WO2017097215A1 - 内嵌脲类结构的wnt通路抑制剂 - Google Patents

内嵌脲类结构的wnt通路抑制剂 Download PDF

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WO2017097215A1
WO2017097215A1 PCT/CN2016/108963 CN2016108963W WO2017097215A1 WO 2017097215 A1 WO2017097215 A1 WO 2017097215A1 CN 2016108963 W CN2016108963 W CN 2016108963W WO 2017097215 A1 WO2017097215 A1 WO 2017097215A1
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group
amide
methyl
bipyridyl
dimethyl
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PCT/CN2016/108963
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English (en)
French (fr)
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王永辉
朱研
周娟
高羽军
王士群
王栋
刘万登
沈锡明
洪彬彬
刘涛
吴耀东
李春启
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杭州雷索药业有限公司
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Publication of WO2017097215A1 publication Critical patent/WO2017097215A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • C07D401/14—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 three or more hetero rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings

Definitions

  • the present invention relates to a WNT pathway inhibitor embedded with a urea structure, which is a compound which modulates the activity of a Wnt signaling pathway, and provides a preparation method of the compound, and the compound is used for preparing a drug antagonizing the Wnt signaling pathway.
  • Wnt signaling pathway plays an important role in life processes such as axis differentiation, tissue and organogenesis, and tumor formation in multicellular organisms.
  • the Wnt gene encodes a protein expressed as a secreted glycoprotein consisting of 19 members, which binds to the Frizzled (Fzd) family of proteins and LDL receptor related protein (LRP) receptors on the cell membrane. It activates a variety of intracellular signaling pathways such as the Wnt/ ⁇ -catenin pathway, the planar polar pathway, and the Wnt/Ca 2+ pathway, regulating a variety of cellular functions including proliferation, differentiation, death, migration, and polarization (Nusse Roel, Varmus Harold E. (1992). Wnt genes. Cell, 69(7), 1073-1087.).
  • Wnt signaling is involved in early tumor evidence derived from the isolation of the oncogene Int1 activated by viral insertion in mouse breast cancer; nearly 10% of patients with colorectal cancer, head and neck cancer, lung cancer, ovarian cancer, melanoma Cancer development is associated with the induction of functional mutations in the R-spondin family and RNF43/ZNRF3, a Wnt signaling regulator (B Madan, Z Ke, N Harmston, et al. (2015). Wnt addiction of genetically defined cancers reversed by PORCN inhibition. Oncogene, 1-11.).
  • LGK974 (ClinicalTrials.gov Identifier: NCT01351103, designed for the upstream target of the Wnt signaling pathway PORCN)
  • the patent publication number is wo2010101849), ETC-1922159 (ClinicalTrials.gov Identifier: NCT02521844, the compound patent publication number is wo2014189466); PRI-724 designed for the downstream target of the Wnt signaling pathway CBP/ ⁇ -catenin (ClinicalTrials.gov Identifier: NCT02413853).
  • the object of the present invention is to provide a novel WNT pathway inhibitor, which synthesizes and screens a series of compounds having antitumor activity through substitution modification of a group.
  • a WNT pathway inhibitor embedding a urea structure which is a compound having the following structural formula: and a pharmaceutically acceptable salt thereof:
  • ring A and ring B are each independently selected from an aromatic ring or an aromatic heterocyclic ring containing 1-2 N atoms or O atoms; and X, Y and Z are each independently selected from one of CR 4 and N atoms.
  • n is selected from any integer value of 1 to 2;
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, halo C 1-6 alkyl, C 1-6 alkoxy, halo C 1-6 alkoxy, hydroxy, amino, cyano, acyl, sulfo, aryl, hetero
  • R 3 is selected from a substituted or unsubstituted aryl group, a heterocyclic group; when R 3 is a substituted aryl group, a heterocyclic group, an aryl group, a substituent on a heterocyclic group
  • the group is selected from the group consisting of halogen, C 1-6 alkyl, halo C 1-6 alkyl, C 1-6 alkoxy, halo C 1-6 alkoxy, hydroxy, cyano, amino, acyl,
  • ring B is selected from an aromatic ring or an aromatic heterocyclic ring containing 1-2 N atoms or O atoms;
  • X, Y, Z are each independently selected from one of CR 4 and N atoms;
  • n is selected from 1 to 1 Any integer value of 2;
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, C 3-6 cycloalkyl, halo C 1-6 alkyl, C 1-6 One or more of an alkoxy group, a halogenated C 1-6 alkoxy group, a hydroxyl group, an amino group, an acyl group, an aryl group, a heterocyclic group; and
  • R 3 is selected from a substituted or unsubstituted aryl group, a heterocyclic group;
  • R 3 is a substituted aryl or heterocyclic group, the substituent group on the aryl group or the heterocyclic group is selected from the group consisting of
  • the heterocyclic group is one or more N, O 3-12-membered heterocyclic ring of S hetero atom.
  • R 1 and R 2 are hydrogen, halogen, C 1-6 alkyl, C a 1-6 alkoxy group, a halogenated C1-6 alkyl group, a halogenated C1-6 alkoxy group, an amide group, a C 1-6 alkyl amide group, or a heterocyclic group;
  • R 3 is One of R 4 is selected from the group consisting of hydrogen, C 1-6 alkyl, halogenated C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkoxy;
  • R 5 selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkoxy, amide; heterocycle
  • the group is selected from a 3-6 membered heterocyclic ring containing one
  • any one of X, Y, and Z is an N atom, and the other is CR 4 ; or any two of X, Y, and Z are N atoms, and the other is CR 4 ;
  • n is selected from 1 or 2; 1 , R 2 is hydrogen, halogen, C 1-6 alkyl, C 1-6 alkoxy, halo C 1-6 alkyl, halo C 1-6 alkoxy, amide, C 1-6 alkyl One of an amide group and a heterocyclic group;
  • R 3 is One of R 4 is selected from the group consisting of hydrogen, C 1-6 alkyl, halogenated C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkoxy;
  • R 5 is selected from one or more of hydrogen, halogen, C 1-6 alkyl, halogenated C 1 - 6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkoxy;
  • the ring group is a 3-6
  • the aryl group is phenyl, naphthyl or anthracenyl;
  • the heterocyclic group is morpholinyl, piperidinyl, pyridyl, pyrimidinyl, pyranyl, thienyl, furyl, pyrrolyl, pyrazolyl, Imidazolyl or thiazolyl;
  • halogen is one of fluorine, chlorine, bromine, and iodine.
  • REX-N-1 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-phenyl-1hydro-pyrazole-1- Amide
  • REX-N-2 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(pyridin-2-yl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-3 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-phenyl-1-hydro-1,2, 3-triazole-1-amide;
  • REX-N-4 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-3-phenyl-1-hydro-pyrazole-1 - amide;
  • REX-N-5 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(3-fluorophenyl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-6 4-(3-Chlorophenyl)-N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-7 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-3-methyl-4-phenyl-1-hydrol -pyrazole-1-amide;
  • REX-N-8 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-5-methyl-4-phenyl-1-hydrol -pyrazole-1-amide;
  • REX-N-9 4-(2-chlorophenyl)-N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-10 4-(4-chlorophenyl)-N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-11 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(pyrazin-2-yl)-1- Hydrogen-pyrazole-1-amide;
  • REX-N-12 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(2-fluorophenyl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-13 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(4-fluorophenyl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-14 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(pyridin-3-yl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-15 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(pyridin-4-yl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-16 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(3-fluoropyridin-2-yl)- 1-hydro-pyrazole-1-amide;
  • REX-N-17 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(5-fluoropyridin-2-yl)- 1-hydro-pyrazole-1-amide;
  • REX-N-18 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(3-fluoropyridin-4-yl)- 1-hydro-pyrazole-1-amide;
  • REX-N-20 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(6-methylpyridin-2-yl) 1-hydrogen-pyrazole-1-amide;
  • REX-N-21 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(5-methylpyridin-3-yl) 1-hydrogen-pyrazole-1-amide;
  • REX-N-22 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(2-methylpyridin-4-yl) 1-hydrogen-pyrazole-1-amide;
  • REX-N-23 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(3-cyanophenyl)-1- Hydrogen-pyrazole-1-amide;
  • REX-N-24 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(pyrimidin-5-yl)-1-hydrogen -pyrazole-1-amide;
  • REX-N-26 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-(pyridazin-3-yl)-1- Hydrogen-pyrazole-1-amide;
  • REX-N-28 4-(4-Acetylpiperazin-1-yl)-N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl) 1-hydro-pyrazole-1-amide;
  • REX-N-29 N-((2',3-dimethyl-[2,4'-bipyridyl]-5-yl)methyl)-4-morpholinyl-1-hydro-pyrazole- 1-amide;
  • Compound as used in the present invention includes all stereoisomers, geometric isomers, tautomers and isotopes.
  • Compounds as used herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers.
  • the compound containing an asymmetric carbon atom in the present invention can be isolated in an optically active pure form or in a racemic form.
  • the optically active pure form can be resolved from the racemic mixture or synthesized by using a chiral starting material or a chiral reagent.
  • the "compound” of the present invention also includes tautomeric forms.
  • the tautomeric form is derived from the exchange of a single bond with an adjacent double bond and accompanied by a proton transfer.
  • the invention also includes atoms of all isotopes, whether in the intermediate or the final compound.
  • the atoms of an isotope include those having the same number of atoms but different mass numbers.
  • isotopes of hydrogen include deuterium and tritium.
  • halogen means fluoro, chloro, bromo or iodo, preferably fluoro, chloro or bromo.
  • cyano refers to -CN.
  • hydroxy refers to -OH.
  • alkyl refers to a straight or branched saturated hydrocarbon group consisting of a carbon atom and a hydrogen atom, such as a C 1-20 alkyl group, preferably a C 1-6 alkyl group, such as methyl, ethyl, or propyl.
  • Base including n-propyl and isopropyl
  • butyl including n-butyl, isobutyl, sec-butyl or tert-butyl
  • pentyl including n-pentyl, isopentyl, neopentyl
  • the alkyl group may be unsubstituted or substituted by one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxyl, aryl, heteroaryl, Amino group, halogen, sulfonyl group, sulfinyl group, phosphoryl group.
  • amino refers to -NH 2 , -NH(alkyl) and -N(alkyl) 2 , and the meaning of the alkyl group is as defined above.
  • -NH (alkyl) is in the form of Specific examples include, but are not limited to, -NHCH 3 , -NHCH(CH 3 ) 2 , -NHC 2 H 5 , etc.;
  • -N(alkyl) 2 has the structural form Specific examples include, but are not limited to, -N(CH 3 ) 2 , -N(CH 3 )C 2 H 5 , and the like.
  • aryl refers to an all-carbon monocyclic or fused ring having a fully conjugated pi-electron system, typically having from 6 to 14 carbon atoms, preferably having from 6 to 12 carbon atoms, and most preferably having six carbon atoms. .
  • the aryl group may be unsubstituted or substituted by one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxyl, aryl, aralkyl, amino, halogen. , sulfonyl, sulfinyl, phosphoryl. Examples of unsubstituted aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl.
  • heterocyclyl refers to a monocyclic or fused ring having from 3 to 12 (integer) ring atoms, wherein one, two or three ring atoms are selected from one or more of N, O, and the remaining rings
  • the atom is C and has a fully conjugated ⁇ -electron system.
  • the heterocyclic group may be a saturated or unsaturated group, or may be unsubstituted or substituted by one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxy, Carbonyl, carboxyl, aryl, aralkyl, amino, halogen, sulfonyl, sulfinyl, phosphoryl.
  • unsubstituted heterocyclic groups include, but are not limited to, pyrrolyl, indenyl, pyrrolidinyl, imidazolyl, pyrazolyl, tetrazolyl, pyridyl, quinolyl, isoquinolinyl, piperidinyl, Pyrimidinyl, pyrazinyl, piperazinyl, furyl, pyranyl, morpholinyl.
  • embedded urea refers to a specific structure formed by the insertion of a certain nitrogen atom of urea into a ring conjugated to a carbonyl group, including but not limited to the following forms:
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound as described above or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers.
  • the "pharmaceutical composition” as used in the present invention refers to a preparation of one or more compounds of the present invention or a salt thereof and a carrier generally accepted in the art for delivery of a biologically active compound to an organism such as a human.
  • the purpose of the pharmaceutical composition is to facilitate delivery of the drug to the organism.
  • pharmaceutically acceptable carrier means a substance which is co-administered with the active ingredient and which facilitates administration of the active ingredient, including but not limited to acceptable for human or animal use as permitted by the State Food and Drug Administration ( Any of the glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, disintegrating agents, suspending agents, stabilizers, for example, livestock) , isotonic agents, solvents or emulsifiers. Examples include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • the pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations such as tablets, pills, and glues.
  • solid, semi-solid, liquid or gaseous preparations such as tablets, pills, and glues.
  • the pharmaceutical composition of the present invention can be produced by a method well known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar coating pill method, a grinding method, an emulsification method, a freeze drying method, and the like.
  • the administration route of the compound of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof including but not limited to oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral , sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous.
  • a preferred route of administration is oral administration.
  • the pharmaceutical composition can be formulated by admixing the active compound with apharmaceutically acceptable carrier which is well known in the art.
  • a pharmaceutical composition for oral administration can be obtained by combining the active ingredient with one or more solid carriers, granulating the resulting mixture if necessary, and adding a small amount of excipient if necessary Processed into a mixture or granule to form a tablet or tablet core.
  • the core may be combined with a coating material which is optionally suitable for enteric processing, in the form of a coating formulation which is more advantageous for absorption by an organism such as a human.
  • the invention also provides the use of a compound as described above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for antagonizing the Wnt signaling pathway.
  • the pharmaceutical use is for the treatment of cell proliferative diseases, digestive diseases associated with abnormal Wnt signaling activity.
  • the pharmaceutical use is for the treatment of cancer, including non-small cell lung cancer, anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, nasopharyngeal carcinoma, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, liver cancer , gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, systemic histiocytosis and neuroblastoma.
  • cancer including non-small cell lung cancer, anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, nasopharyngeal carcinoma, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, liver cancer , gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, systemic histiocytosis and neuroblastoma.
  • the inventors have determined the inhibitory activity of the Wnt pathway STF reporter gene at a molecular level by synthesizing a series of in-situ urea structure-like WNT pathway inhibitors, and found that some compounds have significant inhibitory activity against the Wnt pathway; A zebrafish phenotypic screening experiment was also carried out. It was found that the anti-tumor activity of some compounds in vivo was remarkable by the zebrafish tail-scission regeneration inhibition test and the body axis development inhibition test.
  • the present invention provides an embedded urea structure-like WNT pathway inhibitor, based on a rational drug design of a target, and obtains a series of novel compounds by substitution modification of the group; and combined with the STF report Genetic experiments, zebrafish phenotypic screening experiments, optimized screening of a series of compounds with anti-tumor activity. Therefore, it can be used to develop a new generation of Wnt pathway inhibitors, which has great clinical application value for targeted therapy or prevention of diseases mediated by Wnt pathway, and has a considerable market potential.
  • Figure 1 shows the inhibition of body axis development of AB zebrafish after 48 h of treatment.
  • Figure 2 is a graph showing the dose-effect relationship of body axis development inhibition of AB zebrafish after 48 h treatment (mean ⁇ sd)
  • Figure 3 is a graph showing the inhibition of the tail-type regeneration of AB-type zebrafish after 7dpf treatment.
  • Figure 4 is a graph showing the dose-effect relationship of the inhibition of AB-type zebrafish rear-tail regeneration after treatment of 7dpf (mean ⁇ sd)
  • Figure 5 is a graph showing the growth of tumor volume in each treatment group and control group of colon cancer PDX model CR3150.
  • Figure 6 is a graph showing the changes in body weight in the CR3150 model.
  • liquid chromatography is carried out using a WatersSymmetry C18 column. Thin layer chromatography was performed using GF254 (0.25 mm). Nuclear magnetic resonance chromatography (NMR) was measured using a Bruker-400 NMR spectrometer; LC/MS was performed using a Waters ZQ mass spectrometer (column: WatersSymmetry C18, mm, 5 ⁇ m, 35 ° C) using ESI (+) ion mode .
  • NMR nuclear magnetic resonance chromatography
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • the intermediate REX-P-INT-1 (452.0 mg) was prepared.
  • the synthetic route is as follows:
  • the preparation method of the third embodiment is referred to the second embodiment, wherein the o-chlorobenzene boronic acid in the first step is replaced by p-chlorobenzene boronic acid, and the other preparation methods are the same as those in the second embodiment, and finally the compound REX-N-10 (545.4 mg, 52.3%) is obtained. ).
  • the preparation method of the fourth embodiment is as follows.
  • the o-chlorobenzene boronic acid in the first step is replaced by m-fluorophenylboronic acid, and the other preparation method is the same as that in the second embodiment.
  • the compound REX-N-5 (456.1 mg, 44.7%) is obtained. ).
  • the preparation method of the fifth embodiment is as follows.
  • the o-chlorobenzeneboronic acid of the step one is replaced by m-chlorobenzeneboronic acid, and the other methods are the same, and finally the compound REX-N-6 (502.6 mg, 48.2%) is obtained.
  • the preparation method of the sixth embodiment is as follows.
  • the o-chlorobenzeneboronic acid of the step one is replaced by 2-pyrazineboronic acid, and the other methods are the same, and finally the compound REX-N-11 (531.7 mg, 55.2%) is obtained.
  • the preparation method of the eighth embodiment is as follows.
  • the o-chlorobenzeneboronic acid of the step one is replaced by 2-fluorobenzeneboronic acid, and the other methods are the same, and finally the compound REX-N-12 (50 mg, 13.2%) is obtained.
  • the preparation method of the ninth embodiment is as follows.
  • the o-chlorobenzeneboronic acid of the step one is replaced by 4-fluorobenzeneboronic acid, and the other methods are the same, and finally the compound REX-N-13 (60 mg, 15.2%) is obtained.
  • the preparation method of the tenth embodiment is as follows.
  • the one-step bromopyridine is replaced by m-bromopyridine, and the other methods are the same, and finally the compound REX-N-14 (40 mg, 10.2%) is obtained.
  • the preparation method of the eleventh embodiment is referred to the seventh embodiment, wherein one of the steps of the o-bromopyridine is replaced by p-bromopyridine, and the other methods are the same, and finally the compound REX-N-15 (50 mg, 12.2%) is obtained.
  • the preparation method of the twelfth embodiment is as follows.
  • the o-bromopyridine of the step is replaced by 2-bromo-3-fluoro-pyridine, and the other methods are the same, and finally the compound REX-N-16 (30 mg, 8.5%) is obtained. .
  • the preparation method of the thirteenth embodiment is referred to the seventh embodiment, wherein the one of the steps of the o-bromopyridine is replaced by 3-bromo-5fluoro-pyridine, and the other methods are the same, and finally the compound REX-N-17 (35 mg, 8.9%) is obtained. .
  • the preparation method of the fourteenth embodiment is referred to the seventh embodiment, wherein the one of the steps of the o-bromopyridine is replaced by 3-fluoro-4-bromo-pyridine, and the other methods are the same, and finally the compound REX-N-18 (55 mg, 12.5%) is obtained. .
  • the preparation method of the fifteenth embodiment is referred to the seventh embodiment, wherein the intermediate REX-N-INT-3 of the step one is replaced by the intermediate REX-N-INT-4, and the other methods are the same, and finally the compound REX-N- is obtained. 19 (65 mg, 14.5%).
  • the preparation method of the sixteenth embodiment is referred to the seventh embodiment, wherein the step one of the o-bromopyridine is replaced by 2-bromo-6-methyl-pyridine, and the other methods are the same, and finally the compound REX-N-20 (50 mg, 11.5) is obtained. %).
  • the preparation method of the seventeenth embodiment is referred to the seventh embodiment, wherein the one of the steps of the o-bromopyridine is replaced by 3-bromo-5-methyl-pyridine, and the other methods are the same, and finally the compound REX-N-21 (10 mg, 10.5) is obtained. %).
  • the preparation method of the eighteenth embodiment is referred to the seventh embodiment, wherein one step of the o-bromopyridine is replaced by 2-methyl-4-bromo-pyridine, and the other methods are the same, and finally the compound REX-N-22 (40 mg, 10.3) is obtained. %).
  • the preparation method of the nineteenth embodiment is referred to the seventh embodiment, wherein the one of the steps of the o-bromopyridine is replaced by m-bromophenylacetonitrile, and the other methods are the same, and finally the compound REX-N-23 (100 mg, 20.3%) is obtained.
  • Example 20 The preparation method of Example 20 is as follows. In the seventh embodiment, the one of the steps of the o-bromopyridine is replaced by 5-bromo-pyrimidineacetonitrile, and the other methods are the same, and finally the compound REX-N-24 (80 mg, 15.8%) is obtained.
  • the preparation method of the twenty-first embodiment is the same as in the seventh embodiment, wherein the one of the steps of the o-bromopyridine is replaced by 5-bromo-pyrimidine acetonitrile, and the other methods are the same, and finally the compound REX-N-25 (100 mg, 18.8%) is obtained.
  • the preparation method of the twenty-second embodiment is referred to the seventh embodiment, wherein the one of the steps of the o-bromopyridine is replaced by 5-bromo-pyrimidine acetonitrile, and the other methods are the same, and finally the compound REX-N-26 (100 mg, 18.5%) is obtained.
  • Zebrafish is a vertebrate with up to 85% homology with the human genome. It has similar tissue and organ function and signaling pathways to humans, and has unique advantages such as high egg production, rapid development, and embryonic transparency, making zebrafish Successfully applied to human disease research and high-throughput screening of living drugs.
  • Wnt signaling pathway is widely distributed in invertebrates and vertebrates. It is a highly conserved signaling pathway in the process of evolution. It participates in the regulation of cell growth, apoptosis, self-renewal and survival genes in vivo, and maintains normal in organisms.
  • Physiological functions such as embryonic development and tissue repair regeneration (Wolfram Goessling, Trista E. North, Sabine Loewer, et al.
  • Wnt signaling pathway dysregulation is also closely related to cancer development. For example, 80% to 90% of colorectal cancers contain functional mutations affecting the Wnt signaling pathway gene Adenomatous Polyposis Coli (APC) (Hans Clevers, Roel Nusse. (2012). Wnt/ ⁇ -Catenin Signaling and Disease. Cell, 149(6): 1192-1205.).
  • APC Adenomatous Polyposis Coli
  • RESULTS The 3 hpf (hours post fertilization) type AB zebrafish eggs were selected for the development of the body axis phenotype.
  • the group was administered at a final concentration ranging from 100 ⁇ M to 0.001 ⁇ M and the vehicle control group.
  • the AB eggs were added to a 6-well plate, 3 mL of fish water per well, and the DMSO concentration was ⁇ 1% (v/v).
  • the AB eggs after dosing were imaged at 48 hpf in a 28.5 °C biochemical incubator, and the px values (pixels) of the body length of each group of juvenile fish were measured by NIS-Elements D 3.1 software.
  • the IC 50 value of each compound in the development of AB zebrafish body axis was calculated by nonlinear fitting using GraphPad Prism 6.0.
  • IC 50 is calculated for 50% inhibition concentration
  • the present invention retests the zebrafish tail-splitting phenotype of the compounds in Table 1.
  • the 3dpf (days post fertilization) type AB zebrafish juveniles were selected for shear tail fin modeling, and were randomly divided into groups of 10 ⁇ M to 0.001 ⁇ M and a control group, 15 juveniles per group. Add to 6-well plate, 3 mL fish water per well, DMSO concentration ⁇ 1% (v / v). The AB eggs after dosing were imaged at 7 dpf in a biochemical incubator at 28.5 °C. The px values (pixels) of the caudal fin regeneration length of each group were analyzed by NIS-Elements D 3.1 software.
  • the IC 50 value of each compound in the tail-type regeneration of AB type zebrafish was calculated by nonlinear fitting using GraphPad Prism 6.0.
  • REX-N-23 1.984 REX-N-24 11.1 REX-N-25 3.8 REX-N-26 32.2
  • a series of compounds such as LGK-974 (control) and REX-N-1 (Examples) were tested for Wnt signaling by HEK293T-STF cell line stably transfected with STF reporter gene and L-Wnt3a secreted cell line.
  • pathway inhibitory activity the activity was represented by an index of the IC 50, IC 50 i.e. STF reporter gene Luciferase activity is inhibited by 50% the concentration of the compound.
  • a series of compounds such as REX-N-1 prepared in the examples of the present invention were measured using the Wnt reporter gene platform of Crown Bio, and the results are shown in Table 3. The results indicate that the compounds provided by the present invention have better Wnt signaling pathway inhibitory activity at the molecular level.
  • the compound REX-N-1 prepared in the examples of the present invention was used to carry out human and mouse in vitro liver particle metabolism stability experiments and PK assay in BABL/C mice.
  • the PK test method was as follows: 18 rats were divided into two groups of 9 rats each. One group was administered intravenously at a dose of 5 mg/kg; one group was orally administered at a dose of 10 mg/kg. Each group collected blood through the orbital veins at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 h after administration. About 100 ⁇ L of blood was collected by eyelid blood collection into a clean EP tube containing EDTA (final concentration of EDTA was 0.25 mg/mL). The blood collection tube was quickly inverted at least 5 times after collection to ensure uniform mixing and then placed on ice. Blood collected at each time point was centrifuged at 8000 rpm for 5 minutes at 4 ° C to obtain plasma. Another 1.5 mL centrifuge tube was used to mark the compound name, animal number, and time point, and the plasma was transferred to the tube. Plasma was stored at -80 ° C until analysis.
  • the patient colon tumor CR3150 was cut into 1640 diameter medium pieces into a subcutaneous diameter of 2-4 mm and inoculated into the nude mice, and passaged on nude mice after the tumor grew to 500-700 mm 3 .
  • the tumor was cut into 1640 diameter pieces in a 1640 culture medium for subcutaneous inoculation in experimental nude mice.
  • Female mice were subcutaneously inoculated with tumor fragments on the right side. The tumor growth was observed regularly.
  • the tumor grew to an average volume of about 200 mm 3 , it was randomly divided into three groups according to the tumor size.
  • the solvent, the positive control drug LGK974, and the compound of Example 1 REX-N-1 were administered separately for 5 days at 5 mg/kg and BID. Regular observations of tumor volume and body weight changes.
  • the tumor volume calculation formula is: long diameter ⁇ short diameter 2 /2.
  • TGI (%) (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor growth rate, that is, the percentage of tumor volume or tumor weight relative to the treatment group and the control group at a certain time point.
  • T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively.
  • T/C% T RTV /C RTV ⁇ 100%
  • T RTV treatment group mean RTV
  • C RTV vehicle control group mean RTV
  • RTV V t /V 0
  • V 0 is the tumor volume of the animal at the time of grouping
  • V t is the tumor volume of the animal after treatment.
  • T/C% T TW / C TW ⁇ 100% (T TW : mean tumor weight at the end of the treatment group; C TW : mean tumor weight at the end of the vehicle control experiment).
  • LGK974 and REX-N-1 were administered at a dose of 5 mg/kg for 28 days, and BID showed significant antitumor activity.

Abstract

本发明公开了一种内嵌脲类结构的WNT通路抑制剂，属于调节Wnt信号通路活性的化合物，并提供了该类化合物的制备方法，及该类化合物在制备拮抗Wnt信号通路的药物中的应用。本发明提供的内嵌脲类结构的WNT通路抑制剂，基于靶标的合理药物设计，抗肿瘤活性显著，可用于开发成新一代的Wnt通路抑制剂，具有极大的临床应用价值，市场潜力可观。

Description

内嵌脲类结构的WNT通路抑制剂 技术领域
本发明涉及一种内嵌脲类结构的WNT通路抑制剂，属于调节Wnt信号通路活性的化合物，并提供了该类化合物的制备方法，及该类化合物用于在制备拮抗Wnt信号通路的药物中的应用。
背景技术
Wnt信号通路在多细胞生物体轴分化、组织器官发生、肿瘤形成等生命过程中具有重要作用。Wnt基因编码表达的蛋白是一类分泌型糖蛋白，由19个成员组成，通过与细胞膜上Frizzled(Fzd)家族蛋白及低密度脂蛋白受体相关蛋白(LDL receptor related protein，LRP)受体结合，激活典型Wnt/β-catenin通路、平面极通路、Wnt/Ca2+通路等多种细胞内信号途径，调控着包括增殖、分化、死亡、迁移、极化等多种细胞功能(Nusse Roel,Varmus Harold E.(1992).Wnt genes.Cell,69(7),1073-1087.)。
经研究报道发现，神经性疾病、炎症纤维化疾病、代谢性疾病以及多种类型癌症的发病机制中都有涉及到典型Wnt/β-catenin信号通路β-catenin-TCF/LCF转录复合物的激活的失调(Kahn,M.(2014).Can we safely target the Wnt pathway？Nature reviews.Nat.Rev.Drug.Discovery,13(7),513-532.)。在癌症研究领域中，Wnt信号参与肿瘤早期证据来源于小鼠乳腺癌中分离得到因病毒***而激活的癌基因Int1；近10％的结直肠癌、头颈癌、肺癌、卵巢癌、黑色素瘤患者癌症发生与Wnt信号调控元件R-spondin家族和RNF43/ZNRF3的功能突变诱导相关(B Madan,Z Ke,N Harmston,et al.(2015).Wnt addiction of genetically defined cancers reversed by PORCN inhibition.Oncogene,1-11.)。然而，这类患者目前临床上尚无针对性的Wnt信号通路小分子靶向药物治疗，非选择性的细胞毒制剂及其联合治疗方案所带来的胃肠道反应、骨髓造血抑制等方面的不良反应严重影响患者的生存质量。目前，进入临床试验阶段药物候选物还处于安全性和概念性药效验证的临床I/II期阶段，例如，针对Wnt信号通路上游靶点PORCN设计的LGK974(ClinicalTrials.gov Identifier:NCT01351103，该化合物专利的公开号为wo2010101849)，ETC-1922159(ClinicalTrials.gov Identifier:NCT02521844，该化合物专利的公开号为wo2014189466)；针对Wnt信号通路下游靶点CBP/β-catenin设计的PRI-724(ClinicalTrials.gov Identifier:NCT02413853)。
鉴于此，继续研制具有可以调控Wnt信号通路的药物，寻找到一种作用机制明确、药效显著的化合物，具有重要的临床价值和社会意义。
发明内容
本发明目的在于提供一种结构新颖的WNT通路抑制剂，通过基团的取代修饰，合成并筛选出一系列具有抗肿瘤活性的化合物。
为实现上述发明目的，本发明采取了以下技术方案：
一种内嵌脲类结构的WNT通路抑制剂，为具有如下结构通式的化合物及其药学上可接受的盐：
Figure PCTCN2016108963-appb-000001
其中，环A和环B各自独立地选自芳香环、或含1-2个N原子或O原子的芳香杂环；X、Y、Z各自独立地选自CR4、N原子中的一种；n选自1～2中的任一整数值；R1、R2各自独立地选自氢、卤素、C1-6烷基、C3-6环烷基、C2-6烯基、C2-6炔基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、羟基、氨基、氰基、酰基、磺基、芳基、杂环基中的一种或几种；R3选自取代或未取代的芳基、杂环基；当R3为取代的芳基、杂环基时，芳基、杂环基上的取代基团选自卤素、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、羟基、氰基、氨基、酰基、磺基、杂环基中的一种或几种；R4选自氢、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、C3-6环烷基中的一种；杂环基为含有一个或多个N、O、S杂原子的3-12元杂环。
优选的，为具有如下结构通式的化合物及其药学上可接受的盐：
Figure PCTCN2016108963-appb-000002
其中，环B选自芳香环、或含1-2个N原子或O原子的芳香杂环；X、Y、Z各自独立地选自CR4、N原子中的一种；n选自1～2中的任一整数值；R1、R2各自独立地选自氢、卤素、C1-6烷基、C3-6环烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、羟基、氨基、酰基、芳基、杂环基中的一种或几种；R3选自取代或未取代的芳基、杂环基；当R3为取代的芳基、杂环基时，芳基、杂环基上的取代基团选自卤素、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、羟基、氰基、氨基、酰基、磺基、杂环基中的一种或几种；R4选自氢、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、C3-6环烷基中的一种；杂环基为含有一个或多个N、O、S杂原子的3-12元杂环。
更优选的，结构通式(2)中，
Figure PCTCN2016108963-appb-000003
选自
Figure PCTCN2016108963-appb-000004
Figure PCTCN2016108963-appb-000005
中的一种；X、Y、Z各自独立地选自CR4、N原子中的一种；n选自1或2；R1、R2为氢、卤素、C1-6烷基、C1-6烷氧基、卤代C1-6烷基、卤代C1-6烷氧基、酰胺基、C1-6烷基酰胺基、杂环基中的一种；R3为
Figure PCTCN2016108963-appb-000006
Figure PCTCN2016108963-appb-000007
中的一种；R4选自氢、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基中的一种；R5选自氢、卤素、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、酰胺基中的一种；杂环基为选自含有一个或多个N、O、S杂原子的3-6元杂环。
优选的，为具有如下结构通式的化合物及其药学上可接受的盐：
Figure PCTCN2016108963-appb-000008
其中，X、Y、Z中任意一个为N原子，则另一个为CR4；或X、Y、Z中任意两个为N原子，则另一个为CR4；n选自1或2；R1、R2为氢、卤素、C1-6烷基、C1-6烷氧基、卤代C1-6烷基、卤代C1-6烷氧基、酰胺基、C1-6烷基酰胺基、杂环基中的一种；R3为
Figure PCTCN2016108963-appb-000009
Figure PCTCN2016108963-appb-000010
中的一种；R4选自氢、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基中的一种；R5地选自氢、卤素、C1-6烷基、卤代C1- 6烷基、C1-6烷氧基、卤代C1-6烷氧基中的一种或几种；杂环基为选自含有一个或多个N、O、S杂原子的3-6元杂环。
优选的，芳基为苯基、萘基或蒽基；杂环基为吗啉基、哌啶基、吡啶基、嘧啶基、吡喃基、噻吩基、呋喃基、吡咯基、吡唑基、咪唑基或噻唑基；卤素为氟、氯、溴、碘中的一种。
一种内嵌脲类结构的WNT通路抑制剂，选自如下编号为REX-N-1～REX-N-29的特征化合物：
REX-N-1：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-苯基-1氢-吡唑-1-酰胺；
REX-N-2：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-2-基)-1-氢-吡唑-1-酰胺；
REX-N-3：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-苯基-1-氢-1,2,3-三氮唑-1-酰胺；
REX-N-4：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-3-苯基-1-氢-吡唑-1-酰胺；
REX-N-5：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟苯基)-1-氢-吡唑-1-酰胺；
REX-N-6：4-(3-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺；
REX-N-7：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-3-甲基-4-苯基-1-氢-吡唑-1-酰胺；
REX-N-8：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-5-甲基-4-苯基-1-氢-吡唑-1-酰胺；
REX-N-9：4-(2-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺；
REX-N-10：4-(4-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺；
REX-N-11：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡嗪-2-基)-1-氢-吡唑-1-酰胺；
REX-N-12：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(2-氟苯基)-1-氢-吡唑-1-酰胺；
REX-N-13：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(4-氟苯基)-1-氢-吡唑-1-酰胺；
REX-N-14：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-3-基)-1-氢-吡唑-1-酰胺；
REX-N-15：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-4-基)-1-氢-吡唑-1-酰胺；
REX-N-16：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟吡啶-2-基)-1-氢-吡唑-1-酰胺；
REX-N-17：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(5-氟吡啶-2-基)-1-氢-吡唑-1-酰胺；
REX-N-18：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟吡啶-4-基)-1-氢-吡唑-1-酰胺；
REX-N-19：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-5甲基-4-(吡啶-2-基)-1-氢-吡唑-1-酰胺；
REX-N-20：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(6-甲基吡啶-2-基)-1-氢-吡唑-1-酰胺；
REX-N-21：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(5-甲基吡啶-3-基)-1-氢-吡唑-1-酰胺；
REX-N-22：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(2-甲基吡啶-4-基)-1-氢-吡唑-1-酰胺；
REX-N-23：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氰基苯基)-1-氢-吡唑-1-酰胺；
REX-N-24：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(嘧啶-5-基)-1-氢-吡唑-1-酰胺；
REX-N-25：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(嘧啶-2-基)-1-氢-吡唑-1-酰胺；
REX-N-26：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(哒嗪-3-基)-1-氢-吡唑-1-酰胺；
REX-N-27：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(嘧啶-4-基)-1-氢-吡唑-1-酰胺；
REX-N-28：4-(4-乙酰哌嗪-1-基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺；；
REX-N-29：N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-吗啉基-1-氢-吡唑-1-酰胺；
前述编号为REX-N-1～REX-N-29的特征化合物，具体结构如下：
Figure PCTCN2016108963-appb-000011
本发明所述的“化合物”，包括所有立体异构体、几何异构体、互变异构体和同位素。
本发明所述的“化合物”，可以是不对称的，例如，具有一个或多个立体异构体。除非另有说明，所有立体异构体都包括，如对映异构体和非对映异构体。本发明中含有不对称碳原子的化合物，可以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
本发明所述的“化合物”，还包括互变异构体形式。互变异构体形式来源于一个单键与相邻的双键交换并一起伴随一个质子的迁移。
本发明还包括所有同位素的原子，无论是在中间体或最后的化合物。同位素的原子包括具有相同的原子数、但不同质量数的。例如，氢的同位素包括氘和氚。
含有前述通式结构的化合物，本文中所用的术语具有如下含义：
术语“卤素”，指氟、氯、溴或碘，优选氟、氯或溴。
术语“氰基”，指-CN。
术语“羟基”，指-OH。
术语“烷基”，指由碳原子和氢原子组成的直链或支链的饱和烃基团，如C1-20烷基，优选为C1-6烷基，例如甲基、乙基、丙基(包括正丙基和异丙基)、丁基(包括正丁基、异丁基、仲丁基或叔丁基)、戊基(包括正戊基、异戊基、新戊基)、正己基、2-甲基己基等。所述烷基可以是非取代的、或是被一个或多个取代基所取代，取代基包括但不限于烷基、烷氧基、氰基、羟基、羰 基、羧基、芳基、杂芳基、氨基、卤素、磺酰基、亚磺酰基、磷酰基。
术语“氨基”，指-NH2、-NH(烷基)和-N(烷基)2，烷基的含义如前所述。-NH(烷基)的结构形式为
Figure PCTCN2016108963-appb-000012
具体例子包括但不限于-NHCH3、-NHCH(CH3)2、-NHC2H5等；-N(烷基)2的结构形式为
Figure PCTCN2016108963-appb-000013
具体例子包括但不限于-N(CH3)2、-N(CH3)C2H5等。
术语“芳基”，指具有完全共轭的π电子体系的全碳单环或稠合环，通常具有6-14个碳原子，优选具有6-12个碳原子，最优选具有6个碳原子。芳基可以是非取代的、或被一个或多个取代基所取代，取代基包括但不限于烷基、烷氧基、氰基、羟基、羰基、羧基、芳基、芳烷基、氨基、卤素、磺酰基、亚磺酰基、磷酰基。非取代的芳基的实例包括但不限于苯基、萘基和蒽基。
术语“杂环基”，指具有3-12个(整数)环原子的单环或稠合环，其中有1、2或3个环原子选自N、O中的一个或多个，其余环原子为C，且具有完全共轭的π-电子体系。杂环基可以是饱和的、或非饱和的基团，也可以是非取代的、或被一个或多个取代基所取代，取代基包括但不限于烷基、烷氧基、氰基、羟基、羰基、羧基、芳基、芳烷基、氨基、卤素、磺酰基、亚磺酰基、磷酰基。非取代的杂环基的实例包括但不限于吡咯基、吲哚基、吡咯烷基、咪唑基、吡唑基、四唑基、吡啶基、喹啉基、异喹啉基、哌啶基、嘧啶基、吡嗪基、哌嗪基、呋喃基、吡喃基、吗啉基。
术语“内嵌脲”，指脲的某个氮原子嵌入到和羰基共轭的环中所形成的特定结构，包括但不限于下列几种形式：
Figure PCTCN2016108963-appb-000014
本发明还提供了一种药物组合物，包含如前所述的化合物或其药学上可接受的盐作为活性成份，以及一种或多种药学上可接受的载体。
本发明所述的“药物组合物”，指一种或多种本发明的化合物或其盐与在本领域中通常接受的用于将生物活性化合物输送至有机体(例如人)的载体的制剂。药物组合物的目的是有利于对有机体给药输送。
术语“药学上可接受的载体”，指与活性成份共同给药的、且有利于活性成份给药的物质，包括但不限于国家食品药品监督管理局许可的可接受的用于人或动物(例如家畜)的任何助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。例如包括但不限于碳酸钙、磷酸钙、各种糖和各类淀粉、纤维素衍生物、明胶、植物油和聚乙二醇。
本发明所述的药物组合物，可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶 囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等等。
本发明所述的药物组合物，可以采用本领域熟知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
本发明所述的化合物或其药学上可接受的盐或其药物组合物的给药途径，包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、***内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。优选的给药途径是口服给药。
对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的载体混合，来配制该药物组合物。这些载体能使本发明的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，以用于对患者的口服给药。例如，用于口服给药的药物组合物，可采用如下方式获得片剂：将活性成分与一种或多种固体载体合并，如果需要将所得混合物制粒，并且如果需要加入少量的赋形剂加工成混合物或颗粒，以形成片剂或片芯。片芯可与任选适合肠溶的包衣材料结合，加工成更有利于有机体(例如人)吸收的包衣制剂形式。
本发明还提供了一种如前所述的化合物或其药学上可接受的盐在制备拮抗Wnt信号通路的药物中的应用。
优选的，制药用途为用于治疗与异常的Wnt信号活性相关的细胞增殖性疾病、消化***疾病。
优选的，制药用途为用于治疗癌症，包括非小细胞肺癌、间变性大细胞淋巴瘤、炎性肌纤维母细胞瘤、鼻咽癌、乳腺癌、结直肠癌、弥漫大B细胞淋巴瘤、肝癌、胃癌、食道癌、胰腺癌、卵巢癌、全身组织细胞增生症和神经母细胞瘤。
本发明中，发明人对合成得到的一系列的内嵌脲结构类WNT通路抑制剂，从分子水平测定Wnt通路STF报告基因的抑制活性，发现部分化合物对Wnt通路具有显著的抑制活性；此外，还进行了斑马鱼表型筛选实验，通过对斑马鱼的剪尾再生抑制试验和体轴发育抑制试验，发现部分化合物在体内的抗肿瘤活性显著。
与现有技术相比，本发明提供的一种内嵌脲结构类WNT通路抑制剂，基于靶标的合理药物设计，通过基团的取代修饰，获得了一系列结构新颖的化合物；并结合STF报告基因实验、斑马鱼表型筛选实验，优化筛选出一系列具有抗肿瘤活性的化合物。因此，可用于开发成新一代的Wnt通路抑制剂，对于靶向治疗或预防由Wnt通路介导的疾病具有极大的临床应用价值，市场潜力可观。
附图说明
图1为处理48h后AB型斑马鱼体轴发育抑制图
图2为处理48h后AB型斑马鱼体轴发育抑制量效关系图(mean±sd)
图3为处理7dpf后AB型斑马鱼剪尾再生抑制图
图4为处理7dpf后AB型斑马鱼剪尾再生抑制量效关系图(mean±sd)
图5为结肠癌PDX模型CR3150中各治疗组和对照组小鼠肿瘤体积的生长变化情况图
图6为化合物在CR3150模型中体重变化情况图
具体实施方式
以下是本发明的具体实施例，对本发明的技术方案做进一步的描述，但是本发明的保护范围并不限于这些实施例。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。
本发明提供的目标化合物制备方法中，液相色谱采用WatersSymmetry C18色谱柱。薄层色谱采用GF254(0.25毫米)。核磁共振色谱(NMR)使用Bruker-400核磁共振仪测定；液质连用(LC/MS)使用Waters ZQ质谱检测器(柱子：WatersSymmetryC18，毫米，5微米，35℃)，采用ESI(+)离子模式。
此外，凡涉及易氧化或易水解的原料的所有操作都在氮气保护下进行。除非另有说明，本发明使用的原料都是市售原料、无需进一步纯化可以直接使用。
实施例一、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-苯基-1氢-吡唑-1-酰胺【编号为REX-N-1】的制备
合成路线如下：
Figure PCTCN2016108963-appb-000015
合成方案一：中间体REX-N-INT-1的合成
步骤一：化合物1-2的制备
将化合物1-1(5.0g,24.2mmol)溶解在200ml干燥的四氢呋喃溶液中，在冰水浴中滴加异丙基氯化镁(12.7ml,25.5mmol)，室温搅拌1小时，将N-甲酰基吗啉(2.5ml,24.2mmol)溶解在50ml干燥的四氢呋喃溶液中，室温滴加到反应体系中再搅拌一小时，反应结束后加入冰水淬灭反应，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物1-2(3.0g,79.6％)。
MSm/z[ESI]：156.0[M+1]。
步骤二：化合物1-3的制备
将化合物1-2(3.0g,19.4mmol)、2-甲基-4-吡啶硼酸(3.2g,23.2mmol)溶解在200ml N,N-二甲基甲酰胺中，在氮气保护条件下分别向体系中加入碳酸钾(10.7g,77.6mmol)、四三苯基膦钯(1.1g,1.0mmol)，在100℃条件下搅拌过夜，反应结束向体系中加入冰水200ml，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物1-3(1.8g,44.0％)。
MSm/z[ESI]：213.1[M+1]。
步骤三：化合物1-4的制备
将化合物1-3(1.8g,8.5mmol)溶解在50ml无水乙醇中，分别向体系中加入盐酸羟胺(1.2g,17.0mmol)、醋酸钠(1.4g,17.0mmol)，室温搅拌2小时，反应结束向体系中加入冰水50ml淬灭反应，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物1-4(1.8g,93.4％)。
MSm/z[ESI]：228.1[M+1]。
步骤四：化合物REX-N-INT-1的制备
将化合物1-4(1.8g,7.9mmol)溶解在30ml无水乙醇溶液中，分别加入10％的钯碳(200mg)、盐酸(1.5ml，12.0N)在一个大气压氢气条件下室温反应过夜，反应结束过滤、浓缩，用硅胶柱提纯，得到化合物REX-N-INT-1(1.4g，85.2％)。
MSm/z[ESI]：214.1[M+1]。
合成方案二：化合物REX-N-1的合成
步骤一：中间体REX-N-INT-2的制备
将化合物2-1(5.0g,34.0mmol)、三乙胺(14.1ml,102.0mmol)溶解在100ml干燥的二氯甲烷中，将对甲苯磺酰氯(6.5g,34.0mmol)溶解在50ml干燥的二氯甲烷中滴加到体系中，滴加完毕室温搅拌2小时，反应结束向体系中加入冰水200ml，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物REX-N-INT-2(10.0g,97.6％)。
MSm/z[ESI]：303.0[M+1]。
步骤二：化合物2-3的制备
分别将化合物REX-N-INT-2(10.0g,33.2mmol)、苯硼酸(6.1g，49.8mmol)、碳酸钾(13.7g，99.6mmol)、四三苯基膦钯(1.8g，0.2mmol)溶解在120ml乙二醇二甲醚和24ml水的混合溶剂中，将反应体系在氮气保护、90℃条件下搅拌过夜，反应结束向体系中加入冰水200ml淬灭反应，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物2-3(3.2g,67.3％)。
MSm/z[ESI]：145.1[M+1]。
步骤三：化合物REX-N-1的制备
分别将化合物2-3(3.2g,22.3mmol)、二(三氯甲基)碳酸酯(7.9g,26.8mmol)、N,N-二异丙基乙胺(7.3ml,44.6mmol)溶解在50ml干燥的二氯甲烷中，室温搅拌30分钟，分别将化合物REX-N-INT-1(4.8g,22.3mmol)、N,N-二异丙基乙胺(7.3ml,44.6mmol)溶解在30ml干燥的二氯甲烷滴加到反应体系中，滴加完毕室温搅拌半小时，反应结束向体系中加入冰水200ml，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物REX-N-1(2.5g,29.0％)。
MSm/z[ESI]：384.2[M+1]。
1H-NMR(400MHz,DMSO-d6):δ＝9.24(t,J＝6.0Hz,1H),8.77(s,1H),8.51-8.53(m,2H),8.31(s,1H),7.75(s,1H),7.73(s,2H),7.25-7.43(m,5H),4.53(d,J＝6.0Hz,2H),2.53(s,3H),2.33(s,3H).
实施例二、4-(2-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺【编号为REX-N-9】的制备
合成路线如下：
Figure PCTCN2016108963-appb-000016
中间体REX-N-INT-2的制备：
参照实施例一的制备方法，先制得中间体REX-N-INT-2(1.0克)。
步骤一：化合物1-2的制备
分别将化合物REX-N-INT-2(1.0g,3.3mmol)、邻氯苯硼酸(0.8g，5.0mmol)、碳酸钾(1.4g，9.9mmol)、四三苯基膦钯(180.0mg，0.02mmol)溶解在20ml乙二醇二甲醚和4 ml水的混合溶剂中，将反应体系在氮气保护、90℃条件下搅拌过夜，反应结束向体系中加入冰水50ml淬灭反应，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物1-2(379.5mg,64.6％)。
MSm/z[ESI]：179.0[M+1]。
步骤二：化合物REX-N-9的制备
参照实施例一的制备方法，先制得中间体REX-P-INT-1(452.0毫克)。
分别将化合物1-2(379.5mg,2.1mmol)、二(三氯甲基)碳酸酯(742.8mg,2.5mmol)、N,N-二异丙基乙胺(0.7ml,4.2mmol)溶解在20ml干燥的二氯甲烷中，室温搅拌30分钟，分别将化合物REX-N-INT-1(452.0mg,2.1mmol)、N,N-二异丙基乙胺(0.7ml,4.2mmol)溶解在10ml干燥的二氯甲烷滴加到反应体系中，滴加完毕室温搅拌半小时，反应结束向体系中加入冰水50ml，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物REX-N-9(373.1mg,42.6％)。
MSm/z[ESI]：418.1[M+1]。
1H-NMR(400MHz,CDCl3)δ＝8.58(d,J＝18.4Hz,3H),7.92(s,1H),7.68(s,2H),7.53–7.35(m,5H),7.30(d,J＝7.1Hz,1H),4.69(d,J＝6.1Hz,2H),2.75(s,3H),2.40(s,3H).
实施例三、4-(4-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺【编号为REX-N-10】的制备
合成路线如下：
Figure PCTCN2016108963-appb-000017
实施例三的制备方法参照实施例二，其中步骤一中的邻氯苯硼酸替代为对氯苯硼酸，其余制备方法同实施例二，最终制得化合物REX-N-10(545.4mg,52.3％)。
MSm/z[ESI]：418.1[M+1]。
1H-NMR(400MHz,CDCl3)δ＝8.63–8.52(m,2H),8.45(d,J＝11.9Hz,1H),7.85(s,1H),7.69–7.58(m,2H),7.43(dd,J＝15.5,7.1Hz,4H),7.35(d,J＝8.3Hz,2H),4.68(d,J＝6.2Hz,2H),2.74(s,3H),2.39(s,3H).
实施例四、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟苯基)-1-氢-吡唑-1-酰胺【编号为REX-N-5】的制备
Figure PCTCN2016108963-appb-000018
实施例四的制备方法参照实施例二，其中步骤一中的邻氯苯硼酸替代为间氟苯硼酸，其余制备方法同实施例二，最终制得化合物REX-N-5(456.1mg,44.7％)。
MSm/z[ESI]：402.2[M+1]。
1H-NMR(400MHz,CDCl3)δ＝8.86(t,J＝3.2Hz,1H),8.68(s,1H),8.49(s,1H),7.91(s,1H),7.86–7.80(m,4H),7.39–7.21(m,3H),7.02(t,J＝1.6Hz,1H),4.74(d,J＝6.4Hz,2H),2.91(s,3H),2.47(s,3H).
实施例五、4-(3-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺【编号为REX-N-6】的制备
Figure PCTCN2016108963-appb-000019
实施例五的制备方法参照实施例二，其中步骤一种的邻氯苯硼酸被间氯苯硼酸代替，其余方法相同，最终制得化合物REX-N-6(502.6mg,48.2％)。
MSm/z[ESI]：418.1[M+1]。
1H-NMR(400MHz,CDCl3)δ＝8.66(s,1H),8.50(s,1H),7.91(s,1H),7.82–7.72(m,4H),7.52(t,J＝1.6Hz,1H),7.42–7.28(m,4H),4.73(d,J＝6.4Hz,2H),2.92(s,3H),2.48(s,3H).
实施例六、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡嗪-2-基)-1-氢-吡唑-1-酰胺【编号为REX-N-11】的制备
Figure PCTCN2016108963-appb-000020
实施例六的制备方法参照实施例二，其中步骤一种的邻氯苯硼酸被2-吡嗪硼酸代替，其余方法相同，最终制得化合物REX-N-11(531.7mg,55.2％)。
MSm/z[ESI]：386.2[M+1]。
1H-NMR(400MHz,CDCl3)δ＝9.39(t,J＝1.6Hz,1H),9.19(d,J＝1.6Hz,1H),9.05(s,1H),8.64–8.62(m,1H),8.53–8.51(m,3H),8.45(s,1H),7.74(d,J＝1.6Hz,1H),7.40(s,1H),7.34(dd,J＝4.8,1.2Hz,1H),4.53(d,J＝6.0Hz,2H),2.53(s,3H),2.33(s,3H).
实施例七、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-2-基)-1-氢-吡唑-1-酰胺【编号为REX-N-2】的制备
Figure PCTCN2016108963-appb-000021
步骤一：化合物1-2的制备
分别将化合物REX-N-INT-3(2.46g,12.66mmol)、邻溴吡啶(2g,12.66mmol)、碳酸钾(3.50g,25.32mmol)、四三苯基膦钯(0.88g,0.759mmol)溶解在20ml乙二醇二甲醚和4ml水的混合溶剂中，将反应体系在氮气保护、90℃条件下搅拌过夜，反应结束向体系中加入冰水50ml淬灭反应，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物1-2(600mg,32.7％)。
MSm/z[ESI]：146.0[M+1]。
步骤二：化合物REX-N-2的制备
参照实施例一的制备方法，先制得中间体REX-N-INT-1(1.0克)。
分别将化合物1-2(300mg,2.07mmol)、二(三氯甲基)碳酸酯(417mg,2.07mmol)、N,N-二异丙基乙胺(267mg,0.34ml,2.07mmol)溶解在20ml干燥的二氯甲烷中，室温搅拌30分钟，分别将化合物REX-N-INT-1(441mg,2.07mmol)、N,N-二异丙基乙胺(801mg,1.00ml,6.20mmol)溶解在10ml干燥的二氯甲烷滴加到反应体系中，滴加完毕室温搅拌半小时，反应结束向体系中加入冰水50ml，用乙酸乙酯萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩后用硅胶柱提纯，得到化合物REX-N-2(50mg,6.3％)。
MSm/z[ESI]：385.1[M+1]。 1H-NMR((400MHz,DMSO-d6),δ＝9.33(t,J＝6Hz,1H),8.89(d,J＝0.4Hz,1H)),8.56–8.58(m,1H),8.50–8.54(m,2H),8.39(d,J＝0.8Hz,1H),7.87–7.91(m,1H),7.79–7.84(m,1H),7.73(d,J＝1.6Hz,1H),7.40(s,1H),7.32–7.36(m,1H),7.25–7.29(m,1H),4.52(d,J＝6Hz,2H),2.53(s,3H)2.33(s,3H).
实施例八、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(2-氟苯基)-1-氢-吡唑-1-酰胺【编号为REX-N-12】的制备
Figure PCTCN2016108963-appb-000022
实施例八的制备方法参照实施例二，其中步骤一种的邻氯苯硼酸被2-氟苯硼酸代替，其余方法相同，最终制得化合物REX-N-12(50mg,13.2％)。
MSm/z[ESI]：402.2[M+1]。
1H-NMR(400MHz,DMSO-d6)δ＝9.38(t,J＝1.6Hz,1H),8.68(d,J＝1.6Hz,1H),8.52–8.53(m,2H),8.33(s,1H),7.91(t,J＝1.6Hz,1H),7.74(m,1H),7.42(s,1H),7.25–7.36(m,4H),4.53(d,J＝6.0Hz,2H),2.58(s,3H),2.34(s,3H).
实施例九、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(4-氟苯基)-1-氢-吡唑-1-酰胺【编号为REX-N-13】的制备
Figure PCTCN2016108963-appb-000023
实施例九的制备方法参照实施例二，其中步骤一种的邻氯苯硼酸被4-氟苯硼酸代替，其余方法相同，最终制得化合物REX-N-13(60mg,15.2％)。
MSm/z[ESI]：402.2[M+1]。
1H-NMR(400MHz,DMSO-d6)δ＝9.30(t,J＝1.6Hz,1H),8.81(d,J＝1.6Hz,1H),8.52–8.53(m,2H),8.33(s,1H),7.81–7.83(m,2H),7.73(m,1H),7.42(s,1H),7.33–7.34(m,1H),7.23–7.26(m,1H),4.52(d,J＝6.0Hz,2H),2.58(s,3H),2.34(s,3H).
实施例十、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-3-基)-1-氢-吡唑-1-酰胺【编号为REX-N-14】的制备
Figure PCTCN2016108963-appb-000024
实施例十的制备方法参照实施例七，其中步骤一种的邻溴吡啶被间溴吡啶代替，其余方法相同，最终制得化合物REX-N-14(40mg,10.2％)。
MSm/z[ESI]：385.1[M+1]。
1H-NMR((400MHz,DMSO-d6),δ＝9.33(t,J＝6Hz,1H),8.89(d,J＝0.4Hz,1H),8.50–8.58(m,3H),8.39(d,J＝0.8Hz,1H),7.87–7.91(m,1H),7.79–7.84(m,1H),7.73(d,J＝1.6Hz,1H),7.40(s,1H),7.32–7.36(m,1H),7.25–7.29(m,1H),4.52(d,J＝6Hz,2H),2.53(s,3H),2.33(s,3H).
实施例十一、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-4-基)-1-氢-吡唑-1-酰胺【编号为REX-N-15】的制备
Figure PCTCN2016108963-appb-000025
实施例十一的制备方法参照实施例七，其中步骤一种的邻溴吡啶被对溴吡啶代替，其余方法相同，最终制得化合物REX-N-15(50mg,12.2％)。
MSm/z[ESI]：385.1[M+1]。
1H-NMR((400MHz,CDCl3),δ＝8.63(s,1H),8.55–8.58(m,3H),8.41(d,J＝0.8Hz,1H),7.92(s,1H),7.61–7.65(m,2H),7.52–7.53(m,1H),7.51(s,1H),7.22–7.23(m,1H),7.25–7.29(m,1H),4.68(d,J＝6Hz,2H),2.62(s,3H)2.37(s,3H).
实施例十二、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟吡啶-2-基)-1-氢-吡唑-1-酰胺【编号为REX-N-16】的制备
Figure PCTCN2016108963-appb-000026
实施例十二的制备方法参照实施例七，其中步骤一种的邻溴吡啶被2-溴-3氟-吡啶代替，其余方法相同，最终制得化合物REX-N-16(30mg,8.5％)。
MSm/z[ESI]：403.1[M+1]。
1H-NMR((400MHz,DMSO-d6),δ＝9.42(t,J＝6Hz,1H),8.68(s,1H),8.49–8.52(m,3H),8.38(s,1H),7.84–7.87(m,1H),7.75(s,1H),7.34–7.44(m,3H),4.53(d,J＝6Hz,2H),2.54(s,3H),2.34(s,3H).
实施例十三、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(5-氟吡啶-2-基)-1-氢-吡唑-1-酰胺【编号为REX-N-17】的制备
Figure PCTCN2016108963-appb-000027
实施例十三的制备方法参照实施例七，其中步骤一种的邻溴吡啶被3-溴-5氟-吡啶代替，其余方法相同，最终制得化合物REX-N-17(35mg,8.9％)。
MSm/z[ESI]：403.1[M+1]。
1H-NMR((400MHz,CDCl3),δ＝8.63(s,1H),8.55–8.58(m,3H),8.41(d,J＝0.8Hz,1H),7.92(s,1H),7.53–7.65(m,2H),7.50–7.53(m,1H),7.31(s,1H),7.22–7.23(m,1H),4.39(d,J＝6Hz,2H),2.62(s,3H),2.37(s,3H).
实施例十四、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟吡啶-4-基)-1-氢-吡唑-1-酰胺【编号为REX-N-18】的制备
Figure PCTCN2016108963-appb-000028
实施例十四的制备方法参照实施例七，其中步骤一种的邻溴吡啶被3-氟-4溴-吡啶代替，其余方法相同，最终制得化合物REX-N-18(55mg,12.5％)。
MSm/z[ESI]：403.1[M+1]。
1H-NMR((400MHz,CDCl3),δ＝8.77(s,1H),8.55–8.61(m,3H),8.46(d,J＝0.8Hz,1H),8.09(s,1H),7.76(s,1H),7.49–7.52(m,2H),7.33(s,1H),7.24–7.26(m,1H),4.71(d,J＝6Hz,2H),2.64(s,3H),2.39(s,3H).
实施例十五、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-5-甲基-4-(吡啶-2-基)-1-氢-吡唑-1-酰胺【编号为REX-N-19】的制备
Figure PCTCN2016108963-appb-000029
实施例十五的制备方法参照实施例七，其中步骤一种的中间体REX-N-INT-3被中间体REX-N-INT-4代替，其余方法相同，最终制得化合物REX-N-19(65mg,14.5％)。
MSm/z[ESI]：399.4[M+1]。
1H-NMR((400MHz,CDCl3),δ＝8.55–8.59(m,3H),7.79–7.81(m,1H),7.71– 7.74(m,1H),7.69(s,1H),7.47–7.50(m,2H),7.32(s,1H),7.18–7.28(m,2H),4.65(d,J＝6Hz,2H),2.63(s,3H),2.56(s,3H),2.37(s,3H).
实施例十六、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(6-甲基吡啶-2-基)-1-氢-吡唑-1-酰胺【编号为REX-N-20】的制备
Figure PCTCN2016108963-appb-000030
实施例十六的制备方法参照实施例七，其中步骤一种的邻溴吡啶被2-溴-6-甲基-吡啶代替，其余方法相同，最终制得化合物REX-N-20(50mg,11.5％)。
MSm/z[ESI]：399.4[M+1]。
1H-NMR((400MHz,DMSO-d6),δ＝9.33(t,J＝6Hz,1H),8.85(s,1H),8.52–8.53(m,2H),8.36(s,1H),7.69–7.70(m,1H),7.41(s,1H),7.34–7.36(m,1H),7.13–7.16(m,1H),4.53(d,J＝6Hz,2H),2.54(s,3H),2.50(s,3H),2.34(s,3H).
实施例十七、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(5-甲基吡啶-3-基)-1-氢-吡唑-1-酰胺【编号为REX-N-21】的制备
Figure PCTCN2016108963-appb-000031
实施例十七的制备方法参照实施例七，其中步骤一种的邻溴吡啶被3-溴-5-甲基-吡啶代替，其余方法相同，最终制得化合物REX-N-21(10mg,10.5％)。
MSm/z[ESI]：399.4[M+1]。
1H-NMR((400MHz,CDCl3),δ＝9.36–8.62(m,3H),8.53(s,1H),8.38(s,1H), 7.92(s,1H),7.66(s,1H),7.59–7.62(m,2H),7.32(s,1H),7.23–7.24(m,1H),4.68(d,J＝6Hz,2H),2.63(s,3H),2.39(s,3H),2.37(s,3H).
实施例十八、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(2-甲基吡啶-4-基)-1-氢-吡唑-1-酰胺【编号为REX-N-22】的制备
Figure PCTCN2016108963-appb-000032
实施例十八的制备方法参照实施例七，其中步骤一种的邻溴吡啶被2-甲基-4-溴-吡啶代替，其余方法相同，最终制得化合物REX-N-22(40mg,10.3％)。
MSm/z[ESI]：399.4[M+1]。
1H-NMR((400MHz,DMSO-d6),δ＝9.33(t,J＝6Hz,1H),8.85(s,1H),8.52–8.53(m,2H),8.36(s,1H),7.69–7.70(m,2H),7.41(s,1H),7.34–7.36(m,1H),7.13–7.16(m,1H),4.53(d,J＝6Hz,2H),2.54(s,3H),2.50(s,3H),2.34(s,3H).
实施例十九、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氰基苯基)-1-氢-吡唑-1-酰胺【编号为REX-N-23】的制备
Figure PCTCN2016108963-appb-000033
实施例十九的制备方法参照实施例七，其中步骤一种的邻溴吡啶被间溴苯乙腈代替，其余方法相同，最终制得化合物REX-N-23(100mg,20.3％)。
MSm/z[ESI]：409.4[M+1]。
1H-NMR((400MHz,CDCl3),δ＝8.56–8.60(m,3H),8.27(d,J＝0.8Hz,2H),7.60 –7.67(m,4H),6.44–6.45(m,2H),4.68(d,J＝6Hz,2H),2.64(s,3H),2.37(s,3H).
实施例二十、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(嘧啶-5-基)-1-氢-吡唑-1-酰胺【编号为REX-N-24】的制备
Figure PCTCN2016108963-appb-000034
实施例二十的制备方法参照实施例七，其中步骤一种的邻溴吡啶被5-溴-嘧啶乙腈代替，其余方法相同，最终制得化合物REX-N-24(80mg,15.8％)。
MSm/z[ESI]：386.1[M+1]。
1H-NMR((400MHz,CDCl3),δ＝9.16(s,1H),8.91(s,2H),8.56–8.59(m,3H),7.95(s,2H),7.63–7.65(m,2H),7.31(s,1H),7.22–7.23(m,1H),4.69(d,J＝6Hz,2H),2.63(s,3H),2.38(s,3H).
实施例二十一、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(嘧啶-2-基)-1-氢-吡唑-1-酰胺【编号为REX-N-25】的制备
Figure PCTCN2016108963-appb-000035
实施例二十一的制备方法参照实施例七，其中步骤一种的邻溴吡啶被5-溴-嘧啶乙腈代替，其余方法相同，最终制得化合物REX-N-25(100mg,18.8％)。
MSm/z[ESI]：386.1[M+1]。
1H-NMR((400MHz,CDCl3),δ＝8.96(s,1H),8.55(d,J＝0.8Hz,2H),8.55–8.57(m,2H),8.30(s,1H),7.66(s,1H),7.32(s,1H),7.22–7.23(m,1H),7.14 –7.16(m,1H),4.69(d,J＝6Hz,2H),2.63(s,3H),2.37(s,3H).
实施例二十二、N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(哒嗪-3-基)-1-氢-吡唑-1-酰胺【编号为REX-N-26】的制备
Figure PCTCN2016108963-appb-000036
实施例二十二的制备方法参照实施例七，其中步骤一种的邻溴吡啶被5-溴-嘧啶乙腈代替，其余方法相同，最终制得化合物REX-N-26(100mg,18.5％)。
MSm/z[ESI]：386.1[M+1]。
1H-NMR((400MHz,CDCl3),δ＝9.11(s,1H),8.58(s,1H),8.58(d,J＝0.8Hz,2H),8.35(s,1H),7.66(s,1H),7.50–7.52(m,1H),7.32(s,1H),7.22–7.24(m,1H),4.69(d,J＝6Hz,2H),2.63(s,3H),2.38(s,3H).
实施例二十三、斑马鱼表型筛选实验
斑马鱼是一种脊椎动物，与人类基因组同源性高达85％，与人类有近似的组织器官功能和信号传导通路，并且具有产卵量高、发育迅速、胚胎透明等独特优势，使得斑马鱼成功地应用于人类疾病研究和活体药物高通量筛选。Wnt信号通路广泛存在于无脊椎动物和脊椎动物，是一类在进化过程中高度保守的信号通路，参与生物体内调控有关细胞生长、凋亡、自我更新和生存基因的表达，维持生物体中正常的胚胎发育和组织修复再生等生理功能(Wolfram Goessling,Trista E.North,Sabine Loewer,et al.(2009).Genetic Interaction of PGE2 and Wnt Signaling Regulates Developmental Specification of Stem Cells and Regeneration.Cell,136(6):1136-1147.)。Wnt信号通路失调与癌症发生也密切相关，例如80％～90％的结直肠癌中都含有影响Wnt信号通路基因Adenomatous Polyposis Coli(APC)功能突变(Hans Clevers,Roel Nusse.(2012).Wnt/β-Catenin Signaling and Disease.Cell,149(6):1192-1205.)。
研究表明，斑马鱼在体轴发育和再生修复方面的表型与Wnt信号通路密切相关(Xiaolei Wang,Jesung Moon,Michael E.Dodge,et al.(2013).The Development of Highly Potent Inhibitors for Porcupine.Journal of Medicinal Chemistry,56,2700-2007.)。
因此，利用Wnt信号通路的在不同物种间的保守性以及斑马鱼表型筛选方面的优势，我们考察化合物对正常AB型斑马鱼体轴发育和再生修复的影响，来探讨化合物在体内抗Wnt信号通路活性的强弱。
实验(一)化合物对AB型斑马鱼体轴发育抑制试验
方法：选取3hpf(hours post fertilization)的AB型斑马鱼鱼卵进行体轴发育表型试验，按受试终浓度在100μM～0.001μM范围内给药组以及溶剂对照组进行随机分组，每组20个AB鱼卵加至6孔板内，每孔3mL鱼水，DMSO浓度≤1％(v/v)。加药处理后的AB鱼卵至于28.5℃生化培养箱孵育至48hpf时进行图像采集，利用NIS-Elements D 3.1软件分析测量各组幼鱼体轴长度px值(pixels)。根据不同浓度化合物对各组幼鱼体轴发育生长的抑制率，运用GraphPad Prism 6.0进行非线性拟合计算各化合物在AB型斑马鱼体轴发育上的IC50值。
Figure PCTCN2016108963-appb-000037
结果：本发明实施例制备的REX-N-1等一系列化合物，对AB型斑马鱼体轴发育抑制的活性测定结果见表一；REX-N-1处理AB型斑马鱼48hpf后体轴发育抑制状态见图1，量效关系(mean±sd)见图2。
表一实施例化合物对AB型斑马鱼体轴发育抑制的活性测定
Compounds IC50(μM)
LGK-974 0.583
REX-N-1 0.063
REX-N-5 0.039
REX-N-6 0.366
REX-N-2 1.768
REX-N-11 2.393
REX-N-9 0.015
REX-N-10 >10
REX-N-7 0.491
REX-N-8 0.227
REX-N-12 0.274
REX-N-13 1.055
REX-N-14 3.133
REX-N-15 2.589
REX-N-16 4.58
REX-N-17 0.08
REX-N-18 30.596
REX-N-19 2.57
REX-N-20 1.109
REX-N-21 1.421
REX-N-22 >100
REX-N-23 5.588
REX-N-24 22.3
REX-N-25 13.4
REX-N-26 >100
注：IC50为计算50％抑制率浓度
进一步的，本发明对表1中的化合物进行斑马鱼剪尾再生表型的试验复验。
实验(二)化合物对AB型斑马鱼剪尾再生抑制试验
选取3dpf(days post fertilization)的AB型斑马鱼幼鱼进行剪尾鳍造模处理，按受试终浓度在10μM～0.001μM范围内给药组以及溶剂对照组进行随机分组，每组15个幼鱼加至6孔板内，每孔3mL鱼水，DMSO浓度≤1％(v/v)。加药处理后的AB鱼卵至于28.5℃生化培养箱孵育至7dpf时进行图像采集，利用NIS-Elements D 3.1软件分析测量各组幼鱼尾鳍再生长度px值(pixels)。根据不同浓度化合物对各组幼鱼尾鳍再生的抑制率，运用GraphPad Prism 6.0进行非线性拟合计算各化合物在AB型斑马鱼剪尾再生上的IC50值。
Figure PCTCN2016108963-appb-000038
结果：本发明实施例制备的REX-N-1等一系列化合物，对AB型斑马鱼剪尾再生抑制的活性测定结果见表二；REX-N-1处理3dpf剪尾造模AB型斑马鱼至7dpf尾鳍再生抑制见图3，量效关系(mean±sd)见图4。
表二实施例化合物对AB型斑马鱼剪尾再生抑制的活性测定
Compounds IC50(μM)
LGK-974 0.462
REX-N-1 0.018
REX-N-5 0.044
REX-N-6 0.078
REX-N-2 0.397
REX-N-11 1.819
REX-N-9 0.061
REX-N-10 3.195
REX-N-7 0.317
REX-N-8 0.164
REX-N-12 0.053
REX-N-13 0.429
REX-N-14 3.94
REX-N-15 1.795
REX-N-16 1.631
REX-N-17 0.215
REX-N-18 12.382
REX-N-19 1.523
REX-N-20 0.489
REX-N-21 0.778
REX-N-22 >100
REX-N-23 1.984
REX-N-24 11.1
REX-N-25 3.8
REX-N-26 32.2
实施例二十四、Super-Top-Flash(STF)报告基因试验
化合物LGK-974(对照药)、REX-N-1等一系列化合物(实施例)采用稳定转染STF报告基因的HEK293T-STF细胞株同L-Wnt3a分泌细胞株共培养方式测定其对Wnt信号通路的抑制活性，该活性采用IC50这一指标来表示，IC50即STF报告基因表达的Luciferase活性被抑制50％时的化合物的浓度。
本发明实施例制备的REX-N-1等一系列化合物，利用CrownBio公司的Wnt报告基因平台进行测定，测定结果见表三。结果表明，本发明提供的化合物从分子水平上有较好的Wnt信号通路抑制活性。
表三实施例化合物对Wnt通路STF报告基因抑制的活性测定
Compounds IC50(nM)
LGK-974 0.574
REX-N-1 0.123
REX-N-5 0.845
REX-N-6 3.86
REX-N-2 2.5
REX-N-11 14.2
REX-N-9 18.8
REX-N-10 589.38
REX-N-7 4.01
REX-N-8 1.89
REX-N-12 5.99
REX-N-13 6.98
REX-N-14 3.45
REX-N-15 13.87
REX-N-16 6.258
REX-N-17 0.39
REX-N-18 80.549
REX-N-19 8.602
REX-N-20 0.801
REX-N-21 172.181
REX-N-22 3.779
REX-N-23 3.089
REX-N-24 8.534
REX-N-25 16.103
实施例二十五、体外代谢稳定性及体内PK实验
选取本发明实施例制备的化合物REX-N-1进行了人、小鼠体外肝微粒代谢稳定性实验及在BABL/C小鼠中PK测定。
PK测试方法为：18只大鼠分为两组，每组9只。其中一组通过静脉给药，剂量为5mg/kg；一组通过口服给药，剂量为10mg/kg。每一组在给药后0、0.083、0.25、0.5、1、2、4、8、24h分别通过眼眶静脉收集血液。通过眼眶采血的方式将约100μL血液收集到含EDTA的干净EP管中(EDTA的终浓度为0.25mg/mL)。在收集完成后迅速将采血管倒置至少5次，以确保混合均匀，然后放置在冰上。采集到的各时间点血液在4℃，8000rpm离心5分钟以获得血浆。另取1.5mL离心管标记好化合物名称，动物编号，时间点，将血浆转移至该管中。血浆保存在-80℃直至分析。
具体实验结果如下，结果表明REX-N-1有较好的生物利用度和半衰期：
表四实施例化合物体内PK及体外代谢稳定性实验结果
Figure PCTCN2016108963-appb-000039
实施例二十六、在CR3150裸鼠移植瘤中药效实验
将病人结肠肿瘤CR3150于1640培养液中剪成直径为2-4mm的小块接种到裸鼠的皮下，待肿瘤长至500-700mm3后在裸鼠身上进行传代。待***(P4)肿瘤长至500-700mm3时，将肿瘤在1640培养液中剪成直径为2-4mm的小块用于实验裸鼠皮下接种。雌性小鼠右侧皮下接种肿瘤小块。定期观察肿瘤生长情况，待肿瘤生长至平均体积200mm3左右时，根据肿瘤大小随机分三组。分别给予溶剂、阳性对照药LGK974、实施例1化合物REX-N-1，按照5mg/kg，BID给药28天。定期观测肿瘤体积及体重变化。
肿瘤体积计算公式为：长径×短径2/2。
相对肿瘤抑制率TGI(％)：TGI％＝(1-T/C)×100％。
T/C％为相对肿瘤增值率，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)或瘤重(TW)。
计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)。或，
T/C％＝TTW/CTW×100％(TTW：治疗组实验终结时平均瘤重；CTW：溶媒对照组实验终结时平均瘤重)。
结果见表五、图5和图6，该结果表明：溶剂组、LGK974、REX-N-1分别按照5mg/kg，PO，BID给药28天后，体重变化率分别是1.41％，-9.73％和-8.28％。给药28天后，LGK974，表现出显著抗肿瘤作用，TGI为102.62％，T/C为9.04％(P<0.003)；REX-N-1，也表现出显著的抗肿瘤作用，TGI为99.52％，T/C为11.81％(P<0.003)。
总之，在本次CR3150结肠癌PDX模型中，LGK974和REX-N-1在5mg/kg剂量下，给药28天，BID，均表现出显著的抗肿瘤活性。
表五化合物在CR3150模型中的抗肿瘤药效
Figure PCTCN2016108963-appb-000040

Claims (10)

  1. 一种内嵌脲类结构的WNT通路抑制剂，为具有如下结构通式的化合物及其药学上可接受的盐：
    Figure PCTCN2016108963-appb-100001
    其中，环A和环B各自独立地选自芳香环、或含1-2个N原子或O原子的芳香杂环；
    X、Y、Z各自独立地选自CR4、N原子中的一种；
    n选自1～2中的任一整数值；
    R1、R2各自独立地选自氢、卤素、C1-6烷基、C3-6环烷基、C2-6烯基、C2-6炔基、卤代C1- 6烷基、C1-6烷氧基、卤代C1-6烷氧基、羟基、氨基、氰基、酰基、磺基、芳基、杂环基中的一种或几种；
    R3选自取代或未取代的芳基、杂环基；当R3为取代的芳基、杂环基时，芳基、杂环基上的取代基团选自卤素、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、羟基、氰基、氨基、酰基、磺基、杂环基中的一种或几种；
    R4选自氢、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、C3-6环烷基中的一种；
    所述的杂环基为含有一个或多个N、O、S杂原子的3-12元杂环。
  2. 根据权利要求1所述的内嵌脲类结构的WNT通路抑制剂，为具有如下结构通式的化合物及其药学上可接受的盐：
    Figure PCTCN2016108963-appb-100002
    其中，环B选自芳香环、或含1-2个N原子或O原子的芳香杂环；
    X、Y、Z各自独立地选自CR4、N原子中的一种；
    n选自1～2中的任一整数值；
    R1、R2各自独立地选自氢、卤素、C1-6烷基、C3-6环烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、羟基、氨基、酰基、芳基、杂环基中的一种或几种；
    R3选自取代或未取代的芳基、杂环基；当R3为取代的芳基、杂环基时，芳基、杂环基上的取代基团选自卤素、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、羟基、氰基、氨基、酰基、磺基、杂环基中的一种或几种；
    R4选自氢、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、C3-6环烷基中的一种；
    所述的杂环基为含有一个或多个N、O、S杂原子的3-12元杂环。
  3. 根据权利要求2所述的内嵌脲类结构的WNT通路抑制剂，其特征在于：所述的结 构通式(2)中，
    Figure PCTCN2016108963-appb-100003
    选自
    Figure PCTCN2016108963-appb-100004
    Figure PCTCN2016108963-appb-100005
    中的一种；
    X、Y、Z各自独立地选自CR4、N原子中的一种；
    n选自1或2；
    R1、R2为氢、卤素、C1-6烷基、C1-6烷氧基、卤代C1-6烷基、卤代C1-6烷氧基、酰胺基、C1-6烷基酰胺基、杂环基中的一种；
    R3为
    Figure PCTCN2016108963-appb-100006
    Figure PCTCN2016108963-appb-100007
    中的一种；
    R4选自氢、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基中的一种；
    R5选自氢、卤素、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基、酰胺基中的一种；
    所述的杂环基为选自含有一个或多个N、O、S杂原子的3-6元杂环。
  4. 根据权利要求2所述的内嵌脲类结构的WNT通路抑制剂，为具有如下结构通式的化合物及其药学上可接受的盐：
    Figure PCTCN2016108963-appb-100008
    其中，X、Y、Z中任意一个为N原子，则另一个为CR4；或X、Y、Z中任意两个为N原子，则另一个为CR4；
    n选自1或2；
    R1、R2为氢、卤素、C1-6烷基、C1-6烷氧基、卤代C1-6烷基、卤代C1-6烷氧基、酰胺基、C1-6烷基酰胺基、杂环基中的一种；
    R3为
    Figure PCTCN2016108963-appb-100009
    Figure PCTCN2016108963-appb-100010
    中的一种；
    R4选自氢、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基中的一种；
    R5地选自氢、卤素、C1-6烷基、卤代C1-6烷基、C1-6烷氧基、卤代C1-6烷氧基中的一种或几种；
    所述的杂环基为选自含有一个或多个N、O、S杂原子的3-6元杂环。
  5. 根据权利要求1～4任一项所述的内嵌脲类结构的WNT通路抑制剂，其特征在于：所述的芳基为苯基、萘基或蒽基；所述的杂环基为吗啉基、哌啶基、吡啶基、嘧啶基、吡喃基、噻吩基、呋喃基、吡咯基、吡唑基、咪唑基或噻唑基；卤素为氟、氯、溴、碘中的一种。
  6. 一种内嵌脲类结构的WNT通路抑制剂，选自如下特征化合物：
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-苯基-1氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-2-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-苯基-1-氢-1,2,3-三氮唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-3-苯基-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟苯基)-1-氢-吡唑-1-酰胺；
    4-(3-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-3-甲基-4-苯基-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-5-甲基-4-苯基-1-氢-吡唑-1-酰胺；
    4-(2-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺；
    4-(4-氯苯基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡嗪-2-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(2-氟苯基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(4-氟苯基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-3-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(吡啶-4-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟吡啶-2-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(5-氟吡啶-2-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氟吡啶-4-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-5甲基-4-(吡啶-2-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(6-甲基吡啶-2-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(5-甲基吡啶-3-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(2-甲基吡啶-4-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(3-氰基苯基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(嘧啶-5-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(嘧啶-2-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(哒嗪-3-基)-1-氢-吡唑-1-酰胺；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-(嘧啶-4-基)-1-氢-吡唑-1-酰胺；
    4-(4-乙酰哌嗪-1-基)-N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-1-氢-吡唑-1-酰胺；；
    N-((2',3-二甲基-[2,4'-联吡啶]-5-基)甲基)-4-吗啉基-1-氢-吡唑-1-酰胺。
  7. 一种药物组合物，包含如权利要求1定义的化合物或其药学上可接受的盐作为活性成份，以及一种或多种药学上可接受的载体。
  8. 一种如权利要求1定义的化合物或其药学上可接受的盐在制备拮抗Wnt信号通路的药物中的应用。
  9. 如权利要求8所述的用途，其特征在于：用于治疗与异常的Wnt信号活性相关的细胞增殖性疾病、消化***疾病。
  10. 如权利要求9所述的应用，其特征在于：用于治疗癌症，包括非小细胞肺癌、间变性大细胞淋巴瘤、炎性肌纤维母细胞瘤、鼻咽癌、乳腺癌、结直肠癌、弥漫大B细胞淋巴瘤、肝癌、胃癌、食道癌、胰腺癌、卵巢癌、全身组织细胞增生症和神经母细胞瘤。
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