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WO2016011979A1 - 2，4-二取代7H-吡咯并[2，3-d]嘧啶衍生物、其制法与医药上的用途 - Google Patents

2，4-二取代7H-吡咯并[2，3-d]嘧啶衍生物、其制法与医药上的用途 Download PDF

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Publication number
WO2016011979A1
WO2016011979A1 PCT/CN2015/085089 CN2015085089W WO2016011979A1 WO 2016011979 A1 WO2016011979 A1 WO 2016011979A1 CN 2015085089 W CN2015085089 W CN 2015085089W WO 2016011979 A1 WO2016011979 A1 WO 2016011979A1
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alkyl
compound
group
phenyl
formula
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French (fr)
Inventor
兰炯
金云舟
周福生
雷静
闻冲
张志远
何向宇
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Yangtze River Pharmaceutical Group Co Ltd
Shanghai Haiyan Pharmaceutical Technology Co Ltd
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Yangtze River Pharmaceutical Group Co Ltd
Shanghai Haiyan Pharmaceutical Technology Co Ltd
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Priority to JP2017504050A priority Critical patent/JP6457623B2/ja
Priority to US15/329,183 priority patent/US9890168B2/en
Priority to EP15825250.2A priority patent/EP3173412B1/en
Publication of WO2016011979A1 publication Critical patent/WO2016011979A1/zh
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the invention relates to the technical field of medicine, in particular to a 2,4-disubstituted 7H-pyrrolo[2,3-d]pyrimidine derivative, a preparation method thereof and application as an EGFR tyrosine kinase inhibitor, and Pharmaceutical compositions and pharmaceutical compositions prepared.
  • Lung cancer is the world's highest incidence of cancer. In China, the incidence of lung cancer ranks first among all cancers, and it is also the cancer with the highest morbidity and mortality in China.
  • EGFR mutations of which L858R and exon 19 deletion mutations account for more than 90%. These patients are more sensitive to EGFR inhibitors.
  • 50% of patients who developed resistance to first-generation EGFR inhibitors detected the EGFR T790M mutation.
  • the first generation EGFR inhibitors gefitinib and erlotinib were both greater than 3 uM and were essentially inactive.
  • the second-generation irreversible pan-EGFR inhibitor, afatinib has been approved for marketing, and the drug is significantly better than the first-generation EGFR inhibitor in patients with EGFR-mutant lung cancer.
  • the second-generation inhibitor also has a strong wild-type EGFR inhibitory activity, and the inhibitory activity against wild-type EGFR is significantly higher than that of the resistant T790M mutation.
  • the patient's rash and other toxic side effects are serious and the curative effect on drug-resistant patients is poor.
  • a small number of first-generation EGFR inhibitor-resistant patients respond to these drugs.
  • the object of the present invention is to provide a 2,4-disubstituted 7H-pyrrolo[2,3-d]pyrimidine derivative which is not only resistant to EGFR T790M mutation and sensitive (such as L858R mutation or exon 19 deletion).
  • the mutation has a high inhibitory activity, and the inhibitory activity against wild-type EGFR is significantly reduced, has high selective inhibition, and also has low cytotoxicity.
  • the compounds of the invention also exhibit advantageous physical, toxic and/or metabolic characteristics as compared to other known EGFR mutant inhibitors.
  • Ring A is a substituted or unsubstituted C 3-10 heterocyclic group, a substituted or unsubstituted C 6-10 aryl ring or a substituted or unsubstituted C 4-10 cycloalkenyl group;
  • substituted means that 1 to 6 hydrogen atoms on the ring atom are substituted with a substituent selected from the group consisting of hydroxyl, CN, NO 2 , halogen, C 1-3 alkyl, halogenated C 1- 3- alkyl, -CON(C 1-3 alkyl) 2 , -C(O)OC 1-3 alkyl, -OC(O)C 1-3 alkyl, -COC 1-3 alkyl, -CO -phenyl, -SO 2 C 1-3 alkyl, -SO 2 -phenyl, -S(O)C 1-3 alkyl, -S(O)-phenyl, -N(C 1-3 alkane Base) 2 ;
  • X is a covalent bond, or is NH, O or S;
  • R 0 is hydrogen, C 1-3 alkyl, halogenated C 1-3 alkyl, C 3-6 cycloalkyl, halogenated C 3-6 cycloalkyl, -CHO, -COC 1-3 alkane , -CO-phenyl, -SO 2 C 1-3 alkyl, -SO 2 -phenyl;
  • R 1 and R 2 are each independently hydrogen, halogen, C 1-3 alkyl, halogenated C 1-3 alkyl;
  • R 3 is selected from the group consisting of:
  • phenyl group is unsubstituted or substituted with from 1 to 3 substituents selected from the group consisting of halogen, C 1-3 alkyl.
  • the A ring is:
  • the C 3-10 heterocyclic group is pyrazolyl, morpholinyl, aza C 3-7 cycloalkyl, pyrrolopyridinyl, pyrazolopyridine, carbazole, pyrrole, Mercapto, or pyridyl.
  • the C 4-10 cycloalkenyl group is a cyclopentenyl group, a cyclohexenyl group, or a cycloheptenyl group.
  • the "substituted" means that 1 to 3 hydrogen atoms on the ring atom are substituted with a substituent selected from the group consisting of a hydroxyl group, NO 2 , a halogen, a C 1-3 alkyl group, and a halogen. Substituted C 1-3 alkyl, -SO 2 C 1-3 alkyl, -S(O)C 1-3 alkyl.
  • R 3 is selected from the group consisting of
  • R 0 is hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, monofluoro.
  • R 1 and R 2 are each independently hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, monofluoroethyl, Difluoromethyl, trifluoromethyl.
  • the substituted or unsubstituted C 3-10 heterocyclic group is a substituted or unsubstituted 9 to 10 membered bicyclic ring containing 1, 2 or 3 nitrogen atoms.
  • Heteroaryl ring selected from the group consisting of
  • Z 11 is CR 15 or N
  • Z 12 is CR 16 or N
  • Z 21 is CR 26 or N
  • R 12 , R 13 , R 14 , R 15 , R 16 , R 22 , R 23 , R 24 , R 25 , R 26 are each independently hydrogen, hydroxy, CN, NO 2 , halogen, halogenated C 1- 3- alkyl, C 1-3 alkyl, -CON(C 1-3 alkyl) 2 , -N(C 1-3 alkyl) 2 , -C(O)OC 1-3 alkyl, -OC ( O) C 1-3 alkyl, -COC 1-3 alkyl, -CO-phenyl, -SO 2 C 1-3 alkyl, -SO 2 -phenyl, -S(O)C 1-3 alkane a group, -S(O)-phenyl; said alkyl group, phenyl group being unsubstituted or substituted by one to three substituents selected from the group consisting of fluorine, chlorine, methyl;
  • R 11 and R 21 are each independently hydrogen, halogenated C 1-3 alkyl, C 1-3 alkyl, -COC 1-3 alkyl, -CO-phenyl, -SO 2 C 1-3 alkane.
  • Base -SO 2 -phenyl.
  • the phenyl group is unsubstituted or substituted with from 1 to 3 substituents selected from the group consisting of fluorine, chlorine, and methyl.
  • Z 11 is N;
  • Z 12 is CR 16 ;
  • R 12 , R 13 , R 14 , R 16 are each independently hydrogen, halogen, halogenated C 1-3 alkyl, C 1- 3 alkyl;
  • R 11 is hydrogen, halogenated C 1-3 alkyl, C 1-3 alkyl, -COC 1-3 alkyl, -SO 2 C 1-3 alkyl.
  • Z 11 is N;
  • Z 12 is CR 16 ;
  • R 12 , R 13 , R 14 , and R 16 are each independently hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, Isopropyl, monofluoroethyl, difluoromethyl, trifluoromethyl;
  • R 11 is hydrogen, methyl, ethyl, propyl, isopropyl, monofluoroethyl, difluoromethyl, trifluoromethyl Base, -COCH 3 , -SO 2 CH 3 .
  • Z 21 is CR 26 ;
  • R 22 , R 23 , R 24 , R 25 , R 26 are each independently hydrogen, halogen, halogenated C 1-3 alkyl, C 1-3 alkane
  • R 21 is hydrogen, halogenated C 1-3 alkyl, C 1-3 alkyl, -COC 1-3 alkyl, -SO 2 C 1-3 alkyl.
  • Z 21 is CR 26 ;
  • R 22 , R 23 , R 24 , R 25 , R 26 are each independently hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl , monofluoroethyl, difluoromethyl, trifluoromethyl;
  • R 21 is hydrogen, methyl, ethyl, propyl, isopropyl, monofluoroethyl, difluoromethyl, trifluoromethyl, -COCH 3 , -SO 2 CH 3 .
  • the substituted or unsubstituted C 3-10 heterocyclic group is a substituted or unsubstituted 5 to 6 membered monocyclic heterocyclic ring having 1 to 2 nitrogen atoms.
  • Z 31 is CR 34 or N
  • R 32 , R 33 , R 34 , R 35 , R 36 , R 37 and R 38 are each independently hydrogen, hydroxy, CN, NO 2 , halogen, halogenated C 1-3 alkyl, C 1-3 alkane a group, -CON(C 1-3 alkyl) 2 , -N(C 1-3 alkyl) 2 , -C(O)OC 1-3 alkyl, -OC(O)C 1-3 alkyl, -COC 1-3 alkyl, -CO-phenyl, -SO 2 C 1-3 alkyl, -SO 2 -phenyl, -S(O)C 1-3 alkyl, -S(O)-benzene
  • the phenyl group is unsubstituted or substituted with 1-3 substituents selected from the group consisting of fluorine, chlorine, methyl;
  • R 31 is hydrogen, halogenated C 1-3 alkyl, C 1-3 alkyl, -COC 1-3 alkyl, -CO-phenyl, -SO 2 C 1-3 alkyl, -SO 2 - Phenyl; said phenyl group being unsubstituted or substituted with from 1 to 3 substituents selected from the group consisting of fluorine, chlorine, methyl.
  • Z 31 is N;
  • R 32 and R 33 are each independently hydrogen, halogen, halogenated C 1-3 alkyl, C 1-3 alkyl;
  • R 31 is hydrogen, halogenated C 1-3 alkyl, C 1-3 alkyl, -COC 1-3 alkyl, -SO 2 C 1-3 alkyl.
  • Z 31 is N;
  • R 32 and R 33 are each independently hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, monofluoroethyl, difluoromethyl.
  • R 31 is hydrogen, methyl, ethyl, propyl, isopropyl, monofluoroethyl, difluoromethyl, trifluoromethyl, -COCH 3 , -SO 2 CH 3 .
  • R 35 , R 36 , R 37 , and R 38 are each independently hydrogen, halogen, or C 1-3 alkyl.
  • each of R 35 , R 36 , R 37 and R 38 is independently hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl or isopropyl.
  • the substituted or unsubstituted C 3-10 heterocyclic group is a substituted or unsubstituted 4 to 7 membered saturated monoheterocyclic ring containing 1 nitrogen atom. From the next group:
  • R 41 is hydrogen, C 1-3 alkyl, halogenated C 1-3 alkyl, -COC 1-3 alkyl, -CO-phenyl, -SO 2 C 1-3 alkyl, -SO 2 - Phenyl, -S(O)C 1-3 alkyl, -S(O)-phenyl; said phenyl is unsubstituted or substituted with from 1 to 3 substituents selected from the group consisting of fluorine , chlorine, methyl.
  • the compound of formula (I) is a compound of formula (II), formula (III), formula (IV), formula (V), or formula (VI):
  • R 11 , R 12 , R 13 , R 14 , R 0 , R 1 , R 2 , R 3 , Z 11 , Z 12 are as defined in the specification;
  • R 21 , R 22 , R 23 , R 24 , R 25 , R 0 , R 1 , R 2 , R 3 , Z 21 are as defined in the specification;
  • R 31 , R 32 , R 33 , R 0 , R 1 , R 2 , R 3 , Z 31 are as defined in the specification;
  • R 35 , R 36 , R 37 , R 38 , R 0 , R 1 , R 2 , R 3 are as defined in the specification;
  • R 41 , R 0 , R 1 , R 2 , R 3 , X, n 1 , n 2 are as defined in the specification.
  • X is O
  • substituted or unsubstituted C 3-10 heterocyclic group is selected from the group consisting of:
  • substituted or unsubstituted C 6-10 aryl ring is selected from the group consisting of
  • the compound of formula (I) is a compound of formula (VII) or formula (VIII):
  • R 0 , R 1 , R 2 , R 3 are as defined in the specification.
  • R 0 , R 1 , R 2 , R 3 , X, and A rings described in Formula I are each independently the corresponding group of each specific compound in the Examples.
  • the compound of formula (I) comprises a compound prepared in the examples, for example selected from the group consisting of:
  • a second aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), or a exemplified compound thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, according to the first aspect of the invention, or Stereoisomers, or prodrugs, and also include pharmaceutically acceptable carriers.
  • a compound of the invention in general, can be administered in a suitable dosage form with one or more pharmaceutically acceptable carriers.
  • dosage forms are suitable for oral, rectal, topical, intraoral, and other parenteral administration (eg, subcutaneous, intramuscular, intravenous, etc.).
  • dosage forms suitable for oral administration include capsules, tablets, granules, and syrups and the like.
  • the compound of the present invention contained in these preparations may be a solid powder or granule; a solution or suspension in an aqueous or non-aqueous liquid; a water-in-oil or oil-in-water emulsion or the like.
  • the above dosage forms can be prepared from the active compound with one or more carriers or excipients via conventional pharmaceutical methods.
  • the above carriers need to be compatible with the active compound or other excipients.
  • commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like.
  • Carriers for liquid preparations include water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like.
  • the active compound can form a solution or suspension with the above carriers.
  • compositions of the present invention are formulated, quantified, and administered in a manner consistent with medical practice.
  • the "effective amount" of a compound administered is determined by the particular condition being treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.
  • a compound according to the first aspect of the present invention for use in (i) preparation regulation A medicament for EGFR tyrosine kinase activity, or (ii) for the preparation of a medicament for preventing and/or preparing a medicament for treating EGFR.
  • the regulation is up- or down-regulation.
  • the EGFR-related disease is cancer, diabetes, immune system disease, neurodegenerative disease or cardiovascular disease, and disease having acquired resistance during treatment with an EGFR modulator.
  • the cancer is non-small cell lung cancer, head and neck cancer, breast cancer, kidney cancer, pancreatic cancer, cervical cancer, esophageal cancer, pancreatic cancer, prostate cancer, bladder cancer, colorectal cancer, ovarian cancer, stomach cancer, brain.
  • Malignant tumors include gelatinization A stromal cell tumor, etc., or any combination thereof.
  • the acquired resistance is caused by a T790 mutation encoded by EGFR exon 20 or by a T790 mutation comprising a EGFR exon 20 encoding, such as T790M.
  • the non-small cell lung cancer is caused by a mutation in EGFR, including a sensitive mutation (such as a L858R mutation or exon 19 deletion) and a drug resistance mutation (such as an EGFR T790M mutation).
  • a sensitive mutation such as a L858R mutation or exon 19 deletion
  • a drug resistance mutation such as an EGFR T790M mutation
  • an EGFR modulator refers to a small molecule tyrosine kinase inhibitor that targets EGFR, such as gefitinib, erlotinib, ectinib, lapatinib, afatinib, and the like.
  • a fourth aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compound of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof Or a prodrug, and a drug selected from the group consisting of gefitinib, erlotinib, ectinib, lapatinib, XL647, NVP-AEE-788, ARRY-334543, EKB-569, BIBW2992 , HKI272, BMS-690514, CI-1033, vandetanib, PF00299804, WZ4002, cetuximab, trastuzumab, panituzumab, matuzumab, nimotuzumab, Zaluzumab, Pertuzumab, MDX-214, CDX-110, IMC-11F8, Zemab, Her2 Vaccine PX 1041, HSP
  • “Therapeutically effective amount” means an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug thereof, contained in the pharmaceutical composition or the pharmaceutical composition of the present invention It is preferably 0.1 mg to 5 g/kg (body weight).
  • the pharmaceutical composition can be used to treat EGFR-related diseases such as cancer, diabetes, immune system diseases, neurodegenerative diseases or cardiovascular diseases, diseases with acquired resistance during treatment with EGFR modulators.
  • EGFR-related diseases such as cancer, diabetes, immune system diseases, neurodegenerative diseases or cardiovascular diseases, diseases with acquired resistance during treatment with EGFR modulators.
  • the acquired drug-resistant disease is a disease caused by a T790 mutation encoded by EGFR exon 20, or a disease caused by a T790 mutation encoded by EGFR exon 20.
  • the EGFR exon 20 encodes a T790 of T790M.
  • the compound of the formula (I) of the present invention can be used in combination with other drugs in certain diseases to achieve a desired therapeutic effect.
  • An example of a joint application is to treat advanced NSCLC.
  • the method includes the steps of:
  • the method comprises the steps (i') reacting a compound of formula I-c with a compound of formula I-h in an inert solvent to form a compound of formula (I);
  • R 0 , R 1 , R 2 , R 3 , X and A are as described above; and L 1 is a leaving group.
  • the L 1 includes a triflate; chlorine, bromine, iodine; a sulfonate group (e.g., mesylate, tosylate, p-bromobenzenesulfonate, Tosylate or the like; acyloxy group (e.g., acetoxy group, trifluoroacetoxy group, etc.).
  • a sulfonate group e.g., mesylate, tosylate, p-bromobenzenesulfonate, Tosylate or the like
  • acyloxy group e.g., acetoxy group, trifluoroacetoxy group, etc.
  • the reaction in the step (i), is carried out under acidic conditions.
  • step (i) reduction is carried out with a metal such as iron powder, zinc powder or stannous chloride.
  • step (i) hydrogenation is reduced under palladium carbon catalysis.
  • step (ii) is carried out under basic conditions.
  • a compound of the formula I-g is condensed with a carboxylic acid in the presence of a condensing agent to form a compound of the formula I.
  • the step (i') is carried out in the presence of a catalyst, a ligand or a base.
  • the catalyst in the step (i'), is selected from the group consisting of TFA, p-toluenesulfonic acid, Pd 2 (dba) 3 (tris(dibenzylideneacetone) dipalladium), BINAP(( ⁇ )-2,2'-bis-(diphenylphosphino)-1,1'-binaphthyl), or a combination thereof;
  • the ligand includes: Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethylxanthene); and/or
  • the base includes: cesium carbonate.
  • the inventors have unexpectedly discovered a class of selective inhibitors of EGFR mutations through long-term and in-depth studies, and in vitro experiments have shown that they can inhibit EGFR T790M/L858R double mutant enzyme and cell line H1975 at nanomolar concentrations. Proliferation also had a high inhibitory intensity against the EGFR-sensitive mutant cell line HCC827 (exon 19 deletion), while the inhibition of the wild-type EGFR enzyme and cell line A431 was relatively weak. Therefore, such a structure can be used not only for the treatment of EGFR-sensitive mutant cancer, but also for the case of secondary drug resistance in the current EGFR-TKI treatment; and its mutation selectivity is greatly reduced by the inhibition of wild-type EGFR. In addition, this class of compounds has low cytotoxicity in normal cell lines (such as NIH-3T3 cells), which greatly reduces non-specific side effects and is an ideal replacement for second-generation EGFR-TKI.
  • the "C 3-10 heterocyclic group” means a heterocyclic group having 3 to 10 carbon atoms, and the atom constituting the ring contains at least one hetero atom other than carbon, and is selected from N, S, and O.
  • C 1-3 alkyl group means a linear or branched saturated aliphatic hydrocarbon group having 1 to 3 carbon atoms. For example, methyl, ethyl, propyl, isopropyl.
  • C 3-6 cycloalkyl refers to a cycloalkyl group having 3 to 6 carbon atoms.
  • Examples of the cycloalkyl group are a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group and the like.
  • aza C 3-7 cycloalkyl group means a cycloalkyl group having 3 to 7 carbon atoms, and the atoms constituting the ring contain at least one N atom in addition to carbon.
  • C 6-10 aryl is used interchangeably with “C 6-10 aryl ring” and refers to an aromatic hydrocarbon group having 6 to 10 carbon atoms, such as phenyl, naphthyl and the like.
  • Halogen means fluoro, chloro, bromo or iodo.
  • Heteroaryl ring and “heteroaryl” are used interchangeably to mean having 5 to 10 ring atoms, preferably 5, 6, 9 or 10 ring atoms; sharing 6, 10 or 14 ⁇ in a ring array An electron; and a group having 1 to 5 hetero atoms in addition to a carbon atom.
  • heteroatom refers to nitrogen, oxygen or sulfur.
  • partially unsaturated refers to a pi-electron system that contains one or more unsaturated bonds but does not have a complete conjugation.
  • the "5 to 6 membered monocyclic heteroaryl ring having 1 to 2 nitrogen atoms” means a monocyclic heteroaryl group having 5 to 6 ring atoms, and includes, for example, but not limited to, an imidazole ring, a pyrrole ring, Pyrazole ring, pyridine ring, pyridazine ring, pyrimidine ring, pyrazine ring and the like.
  • a 9- to 10-membered bicyclic heteroaryl ring containing 1, 2 or 3 nitrogen atoms means a bicyclic heteroaryl group having 9 to 10 ring atoms, and includes, for example, but not limited to: anthracene ring, isoindole Anthracene ring, quinoline ring, isoquinoline ring, indazole ring, benzimidazole ring, quinazoline ring, quinoxaline ring, porphyrin ring, pyridazine ring.
  • the 5- to 6-membered monocyclic heteroaryl ring or the 9- to 10-membered bicyclic heteroaryl ring is preferably selected from the group consisting of:
  • a 4 to 7-membered saturated monoheterocyclic ring containing 1 nitrogen atom means 4 to 7 ring atoms and 1 carbon atom is bonded to a nitrogen atom.
  • monoheterocycles include, but are not limited to, piperidine rings, tetrahydropyrrole rings, azetidines, azepanes.
  • 6-membered partially unsaturated monocyclic ring refers to a partially unsaturated, all-carbon monocyclic ring containing 6 ring atoms.
  • 6 ring atoms including but not limited to: 1,3-cyclohexadiene, 1,4-cyclohexadiene, cyclohexene, and the like.
  • active substance of the present invention or “active compound of the present invention” means a compound of the formula (I) of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a precursor thereof
  • the drug has significant EGFR T790M/L858R selective inhibitory activity.
  • the "pharmaceutically acceptable salt” includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” means a salt formed with an inorganic or organic acid which retains the bioavailability of the free base without any other side effects.
  • Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, phosphates, and the like; organic acid salts include, but are not limited to, formate, acetate, propionate, glycolate, gluconate , lactate, oxalate, maleate, succinate, fumarate, tartrate, citrate, glutamate, aspartate, benzoate, methanesulfonate , p-toluenesulfonate and salicylate. These salts can be prepared by methods known in the art.
  • “Pharmaceutically acceptable base addition salts” including but not limited to salts of inorganic bases such as sodium, potassium, calcium and magnesium salts, and the like. These include, but are not limited to, salts of organic bases such as ammonium salts, triethylamine salts, lysine salts, arginine salts and the like. These salts can be prepared by methods known in the art.
  • a compound of formula (I) may be present in one or more crystalline forms, and the active compounds of the invention include various crystalline forms and mixtures thereof.
  • solvate refers to a complex of a compound of the invention with a solvent. They either react in a solvent or precipitate out of the solvent or crystallize out. For example, a complex formed with water is referred to as a "hydrate.” Solvates of the compounds of formula (I) are within the scope of the invention.
  • the compounds of formula (I) of the present invention may contain one or more chiral centers and exist in different optically active forms. When the compound contains a chiral center, the compound contains the enantiomer.
  • the invention includes mixtures of the two isomers and isomers, such as racemic mixtures. Enantiomers can be resolved by methods known in the art, such as crystallization and chiral chromatography. When the compound of formula (I) contains more than one chiral center, diastereomers may be present.
  • the present invention includes resolved optically pure specific isomers as well as mixtures of diastereomers. Diastereomers can be resolved by methods known in the art, such as crystallization and preparative chromatography.
  • the invention includes prodrugs of the above compounds.
  • Prodrugs include known amino protecting groups and carboxy protecting groups which are hydrolyzed under physiological conditions or released via an enzymatic reaction to give the parent compound.
  • Specific prodrug preparation methods can be referred to (Saulnier, MG; Frennesson, DB; Deshpande, MS; Hansel, SB and Vysa, DM Bioorg. Med. Chem Lett. 1994, 4, 1985-1990; and Greenwald, RB; Choe, YH; Conover, CD; Shum, K.; Wu, D.; Royzen, MJ Med. Chem. 2000, 43, 475.).
  • the present invention provides a process for the preparation of a compound of formula (I), which compounds can be readily prepared by a variety of synthetic procedures which are well known to those skilled in the art. Exemplary preparation methods for these compounds can be packaged This includes, but is not limited to, the processes described below.
  • each reaction is mostly carried out in an inert solvent at -20 ° C to 150 ° C (or reflux temperature) (preferably, -5 ° C to 100 ° C, or 0-80 ° C). It is carried out for a period of time (e.g., 0.1-72 hours, preferably 0.5-24 hours).
  • the compounds of formula (I) of the present invention can be prepared by the following schemes and exemplary methods described in the Examples and related publications used by those skilled in the art.
  • the steps in the method can be extended or merged as needed during the specific operation.
  • the compound of the formula (I-a) and the compound of the formula (I-b) can form a compound of the formula (I-c) by a substitution reaction (for example, an affinity substitution reaction or the like);
  • the compound of the formula (Ib) is a boronic acid or a boron ester compound of the various A ring, and the compound of the formula (Ia) and the compound of the formula (Ib) can be produced by a coupling reaction (such as Suzuki coupling).
  • a coupling reaction such as Suzuki coupling.
  • L 1 and L 2 in the compound of formula (Ia) are leaving groups including, but not limited to, triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, toluene An acid ester, a p-bromobenzenesulfonate, a p-toluenesulfonate or the like; an acyloxy group such as an acetoxy group, a trifluoroacetoxy group or the like.
  • a compound of formula (Ic) can be reacted with a compound of formula (Id) by a substitution reaction or a coupling reaction to form a compound of formula (Ie), for example, at a temperature, using a suitable catalyst (or containing a suitable ligand) or The base is carried out with a suitable solvent.
  • the catalyst can be, but is not limited to, TFA or p-toluenesulfonic acid.
  • the palladium catalyst used may be, but is not limited to, Pd 2 (dba) 3
  • the ligand used may be, but not limited to, XantPhos (4,5-bis(diphenylphosphine)-9,9 - dimethylxanthene)
  • the base used may be, but not limited to, cesium carbonate.
  • the compound of the formula (I-e) can be subjected to an amine substitution reaction with various amine compounds to form a compound of the formula (I-e), and the synthesis can be conveniently carried out according to different amine compounds by selecting suitable conditions and methods. For example, it can be carried out using a suitable catalyst (or containing a suitable ligand) or a base and a suitable solvent at a certain temperature. This method is a conventional method used by those skilled in the art.
  • the nitro compound If converted to the corresponding amine compound can be reduced under acidic conditions with a metal (may be, but not limited to, iron powder, zinc powder) or stannous chloride; or under palladium carbon catalysis , hydrogenation reduction.
  • a metal may be, but not limited to, iron powder, zinc powder
  • stannous chloride or under palladium carbon catalysis , hydrogenation reduction.
  • the amine compound I-g can be condensed to the amide under basic conditions with the corresponding acid chloride, or condensed with the corresponding carboxylic acid in the presence of a condensing agent to form an amide.
  • the compound of the formula (Ic) can be reacted with a compound of the formula (Ih) by a substitution reaction or a coupling reaction to form a compound of the formula (I), for example, at a certain temperature, using a suitable catalyst (or containing a suitable ligand) or a base and appropriate The solvent is carried out.
  • a suitable catalyst or containing a suitable ligand or a base and appropriate The solvent is carried out.
  • the catalyst can be, but is not limited to, TFA or p-toluenesulfonic acid.
  • the palladium catalyst used may be, but not limited to, Pd 2 (dba) 3 (tris(dibenzylideneacetone) dipalladium), BINAP(( ⁇ )-2,2'-bis-( Diphenylphosphino)-1,1'-binaphthyl).
  • the ligand used may be, but not limited to, Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethyloxaxime), and the base used may be, but not limited to, cesium carbonate.
  • PG in the compound of the formula (I-h1) is an amino-protecting group.
  • Amino protecting groups include, but are not limited to, tert-butoxycarbonyl (Boc); arylmethoxycarbonyl, benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); benzyl (Bn), trityl (Tr), 1,1-di-(4'-methoxyphenyl)methyl; trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like.
  • Methods for protecting and deprotecting can be referred to conventional methods well known in the art.
  • the compound of the present invention has high inhibitory activity against EGFR T790M mutant (particularly EGFR T790M/L858R double mutant) enzyme and cells, and has low inhibitory activity against EGFR wild type (EGFR WT) enzyme and cells, and thus has high Choose inhibition.
  • the compound of the present invention exhibits high selective inhibition of EGFR double mutant enzymes and cells, and also has low non-specific cytotoxicity.
  • the compounds of the present invention also exhibit advantageous physical properties (e.g., higher water solubility) and favorable toxicity characteristics (e.g., lower hERG blocking tendency) than other known EGFR mutation inhibitors, Favorable metabolic characteristics (eg, better pharmacokinetic characteristics, such as bioavailability).
  • LC-MS Agilent 1200 HPLC System/6140 MS LC/MS (manufacturer: Agilent), column Waters X-Bridge, 150 x 4.6 mm, 3.5 ⁇ m.
  • Preparative high performance liquid chromatography Waters PHW007, column XBridge C18, 4.6*150 mm, 3.5 um.
  • ISCO Combiflash-Rf75 or Rf200 automatic column analyzer Use ISCO Combiflash-Rf75 or Rf200 automatic column analyzer, Agela 4g, 12g, 20g, 40g, 80g, 120g disposable silica gel column.
  • Known starting materials can be synthesized by or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc and Dary Chemicals. the company.
  • the solution in the examples is an aqueous solution unless otherwise specified.
  • the progress of the reaction can be monitored by thin layer chromatography (TLC), and the purification of the compound can be carried out by column chromatography.
  • TLC thin layer chromatography
  • the developer system for column chromatography or TLC may be selected from the group consisting of: dichloromethane and methanol systems, n-hexane and ethyl acetate systems, petroleum ether and ethyl acetate systems, and acetone systems, etc., and the volume ratio of the solvent depends on the polarity of the compound. Adjust differently.
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • DIEA N, N-diisopropylethylamine
  • EA ethyl acetate
  • PE petroleum ether
  • BINAP (2R, 3S )-2,2'-bisdiphenylphosphino-1,1'-binaphthyl.
  • NBS N-bromosuccinimide
  • NCS N-chlorosuccinimide
  • Pd 2 (dba) 3 tris(dibenzylideneacetone) dipalladium
  • Pd(dppf)Cl 2 Pd(dppf)Cl 2 ([1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride).
  • room temperature means about 20-30 °C.
  • reaction substrate a1-1 (10.6 g, 58 mmol) was placed in a 500 mL one-mouth reaction flask, and a mixed solution of tetrahydrofuran/water (100 mL/60 mL) was added to dissolve the substrate.
  • Ammonium chloride (15.5 g, 292 mmol) and reduced iron powder (26 g, 467 mmol) were successively added to the stirred reaction flask under room temperature, and then the reaction system was heated to 65 ° C and stirring was continued for 3 hours. The progress of the reaction was checked by TLC. After the substrate was completely reacted, excess iron powder was removed by filtration, and the filter cake was rinsed three times with ethyl acetate.
  • Step b
  • Compound a1-2 (8.0 g, 43 mmol) was placed in a 500 mL single-mouth reaction flask, and concentrated sulfuric acid (100 mL) was added to dissolve the substrate under constant stirring. Concentrated nitric acid (6.15 mL, 48 mmol) was slowly added dropwise to the stirred reaction flask at minus 20 degrees Celsius and kept at this temperature for 5 minutes. The progress of the reaction was checked by TLC, and after the substrate was completely reacted, it was poured into ice water. While maintaining an ice bath condition of minus 20 degrees Celsius, slowly add a sodium hydroxide/water solution (150 mL/300 mL) to the reaction system to adjust the pH to 8-9.
  • a sodium hydroxide/water solution 150 mL/300 mL
  • step 1
  • reaction substrate tert-butyl 4-fluoro-2-methoxy-5-nitrophenylcarbamic acid tert-butyl ester a2 (11.46 g, 40 mmol) was dissolved in 60 ml of N,N-dimethylacetamide, added N,N,N'-trimethylethylenediamine (4.90 g, 48 mmol), N,N-diisopropylethylamine (7.74 g, 60 mmol) was stirred and stirred at 90 ° C for 6 hours. The progress of the reaction was checked by TLC.
  • the preparation method is the same as that of the compound a, except that the N,N,N'-trimethylethylenediamine in the step 2 of the preparation of the compound a is replaced with 4-dimethylaminopiperidine.
  • step 1
  • step 1
  • Trifluoroacetic acid (1.124 g, 9.86 mmol) was added to a solution of compound k-1 (700 mg, 0.986 mmol) in 10 ml of dichloromethane at 0 ° C. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to give 1.5 g of compound k-2. MS m/z (ESI): 611 [M+H] + .
  • Triethylamine (747 mg, 7.39 mmol) was added to a solution of compound k-2 (750 mg, 0.49 mmol) in 5 ml of dichloromethane at 0 ° C, stirring vigorously at 0 ° C for 30 min and then added methanesulfonyl chloride (56 mg) , 0.49 mmol), vigorously stirred at 0 ° C for 2 hours. After completion of the reaction, the mixture was diluted with water and extracted with EtOAc EtOAc. MS m/z (ESI): 68.93 [M+H] + .
  • step 1
  • step 1
  • n-Butyllithium (576 mg, 9 mmol) was slowly added dropwise to a solution of compound p-1 (800 m, 3 mmol) in 110 ml of THF. After the reaction mixture was stirred for 1 hour, methyl iodide (511 mg, 3.6 mmol) was added at <RTIgt; After the reaction is completed, it is quenched by a saturated ammonium chloride solution at minus 70 degrees Celsius. Extract with ethyl acetate, dry and concentrate under reduced vacuum. Purification by combiflash gave 320 mg of compound p-2. MS m/z (ESI): 202 [M+H] + .
  • the compound p-3 was prepared by referring to the preparation method of the compound c. MS m/z (ESI): 332 [M+H] + .
  • the compound 2-chloro-5-methyl-4-(1-methyl-1H-pyrazol-4-yl)-7-(2) was prepared according to the preparation method of the compound e. -(Trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine p. MS m/z (ESI): 378.2 [M+H] + .
  • the compound c and 2-cyclohexenyloxy-4,4,5,5-tetramethyl-1,3,2-dioxaborane were used as a starting material, and were prepared by referring to the preparation method of the compound g.
  • the compound 2-chloro-4-cyclohexenyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine s was obtained.
  • the preparation method is the same as that of the compound a, except that the N,N,N'-trimethylethylenediamine in the step 2 of the preparation of the compound a is replaced with 1-methylpiperazine.
  • step 1
  • step 1
  • step 1
  • step 1
  • the title compound of the title compound 9 was obtained as a yellow solid.
  • step 1
  • step 1
  • the compound 19-34 is represented by the formula (I), wherein the substituents A, X, R 0 , R 1 , R 2 and R 3 are shown in the following table.
  • Compounds 19-34 can be prepared in a similar manner to compounds 1-18 according to the different structures listed in the table below.
  • the starting materials and intermediates used are those which can be prepared by those skilled in the art according to the prior art.
  • Step 1 Using compound c and ct-2 as starting materials, and preparing according to the method of compound i, the compound ct-3 was obtained in a yield of 65%. MS m/z (ESI): 405 [M+H] + .
  • Steps 2 to 3 The compound ct-3 was used as a raw material, and the compound ct-5 was obtained by referring to the procedures of Steps 3 and 4 in Compound a to give a yield of 55%.
  • Step 4 The compound ct-5 and w were used as starting materials, and the compound was obtained by the procedure of Step 1 in Example 1 to give the compound ct-6, yield 11.7%. MS m/z (ESI): 564 [M+H] + .
  • Steps 5 to 6 The compound ct-6 was used as a starting material, and was prepared by the method of Example 3 to give Comparative Compound 1 in a yield of 11%. MS m/z (ESI): 486 [M+H] + .
  • Test Example 1 Activity inhibition test for wild-type EGFR and mutant EGFR kinase
  • the reagents used in the following z'-lyte test methods were purchased from Invitrogen.
  • the inhibitory activity against T790M/L858R double mutant EGFR kinase (Invitrogen, PV4879), wild type EGFR kinase (Invitrogen, PV3872) was determined by the z'-lyte method.
  • the working concentrations of the components in the 10 ⁇ L T790M/L858R kinase reaction system were: 25 ⁇ M ATP, 0.1 ng/ ⁇ L T790M/L858R kinase, and 2 ⁇ M Tyr04 substrate (Invitrogen, PV3193).
  • the concentration of DMSO after addition of the compound prepared in the above examples of the present invention was 2 vol%.
  • the working concentrations of the components in the 10 ⁇ L wild-type EGFR kinase reaction system were: 10 ⁇ M ATP, 0.8 ng/ ⁇ L wild-type EGFR kinase, and 2 ⁇ M Tyr04 substrate (Invitrogen, PV3193).
  • the concentration of DMSO after adding the test substance was 2 vol%.
  • a 10 mM drug stock solution was dissolved at room temperature and serially diluted with 8 vol% DMSO solution to a final concentration of 4-0.002 [mu]M.
  • 2.5 ⁇ L of the test substance solution and 5 ⁇ L of a mixture of T790M/L858R kinase (or wild-type EGFR kinase) diluted with the reaction buffer and Tyr04 substrate were added to each well, and 2.5 ⁇ L of ATP was added to initiate the reaction.
  • the C1 well was replaced with ATP by a reaction buffer, the C2 well was not added with any drug, and the C3 well was added with a phosphorylated substrate as described in the instructions. After 60 minutes of shaking at 25 degrees on a shaker.
  • the inhibition rate calculation method (refer to the specification of Invitrogen, PV3193) is as follows:
  • Phosphorylation rate (1-((ER ⁇ C3 520nm - C3 450nm ) / ((C1 450nm - C3 450nm ) + ER ⁇ (C3 520nm - C1 520nm )))) ⁇ 100%
  • Inhibition rate (IR) (1 - (phosphorylation rate of test compound) / (phosphorylation rate of C2)) ⁇ 100%
  • the half-inhibitory concentration IC 50 was calculated using XLFIT 5.0 software (IDBS, UK).
  • the compounds of Examples 1-14 of the present invention were EGFR mutant enzymes (T790M/L858R) compared to the positive controls BIBW2992 (Afatinib) and AZD-9291 (see WO2013014448A1 for the preparation method). It showed strong inhibitory activity, while the inhibitory activity against EGFR wild-type enzyme (T790M WT) was weak, and it was found that the compound had almost no activity after the substitution position of the acrylamide group was changed. Therefore, the compound of the present invention has a good selective inhibitory activity against the EGFR mutant enzyme.
  • Test Example 2 Inhibition of EGFR phosphorylation by AEGFR (EGFR wild type) and H1975 (EGFR T790M mutation) cells by EGFR T790M inhibitor (determined by ELISA)
  • the reagents, solution preparation methods, and cell treatment and lysate preparation steps and ELISA detection steps in the following methods were carried out in accordance with the instructions of R&D DYC3570, R&D DYC1095E and R&D DYC1095BE.
  • Cell lysis buffer 1% (w/v) NP-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% (v/v) glycerol glycerol, 1 mM NaVO 3 , 2 mM EDTA.
  • Cell lysate cell lysis buffer + 10 ⁇ g / mL Aprotinin (Sigma) + 10 ug / mL Leupeptin (Sigma), now available.
  • 1x PBS buffer NaCl: 0.137 M, KCl: 0.0027 M, Na 2 PO 4 -12H 2 O: 0.01 M, KH 2 PO 4 : 0.0015 M, pH 7.4.
  • Wash Buffer PBS buffer containing 0.05% (v/v) Tween-20.
  • Antibody dilutions were detected: 20 mM Tris, 137 mM NaCl, 0.05% (v/v) Tween-20, 0.1% (w/v) BSA, pH 7.2-7.4.
  • Blocking solution PBS buffer containing 1% (w/v) BSA.
  • ELISA kits R&D DYC3570, R&D DYC1095E and R&D DYC1095BE.
  • H1975 cells purchased from the Cell Culture Bank of the Chinese Academy of Sciences' Type Culture Collection Committee
  • RPMI 1640 medium was incubated overnight at 37 ° C, 5% (v/v) CO 2 .
  • the R&D capture antibody ((DYC1095BE or DYC1095E)) was diluted with PBS 1:180, and the diluted antibody 100 ⁇ L/well was added to an ELISA reaction plate (Corning costar 42592), and shaken overnight at 25 ° C;
  • the detection antibody is diluted with the detection antibody dilution solution in the prescribed ratio of the kit, and 100 ⁇ L is added to each well, and the mixture is incubated at 25 ° C for 1 hour in the dark;
  • Inhibition rate (%) 100% ⁇ (OD cell- OD drug treatment ) / (OD cell - OD blank )
  • A431 cells purchased from the Cell Culture Bank of the Chinese Academy of Sciences
  • DMEM medium containing 10% FBS per well at 37 ° C. Incubate overnight with 5% CO 2 .
  • the A431 cell culture medium was changed to 90 ⁇ l of serum-free DMEM medium, and cultivation was continued overnight.
  • R&D capture antibody (DYC3570E) was diluted with PBS 1:180, diluted antibody 100 ⁇ L/well was added to an ELISA reaction plate (Corning costar 42592), and shaken overnight at 25 ° C;
  • the detection antibody is diluted with the detection antibody dilution solution in the prescribed ratio of the kit, and 100 ⁇ L is added to each well, and the mixture is incubated at 25 ° C for 1 hour in the dark;
  • Inhibition rate (%) 100% ⁇ (OD EGF - OD drug ) / (OD EGF - OD cells )
  • the compounds of the examples of the present invention have better selective inhibitory activity against EGFR mutant cells than the positive control BIBW2992.
  • Test Example 3 MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) method for detecting cell inhibitory activity
  • MTT test method steps are carried out using methods well known to those skilled in the art, and the reagents used in the methods are commercially available.
  • the medium was removed and 1 mL of 0.25% trypsin/EDTA (Gibco, 25200-056) was added. After washing once, 1.5 mL of trypsin/EDTA was added to digest the adherent cells until the cells were separated, and then 3.5 mL of the medium was added to terminate the digestion.
  • the digested cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 1300 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in fresh medium.
  • the cells were then counted and the cells were diluted to the following concentrations: H1975 cells at 27,800 per ml, A431 cells and NIH3T3 cells at 33,300 per ml.
  • the cells were seeded in a 96-well plate (BD 3072), 90 ⁇ L per well, and cultured overnight.
  • the A431 cell culture medium is: DMEM (Hyclone SH30243.01B) containing 10% FBS (Gibco, 10099-141);
  • NIH3T3 cell culture medium DMEM (Hyclone SH30243.01B) containing 10% FBS (Gibco, 10099-141);
  • H1975 cell culture medium RPMI-1640 (Hyclone SH30809.01B) containing 10% FBS (Gibco, 10099-141);
  • the cells were placed in an incubator. After 72 hours of culture, 10 ⁇ L of 5 mg/ml MTT (Sigma, M5655) solution was added to each well, and then the 96-well plate was incubated in a 37 ° C 5% CO 2 incubator for 4 hours. .
  • the plate was again centrifuged at 2000 rpm for 5 min. After removing the supernatant, 150 ⁇ L of DMSO was added to each well, and the plate was shaken in a shaker until all crystal violet was dissolved. Finally, the absorbance at 492 nm was measured using a microplate reader, and the IC 50 was calculated using XLFIT 5.0 software (IDBS, UK).
  • the compound of the present invention showed strong inhibitory activity against EGFR mutant cells (H1975 cells) and weak inhibition against EGFR wild-type cells (A431 cells) as compared with the positive control BIBW2992.
  • the compound of the present invention therefore has a better selective inhibitory activity against EGFR mutant cells.
  • the compounds of the examples of the present invention have higher IC 50 values for NIH3T3 cells than the positive control BIBW2992, and thus exhibit less toxicity.
  • the compounds of the present invention showed strong inhibitory activity against mutant EGFR enzyme activity, EGFR phosphorylation level, and cell proliferation, while Type EGFR enzyme activity, EGFR phosphorylation level, and cell proliferation show weak inhibitory activity, and thus have good selectivity for EGFR mutant cells.
  • NIH-3T3 cells have a very weak inhibitory effect, so they exhibit low cytotoxicity. Therefore, such compounds have better selective inhibitory activity and lower cytotoxicity against T790M mutant EGFR.
  • the compounds of the invention exhibit good bioavailability.

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Abstract

本发明涉及2，4-二取代7H-吡咯并[2，3-d]嘧啶衍生物、其制法与医药上的用途。具体地，本发明公开了式(I)化合物或其药学上可接受的盐、立体异构体、溶剂化合物或前药，及其制备方法和应用，式中各基团的定义详见说明书。

Description

2,4-二取代7H-吡咯并[2,3-d]嘧啶衍生物、其制法与医药上的用途 技术领域
本发明涉及医药技术领域，特别涉及一种2,4-二取代7H-吡咯并[2,3-d]嘧啶衍生物及其制备方法和作为EGFR酪氨酸激酶抑制剂的应用，以及由其制备的药物组合物和药用组合物。
背景技术
肺癌是全球发病率最高的癌症。在中国，肺癌发病率位居所有癌症中第一位，也是中国发病率和死亡率最高的癌症。
在中国肺癌病人中，30％病人具有EGFR突变，其中L858R和外显子19缺失突变占90％以上，这类病人对EGFR抑制剂更为敏感。现有已上市第一代EGFR抑制剂，例如厄洛替尼、吉非替尼对这类病人治疗效果良好，能够使其中60％以上病人肿瘤缩小，明显延长病人无进展生存期。但绝大多数病人在6-12个月获得耐药，第一代EGFR抑制剂不再起效，而这类病人目前处于无药可用状态。临床发现对第一代EGFR抑制剂产生耐药的病人中有50％检测到EGFR T790M突变。在T790M突变细胞系H1975中，第一代EGFR抑制剂吉非替尼和厄洛替尼均大于3uM，基本没有活性。
目前第二代不可逆pan-EGFR抑制剂阿法替尼已经获批上市，该药对EGFR突变肺癌病人疗效显著好于第一代EGFR抑制剂。但第二代抑制剂同时也具有很强的野生型EGFR抑制活性，对野生型EGFR的抑制活性显著高于耐药T790M突变，病人皮疹等毒副作用严重且对耐药病人疗效较差，仅有小部分第一代EGFR抑制剂耐药的病人对这类药物产生应答。
为了提高对EGFR T790M耐药突变抑制活性的同时降低对野生型EGFR的抑制活性，开发活性更高、选择性更好、毒性更低的第三代EGFR突变体选择性抑制剂具有重要的意义。
发明内容
本发明的目的在于提供一种2,4-二取代7H-吡咯并[2,3-d]嘧啶衍生物，不仅对EGFR T790M耐药突变和敏感型(如L858R突变或外显子19缺失)突变具有较高的抑制活性，而且对野生型EGFR的抑制活性显著降低，具有较高的选择抑制性，同时还具有低细胞毒性。此外与其他已知的EGFR突变体抑制剂相比，本发明的化合物还显示出有利的物理性质，毒性特征和/或代谢特征。
本发明的第一方面，提供一种式(I)所示的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药；
Figure PCTCN2015085089-appb-000001
式中，
A环为取代或未取代的C3-10杂环基、取代或未取代的C6-10芳基环或取代或未取代的C4-10环烯基；
所述的“取代”是指环原子上的1至6个氢原子被选自下组的取代基所取代：羟基、CN、NO2、卤素、C1-3烷基、卤代的C1-3烷基、-CON(C1-3烷基)2、-C(O)OC1-3烷基、-OC(O)C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3烷基、-S(O)-苯基、-N(C1-3烷基)2；
X为共价键，或为NH、O或S；
R0为氢、C1-3烷基、卤代的C1-3烷基、C3-6环烷基、卤代的C3-6环烷基、-CHO、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基；
R1、R2各自独立地为氢、卤素、C1-3烷基、卤代的C1-3烷基；
R3选自下组：
Figure PCTCN2015085089-appb-000002
Figure PCTCN2015085089-appb-000003
其中，所述的苯基为未取代的或被1-3个选自下组的取代基所取代：卤素、C1-3烷基。
在另一优选例中，所述A环为：
(ⅰ)取代或未取代的含1、2或3个氮原子的9至10元双环杂芳基环；
(ⅱ)取代或未取代的含1到2个氮原子的5至6元单环杂芳基环；
(ⅲ)取代或未取代的含1个氮原子的4至7元饱和单杂环；
(ⅳ)取代或未取代的6元部分不饱和单环；
(ⅴ)取代或未取代的C6-10芳基环；或
(ⅵ)吗啉环。
在另一优选例中，所述C3-10杂环基为吡唑基、吗啉基、杂氮C3-7环烷基、吡咯并吡啶基、吡唑并吡啶、吲唑、吡咯、吲哚基、或吡啶基。
在另一优选例中，所述C4-10环烯基为环戊烯基、环己烯基、或环庚烯基。
在另一优选例中，所述的“取代”是指环原子上的1至3个氢原子被选自下组的取代基所取代：羟基、NO2、卤素、C1-3烷基、卤代的C1-3烷基、-SO2C1-3烷基、-S(O)C1-3烷基。
在另一优选例中，在式(I)化合物中，R3选自下组：
Figure PCTCN2015085089-appb-000004
在另一优选例中，在式(I)化合物中，R0为氢、甲基、乙基、丙基、异丙基、环丙基、环丁基、环戊基、环己基、一氟乙基、二氟甲基、三氟甲基、-COCH3、-CO-苯基、-SO2CH3或-SO2-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、溴、甲基、乙基。
在另一优选例中，在式(I)化合物中，R1、R2各自独立地为氢、氟、氯、溴、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基。
在另一优选例中，在式(I)化合物中，所述取代或未取代的C3-10杂环基为取代或未取代的含1、2或3个氮原子的9至10元双环杂芳基环，选自下组：
Figure PCTCN2015085089-appb-000005
其中，Z11为CR15或N；Z12为CR16或N；Z21为CR26或N；
R12、R13、R14、R15、R16、R22、R23、R24、R25、R26各自独立地为氢、羟基、CN、NO2、卤素、卤代的C1-3烷基、C1-3烷基、-CON(C1-3烷基)2、-N(C1-3烷基)2、-C(O)OC1-3烷基、-OC(O)C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3 烷基、-S(O)-苯基；所述的烷基、苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基；
R11、R21各自独立地为氢、卤代的C1-3烷基、C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基。所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基。
在另一优选例中，Z11为N；Z12为CR16；R12、R13、R14、R16各自独立地为氢、卤素、卤代的C1-3烷基、C1-3烷基；R11为氢、卤代的C1-3烷基、C1-3烷基、-COC1-3烷基、-SO2C1-3烷基。
在另一优选例中，Z11为N；Z12为CR16；R12、R13、R14、R16各自独立地为氢、氟、氯、溴、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基；R11为氢、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基、-COCH3、-SO2CH3。
在另一优选例中，Z21为CR26；R22、R23、R24、R25、R26各自独立地为氢、卤素、卤代的C1-3烷基、C1-3烷基；R21为氢、卤代的C1-3烷基、C1-3烷基、-COC1-3烷基、-SO2C1-3烷基。
在另一优选例中，Z21为CR26；R22、R23、R24、R25、R26各自独立地为氢、氟、氯、溴、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基；R21为氢、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基、-COCH3、-SO2CH3。
在另一优选例中，在式(I)化合物中，所述取代或未取代的C3-10杂环基为取代或未取代的含1到2个氮原子的5至6元单环杂芳基环，选自下组：
Figure PCTCN2015085089-appb-000006
其中，Z31为CR34或N；
R32、R33、R34、R35、R36、R37、R38各自独立地为氢、羟基、CN、NO2、卤素、卤代的C1-3烷基、C1-3烷基、-CON(C1-3烷基)2、-N(C1-3烷基)2、-C(O)OC1-3烷基、-OC(O)C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3烷基、-S(O)-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基；
R31为氢、卤代的C1-3烷基、C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基。
在另一优选例中，Z31为N；R32、R33各自独立地为氢、卤素、卤代的C1-3烷基、C1-3烷基；R31为氢、卤代的C1-3烷基、C1-3烷基、-COC1-3烷基、-SO2C1-3烷基。
在另一优选例中，Z31为N；R32、R33各自独立地为氢、氟、氯、溴、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基；R31为氢、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基、-COCH3、-SO2CH3。
在另一优选例中，R35、R36、R37、R38各自独立地为氢、卤素、C1-3烷基。
在另一优选例中，R35、R36、R37、R38各自独立地为氢、氟、氯、溴、甲基、乙基、丙基、异丙基。
在另一优选例中，在式(I)化合物中，所述取代或未取代的C3-10杂环基为取代或未取代的含1个氮原子的4至7元饱和单杂环选自下组：
Figure PCTCN2015085089-appb-000007
其中，n1为1、2或3；n2为1或2；
R41为氢、C1-3烷基、卤代的C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3烷基、-S(O)-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基。
在另一优选例中，n1为1；n2为1；R41为氢、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基、-COCH3、-SO2CH3、-SO2-苯基、-CO-苯基；所述的苯基为未取代的或被1个选自下组的取代基所取代：氟、氯、甲基。
在另一优选例中，式(I)化合物为式(Ⅱ)、式(Ⅲ)、式(Ⅳ)、式(Ⅴ)、或式(Ⅵ)所示的化合物：
Figure PCTCN2015085089-appb-000008
式中，R11、R12、R13、R14、R0、R1、R2、R3、Z11、Z12如说明书中所定义；
Figure PCTCN2015085089-appb-000009
式中，R21、R22、R23、R24、R25、R0、R1、R2、R3、Z21如说明书中所定义；
Figure PCTCN2015085089-appb-000010
式中，R31、R32、R33、R0、R1、R2、R3、Z31如说明书中所定义；
Figure PCTCN2015085089-appb-000011
式中，R35、R36、R37、R38、R0、R1、R2、R3如说明书中所定义；
Figure PCTCN2015085089-appb-000012
式中，R41、R0、R1、R2、R3、X、n1、n2如说明书中所定义。
在另一优选例中，在式(Ⅵ)化合物中，X为O。
在另一优选例中，取代或未取代的C3-10杂环基选自：
Figure PCTCN2015085089-appb-000013
在另一优选例中，取代或未取代的C6-10芳基环选自：
Figure PCTCN2015085089-appb-000014
在另一优选例中，式(I)化合物为式(Ⅶ)或式(Ⅷ)所示的化合物：
Figure PCTCN2015085089-appb-000015
式中，R0、R1、R2、R3如说明书中所定义。
在另一优选例中，式I中所述的R0、R1、R2、R3、X、和A环各自独立为实施例中各具体化合物中相应的基团。
在另一优选例中，所述式(I)化合物包括实施例中所制备的化合物，例如选自下组：
Figure PCTCN2015085089-appb-000016
Figure PCTCN2015085089-appb-000017
本发明第二方面，提供了一种药物组合物，它包括如本发明第一方面所述的式(I)化合物或上述示例化合物、或其药学可接受的盐、或其溶剂化物、或其立体异构体、或前药，并且还包括药学可接受的载体。
通常，本发明化合物或其药学可接受的盐、或其溶剂化物、或其立体异构体、或前药可以与一种或多种药用载体形成适合的剂型施用。这些剂型适用于口服、直肠给药、局部给药、口内给药以及其他非胃肠道施用(例如，皮下、肌肉、静脉等)。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明的化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或辅料经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制剂的载体包括水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。
本发明的组合物以符合医学实践规范的方式配制，定量和给药。给予化合物的“有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。
本发明第三方面，提供了本发明第一方面所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或前药在用于(i)制备调控EGFR酪氨酸激酶活性的药物，或(ii)制备预防和/或制备治疗EGFR相关疾病的药物中的应用。
在另一优选例中，所述调控为上调或下调。
作为优选，所述EGFR相关疾病为癌症，糖尿病，免疫系统疾病，神经退行性疾病或心血管疾病、使用EGFR调节剂治疗期间具有获得性耐药性的疾病。
作为优选，所述癌症为非小细胞肺癌、头颈癌、乳腺癌、肾癌、胰腺癌、子宫颈癌、食道癌、胰腺癌、前列腺癌、膀胱癌、结肠直肠癌、卵巢癌、胃癌、脑恶性肿瘤包括成胶 质细胞瘤等，或它们的任何组合。
作为优选，所述获得性耐药性是由EGFR外显子20编码的T790突变引起的或者是包含EGFR外显子20编码的T790突变所引起的耐药性，如T790M。
作为优选，所述非小细胞肺癌是由EGFR突变引起的，包括敏感型突变(如L858R突变或外显子19缺失)和耐药性突变(如EGFR T790M突变)。
在本发明中，EGFR调节剂是指靶向EGFR的小分子酪氨酸激酶抑制剂，如吉非替尼，厄洛替尼，埃克替尼，拉帕替尼、阿法替尼等。
本发明第四方面，提供了一种药用组合物，包括治疗有效量的本发明第一方面所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或前药，以及选自下组的药物：吉非替尼、厄洛替尼、埃克替尼、拉帕替尼、XL647、NVP-AEE-788、ARRY-334543、EKB-569、BIBW2992、HKI272、BMS-690514、CI-1033、凡德他尼、PF00299804、WZ4002、西妥昔单抗、曲妥珠单抗、帕尼突单抗、马妥珠单抗、尼妥珠单抗、扎鲁木单抗、帕妥珠单抗、MDX-214、CDX-110、IMC-11F8、Zemab、Her2疫苗PX 1041、HSP90抑制剂、CNF2024、坦螺旋霉素、阿螺旋霉素、IPI-504、SNX-5422、NVP-AUY922、或其组合。除本发明的化合物或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或前药以外，上述药用组合物中的其他药物均为本领域技术人员熟知的抗肿瘤药物。
“治疗有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
本发明的所述药物组合物或所述药用组合物中含有的本发明化合物或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药的治疗有效量优选为0.1mg-5g/kg(体重)。
所述的药用组合物可用于治疗EGFR相关的疾病，如癌症，糖尿病，免疫系统疾病，神经退行性疾病或心血管疾病、使用EGFR调节剂治疗期间具有获得性耐药性的疾病。
所述获得性耐药性的疾病是由EGFR外显子20编码的T790突变所引起的疾病，或者是包含EGFR外显子20编码的T790突变所引起的疾病。
在另一优选例中，所述的EGFR外显子20编码的T790为T790M。
本发明的式(I)化合物、或其药学上可接受的盐、或溶剂化物、或其立体异构体、或前药在某些疾病中可以与其他药物联合应用，以达到预期的治疗效果。一个联合应用的例子是用来治疗晚期NSCLC。例如，将治疗有效量的本发明式(I)所示化合物与mTOR抑制剂联用(例如雷帕霉素)；或与Met抑制剂(包括Met抗体MetMAb和Met小分子抑制剂PF02341066)联用；或与IGF1R抑制剂联用(例如OSI-906)；或与热休克蛋白抑制剂联用等。
在本发明的第五方面，提供了第一方面所述的式(I)所示的化合物，或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药的制法，
所述方法包括步骤：
(i)对式I-f化合物进行还原反应，从而形成式I-g化合物；
(ii)将式I-g化合物与丙烯酰氯进行缩合反应，从而形成式(I)所示的化合物；
Figure PCTCN2015085089-appb-000018
或所述方法包括步骤(i’)在惰性溶剂中，将式I-c化合物与式I-h化合物反应，从而形成式(I)所示的化合物；
Figure PCTCN2015085089-appb-000019
上述各式中，R0、R1、R2、R3、X、A环的定义如上所述；L1为离去基团。
在另一优选例中，所述的L1包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基(如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等)；酰氧基(如乙酰氧基、三氟乙酰氧基等)。
在另一优选例中，在所述步骤(i)中，所述反应在酸性条件下进行。
在另一优选例中，在所述步骤(i)中，用金属(如铁粉，锌粉)或者氯化亚锡进行还原。
在另一优选例中，在所述步骤(i)中，在钯碳催化下，加氢还原。
在另一优选例中，所述步骤(ii)在碱性条件下进行。
在另一优选例中，在所述步骤(ii)中，在缩合剂存在下，将式I-g化合物与羧酸进行缩合形成式I化合物。
在另一优选例中，所述步骤(i')在催化剂、配体或碱存在下进行。
在另一优选例中，所述步骤(i')中，所述催化剂选自下组：TFA、对甲苯磺酸、Pd2(dba)3(三(二亚苄基丙酮)二钯)，BINAP((±)-2,2’-双-(二苯膦基)-1,1’-联萘)、或其组合；
所述配体包括：Xantphos(4,5-双(二苯基膦)-9,9-二甲基氧杂蒽)；和/或
所述的碱包括：碳酸铯。
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。
具体实施方式
本发明人经过长期而深入的研究，意外地发现了一类EGFR突变选择性抑制剂，体外实验表明其在纳摩尔浓度下即可抑制EGFR T790M/L858R双突变酶和细胞株H1975的 增殖，同时对EGFR敏感性突变细胞株HCC827(外显子19缺失)也有很高的抑制强度，而对野生型EGFR酶和细胞株A431的抑制则相对较弱。因此，此类结构不但可用于EGFR敏感型突变癌症的治疗，还适用于目前EGFR-TKI治疗中产生继发性耐药的病例；同时其突变选择性大大减小了因抑制野生型EGFR而产生的毒副作用，此外，该类化合物在正常细胞系(如NIH-3T3细胞)中具有较低的细胞毒性，从而大大降低了非特异性毒副作用，是第二代EGFR-TKI的理想替代物。
术语定义
“C3-10杂环基”是指具有3至10个碳原子的杂环基，构成环的原子除碳外，还至少含有一个杂原子，选自N、S、O。例如：吡唑基、吗啉基、杂氮C3-7环烷基、吡咯并吡啶基、吡唑并吡啶、吲唑、吡咯、吲哚基、或吡啶基。
“C1-3烷基”是指具有1至3个碳原子的直链或支链的饱和脂族烃基。例如甲基、乙基、丙基、异丙基。
“C3-6环烷基”指具有3-6个碳原子的环烷基。环烷基实例有环丙基、环丁基、环戊基、环己基等。
“杂氮C3-7环烷基”指具有3-7个碳原子的环烷基，且构成环的原子除碳外，还至少含有一个N原子。
“C6-10芳基”与“C6-10芳基环”可互换使用，是指具有6-10个碳原子的芳香烃基，例如苯基、萘基等。
“卤素”指氟、氯、溴或碘。
“杂芳基环”与“杂芳基”可互换使用，是指具有5到10个环原子，优选5、6、9或10个环原子；环阵列中共享6、10或14个π电子；且除碳原子外还具有1到5个杂原子的基团。术语“杂原子”是指氮、氧或硫。
如本文所用，“部分不饱和”是指含有一个或多个不饱和键，但不具有完全共轭的π电子系统。
“含1到2个氮原子的5至6元单环杂芳基环”是指含5至6个环原子的单环杂芳基，例如包括(但不限于)：咪唑环、吡咯环、吡唑环、吡啶环、哒嗪环、嘧啶环、吡嗪环等。
“含1、2或3个氮原子的9至10元双环杂芳基环”是指含9至10个环原子的双环杂芳基，例如包括(但不限于)：吲哚环、异吲哚环、喹啉环、异喹啉环、吲唑环、苯并咪唑环、喹唑啉环、喹喔啉环、噌啉环、酞嗪环。
在本发明中，所述5至6元单环杂芳基环或9至10元双环杂芳基环优选自下组：
Figure PCTCN2015085089-appb-000020
“含1个氮原子的4至7元饱和单杂环”是指含4至7个环原子且1个碳原子被氮原子 所置换的饱和单环。单杂环的实例包括(但不限于)：哌啶环、四氢吡咯环、氮杂环丁烷、氮杂环庚烷。
“6元部分不饱和单环”是指含6个环原子的部分不饱和的全碳单环。例如包括(但不限于)：1,3-环己二烯、1,4-环己二烯、环己烯等。
药物组合物
术语“本发明的活性物质”或“本发明的活性化合物”是指本发明式(I)化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其具有显著的EGFR T790M/L858R选择抑制活性。
如本文所用，所述“药学上可接受的盐”包括药学可接受的酸加成盐和药学可接受的碱加成盐。
“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其他副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、丙酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、对甲苯磺酸盐和水杨酸盐等。这些盐可通过本专业已知的方法制备。
“药学可接受的碱加成盐”，包括但不限于无机碱的盐如钠盐，钾盐，钙盐和镁盐等。包括但不限于有机碱的盐，比如铵盐，三乙胺盐，赖氨酸盐，精氨酸盐等。这些盐可通过本专业已知的方法制备。
如本文所用，式(I)化合物可以存在于一种或多种晶型，本发明的活性化合物包括各种晶型及其混合物。
本发明中提及的“溶剂化物”是指本发明的化合物与溶剂形成的配合物。它们或者在溶剂中反应或者从溶剂中沉淀析出或者结晶出来。例如，一个与水形成的配合物称为“水合物”。式(I)化合物的溶剂化物属于本发明范围之内。
本发明式(I)所示的化合物可以含有一个或多个手性中心，并以不同的光学活性形式存在。当化合物含有一个手性中心时，化合物包含对映异构体。本发明包括这两种异构体和异构体的混合物，如外消旋混合物。对映异构体可以通过本专业已知的方法拆分，例如结晶以及手性色谱等方法。当式(I)化合物含有多于一个手性中心时，可以存在非对映异构体。本发明包括拆分过的光学纯的特定异构体以及非对映异构体的混合物。非对映异构体可由本专业已知方法拆分，比如结晶以及制备色谱。
本发明包括上述化合物的前药。前药包括已知的氨基保护基和羧基保护基，在生理条件下被水解或经由酶反应释放得到母体化合物。具体的前药制备方法可参照(Saulnier，M.G.；Frennesson，D.B.；Deshpande，M.S.；Hansel，S.B and Vysa，D.M.Bioorg.Med.Chem Lett.1994，4，1985-1990；和Greenwald，R.B.；Choe，Y.H.；Conover，C.D.；Shum，K.；Wu，D.；Royzen，M.J.Med.Chem.2000，43，475.)。
制备方法
本发明提供了式(I)化合物的制备方法，本发明中的化合物可以通过多种合成操作容易地制备，这些操作是所属领域技术人员熟练掌握的。这些化合物的示例性制备方法可以包 括(但不限于)下文所述的流程。
通常，在本发明的制备方法中，各反应大多在惰性溶剂中，在-20℃至150℃(或回流温度)(较佳地，-5℃至100℃，或0-80℃)下，进行一段时间(如0.1-72小时，较佳地0.5-24小时)。
较佳地，本发明式(I)化合物可以通过以下方案及实施例中所述的示例性方法以及本领域技术人员所用的相关公开文献操作完成。
在具体操作过程中，可以根据需要对方法中的步骤进行扩展或合并。
方案1
Figure PCTCN2015085089-appb-000021
方案1中，各取代基和基团如说明书中所定义。
步骤1中，当X为N、O或S时，式(I-a)化合物与式(I-b)化合物可通过取代反应(例如亲和取代反应等)生成式(I-c)化合物；
当X为共价键时，式(I-b)化合物为各类A环的硼酸或硼酯类化合物，式(I-a)化合物与式(I-b)化合物可通过偶联反应(如Suzuki偶联等)生成式(I-c)化合物；
式(I-a)化合物中的L1和L2为离去基团，包括(但不限于)三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等。
步骤2中，式(I-c)化合物可通过取代反应或偶联反应与式(I-d)化合物反应生成式(I-e)化合物，例如在一定温度下，使用合适的催化剂(或含合适的配体)或碱及适当的溶剂进行。如使用酸催化，催化剂可以是但不限于TFA或对甲苯磺酸。使用Buchwald-Hartwig胺化法，所用的钯催化剂可以是但不限于Pd2(dba)3，所用的配体可以是但不限于XantPhos(4,5-双(二苯基膦)-9,9-二甲基氧杂蒽)，所用的碱可以是但不限于碳酸铯。
步骤3中，式(I-e)化合物可与各种胺化合物进行胺取代反应生成式(I-e)化合物，根据不同的胺化合物，可选择合适的条件和方法很方便地进行合成。例如在一定温度下，使用合适的催化剂(或含合适的配体)或碱及适当的溶剂进行。该方法是本领域技术人员所使用的常规方法。
步骤4中，该硝基化合物I-f转化为相应的胺基化合物可在酸性条件下，用金属(可以是但不限于铁粉，锌粉)或者氯化亚锡进行还原；或者在钯碳催化下，加氢还原。
步骤5中，该胺基化合物I-g可在碱性条件下与相应的酰氯缩合成酰胺，或者在缩合剂存在下与相应的羧酸缩合成酰胺。
方案1中，式(I-a)和式(I-b)化合物可市购得到，或采用本领域熟知的方法进行制备。
方案2
Figure PCTCN2015085089-appb-000022
方案2中，各取代基和基团如说明书中所定义。
式(I-c)化合物可通过取代反应或偶联反应与式(I-h)化合物反应生成式(I)化合物，例如在一定温度下，使用合适的催化剂(或含合适的配体)或碱及适当的溶剂进行。如使用酸催化，催化剂可以是但不限于TFA或对甲苯磺酸。使用Buchwald-Hartwig胺化法，所用的钯催化剂可以是但不限于Pd2(dba)3(三(二亚苄基丙酮)二钯)，BINAP((±)-2,2'-双-(二苯膦基)-1,1'-联萘)。所用的配体可以是但不限于Xantphos(4,5-双(二苯基膦)-9,9-二甲基氧杂蒽)，所用的碱可以是但不限于碳酸铯。
方案2中，式(I-h)化合物可通过如下示例性方法进行制备：
Figure PCTCN2015085089-appb-000023
以化合物4-氟-2-甲氧基-5-硝基苯胺为起始原料，依次经过氨基上保护基反应、胺取代反应、硝基还原反应、酰化反应和氨基脱保护反应来制备式(I-h)化合物。上述各步反应均为本领域常规反应。化合物4-氟-2-甲氧基-5-硝基苯胺可市购得到，或通过本领域技术人员已知的方法制备(式(I-h)化合物的合成方法可参见WO2013014448A1)。
式(I-h1)化合物中的PG为氨基保护基。氨基保护基包括但不限于：叔丁氧基羰基(Boc)；芳基甲氧羰基，苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。上保护和脱保护的方法可参考本领域熟知的常规方法。
本发明公开的式(I)化合物以及化合物的制备方法、药物组成和治疗方案，本领域技术 人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的产品、方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
与现有技术相比，本发明的主要优点在于：
(1)本发明化合物对EGFR T790M突变型(特别是EGFR T790M/L858R双突变型)酶和细胞具有高抑制活性，且对EGFR野生型(EGFR WT)酶和细胞具有低抑制活性，因此具有高选择抑制性。
(2)本发明化合物对EGFR双突变型酶和细胞表现高选择抑制性的同时，还具有低非特异性细胞毒性。
(3)与其他已知的EGFR突变抑制剂相比，本发明化合物还显示出有利的物理性质(例如，较高的水溶性)，有利的毒性特征(例如较低的hERG阻断倾向)，有利的代谢特征(例如，较好的药代动力学特征，如生物利用度)。
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，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，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。
除非另行定义，本文所用的术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或同等的方法及材料皆可应用于本发明中。
试剂与仪器
1HNMR：Bruker AVANCE-400核磁仪，内标为四甲基硅烷(TMS)。
LC-MS：Agilent 1200 HPLC System/6140MS液质联用质谱仪(生产商：安捷伦)，柱子WatersX-Bridge，150×4.6mm，3.5μm。
制备高效液相色谱(pre-HPLC)：Waters PHW007，柱子XBridge C18，4.6*150mm，3.5um。
采用ISCO Combiflash-Rf75或Rf200型自动过柱仪，Agela 4g、12g、20g、40g、80g、120g一次性硅胶柱。
已知的起始原料可以采用或按照本领域已知的方法来合成，或可以购自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技 (Accela ChemBio Inc)和达瑞化学品等公司。
若无特殊说明，实施例中的反应均在氮气氛或氩气氛下进行。
若无特殊说明，实施例中的溶液为水溶液。
实施例中，反应进程的监测可采用薄层色谱法(TLC)，化合物纯化可采用柱层析。柱层析或TLC所用的展开剂体系可选自：二氯甲烷和甲醇体系、正己烷和乙酸乙酯体系、石油醚和乙酸乙酯体系和丙酮体系等，溶剂的体积比根据化合物的极性不同而进行调节。
DMF：二甲基甲酰胺，DMSO：二甲基亚砜，THF：四氢呋喃，DIEA：N,N-二异丙基乙胺，EA：乙酸乙酯，PE：石油醚，BINAP：(2R,3S)-2,2'-双二苯膦基-1,1'-联萘。NBS(N-溴代丁二酰亚胺)、NCS(N-氯代丁二酰亚胺)、Pd2(dba)3(三(二亚苄基丙酮)二钯)、Pd(dppf)Cl2([1,1'-双(二苯基磷)二茂铁]二氯化钯)。
如本文所用，室温是指约20-30℃。
化合物a1的制备
Figure PCTCN2015085089-appb-000024
步骤a:
将反应底物a1-1(10.6g,58mmol)放置于500mL的单口反应瓶中，加入四氢呋喃/水(100mL/60mL)混合溶液使底物溶解。在室温条件下，向搅拌的反应瓶中依次加入氯化铵(15.5g,292mmol)和还原铁粉(26g,467mmol)，随后将反应体系加热至65摄氏度并持续搅拌3小时。通过TLC检测反应进度，待底物完全反应后，过滤除去多余的铁粉，滤饼用乙酸乙酯淋洗三次。滤液用乙酸乙酯/水体系萃取三次，分离出有机层，经水洗，饱和食盐水洗涤，无水硫酸钠干燥后，减压浓缩得到化合物a1-2(8.0g)，直接用于下一步反应。产率：93％；纯度：90％；MS m/z(ESI):142.0[M+H]+。
步骤b:
将化合物a1-2(8.0g,43mmol)放置于500mL的单口反应瓶中，在匀速搅拌条件下加入浓硫酸(100mL)使底物溶解。在零下20摄氏度条件下，向搅拌的反应瓶中缓慢滴加浓硝酸(6.15mL,48mmol)，并保持该温度搅拌5分钟。通过TLC检测反应进度，待底物完全反应后，倒入冰水中。保持零下20摄氏度冰浴条件，向反应体系中缓慢加入氢氧化钠/水溶液(150mL/300mL)，调节PH值至8-9。中和完成后的反应液用乙酸乙酯/水体系萃取三次，分离出有机层，经水洗，饱和食盐水洗涤，无水硫酸钠干燥后，减压浓缩得到化合物4-氟-2-甲氧基-5-硝基苯胺a1(8.7g)，直接用于下一步反应。产率：80％；纯度：100％；MS m/z(ESI)：187.0[M+H]+；1H NMR(400MHz,DMSO-d6):δ7.34(d,J＝7.8Hz,1H),7.04(d,J＝13.4Hz,1H),5.25(brs,2H),3.90(s,3H)。
化合物a的制备
Figure PCTCN2015085089-appb-000025
步骤1：
将4-氟-2-甲氧基-5-硝基苯胺a1(11.16g，60mmol)溶于150毫升二氯甲烷，加入二碳酸二叔丁酯(15.60g，72mmol)、三乙胺(12.24g，120mmol)和4-二甲氨基吡啶(0.74g，6mmol)室温下搅拌反应18小时。通过TLC检测反应进度，待底物反应完全后，减压浓缩反应液，柱层析[PE:EA体积比＝80:20]分离纯化得到目标产物a2(12.56g，73％)。MS m/z(ESI):285[M-H]+。(注：以下实验中柱层析均为体积比)
步骤2：
将反应底物叔丁基4-氟-2-甲氧基-5-硝基苯基氨基甲酸叔丁酯a2(11.46g，40mmol)溶于60毫升N,N-二甲基乙酰胺，加入N,N,N'-三甲基乙二胺(4.90g，48mmol)，N,N-二异丙基乙基胺(7.74g，60mmol)，加热至90摄氏度搅拌反应6小时。通过TLC检测反应进度，待底物反应完全后，将反应液冷却至室温，倒入冰水中，用乙酸乙酯萃取，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩得目标产物a3(12.51g，85％)。直接用于下一步反应。MS m/z(ESI):369[M+H]+。
步骤3：
将4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基氨基甲酸叔丁酯a3(12g，32.6mmol)溶于200毫升甲醇，加入1.0g 10％Pd/C。以氢气置换空气后，于室温下以氢气球加氢，搅拌反应1小时。通过TLC检测反应进度，待底物反应完全后，经砂芯漏斗抽滤，以少量甲醇洗涤滤饼，将滤液浓缩得到目标产物a4(10.70g，97％)。直接用于下一步反应。MS m/z(ESI):339[M+H]+。
步骤4：
将5-氨基-4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基苯基氨基甲酸叔丁酯a4(10.1g，30mmol)和三乙胺(6.12g，60mmol)溶于200毫升二氯甲烷，冷却至0摄氏度，加入丙烯酰氯(3.24g，36mmol)，在氮气保护下，室温搅拌3小时。通过TLC检测反应进度，待底物反应完全后，以饱和碳酸氢钠水溶液、饱和食盐水顺次洗涤，无水硫酸钠干燥，抽滤，经减压浓缩得到目标产物a5(9.64g，82％)。直接用于下一步反应。MS m/z(ESI):393[M+H]+。
步骤5：
将5-丙烯酰胺基-4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基苯基氨基甲酸叔丁酯 a5(9.41g，24mmol)溶解在100毫升二氯甲烷中，冷却到0摄氏度，加入20毫升三氟乙酸，在氮气保护下室温搅拌18小时。通过TLC检测反应进度，待底物反应完全后，减压浓缩反应液。将残留物以300毫升二氯甲烷溶解，用饱和碳酸氢钠水溶液、饱和食盐水顺次洗涤，无水硫酸钠干燥，抽滤，减压浓缩得到粗品。经柱层析[DCM:MeOH体积比＝10:1]纯化得到目标产物N-(5-氨基-2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧苯基)丙烯酰胺a(3.26g，46.5％)。MS m/z(ESI):293[M+H]+。
化合物b的制备
Figure PCTCN2015085089-appb-000026
制备方法同化合物a，不同的是将化合物a制法中步骤2中的N,N,N'-三甲基乙二胺换成4-二甲氨基哌啶。
化合物c的制备
Figure PCTCN2015085089-appb-000027
在0摄氏度下，向化合物2,4-二氯-7H-吡咯并[2,3-d]嘧啶c-1(1g，5.32mmol)的DMF(40ml)溶液中加入氢化钠(420mg，10.64mmol)，反应混合物搅拌30分钟后，加入2-(三甲硅基)乙氧基甲基氯(1.34g，7.98mmol)。反应混合物0摄氏度下搅拌3小时。反应结束后，0摄氏度下加水淬灭反应。用乙酸乙酯和水萃取，有机相用水和饱和氯化钠溶液洗涤，减压浓缩有机相得2g油状物2,4-二氯-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶c，不经纯化直接下一步。MS m/z(ESI):318[M+H]+。
化合物d的制备
Figure PCTCN2015085089-appb-000028
将化合物c(1.6g，4.57mmol)、3-羟基氮杂环丁烷-1-羧酸叔丁酯(881mg，5.09mmol)和碳酸铯(3g，9.21mmol)加入30ml乙腈中，反应混合物在氩气氛下80摄氏度搅拌5小时。用TLC板和LC-MS跟踪反应。反应结束后，将反应液过滤，用二氯甲烷洗涤，浓缩滤液得粗产品，粗产品经combiflash(PE:EA体积比＝100:0-80:20)纯化得到化合物3-(2-氯-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶-4-基氧基)氮杂环丁烷-1-甲酸叔丁酯d(1.76g，产率84.53％)。MS m/z(ESI):455.1[M+H]+。
化合物e的制备
Figure PCTCN2015085089-appb-000029
搅拌下，向化合物c(100mg，0.31mmol)的乙腈/水(5/1ml)溶液中加入1-甲基-4-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-1H-吡唑(66mg,0.31mmol)、Pd(dppf)Cl2(23mg,0.031mmol)和碳酸钠(66mg,0.62mmol)，反应混合物经氩气置换三次后在80摄氏度下搅拌4小时。待底物完全反应后，加水淬灭，向反应混合物中加入乙酸乙酯(150ml)分层后，水相用乙酸乙酯(50mL×2)萃取两次，合并的有机相用Na2SO4干燥后浓缩，得到的粗产品经Combi-flash柱层析分离纯化得目标产物2-氯-4-(1-甲基-1H-吡唑-4-基)-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶e(100mg，产率70％)。MS m/z(ESI)：364[M+H]+。
化合物f的制备
Figure PCTCN2015085089-appb-000030
搅拌下，向化合物c(100mg，0.31mmol)的THF(3ml)溶液中加入吗啉(25mg,0.26mmol)和三乙胺(41mg,0.4mmol)，反应混合物室温下搅拌过夜，反应结束后，加水，用乙酸乙酯萃取，有机相用无水Na2SO4干燥，减压浓缩得化合物2-氯-4-吗啉代-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶f(100mg，70％)。不经纯化直接下一步。MS m/z(ESI)：369[M+H]+。
化合物g的制备
Figure PCTCN2015085089-appb-000031
搅拌下，向化合物c(500mg，1.58mmol)的乙腈/水(10/2ml)溶液中加入1-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-1H-吡咯并[2,3-b]吡啶(370mg,1.43mmol)、Pd(dppf)Cl2(106mg,0.14mmol)和碳酸钠(304mg,2.87mmol)，反应混合物在氩气氛下80摄氏度下搅拌4小时。待底物完全反应后，加水淬灭，向反应混合物中加入乙酸乙酯(150ml)分层后，水相用乙酸乙酯(50mL×2)萃取两次，合并的有机相用Na2SO4干燥后浓缩，得到的粗产品经Combi-flash柱层析分离纯化得210mg目标产物2-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶g。MS m/z(ESI)：414.1[M+H]+。
化合物h的制备
Figure PCTCN2015085089-appb-000032
步骤1：
以化合物c(500mg，1.57mmol)和化合物1-(苯基磺酰基)-3-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-1H-吲哚(605mg，1.57mmol)为原料，参照化合物g的制备方法制备，得化合物h-1(400mg，产率60％)。MS m/z(ESI)：539[M+H]+。
步骤2：
向化合物h-1(350mg，0.65mmol)的20ml甲醇溶液中加入氢氧化钾(365mg，6.5mmol)。反应混合物室温搅拌4小时。反应结束后，反应混合物用2M HCl溶液调PH为7。浓缩，加水和乙酸乙酯萃取，有机相用水和饱和食盐水洗涤，减压浓缩得300mg目标物h-2。不纯化直接下一步。MS m/z(ESI)：399.2[M+H]+。
步骤3：
0摄氏度下，向氢化钠(126mg，3.16mmol)的THF(8ml)溶液中加入化合物h-2(630mg，1.58mmol)。反应混合物在0摄氏度下搅拌20分钟。加入碘甲烷(337mg，2.37mmol)。反应混合物室温下搅拌2小时。反应结束后，0摄氏度下加水淬灭，用乙酸乙酯萃取，用水和饱和氯化钠溶液洗涤，浓缩得粗产品。经Combi-flash柱层析分离纯化得340mg目标产物2-氯-4-(1-甲基-1H-吲哚-3-基)-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶h。MS m/z(ESI)：413.3[M+H]+。
化合物i的制备
Figure PCTCN2015085089-appb-000033
向化合物c(500mg，1.58mmol)的乙腈/水(10/2ml)溶液中加入吡啶-3-基硼酸(173mg,1.43mmol)、Pd(dppf)Cl2(116mg,0.14mmol)和碳酸钠(344mg,2.87mmol)，反应混合物100摄氏度下微波反应15分钟。待底物完全反应后，加水淬灭，向反应混合物中加入乙酸乙酯(150ml)分层后，水相用乙酸乙酯(50mL×2)萃取两次，合并的有机相用Na2SO4干燥后浓缩，得到的粗产品经Combi-flash柱层析分离纯化得280mg目标产物2-氯-4-(吡啶-3- 基)-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶i。MS m/z(ESI)：361.1[M+H]+。
化合物j的制备
Figure PCTCN2015085089-appb-000034
以化合物c和苯硼酸为原料，参照化合物i的制备方法制备。MS m/z(ESI):360[M+H]+。
化合物k的制备
Figure PCTCN2015085089-appb-000035
步骤1：
向化合物d(500mg，1.10mmol)和化合物a(321mg，1.10mmol)的15毫升1,4-二氧六环溶液中加入Pd2(dba)3(101mg，0.11mmol)、BINAP(137mg，0.22mmol)和碳酸铯(718mg，2.21mmol)。反应混合物140摄氏度下微波反应25分钟。反应结束后，过滤反应混合物，用二氯甲烷洗涤，滤液减压浓缩后得粗产品，经制备液相分离纯化得700mg化合物k-1。MS m/z(ESI):711.3[M+H]+。
步骤2：
在0摄氏度下,将三氟乙酸(1.124g,9.86mmol)加入到化合物k-1(700mg,0.986mmol)的10毫升的二氯甲烷溶液中,室温下剧烈搅拌2小时。反应结束后,将反应液在减压条件下蒸干,得到1.5g化合物k-2,产物直接用于下一步。MS m/z(ESI):611[M+H]+。
步骤3：
在0摄氏度下,将三乙胺(747mg,7.39mmol)加入到化合物k-2(750mg,0.49mmol)的5毫升二氯甲烷溶液中,0摄氏度下剧烈搅拌30分钟后加入甲磺酰氯(56mg,0.49mmol),0摄氏度下剧烈搅拌2小时。反应结束后,加水稀释,用二氯甲烷/水体系萃取三次,有机层 减压浓缩,经combiflash纯化得到90mg化合物k。MS m/z(ESI):689.3[M+H]+。
化合物m的制备
Figure PCTCN2015085089-appb-000036
向化合物e(800mg，2.2mmol)和化合物a1(410mg，2.2mmol)的10毫升1,4-二氧六环溶液中加入Pd2(dba)3(202mg，0.22mmol)、BINAP(274mg，0.44mmol)和碳酸铯(1.437g，4.41mmol)。反应混合物140摄氏度下微波反应25分钟。反应结束后，过滤反应混合物，用二氯甲烷洗涤，滤液减压浓缩后得粗产品，经combiflash[PE:EA＝100:0]纯化得到840mg化合物N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡唑-4-基)-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶-2-胺m。MS m/z(ESI):514.2[M+H]+。
化合物n的制备
Figure PCTCN2015085089-appb-000037
步骤1：
在氢气氛下，将化合物n-1(5.0g，0.0292mmol)、二甲胺盐酸盐(4.77g，0.0584mmol)、2.1g Pd/C和2.5ml醋酸加入到100ml甲醇中，氢气置换后室温反应48小时。反应结束后过滤，浓缩得粗产品。加饱和NaHCO3和乙酸乙酯萃取，盐水洗涤，浓缩得目标物n-2(5.0g，产率85％)。MS m/z(ESI):201[M+H]+。
步骤2：
0摄氏度下，向化合物n-2(5.0g，0.025mmol)的二氯甲烷(100ml)溶液中加入盐酸/1,4-二氧六环溶液(4M)。反应混合物室温搅拌3小时。反应结束后浓缩，得目标物N,N-二甲基氮杂-3-胺二盐酸盐n(3.5g，82％)。MS m/z(ESI):173[M+H]+。
化合物p的制备
Figure PCTCN2015085089-appb-000038
步骤1：
向化合物c-1(1g，5.32mmol)的80ml二氯甲烷溶液中加入NBS(1.04g，5.85mmol)。反应混合物室温搅拌4小时。反应结束后减压浓缩，经combiflash纯化得到化合物p-1(900mg，产率64％)。MS m/z(ESI):266[M+H]+。
步骤2：
零下70摄氏度下，向化合物p-1(800m，3mmol)的110ml THF溶液中缓慢滴加正丁基锂(576mg，9mmol)。反应混合物搅拌1小时后，在零下70摄氏度加入碘甲烷(511mg，3.6mmol)，继续在该温度下搅拌1.5小时。反应结束后，零下70摄氏度加饱和氯化铵溶液淬灭。用乙酸乙酯萃取，干燥，减压浓缩。经combiflash纯化得到320mg化合物p-2。MS m/z(ESI):202[M+H]+。
步骤3：
以化合物p-2为原料，参照化合物c的制备方法，制备得化合物p-3。MS m/z(ESI):332[M+H]+。
步骤4：
以化合物p-3为原料，参照化合物e的制备方法，制备得化合物2-氯-5-甲基-4-(1-甲基-1H-吡唑-4-基)-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶p。MS m/z(ESI):378.2[M+H]+。
化合物r的制备
Figure PCTCN2015085089-appb-000039
向化合物e(410mg，1.12mmol)的10ml乙腈溶液中加入NCS(180mg，1.35mmol)。反应混合物在70摄氏度下搅拌2小时。反应结束后浓缩，经combiflash纯化得到化合物2,5-二氯-4-(1-甲基-1H-吡唑-4-基)-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶r(300mg，产率67％)。MS m/z(ESI):398[M+H]+。
化合物s的制备
Figure PCTCN2015085089-appb-000040
以化合物c和2-环己烯基氧基-4,4,5,5-四甲基-1,3,2-二氧硼烷为原料，参照化合物g的制备方法制备。得化合物2-氯-4-环己烯基-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶s。MS m/z(ESI):364[M+H]+。
化合物t的制备
Figure PCTCN2015085089-appb-000041
步骤1-2
以化合物e为原料，参照实施例3的类似方法制备得到化合物t-2。MS m/z(ESI):234[M+H]+。
步骤3：
冰浴下，向化合物t-2(23.3mg，0.1mmol)的5ml DMF溶液中一次性加入氢化钠(5mg，0.12mmol)，反应混合物0摄氏度搅拌20分钟后，滴加碘甲烷(28.4mg，0.2mmol)。反应液升至室温搅拌2小时。反应结束后，加水淬灭，乙酸乙酯萃取，有机相无水硫酸钠干燥后浓缩，经combiflash纯化得到化合物2-氯-7-甲基-4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-d]嘧啶t(20mg，0.08mmol)，MS m/z(ESI):248[M+H]+。
化合物u的制备
Figure PCTCN2015085089-appb-000042
步骤1
0摄氏度下，向化合物d(60mg，0.132mmol)的5ml甲醇溶液中加入36％盐酸(0.5ml，4.9mmol)，反应液升至室温搅拌18小时，反应结束后浓缩，得化合物u-1的粗产品，不经纯化直接用于下一步反应。MS m/z(ESI):355[M+H]+。
步骤2
0摄氏度下，向化合物u-1(460mg，1.18mmol)的20ml二氯甲烷溶液中加入N,N-二异丙基乙胺(610mg，4.72mmol)，搅拌5分钟后，0摄氏度滴加甲磺酰氯(203mg，1.77mmol)。反应混合物升至室温搅拌1小时。反应结束后浓缩，得粗品。经combiflash纯化得到化合物2-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)-7-((2-(三甲基甲硅烷基)乙氧基)甲基)-7H-吡咯并[2,3-d]嘧啶u(255mg，产率50％)。MS m/z(ESI):433[M+H]+。
化合物v的制备
Figure PCTCN2015085089-appb-000043
制备方法同化合物a，不同的是将化合物a制法中步骤2中的N,N,N'-三甲基乙二胺换成1-甲基哌嗪。MS m/z(ESI):291[M+H]+。
化合物w的制备
Figure PCTCN2015085089-appb-000044
以化合物w1和N,N,N'-三甲基乙二胺为起始原料，参照化合物a中步骤2和3进行制备，得化合物w，收率98％。MS m/z(ESI):224[M+H]+。
实施例1 N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺(1)的制备
Figure PCTCN2015085089-appb-000045
步骤1：
将化合物e(100mg,0.27mmol)、化合物a(80mg,0.27mmol)、Pd2(dba)3(25mg,0.027mmol)、xantphos(28mg,0.054mmol)和碳酸铯(180mg,0.54mmol)置于4毫升封管反应器中，加入1,4-二氧六环5毫升，以氩气置换空气1分钟，加热至160摄氏度封管反应15分钟。将反应液过滤，以二氯甲烷洗涤滤饼，浓缩滤液得到粗产品，经制备液相分离纯化得目标产物1-a(200mg,产率80％)。MS m/z(ESI):620[M+H]+。
步骤2：
向化合物1-a(100mg，0.16mmol)的5毫升二氯甲烷溶液中加入2ml三氟乙酸，反应混合物室温下搅拌4小时。反应结束后，将反应液在减压条件下蒸干，得到化合物1-b(100mg)，产物直接用于下一步。MS m/z(ESI):520[M+1]+。
步骤3：
向化合物1-b(100mg，0.16mmol)的乙醇/水(10/2ml)溶液中加入碳酸钾(250mg，1.6mmol)，反应混合物室温搅拌2小时。反应结束后，将反应液在减压条件下蒸干，加乙酸乙酯萃取，无水Na2SO4干燥后浓缩，得粗产品。经制备液相分离纯化得标题化合物1(100mg，产率90％)，为黄色固体。
MS m/z(ESI):490[M+H]+。1H NMR(400MHz,CDCl3)δ10.24(s,1H),9.91(s,1H),9.06(s,1H),8.95(s,1H),8.20(s,1H),7.63(s,1H),7.05(dd,J＝3.5,2.0Hz,1H),6.78(s,1H),6.66(dd,J＝3.5,1.8Hz,1H),6.45–6.24(m,2H),5.71(d,J＝11.8Hz,1H),4.06(s,3H),3.90(s,3H),2.91(s,2H),2.71(s,3H),2.26(overlap,8H)。
实施例2 N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-吗啉代-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺(2)的制备
Figure PCTCN2015085089-appb-000046
以化合物f和化合物a为原料，参照实施例1进行制备，得标题化合物2，为黄色固体。MS m/z(ESI):495[M+H]+；1H NMR(400MHz,CDCl3)δ10.04(s,1H),9.62(s,1H),9.12(s,1H),7.24(s,1H),6.74(dd,J＝3.5,2.1Hz,1H),6.68(s,1H),6.35–6.29(m,2H),6.26(s,1H),5.60(d,J＝11.8Hz,1H),3.95–3.89(m,4H),3.82–3.80(m,4H),3.80(s,3H),2.81(t, J＝5.6Hz,2H),2.62(s,3H),2.18(overlap,8H)。
实施例3 N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺(3)的制备
Figure PCTCN2015085089-appb-000047
步骤1：
在0摄氏度下，向化合物k(90mg，0.13mmol)的3ml二氯甲烷溶液中加入3ml三氟乙酸，反应混合物在40摄氏度下搅拌3小时，反应结束后，浓缩反应液得粗产品，不纯化直接进行下一步反应。
步骤2：
在0摄氏度下，向步骤1中的粗产品中加入3ml甲醇溶解，再加入3ml氨水，反应混合物在40摄氏度下搅拌3小时，反应结束后，浓缩反应液得粗产品，经制备液相分离纯化得6mg标题化合物3，为白色固体。MS m/z(ESI):559.2[M+H]+。1H NMR(400MHz,DMSO-d6)δ11.47(s,1H),10.19(s,1H),8.95(s,1H),7.63(s,1H),7.06–7.02(m,1H),7.00(s,1H),6.39(dd,J＝16.9,9.9Hz,1H),6.34(dd,J＝3.5,1.9Hz,1H),6.33–6.25(m,1H),5.76(d,J＝12.0Hz,1H),5.68(s,1H),4.33–4.23(m,2H),3.99(dd,J＝9.7,4.8Hz,2H),3.86(s,3H),3.03(s,3H),2.85(d,J＝6.1Hz,2H),2.70(s,3H),2.27(t,J＝5.6Hz,2H),2.20(s,6H)。
实施例4 N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺(4)的制备
Figure PCTCN2015085089-appb-000048
步骤1：
向化合物g(210mg，0.51mmol)和化合物a(148mg，0.51mmol)的10毫升1,4-二氧六环溶液中加入Pd2(dba)3(46mg，0.05mmol)、BINAP(63mg，0.12mmol)和碳酸铯(332mg，1.02mmol)。反应混合物140摄氏度下微波反应30分钟。反应结束后，过滤反应混合物，用二氯甲烷洗涤，滤液减压浓缩后得粗产品，经combiflash[PE:EA＝90:10]纯化得到270mg化合物4-a。MS m/z(ESI):670.5[M+H]+。
步骤2和步骤3
以化合物4-a为原料，参照实施例3中步骤1和2的方法进行合成，得41mg标题化合物4，为浅黄色固体。MS m/z(ESI):540.2[M+H]+；1H NMR(400MHz,DMSO-d6)δ11.61(s,1H),10.17(s,1H),9.24(s,1H),9.09(d,J＝1.9Hz,1H),8.97(s,1H),7.71(s,1H),7.62(d,J＝3.4Hz,1H),7.29(s,1H),7.02(s,1H),6.80(s,1H),6.61(d,J＝3.4Hz,1H),6.42(d,J＝9.6Hz,1H),6.33(d,J＝14.6Hz,1H),5.80(d,J＝10.0Hz,1H),3.90(s,3H),3.90(s,3H),2.88(s,2H),2.71(s,3H),2.29(s,2H),2.21(s,6H)。
实施例5 N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-甲基-1H-吲哚-3-基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺(5)的制备
Figure PCTCN2015085089-appb-000049
以化合物h和化合物a为原料，参照实施例4的方法进行制备，得标题化合物5的粗品，将得到的粗品经制备液相分离纯化[H2O(0.05vol％甲酸):CH3CN＝85:15～90:10]得到标题化合物5的甲酸盐，为黄色固体。MS m/z(ESI):539[M+H]+；1H NMR(400MHz,CDCl3)δ10.05(s,1H),9.92(s,1H),9.59(s,1H),8.53(s,1H),8.48(d,J＝7.5Hz,1H),8.04(s,1H),7.77(s,1H),7.41–7.37(m,1H),7.32(d,J＝5.8Hz,1H),7.30–7.28(m,1H),7.04(d,J＝3.3Hz,1H),6.84(dd,J＝16.9,10.2Hz,1H),6.68(s,1H),6.65(d,J＝3.3Hz,1H),6.44(dd,J＝16.9,1.6Hz,1H),5.74(dd,J＝10.2,1.5Hz,1H),3.92(s,3H),3.90(s,3H),3.16(t,J＝5.7Hz,2H),2.90(t,J＝5.6Hz,2H),2.63(s,3H),2.62(overlap,6H)。
实施例6 N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(吡啶-3-基)-7H-吡 咯并[2,3-d]嘧啶-2基氨基)苯基)丙烯酰胺(6)的制备
Figure PCTCN2015085089-appb-000050
以化合物i和化合物a为原料，参照实施例4的方法进行制备，得标题化合物6，为浅黄色固体。MS m/z(ESI):487.2[M+H]+；1H NMR(400MHz,DMSO-d6)δ11.87(s,1H),10.31(s,1H),9.51(s,1H),9.31(s,1H),8.88(t,J＝5.8Hz,2H),7.95(s,1H),7.74(dd,J＝7.6,5.0Hz,1H),7.48(s,1H),7.17(s,1H),6.93(d,J＝2.8Hz,1H),6.57(dd,J＝16.9,9.9Hz,1H),6.46(d,J＝16.6Hz,1H),5.94(d,J＝10.0Hz,1H),4.04(s,3H),3.04(s,2H),2.87(s,3H),2.46(s,2H),2.37(s,6H)。
实施例7 N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-苯基-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺(7)的制备
Figure PCTCN2015085089-appb-000051
以化合物j和化合物a为原料，参照实施例4的方法进行制备，得标题化合物7，为 黄色固体。MS m/z(ESI):486.2[M+H]+；1H NMR(400MHz,DMSO-d6)δ11.79(s,1H),10.30(s,1H),9.36(s,1H),8.44(dd,J＝7.4,2.0Hz,2H),7.86(s,1H),7.75–7.69(m,3H),7.44(dd,J＝3.5,2.1Hz,1H),7.16(s,1H),6.88(dd,J＝3.5,1.3Hz,1H),6.58(dd,J＝16.9,9.9Hz,1H),6.47(dd,J＝16.9,2.2Hz,1H),5.94(dd,J＝9.9,2.2Hz,1H),4.05(s,3H),3.03(s,2H),2.86(s,3H),2.45(s,2H),2.37(s,6H)。
实施例8 N-(5-(4-环己烯基-7H-吡咯并[2,3-d]嘧啶-2-基氨基)-2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基苯基)丙烯酰胺(8)的制备
Figure PCTCN2015085089-appb-000052
以化合物s和化合物a为原料，参照实施例4的方法进行制备，得标题化合物8，为黄色固体。MS m/z(ESI):490.3[M+H]+。
实施例9 N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-D]嘧啶-2-基氨基)苯基)丙烯酰胺(9)的制备
步骤1：
将化合物e(100mg,0.275mmol)、化合物b(87mg,0.275mmol)、Pd2(dba)3(26mg,0.028mmol)、xantphos(32mg,0.056mmol)和碳酸铯(185mg,0.56mmol)置于4毫升封管反应器中，加入1,4-二氧六环5毫升，以氩气置换空气1分钟，加热至160摄氏度封管反应15分钟。将反应液过滤，以二氯甲烷洗涤滤饼，浓缩滤液得到粗产品，经制备液相分离纯化得目标产物9-a(200mg,产率70％)。MS m/z(ESI):646[M+H]+。
步骤2和3
以化合物9-a为原料，参照实施例3的步骤1和2，制备得标题化合物9的粗品，将得到的粗品经制备液相分离纯化[H2O(0.05％vol甲酸):CH3CN＝80:20～90:10]得到标题化合物9的甲酸盐，为黄色固体。MS m/z(ESI):516[M+H]+。1H NMR(400MHz,DMSO-d6)δ11.46(s,1H),9.16(s,1H),9.08(s,1H),8.90(s,1H),8.35(s,1H),8.18(s,1H),7.46(s,1H),7.22(dd,J＝3.4,2.3Hz,1H),6.86(s,1H),6.81(dd,J＝3.4,1.5Hz,1H),6.72(dd,J＝17.1,10.0Hz,1H),6.33(d,J＝16.6Hz,1H),5.79(d,J＝11.0Hz,1H),3.97(s,3H),3.91(s,3H), 3.05(d,J＝11.0Hz,2H),2.70(t,J＝11.1Hz,2H),2.56(overlap,1H),2.44(s,6H),1.92(d,J＝10.6Hz,2H),1.77(m,2H)。
Figure PCTCN2015085089-appb-000053
实施例10 N-(2-(2-(二甲基氨基)乙氧基)-4-甲氧基-5-(4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-d]嘧啶-2基氨基)苯基)丙烯酰胺(10)的制备
Figure PCTCN2015085089-appb-000054
步骤1：
0摄氏度下，向2-(二甲基氨基)乙醇(104mg，1.17mmol)的10ml THF溶液中加入氢化钠(69mg，1.72mmol)，0摄氏度搅拌30分钟后，加入化合物m(400mg，0.78mmol)。反应混合物室温搅拌4小时。反应结束后，加入乙酸乙酯和水萃取，有机相用水和饱和氯化钠溶液洗涤，浓缩有机相，经combiflash[二氯甲烷:甲醇＝90:10]纯化得到400mg化合物10-a。MS m/z(ESI):583.2[M+H]+。
步骤2：
将Pd/C(1mg)加入到化合物10-a(10mg，0.017mmol)的5毫升甲醇溶液中。室温下，在H2氛围中搅拌4小时。反应结束后，过滤，滤液浓缩，得10mg化合物10-b，产物直接用于下一步。MS m/z(ESI):553.2[M+H]+。
步骤3：
在0摄氏度下，将丙烯酰氯(137mg，0.74mmol)与三乙胺(203mg，2.01mmol)加入到化合物10-b(60mg，0.117mmol)的15毫升二氯甲烷溶液中，0摄氏度下搅拌2小时。反应结束后，加水稀释，用二氯甲烷/水体系萃取三次，有机层减压浓缩得到粗产物。经制备液相分离纯化得化合物10-c。MS m/z(ESI):607.2[M+H]+。
步骤4和5：
以化合物10-c为原料，参照实施例3中的步骤1和2，制备得标题化合物10，为浅黄色固体。
MS m/z(ESI):490[M+H]+。1H NMR(400MHz,DMSO-d6)δ11.45(s,1H),9.90(s,1H),9.18(s,1H),8.89(s,1H),8.34(s,1H),7.44(s,1H),7.25–7.16(m,1H),6.93(s,1H),6.85–6.77(m,1H),6.48(dd,J＝17.0,10.0Hz,1H),6.39–6.23(m,1H),5.85–5.74(m,1H),4.16(t,J＝5.5Hz,2H),3.97(s,3H),3.91(s,3H),2.55(d,J＝5.6Hz,2H),2.28(s,6H)。
实施例11 N-(2-(3-(二甲基氨基)氮杂环丁烷-1-基)-4-甲氧基-5-(4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺甲酸盐(11)的制备
Figure PCTCN2015085089-appb-000055
步骤1：
将化合物m(400mg，0.78mmol)、化合物n(400mg，2.3mmol)和碳酸钾(861mg，6.24mmol)加入到25ml DMF中，100摄氏度下搅拌2小时，反应结束后过滤，滤液中加入乙酸乙酯，萃取，浓缩有机相。经Combi-flash柱层析纯化后得化合物11-a(310mg，产率67％)。MS m/z(ESI):594[M+H]+。
步骤2-5
以化合物11-a为原料，参照实施例10中步骤2-5进行制备，得标题化合物11，为浅黄色固体。MS m/z(ESI):488[M+H]+。1HNMR(400MHz，DMSO-d6)δ11.45(s,1H),9.33(s,1H),8.65(s,1H),8.28(s,1H),8.23(s,3H),7.34(s,1H),7.20–7.14(m,1H),6.76(d,J＝2.0 Hz,1H),6.54(dd,J＝17.0,10.2Hz,1H),6.32–6.23(m,2H),5.73(dd,J＝10.2,2.0Hz,1H),3.96–3.89(m,8H),3.53(d,J＝6.7Hz,2H),3.07–3.04(m,1H),2.08(s,6H)。
实施例12 N-(4-甲氧基-5-(4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(12)的制备
Figure PCTCN2015085089-appb-000056
以化合物m和1-甲基哌嗪为原料，参照实施例11的方法制备，得标题化合物12，为黄色固体。MS m/z(ESI):488[M+H]+。1HNMR(400MHz，DMSO-d6)δ11.46(s,1H),9.13(s,1H),9.06(s,1H),8.89(s,1H),8.34(s,1H),7.46(s,1H),7.22(dd,J＝3.5,2.3Hz,1H),6.88(s,1H),6.81(dd,J＝3.6,1.7Hz,1H),6.66(dd,J＝17.0,10.2Hz,1H),6.31(d,J＝16.9Hz,1H),5.78(d,J＝11.6Hz,1H),3.97(s,3H),3.92(s,3H),2.85(t,J＝4.6Hz,4H),2.52(s,4H),2.26(s,3H)。
实施例13 N-(5-(5-氯-4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)-2-(4-(二甲基氨基基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺(13)的制备
以化合物r和化合物b为原料，参照实施例9进行制备，得标题化合物13，为黄色固体。MS m/z(ESI):550[M+H]+。1HNMR(400MHz，DMSO-d6)δ11.81(s,1H),9.06(s,1H),8.89(s,1H),8.64(s,1H),8.19(s,1H),7.64(s,1H),7.38(d,J＝2.1Hz,1H),6.85(s,1H),6.71(dd,J＝16.8,10.1Hz,1H),6.30(d,J＝17.0Hz,1H),5.78(d,J＝11.5Hz,1H),3.96(s,3H),3.88(s,3H),3.03(d,J＝11.5Hz,2H),2.67(t,J＝11.1Hz,2H),2.31(s,1H),2.29(s,6H),1.86(d,J＝10.6Hz,2H),1.70(d,J＝8.8Hz,2H)。
Figure PCTCN2015085089-appb-000057
实施例14 N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(5-甲基-4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺(14)的制备
Figure PCTCN2015085089-appb-000058
以化合物p和化合物b为原料，参照实施例9进行制备，得标题化合物14，为浅黄色固体。MS m/z(ESI):530.3[M+H]+。1HNMR(400MHz，DMSO-d6)1H NMR(400MHz,DMSO-d6)δ11.18(s,1H),9.03(d,J＝3.4Hz,2H),8.52(s,1H),8.01(s,1H),7.43(s,1H),6.95(s,1H),6.84(s,1H),6.70(dd,J＝16.9,10.1Hz,1H),6.30(d,J＝16.9Hz,1H),5.78(d,J＝10.3Hz,1H),3.95(s,3H),3.89(s,3H),3.01(d,J＝11.3Hz,2H),2.66(t,J＝10.9Hz,2H),2.30(s,3H),2.24(s,6H),2.23–2.18(m,1H),1.84(d,J＝11.3Hz,2H),1.68(d,J＝11.2Hz, 2H)。
实施例15 N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-甲基-1H-吲哚-3-基)-7H-吡咯并[2,3-D]嘧啶-2-基氨基)苯基)丙烯酰胺(15)的制备
以化合物h和化合物b为原料，参照实施例1的方法制备。得标题化合物15，为黄色固体。MS m/z(ESI):565[M+H]+。1H NMR(400MHz,DMSO)δ11.41(s,1H),9.06(s,1H),8.64(s,1H),8.60(d,J＝7.9Hz,1H),8.42(s,1H),8.23(s,1H),7.65(s,1H),7.51(d,J＝8.1Hz,1H),7.25(t,J＝7.2Hz,1H),7.17(t,J＝6.5Hz,2H),6.85(dd,J＝3.9,2.1Hz,2H),6.68(dd,J＝16.8,10.3Hz,1H),6.20(d,J＝16.9Hz,1H),5.72(d,J＝10.5Hz,1H),3.94(s,3H),3.87(s,3H),3.09(d,J＝10.8Hz,2H),2.71(t,J＝11.2Hz,2H),2.62(s,1H),2.46(s,6H),1.94(d,J＝10.5Hz,2H),1.77(d,J＝9.7Hz,2H).
Figure PCTCN2015085089-appb-000059
实施例16 N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(7-甲基-4-(1-甲基-1H-吡唑-4-基)-7H-吡咯并[2,3-d]嘧啶-2-基氨基)苯基)丙烯酰胺(16)的制备
Figure PCTCN2015085089-appb-000060
以化合物t和化合物a为原料，参照实施例4中步骤1的方法制备。得标题化合物16，为浅黄色固体。MS m/z(ESI):504[M+H]+。1H NMR(400MHz,DMSO)δ10.23(s,1H),9.75(s,1H),8.92(s,1H),8.36(s,1H),7.56(s,1H),7.29(d,J＝4.0Hz,1H),7.04(s,1H),6.85(d,J ＝3.2Hz,1H),6.42-6.29(m,2H),5.79(dd,J＝11.4,1.6Hz,1H),3.97(s,3H),3.93(s,3H),3.79(s,3H),2.87(t,J＝4.2Hz,2H),2.70(s,2H),2.26(t,J＝4.2Hz,2H),2.20(s,6H).
实施例17 N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)-7H-吡咯并[2,3-D]嘧啶-2-基氨基)苯基)丙烯酰胺(17)的制备
以化合物u和化合物b为原料，参照实施例9的方法制备。得标题化合物17，为黄色固体。MS m/z(ESI):585[M+H]+。1H NMR(400MHz,CD3OD)δ9.18(s,1H),6.97(d,1H,J＝3.2),6.89(s,1H),6.59(dd,J＝17.6,9.6Hz,1H),6.44(d,J＝16.8,1H),6.41(d,J＝4.0,1H),5.82(d,J＝10.4Hz,1H),5.82-5.79(m,1H),4.40-4.34(m,2H),4.10-4.05(m,2H),3.93(s,3H),3.22-3.15(m,2H),2.98(s,3H),2.93(s,6H),2.87-2.82(m,2H),2.20-2.13(m,2H),2.06-1.91(m,2H).
Figure PCTCN2015085089-appb-000061
实施例18 N-(4-甲氧基-2-(4-甲基哌嗪-1-基)-5-(4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)-7H-吡咯并[2,3-d]嘧啶-2基氨基)苯基)丙烯酰胺(18)的制备
Figure PCTCN2015085089-appb-000062
以化合物u和化合物v为原料，参照实施例9的方法制备。得标题化合物18，为黄色固体。MS m/z(ESI):557[M+H]+。
实施例19至34
化合物19-34如式(I)所示，其中，取代基A、X、R0、R1、R2、R3如下表所示。化合物19-34可根据下表所列的不同结构按照与化合物1-18相类似的方法制备。所用的起始原料和中间体是本领域技术人员可根据现有方法制备得到的。
Figure PCTCN2015085089-appb-000063
Figure PCTCN2015085089-appb-000064
对比例
Figure PCTCN2015085089-appb-000065
步骤1：以化合物c和ct-2为起始原料，参照化合物i的方法进行制备，得化合物ct-3，收率65％。MS m/z(ESI):405[M+H]+。
步骤2至3：以化合物ct-3为原料，参照化合物a中步骤3和4的方法制备，得化合物ct-5，收率55％。MS m/z(ESI):429[M+H]+。
步骤4：以化合物ct-5和w为起始原料，参照实施例1中步骤1的方法制备，得化合物ct-6，收率11.7％。MS m/z(ESI):616[M+H]+。
步骤5至6：以化合物ct-6为原料，参照实施例3的方法制备，得对比化合物1，收率11％。MS m/z(ESI):486[M+H]+。
测试例1：对野生型EGFR和突变型EGFR激酶的活性抑制测试
以下z’-lyte测试方法中所用试剂均购自Invitrogen。
利用z’-lyte方法测定对T790M/L858R双突变型EGFR激酶(Invitrogen，PV4879)、野生型EGFR激酶(Invitrogen，PV3872)的抑制活性。
10μL T790M/L858R激酶反应体系中各组分的工作浓度为：25μM ATP，0.1ng/μL T790M/L858R激酶，2μM Tyr04底物(Invitrogen，PV3193)。加入本发明上述实施例制备的化合物(即待测物)后DMSO的浓度为2vol％。
10μL野生型EGFR激酶反应体系中各组分的工作浓度为：10μM ATP，0.8ng/μL野生型EGFR激酶，2μM Tyr04底物(Invitrogen，PV3193)。加入待测物后DMSO的浓度为2vol％。
室温溶解10mM的药物储存液，经8vol％DMSO溶液梯度稀释至终浓度4-0.002μM。每孔中加入2.5μL的待测物溶液以及5μL经反应缓冲液稀释的T790M/L858R激酶(或野生型EGFR激酶)与Tyr04底物的混合物，再加入2.5μL的ATP启动反应。其中C1孔用反应缓冲液代替ATP，C2孔不加入任何药物，C3孔按说明书描述加入磷酸化的底物。在25度摇床避光反应60分钟后。加入5μL Development Reagent B(Invitrogen，用TR-FRET稀释缓冲液进行稀释)，于室温摇床反应60分钟。在VictorX5荧光酶标仪(PerkinElmer)上读板，测定激发波长为405nm、发射波长为450nm和520nm的光吸收(例如，C3520nm表示C3孔在520nm的读值)。
抑制率计算方法(参照Invitrogen，PV3193的说明书)如下：
1、ER＝Coumarin Emission(450nm)/Fluorescein Emission(520nm)
2、磷酸化率＝(1-((ER×C3520nm-C3450nm)/((C1450nm-C3450nm)+ER×(C3520nm-C1520nm))))×100％
3、抑制率(IR)＝(1-(测试化合物的磷酸化率)/(C2的磷酸化率))×100％
用XLFIT 5.0软件(英国IDBS公司)拟合计算半数抑制浓度IC50。
表1酶抑制活性与选择抑制活性
Figure PCTCN2015085089-appb-000066
Figure PCTCN2015085089-appb-000067
由表1可知，与阳性对照物BIBW2992(阿法替尼)和AZD-9291(制备方法参见WO2013014448A1，结构如下)相比，本发明的实施例化合物1-14对EGFR突变型酶(T790M/L858R)表现出较强的抑制活性，而对EGFR野生型酶(T790M WT)抑制活性较弱，并且研究发现，将丙烯酰胺基团的取代位置变换后，化合物几乎没有活性。因此本发明的化合物对EGFR突变型酶具有较好的选择抑制活性。
Figure PCTCN2015085089-appb-000068
测试例2：EGFR T790M抑制剂对A431(EGFR野生型)及H1975(EGFR T790M突变)细胞EGFR磷酸化水平的抑制(ELISA法测定)
以下方法中的试剂、溶液的配置方法以及细胞处理和裂解液制备步骤、ELISA检测步骤均按照R&D DYC3570，R&D DYC1095E以及R&D DYC1095BE的说明书进行操作。
一、试剂和溶液
细胞裂解缓冲液：1％(W/V)NP-40，20mM Tris(pH 8.0)，137mM NaCl，10％(V/V)甘油glycerol，1mM NaVO3，2mM EDTA。
细胞裂解液：细胞裂解缓冲液+10μg/mL抑肽酶(Aprotinin)(Sigma)+10ug/mL亮抑蛋白肽酶(Leupeptin)(Sigma)，现配现用。
1xPBS缓冲液：NaCl:0.137M，KCl:0.0027M，Na2PO4-12H2O:0.01M，KH2PO4:0.0015M，pH7.4。
洗涤缓冲液：含有0.05％(V/V)Tween-20的PBS缓冲液。
检测抗体稀释液：20mM Tris，137mM NaCl，0.05％(V/V)Tween-20，0.1％(W/V)BSA，pH 7.2-7.4。
封闭液：含有1％(W/V)BSA的PBS缓冲液。
ELISA试剂盒：R&D DYC3570，R&D DYC1095E和R&D DYC1095BE。
二、H1975细胞
2.1 H1975细胞处理和裂解液制备
(1)将H1975细胞(购自中国科学院典型培养物保藏委员会细胞库)以1×104/孔的密度种到96孔板中，每孔90微升含10％(V/V)FBS的RPMI 1640培养基，37℃、5％(V/V)CO2培养过夜。
(2)将待测化合物按照MTT实验中药物稀释方法稀释，将10μL稀释后的化合物或稀释后的DMSO加入到细胞培板的对应孔中，DMSO终浓度为0.5％(V/V)，37℃、5％(V/V)CO2培养1小时。以纯DMSO处理的细胞培养体系作为细胞对照。
(3)吸掉培养基后加入100μL细胞裂解液，封板模封置于-80℃冰箱中过夜。以细胞裂解缓冲液作为空白对照。
2.2 ELISA检测步骤
按照R&D DYC1095E或R&D DYC1095BE给定说明书进行操作。
(1)R&D捕获抗体((DYC1095BE或DYC1095E))用PBS 1:180稀释，稀释好的抗体100μL/孔加入ELISA反应板(Corning costar 42592)，25℃摇床包被过夜；
(2)360μL洗涤缓冲液洗3次；
(3)加入300μL封闭液，25℃摇床孵育2小时；
(4)360μL洗涤缓冲液洗3次；
(5)加入40μL细胞裂解缓冲液和60μL细胞裂解液，25℃摇床孵育2小时；
(6)360μL洗涤缓冲液洗3次；
(7)检测抗体用检测抗体稀释液以试剂盒说明规定比例稀释，每孔加入100μL，25℃摇床避光孵育1小时；
(8)360μL洗涤缓冲液洗3次；
(9)将TMB底物(R&D DY999)中的A试剂和B试剂以1:1进行混合，每孔100μL，25℃摇床避光孵育20分钟；
(10)2N H2SO4每孔加入50μL；
(11)用酶标仪读板(Thermo Multiskan K3)分别测定细胞对照、空白对照以及药物处理情况下的OD 450值和OD570值，并用相同孔的OD 450值减去相应OD570值分别得到OD细胞、OD空白和OD药物处理。
2.3数据分析
抑制率(％)＝100％×(OD细胞-OD药物处理)/(OD细胞-OD空白)
2.4将计算得到的抑制率用XLFIT 5.0软件计算出IC50值，参见表4。
三、A431细胞
3.1A431细胞的处理和测试步骤
(1)将A431细胞(购自中国科学院典型培养物保藏委员会细胞库)以1×104/孔的密度种到96孔板中，每孔90微升含有10％FBS的DMEM培养基37℃、5％CO2培养过夜。
(2)将A431细胞培养基更换为90微升无血清DMEM培养基，继续培养过夜。
(3)将待测化合物按照MTT实验中药物稀释方法稀释，将10μL稀释后的化合物或稀释后的DMSO加入到细胞培板的对应孔中，DMSO终浓度为0.5％，37℃、5％CO2培养1小时。然后在除细胞对照孔外的每孔中加入10微升2μg/L的EGF(R&D，236-EG-01M)，在细胞孔加入10微升无血清DMEM培养45分钟；以不加入EGF与药物处理的细胞作为 细胞对照，以不加入药物的只加入EGF处理的细胞作为EGF对照。
(4)吸掉培养基后加入100μL细胞裂解液，封板模封置于-80℃冰箱中过夜。
3.2 ELISA检测步骤
参照R&D DYC3570E说明书进行操作。
(1)R&D捕获抗体(DYC3570E)用PBS 1:180稀释，稀释好的抗体100μL/孔加入ELISA反应板(Corning costar 42592)，25℃摇床包被过夜；
(2)360μL洗涤缓冲液洗3次；
(3)加入200μL封闭液，25℃摇床孵育2小时；
(4)360μL洗涤缓冲液洗3次；
(5)加入40μL细胞裂解缓冲液和60μL细胞裂解液，25℃摇床孵育2小时；
(6)360μL洗涤缓冲液洗3次；
(7)检测抗体用检测抗体稀释液以试剂盒说明规定比例稀释，每孔加入100μL，25℃摇床避光孵育1小时；
(8)360μL洗涤缓冲液洗3次；
(9)将TMB底物(R&D DY999)中的A试剂和B试剂以1:1进行混合，每孔100μL，25℃摇床避光孵育20分钟；
(10)2N H2SO4每孔加入50μL；
(11)用酶标仪读板(Thermo Multiskan K3)分别测定细胞对照、空白对照以及药物处理情况下的OD 450值和OD570值，并用相同孔的OD 450值减去相应OD570值分别得到ODEGF、OD药物、OD细胞。
3.3数据分析
抑制率(％)＝100％×(ODEGF-OD药物)/(ODEGF-OD细胞)
3.4将计算得到的抑制率用XLFIT 5.0软件计算出IC50值，参见表2。
表2细胞活性ELISA法测定结果
Figure PCTCN2015085089-appb-000069
由表2可知，与阳性对照物BIBW2992相比，本发明实施例的化合物对EGFR突变型细胞具有较好的选择抑制活性。
测试例3：MTT(3-(4，5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐)方法检测细胞抑制活性
MTT测试方法步骤采用本领域技术人员熟知的方法进行，方法中所用试剂均可市购得到。
首先，移除培养基并加入1mL 0.25％的胰酶/EDTA(Gibco，25200-056)。洗一次后，再加入1.5mL胰酶/EDTA消化贴壁细胞，至细胞分离，然后加入3.5mL培养基终止消化。将消化完的细胞悬浮液移至15mL离心管，1300rpm离心3分钟后弃上清，并用新鲜的培养基重悬细胞。然后细胞计数，并稀释细胞至以下浓度：H1975细胞每毫升2.78万，A431细胞和NIH3T3细胞每毫升3.33万。将细胞种入96孔板(BD 3072)，每孔90μL，培养过夜。
A431细胞培养基为：含10％FBS(Gibco，10099-141)的DMEM(Hyclone SH30243.01B)；
NIH3T3细胞培养基为：含10％FBS(Gibco，10099-141)的DMEM(Hyclone SH30243.01B)；
H1975细胞培养基为：含10％FBS(Gibco，10099-141)的RPMI-1640(Hyclone SH30809.01B)；
取20μL 10mM待测化合物，按照如下浓度梯度(2000，666.67，222.22，74.07，24.69，8.23，2.74，0.91μM)稀释200X药品，再加入无血清培养基(终浓度为：100，33.33，11.11，3.704，1.235，0.412，0.137，0.046μM)，并每孔加入10ul药品到细胞培养板内其中DMSO终浓度为0.5％。
加药后将细胞放入培养箱，培养72小时后，每孔加入10μL的5mg/ml的MTT(Sigma，M5655)溶液，然后将96孔板放入37℃5％CO2培养箱孵育4小时。
再在2000rpm，5min的条件下离心平板，移除上清后，每孔加入150μL DMSO，并在摇床中震荡平板至所有结晶紫溶解。最后使用酶标仪测定492nm光吸收，使用XLFIT5.0软件(英国IDBS公司)计算IC50。
表3化合物对细胞生长的抑制活性和选择性
Figure PCTCN2015085089-appb-000070
由表3可知，与阳性对照物BIBW2992相比，本发明实施例化合物对EGFR突变型细胞(H1975细胞)表现出较强的抑制活性，而对EGFR野生型细胞(A431细胞)表现出较弱抑制活性，因此本发明的化合物对EGFR突变型细胞具有较好的选择抑制活性。
表4化合物对NIH3T3细胞的毒性测试结果
化合物编号 NIH3T3细胞MTT测试(IC50/uM)
1 >10
2 >10
3 >10
4 2.094
5 2.632
6 5.101
7 2.423
9 >10
10 >10
13 >10
BIBW2992 2.750
由表4可知，与阳性对照物BIBW2992相比，本发明实施例化合物对NIH3T3细胞具有较高的IC50值，因此显示出较小的毒性。
从体外激酶活性抑制、细胞内EGFR磷酸化水平抑制、细胞生长抑制实验显示，本发明实施例化合物对突变型EGFR酶活性、EGFR磷酸化水平、细胞增殖表现出较强的抑制活性，而对野生型EGFR酶活性、EGFR磷酸化水平、细胞增殖表现出较弱抑制活性，因此对EGFR突变株细胞具有较好的选择性。细胞毒性实验中对NIH-3T3细胞有极弱的抑制作用，故表现出较低的细胞毒性。因此，此类化合物对T790M突变的EGFR有较好的选择抑制活性和较低的细胞毒性。
同时本发明的化合物表现出良好的生物利用度。
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 (15)

  1. 一种式(I)所示的化合物，或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药：
    Figure PCTCN2015085089-appb-100001
    式中，
    A环为取代或未取代的C3-10杂环基、取代或未取代的C6-10芳基环或取代或未取代的C4-10环烯基；
    所述的“取代”是指环原子上的1至6个氢原子被选自下组的取代基所取代：羟基、CN、NO2、卤素、C1-3烷基、卤代的C1-3烷基、-CON(C1-3烷基)2、-C(O)OC1-3烷基、-OC(O)C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3烷基、-S(O)-苯基、-N(C1-3烷基)2；
    X为共价键，或为NH、O或S；
    R0为氢、C1-3烷基、卤代的C1-3烷基、C3-6环烷基、卤代的C3-6环烷基、-CHO、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基；
    R1、R2各自独立地为氢、卤素、C1-3烷基、卤代的C1-3烷基；
    R3选自下组：
    Figure PCTCN2015085089-appb-100002
    Figure PCTCN2015085089-appb-100003
    其中，所述的苯基为未取代的或被1-3个选自下组的取代基所取代：卤素、C1-3烷基。
  2. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其前药，其中，R3选自下组：
    Figure PCTCN2015085089-appb-100004
  3. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，R0为氢、甲基、乙基、丙基、异丙基、环丙基、环丁基、环戊基、环己基、一氟乙基、二氟甲基、三氟甲基、-COCH3、-CO-苯基、-SO2CH3或-SO2-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、溴、甲基、乙基；和/或
    R1、R2各自独立地为氢、氟、氯、溴、甲基、乙基、丙基、异丙基、一氟乙基、二氟甲基、三氟甲基。
  4. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述取代或未取代的C3-10杂环基为取代或未取代的含1、2或3个氮原子的9至10元双环杂芳基环，选自下组：
    Figure PCTCN2015085089-appb-100005
    其中，Z11为CR15或N；Z12为CR16或N；Z21为CR26或N；
    R12、R13、R14、R15、R16、R22、R23、R24、R25、R26各自独立地为氢、羟基、CN、NO2、卤素、卤代的C1-3烷基、C1-3烷基、-CON(C1-3烷基)2、-N(C1-3烷基)2、-C(O)OC1-3烷基、-OC(O)C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3烷基、-S(O)-苯基；所述的烷基、苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基；
    R11、R21各自独立地为氢、卤代的C1-3烷基、C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、 甲基。
  5. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述取代或未取代的C3-10杂环基为取代或未取代的含1到2个氮原子的5至6元单环杂芳基环，选自下组：
    Figure PCTCN2015085089-appb-100006
    其中，Z31为CR34或N；
    R32、R33、R34、R35、R36、R37、R38各自独立地为氢、羟基、CN、NO2、卤素、卤代的C1-3烷基、C1-3烷基、-CON(C1-3烷基)2、-N(C1-3烷基)2、-C(O)OC1-3烷基、-OC(O)C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3烷基、-S(O)-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基；
    R31为氢、卤代的C1-3烷基、C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基。
  6. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述取代或未取代的C3-10杂环基为取代或未取代的含1个氮原子的4至7元饱和单杂环，选自下组：
    Figure PCTCN2015085089-appb-100007
    其中，n1为1、2或3；n2为1或2；
    R41为氢、C1-3烷基、卤代的C1-3烷基、-COC1-3烷基、-CO-苯基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3烷基、-S(O)-苯基；所述的苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、甲基。
  7. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，式(I)化合物为式(Ⅱ)、式(Ⅲ)、式(Ⅳ)、式(Ⅴ)、或式(Ⅵ)所示的化合物：
    Figure PCTCN2015085089-appb-100008
    式中，R11、R12、R13、R14、R0、R1、R2、R3、Z11、Z12如权利要求4中所定义；
    Figure PCTCN2015085089-appb-100009
    式中，R21、R22、R23、R24、R25、R0、R1、R2、R3、Z21如权利要求4中所定义；
    Figure PCTCN2015085089-appb-100010
    式中，R31、R32、R33、R0、R1、R2、R3、Z31如权利要求5中所定义；
    Figure PCTCN2015085089-appb-100011
    式中，R35、R36、R37、R38、R0、R1、R2、R3如权利要求5中所定义；
    Figure PCTCN2015085089-appb-100012
    式中，R41、R0、R1、R2、R3、X、n1、n2如权利要求6中所定义。
  8. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述取代或未取代的C3-10杂环基选自：
    Figure PCTCN2015085089-appb-100013
  9. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述取代或未取代的C6-10芳基环选自：
    Figure PCTCN2015085089-appb-100014
  10. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，式(I)化合物为式(Ⅶ)或式(Ⅷ)所示的化合物：
    Figure PCTCN2015085089-appb-100015
    式中，R0、R1、R2、R3如权利要求1中所定义。
  11. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述式(I)化合物选自下组：
    Figure PCTCN2015085089-appb-100016
  12. 一种药物组合物，所述组合物包括权利要求1至11中任一项所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药；以及药学可接受的载体。
  13. 权利要求1至11中任一项所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药在(i)制备调控EGFR酪氨酸激酶活性的药物、或者(ii)制备预防和/或治疗EGFR相关疾病的药物中的应用。
  14. 一种药用组合物，所述药用组合物包括：
    权利要求1-11中任一项所述的化合物、或其药学上可接受的盐、或其立体异构体、或其溶剂化物、或其前药；
    以及选自下组药物中的一种或多种：吉非替尼、厄洛替尼、埃克替尼、拉帕替尼、XL647、NVP-AEE-788、ARRY-334543、EKB-569、BIBW2992、HKI272、BMS-690514、CI-1033、凡德他尼、PF00299804、WZ4002、西妥昔单抗、曲妥珠单抗、帕尼突单抗、马妥珠单抗、尼妥珠单抗、扎鲁木单抗、帕妥珠单抗、MDX-214、CDX-110、IMC-11F8、Zemab、Her2疫苗PX 1041、HSP90抑制剂、CNF2024、坦螺旋霉素、阿螺旋霉素、IPI-504、SNX-5422、NVP-AUY922。
  15. 一种权利要求1所述的式(I)所示的化合物，或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药的制法，其特征在于，
    所述方法包括步骤：
    (i)对式I-f化合物进行还原反应，从而形成式I-g化合物；
    (ii)将式I-g化合物与丙烯酰氯进行缩合反应，从而形成式(I)所示的化合物；
    Figure PCTCN2015085089-appb-100017
    或所述方法包括步骤(i’)在惰性溶剂中，将式I-c化合物与式I-h化合物反应，从而形成式(I)所示的化合物；
    Figure PCTCN2015085089-appb-100018
    上述各式中，R0、R1、R2、R3、X、A环的定义如权利要求1中所定义；L1为离去基团。
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EP3173412A4 (en) 2018-02-28
JP6457623B2 (ja) 2019-01-23
US20170233395A1 (en) 2017-08-17
JP2017524703A (ja) 2017-08-31
US9890168B2 (en) 2018-02-13

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