WO2017049992A1 - Egfr激酶抑制剂及其制备方法和应用 - Google Patents

Egfr激酶抑制剂及其制备方法和应用 Download PDF

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WO2017049992A1
WO2017049992A1 PCT/CN2016/089679 CN2016089679W WO2017049992A1 WO 2017049992 A1 WO2017049992 A1 WO 2017049992A1 CN 2016089679 W CN2016089679 W CN 2016089679W WO 2017049992 A1 WO2017049992 A1 WO 2017049992A1
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compound
group
formula
unsubstituted
substituted
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PCT/CN2016/089679
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English (en)
French (fr)
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马大为
俞强
袁钧瑛
夏宏光
蔡东坡
王开亮
张辰
夏尚华
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浙江博生医药有限公司
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Priority to CA2999785A priority Critical patent/CA2999785C/en
Priority to KR1020187011753A priority patent/KR20180096575A/ko
Priority to BR112018005795A priority patent/BR112018005795A2/pt
Priority to RU2018115278A priority patent/RU2018115278A/ru
Priority to EP16847874.1A priority patent/EP3354647B1/en
Priority to JP2018535217A priority patent/JP6752283B2/ja
Priority to US15/763,156 priority patent/US10710979B2/en
Priority to SG11201802470SA priority patent/SG11201802470SA/en
Priority to AU2016327857A priority patent/AU2016327857B2/en
Publication of WO2017049992A1 publication Critical patent/WO2017049992A1/zh
Priority to ZA2018/02141A priority patent/ZA201802141B/en

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    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/48—Two nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • the present invention relates to the field of medicinal chemistry, and in particular, the present invention provides an EGFR kinase inhibitor, and a preparation method and application thereof.
  • EGFR epidermal growth factor receptor
  • HER1 epidermal growth factor receptor
  • Small molecule tyrosine kinase inhibitors block the interaction of ATP with receptor kinase through competitive binding to the tyrosine kinase phosphorylation site in the EGFR intracellular domain, blocking the induction by ligand-receptor binding Autophosphorylation of intracellular kinases and blocking cross-phosphorylation triggered by EGFR receptor dimerization, thereby blocking downstream signaling pathways, is highly efficient and specific.
  • the compound has higher inhibitory activity than the existing tyrosine kinase inhibitory compound.
  • X, Y are each independently selected from the group consisting of N, CH; and X and Y are not simultaneously CH;
  • R is selected from the group consisting of a 5-7 membered heterocyclic ring which is unsubstituted or substituted with 1 to 5 substituents, a C1-C6 alkyl group which is unsubstituted or a 5-7 membered heterocyclic ring substituted with 1 to 5 substituents.
  • a 5- to 7-membered heterocyclic ring having a -NH-unsubstituted or substituted with 1 to 5 substituents, -N(CH 3 )-unsubstituted or substituted with 1 to 5 substituents, wherein Said heterocyclic ring containing at least one hetero atom selected from the group consisting of N, O or S; said substitution means that one or more hydrogen atoms on the group are substituted by an R 1 substituent;
  • R 1 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl, C3-C8 cycloalkyl, unsubstituted or halogenated C1-C6 alkoxy, C3-C8 Cycloalkoxy, -C(O)-R 2 , C2-C8 alkyl acyl group, C2-C8 alkoxy acyl group, -S(O) 2 -R 2 , -SOR 2 ;
  • the R 2 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 ring Alkoxy, -CH 2 -OAc, -NH 2 , -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2 .
  • the X is N and Y is CH.
  • the R is selected from the group consisting of:
  • R 1 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl, C3-C8 cycloalkyl, unsubstituted or halogenated C1-C6 alkoxy, C3-C8 Cycloalkoxy, -C(O)-R 2 , -Boc, -S(O) 2 -R 2 , -CH 2 -OAc;
  • the R 2 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 ring Alkoxy, -CH 2 -OAc, -NH 2 , -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2 .
  • the compound of formula (I) is selected from the group consisting of
  • the compound of formula (I) is selected from the group consisting of
  • R' is selected from the group consisting of a 5-7 membered heterocyclic ring unsubstituted or substituted with 1-5 substituents, a C1-C6 alkyl group-substituted or substituted with 1-5 substituents 5-7 a heterocyclic ring, a 5- to 7-membered heterocyclic ring substituted with -NH-substituted or substituted with 1 to 5 substituents, -N(CH 3 )-unsubstituted or 5-7-membered heterocyclic substituted with 1 to 5 substituents a ring wherein the heterocyclic ring contains at least one hetero atom selected from the group consisting of N, O or S; the substitution means that one or more hydrogen atoms on the group are substituted with an R 1 substituent;
  • R' is a 5-7 membered heterocyclic ring which is unsubstituted or substituted with 1-5 substituents, and the C1-C6 alkyl group is unsubstituted or 1-5 Substituent substituted 5-7 membered heterocyclic ring, -NH-unsubstituted or 5-7 membered heterocyclic ring substituted with 1 to 5 substituents, -N(CH 3 )-unsubstituted or substituted with 1 to 5 substituents a substituted 5-7 membered heterocyclic ring wherein said heterocyclic ring contains at least one hetero atom selected from the group consisting of N, O or S; said substitution means that one or more hydrogen atoms on the group are Substituted by a tert-butoxycarbonyl substituent.
  • reaction is carried out under the catalysis of p-toluenesulfonic acid, trifluoroacetic acid or camphorsulfonic acid, preferably p-toluenesulfonic acid.
  • the inert solvent is selected from the group consisting of NMP, trifluoroethanol, dioxane, or a combination thereof.
  • reaction is carried out at 110-130 °C.
  • reaction time is from 0.1 to 2 h.
  • the method further comprises the steps of:
  • the substituent R 1 is a tert-butoxycarbonyl group.
  • R' is as defined in the second aspect of the invention, and the remaining groups are as defined in the first aspect of the invention.
  • reaction is carried out in the presence of ZnCl 2 or ZnBr 2 .
  • the inert solvent is selected from the group consisting of TEA, DCE, t-BuOH, THF.
  • a pharmaceutical composition comprising: a therapeutically effective amount of a compound of formula (I) according to the first aspect of the invention, or a pharmaceutical thereof, One or more of acceptable salts, tautomers, optical isomers, pharmaceutically acceptable solvates, and optionally pharmaceutically acceptable carriers, excipients, adjuvants, Excipients and / or thinners.
  • the pharmaceutical composition is for the treatment of a disease associated with tyrosine kinase overexpression and/or tyrosine kinase activity.
  • an EGFR inhibitor comprising an inhibitory effective amount of a compound of formula I as described in the first aspect of the invention, or a pharmaceutically acceptable salt thereof, tautomerous One or more of an isomer, an optical isomer, a pharmaceutically acceptable solvate, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant, adjuvant, and/or diluent.
  • the pharmaceutically acceptable salt is a salt of a compound of formula I selected from the group consisting of inorganic acid salts, organic acid salts, alkyl sulfonates, aryl sulfonates, or combinations thereof
  • the salt is selected from the group consisting of hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, propionate, benzoate, horse Acidate, fumarate, succinate, tartrate, citrate, methanesulfonate, ethylsulfonate, besylate, p-toluenesulfonate, or combinations thereof;
  • the pharmaceutically acceptable solvate refers to a solvate of a compound of formula I with a solvent selected from the group consisting of water, ethanol, isopropanol, diethyl ether, acetone, or a combination thereof.
  • the disease associated with epidermal growth factor receptor activity is selected from the group consisting of abnormal cell proliferation, morphological changes, hyperkinesia, angiogenic diseases, tumor growth, tumor metastasis, or a combination thereof.
  • the tumor cell is a H1975 cell.
  • the inhibition has an IC50 value of ⁇ 50 nM.
  • R' is as defined in the second aspect of the invention.
  • R' is as defined in the second aspect of the invention.
  • R' is selected from the group consisting of: Wherein R1 is as defined in the first aspect of the invention.
  • the invention provides a method of treating or preventing a disease associated with EGFR activity and/or expression, the method comprising: administering a therapeutically or prophylactically effective amount of a compound of formula (I) to a subject in need thereof.
  • a method of inhibiting the activity of a tyrosine kinase and/or inhibiting the growth of a tumor cell comprising: administering an inhibitory effective amount of a compound of formula (I) to a subject to be inhibited.
  • Figure 1 shows the effect of the drug of Test Example 2 on the body weight of tumor-bearing nude mice
  • Figure 2 shows the effect of the drug of Test Example 2 on the tumor volume of tumor-bearing nude mice
  • Figure 3 shows the effect of the drug of Test Example 2 on the relative tumor volume of tumor-bearing nude mice
  • Figure 4 is a graph showing the effect of the drug of Test Example 2 on the tumor growth rate of tumor-bearing nude mice;
  • Figure 5 is a graph showing the effect of the drug of Test Example 2 on the tumor weight of tumor-bearing nude mice
  • Fig. 6 shows a photograph of the tumor in Test Example 2.
  • the present inventors After long-term and in-depth research, the present inventors have carried out structural modification of the EGFR inhibitor, and unexpectedly found that the activity of the compound is significantly improved after changing the benzene ring of the piperazine ring in the existing EGFR inhibitor to pyridine or pyrimidine. . Based on the above findings, the inventors completed the present invention.
  • the alkyl group includes a linear or branched alkyl group, and the halogen is F, Cl, Br or I, preferably F or Br.
  • 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-C6 alkyl refers to a straight or branched alkyl group having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, Tert-butyl, or a similar group.
  • the number of carbon atoms of the group when the number of carbon atoms of the group is not limited, it means a group having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms.
  • 5-7 membered heterocyclic ring means a heterocyclic group having 5 to 7 carbon atoms or a hetero atom (selected from N, O, S), and may be a saturated or partially unsaturated cyclic group such as tetrahydrogen. Pyrrolyl, hexahydropyridyl, or the like.
  • C1-C6 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.
  • alkyl acyl refers to a group having the structure "-CO-alkyl", such as methyl acyl, ethyl acyl, propyl acyl, isopropyl acyl, butyl acyl, isobutyl acyl, sec-butyl An acyl group, a t-butyl acyl group, or the like.
  • ester group or "oxy-acyl” are used interchangeably and refer to a group having the structure "-O-CO-alkyl", such as methyl ester, ethyl ester, propyl ester, isopropyl ester. , butyl ester, isobutyl ester, sec-butyl ester, tert-butyl ester, or the like.
  • alkyl ester group or “alkoxy group” are used interchangeably and refer to a group having the structure “alkyl-O-CO-alkyl", such as methyl methyl ester, ethyl methyl ester. , propyl ethyl ester, isopropyl methyl ester, or the like.
  • pharmaceutically acceptable solvate refers to a solvate of the corresponding compound with water, ethanol, isopropanol, diethyl ether, acetone.
  • therapeutically effective amount means a dose which is capable of achieving a desired therapeutic effect in a subject, but which does not cause an excessive adverse effect.
  • the present invention provides a class of compounds of the following formula (I):
  • X, Y are each independently selected from the group consisting of N, CH; and X and Y are not simultaneously CH;
  • R is selected from the group consisting of a 5-7 membered heterocyclic ring which is unsubstituted or substituted with 1 to 5 substituents, a C1-C6 alkyl group which is unsubstituted or a 5-7 membered heterocyclic ring substituted with 1 to 5 substituents.
  • a 5- to 7-membered heterocyclic ring having a -NH-unsubstituted or substituted with 1 to 5 substituents, -N(CH 3 )-unsubstituted or substituted with 1 to 5 substituents, wherein Said heterocyclic ring containing at least one hetero atom selected from the group consisting of N, O or S; said substitution means that one or more hydrogen atoms on the group are substituted by an R 1 substituent;
  • R 1 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl, C3-C8 cycloalkyl, unsubstituted or halogenated C1-C6 alkoxy, C3-C8 Cycloalkoxy, -C(O)-R 2 , C2-C8 alkyl acyl, C2-C8 alkoxy acyl, -S(O) 2 -R 2 , -SOR 2 ;
  • the R 2 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 ring Alkoxy, -CH 2 -OAc, -NH 2 , -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2 .
  • the X is N and Y is CH.
  • the R is selected from the group consisting of:
  • R 1 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl, C3-C8 cycloalkyl, unsubstituted or halogenated C1-C6 alkoxy, C3-C8 cycloalkoxy, -C (O) -R 2, -Boc, -S (O) 2 -R 2, -CH 2 -OAc;
  • the R 2 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 ring Alkoxy, -CH 2 -OAc, -NH 2 , -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2 .
  • the most preferred compounds of the invention include the specific compounds listed above and below in the present invention.
  • the compounds of formula I can be prepared by conventional methods in the art, for example, by reverse synthesis assays, which are within the abilities of those skilled in the art after the present invention discloses the structure of the compounds of formula I.
  • the compounds of the present invention employ a different method from the prior art route in the formation of the mother nucleus.
  • 2,4-dichloro-5-trifluoromethylpyrimidine is first substituted with 3-tert-butoxycarbonylamidoaniline, and then an acryloyl group is attached to the aniline nitrogen, and then The unit reacts to construct a compound skeleton.
  • the inventors have found that by the method according to the invention (2,4-dichloro-5-trifluoromethylpyrimidine first The unit is reacted and then reacted with 3-acrylamidoaniline to provide better selectivity in the synthesis of the compounds of the invention.
  • Step 1 reacting a compound of formula III with a compound of formula 5 in an inert solvent to provide a compound of formula II;
  • Step 2 reacting a compound of formula II with a compound of formula 7 in an inert solvent to provide a compound of formula I';
  • the reaction in the second step, may be carried out by one or more methods selected from the group consisting of:
  • Method 1 In a NMP, a compound of formula II and a compound of formula 7 are reacted in the presence of p-TsOH.H 2 O to provide a compound of formula I'.
  • Method 2 A compound of formula II is reacted with a compound of formula 7 in TFE (trifluoroethanol) in the presence of TFA to provide a compound of formula I'.
  • Method 3 Reaction of a compound of formula II with a compound of formula 7 in the presence of Pd(OAc) 2 , XantPhos and Cs 2 CO 3 in dioxane to give a compound of formula I'.
  • Step 3 the compound of formula I' is deprotected under acidic conditions, and then reacted with the corresponding reagent under basic conditions to obtain formula I;
  • the preparation of the compound of formula I (the starting material is a formula I' from which the tert-butoxycarbonyl group is removed, usually the corresponding amine) is selected from any of the following groups A to G.
  • the pharmaceutical forms of the compounds of the invention may include the compounds themselves, as well as other pharmaceutically acceptable modifications, such as optical isomers, cis and trans isomers, or the like, or pharmaceutically acceptable salts or solvates.
  • the pharmaceutically acceptable salts include, but are not limited to, inorganic acid salts such as hydrochlorides, hydrobromides, nitrates, sulfates, phosphates, etc.; organic acid salts such as Acid salt, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, etc.; alkyl sulfonate, such as methyl sulfonate An ethyl sulfonate or the like; an aryl sulfonate such as a benzenesulfonate or a p-toluenesulfonate.
  • inorganic acid salts such as hydrochlorides, hydrobromides, nitrates, sulfates, phosphates, etc.
  • organic acid salts such as Acid salt, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, etc.
  • the pharmaceutically acceptable solvate includes, but is not limited to, a solvate of the compound with water, ethanol, isopropanol, diethyl ether, acetone, and the like.
  • the compounds of formula I according to the invention have been investigated for their inhibitory activity against the epidermal growth factor receptor (EGFR) and, therefore, the tautomers and racemates of the compounds of the formula I according to the invention or of said derivatives Any one or a mixture of enantiomers, diastereomers, pharmaceutically acceptable salts, pharmaceutically acceptable solvates, for use in the preparation of tyrosine kinase inhibitors It is especially useful for the preparation of EGFR inhibitors.
  • EGFR epidermal growth factor receptor
  • the inhibitor can be applied to a medicament for preventing or treating a disease associated with EGFR. Specifically, it can be applied to the preparation of a medicament for preventing or treating EGFR-related abnormal cell proliferation, morphological changes, hyperkinesia, angiogenesis, and tumor metastasis.
  • the inhibitors are useful in the manufacture of a medicament for the treatment or prevention of tumor growth and metastasis associated with the epidermal growth factor receptor EGFR.
  • the active ingredient of the inhibitor of the present invention is preferably a specific compound shown in the present invention, or a tautomer, a racemate, an enantiomer, a diastereomer, a pharmaceutically acceptable compound. Any one or a mixture of the above acceptable salts, pharmaceutically acceptable solvates.
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the above formula (I), a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer thereof Or one or more of the racemates, and optionally one or more pharmaceutically acceptable carriers, excipients, adjuvants, adjuvants, and/or diluents.
  • the excipient is, for example, an odorant, a flavoring agent, a sweetener or the like.
  • the pharmaceutical composition provided by the present invention preferably contains the active ingredient in a weight ratio of 1-99%, preferably in a ratio of 65 wt% to 99 wt% of the total weight of the compound of the formula I as the active ingredient, and the remainder is pharmaceutically acceptable.
  • the compounds and pharmaceutical compositions provided by the present invention may be in various forms such as tablets, capsules, powders, syrups, solutions, suspensions, aerosols, and the like, and may be present in suitable solid or liquid carriers or diluents. Neutralizes a suitable sterilizing device for injection or drip.
  • compositions of the present invention can be prepared according to conventional methods of preparation in the pharmaceutical arts.
  • the formulation of the formulation comprises from 0.05 to 200 mg of the compound of formula I, preferably from 0.1 mg to 100 mg of the compound of formula I in a unit dosage form of the formulation.
  • the compounds and pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, by routes of administration to the mouth, nose, skin, lungs or the gastrointestinal tract. Most preferably oral. Most preferably, the daily dose is from 0.01 to 200 mg/kg body weight, taken once, or from 0.01 to 100 mg/kg body weight in divided doses. Regardless of the method of administration, the optimal dosage for the individual should be based on the particular treatment. Usually starting with a small dose, gradually increase the dose until the most suitable dose is found.
  • a further aspect of the invention provides an EGFR inhibitor comprising one or more selected from the group consisting of a compound of the above formula I, a pharmaceutically acceptable salt, an isomer thereof or a mixture thereof, and Optionally one or more pharmaceutically acceptable carriers, excipients, adjuvants, adjuvants, and/or diluents.
  • the compounds and compositions of the invention are useful in the treatment and prevention of metabolic diseases associated with EGFR, said diseases Including, but not limited to, diabetes, atherosclerosis, obesity and the like.
  • a further aspect of the invention provides a compound of the above formula I, a pharmaceutically acceptable salt, isomer or mixture thereof, for use in the manufacture of a metabolic system disorder associated with EGFR activity, for example: diabetes Drug use in diseases such as atherosclerosis and obesity.
  • a further aspect of the invention provides a method of treating a metabolic system disorder associated with EGFR activity or expression, such as diabetes, atherosclerosis, obesity, etc., comprising administering to a patient in need of such treatment.
  • a metabolic system disorder associated with EGFR activity or expression such as diabetes, atherosclerosis, obesity, etc.
  • a novel structure of an EGFR inhibitory active compound is provided, which has a lower tumor cell inhibitory concentration than the existing approximate compound, for example, ES071 compared to the control compound 1686 (only in There is a significant increase in the activity of the benzene ring and the pyridine ring. For most tumor cells, the inhibitory concentration is only 1/2 of the former; and the prior art (such as the compound I-4 in CN103269704, the compound in US2012157426) The compounds of the invention also have an extremely unexpected improvement in activity compared to other similar compounds of I-1).
  • the compound of the present invention has physical properties (e.g., water solubility, etc.) different from those of the conventional compound, and thus it is possible to prepare a dosage form different from the conventional compound.
  • a mixed solvent of 2,4-dichloro-5-trifluoromethylpyrimidine (4.2 g, 19.46 mmol) and 100 mL of 1,2-dichloroethane and tert-butanol was added to a 500 mL reaction flask under argon ( Volume ratio 1:1).
  • 1 mol/L of ZnCl 2 diethyl ether solution (42.8 mL, 42.8 mmol) was added dropwise to the ice bath at 0 ° C, and the reaction was stirred at room temperature for 1 hour after the dropwise addition.
  • Compound I' can also be prepared by adding the corresponding intermediate II (10.2 mmol) prepared according to Example 1 to a 100 mL reaction flask, starting material 7 (1.65 g, 10.2 mmol), trifluoroacetic acid (114 mg, 1.0 mmol).
  • Compound I' can also be prepared by adding the corresponding intermediate II (1.0 mmol) prepared according to Example 1 to a 100 mL reaction flask, starting material 7 (0.16 g, 1.0 mmol, 1.0 eq.), palladium acetate (34 mg). , 0.15 mmol, 0.15 eq.), XantPhos (173 mg, 0.3 mmol, 0.30 eq.), cesium carbonate (326 mg, 1.0 mmol, 2.0 eq.) and solvent dioxane (30 mL). Under argon gas protection, the reaction was heated to 100 ° C in an oil bath and stirred for 2 h. The reaction was stopped and cooled to room temperature.
  • This compound can also be used directly for the acylation reaction of the following compound ES-071.
  • Method A The above-obtained amine (1 mmol), triethylamine (202 mg, 2 mmol, 2 eq.) and solvent dichloromethane (40 ml) were sequentially added to a 100 mL reaction flask. A solution of acetyl chloride (1.2 mmol, 1.2 eq.) in 10 ml of dichloromethane was added dropwise with stirring at room temperature, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched by the addition of aq. EtOAc EtOAc.
  • Method B The amine was dissolved in dichloromethane (30 ml), triethylamine (1.0 g, 9.9 mmol) was added at room temperature, acetic anhydride (0.5 g, 4.9 mmol) in 3 ml of dichloromethane was added dropwise, and the mixture was reacted at room temperature for 1 hour. The reaction solution was washed with water (20 mL ⁇ 3), dried over anhydrous sodium sulfate, and evaporated and evaporated.
  • the remaining compound of the formula I can be used according to the fifth embodiment to remove the tert-butoxycarbonyl group under the action of trifluoroacetic acid to prepare the corresponding amine, and the following specific methods are adopted depending on the reactants:
  • Method C can be used to sequentially add the above-obtained amine 8' (1 mmol), triethylamine (202 mg, 2 mmol, 2 eq.) and solvent dichloromethane (40 ml) to a 100 mL reaction flask. ).
  • a solution of sulfonyl chloride R 1 SO 2 Cl (1.2 mmol, 1.2 eq.) in 10 ml of dichloromethane was added dropwise with stirring at room temperature, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched by the addition of a saturated aqueous sodium hydrogen sulfate solution, and then evaporated and evaporated.
  • Method D can be used to sequentially add the above-obtained amine 8' (1 mmol), carboxylic acid (1 mmol, 1 eq.), condensing reagent HATU (1.2 mmol, 1.2 eq.) and solvent DMF to a 100 mL reaction flask. (20ml).
  • a solution of DIEA (2 mmol, 2 eq.) in 5 ml of DMF was added dropwise with stirring at room temperature, and the mixture was stirred at room temperature for 2 hours.
  • the reaction mixture was diluted with ethyl acetate (150m), washed with saturated aqueous sodium chloride (40 mL, 4), dried over anhydrous sodium sulfate, evaporated and evaporated.
  • Method E The above obtained amine 8' (1 mmol), a halogenated hydrocarbon R 1 X (1.5 mmol, 1.5 eq.), DIEA (3 mmol, 3 eq.) can be sequentially added to a 100 mL reaction flask. And solvent DMF (20 ml). The mixture was stirred at 60 ° C for 12 hours, cooled to room temperature, diluted with ethyl acetate (150 ml), washed with saturated aqueous sodium chloride (40 mL ⁇ 4), dried over anhydrous sodium sulfate The final product I.
  • Method F The above obtained amine 8' (1 mmol), halogenated hydrocarbon R 1 X (1.5 mmol, 1.5 eq.), cesium carbonate (3 mmol, 3 eq.) can be sequentially added to a 100 mL reaction flask. And solvent DMF (20 ml). The mixture was stirred at room temperature for 12 hours, diluted with ethyl acetate (150 ml), washed with saturated aqueous sodium chloride (40 mL ⁇ 4), dried over anhydrous sodium sulfate and evaporated.
  • the amine N-methylation reaction can be carried out by the method G: sequentially adding the above obtained amine 8' (1 mmol), 37% aq HCHO (3 mmol), AcOH (1.1 mmol) and solvent methanol (50 ml) to a 100 mL reaction flask. The mixture was heated at 60 ° C for 1 hour, and the reaction liquid was cooled to 0 ° C. Then, 10 ml of dichloromethane was added to the reaction mixture, and sodium cyanoborohydride (1 mmol) was added portionwise, and the mixture was further reacted at 0 ° C for 1 hour to terminate the reaction. It was diluted with 150 ml of dichloromethane, washed with water, dried over anhydrous sodium sulfate, and evaporated, and evaporated, and evaporated,
  • Human lung cancer cells H1975, H522, human hepatoma cells HepG2, human melanoma cells A375 and human epidermal carcinoma cells A431, human colon cancer cells COLO205 were cultured in vitro. After the cells were grown to logarithmic growth phase, the cells were harvested, centrifuged at 1000 rpm for 5 min, and discarded. After clearing, the appropriate amount of the medium was suspended, and the cell concentration was adjusted to 2.5 to 5.5 ⁇ 10 4 /ml (one time was 3.0 to 5.5 ⁇ 10 4 /ml). The cell suspension was inoculated into a 96-well cell culture plate, 100 ⁇ l per well, and placed in a cell culture incubator (37 ° C, 5% CO 2 ) for 24 hours.
  • the drug group was added with 100 ⁇ l of the drug diluted in the cell culture medium per well.
  • the drug was given three replicate wells and the negative control group was 0.5% DMSO medium.
  • 20 ⁇ l of 5 mg/ml of MTT was added to each well and placed at 37 ° C for 3 h.
  • 150 ⁇ l of DMSO was added to each well, shaken at 37 ° C for 5 min, and the absorbance (OD) was measured at 492 nm.
  • IC 50 values were calculated using Prism Graphpad statistical software.
  • the compounds of the present invention have a lower inhibitory concentration for tumor cells.
  • ES071 having only one nitrogen atom in the structure is compared with the control compound 1686 (the former structure is a benzene ring, and the latter is a pyridine ring, Both of them have a significant increase in the activity of the common isosteres in the isostere theory, and the inhibition concentration for most tumor cells is only 1/2 of the former. This shows that the compound of the present invention has achieved expectations based on the prior art. Excellent technical effects outside.
  • Test Example 2 In vivo efficacy test report of ES series compound inhibiting human lung cancer H1975 xenograft
  • BALB/c nude mice female, weighing 17-20 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (license number: SCXK (Beijing) 2012-0001). Breeding environment: SPF animal room, free feeding, 12h light / 12h dark.
  • Human lung cancer H1975 cell culture medium was RPMI 1640 plus 10% FBS, 1% sodium pyruvate, and the culture solution was purchased from GIBCO, serum Biosun.
  • the drug was configured, it was a suspension and precipitated after being placed.
  • H1975 cells in the growth phase were harvested, and the cell concentration was adjusted after digestion, and inoculated into the axilla of nude mice, each inoculated with a volume of 0.1 mL.
  • the diameter of the transplanted tumor was measured with a vernier caliper.
  • 30 tumor-bearing mice were selected and randomly divided into 5 groups according to the tumor size. Each group was administered according to the set experimental protocol. The day of administration was recorded as D1, the negative control group was given the same amount of vehicle, and the long and short diameters of the tumor were measured twice a week, and the body weight of the mice was weighed.
  • the relevant indicators were observed, measured and recorded.
  • the tumor size was measured with a vernier caliper to observe the tumor volume change and growth rate.
  • the tumor-bearing mice were sacrificed and the tumor pieces were dissected and weighed.
  • Efficacy evaluation indicators include: relative tumor proliferation rate and body weight, general status and other related toxicity indicators. At the end of the experiment, the tumor growth curve and body weight curve of each group were plotted.
  • Tumor inhibition rate IR (%) (1-T TW / C TW ) ⁇ 100%, T TW : treatment group TW; C TW : negative control group TW.
  • the subcutaneous tumor volume of the ES series compound and the positive drug group was lower than that of the negative control group, and there were significant differences compared with the negative control group (see Fig. 2, Fig. 3, Fig. 4).
  • the drug is effective, and the anti-tumor effect of ES-072 is better than that of the positive control drug CO-1686.
  • the animals were euthanized, the subcutaneous tumors were dissected and weighed.
  • the ES series compound and the positive drug tumor were significantly smaller than the negative control group, the tumor weight was shown in Fig. 5, and the tumor photograph is shown in Fig. 6.
  • the tumor inhibition rate of CO-1686 was 50.09%, and the inhibition rates of ES-071, ES-072 and ES-075 were 50.93%, 79.42% and 37.27%, respectively.
  • the ES-072 compound inferior tumor inhibition activity in vitro is slightly inferior to ES-071 surprisingly showed a tumor suppressor activity greatly superior to the control compound in the in vivo antitumor experiment, indicating that the compound has Better than the bioavailability of the control compound, it is a promising compound.

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Abstract

本发明提供了一类EGFR激酶抑制剂及其制备方法和应用。具体地，本发明提供了一种如式(I)所示的化合物，其中各基团的定义如说明书中所述。所述的化合物是一类有效的EGFR抑制剂。

Description

EGFR激酶抑制剂及其制备方法和应用 技术领域
本发明涉及药物化学领域，具体地，本发明提供了一种EGFR激酶抑制剂，及其制备方法和应用。
背景技术
EGFR(epidermal growth factor receptor，简称为EGFR、HER1或ErbB-1)是表皮生长因子受体(HER)家族成员之一，该家族成员在细胞生理过程中占据重要位置。EGFR属于酪氨酸激酶型受体，该信号通路在细胞增殖，生长，分化等过程中起重要调节作用，其异常过表达或突变通常会引发肿瘤。
小分子酪氨酸激酶抑制剂通过对EGFR胞内区酪氨酸激酶磷酸化位点的竞争性结合，阻止了ATP与受体激酶的相互作用，阻断由配体-受体结合所诱导的胞内区激酶的自磷酸化，并阻断由EGFR受体二聚所引发的交叉磷酸化，进而阻断下游信号通路，具有高效性和特异性。
使用第一代可逆结合酪氨酸激酶抑制剂如吉非替尼(Gefitinib)和埃罗替尼(Erlotinib)时易发生获得性耐药，Michael J.Eck等发现这种耐药主要是由于ErbB1的gatekeeper残基发生T790M突变引起，该突变通过增强ATP和EGFR酪氨酸激酶的亲和力从而产生耐药性。
Wenjun Zhou等于2009年发现了WZ4002，WZ8040等新型的突变选择性小分子抑制剂：
Figure PCTCN2016089679-appb-000001
Emily J.Hanan等通过环合策略获得了新的激酶抑制剂：
Figure PCTCN2016089679-appb-000002
Kwangho Lee等也在此基础上发展了一系列EGFR激酶抑制剂(US2012157426)，发现I-1具有很好的抑制活性：
Figure PCTCN2016089679-appb-000003
他们进一步设计了化合物I-4(KR20130133202，CN103269704)：
Figure PCTCN2016089679-appb-000004
由于EGFR抑制剂在肿瘤治疗中具有重要应用，因此，本领域迫切需要开发具有酪氨酸激酶抑制活性的药物。
发明内容
本发明的目的是提供一种结构新颖的酪氨酸激酶抑制化合物。所述的化合物与现有的酪氨酸激酶抑制化合物相比，具有更高的抑制活性。
本发明的第一方面，提供了一类如下式(I)所示的化合物：
Figure PCTCN2016089679-appb-000005
其中，
X、Y各自独立地选自下组：N、CH；且X和Y不同时为CH；
R选自下组：未取代或被1-5个取代基取代的5-7元杂环，C1-C6的烷基-未取代或被1-5个取代基取代的5-7元杂环、-NH-未取代或被1-5个取代基取代的5-7元杂环、-N(CH3)-未取代或被1-5个取代基取代的5-7元杂环，其中，所述的杂环含有至少1个选自下组的杂原子：N，O或S；所述的取代指基团上的一个或多个氢原子被R1取代基取代；
其中，所述的R1选自下组：未取代或卤代的C1-C6的烷基、C3-C8的环烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷氧基、-C(O)-R2、C2-C8的烷基酰基、C2-C8的烷氧基酰基、-S(O)2-R2、-SOR2；
所述的R2选自下组：未取代或卤代的C1-C6的烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷基、C3-C8的环烷氧基、-CH2-OAc、-NH2、-NH(C1-C6的烷基)、-N(C1-C6的烷基)2。
在另一优选例中，所述的X为N，Y为CH。
在另一优选例中，所述的R选自下组：
Figure PCTCN2016089679-appb-000006
其中，所述的R1选自下组：未取代或卤代的C1-C6的烷基、C3-C8的环烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷氧基、-C(O)-R2、-Boc、-S(O)2-R2、-CH2-OAc；
所述的R2选自下组：未取代或卤代的C1-C6的烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷基、C3-C8的环烷氧基、-CH2-OAc、-NH2、-NH(C1-C6的烷基)、-N(C1-C6的烷基)2。
在另一优选例中，所述的式(I)化合物选自下组：
Figure PCTCN2016089679-appb-000007
Figure PCTCN2016089679-appb-000008
Figure PCTCN2016089679-appb-000009
在另一优选例中，所述的式(I)化合物选自下组：
Figure PCTCN2016089679-appb-000010
本发明的第二方面，提供了一种如本发明第一方面所述的化合物的制备方法，所述方法包括步骤：
Figure PCTCN2016089679-appb-000011
在惰性溶剂中，用式II化合物和式7化合物反应，得到式I’化合物；
其中，R’选自下组：未取代或被1-5个取代基取代的5-7元杂环，C1-C6的烷基-未取代或被1-5个取代基取代的5-7元杂环、-NH-未取代或被1-5个取代基取代的5-7元杂环、-N(CH3)-未取代或被1-5个取代基取代的5-7元杂环，其中，所述的杂环含有至少1个选自下组的杂原子：N，O或S；所述的取代指基团上的一个或多个氢原子被R1取代基取代；
其余各基团的定义如本发明第一方面中所述。
在另一优选例中，所述的步骤中，R’为未取代或被1-5个取代基取代的5-7元杂环， C1-C6的烷基-未取代或被1-5个取代基取代的5-7元杂环、-NH-未取代或被1-5个取代基取代的5-7元杂环、-N(CH3)-未取代或被1-5个取代基取代的5-7元杂环，其中，所述的杂环含有至少1个选自下组的杂原子：N，O或S；所述的取代指基团上的一个或多个氢原子被叔丁氧羰基取代基取代。
在另一优选例中，所述的反应在对甲苯磺酸、三氟乙酸或樟脑磺酸催化下进行，优选对甲苯磺酸。
在另一优选例中，所述的惰性溶剂选自下组：NMP、三氟乙醇，二氧六环，或其组合。
在另一优选例中，所述的反应在110-130℃下进行。
在另一优选例中，所述的反应时间为0.1-2h。
优选地，所述的方法还包括步骤：
Figure PCTCN2016089679-appb-000012
用式I’化合物制备式I化合物；
其中，R≠R’。
在另一优选例中，所述的R’中，取代基R1为叔丁氧羰基。
在另一优选例中，包括步骤：
Figure PCTCN2016089679-appb-000013
在惰性溶剂中，用式III化合物和式5化合物反应，得到式II化合物；
其中，R’的定义如本发明第二方面中所述，其余各基团的定义如本发明第一方面中所述。
在另一优选例中，所述的反应在ZnCl2或者ZnBr2存在下进行。
在另一优选例中，所述的惰性溶剂选自下组：TEA、DCE、t-BuOH、THF。
本发明的第三方面，提供了一种药物组合物，其特征在于，所述的药物组合物包括：治疗有效量的如本发明第一方面所述的式(I)化合物，或其药学上可接受的盐、互变异构体、光学异构体、药学上可接受的溶剂合物中的一种或多种，以及任选的药学上可接受的载体、赋形剂、佐剂、辅料和/或稀释剂。
在另一优选例中，所述的药物组合物用于治疗与酪氨酸激酶过表达和/或酪氨酸激酶活性过高相关的疾病。
本发明的第四方面，提供了一种EGFR抑制剂，所述的抑制剂含有抑制有效量的如本发明第一方面中所述的式I化合物，或其药学上可接受的盐、互变异构体、光学异构体、药学上可接受的溶剂合物中的一种或多种，以及任选的药学上可接受的载体、赋形剂、佐剂、辅料和/或稀释剂。
在另一优选例中，所述药学上可接受的盐是式I化合物的选自下组的盐：无机酸盐、有机酸盐、烷基磺酸盐、芳基磺酸盐，或其组合；较佳地，所述的盐选自下组：盐酸盐、氢溴酸盐、硝酸盐、硫酸盐、磷酸盐、甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐、甲基磺酸盐、乙基磺酸盐、苯磺酸盐、对甲苯磺酸盐，或其组合；
所述药学上可接受的溶剂合物，是指式I化合物与选自下组的溶剂形成的溶剂合物：水、乙醇、异丙醇、乙醚、丙酮，或其组合。
本发明的第五方面，提供了一种如本发明第一方面所述的式(I)化合物的用途，所述化合物用于选自下组的一个或多个用途：
(a)制备酪氨酸激酶抑制剂；
(b)用于体外非治疗性地抑制酪氨酸激酶的活性；
(c)用于体外非治疗性地抑制肿瘤细胞生长。
在另一优选例中，所述的表皮生长因子受体活性相关的疾病选自下组：细胞异常增殖、形态变化、运动功能亢进、血管新生疾病、肿瘤生长、肿瘤转移疾病，或其组合。
在另一优选例中，所述的肿瘤细胞为H1975细胞。
在另一优选例中，所述抑制的IC50值为≤50nM。
本发明的第六方面，提供了一种如下式II所示的化合物：
Figure PCTCN2016089679-appb-000014
其中，R’的定义如本发明第二方面中所述。
本发明的第七方面，提供了一种如下式III所示的化合物：
Figure PCTCN2016089679-appb-000015
其中，R’的定义如本发明第二方面中所述。
在另一优选例中，所述的R’选自下组：
Figure PCTCN2016089679-appb-000016
其中，R1的定义如本发明第一方面中所述。
本发明的第八方面，提供了一种治疗或预防EGFR活性和/或表达量相关的疾病的方法，所述方法包括：对所需对象施用治疗或预防有效量的式(I)化合物。
本发明的第九方面，提供了一种抑制酪氨酸激酶的活性和/或抑制肿瘤细胞生长的方法，所述方法包括：对待抑制对象施用抑制有效量的式(I)化合物。
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显示了测试实施例2中药物对肿瘤细胞荷瘤裸鼠体重的影响；
图2显示了测试实施例2中药物对荷瘤裸鼠肿瘤体积的影响；
图3显示了测试实施例2中药物对荷瘤裸鼠相对肿瘤体积的影响；
图4显示了测试实施例2中药物对荷瘤裸鼠相对肿瘤增殖率的影响；
图5显示了测试实施例2中药物对荷瘤裸鼠肿瘤重量的影响；
图6显示了测试实施例2中的肿瘤照片。
具体实施方式
本发明人经过长期而深入的研究，对于EGFR抑制剂进行了结构改造，意外地发现，将现有EGFR抑制剂中的哌嗪环相连的苯环改为吡啶或者嘧啶后，化合物活性有明显提高。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，所述的卤素为F、Cl、Br或I，优选为F或Br。
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～C6烷基”指具有1～6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。
特别地，除非特别说明，在本发明中，当未限定基团的碳原子个数时，指具有1-10个碳原子，优选1-4个碳原子的基团。
术语“5～7元杂环”指具有5～7个碳原子或杂原子(选自N、O、S)的杂环基，可以是饱和或部分未饱和的环状基团，例如四氢吡咯基、六氢吡啶基，或类似基团。
术语“C1～C6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“烷基酰基”指具有“-CO-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“酯基”或“氧基-酰基”可以互换使用，指具有“-O-CO-烷基”结构的基团，例如甲酯基、乙酯基、丙酯基、异丙酯基、丁酯基、异丁酯基、仲丁酯基、叔丁酯基、或类似基团。
术语“烷基酯基”或“烷氧基酰基”可以互换使用，均指具有“烷基-O-CO-烷基”结构的基团，例如甲基甲酯基、乙基甲酯基、丙基乙酯基、异丙基甲酯基、或类似基团。
术语“药学上可接受的溶剂合物”指相应的化合物与水、乙醇、异丙醇、乙醚、丙酮的溶剂合物。
在本发明中，“治疗有效量”是指能够在所需的对象中达到所需的治疗效果，但不会造成过度的负面影响的剂量。在本发明的化合物结构被公开后，上述剂量通常可以由本领域技术人员根据实际需要确定。
式I化合物
本发明提供了一类如下式(I)所示的化合物：
Figure PCTCN2016089679-appb-000017
其中，
X、Y各自独立地选自下组：N、CH；且X和Y不同时为CH；
R选自下组：未取代或被1-5个取代基取代的5-7元杂环，C1-C6的烷基-未取代或被1-5个取代基取代的5-7元杂环、-NH-未取代或被1-5个取代基取代的5-7元杂环、-N(CH3)-未取代或被1-5个取代基取代的5-7元杂环，其中，所述的杂环含有至少1个选自下组的杂原子：N，O或S；所述的取代指基团上的一个或多个氢原子被R1取代基取代；
其中，所述的R1选自下组：未取代或卤代的C1-C6的烷基、C3-C8的环烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷氧基、-C(O)-R2、C2-C8的烷基酰基、C2-C8的烷氧基酰基、-S(O)2-R2、-SOR2、；
所述的R2选自下组：未取代或卤代的C1-C6的烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷基、C3-C8的环烷氧基、-CH2-OAc、-NH2、-NH(C1-C6的烷基)、-N(C1-C6的烷基)2。
在另一优选例中，所述的X为N，Y为CH。
在另一优选例中，所述的R选自下组：
Figure PCTCN2016089679-appb-000018
其中，所述的R1选自下组：未取代或卤代的C1-C6的烷基、C3-C8的环烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷氧基、-C(O)-R2、-Boc、-S(O)2-R2、-CH2-OAc；
所述的R2选自下组：未取代或卤代的C1-C6的烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷基、C3-C8的环烷氧基、-CH2-OAc、-NH2、-NH(C1-C6的烷基)、-N(C1-C6的烷基)2。
本发明最优选的化合物包括本发明上文及下文中所列举的各个具体化合物。
式I化合物的合成方法
式I化合物可以通过本领域常规的方法，例如，通过反合成分析法进行制备，在本发明公开了式I化合物的结构后，上述制备方法是在本领域技术人员的能力范围之内的。
值得注意的是，与现有技术的类似化合物的制备方法不同，本发明的化合物在母核骨架的形成过程上采用了与现有技术的路线不同的方法。
在现有技术中，2,4-二氯-5-三氟甲基嘧啶先与3-叔丁氧羰酰胺基苯胺进行取代反应，再在苯胺氮上接上丙烯酰基，再与
Figure PCTCN2016089679-appb-000019
单元进行反应从而构建化合物骨架。
Figure PCTCN2016089679-appb-000020
而发明人发现，通过如本发明所述的方式(2,4-二氯-5-三氟甲基嘧啶先与
Figure PCTCN2016089679-appb-000021
单元进行反应，再与3-丙烯酰胺基苯胺反应，在合成本发明的化合物时具有更好的选择性。
优选的本发明化合物的通用合成方法，如下式所示：
Figure PCTCN2016089679-appb-000022
各步骤的具体实施方式如下：
步骤一、在惰性溶剂中，用式III化合物和式5化合物反应，得到式II化合物；
Figure PCTCN2016089679-appb-000023
步骤二、在惰性溶剂中，用式II化合物和式7化合物反应，得到式I’化合物；
Figure PCTCN2016089679-appb-000024
在另一优选例中，所述的步骤二中，所述的反应可以通过选自下组的一种或多种方法进行：
方法1：在NMP中，在p-TsOH·H2O存在下，用式II化合物和式7化合物反应，得到式I'化合物。
方法2：在TFE(三氟乙醇)中，在TFA存在下，用式II化合物和式7化合物反应，得到式I'化合物。
方法3:在二氧六环中，在Pd(OAc)2、XantPhos和Cs2CO3存在下，用式II化合物和式7化合物反应，得到式I'化合物。
步骤三、式I’化合物在酸性条件下脱除叔丁氧羰基，再在碱性条件下与相应的试剂反应得到式I；
Figure PCTCN2016089679-appb-000025
在另一优选例中，所述的步骤三中，式I化合物的制备(原料为脱除了叔丁氧羰基的式I’，通常为相应的胺)采用选自下组A～G中任一的方法：
A在DCM中，在TEA存在下，用R2COCl与脱除了叔丁氧羰基的式I’化合物反应，得到式I化合物；
B在DCM中，在TEA存在下，用(R2CO)2O与脱除了叔丁氧羰基的式I’化合物反应，得到式I化合物；
C在DCM中，在TEA存在下，用R2SO2Cl与脱除了叔丁氧羰基的式I’化合物反应，得到式I化合物；
D在HATU和DIEA存在下，在DMF或其组合中，用R2COOH与脱除了叔丁氧羰基的式I’化合物反应，得到式I化合物；
E在DIEA存在下，在DMF中，用R1X与脱除了叔丁氧羰基的式I’化合物反应，得到式I化合物；
F在碳酸铯存在下，在DMF中，用R1X与脱除了叔丁氧羰基的式I’化合物反应，得到式I化合物；
G在AcOH存在下，在甲醇中，用HCHO与脱除了叔丁氧羰基的式I’化合物反应，然后加入氰基硼氢化钠继续反应，得到式I化合物；
上述各式中，R2COCl、(R2CO)2O、R2SO2Cl、R2COOH或R1X(X为卤素)为与目标产物式I化合物相应的原料。
药学上可接受的盐和溶剂合物
本发明化合物的药用形式可包括化合物本身，以及药学上可接受的其他变化形式，如光学异构体，顺反异构体等，或者药学上可接受的盐或溶剂合物。
优选地，所述的药学上可接受的盐包括(但并不限于)：无机酸盐，如盐酸盐、氢溴酸盐、硝酸盐、硫酸盐、磷酸盐等；有机酸盐，如甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐等；烷基磺酸盐，如甲基磺酸盐、乙基磺酸盐等；芳基磺酸盐，如苯磺酸盐、对甲苯磺酸盐等。
优选地，所述的药学上可接受的溶剂合物包括(但并不限于)：所述的化合物与水、乙醇、异丙醇、乙醚、丙酮等的溶剂合物。
式I化合物的用途
经研究，本发明所述的式I化合物具有表皮生长因子受体(EGFR)的抑制活性，因此，本发明所述的式I化合物或所述衍生物的互变异构体、外消旋体、对映异构体、非对映异构体、药学上可接受的盐、药学上可接受的溶剂合物中的任意一种或几种的混合物，可应用于制备酪氨酸激酶抑制剂，尤其可应用于制备EGFR抑制剂。
同时，所述的抑制剂可应用于制备预防或治疗与EGFR相关疾病的药物。具体说，可应用于制备预防或治疗与EGFR相关的细胞异常增殖、形态变化、运动功能亢进、血管新生及肿瘤转移疾病的药物。
另外，所述的抑制剂可应用于制备治疗或预防与表皮生长因子受体EGFR相关的肿瘤生长和转移的药物。
本专利所述抑制剂的活性成分优选为本发明中所示的具体化合物，或所示化合物的互变异构体、外消旋体、对映异构体、非对映异构体、药学上可接受的盐、药学上可接受的溶剂合物中的任意一种或几种的混合物。
药物组合物及其用途
本发明的另一方面提供了一种药物组合物，其含有治疗有效量的选自上述通式(Ⅰ)的化合物、其可药用的盐、对映异构体、非对映异构体或外消旋体中的一种或多种，以及任选地，一种或多种可药用的载体、赋形剂、佐剂、辅料和/或稀释剂。所述辅料例如为气味剂、香味剂、甜味剂等。
本发明所提供的药物组合物优选含有重量比为1-99％的活性成份，其优选的比例是，通式I化合物作为活性成分占总重量的65wt％～99wt％，其余部分为药学可接受的载体、稀释液或溶液或盐溶液。
本发明所提供的化合物和药物组合物可以是多种形式，如片剂、胶囊、粉剂、糖浆、溶液状、悬浮液和气雾剂等，并可以存在于适宜的固体或液体的载体或稀释液中和适宜的用于注射或滴注的消毒器具中。
本发明的药物组合物的各种剂型可按照药学领域的常规制备方法制备。其制剂配方的单位计量中包含0.05-200mg通式I化合物，优选地，制剂配方的单位计量中包含0.1mg-100mg通式I化合物。
本发明的化合物和药物组合物可对哺乳动物临床使用，包括人和动物，可以通过口、鼻、皮肤、肺或者胃肠道等的给药途径。最优选为口服。最优选日剂量为0.01-200mg/kg体重，一次性服用，或0.01-100mg/kg体重分次服用。不管用何种服用方法，个人的最佳剂量应依据具体的治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最适合的剂量。
本发明的又一方面提供了一种EGFR抑制剂，其包含选自上述通式I所示的化合物、其可药用的盐、异构体或它们的混合物中的一种或多种，以及任选地一种或多种可药用的载体、赋形剂、佐剂、辅料和/或稀释剂。
本发明的化合物和组合物用于治疗和预防与EGFR相关的代谢系统疾病，所述疾病 包括，但不限于糖尿病、动脉粥样硬化、肥胖症等疾病。
因此，本发明的又一方面提供了上述通式I所示的化合物、其可药用的盐、异构体或它们的混合物在制备用于治疗与EGFR活性相关的代谢系统疾病，例如：糖尿病、动脉粥样硬化、肥胖症等疾病中的药物用途。
本发明的又一个方面提供了一种治疗与EGFR活性或表达量相关的代谢系统疾病，例如：糖尿病、动脉粥样硬化、肥胖症等疾病的方法，其包括向需要该治疗的患者给药选自上述通式I所示的化合物、其可药用的盐、异构体或它们的混合物中的一种或多种。
与现有技术相比，本发明的主要优点包括：
(1)提供了一类结构新颖的EGFR抑制活性化合物，与现有的近似化合物相比，本发明的化合物具有更低的肿瘤细胞抑制浓度，例如，与对照化合物1686相比，ES071(仅在苯环和吡啶环上有差异)的活性具有显著的提升，对于大部分的肿瘤细胞抑制浓度仅为前者的1/2；与现有技术(如CN103269704中的化合物I-4，US2012157426中的化合物I-1)的其他近似化合物相比，本发明的化合物也具有极其意外的活性改善。
(2)与现有化合物相比，本发明的部分化合物对于一些肿瘤株系的选择性提高。
(3)本发明的化合物具有与现有化合物不同的物理性质(如水溶性等)，因此能够制备得到与现有化合物不同的剂型。
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。
制备实施例
实施例一：中间体II的制备
Figure PCTCN2016089679-appb-000026
氩气保护下向500mL反应瓶中加入原料2,4-二氯-5-三氟甲基嘧啶(4.2g,19.46mmol)和100mL 1,2-二氯乙烷和叔丁醇的混合溶剂(体积比1:1)。冰浴0℃下滴加1mol/L的ZnCl2乙醚溶液(42.8mL，42.8mmol)，滴完后室温搅拌反应1小时。继续保持0℃依次滴加另一原料III(6.0g,19.46mmol)的50mL 1,2-二氯乙烷和叔丁醇的混合溶剂(体积比1:1)和三乙胺(2.16g,21.4mmol)。混合物自然升至室温反应10小时。加入200mL二氯甲烷稀释反应液，饱和氯化钠水溶液洗涤(100mL×2),无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析共得产品II及其同分异构体7.61克，收率：80％。其中化合物II的1H NMR(400MHz,CD3OD)：1.46(s,9H),3.40-3.55(m,8H),3.88(s,3H),6.28(d,J＝8.4Hz,1H),7.86(d,J＝8.4Hz,1H),8.49(br s,1H).ESI-MS m/z 488.9(M+H)+.
实施例二：中间体I’(ES-070)的制备
Figure PCTCN2016089679-appb-000027
向100mL反应瓶中依次加入上述原料II及其异构体混合物(5g，10.2mmol)，原料3-丙烯酰胺苯胺(1.65g，10.2mmol)，一水合对甲苯磺酸(0.07g，0.37mmol)和溶剂N-甲基吡咯烷酮(50mL)，氩气保护下，将反应液置于提前预热至120℃的油浴中反应30min，停止反应冷至室温，加入200mL乙酸乙酯稀释，饱和氯化钠水溶液洗涤(100mL×4)，无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得产品I’(ES-070)2.51克，收率：40％。化合物8的1H NMR(400MHz,CD3OD)δ:1.42(s,9H),3.30(br s,4H),3.42(br s,4H),3.81(s,3H),5.70(dd,J＝9.2,2.8Hz,1H),5.90(br s,1H),6.25-6.38(m,2H),7.06(br s,1H),7.26(t,J＝8.0Hz,1H),7.50-7.70(m,2H),7.79(d,J＝8.4Hz,1H),8.13(s,1H).ESI-MS m/z 615.4(M+H)+.
实施例三：中间体I’的制备
化合物I’还可采用如下方法制备：向100mL反应瓶中依次加入根据实施例一制备的相应中间体II(10.2mmol)，原料7(1.65g，10.2mmol)，三氟乙酸(114mg，1.0mmol,0.1eq.)和三氟乙醇(60mL),氩气保护下，升温至80℃油浴中搅拌反应3h，停止反应冷至室温，加入200mL乙酸乙酯稀释，饱和氯化钠水溶液洗涤(50mL×4),无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得产品I’-1，I’-2。
Figure PCTCN2016089679-appb-000028
中间体I’-1，收率50％。1H NMR(500MHz,CD3OD)δ(ppm):1.46(d,J＝10.0Hz,9H),1.86-1.95(m,1H),2.15-2.25(m,1H),3.21-3.24(m,1H),3.39-3.50(m,2H),3.68-3.70(m,1H),3.88(s,3H),4.25-4.35(m,1H),5.74-6.00(m,2H),6.34-6.45(m,2H),7.15-7.25(m,1H),7.30(t,J＝7.5Hz,1H),7.50-7.80(m,3H),8.18(s,1H).ESI-MS m/z 615.6(M+H)+.
Figure PCTCN2016089679-appb-000029
中间体I’-2，收率50％。1H NMR(400MHz,CD3OD)δ(ppm):1.45(s,9H),2.00-2.10(m,2H),2.83(s,3H),3.20-3.35(m,2H),3.48-3.60(m,2H),3.86(s,3H),5.05-5.15(m,1H),5.70-5.90(m,2H),6.30-6.43(m,2H),7.08-7.16(m,1H),7.29(t,J＝7.6Hz,1H),7.55-7.75(m,2H),7.77(d,J＝8.8Hz,1H),8.17(s,1H).ESI-MS m/z 629.7(M+H)+.
实施例四：中间体I’的制备
化合物I’还可采用如下方法制备：向100mL反应瓶中依次加入根据实施例一制备的相应中间体II(1.0mmol)，原料7(0.16g，1.0mmol，1.0eq.)，醋酸钯(34mg，0.15mmol,0.15eq.)，XantPhos(173mg,0.3mmol,0.30eq.),碳酸铯(326mg，1.0mmol，2.0eq.)和溶剂二氧六环(30mL)。氩气保护下，升温至100℃油浴中搅拌反应2h，停止反应冷至室温，加入200mL乙酸乙酯稀释，饱和氯化钠水溶液洗涤(50mL×4),无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得产品I’(ES-073，ES-159)。
Figure PCTCN2016089679-appb-000030
ES-073，收率50％。1H NMR(500MHz,CD3OD)δ(ppm):1.50(s,9H),3.43-3.49(m,4H),3.50-3.58(m,4H),3.84(s,3H),5.77(dd,J＝9.8,1.9Hz,1H),6.32-6.45(m,3H),7.15-7.28(m,2H),7.46(d,J＝7.8Hz,1H),7.78(brs,1H),8.20(s,1H),8.21(s,1H).ESI-MS:m/z 615.6(M+H)+.
Figure PCTCN2016089679-appb-000031
ES-159，收率60％。1H NMR(500MHz,CD3OD)δ(ppm):1.50(s,9H),3.48(br s,4H),3.73(br s,4H),3.88(s,3H),5.77(dd,J＝9.5,2.0Hz,1H),6.32-6.42(m,2H),7.10-7.30(m,2H),7.45(d,J＝7.5Hz,1H),7.60-7.90(m,1H),8.20(s,1H),8.22(s,1H).ESI-MS:m/z616.5(M+H)+.
实施例五：化合物I(ES-071)的制备
Figure PCTCN2016089679-appb-000032
将上述所得产品I’(2g,3.25mmol)溶于20ml二氯甲烷中，室温下滴加4ml三氟乙酸，室温搅拌反应2小时，旋转蒸发移除溶剂后用100ml二氯甲烷稀释，饱和碳酸氢钠水溶液洗涤，饱和氯化钠洗涤，无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得胺ES-069，收率95％，1H NMR(500MHz,CD3OD)δ(ppm):3.04(t,J＝5.5Hz,4H),3.49(br s,4H),3.90(s,3H),5.78(dd,J＝9.5,2.0Hz,1H),6.00(br s,1H),6.34-6.45(m,2H),7.15(br s,1H),7.34(t,J＝8.0Hz,1H),7.60-7.73(m,2H),7.89(d,J＝8.5Hz,1H),8.22(s,1H).ESI-MS m/z 515.4(M+H)+。
该化合物也可直接用于下面合成化合物ES-071的酰化反应。
方法A：向100mL反应瓶中依次加入上述所得胺(1mmol)，三乙胺(202mg,2mmol,2eq.)及溶剂二氯甲烷(40ml)。室温搅拌下滴加乙酰氯(1.2mmol,1.2eq.)的10ml二氯甲烷溶液，滴毕，继续室温搅拌反应2小时。加入饱和碳酸氢钠水溶液淬灭反应，分液，无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得最终产品I(ES-071)。
方法B：将该胺溶于二氯甲烷30ml，室温下加入三乙胺(1.0g,9.9mmol)，滴加乙酸酐(0.5g,4.9mmol)的3ml二氯甲烷，混合物室温反应1小时，反应液水洗(20mL×3)，无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得产品ES-071，1.54克，收率：85％。1H NMR(400MHz,CD3OD)δ(ppm):2.13(s,3H),3.35-3.50(m,4H),3.59-3.66(m,4H),3.87(s,3H),5.75(dd,J＝8.8,2.4Hz,1H),5.95(br s,1H),6.31-6.42(m,2H),7.12(br s,1H),7.31(t,J＝8.0Hz,1H),7.60-7.66(m,2H),7.85(d,J＝8.4Hz,1H),8.19(s,1H).13C NMR(100MHz,DMSO):δ(ppm):21.7,45.3,45.6,53.2,98.2,112.3,116.2,116.7,120.5,127.2,128.8,125.3(q,J＝268Hz),130.1,132.4,135.0,138.9,139.5,154.3,156.2,157.5,161.9,163.6,168.8.ESI-MS m/z 557.6(M+H)+.
实施例六：化合物I的制备
其余式I化合物可根据实施例五在三氟乙酸作用下脱除叔丁氧羰基制备出相应的胺，再根据反应物的不同采用下述具体方法：
当该胺与酰氯反应，可采用实施例五中方法A制备。
当该胺与酸酐反应，可采用实施例五中方法B制备。
当该胺与磺酰氯或者亚磺酰氯反应，可用方法C:向100mL反应瓶中依次加入上述所得胺8’(1mmol)，三乙胺(202mg,2mmol,2eq.)及溶剂二氯甲烷(40ml)。室温搅拌下滴加磺酰氯R1SO2Cl(1.2mmol,1.2eq.)的10ml二氯甲烷溶液，滴毕，继续室温搅拌反应2小时。加入饱和碳酸氢钠水溶液淬灭反应，分液，无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得最终产品I。
该胺与羧酸反应，可用方法D:向100mL反应瓶中依次加入上述所得胺8’(1mmol)，羧酸(1mmol,1eq.)，缩合试剂HATU(1.2mmol,1.2eq.)及溶剂DMF(20ml)。室温搅拌下滴加DIEA(2mmol,2eq.)的5ml DMF溶液，滴毕，室温搅拌反应2小时。加入乙酸乙酯稀释(150m)反应液，饱和氯化钠水溶液洗涤(40mL×4),无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得最终产品I。
该胺与卤代烃反应，可用方法E:向100mL反应瓶中依次加入上述所得胺8’(1mmol)，卤代烃R1X(1.5mmol,1.5eq.)，DIEA(3mmol,3eq.)及溶剂DMF(20ml)。混合物60℃搅拌反应12小时，冷至室温加入乙酸乙酯稀释(150ml)反应液，饱和氯化钠水溶液洗涤(40mL×4),无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得最终产品I。
该胺与卤代烃反应，可用方法F:向100mL反应瓶中依次加入上述所得胺8’(1mmol)，卤代烃R1X(1.5mmol,1.5eq.)，碳酸铯(3mmol,3eq.)及溶剂DMF(20ml)。混合物室温搅拌12小时，加入乙酸乙酯稀释(150ml)反应液，饱和氯化钠水溶液洗涤(40mL×4),无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得最终产品I。
该胺N-甲基化反应，可用方法G:向100mL反应瓶中依次加入上述所得胺8’(1mmol)，37％aq HCHO(3mmol)，AcOH(1.1mmol)和溶剂甲醇(50ml)，混合物加热60℃反应1小时，将反应液冷至0℃后向反应液中加入10ml二氯甲烷，再分批加入氰基硼氢化钠(1mmol)，混合物继续0℃反应1小时结束反应。加入150ml二氯甲烷稀释，水洗，无水硫酸钠干燥，旋转蒸发移除溶剂，快速柱层析得最终甲基化产品I。
各化合物的结构表征数据如下：
For ES-072:
Figure PCTCN2016089679-appb-000033
合成方法按方法G，收率80％。1H NMR(400MHz,CD3OD)δ(ppm):2.32(s,3H),2.51(t,J＝4.8Hz,4H),3.42(br s,4H),3.86(s,3H),5.75(dd,J＝9.2,2.4Hz,1H),5.95(br s,1H),6.31-6.39(m,2H),7.12(d,J＝5.6Hz,1H),7.31(t,J＝8.0Hz,1H),7.55-7.75(m,2H),7.84(d,J＝8.8Hz,1H),8.18(s,1H).13C NMR(100MHz,DMSO)δ(ppm):45.3,46.2,53.1,54.7,98.0,112.0,116.1,116.5,120.6,125.3(q,J＝268Hz),127.3,128.8,129.3,132.3,138.9,139.4,154.6,156.2,157.5,161.9,163.5,172.0.ESI-MS m/z 529.6(M+H)+.
For ES-074:
Figure PCTCN2016089679-appb-000034
合成方法按方法B，收率75％。1H NMR(400MHz,CD3OD)δ(ppm):2.14(s,3H),3.42-3.57(m,4H),3.60-3.72(m,4H),3.82(s,3H),5.75(dd,J＝9.6,1.8Hz,1H),6.26-6.46(m,3H),7.11-7.25(m,2H),7.46(d,J＝7.8Hz,1H),7.74(br s,1H),8.18(s,1H),8.19(s,1H).ESI-MS:m/z 557.5(M+H)+.
For ES-075:
Figure PCTCN2016089679-appb-000035
合成方法按方法G，收率60％。1H NMR(500MHz,CD3OD)δ(ppm):2.57(s,3H),2.77-2.93(m,4H),3.55-3.68(m,4H),3.85(s,3H),5.77(dd,J＝9.9,1.0Hz,1H),6.32-6.48(m,3H),7.14-7.30(m,2H),7.48(d,J＝8.2Hz,1H),7.76(br s,1H),8.21(s,1H),8.23(s,1H).ESI-MS:m/z 529.3(M+H)+.
For ES-123:
Figure PCTCN2016089679-appb-000036
合成方法按方法B，收率65％。1H NMR(400MHz,CD3OD)δ(ppm):1.87-1.99(m,1H),2.03(d,J＝14.9Hz,3H),2.14-2.31(m,1H),3.35-3.77(m,4H),3.85(d,J＝3.1Hz,3H),4.27-4.40(m,1H),5.67-5.87(m,2H),6.29-6.44(m,2H),7.17(br s,1H),7.27(t,J＝8.0Hz,1H),7.45-7.87(m,3H),8.15(s,1H).ESI-MS:m/z 557.6(M+H)+.
For ES-124:
Figure PCTCN2016089679-appb-000037
合成方法按方法E，收率40％。1H NMR(400MHz,CD3OD)δ(ppm):1.73-1.82(m,1H),2.29-2.38(m,1H),2.72-3.06(m,5H),3.16-3.24(m,1H),3.84(s,3H),4.27-4.38(m,1H),4.54(t,J＝4.8Hz,1H),4.66(t,J＝4.7Hz,1H),5.69-5.80(m,2H),6.29-6.46(m,2H),7.18(br s,1H),7.27(t,J＝7.9Hz,1H),7.47-7.76(m,3H),8.16(s,1H).ESI-MS:m/z 561.5(M+H)+.
For ES-125:
Figure PCTCN2016089679-appb-000038
合成方法按方法D，收率60％。1H NMR(400MHz,CD3OD)δ(ppm):1.87-2.03(m,1H),2.12-2.27(m,1H),3.34-3.80(m,4H),3.84(s,3H),4.00-4.18(m,1H),4.23-4.40(m,1H),4.68-4.84(m,4H),5.63-5.97(m,2H),6.29-6.46(m,2H),7.17(brs,1H),7.27(t,J＝7.9Hz,1H),7.46-7.76(m,3H),8.16(s,1H).ESI-MS:m/z 599.5(M+H)+.
For ES-130:
Figure PCTCN2016089679-appb-000039
合成方法按方法A，收率70％。1H NMR(500MHz,CDCl3)δ(ppm):0.79-0.82(m,2H),1.01-1.03(m,2H),1.75-1.80(m,1H),3.41(br s,2H),3.52(br s,2H),3.75(br s,2H),3.79(br s,2H),3.94(s,3H),5.77(d,J＝10.5Hz,1H),6.01(br s,1H),6.21-6.26(m,1H),6.41-6.47(m,1H),7.21(br s,1H),7.33(t,J＝8.5Hz,1H),7.45-7.90(m,2H),8.15(br s,1H),8.28(s,1H).13C NMR(100MHz,CDCl3)δ(ppm):7.51,11.0,41.7,45.1,45.7,45.9,53.2,97.7,113.7,115.1,116.5,119.0,124.8(q,J＝268Hz),127.9,129.3,131.0,138.0,138.5,152.6,153.0,155.7,157.4,160.6,163.6,172.2.ESI-MS m/z 583.5(M+H)+.
For ES-131:
Figure PCTCN2016089679-appb-000040
合成方法按方法A，收率75％。1H NMR(400MHz,CDCl3)δ(ppm):2.85(s,6H),3.25-3.35(m,4H),3.35-3.45(m,4H),3.90(s,3H),5.74(d,J＝10.8Hz,1H),5.97(br s,1H),6.15-6.25(m,1H),6.38-6.45(m,1H),6.84(br s,1H),7.17(br s,1H),7.27-7.40(m,2H),7.45-7.85(m,3H),8.10(br s,1H),8.25(s,1H).ESI-MS m/z 586.5(M+H)+.
For ES-132:
Figure PCTCN2016089679-appb-000041
合成方法按方法C，收率65％。1H NMR(500MHz,CDCl3)δ(ppm):2.87(s,6H),3.30-3.38(m,4H),3.46-3.54(m,4H),3.94(s,3H),5.80(d,J＝10.5Hz,1H),6.00(br s,1H),6.17-6.30(m,1H),6.42-6.48(m,1H),6.86(br s,1H),7.24(br s,1H),7.30-7.40(m,3H),7.45-7.90(m,2H),8.16(br s,1H),8.29(s,1H).ESI-MS m/z 622.6(M+H)+.
For ES-133:
Figure PCTCN2016089679-appb-000042
合成方法按方法C，收率60％。1H NMR(400MHz,CDCl3)δ(ppm):2.78(d,J＝7.2Hz,3H),3.30-3.36(m,4H),3.46-3.54(m,4H),3.94(s,3H),5.80(d,J＝10.5Hz,1H),6.00(br s,1H),6.17-6.30(m,1H),6.42-6.48(m,1H),6.86(br s,1H),7.24(br s,1H),7.30-7.40(m,3H),7.45-7.90(m,2H),8.16(br s,1H),8.29(s,1H).ESI-MS m/z 608.6(M+H)+.
For ES-134:
Figure PCTCN2016089679-appb-000043
合成方法按方法C，收率45％。1H NMR(500MHz,CDCl3)δ(ppm):0.99-1.03(m,2H),1.19-1.22(m,2H),2.27-2.30(m,1H),3.40-3.41(m,4H),3.50-3.60(m,4H),3.94(s,3H),5.80(d,J＝10.5Hz,1H),6.05(br s,1H),6.18-6.30(m,1H),6.44-6.47(m,1H),6.86(br s,1H),7.23(br s,1H),7.30-7.40(m,3H),7.45-7.90(m,2H),8.17(br s,1H),8.29(s,1H).ESI-MS m/z 619.3(M+H)+.
For ES-135:
Figure PCTCN2016089679-appb-000044
合成方法按方法B，收率75％。1H NMR(400MHz,CDCl3)δ(ppm):3.45-3.55(m,4H),3.68-3.80(m,4H),3.94(s,3H),5.80(d,J＝10.5Hz,1H),6.05(br s,1H),6.18-6.30(m,1H),6.41-6.42(m,1H),6.86(br s,1H),7.19(br s,1H),7.30-7.40(m,3H),7.45-7.90(m,2H),8.17(br s,1H),8.29(s,1H).ESI-MS m/z 611.6(M+H)+.
For ES-136:
Figure PCTCN2016089679-appb-000045
合成方法按方法A，收率80％。1H NMR(500MHz,CDCl3)δ(ppm):1.30(t,J＝7.0Hz,3H),3.40-3.50(m,4H),3.55-3.65(m,4H),3.95(s,3H),4.18(q,J＝7.0Hz,2H),5.80(d,J＝10.5Hz,1H),5.95-6.30(m,2H),6.44-6.47(m,1H),6.86(br s,1H),7.22(br s,1H),7.31-7.40(m,3H),7.45-8.00(m,2H),8.15(br s,1H),8.30(s,1H).ESI-MS m/z 587.5(M+H)+.
For ES-137:
Figure PCTCN2016089679-appb-000046
合成方法按方法A，收率85％。1H NMR(500MHz,CDCl3)δ(ppm):1.19(t,J＝7.0Hz,3H),2.40(q,J＝7.0Hz,2H),3.41(br s,2H),3.47(br s,2H),3.58(br s,2H),3.74(br s,2H),3.95(s,3H),5.80(d,J＝10.5Hz,1H),5.95-6.30(m,2H),6.44-6.47(m,1H),6.87(br s,1H),7.22(br s,1H),7.30-7.40(m,3H),7.45-8.00(m,2H),8.15(br s,1H),8.29(s,1H).13C NMR(100MHz,CDCl3)δ(ppm):8.4,28.7,40.3,44.2,44.7,45.0,52.2,97.1,112.7,116.0,118.0,123.0,123.7,125.1(q,J＝268Hz),126.6,128.1,130.2,136.9,137.9,146.5,152.0,154.3,156.7,159.5,163.6,172.4.ESI-MS m/z 571.6(M+H)+.
For ES-138:
Figure PCTCN2016089679-appb-000047
合成方法按方法A，收率70％。1H NMR(500MHz,CDCl3)δ(ppm):1.08(s,9H),2.32(s,2H),3.41(br s,2H),3.46(br s,2H),3.63(br s,2H),3.76(br s,2H),3.95(s,3H),5.79(d,J＝10.5Hz,1H),5.95-6.30(m,2H),6.44-6.47(m,1H),6.86(br s,1H),7.22(br s,1H),7.30-7.40(m,3H),7.45-8.00(m,2H),8.15(br s,1H),8.29(s,1H).ESI-MS m/z 613.7(M+H)+.
For ES-139:
Figure PCTCN2016089679-appb-000048
合成方法按方法A，收率50％。1H NMR(500MHz,CDCl3)δ(ppm):1.33(s,9H),3.43(br s,4H),3.75-3.78(m,4H),3.95(s,3H),5.79(d,J＝10.5Hz,1H),6.05(br s,1H),6.18-6.28(m,1H),6.43-6.48(m,1H),6.86(br s,1H),7.22(br s,1H),7.30-7.40(m,2H),7.45-7.90(m,2H),8.17(br s,1H),8.30(s,1H).ESI-MS m/z 599.6(M+H)+.
For ES-140:
Figure PCTCN2016089679-appb-000049
合成方法按方法A，收率70％。1H NMR(500MHz,CDCl3)δ(ppm):2.21(s,3H),3.44(br s,2H),3.51(br s,4H),3.74(br s,2H),3.94(s,3H),4.78(s,2H),5.80(d,J＝10.5Hz,1H),6.05(br s,1H),6.18-6.28(m,1H),6.43-6.48(m,1H),6.87(br s,1H),7.22(br s,1H),7.30-7.40(m,3H),7.45-7.90(m,2H),8.18(br s,1H),8.30(s,1H).ESI-MS m/z 615.5(M+H)+.
For ES-141:
Figure PCTCN2016089679-appb-000050
合成方法按方法A，收率50％。1H NMR(500MHz,CDCl3)δ(ppm):1.17(d,J＝7.0Hz,6H),2.80-2.90(m,1H),3.41(br s,2H),3.48(br s,2H),3.64(br s,2H),3.75(br s,2H),3.95(s,3H),5.79(d,J＝10.5Hz,1H),6.05(br s,1H),6.18-6.28(m,1H),6.43-6.48(m,1H),6.87(br s,1H),7.22(br s,1H),7.30-7.40(m,2H),7.45-7.90(m,2H),8.18(br s,1H),8.29(s,1H).ESI-MS m/z 619.6(M+H)+.
For ES-142:
Figure PCTCN2016089679-appb-000051
合成方法按方法C，收率50％。1H NMR(500MHz,CDCl3)δ(ppm):2.82(s,3H),3.32-3.34(m,4H),3.57(br s,4H),3.95(s,3H),5.81(d,J＝10.0Hz,1H),6.05(br s,1H),6.18-6.28(m,1H),6.44-6.48(m,1H),6.87(br s,1H),7.22(br s,1H),7.30-7.40(m,2H),7.45-7.90(m,2H),8.18(br s,1H),8.30(s,1H).
For ES-143:
Figure PCTCN2016089679-appb-000052
合成方法按方法C，收率55％。1H NMR(400MHz,CDCl3)δ(ppm):1.38(t,J＝7.6Hz,3H),2.96(q,J＝7.2Hz,2H),3.35-3.38(m,4H),3.51(br s,4H),3.91(br s,3H),5.78(d,J＝10.0Hz,1H),6.00(br s,1H),6.18-6.28(m,1H),6.41-6.45(m,1H),6.84(br s,1H),7.22(br s,1H),7.30-7.40(m,2H),7.45-7.90(m,2H),8.16(br s,1H),8.27(s,1H).ESI-MS m/z607.4(M+H)+.
For ES-144:
Figure PCTCN2016089679-appb-000053
合成方法按方法C，收率50％。1H NMR(400MHz,CDCl3)δ(ppm):1.05(t,J＝7.6Hz,3H),1.80-1.90(m,2H),2.86-2.90(m,2H),3.34-3.36(m,4H),3.51(br s,4H),3.92(s,3H),5.78(d,J＝10.0Hz,1H),6.00(br s,1H),6.15-6.25(m,1H),6.41-6.45(m,1H),6.84(br s,1H),7.22(br s,1H),7.30-7.40(m,2H),7.45-7.90(m,2H),8.15(br s,1H),8.27(s,1H).ESI-MS m/z 621.6(M+H)+.
For ES-145:
Figure PCTCN2016089679-appb-000054
合成方法按方法C，收率40％。1H NMR(400MHz,CDCl3)δ(ppm):1.35(d,J＝6.4Hz,6H),3.60-3.70(m,1H),3.43-3.47(m,8H),3.92(s,3H),5.78(d,J＝10.0Hz,1H),6.00(br s,1H),6.15-6.25(m,1H),6.41-6.45(m,1H),6.84(br s,1H),7.22(br s,1H),7.30-7.40(m,2H),7.45-7.90(m,2H),8.15(br s,1H),8.27(s,1H).ESI-MS m/z 622.3(M+H)+.
For ES-146:
Figure PCTCN2016089679-appb-000055
合成方法按方法E，收率10％。1H NMR(500MHz,CD3OD)δ(ppm):1.99-2.09(m,1H),2.44-2.55(m,1H),2.87(s,3H),3.18-3.30(m,2H),3.43-3.58(m,2H),3.87(s,3H),4.41-4.50(m,1H),5.78(dd,J＝9.9,2.0Hz,1H),5.87(br s,1H),6.33-6.48(m,2H),7.19(br s,1H),7.31(t,J＝8.0Hz,1H),7.46-7.67(m,2H),7.69(d,J＝10.0Hz,1H),8.19(s,1H).ESI-MS:m/z 529.6(M+H)+.
For ES-147:
Figure PCTCN2016089679-appb-000056
合成方法按方法B，收率75％。1H NMR(500MHz,CD3OD)δ(ppm):2.07(d,J＝8.4Hz,3H),2.09-2.27(m,2H),2.87(d,J＝10.4Hz,3H),3.36-3.63(m,2H),3.66-3.77(m,2H),3.89(d,J＝3.8Hz,3H),5.11-5.30(m,1H),5.77(dd,J＝9.7,2.2Hz,1H),5.88(br s,1H),6.31-6.47(m,2H),7.15(br s,1H),7.31(t,J＝8.1Hz,1H),7.54-7.78(m,2H),7.82(dd,J＝8.5,6.7Hz,1H),8.20(s,1H).ESI-MS:m/z 571.6(M+H)+.
For ES-148:
Figure PCTCN2016089679-appb-000057
合成方法按方法E，收率35％。1H NMR(500MHz,DMSO)δ(ppm):1.82-1.90(m,1H),2.10-2.20(m,1H),2.63-3.02(m,9H),3.89(s,3H),4.54(t,J＝5.0,1H),4.64(t,J＝5.0,1H),5.28-5.39(m,1H),5.77(dd,J＝9.7,2.2Hz,1H),5.82(br s,1H),6.32-6.46(m,2H),7.16(br s,1H),7.30(t,J＝8.0Hz,1H),7.52-7.74(m,2H),7.77(d,J＝8.5Hz,1H),8.19(s,1H).13C NMR(100MHz,CD3OD)δ(ppm):28.1,30.3,52.5,53.7,55.0,55.2,57.0,82.8(d,J＝163.4Hz),96.5,109.9,115.5,115.9,120.0,124.9(q,J＝268.3Hz),126.8,128.3,131.9,135.0,138.5,138.9,154.0,155.7,157.0,161.4,163.1.ESI-MS:m/z 575.5(M+H)+.
For ES-149:
Figure PCTCN2016089679-appb-000058
合成方法按方法D，收率60％。1H NMR(500MHz,CD3OD)δ:2.08-2.22(m,2H),2.84-2.88(m,3H),3.37-3.77(m,4H),3.88(d,J＝2.7Hz,3H),4.09-4.21(m,1H),4.79-4.84(m,2H),4.86-4.90(m,2H),5.12-5.25(m,1H),5.77(dd,J＝9.7,2.1Hz,1H),5.88(br s,1H),6.32-6.46(m,2H),7.15(br s,1H),7.31(t,J＝8.1Hz,1H),7.55-7.77(m,2H),7.81(dd,J＝8.6,4.5Hz,1H),8.20(s,1H).ESI-MS:m/z 613.4(M+H)+.
For ES-150:
Figure PCTCN2016089679-appb-000059
合成方法按方法F，收率45％。1H NMR(500MHz,CD3OD)δ(ppm):2.03-2.12(m,1H),2.20-2.31(m,1H),2.72(s,3H),2.89(s,3H),2.95-3.02(m,1H),3.06-3.25(m,3H),3.90(s,3H),5.40-5.48(m,1H),5.78(dd,J＝9.8,2.1Hz,1H),5.88(br s,1H),6.32-6.47(m,2H),7.16(br s,1H),7.31(t,J＝8.1Hz,1H),7.52-7.76(m,2H),7.82(d,J＝8.5Hz,1H),8.20(s,1H).ESI-MS:m/z 543.5(M+H)+.
For ES-151:
Figure PCTCN2016089679-appb-000060
合成方法按方法A，收率70％。1H NMR(500MHz,CD3OD)δ(ppm):2.00-2.11(m,2H),2.87(s,9H),3.35-3.43(m,1H),3.46-3.59(m,3H),3.88(s,3H),5.02-5.13(m,1H),5.77(dd,J＝9.7,2.2Hz,1H),5.87(br s,1H),6.31-6.46(m,2H),7.15(br s,1H),7.31(t,J＝8.1Hz,1H),7.60(br s,1H),7.71(brs,1H),7.80(d,J＝8.5Hz,1H),8.20(s,1H).ESI-MS:m/z600.5(M+H)+.
For ES-157:
Figure PCTCN2016089679-appb-000061
合成方法按方法B，收率80％。1H NMR(500MHz,CD3OD)δ(ppm):2.15(s,3H),3.36-3.51(m,4H),3.60-3.72(m,4H),5.78(dd,J＝9.6,2.2Hz,1H),6.32-6.47(m,2H),6.57(br s,1H),7.15(br s,1H),7.33(t,J＝8.1Hz,1H),7.60(br s,1H),7.74(br s,1H),7.81(dd,J＝9.0,2.0Hz,1H),8.13(br s,1H),8.23(s,1H).ESI-MS:m/z 527.5(M+H)+.
For ES-158:
Figure PCTCN2016089679-appb-000062
合成方法按方法A，收率80％。1H NMR(500MHz,CD3OD)δ(ppm):2.89(s,6H),3.32-3.35(m,4H),3.39-3.44(m,4H),5.78(dd,J＝9.7,2.1Hz,1H),6.32-6.46(m,2H),6.55(br s,1H),7.16(brs,1H),7.33(t,J＝8.1Hz,1H),7.60(br s,1H),7.75(br s,1H),7.81(dd,J＝9.1,2.7Hz,1H),8.12(br s,1H),8.23(s,1H).ESI-MS:m/z 556.5(M+H)+.
For ES-160:
Figure PCTCN2016089679-appb-000063
合成方法按方法B，收率85％。1H NMR(500MHz,DMSO)δ(ppm):2.05(s,3H),3.50(br s,4H),3.66(br s,2H),3.72(br s,2H),3.78(s,3H),5.74(d,J＝9.5Hz,1H),6.21-6.25(m,1H),6.35-6.45(m,1H),7.00-7.40(m,3H),7.65(br s,1H),8.03(s,1H),8.25(br s,1H),8.50(s,1H),10.00(br s,1H).
For ES-161:
Figure PCTCN2016089679-appb-000064
合成方法按方法G，收率50％。1H NMR(500MHz,CD3OD)δ(ppm):2.47(s,3H),2.66(t,J＝5.0Hz,4H),3.83(br s,4H),3.88(s,3H),5.77(dd,J＝9.5,1.5Hz,1H),6.36-6.43(m,2H),7.10-7.30(m,2H),7.46(d,J＝8.5Hz,1H),7.65(br s,1H),8.20(s,1H),8.23(s,1H).ESI-MS m/z 530.5(M+H)+.
For ES-163:
Figure PCTCN2016089679-appb-000065
合成方法按方法D，收率50％。1H NMR(400MHz,CD3OD)δ(ppm):3.60-3.80(m,8H),3.90(s,3H),4.20-4.30(m,1H),4.55(d,J＝9.2Hz,4H),5.74(dd,J＝10.0,2.0Hz,1H),6.28-6.45(m,2H),7.05-7.30(m,2H),7.42(d,J＝7.6Hz,1H),7.60-7.90(m,1H),8.19(s,1H).ESI-MS m/z 600.1(M+H)+.
For ES-164:
Figure PCTCN2016089679-appb-000066
合成方法按方法A，收率60％。1H NMR(400MHz,CD3OD)δ(ppm):2.88(s,6H),3.26(br s,4H),3.75(br s,4H),3.85(s,3H),5.74(dd,J＝10.0,2.0Hz,1H),6.28-6.45(m,2H),7.05-7.30(m,2H),7.42(d,J＝7.6Hz,1H),7.60-7.90(m,1H),8.19(s,1H).ESI-MS m/z587.6(M+H)+.
活性测试实施例
测试实施例1化合物对6株肿瘤细胞增殖的影响
实验方法：
体外培养人肺癌细胞H1975、H522，人肝癌细胞HepG2，人黑色素瘤细胞A375和人表皮癌细胞A431，人结肠癌细胞COLO205，细胞生长至对数生长期后，收集细胞，1000rpm离心5min，弃上清，适量培养基悬浮，调整细胞浓度至2.5～5.5×104/ml(有一次是3.0～5.5×104/ml)。将细胞悬液接种到96孔细胞培养板中，每孔100μl，放置细胞培养箱(37℃，5％CO2)中培养24h后，用药组每孔加入细胞培养基稀释的药物100μl，每种药物设三个复孔，阴性对照组为含0.5％DMSO培养基。培养箱中培养72h后，每孔加入5mg/ml的MTT 20μl，置37℃放置3h。每孔加入150μlDMSO，于37℃摇床振荡5min，于492nm检测吸光度(OD)。运用Prism Graphpad统计软件计算IC50值。
各化合物的编号和结构如下，其中，1686为对照化合物。
Figure PCTCN2016089679-appb-000067
Figure PCTCN2016089679-appb-000068
测试结果见下表：
Figure PCTCN2016089679-appb-000069
Figure PCTCN2016089679-appb-000070
从上述测试结果中可以看出，本发明的化合物对于肿瘤细胞均具有较低的抑制浓度。其中，与本领域中通常所知的电子等排体理论具有差异的是，与对照化合物1686相比，结构中仅相差一个氮原子的ES071(前者结构中为苯环，后者为吡啶环，两者在电子等排体理论中，属于常见的电子等排体)的活性具有显著的提升，对于大部分的肿瘤细胞抑制浓度仅为前者的1/2。这说明本发明的化合物在现有技术的基础上，取得了预期 之外的优异技术效果。
测试实施例2ES系列化合物抑制人肺癌H1975移植瘤的体内药效试验报告
1.试验目的
评价ES系列化合物对裸鼠皮下移植肺癌H1975肿瘤的抑制作用。
2.材料和方法
2.1材料
2.1.1供试品
ES-071、ES-072(微黄)、ES-075，白色粉末，配制前4℃保存
2.1.2阳性药名称：CO-1686
白色粉末，配制前4℃保存。
2.1.3阴性对照品(溶媒)
22％hydroxypropyl-β「-cyclodextrin，100ml纯水中加入22g的hydroxypropyl-β「-cyclodextrin。
2.1.4实验动物
BALB/c nude小鼠，雌性，体重17-20g，购自北京维通利华实验动物技术有限公司(许可证号码：SCXK(京)2012-0001)。饲养环境：SPF级动物房，自由摄食，12h光照/12h黑暗。
2.1.5细胞株
人肺癌H1975细胞培养液为RPMI 1640加10％FBS，1％丙酮酸钠，培养液购自GIBCO，血清Biosun。
实验方法
1.给药方案：如下表1所示：
表1.动物分组和给药方案
Figure PCTCN2016089679-appb-000071
2.药物配制：如下表2中所示：
表2.药物配制表
Figure PCTCN2016089679-appb-000072
药物配置后均为混悬液，放置后有析出。
3.实验步骤
在无菌条件下，取处于生长增殖期的H1975细胞，消化后调整细胞浓度，接种于裸鼠腋窝下，每只接种体积为0.1mL。用游标卡尺测量移植瘤直径，待肿瘤生长至约400mm3时选 取荷瘤鼠30只，根据肿瘤大小随机分成5组。各组按设定实验方案给药，给药当天记为D1，阴性对照组给予等量溶媒，以后每周测量2次肿瘤长径和短径，同时称量小鼠体重。
疗效评价指标：
于首次给药当天开始观察、测量并记录相关指标，实验中用游标卡尺测量肿瘤尺寸以观察瘤块体积变化和生长速度。实验终点时处死荷瘤小鼠，并解剖分离瘤块称重。
疗效评价指标包括：相对肿瘤增殖率及体重、一般状态等相关毒性指标。实验结束时绘制各组肿瘤生长曲线和体重变化曲线。
肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×a×b2，其中a、b分别表示肿瘤长径和短径。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0。其中V0为给药前测量的肿瘤体积，Vt为给药后每一次测量时的肿瘤体积。
抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下：T/C(％)＝(TRTV/CRTV)×100％，TRTV：治疗组RTV；CRTV：阴性对照组RTV，T/C(％)≤40％，并经统计学处理P<0.05为有效。或者瘤重(tumor weight，TW)低于阴性对照组，并经统计学处理P<0.05为有效。肿瘤的抑制率IR(％)＝(1-TTW/CTW)×100％，TTW：治疗组TW；CTW：阴性对照组TW。
统计方法：
数据以Mean±SD表示，组间比较应用t检验。
4.试验结果
4.1药物对动物体重的影响
从各处理组动物体重变化趋势分析，本发明化合物和阳性药物CO-1686组动物与阴性对照组相比体重无显著性差异(图1)，说明本发明的化合物具有较好的安全性。连续灌胃给药14天后，动物进行安乐死，解剖尸检，大体观察各脏器未发现异常。
4.2药物对动物荷瘤大小的影响
连续灌胃给药14天，ES系列化合物和阳性药物组动物皮下肿瘤体积均小于阴性对照组，与阴性对照组相比，均有显著性差异(见图2、图3、图4)，说明药物有效，而且ES-072的抑瘤效果要优于阳性对照药CO-1686组。
试验终点，即给药第14天，动物行安乐死，解剖皮下肿瘤并称重。ES系列化合物和阳性药物肿瘤显著小于阴性对照组，瘤重见图5，肿瘤照片见图6。CO-1686抑瘤率为50.09％，ES-071、ES-072、ES-075抑瘤率分别为50.93％、79.42％和37.27％。其中，ES-072化合物(体外抑瘤活性稍逊于ES-071)在体内抑瘤实验中令人意外地表现出了大大优于对照化合物的抑瘤活性，这表明该化合物在给药后具有优于对照化合物的生物利用度，是一种极具开发前景的化合物。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一类如下式(I)所示的化合物：
    Figure PCTCN2016089679-appb-100001
    其中，
    X、Y各自独立地选自下组：N、CH；且X和Y不同时为CH；
    R选自下组：未取代或被1-5个取代基取代的5-7元杂环，C1-C6的烷基-未取代或被1-5个取代基取代的5-7元杂环、-NH-未取代或被1-5个取代基取代的5-7元杂环、-N(CH3)-未取代或被1-5个取代基取代的5-7元杂环，其中，所述的杂环含有至少1个选自下组的杂原子：N，O或S；所述的取代指基团上的一个或多个氢原子被R1取代基取代；
    其中，所述的R1选自下组：未取代或卤代的C1-C6的烷基、C3-C8的环烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷氧基、-C(O)-R2、C2-C8的烷基酰基、C2-C8的烷氧基酰基、-S(O)2-R2、-SOR2；
    所述的R2选自下组：未取代或卤代的C1-C6的烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷基、C3-C8的环烷氧基、-CH2-OAc、-NH2、-NH(C1-C6的烷基)、-N(C1-C6的烷基)2。
  2. 如权利要求1所述的化合物，其特征在于，所述的X为N，Y为CH。
  3. 如权利要求1所述的化合物，其特征在于，所述的R选自下组：
    Figure PCTCN2016089679-appb-100002
    Figure PCTCN2016089679-appb-100003
    其中，所述的R1选自下组：未取代或卤代的C1-C6的烷基、C3-C8的环烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷氧基、-C(O)-R2、-Boc、-S(O)2-R2、-CH2-OAc；
    所述的R2选自下组：未取代或卤代的C1-C6的烷基、未取代或卤代的C1-C6的烷氧基、C3-C8的环烷基、C3-C8的环烷氧基、-CH2-OAc、-NH2、-NH(C1-C6的烷基)、-N(C1-C6的烷基)2。
  4. 如权利要求1所述的化合物，其特征在于，所述的式(I)化合物选自下组：
    Figure PCTCN2016089679-appb-100004
    Figure PCTCN2016089679-appb-100005
  5. 如权利要求1所述的化合物，其特征在于，所述的式(I)化合物选自下组：
    Figure PCTCN2016089679-appb-100006
  6. 如权利要求1所述的化合物的制备方法，其特征在于，包括步骤：
    Figure PCTCN2016089679-appb-100007
    在惰性溶剂中，用式II化合物和式7化合物反应，得到式I’化合物；
    其中，R’选自下组：未取代或被1-5个取代基取代的5-7元杂环，C1-C6的烷基-未取代或被1-5个取代基取代的5-7元杂环、-NH-未取代或被1-5个取代基取代的5-7元杂环、-N(CH3)-未取代或被1-5个取代基取代的5-7元杂环，其中，所述的杂环含有至少1个选自下组的杂原子：N，O或S；所述的取代指基团上的一个或多个氢原子被R1取代基取代；
    其余各基团的定义如权利要求1中所述。
  7. 如权利要求6所述的制备方法，其特征在于，包括步骤：
    Figure PCTCN2016089679-appb-100008
    在惰性溶剂中，用式III化合物和式5化合物反应，得到式II化合物；
    其中，R’的定义如权利要求6中所述，其余各基团的定义如权利要求1中所述。
  8. 一种药物组合物，其特征在于，所述的药物组合物包括：治疗有效量的如权利要求1所述的式(I)化合物，或其药学上可接受的盐、互变异构体、光学异构体、药学上可接受的溶剂合物中的一种或多种，以及任选的药学上可接受的载体、赋形剂、佐剂、辅料和/或稀释剂。
  9. 一种EGFR抑制剂，其特征在于，所述的抑制剂含有抑制有效量的如本发明第一方面中所述的式I化合物，或其药学上可接受的盐、互变异构体、光学异构体、药学上可接受的溶剂合物中的一种或多种，以及任选的药学上可接受的载体、赋形剂、佐剂、辅料和/或稀释剂。
  10. 如权利要求1所述的式(I)化合物的用途，其特征在于，用于选自下组的一个或多个用途：
    (a)制备酪氨酸激酶抑制剂；
    (b)用于体外非治疗性地抑制酪氨酸激酶的活性；
    (c)用于体外非治疗性地抑制肿瘤细胞生长。
  11. 一种如下式II所示的化合物：
    Figure PCTCN2016089679-appb-100009
    其中，R’的定义如权利要求6中所述。
PCT/CN2016/089679 2015-09-25 2016-07-11 Egfr激酶抑制剂及其制备方法和应用 WO2017049992A1 (zh)

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