WO2016091042A1 - 吡啶甲酰胺类衍生物、其制备方法及其在医药上的应用 - Google Patents

吡啶甲酰胺类衍生物、其制备方法及其在医药上的应用 Download PDF

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WO2016091042A1
WO2016091042A1 PCT/CN2015/094164 CN2015094164W WO2016091042A1 WO 2016091042 A1 WO2016091042 A1 WO 2016091042A1 CN 2015094164 W CN2015094164 W CN 2015094164W WO 2016091042 A1 WO2016091042 A1 WO 2016091042A1
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Prior art keywords
group
compound
formula
romk
cycloalkyl
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PCT/CN2015/094164
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English (en)
French (fr)
Inventor
李心
董庆
孙飘扬
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to MX2017006536A priority Critical patent/MX2017006536A/es
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to KR1020177018177A priority patent/KR20170090477A/ko
Priority to CN201580008517.8A priority patent/CN105980379B/zh
Priority to JP2017528783A priority patent/JP6736816B2/ja
Priority to CA2966788A priority patent/CA2966788A1/en
Priority to RU2017121460A priority patent/RU2694899C9/ru
Priority to US15/531,525 priority patent/US10364234B2/en
Priority to EP15868279.9A priority patent/EP3231802B1/en
Priority to BR112017010645A priority patent/BR112017010645A2/pt
Priority to AU2015361614A priority patent/AU2015361614B2/en
Publication of WO2016091042A1 publication Critical patent/WO2016091042A1/zh
Priority to US16/257,484 priority patent/US10759788B2/en
Priority to US16/257,716 priority patent/US10442794B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4355—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having oxygen as a ring hetero atom
    • 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
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • 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
    • 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
    • A61P9/12—Antihypertensives
    • 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
    • C07D401/12—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 linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • the present invention relates to a class of pyridine carboxamide derivatives, a process for their preparation and pharmaceutical compositions containing the same, and their use as therapeutic agents, in particular as inhibitors of extrarenal medulla secretory potassium channel (ROMK) And its use in the manufacture of a medicament for the treatment and/or prevention of disorders caused by excessive salt and water retention, including hypertension and heart failure.
  • ROMK extrarenal medulla secretory potassium channel
  • the renal outer medullary potassium channel is also called inward-rectifying potassium channel 1.1 (Kir1.1).
  • the ROMK ion channel can coordinate with the Na+-K+-2Cl-cotransporter NKCC2 (responsible for NaCl transport) to regulate Na+ reabsorption through the membranous membrane conductance of the renal medullary ascending segment (TAL).
  • TAL renal medullary ascending segment
  • Batter syndrome is an autosomal recessive hereditary disease characterized by massive loss of salt in the kidneys, hypokalemia, and low blood pressure.
  • Paramyelocytic hyperplasia is mainly caused by mutation of ROMK or Na + -K + -2Cl - cotransporter, except that hypokalemia caused by rotaside cell hyperplasia caused by ROMK mutation is better than Na + -K + - Parathyroid cell hyperplasia induced by 2Cl - cotransporter mutations is greatly alleviated.
  • ROMK suppression function may without causing hypokalemia, effective to inhibit Na + -K + -2Cl - transporter salt reabsorption promoting excretion of urine, functions as a diuretic antihypertensive efficacy.
  • a series of compounds of the ROMK inhibitor have been disclosed, including patent applications such as WO2010129379, WO2012058134, WO2012058116, WO2012058134, WO2013066714, WO2013028474, WO2014085210, WO2014018764, WO2014015495, WO2014085210, WO2013039802, WO2013062892 and WO2012058116, but still need to be developed hERG selective compound.
  • the invention designs a compound having the structure represented by the general formula (I), increases the polar group, reduces the ClogP on the basis of maintaining the ROMK inhibitory activity, improves the hERG selectivity, and increases the safety.
  • R 1 is an alkyl group, wherein the alkyl group is optionally further selected from one of a halogen, a hydroxyl group, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, a carboxyl group, and a carboxylate group. Substituted by a plurality of substituents;
  • R 2 is selected from the group consisting of a hydrogen atom, an alkyl group, a halogen, a cyano group, a nitro group, an alkoxy group, a cycloalkyl group, and a heterocyclic group, wherein the alkyl alkoxy group, cycloalkyl group or heterocyclic group is optionally further Substituted by one or more substituents selected from the group consisting of alkyl, halogen, hydroxy, hydroxyalkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid, and carboxylic acid esters;
  • R 3 is selected from the group consisting of:
  • R 4 or R 5 are each independently selected from the group consisting of a hydrogen atom, an alkyl group, a halogen, a cyano group, a nitro group, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;
  • R 6 is selected from a hydrogen atom, an alkyl group, and a halogen
  • n 0, 1, or 2.
  • a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer thereof, a mixture thereof, or a pharmaceutically acceptable salt thereof wherein R 1 is an alkyl group, wherein the alkyl group is optionally further substituted selected from halogen, hydroxy and alkoxy of one or more substituents; R 1 is preferably a C 1-6 alkyl group, more preferably a methyl group, an ethyl group or a propyl group.
  • a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer thereof, Or a mixture thereof, or a pharmaceutically acceptable salt thereof wherein R 4 is an alkyl group, and R 5 is a hydrogen atom.
  • a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer thereof, Or a mixture thereof, or a pharmaceutically acceptable salt thereof which is a compound of the formula (II):
  • R 1 , R 2 , R 4 , n are as defined in formula (I).
  • a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer thereof, Or a mixture thereof, or a pharmaceutically acceptable salt thereof which is a compound of the formula (III):
  • R 1 , R 2 , R 4 , n are as defined in formula (I).
  • a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer thereof, Or a mixture thereof, or a pharmaceutically acceptable salt thereof which is a compound of the formula (IV):
  • R 1 , R 2 , R 3 , n are as defined in formula (I).
  • a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer thereof, Or a mixture thereof, or a pharmaceutically acceptable salt thereof which is a compound of the formula (V):
  • R 1 , R 2 , R 4 , n are as defined in formula (I).
  • a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer thereof, Or a mixture thereof, or a pharmaceutically acceptable salt thereof which is a compound of the formula (VI):
  • R 1 , R 2 , R 4 , n are as defined in formula (I).
  • Typical compounds of the invention include, but are not limited to:
  • a tautomer a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a compound of the formula (IA) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof. Or a pharmaceutically acceptable salt thereof, as an intermediate for the preparation of a compound of the formula (I):
  • R 1 is an alkyl group, wherein the alkyl group is optionally further selected from one of a halogen, a hydroxyl group, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, a carboxyl group, and a carboxylate group. Substituted by a plurality of substituents;
  • R 2 is selected from the group consisting of a hydrogen atom, an alkyl group, a halogen, a cyano group, a nitro group, an alkoxy group, a cycloalkyl group, and a heterocyclic group, wherein the alkyl group, alkoxy group, cycloalkyl group or heterocyclic group is optionally further Substituted by one or more substituents selected from the group consisting of alkyl, halogen, hydroxy, hydroxyalkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxy, and carboxylate; Can be used to prepare a compound of the formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or An intermediate of a pharmaceutically acceptable salt;
  • n 0, 1, or 2.
  • a compound represented by the formula (IA) or a tautomer thereof, internal elimination a compound represented by the formula (IVA), which is a form of a compound of the formula (IVA): a form of a compound of the formula (IVA): a compound, a pharmaceutically acceptable salt, or a mixture thereof;
  • a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof a compound represented by IV) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof
  • An intermediate wherein: R 1 , R 2 , n are as defined in formula (IA).
  • Typical compounds of the compounds of formula (IA) include, but are not limited to:
  • a tautomer a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof. a method of form, or a pharmaceutically acceptable salt thereof, the method comprising:
  • R 1 -R 3 , n are as defined in the formula (I).
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount such as a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable compound thereof Salts and pharmaceutically acceptable carriers, diluents or excipients.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, in the preparation of a ROMK inhibitor.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the preparation of a medicament for treating or preventing hypertension or heart failure.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the preparation of a medicament for treating or preventing a ROMK-mediated disease, including but not limited to liver cirrhosis, acute and chronic renal insufficiency, nephropathy Syndrome, pulmonary hypertension, cardiovascular disease, myocardial infarction, stroke, cardiac insufficiency, excessive pulmonary tension, atherosclerosis and kidney stones.
  • a ROMK-mediated disease including but not limited to liver cirrhosis, acute and chronic renal insufficiency, nephropathy Syndrome, pulmonary hypertension, cardiovascular disease, myocardial infarction, stroke, cardiac insufficiency, excessive pulmonary tension, atherosclerosis and kidney stones.
  • the present invention also relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, which is used as a ROMK inhibitor.
  • the present invention also relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the treatment or prevention of hypertension or heart failure.
  • the present invention also relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in the treatment or prevention of a ROMK-mediated disease, preferably selected from the group consisting of cirrhosis, acute and chronic renal insufficiency, nephrotic syndrome, pulmonary artery Hypertension, cardiovascular disease, myocardial infarction, stroke, cardiac insufficiency, excessive pulmonary tension, atherosclerosis and kidney stones.
  • a ROMK-mediated disease preferably selected from the group consisting of cirrhosis, acute and chronic renal insufficiency, nephrotic syndrome, pulmonary artery Hypertension, cardiovascular disease, myocardial infarction, stroke, cardiac insufficiency, excessive pulmonary tension, atherosclerosis and kidney stones.
  • the present invention also relates to a method of inhibiting ROMK comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the formula (I) or a tautomer, a mesogen, a racemate, an enantiomer thereof. Isomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.
  • the invention also relates to a method of treating or preventing hypertension and/or heart failure comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer thereof, a mesogen thereof , a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.
  • the present invention relates to a method for treating or preventing a ROMK-mediated disease or condition comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the formula (I) or a tautomer thereof, a mesogen thereof, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a
  • the pharmaceutical composition is preferably selected from the group consisting of cirrhosis, acute and chronic renal insufficiency, nephrotic syndrome, pulmonary hypertension, cardiovascular disease, myocardial infarction, stroke, cardiac insufficiency, hypertonic tone, arteries. Atherosclerosis and kidney stones.
  • the active ingredient-containing pharmaceutical composition may be in a form suitable for oral administration, such as tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or Tincture.
  • Oral compositions can be prepared according to any method known in the art for preparing pharmaceutical compositions, such compositions may contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives, To provide a pleasing and tasty pharmaceutical preparation. Tablets contain the active ingredient and non-toxic pharmaceutically acceptable excipients suitable for the preparation of a tablet for admixture.
  • excipients may be inert excipients such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrating agents such as microcrystalline cellulose, croscarmellose sodium, corn Starch or alginic acid; a binder such as starch, gelatin, polyvinylpyrrolidone or gum arabic and a lubricant such as magnesium stearate, stearic acid or talc.
  • These tablets may be uncoated or may be coated by masking the taste of the drug or delaying disintegration and absorption in the gastrointestinal tract, thus providing a sustained release effect over a longer period of time.
  • water-soluble taste masking materials such as hydroxypropylmethylcellulose or hydroxypropylcellulose, or extended-time materials such as ethylcellulose, cellulose acetate butyrate may be used.
  • hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or in which the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol or an oil vehicle such as peanut oil, liquid paraffin or olive oil.
  • Soft gelatin capsules provide oral preparations.
  • the aqueous suspension contains the active substance and excipients suitable for the preparation of the aqueous suspension for mixing.
  • excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone and acacia; dispersing or wetting agents may be naturally occurring a phospholipid such as lecithin, or a condensation product of an alkylene oxide with a fatty acid such as polyoxyethylene stearate, or a condensation product of ethylene oxide with a long chain fatty alcohol, such as heptadecylethyleneoxy cetyl alcohol (heptadecaethyleneoxy cetanol), or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol, such as polyethylene oxide sorbitol monooleate, or ethylene oxide with derivatives derived from fatty acids and hexitols
  • the aqueous suspensions may also contain one or more preservatives such as ethylparaben or n-propylparaben, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents.
  • preservatives such as ethylparaben or n-propylparaben
  • coloring agents such as ethylparaben or n-propylparaben
  • flavoring agents such as sucrose, saccharin or aspartame.
  • the oil suspension can be formulated by suspending the active ingredient in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • the above sweeteners and flavoring agents may be added to provide a palatable preparation.
  • These compositions can be preserved by the addition of an anti-oxidant such as butylated hydroxyanisole or alpha-tocopherol.
  • the dispersible powders and granules suitable for the preparation of aqueous suspensions can be provided by the addition of water to provide the active ingredient and dispersing or wetting agents, suspending agents or one or more preservatives. Suitable dispersing or wetting agents and suspending agents can be used to illustrate the above examples. Other excipients such as sweetening, flavoring, and coloring agents may also be added. These compositions are preserved by the addition of an anti-oxidant such as ascorbic acid.
  • the pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion.
  • the oil phase may be a vegetable oil such as olive oil or peanut oil, or a mineral oil such as liquid paraffin or a mixture thereof.
  • Suitable emulsifiers may be naturally occurring phospholipids, such as soy lecithin and esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of the partial esters and ethylene oxide, For example, polyethylene oxide sorbitol monooleate.
  • the emulsions may also contain sweeteners, flavoring agents, preservatives, and antioxidants.
  • Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a colorant, and an antioxidant.
  • sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative, a colorant, and an antioxidant.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is then added to a mixture of water and glycerin to form a microemulsion.
  • the injection or microemulsion can be injected into the bloodstream of the patient by a local injection.
  • the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of the invention.
  • a continuous intravenous delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous pump.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration.
  • the suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension, such as a solution prepared in 1,3-butanediol, in a non-toxic parenterally acceptable diluent or solvent.
  • sterile fixed oils may conveniently be employed as a solvent or suspension medium. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be prepared as an injection.
  • the compounds of the invention may be administered in the form of a suppository for rectal administration.
  • These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and thus dissolves in the rectum to release the drug.
  • suitable non-irritating excipient include a mixture of cocoa butter, glycerin gelatin, hydrogenated vegetable oil, polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.
  • the dosage of the drug to be administered depends on a variety of factors including, but not limited to, the activity of the particular compound used, the age of the patient, the weight of the patient, the health of the patient, the conduct of the patient, The patient's diet, time of administration, mode of administration, rate of excretion, combination of drugs, etc.; additionally, the preferred mode of treatment, such as the mode of treatment, the daily dose of the compound of formula (I) or the type of pharmaceutically acceptable salt It can be verified according to traditional treatment options.
  • Alkyl means a saturated aliphatic hydrocarbon group, including straight chain and branched chain groups of 1 to 20 carbon atoms. An alkyl group having 1 to 10 carbon atoms is preferred, and an alkyl group having 1 to 6 carbon atoms is more preferred.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl , 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2- Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl Pentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,
  • lower alkyl groups having from 1 to 6 carbon atoms, non-limiting examples including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl Base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl Base, 2,3-dimethylbutyl and the like.
  • the alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle
  • alkylthio, oxo, amino, haloalkyl, hydroxyalkyl, carboxyl, and carboxylate groups preferably selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl,
  • Cycloalkyl means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably the cycloalkyl ring comprises from 3 to 10 The carbon atom, most preferably the cycloalkyl ring contains from 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptene
  • the alkenyl group, the cyclooctyl group and the like are preferably a cyclopropyl group or a cyclohexenyl group.
  • Polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups.
  • Spirocycloalkyl means a polycyclic group of 5 to 20 members which shares a carbon atom (referred to as a spiro atom) between the monocyclic rings. These may contain one or more double bonds, but none of the rings are fully conjugated. ⁇ electronic system. It is preferably 6 to 14 members, more preferably 7 to 10 members.
  • the spirocycloalkyl group is classified into a monospirocycloalkyl group, a bispirocycloalkyl group or a polyspirocycloalkyl group, preferably a monospirocycloalkyl group and a bispirocycloalkyl group, depending on the number of common spiro atoms between the ring and the ring.
  • spirocycloalkyl groups include
  • fused cycloalkyl means 5 to 20 members, each ring of the system sharing an adjacent carbon atomous all-carbon polycyclic group with other rings in the system, wherein one or more rings may contain one or more Double keys, but no One ring has a fully conjugated pi-electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic fused ring alkyl group, preferably a bicyclic or tricyclic ring, more preferably a 5-membered/5-membered or 5-membered/6-membered bicycloalkyl group.
  • fused cycloalkyl groups include
  • Bridge cycloalkyl means 5 to 20 members, any two rings sharing two carbon-free all-carbon polycyclic groups, which may contain one or more double bonds, but none of the rings have a total The ⁇ -electron system of the yoke. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl group, preferably a bicyclic ring, a tricyclic ring or a tetracyclic ring, and more preferably a bicyclic ring or a tricyclic ring.
  • bridged cycloalkyl groups include
  • the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring to which the parent structure is attached is a cycloalkyl group, non-limiting examples include indanyl, tetrahydronaphthalene Base, benzocycloheptyl and the like.
  • the cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, Hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, amino One or more groups of haloalkyl, hydroxyalkyl, carboxyl and carboxylate groups.
  • Heterocyclyl means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising from 3 to 20 ring atoms wherein one or more of the ring atoms are selected from nitrogen, oxygen or S(O) m ( Wherein m is a hetero atom of 0 to 2, but does not include a ring moiety of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.
  • the heterocyclic ring comprises 3 to 12 ring atoms, wherein 1 to 4 are hetero atoms; more preferably, the heterocyclic ring contains 3 to 10 ring atoms, of which 1 to 3 are hetero atoms; more preferably, the heterocyclic ring contains 5 to 6 ring atoms, of which 1 to 2 are heteroatoms.
  • monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, tetrahydrofuranyl and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • “Spiroheterocyclyl” means a polycyclic heterocyclic group of 5 to 20 members in which one atom (referred to as a spiro atom) is shared between monocyclic rings, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m
  • the hetero atom (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. These may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members.
  • the spirocycloalkyl group is classified into a monospiroheterocyclic group, a dispirocyclic heterocyclic group or a polyspirocyclic group, preferably a monospiroheterocyclic group and a dispiroheterocyclic group, depending on the number of common spiro atoms between the ring and the ring. More preferably, it is 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6-membered monospiroheterocyclic group.
  • Non-limiting examples of spiroheterocyclyl groups include
  • “Fused heterocyclyl” refers to 5 to 20 members, each ring of the system sharing an adjacent pair of atoms of a polycyclic heterocyclic group with other rings in the system, and one or more rings may contain one or more a bond, but none of the rings have a fully conjugated ⁇ -electron system in which one or more ring atoms are selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members.
  • bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group preferably a bicyclic or tricyclic ring, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group.
  • fused heterocyclic groups include
  • “Bridge heterocyclyl” refers to a polycyclic heterocyclic group of 5 to 14 members, any two rings sharing two atoms which are not directly bonded, these may contain one or more double bonds, but none of the rings have a complete conjugation A ⁇ -electron system in which one or more ring atoms are selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members.
  • bridged heterocyclic groups include:
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring wherein the ring to which the parent structure is attached is a heterocyclic group, non-limiting examples comprising:
  • the heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably selected from an alkyl group, Alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy One or more groups of a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, an oxo group, an amino group, a halogenated alkyl group, a hydroxyalkyl group, a carboxyl group, and a carboxylate group.
  • Aryl means a 6 to 14 membered all-carbon monocyclic or fused polycyclic ring (i.e., a ring that shares a pair of adjacent carbon atoms) having a conjugated ⁇ -electron system, preferably 6 to 10 members, more preferably benzene.
  • the base and naphthyl are most preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring wherein the ring to which the parent structure is attached is an aryl ring, non-limiting examples comprising:
  • the aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro , cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl
  • the substituent is preferably selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro , cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocyclo
  • Heteroaryl refers to a 5 to 14 membered aryl group having from 1 to 4 heteroatoms as ring atoms, the remaining ring atoms being carbon, wherein the heteroatoms include oxygen, sulfur and nitrogen. It is preferably 5 to 10 yuan.
  • the heteroaryl group is preferably 5- or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring wherein the ring to which the parent structure is attached is a heteroaryl ring, non-limiting examples comprising:
  • the heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, Hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, One or more groups of a hydroxyalkyl group, a carboxyl group, and a carboxylate group.
  • Alkoxy means -O-(alkyl) and -O-(unsubstituted cycloalkyl) wherein alkyl is as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • the alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a sulfhydryl group, Hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane
  • Haloalkyl means an alkyl group substituted by one or more halogens, wherein alkyl is as defined above.
  • Hydrophilicity refers to an -OH group.
  • Hydroalkyl means an alkyl group substituted by a hydroxy group, wherein alkyl is as defined above.
  • Halogen means fluoro, chloro, bromo or iodo.
  • Carboxy refers to -C(O)OH.
  • Carboxylic acid ester group means -C(O)O(alkyl) or (cycloalkyl) wherein alkyl, cycloalkyl are as defined above.
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may be, but not necessarily, present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3, hydrogen atoms, independently of each other, substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art will be able to determine (by experiment or theory) substitutions that may or may not be possible without undue effort. For example, an amino group or a hydroxyl group having a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and Shape agent.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • R 1 to R 3 and n are as defined in the formula (I).
  • R 1 , R 2 , R 4 , n are as defined in the formula (II).
  • R 1 , R 2 , R 4 , n are as defined in formula (III).
  • R 1 to R 3 and n are as defined in the formula (I).
  • R 1 , R 2 , R 4 , n are as defined in the formula (I).
  • R 1 , R 2 , R 4 , n are as defined in formula (I).
  • Solvents used include, but are not limited to, acetic acid, methanol, ethanol, acetonitrile, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, 1,4-dioxane, water, N,N-dimethylacetamide or N, N-dimethylformamide is preferably a non-polar solvent, more preferably acetonitrile.
  • FIG. 5 Effect of ROMK inhibitor on serum potassium in SD rats.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • the NMR was measured by a Bruker AVANCE-400 nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard was four.
  • DMSO-d 6 dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Methylsilane
  • chemical shifts are given in units of 10 -6 (ppm).
  • the measurement of the MS was carried out using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
  • ESI FINNIGAN LCQAd
  • Thin layer chromatography silica gel plate uses Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate.
  • the specification of silica gel plate used for thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm.
  • the specification for thin layer chromatography separation and purification is 0.4mm. ⁇ 0.5mm silica gel plate.
  • the known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, Companies such as Dare Chemicals.
  • An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon having a volume of about 1 L.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1 L volume.
  • the microwave reaction used a CEM Discover-S Model 908860 microwave reactor.
  • the solution in the reaction means an aqueous solution unless otherwise specified.
  • the temperature of the reaction was room temperature unless otherwise specified.
  • Room temperature is the most suitable reaction temperature, and the temperature range is from 20 ° C to 30 ° C.
  • the progress of the reaction in the examples was monitored by thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the system used for the reaction was: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether And the ethyl acetate system, D: acetone, the volume ratio of the solvent is adjusted depending on the polarity of the compound.
  • the system for the eluent of the column chromatography and the system for the thin layer chromatography of the developer used for the purification of the compound include: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: n-hexane and acetone
  • the volume ratio of the solvent is adjusted depending on the polarity of the compound, and may be adjusted by adding a small amount of triethylamine and an acidic or alkaline reagent.
  • 6-chloro-4-ethoxynicotinonitrile 2b (375 mg, 2.05 mmol), 4-aminopiperidine-1-carboxylic acid tert-butyl ester (422 mg, 2.05 mmol), palladium acetate (23 mg, 0.1 mmol) , 1,3-bis(diphenylphosphino)propane (42 mg, 0.1 mmol), triethylamine (0.57 mL, 4.1 mmol) and 20 mL of acetonitrile, three times of carbon monoxide, pressure 10 bar, warmed to 80 ° C, stirring reaction 16 hours. The reaction mixture was filtered, and the filtrate was evaporated to dryness. Pyridine carboxamide) piperidine-1-carboxylic acid tert-butyl ester 2c (645 mg, white solid).
  • 6-chloro-4-(2-fluoroethoxy)nicotinonitrile 3a (210 mg, 1.05 mmol), 4-aminopiperidine-1-carboxylic acid tert-butyl ester (216 mg, 1.05 mmol), palladium acetate (12 mg, 0.05 mmol), 1,3-bis(diphenylphosphino)propane (22 mg, 0.05 mmol), triethylamine (0.29 mL, 2.1 mmol) and 20 mL of acetonitrile, three times of carbon monoxide, at a pressure of 10 bar, warmed to The reaction was stirred at 80 ° C for 16 hours. The reaction mixture was concentrated under reduced pressure. EtOAcjjjjjjj - tert-butyl formate 3b (140 mg, white solid), yield 34%.
  • 2-Bromo-5-chloropyridine 4a (2 g, 10.4 mmol) was dissolved in 40 mL of tetrahydrofuran and added dropwise at -78 °C. 7.8 mL of a 2 M lithium diisopropylamide solution was stirred for 1 hour, triisopropyl borate (2.94 g, 15.6 mmol) was added, stirred for 30 minutes, warmed to room temperature, and the reaction was stirred for 16 hours.
  • Test Example 1 Determination of ROMK inhibitory activity of human ROMK and rat (rat) by the compound of the present invention
  • Human ROMK/HEK293 cells transfected with human ROMK cDNA (NCBI SEQ ID NO: NM_000220.4) and HEK293 cell line stably expressing ROMK channel
  • Rat ROMK/HEK293 cells transfected with rat ROMK cDNA (NCBI SEQ ID NO: NM_017023.1) and HEK293 cell line stably expressing ROMK channel
  • HEK293 cell line Cell Bank of Chinese Academy of Sciences, GNHu43
  • the inhibitory activity of the compound of the present invention against human ROMK or rat ROMK was measured by the above test, and the measured IC 50 values are shown in Table 1.
  • the compound of the present invention has a significant inhibitory effect on the activity of human ROMK or rat ROMK channel, and the compound has high pharmacological effect.
  • Test Example 2 Determination of hERG inhibitory activity of the compound of the present invention
  • hERG/HEK293 cells transfected with human hERG cDNA (NCBI SEQ ID NO: NM_000238 (RC215928, origene)) and HEK293 cell line stably expressing hERG channel
  • the inhibitory activity of the compound of the present invention on hERG was measured by the above test, and the measured IC 50 values are shown in Table 2.
  • the compounds of the present invention have a weak inhibitory effect on hERG channel activity and low cardiotoxicity.
  • Test Example 3 Effect of electrophysiological manual patch clamp detection on ROMK potassium ion channel
  • the purpose of this experiment was to examine the effect of compounds on ROMK potassium ion channels in HEK293 cells in ex vivo experiments.
  • the ROMK potassium ion channel was stably expressed on the HEK293 cells of the present invention. After the potassium ion current is stabilized, the effect of the compound on the potassium ion channel can be obtained by comparing the potassium ion current before and after the application of different compound concentrations.
  • HEK293 cells Cell Bank of Chinese Academy of Sciences, GNHu43;
  • Human ROMK/HEK293 cells transfected with human ROMK cDNA (NCBI SEQ ID NO: NM_000220.4) and HEK293 cell line stably expressing ROMK channel;
  • Extracellular fluid NaCl, 137; KCl, 4; CaCl 2 , 1.8; MgCl 2 , 1; HEPES, 10; glucose 10; pH 7.4 (NaOH titration);
  • intracellular fluid K Aspartate, 130; MgCl 2 , 5; EGTA 5; HEPES, 10; Tris-ATP 4; pH 7.2 (KOH titration);
  • the compounds were obtained from Sigma (St. Louis, MO) except for the NaOH and KOH used for acid-base titration. buy.
  • Cell culture medium Ham's F12 medium (Invitrogen), 10% (v/v) inactivated fetal bovine serum, 100 ⁇ g/ml hygromycin B, 100 ⁇ g/ml geneticin (Geneticin);
  • Micro-manipulation instrument MP-225;
  • test compound was dissolved in dimethyl sulfoxide (DMSO) and stored at room temperature. On the day of the test, it was diluted with the extracellular fluid to the following final concentration (3, 10, 30, 100, 300 nM) for testing. The final concentration of DMSO in the test compound was 0.3%.
  • DMSO dimethyl sulfoxide
  • Human ROMK/HEK293 cells were grown in a Petri dish containing the above cell culture medium, and cultured in an incubator containing 5% CO 2 at 37 °C. 24 to 48 hours prior to electrophysiological experiments, human ROMK/HEK293 cells were transferred to circular glass slides placed in petri dishes and grown under the same culture medium and culture conditions as above. The density of human ROMK/HEK293 cells on each circular slide requires that most cells be independent, individual requirements.
  • This experiment used a manual patch clamp system for the recording of whole cell currents.
  • a circular slide of surface-grown ROMK/HEK293 cells was placed in an electrophysiological recording cell under an inverted microscope.
  • the extracellular fluid was continuously perfused in the recording tank (about 1 ml per minute).
  • the experimental procedure uses conventional whole-cell patch clamp current recording techniques. Unless otherwise stated, the experiments were carried out at regular room temperature ( ⁇ 25 ° C).
  • the cells were clamped at a voltage of -80 mV.
  • the cell clamp voltage was depolarized to +20 mV to activate the ROMK potassium channel, and after 5 seconds it was clamped to -50 mV to eliminate inactivation and generate tail current.
  • the tail current peak is used as the value of the ROMK current magnitude.
  • the drug to be tested can be superimposed and filled until the inhibition of the ROMK current reaches a steady state.
  • the recent three consecutive current recording lines are recombined as a criterion for judging whether or not the state is stable.
  • rinse with extracellular fluid until the ROMK current returns to the size before the drug is added.
  • One cell can test one or more drugs, or multiple concentrations of the same drug, but need to be flushed with extracellular fluid between different drugs.
  • test data was analyzed by HEKA Patchmaster, XLFit and data analysis software provided by Graphpad Prism, see Table 3.
  • the compounds of the present invention have a strong inhibitory effect on the potassium channel of ROMK.
  • Test Example 4 Effect of electrophysiological manual patch clamp detection on hERG potassium channel
  • the purpose of this experiment was to examine the effect of compounds on hERG potassium ion channels in CHO cells in ex vivo experiments.
  • the hERG potassium ion channel is stably expressed on the CHO cells of the present invention. After the potassium ion current is stabilized, the effect of the compound on the potassium ion channel can be obtained by comparing the potassium ion current before and after the application of different compound concentrations.
  • CHO cell line Sophion Bioscience, Denmark;
  • hERG/CHO cells transfected with human hERG cDNA (NCBI SEQ ID NO: NM_000238 (RC215928, origene)) and CHO cell line stably expressing hERG channel;
  • Extracellular fluid EC 0.0.0 NaCl-Ringer's solution, NaCl, 145; KCl, 4; CaCl 2 , 2; MgCl 2 , 1; HEPES, 10; glucose 10; pH 7.4 (NaOH titration), osmotic pressure ⁇ 305 mOsm;
  • Intracellular fluid IC 0.0.0 KCl-Linger's solution, KCl, 120, CaCl 2 , 5.374, MgCl 2 , 1.75; EGTA 5; HEPES, 10; Na-ATP 4; pH 7.25 ( KOH titration), osmotic pressure ⁇ 295mOsm;
  • the compounds were purchased from Sigma (St. Louis, MO) except for NaOH and KOH for acid-base titration.
  • Cell culture medium Ham's F12 medium (Invitrogen), 10% (v/v) inactivated fetal bovine serum, 100 ⁇ g/ml hygromycin B, 100 ⁇ g/ml geneticin;
  • Micro-manipulation instrument MP-225;
  • CHO hERG cells were grown in a Petri dish containing the above cell culture medium, and cultured in an incubator containing 5% CO 2 at 37 °C. CHO hERG cells were transferred to circular glass slides placed in petri dishes 24 to 48 hours prior to electrophysiological experiments and grown under the same culture and culture conditions as above. The density of CHO hERG cells on each circular slide requires that most cells be independent, individual requirements.
  • This experiment used a manual patch clamp system for the recording of whole cell currents.
  • a circular slide with CHO hERG cells grown on it was placed in an electrophysiology recording trough under an inverted microscope.
  • the extracellular fluid was continuously perfused in the recording tank (about 1 ml per minute).
  • the experimental procedure uses conventional whole-cell patch clamp current recording techniques. Unless otherwise stated, the experiments were carried out at regular room temperature ( ⁇ 25 ° C).
  • the cells were clamped at a voltage of -80 mV.
  • the cell clamp voltage was depolarized to +20 mV to activate the hERG potassium channel, and after 5 seconds it was clamped to -50 mV to eliminate inactivation and generate tail current.
  • the tail current peak is used as the value of the hERG current magnitude.
  • the drug to be tested can be superimposed and filled until the inhibitory effect of the drug on the hERG current reaches a steady state.
  • the recent three consecutive current recording lines are recombined as a criterion for judging whether or not the state is stable.
  • rinse with extracellular fluid until the hERG current returns to the size before the drug is added.
  • One cell can test one or more drugs, or multiple concentrations of the same drug, but need to be flushed with extracellular fluid between different drugs.
  • test data was analyzed by HEKA Patchmaster, XLFit and data analysis software provided by Graphpad Prism, see Table 4.
  • the compounds of the present invention have a weak inhibitory effect on the hERG potassium channel and a low cardiotoxicity.
  • Rats were used as test animals, and the concentration of the drug in plasma at different times after administration of the compound of the example by intragastric administration was determined by LC/MS/MS method.
  • the pharmacokinetic behavior of the compounds of the invention in rats was investigated and their pharmacokinetic characteristics were evaluated.
  • 0.1 mL of blood was collected before administration and 0.5, 1, 2, 4, 6, 8, 11, and 24 hours after administration, placed in an EDTA anticoagulation tube, centrifuged at 3500 rpm for 10 minutes, and plasma was separated and stored at -20 °C. 2 hours after administration food.
  • the content of the test compound in the plasma of rats after intragastric administration of different compounds was determined by LC/MS/MS method. Plasma samples were analyzed by pretreatment with precipitated proteins.
  • Test Example 6 Experimental report on the diuretic effect of ROMK inhibitor on SD rats
  • SD male rats were purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. (Shanghai, China, certificate number 2008001647752, license SCXK (Shanghai) 2013-0016), purchased 120-130g, 5 / cage Feeding, 12/12 hour light/dark cycle adjustment, temperature 23 ⁇ 1°C constant temperature, humidity 50-60%, free access to water. After the animals were purchased, the experiment was started after 7 days of adaptive feeding.
  • the positive control drug structure is as follows:
  • Drug doses are calculated on a base basis.
  • Grouping n Mode of administration normal group 10 0.5% CMC i.g. single
  • Compound 1-0.03mg/kg group 10 0.03mg/kg 150532 i.g. single
  • Compound 1-0.1mg/kg group 10 0.1mg/kg 150532 i.g. single
  • Positive control drug -0.03mg/kg group 10 0.03mg/kg 153111 i.g. single
  • Positive control drug - 0.1mg/kg group 10 0.1mg/kg 153111 i.g. single
  • the experimental method was carried out in accordance with the method of the patent (WO2010129379A1). At least adaptively fed SD rats were fasted overnight in metabolic cages, and were weighed into the following groups after randomization: blank control group, compound 1 test group 0.03 mg/kg group and 0.1 mg/kg group, positive The control group was 0.03 mg/kg and the group was 0.1 mg/kg, and 10 rats in each group. Each rat was given a single gavage (ig, 1 ml/kg) of each compound, and the blank group was intragastrically administered with the corresponding solvent. After the gavage, the rats were placed in a general cage, and 25 ml/kg of physiological saline was administered 30 minutes later, immediately afterwards.
  • the experimental results showed that compared with the blank control group, the urine volume of the positive control drug 0.03mg/kg group and 0.1mg/kg group increased significantly (P ⁇ 0.05), the urine volume increased by 1.41 times and 1.46 times, respectively, and the compound 1 test drug 0.03
  • the urine volume of the mg/kg group and the 0.1 mg/kg group increased significantly (P ⁇ 0.01), and the urine volume increased by 2.76 times and 3.22 times, respectively (see Figure 1); the positive drug and each compound 1 test group had a significant increase in urinary sodium excretion.
  • K+ channels can be divided into the following four types: slow (delayed) K+ channel (K channel), fast (early) K+ channel (A channel), Ca2+ activated K+ channel [K (Ca) channel] And four types of inward rectifying K+ channels.
  • the family of inwardly rectifying K channels (Kir) is further divided into seven types: Kir1 to Kir7, which have different KCNJ gene codes.
  • the renal outer medullary potassium channel (ROMK) is mainly of the Kir1 type. There are at least three subtypes of ROMK channels in the rat kidney: ROMK1, ROMK2 and ROMK3, and ROMK2 is mostly distributed in the medullary sputum.
  • ROMK1 and ROMK3 are mainly expressed on the manifold.
  • ROMK expressed in the thick segment of the medullary ascending branch regulates the secretion and reabsorption of potassium ions together with the Na/K/Cl transporter.
  • ROMK expressed in the cortical collecting tubules regulates the secretion of potassium ions together with the Na/K transporter, blocking
  • the ROMK site can promote the secretion of NaCl into the lumen, and does not cause hyperkalemia caused by hyperkalemia. It is a good diuretic research direction for patients with hypertension. This experiment mainly explores the diuretic effect of ROMK inhibitors.
  • test compound 1 was very soluble, and there was no delamination.
  • the positive control drug had static electricity during the weighing process, which was difficult to weigh, and the agglomeration was not easily dissolved in the initial stage of grinding. Soluble well.
  • results also showed that compared with the normal group, a single oral gavage of Compound 1 and a positive control drug had a significant effect on diuretic sodium excretion in rats, and dose-dependent between the doses of the test compound 1 and the positive control drug. .
  • test drug compound 1 and the test drug positive control drug had obvious diuretic sodium excretion effect and had no effect on blood potassium; however, the diuretic effect of compound 1 was significantly better than that of the positive control drug, and the pharmacodynamics of each group was dose dependent. .

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Abstract

本发明涉及吡啶甲酰胺类衍生物、其制备方法及其在医药上的应用。具体而言，本发明涉及一种通式(I)所示吡啶甲酰胺类衍生物、其制备方法及含有该衍生物的药物组合物，以及其作为治疗剂，特别是作为肾外髓质分泌钾通道(ROMK)抑制剂的用途和其在制备治疗和/或预防高血压和心力衰竭的药物中的用途，其中通式(I)中的各取代基的定义与说明书中的定义相同。

Description

吡啶甲酰胺类衍生物、其制备方法及其在医药上的应用 技术领域
本发明涉及一类吡啶甲酰胺类衍生物、其制备方法及含有该衍生物的药物组合物，以及其作为治疗剂特别是作为特别是作为肾外髓质分泌钾通道(ROMK)抑制剂的用途和其在制备治疗和/或预防由过量的盐和水滞留造成的失调症，包括高血压和心力衰竭的药物中的用途。
背景技术
加强肾脏的盐重吸收功能，会引发高血压风险。相反，抑制肾脏的重吸收功能，可以促进尿液的排泄，起到利尿降压的功效。常见的利尿剂有噻嗪类利尿药，作为美国一线降压药，主要作用于Na+-Cl-转运载体；Loop利尿剂对肾功能受损患者更为有效，主要通过Na+-K+-2Cl-转运蛋白发挥作用。但这两种利尿剂均会引起低血钾症(症状：虚弱、疲劳、肌肉痉挛、便秘和心律问题如心律不齐)，增加心血管疾病的发病率和致死率的风险。
肾外髓质分泌钾通道(renal outer medullary potassium channel，ROMK)也称内向性整流型钾离子通道1.1(inward-rectifying potassium channels 1.1，Kir1.1)。ROMK离子通道可以通过肾脏髓袢升支粗段(TAL)的顶端膜电导，与Na+-K+-2Cl-共转运蛋白NKCC2(负责NaCl转运)协同作用，调节Na+的重吸收。研究发现，ROMK与肾脏的分泌通道有直接的关联，敲除ROMK基因，小鼠TAL和CCD的35-pS离子通道和TAL其它K+离子通道缺失。球旁细胞增生症(Batter syndrome)是一种常染色体隐性遗传性疾病，表现为肾脏大量盐流失，低血钾症，血压较低。球旁细胞增生症主要由ROMK或Na+-K+-2Cl-共转运蛋白的突变引起的，不同的是由ROMK突变引起的球旁细胞增生症的低血钾症较Na+-K+-2Cl-共转运蛋白突变引发的球旁细胞增生症有极大的缓和。综上，抑制ROMK的功能，可以在不引起低血钾症的情况下，有效抑制Na+-K+-2Cl-转运蛋白的盐重吸收功能，促进尿液的排泄，起到利尿降压的功效。
尽管目前已公开了一系列的ROMK抑制剂的化合物，其中包括WO2010129379、WO2012058134、WO2012058116、WO2012058134、WO2013066714、WO2013028474、WO2014085210、WO2014018764、WO2014015495、WO2014085210、WO2013039802、WO2013062892和WO2012058116等专利申请，但仍需开发提高hERG选择性的化合物。本发明设计具有通式(I)所示的结构的化合物，增加了极性基团，在保持ROMK抑制活性的基础上，降低了ClogP，提升了hERG选择性，增加了安全性。
发明内容
本发明的目的在于提供一种通式(I)所示的化合物：
Figure PCTCN2015094164-appb-000001
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：
R1为烷基，其中所述烷基任选进一步被选自卤素、羟基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基和羧酸酯基中的一个或多个取代基所取代；
R2选自氢原子、烷基、卤素、氰基、硝基、烷氧基、环烷基和杂环基，其中所述烷基烷氧基、环烷基或杂环基任选进一步被选自烷基、卤素、羟基、羟烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧酸和羧酸酯中的一个或多个取代基所取代；
R3选自下列基团：
Figure PCTCN2015094164-appb-000002
R4或R5各自独立地选自氢原子、烷基、卤素、氰基、硝基、烷氧基、环烷基、杂环基、芳基和杂芳基；
R6选自氢原子、烷基和卤素；
n为0、1或2。
在本发明一个优选方案中，一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中R1为烷基，其中所述的烷基任选进一步被选自卤素、羟基和烷氧基中的一个或多个取代基所取代；R1优选为C1-6烷基，更优选为甲基、乙基或丙基。
在本发明一个优选方案中，一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中R4为烷基，且R5为氢原子。
在本发明一个优选方案中，一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其为通式(II)所示的化合物：
Figure PCTCN2015094164-appb-000003
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1、R2、R4、n如式(I)中所定义。
在本发明一个优选方案中，一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其为通式(III)所示的化合物：
Figure PCTCN2015094164-appb-000004
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1、R2、R4、n如式(I)中所定义。
在本发明一个优选方案中，一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其为通式(IV)所示的化合物：
Figure PCTCN2015094164-appb-000005
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1、R2、R3、n如式(I)中所定义。
在本发明一个优选方案中，一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其为通式(V)所示的化合物：
Figure PCTCN2015094164-appb-000006
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1、R2、R4、n如式(I)中所定义。
在本发明一个优选方案中，一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其为通式(VI)所示的化合物：
Figure PCTCN2015094164-appb-000007
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1、R2、R4、n如式(I)中所定义。
本发明典型的化合物包括，但不限于：
Figure PCTCN2015094164-appb-000008
Figure PCTCN2015094164-appb-000009
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用盐。
本发明还提供一种通式(IA)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，作为制备通式(I)所示化合物的中间体：
Figure PCTCN2015094164-appb-000010
其中：
R1为烷基，其中所述烷基任选进一步被选自卤素、羟基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基和羧酸酯基中的一个或多个取代基所取代；
R2选自氢原子、烷基、卤素、氰基、硝基、烷氧基、环烷基和杂环基，其中所述烷基、烷氧基、环烷基或杂环基任选进一步被选自烷基、卤素、羟基、羟烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基和羧酸酯基中的一个或多个取代基所取代；可用于制备通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐的中间体；
n为0、1或2。
在本发明一个优选方案中，一种通式(IA)所示的化合物或其互变异构体、内消 旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其为通式(IVA)所示的化合物：
Figure PCTCN2015094164-appb-000011
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐；可用于制备通式(IV)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐的中间体；其中：R1、R2、n如式(IA)中所定义。
通式(IA)化合物的典型的化合物包括，但不限于：
Figure PCTCN2015094164-appb-000012
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用盐。
本发明还提供一种制备通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐的方法，该方法包括：
Figure PCTCN2015094164-appb-000013
通式(IA)化合物与取代的苯并呋喃类衍生物(IB)，优选为与(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮加热反应，得到通式(I)化合物；
其中：R1-R3、n如通式(I)中所定义。
本发明进一步涉及一种药物组合物，所述药物组合物含有治疗有效量的如通 式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐以及药学上可接受的载体、稀释剂或赋形剂。
本发明进一步涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其的药物组合物在制备ROMK抑制剂中的用途。
本发明进一步涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其的药物组合物在制备治疗或预防高血压或心力衰竭的药物中的用途。
本发明进一步涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其的药物组合物在制备治疗或预防ROMK介导的疾病的药物中的用途，所述的疾病包括但不限于肝硬化、急性和慢性的肾功能不全、肾病综合症、肺动脉高血压、心血管疾病、心肌梗塞、中风、心功能不全、肺张力过强、动脉粥样硬化和肾结石。
本发明还涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其的药物组合物，其用作ROMK抑制剂。
本发明还涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其的药物组合物，其用于治疗或预防高血压或心力衰竭。
本发明还涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其的药物组合物，其用于治疗或预防ROMK介导的疾病，所述的疾病优选选自肝硬化、急性和慢性的肾功能不全、肾病综合症、肺动脉高血压、心血管疾病、心肌梗塞、中风、心功能不全、肺张力过强、动脉粥样硬化和肾结石。
本发明还涉及一种抑制ROMK的方法，其包括给予所需患者治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其的药物组合物。
本发明还涉及一种治疗或预防高血压和/或心力衰竭的方法，其包括给予所需患者治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其的药物组合物。
本发明涉及一种治疗或预防ROMK介导的疾病或病症的方法，其包括给予所需患者治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，或包含其 的药物组合物，所述的疾病优选选自肝硬化、急性和慢性的肾功能不全、肾病综合症、肺动脉高血压、心血管疾病、心肌梗塞、中风、心功能不全、肺张力过强、动脉粥样硬化和肾结石。
含活性成分的药物组合物可以是适用于口服的形式，例如片剂、糖锭剂、锭剂、水或油混悬液、可分散粉末或颗粒、乳液、硬或软胶囊，或糖浆剂或酏剂。可按照本领域任何已知制备药用组合物的方法制备口服组合物，此类组合物可含有一种或多种选自以下的成分：甜味剂、矫味剂、着色剂和防腐剂，以提供悦目和可口的药用制剂。片剂含有活性成分和用于混合的适宜制备片剂的无毒的可药用的赋形剂。这些赋形剂可以是惰性赋形剂，如碳酸钙、碳酸钠、乳糖、磷酸钙或磷酸钠；造粒剂和崩解剂，例如微晶纤维素、交联羧甲基纤维素钠、玉米淀粉或藻酸；粘合剂，例如淀粉、明胶、聚乙烯吡咯烷酮或阿拉伯胶和润滑剂，例如硬脂酸镁、硬脂酸或滑石粉。这些片剂可以不包衣或可通过掩盖药物的味道或在胃肠道中延迟崩解和吸收，因而在较长时间内提供缓释作用的已知技术将其包衣。例如，可使用水溶性味道掩蔽物质，例如羟丙基甲基纤维素或羟丙基纤维素，或延长时间物质例如乙基纤维素、醋酸丁酸纤维素。
也可用其中活性成分与惰性固体稀释剂例如碳酸钙、磷酸钙或高岭土混合的硬明胶胶囊，或其中活性成分与水溶性载体例如聚乙二醇或油溶媒例如花生油、液体石蜡或橄榄油混合的软明胶胶囊提供口服制剂。
水悬浮液含有活性物质和用于混合的适宜制备水悬浮液的赋形剂。此类赋形剂是悬浮剂，例如羧基甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、藻酸钠、聚乙烯吡咯烷酮和阿拉伯胶；分散剂或湿润剂可以是天然产生的磷脂例如卵磷脂，或烯化氧与脂肪酸的缩合产物例如聚氧乙烯硬脂酸酯，或环氧乙烷与长链脂肪醇的缩合产物，例如十七碳亚乙基氧基鲸蜡醇(heptadecaethyleneoxy cetanol)，或环氧乙烷与由脂肪酸和己糖醇衍生的部分酯的缩合产物，例如聚环氧乙烷山梨醇单油酸酯，或环氧乙烷与由脂肪酸和己糖醇酐衍生的偏酯的缩合产物，例如聚环氧乙烷脱水山梨醇单油酸酯。水混悬液也可以含有一种或多种防腐剂例如尼泊金乙酯或尼泊金正丙酯、一种或多种着色剂、一种或多种矫味剂和一种或多种甜味剂，例如蔗糖、糖精或阿司帕坦。
油混悬液可通过使活性成分悬浮于植物油如花生油、橄榄油、芝麻油或椰子油，或矿物油例如液体石蜡中配制而成。油悬浮液可含有增稠剂，例如蜂蜡、硬石蜡或鲸蜡醇。可加入上述的甜味剂和矫味剂，以提供可口的制剂。可通过加入抗氧化剂例如丁羟茴醚或α-生育酚保存这些组合物。
通过加入水可使适用于制备水混悬也的可分散粉末和颗粒提供活性成分和用于混合的分散剂或湿润剂、悬浮剂或一种或多种防腐剂。适宜的分散剂或湿润剂和悬浮剂可说明上述的例子。也可加入其他赋形剂例如甜味剂、矫味剂和着色剂。通过加入抗氧化剂例如抗坏血酸保存这些组合物。
本发明的药物组合物也可以是水包油乳剂的形式。油相可以是植物油例如橄榄油或花生油，或矿物油例如液体石蜡或其混合物。适宜的乳化剂可以是天然产生的磷脂，例如大豆卵磷脂和由脂肪酸和己糖醇酐衍生的酯或偏酯例如山梨坦单油酸酯，和所述偏酯和环氧乙烷的缩合产物，例如聚环氧乙烷山梨醇单油酸酯。乳剂也可以含有甜味剂、矫味剂、防腐剂和抗氧剂。可用甜味剂例如甘油、丙二醇、山梨醇或蔗糖配制糖浆和酏剂。此类制剂也可含有缓和剂、防腐剂、着色剂和抗氧剂。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本发明化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1，3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
可按用于直肠给药的栓剂形式给予本发明化合物。可通过将药物与在普通温度下为固体但在直肠中为液体，因而在直肠中会溶化而释放药物的适宜的无刺激性赋形剂混合来制备这些药物组合物。此类物质包括可可脂、甘油明胶、氢化植物油、各种分子量的聚乙二醇和聚乙二醇的脂肪酸酯的混合物。
本领域技术人员所熟知的，药物的给药剂量依赖于多种因素，包括但并非限定以下因素：所用特定化合物的活性、病人的年龄、病人的体重、病人的健康状况、病人的行被、病人的饮食、给药时间、给药方式、排泄的速率、药物的组合等；另外，最佳的治疗方式如治疗的模式、通式化合物(I)的日用量或可药用的盐的种类可以根据传统的治疗方案来验证。
定义
除非有相反陈述，否则下列用在说明书和权利要求书中的术语具有下述含义。
“烷基”指饱和的脂族烃基团，包括1至20个碳原子的直链和支链基团。优选含有1至10个碳原子的烷基，更优选含有1至6个碳原子的烷基。非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1，1-二甲基丙基、1，2-二甲基丙基、2，2-二甲基丙基、1-乙基丙基、2-甲基丁 基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1，1，2-三甲基丙基、1，1-二甲基丁基、1，2-二甲基丁基、2，2-二甲基丁基、1，3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2，3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2，3-二甲基戊基、2，4-二甲基戊基、2，2-二甲基戊基、3，3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2，3-二甲基己基、2，4-二甲基己基、2，5-二甲基己基、2，2-二甲基己基、3，3-二甲基己基、4，4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2，2-二乙基戊基、正癸基、3，3-二乙基己基、2，2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1，1-二甲基丙基、1，2-二甲基丙基、2，2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1，1，2-三甲基丙基、1，1-二甲基丁基、1，2-二甲基丁基、2，2-二甲基丁基、1，3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2，3-二甲基丁基等。烷基可以是取代的或未取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，优选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、疏基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、氨基、卤代烷基、羟烷基、羧基和羧酸酯基中的一个或多个基团。
“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，其包括3至20个碳原子，优选包括3至12个碳原子，更优选环烷基环包含3至10个碳原子，最优选环烷基环包含3至6个碳原子。单环环烷基的非限制性实施例包含环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等，优选环丙基、环己烯基。多环环烷基包括螺环、稠环和桥环的环烷基。
“螺环烷基”指5至20元，单环之间共用一个碳原子(称螺原子)的多环基团，这些可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺环烷基分为单螺环烷基、双螺环烷基基或多螺环烷基，优选为单螺环烷基和双螺环烷基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺环烷基。螺环烷基的非限制性实施例包含
Figure PCTCN2015094164-appb-000014
“稠环烷基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对碳原子的全碳多环基团，其中一个或多个环可以含有一个或多个双键，但没有 一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠环烷基，优选为双环或三环，更优选为5元/5元或5元/6元双环烷基。稠环烷基的非限制性实施例包含
Figure PCTCN2015094164-appb-000015
“桥环烷基”指5至20元，任意两个环共用两个不直接连接的碳原子的全碳多环基团，这些可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥环烷基，优选为双环、三环或四环，更有选为双环或三环。桥环烷基的非限制性实施例包含
Figure PCTCN2015094164-appb-000016
所述环烷基环可以稠合于芳基、杂芳基或杂环基环上，其中与母体结构连接在一起的环为环烷基，非限制性实施例包括茚满基、四氢萘基、苯并环庚烷基等。环烷基可以是任选取代的或未取代的，当被取代时，取代基优选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、疏基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、氨基、卤代烷基、羟烷基、羧基和羧酸酯基中的一个或多个基团。
“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包括3至20个环原子，其中一个或多个环原子选自氮、氧或S(O)m(其中m是0至2的整数)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包括3至12个环原子，其中1～4个是杂原子；更优选杂环基环包含3至10个环原子，其中1～3个是杂原子；更优选杂环基环包含5至6个环原子，其中1～2个是杂原子。单环杂环基的非限制性实施例包含吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基、吡喃基、四氢呋喃基等。多环杂环基包括螺环、稠环和桥环的杂环基。
“螺杂环基”指5至20元，单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。这些可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺环烷基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环 基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性实施例包含
Figure PCTCN2015094164-appb-000017
“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性实施例包含
Figure PCTCN2015094164-appb-000018
“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，这些可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更有选为双环或三环。桥杂环基的非限制性实施例包含：
Figure PCTCN2015094164-appb-000019
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，非限制性实施例包含：
Figure PCTCN2015094164-appb-000020
等。
杂环基可以是任选取代的或未取代的，当被取代时，取代基优选选自烷基、 烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、疏基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、氨基、卤代烷基、羟烷基、羧基和羧酸酯基中的一个或多个基团。
“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，更优选苯基和萘基，最优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，非限制性实施例包含：
Figure PCTCN2015094164-appb-000021
芳基可以是取代的或未取代的，当被取代时，取代基优选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、疏基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氨基、卤代烷基、羟烷基、羧基和羧酸酯基中的一个或多个基团。
“杂芳基”指具有1至4个杂原子作为环原子，其余的环原子为碳的5至14元芳基，其中杂原子包括氧、硫和氮。优选为5至10元。杂芳基优选为是5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，非限制性实施例包含：
Figure PCTCN2015094164-appb-000022
杂芳基可以是任选取代的或未取代的，当被取代时，取代基优选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、疏基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氨基、卤代烷基、羟烷基、羧基和羧酸酯基中的一个或多个基团。
“烷氧基”指-O-(烷基)和-O-(未取代的环烷基)，其中烷基如上所定义。非限制性实施例包含甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基等。烷氧基可以是任选取代的或未取代的，当被取代时，取代基优选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、疏基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫 基、杂环烷硫基、氨基、卤代烷基、羟烷基、羧基和羧酸酯基中的一个或多个基团。
“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
“羟基”指-OH基团。
“羟烷基”指被羟基取代的烷基，其中烷基如上所定义。
“卤素”指氟、氯、溴或碘。
“氰基”指-CN。
“羧基”指-C(O)OH。
“羧酸酯基”指-C(O)O(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本发明的合成方法
为了完成本发明的合成目的，本发明采用如下的合成技术方案：
方案一、
本发明通式(I)所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用盐的制备方法，该方法包括以下步骤：
Figure PCTCN2015094164-appb-000023
通式(IA)化合物与取代的苯并呋喃类衍生物(IB)，优选为与(R)-4-甲基-5-(环氧 乙烷-2-基)异苯并呋喃-1(3H)-酮在有机溶剂中进行加热反应，得到通式(I)化合物；
其中：R1～R3、n如通式(I)中所定义。
方案二、
本发明通式(II)所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用盐的制备方法，该方法包括以下步骤：
Figure PCTCN2015094164-appb-000024
通式(IA)化合物与取代的苯并呋喃类衍生物(IB)，优选为与(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮在有机溶剂中进行加热反应，得到通式(II)化合物；
其中：R1、R2、R4、n如通式(II)中所定义。
方案三、
本发明通式(III)所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用盐的制备方法，该方法包括以下步骤：
Figure PCTCN2015094164-appb-000025
通式(IA)化合物与取代的苯并呋喃类衍生物(IB)，优选为与(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮在有机溶剂中进行加热反应，得到通式(III)化合物；
其中：R1、R2、R4、n如式(III)中所定义。
方案四、
本发明通式(IV)所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用盐的制备方法，该方法包括以下步骤：
Figure PCTCN2015094164-appb-000026
通式(IVA)化合物与取代的苯并呋喃类衍生物(IB)，优选为与(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮在有机溶剂中进行加热反应，得到通式(IV)化合物；
其中：R1～R3、n如通式(I)中所定义。
方案五、
本发明通式(V)所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用盐的制备方法，该方法包括以下步骤：
Figure PCTCN2015094164-appb-000027
通式(IVA)化合物与取代的苯并呋喃类衍生物(IB)，优选为与(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮在有机溶剂中进行加热反应，得到通式(V)化合物；
其中：R1、R2、R4、n如通式(I)中所定义。
方案六、
本发明通式(VI)所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用盐的制备方法，该方法包括以下步骤：
Figure PCTCN2015094164-appb-000028
通式(IVA)化合物与取代的苯并呋喃类衍生物(IB)，优选为与(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮在有机溶剂中进行加热反应，得到通式(VI)化合物；
其中：R1、R2、R4、n如式(I)中所定义。
所用溶剂包括但不限于：醋酸、甲醇、乙醇、乙腈、四氢呋喃、二氯甲烷、二甲基亚砜、1，4-二氧六环、水、N，N-二甲基乙酰胺或N，N-二甲基甲酰胺，优选非极性溶剂，更优选为乙腈。
附图说明
图1：ROMK抑制剂对SD大鼠尿量的影响；
图2：ROMK抑制剂对SD大鼠尿钠排泄的影响；
图3：ROMK抑制剂对SD大鼠尿钾排泄的影响；
图4：ROMK抑制剂对SD大鼠血钠的影响；
图5：ROMK抑制剂对SD大鼠血钾的影响。
具体实施方式
以下结合实施例进一步描述本发明，但这些实施例并非限制本发明的范围。
本发明实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
化合物的结构是通过核磁共振(NMR)或质谱(MS)来确定的。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl3)、氘代甲醇(CD3OD)，内标为四甲基硅烷(TMS)，化学位移是以10-6(ppm)作为单位给出。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo，型号：Finnigan LCQ advantage MAX)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm硅胶板。
柱层析一般使用烟台黄海200～300目硅胶为载体。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH & Co.KG，Acros Organnics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中如无特殊说明，反应均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中如无特殊说明，反应中的溶液是指水溶液。
实施例中如无特殊说明，反应的温度为室温。
室温为最适宜的反应温度，温度范围是20℃～30℃。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂的体系有：A：二氯甲烷和甲醇体系，B：正己烷和乙酸乙酯体系，C：石油醚和乙酸乙酯体系，D：丙酮，溶剂的体积比根据化合物的极性不同而进行调节。
纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂的体系包括：A：二氯甲烷和甲醇体系，B：正己烷和乙酸乙酯体系，C：正己烷和丙酮体系，D：正己烷，E：乙酸乙酯，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和酸性或碱性试剂等进行调节。
实施例1
(R)-5-氰基-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)-4-甲氧基吡啶甲酰胺
Figure PCTCN2015094164-appb-000029
第一步
5-溴-4-甲氧基吡啶甲酸
将5-溴-4-甲氧基吡啶甲酸甲酯1a(250mg，1.01mmol)溶解于10mL甲醇，四氢呋喃和水(V∶V∶V＝3∶3∶1)混合溶剂中，加入氢氧化钠(100mg，2.5mmol)，搅拌反应2小时。反应液减压浓缩，加10mL水，滴加2M盐酸至反应液pH为2，用乙酸乙酯萃取(20mL×3)，用饱和氯化钠溶液(15mL×2)洗涤，无水硫酸钠干燥，过滤， 滤液减压浓缩，得到粗品标题产物5-溴-4-甲氧基吡啶甲酸1b(200mg，白色固体)，产物不经纯化直接进行下一步反应。
MS m/z(ESI)：229.9[M-1]。
第二步
4-(5-溴-4-甲氧基吡啶甲酰胺)哌啶-1-甲酸叔丁酯
将粗品5-溴-4-甲氧基吡啶甲酸1b(150mg，0.65mmol)，4-氨基哌啶-1-甲酸叔丁酯(130mg，0.65mmol)，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(190mg，1mmol)，1-羟基苯并三唑(20mg，0.13mmol)和三乙胺(0.15mL，1mmol)溶解于20mL N，N-二甲基甲酰胺中，升温至50℃，搅拌反应6小时。反应液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物4-(5-溴-4-甲氧基吡啶甲酰胺)哌啶-1-甲酸叔丁酯1c(60mg，淡黄色油状物)，产率：22.4％。
MS m/z(ESI)：414.1[M+1]。
第三步
4-(5-氰基-4-甲氧基吡啶甲酰胺)哌啶-1-甲酸叔丁酯
将4-(5-溴-4-甲氧基吡啶甲酰胺)哌啶-1-甲酸叔丁酯1c(60mg，0.15mmol)，氰化锌(26mg，0.22mmol)和四(三苯基膦)钯(18mg，0.015mmol)溶解于1.5mL N，N-二甲基甲酰胺中，135℃，微波反应40分钟。反应液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物4-(5-氰基-4-甲氧基吡啶甲酰胺)哌啶-1-甲酸叔丁酯1d(32mg，无色油状物)，产率：61.5％。
MS m/z(ESI)：361.2[M+1]。
第四步
5-氰基-4-甲氧基-N-(哌啶-4-基)吡啶甲酰胺
将4-(5-氰基-4-甲氧基吡啶甲酰胺)哌啶-1-甲酸叔丁酯1d(32mg，0.09mmol)溶解于5mL二氯甲烷中，加入1mL三氟乙酸，搅拌反应1.5小时。反应液减压浓缩，加入15mL甲醇，滴加饱和碳酸氢钠溶液至反应液pH为8，反应液减压浓缩。用薄层色谱法以展开剂体系A纯化所得残余物，得到标题产物5-氰基-4-甲氧基-N-(哌啶-4-基)吡啶甲酰胺1e(23mg，白色粘稠物)，产率：100％。
MS m/z(ESI)：261.1[M+1]。
第五步
(R)-5-氰基-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)-4-甲氧基吡啶甲酰胺
将(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮(25mg，0.09mmol，采用专利申请“WO2010129379”公开的方法制备而得)和5-氰基-4-甲氧基-N-(哌啶-4-基)吡啶甲酰胺1e(23mg，0.09mmol)溶解于5mL乙腈中，加热回流搅拌反应15小时。反应液减压浓缩。用薄层色谱法以展开剂体系A纯化所得残余物，得到标题产物(R)-5-氰基-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4- 基)-4-甲氧基吡啶甲酰胺1(4.5mg，淡黄色固体)，产率：11.3％。
MS m/z(ESI)：450.2[M+1]
1H NMR(400MHz，DMSO-d6)：δ 8.88(s，1H)，8.75(d，1H)，7.77(s，1H)，7.71-7.69(m，2H)，5.43-5.40(m，2H)，5.35(s，1H)，5.08(s，1H)，4.09(s，3H)，3.78(s，1H)，2.95(s，3H)，2.38(s，1H)，2.27(s，3H)，2.25(s，2H)，1.72(s，4H)。
实施例2
(R)-5-氰基-4-乙氧基-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺
Figure PCTCN2015094164-appb-000030
第一步
6-氯-4-乙氧基烟腈
将4，6-二氯烟腈2a(500mg，2.89mmol)溶解于20mL四氢呋喃中，0℃下滴加10mL乙醇钠(197mg，2.89mmol)的乙醇溶液，升温至室温，搅拌反应1小时。反应液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物6-氯-4-乙氧基烟腈2b(375mg，白色固体)，产率71％。
MS m/z(ESI)：183.1[M+1]
第二步
4-(5-氰基-4-乙氧基吡啶甲酰胺)哌啶-1-甲酸叔丁酯
高压釜中，加入6-氯-4-乙氧基烟腈2b(375mg，2.05mmol)，4-氨基哌啶-1-甲酸叔丁酯(422mg，2.05mmol)，醋酸钯(23mg，0.1mmol)，1，3-双(二苯基膦)丙烷(42mg，0.1mmol)，三乙胺(0.57mL，4.1mmol)和20mL乙腈，一氧化碳置换三次，压力为10bar，升温至80℃，搅拌反应16小时。反应液过滤，滤液减压浓缩，用薄层色谱法以展开剂体系A纯化所得残余物，得到标题产物4-(5-氰基-4-乙氧基吡 啶甲酰胺)哌啶-1-甲酸叔丁酯2c(645mg，白色固体)，产率84％。
MS m/z(ESI)：373.2[M-1]
第三步
5-氰基-4-乙氧基-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐
将4-(5-氰基-4-乙氧基吡啶甲酰胺)哌啶-1-甲酸叔丁酯2c(100mg，0.27mmol)溶解于5mL二氯甲烷中，加入1mL三氟乙酸，搅拌反应1小时。反应液减压浓缩，得到粗品标题产物5-氰基-4-乙氧基-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐2d(110mg，黄色油状物)，产物不经纯化直接进行下步反应。
第四步
(R)-5-氰基-4-乙氧基-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺
将(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮(50.7mg，0.27mmol)，粗品5-氰基-4-乙氧基-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐2d(110mg，0.27mmol)溶解于15mL乙腈中，加入碳酸钠(56.6mg，0.53mmol)，升温至80℃，搅拌反应48小时。反应液过滤，滤液减压浓缩，用薄层色谱法以展开剂体系A纯化所得残余物，得到标题产物(R)-5-氰基-4-乙氧基-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺2(50mg，淡黄色固体)，产率：40％。
MS m/z(ESI)：465.2[M+1]
1H NMR(400MHz，CD3OD)：δ 8.89(s，1H)，8.74(d，1H)，7.73(s，1H)，7.65(s，2H)，5.41(d，2H)，5.09(br，1H)，4.41(d，2H)，3.71-3.85(m，2H)，2.95(br，2H)，2.41-2.55(m，2H)，2.31(s，3H)，2.12-2.27(m，2H)，1.57-1.81(m，4H)，1.40(t，3H).
实施例3
(R)-5-氰基-4-(2-氟乙氧基)-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺
Figure PCTCN2015094164-appb-000031
Figure PCTCN2015094164-appb-000032
第一步
6-氯-4-(2-氟乙氧基)烟腈
将2-氟乙醇(150mg，2.34mmol)溶解于10mL四氢呋喃中，加入氢化钠(281mg，7.02mmol)，搅拌1小时。将4，6-二氯烟腈2a(405mg，2.34mmol)溶解于25mL四氢呋喃中，0℃下加入反应液，升温至室温，搅拌反应1小时。加入1mL水淬灭反应，反应液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物6-氯-4-(2-氟乙氧基)烟腈3a(210mg，白色固体)，产率45％。
MS m/z(ESI)：201.1[M+1]
第二步
4-(5-氰基-4-(2-氟乙氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯
高压釜中，加入6-氯-4-(2-氟乙氧基)烟腈3a(210mg，1.05mmol)，4-氨基哌啶-1-甲酸叔丁酯(216mg，1.05mmol)，醋酸钯(12mg，0.05mmol)，1，3-双(二苯基膦)丙烷(22mg，0.05mmol)，三乙胺(0.29mL，2.1mmol)和20mL乙腈，一氧化碳置换三次，压力为10bar，升温至80℃，搅拌反应16小时。反应液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物4-(5-氰基-4-(2-氟乙氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯3b(140mg，白色固体)，产率34％。
MS m/z(ESI)：391.1[M-1]
第三步
5-氰基-4-(2-氟乙氧基)-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐
将4-(5-氰基-4-(2-氟乙氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯3b(70mg，0.18mmol)溶解于5mL二氯甲烷中，加入1mL三氟乙酸，搅拌反应1小时。反应液减压浓缩，得到粗品标题产物5-氰基-4-(2-氟乙氧基)-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐3c(80mg，黄色油状物)，产物不经纯化直接进行下步反应。
MS m/z(ESI)：293.1[M+1]
第四步
(R)-5-氰基-4-(2-氟乙氧基)-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋 喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺
将(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮(34mg，0.18mmol)，粗品5-氰基-4-(2-氟乙氧基)-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐3c(80mg，0.18mmol)溶解于20mL乙腈中，加入碳酸钠(38mg，0.36mmol)，升温至80℃，搅拌反应48小时。反应液减压浓缩，用薄层色谱法以展开剂体系A纯化所得残余物，得到标题产物(R)-5-氰基-4-(2-氟乙氧基)-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺3(10mg，白色固体)，产率：12％。
MS m/z(ESI)：481.2[M-1]
1H NMR(400MHz，CD3OD)：δ 8.93(s，1H)，8.79(d，1H)，7.82(s，1H)，7.71(d，2H)，5.41(d，2H)，5.14(br，1H)，4.89(t，1H)，4.77(t，1H)，4.72(t，1H)，4.65(t，1H)，3.71-3.82(m，2H)，2.85-3.15(m，2H)，2.40-2.54(m，2H)，2.31(s，3H)，2.12-2.26(m，2H)，1.61-1.90(m，4H).
实施例4
(R)-5-氰基-4-(二氟甲氧基)-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺
Figure PCTCN2015094164-appb-000033
第一步
(2-溴-5-氯吡啶-4-基)硼酸
将2-溴-5-氯吡啶4a(2g，10.4mmol)溶解于40mL四氢呋喃中，-78℃下滴加 7.8mL 2M的二异丙基氨基锂溶液，搅拌1小时，加入三异丙基硼酸酯(2.94g，15.6mmol)，搅拌30分钟，升温至室温，搅拌反应16小时。反应液中加入50mL 4％的氢氧化钠溶液，搅拌30分钟，分液，取水相，冰浴下滴加6M氢氧化钠溶液至水相pH为3～4，用乙酸乙酯萃取(50mL×2)，合并有机相，用无水硫酸钠干燥，过滤，滤液减压浓缩，得到粗品标题产物(2-溴-5-氯吡啶-4-基)硼酸4b(1.3g，白色固体)，产率53％。
第二步
2-溴-5-氯吡啶-4-醇
将粗品(2-溴-5-氯吡啶-4-基)硼酸4b(1.3g，5.51mmol)溶解于40mL二氯甲烷中，加入双氧水(1.87mL，16.5mmol)，搅拌反应16小时。反应液减压浓缩，得到粗品标题产物2-溴-5-氯吡啶-4-醇4c(1g，白色固体)，产率88％。
MS m/z(ESI)：205.9/207.9[M+1]
第三步
2-溴-5-氯-4-(二氟甲氧基)吡啶
将粗品2-溴-5-氯吡啶-4-醇4c(320mg，1.54mmol)，2-氯-2，2-二氟乙酸钠(470mg，3.08mmol)，碳酸钾(470mg，3.39mmol)溶解于5mL N，N-二甲基乙酰胺中，120℃，微波反应1小时。反应液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物2-溴-5-氯-4-(二氟甲氧基)吡啶4d(950mg，无色油状物)，产率：60％。
第四步
4-(5-氯-4-(二氟甲氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯
高压釜中，加入2-溴-5-氯-4-(二氟甲氧基)吡啶4d(1.03g，3.99mmol)，4-氨基哌啶-1-甲酸叔丁酯(800mg，3.99mmol)，醋酸钯(45mg，0.2mmol)，1，3-双(二苯基膦)丙烷(82mg，0.2mmol)，三乙胺(1.1mL，7.98mmol)和30mL乙腈，一氧化碳置换三次，压力为10bar，搅拌反应16小时。反应液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物4-(5-氯-4-(二氟甲氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯4e(809mg，白色固体)，产率50％。
MS m/z(ESI)：404.1[M-1]
第五步
4-(5-氰基-4-(二氟甲氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯
将4-(5-氯-4-(二氟甲氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯4e(100mg，0.25mmol)，氰化锌(57.6mg，0.49mmol)和四(三苯基膦)钯(88mg，0.07mmol)溶解于5mL N，N-二甲基乙酰胺中，170℃，微波反应30分钟。反应液减压浓缩，用薄层色谱法以展开剂体系B纯化所得残余物，得到标题产物4-(5-氰基-4-(二氟甲氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯4f(83mg，白色固体)，产率：85％。
MS m/z(ESI)：395.0[M-1]
第六步
5-氰基-4-(二氟甲氧基)-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐
将4-(5-氰基-4-(二氟甲氧基)吡啶甲酰胺)哌啶-1-甲酸叔丁酯4f(250mg，0.63mmol)溶解于5mL二氯甲烷中，加入2mL三氟乙酸，搅拌反应1小时。反应液减压浓缩，得到粗品标题产物5-氰基-4-(二氟甲氧基)-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐4g(540mg，黄色油状物)，产物不经纯化直接进行下步反应。
MS m/z(ESI)：297.2[M+1]
第七步
(R)-5-氰基-4-(二氟甲氧基)-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺
将(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮(57.7mg，0.3mmol)，粗品5-氰基-4-(二氟甲氧基)-N-(哌啶-4-基)吡啶甲酰胺2，2，2-三氟乙酸盐4g(260mg，0.3mmol)和N，N-二异丙基乙胺(78.4mg，0.61mmol)溶解于3mL乙醇中，135℃，微波反应1小时。反应液减压浓缩，用薄层色谱法以展开剂体系A纯化所得残余物，得到标题产物(R)-5-氰基-4-(二氟甲氧基)-N-(1-(2-羟基-2-(4-甲基-1-羰基-1，3-二氢异苯并呋喃-5-基)乙基)哌啶-4-基)吡啶甲酰胺4(30mg，白色固体)，产率：20％。
MS m/z(ESI)：487.2[M+1]
1H NMR(400MHz，CD3OD)：δ 9.13(s，1H)，8.87(d，1H)，7.98(t，1H)，7.83(s，1H)，7.63-7.78(m，2H)，5.40(d，2H)，5.08(br，1H)，3.70-3.81(m，2H)，2.96(br，2H)，2.40-2.54(m，2H)，2.28(s，3H)，2.11-2.26(m，2H)，1.61-1.75(m，4H).
测试例：
生物学评价
测试例1、本发明化合物对人(human)ROMK和大鼠(rat)ROMK抑制活性的测定
以下方法用来测定本发明中化合物对人ROMK和大鼠ROMK活性的抑制作用。
1、实验材料及仪器
(1)FluxORTM钾离子通道检测(potassium ion channel assay)(F10016，invitrogen)
(2)乌本苷(O3125-1G，sigma)
(3)flexstation 3酶标仪(MD)
(4)人ROMK/HEK293细胞：转染有人ROMK cDNA(NCBI序列号：NM_000220.4)与稳定表达ROMK通道的HEK293细胞系
(5)大鼠ROMK/HEK293细胞：转染有大鼠ROMK cDNA(NCBI序列号：NM_017023.1)与稳定表达ROMK通道的HEK293细胞系
(6)HEK293细胞系：中国科学院细胞库，GNHu43
2、实验步骤
实验中所用试剂除ddH2O，乌苯甘外均来自FluxORTM钾离子通道检测这个试剂盒，配制方法也参照试剂盒说明书
(1)提前一天在PDL(Poly-D-lysine)包被的培养板中接种人ROMK/HEK293细胞，20000个/孔；
(2)过夜培养后倒掉板中的培养基，按照FluxORTM钾离子通道检测要求操作，加入100μL/孔染料，室温孵育90分钟；
(3)然后倒掉染料，每孔加入100μL含乌本苷(300μM)和丙磺舒的检测缓冲液；
(4)再加入1μL化合物或DMSO到对应的反应孔中，在震荡仪上震荡30s，室温孵育30分钟；
(5)在flexstation3酶标仪中放置培养板，由机器加入25μL/孔刺激缓冲液(K2SO4∶Tl2SO4∶1XFluxOR Chloride-free Buffer∶ddH2O＝3∶12∶40∶125)，立刻在EX/EM为490/525nm处连续读值5分钟；
(6)经Graphpad软件处理prism数据得到化合物抑制人ROMK离子通道的IC50值。
重复以上实验步骤，将人ROMK/HEK293细胞替换成大鼠ROMK/HEK293细胞，测试化合物抑制大鼠ROMK离子通道的IC50值。
本发明化合物对人ROMK或大鼠ROMK的抑制活性通过以上的试验进行测定，测得的IC50值见表1。
表1化合物对人ROMK或大鼠ROMK通道活性抑制的IC50
Figure PCTCN2015094164-appb-000034
结论：本发明的化合物对人ROMK或大鼠ROMK通道活性具有明显的抑制效果，化合物的药效高。
测试例2、本发明化合物对hERG抑制活性的测定
以下方法用来测定本发明中化合物对hERG活性的抑制作用
1、实验材料及仪器
(1)FluxORTM钾离子通道检测(F10016，invitrogen)
(2)flexstation 3酶标仪(MD)
(3)hERG/HEK293细胞：转染有人hERG cDNA(NCBI序列号：NM_000238(RC215928，origene))与稳定表达hERG通道的HEK293细胞系
2、实验步骤
实验中所用试剂除ddH2O外均来自FluxORTM钾离子通道检测试剂盒，配制方法参照试剂盒说明书
(1)提前一天在PDL(Poly-D-lysine)包被的培养板中接种人hERG/HEK293细胞，25000个/孔；
(2)过夜培养后倒掉板中的培养基，按照FluxORTM钾离子通道检测要求操作，加入100μL/孔染料，室温孵育90分钟；
(3)然后倒掉染料，每孔加入100μL含丙磺舒的检测缓冲液；
(4)再加入1μL化合物或DMSO到对应的反应孔中，在震荡仪上震荡30s，室温孵育30分钟；
(5)在flexstation3酶标仪中放置培养板，由机器加入25μL/孔刺激缓冲液(K2SO4∶Tl2SO4∶1XFluxOR Chloride-free Buffer∶ddH2O＝2∶1∶2∶5)，立刻在EX/EM为490/525nm处连续读值5分钟；
(6)经Graphpad软件处理prism数据得到化合物对hERG这一离子通道的IC50值。
本发明化合物对hERG的抑制活性通过以上的试验进行测定，测得的IC50值见表2。
表2化合物对hERG通道活性抑制的IC50
实施例 hERG IC50(μM)
1 43.7
结论：本发明的化合物对hERG通道活性的抑制作用弱，对心脏毒性低。
测试例3、电生理手动膜片钳检测对ROMK钾离子通道的作用
1、实验目的
本实验的目的是为了检测化合物在离体实验中对HEK293细胞中ROMK钾离子通道的影响。ROMK钾离子通道稳定地表达在本发明的HEK293细胞上。在钾离子电流稳定后，比较不同化合物浓度应用前后钾离子电流的大小，可以得到化合物对钾离子通道的影响。
2、实验试剂和材料
(1)HEK293细胞：中国科学院细胞库，GNHu43；
(2)人ROMK/HEK293细胞：转染有人ROMK cDNA(NCBI序列号：NM_000220.4)与稳定表达ROMK通道的HEK293细胞系；
(3)细胞外液(mM)：NaCl，137；KCl，4；CaCl2，1.8；MgCl2，1；HEPES，10；葡萄糖10；pH 7.4(NaOH滴定)；
(4)细胞内液(mM)：K Aspartate，130；MgCl2，5；EGTA 5；HEPES，10；Tris-ATP 4；pH 7.2(KOH滴定)；
化合物除用于酸碱滴定的NaOH和KOH外，均从Sigma(St.Louis，MO)公司 购买。
细胞培养液：Ham’s F12培养基(Invitrogen)，10％(v/v)灭活的胎牛血清，100μg/ml潮霉素B，100μg/ml遗传霉素(Geneticin)；
手动膜片钳系统：HEKA EPC-10信号放大器及数字转换系统，购自德国HEKA Electronics；
微操控仪器：MP-225；
拉制电极仪器：PC-10(Narishige，Japan)。
3、实验方法
测试化合物溶于二甲基亚砜(DMSO)室温储存，测试当天再按要求用细胞外液稀释成下列梯度终浓度(3、10、30、100、300nM)，用于测试。测试化合物的DMSO最终浓度为0.3％。
人ROMK/HEK293细胞生长于含上述细胞培养液的培养皿中，并在37℃、含5％CO2的培养箱中进行培养。电生理实验之前24到48小时，人ROMK/HEK293细胞被转移到放置于培养皿中的圆形玻璃片上，并在以上相同的培养液及培养条件下生长。每个圆形玻片上人ROMK/HEK293细胞的密度需要达到绝大多数细胞是独立、单个的要求。
本实验采用手动膜片钳系统作全细胞电流的记录。表面生长有人ROMK/HEK293细胞的圆形玻片被放置于倒置显微镜下的电生理记录槽中。记录槽内以细胞外液作持续灌流(大约每分钟1毫升)。实验过程采用常规全细胞膜片钳电流记录技术。如无特殊说明，实验都是在常规室温下进行(～25℃)。细胞钳制在-80mV的电压下。细胞钳制电压去极化到+20mV以激活ROMK钾通道，5秒后再钳制到-50mV以消除失活并产生尾电流。尾电流峰值用作ROMK电流大小的数值。上述步骤所记录的ROMK钾电流在记录槽内持续的细胞外液灌流下达到稳定后则可以叠加灌流待测试的药物，直到药物对ROMK电流的抑制作用达到稳定状态。一般以最近的连续3个电流记录线重合作为判断是否稳定状态的标准。达到稳定态势以后以细胞外液灌流冲洗直到ROMK电流回复到加药物之前的大小。一个细胞上可以测试一个或多个药物，或者同一种药物的多个浓度，但是在不同药物之间需要以细胞外液冲洗。
4、数据分析
试验数据由HEKA Patchmaster，XLFit以及Graphpad Prism提供的数据分析软件进行分析，参见表3。
表3本发明化合物对ROMK钾离子通道抑制的IC50
实施例编号 IC50(nM)
1 18.7
结论：本发明的化合物对ROMK钾离子通道抑制作用较强。
测试例4、电生理手动膜片钳检测对hERG钾离子通道的作用
1、实验目的
本实验的目的是为了检测化合物在离体实验中对CHO细胞中hERG钾离子通道的影响。hERG钾离子通道稳定地表达在本发明的CHO细胞上。在钾离子电流稳定后，比较不同化合物浓度应用前后钾离子电流的大小，可以得到化合物对钾离子通道的影响。
2、实验试剂和材料
(1)CHO细胞系：丹麦Sophion Bioscience公司；
(2)hERG/CHO细胞：转染有人hERG cDNA(NCBI序列号：NM_000238(RC215928，origene))与稳定表达hERG通道的CHO细胞系；
(3)细胞外液(mM)：EC 0.0.0 NaCl-林格氏溶液(Ringer’s solution)，NaCl，145；KCl，4；CaCl2，2；MgCl2，1；HEPES，10；葡萄糖10；pH 7.4(NaOH滴定)，渗透压～305mOsm；
(4)细胞内液(mM)：IC 0.0.0 KCl-林格氏溶液，KCl，120，CaCl2，5.374，MgCl2，1.75；EGTA 5；HEPES，10；Na-ATP 4；pH 7.25(KOH滴定)，渗透压～295mOsm；
化合物除用于酸碱滴定的NaOH和KOH外，均从Sigma(St.Louis，MO)公司购买。
细胞培养液：Ham’s F12培养基(Invitrogen)，10％(v/v)灭活的胎牛血清，100μg/ml潮霉素B，100μg/ml遗传霉素；
手动膜片钳系统：HEKA EPC-10信号放大器及数字转换系统，购自德国HEKA Electronics；
微操控仪器：MP-225；
拉制电极仪器：PC-10(Narishige，Japan)。
3、实验方法
在试验之前，首先用DMSO以梯度稀释的方式稀释成30、10、3、1、0.3和0.1mM的贮备液，再用细胞外液稀释成最终的将化合物稀释成下列浓度(30、10、3、1、0.3和0.1μM)来作测试。各浓度测试溶液中DMSO的最终浓度为0.1％。所有的贮备液和测试溶液都经过5-10分钟的超声和振荡以保证化合物完全溶解。
CHO hERG细胞生长于含上述细胞培养液的培养皿中，并在37℃、含5％CO2的培养箱中进行培养。电生理实验之前24到48小时，CHO hERG细胞被转移到放置于培养皿中的圆形玻璃片上，并在以上相同的培养液及培养条件下生长。每个圆形玻片上CHO hERG细胞的密度需要达到绝大多数细胞是独立、单个的要求。
本实验采用手动膜片钳系统作全细胞电流的记录。表面生长有CHO hERG细胞的圆形玻片被放置于倒置显微镜下的电生理记录槽中。记录槽内以细胞外液作持续灌流(大约每分钟1毫升)。实验过程采用常规全细胞膜片钳电流记录技术。 如无特殊说明，实验都是在常规室温下进行(～25℃)。细胞钳制在-80mV的电压下。细胞钳制电压去极化到+20mV以激活hERG钾通道，5秒后再钳制到-50mV以消除失活并产生尾电流。尾电流峰值用作hERG电流大小的数值。上述步骤所记录的hERG钾电流在记录槽内持续的细胞外液灌流下达到稳定后则可以叠加灌流待测试的药物，直到药物对hERG电流的抑制作用达到稳定状态。一般以最近的连续3个电流记录线重合作为判断是否稳定状态的标准。达到稳定态势以后以细胞外液灌流冲洗直到hERG电流回复到加药物之前的大小。一个细胞上可以测试一个或多个药物，或者同一种药物的多个浓度，但是在不同药物之间需要以细胞外液冲洗。
4、数据分析
试验数据由HEKA Patchmaster，XLFit以及Graphpad Prism提供的数据分析软件进行分析，参见表4。
表4本发明化合物对hERG钾离子通道抑制的IC50
实施例编号 IC50(μM)
1 14.95
结论：本发明的化合物对hERG钾离子通道抑制作用较弱，对心脏毒性低。
测试例5、本发明化合物的药代动力学测试
1、摘要
以大鼠为受试动物，应用LC/MS/MS法测定了大鼠灌胃给予实施例化合物后不同时刻血浆中的药物浓度。研究本发明的化合物在大鼠体内的药代动力学行为，评价其药动学特征。
2、试验方案
2.1试验药品
实施例1化合物。
2.2试验动物
健康成年SD大鼠4只，雌雄各半，购自上海西普尔-必凯实验动物有限公司，动物生产许可证号：SCXK(沪)2008-0016。
2.3药物配制
称取适量样品，加入0.5％CMC-Na至终体积，超声制成0.5mg/ml混悬液。
2.4给药
SD大鼠4只，雌雄各半，禁食一夜后分别灌胃给药，剂量为5.0mg/kg，给药体积10mL/kg。
3、操作
于给药前及给药后0.5、1、2、4、6、8、11、24小时采血0.1mL，置于EDTA抗凝试管中，3500rpm离心10分钟，分离血浆，于-20℃保存。给药后2小时进 食。
用LC/MS/MS法测定不同化合物灌胃给药后大鼠血浆中的待测化合物含量。血浆样品经沉淀蛋白预处理后进行分析。
4、药代动力学参数结果
本发明化合物的药代动力学参数见表5。
表5：
Figure PCTCN2015094164-appb-000035
测试例6、ROMK抑制剂对SD大鼠的利尿效果实验报告
1.实验目的
评估ROMK抑制剂阳性对照药物和化合物1对正常大鼠的利尿药效。
2.实验方法和实验材料
2.1.实验动物和饲养条件
实验用SD雄性大鼠，购自上海西普尔-必凯实验动物有限公司(中国上海，合格证编号2008001647752，许可证SCXK(沪)2013-0016)，购入时120-130g，5只/笼饲养，12/12小时光/暗周期调节，温度23±1℃恒温，湿度50～60％，自由进食进水。动物购进后，进行7天适应性饲养后开始实验。
2.2.实验药品
化合物1；
阳性对照药结构如下：
Figure PCTCN2015094164-appb-000036
0.9％氯化钠溶液(500ml：4.5g)。
羧甲基纤维素钠(CMC·Na)：批号为20131022，国药集团化学试剂有限公司。
钠检测试剂盒：批号20150203，由南京建成生物公司提供。
钾检测试剂盒：批号20141112，由南京建成生物公司提供。
药物剂量均按碱基计算。
2.3.实验设计和实验方法
2.3.1.动物分组：
大鼠适应性饲养后，分组如下：
分组 n 给药方式
正常组 10 0.5％CMC(i.g.单次)
化合物1-0.03mg/kg组 10 0.03mg/kg 150532(i.g.单次)
化合物1-0.1mg/kg组 10 0.1mg/kg 150532(i.g.单次)
阳性对照药-0.03mg/kg组 10 0.03mg/kg 153111(i.g.单次)
阳性对照药-0.1mg/kg组 10 0.1mg/kg 153111(i.g.单次)
2.3.2.实验方法：
实验方法按照专利(WO2010129379A1)中方法进行。将至少适应性喂养的SD大鼠置于代谢笼中禁食过夜，称完体重后随机分成以下几组：空白对照组，化合物1受试药0.03mg/kg组和0.1mg/kg组、阳性对照药组0.03mg/kg和组0.1mg/kg，每组大鼠10只。每只大鼠单次灌胃(i.g.，1ml/kg)各化合物，空白组灌胃相应的溶剂，灌胃后将鼠置于一般的笼盒，30min后给予25ml/kg生理盐水，之后立即将大鼠放入代谢笼，禁食禁水，收集并计量4h的总尿量，测定4h的尿钠和尿钾排泄量。并于收集尿液后眼眶采血收集血清检测血钠和血钾浓度。
2.4.实验仪器
室温离心机：型号5417C，由Eppendorf提供。
2.5.数据表达和统计学处理
实验数据表示为平均数(Mean)±标准差(S.D.)。采用excel软件t检验进行统计比较。将给药组与空白对照组数据进行分析比较，是否存在显著数理统计意义，*P＜0.05表示给药组与空白对照组比较具有显著性差异，**P＜0.01表示给药组与空白对照组比较具有高度显著性差异。
3.结果
实验结果显示，与空白对照组比较，阳性对照药0.03mg/kg组和0.1mg/kg组尿量明显增加(P＜0.05)，尿量分别增加1.41倍和1.46倍，化合物1受试药0.03mg/kg组和0.1mg/kg组尿量显著增加(P＜0.01)，尿量分别增加2.76倍和3.22倍(见图1)；阳性药和各化合物1受试药组尿钠排泄显著增加(P＜0.01)，尿钠排泄分别增加1.57倍、 1.65倍、3.12倍和3.31倍(见图2)；和正常对照组相比，阳性药和受试药尿钾虽略有升高但无统计学意义(见图3)；同时，阳性药和各受试药组的血钠和血钾变化不大(P＞0.05)(见图4和5)。
4.讨论
根据功能特性的不同，K+通道可分为以下四种类型：慢(延迟)K+通道(K通道)、快(早期)K+通道(A通道)、Ca2+活化的K+通道〔K(Ca)通道〕和内向整流的K+通道四类。内向整流的K+通道(inwardly rectifying K channels，Kir)家族又分为七种类型：Kir1～Kir7，分别有不同的KCNJ基因编码。肾外髓质分泌钾通道(renal outer medullary potassium channel，ROMK)主要属于Kir1型，在鼠肾中至少存在三种亚型的ROMK通道：ROMK1、ROMK2和ROMK3型，ROMK2大部分分布于髓袢升支粗段；ROMK1和ROMK3主要在集合管上表达。表达于髓袢升支粗段的ROMK与Na/K/Cl转运体一起调节钾离子的分泌和重吸收，表达于皮质集合小管的ROMK与Na/K转运体一起调节钾离子的分泌，阻断ROMK位点既可以促进NaCl分泌到管腔，又不至于使血钾过度降低导致低钾血症，是高血压患者一个良好的利尿剂研究方向，本实验主要探讨ROMK抑制剂的利尿效果。
本次实验过程中，受试药化合物1溶解性很好，没有出现分层现象，但阳性对照药在称取过程中有静电，不易称取，且在研磨初期结团不易溶解，充分研磨后溶解良好。此次结果也显示，与正常组比较，单次口服灌胃化合物1和阳性对照药对大鼠具有明显的利尿排钠的作用，且受试药化合物1和阳性对照药各剂量间呈剂量依赖。
5.结论
受试药化合物1和受试药阳性对照药均具有明显的利尿排钠效果，且对血钾无影响；但化合物1的利尿药效明显优于阳性对照药，各组间药效呈剂量依赖。

Claims (16)

  1. 一种通式(I)所示的化合物：
    Figure PCTCN2015094164-appb-100001
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
    其中：
    R1为烷基，其中所述烷基任选进一步被选自卤素、羟基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基和羧酸酯基中的一个或多个取代基所取代；
    R2选自氢原子、烷基、卤素、氰基、硝基、烷氧基、环烷基和杂环基，其中所述烷基、烷氧基、环烷基或杂环基任选进一步被选自烷基、卤素、羟基、羟烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基和羧酸酯基中的一个或多个取代基所取代；
    R3选自下列基团：
    Figure PCTCN2015094164-appb-100002
    R4或R5各自独立地选自氢原子、烷基、卤素、氰基、硝基、烷氧基、环烷基、杂环基、芳基和杂芳基；
    R6选自氢原子、烷基和卤素；
    n为0、1或2。
  2. 根据权利要求1所述的通式(I)所示的化合物，其中R1为烷基，其中所述的烷基任选进一步被选自卤素、羟基和烷氧基中的一个或多个取代基所取代；R1优选为甲基、乙基或丙基。
  3. 根据权利要求1所述的通式(I)所示的化合物，其中R4为烷基，且R5为氢原子。
  4. 根据权利要求1所述的通式(I)所示的化合物，其为通式(II)所示的化合物：
    Figure PCTCN2015094164-appb-100003
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
    其中：R1、R2、R4、n如权利要求1中所定义。
  5. 根据权利要求1所述的通式(I)所示的化合物，其为通式(III)所示的化合物：
    Figure PCTCN2015094164-appb-100004
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
    其中：R1、R2、R4、n如权利要求1中所定义。
  6. 根据权利要求1～5中任意一项所述的通式(I)所示的化合物，其选自：
    Figure PCTCN2015094164-appb-100005
  7. 一种通式(IA)所述的化合物：
    Figure PCTCN2015094164-appb-100006
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐，
    其中：
    R1为烷基，其中所述烷基任选进一步被选自卤素、羟基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基和羧酸酯基中的一个或多个取代基所取代；
    R2选自氢原子、烷基、卤素、氰基、硝基、烷氧基、环烷基和杂环基，其中所述烷基、烷氧基、环烷基或杂环基任选进一步被选自烷基、卤素、羟基、羟烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基和羧酸酯基中的一个或多个取代基所取代；
    n为0、1或2。
  8. 根据权利要求7所述的通式(IA)所示的化合物，其为：
    Figure PCTCN2015094164-appb-100007
  9. 一种制备根据权利要求1所述的通式(I)所示的化合物的方法，该方法包括：
    Figure PCTCN2015094164-appb-100008
    通式(IA)化合物与取代的苯并呋喃类衍生物(IB)，优选为与(R)-4-甲基-5-(环氧乙烷-2-基)异苯并呋喃-1(3H)-酮加热反应，得到通式(I)化合物；
    其中：R1-R3、n如权利要求1中所定义。
  10. 一种药物组合物，其含有治疗有效量的根据权利要求1～6中任意一项所述的通式(I)所示的化合物以及药学上可接受的载体、稀释剂或赋形剂。
  11. 根据权利要求1～6中任意一项所述的通式(I)所示的化合物或根据权利要求10所述的药物组合物在制备ROMK抑制剂中的用途。
  12. 根据权利要求1～6中任意一项所述的通式(I)所示的化合物或根据权利要求10所述的药物组合物在制备治疗或预防高血压和/或心力衰竭的药物中的用途。
  13. 根据权利要求1～6中任意一项所述的通式(I)所示的化合物或根据权利要求10所述的药物组合物在制备治疗或预防ROMK介导的疾病的药物中的用途，所述的疾病优选选自肝硬化、急性和慢性的肾功能不全、肾病综合症、肺动脉高血压、心血管疾病、心肌梗塞、中风、心功能不全、肺张力过强、动脉粥样硬化和肾结石。
  14. 一种抑制ROMK的方法，其包括给予所需患者治疗有效量的根据权利要求1～6中任意一项所述的通式(I)所示的化合物或根据权利要求10所述的药物组合物。
  15. 一种治疗或预防高血压和/或心力衰竭的方法，其包括给予所需患者治疗有效量的根据权利要求1～6中任意一项所述的通式(I)所示的化合物或根据权利要求10所述的药物组合物。
  16. 一种治疗或预防ROMK介导的疾病或病症的方法，其包括给予所需患者治疗有效量的根据权利要求1～6中任意一项所述的通式(I)所示的化合物或根据权利要求10所述的药物组合物，所述的疾病优选选自肝硬化、急性和慢性的肾功能不全、肾病综合症、肺动脉高血压、心血管疾病、心肌梗塞、中风、心功能不全、肺张力过强、动脉粥样硬化和肾结石。
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CN108290874A (zh) * 2016-06-07 2018-07-17 江苏恒瑞医药股份有限公司 一种肾外髓质分泌钾通道抑制剂的可药用盐
CN108290874B (zh) * 2016-06-07 2021-07-27 江苏恒瑞医药股份有限公司 一种肾外髓质分泌钾通道抑制剂的可药用盐
CN106432071A (zh) * 2016-09-05 2017-02-22 河北正朗制药有限公司 5‑氰基‑4‑甲氧基‑2‑吡啶甲酸的盐酸盐的制备及其应用
CN106432071B (zh) * 2016-09-05 2019-04-02 河北正朗制药有限公司 5-氰基-4-甲氧基-2-吡啶甲酸的盐酸盐的制备及其应用
WO2018093569A1 (en) 2016-11-03 2018-05-24 Bristol-Myers Squibb Company Substituted bicycle heterocyclic derivatives useful as romk channel inhibitors
WO2018222795A1 (en) 2017-06-01 2018-12-06 Bristol-Myers Squibb Company Substituted nitrogen containing compounds
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CN109983008A (zh) * 2017-07-10 2019-07-05 江苏恒瑞医药股份有限公司 一种吡啶甲酰胺类衍生物的制备方法及其中间体
WO2019011200A1 (zh) * 2017-07-10 2019-01-17 江苏恒瑞医药股份有限公司 一种吡啶甲酰胺类衍生物的制备方法及其中间体
CN109879863A (zh) * 2017-12-06 2019-06-14 江苏恒瑞医药股份有限公司 一种肾外髓质分泌钾通道抑制剂的晶型及其制备方法
CN111372929A (zh) * 2017-12-06 2020-07-03 江苏恒瑞医药股份有限公司 一种肾外髓质分泌钾通道抑制剂的晶型及其制备方法
CN109879863B (zh) * 2017-12-06 2020-10-20 江苏恒瑞医药股份有限公司 一种肾外髓质分泌钾通道抑制剂的晶型及其制备方法
WO2019109935A1 (zh) 2017-12-06 2019-06-13 江苏恒瑞医药股份有限公司 一种肾外髓质分泌钾通道抑制剂的晶型及其制备方法
CN111372929B (zh) * 2017-12-06 2022-04-12 江苏恒瑞医药股份有限公司 一种肾外髓质分泌钾通道抑制剂的晶型及其制备方法
WO2020173476A1 (zh) * 2019-02-28 2020-09-03 江苏恒瑞医药股份有限公司 一种含有肾脏钾离子外排通道抑制剂的药物组合物及其制备方法
CN113226315A (zh) * 2019-02-28 2021-08-06 江苏恒瑞医药股份有限公司 一种含有肾脏钾离子外排通道抑制剂的药物组合物及其制备方法

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