WO2020038279A1 - 取代吡唑类化合物、其制备方法、药物组合物及用途 - Google Patents

取代吡唑类化合物、其制备方法、药物组合物及用途 Download PDF

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WO2020038279A1
WO2020038279A1 PCT/CN2019/100835 CN2019100835W WO2020038279A1 WO 2020038279 A1 WO2020038279 A1 WO 2020038279A1 CN 2019100835 W CN2019100835 W CN 2019100835W WO 2020038279 A1 WO2020038279 A1 WO 2020038279A1
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formula
group
compound
preparation
optionally
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PCT/CN2019/100835
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French (fr)
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韩建斌
温翔
李泽东
杨金娜
杨柳
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天津谷堆生物医药科技有限公司
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Priority to JP2021507845A priority Critical patent/JP7474744B2/ja
Priority to US17/267,399 priority patent/US11692004B2/en
Publication of WO2020038279A1 publication Critical patent/WO2020038279A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/02—Heterocyclic radicals containing only nitrogen as ring hetero atoms
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/18—One oxygen or sulfur atom
    • C07D231/20—One oxygen atom attached in position 3 or 5
    • C07D231/22—One oxygen atom attached in position 3 or 5 with aryl radicals attached to ring nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/18—One oxygen or sulfur atom
    • C07D231/20—One oxygen atom attached in position 3 or 5
    • C07D231/22—One oxygen atom attached in position 3 or 5 with aryl radicals attached to ring nitrogen atoms
    • C07D231/26—1-Phenyl-3-methyl-5- pyrazolones, unsubstituted or substituted on the phenyl ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives

Definitions

  • the invention relates to a substituted pyrazole compound, a preparation method thereof, a pharmaceutical composition and an application thereof.
  • Cerebrovascular disease is known for its high incidence, disability, high mortality and recurrence.
  • China is a country with a high incidence of cerebrovascular diseases, and stroke (or cerebral infarction or stroke) is the most important clinical cerebrovascular disease.
  • stroke or cerebral infarction or stroke
  • "China Stroke Prevention Report-2015" statistics show that there are as many as 7 million stroke patients in China, and more than 1.3 million patients die each year due to stroke diseases, which has become the number one cause of death in China.
  • the number of new stroke patients in China exceeds 2 million every year, which continues to increase at a rate of nearly 9% per year.
  • the incidence of stroke among men in China is the third in the world, and the incidence of stroke among women has risen to the global level.
  • the pathological mechanism of stroke is a disease that causes brain tissue damage due to the sudden rupture of blood vessels in the brain or the inability of blood to flow into the brain due to vascular occlusion, including ischemic and hemorrhagic stroke. Stroke disease not only has a high mortality rate, but is also the leading cause of disability in Chinese adults.
  • the pathological mechanism that causes the sequelae of stroke and post-treatment disability is the generation of a large number of reactive oxygen species and free radicals around brain lesions during blood reperfusion after cerebral ischemia. These free radicals can pass through cell membrane damage, protein damage, and nucleic acid / DNA.
  • ALS amyotrophic lateral sclerosis
  • SOD Superoxide Dismutase
  • Mitochondrial encephalomyopathy is a type of disease caused by abnormal mitochondrial structure caused by deletion or mutation of mitochondrial gene or nuclear gene, which causes abnormal brain and striated muscle function.
  • the disease is clinically similar to epilepsy, cerebral infarction, encephalitis, and brain dysplasia, and is a type of disease that seriously affects the health and life of patients.
  • mitochondria are one of the most sensitive organelles to various types of damage, pathological studies have shown that it is attacked by oxygen free radicals, which causes dysfunction and affects the central nervous system to form mitochondrial encephalomyopathy. Progress, Northwest Journal of National Defense Medicine, Vol. 32, No. 2, 2011).
  • the inventors have carefully developed a new type of substituted pyrazole compound that can effectively prevent and / or treat nerve cell damage.
  • the present inventors provide a substituted pyrazole compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof,
  • R 1 represents hydrogen or alkyl
  • X represents -O-, -S- or -NH-;
  • R 2 represents H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted non-aromatic heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl Alkyl, or optionally substituted heteroaralkyl,
  • the "optionally substituted” means unsubstituted or substituted with one or more substituents selected from the group consisting of hydroxyl, alkoxy, aryloxy, arylalkoxy, halogen, alkanoyloxy , Alkoxyacyloxy, unsubstituted or alkyl substituted amino;
  • n is 1 or 2
  • m is 1, 2, 3, or 4
  • Each R 3 is independently of each other a hydroxyl group, a methylol group, an alkanoyloxy group (preferably acetoxy group), a benzoyloxy group (preferably benzoyloxy group, p-chlorobenzoyloxy group) optionally substituted with halogen ), Alkanoyloxymethyl (preferably acetoxymethyl), benzoyloxymethyl (preferably benzoyloxymethyl, p-chlorobenzoyloxymethyl) optionally substituted with halogen , Alkoxy, alkoxymethyl, or unsubstituted or substituted with alkyl, alkanoyl (preferably acetyl), benzoyl (preferably benzoyl, p-chlorobenzoyl) optionally substituted with halogen Group, mono- or di-substituted amino.
  • alkyl and “aralkyl”, “heteroaralkyl”, “alkoxy”, “arylalkoxy”, “alkanoyloxy”, “alkoxyacyl” are each independently a C 1-20 straight or branched alkyl group, optionally, a C 1-17 straight or branched alkyl group, Optionally, C 1-8 straight or branched alkyl, optionally, C 1-6 straight or branched alkyl, optionally, C 1-4 straight or branched alkyl , Optionally, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl Base, n-hexyl, isohexyl, hept
  • cycloalkyl is a C 3-8 cycloalkyl, optionally, a cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
  • non-aromatic heterocyclic group is a non-aromatic C 3-8 heterocyclic group containing 1-2 heteroatoms selected from O, N, and S, and optionally, is an oxiranyl group , Oxetanyl, tetrahydrofuryl, tetrahydropyrrolyl, tetrahydropyranyl, piperidinyl, piperazinyl, or morpholinyl;
  • the aryl group in the "aryl group” or “aralkyl group” is a phenyl group or a naphthyl group;
  • heteroaryl moieties in the "heteroaryl” and “heteroarylalkyl” are each independently a 5-10 membered single containing 1-2 heteroatoms selected from O, N, and S
  • the cyclic or bicyclic fused aromatic heterocyclic group is optionally a pyrrolyl, furyl, pyridyl, pyrazinyl, or pyrimidinyl group.
  • R 1 represents hydrogen or methyl
  • X represents -O- or -NH-
  • R 2 is C 1 -C 17 branched or straight alkyl group attached, C 3 - 6 cycloalkyl, phenyl C 1 -C 6 alkyl, pyridyl optionally substituted with one or more substituents selected from hydroxy, C 1-6 alkanoyloxy, C 1-6 alkoxyloyloxy or -NR′R ′′ -substituted phenyl groups, wherein R′R ′′ are each independently C 1 -C 6 straight or branched Alkyl.
  • n 1 or 2
  • m 2 or 4
  • Each R 3 is independently of each other a hydroxy group, a hydroxymethyl group, or an unsubstituted or mono-substituted amino group with a C 1-6 alkanoyl group.
  • the substituted pyrazole compound represented by the formula (I) is the following formula II, III or IV,
  • R 2 is defined as described above.
  • R ′ 1 is a hydrogen atom, a hydroxymethyl group, and a C 1-4 alkanoyloxymethyl group
  • R ′ 2 is a hydroxy group or a C 1-4 alkanoyloxymethyl group
  • R ′ 3 is a hydrogen group, a hydroxy group, or C 1- 4 alkanoyloxy, amino, or C 1-4 alkanoylamino.
  • the compound represented by the formula (I) is selected from the following compounds:
  • the substituted pyrazole compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof may be in various optical isomer forms.
  • a glycoside compound it may be in an ⁇ configuration or a ⁇ configuration.
  • the aforementioned compounds 33-40 may be in an ⁇ configuration or a ⁇ configuration.
  • the present inventors provide a method for preparing a substituted pyrazole compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof, which comprises combining a compound of the formula (A) with edara Following the steps of reaction under alkaline conditions,
  • R 1 , X, A, R 2 , Is as defined above, Y is halogen, preferably Cl or Br.
  • the compound of formula (I) is a compound represented by formula (II), and the synthetic route of the preparation method is shown below,
  • the preparation method includes:
  • the preparation method of the formula (C) includes reacting a compound of the formula (D) with dichlorosulfoxide;
  • the compound of formula (I) is a compound represented by formula (III), and the synthetic route of the preparation method is shown below,
  • the preparation method includes:
  • the compound of formula (I) is a compound represented by formula (IV).
  • the synthetic route of the preparation method is shown below.
  • the preparation method includes:
  • the compound of formula (I) is a compound represented by the following formula (V).
  • the synthetic route of the preparation method is shown below.
  • the preparation method comprises reacting a compound of formula (M) with edaravone under basic conditions to obtain a compound of formula (V);
  • R 2 , R 3 , m, and n are as defined above.
  • the various optical isomer forms of the substituted pyrazole compounds represented by the above formula (I) or their pharmaceutically acceptable salts, or their solvates can be synthesized by different methods, or isolated obtain.
  • the ⁇ configuration and ⁇ configuration of the above compounds, such as the ⁇ configuration or ⁇ configuration of compounds 33, 34, 35, 36, 37, 38, 39, 40, can be prepared by different synthetic methods, or by separation obtain.
  • the present inventor provides a pharmaceutical composition
  • a pharmaceutical composition comprising one of the substituted pyrazole compounds represented by the above formula (I) or a pharmaceutically acceptable salt thereof, or one of their solvates Or more and optionally a pharmaceutically acceptable carrier.
  • the administration methods of the substituted pyrazole compounds represented by the above formula (I) or their pharmaceutically acceptable salts, or their solvates include: parenteral administration, oral administration, spraying, etc., at least one of which can be used alone Such compounds may be mixed with pharmaceutically acceptable carriers, adjuvants, vehicles, excipients, diluents and the like.
  • the dosage form of the pharmaceutical composition may be, for example, injections, capsules, powders, granules, tablets, pills, syrups, emulsions, suspensions, solutions, etc., and can be formulated according to conventional methods.
  • Parenteral administration includes subcutaneous, intravenous, intramuscular, intraperitoneal or infusion.
  • Injectable preparations such as sterile injectable water or oil suspensions, may be formulated according to known methods with suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or sterile lyophilized powder injection, in which a parenteral non-toxic diluent or solvent such as an aqueous solution is used.
  • Suitable carriers or solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile non-volatile oil can also be used as a solvent or suspension medium. Different types of non-volatile oils or fatty acids can be used for this purpose, including natural, synthetic or semi-synthetic fatty oils or acids, and natural, synthetic or semi-synthetic mono-, di- or triglycerides.
  • Oral solid dosage forms include the aforementioned powders, granules, tablets, pills and capsules.
  • the active compound can be mixed with at least one additive such as sucrose, lactose, cellulose, mannitol, maltose, dextrin, starch, agar, alginate, chitin, chitosan, fruit Gum, gelatin, gelatin, collagen, casein, albumin, and synthetic or semi-synthetic polymers or glycerides.
  • These dosage forms may also include substances other than inert carriers, such as lubricants such as magnesium stearate, preservatives such as parabens and sorbitol, antioxidants such as ascorbic acid, tocopherol, and cysteine, disintegrating Agents, adhesives, thickeners, buffers, sweeteners, flavors and flavoring agents. Tablets and pills can be prepared by additional coating.
  • inert carriers such as lubricants such as magnesium stearate, preservatives such as parabens and sorbitol, antioxidants such as ascorbic acid, tocopherol, and cysteine
  • disintegrating Agents such as as ascorbic acid, tocopherol, and cysteine
  • adhesives such as thickeners, buffers, sweeteners, flavors and flavoring agents.
  • Tablets and pills can be prepared by additional coating.
  • Oral liquid dosage forms include pharmaceutical emulsions, syrups, suspensions, solutions, and contain diluents such as water commonly used in the prior art.
  • the present inventors provide the substituted pyrazole compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof, or the use of the above pharmaceutical composition for the preparation of prevention or treatment.
  • the present inventors provide the substituted pyrazole compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof, or the use of the above pharmaceutical composition for preventing or treating the brain Stroke, cerebral embolism, stroke sequelae, stroke dysfunction, mitochondrial encephalomyopathy, and / or amyotrophic lateral sclerosis.
  • the specific dose for any particular patient depends on a variety of factors, including the activity of the compound used, age, weight, general health, sex, diet, time of administration, mode of administration, rate of secretion, combination of drugs, and the specific condition.
  • the dosage varies with the disease, symptom, subject and the route of treatment.
  • the daily dose is 1-300mg or intravenously 1-100mg, divided into 2-3 times.
  • the active ingredient is used in an appropriate amount, such as about 10-200 mg / time orally, or about 5-100 mg / time by intravenous injection, in two or three equal daily doses.
  • substituted pyrazole compounds represented by the formula (I) of the present invention or a pharmaceutically acceptable salt thereof, or their solvates can effectively prevent nerve cell damage.
  • the substituted pyrazole compound represented by Formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, which is one aspect, has excellent stability.
  • the substituted pyrazole-based compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof has excellent water solubility.
  • the substituted pyrazole compound represented by the formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof has low cytotoxicity.
  • the substituted pyrazole compound or a pharmaceutically acceptable salt thereof or a solvate thereof represented by the formula (I) has a significant neuroprotective effect and can be effectively used for the prevention or treatment of stroke, Cerebral embolism, sequelae of stroke, dyskinesia of stroke, mitochondrial encephalomyopathy, and / or posterior amyotrophic lateral sclerosis.
  • FIG. 1 is the fall latency-time curve of the ALS mice in each group during the wand test.
  • FIG. 2 is the fall latency-time curve of the suspension experiment of ALS mice in each group.
  • MCAO focal cerebral ischemia-reperfusion injury model
  • ALS Amyotrophic Lateral Sclerosis
  • the starting acid chloride can be prepared from the corresponding carboxylic acid, the route is as follows:
  • Step 1 Dissolve anhydrous acetaldehyde and catalytic amount of anhydrous zinc chloride in dry dichloromethane (10mL) at 0 ° C, and then slowly add compound (C). After the dropwise addition is completed, heat to The reaction was performed at 50 ° C for 2.5 hours. After the reaction was completed, 100 mL of dichloromethane was added to the reaction solution, followed by washing with a saturated aqueous sodium hydrogen carbonate solution (1 ⁇ 50 mL). The aqueous phase was extracted with dichloromethane (2 ⁇ 25 mL), and the organic phases were combined. The organic phase was washed successively with distilled water and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography to obtain compound (B).
  • Step 2 Dissolve edaravone (3.6 mmol), potassium iodide (0.4 mmol), and potassium carbonate (10 mmol) in dry DMF (10 mL) and stir at 45 ° C for 10 min. Then, compound (B) (3.3 mmol) dissolved in dry DMF was added, and stirred at 45 ° C. for 1 h. After the reaction was completed, most of the solvent was pumped off with an oil pump, and then 100 mL of ethyl acetate was added to the reaction solution, followed by washing with a saturated ammonium chloride aqueous solution. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined.
  • the organic phase was sequentially washed with distilled water, a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained crude product was separated by semi-preparative high-pressure liquid chromatography and lyophilized using a freeze dryer to obtain the target product compound ( II).
  • the aqueous phase was extracted with ethyl acetate (or dichloromethane), and the organic phases were combined.
  • the organic phase was sequentially washed with distilled water, a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the target product compound (III).
  • Example 19 The product of Example 19 (0.50 g) was dissolved in dry dichloromethane (5 mL) at room temperature, dry triethylamine (0.47 mL) was added, and then ethyl chloroformate (0.35 mL) was slowly added, followed by stirring at room temperature. 2.5h. After the reaction was completed, 100 mL of dichloromethane was added to the reaction solution, followed by washing with a saturated aqueous ammonium chloride solution (1 ⁇ 50 mL). The aqueous phase was extracted with dichloromethane (2 ⁇ 25 mL), and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained oily liquid was purified by silica gel column chromatography (petroleum ether / Ethyl acetate: 3/1), 0.54 g of a pale yellow oily liquid was obtained with a yield of 88.14%.
  • Example 19 The product of Example 19 (0.50 g) was dissolved in dry dichloromethane (5 mL) at room temperature, and dry triethylamine (0.39 mL) was added. Then isobutyryl chloride (0.32 mL) was slowly added, and the mixture was stirred at room temperature for 2.5 h. After the reaction was completed, 100 mL of dichloromethane was added to the reaction solution, followed by washing with a saturated aqueous ammonium chloride solution. The aqueous phase was extracted with dichloromethane, and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained oily liquid was purified by silica gel column chromatography (petroleum ether / ethyl acetate: 3 / 1) 0.48 g of a pale yellow oily liquid was obtained with a yield of 79.48%.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / acetic acid) Ethyl ester: 15/1) to obtain 2.84 g of the product with a yield of 45.55%.
  • the aqueous phase was extracted with dichloromethane (2 ⁇ 25 mL), and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous sodium hydrogen carbonate solution, distilled water, and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained oily liquid was purified by silica gel column chromatography (petroleum ether). / Ethyl acetate: 15/1), 0.55 g of a pale yellow solid was obtained with a yield of 46.31%.
  • Edaravone (0.24 g), potassium carbonate (0.71 g), and potassium iodide (0.34 g) were dissolved in dry DMF (6.00 mL) and stirred at 50 ° C for 20 min.
  • Chloromethyl- (2-benzyloxy-3-N, N-dimethyl) benzoate (0.55g) was dissolved in dry DMF (2.50mL) and added to the above solution, 45 ° C Stir for 3 h. After the reaction was completed, 100 mL of ethyl acetate was added to the reaction solution, followed by washing with a saturated aqueous ammonium chloride solution (1 ⁇ 50 mL).
  • the aqueous phase was extracted with ethyl acetate (2 ⁇ 25 mL), and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained oily liquid was purified by silica gel column chromatography (petroleum ether). / Ethyl acetate: 15/1) to obtain 0.22 g of the product with a yield of 27.87%.
  • P-aminosalicylic acid (8.0 g) was added to anhydrous methanol (80 mL), dichlorosulfoxide (15 mL) was slowly added at 0 ° C, and the reaction was carried out at 60 ° C for 24 h. After the reaction was completed, the remaining methanol was removed by a rotary evaporator, and the pH was adjusted to neutral with an aqueous solution of sodium bicarbonate. Then, 100 mL of dichloromethane was added to the reaction solution for extraction, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a solid. The product was 7.50 g in a yield of 85.91%.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / acetic acid) Ethyl ester: 15/1) to obtain 2.84 g of the product with a yield of 45.55%.
  • the aqueous phase was extracted with dichloromethane (2 ⁇ 25 mL), and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous sodium hydrogen carbonate solution, distilled water, and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained oily liquid was purified by silica gel column chromatography (petroleum ether / acetic acid) Ethyl ester: 15/1), 0.55 g of a pale yellow solid was obtained with a yield of 46.31%.
  • Edaravone (0.24 g), potassium carbonate (0.71 g), and potassium iodide (0.34 g) were dissolved in dry DMF (6 mL) and stirred at 50 ° C. for 20 min.
  • Chloromethyl- (2-benzyloxy-5-N, N-dimethyl) benzoate (0.55g) was dissolved in dry DMF (2.5mL) and added to the above solution, 45 ° C Stir for 3 h. After the reaction was completed, 100 mL of ethyl acetate was added to the reaction solution, followed by washing with a saturated aqueous ammonium chloride solution (1 ⁇ 50 mL).
  • the aqueous phase was extracted with ethyl acetate (2 ⁇ 25 mL), and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained oily liquid was purified by silica gel column chromatography (petroleum ether / acetic acid) Ethyl ester: 15/1) to obtain 0.22 g of the product with a yield of 27.87%.
  • Example 24 The product of Example 24 (0.1 g) was dissolved in dry dichloromethane (5 mL) at room temperature, and dry triethylamine (0.1 mL) was added, then ethyl chloroformate (0.08 mL) was slowly added, and the mixture was stirred at room temperature. 2.5h. After the reaction was completed, 100 mL of dichloromethane was added to the reaction solution, followed by washing with a saturated aqueous ammonium chloride solution (1 ⁇ 50 mL). The aqueous phase was extracted with dichloromethane (2 ⁇ 25 mL), and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained oily liquid was purified by silica gel column chromatography (petroleum ether / acetic acid) Ethyl ester: 3/1) to obtain 0.11 g of a pale yellow oily liquid with a yield of 85%.
  • glucose (4.5 g) and potassium acetate (4.9 g) without crystal water were dissolved in acetic anhydride (25 mL), and then stirred at 90 ° C. for 4 h. After the reaction was completed, the temperature was lowered to room temperature, and the solvent was removed by an oil pump. 100 mL of ethyl acetate was added to the reaction solution, followed by washing with a saturated aqueous sodium hydrogen carbonate solution, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous sodium hydrogen carbonate solution, distilled water, and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain 6.3 g of the product with a yield of 65%.
  • the organic phase was sequentially washed with an aqueous sodium hydrogen carbonate solution, distilled water, and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate: 3 / 1) to obtain 1.7 g of the product in a yield of 64.6%.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate: 4/1), 0.68 g of product was obtained with a yield of 55.4%.
  • Mannose (4.5 g) and potassium acetate (4.9 g) were dissolved in acetic anhydride (25 mL) at room temperature, and then stirred at 90 ° C. for 4 h. After the reaction was completed, the room temperature was returned, and the solvent was removed by an oil pump. 100 mL of ethyl acetate was added to the reaction solution, followed by washing with a saturated aqueous sodium hydrogen carbonate solution, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined.
  • the organic phase was sequentially washed with a saturated aqueous sodium hydrogen carbonate solution, distilled water, and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and recrystallized with ethanol to obtain 6.3 g of the product in a yield of 63%.
  • the organic phase was sequentially washed with an aqueous sodium hydrogen carbonate solution, distilled water, and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate: 5 / 1) to obtain 1.19 g of the product with a yield of 61.31%.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate: 4/1) to obtain 0.64 g of product with a yield of 52.14%.
  • the organic phase was sequentially washed with a saturated aqueous sodium bicarbonate solution, distilled water, and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and recrystallized with ethanol to obtain 11.0 g of the product with a yield of 62%.
  • the organic phase was sequentially washed with an aqueous sodium hydrogen carbonate solution, distilled water, and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate: 5 / 1) to obtain 1.54 g of the product with a yield of 54.47%.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / Ethyl acetate: 4/1) to obtain 0.99 g of the product in a yield of 75.20%.
  • the organic phase was sequentially washed with an aqueous sodium hydrogen carbonate solution, distilled water, and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the organic phase was sequentially washed with a saturated aqueous ammonium chloride solution, distilled water, and a saturated sodium chloride solution, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate: 4/1), 1.07 g of product was obtained with a yield of 84.27%.
  • Xylose (2g, 13.3mmol) was suspended in pyridine (10mL), acetic anhydride (10mL, 106mmol) was slowly added dropwise at 0 ° C, and the reaction was stirred overnight.
  • Ethyl acetate (100mL) was added and 5% dilute hydrochloric acid was added. It was washed with a saturated aqueous sodium hydrogen carbonate solution, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 4 g of the product, which was directly used in the next step.
  • D-ribose (5g, 33.3mmol) was suspended in methanol (80mL), placed in an ice bath, and concentrated sulfuric acid (0.5mL, 9.4mmol) was slowly added dropwise, and the temperature was raised to 60 ° C to react overnight, and the pH was adjusted to 9 with ammonia water. , Filtered, and the solvent was removed under reduced pressure to obtain 5 g of crude product, which was directly used in the next step.
  • Edaravone (870mg, 5mmol) was suspended in acetonitrile (10mL), cesium carbonate (1.7g, 5.25mmol) was added, and the mixture was stirred at room temperature for 20min. 2,3,4-tribenzoyl-D- Ribose bromide was slowly added dropwise. The reaction solution was stirred for 1 hour. Ethyl acetate and a water layer were added. The organic phase was dried over anhydrous sodium sulfate and concentrated by rotary evaporation under reduced pressure. The residue was subjected to silica gel column chromatography (petroleum ether / acetic acid). Ethyl ester: 5/1) gave 1 g of product as a white solid.
  • Edaravone 500mg, 2.9mmol was suspended in acetonitrile (15mL), cooled to 0 ° C, sodium hydride (115mg, 2.9mmol, 60% inoil) was slowly added, and the mixture was stirred at room temperature for 20 minutes.
  • 3,5 -Di-p-chlorobenzoyloxy-2-D-deoxyribose chloride (1.25g, 2.9mmol), continue to stir for 3h at room temperature, add ethyl acetate and water layer, dry the organic phase with anhydrous sodium sulfate, reduce It was concentrated by rotary evaporation, and the residue was subjected to silica gel column chromatography (petroleum ether / ethyl acetate: 5/1) to obtain 450 mg of a crude product.
  • the compound of formula (H) (5.62 mmol) and KOH (0.66 mmol) were dissolved in a 20 mL H 2 O / dioxane (1: 3 v / v) mixed solution, and formaldehyde (35% aqueous solution, 8.43) was added to the above mixed solution. mmol), the reaction solution was reacted at 70 ° C. for 5 min, and then cooled to 25 ° C. and stirred overnight. The pH was adjusted to 7 with 1N HCl and concentrated under reduced pressure. The residue was dissolved in acetone, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the compound of formula (G), which was directly used in the next step.
  • the compound of formula (G) (3.0 mmol) was dissolved in 10 mL of POCl 3 , heated to reflux, and PCl 5 (6.0 mmol) was carefully added in portions. The reaction was refluxed for 2.5 h, and the reaction was detected by TLC. The temperature was lowered to room temperature, and the reaction solution was poured into 200 mL of ice water. The pH was adjusted to neutral with 1N NaOH solution, extracted with CH 2 Cl 2 , dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the compound of formula (F).
  • the content of edaravone is significantly reduced under light conditions.
  • the compounds disclosed in the patent CN107400089 which is directly coupled to edaravone with an ester bond, are exposed to high humidity (25 °C ⁇ 2 °C, 90% ⁇ 5RH), Decomposition occurs under the test conditions of light (25 °C ⁇ 2 °C, 60% ⁇ 5RH, 4500 ⁇ 500LX) and high temperature (60 °C ⁇ 2 °C, 60% ⁇ 5RH).
  • the above compounds in the present invention are superior to the compounds disclosed in edaravone and CN107400089 under the above standard test conditions for pharmaceutical stability.
  • Test example 2 solubility test
  • PC12 cells were resuscitated, RPMI-1640 medium containing 10% horse serum and 5% fetal bovine serum was cultured at 37 ° C and 5% CO 2 for two weeks, and the solution was changed every other day.
  • the compound was diluted to a solution of 12.5, 25, 50, 100, 200, 300 ⁇ M (the compound of Examples 33-40 was additionally added with a solution of 400, 500 ⁇ M) in the medium, and then used.
  • PC12 cells were seeded in a 96-well plate at a density of 1.2 ⁇ 10 4 cells / well, cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours, and then discarded, and the compound solutions of the above series concentrations were added as test solutions. Incubate at 37 ° C in a 5% CO 2 incubator for 48 h.
  • Compound IC 50 ( ⁇ M) Compound IC 50 ( ⁇ M) 3 > 300 32 > 300 4 > 300 33 > 300 5 > 300 34 > 300 7 > 300 35 > 300 9 > 300 36 > 300 13 251.5 37 > 500 16 233.9 38 > 500 17 > 300 39 > 500
  • All tested samples were prepared into a 60 mM stock solution with DMSO, and then diluted 200-fold with cell culture medium to a final concentration of 300 ⁇ M. The sample solution was then diluted with cell culture medium to different concentration gradients for later use.
  • Control group normal laying cells, just like other test cell groups, only add medium, discard the liquid after 24 hours of culture, and then add only medium to continue incubation for 24 hours without compound and hydrogen peroxide treatment. Other operations are the same as for the sample set.
  • Blank group No cells were laid, only medium was added. Other operations are the same as for the sample set.
  • test sample
  • PC12 cells were resuscitated, RPMI-1640 medium containing 10% horse serum and 5% fetal bovine serum was cultured at 37 ° C and 5% CO 2 for two weeks, and the solution was changed every other day.
  • the medium was diluted to 0, 12.5, 25, 50, 100, 200, 300 ⁇ M solutions of the compound, respectively, and used.
  • PC12 cells were seeded into a 96-well plate at a density of 8000 cells / well, and cultured in a 37 ° C, 5% CO 2 incubator for 24 hours. The solution was discarded, and the compound solutions of the above series concentrations were added as test solutions. Incubation was continued for 24 h in a 5% CO 2 incubator.
  • Test example 5 MCAO model for in vivo drug efficacy test
  • the free end of the external carotid artery was then ligated together with the intraluminal nylon filament.
  • the subcutaneous fascia and skin are sutured layer by layer, and penicillin is injected intramuscularly to prevent infection. Animals in the sham operation group only isolated the internal carotid artery.
  • Animals with successful modeling were selected and randomly grouped by random number table method, with 10 animals in each group. They were the model control group, the test drug group, the edaravone group, and 10 sham operation groups.
  • the drug was administered by tail vein infusion for 30 minutes.
  • the sham operation group and the model control group were given an equal volume of physiological saline solution.
  • the brain was taken and cut into 2 mm thick slices along the coronal plane.
  • the above-mentioned half of the brain slices were placed in 2% TTC staining solution at room temperature and incubated at 37 ° C for 10 minutes in the dark to stain. It is stored in the computer, and the infarcted area and the whole brain area are measured with the image analysis system v4.0 software, and the cerebral infarction area is calculated (the area of the infarcted area as a percentage of the total brain area).
  • the above-mentioned compounds of the present invention can have different therapeutic effects by intravenous infusion and single treatment administration, and the effects are shown in Improve the damaged nerve function to varying degrees, reduce the extent of cerebral infarction and reduce the degree of cerebral edema to varying degrees.
  • Test example 6 In vivo pharmacodynamic test II-ALS model
  • mice B6SJL-Tg (SOD1 * G93A) mice were administered intraperitoneally in a single dose, once a day, for 4 consecutive weeks, and the mice were weighed twice a week before and after administration, and fatigue was used.
  • the motor function of the mice was evaluated by a barometer and a suspension test. Plasma was collected at two time points before and four weeks after the administration, and the plasma SOD level of the mice was measured by ELISA.
  • the pharmacological effect of the compound of the example on the ALS mouse model was evaluated by the above indicators, and the effect of the compound of the example on improving the survival time and motor function of the mice was observed.
  • mice were acclimated for more than a week after they arrived at the SPF animal room. Starting 5 days before the experiment, the rod exercise was performed once a day to make the mice used to rod exercise. On the day of the experiment, mice were randomly divided into groups of 8 mice each. Among them, the solvent group was administered the same volume of solvent (normal saline) daily.
  • the administration volume was 10 mL / kg.
  • the drug was administered intraperitoneally once daily for 4 weeks.
  • Onset time of ALS Observed for 2 consecutive days, if limb tremor and / or limb weakness occur in tail suspension state, it is judged to be onset.
  • Method for judging limb weakness The test rat is placed on a 3.5 cm diameter rod-rotator, the rotation speed is adjusted to 15 rpm, and the maximum incubation period of the fall is recorded. The cutoff value is 420 seconds. Less than 420 seconds is regarded as limb weakness.
  • Time of death of the mouse The mouse is placed in a supine position, and it cannot be turned into a prone position within 20 seconds, which is regarded as death.
  • Plasma SOD concentration At two time points before and four weeks after administration, blood was collected with an EP tube containing heparin, mixed thoroughly, centrifuged at 1000g ⁇ 10min, the plasma was collected, placed in a -70 ° C refrigerator after dispensing. Plasma SOD levels were measured using an ELISA kit.

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Abstract

一种式I所示的取代吡唑类化合物、其制备方法、药物组合物及用途。所述化合物具有良好的稳定性、溶解性优异，对细胞毒性小，对神经保护作用效果显著，能够有效预防和治疗神经细胞损伤，是一种理想的预防或治疗：脑卒中，脑栓塞，脑卒中后遗症，脑卒中运动机能障碍，线粒体脑肌病，肌萎缩性脊髓侧索硬化症的药用化合物。(I)

Description

取代吡唑类化合物、其制备方法、药物组合物及用途 技术领域
本发明涉及一种取代吡唑类化合物、其制备方法、药物组合物及用途。
背景技术
脑血管病以发病率高、致残率高、致死率高和复发率高等四高而著称。我国是一个脑血管病高发国家，其中脑卒中(或称脑梗或脑中风)是最主要的临床脑血管疾病。《中国脑卒中防治报告-2015》统计表明，我国脑卒中患者多达700万人，每年因脑卒中疾病死亡的患者人数超过130万，已经成为我国第一死亡原因。并且，我国每年的脑卒中新增患者超过200万人，以每年接近9％的速度持续增加，目前在全球范围内我国男性脑卒中发病率为全球第三位，女性脑卒中发病率跃升为全球第二位(参考文献：国家卫生计生委防治工程委员会.中国卒中流行报告2015.北京：中国协和医科大学出版社，2015；Prevalence,Incidence and Mortality of Stroke in China:Results from a Nationwide Population-Based Survey of 480,687 Adults.Circulation 2017；135:759-771)。
脑卒中的病理机制是由于脑部血管突然破裂或因血管阻塞导致血液不能流入大脑而引起脑组织损伤的一种疾病，包括缺血性和出血性脑卒中。脑卒中疾病不但死亡率高，而且也是我国成年人残疾的首要原因。其中导致脑中风后遗症和治疗后残疾的病理学机制是脑缺血后血液再灌注时在脑部病灶周围产生大量的活性氧和自由基，这些自由基会通过细胞膜损伤、蛋白质损伤、核酸/DNA损害、以及诱导各种炎症介质的产生等形式，对神经细胞，脑细胞形成非可逆伤害，最终导致神经细胞或脑细胞死亡(参考文献：氧自由基与缺血脑卒中，中国中医药咨讯，2010；(10)210-211；Dynamics of free radical processes in acute ischemic stroke:influence on neurological status and outcome.J.Clin.Neurosci.2004,Jun,11(5)501-506)。因此，针对脑卒中的临床治疗如果只强调静脉或者动脉溶栓治疗，很难彻底解决和防止自由基神经损伤给患者带来的后遗症以及残疾的问题和隐患。
与脑卒中类似，肌萎缩侧索硬化症(ALS)也是一种以神经细胞受损萎缩和死亡为病理特征的疾病。ALS也俗称为渐冻症，虽然其发病机制本身目前尚无定论，但生物学证明该疾病至少与超氧化物歧化酶(Superoxide Dismutase,SOD)有关联。SOD是广泛存在于生物体内的金属酶，其中SOD1通过歧化反应将线粒体内的自由基等有害物质氧化并分解成水，从而起到解毒作用。目前科学界普遍认为，SOD1超氧化物歧化酶的突变，是肌萎缩侧索硬化症形成的原因之一(参考文献：Supportive and symptomatic management of amyotrophic lateral sclerosis.Nature Reviews.Neurology.2016,12(9):526– 38.)。
线粒体脑肌病是由于线粒体基因或细胞核基因缺失或突变导致的线粒体结构异常，引起脑部和横纹肌功能异常的一类疾病。该疾病在临床上与癫痫、脑梗塞、脑炎以及脑发育不良等病症类似，是严重影响患者的健康和生命的一类疾病。由于线粒体是对各种损伤最为敏感的细胞器之一，病理学研究表明它受到氧自由基的攻击而导致功能异常进而累及中枢神经系统从而形成线粒体脑肌病(参考文献：线粒体脑肌病影像研究进展，西北国防医学杂志，2011年第32卷第2期)。
我国国家卫生健康委员会，科学技术部，工业和信息化部，国家药品监督管理局以及国家中医药管理局联合发布的《关于公布第一批罕见病目录的通知》(国卫医发【2018】10号)中明确规定肌萎缩侧索硬化症、线粒体脑肌病为我国罕见病也称孤儿病。作为罕见病，ALS虽然发病率很低，但我国人口基数大，据不完全统计我国目前该疾病的患者人数约为10万-20万人，而且目前能够根治该疾病的药物尚未开发成功。线粒体脑肌病在成人中的发病率约为1/4300左右，在儿童中的发病率则高达1/2000。因此，针对该类罕见疾病开发能够改善患者病情的治疗药物是我国乃至全世界生物医药领域科学家的共同奋斗目标和我国医药卫生领域重大攻关课题之一。(参考文献：《肌萎缩侧索硬化症临床诊断进展》[杨琼樊东升.中国现代神经疾病杂志2012.6第12卷第3期；肌萎缩侧索硬化流行病学研究现状[J].中华神经科杂志,2015,48(6):542-544)
发明内容
本发明人精心开发了一种新型的取代吡唑类化合物，能够有效地预防和/或治疗神经细胞损伤。
为此，本发明人提供一种式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，
Figure PCTCN2019100835-appb-000001
其中：
R 1表示氢或烷基；
X表示-O-、-S-或-NH-；
Figure PCTCN2019100835-appb-000002
表示双键，A为O；
R 2表示H、任选取代的烷基、任选取代的环烷基、任选取代的非芳香杂环基、任选取代的芳基、 任选取代的杂芳基、任选取代的芳烷基、或者任选取代的杂芳烷基，
所述“任选取代的”是指未取代或被一个或多个取代基取代，所述取代基选自羟基、烷氧基、芳氧基、芳基烷氧基、卤素、烷酰氧基、烷氧酰氧基、未取代或烷基取代的氨基；
或者，
Figure PCTCN2019100835-appb-000003
表示单键，式(I)中的下式(i)部分
Figure PCTCN2019100835-appb-000004
形成下式(ii)所示的基团，
Figure PCTCN2019100835-appb-000005
式(ii)中，n为1或者2，m为1，2，3或4，
各个R 3相互独立地为羟基，羟甲基，烷酰氧基(优选乙酰氧基)、任选被卤素取代的苯甲酰氧基(优选苯甲酰氧基、对氯苯甲酰氧基)、烷酰氧基甲基(优选乙酰氧基甲基)、任选被卤素取代的苯甲酰氧基甲基(优选苯甲酰氧基甲基、对氯苯甲酰氧基甲基)、烷氧基、烷氧基甲基、或者未取代或被选自烷基、烷酰基(优选乙酰基)、任选被卤素取代的苯甲酰基(优选苯甲酰基、对氯苯甲酰基)的基团、单取代或二取代的氨基。
可选地，所述“烷基”，以及“芳烷基”、“杂芳烷基”、“烷氧基”、“芳基烷氧基”、“烷酰氧基”、“烷氧酰氧基”、“烷酰氧基甲基”中的烷基部分各自独立地为C 1-20直链或支链烷基，可选地，为C 1-17直链或支链烷基，可选地，为C 1-8直链或支链烷基，可选地，为C 1-6直链或支链烷基，可选地，为C 1-4直链或支链烷基，可选地，为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，异戊基、新戊基、叔戊基、正己基、异己基、庚基、正辛基、正壬基、正癸基、十二烷基、十五烷基、或十六烷基；
可选地，所述“环烷基”为C 3-8环烷基，可选地，为环丙基、环丁基、环戊基、或环己基；
可选地，所述“非芳香杂环基”为含有选自O、N、S的1-2个杂原子的非芳香C 3-8杂环基，可选地，为环氧乙烷基、氧杂环丁烷基、四氢呋喃基、四氢吡咯基、四氢吡喃基、哌啶基、哌嗪基、或吗啉基；
可选地，所述“芳基”、“芳烷基”中的芳基为苯基、或萘基；
可选地，所述“杂芳基”、以及“杂芳烷基”中的杂芳基部分各自独立地为含有选自O、N、S的1-2个杂原子的5-10元单环或双环稠合芳香杂环基团，可选地，为吡咯基、呋喃基、吡啶基、吡嗪基、 或嘧啶基。
可选地，上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物中，
R 1表示氢或甲基；
X表示-O-或-NH-；
Figure PCTCN2019100835-appb-000006
表示双键，A为O；
R 2为C 1-C 17的支链或直连烷基，C 3- 6环烷基，苯基C 1-C 6烷基，吡啶基，任选被一个或多个选自羟基、C 1-6烷酰氧基、C 1-6烷氧酰氧基或-NR′R″的基团取代的苯基，其中R′R″各自独立地为C 1-C 6直连或支链的烷基。
或者，
Figure PCTCN2019100835-appb-000007
表示单键，式(I)中的式(i)部分形成式(ii)所示的基团，
式(ii)中，n为1或者2，m为2，3或4，
各个R 3相互独立地为羟基，羟甲基，或者未取代或被C 1-6烷酰基单取代的氨基。
可选地，上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物中，式(I)所示的取代吡唑类化合物为下式II、III或IV所示的化合物，
Figure PCTCN2019100835-appb-000008
其中，R 2定义如上所述。
可选地，上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物中，式(I)中的式(i)部分形成下式(iii)或(iv)所示的基团，
Figure PCTCN2019100835-appb-000009
其中，R′ 1为氢原子、羟甲基、C 1-4烷酰氧基甲基；R′ 2为羟基或C 1-4烷酰氧基；R′ 3为氢、羟基、C 1-4烷酰氧基、氨基、或C 1-4烷酰氨基。
可选地，上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物中，式(I)中的式(i)部分形成下式所示的基团，
Figure PCTCN2019100835-appb-000010
可选地，上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物中，所述式(I)所示化合物选自以下化合物：
Figure PCTCN2019100835-appb-000011
Figure PCTCN2019100835-appb-000012
Figure PCTCN2019100835-appb-000013
Figure PCTCN2019100835-appb-000014
可选地，上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物可以是各种光学异构体形式。对于糖苷类化合物，可以为α构型或β构型。例如，上述化合物33-40可以为α构型或β构型。
另一方面，本发明人提供上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物的制备方法，包括将式(A)化合物与依达拉奉在碱性条件下反应的步骤，
Figure PCTCN2019100835-appb-000015
式中，R 1、X、A、R 2、
Figure PCTCN2019100835-appb-000016
的定义如上所述，Y为卤素，优选为Cl或Br。
可选地，上述制备方法中，
式(I)化合物为式(II)所示的化合物，所述制备方法的合成路线如下所示，
Figure PCTCN2019100835-appb-000017
所述制备方法包括：
1)将式(C)化合物在催化剂的存在下与乙醛反应，制得式(B)化合物；
2)将式(B)化合物在碱性条件下与依达拉奉反应，制得式(II)化合物；
可选的，所述式(C)的制备方法包括将式(D)化合物与二氯亚砜反应；
Figure PCTCN2019100835-appb-000018
或者，
式(I)化合物为式(III)所示的化合物，所述制备方法的合成路线如下所示，
Figure PCTCN2019100835-appb-000019
所述制备方法包括：
1)将式(D)化合物与氯甲基氯磺酸酯反应，制得式(E)化合物；
2)将式(E)化合物在碱性条件下与依达拉奉反应，制得式(III)化合物；
或者，
式(I)化合物为式(IV)所示的化合物，所述制备方法的合成路线如下所示，
Figure PCTCN2019100835-appb-000020
所述制备方法包括：
1)将式(H)化合物与甲醛反应，制得式(G)化合物；
2)将式(G)化合物与PCl 5/POCl 3反应制得式(F)化合物，
3)将式(F)化合物在碱性条件下与依达拉奉反应，制得式(IV)化合物；或者，
式(I)化合物为下式(V)所示的化合物，所述制备方法的合成路线如下所示，
Figure PCTCN2019100835-appb-000021
式中，Y为Cl或Br，
所述制备方法包括将式(M)化合物在碱性条件下与依达拉奉反应制得式(V)化合物；
在上述结构式中，R 2、R 3、m、n定义如上所述。
可选地，上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物的各种光学异构体形式，可以通过不同的方法合成，或者通过分离获得。对于上述化合物的α构型和β构型，例如化合物33、34、35、36、37、38、39、40的α构型或β构型，可以通过不同的合成方法制得，或者通过分离获得。
另一方面，本发明人提供一种药物组合物，所述药物组合物包括上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物中的一种或多种以及任选的药学上可接受的载体。
上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物的给药方式包括：肠胃外给药、口服、喷吸等，可单独使用至少一种这类化合物，或与药用载体，佐药，媒介物，赋形剂，稀释剂等混用。药物组合物剂型可以为例如注射剂、胶囊、粉剂、粒剂、片剂、丸、糖浆、乳剂、悬浮液、溶液等，可按照常规方法配制。
肠胃外给药包括皮下注射、静脉注射、肌肉注射、腹膜内注射或输液。注射制剂，如无菌注射水或油悬浮液可按照已知方法用适宜分散剂或润湿剂以及悬浮剂配制。无菌注射制剂也可为无菌注射液、悬浮液或无菌冻干粉针，其中用肠胃外无毒稀释剂或溶剂，如水溶液。可用的合适载体或溶剂包括水、林格氏溶液和等渗氯化钠液。另外，无菌不易挥发油也可作为溶剂或悬浮介质。为此可用不同类型的不易挥发油或脂肪酸，包括天然的、合成的或半合成的脂肪油或酸，以及天然的、合成的或半合成的单、双或三甘油酯。
口服固体剂型包括上述粉、粒、片、丸和胶囊。在这些固体剂型中，活性化合物可以混用至少一种添加剂，如蔗糖、乳糖、纤维素、甘露糖醇、麦芽糖、糊精、淀粉、琼脂、藻酸盐、几丁质、脱乙酰壳多糖、果胶、阿胶、明胶、胶原、酪蛋白、清蛋白以及合成或半合成聚合物或甘油酯。这些剂型还可包括惰性载体以外的其他物质，例如润滑剂如硬脂酸镁，防腐剂如苯甲酸酯类(Parabens)和山梨醇，抗氧化剂如抗坏血酸、生育酚和半胱氨酸，崩解剂，粘合剂，增稠剂，缓冲剂，增甜剂，香料和增香剂。片和丸可另外加外涂层而制得。
口服液体剂型包括药用乳剂、糖浆、悬浮剂、溶液，含有现有技术中常用的稀释剂如水。
另一方面，本发明人提供上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，或者上述药物组合物的用途，用于制备预防或治疗脑卒中、脑栓塞、脑卒中后遗症、脑卒中运动机能障碍、线粒体脑肌病、肌萎缩性脊髓侧索硬化症等疾病的药物。
另一方面，本发明人提供上述式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，或者上述药物组合物的用途，用于预防或治疗脑卒中、脑栓塞、脑卒中后遗症、脑卒中运动机能障碍、线粒体脑肌病、和/或肌萎缩性脊髓侧索硬化症等疾病。
任何特定病人的具体剂量取决于各种因素，包括所用的化合物的活性、年龄、体重、一般健康状况、性别、饮食、服药时间、服药方式、分泌速度、药物组合和具体病情。剂量随接受治疗的疾病、症状、对象和用药途径而变化，用于成人治疗时，口服日剂量1-300mg或静脉注射1-100mg，分2-3次用药。例如，用于治疗缺血性脑卒中时，以适当量，如约10-200mg/次口服，或者静脉注射约5-100mg/次，每天分2或3次等剂量使用活性成分。
本发明式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物能够有效地防治神经细胞损伤。
作为一方面的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，具有优异的稳定性。
作为另一方面的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，具有优异的水溶性。
作为另一方面的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，细胞毒性小。
作为另一方面的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，具有显著的神经保护作用，可有效地用于预防或治疗脑卒中、脑栓塞、脑卒中后遗症、脑卒中运动机能障碍、线粒体脑肌病、和/后肌萎缩性脊髓侧索硬化症。
附图说明
图1是各组ALS小鼠转棒实验坠落潜伏期-时间曲线。
图2是各组ALS小鼠悬线实验坠落潜伏期-时间曲线。
具体实施方式
以下通过实施例对本发明进行示例性说明，但所提供的实施例不能被理解为是对本发明范围的限 制。
本发明代表性举例化合物列于表1。
表1
Figure PCTCN2019100835-appb-000022
Figure PCTCN2019100835-appb-000023
Figure PCTCN2019100835-appb-000024
Figure PCTCN2019100835-appb-000025
Figure PCTCN2019100835-appb-000026
Figure PCTCN2019100835-appb-000027
Figure PCTCN2019100835-appb-000028
缩略语
TBTU：O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯
DMF：N,N-二甲基甲酰胺
ED：依达拉奉
DBU：1,8-二氮杂双环[5.4.0]十一碳-7-烯
TMSOTf：三氟甲磺酸三甲基硅酯
SOD：超氧化物歧化酶
MCAO：局灶性脑缺血再灌注损伤模型
ALS：肌萎缩侧索硬化症
实施例
实施例1-12
合成路线1：
Figure PCTCN2019100835-appb-000029
其中起始原料酰氯可以由相应的羧酸制备，路线如下：
Figure PCTCN2019100835-appb-000030
通用的合成方法1：
第一步：在0℃下将无水乙醛与催化量的无水氯化锌溶解在干燥的二氯甲烷(10mL)中，然后缓慢加入化合物(C)，待滴加完成后，加热至50℃，反应2.5h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和碳酸氢钠水溶液(1×50mL)洗涤，将水相用二氯甲烷萃取(2×25mL)，合并有机相。有机相依次用蒸馏水、饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品用硅胶柱层析纯化，得到化合物(B)。
第二步：将依达拉奉(3.6mmol)、碘化钾(0.4mmol)、碳酸钾(10mmol)溶解在干燥的DMF(10mL)中，45℃条件下搅拌10min。然后加入溶解在干燥DMF中的化合物(B)(3.3mmol)，45℃条件下搅拌1h。反应完成后，用油泵抽走大部分溶剂，然后向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液洗涤，将水相用乙酸乙酯萃取两次，合并有机相。将有机相依次用蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品用半制备高压液相色谱分离并使用冷冻干燥机冻干得到目标产物化合物(II)。
酰氯制备方法：在0℃，将二氯亚砜慢慢滴加到化合物(D)中，80℃回流2h。反应完成后，用泵将反应液中剩余的二氯亚砜抽走，并将残留物溶解于适量干燥的二氯甲烷中，直接用于下一步。实施例3异丁酸-1-(3-甲基-1-苯基吡唑)氧乙酯的制备
Figure PCTCN2019100835-appb-000031
采用通用合成方法1制备，得到黄色油状液体，产率31.17％。
1H NMR(400MHz,CDCl 3)δ7.68–7.62(m,2H),7.41(t,J＝7.9Hz,2H),7.29–7.23(m,1H),6.46(q,J＝5.3Hz,1H),5.58(s,1H),2.53(dt,J＝14.0,7.0Hz,1H),2.26(s,3H),1.59(d,J＝5.3Hz,3H),1.14(dd,J＝9.4,7.0Hz,6H).
ESI-MS(m/z):[M] +289.20
实施例4特戊酸-1-(3-甲基-1-苯基吡唑)氧乙酯的制备
Figure PCTCN2019100835-appb-000032
采用通用合成方法1制备，得到白色固体，产率为34.28％。
1H NMR(400MHz,CDCl 3)δ7.65(d,J＝7.6Hz,2H),7.41(t,J＝7.9Hz,2H),7.33–7.19(m,1H),6.45(q,J＝5.3Hz,1H),5.57(s,1H),2.26(s,3H),1.58(d,J＝5.3Hz,3H),1.16(s,9H).
ESI-MS(m/z):[M] +303.30
实施例5辛酸-1-(3-甲基-1-苯基吡唑)氧乙酯的制备
Figure PCTCN2019100835-appb-000033
采用通用合成方法1制备，得到黄色油状液体，产率为28.94％。
1H NMR(400MHz,CDCl 3)δ7.64(d,J＝7.7Hz,2H),7.30–7.22(m,2H),7.25(d,J＝8.8Hz,1H),6.47(q,J＝5.3Hz,1H),5.59(s,1H),2.33–2.24(m,5H),1.64–1.54(m,5H),1.33–1.21(m,8H),0.92–0.84(m,3H).
ESI-MS(m/z):[M] +345.19
实施例7苯甲酸-1-(3-甲基-1-苯基吡唑)氧乙酯的制备
Figure PCTCN2019100835-appb-000034
采用通用合成方法1制备，得到黄色油状液体，产率为33.26％。
1H NMR(400MHz,CDCl 3)δ8.02–7.98(m,2H),7.69–7.65(m,2H),7.62–7.56(m,1H),7.47–7.36(m,4H),7.29–7.24(m,1H),6.69(q,J＝5.3Hz,1H),5.65(s,1H),2.23(s,3H),1.72(d,J＝5.3Hz,3H).
ESI-MS(m/z):[M] +323.19
实施例9苯丁酸-1-(3-甲基-1-苯基吡唑)氧乙酯的制备
Figure PCTCN2019100835-appb-000035
采用通用合成方法1制备，得到黄色油状液体，产率为29.87％。
1H NMR(400MHz,CDCl 3)δ7.67–7.61(m,1H),7.40(t,J＝7.9Hz,2H),7.30–7.11(m,7H),6.48(q,J＝5.3Hz,1H),5.60(s,1H),2.61(t,J＝7.5Hz,2H),2.31(t,J＝7.4Hz,2H),2.25(s,3H),1.95–1.88(m,2H),1.58(d,J＝5.3Hz,3H).
ESI-MS(m/z):[M] +365.25
实施例1、2、6、8、10、11、12
采用通用合成方法1制备得到化合物1、2、6、8、10、11、12。
实施例13-32
合成路线2：
Figure PCTCN2019100835-appb-000036
通用合成方法2：
第一步：
在室温条件下将化合物(D)(5.68mmol)溶解在二氯甲烷(15mL)和水(17mL)的混合溶液中，剧烈搅拌的条件下缓慢加入碳酸氢钠(28.45mmol)及四丁基硫酸氢铵(0.56mmol)，然后在0℃下滴加氯甲基氯磺酸酯(8.5mmol)，室温搅拌24h。反应完成后，向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液洗涤，水相用乙酸乙酯萃取，合并有机相。将有机相依次用蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，硅胶柱层析纯化，得到化合物(E)。
第二步：
将依达拉奉(2.93mmol)、碳酸钾(8.84mmol)、碘化钾(3.55mmol)溶解在干燥的DMF(6.00mL)中，50℃条件下搅拌20min。将第一步得到的化合物(E)(2.94mmol)溶解在干燥的DMF(2.50mL)中，并加入到上述溶液中，50℃条件下搅拌3h。反应完成后，向反应液中加入100mL乙酸乙酯(或二氯甲烷)，然后用饱和氯化铵水溶液洗涤，将水相用乙酸乙酯(或二氯甲烷)萃取，合并有机相。 将有机相依次用蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，硅胶柱层析纯化，得到目标产物化合物(III)。
实施例13(3-甲基-1-苯基吡唑-5-)氧甲基特戊酸酯的制备
Figure PCTCN2019100835-appb-000037
采用通用合成方法2制备，得到黄色油状液体，产率为54.64％。
1H NMR(600MHz,CDCl 3)δ7.63(d,J＝7.7Hz,2H),7.40(t,J＝7.9Hz,2H),7.29–7.22(m,1H),5.74–5.68(m,3H),2.29(s,3H),1.20(s,9H).
ESI-MS(m/z):[M] +289.22
实施例14(3-甲基-1-苯基吡唑-5-)氧甲基异丁酸酯的制备
Figure PCTCN2019100835-appb-000038
采用通用合成方法2制备，得到黄色油状液体，产率为53.37％。
1H NMR(400MHz,CDCl 3)δ7.63(d,J＝7.7Hz,2H),7.40(t,J＝7.9Hz,2H),7.29–7.22(m,1H),5.71(d,J＝1.9Hz,3H),2.58(dt,J＝14.0,7.0Hz,1H),2.29(s,3H),1.17(d,J＝7.0Hz,6H).
ESI-MS(m/z):[M] +275.18
实施例15(3-甲基-1-苯基吡唑-5-)氧甲基辛酸酯的制备
Figure PCTCN2019100835-appb-000039
采用通用合成方法2制备，得到黄色油状液体，产率为60.28％。
1H NMR(400MHz,CDCl 3)δ7.63(d,J＝7.9Hz,2H),7.40(t,J＝7.9Hz,2H),7.28–7.21(m,1H),5.70(s,3H),2.36–2.26(m,5H),1.66–1.55(m,2H),1.27(s,8H),0.87(t,J＝6.7Hz,3H).
ESI-MS(m/z):[M] +331.24
实施例16(3-甲基-1-苯基吡唑-5-)氧甲基苯丁酸酯的制备
Figure PCTCN2019100835-appb-000040
采用通用合成方法2制备，得到黄色油状液体1.20g，产率为59.74％。
1H NMR(400MHz,CDCl 3)δ7.62(d,J＝7.9Hz,2H),7.39(t,J＝7.9Hz,2H),7.30–7.11(m,6H),5.69(s,3H),2.62(t,J＝7.6Hz,2H),2.34(t,J＝7.4Hz,2H),2.28(s,3H),1.99–1.89(m,2H).
ESI-MS(m/z):[M] +351.13
实施例17(3-甲基-1-苯基吡唑-5-)氧甲基烟酸酯的制备
Figure PCTCN2019100835-appb-000041
采用通用合成方法2制备，得到黄色固体0.62g，产率为43.28％。
1H NMR(400MHz,CDCl 3)δ9.22(s,1H),8.84(d,J＝4.3Hz,1H),8.34(d,J＝8.0Hz,1H),7.64–7.59(m,2H),7.51–7.45(m,1H),7.38(t,J＝7.9Hz,2H),7.25–7.20(m,1H),5.98(s,2H),5.82(s,1H),2.31(s,3H).
ESI-MS(m/z):[M] +310.15
实施例18(3-甲基-1-苯基吡唑-5-)氧甲基苯甲酸酯的制备
Figure PCTCN2019100835-appb-000042
采用通用合成方法2制备，得到黄色油状液体0.99g，产率为56.21％。
1H NMR(400MHz,CDCl 3)δ8.08–8.02(m,2H),7.68–7.57(m,3H),7.50–7.34(m,4H),7.25–7.20(m,1H),5.96(s,2H),5.81(s,1H),2.30(s,3H).
ESI-MS(m/z):[M] +309.17
实施例19(3-甲基-1-苯基吡唑-5-)氧甲基-(2-羟基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000043
(1)(3-甲基-1-苯基吡唑-5-)氧甲基-(2-苄氧基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000044
采用通用合成方法2制备，得到黄色固体0.37g，产率为48.93％。
(2)(3-甲基-1-苯基吡唑-5-)氧甲基-(2-羟基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000045
(3-甲基-1-苯基吡唑-5-)氧甲基-(2-苄氧基)苯甲酸酯(0.37g)溶解在5mL甲醇中，加入1％催化量10％的钯/碳，氢气环境下室温搅拌1.5h。反应完成后，用硅藻土过滤反应液，用旋转蒸发仪将溶剂蒸干，得到的油状液体用硅胶柱色谱纯化(石油醚/乙酸乙酯:10/1)，得到黄色油状液体0.24g，产率为82.70％。
1H NMR(400MHz,CDCl 3)δ10.36(s,1H),7.79(dd,J＝8.0,1.5Hz,1H),7.61(d,J＝7.6Hz,2H),7.52–7.46(m,1H),7.38(t,J＝7.9Hz,2H),7.27–7.206(m,1H),7.04–6.95(m,1H),6.95–6.86(m,1H),5.95(s,2H),5.80(s,1H),2.30(s,3H).
ESI-MS(m/z):[M] +325.01
实施例20(3-甲基-1-苯基吡唑-5-)氧甲基乙酰水杨酸酯的制备
Figure PCTCN2019100835-appb-000046
采用通用合成方法2制备，得到淡黄色固体0.48g，产率为59.78％。
1H NMR(600MHz,CDCl 3)δ7.99(d,J＝7.8Hz,1H),7.65–7.57(m,3H),7.40(t,J＝7.9Hz,2H),7.33(t,J＝7.6Hz,1H),7.28–7.22(m,1H),7.13(d,J＝8.1Hz,1H),5.90(s,2H),5.77(s,1H),2.30(s,6H).
ESI-MS(m/z):[M] +367.18
实施例21(3-甲基-1-苯基吡唑-5-)氧甲基-(2-乙氧羰基氧基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000047
在室温条件下将实施例19产物(0.50g)溶解在干燥的二氯甲烷(5mL)中，加入干燥的三乙胺(0.47mL)，然后缓慢加入氯甲酸乙酯(0.35mL)，室温搅拌2.5h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和氯化铵水溶液(1×50mL)洗涤，将水相用二氯甲烷萃取(2×25mL)，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的油状液体用硅胶柱色谱纯化(石油醚/乙酸乙酯:3/1)，得到淡黄色油状液体0.54g，产率为88.14％。
1H NMR(400MHz,CDCl 3)δ8.00(dd,J＝7.9,J＝1.6Hz,1H),7.66–7.58(m,3H),7.43–7.32(m,3H),7.27–7.21(m,2H),5.91(s,2H),5.80(s,1H),4.28(q,J＝7.1Hz,2H),2.29(s,3H),1.36(t,J＝7.1Hz,3H).
ESI-MS(m/z):[M] +397.18
实施例22(3-甲基-1-苯基吡唑-5-)氧甲基-(2-异丁酰基氧基)苯甲酸的制备
Figure PCTCN2019100835-appb-000048
在室温条件下将实施例19产物(0.50g)溶解在干燥的二氯甲烷(5mL)中，加入干燥的三乙胺(0.39mL)，然后缓慢加入异丁酰氯(0.32mL)，室温搅拌2.5h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和氯化铵水溶液洗涤，将水相用二氯甲烷萃取，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，用无水硫酸钠干燥，减压浓缩，得到的油状液体用硅胶柱色谱纯化(石油醚/乙酸乙酯:3/1)，得到淡黄色油状液体0.48g，产率为79.48％。
1H NMR(400MHz,CDCl 3)δ8.00–7.93(m,1H),7.65–7.56(m,3H),7.39(t,J＝7.8Hz,2H),7.31(t,J＝7.6Hz,1H),7.29–7.20(m,1H),7.10(d,J＝8.1Hz,1H),5.89(s,2H),5.77(s,1H),2.84(dt,J＝14.0Hz,J＝7.0Hz,1H),2.29(s,3H),1.32(d,J＝7.0Hz,6H).
ESI-MS(m/z):[M] +395.21
实施例23(3-甲基-1-苯基吡唑-5-)氧甲基-(2-羟基-3-N,N-二甲基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000049
(1)2-羟基-3-氨基苯甲酸甲酯的制备
Figure PCTCN2019100835-appb-000050
将2-羟基-3-氨基苯甲酸(8.0g)加入无水甲醇(80mL)中，0℃条件下缓慢加入二氯亚砜(15mL)，60℃条件下反应24h。反应完成后，减压浓缩溶剂，用碳酸氢钠水溶液调节pH至中性，然后向反应液中加入100mL二氯甲烷萃取，无水硫酸钠干燥，减压浓缩，硅胶柱层析纯化，得固体产物7.4g，产率为85.1％。
(2)2-羟基-3-N,N-二甲基苯甲酸甲酯的制备
Figure PCTCN2019100835-appb-000051
室温条件下将2-羟基-3氨基苯甲酸甲酯(2.0g)溶解于甲醇(30mL)中，加入甲醛溶液(6mL)，再加入乙酸(0.5mL)，在低温条件下缓慢加入氰基硼氢化钠(3.72g)，室温条件下反应2h。反应完成后，用旋转蒸发仪浓缩溶剂，残余物用乙酸乙酯(150mL)溶解，水洗，无水硫酸钠干燥，减压浓缩至干，得产物2.1g，产率为85.8％。
(3)2-苄氧基-3-N,N-二甲基苯甲酸甲酯的制备
Figure PCTCN2019100835-appb-000052
将2-羟基-3-N,N-二甲基苯甲酸甲酯(2.0g)溶解于干燥的DMF(7.5mL)中，加入碳酸钾(4.25g)，在室温下加入溴化苄(1.83mL)，90℃条件下反应3h。反应完成后，用油泵将大量的溶剂抽走，然后向反应液中加入100mL乙酸乙酯，用饱和氯化铵水溶液(50mL)洗涤，水相用乙酸乙酯萃取(2×25mL)，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:15/1)，得到产物2.84g，产率为45.55％。
(4)2-苄氧基-3-N,N-二甲基苯甲酸的制备
Figure PCTCN2019100835-appb-000053
将2-苄氧基-3-N,N-二甲基苯甲酸甲酯(2.0g)溶解于甲醇(12mL)中，加入1N氢氧化钠溶液(24mL)，80℃条件下反应2h，反应完成后，用旋转蒸发仪旋走剩余的甲醇溶剂，用盐酸调节pH至有固体析出。加入适量蒸馏水，用二氯甲烷萃取直到水相中不含目标产物，有机相减压浓缩至干，得到黄色固体1.67g，产率为87.72％。
(5)氯甲基-(2-苄氧基-3-N,N-二甲基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000054
在室温条件下将2-苄氧基-3-N,N-二甲基苯甲酸(1.0g)溶解在二氯甲烷(5mL)及水(10mL)的混合溶液中，剧烈搅拌下缓慢加入碳酸氢钠(1.24g)及四丁基溴化铵(0.11g)，然后在0℃下滴加溶解在5mL干燥二氯甲烷中的氯磺酸氯甲酯(0.73mL)，室温搅拌24h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和碳酸氢钠水溶液(50mL)洗涤，将水相用二氯甲烷萃取(2×25mL)，合并有机相。将有机相依次用饱和碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的油状液体用硅胶柱层析纯化(石油醚/乙酸乙酯：15/1)，得到淡黄色固体0.55g，产率为46.31％。
(6)(3-甲基-1-苯基吡唑-5-)氧甲基-(2-苄氧基-3-N,N-二甲基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000055
将依达拉奉(0.24g)、碳酸钾(0.71g)、碘化钾(0.34g)溶解在干燥的DMF(6.00mL)中，50℃条件下搅拌20min。将氯甲基-(2-苄氧基-3-N,N-二甲基)苯甲酸酯(0.55g)溶解在干燥的DMF(2.50mL)中，并加入到上述溶液中，45℃条件下搅拌3h。反应完成后，向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液(1×50mL)洗涤，将水相用乙酸乙酯萃取(2×25mL)，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的油状液体用硅胶柱层析纯化(石油醚/乙酸乙酯:15/1)，得到产物0.22g，产率为27.87％。
(7)(3-甲基-1-苯基吡唑-5-)氧甲基-(2-羟基-3-N,N-二甲基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000056
将(3-甲基-1-苯基吡唑)氧甲基-(2-苄氧基-3-N,N-二甲基)苯甲酸酯(0.22g)溶解在5mL乙酸乙酯中，加入催化量(1％)的钯/碳(10％)，氢气环境下室温搅拌1.5h。反应完成后，用硅藻土过滤反应液，减压浓缩，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:5/1)，得到黄色固体0.17g，产率为79.26％。
1H NMR(400MHz,CDCl 3)δ9.83(s,1H),7.62(d,J＝7.7Hz,2H),7.38(t,J＝7.9Hz,2H),7.28–7.19(m,1H),7.16–7.04(m,2H),6.92(d,J＝9.0Hz,1H),5.96(s,2H),5.81(s,1H),2.88(s,6H),2.29(s,3H).
ESI-MS(m/z):[M] +368.19
实施例24(3-甲基-1-苯基吡唑-5-)氧甲基-(2-羟基-5-N,N-二甲基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000057
(1)2-羟基-5-氨基苯甲酸甲酯的制备
Figure PCTCN2019100835-appb-000058
将对氨基水杨酸(8.0g)加入无水甲醇(80mL)中，0℃条件下缓慢加入二氯亚砜(15mL)，60℃条件下反应24h。反应完成后，用旋转蒸发仪旋走剩余的甲醇，用碳酸氢钠水溶液将pH调节至中性，然后向反应液中加入100mL二氯甲烷萃取，无水硫酸钠干燥，减压浓缩，得固体产物7.50g，产率为85.91％。
(2)2-羟基-5-N,N-二甲基苯甲酸甲酯的制备
Figure PCTCN2019100835-appb-000059
室温条件下将2-羟基-5氨基苯甲酸甲酯(2.0g)溶解于甲醇(30mL)中，向其中加入40％甲醛水溶液(6mL)，再加入乙酸(0.5mL)，在低温下缓慢加入氰基硼氢化钠(3.72g)，室温反应2h。反 应完成后，用旋转蒸发仪旋走剩余的甲醇溶剂，残余物用150mL乙酸乙酯溶解，蒸馏水洗，无水硫酸钠干燥，减压浓缩，得产物2.0g，产率为85.47％。
(3)2-苄氧基-5-N,N-二甲基苯甲酸甲酯的制备
Figure PCTCN2019100835-appb-000060
将2-羟基-5-N,N-二甲基苯甲酸甲酯(2.0g)溶解于干燥的DMF(7.5mL)中，加入碳酸钾(4.25g)，在室温下加入溴化苄(1.83mL)，90℃条件下反应3h。反应完成后，用油泵将大量的溶剂抽走，然后向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液(1×50mL)洗涤，将水相用乙酸乙酯萃取(2×25mL)，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:15/1)，得到产物2.84g，产率为45.55％。
(4)2-苄氧基-5-N,N-二甲基苯甲酸的制备
Figure PCTCN2019100835-appb-000061
将2-苄氧基-5-N,N-二甲基苯甲酸甲酯(2.0g)溶解于甲醇(12mL)中，加入氢氧化钠溶液(24mL)，80℃条件下反应2h，反应完成后，用旋转蒸发仪旋走剩余的甲醇溶剂，用盐酸调节pH至有固体析出，加入适量蒸馏水，用二氯甲烷萃取直到水相中不含目标产物，有机相经减压浓缩得到黄色固体1.67g，产率为87.72％。
(5)氯甲基-(2-苄氧基-5-N,N-二甲基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000062
在室温条件下2-苄氧基-5-N,N-二甲基苯甲酸(1.0g)溶解在二氯甲烷(5mL)及水(10mL)的混合溶液中，剧烈搅拌的条件下缓慢加入碳酸氢钠(1.24g)及四丁基溴化铵(0.11g)，然后在0℃下滴加溶解在5mL干燥的二氯甲烷中的氯磺酸氯甲酯(0.73mL)，室温搅拌24h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和碳酸氢钠水溶液(1×50mL)洗涤，将水相用二氯甲烷萃取(2×25mL)，合并有机相。将有机相依次用饱和碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶液洗涤，无水 硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的油状液体用硅胶柱层析纯化(石油醚/乙酸乙酯：15/1)，得到淡黄色固体0.55g，产率为46.31％。
(6)(3-甲基-1-苯基吡唑-5-)氧甲基-(2-苄氧基-5-N,N-二甲基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000063
将依达拉奉(0.24g)、碳酸钾(0.71g)、碘化钾(0.34g)溶解在干燥的DMF(6mL)中，50℃条件下搅拌20min。将氯甲基-(2-苄氧基-5-N,N-二甲基)苯甲酸酯(0.55g)溶解在干燥的DMF(2.5mL)中，并加入到上述溶液中，45℃条件下搅拌3h。反应完成后，向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液(1×50mL)洗涤，将水相用乙酸乙酯萃取(2×25mL)，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的油状液体用硅胶柱层析纯化(石油醚/乙酸乙酯:15/1)，得到产物0.22g，产率为27.87％。
(7)(3-甲基-1-苯基吡唑-5-)氧甲基-(2-羟基-5-N,N-二甲基)苯甲酸酯的制备
Figure PCTCN2019100835-appb-000064
将(3-甲基-1-苯基吡唑)氧甲基-(2-苄氧基-5-N,N-二甲基)苯甲酸酯(0.22g)溶解在5mL乙酸乙酯中，加入催化量10％的钯/碳，氢气环境下室温搅拌1.5h。反应完成后，用硅藻土过滤反应液，用旋转蒸发仪将溶剂蒸干，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:5/1)，得到黄色固体0.17g，产率为79.26％。
1H NMR(400MHz,CDCl 3)δ9.83(s,1H),7.62(d,J＝7.7Hz,2H),7.38(t,J＝7.9Hz,2H),7.28–7.19(m,1H),7.16–7.04(m,2H),6.92(d,J＝9.0Hz,1H),5.96(s,2H),5.81(s,1H),2.88(s,6H),2.29(s,3H).
ESI-MS(m/z):[M] +368.22
实施例25(3-甲基-1-苯基吡唑-5-)氧甲基-(2-乙氧羰基氧基-5-N,N-二甲基)苯甲酸的制备
Figure PCTCN2019100835-appb-000065
在室温条件下将实施例24产物(0.1g)溶解在干燥的二氯甲烷(5mL)中，加入干燥的三乙胺(0.1mL)，然后缓慢加入氯甲酸乙酯(0.08mL)，室温搅拌2.5h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和氯化铵水溶液(1×50mL)洗涤，将水相用二氯甲烷萃取(2×25mL)，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的油状液体用硅胶柱层析纯化(石油醚/乙酸乙酯:3/1)，得到淡黄色油状液体0.11g，产率为85％。
1H NMR(400MHz,CDCl 3)δ7.62(d,J＝7.7Hz,2H),7.38(t,J＝7.9Hz,2H),7.28–7.19(m,1H),7.16–7.04(m,2H),6.92(d,J＝9.0Hz,1H),5.96(s,2H),5.81(s,1H),4.21(m,2H),1.29(t,J＝7.8Hz,3H),2.88(s,6H),2.29(s,3H).
ESI-MS(m/z):[M] +440.21
实施例26-32
采用通用合成路线2制备化合物26-32。
实施例33 1-苯基-3-甲基-5-O-D-葡萄糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000066
制备方法一：
(1)1,2,3,4,6-O-五乙酰基-D-葡萄糖的制备
Figure PCTCN2019100835-appb-000067
在室温条件下将不含结晶水的葡萄糖(4.5g)及醋酸钾(4.9g)溶解在乙酸酐(25mL)中，然后在90℃下搅拌4h。反应完成后，降温至室温，用油泵将溶剂抽走。向反应液中加入100mL乙酸乙酯，然后用饱和碳酸氢钠水溶液洗涤，将水相用乙酸乙酯萃取，合并有机相。将有机相依次用饱和碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，硅胶柱层析纯化，得产物6.3g，产率为65％。
(2)2,3,4,6-O-四乙酰基-D-溴代葡萄糖的制备
Figure PCTCN2019100835-appb-000068
将1,2,3,4,6-O-五乙酰基-D-葡萄糖(2.5g)溶解在干燥的二氯甲烷(8mL)中，然后在适量醋酸溶液中加入2.5mL 33％的溴化氢水溶液，并在0℃下将其加入上述二氯甲烷溶液中，在室温条件下搅拌反应2h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和碳酸氢钠水溶液洗涤，将水相用二氯甲烷萃取，合并有机相。将有机相依次用碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:3/1)，得产物1.7g，产率为64.6％。
(3)1-苯基-3-甲基-5-O-(2,3,4,6-O-四乙酰基-D-葡萄糖苷)吡唑的制备
Figure PCTCN2019100835-appb-000069
将依达拉奉(0.35g)及碳酸铯(0.79g)溶解在干燥的DMF(10mL)中，在室温下搅拌20min。然后加入2,3,4,6-O-四乙酰基-D-溴代葡萄糖(1.0g)，室温搅拌24h。反应完成后，先用油泵抽走大量的溶剂，然后向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液洗涤，将水相用乙酸乙酯萃取，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:4/1)，得产物0.68g，产率为55.4％。
(4)1-苯基-3-甲基-5-O-D-葡萄糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000070
将(1-苯基-3-甲基-5-O-(2,3,4,6-O-四乙酰基-D-葡萄糖苷)吡唑(0.2g)溶解在3mL甲醇中，加入碳酸钾(0.27g)，在室温条件下搅拌1h。反应完成后，得到的粗产品用半制备高压液相色谱分离并使用冷冻干燥机冻干得到白色固体产物0.11g，产率为79％。(通过该方法制备的化合物33用于以下试验例)。
1H NMR(600MHz,D 2O)δ7.60–7.40(m,5H),5.93(s,1H),5.10(m,1H),3.95–3.86(m,1H),3.79– 3.70(m,1H),3.64–3.42(m,4H),2.23(s,3H)。
ESI-MS(m/z):[M] +337.15
制备方法二：
(1)2,3,4,6-O-四苄基-1-O-三氯亚氨基-D-葡糖糖的制备
Figure PCTCN2019100835-appb-000071
将2,3,4,6-O-四苄基-D-葡糖糖(12g，22.2mmol)和DBU(0.3mL，2.2mmol)溶于二氯甲烷(150mL),搅拌下滴加三氯乙腈(11mL，52.9mmol)。室温反应2h，TLC(石油醚/乙酸乙酯5:1)显示反应完全。减压浓缩除去溶剂，硅胶柱层析纯化(石油醚/乙酸乙酯10:1)得到无色油状物11g，直接用于下一步。
(2)1-苯基-3-甲基-5-O-(2,3,4,6-O-四苄基-D-葡萄糖苷)吡唑的制备
Figure PCTCN2019100835-appb-000072
将依达拉奉(2.8g，16.1mmol)、2,3,4,6-O-四苄基-1-O-三氯亚氨基-D-葡糖糖(11g，16.1mmol)和4A分子筛加入二氯甲烷中，搅拌下滴加TMSOTf(1.1mmol)，室温搅拌1h，TLC(石油醚/乙酸乙酯:4/1)显示反应完全。减压除去溶剂，硅胶柱层析纯化(石油醚/乙酸乙酯5:1)，得到淡黄色固体产品8.4g。
(3)1-苯基-3-甲基-5-O-D-葡萄糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000073
将1-苯基-3-甲基-5-O-(2,3,4,6-O-四苄基-D-葡萄糖苷)吡唑(5g，7.2mmol)溶于甲醇(100mL)，加入Pd/C(10％)，40℃氢化反应6h，过滤，减压除去溶剂。硅胶柱层析纯化(石油醚/乙酸乙酯/甲醇1:1:0.1)得到白色固体1.3g。
1H NMR(400MHz,D 2O)δ7.545-7.419(m,5H),5.950(s,1H),5.599(m,1H),3.683-3.623(m,4H), 3.483-3.348(m,2H),2.233(s,3H)。
ESI-MS(m/z):[M] +337.15
实施例34 1-苯基-3-甲基-5-O-D-甘露糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000074
(1)1,2,3,4,6-O-五乙酰基-D-甘露糖的制备
Figure PCTCN2019100835-appb-000075
在室温条件下将甘露糖(4.5g)及醋酸钾(4.9g)溶解在乙酸酐(25mL)中，然后在90℃下搅拌4h。反应完成后，恢复室温，用油泵将溶剂抽走。向反应液中加入100mL乙酸乙酯，然后用饱和碳酸氢钠水溶液洗涤，将水相用乙酸乙酯萃取，合并有机相。将有机相依次用饱和碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，用乙醇进行重结晶，得产物6.3g，产率为63％。
(2)2,3,4,6-O-四乙酰基-D-溴代甘露糖的制备
Figure PCTCN2019100835-appb-000076
将1,2,3,4,6-O-五乙酰基-D-甘露糖(1.84g)溶解在干燥的二氯甲烷(4.5mL)中，然后在适量醋酸溶液中加入1.5mL 33％的溴化氢水溶液，并在0℃下将其加入上述二氯甲烷溶液中，在室温条件下搅拌反应3.5h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和碳酸氢钠水溶液洗涤，将水相用二氯甲烷萃取，合并有机相。将有机相依次用碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:5/1)，得产物1.19g，产率为61.31％。
(3)1-苯基-3-甲基-5-O-(2,3,4,6-O-四乙酰基-D-甘露糖苷)吡唑的制备
Figure PCTCN2019100835-appb-000077
将依达拉奉(0.35g)及碳酸铯(0.79g)溶解在干燥的DMF(3.50mL)中，在室温下搅拌20min。然后加入2,3,4,6-O-四乙酰基-D-溴代甘露糖(1.0g)，室温搅拌24h。反应完成后，先用油泵抽走大量溶剂，然后向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液洗涤，将水相用乙酸乙酯萃取，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:4/1)，得产物0.64g，产率为52.14％。
(4)1-苯基-3-甲基-5-O-D-甘露糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000078
将1-苯基-3-甲基-5-O-(2,3,4,6-O-四乙酰基-D-甘露糖苷)吡唑(0.2g)溶解在3mL甲醇中，加入碳酸钾(0.28g)，在室温条件下搅拌1h。反应完成后，得到的粗产品用半制备高压液相色谱分离并使用冷冻干燥机冻干得到白色固体产物0.13g，产率为77.28％。
1H NMR(400MHz,D 2O)δ7.58–7.50(m,4H),7.46–7.38(m,1H),5.88(s,1H),5.24(s,1H),4.13–4.06(m,1H),3.97–3.89(m,1H),3.78–3.72(m,1H),3.66–3.59(m,2H),3.53–3.44(m,1H),2.23(s,3H).
ESI-MS(m/z):[M] +337.14
实施例35 1-苯基-3-甲基-5-O-D-半乳糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000079
(1)1,2,3,4,6-O-五乙酰基-D-半乳糖的制备
Figure PCTCN2019100835-appb-000080
在室温条件下将半乳糖(9.0g)及醋酸钾(9.8g)溶解在乙酸酐(50mL)中，然后在90℃下搅拌4h。反应完成后，恢复室温，用油泵将溶剂抽走。向反应液中加入100mL乙酸乙酯，然后用饱和碳酸氢钠水溶液洗涤，将水相用乙酸乙酯萃取，合并有机相。将有机相依次用饱和碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，用乙醇进行重结晶，得产物11.0g，产率为62％。
(2)2,3,4,6-O-四乙酰基-D-溴代半乳糖的制备
Figure PCTCN2019100835-appb-000081
将1,2,3,4,6-O-五乙酰基-D-半乳糖(2.72g)溶解在干燥的二氯甲烷(9mL)中，然后在适量醋酸溶液中加入3mL 33％的溴化氢水溶液，并在0℃下将其加入上述二氯甲烷溶液中，在室温条件下搅拌反应3.5h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和碳酸氢钠水溶液洗涤，将水相用二氯甲烷萃取，合并有机相。将有机相依次用碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:5/1)，得产物1.54g，产率为54.47％。
(3)1-苯基-3-甲基-5-O-(2,3,4,6-O-四乙酰基-D-半乳糖苷)吡唑的制备
Figure PCTCN2019100835-appb-000082
将依达拉奉(0.35g)及碳酸铯(0.79g)溶解在干燥的DMF中，在室温下搅拌20min。然后加入2,3,4,6-O-四乙酰基-D-溴代半乳糖(1.0g)，室温搅拌24h。反应完成后，向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液洗涤，将水相用乙酸乙酯萃取，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的粗产品用硅胶柱色谱纯化(石油醚/乙酸乙酯:4/1)，得产物0.99g，产率为75.20％。
(4)1-苯基-3-甲基-5-O-D-半乳糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000083
将1-苯基-3-甲基-5-O-(2,3,4,6-O-四乙酰基-D-半乳糖苷)吡唑(0.2g)溶解在3mL甲醇中，加入碳酸钾(0.28g)，在室温条件下搅拌1h。反应完成后，得到的粗产品用半制备高压液相色谱分离并使用冷冻干燥机冻干得到白色固体产物0.11g，产率为79.36％。
1H NMR(400MHz,D 2O)δ7.51–7.41(m,4H),7.38–7.31(m,1H),5.84(s,1H),4.97(m,1H),3.89(s,1H),3.79–3.73(m,1H),3.70–3.60(m,4H),2.15(s,3H)。
ESI-MS(m/z):[M] +337.17
实施例36 1-苯基-3-甲基-5-O-D-(2-脱氧)葡萄糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000084
(1)1,3,4,6-O-四乙酰基-D-(2-脱氧)葡萄糖的制备
Figure PCTCN2019100835-appb-000085
将2-脱氧葡萄糖(1.0g，6.1mmol)分散在乙酸酐(5mL)中，在65℃条件下缓慢加入乙酸钾(0.99g，9.1mmol),70℃搅拌反应3.5h，TLC检测反应完毕。反应液冷却至50℃，减压蒸除溶剂，硅胶柱层析纯化，得到白色固体1.2g，产率59％。
(2)3,4,6-O-三乙酰基-D-溴代-(2-脱氧)葡萄糖的制备
Figure PCTCN2019100835-appb-000086
将1,3,4,6-O-四乙酰基-D-(2-脱氧)葡萄糖(2.5g)溶解在干燥的二氯甲烷(8mL)中，然后在适量醋酸溶液中加入2.9mL 33％的溴化氢水溶液，并在0℃下将其加入上述二氯甲烷溶液中，在室温条件下搅拌反应2h。反应完成后，向反应液中加入100mL二氯甲烷，然后用饱和碳酸氢钠水溶液洗涤，将水相用二氯甲烷萃取，合并有机相。将有机相依次用碳酸氢钠水溶液，蒸馏水，饱和氯化钠溶 液洗涤，然后用无水硫酸钠干燥，用旋转蒸发仪将溶剂蒸干，得到的粗产品用硅胶柱色谱纯化(石油醚/乙酸乙酯＝3/1)，得产物1.79g，产率为67.43％。
(3)1-苯基-3-甲基-5-O-(3,4,6-O-三乙酰基-D-(2-脱氧)葡萄糖苷)吡唑的制备
Figure PCTCN2019100835-appb-000087
将依达拉奉(4.93g)及碳酸铯(1.34g)溶解在干燥的DMF中，在室温下搅拌20min。然后加入3,4,6-O-三乙酰基-D-溴代-(2-脱氧)葡萄糖(1.0g)，室温搅拌24h。反应完成后，先用油泵抽走大量溶剂，然后向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液洗涤，将水相用乙酸乙酯萃取，合并有机相。将有机相依次用饱和氯化铵水溶液，蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品用硅胶柱层析纯化(石油醚/乙酸乙酯:4/1)，得产物1.07g，产率为84.27％。
(4)1-苯基-3-甲基-5-O-D-(2-脱氧)葡萄糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000088
将1-苯基-3-甲基-5-O-(3,4,6-O-三乙酰基-D-(2-脱氧)葡萄糖苷)吡唑(1.0g)溶解在15mL甲醇中，加入碳酸钾(1.55g)，在室温条件下搅拌1.5h。反应完成后，得到的粗产品用半制备高压液相色谱分离并使用冷冻干燥机冻干得到白色固体产物0.68g，产率为94.12％。
1H NMR(600MHz,DMSO)δ7.63(d,J＝7.9Hz,2H),7.45(t,J＝7.6Hz,2H),7.27(t,J＝7.2Hz,1H),5.83(s,1H),5.23(m,1H),3.69(d,J＝11.6Hz,1H),3.56–3.47(m,2H),3.26–3.19(m,1H),3.17(s,1H),3.08(t,J＝8.9Hz,1H),2.24–2.17(d,J＝8.0Hz,1H),2.16(s,3H).
ESI-MS(m/z):[M] +321.20
实施例37 1-苯基-3-甲基-5-O-D-(2-氨基-2-脱氧)葡萄糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000089
(1)2-乙酰氨基-1,3,4,6-四乙酰氧基-2-脱氧葡萄糖的制备
Figure PCTCN2019100835-appb-000090
2-氨基-2-脱氧葡萄糖盐酸盐(2.2g,10.2mmol)混悬于吡啶(10mL)中，冷却至0℃，加入乙酸酐(9.4mL，99.6mmol)，室温下搅拌过夜，加入乙酸乙酯(100mL)，用5％稀盐酸，饱和碳酸氢钠水溶液洗涤，饱和食盐水洗涤，无水硫酸钠干燥，减压旋蒸浓缩，残留物用二氯甲烷/乙醚重结晶得3.3g白色固体产物。
(2)2-乙酰氨基-3,4,6-三乙酰氧基-2-脱氧葡萄糖溴的制备
Figure PCTCN2019100835-appb-000091
2-乙酰氨基-1,3,4,6-四乙酰氧基-2-脱氧葡萄糖(1.0g，2.6mmol)溶于二氯甲烷(1mL)中，冷却至0℃，缓慢加入溴化氢乙酸溶液(1.6mL，33％in acetic acid)，室温反应3h，加入二氯甲烷(50mL)，有机相用饱和碳酸氢钠洗涤，饱和食盐水洗涤，无水硫酸钠干燥，减压除去溶剂，残留物直接用于下一步。
(3)5-(2-乙酰氨基-3,4,6-三乙酰氧基-2-脱氧葡萄糖苷)-3-甲基-1-苯基-1H吡唑的制备
Figure PCTCN2019100835-appb-000092
依达拉奉(452mg，2.6mmol)溶于二氯甲烷(5mL)中，加入三乙胺(0.9mL，6.5mmol)，室温搅拌20min，冷却至0℃。将2-乙酰氨基-3,4,6-三乙酰氧基-2-脱氧葡萄糖溴溶于少量二氯甲烷，缓慢滴加入反应液中，室温搅拌继续反应3h，二氯甲烷(50mL)稀释，有机相用饱和氯化铵洗涤，饱和食盐水洗涤，无水硫酸钠干燥，减压除去溶剂，残留物经硅胶柱层析(石油醚/乙酸乙酯:1/1)纯化，得白色固体粗产品，直接用于下一步。
(6)1-苯基-3-甲基-5-O-D-(2-氨基-2-脱氧)葡萄糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000093
上一步粗产物溶于甲醇(15mL)中，加入碳酸钾(538mg，3.9mmol)，室温搅拌反应2h，用醋酸调节pH至中性，半制备(MeOH/H 2O＝2/3)分离，得粉白色固体300mg。
1H NMR(400MHz，D 2O)，δ7.34-7.43(m，5H)；5.87(s,1H)；4.97(m,1H)；3.70-3.88(m,3H)；3.42-3.52(m,3H)；2.15(s,3H)；1.65(s,3H)
ESI-MS(m/z):[M] +378.19
实施例38 1-苯基-3-甲基-5-O-D-吡喃木糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000094
(1)1,2,3,4-O-乙酰基-D-吡喃木糖的制备
Figure PCTCN2019100835-appb-000095
将木糖(2g，13.3mmol)混悬于吡啶(10mL)中，0℃下缓慢滴入乙酸酐(10mL，106mmol)，搅拌反应过夜，加入乙酸乙酯(100mL)，用5％稀盐酸，饱和碳酸氢钠水溶液洗涤，饱和食盐水洗涤，无水硫酸钠干燥，减压旋蒸浓缩得4g产物，直接用于下一步。
(2)2,3,4-三乙酰氧基-D-吡喃木糖溴的制备
Figure PCTCN2019100835-appb-000096
将1,2,3,4-O-乙酰基-D-吡喃木糖(2.5g，7.86mmol)溶于二氯甲烷(2mL)中，冷却至0℃，缓慢滴入溴化氢乙酸溶液(33wt％，8.5mL)，室温反应30min，加入二氯甲烷(50mL)，有机相用饱和碳酸氢钠洗涤，饱和食盐水洗涤，无水硫酸钠干燥，减压除去溶剂，残留物用乙醚和正己烷重结晶得产物2.43g。
(3)5-(2,3,4-三乙酰氧基-D-吡喃木糖苷)-3-甲基-1-苯基-1H吡唑
Figure PCTCN2019100835-appb-000097
将依达拉奉(1.2g,7.1mmol)混悬于乙腈(15mL)中，冷却至0℃，缓慢加入氢化钠(284mg,7.1mmol,60％in oil)，室温搅拌20min，加入2,3,4-三乙酰氧基-D-吡喃木糖溴(2.4g，7.1mmol)，室温继续搅拌反应1h，加入乙酸乙酯和水分层，有机相用无水硫酸钠干燥，减压旋蒸浓缩得粗产物直接用于下一步。
(4)1-苯基-3-甲基-5-O-D-吡喃木糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000098
将上一步粗产物溶于甲醇中，加入碳酸钾(1.5g,10.7mmol)，室温搅拌反应1h，加醋酸调节pH至7左右，旋蒸除去溶剂，残留物经硅胶柱层析(石油醚/乙酸乙酯:8/1)得800mg产物。
1H MNR(400MHz，CD 3OD)，δ7.62-7.64(m,2H)；7.42-7.45(m,2H)；7.29-7.31(m,1H)；5.81(s,1H)；4.96-4.97(m,1H)；3.94(dd，J＝7.7Hz，J＝3.4Hz，1H)；3.53-3.57(m,1H)；3.35-3.42(m,3H)；2.23(s,3H)
ESI-MS(m/z):[M] +307.18
实施例39 1-苯基-3-甲基-5-O-D-核糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000099
(1)1-甲氧基-D-核糖的制备
Figure PCTCN2019100835-appb-000100
D-核糖(5g,33.3mmol)混悬于甲醇(80mL)中，置于冰浴中，缓慢滴加浓硫酸(0.5mL，9.4mmol)，升温至60℃反应过夜，用氨水调节pH至9，过滤，减压除去溶剂得5g粗产品直接用于下一步。
(2)1-甲氧基-2,3,4-三苯甲酰基-D-核糖的制备
Figure PCTCN2019100835-appb-000101
1-甲氧基-D-核糖(2g，12.2mmol)溶于吡啶(30mL)中，冷却至0℃，缓慢滴加苯甲酰氯(8mL，69mmol)，室温搅拌反应过夜，加入乙酸乙酯(100mL)，用5％稀盐酸，饱和碳酸氢钠水溶液洗涤，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，残留物经硅胶柱层析(石油醚/乙酸乙酯:10/1)得4g油状产物。
(3)2,3,4-三苯甲酰基-D-核糖溴的制备
Figure PCTCN2019100835-appb-000102
1-甲氧基-2,3,4-三苯甲酰基-D-核糖(2.4g，5mmol)溶于醋酸(8mL)中，缓慢加入溴化氢乙酸溶液(33wt％，8mL)，室温反应2h，加入二氯甲烷(50mL)，有机相用饱和碳酸氢钠洗涤，饱和食盐水洗涤，无水硫酸钠干燥，减压除去溶剂，残留物直接用于下一步。
(4)5-(2,3,4-三苯甲酰氧基-D-核糖苷)-3-甲基-1-苯基-1H吡唑的制备
Figure PCTCN2019100835-appb-000103
依达拉奉(870mg，5mmol)混悬于乙腈(10mL)中，加入碳酸铯(1.7g，5.25mmol)，室温搅拌20min，用少量乙腈溶解2,3,4-三苯甲酰基-D-核糖溴，缓慢滴加，反应液继续搅拌反应1h，加入乙酸乙酯和水分层，有机相用无水硫酸钠干燥，减压旋蒸浓缩，残留物经硅胶柱层析(石油醚/乙酸乙酯:5/1)得1g白色固体产物。
(5)1-苯基-3-甲基-5-O-D-核糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000104
5-(2,3,4-三苯甲酰氧基-D-核糖苷)-3-甲基-1-苯基-1H吡唑(309mg，0.5mmol)溶于甲醇(3mL)中，加入甲醇钠甲醇溶液(30％，0.25mL)，室温反应2小时，用醋酸调节pH至7左右，减压除去溶剂，残留物经硅胶柱层析(石油醚/乙酸乙酯:1/2)得50mg白色固体产物。
1H MNR(400MHz，CD 3OD)，δ7.53-7.57(m,2H)；7.41-7.47(m,2H)；7.28-7.33(m,1H)；5.80(s,1H)；5.49(s,1H)；4.03-4.16(m,3H)；3.68(dd，J＝12.0Hz，J＝3.3Hz,1H)；3.68(dd，J＝12.0 Hz，J＝5.8Hz,1H)；2.23(s,3H)
ESI-MS(m/z):[M] +307.04
实施例40 1-苯基-3-甲基-5-O-D-(2-脱氧)核糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000105
(1)5-(3,5-二对氯苯甲酰氧基-2-D-脱氧核糖苷)-3-甲基-1-苯基-1H吡唑的制备
Figure PCTCN2019100835-appb-000106
将依达拉奉(500mg,2.9mmol)混悬于乙腈(15mL)中，冷却至0℃，缓慢加入氢化钠(115mg,2.9mmol,60％in oil)，室温搅拌20分钟，加入3,5-二对氯苯甲酰氧基-2-D-脱氧核糖氯(1.25g，2.9mmol)，室温继续搅拌反应3h，加入乙酸乙酯和水分层，有机相用无水硫酸钠干燥，减压旋蒸浓缩，残留物经硅胶柱层析(石油醚/乙酸乙酯:5/1)得450mg粗产物。
(2)1-苯基-3-甲基-5-O-D-(2-脱氧)核糖苷-吡唑的制备
Figure PCTCN2019100835-appb-000107
5-(3,5-二对氯苯甲酰氧基-2-D-脱氧核糖苷)-3-甲基-1-苯基-1H吡唑(450mg，0.8mmol)溶于甲醇和二氯甲烷(1:1，10mL)中，加入甲醇钠甲醇溶液(30％，0.2mL)，室温反应2h，用醋酸调节pH至7左右，减压除去溶剂，残留物经硅胶柱层析(石油醚/乙酸乙酯:1/1.5)得100mg白色固体产物。
1H MNR(400MHz，CD 3OD)，δ7.55(dd，J＝8.7Hz，J＝1.2Hz,2H)；7.44(t，J＝7.9Hz,2H)；7.31(t，J＝7.4Hz,1H)；5.82(m,1H)；5.80(s,1H)；4.35(dd，J＝11.4Hz，J＝6.6Hz,1H)；3.96(dd，J＝10.9Hz，J＝5.3Hz,1H)；3.55(dd，J＝11.7Hz，J＝5.2Hz,1H)；3.45(dd，J＝11.7Hz，J＝6.3Hz,1H)；2.43(dd，J＝13.8Hz，J＝6.7Hz,1H)；2.22-2.26(m,4H)
ESI-MS(m/z):[M] +291.17
实施例41-52
合成路线4：
Figure PCTCN2019100835-appb-000108
通用合成方法4：
第一步：
将式(H)化合物(5.62mmol)和KOH(0.66mmol)溶于20mL H 2O/二氧六环(1:3v/v)混合溶液，向上述混合溶液中加入甲醛(35％水溶液，8.43mmol)，反应液于70℃反应5min，然后降温至25℃搅拌过夜。用1N HCl调节pH至7，减压浓缩。残余物用丙酮溶解，无水硫酸钠干燥，过滤，浓缩，得到式(G)化合物，直接用于下一步。
第二步：
式(G)化合物(3.0mmol)溶于10mL POCl 3，加热至回流，小心分批加入PCl 5(6.0mmol)，回流反应2.5h，TLC检测反应完成。降温至室温，反应液倒入200mL冰水中，1N NaOH溶液调pH至中性，CH 2Cl 2萃取，无水硫酸钠干燥，过滤，浓缩，柱层析纯化，得到式(F)化合物。
第三步：
将依达拉奉(3.6mmol)、碘化钾(0.4mmol)、碳酸钾(10mmol)溶解在干燥的DMF(10mL)中，45℃条件下搅拌10min。然后加入式(F)化合物(3.3mmol)的DMF溶液，45℃条件下搅拌1h。反应完成后，用油泵抽走大量溶剂，然后向反应液中加入100mL乙酸乙酯，然后用饱和氯化铵水溶液洗涤，将水相用乙酸乙酯萃取两次，合并有机相。将有机相依次用蒸馏水，饱和氯化钠溶液洗涤，然后用无水硫酸钠干燥，减压浓缩，得到的粗产品经硅胶柱层析纯化得到目标产物。
实施例41 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基乙酰胺的制备
Figure PCTCN2019100835-appb-000109
采用通用合成方法4制备，得到黄色油状物，收率43.9％。
1H NMR(400MHz,CDCl 3)δ8.03(s,1H),7.60–7.50(m,2H),7.45–7.40(m,2H),7.31–7.26(m,1H),6.08(s,1H),5.84(s,2H),2.32(s,3H),1.83(s,3H)。
ESI-MS(m/z):[M] +246.10
实施例42 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基丙酰胺的制备
Figure PCTCN2019100835-appb-000110
采用通用合成方法4制备，得到黄色油状物，收率41.2％。
1H NMR(400MHz,CDCl 3)δ8.01(s,1H),7.62–7.51(m,2H),7.43–7.41(m,2H),7.32–7.29(m,1H),6.07(s,1H),5.83(s,2H),2.32(s,3H),2.27(m,2H),1.02(t,3H)。
ESI-MS(m/z):[M] +260.17
实施例43 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基异丁酰胺的制备
Figure PCTCN2019100835-appb-000111
采用通用合成方法4制备，得到黄色油状物，收率40.8％。
1H NMR(400MHz,CDCl 3)δ8.03(s,1H),7.61–7.54(m,2H),7.45–7.40(m,2H),7.34–7.28(m,1H),6.05(s,1H),5.86(s,2H),2.53(dt,J＝14.0,7.0Hz,1H),2.33(s,3H),1.22(d,J＝7.0Hz,6H)。
ESI-MS(m/z):[M] +274.16
实施例44 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基三甲基乙酰胺的制备
Figure PCTCN2019100835-appb-000112
采用通用合成方法4制备，得到黄色油状物，收率43.2％。
1H NMR(400MHz,CDCl 3)δ8.02(s,1H),7.62–7.53(m,2H),7.47–7.41(m,2H),7.35–7.27(m,1H),6.03(s,1H),5.82(s,2H),2.33(s,3H),1.20(s,9H)。
ESI-MS(m/z):[M] +288.12
实施例45 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基环丙基甲酰胺的制备
Figure PCTCN2019100835-appb-000113
采用通用合成方法4制备，得到黄色油状物，收率38.1％。
1H NMR(400MHz,CDCl 3)δ8.03(s,1H),7.64–7.53(m,2H),7.45–7.41(m,2H),7.34–7.26(m,1H),6.01(s,1H),5.83(s,2H),2.33(s,3H),1.43(m,1H),0.98(dd,J＝4.42,2.98Hz,2H),0.79(dd,J＝7.91,2.95Hz,2H)。
ESI-MS(m/z):[M] +272.15
实施例46 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基环戊基甲酰胺的制备
Figure PCTCN2019100835-appb-000114
采用通用合成方法4制备，得到黄色油状物，收率39.8％。
1H NMR(400MHz,CDCl 3)δ8.02(s,1H),7.61–7.53(m,2H),7.44–7.40(m,2H),7.32–7.25(m,1H),6.02(s,1H),5.81(s,2H),2.34(s,3H),2.61(q,J＝8.0Hz,1H),1.93-1.84(m,2H),1.82-1.68(m,4H),1.64-1.52(m,2H)。
ESI-MS(m/z):[M] +300.23
实施例47 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基环已基甲酰胺的制备
Figure PCTCN2019100835-appb-000115
采用通用合成方法4制备，得到黄色油状物，收率40.1％。
1H NMR(400MHz,CDCl 3)δ8.03(s,1H),7.62–7.53(m,2H),7.45–7.41(m,2H),7.33–7.24(m,1H),6.03(s,1H),5.82(s,2H),2.32(s,3H),2.13(m,1H),1.89(m,2H),1.78(m,2H),1.66(m,1H),1.42(m,2H),1.24(m,3H)。
ESI-MS(m/z):[M] +314.24
实施例48 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基苯甲酰胺的制备
Figure PCTCN2019100835-appb-000116
采用通用合成方法4制备，得到黄色油状物，收率45.8％。
1H NMR(400MHz,CDCl 3)δ8.02(s,1H),7.84–7.81(m,2H),7.65–7.59(m,2H),7.57–7.52(m,1H),7.49–7.43(m,4H),7.35–7.26(m,1H),6.05(s,1H),5.87(s,2H),2.30(s,3H)。
ESI-MS(m/z):[M] +308.16
实施例49 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基苯乙酰胺的制备
Figure PCTCN2019100835-appb-000117
采用通用合成方法4制备，得到黄色油状物，收率43.4％。
1H NMR(400MHz,CDCl 3)δ8.02(s,1H),7.65–7.59(m,2H),7.45–7.41(m,2H),7.35–7.20(m,6H),6.05(s,1H),5.87(s,2H),3.38(s,2H),2.30(s,3H)。
ESI-MS(m/z):[M] +322.20
实施例50 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基苯丁酰胺的制备
Figure PCTCN2019100835-appb-000118
采用通用合成方法4制备，得到黄色油状物，收率40.5％。
1H NMR(400MHz,CDCl 3)δ8.03(s,1H),7.62–7.53(m,2H),7.45–7.41(m,2H),7.33–7.24(m,3H),7.18(t,J＝7.6Hz,3H),6.03(s,1H),5.82(s,2H),2.68(t,J＝7.5Hz,2H),2.32(s,3H),2.22(t,J＝7.5Hz,2H),2.04–1.93(m,2H)。
ESI-MS(m/z):[M] +350.11
实施例51 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基辛酰胺的制备
Figure PCTCN2019100835-appb-000119
采用通用合成方法4制备，得到黄色固体，收率38.3％。
1H NMR(400MHz,CDCl 3)δ8.02(s,1H),7.61–7.54(m,2H),7.46–7.41(m,2H),7.31–7.25(m,1H),6.02(s,1H),5.81(s,2H),2.38(t,J＝4.0Hz,2H),2.31(s,3H),1.51–1.46(m,2H),1.34–1.25(m,8H),0.90(t,J＝4.0Hz,3H)。
ESI-MS(m/z):[M] +330.18
实施例52 N-((1-苯基-3-甲基-1H-吡唑-5-基)氧基)甲基硬脂酰胺的制备
Figure PCTCN2019100835-appb-000120
采用通用合成方法4制备，得到黄色固体，收率32.7％。
1H NMR(400MHz,CDCl 3)δ8.02(s,1H),7.61–7.54(m,2H),7.46–7.41(m,2H),7.31–7.25(m,1H),6.02(s,1H),5.81(s,2H),2.31(s,3H),2.22(t,J＝7.6Hz,2H),1.65–1.63(m,2H),1.34–1.25(m,28H),0.87(t,J＝6.8Hz,3H)。
ESI-MS(m/z):[M] +470.30
试验例
试验例1：稳定性研究
分别在高湿(25℃±2℃,90％±5RH)、光照(25℃±2℃,60％±5RH,4500±500LX)、高温(60℃±2℃，60％±5RH)条件下考察部分实施例样品的稳定性，样品在特定条件的稳定性试验箱中放置7天，检测有关物质和含量，结果列于表2
表2
Figure PCTCN2019100835-appb-000121
Figure PCTCN2019100835-appb-000122
注：有关物质无显著变化，是指被测试样品的性状、含量与初始测试值相差在仪器测定误差范围内。
结论：依达拉奉在光照条件下含量显著降低，与依达拉奉以酯键直接偶联连的专利CN107400089中公开的化合物分别在高湿(25℃±2℃,90％±5RH)、光照(25℃±2℃,60％±5RH,4500±500LX)及高温(60℃±2℃，60％±5RH)测试条件下产生分解。本发明中的上述化合物在上述药学稳定性标准测试条件下均优于依达拉奉和CN107400089中公开的化合物。
试验例2：溶解度试验
实验方法：用5mL的EP管取大约0.5mL蒸馏水，慢慢加入干燥的化合物至不能溶解(25℃超声震荡，仍出现浑浊)。将溶液过滤至另一个5mL干净且已称重的EP管中，再称重，计算出溶液的重量。将滤液冻干，称重并计算出所剩固体的溶质质量，计算溶剂的质量，进而计算化合物在水中的溶解度。
表3
化合物 溶解度(mg/mL)
33 10.1
34 12.3
35 18.0
36 4.2
37 6.5
38 3.8
39 5.1
40 3.0
依达拉奉 <1.0
结论：本发明中的上述化合物在水中的溶解度远远大于已经上市药物依达拉奉，水溶性能够提高几倍至几十倍。
试验例3细胞毒性考察
1、试验方法
将PC12细胞复苏后，用含10％的马血清和5％胎牛血清的RPMI-1640培养基，37℃、5％CO 2的环境下培养两周，隔一天换一次液。用培养基分别将化合物稀释为12.5、25、50、100、200、300μM的溶液(实施例33-40的化合物另增加400、500μM的溶液)备用。将PC12细胞以1.2×10 4个/孔的密度接种于96孔板内，37℃、5％CO 2培养箱中培养24h后，弃液，分别加入上述系列浓度的化合物溶液作为供试液，37℃、5％CO 2培养箱中继续孵育48h。
孵育结束后，向细胞孔内加入20μL 5mg/mL的MTT溶液，于37℃条件下孵育4h后，小心吸取上层培养基，加入150μL DMSO，置于酶标仪中，震荡5min，检测490nm波长处的吸光度值A 供试液。以无细胞的空白孔加入DMSO后测得的吸光度值A 空白作为空白，以未加药处理的细胞孔同法操作测得的吸光度值A 对照作为对照，计算各孔细胞存活率，细胞存活率(％)＝(A 供试液-A 空白)/(A 对照-A 空 白)×100％。
2、试验结果
表4细胞毒性试验结果
化合物 IC 50(μM) 化合物 IC 50(μM)
3 >300 32 >300
4 >300 33 >300
5 >300 34 >300
7 >300 35 >300
9 >300 36 >300
13 251.5 37 >500
16 233.9 38 >500
17 >300 39 >500
18 >300 40 >500
19 >300 42 >300
24 272.3 47 268.9
25 >300 49 >300
26 >300    
结论：检测化合物在100μM浓度下，均未见明显毒性。
试验例4：体外神经保护作用-I
1、试验方法
样品配制：
所有被测样品用DMSO配制成60mM母液，再用细胞培养基稀释200倍至终浓度300μM。之后用细胞培养基稀释成不同浓度梯度的样品溶液备用。
测试分组：
对照组：正常铺设细胞，与其他被测试细胞组同样只加培养基，培养24h后弃液，再只添加培养基继续孵育24h，不进行化合物和双氧水处理。其他操作与样品组相同。
空白组：不铺设细胞，只添加培养基。其他操作与样品组相同。
样品测试：
将PC12细胞复苏后，用含10％的马血清和5％胎牛血清的RPMI-1640培养基，37℃、5％CO 2的环境下培养两周，隔一天换一次液。培养基分别将化合物稀释为0、12.5、25、50、100、200、300μM的溶液备用。将PC12细胞以8000个/孔的密度接种于96孔板内，37℃、5％CO 2培养箱中培养24h后，弃液，分别加入上述系列浓度的化合物溶液作为供试液，37℃、5％CO 2培养箱中继续孵育24h。孵育后，除空白组和对照组外，其余各孔(包括0μM给药组)加入培养基溶解的终浓度为200μM的H 2O 2(双氧水)溶液，37℃、5％CO 2培养箱中继续孵育24h。
孵育结束后，向细胞孔内加入20μL 5mg/mL的MTT溶液，于37℃条件下孵育4h后，小心吸取上层培养基，加入150μL DMSO，置于酶标仪中，震荡5min，检测490nm波长处的吸光度值A 供试液。以无细胞的空白孔加入DMSO后测得的吸光度值A 空白作为空白，以未加化合物和双氧水处理的对照组同法操作测得的吸光度值A 对照作为对照，计算各孔细胞存活率，细胞存活率(％)＝(A 供试液-A 空白)/(A 对照-A 空白)×100％。
2、试验结果
表5体外神经保护试验结果
化合物 EC 50(μM) 化合物 EC 50(μM) 化合物 EC 50(μM)
1 66.4 21 82.1 37 90.0
3 98.0 22 103.2 39 55.0
4 69.5 23 123.7 40 54.7
6 56.8 24 44.7 41 55.1
7 88.9 25 59.3 42 75.4
8 100.4 26 54.8 43 58.1
9 120.5 28 85.9 44 77.9
10 55.7 30 77.2 45 75.3
12 89.0 31 91.2 46 90.1
13 110.0 32 80.7 47 53.6
14 45.2 33 111.9 48 122.7
15 67.2 34 120.3 50 100.9
16 48.0 35 87.9 52 78.6
17 94.9 36 79.6 ED 170.8
18 75.0        
结论：上述实验结果体现了本发明取代吡唑类化合物针对神经细胞在过氧化物，即氧自由基损伤条件下对神经细胞的有效保护作用。自由基对脑神经细胞、线粒体等的损伤是公认的脑卒中，脑栓塞，脑卒中后遗症，脑卒中运动机能障碍，线粒体脑肌病，以及肌萎缩性侧索硬化症的病理机制。选择和使用针对自由基损伤有效的药物对上述疾病患者实施临床治疗是目前主要的防治手段之一。
试验例5：体内药效试验MCAO模型
1、试验方法
(1)模型制备
SD大鼠，禁食过夜，自由饮水。实验当天用异氟烷气体麻醉并维持。仰卧位固定，沿颈正中线切开皮肤，暴露右侧颈总动脉，小心分离颈总动脉分叉至颅底部血管周围的神经及筋膜，分离颈外动脉分支枕动脉、甲状腺上动脉、舌动脉和上颚上颌动脉，结扎并切断。从颈外动脉游离端插入直径0.260mm尼龙丝线，从颈外动脉远心端将尼龙线导入到颈内动脉，插至Willis环大脑中动脉处，以有效阻断大脑中动脉，插入的尼龙丝线长度距颈总动脉分叉处18～20mm。然后将颈外动脉游离端连同腔内尼龙丝线一并结扎。逐层缝合皮下筋膜和皮肤，肌肉注射青霉素防止感染。假手术组动物仅分离出颈内动脉。
MCAO后2h，小心抽出颈内动脉血管腔内的尼龙丝线，使颈内动脉的血流再灌注，10min后进行神经缺陷评分，有明显神经功能缺陷者(>8分)为造型成功。
(2)分组给药
选取造模成功的动物，采用随机数字表法随机分组，每组10只。分别为模型对照组、受试药组，对照依达拉奉组，另设10只假手术组。
采用尾静脉输注方式给药，给药时间均为30min。假手术组和模型对照组给予等体积生理盐水溶液。
(3)对神经功能的影响
分别于治疗前、治疗后24h，根据表6评分标准，采用盲法对动物进行神经功能缺陷评分，用于评价神经功能损害程度，总分为16分，分数越高，表明动物神经功能损害程度越严重。每组评分与模型对照组比较，减少的百分数体现化合物对神经功能缺陷的改善程度，即：神经功能改善(％)＝(模型对照组—受试药组或依达拉奉组)/模型对照组×100％。结果见表9。
表6大鼠神经功能损害程度评分标准
Figure PCTCN2019100835-appb-000123
(4)脑梗死范围测定
实验结束后取脑，沿冠状面切成2mm厚的切片，间隔取上述一半脑片放入2％TTC染液中，避光37℃温孵10min进行染色，采用数码相机成像系统将数字影象存储于计算机中，并用图象分析系 统v4.0软件测量梗死区和全脑面积，计算脑梗死范围(梗死区面积占全脑面积百分比)。
(5)统计学处理
所有数据均表示为均数±标准差
Figure PCTCN2019100835-appb-000124
表示，单因素方差分析比较组间差异性，两组间比较用LSD法，P<0.05为差异有统计学意义。
2、试验结果
表7 MCAO实验结果
分组 神经功能改善(％) 脑梗死范围(％)
假手术 不评价 0
模型对照 1.25 30.2
ED 15.7 20.1
9 16.0 19.8
13 17.2 17.5
15 15.9 20.0
16 16.2 19.9
33 24.0 14.0
34 21.1 14.5
35 22.2 15.8
36 24.1 14.1
37 17.5 17.6
38 17.0 17.8
39 16.9 18.0
40 16.8 18.5
42 15.9 19.7
43 16.1 19.8
结论：在大鼠大脑中动脉缺血再灌注引起的局灶性脑梗塞损伤模型上，本发明的上述化合物静脉输注、单次治疗给药，可具有不同的治疗效应，其效应表现在能不同程度地改善受损的神经功能，不同程度地减小脑梗死范围，减轻脑水肿程度。
试验例6：体内药效试验Ⅱ-ALS模型
1、试验方法：
采用B6SJL-Tg(SOD1*G93A)小鼠，单剂量腹腔给药，每日给药一次，连续给药4周，给药前及给药后每周称量两次小鼠体重，并采用疲劳转棒仪、悬线试验评价小鼠的运动功能，并在给药前和给药四周后的两个时间点，收集血浆，采用ELISA法检测小鼠血浆SOD水平。通过上述指标评价实施例化合物对ALS小鼠模型的药效作用，观察实施例化合物对小鼠生存期和运动功能的改善作用。
(1)试验分组和给药方法
动物送达SPF动物房后适应一周以上。试验前5天开始，每天一次转棒练习，使小鼠习惯转棒运动。试验开始日按小鼠体重随机分组，每组8只。其中，溶剂组每天给药同体积溶剂(生理盐水)。
(2)给药体积
给药体积为10mL/kg。
(3)给药方式和时间
每日腹腔给药一次，连续给药4周。
(4)指标评价
指标评价时间期限：从试验开始至全部动物死亡为止。
1)ALS发病时间：连续2天观察，在悬尾状态下出现肢体震颤和(或)肢体无力即判为发病。
肢体无力的判断方法：试验鼠放置于直径为3.5cm的转棒仪上，转速调至15rpm，记录坠落的最长潜伏期，以420秒为分界值，不足420秒即视为肢体无力。
2)转棒试验：试验鼠放置于直径为3.5cm的转棒仪上，转速调至12rpm，记录坠落的最长潜伏期，以180秒为分界值，超过180秒按180秒记录，不足180秒按实际时间记录。各组ALS小鼠转棒实验坠落潜伏期的比较结果见图1。
3)悬线试验：将试验鼠置于传统用的鼠笼盖上，轻轻震动鼠笼盖促使试验鼠紧握鼠笼盖，随后迅速翻转鼠笼盖，记录后肢离开笼盖的最长时间，以90秒为分界值，超过90秒按90秒记录，不足90秒按实际时间记录。每次实验重复3次并取其最好成绩记录。各组ALS小鼠悬线实验坠落潜伏期的比较结果见图2。
4)小鼠死亡时间：将鼠置于仰卧位，20秒内不能翻身为俯卧位即判断为死亡。
5)血浆SOD浓度：在给药前和给药四周后的两个时间点，用含有肝素的EP管采血，充分混合，1000g×10min离心，收集血浆，分装后放于-70℃冰箱。用ELISA试剂盒检测血浆SOD水平。
2、试验结果
表8 ALS模型试验结果
Figure PCTCN2019100835-appb-000125
注：①“+”表示与溶剂组相比具有显著性差异，p<0.05。②实施例化合物组与ED组等摩尔给药。③*号表示与溶剂组相比具有统计学差异,p<0.05。
结论：
通过28天药物干预：(1)与溶剂组相比，各组ALS小鼠发病时间不同程度延后；(2)转棒试验和悬线试验结果显示，各给药组坠落潜伏期延长，且实施例24、33组的效果优于已上市药依达拉奉，具有显著药效；(3)各给药组ALS小鼠的存活时间明显延长；(4)血浆SOD结果显示，在给药28天后，给药组小鼠的血浆SOD水平明显高于溶剂组小鼠的血浆SOD水平，给药化合物抑制了SOD值下降。

Claims (10)

  1. 一种式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，
    Figure PCTCN2019100835-appb-100001
    其中：
    R 1表示氢或烷基；
    X表示-O-、-S-或-NH-；
    Figure PCTCN2019100835-appb-100002
    表示双键，A为O；
    R 2表示H、任选取代的烷基、任选取代的环烷基、任选取代的非芳香杂环基、任选取代的芳基、任选取代的杂芳基、任选取代的芳烷基、或者任选取代的杂芳烷基，
    所述“任选取代的”是指未取代或被一个或多个取代基取代，所述取代基选自羟基、烷氧基、芳氧基、芳基烷氧基、卤素、烷酰氧基、烷氧酰氧基、未取代或烷基取代的氨基；
    或者，
    Figure PCTCN2019100835-appb-100003
    表示单键，式(I)中的下式(i)部分
    Figure PCTCN2019100835-appb-100004
    形成下式(ii)所示的基团，
    Figure PCTCN2019100835-appb-100005
    式(ii)中，n为1或者2，m为1，2，3或4，
    各个R 3相互独立地为羟基，羟甲基，烷酰氧基(优选乙酰氧基)、任选被卤素取代的苯甲酰氧基(优选苯甲酰氧基、对氯苯甲酰氧基)、烷酰氧基甲基(优选乙酰氧基甲基)、任选被卤素取代的苯甲酰氧基甲基(优选苯甲酰氧基甲基、对氯苯甲酰氧基甲基)、烷氧基、烷氧基甲基、或者未取代或被选自烷基、烷酰基(优选乙酰基)、任选被卤素取代的苯甲酰基(优选苯甲酰基、对氯苯甲酰基)的基团、单取代或二取代的氨基；
    可选地，所述“烷基”，以及“芳烷基”、“杂芳烷基”、“烷氧基”、“芳基烷氧基”、“烷酰氧基”、“烷氧酰氧基”、“烷酰氧基甲基”中的烷基部分各自独立地为C 1-20直链或支链烷基，可选地，为C 1-17 直链或支链烷基，可选地，为C 1-8直链或支链烷基，可选地，为C 1-6直链或支链烷基，可选地，为C 1-4直链或支链烷基，可选地，为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，异戊基、新戊基、叔戊基、正己基、异己基、庚基、正辛基、正壬基、正癸基、十二烷基、十五烷基或十六烷基；
    可选地，所述“环烷基”为C 3-8环烷基，可选地，为环丙基、环丁基、环戊基、或环己基；
    可选地，所述“非芳香杂环基”为含有选自O、N、S的1-2个杂原子的非芳香C 3-8杂环基，可选地，为环氧乙烷基、氧杂环丁烷基、四氢呋喃基、四氢吡咯基、四氢吡喃基、哌啶基、哌嗪基、或吗啉基；
    可选地，所述“芳基”、“芳烷基”中的芳基为苯基、或萘基；
    可选地，所述“杂芳基”、以及“杂芳烷基”中的杂芳基部分各自独立地为含有选自O、N、S的1-2个杂原子的5-10元单环或双环稠合芳香杂环基团，可选地，为吡咯基、呋喃基、吡啶基、吡嗪基、或嘧啶基。
  2. 根据权利要求1所述的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，其中，
    R 1表示氢或甲基；
    X表示-O-或-NH-；
    Figure PCTCN2019100835-appb-100006
    表示双键，A为O；
    R 2为C 1-C 17的支链或直连烷基，C 3- 6环烷基，苯基C 1-C 6烷基，吡啶基，任选被一个或多个选自羟基、C 1-6烷酰氧基、C 1-6烷氧酰氧基或-NR′R″的基团取代的苯基，其中R′R″各自独立地为C 1-C 6直连或支链的烷基；
    或者，
    Figure PCTCN2019100835-appb-100007
    表示单键，式(I)中的式(i)部分形成式(ii)所示的基团，
    式(ii)中，n为1或者2，m为2，3或4，
    各个R 3相互独立地为羟基，羟甲基，或者未取代或被C 1-6烷酰基单取代的氨基。
  3. 根据权利要求1或2所述的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，其中，式(I)所示的取代吡唑类化合物为下式II、III或IV所示的化合物，
    Figure PCTCN2019100835-appb-100008
    其中，R 2如权利要求1或2中所定义。
  4. 根据权利要求1所述的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，
    其中，式(I)中的式(i)部分形成下式(iii)或(iv)所示的基团，
    Figure PCTCN2019100835-appb-100009
    其中，R′ 1为氢原子、羟甲基、C 1-4烷酰氧基甲基；R′ 2为羟基或C 1-4烷酰氧基；R′ 3为氢、羟基、C 1-4烷酰氧基、氨基、或C 1-4烷酰氨基。
  5. 根据权利要求4所述的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，其中，式(I)中的式(i)部分形成下式所示的基团，
    Figure PCTCN2019100835-appb-100010
  6. 根据权利要求1所述的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，其中，所述式(I)所示化合物选自以下化合物：
    Figure PCTCN2019100835-appb-100011
    Figure PCTCN2019100835-appb-100012
    Figure PCTCN2019100835-appb-100013
    Figure PCTCN2019100835-appb-100014
  7. 权利要求1-6任一项所述的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物的制备方法，包括将式(A)化合物与依达拉奉在碱性条件下反应的步骤，
    Figure PCTCN2019100835-appb-100015
    式中，R 1、X、A、R 2、
    Figure PCTCN2019100835-appb-100016
    的定义如权利要求1-6所述，Y为卤素，优选为Cl或Br。
  8. 根据权利要求7所述的制备方法，其中，
    式(I)化合物为式(II)所示的化合物，所述制备方法的合成路线如下所示，
    Figure PCTCN2019100835-appb-100017
    所述制备方法包括：
    1)将式(C)化合物在催化剂的存在下与乙醛反应，制得式(B)化合物；
    2)将式(B)化合物在碱性条件下与依达拉奉反应，制得式(II)化合物；
    可选的，所述式(C)的制备方法包括将式(D)化合物与二氯亚砜反应；
    Figure PCTCN2019100835-appb-100018
    或者，
    其中式(I)化合物为式(III)所示的化合物，所述制备方法的合成路线如下所示，
    Figure PCTCN2019100835-appb-100019
    所述制备方法包括：
    1)将式(D)化合物与氯甲基氯磺酸酯反应，制得式(E)化合物；
    2)将式(E)化合物在碱性条件下与依达拉奉反应，制得式(III)化合物；
    或者，
    其中式(I)化合物为式(IV)所示的化合物，所述制备方法的合成路线如下所示，
    Figure PCTCN2019100835-appb-100020
    所述制备方法包括：
    1)将式(H)化合物与甲醛反应，制得式(G)化合物；
    2)将式(G)化合物与PCl 5/POCl 3反应制得式(F)化合物，
    3)将式(F)化合物在碱性条件下与依达拉奉反应，制得式(IV)化合物；
    或者，
    其中式(I)化合物为下式(V)所示的化合物，所述制备方法的合成路线如下所示，
    Figure PCTCN2019100835-appb-100021
    式中，Y为Cl或Br，
    所述制备方法包括将式(M)化合物在碱性条件下与依达拉奉反应制得式(V)化合物；
    在上述结构式中，R 2、R 3、m、n定义如权利要求1或2所述。
  9. 一种药物组合物，包括权利要求1-6任一项所述的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物中的一种或多种以及任选的药学上可接受的载体。
  10. 权利要求1-6任一项所述的式(I)所示的取代吡唑类化合物或其药学上可接受的盐、或者它们的溶剂化物，或者权利要求9所述的药物组合物的用途，用于制备预防或治疗脑卒中、脑栓塞、脑卒中后遗症、脑卒中运动机能障碍、线粒体脑肌病、和/或肌萎缩性脊髓侧索硬化症的药物。
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