WO2016008404A1 - 一类吡唑酮化合物及其用途 - Google Patents

一类吡唑酮化合物及其用途 Download PDF

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WO2016008404A1
WO2016008404A1 PCT/CN2015/084006 CN2015084006W WO2016008404A1 WO 2016008404 A1 WO2016008404 A1 WO 2016008404A1 CN 2015084006 W CN2015084006 W CN 2015084006W WO 2016008404 A1 WO2016008404 A1 WO 2016008404A1
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group
compound
formula
alkyl
substituted
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French (fr)
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南发俊
李佳
李静雅
张梅
陈达锴
张丽娜
张润涛
谢治富
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Shanghai Institute of Materia Medica of CAS
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Shanghai Institute of Materia Medica of CAS
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Priority to EP15822833.8A priority Critical patent/EP3170823B1/en
Priority to US15/326,310 priority patent/US10196389B2/en
Priority to JP2017502142A priority patent/JP6408687B2/ja
Publication of WO2016008404A1 publication Critical patent/WO2016008404A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • A61K31/437—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 nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/68—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
    • C07D211/72—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D211/78—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11031—[Hydroxymethylglutaryl-CoA reductase (NADPH)] kinase (2.7.11.31)

Definitions

  • the invention belongs to the field of medicinal chemistry, and relates to a pyrazolone compound and a preparation method thereof, and a pharmaceutical composition comprising the same.
  • Diabetes is a series of metabolic disorders caused by factors such as islet dysfunction and insulin resistance. Genetic factors, immune dysfunction, and mental factors can cause diabetes. According to the World Health Organization, by 2011, there were approximately 346 million people with diabetes worldwide. About 3.4 million people died of hyperglycemia in 2004, and more than 80% of diabetes deaths occurred in low- and middle-income countries.
  • drugs for the treatment of type 2 diabetes including metformin, sulfonylureas, DPP-4 inhibitors, PPAR gamma agonists, alpha glucosidase inhibitors, insulin and GLP-1 analogs, and the like.
  • metformin sulfonylureas
  • DPP-4 inhibitors PPAR gamma agonists
  • alpha glucosidase inhibitors insulin and GLP-1 analogs
  • existing drugs have ineffective effects, short duration of action, and some drugs have side effects such as hypoglycemia, weight gain, edema, fractures, lactic acidosis, and gastrointestinal discomfort.
  • ATP-activated protein kinase plays a leading role in glycolipid metabolism in the body. It is an energy meter and a metabolic main switch that reflect changes in the energy state of cells. Its activation can significantly improve the disorder of glucose and lipid metabolism in type 2 diabetes and enhance the body. Insulin sensitivity has been identified as a new target for the treatment of type 2 diabetes. Studies have shown that many drugs or natural products for the treatment of type 2 diabetes have been found to indirectly activate AMPK in vivo, and the therapeutic effect may be partly due to the activation of AMPK, but these indirect activators have side effects such as gastrointestinal discomfort and weight gain. Therefore, the search for and discovery of AMPK small molecule activators, especially small molecule activators directly acting on AMPK, will be an effective way to seek treatment for type 2 diabetes.
  • R 1 is a group selected from the group consisting of H, C1-C4 alkyl, and a hydrogen atom on the group is optionally a 6- to 12-membered aryl group substituted with one or more substituents selected from the group below.
  • a 5- to 10-membered heteroaryl group halogen, C1-C4 alkyl, C1-C4 haloalkyl, adamantyl, hydroxy, amino, nitro, substituted or unsubstituted acetoxy (AcO), carboxyl, C1 ⁇ C4 alkoxy, C1 ⁇ C4 haloalkoxy group, a cyano group, a substituted or unsubstituted phenyl, -SO 2 -NH 2; wherein the substituents on a group means that one or more hydrogen atoms are selected from Substituents of the lower group are optionally substituted: halogen, trifluoromethyl, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, adamantyl, cyano;
  • R 2 is a group selected from the group consisting of H, C1-C4 alkyl, or R 2 is absent;
  • R 3 is a group selected from the group consisting of H, C1-C4 alkyl, or R 3 is absent;
  • R 4 is a group selected from the group consisting of H, C1-C4 alkyl, C1-C4 haloalkyl, halogen, hydroxy, amino, nitro, acetoxy (AcO), carboxyl, C1-C4 alkoxy, Cyano group
  • R 5 is a group selected from the group consisting of H, C 1 -C 4 alkyl, C 3 -C 7 cycloalkyl, C 1 -C 4 alkylene-C 3 -C 7 cycloalkyl, phenyl, C 1 -C 4 alkylene benzene a group wherein the hydrogen atom on the phenyl or C1-C4 alkylene phenyl group may be optionally substituted by one or more substituents selected from the group consisting of hydroxyl, halogen, C1-C4 alkyl, C1- a C4 haloalkyl group, a C3-C7 cycloalkyl group, a C1-C4 alkoxy group, a C1-C4 haloalkoxy group; wherein when R5 is a phenyl group-containing group, hydrogen on two carbons adjacent to the phenyl group An atom may be optionally substituted with a "-O-(CH 2 )
  • N1 is 0, 1, or 2;
  • N2 is 0, 1, 2, or 3;
  • N3 is 0, 1, or 2;
  • X is selected from the group consisting of O, NR 8 , wherein R 8 is selected from the group consisting of H, C1-C4 alkyl, C1-C4 haloalkyl, C3-C7 cycloalkyl, phenyl, C1-C4 alkylene benzene base;
  • the dotted line is a chemical bond or does not exist.
  • the C1-C4 haloalkyl group includes a C1-C4 fluoro group, preferably a trifluoromethyl group.
  • R 1 is a phenyl group substituted with 1 to 5 substituents selected from the group consisting of halogen and trifluoromethyl.
  • the 6 to 12 membered aryl group is a phenyl group or a naphthyl group.
  • the C1-C4 alkylenephenyl group includes a benzyl group, -C(cyclopropyl)-phenyl group.
  • R 1 is a group selected from the group consisting of a 6- to 12-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with a substituent selected from the group consisting of: Halogen, trifluoromethyl, C1-C4 alkyl, adamantyl, hydroxy, amino, nitro, substituted or unsubstituted acetoxy (AcO), carboxyl, C1-C4 alkoxy, cyano, substituted or Unsubstituted phenyl, -SO 2 -NH 2 ;
  • R 2 is a group selected from the group consisting of H, methyl, or R 2 not present;
  • R 3 is a group selected from the group consisting of H, or R 3 is absent;
  • R 4 is H or methyl
  • N1, n2, n3 are 1;
  • X is O or NR 8 , wherein R 8 is H or methyl.
  • R 1 is an aryl or heteroaryl group selected from the group consisting of phenyl, benzothiazolyl; wherein one or more hydrogen atoms on the aryl or heteroaryl group can be Substituted from one or more substituents selected from the group consisting of halogen, trifluoromethyl, C1-C4 alkyl, hydroxy, amino, nitro, substituted or unsubstituted acetoxy (AcO), carboxyl, C1-C4 Alkoxy, cyano, substituted or unsubstituted phenyl, -SO 2 -NH 2 ; and/or
  • X is NR 8 wherein R 8 is H or methyl.
  • the C1-C4 alkyl group is a deuterated methyl group.
  • the compound is selected from the group consisting of:
  • R 3 , R 4 , R 5 , X, n1, n2 and n3 groups are as described above.
  • -XR 5 is -OCH 3 .
  • the reaction is carried out in the presence of a base, preferably the base is selected from the group consisting of K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 , NaH, or a combination thereof. .
  • the compound of formula Ia is prepared by the method of the third aspect of the invention.
  • R 3 is H.
  • the step (1) is carried out in the presence of a base, preferably in the presence of Et 3 N and/or EtONa; more preferably, the EtONa is a freshly prepared EtONa.
  • the dehydrogenation reaction is carried out in the presence of a reagent selected from the group consisting of palladium on carbon, 2,3-dichloro-5,6-dicyandi
  • a reagent selected from the group consisting of palladium on carbon, 2,3-dichloro-5,6-dicyandi
  • the benzyl-1,4-benzoquinone is preferably carried out in the presence of 10% palladium on carbon.
  • the reaction in the step (3), is carried out in the presence of a base; preferably, the base is selected from the group consisting of LDA, K 2 CO 3 , NaOH, or combination.
  • the method optionally further comprises the steps of:
  • R 5 may be the same or different.
  • the step (i) is carried out in the presence of an alkali metal hydroxide.
  • the alkali metal hydroxide is selected from the group consisting of LiOH, NaOH, or a combination thereof.
  • the step (ii) is carried out in the presence of a condensing agent.
  • the condensing agent is selected from the group consisting of EDCI (1-ethyl-(3-dimethylaminopropyl) Carbide diimine hydrochloride), pyridine/EDC/HOBT, DCC, DCC/HOBt, DCC/DMAP, or a combination thereof.
  • a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains a therapeutically effective amount or a safe and effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition contains 0.0001 to 99% by weight of a compound of formula I or a pharmaceutically acceptable salt thereof; and the balance of a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is for treating a disease associated with AMPK activity.
  • the pharmaceutical composition is for treating a disorder of glycolipid metabolism, and preferably, the pharmaceutical composition is for treating a disease selected from the group consisting of diabetes, obesity.
  • said activation is dose dependent activation when used to non-therapeuticly activate AMPK activity in vitro.
  • said activation is concentration dependent activation when used to non-therapeuticly activate AMPK activity in vitro.
  • the activation when used to non-therapeuticly activate AMPK activity in vitro, is a molecular level activation or a cellular level activation.
  • an AMPK activity activator comprising: an activating effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.
  • the activator is a molecular AMPK activator.
  • the activator is an intracellular AMPK activator.
  • a method of activating AMPK activity wherein a safe or effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof.
  • a method of treating a disease associated with AMPK activity comprising: administering to a subject in need thereof, a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.
  • the subject comprises a mammal (e.g., a human).
  • Figure 1 is an experimental result of a multi-concentration allosteric activation of AMPK ( ⁇ 2 ⁇ 1 ⁇ 1) at the molecular level of each compound;
  • 2A and B show the stimulation of AMPK and ACC phosphorylation by small molecule compounds on L6 myotubes, and the selection of compounds capable of significantly activating AMPK to detect glucose uptake and AMPK dependence;
  • Figure 3 is a graph showing the stimulation of phosphorylation of AMPK and AC by ZM-1 at different concentrations and at different treatment times on HepG2 cells;
  • Figure 4 shows ZM-1 stimulates AMPK and ACC phosphorylation on SD rat primary hepatocytes.
  • the inventors have prepared a class of pyrazolone compounds by long-term and intensive research, and the compounds have direct activation of AMPK activity and can be applied to preparation of drugs for treating obesity and diabetes. Based on the above findings, the inventors completed the present invention.
  • the alkyl group includes a linear or branched alkyl group
  • the alkenyl group includes a linear or branched alkenyl group
  • the alkynyl group includes a linear or branched chain.
  • An alkynyl group, said halogen being F, Cl, Br or I, preferably F or Br.
  • substituted means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4.
  • the referenced atoms include all isotopic forms thereof, for example, when referring to "hydrogen atom", it refers to a hydrogen atom, a deuterium atom, a deuterium atom, or a combination thereof.
  • hydrogen atom it refers to a hydrogen atom, a deuterium atom, a deuterium atom, or a combination thereof.
  • the abundance of various isotopic atoms of an element may be a state in which the element naturally exists in nature, or may be a state in which an isotopic is enriched.
  • C1-C4 alkyl refers to a straight or branched alkyl group having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, Tert-butyl, or a similar group.
  • C3-C7 cycloalkyl refers to a cycloalkyl group having 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or the like.
  • 5 to 10 membered aryl means an aryl group having 5 to 10 carbon atoms, and includes a monocyclic or bicyclic aryl group such as a phenyl group, a naphthyl group, or the like.
  • 5-10 membered heteroaryl refers to a heteroaryl group having 5 to 10 carbon atoms or a hetero atom (selected from N, O, S), such as pyrrolyl, pyridyl, furyl, or the like.
  • C1-C4 alkoxy refers to a straight or branched alkoxy group having from 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso Butoxy, sec-butoxy, tert-butoxy, or the like.
  • C1-C4 acyl means a group having a "-CO-alkyl” structure, preferably having a structure of "-CO-C1-C4 alkyl", such as a methyl acyl group, an ethyl acyl group, a propyl acyl group, and a different group.
  • a methyl acyl group such as a methyl acyl group, an ethyl acyl group, a propyl acyl group, and a different group.
  • C1 ⁇ C4 ester group refers to an alkyl group -COO- structure, preferably a C1 ⁇ C4 alkyl group -COO- structure, e.g. CH 3 COO-, C 2 H 5 COO-, C 3 H 8 COO-, :(CH 3 ) 2 CHCOO-, nC 4 H 9 COO-, tC 4 H 9 COO-, or the like.
  • ester group of C2 to C10 means a group having a structure of 2 to 10 carbon atoms such as "-COO-C1-C9 alkyl", such as CH 3 COO-, C 2 H 5 COO-, C 3 H 8 COO-, (CH 3 ) 2 CHCOO-, nC 4 H 9 COO-, tC 4 H 9 COO-, or the like.
  • halogen refers to F, Cl, Br and I.
  • the present invention provides a compound of the formula I:
  • R 1 is a group selected from the group consisting of H, C1-C4 alkyl, and a hydrogen atom on the group is optionally a 6- to 12-membered aryl group substituted with one or more substituents selected from the group below.
  • a 5- to 10-membered heteroaryl group halogen, C1-C4 alkyl, C1-C4 haloalkyl, adamantyl, hydroxy, amino, nitro, substituted or unsubstituted acetoxy (AcO), carboxyl, C1 ⁇ C4 alkoxy, C1 ⁇ C4 haloalkoxy group, a cyano group, a substituted or unsubstituted phenyl, -SO 2 -NH 2; wherein the substituents on a group means that one or more hydrogen atoms are selected from Substituents of the lower group are optionally substituted: halogen, trifluoromethyl, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, adamantyl, cyano;
  • R 2 is a group selected from the group consisting of H, C1-C4 alkyl, or R 2 is absent;
  • R 3 is a group selected from the group consisting of H, C1-C4 alkyl, or R 3 is absent;
  • R 4 is a group selected from the group consisting of H, C1-C4 alkyl, C1-C4 haloalkyl, halogen, hydroxy, amino, nitro, acetoxy (AcO), carboxyl, C1-C4 alkoxy, Cyano group
  • R 5 is a group selected from the group consisting of H, C1-C4 alkyl, C3-C7 cycloalkyl, phenyl, C1-C4 alkylene phenyl, wherein the phenyl or C1-C4 alkylene
  • N1 is 0, 1, or 2;
  • N2 is 0, 1, 2, or 3;
  • N3 is 0, 1, or 2;
  • X is selected from the group consisting of O, NR 8 , wherein R 8 is selected from the group consisting of H, C1-C4 alkyl, C1-C4 haloalkyl, C3-C7 cycloalkyl, phenyl, C1-C4 alkylene benzene base;
  • the dotted line is a chemical bond or does not exist.
  • the C1-C4 haloalkyl group includes a C1-C4 fluoro group, preferably a trifluoromethyl group.
  • R 1 is a phenyl group substituted with 1 to 5 substituents selected from the group consisting of halogen and trifluoromethyl.
  • the 6 to 12 membered aryl group is a phenyl group or a naphthyl group.
  • the C1-C4 alkylenephenyl group includes a benzyl group, -C(cyclopropyl)-phenyl group.
  • R 1 is a group selected from the group consisting of a 6- to 12-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with a substituent selected from the group consisting of: Halogen, trifluoromethyl, C1-C4 alkyl, adamantyl, hydroxy, amino, nitro, substituted or unsubstituted acetoxy (AcO), carboxyl, C1-C4 alkoxy, cyano, substituted or Unsubstituted phenyl, -SO 2 -NH 2 ;
  • R 2 is a group selected from the group consisting of H, methyl, or R 2 not present;
  • R 3 is a group selected from the group consisting of H, or R 3 is absent;
  • R 4 is H or methyl
  • N1, n2, n3 are 1;
  • X is O or NR 8 , wherein R 8 is H or methyl
  • the dotted line is a chemical bond or does not exist.
  • R 1 is an aryl or heteroaryl group selected from the group consisting of phenyl, benzothiazolyl; wherein one or more hydrogen atoms on the aryl or heteroaryl group can be Substituted from one or more substituents selected from the group consisting of halogen, trifluoromethyl, C1-C4 alkyl, hydroxy, amino, nitro, substituted or unsubstituted acetoxy (AcO), carboxyl, C1-C4 Alkoxy, cyano, substituted or unsubstituted phenyl, -SO 2 -NH 2 ; and/or
  • X is NR 8 wherein R 8 is H or methyl.
  • the compound is selected from the group consisting of:
  • the invention also provides a method for preparing a compound of formula I, characterized in that it comprises the steps of:
  • R 3 is H.
  • the step (1) is carried out in the presence of a base, preferably in the presence of Et 3 N and/or EtONa; more preferably, the EtONa is a freshly prepared EtONa.
  • the dehydrogenation reaction is carried out in the presence of a reagent selected from the group consisting of palladium on carbon, 2,3-dichloro-5,6-dicyandi
  • a reagent selected from the group consisting of palladium on carbon, 2,3-dichloro-5,6-dicyandi
  • the benzyl-1,4-benzoquinone is preferably carried out in the presence of 10% palladium on carbon.
  • the reaction in the step (3), is carried out in the presence of a base; preferably, the base is selected from the group consisting of LDA, K 2 CO 3 , NaOH, or combination.
  • the method optionally further comprises the steps of:
  • R 5 may be the same or different.
  • the step (i) is carried out in the presence of an alkali metal hydroxide.
  • the alkali metal hydroxide is selected from the group consisting of LiOH, NaOH, or a combination thereof.
  • the step (ii) is carried out in the presence of a condensing agent.
  • the condensing agent is selected from the group consisting of EDCI (1-ethyl-(3-dimethylaminopropyl) Carbide diimine hydrochloride), pyridine/EDC/HOBT, DCC, DCC/HOBt, DCC/DMAP, or a combination thereof.
  • reaction materials can be prepared by a method known in the art or can be obtained by a commercially available route.
  • compound of formula Ia is prepared by the following method:
  • the reaction is carried out in the presence of a base, preferably the base is selected from the group consisting of K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 , NaH, or a combination thereof. .
  • a preferred method of preparing a compound of formula I comprises the steps of:
  • the substituted 3-ethoxycarbonyl-4-piperidone ketone hydrochloride A1 is condensed with the substituted benzyl bromide (A2) under basic potassium carbonate to give the intermediate A3.
  • the substituted aryl hydrazine or its hydrochloride (A4) is reacted with the piperidone intermediate A3 to form the pyrazolone compound A5.
  • R 3 is H
  • the compound A5 is dehydrogenated with 10% Pd/C to give a double bond compound A6.
  • R 3 is H
  • the compound A5 is added with the base LDA and the corresponding alkyl iodide to give the compound A7.
  • the compounds A6 and A7 are hydrolyzed to the corresponding acids, which are reacted with the corresponding amines in the presence of a condensing agent such as EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride).
  • a condensing agent such as EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride).
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • the invention also provides the use of the compounds of formula I described.
  • the compound of formula I is for use in the preparation of an AMPK activator;
  • the AMPK activity activator comprises an activating effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.
  • the activator can be a molecular AMPK activator or an intracellular AMPK activator.
  • the compound of formula I is used to prepare an AMPK and/or ACC phosphorylation promoter.
  • the compound of formula I is for non-therapeutic activation of AMPK activity in vitro.
  • the activation is dose dependent or concentration dependent.
  • the activation is a molecular level activation or a cellular level activation.
  • the compound of formula I is used to non-therapeuticly promote AMPK and/or ACC phosphorylation in vitro.
  • the compound of formula I is for use in the preparation of a pharmaceutical composition for the treatment of a disease associated with AMPK activity.
  • the pharmaceutical composition is for treating a disorder of glycolipid metabolism, and preferably, the pharmaceutical composition is for treating a disease selected from the group consisting of diabetes, obesity.
  • NMR was measured with a Mercury-Vx 300M instrument manufactured by Varian, NMR calibration: ⁇ H 7.26 ppm (CDCl 3 ); mass spectrometer with Agilent 1200 Quadrupole LC/MS LC/MS; reagent mainly from Shanghai Chemical Provided by reagent company; TLC thin layer chromatography silica gel plate is produced by Shandong Yantai Huiyou Silicone Development Co., Ltd., model HSGF 254; normal phase column chromatography silica gel used for compound purification is produced by Shandong Qingdao Marine Chemical Plant Branch, model zcx-11, 200-300 mesh.
  • the compound ZM-3 (40 mg, 0.089 mmol) was dissolved in 5 mL of anhydrous THF, cooled to 0 ° C, and LDA (2M, 0.1 mL) was slowly added dropwise. After stirring at this temperature for 10 minutes, MeI (40 L) was added. After stirring at 0 ° C for 0.5 hour, the mixture was stirred at room temperature for 2 hours, and the reaction of the starting material was monitored by TLC. After quenching with saturated ammonium chloride, ethyl acetate (3 ⁇ ), EtOAc (EtOAc)EtOAc. The yield is 60%.
  • Test Purpose To screen small molecule compounds that can allosterically activate AMPK ( ⁇ 2 ⁇ 1 ⁇ 1).
  • the substrate peptide contains a site which can be phosphorylated by AMPK.
  • One end of the reaction system (containing 2mMMgCl 2 , 400 ⁇ M DTT, 160nM substrate peptide, 4 ⁇ M ATP and 1.6nM AMPK) is connected with biotin. .
  • biotin can be linked to streptavidin-XL665 (acceptor) to complete the labeling.
  • the substrate-generated phosphorylated substrate binds to an antibody that recognizes the site's anti-phosphorylation site (labeled Donor), and the other end Biotin can be linked to streptavidin-XL665 (acceptor) to detect the resulting FRET signal using PE's Evision.
  • the final AMPK activity is expressed as the ratio of the 665 nm wavelength reading to the 615 nm wavelength reading (665 nm / 615 nm).
  • Detection reagent Human AMPK ( ⁇ 2 ⁇ 1 ⁇ 1) protein was expressed and purified by E. coli expression system; HTRF KinEASE-STK S1kit purchased from Cisbio; ATP purchased from Sigma.
  • Detection purpose Whether the small molecule compound that significantly activates AMPK at the molecular level has the effect of activating intracellular AMPK.
  • Detection reagents AMPK, phospho-AMPK (T172), phospho-ACC (S79), and Total ACC antibodies were purchased from Cell Signaling Technology.
  • the cells were changed to serum-free medium for 2 hours, and after compound treatment (0.4% DMSO) was added for 1 hour, the cells to be collected were washed once with PBS and then added with 1 ⁇ SDS gel loading buffer (50 mM Tris- The cells were lysed with HCl (pH 6.8), 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromophenol blue (70 ⁇ l per well in a 24-well plate).
  • 1 ⁇ SDS gel loading buffer 50 mM Tris- The cells were lysed with HCl (pH 6.8), 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromophenol blue (70 ⁇ l per well in a 24-well plate).
  • the sample was heated at 100 ° C for 10 minutes, and then centrifuged at 12000 g for 10 minutes, and then the supernatant was taken for SDS-PAGE electrophoresis under the conditions of a layered gel: 70 volts, separation gel: 90 volts.
  • the protein was transferred to a nitrocellulose membrane using a Biorad wet transfer system. After the target band was cut, the strip was placed in a blocking solution (TBST containing 5% BSA) for 1 hour at room temperature. The strips were then incubated overnight at 4 ° C in a primary antibody solution.
  • a blocking solution TST containing 5% BSA
  • the target strip was placed in TBST the next day and washed at room temperature for 3 x 10 minutes. The strips were then incubated in a secondary antibody solution (1:8000 for goat anti-rabbit and goat anti-mouse) and incubated for 1 hour at room temperature, followed by TBST washing for 3 x 15 minutes and exposure with ECL reagent.
  • ZM-1 has the best activation effect on AMPK at the cellular level, and L6 muscle cells can be significantly activated in a dose-dependent manner for 1 hour.
  • AMPK, and its downstream ACC phosphate levels are up-regulated accordingly.
  • HepG2 cells treated with different concentrations of ZM-1 were able to increase the concentration gradient and stimulate the upregulation of AMPK and ACC phosphorylation levels.

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Abstract

本发明提供了一类吡唑酮化合物及其用途，具体地，本发明提供了一种如通式I所示的化合物，其中，各基团的定义如说明书中所述。本发明的化合物具有直接激活AMPK活性，可剂量依赖性地显著促进L6肌细胞和HepG2细胞的AMPK及ACC的磷酸化。

Description

一类吡唑酮化合物及其用途 技术领域
本发明属于药物化学领域，涉及一类吡唑酮化合物及其制备方法，以及包含该类化合物的药物组合物。
背景技术
糖尿病是机体胰岛功能减退、胰岛素抵抗等因素导致的一系列代谢紊乱综合症，遗传因素、免疫功能紊乱、精神因素等多种致病因子均会引发糖尿病。据世界卫生组织统计，到2011年全球大约有3.46亿人患有糖尿病。2004年大约有340万人死于高血糖，而且超过80％的糖尿病死亡发生在低收入和中等收入国家。
现在已经有许多用于治疗Ⅱ型糖尿病的药物，其中包括二甲双胍、磺酰脲类、DPP-4抑制剂、PPARγ激动剂、α葡萄糖苷酶抑制剂、胰岛素和GLP-1类似物等。但是现有药物存在疗效不显著，作用时间短的问题，并且有的药物还存在如低血糖、体重增加、水肿、骨折、乳酸中毒和肠胃不适等副作用。
单磷酸腺苷激活蛋白激酶(AMPK)在体内糖脂代谢中发挥主导作用，是反映细胞内能量状态变化的能量计和代谢主开关，其激活能明显改善2型糖尿病糖脂代谢紊乱，增强体内胰岛素敏感性，已被确证成为治疗2型糖尿病的新靶点。研究证明目前已经发现的许多治疗Ⅱ型糖尿病的药物或者天然产物在体内能够间接激活AMPK，其治疗效果可能部分来源于AMPK的激活，但这些间接激活剂存在肠胃不适、体重增加等副作用。因此寻找和发现AMPK小分子激活剂，特别是直接作用于AMPK的小分子激活剂，将是寻求治疗II型糖尿病的一个有效途径。
综上所述，本领域迫切需要开发副作用少的新型AMPK激活剂。
发明内容
本发明的目的是提供一类副作用少的新型AMPK激活剂。
本发明的第一方面，提供了一种式I化合物，或其药学上可接受的盐：
Figure PCTCN2015084006-appb-000001
其中，R1为选自下组的基团：H、C1～C4烷基、基团上的氢原子任选地被选自下组的一个或多个取代基取代的6～12元芳基或5～10元杂芳基：卤素、C1～C4烷基、C1～C4卤代烷基、金刚烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、C1～C4卤代烷氧基、氰基、取代或未取代的苯基、-SO2-NH2；其中，所述的取代指基团上的一个或多个氢原子被选自下组的取代基任意取代：卤素、三氟甲基、羟基、氨基、C1～C4烷基、C1～C4烷氧基、金刚烷基、氰基；
R2为选自下组的基团：H、C1～C4烷基，或R2不存在；
R3为选自下组的基团：H、C1～C4烷基，或R3不存在；
R4为选自下组的基团：H、C1～C4烷基、C1～C4卤代烷基、卤素、羟基、氨基、 硝基、乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基；
R5为选自下组的基团：H、C1～C4烷基、C3～C7环烷基、C1～C4亚烷基-C3～C7环烷基、苯基、C1～C4亚烷基苯基，其中所述的苯基或C1～C4亚烷基苯基上的氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基，C1-C4卤代烷基，C3-C7环烷基，C1-C4烷氧基，C1-C4卤代烷氧基；其中当R5为含苯基的基团时，所述苯基相邻的两个碳上的氢原子可任选地被“-O-(CH2)n-O-”基团取代，其中n＝1、2或3；
n1为0、1、或2；
n2为0、1、2、或3；
n3为0、1、或2；
X选自下组：O、NR8，其中R8选自下组：H、C1～C4烷基、C1～C4卤代烷基、C3～C7环烷基、苯基、C1-C4亚烷基苯基；
虚线为化学键或不存在。
在另一优选例中，所述的C1～C4卤代烷基包括C1～C4氟代基团，较佳地为三氟甲基。
在另一优选例中，R1为被1-5个选自下组的取代基所取代的苯基：卤素、三氟甲基。
在另一优选例中，所述的6～12元芳基为苯基或萘基。
在另一优选例中，所述的C1～C4亚烷基苯基包括苄基、-C(环丙基)-苯基。
在另一优选例中，R1为选自下组的基团：基团上的氢原子被选自下组的取代基任意取代的6～12元芳基或5～10元杂芳基：卤素、三氟甲基、C1～C4烷基、金刚烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基、取代或未取代苯基、-SO2-NH2；
R2为选自下组的基团：H、甲基，或R2不存在；
R3为选自下组的基团：H，或R3不存在；
R4为H或甲基；
R5为选自下组的基团：C1～C4烷基、C3～C7环烷基或苄基，其中所述的苄基上的任意氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基、C1-C4卤代烷基，C1-C4烷氧基，C1-C4卤代烷氧基，或C2-C4亚烷基；其中，苄基的苯基基团上相邻的两个碳上的氢原子可被“-O-(CH2)n-O-”的基团取代，其中n＝1、2或3；
n1、n2、n3为1；和/或
X为O或NR8，其中，所述的R8为H或甲基。
在另一优选例中，R1为选自下组的芳基或杂芳基：苯基、苯并噻唑基；其中，所述芳基或杂芳基上的一个或多个氢原子可被选自下组的一个或多个取代基取代：卤素、三氟甲基、C1～C4烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基、取代或未取代的苯基、-SO2-NH2；和/或
X为NR8，其中，所述的R8为H或甲基。
在另一优选例中，R5为选自下组的基团：苄基，其中所述的苄基上的任意氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基，C1-C4氟代烷基、C1-C4氟代烷氧基，或C2-C4亚烷基；其中，苄基的苯基基团上相邻的两个碳上的氢原子可被“-O-(CH2)n-O-”的基团取代，其中n＝1、2或3。
在另一优选例中，所述的C1-C4烷基是氘代甲基。
在另一优选例中，所述的化合物选自下组：
Figure PCTCN2015084006-appb-000002
Figure PCTCN2015084006-appb-000003
本发明的第二方面，提供了一种如下式Ia所示的化合物：
Figure PCTCN2015084006-appb-000004
其中，R3、R4、R5、X、n1、n2和n3基团的定义如上文中所述。
本发明的第三方面，提供了一种如本发明第二方面所述的式Ia化合物的制备方法，所述方法包括步骤：
Figure PCTCN2015084006-appb-000005
在惰性溶剂中，用式Ia1化合物与式Ia2化合物反应，得到式Ia化合物；
其中，各基团的定义如本发明第二方面中所述。
在另一优选例中，式中，-X-R5为-OCH3。
在另一优选例中，所述的反应在碱存在下进行，较佳地，所述的碱选自下组：K2CO3、Na2CO3、Cs2CO3、NaH，或其组合。
本发明的第四方面，提供了一种如本发明第一方面所述的式I化合物的制备方法，包括步骤：
(1)在惰性溶剂中，用如式Ia所示的化合物和如式Ib所示的任一化合物反应，得到式I'化合物；
Figure PCTCN2015084006-appb-000006
(b1)任选地，在惰性溶剂中，用如式I'所示的化合物进行脱氢反应，得到式I”化合物；
Figure PCTCN2015084006-appb-000007
(b2)任选地，在惰性溶剂中，用如式I'所示的化合物与R2I进行消除反应，得到式I”'化合物；
Figure PCTCN2015084006-appb-000008
其中，各基团的定义如本发明第一方面中所述。
在另一优选例中，所述式Ia化合物是用如本发明第三方面所述方法制备的。
在另一优选例中，在所述的步骤(2)和步骤(3)中，R3为H。
在另一优选例中，所述步骤(1)在碱存在下进行，较佳地在Et3N和/或EtONa存在下进行；更佳地，所述的EtONa为新制EtONa。
在另一优选例中，在所述步骤(2)中，所述的脱氢反应在选自下组的试剂存在下进行：钯/碳、2,3-二氯-5,6-二氰基-1,4-苯醌，较佳地在10％钯/碳存在下进行。
在另一优选例中，在所述步骤(3)中，所述的反应在碱存在下进行；较佳地，所述的碱选自下组：LDA、K2CO3、NaOH，或其组合。
在另一优选例中，当式I'、I”或式I”'化合物中X为O时，所述的方法还任选地包括步骤：
(i)当R5不为H时，在惰性溶剂中，用式Ic化合物进行水解反应，得到式Id化合物；
Figure PCTCN2015084006-appb-000009
(ii)在惰性溶剂中，用式Id化合物与R5-NH2反应，得到式Ie化合物；
Figure PCTCN2015084006-appb-000010
其中，各基团的定义如权利要求1中所述。
在另一优选例中，在式Ic和式Ie中，R5可相同或不同。
在另一优选例中，所述的步骤(i)在碱金属氢氧化物存在下进行，较佳地，所述的碱金属氢氧化物选自下组：LiOH，NaOH，或其组合。
在另一优选例中，所述的步骤(ii)在缩合剂存在下进行，较佳地，所述的缩合剂选自下组：EDCI(1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐)、吡啶/EDC/HOBT、DCC、DCC/HOBt、DCC/DMAP，或其组合。
本发明的第五方面，提供了一种药物组合物，所述的药物组合物含有式I化合物或其药学上可接受的盐；和药学上可接受的载体。
在另一优选例中，所述的药物组合物含有治疗有效量或安全有效量的式I化合物或其药学上可接受的盐。
在另一优选例中，所述的药物组合物含有0.0001-99wt％式I化合物或其药学上可接受的盐；和余量的药学上可接受的载体。
在另一优选例中，所述的药物组合物用于治疗与AMPK活性相关的疾病。
在另一优选例中，所述的药物组合物用于治疗糖脂代谢紊乱疾病，较佳地，所述的药物组合物用于治疗选自下组的疾病：糖尿病、肥胖。
本发明的第六方面，提供了一种如本发明第一方面所述的化合物的用途，用于：
(a)制备AMPK激活剂；
(b)制备AMPK和/或ACC磷酸化促进剂；
(c)在体外非治疗性地激活AMPK活性；
(d)在体外非治疗性地促进AMPK和/或ACC磷酸化；
(e)制备治疗与AMPK活性相关疾病的药物组合物。
在另一优选例中，当用于体外非治疗性地激活AMPK活性时，所述的激活是剂量依赖性激活。
在另一优选例中，当用于体外非治疗性地激活AMPK活性时，所述的激活是浓度依赖性激活。
在另一优选例中，当用于体外非治疗性地激活AMPK活性时，所述的激活是分子水平激活或细胞水平激活。
本发明的第七方面，一种AMPK活性激活剂，所述的AMPK活性激活剂包括：激活有效量的的式I化合物或其药学上可接受的盐。
在另一优选例中，所述的激活剂是分子AMPK激活剂。
在另一优选例中，所述的激活剂是细胞内AMPK激活剂。
本发明的第八方面，提供了一种激活AMPK活性的方法，对需要的对象施用安全有效量的式I化合物或其药学上可接受的盐。
本发明的第九方面，提供了一种治疗与AMPK活性相关疾病的方法，所述方法包括：对需要的对象施用治疗有效量的式I化合物，或其药学上可接受的盐。
在另一优选例中，所述的对象包括哺乳动物(如人)。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为各化合物在分子水平上多浓度变构激活AMPK(α2β1γ1)的实验结果；
图2A、B为在L6肌管细胞上检测小分子化合物对AMPK及ACC磷酸化的刺激，挑选能够显著激活AMPK的化合物检测其对葡萄糖吸收的促进作用以及对AMPK的依赖性；
图3为在HepG2细胞上ZM-1在不同浓度和不同处理时间下对AMPK和AC的磷酸化刺激；
图4为ZM-1在SD大鼠原代肝细胞上刺激AMPK及ACC磷酸化。
具体实施方式
本发明人经过长期而深入的研究，制备了一类吡唑酮化合物，所述的化合物具有直接激活AMPK活性，可应用于制备治疗肥胖及糖尿病等的药物。基于上述发现，发明人完成了本发明。
术语
在本发明中，所述的烷基包括直链或支链的烷基，所述的链烯基包括直链或支链的链烯基，所述的链炔基包括直链或支链的链炔基，所述的卤素为F、Cl、Br或I，优选为F或Br。
除非特别说明，在本发明中，术语“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：C1～C4烷基、C3～C7环烷基、C1～C4烷氧基、卤素、羟基、羧基(-COOH)、C1～C4醛基、C2～C4酰基、C2～C4酯基、氨基、苯基；所述的苯基包括未取代的苯基或具有1-3个取代基的取代苯基，所述取代基选自：卤素、C1-C4烷基、氰基、OH、硝基、C3～C7环烷基、C1～C4烷氧基、氨基。
特别地，在本文中，除非特别说明，所提到的原子包括其所有同位素的形式，例如，当提到“氢原子”时，指氢原子、氘原子、氚原子或其组合。在本发明中，某元素各种同位素原子的丰度可以是该元素在自然界中天然存在的状态，也可以是某种同位素富集的状态。
术语“C1～C4烷基”指具有1～4个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
术语“C3～C7环烷基”指具有3～7个碳原子的环烷基，例如环丙基、环丁基、环戊基、环庚基、或类似基团。
术语“5～10元芳基”指具有5～10个碳原子的芳基，包括单环或二环芳基，例如苯基、萘基，或类似基团。
术语“5～10元杂芳基”指具有5～10个碳原子或杂原子(选自N、O、S)的杂芳基，例如吡咯基、吡啶基、呋喃基，或类似基团。
术语“C1～C4烷氧基”指具有1-4个碳原子的直链或支链烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、或类似基团。
术语“C1～C4酰基”指具有“-CO-烷基”结构，优选为具有“-CO-C1～C4烷基”结构的基团，例如甲基酰基、乙基酰基、丙基酰基、异丙基酰基、丁基酰基、异丁基酰基、仲丁基酰基、叔丁基酰基、或类似基团。
术语“C1～C4酯基”指具有烷基-COO-结构，优选为具有C1～C4烷基-COO-结构的基团，例如CH3COO-、C2H5COO-、C3H8COO-、：(CH3)2CHCOO-、nC4H9COO-、tC4H9COO-，或类似基团。
术语“C2～C10的酯基”指具有2-10个碳原子的形如“-COO-C1-C9烷基”结构的基团，例如CH3COO-、C2H5COO-、C3H8COO-、(CH3)2CHCOO-、nC4H9COO-、tC4H9COO-，或类似基团。
术语“卤素”指F、Cl、Br和I。
在本文中，各化合物的简称如下表所示：
Figure PCTCN2015084006-appb-000011
式I化合物
本发明提供了一种如下通式I所示的化合物：
Figure PCTCN2015084006-appb-000012
其中，R1为选自下组的基团：H、C1～C4烷基、基团上的氢原子任选地被选自下组的一个或多个取代基取代的6～12元芳基或5～10元杂芳基：卤素、C1～C4烷基、C1～C4卤代烷基、金刚烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、C1～C4卤代烷氧基、氰基、取代或未取代的苯基、-SO2-NH2；其中，所述的取代指基团上的一个或多个氢原子被选自下组的取代基任意取代：卤素、三氟甲基、羟基、氨基、C1～C4烷基、C1～C4烷氧基、金刚烷基、氰基；
R2为选自下组的基团：H、C1～C4烷基，或R2不存在；
R3为选自下组的基团：H、C1～C4烷基，或R3不存在；
R4为选自下组的基团：H、C1～C4烷基、C1～C4卤代烷基、卤素、羟基、氨基、硝基、乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基；
R5为选自下组的基团：H、C1～C4烷基、C3～C7环烷基、苯基、C1～C4亚烷基苯基，其中所述的苯基或C1～C4亚烷基苯基上的氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基，C1-C4卤代烷基，C3-C7环烷基，C1-C4烷氧基，C1-C4卤代烷氧基；其中当R5为含苯基的基团时，所述苯基相邻的两个碳上的氢原子可任选地被“-O-(CH2)n-O-”基团取代，其中n＝1、2或3；
n1为0、1、或2；
n2为0、1、2、或3；
n3为0、1、或2；
X选自下组：O、NR8，其中R8选自下组：H、C1～C4烷基、C1～C4卤代烷基、C3～C7环烷基、苯基、C1-C4亚烷基苯基；
虚线为化学键或不存在。
在另一优选例中，所述的C1～C4卤代烷基包括C1～C4氟代基团，较佳地为三氟甲基。
在另一优选例中，R1为被1-5个选自下组的取代基所取代的苯基：卤素、三氟甲基。
在另一优选例中，所述的6～12元芳基为苯基或萘基。
在另一优选例中，所述的C1～C4亚烷基苯基包括苄基、-C(环丙基)-苯基。在另一优选例中，R1为选自下组的基团：基团上的氢原子被选自下组的取代基任意取代的6～12元芳基或5～10元杂芳基：卤素、三氟甲基、C1～C4烷基、金刚烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基、取代或未取代苯基、-SO2-NH2；
R2为选自下组的基团：H、甲基，或R2不存在；
R3为选自下组的基团：H，或R3不存在；
R4为H或甲基；
R5为选自下组的基团：C1～C4烷基、C3～C7环烷基或苄基，其中所述的苄基上的任意氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基、C1-C4卤代烷基，C1-C4烷氧基，C1-C4卤代烷氧基，或C2-C4亚烷基；其中，苄基的苯基基团上相邻的两个碳上的氢原子可被“-O-(CH2)n-O-”的基团取代，其中n＝1、2或3；
n1、n2、n3为1；
X为O或NR8，其中，所述的R8为H或甲基；
虚线为化学键或不存在。
在另一优选例中，R1为选自下组的芳基或杂芳基：苯基、苯并噻唑基；其中，所述芳基或杂芳基上的一个或多个氢原子可被选自下组的一个或多个取代基取代：卤素、三氟甲基、C1～C4烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基、取代或未取代的苯基、-SO2-NH2；和/或
X为NR8，其中，所述的R8为H或甲基。
其中，优选的活性较好的化合物中，R5为选自下组的芳基：R5为选自下组的基团：苄基，其中所述的苄基上的任意氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基，C1-C4氟代烷基、C1-C4氟代烷氧基，或C2-C4亚烷基；其中，苄基的苯基基团上相邻的两个碳上的氢原子可被“-O-(CH2)n-O-”的基团取代，其中n＝1、2或3。
在另一优选例中，所述的化合物选自下组：
Figure PCTCN2015084006-appb-000013
Figure PCTCN2015084006-appb-000014
Figure PCTCN2015084006-appb-000015
式I化合物的制备
本发明还提供了一种式I化合物的制备方法，其特征在于，包括步骤：
(1)在惰性溶剂中，用如式Ia所示的化合物和如式Ib所示的任一化合物反应，得到式I'化合物；
Figure PCTCN2015084006-appb-000016
(b1)任选地，在惰性溶剂中，用如式I'所示的化合物进行脱氢反应，得到式I”化合物；
Figure PCTCN2015084006-appb-000017
(b2)任选地，在惰性溶剂中，用如式I'所示的化合物与R2I进行消除反应，得到式I”'化合物；
Figure PCTCN2015084006-appb-000018
其中，各基团的定义如上文中所述。
在另一优选例中，在所述的步骤(2)和步骤(3)中，R3为H。
在另一优选例中，所述步骤(1)在碱存在下进行，较佳地在Et3N和/或EtONa存在下进行；更佳地，所述的EtONa为新制EtONa。
在另一优选例中，在所述步骤(2)中，所述的脱氢反应在选自下组的试剂存在下进行：钯/碳、2,3-二氯-5,6-二氰基-1,4-苯醌，较佳地在10％钯/碳存在下进行。
在另一优选例中，在所述步骤(3)中，所述的反应在碱存在下进行；较佳地，所述的碱选自下组：LDA、K2CO3、NaOH，或其组合。
当式I'、I”或式I”'化合物中X为O时，所述的方法还任选地包括步骤：
Figure PCTCN2015084006-appb-000019
(i)在惰性溶剂中，用式Ic化合物进行水解反应，得到式Id化合物；
Figure PCTCN2015084006-appb-000020
(ii)在惰性溶剂中，用式Id化合物与R5-NH2反应，得到式Ie化合物；
其中，各基团的定义如上文中所述；在式Ic和式Ie中，R5可相同或不同。
在另一优选例中，所述的步骤(i)在碱金属氢氧化物存在下进行，较佳地，所述的碱金属氢氧化物选自下组：LiOH，NaOH，或其组合。
在另一优选例中，所述的步骤(ii)在缩合剂存在下进行，较佳地，所述的缩合剂选自下组：EDCI(1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐)、吡啶/EDC/HOBT、DCC、DCC/HOBt、DCC/DMAP，或其组合。
各反应原料可以通过本领域已知的方法制备，或者通过市售途径获得。在一个优选例中，所述式Ia化合物是用以下方法制备的：
Figure PCTCN2015084006-appb-000021
在惰性溶剂中，用式Ia1化合物与式Ia2化合物反应，得到式Ia化合物。
在另一优选例中，所述的反应在碱存在下进行，较佳地，所述的碱选自下组：K2CO3、Na2CO3、Cs2CO3、NaH，或其组合。
一类优选的式I化合物的制备方法包括以下步骤：
Figure PCTCN2015084006-appb-000022
具体而言，取代的3-乙氧基羰基-4-哌啶酮盐酸盐A1与取代的苄溴(A2)在碱碳酸钾条件下缩合，得到中间体A3。取代的芳基肼或者其盐酸盐(A4)与哌啶酮中间体A3反应 生成吡唑酮化合物A5。如R3为H时，化合物A5用10％Pd/C脱氢，得到双键化合物A6。如R3为H时，化合物A5加碱LDA和相应的烷基碘，得到化合物A7。化合物A6、A7经水解转化为相应的酸，该酸在缩合剂如EDCI(1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐)存在下与相应的胺作用得到如通式Ⅰ所示化合物。
上述各反应式中，各基团的定义如上文中所述。
式I化合物的用途
本发明还提供了所述的式I化合物的用途。
在本发明的一个优选例中，所述式I化合物用于制备AMPK激活剂；所述的AMPK活性激活剂包括：激活有效量的的式I化合物或其药学上可接受的盐。所述的激活剂可以是分子AMPK激活剂或细胞内AMPK激活剂。
在本发明的一个优选例中，所述式I化合物用于制备AMPK和/或ACC磷酸化促进剂。
在本发明的另一个优选例中，所述式I化合物用于在体外非治疗性地激活AMPK活性。当用于体外非治疗性地激活AMPK活性时，所述的激活是剂量依赖性激活或浓度依赖性激活。在另一优选例中，当用于体外非治疗性地激活AMPK活性时，所述的激活是分子水平激活或细胞水平激活。
在本发明的另一个优选例中，所述式I化合物用于在体外非治疗性地促进AMPK和/或ACC磷酸化。
在本发明的另一个优选例中，所述式I化合物用于制备治疗与AMPK活性相关疾病的药物组合物。在另一优选例中，所述的药物组合物用于治疗糖脂代谢紊乱疾病，较佳地，所述的药物组合物用于治疗选自下组的疾病：糖尿病、肥胖。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
化合物制备实施例
在下述制备实施例中，NMR用Varian生产的Mercury-Vx 300M仪器测定，NMR定标：δH 7.26ppm(CDCl3)；质谱用Agilent 1200 Quadrupole LC/MS液质联用仪；试剂主要由上海化学试剂公司提供；TLC薄层层析硅胶板由山东烟台会友硅胶开发有限公司生产，型号HSGF 254；化合物纯化使用的正相柱层析硅胶为山东青岛海洋化工厂分厂生产，型号zcx-11，200-300目。
制备实施例1 甲基4-((2-(4-氟-3-(三氟甲基)苯基)-3-氧-3a,4,6,7-四氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酸
(1)乙基1-(4-(甲氧基羰基)苄基)-4-哌啶酮-3-羧酸
Figure PCTCN2015084006-appb-000023
将97mg 3-乙氧甲酰-4-哌啶酮盐酸盐(0.467mmol)和96.3mg 4-溴甲基苯甲酸甲酯(0.420mmol)加入反应瓶，乙腈溶解，加入64.4mg K2CO3(0.467mmol)，室温反应过夜，次日TLC监测反应完全。减压旋除乙腈，水溶后，乙酸乙酯萃取3次，有机相用饱和食盐水洗，无水Na2SO4干燥，减压旋除溶剂，柱层析分离，PE:EA＝5:1，得化合物B10，白色固体119mg，收率89％。
1H NMR(300MHz,CDCl3)δ8.01(dd,J＝8.4,1.8Hz,2H),7.43(d,J＝8.4Hz,2H),4.20(q,J＝7.2Hz,2H),3.91(s,3H),3.68(s,2H),3.19(s,1H),2.60(t,J＝6.0Hz,2H),2.40(t,J＝6.0Hz,2H),1.26ppm(td,J＝7.2,1.8Hz,3H).
(2)甲基4-((2-(4-氟-3-(三氟甲基)苯基)-3-氧-3a,4,6,7-四氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酸
Figure PCTCN2015084006-appb-000024
将化合物B10(10.9g,34mmol)和3-三氟甲基-4-氟苯肼盐酸盐(8.21g,35.7mmol)溶于180mL甲醇，加入三乙胺(5mL,35.7mmol)，加热至60℃，反应1h，TLC监测原料B10反应完全。停止加热，冷却至室温，加入新鲜制备的甲醇钠的甲醇溶液(68mmol，60mL)，室温搅拌过夜。次日，反应液缓慢倒入700mL5％的柠檬酸溶液，析出固体，过滤，固体加水洗3次，收集固体加CaCl2真空干燥，得化合物ZM-3，褐色固体13.5g。(含有色杂质，用甲醇/水体系重结晶后得白色固体，收率85％)。
1H NMR(300MHz,CDCl3)δ8.20–8.08(m,2H),8.03(d,J＝8.1Hz,2H),7.42(d,J＝8.1Hz,2H),7.24–7.15(m,1H),3.93(s,3H),3.74(s,2H),3.62–3.49(m,2H),3.31–3.17(m,1H),2.81–2.53(m,2H),2.35–2.16ppm(m,2H).
制备实施例2 甲基4-((2-(4-氟-3-(三氟甲基)苯基)-3-氧-6,7-二氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酸
Figure PCTCN2015084006-appb-000025
将化合物ZM-3(13g,28.9mmol)悬浮于500mL甲醇和150mL乙酸乙酯中，加入10％Pd/C(7g)，室温搅拌过夜。次日，TLC监测原料反应完全，垫硅藻土过滤，固体多次加二氯甲烷/甲醇＝1/1溶液洗涤，滤液合并浓缩至约100mL，析出固体，过滤得一黄色固体，加少量的DCM洗涤，得黄色固体ZM-2，6.5g,收率50％.
1H NMR(300MHz,CDCl3)δ8.24–8.20(m,2H),8.11(d,J＝7.8Hz,2H),7.98(s,1H),7.42(d,J＝7.8Hz,2H),7.20–7.15(m,1H),4.65(s,2H),3.94(s,3H),3.55-3.50(t,2H),2.92–2.97ppm(t,2H).
制备实施例3 N-(3,4-二甲氧基苄基)-4-((2-(4-氟-3-(三氟甲基)苯基)-3-氧-6,7-二氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酰胺
(1)4-((2-(4-氟-3-(三氟甲基)苯基)-3-氧-6,7-二氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酸
Figure PCTCN2015084006-appb-000026
化合物ZM-2(7.1g,15.8mmol)悬浮于250mL甲醇中，加入LiOH(1.33g,31.6mmol)的水溶液120mL，室温搅拌过夜。次日，溶液澄清，TLC监测原料反应完全，减压除去大部分的甲醇溶，加1N HCl溶液调至PH＝1-2之间，析出固体，减压过滤收集固体，固体加水洗涤3次，真空干燥后得白色固体6.4g,收率90％.
(2)N-(3,4-二甲氧基苄基)-4-((2-(4-氟-3-(三氟甲基)苯基)-3-氧-6,7-二氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酰胺
Figure PCTCN2015084006-appb-000027
化合物433(6.1g,14mmol)和3,4-二甲氧基苄胺(2.8g,16.8mmol)溶解于200mLDMF，依次加入HOBT(100mg)，三乙胺(5ml,35.6mmol)，室温搅拌10分钟后，加入EDCI(6.6g,35.6mmol)，室温搅拌过夜。次日，TLC监测原料反应完全。反应液倒入600mL冰水中，析出固体，收集固体，固体依次用5％柠檬酸溶液和水洗涤，干燥后得7.5g。进一步纯化，固体加二氯甲烷/石油醚＝1/5的溶液100mL搅拌30分钟，收集固体，如此重复3次，得白色固体6.9g，收率84％，HPLC>99.74％。
ZM-1
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.98(s,1H),7.84(d,J＝8.1Hz,2H),7.35(d,J＝8.1Hz,2H),7.21–7.14(m,1H),6.92-6.83(m,3H),6.33(t,1H),4.65(s,2H),4.60(d,J＝5.7Hz,2H),3.88(s,6H),3.55-3.50(t,2H),2.92–2.87ppm(t,2H)
同样的方法，得到化合物：
ZM-6
1H NMR(300MHz,CDCl3)δ8.26–8.24(m,2H),7.95(s,1H),7.54(d,J＝8.1Hz,2H),7.33(d,J＝8.1Hz,2H),7.21–7.10(m,1H),6.93-6.86(m,3H),6.75(t,1H),4.68(s,2H),4.63(d,J＝5.7Hz,2H),3.88(s,6H),3.56-3.510(t,2H),2.92–2.87(t,2H),2.90ppm(s,3H).
ZM-7
1H NMR(300MHz,CDCl3)δ8.14–8.12(m,1H),8.01-7.90(m,4H),7.62(s,1H),7.42-7.40(m,1H),7.33-7.30(m,1H),7.05-7.02(m,1H),6.75-6.71(m,2H),6.64-6.61(m,1H),4.52(s,2H),4.44(d,J＝5.7Hz,2H),3.73(s,3H),3.71(s,3H),3.43-3.38(t,2H),2.63–2.58ppm(t,2H)
ZM-8
1H NMR(300MHz,CDCl3)δ8.25–8.21(m,2H),7.92(s,1H),7.84(d,J＝8.1Hz,2H),7.35(d,J＝8.1Hz,2H),7.33-7.30(m,5H),7.21–7.14(m,1H),6.62(t,1H),4.65(s,2H),4.60(d,J＝5.7Hz,2H),3.53-3.48(t,2H),2.89–2.84ppm(t,2H)
ZM-9
1H NMR(300MHz,CDCl3)δ8.27–8.21(m,2H),7.92(s,1H),7.85(d,J＝8.1Hz, 2H),7.35(d,J＝8.1Hz,2H),7.30-7.28(m,1H),7.20-7.15(t,1H),6.95-6.87(m,3H),6.36(t,1H),4.65(s,2H),4.63(d,J＝5.7Hz,2H),3.81(s,3H),3.54-3.50(t,2H),2.92–2.87ppm(t,2H)
ZM-10
1H NMR(300MHz,CDCl3)δ8.24–8.21(m,2H),7.95(s,1H),7.82(d,J＝8.1Hz,2H),7.33(d,J＝8.1Hz,2H),7.18–7.15(m,1H),6.13(s,1H),4.65(s,2H),3.56-3.50(m,4H),3.02(s,3H),2.92–2.86(t,2H),1.27-1.24ppm(t,3H)
ZM-11
1H NMR(300MHz,CDCl3)δ8.22–8.21(m,2H),7.97(s,1H),7.83(d,J＝8.1Hz,2H),7.34(d,J＝8.1Hz,2H),7.21–7.14(m,1H),6.46(s,2H),6.31(t,1H),4.64(s,2H),4.61(d,J＝5.7Hz,2H),3.90(s,9H),3.54-3.50(t,2H),2.92–2.86ppm(t,2H)
ZM-12
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.92(s,1H),7.84(d,J＝8.1Hz,2H),7.35(d,J＝8.1Hz,2H),7.21–7.14(m,1H),6.85-6.78(m,3H),6.38(t,1H),5.95(s,2H),4.65(s,2H),4.54(d,J＝5.7Hz,2H),3.54-3.50(t,2H),2.91–2.87ppm(t,2H)
ZM-13
1H NMR(300MHz,CDCl3)δ8.24–8.21(m,2H),7.96(s,1H),7.83(d,J＝8.1Hz,2H),7.37(d,J＝8.1Hz,2H),7.21–7.14(m,1H),6.88-6.83(m,3H),6.45(t,1H),5.96(s,2H),4.62(s,2H),4.60(d,J＝5.7Hz,2H),3.55-3.50(t,2H),2.92–2.87ppm(t,2H)
ZM-14
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.92(s,1H),7.82(d,J＝8.1Hz,2H),7.38-7.36(m,5H),7.35(d,J＝8.1Hz,2H),7.21–7.14(m,1H),6.38(d,1H),5.36-5.32(m,1H),4.64(s,2H),3.53-3.48(t,2H),2.90–2.85(t,2H),1.52ppm(d,3H)
ZM-15
1H NMR(300MHz,CDCl3)δ8.24–8.22(m,2H),7.92(s,1H),7.82(d,J＝8.1Hz,2H),7.33(d,J＝8.1Hz,2H),7.21–7.15(m,1H),6.16(t,1H),4.65(s,2H),3.55-3.51(t,2H),3.34-3.29(t,2H),2.91–2.87(t,2H),1.86-1.58(m,5H),1.28-1.21(m,4H),1.06-1.0.98ppm(m,2H)
ZM-16
1H NMR(300MHz,CDCl3)δ8.26–8.25(m,2H),7.92(s,1H),7.83(d,J＝8.1Hz,2H),7.45–7.43(m,1H),7.35(d,J＝8.1Hz,2H),7.33-7.30(m,1H),6.89-6.85(m,3H),6.43(t,1H),4.63(s,2H),4.58(d,J＝5.7Hz,2H),3.88(s,6H),3.53-3.48(t,2H),2.92–2.87ppm(t,2H)
ZM-17
1H NMR(300MHz,CDCl3)δ8.04–8.00(m,3H),7.81-7.78(m,4H),7.28(d,J＝8.1Hz,2H),6.87-6.80(m,3H),4.64(s,2H),4.51(t,1H),3.81(s,6H),3.47-3.44(t,2H),2.83–2.78ppm(t,2H)
ZM-18
1H NMR(300MHz,CDCl3)δ8.06(s,1H),7.91-7.88(d,J＝8.4Hz,1H),7.85-7.82(d,J＝8.1Hz,2H),7.77-7.74(d,J＝8.4Hz,1H),7.42-7.39(t,1H),7.28(d,J＝8.1Hz,2H),6.89-6.80(m,3H),6.30(t,1H),4.65(s,2H),4.56(d,2H),3.85(s,6H),3.55-3.50(t,2H),2.96–2.91ppm(t,2H)
ZM-19
1H NMR(300MHz,CDCl3)δ8.24–8.22(m,2H),7.95(s,1H),7.81-7.77(m,2H),7.38–7.35(m,1H),7.25-7.20(m,1H),6.89-6.85(m,3H),6.43(t,1H),4.63(s,2H),4.58(d,J＝5.7Hz,2H),3.89(s,6H),3.59-3.53(t,2H),2.93–2.87(t,2H),2.37ppm(s,3H)
ZM-20
1H NMR(300MHz,CDCl3)δ8.24–8.21(m,2H),7.94(s,1H),7.83(d,J＝8.1Hz,2H),7.32(d,J＝8.1Hz,2H),7.25-7.20(m,1H),6.91(d,J＝7.2Hz,1H),6.65(d,J＝11.1Hz,1H),6.66(t,1H),4.64(s,2H),4.61(d,J＝5.7Hz,2H),3.85(s,6H),3.54-3.50(t,2H),2.90–2.87(t,2H)
ZM-21
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.93(s,1H),7.81(d,J＝8.1Hz,2H),7.36(d,J＝8.1Hz,2H),7.21–7.14(m,1H),7.02-6.98(m,2H),6.84(d,1h),6.39(s,1H),4.64(s,2H),3.87(s,3H),3.86(s,3H),3.53-3.48(t,2H),2.90–2.85(t,2H),1.84ppm(s,6H)
ZM-22
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.92(s,1H),7.82(d,J＝8.1Hz,2H),7.33(d,J＝8.1Hz,2H),7.21–7.14(m,1H),6.90-6.81(m,3H),6.39(t,1H),4.64(s,2H),4.56(d,J＝5.7Hz,2H),4.12(t,2H),3.86(s,3H),3.54-3.50(t,2H),2.90–2.85(t,2H),1.43ppm(t,3H)
ZM-23
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.91(s,1H),7.85(d,J＝8.1Hz,2H),7.34(d,J＝8.1Hz,2H),7.21–7.14(m,1H),6.90-6.81(m,3H),6.35(t,1H),4.64(s,2H),4.58(d,J＝5.7Hz,2H),4.12(t,2H),3.86(s,3H),3.54-3.50(t,2H),2.91–2.85(t,2H),1.44ppm(t,3H)
ZM-27
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),8.01(s,1H),7.87(d,J＝8.1Hz,2H),7.36(d,J＝8.1Hz,2H),7.20–7.16(m,2H),6.92-6.90(m,1H),6.78-6.25(t,1H),6.58(t,1H),4.65(s,2H)，4.61(d,J＝5.4Hz,2H),3.86(s,3H),3.55-3.50(t,2H),2.91–2.88ppm(t,2H)
ZM-28
1H NMR(300MHz,CDCl3)δ8.24–8.21(m,2H),7.92(s,1H),7.82(d,J＝8.4Hz,2H),7.32(d,J＝8.4Hz,2H),7.17-7.13(m,1H),6.86-6.66(m,3H),6.35(t,1H),4.63(s,2H),3.86(s,3H),3.84(s,3H),3.52-3.47(t,2H),2.89–2.85(t,2H),1.32ppm(s,4H)
ZM-29
1H NMR(300MHz,CDCl3)δ8.23–8.20(m,2H),7.92(s,1H),7.84(d,J＝8.4Hz,2H),7.35(d,J＝8.4Hz,2H),7.20-7.15(m,1H),6.86-6.83(m,3H),6.38(t,1H),4.65(s,2H),4.57(d,J＝6Hz,2H),4.51(m,1H),3.85(s,3H),3.55-3.50(t,2H),2.91–2.86(t,2H),1.36ppm(d,J＝6.3Hz,6H)
ZM-30
1H NMR(300MHz,CDCl3)δ8.19–8.14(m,2H),7.77(d,J＝8.4Hz,2H),7.40(d,J＝8.4Hz,2H),7.30-7.28(m,1H),7.17–7.14(m,1H),6.96-6.91(m,2H),6.86-6.82(m,1H),6.38(t,1H),4.65(d,J＝6Hz,2H),3.81(s,3H),3.60(q,2H),3.19(m,1H),3.00(dd,J 1＝10.5,J2＝1.5Hz,1H),2.72-2.70(m,1H),2.68-2.63(m,1H),2.22-2.19(m,1H),2.07-2.04(m,1H),1.59ppm(s,3H)
ZM-31
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.89(s,1H),7.85(d,J＝8.1Hz,2H),7.35(d,J＝8.1Hz,2H),7.20–7.16(m,2H),6.77-6.25(t,1H),6.75-6.23(t,1H),6.59(t,1H),4.65(s,2H)，4.64(d,J＝5.4Hz,2H),3.55-3.50(t,2H),2.92–2.88ppm(t,2H)
ZM-32
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.90(s,1H),7.85(d,J＝8.1Hz,2H),7.34(d,J＝8.1Hz,2H),7.18–7.13(m,2H),6.95-6.93(m,1H),6.82-6.30(t,1H),6.55(t,1H),4.65(s,2H)，4.58(d,J＝5.4Hz,2H),3.87(s,3H),3.55-3.50(t,2H),2.91–2.88ppm(t,2H)
制备实施例4N-(3,4-二甲氧基苄基)-4-((2-(4-氟-3-(三氟甲基)苯基)-1-甲基-3-氧-2,3,6,7-四氢-1H-吡唑[4,3-c]吡啶-5(4H)-基)甲基)苯甲酰胺
(1)甲基4-((2-(2-(4-氟-3-(三氟甲基)苯基)-11-甲基-3-氧-2,3,6,7-四氢-1H-吡唑[4,3-c]吡啶-5(4H)-基)甲基)苯甲酸
Figure PCTCN2015084006-appb-000028
氩气保护下，化合物ZM-3(40mg,0.089mmol)溶于5mL无水THF，冷却至0℃，缓慢滴加LDA(2M，0.1mL)，此温度搅拌10分钟后，加入MeI(40L)，0℃搅拌0.5小时，升至室温搅拌2小时，TLC监测原料反应完全。加饱和氯化铵猝灭反应，乙酸乙酯萃取(3次)，有机相加水，饱和食盐水洗涤后，无水硫酸钠干燥，浓缩，过柱，DCM/MeOH＝50/1，得25mg，收率60％。
1H NMR(300MHz,CDCl3)δ8.02(d,J＝8.1Hz,2H),7.60-7.58(m,2H),7.45(d,J＝8.1Hz,2H),7.27–7.24(m,1H),3.93(s,3H),3.78(s,2H),3.33(s,2H),2.97(s,3H),2.81-2.77(t,2H),2.63–2.59ppm(t,2H)
(2)4-((2-(2-(4-氟-3-(三氟甲基)苯基)-11-甲基-3-氧-2,3,6,7-四氢-1H-吡唑[4,3-c]吡啶-5(4H)-基)甲基)苯甲酸
Figure PCTCN2015084006-appb-000029
化合物ZM463m(25mg,0.054mmol)溶于5mL甲醇中，加入LiOH(5mg,0.11mmol)的水溶液1mL，室温搅拌过夜。次日，TLC监测原料反应完全，减压除去大部分的甲醇溶，加1N HCl溶液调至PH＝1-2之间，乙酸乙酯萃取(3次)，有机相加水，饱和食盐水洗涤后，无水硫酸钠干燥，浓缩，得产物449，20mg，收率90％。
(3)N-(3,4-二甲氧基苄基)-4-((2-(4-氟-3-(三氟甲基)苯基)-1-甲基-3-氧-2,3,6,7-四氢-1H-吡唑[4,3-c]吡啶-5(4H)-基)甲基)苯甲酰胺
化合物449(20mg,0.045mmol)和3,4-二甲氧基苄胺(15mg，0.089mmol)溶解于5mLDMF，依次加入HOBT(2mg)，三乙胺(20ml，0.14mmol)，室温搅拌10分钟后，加入EDCI(25mg,0.13mmol)，室温搅拌过夜。次日，TLC监测原料反应完全。反应液倒入10mL冰水中，乙酸乙酯萃取(3次)，有机相加水，饱和食盐水洗涤后，无水硫酸钠干燥，浓缩，过柱，DCM/MeOH＝30/1，得20mg,收率76.7％。
ZM-5
1H NMR(300MHz,CDCl3)δ7.75(d,J＝8.1Hz,2H),7.59-7.57(m,2H),7.42(d,J＝8.1Hz,2H),7.30–7.26(m,1H),6.89-6.85(m,3H),6.70(t,1H),4.55(d,J＝5.4Hz,2H),3.84(s,6H),3.72(s,2H),3.25(s,2H),2.96(s,3H),2.81-2.77(t,2H),2.61–2.59ppm(t,2H)
同样方法得到：
ZM-24
1H NMR(300MHz,CDCl3)δ7.73(d,J＝8.1Hz,2H),7.65-7.57(m,2H),7.44(d,J＝8.1Hz,2H),7.30–7.26(m,1H),6.89-6.85(m,3H),6.70(t,1H),4.58(d,J＝5.4Hz,2H),4.12(t,2H),3.86(s,3H),3.76(s,2H),3.30(s,2H),2.97(s,3H),2.81-2.77(t,2H),2.61–2.59(t,2H),1.43ppm(t,3H)
ZM-25
1H NMR(300MHz,CDCl3)δ7.78(d,J＝8.1Hz,2H),7.64-7.57(m,2H),7.42(d,J＝8.1Hz,2H),7.30–7.24(m,1H),6.85-6.78(m,3H),6.35(t,1H),4.58(d,J＝5.4Hz,2H),4.52(m,1H),3.86(s,3H),3.75(s,2H),3.30(s,2H),2.97(s,3H),2.81-2.77(t,2H),2.61–2.59(t,2H),1.36ppm(d,J＝6.3Hz,6H)
ZM-26
1H NMR(300MHz,CDCl3)δ7.76(d,J＝8.1Hz,2H),7.65-7.57(m,2H),7.43(d,J＝8.1Hz,2H),7.30–7.27(m,1H),6.89-6.83(m,3H),6.33(t,1H),4.58(d,J＝5.4Hz,2H),4.12(t,2H),3.86(s,3H),3.76(s,2H),3.30(s,2H),2.97(s,3H),2.81-2.77(t,2H),2.61–2.59(t,2H),1.43ppm(t,3H)
制备实施例5N-(3,4-二甲氧基苄基)-4-((2-(4-氟-3-(三氟甲基)苯基)-3a-甲基-3-氧-3a,4,6,7-四氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酰胺
(1)甲基4-((2-(4-氟-3-(三氟甲基)苯基-3a-甲基-3-氧-3a,4,6,7-四氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酸
Figure PCTCN2015084006-appb-000031
将化合物333(340mg,1.02mmol)和3-三氟甲基-4-氟苯肼盐酸盐(276g,1.02mmol)溶于10mL甲醇，加入三乙胺(0.14mL,1.02mmol)，加热至60℃，反应4h，TLC监测原料反应完全。停止加热，冷却至室温，加入新鲜制备的甲醇钠的甲醇溶液(2mmol,60mL)，室温搅拌过夜。次日，反应液缓慢倒入20mL5％的柠檬酸溶液，乙酸乙酯萃取(3次)，有机相加水，饱和食盐水洗涤后，无水硫酸钠干燥，浓缩，过柱，石油醚/乙酸乙酯＝4/1，得463-2，160mg,收率35％。
1H NMR(300MHz,CDCl3)δ8.19–8.15(m,2H),8.02(d,J＝7.8Hz,2H),7.42(d,J＝7.8Hz,2H),7.20–7.18(m,1H),3.93(s,3H),3.74-3.58(q,2H),3.24-3.19(m,1H),3.00(dd,J 1＝10.5,J2＝1.5Hz,1H),2.75-2.69(m,1H),2.68-2.61(m,1H),2.24-2.19(m,1H),2.07-2.04(m,1H),1.59ppm(s,3H)。
(2)4-((2-(4-氟-3-(三氟甲基)苯基-3a-甲基-3-氧-3a,4,6,7-四氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酸
Figure PCTCN2015084006-appb-000032
化合物463-2(40mg，0.086mmol)溶于5mL甲醇中，加入LiOH(10mg，0.2mmol)的水溶液1mL，室温搅拌过夜。次日，TLC监测原料反应完全，减压除去大部分的甲醇溶，加1N HCl溶液调至PH＝1-2之间，乙酸乙酯萃取(3次)，有机相加水，饱和食盐水洗涤后，无水硫酸钠干燥，浓缩，得产物449-2，38mg，收率85％。
(3)N-(3,4-二甲氧基苄基)-4-((2-(4-氟-3-(三氟甲基)苯基)-3a-甲基-3-氧-3a,4,6,7-四氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酰胺
Figure PCTCN2015084006-appb-000033
化合物449-2(20mg,0.045mmol)和3,4-二甲氧基苄胺(15mg,0.089mmol)溶解于5mLDMF，依次加入HOBT(2mg)，三乙胺(20ml,0.14mmol)，室温搅拌10分钟后，加入EDCI(25mg,0.13mmol),室温搅拌过夜。次日，TLC监测原料反应完全。反应液倒入10mL冰 水中，乙酸乙酯萃取(3次)，有机相加水，饱和食盐水洗涤后，无水硫酸钠干燥，浓缩，过柱，DCM/MeOH＝30/1，得ZM-6，10mg,收率38％。
ZM-33
1H NMR(300MHz,CDCl3)δ8.19–8.14(m,2H),7.78(d,J＝8.4Hz,2H),7.40(d,J＝8.4Hz,2H),7.17–7.14(m,1H),6.92-6.83(m,3H),6.38(t,1H),4.63(d,J＝6Hz,2H),3.81(s,6H),3.68(q,2H),3.18(m,1H),3.00(dd,J 1＝10.5,J2＝1.5Hz,1H),2.72-2.70(m,1H),2.68-2.63(m,1H),2.22-2.19(m,1H),2.07-2.04(m,1H),1.59ppm(s,3H)。
制备实施例6N-(3-甲氧基-4-氘代甲氧基苄基)-4-((2-(4-氟-3-(三氟甲基)苯基)-3-氧-6,7-二氢-2H-吡唑[4,3-c]吡啶-5(3H)-基)甲基)苯甲酰胺
Figure PCTCN2015084006-appb-000034
化合物ZM-30(50mg,0.088mmol)和对甲苯磺酸氘代甲基酯(50mg,0.25mmol)溶解于3mLDMF，加入碳酸铯(300mg)，四丁基碘化铵(10mg)，40°搅拌2小时。TLC监测原料反应完全。反应液倒入10mL冰水中，乙酸乙酯萃取(3次)，有机相加水，饱和食盐水洗涤后，无水硫酸钠干燥，浓缩，过柱，DCM/MeOH＝30/1，得ZM-34，30mg,收率58％
ZM-34
1H NMR(300MHz,CDCl3)δ8.23–8.21(m,2H),7.95(s,1H),7.83(d,J＝8.1Hz,2H),7.31(d,J＝8.1Hz,2H),7.18–7.14(m,1H),6.90-6.83(m,3H),6.56(t,1H),4.64(s,2H),4.56(d,J＝5.7Hz,2H),3.86(s,3H),3.52-3.48(t,2H),2.84–2.89ppm(t,2H)
实验实施例1
1、试验目的：筛选可以变构激活AMPK(α2β1γ1)的小分子化合物。
2、试验原理：底物肽段上含有可被AMPK磷酸化的位点，在反应体系(含2mMMgCl2、400μM DTT、160nM底物肽、4μM ATP和1.6nM AMPK)中其一端接上了biotin。在biotin端，biotin能与streptavidin-XL665(acceptor)相连，完成标记。而在经过45分钟的磷酸化反应之后，该底物生成的磷酸化底物(即产物)便能与识别该位点抗磷酸化位点的抗体(已标有Donor)结合，另一端上的biotin能与streptavidin-XL665(acceptor)相连，从而使用PE公司的Evision来检测产生的FRET信号。最终AMPK活性表示为665nm波长读值和615nm波长读值的比值(665nm/615nm)。
3、检测试剂：利用大肠杆菌表达系统表达、纯化得到人源性AMPK(α2β1γ1)蛋白；购买自Cisbio公司的HTRF KinEASE-STK S1kit；购买自Sigma公司的ATP。
4、实验方法：
1)、200nM AMPK(α2β1γ1)蛋白与200nM CaMKKβ一同在30度水浴4小时，使AMPK完全磷酸化。
2)、使用购买自PE公司的白色浅底板，建立酶反应体系(2mM MgCl2、400μM DTT、160nM底物肽、4μM ATP和1.6nM AMPK)，在30度反应45分钟，加入含识别磷酸化肽段的抗体和XL665的Detection buffer，室温孵育3小时后检测信号。
5、实验结果：如图1及表1所示，小分子化合物对AMPK的激活体现为EC50和激活倍数(Fold)。
表1、针对AMPK(α2β1γ1)小分子变构激活剂的发现
化合物编号 EC50(μM) 激活倍数(Fold)
ZM-1 2.1 3.3
ZM-2 5.8 2.0
ZM-3 9.8 2.8
ZM-4 8.4 3.2
ZM-5 3.3 3.6
ZM-6 1.1 3.0
ZM-7 4.6 2.7
ZM-8 8.6 3.0
ZM-9 5.3 2.8
ZM-10 10.0 2.7
ZM-11 7.5 3.5
ZM-12 5.0 3.0
ZM-13 3.3 2.5
ZM-14 9.7 2.5
ZM-15 5.8 2.2
ZM-16 5.6 3.2
ZM-17 9.5 2.1
ZM-18 6.3 2.8
ZM-19 5.6 2.6
ZM-20 4.4 2.8
ZM-21 4.0 2.2
ZM-22 6.7 2.9
ZM-23 5.4 3.0
ZM-24 2.5 2.8
ZM-25 2.5 3.1
ZM-26 3.0 2.3
ZM-27 5.0 2.3
ZM-28 3.2 2.6
ZM-29 9.7 2.9
ZM-30 8.6 2.7
ZM-31 5.4 3.1
ZM-32 4.1 2.4
ZM-33 7.8 2.7
ZM-34 6.3 2.1
A769662 0.02 3.2
AMP 0.3 2.6
6、结果与讨论：
如表1和图1所示，化合物ZM-1等一系列化合物具有在分子水平上显著的变构激活AMPK的能力。
实验实施例2
1、检测目的：在分子水平显著激活AMPK的小分子化合物是否具有激活细胞内AMPK的作用。
2、实验原理：小分子化合物变构激活AMPK，可引起其底物乙酰辅酶A羧化酶(ACC)Ser-79位的磷酸化。
3、检测试剂：AMPK、phospho-AMPK(T172)、phospho-ACC(S79)、Total ACC抗体购自Cell Signaling Technology。
4、实验方法：
1)、细胞换为无血清培养基饥饿2小时后，加入化合物处理(0.4％DMSO)1小时后，待收样细胞用PBS洗一次后加入1×SDS凝胶加样缓冲液(50mM Tris-HCl(pH6.8)，100mM DTT，2％SDS，10％甘油，0.1％溴酚蓝)裂解细胞(24孔板每孔加70μl)。
2)、样品在100℃加热10分钟，然后12000g离心10分钟后取上清液进行SDS-PAGE电泳，条件为积层胶：70伏，分离胶：90伏。
3)、电泳结束后，用Biorad湿转电转仪系统将蛋白转移至硝酸纤维素膜。将目的条带剪下后，将条带置于封闭液(含5％BSA的TBST)中室温封阻1小时。随后将条带置于一抗溶液中4℃孵育过夜。
4)、第二天将目的条带置于TBST中，室温洗涤3×10分钟。随后将条带置于二抗溶液(羊抗兔与羊抗鼠均为1：8000，溶于TBST)中室温孵育1小时，接着TBST洗膜3×15分钟后，用ECL试剂曝光。
5、实验结果：
如图2A、B所示，在挑选出的在分子水平可以显著激活AMPK的小分子中，ZM-1在细胞水平对AMPK的激活效果最好，处理L6肌细胞1小时可剂量依赖地显著激活AMPK，并使其下游ACC磷酸水平相应上调。
如图3所示，不同浓度ZM-1处理HepG2细胞能够浓度梯度依赖及刺激AMPK、ACC磷酸化水平的上调。
如图4所示，不同浓度ZM-1处理SD大鼠原代肝细胞能够浓度梯度依赖及刺激AMPK、ACC磷酸化水平的上调。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种式I化合物，或其药学上可接受的盐：
    Figure PCTCN2015084006-appb-100001
    其中，R1为选自下组的基团：H、C1～C4烷基、基团上的氢原子任选地被选自下组的一个或多个取代基取代的6～12元芳基或5～10元杂芳基：卤素、C1～C4烷基、C1～C4卤代烷基、金刚烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、C1～C4卤代烷氧基、氰基、取代或未取代的苯基、-SO2-NH2；其中，所述的取代指基团上的一个或多个氢原子被选自下组的取代基任意取代：卤素、三氟甲基、羟基、氨基、C1～C4烷基、C1～C4烷氧基、金刚烷基、氰基；
    R2为选自下组的基团：H、C1～C4烷基，或R2不存在；
    R3为选自下组的基团：H、C1～C4烷基，或R3不存在；
    R4为选自下组的基团：H、C1～C4烷基、C1～C4卤代烷基、卤素、羟基、氨基、硝基、乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基；
    R5为选自下组的基团：H、C1～C4烷基、C3～C7环烷基、C1～C4亚烷基-C3～C7环烷基、苯基、C1～C4亚烷基苯基，其中所述的苯基或C1～C4亚烷基苯基上的氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基，C1-C4卤代烷基，C3-C7环烷基，C1-C4烷氧基，C1-C4卤代烷氧基；其中当R5为含苯基的基团时，所述苯基相邻的两个碳上的氢原子可任选地被“-O-(CH2)n-O-”基团取代，其中n＝1、2或3；
    n1为0、1、或2；
    n2为0、1、2、或3；
    n3为0、1、或2；
    X选自下组：O、NR8，其中R8选自下组：H、C1～C4烷基、C1～C4卤代烷基、C3～C7环烷基、苯基、C1-C4亚烷基苯基；
    虚线为化学键或不存在。
  2. 如权利要求1所述的化合物，其特征在于，
    R1为选自下组的基团：基团上的氢原子被选自下组的取代基任意取代的6～12元芳基或5～10元杂芳基：卤素、三氟甲基、C1～C4烷基、金刚烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基、取代或未取代苯基、-SO2-NH2；
    R2为选自下组的基团：H、甲基，或R2不存在；
    R3为选自下组的基团：H，或R3不存在；
    R4为H或甲基；
    R5为选自下组的基团：C1～C4烷基、C3～C7环烷基、C1～C4亚烷基-C3～C7环烷基或苄基，其中所述的苄基上的任意氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基、C1-C4卤代烷基，C1-C4烷氧基，C1-C4卤代烷氧基， 或C2-C4亚烷基；其中，苄基的苯基基团上相邻的两个碳上的氢原子可被“-O-(CH2)n-O-”的基团取代，其中n＝1、2或3；
    n1、n2、n3为1；和/或
    X为O或NR8，其中，所述的R8为H或甲基。
  3. 如权利要求1所述的化合物，其特征在于，
    R1为选自下组的芳基或杂芳基：苯基、苯并噻唑基；其中，所述芳基或杂芳基上的一个或多个氢原子可被选自下组的一个或多个取代基取代：卤素、三氟甲基、C1～C4烷基、羟基、氨基、硝基、取代或未取代的乙酰氧基(AcO)、羧基、C1～C4烷氧基、氰基、取代或未取代的苯基、-SO2-NH2；和/或
    X为NR8，其中，所述的R8为H或甲基。
  4. 如权利要求1所述的化合物，其特征在于，R5为选自下组的基团：苄基，其中所述的苄基上的任意氢原子可任选被一个或多个选自下组的取代基取代：羟基、卤素、C1-C4烷基，C1-C4氟代烷基、C1-C4氟代烷氧基，或C2-C4亚烷基；其中，苄基的苯基基团上相邻的两个碳上的氢原子可被“-O-(CH2)n-O-”的基团取代，其中n＝1、2或3。
  5. 如权利要求1所述的化合物，其特征在于，所述的化合物选自下组：
    Figure PCTCN2015084006-appb-100002
    Figure PCTCN2015084006-appb-100003
  6. 一种如下式Ia所示的化合物：
    Figure PCTCN2015084006-appb-100004
    其中，R3、R4、R5、X、n1、n2和n3基团的定义如权利要求1中所述。
  7. 如权利要求6所述的式Ia化合物的制备方法，其特征在于，包括步骤：
    Figure PCTCN2015084006-appb-100005
    在惰性溶剂中，用式Ia1化合物与式Ia2化合物反应，得到式Ia化合物；
    其中，各基团的定义如权利要求6中所述。
  8. 如权利要求1所述的式I化合物的制备方法，其特征在于，包括步骤：
    (1)在惰性溶剂中，用如式Ia所示的化合物和如式Ib所示的任一化合物反应，得到式I'化合物；
    Figure PCTCN2015084006-appb-100006
    (b1)任选地，在惰性溶剂中，用如式I'所示的化合物进行脱氢反应，得到式I”化合物；
    Figure PCTCN2015084006-appb-100007
    (b2)任选地，在惰性溶剂中，用如式I'所示的化合物与R2I进行消除反应，得到式I”'化合物；
    Figure PCTCN2015084006-appb-100008
    其中，各基团的定义如权利要求1中所述。
  9. 如权利要求8所述的制备方法，其特征在于，当式I'、I”或式I”'化合物中X为O时，所述的方法还任选地包括步骤：
    (i)当R5不为H时，在惰性溶剂中，用式Ic化合物进行水解反应，得到式Id化合物；
    Figure PCTCN2015084006-appb-100009
    (ii)在惰性溶剂中，用式Id化合物与R5-NH2反应，得到式Ie化合物；
    Figure PCTCN2015084006-appb-100010
    其中，各基团的定义如权利要求1中所述。
  10. 一种药物组合物，其特征在于，所述的药物组合物含有式I化合物或其药学上可接受的盐；和药学上可接受的载体。
  11. 如权利要求1所述的化合物的用途，其特征在于，用于：
    (a)制备AMPK激活剂；
    (b)制备AMPK和/或ACC磷酸化促进剂；
    (c)在体外非治疗性地激活AMPK活性；
    (d)在体外非治疗性地促进AMPK和/或ACC磷酸化；
    (e)制备治疗与AMPK活性相关疾病的药物组合物。
  12. 一种AMPK活性激活剂，其特征在于，所述的AMPK活性激活剂包括：激活有效量的的式I化合物或其药学上可接受的盐。
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