，Ⅰ外构型(exo isomer)。
Figure PCTCN2018080648-appb-000028
优选地，该化合物的内构型异构体和内构型和外构型的混合物，可以抑制神经细胞的损伤和死亡起到治疗脑卒中的作用。
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(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。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1
(1)双环[2,2,1]庚烷甲酸(购买自阿法埃莎)溶于二氯甲烷，加入两当量的草酰氯，两百分之一当量的DMF作为催化剂室温下反应三小时，减压蒸馏除去溶剂制得A环酰氯粗品。
(2)三当量的研磨成粉的氯化铝溶于二氯甲烷中，将A环酰氯和I-a的混合物(1:1)缓缓滴入其中。回流反应五小时，至不在产生氯化氢气体。稀盐酸淬灭反应后，以大量二氯甲烷萃取水相，饱和碳酸氢钠洗二氯甲烷相，硫酸镁干燥后蒸发溶剂得到I-b粗品。
(3)I-b粗品加入到硫酸和醋酸的混合物(3:10)中，100摄氏度反应五小时，加入大量冰块淬灭反应后缓缓加入碳酸氢钠溶液淬灭反应。用乙酸乙酯萃取后，再无水硫酸钠干燥，蒸发溶剂的得到I-c粗品，在用柱层析提纯。
Figure PCTCN2018080648-appb-000029
(4)I-c粗品溶于适量甲苯，加入两当量的十硫化四磷，回流反应五小时。过滤除去剩余硫化剂，旋蒸除去甲苯，柱层析得到I-d。
(5)I-d和五当量盐酸羟胺溶于适量吡啶中，回流反应六小时。滤去不溶物，蒸发吡啶后，柱层析纯化得到I-1粗品。
(6)上一步得到的I-1粗品以沸程60-90度的石油醚分散超声过滤并取滤饼，滤饼为I-1内构型(利用溶解度差异除去其中的外构型产物)。
1H NMR(400MHz,CDCl 3):δ6.37(s,1H),5.99(s,1H),2.94-3.06(m,1H),2.48(s,1H),2.38(s,1H),2.23(s,3H),1.89(t,J＝11.0Hz,1H),1.47-1.77(m,3H),1.20-1.46(m,4H)；MS(EI)m/z 219.1265(M +).
Figure PCTCN2018080648-appb-000030
制备I-1剩余的滤液冷却到零下20度，滤去不溶物，剩余的滤液浓缩即得到I-H。(内构型和外构型的混合物，比例由核磁峰面积之比确定)
1H NMR(400MHz,CDCl 3):δ6.40(s,0.45H),6.37(s,0.55H),6.77(s,0.45H),5.99(s,0.55H),3.45-3.55(m,0.45H),2.94-3.06(m,0.55H),2.76(s,0.45H),2.48(s,0.55H),2.37(s,1H),2.25(s,1.35H),2.22(s,1.65H),1.85-1.97(m,1H),1.12-1.68(m,7H)；MS(EI)m/z 219.1265(M +).
实施例2
Figure PCTCN2018080648-appb-000031
将10g环吡酮溶于100ml四氯化碳中，加入一当量的溴代丁二酰亚胺和百分之五的偶氮二异丁腈作为引发剂。氮气保护下回流反应7h，然后将体系冷却至0度，过滤不溶物。浓缩滤液，柱层析纯化得到粗产品。将粗产品在沸程60-90度的石油醚中分散，超声处理10分钟，过滤并以正己烷清洗滤饼，收集滤饼得到Ⅱ-1，8.25g，收率60％
1H NMR(400MHz,CDCl 3):δ6.09(s,1H),2.98(tt,J＝11.5,3.2Hz,1H),2.34(s,3H),1.95-2.15(m,2H),1.83-1.94(m,2H),1.75-1.93(m,1H),1.18-1.55(m,5H).
实施例3
Figure PCTCN2018080648-appb-000032
除了将NBS替换成NIS外，其余所需原料、试剂及制备方法同实施例2，得到Ⅱ-2，9.1g收率56％。
1H NMR(400MHz,CDCl 3):δ6.10(s,1H),2.97(m,1H),2.39(s,3H),1.95-2.15(m,2H),1.75-1.94(m,3H),1.10-1.58(m,5H)；MS(EI)m/z 333.0225(M +).
实施例4
Figure PCTCN2018080648-appb-000033
将实施例2中石油醚滤液冷却至零下20摄氏度，过滤不溶物后浓缩滤液即得到Ⅱ-3。
1H NMR(400MHz,CDCl 3):δ6.54(s,1H),3.55-3.65(m,1H),2.33(s,3H),2.10-2.30(m,2H),1.85-1.95(m,2H),1.60-1.82(m,2H),1.18-1.55(m,4H)；MS(EI)m/z285.0360(M +)..
实施例5
Figure PCTCN2018080648-appb-000034
(1)将2.85gⅡ-1溶于100ml超干四氢呋喃中，冰浴搅拌下下加入1.5当量的钠氢，待不再释放出氢气后，将反应体系冷却至零下七十八摄氏度。氮气保护下，缓缓滴加3当量的叔丁基锂的正戊烷溶液，体系渐渐变成橙色。当反应体系颜色不再继续加深时，加入6当量的DMF，并逐渐恢复至室温。
(2)反应过夜。后处理时，冰浴搅拌下加入50ml 1N HCl溶液淬灭反应。将四氢呋喃层分离，用50*3ml的乙酸乙酯萃取水相，把四氢呋喃和乙酸乙酯相合并，用饱和食盐水洗三遍后加入无水硫酸钠干燥。蒸发溶液得到粗品。将粗品以甲醇：二氯甲烷＝1:30的流动相硅胶柱层析分离纯化得到，810mg Ⅱ-4，收率34％。
1H NMR(400MHz,CDCl 3):δ6.08(s,1H),3.10-3.25(m,1H),2.56(s,3H),2.00-2.10(m,2H),1.70-1.95(m,3H),1.15-1.52(m,5H)；MS(ES+)m/z 236.1285([M+1] +).
实施例6
Figure PCTCN2018080648-appb-000035
将100mg Ⅱ-4和等当量的甲胺的甲醇溶液溶于5ml 1,2-二氯乙烷中，加入50微升醋酸，常温反应20分钟。然后加入1.2当量的三乙酰氧基硼氢化钠，常温反应过夜。加入5ml饱和食盐水淬灭反应，分离有机相后，将水相用二氯甲烷再次萃取，合并二氯甲烷相和1,2-二氯乙烷相并干燥浓缩得到粗品。粗品柱层析得到Ⅲ-1。
1H NMR(400MHz,CDCl 3):δ8.75-8.98(br s,1H)6.19(s,1H),4.17(s,1H),3.35-3.55(br s,1H),2.65(s,3H),2.27(s,3H),1.55-1.95(m,5H),1.15-1.50(m,5H)；MS(ES+)m/z 251.1758([M+1] +).
实施例7
Figure PCTCN2018080648-appb-000036
除了将甲胺的甲醇溶液替换成正丙胺外，其余所需原料、试剂及制备方法同实施例6，得到Ⅲ-2。
1H NMR(400MHz,CDCl 3):δ8.95-9.20(br s,1H),6.18(s,1H),4.22(s,1H),3.4-3.6(br s,1H),2.68-2.86(br s,2H),2.24(s,3H),1.45-1.95(m,7H),1.10-1.40(m,8H)；MS(ES+)m/z 279.2075([M+1] +).
实施例8
Figure PCTCN2018080648-appb-000037
除了将甲胺的甲醇溶液替换成炔丙胺外，其余所需原料、试剂及制备方法同实施例6，得到Ⅲ-3。
1H NMR(400MHz,CDCl 3):δ5.98(s,1H),3.87(s,2H),3.47(d,J＝2.5Hz,2H),3.05–2.86(m,1H),2.30(s,3H),2.26(s,1H),2.07–1.96(m,2H),1.74-1.94(m,3H),1.56–1.17(m,5H)；MS(ES+)m/z 275.1761([M+1] +).
实施例9
Figure PCTCN2018080648-appb-000038
除了将甲胺的甲醇溶液替换成异丙胺外，其余所需原料、试剂及制备方法同实施例6，得到Ⅲ-4。
1H NMR(400MHz,CDCl 3):δ5.87(s,1H),3.89(s,2H),3.19-3.32(m,1H),2.90-3.10(m,1H),2.24(s,3H),1.65-1.97(m,5H),1.08-1.45(m,11H)；MS(ES+)m/z279.2074([M+1] +).
实施例10
Figure PCTCN2018080648-appb-000039
除了将甲胺的甲醇溶液替换成正庚胺外，其余所需原料、试剂及制备方法同 实施例6，得到Ⅲ-5。
1H NMR(400MHz,CDCl 3):δ6.15(s,1H),4.07(s,2H)3.0-3.1(m,1H),2.97(t,J＝7.7Hz,2H),2.38(s,3H),1.65-1.95(m,7H),1.10-1.45(m,11H),0.75-0.95(m,5H)；MS(ES+)m/z 335.2700([M+1] +).
实施例11
Figure PCTCN2018080648-appb-000040
除了将甲胺的甲醇溶液替换成3-双吖丙啶-6-庚炔胺外，其余所需原料、试剂及制备方法同实施例6，得到Ⅲ-6。
1H NMR(400MHz,CDCl3)δ9.55(s,1H),6.17(s,1H),4.13(s,2H),3.02-3.13(m,1H),2.85(s,2H),2.37(s,3H),2.10-2.25(m,2H),1.95-2.10(m,4H),1.74-1.93(m,4H),1.69(t,J＝7.1Hz,2H),1.52–1.17(m,5H)；MS(ES+)m/z 357.2292([M+1] +).
实施例12
Figure PCTCN2018080648-appb-000041
除了将甲胺的甲醇溶液替换成环戊基胺外，其余所需原料、试剂及制备方法同实施例6，得到Ⅲ-7。
1H NMR(400MHz,CDCl 3):δ5.93(s,1H),3.99(s,2H),3.40-3.60(m,1H),2.90-3.10(m,1H),2.29(s,3H),1.55-2.05(m,9H),1.15-1.47(m,9H)；MS(ES+)m/z305.2224([M+1] +).
实施例13
Figure PCTCN2018080648-appb-000042
除了将甲胺的甲醇溶液替换成苯胺外，其余所需原料、试剂及制备方法同实施例6，得到Ⅲ-8。
1H NMR(400MHz,CDCl 3)δ7.19(t,J＝7.7Hz,2H),7.11(t,J＝7.2Hz,1H),6.76(d,J＝7.5Hz,2H),6.01(s,1H),4.29(s,2H),2.90-3.10(m,1H),2.02(d,J＝12.2Hz,2H),1.72-1.92(m,3H),1.15–1.55(m,5H)；MS(ES+)m/z 313.1917([M+1] +).
实施例14
(1)邻碘苯甲酸溶于二氯甲烷，加入两当量的二氯亚砜，两百分之一当量的N,N-二甲基甲酰胺作为催化剂室温下反应三小时，减压蒸馏除去溶剂制得邻碘苯甲酰氯粗品。邻碘苯甲酰氯和等当量的三乙胺同时加入到甲醇中室温反应20分钟得到邻碘苯甲酸甲酯。
(2)将邻碘苯甲酸甲酯和等当量的环己基乙炔溶于甲苯和三乙胺的混合物(5:1)中，氩气鼓泡十分钟，然后加入百分之一的碘化亚铜和百分之一的PdCl 2(PPh 3) 2作为催化剂，氩气保护下40摄氏度反应4.5h，蒸发溶剂，柱层析得到中间体IV-M。
(3)将IV-M和两当量的盐酸羟胺，四当量的氢氧化钾溶于甲醇中，室温反应过夜。缓缓加入稀盐酸调节pH到3，用二氯甲烷萃取水相，干燥浓缩有机相得到粗产品。粗产品用甲醇：二氯甲烷＝1:20的流通相柱层析得到Ⅳ-1。
Figure PCTCN2018080648-appb-000043
1H NMR(400MHz,CDCl 3)δ8.35(d,J＝8.2Hz,1H),7.63(t,J＝7.4Hz,1H),7.56(d,J＝8.1Hz,1H),7.47(t,J＝7.2Hz,1H),6.45(s,1H),3.16–3.00(m,1H),2.16(d,J＝11.5Hz,2H),1.90(d,J＝11.4Hz,2H),1.81(d,J＝13.5Hz,1H),1.61–1.16(m,5H)；MS(EI)m/z 243.1260(M +).
实施例15
Figure PCTCN2018080648-appb-000044
I-1的乙醇胺盐
将1g I-1溶解于4ml的六十摄氏度的乙酸乙酯中，再加入等当量的乙醇胺。冷却过滤，滤饼用少量冷的乙酸乙酯洗涤两次，干燥除去溶剂残留，即得到I-1的乙 醇胺盐。
1H NMR(400MHz,CDCl 3):δ6.35(d,J＝1.9Hz,1H),5.99(d,J＝2.1Hz,1H),3.62(t,J＝4.0Hz,3H),3.1-3.45(br s,4H),2.95-3.05(dd,J＝9.0,5.6Hz,1H),2.87.(t,J＝4.0Hz,3H),2.47(m,1H),2.37(m,1H),2.20(s,3H),1.88(ddd,J＝12.0,9.0,2.5Hz,1H),1.45-1.72(m,3H),1.20-1.45(m,4H).
实施例16
化合物对氧糖剥夺的SH-SY5Y(人神经母细胞瘤细胞)细胞的改善作用
SH-SY5Y是人神经母细胞瘤细胞，它具有神经细胞的特性。氧糖剥夺(oxygen glucose deprivation，OGD)模型即对培养的细胞剥夺氧气，葡萄糖及血清剥夺的处理，一段时间后再恢复正常的培养条件，继续培养24小时后对细胞活力进行检测。该模型模拟临床上脑缺血性再灌注损伤，采用噻唑蓝(MTT)比色法检测氧糖剥夺对SH-SY5Y细胞活性的影响，初步评价化合物是否具有神经保护作用，尤其是对缺血诱发损伤的改善作用。
实验步骤：
待测化合物溶于DMSO，配成10mM母液，-20℃保存，使用时现用现稀释。SH-SY5Y细胞在含10％胎牛血清，100U/ml青霉素和100U/ml链霉素的MEM/F12培养基中传代培养。细胞经0.25％胰蛋白酶消化后，悬于含10％胎牛血清的MEM/F12培养液中。以3.5×10 5cells/mL的密度将SH-SY5Y细胞接种于96孔培养板上，接种体积为100μL/孔，置于含5％CO 2的37℃恒温培养箱内培养。SH-SY5Y细胞培养24h后，半换液一次，继续培养24h后进行实验。将氧糖缺乏损伤组及给药组均用无糖EBSS平衡盐溶液润洗一遍细胞，随后换成DMEM(无糖)培养液，在给药组中加入相应浓度的待测化合物，氧糖缺乏组加入相应的溶剂对照，置于厌氧仪(含85％N 2，10％H 2，5％CO 2)中培养2h。正常对照组换成含糖及血清的DMEM培养液，放入含5％CO 2的37℃恒温培养箱中培养相同时间。2h后将培氧板从厌氧仪中取出，氧糖缺乏损伤组及给药组补回血清及葡萄糖，葡萄糖的终浓度为1g/L。继续培养24h后，加入5mg/mL MTT(100μL/well)，进行活细胞染色。孵育3h后，弃去培养液，加入DMSO(100μL/well)，并在摇板机上振摇使之充分溶解。490nm的波长下测定各组的OD值。根据下式计算给药后对氧糖剥夺诱发的神经细胞存活率：
存活率＝OD给药组/OD正常组×100％
表1 N-OH吡啶酮类化合物在OGD模型中的药效评价结果
Figure PCTCN2018080648-appb-000045
Figure PCTCN2018080648-appb-000046
正常对照组细胞活力设为100％，数据显示为相比于正常对照组的百分比，为三次独立试验的平均值。试验化合物分两组进行试验，组1的模型组(OGD)细胞活力数值为48.56％，组2的模型组(OGD)细胞活力数值为54.92％。 *p<0.05， **p<0.01， ***p<0.001与模型组(OGD)相比。
表1实验结果表明，前述实施例中的化合物均表现出对OGD损伤的神经细胞的保护活性，取其中活性较优者进行进一步的量效关系研究。
表2 N-羟基吡啶酮类化合物在OGD模型中的浓度-药效评价结果
Figure PCTCN2018080648-appb-000047
Figure PCTCN2018080648-appb-000048
正常对照组细胞活力设为100％，数据显示为相比于正常对照组的百分比，为三次独立试验的平均值。试模型组(OGD组)细胞活力数值为54.23％。 *p<0.05， **p<0.01， ***p<0.001与模型组(OGD组)相比。
表2试验结果表明，复测的六个化合物中，I-1在起效剂量和活性上优于IV-1，II-2，II-3，III7和III5。
实施例17
本发明所涉及化合物的大鼠中动脉栓塞(MCAO，middle cerebral artery occlusion)再灌注(reperfusion)模型药效评价结果：
大脑中动脉(MCA,middle cerebral artery)是人群脑卒中的多发部位，因此阻断MCA所造成的局灶性脑缺血模型是目前应用广泛的动物模型，应用此模型所记录的神经行为学评分和TTC(2、3、5-Triphenyte-trazoliumchloride，2，3，5－三苯基氯化四氮唑)染色，分别能反映脑缺血所引发的神经行为学损伤以及脑缺血区梗死情况，通过评价前两项指标即可反映化合物对脑缺血的保护作用。因此采用该方法对实施例16中测定活性较优的I-1的乙醇胺盐进行体内药效评价。短暂性大脑中动脉栓塞模型主要依据Longa,et al；Stroke，1989所描述的方法进行。大鼠经水合氯醛麻醉(400mg/Kg,i.p.)，行颈部正中切口，分离组织，暴露右侧颈内动脉与颈外动脉分叉处，颈总动脉上细线作活结，结扎颈外动脉，并剪斜切口，将头端烫圆的栓线插入，经颈外动脉进入颈内动脉，沿颈内动脉推进至有阻力感即停止(从颈外动脉与颈内动脉分叉处算起约插入20-22mm)。剪断颈外动脉，放开颈总动脉的动脉夹，缺血2h后退出栓线即为再灌注，24小时后开展行为学评分以及2，3，5－三苯基氯化四氮唑染色。结果如附图1所示。
附图1中TTC染色结果显示：正常脑组织染色鲜红色，梗死部分组织染成白色，与假手术组相比，溶剂对照组损伤侧半脑发生明显梗死(图A)，化合物I-1乙醇胺盐在剂量为0.3mg/kg和1mg/kg时，可以明显降低MCAO动物脑梗死面积体积(图B)，并且1mg/kg I-1可以明显改善大鼠神经行为学功能(图C)。I-1在剂量为1mg/kg时,，实验动物的脑梗死面积和神经学评分较之现有药物依达拉奉10mg/kg给药组更低(图B和C)，说明本发明涉及到的化合物I-1在起效剂量和药效上与现有药物依达拉奉相比，具有一定优势。
本发明提供的新型N-羟基吡啶酮类化合物分子结构较为简单，制备工艺简洁，在SH-SY5Y细胞氧糖剥夺模型实验中显示出较强的神经保护活性，因此有望开发成治疗脑卒中的药物。本发明同时涉及了1-羟基-4-甲基-6-(2-双环[2,2,1]庚烷)-2- 吡啶酮用于治疗脑卒中的新用途，在大鼠短暂性MCAO模型实验中，1-羟基-4-甲基-6-(2-双环[2,2,1]庚烷)-2-吡啶酮能够明显减小脑梗死面积，改善大鼠神经行为学功能，这说明该化合物有望开发成治疗脑卒中的药物。
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. 式I所示的化合物，其立体异构体，药学上可接受的盐，或它们的混合物的用途，其特征在于，用于制备治疗脑卒中的药物，
    Figure PCTCN2018080648-appb-100001
    式中，R 1、R 2各自独立为氢、卤素、C 1-C 8烷基、C 3-8环烷基、C 2-8炔基、C 2- 8烯基、苄基或C 6-10芳基；
    R 3为C 3-C 8的单环环烷基；R为溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；或者R 3为C7-9桥环烷基、H、卤素、C 1-8烷基、C2-8炔基或C2-8烯基，R为氢、溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；
    其中n为1、2、3或4，R 4、R 5独立为氢、C 1-C 10烷基、C 3-C 8的单环环烷基、C 2-8烯基、C 2-8炔基、C 6-10芳基或双吖丙啶取代的C 5-8炔基；
    或者R、R 1与相连的C共同形成取代或未取代的C 6-10芳基，该芳基上的取代基为H、卤素、C 1-8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基或苄基。
  2. 如权利要求1所述的用途，其特征在于，所述治疗是指抑制神经细胞损伤或抑制神经细胞死亡。
  3. 如权利要求1所述的用途，其特征在于，所述的化合物为1-羟基-4-甲基-6-(2-双环[2,2,1]庚烷)-2-吡啶酮：
    Figure PCTCN2018080648-appb-100002
    所述的立体异构体为内构型或外构型；
    Figure PCTCN2018080648-appb-100003
    所述的混合物为内构型和外构型的混合物。
  4. 如权利要求1所述的用途，其特征在于，所述化合物具有以下一个或多个特征：
    (1)R 1、R 2各自独立为氢、卤素、C 1-C 6烷基、C 3-6环烷烃、C 2-5炔烃、C 2-5烯烃、苄基或苯基；
    (2)R为溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；其中n为1、2、3或4，R 4、R 5独立为氢、C 1-C 8烷基、C 3-C 6的单环环烷基、C 2-5烯基、C 2-5炔基、苯基或双吖丙啶取代的C 5-8炔基；
    或者R、R 1与相连的C共同形成苯基；
    (3)C 7-9桥环烷基选自：双环[2,2,1]庚烷基、双环[2,2,2]辛烷基、双环[4，1，1]辛烷基、双环[3,2,1]辛烷基、双环[3,2,2]壬烷基和双环[3,3,1]壬烷基；
    (4)所述药学上可接受的盐为式I所示的化合物的碱土金属、碱金属、铝、过渡金属盐，或者是式I所示的化合物与有机碱形成的盐，所述有机碱选自：乙醇胺、二乙醇胺、N-乙基乙醇胺，N-甲基乙醇胺、三乙醇胺、二乙氨基乙醇、2-氨基-2-甲基-正丙醇、二甲氨基异丙醇、叔丁胺、三异丙醇胺、乙二胺、、吗啉、哌啶、哌嗪、环已胺、三丁胺、十二胺、二甲基十二胺、三乙胺苄胺、二苄胺、N-甲基哌嗪、4-甲基环己胺、N-甲基吗啉、甲胺和乙胺。
  5. 如权利要求1所述的用途，其特征在于，所述化合物为：
    Figure PCTCN2018080648-appb-100004
    Figure PCTCN2018080648-appb-100005
  6. 式I所示的化合物、其立体异构体、药学上可接受的盐或它们的混合物，
    Figure PCTCN2018080648-appb-100006
    式中，R 3为C 3-C 8的单环环烷基、C 7-9桥环烷基、H、卤素、C 1-8烷基、C2-8炔基、C2-8烯基或苄基；
    R 1、R 2各自独立为氢、卤素、C 1-C 8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基、苄基或C 6-10芳基；
    R为溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；其中n为1、2、3或4，R 4、R 5独立为氢、C 1-C 10烷基、C 3-C 8的单环环烷基、C 2-8烯基、C 2-8炔基、C 6-10芳基或双吖丙啶取代的C 5-8炔基；
    或者R、R 1与相连的C共同形成取代或未取代的C 6-10芳基，该芳基上的取代基为H、卤素、C 1-8烷基、C 3-8环烷基、C 2-8炔基、C 2-8烯基或苄基。
  7. 如权利要求6所述的化合物，其特征在于，所述化合物具有以下一个或多个特征：
    (1)R 1、R 2各自独立为氢、卤素、C 1-C 6烷基、C 3-6环烷烃、C 2-5炔烃、C 2-5烯烃、苄基或苯基；
    (2)R为溴、碘、-C(＝O)H或-(CH 2) nNR 4R 5；其中n为1、2、3或4，R 4、R 5独立为氢、C 1-C 8烷基、C 3-C 6的单环环烷基、C 2-5烯基、C 2-5炔基、苯基或双吖丙啶取代的C 5-8炔基；
    或者R、R 1与相连的C共同形成苯基；
    (3)C 7-9桥环烷基选自：双环[2,2,1]庚烷基、双环[2,2,2]辛烷基、双环[4，1，1]辛烷基、双环[3,2,1]辛烷基、双环[3,2,2]壬烷基和双环[3,3,1]壬烷基；
    (4)所述药学上可接受的盐为式I所示的化合物的碱土金属、碱金属、铝、过渡金属盐，或者是式I所示的化合物与有机碱形成的盐，所述有机碱选自：乙醇胺、二乙醇胺、N-乙基乙醇胺，N-甲基乙醇胺、三乙醇胺、二乙氨基乙醇、2-氨基-2-甲基-正丙醇、二甲氨基异丙醇、叔丁胺、三异丙醇胺、乙二胺、、吗啉、 哌啶、哌嗪、环已胺、三丁胺、十二胺、二甲基十二胺、三乙胺苄胺、二苄胺、N-甲基哌嗪、4-甲基环己胺、N-甲基吗啉、甲胺和乙胺。
  8. 如权利要求6所述的化合物，其特征在于，所述化合物为：
    Figure PCTCN2018080648-appb-100007
  9. 一种药物组合物，其特征在于，所述药物组合物包含：
    权利要求6-8任一项所述的化合物、其立体异构体、药学上可接受的盐或者它们的混合物；和
    药学上可接受的载体。
  10. 一种预防和/或治疗脑卒中的方法，其特征在于，向有需要的对象施用权利要求6-8任一项所述的化合物、其立体异构体、药学上可接受的盐或者它们的混合物。
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Citations (3)

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Publication number Priority date Publication date Assignee Title
US4957730A (en) * 1985-12-19 1990-09-18 Hoechst Aktiengesellschaft Antimycotic nail varnish
US20030012749A1 (en) * 2000-01-03 2003-01-16 Karl Kraemer Preparations for the non-traumatic excision of a nail
CN1480132A (zh) * 2002-05-17 2004-03-10 �����ġ����ղ�ʿ��ѧ���޹�˾ 防止局部缺血损伤的保护溶液

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US5846984A (en) * 1996-01-19 1998-12-08 The Trustees Of Columbia University In The City Of New York Use of ciclopirox or a pharmaceutically acceptable salt thereof for inhibiting neuronal cell damage or neuronal cell death
WO2015195474A1 (en) * 2014-06-18 2015-12-23 Biotheryx, Inc. Hydroxypyridone derivatives, pharmaceutical compositions thereof, and their therapeutic use for treating inflammatory, neurodegenerative, or immune-mediated diseases

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Publication number Priority date Publication date Assignee Title
US4957730A (en) * 1985-12-19 1990-09-18 Hoechst Aktiengesellschaft Antimycotic nail varnish
US20030012749A1 (en) * 2000-01-03 2003-01-16 Karl Kraemer Preparations for the non-traumatic excision of a nail
CN1480132A (zh) * 2002-05-17 2004-03-10 �����ġ����ղ�ʿ��ѧ���޹�˾ 防止局部缺血损伤的保护溶液

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